Abstract

ASTROCYTES PROVIDE A SIGNALING CONDUIT FOR UPSTREAM (PIAL ARTERIOLAR) DILATION DURING CORTICAL NEURONAL ACTIVATION IN THE RAT
Dale Pelligrino, Hao-Liang Xu
Neuroanesthesia Research Department, University of Illinois at Chicago, Chicago, IL, USA
BACKGROUND: The tight coupling between neuronal activity and blood flow is fundamental to brain function. Astrocytes play an important role in that process, via their capacity to “sense” neuronal activity and, in turn, transmit that information to parenchymal arterioles. In this study, we examined another aspect of astrocyte-related neurovascular coupling?the ability of astrocytes, in vivo, to act as a signaling conduit for the vitally important process of upstream vasodilation (represented by pial arterioles). Pial arterioles are segregated from the underlying cortical neuropil by the presence of the astrocytic processes comprising the glia limitans (GL). We, therefore, postulated that, irrespective of the strength of cortical neuronal activation, whether excessive (bicuculline-induced seizure [BIS]) or physiologic (sciatic nerve stimulation [SNS]), the associated pial arteriolar dilation involves an astrocytic and GL-related signaling conduit. To test this, we topically applied (via a closed cranial window) L-alpha-aminoadipic acid (L-AAA), an astroglial toxin which selectively damages the GL (without affecting vascular smooth muscle, endothelium, or neurons). Two other hypotheses were addressed: a) inter-astrocytic signaling is dependent upon the gap junctional protein, connexin-43 (Cx43); and b) signaling from the GL to pial arterioles involves a diffusible mediator.
METHODS: Pial arteriolar diameter increases during BIS or SNS, in anesthetized rats, were measured in the absence or presence of L-AAA or Cx43 blockade (via topically-applied gap-27 peptide) and during increased rates of cortical suffusion (tests influence of diffusible mediator). In BIS experiments, EEG power increases were not altered by any experimental intervention, indicating that the changes in pial arteriolar responses were not due to changes in the level of cortical activation.
RESULTS: The predominance of an astrocytic signaling conduit was indicated by data showing that pial arteriolar dilating responses to both neuronal activation paradigms were completely blocked following selective disruption of the superficial GL (fig. 1). Gap-27 elicited a 60–75% reduction in BIS and SNS-induced dilations (fig. 2). Increasing the aCSF suffusion rate led to 40–50% reductions in BIS and SNS-related dilations (fig. 3).
CONCLUSIONS: Astrocytes and the GL play a key role in signaling upstream pial arteriolar dilation during both moderate and excessive cortical neuronal activation. The signaling process may involve Cx43-related gap junctions and/or hemichannels and a diffusible mediator arising from the GL.
CHARACTERIZATION OF A NOVEL POTASSIUM CHANNEL, TWIK-2, CLONED FROM THE RAT MIDDLE CEREBRAL ARTERY
Eric Lloyd, Sean Marrelli, Robert Bryan
Department of Anesthesiology, Baylor College of Medicine, Houston, TX, USA
Background and aims: recently a new family of ion channels, termed two-pore domain potassium channels (K2P), has been discovered and cloned. Although little is known about the expression and function of these channels in the vascular system, we have demonstrated that TWIK-2, a member of the K2P family, is highly expressed in rat middle cerebral artery (MCA) (AJP 291:H770, 2006). The aim of this study was to characterize TWIK-2 and determine if its activation could dilate rat MCAs.
Methods: TWIK-2 was cloned from rat MCA and expressed heterologously (COS and CHO cells) as a fusion protein to green fluorescent protein (GFP) for characterization. Electrical properties of TWIK-2 were determined using patch clamp methods in the whole cell configuration. MCAs were removed from rat brain, mounted on micropipettes in a vessel bath, pressurized, and perfused. MCAs were magnified and the diameter recorded using image analysis software.
Results: Direct fluorescence analysis by deconvolution microscopy in transiently transfected COS cells revealed that GFP-rTWIK-2 localized to the plasma membrane in a punctate pattern by 48 hours after transfection. Stable transfectants of GFP-rTWIK-2 were selected in CHO cells and analyzed by whole-cell electrophysiology. Whole cell currents were 30-fold greater than the nontransfected control group, were non-rectifying in physiological K+, but were inwardly rectifying in symmetrical K+. The observed reversal potentials in different concentrations of extracellular K+ were similar to the theoretical reversal potentials. In physiological K+, membrane potential was −81 +/− 0.5 mv (n=13) for transfected and −51 +/− 2.5 mv (n=16) for control cells. 1 mM BaCl2 inhibited currents by 90 +/− 1% with a calculated IC50 of 86 uM. The TWIK-2 currents were minimally affected by 10 mM tetraethylammonium chloride (TEA), 3 mM 4-aminopyridine (4AP), and 10 uM glibenclimide (glib). 100 uM arachidonic acid increased the currents by 88 +/− 15% (n=6). In isolated pressurized rat MCA, arachidonic acid elicited robust dilations that were not blocked by 10 mM TEA, 3 mM 4AP, or 10 uM glib.
Conclusions: TWIK-2 is a novel potassium channel expressed in cerebral arteries. It is characterized by a non-rectifying current in physiological K+ gradients and is resistant to most potassium channel inhibitors. We conclude that TWIK-2 is an important regulator of vascular tone. (RO1 NS 46666, NS 038660, and AHA Bugher Award 027011N)
TISSUE-TYPE PLASMINOGEN ACTIVATOR CONTRIBUTES TO NEUROVASCULAR COUPLING
Laibaik Park1, Gang Wang1, Josef Anrather1, Sidney Strickland2, Costantino Iadecola1
1Division of Neurobiology, Weill Medical College of Cornell University, New York, NY, USA, 2Laboratory of Neurobiology & Genetics, The Rockefeller University, New York, NY, USA
Introduction
The cerebral blood flow (CBF) increase evoked by synaptic activity, or functional hyperemia, is critical for the brain's functional integrity (1). A substantial component of the CBF response to activation is mediated by NMDA receptors. Tissue plasminogen activator (tPA), a protease involved in intravascular fibrinolysis, participates in NMDA receptor signaling (2), raising the possibility that tPA plays a role in functional hyperemia. Therefore, we investigated whether tPA contributes to the increase in CBF evoked by somatosensory activation.
Methods
CBF was recorded by laser-Doppler flowmetry in anesthetized mice (urethane-chloralose) equipped with a cranial window overlying the somatosensory cortex (n=5/group). Arterial pressure and blood gases were monitored and controlled.
Results
The increase in somatosensory cortex blood flow produced by whisker stimulation was attenuated in tPA-/− mice (Figure A), but the response to topical application of acetylcholine was intact (tPA+/+: 22±3%; tPA-/−: 22±2%; p>0.05; ANOVA). The attenuation in functional hyperemia in tPA-/− mice was rescued by exogenous tPA (rtPA) (Figure A). Functional hyperemia was not attenuated in mice lacking plasminogen (plg), the substrate of tPA (plg+/+: 24±3%; plg-/−: 23±1%; p>0.05). The NMDA receptor inhibitor MK-801 (10 µM; topical application) attenuated functional hyperemia in tPA+/+, but not in tPA-/− mice (Figure B), suggesting that the effect of tPA is mediated through the NMDA receptor. Consistent with this suggestion, the CBF increase elicited by topical NMDA (40 µM), but not kainate (10 µM), was reduced in tPA-/− mice (Figure C). The CBF increase produced by NMDA is mediated by neuronal nitric oxide synthase (nNOS), an enzyme functionally coupled to NMDA receptors through the scaffolding protein PSD-95 (3). A peptide that disrupts the interaction between nNOS and NMDA receptors (NR2B9c), but not its scrambled control (scNR2B9c), blocked the ability of rtPA to rescue functional hyperemia in tPA-/− mice (Figures A & B).
Conclusions
tPA is required for the CBF increase evoked by neural activity. This effect is selective for NMDA receptor-mediated hyperemia and is independent of tPA's proteolytic activity. The results with NR2B9c suggest that tPA exerts its effect by modulating the interaction between the NMDA receptor complex and nNOS through PSD-95. These results unveil a previously unrecognized role of tPA in vital homeostatic responses coupling brain activity with CBF.
NOC/OFQ ANTAGONIST POST INJURY TREATMENT PROTECTS AGAINST CEREBRAL CIRCULATORY COLLAPSE AND HISTOPATHOLOGY AFTER COMBINED PROLONGED HYPOTENSION AND BRAIN INJURY
William M. Armstead, Xiao-Han Chen, Douglas H. Smith
University of Pennsylvania, Philadelphia, PA, USA
Introduction: In humans, traumatic brain injury (TBI) is often accompanied by hypovolemia and hypotension resulting from damage to other organ systems, which increases morbidity and mortality. Basic science animal studies have observed disturbed cerebral autoregulation during acute hypotension after TBI but few have investigated the effects of prolonged hypotension. The opioid NOC/oFQ contributes to impaired cerebral hemodynamics after fluid percussion brain injury (FPI) in the pig. In the context of the neurovascular unit concept, this study addressed the hypothesis that NOC/oFQ contributes to the transition from reversible cerebrovascular dysregulation to the cerebral circulatory collapse and histopathology that occurs during combined prolonged hypotension and FPI.
Methods: FPI (2 atm) was produced in pigs (4 weeks old) equipped with a closed cranial window and CBF determined by radiolabeled microspheres. Acute (10 min) hypotension (45% decrease in arterial blood pressure) was induced at 60 and 240 min post FPI, prolonged hypotension (225 min) was induced at 15 min post FPI, while the NOC/oFG antagonist [F/G]NOC/oFQ(1-13)NH2 (NA) (1 mg/kg iv) was administered 30 min post FPI.
Results: CSF NOC/oFQ was increased significantly more by prolonged hypotension and FPI compared to acute hypotension and FPI or normotensive FPI. CBF and pial artery diameter decreased after normotensive FPI. Pial artery dilation (PAD) was blunted at 60,120, and 240 min during acute hypotension and at 60 min post FPI for prolonged hypotension. In contrast, at 120 and 240 min post FPI and prolonged hypotension, PAD during hypotension was reversed to vasoconstriction. In the pons, CBF was decreased at 60, but not at 120, 240 min post FPI during acute hypotension, but decreased at 60, 120, and 240 min post FPI during prolonged hypotension. In the cerebrum, CBF was decreased more during prolonged compared to acute hypotension at all timepoints post FPI. Hypercapnia (PaCO2 55 or 75 mm Hg) and topical acetylcholine (10-7, 10-5 M) induced PAD was blunted significantly greater during prolonged hypotension compared to acute hypotension or normotension at 60 min post FPI, but was reversed to vasoconstriction during prolonged hypotension at 120 min post FPI. Post FPI administration of the NOC/oFQ antagonist NA blocked the reversal of PAD to vasocontriction, the CBF dysregulation in the cerebrum and pons, and the reversal of PAD to vasoconstriction with hypercapnia and acetylcholine administration during prolonged hypotension at 120, 240 min post FPI. H + E staining of the CA3 hippocampus demonstrated normal neurons in sham control animals, considerable degenerating neurons in FPI and prolonged hypotension animals, but no degenerating neurons in injured animals post treated with NA. In the cortex, significant hemorrhage with edema and high density of degenerating neurons were noted in untreated FPI and prolonged hypotension animals, but small hemorrhage and edema with few degenerating neurons in injured animals that received NA.
Conclusions: These data indicate that NOC/oFQ contributes to the transition from reversible dysregulation to circulatory collapse and histopathology after combined prolonged hypotension and FPI. These observations suggest a novel pharmacotherapeutic intervention for the treatment of CNS disorders resulting from complex hypovolemic TBI origin.
IN VIVO MULTIPHOTON MICROSCOPY OF OLFACTORY GLOMERULI REVEALS ASTROCYTIC CONTROL OF NEUROVASCULAR COUPLING VIA MULTIPLE GLUTAMATERGIC PATHWAYS
Gabor C. Petzold, Dinu F. Albeanu, Tomokazu Sato, Venkatesh N. Murthy
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA
Background and aims: Glutamate is centrally involved in the signaling pathways underlying neurovascular coupling. However, the relevance of presynaptic release, postsynaptic actions and extrasynaptic activity of glutamate have remained controversial. Recent studies have suggested that astrocytic metabotropic glutamate receptors (mGluR) are involved in stimulation-evoked changes of cerebral blood flow (CBF), but the physiological relevance of this and other astrocytic pathways have remained undefined. The aim of this study was to noninvasively investigate these pathways in vivo using physiological stimuli.
Methods: We imaged mice expressing a reporter of neurotransmitter release called synaptopHluorin (spH) in synapses made by the sensory neurons of the olfactory system in vivo using two-photon microscopy. With this probe, presynaptic glutamate release by olfactory sensory synapses in specialized regions called glomeruli can be visualized following physiological odor stimulation (Fig. 1a). Simultaneously, we monitored erythrocytic velocity, flow and linear density within glomeruli by injecting Texas Red dextran into the tail vein and obtaining line scans along the axis of glomerular capillaries (Fig. 1b-d).
Results: We found that CBF increases in glomeruli activated by physiological stimulation with odorants. The CBF increase showed a wide dynamic range (~5–70%), and was highly correlated with the amount of presynaptic glutamate release (Fig. 1e). Surprisingly, odor-evoked CBF increase was not affected by the suppression of postsynaptic activity of the principal output neurons using antagonists of AMPA and NMDA receptors. Glutamate is likely to mediate neurovascular coupling by acting on astrocytes since blockers of astrocytic mGluR-5 reduced the odor-induced CBF increase. Cyclooxygenase (COX) inhibitors partially prevented the effects of mGluR-5 antagonists, indicating the involvement of the previously described phospholipase-A signaling pathway. Unexpectedly, the CBF increase was also inhibited by blockade of glutamate uptake by the broad-spectrum glutamate transport inhibitor DL-threo-β-Benzyloxyaspartic acid and by the astrocyte-specific glutamate transport inhibitor dihydrokainate. This inhibition remained unchanged after pretreatment with COX inhibitors, indicating that astrocytic mGluR and glutamate uptake affect neurovascular coupling via separate pathways.
Conclusions: We have shown that neurovascular coupling in olfactory glomeruli involves presynaptic release of glutamate, but not its postsynaptic actions. Our data also indicate that glutamate increases CBF by acting on astrocytes via separate pathways: stimulation of mGluR and subsequent COX activation, and uptake by astrocytic glutamate transporters.
THE RESPONSE OF THE CEREBRAL MICROVASCULATURE TO REGIONAL CHANGES IN CEREBRAL BLOOD FLOW
Mark G.F. Catherall, Alexander B. Rowley, Stephen John Payne
Department of Engineering Science, University of Oxford, Oxford, UK
Background
The cerebral microvasculature is a highly interconnected network of vessels, which delivers oxygen and other nutrients to brain tissue. Brain tissue is permeated with these vessels, from which no region of brain tissue is very far. The high level of connectivity provides a degree of ‘redundancy’, whereby no individual micro-vessel is crucial for oxygen supply to any region of brain tissue. Even at a higher level in the vascular tree, the effects of reductions or stoppages in blood flow can be ameliorated by this vessel connectivity, although, without detailed knowledge of the structural and connectivity properties of the microvasculature, the regional response to changes in blood flow cannot be estimated accurately. However, recent experimental work has led to the characterisation of the cerebral microvasculature in terms of vessel lengths, diameters and connectivity (Cassot et al., Microcirculation, 13: 1-18, 2006). This enables the response of the microvascular flow, and consequent tissue oxygen concentration, to be analysed under changing input conditions.
Methods
Two and three-dimensional mathematical models of a section of the microvasculature have been constructed, using both regular patterns for vessel distribution and an approach based on random node allocation. The former model has vessels all of the same length, initially with each node connected to its 4 (in 2-D) and 6 (in 3-D) nearest neighbours: the three-connectedness of the microvasculature is obtained by removal of connections based on the flow patterns. These flow patterns are obtained by allocating sources and sinks to individual nodes, to simulate the supplying arterial and draining venous vessels. The latter model generates random nodes and connects the vessels in a way that obtains the correct length distribution and average connectivity. The blood flow through the network is then calculated by solving the pressure-resistance equations in matrix form and the oxygen concentration in the blood vessels is calculated from the mass transport equation. The tissue oxygen concentration is solved using the convection-diffusion equation for a given metabolic consumption.
Results and conclusions
The response of the network to decreases in blood flow has been examined to investigate how the connectivity of the network provides redundancy. It is found that the greater the connectivity, the more robust the network is to local decreases in supplying blood flow. This is also heavily dependent upon the initial flow patterns, as the blood flow in the network can alter significantly upon a local decrease if the initial flow patterns are particularly directional. However, even under extreme conditions, the network is able to provide a high level of ‘protection’ by adjusting the flow patterns and this may play a key role in the response to decreases in cerebral blood flow. It is hoped that this will help in our understanding of the response of the cerebral vasculature in diseased states such as stroke and dementia as well as interpreting the low frequency oscillations that play an important role in resting state networks.
EFFECTS OF EC-IC BYPASS SURGERY ON CEREBRAL PERFUSION IN MOYAMOYA DISEASE AND NON-MOYAMOYA ISCHEMIC STROKE EVALUATED BY OPTICAL TECHNIQUES
Tatsuya Hoshino, Noriaki Yokose, Takashi Awano, Shin Nakamura, Norio Fujiwara, Yoshihiro Murata, Tsuneo Kano, Kaoru Sakatani, Yoichi Katayama
Department of Neurological Surgery, Nihon University School of Medicine, Itabashi-Ku, Tokyo, Japan
Background: EC-IC (extracranial-intracranial) bypass such as superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis has become a standard surgical therapeutic option in moyamoya disease (MD) to prevent recurrent ischemic events. In addition, EC-IC bypass surgery has been performed to prevent stroke in patients with transient ischemic attack caused by hemodynamic compromise. Several studies have demonstrated beneficial effects of bypass surgery on cerebral hemodynamic status in both MD and non-moyamoya ischemic strokes (non-MD). However, little is yet known regarding the difference between these groups of patients in the extent to which the bypass contributes to maintaining adequate cerebral blood oxygenation (CBO), or the temporal changes after surgery. In the present study, we evaluated the CBO changes induced by bypass blood flow in patients with MD and non-MD during the perioperative periods employing optical spectroscopy including visual light spectroscopy (VLS) and near infrared spectroscopy (NIRS).
Methods: We developed a VLS monitoring system which permits continuous monitoring of CBO changes during surgery. Employing the VLS, we evaluated the CBO changes in the MCA territory on the lesion side in 13 patients who underwent STA-MCA anastomosis, including 5 MD and 8 non-MD patients. In addition, employing NIRS, we evaluated the effects of STA blood flow on the CBO at one week after surgery. We measured the changes in CBO during compression of the anastomosed STA; when decreases in oxyhemoglobin concentrations were observed, the bypass was considered to maintain CBO in the MCA territory because decreases in oxyhemoglobin indicate cerebral ischemic changes.
Results: In 4 MD patients and one non-MD patient, the STA blood flow increased the oxyhemoglobin and cortical oxygen saturation (CoSO2), indicating that the bypass supplied blood flow to the ischemic brain; the CBO changes were observed more frequently in MD than in non-MD (p<0.02). The preanastomosis CoSO2 (65.4±5.4%) in MD was significantly lower than that (72.8±7.6%) in non-MD (p<0.05). Postoperative NIRS demonstrated that the bypass began to supply blood flow to the brain in 5 non-MD patients whose bypass did not supply blood flow during surgery.
Conclusion: Although MD has vessels of small diameter as compared to non-MD, the bypass begins to supply blood flow to the ischemic brain earlier in MD than in non-MD after anastomosis. The fact that the CoSO2 in MD was lower than that in non-MD suggested that the perfusion pressure in MD was lower than that in non-MD, and this might account for the difference in the bypass blood supply after anastomosis between MD and non-MD. Our data suggest that, even if the bypass does not supply blood to the brain during surgery in non-MD, the bypass blood flow gradually increases after surgery. The optical spectroscopy is considered useful for evaluating bypass function and facilitates safe and accurate bypass surgery.
ABNORMAL DIAZOXIDE INDUCED VASODILATION IN CEREBRAL ARTERIES FROM ZUCKER OBESE RATS WITH INSULIN RESISTANCE
Prasad Katakam, James A. Snipes, Anna Busija, David W. Busija
Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston Salem, NC, USA
Diazoxide, a mitochondrial KATP channel activator, elicits arterial vasodilation by increasing the generation of mitochondria-derived reactive oxygen species (ROS) that stimulate Ca2+ sparks and Ca2+ activated K+ channels (KCa). We evaluated the diazoxide induced vasodilation in serotonin preconstricted isolated cerebral arteries from 11-13 weeks old Zucker obese (ZO) and lean (ZL) rats by videomicroscopy. The ZO rats were insulin resistant with hyperinsulinemia but were euglycemic and normotensive. Diazoxide induced vasodilation (% maximal relaxation) was diminished in ZO (5.4±5%, p<0.05) compared to ZL (43.2±14%). 3-nitropropionic acid, an activator of mitochondrial ROS generation via inhibition of succinate dehydrogenase, has no affect on vasodilation in ZL (42.7±17%, p=NS), but enhances diazoxide induced vasodilation in ZO (71.2±11, p<0.05). Diazoxide induced vasodilation was partially inhibited by iberiotoxin in ZL (23.6±7%, p<0.05) while it was totally abolished in ZO (1.1±4%). ZO arteries exhibit reduced pressure induced constriction compared to ZL (14.4±4% in ZO versus 28.4±3% in ZL, p<0.05). However, vasodilation to nitroprusside was similar in both ZO and ZL (80.4±6% in ZO versus 87.2±4% in ZO, p=NS). Thus, cerebral arteries from ZO exhibit abnormal diazoxide induced vasodilation mediated by reduced generation of mitochondrial ROS and/or Ca2+ sparks or impaired coupling of Ca2+ sparks with KCa channels.
PRESSURE AND FLOW REGULATION WITHIN THE BRAIN -FACTS, MYTHS AND MISSING LINKS
Mare Czosnyka, Ivan Timofeev, Piotr Smielewski, Andrea Lavinio, Peter Hutchinson, Peter Kirkpatrick, Jonn Pickard
Academic Neurosurgery, Addenbrooke's Hospital, Cambridge, UK
Objective: Continuous assessment of cerebral autoregulation using time- or frequency- domain correlation, phase shift- and transmission between slow waves of MCA blood flow velocity and cerebral perfusion pressure (CPP) or arterial pressure (AP) have been introduced a decade ago. We intend to review clinical applications of two indices describing cerebral autoregulation (1) and pressure reactivity (2) following head injury.
Material and Method: We studied nearly 300 ventilated head injured patients. TCD was recorded digitally along with ICP and AP daily within baseline periods (no interventions) from 10 minutes to 2 hours. ICP and AP were recorded and analysed continuously during whole period of intensive care. Moving correlation coefficient (3 min window), called Mx index, was calculated between low-pass filtered (0.05 Hz) signals of FV and CPP or AP. Similar index, expressing Pressure Reactivity (PRx), was calculated as moving correlation coefficient between AP and ICP.
Results:Facts: Mx correlated strongly with PRx index. Both indices correlated significantly with static autoregulation. Mx showed good agreement with transient hyperaemic response test, leg-cuff test, phase-shift methods, and CO2 reactivity. Poor autoregulation and pressure-reactivity proved to be independent predictor of fatal outcome following head injury. Both indices were significantly disturbed by intracranial hypertension (ICP>25 mmHg). Autoregulation was worse for low CPP (CPP<55 Hg) and for too high CPP(>105 mmHg) than for normal CPP (between 60 and 100 mmHg). Asymmetry of Mx (left-right) was associated with asymmetry of CT scanautoregulation was worse at the side of contusion and at the side of brain expansion causing midline shift. Both Mx and PRx after head injury worsens with age, probably contributing to worse outcome in elderly patients. Mx showed deterioration of cerebral autoregulation in large (>4 ug/ml) plasma concentration of propofol. PRx correlated with a state of low CBF and CMRO2 revealed using PET. PRx is more robust than Mx (no need of fixation of external probes) and seems to be helpful in optimization of CPP-oriented therapy.
Myths: It is probably not true that Mx in head injury patients can be calculated using only AP instead of CPP. Both indices are able to reflect transient changes of autoregulation, like during plateau waves of ICP. Indices can be quite noisy and decent time for averaging (30 minutes minimum) in individual cases is required to assess whether given patient at given time autoregulates or not. Thresholds for functional and disturbed autoregulation dramatically depend on arterial tension of CO2 ?therefore comparison between patients cannot be performed without comparing their PaCO2 concentrations.
Missing Links: Discrepancies between PRx and Mx are still poorly understood. Whether restoration of failing autoregulation can improve outcome, remains to be demonstrated prospectively. Can ‘optimal CPP’ therapy be understood as a consensus between CPP-protocol and Lund-concept ? Can autoregulation after head injury be improved with statins or EPO, like in SAH?
Conclusion: PRx and Mx can be used in many clinical scenarios for continuous monitoring of cerebrovascular regulation, being possibly helpful in predicting outcome and optimizing therapeutical strategies.
NEURONAL REPLACEMENT AND ELECTROPHYSIOLOGICAL CHANGES OF THE NEWLY GENERATED NEURONS IN THE ADULT STRIATUM AFTER ISCHEMIC BRAIN INJURY
Gakushi Yoshikawa1,4, Toshihiko Momiyama2,5, Soichi Oya1,4, Keisuke Takai1,4, Junichi Tanaka1,4, Nobuhito Saito1, Takaaki Kirino3,4, Nobutaka Kawahara1,4
1Department of Neurosurgery, The University of Tokyo Graduate School of Medicine, Tokyo, Japan, 2Laboratory of Cerebral Structure, National Institute for Physiological Sciences, Okazaki, Japan, 3Research Institute International Medical Center of Japan, Tokyo, Japan, 4SORST, Japan Science and Technology Corporation (JST), Osaka, Japan, 5CREST, Japan Science and Technology Corporation (JST), Osaka, Japan
<Introduction> Recent findings have provided evidence that neurons can be generated in damaged regions in which neurogenesis does not occur normally, e.g., hippocampal CA1, striatum, and neocortex. In the present study, we elucidated whether neural progenitors in the subventricular zone (SVZ) are stimulated in situ by the treatment of growth factors, migrate into the damaged striatum, and replace lost neurons following global ischemia. We also followed the time course of the number of these replaced neurons and their neural phenotypes, and investigated the electrophysiological properties using whole-cell patch-clamp recording to characterize the functional development of newly generated neurons in the adult brain. <Materials and methods> Male Wister rats were subjected to 9 minutes global ischemia under the condition of 4-vessel occlusion and hypotension, and the EGF and FGF-2 were infused into the bilateral ventricles for 7 days from Day 2. For labeling the proliferating cells, BrdU was injected intraperitoneally. We also injected GFP-expressing recombinant retroviruses into the ventricles of growth-factor treated animals at Day 2, to determine the migration of proliferating progenitors in the SVZ. <Results> At Day 2 when the treatment of growth factors had not started, severe neuronal loss in the dorso-lateral striatum was observed, where no more than 2.3% of neurons remained. Immunostaining for BrdU and neural progenitor markers such as Pax 6, Mash 1 at Day 7 showed a marked increase of the progenitor cells in the SVZ in the treated animals. At Day 42, a significant increase in the number of striatal neurons, stained with both cresyl-violet and NeuN, was observed in the treated animals (approximately 15% of normal controls, p< 0.05). We observed neurons co-expressing BrdU and NeuN in the lesioned striatum, most of which also expressed phenotypic markers of striatal medium spiny neurons (DARPP-32). Furthermore, we detected that the GFP+−infected cells in the striatum were positively stained by NeuN, indicating that these neurons were derived from dormant neural progenitors residing in the SVZ. These immunohistochemical characteristics of neurons did not change afterwards till Day 70-84, suggesting that the morphological differentiation had matured by 6 weeks. Electrophysiologically, on the other hand, newly generated striatal neurons recorded at 6 weeks showed immature patterns of membrane and firing properties, whereas surviving neurons in the medial striatum of the same specimen showed the mature patterns. Developmental changes in electrophysiological properties were also observed in newly generated striatal neurons until 20 weeks or later. <Conclusion> The treatment of growth factors potentiated proliferation of neural progenitors in the SVZ and enhanced the neuronal replacement in the damaged striatum of the adult brain. Functional development required much longer term than morphological, immunohistochemical maturation. Our present data show that recruitment of progenitors in the SVZ for the repair of ischemic injury may provide a new therapeutic approach for the treatment of stroke.
VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) INCREASES PRODUCTION, MIGRATION AND SURVIVAL OF NEURAL STEM CELLS DURING HYPOXIA AND ISCHEMIA
Hugo H. Marti1, Katja Bieber1, Yaoming Wang2, Kunlin Jin2, David A. Greenberg2
1Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany, 2Buck Institute for Age Research, Novato, CA, USA
Background and aims
Neurons are susceptible to hypoxia and ischemia. However, endogenous adaptive responses aim at protecting the tissue from hypoxic-ischemic injury. Angiogenesis and neuroprotection are two such adaptive responses, and both processes seem to be governed by the same hypoxia-induced growth factors, of which vascular endothelial growth factor (VEGF) is a prominent example. VEGF is upregulated during hypoxia and ischemia and improves outcome after stroke.
New neurons are generated continuously in the subventricular zone (SVZ) and dentate gyrus of the adult brain. Neuropathological processes, including cerebral ischemia, can enhance neurogenesis, as can growth factors and other physiological stimuli. VEGF has been implicated in the hypoxia-induced regulation of neural stem cells (NSC), but it is unknown whether VEGF can enhance migration of newborn neurons toward sites of ischemic injury, where they might be able to replace neurons that undergo ischemic death.
Methods
We previously generated transgenic mice, overexpressing human VEGF165 under the neuron-specific enolase promoter (V1 mice) [1]–[3]. V1 mice were subjected to middle cerebral artery occlusion (MCAO). Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (Brdu) labeling and immunostaining for cell type-specific markers. Effects of VEGF on differentiation, proliferation and apoptosis of isolated NSC were analyzed in vitro.
Results
In V1 mice, brains examined 7-28 days after MCAO showed markedly increased SVZ neurogenesis, chains of neuroblasts extending from the SVZ to the peri-infarct cortex, and an increase in the number of newly generated cortical neurons at 14-28 days after ischemia. In concert with these effects, VEGF overexpression reduced infarct volume and improved postischemic motor function. NSC isolated from the SVZ zone of V1 mice expressed more VEGF and differentiated faster into more mature neuronal cells. Furthermore, VEGF overexpression promoted NSC survival and proliferation under hypoxic conditions and protected NSC from apoptosis, induced by growth factor depletion. Finally, V1 NSC showed an increased phosphorylation of p44/42 MAP kinase and Akt, accompanied by reduced apoptosis under hypoxic conditions which was reversed when the PI3-kinase/Akt pathway was blocked by Wortmannin.
Conclusions
Our results indicate VEGF as a trophic factor for neurogenesis and neuromigration in the adult brain after cerebral ischemia in vivo and for NSC in vitro involving an Akt/PI3-kinase dependent mechanism. Our data suggest that in addition to its neuroprotective effects, which are associated with improved outcome in the acute phase after cerebral ischemia, VEGF enhances postischemic neurogenesis, which could provide a therapeutic target for more chronic brain repair.
VCAM-1 TARGETED INTRACAROTID DELIVERY OF NEURAL STEM CELLS IMPROVES FUNCTIONAL OUTCOME AFTER EXPERIMENTAL STROKE
Raphael Guzman, Alejandro De Los Angeles, Samuel Cheshier, Jason Liauw, Stanley Huang, Raymond Choi, Tonya Bliss, Gary Steinberg
Department of Neurosurgery, Stanford University, Stanford, CA, USA
Background: In acute injury of the brain, transplantation of neural progenitor cells (NPC) is aimed at replacing damaged neural cells. Intravascular delivery of NPC after stroke has been limited by the low efficiency of transendothelial migration. VCAM-1 is one of the endothelial adhesion molecules known to be upregulated early after stroke and is responsible for the firm adhesion of CD49d (VLA-4) expressing inflammatory cells. We hypothesized that selecting CD49d positive NPC would improve their homing to the injured brain and improve functional outcome after stroke.
Methods: Mouse NPC's were analyzed for the expression of the surface integrin CD49d by FACS analysis, immunohistochemistry and static adhesion assay. On the day of intraarterial injection, a CD49d enriched (>90%) and depleted (<1%) NPC population was obtained by magnetic bead enrichement and double FACS sorting. Bl6 mice (20-25g, n=18) were subjected to a left side hypoxia ischemia (20min 8% O2 + temporary right common carotid artery (CCA) occlusion). All mice were tested by rotarod for behavioral deficits and assigned to receive 3×105 CD49d enriched (n=6) or CD49d depleted (n=6) stem cells or vehicle (n=6) injection into the right CCA 48 hours after stroke. Animals were tested by rotarod at 7 and 14 days after stroke and subsequently sacrificed and the brains processed for immunohistochemistry.
Results: FACS analysis revealed approximately 25% CD49d positive NPC at passage six in vitro. Analysis of CD49d cluster like immunoreactivity by confocal microscopy showed a 3-fold (p=0.03) decrease of the integrin expression between passage 6 and 8. In the static adhesion assay NPC adhered to VCAM-1 in a dose dependent manner and adhesion was significantly greater than in uncoated control wells (p<0.005). Two weeks after intracarotid injection significantly more NPC were found in the cortex, hippocampus and subventricular zone adjacent to the stroke in animals receiving CD49d positive as compared to CD49d negative NPC's (1066/cm2 vs. 583/cm2, p<0.05). Behavioral recovery was significantly better for the CD49d positive cells injected group as compared to CD49d negative and vehicle injected animals (p=0.031). The CD49d negative group had a better recovery than the vehicle injected group (ns.).
Conclusions: We show that enrichment of NPC by FACS sorting for the surface integrin CD49d and intracarotid delivery promotes cell homing to the area of stroke in mice and improves sensorimotor recovery. We present two concepts which potentially improve the efficiency of intravascular delivery of stem cells for stroke treatment.
SUPPRESSED BRAIN ANGIOGENESIS DURING MODERATE HYPOXIA IN CYCLOOXYGENASE-2 DEFICIENT MICE
Joseph C. LaManna, Xiaoyan Sun, Mary P. Modesitt, Constantinos P. Tsipis
Department of Anatomy, Case Western Reserve University, Cleveland, OH, USA
Background and Aims: Exposure to chronic moderate hypoxia induces angiogenesis in tissues through cooperation of the HIF-1 transcription factor-vascular endothelial growth factor and the Cyclooxygenase-2(COX-2)-angiopoietin 2 signaling pathways(1, 2, 3). This study demonstrated the significance of COX-2 in hypoxia-induced brain angiogenesis.
Methods: male, 3-month old COX-2 knockout (KO, -/−), wild type (WT) and heterozygote (HET) mice were exposed to hypobaric hypoxia (0.4 ATM, ~8% oxygen) for 3 weeks. Their littermates were kept normoxic in the same location. Mice were fixed for immunohistochemical staining of GLUT-1, an indicator of brain capillaries. Capillary density (N/mm2) was estimated through the full depth of the frontal cortex from 5 sections (4 µm).
Results: Under normoxic conditions there was no difference in capillary density of KO mice compared to their WT and HET littermates. As expected, hypoxic exposure significantly (*) increased the capillary density in the WT and HET mice, compared to that of their normoxic littermates (491 ± 45, n = 4 vs. 416 ± 39, n = 3, mean ± SD, and 496 ± 41, n = 5 vs. 393 ± 16, n = 3, respectively). However, the capillary density in COX-2 -/− mice was unchanged (424 ± 24 vs. 401 ± 13, n = 3 each).
Conclusions: Our data suggest that the COX-2 signaling pathway is required for cerebrovascular adaptation to prolonged moderate hypoxia. However, COX-2 is apparently not necessary for establishing capillary density under normoxic conditions.
ISOLATION AND CHARACTERIZATION OF INJURY-INDUCED NEURAL STEM/PROGENITOR CELLS FROM POST-INFARCT AREA IN MICE
Takayuki Nakagomi1, Akihiko Taguchi2, Orie Saino1, Masatoshi Fujikawa1, Yoshihiro Fujimori1, Tomoyuki Nishizaki3, Tomohiro Matsuyama1
1Institute for Advanced Medical Science, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan, Hyogo, Japan, 2Department of Cerebrovascular Disease, National Cardiovascular Center, Osaka, Japan, Osaka, Japan, 3Department of Physiology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan
Background and aims
The central nervous system (CNS) is known for its limited reparative capacity creating a challenge in the design of effective reparative therapies. It has been reported that injury-induced activation of neural stem/progenitor cells occurs after brain injury. In the present study, we isolated for the first time neural stem/progenitor cells from post-infarct area in mice undergoing permanent MCA occlusion.
Methods
Permanent focal cerebral ischemia was produced in male 6-week-old SCID mice (Clea Japan Inc.) by ligation and disconnection of the distal portion of the left MCA (Taguchi, et al., 2004). The infarct area of this model was limited to the ipsilateral cerebral cortex. For the formation of neurosphere-like cell clusters, cell suspensions were made from ipsilateral post-infarct area or contralateral cerebral cortex on day 7 after stroke. These cell suspensions were incubated in medium promoting formation of neurosphere-like cell clusters. Briefly, cells were incubated in 6 cm2 tissue culture flasks with DMEM/F12 containing EGF, bFGF and N2 supplement. The total number of cell clusters exceeding 80 µm in diameter was counted every other day for 1 month using a phase contrast microscope. To examine whether these clusters give rise to generation of secondary spheres, neurosphere-like cell clusters were dissociated by passage, and incubated again. Neurosphere-like cell clusters after 6 days in culture were characterized by immunohistochemistry. Further, differentiation was induced in the medium by omitting EGF and bFGF for 7 days. The samples were subjected to the analysis by RT-PCR and immunohistochemistry. Neurosphere-like cell clusters after 14 days in culture were characterized by using FACS. Patch-cramp test also was performed for the functional analysis on differentiated neural cells.
Results
After 6 days in culture, formation of neurosphere-like cell clusters in cultures derived from the stroke-affected area was observed. These clusters continued to grow up to day 16 in culture. The total number of cell clusters also was increased (about 50 per dish). These cell clusters revealed the capability of self-renewal and again formed neurosphere-like cell clusters. In contrast, no cell clusters were seen in cell suspensions from the contralateral cerebral cortex. On immunohistochemistry, multiple proliferating cells (Ki67 antigen-positive) were observed in neurosphere-like cell clusters. Cell clusters also expressed the neural stem cell marker (nestin) and notch1. Virtually all of the nestin-positive cells expressed notch1. FACS analysis revealed that ~40% of the cells from the clusters were notch1-positive. Differentiated cells expressed nestin, a marker of neurons (Tuj1, MAP2), as well as astrocytes (GFAP) and oligodendrocyte markers (O4). Expression of transcripts for nestin, MAP-2, GFAP and PLP (a marker for oligodendrocytes) was confirmed by RT-PCR. Patch-cramp analysis showed that some of the isolated cells developed the properties of voltage-gated Na+ channels, indicating the differentiation of neural stem cells to functional neurons.
Conclusions
These results show that neurosphere-like cell clusters from post-stroke area have a character of multipotent neural stem/progenitor cells, thereby providing a potential pool of endogenous precursors to promote repair and accelerate restoration of neural function after brain injury such as stroke.
INHIBITING DEGRADATION OF HYPOXIA-INDUCIBLE FACTOR INCREASES NEURAL STEM CELLS IN MOUSE FOCAL ISCHEMIA
Tsuyoshi Uesugi1, Shunya Takizawa1, Yuko Izuhara2, Yuko Morita1, Shigeharu Takagi1, Toshio Miyata2
1Division of Neurology, Department of Internal Medicine, Tokai University School of Medicine, Kanagawa, Japan, 2Institute of Medical Science, Tokai University School of Medicine, Kanagawa, Japan
Background and purpose: Hypoxia-inducible factor (HIF) is a regulatory factor of transcription that is induced in hypoxic cells. It activates target genes involved in compensation for ischemia, including erythropoietin, VEGF, and glut-1. We examined the cerebroprotective effects of TM6008, a novel inhibitor of the HIF-degrading enzyme prolylhydroxylase (PHD), on infarct volume and intrinsic neural stem cells in mice with permanent focal ischemia.
Methods: The experiments were performed on C57BL/6J mice weighing 20 grams. Briefly, animals were anesthetized using 2.0% isoflurane anesthesia with a mixture of 30% oxygen and 70% nitrous oxide, and the left middle cerebral artery was permanently occluded by cauterizing. Mice were divided into two groups: vehicle (0.2 ml/day of milk alone) and TM6008-treated (200 mg/kg/day of TM6008 + 0.2 ml/day of milk) groups. Mice in both groups were given 50 mg/kg/day of BrdU for 2 days before sacrifice on day 7 or day 14 after occlusion. In all animals, the rotor rod test and blood sampling were performed. Coronal brain sections were fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin. Infarct volume was measured, and immunohistochemical reactions for HIF-1 and BrdU were performed.
Results: No significant difference of rotor rod test results was seen between the TM6008-treated and vehicle groups at any time point. At day 7 after occlusion, there was no statistically significant difference of infarct volume, HIF-1 or BrdU-positive cells between the two groups. At day 14, however, TM6008 significantly reduced brain atrophy and the number of BrdU-positive cells, compared with the vehicle group.
Conclusions: Inhibition of PHD by TM6008 increases intrinsic neural stem cells in the infarcted hemisphere of mice with permanent focal ischemia, accompanied with a reduction of infarct volume. Increasing HIF-1 by blocking its degradation is a novel strategy to treat cerebral infarction, at least in the subacute phase, by promoting neurogenesis.
CIRCULATING ENDOTHELIAL PROGENITOR CELLS AS THE PATHOGENETIC MARKER OF MOYAMOYA DISEASE
Kon Chu, Keun-Hwa Jung, Soon-Tae Lee, Eun-Cheol Song, Dong-In Sinn, Jeong-Min Kim, Hee-Kwon Park, Manho Kim, Jae-Kyu Roh
Stroke and Neural Stem Cell Laboratory, Department of Neurology, Stem Cell Research Center, Seoul National University Hospital, Seoul, South Korea
Backgrounds: Endothelial progenitor cells (EPCs) derived from bone marrow are believed to support the integrity of the vascular endothelium. It has reported that EPC mobilization increases following cerebral ischemia, whereas some data have suggested that the number and function of EPCs correlate inversely with cardiovascular risk factors, outcome and the presence of stroke. Moyamoya disease is an unusual from of chronic cerebrovascular occlusive disease followed by the formation of characteristically abnormal vessel. Objectives: In this study, we attempted to examine the EPC number and function in moyamoya disease. Methods: The stage of steno-occlusive lesions on angiogram was determined according to the modified staging system. MMD patients (stage II-IV) undergoing chronic stroke was included. EPCs were isolated from the peripheral venous blood of 20 patients with moyamoya disease and 20 age-matched healthy volunteers. EPC was counted as colony-forming units (CFU) at the 7-day culture, and the yield of outgrowth endothelial cells were measured during the 3 months of culture. EPC function was measured by Matrigel plate assay.
Results: The number of CFU after 7-day culture was significantly reduced in patients with moyamoya disease versus control subjects (12.34±8.66 versus 70.6±15.35, p < 0.0001). During the long-term culture, OECs was isolated from 5% of healthy volunteer, but more efficiently from 50% of patients with moyamoya disease. Tube formation was reduced in MMD, more in advanced stage. The CFU and OECs yield was not correlated with the angiographic stage.
Conclusions: The presence of moyamoya disease was associated with a reduced number of CFU together with an impairment of functional activity, but higher yield of OECs. These parameters of circulating EPCs might represent the pathogenetic process of Moyamoya disease.
HYPOXIA-INDUCED DAMAGE AND REPAIR IN ADULT MOUSE BRAIN
Marc Uy, Aaron Hirko, Renee Dallasen, Nicole Brandon, Yan Xu
Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA
Background and aims: Hypoxia is associated with a number of clinical conditions such as asthma, altitude sickness, carbon monoxide poisoning, and stroke. It has been postulated that mild hypoxia alone does not cause neuronal death, and hypoxia preconditioning in rodents has been shown to significantly reduce injury in focal and global ischemia. A possible protective mechanism through preconditioning is the pre-activation of neuronal stem cells in the adult brain tissue. We report here an investigation of the long-term physiological responses to a 30-min episode of respiratory hypoxia with 5% O2 in mice, with the ultimate goal of understanding the mechanisms of brain injury and recovery after hypoxia.
Method: Male CD1 mice, weighing 20-30 g, were anesthetized with pentobarbital. Animals were transorally intubated and mechanically ventilated. Hypoxia was induced by adjusting pO2 to 38 mmHg while the animals were paralyzed with pancuronium to prevent spontaneous gasping. Control animals were administered a normal pO2 =138 mmHg for 30 minutes. Body temperature was held constant at 36.8 ± 0.4°C. After the hypoxia, ventilation with room air (pO2 =138 mmHg) was continued until spontaneous breathing was restored. The heart rate, femoral artery pulse distention, blood O2 saturation (spO2), breath distention, and rectal temperature were recorded continuously before, during, and after the hypoxia. Diffusion-weighted MRI, apparent diffusion coefficient (ADC) maps, and angiography were acquired at 14.1 T. Final histology included Nissl staining and NeuN, GFAP, and vimentin immunohistostaining. The percent of damaged neurons and the density of GFAP+ and vimentin+ cells were analyzed.
Results and conclusions: Normal spO2 with room air ventilation was 96 ± 3%. During the 30-min hypoxia, spO2 steadily declined from 82 ± 6% (averaged the first 5 min) to 65 ± 7 % (averaged for the last 5 min). While most animals had normal ADC in the hippocampal CA1 region, some did show a significant decrease in ADC values the second day after the hypoxia, followed by an overshoot in the subsequent days. This is a typical response to ischemia explained by the initial cytosolic edema followed by the interstitial edema in response to injuries. The animals with MRI abnormalities also showed visible neuronal damage in Nissl stained sections. There is a rather strong correlation between the percent of damaged neurons in the CA1/CA3 regions and the density of vimentin+ cells in the same and adjacent regions (r2 =0.90). This result might indicate an influx of neural and astrocytic precursors in response to the cellular damage. At the short and intermediate time points examined after hypoxia, the vimentin+ cells have astrocytic morphology and are NeuN—. At a later stage, these cells are NeuN+ and resemble neurons in structure and orientation. Thus, our results suggest that similar to developing brain, the radial glia in the adult brain from the subventricular zone and dentate gyrus also migrate and transdifferentiate into neurons in response to injury after prolonged hypoxia and reoxygenation. (Funded by NIH R01NS/HL036124).
TRANSPLANTATION OF MOUSE AND PRIMATE ES CELLS INTO ISCHEMIC MOUSE BRAIN
Yasushi Takagi, Motoaki Fujimoto, Makoto Hayase, Junya Hayashi, Jun Takahashi, Nobuo Hashimoto, Kazuhiko Nozaki
Department of Neurosurgery, Kyoto University, Graduate School of Medicine, Kyoto, Japan
Background and aims
Present therapies to stroke are mainly for a primary or secondary prevention and to relieve lost neurons from the cerebral ischemia is expected. Thus cell replacement therapies; embryonic stem cells (ES cells), neural stem cells and bone marrow-derived stem cells have a possibility to offer a novel potential treatment for stroke damage.
Methods
Mouse and monkey ES cells were caused to differentiate into neuronal progenitors by the stromal cell-derived inducing activity method. Focal cerebral ischemia was induced by occluding the middle cerebral artery by the intraluminal filament technique using C57Black6 mice. The donor cells were transplanted into the ischemic lateral striatum at 24 or 48 hours after reperfusion. We analyzed the survival and the differentiation of these cells by immunohistochemical method.
Results
We generated multipotential neural progenitors from ES cells by SDIA method. These cells could differentiate in vitro into neurons, glia, and oligodendrocytes, thus indicating them to be neural stem cells. The cells transplanted into the ischemic brain became located widely around the ischemic area. Moreover, the transplanted cells differentiated into various types of neurons and glias. In the case of primate ES cells, the cells transplanted into the ischemic brain became located widely around the ischemic area. Moreover, the transplanted cells differentiated into various types of neurons and glias. These cells expressed HuC, HuD, GFAP and TuJ1. At 28days after transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into anterior thalamus on the grafted side when compared with the number at 14 days. In the case of mouse ES cells, at 2 weeks after ischemia, the transplanted cells occupied 18.8+2.5% of the hemispheric area. By 4 weeks after it, this percentage rose to 26.5+4.0%. At 4 weeks after tranplantation, green fluorescent protein (GFP)-positive transplanted cells showed a mature neuronal morphology. In addition, we investigated the expression of differentiation markers and that of various neurotransmitters. Transplanted cells were immunopositive for neuronal nuclei (NeuN), tubulin-III (TuJ1), and glial fibrillary acidic protein (GFAP). Of the GFP-positive cells, 33.3+11.5% were glutamate decarboxylase (GAD)-positive, 13.3+5.8% were glutamate-positive, 2.1+2.5% were tyrosine hydroxylase (TH)-positive, 1.8+2.0% were serotonin-positive, and 0.4+0.2% were choline acetyltransferase (ChaT)-positive ones.
Conclusions
Mouse and monkey-derived neuronal progenitors survived and differentiated in the ischemic brain. Monkey-derived neuronal progenitors have the possibility to make the network with host neurons.
PET IMAGING OF AMYLOID DEPOSITIONS IN MCI PATIENTS USING PIB
Anton Forsberg1, Henry Engler4, Gunnar Blomquist5, Ove Almkvist1,2, Goran Hagman2, Anders Wall3, Anna Ringheim3, Bengt Langstrom3,6, Agneta Nordberg1,2
1Neurotec Department, Karolinska Institutet, Stockholm, Sweden, 2Department of Geriatric Medicine, Karolinska University Hospital Huddinge, Stockholm, Sweden, 3Uppsala Imanet, GE Healthcare, Uppsala, Sweden, 4Department of Nuclear Medicine, Uppsala University Hospital, Uppsala, Sweden, 5Department of Oncology, Radiology and Clinical Immunology, Uppsala University, Uppsala, Sweden, 6Department of Organic Chemistry, Uppsala University, Uppsala, Sweden
Background and aims
Mild cognitive impairment (MCI) is a transitional state between normal ageing and dementia, especially early Alzheimer's disease (AD). Patients with MCI have an increased risk of developing AD(1) and early detection of these patients is of great importance in the effort to treat patients at an early stage of the disease. Beta amyloid (Aβ) depositions are one of the major histopathological hallmarks of AD(2) and the presence of Aβ at an early stage of the disease has been reported. Recently the Pittsburgh Compound-B (PIB) labeled with 11C has been demonstrated to image brain Aβ using Positron Emission Tomography (PET) (3) and to discriminate between AD patients and healthy controls.
In this study we aim to investigate amyloid depositions in patients with MCI and relate that to parameters as cerebral glucose metabolism (CMRglc), CSF measurements, as well as neuropsychological investigations. The results from the MCI patients were compared with previous studies of AD patients and healthy controls.
Methods
21 MCI patients were recruited from the Geriatric clinic, Karolinska University Hospital, Stockholm, Sweden, and studied with PIB-PET and FDG-PET at Uppsala Imanet AB, Uppsala, Sweden. The patients also underwent thorough clinical investigations including neuropsychological investigations and CSF sampling of Aβ, total Tau and phosphorylated Tau. For the FDG examinations, parametric maps of CMRglc were generated and normalized to whole brain. For the PIB examination, mean uptake values of the regions-of-interest obtained in a late time interval were normalized to the corresponding uptake in a reference region.
Results
The total group of MCI patients had significantly higher PIB retention in cortical brain areas compared to healthy controls. Seven MCI patients that later converted to AD showed significant higher PIB retention compared to healthy controls and non-converting MCI patients respectively. The PIB retention in the MCI converters was comparable to AD patients. Our results showed significant correlation between CSF Aβ, total Tau, phosphorylated Tau, episodic memory and PIB retention, while no significant correlations were observed between global cognition or CMRglc and PIB retention.
Conclusions
Seven MCI patients that later converted to AD showed significant higher PIB retention compared to healthy controls and non-converting MCI patients respectively. These findings show that it is possible to find amyloid depositions in MCI patients and that PIB probably could serve as a diagnostic marker able to discriminate patients with an increased risk of developing AD. The negative correlation between CMRglc and PIB retention that has previously been observed in patients with mild AD(3) was not present in the MCI group. This suggests that the parallel decline in CMRglc and cognitive performance might occur after the deposition of amyloid.
NORMAL DATABASE OF DOPAMINERGIC NEUROTRANSMISSION SYSTEM IN LIVING HUMAN BRAIN MEASURED BY PET
Hiroshi Ito, Hidehiko Takahashi, Ryosuke Arakawa, Harumasa Takano, Tetsuya Suhara
Clinical Neuroimaging Team, Molecular Neuroimaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan
Introduction: The central dopaminergic system is of interest in the pathophysiology of schizophrenia and other neurodegenerative diseases. Both pre and postsynaptic dopaminergic functions can be estimated by PET with several radiotracers. However, the integrated database of both pre and postsynaptic dopaminergic functions including receptors, transporter, and endogenous neurotransmitter synthesis has not been reported. In the present study, we have built the normal database of pre and postsynaptic dopaminergic functions in the living human brain using PET.
Methods: To measure the dopamine D1 receptor binding, striatal and extrastriatal dopamine D2 receptor binding, dopamine transporter binding, and endogenous dopamine synthesis, PET scans were performed on healthy men after intravenous injection of [C-11]SCH23390, [C-11]raclopride, [C-11]FLB457, [C-11]PE2I, or L-[C-11]DOPA, respectively (10 healthy men for each tracer). The binding potential (BP) was calculated by the reference tissue model method on a voxel by voxel basis with use of the cerebellum as a reference region. For L-[C-11]DOPA study, the dopamine synthesis index (I) was calculated on a voxel by voxel basis with use of the occipital cortex as a reference region from the ratio of area under the time-activity curve of brain regions to a reference region (I = ratio ? 1). All PET images were anatomically standardized using SPM2, and database was built for each radiotracer. Gray matter images were segmented and extracted from all anatomically standardized MR images using the SPM2 and used for partial volume correction.
Results: These databases allow to compare regional distributions of the striatal and extrastriatal dopamine D1 and D2 receptor binding, dopamine transporter binding, and endogenous dopamine synthesis. The BP and I values of each region-of-interest are given in table 1. In the striatum, greatest bindings to dopamine D1, D2 receptors and transporters and greatest dopamine synthesis were observed. For the limbic system, relatively high bindings of dopamine D1 and D2 receptors and relatively high dopamine synthesis were observed in the amygdala and anterior cingulate. Among the neocortical regions, highest binding to dopamine D2 receptors was observed in the temporal cortex, while D1 receptor binding was uniform. The binding to dopamine transporter was very low in the neocortical regions. These distributions were in good agreement with those in the human post mortem studies. The partial volume correction did not change regional distribution of pre and postsynaptic dopaminergic functions in cerebral cortices.
Conclusions: Although a limited spatial resolution of PET scanner might hamper observation of pre and postsynaptic dopaminergic functions in detail, such database must be useful to understand physiology of neurotransmission in the living human brain. These database can also be used to investigate regional abnormality of dopaminergic neurotransmission in neuropsychiatric diseases.
HYPEROXIA AND HYPERCAPNEA INCREASE THE CONCENTRATION OF OXIDISED CYTOCHROME C OXIDASE IN THE HEALTHY HUMAN ADULT BRAIN
Ilias Tachtsidis1, Martin M. Tisdall2, Terence S. Leung1, Caroline A. Pritchard2, Martin Smith2, Clare E. Elwell1
1Department of Medical Physics and Bioengineering, University College London, London, UK, 2Department of Neuroanaesthesia and Neurocritical Care, The National Hospital for Neurology and Neurosurgery, London, UK
Background and Aims
Cytochrome c oxidase (CCO) catalyses over 95% of oxygen metabolism and has been validated as a marker of cellular energy status. The aim of this study is to use broadband near-infrared spectroscopy (BBS) to measure changes in the concentration of oxidised cytochrome c oxidase (delta[oxCCO]) in the healthy adult brain during hyperoxia and hypercapnea.
Methods
10 healthy adult volunteers were studied. We measured cerebral blood flow velocity (Vmca) with transcranial Doppler ultrasonography and changes in concentrations of total haemoglobin (delta[HbT]=delta[HbO2]+delta[HHb]), haemoglobin difference (delta[Hbdiff]=delta[HbO2]-delta[HHb]) and delta[oxCCO] with BBS using optodes fixed to the right side of the forehead. Inspired oxygen concentration (FiO2) was increased using high flow oxygen via a tight fitted face mask and end tidal carbon dioxide (EtCO2) was increased by adding carbon dioxide to the inspired gases. Measured variables were compared with baseline using non-parametric ANOVA.
Results
Median and interquartile range are shown for hyperoxia (figure 1a) and hypercapnia (figure 1b) (*indicates p?0.05).
Discussion
We demonstrate a significant increase in delta[oxCCO] during hyperoxia and hypercapnea in the healthy adult brain. This suggests that oxidation of the mitochondrial electron transfer chain occurs during two physiological paradigms that increase oxygen delivery, as shown by the rise in delta[Hbdiff], via different mechanisms. Hyperoxia increases arterial oxygen content and is associated with a decrease in cerebral blood flow whereas hypercapnea increases cerebral blood flow without affecting arterial oxygen content.
In-vivo studies in animals confirm that cerebral CCO can be further oxidised by increases in oxygen delivery (Springett et al Adv. Exp. Med. Biol. 2003; Quaresima et al Biochim Biophys Acta 1998). Our data support the conclusion that, at normoxic normocapnea, cerebral CCO is not fully oxidised in the human adult and that further oxidation is possible by increasing oxygen delivery. These findings have implications for the monitoring and treatment of brain injury.
PITFALLS IN NIR-SPECTROSCOPY AND FUNCTIONAL MRI FOR MEASURING BRAIN OXIDATIVE METABOLISM WITH SPECIAL REMARKS ON A-V SHUNTS
Minoru Tomita1, Yutaka Tomita2, Takashi Osada1, Miyuki Unekawa1, Haruki Toriumi1, Norihiro Suzuki1
1Department of Neurology, Keio University School of Medicine, Tokyo, Japan, 2Department of Neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki, Gunma, Japan
The interplay between function and oxidative metabolism of the brain has been extensively investigated by spectroscopy (NIRS) and fMRI of metabolic changes responding to somatosensory stimuli. However, concerning optical spectroscopy, we have pointed out that the Lambert-Beer law does not hold during brain activation, since the extinction coefficient of the blood alters due to flow-dependent changes in light scattering by RBC (flow effect: Tomita et al. (2006) NeuroImage 33,1; ibid., 13). In addition, the extinction coefficient per se varies with RBC oxygenation: the concave shape of RBC tends to be spherical with a diameter of ca. 7.2 micrometer, whereas the same RBC become flat and flaccid with a diameter of ca. 7.7 micrometer when deoxygenated. Light scattering at such membranes is significantly affected. The inconstant extinction coefficient jeopardizes quantification of oxy-/deoxyhemoglobin in the tissue. In addition, the Bohr effect in arterial blood produced by respiratory changes in human subjects, e.g., breath-holding or hyperventilation with powerful somatosensory stimuli, shifts the oxygen dissociation curve of hemoglobin, changing cerebral blood oxygenation without any changes in brain metabolism. This paper examines the effect of arterio-venous shunting blood, which could augment dissociation between blood oxygenation and brain oxygenation in both NIRS and fMRI, since the channels transport oxygenated blood from the arteries to the veins without gas exchange. We used 50 rats with a closed cranial window and measured velocities of FITC-labeled RBC in the 50 micrometer deep cortical microvasculature with high speed laser scanning confocal fluorescence microscopy. We found labeled RBC velocity varied widely in capillaries selected with the aid of FITC-dextran staining. The RBC velocity distribution revealed a group of outlier higher velocities (p<0.01) from the normal group which must belong to a different category of vessels: the RBC velocities were extraordinarily high, nevertheless the channels had the same diameter as capillaries (Unekawa, Brain 07). Dynamic observation in vivo revealed the channels run straight and are richly invested with astrocyte endfeet, which were stained in vivo with sulforhodamine 101. The amount of shunted blood without gas exchange through these channels was estimated from the velocity, number of capillaries, and theoretical extraction as 22% of the total perfusion, which agrees with our previous observation of 20% based on the compartmental analyses of inert gas clearance from human brain. In the human study we found that the shunted blood held in reserve was recruited with CO2 inhalation. With CO2, cerebral tissues became equally perfused by blood. When the brain is activated, CO2 in the tissue increases, and the direct shunting blood decreases. NIRS and fMRI falsely estimate the oxidative metabolism to be increased. In conclusion, the shunting blood constitutes a serious pitfall for quantifying brain oxidative metabolism. However, this paper does not intend to deny the possible clinical usefulness of NIRS and fMRI as tools to investigate brain function, since their signals are surely biological, but rather, to emphasize that the interpretation must be cautious.
ACUTE FUNCTIONAL RECOVERY OF CEREBRAL BLOOD FLOW FOLLOWING FOREBRAIN ISCHEMIA IN THE RAT
Chao Zhou1, Tomakazu Shimazu2, Turgut Durduran1,3, Daniel Y. Kimberg2, Guoqiang Yu1, Xiao-Han Chen4, John A. Detre3, Arjun G. Yodh1, Joel H. Greenberg2
1Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, PA, USA, 2Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA, 3Department of Radiology, University of Pennsylvania, Philadelphia, Philadelphia, PA, USA, 4Department of Neurosurgery, University of Pennsylvania, Philadelphia, Philadelphia, PA, USA
Background: Following complete cerebral ischemia the post-ischemic blood flow response to functional activation is severely attenuated for several hours. However, little is know about the spatial and temporal extent of the blood flow response in the acute post-ischemic period following incomplete cerebral ischemia.
Methods: We used laser speckle flowmetry, which provides two-dimensional high spatial and temporal resolution blood flow images, to examine the response of relative cerebral blood flow (rCBF) to controlled vibrissae stimulation during the first few hours following temporary incomplete ischemia. Male Sprague-Dawley rats (n=10) were prepared under halothane anesthesia and then switched to α-chloralose. The bone overlying the right somatosensory cortex was thinned for the laser speckle imaging. Prior to ischemia the left vibrissae (B1-3, C1-3, D1-3) were stimulated for 3 sec at 5 Hz every minute over a 10-minute period while the rCBF data was collected every 200 msec. Fifteen minutes of transient forebrain ischemia was induced by bilateral carotid arterial occlusion along with controlled hypotension (mean arterial blood pressure lowered to 45-50 mmHg by slowly withdrawing blood from a femoral arterial catheter). This reduced cerebral blood flow to 6.6±1.8% (mean±SE) of the pre-ischemic level. The shed blood was re-infused, the carotid ties were released, and the rCBF response to vibrissae stimulation was repeated at 20, 30, 60, 120, and 180 mins after the end of the ischemia. rCBF images were analyzed using a modified general linear model (GLM) with VoxBo (www.voxbo.org). Statistical maps from the analysis were used to determine the spatial extents of the activation at different time points with a fixed Bonferroni-corrected threshold (pth=0.01).
Results: The activation area was significantly reduced 20 mins into reperfusion compared to prior to ischemia (49±12%, p<0.01), and recovered to 77±17% at 2 hours (p=0.36, Figure.1.A). The maximum rCBF response in the activation area determined from the statistical analysis did not change significantly up to three hours post ischemia [ΔrCBF=10.6±0.8% (pre-ischemia); 9.0±0.3% (20 min, p=0.06); 11.1±0.7% (3 hrs, p=0.62)]. However, the time when rCBF reached maximum was significantly delayed from 2.4±0.2 sec prior to ischemia to 3.6±0.1 sec at 20 mins into reperfusion (p<0.001); the delay was reduced to 3.0±0.2 sec after 3 hours, which was still significantly greater than that observed prior to the insult (p<0.05, Figure.1.B).
Conclusions: We have incorporated the GLM method into laser speckle imaging, and showed that although the magnitude of the response to functional stimulation was preserved in the early hours following incomplete cerebral ischemia, the area of activation and the temporal response to activation were significantly altered.
IMAGING BETA AMYLOID DEPOSITION IN VIVO: QUANTITATIVE COMPARISON OF [18F]FDDNP AND [11C]PIB
Nelleke Tolboom1,2, Maqsood Yaqub1, Wiesje van der Flier2, Ronald Boellaard1, Gert Luurtsema1, Albert D. Windhorst1, Frederik Barkhof3, Philip Scheltens2, Adriaan A. Lammertsma1, Bart N.M. van Berckel1
1Department of Nuclear Medicine and PET Research, VU University Medical Centre, Amsterdam, The Netherlands, 2Department of Neurology and Alzheimer Centre, VU University Medical Centre, Amsterdam, The Netherlands, 3Department of Radiology, VU University Medical Centre, Amsterdam, The Netherlands
Introduction
Both [18F]FDDNP and [11C]PIB have been developed as PET tracers for imaging and quantifying beta-amyloid (As) fibril accumulation in Alzheimer's disease (AD) in vivo1,2. The purpose of the present study was to compare the potential value of [18F]FDDNP and [11C]PIB in the (early) diagnosis of AD by performing paired studies in the same patients.
Methods
In this ongoing study, dynamic 90 minutes 3D [11C]PIB and [18F]FDDNP scans were acquired on the same day using an HR+ (Siemens) PET scanner. During both scans, continuous on-line and discrete manual sampling was performed to derive a metabolite corrected arterial plasma input curve. For each subject, volumes of interest (VOI) were defined on an individually acquired, co-registered T1 weighted structural MRI scan using an automated procedure3. Preliminary analysis of time-activity curves was performed using the simplified reference tissue model with cerebellum grey matter as reference tissue. Binding potential (BP) was used as outcome measure, in particular for areas previously associated with AD: frontal, parietal and temporal cortex.
Results
At present, three AD patients, three patients with Mild Cognitive Impairment (MCI) and three age matched normal controls were included. Results are summarised in figures 1 and 2. [11C]PIB showed good contrast between AD patients and normal controls, as described previously1. In addition, the range of BP values in MCI patients was broader, probably due to the known heterogeneity of this group4. [18F]FDDNP provided less contrast between AD patients and normal controls. In AD patients, [11C]PIB BP was in general tenfold higher than [18F]FDDNP BP.
Conclusion
The difference in BP between AD patients and normal controls appears to be more pronounced for [11C]PIB than for [18F]FDDNP. Further studies are needed to substantiate these preliminary findings.
EFFECTS OF EARLY LIFE STRESS ON 5-HT1A RECEPTOR AVAILABILITY IN TWO YEARS OLD RHESUS MONKEYS MEASURED BY [18F]FPWAY AND PET
Simona Spinelli1, Richard E. Carson2, Cristina S. Barr1, Stephen J. Suomi3, Markus Heilig1, J. Dee Higley1, Lixin Lang4, Elliot Stein5, Svetlana I. Chefer5
1Laboratory of Clinical and Translational Studies, National Institute On Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, USA, 2Yale PET Center, New Haven, CT, USA, 3Laboratory of Comparative Ethology, National Institute of Child and Human Development, NIH, Bethesda, MD, USA, 4PET Radiochemistry Group, National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD, USA, 5Neuroimaging Research Branch, National Insitute On Drug Abuse, NIH, Baltimore, MD, USA
Traumatic childhood experiences are associated with increased risk for developing depression, anxiety, posttraumatic stress and substance abuse disorders. Rearing infant macaques with same-aged peers in the absence of adults, has been established as a model of early adversity. This condition has been shown to induce high levels of anxiety-like behavior in monkeys, particularly among females. Chronic early life stress exposures affect 5-HT1A receptor expression and function in various animal models (Leonard, 2005), and dysregulation of the serotonin system is believed to increase vulnerability to developing mood and anxiety disorders. As such, we hypothesized that nursery raised (peer-reared, PR) macaques would exhibit differences in [18F]FPWAY binding potential (BP) relative to those raised with their mothers (mother-reared, MR) and that these changes would not only appear early in life but may be particularly marked among females. To assess the effects of rearing condition on the development of the serotonin system, we compared MR (1 male and 5 female) to PR (5 male and 5 female) rhesus monkeys, assessed at 24-29 months, an age corresponding to 6–7 years in humans. PET data were acquired on an ECAT HR+ scanner over 90-120 min, under isoflurane anesthesia using [18F]FPWAY (Lang et al.,1999;2000) and a bolus-plus-infusion technique (Kbol=60 min) in order to attain constant radioactivity level in all brain regions. PET images were co-registered to individual MRIs using PMOD. A standard region of interest (ROI) template, including prefrontal cortex (PFC), anterior and medial cingulate cortex (ACC and MCC), hippocampus, amygdala, midbrain (including dorsal raphe nucleus) and temporal cortex was constructed from the MRI images for each monkey. BP* was calculated in the ROIs under equilibrium assumption as (CROI?CCER)/CCER. Using sex as a covariate, ANOVA was performed, revealing a main effect of rearing condition (P<0.001). BP* values were elevated in the PFC and hippocampus (increase of 40% and 10%, respectively, P<0.05) in the PR group (BP*=1.85+0.52 in PFC and 3.12+0.35 in hippocampus, mean+SD) compared to MR group (BP*=1.34+0.36 in PFC and 2.83+0.24 in hippocampus, mean+SD). Comparison of only MR and PR female monkeys revealed a 70% (P<0.05) increase in PFC (BP*=2.12+0.58 and 1.24+0.29 in PR and MR group, respectively) and a 20% (P<0.05) elevation in BP* values in ACC in the PR group (BP*=2.94+0.38 and 2.44+0.27 in PR and MR group, respectively). Previous human studies show that depressed subjects have decreased BP (Sargent et al.,2000). Increased BP* values in our study may have been driven mostly by female monkeys whereas the majority of the human studies used male subjects. Studies in humans suggest higher levels of specific distribution volumes in women compared to men (Parsey et al.,2002). In conclusion, these findings suggest that early stress affects the density of 5-HT1A receptors available for radioligand binding during early macaque development. This effect is particularly evident in some sub-regions of the frontal cortex. Elevated BP* values could be related to either an up-regulation of 5-HT1A receptors or to a decrease in tonic serotonin levels.
Support Contributed By: NIDA-, NIAAA-, NICHD- and NIBIB-IRP
IMPROVEMENT OF CEREBRAL OXYGEN METABOLISM AFTER BYPASS SURGERY IN PEDIATRIC MOYAMOYA DISEASE
Satoshi Kuroda1, Tatsuya Ishikawa1, Tohru Shiga2, Naoki Nakayama1, Kiyohiro Houkin3, Nagara Tamaki2, Yoshinobu Iwasaki1
1Department of Neurosurgery, Hokkaido University Hospital, Sapporo, Japan, 2Department of Nuclear Medicine, Hokkaido University Hospital, Sapporo, Japan, 3Department of Neurosurgery, Sapporo Medical University, Sapporo, Japan
Background and aims- Previous studies have clarified that bypass surgery improves cerebral hemodynamics and prevents further ischemic stroke in patients with moyamoya disease. However, the effect of bypass surgery on cerebral metabolism is still unclear. In this study, therefore, the authors evaluated it in both pediatric and adult patients with moyamoya disease, using [15] O-gas positron emission tomography (PET).
Methods- This study included 20 patients who were admitted to our hospital due to moyamoya disease. There were 8 children and 12 adults. All pediatric patients experienced TIA or cerebral infarction. Of 12 adult patients, 11 developed TIA or cerebral infarction and another did intracranial bleeding. Using [15] O-gas PET, cerebral hemodynamics and oxygen metabolism were measured in all patients before and 3 to 4 months after STA-MCA anastomosis and pial synangiosis that widely cover the cerebral hemisphere. By placing 10-mm diameter ROIs on the frontal or temporal cortex, the effects of bypass surgery on cerebral hemodynamics and metabolism were quantitatively analyzed.
Results- In pediatric patients (n=8), cerebral blood flow (CBF) significantly increased from 37.3±9.4 mL/min/100g to 42.8±6.1 mL/min/100g after surgery (P<0.01). Cerebral blood volume (CBV) significantly decreased from 6.2±2.5 mL/100g to 3.6±0.8 mL/100g after surgery (P<0.05). Cerebral metabolic rate for oxygen (CMRO2) significantly increased from 2.84±0.63 mL/min/100g to 3.56±0.53 mL/min/100g after surgery (P<0.01). Pre- and postoperative values of oxygen extraction fraction (OEF) did not differ; 0.43±0.10 and 0.47±0.03, respectively. In adult patients (n=12), CBF significantly increased from 29.4±7.6 mL/min/100g to 37.7±7.8 mL/min/100g after surgery (P<0.01). CBV significantly decreased from 5.3±2.1 mL/100g to 3.6±1.2 mL/100g (P<0.05). OEF significantly decreased from 0.46±0.11 to 0.41±0.05 after surgery (P<0.05). However, CMRO2 did not significantly change after surgery. Thus, pre- and postoperative CMRO2 were 2.37±0.61 mL/min/100g and 2.57±0.54 mL/min/100g, respectively.
Conclusions- These findings strongly suggest that cerebral oxygen metabolism may be depressed in response to prolonged ischemia in pediatric, but not adult, patients and that effective bypass surgery may have the potential to improve cerebral oxygen metabolism by improving cerebral hemodynamics in pediatric patients with moyamoya disease. Improvement of cerebral oxygen metabolism may contribute to not only prevent ischemic stroke but also improve their intellectual outcome.
VALIDITY AND VALUE OF QUANTITATIVE SPECT RECONSTRUCTION IN A MULTI-CENTER CLINICAL STUDY USING I-123 RADIOPHARMACEUTICALS
Iida Hidehiro, Watabe Hiroshi, Nakazawa Mayumi, Hayashida Kohei
Quantitative SPECT Working Group, Japan
Introduction: SPECT has a potential to provide parametric functional images in a “quantitative” manner, as has been widely done with PET in vivo, using several tracers typically labeled with I-123. Due to availability of equipments, as well as well-established delivery/transportation of radio-ligands, SPECT has an advantage in applying it to large-scale clinical studies. It has however been considered that accuracy and inter-institutional reproducibility of SPECT are limited for quantitative assessment of functional parametric images, largely attributed to a lack of general consensus of reconstruction procedures (attenuation/scatter correction) as well as, in case of I-123, significant amount of penetration through collimator from high-energy photons. We have recently developed a novel method to reconstruct SPECT images from existing projection data including appropriate corrections for scatter and attenuation in the object, head contour detection, as well as for collimator-dependent penetration. This study was aimed at evaluating validity and impact of our approach for Multicenter clinical study. We particularly investigated consistency and reproducibility of quantitative values for I-123, at different SPECT systems from different manufacturers.
Materials and methods: The Windows program can handle original projection data to analyze and/or DICOM format obtained from 5 manufactures with essentially 7 different operation systems. Twenty-five institutions provided a series of phantom data for 28 collimators according to a given protocol. Nine are symmetric fanbeam, and rest parallel beam collimators. Experiments were done for a radioactivitycalibrated point source of I-123 in air to determine the absolute sensitivity of the system and to determine the penetration factor for each collimator. Additional experiments were done using a uniform cylindrical phantom filled with the radioactivity mentioned above, and single/multiple rod sources placed in cylindrical water. The scatter/penetration correction was based on the previously proposed transmission dependent convolution subtraction method, and the attenuation correction was implemented during the ordered-subset EM reconstruction. Dynamic SPECT studies were carried out in 9 institutions on 64 patients using I-123 iodo-amphetamine (IMP) to assess quantitative CBF images at rest and during Diamox challenge. Inter- and intra-institutional reproducibility was then evaluated.
Results and discussion: Scatter fraction was consistent among different collimators from different manufacturers as predicted, but the penetration varied largely dependent on the collimator design and manufacturers. Our approach successfully compensated for the penetration and the scatter as well as the attenuation, as demonstrated in reconstructed images of the water-filled single/multiple rod source phantoms, except for one fanbeam collimator. Parallel beam collimator tends to have small penetration than fanbeam. Absolute concentration of the uniform cylindrical phantom was also consistent among institutions, although it was significantly smaller than the true value (0.88+/−0.05). Clinical data also demonstrated that CBF values obtained with I-123 IMP at rest and during Diamox were reproducible among institutions. Normal CBF values obtained in 2 institutions showed also no significant difference.
Conclusion: These data suggested that QSPECT could be a useful tool for quantitative mapping using I-123 compounds, allowing a large scale clinical evaluation using SPECT.
MICROGLIA PROVIDE NEUROPROTECTION AND ENGAGED IN CLOSE CELL-CELL CONTACT WITH NEURONS AFTER ISCHEMIA
Jens Neumann1, Matthias Gunzer2, Oliver Ullrich3, Klaus Dinkel4, Klaus G. Reyman1,4
1Project Group Neuropharmacology, Leibniz Institute for Neurobiology, Magdeburg, Germany, 2German Research Centre for Biotechnology, Immunodynamics, Braunschweig, Germany, 3Institute of Immunology, University Hospital Magdeburg, Magdeburg, Germany, 4Institute for Applied Neuroscience (FAN GGmbH), Magdeburg, Germany
Many neurological insults are accompanied by a marked acute inflammatory reaction, involving the activation of microglia. Using a model of exogenous application of fluorescence-labeled BV2 microglia in pathophysiologically relevant concentrations onto organotypic hippocampal slice injury. Neuronal cell death after oxygen-glucose deprivation (OGD) was determined by propidium iodide incorporation and Nissl staining. Migration and interaction with neurons were analyzed by time resolved 3-D two-photon microscopy. We show that microglia protect against OGD-induced neuronal damage and engage in close physical cell-cell contact with neurons in the damaged brain area. Neuroprotection and migration of microglia were not seen with integrin regulator CD11a-deficient microglia or HL-60 granulocytes. The induction of migration and neuron-microglia interaction deep inside the slice was markedly increased under OGD conditions. Lipopolysaccharide-prestimulated microglia failed to provide neuroprotection after OGD. Pharmacological interference with microglia function resulted in a reduced neuroprotection. Microglia proved to be neuroprotective even when applied up to 4 h after OGD, thus defining a ??protective time window.?? In acute injury such as trauma or stroke, appropriately activated microglia may primarily have a neuroprotective role. Anti-inflammatory treatment within the protective time window of microglia would therefore be counterintuitive.
OVEREXPRESSION OF STAT3 PROMOTES NEURONAL CELL DEATH THROUGH ACTIVATION OF MICROGLIA
Ryuji Hata, Pengxiang Zhu, Fang Cao, Masahiro Sakanaka
Department of Functional Histology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
Background and aims
Previously, we reported that Stat3 was activated following focal cerebral ischemia in mouse [1]. However, little is known about the function of Stat3 in the ischemic brain. Here we investigated the effect of Stat3 overexpression and co-culture with microglia on neurons.
Methods & Results
Recombinant adenovirus expressing Stat3 wild type (St3.Wt), Stat3 dominant negative type (St3.F) and LacZ were constructed as described previously [2].
In vitro : Rat cultured neurons were exposed to adenovirus vector expressing St3.Wt(AdSt3.Wt), St3.F(AdSt3.F) or LacZ(AdLacZ) for 2h and incubated with fresh medium up to 72h. Then, neurons were co-cultured with rat cultured microglia in two different ways.
direct co-culture: microglia were added directly to 24 well plates containing neurons.
insert co-culture: microglia were seeded into cell culture inserts that were placed on the top of the 24 well plates containing neurons.
At 48 hr after co-culture, cells were homogenized in lysis buffer and immunoblotted with antibody against Stat3, p-Stat3(Tyr-705) and MAP2.
In vivo: A fine glass micropipette was introduced into the left caudoputamen in the rats. Twenty microliters of adenovirus vectors, AdSt3.Wt, AdSt3.F or AdLacZ were administered over a 20 min period. Two days later, the left middle cerebral artery (MCA) of the infected animal was occluded. One day after MCA occlusion, infarct volume was evaluated using TTC staining.
Results
As shown in Figs A and B, overexpression of St3.Wt induced p-Stat3 and cell death in neurons after direct co-culture with microglia but not after insert co-culture with microglia. These results suggested that direct contact of microglia with neurons expressing St3.Wt induced stat3 activation and cell death in neurons. Moreover, in vivo experiment revealed that the infarct volume of the AdSt3.Wt-treated group was significant larger than that of AdLacZ-treated group while the infarct volume of the AdSt3.F-treated group was significant smaller than that of AdLacZ-treated group suggesting deteriorative effects of Stat3 on the ischemic brain.
Conclusion
Our data revealed that overexpression of Stat3 promoted neuronal cell death through activation of microglia. These results suggest that activation of Stat3 might play a crucial role in the inflammatory reaction after cerebral ischemia
CORTICAL SPREADING DEPRESSION AND PERI-INFARCT DEPOLARISATION IN HUMAN ISCHEMIC STROKE
Christian Dohmen1,2, Oliver Sakowitz3, Martin Fabricius4, Bert Bosche5, Thomas Reithmeier5, Ralf-Ingo Ernestus5, Gerrit Brinker5, Jens Dreier6, Johannes Woitzik7, Antony J. Strong8, Rudolf Graf2
1Department of Neurology, University of Cologne, Cologne, Germany, 2Max-Planck Institute for Neurological Research, Cologne, Germany, 3Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany, 4Department of Clinical Neurophysiology, Glostrup Hospital, University of Copenhagen, Copenhagen, Denmark, 5Department of Neurosurgery, University of Cologne, Cologne, Germany, 6Department of Neurology, Charite University of Berlin, Berlin, Germany, 7Department of Neurosurgery, University of Mannheim, Mannheim, Germany, 8Department of Clinical Neurosciences, King's College London, London, UK
Objective: Cortical spreading depression (CSD) and peri-infarct depolarisation (PID) have been shown in various experimental models of stroke to cause infarct expansion and secondary neuronal damage. For many years it has been speculated that CSD/PID occur in stroke patients. Recently, CSD and PID have been documented in patients with traumatic brain injury and subarachnoid hemorrhage. In the present study, we report for the first time on CSD/PID in human ischemic stroke measured by subdural electrocorticography (ECoG) in patients with malignant MCA infarction.
Methods: Patients were recruited by centers of the Co-operative Study on Brain Injury Depolarisations (COSBID). The study was approved by the local ethics Committees. All patients suffered from complete or subtotal infarction of the MCA territory and underwent decompressive hemicraniectomy. During the operation, subdural electrode strips with 6 linear contacts were placed over the peri-infarct region of 16 patients. From these electrodes, 4 ECoG channels were aquired in a bipolar fashion; one electrode was used as ground. CSD was defined as gradually developing depression of ECoG activity with simultaneous slow potential changes (SPC) spreading between at least 2 channels. If no local ECoG activity occurred up to the time of the SPC, the event was defined as PID. If some electrodes recorded a local ECoG signal before the SPC, while others did not, the event was defined as CSD/PID.
Results: Monitoring was started between 24 and 109 h after stroke and performed for up to 120 h. The first patient had to be excluded from data analysis due to technical failure during the recording. From the following 15 patients, a total recording time of 1547 h with good data quality was scanned for depolarisation events. In 3 patients, no depolarisation events occurred. In these patients, the electrode strip was placed mainly over infarcted tissue i.e. hypodense tissue on computer tomography images, and no local ECoG activity occurred throughout the measurement. In the remaining 12 patients (80 %), a total of 164 episodes were recorded (127 CSD, 20 CSD/PID, 16 PID). Seizure activity was not apparent in any of the patients.
Conclusion: Similar to findings in experimental stroke models, CSD and PID are found with high incidence in patients with MCA infarction suggesting that this phenomenon is a relevant factor also in the pathophysiology of ischemic tissue damage in humans and thus a target for treatment development.
CEREBRAL BLOOD FLOW, OXYGEN CONSUMPTION AND GLUCOSE UTILIZATION IN HUMANS:A STEREOTAXIC CORRELATION STUDIED WITH PET AND SPM
Kazuhiko Hayashi, Shinichi Takeda, Keisuke Miyake, Kentaroh Fujii, Taku Fujimoto, Yasuhiro Ohsaki, Ashik Bin Ansar, Katsufumi Kajimoto, Yasuyuki Kimura, Hiroki Katoh, Makiko Tanaka, Eku Shimosegawa, Jun Hatazawa
Department of Nuclear Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
We studied local differences in the coupling among cerebral blood flow (CBF), oxygen consumption (CMRO2) and glucose utilization (CMRGlc) by means of statistical parametric mapping (SPM).
Materials and Methods
We measured CBF and CMRO2 in 6 normal subjects (2 males and 4 females, mean age=33.376.6 years old) by PET and the 15O steady-state inhalation (1), while we estimated CMRGlc in the other 6 subjects (6 males, 22.871.9) by PET and 2-deoxy-2-[F-18]fluoro-D-glucose according to the method developed by Phelps et al (2) with standard rate constants and lumped constant of 0.42. We analyzed these data statistically by applying SPM99 (3). We also analyzed absolute values of CBF, CMRO2, CMRGlc, and their relationships.
Results
In SPM analysis, CBF distribution was significantly different from CMRO2 in hippocampus and pons (CBF>CMRO2) and different from CMRGlc in hippocampus and cerebellum (CBF>CMRGlc). In comparison between CMRGlc and CMRO2, the cerebrum showed significantly higher CMRGlc than CMRO2 (Figure 1). Molar ratio of CMRO2/CMRGlc was 4.2 for cerebral cortices and 5.6 for cerebellum.
Discussion
In supply-demand correlation, hippocampus is characterized by having excessive supply of glucose and oxygen, pons having excessive supply of oxygen, cerebellum having excessive supply of glucose. In glucose-oxygen correlation, cerebral cortices showed excessive glucose consumption for oxygen utilization. Shurr hypothesized (4) that the end product of glycolysis would be lactate. Our results may indicate that there is a
lactate reserve
in cerebral cortices excessively produced as a substrate for TCA cycle.
RECOMBINANT HUMAN C1-INHIBITOR PROTECTS AGAINST BRAIN ISCHEMIA/REPERFUSION INJURY WITH A WIDE TIME-WINDOW OF EFFICACY
Claudio Storini1, Raffaella Gesuete1, Emanuela Rossi2, Alessandro Fantin1, Luigi Bergamaschini2, Bertjan Ziere3, Helene Vietsch3, Maria-Grazia De Simoni1
1Mario Negri Institute for Pharmacological Research, Milan, Italy, 2Geriatric Unit, University of Milan, Ospedale Maggiore IRCCS, Milan, Italy, 3Pharming Technologies B.V., Leiden, The Netherlands
We have previously shown that plasma derived C1-INH (pdC1-INH), the endogenous inhibitor of complement and contact/kinin system, has potent neuroprotective effects in ischemic mice, although with a narrow time-window of efficacy(1)–(3). This inhibitor, currently used to manage angioedema attacks in patients with hereditary or acquired C1-INH deficiency (angioedema, HAE or AAE), is purifed from human plasma. Using a plasma derived molecule can be disadvantageous, due to risks for human pathogenic viruses contamination and to difficulties in supply and purification. Due to these limitations, a recombinant approach was chosen to overcome these problems.
Recombinant human C1-INH (rhC1-INH, Pharming Technologies B.V.) has been administrated intravenously to C57Bl/6 ischemic mice at different doses (5, 10 and 15U/mouse) and time-points (0, 3, 6, 18 and 24 hours) from transient ischemia. Forty-eight hours after ischemia, rhC1-INH, at the dose of 15U/mouse, was able to markedly improve the cerebral damage when administrated 0, 3 and 6h from ischemia (13.67±2.59 mm3, 11.71±0.63mm3 and 20.38±2.37mm3, respectively) compared to saline-treated ischemic mice (44.43±5.94mm3). Also when administrated 18 hours after ischemia, rhC1-INH was still significantly effective (27.13±2.58mm3). Fluoro jade staining showed that amelioration of cerebral damage was due to an effective sparing of neurons. Immunohistochemistry for macroglia/macrohages (anti-CD11b) and astrocytes (anti-GFAP) showed that rhC1-INH was able to counteract the microglial and astrocytic activation, as well as macrophage infiltration.
Analysis of rhC1-INH localization in the brain by confocal microscopy revealed that rhC1-INH was confined to cerebral vessels and that it was able to bind endothelial cells as shown by colocalization with Claudin-5 and ICAM-1.
These data demonstrate that rhC1-INH has an important neuroprotective action with a wider time-window of efficacy compared to pdC1-INH.
This favorable profile of rhC1-INH is probably related to a different glycosylation of the native versus recombinant molecules.
EVIDENCE THAT PROTECTION BY HSP70 IS ANTI-INFLAMMATORY IN MODELS OF BRAIN ISCHEMIA
Zhen Zheng, Hualong Ma, Xiannan Tang, Midori A. Yenari
Department of Neurology, VA MED CTR, University of California, San Francisco, CA, USA
Previous studies from our lab and others show that overexpression of Hsp70 in brain cells of transgenic mouse is neuroprotective following transient middle cerebral artery occlusion (tMCAO). While the mechanism of protection has largely been thought to be due to its chaperone functions, preventing protein aggregation and assisting nascent protein folding, other work has shown that Hsp70 is also capable of modulating inflammatory responses, by either potentiating or inhibiting them. Prior work in our lab suggested an anti-inflammatory role for Hsp70 in tMCAO, but since Hsp70 probably protects via several mechanisms, we now directly test the hypothesis that Hsp70 overexpression in inflammatory cells is protective using a bone marrow chimera model. Hsp70 bone marrow chimeras were created by transplanting marrow from male CB6 Hsp70 transgenic mice into female wildtype littermates (Hsp70 chimera). In controls, marrow from male wild-type littermates (Wt chimera), or lacZ transgenic mice were used. Chimeras were generated by lethally irradiating recipients with the heads shielded. Bone marrow cells isolated from donor mice were injected via the tail vein into the irradiated recipient mice (100 × 106 bone marrow cells/recipient). Pilot studies of lacZ chimeras showed that donor marrow engrafted by 1–2 months post transplant (>70% of cells in blood assessed by FACS), and no transplanted cells were visible in the brain. However, 3 months post transplant, a few perivascular transplanted cells were visible. Therefore, 2 months post transplant, mice were subjected to 2 h tMCAO using an intraluminal suture. 48 h later, brains were harvested and stained with cresyl violet. Infarct size was measured using an image analysis system. MHC-class П staining was performed to identify activated inflammatory cells, and the positive cells were counted in 3 random non-overlapping fields within the cortex adjacent to the outer boundary of the infarct. To determine whether Hsp70 expression in microglia may also have an anti-inflammatory effect, cultures of either primary wildtype astrocytes alone or astrocytes with microglial cells (collected from Hsp70 transgenic or wildtype mice) underwent oxygen and glucose deprivation (OGD) for 6 h followed by 24 h reperfusion, then assessed for cell death. Infarct size among Hsp70 chimeras was reduced by 22 % compared to Wt chimeras (P<0.05). MHC Class II expression was significantly decreased in Hsp 70 chimeras (25%) compared to Wt chimeras (37.6%) (P<0.01). In vitro, microglia nearly doubled the extent of cell death due to OGD. The addition of Hsp70 transgenic microglia to astrocytes completely inhibited microglia potentiated cell death (P<0.001). Furthermore, Hsp70 transgenic microglia showed more than 50% decrease in the number of MHC II positive cells following LPS stimulation compared to wildtype microglia using FACS analysis (p<0.005). Our data indicate that Hsp70 overexpression in inflammatory cells is anti-inflammatory, and is protective following brain ischemia. This suggests another novel mechanism of protection by Hsp70.
RBC EXCLUSION WITHOUT CAPILLARY DIAMETRIC CHANGE DURING PASSAGE OF K+−INDUCED CORTICAL SPREADING DEPRESSION IN RATS
Takashi Osada1, Minoru Tomita1, Yutaka Tomita2, Miyuki Unekawa1, Haruki Toriumi1, Norihiro Suzuki1
1Department of Neurology, School of Medicine, Keio University, Tokyo, Japan, 2Institute of Brain and Blood Vessels, Department of Neurology, Mihara Memorial Hospital, Gunma, Japan
[Introduction]In a previous communication we reported K+−induced cortical spreading depression (CSD) caused a few seconds' cessation of capillary flow in association with the wave-ring spread of CSD based on an analysis of tissue hemodilution curves (Neurosci Lett 322: 157, 2002; J Cereb Blood Flow Metab 25: 742, 2005) and segmental constriction/dilatation of arterioles (Neuroreport 17: 1365, 2006). In this paper, we examined whether such changes in RBC movement were related to diametric changes of the pertinent capillaries presumably affected by astrocytic swelling during CSD in rats. [Material and Methods]Nine urethane-anesthetized rats were used. To confirm CSD, DC potential in the cerebral cortex was measured. Images of the movements of individual FITC-labelled RBCs in intraparenchymal single capillaries at an approximate depth of 50 µm in the cortical region of interest (ROI) were acquired employing either a conventional video camera (30 frames/s) or a high-speed camera (250-500/s frame rate) with laser scanning confocal fluorescence microscopy, and analysed with Matlab domain homemade software (KEIO-IS2) to document the RBC tracking and calculate the number and velocity of labelled RBCs appearing in the ROI during a period of CSD wave passage after microinjection of 150 mM K+ solution on the brain surface. In vivo staining of single capillaries with FITC-dextran injected intravenously, and astrocytes stained in vivo with direct application of sulforhodamine 101 on the brain surface (Nat. Methods 1: 31, 2004), respectively. [Result]FITC-labelled RBCs moving in the capillary network appeared like fireflies, and the mean RBC velocity was 2.1 ± 1.8 mm/sec. Within a few minutes after K+ application to the periphery of the ROI, fluctuations of DC potential were initiated with a darkening of the background of the video frames. In 8 out of 9 cases we observed transient RBC exclusion from single capillaries in the ROI during K+-induced CSD: both the number and velocity of RBC were decreased until RBC disappeared from the ROI completely. The disappearance lasted for 3.3±2.3 seconds and the occurrence was statistically significant (p< 0.05). This transient RBC exclusion was followed by recovery of capillary flow to its initial state. The RBC exclusion from capillaries was never observed under control conditions. However, no detectable changes in the diameter of single capillaries were observed in association with the RBC exclusion during CSD passage. Endfeet of astrocytes were detected stained in a brown colour and changed their morphology slowly, but no direct compression effect was observed during CSD passage.[Conclusion]We conclude that RBCs were excluded (de-recruitment) from the capillary network in the ROI during CSD passage with little capillary diametric change. Astrocytes may participate in RBC flow changes but not in the form of direct compression of the capillaries.
SLOWLY EVOLVING INFARCTION IN THE ISCHEMIC PERIPHERY: MRI, BEHAVIORAL AND PATHOLOGICAL ANALYSIS OF RODENT BRAIN AFTER TRANSIENT ISCHEMIA
Toshihiko Kuroiwa1, Liyuan Sun2, Naoyuki Miyasaka3, Ichiro Yamada4, Satoru Ishibashi2, Shu Endo5, Kikuo Ohno6
1Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA, 2Department of Neurology, Tokyo Medical and Dental University, Tokyo, Japan, 3Department of Obstetrics and Gynecology, Tokyo Medical and Dental University, Tokyo, Japan, 4Department of Radiology, Tokyo Medical and Dental University, Tokyo, Japan, 5Animal Research Center, Tokyo Medical and Dental University, Tokyo, Japan, 6Department of Neurosurgery, Tokyo Medical and Dental University, Tokyo, Japan
Infarction evolves slowly in the ischemic periphery after mild ischemia. We examined the time course of post-ischemic peripheral tissue with disseminated selective neuronal changes, reactive astrocytosis and infarction and analyzed the correlation with MRI and behavioral findings in the experimental unilateral forebrain ischemia of rodent.
[Methods] Transient unilateral forebrain ischemia was induced by occlusion of the left common carotid artery for 15 ? 60 min with 8% oxygen inhalation in SD rats (n=57) and by two times 10 min occlusion of the left common carotid artery in Mongolian gerbils (n=36). Intraischemic blood flow was monitored in gerbils by laser Doppler flowmetry (LDF). MRI apparent diffusion coefficient (ADC) mapping and T2 mapping during ischemia and post ischemia was performed using an experimental MRI (4.7T). Neurological deficit was evaluated in gerbil by applying a battery of behavioral tests. The brain removed after perfusion fixation at 1d ? 16w post ischemia was prepared for histological examination. [Results] Intra-ischemic LDF decreased to 22.1±9.5% of baseline in the ischemic core and 37.5±3.5% in the ischemic periphery. Areas with eosinophilic neurons (ENs) appeared in the ischemic center of the cortex at 1d post-ischemia, extended to the peripheral cortex by 4d. The area of high EN density (>80 EN/mm2) transformed into infarction during 2 weeks of post ischemia. Reactive astrocytes (RAs) in this area were involved in the infarction process, indicating occurrence of delayed astrocytic death. Areas with low EN density (<80 EN/mm2) was not transformed into infarcts. Behavioral examination revealed neurological deficit during ischemia, transient recovery shortly after recirculation and secondary neurological deterioration. ADC mapping revealed rapid ADC decrease (6.97±0.4×10-4 mm2/s to 3.97±0.21×10-4 mm2/s, P<0.05) of the ischemic area after onset of ischemia in rats. ADC decrease was transient in the animals with 15 min ischemia. Only selective neuronal death evolved in this group. Animals showing a biphasic ADC decrease ? intra-ischemic decrease followed by a transient recovery and subsequent decrease ? and permanent ADC decrease developed cerebral infarction. [Conclusion] Areas of high, but not low, EN density transformed slowly into infarction after transient forebrain ischemia. Delayed astrocytic death took place in the areas with high EN density. Areas of the slowly evolving infarction were predictable by early repeated ADC mapping. Secondary neurological deterioration was observed in the animals with slowly evolving infarction.
Figure. ADC map 4d post ischemia (left), histological changes 4d (middle) and 16w (right) post ischemia in gerbils
TOWARD A FUNCTIONAL METABOLOMICS OF BRAIN ENERGY METABOLISM AND NEUROTRANSMITTER TRAFFICKING
Paul K. Maciejewski
Magnetic Resonance Research Center, Yale University, New Haven, CT, USA
Although it is widely accepted that glucose is the primary substrate that supports brain function, fundamental questions about pathways and compartmentalization of metabolic processes that accomplish glucose catabolism in brain tissue remain unanswered. Furthermore, a basic understanding of the role of glucose in brain function must recognize that glucose not only serves as a basic energy substrate for brain but also as a substrate for the de novo synthesis of chemical neurotransmitters such as glutamate and GABA.
Metabolomics, an emerging field of biological investigation, is interested in not only the identification and characterization of small-molecule metabolites that are present in biological systems, but also in the identification and characterization of their role within the context of those systems (i.e., what might be called a “functional metabolomics:). Metabolites are material elements that are consumed and produced in metabolic processes. Fluxes of metabolites into and out of metabolic processes provide an indication of the function of metabolites within the context of living systems. Consequently, a functional metabolomics of brain energy metabolism and neurotransmitter trafficking ought to consist of the identification and characterization of metabolic processes that participate in these basic metabolic functions in brain quantified in terms of fluxes of metabolites through those processes.
In principle, brain energy metabolism and other brain metabolic functions (e.g., neurotransmitter synthesis and neurotransmitter transport processes) can be investigated in vivo using isotopic label-tracer methods implemented using Mass Spectroscopy (MS) or Magnetic Resonance Spectroscopy (MRS). However, unlike other metabolomics technologies that identify metabolites and characterize systems in terms of profiles of metabolites, label-tracer methods depend on detailed a priori knowledge about metabolic pathways, knowledge which is used to explain the transfer of label from one metabolite to another. This a prior knowledge about metabolic pathways is provided by metabolic models, that is, by detailed, mathematical descriptions of processes that are presumed to explain the transfer of label from label-enriched precursor substrates to other metabolites. However, existing metabolic models tend to focus on single pathways and do not accommodate the complexity of metabolism and the likelihood that specific metabolic functions may be accomplished via a multiplicity of functionally redundant pathways. This narrow focus limits the design and interpretation of existing in vivo label-tracer studies of metabolism.
Here I will introduce a new “functional metabolomics” approach to the investigation of biochemical pathways for brain energy metabolism and neurotransmitter trafficking in vivo that consists of: 1) a rational method for the specification of alternative, hypothetical biochemical pathways that in principle accomplish these basic metabolic functions in brain, and 2) a method for the design of isotopic label-tracer experiments to determine the extent to which these alternative, hypothetical pathways actually accomplish these specific metabolic functions in vivo. The approach integrates information derived from cellular and molecular biology, metabolomics and proteomics with concepts and techniques derived from engineering, mathematics, and computational sciences.
(Supported by NIH NS-044316)
PROPYLENE GLYCOL AFTER HUMAN TRAUMATIC BRAIN INJURY: NOVEL BIOMARKER OF ALTERED GLUCOSE METABOLISM
Daniel Hirt1, Mujtaba Kadri1, Neil A. Martin1, Shane Que Hee2, Matthew Eliseo1, Jon Berg1, Paul Vespa1, Thomas C. Glenn1
1Division of Neurosurgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA, 2Department of Environmental Health Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
Background
Traumatic brain injury (TBI) metabolism research has indicated that despite increased levels of glucose, conventional cerebral glucose metabolism rarely rises to the expected supra-normal levels to compensate for the injury-induced decrease in oxidative metabolism. In fact, it has been well established that increased plasma glucose (PlGluc) is detrimental following TBI. These observations suggest the activation of alternative glucose metabolic pathways.
Investigations around a novel metabolite, propylene glycol (PG), a compound that we recently identitifed in cerebrospinal fluid (CSF) of TBI patients, suggested that increased PlGluc results in an increased rate of glycation. Glycation is a deleterious metabolic process resulting in the production of toxic PG, advanced glycation end products (AGEs), and methylglyoxal. Both, methylglyoxal and AGEs, have been shown to affect physiological aging, and neurodegenerative diseases, such as Alzheimer's. We hypothesize that the methylglyoxal pathway is likely to be activated following brain injury. As a result, glucose might be involved in unique pathophysiological mechanisms following TBI.
Methods
Daily CSF samples of 34 consented TBI patients (38+/−12 years, GCS 6.5+/−2.1,76% male) and five consented normal pressure hydrocephalus (non-injured) patients were analyzed using proton-nuclear magnetic resonance (H-NMR) spectroscopy and gas chromatography/mass spectroscopy (GC/MS). Additionally, arterial blood samples were collected and analyzed for glucose concentrations using the GM-7 Analox Analyzer.
Results
Statistical analysis of PlGluc levels and PG, an end-product of glycation, revealed a significant correlation (r=0.432; p=0.001). PlGluc higher than 160mg/dl resulted in significantly higher overall PG concentrations (PGGluc<160=1.35mM vs. PlGluc>160=4.38mM; p=0.004). Additionally, propylene glycol correlated significantly with CSF lactate concentrations (r=0.408; p=0.002), which was expected since PG can very easily be converted to lactate.
Most interestingly, the GC/MS analysis of CSF revealed the presence of methylglyoxal. Methylglyoxal was significantly higher in the CSF of TBI patients when compared to the CSF of non-injured patients. On average, TBI patients had 56+/−14ng/g of sample of methylglyoxal present in their CSF, whereas the non-injured patients only had 9.4+/−8.0ng/g of sample (p=0.05).
Conclusion
While there have been numerous studies on glucose metabolism following TBI, to our knowledge, no studies in TBI research have examined the methylglyoxal pathway. This process can be activated after glucose initiates glycation under conditions of hyperglycemia and the presence of free radicals. This pathway has been recently investigated in diabetes research. Considering the physiological similarities between hyperglycemia in diabetes and TBI with regards to the presence of free radicals and the increased susceptibility to developing neurodegenerative diseases, glycation may be an important pathophysiological mechanism following TBI.
The detection of PG and methylglyoxal in the CSF of TBI patients suggests the presence of glycation reactions in the injured brain under conditions of high PlGluc. We speculate that free radicals inhibit glycolysis at the glyceraldehydes-3-phosphate (G3P) step. G3P then gets metabolized to methylglyoxal, AGEs, and PG, all of which can have detrimental effects on the injured brain. In light of these data, we believe that it is of utmost importance to monitor PlGluc and glycation marker levels closely in severely head injured patients to prevent further metabolic distress and cell damage.
INHIBITION OF THE MYOSIN LIGHT CHAIN KINASE PREVENTS HYPOXIA/STROKE-INDUCED BLOOD BRAIN BARRIER DISRUPTION IN VITRO AND IN VIVO
R. Tamaki2,3, C. Kuhlmann1, V. Lessmann1, H. Luhmann1, Oliver Kempski2
1Institute of Physiology and Pathophysiology, Johannes Gutenberg University, Mainz, Germany, 2Institute for Neurosurgical Pathophysiology, Johannes Gutenberg University, Mainz, Germany, 3Department of Neurosurgery, Nara Medical University, Nara, Japan
Background: Increased mortality after stroke is associated with development of brain edema. The aim of the present study was to examine the contribution of endothelial myosin light chain (MLC) phosphorylation to hypoxia/stroke-induced blood brain barrier (BBB) opening.
Methods: Measurements of Trans-Endothelial-Electrical-Resistance (TEER) were performed to analyze BBB integrity in an in-vitro co-culture model (bovine brain microvascular endothelial cells (BEC) and rat astrocytes). Brain water content was analyzed in rats 24 hrs after venous stroke induction using a two-vein occlusion (TVO) model (1). Dihydroethidium was used to monitor intracellular generation of reactive oxygen species (ROS) in BEC. MLC phosphorylation was detected by immunostainings using a confocal laser-scanning microscope.
Results: Hypoxia induced in-vitro by a combined oxygen-glucose deprivation caused a decrease of TEER values by more than 40 %, which was prevented by the application of the MLC-kinase inhibitor ML-7 (10 µmol/l). ML-7 also significantly reduced the elevated brain water content invivo after TVO (80.184± 0.540%, n=8) to 79.714 ± 0.337% (n=10) (sham-op: 78.793± 0.326%, n=8). The NAD(P)H-oxidase inhibitor apocynin (500 µmol/l) prevented the hypoxia-induced TEER decrease. Hypoxia-dependent ROS generation was completely abolished by apocynin and by the Ca2+ chelator BAPTA (10 µmol/l). Interestingly, the hypoxia-dependent decrease of TEER was also blocked by BAPTA. Inhibition of Ca2+ signaling (BAPTA), ROS-formation (apocynin), or the MLC-kinase (ML-7) completely abolished the hypoxia-induced increase of MLC-phosphorylation.
Conclusions: Our data demonstrate for the first time, that MLC phosphorylation contributes to hypoxia/stroke-induced BBB disruption in-vivo and in-vitro. The hypoxia-induced increase of intracellular Ca2+ increases the activity of the NAD(P)H-oxidase. The resulting ROS generation is responsible for an increase of MLC phosphorylation that activates the endothelial contractile machinery leading to paracellular gap formation.
INDUCTION OF AQUAPORIN EXPRESSION IN ISCHEMIC MOUSE BRAIN AFTER THROMBIN PRECONDITIONING
Jerome Badaut1, Melanie Price2, Karine Wiegler2, Nabil Mastour1, Jean-Francois Brunet1, Lorenz Hirt2
1Neurosurgical Research Group, Pavillon 3, CHUV, UNIL, Lausanne, Switzerland, 2Neurology Department, CHUV, UNIL, BH19, Lausanne, Switzerland
Background
Our recent studies have shown that the water channel aquaporin-4 (AQP4) is induced 1h after stroke onset. The role of AQP4 in the evolution of cytotoxic and vasogenic oedema after stroke remains debated. The absence of AQP4 in KO-mice reduces the early cytotoxic oedema formation and in contrast aggravates the later vasogenic oedema. A second aquaporin, AQP9, is induced at a later time-point than AQP4 after stroke onset, and the function of AQP9 is predicted to be in relation with brain energy metabolism(1).
Thrombin at high doses is known to induce oedema while preconditioning is induced by a single injection of a low dose of thrombin (TPC). TPC attenuates lesion size and oedema after middle cerebral artery occlusion (MCAo). This preconditioning protocol is a useful model to understand endogenous mechanisms of protection after brain ischemia and increase our knowledge of the roles of aquaporins in oedema development. We studied the expression of AQPs in ischemic mouse brains after TPC in correlation with the reduction in oedema formation.
Methods
For TPC, mice were injected ICV with 0.01U of mouse thrombin, or with and equal volume of saline (controls), followed 24 hours later by 30 mins of transient MCAo. Expression of AQP4 and AQP9 was evaluated by immunohistochemistry 1h and 48h after ischemia(1) and correlated with oedema.
Results
In TPC animals, oedema formation assessed by measurement of the hemispheric enlargement is significantly reduced at 1h (4.5 ± 2% vs 11.0 ± 5% in controls, p<0.05, n=8) and this was confirmed by the wet-weight/dry-weight ratio. Correlating with the reduction in oedema at 1h after stroke onset, there was a 1.5 fold increase in AQP4 levels compared to controls (P<0.05, n=6) localised in the ischemic striatum. The oedema and the lesion size (5.1 ± 1.6 vs 10.6 ± 1.8 mm2 in controls, n=5) are significantly decreased at 48h after stroke onset in TPC mice, and the level of expression for AQP4 is still higher, but the difference is no longer significant.
At 1h, the number of AQP9 positive astrocytes is also 2 fold higher in TPC animals than in controls (1089 ± 260 vs 480 ± 280 cells in CTL) in the striatum, the hippocampus and the hypothalamus. At 48h after stroke, there is no difference in AQP9 positive cells between controls and TPC mice.
Discussion
In contrast with results obtained in AQP4 KO-mice, the correlation between the early induction of AQP4 and the decrease in oedema formation in TPC mice suggests that the induction of AQP4 prevents the development of the oedema. AQP9 was also induced in TPC mice, throughout the brain and may participate in protection by decreasing the extracellular lactate during ischemia.
Financial supports
Swiss Science Foundation #3100A0-108001, #3200-68306.02, #3100AO-112484 and Swissheart Foundation.
PROBABILISTIC CORTICAL SURFACE MAP OF THE MIDDLE CEREBRALARTERY TERRITORY FOR SINGLE-PHOTON EMISSION COMPUTED TOMOGRAPHY STUDIES
Kohkichi Hosoda, Eiji Kohmura
Department of Neurosurgery, Kobe University Graduate School of Medicine, Kobe, Japan
[Background and aims] The middle cerebral artery (MCA) is the intracranial vessel most frequently affected by cerebrovascular diseases. A more accurate knowledge of the topography of this vessel may have an impact on treatment strategies for ischemic cerebrovascular diseases. The aim of this study was to construct a topographic map of the MCA territory for SPECT using statistical brain mapping. [Methods] The margin of the perfusion deficit associated with infarction due to arterial occlusion, as seen on SPECT imaging, is presumed to approximate the borders of the territory of the artery. Basing the study on this hypothesis, SPECT images obtained in 12 patients with large MCA infarctions due to angiographically confirmed MCA trunk occlusion were selected, anatomically standardized, and compared with SPECT images of healthy volunteers to construct probabilistic cortical surface maps of the MCA territory. Crossed cerebellar diaschisis was used as a primary cutoff marker for creation of the map. This MCA map (Method C) was compared with the conventional ROI method (Method A) and previously reported predefined cortical templates (Method B) for preliminary clinical application. [Results] The probabilistic cortical surface map of the MCA territory showed that regions with the highest ratio of MCA territory included the transverse temporal gyrus (100%), supramarginal gyrus (100%), and inferior parietal lobule (91–92%). For preliminarily clinical application, this map (Method C) was compared with the conventional ROI method (Method A) in predicting hyperperfusion after carotid endarterectomy by performing a receiver operating characteristic (ROC) analysis, which demonstrated the statistically significant superiority of the MCA map (area under the ROC curve [Az] = 0.91) to the ROI method (Az = 0.75; p = 0.025). The ROC analysis also demonstrated a diagnostic value of the MCA map (Az = 0.95) that equaled predefined cortical templates (Method B) (Az = 0.93). [Conclusions] The probabilistic cortical surface map of the MCA territory used for SPECT, which was created using statistical brain mapping techniques, would be useful for an objective assessment of the cerebral perfusion status of patients with cerebrovascular diseases.
LATE VASCULAR ABNORMALITIES FOLLOWING SUBARACHNOID HEMORRHAGE IN THE RAT CORRELATE WITH THE SEVERITY OF ACUTE ISCHEMIA
Radoslaw Michalik1, Anna Domasiewicz1, Roman Gadamski2, Piotr Piotrowski2, Janina Rafalowska2, Zbigniew Czernicki1, Ewa Kozniewska1
1Department of Neurosurgery, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland, 2Department of Experimental and Clinical Neuropathology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland
BACKGROUND AND AIMS. Subarachnoid hemorrhage (SAH) results in acute decrease of cerebral blood flow which is fatal for about 30% of patients. In about 30% of survivors delayed persistent vasospasm develops resulting in morbidity. Despite years of experimental and clinical research on SAH no effective treatment of the delayed vasospasm was found. Majority of experimental studies on SAH aimed to prevent or reverse delayed vasospasm concentrated on this late phase. The aim of this study was to find out whether dysfunction of microcirculation and structural changes in extraparenchymal large, conduit arteries following SAH correlate with severity of acute post-SAH ischemia
METHODS. Adult, anaesthetized male Sprague-Dawley rats were subjected to SAH or sham surgery. SAH was induced by endovascular puncture of the bifurcation of the internal carotid artery. Microflow (LDF) in cerebral cortex was measured bilaterally before, during, and for, at least, 30 min after SAH using laser Doppler flowmeter. Reactivity of microcirculation to CO2, intravenous papaverine, acetylcholine, or thromboxane receptor antagonist (SQ 29,548) administration was assessed at 3, 24, 48, and 72 after SAH. At the same time points ipsi- and contralateral middle cerebral artery and basilar artery were harvested and processed for histological examination and measurement of wall thickness.
RESULTS. Following SAH microflow in the ipsilateral cortex did not respond to the tested vasodilators up to 48 hours. At 72 hours after SAH in some animals the recovery of the response to CO2 and papaverine was noted. This recovery correlated with the severity of acute ischemia and was characteristic for the cases with small SAH (microflow in acute phase below 30% of control for 15-20 min). In all cases with large SAH (microflow in acute phase below 30% of control for more than 40 min) no recovery was observed. In addition, in the animals with large SAH, at 72 hours after the bleeding cerebrovascular resistance decreased after blocking of TXA2/PGH2 receptor. The response to acetylcholine was ebolished irrespective of the severity of SAH and did not recover. Degree of corrugation of the ipsilateral MCA at 3 days after SAH which was chosen as a measure of vasospastic changes also correlated with severity of acute ischemia
CONCLUSIONS. This study demonstrates that duration of the impairment of microvascular vasodilatory capacity following SAH correlates with the severity of acute ischemia. It also shows that critical underperfusion in the acute phase after SAH results in progressive functional deterioration of microcirculation leading to the increase of arachidonic acid metabolites-dependent tone of microvessels in the late phase after SAH.
The study suggests that the therapy of post-SAH states should concentrate on the improvement of microvascular functions.
Supported by grant 3P05A 02421 from the KBN.
NORMOBARIC HYPEROXIA REDUCES BBB OPENING BY INHIBITING MATRIX METALLOPROTEINASE-MEDIATED DEGRADATION OF TIGHT JUNCTION PROTEINS IN A FOCAL ISCHEMIC RAT MODEL
Wenlan Liu, Zhongrui Yuan, Ke Jian Liu
Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, NM, USA
Background and aims: Normobaric hyperoxia treatment has recently been shown by us and other groups to be an effective strategy in reducing stroke lesion volumes and improving neurological function following ischemic stroke in rats. However, the mechanisms of normobaric hyperoxia-induced neuroprotection remain elusive. The disruption of blood brain barrier (BBB) is known to be a critical event during focal cerebral ischemia, which leads to vasogenic edema and hemorrhage. Occludin and claudins are important tight junction proteins regulating paracellular permeability of the BBB by sealing the tight junctions. We have recently shown that matrix metalloproteinases (MMPs) mediate the BBB disruption by degrading tight junction proteins following focal cerebral ischemia in rats. In the present study, we tested the hypothesis that normobaric hyperoxia reduces the opening of blood brain barrier by inhibiting matrix metalloproteinase-mediated degradation of tight junction proteins.
Methods: Rats were subjected to hyperoxia (95% O2) or normoxia (30% O2) during 90 min filament occlusion of the middle cerebral artery, followed by 3-hr reperfusion. At the end of reperfusion, BBB damage was assessed by measuring Evan's blue extravasation in ischemic cortex and caudate. Real-time RT-PCR, immunohistochemistry and western blot were used to analyze the mRNA and protein levels of tight junction proteins in isolated cerebral microvessels. MMP-2 and ?9 were measured by in situ and gel zymography.
Results: We found that extensive BBB opening occurred in ischemic cortex and caudate after 3-hr reperfusion, which was significantly reduced by hyperoxia treatment. The mRNA and protein levels of occludin and claudin-5 were decreased in ischemic cerebral microvessels, which concurrently occurred with increased gelatinolytic activity. Gel zymography showed a 4-fold increase in MMP-9, while no MMP-2 was detected in ischemic microvessels. The reduction of occludin and claudin-5, as well as the increase of MMP-9, was reversed by hyperoxia treatment. To further investigate the involvement of MMP-9 in the mechanism of hyperoxia-mediated neuroprotection, we also carried out parallel experiments using a broad-spectrum MMP inhibitor, BB1101. It was found that treatment of the ischemic rats with BB1101 produced the similar results on MMP-9, occludin, and claudin-5.
Conclusions: These results suggest that normobaric hyperoxia treatment protects early BBB opening by inhibiting MMP-mediated degradation of tight junction proteins. Furthermore, these findings support the notion that hyperoxia treatment could potentially be used as a promising interventional strategy to treat stroke patients.
A VASORELAXANT PEPTIDE IN THE BRAIN AND DELAYED CEREBRAL VASOSPASM AFTER ANEURYSMAL SUBARACHNOID HEMORRHAGE
Masayuki Fujioka1, Kenji Nishio2, Shiro Ueda2, Atsushi Kubo3, Naoto Minamino3, Kenji Kangawa3, Kazuo Kitamura3, Kenichi Mishima4, Takashi Mori5, Takao Asano5, Toshisuke Sakaki6, Kazuo Okuchi2, Akio Asai1, Keiji Kawamoto1, Naoki Otani7, Yasuhiro Yonekawa7, Bo Siesjo8
1Department of Neurosurgery, Kansai Medical University, Hirakata, Osaka, Japan, 2Emergency Medical Center, Nara Medical Univeristy, Nara, Japan, 3National Cardiovascular Center, Suita, Osaka, Japan, 4Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan, 5Department of Neurosurgery, Saitama Medical School and Center, Kawagoe, Saitama, Japan, 6Department of Neurosurgery, Nara Medical University, Kashihara, Nara, Japan, 7Department of Neurosurgery, Zurich University Hospital, Zurich, Switzerland, 8Neuroscience Center, Lund University, Lund, Sweden
Background
Adrenomedullin (AM) is a potent vasorelaxant peptide. Recent studies suggest that circulating and local brain-tissue AM play a role in experimental brain ischemia. We suggested that the AM concentration increased in plasma and cerebrospinal fluid (CSF) in patients after aneurysmal subarachnoid hemorrhage (SAH) (Stroke 2000;31:3082-83).
We report the close relationships between the severity of cerebral vasospasm and the CSF AM increase in SAH patients and between the reactive astrocyte proliferation and the AM increase in CSF.
Methods
We measured plasma and CSF AM concentrations (pAM and cAM) in 58 SAH patients on the day of SAH onset (day 0), then during the first (days 1-3), second (days 4-6), third (days 7-9) and fourth (days 10-12) periods by radioimmunoassay. The pAM was also measured in 13 healthy controls and the cAM in 10. We also investigated the AM immunoreactivities of neurons and astrocytes in the ischemic cerebral cortex of SAH patients and of rat stroke models of 60-minutes middle cerebral artery occlusion (MCAO).
Results
The pAM significantly increased in the SAH patients when compared to 13 healthy controls at anytime within 2 weeks after SAH (p < 0.001). However, there was no significant relationship between pAM and delayed brain ischemia. The cAM increased significantly in patients with transient brain ischemia or with infarction compared to those without ischemic episode (p < 0.05), independently of the pAM. The cAM in patients with cerebral vasospasm increased significantly in the ischemia-onset period more than in the period prior to ischemia-onset (p=0.005). The AM immunoreactivities increased in the astrocytes in SAH patients with vasospasm and in the rats after MCAO.
Conclusions
Our study suggests that plasma AM may play a role in systemic responses of endocrinium and inflammation to SAH. Furthermore, AM in the central nervous system seems to play an essential role in the delayed brain ischemia after SAH. Astrocytes appear to be importantly involved in the increase of CSF AM of SAH patients.
PROSTAGLANDIN D2 PROTECTS NEONATAL MOUSE BRAIN FROM HYPOXIC ISCHEMIC INJURY
Hidetoshi Taniguchi1, Ikuko Mohri2, Hitomi Arahori1, Kosuke Aritake3, Kazuko Wada1, Takahisa Kanekiyo1, Masahiro Nakayama4, Shuh Narumiya5, Keiichi Ozono1, Yoshihiro Urade3, Masako Taniike2
1Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka, Japan, 2Department of Mental Health and Environmental Effects Research, The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Osaka, Hamamatsu, Japan, 3Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka, Japan, 4Division of Clinical Laboratory Medicine and Anatomic Pathology, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan, 5Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan
Prostaglandin (PG) D2, the most abundant prostanoid in the brain, is synthesized by hematopoietic PGD synthase (HPGDS) or lipocalin-type PGDS (L-PGDS) and binds to specific receptors, DP1 or DP2. We investigated the role of PGD2 in the pathogenesis of hypoxic-ischemic encephalopathy (HIE) in neonatal mice at postnatal day 7. In wild-type mice, hypoxia-ischemia increased PGD2 production in the brain up to 90-fold compared with the level in sham-operated brains at 10 min after cessation of hypoxia. Whereas the size of the infarct was not changed in L-PGDS- or DP2-knockout mouse brains compared with that in the wild-type HIE brains, it was significantly increased in HPGDS-L-PGDS double knockout or DP1-knockout mice. The PGD2 level in L-PGDS-, HPGDS-, and HPGDS-L-PGDS-knockout mice at 10 min of reoxygenation was 46%, 7%, and 1%, respectively, of that in the wild-type, indicating the infarct size to be in inverse relation to the amount of PGD2 production. Thus, PGD2-DP1 signaling protected brains from hypoxia-induced ischemic damage in neonatal mice. DP1 receptors were exclusively expressed in endothelial cells after 1 hr of reoxygenation, and cerebral blood flow decreased more rapidly after the onset of hypoxia and did not return to the baseline level after reoxygenation in HPGDS-L-PGDS-knockout mice. Endothelial cells were severely damaged in HPGDS-L-PGDS- and DP1-knockout mice after 1 hr of reoxygenation. In the human neonatal HIE brain, HPGDS-positive microglia were remarkably increased in number. In conclusion, PGD2 protected the neonatal brain from hypoxic-ischemic injury via DP1 receptors by preventing endothelial cell degeneration.
STABILITY AND ABRUPT FALL OF BRAIN TISSUE POTASSIUM IN RAT FOCAL CEREBRAL ISCHEMIA AND THE BLOOD-BRAIN BARRIER
Stephen C. Jones1,2, Weixing Hu3, Yang Wang4, Alejandro D. Perez-Trepichio5, Victor E. Yushmanov1, Alexander Kharlamov1, Fernando E. Boada6
1Department of Anesthesiology, Allegheny Singer Research Institute, Pittsburgh, PA, USA, 2Department of Neurology, Allegheny Singer Research Institute, Pittsburgh, PA, USA, 3Nanjing Medical University, Nanjing, China, 4Intel Corporation, Portland, OR, USA, 5Medical Surgical Specialists, Naples, FL, USA, 6Department of Radiology, MR Research Center, University of Pittsburgh, Pittsburgh, PA, USA
Introduction
Most measurements of brain tissue potassium, [K+]br, in experimental focal ischemia show a gradual fall (1). However, measurements of extracellular [K+] during the 3 h after ischemic onset indicate that [K+]ex is stable (2). Ischemic depolarization at 4-10 min results in an increase in electrolyte permeability, equilibrating [K+]br and [K+]ex. We hypothesize that [K+]br does not fall during the initial 3-h period. In addition, our data suggest that [K+]br falls rapidly between 3–4 h which corresponds to the “classical” 3-h time window. The time of the earliest BBB abnormalities associated with vasogenic edema has been notoriously hard to assess, as permeability agents just start accumulating, and only become detectable after enough material has crossed from the vascular space into brain, usually over 6 h, although Gd-DTPA has been shown to appear in the sulcal spaces at early times (4 h) after human ischemic stroke (3).
Methods
Twenty rats underwent permanent MCA transection with bilateral CCA occlusion. In 18 animals, 1.7–7..5 h after occlusion, MRI-generated ADC maps and autoradiographic CBF (n=7) were determined (4). CBF sections and micro-punch samples for K+ analysis were obtained from the brain at the level of the MR image. In 2 animals, BBB permeability was evaluated using multiple Gd-DTPA injections and T1 imaging at 2.5–5..5 h, quantitated by the intensity difference between the pre- and post-Gd-DTPA images.
Results
Mean normal cortex [K+]br was stable with a mean (±sem) value of 100±1.9 mEq/kg (Figure). In the ischemic core (identified using ADC and CBF) [K+]br showed a sigmoid relation (r2=0.86) with stable [K+]br before (55±3.8 mEq/kg) and after (17±2.0 mEq/kg) an abrupt change in [K+]br at 3.4 h after occlusion. There were no changes in CBF, ADC, H2O%, and [Na+]br accumulation rate before and after the K+ drop. In the Gd-DTPA animals, a thin line of Gd-DTPA enhancement occurred in the CSF space over the ischemic region 3–4 h after occlusion.
Conclusions
The stability of [K+]br during the 3 h after ischemic onset corresponds to previous [K+]ex measurements (2) (p=0.16) and the abrupt drop of [K+]br and Gd-DTPA enhancement occur in the same time period after onset. The drop in [K+]br occurs independently of the other measured parameters suggesting a relation to a change in some aspect of K+ transport. We suggest that this abrupt K+ drop is related to the initial BBB changes observed with Gd-DTPA, and that both signal the onset of vasogenic edema.
Support NIH NS30839
ROLE OF MAPK IN NEUROPROTECTION BY CART IN ISCHEMIC STROKE
Yun Xu1, Jia Jia2, Zichun Hua3, Patricia Hurn4, Nabil J. Alkayed5
1Department of Neurology, The Affiliated Drum Tower (Gulou) Hospital of Nanjing University, Nanjing, China, 2State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University, Nanjing, China, 3Department of Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, OR, USA
BACKGROUND AND SIGNIFICANCE: Our previous work indicated that CART (cocaine and amphetamine regulated transcript) plays an important role in estrogen-mediated neuroprotection in ischemic stroke (Xu Y et al. 2006). Specifically, we previously demonstrated that CART protects primary cultured cortical neurons from cell death induced by oxygen-glucose deprivation (OGD) and decreases infarct size after ischemic stroke induced in mice by middle cerebral artery occlusion (MCAO). The aims of this study are: 1) to further investigate the neuroprotective role of CART in vitro and in vivo using small interfering RNA (siRNA) to downregulate endogenous CART, and 2) to explore a potential mechanism of neuroprotection by CART. CART and its mechanism of action may serve as a novel therapeutic strategy for neuroprotection in ischemic stroke.
METHODS: Five double-stranded RNA fragments corresponding to selected mouse CART mRNA sites were produced using plasmid siRNA expression vector under control of U6 promoter. The vector also co-expresses the coral GFP marker (cGFP) under CMV promoter, which is used to track transfection efficiency. Cortical neurons and HEK293T cells were transfected with CART siRNA or control vector, and interference efficiency was assayed by RT-PCR and ELISA. HEK293 cells were also co-transfected with CART cDNA, since they do not express CART. Finally, CART or vehicle, CART siRNA or control vector in vehicle (cationic polymer polyethylenimine, PEI) were injected into mouse lateral ventricles in vivo 24 hours prior to MCAO. Brains were removed, stained with 2,3,5-triphenyltetrazolium chloride (TTC) for infarct size measurement. Neuronal cell death in vitro was induced by OGD, and assayed by propidium iodide (PI)/Calcein staining, and survival assayed by MTT. The phosphorylation of ERK1/2 was measured by western blot with anti-ERK anti-p(44/42)ERK antibodies.
RESULTS: CART expression was effectively suppressed by siCART. Selected siRNA reduced CART RNA and protein levels in vitro by approximately 80% and 90%, respectively. CART administration reduced infarct size at 24 hrs after MCAO from 39·6% to 22·4% (n=6 per group, p<0.05), while administration of siCART increased infarct size to 48·4% compared to saline-injected mice (23.5·4%, n=6 per group, p<0.05). Neuronal survival by MTT after 2-hr OGD was higher in CART-treated cultures (49±6%, n=3, each in triplicate) compared to saline-treated cultures (35±%, n=3 in triplicate, p<0.05), while survival was significantly reduced in cultures treated with siCART (21±4%, n=3 in triplicate) compared to saline-treated cultures. Similar results were obtained using PI/Calcien staining. Finally, CART increased ERK phosphorylation in cultured neurons after OGD, while siCART inhibited ERK activation after OGD.
CONCLUSIONS: The findings suggest that CART is an important endogenous neuroprotectant against ischemic neuronal cell death in vitro and in vivo, and that mechanism of neuroprotection is in part linked to ERK activation. CART may serve as a novel neuroprotectant against stroke-related brain damage.
INTRATHECAL INJECTION OF EGF AND FGF2 PROMOTES PROLIFERATION OF NEURAL PRECURSOR CELLS IN THE SPINAL CORDS OF ALS MODEL MICE
Yasuyuki Ohta, Makiko Nagai, Tetsuya Nagata, Tetsuro Murakami, Isao Nagano, Hisashi Narai, Tomoko Kurata, Mito Shiote, Yasushi Takehisa, Nobutoshi Morimoto, Kazunori Miyazaki, Tatsushi Kamiya, Koji Abe
Department of Neurology, Graduate School of Medicine, Dentistry and Pharmacy, Okayama University, Okayama, Japan
Background: Endogenous neural precursor cells reside close to the ependyma of the central canal (CC). In injured spinal cord, they are activated to proliferate, but most of them differentiate into glial cells and rarely into neurons. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, which characterized by selective death of motor neuron in the spinal cord. Here, we investigated three steps of their activation, proliferation, migration and differentiation, in the spinal cord of symptomatic ALS model mice treated with intrathecal infusion of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2).
Methods: EGF and FGF2 or vehicle were continuously injected for 7 days using an osmotic minipumps into the lumbar spinal cord region of normal and symptomatic transgenic (Tg) mice with a mutant G93A SOD1 gene at 100 days of age, when they display paralysis. After placement of the minipump, intraperitoneal injections of 5-bromodeoxyuridine (BrdU) began every 6 h (four times per day) for 7 days. 21 days after minipump placement, the lumbar cord (L4-L5) was removed. In order to determine the character of BrdU-labeled cells, we performed immunofluorescence studies using BrdU, nestin, highly polysialylated neural cell adhesion molecule (PSA-NCAM), ionized calcium-binding adapter molecule-1 (Iba1), neuronal nuclear antigen (NeuN), and glial fibrillary acidic protein (GFAP).
Results: We observed that BrdU-labeled cells and BrdU+nestin double-labeled neural precursor cells increased in the spinal cords of Tg mice as compared with non-Tg mice, with a much greater increase produced by EGF and FGF2 treatment. The number of BrdU+nestin double-labeled cells was larger than that of BrdU+Iba1, GFAP or PSA-NCAM double-labeled cells, but none expressed NeuN. On further analysis of the gray matter of Tg mice, the number of BrdU+nestin and BrdU+PSA-NCAM double-labeled cells increased more in the ventral horns than the dorsal horns, which was again greatly enhanced by EGF and FGF2 treatment.
Discussion and conclusions: The present study suggests that cell proliferation becomes active in symptomatic Tg mice and is further enhanced by EGF and FGF2 treatment. Furthermore, the neural precursor cells preferentially differentiate into neuronal precursor cells in Tg mice by EGF and FGF2 treatment.
ALLOPREGNANOLONE REDUCES DAMAGE TO CEREBELLAR PURKINJE CELLS FOLLOWING EXPERIMENTAL CARDIAC ARREST AND CPR IN MICE
N. Taguchi, M.H. Kelley, P.D. Hurn, R.J. Traystman, Paco S. Herson
Department of Anesthesiology and Peri-Op Medicine, Oregon Health and Science University, Portland, OR, USA
Background and Aims: Progesterone has recently been demonstrated to be neuroprotective in animal models of transient cerebral ischemia (MCAO). Using an in vitro neuronal culture model we recently found that progesterone neuroprotection of cerebellar Purkinje cells (PCs) requires metabolism to its GABA-active metabolite allopregnanolone (ALLO). We chose to investigate PCs because they are particularly sensitive to ischemia and surprisingly understudied. The current study evaluated the ability of ALLO to protect cerebellar Purkinje cells in vivo, using a mouse cardiac arrest and CPR (CA/CPR) model of global ischemia. In addition, we utilized an in vitro cerebellar culture model to investigate the mechanism of ALLO protection of PCs.
Methods: Male C57Bl/6 mice were subjected to 10 min. cardiac arrest and resuscitated by ventilation with 100% oxygen in combination with, epinephrine and chest compressions (CPR). Vehicle (20% β-cyclodextran) or ALLO (2 or 8 mg/kg) was administered by i.p. injection 30 min. prior to arrest, and 6 and 24 hours following resuscitation. 48 hours after resuscitation, mice were sacrificed and brains analyzed by FluorJade B staining to identify damaged PCs. The percentage of damaged PCs was determined by counting the number of fluorescent/non-fluorescent PCs in the vermis of each cerebelli.
Neurons were cultured from the cerebellum of embryonic day 18 Sprague-Dawley rats. The culture media was replaced with glucose-free de-oxygenated saline and the cells were placed in an anaerobic incubator for 2 hours in order to perform in vitro ischemia (OGD) experiments. Following OGD, cells were returned to normoxia by incubation in normal culture media for 1–3 hours. In order to determine the effects of ischemia on PC GABA-A receptor activity, whole cell voltage-clamp recordings were made from the somas of Pcs using an Axopatch 200B amplifier interfaced to a computer running pCLAMP9.2. In separate experiments, protein was isolated from cultures and quantitative western blot analysis was performed to determine changes in GABA-A receptor protein.
Results: Our data indicate that ALLO protects cerebellar Purkinje cells following global ischemia (CA/CPR) in a dose-dependent manner. While 2 mg/kg ALLO had no effect on PC damage, 8 mg/kg ALLO provides significant protection of cerebellar PCs, decreasing damage by over 50%. CPR time and epinephrine dose used for resuscitation in vehicle and ALLO treated groups were not different.
In vitro, OGD results in a significant reduction in GABA-activated current in PCs recorded within the first hour after OGD, reducing the average current density by approximately 40%. ALLO (10 nM) prevented the decrease in current after OGD and interestingly, high levels of ALLO (1 µM) caused a 50% increase. Similarly, OGD causes a significant reduction of GABA-A receptor protein 1 hr after OGD, decreasing by approximately 70%. Treatment with 1 µM ALLO maintained the levels of GABA-A receptor protein at control levels following OGD.
Conclusions: These findings indicate that ALLO protects cerebellar PCs in vivo and in vitro and that ALLO prevents OGD-induced loss of GABA-A receptor activity, making it an exciting therapeutic agent that has the ability to both potentiate GABA-A receptors and maintain their activity following ischemia.
IN VIVO STAINING OF ASTROCYTIC ELEMENTS SURROUNDING MICROVESSELS WITH FLOWING RBCS IN MICE
Yutaka Tomita1,2, Minoru Tomita1, Takashi Osada1, Miyuki Unekawa1, Haruki Toriumi1, Ban Mihara2, Norihiro Suzuki1
1Department of Neurology, Keio University School of Medicine, Shinjuku-Ku, Tokyo, Japan, 2Institute of Brain and Blood Vessels, Department of Neurology, Mihara Memorial Hospital, Isesaki, Gunma, Japan
Background and aims: We have previously visualized the astrocytic-vascular interface with sulforhodamine 101 1) in rats (unpublished observation). This study aims to examine the structural relationship of astrocytic endfeet stained with sulforhodamine 101, and endothelial cells stained with FITC dextran in the microvasculature of mouse brain in vivo during continuous recording of FITC-labeled red blood cells (RBCs) flowing through the microvasculature, as observed with a confocal fluorescence microscope.
Methods: The head of a C57BL/6J mouse (n=5) was fixed to a stereotaxic apparatus under isoflurane anesthesia, a skull window was opened over the left parietal cortex 2), and the dura mater was removed carefully. A tail vein was catheterized for injection of FITC-labeled RBCs and FITC-dextran. Images of the movements of individual FITC-labelled RBCs in intraparenchymal single capillaries at a depth of approximately 50µm in the cortical region of interest (ROI) were acquired employing either a conventional video camera (30 frames/s) or a high-speed camera (500 frames/s) through a laser scanning confocal fluorescence microscope. Single capillaries were stained with a small amount of FITC-dextran injected intravenously, and astrocytes were stained with direct application of sulforhodamine 101 on the brain surface in vivo 1). Astrocytic attachment to the intraparenchymal microvasculature and its morphological changes were examined in vivo.
Results: We observed no toxic effects following sulforhodamine application, and the physiological parameters remained unchanged throughout the dye-staining experiments (ca. 3 h). Circulating FITC-labeled RBCs, which had been introduced from the tail vein, were observed like fireflies on the dark background of the cerebral cortex. The astrocytes and their endfeet were visualized in brown, in good contrast to FITC-dextran-stained endothelial cells and flowing, brightly FITC-labeled RBCs in the microvasculature. Arterioles were found to be enveloped densely by a thin endfeet sheath, like a sleeve. The endfeet locally saddled capillaries with occasional protrusions, which pushed the capillary wall towards the lumen and caused marked elongation of RBCs flowing through the narrowed gap. The endfeet tended to cluster at the branching site of capillaries, forming a structure like an ants' nest tunneled with capillaries for RBC traffic. This astrocytic structure changed its morphology slowly but dynamically. In contrast, the astrocytic endfeet had no contact with the venous system. These findings are similar to the results obtained previously in SD rats, except that the capillaries and astrocytic elements appeared to be more clearly visualized in mice.
Conclusions: We successfully visualized astrocytic elements and capillaries of the mouse microvasculature in which FITC-labeled RBCs were actually flowing in vivo. This technique provides a promising tool to investigate neuro-astrocytic control of capillary blood flow.
MODULATION OF THE FUNCTIONAL CEREBRAL BLOOD FLOW RESPONSE BY REDUCTION IN BASELINE BLOOD FLOW
Joy Liau1, Thomas T. Liu2
1Department of Bioengineering, University of California at San Diego, San Diego, CA, USA, 2Department of Radiology, University of California at San Diego, San Diego, CA, USA
Introduction
The effect of reductions in baseline cerebral blood flow (CBF) on the neurovascular coupling mechanism between neural activity and CBF is still poorly understood. While one study [1] reported that the absolute functional change in CBF (ΔCBF) is independent of baseline CBF (additive model), another [2] found that the functional percent increase in CBF (%ΔCBF) is independent of baseline CBF (proportional model). Other studies [3]–[4] have reported decreases in both %ΔCBF and ΔCBF with reductions in baseline CBF. We show here that reductions in baseline CBF due to caffeine lead to functional CBF changes that are more consistent with the additive model.
Methods
Subjects (n=9) refrained from caffeine for 12 hours before the study and ingested a 200mg caffeine pill between sessions (pre-dose and post-dose). Each session had the following scans: (a) anatomical, (b) block design (4cycles, 20s-on/40s-off, visual stimulus), and (c) a cerebrospinal fluid (CSF) scan for CBF quantification. Images were acquired on a 3T GE MRI system with a head coil. Functional runs (3 slices about the calcarine sulcus) used a PICORE QUIPPSII arterial spin labeling sequence with dual echo spiral readout (TE1/TE2=9.1/30ms; TI1/TI2=600/1500ms; TR=2s). The CBF time series was computed from the first echo of the block design run and was used to define a functional region of interest (ROI). The CBF time series was calibrated to absolute values by the CSF scan. After averaging the absolute CBF time series over cycles and voxels in the ROI, the baseline CBF, and ?CBF and %?CBF responses were calculated per subject. Paired two-tailed t-tests were used to assess differences in pre-dose versus post-dose data for the baseline CBF, %?CBF, and ?CBF.
Results
The figure shows pre-dose(blue) and post-dose(red) curves for the (a) %?CBF and (b) ?CBF responses, where vertical bars represent standard error. The caffeine dose led to a significant reduction in baseline CBF(p=0.0003, 35%), a large and significant increase in %?CBF(p=0.0005, 63%), and a small and nearly significant decrease in ?CBF(p=0.08, 15%).
Discussion
Prior studies examining the impact of reductions in baseline CBF on the functional CBF response using either hypocapnia or indomethacin have led to conflicting results that support the additive model [1], the proportional model [2], or neither model [3],[4]. Overall, our findings are more consistent with the additive model as the caffeine-related reduction in ?CBF was small, as compared to the reductions in ?CBF reported in [2]–[4]. The larger %?CBF increase observed with the reduction in baseline CBF is consistent with a relatively greater change in vessel radius when the baseline radius is reduced.
CEREBROVASCULAR EFFECTS OF INHALED NITRIC OXIDE
Nicole Angela Terpolilli, Seong-Woong Kim, Serge Thal, Nikolaus Plesnila
Department of Neurosurgery and Institute for Surgical Research, University of Munich Medical Center, Grosshadern, Munich, Germany
Background and aim: Inhaled nitric oxide (iNO) induces selective pulmonary vasodilatation and has been used as treatment for various lung and heart diseases associated with pulmonary hypertension.(1) Though at first thought to act selectively on the lung increasing evidence for extrapulmonary iNO effects emerged.(2),(3) However, direct evidence for iNO mediated cerebrovascular effects is missing yet. The aim of the current study was therefore to investigate the effect of iNO on cerebral blood vessels by using intravital microscopy.
Results: iNO led to a prompt and lasting dilatation of cerebral venules (10±1% increase vs. baseline, p<0.05) without affecting arteriolar diameter and cerebral blood flow. This effect gradually subsided upon discontiuation of iNO. Direct in vivo imaging of NO with DAF showed signifiantly increased NO concentrations in cerebrovascular endothelial cells (Fig. 1). To further prove that the observed change in vessel diameter was NO dependent we blocked guanylate cyclase with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). This completely abolished the observed effect.
Results: iNO led to a prompt and lasting dilatation of cerebral venules (10±1% increase vs. baseline, p<0.05) without affecting arteriolar diameter and cerebral blood flow. This effect gradually subsided upon discontiuation of iNO. Direct in vivo imaging of NO with DAF showed signifiantly increased NO concentrations in cerebrovascular endothelial cells (Fig. 1). To further prove that the observed change in vessel diameter was NO dependent we blocked guanylate cyclase with 1 abolished the obs H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). This completely erved effect.
Discussion: Inhaled NO is transported from the lung to the brain and leads to dilatation of cerebral venules. Accordingly, this is the first report demonstrating a cerebrovascular effect of inhaled NO. Further studies need to investigate the mechanisms of NO transport from the lung to the brain and additional effects of iNO on the cerebrovasculature during physiological and pathological conditions.
Figure 1: DAF fluoresence in cerebral vessels
BRAIN MODULATION WITH MCS & SCS FOR INTRACTABLE DEAFFERENTATION PAIN; PET ANALYSIS
Haruhiko Kishima1, Youichi Saitoh1, Azuma HIrayama1, Amami Kato1, Masayuki HIrata1, Satoru Oshino1, Naoki Tani1, Masahiko Sumitani2, Jun Hatazawa3, Toshiki Yoshimine1
1Department of Neurosurgery, Osaka University Medical School, Suita, Osaka, Japan, 2Department of Anesthesiology, Osaka University Medical School, Suita, Osaka, Japan, 3Department of Tracer and Kinetics, Osaka University Medical School, Suita, Osaka, Japan
[Background and aims] The mechanisms underlying deafferentation pain are not well understood. motor cortex stimulation (MCS) and spinal cord stimulation (SCS) are useful in the treatment of this kind of chronic pain; however, the detailed mechanisms underlying its effects are also unknown.
[Methods] Six patients with intractable deafferentation pain in the left hand were treated with MCS, and nine patients with this kind of pain in the leg were treated with SCS. For the MCS, 4-array strip stimulating electrode (Resume II, 3587A; Medtronic, Inc., Minneapolis, MN, USA) was placed subdurally at the surface of the right precentral gyrus. For the SCS, 4-array stimulating lead electrode (Pisces Quad, 3487A; Medtronic, Inc.) was placed epidurally in the thoracic-lumbar lesion. These electrodes were controlled by stimulator (Itrel3, Medtronic, Inc.). Both MCS and SCS reduced pain; visual analog scale (VAS) values for pain were 82 ± 20 (mean ± SE) before MCS and 39 ± 20 after MCS, and 76 ± 5 before SCS and 41 ± 5 after SCS. rCBF was measured by PET with H215O before and after MCS and SCS. The obtained images were analyzed with statistical parametric mapping software (SPM).
[Results] Significant rCBF increases were identified after MCS in the left posterior thalamus (PT), left insula, and right orbitatofrontal cortex(OFC), and the right precentral gyrus and right prefrontal cortex (PFC) showed rCBF decrease. In the late post-MCS phase, a significant rCBF increase was detected in the left caudal part of the anterior cingulate cortex (ACC). After SCS, rCBF is increased in the right PT, right OFC, bilateral parietal association cortices. and left PFC.
[Conclusions] These results suggest that MCS modulates the posterior insula and OFC to PT pathways to upregulate the pain threshold and the posterior insula to caudal ACC pathway to control emotional perception, resulting pain relief. Like as MCS, SCS might also modulate brain activation to control of pain threshold and emotion.
ELECTROPHYSIOLOGICAL BASIS OF FUNCTIONAL MRI SIGNALS IN RAT MODELSOF PERMANENT AND TRANSIENT CEREBRAL ISCHEMIA
Maurits Pieter Adriaan Van Meer1,3, Petrus Johannes Stienen2, Kajo van der Marel1, Arie Doornenbal2, Jan Willem Berkelbach Van Der Sprenkel3, Richard Michiel Dijkhuizen1
1Image Sciences Institute, University Medical Center Utrecht, Utrecht, The Netherlands, 2Department of Clinical Sciences of Companion Animals, Utrecht University, Utrecht, The Netherlands, 3Department of Neurosurgery, Rudolf Magnus Institute of Neuroscience, University Medical Center, Utrecht, The Netherlands
Background and Aims
Functional MRI (fMRI) is increasingly applied as a tool to study changes in activation patterns after stroke. However, the electrophysiological basis of fMRI-detected changes in activation responses are unresolved. Here, we measured somatosensory-evoked potentials (SEPs) in the forelimb region of the primary somatosensory cortex (S1fl) at different time-points after permanent and transient stroke in rats. Based on previous fMRI findings1,2, we expected loss of SEPs in ipsilesional S1fl and increased contralesional electrical activity when the affected forelimb is stimulated acutely after stroke.
Methods
Stroke was induced by permanent or transient (60-90minutes) right middle cerebral artery-occlusion (MCA-O) in rats.3
SEP recordings were performed at 2 and 3h (n=5) and at 24h (n=3) after permanent MCA-O, and at 24h after transient MCA-O (n=2). SEPs were simultaneously recorded epidurally from left and right S1fl with increasing and alternating electrical stimulation (0, 0.5, 1, 2, 4 and 8mA) of left and right forelimb under propofol anesthesia. After SEP recordings, animals were sacrificed for TTC staining to assess size and location of the cerebral ischemic lesion. SEP data were digitally low-pass filtered and analysis was done on the averaged SEP for each S1fl out of two sessions for each rat. We calculated the Rate Dispersion Factor (RDF) for the 5-30ms latency range, which provides an index of the overall shape of the SEP waveform.4
Results
Normal SEPs were observed in contralesional S1fl when the unaffected forelimb was stimulated. At 2 and 3h after permanent MCA-O, although reduced, significant SEPs were measured in right, ipsilesional S1fl when the left, affected forelimb was stimulated. At 24 hpost-stroke, ipsilesional SEPs were largely absent. RDF values at 2mA stimulation were 54.0±15.8%, 49.6±16.4% and 8.9±4.3% (percentage of SEP in left, contralesional S1fl during right, unaffected forelimb stimulation; mean±SEM), at 2, 3 and 24h after permanent MCA-O, respectively. At 24h after transient MCA-O, RDF in ipsilesional S1fl was 4.9±3.2%.
Electric activity in contralesional S1fl was also detected when the affected forelimb was stimulated. At 2 and 3h post-stroke, contralesional (left) RDF values at 2mA stimulation of the affected (left) forelimb were 15.6±6.9% and 11.5±3.5%, respectively. These RDF values were diminished at 24h post-stroke (permanent stroke: 5.5±1.8%, transient stroke: 5.9±2.5%).
Conclusions
Our study demonstrates that electrical activity in ipsilesional S1fl is partially preserved at 2-3h after permanent MCA-O, but has largely disappeared at 24h after transient or permanent MCA-O. The latter is in agreement with lack of fMRI activity in rat S1fl during the first days after stroke.1,2 Furthermore, contralesional activation responses upon stimulation of the affected forelimb, as observed in this electrophysiology study, have also been described in fMRI studies in rat stroke models.1,2 Thus, stroke-induced changes in fMRI-detected brain activation, which are based on measurement of hemodynamic responses, correlate with changes in electrophysiological activity.
ANATOMICAL STANDARDIZED STATISTICAL MAPPING OF 123I-IMP SPECT IN BRAIN LESIONS
Yasushi Shibata, Manabu Akimoto, Akira Matsushita, Tetsuya Yamamoto, Shingo Takano, Akira Matsumura
Department of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
Purpose
123I-iodoamphetamine Single Photon Emission Computed Tomography (IMP SPECT) is used to evaluate the cerebral blood flow in patients with either cerebrovascular or neurodegenerative diseases. However, its application for patients with brain tumors has so far been rarely reported. Primary central nervous system lymphoma (PCNSL) is a rare tumor that shows a delayed IMP uptake. The relatively low spatial resolution of SPECT is a clinical problem which is used to diagnose brain tumors. Anatomical standardized statistical mapping is a useful method for improving the diagnostic ability of SPECT. We examined the statistical mapping of IMP SPECT in patients with brain lesions.
Patients
This study included 23 patients with brain lesions: 9 non-Hodgkin B-cell lymphoma, 1 Burkitt lymphoma, 6 glioma, 2 multiple sclerosis, 1 cerebritis, and 3 without any pathological diagnosis but a clinical diagnosis of PCNSL.
Methods
After the intravenous injection of 222MBq of 123I-IMP, early (15 minutes) and delayed (3 hours) images were acquired using a multi-detector SPECT machine (E.CAM, Siemens Medical). All SPECT data were saved in the Digital Imaging and Communications in Medicine (DICOM) format. DICOM data were transferred to a newly developed software program iNeurostat+ (Nihon Medi-physics, Hyogo, Japan) which runs on a Windows personal computer. The SPECT data were anatomically standardized on normal brain images. The increased uptake of IMP were statistically mapped on the tomographic images of the normal brain. The image quality, diagnostic ability and imaging artifacts were evaluated by a visual inspection.
Results
Eight patients showed a high uptake in the delayed IMP SPECT images (6 Diffuse Large B Cell, 2 unknown). All unknown patients were successfully treated with steroids and radiation therapy, so their clinical diagnosis was PCNSL. Glioma, MS and Burkitt lymphoma did not show a high uptake of delayed IMP SPECT. IMP SPECT is useful for the diagnosis of PCNSL, especially for the differentiation between glioma, MS and Burkitt lymphoma. Three patients with pathologically proven PCNSL showed no uptake in IMP SPECT. All these tumors were too small to detect in IMP SPECT. However, statistical mapping revealed the IMP uptake in these tumor. A heterogeneous IMP uptake was seen in homogenous tumors in MRI. For patients with a hot IMP uptake, statistical mapping showed clearer uptake. There were some artifacts on the statistical mapping, however, these artifacts did not result in diagnostic problems due to comparisons of statistical mapping and original SPECT or magnetic resonance imaging.
Discussion
SPECT has some problems, including a low spatial resolution caused by scattering radiation, a partial volume effect and the correction of the absorption and scattering. IMP SPECT is useful for the diagnosis of PCNSL because the uptake is specific.
Statistical mapping is useful for detecting small tumors which cannot be detected by usual IMP SPECT. However, some artifacts also exist in statistical mapping images. The careful interpretation of such image findings is essential.
Conclusion
Anatomical Standardized Statistical Mapping is thus considered to be a useful method for improving the diagnostic sensitivity, spatial resolution and accuracy of IMP SPECT for brain lesions.
CORRELATION OF INTRINSIC OPTICAL SIGNAL AND CEREBRAL BLOOD FLOW DURING ACTIVATION OF RAT SOMATOSENSORY CORTEX
Michael M. Haglund1, Joseph R. Meno2, Al C. Ngai2, H. Richard Winn3
1Department of Neurological Surgery, Duke University, Durham, NC, USA, 2Department of Neurosurgery, University of Washington, Seattle, WA, USA, 3Department of Neurosurgery, Mount Sinai School of Medicine, New York, NY, USA
Background and Aims: Optical imaging of intrinsic signals has been accomplished in vitro and in vivo. The source of the in vitro signal is thought to be related to postsynaptic activation and cellular swelling, while the in vivo signal has been suggested as arising from blood volume changes and oxygen delivery from capillaries. To better define the origin of the optic signal, we decoupled CBF from neuronal activation using adenosine receptor antagonists during contralateral sciatic nerve stimulation (SNS).
Methods: Rats were anesthetized, intubated, ventilated and a cranial window created. The contralateral sciatic nerve was exposed and stimulated as previously described (Ngai et all, 1986). We studied the relationship between arteriolar diameter, intrinsic optical signals (690 nm) and somatosensory evoked potentials (SSEPs), during cortical activation prior to and during application of adenosine receptor antagonists: Theophylline, a non-specific adenosine receptor antagonist (A1 and A2) and CPX (8-cyclopentyl-1, 3-dipropylxanthine), a selective A-1 antagonist.
Results: In mock CSF, pial arterioles in the hindlimb somatosensory cortex dilated 124.7+4.7% during contralateral SNS. Topical application of theophylline (THEO; 5µM, had no effect on resting arteriolar diameter but significantly attenuated SNS-induced vasodilatation (110.3±2.4%; p<0.05 versus mock CSF; n=4). Topical application of (CPX; 1 uM), also had no effect on resting arteriolar diameter but significantly potentiated vasodilatation during SNS (151.9±19.4%; p<0.05; n=5). In the same animals, the optical changes were potentiated during SNS by topically applied THEO, when compared to corresponding mock CSF controls. Topical application of CPX, however, caused a large increase of the intrinsic signals obtained during SNS. In a separate group of animals, SSEP recordings revealed that topical application of THEO had no effect on either the N-1 or P-1 wave. Topical application of CPX significantly attenuated the N-1 wave (n=8; p<0.01).
Conclusion: By adenosine receptor manipulation, we were able to independently alter cortical activity and vessel diameter changes. We demonstrate that change in intrinsic signal, imaged at 690 nm, is not related to the vascular compartment, but rather correlates with changes in parenchymal activity of the brain. Moreover, the present study provides further evidence for a neuromodulatory role of adenosine in regulating cerebral blood flow during increased cortical activity.
[MMH suuppt. by Klingenstein Foundation; HRW suppt. by NS21076]
FOCAL FUNCTIONAL METABOLIC ACTIVITY DURING ACOUSTIC STIMULATION IS UNDERESTIMATED WITH [1- OR 6-14C]GLUCOSE DUE TO METABOLITE TRAFFICKING AND RELEASE
Nancy Cruz, Kelly Ball, Gerald Dienel
Department of Neurology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
Background/Aims. Labeled glucose and its analogs are widely used in imaging and metabolic studies of brain function, astrocyte-neuron interactions, and neurotransmission. Metabolite shuttling among astrocytes and neurons is essential for cell-cell transfer of neurotransmitter precursors, distribution of molecules synthesized or metabolized in one cell type, and elimination of metabolic by-products. However, dispersion and release of labeled compounds from activated cells would reduce signal registration in metabolic labeling studies, causing underestimation of focal functional activation. Processes involved in metabolite trafficking and release were, therefore, assessed in the auditory pathway of conscious rats.
Methods/Results. Unilateral monotonic stimulation increased glucose utilization (CMRglc) in tonotopic bands in the activated inferior colliculus by 35–85% compared to contralateral tissue when assayed with [14C]deoxyglucose (DG), whereas only 20–30% increases were registered with [1- or 6-14C]glucose. Tonotopic bands were not evident with [1-14C]glucose unless assayed during halothane anesthesia (which also blocks gap junctions) or pre-treatment with probenecid (an inhibitor of monocarboxylic acid transport), but were detectable with [6-14C]glucose, consistent with loss of 14C via the pentose shunt pathway during activation. Extracellular lactate levels transiently doubled during acoustic stimulation, suggesting release to interstitial fluid, so metabolite spreading was assessed by microinfusion of [14C]tracers directly into the inferior colliculus from a point source. The volume of tissue labeled by [1-14C]glucose exceeded that by [14C]DG by 3.2- and 1.4-fold during rest and acoustic activation, respectively. During activation, the tissue volume labeled by U-14C-labeled glutamine and lactate rose, whereas that by glucose fell 50% and that by DG was unchanged. Because [14C]DG-6-P is retained intracellularly, [14C]DG trapping represents the volume of tissue within which glucose is phosphorylated by hexokinase, and the higher label distribution by [14C]glucose must be due to spreading of downstream metabolites. Increased release of [14C]glucose metabolites from tissue would cause the low autoradiographic registration of activation, and lower retention of labeled metabolites probably underlies diminished label spreading during activation. Glutamine and lactate, two metabolites known to be released from cells to interstitial fluid, are distributed within tissue more widely during activation compared to rest and contribute to metabolite trafficking from activated cells. Dispersion of [1-14C]glucose and its metabolites during rest was also reduced 50% by pre-infusion of two gap junction blockers, implicating the astrocytic syncytium as a pathway for metabolite diffusion through gap junctional channels along their concentration gradients.
Conclusions. Loss of glucose-derived label from activated cells is due to a number of factors, including decarboxylation reactions in the pentose shunt and, perhaps, other pathways, metabolite spreading within tissue, in part, via the astrocyte syncytium, and metabolite release from activated tissue. Lactate release probably contributes substantially to reduced signal registration during activation for the following reasons, (i) inhibition of monocarboxylic acid transporters by probenecid enhances visualization of tonotopic bands, (ii) extracellular lactate levels rise, (iii) lactate spreading increases, and (iv) labeled lactate has a specific activity half that of glucose. To summarize, dispersion and release of various labeled metabolites contribute to the substantial underestimation of focal CMRglc in conscious subjects when labeled glucose is the tracer.
CEREBRAL PERFUSION PATTERN OF IDIOPATHIC NORMAL PRESSURE HYDROCEPHALUS STUDIED BY SPECT AND STATISTICAL BRAIN MAPPING
Kazunari Ishii1, Hiroki Sasaki1, Atsushi K. Kono1, Naokazu Miyamoto1, Kenichi Shimada2, Shingo Ohkawa2, Tetsuro Kawaguchi3, Etsuro Mori4
1Department of Radiology, Hyogo Brain and Heart Center, Himeji, Hyogo, Japan, 2Institute for Aging Brain and Cognitive Disorders, Hyogo Brain and Heart Center, Himeji, Hyogo, Japan, 3Department of Neurosurgery, Hyogo Brain and Heart Center, Himeji, Hyogo, Japan, 4Department of Behavioral Neurology and Cognitive Neuroscience, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
Background and aims: Normal pressure hydrocephalus (NPH) is characterized by gait disturbance, mental deterioration and urinary incontinence, in association with normal cerebrospinal fluid (CSF) pressure. Although frontal cerebral blood flow (CBF) reduction has been emphasized in the majority of NPH cases, no report has been able to demonstrate a typical CBF pattern because the inclusion of idiopathic normal pressure hydrocephalus (iNPH) and secondary NPH in previous reports has led to heterogeneous CBF patterns. The purpose of this study was to investigate the specific pattern of CBF in subjects with iNPH using voxel-based analysis. Methods: N-isopropyl-p-[I-123] iodoamphetamine (IMP) single photon emission computed tomography (SPECT) images were performed in 30 iNPH patients, who met probable iNPH criteria, 30 Alzheimer disease (AD) patients and 15 normal control (NC) subjects. Inter-group comparisons between iNPH patients and NC subjects and between AD patients and NC subjects were performed using three-dimensional stereotactic surface projection (3D-SSP) analysis. Individual 3D-SSP images of the iNPH patients were assessed by visual inspection. Results: On the Z-score maps, areas of relative hypoperfusion were recognized around the corpus callosum in all 30 iNPH patients, as well as in the Sylvian fissure regions in 19 of 30 iNPH patients which included artifacts by dilated ventricles and the Sylvian fissures. Ten frontal dominant, eight parietotemporal dominant, and 12 diffuse hypoperfusion types were demonstrated. Inter-group comparison between iNPH and NC subjects showed relative hypoperfusion in the frontal and parietotemporal areas and severe hypoperfusion around the corpus callosum and Sylvian fissure regions, while parietotemporal and posterior cingulate CBF reduction was demonstrated between the AD and NC groups (Figure). Conclusion: The voxel-based statistical mapping method revealed an iNPH CBF reduction pattern. Diffuse or frontal dominant CBF reduction was shown and, additionally, severe relative hypoperfusion around the corpus callosum and Sylvian fissures was demonstrated, which probably reflects dilatation of the ventricle systems and Sylvian fissures and may represent characteristic patterns of iNPH.
QUANTIFIED T1 AND T2 AS USEFUL ADJUNCTS TO DWI FOR EARLY INFARCT DETECTION IN PERMANENT AND TRANSIENT MCAO
Jaspreet Kaur1,2, Ursula Tuor1,2,3, Zonghang Zhao1, Hisham Assem4, Jodie Lee-Petersen1,2, Tadeusz Foniok3, Phillip Barber1,2
1Hotchkiss Brain Institute and Clinical Neurosciences, University of Calgary, Calgary, AB, Canada, 2Experimental Imaging Centre, University of Calgary, Calgary, AB, Canada, 3MR Technology, Institute for Biodiagnostics West, Calgary, AB, Canada, 4O'Brien Centre for The Bachelor of Health Sciences Program, University of Calgary, Calgary, AB, Canada
Introduction
Ischemic stroke is a leading cause of death and disability. Our knowledge of differences in acute MR imaging changes between transient and permanent ischemia remain incomplete. We hypothesized that quantified T1 and T2 can be useful adjuncts to DWI for early detection of reperfusion injury or infarction. Therefore, we investigated the differences in MR changes between transient and permanent MCAO, by applying sequential high-resolution quantified T1, T2 and ADC measurements at different time points following ischemia.
Methods
Rats underwent permanent or transient MCAO in tandem with temporary occlusion (90 min) of common carotid arteries. Within 30 min of occlusion, MR sequence images were acquired, using a 3×3 cm2 field of view and a 256×128 matrix for 7 slices 1.0 mm thick through the cerebrum, that included a multi-echo a T1 map (inversion recovery Snapshot-FLASH imaging scans), T2 weighted images and an ADC map at 1.5h, 2.5 h and 24 h following MCAO. T1, T2 and ADC were measured in the infarct area, and represented as percentage of whole brain area. Data was compared using one way ANOVA for multiple comparisons.
Results
At early (1.5 and 2.5 h) and delayed (24 h) time points, changes in T1 were observed in permanent and transient MCAO. Early extensive changes in T1 signatures in the permanent ischemia were observed at a time when no notable T2 or ADC changes existed. T1 changes in transient MCAO were statistically higher than those observed in permanent MCAO at 2.5 and 24 hrs. In permanent MCAO, quantified T2 signatures demonstrated small statistically insignificant increases between 1.5 to 2.5 h, while in transient MCAO, following reperfusion; significant early changes in T2 were observed that evolved over time. No significant changes occurred in ADC overtime in permanent ischemia, while changes in ADC following transient MCAO were statistically significant at 1.5 and 24 h time points. Infarct areas calculated within T1 maps, compared over different time points in permanent and transient ischemia showed a significant evolution over time. For T2 maps, significant infarct evolution was observed at 2.5–24. h in permanent and transient MCAO. Infarct progression observed in ADC became statistically significant at 2.5 h, and was similar to that observed in T2. In both models, infarct area calculated early (1.5 h) with the T1 sequence was significantly greater than that within T2 and ADC maps.
Conclusion
An early extensive and distinct increase in T1 occurred in permanent and transient MCAO, prior to the onset of cytotoxic or vasogenic edema. It is likely that T1 changes are related to reduced blood flow and an early increase in total water content with an intact BBB that occurs within the first few hours following cerebral ischemia. Quantified T1 may be a useful adjunct for detection of early ischemic injury and help in defining ischemic penumbra. Further studies on changes in BBB permeability are warranted to establish the relationship between MR imaging changes and BBB dysfunction.
CYTOKINE BIOLOGY, LIPID METABOLISM AND CITICOLINE IN STROKE
Rao Muralikrishna Adibhatla, James F. Hatcher, Eric C. Larsen
Department of Neurological Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA
Background and aims: Phosphatidylcholine (PC) is a major membrane phospholipid (~50% of the total phospholipid) and even 10% loss is sufficient in itself to induce cell death. PC homeostasis is regulated by a balance between hydrolysis and synthesis. PC is hydrolyzed by phospholipase A2 (PLA2), PC-phospholipase C (PC-PLC) and phospholipase D (PLD). PC synthesis is rate-limited by cytidine triphosphate:phosphocholine cytidylyltransferase (CCT) that makes CDP-choline in the brain. TNF-α and IL-1 (IL-1a/s) are pro-inflammatory cytokines that are up-regulated in the brain after stroke. Phospholipases are induced by TNFa/IL-1 and mediate their cytotoxicity. TNF-α/IL-1 also down-regulate CCT. Thus, TNF-α/IL-1 alter lipid metabolism by increasing PC hydrolysis (stimulate phospholipases) and inhibiting PC synthesis (decrease CCT). The aim of the study is whether citicoline counteract the lipid alterations caused by cytokines after stroke.
Methods: Focal cerebral ischemia was induced for 1 hr in male spontaneously hypertensive rat (SHR) using middle cerebral artery occlusion (MCAO) by intraluminal suture technique. Citicoline liposomes were prepared by Lipex thermobarrel extruder pressurized with argon. Treatments: TNF-α ab (0.36 mg/kg i.v.), IL-1ra (20 µg/4 µL icv), free CDP-choline (500 mg/kg 0 & 3 h i.p.) and CDP-choline liposomes (18 mg/kg 0 & 3 h i.v.). TNF-α and IL-1s levels were measured by ELISA. mRNA expression by RT-PCR; Protein expression by Western blotting and infarction by TTC staining. PLA2 activity by radioactive assay, PC-PLC activity was by AMPLEX Red PC-PLC assay.
Results: tMCAO significantly increased sPLA2 mRNA and protein levels. CDP-choline, TNF-α antibody (TNF-α ab), or interleukin-1 receptor antagonist (IL-1ra) significantly attenuated sPLA2 protein levels. tMCAO resulted in loss of CCT protein as well as activity that were partially restored by citicoline. tMCAO up-regulated PLD1 and PLD2 over 7 d reperfusion. However, there were no changes in the cPLA2 and iPLA2 protein expression after tMCAO. PC-PLC activity was increased after tMCAO. The up-regulation of phospholipases (PLA2, PC-PLC, PLD) and down-regulation of CCT collectively resulted in the loss of PC and total phospholipids. Citicoline attenuated sPLA2 up-regulation and CCT down-regulation after tMCAO. TNF-α antibody and IL-1ra also significantly attenuated sPLA2 expression. Citicoline attenuated stroke-induced up-regulation of TNF-α and IL-1s levels. Liposome encapsulated CDP-choline significantly decreased infarction (by 62%) compared to equivalent dose of free CDP-choline (26% reduction) after tMCAO.
Conclusions: These results suggest that citicoline restores PC levels by differentially affecting phospholipases and CCT through cytokine alterations after stroke. Since all cells (neurons, astrocytes, endothelial cells etc., called the ‘neurovascular unit’) contain PC as a membrane building block, citicoline protection may arise from stabilizing the ‘neurovascular unit’. Advantages of citicoline liposomes in stroke include: 1) effective at low doses (18 mg/kg citicoline liposomes vs 500 mg/kg free citicoline), 2) the presence of GM1 ganglioside confers long-circulating properties by suppressing phagocytosis, 3) brain uptake of the drug is increased to ~23% of the injected dose, and 4) Citicoline liposomes deliver the agent intact to the brain, circumventing the rate-limiting CCT step. Our studies provide an integration of citicoline effects with cytokine biology and lipid metabolism in cerebral ischemia.
PHARYNGEAL COOLING DECREASES BRAIN TEMPERATURE AND INTRACRANIAL PRESSURE DURING RESUSCITATION IN MONKEYS
Yoshimasa Takeda, Motomu Kobayashi, Hideki Taninishi, Toshihiro Sasaki, Minako Arai, Kiyoshi Morita
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan
Introduction The development of a technique that enables brain temperature to be immediately and selectively reduced without affecting systemic circulation is needed to perform resuscitative hypothermia in a clinical situation. Since bilateral common carotid arteries exist at about 1 centimeter (C) from the pharynx, cooling the pharyngeal region decreases the carotid arterial temperature first and subsequently decreases brain temperature before lowering cardiac temperature. The present study was designed to evaluate the effects of pharyngeal cooling on brain temperature, systemic temperature, intracranial pressure and extracellular glutamate concentration during resuscitation in monkeys.
Methods Japanese monkeys (7.5+−2.4 kg) were divided into a control group (n=6) and a pharyngeal cooling group (n=3) and were anesthetised with 1% isoflurane. Cardiac arrest (12 minutes) was initiated with electrical stimulation. Changes in intracranial pressure (parietal region) and epidural (parietal region), sub-cortical (parietal region, 3 cm below the cortical surface) and rectal temperatures were monitored during a 30-minute resuscitation period. A micro dialysis probe was inserted into the parietal cortex for the measurement of glutamate concentration. In the pharyngeal cooling group, a pharyngeal cuff was inserted into the pharynx and was perfused with water (5 C degree) at the rate of 500 ml/minute from the onset of resuscitation for 30 minutes.
Results Cortical (33.3+−0.7 C degree) and sub-cortical (35.8+−0.5 C degree) temperatures decreased during cardiac arrest in both groups. In the control group, however, cortical and subcortical temperatures increased with the initiation of resuscitation and reached 35.1+−0.5 C degree and 36.5+−0.2 C degree, respectively, 30 minutes later. In the pharyngeal cooling group, cortical and sub-cortical temperatures decreased and reached 31.1+−1.5 C degree and 32.4+−1.3 C degree, respectively, 30 minutes later. Rectal temperature was unchanged in both groups. During the resuscitation period, the intracranial pressure increased to 30+−16 mmHg (control) and 14+−6 mmHg (pharyngeal cooling) and took 23+−9 minutes (control) and 6+−1 minutes (pharyngeal cooling) to become less than 10 mmHg. Extracellular glutamate concentration was unchanged during cardiac arrest in both groups and increased after the onset of resuscitation. The maximum concentration of extracellular glutamate was decreased in the pharyngeal cooling group (13 micro mol/l) compared to that in the control group (39 micro mol/l). The periods required for recovery of mean arterial blood pressure (>50 mmHg) were the same (4+−1 minutes after onset of resuscitation) in the two groups.
Conclusions Cortical and sub-cortical temperatures were decreased with the initiation of pharyngeal cooling without lowering of rectal temperature during the resuscitation period in monkeys. Intracranial pressure and extracellular concentration of glutamate were lower in the pharyngeal cooling group without affecting time required for recovery of mean arterial blood pressure (>50 mmHg).
NEUROPROTECTIVE EFFECTS OF INTRANASAL ERYTHROPOIETIN AFTER PERMANENT UNILATERAL ISCHEMIA IN MONGOLIAN GERBILS, WITH A THERAPEUTIC WINDOW UP TO 18 HOURS
Jorge Daniel Garia Salman1, Iliana Sosa Teste2, Yuneidys Mengana Tamos2, Nelvis Subiros1, Adriana Munoz4, Yanier Nunez4, Yamila Rodriguez Cruz5, Albertina Thomas2, Barbara Gonzalez2, Julio Cesar Garia Rodriguez2, Rosa Maria Coro Antich1, Katiuska Rodriguez2
1INN Neurology National Institute, Havana, Cuba, 2CENPALAB, Havana, Cuba, 4CIDEM, Havana, Cuba, 5ISCM, Havana, Cuba
Human recombinant erythropoietin with low content of sialic acids was administered intranasally after permanent unilateral common carotid occlusion in Mongolian gerbils. Four ischemic treated groups were formed, in which the treatment began immediately, at 6, 12 and 18 hours after surgery. Treatment (120 IU/animal), lasts for 4 days, 30 IU/day. Habituation behavior of the exploratory activity was measured one day before and seven days after surgery. Survival rate at day 7 and behavioral test were compared with ischemic untreated and sham-operated animals. Survival rate was higher in immediately treated (68%), 6h (72%), and 12h (65%) than in ischemic untreated (46%) gerbils. Comparison between the habituation behavior before and after surgery revealed an increased habituation in shame-operated animals, as well as in all ischemic treated groups, indicating an erythropoietin-induced protection of learning and long term memory in survivors. When comparing the behavioral performance among groups seven days after surgery, was observed a loss of habituation in the ischemic-untreated group only. Results suggested that therapeutic window of intranasal erythropoietin with low content of sialic acids, could be extended to the first 12 hours of ischemia, although neuroprotective properties were observed even at 18h in less injured animals.
LONG-TERM OBSERVATION OF FREE RADICAL SCAVENGER EFFECTS ON CEREBRAL INFARCTION INDUCED BY NON-INVASIVE PHOTOTHROMBOSIS IN MICE
Shinji Nakajima1,2, Syojiroh Sawada1, Yuhji Morimoto3, Masashi Nibuya2, Souichiroh Nomura2
1Departments of Neurology and Psychiatry, National Defence Medical College, Tokorozawa, Saitama, Japan, 21st Department of Internal Medicine, National Defence Medical College, Tokorozawa, Saitama, Japan, 3Department of Integrative Physiology and Nano-Medicione, National Defence Medical College, Tokorozawa, Saitama, Japan
Background and aims: Various drugs are used to remedy adverse events with spatiotemporal evolution in the acute phase of stroke, and recent clinical studies set the therapeutic assessment to the chronic stage with recovery. Longer experimental observation is also desirable in animal studies to assess therapeutic effects for the restoration of damaged tissues and neural functions. The aim of this study is to examine effects of free radical scavenger (edaravone, MCI-186) administered in the hyperacute stage of stroke using relatively small number of animals longitudinally.
Methods: C57BL/6J mice were anesthetized by the intra-peritoneal injection of 1% chloralose and 10% urethane mixture, keeping rectal temperatures at 37.5 degrees on the heating pad (Nihon Kohden, ATB-1100). Pphotothrombosis (PT) was created in the right parietal lobe by the stereotactic transcranial irradiation of He-Ne laser light (532 nm, 5 mW) after the injection of rose-bengal solution via the tail vein. Cerebral blood flow was measured using the laser-Doppler flow meter (Advance, ALF21). Animals were divided into the ischemic control (C) group, in which the laser was irradiated for 3-, 5, 10-, 15-, and 30-minute (N=6, in each group) to compare time-dependencies of PT formation, and edaravone (E) group (N=6) with 10-minute irradiation, where edaravone was administered twice, immediately before and 30-minute after PT (3 mg/kg IV, each time). Cerebral images were taken by the 9.4 T MRI (Bruker, AVANCE 400WB) at 1hour, 1- and 3-day, followed by 1- and 12-week sequentially in individual mice. Ischemic changes were recorded in T1+Gd-DTPA (PWI), Diffusion (DWI), and T2WIs (20mm FOV, 256×256 matrix, coronal slices), and sizes of ischemic loci were measured by the ROI analysis program. Microscopic observations were performed through the lubricated skull intermittently to analyze microvasculature changes after the acute ischemic load.
Results: MRI showed ischemic changes in PWI and DWI, and T2WIs at 1-hour with enlarged lesions dependent to irradiation time over 5-minute. T2WI lesions in 10-minute irradiation occupied 20.4±1.7 % (M±SEM) of the ipsilesional hemisphere in the C group, and decreased to 4.8±0.5 % (p<0.005) in the E group at 1-hour. Ischemic changes enlarged with edema to 27.7±4.5 % in the C group and 8.1±1.0 % (p<0.005) in the E group at 1-day. These edematous changes subsided at 1-week, and shrunk to small infarctions at 12-week. Microscopic observation showed extensive occlusions including arterioles, capillaries, and venules by platelets-thrombi immediately after PT with very gradual resolution and recanalization thereafter.
Conclusions: Laser irradiation in the present study created stereotypic ischemic lesions by multi-vessel PT in an irradiation-time dependent manner, which might be different from selective arterial PT with variable MRI lesions in cases of early recanalization. Free radical scavenger, edaravone, significantly decreased the size of photothrombotic lesions when observed by sequential MRI, showing the direct scavenging effect against singlet oxygen formed by photochemical reactions, as we already demonstrated in vitro and in vivo studies. Suppression of cerebral edema may be due to the reduction of the postischemic inflammatory process around venules through blocking the enhanced dissociation of NFkB and IkB by free radical oxygen species.
ISCHEMIC PRECONDITIONING RECOVERS NERVE GROWTH FACTOR, BRAIN-DERIVED NEUROTROPHIC FACTOR AND THEIR HIGH AFFINITY RECEPTORS AFTER FOREBRAIN ISCHEMIA
Tsong-Hai Lee1, Jen-Tsung Yang2, Yu-Shien Ko3
1Department of Neurology, Chang Gung Memorial Hospital, Linkou Medical Center and Chang Gung University School of Medicine, Taoyuan, Taiwan-R.O.C., 2Department of Neurosurgery, Chiayi Chang Gung Memorial Hospital and Chang Gung University School of Medicine, Chiayi, Taiwan-R.O.C., 3Department of Cardiology, Chang Gung Memorial Hospital, Linkou Medical Center and Chang Gung University School of Medicine, Taoyuan, Taiwan-R.O.C.
Background and aims
Preconditioning of gerbil brain with a sublethal forebrain ischemia is known protective to hippocampal CA1 neurons following a subsequent lethal ischemia (the second ischemia) which normally damages neurons (ischemic tolerance). The role of neurotrophic factor in the mechanism of ischemic tolerance has not been well discussed.
Methods
Male Mongolian gerbils, 12-13 weeks old and weighing 65-85 g, were used for study. To induce ischemic tolerance, a 2-min period of occlusion of bilateral common carotid arteries (ischemic preconditioning) was followed by 3 days of reperfusion, and then a 3-min period of occlusion (the second ischemia) was induced. Animals were decapitated at 3 days of reperfusion after sham operation or 2-min ischemic preconditioning and at 4 hours, 1 day, 3 days and 7 days after the second ischemia (n= 5, in each time point). Neuronal apoptosis was detected by using an in situ apoptosis detection kit. Hematoxylin and eosin (H&E) staining and 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) staining were used to calculate the survived CA1 neurons. Immunofluorescent detection of NGF, TrkA, BDNF and TrkB was performed in brain sections. Confocal laser scanning microscopy of immunolabeled sections was carried out with use of a Leica TCS SP2 system. Western blot analysis was also studied.
Results
Using confocal laser scanning microscope, we found that the immunofluoresence of NGF and BDNF and their high affinity receptors (TrkA and TrkB) was present in the hippocampal cells of sham-operation gerbils. A 2-min ischemic preconditioning caused little change of these proteins (ANOVA test, P> 0.05). After the second lethal ischemia, in the CA1 area with ischemic preconditioning, only BDNF but not NGF and their high affinity receptors showed a transient reduction at 4 hours (ANOVA test, P< 0.01) and improved from 1 day (ANOVA test, P> 0.05). In the CA1 area with no ischemic preconditioning, NGF and its high affinity receptor, TrkA, showed a consistent reduction from 4 hours to 7 days (ANOVA test, P< 0.05); BDNF and TrkB decreased transiently from 4 hours to 1 day (ANOVA test, P< 0.05) but were recovered in the survived neurons from 3 days. At 3 and 7 days after the second lethal ischemia, apoptotic cell injury could be seen in the CA1 area with no ischemic preconditioning, and was sparsely seen in the CA1 area with ischemic preconditioning. In the ischemia-resistant CA3 and dentate gyrus areas, only BDNF decreased significantly at 7 days in the CA3 area with no ischemic preconditioning. However, there was no significant change of NGF, TrkA and TrkB immunofluorescence from 4 hours to 7 days after the second lethal ischemia in the gerbils with or without ischemic preconditioning. Western blot study showed a similar change to the confocal immunofluorescent study.
Conclusions
Our study showed that ischemic preconditioning recovers the initial decline of NGF and BDNF and their corresponding receptors in the vulnerable CA1 neurons after the second lethal ischemia, suggesting a possible role of growth factors in the protective mechanism of ischemic tolerance.
OXIDATIVE STRESS CONTRIBUTES TO MEMORY IMPAIRMENT AND NEURONAL DAMAGE IN EXPERIMENTAL CEREBRAL HYPOPERFUSION
Eszter Farkas1, Eva Mracsko2, Monika Krecsmarik1,2, Eszter Karg3, Botond Penke4, Ferenc Bari2
1Department of Anatomy, School of Medicine, University of Szeged, Szeged, Hungary, 2Department of Physiology, School of Medicine, University of Szeged, Szeged, Hungary, 3Department of Pediatrics, School of Medicine, University of Szeged, Szeged, Hungary, 4Department of Medical Chemistry, School of Medicine, University of Szeged, Szeged, Hungary
The permanent, bilateral occlusion of the common carotid arteries of rats (2VO) has been widely used as a model for chronic cerebral hypoperfusion as it occurs in aging and dementia. 2VO in experimental animals leads to memory dysfunction and a progressive loss of hippocampal pyramidal neurons. However, the potential mechanisms of neuronal damage after the onset of 2VO have not been identified. The present investigation set out to determine whether oxidative stress may contribute to the 2VO-induced memory impairment and neurodegeneration. Treatments with the antioxidant alpha-tocopherol were employed as pharmacological means to counteract and, thus, confirm the incidence of oxidative stress in the 2VO model.
2VO was imposed on male Wistar rats (n=70). Sham-operated animals (SHAM) served as controls (n=66). Half of the animals were pre- or post-treated repeatedly with alpha-tocopherol (5 × 100 mg/kg daily, i.p.), the other half received soybean oil, the vehicle of alpha-tocopherol. One week after the onset of 2VO, the spatial learning capacity of the animals was assessed in the Morris water maze (n=65). After testing, hippocampal slices were stained with cresyl violet in order to examine the pyramidal cell layer integrity. The density of MAP-2-positive dendrites and the level of OX-42-labeled microglial activation were determined immunocytochemically. The amount of nitrotyrosine (generated by the free radical peroxinitrite) was determined with Western blotting (n=32). Alpha-tocopherol concentration was determined in the peripheral tissues, blood and brain with HPLC (n=39).
Alpha-tocopherol attenuated the 2VO-induced learning impairment. The treatments with alpha-tocopherol, and particularly the post-treatment, prevented the 2VO-induced pyramidal cell death and the activation of microglia in the hippocampus CA1 region, and the degeneration of MAP-2-positive dendrites in the CA3 region, determined 2 weeks after the onset of 2VO. The amount of nitrotyrosine in the hippocampus was moderately elevated as compared with SHAM controls on days 3 and 7 (relative optical density: 0.87±0.11 vs. 0.75±0.10, and 0.98±0.03 vs. 0.86±0.10, respectively), but was not affected on day 1 after the initiation of 2VO. The alpha-tocopherol concentration was considerably elevated in the peripheral tissues and the blood, while only moderate accumulation of alpha-tocopherol was detected in the hippocampus (31.99±1.79 vs. 28.98±1.36 nmol/g).
The data suggests that oxidative neuronal injury plays a role in the 2VO-related neurodegenerative processes. Further, alpha-tocopherol appears beneficial in combating the cerebral hypoperfusion-related neuronal damage. Finally, the observation that the post-treatment achieved better neuroprotection than the pretreatment may imply that the increased availability of alpha-tocopherol at the time of an ongoing ischemic injury may be required for a better neurological outcome from the insult.
Grant information: This work was supported by the Hungarian Scientific Research Fund (OTKA) grants F042803 and K063401, and the Hungarian National Office for Research and Technology (NKTH).
ISCHEMIC PRECONDITIONING CONFERS NEUROPROTECTION VIA GABA SYNAPSES
Richard Anthony DeFazio, Ami P. Raval, Miguel A. Perez-Pinzon
Department of Neurology, UM Miller School of Medicine, Miami, FL, USA
Preconditioning activates endogenous neuroprotective mechanisms against ischemia. Mild ischemic insults (ischemic preconditioning) confer protection against subsequent insults that would otherwise be lethal. Understanding the mechanisms of these endogenous protective mechanisms could lead to novel therapeutic strategies. Previous studies showed ischemic preconditioning increased the release of GABA during ischemia (Dave et al. 2005). We tested the hypothesis that preconditioning acts via GABA synapses. To define the changes in GABA synapses, whole cell patch clamp recordings of GABA-A receptor mediated postsynaptic currents were obtained 48 hours after preconditioning in the presence of tetrototoxin and glutamate receptor antagonists (miniature postsynaptic currents). Ischemic preconditioning significantly decreased the interval between synaptic currents (29.4 +/− 4.0 msec vs. 65.8 +/− 8.4 msec; p<0.01, N=8), suggesting an increase in the number of active synapses. Ischemic preconditioning depends on activation of the epsilon PKC signaling pathway (Raval et al. 2003). Preconditioning can be mimicked by epsilon PKC activators (pharmacological preconditioning) and abolished by antagonists of epsilon PKC. In contrast to ischemic preconditioning, pharmacological preconditioning increased the amplitude of GABA synaptic currents (−92.8 +/− 14.1 pA vs. −58.0 +/− 5.1 pA; p<0.05, N=7) without affecting the frequency. Thus, both forms of preconditioning appear to converge on the GABA synapse. We confirmed this conjecture by testing the hypothesis that partial blockade of GABA-A receptors abolishes neuroprotection conferred by preconditioning. Co-application of the GABA-A receptor antagonist bicuculline (100 nM; IC50 3 uM) during lethal ischemia abolished both forms of preconditioning (N=5). We conclude that modifications of GABA synapses represent a major target of endogenous neuroprotective signaling pathways evoked by preconditioning.
Supported by NIH NINDS R01 NS34773, R01 NS54147, R01 NS45676, P01NS005820 and the Stanely J. Glaser Research Foundation.
REDUCTION OF SECONDARY NEURONAL DAMAGE BY THE IMMUNOSUPPRESSANT MYCOPHENOLATE MOFETIL AFTER CONTROLLED CORTICAL IMPACT INJURY IN RATS
F. Dehghani2, Beat Alessandri1, M. Nazari2, N.P. Hailer3, H.-W. Korf1, Oliver Kempski1
1Insitute for Neurosurgical Pathphysiology, Johannes Gutenberg-University of Mainz, Mainz, Germany, 2Institute of Anatomy II, Johann Wolfgang Goethe-University, Frankfurt a.M., Germany, 3Institute for Surgical Sciences, University Hospital Uppsala, Department of Orthopaedics, Uppsala, Sweden
Background/Aim: Lesions of the CNS induce specific morphological and functional changes of glial cells such as proliferation, cell shape transformation and secretion of proinflammatory cytokines and NO. It has been suggested that suppression of glial cell activation in the aftermath of the neuronal injury inhibits the development of secondary damage and contributes to improved neuronal survival. In previous studies with organotypic hippocampal slice cultures and glial single cell cultures we have shown that mycophenolate mofetil (MMF) reduced the number of degenerating neurons after excitotoxic injury, inhibited the proliferation of glial cells and suppressed the activation state of microglial cells. To prove that MMF exerts equally potent neuroprotective effects in vivo, we tested this compound in a model of traumatic brain injury in rats.
Method: To inflict a focal injury on brain tissue, we used a model of controlled cortical impact (CCI). 8-12-week-old Sprague-Dawley rats (350g-450g) received a unilateral CCI injury (5 m/sec velocity, 2.5mm depth, 200 ms dwell time) and vital parameters were monitored for up to 2h after the injury. Injured animals were treated with vehicle or MMF up to 14 days. Animals were sacrificed 4h (n=4/group), 1 (n=4/group), 3 (n=4/group) or 14 days (n=8/group) after the trauma. The lesion volume was calculated by quantitative morphometry on serial sections stained with hematoxylin/eosin. Sections were also stained with GFAP and NeuN for glial and neuronal cell types, respectively, with IB4 (isolektin B4) for microglia and with ki-67 for proliferating cells.
Results: In vehicle treated animals CCI caused a moderate cortical contusion, swelling (1 day) and massive neuronal cell loss underneath the impact site. The lesion volume showed an average of 31.41 mm3 and the lesion edges were poorly defined. Immunohistochemistry showed microgliosis, astrogliosis, massive invasion of blood borne monocytes and a high number of proliferating glial cells. In contrast, MMF treatment led to a significantly reduced lesion volume of 19.41 mm3. The lesion was sharply demarcated against neighboring NeuN immunoreactive neurons. Immunohistochemically a strong suppression of glial activation and proliferation was visible and the number of proliferating glial cells was attenuated.
Conclusion: Our findings show that the immunosuppressant MMF protects in vivo neurons from secondary neuronal damage, inhibits the proliferation of astrocytes and reduces the activation state of glial cells resulting in a smaller lesion volume. MMF may be used as a neuroprotective agent in treatment of acute traumatic CNS pathologies. This study was supported by Dr. August Scheidel-Stiftung.
ERK1/2-P90RSK-MEDIATED PHOSPHORYLATION OF NA+/H+ EXCHANGER ISOFORM 1 IN ISCHEMIC NEURONAL DAMAGE
Jing Luo1,2, Douglas B. Kintner2, Gary E. Shull3, Dandan Sun1,2
1Department of Physiology, University of Wisconsin Medical School, Madison, WI, USA, 2Department of Neurosurgery, University of Wisconsin Medical School, Madison, WI, USA, 3Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, OH, USA
Background and Aims: We recently reported that Na+/H+ exchanger isoform 1 (NHE1) activity plays a role in ischemic cell damage and inhibition of NHE1 activity is neuroprotective in both in vitro and in vivo ischemic models (J. Neurosci., 2005). However, the cellular mechanisms underlying activation of NHE1 activity remain unknown. In this study, we investigated ERK1/2-mediated stimulation of NHE1 in neurons following in vitro ischemia.
Methods and Results: Two hours of oxygen and glucose deprivation (OGD) did not change expression of phosphorylated ERK1/2 (p-ERK1/2). In contrast, 10-60 min reoxygenation (REOX) triggered a significant increase in p-ERK1/2 levels. The increase in p-ERK1/2 was accompanied by translocation of p-ERK1/2 from cytosol to nuclei following OGD/REOX. The MEK inhibitor U0126 (10 µM) abolished elevation of p-ERK1/2 expression and its nuclear translocation. Moreover, NHE1 activity was significantly increased at 10-60 min REOX after 2 h OGD. OGD/REOX not only increased Vmax of NHE1 for H+ but also shifted Km toward a higher pHi. OGD/REOX-mediated changes in NHE1 kinetics were absent when MEK was inhibited by U0126. We speculated that changes in NHE1 kinetics result from phosphorylation of NHE1 (p-NHE1) via ERK-p90RSK signaling pathways. After 10 min REOX following 2 h OGD, not only was p-NHE1 increased, but there was a concurrent increase in phosphorylation of p90RSK (p-p90RSK), a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished elevation of both p-NHE1 and p-p90RSK. Either genetic ablation or pharmacological inhibition of NHE1 activity reduced neuronal death following OGD/REOX. U0126 provided a similar level of neuroprotection.
Conclusions: Taken together, these results suggest that NHE1 is phosphorylated via activation of ERK1/2- p90RSK pathways and contributes to ischemic neuronal damage.
Key words: oxygen and glucose deprivation, HOE 642, p90RSK, protein phosphorylation, U-0126
GREATER OXIDATIVE STRESS SELECTIVELY IN CA1 ASTROCYTES IS CENTRAL TO THE SELECTIVE VULNERABILITY OF CA1 NEURONS AFTER TRANSIENT FOREBRAIN ISCHEMIA
Yi-Bing Ouyang, Ludmila A. Voloboueva, Li-Jun Xu, Rona G. Giffard
Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA
Background: Transient global ischemia, as with cardiac arrest, causes loss of CA1 hippocampal neurons 2–4 days later, while nearby dentate gyrus (DG) neurons are relatively resistant. For many years selective neuronal vulnerability has been presumed to reflect the inherent vulnerability and connectivity of the neuronal population. Whether differential astrocyte vulnerability contributes to CA1 neuronal death has been much less studied, and the role of astrocytes in the different hippocampal regions in ischemic injury is uncertain. We investigated the role of astrocytes in selective neuronal loss using in vivo and in vitro preparations. Methods: Rat 2-vessel occlusion plus hypotension to 45 mm Hg for 10 minutes forebrain ischemia was used. Following different reperfusion intervals from 2 hr to 72 hr, brains were harvested for immunohistochemistry, or for western analysis. Primary cultures of astrocytes isolated from either CA1 or the DG region, and hippocampal slice cultures were also used. Protein expression was determined by western blot or immunohistochemistry, and live cell imaging was performed to assess ROS with hydroethidine or mitochondrial membrane potential with TMRE. Results: CA1 astrocytes were found to be more sensitive to ischemia than DG astrocytes. In rats subjected to transient forebrain ischemia, CA1 astrocytes lose immunoreactivity for GFAP, S100a and glutamate transporter GLT-1, as well as glutamate transport activity, within a few hours of reperfusion, but without astrocyte cell death. This occurs well before any evidence of neuronal injury. Using subregion specific astrocytes cultures we observed that CA1 astrocytes show early increases in mitochondrial free radicals and reduced mitochondrial membrane potential after stress, while similar changes were not observed in DG astrocytes. Thus greater oxidative stress may contribute to the selective CA1 astrocyte changes, including early loss of GLT-1, which is sensitive to oxidation. The respiratory chain inhibitor rotenone was used to increase mitochondrial production of ROS in culture and this resulted in rapid decrease of GLT-1 immunostaining in CA1 astrocytes. To determine whether the early loss of GLT-1 contributes to CA1 neuronal loss we increased GLT-1 expression in astrocytes with ceftriaxone. This significantly reduced the extent of CA1 neuronal loss from forebrain ischemia in vivo and in slice cultures. Discussion: Oxidative stress is a well recognized contributor to postischemic injury. In the case of forebrain ischemia, while some ROS may originate from neurons, it also appears that some ROS may originate from astrocytes. An important consequence of the toxicity of the ROS produced from either source is regionally selective astrocyte impairment. A clear difference was seen both in vivo and in vitro between the vulnerability of CA1 and DG astrocytes. We suggest that greater oxidative stress and loss of GLT-1 function selectively in CA1 astrocytes is central to the well known selective vulnerability of CA1 neurons.
IN VIVO DETECTION OF SUPEROXIDE-ANION RADICAL BY A NEWLY DEVELOPED ELECTROCHEMICAL BIOSENSOR IN BRAIN ISCHEMIA-REPERFUSION MODEL OF RATS
Motoki Fujita1, Susumu Yamashita1, Kenji Fujimoto1, Kazumi Kumagai1, Kotaro Kaneda1, Yasutaka Oda1, Ryosuke Tsuruta1, Shunji Kasaoka2, Makoto Yuasa3, Tsuyoshi Maekawa1,2
1Advanced Medical Emergency and Critical Care Center, Yamaguchi University Hospital, Ube, Yamaguchi, Japan, 2Department of Emergency and Critical Care Medicine, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan, 3Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, Tokyo, Japan
Introduction: The reactive oxygen species (ROS) play an essential role in vivo, but the excessive production of it leads to an oxidative stress and a tissue injury. In ROS, a superoxide anion radical (O2-?) is the key radical because the generation of almost ROS derives from O2-?. Recently, we exploited an all-synthetic biochemical sensor that can detect O2-? specifically. This sensor is coated with Bromoiron (III) meso-tetra(3-thienyl)porphyrin ([FeBr(ttp)]) that mimics cytochrome c and is stable in vivo. To evaluate the production and the attenuation of O2-? in vivo, a newly developed electrochemical biosensor was used in forebrain ischemia-reperfusion rats with or without superoxide dismutase (SOD).
Materials and Methods: Fourteen Male Wister rats (260-280g) were randomly assigned to two groups, i.e., the reperfusion group (n=7) and the SOD group (n=7). A microdialysis probe for glutamate was embedded in the forebrain parenchyma on the previous day of the experiment under pentobarbital anesthesia. The rats were anesthetized by 4% of isoflurane and 100% of O2. They were subjected to tracheostomy and ventilated mechanically. The top of electrochemical O2-? sensor was inserted into the left internal jugular vein through the thyroid vein and the other venous branches were ligated. After the operation, the anesthesia was changed to 0.9% of isoflurane and 40% of O2 and the O2-? value was measured with Active O2-?TM (BORON JAPAN CO.LTD., Noda, Japan) continuously. After the stabilization of the blood gas and O2-? value, the forebrain ischemia-reperfusion was made by bilateral common carotid artery occlusion with hemorrhagic hypotension for 20 min and by releasing the occlusion with returning the shed blood. In the SOD group, SOD (5 mg/kg) was administrated intravenously at 20 minutes after reperfusion. The measurements were continued for 1 hour after reperfusion.
Results: The detection current of O2-? increased after the reperfusion in both groups. In the SOD group, the elevation of O2-? current attenuated after the administration of SOD (Figure 1). The electric charge of O2-? (Q value) was integrated in every 20 minutes after reperfusion. In the SOD group, Q value was decreased significantly during 40-60 minutes after the reperfusion (23.7±8.8 v.s. 13.1±5.9 micro C, p<0.05)
Conclusions: The excess production of superoxide anion radical was detected by a newly developed electrochemical biosensor in vivo and rapid reduction of superoxide by intravenous administration of SOD was confirmed. This is the first report of the real-time measurement of superoxide in vivo.
NEUROPILIN-1 IS A DIRECT TARGET OF THE TRANSCRIPTION FACTOR E2F1 IN ISCHEMIC NEURONAL DEATH
Susan Jiang1, Melissa Sheldrick1,2, Angele Desbois1, Jackie Slinn1, Sheng T. Hou1,2
1NRC Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada, 2Department of Biochemistry, University of Ottawa, Ottawa, ON, Canada
Background and aims
Molecular mechanisms of neuronal death caused by cerebral ischemia are still subjected to intense investigations. Previous studies, including those of our own, have shown that the transcription factor E2F1 plays an important role in modulating ischemia-induced neuronal death. Ample studies have also shown that cerebral ischemia in animal models and human patients are associated with growth of new blood vessels and with neuronal remodeling. On the molecular level, these processes may be regulated by common mediators, such as the neuropilin family members (NRPs). In the present study, we identified E2F1 target genes using custom DNA microarrays and determined that NRP-1 is a bona fide transcriptional target of the transcription factor E2F1 during neuronal death caused by cerebral ischemia.
Methods
All procedures for cerebral ischemia produced by middle cerebral artery occlusion (MCAO) using C57/black mice were approved by the local Animal Care Committee. Under temporary isofluorane anesthesia, mice were subjected to MCAO using an intraluminal filament as previously described. After 1 h of MCAO, the filament was withdrawn, blood flow restored to normal by laser Doppler flowmetry and wounds sutured. Animals were sacrificed after 24 h of reperfusion. Brain infarction was measured and brain tissues were subjected to RNA and protein extraction for chromatin immunoprecipitation, DNA microarray, EMSA, PCR, Western blotting, and immunostaining analysis.
Results
Ccomparison of gene expression between brain cortices from E2F1+/+ and -/− mice using a custom high-density DNA microarray, we identified a group of E2F1 putative target genes which might be responsible for ischemia-induced E2F1-dependent neuronal death. Neuropilin-1 (NRP-1), a receptor for semaphorin 3A-mediated axon growth cone collapse and retraction, was confirmed to be a direct target of E2F1 based on the fact that (1) NRP-1 promoter sequence contains an E2F1 binding site, (2) re-activation of NRP-1 expression in E2F1-/− neurons when E2F1 gene was replaced, (3) activation of NRP-1 promoter by E2F1 in a luciferase reporter assay, (4) electrophoretic mobility gel shift analysis (EMSA) confirmed the presence of an E2F-binidng sequence in NRP-1 promoter, and (5) chromatin immunoprecipitation assay (ChIP) showed that E2F1 binds directly to the endogenous NRP-1 promoter. Interestingly, the temporal induction in cerebral ischemia-induced E2F1 binding to the NRP-1 promoter correlated with the temporal induction profile of NRP-1 mRNA, confirming that E2F1 positively regulates NRP-1 during cerebral ischemia. Functional analysis also showed that NRP-1 receptor expression was extremely low in E2F1-/− neurons which led to the diminished response to semaphorin 3A-induced axonal shortening and neuronal death. A NRP-1 selective peptide inhibitor provided neuroprotection against oxygen-glucose-deprivation.
Conclusions
Taken together, these findings support a model in that E2F1 targets NRP-1 to modulate axonal damage and neuronal death in response to cerebral ischemia.
This study was supported by a grant from the Heart and Stroke Foundation of Ontario to STH. MS holds a HSFO MSc scholarship
ROSIGLITAZONE PROTECTS BRAIN FROM ISCHEMIC INSULT BY ACTIVATING PEROXISOME PROFERATOR-ACTIVATED RECEPTOR GAMMA (PPAR-R) AND ATTENUATING APOPTOTIC CELL DEATH
Jui-Sheng Wu, Yi-Tong Chen, Wai-Mui Cheung, Teng-Nan Lin
Neuroscience Division, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan-R.O.C.
Background and Aims: Peroxisome proliferator-activated receptor gamma (PPAR-r) is a nuclear membrane-associated transcription factor that governs various genes, including lipid biosynthesis, glucose metabolism and inflammatory. It has been shown that activation of PPAR-r attenuates myocardial ischemia/reperfusion damage. Rosiglitazone (Rosi), a TZD and synthetic agonist of PPAR-r, is a clinical drug for type 2 diabetes. Several lines of evidence suggest that TZDs may protect brain against ischemia/reperfusion injury; however, the underlying mechanisms are not clear. Previously, we have shown that intra-ventricular infusion of 15-deoxy-Delta (12, 14)-PGJ2 (15d-PGJ2; a stable PGD2 metabolite and physiological agonist of PPAR-r) reduced cerebral infarction, and this protective effect were abrogated by inhibitor of PPAR-r. The aim of this study is to investigate the cellular and molecular mechanisms underlying the protective effect of Rosi-PPAR-r against ischemic cerebral infarction.
Methods: Rats were subjected to 30 minutes of transient middle cerebral artery occlusion and intra-cerebral ventricular infusion of Rosi. For in vitro study, Rosi was added to Murine N2-A neuroblastoma cells either alone or in various combinations before subject to OGD. Western blot analysis was used to study the temporal profiles of proteins. Apoptotic cell death was analyzed by flow cytometry. PPAR-r activity was analyzed by luciferase reported assay. Plasmid and siRNA transfection were used to over-express or silence PPAR-r.
Results: Our results indicated that Rosi significantly reduced infarct volume, even when Rosi was administered 2 hours after a 30-min transient cerebral ischemia, via attenuating ischemia-induced caspase-3 activation and PARP-1 degradation. Moreover, Rosi also protects N2-A against oxygen-glucose deprivation (OGD)-induced cell death by reducing OGD-induced ROS production, depolarization of mitochondrial membrane potential (MMP), caspase-3 activation, PARP-1 degradation and apoptosis. The protective effects of Rosi were abolished in the present of PPAR-r antagonist, were mimicked by PPAR-r over-expression and were abolished in the presence of siRNA against PPAR-r.
Conclusions: In summary, our results suggest that Rosi, by activating PPAR-r, protects neuronal cells against ischemic-induced apoptosis.
HYPERLIPIDEMIA EXACERBATES ISCHEMIC BRAIN INJURY VIA PROMOTING CD36-MEDIATED FOAM CELL FORMATION
Sunghee Cho1,2, Aaron Tolhurst1, Maria Febbraio3, Eunhee Kim1
1Burke Medical Research Insitute, White Plains, NY, USA, 2Department of Neuroscience, Weill Medical College of Cornell Univesity, New York, NY, USA, 3Department of Cell BIology, Cleveland Clinic, Cleveland, OH, USA
Oxidative stress is implicated in the pathogenesis of ischemia-reperfusion injury. Oxidative modification of low-density lipoprotein (oxLDL) is a key step for the generation of pro-atherogenic ligands. Subsequent uptake of oxLDL by scavenger receptors leads to macrophage foam cell formation, atheroma, and a chronic pro-inflammatory state. Among multiple scavenger receptors, CD36 is one of the major scavenger receptors responsible for the uptake of oxLDL and foam cell formation. A recent study demonstrated that CD36 mediates free radical production and ischemic injury (Cho et al., 2005). It has been suggested that ischemic stroke shares common risk factors with coronary heart diseases. Among the risk factors, hypercholesterolemia is common in humans and a known risk factor for stroke. Based on the fact that CD36 i) is involved in ischemic injury and ii) functions in the uptake of oxLDL, we investigated whether the hyperlipidemic condition exacerbates ischemia-induced injury and whether the exacerbation occurs via up-regulating CD36 pathways.
To produce hyperlipidemic (HL) mice, 5 week-old ApoE null mice, which are spontaneously hyperlipidemic, were fed a high fat diet (TD88137, Halen Taklad) for 8 weeks. Age matched wild-type mice fed standard chows were used as normal lipid (NL) mice. Weight and cholesterol levels were determined at the end of diet and then mice were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min using an intraluminal filament. Infarct volume and swelling were determined 3 days after ischemia in cresyl violet stained sections with correction for brain swelling. CD36 gene and protein expression were determined either in the post-ischemic brain tissue or mouse peritoneal macrophages by real-time RT-PCR and Western blot analysis. In addition, foam cell formation, a CD36-mediated function, was assessed in the post-ischemic brain sections.
Compared to the NL mice, significant increases in body weight and plasma cholesterol levels in HL mice were observed (weight, 35.8±0.6 vs 25.5±0.08 gr: Cholesterol: 725.80±54.82 vs 119.22±4.96 mg/dl, p<0.05, n=15). HL mice also exhibited larger infarct size and increased % hemispheric swelling (infarct, 64.9±2.9 vs 42.5±4.3 mm3 p<0.05: Swelling, 29.8±4.4 vs 8.5±1.3%, p<0.05 n=7). Because CD36 expression mainly occurs in microglia/macrophages within the infarct territory, CD36 expression was determined in peritoneal macrophages harvested from NL and HL mice. Compared to NL mice, CD36 expression was increased in HL mice prior to and 3 days after ischemia (Pre (n=4): 1.56±0.19 vs 1.00±0.13: Post (n=5): 2.74±0.41 vs 1.00±0.27, p<0.05). Importantly, in contrast to NL mice, numerous lipid-laden foam cells were found in the penumbra in HL mice (Figure), indicating that hyperlipidemic condition promotes foam cell formation in the post-ischemic brain.
The data suggest that hyperlipidemia exacerbates ischemia injury via upregulation of CD36 expression/function in post-ischemic brain. Targeting CD36 receptors may be a useful strategy to reduce ischemic injury in the hyperlipidemic condition. Supported by NIH grants HL82511.
IN VITRO PRECONDITIONING OF EMBRYONIC STEM CELLS PROMOTES CELL SURVIVAL AND FUNCTIONAL RECOVERY AFTER TRANSPLANTATION INTO THE ISCHEMIC RAT BRAIN
Ling Wei1, Michelle Theus1, Shan Ping Yu1,2
1Department of Pathology, Medical Unviversity of South Carolina, Charleston, SC, USA, 2Department of Pharmaceutical Sciences, Medical Unviversity of South Carolina, Charleston, SC, USA
Hypoxic preconditioning (HP) and stem cell transplantation have been studied as two individual therapies. We now combine these methods to achieve increased therapeutic effects after ischemic stroke. Cultured embryonic stem (ES) cells during neural induction with retinoic acid (RA) forms embryoid bodies (EBs). In HP group, EBs were exposed to low oxygen (1% O2, 8 h) during the final stage of RA induction. The sublethal in vitro hypoxia pre-treatment significantly enhanced the tolerance of ES cell derived neural progenitor cells (ES-NPCs) and bone marrow mesenchymal stem cells (BMSC) to serum deprivation-induced cell death, decreased caspase-3 activation, increased secretion of erythropoietin (EPO) and upregulated expression of bcl-2, glial-derived neurotrophic factor (GDNF), HIF-1a, erythropoietin receptor (EPOR), neurofilament (NF), and synaptophysin in ES-NPCs. One week after RA, expression of neurofilaments and synaptophysin were noticeably higher in HP-treated ES-NPCs compared to control cells. The HP-initiated protection lasted for at least 6 days; it was diminished by blocking EPOR. Instead of hypoxia, rhEPO pre-treatment also significantly reduced caspase-3 activation and cell death. In vivo experiments confirmed that transplanted HP-primed ES-NPCs survived better in the ischemic brain. Fourteen days later, transplanted cells were labeled by NeuN or GFAP. The HP treated, ES-NPCs-derived neuronal cells showed extended neurite processes and formed neuronal networks 2 weeks after transplantation. HP-primed ES-NPCs conferred accelerated recovery in a number of sensorimotor activities. Transplantation of ES-NPCs improved the rotarod test score 7 to 35 days after transplantation. The enhanced motor functional benefit lasted for at least 35 days. We propose that the HP/EPO-priming strategy presents a unique and beneficial method for improving clinical potential of cell transplantation therapy.
INTRAVITAL IMAGING REVEALES THAT TRANSIENT FOREBRAIN ISCHEMIA INDUCES LONG-LASTING INCREASE IN INTRACELLULAR CALCIUM ION CONCENTRATION IN RAT HIPPOCAMPAL CA1
Seiji Yamamoto, Yong Wang, Atsuo Miyakawa, Takashi Sakurai, Kyoko Ibaraki, Susumu Terakawa
Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan
INTRODUCTION: In cultured neurons, the increase in intracellular Ca2+ concentration ([Ca2+]i) plays a crucial role in excitotoxicity. However, in whole animals, calcium response following ischemia remains to be established. Especially, change in [Ca2+]i in the hippocampus upon ischemia remains unclear, since no method has been available to measure the [Ca2+]i in the deep structure of brain. In the present study, we sought to determine, in rats, the [Ca2+]i changes following transient forebrain ischemia in hippocampus.
METHODS: In anesthetized, spontaneously ventilated SD rats (300 g), bilateral vertebral arteries were coagulated, and transient forebrain ischemia was induced by occluding bilateral common carotid arteries with balloon occluders. To obtain the intravital fluorescence image, we employed an imaging fiber bundle (1.0 mm outer diameter) including 20,000 fibers (4.5 µm outer diameter) coupled to the microlens-attached Nipkow-disk scanner (CSU-21, Yokogawa, Japan) equipped with 10x objective lens. This fiber-coupled confocal microscope (FCM) is capable to observe the confocal image just under the fibers (1). FCM was inserted stereotaxically into CA1 and CA3 regions, respectively. Fluo-3/AM (440 µM, 2.5 µl), a [Ca2+]i sensitive fluorescent dye, was microinjected into the brain, and observed every 30 s before and after 10-min forebrain ischemia with FCM. Since the hemoglobin absorbs both excitation and emission light, ischemia may affect the fluorescent intensity of Fluo-3 inside the brain. To analyze the effects of ischemia on the fluorescence intensity, the fluorescent beads (0.2 µm diameter, similar excitation and emission to those of Fluo-3) were microinjected into hippocampus and the images were taken before and after 10-min ischemia in the same manner as did in [Ca2+]i imaging.
RESULTS: Four-vessel occlusion decreased CBF (mean±SD%) measured by laser-Doppler flowmetry, to 25±18 (n=6) in CA1 and to 20±11 (n=6) in CA3. They did not significantly differ. 10-min forebrain ischemia increased fluorescence intensity (mean±SD%) of the beads in the hippocampus up to 108±5 (n=4), which declined to the baseline level following reperfusion. In all measurements, the ischemia showed significant increase (102±2, n=12 to 106±4, n=84, p<0.01). In [Ca2+]i imaging, therefore, the change in fluorescence during ischemia was corrected. During 10-min ischemia, the fluorescence intensity of Fluo-3 slightly increased up to 110±9, and then increased to 134±19 at the end of 20-min reperfusion in CA1 (n=4). In contrast, in CA3 (n=5), the intensity did not change upon ischemia, and at reperfusion slightly elevated to 108±4 without long-lasting increase. During 14-20 min reperfusion, the [Ca2+]i increase in CA1 was statistically larger (p < 0.05) than that in CA3.
COMMENTS: In CA1, but not in CA3, transient forebrain ischemia induced long lasting Ca2+ increase in situ, whereas it produced similar CBF changes in these regions. Since the [Ca2+]i increase was significantly larger in CA1 during reperfusion period, it might reach to toxic levels and then mediate the vulnerability of CA1 against ischemia.
Supported by the grants #16390407(SY), #16047212(SY), #18053010(SY)
PROTEOMIC ANALYSIS OF APOPTOTIC CELL DEATH
Duncan Short, Ian Heron, Jui-Lee Birse-Archbold, Lorraine Kerr, John Sharkey, James McCulloch
ACE and Centre for Neuroscience Research, University of Edinburgh, Edinburgh, Scotland, UK
Apoptosis contributes to cell death after cerebral ischaemia. A quantitative proteomics approach has been employed to define alterations in protein levels in apoptosis induced with staurosporine. Human neuroblastoma derived SH-SY5Y cells were treated with staurosporine (500nM for 6 hours) to induce apoptosis. Cell viability, morphology and cytochrome C release was defined in this model system prior to proteomic analysis. Quantitative two dimensional gel electrophoresis was used to determine the altered protein levels with MALDI-TOF mass spectrometry identification of proteins. For each treatment condition(vehicle or staurosporine),8-9 gels were prepared independently and means+/−SD computed. Of the 154 proteins analysed, thirteen proteins were significantly altered as a result of the apoptotic stimulus. Ten of the proteins showed a statistically significant increase in level with staurosporine and these were identified as heat shock cognate 71 (Hsc71)(increased by 42%), two isoforms of heat shock protein 70 (Hsp70)(increased by 77&56%), glucose regulated protein 78 (GRP78)(increased by 81%), F-actin capping protein(increased by 151%), stress-induced phosphoprotein 1(increased by 64%), chromatin assembly factor 1 (CAF 1)(increased by 155%), protein disulphide isomerase A3 precursor (PDI A3)(increased by 69%), transitional endoplasmic reticulum ATPase (increased by 82%)and actin interacting protein 1 (AIP 1)(increased by 64%). Three proteins which displayed significant decrease in levels with staurosporine were identified as tubulin(decreased by 45%), vimentin(decreased by 62%) and glucose regulated protein 94 (GRP94)(decreased by 27%). The functional roles ascribed to the proteins which were significantly altered during staurosporine induced apoptosis were cytoskeletal (2 proteins), cytoskeletal assembly (2 proteins), protein folding (7 proteins), mitosis (1 protein) and chromatin assembly (1 protein) and by subcellular location into cytoplasmic (6 proteins), cytoplasmic and nuclear (2 proteins), endoplasmic reticular (4 proteins) and nuclear (1 protein). The functional roles and subcellular locations of these proteins collectively indicate that staurosporine induced apoptosis provokes induces an unfolded protein response involving molecular chaperones, co-chaperones and structural proteins indicative of Endoplasmic Reticulum (ER) stress.
VASOPROTECTIVE EFFECTS OF INOS-DERIVED NO AND NOX2-DERIVED SUPEROXIDE IN ISCHEMIC PRECONDITIONING
Alexander Kunz, Laibaik Park, Takato Abe, Josef Anrather, Ping Zhou, Costantino Iadecola
Division of Neurobiology, Weill Medical College of Cornell University, New York, NY, USA
Background: Ischemic preconditioning (PC) is a phenomenon in which a sublethal stimulus confers tolerance toward a subsequent lethal ischemic insult. For example, systemic administration of lipopolysaccharide (LPS) reduces the injury produced by focal cerebral ischemia (JCBF&M 25:493,2005). LPS-induced PC requires iNOS for its expression, but the mechanisms by which iNOS-derived NO mediates the tolerance remain unclear. In systemic vessels, LPS improves vascular reactivity (EJP 377:209,1999), raising the possibility that LPS acts by improving CBF. NO exerts many of its actions by reacting with the radical superoxide to form peroxynitrite (PN). Therefore, we investigated whether vasoprotective effects of NO play a role in LPS-induced PC and, if so, whether reactive oxygen species (ROS) and PN are involved.
Methods: Male C57Bl/6 mice (n=5-7/group) were treated with LPS (0.5 mg/kg; ip) and subjected to transient middle cerebral artery occlusion (MCAO) 24hrs later. Ischemic CBF was monitored by laser-Doppler flowmetry in the neocortex spared from infarction by PC. CBF reactivity to whisker stimulation or neocortical application of acetylcholine (ACh; 10µM), was tested in the post-ischemic neocortex 1h after reperfusion. Infarct volume was measured 72hrs after MCAO in thionine-stained sections.
Results: LPS improved CBF (Fig. A) and reduced infarct volume (Fig. B). Following MCAO, the increase in CBF induced by whisker stimulation (Fig. C) and Ach were attenuated (Ach: −56±8%, p<0.05) However, LPS prevented these cerebrovascular abnormalities (Fig. C). The beneficial effects of LPS on CBF, infarct volume and CBF reactivity were not observed in wild type (WT) mice treated with the iNOS inhibitor aminoguanidine (Fig. A-C) or in iNOS-null mice (e.g. infarct volume: Veh-iNOS-/−: 25±5; LPS-iNOS-/−: 28±2). LPS increased ROS production (53±24%; p<0.05), assessed with hydroethidine and upregulated the immunoreactivity for the PN marker 3-nitrotyrosine (69±14%; p<0.05). These effects were not observed in mice lacking the nox2 subunit of the ROS-producing enzyme NADPH oxidase (p>0.05). Furthermore, 3-nitrotyrosine immunoreactivity was abolished by treatment of WT mice with the PN decomposition catalyst FeTPPS (20mg/kg; i.v.). The beneficial LPS effects on CBF and infarct volume were not observed in nox2-null mice (p>0.05) and were attenuated in WT mice treated with FeTPPS (Fig. A-C).
Conclusions: LPS PC is associated with marked neuroprotection and preservation of penumbral CBF. The CBF improvement in regions at risk for infarction can be attributed to a beneficial effect of LPS on cerebrovascular reactivity, leading to improved intraischemic CBF. The vasoprotective effect of LPS is mediated by PN formed by iNOS-derived NO and NADPH oxidase-derived superoxide. Thus, PN, in addition to its deleterious roles, can also be protective by improving cerebrovascular function and ischemic CBF.
THE ROLE OF ACTIVATED MICROGLIA/MACROPHAGES IN NEONATAL FOCAL STROKE
Zinaida Vexler1, Andra Dingman1, Sarah Lee1, Joel Faustino1, Nikita Derugin2, Takashi Wada4, Michael Wendland3
1Department of Neurology, UCSF, San Francisco, CA, USA, 2Department of Neurosurgery, UCSF, San Francisco, CA, USA, 3Department of Radiology, UCSF, San Francisco, CA, USA, 4Kanazawa University, Kanazawa, Japan
Background and aims: Microglia - resident macrophages - are the main cell type that provide immuno-surveillance in the brain. In vivo data from cerebral ischemia and trauma models have suggested a general rule that more severe brain injury is associated with higher macrophage densities and that strategies that limit microglial activation in general, or that specifically inhibit synthesis of toxic molecules produced in these cells can be neuroprotective. These cells undergo a graded process of activation in response to injury, including morphological transformation, increased migratory activity, proliferatio, secretion of cytokines, and antigen presentation. Using a focal transient middle cerebral artery (MCA) occlusion model in postnatal day 7 (P7) rats, we have previously shown a major transient accumulation of inflammatory cytokines, rapid microglial activation, and minimal invasion of peripheral monocytes/macrophages in acutely injured regions (Fox et al JCBFM; 2005, Dingman et al JNC, 2006; Denker et al JNC 2007). The anti-inflammatory drug minocycline administered acutely, while protective, did not diminish the number of activated microglia/macrophages (ED1+ cells), or increased levels of IL-1b, MCP-1 and CINC-1 in ischemic-reperfused brain (Fox et al, JCBFM; 2005). We asked whether neuroinflammation after neonatal stroke can be attenuated and protection achieved by limiting migration of cells of monocyte lineage to injured region.
Methods: P7 rats were subjected to a 1.5 hr MCA occlusion, treated with either a neutralizing anti-MCP-1 antibody or propagermanium, a compound that attenuates monocyte migration by targeting of glycosylphosphatidylinositol (GPI)-anchored proteins closely associated with CCR2. A neutralizing anti-MCP-1 or vehicle was administered i.p. 30 min prior to occlusion. IL1a, IL1b, TNF-a, IL-6, IL-18, CINC-1 and MCP-1 were measured in the same sample (Multi-plex cytokine assay) in the plasma, injured tissue and matching tissue from contralateral hemisphere at 4 and 8 hours; histological outcome was determined at 24 hr post reperfusion. 8mg/kg propagermanium or the corresponding vehicle (1% gelatin) were administered orally 30 min prior to occlusion and daily for 3 days post-ischemia. Animals were sacrificed 7 days later for histology and quantification of macrophage densities.
Results: MCP-1 neutralization reduced ischemia-induced increases in IL1b, IL-6, and CINC-1 in the systemic circulation, but did not diminish the elevated cytokine production locally in the injured brain tissue. Administration of an anti-MCP-1 antibody did not affect the injury volume at 24 hr post-reperfusion (5511% Vs. 5611%, injured/contralateral hemisphere in anti-MCP-1 treated Vs. vehicle-treated group, respectively, n=5-6/group). Treatment with propagermanium substantially reduced accumulation of IB4+ cells in injured regions. The volume of remaining tissue was 7612% in treated Vs. 6915% in vehicle-treated group (n=4-6/group, p>0.05).
Conclusions: Our findings suggest the existence of a relatively independent pool of cytokine production in the injured brain that is not modulated by the systemically produced MCP-1, thus limiting the ability of peripheral MCP-1 to affect acute ischemic injury in neonatal brain. Our data also suggest that blocking the migration of peripheral macrophages may not be sufficient to protect the neonatal brain from stroke and that activated microglial cells play a profound role in neonatal stroke.
NIH NS44025, UCP R766-04, AHA GIA 0655236Y
INJURY TO CORPUS AND CINGULATE CALLOSUM DEVELOPMENT WITH HYPOXIA: ABEHAVIORAL DTI STUDY ON C57B/L6 MICE
Halima Chahboune1,2,7,8, Laura R. Ment2,3, William B. Stewart4, Douglas L. Rothman1,6,7,8, Michael L. Schwartz5, Fahmeed Hyder1,6,7,8
1Department of Diagnostic Radiology, Yale University, New Haven, CT, USA, 2Department of Pediatrics, Yale University, New Haven, CT, USA, 3Department of Neurology, Yale University, New Haven, CT, USA, 4Department of Neurosurgery, Yale University, New Haven, CT, USA, 5Department of Neurobilogy, Yale University, New Haven, CT, USA, 6Department of Biomedical Engineering, Yale University, New Haven, CT, USA, 7Core Center for Quantitative Neuroscience With Magnetic Resonance, Yale University, New Haven, CT, USA, 8Magnetic Resonance Research Center, Yale University, New Haven, CT, USA
INTRODUCTION
Diffusion tensor imaging (DTI) of brain has gained wide acceptance as a tool for non-invasive microimaging of anatomical connectivity and morphology. The purpose here was to use DTI to assess potential alterations in morphology of developing axon pathways in a clinically-relevant neonatal rodent model of chronic sublethal hypoxia (CSH) injury to developing brain. Here we show local modifications in brain development due to hypoxia, in agreement with histology and behavior.
MATERIALS and METHODS
C57B/L6 litters (P15 to P51), fostered by CD-1 dams, were reared under hypoxic 9.5±1.0%) or normoxic conditions from postnatal day 3. Similarly reared groups of mice reared under either hypoxic or normoxic conditions were behaviorally assessed. Spontaneous activity and motor coordination and learning were assessed. The mice were anesthetized with urethane and MRI experiments were performed on a 9.4T Bruker horizontal-bore system with custom-made surface coils. Diffusion weighting (b ? 1000 s/mm2) in 16 different directions were acquired with one reference image (b ? 0 s/mm2). Maps of mean diffusivity (ADC), fractional anisotropy (FA) were calculated, and from which the primary eigenvectors were used to calculate diffusion (DEC). The data were used to get trajectory maps in medial-lateral, dorsal-ventral, and anterior-posterior.
RESULTS and DISCUSSION
Behavioral studies revealed significant levels of spontaneous activity for CSH mice than normal mice for total distance traveled and the velocity of movements during each 15 minute observation session. In contrast no differences were observed between the two groups on the Rotorod performance. These behavioral results suggest that CSH mice show no gross impairment in either the movement quality or simple motor learning as assessed by session improvement on the Rotorod task. Whole brain in vivo DTI studies through development showed morphological changes in different brain regions. There were no change in anisotropy in the forelimb and whisker barrel areas for either group (data not shown), which may partly support the behavioral results of no significant differences observed between the two groups on the Rotorod performance. The medial-lateral anisotropy in the corpus callosum and the anterior-posterior anisotropy were significantly altered by the hypoxic challenge early in development: (Fig. 1). In conclusion, our results here demonstrate that in vivo DTI at high magnetic field can detect normal developmental changes and differentiate changes in development as a consequence of hypoxia, which is a moderate perturbation in comparison to genetic manipulations.
ACKNOWLEDGEMENTS
Support by R01 DC-003710, R01 MH-067528, P01 NS-35476, and a gift from Pfizer
ROLE OF CYCLOPHILIN D IN HYPOXIA-ISCHEMIA INDUCED BRAIN INJURY DURING DEVELOPMENT
Xiaoyang Wang1, Michael A. Forte2, Paolo Bernardi3, Catherine Rousset1, Changlian Zhu4, Klas Blomgren4, Ylva Carlsson1, Carina Mallard1, Henrik Hagberg1,5
1Perinatal Center, Department of Physiology, Goteborg University, Goteborg, Sweden, 2Vollum Institute, Oregon Health and Sciences University, Portland, OR, USA, 3Department of Biomedical Sciences and Consiglio Nazionale Delle Ricerche Institute of Neuroscience, University of Padova, Padova, Italy, 4Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, Goteborg University, Goteborg, Sweden, 5Perinatal Center, Department of Obstetrics and Gynecology, Goteborg University, Sahlgrenska University Hospital, Goteborg, Sweden
OBJECTIVE Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. Cyclophilin D (CyD), a component of mitochondria membrane permeability transition pore (PTP) is important in Ca 2+ induced membrane permeability and necrotic cell death trigged by ischemia/reperfusion injury in adult brain and heart (1)–(4), whereas the role of CyD in hypoxic-ischemic (HI) injury in the immature brain is unknown. Caspase-dependent and non-dependent apoptotic mechanisms appear to be more critical in neonatal vs. adult brain (Zhu et al. 2005) and we hypothesized that CyD may play a different role in the developing brain. We crossed CyD deficient mice (KO) and wild-type (WT) mice to produce heterozygotes (Het), which were bred further to produce littermate animals with mixed genotypes.
METHODS At postnatal day (PND) 9 or 60, littermates of CyD KO, WT and Het were subjected to left carotid artery ligation and 10% O2 for 40 minutes or 30 minutes, respectively. Mice were sacrificed at 7 days after HI. Brain sections were stained with micro tube protein-2 (MAP-2) and the infarct volume and the total tissue loss was calculated.
RESULTS 1) At PND9, brain injury was more prononuced in CyD deficient mice than in WT mice. The total tissue loss was 20.6 ± 3.5 mm3 in CyD KO mice (n=19) vs 10.5 ± 1.7 mm3 in WT mice (n=16) (p=0.017). Regional neuropathology score analysis showed that injury in cortex, hippocampus, thalamus and striatum were 59%, 17%, 53% and 31% lower in WT compared to KO. 2) In PND60 adult brain, CyD deficiency resulted in a significantly reduced HI brain injury. The total tissue loss in PND60 mice was 2.3 ± 1.9 mm3 in CyD KO mice (n=13) vs 14.3 ± 4.8 mm3 in WT mice (n=18) (p=0.035). Brain injury was reduced in KO vs. WT by 68% in cortex, 50% in hippocampus, 90% in thalamus and 73% in striatum.
CONCLUSIONS 1) CyD-dependent PTP appears to play a different role in neontal and adult HI brain injury. 2) The present results might indicate that the known pro- and antiapoptotic actions of CyD do not have the same weight in immature as in the adult brain. These results agree with previous studies suggesting that cell death mechanisms especially those related to the apoptotic machinery are different in the developing CNS (5), (6) (7).
ROLE OF URACIL MISINCORPORATION FOR NEUROGENERATION AND BRAIN DYSFUNCTION INCUDED BY CHRONIC FOLATE DEFICIENCY
Golo Kronenberg1,2, Christoph Harms1, Robert W. Sobol3, Fernando Cardozo-Pelaez4, Heinz Linhart5, Mustafa Balkaya1, Karen Gertz1, Sarah Eckart2, Rainer Hellweg2, Heide Hoertnagl6, Rudolf Jaenisch5, Matthias Endres1
1Klinik Und Poliklinik Fuer Neurologie, Charite-Unversitaetsmedizin Berlin, Campus Mitte, Berlin, Germany, 2Klinik Und Hochschulambulanz Fuer Psychiatrie Und Psychotherapie, Charite-Universitaetsmedizin Berlin; Campus Benjamin Franklin, Berlin, Germany, 3University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA, USA, 4Department of Biomedical and Pharmaceutical Sciences, Center for Environmental Health Sciences, University of Montana, Missoula, MT, USA, 5Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA, USA, 6Institut Fuer Pharmakologie Und Toxikologie, Charite-Universitaetsmedizin Berlin, Campus Mitte, Berlin, Germany
Background and aims: Folate deficiency contributes to neurodegenerative and psychiatric disease such as dementia and depression. We hypothesized that the pathogenetic mechanisms include uracil misincorporation.
Methods: We analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (Ung-/−) vs. wildtype controls.
Results: Folate depletion increased nuclear mutation rates in Ung-/− embryonic fibroblasts, and conferred death of cultured Ung-/− neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in Ung-/− but not Ung+/+ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione (Figure). Further, FD induced cognitive and mood alterations such as depressive and anxious behavior that were aggravated in Ung-/− mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism and inhibited adult hippocampal neurogenesis.
Conclusions: These results indicate that impaired uracil repair is involved in neurodegeneration and brain dysfunction induced by folate deficiency.
Fig legend: (A) Representative images of 10mm thick H&E-stained sections illustrating reduced neuronal density in CA3 of Ung-/− animals as a consequence of chronic folate deficiency. (B) Density of CA3 pyramidal neurons; ANOVA for genotype: F1,16=22,8; p<0.0005; treatment: F1,16=11.3; p<0.005, interaction: F1,16=3.8; p=0.068; 1-b=0.44. (C) Uracil misincorporation in DNA, ANOVA for genotype: F1,29=5.6, p=0.02 (D) BDNF protein levels in hippocampus, ANOVA for interaction: F1,31=16.6, p<0.0005. (E) GSH levels in whole cerebral hemispheres; ANOVA for interaction: F1,20=5.6, p=0.03. (F) GSH levels in cerebellum; ANOVA for interaction: F1,20=20.4, p=0.0002. *p<0.05 ND vs. FD, #p<0.05 Ung+/+ vs. Ung-/−, +p<0.05 for interaction; two-way ANOVA and Tukey post-hoc, n=5-9 animals per group
ERYTHROPOIETIN PROMOTES NEURONAL REPLACEMENT VIA ANGIOGENESIS AND NEUROGENESIS AFTER HYPOXIA/ISCHEMIA IN NEONATAL RATS
Masanori Iwai1, Wei Yin1, Guodong Cao1, R. Anne Stetler1,2, Jun Chen1,2
1Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA, 2Geriatric Research, Education and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, PA, USA
Background and aims: Erythropoietin (EPO) has shown potent neuroprotective effects against ischemic injury in both adult and neonatal brain via its anti-apoptotic actions. Recent evidence has suggested that EPO may have additional effects on post-ischemic adult brain by promoting neurovascular unit remodeling. Indeed, endogenously expressed EPO and its receptors appear to be essential for ischemia-induced neurogenesis in adult brain. The current study was conducted to determine the effect of exogenous administration of EPO on angiogenesis and neurogenesis, the two major events believed to contribute to neuronal replacement, in the neonatal brain after hypoxia/ischemia (HI).
Methods: HI was induced by left-CCA ligation followed by 130 minutes exposure to 8 % oxygen in seven-day-old rats. They were then treated with recombinant human EPO (rhEPO; 1000U/kg) at 20 minutes after HI, and again at 2, 4 and 6 days. Rats were euthanized on day 7 after HI for evaluation of infarct volume, angiogenesis and neurogenesis, and on day 28 for evaluation of neuronal replacement. Neurogenesis and angiogenesis were analyzed using bromodeoxyuridine (BrdU) labeling, and by tomato lectin labeling to visualize functional vessels. The newly generated neurons were also classified as either neuronal progenitors, using anti-doublecortin (DCX) antibody, or mature neurons, using anti-neuronal nuclei (NeuN) antibody. At 7 (n=28) and 28 days (n=19) after HI, neurogenesis was evaluated using double immunofluorescence for BrdU/DCX or BrdU/NeuN. Neurological outcomes were also evaluated until 28 days after HI.
Results: Treatment with rhEPO significantly reduced the infarct volume both in the cortex (47.8%, p<0.01) and in the ST (42.1%, p<0.01). Enhancement of angiogenesis with rhEPO treatment was observed in the ischemic boundary of cerebral cortex. Compared with vehicle treatment, rhEPO treatment significantly increased BrdU-labeled cells on the vessels (114 ± 45 vs 55 ± 15/mm2, p<0.01) as well as the number of vessels (178 ± 67 vs 141 ± 61/mm2, p<0.05). In addition, rhEPO treatment significantly increased the number of BrdU-labeled cells in the ipsilateral subventricular zone (486 ± 88 vs. 381 ± 87/section in the vehicle, p < 0.01) and the number of doublecortin positive cells in the cerebral cortex (29 ± 10 vs. 17 ± 6/mm2, p < 0.01) and striatum (55 ± 27 vs. 42 ± 13 / mm2, p < 0.05) of ischemic hemisphere. Finally, rhEPO treatment significantly increased the number of BrdU/NeuN-double positive cells in the ischemic boundary of cerebral cortex (16 ± 11 vs. 3 ± 4/mm2, p < 0.01) and striatum (16 ± 11 vs. 6 ± 7 / mm2, p < 0.01) at 28 days after HI. Treatment with rhEPO significantly improved neurobehavioral development.
Conclusions: Our data suggests that treatment with rhEPO contributes to neurovascular unit remodeling by promoting tissue protection, angiogenesis and neurogenesis in neonatal HI injured brain and produces improvement of neurobehavioral development. However, EPO appears to have limited role in supporting new born neuron survival after HI, as only modest amounts of newborn cells with a mature neuronal phenotype (NeuN positive) eventually survived.
IMAGING OF AMYLOID-PLAQUES AND CEREBRAL GLUCOSE METABOLISM IN SEMANTIC DEMENTIA AND ALZHEIMERS DISEASE
Alexander Drzezga1, Timo Grimmer2, Gjermund Henriksen1, Isabelle Stangier1, Robert Perneczky2, Janine Diehl-Schmid2, Chester A. Mathis3, William E. Klunk4, Hans-Jurgen Wester1, Markus Schwaiger1, Alexander Kurz2
1Department of Nuclear Medicine, Technical University of Munich, Munich, Germany, 2Department of Psychiatry, Technical University of Munich, Munich, Germany, 3Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA, 4Department of Psychiatry, Western Psychiatric Institute & Clinic, Pittsburgh, PA, USA
Background and aims: Semantic dementia (SD) is a rare clinical syndrome, assigned to the group of frontotemporal lobar degenerations (FTLD). Histopathological analysis has not revealed the deposition of amyloid-plaques in the majority of SD-cases, in contrast to dementia of the Alzheimer type (AD). However, some overlap of clinical symptoms and imaging findings between AD and SD has been shown and a reliable differentiation of the underlying pathology can not be guaranteed based on clinical examination alone. Our aim was to determine, whether differences between AD and SD can be found by means of in vivo amyloid-plaque imaging, providing complementary information to results obtained by measurement of cerebral glucose metabolism.
Methods: Patients with AD (n=8, 4 female) and with SD (n=8, 4 female), matched for gender, age and overall degree of cognitive impairment, were recruited using established clinical criteria. Cerebral glucose metabolism was examined with [F-18]Fluorodeoxyglucose (FDG)-PET, cerebral amyloid-plaque load was assessed using [C-11]6-OH-BTA-1 (PIB)-PET. A volume-of-interest analysis (VOI), using the cerebellum as a reference region, and voxel-based statistical group comparisons (SPM2) were carried out between the patient groups (FDG- and PIB-PET data) and a group of healthy controls (FDG-PET data).
Results: Characteristic patterns of hypometabolism could be demonstrated in both clinically defined AD and SD (AD: bilateral temporoparietal and frontal cortex; SD: left>right temporal, frontal mesial cortex) with some regional overlap, particularly in temporal cortices. In contrast, strong [C-11]PIB amyloid-plaque tracer binding was observed only in patients with AD in bilateral temporoparietal, frontal and posterior cingulate cortex and the precuneus. This increased amyloid-plaque deposition, observed in AD, clearly extended the metabolic differences between the groups, even in regions with comparable metabolic deficits.
Conclusions: These findings support the common notion hat neuronal dysfunction is associated with amyloid-plaque deposition in AD. In contrast, in the examined cases of typical SD, neuronal dysfunction was apparently based on pathology other than amyloid-plaque deposition. Amyloid-plaque imaging may be valuable to clearly characterize dementias in vivo, based on the underlying pathology rather than on neuropsychological findings. This may be important for definition of individual prognosis and for the selection of patients for scientific trials.
OMENTUM TRANSPOSITION IN THE TREATMENT OF ALZHEIMER DISEASE
Harry Goldsmith
Department of Surgery, University of Nevada School of Medicine, Reno, NV, USA
Background and aims: It has been shown that the omentum is rich in biological substances and especially in the amount of angiogenic material incorporated in the tissue (VEGF). The aim of the presentation is to show that the omentum can introduce large amounts of blood, neurotransmitters, growth factors, and stem cells into the brain, which have been shown in some patients to reverse the cognitive changes of Alzheimer disease.
Methods: The omentum is separated from the transverse colon and the proximal portion of the stomach, leaving the gastroepiploic vessels within the omental apron. The omentum is then surgically lengthened and brought up through a subcutaneous tunnel developed along the chest and neck. A craniotomy is performed and the dura mater opened. The omentum is then laid directly on the brain.
Results: Several publications in neurosurgical journals have reported that in biopsy-proven Alzheimer-disease patients, of the twenty-five who were operated upon, six showed no improvement, nineteen showed some degree of improvement with nine patients showing significant reversal of their cognitive and neurological conditions.
Conclusions: It is possible to reverse cognitive symptoms of Alzheimer disease as a result of omental transposition to the brain.
CEREBRAL NICOTINIC ACETYLCHOLINE RECEPTORS IN PATIENTS WITH ALZHEIMERS DISEASE AND AMNESTIC MILD COGNITIVE IMPAIRMENT, COMPARISON TO GLUCOSE METABOLISM
Kai Kendziorra1, Philipp Meyer1, Henrike Wolf2, Henryk Barthel1, Swen Hesse1, Anita Seese1, Dietlind Sorger1, Marianne Patt1, Georg Becker1, Andreas Schildan1, Peter Brust3, Hermann Josef Gertz2, Osama Sabri1
1Department of Nuclear Medicine, University of Leipzig, Leipzig, Germany, 2Department of Psychiatry, University of Leipzig, Leipzig, Germany, 3Institute of Interdisciplinary Isotope Research, Leipzig, Germany
Background and Aims
Recent studies indicate, that cholinergic marker enzymes might be decreased only in advanced Alzheimer's disease (AD) and may even be upregulated in earlier disease stages. However, post mortem studies indicate a loss/dysfunction of nicotinic acetylcholine receptors (nAChRs) already in mildly symptomatic AD. 2-[18F]F-A-85380 (2-FA) binds specifically to the α4β2 nAChR, which is the most severely reduced subtype at the onset of AD. To clarify whether 2-FA-PET is of use for the early diagnosis of AD, we examined patients with mild AD and with amnestic mild cognitive impairment (MCI).
Methods
20 AD-patients, 11 MCI-patients and 10 healthy controls (NC) underwent 2-FA-PET. All of them had never taken any cholinergic medication and were non-smokers. Distribution volume (DV) images of 2-FA were calculated using the Logan Plot with an arterial input function corrected for decay, plasma protein binding and radiolabelled metabolites. Irregular ROIs were defined after co-registration of DV images to an individual MRI for calculating the binding potential (BP, reference region: corpus callosum). F-18-FDG-PET was performed in 9 MCI and 16 AD patients, respectively. FDG-data were analysed visually and by voxel-based analysis (BRASS; Hermes Medical Solutions, and 3D-SSP; University of Washington). Patients were followed up over a mean time of 17 months.
Results
Compared to NC, both the MCI- and AD-patients showed significant reductions in 2-FA-BP in the caudate head, hippocampus, temporal, frontal, anterior and posterior cingulate cortex (all p<0.05). Individual analyses yielded significant reductions (below mv ? 2sd) in 2-FA-BP in nine MCI-patients, of whom six converted to AD in the follow-up period (converters). Only one MCI-patient (converter) had a pathological FDG-PET. Two MCI patients yielded 2-FA-BPs in the range of the NC in all ROIs and had a normal FDG-PET. In keeping with the PET findings, these two patients did not convert to AD during the follow up period. Figure: 2-FA-BP in NC (A), AD-patient (B) and two MCI-patients (C; D). Clear reduction in (B and D), while(C) is comparable (A).
Conclusions
MCI and AD patients show significant reductions of α4β2-nAChR availability in the same brain areas. The α4β2-nAChR reduction seems to be present before changes in glucose metabolism occur. These findings indicate a potential role of 2-FA-PET in the early diagnosis of AD. Whether 2-FA-PET is able to predict conversion from MCI to AD with higher accuracy than FDG-PET has to be investigated in larger cohorts.
EFFECTS OF CEREBRAL ISCHEMIA ON EVOKED CEREBRAL BLOOD OXYGENATION RESPONSES AND BOLD CONTRAST FUNCTIONAL MRI IN STROKE PATIENTS
Norio Fujiwara, Noriaki Yokose, Tatsuya Hoshino, Yoshihiro Murata, Tsuneo Kano, Kaoru Sakatani, Yoichi Katayama
Department of Neurological Surgery, Nihon University School, Tokyo, Japan
Background: Blood oxygenation level dependent (BOLD) contrast functional MRI (BOLD-fMRI) has been used in functional studies on stroke patients, under the assumption that these patients have normal neurovascular coupling. Recent studies have revealed, however, that BOLD-fMRI does not correctly image activation areas in stroke patients. It was suggested that impairments of neurovascular coupling may alter the evoked cerebral blood oxygenation (CBO) responses and hemodynamic changes, and this could result in failure of BOLD imaging in stroke patients. BOLD-fMRI alone, however, cannot elucidate the precise mechanisms in the failure of BOLD imaging, since BOLD-fMRI provides information mainly about concentration changes of deoxyhemoglobin (deoxy-Hb), which is paramagnetic. In this study, in order to evaluate these issues, we compared the evoked CBO responses measured by near infrared spectroscopy (NIRS) and BOLD imaging in chronic stroke patients with moderate and severe cerebral ischemia.
Methods: We measured the evoked CBO responses in the primary sensorimotor cortex (PSMC) by means of NIRS (NIRO-300, Hamamatsu Photonics KK, Japan) during contralateral motor tasks. We compared the AV of BOLD-fMRI in the PSMC on the non-lesion and lesion sides. SPECT was employed to classify the ischemic status as moderate (slight reduction of rCBF and cerebrovascular reserve capacity (CVRC)) or severe (marked reduction of rCBF and CVRC; i.e., misery perfusion).
Results: In age-matched controls, deoxyhemoglobin concentration decreased with concomitant increases in oxyhemoglobin and total hemoglobin concentrations during activation. The PSMC on the non-lesion side exhibited a normal CBO response pattern. On the lesion side, moderate cerebral ischemia did not affect the CBO response pattern, but severe cerebral ischemia caused an increase of deoxyhemoglobin during the task, associated with increases of oxyhemoglobin and total hemoglobin. Moderate cerebral ischemia induced only a slight reduction of the AV on the lesion side; however, severe cerebral ischemia markedly reduced the AV on the lesion side. The BOLD signal did not change in some areas of the PSMC on the lesion side in severe cerebral ischemia, while it tended to decrease in other areas during the tasks (Figure 1).
Conclusions: Misery perfusion caused a marked reduction of the AV in BOLD imaging, associated with an increase of deoxyhemoglobin concentration during activation. BOLD-fMRI investigations of stroke patients should be performed while giving consideration to the baseline circulatory status. Functional NIRS could be an alternative means to assess the CVRC.
CORTICAL SPREADING ISCHEMIA IS INDUCED BY COMBINED INHIBITION OF NA, K-ATPASE AND NO SYNTHASE AND DEPENDS ON INTRACELLULAR CA2+ STORES
Sebastian Major1,2, Georg Royl1,2, Ute Lindauer1, Jens Dreier1,2
1Department of Experimental Neurology, Charite, Universitaetsmedizin Berlin, Berlin, Germany, 2Department of Neurology, Charite, Universitaetsmedizin Berlin, Berlin, Germany
Background: Delayed ischemic neurological deficit (DIND) is one of the major complications after subarachnoid hemorrhage (SAH). It occurs in up to 40% of patients who survive the initial bleeding. About 13% develop delayed cerebral infarcts on CT.
Based on experiments in rats, it has been proposed that cortical spreading ischemia (CSI) is a mechanism of DIND. CSI is a long-lasting, spreading wave of an ischemic blood flow change leading to widespread cortical infarcts. It represents an inverse hemodynamic response to cortical spreading depolarization. CSI occurs when the baseline extracellular K+ concentration ([K+]o) is increased and the NO concentration is reduced (e.g. due to nitric oxide synthase (NOS) inhibition by L-NNA) in the subarachnoid space.
In cell culture experiments increased baseline [K+]o is associated with a decrease of Na, K ATPase activity. To test whether this ubiquitous ATPase is involved in the mechanism of CSI, we investigated if CSI occurs after combined brain topical administration of L-NNA and the Na, K-ATPase inhibitor ouabain.
Methods: A closed cranial window was implanted in rats. Cerebral cortex was superfused with artificial cerebrospinal fluid (ACSF) containing L-NNA (1 mM). Subsequently, increasing concentrations of ouabain (0.5, 5, and 50 µM) were added to the ACSF. The CBF changes were monitored either by Laser-Doppler-Flowmetry or LAser Speckle Contrast Analysis imaging, the depolarization by subarachnoid Ag-AgCl electrode.
Results: Compared to L-NNA or ouabain alone the combination induced CSI in a dose dependent manner (duration of hypoperfusion under L-NNA and ouabain (5 µM): 3:50 (3:00 ? 5:26) min and L-NNA and ouabain (50 µM): 6:55 (5:20 ? 22:24) min). CBF fell to about 50% of baseline value (=100%). Using a digital camera we recorded a propagating progressive constriction of arteries simultaneously with the CBF decrease.
At least at the lower applied ouabain concentration (up to 5 µM), only the α2 and α3 isoforms of the Na, K ATPase are inhibited in rats. Their inhibition leads to increase of the cytoplasmic Ca2+ concentration and subsequently to filling of the Ca2+-stores in the sarco/endoplasmic reticulum. Thus we tested, if depletion of these internal stores has an influence on the CSI. Therefore we added 100 µM of thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase, to the ACSF 30 min before applying ouabain (50 µM). This did not prohibit hypoperfusion, but shortened it significantly to 1:16 (0:58 ? 3:18) min (vehicle control 4:31(2:07 ? 8:47)).
Conclusion: Combined NOS and Na, K-ATPase inhibition produce CSI in a dose-dependent manner. Ca2+ stored in the sarco/endoplasmic reticulum appears to be necessary for maintaining the vasoconstriction during CSI.
CEREBRAL HEMODYNAMICS AND CLINICAL COURSE AFTER SUPERFICIAL TEMPORAL ARTERY-MIDDLE CEREBRAL ARTERY ANASTOMOSIS IN ADULT ISCHEMIC-TYPE PATIENTS WITH MOYAMOYA DISEASE
Masaru Idei, Kanji Yamane, Hirohumi Okii, Kiyoshi Kumano, Hidetaka Onda
Department of Neurosurgery, Chugoku Rousai Hospital, Kure, Hiroshima, Japan
[Background and aims] We measured regional cerebral blood flow (rCBF) using xenon-enhanced CT and assessed overall outcome after superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis in adult ischemic-type patients with moyamoya disease.
[Methods] Our subjects consisted of 16 (25 sides) cases who underwent STA-MCA double anastomosis from April, 1994 to February, 2006. Clinical features and rCBF were investigated before surgery, two weeks, half a year, one year, two years, three years and four years after surgery. rCBF was compared with normal control value (control rCBF) in our hospital. The region of interest was conducted in the anterior cerebral artery (ACA), middle cerebral artery (MCA) and posterior cerebral artery (PCA) territories.
[Results] Before surgery, rCBF in the ACA and MCA territories was found to be significantly lower than control rCBF, but in the PCA territory was preserved. During follow-up, rCBF in the ACA territory did not changed, but on the other hand rCBF in the MCA territory significantly increased. rCBF was preserved in the PCA territory and gradually decreased during follow-up. Only one patient had infarction in the ACA territory 9 years after surgery, but the remaining 15 patients had no recurrence of stroke.
[Conclusions] Our results showed that dominant posterior distribution of the rCBF before surgery gradually became to be the normal distribution after STA-MCA anastomosis. It suggested that STA-MCA anastomosis could prevent a recurrence of stroke in the MCA region, but not completely in the ACA region.
FACTORS OF HYPERPERFUSION SYNDROME AFTER CAROTID ENDARTERECTOMY AND INTRAOPERATIVE NEAR INFRARED SPECTROSCOPY
Norikazu Miura1, Kenji Yoshitani2, Masakazu Kuro2
1Anesthesiology Department, Yokohama City University Graduate School of Medicine, Yokohama, Japan, 2Anesthesiology Department of National Cardiovascular Center, Osaka, Japan
Background: Postoperative cerebral hyperperfusion syndrome (HS) after carotid endarterectomy (CEA) causes miserable adverse cerebral complication, such as intracranial hemorrhage. Furthermore, the prognosis of patients with HS is poor. Previous studies reported that impaired cerebral vasoreactivity was implicated in HS (1). However, Powers' staging (2), one of clinical indices of cerebral hemodynamic impairment, has not been tested whether that is a factor of HS. In addition, near infrared spectroscopy (NIRS) said to reflected cerebral vasoreactivity (3). Therefore, we tested the hypothesis whether Power's staging was a factor of HS, or intraoperative NIRS measurement predicted HS.
Methods: The records of 116 consecutive patients with CEA were reviewed. Logistic regression analysis was used to assess the effects of Power's staging and following factors on HS; age, sex, history of hypertension, diabetes mellitus, smoking, atrial fibrillation, symptom for internal carotid artery stenosis, history of ischemic heart disease, severity of carotid artery stenosis, stump pressure, and systemic blood pressure after declamping. Tissue oxygen index (TOI, were measured using NIRO 200 (Hamamatsu Photonics, Hamamatsu) during CEA in 5 patients preliminary.
Results: Sixteen patients (18%) had HS after CEA. Significant predictors with univariate logistic regression analyses were Powers' staging, ipsilateral carotid artery stenosis (>80%), contralateral carotid artery stenosis (>50%), and systolic arterial pressure (>120mmHg) after declamping of carotid artery. Multivariate logistic regression analysis demonstrated that the Powers' staging (p=0.001), contralateral internal carotid artery stenosis more than 50% (p=0.003) and systolic arterial pressure (>120mmHg) (p=0.01) were significant predictive variables for postoperative HS. During CEA, tissue oxygen index (TOI) was measured in 5 patients of 16 patients who suffered from HS. The values of TOI after declamping of internal carotid artery were significant higher than that during clamping (61 ± 5 to 68 ± 8%, p<0.05).
Conclusion: The results in our retrospective study demonstrated that the Powers' staging, as an index of hemodynamic state, contralateral carotid artery stenosis more than 50% and systolic arterial pressure (>120mmHg) after carotid artery declamping were predictive factors for postoperative HS. NIRS monitoring, TOI could be effective to predict postoperative HS as an intraoperative index.
MAGNETIC RESONANCE MOLECULAR IMAGING OF ACTIVATED ENDOTHELIUM IN ACUTE STROKE USING A TARGETED IRON-OXIDE NANOPARTICLE CONTRAST AGENT
Albert Jin1, David Rushforth2, Rana Filfil3, Jaspreet Kaur1, Ursula Tuor2, Tadeusz Foniok2, Kumud Deka2, David Kirk2, Jody Lee-Petersen1, Boguslaw Tomanek2, Feng Ni3, Philip Barber1
1Experimental Imaging Centre, University of Calgary, Calgary, AB, Canada, 2Institute of Biodiagnostics (West), National Research Council of Canada, Calgary, AB, Canada, 3Biotechnology Research Institute, National Research Council of Canada, Montreal, QC, Canada
Background: Because of the inherent complexity of neuroinflammation in acute stroke, critical aspects of the early evolution of stroke injury are poorly understood. Currently, MRI cannot discriminate pathological processes such as endothelial activation in neuroinflammation. The aim of this study was to use MRI to visualize endothelial activation in a mouse model of stroke using an iron oxide nanoparticle contrast agent (MNP-Psel) that was modified to bind with high affinity to P-selectin.
Methods: MNP-Psel was compared to the iron oxide agent Feridex (Berlex), which does not have any binding specificity. As a model for focal cerebral ischemia and reperfusion, the left middle cerebral artery (MCA) was transiently occluded with a silicon-coated nylon suture to effect a 75% or greater reduction in regional cerebral blood flow for 30 minutes in male C57 Black 6 mice. Normothermia was maintained throughout the procedure and afterwards. 24 hours post-reperfusion, mice received 2.8 Fe mg/kg of either Feridex or MNP-Psel. MRI scans at 9.4T acquired prior to and after contrast injection included: multi-echo T2 weighted images, T2*-weighted images, and a T1 map. Subtraction maps (pre-contrast ? post-contrast) were used to identify areas with differences in T1, T2 and T2*. The T2 effect in the contralateral side was subtracted from the effect in the stroke side to remove confounding from non-specific contrast agent accumulation: [1? Percent T2 decrease on contralateral side/Percent T2 decrease on stroke side] × 100. After imaging was complete, mice were euthanised by sodium pentobarbital overdose and the brain was perfusion fixed in 4% paraldehyde. The brain was then embedded in paraffin and sections through the cortex and striatum were taken for iron histochemical staining by the Prussian blue method.
Results: T1 values were unaffected by MNP-Psel or Feridex. T2 decreased nonspecifically in both hemispheres after Feridex injection but preferentially in the stroke hemisphere with MNP-Psel, indicating that MNP-Psel accumulation was mediated through an interaction specific to the infarct and peri-infarct regions. Prolonged lowering of T2* after MNP-Psel was seen preferentially in the stroke hemisphere. The presence of T2 and T2* effects of MNP-Psel in the peri-infarct regions indicated endothelial activation beyond the infarction boundary noted on T2 maps. Brain sections from MNP-Psel-injected mice showed iron accumulation on endothelium in the infarct and peri-infarct regions.
Conclusion: In this study, there is a prolonged shortening of T2 and T2* values within the infarct and peri-infarct region after injection of MNP-Psel that is not observed with Feridex. Additionally, histological examination of the mouse brain after injection with MNP-Psel showed an accumulation of iron on endothelium in the infarct and peri-infarct regions. These results suggest that MNP-Psel binds to P-selectin expressed on activated endothelium in the stroke hemisphere. This demonstrates the potential use of targeted iron oxide contrast agents to visualize in vivo endothelial activation in acute stroke.
NEUROVASCULAR COUPLING AND CEREBRAL METABOLIC RATE OF OXYGEN: EFFECTS OF BRAIN HYPOTHERMIA
Martina Fuechtemeier1, Nikolas Offenhauser1, Christoph Leithner1, Jens Steinbrink1, Matthias Kohl-Bareis2, Ulrich Dirnagl1, Ute Lindauer1, Georg Royl1
1Department of Experimental Neurology, Charite Universitaetsmedizin Berlin, Berlin, Germany, 2University of Applied Sciences Koblenz, RheinAhr Campus, Remagen, Germany
Background: Neuronal activation is accompanied by a local increase in cerebral blood flow (CBF) and in cerebral metabolic rate of oxygen (CMRO2). From combined measurements of CBF and hemoglobin concentration, relative changes in CMRO2 during functional challenges can be calculated. The quantitative relationship between basal CMRO2 and brain temperature changes over a range of 10°C (Q10) has not yet been determined with this method and can serve as a validation. In addition, recent studies have raised the question whether neurovascular coupling serves local cooling of activated brain tissue. Then again, the influence of hypothermia on neurovascular coupling is not known. We addressed these issues by studying the effects of graded hypothermia in a rat model of neurovascular coupling in the somatosensory cortex.
Methods: Anesthetized Wistar rats (n=6) underwent surgical preparation of a closed cranial window over the somatosensory cortex. Using Laser Doppler flowmetry and optical spectroscopy, changes in CBF and in hemoglobin concentration were measured continously. Furthermore, an electrocorticogramm (ECoG) was simultaneously recorded from the measurement site. By the application of ice packs, whole-body hypothermia (masseter temperature from 35°C down to 25°C in 2 hours) was generated, followed by re-warming. While maintaining anesthesia with alpha-chloralose, spontaneous ongoing activity was measured, interleaved by blocks of functional measurements during electrical forepaw stimulation (4sec, 1.6mA, 4Hz).
Results: Hypothermia led to a decrease of spontaneous CBF and CMRO2. The calculated Q10 value for CMRO2 was 3.0 (95% Confidence Interval: 2.6 ? 3.4). Parallel to CBF and CMRO2, the power of the ECoG low-frequency-band decreased. Functional changes of CBF and CMRO2 were reduced during hypothermia as well as the amplitudes of somatosensory evoked potentials.
Conclusion: 1. With optical methods, spontaneous changes in relative CMRO2 can be quantified, revealing a Q10 for CMRO2 that is well in the range of literature values. 2. During hypothermia, neurovascular coupling is preserved. This applies to CBF changes accompanying spontaneous ongoing activity as well as CBF changes during functional activation. Thus, a possible role of activity-induced neurovascular coupling in local temperature regulation cannot be excluded.
Keywords: hypothermia; CMRO2; Q10 value; neurovascular coupling; rat
Supported by DFG and Hermann and Lilly Schilling Foundation.
MAGNETIC RESONANCE AND NEAR-INFRARED SPECTROSCOPY MEASUREMENTS OF ENERGY METABOLITES AND CEREBRAL OXYGEN CONSUMPTION FOLLOWING HYPOXIA ISCHEMIA
Jeff D. Winter1,2, Ken M. Tichauer1,2, Neil Gelman1,2, R. Terry Thompson1,2, Ting Y. Lee1,2, Keith St. Lawrence1,2
1Imaging Division, Lawson Health Research Institute, London, ON, Canada, 2Department of Medical Biophysics, University of Western Ontario, London, ON, Canada
Background and Aims
Hypoxia ischemia (HI) is a major cause of perinatal brain injury. 31P magnetic resonance spectroscopy (MRS) studies have shown that tissue damage develops 1–3 days after the initial hypoxic ischemic insult (1). This delay offers the opportunity to reduce tissue damage by preventing delayed brain injury. However, there is currently no reliable method for detecting the onset of delayed injury. Our group recently developed a near-infrared spectroscopy (NIRS) method for measuring the cerebral metabolic rate of oxygen (CMRO2) to detect early reductions in CMRO2 following HI in newborn piglets (2)–(4). The purpose of the present study was to combine NIRS with MRS to determine the temporal relationship between CMRO2 and energy metabolites following HI.
Methods
Simultaneous NIRS, 31P- and 1H-MRS data were acquired at 3 Tesla on six hypoxic-ischemic and six control piglets (< 36 hr of age). HI was induced by clamping the carotids and reducing the inspired oxygen to 8% for 30 min. Data were collected before (baseline) and for six hours following HI. The ratios of phosphocreatine to inorganic phosphate (PCr/Pi) and nucleotide triphosphate to exchangeable phosphates (NTP/EPP), where EPP = (Pi + PCr + 3NTP), were measured from 31P spectra. The lactate to choline ratio (lac/cho) was measured from 1H spectra. CMRO2 was determined using NIRS measures of deoxy-hemoglobin and cerebral blood flow (CBF), i.e., CMRO2 = CBF* (arterial O2 ? venous O2).
Group differences between HI and control piglets were determined using a one-way ANOVA for each time point for NIRS/MRS parameters with a significant time-by-group interaction.
Results
The PCr/Pi and NTP/EPP ratios were significantly reduced in the HI group compared to controls up to 90 and 150 min post insult, respectively. No differences between the groups were observed at later time points. The absolute changes in PCr and NTP relative to baseline showed similar trends. The Lac/Cho ratio was significantly elevated at 30 min after HI, but returned to control levels for all later time points. CMRO2 decreased following HI and was significantly lower than controls for all times after 150 min.
Conclusions
As we previously observed, CMRO2 was significantly decreased following HI (4). In this study, the NTP and PCr levels returned to baseline during the same period, which implies that reduced CMRO2 is not caused by immediate cell death, but reflects a reduction in oxidative metabolism. This reduction could either be due to impaired mitochondrial function, with a compensatory increase in aerobic glycolysis to maintain NTP levels, or reduced NTP utilization following HI (5),(6). Our group is currently incorporating measurements of cerebral glucose metabolism in order to address this issue.
SUSTAINED ALTERATION OF NEUROMETABOLIC, -VASCULAR COUPLING BY CORTICAL SPREADING DEPRESSION IN RAT SOMATOSENSORY CORTEX
Nikolas Offenhauser, Georg Royl, Christoph Leithner, Ulrich Dirnagl, Ute Lindauer
Department of Experimental Neurology, Charite, Berlin, Germany
Neuronal activity increases blood flow and oxygen consumption in the brain. The mechanisms by which increased metabolic activity during local increases in neuronal activity are coupled to hemodynamic changes are incompletely understood. Especially, there is only limited knowledge, if pathophysiological events influence this coupling relationship and how baseline state alterations of brain blood flow or metabolism are related to activity induced changes in regional cerebral blood flow (rCBF), oxygen consumption and neuronal activity. We tested the hypothesis that this neurometabolic, neurovascular coupling relationship is variable.
Cortical spreading depression (CSD) was used to induce a transient alteration in neuronal activity and vascular hemodynamic responsiveness, to investigate the variability in the relationship between neuronal, metabolic and vascular responses under functional activation (forepaw stimulation, 5Hz, 10s). Alpha-chloralose-urethane anaesthetized, male Wistar rats where used. Activity induced changes in rCBF, regional blood oxygenation (rCHo: oxy-, deoxyhemoglobin concentration) and saturation, by a combined microfiber optical hemoglobin spectroscopy and laser-Doppler flowmetry probe, where recorded and oxygen consumption during activation was calculated. ECoG and SEPs were recorded by a silver ball surface electrode as indicators of baseline and evoked neuronal activity. Averaged responses prior to single (n=6) or repetitive (n=3) CSDs were compared with blocks of averaged responses up to 1.5h.
Baseline rCBF after a single CSD was initially significantly reduced (up to 35%, +/− 4, SEM) and accompanied by a significant increase in deoxy-hemoglobin concentration (Hbr; 16%, +/− 3) and decrease of oxy-hemoglobin concentration (Hbo; 36%; +/− 6). These changes gradually returned to baseline at the end of recording (90 min. post CSD).
After CSD, rCBF responses and SEPs were significantly attenuated (by 45%, +/− 5 and 20%, +/−4, respectively). Hbr responses after CSD ranged from biphasic increase-decrease patterns to entirely positive responses, mirrored by the concomitant Hbo response.
Most interestingly, the individual parameters showed a different recovery pattern after CSD. The Hbr response continuously increased after CSD. An effect found to be more pronounced after repetitive CSDs. In general, SEPs and activity induced CMRO2 changes after CSD where significantly different from the control condition only up to 45 min. after CSD, while activity induced rCBF responses remained significantly reduced up to the end of the recording period (90 min). At this time, baseline parameters of neuronal activity (indicated by ECoG), CBF and rCHo had returned to baseline. Additionally, was the change in the Hbr response not explained by a change in the CBF response onset timing.
The observed divergent modification of activity induced changes in rCBF, neuronal activity and oxygen consumption by CSD, indicates a long term change in neurometabolic, -vascular coupling and suggests a variable relationship between neuronal activity, oxygen consumption and CBF response in rat cerebral cortex. Taken together, these findings provide in vivo evidence for the concept of plasticity in neurometabolic -vascular coupling.
Supported by the DFG and the Hermann and Lilly Schilling Foundation.
ENDOTHELIAL NITRIC OXIDE SYNTHASE S1179 PHOSPHORYLATION IMPROVES CEREBRAL BLOOD FLOW AND REDUCES INFARCT SIZE IN FOCAL CEREBRAL ISCHEMIA
Hwa Kyoung Shin1, Dmitriy N. Atochin2, Sae-Won Lee1, Paul L. Huang2, Michael A. Moskowitz1, Cenk Ayata1,3
1Stroke and Neurovascular Regulation Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA, 2Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA, 3Stroke Service and Neuroscience Intensive Care Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA
Background and aims: Endothelial nitric oxide synthase (eNOS) is an important enzyme regulating vascular tone, platelet aggregation and inflammatory cell migration in cerebral ischemia. eNOS activity is regulated by multiple mechanisms; among these, eNOS phosphorylation appears to be an important regulatory step. Phosphorylation of eNOS at serine 1179 (S1179) by protein kinase Akt increases its catalytic activity at all intracellular calcium concentrations. S1179 phosphorylation is induced by a number of physiological stimuli such as shear stress, and inhibited in diabetes, hyperlipidemia, and hypertension, and thus appears to be critical in both physiological and disease states. However, the impact of eNOS phosphorylation on vascular function and disease pathogenesis has not been studied, in vivo.
Methods: To determine the effect of eNOS S1179 phosphorylation on cerebral hemodynamic and metabolic response to ischemia, we subjected wild-type (WT, n=10), eNOS knockout (eNOS-/−, n=10), and transgenic mice expressing only phosphomimetic (S1179D, n=12) or unphosphorylatable (S1179A, n=11) mutant forms of eNOS (on eNOS-/− background) to distal or filament middle cerebral artery occlusion (dMCAO or fMCAO). Cerebral blood flow (CBF) was imaged non-invasively in ischemic core and penumbra using simultaneous laser speckle flowmetry.
Results: Total eNOS protein expression was similar between S1179A and S1179D lines, although both transgenic lines had reduced eNOS expression compared to wild type littermates by western blotting. The S1179 phospho-eNOS was not detectable in brain or heart in either transgenic line. Mean arterial blood pressures were elevated in eNOS-/−, S1179A, and S1179D mice compared to WT (p<0.05); arterial blood gas values did not differ. Upon dMCAO, the area of cortex with severe CBF deficit (i.e., ?20% CBF) was significantly greater in eNOS-/− mice compared to WT (p<0.05). Expression of the S1179A transgene in eNOS-/− mice did not improve the CBF deficit, while expression of the S1179D transgene reduced the CBF deficit close to that seen in WT (p<0.05 vs. eNOS-/−). Upon fMCAO, infarct volumes were smaller in S1179D and WT compared to S1179A transgenic and eNOS-/− mice (p<0.05).
Conclusions: These data provide the first in vivo evidence suggesting that phosphorylation of eNOS at S1179 is an important determinant of cerebral hemodynamic response to ischemia, and may be a useful target for the treatment and prevention of stroke.
This work was supported by the American Heart Association (0335519N, Ayata), NIH (P50 NS10828, Moskowitz; NS048426, Huang).
EFFECTS OF PROSTAGLANDIN E (PGE) ON REACTIVITY OF NON-PRESSURIZED CEREBRAL ARTERIOLES
JiSuk K. Yoo, David K. Kung, Leonid Groysman, Adam L. Sandler, H. Richard Winn
Department of Neurosurgery, Mount Sinai School of Medicine, New York, NY, USA
Background and Aims: Recent publications using in vitro brain slices have documented changes in intracerebral arterioles during astrocytic and neuronal activation. These in vitro intracerebral vessels studied in slices are non-pressurized and non-perfused. However, the physiological relevance of the observed changes in these arterioles is uncertain. We have previously shown that un-pressurized penetrating arterioles do not react to standard stimulants (changes in extra-luminal pH and adenosine) in an expected manner. Moreover, the addition of epinephrine (10-6M), a substance frequently used in cortical experiments, did not normalize the vascular response of un-pressurized arterioles. In the present study, we evaluate the potentiating effects of PGE.
Methods: Isolated penetrating rat arterioles were freshly harvested, cannulated, perfused, and pressurized as previously described (Ngai and Winn, 1995). Lumen diameter was measured with a video dimension analyzer. After a period of equilibration, we evaluated the response of these arterioles to change in the abluminal pH (6.8 & 7.6) and to adenosine (10-5 M). Between these challenges, the extra-luminal fluid was perfused with buffer and the vessel returned to baseline diameter. The reactivity of penetrating arterioles was then studied in absence and presence of PGE (7.5 ×10 −8 M) in two conditions: 1. Pressurized and perfused, simulating the in vivo state; group 2. Un-pressurized and non-perfused simulating conditions found in vitro in hippocampal and cortical slices.
Results: As indicated in Fig 1, pressurized and perfused arterioles reacted to stimulation in an expected manner. In the absence of intra-luminal pressure and flow, simulating conditions in a slice preparation, arterioles failed to dilate to a low pH or extraluminal Ado. The addition of PGE to the bath increased resting tone and restored reactivity in un-pressured and un-perfused vessels. We saw a similar trend in uncannulated, unpressurized vessels (data not shown).
Conclusion: In studying arteriole-neuronal interactions in brain slice, PGE can be used to simulate in vivo conditions.
[Supported by NS 021076]
PHOTOTHROMBOTIC OCCLUSION OF PENETRATING ARTERIOLES GENERATES SEVERE ISCHEMIA
Nozomi Nishimura1, Chris B. Schaffer1, Beth Friedman3, Patrick D. Lyden3,4, David Kleinfeld2,4,5
1Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA, 2Department of Physics, University of California at San Diego, San Diego, CA, USA, 3Department of Neurosciences, University of California at San Diego, San Diego, CA, USA, 4Graduate Program in Neurosciences, University of California at San Diego, San Diego, CA, USA, 5Center for Theoretical Biological Physics, University of California at San Diego, San Diego, CA, USA
Background and aims: Penetrating arterioles bridge the communicating arterioles on the surface of cortex to the subsurface microvascular bed that feeds the neurons. Optical methods were used to study ischemia due to clots in these vessels.
Methods: We used photochemically-triggered thrombosis to produce an occlusion in a single penetrating arteriole in the cortex of anesthetized rat. Two-photon microscopy was used to map the vasculature and measure red blood cell (RBC) velocity in vessels as deep as 400 µm before and after occlusion.
Results: We found that occlusions of penetrating arterioles caused severe ischemia and analyzed the spatial extent of the effect. Immediately downstream vessels dropped to 3% of initial speed. The blood flow increased with distance from the occluded arteriole and approached baseline values about 350 µm away from the occlusion. This suggests that the penetrating arterioles in rat parietal cortex primarily serve territories approximately 700 µm in diameter.
Conclusions: The ischemia produced by occlusions in penetrating arterioles is more severe than ischemia after occlusions in surface vessels and subsurface microvessels. The severe ischemia from occlusions of penetrating arterioles and arteries may be a cause of small lesions that are found in the brains of elderly humans. Such lesions are thought to play a role in neurodegenerative diseases.
Figure. (a) Schematic of photothrombosis of penetrating arterioles. (b) Fraction of the baseline speed is plotted versus the radial distance of the measured vessel from the occluded arteriole. Red line is average of 50 vessels, black lines show SEM.
POST-ISCHEMIC VASCULAR ADHESION PROTEIN-1 (VAP-1) BLOCKADE PROVIDES NEUROPROTECTION IN RATS SUBJECTED TO TRANSIENT MCAO
Lizhen Mao, Hao-Liang Xu, Dale Pelligrino
Neuroanesthesia Research Department, University of Illinois at Chicago, Chicago, IL, USA
BACKGROUND: There is no clear consensus regarding a role for leukocytes in the post-ischemic neuropathology associated with cerebral ischemia and reperfusion models. One example of this lack of agreement derives from studies employing neutrophil/leukocyte depletion paradigms, where both beneficial (1) and ineffectual (2) influences have been reported. However, the potential for “off-target” actions limits the usefulness of such strategies. We recently obtained evidence that VAP-1 (also called semicarbazide-sensitive amine oxidase [SSAO]), an enzyme concentrated in cerebral vascular cells, plays an important role in the process of post-forebrain ischemia neutrophil infiltration. In that study (1), we delayed treatment with the highly-selective inhibitor of VAP-1/SSAO (LJP-1207) until a time-point just prior to the onset of infiltration (6h reperfusion). Substantial reductions in post-ischemic neutrophil infiltration and significant neuroprotection (at 72h) were observed. However, the rats used in that study (diabetic, estrogen-treated ovariectomized females) exhibit an exaggerated post-forebrain ischemia neutrophil infiltration response. Such findings, therefore, do not necessarily indicate a beneficial role for VAP-1/SSAO inhibition in all cases of ischemia/reperfusion.
METHODS: In the present study, we examined the effects of LJP-1207 post-treatment (2h reperfusion) in a more commonly-used model; i.e., non-diabetic adult male rats subjected to 1h MCAO (intraluminal suture) and 3 days reperfusion. Prior to these experiments (in separate rats), we monitored, using cranial window-based intravital microscopy of pial venules overlying the ischemic cortex, patterns of adhesion and infiltration of neutrophils over the initial 10h of reperfusion following 1h MCAO.
RESULTS: Perivascular accumulation of leukocytes was seen to gradually rise up to 10h (expressed as the percentage of the viewed venular area-fig.1), with signs of extravasation seen (in 50% of the rats) after 8h. This represents a more modest neutrophil response than in the diabetic females cited earlier. Nevertheless, VAP-1/SSAO-inhibited rats displayed a 53% reduction in infarct volume, relative to controls (fig. 2A), and a significant improvement in neurologic outcome (fig. 2B).
CONCLUSIONS: These findings suggest that VAP-1/SSAO inhibition may also represent an effective intervention for limiting neuropathology following transient focal ischemia. The mechanism may relate to preventing generation of SSAO products like formaldehyde and hydrogen peroxide, thereby limiting leukocyte/vascular interactions. References: (1) J Pharmacol Exp Ther 317:19, 2006; (2) Stroke 37:1288, 2006.
DETECTION OF EXTRASTRIATAL DOPAMINE RELEASE IN HEALTHY HUMAN VOLUNTEERS
Rajendra Badgaiyan, Alan Fischman, Nathaniel Alpert
Division of Nuclear Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA
Detection of task-induced release of dopamine in the extrastriatal brain areas is critical for understanding of the brain mechanisms that control human cognition. The detection in these areas however has been a challenge because of relatively low receptor density. We have earlier demonstrated that task-induced release of striatal dopamine can be detected using a molecular imaging technique. This demonstration however, is of limited use because it cannot be used to detect dopamine released in the extrastriatal areas, where most of human cognitive processing takes place. Since the high affinity dopamine receptor ligand fallypride has been shown to bind in detectable quantities in the human extrastriatal areas, we used this ligand to detect task-induced release of extrastriatal dopamine. In this experiment, healthy volunteers performed an emotional memory task in the scanner after receiving a single intravenous bolus of 18F-fallypride. The task consisted of a control (no emotional memory) and a test condition. The control condition was started after the ligand administration. The test condition was started 25 min after the ligand injection. The ligand concentration was measured using an ECAT HR+ camera in 60 sec intervals. Analyses of behavioral data suggested that emotional memory was elicited in the test condition. Immediately after initiation of this condition the rate of ligand displacement increased significantly in the extrastriatal brian areas that have previously been implicated in the processing of emotional memory. These areas included amygdala, medial temporal lobe and ventral prefrontal cortex. The displacement rate did not change significantly in the reference region (cerebellum). Since the experiment found evidence of significant dopamine release in the extrastriatal areas that have previously been implicated in the processing of emotional memories, the results suggest that the ligand 18F-fallypride can be used to detect dopamine released in the extrastriate brain areas during performance of a cognitive task.
CILOSTAZOL MAINTAINS DOPAMINE AND SUBSTANCE P LEVELS AND IMPROVES SWALLOWING REFLEX IN A RAT CHRONIC CEREBRAL HYPO-PERFUSION MODEL
Ning Zhang1,2, Terubumi Watanabe1, Nobukazu Miyamoto1, Ryota Tanaka1, Nobu Hattori1, Takao Urabe1
1Depertment of Neurology, Juntendo University School of Medicine, Tokyo, Japan, 2Research Institute for Disease of Old Age, Juntendo University School of Medicine, Tokyo, Japan
Background and aims: Pneumonia is a common complication with the highest attributable proportion of death in the entire stroke population. Dysphagia with aspiration and the depression of cough reflex caused by central nervous disorder are believed the most important factors contributing to the risk of post-stroke pneumonia. Cilostazol is a potent inhibitor of type III phosphodiesterase used as an anti-platelet agent for the treatment of chronic cerebral infarction. The present study was designed to assess the pneumonia prevent mechanism of Cilostazol through its effects on regulating the dopamine and substance P levels as well as on improving the swallowing reflex in rat model of chronic cerebral hypo-perfusion.
Methods: Rats were subjected to bilateral common carotid artery ligation (LBCCA) and divided randomly into the vehicle group (n=72) and the Cilostazol group (n=72). Saline elicited swallowing reflex was evaluated at baseline and at days 14, 28 and 42 days after LBCCA. The levels of dopamine and substance P in the striatum were detected. The immunohistochemistry and immunoblotting analyses for tyrosine hydroxylase (TH), cyclic adenosine monophosphate responsive element binding protein (CREB) and phosphorylated CREB (p-CREB) in the substantial nigra were also performed at the investigated time points.
Results: Cilostazol significantly improved the elicited swallow reflex by shortening the latent time (3.7±0.2 vs 4.3±0.24 sec; P< 0.05) and increasing the numbers of swallow reflex (8±0.3 vs 6±0.8; P<0.05) at 14 days of hypo-perfusion. Compared with the vehicle group, Cilostazol up-regulated CREB phosphorylation and increased TH expression in the substantial nigra; in the striatum, Cilostazol markedly maintained dopamine (84.71±2.26 vs 79.2±4.13 % control; P<0.05) and substance P levels (86.65±7.89 vs 73.88±6.52% control; P<0.001) at 14 days of hypo-perfusion.
Conclusions: Our results indicate that based on up-regulating the expression of TH through cell signaling pathway of CREB phosphorylation, Cilostazol modulates the striatal dopamine and substance P levels and improves the swallowing reflex, and suggest that Cilostazol is an adequate intervention to sufficiently prevent pneumonia in the patients of chronic cerebral ischemia.
INFLUENCE OF SURGICAL TREATMENT FOR BRAIN AND ITS FUNCTION IN PATIENTS WITH UNRUPTURED CEREBRAL ANEURYSMS
Yoshiaki Kumon, Hideaki Watanabe, Keiji Igase, Hiroaki Matsumoto, Takanori Ohnishi
Department of Neurosurgery, Ehime University Graduate School of Medicine, Toon City, Ehime, Japan
Background and aims: We evaluated the influence of direct surgical treatment in patients with unruptured cerebral aneurysms of the anterior circulation.
Methods: 133Xe-SPECT and Wechsler Adult Intelligence Scale - Revised (WAIS-R) examination were done in 30 patients (31 operations), before and 1 month after surgery.
Results: Deterioration of total IQ (more than 3 scores) was recognized in 8 operations (26%), and resting CBF decreased significantly in the ACA and anterior border zone (BZ) areas. In 10 operations for ACoA aneurysm, WAIS-R results deteriorated in 4, and resting CBF decreased in the ACA and anterior BZ areas. In 9 operations for MCA aneurysm, WAIS-R results deteriorated in 4, and cerebrovascular response decreased in the anterior and posterior BZ areas. In 12 operations for ICA aneurysm, no patient showed deterioration in WAIS-R results and CBF. In 4 patients older than 70 years old, WAIS-R results deteriorated in 3.
Conclusions: WAIS-R and CBF results deteriorated in considerable number of patients with ACoA or MCA aneurysm, and aged patients. We should recognize these baneful influences of direct surgery.
PLATELET AGGREGABILITY USING WHOLE BLOOD AFTER ANTIPLATELET AGENTS IN PATIENTS WITH NON-CARDIOEMBOLIC STROKE
Daisuke Furuya, Norio Tanahashi, Nobuo Araki, Tomokazu Shimazu, Harumitsu Nagoya, Yuji Kato, Yasuo Ito, Mikiko Ninomiya, Kunio Shimazu
Department of Neurology, Saitama Medical School, Moroyama, Saitama, Japan
Background and aims: The purpose of this study was to measure platelet aggregability using a new method of analysis, whole blood aggregation (WBA), and to compare inhibitory effects of antiplatelet agents.
Methods: Subjects comprised 52 consecutive patients with non-cardioembolic stroke. Mean age was 66±15 (mean±SD) years (range, 53-88 years). Platelet function was measured using WBA-neo (ISK Co, Japan) after pharmacotherapy. Four concentrations of adenosinediphosphate (ADP) and collagen were used as aggregating agents. Participants were retrospectively divided into three groups, receiving aspirin (100mg/day, n=30), cilostazol (200mg/day, n=13) and clopidogrel (75mg/day, n=9). Differences in platelet aggregations among three groups were compared using thresholds showing 50 % of maximum aggregating rate as inhibitory parameters.
Results: ADP-induced platelet aggregation showed significantly (p<0.05) greater inhibition in clopidogrel group (6.8±2.2 micromol/L) than in aspirin and cilostazol group (4.2±2.4, 2.8±1.9; figure A). Collagen-induced platelet aggregation displayed significantly (p<0.05) stronger inhibition in aspirin group than in cilostaazol and clopidogrel group (figure B).
Conclusions: Clopidogrel reduced ADP-induced platelet aggregation (PA) specifically, and aspirin reduced collagen induced PA.
INCREASING DOSES OF RT-PA REDUCE MMP-3 ACTIVITY FOLLOWING EXPERIMENTAL FOCAL ISCHEMIA
Dorothe Burggraf1, Sebastian Schuhmacher1, Martin Dichgans1, Gerhard Hamann2
1Department of Neurology, Ludwig-Maximilians University, Klinikum Grosshadern, Munich, Germany, 2Department of Neurology, HSK Dr. Horst Schmidt Klinik, Wiesbaden, Germany
Background and aims: Focal cerebral ischemia results in gradual breakdown of the extracellular matrix. Matrix metalloproteinases (MMPs) especially MMP-9 are important in this process. Recombinant tissue plasminogen activator (rt-PA) results in up-regulation of MMP-9. Pro MMP-9 is activated through active MMP-3 (stromelysin), to active MMP-9. We therefore hypothesized that rt-PA increases the activation of MMP-9 by activating MMP-3. To convert the precursor protein pro-MMP-3 into the active form, a proteolytic cleavage and the cleavage of the pro-form to lower molecular weight active froms (45, 28, and 21 kDa) is essential. Methods: In this study we quantified MMP-3 expression following ischemia (3h)/reperfusion (24h) in a suture model in rats. We observed changes of this activation after the treatment of the animals with rt-PA in different doses. Each group (n=6) received either saline, 0.9, 9 or 18 mg/kg bw rt-PA at the end of the ischemic period. MMP-3 was determined by western blot of the ischemic and non ischemic basalganglia and cortex separately. Zymography was used to confirm the results.
Results: Active MMP-3 (45kDa form) was slightly increased in the ischemic region compared to the contralateral area (cortex: 120·7%; basalganglia: 109·9%; p<0.05 versus sham operated rats). In contrast, the 21 kDa degradation fragment was significantly (p<0.05) decreased. With rising rt-PA doses a decrease of active MMP-3 was shown in ischemic cortical and basal ganglia region respectively (0.9?9-18mg: cortex: 86 ·7%; 85·7%;76·14%, p<0.001; basal ganglia: 97·8%;82·9%;77·10%; p<0.001) joint to a steady increase of the degraded form. Zymographic analysis showed that the active form of MMP-9 is rising with increasing rt-PA doses.
Conclusions: Our results suggest that active MMP-3 was depleted during the 3h ischemia and 24 h reperfusion period. The amount of MMP-3 seems to be a limiting factor in the activation cascade of MMP-9. So active MMP-3 could be a target to inhibit the MMP-9 destructive upregulation following rt-PA treatment.
ENERGY METABOLISM OF LYMPHOCYTES FROM ACUTE STROKE PATIENTS
Roberto Federico Villa1, Antonella Gorini1, Diego Geroldi2, Guglielmo Pero3, Francesca Brera1
1Department of Physiological-Pharmacological Sciences, University of Pavia, Pavia, Italy, 2Department of Internal Medicine, University of Pavia, Pavia, Italy, 3Department of Neuroradiology, Niguarda Ca’ Granda Hospital, Milano, Italy
BACKGROUND AND AIMS - Systematic studies on energy metabolism of lymphocytes during ageing in male and female subjects are lacking, especially in patients with CNS Diseases. A study on cytoplasmic and mitochondrial enzymes of lymphocytes, as biological indicators of energy metabolism, was performed in healthy blood donors and in patients with acute stroke.
METHODS - The controls and patients were divided according to the: (a) sex, to study gender factor; (b) ages (from 20-30 to 60-70 up to 86 years groups) to study age factor; (c) patients with acute stroke (female: 31-91 years and male 53-86 years groups). On purified lymphocytes, the catalytic properties of the following enzymes were assayed: for glycolysis, hexokinase (HK), lactate dehydrogenase (LDH); for Krebs' cycle (TCA), citrate synthase (CS), malate dehydrogenase (MDH); for Electron Transfer Chain (ETC), NADH-cytochrome c reductase (Complex I, Complex I-III), succinate dehydrogenase (SDH, Complex II), cytochrome oxidase (COX, Complex IV); for glutamate and related amino acids metabolism, glutamate dehydrogenase (GlDH), glutamate-oxaloacetate- (GOT) and glutamate-pyruvate- transaminases (GPT); some enzyme activities were assayed also in presence of Triton X-100 (TX).
RESULTS - The enzyme activities of lymphocytes differ for gender, particularly during ageing (ANOVA test): HK increased (male); CS increased starting at 31 (male) and 51 (female) years, SDH at 51 (male) or 61 (female) years; ETC enzymes increased starting at 31 years, only in male. Amino acid linked enzymes were unaffected by age (male), only GPT increased at 31-50 years (female). In stroke male patients, LDH, CS activities increased, while COX, GlDH and GlDH-TX decreased; in female, HK, CS, MDH, Complex I, Complex I-III, GPT activities increased, while Complex IV, GlDH and GlDH-TX activities decreased and also proteins concentration.
CONCLUSIONS - This systematic study on functional proteomics of lymphocytes during physiological ageing, allows to elaborate a model to study the CNS Diseases (cerebral ischemia in this case) and, eventually, the drugs' actions. The most representative enzyme activities related to energy trasduction, were used as biological markers to monitor the energy metabolism on peripheral cells, reflecting that of the brain, as the literature suggests.
In control subjects, the cytosolic and mitochondrial enzyme activities are much more changed in their catalytic activities by ageing in female respect to male. Stroke strongly affected the energy-linked enzyme systems of lymphocytes, but differently for gender and age. Glycolysis, TCA, ETC and glutamate enzymes are particularly affected in female than in male, demonstrating that the glucose metabolism and ATP synthesis are primary affected in lymphocytes, suggesting the brain metabolic dysfunction. In fact, during the stroke, these results suggest an increased energy requirement that is not compensated by cytochrome oxidase activity, which decreased in male and female patients. In addition, in female patients, there is an increased metabolism of pyruvic acid via transamination reaction by GPT and a decreased capacity to metabolize glutamate (GlDH decrease), while in male patients there is an increased anaerobic metabolism (LDH increase). These metabolic modifications may play an important physiopathological role, according to the observed cerebral dysfunctions of patients with acute cerebral ischemia.
REGIONALLY INCREASED TEMPORAL LOBE EFFLUX OF R-[11C]VERAPAMIL OCCURS IN SOME PATIENTS WITH THERAPY REFRACTORY EPILEPSY
Martin Bauer1, Oliver Langer1,2, Alexander Hammers3,4, Rudolf Karch5, Ekaterina Pataraia6, Matthias J. Koepp4, Aiman Abrahim1, Gert Luurtsema7, Kurt Kletter8, Christoph Baumgartner6, Markus Muller1
1Division of Clinical Pharmacokinetics, Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria, 2Department of Radiopharmaceuticals, Austrian Research Centers, Seibersdorf, Austria, 3Division of Neuroscience, Faculty of Medicine, Imperial College, and MRC Clinical Sciences Centre, Hammersmith Hospital, London, UK, 4Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College London, London, UK, 5Department of Medical Computer Sciences, Medical University of Vienna, Vienna, Austria, 6Department of Neurology, Medical University of Vienna, Vienna, Austria, 7Department of Nuclear Medicine and PET Research, VU University Medical Center, Amsterdam, The Netherlands, 8Department of Nuclear Medicine, Medical University of Vienna, Vienna, Austria
Background and aims: R-[11C]verapamil, a radiolabelled substrate of the multidrug transporter P-glycoprotein (P-gp), has been developed as a probe for in vivo evaluation of P-gp function at the human blood-brain barrier with PET (Lubberink et al., 2006). Because regional overexpression of P-gp in epileptic brain tissue is discussed as a factor that contributes to pharmacoresistance in epilepsy, we studied brain R-[11C]verapamil distribution in 7 patients with drug-resistant unilateral therapy-refractory temporal lobe epilepsy.
Methods: After i.v. bolus injection of about 400 MBq of R-[11C]verapamil (specific activity: >20 GBq/µmol) dynamic brain PET imaging and arterial blood sampling were performed for 60 min. Volumes of interest (VOIs) were defined on MRI scans by using a frequency-based anatomical atlas (Hammers et al., 2003). R-[11C]verapamil kinetics were compared in homologous brain VOIs located ipsilateral and contralateral to the seizure focus. PET data were modeled by a previously described 1-tissue compartment model using a partially metabolite-corrected arterial input function (Lubberink et al., 2006).
Results: Among different VOIs, radioactivity was highest in the choroid plexus. The hippocampal VOI could not be used for data analysis because it was contaminated by spill-in of radioactivity from the adjacent choroid plexus. In several other temporal lobe regions that are known to be involved in seizure generation and propagation ipsilateral influx rate constants K1 and efflux rate constants k2 of R-[11C]verapamil were increased as compared to the contralateral side. Parameter asymmetries were most prominent in parahippocampal and ambient gyrus (K1, range among patients: −3.8% to +22.3%; k2, range: −2.3% to +43.9%), amygdala (K1, range: −20.6% to +31.3%; k2, range: −18.0% to +38.9%) and medial anterior temporal lobe (K1, range: −8.3% to +14.5%; k2, range: −14.5% to +31.0%). In contrast to temporal lobe VOIs, asymmetries were minimal in a region not involved in epileptogenesis located outside the temporal lobe (superior parietal gyrus, K1, range: −3.7% to +4.5%; k2, range: −4.2% to +5.8%). In five of seven patients, ipsilateral efflux (k2) increases were more pronounced than ipsilateral influx (K1) increases, which resulted in prominent ipsilateral reductions (10–26%) of R-[11C]verapamil distribution volumes.
Conclusion: Our pilot data suggest that regionally enhanced P-gp activity might contribute to pharmacoresistance in some patients with temporal lobe epilepsy.
THE USE OF OXYGEN AS A CONTRAST AGENT AND METABOLIC BIOTRACER IN ACUTE STROKE PATIENTS
Krishna Dani1, Keith Muir1, David Brennan2, I. Mhairi Macrae1, Barrie Condon2, Celestine Santosh3
1University of Glasgow, Glasgow, Scotland, UK, 2Department of Clinical Physics, Institute of Neurological Sciences, Glasgow, Scotland, UK, 3Department of Neuroradiology, Institute of Neurological Sciences, Glasgow, Scotland, UK
Introduction
Blood Oxygen Level Dependent Magnetic Resonance Imaging (BOLD MRI) exploits the properties of oxyhaemoglobin and deoxyhaemoglobin. Deoxyhaemoglobin is paramagnetic therefore reduces T2* value in the water molecules of tissue. Oxygen dissolved in plasma has a similar effect. However, oxygen can also combine with deoxyhaemoglobin, which alters signal, since oxyhaemoglobin is diamagnetic. Therefore oxygen may be used both as a contrast agent and metabolic biotracer.
Methods
We recruited 8 patients with cortical stroke. 6 patients had a Total Anterior Circulation Infarct, 1 patient had a Partial Anterior Circulation Syndrome and 1 patient had a Posterior Circulation Syndrome. Mean age was 64 (s.d. 8.2, range 52-76)), median NIHSS 9 (i.q. range 6-17). Median time from onset was 20h (i.q. range 18.5–24..5).
A 3T MRI scanner (GE) was used with an EPI sequence sensitive to T2*. Oxygen (15l/min) was administered via a face mask; vector of onsets 180s, duration 300s. Statistical colour maps were produced using SPM and MRIcro software.
Results and Discussion
In all cases there was a positive response to oxygen challenge in normal brain grey matter. In areas of restricted diffusion we saw either predominantly negative responses (n=3) or no response to oxygen challenge (n=2). There were, however, small areas of positive response to oxygen challenge within areas of restricted diffusion (Figure 1). In two patients with small stroke current resolution of the scan was insufficient to allow assessment. In two patients microhaemorrhages were seen to alter tissue paramagnetism and scans excluded.
In normal and metabolising tissue where there is generation of deoxyhaemoglobin, oxygen given as a ‘challenge’ will combine with deoxyhaemoglobin to form oxyhaemoglobin, thus increasing the signal from baseline. In poorly metabolising tissues where there is a limited or reduced generation of deoxyhaemoglobin there will be an excess of free oxygen thereby reducing the signal from baseline. This explains a reduced signal in the ‘infarct core’ and an increased signal in normal areas, after oxygen challenge. Areas within regions of restricted diffusion showing a positive response to oxygen challenge may represent potentially viable tissue.
We believe that this technique may help stratification of metabolism in different tissues in acute stroke. We conclude that oxygen can be used as a contrast agent and a metabolic biotracer.
Funding
This work was supported by The Neurosciences Foundation, Glasgow.
Figure 1.
DWI scan and corresponding slice from oxygen challenge in a patient scanned at 18h30m. This patient was one of three whose T2* signal within areas of restricted diffusion was predominantly reduced with oxygen challenge (Red = positive signal change. Blue = negative signal change.)
COMPARISON BETWEEN CEREBRAL OXYGEN METABOLISM AND C-11 FLUMAZENIL PET IN THE PATIENTS WITH TRAUMATIC BRAIN INJURY
Tohru Shiga1, Katsunori Ikoma3, Chietsugu Katoh4, Hirotaka Isoyama3, Yuji Kuge1, Tomoya Kohno2, Satoshi Terae5, Nagara Tamaki2
1Department of Molecular Imaging, Hokkaido University, Sapporo, Japan, 2Department of Nuclear Medicine, Hokkaido University, Sapporo, Japan, 3Department of Rehabilitation, Hokkaido University, Sapporo, Japan, 4Department of Health Science, Hokkaido University, Sapporo, Japan, 5Department of Radiology, Hokkaido University, Sapporo, Japan
Objective
The traumatic brain injury causes brain dysfunction in many cases. Some patients have moderate - severe brain dysfunction despite with slight or no abnormality in MRI. The purpose of this study is to investigate whether the loss of neuronal integrity is cause of brain dysfunction in the patients with traumatic brain injury using C-11 flumazenil PET (FMZ-PET) comparing with O-15 gas PET.
Method
This study included twenty-one patients with traumatic brain injury, posttraumatic brain dysfunction. All of the studies were examined at least six month later after injury.
The dynamic flumazenil PET and O-15 labeled CO, CO2, O2 gas PET was studied in all patients. The binding potential of flumazenil (BP), cerebral blood volume (CBV), the cerebral blood flow (CBF), the oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2) were calculated. Axial T1WI, T2WI, FLAIR and T2*WI images were obtained with 1.5 tesla scanner. Coronal images were added in some cases.
The BP and CMRO2 images were visually interpreted by at least two experienced nuclear physicians by consensus. The lesions were determined as positive if a definite localized area of decreased distribution with respect to the surrounding normal tissue was present except for physiological distribution.
Results
Eleven out of twenty two patients had normal T1 and T2 MRI. Eight patients showed the brain contusion and two showed diffuse axonal injury in MRI. All of them showed abnormal finding in CMRO2 image. Hypometabolism was showed in the temporal lobe, the frontal lobe, the parietal lobe and the occipital lobe in fifteen, ten, one and one patient respectively. Five patients showed hypo metabolism in the bilateral cerebral cortex. Fifteen, four and two patients showed decreased uptake of FMZ-PET in the temporal lobe, frontal lobe and the occipital lobe. There was no lesion that showed low uptake in FMZ-PET without hypometabolism in CMRO2.
Conclusion
These results indicated that the loss of the neuronal integrity was one of the causes of the brain dysfunction in patients with traumatic brain injury. The finding of C-11 FMZ is useful to distinguish hypometabolism caused by neuronal loss from functional hypometabolism.
NMDA RECEPTOR ANTAGONIST SM-31900 PROTECTS AGAINST DELAYED NEURONAL DEATH AND REDUCES NEUROGENESIS INDUCED BY TRANSIENT GLOBAL ISCHEMIA IN RATS
Taku Sugawara1, Suguru Yamaguchi1, Masaya Oda1, Tomoya Omae1, Kazuo Mizoi1, Hiroyuki
Kinouchi2
1Department of Neurosurgery, Akita University School of Medicine, Akita, Japan, 2Department of Neurosurgery, University of Yamanashi, Faculty of Medicine, Kofu, Japan
The N-methyl-D-aspartate receptor (NMDAR) is known to play an important role in ischemic neuronal injury in the hippocampus, and also implicated in modulation of neurogenesis in the hippocampal dentate gyrus (DG). Recent evidence confirmed that amino acid neurotransmitter glycine acts as a coagonist in excitatory neurotransmission at NMDAR. The aim of this study is to investigate the role of glycine in ischemic injury and neurogenesis in the hippocampus in the rat global ischemia model. Global ischemia was induced with or without the treatment with NMDAR antagonist or NMDAR glycine site blocker. Neuronal injury was morphologically examined and neurogenesis was assessed by bromodeoxyuridine uptake. Global ischemia induced severe injury in the hippocampal CA1 neurons, but pharmacological antagonism of NMDAR or glycine site ameliorated the injury. Global ischemia enhanced neurogenesis in DG, but these pharmacological interventions resulted in attenuation of neurogenesis. These results suggest that glycine plays an important role in NMDAR-mediated neuronal injury and regulation of neurogenesis in the hippocampus.
NITRIC OXIDE PRODUCTION IN RAT BRAIN OF DIABETES MELLITUS TYPE 1 AND 2 MODELS DURING FOREBRAIN ISCHEMIA AND REPERFUSION
Tomokazu Shimazu, N. Araki, T. Ohkubo, Y. Asano, D. Furuya, H. Nagoya, Y. Ito, Y. Kato, M. Ninomiya, K. Shimazu
Division of Neurology, Deperment of Internal Medicine, Saitama Medical University, Saitama, Japan
Background and Aims: Experimental and clinical studies suggest that oxidative stress plays an important role in pathogenesis of diabetes mellitus type 1 and 2. Hyperglycemia leads to free radical generation and causes neuronal injury. The purpose was to investigate the nitric oxide (NO) production in rat brain of diabetes mellitus type 1 and 2 models during forebrain ischemia and reperfusion.
Methods: Wistar rats were divided into two groups: control (n=7) and type 1 diabetic group (n=7) which was induced by injection of streptozotocin (STZ) (40 mg/kg). Otsuka Long-Evans Tokushima Fatty (OLETF; n=6) and Long-Evans Tokushima Otsuka (LETO: n=5) rats were used as type 2 diabetic and control groups. The animals were anesthetized by pentobarbital sodium (i.p.). NO production was continuously monitored by in vivo microdialysis. Microdialysis probes were inserted into the left striatum and hippocampus. The probes were perfused with Ringer's solution at a constant rate of 2 µl/min. Laser Doppler probes were inserted into the right striatum and hippocampus. After a 2-hours equilibrium period, fractions were collected every 10min forebrain cerebral ischemia was induced by occlusion of both common carotid arteries, and systemic hypotension (MABP<50mmHg) which was induced by hemorrhage. After 20min, the loops around both common carotid arteries were released and the blood was reinfused. Levels of NO metabolites, nitrite and nitrate, in the dialysate were determined using the Griess reaction.
Results: (1) Cerebral Blood Flow (CBF): In all groups, CBF decreased rapidly after occlusion and systemic hypotension, and rapidly increased after reperfusion. There were no significant differences in CBF between the each two groups. (2) NO production during ischemia and reperfusion: during ischemia the changes in total NO (sum of nitrite and nitrate) in striatum in STZ group (91.4±4.7%; mean±SE, 63.7±2.0% of the baseline level) were significantly higher than those of the control group (64.4±6.8%, 48.6±6.5% of the baseline level) (p<0.05) (Fig.1.). There were no significant differences in hippocampus. In the OLETF groups, no significant differences were obtained in striatum. NO production during 10min after reperfusion the change in total NO in hippocampus of OLETF group was higher than that of LETO group (p<0.1) (Fig.2).
Conclusion: These data indicate that NO may be an important mediator in the exacerbation of post-ischemic brain injury in the striatum in STZ rat, and in the hippocampus in OLETF rat.
INCREASED BASELINE K+ DOES NOT CONTRIBUTE TO CORTICAL SPREADING DEPOLARIZATION (CSD)-INDUCED MICROVASCULAR SPASM VIA INCREASED PEAK K+ DURING CSD
Mesbah Alam, Sebastian Major, Jens P. Dreier
Department of Neurology and Experimental Neurology, Charite Hospital, Berlin, Germany
Introduction: It has been proposed that CSD-induced microvascular spasm (cortical spreading ischemia, CSI) is a mechanism of delayed ischemic neurological deficits after subarachnoid hemorrhage (Dreier et al., 1998; 2000; 2006). CSD/CSI is, e.g., induced by brain topical application of artificial cerebrospinal fluid (ACSF) containing increased K+ combined with either the NO scavenger hemoglobin or the NO synthase inhibitor N-nitro-L-arginine (L-NNA). The role of artificially increased baseline extracellular K+ concentration ([K+]o) for CSD/CSI has remained unclear. [K+]o rises to ~50mM during CSD and acts as a vasoconstrictor in concentrations above 20mM. Therefore, if artificial increase of baseline [K+]o caused increased peak [K+]o during CSD, it would provide a simple explanation how increased baseline [K+]o contributes to CSD-induced microvascular spasm. In the present study, we compared peak [K+]o between CSD/cortical spreading hyperemia (CSH) under physiological ACSF and CSD/CSI under L-NNA (1mM) with increased K+ (35mM).
Methods: An open cranial window was implanted in 26 rats under thiopental. Cerebral blood flow (CBF) was measured with laser-Doppler flowmetry, and the subarachnoid direct current (DC)-potential (DCsa) with an AgCl electrode. Intracortical DC-potential (DCic) and [K+]o were recorded with ion-sensitive microelectrodes (cortical depth, 100 µm).
Results: CSD/CSI was associated with a negative shift of DCsa (−4.0±1.8mV) and DCic (−19.7±1.9mV) lasting for 5.8±3.9min and 6.2±3.6min, respectively. Immediately before CSD/CSI, CBF was 94±27% of baseline (= 100%) caudally and 125±74% rostrally. CSI consisted of a sharp CBF decrease which spread between caudal and rostral laser probes with a latency of 21.1±13.3s. The minimal CBF value was 21±11% lasting for 4.4±4.2min. Immediately before CSD/CSI, [K+]o was 9.3±3.7mM and rose to 49.5±8.0mM during CSD/CSI. The negative shift of DCsa started 11.2±14.4s before the sharp CBF decrease in CSD/CSI whereas, after cardiac arrest, a simultaneous sharp CBF decrease at both laser probes preceded DCsa by 41.0±22.2s. CSD/CSH was associated with a negative shift of DCsa (?1.6±0.8mV) and DCic (−18.0±3.0mV) lasting for 1.6±0.8min and 1.6±0.5min, respectively. Immediately before CSD/CSH, CBF was 110±13% of baseline (=100%) caudally and 105±19% rostrally. CSH consisted of a rise in CBF to 285±65% spreading with a latency of 47.8±28.5s and lasting for 1.9±0.4min. Immediately before CSD/CSI [K+]o was 2.5±0.8mM and rose to 46.3±13.1mM during CSD/CSH. The negative shift of DCsa started 15.7±23.9s before the CBF rise in CSD/CSH whereas, after cardiac arrest, a simultaneous sharp CBF decrease at both laser probes preceded DCsa by 41.0±22.2s.
Conclusion: In contrast to intracortical baseline [K+]o before CSD, no significant difference of peak [K+]o was found between CSD/CSH and CSD/CSI. In conclusion, rise of baseline [K+]o before CSD/CSI does not contribute to CSD-induced microvascular spasm via increase of peak [K+]o during CSD.
BLOOD FLOW DEPENDENT CHARACTERISTICS OF OPTICAL INTRINSIC SIGNALS DURING CORTICAL SPREADING DEPRESSION IN RATS
Zhen Wang, Pengcheng Li, Weihua Luo, Qingming Luo
Wuhan National Laboratory for Optoelectronics, Britton Chance Center for Biomedical Photonics, Huazhong University of Science and Techonlogy, Wuhan, China
This study was aimed to investigate the potential influence of the residual cerebral blood flow (rCBF) on the spatiotemporal characteristics of optical intrinsic signals (OIS) induced by spreading depression (SD) in local ischemic rat cortex. For this purpose, SD waves were induced by K+ on rat cortex with small cerebral ischemia focal, and residual CBF and IOS were accessed by combining laser speckle flowmetry with optical intrinsic signal imaging. Adult male Sprague-Dawley rats (n=10) weighing 200 to 300 g were anesthetized with α-chloralose/urethane and the parietal-temporal cortex was exposed through craniectomy. Small branches of middle cerebral artery which serve targeted cortex were carefully ligated by passing short pieces of 11-0 suture through the dura and under the vessels to cause a small local cerebral ischemia (around 3 mm in diameter). A second cranial window located in the frontal bone was made for the induction of SD. For the CBF measurement using laser speckle flowmetry, a He-Ne laser (632.8nm, 17mW, Melles Griot, U.S.A) was used to illuminate the cortex, and a fixed wavelength (550±10 nm) light source was used for illumination when performing optical intrinsic signal imaging. Both the two techniques employed the same optics (Microscope SZX12, Olympus, Japan) and CCD camera (12-bit, 480×640 pixel array, Pixelfly VGA, Germany) for image acquisition. Six hours after ministroke, SD was induced by applying 3M KCl (ca. 50µl) into the cranial window located at the frontal cortex.
In the experiments, four kinds of temporal pattern of changes in OIS are observed at the cortical locations with different level of rCBF perfusion. At rCBF ranging from 40?60% of baseline, OIS expressed more significant initial rise in light reflectance than that observed at the cortical region with rCBF greater than 60% of baseline. More interestingly, for the cortical region where rCBF dropped to 30–40% of baseline, the duration of changes in OIS caused by SD was significantly prolonged and the number of SD waves decreased. For the region with rCBF below 30% of baseline, there were no SD occurred and also no significant changes in optical reflectance observed. Our experimental results indicate a tight coupling between the characteristics of OIS and CBF levels during cortical spreading depression. This new finding suggests that OIS induced by SD might be used as a reflection of blood flow levels to some extent.
Keywords: cerebral blood flow, spreading depression, optical intrinsic signals, laser speckle imaging
DELAYED NEURONAL CELL DEATH IN THE BRAINSTEM AFTER TRANSIENT ISCHEMIA IN GERBILS
Fang Cao, Ryuji Hata, Pengxiang Zhu, Masahiro Sakanaka
Department of Functional Histology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
Background and aims
In the central nervous system, certain areas are selectively damaged even by a brief ischemic insult, and this topographical heterogeneity is known as “selective vulnerability”. The mechanism responsible for this vulnerability is of particular importance to establish therapeutic procedures, because the same approach may help to ameliorate ischemic damage. Since little is known about selective vulnerability in the brainstem, we investigated selective vulnerability in the brainstem after transient brainstem ischemia.
Methods
Transient brainstem ischemia was induced in gerbils by occlusion of the bilateral vertebral arteries for 15 min. The animals were divided into the three groups subjected to brainstem ischemia and reperfused for 0, 1d, and 3d (n=4 in each group). After deep anesthesia, the gerbils were perfused with 4%paraformaldehyde. Their brains were dissected out and embedded in paraffin. Serial 5-µm coronal sections (including lateral vestibular nucleus that has been reported to be most vulnerable during ischemia [Neuroscience 56, 423-439, 1993]) were cut and immunostained with antibodies against MAP2 and Iba1.
Results
As shown in Fig.A, immunostaining for MAP2 revealed that ischemic lesions were detected in the lateral vestibular nucleus (LVe) and the ventral part of the spinal trigeminal nucleus (Sp5) in all four animals. One day after reperfusion, however, these ischemic lesions disappeared (Fig.B). Three days after reperfusion, ischemic lesions reappeared again in these two areas in all animals and new ischemic lesions were detected in the dorsal part of Sp5 and ventral cochlear nucleus (VC) in two animals (50%). Furthermore, immunostaining for Iba1 revealed that clusters of ameboid microglia/macrophage were detected in the same areas where MAP2 staining was lost at 3 days after reperfusion (Figs. D-E).
Conclusions
Our results suggest that delayed progression of ischemic neural death takes place in the brainstem as well as forebrain. Furthermore, our results also suggest that activated microglia/macrophage play a crucial role in this delayed neuronal cell death in the brainstem.
INFLUENCE OF ENOS-GLU298ASP-POLYMORPHISM ON L-ARGININE INDUCED CEREBRAL BLOOD FLOW REACTIVITY
Karen Gertz1, Peter Brunecker1, Jorg Schultze1, Stephan Schreiber1, Florian Doepp1, Jens Steinbrink1, Constanze Jung2, Karl Stangl2, Christoph Naber3, Matthias Endres1
1Department of Neurology, Charite Universitatsmedizin Berlin, Berlin, Germany, 2Department of Cardiology, Charite Universitatsmedizin Berlin, Berlin, Germany, 3Department of Cardiology, University Essen, Essen, Germany
Background: Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a key role in vascular regulation and is an important target for stroke protection. Epidemiological evidence links DNA variants in the eNOS gene to increased vascular risk in humans. Here, we studied the impact of the Glu298Asp (G894T) polymorphism which generates a protein with different susceptibility to cleavage and impaired enzymatic activity on cerebral blood flow (CBF).
Methods: The study was approved by institutional review boards and all participants gave informed consent. The study population consisted of 20 healthy volunteers which were matched according to Glu298Asp polymorphism determined by polymerase chain reaction (PCR): G/G (homozygous “normal” alleles), G/T (heterozygous) and T/T (homozygous polymorphism) genotype (5, 9 and 6 individuals, respectively). CBF was stimulated via infusion of the NOS substrate L-Arginine (500mg/kg body weight). Immediately before and after the infusion T2*-weighted dynamic susceptibility contrast (DSC) MRI was performed. CBF, cerebral blood volume (CBV), and mean transit time (MTT) were voxelwisely estimated by a common deconvolution analysis. Maps of time to peak (TTP) were calculated from the bolus data including the peripheral transport.
Results: To detect small changes in cerebral perfusion, we optimized the post-processing of DSC-MRI using a novel correction algorithm, which accounts for saturation effects in the arterial input curve (AIC) reducing variations in the estimated parameters (Brunecker et. al., ISMRM 2003). As a result of this analysis maps of tissue arrival time (TAT) as the first moment of an analytical tissue response function were determined. All above mentioned parameters were measured in different regions of interest (ROIs) including white matter, foramen semiovale, thalamus, caudatum and over a whole transversal slice.
TTP was significantly reduced after infusion with L-Arginine in individuals with G/G genotype in all measured ROIs (1,23; 1,42; 1,69; 2,12; 2,58 sec, respectively), but was not shortened in individuals with G/T and T/T alleles. Furthermore we found that in individuals with G/G or G/T genotypes TAT was significantly reduced after L-Arginine over the whole slice (0,24 and 0,21 sec, respectively) and in foramen semiovale (0,23 and 0,44 sec, respectively). In contrast, in individuals with T/T genotype L-Arginine had no significant effect on TAT.
Conclusion: Our study has the following major findings. 1) Perfusion changes in the brain induced by L-Arginine can be measured by DSC-MRI. 2) The effect of L-Arginine on different blood flow parameters measured by DSC-MRI is more pronounced in TTP and TAT as compared to MTT and CBF. Whereas TTP is more related to peripheral flow TAT reflects cerebral flow changes within the arterial vascular bed. In contrast, there was no apparent effect on CBF, which represents blood flow over the whole vessel supply from arterioles to venules. 3) Importantly, in individuals homozygous for the Glu298Asp polymorphism the blood flow response to L-Arginine was blunted or absent (ie, no reduction of TTP and TAT). In conclusion, our findings show reduced endothelial function in patients homozygous for Glu298Asp polymorphism.
MITOCHONDRIAL OXYGENATION IN THE RAT CEREBRAL CORTEX
Roger Springett, Michelle Abajian, Maureen Ripple
Department of Radiology, Dartmouth Medical School, Hanover, NH, USA
Introduction: The oxygen limitation model (OLM) is the prevailing model to explain why increases in cerebral blood flow (CBF) are much greater than increases in cerebral metabolic rate for oxygen (CMRO2) during functional activation in the brain. Within the framework of the OLM, oxygen diffuses from the capillary bead to the mitochondria where it is consumed. The rate of oxygen diffusion (DO2), which is equal to CMRO2, is given by:
DO2 = CMRO2 = L(PcO2-PmO2)
where PcO2 and PmO2 are the mean capillary bed and mitochondrial oxygen tension respectively and L is the effective permeability constant.
A fundamental assumption of the OLM is that PmO2 is zero, or near zero. Under these conditions, large increases in CBF are necessary to raise PcO2 and increase the oxygen gradient between capillary bed and mitochondria to support a modest increase in CMRO2. The major challenge in testing this model is that there are no methods of measuring PmO2.
We have developed and validated a visible spectroscopy system that can measure the oxidation state of mitochondrial cytochrome c (Cytc), which is sensitive to PmO2, as well as the absolute hemoglobin concentration and hemoglobin saturation (SmcO2) from the rat cerebral cortex using a thinned skull preparation. We have integrated this system with laser Doppler flowmetry and, using SmcO2 as a surrogate of local venous saturation, we are able to calculate CMRO2. The goal of this study is to use this system critically test the OLM.
Methods: Cortical Spreading depression (CSD) is a wave of depolarization that propagates across the cortex at a rate of 2-4mm/minute. At the wave front there is a large increase in CMRO2, presumably to generate ATP to restore membrane polarity, which drives PmO2 to low levels and results in a reduction of Cytc. Cytc cannot be used to measure PmO2 directly because the relationship between PmO2 and Cytc is not precisely known. Instead, we approximate PmO2 as zero at the maximum reduction in Cytc and use PcO2, derived from SmcO2 using the rat hemoglobin disassociation curve, to calculate L. Once L is known, baseline PmO2 can be calculated from the baseline PcO2 and CMRO2.
Sprague Dawley rats were anesthetized with isoflurane, the skull thinned for optical access and the animals artificially ventilated. CSD was induced by applying saturated KCL to the dura close to the measurement site.
Results: Baseline SmcO2 was 73.7±3.1% (mean±SD, n=6) giving a PcO2 of 54±3mmHg. CSD increased CMRO2 and CBF by 75±18% and 8±19% respectively, decreased SmcO2 and PcO2 to 59.7±3.0% and 42.8±2.0mmHg respectively and reduced Cytc by 29.6±8.9%. Assuming PmO2 was zero at this point results in a value for L of 0.260±0.028 mmHg/%CMRO2 and a baseline PmO2 of 28.4±4.6mmHg.
Discussion: The calculated value of PmO2 is not consistent with the fundamental assumption of the OLM. Furthermore, we observe an increase in CMRO2 of 75% at almost constant CBF in contradiction to the OLM. We conclude that the OLM cannot explain why CBF is always much greater than CMRO2 in functional activation studies.
CEREBRAL HEMODYNAMICS AND OXYGEN METABOLISM IN PATIENTS WITH MOYAMOYA VESSELS ASSOCIATED WITH ATHEROSCLEROTIC STENO-OCCLUSIVE ARTERIES
Hiroki Kato1, Eku Shimosegawa1, Yasuyuki Kimura1, Katsufumi Kajimoto1, Makiko Tanaka1, Kazuo Kitagawa2, Jun Hatazawa1
1Department of Nuclear Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine, Osaka, Japan, 2Department of Internal Medicine, Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
Background and aims
Moyamoya disease is characterized by progressive steno-occlusive lesions of internal carotid artery (ICA), middle cerebral artery (MCA), or anterior cerebral artery (ACA) associated with moyamoya vessels. The development of basal moyamoya vessels was reported to be a sign of severe hemodynamic impairment in adult patients with ischemic moyamoya disease [1]. On the other hand, we often encounter the patients with intracranial atherosclerotic steno-occlusive lesions demonstrating secondary collateral networks (atherosclerotic moyamoya vessels, AMVs). Although the angiographic appearance was similar, hemodynamic and metabolic back ground of the AMVs has not been revealed yet. The aim of the present report was to evaluate the hemodynamic and metabolic status in relation to the development of the AMVs.
Methods
Twenty-one patients in chronic phase (64 +/− 8.5 yo) (male/female: 14/7) had unilateral atherosclerotic steno-occlusive lesions in ICA or MCA. They underwent O-15 gas PET to measure cerebral blood flow (CBF), cerebral metabolic rate of oxygen (CMRO2), oxygen extraction fraction (OEF), and cerebral blood volume (CBV). Measurement of these parameters was executed in the ipsilateral MCA area by automated constant ROI analysis software FineSRT [2]. The angiography was read by an experienced neuroradiologist without any clinical information. Based on the angiographic findings of collateral vessels, the patients were classified into two groups; i.e. AMVs group or no-AMV group. Seven patients were classified into AMVs group (67 +/− 5.3 yo, male/female:4/3), and 14 patients into no-AMV group(63 +/− 9.6 yo, male/female:10/4).
Results
There was no significant difference in CBF and CMRO2 in the ipsilateral MCA area between the two groups. OEF and CBV in the ipsilateral MCA area except for basal ganglia were significantly higher in the AMVs group than in the no-AMV group (P<0.001, P=0.006). In the ipsilateral basal ganglia, although CBV was significantly higher in the AMVs group than in the no-AMV group (P=0.012), no significant difference in CBV was detected between them.
Conclusions
The presence of AMVs in the angiography suggests severer hemodynamic failure in patients with atherosclerotic steno-occlusive lesions.
EFFECTS OF GROUP II METABOTROPIC GLUTAMATE RECEPTOR ACTIVATION ON NEUROPROTECTION AND EXTRACELLULAR GLUTAMATE CONCENTRATION AFTER TRANSIENT FOREBRAIN ISCHEMIA
Hideyuki Yoshioka, Masao Sugita, Takashi Yagi, Toru Horikoshi, Hiroyuki Kinouchi
Department of Neurosurgery, Faculty of Medicine, University of Yamanashi, Chuou, Yamanashi, Japan
BACK GROUND AND PURPOSE
The activation of group II metabotropic glutamate receptors (mGluR) inhibits the excessive release of glutamate from the presynaptic terminals. To clarify this neuroprotective effect on cerebral ischemic injury, we investigated whether group II mGluR agonist (DCG-IV) can attenuate neuronal degeneration and extracellular glutamate concentration ([Glu]e) in a rat model of transient forebrain ischemia.
METHODS
Adult male SD rats were anesthetized with halothane in nitrous oxide / oxygen, and transient forebrain ischemia was induced by bilateral carotid artery occlusion with blood-extracted hypotension (40-45 mmHg) for 5 min. DCG-IV was administered intraventricularly 36, 24, 12, 0hr before ischemia and animals were divided into 4 groups according to the single dosage of DCG-IV; artificial CSF (aCSF) (n=6), 10 pmol (n=7), 100 pmol (n=8), 250 pmol (n=8). Seven animals treated with 250 pmol of DCG-IV were also received intraperioneal injections of LY341495 (10mg / kg), competitive selective antagonist of group II mGluR, 1hr before ischemia. The survival rate of hippocampal CA1 neurons was evaluated by cresyl- violet staining 5 days after ischemia. [Glu]e in the CA1 subfield was measured by a microdialysis biosensor from the onset of ischemia to 15min after reperfusion in the aCSF and 250 pmol group. The survival rate was statistically analyzed by Kruskal- Wallis test, and microdialysis data were analyzed by 2 way repeated measures analysis of variance. Values of P<0.05 were considered as significant.
RESULTS
The survival rate of CA1 neurons in the aCSF group was 8.5%±1.6% (mean ± SEM), while those in the DCG-IV treated groups with 10, 100 and 250pmol of single dosage were 7.2%±0.9%, 16.2%±2.6% and 33.6%±4.2% (P<0.05), respectively. DCG-IV treatment improved neuronal survival in the concentration depending manner. The co-injection of LY3414195 abolished this neuronal protective effect of DCG-IV, and the survival rate was significantly reduced to 20.7±1.8% (P<0.05).
[Glu]e increased steeply after ischemia and reached the maximum at 1 min after the reperfusion, then gradually reduced to the base line (Fig.1). Average of maximum [Glu]e was significantly attenuated in the DCG-IV 250pmol group (51.5±15.7µM) than in the control group (137.7±26.1 µM, P<0.05).
CONCLUSION
The present study clearly demonstrated that DCG-IV has the neuroprotective effect against ischemic neuronal injuries by the inhibition of the excessive release of glutamate through the activation of group II mGluR.
INHIBITION OF THE PI3K/AKT PATHWAY ABOLISHES THE PROTECTIVE EFFECT OF POSTCONDITIONING AGAINST FOCAL ISCHEMIA IN RAT
Xuwen Gao1, Hanfeng Zhang1, Heng Zhao1,2
1Department of Neurosurgery, Stanford University, Stanford, CA, USA, 2Stanford Stroke Center, Stanford University, Stanford, CA, USA
Backgrounds and Aims: We recently demonstrated that postconditioning with a series of mechanical interruptions of reperfusion significantly reduces ischemic damage 2 days after stroke. We extend these studies by asking if postconditioning improves neurological function and whether postconditioning has a long-term protective effect. In addition, we addressed whether the PI3K/Akt signaling pathway contributes to the protection afforded by postconditioning. The PI3K/Akt pathway plays a critical role in neuronal survival after stroke and is regulated by a cascade of phosphorylation events. PTEN is a negative regulator of the Akt pathway, and phosphorylation of PTEN blocks its pro-apoptotic effect. Upon activation, Akt phosphorylates GSK3β and downregulates its activity. Phosphorylation of β-catenin by GSK3β leads to degradation of β-catenin and apoptosis. By examining the activation state of the Akt pathway and modifying its activity during stroke with or without postconditioning, we found that postconditioning reduces infarction partially through the Akt pathway.
Methods: Focal ischemia was generated by permanent distal MCA occlusion plus 30 min of transient bilateral CCA occlusion in male SD rats. Ischemic postconditioning was performed by occluding CCAs for 10 seconds and releasing for 30 seconds for total of three cycles starting from 30 seconds after reperfusion. Behavioral tests using three standard methods were performed from 2 to 30 days after stroke, and ischemic damage was evaluated at 30 days. For Western blots, ischemic brains were harvested 1, 5 and 24 h after stroke. Whole cell homogenates from the ischemic core and penumbra were prepared for Western blots. Antibodies detecting Akt phosphorylated on Ser473 (P-Akt), P-PDK1, P-PTEN, pan-β-catenin and activated (non-phosphorylated on Ser37 and Thr 41) β-catenin were used. A PI3K/Akt inhibitor, LY 294002, was injected before ischemic onset to test the effect of inhibiting the PI3K/Akt in postconditioning. In vitro Akt kinase assays were also performed.
Results: We found that postconditioning not only improves neurological functions, but also reduces ischemic tissue loss up to 30 days after stroke. P-Akt (active Akt) increased from 1 to 24h in the ischemic penumbra, (P<0.05, n=5-7, Two way NOVA). In the ischemic core, P-Akt slightly decreased at 1h but increased at 24h. Postconditioning enhanced levels of P-Akt in both the penumbra and core (P<0.01). Akt kinase assays directly showed that postconditioning attenuated the decrease in Akt activity (P<0.05). Treatment with LY 294002 significantly enlarged infarct size in ischemic rat brains treated with postconditioning (P<0.05, n=7-8). The levels of P-PTEN (inactive PTEN) significantly decreased after stroke with or without postconditioning. Cleaved β-catenin was detected as early as 1h and the levels of cleavage products further increased at 24h in both the penumbra and core. Postconditioning attenuated β-catenin cleavage at 24h in the penumbra but not in the core. Changes in cleaved active β-catenin were similar compared with total β-catenin.
Conclusion: Postconditioning has a long-term protective effect on neurological function and the Akt survival pathway contributes to the neuroprotective effect of postconditioning.
INTEGRATING COMPREHENSIVE AND ALTERNATIVE MEDICINE INTO STROKE: COMBINATION OF CHINESE HERBS ATTENUATED DAMAGE AND ACCELERATED SELF-REPAIR FOLLOWING FOCAL CEREBRAL ISCHEMIA
Baowan Lin1, Weizhao Zhao1, Guillermo Fernandez1, Xuming Ji2, Lin Li1, Raul Busto1
1Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA, 2Department of Neurosurgery, Xuanwu Hospital, Beijing, China
Background: Bedside herbal therapy has been using in stroke patients in China for generations. The present study used a specific combination of 9 herbs including Huang Qin (Secutellaria baicalensis) in animal experiment to investigate if and how it works. The therapeutic strategies are encouraging blood circulation and self-healing, inhibiting inflammation and scavenging free oxygen radicals.
Methods: Cerebral infarction was produced in overnight-fasted Male Sprague-Dawley rats (280-315g) by 2-h intraluminal occlusion in middle cerebral artery (MCA) with a suture. Head temperature was maintained at 36-36.5oC and body temperature at 37-37.5oC. All rats received behavioral test. Rats with full neurological deficit scores ranging10 −12 at 2h after MCAo participated in the study. Rats survived for 3 or 28 days before histopathological analysis. Treated rats had the herb cocktail-style (HC) soup, which was initiated at 2 hrs after reperfusion and daily administrated by gavage. Nontreated rats received drink water (DW).
Results
Infarct sizes were 194 ± 70 (DW, n=10) vs. 91 ± 69 mm3 (HC, n=9) on day 3; 101 ± 51 (DW, n=9) vs. 38 ± 22 mm3 (HC, n=11) on day 28. Infarct volumes decreased by 53 ? 62 % in HC groups (mean ± S.D.; ANOVA, p?0.01). Protection was in penumbra in cortex. On day 28, 8 of 9 DW rats showed cavitation; 3 of 11 HC rats had cavities. Prominent and huge cavities were in the infarcts in DW rats while small ones in HC rats.
Inflammation in infarcts was attenuated in HC group. Numbers of neutrophils were 548 ± 252 (HC) vs.1343 ± 431 (DW) (p=0.008); macrophages were 4485 ± 1487 (HC) vs. 7374±1824 (DW) (p<0.05) in bregma level ~ −0.3 mm on day 3.
Normal endothelia (NE) in infarcted hemisphere decreased in DW group on day 3. However, amounts of NE in parietal and cingulate cortex were normal in HC rats. The occupied field-areas by NE profiles were 2.1 ± 1 % (HC) vs. 1 ± 1 % (DW) (p=.01) in parietal II region and 1.6 ± .03 % (HC) vs. 1.1 ± .05 % in cingulate cortex (DW) (p=.01). On day 28, NE were 0.8 ± 0.4 % (HC) vs. 0.3 ± 0.2 % (DW) in the infarct in striata (p=0.001). NE increased in striata in HC group, but not DW rats. Normal blood vessels were existed in the infarct core in HC rats while absent in DW rats.
Neurogenesis: on day 28, the numbers of newborn neurons detected by anti-βIII tubulin in peri-infarct zone and inside infarct were 143 ± 98 (HC) vs. 56 ± 48 (DW) (p=0.02) [sum of the neurons in 3 standard levels (bregma levels ~ 2.7, −0.3 and −3.8 mm)].
Neuronal deficit recovered earlier and faster in HC rats; significant recovery began at day 1 and no paralysis on day 28 while DW rats recovered later and with some deficits on day 28 (p<0.05).
Conclusion: The 3-28 days of 9-herbal therapy protected brain from focal ischemia and encouraged post-ischemic neuronal function recovery and brain self-repair.
THE EFFECT OF HYPERBARIC OXYGEN PRECONDITIONING ON EARLY APOPTOSIS IN A RAT MODEL OF TRANSIENT GLOBAL CEREBRAL ISCHEMIA
Robert P. Ostrowski1, Gerhart Graupner2, Elena Titova1, Jennifer Zhang1, Jeffrey Chiu1, Neal Dach1, Dalia Corleone1, John H. Zhang1,3,4
1Department of Physiology, Loma Linda University, Loma Linda, CA, USA, 2Department of Pediatrics, Loma Linda University, Loma Linda, CA, USA, 3Department of Neurosurgery, Loma Linda University, Loma Linda, CA, USA, 4Department of Anesthesiology, Loma Linda University, Loma Linda, CA, USA
Background and aims: We hypothesized that the brain protective effect of hyperbaric oxygen (HBO) preconditioning is mediated by the inhibition of early apoptosis, and the anti-apoptotic effect is prolonged to the later phase post-ischemia. We also tested whether inhibition of apoptosis due to HBO preconditioning may be mediated by the suppression of p38 MAP kinase and increases in brain-derived neurotrophic factor (BDNF) early after ischemia.
Methods: We used one-stage four-vessel occlusion (4VO) global ischemic model (10 min. of occlusion). A total of 106 male Sprague-Dawley rats (300-350 g) were assigned into 4 groups: sham, global ischemia (4VO) untreated and ischemic group preconditioned with either 3 or 5 courses of hyperbaric oxygenation (3HBO+4VO or 5HBO+4VO). HBO was applied at 2.5 atmospheres absolute for 1 h per treatment. Five times HBO preconditioning included one treatment per day for five days; the last treatment was applied 24 h before induction of ischemia. Triple HBO regimen comprised of treatments at 24, 12 and 4 h before ischemia. Rats were sacrificed at 3, 6, 16, 24 and 72 h and 7 days after ischemia.
We developed in vivo fluorescent staining with annexin V ? an early apoptotic marker. The neuronal activation of p-38 was assessed by anti-phospho p38 (p-p38) and anti-NeuN double immunofluorescent stain. Cell counts were performed by observers blinded to the study. Brain BDNF levels were determined by ELISA. For evaluation of neurological outcomes sensorimotor and T-maze tests were used.
Results: The number of apoptotic neurons in the cerebral cortex was significantly greater in 4VO group than in the sham group. In parallel, the increased neuronal immunoreactivity of p-p38 was noted. HBO preconditioning provided reduction in early apoptosis in cerebral cortex and the hippocampus, associated with a suppression of p-p38 and increase in BDNF brain content. This protection was followed by a reduction in TUNEL-positive cells and good preservation of neurons (Nissl stain) at 24 h and 3 and 7 days.
However, more annexin-positive cells were observed in 3HBO+4VO group than in 5HBO+4VO group. Also p-p38 suppresion positively correlated with a degree of protection and was less remarkable in the 3HBO+4VO group. We observed multiple double annexin/TUNEL positive cells at 16 h after global ischemia in the untreated group but not in the preconditioned groups. Several annexin-positive cells in HBO groups were not TUNEL positive. Mortality and neurological outcomes were ameliorated in both HBO preconditioning groups.
Conclusions: This study provided evidence that neuronal apoptosis occurs in the early phase after global cerebral ischemia. Cells showing early signs of apoptosis displayed DNA fragmentation later on. Therefore, the HBO preconditioning-induced reduction of early apoptosis and the inhibited conversion of early to late apoptosis may prevent delayed cell injury from developing. The reduction of early post-ischemic apoptosis in the brain due to HBO preconditioning may be mediated by increasing BDNF and suppressing p38 activation. Our study also suggests that the fast track of HBO preconditioning can provide satisfactory protection against anticipated brain insults.
CARBON MONOXIDE PROTECTS AGAINST BRAIN ISCHEMIA
Emil Zeynalov1, Sylvain Dore1,2
1Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, SOM, Baltimore, MD, USA, 2Department of Neuroscience, Johns Hopkins University, SOM, Baltimore, MD, USA
Background: Historically, carbon monoxide (CO) gas has been generally considered to be toxic. Here, we postulate that physiological levels or low amounts of CO can in fact be protective in the brain. Using both in vivo and in vitro approaches, we have previously demonstrated that the rate-limiting enzyme for the endogenous synthesis of CO, heme oxygenase, is highly neuroprotective against various types of insults. Our goal was to determine whether CO can prevent brain damage and neurological deficits following cerebral ischemia.
Method: To test this hypothesis, C57Bl/6 male mice (22-28g) were submitted to 90-min transient brain ischemia. Successful occlusion was confirmed by an 87–90% reduction in cerebral blood flow as measured by laser-Doppler flowmetry. The mice were then exposed to air or various levels of CO: 125, 250, or 500ppm (n/group = 11, 10, 8, and 6, respectively), amounts which can be present at street levels. Mice were survived 48 hours after reperfusion; brains were harvested, sliced into 2-mm thick sections, and stained with 1% triphenyltetrazolium chloride. To measure the brain water content, brains of sacrificed animals were harvested, weighed, and dried at 100°C for 72 hours. Brains were weighed again immediately after removal from the oven, and wet-to-dry ratio was calculated. Experiments were carried out in accordance with the guidelines of the NIH for the care and use of animals in research and were approved by the institutional animal care and use committee of Johns Hopkins University.
Results: We found that mice exposed to 125ppm CO developed a significantly smaller infarct size than those exposed to air only (p<0.05). Exposure to 250ppm appeared to produce the maximal beneficial effect. In mice subjected to unilateral transient focal ischemia and exposed to 250ppm CO after reperfusion, the ipsilateral hemisphere had lower brain water content at 48 hours than that of the control animals. Water content in the contralateral hemispheres remained unchanged in both CO- and air-exposed groups.
Discussion: To our knowledge, this is the first study reporting that CO can reduce infarct volume and limit brain edema. Various mechanisms of action are being postulated and investigated. Physiological amounts of CO have been shown to play an important role in many cytoprotective mechanisms. It has been shown that low concentrations of exogenous CO can have anti-inflammatory and anti-apoptotic effects, which would protect tissues and organs against various insults. In addition, CO can mediate vascular signaling that leads to vasodilatation, improving cerebral blood flow and brain perfusion. And by suppression of inflammatory and cell death pathways, CO permits protection of tissues and organs against various insults. Exposure of CO at 250ppm would be the optimal level for studying the neuroprotective effects and can be used for the treatment of experimental ischemic stroke in mice. Thus, development of a protocol to maintain CO within these optimal levels could be a novel therapeutic tool to fight brain damage and neurological outcome associated with stroke and other age- and vascular-related neurodegenerative disorders. (This work was supported by NIH grants.)
AN ELECTRIC FIELD INCREASES BRAIN-DERIVED NEUROTROPHIC FACTOR LEVELS IN THE BRAIN, ENHANCES SPATIAL LEARNING AND RESISTANCE TO FOCAL ISCHEMIA
Hiroji Yanamoto1,2, Susumu Miyamoto2, Yukako Nakajo1,3, Yoshikazu Nakano1
1Laboratory for Cerebrovascular Disorders, Research Institute of National Cardiovascular Center, Suita, Osaka, Japan, 2Department of Neurosurgery, National Cardiovascular Center, Suita, Osaka, Japan, 3Laboratory for Basic Science, Rakuwakai Otowa Hospital, Kyoto, Japan
Background and aims: Brain-derived neurotrophic factor (BDNF) is known to enhance learning and memory acquisition or to promote neuronal survival protecting the brain from ischemic injuries. Recently, we reported that intracerebral infusion of recombinant BDNF protects the brain from focal ischemia, and that genetically engineered BDNF overexpression mice; heterozygous BDNF (+/−) transgenic mice demonstrate significantly enhanced spatial learning compared to the wildcard littermates. Development of a safe method that can increase endogenous BDNF levels has been expected to enhance learning, or to protect the brain against ischemic stroke. Specific types of electric stimulation that have been shown to be associated with elevated levels of BDNF in the brain include the following: a brief period of direct current electrification, an epileptiform discharge, and chronic electroconvulsive therapy (ECT). Here, we investigated the effects of whole-body exposure to alternating high voltage electric potential (HELP) on BDNF levels in the brain, spatial learning, and resistance to cerebral infarction development in mice.
Methods: An electric field generated with HELP; 5.8 kVrms or 0 Vrms (untreated control) was applied to normal adult mice (8-9-week old, C57BL/6J), for 5 hours a day for 1 or 3 weeks, using a mattress-type working electrode (HELP supplier) which was placed directly beneath the mouse cage, under the condition that the unit composed with the cage and HELP supplier was insulated from its surroundings to make indirect contact (through room air > 20 cm) with ground level. Estimated electric field generated in the cage was < 29 kV/m during the HELP supply. After the treatment, we analyzed alterations in BDNF levels in the brain using ELISA, or performance in a modified Morris water maze test (MWM). In a separate set of animals after the treatment, we also analyzed alterations in infarcted lesion volume caused by transient focal ischemia using the 3-VO technique.
Results: After HELP treatment for 1 or 3 weeks, the BDNF levels in the corticostriatum increased (205 % or 240 %, respectively), and escape latency in MWM improved (54 % or 31 %, respectively), compared with untreated the corresponding control animals (n=18-20 each, P < 0.05, one-way ANOVA). The infarcted lesion volume measured 24 hours after the induction of ischemia was decreased when ischemia was induced after HELP treatment for 3 weeks, compared with controls (15.7 ± 7.7 mm3 vs. 25.4 ± 8.7 mm3, mean ± SD, n=7 each, P < 0.05, one-way ANOVA).
Conclusions: It is here reported for the first time that repetitive HELP application increases BDNF levels in the brain, enhances spatial learning, and protects the brain from ischemic stroke. Use of repetitive HELP (< 30 kVrms) was approved 40 years ago by the Ministry of HLW (Japan) as an effective therapy for improving shoulder muscle stiffness, chronic constipation, insomnia, or headache in humans.
NORMOBARIC HYPEROXIA EXERTS ITS NEUROPROTECTIVE EFFECT IN ISCHEMIC RATS BY DELAYING AND ATTENUATING THE EARLY NOS-DERIVED NO PRODUCTION
Zhongrui Yuan, Wenlan Liu, Aaron Schnell, Ke Jian Liu
Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, NM, USA
Background and aims: Neuronal release of nitric oxide (NO) plays a prominent role in excitotoxicity, an important trigger and executioner of tissue damage in focal cerebral ischemia. Our recent study has shown that normobaric hyperoxia (NBO) treatment during ischemia can maintain penumbral oxygenation at a physiological level, which leads to a significant reduction in stroke lesion volumes and improved neurological function in a rat model of transient focal cerebral ischemia. The mechanism for the observed neuroprotection of NBO treatment is, however, unclear. In the present study, we tested the hypothesis that NBO treatment can attenuate neuronal nitric oxide synthase (nNOS)-derived NO production.
Methods: NO production and the formation of 3-nitrotyrosine (3-NT) were measured in the ischemic cortex at 10 min., 30 min., 60 min., and 90 min. after onset of ischemia. Total amount of NO production, as determined by the concentration of nitrate and nitrite, was quantified by the Griess method. HPLC-EC analysis was performed to measure 3-NT.
Results: We found that in experimental groups with normoxia (30%O2:70%N2), ischemia caused a time-dependent production of NO in the ischemic cortex, reached peak at 10 min (831 ± 172 nmol/mg protein, n=6) after onset of ischemia, which then returned to baseline level at 60 min (327 ± 137 nmol/mg protein, n=6). On the other hand, the NO production in the NBO (95%O2:5%CO2) treatment group was significantly lower (691 ± 94 nmol/mg protein, n=6), and peaked much later (at 30 min). For the formation of 3-NT, compared with a normoxia group, NBO treatment reduced the formation of 3-NT by 30 ± 4% (p<0.05). In order to understand the source of the NO and 3-NT production, we also carried out parallel experiments in animals pre-treated with 7-nitroindazole (7-NI), which is an nNOS selective inhibitor. We found that 7-NI pre-treatment has similar effects on NO and 3-NT production as does NBO treatment. Combined treatments with both 7-NI and NBO did not reduce NO and 3-NT production further than the individual treatments alone. These findings indicate that that NO, and the resulting 3-NT, was derived from nNOS.
Conclusions: Taken together, our findings provide direct evidence that NBO exerts its neuroprotective effects through both delaying and attenuating the early nNOS-derived NO production, in a similar manner to the beneficial effect of nNOS selective inhibitors. These results also suggest that NBO could potentially be used to expand the therapeutic time window for stroke patients.
APOCYNIN REDUCES BLOOD-BRAIN BARRIER DISRUPTION BY INHIBITING NADPH OXIDASE ACTIVATION AND MMP-9 EXPRESSION IN EXPERIMENTAL STROKE
Xiannan Tang1,2, Maya Koike1,2, Lijun Xu2, Nick Cairns3, Belinda Cairns3, Rona Giffard2, Midori Yenari1
1Department of Neurology, UCSF and SF VAMC, San Francisco, CA, USA, 2Department of Anesthesia, Stanford University, Palo Alto, CA, USA, 3Combinix, Inc., Mountain View, CA, USA
Introduction: Inflammation accompanying stroke has received much attention in recent years, as it is now believed that this response contributes to secondary ischemic injury. We previously showed in vitro that microglia worsen injury to blood-brain barrier (BBB) components subjected to ischemia-like insults, and that this was largely due to the generation of superoxide by NADPH oxidase. Here we study whether NADPH oxidase contributes to BBB disruption and brain hemorrhage in vivo, and whether apocynin, an inhibitor of NADPH oxidase could reduce BBB disruption following experimental stroke. Methods: Male C57 mice were subjected to 2h transient middle cerebral artery occlusion (tMCAO) were treated with: apocynin 0 (n=5), 2.5 (n=7) or 5 (n=8) mg/kg IV dissolved in pyrogen-free 0.1% DMSO 30 minutes after reperfusion. Following 22h reperfusion, animals were evaluated for neurological deficits, and then sacrificed. Brains were assessed for infarct volume, hemorrhage, and Evans blue dye extravasation. To determine whether apocynin enters the brain after parenteral administration, 2 mice were intravenously injected with Texas Red conjugated apocynin. In order to further determine whether apocynin could reduce the superoxide generation, another 6 C57BL male mice were subjected to tMCAO and treated with apocynin (2.5 mg/kg) and 0.1% DMSO (n=3 respectively) In situ detection of superoxide anion was performed using hydroethidine (HEt). For MMP-9 expression study, also 6 C57BL male mice were subjected to tMCAO and treated with apocynin (2.5 mg/kg) and 0.1% DMSO (n=3 respectively). TTC staining was performed to delineate the infarct; brain tissue around the infarct core was sampled and used for Western blot assay. Results: Apocynin was found to be able penetrate the intact BBB. Apocynin treatment (2.5 mg/kg) reduced infarct volume by 37% (P<0.05) and improved neurological deficits (1.5 vs. 2.5, P<0.05). There was a trend of decreased cerebral hemorrhage rate (16.7% vs. 58.3%, P=0.09) and it reduced the extent of Evans blue dye extravasation by 74% (P<0.001). In contrast, 5 mg/kg apocynin did not decrease the hemorrhage rate (60% vs. 58.3%, NS), nor reduce infarct volume (140±35 vs. 144±36 cm3, NS), nor improve neurological function (2.48 ±0.42 vs. 2.5±0.56, NS). Apocynin markedly reduced superoxide generation. Superoxide generation as estimated by the relative fluorescence intensity (RFI) per microscopic field, increased 4.5 fold following tMCAO compared to sham operated controls. Apocynin treatment almost completed suppressed superoxide generation (4.5 fold less RFI compared to vehicle treated controls, P<0.01). Furthermore, number of cells positive for superoxide were increased by tMCAO (160±3 vs. 31±3 per 400X HPF in sham operated controls, P<0.05), and this was also decreased by apocynin treatment (3±1, P<0.05). Immunohistochemistry showed most of the superoxide were generated by neurons and in part by microglia, but very few by cerebrovascular endothelial cells. Compared to the vehicle treated animals, the expression of MMP-9 in treated animals (apocynin 2.5mg/kg) reduced significantly (P<0.05). Conclusion: NADPH oxidase plays an important role in worsening BBB disruption and hemorrhage following experimental stroke. Apocynin improves outcome, but appears to have a narrow therapeutic window. Apocynin reduces superoxide generation and MMP-9 expression.
CALCIUM CHANNEL BLOCKERS PREVENT NEURONAL DAMAGE AND HAVE AN ANTIOXIDATIVE EFFECT AFTER TRANSIENT FOCAL ISCHEMIA IN RATS
Violeta Lukic Panin, HanZhe Zhang, Takeshi Hayashi, Atsushi Tsuchiya, Yoshihide Sehara, Toru Yamashita, Kentaro Deguchi, Tatsushi Kamiya, Koji Abe
Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
BACKGROUND: Cerebral ischemia is a third leading cause of death and at first place cause of disability all over the world. There are a lot of drugs that are in experimental stage for treatment of stroke. Among them are Calcium Channel Blockers (CCB) that have, in animal models, different effectiveness in healing of ischemic damage in brain. Mechanism of CCB action in cerebral ischemia is still unclear, but antioxidative property is supposed to be implicated. In the present study, we investigated antioxidative and neuroprotective properties of two CCB, azelnidipine and amlodipine.
METHODS: Male Wistar Kyoto rats were subjected to 90 minutes of transient middle cerebral artery occlusion (MCAO) by a nylon thread. Animals were divided into 3 groups, vehicle, azelnidipine and amlodipine group. In azelnidipine and amlodipine group, rats were treated with azelnidipine (1mg/kg) and amlodipine (1mg/kg) by gastric gauge for two weeks before MCAO. Vehicle group was treated by solution of methyl cellulose for two weeks. Rats were killed 24hs after MCAO. Clinical parameters (mean arterial pressure, heart rate, body weight), infarct volume, oxidative stress markers which are HEL, 4-HNE, AGE and 8-OHdG, and evidence of apoptosis by TUNEL, were investigated.
RESULTS: There were no significant difference among groups in mean arterial pressure, heart rate and body weight. Treatment with azelnidipine and amlodipine reduced infarct volume. Azelnidipine treated group showed more marked infarct volume reduction than amlodipine group (figure). The number of HEL, 4-HNE, AGE and 8-OHdG positive cells were significantly decreased in CCB treated groups. These molecules were again fewer in azelnidipine group than in amlodipine group. In TUNEL staining, the number of positive cells was smaller in the CCB treated group, especially in azelnidipine group.
CONCLUSIONS: Pretreatment of azelnidipine and amlodipine had a neuroprotective effect in ischemic brain. Antioxidative property is one of the important profiles of CCB that is implicated in brain protection.
CAROTID IMT AND RISK OF CARDIOVASCULAR EVENTS IN HIGH-RISK PATIENTS
Kazuo Kitagawa1, Hidetaka Hougaku1, Hiroshi Yamagami1, Hiroyuki Hashimoto2, Taku Hoshi1, Shigetaka Furukado1, Yuko Abe1, Manabu Sakaguchi1, Masayasu Matsumoto3, Masatsugu Hori1
1Stroke Division, Department of Medicine, Osaka University Hospital, Suita, Osaka, Japan, 2Division of Stroke and Hypertension, Department of Medicine, National Osaka Hospital, Osaka, Japan, 3Department of Clinical Neuroscience, Hiroshima Graduate School of Biomedical Sciences, Hiroshima, Japan
Background and Purpose: An increased carotid intima-media thickness (IMT) has proved to be associated with the presence of cardiovascular risk factors, cardiovascular disease (CVD), and atherosclerosis elsewhere in the arterial system. Prospective follow-up studies in the population at large have shown a positive correlation between increased carotid IMT and the risk of myocardial infarction and stroke. The purpose of this study was to determine the usefulness of carotid IMT measurement in such patients.
Methods: The study comprised 900 outpatients with cardiovascular risk factors or established atherosclerosis. Carotid IMT was calculated as the mean bilateral IMT of the common carotid artery (CCA), bifurcation, and internal carotid artery (ICA). Baseline vascular risk factors, medications, and history of CVD were recorded at the time of enrollment. The incidence of CVD events was determined prospectively.
Results: During a mean follow-up period of 2.6 years, there were 64 CVD events. The relative risk of a CVD event increased with increased IMT. Association between CVD events and carotid IMT was significant after adjustment for risk factors and history of CVD, showing an increased risk per IMT tertile from the middle tertile (relative risk, 2.5; 95% confidence interval [CI]: 1.0–6..3) to the highest (relative risk, 3.6; 1.4–9..0). When patients with a history of CVD were excluded (n=574), the predictive value of IMT was significant even after adjustment for risk factors (hazard ratio per 1 SD IMT increase was 1.57 [95% CI: 1.11 to 2.20]).
Conclusions: We have shown that carotid IMT independently predicts vascular events in high-risk patients. Carotid IMT measurement is of value for clinical stratification of patients at high risk of vascular events.
HYPOTHERMIA STRENGHTS INHIBITORS OF MATRIX DEGRADING PROTEASES FOLLOWING EXPERIMENTAL FOCAL CEREBRAL ISCHEMIA IN MICE
Jan Burk1, Dorothe Burggraf1, Milan Vosko1, Martin Dichgans1, Gerhard F. Hamann2
1Department of Neurology, Ludwig-Maximilians University, Munich, Germany, 2HSK Dr. Horst Schmidt Klinik, Wiesbaden, Germany
The treatment of an ischemic stroke under hypothermic conditions resulted in a better outcome in clinical studies. In this experiment we investigated the microvascular damage and proteolytic systems caused by ischemia in mice using hypothermia.
Focal cerebral ischemia was induced by occlusion of the arteria cerebri media (MCAO) using an intraluminal filament technique. The hypothermia model was established in our laboratory in mice. A first set of hypothermia experiments contained 16 mice. Eight mice received normothermia (37°C, NT) and eight received hypothermia (32?34°C, HT) treatment during 24 hours of reperfusion.
The analysis of the hypothermic group in comparison with the normothermic group resulted in a significantly reduced infarct volume (NT: 56±6mm3 SEM, HT: 33±5mm3 SEM; p<0.05) and showed considerable less neurological deficits (Garcia score) after 24 hours (p=0.002). In addition, the degradation of the basal lamina collagen type IV in hypothermia was strongly reduced (p<0.05) as well as the extravasation of haemoglobin (p<0.05).
In the biochemical analysis we found in the hypothermia group a significant decrease of the urokinase plasminogen activator (uPA) (p<0.05) and its inhibitor PAI-1 (p<0.05) compared to normothermia group. Conspicuous, during hypothermia as opposed to normothermia, there was a shift in the plasminogen-plasmin system from the proteases to their inhibitors. The ratio of uPA/PAI-1 (NT: 11:1; HT: 5:1) decreased significantly (p<0.05) during hypothermia.
In conclusion we demonstrated that hypothermia reduces the severe consequences of cerebral ischemia, protects the basal lamina and reduces the infarct volume and hemorrhage. Furthermore, one possible explanation for the beneficial effects of hypothermia might be the shift of the matrix degradading proteolytic systems to the inhibitoric side.
COMPARISON BETWEEN ANTIPLATELET THERAPY AND COMBINATION OF ANTIPLATELET AND ANTICOAGULATION THERAPY FOR SECONDARY PREVENTION IN STROKE PATIENTS WITH ANTIPHOSPHOLIPID SYNDROME
Yasuhisa Kitagawa1, Naoki Yuasa1, Noriko Takahashi1, Tatsuya Ishkawa1, Kentaro Tokuoka1,
Takashi Yasuda2, Shigeharu Takagi2
1Department of Neurology, Tokai University Hchioji Hospital, Tokyo, Japan, 2Department of Neurology, Tokai University, Kanagawa, Japan
Satisfactory results have not yet been obtained in therapy for secondary prevention in ischemic stroke patients with antiphospholipid syndrome (APS). We therefore compared single antiplatelet therapy and a combination of antiplatelet and anticoagulation therapy for secondary prevention in ischemic stroke patients with APS.
The subjects were 20 ischemic stroke patients with antiphosholipid antibody (10 males and 10 females, mean age 48 years), 13 with primary antiphospholipid syndrome and 7 with SLE-related antiphospholipid syndrome. Diagnosis of APS was based on the 1998 Sapporo Criteria. Eligible patients ware randomly assigned to either single antiplatelet therapy (aspirin 100 mg or ticlopidine 200 mg) or a combination of antiplatelet and anticoagulation therapy (INR: 2.0–3..0; mean 2.4±0.3) for the secondary prevention of stroke according to a double-blind protocol. There was no significant difference between the two groups in age, gender, NIHSS on admission, mRS at discharge, or rates of hypertension, diabetes mellitus, hyperlipidemia, and cardiac disease. We examined the Kaplan-Meier survival curves in each treatment. The primary outcome was the occurrence of stroke. The mean follow-up time was 3.9±2.0 years. The cumulative incidence of stroke in patients with single antiplatelet treatment was statistically significantly higher than that in patients receiving the combination of antiplatelet and anticoaglation therapy (log-rank test, p-value=0.026). The incidence of hemorrhagic complications was similar in the two groups. The recent APASS study did not show any difference in effectiveness for secondary prevention between single antiplatelet (aspirin) and single anticoagulant (warfarin) therapy. Our results indicate that combination therapy may be more effective in APS-related ischemic stroke.
EVALUATION OF SEE-JET METHOD ON HEMODYNAMIC CEREBRAL ISCHEMIA
Tetsu Kurokawa1, Shoichi Kato1, Kei Harada1, Hisato Nakayama1, Hirosuke Fujisawa1, Hideyuki Ishihara1, Hiroyasu Koizumi1, Tatsuo Akimura1, Nakagawara Joji2, Michiyasu Suzuki1
1Department of Neurosurgery, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan, 2Department of Neurosurgery, Nakamura Memorial Hospital, Sapporo, Hokkaido, Japan
[Background and Aims] The indication of cerebral revascularization such as extracranial-intracranial bypass has been determined by the severity of hemodynamic ischemia. And the result of Japanese Extractranial-Intracranial Bypass Trial (JET study) suggests that appropriate selection of patient based on severity of cerebral hemodynamic ischemia is important. To evaluate the severity, the cerebral blood flow (CBF) and cerevrovascular reserve (CVR) had been evaluated by setting of region of interest (ROI). However, this ROI setting method highly depends on the examiner's bias. To avoid this, we have utilized the stereotactic extraction estimation (SEE) ?JET software. Using this software, the cortical CBF and CVR can be evaluated in the every pixel automatically. We evaluated whether the severe hemodynamic stage (stage II) in the motor area detected by SEE-JET correlate with the clinical symptoms and compared with the ROI setting method. [Methods] Between December 2004 and November 2005 in our institute, 210 (142 male and 68 female) patients aged 64.8±13.7 years (mean±s.d.) with cerebral major vessel disease were recruited in this study. Resting CBF and CBF after acetazolamid challenge were measured by the Dual table autoradiographic (DT-ARG) method. The ROI of a middle cerebral artery area was manually drawn on bilateral cerebral cortex of the basal ganglia level. In the SEEJET evaluation, the motor area was determined by automatically based on the anatomical map. We evaluated whether the stage II was included or not in the motor area. The sensitivity and the specificity were compared between the SEE-JET and ROI setting method. [Results] The sensitivity and the specificity to the clinical symptoms (motor deficits) was 0.17 and 0.91 respectively with ROI setting method. In the SEE-JET method, the sensitivity and the specificity to stage II with motor area and motor deficit were 0.55 and 0.81 respectively. [Conclusions] To compare the ROI setting method, stage II in the motor area detected by the SEE-JET method is more sensitive to the clinical symptom and therefore this modality can be strongly recommended to determine the indication of the cerebral revascularization.
RECURRENCE WITHIN 30 DAYS AFTER ISCHEMIC STROKE IN JAPANESE PATIENTS: THE JAPAN STANDARD STROKE REGISTRY STUDY
Kazunori Toyoda1, Yasushi Okada2, Shotai Kobayashi3
1Cerebrovascular Division, National Cardiovascular Center, Suita, Japan, 2Department of Cerebrovascular Disease, National Hospital Organization Kyushu Medical Center, Fukuoka, Japan, 3Shimane University Hospital, Izumo, Japan
Background and aims: Stroke in Japan differs much from Western patterns in incidence and frequency of stroke subtypes, and has many unique characteristics. We determined the contributory factors and outcome of early recurrence of ischemic stroke in Japanese patients.
Methods: A multicenter stroke registration study (the Japan Standard Stroke Registry Study) based on a computerized database from 54 Japanese institutes, using 8,036 patients with brain infarction who were hospitalized within 48 hours after the symptom onset between January 2000 and March 2004.
Results: Within 30 days after the initial stroke, 395 patients (4.9%) suffered from recurrent stroke. Recurrence was most frequently occurred in atherothrombotic patients (6.6%), followed by cardioembolic patients (6.2%). Compared to patients who did not have a recurrent stroke, patients who had an early recurrence more frequently had: a previous ischemic stroke (p=0.0222); hypertension (p=0.0188); atrial fibrillation (p=0.0001); antiplatelet (p=0.0052) and antihypertensive (p=0.0017) medication prior to the initial stroke; and progression of symptoms within 48 hours (p<0.0001). After multivariate analysis, hypertension (OR 1.35, 95% CI 1.07 ? 1.70) and atrial fibrillation (OR 1.50, 95% CI 1.18 ? 1.92) were independently predictive of early recurrence of overall patients. In atherothrombotic patients, diabetes mellitus (OR 1.49, 95% CI 1.06 ? 2.09) and atrial fibrillation (OR 2.00, 95% CI 1.23 ? 3.24) were independently related to early recurrence; in cardioembolic patients, hypertension (OR 1.53, 95% CI 1.05 ? 2.22) and aortic aneurysm (OR 4.07, 95% CI 1.32 ? 12.57) were related. Among stroke subtypes, atherothrombotic stroke (OR 1.94, 95% CI 1.54 ? 2.45) and stroke of other etiology (OR 1.50, 95% CI 1.03 ? 2.19) were positive independent predictors of early recurrence after adjustment of risk factors. Cardioembolic stroke was no longer positively related to recurrence after adjustment for atrial fibrillation On discharge from our institute, modified Rankin Scale was higher in patients with than without early recurrence [3 (0 ? death) vs. 2 (0 ? death), p<0.0001]
Conclusions: This study using a large Japanese population reproduced the previous consensus that atherothrombotic stroke, hypertension, and atrial fibrillation contributed to early recurrence of ischemic stroke. In addition, analysis according to each stroke subtype clarified that diabetes mellitus was independently related to early recurrence for atherothrombotic patients.
REDUCTION IN CAROTID ARTERIAL PLAQUE AFTER FLUVASTATIN ASSESSED BY 3D ULTRASONOGRAPHY
Hideki Ohba, Kiyofumi Mori, Masato Kin, Masanori Mizuno, Yasuhiro Ishibasi, Yasuo Terayama
Department of Neurology, Iwate Medical University, Morioka, Iwate, Japan
Background and aims
Detection and evaluation of carotid plaque using conventional 2D (two-dimentional) carotid ultrasonography is not sufficient to evaluate three-dimensional extension of carotid plaque. We have been applying the latest 3D (three-dimensional) ultrasonography to observe three-dimensional aspects of carotid plaque. The present study compares morphological changes of carotid plaque before and after administration of fluvastatin using the 3D ultrasonography.
Methods
The subjects consist of 5 patients with carotid plaque (M:F = 4:1, 66.4±17.1years old). Serum LDL cholesterol varies from 144 to 152mg/dl. Three-dimensional plaque images were acquired using VOLUSON 730 expert (GE Healthcare) with 3D/4D probe. Three-dimensional images and volume of plaque among 5 subjects were compared with those of subjects 3 months after administration of 30mg of fluvastatin.
Results
Among 4 subjects, serum LDL cholesterol was decreased from 148.8±3.4 to 97.8±22.2 mg/dl while LDL cholesterol was increased from 152 to 161mg/dl in one subject after fluvastatin. Plaque contour were markedly improved in all subjects. Volume of plaque among all subjects significantly diminished from 0.72±1.17 to 0.35±0.63 cm3 after fluvastatin (p<0.001).
Conclusions
The 3D ultrasonography is valid and useful tool superior to conventional 2D method for evaluation and quantitative measurement of carotid plaque with high reliability.
The present study indicates that fluvastatin improves morphology of atherosclerotic carotid plaque reducing its volume within 3 months.
NON-INVASIVE MEASUREMENT OF ADVANCED GLYCATION END PRODUCTS IN PATIENTS WITH CEREBRAL INFARCTION
Yuko Morita1, Reiko Nagano1, Yuko Izuhara2, Shunya Takizawa1, Shigeharu Takagi1, Toshio Miyata2
1Division of Neurology, Department of Internal Medicine/Tokai University School of Medicine, Isehara, Kanagawa, Japan, 2Institute of Medical Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan
Background and purpose: Oxidative modification of carbohydrates and lipids enhances the formation of advanced glycation end products (AGEs). Although AGEs formation is markedly accelerated in hyperglycemia, its formation occurs in normoglycemia (e.g., during ischemic stroke) (1) and initiates a range of inflammatory responses (2). Recently, an AGE Reader (DIAGNOPTICS, USA) has become available to measure skin autofluorescence non-invasively. We used it to test our hypothesis that skin AGEs level increases in proportion to the progression of systemic atherosclerosis, even in patients with cerebral infarction, and is therefore available as a marker for predicting recurrence of ischemic stroke. In this study, we investigated the levels of skin AGEs in patients with chronic cerebral infarction, patients with silent infarction and age-matched controls. We also examined the effect of angiotension II receptor blocker (ARB) on the level of advanced glycation (3).
Methods: We measured skin AGEs levels in patients with chronic symptomatic cerebral infarction (n=107) and asymptomatic cerebral infarction (n=26), as well as age-matched controls (n=25), using an AGE Reader. Patients with chronic symptomatic cerebral infarction were separated into two groups; those who received ARB and those who did not.
Results: Skin AGEs levels in patients with symptomatic cerebral infarction were significantly increased compared with the control group (p<0.05; 2.1127±0.5355 v.s. 2.0202±0.4005). AGEs in patients with asymptomatic cerebral infarction were not increased versus the controls (p<0.05; 1.9781±0.3732 v.s. 2.0202±0.4005). However, AGEs in the symptomatic and asymptomatic groups were not significantly different. AGEs in the groups that did and did not receive ARB were also not significantly different.
Conclusion: Our data suggest that increased formation of AGEs may be an indicator of oxidative stress, not only in diabetes and renal failure, but also in chronic cerebral infarction. Further study will be required to establish whether skin AGE level is a useful parameter for predicting recurrence of ischemic stroke.
KNOCKING-DOWN AND KNOCKING-OUT OF NCKX2 GENE EXPRESSION EXACERBATE THE BRAIN INSULT INDUCED BY FOCAL ISCHEMIA
Ornella Cuomo1, Giuseppe Pignataro1, Rosaria Gala1, Francesca Boscia1, Anna Tortiglione1, Valeria Valsecchi1, Gianfranco Di Renzo1, Jonathan Lytton2, Lucio Annunziato1
1Division of Pharmacology, Department of Neuroscience, Federico II University of Naples, Naples, Italy, 2Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada
Sodium/Calcium exchangers are neuronal plasma membrane transporters which, by coupling Ca2+ and Na+ fluxes across neuronal membranes, may play a relevant role in brain ischemia. The exchanger gene superfamily comprises two arms: the K+-independent (NCX) and K+-dependent (NCKX) exchangers. In the brain, 3 different NCX and NCKX family members have been described (Annunziato et al., 2004; Cai and Lytton, 2004, Visser et al; 2006).
Previous studies showed that more than 60% of calcium extrusion was mediated by sodium calcium exchangers and that 90% of this exchange was NCKX-mediated (Lee et al., 2002). Recently, it has been shown that NCX blockade induces a worsening in neuronal damage, whereas its activation may provide neuroprotection in an in vivo model of focal ischemia (Pignataro et al., 2004). By contrast, the function of NCKX in cerebral ischemia has not been determined. Thus, the aim of the present study was to investigate the role played by NCKX2 in rodents subjected to permanent or transient middle cerebral artery occlusion, pMCAO and tMCAO.
To this purpose, NCKX2 mRNA and protein expression were evaluated in the ischemic core and in the remaining ipsilateral non-ischemic area at different times after pMCAO in rats, by means of real-time RT-PCR, in situ hybridization, Western Blot, and immunohistochemistry analysis. The results showed that, both in the ischemic core and in the periinfarctual area, NCKX2 mRNA and protein were downregulated. Interestingly, immunohistochemical experiments revealed that, although the NCKX2 immunoreactivity was globally reduced, some surviving cells, mainly localized in the pheripheral zone of the ischemic core strongly expressed NCKX2 protein.
The role of this protein in the development of ischemic damage was also assessed in ischemic rats in which NCKX2 expression was knocked-down by a specific antisense oligodeoxynucleotide (ODN) intracerebroventricularly injected or in ischemic knocked-out nckx2-/− mice. Both knocking-down and knocking-out NCKX2 expression by antisense strategy or genetic deletion drammatically increased the extent of the ischemic lesion in rats and mice subjected to pMCAO and tMCAO, respectively, showing that NCKX2 plays a pivotal role in the development of cerebral ischemia.
Overall, these results suggest that NCKX2 is involved in the progression of the ischemic lesion and may represent a potential target in the development of new therapeutic strategies for stroke treatment.
SRC KINASE INHIBITION IMPROVES ACUTE OUTCOMES FOLLOWING EXPERIMENTAL INTRACEREBRAL HEMORRHAGE
Timothy D. Ardizzone, Xinhua Zhan, Brad Ander, Frank R. Sharp
Department of Neurology, MIND Institute, University of California at Davis, Sacramento, CA,
USA
• BACKGROUND AND AIMS. The mechanisms by which intracerebral hemorrhages produce changes of blood flow and metabolism, cell death, and behavioral abnormalities are complex. In this study we begin to test the hypothesis that intracerebral hemorrhage activates Src kinases that phosphorylate other molecules to produce cell injury and behavioral deficits following ICH. The study was prompted by observations that thrombin (Xi, Keep, Hoff and colleagues) appears to mediate the acute edema that occurs following intracerebral hemorrhage. One way in which thrombin might produce edema would be for thrombin to cleave thrombin receptors (called PAR receptors) that in turn activate Src kinase family members that phosphorylate target proteins like NMDA receptors, VEGF, HIF, metalloproteinases, tight junction proteins and others. Altered functions of these molecules could contribute to local changes of flow and metabolism around ICH; and to edema, cell death and behavioral abnormalities associated with ICH.
• METHODS. Intracerebral hemorrhage (ICH) was produced in adult male Sprague Dawley rats by direct injection of autologous blood (50 microliters) into striatum. Src kinase activity, glucose hypermetabolic areas around the ICH, TUNEL stained cells, and apomorphine induced rotational behaviors were assessed in animals with ICH pretreated with the Src kinase inhibitor, PP1, or with vehicle. These initial studies were aimed at determining whether a Src kinase inhibitor would improve outcomes following experimental ICH.
• RESULTS. PP1 (3mg/kg) blocked increases of Src kinase activity (5 fold) at 3h after ICH. PP1 also blocked the areas of glucose hypermetabolism (p<0.05) and decreased the numbers of TUNEL stained cells (p<0.05) surrounding the ICH at 24h. Finally, apomorphine (1mg/kg) induced rotation at 24h following ICH was markedly attenuated by prior treatment with PP1 (3mg/kg i.p.) (p<0.05).
• CONCLUSIONS. PP1 decreases Src kinase activation, glucose metabolic activation, cell death and behavioral abnormalities following ICH in striatum of adult rats. It is hypothesized that intracerebral hemorrhage, possibly via thrombin activation of PAR receptors, activates Src that phosphorylates NMDA receptors, MMPs and other proteins that mediate injury following ICH. Future studies will determine whether thrombin activates Src and whether this occurs via PAR receptors and whether ICH in fact stimulates Src mediated phosphorylation of NMDA and AMPA receptors, HIF and other potential target molecules following ICH.
DANTROLENE DECREASES ISCHEMIC BRAIN DAMAGE THROUGH REDUCING ENDOPLASMIC RETICULUM INJURY
Takeshi Hayashi, Feng Li, Guang Jin, Kentaro Deguchi, Shoko Nagotani, Hanzhe Zhang, Yoshihide Sehara, Atsushi Tsuchiya, Toru Yamashita, Violeta Lukic, Tatsushi Kamiya, Koji Abe
Department of Neurology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
Background: The endoplasmic reticulum (ER) plays an important role in ischemic neuronal cell death. Calcium depletion from the ER lumen is the key step in injury of this organelle, and therefore, prevention of this step may reduce the ischemic brain damage. Dantrolene is a ryanodine receptor antagonist, which prevents calcium release from the ER lumen. In order to determine the effect of dantrolene on ischemic brain damage and ER stress response, we investigated changes in ER stress-related molecules expression and brain infarction volume using rat middle cerebral artery (MCA) occlusion model.
Methods: Under anesthesia by isoflurane, the right MCA of male Wistar rat was occluded for 90 minutes by a nylon thread. Just after the restoration of cerebral blood flow (CBF), twenty µg of dantrolene, which was dissolved in dimethyl sulfoxide (DMSO) at 5 µg/µl, was slowly injected into left lateral ventricle. Control animals underwent the same surgery but only DMSO was injected. At 24 hrs after the CBF restoration, the animals were sacrificed and coronal brain sections of 2 mm thickness were prepared, with which infarction volume was measured by a 2,3,5-triphenyltetrazolium method. At 1, 4, and 24 hrs after the CBF restoration, the animals were sacrificed and frozen brain sections were prepared, with which ER stress responses were investigated. As ER stress markers, we used phosphorylated form of double-stranded RNA-activated protein kinase (PKR)-like ER kinase (p-PERK), phosphorylated form of eukaryotic initiation factor 2α (p-eIF2α), activating transcription factor-4 (ATF-4), and C/EBP-homologous protein (CHOP). We also performed western blotting study for these molecules using brain samples at the same time points.
Results: As shown in the figure, the infarction volume was significantly decreased by dantrolene (*p<0.05). Immunohistochemical and western blotting studies showed that p-PERK and p-eIF2α were induced in the ischemic peripheral region at 1 and 4 hrs, which were prevented by dantrolene. ATF-4 and CHOP were induced in the ischemic peripheral region from 1 hr and peaked at 24 hrs, but both of them were again inhibited by dantrolene.
Conclusions: It was indicated that dantrolene reduced ischemic brain damage through reducing ER stress response. Calcium depletion from ER lumen should be the key step in ischemic neuronal cell death in vivo.
THE ROLE OF BRAIN AND HEMATOMA-DERIVED COMPLEMENT C3 IN HEME OXYGENASE-1 UPREGULATION AND MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION FOLLOWING INTRACEREBRAL HEMORRHAGE
Y. He1, S. Wan1, Y. Hua1, J.G. Younger2, R.F. Keep1, J.T. Hoff1, Guohua Xi1
1Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA, 2Department of Emergency Medicine, University of Michigan, Ann Arbor, MI, USA
Background and Aims: Complement activation, heme degradation and iron toxicity contribute to intracerebral hemorrhage (ICH)-induced brain injury. Heme oxygenase-1 (HO-1), also called heat shock protein 32, is a key enzyme of heme degradation. Brain HO-1 levels are increased following ICH and inhibition of heme oxygenase reduces ICH-related brain damage. Activation of mitogen-activated protein kinases (MAPKs) occurs in the brain after cerebral ischemia and is associated with ischemic brain injury, but it is not clear if MAPKs are activated following ICH. The present study investigated if MAPK pathways in the brain are activated following ICH and whether hematoma-derived complement C3 plays a role in HO-1 upregulation and MAPK activation induced by ICH.
Methods: Male complement C3-sufficient and C3-deficient mice were used in this study. Mice received an intracerebral infusion of either 30-mL of autologous or donor blood. There were four groups: C3-sufficient blood injected into C3-sufficient brain, C3-deficient blood injected into C3-deficient brain, C3-deficient blood injected into C3-sufficient brain and C3-sufficient blood injected into C3-deficient brain. All mice were killed 1 or 3 days later and the brains were used to measure HO-1 and MAPKs (p44/42 and p38) by Western blot analysis and immunohistochemistry.
Results: Western blot analysis showed HO-1 levels in the ipsilateral basal ganglia after 3 days were lower in C3-deficient brains receiving C3-deficient blood (2081±987 pixels) compared to C3-sufficient brains injected with C3-sufficient blood (3939±505 pixels, p<0.05). There were also lower HO-1 levels in the ipsilateral basal ganglia of C3-deficient mice receiving C3-sufficient blood (1951± 1144 pixels) compared to C3-sufficient mice receiving C3-deficient blood (4618±515, p<0.05). Most of HO-1 positive cells in the perihematomal zone were glia-like cells.
For MAPK measurements, the ratio of phosphorylated MAPK to total MAPK levels was determined at 24 hours. C3-sufficient mice infused with their own blood had higher levels of activated p44/42 MAPK in the ipsilateral basal ganglia (0.69 ± 0.34) compared to C3-defficient mice infused with their own blood (0.04 ± 0.08, p<0.05). Also, there were significant higher activated p44/42 MAPK levels in C3-sufficient mice injected with deficient blood (1.23 ± 0.13) compared to C3-defficient mice infused with C3-sufficient blood (0.38 ± 0.33, p<0.05). The effect of C3 sufficiency/deficiency on p38 MAPK activation was similar to that on p44/42 MAPK.
Conclusion: These results indicate that complement factor C3 has a central role in HO-1 upregulation and p38 and p44/42 MAPK activation following ICH. The source of that complement does not appear to be the hematoma but rather local brain production or entry across the blood-brain barrier.
CONGENIC REMOVAL OF HYPERTENSION QTL ATTENUATES FOCAL ISCHEMIC INJURY
Hiroshi Yao1, Zong-Hu Cui2, Junichi Masuda3, Toru Nabika2
1Center for Emotional and Behavioral Disorders, National Hospital Organization Hizen Psychiatric Center, Saga, Japan, 2Department of Functional Pathology, Shimane University School of Medicine, Izumo, Japan, 3Department of Laboratory Medicine, Shimane University School of Medicine, Izumo, Japan
Background and aims: A genome-wide screen found a blood pressure quantitative trait locus (QTL) on rat chromosome 1 in stroke-prone spontaneously hypertensive rats of a Japanese colony (SHRSP/Izm). Congenic strategy using inbred rat strains that display genetic hypertension is a powerful method to examine the effects of polygenetic traits on the stroke phenotype. In the present study, we investigated the effects of congenic removal of this QTL from SHRSP/Izm on infarct size produced by middle cerebral artery (MCA) occlusion. Methods: To establish the congenic strain (SHRSPwch1.0), the blood pressure QTL was introgressed from Wistar-Kyoto/Izm to SHRSP/Izm by repeated backcrossing. Male SHRSP/Izm (10-12 weeks old [young adult] n=8, 5 months old [adult] n=17) and SHRSPwch1.0 (young adult n=7, adult n=15) were randomly assigned to distal MCA occlusion as previously described (Stroke 1996;27:333-336). Briefly, a krypton laser operating at 568 nm (643-Y-A01, Melles Griot Inc.) was used to irradiate the distal MCA at a power of 20 mW for 4 min. The photosensitizing dye rose bengal (15 mg/mL in 0.9% saline; Wako) was administered intravenously to a body dose of 20 mg/kg over 90 sec starting simultaneously with 4 min of laser irradiation. Cerebral blood flow (CBF) was determined with laser-Doppler flowmetry 30 minutes after MCA occlusion in adult SHRSP/Izm (n=5) and SHRSPwch1.0 (n=5). A laser-Doppler flowmetry probe was laterally scanned, and CBF of the distal MCA cortical territory was measured at 5 points (2 mm posterior and 2.0, 2.5, 3.0, 3.5, and 4.0 mm lateral to the bregma) as previously described (Stroke 2003;34:2716-2721). Results: Resting mean arterial blood pressure (MABP) was 212±23 mmHg in adult SHRSPwch1.0, which was significantly lower than 241±22 mmHg in SHRSP/Izm. Infarct volume in the congenic rats was significantly decreased compared with that in SHRSP/Izm (66.4±21.5 mm3 vs. 103.4±24.8 mm3). CBF, determined at collaterally-perfused cortex with laser-Doppler flowmetry after MCA occlusion, was significantly greater in adult SHRSPwch1.0 compared with CBF in adult SHRSP/Izm. In young adult rats, there were no significant differences in MABP or in infarct volume between SHRSPwch1.0 and SHRSP/Izm. Conclusions: The congenic removal of a blood pressure QTL lowered blood pressure and caused a substantial reduction in infarct volume (−36%) with increased collateral CBF after MCA occlusion in the congenic rat. We demonstrated for the first time that the congenic strategy is useful to investigate the effects of genetic hypertension on focal ischemia or stroke.
DELETERIOUS EFFECTS OF DIHYDROTESTOSTERONE ON CEREBRAL ISCHEMIC INJURY
Jian Cheng, Nabil J. Alkayed, Patricia D. Hurn
Department of Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, OR, USA
Outcome from cerebral ischemia is sexually dimorphic in many experimental models. Male animals display greater sensitivity to ischemic injury than do their female counterparts, however the underlying mechanism is unclear. The present study determined if the potent and non-aromatizable androgen, dihydrotestosterone (DHT), exacerbates ischemic damage in male rats and alters post-ischemic gene expression after middle cerebral artery occlusion (MCAO). Adult male rats were castrated and hormone-replaced with either testosterone (T) or DHT (50 mg subcutaneous implant) one week before MCAO. 2h MCAO was achieved by intraluminal filament techniques and uniformity of MCAO was confirmed by laser Doppler flowmetry. The effect of DHT on cerebral infarct damage was quantified at 22h reperfusion by 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) histology and compared to that of T. Micropunches (1 mm diameter) from cortical peri-infarct zones and corresponding contralateral tissue were isolated and analyzed for early gene expression at 6 h of reperfusion using DNA microarrays (Affymetrix). Quantitative real-time RT-PCR (qPCR) was used for subsequent confirmation of differential expression pattern of selected gene candidates, including anti-apoptosis gene bcl-2 and pro-apoptosis gene bax. As expected, castration reduced plasma T and DHT to below the level of assay detection (Radioimmunology Assay), while hormone replacement restored total T and DHT plasma levels (37±7 and 3±1 ng/ml, respectively) to the physiologically relevant ranges. Castrated males (CAST) sustained smaller damage than gonadally intact males (Intact) or DHT-treated animals. (Cortex: CAST 15±6% of ipsilateral structure; Intact 34±4%; DHT 42±4%; Striatum: CAST 43±6 %; Intact 75±5%; DHT 88±4%). T increased infarction in striatum (82±5%) as compared to CAST, but not in cortex. Microarray studies identified transcripts that were altered by DHT alone or DHT with MCAO/ 6 h reperfusion. In DHT-treated animals, 422 genes/ESTs were increased by MCAO and 358 were decreased. In castrated animals, 271 genes/ESTs were increased and 350 were depressed by ischemia. When ischemic cortex was compared between DHT and CAST groups (DI-CI), 421 genes were increased by DHT treatment and 239 genes were decreased. Functional analysis of the genes with differential expression patterns between DI-CI indicated that a larger number of genes were elevated by DHT vs castration after ischemia, many of which could account for cell death through enhanced inflammation, dysregulation of blood-brain barrier and the extracellular matrix, apoptosis, and ionic imbalance. We used qPCR and immunoblotting to confirm the microarray data. Seven of ten gene candidates were confirmed by qPCR and the differential expression pattern of cyclooxygenase-2 was additionally confirmed by immunoblotting. By using qPCR techniques we also found that intact and DHT groups demonstrated higher levels of cortical bax mRNA relative to CAST at 22h reperfusion, but bcl-2 was similar in all groups. Our data suggest that DHT is an important androgenic mediator of ischemic damage in male brain and that transcriptional mechanisms should be considered as we seek to understand innate male sensitivity to cerebral ischemia. Supported by NIH NS 49210, NS33668, N03521
EARLY INCREASE OF BLOOD OXYGEN TRANSPORT CAPACITY ALLEVIATES ISCHEMIC BRAIN DAMAGE
Clemens Heiser, Lothar Schilling
Division of Neurosurgical Research, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany
Background and aims: In the pathophysiological sequelae of cerebral ischemia a number of reaction pathways are thought to be involved including excitotoxic, apoptotic, and inflammatory mechanisms. However, the contribution of the individual pathways is still a matter of debate. In the present study we sought to characterize the role of tissue hypoxia on structural brain damage in a model of focal brain ischemia.
Methods: Male Sprague-Dawley rats received an intravenous bolus injection of pimonidazole (60 mg/100 g bw) and 1 h later underwent a permanent right-sided middle cerebral artery occlusion (MCAO) using an intraluminal filament. After establishing focal ischemia NaCl (1 ml/100 g bw, experimental groups I, II) or an artificial O2 carrier (Oxycyte, a second generation perfluorocarbone solution, 1 ml/100 g bw; group III) was injected followed by breathing air (group I) or pure oxygen (normobaric hyperoxygenation [NBH]), groups II, III) for a maximum of 8 h. The animals were killed 2, 4, 8 h (group I) or 2, 4, 8, 24 h (groups II, III) after MCAO, the brain was removed and serially cut in coronal slices for immunohistochemical detection of pimonidazole adducts indicative of cellular hypoxia or silver nitrate staining for outlining structural brain damage, respectively. The sections were scanned for volumetric analysis with appropriate correction for brain swelling.
Results: Even after 2h of MCAO areas of ischemic brain damage were clearly detectable and could be quantified in each animal. The results (mean+SD) are listed in the table with swelling given in % of contralateral hemisphere and damage and hypoxia indicated in mm3 (+p<0.05 vs. group II; *p<0.05 vs. group III; n.d., not determined).
Conclusions: Improving the O2 transport capacity by an artificial carrier early after onset of focal cerebral ischemia resulted in a significant decrease of tissue damage. This effect was sustained even after ceasing this therapy. These results suggest that an early tissue hypoxia plays a significant role in the development of post-ischemic brain damage.
NEURONS OF THE RODENT BRAIN EXPRESS HEMOGLOBIN
Dominik W. Schelshorn, Armin Schneider, Heinrich F. Burgers, Maria Harlacher, Mathilde Lorenz, Wolfgang Kuschinsky, Martin Henrik Maurer
Dept. of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany
The role of hemoglobin in oxygen transport of red blood cells is well known. In vertebrates the expression of neuroglobin and cytoglobin has been demonstrated in neurons, whereas the existence of hemoglobin in these cells has so far not been verified. While the presence of hemoglobin mRNA and protein has been assessed in brain homogenate, these findings have been interpreted as artifacts derived from blood residues in the brain samples.
In this study, we systematically investigated the in vivo expression of hemoglobin and its mRNA in the adult rodent brain. To exclude red blood cell contamination, we also assessed the basic in vitro expression of hemoglobin and its mRNA in rat neuronal cultures. The cultures were also treated with known inductors of hemoglobin synthesis to identify potential regulators of expression.
Whole brain experiments
We perfused adult mice and rats to remove blood from the brain and analysed mRNA and protein from homogenate.
The expression of hemoglobin subunit alpha and beta mRNA and protein was confirmed by Real Time RT-PCR as well as by 2D gel electrophoresis and mass spectrometry. Immunohistochemistry stainings of neurons in brain sections were positive for hemoglobin. This staining was blocked by pre-incubation of the hemoglobin-specific antibody with purified rat hemoglobin.
Neuronal cultures
Rat neuronal cultures were prepared from cortex (E18), whole brain (P1), and cerebellum (P6) and cultured for 1–3 weeks. Hemoglobin expression of untreated cells was compared to the expression levels of cells treated with sodium butyrate or hemin.
Both alpha and beta chain were detected by RT-PCR. Densitometric analysis of the PCR bands revealed an increase of the transcripts after stimulation with sodium butyrate or hemin in cultures from whole brain (P1) and cerebellum (P6). Protein analysis by image warping and gel matching of 2-D gels confirmed the presence of hemoglobin.
Conclusion
Our results provide evidence that hemoglobin is expressed in neurons of the rodent brain, indicating a function in the vertebrate brain beyond oxygen transport in the blood. Hemoglobin might serve as an intermediary O2 storage or regulate NO chemistry.
Acknowledgements: Supported by the German Research Foundation (DFG, MA 2492/2-2, MHM and WK)
FOCAL CEREBRAL ISCHEMIA IN THE TNF-TRANSGENIC RAT: REGIONAL EXPRESSION OF TNF PROTEIN, TNF CONVERTING ENZYME, AND TNF RECEPTOR 1
Luther Creed Pettigrew1,2, Xiao-Hong Song1, Susan D. Craddock1, Mark S. Kindy3,4
1Stroke Program, Sanders-Brown Center On Aging, and Department of Neurology, University of Kentucky Medical Center, Lexington, KY, USA, 2Department of Veterans Affairs Medical Center, Lexington, KY, USA, 3Department of Physiology and Neurosciences, Medical University of South Carolina, Charleston, SC, USA, 4Department of Veterans Affairs Medical Center, Charleston, SC, USA
Background and Aims: Tumor necrosis factor-alpha (TNFa) is an inflammatory mediator that becomes elevated in ischemic brain. TNF converting enzyme (TACE) cleaves active TNFa from a transmembrane precursor. Active TNFa will bind to its receptor, TNFR1, and may promote apoptosis through activation of caspase 8. We tested the hypothesis that overexpression of the murine TNFa gene in the brain of a transgenic (TNFa-Tg) rat will affect TNFa protein levels and alter regional expression of TACE and TNFR1 after focal ischemia.
Methods: TNFa-Tg and wild type (WT) rats underwent middle cerebral artery occlusion (MCAO) for 1 hr. Samples of ischemic core (IC) and penumbral (PN) tissue obtained after 30 min or 3/24 hr of reperfusion were assayed for TNFa (Linco multiplex) and expression of TACE and TNFR1 (western blot).
Results: The TNFa levels in IC and PN of TNFa-Tg rat brain were unchanged at 30 min and 3 hr but fell below non-ischemic control levels at 24 hr (p<0.02; n=3 ? 5 per group; Fisher's test; Table 1). The TNFa levels in IC and PN of WT rats could be measured only at 24 hrs (p=NS compared to non-ischemic WT controls). TNFa levels were significantly higher in IC (p<0.003) and PN (p<0.01) of the TNFa-Tg rat than in WT animals at all 3 sampling times.
Non-ischemic TNFa-Tg and WT rats were equivalent in expression of active TACE isotype and TNFR1. After MCAO, TACE expression increased in both IC and PN for TNFa-Tg and WT rats at 30 min (p<0.003) and at 24 hrs (p<0.03) but only in TNFa-Tg animals at 3 hrs (p<0.03). In ischemic TNFa-Tg rats, TNFR1 expression increased in both IC and PN at 30 min (p<0.004) and remained upregulated at 3 (p<0.05) and 24 (p<0.0001) hrs in IC. For ischemic WT animals, TNFR1 expression remained unchanged at 30 min but was increased in both IC and PN at 3 (p<0.01) and 24 hrs (p<0.006).
Conclusions: TNFa increased in both IC and PN after MCAO in TNFa-Tg rats and remained several fold higher than in WT rats 24 hrs afterward. TACE was not constitutively upregulated but became increased in response to ischemic stress more robustly than in WT animals. TNFR1 expression became elevated in the TNFa-Tg animal during early reperfusion and showed regional heterogeneity not observed in WT rats. We conclude that cerebral ischemic injury in the TNFa-Tg rat may amplify basal TNFa synthesis through upregulation of TACE and could augment cellular death through enhanced TNFR1 activation. This research was supported by NIH/NINDS NS047395 (LCP).
MICROCIRCULATORY RESPONSE TO FUNCTIONAL BRAIN STIMULATION
Bojana Stefanovic, E. Hutchinson, V. Yakovleva, L. Belluscio, A.P. Koretsky, A.C. Silva
NINDS, NIH, Bethesda, MD, USA
Background
On the mesoscopic scale accessible by hemodynamically weighted modalities such as PET, fMRI, and OIS, the vascular response to brain stimulation is spatially restricted and temporally locked to neuronal activity under a wide range of conditions. However, little is known about the hemodynamic changes on the microscopic scale. Prior studies (Kleinfeld, 1998; Schulte,2003; Chaigneau,2003) suggest a highly heterogeneous system, but suffer from very sparse sampling and use of a wide range of functional paradigms. This work aims to extend earlier investigations by employing a well-described functional paradigm and punctilious analysis to allow direct quantification of vessel volumes.
Methods
Ten male SD rats were intubated, ventilated, and anesthetized with α-chloralose. Electrical pulses (0.3-ms, 2-mA, at 3Hz) were delivered to the forepaw. Two-photon microscopy was performed in a closed cranial window over the S1FL using a Ti:S fs pulsed laser, with 805nm excitation. The passage of 150?300µL iv. bolus of rhodamine-labeled dextran (5mg/ml in PBS) was tracked using a single 1024×1024-µm2 slice, with a 8.0×8.0µm resolution, at 225ms/frame. These data were segmented into extra- and intra-vascular spaces, and a gamma variate fitted to each intravascular voxel's time course via NLS optimization to estimate transit times. Sixty high resolution slices (1–2µm in-plane, 1.5–3µm through-plane) were acquired during rest and activation to estimate steady-state intravascular volume changes. Exponential correction for depth intensity attenuation was performed on these stacks and followed by semi-automated segmentation based on region growing from manually defined seeds. Capillary line scans (1.7ms/line) were done to estimate RBC speeds.
Results
The transit time across the imaged vasculature decreased by 20±8%: from 1.6±0.3s at rest to 1.2±0.2s during stimulation. Figure 1 displays representative transit time maps. The mean CBV increased by 10.2±0.6%. A result of semi-automatic segmentation of vasculature is shown in Figure 2. Smaller vessels dilated by 11.4±0.7% on average, a figure that was significantly (p<10-6) larger than the average 7.8±1.1% dilation of medium size vessels. Figure 3 shows histograms of ACBV in small and medium caliber vessels. No mean change in the capillary RBC speed was observed following stimulation (AvRBC~−8.1±8.7%).
Conclusion
The findings support very heterogeneous microcirculatory changes following stimulation. Average decrease of 0.5±0.1s (20±8%) in the transit time provides the all-important validation of the ROI. For the first time, a direct measurement of individual vessel volumes, rather than point estimation of the vessel diameter, was afforded by the present study. Larger dilatation of the smaller vessels underlay the mean stimulation-induced increase in blood volume of 10.2±0.6%, suggesting increased vascular homogeneity at activation relative to rest.
THE ROLE OF THE BLOOD FLOW RESPONSE DURING FUNCTIONAL ACTIVATION IN THE SOMATOSENSORY CORTEX
Roger Springett, Michelle Abajian, Maureen Ripple
Department of Radiology, Dartmouth Medical School, Hanover, NH, USA
Introduction: Optical spectroscopy has been extremely successful in measuring the vascular response to functional activation in the brain. However, current optical systems only measure the change in hemoglobin concentration and cannot measure the baseline state. We have developed and validated a visible spectroscopy system to measure the absolute concentration and saturation of hemoglobin as well as the oxidation state of cytochrome c (Cytc). Cytc is a component of the mitochondrial electron transport chain and is sensitive to oxygenation at the mitochondrial level. This study is examines mitochondrial oxygenation during forepaw activation in the rat.
Methods: Sprague Dawley rats were anesthetized with isoflurane, the skull thinned for optical access and electrodes were inserted in the forepaw. The inspired oxygen fraction (FiO2) was set to 0.30 and the animals artificially ventilated. The anesthetic was then switched to alpha-chloralose, the animals allowed to stabilize for 1 hour and then the forepaw was stimulated with a pulse train that consisted of 10 repeats of 4 seconds of 0.4ms pulses at 5Hz with amplitude of 1mA. FiO2 was then decreased to 0.16 and the pulse train repeated.
Results: Baseline hemoglobin saturation (SmcO2) was 75% under isoflurane when FiO2 was 0.30, it decreased to 55% under alpha-chloralose anesthesia and increased to 70% during stimulation. When FiO2 was decreased to 0.16, SmcO2 decreased to 45% and increased to 55% during stimulation. Switching to alpha-chloralose anesthesia resulted in small reduction of Cytc amounting to 3% of total Cytc. During activation, Cytc reoxidized to isoflurane baseline values. When FiO2 was decreased to 0.16, there was a further reduction in Cytc by an additional 6% and activation resulted in an oxidation back to 3%.
Discussion: Previously, we have observed that Cytc is independent of small changes in cerebral oxygenation under isoflurane anesthesia in the rat and that SmcO2 must fall below 60% before there is a reduction in Cytc. Alpha-chloralose is the preferred anesthetic for small-animal activation studies and it reduced Cytc by 3% consistent with the decrease in SmcO2. During activation, there was a small oxidation in Cytc as the blood flow response increased SmcO2 and relieved this reduction. This oxidation is consistent with the oxidation observed in cytochrome oxidase during activation in the human brain using near infrared spectroscopy and suggests that alpha-chloralose anesthesia in the rat accurately models cerebral oxygenation in the human brain. When the relationship between Cytc and SmcO2 was compared during activation and during hypoxia, it was observed that Cytc reacted passively to changes in SmcO2.
The results suggest that mitochondrial oxygen tension in the brain is in the range where the mitochondria have to adapt their metabolic state to maintain oxygen consumption and rate of ATP production. This adaptation is observed as a small reduction in Cytc under otherwise normoxic conditions. We hypothesize that the role of the blood flow response during activation is to increase mitochondrial oxygenation and drive the mitochondria out of this adaptive-range of oxygen tension into the range where the mitochondrial metabolic state is independent of oxygen.
SPECIFIC SUBSETS OF CORTICAL GABA INTERNEURONS ARE INVOLVED IN THE NEUROVASCULAR COUPLING RESPONSE TO WHISKER STIMULATION IN THE RAT
Priscilla Fernandes1, Ara Kocharyan1, Elvire Vaucher2, Edith Hamel1
1Laboratory of Cerebrovascular Research, Montreal Neurological Institute, McGill University, Montreal, QC, Canada, 2Ecole D'Optometrie De L'Universite De Montreal, Montreal, QC, Canada
Introduction: The rat whisker-to-barrel pathway has a well-defined topography and displays well-delineated changes in contralateral cerebral blood flow (CBF) upon specific whisker stimulation. However, the cellular basis of this neurovascular coupling response remains largely unknown. In this study, we identified the neurons that are activated in the somatosensory cortex by whisker stimulation using double-immunocytochemistry. Further, we investigated the contribution of neuronal and glial messengers in this hemodynamic response by CBF monitoring with laser Doppler flowmetry (LDF).
Methods: Adult male Sprague-Dawley rats (280-320 g) were handled for one week prior to the experiment to habituate them to the stimulation procedure. Three days prior to the experiment all but three whiskers (C1-C3) on the right side of the snout and all whiskers on the left side were clipped under anesthesia. On the experimental day, unanesthetized rats were lightly restrained, and the C1-C3 vibrissae gently stroked (20 min). The identity of the activated cells (c-Fos positive) was determined by double-immunostaining for COX-2 (pyramidal cells), glial fibrillary acidic protein (GFAP, astrocytic marker), COX-1 (microglia) or, for the different subsets of GABA-interneurons, their co-localized peptides (somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP)) or enzymes (nitric oxide synthase (NOS), choline acetyltransferase (ChAT)) in 25 um coronal and 30 um tangential sections from perfusion-fixed brains 60 min after stimulation, in both the ipsilateral (control side) and contralateral (activated side) hemispheres. In another set of experiments in anesthetized rats, the mediators of the hemodynamic response evoked by 20 sec whisker stimulation were assessed by LDF monitoring of CBF after intracisternal injections (3 µl of a 10-4 M, pH 7.4 buffered solution) of either vehicle, a GABA-A receptor antagonist (picrotoxin), or selective inhibitors of COX-1 (SC-560) or COX-2 (NS-398).
Results: Whisker stimulation elicited CBF increases (35.3± 2.8%, from baseline, p<0.05, t-test, n=15), and a strong activation of c-Fos expressing cells in the contralateral barrel cortex corresponding to the activated whiskers. There was a selective activation of c-Fos positive nuclei in COX-2 positive pyramidal cells (25.4% of all labeled cells, p<0.01, t-test), and in GABA interneurons that contain VIP and/or ChAT (18.6 and 13.9% of all labeled cells, respectively, p<0.01, t-test), but not SOM, NPY or NOS. Astrocytes and microglia did not display c-Fos immunoreactivity. Tangential sections with cytochrome oxidase-delineated barrels demonstrated activation of VIP or ChAT interneurons and COX-2 pyramidal cells within the hollows of the activated barrels. The induced CBF response was significantly decreased by picrotoxin (−38.0%, p<0.01, n=7) and, in agreement with previous studies, by NS-398 (1) (−44.6%, p<0.05, n=4) but not by SC-560 (2) (−4.2%, ns, n=4).
Conclusions: These results show that GABA, most likely released from cortical GABA interneurons that specifically colocalize VIP and/or ChAT, contributes to the hemodynamic response to whisker stimulation, together with mediators released from COX-2 pyramidal cells (1). Whether the effects are direct through dilatory cerebrovascular GABA-A receptors or indirect via astroglial intermediaries is currently under investigation. Supported by CIHR grant (MOP-53334, EH).
EVIDENCE FOR CAPILLARY VASOCONSTRICTION IN CEREBRAL CORTEX OF MICE
Francisco Fernandez-Klett, Nikolas Offenhauser, Ulrich Dirnagl, Josef Priller, Ute Lindauer
Department of Experimental Neurology, Charite, Universitaetsmedizin Berlin, Berlin, Germany
The role of capillaries in the regulation of cerebral blood flow is still under debate. Pericytes, the second cell type present in capillaries beneath endothelial cells, possess contractile properties in vitro and have recently been shown to contract and dilate capillaries in cerebellar slices and the retina of mice (Peppiatt et al., 2006), but the physiological significance in vivo is still unclear.
Aim of our study was to investigate the ability of cortical capillaries to actively constrict in vitro and to elucidate the impact on blood flow in vivo by application of the thromboxane A2 (TXA2) mimetic U46619. TXA2 is a well known vasoconstrictor which can be released by platelets, endothelial cells and microglia and has been involved in the pathophysiology of stroke, subarachnoidal hemorrhage and CNS trauma. We have used the beta-actin-eGFP mouse, in which a large expression of GFP in endothelial cells and pericytes enables a detailed depiction of capillary morphology and morphometric changes by means of two photon fluorescence microscopy. In brain slices of the sensory cortex we observed a significant constriction in near all capillaries studied with 100 nM U46619 (74±8 % of outer diameter, n=7). The constriction was concentration-dependent. Prior incubation with the competitive TXA2-receptor antagonist SQ 29,548 (1 µM) totally prevented the constriction (n=5). In 4 out of 6 cases, SQ 29,458 also elicited a partial reversal of the reduction of capillary diameters induced by U46619. Hovewer, the nitric oxide donor spermine-NO (0,1 mM) failed to revert the constriction in 6 out of 7 cases. Morphologically, the constriction showed mostly a sphincter-like pattern and often occurred at capillary junctions. Occasionally, changes in the shape of the soma of pericytes were observed, suggesting that these cells are responsible for the constriction. This results demonstrate that cortical capillaries are capable of actively changing their lumen size, independently of perfusion changes at higher order vessels.
We have further used the closed cranial window preparation over the cerebral cortex of anesthetized, mechanically ventilated beta-actin-eGFP mice to study the vascular response to superfusion of U46619 in vivo. By means of two photon microscopy we have assessed the erythrocyte flow in single capillaries as well as diameter changes in capillaries and pial vessels. In parallel, laser-Doppler flowmetry was used to measure cerebral blood flow. Our preliminary results (n=3) show a decrease of blood flow in response to the TXA2 mimetic U46619 as measured by LDF. In single capillaries, flow reductions up to flow stops occured, together with a slight reduction in capillary diameters. Interestingly, this was not paralleled by a net constriction of neither pial arterioles nor venules.
Taken together, our data support the notion of capillaries as active contributors to blood flow changes in the cerebral cortex. This mechanism could be relevant in pathological conditions with increased TXA2 release.
Supported by DFG and Hermann and Lilly Schilling Foundation.
THE CEREBRAL SYMPATHETIC NORADRENERGIC SYSTEM AND REGIONAL CEREBRAL ENERGY METABOLITES IN AVM-PATIENTS - A MICRODIALYSIS STUDY
Carsten Stueer1, Toshiki Ikeda2, Michael Stoffel1, Oliver Sakowitz3, Gerd Luippold4, Carlo Schaller2, Bernhard Meyer1
1Neurosurgical Department, Munich Tech University, Munich, Germany, 2Neurosurgical Department, Bonn Medical Center, Bonn, Germany, 3Neurosurgical Department, University of Heidelberg, Heidelberg, Germany, 4Institute of Pharmacology, University of Tuebingen, Tuebingen, Germany
Background and aims: To test the hypothesis that the activity of the sympathetic nervous system (SNS) and regional metabolism around resection for arteriovenous malformations differs from those measured in surgery for nonvascular lesions.
Methods: Cortical microdialysis (MD) was performed intraoperatively before and after resection in n = 12 patients with small/medium-sized AVM and transsylvian dissection in n = 15 patients with nonvascular lesions (= controls). Serum Norepinephrine (NE) concentrations were assessed before and after resection/dissection to control for systemic NE alterations. All data were pooled according to groups and times, and compared via ANOVA (P<0.05) plus a post-hoc Dunnet correction for multiple comparisons. All data (given below as means ± SD) were collected in the context of a prospective study.
Results: Activity of the sympathetic nervous system as assessed with NE microdialysate concentrations were significantly higher in non-AVM patients (p<0.05) prior to sylvian transsection (5.4 ± 1.4 nmol/L) compared to post dissection (4.2 ± 1.1 nmol/L). NE levels were significantly lower in the AVM group, while there was no significant difference between pre- and post conditions (AVM pre: 3.3 ± 1.2 nmol/L, AVM post: 2.9 ± 1.7 nmol/L). Serum NE levels remained constant throughout surgery in both groups (Control pre: 1.0 ± 0.8 nmol/L, post: 0.9 ± 0.7 nmol/L; AVM pre: 0.7 ± 0.6 nmol/L, post: 1.1 ± 0.9 nmol/L), Concentrations of lactate, glutamate, glycerol as well as lactate/pyruvate ratio referred to as regional cerebral energy metabolites were without any significant differences.
Conclusions: Chronic arterial non-ischemic hypoperfusion in human AVM correlates with a down-regulation of the activity of the cerebral sympathetic nervous system. The postresection, protective capacity of the SNS seems sole impaired in AVM patients, which might be the basis for a hyperperfusion syndrome in larger and complex AVM.
INTRAMICROVASCULAR BEHAVIOR OF LEUKOCYTES IN MOUSE BRAIN OBSERVED BY HIGH SPEED LASER CONFOCAL FLUORESCENCE MICROSCOPY
Norio Tanahashi1, Kimihiko Hattori1, Nobuo Araki1, Kunio Shimazu1, Tatsuya Saitoh2, Minoru Tomita3
1Department of Neurology, Saitama Medical University, Saitama, Japan, 2Department of Neurosurgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan, 3Department of Neurology, Keio University, Tokyo, Japan
Background and aims
It is well known that leukocytes play an important role in the pathogenesis of cerebral ischemia and reperfusion. However, leukocytes behavior in brain microcirculation especially in the intraparenchymal vessels has not been fully investigated because of methodological difficulties. The purpose of this study is to observe leukocytes behavior in microvasculature of the anesthetized mouse cerebral cortex, using a high-speed laser-scanning confocal fluorescence microscope and image analysis system.
Methods
Fifteen C57 BL/6J mice anesthetized with chloral hydrate were fixed with a head holder After removal of the skull bone, leucocytes were labeled with a bolus of 3 microgram of rhodamine 6G (absorption peak, 526 nm; emission peak, 555 nm; Sigma) in 0.2 mlL 0.9% saline, which was injected intravenously, followed by a continuous infusion of 0.2 mL/h at the same concentration. Labeled leukocytess in the microvasculature of living mouse brain cortex were observed continuously under fluorescence microscopy. Leukocytes were tracked with a laser-scanning confocal fluorescence microscope (1000-5000 frames/s) and obtained images were analyzed on computer for the measurement of velocity. Leukocytes behavior was observed under physiological and pathological condition.
Results
In the pial veins, capillaries, and arteries/arterioles, flowing leukocytes were detected at a high speed laser confocal fluorescent microscopy (1000-5000 frames per sec). Leukocytes velocities in the physiological state were variable and mean velocities were 5.0±0.94 mm/s in arterioles and 1.56±0.21 mm/s in venules. In the physiological state, rolling and adhesion of leukocytes were rarely seen in the microvasculature of brain cortex. Following transient global ischemia (5 minutes of bilateral common carotid artery occlusion), rolling of leukocytes and adhesion in the pial veins were occasionally seen (Figure), but plugging of leukocytes in the capillaries were rarely seen. Rolling or adhesion of leukocytes was not seen in the pial arteries.
Conclusions
Leukocyte velocities at physiological state closely approximate the velocities of platelets and erythrocytes. Transient cerebral ischemia for a short period affected leukocyte behavior only in the venular side. The high speed laser confocal fluorescent microscopy is a promising tool for investigating leukocytes behavior in brain microcirculation.
PLATELET BEHAVIOR AND THROMBUS FORMATION IN MOUSE BRAIN MICROVASCULATURE OBSERVED ON HIGH SPEED LASER CONFOCAL FLUORESCENCE MICROSCOPE
Kimihiko Hattori1, Norio Tanahashi1, Nobuo Araki1, Kunio Shimazu1, Tatuya Saitoh2, Minoru Tomita3
1Department of Neurology, Saitama Medical University, Saitama, Japan, 2Department of Neurosurgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan, 3Department of Neurology, Keio University, Tokyo, Japan
Background and aims
Interaction between platelets and vessel walls plays an important role in the pathogenesis of cerebral ischemia. However, platelet behavior and thrombus formation in the microvasculature of the brain has not been fully investigated due to methodological difficulties. The purpose of this study was to measure platelet velocity in the brain microvasculature of anesthetized mice, and to observe the process of thrombus formation and platelet behavior in the brain microvasculature using high-speed laser-scanning confocal fluorescence microscope.
Methods
Eighteen C57 BL/6J mice were anesthetized with chloral hydrate; their heads were fixed with a head holder, and a cranial window was made in the parietal region. Platelets, labeled by carboxylfluorescein succinimidylester (CFSE) in vitro, were injected intravenously. A thread moistened with FeCl3 was placed for a short period of time on microvessels located on the brain surface to induce thrombus formation. Rhodamine 6G (Sigma) was intravenously injected to observe the process of thrombus formation. Labeled platelets and thrombus were observed continuously with a fluorescence microscope. Platelets were tracked with a laser-scanning confocal fluorescence microscope (1000-5000 frames/s)(Figure 1) and obtained images were analyzed on computer.
Results
Platelet adhesion to endothelial cells was rarely seen in arterioles in the physiological state. On the contrary, platelet adhesion was occasiollaly seen. Platelet velocities in the physiological state were variable and mean velocities were 4.20 ·0.46 mm/s in arterioles and 2.13 ·0.62 mm/s in venules.
After exposure of FeCl3 to the pial artery, thrombus formed along the vessel wall, and platelets accumulated on the surface of the thrombus (Figure 2). Fragmentation of thrombus was occasionally seen.
Conclusions
Mean platelet velocity in arterioles was twofold faster than that in venules in the physiological state. Induction of thrombus formation by FeCl3 is a promising method by which to investigate the pathophysiology of the interaction between platelets and endothelial cells.
NON-INVASIVE HYPOTENSIVE FMRI STUDY IN RAT SOMATOSENSORY CORTEX
Peter Herman1,2, Basavaraju Sanganahalli1, Fahmeed Hyder1,3, Andras Eke2
1Department of Diagnostic Radiology, Yale University, New Haven, CT, USA, 2Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary, 3Department of Biomedical Engineering, Yale University, New Haven, CT, USA
INTRODUCTION
The functional BOLD response is dependent on various physiological parameters, such as CMRO2, CBF, CBV, and arterial hematocrit. Experimental data on how systemic physiological changes (e.g., blood pressure) influence the BOLD response are needed. Prior studies induced hypertension and hypotension in rat by vasoactive drugs and evaluated the changes in BOLD and CBF during electrical forepaw stimulation [1]. However these drugs not only alter the systemic blood pressure but in addition they exert influence on the behavior of cerebral arteries. Our aim was, therefore, to study the BOLD response under non-chemically induced levels of arterial hypotension.
METHODS
Six artificially ventilated rats were anesthetized by α-chloralose (40 mg/kg/hr, i.p.). An arterial line was used for monitoring blood pressure and taking samples for blood pH, pO2, pCO2 throughout the experiment. All fMRI data were obtained on a modified 9.4T. Spin echo EPI data were acquired with TR of 2 s. The hypotensive steps (of 100, 80, 60, and 40 mmHg) were maintained by a computer-controlled negative lower body pressure method which has been described previously [2]. The duration of each hypotensive step was ~10 min and after each step the pressure was allowed to rise spontaneously. Data were collected in 90 s windows: 30 s before and after the 30 s forepaw stimulation (3 Hz, 2 mA, 0.3 ms) of both paws in every hypotensive step.
RESULTS
The BOLD responses were measured only in those voxels where the Student t statistics showed significant change at P<0.05. The BOLD responses showed significant increases at 60 and 40 mmHg as compared with the control, 100 and 80 mmHg pressure levels. There were no significant differences across the hypotensive steps of 100, 80, and 60 mmHg for baseline CBF levels, indicating the presence of CBF autoregulation. Prior studies using chemically induced hypotension have found that the BOLD response to stimulation did not change during hypotension [1] despite concomitant changes in CBF and CBV produced by hemorrhagic hypotension within range of 40-70 mmHg [3]. Our results also demonstrate that below the threshold of CBF autoregulation, the BOLD response is significantly higher than in the control period. Consequently, the BOLD response alone cannot be used to accurately monitor brain function in experimental settings when blood pressure drops below the threshold of CBF autoregulation.
ACKNOWLEDGEMENTS
Supported by NIH (DC-003710, MH-067528) and OTKA (T34122) grants.
HYPERCHOLESTEROLEMIA WORSENS THE BLOOD FLOW DEFICIT IN FOCAL CEREBRAL ISCHEMIA IN THE ABSENCE OF ATHEROSCLEROSIS
Hwa Kyoung Shin1, James A. Lapointe li2, Dmitriy N. Atochin2, Michael A. Moskowitz1, Paul L. Huang2, Cenk Ayata1,3
1Stroke and Neurovascular Regulation Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA, 2Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA, 3Stroke Service and Neuroscience Intensive Care Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA
Background and aims: Hypercholesterolemia may increase stroke risk by accelerating atherosclerosis and narrowing luminal diameter in cerebral vessels as well as disrupting vascular endothelial and smooth muscle function. We tested the hypothesis that that hypercholesterolemia impairs cerebrovascular function and worsens cerebral blood flow (CBF) deficit and tissue outcome after focal ischemia even in the absence of hemodynamically significant vessel narrowing.
Methods: We tested this hypothesis in ApoE knockout (KO) mice using high spatiotemporal resolution laser speckle flowmetry through intact skull noninvasively, and studied the hemodynamic impact of hypercholesterolemia on the evolution of CBF deficit in focal cerebral ischemia. Wild type (WT, C57BL/6), ApoE KO or ApoE/eNOS double KO mice were fed high-fat “western” diet for 4, 8, or 10 weeks starting at 4 weeks of age. Histopathological examination of extracranial arteries in 14 week-old ApoE KO showed only minimal atherosclerotic changes such as fatty streaks with subendothelial foam cells. Intracranial arteries did not develop atherosclerotic lesions even after 12 months, despite severe extracranial atherosclerosis.
Results: Mean arterial blood pressure was higher in ApoE KO compared to WT in all age groups (94+/−2 vs. 85+/−2 mmHg, respectively; n=17 each, p<0.05); arterial blood gas values did not differ. Distal middle cerebral artery occlusion (dMCAO) resulted in significantly larger area of severe CBF deficit (?20% residual CBF) in ApoE KO after 8 and 10, but not 4 weeks of high-fat diet compared to age- and diet-matched WT (n=4-7; see table). Ten weeks of high-fat diet also tended to increase the CBF deficit in WT mice compared to 4 or 8 weeks of high-fat diet (p=0.1). ApoE/eNOS double KO mice (8 weeks high-fat diet) developed 80% larger area of severe CBF deficit compared to age- and diet-matched ApoE KO (n=6 and 7, p<0.05). Unexpectedly, the frequency of peri-infarct depolarizations was significantly reduced in all groups of mice on high-fat diet compared to mice on regular diet (p<0.05), regardless of diet duration. Consistent with larger CBF deficits, infarct volumes (48h after 1h dMCAO) were 60% larger in ApoE KO mice compared to wild type after 8 weeks of high-fat diet (n=5 each, p=0.1).
Conclusions: These data suggest that hypercholesterolemia impairs cerebrovascular response to focal ischemia in the absence of hemodynamically significant atherosclerosis.
This work was supported by the American Heart Association (0335519N, Ayata), NIH (P50 NS10828, Moskowitz; NS048426, Huang).
REDUCTION IN 5HT2A BINDING POTENTIAL USING [11C] MDL 100907 IN SCHIZOPHRENIA
Dean F. Wong1,2, Nicola Cascella2, Yun Zhou1, James R. Brasic1, Mohab Alexander1, Weiguo Ye1, Ayon Nandi1, Anil Kumar1, Marika Maris1, Suzana Corritori3, Daniel P. van Kammen3
1Department of Radiology, Johns Hopkins Medical Institutions, Baltimore, MD, USA, 2Department of Psychiatry, Johns Hopkins Medical Institutions, Baltimore, MD, USA, 3ACADIA Pharmaceuticals Inc., San Diego, CA, USA
Background and Aims: Schizophrenia has been associated not only with altered dopamine receptors but also serotonin because of the possibility of serotonin's relationship in the therapeutic action of atypical antipsychotics. Some previous PET studies in drug naive patients using [18F]setoperone or in subjects at enhanced risk for schizophrenia using [18F]altanserin have demonstrated reductions in 5HT2A receptors in frontal and pre-frontal cortex, respectively. We now present the results of a study in drug free schizophrenia patients using the selective 5HT2A PET radioligand [11C] MDL 100907 (MDL).
Methods: Ten medically healthy subjects with schizophrenia or schizoaffective disorder (SS) (9 males, 1 female, average age 41 + 9) and nine healthy normal controls (CS) (4 females, 5 males average age 32 + 8) were studied. Exclusion criteria for the schizophrenia population included any other DSM-IV Axis I diagnosis other than schizophrenia/schizoaffective disorder, evidence of a current acute psychotic episode or any neurological disorders. The schizophrenia population was given a washout period of 2–3 weeks from any neuroleptic medication and any medication with a known effect on serotonin receptors.
In order to obtain measurements of 5-HT2A receptor levels, we administered 18-20 mCi IV bolus of the radiotracer [11C]MDL 100907 (MDL) followed by a 95-min PET scan. A parametric imaging approach (Zhou et al, 2003) was used to estimate MDL binding potential (BP)
Results: When the 10 SS were compared with the nine healthy CS, we found that SS had lower 5HT2A binding potential (BP) by up to 50% in all cortical regions as compared to CS (p = 0.001). However, since several studies have shown an age-related decrease in 5HT2A receptor levels (Adams, et al, 2004), we also examined an age-matched subset of the population, which included 6 patients and 6 controls, matched to an average age of 33 +7 years for both populations. In the age matched set, SS had significantly lower BP than CS in the following regions: cingulate cortex (p= 0.02), orbito-frontal cortex (p= 0.04), pre-frontal cortex (p=0.02), and temporal cortex (p=0.03).
Prior exposure to neuroleptics and other serotonin-affecting medications, though terminated at least two weeks before the PET study in this case, may have been a confound. However, when we grouped the subjects based on drug free period and dose of antipsychotics taken and analyzed the BP data in terms of these groups, we could find no significant relationship between drug free period or type and dose of antipsychotics and BP, using both parametric and non-parametric (rank-order) analyses.
Conclusions: This study showed significant reductions in BP with MDL in cingulate, orbito-frontal, pre-frontal and temporal cortices, matching findings from previous PET studies of 5HT2A binding with PET as well as post-mortem studies (Meltzer, et al, 1999; Adams, et al, 2004). In addition, we show additional cortical regions that were not observed in those studies, which focused only on the frontal and pre-frontal cortices. In summary MDL is a suitable approach and practical for not only investigating the pathophysiology of schizophrenia, but providing a convenient short-lived selective radiotracer for drug development studies.
COMPENSATORY INVOLVEMENT OF IPSI- AND CONTRALATERAL CORTEX AREAS IN APHASIA
Wolf-Dieter Heiss1, Alexander Thiel2, Lutz Winhuisen2
1Max Planck Institute for Neurological Research, Cologne, Germany, 2Department of Neurology, University of Cologne, Cologne, Germany
Objective
Recovery after focal brain lesions is dependent on the adaptive plasticity of the non-affected elements of the functional network. Language function is extremely lateralized to the left hemisphere. A further topological specialization concerns the various components of language comprehension and production within a complex bilateral network. This study analyzed the impact of various regions ipsi- and contralateral to a cortical lesion on recovery of aphasia.
Methods
Repetitive transcranial magnetic stimulation (rTMS, 4 Hz at resting motor threshold for 30 sec), which interferes with normal language function, was combined with measurements of regional cerebral blood flow at rest and during verb generation applying O15-water (370 MBq) and positron emission tomography (PET). Eight male volunteers, 11 patients with predominantly non-fluent aphasia 2 weeks after left sided middle cerebral artery infarction and 17 patients with mild to moderate aphasia due to gliomas in the left hemisphere were included in the study.
Results
All normal subjects exhibited longer latencies during rTMS over the left inferior frontal gyrus but not during stimulation over the right IFG. At rest, rTMS decreased blood flow ipsilateral and contralateral. During verb generation, rCBF was decreased during rTMS ipsilateral under the coil, but increased ipsilateral outside the coil and in the contralateral homologous region, indicating collateral and transcallosal inhibition.
Three stroke patients activated rCBF in the left inferior frontal gyrus, 8 activated both sides during verb generation. RTMS resulted in increased reaction time latency or error rate with right IFG stimulation in 5 patients indicating essential language function. In the verbal fluency task these patients had a lower performance than patients with effects of rTMS only over the left IFG, suggesting a less effective compensatory potential of right sided network areas.
All 17 glioma patients showed a TMS effect during stimulation over the left IFG. During stimulation over the right IFG 5 patients had longer reaction time latencies. In accordance with these rTMS effects the right TMS negative patients had significantly higher flow activation in the left IFG during verb generation. Right IFG flow activation was significantly more intense in right TMS-positive patients than in right TMS-negative patients and controls.
Conclusion
The different dynamics of language recovery after stroke and in tumours suggest a hierarchy for effective recovery: best recovery can usually only be achieved by restoration of the original activation pattern within the dominant hemisphere; if primary functional centres are damaged, reduction of collateral inhibition leads to activation of areas around the lesion permitting intrahemispheric compensation involving ipsilateral secondary centres; if ipsilateral network components are severely damaged reduction of transcallosal inhibition causes activation of contralateral homotopic areas leading to interhemispheric compensation which is less effective. As a therapeutical consequence, inhibition of contralateral activity, e.g. by rTMS may improve the efficacy of speech therapy.
CBF DURING CHANGES IN HEAD OF BED POSITION IN ACUTE, ISCHEMIC STROKE MONITORED AT THE BED-SIDE: HEMISPHERIC EFFECT OF INFARCT
Turgut Durduran1,2, Chao Zhou2, Brian L. Edlow3, Guoqiang Yu2, Regine Choe2, Brett L. Cucchiara3, Mary Putt4, Qaisar Shah3, Scott E. Kasner3, Arjun G. Yodh2, Joel H. Greenberg3, John A. Detre3
1Department of Radiology, University of Pennsylvania, Philadelphia, PA, USA, 2Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, PA, USA, 3Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA, 4Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, PA, USA
Introduction: In acute ischemic stroke, a flat head of bed (HOB) position is often used to improve perfusion to regions with presumably impaired autoregulation of CBF. Direct measurement of CBF at the microvascular level and at the bed-side is currently not feasible in intensive care units. We have developed an optical technique ? diffuse correlation spectroscopy ? that has enabled us to measure CBF changes in acute, ischemic stroke during changes in head of bed position (HOB). Hypothesis: CBF response to changes in HOB position on the infarcted hemisphere will be large compared to the “healthy” hemisphere providing evidence of impaired autoregulation in ischemic or penumbral cortex. Methods: Optical measurements of relative CBF during HOB manipulations were obtained from patients with acute hemispheric ischemic stroke (n=17) with probes placed on the forehead measuring CBF near the frontal poles. CBF was measured sequentially for 5 min each at HOB positions: 30, 15, 0, −5 and 0 degrees and was normalized to its value at 30 degrees. A linear mixed-effects model was used to analyze the statistical significance of relevant parameters. Results: A representative figure of a SF patient with unilateral ischemic stroke is shown. HOB position was found to be a significant factor (p<0.05) in both hemispheres, but was a stronger factor in the infarcted area (p<0.02) which also showed a larger variation. However, “time after stroke” was not a significant factor (p=0.45). Repeated measurements at flat HOB showed excellent repeatability at each study day (R>0.9). Conclusion: Diffuse optical correlation spectroscopy permits the measurement of relative CBF at the bedside. With further improvements in this technology, these measurements will provide valuable information permitting the manipulation of hemodynamic parameters so as to maximize the management of acute stroke patients.
IMAGING APOPTOSIS BY SPECT AFTER STROKE AND THE EFFECT OF MINOCYCLINE ON REDUCING APOPTOTIC CELL DEATH
Xiannan Tang1,2, Francis Blankenberg3, Danye Cheng3, Midori Yenari1
1Department of Neurology, UCSF and SF VAMC, San Francisco, CA, USA, 2Department of Anesthesia, Stanford University, Palo Alto, CA, USA, 3Department of Radiology, Stanford University, Palo Alto, CA, USA
Introduction: Programmed cell death (apoptosis) is thought to play a role in brain injury following stroke. There are obvious advantages to being able to image apoptosis and the progression of stroke noninvasively. Annexin V is an endogenous protein with high affinity to phosphatidylserine (PS). Externalization of membrane PS is an early sign of apoptosis. We previously showed that radiolabeled annexin V can be used to noninvasively detect apoptosis in stroke models, and that annexin V binding occurs primarily in ischemic neurons. Minocycline is a common antibiotic that has strong anti-apoptotic and anti-inflammatory properties. Because of these properties minocycline may be of benefit in reducing neuronal apoptosis from ischemia and subsequent post-ischemic inflammation if administered soon after a stroke. In this study we explored whether 99mTc-annexin V imaging using SPECT (single photon emission computed tomography) could be used to monitor a potential treatment response to minocycline in experimental stroke. Methods: CB6/F1 adult male mice underwent unilateral distal middle cerebral artery occlusion (dMCAO) and were survived 1, 3, 7 and 30 days. Animals were given 45 mg/kg minocycline (or vehicle) i.p. 30 min and 12 hour after dMCAO, then 22.5 mg/kg twice daily up to the time of sacrifice. In each group, mice were injected with 5-10 mCi of 99mTc Annexin V 2 hours before undergoing SPECT. Sensorimotor function were studied using the cornerand ladder tests, then brains were collected for study of infarct volume and assessed for apoptosis using TUNEL staining and activated microglia using isolectin B4 (IB4) histochemistry. Double labeling was performed to identify neurons (NeuN), microglia (isolectin B4 or tomato lectin) and astrocytes (GFAP) that bound annexin V. Results: 99mTc-annexin V uptake in the injured (left) hemisphere was significantly increased by ischemia, and was decreased 2–3 fold by minocycline at all time points. Minocyline reduced infarct size as seen histologically and improved behavioral indices up to 30 d. The pattern of infarct volume histologically measured at different time points after stroke is highly consistent with that measured by SPECT. This was correlated to reduced numbers of TUNEL positive cells (P<0.05) & microglia (P<0.01) positive cells among treated mice. Double labelling studies demonstrated that annexin V bound mostly neurons at 1 and 3 d, and microglia at 7 and 30 d. Conclusions: Minocycline significantly reduces neuronal apoptosis and infarct size, and improves neurological outcome in mice following acute focal cortical ischemia. This correlated to patterns observed on annexin V SPECT imaging, and suggests that this may be a novel way to noninvasively monitor the progression of stroke and response to therapy.
CENTRAL ROLE OF ASTROCYTIC GLUTAMATE UPTAKE ACTIVITY IN ODOR-EVOKED FUNCTIONAL SIGNALS IN OLFACTORY GLOMERULI
Hirac Gurden1,2,3, Barbara L'Heureux1, Frederic Pain1, Martine Guillermier2, Laurent Pinot1, Francoise Lefebvre1, Albertine Dubois2, Thierry Delzescaux2, Roland Mastrippolito1, Philippe Hantraye2, Gilles Bonvento2, Zach Mainen3, Philippe Laniece1
1Cnrs Umr8165, Imnc, Orsay, France, 2CNRS-CEA URA221, MIRCen, Fontenay-Aux-Roses, France, 3Cshl, New-York, NY, USA
Functional imaging signals are linked to metabolic and vascular activity, but how these processes are related to neural activity is not well understood. Recent data suggest that astrocytes play a key role in the mechanisms leading from neural transmission to functional signals. To provide direct insights into this issue, we studied the olfactory glomeruli (the functional modules allowing the first step of olfactory coding in the brain) where astrocytes express both GLT1 and GLAST glutamate transporters. To record odor-evoked metabolic and vascular changes in rat and mouse glomeruli, we used 1) imaging of intrinsic optical signals (IOS) 2) blood flow measurement by laser-Doppler flowmetry (LDF) 3) [14C]-2-Deoxy-D-Glucose (2DG) uptake.
First, we recorded IOS maps in both species in response to different odorants (phenol and benzaldehyde, 0.2 to 2% of saturated vapor) with changes in reflectance up to 1%. Second, using LDF, we measured more specifically blood flow in the local vascular network of the mouse glomeruli in response to benzaldehyde. LDF showed stimulus onset-locked and odor-specific blood flow increases from 20 to 30% in both species. Finally, we observed ~20% increase in glucose uptake among the subset of lateral and medial glomeruli activated bilaterally by benzaldehyde in awake mice.
We report that 1) blocking astrocytic glutamate transporters GLT1 and GLAST by TBOA blocks IOS (Gurden et al., 2006): this optical signal is reduced by about two-thirds, 36 ± 6% of control, P < 0.005 (t test, “control vs treated bulb”, n=5) but 2) 2DG uptake is not significantly different in the olfactory bulb of GLT1 knock-out mice compared to wild-types: respectively 17 ± 4% vs 20 ± 3% of relative 2DG uptake increase, n=6 in each group (the % represents relative changes calculated by the ratio of optical density values in activated / non-activated glomeruli). Thus, instead of GLT1, GLAST might have a central role in the neuro-energetic coupling in the olfactory glomeruli leading us to start the study of GLAST knock-out mice.
In conclusion, activation of olfactory glomeruli in rat and mouse is a suitable experimental system for the study of physiological neurovascular and neurometabolic couplings in vivo. According to our results, astrocytic transporter activity is crucial for the induction of functional optical signals. Finally, we believe that our current work on astrocytic transporter knock-out will help to determine whether glutamate uptake activity is the central signaling pathway leading from synaptic transmission to functional signals.
Supported by a Neuroscience grant from the ≪ Agence Nationale de la Recherche ≫ (Astroglo 2006).
IN VIVO DETECTION OF ADULT NEURAL STEM CELLS LABELLED IN SITU USING FERUMOXIDE-POLYCATION COMPLEX
Rachael Dobson1,2, David L. Thomas3, David G. Gadian1, Patrizia Ferretti2, Mark F. Lythgoe1
1RCS Unit of Biophysics, UCL Institute of Child Health, University College London, London, UK, 2Developmental Biology Unit, UCL Institute of Child Health, University College London, London, UK, 3Wellcome Trust High Field Laboratory, Department of Medical Physics and Bioengineering, University College London, London, UK
Introduction: Neural stem cells (NSC) are located in the subventricular zone (SVZ) of the lateral ventricles in the adult brain. This population migrates towards the olfactory bulb along the rostral migratory stream (RMS), and has been shown to respond to ischaemic injury through increased neurogenesis and migration toward the infarct. Monitoring the timecourse of this behaviour in vivo has not been achieved with any imaging modality. Endogenous labelling with MRI contrast agents has been problematical due to diffusion of free agent outside the ventricles and low labelling efficiency. The polycation protamine sulphate enhances viral gene transfer through increased cell membrane contact and aggregation of virus. When complexed with Endorem, a ferumoxide contrast agent, protamine sulphate increases NSC labelling efficiency four-fold in vitr0. This complex, FePro, has been used to label stem cells in vitro. Here we demonstrate the utility of FePro to label adult NSC in situ in the SVZ, and we assess its labelling efficiency and regional distribution using MRI compared to Endorem alone.
Methods: Rats were injected with 2µl FePro or Endorem into the left lateral ventricle. FePro injected rats were sacrificed and perfused fixed with 4% paraformaldehyde at 24 hours, 7 days, and 28 days for histology, and scanned at 7 days and 28 days. Endorem-injected rats were scanned at 7 days and sacrificed. Animals were scanned using a 2.35T magnet interfaced to a SMIS console, with 2DFT coronal and sagittal spin echo sequences (TR 1500 ms; TE 120 ms; FOV 30 mm), and 3D gradient echo sequence (TR 80 ms; TE 40 ms; FOV 30 mm). Histological sections were stained with Prussian blue (PB) for iron detection and with Hematoxylin/Eosin, or for fluorescence immunohistochemistry with doublecortin and nestin.
Results: In Endorem-injected animals at 7 days, the injected contrast agent had diffused across the septal region into the right ventricle and along the RMS but did not localise with cells. In FePro-injected animals at 24hr, 7 days and 28 days, the contrast agent was localised to the left ventricle and some diffusion caudally into the left third and fourth ventricles occurred at later timepoints. At 28 days, signal was detected in the RMS. Immunohistochemistry confirms the presence of doublecortin-positive migrating cells in the SVZ and RMS. Histological sections show the FePro localising with cells in the SVZ, and with cells in the RMS.
Conclusions and discussion: Directional movement of free endorem along the RMS may be caused by the directional flow of CSF created by ciliary epithelium in the SVZ. The FePro complex prevents diffusion of endorem outside of the ventricles over a long period of time and enhances cell labelling. This may be due to restricted diffusion of the FePro complex through increased cell membrane interaction and increased internalisation of the complex. This labelling technique may provide an important methodology for monitoring the behaviour of endogenous stem cells in response to CNS injury using MRI.
CEREBRAL BLOOD FLOW AND METABOLISM IN PATIENTS WITH SILENT BRAIN INFARCTION
Ashkan Mowla1, Reza Gharebaghi2
1School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran, 2Shahed University, Tehran, Iran
OBJECTIVES: To evaluate CBF and metabolism in patients with Silent brain infarction (SBI) in relation to symptomatic stroke.
METHODS: The patients underwent PET and were separated into three groups; control group, with no lesions on CT (n=10, mean age 55), SBI group, with no neurological signs or history of stroke, but with ischaemic lesions on CT (n=10, mean age 61), and brain infarction group (BI group), with neurological deficits and compatible CT lesions in the area supplied by perforating arteries (n=20, mean age 56). Regional CBF, oxygen extraction fraction (OEF), cerebral metabolic rate for oxygen (CMRO2), and cerebral blood volume (CBV) were measured by PET.
RESULTS: Mean values for CBF to the cerebral cortex and deep grey matter were lower in the SBI group (32.5 (SD 5.8) and 33.3 (SD 6.9) ml/100 g/min, respectively) and in the BI group (30.8 (SD 5.2), 32.9 (SD 5.8), respectively) than in the Control group (36.0 (SD 6.6) and 43.5 (SD 9.5), respectively). Although mean CMRO2 of deep grey matter (2.36 (SD 0.52) ml/100 g/min) was significantly decreased in the SBI group compared with the control group (2.76 (SD 0.480), p<0.01), CMRO2 of the cortical area was as well preserved in the SBI patients (2.36 (SD 0.39)) as in the controls (2.48 (SD 0.32)) with a compensatory increase of mean OEF (0.45 (SD 0.06) and 0.41 (SD 0.05), respectively).
CONCLUSIONS: Patients with SBI showed decreased CBF and CMRO2 in deep grey matter. On the other hand, decreased CBF with milder increased OEF, resulting in preserved CMRO2 in the cerebral cortex indicates the presence of occult misery perfusion, suggesting that patients with SBI have reduced cerebral perfusional reserves.
MULTIVARIATE SYSTEM IDENTIFICATION OF CEREBRAL AUTOREGULATION
Tingying Peng1, Alexander. B. Rowley1,2, Stephen. J. Payne1
1Department of Engineering Science, University of Oxford, Oxford, UK, 2Computing Laboratory, University of Oxford, Oxford, UK
Background
Blood flow in the brain is controlled by numerous processes in both normal and pathological conditions. Techniques to assess the control of cerebral blood flow by systemic variables (such as arterial blood pressure, ABP) frequently attempt to use a linear time invariant system identification approach to assess the extent to which low frequency content in variables related to cerebral oxygenation (cerebral blood flow velocity, CBFV) can be explained by a linear dynamical system acting upon low frequency content in systemic variability (for example oscillations in mean arterial blood pressure). In the case of a univariate input-output relationship, the coherence function is typically found to be very low at low frequencies, which means that the validity of the procedure in identifying the system dynamics at these frequencies has been questioned. Many studies argue that the presence of low coherence indicates strong non-linearity of cerebral autoregulation.
However, CBFV variation is not entirely determined by pressure. Other mechanisms responsible for controlling blood flow include the reactivity of cerebral vessels to arterial CO2 and O2 levels. The observed low value of univariate coherence between ABP and CBFV in the low frequency region might be due to the additional input of CO2 and O2, rather than the strong non-linearity of the transfer path from ABP to CBFV. To test this hypothesis, the method of multiple coherence analysis has been used to investigate the contribution of CO2 and O2 to CBFV variability. Multiple coherence quantifies the extent to which the proposed output of the system can be explained by a linear combination of the input variables.
Methods
Data from 13 healthy subjects, with measurements of beat-to-beat spontaneous fluctuations in mean arterial blood pressure (ABP) and cerebral blood flow velocity (CBFV), breath-to-breath arterial CO2 (PaCO2) and O2 (PaO2) pressure fluctuations was used. The multiple coherence function of the relationship between an assumed CBFV output driven by input ABP, PaCO2 and PaO2 was computed using non-parametric system identification (division of cross and power spectra).
Results and conclusion
The additional inputs of important physiological variables, PaCO2 and PaO2, resulted in significantly higher values of multiple coherence for frequencies <0.05Hz than the corresponding value obtained for univariate coherence with only a single assumed input of ABP. This illustrates that when additional variables that could be thought to act in the control of cerebral blood flow are accounted for using multiple coherence, the reliability of linear system identification at low frequencies is improved. Moreover, it is also found that the transfer function between ABP and CBFV time series at low frequencies can be modified by CO2 and O2 reactivity and no longer represents pressure autoregulation only. The effect of this variability can be illustrated by demonstrating how autoregulation index as measured by Tiecks using univariate system identification techniques is increased when multiple variability is accounted for.
ACID-BASE MANAGEMENT AFFECTS ENDOTHELIUM-DEPENDENT CEREBRAL VASOREACTIVITY IN MODERATELY HYPOTHERMIC RATS
Izumi Yuzawa, Masaru Yamada, Kiyotaka Fujii
Department of Neurosurgery, Kitasato University School of Medicine Sagamihara, Kanagawa, Japan
Background and aims: The effect on the cerebral circulation of acid-base management under moderate therapeutic hypothermia is not fully understood. We examined cerebral vasoreactivity to topically administered vasodilators and hypercapnia under conditions of normothermia (37°C), induced hypothermia (30°C) with alpha- and pH-stat management, and rewarmed normothermia in rats.
Methods: Using laser-Doppler flowmetry and the closed cranial window method, we measured changes in regional cerebral blood flow (rCBF) and pial arteriole reactivity. rCBF changes during carbon dioxide (CO2) inhalation, and vasoreactivity to acetylcholine (Ach), an endothelium-dependent vasodilator, sodium nitroprusside (SNP), an endothelium-independent nitric oxide (NO) donor, and cilostazol, a phosphodiesterase III inhibitor were compared in normothermic rats, hypothermic rats subjected to alpha- and pH-stat management, and in rats rewarmed to normothermia.
Results: Ach-induced vasodilation was significantly attenuated under alpha-stat- but not pH-stat management and rewarming completely restored the normal vasodilatory response. CO2 reactivity was preserved under both acid-base management protocols.
Conclusion: At moderate hypothermia, endothelium-dependent vasodilation is reversibly impaired under alpha-stat- but not pH-stat conditions, indicating that acid-base management significantly affects endothelial cell function under moderate hypothermia.
LINEARIZABILITY OF NEURO-VASCULAR COUPLING RESULTING FROM LOW-FREQUENCY INTERMEDIATE-DURATION STIMULI
Tina Rasmussen1,2,3,4, Martin Lauritzen3,4, Ryuta Kawashima1
1Institute of Development Aging and Cancer, Tohoku University, Sendai, Japan, 2Department of Control Engineering, Institute of Electronic Systems, Aalborg University, Aalborg, Denmark, 3Department of Medical Physiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark, 4Department of Clinical Neurophysiology, Glostrup University Hospital, Glostrup, Denmark
Previous studies [Ances et al. 2000, Martindale et al. 2003 and 2005] have investigated neuro-vascular coupling in rat somato-sensory cortex resulting from stimulation with a single frequency and a single duration. They have shown that this system can be approximated with a linear dynamic mathematical model when the stimulation evoking the system dynamics has a low frequency, that is, 5 Hz or less, and a short duration, that is, 5 s or less.
We investigate the limits of linearizability in rat cerebellar cortex. We present evidence that this system can be approximated with a linear dynamic mathematical model when the stimulation frequency is kept below 7 Hz and the stimulation duration is fixed at 15 s (an intermediate duration). Specifically, when the stimulation duration is fixed to 15 s, a single model is able to cover the system dynamics resulting from multiple frequencies in the range 0.5–7. Hz.
We furthermore investigate the coupling during application of stimulation with frequencies in the range 2-30 Hz and durations of 60 s and 600 s (long durations). Based on the characteristics of the deviations of the experimental data from a linear dynamic model, we hypothesize that the neuro-vascular system in rat cerebellar cortex can be linearized for durations up to 20 s and frequencies up to 7 Hz.
CNTF-ACTIVATED ASTROCYTES DISPLAY METABOLIC PLASTICITY AND INCREASED RESISTANCE TO METABOLIC INSULTS
Carole Escartin1,4, Karin Pierre4, Emmanuel Brouillet1, Thierry Delzescaux2, Martine Guillermier1, Marc Dhenain1, Nicole Deglon1,2,3, Philippe Hantraye1,2,3, Luc Pellerin4, Gilles Bonvento1
1CEA, DSV, DRM, CNRS URA 2210, Service Hospitalier Frederic Joliot, Orsay, France, 2CEA, DSV, DRM, U2IBP, Service Hospitalier Frederic Joliot, Orsay, France, 3MIRCen, CEA, Fontenay-aux-Roses, France, 4Departement De Physiologie, Universite De Lausanne, Lausanne, Switzerland
Introduction: High energy demands of neurons make them vulnerable to adverse effects of energy impairment. Astrocytes have been shown to regulate the flux of energy substrates to neurons. In pathological situations, astrocytes are activated but the consequences on brain energy metabolism are still poorly characterized.
Materials and Methods: To characterize the metabolic profile of reactive astrocytes, we used our two previously described models of astrocyte activation through either lentiviral-gene transfer of the cytokine ciliary neurotrophic factor (CNTF, a cytokine known to activate astrocytes) in the rat striatum in vivo or exposure of primary neurone astrocyte mixed cultures to recombinant CNTF (1). We assessed 2-[18F]-deoxy-D-glucose (FDG) uptake in vivo using autoradiography and micro-PET, the level of expression of different proteins by immunoblots and fluorescent immunohistochemistry, the rate of oxidation of both [14C]-3-hydroxybutyrate (BHB) and [14C]-palmitate in vitro and finally the effects of toxic levels of extracellular fatty acids (0.2mM palmitate for 24h) and glycolysis (0.4 mM iodoacetate for 10 min) inhibition upon neuronal survival in vitro.
Results: We found that CNTF-induced astrocyte activation in the striatum was associated with a marked rearrangement of metabolic features in absence of any pathological process. Glycolysis was stably reduced, AMP-activated protein kinase (AMPK, a central regulator of energy metabolism) was activated both in vivo and in vitro and key proteins of ketolysis (monocarboxylate transporter 1 MCT1, succinyl-CoA:3-oxoacid CoA-transferase, SCOT and beta-hydroxybutyrate dehydrogenase, BDH) and β?oxidation (carnityl-palmityl transferase, CPT1, the rate-limiting enzyme for β-oxidation of fatty acids) were overexpressed by CNTF-activated astrocytes in vivo. In vitro, the oxidative rate of both fatty acids and ketone bodies was significantly enhanced by 100 and 30%, respectively. This metabolic plasticity of CNTF-activated astrocytes significantly improved their ability to resist to glycolysis inhibition, ultimately providing protection to neurons. Such an effect was partially mediated by the transport of monocarboxylates. We finally found that CNTF-activated astrocytes, which perform β-oxidation two times more efficiently than controls, were totally resistant to toxic palmitate levels in vitro.
Conclusion: These results show that CNTF-activated astrocytes, with their increased capacity to use alternative energetic substrates besides glucose, have a strong protective potential to face severe metabolic insults and confirm their crucial role as ‘metabolic nurses' for neurons.
NEUROPROTECTIVE EFFECT OF XG-102 ADMINISTERED ALONE OR IN COMBINATION WITH TISSUE PLASMINOGEN ACTIVATOR
Karine Wiegler1, Christophe Bonny2, Didier Coquoz3, Lorenz Hirt1
1Department of Neurology, CHUV, Lausanne, Switzerland, 2Department of Medical Genetics, CHUV, Lausanne, Switzerland, 3Xigen Pharmaceuticals, Lausanne, Switzerland
Background
XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide which inhibits the c-jun N-terminal kinase (JNK), has been shown so far to be a powerful neuroprotective drug in different rodent models of cerebral ischemia (Borsello et al., Nat Med, 2003; Hirt et al., Stroke, 2004) when administered by intra-cerebro-ventricular injection (i.c.v.). A high degree of protection was reached even with delayed treatment, as late as 6 hours after 30 minute MCAo. As i.c.v. administration is not convenient in stroke patients, it was necessary to determine the feasibility of systemic administration. We also evaluate in an in vitro model of ischemia, the effect of combined administration of XG-102 and tissue plasminogen activator (TPA), used to restore blood flow in ischemic stroke patients but known to exacerbate excitotoxicity.
Methods
Young adult male mice ICR-CD1 were subjected to 30 min transient suture MCAo followed by reperfusion, with laser Doppler monitoring of regional cerebral blood flow. XG-102 at different concentrations, or vehicle, was administered intravenously (i.v.) 6 hours after ischemia onset firstly to determine the dose range and the efficacy of the protection at different doses; then a single dose was chosen for a blinded experiment. Efficacy of protection was also evaluated at 9 hours after MCAo. Lesion size was determined after 48 hours by HE staining. Neurological outcome was evaluated by neurological scores and rotarod tests. We used a model of rat organotypic hippocampal slice cultures subjected to oxygen (5%) and glucose deprivation (OGD) for 30 minutes (de Castro et al., Exp Neurol, 2006) to determine whether it is possible to combine XG-102 and TPA. Cell death was evaluated by PI staining at 48h.
Results
XG-102 administered i.v. 6 hours after ischemia onset significantly reduced the infarct volume at 48 hours. The lowest dose with maximal neuroprotection, was 0.3 µg/kg, which reduced the infarct volume from 62+/−19mm3 (n=18) for the vehicle-treated group to 18+/−9mm3 (P<0.001, n=5). A dose of 1 mg/kg tested blindly provided a very significant reduction (approximately 70%) of the lesion volume, from 52+/−22mm3 (n=8) to 13+/−2mm3 (P<0.0001, n=9). The behavioural outcome after transient MCAo was also considerably improved. Administration of XG-102 9 hours after MCAo did not significantly reduce the infarct.
In organotypic slices, TPA alone (0.9 and 9 µg/ml) administered immediately after OGD, increased cell death due to its excitotoxic properties. However the addition of XG-102 (12 nM), 6 hours after OGD onset, induced a strong reduction (P<0.001) of cell death compared with vehicle treated slices, in the presence of TPA (49+/−19%, n=20 vs 12+/−14%, n=24).
Conclusion
XG-102 is a powerful neuroprotectant in our mouse stroke model, and can be administered i.v. up to 6 hours after MCAo. The feasibility of systemic administration is an important step towards the development of XG-102 as a candidate drug in human ischemic stroke. The fact that XG-102 does not interact with TPA in vitro, potentially opens the feasibility to combine these two drugs to treat stroke patients.
Financial support
CTI #7057.2
A NOVEL INHIBITOR OF ADVANCED GLYCATION AND ENDOPLASMIC RETICULUM STRESS REDUCES INFARCT VOLUME IN RAT TRANSIENT FOCAL ISCHEMIA
Shunya Takizawa1, Yuko Izuhara2, Yuko Morita1, Tsuyoshi Uesugi1, Shigeharu Takagi1, Toshio Miyata2
1Division of Neurology, Department of Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan, 2Institute of Medical Sciences 2, Tokai University School of Medicine, Isehara, Kanagawa, Japan
Background and aims: Angiotensin II receptor blockers inhibit formation of advanced glycation end products (AGEs) and reduce oxidative stress, and should therefore ameliorate ischemic cerebral damage, but their hypotensive effect limits their clinical use. We have discovered a novel non-toxic inhibitor of advanced glycation and oxidative stress, TM2002, by means of high-throughput screening of the anti-oxidative and AGEs-lowering activities of over 1300 compounds. We here demonstrate its in vivo effectiveness in both transient and permanent focal ischemia models in rats.
Methods: TM2002 (5.58 mg/kg twice a day) was administered intravenously in a rat transient focal ischemia model, and we evaluated its effect on infarct volume and neurological function. Brain tissues were examined immunohistochemically for heme oxygenase-1, advanced glycation end products (AGEs), oxygen regulated protein (ORP) 150, heat shock protein (HSP) 70i, HSP60, and TUNEL. Further, in a photothrombotic occlusion model, we evaluated the dose-dependence of the effect of TM2002 on infarct volume and neurological functions, conducted time-window studies of its neuroprotective efficacy, and compared its effect on infarct volume with that of the known neuroprotective agent NXY-059.
Results: There was no statistically significant difference in any physiological parameter, including mean arterial blood pressure, between the TM2002- and vehicle-treated groups throughout the experiment. TM2002 significantly reduced infarct volume compared with the vehicle (79.5 ± 52.8 vs. 183.3 ± 68.7 mm3, p < 0.01), and reduced the numbers of cells immunohistochemically positive for heme oxygenase-1, AGEs, ORP150, and TUNEL, especially in the penumbra, as compared with the vehicle-treated group. Further, in the rat photothrombotic occlusion model, TM2002 (2.79, 5.58, and 11.16 mg/kg twice a day) dose-dependently reduced the infarct volume (to 242.1±102.3, 201.3±47.7, and 171.3±48.0 mm3, respectively), and improved the neurological deficits. In this model, TM2002 reduced infarct volume even when administered 1 hour after occlusion, and the efficacy of TM2002 was equivalent to that of NXY-059.
Conclusions: Our novel synthetic compound significantly ameliorated ischemic cerebral damage through reduction of ER stress, advanced glycation, and oxidative stress, independently of blood-pressure lowering. Since the efficacy of TM2002 was equivalent to that of NXY-059 in the photothrombotic occlusion model, a clinical trial of TM2002 in stroke patients might be warranted.
ISCHEMIC TOLERANCE INDUCED BY REPEATED HYPERBARIC OXYGEN IN RAT BRAIN: TIME WINDOW AND GENOME-WIDE GENE AND PROTEIN EXPRESSION
Takao Hirata1, Ying Jun Cui1, Takeshi Funakoshi2, Yoichi Mizukami2, Yu-ichiro Ishikawa3, Futoshi Shibasaki4, Mishiya Matsumoto1, Takefumi Sakabe1
1Department of Anesthesiology and Resuscitology, Yamaguchi University School of Medicine, Yamaguchi, Japan, 2Center for Gene Research, Yamaguchi University, Yamaguchi, Japan, 3Center for Microarray Analysis, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, 4Department of Molecular Cell Physiology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
Background and Aims
Central nervous system acquires tolerance against ischemic insult when the neuronal tissues are exposed to a brief period of ischemia before they are subjected to otherwise lethal ischemia. If clinically applicable measure other than ischemic preconditioning to induce tolerance (cross-tolerance) against ischemia is established, it will provide robust therapeutic strategy in ischemic conditions. Among the various preconditioning stimuli that might produce cross-tolerance, Hyperbaric oxygen (HBO) is attractive because it is safe and has been already used for various diseases. In the present study we examined the effects of repeated HBO on neuropathological changes in hippocampal CA1 after forebrain ischemia as well as on genome-wide gene and protein expression in rats.
Methods
Wistar rats were used. We first examined the effects of HBO (3.5 ATA, 100% oxygen, 1h each day for 5 days) with various intervals (6h, 12h, 24h, and 72h, n=8, in each) between the last HBO and forebrain ischemia (8 min) on neuropathological outcome in the hippocampal CA1 (7 days after ischemia). The rats in the untreated group (n=8) were only exposed to 21% O2 (1 ATA) (sham treatment) for 5 consecutive days and exposed to forebrain ischemia 24h after the last HBOexposure.
Then, we conducted genome-wide gene expression analysis of hippocampal CA1 region using an oligonucleotide-based DNA microarray containing 20,500 probe sets at the same time points after HBO or sham treatment sessions (n-5, in each) without subjecting animals to ischemia. Based on microarray dataset, we then selected genes, whose expression patterns correspond to time course of histological protection, and examined their translational level by Western blotting.
Results
Repeated exposure to HBO induced tolerance, which was achieved with an interval of 6h, 12h, and 24h (survived CA1 neurons 55%, 75%, and 53%, respectively), but faded when the interval was elongated to 72h (survived neurons only 6%). Analysis on microarray data showed significant up-regulation in 60 probe sets including 7 annotated genes (p75NTR:p75 neurotrophin receptor; C/EBP Δ:CCAAT enhancer binding proteinΔ; CD74:CD74 antigen; Edg2, Trip10, Nrp1, and Igf2), whose time-course expression corresponded to HBO-induced neuroprotection. The protein levels of p75NTR, C/EBPΔ, and CD74 were significantly increased (maximum fold-changes 2.9, 2.0, and 7.9, respectively).
Conclusions
These results suggest that HBO induces cross-tolerance against ischemic injury, which exhibits time window, protective at 6h, 12h and 24h but not protective at 72h. Although the precise interaction is to be determined, the genes/proteins relevant to neurotrophin and inflammatory-immune system may be involved in HBO-induced neuroprotection.
THE PROTECTIVE EFFECT OF RAPID ISCHEMIC PRECONDITIONING ON THE PI3K/AKT PATHWAY IN FOCAL ISCHEMIA IN RAT
Hanfeng Zhang1, Xuwen Gao1, Gary Steinberg1,2, Heng Zhao1,2
1Department of Neurosurgery, Stanford University, Stanford, CA, USA, 2Stanford Stroke Center, Stanford University, Stanford, CA, USA
Background and Aims: Two protective time windows for preconditioning have been identified: rapid preconditioning, performed 30 min to one hour before onset of a subsequent severe ischemia, and delayed ischemic tolerance, performed one to several days before a severe ischemia. Only a few reports are available for rapid ischemic tolerance, and the protective mechanisms of rapid tolerance are largely unexplored. Here we investigate whether rapid preconditioning reduces infarction in a focal ischemic model generated by permanent distal MCA occlusion plus transient bilateral CCA occlusion and study its potential protective mechanisms. It is well documented that the Akt/PKB survival pathway is involved in the protective effect of delayed preconditioning in global ischemia. However, few studies have addressed its role in focal ischemia and none have studied its role in rapid preconditioning. Akt activity is regulated by phosphorylation via several molecules upstream of the Akt kinase. Akt is activated via phosphoryation by PDK1 (either directly or indirectly). Conversely, PTEN dephosphorylates and inactivates Akt. Upon activation, Akt phosphorylates GSK3β and downregulates its activity. Phosphorylation of β-catenin by GSK3β leads to degradation of β-catenin and apoptosis. Here we report that rapid preconditioning reduces infarction in a distal permanent MCA occlusion model and this protection correlates with activation of the Akt pathway.
Methods: Focal ischemia was generated by permanent distal MCA occlusion plus 30 min of transient bilateral CCA occlusion in male SD rats. Rapid ischemic postconditioning was performed by occluding the left distal MCA for 15 min. Test ischemia was induced 1h later. For Western blot, ischemic brains were harvested 1, 5 and 24 h after stroke. Whole cell homogenates from the ischemic core and penumbra were prepared for Western blots. Protein levels of phosphorylated-Akt (Ser473) (P-Akt), P-PDK1, P-PTEN, activated β-catenin and pan-β-catenin were measured. In vitro Akt kinase assays were also performed to evaluate Akt activity.
Results: Rapid ischemic preconditioning significantly reduced infarct size as measured 2 days post-stroke (P<0.05). P-Akt transiently increased from 1 to 5h in the ischemic penumbra (P<0.05, n=5-7, Two way NOVA) but not in the ischemic core. Preconditioning did not further enhance protein level of P-Akt at 1 and 5h, but attenuated its decreases at 24h in both the penumbra and core. Akt kinase assays showed that preconditioning attenuated decreases in Akt activity at 5 and 24h in the penumbra (P<0.05). Levels of P-PDK1 decreased from 5h after stroke, but there was no difference between animals treated with and without preconditioning. The levels of P-PTEN (inactive PTEN) decreased after stroke, and preconditioning attenuated its decreases at 24h in both the penumbra and core. Cleaved β-catenin appeared as early as 1h and the levels of cleavage products further increased at 24h in both the penumbra and core. Preconditioning attenuated β-catenin cleavage at 24h in both the penumbra and core.
Conclusion: Rapid preconditioning reduces infarction in a distal permanent MCA occlusion model, and this correlates with activation of the Akt survival pathway.
THE BK CHANNEL OPENER NS1619 INDUCES NEURONAL PRECONDITIONING IN VITRO
Tamas Gaspar1, Ferenc Domoki1,2, Keita Mayanagi1, James A. Snipes1, Ferenc Bari2, David W. Busija1
1Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston Salem, NC, USA, 2Department of Physiology, Faculty of Medicine, University of Szeged, Szeged, Hungary
Background and aims. Targeting the large conductance Ca activated potassium (BK) channels is a novel approach to induce preconditioning (PC). NS1619 (NS) is a selective opener of the BK channel and was shown to protect the heart against an otherwise lethal ischemic insult. Recently, the BK channel has been reported to be present in the inner mitochondrial membrane of rat neurons, but its role in tolerance induction in the central nervous system has not yet been explored. The aim of our in vitro study was to examine whether NS can induce PC and protect rat cortical neuronal cultures against toxic stimuli. We also investigated its effect on reactive oxygen species (ROS) production, plasma membrane potential, and expression and activity of cytoprotective proteins.
Methods. Cortical neurons of 18-day old Sprague-Dawley rat fetuses were cultured in B27 supplemented Neurobasal medium. One week after plating, a 6-hour daily treatment with different doses of NS (10 ? 100 µM) for 3 days was introduced. Eighteen hours after the last treatment, neuronal cultures were exposed to 180 min of oxygen and glucose deprivation (OGD), 6 hour of exogenous hydrogen peroxide toxicity (80 µM), or 60 min of glutamate excitotoxicity (200 µM). Cell viability was evaluated with CellTiter 96 AQueous One Solution assay. ROS generation, plasma and mitochondrial membrane potentials were monitored using hydroethidine, di-8-ANEPPS, and tetramethylrhodamine ethyl ester, respectively. The expression of manganese dependent superoxide dismutase (MnSOD), cupper and zinc dependent superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), and catalase was examined using Western blotting. Enzyme activity of antioxidants was assessed with a microplate reader using commercially available kits.
Results. Application of NS increased ROS generation, depolarized mitochondria, and hyperpolarized the cell membrane of neurons. Three-day treatment with NS dose-dependently protected the cell cultures against OGD, H2O2, and glutamate, although higher concentrations of the compound were needed to elicit PC than previously reported in the heart. The highest tolerance was observed against H2O2 where a dose of 40 µM provided complete protection (viability: control, 100.0±1.97%; untreated, 50.1±2.81%*; NS 40 µM, 93.2±3.25%#; *p<0.05 vs. control; #p<0.05 vs. untreated), whereas even higher doses were required against OGD (viability: control, 100.0±1.61%; untreated, 45.9±0.58%*; NS 100 µM, 88.4±0.56%*#; *p<0.05 vs. control; #p<0.05 vs. untreated), and the protection was found to be incomplete against glutamate (viability: control, 100.0±1.21%; untreated, 48.2±0.42%*; NS 100 µM, 74.4±1.06%*#; *p<0.05 vs. control; #p<0.05 vs. untreated). Co-treatment with the BK channel antagonist Paxilline did not block ROS generation and did not antagonize the neuroprotective effect of NS. Despite the protection afforded by 40 µM NS against H2O2, this concentration of the compound did not change the activity and/or expression of ROS scavengers.
Conclusion. Our results demonstrate that NS1619 is a potent neuroprotective agent in vitro. However, the need of relatively high doses of NS1619, and the effects on the cell membrane potential indicate the involvement of non-mitochondrial mechanisms in the induction of neuroprotection.
NEUROPROTECTIVE EFFECTS OF GLNVA ON INFLAMMATION-INDUCED DOPAMINERGIC NEURONAL CELLS DEATH
Yi-Ching Lo, Yi-Chin Lin
Department and Graduate Institute of Pharmacology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan-R.O.C.
Background and aims: Glyceryl nonivamide (GLNVA) has been reported to prevent subarachnoid hemorrhage-induced cerebral vasospasm. In this study, the anti-inflammatory and neuroprotective effects of GLNVA were investigated in mouse microglial BV-2 cells and human dopaminergic neuron SH-SY5Y cells.
Methods: we characterize the anti-inflammatory and neuroprotective effects of GLNVA on lipopolysaccharide (LPS)-induced neuronal cytotoxicity by measuring the ratio of dead cells, production of inflammatory cytokines? anti-inflammatory cytokine and the inflammatory protein expression.
Results: The results showed that pretreatment of BV-2 with GLNVA diminished LPS-induced nitric oxide production, overexpression of iNOS and gp91phox and intracellular ROS. GLNVA also reduced COX-2 expression, IκBα / IκBβ degradation and the overproduction of TNF-α, IL-1β and PGE2. However, GLNVA augmented anti-inflammatory cytokine IL-10 production on LPS-stimulated BV-2 cells. In co-culture conditions, we employed LPS stimulated BV-2 as a reactive microglial model and GLNVA decreased the reactive microglial neurotoxicity-induced cell death and overexpression of nNOS and gp91phox on SH-SY5Y cell.
Conclusions: GLNVA possesses neuroprotective effect by inhibiting oxidative stress and inflammation induced by LPS.
CYCLOSPORIN-A PREVENT MITOCHONDRIAL PERMEABILITY TRANSITION SIMILARLY IN BOTH BRAIN CORTEX AND SPINAL CORD
Saori Morota1,2, Magnus Hansson J.3, Yoshihisa Kudo1, Eskil Elmer3, Nagao Ishii2, Hiroyuki Uchino2
1Laboratory of Cellular Neurobiology, Tokyo University of Pharmacy and Life Science, Hachioji, Japan, 2Department of Anesthesiology, Tokyo Medical University Hachioji Medical Center, Tatemachi, Department of Anesthesiology, Tokyo Medical University Hachioji Medical Center, Hachioji, Japan, 3Laboratory for Experimental Brain Research, Department of Clinical Sciences, Lund University, Lund, Sweden
The mitochondrial permeability transition (mPT) is defined as sudden increase in permeability of the inner mitochondrial membrane and is primarily caused by calcium overload. mPT has been increasingly implicated as a potential pathogenetic mechanism in neurodegeneration. Varying sensitivity to mPT has been demonstrated for regions within CNS as well as between tissues. This variance has been thought that possibly contributes to different efficacy of mPT inhibitor Cyclosporin-A (CsA) on different region e.g. brain and spinal cord, and selective vulnerability. However, comparison of mitochondrial function and mPT sensitivity is possibly hampered by confounding influence due to different tissue handling. The objectives of present study were to stringently compare mitochondria from the brain cortex and spinal cord with respect to mPT sensitivity, calcium retention capacity (CRC) and respiratory function, and further, to re-evaluate the efficacy of CsA on mitochondria isolated from brain cortex and spinal cord. In present study, we induced whole-body hypothermia in animals before tissue extraction to counteract differential tissue degeneration during extraction. Overall, the features of mPT and cyclosporin-A sensitivity on administration of calcium bolus loads were similar. CsA also demonstrated equal relative improvement of CRC in these mitochondria although spinal cord mitochondria were able to retain less calcium than brain cortex mitochondria. Further we evaluated whole-body hypothermia of animal as a technique to minimize possible confounding influence by differential tissue handling and found it improve active phosphorylating rates of respiration. The present findings imply that the general features of mPT and the effect of CsA as mPT inhibitor are similar in brain cortex and spinal cord mitochondria.
A RCT OF MILD HYPOTHERMIA THERAPY IN SEVERE TRAUMATIC BRAIN INJURY IN JAPAN - A PRELIMINARY REPORT
Tsuyoshi Maekawa, Nariyuki Hayashi, Keiki Ogino, Jun Takezawa, Seigo Nagao, Yasuo Ohashi, Susumu Yamashita, Kotaro Kaneda, Ryosuke Tsuruta, Shunji Kasaoka
Hypothermia for Head Injury Study Group in Japan
Background and aims
Protection against brain insults is one of the hardest aspect in the clinical practice. Recently, mild hypothermia (MH) was applied to cardiopulmonary resuscitated victims and proved to have brain protective effects by two randomized clinical trials (RCT). While Clifton's group had failed in traumatic brain injury (TBI patients GCS :less than 8). To prove the effectiveness of MT therapy in TBI, the RCT has been continued in Japan Since 2001.
Methods
We have applied MH therapy in severe TBI patients and aimed to get 300 cases in 38 medical centers in Japan. Inclusion criteria are (1) GCS : 4–8 except best motor response of 6, (2) core body temperature must be controlled to 35.5 °C at 6 hours after head injury in the MH group, (3) age : 15-70 yo. Patients are randomized into either control (35.5~37.0 °C) group or MH (32.0~34.0 °C) group. Core body temperature must be controlled for at least 72 hr and the duration could be prolonged, if necessary. Their physiologic parameters, such as cardiac index, mixed venous and internal jugular venous blood oxygen saturation and temperature, and so on (8 parameters) are quantified and stored in every one minute until 72 hr after the onset of TBI. Outcome of MH therapy is evaluated by GOS at 3 and 6 months, and by biochemical parameters such as glutamate, heat shock protein, cytokines, and the others. At this point, 143 cases are enrolled. The key has not opened. The treatments (control of MH) blinded CSF samples were analyzed in the good outcome and the poor outcome cases in some institutes.
Results
Complications such as pneumonia during MH therapy seems to be much less than those of Clifton's RCT. Results of biochemical substrates in CSF are shown in the figure between the good outcome and the poor outcome cases.
Conclusion
RCT has been continued in Japan since 2001 and so far, 143 cases are enrolled. Although, the key has not open, the biochemical substrates in CSF are quite different between the good outcome and the poor outcome cases.
NEUROPROTECTIVE EFFECTS OF INCREASED FOREBRAIN MINERALOCORTICOID RECEPTOR EXPRESSION
Malcolm Macleod1, Karen Horsburgh2, Sung-Eun Bae3, Roderick Carter3, Dirk Stenvers3, Jill Fowler2, Joyce Yau3, Celso Gomez-Sanchez4, Megan Holmes3, Christopher Kenyon3, Jonathan
Seckl3, Maggie Lai3
1Department of Clinical Neurosciences, University of Edinburgh, Edinburgh, Scotland, UK, 2Centre for Neuroscience Research, University of Edinburgh, Edinburgh, Scotland, UK, 3Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, Scotland, UK, 4Division of Endocrinology, University of Mississippi Medical Center, Jackson, MS, USA
Background and Aims: Glucocorticoid control of neuronal function requires actions mediated through both the mineralocorticoid (MR) and glucocorticoid receptor (GR). Chronic overactivation of GR is detrimental, targeting some neurons for apoptosis, disturbing neuroendocrine control and impairing cognition and behaviour. In contrast, the effects of increased MR signalling have yet to be explored in detail, perhaps because MR is largely occupied at basal steroid levels so modulation of signalling is more dependent on changes in receptor density than on ligand concentration. What data there are suggest a possible role in the promotion of neuronal survival and improved learning and memory. We have previously shown that neuronal MR induction occurs early in response to cellular injury in vivo and in vitro and is associated with enhanced survival. However, the causality of this association and any relevance for human health has yet to be established.
Methods: We therefore (i) generated a forebrain specific MR overexpressing transgenic mouse (MR-Tg) under the control of the Cam KII alpha promoter and measured the extent of neuronal death following transient global cerebral ischaemia; (ii) measured the expression of MR and GR in human hippocampal sections following initially non-fatal cardiac arrest. Given the localization of the transgene, MR-Tg mice also served as a useful model in assessing the role of MR in hypothalamic-pituitary-adrenal (HPA) axis regulation, in cognition and behaviour.
Results: Neuronal death measured 3 days after 20 minutes of bilateral common carotid artery occlusion was reduced by 33.4 ± 4.2% in the caudate nucleus, CA-1 and CA-2 regions of the hippocampus compared to wildtype littermate controls (F(1,68)=10.4, p=0.002). MR-Tg mice also demonstrated improved memory retention in the Morris water maze (p<0.02), spent significantly more time in the anxiogenic central zone of the open field test (p<0.05) and displayed decreased latency to enter (p<0.05) and spent more time in the light arena (p<0.05) in the dark-light exploration test compared to wildtype controls. Basal plasma corticosterone levels, GR expression, the glucocorticoid response to restraint stress, basal blood pressure and general locomoter activity was not affected by genotype. Patients dying 4 days to 8 weeks after initially non-fatal cardiac arrest had increased hippocampal expression of MR but not GR, suggesting modulation of MR expression observed in experiments in vitro and in vivo is also seen in humans.
Conclusions: Increased neuronal MR attenuates neuronal loss in cerebral ischaemia and has beneficial effects on memory and mood ?which are often compromised in humans post-stroke. These effects are not due to an effect of circulating glucocorticoid levels, which are unchanged, and identify increased MR expression as a therapeutic target not only in cerebral ischaemia but also in disorders of mood.
PROTECTIVE EFFECTS OF DIHYDROGINSENOSIDE RB1 ON ISCHEMIC BRAIN DAMAGE AND COMPRESSIVE SPINAL CORD INJURY THROUGH UPREGULATION OF VEGF AND BCL-XL
Ryuji Hata, Pengxiang Zhu, Fang Cao, Keiichi Samukawa, Masahiro Sakanaka
Department of Functional Histology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
Background and aims
Previously, we showed that intravenous infusion of ginsenoside Rb1(gRb1) ameliorated ischemic brain damage through upregulation of Bcl-xL [JCBFM 26:708-721, 2006]. In the present study, we produced dihydroginsenoside Rb1(dgRb1), a stable chemical derivative of gRb1, and investigated the effects of dgRb1 on ischemic brain damage and spinal cord injury (SCI) in rats.
Methods & Results
Intravenous infusion of dgRb1 to rats with permanent occlusion of the middle cerebral artery significantly ameliorated ischemia-induced place navigation disability (Figs A and B; n=6 in each group) and caused an approximately 50% decrease in the infarct volume (Fig C; n=6 in each group). We also showed that dgRb1 improved locomotor activity (Fig D), rearing activity (Fig E) and BBB score (Fig F) after SCI (n=8 in each group). In subsequent in vitro studies, we showed that dgRb1 could upregulate the expression of not only Bcl-xL, but also VEGF in neurons (n=5 in each group). We also showed that dgRb1-induced expression of bcl-xL and VEGF mRNA was HRE (hypoxia response element) and STRE (signal transducers and activators of transcription 5 (Stat5) response element) dependent, respectively.
Conclusions
Our results suggest that dgRb1 can be applied in patients with SCI or acute cerebral stoke. DgRb1, that upregulates VEGF and Bcl-xL, may also be useful for the treatment of neurodegenerative diseases such as Parkinson disease and Alzheimer disease.
A RHO-KINASE INHIBITOR PREVENTS APOPTOSIS-INDUCED NEURONAL CELL DEATH FOLLOWING TRANSIENT FOCAL ISCHEMIA IN RATS
Tatsushi Kamiya1, Toshiki Inaba2, Chikako Nito2, Masayuki Ueda2, Kengo Kato2, Satoshi Suda2, Takeshi Hayashi1, Kentaro Deguchi1, Toru Yamashita1, Yoshihide Sehara1, Atsushi Tsuchiya1, Han Zhe Zhang1, Violeta Lukic1, Yasuo Katayama2, Koji Abe1
1Division of Neuroscience, Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama, Japan, 2Second Department of Internal Medicine, Nippon Medical School, Tokyo, Japan
Background and aims: Recently, it has been reported that rho-kinase that participates in the regulation of vascular tonus, spasm, and remodeling in response to smooth muscle constriction, is involved in the atherosclerosis, inflammation, and hypertention (1). Furthermore, rho-kinase is involved in nitric oxide (NO), free radical, resulting in the neuronal cell death (2). Nevertheless, the mechanism of rho-kinase on the ischemic brain have not been fully understood. The aim of this study is, therefore, to determine whether a newly rho-kinase inhibitor, fasudil, which is a myosin light chain kinase inhibitor and have strong inhibition of rho-kinase, would prevent neuronal cell death following transient focal ischemia in rats compared to a free radical scavenger, edaravone, which is the first clinical neuroprotective agent used in Japan for the treatment of acute stroke patients.
Methods: Sprague-Dawley rats were subjected to MCAo using an intraluminal suture technique for 90 minutes (3). The rats were reperfused for 24 hours and decapitated for infarct and edema analysis (4). Animals were randomly divided into the following four groups. (I) vehicle-treated control group (II) low dose of fasudil (3.0 mg/kg)-treated group (III) high dose of fasudil (10.0 mg/kg)-treated group (IV) edaravone-treated group. A rho-kinase inhibitor-treated animals received a continuous injection of fasudil (3.0 or 10.0 mg/kg) intravenously for 60 minutes after the onset of ischemia, while vehicle-treated groups received same dose of vehicle. Edaravone-treated animals received twice injection of edaravone (3.0 mg/kg) intravenously immediately after the reperfusion and 30 minutes after reperfusion. During ischemia, temporal muscle and rectal temperatures were monitored and maintained at 37 degrees in the experimental animals. Neurological symptom evaluations (posture and hemiplegia) were performed immediately before infarct and edema analysis. Furthermore, cortical cerebral blood flow was measured during ischemia and reperfusion.
Results: The cortical and striatal infarct volume in the edaravone-treated group was significantly less than those in the vehicle-treated control group I (p<0.001, p<0.01 respectively). Fasudil (group II, III) decreased the cortical or striatal infarct volume significantly compared with those of groups I (p<0.001, p<0.05 respectively). The cortical or striatal edema significantly was also improved in the low dose (3.0 mg/kg) fasudil-treated groups significantly, compared with those of groups I (p<0.05, p<0.05, respectively). Moreover, low dose or high dose (10.0 mg/kg) fasudil significantly improved neurological symptoms (p<0.001, p<0.01 respectively).
Conclusions: These results demonstrate that a rho-kinase inhibitor, fasudil prevents neuronal cell death, compared to a free radical scavenger, edaravone. Furthermore, it is suggested that this rho-kinase inhibitor may be a candidate for new neuroprotectant for the treatment of acute stroke in future.
NO FURTHER DECREASE OF 5-HT2A RECEPTORS IN PATIENTS WITH MILD COGNITIVE IMPAIRMENT: A TWO-YEAR FOLLOW-UP STUDY
Lisbeth Marner1, Gitte Moos Knudsen1, Karine Madsen1, Steven Haugbol1, Soren Holm3, Willian Baare4, Steen G. Hasselbalch1,2
1Neurobiology Research Unit, The Neuroscience Center, Rigshospitalet, Copenhagen, Denmark, 2Memory Disorders Research Unit, The Neuroscience Center, Rigshospitalet, Copenhagen, Denmark, 3Department of Clinical Physiology and Nuclear Medicine, Rigshospitalet, Copenhagen, Denmark, 4Danish Research Center for Magnetic Resonance, Hvidovre Hospital, Copenhagen, Denmark
Background and aims. Previous postmortem studies have revealed a profound reduction in the serotonin 2A (5-HT2A) receptor binding in patients with Alzheimer's disease (AD). In vivo studies of patients with AD with assessment of the 5-HT2A receptors using [18F]-Altanserin positron emission tomography (PET) have confirmed this finding. In patients with mild cognitive impairment of the amnestic type (MCI), we have previously demonstrated a 20–30% reduction in cortical 5-HT2A-receptor binding as compared to healthy subjects1. Here we present a two-year follow-up of both patients and healthy subjects.
Methods. At baseline (n= 16 patients, 17 healthy subjects) and at two-year follow-up (n= 14 patients, 12 healthy subjects), all individuals were investigated with magnetic resonance imaging (MRI) and [18F]-Altanserin PET using a bolus-infusion approach. Volumes of interest (VOI's) were delineated automatically on coregistered MRI images, and partial volume (PV) corrected and non-partial volume (nonPV) corrected VOI values were extracted from the PET images. The binding potential of specific binding (BP1) was calculated using cerebellum as a reference region devoid of 5-HT2A-receptors (CREF) and metabolite corrected plasma values (CPLASMA) according to BP1=(CVOI-CREF)/CPLASMA where CVOI is the radioactivity concentration in the VOI. As the temporal and parietal cortical regions often show hypometabolism in early AD and at the same time have a high 5-HT2A-receptor density, they were chosen for the present analysis.
Results. In the two-year follow-up period, 9 out of 14 patients with MCI had progressed to fulfill AD criteria. In both MCI patients and healthy subjects, no significant change in binding was found as compared to the investigation carried out two years earlier. In MCI patients, the average PV-corrected BP1 was 2.16 at baseline and 2.09 at two-year follow-up (95% C.I. for difference: [-0.48 0.19]) and in healthy subjects BP1 was 2.62 and 2.54 (95% C.I. for difference: [-0.35 0.20]). The average nonPV-corrected BP1 MCI patients was 0.70 at baseline and 0.62 at two-year follow-up (95% C.I. for difference: [-0.21 0.05]) and in healthy subjects 0.97 and 0.89 (95% C.I. for difference: [-0.18 0.04]). The patients that converted to AD during the follow-up period did not differ significantly from the patients that did not convert.
Conclusions. We conclude that the reduced levels of 5-HT2A-receptor binding in MCI patients are relatively stable, even in patients that convert to AD. Our finding suggests that the lower cortical 5-HT2A-receptor binding in MCI patients is due to brain changes that take place in the very early stages of AD before symptoms are present or perhaps even constitute a permanent trait marker for the risk of developing AD.
MALE-SPECIFIC ISOFLURANE PRECONDITIONING NEUROPROTECTION IN EXPERIMENTAL FOCAL STROKE IS TESTOSTERONE DOSE-SPECIFIC AND ANDROGEN RECEPTOR-DEPENDENT IN MICE
Lan Wang, P.D. Hurn, S.J. Murphy
Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, OR, USA
Background and Aims: We have shown that neuroprotection due to isoflurane preconditioning (IsoPC) 24 hours before experimental stroke is male-specific, with the neuroprotective benefits of IsoPC being lost in females and estradiol-treated animals. We evaluated the testosterone (T) dose relationship and the role of the androgen receptor (AR) in the ischemic sensitivity of IsoPC male mouse brain.
Methods: Selected groups of male and castrated (CAST) mice were treated with 21-day release subcutaneous implants containing T (1.5 or 5 mg) or flutamide (FLUT, 5 mg), an AR antagonist, 1 week before preconditioning. Males ± FLUT, CAST ± FLUT, CAST + 1.5T ± FLUT, and CAST + 5T ± FLUT were preconditioned for 4 hours with air (sham preconditioning, Sham PC) or 1.0% IsoPC. Mice then underwent 2 hours of middle cerebral artery occlusion 24 hours after preconditioning. Laser-Doppler flowmetry (LDF) was used to monitor cortical perfusion. Brains were collected at 22 hours reperfusion. Cortical and striatal infarct volumes (% contralateral structure) were determined by digital image analysis of 2 mm thick coronal brain slices stained with 2,3,5-triphenyltetrazolium chloride.
Results: Relative LDF changes were equivalent among groups. In contrast to corresponding Sham PC groups, IsoPC decreased cortical and striatal infarct volumes in males and CAST + 1.5T, and striatal infarct volumes in CAST + 5T (Table). IsoPC had no effect on cortical and striatal infarct volumes in males + FLUT, CAST ± FLUT and T-treated CAST + FLUT mice, and on cortical infarct volume in CAST + 5T as compared to corresponding Sham PC groups (Table).
Conclusions: IsoPC neuroprotection in experimental stroke is testosterone-dependent and dose-specific, with IsoPC neuroprotection being lost in androgen-deficient CAST, males and CAST + 1.5T being completely protected, and CAST + 5T only showing reduced striatal injury. Testosterone's effects in IsoPC ischemic male brain are likely AR-mediated as FLUT blocked IsoPC neuroprotection in male and T-treated CAST ischemic brain. Further studies are needed to explore the role of other androgens like dihydrotestosterone and to confirm an AR-dependent mechanism in male-specific IsoPC neuroprotection.
NICOTINAMIDE REDUCES THE INTRACEREBRAL CELLULAR INFLAMMATORY RESPONSE FOLLOWING TRANSIENT FOCAL CEREBRAL ISCHEMIA IN RATS
Tsung-Ying Chen1,2, Yu-Hsiang Kuan1, Hung-Yi Chen1,3, Ming-Yang Lee1, E-Jian Lee1
1Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical Center and Medical School, Tainan, Taiwan-R.O.C., 2Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan-R.O.C., 3Institute of Pharmacy, China Medical University, Taichung, Taiwan-R.O.C.
We have previously shown that nicotinamide protects against energy depletion and decreases infarction in a rat model of cerebral ischemia [1], [2]. It is known that postischemic cell-mediated neuroinflammation exaggerates the initial brain insult caused by ischemia. Herein, we explored whether nicotinamide mitigates the cellular inflammatory response after transient focal cerebral ischemia for 90 min in rats. Nicotinamide (NICO, 500 mg/kg) or vehicle was given intravenously at reperfusion onset. Immunohistochemistry and flow cytometric analysis were used to evaluate the cellular inflammatory response at 48 hr after reperfusion. Relative to controls, NICO-treated animals resulted in significantly decreased macrophages (4.57(x105)±3.00 vs. 2.83(x105)±1.42) and T lymphocytes (0.74(x105)±0.57 vs. 0.28(x105)±1.42) in the bloodstream (P>0.05). Treatment with NICO also significantly decreased the cellular inflammatory response in the ischemic hemisphere. Specifically, melatonin effectively decreased the extent of neutrophil emigration (Ly6G-positive/CD45-positive) and macrophage/activated microglial infiltration (CD11b-positive/CD45-positive) by 55% (P<0.01) and 66% (P<0.01), respectively. In addition, NICO significantly decreased the population composition of T lymphocyte (CD3-positive/CD45-positive; P<0.001) by 39%. This NICO-mediated decrease in the cellular inflammatory response was accompanied by both reduced brain infarction by 34% (P<0.05) and improved surviving neurons in penumbral cortex and caudoputamen by 112% (P<0.005) and 250% (P<0.001), respectively. Thus, intravenous administration of NICO upon reperfusion effectively decreased the emigration of circulatory neutrophils, T-lymphocytes and macrophages/monocytes into the injured brain and inhibited focal microglial activation following cerebral ischemia-reperfusion. The finding demonstrates nicotinamide's inhibitory ability against the cellular inflammatory response after cerebral ischemia-reperfusion, and further supports its pleuripotent neuroprotective actions suited as a therapy of candidate for further clinical trials in the context of ischemic stroke.
TETRACYCLINE DERIVATIVES INHIBIT CALPAINS AND PROTECT MOUSE NEURONS AGAINST CEREBRAL ISCHEMIA
Susan Jiang1, Jittiwud Lertvorachon2, Sheng Tao Hou1, Yasuo Konishi2, Jacqueline Webster1, Geoff Mealing1, Eric Brunette1, Joseph Tauskela1, Edward Preston1
1NRC Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada, 2NRC Biotechnology Research Institute, National Research Council Canada, Montreal, QC, Canada
Background and aims
Stroke is one of the most common life-threatening neurological diseases. Despite significant advances in the understanding of the molecular events following cerebral ischemia, there are still no potent neuroprotective therapeutics against stroke-induced brain damage. The ischemia-induced excessive release of neurotransmitter glutamate causes excitotoxicity, which is believed to be the major cause of toxicity to neurons. Glutamate over-activates NMDA receptors, causing increased intracellular Ca2+ influx leading to the accumulation of toxic levels of intracellular calcium ions. Elevation in intracellular Ca2+ concentrations activates Ca2+-dependent proteases, such as calpains, which break down critical structural proteins causing neuronal death. In the present study, we tested the protective effects of two tetracyclines against glutamate-mediated excitotoxicity and cerebral ischemia-induced brain damage. The potential molecular mechanisms of such neuroprotection were also investigated.
Methods
All procedures for cerebral ischemia produced by middle cerebral artery occlusion (MCAO) using C57/black mice were approved by the local Animal Care Committee. Under temporary isofluorane anesthesia, mice were subjected to MCAO using an intraluminal filament as previously described. After 1 h of MCAO, the filament was withdrawn, blood flow restored to normal by laser Doppler flowmetry and wounds sutured. Animals were sacrificed after 24 h of reperfusion. Brain infarction was measured and brain tissues were subjected to RNA and protein extraction for PCR, Western blotting, and immunostaining analyses.
Results
We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized NMDA receptor activities causing 16% and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity.
Conclusions
Taken together, these results demonstrated that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.
LYSINE 134 OF BASIC FGF IS A PREREQUISITE FOR ITS NEUROPROTECTIVE ACTIVITY
Susanne Klumpp, Karsten Rose, Dorothee Kriha, Stefanie Pallast, Vera Junker, Josef Krieglstein
Institut Fuer Pharmazeutische Und Medizinische Chemie, Westfaelische Wilhelms Universitaet Muenster, Muenster, Germany
Introduction: Basic fibroblast growth factor (bFGF) is involved in various cellular processes, e.g. cell proliferation, angiogenesis and neuroprotection. In previous work we could provide evidence that binding of ATP to or phosphorylation of bFGF is essential for its biological activity [1]. Therefore, site-directed mutagenesis was used to identify the amino acids of bFGF responsible for ATP binding or phosphorylation. By doing so we discovered that heparin binding and, correspondingly, the neuroprotective potency of bFGF was abolished in one of the mutants generated.
Methods: Escherichia coli TG1 cells carrying the plasmid pCytexp1-bFGF were used for overexpression of bFGF. Site-directed mutagenesis was performed according to the manual of the QuikChange mutagenesis kit (Stratagene). The neuroprotective effect of bFGF and mutants was tested in primary cultures of embryonic (E19) hippocampal neurons damaged by staurosporine (200 nM) and in a mouse model of focal cerebral ischemia.
Results: Replacing lysine 134 of bFGF by alanine resulted in a growth factor protein that could be labeled with [γ−32P]ATP to the same extent as the wildtype (wt) protein. However, this mutant bFGF(K134A) did not protect the cultured hippocampal neurons against damage whereas wt-bFGF and various other mutants did (Fig. 1). Furthermore, phosphorylation of Akt and ERK-1,2 was significantly diminished in cultured neurons treated with bFGF(K134A) indicating decreased cellular signalling compared to neurons treated with wt-bFGF. Notably, only wt-bFGF, but not bFGF(K134A), was capable of reducing the infarct area after occlusion of the middle cerebral artery of mice. A heterotrimeric complex consisting of one heparin and two bFGF molecules is known to interact with the bFGF receptor. Lysine 134 of bFGF is located within the heparin binding site. Indeed, binding of bFGF(K134A) to heparin acrylic beads was markedly reduced in comparison to that of wt-bFGF.
Conclusion: Our results demonstrate that lysine 134 plays a pivotal role for the neuroprotective activity of bFGF. Since labeling with [γ−32P]ATP is unaffected by this mutation but heparin binding is diminished we suggest that the single amino acid substitution K134A is detrimental to neuroprotection of bFGF because of the reduced heparin binding affinity.
Fig. 1: Replacement of lysine 134 in bFGF abolished the neuroprotective effect. Scheffe test: *p<0.001.
THROMBIN, FACTOR XA AND ACTIVATED PROTEIN C CAN PROTECT THE RAT CORTICAL AND HIPPOCAMPAL NEURONS FROM GLUTAMATE-INDUCED DEATH
Lubov Gorbacheva1, Tatyana Storozhevykh2, Vsevolod Pinelis3, Svetlana Strukova4
1Lomonosov Moscow State University, Department of Human and Animal Physiology, Moscow, Russia, 2The Scientific Centre for Children Health, Russian Acad. Med. Sci., Moscow, Russia, 3The Scientific Centre for Children Health, Russian Acad. Med. Sci., Moscow, Russia, 4Lomonosov Moscow State University, Department of Human and Animal Physiology, Moscow, Russia
Background and aims. Glutamate-induced damage of calcium homeostasis in neurons is one of the mechanisms of brain injury (hemorrhagic insults, ischemia, neurodegenerative diseases and etc.) (Hossain MA., 2005). It is known, that serine proteinases can participate in regulatory process in CNS (Xi, G. et al, 2003; Kawabata, A. et al, 2005; Zlokovic, B. et al, 2005). We investigated the role of thrombin (Th), factor Xa (FXa) and activated protein C (APC) in cell survival and calcium homeostasis in cultured rat cortical and hippocampal neurons.
Methods. Studies were performed using 7-10-day primary culture of neurons from cortex or hippocampus obtained from brain of 1-3-day-old Wistar rats. 24 h after the cell exposure (30 min) to 100µM glutamate (Glu) or serine proteases and glutamate neuron death in culture was estimated. Cell death was determined using two approaches, biochemical (MTT-test and the measurement of lactate dehydrogenase activity in the medium) and morphological (Hoechst 33342, Ethidium bromide and Syto-13). Using fluorescent probes Fura-2/AM or Fura-2FF/AM intracellular calcium concentration ([Ca2+]i) was measured. Student,s t-test was used for statictical treatment.
Results. We observed that exposure neurons to Glu led to the increase of apoptotic cells (to 33%) at 24 h. The pretreatmant of culture with Th (0,1-10 nM), FXa (1-10 nM) and APC (Sigma) (0,05-10 nM) significantly (on average in two times) reduced the Glu-induced neuronal death. But the most effective protective concentrations of APC were different for cortical and hippocampal neurons (0,1nM and 0,05 nM, accordingly). High concentrations of serine proteases (100nM Th and 100nM APC) have not neuroprotective effect at Glu-induced neuron death and caused cell death in 32% of cells. Inactivated with PMSF Th, FXa and APC did not protect neurons from Glu-induced toxicity.
As known, the apoptotic effect of glutamate is based on the overloading of cells by calcium and disruption in the regulation of calcium homeostasis. In our research, Th- and FXa- (but not APC) induced decrease of the cell number in which the basal level of intracellular calcium was recovered after removal of glutamate, was revealed.
At the same time only Th elicited the rapid transient increase in intracellular calcium in hippocampal and cortical neurons. Using the blockers of IP3 and ryanodine receptors, 2-ABP and dantrolene, was shown the predominant role of the IP3 receptors in the calcium response of neurons to Th.
Conclusions. The present findings suggest that Th, FXa and APC at low concentrations (< 10 nM) has the protective effect on cortical and hippocampal neurons survival at Glu-induced toxicity. Hippocampal neurons sensitivity to APC was higher than one to cortical neurons. However, most likely these protective effects have not identical mechanisms.
FUNCTIONAL VARIATION IN THE SEROTONIN TRANSPORTER GENE DECREASES IN VIVO BRAIN SEROTONIN 2A RECEPTOR BINDING
Steven Haugbol1, Lars H. Pinborg1, Vibe G. Frokjaer1, David Erritzoe1, Lisbeth Marner1, Steen G. Hasselbalch1, Claus Svarer1, Lis Hasholt2, Soren Holm3, Olaf B. Paulson4, Gitte M. Knudsen1
1Neurobiology Research Unit, Rigshospitalet, Copenhagen University Hospital and Center for Integrated Molecular Brain Imaging, Copenhagen, Denmark, 2Section of Neurogenetics, Department of Medical Biochemistry and Genetic, University of Copenhagen, Copenhagen, Denmark, 3PET and Cyclotron Unit, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 4Danish Research Centre for Magnetic Resonance, Copenhagen University Hospital, Copenhagen, Denmark
BACKGROUND AND AIMS: There is increasing evidence that the developing brain is under profound serotonergic control. Various polymorphisms of the serotonin transporter (5-HTT) gene are known to be associated with anxiety, depression, and vulnerability to affective disorders. The postsynaptic serotonin 2A receptor (5-HT2A) receptor is widely distributed in the brain with highest density in cortical brain regions. The receptor is down-regulated both in genetically modified mice lacking the 5-HTT and after chronic blockade of the serotonin reuptake site with SSRIs. These observations support that 5-HT2A receptor density is regulated by brain serotonin levels that in turn are adjusted by the 5-HTT. Clinically, the 5-HT2A receptor has been implicated in psychosis, depression and different behavioral traits. The 5-HT2A receptor agonists elicit hallucinations and antagonists are useful both as antipsychotics and as adjuvant treatment in depression. AIMS: Here we investigate the effects of two frequent functional polymorphisms in the 5-HTT gene (SLC6A4) on the 5-HT2A receptor binding. METHODS: 104 healthy volunteers (39 women and 65 men; mean: 41 years; age range: 18 ? 80 years) underwent [18F]altanserin PET scanning for assessment of the 5-HT2A receptor binding in the brain. The genotypes were assessed by polymerase chain reaction, oligonucleotide primers, and electrophoreses. One polymorphism is positioned in the promoter region (5-HTTLPR) and is fixed in a short allele and a long allele. The other is located in the second intron (STin2) and the alleles most frequently contain 10 and 12 tandem repeats. We applied multivariate repeated measures ANOVA (SAS statistical software package) to analyze the effect of the length polymorphisms. RESULTS: Genotype frequencies of 5-HTTLPR and STin2 all conformed to the Hardy-Weinberg equilibrium. In all examined brain regions, 5-HT2A receptor binding was lower in subjects with STin2 10/10 and 10/12 in comparison to subjects carrying 12/12. Multivariate repeated measures ANOVA of the group allelic effect of STin2 on 5-HT2A receptor binding were significant, F = 4.54, P = 0.03. The allelic effect of 5-HTTLPR itself showed no significant effect on 5-HT2A receptor binding, F = 2.56, P = 0.11. However, when adding both class variables (genotypes) to the repeated ANOVA model the effect of the STin2 polymorphism became more significant, F = 6.29, P = 0.01. CONCLUSION: The finding adds further evidence to the generally accepted view that differential expression of the 5-HTT governs serotonergic neurotransmission. Here, we suggest that the combination of two 5-HTT polymorphisms, the 10 and the s allele, leads to a lifelong increase in endogenous 5-HT tone and a subsequent desensitization of 5-HT2A receptors. This finding is consistent with the understanding of serotonin being profoundly involved in neurodevelopment and its possible implications in neuropsychiatric diseases via the 5-HT2A receptor.
SEROTONIN TRANSPORTER (SERT) AVAILABILITY MEASURED WITH [11C]DASB PET IS REDUCED IN OBSESSIVE-COMPULSIVE DISORDER INEDEPENTLY FROM THE SERT GENE PROMOTER VARIANT
Swen Hesse1, Katarina Stengler-Wenzke2, Ralf Regenthal3, Georg-Alexander Becker1, Marianne Patt1, Anita Seese1, Daniel Teupser4, Henrik Roth5, Henryk Barthel1, Kai Kendziorra1, Wolfgang Heinke6, Ulrich Hegerl2, Osama Sabri1
1Department of Nuclear Medicine, University Hospital, University of Leipzig, Leipzig, Germany, 2Department of Psychiatry, University Hospital, University of Leipzig, Leipzig, Germany, 3Institute of Clinical Pharmacology, University of Leipzig, Leipzig, Germany, 4Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Leipzig, Germany, 5Department of Diagnostic and Interventional Radiology, University Hospital, University of Leipzig, Leipzig, Germany, 6Department of Anaesthesiology and Intensive Care Therapy, University Hospital, University of Leipzig, Leipzig, Germanty
Introduction: Derived from the clinical effectiveness of selective serotonin reuptake inhibitors (SSRIs) it is currently under debate whether the brain serotonin transporters (SERT) are altered in patients with obsessive-compulsive disorder (OCD). However, first in-vivo imaging studies on SERT employing SPECT/PET did not reveal consistent results (Hesse et al., 2004). The aim of the present study was therefore to measure SERT availability in OCD patients using the SERT-selective PET radiotracer [11C]DASB. Since a polymorphism of the SERT gene promoter might have an impact on the in vivo SERT availability in OCD, we furthermore compared the SERT PET measures of promoter variants (S, short allele, L, long allele).
Methods: Ten drug-naive OCD patients (ICD 10 F42.0–42..2; 2 females, age 39.1 ± 14.3 years; age of onset 24.5 ± 15.5 years; genotype S/L:L/L = 1:1.5) underwent dynamic PET for 2 hours after intravenous bolus injection of 370 MBq [11C]DASB. OCD severity was assessed using the Yale-Brown Obsessive Compulsive Scale (Y-BOCS). Only patients with Y-BOCS >/= 20 points were included into the study. Depressive symptoms were rated using the Beck Depression Inventory (BDI). Parametric images of SERT binding potential (BP) were generated from the PET data by the multi-linear reference tissue model with 2 parameters and the cerebellum as the reference tissue (Ichise et al., 2003) after model validation with arterial input function determination and regional analyses of BP values was performed. The BP images were co-registered with 3D MRI data using the MultiModality software (HERMES Medical Solutions) for delineating the regions of interest (ROI, striatum and sub-regions, thalami, midbrain/brainstem raphe, and cortical areas). For considering probable effects of nicotine on SERT BP, subjects were classified as 0, non-smokers, to 4, heavy smokers.
Results: In OCD patients, the BPs were significantly reduced in the SERT-rich brain region, e.g. the (left) thalamus and the midbrain raphe (2.03 ± 0.27 and 1.88 ± 0.24 versus 2.81 ± 0.26 and 2.70 ± 0.10 in age-matched controls, p = 0.005 and p = 0.001) and in limbic areas (hippocampus) but not in cortical areas. The BP values did neither correlate with YBOCS, smoking status, age nor differ between S/L and L/L carriers. There was an overall trend toward a negative association between BDI and BP values, which became significant in the thalamus (e.g. right: R = -.687, p = 0.041), the caudate (right: R = -.743, p = 0.022), and frontal regions (e.g. the right medial frontal cortex: R = -.712, p = 0.031).
Conclusion: The SERT availability is reduced in OCD patients confirming own previous [123I]s-CIT SPECT studies (Hesse et al., 2004). This reduction seems to involve not only SERT-rich brain regions but also limbic areas (hippocampus) but does not depend on SERT gene promoter polymorphism. We assume that, in OCD; SSRIs act mainly via SERT-dependant anti-depressant effect. Verification of this hypothesis, however, will require comparative on-medication versus off-medication studies.
BOLD SIGNAL AMPLITUDE SHOWS AN INVERSE DEPENDENCE ON BASELINE CBF
Joy Liau1, Thomas T. Liu2
1Department of Bioengineering, University of California at San Diego, San Diego, CA, USA, 2Department of Radiology, University of California at San Diego, San Diego, CA, USA
Introduction
Recent studies have shown that modulation of the baseline level of cerebral blood flow (CBF) with pharmacological agents can alter the amplitude of the blood oxygenation level dependent (BOLD) response [1]–[2]. However, the effect of naturally occurring inter-subject variations in CBF on the BOLD response has not been completely characterized. Here, we show that measures of baseline CBF can account for a significant portion of the inter-subject variance in the BOLD response amplitude.
Methods
Nine subjects participated in the study, which consisted of a resting-state scan (3min fixation) and a block design run (4 cycles, 20s-on/40s-off, visual stimulus). Images (3 slices about the calcarine sulcus) were acquired on a 3T GE whole body MRI system with a head transmit coil and an 8 channel receive head coil using a PICORE QUIPPS II arterial spin labeling sequence with dual echo spiral readout (TE1/TE2=9.1/30ms; TI1/TI2=600/1500ms; TR=2s). The CBF time series was computed from the first echo of the block design run and the BOLD times series from the second echo. A functional region of interest (ROI) was defined using both series. For each subject, the CBF block response amplitude (%?CBF) and BOLD block response amplitude (%?BOLD) were found within the ROI. Data from the resting-state scan were calibrated [3], and the average baseline CBF within the ROI was computed on a per-subject basis.
Results
The figure shows scatter plots of (a) %?BOLD and (b) %?CBF versus baseline CBF. Linear fits are shown by the dotted lines. There was a significant correlation between baseline CBF and %?BOLD (r=-0.84, p=0.0045) as well as with %?CBF (r=-0.77, p=0.016).
Discussion
The BOLD response arises from changes in oxygen metabolism (CMRO2), CBF, and blood volume as described in a physiological model of BOLD [4]. From this model, increases in %?CBF lead to increases in %?BOLD. Our finding of an inverse relationship between %?CBF and baseline CBF is therefore consistent with the inverse relation between BOLD response amplitude and baseline CBF. Assuming typical parameters (Grubb's exponent ?=0.38, deoxyhemoglobin(dHb) exponent ?=1.5; coupling constant between CBF and CMRO2 of n=3) [4] and using %?CBF and baseline CBF values derived from the linear fit in (b), the predicted dependence (red line in (a)) shows good agreement with the data. Finally, the results presented here indicate that measures of baseline CBF may be useful as a covariate in statistical analyses that aim to minimize its contribution to inter-subject variability in BOLD amplitude measures.
BRAIN ACTIVATION OF ACTUAL BUT NOT SIMULATED CAR-DRIVING USING PET AND [18F]FDG
Myeonggi Jeong1, Manabu Tashiro1, Keiichiro Yamaguchi1, Laxsmi Singh1, Masayasu Miyake1, Ren Iwata2, Masatoshi Itoh1
1Division of Nuclear Medicine, Tohoku University Cyclotron and Radioisotope Center, Sendai, Miyagi, Japan, 2Division of Radiopharmaceutical Chemistry, Tohoku University Cyclotron and Radioisotope Center, Sendai, Miyagi, Japan
Background and Aims
Car-driving is a combination of complex neural tasks such as attention, perception, integration of visual and somatosensory inputs, generation of motor outputs and action controls. Though the car-driving is not a difficult task for many experienced drivers, all drivers might sometimes encounter potentially-dangerous situations induced by cognitive and psychomotor deficits due to aging, neurological disorders, sedative drugs and mobile phone use. Therefore, elucidation of the brain mechanism during car-driving is important and might lead to development of an effective system to prevent accidents. The present study aims at identifying the brain activation during actual car-driving on the road, and at comparing the result to those of previous studies on simulated car-driving.
Methods
Thirty normal volunteers, aged 20 to 56 years, were divided into three subgroups, active driving, passive driving and control groups, for examination by positron emission tomography (PET) and [18F]2-deoxy-2-fluoro-D-glucose (FDG). The active driving subjects (n=10) drove for 30 minutes on quiet normal roads with a few traffic signals. The passive driving subjects (n=10) participated as passengers on the front seat. The control subjects (n=10) remained to be seated in a lit room with their eyes open. Voxel-based t-statistics were applied using SPM2 to search of brain activation among the subgroups mentioned above.
Results
Significant brain activation was detected during active driving in the primary and secondary visual cortices, primary sensorimotor areas, premotor area, the parietal association area, the cingulate gyrus, the parahippocampal gyrus as well as in the thalamus and cerebellum. The passive driving manifested a similar-looking activation pattern, lacking activations in the premotor area, the cingulate and parahippocampal gyri and in the thalamus. Direct comparison of the active and passive driving conditions revealed activation in the cerebellum.
Conclusions
So far, several activation studies during simulated car-driving have been conducted using functional MRI and PET with [15O]H2O. The result of actual driving seemed to be similar to that of simulated driving, suggesting that visual perception and visuomotor coordination were the main brain functions while driving. In terms of attention and autonomic arousal, however, it seems there was a significant difference between simulated and actual driving possibly due to risk of accidents. Autonomic and emotional aspects of driving should be studied using actual driving study-design. Functional neuroimaging using actual and simulated car-driving task will be a useful tool in the filed of transportation medicine.
IMPAIRED SENSITIVITY TO PAIN IN MULTIPLE FUNCTIONAL DISORDER
Ruta Kuzminskyte1,2, R. Kupers2,3, J. Geday3, P. Videbech4, P. Fink1, A. Gjedde2,3
1Research Clinic for Functional Disorders and Psychosomatics, Aarhus University Hospitals, Aarhus, Denmark, 2Center of Functionally Integrative Neuroscience, Aarhus University Hospitals, Aarhus, Denmark, 3Pathophysiology and Experimental Tomography Center, Aarhus University Hospitals, Aarhus, Denmark, 4Institute of Biological Psychiatry, Aarhus University Hospital, Aarhus, Denmark
Background and aim: Some patients complain of physical symptoms from various organ systems that are not attributable to any known conventionally defined disease and are not adequately supported by clinical or paraclinical findings. Such patients are suffering from Multiple Functional Disorder (MFD) and their symptoms are functional somatic symptoms. Functional somatic symptoms are painful, severe, and persistent and have a significant impact on patients' well-being and functioning.
Given the lack of peripheral pathology in MFD patients, research focuses on the central nervous system as the potential source of the problem. The aim of this study was to examine cerebral responses to painful heat stimulation in MFD patients and compare these with responses in healthy controls.
Methods: Fourteen MFD patients and 14 healthy controls participated in the study. All subjects underwent a psychiatric (SCAN) interview, a thorough somatic check-up and quantitative sensory testing, during which individual thermal pain thresholds were determined. We used water (H215O) uptake detected with PET to measure changes in regional cerebral blood flow (rCBF). Measurements were made in triplicate at baseline and during painful stimulation. Stimulus strengths were determined individually as each subject's pain threshold. After every tomography session, the subjects rated the intensity of the induced pain.
Results: At baseline, the rCBF were similar in MFD patients and healthy controls, and mean stimulus temperatures and mean ratings of pain during painful stimulation did not differ between the two groups. However, differences in rCBF (both increases and decreases) were observed between the two groups during pain stimulation. In pain, the MFD patients had significantly lower increase of rCBF contralaterally in inferior parietal lobe (BA 40), and bilaterally in inferior frontal gyri (BA 10) when compared to controls.
Conclusions: The MFD patients and the controls reported similar perception of painful stimuli induced by similar stimulus temperatures. Nevertheless, the MFD patients exhibited significantly lower increase in rCBF during painful stimulation contralaterally in secondary somatosensory cortex and bilaterally in prefrontal cortices when compared to controls, suggesting that pain perception is impaired in the MFD patients.
EFFECT OF BRAIN HYPOTHERMIA ON CEREBRAL GLUCOSE METABOLISM IN ACUTE BRAIN INJURY: EVALUATION WITH FLUORODEOXYGLUCOSE POSITRON EMISSION TOMOGRAPHY
Nobuyuki Kawai1, Yoshihiro Nishiyama2, Yuka Yamamoto2, Takashi Tamiya1, Seigo Nagao1
1Department of Neurological Surgery, Kagawa University School of Medicine, Kita-Gun, Kigawa, Japan, 2Department of Radiology, Kagawa University School of Medicine, Kita-Gun, Kagawa, Japan
Background and aims: Brain hypothermia (BH) has been used for brain protection in patients with acute brain damage. Suppression of the metabolism and subsequent lowered blood flow in the brain has been considered as a major protective mechanism of BH. To evaluate the effect of BH on glucose metabolism, we examined cerebral glucose metabolism during and after BH in patients with acute brain damage.
Methods: We investigated 4 patients (3 severe head injury and 1 cardiac arrest) who were treated with mild BH (34-34.5?C) for 3–5 days. All patients were sedated with midazolam and buprenorphine and paralyzed with vecuronium during BH. Cerebral metabolic rate for glucose (CMRglc) was determined quantitatively with 18F-fluoro- deoxyglucose positron emission tomography (FDG-PET) kinetic analysis during and after BH.
Results: Case 1 was 43 year-old female with diffuse brain injury (GCS 8). PDG-PET during BH showed diffuse glucose hypometabolism (CMRglc 25.5 µmol/100g/min) in the brain. Follow-up FDG-PET examined on the 28th hospital day showed markedly increased glucose metabolism (CMRglc 53.3 µmol/100g/min) except for the contusional brain. The patient had moderate disability at discharge. Case 2 was 69 year-old male with focal brain injury (GCS 5). The patient received craniotomy for delayed traumatic intracerebral hematoma. PDG-PET during BH showed diffuse glucose hypometabolism (CMRglc 34.9 µmol/100g/min) in the brain. Follow-up FDG-PET examined on the 30th hospital day showed mildly increased glucose metabolism (CMRglc 45.5 µmol/100g/min). The patient had severe disability at discharge. Case 3 was 57 year-old male with diffuse brain injury (GCS 6). PDG-PET during BH showed diffuse glucose hypometabolism (CMRglc 35.4 µmol/100g/min) in the brain. Follow-up FDG-PET examined on the 60th hospital day showed further decreased glucose metabolism (CMRglc 28.2 µmol/100g/min). The patient did not show any improvement in his consciousness. Case 4 was 45 year-old male with out-of-hospital cardiac arrest. After successful resuscitation, he was semicomatous with GCS of 8. PDG-PET during BH showed diffuse glucose hypometabolism (CMRglc 35.3 µmol/100g/min) in the brain. Follow-up FDG-PET examined on the 15th hospital day showed markedly increased glucose metabolism (CMRglc 58.8 µmol/100g/min). The patient fully recovered with no neurological deficits.
Conclusions: In two patients who showed favorable recovery (Case 1&4), BH suppressed the cerebral glucose metabolism approximately 40–50%. Considering the effect of sedative drugs on the metabolism using during BH, the suppressive effect of BH on the cerebral glucose metabolism is mild. In two patients who showed unfavorable recovery (Case 2&3), follow-up FDG-PET study showed decreased or mildly increased glucose metabolism in the brain. Temporal changes in the cerebral glucose metabolism using FDG-PET may be a useful indicator of neurological recovery in patients with severe acute brain damage.
CLINICAL APPLICATION OF REAL-TIME CEREBRAL PERFUSION IMAGING USING DIGITIZED DSA IMAGE DATA
Yoshikazu Nakajima1, Masayuki Hirata2, Amami Kato2, Toshiyuki Fujinaka2, Yoshifumi Teramoto1, Mamoru Ito1, Toshiki Yoshimine2
1Department of Neurosurgery, Rinku General Medical Center, Izumisano, Osaka, Japan, 2Depatment of Neurosurgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
Introduction: We have previously reported the utility of a perfusion imaging (PI) method that displays color-coded images of global and local cerebral perfusion based on DSA image data (1)–(3). The previous system processed digitized data converted from an analog signal on videotape. The aim of this study was to evaluate the clinical utility of a new system that can directly analyze digitized data from serial DSA images in a DICOM format on a portable personal computer (PC).
Methods: We created the new system program using the Code Warrior C language image (Metrowerks, USA). The system was set up to operate on Windows XP (Microsoft Corp., USA) using Scion Image for Windows (Scion Corp, USA). It can process DSA image data in a DICOM format, provided by the workstation with DSA equipment (CRS-PC, Cardiac View Station; GE OEC Medical Systems, Inc.). We applied this system to assess changes in cerebral perfusion in the treatment of acute apoplexy.
Results: This system was able to visualize cerebral perfusion using 20 DSA images (512 × 512-pixels) in less than 1 second. Information regarding changes in flow velocity of the cerebral vessels and global perfusion status could be provided in semi-real time. Moreover, as compared to our previous system, the new system can detect smaller differences in signal density in the high-density range (Table 1). This is important for clinical use because the signal density of cerebral vessels on DSA is around this high range.
Conclusion: The system described here can provide indispensable information for objectively evaluating DSA image data in semi-real time. It could be especially beneficial for intra-operative monitoring of cerebral perfusion during endovascular surgery.
DOES BOLD FUNCTIONAL MRI HAVE THE CAPABILITY OF MAPPING SUB-MILLIMETER-SCALE FUNCTIONAL COLUMNS?
Chan-Hong Moon, Hiro Fukuda, Sung-Hong Park, Seong-Gi Kim
Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
[Background and aims] The ability of blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) in mapping sub-millimeter-scale functional columns still remains debatable mainly due to the spatially non-specific large vessel contribution, poor sensitivity and reproducibility, and lack of independent evaluation. Furthermore, regions with the highest conventional BOLD signals may indicate neurally inactive domains rather than active columns if, for instance, the results from optical intrinsic signal (OIS) are directly applicable. To examine these issues, here we performed BOLD fMRI at an ultrahigh magnetic field to map orientation-selective columns of isoflurane-anesthetized cats.
[Methods] Gradient-echo (GE) BOLD fMRI (TE = 18 ms, TR = 500 ms) and spin-echo (SE) BOLD fMRI (TE = 40 ms, TR = 2 s) with 0.3×0.3×1.0 mm3 resolution were obtained in six anesthetized cats (10 hemispheres) at 9.4 Tesla. SE BOLD technique at high magnetic fields is mostly sensitive to microvessels including capillaries, and has been shown to improve spatial localization to the parenchyma. As a reference for the known neural interpretation, cerebral blood volume (CBV)-weighted fMRI (TE = 10 ms, TR = 1 s) was also obtained after the injection of monocrystalline iron oxide nanoparticles (MION). Visual responses were induced by 800-sec-long full-field moving gratings with eight orientations (10 s for each orientation, 10 repetitions) without inter-stimulation gap. Fourier analysis was applied to obtain maps of orientation-specific signal (1/80 Hz). For neural interpretation, BOLD iso-orientation maps were directly compared to MION-based CBV fMRI maps and correlation coefficients (R) were calculated. For further evaluation (n = 3 of 6 cats, 4 hemispheres), the BOLD maps were also compared to the isoorientation maps in the same cats from CBV-weighted OIS.
[Results] Temporally-encoded continuous cyclic orientation stimulation with Fourier analysis reduces orientation-non-selective signals such as draining artifacts and allows us to obtain conventional GE BOLD iso-orientation maps with high reproducibility (R = 0.74 ± 0.12, n = 10 hemispheres). Further reduction of large vessel contribution was achieved using, SE BOLD, but with overall decreased sensitivity; the ratio of SE BOLD contrast-to-noise ratio (CNR) to GE BOLD CNR was approximately 1 to 3. The both GE and SE BOLD iso-orientation maps were significantly correlated with maps determined by MION-based CBV fMRI (R = 0.78 ± 0.06 for GE and 0.76 ± 0.08 for SE). Furthermore, the average least-distance for local maxima of isoorientation domains between GE BOLD and OIS maps was 0.22 ± 0.02 mm (n = 4 hemispheres). Since these distances are shorter than the column width of 0.70 mm (i.e., a half of the measured inter-columnar distance), we consider that the BOLD iso-orientation maps match with OIS isoorientation maps. These results suggest that the highest BOLD signals are localized to the sites of increased neural activity when column-non-selective signals are suppressed.
[Conclusions] Thus, sub-millimeter-scale functional columns can be reliably mapped using conventional BOLD fMRI.
[Acknowledgements] We thank Michelle Tasker and Ping Wang for animal preparation. This study was supported by NIH (NS44589, EB003324) and the McKnight Foundation.
DECREASED SEROTONIN 5-HT1A RECEPTOR AVAILABILITY IN MEDICATION-NAIVE PATIENTS WITH MAJOR DEPRESSIVE DISORDER ? A PET STUDY USING [CARBONYL-11C]WAY-100635
Jussi Hirvonen1,2, Hasse Karlsson1,3, Jaana Kajander1, Antti Lepola2, Juha Markkula1, Helena Rasi-Hakala1, Kjell Nagren2, Sargo Aalto4, Jouko Salminen5, Jarmo Hietala1,2
1Department of Psychiatry, University of Turku, Turku, Finland, 2Turku PET Centre, University of Turku and Turku University Central Hospital, Turku, Finland, 3Department of Psychiatry, University of Helsinki, Turku, Finland, 4Department of Psychology, Abo Akademi University, Turku, Finland, 5The Social Insurance Institution of Finland, Turku, Finland
BACKGROUND AND AIMS
Serotonin 5-HT1A receptor function is implicated in the pathophysiology of major depressive disorder (MDD). Previous studies have examined 5-HT1A availability mainly in medicated or medication-free relatively chronic patients with MDD, or patients suffering from familial depression. In this study, we assessed the 5-HT1A receptor availability in vivo in antidepressant-naive primary care patients with MDD using positron emission tomography (PET) and the selective antagonist radioligand [carbonyl-11C]WAY-100635.
METHODS
21 patients diagnosed with DSM-IV-defined MDD (8 male, 13 female, aged 40 ± 9 years, mean ± SD, total HAM-D scores 13?23, total BDI scores 14?40, duration of illness 4?104 weeks) were compared against 15 healthy volunteers (5 male, 10 female, aged 33 ± 8 years, mean ± SD). 20 patients were drug-naive, and 1 had been drug-free for 5 years. Regional tissue distribution volumes (VT) were estimated using ROI-based tissue time-activity curves and the metabolite-corrected arterial plasma curve as input function. Binding potential was calculated indirectly from VT values: BP1=VT[ROI]-VT[REF] and cerebellar white matter as a reference region. A repeated measures analysis of variance (rmANOVA) was fitted to the natural logarithm of BP1 using group status and sex as between-subject predictors and hemisphere and region as within-subject predictors.
RESULTS
The rmANOVA demonstrated a significant main effect of group (F=5.00, p=0.032) but no main effect of sex. Group by region and group by hemisphere interactions were not significant. In all regions, MDD subjects had lower BP1 values compared with healthy controls (average −18.7 %, range −24.9 % to −8.5 %) (Figure 1). The group difference in the raphe nuclei did not reach statistical significance (t=1.41, p=0.168). BP1 did not correlate with BDI total scores, HAM-D total scores, or duration of illness. In a post-hoc analysis of HAM-D subscores, lower BP1 was associated with higher likelihood of insomnia.
CONCLUSIONS
This study demonstrated a wide-spread decrease in the availability of serotonin 5-HT1A receptors in a relatively large sample of drug-naive primary care patients with MDD, suggesting the involvement of 5-HT1A receptors in the pathophysiology of the illness. Lack of correlation with overall severity of the illness might be explained by trait-like nature of this abnormality, non-specificity to the depressive phenotype, or a threshold effect.
TRANSIENT HYPERACTIVATION IN BILATERAL MOTOR RELATED AREAS DURING RECOVERY FROM POSTSTROKE MILD HEMIPARESIS-A STUDY WITH NIRS
Kotaro Takeda1,2, Itsuki Imai3, Kimiko Maruyama1, Yukihiro Gomi1, Nobuaki Shimoda1,2, Masao Hiwatari1,2, Hiroyuki Kato1,2
1Center for Clinical Medicine and Research, International University of Health and Welfare, Nasushiobara, Japan, 2CREST, Japan Science and Technology Agency, Kawaguchi, Japan, 3Department of Rehabilitation, Nasu Neurosurgical Center, Nasushiobara, Japan
[Background and aims] Hemiparesis is the most common neurological deficit after stroke. It has been demonstrated that several motor related cortical areas in both hemispheres contribute to motor recovery from the poststroke dysfunction. Although recent fMRI and PET studies have suggested that a transient involvement of ipsilateral hemisphere plays an important role for the recovery, the mechanisms and the recovery process have been imcompletely understood. We used near-infrared spectroscopy (NIRS), which can noninvasively monitor the changes in concentrations of oxygenated hemoglobin (Oxy-Hb) and deoxygenated Hb (Deoxy-Hb), for investigating the change of cerebral activation areas in the patients with mild hemiparesis from acute to chronic phases after stroke. [Methods] NIRS recordings were performed using a 44-channel optical topographic system (ETG-4000, Hitachi Medical) during 5 cycles of 15 s hand grasping and 30 s resting periods while sitting on a chair. According to the international 10-20 system, 30 probes (22 channels on each hemisphere) were mounted on head shells and were placed on the scalp overlying the primary sensorimotor cortex (SM1) and the dorsolateral premotor cortex (PMd). The activation areas in 6 motor recovered patients (52-73 years old; 4 females and 2 males) with a cerebral infarction were examined longitudinally through acute to chronic phases after stroke. [Results] The 6 patients showed the same NIRS findings (Figure). During affected hand movements, the amounts of Oxy-Hb increased significantly in bilateral SM1 and PMd extensively at the acute phase. At the chronic phase, in contrast, significant activation was observed predominantly in the contralateral SM1, which pattern was similar to that seen in normal subjects. This change occurred sigmoidally about 30 days after stroke. [Conclusions] The activation pattern during affected hand grasping changed from the bilateral extensive activation pattern to the contralateral predominant pattern. In addition, the activation areas were focused on the contralateral SM1. The present findings suggest that motor recovery from mild hemiparesis is facilitated by an early, transient recruitment of bilateral hemispheres including SM1 and PMd, and that this period is critical for the therapeutic interventions that are aimed at functional recovery.
TRANSCRANIAL ULTRASONIC BRAIN TISSUE PERFUSION IMAGES BY POWER MODULATION IMAGING IN COMPARISON WITH SECOND HARMONIC IMAGING
Toshiyuki Shiogai1, Kaori Ikeda1, Masami Matsumoto1, Aki Morisaka1, Yoshinari Nagakane2, Toshiki Mizuno2, Masanori Nakagawa2, Hiroshi Furuhata3
1Departments of Clinical Neurosciences, Kyoto Takeda Hospital, Kyoto, Japan, 2Department of Neurology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 3ME Laboratory, Tokyo Jikei University School of Medicine, Tokyo, Japan
Objective: In order to improve temporal resolution of ultrasonic tissue perfusion images, harmonic imaging with an increased reception frequency has frequently been utilized. For the introduction of transcranial brain tissue perfusion imaging, fundamental imaging without increased harmonic frequency reception and with transient response high transmitting acoustic power images, appears suitable while taking bone- and depth-dependant ultrasound attenuation into consideration. Power modulation imaging (PMI) uses a fundamental frequency and transmits pulses of alternating amplitude and is introduced into continuously transmitted real-time low power images by means of second-generation ultrasound contrast agents. The objective is to evaluate transcranial brain tissue perfusion images utilizing high power images transmitted by transient response, and to compare between PMI and conventional second harmonic imaging (SHI).
Methods: The subjects were 10 neurological patients (ages 34-92, mean 70; 8 male, 2 female). Utilizing a SONOS5500 S3 transducer (Philips) after a bolus intravenous LevovistR injection (400mg/ml, 2.5g), transient response images utilizing PMI (1.7MHz) and SHI (transmitting/receiving frequencies: 1.3/2.6MHz) were created every 2 seconds at the bilateral transtemporal diencephalic horizontal plain. The mechanical index of both images was 1.6. The investigation depth was 16cm and the focus was 8 cm. Five regions of interest (ROI) were placed in the bilateral basal ganglia and thalamus, and contra-lateral temporal lobe. Peak intensity (PI) and time to PI (TPI) based on time-intensity curves of the ROIs as analyzed by QLAB software (Philips), were compared between both images (n = 18).
Results: 1) Significant depth dependant PI attenuation was observed in both images. 2) PI in all PM ROIs tended to be higher than those of SHI, significantly in the contra-lateral side. 3) Significant depth dependant TPI prolongation was not observed in either image type.
Conclusions: Transcranial brain tissue perfusion transient response images utilizing PMI are superior to SHI in contra-lateral cerebral hemisphere visualization. This will contribute to shorter examination times by visualizing bilateral hemispheres via unilateral temporal windows. For quantitative evaluation of brain tissue perfusion, TPI is a more suitable parameter than PI.
INCOMPLETE BRAIN INFARCTION IN PATIENTS WITH MOYAMOYA DISEASE: STATISTICAL IMAGING ANALYSIS USING 123I-IMZ-SPECT
Jyoji Nakagawara, Kenji Kamiyama, Toshiaki Osato, Toshiiti Watanabe, Hirohiko Nakamura
Department of Neurosurgery, Nakamura Memorial Hospital, Sapporo, Hokkaido, Japan
Background and aims
The extent of ischemic cerebral damage could be graded by magnitude and duration of cerebral ischemia. Incomplete brain infarction defined by reduction of central benzodiazepine receptor (BZR) density using 123I-iomazenil (IMZ)-SPECT had been observed within both ischemic penumbra salvaged by restored CBF in acute stroke (1) and misery perfusion proved in chronic hemodynamic stroke. In this study, relationship between long-standing hemodynamic ischemia and the occurrence of incomplete brain infarction in patients with moyamoya disease was estimated by CBF-SPECT and IMZ-SPECT.
Methods
Ten patients with moyamoya disease (7 with TIA or infarction, 3 with ICH) were enrolled in this study. The stage of hemodynamic ischemia (0~II) was estimated by quantified resting CBF and acetazolamide-activated CBF using 123I-IMP-ARG method (2), and the stage was classified as follows; Stage 0: cerebrovascular reserve (VR) [(acetazolamide-activated rCBF-Resting rCBF)/Resting rCBF×100%] >30%, Stage I: resting CBF > 80% of normal mean or 10% < VR < 30%, Stage II: resting CBF < 80% and VR < 10%. The stage of hemodynamic ischemia in cerebral cortex was displayed on the platform of 3-dimensional stereotactic surface projections (3D-SSP) (3). Using stereotactic extraction estimation (SEE) analysis (4), quantified resting and acetazolamide-activated cortical blood flow data were unfold over the platform of 3D-SSP pixel by pixel. VR and hemodynamic stage in cortex are also assessed pixel by pixel. On the other hands, incomplete brain infarction was defined as significant reduction of BZR density by 3D-SSP analysis of IMZ-SPECT. Delayed image 3-hour after tracer injection was dealt as equivalent map to BZR density (5).
Results
The areas of incomplete brain infarction were displayed on 3D-SSP by cluster of pixels which consisted of Z-score value more than 2 S.D. Incomplete brain infarction defined by 3D-SSP analysis of IMZ-SPECT was observed not only in Stage II ischemic area but also in part of Stage I ischemic area. In case with ICH, selective loss of cortical neuron was occasionally observed by undercutting of cortex (white matter lesions).
Conclusions
In patients with moyamoya disease, long-standing hemodynamic ischemia could result in selective loss of cortical neuron (incomplete brain infarction). Cortical neuron could be damaged by long-standing hemodynamic ischemia, even though degree of hemodynamic ischemia has been mildly reduced. Statistical imaging analysis of incomplete brain infarction in moyamoya disease could be valuable for assessing the prognosis of surgical revascularization and the evidence of higher brain dysfunction.
COMPARISON OF C-11 ACETATE, C-11 METHIONINE AND F-18 FDG PET IN PATIENTS WITH BRAIN MALIGNANT AND BENIGN TUMORS
Yuka Yamamoto1, Yoshihiro Nishiyama1, Nobuyuki Kawai2, Motoomi Ohkawa1
1Department of Radiology, Faculty of Medicine, Kagawa University, Kita-Gun, Kagawa, Japan, 2Department of Neurological Surgery, Faculty of Medicine, Kagawa University, Kita-Gun, Kagawa, Japan
Background and aims: F-18 FDG (FDG) PET has been used in brain tumor imaging. However, several studies have demonstrated diagnostic limitations of FDG PET for imaging brain tumors because of the high basal glucose metabolic rate of normal brain tissue. C-11 methionine (MET) PET has been reported to be useful for evaluating brain tumors. C-11 acetate (Acetate) is currently being investigated as a new tracer for imaging neoplasms, most notably prostate cancer. Acetate PET has promising potential for the detection of brain tumors, because its uptake in normal brain is low. We compared the uptake of Acetate, MET and FDG in patients with brain tumor.
Methods: Twenty-two lesions in 20 patients with known brain tumor were evaluated regarding the uptake of Acetate, MET and FDG. The diagnoses were 12 primary malignant tumors, 2 metastases, 8 primary benign tumors. All patients underwent PET with Acetate (15 min p.i.), MET (15 min p.i.) and FDG (40 min p.i.). PET results were evaluated by visual and semiquantitative analysis. For semi-quantitative analysis, the standardized uptake value (SUV) and tumor to contralateral normal brain tissue (T/N) ratio were determined by region-of-interest analysis.
Results: The mean value of SUV in the contralateral normal brain tissue of Acetate (1.5±0.3) was lower than that of MET and FDG (1.9±0.4 and 7.1±1.9, respectively) (P<0.001). The mean value of SUV in the contralateral normal brain tissue of MET was also lower than that of FDG (P<0.001). MET uptake was observed in all 14 malignant tumors, Acetate in 12 malignant tumors and FDG in 5 malignant tumors. The mean value of T/N ratio of MET (2.5±1.3) and Acetate (1.8±1.0) in malignant tumors was higher than that of FDG (1.3±1.0) (P<0.02). There was also a significant difference between T/N ratio of MET and that of Acetate in malignant tumors (P<0.02). Both MET and Acetate uptake was observed in 6 of 7 benign tumors and FDG in 1 benign tumor. The mean value of T/N ratio of MET (2.0±0.7) and Acetate (2.1±0.8) in benign tumors was also higher than that of FDG (0.7±0.1) (P<0.02). There were no significant differences between T/N ratio in malignant and benign tumors on MET, Acetate and FDG PET.
Conclusions: MET is more sensitive than Acetate or FDG for detecting brain tumors although Acetate may be a promising tracer for the detection of brain tumors.
GD-CHELATE MRI CONTRAST AGENTS DELIVERED TO BRAIN PARENCHYMA BY INTRA-CEREBRAL VENTRICULAR INFUSION ARE LOCALIZED TO THE EXTRACELLULAR SPACE
Paul Schornack, Ping Wang, Seong-Gi Kim
Department of Radiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
This study ventures to demonstrate that Gd-chelates are localized to extracellular space in brain parenchyma. Distribution characteristics of uncharged Gd-chelates, GdDTPA-BMA(Omniscan) and GdHPO3A(Prohance), are quantitatively compared during prolonged “incubation? in rat brain, in vivo, treating Prohance as a good extracellular marker. Since the blood brain barrier(BBB) precludes delivery by vascular routes, achieving mM agent concentrations throughout brain parenchyma is accomplished by infusing agents over several hours directly into lateral ventricle CSF(ICVinfusion). Millimolar agent concentrations occur throughout the brain for up to 20hr post-infusion, due to the limited clearance pathways of the BBB/CSF systems. With vascular infusion, agents are typically only present in tissues at mM concentrations for seconds to minutes and have essentially no contact with brain parenchyma cells due to the BBB. Conversely, the conditions of the ICV infusion experiment are very similar to those that exist in the studies of Cabella(2006) exploring intracellular labeling of cultured cells with different Gd-chelates by incubation(4hr) with 1-50mM agent. While both Gd-chelates used in the present study are uncharged and possess the same MW and size, their thermodynamic stabilities are markedly different; the macrocyclic GdHPCO3A logK=23.8 is 10^7 greater than the logK=16.5 of GdDTPA-BMA. The more thermodynamically stable GdHPCO3A was observed to not enter cells while GdDTPA-BMA appears to be taken up into cells (due to Gd3+ release from GdDTPA-BMA induced by phosphate).
Experiments used male Sprague-Dawley rats(390-440gm) with Isofluorane anesthesia. The ICV infusion cannula was positioned in the left lateral ventricle using a stereotaxic frame for delivery of identical quantities of either agent (30µL of 250mM at 6µL/hr over 5hr). T1 maps were obtained by inversion recovery gradient echo(Turboflash) MRI between 4 and 16hr after agent infusion using a 9.4T Varian system.
Agent clearance rate constants(kclearance) from ROIs of the R1(1/T1) maps throughout the brain were obtained. R1 histograms shown indicate similar distribution kinetics in the brain for both agents. Plots of kclearance with z-position show very similar clearance rates of these uncharged Gd-chelates throughout the entire depth of that ROI. The Gd-chelates in the present study clear out of the brain with time constants of 7-15hr with no agent apparent after 36hr; inconsistent with the observation that Gd-chelates taken up by pinocytosis in stem cells have been observed for up to 14 days in endosomes! Taking GdHPCO3A as a reliable extracellular marker under the conditions of this experiment, these results indicate that Gd-chelates remain predominantly in the extracellular space and do not enter cells during their prolonged presence in brain parenchyma.
EFFECTS OF PVH, ANTICLOTTING, ANTIPLATELET DRUGS, AND ANTIHYPERTENSIVES ON MICROBLEEDS
Yuichi Komaba1, Kazumasa Arii2, Noriko Tanaka2, Yasuo Katayama1
1Department of Neurology, Nippon Medical School, Tokyo, Japan, 2Department of Neurology, Hakujikai Memorial Hospital, Tokyo, Japan
Introduction?
Gradient-echo T2*-weighted MRI is sensitive to detect old microbleeds (Mbs) due to microangiopathy. Therfore, microbleeds on T2*-weighted MRI are strtongly related with grade of periventricular hyperintensities (PVH). Moreover old microbleeds are reported to be a potential risk factor for cerebral bleeding after ischemic stroke. Anticlotting and/or antiplatelet drugs are recommended for treatments of ischemic stroke. Thought patients with old microbleeds after ischemic stroke happen to receive anticlotting and/or antiplatelet drugs, effects of anticlotting and/or antiplatelet drugs on old microbleeds have been almost unkown. Angiotensin receptor blocker (ARB) is thought to have brain protective effect, effects of ARB on old microbleeds have been also unkown.
Methods?
We performed retrospective analysis on clinical data for outpatients with old ischemic stroke. We selected outpatients with old ischemic stroke who underwent routine follow-up MRI concluding with Gradient-echo T2*-weighted MRI (male= 46, female= 90; age 71.5+/−13.5yo). Patients were divided into 2 groups depending on the presence or absence of Mbs due to findings of Gradient-echo T2*-weighted MRI. We used chi-square and Mann-Whitney analysis to compare differences in presence or absence of Mbs between these 2 groups. Multiple logistic analysis was performed to determinate whether grade of PVH, anticlotting drugs, antiplatelet drugs, or antihypertensives including ARB were risk factor for Mbs, adjusting a variable for age, gender, hypertension, hyperlipidemia, diabetes, ischemic heart disease, and arterial fibrillation.
Results?
The Mbs(+) group included 23 female (67.51%) and 11 male (32.5%) subjects, compared with 67 female (65.7%) and 35 male (34.3%) subjects in the Mbs(−) group (no significant sex difference between groups). Univariate associations between the potential determinants and Mbs according to chi-square and Mann-Whitney statistics are presented in Table 1. Mbs were significantly associated with diabetes and grade of PVH. We used multiple logistic regression to confirm independent relationships between grade of PVH, anticlotting drugs, antiplatelet drugs, antihypertensives and Mbs. The OR for Mbs according to grade of PVH was 11.582 (95% CI, 3.106 to 43.192). Anticlotting drugs, antiplatelet drugs, and antihypertensives were not independent determinants of occurrence of Mbs.
Conclusions?
We reveal safety of antiplatelet drugs and antihypertensives to Mbs. Effects of antihypertensives on Mbs is not been demonstrated.
INITIAL EXPERIENCE OF NEW COREGISTRATION SOFTWARE BRAIN EASY ANALYSIS TOOL FOR 201TL SPECT IN BRAIN TUMORS
Yasushi Shibata, Akira Matsushita, Manabu Akimoto, Tetsuya Yamamoto, Shingo Takano, Akira Matsumura
Department of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
Purpose
201Thallium Single Photon Emission Computed Tomography (Tl SPECT) is a useful functional imaging modality for the diagnosis of malignancy and activity of brain tumors. One of the drawbacks of Tl SPECT is its low spatial resolution. The coregistration of functional SPECT images and high resolution anatomical MRI is considered to be a reasonable method to improve the low spatial resolution of SPECT. We used a newly developed software Brain Easy Analysis Tool for 201Tl-SPECT (BEAT-Tl, Daiich Radioisotope Labs. Tokyo, Japan) for the coregistration of Tl-SPECT and MRI in patients with brain tumors. We herein report our initial experience in using BEAT-Tl.
Patients and Methods
All patient had been admitted to Tsukuba University Hospital. MRI was performed using the 1.5 Tesla clinical MRI scanner (Gyroscan NT Intera, Philips). T1 weighted images with or without Gadolinium-enhancement, T2 weighted images and Fluid attenuated inversion recovery images (FLAIR) were routinely acquired. Tl-SPECT images were acquired 15 minutes (early image) and 3 hours (delayed image) after the intravenous injection of 74 MBq of Thallium Chloride using multi-detector SPECT machine (E.CAM, Siemens Medical).
MRI and SPECT data were saved as Digital Imaging and Communications in Medicine (DICOM) formats. DICOM data were converted to analyze format using File Converter Ver. 2.5 (Daiich Radioisotope Labs. Tokyo, Japan). Both the MRI and SPECT data of analyze format were transferred to the BEAT-Tl program and then coregistered images were created. The BEAT-Tl program is based on Statistical Parametric Mapping (SPM) and it can be used on a Windows personal computer. The imaging quality, clinical usefulness and the artifacts of the coregistered images were evaluated by visual inspection.
Results
The coregistered images were easily and promptly created. The image quality was sufficient. There were no large displacements of the MRI and SPECT images and no major artifacts. Small hot uptake in Tl SPECT always coregistered with an enhanced tumor in MRI. The coregistered images demonstrated metabolic heterogeneity on Tl SPECT in relatively homogeneous large tumors on MRI.
Discussion
The major disadvantage of SPECT is its low spatial resolution caused by scattering radiation. Because no anatomical landmarks were seen in the Tl SPECT images, it is sometimes difficult to precisely understand the lesion locations and the relationships with normal structures based on only Tl SPECT images. The coregistration of Tl SPECT and MRI has thus completely solved these problems. In addition, even when tumors were found to be homogenous in MRI, Tl SPECT showed metabolic heterogeneity. Further pathological and clinical studies are expected to reveal the clinical significance of these imaging modalities. SPECT/CT or PET/CT are thus considered to be alternative methods to create coregistration images. At present, these machines are expensive and not available in most clinical settings. As a result, the coregistration software is both a convenient and sufficiently accurate method to create coregistration images in most clinical facilities.
Conclusion
BEAT-Tl is a useful coregistration software program to evaluate brain tumors. It improve the spatial resolution of SPECT images and such coregistration images demonstrate a metabolic heterogeneity in tumors.
IN VIVO IMAGING OF THE PENUMBRA IN MICE USING SMALL ANIMAL 18F-FDG PET
Benjamin Klaesner1, Uta Mamrak2, Marc Huisman1, Markus Schwaiger1, Alexander Drzezga1,
Nikolaus Plesnila2
1Department of Nuclear Medicine, Klinikum Rechts Der Isar, Technical University of Munich, Munich, Germany, 2Laboratory of Experimental Neurosurgery, Department of Neurosurgery & Institute for Surgical Research, University of Munich Medical Center, Grosshadern, Munich, Germany
Background and aims
Positron emission tomography (PET) is used to image the ischemic penumbra after stroke in humans. Until recently, however, the spatial resolution of these techniques was not high enough to properly image the brain and small rodents. The aim of the current study was therefore to investigate whether the ischemic penumbra can be identified and studied in a murine model of ischemic stroke using the latest generation of high resolution small animal PET scanners.
Methods
Male C57/B16 mice (n=5) were investigated and subjected to laser Doppler-controlled left MCA occlusion (MCAo) by an intraluminal filament. As a measure of cerebral glucose metabolism 14 MBq 18F FDG were injected i.v. 20 min after induction of ischemia. A small animal PET scan was acquired (30-50 min p.i., microPET FOCUS F120 scanner (Siemens Medical Solutions, USA)), followed by a small animal whole body CT-scan (Siemens MicroCAT 2) for anatomic allocation. PET data were reconstructed with FBP (ramp filter with a cut-off at the Nyquist frequency). The image volume consisted of 128×128×95 voxels, with a size of 0.866 × 0.866 × 0.796 mm3 per voxel.
Results
The acquired FDG PET images showed a significantly reduced cerebral metabolism (50% ± 11% of contralateral hemisphere) in the MCA territory, i.e. the area of cerebral ischemia (p<0,05, Fig 1, white arrow). Furthermore, we could demonstrate that the small animal PET can identify the increased glucose metabolism in the ischemic penumbra (114% ± 6% of contralateral hemisphere, Fig. 1, grey arrow).
Conclusions
These results demonstrate the feasibility of PET imaging in murine models of ischemic stroke. The glucose metabolism of the infarct core as well as of the ischemic penumbra can be imaged with high resolution and accuracy. This method may therefore open new possibilities for the investigation of the brain metabolism in transgenic mice and for non invasive evaluation of neuroprotective substances.
Figure 1
PET/CT-Fusion: left hemispheric ischemic lesion (white arrow). Adjacent penumbra with relatively higher FDG uptake (grey arrow).
OXYGEN METABOLISM CHANGES BEFORE AND AFTER SHUNTING OPERATION IN PATIENTS WITH IDIOPATHIC NORMAL PRESSURE HYDROCEPHALUS (I-NPH)
Junichi Miyamoto, Kazunori Tatsuzawa, Yasuo Inoue, Katsuyoshi Mineura
Department of Neurosurgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
Objective - The aim of the present study is to evaluate the changes in cerebral oxygen metabolism before and after ventriculo-peritoneal shunt (VPS) in patients with idiopathic normal pressure hydrocephalus (i-NPH).
Methods - Eight patients with i-NPH underwent VPS and an 15O-positron emission tomography (15O-PET) study before and approximately 3 months after VPS. The relationship between the changes in oxygen metabolism before and after VPS was analyzed.
Results - In five patients, the symptoms improved based on the classification by Krauss (1) (good responders) after VPS. In three patients, the symptoms improved subjectively following VPS (poor responders). The postoperative regional metabolic rate of oxygen (rCMRO2) of the good responders increased significantly. The postoperative regional oxygen extraction fraction (rOEF) in reduced in the poor responders.
Conclusion - The changes in oxygen metabolism imply metabolic pathogenesis and predict responsiveness to shunting operation in iNPH.
Figure - The graphs show the results of the 15O-PET study in the frontal white matter (A), putamen (B), and thalamus (C). Graphs show the results of rCBF (a), rCBV (b), rOEF (c), and rCMRO2 (d). The continuous line shows the results of good responders and the broken line shows the poor responders. rCMRO2 increases apparently after VPS in the good responders. In the poor responders, the change of rOEF is apparent without any improvements of rCMRO2.
BRAIN HISTAMINE H1 RECEPTOR OCCUPANCY OF A NEW ANTIHISTAMINE, BEPOTASTINE, MEASURED BY PET AND [11C]DOXEPIN
Manabu Tashiro1, Duan Xudong1, Motohisa Kato2, Masayasu Miyake2, Shouichi Watanuki2, Youichi Ishikawa3, Yoshihito Funaki3, Ren Iwata3, Masatoshi Itoh1, Kazuhiko Yanai2
1Division of Nuclear Medicine, Tohoku University Cyclotron and Radioisotope Center, Sendai, Miyagi, Japan, 2Department of Pharmacology, Graduate School of Medicine, Tohoku University, Sendai, Miyagi, Japan, 3Division of Radiopharmaceutical Chemistry, Tohoku University Cyclotron and Radioisotope Center, Sendai, Miyagi, Japan
Background and aims
Histamine H1 receptor (H1R) antagonists, or antihistamines, are often used for treatment of allergic disorders such as seasonal rhinitis. Antihistamines mainly act on the peripheral tissues but can induce sedation. This undesirable central side effect is caused by blockade of nerve transmission in the histaminergic neuron system. First-generation antihistamines such as diphenhydramine can easily penetrate blood-brain barrier (BBB) and tend to occupy a large proportion of post-synaptic H1Rs. Second-generation antihistamines such as fexofenadine and olopatadine can slightly penetrate BBB and H1Rs are slightly occupied. Thus, variation in cerebral H1R occupancy (H1RO) of antihistamines results mainly from their different BBB permeability. The aim of the present study was to compare the sedative property of a new antihistamine, bepotastine, in terms of H1RO using PET and [11C]doxepin.
Methods
Eight healthy male volunteers (mean age +/− s.d.: 24.4 +/− 3.3 years old) were studied after single oral administration of bepotastine 10 mg, diphenhydramine 30 mg (a typical sedative antihistamine), or placebo, using PET and [11C]doxepin in a crossover study-design. Binding potential ratio and H1R occupancy values were calculated using placebo data, and were compared between bepotastine and diphenhydramine. PET brain images were reconstructed with a filtered back projection algorithm. The brain images were then normalized by plasma radioactivity at 10 min post-injection to yield static distribution volume (DV) images. H1RO of bepotastine was compared to that of diphenhydramine. The present study was approved by the Committee on Clinical Investigation at Tohoku University Graduate School of Medicine, Japan. All experiments were performed at the Cyclotron and Radioisotope Centre, Tohoku University.
Results
Brain images following administration of bepotastine demonstrated slightly lower binding potential in comparison to those following placebo, and images following diphenhydramine administration demonstrated significantly lower binding potential in comparison to both placebo and bepotastine. Overall cortical mean H1RO of bepotastine and diphenhydramine were 15% and 57%, respectively. H1R occupancy of both bepotastine and diphenhydramine correlated well with their respective drug plasma concentration (p< 0.001).
Conclusions
In the present study, H1ROs following oral administration of bepotastine 10 mg or diphenhydramine 30 mg were calculated as 14.8% and 56.8%, respectively. In our previous studies, our previous PET studies demonstrated that first-generation antihistamines occupied more than 50% of available H1Rs. The result of bepotastine (15%) is in accordance with the categorization of bepotastine as a second-generation antihistamine. Previous studies have demonstrated H1ROs of other second-generation antihistamines such as terfenadine 60 mg (12–17%), fexofenadine 120 mg (1%) and ebastine 10 mg (10%). As a whole, second-generation antihistamines seem to occupy around 0 to 20% of brain H1Rs.
In conclusion, PET and [11C]doxepin is useful for evaluating sedative side effects of various psychoactive drugs with antihistamine effects. Collection of more H1RO data is encouraged for establishment of a reliable international database for evaluation of the sedative profile of psychoactive drugs.
A NEAR-INFRARED SPECTROSCOPY (NIRS) STUDY OF LEXICAL PITCH ACCENT PROCESSING IN JAPANESE SPEAKERS
Yutaka Sato1, Reiko Mazuka1,2, Yuko Sogabe1
1Laboratory for Language Development, BSI, RIKEN, Wako, Saitama, Japan, 2Department of Psychology and Neuroscience, Duke University, Durham, NC, USA
Background and aims
The aim of this study is to investigate the neural mechanisms underlying the processing of Japanese lexical pitch accent. The left and right cerebral hemispheres work together but differently for auditory language processing in human adults. The left side activates dominantly for processing most of the linguistic aspects of speech, including phonemic, lexical and syntactic analyses (Purves, 2001; Zatorre et al., 1992). On the other hand, the right dominant or nondominant activations are observed during processing of prosodic cues such as intonational pitch characterized by changing fundamental frequencies (Imaizumi et al., 1998; Zatorre et al., 1992). Although prosody is usually associated with the melodic features of spoken language, lexical level prosody such as Japanese pitch accent can be used to distinguish word meanings. For example, a pair of homophones with two syllables is distinguished by the pitch accent pattern which is either high-low (HL) or low-high (LH) such as a'me (HL: “rain”) vs. ame’ (LH: “candy”). A question then arises with regard to how the lexical pitch accent is processed, especially in terms of the functional lateralization.
Methods
We employed 44-channel near-infrared spectroscopy (NIRS), which can noninvasively measure relative changes in the concentration of hemoglobin (Hb) in the localized brain tissue. The subjects were healthy right-handed Japanese speakers (n=18, nine male and nine female, 20-22 years). We used a'me and ame’ tokens (14 pairs), ka'me (turtle) and kame’ (ceramic jar) tokens (14 pairs), and bisyllabic Japanese words differing by pitch accent pattern (HL vs. LH) (14pairs). In addition, pure tone stimuli were created by extracting fundamental frequencies from the a'me and ame’ words. Four conditions were performed in a block design paradigm. In the ame condition, the baseline block (20 s or 25 s) contained only a'me or ame’ which was repeated approximately every 1.25 s. The test block (10 s) contained both a'me and ame’ presented in a pseudo-random order with the equal probabilities. The kame condition was included to compare the phonemic change (/a/ to /ka/) to the lexical pitch-accent. In this condition, a'me or ame’ was presented during the baseline, but ame and kame were presented in the test block. Similarly, in the variable words and the pure tone conditions, the baseline block contained either HL or LH pattern stimuli, the test block consisted of both pitch pattern stimuli. In order to assess cerebral lateralization, a laterality index, LI = (L ? R) / (L + R), was calculated from the peaks of the Oxy-Hb responses in the left (L) and the right (R) temporal areas in each condition.
Results and conclusions
The results showed that the LI for the pure tone condition was significantly smaller than those for other three conditions, indicating that the lexical pitch accent is processed with more leftward shift compared with the processing of non-linguistic pure tone stimuli. Our results suggest that unlike more global prosody, which has been reported to be processed by the right hemisphere, lexical pitch accent is processed “linguistically” by Japanese native speakers.
HEMODYNAMIC CHANGES AFTER SPECTACULAR SHRINKING DEFICIT IN PATIENTS WITH CEREBRAL INFARCTION IN COMPARISON TO MRI FINDINGS
Yoshiharu Taguchi, Shutaro Takashima, Kouzi Hirano, Nobuhiro Dougu, Shigeru Toyota, Kortaro Tanaka
The Department of Neurology, Toyama University Hospital, Toyama, Japan
Introduction: Spectacular shrinking deficit (SSD) refers to a rapid recovery from a major hemispheric syndrome within a few hours after onset of stroke (1). SSD is considered to result from rapid embolic lysis, fragmentation and migration of embolus before irreversible damage occurs (1). However, little is known about the cerebral hemodynamic changes after SSD (2). We evaluated changes in regional cerebral blood flow (rCBF) using 99mTc-ECD single-photon emission computed tomography (SPECT) in patients with SSD, and compared these findings with MRI images.
Methods: We examined rCBF by 99mTc-ECD SPECT in 15 patients after SSD. The SPECT study was performed within 8 days after SSD and the follow-up SPECT study was done if patients revealed hyperperfusion. The obtained SPECT data were analyzed semiquantitatively using a three-dimensional stereotaxic region-of-interest template (3DSRT) reported by Takeuchi et al. (3). Brain MRI or CT was examined in all patients to evaluate the size of lesion.
Results: The examined patients were 8 men and 7 women, aged 70.8±11.9 years (mean±SD). In regard to type of cerebral infarction, 9 patients were cardiogenic embolism, 3 patients were artery to artery embolism and 3 patients had unknown embolic source. The time interval between the onset of cerebral hemispheric syndrome and the subsequent rapid improvement was 2.25±3.73 hours (mean±SD). SPECT showed hyperperfusion (111.45±6.03%) in the reperfused territory compared to the contralateral side in 6 patients, hypoperfusion (93.83±3.55%) in 6 patients and normoperfusion (100.74±2.95%) in 3 patients. The follow-up SPECT showed hypoperfusion (87.74±3.55%) in 4 patients and normoperfusion (100.74±3.95%) in 2 patients out of the 6 patients who revealed hyperperfusion on the first SPECT study. The SPECT study was performed significantly earlier in the patients revealing hyperperfusion (3.2±1.2 days after SSD) than in those revealing hypoperfusion (6.0±1.8 days)(p<0.05). In patients without any abnormal areas in diffusion-weighted MRI, the above SPECT findings were very helpful to assess the size of initial ischemic area.
Conclusion: The present study indicated that the initial ischemic areas become hyperperfused during the first few days after SSD, followed by a gradual decline of CBF. Measurement of rCBF by SPECT is very helpful to assess the size of initial ischemic area, especially in patients with no abnormality in diffusion-weighted MRI.
BRAIN METHIONINE METABOLISM IN HUMANS:A CORRELATION WITH CEREBRAL OXYGEN UTILIZATION STUDIED WITH PET AND SPM
Ryo Ashida, Kunihiro Kawanishi, Nobuhiko Shiraki, Haruka Maeda, Taku Fujimoto, Yasuhiro Ohsaki, Ashik Bin Ansar, Katsufumi Kajimoto, Yasukazu Kimura, Hiroki Kato, Makiko Tanaka, Eku Shimosegawa, Jun Hatazawa
Department of Nuclear Medicine and Tracer Kinetics, Osaka Univesity Graduate School of Medicine, Suita, Osaka, Japan
A number of experimental studies indicated that methionine (MET) is metabolized by neurons, glial cells, and blood vessels. Although normal distribution of MET brain uptake was demonstrated in experimental animals (1), it is still unknown in humans. In this study, we measured local brain uptake of [11C]-MET in neurologically normal subjects using PET. By applying SPM99, we created a standardized map of MET uptake in humans. We compared a pattern of MET uptake with that of cerebral metabolic rate of oxygen (CMRO2).
Materials and Methods
We measured [11C]-MET distribution in 2 normal volunteers and 4 neurologically normal patients (4 males and 2 females; mean age 63.3±15.6 years; 42~81 years). The PET scan started 20 min after [11C]-MET injection and continued for 10min. In each subject, we morphologically normalized and smoothed [11C]-MET images using the PET template installed in the SPM99. In other 6 normal volunteers (2 males and 4 females, mean age 33.3/6.6 years; 26~43 years), we measured cerebral blood flow and CMRO2 by 15O steady-state inhalation method. The SPM analysis was employed to examine regional differences between MET uptake and CMRO2.
Results
The cerebellum showed the highest accumulation of [11C]-MET with cerebellum to cerebral white matter ratio of 2.22, followed by occipital cortex (2.10), basal ganglia (1.88), pons (1.87), and temporal cortex (1.87). Parahippocampal gyrus (1.53) and anterior cingulated gyrus (1.53) showed the lowest. In comparison with CMRO2, MET uptake was significantly lower in anterior cingulate gyrus and insular cortices, and significantly higher in parahippocampal gyrus and pre-and postcentral gyrus.
Discussion
We created the first standardized PET image of [11C]-MET in human brain. In most brain structures, the MET accumulation was similar to that in rats (1) except basal ganglia. The basal ganglia showed the third highest in humans (1.88) but the lowest in rats (1.06). The pituitary gland was visually detected in the MET standard map. Elevated MET accumulation in pituitary gland would reflect active protein synthesis for pituitary hormones.
We found remarkable difference between MET uptake and CMRO2 in resting conditions. Anterior cingulated gyrus showed elevated CMRO2 whereas MET uptake was lowest among cerebral cortices. Postcentral and precentral gyri showed lower CMRO2 than MET accumulation. Kajimoto et al. recently demonstrated that brain MET uptake is independent of neuronal electric activity. In crossed cerebellar diaschisis, cerebellar glucose metabolism was suppressed by 17%, but MET uptake was not reduced. Their observation and the present findings indicated that MET uptake is independent of neuronal electric activity during physiological conditions.
COMPARISON OF HIPPOCAMPAL ATROPHY ASSESSED USING NEW MRI SOFTWARE (VSRAD) IN ALZHEIMER'S DISEASE, VASCULAR DEMENTIA, AND DEMENTIA WITH LEWY BODIES
Kazunori Okada1, Nobuyuki Umegae1, Takuya Yamaguchi1, Shuhei Yamaguchi2
1Department of Neurology, Oda Municipal Hospital, Oda City, Shimane Prefecture, Japan, 2Department of Internal Medicine III, Shimane University School of Medicine, Oda City, Shimane Prefecture, Japan
[Introduction] The early diagnosis of Alzheimer's disease (AD) is important for determining when to administer drugs such as cholinesterase inhibitors, and for estimating the prognosis. Matsuda et al. have recently developed new magnetic resonance imaging (MRI) analysis software, the Voxel-based Specific Regional Analysis System for Alzheimer's Disease (VSRAD), which analyzes brain volume by voxels and yields a hippocampal atrophy index using the Z score. To clarify the usefulness of VSRAD, we compared the severity of disease, intellectual function, and hippocampal atrophy in subjects with AD, vascular dementia (VD), and dementia with Lewy bodies (DLB).
[Subjects] All of the subjects were outpatients and they could walk without assistance. The subjects consisted of 30 probable AD patients (mean age 80.2 yr), 10 VD patients (76.3 yr), 3 DLB (71.0 yr), and 8 normal controls (NC; 66.4 yr). Each disease was diagnosed according to the DSM-IV and the guidelines for DLB.
[Methods] Neuropsychological examinations administered consisted of the Mini-Mental State Examination (MMSE) for all subjects and the Alzheimer's Disease Assessment Scale (ADAS-J cog.) for AD. The impairment of daily life was assessed using the Clinical Dementia Rating (CDR). Head MRI images were obtained in 1.5-mm-thick sagittal slices and converted into DICOM files. The images were analyzed on a personal computer using the VSRAD system.
[Results]
The average Z score, which indicates the degree of parahippocampal atrophy compared to age-matched healthy controls, was 3.0±1.3 for AD, 1.7±0.7 for VD, 1.2±0.3 for DLB, and 1.1±0.5 for NC. The Z score for AD was significantly higher than for VD and NC, and the Z score for VD was significantly higher than for NC [Fig. 1].
Only in the AD group were significant correlations observed between the Z score and the MMSE (r2=0.35, p<0.001) and ADAS (r2=0.17, p<0.05).
[Conclusions] The VSRAD system provides useful information about hippocampal atrophy that distinguishes AD from VD. In addition, it indicates the severity of AD patients.
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EXOGENOUS GROWTH FACTORS AMELIORATE BEHAVIORAL DEFICITS THROUGH ACTIVATION OF ENDOGENOUS NEURONAL REGENERATION IN YOUNG AND AGED RAT HUNTINGTON MODELS
Jun-ichi Tanaka1,3, Gakushi Yoshikawa1,3, Soichi Oya1,3, Nobuhito Saito1, Takaaki Kirino2,3, Nobutaka Kawahara1,3
1Department of Neurosurgery, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 2Research Institute International Medical Center of Japan, Tokyo, Japan, 3Solution Oriented Research for Science and Technology, Japan and Science Technology Corporation, Saitama, Japan
Introduction: We previously reported that exogenously administrated growth factors (GFs) activated neural progenitors and recruited them to the hippocampal CA1 region in rat global ischemia, which was accompanied by improvement of impaired functions. Here rises a question whether these self-repair mechanisms also occur in other non-neurogenic sites not only in young but in aged adults. To address these issues, this study was conducted using a rat model of Huntington's disease. Methods: Young (2 months of age) and aged (more than 18 months of age) male Wistar rats were given quinolinic acid (100 nMol/1ul) or PBS directly into the right striatum. Two days later, the treated groups were given either a cocktail of EGF and FGF-2 or vehicle solution intraventricularly for 7 days, with simultaneous administration of BrdU (100mg/kg, ip.) twice daily for the last 3 days. Some of non-treated animals were sacrificed just after injuries. Brains were removed after 6 to 7 weeks, and immunohistochemical investigations were carried out on serial coronal sections. Histological quantification was conducted in 3 rectangular areas with sizes of 0.5 × 2 mm2, L: lateral adjacent to the lesion core, IM: intermediate and M: medial close to the lateral ventricle. To confirm newborn cells as neurons, double staining was performed using markers for NeuN, Hu, Dcx or GFAP together with BrdU. Fluorogold, a retrograde tracer, was injected for 3 GF-treated rats into the ipsilateral globus pallidus 3 days prior to sacrifice. Phenotypes of new neurons were classified by antibodies against choline-acetyltransferase (ChAT), parvalbumin, neuropeptide Y and DARPP-32. Behavioral study was carried out using the apomorphine (1.0 mg/kg)-induced rotation test. In the younger groups, the staircase test was also conducted. Difference with P<0.05 were considered statistically significant. Results: In the young adults, by cresyl violet staining, the number of neurons increased by 5.8% without treatment compared with that at 2 days, which was further augmented significantly by the GFs (42.8%) especially in the area L and IM as compared to vehicle control (3.2 %) (unpaired t-test, n=5-9/group). These cells were confirmed as mature neurons by double staining for BrdU and NeuN. Although the same trends were observed in aged animals, they did not reach a significant difference (P=0.08, n=4, each). Intriguingly, immunostaining revealed a relatively large increase of ChAT-positive newborn cells (from 12 to 39%). Double staining for Fluorogold and BrdU identified colabeled cells in the rostral striatum, indicating the integration of these newborn cells into the existing circuits. Rotometer test revealed the significant recovery in GFs-treated groups in both young and aged animals (paired t-test, n=7-11). Staircase test in young GF-treated animals showed good recovery of an impaired forelimb function equal to the unlesioned side; while vehicle-treated animals remained unskillful (n=16 and 9). Conclusions: Exogenously administered GFs augmented the endogenous regenerative capacity in the striatum, and improved behavioral functions in both young and aged adult Huntington's disease rats. This novel regenerative strategy without transplantation may have a therapeutic potential in various neuronal injuries in the striatum even in the aged adults.
ENGRAFTMENT AND DISPERSION OF HUMAN EMBRYONIC STEM CELL-DERIVED MULTIPOTENT NEURAL STEM CELLS IN EXPERIMENTAL STROKE MODELS
Marcel Daadi, Gary Steinberg
Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA
Cellular therapy for stroke has demonstrated promising functional recovery in experimental stroke models. However, the current sources of stem cells are not amenable for the needs of large-scale production and multi-centered clinical trials. Human embryonic stem cells (hESCs) have the potential to provide an indefinite and renewable source of a wide range of normal cell types. However, the isolation of homogenous and self-renewable sources of neural progenitors capable of differentiating into the principal neural lineages from hESCs remains largely unexplored.
We carried out a series of experiments to isolate stable, homogenous and expandable multipotent neural stem-progenitor cell lines (NSPCs) from hESCs. The hESCs were cultured in serum free medium containing epidermal growth factor, basic fibroblastic growth factor and leukemia inhibitory growth factor. After 3 days in vitro (DIV), the cells appeared aggregated in clusters or spheres. These primary spheres were collected and replated in fresh culture media supplemented with factors. Secondary spheres were then generated and expanded for 5 to 6 passages. To ascertain self-renewal ability under clonal conditions, a single cell suspension made from dissociated spheres were plated at clonal density (10 cell/µl). After 48 hrs, plated single cells underwent a first cell division and gave rise to progeny expressing nestin, a neurepithelial stem cell marker. The single cell-derived spheres continued to grow over the 7 DIV. At this stage, all the neurospheres generated were nestin+ and did not express the pluripotency markers SSEA4, OCT4 or Nanog. The NSPCs showed stable growth and a consistent 2.7±0.2 fold increase every 5 to 7 days. The population doubling at each passage averaged at 1.4±0.1. When induced to differentiate, 36.6±2.7 of the cells expressed nestin, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% were GFAP+ astrocytes and 7.1±0.4% were oligodendrocytes and expressed galactocerebroside.
The NSPCs were then evaluated in the rat middle cerebral artery occlusion (MCAO) model of stroke. Sprague Dawley rats (n=10) were subjected to 1.5 h MCAO. One week later, immunosuppressed rats were transplanted with NSPCs (2 × 105) into the ischemic boundary zone in the striatum. Animals were sacrificed 1 week later and the brains processed for histo-pathology and immunocytochemistry. Grafted NSPCs, identified with a human-specific nuclear marker, demonstrated a 14.2±3.1% survival rate and 61.2±4.7% migrated at least 200 µm away from the graft core and penetrated an average distance of 806.4±49.3 µm into the stroke-damaged tissue. They extended neurite outgrowth into the host parenchyma and did not show tumor formation. Characterization of the grafted cells demonstrated the absence of pluripotency markers and the presence of neural-specific markers. Transplanted cells expressed nestin and differentiated into TuJ1+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes with 6.5±0.9% of the neurons expressing doublecortin.
We have demonstrated that under defined culture conditions, pluripotent hESCs can give rise to homogenous, expandable multipotent NSPCs. Transplantation of NCPSs into infarcted neural tissue resulted in good survival, migration and neural differentiation into the principal CNS cell types. These findings provide preliminary evidence of potential use of hES derivatives in cell therapy for stroke.
MICROGLIA PROMOTES ASTROGLIOGENESIS OF THE NEURAL STEM CELLS THROUGH ACTIVATING JAK/STAT SIGNALING
Pengxiang Zhu, Ryuji Hata, Fang Cao, Masahiro Sakanaka
Department of Functional Histology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
Background and aims
Neural stem cells (NSC) are thought to undergo differentiation into neurons and astrocytes under the influence of microenvironment. In vivo and in vitro evidence suggested astrocytes promoted neurogenesis of NSC [1]. However little is known about the effects of microglia on neural stem cell fate.
Methods & Results
Microglia from the newborn rats and NSC from the 17-day-old rat embryos were cultured as described elsewhere [2,3]. Medium in the 24 well plate containing NSC was changed to the microglia-condition medium (MCM, condition medium from 24-h microglia cultures at the density of 0.7×105/well or 2.0×105/well). Three days later, western blot analysis revealed that MCM promoted astrogliogenesis of NSC (Figs A-C). We next investigated mRNA levels of several secreted factors that were supposed to modulate NSC fate. As shown in Fig D, expression of LIF and FGF2 mRNA in microglia was prominent. These data suggested that LIF and FGF2 were involved in the mechanism of microglia-induced astrogliogenesis in the NSC. Since LIF can confer Jak/Stat signaling pathway, we applied a Jak2 kinase inhibitor AG490(1µM) and demonstrated AG490 could inhibit the microglia-induced astrogliogenesis of NSC. Furthermore, promoter assay revealed that MCM could actually activate Stat3 function in NSC.
Conclusions
We showed that microglia promoted astrogliogenesis of the NSC. Our data suggest that activation of Jak/Stat signaling pathway play a crucial role in the astrogliogenesis of NSC mediated by microglia.
ANOXIA INCREASES THE NUMBER OF ADULT NEURAL STEM CELLS BY INHIBITION OF APOPTOSIS
Heinrich F. Burgers1, Martin Henrik Maurer1, Dominik Schelshorn1, Armin Schneider2, Wolfgang Kuschinsky1
1Department of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany, 2SYGNIS Pharma AG, Heidelberg, Germany
Introduction: Neurons are damaged by deprivation of oxygen and glucose. It is not known whether their precursors, i.e. adult neural stem cells are likewise damaged by oxygen and glucose deprivation. We have investigated the influence of anoxia, glucose deprivation and oxygen glucose deprivation on proliferation and survival of adult neural stem cells in vitro. Since erythropoietin is known as neuroprotective agent, we also tested its effect.
Methods: We cultured adult neural stem cells (NSC) from the subventricular zone of adult rat brains and exposed them to normoxia (controls) or anoxia (5% CO2 in N2) for 24h. Glucose deprivation was induced adding 5mM 2-deoxy-D-glucose. Cell viability was determined using a colorimetric assay (CellTiter96 AQueous One; Promega, Mannheim, Germany). Total caspases activity was recorded using a homogeneous caspases assay (Roche, Penzberg, Germany). Caspase-3/7 activity was determined using the Apo-ONE Homogeneous Caspase-3/7 Assay (Promega). Proliferative activity was determined by BrdU uptake and subsequent immunostaining of fixed cells on coverslides. Proliferation rates were determined by the proliferation dependent dilution of a fluorescent intracellular dye, CFSE (Sigma-Aldrich, Munchen, Germany) using FACS (BD Bioscience, Heidelberg, Germany). Erythropoietin (Janssen-Cilag, Neuss, Germany) concentrations ranged from 0.1 to 100 u/mL.
Results: Cell viability of NSC increased by more than 50% during anoxia compared to normoxic controls (n=240, p<0.01). This increased cell viability could be caused by either (1) reduced apoptosis or (2) by increased proliferation.
(1) The two caspases assays of apoptosis yielded different results: The general caspases activity was significantly reduced by about 50% (n=15, p<0.05) whereas the specific activity of caspases-3/7 of anoxic cells was not affected (n=18, n.s.), indicating a pathway of apoptosis different from caspases-3/7. (2) Proliferation activity was tested by BrdU incorporation (24h). About 99% of the normoxic cells had incorporated BrdU, whereas only 71% of the anoxic cells were found BrdU-positive, indicating decreased proliferation activity in anoxic cells. CFSE staining showed identical effects.
Under glucose deprivation cell viability was reduced to 44% (n=36, p<0.05) regardless of anoxia or normoxia (n.s.).
Erythropoietin did not affect cell viability in normoxia and anoxia, whereas under oxygen glucose deprivation, erythropoietin moderately increased cell viability by up to 6% (n=216, p<0.05). Again, total caspases activity was reduced in a dose dependent manner by 25% (n=24, p<0.05) in normoxia and anoxia whereas caspase-3/7 activity of anoxic cells was not affected.
Conclusions: The main finding of the present study is an increased viability of adult neural stem cells during anoxia. This effect, which is known from other types of stem cells, can be explained by reduced overall (homogeneous) caspases activity, but not by specific effects on caspases-3/7 or on cell proliferation. Glucose deprivation is detrimental to cell viability, even during anoxia.
The erythropoietin-induced inhibition of homogeneous caspases activity did not result in an increased viability during the short experimental period (24h). However, we cannot exclude a positive effect of erythropoietin during longer observation periods.
Support: German Ministry of Education and Research (National Genome Research Network, NGFN2), Dell GmbH, Estate of Friedrich Fischer.
ZINC CHELATION BY CLIOQUINOL OR VESICULAR ZINC DEPLETION BY TARGETED DELETION OF ZNT3 REDUCES HIPPOCAMPAL NEUROGENESIS FOLLOWING HYPOGLYCEMIC INSULTS
Sang Won Suh1,3, Seok Joon Won2,3, Aaron Hamby1,3, Christian Sheline4, Raymond Swanson1,3, Jialing Liu2,3
1Department of Neurology, UCSF, San Francisco, CA, USA, 2Department of Neurosurgery, UCSF, San Francisco, CA, USA, 3Department VAMC, San Francisco, San Francisco, CA, USA, 4Dpepartment of Neurology, Washington University, St. Louis, MO, USA
Neurogenesis after brain injury not only leads to the replacement of damaged cells but might also contribute to functional recovery, suggesting the possibility of endogenous neural repair. In the brain, neurogenesis occurs in the subgranular zone (SGZ) of the dentate gyrus (DG) where high amount of vesicular zinc is localized in the presynaptic terminals. The aim of this study is to determine whether the vesicular zinc plays a modulating role in neurogenesis either under normal or pathological conditions. In this study, we investigated the role of zinc on hippocampal neural regeneration in a rodent model of hypoglycemia using cell permeable zinc chelator, clioquinol (CQ) or targeted deletion of the zinc transporter 3 (ZnT3 KO). In the rodent model of hypoglycemia induced by insulin followed by 30-minute isoelectric electroencephalogram (EEG), extensive neuronal loss was observed in the hippocampus including area CA1 and the DG. At this time, a transient increase in progenitor cell proliferation in the DG SGZ was detected, leading to an increase of immature neuroblasts. Most of the surviving newborn cells assumed a neuronal phenotype within one week in the DG. Animals treated with CQ (30 mg/kg) twice per day for 1 week after hypoglycemia showed decreased progenitor cell proliferation in the SGZ compare to vehicle treated ones, with a parallel reduction in the number of neuroblasts. Using a mice model of hypoglycemia, wild type mice showed substantial increase of progenitor cell proliferation one week after hypoglycemia. However, ZnT3 KO mice showed less number of neuroblasts in the SGZ both under normal and hypoglycemic conditions. Our data suggest that synaptic zinc chelation or genetic depletion of vesicular zinc significantly reduces progenitor cell proliferation in SGZ. For the first time, our present study demonstrates that vesicular zinc in the hippocampus modulates neurogenesis in the adult brain not only under normal condition but also following insulin-induced hypoglycemia. The mechanisms involved in the Zn-mediated inhibition of hippocampal neurogenesis warrant further investigation.
Supported by Juvenile Diabetes Research Foundation (SWS, JDRF2-2006-113; JL NIHR21 NS051576)
IMPLANTATION OF A NEW POROUS GELATIN-SILOXANE HYBRID INTO A BRAIN LESION AS A POTENTIAL SCAFFOLD FOR TISSUE REGENERATION
Kentaro Deguchi1, Takeshi Hayashi1, Kanji Tsuru3, Mikiro Takaishi2, Mitsuyuki Nagahara3, Shoko Nagotani1, Yoshihide Sehara1, HanZhe Zhang1, Satoshi Hayakawa3, Tatsushi Kamiya1, Masahiro Miyazaki2, Akiyoshi Osaka3, Nam-ho Huh2, Koji Abe1
1Departments of Neurology, Graduate School of Medicine, Dentistry and Pharmacy, Okayama University, Okayama, Japan, 2Departments of Cell Biology, Graduate School of Medicine, Dentistry and Pharmacy, Okayama University, Okayama, Japan, 3Biomaterial Laboratory, Faculty of Engineering, Okayama University, Okayama, Japan
Background: For brain tissue regeneration, a scaffold for migrated or transplanted cells with vessels is important if the tissue become pannecrotic. The present study was designed to determine whether the implantation of porous gelatin-siloxane hybrids can serve as a scaffold for brain tissue regeneration.
Methods: We used male Wistar rats of 12-weeks old. A new porous gelatin-siloxane hybrid derived from the integration of gelatin and 3-(glycidoxypropyl) trimethoxysilane (GPSM) was implanted into a defect of the cerebral cortex, which was located at 3.0 mm anterior to 2.0 mm posterior of the bregma (5 mm in length) and 2.0–4..0 mm (2 mm in width) lateral to the midline, and the depth of the lesion was approximately 2.0 mm below the skull surface. The block of GPSM was presoaked in basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) or only vehicle, and the time of implantation was just after or at 30d of brain defect making. The animal was decapitated and the frozen sections were prepared. In order to identify the cell types in the newly formed tissue, immunohistochemical analysis was performed for N-acetylglucosamine oligomers (NAGO), an endothelial cell marker, GFAP, ionized calcium-binding adapter molecule-1 (Iba1), NeuN and bromodeoxyuridine (BrdU). Additionally, in order to determine whether BrdU-labeled cells differentiate into vascular endothelial, astroglial and microglial cells, double immunofluorescent studies were performed for BrdU and NAGO, GFAP or Iba1. Furthermore, we observed axonal growth into the scaffold by double staining for BrdU and MAP2.
Results: The control brain showed distortion of the cerebral cortex and enlargement of the ipsilateral lateral ventricle (Fig.1 A, C). On the other hand, the GPSM implanted into the lesion remained at the same site for 60 days, kept integrity of the brain shape, and attached well to the surrounding brain tissues (Fig.1 B, D). Marginal cavities of the scaffolds were occupied by newly formed tissue (Fig.1 E), where newly produced vascular endothelial, astroglial and microglial cells were found with BrdU positivity. The numbers of those cells were dose-dependently increased with bFGF and EGF. Extension of dendrites was also found from the surrounding cerebral cortex to the newly formed tissue, especially with addition of bFGF and EGF.
Conclusion: The present study showed that GPSM had biocompatibility. It can thus serve as a potential scaffold for cell migration, angiogenesis and dendrite elongation with dose-dependent effects of bFGF and EGF.
NEURAL STEM CELLS EXERT A NEUROPROTECTIVE ACTION BY CHANGING THE ISCHEMIC MICROENVIRONMENT
Principato Maria-Cristina1, Carmen Capone1, Simona Frigerio2, Stefano Fumagalli1, Maurizio Gelati2, Claudio Storini1, Mery Montinaro1, Marco De Curtis3, Eugenio Parati2, De Simoni Maria-Grazia1
1Laboratory of Inflammation and Nervous System Diseases, Mario Negri Institute, Milano, Italy, 2Laboratory of Neurobiology and Neuroregenerative Therapies, Carlo Besta Neurological Institute, Milano, Italy, 3Clinical Epileptology and Experimental Neurophysiology Unit, Carlo Besta Neurological Institute, Milano, Italy
Cell replacement strategies are particularly promising candidate therapies for stroke, a condition in which a widespread loss of different cell types occurs that in principle may benefit from exogenous stem cells replacement of lost or damaged cells. In the present study we have used a murine model of focal, transient ischemia to investigate if: 1) infused NSC are able to decrease ischemic injury and restore brain function; 2) these cells induce changes in the environment in which they are infused; 3) changes in brain environment consequent to transient ischemia are relevant for NSC action.
Neurosphere-derived stem cells were obtained from brains of C57BL/6 newborn mice. NSC were infused intracerebroventricularly 4h or 7d after 30min focal ischemia obtained by intraluminal MCAO. Behaviour was evaluated in an open field paradigm at day 7 or 14 after ischemia/reperfusion. The impairment in the numbers of rears and of contacts with objects, measures related to exploratory behavior and sensory-motor activity respectively, observed in ischemic mice, was significantly improved in ischemic mice receiving NSC. Neuronal loss, evaluated in striatum and in cortex, was significantly reduced at day 7 and day 14 in ischemic mice infused with NSC at 4h compared to controls and to ischemic mice receiving NSC at 7d. Infusion of adult fibroblasts in the same experimental conditions was not effective. Assessment of infused NSC distribution revealed that they migrated from the ventricle to the brain parenchyma, they were observable up to day 14, but they progressively decreased in number. In mice receiving NSC at day 7 and in sham-operated mice, few cells located near the injection site could be observed only at 24h, indicating that the survival of these cells in brain tissue relates to the ischemic environment. The mRNA expression of trophic factors such as IGF-1, VEGF-A, TGF-alpha1, BDNF and SDF-1beta increased 24h after NSC infusion in ischemic mice treated with NSC at 4h compared to sham-operated and to mice receiving NSC at 7d. Ischemic brains infused with NSC showed higher activation of microglia/macrophages compared to sham-operated mice receiving NSC or to ischemic brains receiving saline injection showing that microglia/macrophages activation is required for NSC action.
In conclusion, neurosphere-derived NSC are able to reduce functional impairment and neuronal damage after ischemia/reperfusion injury. The reciprocal interaction between NSC and the ischemic environment is crucial for NSC protective actions. Indeed NSC survival is enhanced by the ischemic environment and NSC can induce the activation of chemokine and neurotrophic factors. NSC protective effects also relates to the interaction with activated microglia/macrophage. Based on these observations we propose that NSC exert their protective effect by a bystander control of the ischemic environment.
THERAPEUTIC TIME WINDOW FOR INTRAVENOUS TRANSPLANTATION OF HUMAN NEURAL STEM CELLS AFTER CEREBRAL ISCHEMIA IN ADULT RATS
Miyeoun Song, Yong-Ju Kim, Yoon-Ha Kim, Jina Roh, Byung-Woo Yoon
Department of Neurology, Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea
Introduction: It was reported that human neural stem cells (hNSCs) improve functional recovery in the adult rats with experimental focal ischemia when administered one day after stroke. However, the optimal time to administer these cells after cerebral ischemia has not been established. In this study, we intravenously delivered hNSCs at various time after induction of ischemia in the rat middle cerebral arterial occlusion (MCAo) model to determine specific therapeutic time for efficacy.
Methods: After MCAo, Sprague-Dawley male rats weighing 250-300 g were divided into ischemia-only and ischemia-hNSC groups. In ischemia-hNSC group, hNSCs (4×106 cells) were intravenously injected at different time points (3hr, 1d, 3d, 7d, 14d, and 28d after MCAo, respectively). After transplantation, rats were evaluated for 4 weeks (at 1 day before and after ischemia, and weekly thereafter) using modified neurological severity score (mNSS) and cylinder test. To measure the infarction size, Nissl staining was used.
Results: Histological analysis at 28 days after transplantation revealed reduced infarction volume comparing with the ischemia-only group. Moreover, infarction volume of 1d-ischemia-hNSC group (150±37 mm3, n=6) was significantly decreased (p=0.02) as compare with these of 28d-hNSCs-ischemia group (202±39 mm3, n=6). On mNSS, 3hr (4.5±2.2, n=8, p=0.028) and 1d-ischemia-hNSC groups (3.5±2.3, n=6, p=0.014) showed better performance from 4 weeks after the transplantation than control group (6.9±1.5, n=7). Conclusion: Our study demonstrates that intravenous administration of hNSCs 3hr to 1 day after stroke could reduce the size of infarction, and result in functional improvement.
Grant support: This study was supported partly by a grant from the Korea Health 21 R & D Project, Ministry of Health & Welfare (Project No.: A040004), and partly by a National R & D Program Grant of The Ministry of Science and Technology. (Project No. SC-3111), Republic of Korea.
CHARACTERIZATION OF A FOCAL EAE-MOG MODEL FOR THE STUDY OF REACTIVATION OF CNS LESIONS BY A SYSTEMIC INFLAMMATORY RESPONSE
Sebastien Serres1, Yanyan Yiang2, Sandra Amor3, Daniel C. Anthony2, Nicola R. Sibson1
Experimental Neuroimaging Group, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK, Experimental Neuropathology Laboratory, Department of Pharmacology, University of Oxford, Oxford, UK, Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands
Multiple sclerosis (MS) is a chronic inflammatory disorder in which 70% of MS patients display a relapsing-remitting form of the disease. It is thought that bacterial and viral infections may trigger relapses and progression of MS. In this study, our aim was to characterise a focal EAE-MOG model targeted in the brain by intracerebral injection of cytokines, and to determine whether this lesion could be reactivated by a systemic inflammatory response. The lesions were monitored by Magnetic resonance imaging (MRI) and immnunostaining.
Male Lewis rats were immunised by subcutaneous injection of MOG peptide (35-55) or MOG protein. Three weeks later, a solution containing 1.45µg of TNFα + 1µg of IFNγ (in 2 · 1 sterile saline) was injected into the corpus callosum of the same animals. Subsequently, animals underwent MRI at days 0, 1, 7, 14, 21 and 28. T1- (pre- and post-gadolinium) and T2-weighted images, as well as apparent diffusion coefficient and regional cerebral blood volume (rCBV) maps were obtained using a 7T horizontal bore magnet. Four weeks after cytokine injection, the animals were challenged systemically by intraperitoneal injection of lipopolysaccharide (LPS; 100 µg/kg) and underwent further MRI examinations 6 and 24h later. Further animals were perfusion-fixed at the same timepoints as the MRI for immunohistochemical analysis.
At 1 day post injection, we observed an acute effect of the cytokines with breakdown of the BBB, increased rCBV and ADC changes in the injected hemisphere. We also observed a significant disruption of the signal on T2-weighted images in the corpus callosum at the injection site. The longitudinal study of T2-weighted images revealed the presence of a chronic hypointense signal in the striatum below the injection site and an increase in ventricle size with incomplete recovery of the signal from the corpus callosum [Figure 1a]. ED1 immunostaining correlated with this hypointense signal, suggesting involvement of activated macrophage in this effect. The disruption and remission of the corpus callosum structure is correlated to an inflammation process as revealed by ED1 staining. The study of the ongoing disease also revealed an increase in the left/right ratio of rCBV followed by recovery to normal values by 28 days [Figure 1b].
Induction of a systemic inflammatory response with LPS challenge, resulted in an increase in the left/right rCBV ratio at both 6 and 24h after LPS injection [Figure1b], together with recruitment of both macrophages as revealed by ED1 staining [Figure1c]. These findings show that the reactivation of inflammatory processes by a bacterial endotoxin within a previously quiescent CNS lesion can be obtained in a clinically-relevent model of MS.
EXPRESSION OF GLUCOSE TRANSPORTER MOLECULES IN ADULT NEURAL STEM CELLS: INFLUENCE OF HYPOXIA AND HYPERGLYCEMIA
Martin Henrik Maurer, H. Kenny Geomor, Heinrich F. Burgers, Dominik W. Schelshorn, Wolfgang Kuschinsky
Department of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany
Background and aims: Glucose constitutes the main substrate for the nervous tissue. It is transported by specialized glucose transporter molecules, the GLUT family. The facilitative glucose transport protein GLUT1 is expressed at the blood-brain barrier and in astrocytes, whereas the GLUT3 isoform is responsible for the neuronal glucose uptake. Almost nothing is known about the energy metabolism of neural stem cells in the adult brain with regard to functional similarities and differences compared to neurons and astrocytes. In the current study, we addressed the questions, whether neural stem cells express glucose transporter molecules and whether this expression can be regulated by typical pathophysiological stimuli such as hypoxia and exposure to high glucose concentrations.
Methods: Neural stem cells were isolated from the subventricular zone of adult rat brains and cultured using standard protocols. For hypoxia, neural stem cells were incubated in a hypoxia chamber for 24 h in 5% CO2, 95% nitrogen. For hyperglycemia, the glucose concentration in the cell culture media was doubled. GLUT1 and GLUT3 expression was compared by RT-PCR, Western blotting, and immunocytochemistry.
Results: Neural stem cells expressed mRNA for GLUT1 and GLUT3, but not for GLUT2 and GLUT4. We found co-expression of GLUT1 and GLUT3 in the same cells throughout, indicating that there are no subpopulations of neural stem cells expressing only one glucose transporter type. Both hypoxia and high glucose concentrations increased GLUT1 and GLUT3 expression on the mRNA and protein levels. Whereas GLUT1 mRNA concentrations increased moderately (about 1.4-fold), GLUT3 mRNA expression increased strongly (about 10-fold) in hypoxia, hyperglycemia, or the combination thereof. On the protein level, GLUT1 showed a high baseline expression which did not change under the experimental conditions, whereas GLUT3 increased about 2-fold.
Conclusions: The data show the existence of GLUT1 and GLUT3 in neural stem cells and a moderate degree of regulation in their expression. Since neural stem cells are capable of differentiating both into astrocytes (expressing GLUT1), and neurons (expressing GLUT3), we interpret the co-expression of GLUT1 and GLUT3 in the neural stem cells as sign of maintained multipotency.
Acknowledgements: Supported by the German Ministry of Education and Research (BMBF) within the National Genome Research Network NGFN-2 (01GS0496, MHM and WK), the German Research Foundation (DFG, MA 2492/2-2, MHM and WK) and the Estate of Friedrich Fischer (MHM).
NEURODEVELOPMENTAL STUDIES IN C57B/L6 MICE ASSESSED BY IN VIVO DTI
Halima Chahboune1,2, Laura. R. Ment2, William. B. Stewart3, Douglas. L. Rothman4, Fahmeed Hyder5
1Department of Diagnostic Radiology, Yale University, New Haven, CT, USA, 2Department Of Pediatrics, Yale University, New Haven, CT, USA, 3Department of Surgery, Yale University, New Haven, CT, USA
DTI is a noninvasive technique that allows studies of neurodevelopment in transgenics or mutants. Prior in vitro mouse brain DTI studies at high magnetic fields that have revealed tissue anisotropy variations were conducted in fixed brain with DTI signal acquisitions requiring several hours to a day. The purpose of this study was to assess the feasibility of in vivo DTI to quantify maturation.
METHODS
Normal C57B/L6 mice, from P15 to P45, were anesthetized with urethane and MRI experiments were performed at 9.4T with custom-made surface coils. The animals were kept warm by a hot water blanket and whenever possible, blood samples were removed at the end of the experiment to verify physiology. DTI data were collected using a modified Stejskal-Tanner spin-echo diffusion-weighted sequence (Δ = 5 ms; Δ = 8 ms; TR/TE = 1000/18; NEX = 2; matrix = 128×128; FOV = 20×20 mm; slice thickness = 0.25 mm). Diffusion weighting (b ? 1000 s/mm2) in 16 different directions were acquired with one reference image (b ? 0 s/mm2). Maps of mean diffusivity (ADC), fractional anisotropy (FA), and volume ratio (VR) were calculated, and from which the primary eigenvectors were used to calculate diffusion encoded color (DEC) maps. The data were used to get trajectory maps in medial-lateral, dorsal-ventral, and anterior-posterior.
RESULTS
The brain regions quantified by the current in vivo DTI studies through development (P15 to P45) showed some morphological changes. In the corpus callosum, the ADC decreased slightly with age (0.8×10-3 to 0.5×10-3 mm2/s), the FA gradually increased with age (0.4 to 0.6) which implied maturation of medial-lateral fibers (R2=0.99) with marginal or no significant changes in anterior-posterior or dorsal-ventral fibers. The high FA value in corpus callosum in early age may be attributed to tightly packed axons that allow water diffusion primarily along the fiber direction as well as the myelination of axons. In the cingulate, the ADC values decreased slightly with age (0.8×10-3 to 0.5×10-3 mm2/s), the FA increased with age (0.3 to 0.4), which implied maturation of anterior-posterior fibers (R2=0.80) with marginal or no significant changes in medial-lateral or dorsal-ventral fibers. The high FA value in the cingulated may be attributed to glial morphological changes. In the forelimb and whisker barrel areas, no significant differences were observed. In the future, localized changes somatosensory areas may have to be studied before P15.
ACKNOWLEDGEMENTS
Supported by NIH grants (R01 MH-067528, P01 NS-35476) and a Pfizer gift.
VOXEL-WISE DETECTION OF AMYLOID DEPOSITION WITH PITTSBURGH COMPOUND B (PIB) IN NORMAL AGING: A PARTIAL VOLUME CORRECTION-BASED APPROACH
Hiroto Kuwabara1, Anil Kumar1, Jitka Sojkova1,2, Olivier Rousset1, Mohab Alexander1, Weiguo Ye1, Daniel Holt1, Robert Dannals1, Susan M. Resnick2, Dean F. Wong1,3
1Department of Radiology, Johns Hopkins University, Baltimore, MD, USA, 2National Institute On Aging, Baltimore, MD, USA, 3Department of Psychiatry, Johns Hopkins University, Baltimore, MD, USA
Background and Aims: Identification of voxel-wise amyloid deposition in normal aging may be hampered by high inter-subject variability and the apparent greater retention of PIB in white matter than gray matter. We employed a new approach to compare observed binding potential (BP) maps to partial volume correction (PVC)-predicted BP maps that served as individuals' own control, to cope with inter-subject variability while accounting for gray-white matter composition at the voxel level.
Methods: Twenty-eight nondemented participants of Baltimore Longitudinal Study of Aging (age: 79.2 ± 8.0; 11 males and 17 females) underwent one 90-min dynamic PIB-PET scan. BP maps of PIB scans were constructed by the bolus-plus-infusion transformation (BPIT; Kuwabara et al., 2002). Gray matter, white matter, and cerebro-spinal fluid space segments were identified on MRI, transferred to PET space, and smoothed by PET camera specific Gaussian kernels to reproduce the partial volume effect. The resulting geometric transformation matrix (M; Rousset et al., 1998), is an n by 3 matrix where n is the number of voxels covering the three segments. PV-corrected BP values of the three segments (T, a 3 by 1 matrix) were obtained by left matrix division using Gaussian elimination. The PVC-predicted BP map was constructed by inserting the M*T product to the PVC voxels and −1 to the remaining voxels. Thus, PVC-predicted BP maps represent weighted mean BP values of the three segments at the voxel level assuming a homogenous BP value for each segment throughout the brain. Comparison of observed BP maps to PVC-predicted BP maps, using SPM2, allows for exploring regions that show significant deviation from the homogeneity assumption.
Results: PV-corrected BP values were 0.14 ± 0.15 (unit-less; mean ± SD) for gray matter, 0.54 ± 0.10 for white matter, and −0.73 ± 0.12 for cerebro-spinal space. Table lists four clusters (observed BP values > PVC-predicted BP values) identified with SPM (p<0.05, corrected; volume > 1 ml) across the 28 subjects.
Conclusions: The approach successfully identified four regions of relative increases in PIB binding in nondemented older adults with stringent statistical criteria. The results suggest the PVC-based approach can detect subtle increases in PIB binding in nondemented subjects with a low cortical BP value despite a several fold higher white matter BP value. Further studies on demented populations will be required to understand the clinical significance of PIB accumulation at this early stage of amyloid deposition.
Grant Support: Intramural Research Program, NIA, NIH; N01-AG-3-2124; K24 DA00412 (DFW)
CEREBRAL ACTIVATION AND DEACTIVATION DURING NAVIGATION IN A VIRTUAL REALITY ENVIRONMENT IN PATIENTS WITH MCI. AN O15-WATER PET ACTIVATION STUDY
Marc Daniel Wermke1, Timo Grimmer2, Isabell Stangier1, Stephan Nekolla1, Alexander Kurz2, Markus Schwaiger1, Alexander Drzezga1
1Nuclear Medicine Department, Technische Universitat, Munich, Germany, 2Psychiatry Department, Technische Universitat, Munich, Germany
Background and aims: In patients with Alzheimer's disease (AD), characteristic abnormalities of cerebral activation during cognitive performance have been previously observed. The aim of the current study was to assess the cerebral activation and deactivation patterns during navigation in a virtual reality (VR) environment in healthy volunteers and patients with mild cognitive impairment (MCI) and to correlate the cerebral activation with navigation speed.
Methods: A healthy control group (n=11, right handed, 3 female, age 67+/−10 years) and a matched group of 11 MCI-patients (4 female, 71+/−9 years) underwent a navigation task in a computer-based 3D VR-labyrinth, based solely on visual clues. During active navigation 8 O15-water PET-scans were performed (370 MBq i.v.-bolus). In each subject navigation speed (time from start to destination) was measured. In addition, an FDG-PET-scan at rest condition was performed in all subjects. Statistical analysis (correlation analysis of O15 water activation with navigation speed and group comparison of O15-water and F-18 FDG PET scans) was carried out, using SPM02 software (Wellcome Inst., London, UK), p<0.05, FDR-corrected.
Results: Analysis of FDG PET-data revealed a significantly lower metabolism exclusively in the posterior cingulate cortex in the MCI-group compared to controls. Controls showed a significantly better navigation performance (required time: 67±46 sec, MCI-group 139±53 sec). Analysis of O15 water PET showed a significantly different pattern of activation in MCI and controls. Stronger activations in controls were found in the left cerebellum, the occipital lobe (Brodman area [BA] 18,19) and the sensorimotor cortex (BA 3,4) and in MCI in the prefrontal cortex (BA 9), the SMA (BA 6) and the anterior cingulate cortex (BA 32). Relevant deactivation of auditory cortical regions (BA 41,42,22) ?representing cross-modal inhibition- was detected exclusively in controls. In addition different patterns of correlation between navigation speed and regional brain activation were found in the two groups. Navigation speed correlated with activation in left cerebellum, left premotor cortex (BA 6) and right thalamus in controls and in MCI with activation in left temporal cortex (BA 20), left posterior cingulate cortex (BA 31) and right cerebellum.
Conclusions: Several cortical key regions have already been associated with human navigation in healthy subjects. In our study MCI showed activations differing from the activations found in controls during navigation. Also the deactivation of task-irrelevant cerebral regions observed in controls, was absent in MCI. This suggests that MCI is characterized by a progressive impairment of cross-modal cerebral deactivation-functions, resulting in a decreased ability to direct attention primarily to the relevant cognitive modality. In addition, a correlation analysis revealed a significantly different network associated with successful navigation-performance in MCI compared to controls. These results might indicate compensatory changes or functional deficits in MCI due to ongoing neurodegeneration.
FAMILIAL ALS WITH A NOVEL L126S MUTATION IN THE SOD1 GENE SHOWING MILD CLINICAL FEATURES AND LOW PENETRANCE IN FEMALES
Yasushi Takehisa, Tetsuro Murakami, Kentaro Ohta, Tomoko Kurata, Nobutoshi Morimoto, Makiko Nagai, Tatsushi Kamiya, Koji Abe
Department of Neurology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Okayama, Japan
Back ground: Mutations in the SOD1 gene are responsible for approximately 25% of familial amyotrophic lateral sclerosis (ALS) cases. However, the correlation between the clinical and pathological features and the various SOD1 gene mutations has not been well characterized.
Objective: To screen the SOD1 gene in search of potential mutations and to obtain clinical and pathological datas for three Japanese families with ALS.
Design: Clinical histories and neurological findings, gross and microscopic pathological features, and DNA analysis of the SOD1 gene.
Results
The two families with ALS showed a novel missense mutation in the SOD1 gene, which was heterozygous for a point mutation TTG to TCG causing substitution of leucine for serine at codon 126 (Leu126Ser) in exon 5.
Clinically the patients showed slower disease progression and lack of upper motor neuron (UMN) signs.
Neuropathologically, the autopsied patient showed the form of familial ALS with posterior column involvement, and pontocerebellar tract and the dentate nuclei of the cerebellum are also involved. Furthermore abundant Lewy body-like hyaline inclusions (LBHIs) were observed in the affected motor and non-motor neurons.
Conclusion: Familial ALS with a novel mutation Leu126Ser in the SOD1 gene showed mild clinical features and lack of UMN signs. We assume that Leu126Ser may be associated with the clinical features, and that both the mutation site in the SOD1 gene and disease duration may be associated with the formation of LBHIs. The mean age of onset was 50.3 (28-74), and the mean duration was 7.7 (2-14) years. The patients showed similar mild progression of illness compared with the duration in familial ALS (FALS) patients with the SOD1 mutation (mean duration, 3.9 years), and a relatively low penetrance in females.
CEREBRAL GLUCOSE METABOLISM BY PET IN MILD COGNITIVE IMPAIRMENT
Jyrki Kuikka1,4, Anne Hamalainen2, Tuija Kangasmaa1, Minna Rusanen2, Eini Niskanen2, Ritva Vanninen3, Hilkka Soininen2,4
1Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, Kuopio, Finland, 2Department of Neurology, Kuopio University Hospital, Kuopio, Finland, 3Department of Clinical Radiology, Kuopio University Hospital, Kuopio, Finland, 4Kuopio University Neuroscience Centre, Kuopio, Finland
Background and aims
Mild cognitive impairment (MCI) is considered as a transitional state between healthy aging and dementia. However, not all patients develop Alzheimer's disease (AD) but remain stable over time, even revert to a normal state, or progress to another dementia. Clinical diagnosis of early AD is often difficult and positron emission tomography (PET) evaluations are used to support the clinical diagnosis at the early stage. The aim of our study was to investigate the medial temporal lobe (MTL) FDG uptake in the classification of normal and MCI patients.
Methods
Fourteen patients with MCI (7 females and 7 males with mean age of 76.5 y; range: 67-84 y; education 7.8 y; MMSE 25.6) and 13 controls (11 females, 2 males; mean age 74.4 y; range 68-81; education 8.8 y; MMSE 27.9) were studied with a mobile PET/CT scanner (GE Discovery: www.GEhealthcare.com). A dose of 250 MBq of 18F-FDG (supplied by MAP Medical Technologies Oy, Helsinki, Finland) was intravenously injected, and 30 minutes later PET imaging (3-D mode) was acquired. The images were reconstructed and were further analyzed with the Statistical parametric mapping method (SPM2: www.fil.ion.ucl.ac.uk/spm). No partial volume correction was performed. Both voxel-based and regions of interest (ROI) analyses were computed with SPM2. The ROI analyses were normalized to the cerebellar FDG count density. Whole-brain structural analysis was performed with voxel-based morphometry (VBM).
Results
The MMSE scores, but not age or education, revealed significant differences between the controls and MCI subjects. Highly significant FDG hypometabolism was found bilaterally in the MTL regions in MCI patients (Fig. 1). No significant metabolic reductions were seen in the other cortical regions. However, the left and right inferior, middle and superior frontal regions revealed a trend towards FDG reductions in MCI patients when compared to control subjects. No significant clusters of atrophy were detected between the controls and MCI subjects.
Conclusions
The results support the previous findings revealing that reduced FDG levels are seen in MCI with early stage and as previously shown (Mosconi L et al. Early detection of Alzheimer's disease using neuroimaging. Exp Gerontol 2006; Jul 11: Epub ahead of print), especially entorhinal cortex as a part of the MTL predicts decline from normal aging to MCI.
STATINS PROTECT NEURONS AND ASTROCYTES AGAINST ISCHEMIA-LIKE INSULTS, BUT NOT MICROGLIA-INDUCED TOXICITY
Hualong Ma1,2, Zhen Zheng1,2, Maya Koike1,2, Scott Zamvil1, Midori Yenari1,2
1Department of Neurology, University of California San Francisco, San Francisco, CA, USA, 2Department of Neurology, Veteran Affairs Medical Center, San Francisco, CA, USA
It has been reported by several groups that statins, a class of cholesterol lowering drugs, are neuroprotective. The underlying mechanism for such protection is thought to be due, in part, to its ability to enhance cerebral blood flow by upregulating endothelial nitric oxide synthase (eNOS). However, recent work has also suggested an anti-inflammatory effect. We evaluated the effects of atorvastatin on neuronal and astrocyte cell death as well as on microglial activation in an in vitro ischemia model. Primary neuronal cultures were prepared from cortices of E16 Swiss Webster mice and used for experiments at 12 DIV. Primary microglial and astrocyte cultures were obtained from 1 day old mice. Co-cultures of astrocytes with neurons (AN) with or without microglia (NAM) were prepared by seeding 2×104 cells/ml (neuron), and 2×105 cells/ml (microglia) on top of astrocytes. Cocultures were used for experiments 48 hours (microglia) and 12 days (neuron) later. Cultures were subjected to oxygen and glucose deprivation (OGD) for 90 minutes (neuron containing cultures) and 6 hours (astrocytes alone) followed by reperfusion for 24 hours. Overall cell death was evaluated by using LDH assay and trypan blue staining. Neuronal death was estimated using a MAP-2 ELISA based assay. Following OGD, treatment of primary astrocytes (A) with 1µM atorvastatin led to less cell death by 90% (P=0.001) compared to control. Coculture with microglia (AM) showed increased cell death by 2 fold (P<0.01). However, atorvastatin failed to affect the extent of microglia-exacerbated cell death (NS). Atorvastatin treatment of primary neuron cultures (N) also showed reduced cell death by 23% (P<0.001). Interestingly, the addition of microglia to neuron cultures (NM) did not increase cell death, as was seen with astrocytes. Atorvastatin treatment of NM cultures failed to affect cell death compared to untreated NM and N cultures. In cocultures of neuron and astrocytes (AN), statin treatment decreased cell death by 38% (p < 0.01). Although addition of microglia to AN coculture (ANM) increased neuron cell death by 2 fold (P <0.005), statin treatment again showed no effect on microglia-potentiated neuronal cell death. In another experiment, we measured the expression of MHC-class II molecules on cultured BV2 cells (murine microglia) using FACS analysis. After stimulation with LPS (10 µg/ml) for 24 h, microglia showed increased activation by FACS analysis of MHC-class II antigen expression. Treatment of these cells with atorvastatin failed to inhibit MHC-II expression. In conclusion, our data suggest a mechanism of direct protection of neuron and astrocytes against ischemia-like insults in culture by atorvastatin. This protection appears to be independent of vascular changes/cerebral blood flow or microglial activation/toxicity. We further show that microglia alone are not toxic to neurons under conditions of OGD, but appear to require the presence of astrocytes to increase neuronal death.
TISSUE SODIUM CONTENT AND IRREVERSIBLE ISCHEMIC TISSUE DAMAGE DURING FOCAL BRAIN ISCHEMIA
George LaVerde1, Edwin Nemoto1, Charles Jungreis3, Alexander Kharlamov2, Steven Jones2, Costin Tanase1, Fernando Boada1
1Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA, 2Department of Anesthesiology, Allegheny General Hospital, Pittsburgh, PA, USA, 3Department of Radiology, Temple University, Philadelphia, PA, USA
BACKGROUND
In vivo Sodium MRI has been proposed as a means for assessing tissue viability during acute brain ischemia. Ischemic brain tissue sodium concentration (TSC) increases linear leading to the hypothesis that a threshold in TSC could identify irreversible tissue damage. In this study, we demonstrate, using an non-human primate model, that TSC accumulation rates, and therefore time to irreversible tissue damage, can vary from subject to subject, and within the ischemic tissue of a single subject.
METHODS
Focal brain ischemia was induced in pig-tail monkeys (n=4) through embolization of the right posterior cerebral artery and balloon catheter occlusion of the right middle cerebral artery (MCA) (1). Animals were kept under anesthesia (fentanyl infusion), temperature maintained at 37oC and blood pressure and exhaled gases monitored continuously. Post mortem immunohistochemical staining of microtubule associated protein 2 (MAP2) was performed to identify ischemic damage (2).
Sodium/proton images were acquired on a 3 Tesla scanner, using custom-built 23Na and 1H quadrature RF coils. Proton 3D spoiled gradient recalled (pSPGR) and MR angiogram data were acquired. Serial sodium single quantum (SQNa) 3D sodium images were acquired throughout the duration of the experiment using a twisted projection imaging sequence (3) (5min each, TE/TR=0.5/100ms) and image intensities converted to TSC using sodium calibration standards. Pixel by pixel linear regression analysis of the images was performed to generate TSC slope's spatial maps.
RESULTS
Figure 1 shows (a) pSPGR, (b) SQNa, (c) TSC slope map (from 25 SQNa images, hours 1-4), (d) MAP2 histological section showing infarcted region (decreased MAP2 staining), (e) overlay ((d) and (a)), and (f) overlay of (a), (d) and (c). Linear regression of the ischemic brain TSC time course demonstrated that the TSC slope during MCA occlusion ranged from 5.44%/hr to 7.15%/hr across the four animals. This result was lower than the 16–35%/hour reported for rodent models of focal brain ischemia (5), presumably due their different cerebral metabolic rate.
CONCLUSIONS
Our results demonstrated that TSC increases linearly during evolving brain ischemia and that the TSC accumulation rate varies across individual subjects and within the ischemic defect of individual subjects. These results support the study by Jones et al. (4), which suggests that sodium MRI could predict stroke onset time for patients unsure of their symptom's duration. Our findings suggest sodium MRI could facilitate thrombolytic therapy clinical decision making based on the TSC content of the ischemic tissue.
Support: NIH R01NS044818.
HMG COA REDUCTASES REDUCES ISCHEMIC BRAIN INJURY OF WISTAR RATS THROUGH DECREASING OXIDATIVE STRESS ON NEURONS
Shoko Nagotani, Takeshi Hayashi, Kentaro Deguchi, Hanzhe Zhang, Yoshihide Sehara, Atsushi Tsuchiya, Toru Yamashita, Violeta Lukic, Tatsushi Kamiya, Koji Abe
Department of Neurology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
Background: Statins possess many effects besides cholesterol lowering effect, which is called pleiotrophic effects. They show neuroprotective effect against ischemic damage, but how they protect neurons is not exactly made clear. As they have anti-oxidative property and oxidative stress is implicated in ischemic brain damage, we speculated that anti-oxidative property of statins plays am important role. We therefore investigated statins' influences on the oxidative neuronal damage in the brain after ischemia.
Methods: We used male Wistar rats (10 weeks old, 230 g). Atorvastatin (20 mg/kg), pitavastatin (10 mg/kg), simvastatin (20 mg/kg), or vehicle (methyl cellulose) was per-orally administered for 14 days, and then 90 min of middle cerebral artery occlusion was imposed on the rats. At 24 hr after CBF restoration, the animals were anesthetized and blood samples were collected. Triglyceride (TG), total-cholesterol (T-cho), high-density lipoprotein-cholesterol (HDL-cho), and low-density lipoprotein-cholesterol (LDL-cho) were measured using these samples. Just after blood collection, the animals were decapitated and the brains were removed. In order to evaluate oxidative stress, production of 4-hydroxynonenal (HNE) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were investigated by immunohistochemistry. Infarction volume was investigated using same sections by cresyl violet staining.
Results: In the vehicle group, massive infarction was confirmed and HNE and 8-OHdG are robustly produced. In the statins-treated group, the infarction was smaller and the HNE and 8-OHdG production was less prominent than the vehicle group (Figure). Among the statins investigated, simvastatin was most effective for reducing oxidative stress and infarction volume. On the other hand, statin-treatment did not decrease the cholesterol levels in the rats.
Conclusion: Statins possess anti-oxidative proterty, which is implicated in neuroprotection against ischemic injury. Drug's lipophilic property correlated with their neuroprotective property, suggesting that entering into brain is critical for exerting their effects.
PGE2-EP3 RECEPTOR STIMULATION LEADS TO EXACERBATION OF STROKE AND EXCITOTOXIC INJURY
Muzamil Ahmad1, Abdullah Ahmad1, Sylvain Dore1,2
1Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, SOM, Baltimore, MD, USA, 2Department of Neuroscience, Johns Hopkins University, SOM, Baltimore, MD, USA
Background: Inflammation has been shown to play a major role in the pathological response and outcome of stroke and other central nervous system disorders. Inflammation is mediated at least in part by prostaglandins (PGs), which are produced through the cyclooxygenase (COX) pathway. PGE2 mainly binds to a family of G-protein-coupled receptors known as EP receptors. The EP3 receptor mediates the activation of several signaling pathways, leading to changes in cAMP levels, calcium mobilization, and activation of phospholipase C. Our goal in this study was to determine the role of EP3 in brain injury induced by ischemia-reperfusion injury.
Method: Using 25-30g male C57BL/6 mice, we investigated the effects of a selective EP3 agonist on 90-min middle cerebral artery occlusion (MCAO)-induced infarct volume, relative cerebral blood flow (CBF), mean arterial blood pressure (MABP), and other physiological parameters. Furthermore, since excitotoxicity is involved in the resulting injury caused by ischemia/reperfusion, we went on to determine the effect of EP3 agonist on lesion size after NMDA-induced toxicity. All animal protocols were in accordance to NIH guidelines and approved by the JHU Animal Care and Use Committee.
Results: Intracerebroventricular injection of the EP3 agonist before MCAO significantly enhanced hemispheric infarct volumes. Infarct volume increased in a dose-dependent manner as compared with the vehicle-treated group (n = 9/dose). As estimated by laser-Doppler flowmetry, relative CBF decreased significantly in each group of mice after insertion of the nylon monofilament but the differences in CBF reduction among the treated groups were not significantly different. No statistically significant differences in pH, PaCO2, PaO2, or MABP were detected before ischemia, during ischemia, or during reperfusion among the groups of mice. On the other hand, mouse pretreatment with the EP3 agonist dose dependently aggravated the brain injury caused by NMDA injection.
Conclusion: Some conflicting findings have been reported in the literature in regard to the role of EP3 receptors in health and disease; some investigators have reported that EP3 affords protection, whereas others state that it aggravates toxicity. Considering that PGE2 is often called the “pro-inflammatory” prostaglandin, we have used the mouse models of cerebral ischemia and induced excitotoxicity to elucidate and better understand the EP3 function. We found that stimulation of the EP3 receptor with a selective agonist significantly exacerbated the lesion volume following NMDA injection and the infarct volume after transient ischemia, while none of the physiological parameters studied were significantly affected. To our knowledge, this is the first report to show that stimulation of the EP3 receptors exacerbates brain damage; thus deletion or pharmacologic blockade of this receptor might limit brain damage induced by pro-inflammatory COX metabolites, and these receptors could be used as therapeutic targets for the prevention or reduction of ischemia-reperfusion-associated brain injury. (This study was supported by an AHA postdoctoral fellowship and by NIH grants.)
BENEFITS OF PRE-TREATMENT WITH EDARAVONE IN TPA INTRAVENOUS THERAPY FOR ACUTE CEREBRAL INFARCTION
Yoshifumi Teramoto1, Yoshikazu Nakajima2, Mamoru Ito2, Makoto Dehara2, Takao Soda3, Tomonari Yabuuchi2, Koji Tokiyoshi2, Toshiki Yoshimine4, Mamoru Taneda1
1Department of Neurosurgery, Kinki Universiy School of Medicine, Osaka-Sayama, Japan, 2Department of Neurosurgery, Rinku General Medical Center, Izumisano, Japan, 3Department of Neurology, Rinku General Medical Center, Izumisano, Japan, 4Department of Neurosurgery, Osaka Universiy School of Medicine, Suita, Osaka, Japan
[Objective] In thrombolytic therapy, the administration of edaravone, a free radical scavenger, before thrombolysis can potentially remove the free radicals that are generated upon effective recanalization, inhibit hemorrhagic infarct and severe cerebral edema, and improve vital and functional prognosis.
[Cases and Methods] Of the 60 patients who have been treated for acute cerebral infarction at our clinic since November 2005, when tPA intravenous therapy first became available for this condition in Japan, and who ranged between 5 and 22 on the National Institute of Health Stroke Scale (NIHSS) at the time of first visit, 13 patients (seven men and six women aged between 56 and 79 years; mean 71.5 years) who underwent tPA therapy were examined in this study. Of these 13 patients, 54% had cardiogenic cerebral infarction, 38% had cerebral atherothrombosis, and 8% had lacunar infarction (according to TOAST classification). Towards evaluating the benefits of pre-treatment with edaravone, we used MRI and CT to assess the change in cerebral edema over time in patients who underwent tissue-type plasminogen activator (tPA) intravenous therapy with antecedent intravenous administration of edaravone.
[Results] The tPA intravenous therapy was started 48 to 147 (mean: 108) minutes after the onset of stroke symptoms. Recanalization was achieved in 38% of the patients, was not achieved in 31%, and the status of recanalization remained unknown in 31%. Symptoms improved in five patients, did not change in four patients and were aggravated in four patients (including one patient with the complication of hemorrhage). In all 11 patients who received edaravone (30 mg) intravenously before (5 to 45 minutes before; mean 20.7 minutes) tPA, cerebral edema was limited to the area surrounding the infarction lesion and there was no rapid exacerbation of consciousness or neurologic symptoms secondary to extensive brain swelling with midline-shift or severe cerebral edema secondary to hemorrhagic infarct. Cerebral hemorrhage at a site distinct from the infarction lesion was observed in one patient with midline-shift developed in one of the two patients who received edaravone after (5 and 25 minutes after) intravenous injection of tPA.
[Discussion] Evaluation of the clinical cases of acute cerebral infarction treated with tPA intravenous therapy suggests that pre-treatment with edaravone can inhibit white matter injury or cerebral edema due to recanalization. Future studies including randomized controlled trials on a large number of cases are desired.
CEREBROVASCULAR CO2 REACTIVITY EXAMINED AFTER CHRONIC PRETREATEMENT OF AN ANGIOTENSIN II TYPE 1 ANTAGONIST IN RATS WITH ACUTE ISCHEMIC STROKE
Shigenori Mizusawa1, Yasushi Kondoh1, Kazuhiro Nakamura1, David Wright1, Svend Strandgaard2, Iwao Kanno3,1
1Akita Research Institute for Brain and Blood Vessels, Akita, Japan, 2Department of Nephrology, Herlev University Hospital, Copenhagen, Denmark, 3Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan
INTRODUCTION: Pretreatment of Angiotensin II type 1 (AT1) antagonist, candesartan, reduces the infarct size in middle cerebral artery occlusion (MCAO) model rats [1]. Candesartan is thought to affect larger cerebral resistance vessels and lower the limit of cerebral blood flow (CBF) autoregulation. The reactive site of CO2 is smaller resistance vessels, therefore treatment with candesartan should not affect cerebrovascular CO2 reactivity. We examined CO2 reactivity under chronic pretreatment of candesartan to test this relationship.
METHOD: Twelve Sprague-Dawley rats were administrated with candesartan (0.3mg/kg/day;n=6) or vehicle (0.1N Na2CO3;n=6) for 14 days prior to experimentation by using subcutaneously implanted osmotic minipumps. Ischemic regions were induced by occluding the left middle cerebral artery with embolic thread and an endotracheal tube was inserted for artificial ventilation and CO2 inhalation. After preparation, the animals were set in the bore of a 4.7T MR spectrometer and a CBF image was acquired using a continuous arterial spin labeling (CASL) method 3 hours after the MCAO [2]. Inspired CO2 was then adjusted to 0.8% and 1.6% of ventilated volume and CASL scans were acquired after CBF stabilized. The CO2 reactivity was calculated by exponential curve fitting using the least squares method and the ischemic area was defined using the apparent diffusion coefficient (ADC).
RESULTS: The figure shows mean CBF values measured at various PaCO2 levels. In the healthy hemisphere, the CO2 reactivity in animals treated with candesartan (2.1%/mmHg) was significantly lower than vehicle (3.3%/mmHg). CBF response to PaCO2 was well conserved in the ischemic penumbra (70 % < ADC < 90% of mean value in control hemisphere) especially in the candesartan group.
DISCUSSION: Vasodilatation limits further vessel extension resulting in decreased CO2 reactivity. The results suggest that candesartan induced persistent vasodilation of smaller resistance vessels hence decreased response to CO2. A smaller infarction observed in MCAO animals treated with candesartan may be due to persistent vasodilation of smaller resistance vessels in the ischemic penumbra.
MECHANISMS OF ANGIOTENSIN II-INDUCED ENDOTHELIAL DYSFUNCTION: ROLE OF INFLAMMATION, INTERLEUKIN-6 AND INTERLEUKIN-10
Sean P. Didion1, Dale A. Kinzenbaw1, Laura I. Schrader1, Frank M. Faraci1,2
1Department of Internal Medicine, University of Iowa, Iowa City, IA, USA, 2Department of Pharmacology, University of Iowa, Iowa City, IA, USA
Background and Aims: Hypertension has an enormous negative impact on the carotid artery and cerebral circulation. Angiotensin II (Ang II) plays a major role in hypertension, in part by promoting vascular inflammation. The goal of this study was to examine the role of interleukin 6 (IL-6, a pro-inflammatory cytokine) and IL-10 (an anti-inflammatory cytokine) in vascular dysfunction produced by Ang II.
Methods: Responses of carotid arteries from wild-type, IL-6 and IL-10 deficient mice (IL-6 -/and IL-10 -/−, respectively) were examined following either overnight incubation with Ang II (1 or 10 nmol/L) or chronic infusion of Ang II in vivo (1.4 mg/kg per day for 10-14 days).
Results: Baseline blood pressure and Ang II-induced hypertension were similar in control and IL-10 -/− mice. Ang II-infusion for 10 days produced modest impairment of endothelial function in control mice but marked impairment in IL-10 -/− mice [e.g., 100 µmol/L acetylcholine (ACh), an endothelium-dependent agonist, produced 102±3 and 55±1% relaxation in vessels from vehicle-and Ang II-infused IL-10 -/− mice, respectively]. Overnight treatment with 1 nmol Ang II had no effect on responses to ACh in vessels from control mice but markedly reduced responses to ACh in IL-10 -/− (e.g., 100 µmol/L ACh produced 82±8 and 37±5% relaxation in vehicle- and Ang II-treated arteries, respectively). This effect of Ang II in IL-10 -/− mice was reversed by PEG-SOD, a scavenger of superoxide. Vascular superoxide levels increased following local treatment with Ang II in IL-10 -/− but not control mice. In studies of IL-6, baseline blood pressure and Ang II-induced hypertension were similar in control and IL-6 -/− mice. Ang II-infusion (14 days) produced impairment of responses to ACh in arteries from control, but not IL-6 -/− mice (e.g., 100 µmol/L ACh produced 51±7 and 80±5% relaxation in vessels from control and IL-6 -/− mice, respectively). IL-6 deficiency also protected against endothelial dysfunction produced by acute treatment with 10 nmol/L Ang II, a concentration that produced endothelial dysfunction in arteries from control mice. Endothelial dysfunction could be reproduced in vessels from IL-6 -/mice following overnight incubation with Ang II plus recombinant IL-6 (0.1 nmol/L). Responses to nitroprusside, an endothelium-independent agonist, were not affect by either genotype or treatment, suggesting that the effect of Ang II for endothelium was selective. Consistent with this functional data, Ang II-induced increases in vascular superoxide levels were attenuated in IL-6 -/mice.
Conclusions: Taken together, our findings provide the first evidence that IL-6 promotes and IL-10 limits Ang II-mediated oxidative stress and endothelial dysfunction. These effects were found to occur both in vivo and in vitro suggesting effects of both cytokines occur within the vessel wall independent of changes in arterial pressure. These findings have implications for mechanisms of hypertension-induced vascular dysfunction and carotid artery disease, a major risk factor for ischemic stroke.
OVEREXPRESSION OF COPPER-ZINC SUPEROXIDE DISMUTASE ATTENUATES CYTOSOLIC PHOSPHOLIPASE A2 ACTIVATION BY BLOCKING THE P38 MAPK PATHWAY AFTER FOCAL CEREBRAL ISCHEMIA
Chikako Nito, Deborah J. Myer, Kuniyasu Niizuma, Pak H. Chan
Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA
Background and purpose: Cytosolic phospholipase A2 (cPLA2) is a key enzyme that mediates arachidonic acid (AA) metabolism and thus causes cerebral ischemia-induced oxidative injury, blood-brain barrier (BBB) dysfunction and edema. Recent reports have shown that p38 mitogen-activated protein kinase (MAPK) is related to phosphorylation and activation of cPLA2 and AA release in a Ca2+/calmodulin-dependent kinase II. However, the involvement of the p38 MAPK pathway in cPLA2 activation, and the role of reactive oxygen species (ROS) in the expression of p38 MAPK/cPLA2 after ischemia-reperfusion (IR) injury in the brain remain unclear.
Methods: To investigate these issues, we used a transient middle cerebral artery occlusion (MCAO) model with rats that overexpress copper-zinc superoxide dismutase (SOD1) and with wild-type rats. After 90 min of MCAO, the cortex of the affected side was removed at 6 and 24 h and 3 and 7 days. We also administered SB203580, a potent p38 MAPK inhibitor, into the rat brains and examined phospho-p38 MAPK expression and cPLA2 levels measured by Western blot analysis and activity assays. Infarct or edema volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining.
Results: Western blot analysis and activity assays demonstrated that expression of phospho-p38 MAPK was increased after reperfusion, maximally on day 1, and that phospho-cPLA2 significantly increased at 1 day (Figure 1; *p< 0.05,). The intraventricular administration of SB203580 provided a significant suppression of phosphorylation and activation of cPLA2 (p< 0.05). Moreover, SOD1 overexpression significantly diminished phosphorylation and activation of both p38 MAPK and cPLA2 (Figure 2; *p<0.05, ##p<0.05, respectively) and reduced cortical infarct or edema volume at 3 days (p < 0.05 and p< 0.01, respectively) after reperfusion.
Conclusions: These results suggest that ROS may induce cPLA2 activity through the p38 MAPK pathway and promote BBB disruption in the brain cortex after IR injury.
APPEARANCE OF INTRA-NUCLEAR MATRIX METALLOPROTEINASES IS INVOLVED IN THE EARLY STAGES OF NEURONAL DEATH FOLLOWING BRAIN ISCHEMIA
Anna Rosell, Eloy Cuadrado, Maria Borrell-Pages, Miriam Navarro, Mar Hernandez-Guillamon, Anna Penalba, Laura Ortega, Joan Montaner
Neurovascular Research Laboratory, Institut De Recerca, Hospital Vall D'Hebron, Barcelona, Spain
Background and aims: Matrix Metalloproteinases (MMPs) are known to play a critical role increasing brain damage after brain ischemia both in humans and in animal models. Although MMPs are best known as being either secreted or transmembrane proteins, recent publications have identified unexpected locations for different MMPs. A variant of MMP-11 has been described as an intracellular active protease and MMP-14/MT1-MMP has been identified in the centrosome. Moreover, nuclear MMP-2 has been identified in cardiac myocytes and nuclear location of MMP-3, and the fact that can induce apoptosis via its catalytic activity, has been recently described in human liver cells. In the present study we aimed to search for MMP expression and cellular location following brain ischemia.
Methods: Several samples from patients who had an ischemic stroke within the previous 5 days were included in the study. On autopsy, by an experienced neuropathologist, morphological features and last available neuroimages were used to guide brain tissue sampling from ischemic ipsilateral hemisphere or from contralateral hemisphere. Finally, 19 samples from 7 ischemic stroke patients (12 infarct samples and 7 contralaterals) were studied. Brain tissue samples from 2 patients that died due to a non-inflammatory pathology were used as controls.
To detect MMPs in human brain homogenates we used a protein array technology (Searchlight), which allowed us to measure MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10 and MMP-13 concentrations.
To study the temporal profile of MMP activation in cells, in vivo experiments were conducted in a rat permanent focal MCAO model, occlusion times: 30, 60, 90, 120 minutes and 48 hours (n=3 per time-point).
In situ zymography that detects gelatinase/collagenase activity was carried out on human and rat brain sections, including some controls using a metalloproteinase inhibitor (phenanthroline). Reaction products were examined with fluorescence microscope and using confocal microscopy.
Results: Brain tissue from ischemic patients showed higher levels of MMP-2, MMP-3, MMP-8, MMP9, MMP-10 and MMP-13 in the infarct area as compared with contralateral area (p=0.005, p=0.049, p=0.041, p=0.031, p=0.021 and p=0.001, respectively) while in situ zymography showed a clear collagenase/gelatinase activity at the nucleus of neuronal cells.
Regarding the temporal profile of MMP activation we observed an early nuclear gelatinase/collagense activity (at 30 minutes) that increased together with the occlusion time. This time-dependent response was perceived on the number of cells with nuclear activity and on the intensity. After 60 minutes of occlusion the gelatinase/collagenase activity also appeared in the cytoplasm of the cells following again a time-dependent response.
Finally our controls confirmed our findings since the sham rats did not show nuclear activity and gelatinase/collagenase activity was blocked after treatments with phenanthroline both in human and rat sections.
Conclusions: Several MMPs are up-regulated after stroke as well as a new location of gelatinase/collagenase activity is identified into the nucleus of brain cells at the early stages of brain hypoxia/ischemia. The unexpected location of this activity merits further research in order to identify the responsible matrix metalloproteinases, to understand their contribution to neuronal death and to explore new treatments for stroke.
ISCHEMIC PRECONDITIONING VIA EPSILON PKC INDUCES ERK1/2 AND STAT3 PATHWAYS IN MIXED CORTICAL NEURONS/ASTROCYTES
Eun Joo Kim1, Jesus Purroy1,2, Anna M. Planas2, Miguel A. Perez-Pinzon1
1Department of Neurology and Neuroscience, Cerebral Vascular Disease Research Center, University of Miami Miller School of Medicine, Miami, FL, USA, 2Institut D Investigacions Biomediques De Barcelona, Consejo Superior De Investigaciones Cientificas-Institut D Investigacions Biomediques August Pi I Sunyer, Barcelona, Catalonia, Spain
Ischemic preconditioning (IPC) is a protective mechanism against ischemia/reperfusion injury in brain and heart. Our previous studies have shown that IPC via epsilon PKC activation induced the phosphorylation of extracelluar signal regulated kinase1/2 (ERK1/2) and cyclooxygenase-2 (COX-2) leading to the neuroprotection (1). However, transcriptional signal pathways by which IPC regulates COX-2 in brain remain undefined. The signal transducers and activators of transcription (STATs) upregulate COX-2 expression during cardioprotection (2). A previous study with epsilon PKC knock out mouse model suggests that serine-727-STAT3 phosphorylation was mediated by epsilon PKC activation (3). Therefore, in this study, we tested the hypothesis that IPC via epsilon PKC activates STATs transcriptional signaling pathways.
Methods: Mixed cortical neuron/astrocyte cell cultures are prepared from rat embryo/neonatal rat (18-19 days old/1-2 days old) respectively. Cell cultures were exposed to 1 h of IPC and 48 h of reperfusion later. Epsilon PKC specific activating peptide (100 nM) was administrated to cell culture for 1 h. Cell lysates were isolated from different intervals of reperfusion after IPC or epsilon PKC specific activating peptide treatment for immunoblotting with antibody against p-ERK1/2 and P- ser727-STAT3.
Results: Ischemic preconditioning induced the serine-727 phosphorylation of STAT3 from 15 min and was sustained for 2 h of reperfusion. STAT3 phosphorylation was accompanied by the phosphorylation of ERK1/2 (15 min-1 h of reperfusion). Similarly, epsilon PKC specific activating peptide (100 nM) also induced the phosphorylation of STAT3 and ERK1/2. To test whether IPC-induced STAT3 activation is mediated by ERK1/2 activation or by directly PKC activation, PD98059 (MAPK-K inhibitor,0.01 mM) was administrated to cell cultures during and after IPC. PD98059 prevented the IPC-induced phosphorylation of STAT3. Our previous studies showed that Inhibition of ERK1/2 prevented IPC-induced COX-2 expression (1). Based on our findings in future studies, we will further define whether STAT3 activation induces COX-2 expression after IPC.
Conclusion: Our findings suggest that IPC via epsilon PKC activation is mediated by ERK1/2->STAT3 pathway and the signaling pathways of epsilon PKC->ERK1/2->STAT3 might be involved COX-2 expression following IPC.
This study was supported by PHS grants NS34773, NS05820, NS045676 and AHA Florida& Puerto Rico Affiliate grant 0525331B.
PKC MEDIATES THE SERINE-727 PHOSPHORYLATION OF STAT1 AND STAT3 IN ASTROCYTES FOLLOWING OXIDATIVE INJURY
Jesus Purroy1,2, Roser Gorina1, Eun Joo Kim2, Anna M. Planas1, Miguel A. Perez-Pinzon2
1Institut D'Investigacions Biomediques De Barcelona, CSIC-IDIBAPS, Barcelona, Catalonia, Spain, 2Cerebral Vascular Disease Research Center, Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA
Signal transducers and activators of transcription (STATs) are transcription factors which reside in the cytoplasm until they are activated by a tyrosine phosphorylation. A second phosphorylation on Ser-727 is believed to induce maximal transcriptional activation of STAT1. Both STAT1 and STAT3 are quickly phosphorylated in serine-727 in astrocytes following oxidative stress with H202. We set out to test whether PKC was the kinase involved in the serine-727 phosphorylation of either STAT1 or STAT3.
Methods: Astrocytes purified from rat neonates were cultured in 60mm dishes for 12 days prior to treatment. Ten micromolar chelerythrine, 50 µM NSC-87877 or 30 µM PD08059 were added to the plates 30 minutes prior to treatment with 300 µM H202 for times ranging from 5 minutes to 90 minutes. Cell lysates were obtained either whole or fractioned (cytosolic/membrane). PVDF membranes were probed with antibodies against delta-PKC, epsilon-PKC, P-Ser727-STAT1, P-Ser727-STAT3, STAT1 and STAT3.
Results: Inhibition of PKC activity with chelerythrine prevented the serine-727 phosphorylation of both STAT1 and STAT3. Analysis of the translocation of two PKC isozymes showed that epsilon-PKC (ePKC) is deactivated immediately following oxidative stress, whereas delta-PKC is still in the membrane fraction 20 minutes after incubation of cultured astrocytes with 300 µM hydrogen peroxide. We then tested whether PKC is a direct kinase of STATs or whether it mediates this phosphorylation by either activating a downstream kinase or inhibiting a phosphatase. The serine-727 phosphorylation of STAT1 and STAT3 is not prevented by either the inhibition of phosphatase Shp2 with NSC-87877 or the ERK1/2 pathway with PD08059. We are currently testing whether other kinases or phosphatases are part of the phosphorylation pathway between one PKC isozyme and STAT1.
Conclusions: A PKC isozyme mediates the phosphorylation of both STAT1 and STAT3 in serine-727 following oxidative injury in astrocytes. Preliminary data suggest that delta-PKC may be the PKC isozyme involved, either directly or via a signaling cascade.
Grant support: Grant 0525393B from the American Heart Association and CICYT (SAF2005-05793-CO2).
EFFECT OF CILOSTAZOL ON CEREBRAL PRODUCTION OF NITRIC OXIDE DURING CEREBRAL ISCHEMIA AND REPERFUSION IN MICE
Harumitsu Nagoya, Nobuo Araki, Takeshi Ohkubo, Yoshio Asano, Daisuke Furuya, Kimihiko Hattori, Tomokazu Shimazu, Yasuo Ito, Yuji Kato, Kunio Shimazu
Department of Neurology, Saitama Medical University, Iruma-Gun, Saitama, Japan
Background and aims: It is suggested that cilostazol prevents a recurrence of cerebrovascular disorders, especially lacunar stroke. Recently, it is reported that cilostazol reduces cerebral infarction volume by scavenging free radical and inhibiting apoptosis. We investigated the effect of cilostazol on the cerebral nitric oxide production in mice in steady state and during ischemia and reperfusion.
Methods: Thirty-one male C57BL/6 mice were used: control group (n=12), and cilostazol group (n=19). Rectal temperature was kept between 37.0 and 37.5 °C under halothane inhalation anesthesia. A catheter was inserted into a femoral artery for measuring blood pressure (BP). Ringer's solution was perfused at a constant rate 2 µL/min through a microdialysis probe stereotaxically inplanted into the left striatum. We collected perfusate every 10 minutes. A laser Doppler probe was fixed on the surface of contralateral skull and measured cerebral blood flow (CBF) continuously. Forebrain cerebral ishemia was made by clipping both common carotid arteries, using Zen clips for 10 minutes and afterward clips were removed and reperfused. Levels of nitric oxide (NO) metabolites, nitrite and nitrate, in the dialysate were determined using the Griess reaction. Results: [I] Effect of Cilostazol treatment on the baseline of BP, CBF, and NO metabolites; Mean BP decreased from the baseline level (65mmHg) to 62mmHg 20 minutes after the administration of cilostazol (p<0.05). CBF also decreased from the baseline level to 93.5% and 91.0% in 20 minutes and 30 minutes after the administration of cilostazol, respectively (p<0.05). The level of nitrite decreased from 2.1 µmol/L to 1.8 µmol/L in 30 minutes after the administration of cilostazol (p<0.01). The level of nitrate was not changed after the administration of cilostazol. [II] Effect of cilostazol on the change in BP, CBF, and NO metabolites during cerebral ischemia and reperfusion; (1) BP: Mean BP in the cilostazol group after ischemia/reperfusion was significantly lower than that of control group (Fig. 1). (2) CBF: During ischemia, CBF decreased to 7.8%, and 4.8% of the baseline level in the cilostazol group and the control group, respectively. There was no significant difference between the two groups. (3)%change of nitrite : The level of nitrite in the cilostazol group after 50, 60, and 70 minutes from the start of reperfusion were lower than those of the control group (p<0.05) (Fig. 2). (3)%change of nitrate : No significant difference was seen between the two groups. Conclusion: These data suggested that cilostazol reduces the nitric oxide production in the reperfusion phase and may protect ischemic neuronal injury.
NADPH OXIDASE IS ACTIVATED, BUT NOT DIRECTLY INVOLVED IN NEURONAL DEATH INDUCED BY HYPOGLYCEMIA-GLUCOSE REPERFUSION
Kazuki Nagasawa, Taichi Kakuda, Sadaki Fujimoto
Department of Environmental Biochemistry, Kyoto Pharmaceutical University, Kyoto, Japan
[Background and aim] In diabetic patients receiving insulin therapy, hypoglycemia followed by glucose supplementation induces significant neuronal death in the central nervous system, and its consequences are severe clinical problems. Although reactive oxygen species (ROS) was reported to be involved in this type of neuronal death, detail mechanism for the ROS generation has not been understood well so far. The aim of this study was to clarify whether activation of NADPH oxidase, a well known ROS generating enzyme in phagocytic cells, was involved in neuronal death induced by the treatment of glucose deprivation followed by glucose reperfusion (GD/R) using primary cultured rat cortical neurons.
[Methods] Neurons were obtained from E18 rat embryo, cultured in B-27-supplemented EMEM, and used at 8-10DIV. GD/R treatment was performed as follows: ‘glucose deprivation’ was initiated by twice washes of neurons with glucose-free BSS, and they were incubated in glucose-free BSS for the designated times, and then cultured for an appropriate time interval in BSS (for the short time incubation) or B-27 minus-AO-supplemented EMEM (for long time incubation) as ‘glucose reperfusion’. Expression of proteins and mRNAs were determined by western blot and real-time PCR, respectively. Activation of NADPH oxidase was assessed by translocation of p47phox, its cytosolic component under resting conditions, to membrane fraction with an immunocytochemical analysis. ROS generation was monitored by staining with 5-(and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF) using a fluorescence microscopy, and cell viability was determined by an MTT asssay and/or a trypan blue exclusion test.
[Results] The GD/R treatment decreased the intracellular NAD+ concentration, and induced the accumulation of poly(ADP-ribose) (PAR) and nitrotyrosine, and decrease of neuron viability, which was accompanied with translocation of apoptosis inducing factor, but no cleavage of poly(ADP-ribose) polymerase-1, and was recovered by coadministration of N-acethyl-L-cycteine, a nonspecific ROS scavenger, and 3-aminobenzamide, a PARP inhibitor, indicating it was confirmed to be mediated by ROS generation and extensive PARP-1 activation. A nonspecific NMDA receptor antagonist, MK801, had no effect on the GD/R-induced decrease of neuron viability, its contribution being negligible. The neurons expressed functional NADPH oxidase and it was activated by the GD/R treatment, but did not generate detectable ROS with DCF staining, and its pharmacological inhibition by apocynin deteriorated the GD/R-induced decrease of neuron viability. On the other hand, a chelator for extracellular zinc, CaEDTA, almost completely abolished the GD/R-induced ROS generation and PAR accumulation, and improved the neuron viability.
[Conclusion] These findings suggested that NADPH oxidase is not directly involved in the GD/R-induced neuronal death, and its activation of neuronal NADPH oxidase might contribute to maintain the intracellular redox balance in oxidative stress-loaded neurons, and furthermore demonstrated that extracellularly chelatable zinc plays a central role in GD/R-induced neuronal events.
HYPEROXIDIZED PEROXIREDOXINS AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IMMUNOREACTIVITY AND PROTEIN LEVELS ARE CHANGED IN THE GERBIL HIPPOCAMPAL CA1 REGION AFTER ISCHEMIC INSULT
Ki-Yeon Yoo1, In Koo Hwang1, Dae Won Kim2, Jung Hoon Choi3, Seoung Uk Lee1, Ok Kyu Park1, In Se Lee3, Soo Young Choi2, Moo Ho Won1
1Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chuncheon, South Korea, 2Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chuncheon, South Korea, 3Department of Anatomy and Cell Biology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Seoul, South Korea
Oxidative stress is a major pathogenic event occurring in several brain disorders and is a major cause of brain damage due to ischemia/reperfusion. Thiol proteins are easily oxidized in cells exposed to reactive oxygen species (ROS). In the present study, we investigated transient ischemia-induced chronological changes in hyperoxidized peroxiredoxins (Prx-SO3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH-SO3) immunoreactivity and protein levels in the gerbil hippocampus induced by 5 min of transient forebrain ischemia. Weak Prx-SO3 immunoreactivity is detected in the hippocampal CA1 region of the sham-operated group. Prx-SO3 immunoreactivity was significantly increased 12 h and 1 day after ischemia/reperfusion, and the immunoreactivity was decreased to the level of the sham-operated group 2 days after ischemia/reperfusion. Prx-SO3 immunoreactivity in the 4 days post-ischemia group was increased again, and the immunoreactivity was expressed in glial components for 5 days after ischemia/reperfusion. GAPDH-SO3 immunoreactivity was highest in the CA1 region 1 day after ischemia/reperfusion, the immunoreactivity was decreased 2 days after ischemia/reperfusion. Four days after ischemia/reperfusion, GAPDH-SO3 immunoreactivity increased again, and the immunoreactivity began to be expressed in glial components from 5 days after ischemia/reperfusion. The patterns of changes in Prx-SO3 and GAPDH-SO3 protein levels in the ischemic CA1 region were similar to the patterns of the immunohistochemical changes in the ischemic CA1 region. In conclusion, Prx-SO3 and GAPDH-SO3 immunoreactivity and protein levels in the gerbil hippocampal CA1 region are significantly increased 12 h-24 h after ischemia/reperfusion and their immunoreactivity begins to be expressed in glial components from 4 or 5 days after ischemia/reperfusion.
CAVEOLIN EXPRESSION IS ESSENTIAL FOR NMDA-MEDIATED ERK1/2 PHOSPHORYLATION IN CULTURED NEURONS
Brian Head, Hemal Patel, Yasuo Tsutsumi, John Drummond, David Roth, Piyush Patel
Department of Anesthesia, University of California, VA San Diego, San Diego, CA, USA
Ischemic preconditioning (PC) is a phenomenon whereby various stimuli significantly reduce the vulnerability of the brain to subsequent lethal ischemia. Although the endogenous signaling mechanisms remains unknown, previous work suggests that NMDA receptor (NMDAR) mediated signaling plays a critical role in neuronal PC. NMDAR activation leads to downstream activation of pro-survival kinases Src and ERK1/2. Caveolin (Cav), a marker of caveolae, scaffolds signaling complexes in discrete microdomains in most cells, including the Src tyrosine kinase and ERK1/2 and recent work from our laboratory has shown that the NR2B subunit of the NMDAR is scaffolded by caveolin-1. Src has been demonstrated to be activated by NMDA and by sub-lethal ischemia, both of which induce PC. It is therefore conceivable that transient activation of Src and ERK1/2 is necessary to achieve PC and that Cav-1 expression and scaffolding of Src and ERK1/2 is essential for maintaining the fidelity of this signaling cascade during neuronal preconditioning. The present study was conducted to test this hypothesis. Neurons were subjected to sublethal oxygen glucose deprivation (OGDSL; 25 min) or exposed to NMDA (10 µM; 25 min) 24 hr prior to lethal OGD (OGDL; 60 min) and neuronal cell death was assessed after 24 hr with trypan blue. NMDA or OGDSL blocked OGDL-mediated neuronal cell death (one-way ANOVA P<0.0001). The NMDAR antagonist, MK801, and the selective Src kinase inhibitor, PP2, attenuated both NMDA and OGDSL-mediated neuronal preconditioning in vitro. Immunoblot analysis of OGDSL-treated or NMDA-stimulated neurons revealed a significant enhancement in phosphorylated Cav-1 (P-Cav-1), P-Src, P-ERK1/2, and P-p38 (P<0.05). Treatment of neurons with Cav-1-siRNA (72 hr) reduced Cav-1 protein expression; in these neurons, Src and ERK1/2 phosphorylation in response to NMDA exposure was not observed by immunoblot and fluorescence microscopy. In neurons harvested from Cav-1 null mice, NMDA or OGDSL failed to enhance P-Src or P-ERK1/2. Collectively, these data indicate that Src activation plays an important role in PC. The data are consistent with the hypothesis that Cav-1 expression is required for NMDA and OGDSL-mediated activation of Src and ERK1/2, two pro-survival kinases involved in neuronal preconditioning.
MECHANISMS OF OXIDATIVE STRESS-INDUCED CALCIUM SIGNALING IN HUMAN CNS PERICYTES
Masahiro Kamouchi, Takanari Kitazono, Junya Kuroda, Kuniyuki Nakamura, Yuji Shono, Noriko Hagiwara, Hiroaki Ooboshi, Setsuro Ibayashi, Mitsuo Iida
Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Background and aims
Central nervous system (CNS) pericytes encircle abluminal surface of endothelial cells in brain microvasculature, such as capillaries, arterioles and venules where smooth muscle cells are sparse or lacking. The pericytes closely communicate with the endothelial cells and play a pivotal role in angiogenesis, vascular stability, vascular permeability and regulation of blood brain barrier. We have reported previously that oxidative stress may cause dysregulation of intracellular Ca2+ in rat CNS pericytes. The aims of the present study were to elucidate the mechanisms of reactive oxygen species (ROS)-induced Ca2+ dysregulation in the pericytes.
Methods
Using cultured human brain microvascular pericytes, cytosolic Ca2+ concentration was measured by means of fura-2 fluorescence. Production of ROS was detected by fluorescence of dihydroethidium. Reverse transcription and polymerase chain reaction (RT-PCR) was used to examine expression of mRNA. Knockdown of Na+/H+ exchanger1 (NHE1) was done by transfecting the cells with a specific double-strand siRNA for NHE1.
Results
Dihydroethidium fluorescence revealed that human CNS pericytes produced ROS at rest without stimulation. Externally applied hydrogen peroxide dose-dependently (10µM-10mM) increased cytosolic Ca2+ in human CNS pericytes. Cytosolic Ca2+ remained high after wash out of hydrogen peroxide; however, addition of dithiothreitol (5mM) rapidly reversed cytosolic Ca2+ to the resting level, indicating that the Ca2+ increase is related to oxidation of thiol. Hydrogen peroxide-induced Ca2+ increase was not inhibited by nicardipine (1µM), Gd3+ (10µM), La3+ (10µM) or omission of external Ca2+. Neither thapsigargin (1µM), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, nor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (10µM), a mitochondria uncoupler, could attenuate the Ca2+ rise. Amiloride (3–30µM) and its derivatives, benzamil (3–30µM) or hexamethylene amiloride (3–30µM) inhibited hydrogen peroxide-induced Ca2+ increase. Human CNS pericytes expressed Na+/Ca2+ exchanger1 (NCX1), NHE1 and NHE7. However, removal of external Na+ did not affect the Ca2+ increase. Moreover, neither KB-R 7943 (10µM), selective inhibitor of NCX, nor ablation of NHE1 by RNA interference inhibited hydrogen peroxide-induced Ca2+ increase.
Conclusions
Oxidative stress releases Ca2+ from intracellular store sites via oxidation of thiol in human CNS pericytes. Amiloride sensitive protein other than NCX or NHE is involved in the hydrogen peroxide-induced Ca2+ increase. Oxidative stress may exert its actions at least partially via release of intracellular Ca2+ in human CNS pericytes.
TETRAHYDROBIOPTERIN DEFICIENCY RESULTS IN OXIDATIVE STREAA AND ENDOTHELIAL DYSFUNCTION IN CAROTID ARTERIES OF SALT-SENSITIVE HYPERTENSION
Hui-Wen Yu1,2, Alex Chen1
1Departments of Pharmacology and Neurology, Neuroscience Program and Cell and Molecular Biology Program, Michigan State University, East Lansing, MI, USA, 2Department of Medicine, Guangzhou Red Cross Hospital, Guangzhou, China
Introduction: Tetrahydrobiopterin (BH4) is an essential cofactor of endothelial nitric oxide (NO) synthase (eNOS). When BH4 levels are decreased, eNOS becomes uncoupled to produce superoxide anion (O2-) instead of NO, contributing to endothelial dysfunction. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for de novo BH4 synthesis. The hyperphenylalaninemic mouse mutant (hph-1 mice) displays 90% deficiency of the endothelial-specific GTPCH-?. This study tested the hypothesis that BH4 deficiency leads to oxidative stress and endothelial dysfunction in carotid arteries of deoxycorticosterone acetate (DOCA)-salt hypertension, a salt-sensitive model characterized by suppressed plasma renin level due to sodium retention, but manifests in eNOS uncoupling.
Methods: DOCA-salt hypertension was created in 8-10 week-old male hph-1 and wild-type mice (both of C57BL/6 background). Systolic blood pressure was measured by tail-cuff method. Carotid arteries were obtained three weeks after surgery. Endothelium-dependent relaxations in carotid arteries in response to acetylcholine (ACh, 1 nM −10 µM) were assessed by isometric tension recordings. Vascular superoxide levels were assayed by lucigenin-enhanced chemiluminescence.
Results: There was no significantly difference in systolic blood pressure between wild-type and hph-1 mice (104.50±2.58 vs. 104.67±2.62 mmHg, n=6-8, p>0.05). However, systolic blood pressure was significantly increased in wild-type DOCA-salt mice when compared to Sham-operated control mice (136.25±3.71 vs. 104.50±2.58 mmHg, n=8, p<0.001), which was further elevated in DOCA-salt treated hph-1 mice (136.25±3.71 vs. 156.17±3.72 mmHg, n=6-8, p<0.01). Consistently, vascular superoxide levels were significantly increased in DOCA-salt treated hph-1 mice when compared to the Sham controls (1.91±0.25 vs.0.56±0.10 nmol/min/mg tissue, n=6, p<0.01). Endothelium-dependent relaxations in carotid arteries in response to ACh (1 nM-10 µM) were significantly decreased in DOCA-salt treated hph-1 mice as compared to Sham controls in the presence of PEG-catalase (250U/mL, n=4-8, p<0.05), a membrane-permeable scavenger of hydrogen peroxide (n=5-8, p<0.05). The relaxation responses were abolished by NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 0.3 mM, n=4-8, p<0.001).
Conclusions: These findings demonstrate that deficiency of eNOS cofactor BH4 leads to oxidative stress-induced endothelial dysfunction of carotid arteries, parallel with enhanced blood pressure level in DOCA-salt hypertensive mice.
EFFECTS OF FENOFIBRATE, A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA AGONISIT, ON NITRIC OXIDE PRODUCTION DURING CEREBRAL ISCHMIA AND REPERFUSION IN MICE
Takeshi Ohkubo, Nobuo Araki, Yoshio Asano, Daisuke Furuya, Tomokazu Shimazu, Harumitsu Nagoya, Yasuo Ito, Yuji Kato, Mikiko Ninomiya, Kunio Shimazu
Division of Neurology, Department of Internal Medicine, Saitama Medical University, Iruma-Gun, Saitama, Japan
Background and aims: Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. Fenofibrate, used clinically as a lipid-lowering agent, activates peroxisome proliferator-activated receptor α (PPARα). It has been reported that fenofibrate reduces cerebral infarct size, but its direct action for NO production has not been clarified. We investigated the effects of fenofibrate on NO production during cerebral ischemia and reperfusion.
Methods: Twenty one C57BL/6 mice were used in the study. Fenofibrate (50mg/kg/day) was orally administered for 4 days before each brain ischemia in 9 mice (fenofibrate group), and the drug was not administered in the remaining 12 mice (control group). The animals were anesthetized with 2% halothane and maintained with 0.5–1% halothane. NO production was continuously monitored by in vivo microdialysis. A microdialysis probe was inserted into the left striatum and perfused with Ringer's solution at a constant rate of 2µl/min. After 2 hours equilibrium period, fractions were collected every 10 minutes. A laser Doppler probe was placed on the right skull surface. Global ischemia was produced by clipping both common carotid arteries using Zen clips for 10 minutes. The levels of nitrite (NO2-) and nitrate (NO3-) in the dialysate samples were measured by the Griess reaction.
Results: (1) Blood Pressure: No significant differences were observed between fenofibrate group and control group. (2) Cerebral Blood Flow (CBF): CBF decreased to 4.6±0.9% (mean±SE) in fenofibrate group and 4.9±0.7 in control group during ischemia. After reperfusion, CBF transiently returned to baseline values and then gradually decreased significantly in both groups. There were no significant differences between the two groups. (3) NO Metabolites (Fig1, Fig2, Fig3): In fenofibrate group, NO2- levels at baseline (2.12±0.26*µmol/L) and at from 30 (2.23±0.26*) to 60 minutes (2.10±0.27*) after reperfusion were significantly higher than those in control group (1.50±0.16, 1.43±0.16, 1.32±0.14, respectively). The levels of NO3- during ischemia (1.34±0.20*) was significantly higher than that in control group (0.89±0.091), but no significant differences were observed between the two groups after reperfusion. Total NO (NO2-+NO3-) levels at baseline (3.99.0±0.28*) and at 30 (4.48±0.27*) and 40 minutes (4.97±0.24**) in fenofibrate group were significantly higher than those in control group (2.93±0.25, 3.56±0.28, 3.92±0.23, respectively). [*: p<0.05, **: p<0.01]
Conclusions: The above data indicate that fenofibrate affects the whole NO production in baseline and in early phase after reperfusion. It has been reported that PPARα activators upregulates eNOS expression, mainly through mechanisms of stabilization eNOS mRNA. These data suggest fenofibrate may exert the brain protective effect through the influence on NO metabolism.
NEUROPROTECTIVE ROLE OF PRAS IN APOPTOTIC NEURONAL CELL DEATH AFTER STROKE
Atsushi Saito1, Pak H. Chan2
1Department of Neurosurgery, Aomori Prefectural Central Hospital, Aomori, Japan, 2Department of Neurosurgery, Stanford University, Palo Alto, CA, USA
The Akt signaling pathway contributes to regulation of apoptosis after a variety of cell death stimuli. A novel proline-rich Akt substrate (PRAS) was recently detected and found to be involved in apoptosis. However, the role of the PRAS pathway in apoptotic neuronal cell death after ischemia remains unknown. Phosphorylated PRAS (pPRAS) and the binding of pPRAS/phosphorylated Akt (pPRAS/pAkt) to 14-3-3 (pPRAS/14-3-3) were detected, and their expression transiently decreased in mouse brains after transient focal cerebral ischemia (tFCI). Liposome-mediated pPRAS cDNA transfection induced overexpression of pPRAS, promoted pPRAS/14-3-3, and inhibited apoptotic neuronal cell death after tFCI. These results suggest that PRAS phosphorylation and its interaction with pAkt and 14-3-3 might play an important role in neuroprotection mediated by NGF in apoptotic neuronal cell death after tFCI.
CORTICAL COLD LESIONS AND BRAIN PRECONDITIONING. DISTINCT SIZE THRESHOLDS FOR PROTECTION AND EFFECTS ON RESTING CBF
Liang Zhao, Thaddeus S. Nowak Jr.
Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, USA
Background and Aims: Recent studies indicate that preconditioning (PC) effects of brief middle cerebral artery (MCA) occlusion may be mediated by early CBF recovery in penumbral regions during subsequent occlusions (1). CBF improvement was also noted after PC by NaCl-induced cortical lesions (2). Both studies identified variable effects of such procedures on resting CBF in the hemisphere subjected to manipulations, suggesting that injury-associated reduction in metabolism could also contribute to the protected state. This study compared the impact of lesion size on resting CBF and protection after cortical cold injury.
Methods: Spontaneously Hypertensive Rats under halothane anesthesia were subjected to cortical cold lesions by brief application of a liquid nitrogen-chilled metal rod to thinned skull overlying frontal cortex. Rod size and exposure times were varied to produce lesions of different sizes. In one group of rats CBF was evaluated the following day by a quantitative autoradiographic method using [14C]iodoantipyrine as the diffusible tracer. The same series of serial frozen sections was later stained with hematoxylin/eosin to assess cold lesion volume. Another group was subjected to permanent tandem occlusion of the right MCA and common carotid artery, with infarct and cold lesion volumes evaluated 24 h later.
Results: Mean cortical CBF relative to the contralateral hemisphere was not impacted until cold lesion volume exceeded 5 cubic mm (Figure). In contrast, reductions in infarct volume were evident in association with the smallest cold lesions detected. Sham surgical procedures had no effect (not shown).
Conclusions: These results indicate that even small cortical lesions that have no impact on resting CBF have robust preconditioning effects against subsequent focal ischemia. Prior reductions in brain CBF and metabolism are therefore not required for such protection. Further experiments are necessary to fully assess the efficacy of cold lesion PC, but the reductions in infarct volume observed here approach the maximum seen after ischemic PC (1). This suggests that cold lesions provide an alternative, easily titrated PC method that permits independent assessment of the insult to frontal cortex, avoiding potential confounds of variable MCA territory injuries that arise after ischemic PC.
Supported by USPHS grant NS42267 (TSN)
PROSTANOIDS, NOT REACTIVE OXYGEN SPECIES, MEDIATE COX-2-DEPENDENT NEUROTOXICITY
Yasuhiro Manabe1, Josef Anrather2, Takayuki Kawano2, Kiyoshi Niwa2, Ping Zhou2, Elizabeth Ross2, Alexander Kunz2, Koji Abe3, Costantino Iadecola2
1Okayama Medical Center, Okayama, Okayama, Japan, 2Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY, USA, 3Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan
Background and aims: The prostaglandin synthesizing enzyme cyclooxygenase-2 (COX-2) has emerged as a critical pathogenic factor in brain diseases associated with activation of N-methyl-D-aspartate (NMDA) receptors. However, the COX-2 reaction products responsible for these deleterious effects have not been identified. Methods and results: We found that the brain damage produced by direct injection of NMDA into the somatosensory cortex is attenuated by the COX-2 inhibitor or in COX-2-null mice, but that the associated production of free radicals is not. Conclusions: This study directly implicates COX-2-derived prostanoids, rather then radicals, in the COX-2-dependent component of the damage mediated by NMDA receptors and strengthen the rationale for using COX-2 inhibitors in the treatment of neurological diseases associated with glutamate neurotoxicity.
INDUCTION OF UBIQUITIN, UCH-L1 AND PARKIN AND SELECTIVE VULNERABILITY OF MOTOR NEURON IN SPINAL CORD AFTER TRANSIENT ISCHEMIA
Takashi Yamauchi1, Masahiro Sakurai2, Koji Abe3, Yoshiki Sawa4
1Department of Cardiovascular Surgery, Sakurabashi Watanabe Hospital, Osaka, Osaka, Japan, 2Department of Cardiovascular Surgery, National Hospital Organization Sendai Medical Center, Sendai, Miyagi, Japan, 3Department of Neurology, Okayama University Graduate School of Medicine, Okayama, Okayama, Japan, 4Department of Cardiovascular Surgery, Osaka Graduate School of Medicine, Suita, Osaka, Japan
Background and aims: Vulnerability of motor neuron in spinal cord against ischemia is considered to play in the development of delayed paraplegia after operation of thoracic aorta. However, the reasons of such vulnerability are not fully understood. Recently, ubiquitin system has been reported to participate in neuronal cell death. In the present study, we investigated the expression of ubiquitin system molecules and discussed the relationship between the vulnerability and ubiquitin system after transient ischemia in spinal cord.
Methods: Fifteen minutes spinal cord ischemia of rabbits was applied with use of a balloon catheter. In this model, spinal motor neuron shows selectively delayed neuronal death whereas other spinal neuron such as inter neurons survive. Immunohistochemical analysis and western blotting for ubiquitin system molecules, ubiquitin, deubiquitylating enzyme ubiquitin (carboxy-terminal hydrolase 1;UCH-L1) and E3 ubiquitin ligase parkin, were examined.
Results: In cytoplasm, ubiquitin and UCH-LI were induced strongly both in inter neuron and motor neuron at early stage of reperfusion, but the prolonged expression was observed in motor neuron. Parkin was induced strongly at 3 hr after reperfusion, but the immunoreactivity returned to almost the same level of sham group at 6 hr in both neurons. In neuclei, ubiquitin, UCH-L1 and parkin were strongly induced in inter neuron whereas no upregulation of these proteins were observed in motor neuron.
Conclusions: These results indicate that the vulnerability of motor neuron of spinal cord might be partially attributed to the different response in ubiquitin and its related molecule response after transient ischemia.
MECHANISMS UNDERLYING THE BENEFIT OF REPEATED PRECONDITIONING INSULTS IN A RAT 4-VESSEL OCCLUSION MODEL OF GLOBAL ISCHEMIA
Masayuki Ueda, Thaddeus S. Nowak Jr.
Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, USA
Introduction: Repeated priming insults can increase the efficacy of ischemic preconditioning (1), but underlying mechanisms are unknown. This study compared hippocampal CA1 neuron protection after single and repeated priming insults monitored by DC potential measurement in a rat 4-vessel occlusion (4-VO) model (2).
Methods: All procedures were carried out under halothane anesthesia. Vertebral arteries were first cauterized, with recovery overnight. Common carotid arteries were occluded for 2 min and the duration of ischemic depolarization was recorded in each hippocampus (n=15), and this was repeated 2 days later. A comparison group (n=18) received single priming insults yielding depolarizations of varied duration. Test occlusions were produced 2 days after the final priming insult, and hippocampi experiencing standard insults of 7.5–9..5 min depolarization were included in subsequent analyses (14 of 15). Rats were perfusion-fixed 7 days after the test insult and CA1 neurons were counted in paraffin sections stained with hematoxylin/eosin.
Results: The first priming occlusions resulted in depolarizations of 50-104 sec. The second occlusion produced depolarizations of comparable duration (54-106 sec), most within 20 sec of that produced during the first occlusion (6 longer and 4 shorter), but 4 hippocampi failed to depolarize. The lag between occlusion and depolarization was 88 / 15 and 98 / 16 sec after first and second occlusions, respectively (excluding depolarization failures), but this delay increased to 133 / 35 seconds for the test insult. The magnitude of tolerance induction after repeated priming occlusions was well correlated with the duration of the longer of the two depolarization produced during priming insults, following the relationship defined for single priming insults in previous studies (Figure), although none reached the optimal preconditioning range (2-3 min).
Conclusion: There was no obvious cumulative effect of repeated insults that each produced short, suboptimal depolarizations. Efficacy may have been somewhat improved by increasing the probability of achieving a longer priming insult. There was no other advantage to repeated priming in this model, in which depolarization defined test insult severity. However, the delay in ischemic depolarization would reduce the severity of insults defined by occlusion duration. Since such depolarization delay appears to be more prominent in rats than in gerbils (2),(3) there may be species differences in this effect.
Supported by USPHS grant NS32344 (TSN)
ESTROGEN REPLACEMENT THERAPY POTENTIATES POST-ISCHEMIC CEREBRAL NEUTROPHIL ACTIVITY IN DIABETIC OVARIECTOMIZED RATS VIA RAGE
Hao-Liang Xu, Hae-Kyung Lee, Dale Pelligrino
Neuroanesthesia Research Department, University of Illinois at Chicago, Chicago, IL, USA
BACKGROUND: Although neuroprotective benefits of estrogen replacement therapy (ERT) have been established in multiple experimental ischemia models, the results of large-scale clinical studies have not supported the animal findings, and even hinted that ERT may be detrimental. This implies that conditions may exist whereby ERT may exacerbate rather than limit ischemic neuropathology. We recently reported that, in the diabetic, ovariectomized (OVX) rat, ERT promotes neutrophil adhesion and infiltration in the post-ischemic brain (rather than the reductions seen in non-diabetics), and exacerbates ischemic brain damage (1). In that study, a linkage between enhanced neutrophil infiltration and increased neuropathology was established. In the present study, we hypothesized that ERT-related exacerbation of post-ischemic inflammation in diabetics is linked to an increase in the presence of advanced glycation end-products (AGE) and the AGE receptor (RAGE) (2).
METHODS: Two principal strategies were used to test this hypothesis: a) RAGE inhibition (using a soluble RAGE ectodomain decoy inhibitor [sRAGE]; 425 ng in aCSF, injected icv 24h prior to study); or b) AGE disruption (4-6 wk treatment with the anti-AGE drug, ALT-711, via drinking water). To that end, we monitored post-ischemic neutrophil (rhodamine-tagged) adhesion/infiltration (intravital microscopy/closed cranial window system) in diabetic (streptozotocin [STZ]-injected; studied at 4–6 weeks post-STZ) OVX female rats, with or without chronic ERT (17B-estradiol [E2], 0.1 mg/kg/day for 1 week). All rats were subjected to 20 min of transient forebrain ischemia (carotid occlusion + hypotension) and 10 h reperfusion.
RESULTS: The data summarized in fig. 1 (sRAGE treatment) and fig. 2 (ALT-711 treatment) is taken from OVX + E2 rats and is expressed as the total area of rhodamine reactivity (intra- plus extravascular neutrophils) divided by the viewed pial venular area (means +/− SE). Extravasation of neutrophils in the vehicle-treated rats was seen starting at 8h reperfusion, while no infiltration was seen in the inhibitor-treated rats. In OVX rats, no infiltration was observed. Moreover, no effects of sRAGE or ALT-711 (vs vehicle) were seen (not shown). In ERT females, sRAGE and ALT-711 treatments were associated with substantial reductions in post-ischemic pial venular leukocyte adhesion and infiltration.
CONCLUSIONS: These results suggest that the exacerbation of post-ischemic leukocyte adhesion by chronic ERT in diabetic OVX females involves a RAGE-related process.
REGULATORY CD4FOXP3+ T-LYMPHOCYTES PREVENT DELAYED INFARCT GROWTH IN MURINE FOCAL ISCHEMIA
Roland Veltkamp1, Elisabeth Suri-Payer2, Clemens Sommer3, Claudia veltkamp4, Henrike Doerr3, Thomas Giese5, Arthur Liesz1
1Department of Neurology, University of Heidelberg, Heidelberg, Germany, 2Department of Immunogenetics, DKFZ, Heidelberg, Germany, 3Department of Neuropathology, University of Mainz, Mainz, Germany, 4Department of Internal Medicine IV, University of Heidelberg, Heidelberg, Germany, 5Department of Immunology, University of Heidelberg, Heidelberg, Germany
Background and Aims: Inflammatory cascades contribute substantially to secondary ischemic brain damage. Regulatory CD4+CD25+Foxp3+ T lymphocytes (Treg) are important anti-inflammatory modulators in various inflammatory diseases. We studied the role of Tregs in the progression of brain damage after ischemic stroke.
Methods: Permanent focal cerebral ischemia was induced by transtemporal middle cerebral artery occlusion (MCAO). Tregs were eliminated either by preischemic depletion with a mAb (clone PC61) in C57Bl/6 mice or by adoptive transfer of CD4+CD25- into rag2-/− mice. Infarct volume and cerebral cytokine expression (RT-PCR) were measured at various time points after MCAO. Tcell invasion was shown by flow cytometry and immunohistochemistry, respectively.
Results: Depletion of Tregs had no effect on infarct size 24 h after MCAO. Treg-depleted mice tended to have greater infarcts 72 h, and had significantly larger infarct volumes 7 d after MCAO (control: 7.4mm3; antibody-treated mice: 12.1mm3). Thus, Tregs prevented delayed infarct growth. Correspondingly, transfer of MACS sorted CD4+25- T cells into lymphocyte-deficient rag2-/− mice resulted in larger infarcts (12.9mm3) than transfer of CD4+ cells (6.8 mm3; p<0.05). In mice lacking Tregs, RNA levels of proinflammatory cytokines were significantly more elevated in the ischemic hemisphere compared to control (6 h after MCAO: TNF?: 4fold and IL-1? 2fold; 72 h after MCAO: IFN?: 5 fold). Treg invasion into the ischemic brain became first detectable 72 h after MCAO by immunohistochemistry and FACS.
Conclusions: Tregs are master anti-inflammatory modulators in ischemic stroke which substantially reduce secondary infarct progression by downregulating proinflammatory cytokine ?induced cell death. Based on our findings, this effect is probably mediated by early humoral signalling and by delayed Treg invasion. Better understanding of this postischemic crosstalk between immune cells and the damaged CNS may lead to new therapeutic strategies for ischemic stroke.
EFFECTS OF 24-HOUR EXPOSURE TO HIGH GLUCOSE CONCENTRATIONS ON OXIDATIVE METABOLISM IN RAT ASTROGLIAL CULTURES
Shinichi Takahashi, T. Abe, N. Suzuki
Department of Neurology, Keio University School of Medicine, Shinjuku-Ku, Tokyo, Japan
BACKGROUND AND AIMS
Not only a sustained hyperglycemic state, but also a fluctuating plasma glucose concentration has been implicated in the derangement of brain energy metabolism. Acute hyperglycemia is not thought to alter the cerebral metabolic rate of glucose (CMRglc). However, whether chronic hyperglycemia enhances or suppresses the CMRglc remains controversial, and the effect of a fluctuating plasma glucose concentration on the CMRglc has not been examined extensively. Astrocytes are thought to be more dependent on glycolytic, rather than oxidative, glucose metabolism for energy production. Recent findings, however, have revealed that oxidative metabolism in astrocytes might be comparable to that in neurons in vivo. Recently, we reported that oxidative metabolism in astrocytes depended on the environmental glucose concentration in vitro (Abe et al., JCBF&M 26: 153-160, 2006). In the present study, we examined the effects of short-term exposure to a high glucose concentration on the oxidative metabolism of astroglias, as assessed by the oxidation of [14C]lactate or [14C]acetate.
METHODS
Primary cultures of neurons or secondary cultures of astroglia were prepared from SD rats, as described previously (Takahashi et al, PNAS 92: 4616-4620, 1995). The cells were cultured in the presence of a high (22 mM) or low (5 mM) concentration of glucose. Twenty-four hours prior to the 14C assay, the culture media of replaced with media containing a high (22 mM) or low (5 mM) concentration of glucose and the cells were further incubated for 24 hours. On the day of the assay, the cells were washed twice with PBS without glucose and were assayed for [14C]deoxyglucose phosphorylation and [14C]lactate or [14C]acetate oxidation.
RESULTS
The high or low glucose media did not alter the rates of [14C]deoxyglucose phosphorylation. The low glucose medium increased the oxidation of [14C]lactate only in astroglia, as evidenced by the increased production of 14CO2. Twenty-four-hour exposure to a high glucose concentration resulted in a significant decrease in [14C]lactate oxidation in the astroglia (2.9 ± 0.5 pmol lactate/µg protein/60 min, mean ± SD, n=4), compared with that in cells cultured in the presence of a low glucose concentration (4.8 ± 0.4, p < 0.001). In contrast, 24-hour exposure to a low glucose concentration after cultivation in a high glucose medium did not affect [14C]lactate oxidation in the astroglia. [14C]acetate, an astrocyte-specific reporter molecule was also employed. [14C]acetate oxidation in neurons was negligible. [14C]acetate oxidation in the astroglia that were cultured in a high glucose medium (1.7 ± 0.1 pmol acetate/µg protein/60 min, n=4) was lower than that in the cells that were cultured in a low glucose medium (2.8 ± 0.4). Similarly, 24-hour exposure to a high glucose medium significantly reduced [14C]acetate oxidation from 1.9 ± 0.3 to 1.2 ± 0.3 (p < 0.05).
CONCLUSIONS
Fluctuations in the environmental glucose concentration do not alter glucose metabolism in neurons. In contrast, increasing the concentration of glucose, either acutely or chronically, suppressed oxidative metabolism in astroglia. These different responses of neurons and astroglia may shed new light on brain energy metabolism in diabetic patients with fluctuating plasma glucose concentrations.
CEREBRAL BLOOD FLOW IN WILD & ADENOSINE A2A KNOCK-OUT MICE DURING ACUTE HYPOXIA: EFFECTS OF A2A ANTAGONIST, ZM 241385
Grzegorz J. Miekisiak, Alexander Beylin, David K. Kung, JiSuk K. Yoo, Adam L. Sandler, H. Richard Winn
Department of Neurosurgery, Mount Sinai School of Medicine, New York, NY, USA
Background and Aims
ADO has been suggested as being involved in the regulation of cerebral hypoxic hyperemia via Ado 2a receptor. Consequently we evaluated the CBF response to hypoxia in WT and A2a KO mice in the absence and after administration of the A2a antagonist, ZM 241385, during 30 s. of hypoxia.
Methods
Mice (22-30 gm) were intubated, anesthetized, and ventilated. We continuously monitored end-tidal CO2, arterial blood pressure (BP), & CBF (laser Doppler). Before and after administration of ZM 241385, WT and KO animals were randomly challenged for 30s.with 10% 02/90% N2. This degree of hypoxia resulted in a PaO2~30-35mmHg. Since ZM prevented hypotension during hypoxia, in another group of animals, we stabilized the changes in MABP during hypoxia.
Results
WT vs. KO
Because MABP decreased significantly more during hypoxia in the KO than the WT mice, we controlled the changes in MABP. As indicated in Fig. 1, CBF during hypoxia increased to 181.9 · 7.8% 182 ± % in the WT as compared to 115.8 ± 3.0 % (p< 0.01)
Effects of ZM 241385
In the KO mice, ZM had no effect on the response of CBF (data not shown). In contrast, as indicated in Fig 2, ZM significantly (p<0.01) attenuated the CBF response in the WT mice (160.4 ± 5.4% vs 132.1 ± 2.8%).
Conclusions: The present study indicates that a significant component of the increase in CBF during moderate hypoxia is related to A2a receptor mediated vasodilatation. The persistence of some vasodilatation during hypoxia may be secondary to non A2a adenosine receptor mechanisms (i.e., A2b), by the adaptation of knockout animals and/or to non-adenosine related mechanism.
[Supported by NS021076]
CAUDATE PHOTOTHROMBOTIC INFARCTION OF RODENT: NEUROPATHOLOGICAL AND BEHAVIORAL STUDY
Toshihiko Kuroiwa, Guohua Xi, Masanobu Okauchi, Ya Hua, Timothy Schallert, Julian T. Hoff, Richard F. Keep
Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA
Basal ganglia infarction is typically caused by small end-artery occlusion, which is different from cortical infarction typically caused by large artery occlusion. We have developed a model of caudate infarction in rodent induced by photothrombotic occlusion of parenchymal small vessels using a stereotaxically placed optic fiber, and analyzed the time course of behavioral and pathological changes.
[Methods] Male SD rats (n=20) and male Mongolian gerbils (n=24) were used in this study. Optic fiber 0.5 mm or 0.75 mm in diameter coated with fluorocarbon resin was placed stereotaxically to the left caudate nucleus under isoflurane anesthesia, Rose Bengal dye (20mg/kg) was intravenously injected and caudate nucleus was exposed to cold white light (lighting intensity, 900 ? 2400 Lux) via the optic fiber for 3, 5 or 10 min. A battery of neurological tests was performed repeatedly during 7 days after light exposure. The brain was removed after transcardiac perfusion with fixative at different post-exposure time-intervals, and prepared for light-microscopical and electron- microscopical examination. Vascular perfusion was examined in the brain removed shortly after intravenous injection of FITC-dextran. BBB opening was detected by Evans Blue extravasation, and ischemic brain edema was quantified by tissue gravimetry. [Results] Round infarction with thrombosed parenchymal small vessels was produced in the caudate nucleus. The infarct size increased in parallel the light exposure time (1.5mm ? 3.5mm in diameter). The infarcted tissue was surrounded by a thin layer (0.12mm ? 0.14mm) of selective neuronal death with interstitial microvacuolation (ischemic edema) with BBB opening to serum proteins. The density of vessels with FITC-dextran was decreased in the peri-infarct tissue where dilated vessels were scattered. Electron microscopy revealed platelet thrombi occluding parenchymal small vessels, astrocytic swelling, dark neuronal change and hydropic oligodendrocyte with pyknosis. Neurological test revealed circling toward left, left corner turn, impaired right-hindlimb placing, which was milder and transient (Foot-fault placing score= 14.2 ± 9.0) in 3–5 min exposure group and more severe (22.5 ± 10.0, p<0.05) and lasted longer in 10 min exposure group. [Conclusion] Small infarction localized in the caudate nucleus was produced by photothrombotic occlusion of the parenchymal small vessels using a stereotaxically placed optic fiber in rodents. The infarction was surrounded by a thin layer of selective neuronal death and hypoperfusion. Severity and duration of neurological deficit parallels the infarction size. We have established a rodent model of basal ganglia infarction induced by photothrombotic occlusion of the parenchymal small vessels.
Figure. Photothrombotic infarction of caudate nucleus in rat (left) and parenchymal small vessels occluded with platelet thrombus
IMAGING OF EXPERIMENTAL EPILEPSY SURGERIES: CALLOSOTOMY, MULTIPLE SUBPIAL TRANSECTION AND DEEP BRAIN STIMULATION
Tatsuya Tanaka, Kiyotaka Hashizume, Akira Hodozuka, Seiji Takebayashi, Masayuki Kunimoto
Department of Neurosurgery, Asahikawa Medical College, Asahikawa, Japan
Bakground and Aim: In various epilepsy surgery, seizure-free rate of each surgery is still controversial. And cost-effectiveness of surgery is still discussing. In the present study, experimental epilepsy surgeries were performed in cats and in rats. Electrophysiological and metabolic approaches were made.
Methods: Under intraperitoneal pentobarbital anesthesia, electrodes or chemitrodes were placed and chronic preparation was made. (1) After seizure induction, deep brain stimulation(DBS) was performed. (2) For multiple subpial transaction(MST) or callosotomy, craniotomy was made in all animals and the dura was opened unilaterally. Bipolar electrodes were implanted and EEG recording was made. Two micrograms of kainate was injected into unilateral sensori-motor cortex and focal cortical seizures were elicited. MST or callosal section was performed using stereotactic coordinates. EEG analysis was made. (3) Under intraperitoneal pentobarbital anesthesia, femoral artery and femoral veins were canulated. Craniotomy was made in all rats and the dura was opened unilaterally. Two micrograms of kainite was injected into unilateral sensori-motor cortex and focal cortical seizures were elicited. MST or callosal section was performed using stereotactic coordinates. Intravenous injection of 14C-deoxyglucose was made and the rats were processed for autoradiography.
Result: All cats and rats exhibited focal cortical seizures after kainite injection. DBS suppressed seizure propagation. Seizure activity between two MSTs was suppressed and seizure propagation to contralateral cortex was clearly suppressed after callosotomy. But, kainite-induced seizures remained in the focus and seizure propagation was observed to the subcortical structures in both cases. (3) After MST or callosotomy, seizure propagation to contralateral cortex(hypermetabolic area) was suppressed. Hypermetabolic area in the focus, ipsilateral basal ganglia and thalamus, and contralateral basal ganglia remained even after MST or callosotomy.
Conclusion: MST or callosotomy may be a palliative surgical option only to reduce seizure activities in the cortex. But seizure activities of the focus remained even after the procedures. DBS may be the promising procedure when epileptic focus locate in the eloquent cortex.
ADAPTIVE GLIAL STRESS RESPONSES IN CHEMICAL PRECONDITIONING: FOCUS ON ASTROCYTIC GLUTAMINE SYNTHETASE BUFFERING GLUTAMATE-MEDIATED NEUROTOXICITY
Akihiko Hoshi, Toshiki Nakahara, Keiko Shimizu, Kazuhiro Endo, Teiji Yamamoto
Department of Neurology, Fukushima Medical University, Fukushima, Japan
Astrocytic control of glutamatergic activity through glutamate transporters plays a pivotal role in neuroprotection. In astrocytes, approximately two-thirds of glutamate is converted by glutamine synthetase (GS) into glutamine (glutamate-glutamine cycle), that is, astrocytes serve as a sink to take up the released glutamate from neurons and utilize it to synthesize glutamine. 3-Nitropropionic acid (3-NPA) is an irreversible inhibitor of succinate dehydrogenase in the tricarboxylic-acid cycle, and provides ischemic tolerance to the brain. So far, there have been no reports on the relationship of astrocytic GS and ischemic tolerance by chemical preconditioning. We have investigated the temporal profile of GS expression parallel with glial fibrillary acidic protein and heat-shock protein 70 (HSP70) in order to test the hypothesis that astrocytes have a major role in 3-NPA-induced chemical preconditioning. In our rat model of permanent focal ischemia, preconditioning with 3-NPA singnificantly reduced the subsequent neurological deficits and infarct volume within 24-72 hours after treatment. Immunohistochemically, protoplasmic astrocytes in the cortex and striatum were activated in terms of upregulation of GS and more abundant protoplasmic processes with 3-NPA preconditioning, however, HSP70 expression could not be induced. Thus, the activation of astrocytes and upregulation of GS play an important role in 3-NPA-induced preconditioning. In view of glutamate being imposed on the cerebral ischemic damage, the astrocytic GS may contribute to 3-NPA-induced ischemic tolerance.
EFFECTS OF MAGNESIUM TREATMENT IN A MODEL OF INTERNAL CAPSULE LESION IN SPONTANEOUSLY HYPERTENSIVE RATS
Clotilde Lecrux1, Chris McCabe2, Chris J. Weir3, Lindsay Gallagher2, Jim Mullin4, Omar Touzani1, Keith W. Muir5, Kennedy R. Lees6, I. Mhairi Macrae2
1University of Caen, Centre CYCERON, CNRS UMR 6185, Caen, France, 27TMRI Facility and Wellcome Surgical Institute, Division of Clinical Neuroscience, University of Glasgow, Glasgow, Scotland, UK, 3Robertson Centre for Biostatistics, University of Glasgow, Glasgow, Scotland, UK, 4Department of Clinical Physics and Bioengineering, Southern General Hospital, Glasgow, Scotland, UK, 5Division of Clinical Neurosciences, University of Glasgow, Institute of Neurological Sciences, Southern General Hospital, Glasgow, Scotland, UK, 6University Department of Medicine and Therapeutics, Gardiner Institute, Western Infirmary, Glasgow, Scotland, UK
Background and aims - Previous studies have demonstrated that magnesium is neuroprotective in several models of cerebral ischemia in rats. The Intravenous Magnesium Efficacy in Stroke (IMAGES) trial found no significant effect of magnesium sulphate overall but subgroup analysis detected benefit in patients with lacunar stroke syndromes and/or with higher than median blood pressure. We therefore aimed to assess the effects of magnesium sulphate administration following white matter damage in vivo in spontaneously hypertensive rats (SHR).
Methods - We developed a model involving a lesion of the left internal capsule by a local injection of endothelin-1 (ET-1) (200 pmol) in adult, male SHR. We evaluated the effect of subcutaneous injections of magnesium sulphate (1×300 mg/kg 30 mins before + 4×200 mg/kg each hour after) on functional recovery, 3 and 10 days after ET-1 injection. Group sizes were n=15 vehicle, n=13 magnesium. We assessed lesion size after 2 days using MRI T2-weighted images and after 11 days using histological staining specific for white matter (luxol fast blue). Infarct volumes were statistically analysed using Student's unpaired t-test and behavioural data using ANCOVA, ANOVA and PLSD Fisher tests.
Results ? ET-1?induced lesion in the internal capsule produced significant motor deficits on both the forelimb and the hindlimb for 10 days in the vehicle group (figure), which were significantly ameliorated by the magnesium treatment in both the cylinder (left forelimb use, p<0.01 and both forelimb use, p<0.03 versus vehicle) and the walking-ladder (right hindlimb score, p<0.02 versus vehicle) tests. Infarct volumes (expressed as percentage of ipsilateral hemisphere) were not significantly different between animals receiving vehicle or magnesium sulphate treatments: 1.2 ±0.1% and 2.0±0.5%, respectively using MRI and 0.3±0.1% and 0.5±0.1%, respectively from histology at 11 days post-stroke.
Conclusions - We have developed a new model of white matter ischaemia in SHR in which we were able to assess specific behavioural deficits persistent for 10 days. Furthermore, we demonstrate that magnesium treatment can provide an improvement of motor abilities, suggesting that magnesium remains an appealing protective substance in the case of white matter ischaemic damage in the rat.
REDUCTION IN OXIDATIVE STRESS BY SOD1 OVEREXPRESSION ATTENUATES ACUTE BRAIN INJURY AFTER SUBARACHNOID HEMORRHAGE VIA ACTIVATION OF AKT/GSK3B SURVIVAL SIGNALING
Hidenori Endo1,2, Kuniyasu Niizuma1, Miki Fujimura2, Teiji Tominaga2, Pak H. Chan1
1Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Stanford, CA, USA, 2Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai, Japan
Introduction: Subarachnoid hemorrhage (SAH) results in a high mortality rate, despite sophisticated medical management and neurosurgical techniques. Recent studies have emphasized the importance of acute brain injury after SAH. Oxidative stress plays important roles in the pathogenesis of acute brain injury after SAH. We have reported that copper/zinc-superoxide dismutase (SOD1) is a crucial endogenous enzyme responsible for eliminating superoxide, and that overexpression of SOD1 reduces cell injury after SAH. In cerebral ischemia, SOD1 overexpression decreased ischemic neuronal death through activation of the Akt/GSK3b survival pathway. In this study, we investigate the relationship between SOD1 and the Akt/GSK3b pathway in acute brain injury after SAH. We address this issue by examining apoptotic cell death, superoxide production, and phosphorylation of Akt/GSK3b after SAH, using both wild-type (Wt) and SOD1 transgenic (Tg) rats.
Materials and methods: To examine the relationship between SOD1 and the Akt/GSK3b pathway following SAH, we used a perforation SAH model in both Wt and SOD1 Tg rats. One, 6 and 24 hours after SAH, samples were taken from the cerebral cortex, and used for Western blot analysis and immunohistochemical analysis. To investigate apoptotic cell death in acute brain injury after SAH, DNA fragmentation was analyzed with a commercial enzyme immunoassay. Early production of superoxide anions (O2?) during SAH was investigated with the use of hydroethidine (HEt).
Results: The mortality rate at 24 hours for the SOD1 Tg rats (7.69%) was significantly decreased compared with the Wt rats (31.4%). DNA fragmentation in the SOD1 Tg rats was significantly decreased at 24 hours compared with the Wt rats at the same time point. O2? production shown by oxidized HEt signals was observed in the cerebral cortex at 1 hour in the Wt rats, and was markedly decreased in the SOD1 Tg rats. Western blot analysis showed that immunoreactivity of phospho-Akt and phospho-GSK3b was significantly increased at 6 hours compared with the control brain samples in the Wt rats. In the SOD1 Tg rats, the increase in phospho-Akt and phospho-GSK3b levels was more prominent and persistent. In a comparison between the two groups, phospho-Akt and phospho-GSK3b were significantly increased at 24 hours in the SOD1 Tg rats. An immunohistochemistry study showed slight immunoreactivity of phospho-Akt and phospho-GSK3b in the cerebral cortex of the control brains. This immunoreactivity became stronger at 24 hours in both groups. In the SOD1 Tg rats, phospho-Akt and phospho-GSK3b expression at 24 hours was more prominent than in the Wt rats. A double immunofluorescent study demonstrated that phospho-Akt-positive cells colocalized with phospho-GSK3b-positive cells in the cerebral cortex 6 hours after SAH. Moreover, phospho-GSK3b-positive cells colocalized with neurons at the same time point.
Conclusions: This study suggests that oxidative stress plays a significant role in acute brain injury after SAH, and that the neuroprotection of SOD1 is partly mediated by activation of the Akt/GSK3b survival pathway. Reducing oxidative stress, thereby activating the survival pathway, could be a therapeutic target for acute brain injury after SAH in a clinical situation.
TEMPORAL AND REGIONAL EVOLUTION OF AQUAPORIN 4 EXPRESSION AND MAGNETIC RESONANCE IMAGING IN A RAT PUP MODEL OF NEONATAL STROKE
Jerome Badaut1, Stephen Ashwal2, Beatriz Tone2, Amber Wang3, Luca Regli1, Hui R. Tian2, Andre Obenaus3
1Neurosurgery Research Group, CHUV, UNIL, Lausanne, Switzerland, 2Department of Pediatrics, Loma Linda University School of Medicine, Loma Linda, CA, USA, 3Department of Radiation Medicine, Loma Linda University School of Medicine, Loma Linda, CA, USA
Aim : Cerebral edema contributes to the morbidity and mortality and can readily be observed using magnetic resonance imaging (MRI). Recent studies show that aquaporin 4 (AQP4, water channels) is induced early after stroke and potentially participates in the development of brain edema(1). We studied whether induction of AQP4 correlated with edema development in the 10-day old rat pup undergoing transient middle cerebral artery occlusion (tfMCAO) using high field (11.7 Tesla) MRI with T2WI and DWI followed by immunohistochemical investigation of AQP4 protein expression.
Methods : Focal ischemia in the rat pup was accomplished using a filament model of temporary unilateral occlusion of the left MCA(2). MRI was performed at 24-hours, 72-hours, and 28-days after tfMCAO. AQP4 immunolabeling was evaluated on slices corresponding to the MRI acquisitions using an infrared-scanner (LiCor) for quantification and epifluorescence microscopy.
Results : At 24-hours, we observed increased T2 values and decreased ADC within injured cortical and striatal regions that reflected the onset of edema formation. Coincident with these MR changes were significant increases in AQP4 expression in the border regions of injured tissues. Increased AQP4 expression was observed on astrocyte end-feet in contact with blood vessels. Striatal imaging findings were still present at 72-hours with a slow normalization of AQP4 expression in the peri-infarct regions. At 28-days AQP4 expression normalized throughout the brain except striatal T2 and ADC values which remained increased. Our findings show that induction of AQP4 is increased during the period of active edema formation in the peri-lesional region but at 28 days does not correlate with brain water content as observed with MRI.
Conclusion : Induction of AQP4 on astrocyte end-feet at 24 and 72 hours could participate in the clearance of water and solutes from brain parenchyma after ischemia in the immature rat brain.
Figure : A) Macroscopic comparison of MRI data and AQP4 immunohistochemistry at 24 hrs after tfMCAO occlusion : A1) A representative T2 image and A2) ADC-image show respectively an increase and decreased signal intensities within the ischemic region which are suggestive of edema formation. A3) Illustration of the AQP4-IR shows that the intensity of AQP4 is increased in border of the red line.
B) The intensity of the AQP4 staining was significantly increased around blood vessels (arrowheads) in cortex tissues in vicinity the regions where ADC was decreased and T2WI-values were increased. C) In frontal cortex of the contralateral hemisphere the intensity of AQP4-IR around blood vessels (arrow-heads, D) was higher than in the contralateral parietal cortex (arrow-heads, D). Bar : B-D=40 micrometer.
PREFERRED BLOOD-BRAIN TRANSFER OF O-(2-[18F]FLUOROETHYL)-D-TYROSINE (D-FET) INTO THE PORCINE BRAIN
Peter Brust1, Victoria Makrides2, Rainer Hinz5, Wolfgang Weber4, Hans-Jurgen Wester4, Steffen Fischer1, Katja Huggel2, Vadivel Ganapathy6, Francois Verrey2, Reinhard Bauer3
1Institute of Interdisciplinary Isotope Research, Leipzig, Germany, 2Institute of Physiology, University of Zurich, Zurich, Switzerland, 3Institute of Molecular Cell Biology, University Hospital Jena, Jena, Germany, 4Department of Nuclear Medicine, Technische Universitat Munchen, Munchen, Germany, 5Wolfson Molecular Imaging Centre, The University of Manchester, Manchester, UK, 6Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA
Large neutral amino acids (LNAA) are transported by a variety of transporters, among which the SLC7A5/LAT1 transporter is regarded as the predominant LNAA transporter at the blood-brain barrier (BBB). LAT1 has broad substrate specificity, accepting even the D-isomers of amino acids as high affinity substrates. Also, LAT1 is frequently overexpressed in malignant tumors providing the rationale for the use of radiolabeled LNAA such as L-FET for imaging intracerebral tumors with PET. Recently, experimental evidence was obtained from various authors that D-enantiomers of some LNAA are superior to L-enantiomers with regard to the imaging of certain peripheral tumors. Therefore, in this study the transport of D-FET across the blood-brain barrier was studied with PET in anaesthetized piglets and patients after subtotal resection of brain tumors and compared with L-FET.
Ten piglets of mixed German domestic breed were anesthetized with 0.25% isoflurane in 65% nitrous oxide and 35% oxygen. L-[18F[FET (98 ± 11 MBq, n=4) or D-[18F]FET (128 ± 34 MBq, n=6) in 10 ml saline was intravenously infused within 2 min. Compartmental modeling of PET data was used to estimate the rate constants for the blood-brain (K1) and the brain-blood (k2) transfer of both radiotracers. Six patients were investigated with L-FET-PET and D-FET-PET after subtotal resection of brain tumors. Standardized uptake values (SUV) were calculated in brain cortex and lesions. Additionally, two different approaches were used to assess the interaction of L-FET and D-FET with transporters known to have affinity for LNAA. First, we studied the hLAT1 transporter in HRPE cells (human retinal pigment epithelial cells). Second, hLAT1, LAT2, XPCT and PAT1 were studied in Xenopus laevis oocytes.
The normalized brain uptake in piglets of D-FET 5 min after injection is about 140% higher than that of L-FET. The rate constants for the blood-brain and brain-blood transfer of D-FET in piglets are significantly higher (by factor 3-4) than those for L-FET. Both findings indicate that in piglets the BBB transport of D-FET is significantly faster than that of L-FET. This observation is in contrast with previous studies demonstrating high and stereoselective uptake of L-FET in mouse brain, and low uptake of D-FET into mouse brain (Wester et al., 1999). Also, the studies in humans revealed an initial uptake of D-FET which was similar to L-FET but followed by a rapid washout from brain resulting in a significantly higher uptake of L-FET at later time points. Thus, the transport kinetics for D-amino acids seems to differ among species. The differences in brain uptake of L-FET and D-FET can only be explained by differences in their affinity to specific transport systems. L-FET showed at least 20-fold higher in vitro affinity than D-FET towards LAT1 and LAT2 whereas both enantiomers have similar affinities to XPCT and PAT1 transporters.
In conclusion, we have demonstrated specific transport of L-FET and D-FET across the BBB which cannot be fully explained by known transport systems. The study provides further evidence for the presence of an unknown, D- amino acid preferring transporter / transport system at the blood-brain barrier.
DIFFERENCES IN POSTOPERATIVE GEOMETRICAL AND HEMODYNAMIC CONDITIONS OF CAROTID ARTERIES BETWEEN CAROTID ARTERY STENTING AND CAROTID ENDARTERECTOMY WITH PATCH ANGIOPLASTY
Koji Tokunaga, Kenji Sugiu, Ayumi Nishida, Shigeki Ono, Keisuke Onoda, Isao Date
Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
Background and aims: Carotid endarterectomy with a patch graft (Patch CEA), allowing marked increase in vessel diameters and reduction of the short- and long-term risk for ischemic stroke, has been our treatment of choice for patients with carotid artery stenosis. Recently, carotid artery stenting (CAS) has emerged as an alternative to CEA, particularly for patients at high surgical risk. However, postoperative geometrical and hemodynamic differences between these two treatment modalities have been rarely investigated. The purpose of this study was to evaluate the postoperative changes in configurations and flow velocities of carotid arteries after Patch CEA compared to CAS. Methods: Thirty-eight consecutive patients with carotid artery stenosis treated by CAS or Patch CEA were included. Pre- and postoperative configurations of carotid arteries were evaluated by conventional digital subtraction angiography and/or 3D-CT angiography. Ultrasonographic examinations were performed using a color duplex scanner with a 7.5(7.2–8..6)-MHz linear probe or a 5-MHz sector probe (Aplio, Toshiba Medical Systems Corp., Tokyo). Doppler waveforms were recorded in the middle portion of the common carotid artery (CCA) and the internal carotid artery (ICA) bulb to measure parameters of flow velocities including peak flow velocities (PFV), end diastolic velocities (EDV) and mean velocities (Vmean). Results: Twenty-five patients were treated by CAS, and Patch CEA was performed for 13 patients. There were no differences in preoperative degrees of stenosis (83% in CAS group vs 79% in CEA group) and flow velocities of CCA and ICA between two groups. The postoperative mean degree of stenosis was 12% and 0% in CAS group and Patch CEA group, respectively. Mild stenoses tended to be left at the severely calcified plaque after CAS. After Patch CEA, the configurations of the ICA bulbs were long and widely restored. The averages of diameters of the postoperative ICA bulbs measured by ultrasonography were 4.5 mm in CAS group and 7.0 mm in Patch CEA group (P<0.01). There were no differences in the averages of postoperative PFV, EDV and Vmean of CCA between two groups, whereas the averages of PFV, EDV and Vmean of ICA in CAS group were 80 cm/s, 25 cm/s and 42 cm/s, respectively, and significantly greater than those (53 cm/s, 16 cm/s and 28 cm/s, respectively) in Patch CEA group (P<0.01). Conclusions: There are significant differences in postoperative geometrical and hemodynamic conditions between CAS group and Patch CEA group. Suboptimal resolution of carotid stenosis may lead to acute/subacute occlusion or restenosis in the chronic stage. On the other hand, excessive increase in vessel diameter may cause recurrent atheromatous plaque because of atherogenic low and disturbed shear stress. Further studies including long-term clinical follow-up and computer flow dynamics are needed to clarify the impact of geometrical and hemodynamic differences between CAS and Patch CEA on clinical courses of patients with carotid stenosis.
GENDER-RELATED TEMPOROSPATIAL DIFFERENCES IN BLOOD-BRAIN BARRIER PERMEABILITY FOLLOWING EXCITOTOXIC LESIONS IN THE RAT
Karim Khallout1, Jerome Toutain1, Nicolas Delcroix2, Eric T. MacKenzie1
1Centre Cyceron UMR 6185, Caen, Lower Normandy, France, 2Centre Cyceron UMR 6194, Caen, Lower Normandy, France
GENDER-RELATED TEMPOROSPATIAL DIFFERENCES IN BLOOD-BRAIN BARRIER PERMEABILITY FOLLOWING EXCITOTOXIC LESIONS IN RATS
Khallout K.1., Toutain J.1, Delcroix N.2, MacKenzie E.T.1
University of Caen, CNRS UMR 6185 1, UMR 6194 2 Centre CYCERON Bd H. Becquerel 14074 BP 5229 Caen, FRANCE,
Introduction: The object of these investigations was to study brain permeability in male and female rats following excitotoxics lesions as a function of time, so as to better understand the effects of gender-related hormones on the blood-brain barrier (BBB). We examined, through the use of Evans blue albumin (EBA) as a tracer, the differences in extravasation between the two sexes.
Methods: All striatal lesions were provoked by the intrastriatal injection of 75 nmoles NMDA in 3 µL. EBA extravasation was quantified by fluorescent microscopy over serial coronal sections as was the volume tissue necrosis (thionine staining cf. fig. 2).
Results (cf. fig. 1): The insult provoked a volume of EBA extravasation, which correlated with the tissue necrosis; the lesions volumes were also highly sex and time-dependent (p<0.001). Statistically, the data were analyzed by polynomial two degree equations followed by ANOVA.
Conclusions: While the disruption of BBB during ischaemia has been frequently studied, there are few time-based publications. Excitotoxic lesions have been much less examined. We clearly demonstrate a quadratique time-dependent dysfunction of the BBB following intrastriatal lesions and differences in time-related (p<0,001) volumes between the two genders (males : R2 = 0,4532; p<0,02 and females : R2 = 0,7008 p<0.001). The quantification of the extravasation between the two sexes has been analyzed on a basis of the consolidated lesions. This study merits to be continued with the use of ovariectomized rats in order to examine the reasons for the exacerbation and delayed resolution between the two sexes. Such approaches are underway.
Acknowledgements: The authors thank many other members of the group that have contributed to this study.
C1-INHIBITOR ATTENUATES NEUROBEHAVIORAL DEFICITS FOLLOWING CONTROLLED CORTICAL IMPACT BRAIN INJURY IN MICE
Luca Longhi1,2, Carlo Perego1, Elisa Zanier1,2, Fabrizio Ortolano1,2, Paolo Bianchi1, Luigi Bergamaschini3, Nino Stocchetti1, Maria Grazia De Simoni2
1Department of Anesthesia and Critical Care Medicine, Milan University, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli E Regina Elena, Milano, Italy, 2Mario Negri Institute, Milano, Italy, 3Department of Internal Medicine, Milan University, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli E Regina Elena, Milano, Italy
Background and aims: The goal of the study was to evaluate the neurobehavioral effects of the C1-inhibitor (C1-INH), an endogenous inhibitor of complement and contact-kinin pathways, following controlled cortical impact (CCI) brain injury in mice.
Methods: Mice (n = 36) were anesthetized with intraperitoneal administration of sodium pentobarbital (65 mg/kg) and subjected to CCI brain injury. At 10 minutes postinjury, animals randomly received an intravenous infusion of either C1-INH (15 U) or saline (equal volume, 150 µl). A second group of mice received identical anesthesia, surgery, and saline to serve as uninjured controls. Neurobehavioral motor outcome was evaluated weekly from 1 to 4 weeks postinjury by performing Neuroscore. Cognitive function was evaluated at 4 weeks postinjury using the Morris water maze (MWM) test of learning ability.
Results: Consistently, brain-injured mice receiving C1-INH showed attenuated neurological motor deficits during the 4-week period compared with injured mice receiving saline (Figure).
At 4 weeks postinjury all mice were able to swim in the MWM without notable physical impairment and demonstrated an ability to learn the visuo-spatial task, as reflected by decreasing latencies to find the platform over the three-day period. Brain-injured mice receiving C1-INH showed an average latency of 30.7 ± 1.9 seconds which was significantly faster (better performance) than the average latency of brain-injured animals receiving saline (42.9 ± 3.1 seconds, p < 0.01).
Conclusion: Post-traumatic administration of the endogenous complement inhibitor C1-INH significantly attenuates neurobehavioral motor and cognitive deficits associated with traumatic brain injury. C1-INH confirms to possess clear neuroprotective actions as previously shown in ischemia/reperfusion brain injury (1).
AGMATINE ATTENUATE BRAIN EDEMA THROUGH LESSENING THE EXPRESSION OF AQUAPORINS AFTER TRAUMATIC BRAIN INJURY
Yong Woo Lee1,2, Jae Hwan Kim1,2, Jin Woo Chang1,3, Kyung Ah Park2, Won Taek Lee2, Jong Eun Lee1,2
1Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, South Korea, 2Department of Anatomy, Yonsei University College of Medicine, Seoul, South Korea, 3Department of Neurosurgery, Yonsei UniversityCollege of Medicine, Seoul, South Korea
Background and aims: Brain edema is a pathophysiological condition of increased water content due to a variety of brain injuries, including trauma, ischemia and progression results in major focal brain dysfunction. The cold injury model is one of the established models for the study of vasogenic brain edema development and secondary rain injury[1]. The aquaporins are a family of water-selective membrane channels. Aquaporin-1, −4 and −9 have been identified as the three major water channels in the brain[2]. Agmatine is a primary amine formed by the arginine decarboxylase and is known as an antagonist at N-methyl-D-aspartate (NMDA) receptors and an inhibitor for NOS. Previously we reported the neuroprotective effect of agmatine on infarct volume and brain edema following cerebral ischemia[3]. In this study, we investigate that administration of agmatine regulates aquaporin expression and attenuates brain edema after TBI.
Materials and methods: For the cold traumatic injury model, the skull of the rat was mounted in a stereotactic device for trephination (diameter for 4mm) above the right parietal cortex leaving dura mater intact. Cortical lesion was induced by attachment of metal probe (diameter 3mm) cooled by liquid nitrogen onto the brain surface 5 times for 30 seconds. Agmatine was treated 30min after traumatic cold injury (100mg/kg, IP). Water content was analyzed in 24hours after TBI. The expression of aquaporins was examined with immunohistochemistry and immunoblotting.
Results: The physiological parameters before cold traumatic injury were monitored and maintained throughout the experiment. Trauma-induced loss of brain volume and the number of eosinophilic neurons were reduced and improved neurological deficits in agmatine treatment group. The water content in cold traumatic injured brains at 24hours after insults was measured and decreased in agmatine treatment group. We examined the expression of aquaporin-1,-4 and −9 using immunohistochemistry and immunoblotting in cerebral cortex and hippocampus. In all time point, aquaporin-1 was decreased in agmatine treatment group compared to experimental control after TBI. Aquaporin −4, −9 were similar expressed in agmatine treatment group and experimental control 1, 2 day after TBI. But 1week after TBI, the expressions of aquaporin −4,-9 were decreased in agmatine treatment group.
Conclusion: Agmatine could attenuate brain edema through lessening water content by suppression of the expression of aquaporins. These results may suggest agmatine as a novel therapeutic material for vasogenic and cytosolic edema including traumatic brain injury and brain tumor.
CEREBRAL VASOREACTIVITIES IN PATIENTS WITH CAROTID STENOSIS HELP TO PREDICT HYPERPERFUSION AFTER CAROTID INTERVENTION
Ting-Yu Chang1, Ho-Ling Liu2,3, Tai-Cheng Wu1, Chien-Hung Chang1, Hsiu-Chuan Wu1, Yeu-Jhy Chang1, Tsong-Hai Lee1
1Stroke Section, Department of Neurology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan-R.O.C., 2Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan-R.O.C., 3MRI Center, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan-R.O.C.
Background: Carotid arterial stenting andendarterectomy are both common carotid interventions in managing patients with critical or symptomatic stenosis. Among various complications of carotid intervention, hyperperfusion syndrome is rare but the most devastating one leading to poor outcome. It is important to predict and to discover hyperperfusion early before and after such interventional procedures. The aim of this study is to investigate whether cerebral vasoreactivity (CVR) is a useful parameter to identify patients at risk for hyperperfusion after receiving carotid intervention. We also compare the influences on CVR in patients with either unilateral or bilateral carotid stenosis.
Method: Six patients with carotid stenosis were enrolled and divided into two groups: one is unilateral ICA tight stenosis (stenosis>= 70%), and the other is bilateral ICA tight stenosis (including ICA occlusion). Before intervention, baseline CVRs of patients were evaluated under breath-holding condition by functional MRI. Evaluations of cerebral perfusion were done by perfusion MRI before and 3–5 days after carotid intervention. Cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), and time to peak (TTP) were generated following deconvolution of an arterial input function as parameters. Clinical hyperperfusion was defined as patients with one of following symptoms or signs after procedures: dizziness without hypotension, headache, seizures, and new onset cerebral hemorrhage.
Result: In unilateral ICA tight stenosis group, patients with reduced CVR (CVR<=10%) showed remarkable changes on post-procedural MR perfusion with increased CBF and decreased TTP. Clinical hyperperfusion syndrome was identified in one patient, who had reduced CVR. In bilateral ICA tight stenosis group, CVR was reduced over the hemisphere ipsilateral to the artery with higher grade of stenosis. The CVR did not show correlation with the MR perfusion change after carotid intervention.
Conclusion: Functional MRI is a useful and reproducible tool in evaluating CVR. Perfusion MRI is sensitive in detecting changes of cerebral perfusion. In patients with unilateral tight stenosis of carotid artery, CVR may help to identify subjects at risk for hyperfusion after carotid intervention.
CPP, BLOOD GLUCOSE, AND BRAIN EXTRACELLULAR GLUTAMATE AND GLUCOSE IN SEVERE TRAUMATIV BRAIN INJURY
Yasuhiro Kuroda1, Susumu Yamashita1, Kazumi Kumagai1, Yuko Abe1, Ken-Ya Kawakita1, Shiro Yamashita1, Takehiro Nakamura1, Nobuyuki Kawai2, Nobuhiro Maekawa1, Seigo Nagao2
1Department of Emergency and Critical Care Medicine, Kagawa University Hospital, Miki, Kagawa, Japan, 2Department of Neurological Surgery, University of Kagawa, Faculty of Medicine, Miki, Kagawa, Japan
Objective; Using brain microdialysis in severe traumatic brain injury, we investigated the time course of brain extracellular glutamate and glucose concentrations to find the relationship between Microdialysate (MD) concentrations and physiologic variables (cerebral perfusion pressure: CPP, blood glucose).
Material/Method; The study was approved by the ethic committee of Kagawa University Hospital and informed consent was obtained by the relatives. Six patients (21-69 years, 5 male. 1 female) with severe traumatic brain injury were enrolled. Neuromonitoring was performed by the consists of cerebral microdialysis (CMA60) and intracranial pressure (ICP) measurements (Codman). These probes were placed in a penumbra area (in a case with craniotomy) or right frontal lobe (in a case with burr holing) as confirmed by following-up cranial computed tomography. MD concentrations of glutamate and glucose, and value of ICP were measured every one hours.
Results; MD glutamate concentration was maximal (47 uM) immediately after probe insertion and then decreased to under 10 uM, 24 hours after brain injury in five cases with preserved CPP. MD glucose concentrations (normal value: 1.7 mM) were variable with blood glucose level (70 ? 160 mg/dl) in these patients. In one patient with no CPP, MD glutamate was sustained to increase over 300 uM and MD glucose decreased to zero mM irrespective with the value of blood glucose level.
Discussion; Initial increase of MD glutamate may reflect the degree of primary brain damage if CPP was preserved. MD glucose may change due to the blood glucose if CPP was preserved. Decrease of MD glucose in bad outcome patient could be related with the hypoxic/ischemic episode (lack of delivery of glucose and/or anaerobic glycolysis).
ACUTE SYSTEMIC ERYTHROPOIETIN THERAPY REDUCES CEREBRAL VASOSPASM AND DELAYED ISCHEMIC DEFICITS AFTER ANEURYSMAL SUBARACHNOID HEMORRHAGE: A DOUBLE-BLIND, RANDOMIZED-CONTROLLED TRIAL
Ming-Yuan Tseng, Hugh Richards, Marek Czosnyka, Peter J. Hutchinson, John D. Pickard, Peter J. Kirkpatrick
Department of Neurosurgery, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK
Background: In vivo studies have demonstrated neuroprotective properties of erythropoietin (EPO), similar to ischemic preconditioning. Furthermore, acute therapy with recombinant human EPO appears to be both safe and potentially beneficial after ischemic stroke. In this phase-II randomized controlled trial, we wished to investigate whether high-dose EPO would protect against vasospasm-related delayed ischemic deficits (DIDs) after aneurysmal subarachnoid hemorrhage (aSAH).
Methods: Eighty aSAH patients (age mean 58.6, range 24-82 years) were randomized to receive iv EPO (30,000 IU Epoetin-beta, NeoRecormon, Roche, Welwyn Garden City, UK) or 0.9% saline on days 0, 2, 4 following admission starting within 72 hours of the bleed. Primary endpoints were the incidence, severity, and duration of cerebral vasospasm, and the duration of impaired autoregulation estimated from transcranial Doppler. Secondary endpoints were the incidence of vasospasm-related DIDs, and the modified Rankin scale (MRS) and National Institutes of Health Stroke Scale (NIHSS) scores at discharge.
Results: No trial-related complications were observed. Pre- and post-randomization characteristics were well balanced between the trial arms. The incidence and duration of vasospasm were significantly reduced in the EPO arm from 65% to 40% (p=0.025, Figure 1) and from 4.0 to 1.6 days (p=0.007) respectively. Impaired autoregulation was shortened from 6.6 to 3.5 days (p<0.001) for ipsilateral recordings, and vasospasm-related DIDs were reduced from 40 to 7.5% (p=0.001, Figure 2) in the EPO arm. Patients treated with EPO also demonstrated a more favorable MRS (MRS 1& 2, 40.0 vs. 67.5%, p=0.014) and lower NIHSS scores (11.7 vs. 5.1, p=0.005).
Conclusions: Acute systemic treatment with high-dose EPO appears to be safe and reduces the physiological and clinical manifestation of cerebral vasospasm after aSAH. Long-term follow-up is required to confirm that the EPO therapy improves early outcome.
NEWLY DEVELOPED FOCAL CEREBRAL ISCHEMIC AND REPERFUSION MODEL USING ENDOVASCULAR TECHNIQUE IN PRIMATES
Mitsuhito Mase1, Fumiko Ono2, Naoyuki Saito2, Yuko Katakai2, Keiji Terao3, Yasuhiro Yoshikawa4, Kazuo Yamada1
1Department of Neurosurgery and Restorative Neuroscience, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, 2The Corporation for Production and Research of Laboratory Primates, Tsukuba, Japan, 3Tsukuba Primate Center for Medical Science, National Institute Biomedical Innovation, Tsukuba, Japan, 4Department of Biomedical Science, Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo Tokyo, Japan
Background and Purpose: Control of focal cerebral ischemia and reperfusion injury is very important clinical issue. There has been many studies using rodent models, but frequently, the results could not be directly applied to human due to difference of species. The purpose of this study is to establish an easy and reproducible focal cerebral ischemia and reperfusion model in primate with following condition; 1) less-invasive procedures, 2) estimation of the severity and extent of ischemia using MRI during and after the ischemia, 3) long term survival, 4) monitoring higher cognitive function.
Materials and methods: Macaca fascicularis (n=9, BW: 3-5kg) were used. Under general anesthesia, an infusion catheter (4Fr) was introduced into the right internal carotid artery through the right femoral artery. A micro-catheter (0.021inch) was inserted into the right meddle cerebral artery (MCA) using micro-guide wire (0.010inch). The tip of the micro-catheter was at the MCA bifurcation to occlude the MCA completely. After the three-hour ischemia, a reperfusion was made by a withdrawal of the micro-catheter. Serial MR images (T1, T2, diffusion, and perfusion weighted images) were obtained using 3.0T MRI (Allegra, SIEMENS) during and after the ischemia. Regional cerebral blood flow (CBF) and volume (CBV) were calculated from the perfusion data. Neurological examinations (motor, sensory) were monitored daily, and performance in a food retrieval task (1) were assessed twice a week for 5 weeks after the ischemia. A food retrieval task is a test to estimate the capacity of spatial memory. Each animal has already been trained to complete the task for two months before the stroke.
Results: At first, preliminary experiments were performed using 5 animals to decide the endovascular occlusion method, the duration of ischemia, anesthetic methods, and suitable acquisition parameters of MR images. Afterwards, MCA occlusion and reperfusion model was made using 4 animals, which could survive until sacrifice at 5 weeks. The severity and extent of ischemia can be visualized using diffusion and perfusion weighted MR images during ischemia. Ischemic core was seen in the basal ganglia, which was extended to the frontal and temporal cortices to various extents. Severe brain swelling (edema) was shown in the right hemisphere at day 3 in all cases. At that time CBV and CBF also significantly increased, which were normalized until two weeks. All animals had left mild hemiparesis, improving quickly. Performance in a food retrieval task was severely disturbed until 2 or 3 weeks after the stroke, which was not correlated to the severity of hemiparesis, and gradually improved. Histological examination confirmed that permanent infraction existed mainly in the caudate nucleus, putamen, and temporal cortex.
Conclusion: We have developed MCA occlusion and reperfusion model in primates. This model is made by less-invasive endovascular techniques, and can allow long-term survival with estimating higher cortical function. Difference of severity and extent of ischemia of each animal can be shown on MRI. This model could be very useful to evaluate new therapeutic drugs for human cerebral ischemia.
EXACERBATED EDEMA, INFLAMMATION AND BRAIN DAMAGE IN TYPE-2 DIABETIC MICE SUBJECTED TO FOCAL ISCHEMIA
Raghu Vemuganti1, Kellie K. Bowen1, Jin Liang1, Douglas L. Feinstein2
1Department of Neurological Surgery, University of Wisconsin, Madison, WI, USA, 2Department of Anesthesiology, University of Illinois-Chicago, Chicago, IL, USA
One of the limiting factors in stroke therapeutic development is the use of healthy rodents to understand the mechanisms of ischemic neuronal death. In humans, diabetes increases the risk of stroke incidence as well as stroke-induced mortality. The db/db mouse (C57BLKS/J-m+/+Leprdb/db) is a model of type-2 diabetes with 4 times higher blood sugar than its normoglycemic genetic control (db/+ mouse). To understand the mechanisms that render diabetics to increased brain damage, we evaluated the effects of transient middle cerebral artery occlusion (MCAO) in adult db/db mice. Following a 2h transient MCAO, the mortality rate within the first day was 7-fold higher in the db/db mice over db/+ mice (n = 11/group; p<0.05). Following a 45 min transient MCAO and 3 days of reperfusion, there was a significant increase in the size of infarct (by 52±8%; p<0.05), cerebral edema (by 52±8%; p<0.05) and neurological dysfunction in db/db over db/+ mice (n = 9/group). The db/db mice also showed significantly higher post-ischemic inflammatory markers (ICAM1+ capillaries, extravasated macrophages/neutrophils and expression of inflammatory genes IL-1β, IL-6, ICAM1, MCP1 and P-selectin) compared to db/+ mice. When db/db mice were fed with a chow enriched with rosiglitazone (a PPARγ agonist known to control blood glucose levels and inflammation in diabetics), transient MCAO-induced infarction, edema, neurological dysfunction and inflammatory gene expression decreased significantly indicating the potential of PPARγ as a future stroke therapeutic target in diabetics. Funded in part by US NIH and AHA.
THE USE OF CEREBROLYSIN IN THERAPY OF STROKE-INDUCED HERPETIC INFECTION
Andrew E. Kulchikov1, Alexander N. Makarenko2, Irina G. Vasileva3
1Orlov Regional Clinical Hospital, Orel, Russia, 2Orlov State University, Orel, Russia, 3Institute of Pharmacology and Toxicology of AMS of Ukraine, Kiev, Ukraine
Background and aims: the study of possibility of reactivation of latent herpetic infection in patients with acute ischemic stroke (AIS) and its correction by Cerebrolysin (“Ebewe”, Austria).
Methods: the patients of both sexes in the age from 55-85 years with primary AIS, in which the DNA of herpes simplex virus (HSV) was marked by means of polymerase chain reaction (PCR), were included in the study. These patients were divided in 2 groups: basic (receiving standard therapy and Cerebrolysin) and control (receiving standard therapy). Neurologic deficit was assessed by means of NIH-NINDS, modified Rankin scale and Barthel index. Infectious complications were defined by SIRS. HSV in blood was assessed by PCR method and enzyme multiplied immunoassay (EMIA). The immune status was assessed, CT/MRI of brain and laboratory tests were made.
Results: PCR on HSV in blood was positive in 98 (27,7%) from 353 patients with acute ischemic stroke. Exactly these 98 patients were included in the study and divided in 2 groups: basic and control. Cerebrolysin was injected in the dose of 10 ml intravenously during 10 days, in the first 12 hours from the onset of the disease. To the 10th day of AIS in the basic group the accelerated restoration of neurologic dysfunctions was observed (according to NIH-NINDS scale, modified Rankin scale and Barthel index), the reduction of infectious syndrome level by SIRS scale, the reduction of quantity of positive PCR and EMIA on HSV, normalization of the disturbed indexes of immune system (p<0,01) were marked.
Conclusions: in AIS in patients in terms of immunodepression the reactivation of latent herpetic infection is marked in 27,7 %, Cerebrolysin is the effective drug in therapy of this complication of stroke due to neuroimmunoresolving action.
A PROTECTIVE INVOLVEMENT OF BIP, AN ER-RESIDENT PROTEIN, ON NEURONAL DEATH INDUCED BY TRANSIENT FOREBRAIN ISCHEMIA IN GERBIL
Yasuhisa Oida1, Masamitsu Shimazawa1, Atsushi Oyagi1, Takashi Kudo2, Kazunori Imaizumi3, Hideaki Hara1
1Department of Biofunctional Molecules, Gifu Pharmaceutical University, Gifu, Japan, 2Department of Clinical Neuroscience, Psychiatry, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan, 3Division of Molecular and Cellular Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki, Japan
Background and aims: Endoplasmic reticulum (ER)-stress is caused by the accumulation of unfolded proteins in the ER lumen, and is associated with stroke and neurodegenerative diseases such as Parkinson's and Alzheimer diseases. The purpose in the present study was to investigate the involvement of ER-stress in the execution of cell death in transient forebrain ischemia model of gerbil. In addition, we evaluated the effects of a selective inducer of BiP to examine whether induction of BiP inhibits the neuronal cell death induced by the ischemia.
Methods: We assessed the expression patterns of immunoglobulin heavy chain binding protein (BiP)/glucose-regulated protein (GRP) 78, an ER-resident molecular chaperone whose expression serves as a good marker of ER-stress, activating transcription factor (ATF)-4, and C/EBP homology protein (CHOP) by immunohistochemistry and/or Western blotting after transient forebrain ischemia in gerbils. Double-fluorescent staining of CHOP immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method were performed to clarify the involvement of CHOP in cell death.
Results: Immunohistochemical and Western blot analyses of the CA1 showed that BiP expression increased at 12 hr, reached a peak at 3 days, and gradually decreased thereafter as compared with that of control group. A transient increase was shown in CA3 at 1 day after ischemia, but unchanged in dentate gyrus and cortex. Furthermore, the signal for ATF-4 and CHOP was increased at 1 day and 3 days in CA1, and 12 hr in CA3. Co-localization of CHOP immunoreactivity and DNA fragmentation were detected by the TUNEL method 3 days after ischemia in CA1, but not in CA3. Moreover, we evaluated the effect of a selective inducer of BiP (BiP inducer X; BIX) against global transient forebrain ischemia. BIX significantly induced the expression of BiP mRNA in in vivo. Furthermore, BIX protected the cell death and the increase of TUNEL-positive cells induced by the ischemia.
Conclusions: These findings indicate that ER stress may play a pivotal role in the post-ischemic neuronal death in the hippocampal CA1 subfield and that the induction of BiP could prevent the neuronal damage.
SELECTIVE OCCLUSION OF THE MIDDLE CEREBRAL ARTERY IN MICE BY AN INTRALUMINAL APPROACH
Jianya Ma, Thaddeus S. Nowak Jr.
Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, USA
Background and Aims: A significant limitation in experimental stroke research is the heterogeneity of models employed, which complicates comparison of results among laboratories. This is a particular concern with intraluminal filament methods, both because filament design and fabrication vary and because the internal carotid artery (ICA) obstruction and diffuse ischemia they produce limit their application to short occlusion intervals, often in strains with appreciable collateral perfusion. Technical issues and strain variation concerns are amplified in mice. This report describes implementation of an occluding device for mice consisting of a silicone cylinder molded onto nylon filament that can traverse the ICA and enter the middle cerebral artery (MCA) to produce selective, reversible occlusion, extending recently published methodology for rats (1).
Methods: Silicone cylinders of either 150 or 200 micron diameter and averaging 500 microns in length were molded onto 8–0 nylon filaments 10 mm in length. These were introduced into the cranial circulations of halothane-anesthetized adult (22-25 g) male mice of BALB/c, C57BL/6 and DBA/2J strains via the left external carotid artery as routinely described for intraluminal occlusions. Filaments were heparinized and mice received 100 units/kg subcutaneous heparin to reduce risk of secondary occlusion due to clot formation along the filament. After 24 hours brains were removed to assess filament position and circle of Willis anatomy, and to measure infarct volumes after triphenyltetrazolium staining.
Results: Successful MCA entry was achieved in 70% (7/10) of BALB/c and 81% (47/58) of C57BL/6 using 200 micron devices, and 88% (7/8) of DBA/2J after diameter reduction to 150 microns. Failures resulted from acute branch angles at the origin of the MCA. Cortical infarct volumes were 86 +/− 14 and 39 +/− 4 cubic mm (mean +/− SD) in BALB/c and C57BL/6 mice, respectively, with no infarcts seen in DBA/2J mice, in agreement with previous data on relative vulnerability to focal ischemia in these strains (2),(3). No mortality was observed.
Conclusions: These results demonstrate the application of a refined intraluminal occlusion method in mice, using filaments manufactured to close tolerances. Dimensions can be modified to accommodate variations in vascular caliber among strains. Selective occlusion of the MCA permits the study of long durations of focal ischemia while maintaining the advantages of the widely used intraluminal approach.
Supported by USPHS grant NS42267 (TSN)
EARLY CHANGES OF GENE EXPRESSION IN THE PERIFOCAL BRAIN TISSUE AFTER TRAUMATIC INJURY
Marc Schuerings1, Markus Haak2, Li Li2, Felix Engel3, Norbert Gretz2, Lothar Schilling1
1Division of Neurosurgical Research, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany, 2Medical Research Center, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany, 3Department of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany
Background and aims
A focal brain lesion often goes along with a disruption of blood vessels and/or damage of the blood-brain barrier (BBB) leading to extravasation of plasma constituents. This vasogenic edema spreading into the surrounding tissue may contribute to the delayed growth of the tissue damage. However, the mechanism involved are still unclear. Therefore, we have studied in a model of cortical traumatic brain injury the alterations of gene expression in the perilesional edematous brain tissue.
Methods
A cortical brain lesion of moderate size was produced by applying a cold probe (diameter, 4mm, −68°C, 1 min) onto the exposed dura over the right parietal cortex of male Sprague-Dawley rats. The extent of cell death was followed by intravenous (iv) injection of propidium iodide (PI; 0.5%, 1 ml/100g bw before and 6h after lesion) and the spread of edema by iv injection of fluorescein (5%, 1 ml/100g bw 2h before sacrifice). In-situ perfusion was performed 12, 24, 48, or 72 h after trauma to remove the blood. From the isolated brain serial coronal cryosections (thickness, 300 µm) were obtained which were used for microdissection of the viable perifocal tissue (PI negative, fluorescein positive). Total RNA was extracted, subjected to reverse transcription and preparation of cRNA using biotinylated nucleotides (cytosine and uracil) for gene chip analysis (Affymetrix RAE-230A containing 13115 genes).
Results
To determine the significance of alterations of gene expression a cutoff value of 10E-5.92829 was chosen (p<0.1 with Bonferroni adjustment for multiple testing). The numbers of genes significantly regulated were 636 (12h), 598 (24h), 711 (48h) and 3009 (72h). The dendrogram analysis revealed the largest degree of similarity between 12 and 24 h after trauma while the 72h tissue differed most from all time points. Pathway analyses based on pathways taken from the Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) were performed using Fisher's exact test or permutation analysis methodology. The analyses of KEGG pathways showed similar results with both statistical methods with significant alterations (p<0.05) in the prion disease-, hematopoetic cell lineage-, biosynthesis of steroids-pathway at the early time points and metabolic pathways mainly at the latest time. Based on GO pathways immunological terms were significantly altered during the entire observation period, while apoptosis- and cell cycle-associated terms were altered up to 48h after trauma (Fisher's exact test, p<0.05). Permutation analysis confirmed the upregulation of immunological GO terms and also showed marked alterations of neuron-specific terms over all 4 time points.
Conclusion
A moderate cortical trauma leads to marked alterations of gene expression in the perifocal viable tissue which were initially related to cell cycling as well as immunological and apoptotic pathways the latter suggesting activation of local inflammatory response. The down-regulation of neuron-specific genes and the activation of metabolic pathways may reflect self protection of the endangered cells to limit the spread of secondary brain damage. The early upregulation of cell cycling genes and delayed stimulation of cell metabolism-related genes probably indicate early processes of tissue reconstitution, such as glial scaring.
DIFFERENTIAL REGULATION OF ANGIOTENSIN RECEPTOR EXPRESSION AFTER EXPERIMENTAL BRAIN TRAUMA
Serge Thal1, Sebastian Wyschkon1, Raimund Trabold2, Nikolaus Plesnila2, Kristin Engelhard1, Christian Werner1
1Department of Anaesthesiology, Johannes Gutenberg University, Mainz, Germany, 2Laboratory of Experimental Neurosurgery, Department of Neurosurgery and Institute for Surgical Research, University of Munich Medical Center, Grosshadern, Munich, Germany
*Introduction: *Several studies have investigated the influence of the renin-angiotensin system on secondary brain damage after cerebral ischemia. Angiotensin II receptor type 1 (AT1) seems to increase cerebral damage while angiotensin II receptor type 2 (AT2) may offer protection, preventing secondary brain damage. No studies have investigated the modulation of AT1 and AT2 after brain trauma. We, therefore, measured the expression of AT1 and AT2 after brain trauma in mice to evaluate the potential time window of a therapeutic intervention.
*Methods:* Male C57Bl/6 mice were anaesthetized with isoflurane (0.8 %) and a pneumatic brain trauma was induced on the right parietal cortex (controlled cortical impact (CCI), 8 m/s, 1 mm depth). Brain tissue was removed 15 min, 3, 6, 12 and 24 hr (n=6 each) after CCI and from naive animals (n=7). The absolute amount of AT1 and AT2 mRNA was determined by real-time RT-PCR (Lightcycler^R) in the traumatised and the contralateral hemisphere. The results were normalized against beta-2-microglobin as control gene. Statistics: ANOVA on RANKS, p<0.05.
*Results:* The expression of AT1 was significantly reduced at the site of contusion (15 min: −10 %, 3 hr: −10 %, 6 hr: −20%, 12 hr: −30 %*, 24
hr: −60 %* vs. naive, p<0.05). In the contralateral, non-traumatised tissue the AT1 expression was reduced 15 min after trauma, returning to baseline 3-12 hr after trauma, and finally dropping again at 24 hr (15 min: −20 %, 3 hr: 0 %, 6 hr: 0 %, 12 hr: −10 %, 24 hr: −20 % vs. naive).
The expression of AT2 in the contusion area was increased in comparison to healthy animals 15 min until 12 hr after CCI (+150 to 200 %), finally dropping to −20 % vs. naive 24 hr after CCI. In the contralateral hemisphere the AT2 expression was also increased by 200 %, dropping to 150 % 24 hr after CCI.
*Conclusion:* Brain trauma results in an immediate up-regulation of AT2 and down-regulation of AT1 in the injured hemisphere. The AT1/2 expression profile is similar to data in brain ischemia. The beneficial regulation of AT1 and AT2 in injured tissue might represent a cellular rescue mechanism. This suggests that a therapeutic intervention at AT2 within a window of opportunity (3-12 hr) after CCI would encounter increased expression of AT2 with a potential reduction of secondary brain damage.
REGULATION OF CALCIUM IN CEREBROVASCULAR ENDOTHELIUM BY TRANSIENT RECEPTOR POTENTIAL (TRP) CHANNELS
Sean Marrelli1,2, R. Bryan1,2
1Department of Anesthesiology, Baylor College of Medicine, Houston, TX, USA, 2Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA
Background and Aims
We recently demonstrated that endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations of cerebral arteries could be blocked by inhibitors of phospholipase A2 (PLA2). The present studies extend this finding by elucidating the mechanism by which PLA2 inhibition blocks dilation. We hypothesized that PLA2 activation critically regulates endothelial cell (EC) Ca2+ influx via TRPV4 channels in the initiation of EDHF-dependent dilations.
Methods
Male rat middle cerebral arteries (MCA) were mounted in an isolated vessel chamber. Arteries were cannulated and pressurized (85 mm Hg pressure) and luminally perfused (100 ul/min). For measurement of EC intracellular free calcium concentration ([Ca2+]i), the endothelium was selectively loaded with fura 2-AM. Luminal delivery of UTP was used to elicit EDHF-dependent dilations. In some experiments, MCA endothelial cells were enzymatically dissociated and used within 4 hours of isolation. L-NAME and indomethacin were present throughout to block production of NO and prostaglandins, respectively.
Results
Luminal UTP (10-7 to 10-5 M) produced a dose-dependent increase in endothelial Ca2+ and maximal dilation of the artery at 10-5 M UTP. Pre-incubation with PACOCF3 (a PLA2 inhibitor) abolished the endothelial Ca2+ response and the dilation to UTP. Manganese quenching experiments demonstrated Ca2+ influx across the luminal and abluminal face of the endothelium in response to UTP. Interestingly, PLA2-sensitive Ca2+ influx occurred primarily across the abluminal surface. Luminal application of arachidonic acid, the primary product of PLA2 and a demonstrated activator of certain TRPV channels, increased both EC [Ca2+]i and MCA diameter. TRPV4 mRNA and protein was demonstrated specifically in endothelium by RT-PCR and immunofluorescence, respectively. Lastly, application of 4 alpha-phorbol 12,13-didecanoate (4a-PDD; a TRPV4 channel activator) produced an increase in [Ca2+]i of freshly isolated EC that was significantly reduced in the presence of ruthenium red (a TRPV channel blocker).
Conclusions
We conclude that PLA2 is involved in EC Ca2+ regulation through production of arachidonic acid that produces subsequent activation of TRPV4 channels. Furthermore, we present evidence that the PLA2-sensitive component of Ca2+ influx is polarized to the abluminal face of the endothelium.
This work was supported by AHA grants 0230353N (SPM), 0665100Y (SPM), 0270110N (RMB) and NIH grant R01 NS46666 (RMB).
IN VIVO SIMULTANEOUS OBSERVATION OF CEREBRAL MICROVESSELS DURING REPERFUSION AFTER BRAIN ISHCHEMIA IN RATS USING SYNCHROTRON RADIATION
Masanori Morita1, Motohisa Ohkawa1, Syuhei Miyazaki1, Keiji Umetani2, Kouichirou Suzuki1
1Department of Emergency Medicine, Kawasaki Medical School and Kawasaki Medical School Hospital, Kurashiki, Okayama, Japan, 2Japan Synchrotron Radiation Research Institute, SPring-8, Sayo-Gun, Hyogo, Japan
BACKGROUND AND PURPOSE: To clarify cerebral circulation disorders after ischemia and reperfusion, evaluation in vivo by visualization of intracerebral microvessels is appropriate. However, usual method is difficult to examine the blood vessels in the entire cerebrum including the striate arteries distributed in the cerebrum. A new angiography system, which consists of monochromatic synchrotron radiation (MSR) as an X-ray source and a high-resolution camera with a video system as a detector, has been developed in Hyogo, Japan. This angiography system has nearly parallel X-rays, and consequently can visualize microvessels of 20-30 µm in diameter. We aimed at in vivo simultaneous observation of superficial cortical and intracerebral microvessels during reperfusion after transient forebrain ischemia.
METHODS: Transient brain ischemia was induced by the method of ten-minute four-vessel occlusion in rats under general anesthesia. Angiographic images were then sequentially obtained at 3 frames/second. Changes in the cerebral microvessels were observed before and 1, 5, 10, 15, 20, and 30 min after transient cerebral ischemia using the MSR angiography system.
RESULTS: Diameters of ICA, MCA, and striate artery significantly increased 1 minute after reperfusion, while the pial arteriole caliber significantly decreased (80% of baseline). Thereafter, MCA, pial arteriole and striate artery significantly dilated 5 and 10 minutes after reperfusion. Although the ICA caliber of 30-minute reperfusion significantly decreased as compared with the basal value, the other 3 vessels' calibers maintained above basal value throughout the experiment. Early venous filling was observed at 5 and 10 minutes after reperfusion.
CONCLUSION: We herein describe and discuss the use of an in vivo experimental model in which changes in transient cerebral ischemia in the rat cerebral microcirculation were clearly depicted with an MSR angiography system. The MSR angiography system is useful for investigating morphological changes in both cortical and central branches of cerebral vessels in rats during reperfusion after cerebral ischemia. The pathophysiologic basis of early venous filling is incompletely understood. Clear detection of early venous filling in rats with the MRS angiography system could help us clarify it.
ENDOTHELIN RECEPTOR ANTAGONISM BLOCKS CALPONIN PHOSPORYLATION FOLLOWING BRAIN TRAUMA
Christian Kreipke, Randy Morgan, Jose Rafols
Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, Detroit, MI, USA
Background and AIMS
Previous work from our laboratory has shown that rat cerebral cortical microvessels undergo significant ultrastructural alterations including a nearly 40% reduction in the mean cross sectional luminal from 4 to 24 hours after traumatic brain injury (TBI). Such microvascular alterations were temporally correlated with an impaired microcirculation as expressed by a 37% reduction in cerebral cortical blood flow as detected by both laser Doppler flowmetry (LDF) and Susceptibility Weighted Imagining (SWI). In addition the alterations were accompanied by direct ultrastructural observation of enhanced contractility of smooth muscle (SM) of the wall of reacting arterioles which suggests that the increased vascular muscle tone and vasoconstriction of these vessels after TBI are causally related to the dysfunctional microcirculation. However the molecular mechanism underlying the enhanced vasoreactivity after TBI remains unclear. Endothelin-1 (ET-1), acting through its G-protein coupled receptors, A and B, signals downstream cellular and molecular changes in SM ultimately leading to vasoconstriction. Our laboratory has shown upregulation of both ET-1 protein and ET-1 mRNA temporally coinciding with the state of sustained hypoperfusion after TBI. A potential target of ET-1 is the contractile protein, calponin (Cp), which, upon phosphorylation, disinhibits acto-myosin cross bridging leading to enhanced SM contractility. We have reported that following TBI Cp both migrates from a central to a more peripheral region of SM cytosol immediately subjacent to the cell membrane. This Cp migration coincides temporally both with its sustained phosphorylation and the enhanced vasoconstriction. While the link between ET-1 and Cp has only been speculative, we have attempted, here, to provide direct evidence for a causal relationship between activation of ET receptors and Cp phosphorylation.
Methods
Using Marmarou's model of diffuse brain injury, adult male rats (250-450 g) were subjected to TBI. Directly following injury, we injected via intraventricular cannulae, the ET receptor A (ETRA) antagonist, BQ123. At 4, 24, and 48 hours post TBI tissue was collected from the dorsal hippocampus (hipp) and sensorimotor cortex (smCx), areas known to be important for cognitive and motoric behaviors and subjected to either double immunofluorescence microscopy for alpha-smooth muscle actin (SMA), a marker for SM, and Cp, or for two-dimensional nonequilibrium pH gradient gel electrophoresis (NEPHGE)/SDS-PAGE for detection of Cp phosphorylation.
Results
ETRA blockade resulted in a decrease in Cp phosphorylation and prevented its recompartmentalization along the SM wall.
Conclusions
These results indicate a direct association between the previously observed alterations in Cp following TBI and activation of ETRA. These findings may provide a rationale for targeted therapy to improve the defective microcirculation following TBI.
INCREASE IN PO2 ON THE RAT BRAIN SURFACE BY PHTOACTIVATION
Satoshi Kimura, Katuyoshi Mineura
Department of Neurosurgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
Introduction: The phenomenon that photostimulation induced vascular relaxation was reported previously (Ref. 1). In preliminary experiments, we found that it induced a rapid arteriolar vasodilation on rat brain surface (Fig.1). We developed the novel technique that we could measure the PO2 on the brain surface, using the oxygen-sensitive fluorescent membrane (Brain 05). We modified this technique to measure the PO2 on the brain and to photostimulate it simultaneously. The aim of this study was to investigate that the effect of photostimulation influences the oxygenation of rat brain.
Methods: An oxygen-quenching fluorescence dye, tris (1,10-phenanthroline) ruthenium (II) chloride hexahydrate, was embedded in a highly gas-permeable, opaque silicone-rubber film formed on a microscope coverslip. In this study, we modified the membrane to allow the one-tenth excitation light to reach brain surface. This modification enabled us to evaluate the PO2 on the brain surface and to photostimulate it simultaneously. Under anesthesia, the rat brain was subjected to 1 minute photostimulation after 1 minute shading. The trends of the PO2 on the brain were measured and analysed.
Results: We obtained 13% elevation of PO2 after stimulation, but then the PO2 of control period was unchanged. The difference of oxygenation was statistically significant.
Conclusion: We detected the increasing oxygen tension of the brain surface by photoactivation.
EXTRAORDINARY HIGH FLOW CAPILLARIES IN THE CEREBRAL MICROVASCULAR NETWORK
Miyuki Unekawa1, Minoru Tomita1, Yutaka Tomita2, Takashi Osada1, Haruki Toriumi1, Norihiro Suzuki1
1Department of Neurology, Keio University School of Medicine, Tokyo, Japan, 2Department of Neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Gunma, Japan
Capillary RBC velocity in the brain is temporally and spatially heterogeneous. Recent advances in technology with a high-speed laser-scanning confocal fluorescence microscope and Matlab domain home-made software (KEIO-IS2) developed by us have made it possible to examine the vast number of RBC appearing in the cortical microvasculature quantitatively. In this paper, we attempted to examine the frequency distribution function of RBC velocities in intraparenchymal tissue capillaries of the rat cerebral cortex. The method has been reported elsewhere (Tomita at al., JCBFM 25: S164, 2005). Briefly, we used 37 urethane-anesthetized Wistar rats with a closed cranial window in the temporo-parietal region. After FITC-labeled RBCs were injected into the circulating blood, the labeled RBCs observed in the ROI at 50 micrometer in depth below the brain surface were tracked at 500 frames/s and their velocities calculated with KEIO-IS2. Capillaries were identified one by one on FITC-dextran stained small vessels with a criteria of a diameter less than 10 micrometer. We obtained 4252 RBC velocities of single capillaries from approximately 100 samples each in 37 rats. The velocity ranged from 0.15 to 9.40 mm/s with a mean of 2.05±1.59 mm/s. The RBC number (N) was plotted in units 0.5 mm/s apart (Fig. 1). As shown in the frequency distribution function, capillary velocities were clustered at around 2.0 mm/s, smearing at higher velocities. Although RBC velocity increases were continuous, we tested the outlying value X statistically based on (x(mean)?X)/s, where s the standard deviation (Grubbs FE, Ann Math Stat 21: 27, 1950). We found that the capillaries having RBC velocity higher than 6.5 mm/s can be excluded statistically significantly (p<0.01). They must belong to vessels in a different category. The amount of oxygen delivered by arterial blood (D) is estimated by c×n×f, where c is the arterial concentration, n the capillary number (N in Fig.1), and f the flow linearly related to the velocity. Oxygen extracted at the place capillaries enter into the tissue is expressed as E = 1 ? e(exp)-B/f, where B is a constant related to the capillary surface area and length. The equation implies that gas in fast flowing blood is less extracted, with little gas exchange in the tissue and a large amount of gas reaching the vein. When E was subtracted from D, the amount of shunting blood can be obtained. Broad calculation of the shunting blood over the total arterial blood yielded 22% of the blood appearing in the vein without gas exchange. This might result from what have been called thoroughfare channels or non-nutritional capillaries carrying 22 % of the blood in reserve.
INTERINDIVIDUAL VARIATION OF BASELINE CBF: RELATION WITH CEREBRAL VASCULAR TONE ESTIMATED BY PET
Hiroshi Ito1, Iwao Kanno1, Masanobu Ibaraki2, Kazuhiro Takahashi1, Tetsuya Suhara1, Shuichi Miura2
1Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan, 2Department of Radiology and Nuclear Medicine, Akita Research Institute of Brain and Blood Vessels, Akita, Japan
Introduction: Inter-individual variations in normal human CBF at rest condition have been reported. Inter-individual variation of cerebral vascular tone it considered to contribute to this, and several determinants of cerebral vascular tone have been proposed. In the present study, the relation of inter-individual variation between CBF and cerebral vascular tone at rest condition was investigated by PET.
Methods: CBF and CBV at rest condition were measured by PET with H215O and 11CO, respectively, in 20 normal subjects. CBF at rest condition and cerebral vascular response to changes in PaCO2, which was calculated as the percentage changes in CBF per absolute change in PaCO2 (%/mm Hg), were measured by PET with H215O in another 20 normal subjects. Regions-of-interest were defined for the neocortical regions.
Results: The relation between CBF and CBV at rest condition was CBV = 1.00CBF^0.30. A significant negative correlation between baseline CBF and the vascular response to hypocapnia was observed (P<0.05). A tendency of negative correlation, but not significant, between baseline CBF and the vascular response to hypercapnia was also observed.
Conclusions: Because CBV is an indicator of cerebral vessel diameter if vessel length does not change, the relation between CBF and CBV, which is in good agreement with Poiseuille's law, indicates that inter-individual differences in CBF are related to those in cerebral vessel diameter corresponding to cerebral vascular tone. The correlations between baseline CBF and vascular responses support the assumption that cerebral vascular tone might incline toward vasoconstriction and vasodilatation when CBF is low and high between individuals, respectively. Although several determinants of cerebral vascular tone have been proposed, the mechanism of such inter-individual differences in cerebral vascular tone is unknown.
DILATION OF THE MIDDLE CEREBRAL ARTERY OF THE RAT DURING ACUTE HYPONATREMIA IS ENDOTHELIUM-DEPENDENT
Katarzyna Romaniuk, Ewa Kozniewska
Department of Neurosurgery, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland
BACKGROUND AND AIMS. Hyponatremia is defined as an electrolyte disorder occurring when serum sodium level falls below 135 mmol/l. It is frequently observed after brain trauma or subarachnoid bleeding and is typically associated with increased plasma level of vasopressin (AVP). Although hyponatremia has been reported to impair the recovery of neurosurgical patients its effect on brain vasculature is practically unknown. Therefore, present study was carried out to determine the effect of low sodium and increased AVP concentration in the extracellular fluid on the calibre of the middle cerebral artery (MCA) of the rat.
METHODS. MCAs (n=38) harvested from the brains of the adult, male Wistar rats were used for this study. The vessels were mounted in the small organ chamber filled with 3-(N-morpholino) propanesulfonic acid (MOPS) ? buffered saline solution and pressurized. The chamber was placed on an inverted microscope equipped with videocamera. Changes in the diameter of MCA were observed on the monitor and analyzed with a help of a customer made software.
RESULTS. Decrease of the extracellular concentration of Na+ from 140 to 120 mM resulted in the dilation of MCA by 17% (from 143±10 to 167±10 micrometers, p<0.01). Dilation was abolished when endothelial cells were mechanically removed or NO synthesis was inhibited. Addition of 15 pg/ml of AVP to the extraluminal fluid during normonatremia prior to the administration of low sodium buffer did not affect the diameter of MCA. However, when low sodium buffer was given together with AVP, constriction of MCA by about 16% (p<0.02) instead of dilation was observed.
CONCLUSIONS. These results demonstrate for the first time that during acute hyponatremia healthy cerebral blood vessels dilate through endothelium- and NO-dependent mechanism. However, when endothelial control is abolished or impaired as following head trauma or subarachnoid hemorrhage, hyponatremia may result in vasoconstriction, particularly when associated with elevated plasma vasopressin levels. This finding may explain adverse vascular effects of AVP-associated hyponatremia observed in vivo.
THE HEMODYNAMIC RESPONSE DURING FUNCTIONAL ACTIVATION REFLECTS COUPLING RATHER THAN UNCOUPLING
Olaf B. Paulson1,2, Egill Rostrup2
1Neurobiology Research Unit, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark, 2MR Department, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark
Cerebral blood flow and cerebral metabolism is normally coupled, i.e., an increase in metabolic demand in the tissue and will lead to an increase of flow. However, during functional activation flow and glucose metabolism remain coupled as they increase proportionally, whereas oxygen metabolism only increases to a minor degree ? the so-called uncoupling of flow and oxidative metabolism. This physiological phenomenon forms the basis for studies of cerebral activation using PET or fMRI. Several studies have dealt with these aspects and theories have been forwarded regarding the mechanisms underlying the changes. Special attention and speculations and have been directed towards a potentially deficient oxygen supply to the tissue most distant from the capillaries.
In the present presentation we forward the theory that the essential mechanism responsible for the flow increase during functional activation is a tight coupling between flow and glucose metabolism. The uncoupling of flow and oxidative metabolism then remains a simple consequence of a less pronounced increase in oxygen consumption. Based in the literature on the cerebral microcirculation it is emphasized that physiological capillary heterogeneity and physiological recruitment of capillaries is mandatory in order to accomplish the increased demand for glucose supply to the cerebral tissue. These basic aspects of the cerebral microcirculation will result in an increased physiological density of capillaries in the cerebral tissue and thereby also to surplus in oxygen supply. The basic literature underlying these concepts will be discussed and related to the theories advanced in the field.
ESTIMATION OF OEF USING SPECT AND PERFUSION CT
Yoshikatsu Kawata
Department of Neurosurgery, Rumoi City Hospital, Rumoi, Hokkaido, Japan
[Objectives]The oxygen extraction fraction (OEF) is a very useful indicator of cerebral circulatory insufficiency. At present, PET is the only method to measure OEF, and the measurement of OEF is not available in general institutions without PET facilities. Therefore, we attempted to estimate OEF values from CBF and mean transit time (MTT) measured by SPECT and perfusion CT, which are available in general institutions. [Subjects and methods] We examined 444 areas (222 pairs), for which CBF and MTT were measured by SPECT and Perfusion CT at our institution, and 132 areas (66 pairs), for which CMRO2, OEF, CBF, and MTT values were available in the literature. The areas without cerebral infarctions on brain CT, and those whose cerebral circulatory dynamics were impaired unilaterally were subjected to the measurement at our institution. The results were then evaluated by a ratio of the measurements to those on the unaffected side. [Theoretical background]Assuming that CMRO2 values are normal (i.e. CMRO2 ratio = 1, ratio: affected side/unaffected side), then the OEF ratio equals the (1/CBF) ratio. Therefore, the theoretical OEF ratio can be estimated. However, when CMRO2 values are not normal, the OEF ratio cannot be estimated without further information. In this study, we determined the relationship among the CBF values, the MTT values, and the theoretical OEF values in the areas that are supposed to have normal CMRO2 values measured at our institution, and examined the deviation of the measured CBF and OEF values in the areas with abnormal CMRO2 values described in the literature from the above relationship. We considered that, if some relationship can be found between them, the OEF ratio may be estimated without measuring OEF. [Results] In the study of subjects measured at our institution (n=222 pairs), the CBF ratio showed a tendency to decrease linearly as the MTT ratio increased. The relationship can be expressed as the following formula: CBF ratio = −0.321(MTT ratio)+1.267 (R^2 = 0831, n = 222)(Formula 1). Assuming that the CMRO2 values are normal, then the OEF ratio = (MTT ratio)/{1.267(MTT ratio)-0.321} (Formula 2). The relationship between the CBF ratio and the OEF ratio was examined including the areas with abnormal CMRO2 in the literature. The ratio of the measured CBF ratio to the CBF ratio obtained from Formula 1 after measuring the MTT ratio (CBF’), and the ratio of the measured OEF ratio to the OEF ratio obtained from Formula 2 (OEF’) showed highly significant negative correlation {OEF'= −682CBF'+ 1.58 (R^2 = 0.658)}. When this regression line is used as a calibration curve, the OEF ratio can be estimated if the CBF ratio and the MTT ratio are known. Based on this, we calculated the theoretical OEF ratios of the subjects in the literature only from the CBF ratios and the MTT ratios, and they coincided with the measured OEF ratios very well. [Conclusion] It was considered that the OEF values can be estimated accurately from CBF and MTT.
ASSOCIATION OF POSTOPERATIVE CEREBRAL HYPERPERFUSIION WITH PREOPERATIVE HEMODYNAMIC IMPAIRMENT AND INTRAOPERATIVE ISCHEMIA
Nobukazu Komoribayashi1, Kuniaki Ogasawara1, Masakazu Kobayashi1, Takeshi Fukuda1, Hideo Saitoh1, Kazunori Terasaki2, Takashi Inoue1, Akira Ogawa1
1Deparment of Neurosurgery, Iwate Medical University, Morioka, Japan, 2Cyclotron Research Center, Iwate Medical University, Morioka, Japan
? Background and aims
The aim of this study was to investigate whether postoperative hyperperfusion is associated with pereoperative cerebral hemodynamic impairment due to chronic ischemia and with acute cerebral ischemia during clamping of the internal carotid artery (ICA) during carotid endarterectomy (CEA).
? Methods
Transcranial cerebral oxygen saturation (SO2) was monitored intraoperatively using near-infrared spectroscopy in 89 patients undergoing CEA for ipsilateral ICA stenosis (>70%). Cerebral blood flow (CBF) and cerebrovascular reserve capacity (CVRC) to acetazolamide were also measured using single photon emission computed tomography (SPECT) before CEA. In addition, CBF was measured immediately after CEA and on the third postoperative day.
? Results
Hyperperfusion (CBF increase > 100% compared with preoperative values) was observed immediately after CEA in 10 of 18 patients (56%) with reduced preoperative CVRC. Also, post-CEA hyperperfusion was observed nine of 16 patients (56%) whose SO2 during clamping of the ICA decreased to less than 90% of the preclamping value. Logistic regression analysis showed that reduced preoperative CVRC and reduced SO2 during ICA clamping were significant independent predictors of the development of hyperperfusion immediate after CEA. In fact, all patients with reduced preoperative CVRC and reduced SO2 during ICA clamping developed post-CEA hyperperfusion, and two of these patients developed cerebral hyperperfusion syndrome.
? Conclusions
These data suggest that development of cerebral hyperperfusion after CEA is associated with preoperative hemodynamic impairment and intraoperative cerebral ischemia.
EPSILON PKC INTERACTS WITH MITOCHONDRIAL ATP-DEPENDENT POTASSIUM CHANNEL DURING ISCHEMIC PRECONDITIONING IN ORGANOTYPIC SLICE CULTURES
Miguel A. Perez-Pinzon, Ami Raval, Kunjan R. Dave
Department of Neurology and Neuroscience, Cerebral Vascular Disease Research Center, University of Miami School of Medicine, Miami, FL, USA
Background: Mitochondrial ATP-dependent potassium (mtK+ATP) channel activation has been reported to mediate cardiac ischemic preconditioning (IPC) via generating reactive oxygen species (ROS). The role of the mtK+ATP channel in IPC induction in the brain is still not well defined. Previously we demonstrated that IPC-induced neuronal survival in the hippocampal CA1 required epsilon protein kinase C (εPKC) activation (1), (2). Thus, the goal of the present study was to define whether mtK+ATP channel activation is required during the triggering phase of IPC. Further, we hypothesize that εPKC-mediated neuroprotection requires mtK+ATP channel phosphorylation.
Methods: Hippocampal slices were obtained from 9-11 days old Sprague Dawley rats and cultured for 14-15 days. Slices exposed to oxygen/glucose deprivation (OGD) for 40 min ('test’ ischemia) were used as the ischemia group. Slices exposed to OGD for 15 min (or a pharmacological agent), 48 h prior to ‘test’ ischemia were used as the IPC/pharmacological preconditioned (PPC) groups. Propidium Iodide (PI) fluorescence images were obtained using a SPOT CCD camera and were digitized using SPOT advanced software. Percentage of relative optical intensity was used as an index of cell death. We confirmed the presence of mtK+ATP channel specific subunits (Kir 6.1 and Kir 6.2) in hippocampal mitochondria by Western blotting. Immunoprecipitation was carried out to study phosphorylation of Kir 6.2 in different experimental conditions. Results are expressed, as mean ± SEM. Statistical significance was determined with an ANOVA test followed by a Bonferroni's post-hoc test.
Results: Opening of mtK+ATP channel (diazoxide, 50 µM) 48 h prior to ischemia protected the hippocampal CA1 and mimicked neuroprotection mediated by IPC/PPC with the εPKC agonist. PI fluorescence level of ischemic-, IPC-, and mtK+ATP channel agonist (diazoxide) treated groups were 50.6 ± 5.2 (n = 6); 28.5 ± 4.9 (n = 4); 16.8 ± 7.6 % (50 mM; n = 8; p < 0.001, compared to ischemia) respectively. Blockade of the mtK+ATP channel using 5-hydroxydecanoic acid (HD) abolished IPC/εPKC mediated neuroprotection. PI fluorescence values of HD-treated and IPC groups were 58.13 ± 15.9 % (n =12) and 29.7 ± 15.7 % (n =8), respectively (p < 0.001). Similarly, PI fluorescence values of εPKC agonist and εPKC agonist plus HD-treated groups were 33.5 ± 6.7 % (n = 5), and 54.3 ± 3.3 % (n = 5), respectively. Finally, we determined if ischemic and εPKC agonist-mediated PPC leads to phosphorylation of mtK+ATP channel specific subunits Kir 6.2. The levels of Kir 6.2 phosphorylation were higher by 79 (n = 3, p<0.01) and 104% (n = 3, p<0.01) in IPC and PPC groups, respectively, when compared with mitochondria from sham treated slices (n = 3). Inhibition of εPKC activation after IPC prevented Kir 6.2 phosphorylation, suggesting that εPKC-mediated Kir 6.2 phosphorylation following IPC.
Conclusion: The signal transduction pathway that ensues following IPC/εPKC agonist-mediated PPC requires opening of the mtK+ATP channel.
Grant support: Supported by PHS grants NS34773, NS045676, NS054147
SEX-SPECIFIC ANTI-OXIDANT CAPACITIES IN ASTROCYTES
Mingyue Liu, Nabil J. Alkayed, Esteban Oyarzabal, Patricia D. Hurn
Department of Anesthesiology and Peri-Operative Medicne, Oregon Health and Science University School of Medicine, Portland, OR, USA
Introduction: Neuronal cell death after exposure to various cytotoxins is sex-specific; male cells being more sensitive to nitrosative stress and excitotoxity than female neurons (1). We previously demonstrated that astrocytes cultured from female brain (XX) are less susceptible to cell death induced by oxygen-glucose deprivation (OGD) and hydrogen peroxide (H2O2) than male (XY) astrocytes (2). In the current study, we tested the hypothesis that male and female astrocytes respond differently to oxidative stress, in part due to differences in antioxidant capacity between male and female astrocytes.
Methods: Primary cultured cortical astrocytes were prepared from 1-3-day old male and female rat pups separately and grown to confluency in steroid-free medium. Male and female rat pups were distinguished by a larger genital papilla and longer ano-genital distance in male vs. female pups. Sex was confirmed by inspecting internal organs and gonads after laporatomy, e.g. uterine horns in females and ductus deferens in males, and by multiplex polymerase chain reaction (PCR) using male-specific marker SRY (sex determination region on the Y chromosome, responsible for testes formation) and the universal marker myogenin (Myog), which is expressed in both males and females. At day 14 in vitro (div), cultures were homogenized for protein and mRNA isolation. The protein levels of three antioxidant enzymes: glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), were determined by Western blotting in male and female astrocytes. Furthermore, GPx mRNA level was determined by RT-PCR, and GPx activity was measured in male and female astrocytes using a glutathione peroxidase activity assay kit measures GPx activity indirectly by a coupled reaction with glutathione reductase.
Results: The protein levels of SOD and CAT were not different between male and female astrocytes. However, the level of GPx protein was 3-fold higher in female compared to male astrocytes (3±0.6 fold, n=4, p<0.05). Similarly, GPx mRNA and activity were significantly higher in female compared to male astrocytes in female compared to male astrocytes (2±0.2 fold, n=3, p<0.05 for the mRNA and 2.4±0.1 fold, n=3, p<0.05 for the activity).
Conclusions: We conclude that female astrocytes express higher levels of GPx and exhibit higher GPx activity than male astrocytes. The sex difference in GPx expression and activity may afford female astrocytes with higher anti-oxidant capacity, and may underlie the difference in ischemic cell death between male and female astrocytes.
ESTRADIOL IS PROTECTIVE AGAINST ISCHEMIC BRAIN DAMAGE THROUGH SIGNAL TRASDUCER AND ACTIVATOR OF TRANSCRIPTION 3 (STAT3)
Suzan Dziennis1, Taiping Jia2, Oline K. Ronnekleiv2, Patricia D. Hurn1,2, Nabil J. Alkayed1,2
1Department of Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, OR, USA, 2Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR, USA
Background and Aims: Estradiol is neuroprotective in animal models of cerebral ischemia (1). Although the precise mechanisms are unknown, estrogen's beneficial properties are in part linked to rapid activation of signal transduction pathways that lead to the transcription of neuroprotective genes (2). Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated in response to both estrogen (3) and ischemia (4). Upon activation, STAT3 translocates to the nucleus where it upregulates neuroprotective genes, such as bcl-2 (5). We wished to determine whether estradiol replacement enhances STAT3 activation in female rat brain after focal cerebral ischemia and if STAT3 activation plays a protective role against ischemic brain injury. Methods: Adult female rats were ovariectomized (OVX) at 10-12 weeks and implanted with 25 µg 17s-estradiol (EST) 7 days prior to middle cerebral artery occlusion (MCAO). A 2-hour MCAO was induced by the intraluminal filament technique. Laser-Doppler cerebrocortical perfusion, mean arterial pressure, PO2, PCO2, pH, glucose and terminal plasma estrogen were measured. A single intraperintoneal injection of the STAT3 inhibitor JSI-124 (1mg/kg, was given 15 minutes after MCAO. Brains from separate groups were removed and processed for one of the following analyses: 1) brains were subdissected and rapidly frozen to prepare cortical nuclear and cytosolic extracts for phospho-STAT3 (P-STAT3) and total-STAT3 (T-STAT3) immunoblot analysis, 2) brains were sectioned and stained with 2,3,5-triphenyltetrazolium chloride (TTC) for infarct size measurement, or 3) brains were fixed in 4% paraformaldehyde for immunohistochemistry (IHC) with P-STAT3, Bcl-2 and MAP2 antibodies. Phospho-STAT3 was measured at 3 and 22 hours post reperfusion. IHC and infarct volume were measured at 22 hours post reperfusion. Results: Physiological variables were not different among groups. Estradiol increased P-STAT3 in the cytosolic and nuclear fractions at 3 hours following MCAO. In estradiol replaced animals, P-STAT3 remained elevated in nuclear fractions at 22 hours in ischemic cortex. Furthermore, the nuclear P-STAT3 co-localized with cells expressing neuronal and survival markers MAP-2 and Bcl-2. The P-STAT3 inhibitor JSI-124 abolished the effect of estradiol to reduce infarct size after MCAO in ovariectomized rats. Conclusion: Estradiol enhances STAT3 phosphorylation in ischemic brain, and activated STAT3 contributes to the hormone's neuroprotective property.
Grant support: Supported by F32 NS053065 (SD), NS 33668 (PDH) PO1 NS049210 (OKR, PDH and NJA)
DELAYED NEURONAL PRECONDITIONING WITH BMS-191095 IS MEDIATED BY CATALASE
Tamas Gaspar1, Anna Busija1, Bela Kis1, Keita Mayanagi1, Christina Tulbert1, James A. Snipes1, Ferenc Bari2, David W. Busija1
1Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston Salem, NC, USA, 2Department of Physiology, Faculty of Medicine, University of Szeged, Szeged, Hungary
Background and aims. Selective opening of the mitochondrial ATP sensitive potassium (mitoKATP) channels with BMS-191095 (BMS) is an effective way to induce tolerance against toxic stimuli. Previous works from our laboratory demonstrated that stress-induced free radical production was completely abolished in BMS-preconditioned neurons, suggesting that improved free radical scavenging capacity plays an important role in the neuroprotective effect of BMS. The aim of our present in vitro study was to identify the mechanisms of delayed preconditioning induced by BMS.
Methods. Cortical neurons of 18-day old Sprague-Dawley rat fetuses were cultured in B27 supplemented Neurobasal medium. To induce preconditioning, the neuronal cultures were treated with BMS (30 µM) once a day for 1, 2, or 3 days. Twenty-four hours after the last treatment, neuronal cultures were exposed to glutamate (200 µM) for 60 min. In some experiments, different concentrations of the catalase inhibitor 3-aminotriazole (3AT; 1, 10, 100 mM) were applied to the cultures during and for 3 hours after glutamate excitotoxicity. Cell viability was evaluated with CellTiter 96 AQueous One Solution assay on the next day. ATP levels in cortical neurons were measured using CellTiter-Glo luminescent assay. The expression of manganese dependent superoxide dismutase (MnSOD), cupper-zinc dependent superoxide dismutase (CuZnSOD), catalase, Bcl-2, and heat shock protein 70 (HsP-70) was analyzed using Western blotting. The enzymatic activity of SODs and catalase was measured with a microplate reader using commercially available kits.
Results. BMS treatment resulted in higher ATP levels in resting cells (in 1E-11 mol/well: untreated, 10.65 ± 0.21; BMS 30 µM, 11.84 ± 0.22*; *p<0.05 vs. untreated). Neurons preconditioned with BMS could preserve ATP during exposure to glutamate (in 1E-11 mol/well: untreated, 4.4 ± 0.04; BMS 30 µM, 7.01 ± 0.10*; *p<0.05 vs. untreated). Treatment with BMS resulted in a dose-dependent elevation of catalase expression in both transcriptional and translational levels whereas the protein levels of MnSOD, CuZnSOD, Bcl-2, and HsP-70 did not change. A parallel increase in the enzymatic activity of catalase was observed while the activity of SODs did not change. Catalase overexpression could not be blocked with the putatively selective mitoKATP antagonist 5-hydroxydecanoate. The neuroprotective effect of BMS, however, could be partially and dose-dependently antagonized using 3-AT (cell viability after exposure to glutamate: untreated, 54.5 ± 1.32%; BMS 30 µM, 90.6 ± 3.12%*; BMS 30 µM + 3-AT 1 mM, 81.3 ± 1.32%*#; BMS 30 µM + 3-AT 10 mM, 69.9 ± 1.70%*#; BMS 30 µM + 3-AT 100 mM, 67.3 ± 2.01%*#; *p<0.05 vs. untreated; #p<0.05 vs. BMS-treated group).
Conclusion. Our results indicate that improved energy homeostasis and increased catalase expression, and activity play important roles in the neuroprotective effect of the mitoKATP opener BMS-191095.
MICROPLASMIN REDUCES DOSE-DEPENTLY MICROVASCULAR BASAL LAMINA DAMAGE IN EXPERIMENTAL FOCAL CEREBRAL ISCHEMIA
Dorothe Burggraf1, Milan Vosko1, Marion Schubert1, Martin Dichgans1, Gerhard Hamann2
1Department of Neurology, Ludwig-Maximilians University Munich, Klinikum Grosshadern, Munich, Germany, 2Department of Neurology, HSK Dr. Horst Schmidt Klinik, Wiesbaden, Germany
Background and aims: Until now the only approved pharmacological therapy for acute stroke patients remains thrombolysis, by using recombinant tissue-type plasminogen activator (rt-PA, Alteplase). Thrombolytic therapy of ischemic stroke with rt-PA improves clinical outcome, but rt-PA also causes secondary intracerebral bleeding. Novel agents that achieve higher recanalization rates, lower hemorrhage rates, or both, might be able to improve the delicate balance of benefit and risk. An alternative substance could be microplasmin, a derivative of plasmin lacking the 5 “kringle” domain. We examined the effect of different microplasmin doses on microvascular damage, blood brain barrier disruption and the alternation from MMP-2 and MMP-9 and their endogenous inhibititors after focal cerebral ischemia in rats.
Methods: The suture model was used to induce a constant period of ischemia (3h) followed by reperfusion for 24h in rats. Microplasmin was injected with the chosen dose (0.5 mg, 1 mg, 2.5 mg, 5 mg or 10 mg; each N=6) at the end of ischemia. A control group was treated with saline. Another group was sham-operated.
Structural injury was detected by Nissl staining. The loss of collagen type IV, a major component of the basal lamina, was quantified by collagen western blotting. The amount of both gelatinases MMP-2 and MMP-9 was quantified by zymography. Their endogenous inhibitors (tissue inhibitor of MMPs: TIMPs) were analyzed by reverse zymography.
Results: We detected a significant reduction (p=0.01) of infarct volume in 10 mg microplasmin group (51.0 ± 22.6mm3) compared with the control group (167.3 ± 13.1mm3). A significant reduction (p<0.05) of basal lamina damage in basal ganglia region could be determined in 10 mg microplasmin animals in comparison to control group: 87±4% vs. 75±2%. With decreasing doses of microplasmin the infarction volume and the microvascular damage is decreasing (p<0.05; ANOVA). The extravasation of hemoglobin in basal ganglia was articulately reduced in 10 mg microplasmin (154±24%) treated rats compared with control group (442±124%). The MMP-activity (MMP-2 and MMP-9) was decreasing with rising microplasmin doses (p<0.05). However, the amount of the corresponding TIMPs remained unchanged by different doses microplasmin. Thus the balance between the proteases (MMP) and their inhibitors was shifted towards the inhibitory side.
Conclusions: Microplasmin showed a dose-dependent protective effect on microvascular basal lamina and blood brain barrier. With the high microplasmin dose (10 mg) the best neurological and microvascular protection could be detected. These results suggested that microplasmin might carry less risk for hemorrhage than tPA treatment of ischemic stroke. Thus, microplasmin may be safer than tPA in terms of damage of the microvasculature associated with thrombolytic therapy of ischemic stroke.
ROLE OF SPECIFIC MITOCHONDRIAL ATP-DEPENDANT POTASSIUM CHANNEL IN ISCHEMIC TOLERANCE
Megumi Watanabe1,2, Ken-ichiro Katsura1, Genki Mizukoshi1, Ikuroh Ohsawa2, Sadamitsu Asoh2, Shigeo Ohta2, Yasuo Katayama1
1Division of Neurology, Nephrology and Rheumatology, Department of Internal Medicine, Nippon Medical School, Tokyo, Japan, 2Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, Kanagawa, Japan
Background and aims: When brain cells are subjected to brief ischemia, they become resistant to a subsequent lethal ischemic insult. We have reported roles of mitochondrial ATP dependent potassium (mito-KATP) channels in ischemic tolerance phenomenon for 2 different in vivo ischemic model. Damages in CA1 neuronal cells of gerbil global ischemia model, or infarct volumes of rat transient MCA-occlusion model, were attenuated because of ischemic tolerance phenomenon after preceding brief ischemic insult. The mito-KATP channel inhibitor, 5-hydroxydecanoate (5HD), canceled the tolerance, and mito-KATP channel opener, diazoxide (DZX), enhanced the tolerance. To clarify the mechanism of ischemic tolerance, we studied roles of mito-KATP channels on the ischemic tolerance phenomenon by using brain primary culture.
Methods: To mimic a in vivo ischemic condition, cells were exposed to oxygen-glucose deprivation (OGD). Cells were placed in OGD medium (without glucose, gassed with 95% N2/5% CO2), and incubated for the desired time with a chamber gassed with 95% N2/5% CO2. Survival neuron was distinguished with its morphological changes. At first, Cells were subjected to 10 min of OGD as a sublethal ischemia (PC), and 24 hours later, subjected to 80 min of OGD as lethal ischemia. 5HD and DZX were administrated 30 min before the lethal ischemia in the medium. We also measured mitochondrial membrane potential with tetra-methyl-rhodamine methyl ester (TMRM) under confocal micrography.
Results: In preconditioned groups cells were significantly more survived than in control group. In 5HD administrated group, the number of surviving cells were significantly reduced. DZX administration did not reduced the number of surviving neurons. Preconditioned cells were relatively depolarized, and 5HD administration inhibited the depolarization induced by the precondition.
Conclusions: Our results may suggest that mito-KATP channels play a key role in neuroprotection in ischemic tolerance phenomenon through mitochondrial membrane potential. Mitochodrial depolarization generated by ischemic tolerance phenomenon, may lead to inhibiting mitochondrial calcium overload upon lethal ischemia, and thereby protecting neurons.
CINNAMOPHILIN ATTENUATES GRAY AND WHITE MATTER DAMAGE AND IMPROVES NEUROBEHAVIORAL AND ELECTROPHYSIOLOGICAL RECOVERIES FOLLOWING TRANSIENT FOCAL CEREBRAL ISCHEMIA
Hung-Yi Chen1,2, Tsung-Ying Chen1,3, Chieh-Chih Huang1, Yu-Hsiang Kuan1, E-Jian Lee1, Tian-Shung Wu1,4
1Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical Center and Medical School, Tainan, -R.O.C., 2Institute of Pharmacy, China Medical University, Taichung, Taiwan-R.O.C., 3Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan-R.O.C., 4Department of Chemistry, National Cheng Kung University, Tainan, Taiwan & National Research Institute of Chinese Medicine, Taipei, Taiwan-R.O.C.
Introduction: Optimal functional integration requires not only the preservation of the cortex and the subcortical cytoarchitectures, but also an integrity of the anatomical, electrophysiological and metabolic links between gray and white matter [1]–[2]. Cinnamophilin (CINN; 20-80 mg/kg) dose-dependently protects against ischemic lesion via decreasing postischemic oxidative damage in mice [2]. We herein evaluate whether CINN could decrease gray and white matter damage and, therefore, enhance the restoration of neurobehavioral and electrophysiological outcomes following transient focal cerebral ischemia.
Methods: Male Sprque-Dawley rats, weighing 240-280 gm, were subjected to intraluminal suture occlusion for 60 minutes. CINN (80 mg/kg) or vehicle was given intravenously at 1 hour after reperfusion. Physiological parameters, including core temperature, local cortical perfusion and arterial blood gases, were measured before, during and after cerebral ischemia-reperfusion, whereas the somatosensory potentials evoked from each forepaw and hindpaw region were recorded prior to the ischemic insult and at 7 days after reperfusion. Postmortem gray matter damage was determined by quantitative image analysis of Nissl-stained sections. White matter damage was evaluated immunohistochemically by SMI-31 and myelin basic protein (MPB).
Results: CINN (80 mg/kg, i.v., n=9) given 2 h after the ischemic insult significantly reduced infarct volumes (27.3±7.3% vs. 39.9±5.8%, P<0.005) and improved neurobehavioral outcome by 47.4% (P<0.001), compared with those in control animals (n=9). CINN treatment was also noted to result in more surviving neurons in cortical (582±203 vs. 207±100, per mm2, P<0.001) and subcortical (201±67 vs. 646±153, per mm2, P<0.001) regions, compared to controls. In addition, CINN not only significantly improved somatosensory responses evoked from the contralateral, non-affected fore- and hindpaw by 33.2% and 26.9% (P<0.001, respectively), but also significantly improved contralateral transcallosal diaschisis evoked from the affected forepaw by 8% (P<0.05). Furthermore, we have observed that CINN significantly preserved more phosphorylated component-H of neurofilaments by 33.1% (52.6±12.4% vs. 19.6±12.7%, P<0.005) and improved the integrity of myelin by 28.2% (61.4±7.4% vs. 44.1±16.2, P<0.05) in the ischemic hemisphere.
Conclusion: Delayed treatment with CINN not only protects against gray and white matter damage, but also offers the advantage of enhancing neurohavioral and electrophysiological outcomes following transient focal cerebral ischemia in rats. These findings support that the agent may be worthwhile for further investigation for its clinical implications in the treatment of ischemic stroke.
THE SELECTIVE ESTROGEN RECEPTOR MODULATOR LY362361 IS NOT NEUROPROTECTIVE IN A RAT MODEL OF TRANSIENT FOCAL ISCHEMIA
Tracy Deanne Farr, Hilary V.O. Carswell, I. Mhairi Macrae
Wellcome Surgical Institute, University of Glasgow, Glasgow, Scotland, UK
The results from the Women's Health Initiative trial indicated that women receiving estrogen were 39% more likely to suffer a stroke than women receiving placebo. This has raised concern regarding the health risks of chronic estrogen prescription, and surprised the pre-clinical research community, since estrogen has consistently been shown to provide neuroprotection from stroke. However, there are some reports that reveal estrogen's capacity to exacerbate ischaemic injury (Bingham, et al., 2005. JCBFM. 25: 414; Theodorsson and Theodorsson, 2005. Peptides. 26: 2257; Harukuni, et al., 2001. Brain Res. 900: 127). In light of this, emphasis has been placed on selective estrogen receptor modulators (SERMs), which may offer improved alternatives to estrogen as neuroprotectants. The present study investigated the novel, brain penetrable SERM, LY362321, in a rat model of transient middle cerebral artery occlusion (MCAO). Female Sprague Dawley rats were ovariectomized and randomized to receive daily subcutaneous injections of 1mg/kg (n=11), 10mg/kg (n=10) of LY362321, or vehicle (n=11); the experimenter was blinded to the treatments. The left MCA was temporarily occluded (90minutes), with cortical blood flow monitoring, at 12 days post ovariectomy. Sensorimotor function was assessed using a neurological score prior to and daily following MCAO. Infarct size was assessed at 3 days post-MCAO on 1% 2, 3, 5-triphenyltetra-zolium chloride (TTC) stained sections. The results indicated that there were no significant differences amongst groups in cortical blood flow during the MCAO. Furthermore, there was no significant difference in infarct size (one way ANOVA (F(2,32) = 0.542, p = 0.587) amongst vehicle, 1, and 10mg/kg treated animals (22.9 · 16.4, 16.7 · 14.6, and 21.1 · 12.9, respectively, mean ± SD of % total brain area) (Figure 1). The MCAO induced a significant decline in neurological score in the vehicle group (from 14 down to 7 at 24hrs post-MCAO) but this was not affected by LY362321 at either dose. In conclusion, pretreatment with a low or high dose of the novel SERM, LY362321, did not significantly influence cerebral blood flow, infarct volume, or sensorimotor function in rats exposed to transient MCAO.
Acknowledgements
The authors wish to acknowledge Drs. Mike O'Neill and Jeff Dodge (Eli Lilly) for supply of LY362321. This work was supported by a University of Glasgow Postgraduate Studentship, Overseas Research Studentship, and Canadian Stroke Network Postgraduate Studentship to TDF.
CEREBRAL METABOLIC RATE OF OXYGEN IS A POTENTIAL MARKER OF INSULT SEVERITY FOLLOWING HYPOXIA-ISCHEMIA IN NEWBORNS
Kenneth M. Tichauer1, Jennifer Hadway1, Daisy Y.L. Wong2, R. Jane Rylett2, Ting-Yim Lee1,3, Keith St. Lawrence1
1Imaging Program, Lawson Health Research Institute, London, ON, Canada, 2Cell Biology Research Group, Robarts Research Institute, London, ON, Canada, 3Imaging Research Laboratories, Robarts Research Institute, London, ON, Canada
Background and Aims
Impaired oxidative metabolism after hypoxia-ischemia (HI) is an indicator of delayed brain injury (1). We have recently developed a near-infrared spectroscopy (NIRS) technique for bedside measurements of the cerebral metabolic rate of oxygen (CMRO2) in critically ill newborns. The method requires measurement of cerebral blood flow with indocyanine green as the tracer as well as measurement of deoxy-hemoglobin concentration in the blood of brain tissue (2)–(3). We have shown previously that there was an immediate and persistent decrease in CMRO2 following 30 minutes of HI in piglets (4). In the current study, the severity of HI was varied to assess the prognostic value of the NIRS technique.
Methods
Four piglets were subjected to 20 minute (moderate) and two piglets to 30 minute (severe) HI insults by occluding both carotid arteries and reducing the fraction of inspired oxygen to 8 %. An additional three piglets served as sham-operated controls. Measurements of CMRO2 were acquired with NIRS at baseline and periodically for 24 hours following resuscitation. At 24 hours, brains were collected for TUNEL staining and the stained brain sections were analyzed to quantify the extent of delayed (apoptotic) cell death.
Results
Average CMRO2 followed significantly different trends after HI depending on the duration of the insult (p < 0.05). The control CMRO2 was 1.90 ± 0.17 mlO2/min/100g throughout the 24-hour period. In the first four hours after, CMRO2 was 1.91 ± 0.39 and 1.43 ± 0.15 mlO2/min/100g for the moderate insult (p > 0.05) and severe insult (p < 0.05) group compared to controls, respectively. By 6 hours and for the remainder of the study, CMRO2 of the moderate group further dropped to 1.35 ± 0.23 mlO2/min/100g and was not significantly different from the severe HI group (1.44 ± 0.16 mlO2/min/100g); both experimental groups were significantly different from control during this period. Preliminary TUNEL analysis from one severe insult and one control piglet revealed that the number of apoptotic cells in the cortex of the injured brain was 3 times greater than in the control.
Conclusions
These preliminary results suggested that 1) NIRS is sensitive to varying degrees of HI at early times after the primary insult; and, 2) the observed reductions in CMRO2 anticipate exaggerated levels of delayed (apoptotic) cell death at 24 hours post-insult. The potential for NIRS to differentiate between varying severities of HI injury coupled with the ability of applying it safely and rapidly at the bedside argues strongly for its capacity to improve prognosis and aid treatment strategy of HI in the neonatal intensive care unit.
HEMODYNAMIC CHANGES AND OXYGEN METABOLISM DURING EVENT RELATED AND PROLONGED ACTIVATION OF A DISTINCT CORTICAL COLUMN IN RAT SOMATOSENSORY CORTEX
Georg Royl1, Christoph Leithner1, Heike Kaasch1, Jan Philipp Mueller1, Dirk Megow1, Jens Steinbrink1, Matthias Kohl-Bareis2, Ulrich Dirnagl1, Ute Lindauer1
1Department of Experimental Neurology, Charite - Universitaetsmedizin Berlin, Berlin, Germany, 2University of Applied Sciences Koblenz, RheinAhrCampus Remagen, Remagen, Germany
2D optical imaging of hemoglobin concentration and oxygenation was combined with cerebral blood flow (CBF) imaging with Laser Speckle Contrast Analysis to investigate the neurovascular response of the somatosensory cortex to mechanical deflection of a single whisker hair in the anesthetized rat. After optical imaging, a multi-electrode array was inserted into the area of activation and the experiment was repeated while measuring multiunit activity to accurately localize the site of neuronal activation (the activated whisker barrel). Two different stimulation paradigms were employed. One paradigm (single event) consisted of a single whisker hair deflection, randomized with a minimal interstimulus time of 15s. For the other paradigm (prolonged activation) the whisker hair was deflected periodically (4Hz) for 16s, with a minimal interstimulus time of 60s. Within the activated whisker barrel, this prolonged whisker stimulation led to a transient increase in CBF (5.0 ± 1%), total Hemoglobin (HbT, 0.7± 0.2%) and cerebral metabolic rate of oxygen (CMRO2, 3.7% ± 1%) and a decrease in deoxygenated Hemoglobin (Deoxy-Hb, −0.6 ± 0.3%), relative to baseline values. However, a single whisker deflection did not lead to a significant change in HbT and Deoxy-Hb while still causing an increase in CBF and CMRO2 by ~1.8% ± 0.7%. This dissociation of flow and hemoglobin related signals can be attributed to a short velocity related CBF increase without a significant change of cerebral blood volume or oxygenation. In conclusion, oxygenation related functional imaging like BOLD fMRI might be inferior to CBF based techniques when investigating fast single event related neuronal activation. Implications from this study considering the temporal and spatial profile of neurovascular and neurometabolic coupling will be discussed.
VASCULAR TERRITORIES MEASUREMENT USING ARTERIAL SPIN LABELING WITH A LOCALIZED LABELING RF COIL
Fernando F. Paiva1,2, George C. Nascimento1, Alberto Tannus2, S. Lalith Talagala1, Afonso C. Silva1
1National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA, 2Instituto De Fisica De Sao Carlos, Universidade De Sao Paulo, Sao Carlos, SP, Brazil
INTRODUCTION
Arterial spin labeling (ASL) techniques have proven to be an excellent tool to obtain quantitative maps of perfusion non-invasively. Recently, there has been increased interest in using ASL to map vascular terri-tories of major cerebral arteries, as a way to provide complimentary identification of the source and to predict and verify the extent of vascular occlusions. In the present study, perfusion territory maps of the major feeding arteries to the brain were obtained using a separate RF coil for localized ASL.
METHODS
Animal Studies: Sprague-Dawley rats (200-350g), orally intubated and maintained anesthetized under isoflurane, were scanned in a 7T/30cm magnet (Bruker-Biospin, Billerica, MA) equipped with gradients capable of 450mT/m amplitude (Resonance Research Inc, Billerica, MA). A home-built, transmit-only birdcage coil, and a receive-only quadrature surface coil (RAPID Biomedical GmbH, Rimpar, Germany), were used for all image acquisition. A small figure-8 shaped labeling coil was positioned under the neck of the animal, approximately 2cm away from isocenter.
Human Studies: healthy volunteers were scanned in a 3T GE Signa system equipped with gradients capa-ble of 40mT/m amplitude. A detunable, transmit/receive coil (Nova Medical Inc, Wakefield, MA) was used for image acquisition and a surface labeling coil was placed on the neck.
Continuous arterial spin labeling (CASL) was obtained with adiabatic flow-driven inversion of the chosen feeding arteries. A labeling plane was defined by rotating the labeling gradient Geff in the horizontal plane by an angle θ with respect to the longitudinal axis (Fig. 1). Because of the confined excitation profile of the labeling coil, only the arteries passing over the intersection of the labeling plane with the labeling coil undergo the ASL inversion.
RESULTS
Figure 2 shows anatomical (Fig. 2a) and perfusion-weighted images (Fig. 2b) for selected coronal slices in rats. Perfusion images of the left and right hemispheres are shown in Fig. 2c-d, achieved by labeling only the left and the right CCA, respectively. Figure 3 shows a color-coded map of the territories of the major feeding brain arteries in humans, depicting a nice demarcation of each territory.
CONCLUSIONS
The combination of a separate labeling coil with proper placement and direction of the labeling plane al-lows for demarcation of the perfusion territory of major cerebral feeding arteries with reduced magnetization transfer effects and high SNR. The method described explores the advantages of the separate labeling coil and allows the labeling of different arteries without repositioning of the coil or the subject. It can be used to evaluate different perfusion territories including those from arteries that are not uniquely accessi-ble, a useful feature for the diagnosis of cerebrovascular diseases.
PREDICTION OF HYPERPERFUSION SYNDROME AFTER CAROTID STENTING BY PERFUSION CT
Chien-Hung Chang1, Chi-Jen Chen3, Shy-Chyi Chin2, Yeu-Jhy Chang1, Ting-Yu Chang1, Tai-Cheng Wu1, Shan-Jin Ryu1, Tsong-Hai Lee1
1Department of Neurology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan-R.O.C., 2Department of Diagnostic Radiology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan-R.O.C., 3Department of Diagnostic Radiology, E-Da Hospital and I-Shou University, Kaohsiung, Taiwan-R.O.C.
Background and Purpose
Hyperperfusion syndrome is a rare complication after endarterectomy or stenting in the carotid circulation. The parameter in predicting hyperperfusion syndrome has not been well understood.
Method
We prospectively studied 54 patients with symptomatic carotid stenosis who underwent angioplasty and stent placement in our hospital during 2001-2005. The hyperperfusion syndrome is defined according to clinical features: headache, seizure, focal neurological deficit or intracranial hemorrhage. Pre-stenting cerebral blood volume (CBV), cerebral blood flow (CBF) and time-to-peak (TTP) were quantitatively measured by perfusion CT to evaluate cerebral hemodynamic status. Brain computed tomogram (CT) within seven days after stenting was examined to study the possible etiology of hyperperfusion syndrome.
Result
Among the 54 patients, six patients had cerebral hyperperfusion phenomenon after carotid stenting. The significant variables related to hyperperfusion syndrome included critical contralateral stenosis (? 90%) (hyperperfusion vs. no hyperperfusion: 66.7% vs. 20.5%, P= .015), symptomatic ischemic heart disease (66.7% vs. 18.8%, P= .010), and post-stenting mean arterial blood pressure (171 +/− 16 mmHg vs. 139 +/− 14 mmHg, P= .05). In the perfusion CT study before stenting, we considered normal study if perfusion difference was ? 10% between bilateral hemisphere. The pre-stenting CBV and CBF had no significant difference between bilateral hemisphere. However, TTP index (ipsilateral TTP/contralateral TTP) of ? 20% was found more common in patients with hyperperfusion syndrome (hyperperfusion vs. no hyperperfusion: 83.3% vs. 31.1%, P= .013).
Conclusion
Our study suggests that perfusion CT study before carotid stenting may aid in the prediction of patients at risk for hyperperfusion syndrome.
INTERLEUKIN-6 STIMULATES CIRCULATING HUMAN ENDOTHELIAL PROGENITOR CELL (EPC) ANGIOGENESIS IN VITRO
Y. Fan1,2, J. Ye5, F. Shen1,2, Y. Zhu1,2, Y. Yeghiazarians4, W. Zhu1,2, Y. Chen1,2, M. Lawton3, W. Young1,2,3,4, Guo-Yuan Yang1,2,3
1Center for Cerebrovascular Research, 2Anesthesia and Perioperative Care, 3Department of Neurological Surgery, 4Department of Neurology, 5Department of Internal Medicine
Introduction: Endothelial progenitor cells (EPCs) are defined as cells in bone marrow, circulating blood or adjacent tissue, which are positive for both stem cell markers CD34 and CD133, and endothelial cell markers such as VWF and KDR. EPCs contribute to postnatal vasculogenesis during wound healing, ischemic injury or tumor growth. EPCs respond to many growth factors and cytokines; however, it is still unclear if interleukin-6 (IL-6), a multifunctional cytokine that plays an important role in angiogenesis and vascular remodeling, has effects on EPCs. Here, we provide evidence that IL-6 stimulates EPC angiogenic changes in vitro.
Methods: For EPC culture, mononuclear cells were isolated from circulating blood using gradient centrifuge with Histopaq 1077 and seeded in fibronectin-coated plates. Dil-acLDL uptake, FITC-UEA binding assay and immunofluorescence of CD34, VWF, KDR and VE-cadherin were used to characterize the cultured EPCs. IL-6 receptor CD126 was detected on EPCs using immunofluorescence, and further identified by phosphorylation of ERK1/2 and STAT3 in response to IL-6 stimulation using Western blot. Finally, to determine if IL-6 stimulation causes EPC angiogenic changes, we performed EPC proliferation assay using BrdU incorporation assay, EPC migration assay using transwell system, and EPC tube formation using MetraGel. All the results were analyzed using a one-way ANOVA with Fisher's comparison tests. A random p< 0.05 is considered statistically significant.
Results: EPC colonies formed after 2 weeks in culture. The cultured EPCs grew like cobble stones and were able to uptake Dil-acLDL, bind FITC-UEA functionally and staining positive for cell markers CD34, KDR, VWF, and VE-cadherin. CD126 was expressed on EPCs. After stimulation with IL-6, two IL-6 downstream signal molecules ERK1/2 and STAT3 were phosphorylated in a dose-dependent manner and were inhibited by antibodies against IL-6 and IL-6 receptor. IL-6 induced EPC proliferation with a maximal effect at 25 ng/ml. Enhanced EPC migration had an optimal effect at 50 ng/ml compared with non-treated control groups (p<0.01), and showed a dose-dependent manner. Furthermore, EPC tube formation on MetraGel increased after different concentrations of IL-6 stimulation (p<0.01), with an optimal dose of 50 ng/ml. These effects were blocked significantly after using both IL-6 neutralizing antibody and IL-6 receptor antibody (p<0.01). The results demonstrated that IL-6 stimulation induced EPC angiogenic changes in vitro.
Conclusion: We successfully cultured EPCs form human circulating blood. We provide, for the first time, the evidence that EPCs express functional IL-6 receptor and that IL-6 can stimulate EPC proliferation, migration and tube formation upon binding of its receptor. These results provide a novel clue that EPCs may be involved in IL-6-related diseases.
CORRELATIONS BETWEEN FUNCTIONAL AND STRUCTURAL OBSERVATIONS AFTER TRANSPLANTATION OF HUMAN EMBRYONIC STEM CELL-DERIVED PROGENITORS INTO THE ISCHEMIC RAT BRAIN
Christine Seminatore1, Jerome Polentes1, Valerie Itier3, Laetita Aubry2, Aurore Bugi2, Thierry Delzescaux4, Marc Dhenain4, Philippe Hantraye4, Anselme Perrier2, Brigitte Onteniente1
1Inserm Umr 549, Paris, France, 2Inserm Umr 861, Istem, Evry, France, 3Inserm Emi 0011, Creteil, France, 4Cea, Cnrs 2210, Orsay, France
Cell therapy with stem cell-derived neural progenitors has become a challenging medical perspective. Neural progenitors from a variety of stem cell sources can differentiate into neural cell types after transplantation into the ischemic brain. In some instances graft survival and cell differentiation into the required neural cell types are correlated with improvements of functional deficits. However, little is known on the possibilities to evaluate the efficacy of cell therapy in stroke in settings closer to the clinic, i.e. over a long period of time without sacrifice of the animals.
The aim of this study was to validate non invasive investigation techniques for embryonic stem cells (ESC) therapy in stroke using human ESC (hESC). We analyzed the differentiation and survival properties of early neural progenitors derived from the SA-001 hESC line (Cellartis AB, Sweden) over 4 months after transplantation into the striatum of adult male Sprague-Dawley rats 7 days after a 2 hours occlusion of the middle cerebral artery (tMCAO). Rats were analyzed with T2 magnetic resonance imaging (MRI, 3 Tesla) and two behavioral tests, the apomorphin-induced rotation test (Apo) for motor impairments, and the adhesive removal test (ADR) for sensory-motor impairments. Animals received an immunosuppressive tri-therapy containing cyclosporine A, azathioprin and methyl-prednisolone for 5 weeks.
MRI allowed identification of the graft as an hypo dense signal. 3D reconstitution was used to measure infarct and graft volumes. No correlations were found between infarction severity and graft volumes, nor between infarction severity and body weight modifications. Infarction severity correlated with Apo and ADR deficits. Transplantation into the peri-lesional parenchyma resulted in good survival of cells with neural (Pax6, GFAP, nestin) phenotype, although some non-neural cells (e.g. cytokeratin-14 positive) were also found. Grafted cells were surrounded by DARPP-32 fibers specific for the medium-spiny GABAergic host striatal neurons. When transplanted into the lesion cavity, hESC-derived progenitors gave rise to few neural cells and mainly to teratoma-like structures. No teratoma were formed when progenitors were transplanted into the unlesioned striatum, but the number of cytokeratin-14-labeled cells increased significantly. Teratoma-like structures could not be differentiated from neural grafts on 3T T2 MRI, indicating the limits of the technique. MRI also revealed a stabilization of the lesion at 1 month. Evolution of the lesion until 1 month was confirmed post-mortem by the presence of conspicuous inflammatory reaction at the lesion border in the absence of graft.
Partial (up to 40%) recovery of motor and sensory-motor functions were observed at 4 months in animals transplanted into the parenchyma. Motor deficits displayed a bi-phasic curve, with a partial recovery at 1 month followed by degradation of Apo test scores. This was not observed for the ADR test, suggesting an increased D2-receptors activity of GABAergic cells remaining on the lesioned side.
SC and PJ were equally involved in the work
HYPOFRONTALITY INDUCED BY VIOLENT VIDEO GAME PLAYING IN YOUNG ADULTHOOD
Yuan-Hwa Chou1, Tung-Ping Su1, Bang-Hung Yang2, Shyh-Jen Wang2
1Department of Psychiatry, Taipei Veterans General Hospital, Taipei, Taiwan-R.O.C., 2Department of Nuclear Medicine, Taipei Veterans General Hospital, Taipei, Taiwan-R.O.C.
Background and Aims: Violent video game playing is popular worldwide. It has been reported that brief exposure to a violent video game increased aggressive behavior. However, the effect of violent video game playing on human brain function is still unclear. The purpose of the study was to determine the effect of violent video game playing on cerebral blood flow (CBF) in young adulthood.
Methods: Twelve healthy subjects (averaged age was 22±3.2 years) who received three times single photon emission computed tomography (SPECT) examination with radiotracer 99mTc ECD for measuring CBF were recruited. The first SPECT measurement was measured as baseline, second and third SPECT measurements as study tasks. Each task measurement was performed after 30 min violent and mentality video game playing. There was two-week interval between SPECT measurements. Statistic Parametric Mapping (SPM2) was used for the mapping of CBF.
Results: The results showed that CBF in left frontal lobe was decreased (green area) significantly, whereas CBF was increased (red area) in occipital cortex after violent video game playing (Fig 1). However, the effect can not be found in CBF after mentality video game playing. To our knowledge this is the first data demonstrated hypofrontality induced by violent video game playing in young adulthood. The finding of hypofrontality is similar to those previous studies which showed hypofrontality in patient with major depression or schizophrenia.
Conclusion: As frontal lobe function is crucial in our cognitive function, executive function and emotion, we suggest that clinical study regarding relationship of violent video game playing and mental disorders has to be conducted in adolescence or young adulthood urgently.
THE FEASIBILITY OF 3D TENSOR TRACTOGRAPHY AND 3DAC AS A MODALITY TO ASSESS MOTOR FUNCTION IN PATIENTS WITH CEREBRAL INFARCTION
Keiji Igase1, Yoshiaki Kumon1, Shigeyuki Nagato1, Kazuhiko Sadamoto2, Takanori Ohnishi1
1Department of Neurosurgery, Ehime University, Toon, Ehime, Japan, 2Department of Neurosurgery, Washo-Kai Sadamoto Hospital, Ehime, Japan
Background and aims: In recent years 3D tensor tractography (DTT) has been applied for many CNS diseases to depict neuronal fibers through measuring a fractional anisotropy, meanwhile 3 dimensional anisotropic contrast (3DAC) has been recognized as an imaging method for delineating true neuronal fibers just as they were. However, there seems to be only a few reports that aim to evaluate the feasibility of both DTT and 3DAC to predict a motor recovery in patients suffering from cerebrovascular diseases. Thus we have embarked on investigating the possibility of these 2 methods focusing on a prediction in motor outcome in patients with cerebral infarction.
Methods: Nineteen patients with a brand-new lacunar infarct, who underwent DTT at least twice in each acute (mean 3.8 days) and subacute phase (mean 19.5 days), were enrolled, out of which only 4 patients underwent 3DAC study. Patients were separated to 2 groups, recovery and nonrecovery. Patients categorized to former group had almost complete recovery in motor function 3 month later, while those to latter had a remaining paresis even 3 month after the onset. DTT was implemented with 3 tesra MRI (SignaExcite; GE) and analyzed with dTV.IISR produced by the department of Radiology, Tokyo University. Pyramidal tract was delineated by setting each ROI, the cerebral peduncle for seed point and the motor cortex for target point. The number of pyramidal fibers was recognized as drawn lines obtained from a result display in this software, the ratio of numbers in the injured side to those in the intact side was measured. Meanwhile 3DAC image was produced with an imaging software through assigning 3 directions (anterior-posterior: A-P, right-left: R-L, superior-inferior: S-I) to RGB color.
Results: In acute phase mean ratio of both recovery and non-recovery groups were 70.8±21.6% and 63.5±23.4% respectively, resulting in no significant difference between two groups, meanwhile in subacute phase the ratio of recovery group was 100.5±28.3%, which revealed a significant difference compared with that of non-recovery group 46.8±10.3% (p<0.05). Furthermore, in a recovery group there was a significant difference between mean ratio in acute and that in subacute phases (p<0.001). 3DAC image obtained in acute phase disclosed that fibers in S-I direction appeared to be predominantly injured in non-recovery group.
Conclusions: The feasibility of DTT in predicting a recovery of motor function in patients with cerebral infarction has been shown, and the possibility that 3DAC image can estimate a motor outcome even in faster phase than DTT study has been suggested as well. Collecting cases might make an apparent answer to these questions.
ROLES OF BMPS IN THE DEVELOPMENT OF GLIOVASCULAR UNITS
Tetsuya Imura, Shinji Fushiki
Department of Pathology and Applied Neurobiology, Kyoto Prefectural University of Medicine, Kyoto, Japan
Background and aims
Precise mechanisms in the establishment of gliovascular units still remain unclear, but have important implications for both brain development and injury. Since gliogenesis and angiogenesis are temporally and spatially associated during development, cellular interactions between these two systems have been postulated to play a crucial role. In this study, using a combined in vitro and in vivo approach, we investigated how endothelial cells and neural progenitor cells interact with each other.
Methods
Neural progenitor cells isolated from neonatal mouse cortical tissues were cocultured with endothelial cells using the membrane-separated coculture system. Capillary vessels were isolated from neonatal mouse cortical tissues by the density-dependent centrifugation.
Results
Endothelial cells promoted astrocyte differentiation but inhibited oligodendrocyte differentiation of neural progenitor cells. Endothelial cells secreted bone morphogenetic proteins (BMPs) that activate Smad signaling in neural progenitor cells. Treatment with the BMP antagonist Noggin blocked the effects of endothelial cells on glial differentiation. Neural progenitor cells promoted endothelial cell growth under immature proliferating conditions, but once differentiated into astrocytes they inhibited endothelial cell growth and changed gene expression patterns from proangiogenic to antiangiogenic. In vivo, Smad signaling in cortex was transiently activated in neural progenitor cells and capillary vessels in association with the development of astrocytes, but was inactivated before that of oligodendrocytes. Isoalted capillary vessels of the developing cortex expressed high levels of BMPs.
Conclusion
Together, these results suggest that endothelial cell-derived BMPs play a role in the establishment of gliovascular units during development.
SURVIVAL AND DIFFERENTIATION INTO NEURONS OF TRANSPLANTED ES CELL-DERIVED PRECURSORS WITHIN STROKE LESION
Klaus G. Reymann1, Claudia Buehnemann1, Christian Bernreuther2, Marcel Dihne2, Holger Braun1, Christoph Y. Malik3, Andreas Scholz3, Melitta Schachner2
1Leibniz Institute for Neurobiology, Project Group Neuropharmacology, Magdeburg, Germany, 2Centre for Molecular Neurobiology, Universitaetskrankenhaus Hamburg-Eppendorf, Hamburg, Germany, 3Institute for Physiology, Giesen, Germany
Background and aims
Stroke represents one of the leading causes of death and disability in Western countries, but despite intense research, only few options exist for the treatment of stroke-related infarction of brain tissue. In experimental stroke, cell therapy can partly reverse some behavioural deficits. However, the underlying mechanisms have remained unknown as most studies revealed only little if any evidence for neuronal replacement and the observed behavioural improvements appeared to be related rather to a graft-derived induction of a positive response in the remaining host tissue than to cell replacement by the graft itself. The present study was performed to test a murine embryonic stem cell-based approach in rats subjected to experimental stroke.
Methods
Enhanced green fluorescent protein+ (GFP+) embryonic stem cells were pre-differentiated into nestin+ neural precursor cells before they were injected into rats subjected to a focal cerebral ischemia which was induced by injection of the vasoconstrictor endothelin near to the middle cerebral arteria (MCA) of adult rats. Efficacy of cell therapy regarding graft survival, neuronal yield and diversity, and electrophysiological features of the grafted cells were tested after transplanting embryonic stem cell-derived neural precursors one week after ischemia into the infarct core and periphery. Rats were immunosuppressed by daily injections of 10 mg/kg cyclosporine A.
Results
In the case of contralateral application we found no migration into the damaged area along the corpus callosum. However, grafted cells can survive, albeit not entirely, most probably as a consequence of an ongoing immune response, within the infarct core for up to 12 weeks after transplantation. These cells survive and differentiate with high yield into immunohistochemically mature glial cells and neurons of diverse neurotransmitter-subtypes. Most importantly, transplanted cells demonstrate characteristics of electrophysiologically functional neurons with voltage gated sodium currents that enable these cells to fire action potentials. Additionally, during the first seven weeks after transplantation we observed spontaneous excitatory post-synaptic currents in graft-derived cells indicating synaptic input.
Conclusions
Our observations show that embryonic stem cell-based regenerative approaches may be successful in an acutely necrotic cellular environment.
NEUROGENESIS FOLLOWING CEREBRAL ISCHEMIA IN CEREBRAL CORTEX AND WHITE MATTER
Yasuaki Kokubo, Yasuhiko Matsumori, Atsushi Kuge, Sunao Takemura, Shinya Sato, Takamasa Kayama
Department of Neurosurgery, Yamagata University Faculty of Medicine, Yamagata, Japan
Backgrounds: Although cerebral cortex and white matter play an important role in brain function, there are few reports on neurogenesis in cerebral cortex and white matter following cerebral ischemia. Therefore we investigated cortical neurogenesis following focal cerebral ischemia (study1) and the dynamics of oligodendrocyte in corpus callosum with correlation of cognitive and motor function following chronic cerebral ischemia (study2) in rats.
Materials and Methods: (study 1) Male Sprague-Dawley (SD) rats (270-300 g) were subjected to 60-min middle cerebral artery occlusion. Proliferating cells were labeled by the cumulative administration of 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU) at 1, 2, 3, 4, 6, and 8 weeks after ischemia. We evaluated BrdU, NeuN positive cells in hippocampal dentate gyrus (DG) and the subventricular zone of the lateral ventricle (SVZ), and cerebral cortex by immunohistochemistry. (study 2) Male SD rats (210-250g) were subjected to permanent occlusion of bilateral common carotid arteries. We evaluated NG2 (maker of premature olidogendrocyte) and GST-π(maker of mature olidogendrocyte)positive cells in corpus callosum by immunohistochemistry after 2, 4, 6, 8 weeks of ischemia. For evaluation of cognitive and motor function, Morris's water-maze test and rotarod test were performed, respectively.
Results: (study1) BrdU-positive cells co-expressed NeuN in peri-infarct zone of cerebral cortex at 3 to 8 weeks after cerebral ischemia. BrdU-positive cells proliferated in the DG and SVZ. Their proliferation peaked at 3 weeks in the SVZ and at 4 weeks in the DG. (study 2) NG2 positive cells were increased at 4weeks of ischemia and GST-π positive cells were decreased at 2weeks of ischemia and then increased in corpus callosum, significantly. Deterioration of cognitive function was observed after 2weeks of ischemia. There was no motor dysfunction after ischemia. Although regeneration of oligodendrocyte was observed after 4 weeks of ischemia, deterioration of cognitive function was still remained.
Conclusions: We found no obvious migration of neural progenitor cells in cerebral cortex after ischemia. Therefore we postulate that endogenous dormant neural stem cells residing in the cortex are activated by ischemic insult to induce the proliferation of neural progenitors and differentiation into mature neurons. On the other hand, GST-π positive cells were increased after 4weeks of ischemia following decrease at 2 weeks. This result suggests that regeneration of oligodendrocyte occurs following chronic cerebral ischemia. However, we need further investigation regarding correlation with cognitive function. Although the issue is still controversial, progress of research on neurogenesis in cerebral cortex and white matter is required to develop the treatment for functional recovery of stroke patients.
GLUCOSE ACCELERATES DEATH AT REOXYGENATION IN A MODEL OF IN VITRO ISCHEMIA
Anna Serra-Perez, Anna M. Planas, Tomas Santalucia
Department of Pharmacology and Toxicology, IIBB-CSIC/IDIBAPS, Barcelona, Catalonia, Spain
Background and aims: Reduction of glucose and oxygen supply to the brain parenchyma can lead to neuronal cell death. Despite the immediate deleterious effects of ischemia, part of the subsequent tissue damage might be due to reperfusion. We have investigated the role of glucose in the induction of death after oxygen and glucose deprivation (OGD) followed by reoxygenation in an in vitro model.
Methods: Our model consisted of human neuroblastoma-derived SH-SY5Y cells differentiated with retinoic acid, which were subjected to 15h of OGD followed by a reoxygenation period of variable length, in which cells were incubated in control glucose concentrations.
Results: Counting of propidium iodide-positive nuclei in photomicrographs showed a significantly higher proportion of dead cells at 15h of OGD compared to cells that had been kept in the presence of glucose during anoxia for the same length of time. The difference widened in the hours following restoration of the oxygen and glucose supply. Nevertheless, incubating the cells with the glucose transport inhibitor cytochalasin B during reoxygenation or omitting glucose from the reoxygenation medium delayed cell death. This indicated that the utilization of glucose during reoxygenation impaired cell survival. The effects of glucose do not seem to depend on the availability of ATP, since analysis of the cellular ATP content showed that, although it was severely depleted at the end of the 15h-OGD, levels were eventually restored to almost normal after reoxygenation, regardless of the presence of glucose. We observed that by the end of the 15h-OGD, both cytochrome C and AIF were released from the mitochondrial fraction into the cytosol, which hinted at possible mechanisms mediating cell death after OGD and reoxygenation and that could involve the activation of apoptotic pathways. Nonetheless, we found no activation of caspase 3 during either the OGD or the reoxygenation periods, but an increase in the activity of calpain (as deduced from the analysis of the cleavage fragments of α-spectrin), which is often associated with necrotic death. Although nuclear import of AIF released from mitochondria has been associated with the development of caspase-independent apoptosis, we found no evidence for such import in our model.
Conclusions: In all, our data suggest that under these conditions OGD commits cells to a delayed death that is executed during reoxygenation in a way that requires the utilization of glucose by an as yet unknown mechanism.
TIME COURSE OF HUMAN SALIVARY STRESS PEPTIDES (AMYLASE, IGA, CHROMOGRANIN A) AFTRE PSYCHOLOGICAL STRESSOR EXAMINATION, UCHIDA-KRAEPELIN TEST
Koreaki Sugimoto1, Aya Kanai1, Noriaki Shoji2
1Department of Psychosomatic Medicine, Tohoku Fukushi University, Sendai, Japan, 2Department of Oral Diagnosis, Tohoku University Graduate School of Dentistry, Sendai, Japan
[BACKGROUND AND AIM]Salivary peptide such as salivary amylase, IgA, and chromogranin A are known to be related to stress response. These peptides are secreted with noradrenalin exocytosis in the salivary, and show up-regulation under stressful conditions. So these are considered stress markers. Salivary is easy to collect without invasion. But the time course of stress response of these peptides is not clear. In this study we measured the human salivary α-amylase, IgA, and chromogranin A from the same sample which are taken from volunteers who were stressed by Uchida-Kraepelin test (U-K test). [METHODS] Volunteers (n=8) are all healthy women, aged 20-30 years old, and have had no medications, pregnant, oral disease and habituation of alcohol drunk. They didn't have food and drink for 30 min before the test. U-K test, psychological stressor as well as personality examination, was performed to these volunteers for 30 min at room temperature in routine way. Their salivary were collected before the test and 5, 10, 15, 20, 25, 30, 40, 50, 60 min after the test. Salivary α-amylase was examined by chemiluminescence's method (dry chemistry system), and IgA and chromogranin A were examined by enzyme immuno assay. [RESULTS AND CONCLUSIONS] Salivary α-amylase did not increased after U-K test, however, salivary IgA and chromogranin A were increased. A previous report showed that salivary α-amylase did not increase after U-K test, but increased and responded quickly while U-K test performance. Salivary IgA and chromogranin A are good stress markers after psychological stress conditions. Moreover we analyzed the relationship between salivary stress response and person's character obtained by U-K test.
This work was carried out in the Kansei Fukushi Research Center of Tohoku Fukushi University, Sendai, Japan, supported by “Academic Frontiers” Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology(2004~2008).
EXPRESSION OF GNRH BY ELECTROACUPUNCTURE IN RATS IN DIFFERENT ESTROUS CYCLE
Shaojun Wang, Bing Zhu, Zhigao Jin
Institute of Acupuncture & Moxibustion, China Academy of Chinese Medical Sciences Beijing,100700, China
PACAP has been found neuroprotective in numerous models of cerebral ischemia. PACAP-induced neuroprotection in vivo is partially based on its vasorelaxant effect. Cerebral ischemia occurring during the perinatal period often result in neurological sequelae. One possible contributor to the neurological damage is the cerebrovascular dysfunction, e.g. hypercapnia-induced arteriolar vasodilation is severely attenuated after ischemia reperfusion (I/R). The mechanism of PACAP induced cerebrovascular changes in newborns is unexplored.
We sought to characterize the vascular reactivity to PACAP-27 and −38 (naturally existing forms), as well as to shorter PACAP sequences in a piglet model that correlates well with the vascular physiology of newborn babies. Using the non selective cyclooxygenase (COX) inhibitor indomethacin (5 mg/kg, iv), the selective COX-1 inhibitor SC 560 (1 mg/kg, iv) and the selective COX-2 inhibitor NS 398 (1 mg/kg, iv), we investigated if COX-derived metabolites play a role in the cerebrovascular effects of PACAP. We also determined the effect of the nitric oxide synthase (NOS) inhibition by N-nitro-l-arginine methyl ester (L NAME, 15 mg/kg, iv). We examined the influence of short PACAP fragments (10-5 M topically) on PACAP-27 and-38 induced vasodilation. Further, we tested whether application of PACAP preserves hypercapnia-induced cerebrovascular dilator responses after I/R. Anesthetized (Na thiopenthal 40 mg/kg, ip, followed by α chloralose 40 mg/kg, iv), ventilated piglets (1 day old, 1 2 kg, n=104) were equipped with closed cranial windows. Pial arteriolar diameters were determined via intravital microscopy.
Topical PACAP-27 and −38 elicited similar, repeatable, dose dependent pial arteriolar dilations. Percent changes in diameters to 10-8, 10-7, and 10-6 M PACAP-38 and-27 were 6±1, 16±2, and 40±4, and 9±2, 19±3, 36±4 (mean±SEM), respectively. In contrast, the shorter segments of the peptides (6-27, 6-38, 6-15, 20-31, and 1-15) did not display any vasoactivity. Arteriolar dilations to PACAP-38 were abolished 20 min after indomethacin and SC-560 (to 10-6 M 35±4% vs. −1±1%*, and 36±6% vs. 1±1*, *p<0.05), but the responses were unaffected by NS-398 (to 10-6 M 39±4% and 45±8%, before and after the treatment, respectively). L NAME was also ineffective (34±5% vs. 26±5%). Co-application with PACAP 6-27 and 6-38 (putative PACAP receptor antagonists) reduced the PACAP induced caliber changes efficiently (e.g. changes to 10-6 M PACAP-38 were 46±10% vs. 7±3%* without and with PACAP 6-27), however other short segments had no impact on this response. Graded hypercapnia resulted in large, concentration-dependent, reversible increases in pial arteriolar diameters. Pretreatment with PACAP (10-8 M, topically for 30 minutes) preserved the hypercapnia-induced vasodilation after I/R (62±5% and 56±3% to 10% CO2 before and after I/R).
In summary, PACAP is a potent vasodilator in the neonatal cerebral circulation, and this vascular reaction depends critically on COX-1 derived prostanoids. The role of NO seems to be ancillary. Preservation of arteriolar dilator responsiveness to hypercapnia by PACAP may contribute to neuroprotection.
Supported by the Hungarian Research grants: OTKA-T046531, ETT-2006 and NKTH.
FRACTIONATED MANGANESE-ENHANCED MAGNETIC RESONANCE IMAGING
Nicholas A. Bock, J.B. Mackel, A.C. Silva
Cerebral Microcirculation Unit, Laborartory of Functional and Molecular Imaging, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
Introduction: In manganese-enhanced MRI (MEMRI), manganese is administered systemically to an animal to improve contrast in the neuroarchitecture and to probe neuronal function. To maximize contrast, a large dose is administered at the risk of eliciting acute toxic side-effects. In the present work, we investigate administering manganese in small, fractionated doses over time to 1 lessen the acute toxicity of the Mn2+ for susceptible organs like the liver and heart, and 2 provide multiple periods of Mn2+ exposure in an animal to perform functional stimulations or pharmacological challenges.
Methods: Twenty male Sprague-Dawley rats (222-286 g) were used under the guidelines of the NINDS/NEI, NIH ACUC. For single-dose studies, animals received a tail-vein injection of 180 mg/kg MnCl2?4H2O as a 100mM solution pH-buffered in bicine and infused at 1.25 ml/h. The other rats received fractionated intraperitoneal doses of 30 mg/kg MnCl2?4H2O as a 25 mM solution every 48 hours to allow the Mn2+ to clear the heart and liver before the next dose. Animals were imaged live at 48 hours post-injection on a 7T Bruker animal MRI with a T1-weighted 3D sequence (FSE, rare factor = 2, TE = 12.3 ms, TR=150 ms, isotropic resolution = 167 µm). B1 inhomogeneity in the surface coil images was corrected using a transmit coil reference image, and the signals from various regions in the brain were measured and normalized to the signal from muscle in the head for inter-animal comparisons.
Results: The signal in the majority of brain regions saturated around a total cumulative dose of 180 mg/kg in rats receiving fractionated doses up to 270 mg/kg. The figure shows representative images at various fractions (top) and for the single 180 mg/kg dose (bottom). Both the contrast and SNR are better in the single-dose image and fine details of the neuroarchitecture, such as cortical lamination, are better visible. Regions that significantly enhanced at the p=0.05 level in the single-dose rats included the thalamus, CA1 and CA3 of the hippocampus, the hypothalamus, and the striatum.
Conclusion: Fractionating the Mn2+ dose greatly lessened the toxic burden; all fractionated rats appeared healthy after 18 days of injections, while the single-dose rats were euthanized after imaging because of necrosis in their tails and lethargic behaviour. The low signal change and faster saturation in the cortex suggests it receives Mn2+ via anterograde transport from other regions closer to the ventricles. Finally, the lower final signal in the fractionated rat images suggests that the cumulative manganese uptake is affected by a lower driving gradient following the 30 mg/kg versus the 180 mg/kg dose. Overall, fractionated MEMRI produced good images with a healthier outcome for the animals.
REGIONALITY AND RATE OF SODIUM ACCUMULATION IN RAT FOCAL CEREBRAL ISCHEMIA BY SODIUM MRI
Victor E. Yushmanov1, Alexander Kharlamov1, Boris Yanovski1, George LaVerde3, Fernando E. Boada3, Stephen C. Jones1,2
1Department of Anesthesiology, Allegheny Singer Research Institute, Pittsburgh, PA, USA, 2Department of Neurology, Allegheny Singer Research Institute, Pittsburgh, PA, USA, 3Department of Radiology, MR Research Center, University of Pittsburgh, Pittsburgh, PA, USA
Background and Aims
The apparent diffusion coefficient (ADC) is a marker for the extent but not severity of ischemic stroke. We proposed 23Na MRI to characterize ischemia by brain sodium concentration ([Na+]br) and to determine precisely the stroke onset time to establish eligibility for thrombolytic therapy (1). Herein, we hypothesize that 1) the rate of [Na+]br elevation ('slope’) in the rat model of focal ischemic stroke is the same in the cortex (where collateral circulation is present) and caudate putamen (CP) without collateral circulation; 2) regional [Na+]br is more sensitive than ADC for assessing ischemic damage.
Methods
ADC maps reconstructed from diffusion-weighted MRI showed ischemic lesions in 9 rats with MCAO. 23Na MRI was performed at 3T using twisted projection imaging (2). The infarct size and location were verified by the change in surface reflectivity of ischemic tissue and MAP2 immunohistochemistry (3). [Na+]br and [K+]br in half-milligram brain samples were determined by flame photometry and histochemical K+ staining (4).
Results
Frontal cortex, parietal cortex, CP, and the site of the maximum [Na+]br slope were analyzed for ADC deficit and slope using homotopic contralateral regions as reference. The ADC deficit was uniform over time and between different ischemic regions (ADCi/ADCc mean 0.63·0.02). The slopes in frontal, parietal cortex and CP were not different (p>0.3, mean 16·2%/h). Within an ADC-homogeneous infarct region (p>0.7), sites of significantly higher slope (mean 22·4%/h, p<0.005) were observed. They were located near the stroke periphery in 7 animals. In the ADC-deficient regions, there was no slope/ADC correlation (Fig. 1). In four instances, Na accumulation occurred in the areas with normal ADC adjacent to the ischemic cortex.
Conclusions
The slope differences observed within the ischemic area suggest that 23Na MRI can characterize the ischemic damage. 23Na MRI is related to regional metabolic changes, in particular, the functionality of Na/K-ATPase and other ion pumps, and thus provides physiological information not available by ADC. The rate of [Na+]br increase is independent of the tissue potential for collateral arterial supply with maximum slopes observed at the periphery of the ischemic core.
Support: NIH R01-NS30839
SEGMENTATION OF MR PERFUSION IMAGES FOR PATIENTS WITH CAROTID STENOSIS USING INDEPENDENT COMPONENT ANALYSIS
Yi-Hsuan Kao1, Michael Mu-Huo Teng2,3, Yu-Ching Lin1, Io-Pong Lam1, Yi-Hui Hsiao1
1Department of Biomedical Imaging and Radiological Sciences, National Yang Ming University, Taipei, Taiwan-R.O.C., 2School of Medicine, National Yang Ming University, Taipei, Taiwan-R.O.C., 3Department of Radiology, Taipei Veterans General Hospital, Taipei, Taiwan-R.O.C.
Background and aims: Hemodynamic parametric images calculated from magnetic resonance (MR) perfusion images are used to evaluate cerebrovascular diseases. However, it is difficult to assimilate all available information on the parametric images. We report the segmentation of MR perfusion images for carotid stenosis patients using independent component (IC) analysis (1),(2).
Methods: MR perfusion images acquired from carotid stenosis patients were retrospectively segmented using the FastICA algorithm (1). For quantitative analysis, regions of interest for different tissue types were selected from the segmentation results and various parametric images were calculated.
Results: Figures A - F illustrate the IC segmentation results for a patient with a 95% right internal carotid stenosis and bad collateral blood flow. Figure (A) displays the normalized signaltime curves for the five output IC images shown in (B)-(F). The arterial-phase IC1 image (B) demonstrates a lack of the right-side middle and anterior cerebral arteries. The delay-supplied arteries, immediate gray matter, and collateral normal gray matter appear on IC2 image (C). Severely delay-perfused brain parenchyma at the stenotic side appears on the venous-phase IC3 image (D). Choroid plexus is separately displayed on IC4 image (E). Cerebrospinal fluid and white matter are shown on IC5 image (F). The relative cerebral blood volume image (G) demonstrates a slight hyper intensity area at the lesion site and this area corresponds to the delay-perfused area seen on the time to peak (H), Tmax (I), and mean transit time (J) images. Quantitative measurements on time to peak of the concentration-time curves for arteries at normal and stenotic sides were found to be related to the severity of stenosis observed on angiograms.
Conclusion: The IC analysis technique can simultaneous provides spatial and temporal information on local blood perfusions for carotid stenosis patients. The information correlates well with parametric images as well as angiograms.
DIFFERENCES IN HIPPOCAMPAL MR FINDINGS WITH WHITE MATTER SUPPRESSION SEQUENCE IN MCI AND ALZHEIMER DISEASE USING 3 TESLA MRI
Junko Takahashi1, Satoshi Takahashi1, Hisashi Yonezawa1, Masako Kudo1, Satoko Obara1, Toshihide Shibata1, Yasuo Terayama1, Makoto Sasaki2, Takashi Inoue3
1Department of Neurology, Iwate Medical University, Morioka, Iwate, Japan, 2Department of Radiology, Iwate Medical University, Morioka, Iwate, Japan, 3Department of Neurosurgery, Iwate Medical University, Morioka, Iwate, Japan
Background and aims: Patients with Alzheimer disease(AD)decline their cognitive ability in almost same manner as the disease progresses. The pathology of AD have been throughly investigated, while neuroimaging techniques have been limited its possibility mainly due to their resolution. Recently the advent of 3 Tesla (3T) MRI provides fine anatomical details with high spacial and contrast resolutions to visualize regional cell loss in minute structures. To visualize and compare regional gray matter cell density of hippocampus, entorhinal area and substantia innominata using 3T MRI in patients with AD or mild cognitive impairment (MCI), inversion recovery (IR) sequence was employed. Methods: Subjects were 43 patients with AD (16 males and 27 females), 10 patients with MCI (3 males and 7 females; 74.3 ± 6.7 years old), and 12 controls (6 males and 6 females, 72.5 ± 13.8 years old) without neurological deficits whose MRI were normal. Functional Assessment Staging (FAST) among AD patient was IV for 27 patients (9 males and 18 females; 73.4 ± 8.8 years old), V for 9 patients (3 males and 6 females; 74.1 ± 7.2 years old), VI for 7 patients (4 males and 3 females; 68.3 ± 10.1 years old). All subjects underwent Mini-Mental State Examination (MMSE), Wechsler Adult Intelligence Scale-Revised (WAIS-R), Wechsler Memory Scale-Revised (WMS-R), Modified Wisconsin Card Sorting Test and neurological examination. All scans were performed using a Signa VH/I 3.0 Tesla MR imaging system (GE Medical Systems, Milwaukee, WI) and a standard head coil. We employed IR sequence (TR 4000ms, TE 20ms, TI 250 ms, matrix 256×256, FOV 220×220 mm, 3 mm thickness and 1 mm gap) to suppress white matter signals, words for emphasizing gray matter signals. Using gray matter emphasized image, ROIs were positioned on CA1, subiculum, entorhinal area and substantia innominata and their intensity were measured. Prior to IR sequence, all cases underwent conventional spin echo T2 weighted sequence (TR 3800ms, TE 80 ms, matrix 512×256, FOV 220×220 mm, 5 mm thickness and 1.5 mm gap). Results: The intensity in subiculum and entorhinal area were significantly decreased compared with CA1 (p<0.05) from MCI stage of each AD patients. On the other hand, intensity of substantia innominata, which was unchanged during MCI stage, declined after FAST IV stage of AD (p<0.05). Conclusions: By using 3T MRI, we can visualize detailed morphological changes of hippocampus, entorhinal area and substantia innominata, and furthermore observe longitudinal changes of those structures from MCI to AD patients. This method is a powerful tool to elucidate AD etiology and to evaluate the efficacy of anti-AD drugs including acetylcholinesterase inhibitor.
EVIDENCE FOR A VASCULAR CORRELATE OF THE BEREITSCHAFTPOTENTIAL USING NEAR-INFRARED SPECTROSCOPY
Christoph Drenckhahn1,2,3,4, Hellmuth Obrig2,4, Jens Steinbrink2,4, Hannes Duemmler1, Matthias Kohl-Bareis4,5, Arno Villringer3,4, Dreier Jens1,2,4
1Department of Experimental Neurology, Charite, Berlin, Germany, 2Department of Neurology CCM, Charite, Berlin, Germany, 3Department of Neurology CBF, Charite, Berlin, Germany, 4Berlin Neuroimaging Center, Berlin, Germany, 5RheinAhr Campus, University of Applied Sciences Koblenz, Remagen, Germany
Aim: Using near-infrared spectroscopy (NIRS) in human healthy subjects we investigated alterations in deoxy-hemoglobin (deoxy-Hb) concentrations as an indirect parameter for a hemodynamic response in the primary motor cortex (M1) and the frontal medial wall areas (FMWA). The FMWA comprises higher-order motor areas including the supplementary motor area, which are involved in preparation and initiating of voluntary movement. M1 and FMWA were measured simultaneously in order to identify a correlate of the Bereitschaftspotential following voluntary movement.
Method: 16 strongly right-handed volunteers underwent different finger tapping tasks which are known to be associated with increase in cerebral blood flow in M1 and FMWA. The tasks were designed as a block with repetitive finger tapping for 20s or as single trials with finger tapping either self-paced or externally triggered. 8 light sources and 8 detectors were positioned over the left M1 and the FMWA covering an area of 12.5×7.5 cm. Time course and amplitude were analysed for the concentration of deoxy-Hb. For spatial analysis, the recording points with significant decrease in deoxy-Hb were determined.
Results: We detected a circumscribed decrease in deoxy-Hb at two different locations corresponding to FMWA and M1 during functional activation. The FMWA showed significantly earlier decrease of deoxy-Hb compared with the M1 (self paced: ?†t=0.78 s, p<0.01 / cued: ?†t=0.54 s, p<0.01) demonstrating a vascular correlate of the Bereitschaftspotential. Onset in SMA started significantly earlier following self initiated movements than externally triggered movements (?†t=0.57 s, p<0.01) whereas the M1 failed to reach significance (?†t=0.32 s, p=0.12). Significant changes in deoxy-Hb were located within an area of ~10 cm2 in projection on M1 and ~15 cm2 in projection on SMA, respectively.
Conclusion: We established a non-invasive bedside method to measure changes of deoxy-Hb reflecting alterations in cerebral blood flow in primary and secondary motor areas with a high temporal resolution during functional activation in healthy subjects. Simultaneous investigation of M1 and SMA indicated a change of deoxy-Hb related to the Bereitschaftspotential.
SIGNIFICANCE OF HYPOINTENSE LEPTOMENINGEAL VESSELS ON T2*-WEIGHTED GRADIENT ECHO IMAGING FOR PREDICTION OF ACUTE ISCHEMIC LESION GROWTH
Kyung-Hee Cho, Hye J. Kim, A-Hyun Cho, Sun U. Kwon, Jong S. Kim, Dong-Wha Kang
Department of Neurology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea
Background and Purpose
For the response to reduced cerebral perfusion pressure, oxygen extraction fraction (OEF) increases in brain regions with reduced cerebral blood flow to preserve oxygen metabolism. T2*-weighted gradient echo imaging (GRE) is sensitive to blood deoxygenation, which results in hypointense signals in blood vessels. Thus, hypointense leptomeningeal vessels (HLV) on GRE in hypoperfused region may indicate the presence of tissue with increased OEF. The relationship of this vessel sign with regional tissue fate remains unknown in acute ischemic stroke. We sought to examine the prognostic value of the HLV on GRE for the prediction of acute ischemic lesion growth.
Methods
We included consecutive acute ischemic stroke patients who 1) had an acute infarction in the territory of middle cerebral artery (MCA), 2) underwent diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI) and GRE within 24 hours of stroke onset and follow-up DWI 5 days later, and 3) had a baseline PWI-DWI mismatch (mean transit time lesion greater than DWI lesion >20%). Patients receiving thrombolysis or neurointerventional therapy were excluded. The presence of HLV on GRE within the territory of mean transit time deficit was determined. Patients were divided into 3 groups: Group A, no HLV; Group B, HLV with increased cerebral blood volume (CBV); and Group C, HLV without increased CBV. The ischemic lesion volume was measured blind to the clinical and other imaging information. The infarct volume on baseline and follow-up DWIs and the difference between the two DWIs were compared among 3 groups. The tissue fate of the area with HLV was determined blind to the information of CBV.
Results
Of the 81 patients who met the inclusion criteria, HLV was observed in 50 (61.7%). CBV was increased in 34 of these 50 patients (68.0%). The ischemic lesion growth (18.4 cc in Group A (n=31), 23.2 cc in Group B (n=34), and 28.7 cc in Group C (n=16)) was significantly different among 3 groups (p=0.030, Kruskal-Wallis test), with infarct growth being the greatest in Group C: there was no difference between Groups A and B (p=0.46), while significant difference existed between Groups A and C (p=0.020) and between Groups B and C (p=0.017). In terms of tissue fate of the area with HLV, progression to infarction was more frequent (p=0.005) in Group C (9 of 16, 56.3%) compared to Group B (5 of 34, 14.7%).
Conclusions
The presence of HLV on GRE, when associated with no increase of CBV, may indicate a hemodynamic marker of impending irreversible ischemic injury.
STRIATAL AND EXTRASTRIATAL DOPAMINE D2 RECEPTOR OCCUPANCY BY PALIPERIDONE EXTENDED-RELEASE TABLETS: A PET STUDY
Ryosuke Arakawa1,2, Hiroshi Ito1, Akihiro Takano1, Hidehiko Takahashi1, Takuya Morimoto1, Takeshi Sassa3, Katsuya ota4, Motoichiro Kato5, Yoshiro Okubo2, Tetsuya Suhara1
1Molecular Neuroimaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan, 2Department of Neuropsychiatry, Nippon Medical School, Tokyo, Japan, 3Asai Hospital, Chiba, Japan, 4Onda-Daini Hospital, Chiba, Japan, 5Department of Neuropsychiatry, Keio University School of Medicine, Tokyo, Japan
Background and aims: Paliperidone is a novel antipsychotic drug in development for the treatment of schizophrenia. It shows almost the same pharmacological profile as risperidone of which it is the active metabolite with high affinity for dopamine D2 receptor and serotonin 5-HT2 receptor. Paliperidone ER is the extended-release (ER) formulation of paliperidone, which enables low peak to trough fluctuations and it was approved by FDA (USA) for the treatment of schizophrenia in mid December 2006. In this study, we evaluated the therapeutic dose range of paliperidone ER in the patients with schizophrenia using positron emission tomography (PET). The difference in dopamine D2 receptor occupancy by paliperidone ER between the striatal and extrastriatal regions was also investigated.
Methods: Thirteen male patients (age range 22-40 yr; mean ± S.D., 29.4 ± 5.4 yr) who were diagnosed with schizophrenia according to the DSM-IV criteria participated in this study. They received no more than one oral antipsychotic drug prior to the entry. This study was conducted as a part of an open-label phase IIa trial of paliperidone ER in Japan (Sponsored by Janssen Pharmaceutical K.K.). Before the study initiation, a written informed consent (IC) was obtained from all patients participated. The paliperidone ER doses were 3 mg/day in 6 patients, 9 mg/day in 4 patients and 15 mg/day in 3 patients given once daily. The clinical symptoms were assessed with the positive and negative symptom scale (PANSS) at baseline (pre-dose) and at endpoint. The extrapyramidal symptoms were assessed by clinical observations. Two PET scans per patient were performed at least 2 weeks after the initiation of treatment with paliperidone ER on a same day with [11C]raclopride and [11C]FLB 457 for measurement of striatal and extrastriatal dopamine D2 receptor occupancy, respectively. This study was approved by the Ethics and Radiation Safety Committee of National Institute of Radiological Sciences, Chiba, Japan.
Results: The dopamine D2 receptor occupancy in the striatum measured with [11C]raclopride was 54.2% to 85.5% and that in the temporal cortex measured with [11C]FLB 457 was 34.5% to 87.3%. ED50 in the striatum was 2.38 mg/day and 6.65 ng/ml and that in the temporal cortex was 2.84 mg/day and 7.73 ng/ml. There was no significant difference of dopamine D2 receptor occupancy between the striatum and the temporal cortex at any doses. Average PANSS scores of all patients were 62.9 ± 16.5 at baseline and 58.5 ± 16.8 at endpoint. Three patients, 2 patients in 15mg and 1 patient in 9mg, showed extrapyramidal symptoms during the course of the study.
Conclusions: The data from this study suggests that about 5-10 mg of paliperidone ER has a comprehensive benefit-risk ratio. No significant difference of dopamine D2 receptor occupancy was observed between striatal and extrastriatal regions, indicating no regional selectivity of paliperidone.
THE EFFECT OF CAROTID ANGIOPLASTY AND STENTING (CAS) ON CEREBRAL ACTIVATION DURING FINGER MOVEMENTS: A FUNCTIONAL MAGNETIC RESONANCE IMAGING STUDY
Tai-Cheng Wu1, June Hung1, Jiun-Jie Wang2, Yeu-Jhy Chang1, Chien-Hung Chang1, Ting-Yu Chang1, Hsiu-Chuan Wu1, Tsong-Hai Lee1
1Department of Neurology, Chang Gung Memorial Hospital, LinKou Medical Center, Taoyuan, Taiwan-R.O.C., 2Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan-R.O.C.
Background Previous studies in patients who underwent carotid angioplasty and stenting (CAS) due to severe carotid stenosis have observed a significant hemodynamic increase in peak systolic and mean flow velocity by transcranial Doppler imaging associated with changes in cerebrovascular reactivity at 2 days and at 2 to 4 months postoperatively. However, it remains unclear whether task-related brain activation could be altered soon after CAS. Here we used functional magnetic resonance imaging (fMRI) to examine the effect of CAS on neural correlates of a motor task.
Methods Three male patients (median age 68 years, range 56-84) who underwent unilateral CAS participated this study. Each patient attended two separate fMRI sessions, on the day prior to CAS and on the day no later than 5 days after CAS respectively. During each scanning session, blood oxygenation level dependent (BOLD) sensitive images were acquired whilst patients were performing a finger tapping task. Image data were analyzed with statistical parametric mapping 2 (SPM2).
Results Prior to CAS, unilateral finger tapping activated contralateral and/or ipsilateral motor association areas, suggesting incomplete motor lateralization for some individuals. After CAS, all patients showed variable degrees of increase in BOLD signals in the motor association areas on the same side of CAS, specifically in response to movements of contralateral fingers only (cases no. 1 and no. 3) or ipsilateral fingers only (case no.2).
Conclusion Our study suggests that unilateral CAS may rapidly alter the pattern of cerebral activations, in response to the movement of one specific hand only. These effects on task-related cerebral activation may reflect the improvement of vasomotor reactivity following CAS.
EVOLUTION OF CMRO2 DURING STROKE RECOVERY IN RAT MODELS
Young R. Kim1, Emiri Tejima2, Bruce R. Rosen1
1MGH, MIT, HMS Athinoula A. Martino's Center for Biomedical Imaging, Harvard School of Medicine, Charlestown, MA, USA, 2Neuroprotection Laboratory, MGH, Harvard School of Medicine, Charlestown, MA, USA
Stroke impairs neuro-vascular functions at the cellular level and causes sensorimotor impairments. Some degree of sensorimotor recovery subsequently occurs, yet the changes in basal level function that are directly linked to this recovery are not well understood. Moreover, understanding and monitoring stroke recovery in humans using non-invasive brain imaging of Blood Oxygenation Level Dependent (BOLD) functional magnetic resonance imaging (fMRI) is hampered by uncertainties surrounding the relationship between restoration and remodelling of all of the functional components of the neurovascular unit (NVU) during the recovery process. Our data, based on the fMRI using electrical forelimb stimulation in a rat stroke model suggest a differential evolution in hemodynamic signals and metabolic measures during the period of sensorimotor recovery. In particular, early after stroke (~1day), when sensorimotor impairment is most severe, the ipsilesional sensorimotor cortex exhibits proportionately greater loss in CMRO2, than CBF and CBV, resulting in a smaller loss in BOLD (Figure 1). Thereafter, as sensorimotor recovery occurs post-stroke, the ipsilesional sensorimotor cortex exhibits early and progressive increases in CMRO2, with delayed increases in CBV and CBF responses. As a result, it is suggested that the level of sensorimotor function throughout the recovery process is strongly correlated with CMRO2 in the ipsilesional sensorimotor cortex, and less correlated with BOLD (Figure 1).
Figure 1. Representative functional activation maps of rat brains 1 day and 14 days (left and right two columns, respectively) after the 2 hour-occlusion of MCAO. First row: T2-wighted images and T2 maps. Second-fifth row: BOLD, CBF, CBV, and CMRO2 activation maps in response to electrical stimulation of unaffected and affected sides. Note decrease in BOLD, but increase in CMRO2, between 1 and 14 day time points.
AMYLOID IMAGING IN ALZHEIMER'S DISEASE AND MCI BY [11C]-PIB PET
Hitoshi Shimada1,2, Hitoshi Shinoto1, Shigeki Hirano1, Kouichi Sato1, Noriko Tanaka1, Tsuyoshi Ota1, Kiyoshi Fukushi1, Tetsuya Suhara1, Takamichi Hattori2, Toshiaki Irie1
1Molecular Probe Group, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba-Shi, Chiba, Japan, 2Department of Neurology, Chiba University Faculty of Medicine, Chiba-Shi, Chiba, Japan
Background and aims: Deposits of beta-amyloid plaques in the brain are one of the most characteristic neuropathological changes of Alzheimer's disease (AD). Pittsburgh Compound-B (PIB) is an amyloid-binding radiotracer for Positron emissipn tomography (PET), and using it can estimate the beta-amyloid deposition in vivo. In this study, we investigated the pattern of beta-amyloid deposition in patients with AD and mild cognitive impairment (MCI) by PET. Methods: Participants were 12 patients with AD (mean age: 69+/−8 years; male/female: 3/9; MMSE: 18+/−4), 8 MCI (mean age: 75+/−4 years; male/female: 5/3; MMSE: 27+/−2) and age-matched 13 healthy controls (mean age: 64+−9 years; male/female: 4/9; MMSE: 28+−2). A dose (about 10mCi) of [11C]-PIB was i.v. injected and sequential PET scans were performed for 90min PET scans with arterial blood sampling. Data were analyzed using Logan plot graphical analysis with the cerebellum as a reference region. Beta-amyloid deposition were compared between AD group and healthy control group using t-test in statistical parametric mapping 5 (SPM). Same statistical analysis was performed between MCI group and healthy control group. RESULTS: The SPM showed that amyloid deposition was significantly higher in parietal association area, cingulate, frontal association area and lateral temporal cortex in the AD group (uncorrected p<0.0001, extent>50), though no significant deposition were shown in sensorimotor, medial temporal and occipital cortexes. Five of eight patients with MCI showed the AD-like pattern and the rest showed healthy control-like pattern. Conclusion: The characteristic brain amyloid deposition was shown in AD by PET. The pattern of the amyloid deposition in MCI group was devided into two group, AD-like group and healthy control-like group. It may suggest that the former (AD-like group) will convert to AD and the latter (healthy control-like group) will remain the amnestic MCI. Follow-up study is needed.
RECOVERY OF CEREBRAL OXYGEN METABOLISM AFTER COGNITIVE REHABILITATION IN PATIENTS WITH HIGHER CORTICAL DYSFUNCTION DUE TO TRAUMATIC BRAIN INJURY
Mitsuhito Mase1, Kazuo Yamada1, Hidehiro Kabasawa2, Tetuso Ogawa2, Junko Abe2, Yuri Nagano2, Akihiko Iida3
1Department of Neurosurgery and Restorative Neuroscience, Nagoya City University Graduate School of Medical Science, Nagoya, Japan, 2Departments of Rehabiliation, Nagoya City Rehabilitation and Sports Center, Nagoya, Japan, 3Departments Radiology, Nagoya City Rehabilitation and Sports Center, Nagoya, Japan
Background and Purpose: Neuropsychological impairment is one of the most crucial sequelae after mild traumatic brain injury (TBI), disturbing resumption of one's works or previous daily activity. We have already shown that the severity of this higher cortical dysfunction correlated to a decrease of cerebral metabolic rate of oxygen (CMRO2) and the severity of diffuse axonal injury [1]. After cognitive rehabilitation, these symptoms could improve to a certain extent by one or two years. However, the mechanism and time-course of the recovery have not been clearly shown. In order to clarify this, we studied cerebral blood flow (CBF) and metabolism using positron emission computed tomography (PET) in patients with these sequelae before and after cognitive rehabilitation.
Methods: Four patients (male 3, female 1, mean 26.0 year old) with neuropsychological impairments such as disturbances of memory, attention, and / or information processing after TBI due to traffic accidents were included in this study. The mean interval between the injury and the estimation was 6–9 (mean: 7.25) months. CBF, CMRO2, oxygen extraction fraction (OEF), cerebral metabolic rate of glucose (CMRGlu), and distribution of benzodiazepine receptors of brain (BZR) were measured before and after the 6-month cognitive rehabilitation. Higher cortical dysfunction was evaluated using Wechsler Adult Intelligence Scale-Revised (WAIS-R), Miyake's memory test (Miyake MT), Rey-Osterrieth complex figure test (Rey's Figure), Rivermead Behavioral Memory Test (RBMT), and paced auditory serial addition task (PASAT).
Results: After cognitive rehabilitation, scores of WAIS-R and Miyake's MT significantly improved in all cases. Rey's Figure, RBMT, and PASAT also improved, but the extent of recovery was varied in each patient. In all patients, CBF and OEF were within normal ranges, however, CMRO2 slightly decreased (luxury perfusion state) in almost all area before rehabilitation. The area where CMRO2 decreased was much wider than the area where cerebral contusion existed on MRI. CMRO2 improved in the whole brain in all patients after rehabilitation. CMRGlu also improved slightly after recovery of symptoms in 3 of the 4 patients, but one patient with decreased CMRO2 showed normal CMRGlu. BZR activity decreased in two cases before, which did not change after the rehabilitation.
Conclusions: Relative luxury perfusion state (decrease of CMRO2 with normal CBF and OEF) was observed in patients with cognitive impairments in almost 7 months after mild TBI. These symptoms improved after cognitive rehabilitation, and CMRO2 improved simultaneously. Changes of CMRGlu showed the similar tendency to changes of CMRO2. However, the sensitivity of the change was not good. BZR activity is not suitable to estimate cognitive impairment. We conclude that CMRO2 using PET can become a useful and objective marker for estimate severity and recovery of cognitive impairment due to diffuse axonal injury.
REACTIVITY OF CEREBRAL BLOOD VESSEL IN CADASIL
Yoshinari Nagakane1, Mizuno Toshiki1, Hideaki Tomimoto2, Makaki Kondo1, Noriko Ishigami1, Aiko Tamura1, Takahiko Tokuda1, Yo Ushijima3, Tuneo Nishimura3, Masanori Nakagawa1
1Department of Molecular Neurology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Department of Neurology, Kyoto University, Kyoto, Japan, 3Department of Radiology, Kyoto Prefectural University of Medicine, Kyoto, Japan
<Purpose>
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary microangiopathy due to a mutation in the Notch3 gene, leading to cerebral white matte lesions, infarctions and finally vascular dementia. Reactivity to acetazolamide in CADASIL patients reduced in the previous reports using MRI or transcranial doppler sonography (TCD). To test whether reactivity of cerebral blood flow reduce in a CADASIL patient, we evaluated CADASIL patients before and after acetazolamide injection using Iodine123- iodoamphetamine single photon emission computed tomography (IMP-SPECT).
<Method>
Two Cases diagnosed as CADASIL by family history, neurological examination, MRI studies and skin biopsy enrolled to this study. Genetic analysis revealed a heterozygous missense mutation in the Notch 3, producing an Arg141Cys substitution.
IMP-SPECT was performed with a single blood sample before, and after the injection of acetazolamide. Cerebral blood flow in the ROI was calculated quantitatively. ROI was set frontal, temporal, parietal, occipital, basal ganglia, brainstem and cerebellar hemisphere.
<Results>
The average of cerebral blood flow increased more than 100% in the cortex except temporal lobe in CADASIL patient. Non-demented CADASIL patients showed normal cerebral blood flow and dynamic increase of cerebral blood flow in the cerebral white matter after injection of acetazolamide.
<Discussion>
Although a demented CADASIL patient showed low cerebral blood flow in the whole cortex, reactivity to acetazolamide was preserved in the cortex except recently infarcted area. A non-demented CADASIL patient showed normal cerebral blood flow at rest and hyperreactivity was observed only in the white matter. Our data suggests that quantitative IMP-SPECT is a useful tool for evaluating and monitoring reactivity of cerebral vessel in CADASIL patients.
IMPROVING THE VISUALIZATION OF SPATIOTEMPORAL CHANGES IN CEREBRAL BLOOD FLOW THROUGH INTACT RAT/MOUSE SKULL BY LASER SPECKLE TEMPORAL CONTRAST ANALYSIS
Pengcheng Li, Jianjun Qiu, Shaoqun Zeng, Qingming Luo
Wuhan National Laboratory for Optoelectronics, Britton Chance Center for Biomedical Photonics, Huazhong University of Science and Technology, Wuhan, China
Laser speckle flowmetry based on the laser speckle contrast analysis has been used to assess two-dimensional (2-D) cerebral blood flow (CBF) with high spatial and temporal resolution. However, it can only be applied to the subjects whose skull were removed or thinned to transparency, or for the subjects whose skull were thin and transparent, such as mouse, but fails to reveal the CBF through the thick scattering skull. Here we describe a method that is able to reduce the influence of the static superficial scattering tissue on imaging the CBF and thus improve the visualization of CBF through the intact rat skull. It is based on the temporal statistics analysis of sequences of laser speckle images. Both physical model experiments and animal experiments were performed to demonstrate the feasibility of this method, and also its limitation. In the animal experiments, the spatiotemporal patterns of the propagation of changes in CBF during cortical spreading depression induced by KCl were investigated with the adult male rat and mouse, which were anesthetized and fixed in a stereotactic frame. The skin over the skull was removed and kept the skull intact. The experimental results show that comparing with the conventional laser speckle contrast analysis technique, our method significantly improve the resistance to the static superficial structure when the image area contains static scattering. Even for the case imaging CBF through mouse skull which is relative transparent, the results obtained by laser speckle temporal contrast analysis also show better image quality than conventional laser speckle contrast analysis.
Keywords: Cerebral blood flow, Laser speckle imaging, Intact skull
LARMINAR-DEPENDENT CBF RESPONSES INDUCED BY VISUAL AND HYPERCAPNIC STIMULATION
Tae Kim, Ping Wang, Seong-Gi Kim
Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
Background and aims
Metabolic changes accompanying neural activity modulate hemodynamic responses including CBF and CBV. Recently, relative CBF and CBV responses induced by neural activity are the highest in middle cortical layer IV where the highest capillary density and highest metabolic responses are observed, suggesting that hemodynamic responses are highly correlated to metabolic responses induced by neural activity (1),(2). However, it is still possible that laminar-dependent hemodynamic responses may not relate to metabolic responses, but merely reflect intrinsic laminar-scale regulation. To evaluate this issue, we measured laminar-dependent CBF responses induced by global, non-metabolic hypercapnic stimulation in addition to visual stimulation in isoflurane-anesthetized cats.
Methods
Four studies in three female adolescent cats were performed on a 9.4T MRI (Varian) system. The cat was orally intubated and mechanically ventilated under 0.8–1..2% isoflurane anesthesia in a 2:1 air:O2 mixture. Pancuronium bromide (~0.2 mg/Kg/h) was delivered through the cephalic vein. The end-tidal CO2 level was maintained in the range of 3.4 ? 3.6% by adjusting the respiration rate and volume, and the rectal temperature was controlled at 38.5 +/− 0.5 C with a feedback regulated hot water circulator. The animal's head was fixed with an in-house head frame. Binocular full-field visual stimuli were presented with square-wave high-contrast moving gratings (2 cycles/s) with 0.15 cycles/degree of spatial frequency during 30-s stimulation. For hypercapnic stimulation, a gas mixture with ~6% CO2 was inhaled during 30-s without changing other inhalation levels. CBF fMRI images were measured by the slice-selective (SS) and non-slice-selective (NS) inversion recovery images of a flow alternating inversion recovery (FAIR) method (TE = 20 ms, TR = 3 s including TI = 1.5 s) using a gradient echo echo-planar imaging pulse sequence on a single slice with matrix size = 64 * 64, field of view = 2 * 2 cm, and slice thickness = 0.2 cm. The acquisition order of NS and SS was alternatively changed. Cortical depth profile analysis for layer specificity was performed in area 18 within the visual cortex.
Results and discussion
CBF values in baseline conditions (without stimulation) were consistent in both visual and CO2 stimulation (R = 0.90 +/− 0.05). Large baseline CBF signal was observed in upper cortical layers, possibly due to the signal contribution from large arterial vessels. Functional CBF maps were successfully obtained during both visual and CO2 stimulation. Consistent with previous observations during neural activation, the largest relative changes in CBF occur at the middle of the cortex (~1 mm depth from the surface of the cortex) during visual stimulation. Interestingly, the similar observation was also found during hypercanic stimulation, which differs from our expectation of homogeneous CBF responses across layers. Our data indicate that larger CBF changes in middle cortical layers due to visual stimulation may not be caused by metabolic demand, but purely intrinsic layer-dependent vascular regulation.
QUANTITATIVE CEREBRAL BLOOD FLOW AND PERFUSION TERRITORIES MEASUREMENT IN NORMOTENSIVE AND HYPERTENSIVE RATS USING ARTERIAL SPIN LABELING
Fernando F. Paiva1,2, Erica C. Henning1, Alberto Tannus2, Afonso C. Silva1
1National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA, 2Instituto De Fisica De Sao Carlos, Universidade De Sao Paulo, Sao Carlos, SP, Brazil
INTRODUCTION
The spontaneously hypertensive rat (SHR) is a strain that exhibits spontaneous elevated blood pressure and that has been widely used as an experimental model of hypertension and stroke. However, a detailed characterization of resting CBF and angiographic morphology has not been yet performed in these rats. In the present study, a three-coil system was employed to obtain CBF and perfusion territory maps of the major feeding arteries to the brain in normotensive and hypertensive rats. In addition, MRAs were acquired to assess major differences in the vasculature between the two groups as well as variations in the formation of the Circle of Willis that are related to the vascular territories.
METHODS
Sprague-Dawley (SD) (N=6) and SHR (N=6) rats (300-350g), were anesthetized under isoflurane and scanned in a 7T/30cm magnet (Bruker-Biospin, Billerica, MA) equipped with gradients capable of 450mT/m amplitude (Resonance Research Inc, Billerica, MA). A home-built, transmit-only volume RF coil, and a receive-only quadrature surface coil (RAPID Biomedical GmbH, Rimpar, Germany), were used for image acquisition. A small figure-8 shaped labeling coil was positioned under the neck of the animal, approximately 2cm away from isocenter. Multi-slice, single-shot EPI were obtained during CASL: TR/TE=5115/38ms, FOV=2.56×2.56cm, matrix=96×96, slice thickness=1.5mm, 10 slices and 32 averages. Whole brain MRAs were acquired by using a 2D flow compensated gradient echo sequence: TR/TE=30/4.5ms, flip angle=90o, FOV=1.92×1.92cm, matrix=192×192, 218 slices, slice thickness=1mm and interslice distance=0.1mm.
RESULTS
Figure 1 shows multislice CBF maps obtained from an SHR rat labeling both (Fig. 1b) and only the right and left CCAs, respectively (Fig. 1c). Whole brain CBF values in SHR rats (147±25 ml/100g/min) were significantly higher than in normotensive rats (112±15 ml/100g/min, p<0.05). Regional variation in CBF in both groups is shown in Fig. 2 and was significantly higher in SHR rats in the sensory-motor cortex, but not in caudate putamen.
Asymmetry in the cingulate cortex corresponding to the territory of the anterior cerebral artery (ACA) is shown in Fig. 1c-d. CBF contrast in both sides was obtained by labeling the right CCA, but not the left. Fig. 3a shows a whole-brain MRA in this rat, where a dominance in the left A1 was observed (Fig. 3b), explaining the asymmetric pattern observed in the perfusion territories (Fig. 3c-d).
CONCLUSIONS
In this study we used CASL with a dedicated labeling coil to compare resting whole-brain CBF in nor-motensive and hypertensive rats. Higher CBF values were found in hypertensive rats, and asymmetric perfusion territories could be explained by higher lateral dominance of arteries in the circle of Willis. The CBF characterization in SHR rats will allow studies of vascular reactivity and stroke in these animals.
DIFFERENCIAL BRAIN ACTIVATION DUE TO EXPERIENCE OF VISCERAL STIMULI IN HUMANS: A PET STUDY
Toyohiro Hamaguchi1,2, Michiko Kano2, Motoyori Kanazawa2, Hisashi Rikimaru3, Satoshi Watanabe2, Masatoshi Ito3, Kazuhiko Yanai4, Shin Fukudo2
1Department of Occupational Therapy, Niigata University of Health and Welfare, Niigita, Japan, 2Department of Behavioral Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan, 3Division of Nuclear Medicine, Tohoku University, Cyclotron Radio Isotope Center, Sendai, Japan, 4Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan
Irritable bowel syndrome is considered to be a disorder of brain-gut interactions, particularly of visceral hypersensitivity. However, the brain area related to initial sensitization provoked by the programming of visceral stimulation is still unknown. The hypothesis of this study is that brain activation due to the colonic distention is different between with initial intense stimulation and with intense stimulation after the mild stimulation, and its also hypothesized that brain activation due to sham colonic distention is different between before intense stimulation and after intense stimulation.
Forty-five normal volunteers participated in the study. Using a colonoscope, a bag was inserted into the descending colon and the colon was distended with barostat. Orders of stimuli was set with six different patterns. Regional cerebral blood flow was measured by PET. Brain image was analyzed with SPM 2.
The main effect of the sham stimulation (0 mm Hg) after 40 mm Hg distention denoted activation of the right cingulate gyrus (Brodmann area; BA 24, Z = 4.16 uncorrected p < 0.001), right insula (BA 13, Z = 4.08), and right medial frontal gyrus (BA46, Z = 3.92).
These findings suggest that the brain may behave differently in response to the different order of visceral stimulation. The determinants of the modulation of visceral perception and emotion may include the combined effects of experience of the stimuli.
123I-IOMAZENIL SPECT PERFORMED DURING THE ACUTE STAGE FOR PATIENTS WITH TRAUMATIC BRAIN INJURY: A NEW APPROACH TO EVALUATING CORTICAL DAMAGE
Hiroyasu Koizumi, Hirosuke Fujisawa, Tetsu Kurokawa, Shoichi Kato, Koji Kajiwara, Tatsuo Akimura, Sadahiro Nomura, Masami Fujii, Michiyasu Suzuki
Department of Neurosurgery, Yamaguchi University, School of Medicine, Ube, Yamaguchi, Japan
<Background> 123I-iomazenil were developed as radioligands of central benzodiazepine receptors distributed mainly in the cerebral cortex neurons. Because of the specific distribution of benzodiazepine receptors, these radioligands have been successfully applied to detect neuronal integrity in acute brain strokes.
Also, in the case of traumatic brain injury, neurons were irreversibly damaged in the cerebral cortex following cortical contusion injury. In addition to the morphological changes detected by CT/MRI scans, neuronal damage to the cortex post-contusion was also functionally visualized on 123I-iomazenil (IMZ) SPECT as a region of defects for RI accumulation.
<Methods> In this presentation, IMZ SPECT was performed for the patients with traumatic brain injury during the acute stage to functionally evaluate the cortical damage in comparison with morphological images detected by CT/MRI. In twelve patients, IMZ bindings were studied by SPECT during the acute stage following traumatic brain injury. Written informed consent was received from all patients beforehand. Three of the twelve patients were diagnosed with diffuse axonal injuries, while eight suffered focal brain injuries, such as cerebral contusions and acute subdural hematomas. One patient was suffered from hypoxic ischemic encephalopathy following an acute subdural hematoma.
<Results> In the case of hypoxic ischemic encephalopathy, IMZ SPECT revealed diffuse cortical disorders expressed in the form of a broad defect with IMZ accumulation. The cerebral cortex of intact with IMZ accumulation emerged in three patients; diagnosed as diffuse axonal injury. In eight patients diagnosed with cerebral contusion meanwhile, areas with markedly decreased IMZ binding, corresponding to regions on the CT/MRI scans, were detected on SPECT.
Surprisingly, in four of the eight patients diagnosed with cerebral contusion, areas with decreased IMZ bindings, expected to represent irreversible changes, had markedly reduced in size one month after the induction of the traumatic brain injury. However, in the remaining four patients, the size of areas with decreased IMZ binding remained unchanged at the chronic stage.
The reduction in the areas of defect for RI accumulation suggests that benzodiazepine receptors were additionally detected with IMZ binding at the chronic stage in the cortical lesion, where receptors were not detected at the acute stage. The mechanisms of this phenomenon are unclear and moreover, further investigations to add cases should be necessary.
MELODY PERCEPTION IN NONMUSICIANS - A FUNCTIONAL MRI STUDY
Miho Yamauchi, Takuya Hayashi, Akihide Yamamoto, Hiroshi Sato, Hidehiro Iida
Advanced Medical Engineering Center, Research Institute, National Cardiovascular Center, Suita, Osaka, Japan
Introduction
Melody, one of main components of music, is a series of pitches sounding in succession, possibly perceived as a single entity (Gestalt) in brain. For most of listeners, changing the key of a melody can be perceived as change in feel, but not as change in melody itself, suggesting that the relative relationship of the individual pitches to each other is fundamental in melody perception. In the current study, we performed a melody discrimination paradigm in which melodies were changed in the key (a center of gravity of pitches) or/and in a part of pitches in the melody, and correlated the task difficulty with the BOLD response measured by fMRI.
Methods
Eight healthy right-handed subjects, who had not received musical instruction except compulsory education (mean age: 22 years) were recruited. Subjects received two sessions of 6-min fMRI scan during which they sequentially listened to a total of 32 pairs of two melodies. After each pair of melodies, subjects discriminated whether the second melody was same or not as the first one and answered it by the button in the right hand. Each melody consisted of 8 tones of sine wave in equal temperament in duration of 4.8sec. Among 32 pairs of melodies, 8 pairs consisted of the same melodies for the first and second, but the rest of pairs consisted of the second melody that were different from the first in all pitches (all notes were 2-degree higher than the first) or/and a part of pitches (only the sixth note was 1-degree higher or lower than the first). The order of 32 pairs of melodies was pseudo-randomized across two fMRI sessions. We used an MRI-compatible headphone to present melody pairs, a 3-Tesla MRI scanner (GE, Milwaukee, USA) for fMRI scanning, and SPM2 for data analysis.
Results and Discussions
An overall average rate of correct answers was 77.0%, however, the rate of correct answers was lower in either task with total or/and partial change in pitches, compared to that without. No interaction effect was found between total and partial changes. During a period of the second melody listening, we found larger BOLD signals at frontal operculum/right inferior frontal gyrus (BA45), superior frontal gyrus inside (BA8), and right supramarginal gyrus (BA40) compared to that during the first melody. When this contrast (2nd melody > 1st melody) was compared between tasks with or without partial pitch change, we found a larger BOLD signals at the right inferior frontal gyrus (BA45) in the task partially changed, compared to that without. In contrast, in the task with total pitch change, we did not find significant BOLD changes at this area but slight increase in the right supramarginal gyrus (BA40), left middle frontal gyrus (BA8), and left inferior temporal gyrus (BA18). These results suggest that the right inferior frontal gyrus (BA45), a region contralateral to the motor language area, is associated with perception of relative relationship of a series of pitches, while the perception of the key is processed by different neural systems.
DIRECT COMPARISON OF 201TL AND 99TC-MIBI SPECT OF GLIOMA BY A RECEIVER OPERATING CHARACTERISTIC ANALYSIS
Yasushi Shibata, Wataru Katayama, Tetsuya Yamamoto, Shingo Takano, Akira Matsumura
Department of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
Purpose
Single photon emission computed tomography (SPECT) is a valuable diagnostic modality for brain tumor malignancy and activity, however, the results of SPECT examinations always include some false positive or false negative findings. The diagnostic value of brain tumor SPECT has been evaluated using sensitivities and specificities with arbitrary cut off values. A receiver operating characteristic (ROC) analysis is useful for evaluating the diagnostic ability of examinations that include some errors. We directly compared the diagnostic ability of 201Thallium (Tl) SPECT and 99mTechnetium methoxyisobutylisonitrile (Tc-MIBI) SPECT for patients with initial glioma using an ROC analysis.
Patients and Methods
The study population included 59 patients with glioma who were admitted Tsukuba University hospital between 1999 and 2005. All pathological diagnoses were confirmed after tumor removal surgery. The benign group included low grade astrocytoma (n=24) while the malignant group included anaplastic astrocytoma (n=10) and glioblastoma (n=25).
201Tl SPECT and 99mTc-MIBI SPECT images were obtained 15 min (early) and 3 hours (delayed) after the intravenous injection of 74 MBq of 201Tl chloride or 740 MBq of 99mTc-MIBI using a multi-detector SPECT machine (E.CAM, Siemens Medical). The Regions of Interest (ROI) were set on tumor and contra-lateral normal white matter in SPECT images and Tumor/Normal (T/N) ratios were calculated as the ratios of radioactivity in the ROIs.
The diagnostic abilities of T/N ratios for malignancy were analyzed by an ROC analysis using the ROCKIT1.1B2 Beta and PlotROC.xls software programs (University of Chicago). The Az values were calculated from the areas under the ROC curves and the diagnostic accuracy was compared between each imaging modality. Any differences in the Az values were analyzed using the bivariate x2 test.
Results
The Az values for early Tl, delayed Tl, early MIBI, delayed MIBI were 0.90, 0.91, 0.87, and 0.93, respectively. All Az values were high and no statistical difference was observed between each modality. The delayed MIBI had the highest Az while the early MIBI had the lowest Az. The delayed Az values were higher than the early Az values, especially in MIBI.
Discussion
Some facilities omitted the acquisition of delayed images due to their busy schedule. However, our results clearly demonstrated the superior diagnostic value of delayed images in both of Tl and MIBI SPECT. These results revealed almost equal usefulness of Tl and MIBI SPECT for the diagnosis of gliomas. However, the SPECT study is not perfect, there are still some false positive and false negative findings. Multi-modality imaging studies may therefore help to diagnose brain tumors correctly. If we must limit SPECT examinations due to budget limitations related to health insurance policy, then MIBI SPECT with a delayed acquisition is therefore considered to be the most reasonable choice.
Conclusion
Both of Tl and MIBI SPECT are useful imaging modalities for the detection of glioma malignancies. Delayed MIBI SPECT demonstrated the best diagnostic value in our patients with gliomas based on an ROC analysis.
CALCIFICATION OF INTRACRANIAL ARTERIES-DETECTION AND EVALUATION USING MULTI-DETECTOR ROW CT (MDCT)
Masato Kin1, Hideki Ohba1, Masanori Mizuno1, Yasuhiro Ishibashi1, Makoto Sasaki2, Yasuo Terayama1
1Department of Neurology, Iwate Medical University, Morioka, Iwate, Japan, 2Department of Radiology, Iwate Medical University, Morioka, Iwate, Japan
Background and purpose— The importance of calcification associated with atherosclerotic plaque in intracranial arteries has been debated. The purpose of the present study is to clarify the relation between calcification of intracranial arteries detection and evaluation using MDCT and risk factors for cerebral infarction.
Method— One hundred and thirteen (113) subjects (M:F=71:42, 67.0+10.7 years) including 88 with cerebral infarction and 25 without cerebral infarction were included. Detection and evaluation of calcification was performed using 16 lines of MDCT made by Toshiba Corporation. Analysis of the arterial image was performed using DICOM viewer software “ExaVision LITE” (ZIOSOFT, Inc.). Calcification of 10 different region of intracranial arteries were evaluated by calcification score (CS). Statistical analysis was performed using Dra??s SPSSa!.
Result— CS was significantly correlate with aging (p<0.001) and hypertension (p<0.05). CS of anterior circulation of intracranial arteries was significantly correlate with aging (p<0.001). CS of posterior circulation of intracranial arteries was significantly correlate with aging (p<0.01) and hypertension (p<0.05).
Conclusions— Aging and hypertension contribute to the calcification of intracranial arteries including both anterior and posterior circulation. Hypertension does not contribute to the calcification of anterior circulation. On the other hand, it contributes to the calcification of posterior circulation in the same way as coronary arterial system. The above data suggests that a factor contributing to calcification varies in intracranial arterial system.
TIMING OF CELL TRANSPLANTATION AFTER STROKE SIGNIFICANTLY INFLUENCES THE CELL-HOST INTERACTION
Hideo Shichinohe, Marcel Daadi, Anne-Lise Maag, Nobutaka Horie, Theo Palmer, Tonya Bliss, Gary K. Steinberg
Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA
Background and aims: Growing evidence suggests that cell transplantation holds great potential as a therapy for stroke. Several groups have shown that transplanted cells can survive in the stroke brain, migrate towards the injured tissue and improve neurological function. However, many questions must be answered before moving this therapy to the clinic. One fundamental variable that needs to be defined is the optimal time after stroke for transplantation. This study looks at two time points of cell delivery and describes the difference that the time of transplantation makes to the host response and graft biology.
Methods: Primary medial ganglionic eminence (MGE) neural precursors were isolated from E15 rat embryos carrying the transgene for enhanced green fluorescent protein. Stroke was induced in rats by distal middle cerebral artery occlusion (dMCAo). A suspension of MGE cells was transplanted into the rat cortex at day 2 (n=2) or day 14 (n=3) after stroke. Animals were sacrificed at day 42 post-dMCAo and brain sections stained for neural, monocytic and blood vessel markers.
Results: Cell survival differed drastically between the two transplant groups. Cells transplanted at day 2 post-stroke showed very little survival at day 42; the cells in the core appeared to be dead but a thin layer of cells survived around the periphery of the graft. In contrast, when examined at day 42, the cells transplanted at d14 exhibited much more robust survival throughout the graft and formed a much more elongated graft. There was a significant difference in transplanted cell survival between the two transplant groups ('day-2′ group; 0.7 ± 0.2 ×10^5 cells/mm3, ‘day-14′ group; 2.0 ± 0.3 ×10^5 cells/mm3, P=0.02). No difference in blood vessel density in and around the grafts was found between the groups. Therefore, differential vasculature did not account for the difference in cell survival. There was, however, a significant difference in the inflammatory response between the two transplant groups. The day-2 group showed a robust inflammatory response at 6 weeks after transplantation, with Iba1-positive (monocytic) cells both within and surrounding the graft. In the day-14 group at six weeks post transplantation, there were very few Iba1-positve cells in the graft and a dramatic lack of Iba1 cells immediately surrounding the graft, much fewer than in the rest of the hemisphere. However, in this Iba1-sparse area there were many host astrocytes which appeared to form a barrier around the graft precluding the monocytes.
Conclusions: Fetal neural precursors derived from the MGE survive poorly when transplanted two days post-dMCAo, possibly due to a prolonged host immune response. However, cells transplanted at 14 days post-dMCAo survived to a greater degree; they appeared to be protected from the monocytes by a wall of host astrocytes. Thus, the timing of transplantation after stroke has a long-term effect on the host response to the graft. This response can dramatically affect the graft's microenvironment and ultimately determine the success of cell-based therapy.
IDENTIFICATION OF NEURAL OUTGROWTH CELLS FROM PERIPHERAL BLOOD OF STROKE PATIENTS AND THEIR CONTRIBUTIONS TO ISCHEMIC BRAIN REGENERATION
Kon Chu, Keun-Hwa Jung, Soon-Tae Lee, Eun-Cheol Song, Dong-In Sinn, Jeong-Min Kim, Hee-Kwon Park, Manho Kim, Jae-Kyu Roh
Stroke and Neural Stem Cell Laboratory, Department of Neurology, Stem Cell Research Center, Seoul National University Hospital, Seoul, South Korea
Background: Bone marrow harbors tissue-committed stem cells that may migrate to the circulation and exert beneficial effects on regeneration. Recent studies have identified a subset of outgrowth cell population with endothelial phenotype in longterm cultures of peripheral blood mononuclear cells. The concept that peripheral blood-derived cells participate in neural regeneration remains highly controversial and no specific cell type has been identified. In this study, we undertook to characterize outgrowth cells (OCs) in peripheral blood culture from stroke patients and from healthy subjects, and to demonstrate their therapeutic efficacy against experimental ischemia.
Methods: Patients undergoing acute stroke was included. EPCs were isolated from the peripheral venous blood of 25 patients and 15 age-matched healthy volunteers. The yield of outgrowth endothelial cells was measured during the 3 months of culture. We transplanted the OCs in the ischemic cortex of adult rats and analyzed the functional recovery and engraftment pattern.
Results: OCs were isolated efficiently from stroke patients, and demonstrated heterogenous populations with different morphologies, e.g., cobblestone, palisading, or branching features. Two different types of OCs were identified: endothelial OCs; neuronal OCs, according to their morphology and differentiation patterns toward endothelial or neural lineages. The transplanted neuronal OCs survived in the ischemic brains of a rat model and improved functional recovery over 5 weeks post-transplantation. The majority of migrating, transplanted cells in ischemic rats had neuronal or endothelial phenotypes.
Conclusion: The feasibility of extracting and culturing neural progenitor cells in large numbers suggests that such autologous progenitor cells will be useful for applications ranging from basic research to cell-based therapy.
VEGFR2 ANTAGONISM ATTENUATES BEHAVIORAL IMPROVEMENTS ON A RADIAL ARM MAZE FOLLOWING TRAUMATIC BRAIN INJURY (TBI)
Randy Morgan, Christian Kreipke, Jose Rafols
Department of Anatomy and Cell Biology, Wayne State University, School of Medicine, Detroit, MI, USA
Background and AIMS
In a rodent acceleration impact model of diffuse brain injury (450 g weight dropped from a height of 2 m onto the skull) we previously reported a nearly 40% reduction in cerebral cortical blood flow that persisted for at least 48 h after impact. In addition, compared to controls, animals subjected to traumatic brain injury (TBI) significantly underperformed on a radial arm maze, however after a prolonged period of poor performance (21 days) these animals recovered and performed at control levels. While the cognitive behavioral improvement may have been due to reconnection of neural circuits previously disconnected as a result of brain injury, an alterative explanation is that improvement of the cerebral microcirculation through angiogenesis after TBI may have been responsible for he behavioral recovery. As such we and others have documented marked increases in angiogenic factors including vascular endothelial growth factor (VEGF) and its receptor (VEGFR2), as well as increased capillary density in areas of the brain involved both in motoric and cognitive behaviors after TBI. Therefore here we aimed to test whether blockade of angiogenesis via VEGFR2 antagonism could attenuate the behavioral improvement seen on the radial arm maze after TBI.
Methods
Two days prior to injury, rats were given intraperitoneal (IP) injections of vehicle (control) or the VEGFR2 antagonist, SU5416. Animals were then subjected to TBI using Marmarou's model of diffuse brain injury. Following recovery from injury (usually one hour) behavioral testing commenced and continued for 30 days post injury. Animals continued to receive IP injections of vehicle or SU5416 for the duration of the testing period.
Results
Our results show that blockade of VEGFR2 caused a marked increase in the time needed to reach control level performance on the radial arm maze.
Conclusion
These results indicate that VEGFR2 antagonism attenuates cognitive function. Moreover, these findings indicate that angiogenesis serves as a natural mechanism to improve behavioral outcome following TBI. In subsequent experiments, we will stimulate VEGFR2 to determine whether an increased rate and magnitude of angiogenesis can improve cognitive outcome following TBI.
ISOFLURANE PRECONDITIONING ENHANCES POST-ISCHEMIC NEUROGENESIS IN ADULT MICE
Wenri Zhang, Lan Wang, Stephanie J. Murphy, Patricia D. Hurn, Ansgar M. Brambrink
Department of Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, OR, USA
Background and Aims: Exposure to isoflurane prior to brain ischemia (“anesthetic preconditioning”) improves functional outcome and results in higher numbers of viable neurons after the insult, which has been attributed to neuroprotective effects of the volatile anesthetic. We tested the hypothesis that isoflurane preconditioning (isoPC) induces an up-regulation of post-stroke neurogenesis in adult male mice.
Methods: Adult C57BL/6 mice were preconditioned for 4 hours with air (sham preconditioning, shamPC) or 1.0% isoflurane (isoPC). Twenty-four hours after preconditioning, mice underwent 60 min of reversible middle cerebral artery occlusion (suture model). Animals were survived for 3 or 21 days, and received twice daily intraperitoneal injections of 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU, 50 mg/kg). At designated times, brains were processed (intravital perfusion-fixation, 4% PFA, free-floating 40 µm-cryosections) and stained for BrdU (proliferated cells), doublecortin (neuroblasts), and NeuN (mature neurons). Confocal microscopy in coronal sections was used to quantify double-labeled cells in dentate gyrus (DG), subventricular zone (SVZ), striatum, and peri-ischemic neocortex (overlay images; cells per tissue volume). Representative sections are stained for morphologic evaluation (MAP-2, Fluoro-Jade).
Results: We observed an increase in postischemic neurogenesis after isoPC (fig. 1). Maturation and survival of newborn cells were also enhanced by isoPC in ipsilateral dentate gyrus at 21 days after stroke (9959±423 vs. 2657±0 BrdU+ neuroblasts; 4310±1358 vs. 2030±0 BrdU+ mature neurons, n=1-4/group). Moreover, enhanced migration of newborn cells into damaged areas after ischemia was found in isoPC vs. shamPC animals.
Conclusions: The extent of functional impairment resulting from cerebral ischemia is generally considered to reflect the volume of lost brain tissue. However, newborn neurons may contribute profoundly to the amazing capacity for rehabilitation after cerebral ischemia, which apparently is enhanced after anesthetic preconditioning.
ENFORCED PHYSICAL TRAINING PROMOTES NEUROGENESIS IN THE SUBGRANULAR ZONE AFTER FOCAL CEREBRAL ISCHEMIA
Seung-Hoon Lee, Yun-Hee Kim, Young-Ju Kim, Miyeoun Song, Byung-Woo Yoon
Department of Neurology, Seoul National University Hospital, Seoul, South Korea
Background and Purpose: Cerebral ischemia increases neurogenesis in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus, which might be altered by enriched environment including voluntary physical activity. We tested whether enforced physical training (EPT) influences differentially on the neurogenesis of the SVZ and the SGZ after cerebral ischemia. Methods: Adult male Sprague-Dawley rats were subjected to focal cerebral ischemia for 2 hours, and were divided into EPT and non-EPT group. All rats in the EPT group were trained using rota-rod for 14 days. 5-bromo-2′-deoxyuridine (BrdU) was injected to determine levels of cell proliferation. Functional recovery was assessed by a set of behavioral test batteries. Extent of endogenous neurogenesis in the SVZ and the SGZ was quantified using immunofluorescence staining. Results: Although final infarction volume was not significantly different between the groups, functional recovery was better in the EPT group at 10 and 17 days after ischemia. In the SVZ, BrdU-labeling and double-labeling of BrdU/Dcx or BrdU/NeuN were not significantly between the groups. In the SGZ, EPT enhanced significantly BrdU-positive cells (EPT vs. non-EPT: 159.1±19.9 vs. 101.8±7.8, p=0.04), and the number of BrdU/Dcx doublelabeled cells was also increased in the EPT group (130.6±16.9 vs. 73.6±7.2, p=0.01). Conclusion: Our results indicated that EPT promotes neurogenesis in the SGZ of the dentate gyrus, but not in the SVZ after ischemia. Biochemical mechanism of the differential effects of EPT remains to be clarified.
IGF-1 AND FGF2 SYNERGISTICALLY PRMOTE ADULT NEURAL PROGENITOR PROLIFERATION UNDER NORMAL AND ISCHEMIC CONDITIONS
Robert Dempsey1, Haviryaji Kalluri1, Yi-ping Yan1, Kurt Sailor2, Raghu Vemuganti1
1Department of Neurological Surgery, University of Wisconsin, Madison, Madison, WI, USA, 2Department of Neurosurgery, Johns Hopkins Medical School, Baltimore, MD, USA
Transient focal ischemia (stroke) is a potent stimulator of neurogenesis in adult rodent brain. We previously observed that following transient middle cerebral artery occlusion (MCAO) in adult rats, the expression of IGF-1 is significantly elevated in the ischemic hemisphere. The neural progenitors in the subventricular zone (SVZ) and dentate gyrus (DG) expressed IGF-1 receptor. Inhibiting the IGF-1 activity by infusion of IGF-1 antibody into the lateral ventricle significantly prevented the ischemia-induced increase of BrdU positive cells in the both SVZ and DG. On the contrary, infusion of IGF-1 resulted in a significant elevation of BrdU positive cells in the both SVZ and DG of the post-ischemic brain. These results indicate that IGF-1 upregulation in the ischemic brain may increase neural progenitor proliferation or enhance their survival. Therefore, we used an in vitro culture system to dissect the mechanisms of IGF-1 effects on adult neural progenitor cells. IGF-I activated PI-3kinase/Akt pathway but not ERK pathway in the adult neural progenitor cells in vitro. Furthermore, LY294002 (PI-3 kinase inhibitor), but not U0126 (ERK inhibitor) inhibited the IGF-I induced survival of cells, while FGF2 (ERK activator) enhanced the IGF-I mediated survival of the cells as measured by cell proliferation assay. In addition, stimulation of ERK pathway by FGF2 induced the expression of cyclin D1, which is essential for the entry of cells into cell cycle, while IGF-I in the presence of FGF2 upregulated the expression of cyclin D1. Likewise, IGF-I in the absence or presence of FGF2 increased the phosphorylation of GSK supporting its role in the survival of NPCs. To further confirm the role of ERK activation in the proliferation, we cultured cells in FGF2 + IGF-I containing medium in the presence and absence of U0126 (ERK inhibitor) and show the inhibition of nestin expression in U0126 treated cells. The decrease in the cyclin D1 content in conjunction with the inhibition of nestin expression by ERK inhibitor, confirms the role of ERK in the proliferation of cells. Thus, these studies show that following focal ischemia, IGF-I might enhance the survival of the neural progenitor cells so that other mitogens like FGF2 might enhance their proliferation.
These studies were funded by NIH and AHA
INTRA-ARTERIAL TRANSPLANTATION OF BONE MARROW MONONUCLEAR CELLS IMMEDIATELY AFTER REPERFUSION AMELIORATES BRAIN INJURY FOLLOWING TRANSIENT FOCAL ISCHEMIA IN RATS
Nobuo Kamiya1, Hironaka Igarashi2, Yasuhiro Nishiyama1, Masayuki Ueda1, Yasuo Katayama1
1Division of Neurology, Nephrology and Rheumatology, Department of Internal Medicine, Nippon Medical School, Tokyo, Japan, 2Center for Integrated Human Brain Science, Brain Research Institute, University of Niigata, Niigata, Japan
Introduction: Transplantation of bone marrow cells on experimental cerebral ischemia has been reported to possess neuroprotective and regenerative effects (1),(2), however neuroprotective effect of the cells administered immediately after reperfusion has been rarely investigated. The present study was designed to examine whether ischemic cerebral injury can be ameliorated by bone marrow mononuclear cells (MMCs) administered immediately after reperfusion on transient focal ischemia in rat, and if so, to determine whether difference of the effectiveness is seen dependent on route of administration (intra-arterial and intravenous MMC administrations).
Methods: Male Sprague?Dawley rats, weighing 250?300g, were used in the study. Bone marrow was obtained autologously from the right femur of each animal, and MMCs were isolated prior to ischemia. Rats were subjected to transient focal ischemia for 90 min using the intraluminal suture technique, followed by MMC administration (1.0>10E7 cells) through the ipsilateral carotid artery (IA group) or the femoral vein (IV group) immediately after reperfusion. Several animals were received same procedure except for intravenous vehicle injection instead of MMC transplantation (control group). Animals were deeply anesthetized and decapitated 24 hrs after reperfusion. The brains were carefully removed and dissected into 2 mm-thick coronal sections, and the slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC). The infarct volume was calculated by summing the infarct area of the TTC-stained sections from each animal.
Results: Averaged 1.13±0.25>10E7 MMCs were harvested from each rat. Total infarct volume was reduced in IA group (78.3±13.3 mm3), but not in IV group (119.0±35.7 mm3), compared with that in control group (140.6±47.8 mm3), with a statistical significance (p<0.05). The cortical infarct volume was also significantly decreased in IA group compared to control group, although striatal infarct volume was not statistically different among the groups.
Conclusion: The present data confirm significant neuroprotection following transient focal ischemia by autologous MMCs administered intra-arterially, but not intravenously, immediately after reperfusion in rats.
IN VIVO IMAGING OF NEUROKININ 1 RECEPTOR DISTRIBUTION WITH 18F-L829165 POSITRON EMISSION TOMOGRAPHY IN PARKINSON'S DISEASE AND LEVODOPA-INDUCED DYSKINESIAS
Yasuyuki Kimura1,2, Alan Whone1, Nicola Pavese1, Richard Hargreaves3, Don Burn3, David J. Brooks1
1MRC Clinical Sciences Centre and Division of Neuroscience, Faculty of Medicine, Imperial College, Hammersmith Hospital, London, UK, 2Department of Nuclear Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine, Suita, Osaka, Japan, 3Merck & Co., Inc.
Background: The majority of Parkinson's disease (PD) patients receving long-term replacement therapy with levodopa develop adverse events of motor fluctuations and dyskinesias. Alterations of substance P (SP) levels and neurokinin 1 (NK1) receptor availability have been reported to be associated with levodopa-induced dyskinesias (LID) in animal lesion and postmortem studies.
Methods: To determine whether changes in NK1 receptor binding are involved in the pathophysiology of human PD and LID, we measured in vivo NK1 receptor availability in normal subjects and dyskinetic and nondyskinetic PD patients using positron emission tomography (PET) and the NK1 receptor ligand 18F-L829165. Patlak linear graphical analysis was used to create parametric images of 18F-L829165 influx constants (Ki). Regional 18F-L829165 Ki's were calculated using region of interest (ROI) analysis. We also used statistical parametric mapping (SPM) to localise changes in 18F-L829165 Ki at a voxel level.
Results: In normal subjects, the highest 18F-L829165 Ki's were observed in the caudate nucleus and putamen. The globus pallidum and neocortex showed lower 18F-L829165 Ki's. Using ROI analysis, we found significantly lower 18F-L829165 Ki's in the caudate nucleus, ventral striatum, globus pallidus lateral segment, locus coeruleus, and parietal, temporal, and occipital lobes of PD patients compared with normal subjects. SPM confirmed the presence of decreased bilateral striatum, neocortex, and dorsal midbrain 18F-L829165 Ki in the PD patients. Although the ROI analysis did not reveal any significant difference in 18F-L829165 Ki between the non-dyskinetic and dyskinetic PD patients, SPM revealed relatively decreased right putamen and dorsal midbrain 18F-L829165 Ki's in the dyskinetic PD patients.
Conclusions: 18F-L829165 PET reveals in vivo the distribution of the cerebral NK1 receptor availability in normal subjects. The NK1 receptor binding is reduced in the striatum and cortex of PD patients and the presence of dyskinesias is associated with further striatal and midbrain reductions. The SP and NK1 receptor systems could, therefore, play a role in the pathophysiology of PD and levodopa-induced dyskinesias.
AGE-DEPENDENT CEREBROVASCULAR DYSFUNCTION IN TG2576 TRANSGENIC MOUSE MODEL OF HUMAN CEREBRAL AMYLOID ANGIOPATHY
Hwa Kyoung Shin1, Monica Garcia-Alloza2, Phillip B. Jones2, Laura Borrelli2, Brian J. Bacskai2, Matthew P. Frosch2, Bradley T. Hyman2, Michael A. Moskowitz1, Cenk Ayata1,3
1Stroke and Neurovascular Regulation Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA, 2Alzheimers Disease Research Laboratory, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 3Stroke Service and Neuroscience Intensive Care Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA
Background and aims: The Tg2576 transgenic mouse model of human cerebral amyloid angiopathy (CAA) is characterized by age-dependent cerebrovascular deposition of amyloid-beta starting from 9 months of age and progressively worsening to involve most pial arterioles by 18 months. Here, we tested whether vascular amyloid-beta deposition impairs physiological CBF responses to 5% inhaled CO2, whisker stimulation or cortical spreading depression (CSD) in aged Tg2576 mice using laser speckle flowmetry non-invasively through intact skull.
Methods: We tested 8 month-old Tg2576 mice with elevated soluble amyloid-beta but no vascular amyloid-beta deposition, and 19 month-old Tg2576 mice with widespread and severe vascular amyloid-beta deposits, and compared these to wild type littermates.
Results: Hypercapnic hyperemia (5% CO2) was attenuated by 39% in 19, but not 8 month-old Tg2576 mice (p<0.05). The slope of the CBF increase was significantly attenuated in 19 month-old Tg2576 mice only (18±3 vs. 9±1%/min in wild type and Tg2576, respectively; p<0.05). The response to hypercapnia was also attenuated in aged wild type mice (p<0.05) but this did not explain the difference between the mutant strains. The CBF response to vibrissal stimulation was attenuated by 48% in 19, but not 8 month-old Tg2576 compared to wild type littermates (p<0.05). Aging alone also tended to attenuate the functional hyperemic response to vibrissal stimulation in 19 month-old wild type mice but the data were not significant (p=0.1). Both the transient hypoperfusion during CSD and the subsequent post-CSD oligemia were diminished in 19 but not 8 month-old Tg2576 compared to wild type littermates, suggesting that vasoconstrictor responses were also impaired in 19 month-old Tg2576 mice (p<0.05). Aging alone did not attenuate the vasoconstrictive CBF response to CSD in wild type mice. Anesthetic-induced changes in blood flow (i.e., Isoflurane-induced increase and alpha-chloralose-induced decrease in resting CBF) were both attenuated in 19 but not 8 month-old Tg2576 compared to wild type littermates, further suggesting that CAA impacts both vasodilator and vasoconstrictor responses.
Conclusion: Our data suggest that vascular amyloid-beta deposition rather than elevated soluble amyloid-beta is associated with cerebrovascular dysfunction when imaged non-invasively in these physiological models. Furthermore, advancing age is independently associated with impaired physiological CBF responses to hypercapnia and functional activation.
This work was supported by the American Heart Association (0335519N, Ayata), NIH (P50 NS10828, Moskowitz; AG08487, Hyman)
EFFECT OF AGE ON (R)-[11C]PK11195 BINDING IN HEALTHY SUBJECTS
Alie Schuitemaker1,2, Bart N.M. van Berckel2, Ronald Boellaard2, Marc A. Kropholler2, Philip Scheltens1, Adriaan A. Lammertsma2
1Department of Neurology, VU Medical Center, Amsterdam, The Netherlands, 2Department of Nuclear Medicine and PET Research, VU Medical Center, Amsterdam, The Netherlands
Introduction
The presence of activated microglia has been studied in vivo in several neurodegenerative diseases using (R)-[11C]PK11195 and PET. During normal aging, increased (R)-[11C]PK11195 binding in the thalamus has also been described1,2, but other regions in the normal brain have not been evaluated extensively. The objective of the present study was to investigate to what extent microglial activation occurs during normal aging.
Methods
Eleven elderly (age > 63) and 7 young (age < 45) healthy subjects were included. Dynamic PET scans were acquired in 3D mode following bolus injection of 259 ± 32 MBq (R)-[11C]PK11195 with a specific activity of 80 ± 34 GBq/µmol for young and 313 ± 80 MBq with a specific activity of 104 ± 37 GBq/µmol for elderly controls, respectively. (R)-[11C]PK11195 time activity curves (TAC) were generated using a standard probabilistic volume of interest (VOI) template3. These TAC were analysed using the simplified reference tissue model4, with cerebellum as reference tissue and binding potential (BP) as outcome measure. In addition, parametric BP images were generated using receptor parametric mapping5. These images were compared at the group level using SPM with a two sample t-test per voxel and a threshold of p = 0.01, uncorrected for multiple comparisons and without proportional scaling6.
Results
Using the VOI approach, in elderly subjects increased BP was found in orbital frontal and medial inferior temporal lobes, putamen, enthorhinal cortex and hippocampus. Parametric BP images showed increased BP in elderly subjects in left and right frontal cortex, left and right medial temporal cortex, left and right thalamus, and midbrain.
Discussion and conclusions
Increased (R)-[11C]PK11195 binding in the thalamus with aging is in agreement with previous findings1,2 The discrepancy between results obtained by SPM and VOI analyses in the thalamus might be caused by the poor gray/white matter segmentation in this area. The distribution of increased (R)-[11C]PK11195 binding in elderly subjects observed in the present study indicates more widespread microglia activation during normal aging.
DENDRITIC ARBORIZATION AND ANCHORINGS OF NMDA AND AMPA RECEPTORS IN PRIMARY HIPPOCAMPAL NEURONS ARE CONTROLLED BY SIGMA-1 RECEPTORS AT ER
Shang-Yi Tsai, Teruo Hayashi, Tsung-Ping Su
Cellular Pathobiology Unit, CNRB, IRP, National Institute On Drug Abuse, NIH, DHHS, Baltimore, MD, USA
The endoplasmic reticular (ER) protein sigma-1 receptor (Sig-1R) has been implicated in many CNS diseases including depression, amnesia, pain, as well as in neuroplasticity associated with cocaine or methamphetamine abuse. Functionally, Sig1-Rs have been shown by us to affect differentiation in neuronal cell lines. This study, by employing the siRNA technique, examined the involvement of Sig-1Rs in the development of dendritic arborization and the associated anchorings of NMDA and AMPA receptors in rat hippocampal primary neurons. Hippocampal neurons treated with the siRNA against Sig-1Rs form fewer dendritic spines whereas possess more filopodia. The MAP-2B staining, which marks neuronal dendrites, shows that the siRNA treatment also impairs the growth and development of dendrites. Morphometric measurements indicate that siRNA-transfected neurons have shorter dendrites and possess fewer branches on the dendrite. The knockdown of Sig-1Rs by the siRNA not only reduces the level of marker proteins for synapse, including PSD-95 and synaptophysin, but also changes the localization of NMDA and AMPA receptors in hippocampal neurons. In siRNA-transfected neurons, NR-1 and GluR2/3 respectively form clusters and localize mainly at the dendritic shafts and not at spine heads where they normally exist. siRNAs also retard the extension of axons in hippocampal neurons. These results suggest that Sig-1Rs are related to a common mechanism critical at multiple steps in the development and maturation of dendritic arbors and, as a consequence, affect the anchoring of receptors or ion channels to the postsynaptic region. The exact mode of action of Sig-1Rs in this regard is under investigation.
SEROTONIN TRANSPOTER BINDING IN PATIENTS WITH OBSESSIVE COMPULSIVE DISORDERS: A PET WITH [11C] DASB
Ryohei Matsumoto1,2, Hiroshi Ito1, Yota Fujimura1, Yoko Ikoma1, Chie Seki1, Jun Kosaka1, Ryosuke Arakawa1, Tomomichi Ando1, Akihiro Takano1, Hidehiko Takahashi1, Kenji Fukui2, Tetsuya Suhara1
1Department of Molecular Neuroimaging, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan, 2Department of Psychiatry, Graduate School of Kyoto Prefectural University of Medicine, Kyoto, Japan
Background and aims
Obsessive compulsive disorder (OCD) is psychiatric disorder characterized by recurrent obsessions and compulsions. Based on the selective efficacy of serotonin reuptake inhibitors (SRIs) and comorbid depression that is often observed in OCD, the serotonergic system including serotonin transporter (SERT) has been of interest in pathophysiology of OCD. Previous study using positron emission tomography (PET) with a radiotracer [11C]McN5642 for SERT failed to changes in density of SERT in OCD patients. The aim of the present study is to investigate SERT density using [11C]DASB which shows high selectivity and affinity to SERT as compared with [11C]McN5642.
Methods
Ten patients with OCD, free of psychiatric medication and comorbid depression, and 18 gender and age-matched healthy subjects participate in this study. OCD was diagnosed according to the DSM-IV. OCD severity was assessed by Yale?Brown Obsessive?Compulsive Scale (Y-BOCS, scale range: 0?40). After intravenous infusion of [11C]DASB, a dynamic PET scanning was performed for 90 min. Regions-of-interest were defined on the frontal cortex, thalamus, striatum and midbrain on the coregistered magnetic resonance images. The binding potential (BP) of [11C]DASB was calculated using a two-parameter multilinear reference tissue model (MRTM2) developed by Ichise et at al.
Results
As shown that Y-BOCS score was 22±7.6 (range: 7-32), the degree of severity in the present subjects varied relatively widely. BP values for OCD patients were 0.17±0.05, 1.48±0.10, 1.20±0.09, and 1.35±0.28, and values for healthy controls were 0.22±0.10, 1.60±0.24, 1.53±0.33, and 1.24±0.20 in the frontal cortex, thalamus, striatum and midbrain, respectively. No significant group differences were observed in BP of [11C]DASB in any of brain regions. No significant correlations were observed between severity of OCD and BP.
Conclusions
Although SERT has been of interest in pathophysiology of OCD, no significant differences were observed in SERT density between OCD and healthy controls, same as the previous study using [11C]McN5642. OCD without comorbid depression may not be associated in only serotonergic system, but also other neurotransmission systems.
BENEFICIAL EFFECTS OF INTRATHECAL IGF-1 ADOMINISTRATION IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Nobutoshi Morimoto, Isao Nagano, Tetsuro Murakami, Makiko Nagai, Mito SHiote, Yasuyuki Ota, Kazunori Miyazaki, Tomoko Kurata, Mizuki Ishikawa, Yasushi Takehisa, Tatsusi Kamiya, Kouji Abe
Department of Neurology Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan
Background: Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by degeneration of the upper and lower motor neurons in the brain and spinal cord resulting in progressive paralysis of voluntary muscles. However, there is currently no effective pharmacological treatment for amyotrophic lateral sclerosis. In a transgenic mouse model of ALS, intrathecal infusion of insulin-like growth factor-1 (IGF-1) showed a promising increase in survival.
Objectives: To examine whether IGF-1 is effective for the prevention of disease progression, we performed a double-blind clinical trial to assess the effect of intrathecal administration of IGF-1 in patients with ALS.
Methods: Nine patients with ALS were randomly assigned to receive either a high dose (3 g/kg of body weight) or low dose (0.5 g/kg of body weight) of IGF-1 every 2 weeks for 40 weeks. The outcome measurements were the rate of decline of bulbar and limb functions (Norris scales) and forced vital capacity.
Results: The high-dose treatment slowed a decline of motor functions of the ALS patients in total Norris and limb Norris scales, but not in bulbar Norris or vital capacity. The intrathecal administration of IGF-1 had a modest but significant beneficial effect in ALS patients without any serious adverse effects.
Discussion: Intrathecal IGF-1 treatment could provide an effective choice for ALS although further studies in more patients are needed to confirm its efficacy and optimize dosages of IGF-1.
BRAIN ELECTRIC SIGNATURE OF DE NOVO PARKINSON'S DISEASE WITH LOW RESOLUTION BRAIN ELECTROMAGNETIC TOMOGRAPHY
Hideaki Tanaka1, Mio Arai2, Yuya Hoshino1, Tomohiro Ogawa1, Mikio Okayasu1, Yuka Watanabe1, Hidehiro Takekawa1, Akinori Hozumi1, Koichi Hirata1
1Department of Neurology, Dokkyo Medical University, Mibu, Tochigi, Japan, 2Department of Neurology, Tokyo Medical University, Kasumigaura Hospital, Kasumigaura, Ibaraki, Japan
Background and aims: The present study aimed to search for the brain electric signature of drug naive de-novo Parkinson's disease patients (PDPs) without dementia compared with normal controls (NCs).
Methods: We studied 16 drug naive de-novo PDPs and 16 age-, sex- and education- matched NC subjects. We recorded 20-channel spontaneous eyes closed resting EEGs (BioLogic Brain Atlas, 0.53–30.Hz band pass) according to the international 10/20 system. After recording, 20 artifact-free EEG epochs consisting of 2-second were selected for each subject. Global field power (GFP) for EEG frequency were determined by means of a Fast Fourier Transform (FFT) and compared between the two groups. Low Resolution Electromagnetic Tomography (LORETA) was used to compute the three-dimensional intracerebral distribution of electric activity for 7 EEG bands: delta (1.5–6.Hz), theta (6.5–8.Hz), alpha1 (8.5–10.Hz), alpha2 (10.5–12.Hz), beta1 (12.5–18.Hz), beta2 (18.5–21.Hz), beta3 (21.5–30.Hz). Images of two groups were statistically compared using non-parametric randomization tests with correction for multiple testing. To assess the cognitive functions, the Japanese version of the Mini-Mental State Examination (MMSE) and the New Modified Wisconsin Card Sorting test (WCST) were recorded.
Results: MMSE score revealed no significant differences between two groups, however PDPs showed significant reduction of achieved categories and significant increase of preservative errors in WCST. The EEGs of patients showed significant increase in slow activity (delta and theta) and significant reduction in fast activity (alpha2 and beta1). And alpha peak frequency of PDPs decreased. LORETA analysis indicated significant increase of slow activity (delta and theta) in the bilateral occipital lobe and significant decrease of fast activity (alpha2 and beta1) in the limbic lobe in PDPs compared with NCs. indicated occipital and limbic hypoactivities perfusion in denovo PDPs without dementia. This findings confirmed SPECT data which demonstrated occipital hypoperfusion corresponding to impairment of visual cognition.
CORTICAL NEURONAL AND GLIAL PATHOLOGY IN TGTAUP301L TRANSGENIC MICE: NEURONAL DEGENERATION, MEMORY DISTURBANCE AND PHENOTYPIC VARIATION
Tetsuro Murakami1, T. Kawarabayashi2, M. Ikeda3, E. Matsubara4, M. Nagai1, T. Kurata1, N. Morimoto1, Y. Ohta1, Y. Takehisa1, K. Abe1, D. Westaway5, P. St. George-Hyslop5, M. Shoji2
1Department of Neurology, Okayama University Medical School, Okayama, Japan, 2Department of Neurology, Hirosaki University School of Medicine, Hirosaki, Japan, 3Department of Neurology, Gunma University Medical Shool, Gunma, Japan, 4Department of Alzheimer's Disease Research, National Institute of Longevity Sciences, National Center for Geriatrics and Gerontology, Aichi, Japan, 5Departments of Medicine (Neurology) and Laboratory Medicine and Pathobiology, Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, ON, Canada
Recapitulation of tau pathologies in an animal model has been a long-standing goal in neurodegenerative disease research. We generated transgenic (TgTauP301L) mice expressing a frontotemporal dementia with parkinsonism linked to chromosome 17 (FTPD-17) mutation within the longest form of tau (“2N, 4R”). TgTauP301L mice developed florid pathology including neuronal pretangles, numerous Gallyas-Braak-positive neurofibrillary tangles and glial fibrillary tangles in the frontotemporal areas of the cerebrum, in the brainstem, and to a lesser extent in the spinal cord. These features were accompanied by gliosis, neuronal loss and cerebral atrophy. Accumulated tau was hyper-phosphorylated, conformationally changed, ubiquitinated and sarkosyl-insoluble, with electron microscopy demonstrating wavy filaments. Aged TgTauP301L mice exhibited impairment in hippocampally-dependent and -independent behavioral paradigms, with impairments closely related to the presence of tau pathologies and levels of insoluble tau protein. We conclude that TgTauP301L mice recreate the substantial phenotypic variation and spectrum of pathologies seen in FTDP-17 patients. Identification of genetic and/or environmental factors modifying the tau phenotype in these mice may shed light on factors modulating human tauopathies. These Tg mice may aid therapeutic development for FTDP-17 and other diseases featuring accumulations of four-repeat tau, such as Alzheimer's disease, corticobasal degeneration and progressive supranuclear palsy.
AGE-DEPENDENT PLAQUE DEPOSITION CORRELATES WITH A MORE EXPLORATORY PHENOTYPE IN ELDERLY TRANSGENIC APPSWE/PS1DE9 MICE WHEN ADMINISTERED WITH DOI
Ruben Christensen1, Gitta Wortwein2, Anders Brandt Jorgensen3, Tine Bryan Stensbol3, Gitte
Moos Knudsen1, Susana Aznar1
1Neurobiology Research Unit, Copenhagen University Hospital, Copenhagen, Denmark, 2The Laboratory of Neuropsychiatry, Copenhagen University Hospital, Copenhagen, Denmark, 3H. Lundbeck A/S, Copenhagen Valby, Denmark
The transgenic APPswe/PS1dE9 animal model is regarded as a solid model of Alzheimer's disease (AD). These transgenic mice display an age-dependent increase in brain beta-amyloid (Abeta) plaque deposition, which occurs early (4-6 months) and is accelerated during their lifespan. Several PET studies have shown that cerebral 5-HT2A receptor binding is decreased in Alzheimer patients, correlating with the cognitive decline and neuropsychiatric symptoms, i.e. mood disturbances and depression. Furthermore, newer studies show that the 5-HT2A receptor is involved in anxiety modulation. We made use of these animals to study the association between plaque deposition and anxiolytic and changes in exploratory behaviour.
Different groups of APPswe/PS1dE9 transgenic and wild type mice, corresponding to age 4, 7 and 10 months, defined as pre-, mid- and late plaque stages respectively, were tested in the elevated plus maze and open field test. In the later test, animals were injected with a single injection of the 5-HT2A serotonin receptor agonist, DOI, to see whether the exploratory behaviour of the transgenic mice would differ by pharmacological activation of this receptor.
When tested in the elevated plus maze test, the transgenic APPswe/PS1dE9 mice did not show higher anxiety than the age-matched wild type mice. Both the transgenic and wild-type mice showed a decrease in time spending on the open arm with aging. In the open field test, transgenic animals showed a significant effect of DOI in the 7 months group. When stimulated with DOI the transgenic mice displayed a more active exploratory behaviour than their age-matched wild-type. In the 10 month old group no significant difference between the transgenic and wild-type was observed, probably due to the heavy plaque burden.
These preliminary results indicate an alteration in the responsiveness to the 5-HT2A agonist in the 7 months transgenic APPswe/PS1dE9, suggesting changes in the levels of this receptor in animals with beta-amyloid plaque deposition. Furthermore, these results indicate the involvement of the 5-HT2A in exploratory behaviour.
DELAYED ADMINISTRATION OF CYCLIC G-2ALLYLP REDUCED THE LONG-TERM FORELIMB AKINESIA INDEPENDENT STRIATAL DOPAMINE DEPLETION IN RATS
Jian Guan, Sam Mathai, Helena Kim, Rong Zhang, Rita Krishnamurthi
Liggins Institute, University of Auckland, Auckland, New Zealand
Background: Parkinson's disease (PD) is a neurodegenerative disorder due to progressive substantia nigra-striatum dopamine depletion. PD patients only experience bradykinesia, akinesia and loss of motor coordination after the loss of majority dopamine neurons in substantia nigra. During the pre-symptomatic period the brain appeared to be able to compensate and delay impending motor deficits, probably through endogenous reparative mechanisms including neurogenesis and the plasticity within cortical - basal ganglia circuit in both dopamine and non-dopamine neurotransmission. Cyclic glycol-2allyl-proline (cG-2allylP), derived from an endogenous DKP protects neurons from ischemic brain injury. Current study investigated the possible treatment effect of cG-2allylP in preventing 6-OHDA induced Parkinson motor deficit and dopamine depletion in rats.
Methods: Unilateral dopamine depletion was induced in adult rats. Two microinjections of 6-OHDA (7µg × 2 sites) were administered into right side striatum stereotaxically. 6-OHDA induced forelimb akinesia was examined using adjusting step test two weeks after the lesion. To have a comparable baseline crossed all groups, the rats were allocated into four treatment groups based on the number of adjusting steps made. Two weeks daily treatment of cG-2allylP (0 or 0.2 or 1 or or 5mg/kg/day, s.c) were initiated 14 days after the lesion. Parkinsonian deficit was examined at 6, 8, 10 and 12 weeks after the lesion using adjusting step tests. The nigral-striatal dopamine depletion was examined using tyrosine hydroxylase (TH) immunihistochemical staining 12 weeks after the lesion.
Results: 6-OHDA injection resulted to an approximately 90% loss of TH positive neurons in the SNc and around 80% loss of TH terminal staining in the striatum 12 weeks after the injury. A loss of ChAT positive neurons was also seen in the medial-ventral striatum, where 6-OHDA was injected. The number of adjusting steps was significantly reduced by approximately 80% 14 day after 6-OHDA lesion in all the groups. While the deficit remained in the vehicle treated group throughout the tests, the treatment of cG-2allylP significant increased the number of adjusting steps by 50%, suggesting a functional recovery. The treatment effect was not dose dependent with three doses tested showing similar effects. A trend towards increase in number of TH positive neurons was found in the SNc, however there was no improvement in TH terminal staining in the striatum. Cyclic G-2allylP treatment increased the number of doublecortin positive neuroblasts in the sub-ventricular zone compared to the vehicle treated group and without altering cell proliferation. A partial restoration in striatal ChAT neurons was also seen in the group treated with cG-2allylP.
Conclusion: Delayed treatments with cG-2allylP after the onset of motor deficit improved the long-term motor function following 6-OHDA induced striatum dopamine depletion. The functional recovery of cG-2allylP was independent on dopamine restoration in the striatum. The improved neurogenesis and plasticity in non-dopamine neurotransmission may have a role in cG-2allylP associated functional recovery, which need to be further examined.
IN VIVO DETECTION OF AMYLOID DEPOSITS IN ALZHEIMER'S DISEASE PATIENTS USING [11C]BF-227 PET
Nobuyuki Okamura1, Yukitsuka Kudo2, Shozo Furumoto2, Manabu Tashiro3, Katsutoshi Furukawa4, Yoshihito Funaki3, Motohisa Kato1, Satoshi Ito1, Yoichi Ishikawa3, Masahiro Maruyama4, Hiroyuki Arai4, Hiroshi Fukuda5, Ren Iwata3, Masatoshi Itoh3, Kazuhiko Yanai1
1Department of Pharmacology, Tohoku University School of Medicine, Sendai, Japan, 2Tohoku University Biomedical Engineering Research Organization, Sendai, Japan, 3Cyclotron and Radioisotope Center, Tohoku University, Sendai, Japan, 4Department of Geriatrics and Gerontology, Tohoku University School of Medicine, Sendai, Japan, 5Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
Background and aims: Progressive deposition of amyloid plaques is a critical event for the pathogenesis of Alzheimer's disease (AD). In vivo detection of amyloid deposits using positron emission tomography (PET) would thus prove useful for early diagnosis of AD and tracking disease progression. The purpose of this study is to evaluate the diagnostic utility of a novel PET imaging probe, 2-[2-(2-Dimethylaminothiazol-5-yl)ethenyl]-6-[2-(fluoro)ethoxy]benzoxazole (BF-227), in AD patients. Methods: Binding affinity of BF-227 to amyloid-β (Aβ) fibrils was calculated. Binding property of BF-227 to amyloid plaques was evaluated by neuropathological staining of AD brain sections. For clinical evaluation of [11C]BF-227, 10 normal subjects, 10 subjects with mild cognitive impairment (MCI) and 10 patients with AD participated in this study. Dynamic PET images were obtained for 60 min after administration of [11C]BF-227. Regional standardized uptake value (SUV) and the ratio of regional to cerebellar SUV were calculated as an index of [11C]BF-227 retention. Regional tracer distribution of AD patients was statistically compared with normal subjects on a voxel-by-voxel basis. Results: BF-227 displays high binding affinity to Aβ fibrils (Kd = 0.73 nM). BF-227 specifically binds amyloid plaques in AD brain sections. The clinical PET study using [11C]BF-227 demonstrated the retention of this tracer in cerebral cortices of AD patients, but not in those of normal subjects. All AD patients were clearly distinguishable from normal individuals using temporal SUV ratio. Cortical BF-227 retention in MCI subjects appeared either AD-like or control-like. Patient with frontotemporal dementia (FTD) showed no cortical retention of BF-227. Voxel-by-voxel analysis of PET images revealed that cortical BF-227 retention in AD patients is mainly distributed to posterior association area of the brain and corresponded well with the predilected site for amyloid plaque depositions containing dense Aβ fibrils. Conclusions: These findings suggest that [11C]BF-227 is a promising PET probe for in vivo imaging dense amyloid deposits in AD patients.
AGE-RELATED WHITE MATTER CHANGES, CEREBRAL MICROBLEEDS AND THEIR CORRELATION WITH COGNITIVE FUNCTION AND DEPRESSION
Monzurul Hasan Chowdhury1, Atsushi Nagai2, Kazuo Takahashi1, Hirokazu Bokura1, Shuhei Yamaguchi1, Shotai Kobayashi1
1Deparment of Internal Medicine III, Shimane University School of Medicine, Izumo, Shimane, Japan, 2Deparment of Laboratory Medicine, Shimane University School of Medicine, Izumo, Shimane, Japan
Background and aims: Aging and hypertension are the two major contributory risk factors for the white matter changes. Hypertension also plays a leading role for the occurrence of cerebral microbleeds. In healthy people, without any cerebrovascular disease (CVD) risk factors, aging and cerebral amyloid angiopathy sometimes progress simultaneously and silently. In our study, the relationship of their symptoms, cognitive functions, and MRI findings was evaluated in normal population.
Methods: We searched the database of 1120 subjects without hypertension, diabetes mellitus and hyperlipidemia (as defined by the standard regulatory body), visited the Institute of Shimane Health Science for health screening of the brain over 11 years from 1995 to 2006. The mean age of our study population was 60.3 ± 10.0 years and sex ratio M/F was 608/512. We examined all the physical parameters and blood levels available in the database and also examined both the conventional and T2* weighted MRI to define the cerebral microbleeds, cerebral amyloid angiopathy and age related brain changes in our study population. The diagnosis of periventricular hyperintensity was performed on MRI and was graded from 0-4, where grade 0–2 were defined as mild PVH and grade 3–4 were defined as marked PVH. After grading of PVH, we performed the logistic regression analysis to evaluate the outcome of these 2 groups in respect of their age, blood pressure, blood values, cognitive functions with word fluency, Okabe's intelligence evaluation test (Okabe's test: a shortened version of WAIS), Kohs' block design test (Kohs' test) and Zung's self rating depression scale (SDS).
Results: In our study population, 38 subjects (3.4%) had PVH score 3-4. Logistic regression analysis showed that age and memory were significantly related with PVH (p < 0.0001, p < 0.001). T2* weighted MRI revealed 9 subjects had cerebral cortical microbleeds and 9 subjects had deep gray matter microbleeds. Comparisons of these two groups revealed that the cerebral cortical microbleeds group had higher age, larger deep white matter hyperintensity and hippocampal atrophy, associated with less memory score (Okabe's test) as compared with the deep gray matter microbleeds group.
Conclusion: We demonstrated that PVH is related with aging and memory impairment in healthy elderly individuals. It is suggested that when cerebral cortical microbleeds occur in elderly individuals without any known CVD risk factors, cerebral amyloid angiopathy should be considered.
ACCUMULATION OF PARKINSON'S-DISEASE-RELATED MOLECULES IN LEWY BODIES AND GLIAL CYTOPLASMIC INCLUSIONS
Tetsuro Murakami1, Y. Imai2, H. Inoue3, T. Kawarabayashi4, M. Nagai1, T. Kurata1, Y. Takehisa1, Y. Harigaya5, M. Shoji4, R. Takahashi3, K. Abe1
1Department of Neurology, Okayama University Medical School, Okayama, Japan, 2RIKEN, Brain Science Institute, Saitama, Japan, 3Department of Neurology, Kyoto University Medical School, Kyoto, Japan, 4Department of Neurology, Hirosaki University of Mediciene, Aomori, Japan, 5Department of Neurology, Maebashi Red Cross Hospital, Gunma, Japan
Objective: To clarify the involvement of molecules that have been shown to be related to Parkinson's disease in the formation of Lewy bodies and glial cytoplasmic inclusions.
Background: The appearance of Lewy bodies (LBs), as well as neuronal loss in the substantia nigra, is a hallmark of Parkinson's disease (PD). Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized clinically by parkinsonism and ataxia, and pathologically by accumulation of alpha-synuclein in oligodendrocytes (glial cytoplasmic inclusions, GCIs). Recent progress in molecular biology has revealed several proteins that are genetically or experimentally related to PD. PD and MSA are now summarized as alpha-synucleinopathy, and a common cascade arising from the aggregation and accumulation of alpha-synuclein to the formation of LBs or GCIs leading to cell death is suggested.
Methods: We immunohistopathologically examined the localization of Parkinson's-disease-related molecules (alpha-synuclein, Pael-R, Parkin, Glup, and ubiquitin) in LBs and GCIs in PD, MSA, and normal controls (n=6, respectively).
Results: All these molecules examined were shown to be accumulated in LBs of PD. In GCIs of MSA, however, only alpha-synuclein and ubiquitin were shown to be present, and Pael-R, Parkin, and Glup were not accumulated.
Conclusions: Alpha-synuclein, Pael-R, Parkin, Glup, and ubiquitin are actually accumulated in LBs, and might be involved in the inclusion formation. However, in GCIs of MSA, only alpha-synuclein and ubiquitin was present, suggesting that the presence of a complete distinct pathway involved in the formation of LBs and GCIs in PD and MSA.
DISTRIBUTION OF AMYLOID DEPOSITION IN ALZHEIMER'S DISEASE AND NORMAL AGING DEMONSTRATED BY [11C]PIB PET
Hitoshi Shinotoh, Kiyoshi Fukushi, Shigeki Hirano, Hitoshi Shimada, Noriko Tanaka, Tsuneyoshi Ota, Koichi Sato, Tetsuya Suhara, Toshiaki Irie
Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan
Background and aims: Beta-amyloid plaques are the hallmark of Alzheimer's disease (AD) and the prevalence of AD increases in general population as aging. A previous [11C]PIB PET study showed that four of the 41 non-demented subjects already had amyloid deposition in the brain similar to those in AD patients1). We investigate age-associated amyloid deposition in middle-aged to elderly healthy subjects in an attempt to explore the initial distribution of amyloid deposition in presymptomatic phase of AD by [11C]PIB PET.
Methods: The subjects consisted of 15 healthy subjects ranging in age from 48 to 91 years with a mean age of 66 years and 12 patients with AD ranging in age from 58 to 85 years with a mean age of 69 years. The mean MMSE scores of the AD group was16.5 ± 5.3. [11C]PIB -PET acquisition was a dynamic sequence of 19 PET scans for 90 min. Spatial normalization of PET images were performed using a specific [11C]PIB template, which was created from 24 [11C]PIB scans. The Logan graphical analysis using arterial input function was employed to calculate distribution volume (DV). The target tissue to the cerebellum DV ratio (DVR) was calculated as an index of amyloid deposition in each voxel. Voxel-based statistical analysis was performed using SPM2.
Results: Two-tailed t-test showed significant amyloid deposition in the posterior cingulate, precunate, lateral parietal and temporal cortices, and frontal cortex in the AD patients compared with healthy controls (p<0.001, extent >100 voxels). Simple regression analysis showed a significant correlation between age and amyloid deposition in the posterior cingulated, precunate, upper part of parietal cortex, lower lateral temporal cortex, gyrus rectus, and occipital cortex (p <0.01, extent >100 voxels).
Discussion and Conclusion: The age-associated amyloid deposition may represent the amyloid deposition in the early phase of presymptomatic AD. The distribution of the age associated amyloid deposition mostly overlapped with those in AD. However, the age-associated amyloid deposition was seen in the occipital cortex, where no significant amyloid deposition was seen in the AD patients. The results suggest that the occipital cortex may be one of brain regions with amyloid deposition in the early phase of AD but the amyloid may not deposit further in the occipital cortex in the subsequent phases of AD.
CHANGES IN CBF DISTRIBUTION DURING ADOLESCENCE:A STUDY IN NEUROLOGICALLY NORMAL PATIENTS BY MEANS OF BRAIN SPECT AND SPM99
Taku Fujimoto, Kentaro Fujii, Hironori Otomune, Takuya Uehara, Yasuhiro Osaki, Ashik Bin Ansar, Yasuyuki Kimura, Katsufumi Kajimoto, Hiroki Katoh, Makiko Tanaka, Eku Shimosegawa, Jun Hatazawa
Department of Nuclear Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
Objectives: The changes in regional cerebral blood flow (CBF) distribution during adolescence have been little studied because of ethical reasons. We analyzed regional CBF distribution in neurologically normal patients age ranged from 8 to 41 years.
Patients and Methods: Twenty-two patients were enrolled in the present study. They were suspected to have neurological defects, and it was finally concluded that they were neurologically normal. All underwent brain perfusion SPECT with 99mTc-ECD under resting condition and no medications. The patients were subdivided into the following age groups: the 8-11 years-old group (early adolescence, 4 men and 3 women), the 16-18 years-old group (late adolescence, 4 women), and the 20-40 years-old group (Adult, 5 men and 6 women). By means of the SPM99, the images were anatomically normalized and smoothed. The between-group differences in CBF distribution were estimated.
Results: Between early and late adolescence, significant relative CBF difference (late-early) was found in right hippocampus (x=24, y=6, z=20, p<0.01, uncorrected) and anterior cingulate gyrus (x=10, y=26, z=-12, p<0.05, uncorrected). Between late adolescence and adult group, significant CBF difference (adult-late) was found in bilateral precuneus and posterior cingulate gyrus (x=22, y=76, z=40, p<0.01, uncorrected).
Discussion: The CBF distribution in the right hippocampus and anterior cingulate gyrus was increased during early adolescence, and the CBF distribution in the bilateral precuneus and posterior cingulate gyrus was increased during late adolescence. The hippocampus is responsible for storage and retrieval of general declarative memory [1]. The precuneous plays main part in retrieval of episodic memory [2]. The anterior cingulate gyrus mediates processing selection of extraordinary stimulation such as visual or olfactory stimulation [3]. The posterior cingulate cortex is involved in spatial orientation and memory, and connections between this area and parahippocampal cortices contribute to these processes [4]. Our findings indicate that the functional developments of memory system in human brain occur during adolescence.
DYSFUNCTION OF MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION IN ALS RAT MODEL
Makiko Nagai1, Tetsuro Murakami1, Yasuyuki Ohta1, Yasushi Takehisa1, Nobutoshi Morimoto1, Kazunori Miyazaki1, Tomoko Kurata1, Tatsushi Kamiya1, Koji Abe1, Serge Przedborski2
1Department of Neurology, Okayama University, Okayama, Japan, 2Center for Motor Neuron Biology and Disease, New York, NY, USA
Background and aims. Amyotrophic lateral sclerosis (ALS) is caused by the selective degeneration of motoneurons in spinal cord, brain stem and cerebral cortex. The degeneration of motoneurons leads to skeletal muscle atrophy, paralysis and death. About 10 % of ALS cases are familial and mutation of Cu, Zn superoxide dismutase (SOD1) has been demonstrated to be a culprit among 20 % of the familial cases. Although contraversial, mitochondrial abnormalities have been reported in ALS-patients and in mouse model such as mutated human SOD1 transgenic mice and mnd mouse. To address this issue we assess mitochondrial function in transgenic rats overexpressed a human mutated SOD1 associated with ALS.
Material and methods. Spinal cords and cerebellum from G93A and non-transgenic rats were homogenized in isolation buffer. The homogenate was centrifuged at 1,000 g for 10 min, and the resulting supernatant was layered onto 7.5% Ficoll medium on top of the 10% Ficoll medium and centrifuged at 79,000 g for 30 min. The mitochondiral pellet was resuspended in isolation buffer. 10 µg isolated mitochondria were separated on 15% SDS-PAGE and transferred to a nitorocellulose membrane. The membrane was immunostained rabbit anti-human SOD1 antibody or mouse anti-cytochrome c oxidase antibody. Secondary antibodies conjugated with HRP were used. Mitochondria were suspended in respiration buffer at 28 oC, and oxygen-consumption rates were measured in a closed-chamber cuvette with a mini-stirring bar using Clark-type electrode. To assess mitochondrial respiration mediated through complex I, mitochondria were preincubated with 10 mM glutamate and 5 mM for 2.5 min before 500 µM ADP was added to induce state 3 respiration. For complex II mediated respiration, mitochondria were preincubated for 2.5 min with 10 mM succinate before 500 µM ADP was added. For complex IV mediated respiration, mitochondria were preincubated for 2.5 min before 10 mM ascorbate and 0.2 mM N,N,N',N'-tetramethyl-benzendiamine were added to induce oxygen consumption.
Results. Significant level of human mutated SOD1 was detected in purified mitochondria of G93A rat, but not endogeneous rat SOD1. SOD1 aggregates were detected in G93A spinal cord mitochondria. Significant reduction of O2 consumption using complex I substrates were observed in the spinal cord mitochondria from G93A rats. On the other hand, enzyme activities, which are normalized against citrate synthase activities showed no significant changes in complex I. Polarographical study of mitochondrial respiration via complex II or complex IV showed no significant difference between nontransgenic and G93A rat mitochondria. difference between non-transgenic and G93A rat mitochondria.
Conclusion. In transgenic G93A rat model of ALS, we showed a specific reduction of mitochondria respiration mediated through complex I, but not complex II and IV. This functional abnormality was only detected in intact mitochondria of G93A spinal cord, but not in freeze-thawed lysed mitochondria. This study supports the role of mitochondial defects in the pathogenesis of ALS.
EXTENDED POLYGLUTAMINE REPEATS TRIGGER A FEEDBACK LOOP INVOLVING THE MITOCHONDRIAL COMPLEX III, THE PROTEASOME, AND HUNTINGTIN AGGREGATES
Hirokazu Fukui1, Carlos Moraes1,2,3
1Neuroscience Graduate Program, University of Miami Miller School of Medicine, Miami, FL, USA, 2Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA, 3Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL, USA
Mitochondrial abnormalities represent a major cytopathology in Huntington's disease, a fatal neurodegenerative disease caused by CAG repeat expansions in the gene encoding huntingtin. In the present study, we investigated whether defects in the mitochondrial respiratory function are consequences of the expression of mutant huntingtin or whether they promote the formation of huntingtin aggregates. To take advantage of existing mitochondrial DNA mutants, we developed human osteosarcoma 143B cells expressing mutant huntingtin in an inducible manner and found that cells expressing mutant huntingtin but not wild type huntingtin exhibited a reduced activity of Complex II + III and an increased activity of Complex IV. Conversely, pharmacological treatments that inhibited Complex III activity significantly promoted the formation of huntingtin aggregates. This effect of Complex III inhibition on the huntingtin aggregates appeared to be mediated by the inhibition of proteasome activity but not by ATP depletion or production of reactive oxygen species. Accordingly, complex III mutant cells also showed decreased proteosome activity. These results suggest the presence of a feedback system connecting the mitochondrial respiratory Complex III and the production of huntingtin aggregates. Our results suggest that therapeutic interventions targeting complex III and/or proteasome could ameliorate the progress of Huntington's disease.
DIFFERENCES OF NEURAL INTERACTIONS BETWEEN EARLY AND LATE ONSET ALZHEIMER'S DISEASE: A NETWORK ANALYSIS
Sang Soo Cho1, Heejung Kim1, Hey Jin Kang1, Eun Joo Kim2, Dong Soo Lee1, Yu-kyeong Kim1, Duk L. Na2, Sang Eun Kim1
1Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, South Korea, 2Department of Neurology, Sungkyunkwan University School of Medicine, Seoul, South Korea
Background and aims: Early-onset (EO) and late-onset (LO) Alzheimer's diseases (ADs) are known to have different cognitive decline profiles and pathophysiology. The present study was designed to determine and compare the neural interactions underlying general cognitive function for the two subtypes of Alzheimer's disease using FDG PET and path analysis.
Methods: FDG PET imaging was performed in 73 patients with EO AD (AD onset before 65 yr) (age 59.1± 5.7 y, 49 females/24 males, MMSE score 17.5±6.9), 47 patients with late onset AD (age 74.1±4.7 y, 33 females/14 males, MMSE 18.9±6.5) and 15 young (54.0±2.8 y, 6/9) and 20 aged (69.0±4.1 y, 12/8) healthy controls. For path analysis, five brain regions (middle and inferior frontal, inferior temporal gyri, inferior parietal lobule, and posterior cingulate) were selected in each hemisphere based on the results of the correlation analysis between MMSE score and regional brain glucose metabolism using SPM99. Hippocampus was selected additionally because this area is important for the pathology of AD. Voxel values for each coordinate were extracted from the data set of each subject. For the model construction, anatomical connection was defined based on the previous researches. First, we tested the goodness-of-fit to assess the suitability of the constructed model. Second, using this model, we evaluated the differences in the path model between two patients groups. All the model construction and path analysis processing was done by AMOS 5.0 (SmallWaters Co.)
Results: Goodness-of-fit of final constructed model was selected [X2 (4) =3.12, P=0.54 in left and X2 (4) = 1.88, P=0.76 in right]. In both hemispheres, the intra-hemispheric functional networks proved to be significantly different between the two AD groups [left: X2 diff (13) = 28.38, P < 0.01; right: X2 diff (13) = 27.65, P < 0.05]. Comparing with functional network of age-matched normal controls, several inter-regional connections were weakened in both AD group. In EO AD, especially the neural connections from frontal to lateral and medial temporal area were changed while in LO AD, those of frontal to posterior cingulate and hippocampus to posterior cingulate were changed.
Conclusion: Our data demonstrate the different patterns of dysorganization in functional network between EO and LO AD patients. They provide additional insight into the pathophysiology of AD subtypes and neural functioning for cognitive processes.
EFFICACY OF MINOCYLINE FOR DELAYED TISSUE PLASMINOGEN ACTIVATOR TREATMENT AFTER EMBOLIC FOCAL ISCHEMIA IN RATS
Yoshihiro Murata1, Anna Rosell1, Xiaoying Wang1, Robert H. Scannevin2, Kenneth Rhodes2, Yoichi Katayama3, Eng H. Lo1
1Neuroprotection Research Laboratory, Departments of Neurology and Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, 2Johnson and Johnson Pharmaceutical Research and Development, Spring House, PA, USA, 3Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan
INTRODUCTION: Tissue plasminogen activator (tPA) therapy for acute stroke patients must be administrated within 3 hours after symptom onset. The narrow therapeutic time window (TTW) may be related to the elevated risks of intracranial hemorrhage after thrombolysis. In the present study, we investigated whether minocycline can prevent delayed tPA- associated intracranial hemorrhage and extend the TTW of tPA treatment. METHODS: Male spontaneously hypertensive rats were subjected to embolic middle cerebral artery (MCA) occlusion. Regional cerebral blood flow was monitored by laser-Doppler flowmetry. Five experimental groups were used. Gtoup1; Saline at 1hour after ischemia (untreated group), Group2; tPA at 1hour after ischemia, Group3; tPA at 6hr after ischemia (delayed tPA), Group4; minocycline at 4hours after ischemia (minocycline alone), Group5; minocycline and tPA were administrated respectively at 4hours and at 6hours (combination treatment with minocycline and delayed tPA). All drugs were administrated intravenously. At 24hours after ischemia, we evaluated mortality rates, infarct volumes with TTC (2,3,5-triphenyltetrazolium chloride) staining, and hemorrhage volumes with spectrophotometric assay. Blood samples for MMP zymography were taken from Group 3 and Group 5 to investigate the effect of minocycline for delayed tPA treatment. RESULTS: Early 1 hr thrombolysis with tPA restored blood flow and significantly reduced infarct volumes. However, late 6 hr tPA did not work, and instead increased mortality and induced hemorrhagic conversion. Treatment with minocycline re-established late tPA's protective effects by decreasing mortality, reducing infarction volumes in surviving rats, and ameliorating brain hemorrhage severity. Quantitative data are presented in the Table below:
In Group 3, plasma MMP-9 was upregulated after ischemia and tPA treatment further amplified these ischemic responses in MMP-9. Combination treatment with minocycline plus tPA significantly reduced the increment of MMP-9 post-ischemia and post-tPA, in concert with the improved outcomes described above. CONCLUSIONS: Our initial results indicate that (1) minocycline can reduce infarct volume even though tPA is administrated at 6 hours after ischemia, (2) minocycline can prevent intracranial hemorrhage associated with tPA treatment, (3) minocycline can reduce the mortality rate associated with delayed tPA treatment, and (4) these improvements in outcome after delayed tPA plus minocycline therapy may be related to the ability of minocycline to ameliorate ischemic and tPA-induced MMP-9 responses. Therefore, minocycline may be a promising candidate to extend the TTW of tPA treatment in part by preventing tPA-associated intracranial hemorrhage. Supported in part by NIH grants R01-NS37074, R01-NS48422, R01-NS56548, and P50-NS10828.
ELEVATION OF INTERLEUKIN-6 LEVELS IN THE ASPIRATION FROM THE STENOTIC LESION AFTER PERCUTANEUS CAROTID ARTERY STENTING
Yuko Abe1, Kazuo Kitagawa1, Manabu Sakaguchi1, Taku Hoshi1, Shigetaka Furukado1, Hidetaka Hogaku1, Masatsugu Hori2, Toshiyuki Fujinaka1, Toshiki Yoshimine1
1Stroke Center, Osaka University Hosipital, Suita, Osaka, Japan, 2Department of Cardiovascular Medicine, Osaka University, Suita, Osaka, Japan
Background: Inflammation plays a fundamental role in the initiation, development, progression, and eventual rupture of atherosclerotic plaques. A variety of circulating markers of inflammation such as high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6) and interleukin-18 (IL-18) have been predictors of future vascular events. However, it remains unclear as to which markers of inflammation originate locally from the atherosclerotic plaque. Carotid artery stenting (CAS) with a distal protection device causes the mechanical plaque rupture and releases the components of atheromatous plaque. Therefore, we investigated inflammatory marker levels by aspiration at the site of the stenotic lesion after CAS together with those in the descending aorta.
Methods: The study involved 23 patients with 27 severe carotid artery stenosis vessels that underwent CAS. In all patients, a balloon based embolization protection device and aspiration catheter were used during CAS. We took the preprocedual blood sample from the descending aorta and the postprocedual blood sample at the site of the plaque under distal balloon occlusion. Serum concentrations of IL-6, IL-18, and hsCRP were measured from these samples.
Results: The preprocedual concentrations of IL-6, IL-18, and hsCRP amounted to 2.14 ng/L (interquartile ranges (IQR), 1.09 to 4.51 ng/L), 239 ng/L (IQR, 192 to 319 ng/L), and 0.09 mg/dL (IQR, 0.05 to 0.60 mg/dL). The postprocedual concentrations of IL-6, IL-18, and hsCRP became 3.34 ng/L (IQR, 1.99 to 5.56 ng/L), 250 ng/L (IQR, 181 to 330 ng/L), and 0.09 mg/dL (IQR, 0.03 to 0.46 mg/dL). The local level of IL-6 significantly increased at the site of plaque after CAS (P<0.001). In contrast, local hsCRP slightly decreased (P=0.002) while IL-18 remained unchanged (P=0.355).
Conclusion: The fact that local level of IL-6 increased after CAS relative to the systemic circulation supports our hypothesis that local IL-6 production was present in carotid atherosclerotic plaque. These results also suggested that serum IL-18 and hsCRP levels may be a systemic origin.
IMPLANTED HUMAN NEURAL STEM CELL PROMOTE NEUROGENESIS OF ENDOGENOUS STEM CELLS AFTER FOCAL CEREBRAL ISCHEMIA
Sun Ryu1, Seung-Hoon Lee1, Miyeoun Song1, Seung up Kim2, Byung-Woo Yoon1
1Department of Neurology, Seoul National University and Neuroscience Research Institute, Clinical Research Institute SNU Hospital, Seoul, South Korea, 2Brain Disease Research Center, Ajou University, Seoul, South Korea
Background and Purpose Transplantation of human neural stem cell (hNSC) have been previously shown to improve functional recovery of ischemic rats. To extend these findings, we examined the endogenous stem cell proliferation in the presence of intraventricular implanted hNSC after focal cerebral ischemia.
Methods Transient focal ischemia was induced by middle cerebral artery occlusion (MCAo) for 60 minutes with a nylon filament in rat. Twenty four hours after MCAo, either 6 × 105 hNSC or phosphate-buffered saline was injected into the lateral ventricle on the ischemic side. All animals received daily injection of bromodeoxyuridine (BrdU; 100mg/kg, i.p.) for 7days after treatment for identification of newly synthesized DNA. On the 14th day after MCAo, we evaluated infarct volume and endogenous neurogenesis histologically. Functional recovery was serially evaluated by the modified Neurological Severity Score after MCAo.
Result At 14 days after transplantation, there was a significant recovery in behavioral performance (p < .05). Cerebral infarcts in hNSC-implanted rats were reduced in size (p < .05). hNSCs were found to survive and preferentially localize to ipsilateral ischemic hemisphere. Cell proliferation and endogenous neurogenesis were assessed with BrdU labeling and immunostaining for cell type specific markers.
Significantly more BrdUrd-labeled endogenous cells were located in the the subventricular zone and hippocampus (p < .05), and more newly generated cells in both region were immunoreactive for Dcx, NeuN and GFAP in hNSC- implanted rats than in vehicle-injected rats.
Conclusion Implantation of human NSCs promoted the proliferation of endogenous neural stem cells, and that the proliferated cells differentiated into more mature neural cells after stroke in rats.
EFFECTS OF LONG-TERM ADMINISTRATION OF HMG-COA REDUCATESE INHIBITOR, ATROVASTATIN, ON STROKE EVENTS AND L-CBF IN SHRSP
Noriko Tanaka, Yasuo Katayama, Toshiya Katsumata, Tatsuo Otori, Yutaka Nishiyama
Division of Neurology, Department of Internal Medicine, Nippon Medical School, Tokyo, Japan
[Background and aims] To determine the effect of long-term administration of a HMG-CoA reductase inhibitor, atorvastatin, on stroke events in stroke-prone spontaneously hypertensive rats (SHRSPs), and its possible neuroprotective effect.
[Methods] Atorvastatin (2 mg/kg, 20 mg/kg) or vehicle was orally administered to 8-week-old SHRSPs for 11 weeks. The survival ratio and stroke incidence were calculated, and plasma lipids and plasma levels of ADMA (asymmetric dimethylarginine, a marker of endothelial dysfunction) were measured after sacrifice. The effect of atorvastatin on local cerebral blood flow (l-CBF) was also determined in 13-week-old SHRSPs after treatment with 20 mg/kg atorvastatin daily for 5 weeks.
[Results] Survival ratios at 19 weeks of age were 15, 30 and 50% in the vehicle, low-dose (2 mg/kg) and high-dose groups (20 mg/kg), respectively (Fig.1). Survival ratio was significantly higher in the high-dose group compared to the vehicle group. Stroke incidence was significantly lower in the high-dose group compared to the vehicle group. ADMA levels were 0.81±0.18 (mean± S.D.), 0.62±0.09 and 0.61±0.06 µmol/L in the vehicle, low-dose and high-dose groups, respectively. Atorvastatin significantly reduced the ADMA levels without affecting plasma lipid levels. L-CBF tended to be higher in the treated group compared to control, but without significance. The l-CBF decreases in SHRSPs as hypertension develops. Statin administration may attenuate the reduction of CBF, arterial endothelial injury and disruption of the blood-brain barrier in SHRSPs. Thus, atorvastatin was determined to provide a protective effect against hypertension-based stroke. The data suggest that increased NO production may take part in the efficacy of the statin for stroke protection. In addition to the lipid-lowering effects of statins, pleiotropic effects may play important roles in stroke prevention.
[Conclusion] Statins may exert protective effects, not only against atherosclerosis-based stroke, but also hypertension-based stroke.
POSTISCHEMIC ADMINISTRATION OF THE CANDESARTAN REDUCES INFARCT SIZE AFTER TRANSIENT CEREBRAL ISCHEMIA IN HYPERTENSIVE RATS
Emi Omura-Matsuoka, Kazuo Kitagawa, Yoshiki Yagita, Tsutomu Sasaki, Yasukazu Terasaki, Naoki Ohyama, Kenichi Todo, Masatsugu Hori
Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
Background and aims: In the present study, we investigated the ability of the orally administered angiotensin II type 1 receptor blocker, candesartan, to ameliorate stroke outcome in spontaneously hypertensive rats after focal cerebral ischemia.
Methods: Adult male spontaneously hypertensive rats weighing 250 to 350 g were used in this study. Focal cerebral ischemia was induced by middle cerebral artery occlusion permanently or transiently, lasting for 60 minutes following 48 hours reperfusion. In rats with permanent MCA occlusion, vehicle (n = 8) or candesartan (n = 8 per group) was applied orally by gavage at three different doses (0.1, 1 and 10 mg/kg), three hours after ischemia. In rats with transient MCA occlusion, vehicle (n = 12) or candesartan (n = 12 per group) were administered twice (3 and 24 hours after MCA occlusion). We measured systolic arterial blood pressure in conscious rats with permanent MCA occlusion by the tail-cuff method before ischemia and 3, 6 and 24 hours after ischemia. In rats with transient MCA occlusion, time point of 48 hours after ischemia was added. Evaluation of neurological deficits was performed before ischemia and 3, 6, 24 and 48 hours after ischemia. Twenty-four or forty-eight hours after MCA occlusion, the brain was dissected into coronal 2-mm sections and the brain slices were incubated in saline containing 2% 2,3,5-triphenyltetrazolium chloride (TTC). The ischemic volume was determined by integrating the appropriate area and the section thickness.
Results: 1. Blood Pressure; In both permanent and transient MCAO groups, treatment with 1 and 10 mg/kg candesartan significantly reduced systolic blood pressure as early as 3 hours after administration compared with vehicle, and the reduction was maintained till 21 hours after treatment. In transient MCAO group, the reduction in systolic blood pressure was more pronounced after 24 hours after second administration in a dose-dependent manner. There was no significant difference in blood pressure between candesartan 0.1 mg/kg-treated and vehicle-treated rats. 2. Ischemic Lesions; A. Permanent ischemia; Lesion areas detected with TTC staining were occupied almost of all left hemisphere supplied by the MCA (vehicle, 315.4 ± 56.1 mm3). There was no difference in lesion size among the treatment groups (0.1 mg/kg, 309.3 ± 54.9 mm3; 1 mg/kg, 346.2 ± 48.1 mm3; 10 mg/kg, 322.1 ± 31.5 mm3). B. Transient ischemia; Cerebral cortex and caudoputamen were also affected in transient MCA occlusion, but the extent of ischemic lesion were smaller than that of permanent MCA occlusion group. The total lesion volume was significantly smaller in rats treated with candesartan at dose of 0.1 mg/kg than those treated with vehicle (109.6 ± 86.6 mm3 versus 234.9 ± 59.1 mm3; P < 0.05). There was no difference between candesartan 1 or 10 mg/kg treated-rats and vehicle-treated rats. 3. Neurological Findings; In both permanent and transient MCAO groups, there was no difference among the treatment groups.
Conclusions: Our results demonstrate that the angiotensin II type 1 receptor blocker, candesartan orally applied after transient focal ischemia reduced infarct volume, at doses with no effect on blood pressure.
TISSUE-TYPE PLASMINOGEN ACTIVATOR ENHANCED THE PERMEABILITY OF BLOOD BRAIN BARRIER
Yasuhiro Suzuki1, Nobuo Nagai2, Desire Collen2, Kazuo Umemura1
1Department of Pharmacology, Hamamatsu University School of Medicine, Hamamatsu, Japan, 2Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
[Background] Although thrombolytic therapy of ischemic stroke with tissue-type plasminogen activator (tPA) improves clinical outcome, it increases a risk of intracerebral hemorrhage, which has been thought to be via plasmin activation. Recently, however, it has been reported that tPA affects the permeability of blood brain barrier (BBB) without plasmin activation, and then that may be result in intracerebral hemorrhage and brain damage.
[Objectives] We investigate whether tPA may affect the permeability of BBB in a middle cerebral artery (MCA) occlusion model in mice.
[Methods] The permanent MCA occlusion was performed by double ligations and cutting the MCA between them. Permeability of BBB was measured by leakage of Evans Blue (EB) to the brain tissue. EB was intravenously injected 2 hours before sacrifice. An intravenous bolus of 10 mg/kg of tPA was given at various time (either 1, 4 or 70 hours) from MCA occlusion. Instead of tPA, same dose of inactivated tPA obtained by incubation with D-Phe-Pro-Arg-choloromethylketone was also administered. To investigate possible mechanism(s) of tPA effects, one or two mg/kg of receptor associated protein (RAP), an antagonist of low-density lipoprotein (LDL) receptor family, was intravenously administered 5 min before injection of tPA.
[Results] tPA given 4 hours after MCA occlusion significantly increased EB extravasation 2 hours after tPA administration (1.0 [<0.01?6.8] µg in median and range; n=8, P<0.05 vs. 0.44 [<0.01?0.84] in controls, n=8), whereas tPA given at 4 hours did not increased EB extravasation at 5 hours after tPA treatment (0.27 [<0.01?0.77] µg; n=6, NS vs. 0.06 [<0.01?0.58] µg in controls; n=6). Furthermore, tPA given 1 hour after MCA occlusion did not alter EB extravasation 2 hours after tPA administration (0.08 [<0.01?6.8] µg; n = 6, NS vs. 0.01 [<0.01?0.66] in controls, n=6), whereas tPA given 70 hours after MCA occlusion significantly increased EB extravasation 2 hours after tPA administration (4.1 [1.2?8.0] µg; n=6, P<0.05 vs. 0.96 [0.03?0.84] in controls, n=8). Administration of inactivated tPA 4 hours after MCA occlusion did not increase the EB extravasation 2 hours after tPA treatment (0.17 [<0.01?0.48]; n=6). Co-treatment with 1 or 2 mg/kg of RAP 4 hours after MCA occlusion significantly suppressed to the EB extravasation 2 hours after tPA treatment in a dose-dependent manner; a median value of 0.88 µg (0.08–2..9) with 1 mg/kg or 0.10 µg (<0.01?0.91) with 2 mg/kg of RAP (P<0.05 in each vs. without RAP treatment, n=7).
[Conclusions] tPA given 4 hours after MCA occlusion transiently enhanced the permeability of BBB 2 hours after the treatment, which reduced to the basal level at 5 hours. The enhancement by tPA treatment was specific beyond 4 hours after MCA occlusion. The increase of BBB permeability by tPA required its protease activity and was through LDL receptor family. Therefore, the temporal increase of BBB permeability after delayed tPA treatment may be related to the intracranial hemorrhage and brain damage.
DETECTION OF MISERY PERFUSION BY TRANSCRANIAL COLOR SONOGRAPHY IN CAROTID OCCLUSIVE DISEASE: COMPARISON WITH POSITRON EMISSION TOMOGRAPHY
Yukio Sugiyama1, Takuya Hayashi2, Kazuyuki Nagatsuka1, Hiroshi Moriwaki1, Kotaro Miyashita1, Susumu Miyamoto3, Kazuo Minematsu1, Hiroaki Naritomi1
1Department of Cerebrovascular Medicine, National Cardiovascular Center, Osaka, Japan, 2Department of Investigative Radiology, National Cardiovascular Center Research Institute, Osaka, Japan, 3Department of Cerebrovascular Surgery, National Cardiovascular Center, Osaka, Japan
[Background and aims]
Increased oxygen extraction fraction (OEF) measured by positron emission tomography (PET), known as an indicator of misery perfusion, is an independent predictor of subsequent stroke in patients with carotid occlusive disease. This study tested whether transcranial color sonography (TCCS), a widely available, noninvasive, and cost-effective modality, can detect hemispheres with misery perfusion.
[Methods]
In 27 carotid arteries of 24 patients (67.8 years of mean age) with carotid occlusive disease, we measured mean flow velocity (MFV) of middle cerebral artery (MCA) at a depth of 50 mm using TCCS while subjects were normally breathing or breath-holding, and calculated the breath-holding index (BHI) to evaluate cerebrovascular reactivity (Stroke 23: 668, 1992). We excluded carotid arteries which accompanied the ipsilateral MCA occlusive lesion. We correlated MFV and BHI with cerebral blood flow (CBF), cerebral blood volume (CBV), oxygen extraction fraction (OEF) measured by PET in the MCA area of the corresponding hemispheres.
[Results]
MFV was positively correlated with CBF (r=0.44, p<0.05). BHI ranged from 0.09 to 1.58 (mean ± SD, 0.65 ± 0.36) and was negatively correlated with CBV/CBF (r=-0.57, p<0.01) and OEF (r=-0.58, p<0.01). If the cut-off point of BHI was set to 0.45, hemispheres with increased OEF (>0.52, the mean + 2SD calculated from five normal volunteers) was detected with sensitivity of 100% and specificity of 94%.
[Conclusions]
Despite small number of observations, the current study suggests that the TCCS could be useful for screening misery perfusion in carotid occulusive disease.
TRENDS IN THE INCIDENCE OF STROKE IN YAMAGATA, JAPAN
Rei Kondo, Yasuhiko Matsumori, Daisuke Tsuchiya, Naoki Kato, Yasuaki Kokubo, Shinya Sato, Takamasa Kayama
Department of Neurosurgery, Yamagata University Faculty of Medicine, Yamagata, Japan
The purpose of this study was to clarify the incidence and the features of acute cerebrovascular diseases in Yamagata Prefecture where the population is about 1.22 million, by comparing with those in other prefectures reported in previous studies.
Consecutive 13,641 cases of acute stroke were prospectively registered to Yamagata Society in Treatment for Cerebral Stroke (YSTCS) between January 1, 1998 and December 31, 2002. Cerebral infarction, intracerebral hemorrhage, subarachnoid hemorrhage and transient ischemic attack were observed in 58.4%, 25.8%, 11.4% and 4.4% of the patients, respectively. The frequencies of intracerebral hemorrhage and subarachnoid hemorrhage (37.2%) in Yamagata Prefecture were significantly higher than those reported in other studies (p < 0.01). In regards to the time from the onset to medial contact, the rate of the time within 3 hours and the time from 3 to 6 hours were 21% and 15%, respectively. There was the significant longer time in Yamagata Prefecture than in others (p < 0.01).
This study was one of the largest stroke registration studies in Japan enrolling 13,641 patients. In all patients, computed tomography (CT) and/or magnetic resonance image (MRI) was performed on admission. In addition, diagnosis was especially made by experienced neurologists or neurosurgeons in stroke based on CT and/or MRI findings. Our results could show current status of stroke patients in Yamagata Prefecture.
RISK FACTORS OF CEREBRAL INFARCTION: COMPARISON BETWEEN LACUNAR THROMBOSIS AND ATHEROSCLEROTIC THROMBOSIS
Kiwami Kidana, Mieko Otsuka, Akira Ueki
Department of Neurology, Jichi Medical University Omiya Medical Center, Saitama-City, Saitama, Japan
Purpose: The major risk factor for lacunar thrombosis is hypertension, and those for atherosclerotic thrombosis are diabetes, and hyperlipidemia. Incidence of lacunar thrombosis has been decreased in recent Japan mainly due to treatment of hypertension. On the contrary, rapidly growing incidence of diabetes and/or hyperlipidemia in Japan may contribute the increased incidence of atherosclerotic thrombosis. To test this explanation, we compared risk factors between patients with lacunar thrombosis, atherosclerotic thrombosis, and cardiogenic embolism.
Methods: Fifty nine patients who admitted to our neurological ward from July, 2005 to August, 2006 were recruited. Clinical diagnosis of three groups was made by history of present illness (the mode of onset, clinical course), neurological examination, and cranial CT, MRI, and MRA. Frequencies of hypertension, diabetes, hyperlipidemia, obesity, smoking, dehydration., alcohol consumption were compared between three subgroups.
Results: We picked out 59 patients in all- lacunar thrombosis was 22 patients (37.3%), atherosclerotic thrombosis was 14 patients (23.7%), and cardiogenic embolism was 23 patients(38.9%). The average age of embolic infarction was 72.2±9.4 years old, lacunar was 62.2±8.9 years old, and atherosclerotic thrombosis was 62.6±11years old. The male ratio of lacunar thrombosis was 90.9%, atherosclerotic thrombosis was 64.3%, and cardiogenic embolism was 60.9%. The ratio of diabetes was 50% in lacunar thrombosis, 14.3% in atherosclerotic thrombosis, and 34.8% in cardiogenic embolism. The ratio of hypertension was 86.4% in lacunar thrombosis, 57.1% in atherosclerotic thrombosis, and 52.2% in cardiogenic embolism. The ratio of hyperlipidemia was 45.5% in lacunar thrombosis, 28.6% in atherosclerotic thrombosis, and 21.7% in cardiogenic embolism. The average of BMI was 23.6±3.0 in lacunar thrombosis, 20.7±2.9 in atherosclerotic thrombosis, and 23.0±5.5 in cardiogenic embolism.
Conclusion: Unexpectedly, frequencies of diabetes, hyperlipidemia, and obesity was lower in atherosclerotic thrombosis than lacunar thrombosis. Higher consumption of alcohol and probably concomitant malnutrition might contribute the genesis of atherosclerotic thrombosis.
REGULATION OF FOXO3A TRANSCRIPTION FACTOR BY PHOSPHOINOSITIDE 3-DEPENDENT SIGNALING IN ISCHEMIC NEURONAL DEATH
Gyeong-Moon Kim, Kyung-Sil Jin, Kwang-Yeol Park
Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
Backgound and aims: FOXO3a, a forkhead family of transcription factor, has been known to regulate apoptosis, cell cycle, cell metabolism, or the activation of genes involved in DNA repair and oxidative stress. Akt deactivates FOXO3a by direct phosphorylation and cytoplasmic retention. Survival factor withdrawal leads to FOXO3a dephosphorylation, nuclear translocation, target gene activation and apoptosis eventually. We sought to evaluate the relationship between FOXO3a activation and its target gene expression in neuronal cell death after oxygen-glucose deprivation (OGD). Methods: Cultured neurons from rat embryonic cortex were subjected to OGD for 120 minutes. At periods of 30 minutes, 2, 6, and 24 hours after reoxygenation, proteins were purified and subcellular fractionization was performed. The levels of Akt, phospho-Akt, Bim and Fas ligand (FasL) expression were measured by Western blot and immunohistochemistry. DNA binding activity of FOXO3a was determined by electromobility gel shift assay (EMSA). We also evaluated the amount of cell death after specific Akt/PI3K inhibitor LY294,002 and evaluate the changes of protein binding of FOXO3a and the expression of Bim and FasL. Results: p-Akt activity was increased in the early phase of reoxygenation period in both cytoplasmic and nuclear extracts. FOXO3a target genes, Bim and FasL, showed increased expression after OGD. EMSA revealed significant elevation of DNA binding activity for FOXO3a after OGD and it peaked at 3–6 hours of reoxygenation. In addition, subcellular localization of FOXO3a by double immunofluorescence staining with neuronal marker (NeuN) revealed nuclear translocation of FOXO3a in morphologically apoptotic neurons. Furthermore, LY294,002 treatment promoted ischemic neuronal death through increased DNA binding of FOXO3a and enhanced expression of FasL. Conclusions: These results suggest that the activation of FOXO3a transcription factor may be mediated, at least in part, by the PI3K-dependent signaling pathway in ischemic neuronal death. Transcriptional regulation of FOXO3a maybe targeted as therapeutic approach to prevent neuronal apoptosis after acute ischemic insult.
OXIDATIVE STRESS INCREASES THE PHOSPHORYLATION OF SER10 HISTONE H3 THROUGH IKAPPAB KINASE ALPHA IN ENDOTHELIAL CELLS AFTER ISCHEMIA AND REPERFUSION
Yun Seon Song, Purnima Narasimhan, Carolina M. Maier, Pak H. Chan
Departments of Neurosurgery, Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Stanford, CA, USA
Introduction: Histone modification is one of the key regulatory steps essential for transcriptional activation. Phosphorylation of histone H3 activates the transient expression of immediate-early genes. Stimulation of both the ERK pathway and the stress-activated p38 pathway also results in an increase in phosphorylation of histone (Ser10) during activation of immediate-early gene expression (Strahl and Allis 2000). It has been reported that IKK alpha has histone H3 kinase activity after cytokine exposure. The cytokine-induced phosphorylation of Ser10 by IKK alpha in histone H3 seems to be especially important for the subsequent acetylation of Lys14 by cAMP-response element binding protein for transcriptional activation (Yamamoto et al. 2003). However, the cellular role of IKK alpha associated with oxidative stress in cerebral ischemia is unknown.
Materials and methods: To clarify the role of IKK alpha in oxidative stress, we investigated the IKK alpha-related proteins after transient focal cerebral ischemia (tFCI) and oxygen-glucose deprivation (OGD) of endothelial cells. Samples were taken 0, 1, and 2 hours after tFCI and OGD. Cytosolic and nuclear fractions were prepared from ischemic brain and endothelial cells using ProteoExtract(Calbiochem) to study the nuclear translocation of protein. Changes in protein were investigated with the use of Western blot analysis and immunohistochemical analysis.
Results: We observed nuclear accumulation of IKK alpha 1 hour after 30 minutes of transient focal ischemia using Western blotting. Immunohistochemistry also showed nuclear accumulation of IKK alpha, and showed increased expression and phosphorylation of IKK alpha in endothelial cells after tFCI. To gain further insight into nuclear function of IKK alpha, we examined nuclear changes in histone H3 and its modified H3 after in vitro OGD of endothelial cells. Immediately after OGD of endothelial cells, the phosphorylated Ser10 histone H3 was increased 1.8-fold compared with the control, in accordance with nuclear accumulation of IKK alpha in endothelial cells after OGD. Acetylated (Lys14) and phospho (Ser10) histone H3 also increased 4.8-fold compared with the control. An upstream kinase of IKK alpha, NF-kappaB-inducing kinase (NIK), was also increased up to 3.5-fold in endothelial cytosol after OGD. We found increased expression of pro-inflammatory COX-2 and MMP-9 after OGD of endothelial cells.
Conclusion: Our results show that the histone H3 kinase activity of IKK alpha is strongly induced via NIK after ischemic oxidative stress. Nuclear IKK alpha may play a role in phosphorylation of Ser10, which might be important for the subsequent acetylation of Lys14 in histone H3. It is critical in facilitating the rapid oxidative stress-induced expression of harmful downstream genes, such as COX-2 and MMP-9, which cause neuronal cell death. Blocking IKK alpha activation by oxidative stress is a new potential therapeutic target for clinical application in stroke.
HYPOXIA-INDUCED UPREGULATION OF ENDOTHELIAL SMALL G PROTEIN RHOA AND RHO-KINASE/ROCK2 INHIBITS ENOS EXPRESSION
Hong Guo Jin1, Hiroshi Yamashita1, Yoshito Nagano1, Hiromasa Fukuba1, Masanori Hiji1, Toshiho Ohtsuki1, Tetsuya Takahashi1, Tatsuo Kohriyama1, Kozo Kaibuchi2, Masayasu Matsumoto1
1Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan, 2Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Showa, Nagoya, Japan
The small G protein RhoA and its downstream effector Rho-kinase/ROCK2 play an important role in regulation of various vasculature cellular functions. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is an important mediator of vascular homeostasis and cerebral blood flow. Using the human endothelial cell line HUVEC, the present study investigated the role of RhoA and Rho-kinase in endothelial eNOS protein expression under hypoxic conditions as an in vitro model of ischemia. RhoA protein levels in HUVEC were low under normoxic conditions, but were significantly increased after 5 h of hypoxia. Endothelial Rho-kinase expression was not detected until after 3 h of hypoxia; such expression remained significantly increased after 5 h. On the other hand, endothelial eNOS expression was similar after 3 h of hypoxia, but was significantly decreased after 5 h. The hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of RhoA and significantly inhibited by suppression of RhoA expression by small interfering RNA. The hypoxia-induced decrease in eNOS expression was significantly inhibited when endogenous Rho-kinase activation was inhibited by Rho-binding domain expression. Furthermore, the hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of Rho-kinase. Since expression and activation of RhoA and Rho-kinase inhibit eNOS expression in endothelial cells, attempts to down-regulate RhoA and Rho-kinase by multiple drugs, such as statins or Rho-kinase inhibitors, might provide endothelial and cardiovascular benefits through upregulation of eNOS.
ALPHA-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLE PROPIONIC ACID (AMPA) RAPIDLY INDUCES DNA FRAGMENTATION OF THE HIPPOCAMPAL NEURONS IN THE INITIAL STAGE OF NECROSIS
Kyoko Ibaraki, Seiji Yamamoto, Yong Wang, Atsuo Miyakawa, Takashi Sakurai, Susumu Terakawa
Photon Medical Reseach Center, Hamamatsu University School of Medicine, Hamamatsu, Japan
INTRODUCTION: Under the video enhanced contrast-differential interference contrast (VEC-DIC) microscope, the intact cultured neurons show large nuclei containing an amorphous nucleoplasm, and an application of glutamate induces granular structure inside the nucleus within 20 minutes (1). This nuclear change corresponds to DNA fragmentation, indicating that the extracellular stimuli would affect the nuclear DNA in the very early stage of excitotoxicity (2). However, whether alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) could produce the comparable change remains unclear. In the present study, we sought to determine the sole effects of AMPA application on the hippocampal neurons.
METHODS: Dissociated hippocampal cultures were prepared from one-day old Wister rats, and used for the experiment three weeks later. Morphological changes in hippocampal neurons induced by AMPA were observed under the VEC-DIC microscope. To detect the type of cell death, that is, necrosis or apoptosis, hippocampal neurons were stained following AMPA application with propidium iodide (PI) and fluorescein-4-isothiocyanate (FITC) conjugated annexin V (annexin V-FITC), and examined under the fluorescence microscope. DNA fragmentation was assessed using comet assay, a single cell gel electrophoresis assay. To assess the severity of DNA fragmentation, we classified DNA damage from grade 1 (no damage) to 5 (severe damage) by visual scoring.
RESULTS: Under the VEC-DIC microscope, continuous exposure to higher concentration of AMPA (100 µM, 1 mM) dose-dependently showed the nuclear granulation and the cellular swelling within 5 minutes. Although 10 µM-AMPA also induced the cellular swelling, it did not produce the marked nuclear granulation, suggesting that the pathway inducing cellular swelling may be distinct from that inducing nuclear granulation. Non-competitive AMPA receptor antagonist, GYKI-52466 (50 µM), blocked these morphological changes after continuous exposure to 100 µM-AMPA for 1 h, while N-methyl-D-aspartate (NMDA) receptor blocker, MK-801 (20-200 µM), did not do so, indicating that AMPA mediated toxicity independently of NMDA receptor activation. The staining with PI and annexin V-FITC revealed that AMPA (100 µM, 1 mM) induced necrosis but not apoptosis in dose dependent manner. The treatment with AMPA (100 µM, 1 mM) for 1 h significantly increased the percentage of grade 4, severer DNA fragmentation in comet assay.
COMMENTS: In hippocampal neurons, AMPA receptor stimulation alone, independently of NMDA receptor, induces the cellular swelling and the nuclear granulation in the very early stage of necrosis. The granular change in the nucleus observed under the VEC-DIC microscope corresponds to DNA fragmentation. Our results suggest that: 1) even in the process of necrosis, the extracellular stimuli to the AMPA receptor rapidly produce the nuclear signal to induce DNA fragmentation; and 2) the pathway inducing DNA fragmentation may be less sensitive to AMPA receptor activation as compared with that inducing cellular swelling.
Supported by the grants from JSPS (#18591584), and from Japanese COE program.
SELECTIVE VULNERABILITY OF MOTOR NEURON AND ENDOPLASMIC RETICULUM STRESS RESPONSE AGAINST TRANSIENT ISCHEMIA IN SPINAL CORD
Takashi Yamauchi1, Masahiro Sakurai2, Koji Abe3, Yoshiki Sawa4
1Department of Cardiovascular Surgery, Sakurabashi Watanabe Hospital, Osaka, Osaka, Japan, 2Department of Cardiovascular Surgery, National Hospital Organization Sendai Medical Center, Sendai, Miyagi, Japan, 3Department of Neurology, Okayama University Graduate School of Medicine, Okayama, Okayama, Japan, 4Department of Cardiovascular Surgery / Osaka Graduate School of Medicine, Suita, Osaka, Japan
Background and aims: Delayed paraplegia after operation of thoracic aorta is considered to be related to vulnerability of motor neuron against ischemia. However, the mechanisms of such vulnerability are not fully understood. Recently, endoplasmic reticulum (ER) stress has been reported to participate in neuronal cell death. In the present study, we investigated the expression of ER stress related molecules and discussed the relationship between the vulnerability and ER stress after transient ischemia in spinal cord.
Methods: Rabbit spinal cord ischemia model was applied with use of a balloon catheter. In this model, spinal motor neuron shows selectively delayed neuronal death whereas other spinal neuron such as inter neurons survive. Histological changes with hematoxylin-eosin staining and immunohistochemical analysis and western blotting for ER stress related molecules, which are glucose regulated protein 78 (GRP78), phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2α) and inositol requiring ER transmembrane RNAse α isoform (IRE1α), were examined.
Results: The immunohistochemical study showed that GRP78, which is supposed to be molecular chaperone, was induced strongly both in inter neuron and motor neuron at early stage of reperfusion, but the prolonged expression was observed in inter neuron. P-eIF2α, which inhibits protein synthesis and modulate ER stress, was induced only in inter neuron at 6 hr of reperfusion. IRE1α, which is supposed to transduce ER stress related death, was more strongly and prolonged expressed in motor neuron.
Conclusions: These results indicate that the vulnerability of motor neuron of spinal cord might be partially attributed to ER stress response for transient ischemia.
SIAH-1 FACILITATES UBIQUITINATION AND DEGRADATION OF FACTOR INHIBITING HIF-1ALPHA (FIH)
Hiromasa Fukuba, Hiroshi Yamashita, Yoshito Nagano, Jin Hong Guo, Masanori Hiji, Toshiho Ohtsuki, Tetsuya Takahashi, Tatsuo Kohriyama, Masayasu Matsumoto
Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan
Hypoxia-inducible factor-1alpha (HIF-1alpha) has a central role in neuroprotective responses to hypoxia in the brain. Hydroxylation of HIF-1alpha by prolyl-hydroxylase PHD and aspargynyl-hydroxylase FIH (factor inhibiting HIF-1alpha) causes proteasomal degradation and transcriptional inhibition of HIF-1alpha. Siah ubiquitin ligases regulate the abundance of PHD via targeting for proteasomal degradation. The present study identified Siah-1 as a binding partner for another hydroxylase FIH. Siah-1 and FIH coimmunoprecipitated each other in mammalian cells. Siah-1 was found both to interact with the JmjC domain of FIH through its substrate-binding domain and to specifically ubiquitinate FIH via its RING finger domain. Siah-1 facilitated FIH degradation via the ubiquitin-proteasome pathway under hypoxic conditions. Such findings suggest that Siah ubiquitin ligases might play a role as up-stream regulators of both hydroxylases for HIF-1alpha, i.e., PHD and FIH, by targeting them for proteasomal degradation, leading to increased HIF-1alpha abundance, and transcriptional activity in hypoxia.
CYCLING PROPAGATION OF SPREADING DEPOLARIZATION AROUND ISCHEMIC FOCI IN THE GYRENCEPHALIC BRAIN OF CATS
Hajime Nakamura1, Anthony J. Strong2, Stefan Vollmar1, Michael Sue1, Andrew K. Dunn3, Rudolf Graf1
1Max-Planck Institute for Neurological Research, Cologne, Germany, 2Department of Clinical Neurosciences (Neurosurgery), King's College London, London, UK, 3Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA
[INTRODUCTION]
In focal brain ischemia, cortical spreading depression (CSD) and peri-infarct depolarization (PID) contribute to infarct expansion. The temporal and spatial propagation of these waves of spreading depolarization, however, remain obscure because detection methods used so far have assessed single cortical spots and not the territory around ischemic foci as a whole. Since waves of reactive changes of cerebral blood flow (CBF) follow the depolarization waves, we used Laser Speckle Flowmetry (LSF) to characterize propagation pathways of CSD/PID as CBF waves in two lesion models in cats.
[METHODS]
Model 1: In seven α-chloralose anesthetized cats, the right cerebral hemisphere was exposed to measure dynamic perfusion by LSF, and microelectrodes measured the intracortical DC potential to study coupling between DC and CBF changes. The MCA was occluded transorbitally and spontaneous CSD/PID was monitored for around 16 hours.
Model 2: In seven cats, 1M KCl was injected via a micropipette to produce small lesions. In LSF images, these lesions were visuable as persisting small hypoemic areas. Thus, KCl injection sites were regarded as small ischemic/traumatic lesions.
[RESULTS]
In model 1, 27 CBF waves (velocity ~3 mm/min) hit electrode locations, and 22/27 waves (81%) were accompanied by DC shift (amplitude > 10 mV). CBF waves were thus considered to represent spreading depolarizations.
In model 1, two types of CBF waves were detected; 1) 45/95 waves propagated from core to peripheral (RADIAL) and 2) 50/95 waves circumvented the infarct border (CIRCUMFERENTIAL) within the field of view. Circumferential waves occurred sometimes periodically leading to the speculation that CSD cycles around the lesion not to be seen completely (see figure).
In model 2, 36 KCl injections induced 31 CBF waves. 11/31 waves completely observable in the field of view cycled one or multiple times around the smaller lesions (mean: 2.8±1.8 turns/event).
[CONCLUSION]
By means of LSF, periodic circumferential CBF waves were detected around the MCAo induced lesions in model 1, and cyclic propagation around the smaller KCl induced lesions in model 2. These results imply the existence of cycling waves of CSD/PID covering the penumbral surrounding of focal lesions. We consider cyclic propagation of CSD/PID a significant factor of energy exhaustion and therefore of secondary lesion expansion.
WILD-TYPE BONE MARROW TRANSPLANTATION AGGRAVATES BRAIN INFARCT AFTER MIDDLE CEREBRAL ARTERY OCCLUSION/REPERFUSION IN MMP-9 KNOCK-OUT MICE
Qingmin Guo1, Mohammed Hossain2, Damir Janigro2, Marc R. Mayberg3, Shobu Namura1
1Department of Anatomy and Neurobiology, Neuroscience Institute, Morehouse School of Medicine, Atlanta, GA, USA, 2Department of Neurological Surgery, Cerebrovascular Research Center, Cleveland Clinic Foundation, Cleveland, OH, USA, 3Seattle Neuroscience Institute, Seattle, WA, USA
[Background and aims] Matrix metalloproteinases (MMPs) are implicated in post-ischemic brain injury. Although MMP-9 activity is demonstrated in brain tissue after focal ischemia, its critical cellular source remains unclear. In this study, we examined MMP-9 activity and brain tissue damage in the following four chimeric mouse groups with combinations of wild-type (WT) and MMP-9 knockout (KO) mice as donor and recipient: WT/WT, WT marrow transplanted into WT recipients; WT/KO, WT marrow transplanted into KO recipients; KO/WT, KO marrow transplanted into WT recipients; KO/KO, KO marrow transplanted into KO recipients.
[Methods] The chimeric animals were generated by irradiation (9 Gy) and bone marrow transplantation (3-5 million cells). Six weeks after bone marrow transplantation, these mice were subjected to 90 min of transient middle cerebral artery occlusion (filamentous MCAO) and reperfusion under general anesthesia with isoflurane. Brain was collected at 24 hr after reperfusion and cut into coronal sections by using a cryostat. Brain infarct was assessed by Nissl staining and expressed as % of contralateral hemispheric volume. Data were expressed as mean +/− SD. Statistical comparisons were performed using Kruskal-Wallis non-parametric test followed by Mann-Whitney test. P < 0.05 was considered as significant. In separate animals, each brain hemisphere was collected at 24 hr after reperfusion, homogenized, and thereafter analyzed for gelatinolytic enzymatic activity by gel zymography.
[Results] The ischemic hemispheres of WT/KO demonstrated comparable MMP-9 activity levels to those in WT/WT. In contrast, KO/WT and KO/KO were devoid of MMP-9 activity. MMP-2 activity levels were similar in all groups. KO/KO demonstrated significantly smaller infarct (41.1+/−12.5, n=5) compared with those in WT/WT (58.9+/−5.9, n = 5) and WT/KO (56.1+/−10.6, n=5). KO/WT demonstrated comparable infarct size (50.4+/−7.8, n=5) to that in WT/KO.
[Conclusions] These findings suggest that bone marrow-derived cells contribute as the main cellular source of MMP-9 to brain infarct formation.
Support Contributed By: National Institute of Neurological Disorders and Stroke Grant NS048532, NS034194, NS046513.
IMPACT OF SELECTIN LIGANDS ACTIVITIES ON LEUKOCYTE RECRUITMENT AND ISCHEMIC DAMAGE AFTER TRANSIENT FOCAL CEREBRAL ISCHEMIA
Hiroharu Kataoka1,2, Seong Woong Kim2, Nikolaus Plesnila2
1Department of Neurosurgery, Kyoto University, Kyoto, Japan, 2Institute for Surgical Research, Ludwig-Maximilians University, Munich, Germany
Background and aims: Leukocytes contribute to secondary brain damage after transient focal cerebral ischemia, and the mechanism of leukocyte accumulation in the ischemic brain is not fully understood. Because selectins mediate rolling, the first step of leukocyte endothelium interaction (LEI), we investigated the effect of the deficiency of selectin ligand activity on LEI and ischemic damage after transient focal cerebral ischemia in the present study.
Methods: We used alpha (1,3) fucosyltransferase IV and VII double knockout (FucT-IV-/−/FucT-VII-/−) mice, in which terminal fucosylation of selectin ligands is impaired and thus all selectin-dependent rolling disappear. FucT-IV-/−/FucT-VII-/− and the wild type mice were subjected to 60 minutes of transient middle cerebral artery occlusion (MCAO) by an intraluminal filament. Leukocyte-endothelium interactions were assessed by intravital microscopy (IVM) 5 hours after reperfusion. Infarction volume, neuroscore and leukocyte infiltration into the brain parenchyma were examined after 24 hours of MCAO.
Results: IVM disclosed almost no rolling leukocytes in pial venules of FucT-IV-/−/FucT-VII-/−mice (1.3±2.0/100mm × min, P<0.01 vs. control). However, the decrease of rolling lead to neither reduced leukocyte adhesion to endothelium nor reduced leukocyte infiltration in the brain parenchyma. And infarction volume and motor function after 24 hours of MCAO were not different between FucT-IV-/−/FucT-VII-/− mice and wild type mice.
Conclusion: The current results suggest that selectin blockade is not enough to prevent leukocyte recruitment and to ameliorate ischemic damage after transient focal cerebral ischemia.
SINGLE-CELL RESOLUTION MAPPING OF POTASSIUM METABOLISM IN THE HIPPOCAMPAL FORMATION AFTER GLOBAL ISCHEMIA IN RATS
Monika Riek-Burchardt1, Petra Henrich-Noack2, Claudia Pforte2, Henning Scheich2, Klaus G. Reymann1,2, Juergen Goldschmidt2
1FAN-Research Institute for Applied Neurosciences GmbH, Magdeburg, Germany, 2Leibniz Institute for Neurobiology, Magdeburg, Germany
Transient global ischemia results in delayed death of vulnerable neurons in the hippocampal formation. Different methods exist for detecting and mapping ischemia-induced biochemical and morphological alterations, but is has remained impossible so far to map, with single-cell resolution, changes in metabolism and neuronal activity in the post-ischemic hippocampal formation.
We here used a novel technique, thallium autometallography (TlAMG), for mapping patterns of neuronal activity and metabolism in the hippocampal formation of rats at different time points after ischemia (6h, 1d, 2d, 3d, 7d, 14d and 8weeks after transient two-vessel occlusion with hypotension). TlAMG is a histochemical method for non-radiaoactive high-resolution mapping of the uptake and distribution of the monovalent thallium ion (Tl+) in the brain. Tl+ is a well-established K+-probe. Since in neurons K+-uptake strongly depends on Na,K-ATPase activity, which in turn is increased in active versus less active neurons, TlAMG provides information about both, potassium metabolism and neuronal activity.
With TlAMG we find complex patterns of neuronal and glial Tl+ uptake in the post-ischemic hippocampal formation. At time points later than 3d after ischemia we find pyramidal neurons in CA1 which are no longer able to accumulate Tl+ indicating the breakdown of Na,K-ATPase activity and K+-gradients. In CA1 neurons with morphological characteristics of interneurons, in contrast, Tl+-uptake can be high even at very late time points after ischemia. At time points earlier than three days, and before morphological alterations are detectable, there is an increase in Tl+-uptake in CA1 pyramidal neurons. Interestingly in both cases ? the increased uptake before cell death and the early phase of decreased uptake reflecting breakdown of K+-gradients ? pathological alterations in Tl+-uptake appear to affect spatially clustered groups of neighboring pyramidal cells in CA1 to different degrees.
CYSTATIN C EXPRESSION IN ISCHEMIC WHITE MATTER LESIONS
Atsushi Nagai1, Nobuyuki Umegae2, Masaharu Terashima3, Seung U. Kim4, Junichi Masuda1, Shuhei Yamaguchi2
1Deparment of Laboratory Medicine, Shimane University School of Medicine, Izumo, Shimane, Japan, 2Deparment of Internal Medicine III, Shimane University School of Medicine, Izumo, Shimane, Japan, 3Deparment of Biochemistry and Molecular Medicine, Shimane University School of Medicine, Izumo, Shimane, Japan, 4Deparment of Neurology, University of British Columbia, Vancouver, BC, Canada
Background and aims: Cystatin C, an inhibitor of cysteine proteases including cathepsins B, H, L and S, is secreted from various kinds of cells in the body and involved in the regulation of local inflammation, tumor invasion and metastasis. It also exists in cerebrospinal fluid (CSF) 5.5 times higher concentration than in the serum and might function as a major cysteine-protease inhibitor in CSF. There is growing evidence that cystatin C level in CSF is one of the indices to estimate the condition of cystein protease system in various neurological diseases. To study whether cystatin C is involved in the progression of ischemic white matter lesions (WMLs), cystatin C levels in CSF were analyzed in patients with leukoencephalopathy. Furthermore, expression of cystatin C was investigated pathologically in the white matter tissues with severe WMLs as well as in the established neuronal culture system.
Materials and Methods: Cystatin C levels in CSF were measured in 23 cases with severe WMLs of Fazekas' grade 3 (14 with hypertension; W/HT(+) and 9 without hypertension; W/HT(−)) and compared with those obtained in 38 cases with slight WMLs of grade 0-1. In the pathological study, cystatin C immunoreactivity was investigated in the white matter of 3 patients with severe WMLs compared to 3 patients with slight WMLs and 4 patients without WMLs. Expression and secretion of cystatin C were analyzed in the neuronal culture system by using the stimuli of some proteases and cytokines.
Results: Cystatin C levels in CSF in patients with W/HT(+) and W/HT(−) were lower than the patients with grade 0–1 (p < 0.005 and p < 0.0001, respectively). The immunohistochemical study showed that expression of cystatin C was detected in astrocytes, and the number of astrocytes in the severe WMLs was decreased compared to the controls (p < 0.005) and slight WML group (p < 0.005). In neuronal cultures, the stimuli with thrombin, matrix metalloproteinases and interleukin 1beta increased the mRNA expression of cystatin C in human astrocytes and hybrid neuron cultures, but ELISA revealed that only thrombin significantly increased production and secretion of cystatin C in astrocytes. The stimulus with more than 10 U/ml of thrombin induced cell death of astrocytes.
Conclusion: These results suggest that low cystatin C levels in CSF of patients with ischemic WMLs might depend on the decreased number of astrocytes that secrete cystatin C in response to the stimuli of proteases and inflammatory cytokines. Cystatin C secretion from astrocytes may influence the level in CSF and modify the progressing process of WMLs.
MILD EMBOLIC ISCHEMIA-INDUCED NEUROVASCULAR INJURY IS ASSOCIATED WITH ACCUMULATION OF BETA-AMYLOID IN THE RAT CEREBRAL MICROVESSELS
Feng Han1, Kohji Fukunaga1,2
1Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan, 2Tohoku University 21st Century COE Program, Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation, Sendai, Japan
Dysfunction of neurovascular system contributes to neurodegeneration and cognitive decline in Alzheimer's disease (AD) patients. In the present study, immunohistochemical studies revealed that immunoreactivity against β-amyloid peptide, which is consider to be causal factor of AD, significantly increased in the microvessels 12 weeks after mild embolic ischemia injury in aged male rats. Likewise, Western blotting analyses using extracts from microvessels demonstrated that β-amyloid peptide level elevated one week after mild ischemic injury in aged rats, and more pronounced increase was found 8 and 12 weeks after ischemia. The β-amyloid peptide deposition in microvessels following embolic ischemia was apparently in part due to increase in peroxynitrite formation after ischemic injury. Indeed, protein tyrosine nitration in microvessels observed 6-24 hours after ischemia, preceded production of β-amyloid peptide in the microvessels. In 8-12 weeks after ischemic injury, β-amyloid peptide deposition also coincided with GSK3β activation, tau hyperphosphorylation and Fas-ligand overexpression. At the same period, CaMKII activity which required for memory formation was also largely attenuated. Taken together, mild ischemia-induced peroxynitrite formation in the microvessels likely triggers the early events of AD such as β-amyloid accumulation, thereby leading to neurodegeneration.
Key words: β-amyloid peptide; Microvascular; Peroxynitrite; Cerebral ischemia; Alzheimer's disease
IN VIVO 19F CSI DETECTION OF FDG AND FGD-6P IN RAT BRAIN
Daniel Coman1, Basavaraju Sanganhalli1, David Cheng1, Timothy McCarthy2, Douglas Rothman1, Fahmeed Hyder1
1Department of Diagnostic Radiology, Yale University, New Haven, CT, USA, 2Pfizer Inc., Groton, CT, USA
INTRODUCTION
19F MRS studies of 2-fluoro-2-deoxy-D-glucose (FDG) and 2-fluoro-2-deoxy-D-glucose-6-phosphate (FDG-6P) may be used for assessing tumor metabolism [1, 2]. To date, 19F MRS measurements of FDG metabolism in the brain have either been achieved ex vivo from extracted tissue [3, 4] or in vivo by unusually long acquisition times, e.g., 5 hours [5]. Here we report in vivo detection of FDG and FDG-6P in real-time in rat brain with 19F CSI. Detection of FDG and FDG-6P by 19F CSI within a few minutes of infusion represents a very important step towards exciting applications in assessing tumor pathophysiology.
METHODS
Non-decoupled, in vitro 19F NMR spectra of samples containing FDG and FDG-6P were acquired on an 11.7T system. Male rats (Sprague-Dawley, Fisher) were anesthetized with halothane and an intravenous line was used for administration of D-tubocurarine chloride or FDG (A). Various infusion times (10?20 min) were used such that the total infusion dose was between 200 and 1000 mg/kg. 19F 2D CSI data were obtained with a 1H/19F resonator/surface probe with 8×8 encoding steps. All spectra were broadened by 10 Hz and baseline corrected.
RESULTS
1H decoupling of 19F NMR spectra was not used as decoupling can inject additional noise to reduce SNR and high power deposition (due to rapid recycling) can alter brain temperature. In vitro 19F spectra of FDG (B; top) shows two conformations, α and β. The presence of two non-equivalent 1H nuclei adjacent to the 19F nucleus (A) results in splitting of each α and β resonances in four peaks (B; top). In vitro 19F NMR spectra of the mixture of FDG and FDG-6P (B; bottom) indicates that resonances of the β conformation are overlapping, while the two rightmost resonances of the α conformation are separated by 0.1 ppm (47 Hz) and therefore well resolved. With additional line-broadening of about 25 Hz to the in vitro data (to mimic the situation in vivo), the FDG and FDG-6P resonances can still be resolved (C; arrows). In vivo 19F 2D CSI from a rat's brain (D,E) shows the localized detection of both FDG and FDG-6P resonances in real-time (27 min) to allow determination of glucose metabolism.
ACKNOWLEDGEMENTS
Supported by grants from NIH (MH-067528, DC-003710) and by a Pfizer gift.
IN VIVO MAPPING OF ACUTE TISSUE HYPOXIA AFTER MIDDLE CEREBRAL OCCLUSION USING 18F-FLUOROMISONIDAZOLE AND POSITRON EMISSION TOMOGRAPHY IN THE RAT
Masashi Takasawa1,2, John S. Beech3, Tim D. Fryer4, P. Simon Jones1, Young T. Hong4, Rob Smith4, David K. Menon3, Franklin I. Aigbirhio4, Jean-Claude Baron1
1Stroke Research Group, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK, 2Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK, 3Division of Anaesthesia, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK, 4Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
Background: 18F-fluoromisonidazole (FMISO) is gaining momentum as a PET marker of tissue hypoxia after stroke in man. We previously showed in rat microPET studies that FMISO is specific for the hypoxic viable tissue (1). However, no previous study has attempted to map the topography of hypoxia after stroke. Invasive PO2 probe and electron paramagnetic resonance studies have reported a gradient of tissue PO2 following acute occlusion, showing that the hypoxia is severe in the ischemic core but less so in peripheral areas (2), (3). However, the actual 3D topography of hypoxia was not provided by these point measurements. In the present study, we aim to map the topography of hypoxia using FMISO microPET in the acute stage of middle cerebral artery occlusion (MCAo).
Methods: As described in detail elsewhere (1), we used Buchan's distal MCA clip model in male spontaneously hypertensive rats (SHRs) (n=3), and also studied normal SHRs (n=5). Around 80MBq 18F-FMISO was iv injected mean 64.7 minutes after the clip was placed, and PET scanning (microPET P4 scanner, Concorde Microsystems, USA) was continued for three hours. The brain was harvested two days later for histology. Corrections for randoms, dead time, background, normalization, attenuation, sensitivity and decay were applied to the data during reconstruction; final image resolution was ~2.3mm FWHM. We created 3D standardized uptake value (SUV) maps using the average of the last ten frames (130-180mins) in each rat, which was coregistered onto normal SHR T2 MRI template using SPM2. The mean SUV distribution map for each rat group was produced by averaging the SUV maps. A MCA cortex ROI was created based on the anatomy of the MRI template and MCA infarct literature.
Results: In all three stroke rats, significant trapping was found over the affected MCA cortex, with continuous increases of PET activity over time until end of scan. The SUV of MCA cortex ROI in stroke rats was 1.15±0.33 (mean ± SD) in affected side as compared to 0.62±0.35 in the unaffected side (p<0.05), and 0.61±0.10 in normal rats (p<0.05).
In the mean SUV map, FMISO trapping was highest in the anterior MCA cortex (proximal relative to clipping site), and gradually decreased towards posterior areas of the MCA territory (SUV: 1.36 and 0.93 in peak and posterior areas respectively). This distribution was identical in each rat. Preliminary data from one rat suggest the acute-stage distribution of FMISO trapping matched the final infarct topography.
Conclusion: The topography of FMISO trapping observed here, with a clear antero-posterior gradient, is consistent with the classical core-penumbra concept and with available point measurements of tPO2 (2),(3). The assumption that the degree of tracer trapping reliably represents tPO2 will need to be addressed. Serial FMISO studies would be of interest to understand the temporal profile of hypoxia, and to relate acute hypoxia to final tissue outcome.
LACTATE IS OXIDIZED BY THE HUMAN BRAIN FOLLOWING TRAUMATIC BRAIN INJURY
Thomas Glenn1, Gareth Wallis2, Daniel Hirt1, Paul Vespa1, Josh Dusick1, George Brooks2, David Hovda1, Neil Martin1
1Department of Neurosurgery, UCLA, Los Angeles, CA, USA, 2Department of Integrative Biology, UC Berkeley, Berkeley, CA, USA
Background and Aims
We have previously shown in patients with traumatic brain injury (TBI) that the brain takes up rather than releases lactate (Glenn, 2003). However, the metabolic fate of lactate entering the brain in these patients has not been determined. Therefore, we infused the stable isotope tracer [3-13C]lactate into three severely injured TBI patients, and measured cerebral 13CO2 production to determine the extent to which consumed lactate is oxidized by the injured brain. As well, we infused [6,6-2H2]glucose simultaneously, and in separate experiments infused [1-13C]- and [6,6-2H2]glucose to facilitate comparison of cerebral lactate and glucose metabolism following TBI.
Methods
Three severely injured TBI patients (age 58 +/−15 years, all male) were consented for the study. A primed continuous infusion of [3-13C]lactate and [6,6-2H2]glucose, or [1-13C]- and [6,6-2H2]glucose, was administered intravenously for 120 minutes, with arterial and jugular bulb venous blood samples collected at time 0 and every 30 minutes thereafter. Experiments were performed within 24 hours of each other. Blood samples were analyzed for isotope enrichments (IRMS or GCMS) and concentrations of CO2, lactate and glucose. Cerebral flux and oxidation rates were calculated using the combination of blood flow (133Xenon clearance technique), isotope and mass balance measurements. Absolute and relative flux or oxidation rates are presented.
Results
Patients exhibited a small net cerebral lactate release during the [3-13C]lactate infusion trial (0.15±0.07 mg/100g tissue/min), but considerable tracer-measured lactate uptake was observed (1.84±0.64 mg/100g/min). Thus, total lactate production (net release + tracer-measured uptake) was far greater than net release alone (1.99±0.70 mg/100g/min). Lactate uptake exceeded net glucose uptake (1.39±0.30 mg/100g/min) in this trial. When [3-13C] lactate was infused, lactate oxidation was calculated to be 0.16±0.06 mg/100g/min which represents 15±4 % of arterial lactate extracted. This accounted for up to 16±8 % (range 4–32%) of cerebral oxidative CO2 production. Extra-cerebral [3-13C] lactate incorporation into glucose, and thus glucose contribution to cerebral 13CO2 production was considered neglible as judged by minimal arterial 13C glucose enrichment (0.006% vs. 1.373% for arterial lactate). Data for 2 patients is presented for the [1-13C]glucose infusion trial, during which net glucose uptake was also observed (3.45±0.48 mg/100g/min). Cerebral glucose oxidation was calculated to be 1.27±0.44 mg/100g/min, which accounted for 62±20% of cerebral oxidative CO2 production assuming no astrocyte-neuron lactate shuttling.
Conclusions
This study is the first of its kind to show that, in addition to glucose, energy production from arterial lactate can be an important contributor to oxidative metabolism in the injured human brain. As well, the occurrence of a small net cerebral lactate release during substantial cerebral lactate uptake indicates, for the first time, dynamic cerebral lactate turnover in the injured brain (i.e. simultaneous lactate uptake and production). While inter-patient variability was high, due in part to the time after injury that the study was conducted and the differences in injury severity, the novel application of these techniques and the preliminary findings observed may have important implications for understanding both the metabolic consequences of TBI and directing future metabolically based therapies.
VISUALIZATION OF THE PENUMBRA: COMBINATION OF IMAGING TECHNIQUES PROVIDES INSIGHTS INTO THE PATHOPHYSIOLOGY OF INFARCT DEVELEPMENT IN FOCAL ISCHEMIA
Axel Heimann, Kolamal Nageswari, Beat Alessandri, Oliver Kempski
Institute for Neurosurgical Pathophysiology, Johannes Gutenberg-University, Mainz, Germany
Background and aims
The infarct core expands at the cost of the penumbra. Thus, temporal changes are essential to understand and to treat ischemic events. The aim of this study was to visualize regional flow, hemoglobin oxygen saturation and microcirculatory changes after permanent focal cerebral ischemia.
Material and Methods
8 Mongolian gerbils (60?70g) were anaesthetized with halothane and underwent a permanent occlusion of the middle cerebral artery (MCAO). After stable baseline conditions MCAO was induced by insertion of an endovascular 5–0 nylon suture. At baseline, 30, 60, 90 and 120 min after MCAO regional cerebral blood flow (rCBF) was assessed by laser Doppler scanning, and tissue HbO2-saturation by non-invasive micro-lightguide spectroscopy using a computer assisted scanning technique (1). Microcirculation was studied in an additional setup by intravital microscopy 60, 90,120 and 150 minutes after MCAO. To do so, gerbils were anaesthetized with urethane and divided into a MCAO-group (n=6) and a sham-operated group (n=8). Leucocyte-endothelial-interactions and velocity of red blood cells (RBCs) were recorded from 6–8 different parietal locations in each hemisphere after injection of Rhodamine 6G and FITC-labeled erythrocytes, respectively.
Results
The scanning technique demonstrated that rCBF decreased significantly 30 min after MCAO from identical baseline values (ipsilateral: 42.0±6.4; contralateral: 44.7±6.8 LDU) ipsilaterally to 23.4±9.4% baseline and did not recover during the observation period (60′min: 27.8±8.9%; 90′min: 31.5±10.7%; 120′min: 32.8±11.6% baseline), while contralaterally rCBF remained unchanged. Likewise, there was no difference between the hemispheres in baseline tissue HbO2-saturation (68±2 % ipsilateral; 70.0±2% contralateral). MCAO induced a significant reduction of the ipsilateral HbO2-saturation (30′min: 24.8±6.3%) which then improved again (60′min: 35.5±7.1%; 90′min: 41.9±7.4%; 120′min: 41.2±7.7 %). Contralateral tissue HbO2-saturation never fell below 58.8±4.2%. In the intravital microscopic investigation RBC-velocity in ipsilateral capillaries ranged between 0.342 and 0.405 mm/s, and always was significantly slower compared to contralateral (1.1±0.03) or control animals (1.3±0.13). In some capillaries stasis was detected by non-moving FITC-labeled RBC's in the ipsilateral hemisphere only. In post-capillary venules rollers were massively increased at 60,90,120, and 150 min after MCAO in the ipsi-(10.0±3.1;11.8±2.3;16.2±5.0;19.20±6.7 rollers/100:m/min) and also in the contralateral hemisphere (0.4±0.4;2.6±1.9;4.8±2.8;5.40±1.5 rollers/100:m/min, respectively). At the same time points the number of stickers was 10.5±2.2, 14.2±3.5, 21.0±9.7, and 17.5±7.5 ipsilaterally and 0.8±0.6, 2.2±0.7, 2.8±1.7, and 5.0±2.1 contralaterally. There were no rollers in control animals, except for 150 min after MCAO (1.3 rollers/100:m/min) and the number of stickers was 0.3±0.3 at 90 min, 0.7±0.3 at 120 min, and 1.3±1.3 stickers/100:m/min at 150 min.
Conclusion: Increasing tissue HbO2-saturation with no recovery of rCBF and RBC velocity indicate decreasing oxygen consumption as a sign of progressing cell death in the penumbra which is currently under study.
THE IMPACT OF ANESTHETICS AND NORMOBARIC HYPEROXIA ON SUSCEPTIBILITY TO CORTICAL SPREADING DEPRESSION
Chiho Kudo, Michael A. Moskowitz, Cenk Ayata
Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA
Cortical spreading depression (CSD) is a transient neuronal and glial depolarization and disruption of membrane ionic gradients that propagates slowly across the cerebral cortex. CSD susceptibility can be experimentally assessed by recording the frequency of CSDs evoked upon continuous topical application of KCl (1M). We systematically studied the influence of different anesthetic types and the impact of normobaric hyperoxia on CSD susceptibility in this model. Male Sprague-Dawley rats were intubated and mechanically ventilated, and femoral arteries were cannulated for arterial pressure and blood gas measurements. CSD occurrence and propagation speed were recorded using two intracortical glass micropipettes (350 micron depth). One of 3 different anesthetics was tested in each rat: isoflurane (1%), alpha-chloralose (60-150mg/kg/h with nitrous oxide, 100-180mg/kg/h without nitrous oxide), urethane (0.4–0..65g/kg/h with nitrous oxide, 0.6–0..76g/kg/h without nitrous oxide). Each anesthetic was used either alone (30%O2, 70%N2) or in combination with nitrous oxide (30%O2, 70%N2O) or normobaric hyperoxia (100%O2). We found that highest CSD frequency was observed under urethane anesthesia (p<0.05 vs. all other groups except alpha-chloralose+100%O2; Table). Isoflurane significantly reduced the frequency of CSDs compared to urethane (p<0.05), especially when combined with nitrous oxide (p<0.05 vs. all other groups). Nitrous oxide also suppressed CSDs under urethane (p<0.05) but not alpha-chloralose anesthesia. Normobaric hyperoxia tended to increase CSD frequency under alpha-chloralose (p=0.1) but not isoflurane anesthesia. We were unable to test the impact of nitrous oxide alone on CSD susceptibility, because, nitrous oxide does not provide surgical depth of anesthesia. The propagation speed, amplitude and duration (at half-maximal amplitude) of CSDs did not differ among groups. In summary, CSD susceptibility is modulated by the choice of anesthetic. The mechanism of CSD suppression by isoflurane may involve blockade of gap junctions, whereas the inhibitory effect of nitrous oxide likely involves blockade of NMDA receptors or voltage-gated calcium channels. Our data underscore the importance of proper anesthetic choice for experimental studies of CSD susceptibility (Guedes and Barreto 1992; Verhaegen, Todd et al. 1992; Saito, Graf et al. 1995; Piper and Lambert 1996), and suggest that nitrous oxide may have efficacy as an abortive agent in the clinic for acute migraine, as previously reported (Triner, Bartfield et al. 1999). Furthermore, the lack of CSD suppression by normobaric hyperoxia argues against a pathophysiological role for CSD in cluster headache where normobaric hyperoxia is an effective therapy.
Acknowledgements: This study was supported by NIH Migraine Program Project (1P01NS35611, MAM) and AHS/Merck US Human Health Migraine and Women's Health Research Award (2006, CK).
MILD HYPOTHERMIA (33°C) AMELIORATES BLOOD BRAIN BARRIER DYSFUNCTION AND IMPROVES NEUROLOGICAL OUTCOME FOLLOWING SUBARACHNOID HEMORRHAGE IN RATS
Elisabeth Torok1,3, Andreas Trinkl2, Stefan Zausinger3, Nikolaus Plesnila1,3, Karsten Scholler3
1Institute for Surgical Research, University of Munich, Medical Center Groshadern, Munich, Germany, 2Department of Neurology, University of Munich, Medical Center Groshadern, Munich, Germany, 3Department of Neurosurgery, University of Munich, Medical Center Groshadern, Munich, Germany
Objective: Acute brain injury is the primary cause of mortality after SAH. Therapeutic options for the inhibition of the early pathophysiological mechanisms after SAH are quite limited. Hypothermia has a neuroprotective effect in severe head injury and cerebral stroke. Therefore the aim of the current study was to investigate acute and chronic effects of mild hypothermia after SAH.
Methods: Male Sprague-Dawley rats were subjected to SAH induced by an endovascular filament. Animals were randomly assigned to one of the following three groups: 1) normothermia, 2) two hours of hypothermia starting 60 minutes after SAH, and 3) two hours of hypothermia starting 180 minutes after SAH. Cortical laser Doppler flux (rCBF) and ICP were continuously measured up to 6 hours after SAH. Brain water content was measured 24 hours after SAH by the wet-dry method. Neurological deficits were assessed from day 1?7. On Day 7 we quantified the permeability of the BBB by Bovine serum albumin Western Blot, the alteration of the microvascular basal lamina by Collagen-type-IV-Western Blot, and the activity of matrix metalloproteinases (MMP) 9 & 2 by gelatine zymography.
Results: 2 hours of mild hypothermia, induced 60 minutes after SAH improved the ipsilateral rCBF (4h, p<0,05), the neurological outcome (24h, p<0,05), and the integrity of the BBB (7d, p<0,05) and the microvascular basal lamina (7d, p<0,05). Mild hypothermia also reduced the activity of MMP-2. Induction of hypothermia 180 minutes after SAH had no neuroprotective effect. Mild hypothermia had no effect on post-ictal ICP and brain water content.
Conclusion: Two hours of mild hypothermia, induced 60 minutes after SAH have a positive effect on the neurological function and the integrity of the BBB, although it does not affect brain edema formation. Activation of MMP-2 may play a role for the damage of the microvascular basal lamina. Over all hypothermia seems to be an effective strategy for the treatment of early brain injury after SAH.
MICROARRAY ANALYSIS OF BBB-DAMAGED VESSELS EXTRACTED BY MICRODISSECTION
Masaki Ueno1, Yasuyuki Iwanaga2, Masaaki Ueki2, Haruhiko Sakamoto1
1Department of Pathology and Host Defense, Fac. of Medicine, Kagawa University, Kagawa, Japan, 2Department of Anesthesilogy and Emergency Medicine, Faculty of Medicine, Kagawa University, Kagawa, Japan
Background and aims: We previously reported (1) that the blood-brain barrier (BBB) function was deteriorated in vessels located along hippocampal fissures in 3-month-old stroke-prone spontaneously hypertensive rats (SHRSP). In this study, vascular samples were microdissected from the hippocampi of SHRSP and WKY as a control and the difference in gene expression between the BBB-damaged vessels in SHRSP and vessels without BBB damage in WKY was examined by a microarray.
Methods: Three-month-old SHRSP and WKY rats were used in this study. Microarray analysis was performed in vascular samples extracted by microdissection. In addition, the differences in the expression of some genes and proteins between brain tissues in the two strains of rats were examined using real-time PCR, Western blotting and immunohistochemistry.
Results: The microarray assay revealed that the ratio of osteopontin gene expression in the vascular tissue of the hippocampi of SHRSP to that of WKY was the highest among 8,435 genes examined. Real-time PCR analysis revealed that the gene expression of osteopontin was significantly upregulated in the hippocampal samples of SHRSP compared with that in the hippocampal samples of WKY rats or with that in the cortical samples of SHRSP. Immunohistochemical and Western blot analyses showed that the osteopontin protein expression was seen in perivascular ED1-positive macrophages/microglial cells located around hippocampal fissures and significantly upregulated in the hippocampi of SHRSP compared with that of WKY.
Conclusions: These findings indicate that the expression of osteopontin is upregulated in BBB-damaged vessels in hypertensive SHRSP, suggesting a novel role of osteopontin in BBB function.
LOSS OF CO2-REACTIVITY IN CEREBRAL RESISTANCE VESSELS AFTER SUBARACHNOID HEMORRHAGE IN MICE
Benjamin P. Friedrich2, Sergej Feiler2, Sebastian Sonanini2, Nikolaus Plesnila1,2
1Laboratory of Experimental Neurosurgery, Department of Neurosurgery, University of Munich Medical Center, Grosshadern, Munich, Germany, 2Institute for Surgical Research, University of Munich Medical Center, Grosshadern, Munich, Germany
Introduction: Severe impairment of cerebral blood flow (CBF) is one of the major acute sequels of subarachnoid hemorrhage (SAH). Underlying mechanisms are, however, poorly understood. Because CO2 plays a major role in the regulation of CBF we investigated the CO2-reactivity of cerebral resistance vessels in the brain following experimental SAH by intravital microscopy (IVM).
Methods: Male C57/Bl6 mice were anesthetized with medetomidine, fentanyl, and midazolam (i.p.), intubated, and mechanically ventilated. Endtidal pCO2 and arterial blood pressure were measured by microcapnometry and a pressure transducer connected to a femoral line, respectively. SAH was induced by an intraluminal filament. Three or 24 hours later the cerebral microcirculation was visualized by intravital fluorescence microscopy through an opened cranial window using FITC-dextran. In four groups (no surgery, sham surgery, SAH 3h, SAH 24h; n=6 each) vessel diameters of cerebral arterioles were assessed 1 h before and 15 min after ventilation with 10% CO2. In two other groups (n=6 each) animals where successively normo-/hyper-/normo- and finally hypoventilated (15 min each) in order to assess vessel reactivity to hypo- and hypercapnia.
Results: Under baseline conditions arterial diameters were constant in all groups (baseline diameters were normalized to 100%). Ventilation with 10% CO2 (paCO2 = 274 +/− 11 mmHg) induced rapid and significant vasodilation in control and sham animals (164 +/− 16%; p<0.001). After SAH CO2 did not induce any vascular reaction (99 +/− 10% and 107 +/− 4% after 3 and 24 h, respectively). In the hyper- and hypoventilated sham operated animals arterial diameters shrank after hyperventilation (86 +/− 8%; p<0.05; paCO2 = 22 +/− 2 mmHg), returned to normal when animals were normoventilated (102 +/− 12%), and dilated after hypoventilation (134 +/− 18%; p<0.05; paCO2 = 49 +/− 2 mmHg). After SAH vessels did not show any reaction to different paCO2 levels (101 +/− 8% after hyperventilation; 105 +/− 8% after hypoventilation; p>0.05 each). All data are given as means +/− SD.
Discussion: Already within the first few hours following SAH - when no vasospasm of the large arteries is observed - cerebral resistance vessels, i.e. cerebral arterioles, do not react to CO2. Our data therefore suggest, that quite rapidly after SAH cerebral vasoreactivity is dramatically impaired thereby possibly contributing to the early decrease of CBF and high mortality after SAH. The mechanisms of the abrogated CO2-reactivity after SAH, however, are yet unclear and require further investigation.
UNILATERALLY SYMPTOMATIC MOYAMOYA DISEASES IN CHILDREN: LONG-TERM FOLLOW-UP OF 20 PATIENTS WITH PET AND SPECT
Shinji Nagata1, Koichiro Matsukado1, Takato Morioka1, Futoshi Mihara2, Tomio Sasaki1
1Department of Neurosurgery, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan, 2Departmetn of Radiology, Fukuoka Higashi Medical Center, Fukuoka, Japan
Background and aims: In unilaterally symptomatic moyamoya disease in children, it remains controversial whether bypass surgery should be performed on the asymptomatic side. Here, we verify the validity of our strategy of only performing bypass surgery on the symptomatic side.
Methods: Among 91 pediatric moyamoya patients who underwent bypass surgery in our department between 1980 and 2004, 20 patients with unilateral ischemic symptoms who were followed up for more than 60 months were analyzed in the present study. Initially, we only performed bypass surgery on the symptomatic side for all 20 patients. Among these 20 patients, 5 developed frequent transient ischemic attacks (TIAs) in the initially asymptomatic side and underwent a second bypass surgery on that side (Group A), 8 developed sporadic TIAs and were followed up without surgery (Group B), and 7 did not experience any ischemic symptoms on the asymptomatic side (Group C).
Results: In total, 18 patients progressed well without cerebral infarctions after their last surgery, although some of them showed deterioration of angiographic stenosis and a transient decrease in the regional cerebral blood flow and/or cerebral perfusion reserve. One patient in Group A had an intraventricular hemorrhage 5 years after the second operation and one in Group B had a minor stroke on the initially asymptomatic side.
Conclusion: In unilaterally symptomatic moyamoya disease, we can delay bypass surgery for the asymptomatic side until the development of ischemic symptoms.
LACK OF GLUTAMATE EXCITOTOXICITY IN PRIMARY RAT AND PIGLET CEREBROVASCULAR ENDOTHELIAL CELL CULTURES
Ferenc Domoki1,2, Bela Kis1, Tamas Gaspar1, Ferenc Bari2, David W. Busija1
1Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC, USA, 2Department of Physiology, University of Szeged, Szeged, Hungary
Background and aims: Cerebrovascular endothelial cells (CECs) have recently been implicated as targets of excitotoxic injury, contributing to stress-related blood brain barrier (BBB) disruption. Thus, we sought to determine whether primary cultures of CECs are indeed sensitive to a pharmacological dose of L-glutamate (L-glut) or N-methyl-D-aspartate (NMDA).
Methods: Primary CEC cultures were obtained from 2 week old Wistar rat pups and 1 day old piglets. Cells were seeded onto collagen type IV/fibronectin coated dishes, 12 mm circular cover slips, 96-well plates, or 24mm diameter, 3µM pore size TranswellR inserts to study cell morphology, intracellular Ca2+ levels (Fluo4-fluorescence, confocal microscopy), CEC viability (MTS-formasan assay), or transendothelial electrical resistance (TER), respectively. CECs produced confluent cultures and were used within 4–5 days after isolation.
Results: L-glut (Fig A) and NMDA (1-1mM) did not elevate Ca2+ levels in rat CECs in contrast to the profound effect of the Ca2+ ionophore calcimycin. Neither L-glut nor NMDA (1-1mM, 8h) caused visible changes in the neat cobblestone appearance of CECs. They also unaffected rat (Fig B) or piglet CEC viability and did not augment cell death induced with 8h medium withdrawal (Eagle's balanced salt solution (EBSS) supplemented with 1g/l glucose) or 12h oxygen-glucose deprivation (OGD). TER was also unaffected by both L-glut (Fig C) and NMDA application.
Conclusions: Our results indicate that primary rat and piglet CEC cultures are resistant to glutamatergic stimuli and maintain prominent in vitro BBB features even at doses exceeding the physiological range. Our findings are consistent with previous studies in piglets showing intact pial arteriolar responses in vivo after prolonged exposure to L-glut/NMDA, and no effect of NMDA on pial arterioles in vitro. The present findings challenge previous studies claiming CECs are vulnerable to excitotoxicity.
Support: HL-50587, HL-65380, HL-77731, and OTKA T046531.
BENEFIT OF CONTINUOUS CARDIAC OUTPUT MONITORING IN NEUROINTENSIVE CARE OF TRAUMATIC BRAIN INJURED PATIENTS
Tadashi Kaneko1, Kotaro Kaneda1, Yasutaka Oda1, Hiroyasu Koizumi2, Takeshi Inoue1, Ryosuke Tsuruta1, Shunji Kasaoka1, Michiyasu Suzuki2, Tsuyoshi Maekawa1
1Advenced Medical Emergency and Critical Care Center, Yamaguchi University Hospital, Ube, Yamaguchi, Japan, 2Department of Neurosurgery, Yamaguchi University Hospital, Ube, Yamaguchi, Japan
Introduction: It is reported that maintenance of cerebral blood flow (CBF) is essentioal in neurointensive care. The guidelines of traumatic brain injury (TBI) remarked only mean arterial pressure (MAP), intracranial pressure (ICP) and cerebral perfusion pressure (CPP). If CBF should be maintained, more advanced hemodynamic monitoring is required. In this study, we investigated whether hemodynamic parameters could be risk factors in TBI patients.
Methods: Hemodynamic parameters were retrospectively investigated in 13 severe TBI patients who were started pulmonary artery catheter monitoring within 24 hours after admission. Two cases caused cerebral herniation (CH group), in spite of initial ICP were low. Hemodynamic parameters were collected up to 72 hours after admission, and were compared between CH and non-CH groups.
Results: Table shows the average of 72 hours sequential changes of MAP, ICP, CPP, SvO2, CCI and Hb in CH and non-CH group. MAP, ICP, CPP and CCI had the significantly difference between the two groups. TBI treatment guidelines show that CPP, calculated by MAP and ICP, is emphasized on hemodynamic treatment of severe TBI patients. In our study, average CPP was kept more than 60 mmHg defined by TBI guidelines in both, CH group showed significantly lower CCI. These data suggests that both CPP and CCI should be maintained to keep enough CBF in severe TBI patients. Recently, it was reported that hypertonic saline reduced intracranial pressure and improved cardiac index. There is a possibility that improving CPP and CCI without hypertonic saline could be maintain intracranial pressure.
Conclusion: In severe TBI patients, CCI monitoring has essential role in neuro-intensive care. From our retrospective data, improving both CPP and CCI could keep sufficient CBF and prevent cerebral herniation. Eligible CCI is probably more than 3.0 l/min/m2 from our data.
ENHANCEMENT OF CEREBRAL BLOOD FLOW BY SYSTEMIC HYPERTONIC SALINE THERAPY CORRELATES WITH OUTCOME IN PATIENTS WITH POOR-GRADE SPONTANEOUS SUBARACHNOID HEMORRHAGE
Ming-Yuan Tseng, Pippa G. Al-Rawi, Marek Czosnyka, Peter J. Hutchinson, Hugh Richards, John D. Pickard, Peter J. Kirkpatrick
Department of Neurosurgery, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK
Background: Systemic administration of 23.5% hypertonic saline enhances cerebral blood flow (CBF) in patients with poor-grade spontaneous subarachnoid hemorrhage (SAH). Whether the increment in CBF correlated with changes in autoregulation indices (Mx, PRx) or outcome at discharge is unknown.
Methods: Thirty-five poor-grade aSAH patients received intravenous 23.5% hypertonic saline, 2 mL/Kg, and bedside monitoring, including transcranial Doppler (TCD) and intracranial pressure (ICP) monitoring. Seventeen of them underwent xenon-enhanced computerized tomography (XeCT) for quantifying CBF. Outcome was measured by modified Rankin Scale (MRS) at discharge. Data were analyzed using repeated measurement ANOVA and Dunnett's correction. Comparison was made between patients with favorable/unfavorable outcome using multivariate logistic regression.
Results: A maximum rise in blood pressure by 10.27% (p<0.05) and cerebral perfusion pressure (CPP) by 21.24% (p<0.01) at 30 minutes, followed by a maximum fall in ICP by 93.06% (p<0.01) at 60 minutes was observed. Changes in ICP and CPP persisted for a minimum of 180 minutes. TCD showed that the baseline autoregulation (Mx) was impaired on the ipsilateral side of ruptured aneurysm and increments in flow velocities were higher and longer on the contralateral side (48.75 vs. 31.96%, p=0.045; 180 vs. 90 minutes, p<0.05). The autoregulation was briefly impaired on the contralateral side during the infusion. XeCT showed a dose-dependent effect of CBF increments on favorable outcome (odds ratio for MRS 1 to 3, 1.27/ml/100gm tissue*min, p=0.045, Figure)
Conclusions: Bolus systemic hypertonic saline therapy may be used for rescuing cerebral ischemia in patients with poor-grade subarachnoid hemorrhage.
EXPERIENCE WITH ALPHA-CHLORALOSE IN RAT MIDDLE CEREBRAL ARTERY OCCLUSION
Janos Luckl, Joel Greenberg
Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA
Introduction: Alpha-chloralose is widely used as an anesthetic in studies of the cerebro-vasculature because of its minimal depression of autonomic function. However, there have been no controlled studies of focal ischemia in rat under alpha-chloralose anesthesia because of its potential convulsant properties and its long-lasting action. In the current study we demonstrate that alpha-chloralose (AC) has a fairly wide margin of safety and we compare its effect with isoflurane (ISO) on blood flow, infarct size, EEG and peri-infarct depolarization (PID) during transient middle cerebral artery (MCA) occlusion in the rat.
Methods: Artificially ventilated rats were prepared using 1% ISO anesthesia for occlusion of the right MCA. Anesthesia was either switched to AC (60 mg/kg bolus, 30 mg/kg/hr; n=7) or was maintained on ISO (n=8). The MCA was occluded for 90 minutes using the filament technique and physiological parameters were maintained and monitored during ischemia and the reperfusion (30 min.) phase. EEG was monitored from a screw electrode and changes in cerebral blood flow (rCBF) from a laser Doppler probe (5 mm lateral, 2 mm posterior to bregma) placed over the ischemic cortex. The animals of the AC group required 3 hrs post-surgical observation including weaning from the respirator, extubation and monitoring core temperature. Neurological examinations were performed at 24 and 72 h after ischemia at which point all animals were sacrificed and the brains removed for determination of infarct size.
Results: The experimental protocol and treatment with the two different anesthetics did not cause significant alterations in most of the physiological variables. Significantly higher pre - and post-ischemic blood pressure were detected in the AC animals. While the initial drop in flow and the intraischemic rCBF were almost identical in the two groups (AC:33¬+/−9.7%, ISO:36+/−7.6%), the animals under AC anesthesia showed a significantly better reperfusion (AC:154+/−35%, ISO:113+/−38%; p<0.05). Analysis of infarct areas at six stereotaxic levels and subsequent calculation of infarct volumes revealed that the cortical and total ischemic injury were larger under AC (177+/−35.1 and 238+/−40 mm3) than under ISO (137+/−16.2 and 194+/−16.9 mm3) (p<0.05). The reduction in the EEG power was greater in the AC group early into ischemia but by 60 min this difference disappeared. The incidence and total duration of non-convulsive (electric) seizures were similar in both groups, and occurrence of clinically manifested tonic/clinic seizures on the contralateral limbs was also similar. The number and duration of peri-infarct depolarizations were calculated from the “hyperemic” flow changes. The averaged duration (132+/−14 sec.) and frequency (8/90 min.) of these “flow peaks” under isoflurane anesthesia is consistent with electrophysiological data in the literature. The total duration and frequency of PID was significantly higher in the AC animals supporting the hypothesis that isoflurane inhibits not only the formation of spreading depression, but also PID.
Conclusion: We conclude that alpha-chloralose is a safe anesthetic in stroke studies with excellent postoperative recovery and survival. Its features of not depressing cardiovascular and cerebrovascular reflexes and presumably not exerting neuroprotective effects make it an excellent anesthetic for stroke studies in the rat.
PREISCHEMIC NOT POSTISCHEMIC INSULIN AT A SINGLE CLINICALLY RELEVANT DOSE PROTECTS AGAINST ISCHEMIC SPINAL CORD INJURY IN RABBITS
Mishiya Matsumoto, Daisuke Nagamizo, Shunsuke Tsuruta, Hiroaki Matayoshi, Atsuo Yamashita, Takefumi Sakabe
Department of Anesthesiology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan
Background and aims: A recent clinical trial of intensive insulin therapy in critically ill patients has aroused interest in brain protection by insulin in acute ischemic stroke. However, it has not been well determined whether insulin protects against ischemic spinal cord injury. We examined the effects of single dose of insulin that prevents mild hyperglycemia either in the preischemic or postischemic period on the outcome after transient spinal cord ischemia in rabbits.
Methods: We assigned rabbits to 4 groups (n = 8 in each); untreated control (C) group, preischemic insulin (Pre-I) group, preischemic insulin with glucose (GI) group, and postischemic insulin (Post-I) group. Insulin (0.5 IU/kg) was administered 30 min before ischemia in the Pre-I and GI groups, and just after reperfusion in the Post-I group. The blood glucose concentrations in the GI group were maintained comparable to those in the C group. Spinal cord ischemia was produced by occluding the abdominal aorta for 13 min under general anesthesia with isoflurane. Rabbits were neurologically assessed daily for 7days after reperfusion using 5-point score system: 4 = normal motor function, 3 = ability to draw legs under body and hop but not normally, 2 = some lower-extremity function with good antigravity strength but inability to draw legs under body and/or hop, 1 = poor lower-extremity function but weak antigravity movement only, 0 = paraplegic with no lower-extremity function. After final neurological assessment, the lumbar spinal cord was histopathologically evaluated by hematoxylin and eosin staining.
Results: The mean blood glucose concentration before ischemia in the Pre-I group (118 ± 17 mg/dL, mean ± SD) was significantly lower than in the C (159 ± 21 mg/dL), GI (180 ± 30 mg/dL), and Post-I (158 ± 16 mg/dL) groups and those of 30 min after reperfusion in the Pre-I (92 ± 17 mg/dL) and Post-I (100 ± 12 mg/dL) groups were significantly lower than in the C (148 ± 31 mg/dL) and GI (140 ± 22 mg/dL) groups. The mean motor function score in the Pre-I group (3.8 ± 0.7) was significantly better than in the C (1.1 ± 0.4), GI (1.6 ± 1.5), and Post-I (1.6 ± 1.6) groups. The number of normal neurons in the anterior lumber spinal cord in the Pre-I group (60 ± 14) was significantly greater than in the C (18 ± 18), GI (29 ± 26), and Post-I (24 ± 31) groups.
Conclusions: These results suggest that a relatively small dose of preischemic insulin protects against ischemic spinal cord injury. Although insulin has been reported to have a direct neuroprotective effect, the protective effects demonstrated in the present study are mainly due to its glucose-lowering effect.
NITROUS OXIDE ADMINISTRATION INCREASED NEURONAL DAMAGE DURING CEREBRAL ISCHEMIA WITHOUT AFFECTING EXTRACELLULAR GLUTAMATE CONCENTRATION IN GERBILS
Hideki Taninishi, Yoshimasa Takeda, Motomu Kobayashi, Minako Arai, Toshihiro Sasaki, Kiyoshi Morita
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama City, Japan
[Background] It has been conflicting that nitrous oxide affected deteriorative or neuroprotective during cerebral ischemia, no conclusion has been evaluated. Therefore, in the present study, the effect of nitrous oxide on neuronal cell damage during cerebral ischemia was quantitatively evaluated by the ischemic duration causing 50% neuronal damage, and the relationships between ischemic depolarization and histological outcome were determined. Furthermore, the effect of nitrous oxide on dynamic change in extracellular glutamate concentration was also determined. [Materials and methods] Under 1% halothane anesthesia in 30% oxygen, fifty gerbils (weighing 71.0±5.6 g) were randomly assigned to a group receiving 70% nitrous oxide (N2O group, n=25) and a group receiving 70% nitrogen (N2 group, n=25). In the first experiment, forebrain ischemia was performed by occlusion of bilateral common carotid arteries for 3, 5 or 7 minutes (n=6 in each group). During ischemia, direct current potential was recorded from the bilateral hippocampal CA1 regions. Histological evaluation of bilateral hippocampal CA1 regions was performed 5 days after the ischemia. Relationships of neuronal damage with ischemic duration and duration of ischemic depolarization were determined by logistic regression curves. In the second experiment, forebrain ischemia was performed for 5 minutes by the same procedure as that used in the first experiment (n=7 in each group). Right hippocampal extracellular glutamate was measured using microdialysis and high-performance liquid chromatography every minute from 5 minutes before to 30 minutes after the ischemia. During the experimental period, nitrous oxide or nitrogen administration was continued, cerebral and rectal temperatures were maintained at 37.0±0.5 °C. [Results] Onset time of ischemic depolarization was shorter in the N2O group (1.65±0.46 minutes) than in the N2 group (1.92±0.38 minutes, p<0.01). In the case of 5-minute ischemia, the percentage of damaged neurons in the N2O group (81.5±22.4 %) was higher than that in the N2 group (38.9±13.3 %, p<0.01). Logistic regression curves in the N2O group shifted to the direction that neuronal damage was increased, ischemic durations causing 50% neuronal damage in the N2O group and N2 group were 4.43 minutes and 5.29 minutes, respectively (p<0.05). The 95% confidence intervals did not overlap from 3.80 to 5.85 minutes of ischemic duration and from 5.35 to 7.97 minutes of ischemic depolarization. There was no difference in both groups on dynamic change in extracellular glutamate concentration. Peak extracellular glutamate concentrations in the N2O group and N2 group were 61.0±39.6 mcM and 70.0±31.3 mcM, respectively (p=0.64). [Conclusion] Nitrous oxide accelerated neuronal damage during cerebral ischemia. In the N2O group, onset time was shorter and logistic regression curves were shifted to the direction that neuronal damage was increased compared with those in the N2 group. However, dynamic changes in extracellular glutamate concentration had no difference in the both groups. These results suggested that the acceleration of neuronal damage by nitrous oxide was predominantly affected by a factor other than the amount of extracellular glutamate release during ischemic depolarization.
EFFECTS OF FOCAL ISCHEMIA ON ENDOTHELIUM-DEPENDENT RELAXATION AND GENE EXPRESSION IN CEREBROARTERIAL ENDOTHELIAL CELLS: FOCUS ON THE ENDOTHELIN SYSTEM
Lothar Schilling, Benedikt Brosch, Gerold Koch, Nina Weinzierl, Clemens Heiser, Ralf Erber
Division of Neurosurgical Research, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany
Background and aims: In the rat middle cerebral artery (MCA) endothelin (ET)-1 may induce contraction by activating ET(A) receptors located on smooth muscle cells and relaxation activating by ET(B) receptors on the endothelium. This relaxation is mediated by release of nitric oxide. Transient focal brain ischemia in rats has been described to result in a de novo gene expression of the ET(B) receptor in the smooth muscle cells mediating contraction [1]. The aim of the present study was to determine whether there is also an increase of ET(B) receptor-mediated relaxation and an upregulation of the ET(B) receptor-mRNA in the endothelial cells.
Methods: Adult male Sprague-Dawley rats underwent a 2 h transient MCA occlusion (MCAO) and were sacrificed 22 or 46 h later. Naive animals were used for control. Cerebrovascular reactivity to sarafotoxin 6c (S6c, a selective ET(B) receptor agonist) was studied in ring segments of the MCA. Gene expression was studied selectively in endothelial cells which were disrupted in situ using a microperfusion protocol. The total mRNA was sampled and underwent reverse transcription followed by quantitative polymerase chain reaction (qPCR) analysis of the mRNA for Tie-2 and smooth muscle specific actin, for ET(B), ET(A), ET-1, endothelin converting enzyme (ECE), and endothelial nitric oxide synthetase (eNOS) using elongation factor (EF)-1 as house-keeping gene. Analysis was performed using the ΔΔCT methodology.
Results: After ischemia/reperfusion endothelium-dependent relaxation was significantly enhanced. The pD2 values (mean±SD) obtained for S6c are: 9.3±0.4 (24h) and 9.7±0.4 (48h) vs. 8.9±0.3 for controls; the maximum relaxation (% of precontraction, induced by U46619, 3E-7 M) obtained after MCAO are 76.0±20.3% (24h) and 79.7±18.0% (48h) vs. 43.6±20.7% (control). However, S6c did not induce contraction when applied on resting tone, neither in control animals nor after ischemia/reperfusion injury.
Analysis of gene expression in the endothelial cells showed that compared with the intact vessel wall the Tie-2 mRNA was approximately 3.5 fold increased and actin mRNA content was only 6.5%. The ΔΔCT values for the remaining genes are listed in the table (*p<0.05 vs. control).
Conclusion: The present results indicate a significant upreguation of the gene expression of the ET-system in cerebral endothelial cells following ischemia/reperfusion injury. These alterations, underlining the importance of the vascular ET-system in the pathophysiology of ischemia/reperfusion injury are more pronounced after 24 than after 48 h. Furthermore, eNOS expression is transiently upregulated at 24h after MCAO. However, ET(B) receptor-mediated relaxation is significantly enhanced even at 48h after ischemia. Therefore, endothelium-dependent relaxation may involve more than the NO pathway in regulation of vessel tone after ischemia/reperfusion injury.
PERMANENT MCAO IN SHEEP - A NOVEL MODEL OF FOCAL CEREBRAL ISCHEMIA
Johannes Boltze1,2, Annette Foerschler3, Henryk Barthel4, Andrea Barbara Lang1, Christiane Maria Boltze1, Osama Sabri4, Frank Emmrich1,2, Uwe Gille5,6
1Fraunhofer Institute of Cell Therapy and Immunology, Leipzig, Germany, 2Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany, 3Department of Neuroradiology, University Clinic of Leipzig, Leipzig, Germany, 4Department of Nuclear Medicine, University of Leipzig, Leipzig, Germany, 5Institute for Veterinary Anatomy, University of Leipzig, Leipzig, Germany, 6VITA34 INc., Leipzig, Germany
Background and aims
In stroke, the effectiveness of experimental cell therapy has recently been demonstrated in rodent models. There is optimism that cell therapy has the potential to overcome a current dilemma (apart from thrombolysis in acute stage) in stroke therapy: so far most alternative therapeutic strategies including about 60 neuro-protective drugs which had likewise proven efficacious in pre-clinical rodent studies, subsequently failed in clinical trials. This demonstrates that translation of new stroke therapies into clinical application requires close-to-practice models, i.e. large animal models of focal cerebral ischemia. Existing primate models are cost intensive, restricted to highly specialized breeding facilities and limited by ethical considerations. Therefore, we developed a new, cost-effective stroke model in sheep that allows to create variable, but reproducible lesion sizes / neurological dysfunctions.
Methods
25 adult Merino rams were randomly assigned to one of five groups: transcranial 1-branch- (n=5), 2-branch- (n=5) or total middle cerebral artery occlusion (MCAO, n=5), sham operation (n=5) and control group (no operation, n=5). Following exposure of the left MCA, the vessel was occluded or touched (sham) by high frequency bipolar forceps. Animals underwent an analgesic and antibiotic treatment for 7 days. Neurological investigation was performed before surgery and at days 1, 4, 7, 10, 16, 25, 32 and 42 following MCAO according to a house-designed score point system. Further, effects of MCAO were evaluated by magnetic resonance imaging (MRI) including T1-, T2-, T2*-sequences, diffusion weighted (DW) imaging and magnetic resonance angiography (MRA) using a Philips 1,5T clinical scanner at days 1, 14 and 42. In addition, positron emission tomography (PET) was performed at days 14 and 42. Animals were killed at day 43 post operation and brains were removed for neuropathological investigation.
Results
Total MCAO results in a large cortical lesion and severe neurological dysfunctions including absent/delayed startle reflexes, ataxic movement, circling and torticollis. 1- and 2-branch MCAO caused smaller cortical lesions and limited functional deficits. The MCA was clearly occluded as confirmed by MRA in all cases. Further, sheep with occluded MCA showed neurofunctional disabilities and clear signs of focal cerebral ischemia in MRI, PET and after brain removal.
Two sham operated animals showed very mild, transient dysfunctions lasting for 2 days and minor cortical contusion damage at the day 1 while controls did not show any sensomotoric disabilities. Ischemic lesions were not observed using MRI in the sham operated animals.
Conclusion
Our data show that the developed model is feasible to study the mechanisms of focal cerebral ischemia in a large animal model. Reproducibility of lesion size and functional deficits allows detailed studies of new therapeutic strategies like cell treatment. Because of its reasonable size, the sheep brain can reliably be imaged using clinical scanners, i.e. even if dedicated animal scanners are not available. Since MCAO causes a severe, but not total loss of motor functions in sheep, the animals can easily be housed in a species-appropriate environment at low costs. We have subsequently used this model to evaluate the efficacy of autologous bone marrow administration upon stroke.
POSTISCHEMIC NORADRENERGIC STIMULATION WITH NOREPINEPHRINE REUPTAKE INHIBITORS REDUCES BRAIN INJURY AND FACILITATES RECOVERY FOLLOWING PHOTOTHROMBOTIC CORTICAL ISCHEMIA IN MICE
Sohee Park, Jooyeon Lee, Jinho Jang, Wonsuk Lee
Department of Pharmacology, Pusan National University School of Medicine, Busan, South Korea
This study aimed to investigate whether norepinephrine (NE) reuptake inhibitors reduces brain injury and facilitates the recovery following photothrombotic cortical ischemia in mice. Male ICR mice were anesthetized and systemically administered Rose Bengal. Permanent focal ischemia was induced in the medial frontal cortex and somatosensory cortex by irradiating the skull with a cold light laser. Animals were subacutely treated with NE reuptake inhibitor (desipramine or maprotiline) for 10 days starting on the second day after ischemia induction. The light-irradiation after Rose Bengal injection caused well-demarcated tissue damage in the cerebral cortex, which was located in the sensorimotor cortex and extended to the corpus callosum with no evidence of striatal damage. The infarct size including infarct area and infarct volume was significantly reduced at 14 days after the first dose of desipramine as well as maprotiline, and this infarct-reducing ability was further preserved until 21 days after the first dose. The Evans blue extravasation index of desipramine- and maprotiline-treated group was significantly lower than that of vehicle-treated group. The lower limit (LL) of mean arterial blood pressure for cerebral blood flow autoregulation was shifted toward a normal value by subacute treatments with desipramine and maprotiline. This LL-restoring ability of desipramine and maprotiline was preserved until 21 days after the first dose. Taken together, it is suggested that postischemic noradrenergic stimulation with NE reuptake inhibitors could facilitate the recovery following photothrombotic cortical ischemia, and thereby indicating advantages in the therapeutic strategy for cerebral ischemia. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. R01-2004-000-10570-0).
A COMPARISON OF GENE AND PROTEIN REGULATION CHANGES BETWEEN HUMAN AND A RAT MCAO MODEL OF STROKE USING CDNA MICROARRAYS
Nick Mitsios1, Mohamad Saka1, Roberta Pennucci1, Jerzy Krupinski2, Coral Sanfeliu3, John Gaffney1, Pat Kumar1, Shant Kumar4, Matthew Sullivan1, Mark Slevin1
1Department of Biological Sciences, Manchester Metropolitan University, Manchester, Lancs., UK, 2Department of Neurology, University Hospital De Bellvitge, Barcelona, Spain, 3Department of Pharmacology and Pathology, Institute of Biomedical Investigations, Barcelona, Spain, 4Department of Pathology, Manchester University and Christie Hospital, Manchester, Lancs., UK
Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. An analysis of gene expression profiles after stroke in humans and a comparison with those in animal models could identify novel gene expression changes and lead to a better understanding of stroke pathophysiology.
We have used microarrays to investigate the dynamic changes in gene expression at various timepoints after MCAO in human and rat brain.
Our results demonstrated a significant difference in the total numbers of genes affected and the time-course of gene expression between the two models. The total number of deregulated genes in the rat model was 335 versus 126 in the human, while out of a total number of 393 overlapping genes, 184 were changed only in the rat and 36 only in the human with a total of 41 genes deregulated in both models. Interestingly, the mean fold changes were much higher in the human stroke study. Some genes have not been previously studied in association with ischemia and their expression patterns were further analyzed by RT-PCR, Western blotting and immunohistochemistry at various time points for the validation of the cDNA array results. These genes included cyclin-dependent kinase 5, p21-activated kinase 1, matrix metalloproteinase 11 and leukaemia inhibitory factor.
These findings confirmed previous studies reporting that parallel screening of gene expression can detect both previously known and novel transcriptional features of cerebral ischemia, leading to a better understanding of this complex process. Moreover, our work highlights the importance of research using human stroke tissue in the search for novel therapeutic agents, which may be useful in the treatment of stroke.
LINKAGE STUDIES FOR FAMILIAL ARTERIOVENOUS MALFORMATION IN THE TAKAYAMA COMMUNITY
Katsunobu Takenaka1, Sumiko Inoue2, Wanyang Liu2, Kayoko Inoue2, Youhei Mineharu2, Hiroyasu Yamakawa3, Masamitsu Abe4, Jafar J. Jafar5, Roman Herzig6, Hidenori Kitajima1, Katsuhiko Hayashi1, Akio Koizumi2
1Department of Neurosurgery, Takayama Red Cross Hospital, Takayama, Gifu, Japan, 2The Department of Health and Environmental Sciences, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan, 3Department of Emergency Medicine, Chunou Kousei Hospital, Seki, Gifu, Japan, 4Department of Neurosurgery, Faculty of Medicine, Saga University, Saga, Saga, Japan, 5Department of Neurosurgery, New York University Medical Center, New York, NY, USA, 6Stroke Center, Department of Neurology, Palacky University Medical School and University Hospital, Olomouc, Czech Republic
Background and aims: Genetic factors for brain arteriovenous malformation (AVM) are unexplored due to the low incidence of familial cases, albeit local and familial clustering 1. To circumvent such difficulties, we conducted a combination of a pedigree-based linkage analysis and a population-based association analysis in the Takayama community2. Subjects: The study was approved by the Ethics Committee of Kyoto University Institutional Review Board. A total of 12 patients with AVM, from 6 families, participated in the linkage analysis. We recruited 26 unrelated cases (17 men and 9 women) and 30 controls (14 men and 16 women) for the association analysis. Two sets of identical twins from Takayama, with discordant phenotypes, also participated. Methods: Linkage Analysis: A genome-wide linkage analysis was performed in 12 patients from 6 unrelated families using the GENEHUNTER program3. Association Analysis: A genome-wide association analysis of 26 cases and 30 controls was performed using a GeneChip 10K mapping array. Statistical Criteria for the Selection of Candidate Genes: In the linkage analysis, we considered p<0.05 to be an evidence of suggestive linkage. In the single SNP association analysis, p<0.0001 was considered to be statistically significant. Results: With a dominant model, the linkage analysis revealed 7 candidate regions (Table 1). With a recessive model, a significant p value was only obtained at 6q25 (p=0.005, data not shown). The highest LOD score of 1.88 was obtained at D6S1581 with a dominant model (Table 1). Association analyses revealed 4 SNPs and 2 haplotypes with a significant association (Table 2). Conclusions: We minimized the population stratification by selecting a study population within the Takayama community, where clusters of sporadic AVM and familial AVM have been reported4. However, the present study failed to identify genetic factors for AVM, although the low statistical power may have resulted in such evidence being missed.
CHINESE HERBAL MEDICINE SERVE AS NEUROPROTANT FOR NEURODEGENERATIVE DISEASES
Hsin-Hsueh Lee1, Yi-Hsuan Lee2, Ling-Ling Yang1
1Department of Pharmacognosy, College of Pharmacy, School of Pharmacy, Taipei Medical University., Taipei, Taiwan, 2Department of Physiology, Taipei Medical University, Taipei, Taiwan
Recently the herbal nutrition supplements are very popular applied for chronic disease prevention. In this study, we want to assess the role of the Chinese herbal medicines on neuroproective effects in rat cortical neurons. Controlling or modifying risk factors hypertension, high blood cholesterol, smoking, overweight/obesity, diabetic, and physical inactivity can prevent stroke. The polyphenols isolated from Scutellaria baicalensis Georgi, including baicalin and baicalein were investigated for their neuroprotective effects against glutamate/NMDA(Glu/NMDA) stimulation and glucose deprivation in Sprague-Dawley (SD) rat primary cultured rat brain neurons. Cell death was accessed by lactate dehydrogenase (LDH) release assay for necrosis, and mitochondrial activity was accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity assay. The Ca2+ influx was assayed as described using 45Ca and investigated by confocal microscopy. S. baicalensis exerted neuroprotective effects. Baicalein is the most effective polyphenolic compound tested in preventing neurotoxicity induced by both glutamate and GD, whereas baicalin was only effective in preventing glutamate toxicity. Chinese herbal medicines exerted neuroprotective polyphenolic compound with novel actions on both activity-dependent of [Ca2+]i activity. Further studies are needed not only to elucidate its neuroprotective mechanism, but also to reveal its role in intracellular calcium signaling for cortical functions.
Key words: S. baicalensis, Baicalein, glutamate, glucose-deprivation, neuroprotection
PREVENTION OF SPINAL MOTOR NEURON DEATH BY IGF-1 ASSOCIATING WITH THE SIGNAL TRANSDUCTION SYSTEMS IN SODG93A TRANSGENIC MICE
Hisashi Narai1, Yasuhiro Manabe1, Tetsuro Murakami2, Makiko Nagai2, Tatsushi Kamiya2, Koji Abe2
1National Hospital Organization Okayama Medical Center, Okayama, Japan, 2Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan
Background and aims
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease that is characterized by selective loss of central and peripheral motor neurons. There are many hypotheses about the underlying cause of this disease: one theory is that motor neurons lack crucially needed trophic factors, resulting in neuronal degeneration, cell death, and atrophy of target muscles. The role of insulin-like growth factor-1 (IGF-1) in ALS and its mechanism of action are important from both pathogenic and therapeutic points of view.
Methods
The present study investigated the changes of IGF-1Rβ and the key intracellular downstream protein insulin receptor substrate-1 (IRS-1) using SOD1 G93A transgenic mice with continuous intrathecal IGF-1 injection. We made up the number of mice for the histological studies, non-Tg littermates and Tg mice treated with vehicle, low-dose (0.1mg/kg) or high-dose (1mg/kg) IGF-1 at 240 days of age.
Results
The number of lumbar spinal motor neurons was preserved with IGF-1 treatment in a dose dependent manner (1215.0 ± 97.2 (/mm thickness, mean ± SD) in wild type mice, 281.5 ± 36.4 in Tg mice, 715.0 ± 46.5 in Tg mice with low dose IGF-1 treatment and 900.0 ± 346.7 in Tg mice with high dose). The number of immunopositive motor neurons in the anterior horn for IGF-1Rβ was not significantly different between wild type and Tg mice. However, treatment of Tg mice with IGF-1 decreased the number of immunopositive motor neurons in a dose-dependent manner with low dose (74.0 ± 9.2) and high dose (20.0 ± 1.6). On the other hand, the percentage of immunopositive motor neurons per total living motor neurons in vehicle treatment mice (69.3 ± 11.6%) was greatly increased as compared with wild type mice (12.3 ± 3.5%). With IGF-1 treatment, the ratios were dramatically decreased in a dose-dependent manner with low dose (10.3 ± 1.4%) and high dose (2.2 ± 1.0%). The number of immunopositive motor neurons in the anterior horn for IRS-1 was 196.5 ± 25.9 in wild type, which were not different in Tg mice (217.5 ± 12.1). In contrast, treatment with IGF-1 decreased the number of immunopositive motor neurons of Tg mice in a dose-dependent manner with low dose (52.8 ± 15.3) and high dose (45.0 ± 7.2). The ratio of immunopositive motor neurons per the total number of living motor neurons for IRS-1 in wild type mice was 16.7 ± 2.6%, which greatly increased to 74.6 ± 13.1% in Tg mice. With IGF-1 treatment, the ratios of immunopositive motor neurons were again dramatically decreased in a dose-dependent manner with low dose (7.3 ± 2.2%) and high dose (5.0 ± 2.4%).
Conclusions
We investigated signal transduction systems in SODG93A Tg mice that received continuous intrathecal injection of IGF-1 and observed the percentage of IGF-1Rβ and IRS-1 immunopositive motor neurons in Tg mice increased then decreased with IGF-1 treatment. These results suggest that IGF-1 treatment prevents motor neuron loss by affecting the signal transduction system through IGF-1R and the main downstream signal IRS-1.
COUPLING OF GLYCOLYSIS TO SYNAPTIC TRANSMISSION: ROLE OF SYNAPTIC VESICLE-ASSOCIATED PYRUVATE KINASE IN NEUROTRANSMITTER UPTAKE INTO SYNAPTIC VESICLES
Atsuhiko Ishida1,5, Yasuko Noda1, David G. Bole1, Hisashi Umemori1, Tetsufumi Ueda1,2,3,4
1Molecular and Behavioral Neuroscience Institute, Medical School, University of Michigan, Ann Arbor, MI, USA, 2Ann Arbor, MIDepartments of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, MI, USA, 3Department of Pharmacology, Medical School, University of Michigan, Ann Arbor, MI, USA, 4Department of Psychiatry, Medical School, University of Michigan, Ann Arbor, MI, USA, 5Department of Biochemistry, Asahikawa Medical Col., Asahikawa, Japan
Glucose metabolism is of vital importance in synaptic transmission. We have focused on the potential role of glycolysis on glutamate accumulation into synaptic vesicles. Previously, we showed that the ATP-generating glycolytic enzymes glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase, which were found to be bound to synaptic vesicles, provide energy required for glutamate transport into synaptic vesicles (J. Biol. Chem. 278, 5929-5940, 2003). Here we report that ATP generated by the vesicle-associated pyruvate kinase, another ATP-generating glycolytic enzyme, is harnessed to transport glutamate into synaptic vesicles. When synaptic vesicles isolated by sucrose-density gradient centrifugation were incubated with [3H]Glu in the presence of phosphoenol pyruvate (PEP) and ADP, a marked incorporation of [3H]Glu was observed. The uptake was dependent on a low concenrtaration of Cl- in the medium, and was inhibited by FCCP, Bafilomycin, Rose Bengal, and trans-ACPD, but not by cis-ACPD. These results show that it occurs through vesicular glutamate transporters (VGLUT), in the presence of an electrochemical proton gradient generated by V-type H+-ATPase (V-ATPase). The pyruvate kinase activators K+ and Tl+ stimulated both vesicle-associated pyruvate kinase activity and PEP/ADP-dependent glutamate uptake. The highly selective pyruvate kinase inhibitor (Z)-3-chlorophosphoenolpyruvate diminished both vesicle-associated pyruvate kinase activity and PEP/ADP-dependent glutamate uptake. Two-dimensional Western blotting analysis revealed that a unique subunit species of pyruvate kinase, not detected in the cytosol, is associated with synaptic vesicles. These data demonstrate that glutamate uptake is supported by activation of vesicle-associated pyruvate kinase. ATP-trapping experiments using hexokinase have indicted that ATP endogenously produced by the vesicle-associated pyruvate kinase is used more efficiently by V-ATPase than is exogenously added ATP. This suggests that pyruvate kinase is functionally coupled to V-ATPase. In support of these biochemical studies indicating the occurrence of pyruvate kinase in synaptic vesicles, we also provide immunocytochemical evidence that a population of synaptic vesicles is associated with pyruvate kinase. Furthermore, we have found that not only Glu but also other neurotransmitters such as GABA, dopamine, and serotonin were taken up into crude synaptic vesicles in an ADP/PEP-dependent manner. These observations support the notion that glycolysis is coupled to synaptic transmission through its linkage to neurotransmitter accumulation in synaptic vesicles. Supported by NIH Grant 042200 and NIMH Grant 071384 (T.U.)
GLP-1 REDUCES BLOOD-BRAIN GLUCOSE TRANSPORT
Susanne Lerche1, Birgitte Brock1, Joergen Rungby1, Hans E. Boetker1, Niels Moeller1, Anders Rodell2, Jens J. Holst3, Ole Schmitz1, Albert Gjedde2
1Department of Pharmacology, Aarhus University, Aarhus, Denmark, 2PET Center, Aarhus University Hospital, Aarhus, Denmark, 3Department of Medical Physiology, Copenhagen University, Copenhagen, Denmark
Glucagon-like-peptide-1 (GLP-1) has many effects regarding glucose homeostasis. It stimulates insulin secretion in a glucose dependent manor, inhibits glucagon secretion and gastric emptying and has trophic effects on pancreatic β-cells, making it a potent antidiabetic hormone. GLP-1 receptors are broadly represented in the brain. Recent research in rodents has suggested a potentially protective effect of GLP-1 in the brain. The mechanism behind this effect is unknown. The possible role of GLP-1 in the human brain glucose metabolism is not well characterized. To gain more insight into changes of glucose delivery and/or metabolism, which may explain neuroprotective effects, we explored glucose transport and consumption in 10 healthy men in a randomized, double-blinded placebo-controlled cross-over experimental design. The acute effect (independent of insulin) of GLP-1 on glucose uptake in the brain was visualized by Positron Emission Tomography (PET) during a pituitary-pancreatic normoglycaemic (plasma glucose ? 4.5mM) clamp with FDG (18-fluoro-deoxy-glucose) as a tracer of glucose. Insulin infusion rate was 0.12mU/kg/min, growth hormone and glucagon were replaced to baseline levels. Total GLP-1 levels were 258 ± (SEM) 23 vs. 16 ± 0.09 pmol/l (GLP-1 vs. placebo). In total cerebral grey matter, GLP-1 reduced initial glucose clearance, K1(glc) by 29%, 21±2 ml/(100cm3)/min on placebo vs. 15±1 ml/(100cm3)/min on GLP-1 (P= 0.04). In the cerebral cortex, reduction was 29%, 21±2 vs. 15±1 (P=0.04), in white matter 33%, 12±1 vs. 8±0.3 (P=0.03), in thalamus 29% 24±3 vs. 17±1 (P=0.07), in striatum 29%, 21±2 vs. 15±1 (P=0.05), in cerebellar cortex 28%, 25±3 vs. 18±1 (P=0.07) and in brainstem 31%, 16±2 vs. 11 ± 0.6 (P=0.02). The efflux coefficient, k2(glc) showed similar reduction. The regions showed a uniform trend towards reduced cerebral glucose metabolic rate and increased lumped constant. As a result, the intracerebral glucose concentration remained the same in all regions, with and without GLP-1. For the first time, we demonstrate that a hormone involved in postprandial glucose regulation also limits glucose delivery to brain tissue and hence provides evidence that GLP-1 has an important role in brain glucose regulation. As GLP-1 reduces glucose transport across the intact blood brain barrier at normoglycaemia, GLP-1 may also protect the brain by limiting intracerebral glucose fluctuations when plasma glucose is increased.
CEREBRAL BLOOD FLOW IN CIRRHOTIC PATIENTS WITH ACUTE HEPATIC ENCEPHALOPATHY
Peter Iversen1, Per Borghammer2, Michael Sorensen1, Susanne Keiding1
1PET Centre and Department of Medicine V, Aarhus University Hospital, Aarhus, Denmark, 2PET Centre, Aarhus University Hospital, Aarhus, Denmark
The study compared cerebral blood flow (global and regional) in cirrhotic patients (n=17) with an episode of acute hepatic encephalopathy (HE), New Haven criteria grade I (n=6), grade II (n=5), grade III (n=4), or grade IV (n=2) with cirrhotic patients without HE (n=20), and age and sex matched healthy controls (n=15). The aim was to test the hypothesis that the neurological symptoms defined by the New Haven criteria are related to changes in cerebral blood flow.
Global and regional cerebral blood flow rates (CBF) were measured with dynamic 15O-water PET. Parametric maps of CBF were created using measured arterial inputs. The maps were registered to an MRI-template in a common stereotactic space and smoothed with a Gaussian filter to a final resolution of 14 mm (FWHM). Voxel-based statistical analysis was used to test differences of CBF in selected regions of interest in the grey matter tissue.
When compared to controls, both groups of patients with cirrhosis had significantly reduced global perfusion rates. There was no significant difference between the group of patients with HE and without HE (Table 1). After correcting for effects of global decreases by normalization of individual scans to the global grey matter perfusion rate, the regional CBF rates between the patient groups were found significantly reduced in the frontal lobe, occipital lobe, and the posteriomedial part of the parietal lobe, with exception of sensorimotor cortex (M-I and S-I) and supplementary motor area (SMA) (data not shown).
Due to hyperventilation, the patients with HE were in a state of respiratory alkalosis with reduced arterial partial pressure of CO2 (PCO2) (Table 1); in patients with cirrhosis with no HE, PCO2 was normal. However, there was no significant correlation between CBF and the degree of HE, suggesting that hyperventilation had minimal influence on CBF in the HE patient group.
In conclusion, the findings do not support the hypothesis that low cerebral blood flow is exclusive to the complication of cirrhosis.
REGION SPECIFIC EFFECTS OF HYPOXIC PRECONDITIONING ON CEREBRAL BLOOD FLOW IN THE ADULT RAT BRAIN
Susanne Wegener1,2, Khaled Restom1, Eric C. Wong3
1Department of Radiology, University of California San Diego, La Jolla, CA, USA, 2Department of Neurology, Berlin Neuroimaging Center, Berlin, Germany, 3Department of Radiology and Psychiatry, University of California San Diego, La Jolla, CA, USA
Background and aims: Neuroprotection induced by hypoxic preconditioning (HP) results in a more efficient cellular and vascular response to injury. As part of this response, the attenuation of a cerebral blood flow (CBF) decrease has been identified in hypoxic/ischemic injury models in the immature rat brain. Not much is known, however, about the mechanisms of HP in the mature brain. Our goal was to characterize HP induced changes in 1) baseline CBF, 2) vasoreactivity to a 5% CO2 challenge and 3) the CBF response to a global hypoxic stress.
Methods: 14 adult spontaneously breathing Wistar rats were exposed to either room air (sham) or 8% O2 (HP) for 3h. Two days later, rats were anesthetized with 1.5% Isoflurane and CBF maps were acquired using quantitative pulsed arterial spin labeling methods along with anatomical images on a 3T MRI scanner during inhalation of room air, 12% O2 or 5% CO2 in air. Arterial blood gases and blood pressures were obtained under the same anesthetic regimen. Region of interest as well as voxel wise statistics (corrected for multiple comparisons) were applied for CBF analysis.
Results: CBF showed previously described regional variations when rats were breathing room air. Interestingly, discrete shifts in the distribution of CBF in air were observed in HP as compared to sham animals. 12% O2 lead to mild increases in blood flow to subcortical structures, e.g. the hippocampus, thalamus, caudate putamen, and less to the parietal cortex in sham animals (Fig.1). In HP animals, the CBF response to hypoxia was attenuated. Clusters of voxels exhibiting significant differences in hypoxia CBF maps in HP animals were detected in the ventral caudate putamen and insular/parietal cortex. The response to 5% CO2 was a robust CBF increase in most brain regions and was not different between groups.
Discussion: The increase in CBF to vulnerable brain regions such as the hippocampus with hypoxia most likely reflects a compensatory mechanism to redistribute blood flow for maintenance of energy metabolism. The region dependent attenuation of this response in HP suggests that the hypoxic stimulus imposed less stress on certain areas of the brain, possibly due to cellular or vascular adaptation processes. Furthermore, preconditioning induced discrete local changes in baseline CBF might indicate a role of these brain regions in cerebral tolerance formation.
FLOW / METABOLISM COUPLING IN NEURODEGENERATIVE DISORDERS: A PRELIMINARY REPORT
Eric A. Schmidt1, Pierre Payoux2,3, Julien Darreon3, Thierry Voisin4, Jean Paul Esquerre2, Pierre Celsis2,3
1Service De Neurochirugie, Hopital Purpan, Toulouse, France, 2Centre TEP, Hopital Purpan, Toulouse, France, 3Unite INSERM 825, Hopital Purpan, Toulouse, France, 4Federation De Geriatrie, Casselardit Toulouse, Toulouse, France
Background and aims
Coupling and uncoupling between cerebral blood flow and metabolism is known in physiology and pathology. However, imaging of coupling has not been studied as a tool. We aim at defining new imaging techniques which might become a surrogate disease marker; uncoupling might herald pathological conditions. Two groups were studied: patients suffering of normal pressure hydrocephalus (NPH), and those complaining of chronic fatigue syndrome (CFS).
Methods
6 patients suspected of NPH (mean age 72), and 8 patients suspected of CFS (mean age 40) were enrolled. Each patient underwent the same day H215O PET and 18F-FDG PET. Firstly 2 injections of 300 Mbq of H215O were performed to measure rCBF in baseline condition. After 10 minutes, subjects underwent FDG PET, 20 minutes after an injection of 300 Mbq of 18F-FDG.
To perform a voxel-based analysis we realigned the scans using a H215O and 18F-FDG normalized templates implemented in SPM2. A voxelwise unpaired t test was performed generating a t statistic map for the contrast condition effect after the specification of a design matrix. This was then converted to a Z map to assess statistical significance at a P level of 0.05. Uncoupling in regional cerebral CMRglu and rCBF, were investigated according to the general linear model in each voxel. Proportional scaling was applied by adjusting the mean global activity of each scan to 50 ml/100ml/min and the threshold of gray/white matter to 0.8. A voxel level threshold family wise error (FWE) was used with multicomparaison test. We created an hybrid template (H215O and 18F-FDG) for this study, using a mathlab function with all the FDG and H215O images of the subjects. Finally we compare FDG and H215O as contrast.
Results
Using SPM2 at a FWE threshold with an hybrid template, we have been able to normalize our results especially irrespective of the ventricular dilation. We identified many uncoupling area in our group of NPH patients. In NPH hypermetabolism relative to rCBF was detected in various regions mainly bilateral occipital cortex. In CFS no specific pattern was identified. In both pathological conditions, hyperperfusion relative to metabolism was detect mainly around the vessels.
Conclusion
We have been able to perform coupling and uncoupling images. Our preliminary results suggest that i) regions of relative hypermetabolim patterns can be identified within the brain, and ii) relative hyperperfusion is detected around the vessels, which is expected.
CEREBRAL AUTOREGULATION REMAINS INTACT DURING AVM-RESECTION
Carsten Stueer1, Toshiki Ikeda2, Michael Stoffel1, Carlo Schaller2, Bernhard Meyer1
1Neurosurgical Department, Munich Tech University, Munich, Germany, 2Neurosurgical Department, Bonn Medical Center, Bonn, Germany
Background and aims: To test the hypothesis that function of cerebral autoregulation in AVM patients undergoing resective surgery differs to the one observed in nonvascular patients.
Methods: Dynamic autoregulation (AR) via correlation coefficient index method (CCI) was assessed before and after resection in n = 12 patients with small/medium-sized AVM and transsylvian dissection in n = 15 patients with nonvascular lesions (= controls). MAP, CBF (thermal dilution method), and routine Intraoperative physiological parameters were continuously measured. All data were pooled according to groups and times, and compared via ANOVA (P<0.05) and post-hoc Wilcoxon correction for multiple comparisons. All data (given below as means ± SD) were collected in the context of a prospective study.
Results: CCI did not show any significant within-group or between-group differences regarding the controls (control pre: 0.06 ± 0.4, post: −0.04 ± 0.5) and AVM-group (pre: 0.04 ± 0.4, post: 0.04 ± 0.5)), respectively.
Conclusions: Dynamic autoregulation results indicate that the status of autoregulation is not altered in patients with uncomplicated small or medium AVM compared to non-AVM conditions. During resective surgery, autoregulation remains intact in AVM patients.
ADENOSINE (ADO) IS NOT INVOLVED IN THE INCREASE IN CBF INDUCED BY HYPERCARBIA
Grzegorz J. Miekisiak, Alexander Beylin, David K. Kung, H. Richard Winn
Department of Neurosurgery, Mount Sinai School of Medicine, New York, NY, USA
Background and Aims: ADO has been suggested as contributing to cerebrovascular dilatation during hypercarbia. Consequently, we evaluated the CBF response to 60 s. of hypercarbia (7%) in WT and A2a Knock-Out (KO) mice in the absence and presence of the non-specific Ado receptor antagonist, theophylline (THEO, as aminophylline= 50mg/kg, ip) as well as the specific A2a antagonist, ZM 241385 (1 mg/kg, iv). Ado induced vasodilatation of cerebral vessels occurs primarily through activation of the A2a receptor.
Methods: Mice (22-30 gm) were intubated, anesthetized, and ventilated. We continuously monitored end-tidal CO2, arterial blood pressure (BP), & CBF (laser Doppler). WT and KO animals were blindly challenged with 7% CO2 (40%O2 balanced with nitrogen) for 60s and CBF measured by laser Doppler.
Results: As indicated in Fig 1, inhalation of 7% CO2 for 60 s. resulted in a 29.5 ± 2.4 % increase in the WT animals and a 34.55 ± 3.3 % in the KO animals. Pre-treatment of each group with nonspecific Ado receptor antagonist, THEO, did not statistically affect the CBF response. Similarly, pre-treatment with ZM 241385, the specific A2a receptor antagonist, did not alter the increase in CBF during hypercarbic challenge (data not shown).
Conclusions: These data are not consistent with the hypothesis that adenosine is involved in hypercarbic cerebral vasodilatation.
[Supported by NS 021076]
HYPERTENSION ? SEQUENCE OF STRUCTURAL CHANGES IN THE ARTERIOLES OF THE BRAIN MICROCIRCULATION
Roland Auer1, Garnette Sutherland2
1Department of Pathology, University of Calgary, Calgary, AB, Canada, 2Department of Clinical Neurosciences, University of Calgary, Calgary, AB, Canada
BACKGROUND: Hypertension is common. The brain microvasculature undergoes sequential structural changes which have not been clearly delineated.
METHODS: 20 sequential autopsies of hypertensives were examined, with heart weights ranging from 450 to 650 grams.
RESULTS: Arterioles initially undergo smooth muscle hyperplasia. Tortuosity ensues, producing glomeruloid vascular formations. Eventually smooth muscle cells die by apoptosis, either slowly or rapidly, with macrophage accumulation. The resultant acellular wall consists totally of collagen which is laid down in reaction to smooth muscle cell loss. Two changes can then occur ? ectasia or stenosis. Ectasia occurs according to P=2T/R and becomes self-sustaining. If collagen deposition is insufficient, eventual rupture occurs as thinning of a microaneurysm leads to a wall rent and a brain hemorrhage. If collagen deposition is exuberant, stenosis and lacunar infarction can occur. This sequence of arteriolar changes in the white matter is associated with imaging alterations in the centrum semiovale. Lacunes occur in spinal cord and hippocampus, not just deep telencephalon. Bleeds can be treated with recombinant factor VIIa because the size of the leaking vessel is so small, unlike berry aneurysms.
ABNORMAL DIAZOXIDE-INDUCED VASODILATION OF MIDDLE CEREBRAL ARTERIES FOLLOWING TRANSIENT FOCAL CEREBRAL ISCHEMIA IN RATS
Prasad Katakam1, Gabor Lanzser1,2, Keita Mayanagi1, James A. Snipes1, David W. Busija1
1Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winsto Salem, NC, USA, 2Institute of Clinical Experimental Research and Human Physiology, Semmelweis University, Budapest, Hungary
Diazoxide, a mitochondrial KATP channel activator, elicits arterial vasodilation by increasing the generation of mitochondria-derived reactive oxygen species that stimulate calcium (Ca2+) sparks and calcium activated K+ (KCa) channels. We evaluated the diazoxide-induced vasodilation in rat middle cerebral arteries (MCA) following transient focal cerebral ischemia. Male Wistar rats were subjected to cerebral ischemia by MCA occlusion (MCAO, 90 minutes) followed by reperfusion (48 hours). Vascular reactivity of isolated MCAs was assessed by videomicroscopy. Diazoxide (100 micromol/L) induced vasodilation was intact in ipsilateral MCAs while it was diminished in contralateral MCAs (% relaxation: 35±6 in contralateral MCAs versus 62±7 in ipsilateral MCAs and 69±6 in MCAs from Wistar rats not exposed to MCAO, p<0.05). Similarly, vasodilation to bradykinin (10 micromol/L) was reduced in contralateral MCAs (27±8%, p<0.05) compared to ipsilateral (68±6%). Nitroprusside (100 micromol/L) induced vasodilation was preserved in all MCAs (95±3 in ipsilateral versus 96±3 in contralateral MCAs, p<0.05). In contrast, pressure induced constriction was decreased in ipsilateral MCAs compared to contralateral (% constriction at 80 mmHg: 4±2 in ipsilateral versus 22±6 in contralateral MCAs, p<0.05). Similarly, endothelin-1 (10 nanomol/L) induced constriction was also diminished in ipsilateral MCAs (23±9%, p<0.05) compared to contalateral MCAs (56±6%). Thus, cerebral ischemia diminishes Diazoxide induced as well as endothelium-dependent vasodilation in contralateral MCAs despite intact myogenic tone and smooth muscle dependent vasodilation. Diminished generation of mitochondria-derived ROS and/or Ca2+ sparks or impaired coupling of Ca2+ sparks to KCa channels may mediate diminished vasodilation to diazoxide.
MULTI-MODAL STUDY OF STIMULUS HETEROGENEITY IN RAT SOMATOSENSORY CORTEX
Peter Herman1,2, Basavaraju Sanganahalli1, Fahmeed Hyder1,3
1Department of Diagnostic Radiology, Yale University, New Haven, CT, USA, 2Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary, 3Department of Biomedical Engineering, Yale University, New Haven, CT, USA
INTRODUCTION
To expand the heterogeneity examination of the neurometabolic-neurovascular coupling, multimodal data are needed to describe the effects of the various stimuli to the functional activation. We have measured neurometabolic-neurovascular responses (BOLD, CBF, pO2) and electrophysiological responses (MUA, LFP) in rats during 30 s forepaw stimulation in the layer 4 of the somatosensory cortex, as a function of stimulation parameters.
METHODS
Artificially ventilated rats (n = 43) were anesthetized with α-chloralose (40 mg/kg/hr). All fMRI experiments were conducted at 11.7T. Gradient-echo EPI data (TE=15ms) were acquired with TR of 1 s. Data were collected in 120 s windows: 30 s before and 60 s after the 30 s forepaw stimulation. Electrical activities (MUA, LFP) were measured with high impedance microelectrodes (2-4 MΩ) simultaneously with laser Doppler flowmetry and pO2 responses with optical methods. Responses were measured in during 30 s forepaw stimulation and as a function of stimulation parameters (0.2, 2.0, and 10.0 mA; 1, 3, and 9 Hz), where the duration of each stimulus pulse was 0.3 ms.
RESULTS
Generally, all measured responses increased with stimulation amplitude, but at stimulation amplitude of 0.2 mA all measured responses were quite variable with frequency. The best stimulation frequency and stimulation amplitude correlations were with the BOLD response. For activated BOLD pixels at P<0.05, we found that increasing the stimulation amplitude elevated the response, whereas increasing the stimulation frequency decreased the response. For the CBF response, a similar trend was observed with stimulation amplitude, but no correlations could be drawn with changes in the stimulation frequency. For the tissue pO2 response, we found significant changes only with specific stimulus conditions (i.e., 3Hz/2mA, 3Hz/10mA, 9Hz/10mA) suggesting that the tissue hyperemic changes are less pronounced. For the neurophysiologic responses (MUA, LFP), the most significant changes were detected with stimulation frequency >3Hz and stimulation amplitude >2mA. To describe the dynamic correlation across the different responses, we compared the percent of the averaged normalized differences (PAND), where 0% and 100% indicate perfect and imperfect matches, respectively. The correlations for the BOLD or CBF responses with the LFP responses were slightly lower (i.e., PAND values BOLD-LFP and CBF-LFP were 39% and 23%, respectively) than with the MUA responses (i.e., PAND values BOLD-MUA and CBF-MUA were 30% and 18%, respectively). One of the highest correlation was found between the BOLD and CBF responses (i.e., PAND value for BOLD-CBF was 21%). Generally, most PAND values were below 50% suggesting that there was good dynamic correlation across the different measured responses.
ACKNOWLEDGEMENTS
Supported by NIH (DC-003710, MH-067528) and OTKA (T34122) grants.
EFFECT OF CELL-FREE HEMOGLOBIN TRANSFUSION AND 20-HETE SYNTHESIS INHIBITION ON PIAL ARTERIOLAR DIAMETER DURING MIDDLE CEREBRAL ARTERY OCCLUSION
Suyi Cao1, Herman Kwansa1, Richard Roman2, Raymond Koehler1
1Department of Anesthesiology, Johns Hopkins University, Baltimore, MD, USA, 2Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA
Background and aims: Hemodilution can increase cerebral blood flow during middle cerebral artery occlusion (MCAO), but neuroprotection is limited by the decrease in oxygen carrying capacity. Exchange transfusion of cell-free hemoglobin polymers (Hb) during MCAO can decrease infarct volume. Without MCAO, exchange transfusion of a 5% albumin solution produces pial arteriolar dilation, whereas exchange transfusion with a Hb solution produces pial arteriolar constriction that is blocked by the 20-HETE synthesis inhibitor HET0016 (Qin et al. J Appl Physiol 2006; 100:336-42). Because 20-HETE synthesis is oxygen dependent, the constriction after Hb transfusion may represent an autoregulatory response to limit over-oxygenation at decreased blood viscosity. We tested whether HET0016 would augment pial arteriolar dilation selectively after Hb exchange transfusion compared to albumin transfusion during MCAO.
Methods: In isoflurane-anesthetized and mechanically ventilated rats, cranial windows were constructed over the MCA-ACA border region for measurement of pial arteriolar diameter. MCAO was produced for 2 h by the intraluminal filament technique. At 15 min of MCAO, the window was superfused with 0.1% ethanol vehicle or 1 uM HET0016 for the remainder of the protocol. At 35-50 min of MCAO, an exchange transfusion with a 5% albumin solution or 5% albumin + 6% Hb solution was performed. The Hb polymer had a nominal molecular weight of ~20 MDa and a P50 of 4 mmHg.
Results: MCAO initially produced 36±10% (±SD) dilation which subsided to 19±6% dilation at 30 min of MCAO in the MCA border region. Subsequent exchange transfusion with the albumin solution resulted in no further change in diameter (22±16% of preischemia at 2h MCAO; n=8). In a group with initial dilation of 36±16%, HET0016 superfusion did not attenuate the loss of dilation at 30 min (22±14%) or the level of dilation after albumin transfusion (27±22% at 2 h MCAO; n=8). In a third group in which initial dilation after MCAO (25±9%) subsided to 12±12% at 30 min with superfusion of vehicle, subsequent exchange transfusion of Hb resulted in a loss of sustained dilation (4±19% of preischemia at 2h MCAO; n=8). In a fourth group with initial dilation of 35±12%, superfusion of HET0016 failed to block this loss of dilation (6±17%; n=9) after Hb transfusion.
Conclusion: Initial vasodilation after MCAO is not fully sustained by either albumin or Hb transfusion. However, loss of vasodilation is greater after Hb transfusion than after albumin transfusion and may limit efficacy of this oxygen carrier during MCAO. Blunted vasodilation after albumin or Hb transfusion is not dependent on 20-HETE synthesis, possibly because low oxygenation during MCAO limits 20-HETE synthesis. (Supported by NIH NS38684)
TIME VARYING MULTIVARIATE SYSTEM IDENTIFICATION OF CEREBRAL AUTOREGULATION
Alexander Brian Rowley1,2, T. Peng1, S.J. Payne1
1Department of Engineering Science, University of Oxford, Oxford, UK, 2University Computing Laboratory, University of Oxford, Oxford, UK
BACKGROUND
Blood flow in the brain is controlled by systemic variability (heart rate, blood pressure, and CO2) as well as mental activation. Numerous studies attempt to assess the dynamical properties of the various relationships using low frequency oscillations that can be observed in all of the variables, sometimes at very similar frequencies.
Recent studies attempting to assess control of cerebral blood flow by systemic blood pressure have demonstrated significant changes in the dynamical relationship during mental activation paradigms. It is also reasonable to expect that attempts to assess changes in blood flow due to mental activation may be confounded by systemic variability.
We have shown that under some circumstances, measurement of additional systemic variability (for example CO2 measurements) can improve the reliability of estimating a dynamical relationship between blood pressure and cerebral blood flow. It could also be expected that measuring variability related to mental activation would further improve the reliability of this process. Difficulties arise because the factors leading to a change in cerebral blood flow during mental activation are still a topic of active research. When it is not possible to measure all variability leading to a dynamic response of a system it could be expected that any system relationship that is identified would vary due to the unmeasured variability. This artefact in the system identification process is termed ‘non-stationarity’ and might be expected to lead to the low values of coherence found during studies which attempt to perform linear system identification to understand cerebral blood flow control.
METHODS
We have developed a technique for performing non-parametric system identification of cerebral autoregulation using the maximal overlap wavelet packet transform to carry out time varying estimation of cross and power spectral densities. This allows assessment of a multivariate linear relationship between inputs: blood pressure and CO2 variability; and outputs: cerebral blood flow velocity measured using transcranial Doppler ultrasonography (TCD) or near infrared spectroscopy (NIRS) measurements.
This technique is applied to combined measurements of arterial blood pressure, CO2 variability and TCD measured cerebral blood flow velocity in the resting state, and combined measurements of arterial blood pressure and Near Infrared Spectroscopy during mental activation paradigms.
RESULTS AND CONCLUSIONS
In the resting state it appears that a significant proportion of low frequency cerebral blood flow variability can be explained by a linear system acting upon low frequency variability in CO2 and ABP. This relationship appears constant with time and multiple coherence function estimation using traditional stationary spectral techniques appears to work well. It appears that during mental activation this assumption of stationarity breaks down, however, in using time frequency analysis to estimate the properties of the underlying system the extent to which this occurs can be reliably quantified, providing a more general technique of discovering the behaviour of the system from experimental data.
QUANTIFYING NONLINEAR COUPLING IN CEREBRAL HAEMODYNAMICS USING INFORMATION TRANSFER
Alexander Brian Rowley1,2, S.J. Payne1
1Department of Engineering Science, University of Oxford, Oxford, UK, 2University Computing Laboratory, University of Oxford, Oxford, UK
BACKGROUND
The information theoretical concept of transfer entropy [Schreiber, T. Phys Rev Lett. 2000] was recently used [Katura et al. NeuroImage. 2006.] to assess the influence of systemic variability on spontaneous low frequency oscillations measured in the human brain using diffuse optical tomography. Transfer entropy is a probabilistic technique of assessing general coupling between time series. It has considerable advantages over traditional techniques (such as correlation) as it makes no assumptions about the linearity of the underlying relationship.
It was recently shown [Achard, S. Journal of Neuroscience. 2006] that correlation of coefficients of the maximal overlap wavelet packet transform of functional magnetic resonance imaging (fMRI) time series in humans can be used to characterise functional connectivity between regions of the brain by assessing similarity between time series of low frequency (<0.1Hz) oscillations. Importantly, the wavelet packet transform provides a means of separating the time series into components at different scales (frequency bands). When this decomposition has been performed, correlation provides a measure of linear similarity between scales. Replacing this correlation step in the algorithm with a similarity measure based upon transfer entropy relaxes the assumption of linearity and can be used to estimate a directed measure of information transfer between inter scale time series.
METHODS
We have developed an estimator for transfer entropy that uses a recently developed unsupervised learning algorithm [Figueiredo et al. IEEE Trans. on Pattern Analysis and Machine Intelligence 2002.] for estimation of the probability density functions of the time series embedding from relatively short duration records. This estimator is applied to quantification of the interrelationship between wavelet transform coefficients of low frequency oscillations measured using near infrared spectroscopy (NIRS) and those observed systemically (for example mean arterial blood pressure (ABP)).
RESULTS AND CONCLUSIONS
Use of a Gaussian mixture model to converge on the underlying probability density functions provides a practical estimator for transfer entropy between time series that may be measured using near infrared spectroscopy. The maximal overlap wavelet packet transform is an efficient algorithm for decomposing a time series into coefficients that have highly localised frequency content. Applying the technique to quantification of coupling between NIRS and systemically measured variability provides a measure of the extent to which low frequency behaviour measured using NIRS can be attributed to systemic variability.
It is expected that this technique will also be useful to investigate functional connectivity between regions of the brain by assessing scale dependent similarity of low frequency oscillations measured using FMRI without making the assumption of linearity of the underlying coupling relationship
HETEROGENOUS RESPONSE IN CBF DURING AUTOREGULATION: A NONINVASIVE LASER SPECKLE STUDY IN THE RAT BRAIN CORTEX
Peter Herman, Laszlo Kocsis, Istvan Portoro, Andras Eke
Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary
INTRODUCTION
Blood flow autoregulation is a spatio-temporal phenomenon. A fractal cerebrovascular system distributes flow among great number of microregions in the brain cortex [1]. Data is needed how each of these microregions deal with the hypotensive challenge. We report on a heterogenous pattern of microregional autoregulatory threshold.
METHODS
Animal preparation: Six artificially ventilated Wistar rats were anesthetized by Urtehane (1.3 g/kg, i.p.). An arterial line was used for monitoring blood pressure and taking samples for blood pH, pO2, pCO2 throughout the experiment. The bone was thickened to translucency over the parietal cortex 2 days prior the experiment. A metal platform was fixed to the skull to secure the head's position during the experiment. Laser Speckle measurements: The speckle contrast images (4096 images/150s) were collected with a Coolsnap CF camera with 256×256 resolution (voxelsize: 16 µm2) at 655nm. A 5×5 kernel was used to calculate flow velocity. The hypotensive steps (of 90, 80, 70, 60, 50 and 40 mmHg) were maintained by a computer controlled hypobaric method which has been described previously [2]. The duration of each hypotensive step was ~2.5 min. During the transient between steps, pressure slowly ramped to target level at a rate of ~0.07mmHg/s. The calculated velocity images were averaged for each hypotensive period. The autoregulatory threshold for CBF was obtained for each microregion within the mapped area at 80% of the respective control level.
RESULTS and DISCUSSION
The lower threshold of CBF autoregulation proved spatially heterogeneous (see figure for a representative experiment). On the left side the anatomical image on the right side the autoregulatory threshold map are shown. The color coded bar represents the latter in mmHg. In terms of flow velocity, the pial vessels appear to be more exposed to the autoregulatory challenge than the parenchymal vascular system.
ACKNOWLEDGEMENTS
Supported by OTKA (T34122) and EuroBloodSubstitues Consortium (LSHB-CT-2004-503023) grants.
THE INFLUENCE OF HYPOGLYCEMIA ON REGIONAL CEREBRAL BLOOD FLOW IN CASES OF HYPOGLYCEMIC COMA
Yuzo Terakawa1, Naohiro Tsuyuguchi1, Katsuyuki Nunomura2, Naoaki Murayama2, Masahito Fujishige2, Akinori Yamamura2, Toshio Nakagawa2, Kazuo Hashi2
1Department of Neurosurgery, Osaka City University Graduate School of Medicine, Osaka, Japan, 2Department of Neurosurgery, Shinsapporo Neurosurgical Hospital, Hokkaido, Japan
Background: Recently, diffusion-weighted imaging (DWI) has been reported to show hyperintensity lesions in the acute stage of hypoglycemia. However, changes of regional cerebral blood flow (CBF) in hypoglycemic coma in human patients have received scant attention.
Methods: We report two cases of hypoglycemic coma in which both DWI and single photon emission computed tomography (SPECT) were used.
Results: Case 1. A 74-year-old woman with a history of diabetes mellitus controlled using oral medication was admitted to our hospital after being found unconscious at home. Her family had noticed the patient had been drowsy the day before admission. On admission, the patient had slight hypertension (blood pressure 158/80mmHg), regular heart rate, and irregular respiration but no hypoxia. Neurologically, the patient was in a deep coma (Japan Coma Scale 200, Glasgow Coma Scale 5) with nonreactive pinpoint pupils. Oculocephalic reflex was intact and Babinski reflex was positive bilaterally. The patient showed tetraplegia with decerebral posturing. The DWI obtained immediately after admission revealed bilaterally symmetrical hyperintensity lesions in the middle cerebral peduncles, temporoparietal cortex, corona radiate, and the splenium of the corpus callosum. The blood glucose level on admission was 20 mg/dl. Although the blood glucose was rapidly corrected to within the normal range by intravenous administration of glucose, the level of consciousness remained unchanged. SPECT obtained on the following day revealed diffuse hyperperfusion in both hemispheres, especially in the bilateral temporoparietal regions. Case 2. An 89-year-old woman with medically controlled diabetes mellitus and hypertension was admitted to our hospital for consciousness disturbance. On admission, the patient had slight hypertension (blood pressure 150/70mmHg) but otherwise normal vital signs. Neurological examination on admission showed the patient was in a coma (Japan Coma Scale 100, Glasgow Coma Scale 6) and tetraplegic. Babinski reflex was positive bilaterally. The DWI obtained on admission revealed bilaterally symmetrical hyperintensity lesions in the temporoparietal cortex (greater on the right side), internal capsules, deep white matter at the level of centrum semiovale, and the splenium of the corpus callosum. The laboratory data on admission showed the blood glucose level was 45 mg/dl. Despite the glucose infusion, the level of consciousness did not recover. SPECT obtained two days after admission revealed relative hyperperfusion in the temporoparietal area.
Conclusions: These radiological findings indicate that severe hypoglycemia may lead to an increase in CBF in the acute stage of hypoglycemic coma.
HEMODYNAMIC RESPONSE TO FOREPAW STIMULATION IN ANESTHETIZED MICE SOMATOSENSORY CORTEX
Kazuto Masamoto, Michelle Tasker, Ping Wang, Seong-Gi Kim
Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
Aim: Genetically altered mice are a potentially powerful tool to elucidate the molecular basis of neuro-vascular coupling mechanism. However, neural activity-induced hemodynamic responses in anesthetized murine cortex remains uncharacterized, which hampers the use of a mice model for functional imaging studies (e.g., fMRI and optical imaging). In the present study, hemodynamic responses to forepaw stimulation in the mice somatosensory cortex were characterized under pentobarbital, ketamine-xylazine, and isoflurane anesthesia, all of which are generally used for repeated animal experiments.
Materials and methods: A total of thirteen male C57B6J mice (23-35 g) were divided into three groups. First group (N = 4) was induced with pentobarbital anesthesia (an initial dose of 90 mg/kg i.p. and supplemental dose of 30-45 mg/kg/h i.p.). Second group (N = 6) was induced with a cocktail of ketamine and xylazine (an initial dose of 60 mg/kg and 10 mg/kg i.m., respectively, and supplementally injected with only ketamine 20 mg/kg i.m. every 20-40 min.). The animals in the third group (N = 3) were ventilated with a mixture of isoflurane (4% for induction and 1.3–1..5% for experiments) and air with supplemental oxygen (a total of 30–35%). Animals in the isoflurane group were intubated and mechanically ventilated, whereas the other two groups breathed spontaneously. After the skin covering the skull was removed, oil was immediately placed to preserve the integrity of the skull. First, intrinsic optical imaging (620-nm wavelength) was performed for the localization of the forepaw area in the primary somatosensory cortex. Then, hemodynamic response was measured with laser-Doppler flowmetry (LDF) on the activation focus induced by electrical forepaw stimulation (rectangular pulses with 0.5-ms pulse width and 0.8-mA current). Stimulation duration was fixed at 10 sec for all experiments and stimulation rate (frequency) was varied (2, 4, 6, 8 and 12 Hz). Preliminary experiments determined a pulse width and current that would not elicit a pain response from the animal.
Results and discussion: All animal groups showed clear localization of forepaw area in 620-nm optical imaging. The clearest contrast of activation area was observed with ketamine-xylazine group. Under pentobarbital anesthesia, the hemodynamic response increased with an increase in stimulation frequency. The peak of 10 ± 12% (Mean ± SD, N = 4) signal change in LDF was observed at stimulation with 12 Hz. Under ketamine-xylazine anesthesia, the highest signal change (13 ± 4% at peak) was observed at stimulation with 6 Hz. These results indicate that the optimum frequency differs depending on the anesthesia used, which was consistent with our previous results obtained in rats (Masamoto et al., 2006). In isoflurane-anesthetized group, however, no significant hemodynamic response was observed for any stimulation frequency. This result indicates that the isoflurane may not be a good agent for functional imaging studies with mice, which was not the case in rats anesthetized with isoflurane (Masamoto et al., 2006). In conclusion, mice anesthetized with pentobarbital or ketamine-xylazine can be used for repeated functional imaging studies, which make possible to probe the mechanism of neurovascular coupling with a specific molecular basis.
FUNCTIONAL UNCOUPLING OF PERIVASCULAR NITRIC OXIDE PRODUCTION FOLLOWING NEONATAL HYPOXIA/ISCHEMIA
Thomas Kent1, Regino Perez-Polo2, Roderic Fabian1
1Department of Neurology, Baylor College of Medicine / Michael E Debakey VAMC, Houston, Texas, USA, 2Department of Human Biochemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA
Background and Aims: Compromise of cerebral vascular function due to a loss of vascular reactivity may be a cause of cerebral injury after hypoxia-ischemia. A phenomenon that has gained increasing attention as a possible determinant of blood flow and a cause of vascular injury under pathological conditions is the uncoupling of endothelial cell nitric oxide synthase (eNOS, NOS3), which reduces NO production and results in the production of superoxide anion (O2-) by the enzyme. There have been few direct assessments of this phenomenon in the CNS, although eNOS coupling appears to be developmentally regulated in the neonate in other vascular beds. Using a histofluorescence method, we characterized acute changes in cerebral blood flow, vascular O2- anion, and nitric oxide (NO) in neonatal rat cerebral hypoxic/ischemic injury and their response to pharmacological manipulation and the eNOS substrate, tetrahydrobiopterin (BH4).
Methods: Neonatal P7 rats were subjected to cerebral hypoxia ischemia by unilateral carotid ligation followed by hypoxia with hyperoxic resuscitation. Laser Doppler flowmetry was used to monitor CBF, and cerebral vascular wall NO and O2- were determined using i.p.-injected fluorescent dyes (DAF-2AC and DHE respectively) and stimulation with acetylcholine, assessed with fluorescence microscopy. The effect of the NADPH inhibitor apocyanin and the NOS inhibitor, L-NAME, injected i.p. at the onset of resuscitation on fluorescence patterns, and the functional effect of tetrahydrobiopterin supplementation were determined.
Results: We found a progressive decline in perivascular NO production and an increase in perivascular O2- in the hours following hyperoxic resuscitation (Figure: ratio between ischemic/contralateral hemisphere for DAF-2AC and DHE fluorescence; p=0.01 at 24 hours). Reperfusion CBF declined by up to 30% in the first 3 hours relative to baseline. The ratio between perivascular O2- and NO fluorescence was restored by both apocyanin and L-NAME. Administration of tetrahydrobiopterin increased vascular wall NO, reduced vascular wall superoxide anion levels (Figure), and normalized CBF.
Conclusions: These results demonstrate vascular dysfunction follows hyperoxic resuscitation after neonatal hypoxic/ischemic injury manifested by reduced CBF, a reduction in perivascular production of NO and an increase in O2-. Inhibitor studies implicate both NADPH oxidase and NOS and indicate these enzymes functionally disrupt vascular function. That tetrahydrobiopterin normalized these abnormalities suggests a possible role of substrate oxidation leading to uncoupling of eNOS due to tetrahydobiopterin deficiency with resultant implications for propagation of injury.
THE SPHINGOSINE-1-PHOSPHATE ANALOGUE FTY720 IS NEUROPROTECTIVE IN A RODENT MODEL OF BRAIN ISCHEMIA
Hyung-Hwan Kim2, Hwa Kyoung Shin1, Nicolas Blondeau3, James Liao2, Christian Waeber1
1Department of Radiology, Massachusetts General Hospital, Charlestown, MA, USA, 2Department of Vascular Medicine Research, Brigham & Women's Hospital, Cambridge, MA, USA, 3I.P.M.C., UMR 6097, C.N.R.S, U.N.S.A., Valbonne, France
Sphingosine-1-phosphate (S1P) is a lipid mediator exerting pleitropic effects on a wide variety of cell types, including glial cells, neurons and endothelial cells. In particular, S1P is known to be a survival factor for various cell types and is a key player in protective mechanisms against hypoxia- or ischemia-mediated insults. FTY720 has shown significant protective effects after controlled bilateral kidney ischemia, and attenuates ischemia-reperfusion injury in both normal and cirrhotic livers by activation of cell survival Akt signaling. The purpose of this study was to evaluate the effect of FTY720 administration on in vivo and in vitro models of brain ischemia. Halothane-anesthetized C57Bl/6 mice (20-25 g) underwent 90-min focal cerebral ischemia induced with an 8.0 nylon monofilament coated with a silicone resin/hardener mixture, introduced into the left internal carotid artery up to the anterior cerebral artery. Regional cerebral blood flow was measured using laser-Doppler-flowmetry. Mice treated with 3 mg/kg FTY720 orally once a day for 3 days starting after reperfusion showed a significantly reduced infarct 3 days after reperfusion, as visualized by TTC staining (108 ± 14 vs. 68 ± 8 mm3 for vehicle-treated mice; p=0.04), and significantly attenuated neurological deficit (5-point scale) 1, 2 and 3 days after reperfusion (Q1, median, Q3: 1, 1, 1.5 vs. 2, 2, 3 on day 3; p=0.02). Intravenous 0.5 mg/kg FTY720 did not affect systemic blood pressure and CBF (assessed with laser speckle flowmetry) for up to 60 minutes. We then tested whether the effects of FTY720 on infarct size and neurological deficit were due to increased cerebral blood flow during ischemia using noninvasive laser speckle flowmetry. FTY720 (3 mg/kg) did not alter the CBF deficit when administered orally to isoflurane-anesthetized mice 1h before distal middle cerebral artery occlusion (dMCAO): the area of severely ischemic cortex (? 20% residual CBF) was 3.7±2.0mm2 in FTY-treated mice, compared to 4.2±0.9 mm2 in the saline-treated group. Because S1P is a potent anti-apoptotic factor, we then examined the effects of FTY720 in vitro, using a oxygen-glucose deprivation (OGD) cell death model. Mouse primary cortical neurons were exposed to 2-hr OGD/24-hr reoxygenation, decreasing neuron viability by more than 40% with respect to basal cell death (as assessed using Hoechst staining). 3 microM S1P and 100 nM FTY (added at the time of re-oxygenation) restored viability to control levels. Similarly, in rat brain primary endothelial cells subjected to 4-hr OGD/24hr recovery, 3 microM S1P and 30 nM FTY720 increased cell viability to levels observed in control cells. The presence of an Akt inhibitor (20 microM 1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate) or NOS inhibitor (100 microM L-NAME) reversed the protective effects of 3 microM S1P on rat brain endothelial cells following 4hr OGD/24hr re-oxygenation. Taken together, these results suggest that the S1P receptor/Akt/NOS pathway may play an important role in neuroprotection following brain ischemia.
NEUROPROTECTIVE EFFECT OF THE ROOTS POLYGONUM CUSPIDATUM ON TRANSIENT FOCAL CEREBRAL ISCHEMIA IN RATS
Youngmin Bu1, Jinmo Kim1, Mi-Yeon Kim2, Yun Tai Kim2, Jinhee Jung2, Young Mi Park3, Zhen Hua Jin3, Kiok Sung3, Hoyoung Choi2, Hocheol Kim2,3
1Department of Herbal Pharmacology, College of Pharmacy, Woosuk University, Chonbuk, South Korea, 2Korea Institute of Science and Technology of Eastern Medicine (KISTEM), NeuMed Co. Ltd., Seoul, South Korea, 3Department of Herbal Pharmacology, College of Oriental Medicine, KyungHee University, Seoul, South Korea
Objectives: The roots of Polygonum cuspidatum has been widely used in traditional Korean medicine for circulatory diseases. It contains significant amount of phenols, such as resveratrol and is known to have various biological activities. The purpose of the present study is to development of neuroprotective antioxidant agents. For the purpose, we investigated the neuroprotective effect of antioxidant herb, the root of Polygonum cuspidatum on transient focal cerebral ischemia in rat.
Methods: The roots of Polygonum cuspidatum were extracted by 85% MeOH (PC). PC was fractionated by hexane (PCH), ethyl acetate (PCE), butanol (PCB), and water (PCW). Radical scavenging effects were investigated using DPPH assay and MDA assay in brain homogenates. Neuroprotective effect was invesigated using transient focal cerebral ischemia rat model (2 h of ischemia, 22 h of reperfusion). Sensory motor function was investigated by rotarod test, balance beam test at 24 h after ischemia. The brain slices were stained by 2 % 2, 3, 5-triphenyltetrazolium chloride and the infarct volume was measured by graphic analyzer at 24 h after ischemia.
Results: PC showed dose-dependent radical scavenging effects on DPPH and MDA assay. PCE was most effective fraction among fractions (Figure). Oral administration of PC 300 mg/kg reduced brain infarct by 30.8% and improved sensory motor function. PCE was aloso most effective fraction on focal cerebral ischemia rat model. PCE improved the rotarod test by 33%, the balance beam test by 32% and reduced brain infarct volume by 35.8% compared with vehicle treated group. Quantative study of phenol contents in fractions showed that PCE contained phenols more than other fractions.
Conclusion: Polygonum cuspidatum could be a candidate herb for the development of neuroprotective agents. According to the active guided fractionation and HPLC analysis, polyphenols like resveratrol could be the major compounds of neuroprotection.
Grant support: This work was supported by grant from the Brain Korea 21 Project (Ministry of Education, Korea), and by a grant (PF002102-00) from the Plant Diversity Research Center of the 21st Frontier Research Program funded by the Korean Ministry of Science and Technology.
ALTERED EXPRESSIONS OF IL-1BETA AND IL-1RA BY ESTROGEN IN ORGANOTYPIC HIPPOCAMPAL SLICES EXPOSED TO OXYGEN-GLUCOSE DEPRIVATION
Ji-Seung Choi, Soo-Jeong Kim, Kyoung-Eun Lee, Eun-Mi Park
Department of Pharmacology, Ewha Medical Research Institute, College of Medicine, Ewha Womans University, Seoul, South Korea
Background and aims : Anti-inflammatory mechanisms have been implicated in beneficial effects of estrogen. However, it is not clearly defined how estrogen regulates mediators related to inflammation. The balance of IL-1β and IL-1 receptor antagonist (IL-1ra) plays a role in the development of inflammation. This study was performed to evaluate whether estrogen affects the expression of IL-1β and IL-1ra in ischemic hippocampus.
Methods : Hippocamapal slices obtained from 10 days old rats were exposed to oxygen-glucose deprivation (OGD) for 30 min and then were reperfused for 72 h. 17-β estradiol (E2, 1 nM) was treated for 7 days before OGD. CA1 neuronal death was quantified by propidium iodide (PI) staining after reperfusion. Expressions of IL-1β and IL-1ra in slices were studied by real-time PCR and immunofluorescence.
Results : PI intensity of CA1 in slices treated with E2 was significantly reduced at 24 h and 72 h after reperfusion (p<0.05 vs. no treatment, fig. 1). E2 also reduced mRNA expression of IL-1β at 2h and 24h after reperfusion (p<0.05, fig.2). Fewer IL-1β positive cells were detected in E2 pretreated slices compared to no treatment. However, mRNA expression of IL-1ra was markedly enhanced by E2 up to 72h after reperfusion (p<0.05 vs. no treatment, fig.3).
Conclusions : These data demonstrate not only suppression of IL-1β but also enhancement of IL-1ra by estrogen pretreatment in ischemic hippocampus, suggesting that modulation of the balance between IL-1β and IL-1ra may be associated with anti-inflammatory mechanisms of estrogen, in part.
“This work was supported by the Korea Research Foundation Grant funded by the Korean Government(MOEHRD)” (KRF-2005-204-E00075).
ISOFLURANE PRECONDITIONING NEUROPROTECTION IN EXPERIMENTAL FOCAL STROKE IS GENDER-SPECIFIC IN YOUNG AND MIDDLE-AGED MICE
Hideto Kitano1, Lan Wang2, Vanessa R. Anderson2, Jennifer M. Young2, Ann E. Meinke2, Patricia D. Hurn2, Stephanie J. Murphy2
1Department of General Internal Medicine, Hyogo College of Medicine, Hyogo, Japan, 2Department of Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, OR, USA
Background and Aims: We have previously shown that neuroprotection due to isoflurane preconditioning (IsoPC) 24 hours before experimental focal cerebral ischemia is specific to young males. However, it is unknown if males and females respond differently to IsoPC in experimental stroke at different ages. We evaluated IsoPC in male and female ischemic brain in young (8 to 14 weeks old) and middle-aged (35 to 40 weeks old) mice and determined if gender-and age-specific responses are linked to differences in Akt activation.
Methods: Young and middle-aged male and female C57BL/6 mice were preconditioned for 4 hours with air (sham preconditioning, Sham PC) or 1.0% IsoPC. Mice subsequently underwent either 2 hours of reversible middle cerebral artery occlusion or were used for Akt determinations 24 hours after preconditioning. For infarction volume analysis, brains were collected at 22 hours reperfusion. Cortical and striatal infarct volumes (% contralateral structure) were determined by digital image analysis of 2 mm thick coronal brain slices stained with 2,3,5-triphenyltetrazolium chloride. For Akt analysis, cortices and striata were subdissected from the left hemisphere of preconditioned brains and pooled together from 3 mice (n=4 pooled samples per group) for western blot analysis. Akt activation was expressed as the ratio of normalized phosphorylated Akt (p-Akt) to total Akt (t-Akt).
Results: In contrast to corresponding Sham PC mice, cortical and striatal infarct volumes following IsoPC were decreased in young and middle-aged males; no significant differences in middle-aged females; and increased in young females. As compared to corresponding Sham PC mice, cortical and striatal p-Akt/t-Akt was increased by IsoPC only in young males, with no changes in p-Akt/t-Akt by IsoPC in middle-aged males and in females irrespective of age (Table).
Conclusions: Regardless of age, IsoPC neuroprotection in experimental stroke is male-specific, with the neuroprotective benefits of IsoPC being lost in females. Gender and age can alter cortical and striatal Akt activation in response to IsoPC. However, further study is needed to determine other possible divergent mechanisms underlying the sex-and age-specific responses to IsoPC in ischemic stroke.
NEUROPROTECTIVE AND ANTIOXIDANT EFFECTS OF TFM-007, A TAIWAN FOLK MEDICINE
Hsin-Hsueh Lee, Ling-Ling Yang
Department of Pharmacognosy, College of Pharmacy, School of Pharmacy, Taipei Medical University, Taipei, Taiwan
Oxidative stress is implicated in neuronal apoptosis that occurs in physiological settings and in neurodegenerative disorders. Taiwan is located in subtropical area and abundant of natural resources and ethnobotanies. In this study, two hundred of the common anti-inflammatory ethnobotanies of ethanol extracts were detected. From anti-oxidantive activities survey, we found the ethanol extract of TFM-007 is one of the most potential bio-resource. The dried flower of TFM-007 has been traditionally used in oriental medicine for the anti-tussive and anti-inflammatory. In the present study, we investigated the effects of free radical scavenging and anti-oxidative activities of TFM-007. Its antioxidant activities were evaluated by cell-free bioassays. TFM-007 with IC50=65 µg/ml) was shown to inhibit lipid-peroxidation initiated by Fe2+/ascorbic acid in rat brain, liver, heart and kidney homogenates. In addition, TFM-007 (IC50=10 µg/ml) on FRAP and scavenge DPPH radicals ability. At the same time, the neuroprotective activity will be displayed on different insults in Wistar cortical neurons. Furthermore, the results showed TFM-007 at micro morality concentration attenuated the arachidonic acid and glutamate-induced neurotoxicity. This is the first demonstration of neuroprotective and anti-oxidative effects of TFM-007. Although complex mechanisms may be involved in the neuroprotective actions, TFM-007 may be useful for the management of neurodegenerative disorders associated with excitotoxicity and oxidative stress and the effects with anti-apoptosis and survival related proteins will further investigated.
Key words: Ethnobotany, TFM-007, arachidonic acid, glutamate, oxidative stress, neuroprotection
GELDANAMYCIN PROTECTS RAT BRAIN THROUGH OVEREXPRESSION OF HSP70 AND REDUCING BRAIN EDEMA AFTER CEREBRAL FOCAL ISCHEMIA
Hyung-Min Kwon1, Yong-Ju Kim2, Seung-Hoon Lee2, Byung-Woo Yoon2
1Department of Neurology, Seoul National University Boramae Hospital, Seoul, South Korea, 2Neuroscience Research Institute, SNUMRC and Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea
Background and aims: Geldanamycin (GA), a benzoquinone ansamycin, binds HSP90, releases heat shock factor 1 and induces heat shock proteins (HSPs). HSP70, a major molecular chaperone, protects the brain against ischemic injury through inhibition of apoptotic pathways in vivo and reduced matrix metalloproteinase 9 (MMP-9) after ischemia in vitro. We hypothesized that GA would protect brain from focal ischemia via induction of HSP70 and MMP suppression in vivo.
Methods: GA or vehicle was injected into the lateral cerebral ventricles of adult male Sprague-Dawley rats using the stereotatic frame 24 hour before ischemia. Rats were subjected to 2-hour middle cerebral artery occlusions using the suture technique followed by 22-hour reperfusions. One day after ischemia, we evaluated infarction volume, brain swelling, behavioral scores, and immunohistochemistry.
Results: Western blots showed that GA at 2 µg/kg induced HSP70 by 24 hour following administration. GA decreased infarct volumes (219.8 ± 42.8 mm3 vs 63.5 ± 23.5 mm3)and brain edema (20.4 ± 6.3% vs 10.9 ± 3.8%), and improved behavioral outcomes (P < 0.05). Immunohistochemistry showed that GA induced HSP70 and decreased MMP-9.
Conclusions: GA protects brain from focal ischemia and reduces edema. This may be due, at least in part, to GA overexpression of HSP70.
ASIATIC ACID IS NEUROPROTECTIVE IN A MOUSE MODEL OF STROKE
Rajanikant Krishnamurthy, Daniel Zemke, Arshad Majid
Department of Neurology and Ophthalmology, Michigan State University, East Lansing, MI, USA
Background and aims - Asiatic acid is a plant-derived compound with anti-inflammatory and anti-oxidant properties that is currently used as a wound healing agent. Recent studies have shown that asiatic acid protects cultured neurons against both glutamate and beta-amyloid induced toxicity by reducing oxidative stress. Oxidative stress is believed to play an important role in neuronal damage associated with stroke, and many anti-oxidant drugs have been shown to be neuroprotective in models of stroke. Furthermore, some derivatives of asiatic acid have been found to have anti-apoptotic effects, and oral administration of asiatic acid enhances nerve regeneration in animals. Both of these effects may be beneficial for the treatment of stroke as well. The purpose of this study was therefore to determine if asiatic acid is protective in permanent focal cerebral ischemia in mice, a model of stroke.
Methods - Mice were administered 4 doses of 75 mg/kg asiatic acid or vehicle orally at 1 hour before and 3, 10, and 20 hours after the induction of ischemia by permanent occlusion of the middle cerebral artery.
Results - Mice treated with asiatic acid showed an approximately 50% decrease in infarct size at 24 hours after induction of ischemia compared with vehicle-treated controls.
Conclusions - These results suggest that asiatic acid may be an effective treatment for the reduction of damage after stroke.
PYRUVATE PROTECTION IN EXPERIMENTAL STROKE INVOLVES AN ANTI-INFLAMMATORY MECHANISM VIA INHIBITION OF NF-KB AND MMP9
Qing Wang1,2, Xiannan Tang1,2, Raymond A. Swanson1, Midori A. Yenari1
1Department of Neurology, University of California, San Francisco and The San Francisco Veterans Affairs Medical Center, San Francisco, CA, USA, 2Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA
Stroke is the third leading cause of death in the United States after cancer and heart disease. Existing therapy for ischemic stroke, intravenous rt-PA, is limited. Increasing attention for protective therapy is focused on preserving the ischemic penumbra and inhibiting post-ischemic inflammation. Pyruvate is a key intermediate in glucose metabolism, but is also a free radical scavenger. Prior work has shown that ethyl pyruvate is neuroprotective in a rat model of middle cerebral artery occlusion (MCAO). The purpose of this study is to assess the neuroprotective and anti-inflammatory effects of pyruvate following transient MCAO in rats. Male Sprague-Dawley rats (n=8/group) were subjected to 2 hours of MCAO, and pyruvate (500mg/kg, IP) was administered 10 minutes before reperfusion. 24 hours later, infarct size, neurological deficits, physiological parameters, NF-kB translocation, microglial activation and neutrophil infiltration were evaluated. Pyruvate did not affect physiological parameters but significantly reduced infarct volume by 71.25 % (46.20 ± 10.1 mm2 vs 160.49 ± 21.6 mm2 P<0.01). This was accompanied by improvement in behavioral tests, including forelimb-placement, elevated body swing test, cylinder test, and Bederson score (P<0.05). Pyruvate treatment also reduced the number of neutrophils stained by MPO by 45% (46±2.68/HPF vs 25± 1.69/HPF, P<0.001, n=7/group) compared with the vehicle-treated group. Microglial activation as measured by ED1 staining was reduced by 40% (30± 2.5/HPF vs 18±1.2/HPF, P<0.001). Isolectin B4 positive cells (to identify all microglia) were also reduced by 52% (52±2.57/HPF vs 25±1.7/HPF, P<0.001). Immunohistochemistry showed that pyruvate reduced numbers of brain cells with nuclear NF-kB staining from 31% (MCAO) to 7% (MCAO+pyruvate) (P<0.001). As a positive control, a separate set of rats (n=3/group) was given LPS (5mg/kg, IV) to cause brain inflammation without cell death. LPS increased nuclear staining of NF-kB and this was almost completely abolished by pyruvate (19% for LPS vs 0.5% for LPS+pyruvate, P<0.001). A NF-kB DNA binding assay was used to test activity. NF-kB's DNA binding capacity increased by 4.57 and 4.80 folds for LPS and MCAO treatment respectively compared with control, uninjured rats (7.68% for control vs 39.13% and 44.56% for LPS and MCAO, respectively, P<0.001). Pyruvate treatment significantly reduced NF-kB activation in LPS and MCAO groups (18.7 ± 1.5% for MCAO + pyruvate and 17.53 ± 1.4% for LPS+ pyruvate vs 44.56 ± 3.9% for MCAO and 39.13 ± 2.4% for LPS, P<0.001, n=7/group). Western blots of MMP9, a NFkB regulated gene, showed increased MMP9 protein by 4 and 3.70 fold for LPS and MCAO treatment, respectively compared with shams (9.36±0.73 % for shams vs.37.39±11.97% and 34.86±8.21% for LPS (P<0.01) and MCAO (P=0.01), respectively, n=3/grp). Pyruvate treatment profoundly reduced MMP9 increases due to LPS administration and MCAO (11.54 ± 1.27% for MCAO + pyruvate and 10.78 ± 1.32% for LPS+ pyruvate, P<0.01 vs. no treatment). Taken together, these results strongly suggest that neuroprotective effects of pyruvate on MCAO may be closely correlated to its anti-inflammatory effect, possibly at the transcriptional level.
Key Words: middle cerebral artery occlusion, pyruvate, cerebral ischemia, stroke, inflammation, neuroprotection.
This work was funded by NINDS, AHA.
Specific grants:
R01 NS40516
P50 NS14543
AHA EIA 0540066N.
CAPSAICIN PRE-TREATMENT IN EXPERIMENTAL CEREBRAL ISCHAEMIA
Renee Turner, Robert Vink
Discipline of Pathology, University of Adelaide, Adelaide, SA, Australia
Background and Aims: Few studies have examined the role of neuropeptides following cerebral insults such as stroke and traumatic brain injury. However, our laboratory has previously shown that the neuropeptide substance P (SP) is increased following experimental stroke, indicative of neurogenic inflammation, and is associated with significant functional deficits, cerebral oedema and breakdown of the blood brain barrier. Blocking the action of SP with a SP receptor antagonist significantly improves outcome, in particular, functional outcome. Capsaicin, an agent isolated from chilli peppers, causes the release of neuropeptides, including SP, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), amongst others, to the point of depletion. Given the beneficial effects of SP receptor blockade, the removal of neuropeptides may also provide significant protection from the ischaemic event. In the present study, we investigated whether capsaicin pre-treatment was more protective than a SP antagonist following stroke.
Methods: Animals were randomly assigned to treatment and control groups. Capsaicin (125mg/kg) or equal volume of vehicle was administered subcutaneously over a 3 d period. From our trauma studies, 14 d was shown to be the time-point where neuropeptides were depleted, as such at14 d following pre-treatment experimental stroke was induced under Isoflurane anaesthesia using the reversible thread model of middle cerebral artery occlusion. Reperfusion was achieved at 2 h post-stroke onset. Those animals not exhibiting anti-clockwise circling were excluded. The motor function of the animals was assessed using a rotarod device for 7 d following stroke.
Results: Animals pre-treated with capsaicin demonstrated a significantly improved motor function, as assessed by the rotarod, compared to vehicle pre-treated animals. However, neither group achieved normal functional levels during the post-stroke assessment period. We compared the performance of capsaicin-treated animals to SP antagonist-treated animals. Interestingly, the performance of SP antagonist-treated animals was superior to capsaicin-treated animals.
Conclusions: It was expected that ablation of neuropeptides would provide a greater deal of protection form the ischaemic event than a SP antagonist. However, capsaicin pre-treatment does not provide any more protection from the ischaemic event. Given the fact that the capsaicin group performed below that of the SP antagonist group suggests that some neuropeptides may in fact have beneficial roles following stroke. CGRP, a known potent vasodilator, may be one such neuropeptide.
EFFECTS OF LACTATE LOADING ON BRAIN GLUCOSE METABOLISM: INVESTIGATION WITH [18F]FDG-PET IN CONSCIOUS RATS
Mikako Ogawa1, Y. Ouchi2, T. Fuchigami1, T. Torizuka2, M. Futatsubashi2,3, Y.B. Jin1, Y. Magata1
1Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan, 2Positron Medical Center, Hamamatsu Medical Center, Hamamatsu, Japan, 3PET Medical Application Group, Hamamatsu Photonics K.K., Hamamatsu, Japan
Backgrounds and aims: Glucose has been thought as a principal metabolic substrate of the brain. However, Magestritti et al. proposed an astrocyte-neuron lactate shuttle hypothesis, which claims the metabolic role of lactate in the brain, especially in neurons(1). This model hypothesizes that lactate produced in astrocytes is then transferred to the neuron, which then uses the lactate as an energy substrate aerobically. Recently, Kemppainen et al. reported that high intensity exercise decreased brain glucose uptake(2). They speculated that lactate is produced during the exercise and utilized in the brain. To elucidate this phenomenon in terms of the role of lactate in brain energy metabolism, we investigated the effect of lactate administered systemically on the brain glucose metabolism in conscious rats by [18F]FDG-PET.
Methods: Each conscious rat (n=8) was firmly placed in a cone-shaped thin polyethylene holder on the scanner bed. Rats were fasted 12hr before the PET scan. Dynamic PET scans were performed after a bolus intravenous injection of 7.4 MBq [18F]FDG for 45 min using small animal PET scanner (FX, Gamma Medica-Ideas Inc.). Arterial blood sampling was performed throughout the scanning period, and plasma was separated by centrifugation and counted by a γ-counter. Blood glucose concentration was measured at 45 min after [18F]FDG injection. For the lactate group (n=4), 100 mM lactate was infused intravenously (0.65 mL/hr) throughout the measurement from 10min prior to [18F]FDG injection. The regions of interest (ROIs) were placed on the coronal sections of serial PET images generated every 1 min. The rate constants (K1, k2, k3, k4) were estimated by OLS to calculate CMRglc. We used 0.625 for LC (3).
Results: Lactate infusion reduced the value of Kcomp (= K1*k3/(k2+k3)) (control group: 0.043 min-1, lactate group: 0.034 min-1), and the CMRglc level was found to be higher in the lactate group (52.8 µmol/100g/min) than in the control group (42.7 µmol/100g/min). The blood glucose concentration was elevated after lactate treatment.
Conclusion: The decrease of Kcomp may indicate a possibility of greater utilization of lactate than glucose in the experimental setting of intravenous lactate infusion. Thus, it was suggested that lactate might be a favorable fuel for brain energy metabolism than glucose in such circumstances. The unexpected finding of CMRglc elevation in the lactate group might be due to an increase of serum glucose through glyconeogenesis in the liver activated by the lactate loading. Using this technique, the dynamics of lactate in the brain ischemia can be further clarified using living rats with MCA occlusion.
A ROLE OF DORSOLATERAL PREFRONTAL CORTEX IN DRUG-CUE INDUCED NEURAL RESPONSE
Takuya Hayashi1,2, Ji Hyun Ko1, Antonio Strafella1, Bruce Pike1, Alain Dagher1
1McConnell Brain Imaging Center, Montreal Neurological Institute, Montreal, QC, Canada, 2Investigative Radiology, National Cardiovascular Center Research Institute, Osaka, Japan
Background and aims: The drug cue-reactivity paradigm has been among the most prominent tools for investigating craving and relapse. Previous neuroimaging studies have associated cue-reactivity with activations of frontal areas, such as anterior cingulate (ACC) and dorsolateral prefrontal (DLPFC) cortices, in a manner dependent on the context of drug use. We had previously shown that an area of DLPFC responded to cigarette cues in a manner that was modulated by drug availability, and suggested that this region may modulate the response of other brain areas to cues. The current study used repeated transcranial magnetic stimulation (rTMS) to reduce excitability of this DLPFC area and investigated the context-dependent neural response to smoking cues using fMRI.
Methods: Ten healthy smokers (8 men; mean age: 23 years; average smoking: 19 cigarettes/day) were recruited. Each subject was scanned for structural MRI to localize the target of rTMS, followed by four fMRI scans on different days, during which they viewed video clips of smoking-cue (individuals smoking cigarettes) or control (without smoking). Each of four fMRI scans was performed under different conditions of expectancy (i.e. non-expectant: no smoking allowed for 4 hours after the scan, verified by CO monitoring; expectant: cigarette available immediately after the scan) and rTMS (true vs. sham) targeting the left DLPFC region identified in our previous study. The target for the rTMS was identified by linearly transforming the MNI template brain into each user's native space. Using frameless stereotaxy, we placed the TMS coil over each subject's target, and gave 1800 pulses of 1Hz rTMS with intensity of 59% (of stimulator maximum) which corresponded to the average resting motor threshold. Immediately after receiving true or sham rTMS, subjects underwent a 15-min fMRI scan while they watched alternate 2-minute video clips of the smoking-cue and control. They answered a craving questionnaire after each clip. Image data was analyzed using FSL.
Results: The self-reporting cue-induced craving tended to be lower in the true rTMS condition compared to sham rTMS. In the sham rTMS conditions, we found larger cue-induced BOLD response in the left DLPFC and ACC during the expectant compared to the non-expectant condition, as well as a correlation between craving and BOLD signal in the left DLPFC, as in our previous study2. In contrast, in the true rTMS conditions, neither cue-induced BOLD response, nor correlation between BOLD and craving were affected by expectancy in either frontal area. Analysis of effective connectivity revealed that the stimulated part of left DLPFC became less responsive to input from the contralateral DLPFC and medial temporal regions, but not to ACC (thresholded at uncorrected p = 0.001).
Conclusions: These findings indicate that in cue-reactivity the DLPFC facilitates ACC activity based on inputs from areas other than ACC, suggesting a key role of DLPFC in associating perceived drug availability with craving in a positive feed-forward manner.
DELINEATION OF VOI'S AT HMPAO SPECT IMAGES FOR AUTOMATIC CLINICAL EVALUATION OF NEW IMAGES
Claus Svarer1, Gerda Thomsen1, David Erritzoe1, Steen G. Hasselbalch2, Lisbeth Marner1, Vibe Frokjaer1, Lars H. Pinborg1, Karine Madsen1, Gitte M. Knudsen1
1Neurobiology Research Unit, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark, 2Memory Disorders Research Unit, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
Background and aims: In daily clinical routine, evaluation of 99mTc-HMPAO SPECT images is generally performed by simple visual inspection. However, this evaluation is critically dependent on experienced reviewers. In this study an automatic method for delineation of Volumes of Interest (VOI's) at HMPAO SPECT images is presented. The method does not require a MR image but works directly at the HMPAO images. It can be used for automatic quantification of flow values for different brain regions in clinical SPECT scanning and may be a helpful additional tool for HMPAO SPECT evaluation.
Methods: SPECT HMPAO images together with high resolution (?1.2 mm) T1-weighted MRI were acquired for 10 normal healthy subjects. Using the approach described in (Svarer, 2005) a VOI set consisting of 11 VOI's (left and right) was generated automatically for each of the HMPAO images. These HMPAO images together with the VOI's were used as templates. For a normal database of 36 HMPAO images (age 42-84 year) the spatial transformation between HMPAO template spaces and the individual images was estimated by the method described by Svarer et al. (2005) though using HMPAO templates. A VOI set was therefore available for each of the 36 images in the database. Semi-quantitative estimates of rCBF relative to cerebellum were obtained by dividing each VOI value with the corresponding cerebellar mean VOI value. For ten subjects (five healthy subjects and five patients) the same approach was used for extraction of mean VOI values from their respective HMPAO images. The database mean VOI values was detrended for age and similar transformations were performed for the VOI values for the ten subjects. Student's t-test was used to test if the mean VOI values were significant different (p<0.05) from the normal database, and similar tests were performed between left and right side for five of the VOI's and between a posterior and anterior cortical VOI.
Results: The outcome of the automatic approach was in perfect accordance with the overall assessment of three trained clinicians. One of five presumably healthy subjects unexpectedly proved to have an abnormal rCBF pattern; this image was also judged as abnormal on the basis of the automatic approach. In addition, the automatic approach identified a slight left-right side difference in one of the other healthy subjects, that was left unnoticed by the observers. Further, the images for the five patients were all identified as abnormal by the automatic approach where one of these was rated as normal by visual inspection.
Conclusion: Using this automatic approach it was possible objectively to get semi-quantitative estimates of rCBF for a number of brain regions, thus quantitatively assessing abnormalities in relation to a database of age-matched healthy subjects. In the limited number of patients presented, the automatic approach performed equally well to experienced observers and, in addition, gave rCBF values relative to healthy controls.
ANALYSIS OF BRAIN SPECT IMAGE OF MOYAMOYA DISEASE BY STEREOTACTIC EXTRACTION ESTIMATION (SEE). -COMPARISON OF ISCHEMIC AND HEMORRHAGIC PATIENTS-
Masaru Yamada1, Izumi Yuzawa1, Sachio Suzuki1, Kazuhisa Iwamoto1, Kiyotaka Fujii1, Yuji Asano2
1Department of Neurosurgery, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan, 2Department of Radiology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan
Background and aims: Cerebral blood flow (CBF) characteristics and differences of Moyamoya disease in ischemic and hemorrhagic patients are not fully understood. To elucidate CBF characteristics between different onset symptoms of Moyamoya disease, we analyzed SPECT data by using stereotactic extraction estimation (SEE).
Subjects and Methods: Preoperative 32 moyamoya disease patients were subjected to quantitative 123I-IMP-SPECT (ARG method) with one-time arterial sampling at rest and after acetazolamide-challenge. Data were analyzed by 3D-SSP method using SEE software. All the SPECT data were given anatomical coordinates. “Extent” of CBF dectrease is defined as number of pixels whose value is below 2SD of the normal database in each identical anatomical area. “Severity” of CBF decrease is defined as mean value of such pixels.
Results: Extent of CBF decrease was statistically larger in patients with ischemic presentation who needed bypass surgery compared to those of conservatively treated ischemic patients and hemorrhagic patients. Severity of CBF decrease was statistically larger in patients with ischemic presentation who needed bypass surgery compared to those of hemorrhagic patients. Limit of this method was difficulty in precise exclusion of SPECT data of damaged brain.
Conclusion: SEE analysis using whole brain SPECT data clearly showed CBF decrease in extent and severity in hemorrhagic patients with moyamoya disease is significantly smaller than that of ischemic patients. This may suggest pathophysiological mechanism for hemorrhage is not directly related to CBF decrease.
DIAGNOSTIC VALUE OF KINETIC ANALYSIS USING DYNAMIC FDG PET IN PATIENTS WITH MALIGNANT BRAIN TUMOR
Yoshihiro Nishiyama1, Yuka Yamamoto1, Nobuyuki Kawai2, Motoomi Ohkawa1
1Department of Radology, Faculty of Medicine, Kagawa University, Kita-Gun, Kagawa, Japan, 2Department of Neurological Surgery, Faculty of Medicine, Kagawa University, Kita-Gun, Kagawa, Japan
Background and aims: The most frequent malignant primary brain tumors are high grade astrocytoma (anaplastic astrocytoma and glioblastoma) and central nervous system (CNS) lymphoma. MRI detects small intracranial lesions, but has difficulties in making a differential diagnosis. FDG PET is widely applied to the study of brain tumors. However, several studies have demonstrated diagnostic limitations of FDG PET for imaging brain tumors because of the high basal glucose metabolic rate of normal brain tissue. We evaluated the quantitative imaging of glucose metabolism of malignant primary brain tumors with PET using kinetic analysis.
Methods: Twenty-eight lesions in 25 patients with malignant primary brain tumor (4 anaplastic astrocytoma, 10 glioblastoma and 14 CNS lymphoma were examined by FDG dynamic PET. Dynamic emission data were acquired for 60 min immediately following injection of FDG. Applying a three-compartment four-parameter model, metabolic rate constants [K1 (from plasma to brain), k2 (from brain to plasma), k3 (phosphorylation)] and cerebral metabolic rate of glucose (CMRGlc) were quantitatively measured in tumor lesions and the contralateral gray matter.
Results: Tumor K1 was lower than in the contralateral gray matter in all 4 anaplastic astrocytomas, in 8 glioblastomas (80%) and in 7 CNS lymphomas (50%). Tumor k2 was lower than in the contralateral gray matter in 2 anaplastic astrocytomas (50%), in 7 glioblastomas (70%) and in 11 CNS lymphomas (79%). Tumor k3 was higher than in the contralateral gray matter in 1 anaplastic astrocytoma (25%), in 6 glioblastomas (60%) and in 12 CNS lymphomas (86%). Tumor CMRGlc was higher than in the contralateral gray matter in no anaplastic astrocytoma, in 4 glioblastomas (40%) and in 12 CNS lymphomas (86%). The CMRGlc value (mean±S.D.) of CNS lymphoma (69.6±32.6) was significantly higher (p<0.03) than that of anaplastic astrocytoma (35.7±7.7) and glioblastoma (43.3±23.5).
Conclusions: The kinetic analysis, especially CMRGlc, from dynamic FDG PET might be useful for differential diagnosis of high grade astrocytoma and CNS lymphoma.
FUNCTIONAL NEUROIMAGING OF AUTONOMIC NERVOUS RESPONSES DURING LAVENDER-AROMA UNING [18F]FDG PET
Xudong Duan1, Manabu Tashiro1, Tomoyuki Yambe2, Sayuri Seto3, Masatoshi Itoh1
1Division of Nuclear Medicine, Cyclotron and Radioisotope Center, Tohoku University, Sendai, Japan, 2Department of Medical Engineering and Cardiology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan, 3Teikoku Seiyaku CO., Ltd., Kagawa, Japan
? Background and aims
Changes in the autonomic nervous activity can be induced by various sensory and emotional stimuli. The authors examined whether the power spectral analysis of heart rate variability (HRV) could detect changes in autonomic tone following a lavender aroma treatment or not.
? Methods
10 healthy young women underwent continuous electrocardiographic (ECG) monitoring before and after the lavender fragrance stimulation.
? Results
Increases in the parasympathetic tone were observed after the lavender fragrance stimulus as seen as increases in the HF component and decreases in the LF/HF. Additional measurement with positron emission tomography (PET) demonstrated the regional metabolic activation in the orbitofrontal, posterior cingulate gyrus, brainstem, thalamus and cerebellum, as well as the reductions in the pre/post-central gyrus and frontal eye field.
? Conclusions
Lavender fragrance can promote relaxation by depressing sympathetic activity while augmenting parasympathetic activity in normal adults. Our findings suggest a possible use of lavender fragrance to treat patients with various types of autonomic dysfunctions.
THE THERAPEUTIC INTERVENTION FOR PERSONS WITH PROFOUND MULTIPLE DISABILITIES - CASE STUDY USING NEAR-INFRARED SPECTROSCOPY -
Daisuke Hirano1, Takamichi Taniguchi2, Kotaro Takeda3, Hiroyuki Iwasaki1, Hideo Shimoizumi1, Motoko Sugihara2
1International University of Health and Welfare Rehabilitation Center, Otawara, Tochigi, Japan, 2Department of Occupational Therapy, The School of Health Science, International University of Health and Welfare, Otawara, Tochigi, Japan, 3CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
[Background and aims]
Many of persons with profound multiple disabilities (PMD) have difficulty in responding verbally to their families and therapists. Families and therapists recognize their responses through their facial expression, eyes and muscle tones. Moreover, they try to interpret the meaning of their physical responses.
The purpose of this research is to clarify the meaning of individual responses expressed by PMDs from an aspect of brain function, and to examine the methodology of therapeutic intervention for them. The researchers also examined the individual task based on the information of their developmental and medical histories as well. The experiments using Near-infrared spectroscopy (NIRS) were conducted in accordance with their individual tasks. In this research, two cases are studied.
[Methods]
NIRS has been used to enable the researchers to monitor non-invasively variations of concentrations of oxygenated hemoglobin (Oxy-Hb) and deoxygenated Hb (Deoxy-Hb).
First case is a 31-year-old male who has cerebral palsy and mental retardation. He can rotate his neck, and flex and abduct his left shoulder joint. His father is a truck driver, and he used to play cassette tapes constantly whenever he drove with his son on the passenger's seat. The tapes were of Enka (Japanese traditional popular songs). In addition, he can make a vivid response to some particular call. Tasks to be measured are (1) Enka songs which are very familiar to him, (2) the particular call to which he had responded vividly, and (3) other calls to which he has not cared. Data on NIRS were recorded using optical topography system (ETG-4000, Hitachi Medical) with 22 channels placed on the frontal lobe.
Second case is a 5-year-old girl with hypoxic brain damage sequela, mental retardation, and epilepsy. She has visual acuity of 0.03 (naked-eye) and 0.4 (with glasses), and auditory acuity of Rt60db and Lt70db. Cervical rotation is possible for her. Her therapists can not observe the change in her everyday life with or without glasses on. The task to be measured is her responses to the therapists' varied voices with eye contact with or without her glasses. Data on NIRS were recorded using optical topography system with 24 channels placed on both lobes-frontal and occipital.
[Results]
In case 1, an increase of Oxy-Hb was measured in the same specific channel for Enka and voice clearly given. However, and increase of Oxy-Hb was not noticed for voice indefinitely given.
In case 2, with glasses on, an increase of Oxy-Hb was measured with most of the channels compared with naked eyes.
[Conclusion]
The data of NIRS can indicate the task for a necessary therapeutic intervention of PMDs.
The task to be measured for NIRS was specified through close observation by family members and therapists, developmental history, medical information and other significant factors.
The result of this measurement should be adopted to the therapeutic intervention in daily life. By embracing those results, family members and therapists can easily recognize the subjects' (the man and the child) behavioral changes to bring forth better understanding.
DISTAL MIDDLE CEREBRAL ARTERY OCCLUSION MODEL FOR REAL TIME OPTICAL MONITORING OF CEREBRAL BLOOD FLOW AND METABOLISM
Hwa Kyoung Shin1, Sean I. Savitz1, Christian Waeber1, Michael A. Moskowitz1, Cenk Ayata1,2
1Stroke and Neurovascular Regulation Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA, 2Stroke Service and Neuroscience Intensive Care Unit, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA
Background and aims: Optical imaging of cerebral cortex has recently gained momentum with the advent of novel technologies such as laser speckle flowmetry (LSF), multispectral reflectance imaging (MSRI), intrinsic optical signal imaging, and multiphoton confocal microscopy. Here we describe the technique and characterize the hemodynamic, electrophysiological, histological and behavioral outcome features of a distal middle cerebral artery occlusion (dMCAO) model where ischemia and reperfusion can be reproducibly achieved during real-time optical imaging.
Methods: Isoflurane-anesthetized, intubated and ventilated mice (C57BL/6) with arterial blood gas and pressure monitoring are placed in a stereotaxic frame, and dorsal skull surface is exposed. After stripping of the temporalis muscle, distal MCA is exposed via a small burr hole in the temporal bone just above the zygomatic arch, and occluded using a microvascular clip. LSF and MSRI are simultaneously and continuously performed before and during dMCAO. Reperfusion is achieved 60 min later by careful removal of the clip, and confirmed by imaging. Infarct volume and tissue swelling are assessed at 24 and 48 hr (TTC), and 1 month (H&E). Neurological deficits are longitudinally assessed using 5-point, corner and cylinder tests (n=5).
Results: Infarcts were limited to the dorsolateral cortex sparing the striatum. Infarct volumes were significantly larger at 48h compared to 24 hr after reperfusion (21+/−2 vs. 10+/−2 mm3, n=5 and 8, respectively, p<0.05; +/−SEM). By overlying the dorsal view of the cortical infarct at 48h (TTC) and the dorsal CBF map 60 min after dMCAO (LSF), we determined that residual CBF less than 28+/−2% of preischemic baseline resulted in cerebral infarction (Fig). Ischemic tissue swelling (ipsilateral-contralateral hemisphere) was detectable at 24h (6.2+/−1.0 mm3) and tended to increase at 48h (8.1+/−2.4 mm3). There were 2.8+/−0.4 peri-infarct depolarizations/h, as determined by spreading hypoperfusion waves on LSF and confirmed by electrophysiological recordings using glass micropipettes. Neurologically, cylinder test showed reduced use of contralateral paw during a full rear; this motor deficit fully recovered between days 7 and 14. Corner test was more sensitive at later time points, where an ipsilateral preference was detected at 28d. The traditional 5-point grading scale did not demonstrate deficits at any time point after occlusion.
Conclusions: Real time optical and electrophysiological monitoring of focal cerebral ischemia is feasible in a dMCAO mouse model and may be useful for examining the impact of specific genes on cerebrovascular function.
This work was supported by the American Heart Association (0335519N, Ayata; 0475008N, Savitz), NIH (P50 NS10828, Moskowitz).
DOPAMINERGIC REGULATION OF CONSCIOUS EXPERIENCE
Hans Lou1, Pedro Rosa1, Joshua Skewes1, Troels Kjaer2, Albert Gjedde1
1CFIN, Aarhus University, Aarhus, Denmark, 2Department Clinical Neurophysiol, Copenhagen University, Copenhagen, Denmark
In attention deficit hyperactivity disorder (ADHD), both conscious experience and dopaminergic activity are low (1), and in yoga nidra meditation high (2). We propose that the link is causal, and that dopaminergic activity regulates conscious experience. We show with [11C]raclopride PET in eight healthy, young, right-handed female volunteers that visuo-verbal conscious experience is linked to dopamine release in the striatum: Presenting each of 24 words 12 times for 43 ms did not result in conscious experience from viewing words, as ascertained by interview after the scanning. Conscious experience arose when subjects viewed each of 24 words six times for 86 ms during 60 min scanning session. The order was counterbalanced, and word presentation was terminated by masking. The mean pB of the five striatal regions tested was 3.47 for the “unaware” condition and 2.38 for the “aware” condition, reflecting a rise in dopamine D2 receptor occupancy from 12.5 % in the unaware condition to 16% in the aware condition (P=0.03, two-tailed t-test).
The effect on visual awareness of D1/D2 receptor stimulation with oral pergolide was examined in a double blind study of 14 healthy, young, right-handed female volunteers. We recorded how well 3–4 letter words were subjectively experienced as seen on a four-point scale from 0 (not seen at all) to 3 (completely and clearly seen), with 1 and 2 in between. Recorded immediately after each test, the ratings for 16, 30, and 50 ms presentation times, terminated by masking (placebo in brackets), were 1.62 (1.44), 2.11 (1.90), and 2.58 (2.38). Pergolide differed from placebo with P= 0.05 (repeated measures ANOVA). Each data point included 40 words. The forced choice measure of recognition memory was unchanged on pergolide. The test upheld the hypothesis of dopaminergic regulation of conscious experience. The hypothesis explains how disturbed conscious experience with delusions and hallucinations is prevalent in conditions with dysregulation of synaptic dopamine concentration, such as may be the case in schizophrenia and drug abuse.
PATHOPHYSIOLOGY OF TRANSIENT DIFFUSION-WEIGHTED MRI LESIONS IN POST-STROKE SEIZURES: CHANGES IN LOCAL CEREBRAL BLOOD FLOW AND GLUCOSE METABOLISM
Hiroshi Moriwaki1, Naoaki Yamada2, Takuya Hayashi2, Yukio Sugiyama1, Kazuhito Fukushima2, Kotaro Miyashita1, Hiroaki Naritomi1
1Department of Cerebrovascular Medicine, National Cardiovascular Center, Suita, Osaka, Japan, 2Department of Radiology, National Cardiovascular Center, Suita, Osaka, Japan
BACKGROUND AND AIMS: Following seizures, focal abnormality may be observed transiently on diffusion-weighted MRI (DWI). However, detailed mechanisms of such transient high signal intensity lesion (THL) manifestation have yet remained unclear. In order to clarify the clinical significance and pathogenetic mechanism of THL, we performed SPECT/PET studies in association with MRI in patients with post-stroke seizures.
METHODS: During the last 8 years, 75 patients with post-stroke seizures underwent MRI studies in association with Tc-99m HMPAO SPECT and/or F-18 FDG PET in the ictal and/or peri-ictal phases. In 21 of them (12 males, mean age 69.8 yrs), THL was observed on DWI. Thirteen of 21 patients had status epilepticus. MRI was performed repetitively after adequate control of seizures. Tc-99m HMPAO SPECT was performed at the same period as initial MRI in all the patients, and F-18 FDG PET was undertaken in 9 of them. On both HMPAO and FDG images, the count ratio of abnormal high-uptake area to the contralateral homologous area (L/N ratio) was calculated.
RESULTS: A total of 43 THLs were observed in 21 patients. In 10 patients, single THL was located in areas surrounding the old cerebral infarction or hemorrhage. In the other 11 patients, multiple THLs were observed; 29 of 33 lesions were located remote from the old stroke area, such as the thalamus (n=6), hippocampus (n=8), cingulate gyrus (n=5) and cerebral cortices (n=10). Multiple THLs were more frequently associated with status epilepticus than single THL (10/11 vs. 3/10, P<0.01) and eventually resulted in infarct-formation at the hippocampus in 4 patients. HMPAO demonstrated flow increase in all patients. The L/N ratio of FDG was significantly higher than that of HMPAO (1.47+/−0.21 vs. 1.17+/−0.14, P<0.001).
CONCLUSION: The present results suggest that multiple THLs following seizures usually manifest in the limbic system such as the thalamus or hippocampus relating with the severity of seizures. The manifestation of transient DWI lesions may be attributable to excessive metabolic increase accompanied by flow increase.
SPECTRAL CONTENT OF THE ONGOING BOLD FMRI ACTIVITY IS MODULATED BY RESTING STATE-TYPE
Mark McAvoy, Giovanni d'Avossa, Marcus E. Raichle, Linda Larson-Prior
Washington University School of Medicine, Saint Louis, MO, USA
Background
In functional imaging, activations are defined against a resting state of eyes closed, eyes open, or maintained fixation. It has become clear that baseline BOLD signals display periodic fluctuations. Furthermore, it is well known that ongoing neural activity in the resting state is modulated by state-type. Specifically the spectra of alpha band EEG/MEG signals are modulated by resting state-type [1]. This abstract reports effects of state-type on the spectral content of ongoing fMRI BOLD activity.
Methods
Ten subjects participated in a BOLD fMRI experiment comprised of nine 5.5 minute runs: 3 eyes open, 3 eyes closed, 3 fixation. A general linear model was fit to each subject's detrended data. Regressors included a constant term and cosinusoids modeling frequencies from one full cycle to the Nyquist limit. For each subject, root mean square amplitude power was computed for each frequency and entered into a group analysis: voxel-wise repeated measures ANOVA with random factor subject and fixed factors frequency and state-type. The voxel-wise main effects (frequency, state-type) and interaction were corrected for multiple comparisons [2].
Results
The voxel-wise main effect of frequency showed the spectra of the entire brain to be nonwhite, replicating a known BOLD phenomenon. The main effect of state-type highlighted a subset of regions found within the state-type by frequency interaction, thus we report the latter. Regions whose spectra were modulated by state-type were found in primary sensorymotor cortex, striate and extrastriate visual cortex, auditory cortex, and parahippocampal areas (Figure 1). Across all regions, the eyes closed condition was associated with greater modulation of the ongoing BOLD signal than eyes open and fixation conditions in three frequency bands: 0.006–0..02 Hz, 0.02–0..035 Hz, and 0.035–0..045Hz. Representative spectra shown in Figure 2.
Conclusions
The power spectrum of ongoing BOLD activity shows significant effects of resting state-type. Furthermore, these effects are similar to effects of state-type on the spectra of alpha band EEG/MEG signals [1]. This suggests a possible electrophysiological correlate of ongoing resting state BOLD activity.
Regions with significant effects of frequency and state-type on the amplitude of the ongoing BOLD signal were limited to early sensory, motor regions and limbic structures. Ongoing activity in higher order regions was not significantly modulated by resting state-type.
ELECTROPHYSIOLOGICAL EVIDENCE FOR LONG_TERM DEPRESSION IN THE CONTRALATERAL, INTACT BRAIN FOLLOWING TRANSIENT FOCAL CEREBRAL ISCHEMIA IN RATS
E-Jian Lee1, Sheng-Yang Huang1, Yu-Hsiang Kuan1, Tsung-Ying Chen2, Hung-Yi Chen3, Tian-Shung Wu4
1Department of Surgery, National Cheng Kung University Medical Center, Tainan, Taiwan-R.O.C., 2Department of Anesthesiology, Buddhist Tzu-Chi University and Buddhist Tzu Chi General Hospital, Hualien, Taiwan-R.O.C., 3Institute of Pharmacy, China Medical University, Taichung, Taiwan-R.O.C., 4Department of Chemistry, National Cheng Kung University, Tainan, Taiwan & National Research Institute of Chinese Medicine, Taipei, Taiwan-R.O.C.
Introduction: Focal brain injury may induce synapto-functional, metabolic and electrophysiological dysfunction in the contralateral areas [1]. We hypothesize that contralateral electrophysiological diaschisis may represent a form of long-term depression after injury.
Methods: Male Sprque-Dawley rats, weighing 240-280 gm, were subjected to intraluminal suture occlusion for 60 minutes or sham-occlusion. Physiological parameters, including core temperature, local cortical perfusion and arterial blood gases, were measured, whereas the somatosensory potentials (SSEPs) evoked from individual forepaw and hindpaw regions were recorded prior to the ischemic insult and at 1, 3, 7, 21 or 28 days after reperfusion. Postmortem brain ischemic damage was determined by quantitative image analysis of Nissl-stained sections.
Results: The sham-operated group did not cause a significant change in the physiological parameters or the SSEP waveforms recorded either in the ipsilateral or the contralateral brain hemispheres at various intervals of recording. There was no difference in brain infarction volumes among each time interval of the ischemic groups (p > 0.05). Relative to the baseline data, at 24-72 hours of reperfusion the SSEPs recorded from ischemic fore- and hindpaw cortical fields were depressed and the amplitudes decreased to 36.4–41..3% and 41.9–44..5% of baseline, respectively (p < 0.001). On contrast, the SSEPs recorded from non-ischemic fore- and hindpaw cortical fields decreased. The amplitudes decreased to 76.0–83..0% and 74.5–81..8% of baseline, respectively (p < 0.001). At 7-28 days of reperfusion improved SSEPs recorded from ischemic fore- and hindpaw cortical fields were noted. The amplitudes recovered to 49.9–54..7% and 45.5–58..5% of baseline, respectively (p < 0.001). On contrast, the SSEPs recorded from non-ischemic fore- and hindpaw cortical fields were also noted to improve. The amplitudes recovered to 88.6–96..2% and 85.5–96..3% of baseline, respectively (p < 0.001 up to 21 days of reperfusion). Relative to the pooled sham-operated controls, the amplitudes of SSEPs recorded from both ischemic fore-and hindpaw cortical field in the ischemic animals significantly but gradually improved by 18.3% (p < 0.001) and 16.6% (p < 0.001) of baselines, respectively, during a course of 28-day recovery. On contrast, the amplitudes of SSEPs recorded from both non-ischemic fore- and hindpaw cortical field in the ischemic animals also significantly improved by 17.9% (p < 0.001) and 10.4% (p < 0.001) of baselines, respectively, up to 21 days of reperfusion.
Conclusions: Cerebral ischemia-reperfusion induces time-dependent electrophysiological depression not only in the ischemic, but also in the contralateral, intact brain, and both the events, however, improve over time. This electrophysiological evidence suggests long-term depression in the contralateral, intact brain and functional re-organization following cerebral ischemia-reperfusion.
NON-MAGNETIC WHISKER STIMULATION FOR FMRI AND NEUROPHYSIOLOGICAL STUDY
Basavaraju G. Sanganahalli, Peter Herman, Fahmeed Hyder
Department of Diagnostic Radiology, Yale University, New Haven, CT, USA
The use of animal models for simultaneous fMRI and neurophysiology has become important for neuroscience studies. In the rat, many studies have utilized different stimulation devices to characterize the whisker deflection to barrel response for fMRI and elctrophysiological studies. It is extremely important to generate stimuli with identical sensory effects both inside (fMRI) and outside (neurophysiology) the magnet. Air-puffs were generated from pulses of compressed air, which could be delivered in a computer-controlled way by inbuilt solenoid unit. Under these conditions, the air puff stimuli deflected all the whiskers by 2 mm (rostral-caudal) which were glued with a tiny adhesive tape. Whiskers stimuli were presented at 4-40Hz for 30 s duration. All fMRI data were obtained on a modified 11.7T Bruker horizontal-bore spectrometer. The images were acquired with gradient echo EPI sequence (TR/TE= 1000/12 ms). Whisker stimulation induced positive BOLD response in the contralateral barrel cortex (Fig) with a maximum response at 12 Hz (top; 4, 8, 12 Hz and bottom; 20, 30 and 40 Hz). For approximately the same location in the brain, the same stimulus (each frequency) produced similar activation patterns across different subjects. The fMRI results were in agreement with neurophysiology data from pO2 and LDF probes reflecting tissue oxygenation and perfusion changes, which together give rise to the BOLD signal change. While this device provides easy control of frequency of stimulation, we are currently investigating amplitude variations. In summary, unilateral whiskers stimulation with air puffs resulted in significant increases in neurophysiologic signals in the contralateral barrel field in agreement with prior findings.
ACKNOWLEDGEMENTS
Supported by NIH (DC-003710, MH-067528) and OTKA (T34122) grants.
WHICH IS CORRELATED WITH CLINICAL FEATURES, SEVIERITY OR EXTENT OF LESION IN ALZHEIMER'S DISEASE IN SPECT?
Akiko Ishiwata1, Sunao Mizumura2, Shin Kitamura1, Yasuo Katayama1
1Department of Neurology, Nippon Medical School, Tokyo, Japan, 2Department of Radiology, Nippon Medical School, Tokyo, Japan
BACKGROUND AND AIMS: We have reported perfusion decline in the posterior cingulate gyrus, the precuneus and the hippocampus in the very early stage of Alzheimer's disease (AD) as a predictive marker for progression. This study is to evaluate whether clinical severity is correlated with extent and severity of these lesions in SPECT in AD. METHODS: Forty-one patients (13 males, age 71 +/− 7.8 yrs, mean +/− SD) with AD according to NINCDS-ADRDA underwent 123I-IMP SPECT and neuropsychological examinations including Mini Mental State Examination (MMSE), Alzheimer's Disease Assessment Scale Japanese version (ADAS-J cog), Wechsler Adult Intelligence Scale-Revised (WAIS-R) at initial visit. Image sets were transformed to the bicommissural stereotactic coordinate system, and regional anatomical activities were examined by three-dimensional stereotactic surface projections (3D-SSP). An abnormal Z-score was defined as >1.64 (corresponding to p<0.05). The extent of lesions was defined as a total number of the coordinates with abnormal Z-score in the cerebral hemisphere, and severity as an average Z-value in the same region. Spearman correlation coefficients were used to evaluate whether clinical severity was correlated with extent and severity of these lesions on 3D-SSP. RESULTS: All patients underwent MMSE (mean score 16.3, range 3-25) and WAIS-R (mean score 79.3, range 43-110), and 21 patients underwent ADAS-J cog (mean score 21.0, range 8.3–40..7). The extent was significantly correlated with ADAS-J cog score (r = 0.62, p = 0.02), however, the severity showed no correlation (r = 0.31, p = 0.24). CONCLUSIONS: Diffuse rather than focal perfusion abnormality reflects progression of clinical symptom in AD.
SELECTIVE NEURONAL DEATH AND PARAMAGNETIC EFFECT AFTER ISCHEMIC STRESS
Masayuki Fujioka1, Toshiaki Taoka2, Yoshiyuki Matsuo3, Kenichi Mishima4, Kumiko Ogoshi5, Yoichi Kondo6, Masakazu Tsuda4, Michihiro Fujiwara4, Takao Asano7, Toshisuke Sakaki8, Akihiro Miyasaki8, Akio Asai1, Keiji Kawamoto1, Darren Park9, Bo Siesjo10
1Department of Neurosurgery, Kansai Medical University, Hirakata, Osaka, Japan, 2Department of Radiology, Nara Medical Univeristy, Kashihara, Nara, Japan, 3Developmental Research Laboratories, Shionogi & Co., Ltd., Toyonaka, Osaka, Japan, 4Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan, 5Department of Public Health, Nara Medical Univeristy, Kashihara, Nara, Japan, 6Department of Neuroscience, Okayama University Medical School, Shikata, Okayama, Japan, 7Department of Neurosurgery, Saitama Medical School and Center, Kawagoe, Saitama, Japan, 8Department of Neurosurgery, Nara Medical University, Kashihara, Nara, Japan, 9Neuroscience Center, The Queen'S Medical Center, University of Hawaii, Honolulu, HI, USA, 10Neuroscience Center, Lund University, Lund, Sweden
The delayed neuronal death after brain ischemia has been well investigated in the pyramidal neuronal death in the hippocampal CA1 region and in the neurodegeneration of the thalamus and substantia nigra after cerebral cortical ischemia (known as remote effect). Recently, we have reported the novel type of delayed ischemic neuronal death that is indicated by magnetic resonance imaging (MRI) (Ann Neurol 2003;54:732-747, Stroke 2000;31:797-798, Stroke 1999;30:1975-1977, Stroke 1999;30:1038-1042, Stroke 1999;30:1043-1046, Stroke 1997;28:584-587, Stroke 1994;25:2091-2095).
Transient global brain ischemia, hypoglycemic coma, and brief focal ischemia lead to the primary neuronal damage in the areas that are subjected to the transient energy failure in humans and rats (hippocampus, striatum, cerebral cortex, substantia nigra and thalamus). Subsequently, the slowly-progressive secondary neurodegeneration and chronic glial activation (astrocyte and microglia) develop in these areas. The chronic brain inflammation, long-lasting oxidative stress and extracellular accumulation of amyloid precursor protein (APP) are involved in this slowly-progressive neurodegeneration. Furthermore, manganese (Mn), a paramagnetic metal, increases in the ischemic areas. The Mn is neurotoxic and can provoke the secondary neurodegeneration. MRI detects the on-going process of this delayed ischemic neurodegeneration associated with paramagnetic effect. We report the experimental and clinical data regarding this delayed ischemic neurodegeneration that results in the cognitive decline of late-onset in humans and rats.
We suggest that 1) the hyperintensity on T1-weighted MRI after ischemic stress involve tissue Mn accumulation accompanied by Mn-superoxide dismutase and glutamine synthetase inductions in reactive astrocytes, 2) the MRI changes correspond to slow neurodegeneration with a chronic inflammatory response and signs of oxidative stress, and 3) the subjects with these MRI changes are at risk for showing a late impairment of brain function even though the transient ischemia is followed by total neurological recovery.
A PET [18-F]ALTANSERIN STUDY OF 5-HT2A RECEPTOR BINDING IN THE HUMAN BRAIN AND RESPONSES TO PAINFUL HEAT STIMULATION
Ron Kupers1,2, Vibe Frokjaer3, Arne Naert1, David Erritzoe3, Henrik Kehlet1, Gitte Knudsen3
1Department for Surgical Pathophysiology, Rigshospitalet, Copenhagen, Denmark, 2PET Unit, Rigshospitalet, Copenhagen, Denamrk, 3Neurobiology Research Unit, University Hospital Rigshospitalet, Copenhagen, Denmark
Background: There is strong evidence that serotonin plays an important role in pain regulation. One source of serotonin pathways in the CNS arises from the raphe nuclei from which the major descending pain modulatory pathways originate. A second source arises from the medullary, pontine and mesencephalic reticular formation and innervates an extensive (sub)cortical network, including the thalamus, basal ganglia, anterior cingulate and prefrontal cortex, amygdala and hippocampus. This places the serotonin pathways in a very strategical position to regulate pain processing. No PET study has yet investigated possible correlations between serotonin binding and pain responsiveness. In this study we assessed the relation between baseline 5HT2A binding and response to noxious heat stimulation in a group of young healthy volunteers.
Methods: Twenty-two healthy subjects (32.2+8.9 y), 12 males and 8 females underwent a 40 min PET scan using a steady-state regime with the 5-HT2A antagonist, [F-18]altanserin. PET images were co-registered to 3 Tesla MRI's that were acquired for each individual subject, and ROIs were applied automatically by applying a predefined MRI-based template onto the individual MRIs and PET images. Cerebellum was used as a reference region valid for non-specific binding alone. The binding potential of specific tracer binding (BP1) was used as the outcome measure and calculated for 19 regions of interest (ROI). Response to painful stimulation was tested in a separate quantitative sensory testing session, carried out several months after the PET scanning. Responses to both phasic and tonic heat stimulation were assessed. Heat stimuli were administered to the upper leg with a peltier element-based thermal stimulator (probe surface 3×3cm). In each participant we measured the pain threshold, pain tolerance, response to a series of phasic heat stimuli of 5 s and response to a 7-minute tonic painful heat stimulus. Pain intensity and pain unpleasantness were rated following (phasic) or during (tonic) stimulation.
Results: None of the regions showed a significant correlation between [F-18]altanserin binding and pain threshold. In contrast, [F-18]altanserin BP in sensory-motor (r=0.59;P=0.01), parietal (r=0.48;P=0.05) and posterior cingulate (r=0.52;P=0.03) cortices correlated significantly with pain ratings on the 47oC phasic stimulus. In addition, a negative correlation (r=-0.69;P=0.002) was found between [F-18]altanserin BP in the caudate nucleus and pain tolerance. Significant correlations were also found between tonic pain ratings and [F-18]altanserin binding in orbitofrontal (r=0.52;P=0.03) and sensory-motor (r=0.56;P=0.02) cortices or response to tonic heat pain.
Conclusion: Our data show suggest that 5-HT2A receptors in both cortical and subcortical structures may co-determine an individual's response to pain. The correlation between [F-18]altanserin binding in the caudate nucleus and pain tolerance provides a neurochemical basis for behavioral findings of a role of this area in pain tolerance. The correlation between [F-18]altanserin binding and tonic pain ratings suggests a possible role of this area in pain coping mechanisms.
IN VIVO CHARACTERIZATION OF NEURONAL METABOLISM AFTER STROKE IN RATS AS STUDIED BY 1H/13C MRS
Jet P. van der Zijden1, Robin A. De Graaf2, Rick M. Dijkhuizen1
1Image Sciences Institute, University Medical Center Utrecht, Utrecht, The Netherlands, 2Department of Diagnostic Radiology, Magnetic Resonance Research Center, Yale University School of Medicine, New Haven, CT, USA
Background and Aims
Acute loss of sensorimotor function after stroke is often followed by spontaneous functional recovery, which may be correlated with survival or recovery of neuronal tissue. Metabolic changes in neurons play a major role in brain pathophysiology, however, these changes have been incompletely characterized in relation to neuronal death, survival and recovery after stroke.
Dynamic in vivo 1H/13C magnetic resonance spectroscopy (MRS) of incorporation of 13C-label into MRS-detectable glutamate (Glu) and glutamine (Gln) during infusion of 13C -labeled glucose can provide unique information on changes in glucose metabolism and glutamergic neurotransmission.1 The aim of the study was to assess differences in brain metabolism in lesion core, lesion borderzone and unaffected brain areas after experimental stroke in rats, using in vivo 1H/13C MRS.
Methods
Transient focal cerebral ischemia was induced in male Wistar rats (n = 3) by 90-minutes intraluminal occlusion of the right middle cerebral artery.2 At three weeks after stroke, in vivo MR was performed on an 11.7 T horizontal bore MR sytem (Bruker BioSpin). Ischemic lesions were characterized using T2-weighted MRI. In addition, repetitive two-dimensional 1H-observed/13C-edited MRS imaging (MRSI)1 of a column through dorsal ipsi- and contralateral cerebral cortex was performed during infusion of 13C ?labeled glucose (spatial resolution: 1 ′ 2 ′ 5 mm3; temporal resolution: 11.1 minutes). From these experiments, dynamic incorporation of the 13C label into metabolic products Glu and Gln was measured.
For data analysis, ipsilesional MRSI voxels were divided in three different regions based on T2 data: lesion core (T2 > mean contralateral T2 + 2SD); lesion border (voxel adjacent to T2-lesion area); healthy tissue. Homologous contralateral MRSI voxels served as control areas. Good sensitivity and optimal spectral resolution of the 1H/13C MR spectra allowed separate detection of [4-13C]Glu and [4-13C]Gln signals using the LCModel approach.3 Glu and Gln turnover were determined from [4-13C]Glu and [4-13C]Gln time curves in ipsi- and contralateral MRSI voxels.
Results and Conclusions
In contralesional volumes, clear Glu and Gln turnover was observed. At three weeks after stroke, there was significant loss of Glu or Gln turnover in the lesion core (9 ± 14% and 25 ± 25%, resp., as compared to contralateral), reflective of severe neuronal damage. In perilesional regions, i.e. outside the T2 lesion area, Glu and Gln turnover were reduced (76 ± 11% and 74 ± 23%, resp., relative to contralateral). Thus, despite normal appearance on MRI, diminished Glu and Gln turnover point toward disturbed neuronal metabolism in perilesional tissue.
Our study shows that dynamic in vivo 1H/13C MRSI can detect metabolically compromised tissue that appears unaffected on conventional MRI. Thereby, it may offer a unique tool to obtain detailed insights into neuronal tissue status in relation to functional loss and recovery after stroke.
DOPAMIINERGIC FUNCTIONS IN IDIOPATHIC NORMAL PRESSURE HYDROCEPHALUS
Yasuomi Ouchi1, Teiji Nakayama2, Toshihiko Kanno1, Etsuji Yoshikawa3, Masami Futatusbashi3, Yasuo Tanizaki1, Genichi Sugiura4, Tatsuo Torizuka1
1Positron Medical Center, Hamamatsu Medical Center, Hamamatsu, Japan, 2Department of Neurosurgery, Hamamatsu Medical Center, Hamamatsu, Japan, 3Hamamatsu Photonics KK, Hamamatsu, Japan, 4Department of Psychiatry, Hamamatsu Medical University, Hamamatsu, Japan
Idiopathic normal pressure hydrocephalus (iNPH) is a brain disorder with unique entity, characterized by the clinical triad; gait impairment, dementia and urinary incontinence. Gait disturbance is sometimes reminiscent of parkinsonism, and some patients are indeed responsive to levodopa to some degree. These findings indicate the presence of dopaminergic dysfunction in iNPH, and if so, non-surgical therapeutic approach may be replaced for shunting. Here, we investigated alterations in dopaminergic function by examining the levels of binding of pre- and postsynaptic dopamine radiotracers using PET before and after drainage of cerebrospinal fluid (CSF).
[Methods]
Eight iNPH patients and eight age-matched healthy subjects were examined. The diagnostic criteria for iNPH were based on the presence of clinical triad with different severity, enlarged ventricles revealed by magnetic resonance imaging (MRI), and lumber cerebrospinal pressure less than 20 cm Hg. Using a brain-purpose PET camera (SHR12000, Hamamatsu Photonics, japan), we scanned each subject twice using two radiotracers, [11C]CFT and [11C]raclopride within the same day. After tracer injection, serial PET scans were performed with periodical arterial blood sampling in the [11C]CFT study, and without it in the [11C]raclopride study. Estimation of each binding potential (BP) was based on three compartment curve-fitting analysis using regions of interest method applied on MR and PET images. Analysis of variance was used for within-group comparison, and correlation analysis was performed to examine clinico-pathophysiogical relevance. Three patients in the iNPH group were re-scanned in the same way one week after the continuous drainage of CSF.
[Results]
The [11C]CFT BP in the striatum was the same between groups, whereas [11C]raclopride BP levels in the putamen and nucleus accumbens were significantly lower in the iNPH group than those in the healthy group. There was not a significant difference between the bilateral values. In the iNPH group, [11C]raclopride BP in the dorsal putamen correlated negatively with gait severity (r=0.720, p<0.05), and[11C]raclopride BP in the nucleus accumbens correlated positively with emotional recognition score (r=0.727, p<0.05). MRI-based structural changes in iNPH patients failed to correlate with [11C]raclopride BP in any regions. Putaminal [11C]raclopride BP after drainage in iNPH patients was higher than that before the treatment.
[Discussion]
The present study indicated a preservation of the dopaminergic presynaptic function and in contrast a reduction of postsynaptic function in iNPH. This observation is interestingly opposite to the finding in early-staged PD patients with reduced [11C]CFT and normal or higher [11C]raclopride binding. Because the putaminal [11C]raclopride binding correlated with gait difficulty, it was suggested that the postsynaptic dysfunction of the dorsal putamen plays an important role in gait impairment in iNPH. Reduction in CSF by drainage may disinhibit the suppressed postsynaptic dopaminergic function and exert a stimulating effect on gait performance. An investigation of [11C]raclopride BP in the putamen may be a good indicator for evaluating the severity of gait dyspraxia seen in NPH.
[Conclusion]
Alteration in the postsynaptic dopamine function may relate to the pathophysiology of iNPH.
MULTI-MODAL IMAGING OF ACUTE AND CHRONIC UP-REGULATION OF HYPOXIA-INDUCIBLE FACTOR-1 IN TUMORS
Tadashi Miyagawa, Hiroki Namba, Ronald Blasberg
Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Objective: Detection and assessment of tumor hypoxia, blood flow dependent hypoxia (acute) and oxygen diffusion dependent hypoxia (chronic), are important to understand tumor biology for diagnosis and treatment. This study addresses whether in vivo multi-modal imaging systems can be used for assessment of acute and chronic up-regulation of hypoxia-inducible factor-1 (HIF-1) in tumors.
Methods: Multi-modality reporter gene system for imaging using fluorescence, bioluminescence (BLI) and nuclear imaging technique controlled by hypoxia responsive elements (HRE/TGL) was developed and transduced to C6 and RG2 glioma cells. After selection by FACS, the tumor cells were implanted into mice subcutaneously. Constitutively TGL expressing tumor cells were also implanted in the same animals as a positive control. Acute tumor hypoxia was induced by i.p. injection of Hydralazine (HYZ). To investigate the feasibility of BLI to image up-regulation of HIF-1, time-scheduled sequential BLI was performed with or without HYZ treatment. Thereafter, autoradiographic imaging study was applied in the same animals with [18F]-FDG, [3H]-Xanthine and [14C]-FIAU in order to investigate quantitative and special relationship among glucose metabolism, tumor blood flow and HIF-1 expression.
Results: Regional assessment of BLI expressed by relative photon (ratio to 15min value) in HRE/TGL C6 tumor in vivo showed no differences between large and small tumors and plateau phase 10 min after i.p. injection of luciferase, lasting 10 min and decreasing thereafter. HYZ treatment increased the BLI relative photon in the HRE/TGL tumors whereas an increase of BLI relative photon in positive control tumor was marginal. Quantitative evaluation revealed that HYZ reduced tumor blood flow measured by Xanthine from 0.36 to 0.10 (ml/min/g), suggesting that HYZ can induce acute tumor hypoxia by decreasing blood flow. Autoradiographic images showed there was a good spatial relationship between FDG and FIAU uptake in the tumors, indicating that glucose metabolism was enhanced in hypoxic lesion and correlated with up-regulation of HIF-1 in the tumors. Immunostaining with pimonidazole and oxygen regulating protein 150 also revealed tumor hypoxia in the lesion of both increased FDG and FIAU uptake.
Conclusion: Cells transduced with the multimodality reporter system can be used to visualize hypoxic conditions with fluorescence, BLI and radiotracer. Under acute and/or chronic hypoxic conditions, HIF-1 expression in tumors measured by FIAU uptake was observed in blood flow dependent manner. FDG uptake was spatially correlated with HIF-1 expression.
PARIETAL ACTIVATION MODULATES TIME DELAY IN INTER-TEMPORAL CHOICES
Jon S. Wegener1,2, Kristoffer Madsen1,3, Mark S. Christensen1,4, Julian Jamison5
1Danish Research Centre for MR, Copenhagen University Hospital, Hvidovre, Denmark, 2Center for Integrated Molecular Brain Imaging, Copenhagen University Hospital, Hvidovre, Denmark, 3Informatics and Mathematical Modelling, Technical University of Denmark, Denmark, 4Institute of Exercise and Sport Sciences, Copenhagen University, Copenhagen, Denmark, 5Brain and Creativity Institute, University of Southern California, Los Angeles, CA, USA
Humans make many choices that involve evaluating outcomes occurring at different points in time. These evaluations of inter-temporal choices have large individual differences and have many implications for outcomes in health and elsewhere. Although inter-temporal choices have been studied behaviorally in several species, the neural underpinnings have only recently been addressed in two fMRI studies (McClure et al. 2004, 2006). Our aim was to investigate the pre-decision (evaluation) phase of inter-temporal choices to elucidate how the time delay component, independent of choice and reward, is processed in the brain. We hypothesized a graded activation of bilateral parietal areas in response to the processing of longer time delays. We used a simple event related fMRI paradigm with binary choices. Option A was a fixed 700 DKK (1USD=approx. 6 DKK) reward immediately after the scanning session, and option B was either a control condition with an immediate reward of 700 + random noise (+/− 10 DKK), or an equal distribution across three different time delays: one week, one month, or one year. For each time point independently, the amounts for the future options varied adaptively according to the previous choices made by the subject. Subjects were instructed that the computer would randomly pick one of their approx. 200 choices. This amount would be transferred to their bank account at the associated point in time. 20 healthy subjects (15F/5M) were scanned in a Siemens 3T Trio (3×3×3 mm voxels, 40 slices, TR=2.35), measuring pulse and respiration. The paradigm consisted of 2 s trials followed by a 1–6 s. ISI. The scanning session consisted of a structural scan followed by an approx. 20 min trial run. Individual data were analyzed using separate regressors specifying (1) the onsets of the presentation of the control condition, one week, one month, and one year, and (2) first and second order parametric modulations of time and reward, and (3) a set of nuisance regressors based on pulse, respiration and residual movement. Results from group level random effects analysis of the first order modulations of time (n=19, p<=0.05 (FDR)), confirmed our hypothesis, showing graded activations in parietal areas bilaterally. Additional activations were observed in R temporal areas, inferior frontal areas bilaterally, and PCC. The activations in these areas got progressively stronger the shorter the time delay was between option A and B. Finally, we performed exploratory correlation analyses between the discount factor at one year (i.e. the inverse of the option B reward level at which the subject was indifferent to option A) and the slope of the graded activations in the regions identified above. This was significant for multiple areas, including the R parietal (r=-0.74), L parietal (r=-0.44), and the R temporal (r=-0.43). In conclusion, our results indicate that the parietal areas participate both in the evaluation of time delays in inter-temporal choices and that the steepness of activation correlates with the steepness of the individual time-discount function.
OPTICAL PHARMACOKINETICS OF INTRAARTERIAL MITOXANTRONE
Shailendra Joshi1, Roberto Reif2, Mei Wang1, Irving J. Bigio2
1Department of Anesthesiology, Columbia University, New York, NY, USA, 2Department of Biomedical Engineering, Boston University, Boston, MA, USA
Introduction: Intraarterial drug delivery is associated with rapid changes in tissue drug concentrations. The drug transits the cerebral circulation in 1–5 seconds, depending on the animal species. Measurements of the changes in tissue drug concentrations are beyond the time resolution of conventional laboratory techniques such as microdialysis or tissue biopsy. Optical Pharmacokinetics (OP) is a specialized form of diffuse reflectance spectroscopy, which enables the noninvasive real-time measurement of drug concentrations in-situ. We hypothesized that intraarterial delivery of mitoxatrone (MTO) will be enhanced if the drug was injected during cerebral hypoperferfusion (CPH).
Method: The OP method determines drug concentrations by measurement of the wavelength-dependent optical absorption coefficient of the tissue, and can be used for drugs that have an optical absorption band within the wavelength range from near UV to near-IR (300-1900 nm). An optical fiber probe, comprising separate illumination and collection fibers, is placed in gentle contact with the tissue surface, and the tissue beneath the surface is sampled by the diffusely back-scattered light.
Experimental protocol: Preliminary experiments in four rabbits each revealed that there was a need to disrupt the BBB for effective delivery. Ten rabbits were anesthetized by intravenous propofol infusions. Surgical preparation included the cannulation of the ear lobe vein, femoral and internal carotid arteries, skull shaving for the placement of OP and Laser Doppler probes and skull screws to attached the EEG leads. Hemodynamic data was continuously recorded. After the baseline measurements, the BBB of the animals was disrupted by intracarotid injection of mannitol (8 ml, 25% over 40 seconds). Thereafter animals were randomized to receive 3mg of MTO injection with normal blood flow (normal perfusion group) or when the blood flow was significantly decreased by contralateral ICA occlusion and severe systemic hypotension (CPH group). with bolus intravenous injection of esmolol (30 mg) and adenosine(30 mg).
Results: Figure 1 illustrates the changes in hemodynamics and tissue concentrations of MTO in the two groups of animals. Bolus injection of MTO during cerebral hypoperfusion increased the area under the concentration time curve but did not result in a sustained increase in tissue concentrations.
Discussion and Conclusions: The experiments demonstrate the feasibility of measuring tissue drug concentrations using diffuse reflectance spectrometry. OP measurements revealed that tissue concentration of MTO did not significantly increase with induced cerebral hypoperfusion. This is in contrast to dramatic increase in tissue concentrations of lipid soluble drugs like propofol, pentothal, and carmustine that we have observed in the past. Reduction of arterial pressure could adversely effect tissue deposition of water soluble compound by decreasing or even revering the fluid efflux in the capillary bed.
EVALUATION OF SIGMA1 RECEPTORS IN ALZHEIMER'S DISEASE USING [11C]SA4503 PET
Masahiro Mishina1,2,3, Kiichi Ishiwata2, Masashi Ohyama2, Shin Kitamura3,4, Yuichi Kimura2, Kei-ichi Oda2, Shiro Kobayashi1, Yasuo Katayama3, Kenji Ishii2
1Neurological Institute, Nippon Medical School Chiba-Hokusoh Hospital, Imba-Gun, Chiba, Japan, 2Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Itabashi-Ku, Tokyo, Japan, 3Department of Neurology, Nephrology and Rheumatology, Nippon Medical School, Bunkyo-Ku, Tokyo, Japan, 4Department of Internal Medicine, Nippon Medical School Musashi Kosugi Hospital, Kawasaki-Shi, Kanagawa, Japan
OBJECTIVE: To investigate the mapping of sigma1 receptor in Alzheimer's disease (AD) using [11C]SA4503 PET.
METHODS: We studied five AD patients and seven volunteers. A dynamic series of decay-corrected PET data acquisition was performed for 90 min starting at the time of the injection of 500 MBq of [11C]SA4503. A two-tissue two-compartment model was used to estimate K1, k2, k3 and k4. The ratio of k3 to k4 was computed as the binding potential (BP). Unpaired t-tests were used to compare the K1 and BP in patients with AD and normal subjects.
RESULTS: To compare with normals, BP of AD was significantly lower in the frontal, temporal and occipital lobe, cerebellum and thalamus (Figure B), while K1 was significantly lower in parietal lobe (Figure A).
DISCUSSION: Distribution of cortical sigma1 receptors was reduced in patients with AD.
CONTEXTUAL MODULATION OF THE HIGH-RESOLUTION 3T GE BOLD FMRI RESPONSE
Cheryl Olman
Departments of Psychology and Radiology, University of Minnesota, Minneapolis, MN, USA
Background and Aims. Inhibitory neural processes play a key role in human behavior: it is hard to find a perception or behavior that is not modulated by context, and the mechanisms of contextual modulation are frequently, if not dominantly, inhibitory. In human vision, contextual modulation is apparent in the suppression of visual features that are not distinguished from a scene background, those which lack contrast along low-level dimensions such as brightness, orientation or color. We have begun a series of experiments measuring the hemodynamic response to low-level contextual modulation in early visual areas.
Methods. In this particular experiment, four target stimuli (Gabor patches) were presented at 3 degrees eccentricity (one in each quadrant of the visual field). Subjects were required to perform a contrast discrimination task on the target stimuli: the contrast of one of the four Gabors was augmented during one of two stimulus presentation intervals (a standard two-interval forced-choice task). In preparatory studies before the fMRI experiment, this psychophysical measurement allowed us to infer the early visual contrast response function to the target under three conditions: targets alone, targets with parallel flanking Gabor patches that suppressed the early visual response (measured as increased discrimination thresholds), and targets with orthogonal flanking Gabor patches (which did not suppress the early visual response). Responses to the targets were suppressed by 30% when targets presented at 35% contrast were surrounded by 50% contrast parallel flankers. In the corresponding fMRI experiment, the contrast discrimination task motivated a constant level of effort and alertness during all three experimental conditions and allowed experimental verification of the presence of surround suppression during the scanning. For the fMRI experiment, the three experimental conditions were presented in an event-related design with an average inter-trial-interval of 4.5 s. The posterior occipital cortex was imaged using a gradient echo (GE) EPI pulse sequence at 3 Tesla, with 1.5 mm isotropic resolution and a volume repetition time of 1.5 s. A general linear model was used to estimate the event-related response in each voxel to each stimulus type.
Results. The amplitude of the BOLD response to the targets with parallel flankers was not decreased, even though both the psychophysical experiments and the electrophysiological literature indicate neural activity in this region of primary visual cortex is suppressed.
Conclusions. Even considering the possible confounds of hemodynamic blurring, this finding questions the utility of standard GE BOLD techniques for studying contextual modulation on a fine scale. Although there was no observed correlation between BOLD peak amplitude and inferred neural activity in primary visual cortex, the different stimulus configurations elicited interesting differences in the shape of the hemodynamic response: the addition of suppressive flankers modulated the relative contributions of positive and negative portions of the response. Further experiments are investigating what the shape of the hemodynamic response function can tell us about contextual interactions in high resolution BOLD fMRI.
CLINICAL USEFULNESS OF THREE-DIMENTIONAL PREOPERATIVE PLANNING IN NEURONAVIGATION SYSTEM WITH MULTIMODAL IMAGE FUION TECHNIQUE
Noritaka Masahira, Shoko Yamada, Naoki Ikawa, Shinichi Toyonaga, Hiromichi Nakabayashi, Kae Chang Park, Keiji Shimizu
Department of Neurosurgery, Kochi University Medical School, Nankoku, Kochi, Japan
Background: Surgical approach to a brain tumor is usually decided preoperatively based on conventional computed tomography (CT) scan, magnetic resonance imaging (MRI), and angiography, but individualized preoperative assessment of the relationship between critical structures is difficult because of poor visualization of these examinations.
Methods: MRI was SIGNA Horizon 1.5T (GE, Milwaukee, WI) and scanning conditions were as follows; T1-weighted-imaging (T1WI), scanning sequence of Fast SPGR, 1.5mm slice, slice gap of 0mm, TE/TR = 2.8/12 or 4.2/13, Matrix of 256×256, and 240mm field of view. CT scan was Aquilion TSX-101A (TOSHIBA medical systems, Tokyo, Japan) and scanning conditions were as follows; helical multi-slice scanning, 1.0mm slice, helical pitch of 15.0. Navigation system was Vecter Vision2 with plannging software of iPlan Cranial version 2.5 (Brain LAB, Heimstetten, Germany).
Case presentation: Intracranial metastasis from renal cell carcinoma was identified at the pituitary stalk compressing chiasma upward in a 72-year-old woman who complained of visual disturbance. The patient underwent tumor resection in the left pterional approach considering the tumor location of the left side dominancy, however, the surgery finished without touching the tumor due to too small working space. Then, we used preoperative three-dimensional (3D) planning of neuronavigation system with multimodal image fusion soft. The 3D image showed right side deviation of anterior communicating artery with elevated right A1 portion causing enough working space for tumor removal surrounded by right internal carotid artery, A1 segment, and optic nerve. The 3D image also proved the necessity to remove the right orbital roof and zygomatic arch for recognization of the highest attachment of the tumor to the brain tissue, and we performed right orbitozygomatic approach for this case. The operation finished as we planned preoperatively and total resection of the tumor was completed. The visual acuity of the patient recovered very well when she was discharged our hospital one month after the surgery.
Conclusions: Neuronavigation system is important part of both planning and performing intracranial surgery.
THROMBIN AND INTRACEREBRAL HEMORRAHGE-INDUCED NEUROGENESIS IN ADULT RATS
Shuxu Yang, Shuijiang Song, Guohua Xi, Takehiro Nakamura, Richard F. Keep, Julian T. Hoff, Ya Hua
Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA
Background and Aims: Neurogenesis is modulated by both physiological stimuli and pathological conditions, where it may contribute to brain recovery. However, neurogenesis in intracerebral hemorrhage (ICH) has not been investigated. Thrombin formation causes acute brain injury after ICH, but thrombin also can stimulate cell proliferation. The present study examined if neurogenesis takes place in ICH and the role of thrombin in ICH-related neurogenesis.
Methods: Male adult Sprague-Dawley rats were used in this study. This study was divided into four parts. In the first part, rats received either an intracaudate injection of 100-µl autologous whole blood (ICH) or a needle insertion (sham). The rats were killed 1, 3, 7, 14, 30 and 60 days later for Western blot analysis and immunohistochemistry of doublecortin (DCX). In the second part, rats had an ICH or a sham operation, and then received intraperitoneal injections of 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg) at day 7 and day 9 after ICH or sham operation. Brains were perfused at 14 days after ICH to identify BrdU-positive cells. In the third part, rats had an intracaudate injection of 50-µl thrombin (1 U) and brains were sampled for Western blots 3, 7, 14 and 30 days later. In the last part, rat received an injection of 100-µl blood with or without a thrombin inhibitor, hirudin (5 U). The brains were sampled at 14 days for DCX quantitation.
Results: The timecourse of DCX was examined by Western blot analysis. DCX levels in the ipsilateral basal ganglia started to increase as early as seven days after ICH (170 ± 49 % of sham), peaked at 14 days (805 ± 241 % of sham, p < 0.01) and then gradually decreased at one month (437 ± 161 %, p < 0.05) and two months (360 ± 104 %, p < 0.05). At two weeks after ICH, DCX protein levels in the ipsilateral basal ganglia were strongly increased (2722 ± 814 vs. 383 ± 50 pixels in sham, p < 0.01). Immunohistochemistry also demonstrated that DCX immunoreactivity was markedly increased in the ipsilateral subventricular zone and basal ganglia at two weeks after ICH. To confirm if the neuroblasts were newly generated after ICH, we performed immunofluorescence doublestaining for DCX and BrdU. We found that DCX positive cells were BrdU positive. To test the role of thrombin in neurogenesis, one unit thrombin, which does not cause marked brain injury, was injected into the caudate. Thrombin increased DCX levels in the ipsilateral basal ganglia at day 7 (p<0.05). We also found hirudin blocked ICH-induced upregulation of DCX in the ipsilateral basal ganglia (151±94 vs. 2689±837 pixels in the vehicle-treated group, p<0.05).
Conclusions: Our results demonstrated that neurogenesis occurs in the brain following ICH and that thrombin may play a role in ICH-induced neurogenesis.
NOTCH SIGNALING IS ACTIVATED IN ADULT HIPPOCAMPAL NEURONAL REGENERATION AFTER THE TRANSIENT GLOBAL ISCHEMIA
Soichi Oya1, Gakushi Yoshikawa1, Nobuhito Saito1, Takaaki Kirino2,3, Nobutaka Kawahara1,3
1Department of Neurosurgery, The University of Tokyo Graduate School of Medicine, Tokyo, Japan, 2Research Institute International Medical Center of Japan, Tokyo, Japan, 3SORST, Japan Science and Technology Corporation (JST), Tokyo, Japan
Introduction. Notch signaling has been shown to regulate neural stem cells in the developing brain. In general, activation of Notch signaling has two main effects. One is proliferation and maintenance of neural stem cells. The other is inhibition of neural differentiation of neural stem cells (NSC) in favor of their taking glial fate. While these facts have been confirmed first in in vitro settings and developing brains, the function of Notch signaling in the adult neurogenesis is recently becoming a big issue to be explained. Along with this idea, we were encouraged to investigate whether Notch signaling is involved in adult neurogenesis in the hippocampal CA1 after transient global ischemia. We also studied whether we could boost the therapeutic effect of growth factor treatment by manipulation of Notch signaling.
Materials and methods. Adult male Wister rats were subjected to 6-minute global ischemia using 4-vessel occlusion and hypotention. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were injected into the lateral ventricles from Day2 to Day5 to promote proliferation of NSCs responding to the ischemic insult. BrdU was injected intraperitoneally to label proliferating cells during this period. Subsequent intraventricular administration of the gamma secretase inhibitor was performed from Day5 to Day11 aiming at Notch signaling inhibition and augmented neuronal differentiation of NSCs. Rats were sacrificed at Day5 and Day28 to evaluate the change of Notch1 positive cells around the posterior periventricle and the NeuN positive cells in the CA1 region, respectively.
Results. The number of NeuN positive cells in the postischemic CA1 region decreased to 6% of that of control rats. The EGF/FGF2 treated rats, however, showed significant 22% increase in the number of NeuN positive cells in the CA1 region. At Day5, triple-fold increase of Notch1 positive cells were observed between the posterior periventricle and CA1 region in treated rats compared to non-treated rats. These cells co-expressed BrdU, Nestin, and Notch1. In rats subjected to gamma secretase therapy subsequent to EGF/FGF2, the number of NeuN positive cells showed small but significant increase of 28% compared to rats treated only by EGF/FGF2.
Conclusion. Increase of Notch1 positive cells along with growth factor treatment in the acute phase of ischemia indicates Notch signaling is involved in neuronal regeneration in the rat hippocampal CA1 after transient global ischemia. We also confirmed administration gamma secretase inhibitor in the subacute stage leads to improvement of cell regeneration. We further need to clarify how this treatment affects neuronal regeneration in adult neurogenesis in the CA1 region to assess whether Notch signaling could be a possible target for the cell regeneration therapy.
CELL THERAPY OF STROKE IN SMALL AND LARGE ANIMALS
Johannes Boltze1,2, Annette Foerschler3, Uwe Richard Schmidt1,2, D.C. Wagner1,2, Daniel Christoph Schwarz4, Christiane Maria Boltze1, Larissa Bulawina2, Andrea Barbara Lang1, Osama Sabri5, Henryk Barthel5, Frank Emmrich1,2, Uwe Gille6,7
1Fraunhofer Institute of Cell Therapy and Immunology, Leipzig, Germany, 2Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany, 3Department of Neuroradiology, University Clinic of Leipzig, Leipzig, Germany, 4Department of Neurology, University Clinic of Leipzig, Leipzig, Germany, 5Department of Nuclear Medicine, University Clinic of Leipzig, Leipzig, Germany, 6Institute of Veterinary Anatomy, University of Leipzig, Leipzig, Germany, 7VITA 34 INc., Leipzig, Germany
Background and aims
Human umbilical cord blood (HUCB) and bone marrow (BM) stem cells as a stroke treatment is a recent scientific topic. However, little is known about the cellular mechanisms leading to neurofunctional improvement. We evaluated the necessity of CD34+ cells among the transplant and the time window of HUCB cell administration. A comparison between adult vs. fetal neural stem cells (NSC) and local vs. systemic administration was performed. Finally, efficacy of autologous BM transplantation was studied in a novel sheep model.
Methods
139 male rats were assigned to groups as given in figure 1. Stroke was induced by middle cerebral artery occlusion (MCAO). 106 cells were administered intravenously or 3×105 cells were injected locally (striatum). Behavioural outcome was observed until day 29 using common tests (RotaRod, Beamwalk). MRI was performed at days 1, 8 and 29 followed by immunohistochemistry.
19 Merino rams were assigned either to a cell therapy (n=8) or control group (n=11). Cell therapy group received 4×106 autologous mononuclear BM cells per kilogram bodyweight 24 hours upon MCAO. Behavioural phenotyping and MRI were performed until day.
Results
Surprisingly, CD34- and CD34+ samples had nearly the same therapeutic effect. HUCB and BM derived cells were detected in the border zone of the lesion. Cell treatment was superior to saline injection (p<0.01). T-cell transplantation and HUCB cell treatment after 14 days failed to induce functional improvement. T-cell administration was even inferior as compared to controls (p<0.05). Local administration did not show superiority. Neural differentiation was only observed upon local NSC administration while reduction of gliosis/lesion size and neurofunctional improvement were observed in all groups receiving sufficient cell treatment, also in the large animal arm.
Conclusion
Results are consistent with the hypothesis that HUCB cells, BM cells and NSCs provide an excellent cell-based treatment for stroke. CD34+ is not an appropriate marker to characterise the responsible cells. Neural differentiation or local administration are not necessary for functional improvement and lesion size reduction. In sheep autologous BM cell therapy is beneficial up to 24 hours after MCAO. Therapeutic success in rodents was observed in a time window of 72 hours.
THE LOCALIZATION OF PHOSPHO-CREB IN NEUROBLASTS IN ISCHEMIA-INDUCED STRIATAL NEUROGENESIS
Tsutomu Sasaki, Kazuo Kitagawa, Emi Omura-Matsuoka, Kenichi Todo, Yasukazu Terasaki, Shiro Sugiura, Yoshiki Yagita, Masatsugu Hori
Division of Stroke Research, Department of Cardiovascular Medicine, Osaka, Japan
Background: Stroke leads to markedly enhanced neuronal progenitor proliferation in the subventricular zone and induce migration toward injured striatum. In hippocampal neurogenesis, the cAMP-CREB pathway regulates multiple aspects of adult hippocampal neurogenesis. Also, in the normal SVZ-Olfactory bulb, the implication of cAMP-CREB cascade has been elucidated. Neurotrophins play crucial roles in adult neurogenesis following ischemia. Brain-derived growth factor and insulin-like growth factor promote adult neurogenesis, and both growth factors possess the CRE sequence in their promoters. Therefore, we examined the profiles of phospho-CREB (pCREB) expression in stroke-induced striatal neurogenesis.
Subjects: 1) Transients focal ischemia: Middle cerebral artery was occluded by the intraluminal filament for 30 min. 2) BrdU labeling protocols: BrdU was used to label proliferating cells. BrdU was daily injected starting 2 days after ischemia for 2 weeks. Mice were killed at 1, 7, 14, and 42 days after BrdU injection, transcardially perfused with 4% PFA and immunohistochemistry was performed. Expression of phospho-CREB was evaluated. 3) Rolipram treatment: Rolipram (3 mg/kg, or vehicle, i.p.) was administered 3 days after BrdU labeling for 3 weeks. CREB-CRE cascade activation by rolipram was evaluated by CRE-lacZ transgenic mice. At 42 days after BrdU labeling, the striatal and hippocampal neurogenesis were compared.
Results: Msi-1-positive progenitor cells in the SVZ did not stain for pCREB. Most DCX- or PSA-NCAM-positive neuroblasts in the SVZ showed colocalization of pCREB staining. Also, the marked enhancement of phospho-CREB immunoreactivity was observed in the migrating neuroblasts toward the injured striatum. The treatment of rolipram activated the CREB-CRE signaling, and significantly enhanced the survival of nascent BrdU-positive neurons in the hippocampus, but rolipram only did not significantly enhance the striatal neurogenesis.
Conclusion: The enhanced phospho-CREB expression in neuroblasts plays a crucial role in both hippocampal and injured striatal neurogenesis. These results indicate that activation of cAMP-CREB signaling in combination with neurotrophins may have implications for therapeutic intervention to enhance neurogenesis after ischemia.
INTRAVENOUS TRANSPLANTATION OF MESENCHYMAL STEM CELLS DERIVED FROM PERIPHERAL BLOOD PROTECTS AGAINST CEREBRAL ISCHEMIA
Osamu Honmou, Ryo Ukai, Kiyohiro Houkin
Department of Neurosurgery, Sapporo Medical University, Sapporo, Hokkaido, Japan
Transplantation of mesenchymal stem cells prepared from adult bone marrow (BMSCs) has been reported to ameliorate functional deficits in several CNS diseases in experimental animal models. Despite the fact that the stem/precursor cells in peripheral blood are widely used for reconstruction in the hematopoietic system, it is not fully understood whether peripheral blood-derived precursor/stem cells (PMSCs) can differentiate into a neural lineage. To test the hypothesis that treatment with PMSCs may have a therapeutic benefit in stroke, we compared the efficacy of systemic delivery of BMSCs and PMSCs. A permanent middle cerebral artery occlusion in rat was induced by intraluminal vascular occlusion with a microfilament. Rat BMSCs and PMSCs were prepared in culture and intravenously injected into the rats 6 hours after MCAO. Lesion size was assessed at 6 hours, 1, 3 and 7 days using MR imaging and histology. The hemodynamic change of cerebral blood perfusion on stroke was assessed the same times using perfusion weighted image (PWI). Functional outcome was assessed using the treadmill stress test. Both BMSCs and PMSCs treated groups had reduced lesion volume, improved regional cerebral blood flow, and functional improvement compared to the control group. The therapeutic benefits of both MSC-treated groups were similar. These data suggest that PMSCs derived from peripheral blood could be an important cell source of cell therapy for stroke.
EXPRESSION OF NETRIN-1 AND ITS RECEPTORS DCC AND NEOGENIN IN RAT BRAIN AFTER ISCHEMIA
Atsushi Tsuchiya, Takeshi Hayashi, Kentaro Deguchi, Yoshihide Sehara, Toru Yamashita, HanZhe Zhang, Violeta Lukic, Makiko Nagai, Tatsushi Kamiya, Koji Abe
Department of Neurology, Graduate School of Medicine, Dentistry and Pharmacy, Okayama University, Okayama, Japan
Background: Once the central nervous system (CNS) was severely damaged, neurological deficit would permanently continue because tissue regeneration is quite limited. However, even if new neurons were not provided, a functional recovery should take place if axons grew after injury. It is therefore very important to investigate the mechanism of axonal growth in the ischemic brain in order to consider a novel mean of therapy for stroke. Chemotropic factors are important for axon pathfinding, and a growing number of chemotropic factors have been identified in the past several years. Netrins are chemotropic factors for axon with chemoattractant or chemorepellant guidance activities, and deleted in colorectal cancer (DCC) and neogenin are receptors for netrins.
Method: We examined expression of netrin-1, DCC, and neogenin in rat brain after 90 min of transient middle cerebral artery occlusion (tMCAO). The animals were sacrificed at 1, 7, 14, and 28 days after the ischemia, and immunohistochemistry for these molecules were carried out. In order to identify the cell phenotype which expressed them, we performed double staining for these molecules and cell type markers, which are NeuN, GFAP, and NAGO.
Results: Netrin-1 was expressed in the peri-ischemic area with a peak at 14 days (fig 1). Because axonal regeneration takes place at 14 days after injury, such expression could be involved in promotion of axonal growth after tMCAO. Although the origin of netrin-1 in normal adult brain had remained poorly understood, we showed it was neuron but not astroglial cell after the tMCAO. DCC was expressed both in neurons and astrocytic feet which peaked at 14days (fig 2). Although DCC expressed in neurons plays a role in promoting axonal growth, the role of DCC expressed in astrocytes remains uncertain. It could also be implicated in axonal growth under ischemia. The expression of netrin-1 and its receptor DCC were overlapped both in the spatial and temporal patterns, which indicate that netrin-1 bound to DCC and then could work for axonal growth. Neogenin was expressed in endothelial cells at the MCA territory, which was different from the cell phenotype with netrin-1 expression (fig 3). This suggests that neogenin's angiogenic activity may not have relevance with netrin-1, but rather some other molecules might be implicated.
Conclusion: Netrin-1-DCC system was activated in the ischemic brain, and should be involved in axon growth after injury.
SUBVENTRICULAR ZONE-DERIVED NEUROBLASTS MIGRATE AND DIFFERENTIATE INTO MATURE NEURONS IN THE POST-STROKE STRIATUM
Toru Yamashita1, Mikiko Ninomiya2, Takeshi Hayashi1, M. Nagai1, Tatsushi Kamiya1, Nobuo Araki2, Hideyuki Okano3, Kazunobu Sawamoto4, Koji Abe1
1Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, 2Department of Neurology, Saitama Medical School, Saitama, Japan, 3Department of Physiology, Keio University School of Medicine, Tokyo, Japan, 4Bridgestone Laboratory of Developmental AndRegenerative Neurobiology, Keio University School of Medicine, Tokyo, Japan
In the normal brain, GFAP-expressing cells in the subventricular zone (SVZ) include a neurogenic cell population that gives rise to olfactory bulb neurons only. Herein, we report evidence that these cells can produce new neurons outside the olfactory bulbs after a stroke. SVZ GFAP-expressing cells labeled by a cell-type-specific viral infection method were found to generate neuroblasts that migrated toward the injured striatum. Long-term tracing experiment using a Cre-loxP system revealed the SVZ-derived neuroblasts differentiated into mature neurons in the striatum, in which they formed synapses with neighboring striatal cells.
TRANSFECTINON OF NOTCH-ICD INTO BONE MARROW STROMAL CELLS LEADS TO FUNCTIONAL RECOVERY IN A RAT MODEL OF STROKE
Makoto Hayase1,2, Yasushi Takagi1, Mari Dezawa2, Kazuhiko Nozaki1, Nobuo Hashimoto1
1Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan, 2Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan
OBJECTIVE: We previously demonstrated that Neurons can be specifically induced from bone marrow stromal cells (MSCs) with extremely high efficiency using gene transfection of the Notch intracellular domain (Notch-ICD) and trophic factor administration. We investigated the efficacy of Notch-ICD transfected MSCs transplantation into rats subjected with focal cerebral ischemia. METHODS: Three days before transplantation, permanent focal cerebral ischemia was induced on Wistar rats. Notch-ICD transfected MSCs were locally transplanted into ischemic lateral striatum and cortex. The behavioral analyses were performed on days 4, 7, 10, and 14 after transplantation. RESULTS: transplantation of Notch-ICD transfested MSCs improved the score of modified limb placing test. Histological analysis revealed that transplanted cells expressed Tuj-1 and NeuN as neuronal marker. CONCLUSION: These results suggest that the use of MSCs transfected with Notch-ICD may be a promising therapeutic strategy for cerebral infarction.
CRITICAL ROLE OF PCREB SIGNALING FOR NEUROGENESIS AFTER CHRONIC CEREBRAL HYPOPERFUSION
Ryota Tanaka, Nobukazu Miyamoto, Yuji Ueno, Ning Zhang, Takao Urabe
Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
Background
New neurons are generated throughout life in mammals in the dentate gyrus of the hippocampus and in the subventricular zone (SVZ) of forebrain. The olfactory bulb (OB) of adult mammals is the target of neuroblasts migrating from the SVZ. Newborn cells in the SVZ migrate tangentially in chains toward the OB. The present study was designed to assess the time course of newborn neurons in the SVZ and OB after chronic cerebral hypoperfuions and whether pCREB signaling affects newborn neurons after ischemic insults by the administration of cilostazol, a potent inhibitor of type III phosphodiesterase.
Methods
Rats underwent permanent bilateral common carotid artery ligation (LBCCA). They were divided into three, sham operated (n=45), vehicle (n=45), and the cilostazol treated (n=45) groups. Performance at the Morris water maze task and immunohistochemistry for 5-Bromodeoxyuridine (BrdU), Doublecortin (DCX), microtubule associated protein 2 (Map-2), single stranded DNA (ssDNA) and cAMP-responsive element binding protein phosphorylation (pCREB) were analyzed at 7, 14, 21, and 28 days after cerebral hypoperfusion.
Result
The reduction of OB size were gradually progressed in vehicle group, but not in sham and cilostazol group. At Morris water maze task, the vehicle group showed a significantly longer escape latency than the sham group (p<0.05). Interestingly, the escape latency of the cilostazol group gradually decreased over the testing period compared with the vehicle group (p<0.05). In vehicle group, neuroblasts labeled by DCX and newly generated cells labeled by BrdU in the SVZ significantly increased after ischemia compare with sham operated animals (p<0.05). However, those newly generated cells in the SVZ did not correctly migrate into the rostral migratory stream (RMS) and reach to OB. Apoptotic cells labeled by the ssDNA staining were significantly increased in SVZ, RMS and OB (p<0.05) in vehicle group. In cilostazol group, significant higher number of neuroblasts were seen at RMS and OB compare with vehicle group, moreover, there were fewer number of apoptotic cells compare with vehicle group (P<0.05). CREB phosphorylation in neuroblasts dramatically decreased within the SVZ, RMS and OB in vehicle group (p<0.05), while those expression of pCREB were maintained in cilostazol group as well as sham operated control group.
Conculusion
Previous report showed transient forebrain ischemia increased newly generated cells in the SVZ. Our result suggest that chronic cerebral ischemia also promote the neurogenesis in forebrain. However, those effect did not persist until later period after ischemia. Also our finding indicated that decreased migration and survival of newly generated neuroblasts were result from down regulation of CREB phosphorylation after cerebral hypoperfusion.
VASCULAR ENDOTHELIAL GROWTH FACTOR PROMOTES BRAIN TISSUE REGENERATION WITH A NOVEL BIOMATERIAL POLYDIMETHYLSILOXANE-TETRAETHOXYSILANE
HanZhe Zhang1, Takeshi Hayashi1, Kanji Tsuru2, Kentaro Deguchi1, Mitsuyuki Nagahara2, Satoshi Hayakawa2, Makiko Nagai1, Tatsushi Kamiya1, Akiyoshi Osaka2, Koji Abe1
1Department of Neurology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan, 2Biomaterial Laboratory, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan
Background: Injury of the central nervous system (CNS) of mammals may cause irreparable damage due to the limited regeneration capacity of the tissue. In order for the CNS tissue to regenerate, to supply a scaffold for the newly produced cells to adhere is necessary. Application of biodegradable polymers such as collagen, gelatin, fibrin and alginates has been investigated for organ damage other than the CNS. In this context, we recently produced a novel biomaterial from polydimethylsiloxane (PDMS) and tetraethoxysilane (TEOS). We added the most potent angiogenic factor vascular endothelial growth factor (VEGF) and observed the difference in brain regenerative process in the PDMS-TEOS scaffold.
Methods: Male Wistar rats of 12 weeks old were anesthetized and placed in a stereotaxic apparatus. A piece of bone over the right frontal cortex was excised, and 2 × 5 mm defect in the brain was made by cutting and aspiration. The defect was positioned from 1.0 mm anterior to 4.0 mm posterior of the bregma and 2.0 to 4.0 mm lateral to the midline. The depth of the lesion was 2.0 mm from the brain surface. The block of PDMS-TEOS scaffold, with or without VEGF, was placed into the lesion immediately after making the defect. The animals were sacrificed at 30 days after PDMS-TEOS scaffold implantation. For histological analysis of the newly formed tissue, the sections were stained with HE. In order to identify cell types in the newly formed tissue, we performed immunohistochemical analysis for glial fibrillary acidic protein (GFAP), an astrocyte marker, and N-acetylglucosamine oligomer (NAGO), an endothelial cells marker. In order to determine whether the cells in PDMS-TEOS scaffold were under mitosis, we carried out double fluorescent study for cell phenotype markers and Ki67.
Result: The PDMS-TEOS scaffold remained at the implanted site for 30 days and kept the integrity of brain shape (Fig. A and B). HE staining showed that a small number of cells infiltrated into the PDMS-TEOS scaffold even without VEGF at 30 days after the implantation, but that addition of VEGF significantly increased the number of infiltrated cells NAGO staining showed only a few positive cells in the PDMS-TEOS scaffold without VEGF, but significant increase of endothelial cells with VEGF was confirmed. Immunohistochemical study for GFAP demonstrated that only a few astrocytes were found in the PDMS-TEOS scaffold without VEGF, which was again significantly increased with VEGF. Double staining with proliferation maker Ki67 demonstrated that VEGF significantly increased newly formed astrocytes and endotheial cells, indicating that addition of VEGF accelerated tissue restoration and angiogenesis.
Conclusions: The present study showed that a new porous PDMS-TEOS is a good candidate biomaterial for brain tissue restoration. Endothelial as well as glial cells successfully infiltrated into this biomaterial. Addition of VEGF further promoted new tissue formation.
ENCAPSULATED BDNF-PRODUCING CELL GRAFT HAS A NEUROPROTECTIVE EFFECT AND ENHANCES NEUROGENESIS IN THE DAMAGED HIPPOCAMPUS
Tetsuro Shingo, S. Kuramoto, W. Yuan, A. Kondo, T. Kadota, N. Tajiri, T. Morimoto, H. Yoshida, Y. Miyoshi, I. Date
Department of Neurological Surgery, Okayama University Graduate School, Okayama, Japan
[Background] It has been well known for that brain-derived neurotrophic factor (BDNF) influences the birth of granule cells in the dentate gyrus, which is one of the areas in the brain that demonstrates neurogenesis throughout life, and also contributes to the development of temporal lobe epilepsy (TLE). Encapsulation of cells has the potential to provide a protective barrier against host immune cell interactions after grafting and to supply the neurotrophic factors or neurotransmitters in the stable fashion throughout the brain. We investigated the effect of the continuous and low dose BDNF supply to the hippocampus of the epilepsy model rat using the encapsulated BDNF-producing cells that were implanted in the striatum.
[Methods] We introduced BDNF complementary DNA into baby hamster kidney (BHK) cells and established a cell line that produces BDNF continuously. BHK-BDNF cells and BHK cells that had been transfected with an expression vector that did not contain BDNF cDNA (BHK-control) were encapsulated into the hollow fibers. Each capsule was implanted into the striatum of Fisher 344 rat. Seven days after capsule implantation, bromodeoxyuridine (BrdU), which labeled the proliferated cells in the subventricular zone or dentate gyrus of hippocampus, was administered seven times in a day. Next day we injected kainate 1 microgram solved in PBS, or PBS only into the rat lateral ventricle stereotactically. The evolutions of epilepsy following kainate treatment were monitored by video-EEG. Two weeks after lesion, rats were processed immunohistochemically.
[Results] Prior to implantation, we checked, using ELISA, to see whether BDNF was continuously released at a rate of more than 5 ng/capsule/day from encapsulated BHK-BDNF cells. Although the level of BDNF from the BHK-BDNF capsules following explantation was lower than that prior to implantation, the retrieved BHK-BDNF capsules persisted in producing approximately 3 ng of BDNF per day up to 3 weeks after transplantation. BDNF-producing capsule transplantation enhanced the proliferation of BrdU/PSA-NCAM positive cells in the subventricular zone and subgranular zone of the normal rats. In epilepsy rats, low dose of BDNF protected hippocampal neurons (CA1 and 3) against kainate administrations, and epileptic spikes were improved on the EEG. BrdU positive cells also increased in BHK-control group, but most of the divided cells did not differentiate into neurons or neuronal progenitor cells. Some of BrdU positive cells in the granule cell layer migrated toward the CA3 regions.
[Conclusion] These results suggest encapsulated BDNF-producing cells promote neurogenesis and assist the repair of hippocampal neurons from damage.
A NOVEL STIMULANT OF NEUROGENESIS FOLLOWING MOUSE BRAIN ISCHEMIA VIA PI3K/AKT AND ERK ACTIVATION IN THE SUBGRANULAR ZONE OF HIPPOCAMPUS
Kohji Fukunaga, Norifumi Shioda
Department of Pharmacology, Graduate School of Pharmaceutical Sciences and The 21st Century COE Program, CRESCENDO, Tohoku University, Sendai, Japan
Neurogenesis is well documented in the subgranular zone (SGZ) of hippocampus. Especially, in the hippocampal, crucial role of neurogenesis in learning and memory formation has been addressed. The neurogenesis in the hippocampus is regulated by environmental stress, seizure and stroke. However the mechanism underlying neurogenesis in the adult brain remains to be unclear. We here assessed whether a novel stimulant of phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways promotes brain ischemia-induced neurogenesis in a mouse transient middle cerebral artery occlusion (MCAO) model. Intraperitoneal administration of a vanadyl compound, which is a potent inhibitor for protein tyrosine phosphatases, stimulated neurogenesis in SGZ following brain ischemia. The drug led to activation of PI3K/Akt and ERK pathways in SGZ and those activations were inhibited by treatment with specific inhibitor, wortmannin or U-0126, respectively. Likewise, wortmannin or U-0126 treatment totally abolished both ischemia- and the vanadyl compound-induce neurogenesis. In behavioral pharmacological studies, the vanadyl compound-induced neurogenesis was closely associated with improvement of memory deficits following brain ischemia. Taken together, peripheral administration of a novel vanadyl compound stimulates neurogenesis following brain ischemia through PI3K/Akt and ERK activation in the SGZ, thereby improving ischemia-induced memory deficits.
DISTRIBUTION OF INDUCIBLE NITRIC OXIDE SYNTHASE AND CELL PROLIFERATION IN RAT BRAIN AFTER TRANSIENT MIDDLE CEREBRAL ARTERY OCCLUSION
Yoshihide Sehara, Takeshi Hayashi, Kentaro Deguchi, Atsushi Tsuchiya, Toru Yamashita, Hanzhe Zhang, Violeta Lukic, Tatsushi Kamiya, Koji Abe
Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
Background: Nitric oxide (NO) is a physiological messenger synthesized by NO synthase (NOS), including neuronal nNOS, endothelial eNOS, mitochondrial mtNOS, and inducible iNOS. All central nervous system (CNS) cells synthesize NO by oxidation of L-arginine, in a reaction catalyzed by NOS. NO can be neuroprotective or neurotoxic in cerebral ischemia, depending on the NOS isoform involved. In addition to direct neurotoxic effect in ischemic brain, iNOS adversely affect neurological outcome by blocking neurogenesis. In the present study, therefore, we studied the chronological and spatial change of the distribution of iNOS and cell proliferation in subventricular zone (SVZ) after transient focal cerebral ischemia. Methods: Adult male Wistar rats (12 weeks old) were used in this study. The right middle cerebral artery (MCA) was transiently occluded through the common carotid artery for 90 min (tMCAO). In order to estimate the number of proliferating cells, 50 mg/kg of bromodeoxyuridine (BrdU) were intraperitoneally injected at 12 and 24 hours before decapitation. The rats were sacrificed at 1, 3, 7 and 21 days after tMCAO (n = 6, each). Sham-operated rats were sacrificed at 7 days after the sham operations (n = 6). The brains were quickly removed, frozen in powdered dry ice and coronal sections of 10 µm thickness were prepared. These sections were obtained from caudoputamen level, and were identical among the investigated animals. Results: After 90 min of tMCAO, iNOS positive cells decreased in the ischemic core at 1 to 21 d, and increased in the ipsilateral periischmic area at 1 and 3 d. BrdU positive cells appeared in the ischemic core at 3 to 21 d, in the periischemic area at 3 and 7 d, and increased in the ipsilateral SVZ at 7 d. ED-1 positive cells appeared in the ischemic core at 3 to 21 d, and some of them were double positive for BrdU or iNOS. It was thus indicated that the major source of iNOS in the ischemic core was macrophages. In the periischemic region, on the other hand, iNOS expressing cells were positive either for NeuN or GFAP, indicating that neurons and astrocytes expressed iNOS. Conclusions: The chronological change of iNOS expression was summarized in the Figure. iNOS expression preceded appearance of BrdU positive cells. It is possible to consider that iNOS inhibits cell proliferation in the ischemic brain.
UPREGULATION OF SYNTAXIN1 IN THE ISCHEMIC CORTEX FOLLOWING PERMANENT FOCAL ISCHEMIA IN RATS
Fang Cao, Ryuji Hata, Pengxiang Zhu, Masahiro Sakanaka
Department of Functional Histology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
Background and aims
Syntaxin1 is a 35kDa protein that interacts with the synaptic-vesicle protein Synaptotagmin. Syntaxin1 mediates vesicle fusion in diverse vesicular transport processes along the exocytic and the endocytic pathway. In this study, we investigate effects of ischemia on expression of these two proteins.
Methods & Results
Adult male spontaneously hypertensive rats stroke-prone (SHR-SP) were used. Rats were anesthetized and the left MCA was coagulated and cut. At predetermined time periods (0h, 2h, 6h and 1d after ischemia), animals were sacrificed after deep anesthesia. Their brains were removed and divided into the areas of ischemic cortex, penumbral cortex and contra-lateral control cortex. Samples were homogenized in lysis buffer and immunoblotted with antibody against s-actin, MAP2, Synaptotagmin, and Syntaxin1. As shown in Fig A, the level of Syntaxin1 was upregulated in the ischemic cortex at 1d after occlusion while the level of Synaptotagmin was not changed during ischemia. Immunohistochemical investigation revealed that syntaxin1 was upregulated in the ischemic cortex where MAP2 immunoreactivity was lost (Fig B).
Conclusions
Since Syntaxin1 is known to be a marker of synaptogenesis, our results suggest that Syntaxin1 can play a crucial role in re-synaptogenesis after cerebral ischemia.
THERAPEUTIC EFFECT OF PIGF GENE-MODIFIED HUMAN MESENCHYMAL STEM CELLS ON STROKE MODEL
He Liu1, Osamu Honmou1, Kuniaki Harada1, kiminori Nakamura2, Kiyohiro Houkin1, Hirofumi
Hamada2, J.D. Kocsis3
1Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo, Japan, 2Department of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan, 3Neuroscience Research Center, VA Medical Center, West Haven, CT, USA
Background: Intravenous delivery of mesenchymal stem cells (MSCs) into rat cerebral ischemia models has demonstrated reduced infarction size and improved functional outcome. Placental growth factor (PlGF) is angiogenic to impaired non-neural tissue. We transfected hMSCs with the PlGF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether PlGF gene-modified hMSCs (PlGF-hMSCs) could contribute to the functional repair in a rat permanent middle cerebral artery occlusion (MCAO) model.
Method: hMSCs and PlGF-hMSCs were intravenously infused into the rats 3 hours after MCAO. MRI studies, histological examination and behavioral studies were performed to evaluate the therapeutic benefits with treatment.
Results: ELISA analysis of the infarcted hemisphere revealed an increase in PlGF in both hMSC groups, but a greater increase in the PlGF-hMSC group. After 7 days of MCAO, both hMSCs and PlGF-hMSCs reduced lesion volume and apoptosis, induced angiogenesis, and elicited functional improvement compared to the control sham group, but the effect was greater in the PlGF-hMSC group.
Conclusion: Thus we suggest that the hypothesis that PlGF contributes to neuroprotection and angiogenesis in cerebral ischemia and cellular delivery of PlGF to the brain can be achieved by intravenous delivery of hMSCs.
CELL THERAPY FOR CEREBRAL ISCHEMIA IN SUBACUTE STAGE: TRANSPLANTATION OF EMBRYONIC STEM CELLS-DERIVED NEURAL PROGENITORS SORTED BY FLUORESCENCE-ACTIVATED CELL SORTING
Motoaki Fujimoto, Hideki Hayashi, Yasushi Takagi, Jun Takahashi, Kazuhiko Nozaki, Nobuo Hashimoto
Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
Cerebral ischemia causes neuronal death and disruption of neural circuit in the central nervous system. Various neurological disorders caused by cerebral infarction can be potentially fatal and keep impairing the quality of life. Recent treatment strategy for ischemia focuses just on early reperfusion and prevention of neuronal death in acute stage of infarction. Functional recovery in chronic stage mainly depends on physical treatment and rehabilitation. We aim to establish the cell therapy for cerebral ischemia, using embryonic stem (ES) cells. ES cells have self-renewaling and pluripotentive capacity. We reported the transplanted monkey and mouse-ES cells could survive and differentiate into various types of neurons and glial cells, and make the neuronal network in basal ganglia. In this report, we transplanted the neural progenitors derived from ES cells into the ischemic mouse putamen at 1week after transient middle cerebral artery occlusion. We inspired the differentiation of the neural progenitors from mouse ES cells using the serum-free suspension culture (SFEB culture) method. In this method, telencephalic neural progenitors could be effectively induced from the floating aggregates of ES cells. Furthermore, to prevent tumor formation, we purified the progenitors expressing the neural progenitor marker Sox1 by fluorescence-activated cell sorting (FACS). At 1 month after transplantation we analyzed the survival and differentiation of the transplanted cells. In addition we estimated the network formation and the functional recovery from ischemic damage.
ENDOGENOUS THROMBOSPONDINS 1 AND 2 ARE NECESSARY FOR SYNAPTIC PLASTICITY AND SPONTANEOUS FUNCTIONAL RECOVERY AFTER STROKE
Jason Liauw1, Stan Hoang1, Cagla Eroglu2, Michael Choi1, Raphael Guzman1, Tonya Bliss1, Ben
Barres2, Gary K. Steinberg1
1Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA, 2Department of Neurology and Neurological Sciences / Stanford University School of Medicine, Stanford, CA, USA
Background/Aims: Thrombospondins 1 and 2 (TSP-1/2) belong to a family of secreted multimeric extracellular glycoproteins and have angiostatic as well as synaptogenic properties. While the robust expression of TSP-1/2 that occurs after stroke has been postulated to drive the resolution of post-ischemic angiogenesis, little is known about the role TSP-1/2 may play in synaptogenesis and subsequent functional recovery after stroke. In this study, we investigate whether TSP-1/2 is necessary for the spontaneous synaptic and motor recovery that occurs after stroke.
Methods: An ischemia model was generated in 8 to 12 weeks old wild-type (n=10) and TSP-1/2 knockout (KO) mice (n=8) by unilateral occlusion of the distal middle cerebral artery. Spontaneous recovery of motor functions was assessed through rotarod, tongue protrusion, sticky-tape, and limb asymmetry behavioral paradigms for four weeks after stroke. At 4 weeks post-stroke, synaptic quantification was performed by staining sections with mouse anti-synaptophysin (pre-synaptic) and rabbit anti-PSD-95 (post-synaptic). Synaptic puncta, defined as co-localization of pre-and post-synaptic markers, were identified using ImageJ. Synaptic densities were compared in the penumbra region between wild-type and TSP-1/2 knock out mice (n=5 per group). Biotinylated dextran-amine (BDA) was injected into the contralesional homotopic cortex to quantify axonal sprouting from the contralesional homotopic cortex to the ipsilesional penumbra and striatum.
Results: TSP-1/2 KO mice exhibited significant deficits in their ability to recover motor function following ischemic brain injury as compared to their WT counterparts in the tongue protrusion test at week 4 (p<0.05). Moreover, synaptic staining revealed reduced synaptogenesis in the penumbra of TSP-1/2 KO mice as compared to that seen in homologous areas of WT mice (p<0.05). BDA staining also showed reduced axonal sprouting in the penumbra region from the contralesional cortex in TSP-1/2 KO mice compared to that in wild-type.
Conclusion: These results demonstrate that functional recovery after stroke requires TSP-1/2 expression. TSP-1/2 expression may also play a necessary role in neuroplasticity after stroke given that a deficiency of TSP-1/2 results in a reduction of synaptogenesis and axonal sprouting. An understanding of the mechanisms underlying post-stroke neuroplasticity may improve on therapies aimed at augmenting recovery after stroke.
IMPROVED BRAIN OXYGEN UTILIZATION DURING GRADED ASPHYXIA IN NEWBORN PIGLETS WITH INTRAUTERINE GROWTH RESTRICTION
Reinhard Bauer1,3, Bernd Walter2, Ulrich Brandl3
1Institute of Molecular Cell Biology, University Hospital, Friedrich Schiller University, Jena, Germany, 2Department of Neurosurgery, Zentralklinik Bad Berka, Bad Berka, Germany, 3Department of Neuropediatrics, University Hospital, Friedrich Schiller University, Jena, Germany
Background and aims: There is insufficient knowledge referring to the effects of intrauterine growth restriction (IUGR) on metabolic regulation of cerebral blood flow (CBF) in newborns. Recently, we have shown that IUGR resulted in an improved ability to withstand critical periods of gradual oxygen deficit as shown by improved cerebrovascular autoregulation at hemorrhagic hypotension in an animal model of naturally occurring IUGR in term born piglet*. Furthermore, IUGR is associated with an increased incidence of perinatal asphyxia, which results in cardiocirculatory redistribution. However, it is unknown whether IUGR can affect brain O2 availability under asphyxic conditions.
Methods: Studies were conducted to examine the effects of IUGR on CBF regulation and oxygen consumption (CMRO2) at different stages of asphyxia (hypercapnic hypoxemia) and pure hypoxia (normocapnic hypoxemia). We used 1-day old anesthetized piglets (n=85). Animals were divided into normal-weight piglets (NW, n=42; BW:1522 ± 125g) and IUGR piglets (n = 43, BW:826 ± 51g) according to their birth weight. Correct allocation was confirmed afterwards by estimation of the brain to liver ratio. CBF was measured by colored microspheres. Brain AVDO2 was determined using arterial and sagittal sinus blood samples (CMRO2 = CBF × AVDO2). Different stages of hypoxemia were induced for 1-h by appropriate FiO2 lowering (moderate hypoxia: paO2=31-34 mmHg, severe hypoxia: paO2=20-22 mmHg). Fourteen NW and sixteen IUGR piglets received additionally 6% CO2 to the breathing gas, so that a paCO2 of 74-80 mmHg resulted (asphyxia groups). Eight NW and nine aIUGR animals served as untreated control. In addition, three NW and three IUGR piglets were used for brain COX-2 and eNOS estimation by Western blotting. Furthermore, affinity of hemoglobin for oxygen under hypoxic and asphyxic conditions were measured in blood samples from fifteen NW and fourteen IUGR newborn piglets by estimation of individual oxygen dissociation curves and calculation of halfsaturation oxygen pressure (P50) values from linear interpolations using the Hill plot.
Results: Systemic cardiovascular and blood gas parameters, as well as hypoxia- related effects on alteration in CBF regulation and CMRO2 were similar in NW and IUGR piglets: At pure moderate and severe O2 deficit a marked CBF increase, maintained O2 availability and aligned O2 utilization resulted in maintained O2 consumption. In contrast, during asphyxia cerebral oxygen extraction was markedly increased in IUGR animals (P < 0.05). This resulted in a significantly diminished CMRO2-related increase of CBF at gradually reduced arterial oxygen content (P < 0.05). A marked difference in brain COX-2 and eNOS content could not be verified between NW and IUGR piglets. However, blood derived from IUGR piglets showed an increased O2 affinity by 17% at asphyxic conditions (P < 0.05).
Conclusions: An enhanced effectivity in oxygen availability appeared in newborn IUGR piglets under graded asphyxia by improved cerebral oxygen utilization. This is connected with altered O2 affinity of hemoglobin leading to increased oxygen transport capacity under asphyxic conditions. We suggest that IUGR newborns are more capable of protecting the brain against O2 loss during asphyxia than NW neonates.
POSTNATAL MATURATION ENHANCES CALCIUM INFLUX, AND REDUCES MYOFILAMENT CALCIUM SENSITIZATION IN MYOGENIC RESPONSES TO CEREBRAL ARTERY STRETCH
Renan Sandoval, Elisha Injetti, James Williams, William Pearce
Department of Physiology, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, CA, USA
Background & Aims
Cerebrovascular tone is determined by both cytosolic [Ca++] and myofilament Ca++ sensitivity, which in turn, reflects the additive relations between [Ca++] and myosin light chain (MLC) phosphorylation (thick-filament reactivity), and between MLC phosphorylation and contractile force (thin-filament reactivity). Previous studies have demonstrated that postnatal maturation dramatically alters the relative contributions of cytosolic [Ca++], thick-filament reactivity, and thin-filament reactivity to agonist-induced contractions. The present study tests the hypothesis that the contributions of each of these variables to stretch-induced myogenic contractions also change during postnatal maturation of ovine cerebral arteries.
Methods
Segments of posterior communicating arteries from term fetal and non-pregnant adult sheep were wire-mounted and incrementally stretched from 1.3 to 2.7 times the resting, unstressed diameter. At each of 8 stretch levels, myogenic tone was determined as the difference between total tension and tension in the presence of 3 mM EGTA. Cytosolic [Ca++] was also determined at each stretch via fura-2 fluorescence photometry. In parallel, arteries were frozen at each stretch level and analyzed for MLC phosphorylation using urea gel Western blots. In other experiments, arteries were permeabilized using beta-escin, and then held at pCa values of 5.5 or 7.0 during incremental stretch.
Results
Fetal and adult cerebral arteries exhibited similar myogenic responses with peak tensions averaging 23.9±6.8% and 26.0±4.8% of Kmax at stretch ratios of 2.19±.04 and 2.23±.02, respectively. Graded stretch increased cytosolic [Ca++] at stretch ratios >2.0 in adult cerebral arteries, but increased [Ca++] only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, stretch increased myogenic tone with a peak response at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %Kmax values at peak myogenic tone were not significantly different at either pCa 7.0 (22.7±3.0%) or pCa 5.5 (24.8±5.0%). Conversely, adult %Kmax values at peak myogenic tone were significantly less at pCa 7.0 (15.7±4.2%) than at pCa 5.5 (26.6±3.0%). Regarding stretch-induced MLC phosphorylation in intact cerebral arteries, there were no significant differences in the maximal extent of stretch-induced MLC phosphorylation between fetal (19.9±4.9%) and adult (17.1±2.7%) cerebral arteries. The stretch-ratios that produced half-maximal (EC50) MLC phosphorylation were also highly similar in fetal (1.68±0.10) and adult (1.64±0.18) cerebral arteries, respectively.
Conclusions
Overall, the data demonstrate that the myogenic response is highly conserved in cerebral arteries during postnatal maturation. Nonetheless, the relations between stretch and cytosolic calcium, and between stretch and myofilament calcium sensitization, appear to be regulated very differently in fetal and adult cerebral arteries. This pattern of age-related differences suggests that stretch-induced myofilament calcium sensitization is upregulated in fetal compared to adult arteries. In particular, the data imply that stretch-induced modulation of thin-filament reactivity may be more important for the myogenic response in fetal compared to adult cerebral arteries. Conversely, the ability of stretch to increase cytosolic calcium appears to improve during postnatal maturation. Such differences should be carefully considered when designing strategies for management of NICU patients at risk for, or recovering from, cerebrovascular injury. Supported by NIH HD31226, HL54120, and HL64867.
CEREBRAL BLOOD FLOW, CEREBRAL OXYGEN METABOLISM, AND ATROPHY IN HEALTHY AGING
Manouchehr S. Vafaee1, Per Borghammer1, Joel Aanerud1, Paul Cumming2, Albert Gjedde1,2
1PET Center, Aarhus University Hospital, Aarhus, Denmark, 2CFIN, Aarhus University, Aarhus, Denmark
Introduction: It is debatable where and to what extent cerebral blood flow (CBF) and cerebral oxygen metabolism (CMRO2) decline with healthy aging. It is possible that neocortical atrophy could affect the measurements of these physiological factors in addition to the direct effects of aging on blood flow and metabolism. To tackle these issues and to shed some light on the cerebrometabolic changes following normal aging, we completed a series of CBF/CMRO2 PET studies on a healthy group.
Material and methods: 12 healthy young males and females, aged (26±4 yrs), and 16 elderly healthy subjects, aged (58y±5 yrs), underwent one CBF and two identical CMRO2 quantitative 3-min PET scans in a state-of-rest. The cortical grey matter was extracted from the subjects MRI scans by means of an automated segmentation algorithm. We also performed a voxel-based subtraction of young and old and a voxel-wise regression of CBF and CMRO2 versus age. The magnitudes of CMRO2 and CBF were determined with the two-compartment weighted-integration methods. Mean subtracted image volumes (elderly minus young) were converted to z-statistic volumes. Significant focal differences of CBF and CMRO2 and grey matter intensity were identified on the basis of the 3D Gaussian random field theory and cluster analysis.
Results: The average whole brain CBF in young subjects (53±5ml/min.hg) did not differ from the elderly population (52±6ml/min.hg). However, the average global CMRO2 in aged subjects (154±24µmol/min.hg) was lower than in the young subjects (166±13µmol/min.hg) but did not reach significance (p=0.1). Oxygen consumption had decreased more progressively than CBF in some regions and predominantly so in the neocortex, although clusters of CBF and CMRO2 decline overlapped extensively. CBF and CMRO2 decreased in most of the frontal lobe (except orbitofrontal cortex, M1, PMA and SMA), and L/R lateral parietal cortex and L posterior cingulate. Oxygen decline was more pronounced in the pre-central gyrus than CBF. A somewhat similar and significant pattern of decrease in grey matter intensity was seen, primarily in the frontal and lateral parietal cortices.
Discussion: While it is accepted that many changes occur in the brain as a result of normal aging, it is not fully agreed to what degree and in which regions of the brain those changes take place. Our data indicate that global CMRO2 decreases (although not significantly) without concomitant decrease in global CBF. If so, the finding confirms that oxygen consumption is a more accurate index of neuronal work than blood flow. We observe overlapping areas of decreased CBF and CMRO2 and decline in grey matter intensity. It is thus likely that neocortical atrophy contributes to the decreased signal in these PET scans. However, the data also revealed non-overlapping areas. The decreases of CBF and CMRO2 in healthy aging are most likely due to combinations of atrophy and direct changes, such as functional deafferentation, changes in neurotransmitter systems, and cell death following healthy aging. The metabolic depression as a result of healthy aging suggests a dysfunction of neuronal circuits that underlie some age-related changes in cognitive function.
EVIDENCE OF ENDOTHELIAL DYSFUNCTION IN PRIMARY LATERAL SCLEROSIS (PLS) WITH COGNITIVE IMPAIRMENT AS MEASURED BY CT PERFUSION
Matthew Murphy1,2,3, Gloria Grace4, Michael Strong4, Carmela Tartaglia4, J.B. Orange5, Xiaogang Chen2, Ann Rowe4, Karen Findlater4, Ting-Yim Lee1,2,3
1Department of Medical Biophysics, University of Western Ontario, London, ON, Canada, 2Imaging Research Laboratories, Robarts Research Institute, London, ON, Canada, 3Imaging Division, Lawson Health Research Institute, London, ON, Canada, 4Department of Clinical Neurological Sciences, London Health Sciences Centre, London, ON, Canada, 5School of Communication Sciences and Disorders, University of Western Ontario, London, ON, Canada
BACKGROUND: Primary lateral sclerosis (PLS) is a rare neurodegenerative disorder of the upper motor system that may also present with clinically significant cognitive impairment. We propose that mean transit time (MTT), or the ratio of cerebral blood volume (CBV) to cerebral blood flow (CBF), a surrogate indicator of cerebral perfusion pressure and hence vascular tone, can be used to investigate endothelial dysfunction in PLS patients with cognitive impairment.
METHODS: Nine male and nine female PLS patients aged 44 to 72 (mean, 59.11 ± 8.67) years were studied with CT Perfusion, a contrast enhanced dynamic CT scanning method which measures cerebral blood flow (CBF), blood volume (CBV) and MTT, on the same day of neuropsychological testing. Eight of these patients had concomitant cerebrovascular disease and/or diabetes and were excluded from the analysis. The remaining 10 patients, (5 men, 5 women) with an age range of 44 to 69 years (mean, 58.4 ± 9.6 years), were then stratified into two groups based on neuropsychological testing: (1) cognitively-intact PLS patients (PLSn; those having zero or one abnormal scores (n=7)); and, (2) cognitively-impaired PLS patients (PLSci; those having greater than 2 abnormal test scores (n=3)). These data were then compared with 4 male and 2 female normal controls aged 34 to 63 (mean, 52.9± 9.9) years. CT images were segmented into the four lobes of the brain with each lobe further subdivided into grey and white matter (GM and WM, respectively). CBF, CBV and MTT for GM and WM in each lobe were compared across groups using ANOVA with post-hoc Tukey tests and significance declared at P < 0.05.
RESULTS: PLSci showed increased CBV and MTT with reduced CBF relative to PLSn. MTT was significantly increased in the PLSci group in all regions and the increase affected both grey and white matter, with the exception of sparing of the temporal lobe white matter. No differences in MTT existed upon inclusion of the full cohort of patients including those with concomitant cerebrovascular disease and/or diabetes.
CONCLUSIONS: As demonstrated by the current study, cognitive impairment in the background of neurodegeneration found in PLS may affect neurovascular coupling leading to disturbances in local cerebral hemodynamics and the increase in MTT of PLSci patients. MTT, being the ratio of CBV to CBF, is sensitive to changes in vascular tone. The observed increase in MTT, therefore, may signify abnormal vascular tone and hence endothelial dysfunction in PLSci patients.
SEROTONIN TRANSPORTER BINDING IN HEALTHY TWINS AT HIGH RISK OF DEVELOPING MOOD DISORDER IN COMPARISON WITH TWINS AT LOW RISK
Vibe G. Frokjaer1, Maj Vinberg2, David Erritzoe1, William Baare3, Jacob Madsen4, Lars V. Kessing2, Gitte M. Knudsen1
1Neurology Research Unit, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark, 2Psychiatric Department, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark, 3Danish Magnetic Resonance Center, Hvidovre University Hospital, University of Copenhagen, Copenhagen, Denmark, 4PET and Cyclotron Unit, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
Background and aims
Mood disorders are thought to be caused by a combination of genetic and environmental stress factors and disturbances in serotonergic transmission seem to be an important pathophysiological component. Previous postmortem and PET studies in depressive patients have identified changes in the serotonin transporter (SERT) binding; most often a lower binding has been found in depressive and in bipolar patients, but it is not clear whether those changes represent state or trait markers of the disorders. We tested the hypothesis that cerebral SERT binding act as a trait marker for mood disorders by comparing SERT-binding in twins genetically predisposed to mood disorder with a control group of non-predisposed twins.
Methods
Nine healthy twins, (5 DZ and 4 MZ, mean age 32.2±4.2 years (range 27-40) genetically predisposed to affective disorder (6 unipolar, 3 bipolar) and 9 non-predisposed age and gender matched controls (5 DZ, 3 MZ, and 1 non-twin), mean age 31.6+4.9 years (range 25-40) were investigated with [11C]DASB PET and MRI. Female:male ratio was 6:3 in both groups reflecting the profile in depression. Subjects with and without genetic predisposition to affective disorder were identified by linking information from the Danish Twin Register and the Danish Psychiatric Central Register. Imaging: The SERT binding was imaged with [11FC]DASB PET based on 90 minutes dynamic acquisition starting immediately after bolus injection of 5.0±1.9 (cases) and 5.6±1.7 (controls) Mbq [11FC]DASB/kg body weight. The following volumes of interest were investigated: thalamus, caudate, putamen, midbrain, and anterior cingulate. Quantification of SERT: The binding potential of specific tracer binding (BP1) was calculated using MRTM2 with cerebellum as a reference region and fixing of k2′ (clearance rate constant from cerebellum) by applying MRTM on data from striatum (volume-weighted average of caudate nucleus and putamen). Symptom assessment: All subjects completed Major Depression Inventory, Becks Depression Inventory (self-reported questionnaires) and Hamilton Depression Rating Scale (interview), which evaluate symptoms of depression, anxiety and mania. Statistics: Group comparisons between regional SERT binding were performed using unpaired t-test.
Results
Clinical data and symptom scores: The predisposed group smoked more and tended to be less educated. The depression and anxiety symptom profiles of the predisposed and control group did not differ significantly. Regional SERT binding: We found a lower SERT binding in the putamen in the predisposed group in comparison with the controls (p=0.03) whereas no significant differences between the groups were identified for thalamus, caudate, midbrain, or anterior cingulate.
Conclusion
We tested whether genetic predisposition to mood disorder, in the absence of depressive and anxiety symptoms, is related to specific changes in regional SERT binding. We found that healthy twins at high risk of developing a mood disorder have lower putaminal serotonin transporter binding in comparison with twins at low risk. Our data suggest that low serotonin transporter binding may represent a trait marker for mood disorder.
STATISTICAL PARAMETRIC MAPPING ANALYSIS OF RELATIONSHIP BETWEEN REGIONAL BLOOD FLOW AND SYMPTOM CLUSTERS OF DEPRESSIVE MOOD IN CHRONIC KIDNEY DISEASE
Sang Heon Song1, Seong-Jang Kim2, Ji Hoon Kim3
1Department of Internal Medicine, Pusan National University Hospital, Busan, South Korea, 2Department of Nuclear Medicine, Pusan National University Hospital, Busan, South Korea, 3Department of Psychiatry, Pusan National University Hospital, Busan, South Korea
Background: To investigate the relationship between regional cerebral blood flow (rCBF) and symptom clusters of depressive mood in pre-dialytic chronic kidney disease (CKD). Methods: Twenty seven patients with stage 4–5 CKD were performed statistical parametric mapping (SPM) analysis of brain SPECT. Correlation analyses between separate symptom clusters of depressive mood and rCBF were done. Results: The first factor, depressive mood, was negatively correlated with rCBF in right insula, posterior cingulated, and left superior temporal gyrus, and positively correlated with rCBF in left fusiform gyrus. The second factor, insomnia, was negatively correlated with rCBF in right middle frontal gyrus, both cingulate gyrus, right insula, right putamen, and right inferior parietal lobule, and positively correlated with rCBF in left fusiform gyrus and both cerebellar tonsil. The third factor, anxiety and psychomotor aspect, was negatively correlated with rCBF in left inferior frontal gyrus, right superior frontal gyrus, right middle temporal gyrus, right superior temporal gyrus, and left superior frontal gyrus, and positively correlated with rCBF in right ligual gyrus and right parahippocampal gyrus. Conclusion: In this study, the separate symptom clusters were correlated with specific rCBF pattern similar to areas that involved in major depressive disorder patients without CKD. However, discordant areas with rCBF of major depressive disorder patients were revealed, too. Further investigation of larger scale should be needed.
VOXEL BASED STATISTICAL ANALYSIS OF REGIONAL CEREBRAL GLUCOSE METABOLISM IN CHILDREN WITH ATTENTION-DEFICIT HYPERACTIVITY DISORDER
Jihoon Kim1, Seong-Jang Kim2, Jung Sub Lee3
1Department of Child-Adolescent Psychiatry, Pusan National University Hispital, Busan, South Korea, 2Department of Nuclear Medicine, Pusan National University Hispital, Busan, South Korea, 3Department of Orthopedic Surgery, Pusan National University Hispital, Busan, South Korea
Voxel based statistical analysis was conducted to identify cerebral metabolic differences between children with Attention-deficit Hyperactivity disorder (ADHD) and normal controls. F-18 FDG-PET brain scans were compared between 14 drug- naive children with ADHD and 15 drug-naive normal controls using statistical parametric mapping (SPM). Decreased glucose metabolism were found in left parahippocampal gyrus, left hippocampus, left anterior cingulate gyrus, right anterior and posterior lobes of cerebellum, left superior temporal gyrus, left insula, left medial and middle frontal gyri, right medial frontal gyrus, left basal ganglia (putamen, amygdala and caudate nucleus) in children with ADHD. Also, ADHD patients showed hypermetabolism in both supramarginal gyri. Current study suggests that children with ADHD have more dysfunction relatively large area of brain compared to normal controls.
DOES ALZHEIMER'S DISEASE REALLY EXIST?
Frederick Peng
Department of Neurosurgery & Neurological Institute, Taipei VGH, Taipei, Taiwan-R.O.C.
Background and Aims: This presentation aims to rectify the historical mistake committed by Kraepelin; it has come down as Alzheimer's disease for almost a century. The eponym was not based on scientific merits but, rather, on power play. There were pieces of evidence: (1) the patient had at least four organic brain diseases; (2) Alzheimer did not treat the patient, nor did he perform the autopsy and prepare the autopsied materials for oral presentation and publication, but (3) Oskar Fischer (1907) had a far superior contribution which has been either overlooked or misunderstood.
Methods: I have undertaken a critical literature review from a historical perspective, including Alzheimer (1907) and Fischer (1907)along with Bonfiglio (1908), Perusini (1910;1911), Kraepelin (1910) and many other selected publications
Results: Alzheimer's disease (AD) has been known since 1910. It is claimed to consist of two hallmarks: senile plaques and neurofibrillary tangles. Upon reviewing the literature, I have found that this claim is utterly false; instead, the patient had at least four brain diseases: arteriosclerosis, evenly atrophic brain, senile plaques, and neurofibrillary tangles; I also suspect that she had DM and decubitus angina.
The false claim has been taught at every medical school throughout the world. However, the historical facts indicate that Alzheimer NEVER made any original histopathologic description of senile plaques in the patient, although he did briefly describe “the transformation of fibrils.” In fact, he did not even treat the patient; instead, he assigned her to his junior colleague, Gaetano Perusini, who treated her for just seven months (November 1901-June 1902), although most subsequent investigators believe that Alzheimer treated her from 1901 till her death in 1906. The reason is that Alzheimer quit his job in Frankfurt in June 1902 to join Kraepelin in Heidelberg; the two then moved to Munich in 1903. In other words, the materials for his oral presentation in November, 1906 at a conference of German psychiatrists in Tubingen and for his 1907 paper were entirely collected and prepared by Perusini. On the other hand, Oskar Fischer made the most extensive and detailed description in 1907 of 16 cases with eight histopathologic illustrations of both diseases.
My thorough examination of the two publications in 1907 by Fischer, whose article was a lengthy one of 12 pages, and Alzheimer, whose case report was barely two-page long, has enabled me to compare them point-by-point in this presentation. I declare that it is unfair to Fischer and the general public to allow the false claim to stand unchallenged. Even Perusini changed his mind in 1911 and followed Vedrani (1910;1911), by attributing senile plaques and presbyophrenia to Fischer as Fischer's Plaques and Fischer's Presbyophrenia.
Conclusions: The eponym, AD, is a misnomer, created by Kraepelin for his self interest, which has misled the entire medical field for almost a century. In fairness to Fischer and to do justice to the scientific community, I call for a new term to replace AD which never existed, does not exist, and will never exist.
SIGNIFICANT ASSOCIATION BETWEEN LEUKOARAIOSIS AND METABOLIC SYNDROME IN HEALTHY PEOPLE
Kaechang Park, Shoko Yamada, Hiromichi Nakabayashi, Shinichi Toyonaga, Noritaka Masahira,
Eiiichi Nakai, Naoki Ikawa
Department of Neurosurgery, Kochi Unversity Medical School, Kochi, Japan
Introduction: LA has been considered as an intermediate substitute of ischemic brain damages. MetS attracts attention as a risk factor for cardiovascular and cerebrovascular diseases. However, no study has described the relationship of MetS to LA in healthy individuals derived from various age groups.
Methods: We studied 3,070 persons at ages between 28 and 78 years (mean, 52.7 years) with no past history of stroke who visited a healthcare facility in Kochi prefecture, Japan, from September 2005 through December 2006 and underwent a questionnaire survey, blood analysis, and magnetic resonance imaging of the brain as part of their routine health checkup at the facility. MetS was defined using the criteria of the National Cholesterol Education Program Adult Treatment Panel III. LA was assessed using the visual rating scale of the Atherosclerosis Risk in Communities study. Multiple logistic regression analysis was performed to examine associations between LA and MetS and its components while considering influences of potential confounding variables.
Results: 752 (24.2%) subjects had LA on MRI. MetS was significantly associated with the presence of LA (multivariate adjusted odds ratio [OR], 3.40; 95% confidence interval [CI], 2.34–4..95). When five components were entered simultaneously into a model, significant associations were observed for elevated blood pressure (BP) (adjusted OR, 2.13; 95% CI, 1.54 −2.95), impaired fasting glucose (IFG) (adjusted OR, 1.16; 95% CI, 1.14–2..41), and hypertriglyceridemia (hyper-TG) (adjusted OR, 1.55; 95% CI, 1.07–2..26). Adjusted ORs of LA for individuals with MetS were relatively constant across grades of LA; the adjusted OR obtained from a multinomial logistic regression model were 3.41 (95%CI, 2.30–5..06) for minimal LA, 3.07 (95%CI, 1.75–5..38) for mild, moderate, and severe LA, and 3.33 (95%CI, 2.30–4..84) for all grades of LA.
Conclusions: MetS was significantly associated with all grades of LA, including the minimal grade of LA. Elevated BP, IFG, and hyper-TG of MetS components were independent risk factors of LA. MetS can play an important role in identifying healthy individuals who have an increased risk of LA.
HEMODYNAMIC CHANGES IN FOCAL BRAIN ISCHEMIA OF RATS ASSESSED BY LASER SPECKLE FLOWMETRY: ROLE OF ISCHEMIC DEPOLARIZATIONS
Hajime Nakamura1,3, Anthony J. Strong2, Guenter Mies1, Heike Endepols1, Stefan Vollmar1, Michael Sue1, Toshiki Yoshimine3, Andrew K. Dunn4, Rudolf Graf1
1Max-Planck Institute for Neurological Research, Cologne, Germany, 2Department of Clinical Neurosciences (Neurosurgery), King's College London, London, UK, 3Department of Neurosurgery, Osaka University, Osaka, Japan, 4Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA
[INTRODUCTION]
Following the onset of focal ischemia, pathogenetic processes such as peri-infarct depolarizations are triggered but their relationship to hemodynamic alterations remains to be established. Laser speckle flowmetry (LSF) allows to follow hemodynamics with high spatial and temporal resolution. We used LSF in a distal MCA occlusion model in rats to characterize perfusion patterns around the infarct core and thus to provide clues on the fate of penumbral tissue.
[METHOD]
Six isoflurane anesthetized Wistar rats were tracheotomized and artificially ventilated. Blood pressure was continuously monitored. Hemicraniectomies (1.5×1.0 cm) exposed the left MCA/ACA territory. After dura removal, the cortex was covered with mineral oil and kept at 37°C. A LSF system (laser diode & detecting camera) was placed over the exposed cortex to acquire images at a rate of 12 frames/min. The frontoparietal MCA branch was coagulated and perfusion was monitored for up to 18 hours. Post- experimental pixel by pixel evaluations of LSF images allowed two-dimensional processing of perfusion changes to assess hemodynamic patterns around the infarct core.
[RESULTS]
In 3/6 rats, the initial ischemic core increased during the observation time following distal MCA occlusion without alteration of autoregulation. This growth was regularly related to the occurrence of ischemic depolarizations as observed in the LSF videos by propagating waves of reactive hyperemia. The figure shows a most dramatic example of such alteration: within 1 hour, 7 depolarizations occurred, and the initial ischemic core grew by around 100% at the expense of the peri-infarct surrounding.
[CONCLUSION]
LSF allows to establish hemodynamic patterns after onset of focal cerebral ischemia related to the occurrence of ischemic depolarizations.
A UNIFIED NEUROBIOLOGICAL FRAMEWORK FOR FUNCTIONAL NEUROIMAGING USING A MODEL INTEGRATING COMPARTMENTALIZED ENERGY METABOLISM AND HEMODYNAMICS
Agnes Aubert1, Luc Pellerin2, Pierre Magistretti3, Robert Costalat4
1URA CEA CNRS 2210, Service Hospitalier Frederic Joliot, Orsay, France, 2Departement De Physiologie, Universite De Lausanne, Lausanne, Switzerland, 3Brain and Mind Institute, Ecole Polytechnique Federale De Lausanne and Centre De Neurosciences Psychiatriques, Department of Psychiatry, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 4Universite Pierre Et Marie Curie-Paris6, Institut National De La Sante Et De La Recherche Medicale U678, Paris, France
Background: Brain functional imaging methods, including functional magnetic resonance imaging (fMRI) and positron emission tomography (PET), have dramatically evolved in recent years. However, their biochemical and physiological bases remain unclear, especially the cellular and molecular mechanisms taking place upon activation in neurons and astrocytes. On the basis of a previously developed modeling approach, we developed a coherent neurobiological framework that not only explains several in vitro and in vivo experimental results but also provides a basis to interpret imaging signals, especially fMRI and Magnetic Resonance Spectroscopy (MRS) data.
Methods: System of ordinary differential equations has been numerically solved using MATLAB software version 7.
Results: First, using a model of compartmentalized energy metabolism between astrocytes and neurons, we show that complex NADH kinetics observed in vitro by Kasischke and colleagues (2004, Science 305, 99-103) can be accounted for by distinct metabolic responses in neurons and astrocytes. Second, extension of this model to an in vivo situation, including hemodynamic aspects, allowed us to predict the cerebral metabolic rate of oxygen consumption, and, combined with the changes in the cerebral blood flow, we could reproduce the time evolution of intraparenchymal oxygen concentration upon activation as measured experimentally in animal, for instance by Ances and colleagues (2001, Neuroscience Letters 306, 106-110). Third, applying the same kind of model to fMRI in humans, we could determine that the initial dip of the blood oxygen level dependent (BOLD) signal in fMRI, is critically dependent on the oxidative response of highly oxidative cells, for instance highly oxidative neurons, while the late features of the BOLD signal correspond to a combination of responses from two cell types: highly oxidative cells ('red’ neurons) and poorly oxidative cells (astrocytes and ‘white’ neurons). Moreover, the model allows to suggest a set of rules for (i) the presence of more or less oxidative cells within a voxel, (ii) the influence of the kind of stimulation, upon the fMRI signal.
Conclusion: Our results, using a versatile modeling approach, suggest new rules for the interpretation of fMRI signals, which could be tested by comparing biochemical and physiological data obtained in vitro and in vivo in animals to fMRI and MRS data in humans.
ACUTE TREATMENT WITH ROSUVASTATIN PROTECTS OBESE MICE AGAINST TRANSIENT CEREBRAL ISCHEMIA
Keita Mayanagi, Prasad V. Katakam, Tamas Gaspar, Ferenc Domoki, David W. Busija
Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC, USA
Background and aims
Obesity is associated with significantly higher morbidity and mortality in stroke. Recent evidence indicates that acute treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) is protective in stroke. Previously, we showed that 1-day treatment with rosuvastatin (RSV) can reverse impaired cerebrovascular responsiveness in insulin-resistant rats as effectively as with 4-weeks treatment (1). Whether short term statin therapy can reduce infarct volume following experimental stroke in insulin resistant or diabetic animals is unclear. The purpose of this study was to investigate the acute effects of RSV against transient focal cerebral ischemia in mice suffering from insulin resistance and obesity.
Materials and Methods
Male 8 weeks old C57BL/6J ob/ob, lean and wild-type (WT) mice were treated with RSV (10 mg/kg/day, intraperitoneally) or vehicle for 3-days. Ischemia was induced by 60 minutes of middle cerebral artery occlusion (MCAO) using intraluminal suture method. Infarct volumes were measured 24 hours after reperfusion with TTC staining. In additional groups of animals, blood pressure and regional cerebral blood flow (rCBF) were monitored continuously through the 60 min MCAO procedure. Blood samples were withdrawn for measurement of blood glucose, total cholesterol, and insulin levels after the monitoring.
Results
Blood glucose, total cholesterol and insulin levels were significantly elevated in ob/ob mice but were not affected by RSV. The rCBF reductions during MCAO were similar among all groups and were not affected by RSV. However, ob/ob mice exhibited a higher infarct volume compared to lean or WT. Furthermore, RSV significantly reduced infarct volume only in ob/ob mice (40.2 ± 2.7% vs. 31.7 ± 2.7%, p<0.05).
Conclusion
Insulin resistance and obesity lead to vascular dysfunction (1) and elevated damage following strokes. However, treatment with statins provides rapid, beneficial effects prior to reductions in blood cholesterol levels.
Figure
Effect of acute treatment with RSV on infarction volume in WT, lean and ob/ob mice.
(WT - vehicle; n=8; WT - RSV: n=8; Lean - vehicle: n=11; Lean - RSV: n=8; ob/ob - vehicle: n=11, ob/ob - RSV: n=13)
*P<0.05 WT, Lean - vehicle vs. ob/ob - vehicle, **P<0.05 ob/ob - vehicle vs. ob/ob - RSV.
ATTENUATION OF ISCHEMIC BRAIN INJURY AFTER FOCAL CEREBRAL ISCHEMIA IN RAT BY DOUBLE-STRANDED ADENO-ASSOCIATED VIRUS 2-DELIVERED VASCULAR ENDOTHELIAL GROWTH FACTOR
Kai-Yun Chen, Cheng-Fu Chang, Yung-Hsiao Chiang
Department of Neurological Surgery, Tri-Service General Hospital, Taipei, Taiwan-R.O.C.
Background and aims: Vascular endothelial growth factor (VEGF) has been shown to display neuroprotective effects in hypoxic-ischemic injury. We investigated the neuroprotective capacity of VEGF in a rat model of stroke.
Methods: To increase the in vivo transduction efficiency in brain, we used a double-stranded adeno-associated virus 2 vector to deliver VEGF. Seven days after gene transfer, the rats underwent 60 minutes of middle cerebral artery occlusion (MCAO) followed by 24 hours of reperfusion. Infarct volume was determined by TTC staining, neuronal injury was examined by TUNEL, cleaved caspase3 and fluoro-Jade B immunostaining. To further examined the signaling pathway of VEGF's neuroprotective activity, the expression and phosphorylation of Akt, ERK-1/−2 and p38 were analyzed by western blot.
Results: A single administration of dsAAV2 vector carrying VEGF (dsAAV2-VEGF) 7 days before the onset of ischemia, effectively decreased the infarct volume, TUNEL-positive, cleaved caspase3-positive and fluoro-Jade B-positive neurons. Next, we explored whether the dsAAV2-VEGF protected primary cultures of cortical neurons from hypoxia. After infection of dsAAV2-VEGF in primary cortical neurons 3 days before 24 h of hypoxia, the number of surviving cells was significantly increased and the activation of caspase3 was reduced. Both ischemic brains and cortical neurons infected with dsAAV2-VEGF showed increased phosphorylated Atk, while the expressions of phosphorylated ERK-1/−2 and p38 were without differences.
Conclusion: Exogenous expression of VEGF by dsAAV2 vector could attenuate ischemic brain injury through activation of Akt. In vitro experiments demonstrated that dsAAV2-VEGF could protect cortical neurons from hypoxic injury. Our results suggest that dsAAV2-VEGF is a highly potent neuroprotective tool for stroke therapy.
PRECLINICAL SAFETY PROFILE OF THE RECOMBINANT DIRECT-ACTING FIBRINOLYTIC, ALFIMEPRASE
Yuko Kurosawa1, Joseph Clark1, Aigang Lu1, Kenneth Luskey3, Gail Pyne-Geithman1, Thomas Tomsick2
1Department of Neurology, University of Cincinnati, Cincinnati, OH, USA, 2Department of Neuroradiology, University of Cincinnati, Cincinnati, OH, USA, 3Nuvelo Inc., San Carlos, CA, USA
Purpose. To assess safety of alfimeprase in a rat acute ischemic stroke reperfusion model. Alfimeprase, a rapid direct-acting fibrinolytic, was supplied by Nuvelo. Safety was compared to tissue plasminogen activator (tPA) with hemorrhagic transformation (HT) as primary endpoint and behavioral changes and mortality as secondary endpoints. Hemorrhage is evaluated to provide hemorrhage rate and volume in the brain.
Methods. Five SD rat groups were studied in blinded fashion: alfimeprase at doses of 0.03 (N = 8), 0.1 (N = 11), and 0.3 mg/kg (N = 8); tPA at 1 mg/kg (N = 9); and carrier infused controls (N = 9). The carrier control was the carrier for the alfimeprase. The middle cerebral artery was occluded using a 3–0 suture for 5 hours followed by removal of the suture for reperfusion. Drug or carrier solutions were infused immediately following reperfusion through PE 10 tubing over a 10 minute period. The animals were anesthetized, decapitated and the brains rapidly harvested and frozen approximately 24 hours later. Serial brain sections were obtained (typically 6 sections per brain) and inspected for the number and volume of hemorrhages.
Results. The HT number, severity and the mortality rate for the 0.03 mg/kg dose of alfimeprase were significantly lower than for tPA. In a pooled analysis of all doses, alfimeprase was not significantly different than control for all parameters studied.
Conclusion. Alfimeprase at 0.03 mg/kg dose caused less HT and lower mortality than tPA. When compared to tPA, all other doses of alfimeprase were not significantly worse for all parameters tested. Alfimeprase is a new fibrinolytic agent, with a favorable safety profile based on phase II clinical data, that is appropriate for further evaluation in human stroke revascularization studies.
This work supported by Nuvelo.
LOW FREQUENCY (60 KHZ) TRANSCRANIAL THERAPEUTIC ULTRASOUND IS DETRIMENTAL AND LEADS TO INFARCT ENLARGEMENT IN MCAO IN RATS
Peter Reuter1, Max Nedelmann2,3, Maureen Walberer2,3, Clemens Sommer4, Beat Alessandri5, Oliver Kempski5, Marianne Dieterich1, Holger Kuntze1, Clemens Mueller3, Georg Bachmann3, Manfred Kaps2, Tibo Gerriets2,3
1Department of Neurology, Johannes Gutenberg-University, Mainz, Germany, 2Department of Neurology, Justus Liebig-University, Giessen, Germany, 3Department of Radiology, Experimental Neurology Research Group, Kerkhoff Clinic, Bad Nauheim, Germany, 4Department of Neuropathology, Johannes Gutenberg-University, Mainz, Germany, 5Institute for Neurosurgical Pathophysiology, Johannes Gutenberg-University, Mainz, Germany
Background and aims: Recent experimental and clinical studies have raised concerns about the safety of very low frequency ultrasound in transcranial therapeutic application in cerebral ischemia. This study was designed to evaluate safety aspects and potential deleterious effects of 60 kHz ultrasound in experimental middle cerebral artery occlusion (MCAO) in rats and to gain insight into the nature of observed side effects.
Methods: 45 Male Wistar rats were submitted to either temporary (90 min; groups I and II) or permanent MCAO (groups III and IV) using the suture technique. All animals received rt-PA in a 1 hour infusion starting 90 min after the beginning of occlusion. Groups I and III were additionally treated with 60 kHz ultrasound (0.2 W/cm2, duty cycle 50%). Outcome assessment consisted of MRI and clinical evaluation after 5 and 24 hours, and histology (perfusion fixation after 24 hours).
Results: Mortality was higher in the groups treated with ultrasound, as compared to controls. In groups I and III, most of these animals died during the insonation period. Histology revealed disseminated microscopic bleeding as one possible cause of death. In comparison, all control animals survived the corresponding sham insonation period (p<0.05). In temporary occlusion, the ischemic lesion volume in surviving animals was significantly larger after insonation (p<0.05). No difference in lesion volume was seen after permanent MCAO. Neurological assessment showed impairment of hearing as an additional specific side effect in ultrasound treated animals (p<0.0001).
Conclusions
The results of this study underline the potentially harmful effects of low frequency ultrasound. Different patterns of biological deleterious effects are described, including bleeding, hearing loss and a specific damaging effect of the ischemic penumbra resulting in increased ischemic lesion volume. As the applied ultrasound intensities were low, the results clearly demonstrate the potential limitations of low frequency therapeutic ultrasound and the importance of pre-clinical safety assessment.
LONG-TERM EFFECTS OF MILD HYPOTHERMIA ON GRAY AND WHITE MATTER INJURY AFTER SPINAL CORD ISCHEMIA IN RATS
Toshinori Horiuchi, Masahiko Kawaguchi, Naoko Kurita, Satoki Inoue, Hitoshi Furuya
Department of Anesthesiology, Nara Medical University, Kashihara, Nara, Japan
Background and aims: The short-term (in a several days) effects of mild hypothermia on gray matter injury following spinal cord ischemia (SCI) have been well evaluated. However, its long-term effect remained undetermined. In this study, we assessed long-term effects of intraischemic mild hypothermia on white matter injury as well as gray matter injury after spinal cord ischemia in rats. Methods: Ninety-five male Sprague-Dawley rats were randomly allocated to one of the following 8 groups (n=10-13/each) according to body temperature (degrees C) during SCI and a reperfusion period of the days; 38-2d, 35-2d, 32-2d, sham-2d, 38-28d, 35-28d, 32-28d, and sham-28d. SCI was induced by a balloon catheter and blood withdrawal for 15 minutes. In hypothermic groups, the body temperature was maintained at 35 or 32 decrees C during ischemia and rewarmed in 30 minutes after SCI. The rats were sacrificed 2 or 28 days after SCI for histological assessments. The hind-limb motor function was assessed using the Basso-Beattie-Bresnahan scale scoring. Gray matter injury was assessed on the basis of the number of normal neurons in the ventral horn. White matter injury was assessed on the basis of the extent of vacuolation in the ventral white matter. Results: Although motor function gradually recovered over 28 days, motor function was significantly better in hypothermic groups than in the normothermic group at 2 and 28 days after reperfusion. The gray matter and white matter injuries were significantly lower in hypothermic groups that in the normothermic group at 2 and 28 days after reperfusion. However, the motor function, and gray and white matter injuries were similar between the 32-28d and 35-28d groups. Conclusions: Intraischemic mild hypothermia attenuated hind-limb motor function, and white and gray matter injuries following spinal cord ischemia (SCI) in rats, and the effects sustained for long-term period of 28 days.
THE IMPORTANCE OF CLOT CONSTITUTION IN A RAT EMBOLIC STROKE MODEL
Ivo A.C.W. Tiebosch1, Annette van der Toorn1, Caspaar Bijleveld2, Rick M. Dijkhuizen1
1Image Sciences Institute, University Medical Center Utrecht, Utrecht, The Netherlands, 2Department Biochemistry and Cell Biology, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands
Background and aims: This study was conducted to compare white and red autologous blood clots in their ability to induce experimental embolic stroke in rats, which can be successfully dissolved by thrombolytic therapy.
Methods: Eight male Wistar rats (347+/−13.2 g) were used. Autologous blood was withdrawn from the tail vein. To prepare white emboli, blood was mixed with a 10% volume of 4% sodium citrate and centrifuged. 200 µl of plasma was separated and mixed with 0.5 µl of the pellet and coagulation was initiated with 20 µl of 3% calcium chloride. Red emboli were prepared without separating plasma. All emboli were allowed to coagulate spontaneously during 15-20 h within a PE-50 tube (inner diameter: 0.58 mm), without the use of thrombin.
Experimental stroke was induced by injecting a 25-mm long embolus into the right internal carotid artery through a PE-50 tube connected to a syringe filled with 1 mg/ml rat albumin in phosphate-buffered saline. Two hours following stroke induction, rats received rtPA (10 mg/kg, i.v.).
Serial MRI was done on a 9.4 T MR system. Lesion volume was measured with diffusion- and T2-weighted MRI at 1–2 h, 24 h, and 7 or 8 days after stroke induction. In addition, blood-brain barrier leakage was assessed with T1-weighted MRI before and 30 min after Gd-DTPA injection (0.2 mmol/kg, i.v.), at 24 h and 7/8 days after stroke induction.
Neurological deficit scores (NDS: 0-21) were taken at 1, 3 and 7/8 days after stroke.
Results: Rats that had received white emboli (n=3) had no or a small lesion in the middle cerebral artery (MCA) territory (apparent diffusion coefficient (ADC) lesion volume: 0+/−0 mm3 at 1–2 h (i.e., pre-rtPA); T2 lesion volume: 4.6+/−5.5mm3 at 24 h post-stroke (i.e., post-rtPA)). Also, neurological deficiency was minor (NDS: 1.3+/−0.6 at 3 days post-stroke). Visual analysis of white clots showed that these were irregularly shaped and compressible, as described by Kirchof (ref1).
Occlusion of the MCA with red emboli (n=4) resulted in larger lesions depending on the quality of the injected red clot. Emboli with a relatively low amount of erythrocytes entrapped in the fibrin network induced smaller lesions (ADC lesion volume: 1.3+/−1.8 mm3 at 1–2 h (n=2); T2 lesion volume: 2.6 mm3 at 24 h post-stroke (n=1)). Emboli closely packed with erythrocytes induced large lesions (ADC lesion volume: 99.2+/−101.5 mm3 at 1–2 h; T2 lesion volume: 163.3+/−70.2 mm3 at 24 h post-stroke (n=2)), clear Gd-DTPA leakage and increased NDS (8.5+/−2.1 at 3 days post-stroke).
Conclusions: Our findings suggest that close packing of erythrocytes within the fibrin network is of importance for the preparation of stable, spontaneously coagulated emboli that can effectively occlude the MCA in rat embolic stroke models. Suitable red emboli can be prepared without additional thrombin, which has been shown to affect efficacy of thrombolytic treatment (ref.2). However, experimental conditions should allow rapid coagulation to prevent sedimentation of erythrocytes.
Funded by the Netherlands Heart Foundation (2005B156).
SIMPLE AND RAPID METHOD FOR DETECTION OF PLATELET AGGREGABILITY WITH HEMATOLOGY ANALYZER IN CEREBRAL INFARCTION
Mie Shimizu1, Yasuhisa Kitagawa1, Saori Kohara1, Kentaro Tokuoka1, Yukito Shinohara2, Shigeharu Takagi1
1Department of Neurology, Tokai University School of Medicine, Isehara, Kanagawa, Japan, 2Kyosai Tachikawa Hospital, Tachikawa, Tokyo, Japan
In order to monitor platelet function and select appropriate antiplatelet therapy for the prevention of stroke, platelet activation can be measured by means of flow cytometry, but this method is not suitable for routine clinical use. We therefore developed a simple and rapid method to detect aggregates (clumps) of platelets using a hematology analyzer (Am J Hematol 2003;72:282-283). The purpose of this study is to investigate the clinical usefulness of our method in patients with cerebral infarction.
The subjects were 76 patients with chronic cerebral infarction (58 with lacunar infarction, 18 with atherothrombotic infarction; mean age 66 years) and 59 controls (mean age, 55 years) without cerebral infarction on MRI. Controls were divided into two subgroups according to the presence or not of at least one of hypertention, hyperlipidemia, diabetes mellitus or the predictive factors such as carotid stenosis. Blood samples were drawn into two plastic tubes, one containing sodium citrate and one containing K2-EDTA powder. Both plastic tubes were placed in the CELL-DYN 4000R hematology analyzer (Abbott Diagnostics, Abbott Park IL), which employs multi-angle argon laser light scattering. Platelet aggregates can be identified from a 90- vs. 0-degree light scattering plot. If platelets clumps exist, they appear on the scatter plot as a population of events extending diagonally through a region between mononuclear and polymorphonuclear cells. The appearance rates were 45% in lacunar infarction (26 of 58 patients) and 56% in atherothrombotic infarction (10 of 18 patients). On the other hand, the appearance rate of platelet aggregates in controls without infarction was 48% (28 of 59 patients). Platelet aggregates were not detected in controls without any risk or predictive factors. However, platelet aggregates appeared in 69% of controls (27 of 39 subjects) with one more risk or predictive factors. We suggest that detection of platelet aggregates is useful for estimation of the presence of risk factors for stroke, and for predicting the risk of cerebral infarction. This method is simple and automated, and is suitable for screening platelet function in high-risk patients.
ROLE OF NHE1 IN CALCIUM OSCILLATION AND CELL PROLIFERATION IN HUMAN CNS PERICYTES
Kuniyuki Nakamura1, Masahiro Kamouchi1, Takanari Kitazono1, Junya Kuroda1,2, Ryu Matsuo1,2, Noriko Hagiwara1, Hiroaki Ooboshi1, Setsuro Ibayashi1, Mitsuo Iida1
1Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 2Department of Medicine, Seiai Rehabilitation Hospital and Fukuoka Institute of Neurogenetics and Stroke, Fukuoka, Japan
Background and aims
Central nervous system (CNS) pericytes located at the abluminal side of microvessels, such as arterioles, venules, and particularly capillaries, appear to play an important role in angiogenesis, regulation of blood flow, immune responses, and maintenance of blood-brain barrier (BBB). Na+/H+ exchanger isoform 1 (NHE1) ubiquitously expressed in plasma membrane has been implicated to have a role in proliferation of non-vascular cells through regulation of intracellular pH, Na+ and cell volume; however, the relationship between NHE1 and pericyte growth is not known. Thus, the aim of the present study was to elucidate the role of NHE1 in the proliferation of human CNS pericytes. We have found on intracellular Ca2+ change, an essential signal of cell growth.
Methods
Human brain microvascular pericytes were cultured and cytosolic Ca2+ concentration and pH were measured by fluorescent indicators, fura-2 and 2′, 7′-bis-2-carboxyethyl-5-(6)-carboxyfluorescein (BCECF), respectively. Reverse transcription and polymerase chain reaction (RT-PCR) was used to examine expression level of each mRNA. Knockdown of NHE1 mRNA was done by RNA interference (RNAi) with the double-strand siRNAs targeting NHE1 specifically. Cell proliferation was evaluated by cell count.
Results
Human microvascular pericytes showed spontaneous Ca2+ oscillation in the presence of extracellular Na+ (132mM). A decrease in extracellular Na+ (0-99mM) evoked transient Ca2+ rise followed by Ca2+ plateau or Ca2+ oscillation, whereas increase in extracellular Na+ to 166mM eliminated the Ca2+ responses. A decrease in extracellular pH to 6.5 induced similar cytosolic Ca2+ change as that by low extracellular Na+. Low Na+-induced Ca2+ oscillation was inhibited by hexamethylene amiloride (HMA, an inhibitor of Na+/H+ exchanger) dose-dependently (5–50µM). The Ca2+ oscillation was also inhibited by amiloride (50µM) or benzamil (50µM). Nicardipine (1µM), Gd3+ (100µM), La3+ (100µM) or omission of external Ca2+ did not affect the Ca2+ oscillation. KB-R7943 (10µM; a selective inhibitor of Na+/Ca2+ exchanger), carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (1µM; FCCP, the mitochondrial uncoupler), or changes in the external osmolarity did not affect the Ca2+ oscillation. On the other side, the Ca2+ oscillation was completely abolished by pretreatment with thapsigargin (1µM; an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase), suggesting that Ca2+ oscillation was originated from endoplasmic reticulum. RT-PCR revealed that human CNS pericytes expressed NHE1 and NHE7. Low extracellular Na+ could not induce the Ca2+ oscillation in the cells transfected with specific siRNA targeting NHE1. Proliferation of the pericytes was significantly attenuated by addition of HMA (5µM) to the medium. Knockdown of NHE1 by transfecting mRNA also inhibited the proliferation of pericytes.
Conclusions
These results indicate that NHE1 plays an important role in Ca2+ signaling via modulation of endoplasmic reticulum and thereby contributes to the regulation of proliferation in CNS pericytes.
This novel role of NHE1 in the pericytes may have pathophysiological relevance to angiogenesis or BBB disruption in the cerebral ischemia.
HYPOTHERMIA'S NEUROPROTECTION AGAINST ISCHEMIC REPERFUSION INJURY IS MEDIATED BY P-PTEN
Sang Mi Lee, Heng Zhao, Guo H. Sun, Gary K. Steinberg
Department of Neurosurgery, Stanford Medical Center, Palo Alto, CA, USA
Introduction: Hypothermia is protective against stroke, possibly by lowering levels of reactive oxygen species (ROS). We now explore the mechanistic relationship between ROS and hypothermia by looking at the effects of post-ischemic hypothermia. We previously showed that negative regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) might contribute to the protective effects of intra-ischemic hypothermia. Here, we investigated the interplay between post-ischemic hypothermia, ROS activity and PTEN regulation in a focal ischemia model in rat. We also employ a genetic model of elevated ROS, SOD2 knockout mice.
Methods: SD rats were subjected to 1 hr occlusion of the left MCA and bilateral CCAs. Body temperature was either 37oC (group 1), lowered to 30oC immediately upon reperfusion (group 2), or lowered to 30oC 20 min after reperfusion (group 3). In mice, ischemia was generated by intraluminal suture occlusion of the MCA for 30 min, and mild hypothermia (33 oC) was induced at ischemic onset and maintained during the occlusion. Infarct volume was measured by TTC staining. ROS was detected in situ by injecting hydroethidine. PTEN and p-PTEN were detected by western blot and immunohistochemistry. N-tert-Butyl-a-(2-sulfophenyl) nitrone (PBN) was used to inhibit ROS.
Results: Hypothermia initiated immediately after reperfusion significantly reduced infarct volume (n=8, ***p<0.0001), but hypothermia delayed 20 min after reperfusion did not provide protection. Hypothermia suppressed ROS generation (n=4, *p<0.05) and maintained p-PTEN levels with immediate hypothermia (n=4, *p<0.005), but not in with delayed hypothermia. Inhibiting ROS blocked the reduction in p-PTEN after reperfusion (n=3, *p<0.05), indicating that reduction in p-PTEN might be caused by ROS after ischemia. Hypothermia reduced ROS generation after stroke in Wt and SOD2K/O mice. However, p-PTEN levels were significantly lower in SOD2K/O mice than in Wt after stroke (n=4, *p<0.05) Hypothermia blocked the decrease of p-PTEN in both cases.
DOPAMINE MODULATION ON EXCITATORY SYNAPTIC TRANSMISSIONS IN STRITAL NEURONS AFTER TRANSIENT CEREBRAL ISCHEMIA
Yuchun Zhang, Ping Deng, Zao C. Xu
Department of Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA
Neurons in the neostriatum receive massive glutamatergic inputs from cerebral cortex and dopaminergic inputs from substantia nigra. Spiny neurons in the neostriatum are highly vulnerable to cerebral ischemia. Enhancement of glutamatergic excitatory synaptic transmissions has been implicated in ischemia-induced excitotoxic neuronal death. Dopamine concentration dramatically increases during ischemia. Despite enormous studies suggest that dopamine is involved in the ischemia-induced neuronal loss in the striatum, it remains unclear how dopamine interacts with glutamatergic excitotoxicity. The present study investigates the dopamine modulation on excitatory synaptic transmission in spiny neurons after ischemia.
Transient forebrain ischemia was induced in male adult Wistar rats for ~20 minutes using 4-VO model. Brain slices were prepared at different intervals after reperfusion. Whole-cell voltage-clamp recordings were performed on medium-sized neurons in the neostriatum to examine locally evoked excitatory postsynaptic currents (EPSCs) or spontaneous miniature EPSCs.
Evoked EPSCs in spiny neurons were potentiated at 9 h after ischemia. The ischemia-induced potentiation in synaptic efficacy was associated with an enhancement of presynaptic release as demonstrated by an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and a decrease in the paired-pulse ratio. The amplitude of inward currents evoked by exogenous application of glutamate did not show significant changes after ischemia, suggesting that postsynaptic mechanism is not involved. The ischemia-induced increase in mEPSCs frequency was not affected by blockade of voltage-gated calcium channels, but it was eliminated in the absence of extracellular calcium. Bath application of ATP P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) significantly reduced mEPSC frequency in ischemic neurons but had no effects on the control ones. Furthermore, the inhibitory effect of PPADS on ischemic neurons was abolished in Ca2+ -free external solution.
We found that dopamine depressed EPSCs in post-ischemic neurons through D1-like receptors (D1Rs). D1Rs activation also reduced the frequency of miniature EPSCs and increased the paired-pulse ratio, suggesting a presynaptic mechanism. 8-Br-cAMP, an activator of cAMP-dependent protein kinase (PKA), mimicked the depressive effects of D1Rs activation. Bath application of Rp-cAMP, an inhibitor of PKA, blocked the D1Rs-induced depression of EPSCs in post-ischemic spiny neurons, although intracellularly applied Rp-cAMP had no effects. In addition, D1Rs failed to reduce EPSCs amplitude in the presence of adenosine A1 receptors (A1Rs) antagonist.
These results indicate that excitatory synaptic transmissions in spiny neurons are potentiated after ischemia, which might be associated with excitotoxic cell death. However, dopamine, through D1Rs activation, depresses excitatory synaptic transmission in spiny neurons, suggesting that dopamine modulation on excitatory transmission does not contribute to the excitotoxicity after ischemia.
NEUROPROTECTIVE EFFECT OF EDARAVONE IN RAT TRAUMATIC BRAIN INJURY MODEL - THE KINETICS OF OXIDATIVE STRESS AND ANTIOXIDANT POTENCY-
Kenji Dohi1, Kazue Satoh2, Sachiko Yofu2, Tomoya Nakamachi2, Shunsuke Nakamura1, Seiji Shioda2, Tohru Aruga1
1Department of Critical Care and Emergency Medicine, Showa University, Tokyo, Japan, 2Department of Anatomy, Showa University, Tokyo, Japan
INTRODUCTION
A large number of studies have reported that oxidative stress plays a key role in the development of TBI. Consequently, one of the keys to managing TBI may be to control lipid peroxidation. Edaravone (MCI-186), a novel free radical scavenger, was developed to prevent lipid peroxidation in neurological conditions, and is currently the only such drug approved for use on cerebral infarction patients, having been shown to improve the functional outcome as compared to placebo following ischemic stroke. Since ischemic damage contributes to the complicated pathophysiology of TBI, edaravone was also thought to have therapeutic potential in such cases. However, it has not yet been determined whether it exerts a protective effect in TBI.
In the present study, we investigated the neuroprotective effect of edaravone in a rat TBI model. We also analyzed its effect on alkoxyl radicals (RO?), hydroperoxide and antioxidant potency by assaying jugular vein serum.
MATERIALS AND METHODS
Animals were anesthetized with a 50 mg/kg of sodium pentobarbital. After craniectomy, a sterile metal rod 4 mm in diameter which is the device for cold injury, was chilled in liquid nitrogen, then applied to the surface of the parietal cortex for 1min. After a 10 min injury period, edaravone (3 mg/kg i.v) was administered via the right femoral artery (edaravone (+) group). Blood alkoxyl radical intensities were measured with electron spin resonance (ESR). Blood hydroperoxide levels (d-ROM) and bio antioxidant potential (BAP) were evaluated using using the free radical electron evaluator (FREE).
RESULTS
The damaged area was significantly smaller in animals treated with edaravone. Jugular blood alkoxy radical intensity was suppressed 24 hours after TBI. Edaravone also suppressed serum ROM concentrations in the jugular vein at 6 and 12 hours. Serum antioxidant potency and α-tocopherol concentrations were higher in edaravone-treated animals 6 hours after TBI.
CONCLUSION
In conclusion, this study provides that edaravone, a free radical scavenger, can exert neuroprotective and antiradical effects following TBI in the rat. Given that lipid peroxidation plays an important role in neurotrauma, the investigation of free radical generation, as well as determining antioxidant abilities and the effects of antioxidants including α-tocopherol, may be important for evaluating the overall balance of oxidative stress following acute brain injury.
EFFECTS OF PIOGLITAZONE ON ISCHEMIC CHANGE OF HIPPOCAMPAL CA1 FOLLOWING CEREBRAL ISCHEMIA AND REPERFUSION IN A TYPE2 DIABETIC ANIMAL MODEL
Yasuo Ito, Nobuo Araki, Takeshi Ohkubo, Yoshio Asano, Daisuke Furuya, Kimihiko Hattori, Tomokazu Shimazu, Harumitu Nagoya, Yuji Kato, Kunio Shimazu
Department of Neurology, Saitama Medical University, Moroyama, Saitama, Japan
Introduction: We have reported that peroxisome proliferator-activated receptor (PPAR gamma) agonist, pioglitazone, protects against cerebral injury by anti-oxidant mechanisms[1]. It is suggested that hyperglycemia during acute cerebral ischemia is associated with poor prognosis. The purpose of this study is to investigate the effects of pioglitazone on ischemic change of hippocampal CA1 following cerebral ischemia and reperfusion in Otsuka Long Evans Tokushima Fatty (OLETF) rats, an animal model of obesity and type 2 diabetes.
Methods: (1) Male OLETF rats (n=11) were used. Pioglitazone 20 mg/kg/day was given in 6 rats for 4 days (pioglitazone group), and 5 rats were used as contol group. NO production was continuously monitored by in vivo microdialysis. Microdialysis probes were inserted into the left striatum and hippocampus and were perfused with Ringer's solution at a constant rate 2 µl/min. Laser Doppler probes were also inserted into the right striatum and hippocampus. Blood pressure, blood gases and temperature were monitored and maintained within normal ranges throughout the procedure. After 2 hours equilibrium period, fractions were collected every 10 minutes. Forebrain cerebral ischemia was produced by occlusion of both common carotid arteries, and systemic hypotension (MABP< 50mmHg) was induced by hemorrhage. After 20 minutes, the loops around both common carotid arteries were released and the blood was reinfused. Levels of NO metabolites, nitrite (NO2-) and nitrate (NO3-), in the dialysate were determined using the Griess reaction. (2) Analysis of hippocampus CA1 neurons: Two hours after the start of reperfusion, animals were perfused with 4% paraformaldehyde. We divided hippocampal CA1 neurons into three phases (a group of severe ischemia, a group of moderate ischemia, a group of survive), and the ratio of the number of surviving neurons in comparison to the degenerated ones was calculated (survival rate).
Results:
Blood Pressure: There were no significant differences between the both groups. Cerebral Blood Flow (CBF): There were no significant differences between the both groups in hippocampus. Nitric oxide metabolites in hippocampus: i) NO2-; There were no significant differences between the both groups. ii) NO3-; pioglitazone group (0.69 ± 0.15µmol/L; mean ± SD, 1.11± 0.35) showed significantly higher than that of control group (0.54 ± 0.52, 0.74 ± 0.72) during ischemia, and pioglitazone group showed significantly higher levels than the control group in 20~70 minutes after the start of reperfusion (Fig.1). Survival rate in hippocampus CA1 area: pioglitazone group (79.4 ± 5.9%; mean ± SD) showed significantly higher than that of control group (52.9 ± 1.7) (Fig.2).
Conclusion: These in vivo data suggest that pioglitazone influences on the NO production in hippocampus in a type 2 diabetes mellitus model rat, and may protect against cerebral ischemic injury following ischemia and repurfusion.
OLIGODENDROGENESIS AFTER CHORNIC CEREBRAL HYPOPERFUSION IN RATS
Nobukazu Miyamoto, Ryota Tanaka, Ning Zhang, Yuji Ueno, Takao Urabe
Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
Background and purpose
Although white matter lesions contribute to cognitive impairment in post-stroke patients, tissue repair in those lesions is not clear. The present study was designed to assess the oligogenetic potential for tissue repair, against white matter damage caused by cerebral ischemia and that such effect is enhanced by cilostazol, a potent inhibitor of PDE III through bFGF activation in a rat chronic cerebral hypoperfusion model.
Methods
Rats underwent bilateral common carotid artery ligation (LBCCA). They were divided into three, sham operated (n=45), vehicle (n=45), and the cilostazol treated (n=45) groups. 5-Bromodeoxyuridine (BrdU, 50mg/kg) was injected for analysis of newly generated cells in white matter. Performance at the Morris water maze task and immunohistochemistry for BrdU, glutathione S transferase-pi (GST-pi), platelet derived growth factor receptor-α (PDGFR-α), basic fibroblast growth factor (bFGF), and phosphorylated cAMP-responsive element binding protein (pCREB) were analyzed at 7, 14, 21, and 28 days after hypoperfusion.
Result
At Morris water maze task, the vehicle group showed a significantly longer escape latency than the sham group (p<0.05). Interestingly, the escape latency of the cilostazol group gradually decreased over the testing period compared with the vehicle group (p<0.05). The white matter lesions were progressed in the corpus callosum in time dependent manner in vehicle group (p<0.05), but not progressed in sham and cilostazol group. Oligo progenitor cells double labeled by BrdU/PDGFR-α in corpus callosum significantly increased after ischemia compared with sham operated animals (p<0.01). Newly generated oligo progenitor cells also differentiated into more mature phenotype in early phase after ischemia, but this discontinued in later period (p<0.05). Cilostazol treatment did not increase the number of BrdU positive cells after ischemia compared with vehicle group (p<0.05), while it markedly promoted the differentiation of oligo progenitor cells into more mature states (p<0.01). The treatment of cilostazol upragulated bFGF signaling and subsequent activation of pCREB.
Conclusion
Our result indicated that chronic cerebral hypoperfusion trainsiently promoted generation of oligo progenitor cells, and application of cilostazol resulted in generation of mature oligodendrocyte even in later period of ischemia. Cilostazol could be a useful treatment for recovery of cognitive impairment in post stroke patients.
EFFECT OF EDARAVONE ON BRAIN EDEMA AND NEUROLOGICAL DEFICITS AFTER EXPERIMENTAL INTRACEREBRAL HEMORRHAGE
Takehiro Nakamura1, Yasuhiro Kuroda2, Osamu Miyamoto1, Toshifumi Itano1, Seigo Nagao3, Guohua Xi4, Richard Keep4
1Department of Neurobiology, Kagawa University Faculty of Medicine, Miki, Kagawa, Japan, 2Department of Emergency and Critical Care Medicine, Kagawa University Faculty of Medicine, Miki, Kagawa, Japan, 3Department of Neurological Surgery, Kagawa University Faculty of Medicine, Miki, Kagawa, Japan, 4Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA
Background and aims: Our previous studies have indicated that iron accumulation and oxidative stress contribute to secondary brain injury in the brain after intracerebral hemorrhage (ICH). We, therefore, examined whether edaravone, free radical scavenger, can reduce ICH-induced brain injury. Methods: There were three sets of experiments in this study. Pentobarbital anesthetized male Sprague-Dawley rats were anesthetized received an infusion of 100-µl autologous whole blood into the right basal ganglia or just a needle insertion (shams). The rats were sacrificed 24 hours later. In the first set of experiments, iron distribution was examined histochemically (enhanced Perl's reaction). In the second set, the effects of edaravone on ICH-induced brain injury were examined by measuring brain edema and neurological deficits. Edaravone or vehicle were administered (6mg/kg, immediate, two or six hours after ICH, ip, n=5 in each group). Behavioral test was used forelimb placing test. In the third set, apurnic/apyrimidinic abasic sites (AP sites) and 8-Hydroxyl-2′-deoxyguanosine (8-OHdG), which are hallmarks of DNA oxidation, were investigated by ELISA after treatment for ICH (n=3). Results: Iron accumulation was observed in the perihematomal zone 24 hours after ICH. Systemic administration of edaravone immediate and 2 hours after ICH reduced brain water content in the ipsilateral basal ganglia 24 hours after ICH compared with vehicle (80.9 ± 0.8% vs. 84.1 ± 1.5%, 81.3 ± 0.8 % vs. 83.8 ± 1.7 %, mean ± SD, respectively, p < 0.05). Delayed edaravone treatment 6 hours after ICH failed to reduce brain edema. Edaravone treatment immediate and 2 hours after ICH also ameliorated neurological deficits (Forelimb placing score: 57 ± 17 % vs. 12 ± 13 %, p < 0.05). Edaravone attenuated ICH-induced changes in AP-sites and 8-OHdG (Figure 1A&B). Conclusions: Edaravone and other free radical scavengers may be potential therapeutic agents for ICH. They may act by reducing the oxidative stress caused by the release of iron from the hematoma.
EDARAVONE MAY PROTECT WHITE MATTER DISORDERS IN A CHRONIC RAT HYPOPERFUSION
Yuji Ueno, Nobukazu Miyamoto, Ryota Tanaka, Nobutaka Hattori, Takao Urabe
Department of Neurology, Juntendo University, Bunkyo-Ku, Tokyo, Japan
Background and purpose
Edaravone, free-radial scavenger, is effective to protect against brain ischemia. Using a rat model of transient middle cerebral artery occlusion, it was revealed that edaravone exerted neuroprotive effect through the early scanvenging pathway and late anti-inflammatory effect. However, in a chronic hypoperfusion rat model, little information is available on its role. In human studies, edaravone contributed to good functional outcome in stroke patients. We hypothesized that edaravone played an role of neuroprotection in a chronic rat hypoperfusion.
METHODS
Adult males Wister rats (9 weeks), weighting 270 to 300 g, underwent bilateral common carotid artery ligation. They were divided into the edaravone group (n=15) and the vehicle (control) group (n=15). Immediately and 30 minutes after ligation of both carotid arteries, 3mg/kg of edaravone were administered intravenously. Klüver?Barrera staining and immunohistochemistry of glutathione-S-transferase-pi (GST-pi) were analyzed at baseline and at 3, 7, 14, 21, and 28 days after hypoperfusion. Bilateral corpus callosum were evaluated in each rat.
Results
The number of GST-pi positive oligodendrocytes tends to be higher in the edaravone group compared with the vehicle group on each day. In particular, on 21day, there was significant difference of the number of GST-pi positive oligodendrocytes in the edaravone and vehicle group (211±21 vs 176±16 (cells/0.25 cm2), P=0.019) (FIgure 1). On Klüver?Barrera staining, the vehicle group predominantly showed the disarrangement of the nerve fibers and formation of marked vacuoles on 21 and 28 days (Figure 2).
Conclusion
Our study identified that white matter damages were alleviated in the edaravone group rather than the vehicle group. Therefore, it was suggested that edaravone might be effective to protect white matter against chronic hypoperfusion. Previous study reported that edaravone mediate via its antioxidant effect through the suppression of lipid peroxidation and oxidated DNA damage. Our findings suggest these mechanisms might be found in chronic hypoperfusion. Further study using other immunohistochemistry and much larger sample size should be needed.
IN VIVO 1H CSI MEASUREMENT OF TEMPERATURE AND PH IN RAT BRAIN
Daniel Coman1, Hubert Trubel1,2, Fahmeed Hyder1
1Department of Diagnostic Radiology, Yale University, New Haven, CT, USA, 2University Witten, Herdecke, Wuppertal, Germany
INTRODUCTION
The proton chemical shifts from the complex between the thulium ion and the macrocyclic chelate DOTP8- are strongly dependent on temperature and pH [1]. Here we have been extended our past work with TmDOTP5- to exploit the abundant temperature and pH information that is available in the observable TmDOTP5- resonances. As a consequence the new TmDOTP5-model was calibrated for accurate temperature (±0.05 °C) and pH (±0.02) measurements.
METHODS
The data base for the model were obtained from samples of 5 mM Na(TmDOTP5-) and TSP at different pH values (6.5?8.0) and CaCl2 concentrations (0?5 mM). In vitro 1H NMR spectra were acquired on an 11.7T system at various temperatures (25?45 °C). 2D CSI was done with 16 (in vivo) or 32 (in vitro) encode steps in each direction.
RESULTS
A parametrized solution exists in which a given chemical shift of TmDOTP5- (A) depends on specific values of temperature, pH, and [Ca2+]. The maximum number of protons that can be used in the proposed model is three (H2, H3 and H6) excluding the low SNR observable resonance (H1). Using in vitro samples with high SNR and uniform temperature/pH within an NMR tube, at a given [Ca2+], the equations below describe temperature/pH dependencies
T=a1+a2pH+a3ln(Δ2)+a4ln(Δ3)+a5ln(Δ6) and pH=b1+b2T+b3ln(Δ2)+b4ln(Δ3)+ b5ln(Δ6)
where T is the temperature and Δ2, Δ3 and Δ6 are the chemical shifts of H2, H3 and H6 protons, respectively. The parameters ai and bi were obtained at each [Ca2+] by multiple linear regressions. This approach to calculate temperature/pH distributions is demonstrated by an in vitro 2D CSI experiment on three plastic tubes containing TmDOTP5- (B) at various pH values, all at room temperature. The results of the 2D CSI experiment (C) were used to calculate temperature/pH maps with high accuracy, where the small variations in temperature/pH values within each tube are not a result of the parameterization model used, but rather SNR limitations which can be improved upon further. In vivo results from a rat (D) demonstrate homogenous temperature/pH distribution within expected range [1].
ACKNOWLEDGEMENTS
Supported by grants from NIH (MH-067528, DC-003710).
EXOGENOUS ETHYLPYRUVATE VS PYRUVATE DURING METABOLIC RECOVERY AFTER OXIDATIVE STRESS IN NEONATAL RAT CEREBROCORTICAL SLICES
Lawrence Litt1, Jianying Zeng1, Guo-Yuan Yang1, Mark Kelly2
1Anesthesia Department, University of California, San Francisco, CA, USA, 2UCSF Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA
Exogenous ethylpyruvate (EP) is known to provide better outcomes in ischemia and oxidative stress than equimolar doses of exogenous pyruvate (pyr). In solution pyr spontaneously forms parapyruvate, a very potent metabolic inhibitor. Exogenous EP's superiority has been attributed to the absence of parapyruvate, better radical scavenging, and improved boosting of TCA Cycle metabolism via pyr delivery after hydrolysis of the ester linkage. Recent in vivo studies found a wide neuroprotection window for EP in cerebral ischemic injury and H2O2 challenges. We wanted to learn the relative importance of EP's additional radical scavenging compared to its metabolic assistance via provision of pyr as a TCA substrate. Oxygenated ex vivo neonatal rat cerebrocortical slices, 350µ thick from P7 Sprague-Dawley rats, were acutely exposed for 1 hr to a 2 mM H2O2 stress, and then recovered during superfusion with oxygenated artificial ACSF preparations having 2 mM glc and one of three unique ACSF components: a) 20 mM exogenous EP; b) 20 mM exogenous pyr; and, c) 1 mM of the non-metabolizable radical scavenger N-tert-butyl-α-phenylnitrone (PBN). Each study was repeated three times along with control runs. Slices were taken and frozen at t=0h (just before the H2O2 stress), and again at t=5h (four hrs after the H2O2 stress.) PCA extracts for the two time points were studied with 14.1 Tesla 31P/1H NMR spectroscopy. In a special study 13C-EP was administered, with the label being at the methyl group (C3) of the pyruvate moiety. 2D[1H-13C] HSQC NMR spectra were acquired to look for intracellular metabolism of EP's pyr, and also for a proposed radical scavenging pathway that has EP being turned into formate and acetate. Acute cell injury was assessed by counting TUNEL stained cells. RESULTS: For each rescue solution 1H NMR spectroscopy provided accurate changes for the following metabolites: lactate, alanine, NAA, GABA, glutamate, glutamine, succinate, PCr/Cr, taurine, and myoinositol. NAA and alanine, measured at t=5h for the EP group were significantly greater than values for the no treatment group. EP of 20 mM was more effective than PBN or pyr, achieving higher levels of Cr/PCr and NAA, the neuron marker. 31P data showed that, compared to other rescue regimens, ethylpyruvate also provided the best ATP preservation, but levels were higher by only 12%−29%, although ADP/ATP ratios were lowered by 30%−45% (p < 0.05). Overlaid HSQC spectra for 13C-pyr and 13C-(ethylpyruvate) showed the same TCA metabolites being generated in both groups. The acetate/formate pathway was not seen. TUNEL positive cell percentage in the ethylpyruvate group was significantly less than half of that in the PBN or pyruvate rescue groups (p<0.05), and less than a third of that in the “no treatment” group. CONCLUSION: Ethylpyruvate easily entered cells and provided pyr as a TCA substrate. It was more protective than pure pyruvate or a pure radical scavenger, and thus appears to work via both mechanisms: substrate addition and antioxidant augmentation. Supported by NIH grant R01 GM034767.
SINGLE-CELL RESOLUTION MAPS OF POTASSIUM UPTAKE IN THE RODENT CNS - IMPLICATIONS FOR CEREBRAL ENERGY METABOLISM AND FUNCTIONAL NEUROIMAGING
Juergen Goldschmidt, Holger Lison, Henning Scheich
Leibniz Institute for Neurobiology, Magdeburg, Germany
BACKGROUND AND AIMS: The cerebral metabolic rates of oxygen and glucose in awake animals and humans in the resting state differ in different brain regions, most likely reflecting, at least in part, differences in neuronal activity. The degrees to which excitatory neurons, inhibitory neurons or astrocytes, and different subcellular compartments such as axons or dendrites contribute to these regional differences in brain metabolism are largely unknown since no methods exist to map, cell-by-cell, either the electrical discharges of neurons or oxygen- or glucose-consumption in the brain.
In order to gain insight into the patterns of brain metabolism and neuronal activity in the resting state at the single-cell level we here made use of a recently introduced technique, thallium autometallography, that makes it possible to non-radioactively map patterns of potassium (K+) uptake in the brain of experimental animals.
Thallium autometallography is a tracer technique similar in rationale to the 2-deoxyglucose method. It is based on the histochemical detection of thallium (Tl+), which has been used for decades as a K+-probe in vitro and in myocardial imaging in humans in vivo.
METHODS: We injected awake behaving unstimulated rodents with Tl+ and mapped the distribution of the tracer in the central nervous system.
RESULTS: We find complex distributions of the tracer at the regional, cellular and subcellular level throughout the central nervous system. Different areas of cerebral cortex, for instance, differ in laminar and cellular uptake patterns. Areas that are known for high metabolic rates in awake resting humans, in particular the cingulate cortical areas, also have high rates of Tl+-uptake in rodents. Tl+-uptake in these areas is especially high in upper cortical layers. In sensory cortical areas Tl+-uptake is high in cortical layers III and IV, and especially in inhibitory interneurons in these layers. Uptake in GABAergic neurons in the CNS in general differs in different cell types. Tl+-uptake in cerebellar Purkinje cells, for example, is usually low.
In many brain regions, e.g. cerebral cortex and brainstem, the tracer is found predominantly in the somatodendritic compartments of neurons. Since neuronal K+-uptake is mediated to a large degree by the Na,K-ATPase this distribution argues for predominant somatodendritic energy consumption. In other regions, e.g. thalamic relay nuclei, neuronal uptake is comparatively low despite high glucose consumption, and astrocytic uptake is much more pronounced.
CONCLUSIONS: We conclude from this highly complex distribution that, in the mammalian CNS, no general rules exist with respect to the metabolic demands of neuronal versus astrocytic processing or excitation versus inhibition. Instead, region-specific intricate networks seem to exist in which the metabolic demands of the constituent elements vary in different ways depending on the different modes of neuronal processing.
IMMUNOHISTOCHEMICAL STUDY OF THE OREXIN SYSTEM IN THE RAT BRAIN.-POSSIBLE INVOLVEMENT IN MIGRAINE PATHOGENESIS
Kenzo Koizumi1, Junichi Hamada1, Michinari Fukuda2, Junko Yonekura1, Naomi Kanazawa1,
Fumihiko Sakai1
1Department of Neurology, Kitasato University School of Medicine, Kanagawa, Japan, 2Kitasato University School of Allied Health Sciences, Kanagawa, Japan
The orexin system is known to project from neurons of lateral hypothalamus area to all over the central nervous system except cerebellum and modulates sleep and vigilance. Clinically, migraine patients often experience that migraine could be induced either by lack of sleep or too much sleep, yet sleep may relieve headache. These suggest that sleep plays a role in the migraine pathogenesis.
To elucidate the role of the orexin system in the pathophysiology of migraine, we investigated the distribution of orexin-A and orexin-1 receptors in dura mater, trigeminovascular system and hypothalamus and periaqueductal gray and medulla oblongata and upper cervical spine and autonomic ganglia (superior cervical ganglion, otic ganglion, sphenopalatine ganglion) of rat.
A total of 5 Sprague Dawley rats, weighing 350-450g, were perfused with ice-cold Zamboni's fixative. Hypothalamus and periaqueductal gray and medulla oblongata and trigeminal ganglia, dorsal root ganglia, autonomic ganglia and upper cervical spinal cord as well as circle of Willis with its major branches and dura mater were dissected. Specimens were sectioned in 5µm thick, and dura mater and pial arteries were processed as the whole mount preparation. The orexin-A was detected by rabbit anti-orexin-A polyclonal antibody (Chemicon International Inc.), and orexin-1 receptors were also detected by rabbit anti-orexin-1 receptor affinity purified polyclonal antibody (Chemicon International Inc.). Immunofluorecence staining method was utilized.
In the lateral hypothalamic area, both orexin-A and orexin-1 receptor-immunoreactive (IR) neurons and nerve fibers were observed. In periaqueductal gray and dorsal raphe, orexin-A-IR nerve fibers and orexin-1 receptor-IR neurons were also observed. In trigeminospinal tract orexin-A-IR fibers were observed. In the spinal trigeminal nucleus orexin-1 receptor-IR neurons were observed. Neither orexin-A nor orexin-1-receptors immunoreactivity was observed around the pial arteries. In dura mater and dural arteries orexin-1 receptor-IR nerve fibers were observed. Any orexin-A-IR neuron was not observed in these ganglia. The immunoreactivity of orexin-1 receptors was considered to be nonspecific outcome.
This study demonstrates that the orexin system innervates intracranial structures which may be involved in the migraine pathogenesis. Particularly, it has never been reported that orexin-1 receptors express on dura mater and dural arteries. It is suggested that the orexin system acts on dura and dural arteries directly and via noradrenergic and serotonergic systems.
ROLE OF MCT1 AND MCT2 IN LACTATE UPTAKE BY PRIMARY CORTICAL ASTROCYTES AND NEURONS IN VITRO: A RNA INTERFERENCE STUDY
Fumihiko Maekawa, Luc Pellerin
Department of Physiology, University of Lausanne, Lausanne, Switzerland
Background and aims
Supply of lactate by astrocytes to neurons during activation of specific brain areas has been suggested to be an important mechanism to support neuronal activity. Monocarboxylate transporters expressed in astrocytes and neurons are proposed to play pivotal roles in this process of lactate shuttling. In mouse cerebral cortex, the monocarboxylate transporters (MCTs) known as MCT1 and MCT2 are found specifically on astrocytes and neurons, respectively. Despite such a suggestive distribution, it remains to be functionally demonstrated whether endogenous MCT1 and MCT2 indeed serve such a lactate shuttling role. As a first step to clarify this point, we used the recent advanced RNA interference technology in order to obtain knockdowns of MCT1 and MCT2 and investigated the impact on lactate uptake by astrocytes and neurons.
Methods
Astrocyte-enriched and neuron-enriched cultures were made from mouse cerebral cortices taken on postnatal day 1 and embryonic day 17, respectively. Astrocytes were treated with the small interfering RNA (siRNA) against MCT1 mRNA at 9 days in vitro (DIV9) and lactate uptake assay was performed at DIV11. The siRNA against MCT2 mRNA was applied on cultured neurons at DIV3 and lactate uptake assay was performed at DIV8. In both cases, non-silencing siRNA with no homology to any known mammalian gene was used as negative control. Quantitative measurements of MCT1 and 2 proteins were performed by western blot. For lactate uptake assay, astrocytes and neurons were incubated with [14C]-labeled lactate in MES buffer (pH6.0) for 10 sec and for 30 and 60 sec, respectively. After incubation, cells were washed with MES buffer and homogenized in 0.1N NaOH and 0.1% Triton-X. Radioactivity was measured by scintillation counting and values were normalized by the amount of total protein measured with the Bradford method.
Results
We observed that treatment of astrocytes with the siRNA against MCT1 mRNA and of neurons with the siRNA against MCT2 mRNA inhibited the expression of each protein, respectively. Expression levels of MCT1 in astrocytes treated with siRNA against MCT1 mRNA were less than 10% while MCT2 protein levels in neurons treated with siRNA against MCT2 mRNA were less than 40%, in comparison with cells treated with negative control siRNA. As a consequence of reduced MCT1 expression, lactate uptake was significantly decreased in astrocytes (36 % vs negative control). Similarly, lactate uptake was significantly decreased in neurons with reduced MCT2 expression (62 % vs negative control).
Conclusions
These results suggest that MCT1 and MCT2 are necessary for lactate uptake by astrocytes and neurons, respectively. It seems that these transporters may facilitate cellular energy substrate supply under basal conditions. In addition, these transporters might be necessary for lactate shuttling between astrocytes and neurons under conditions of excitation in specific brain areas, although assessment of lactate release by astrocytes following treatment with siRNA against MCT1 mRNA should be performed in a future study. Further studies using this approach should allow us to better characterized the roles of these transporters in brain energy homeostasis.
TRANSIENT ENERGETICS FROM FMRI: SINGLE EVENT TO BLOCK DESIGN PARADIGMS
Basavaraju G. Sanganahalli, Peter Herman, Fahmeed Hyder
Department of Diagnostic Radiology, Yale University, New Haven, CT, USA
Correlation and/or deconvolution methods have been used to examine the “dynamic” coupling between neuronal activity and fMRI signals. These approaches have resulted in heuristically-based mathematical models for describing BOLD and its underlying physiology. We are investigating the relationships between the complex components of the BOLD signal (e.g., CBF, CBV, CMRO2) and neuronal activity (e.g., MUA, LFP), both at steady-state and transiently. The aim of this study was to examine the degrees of correlation from the single event to the block design paradigm. By control of the stimulus frequency and the number of stimulus impulses, we hypothesized that it should be possible to extrapolate transients of CMRO2 using the time-dependent behaviors of non-energetic components of BOLD (i.e., CBF, CBV) and neuronal activity (e.g., MUA, LFP). All studies were conducted at 11.7T and concurrent extracellular recordings in an anesthetized rat model with electrical stimulation of the forepaw. By conducting experiments at different frequencies, we found variable dependencies of each component. Because of the fact that the BOLD and CBF (or CBV) relationships are significantly different under these range of conditions, we infer that CMRO2 ? the unmeasured dominant component of BOLD ? has a strong dependence for stimulation frequencies ranging from 1.5 to 6.0 Hz. This indirect interpretation of the high time resolution changes in CMRO2 is supported by relationships with neuronal activity (e.g., MUA, LFP) data, thus providing a pseudo validation of transient neuronal energetics that underlying BOLD. These preliminary results suggest that significant oxidative changes can occur as early as ~200 ms following stimulation onset for the anesthetized rat.
ACKNOWLEDGEMENTS
Supported by NIH (DC-003710, MH-067528) and OTKA (T34122) grants.
CAN REPOLARIZATION OF THE AREA SHOWING PERMANENT DEPOLARIZATION BE INDUCED BY INTRAISCHEMIC HYPOTHERMIA IN A FOCAL ISCHEMIC RAT MODEL?
Toshihiro Sasaki, Yoshimasa Takeda, Minako Arai, Hideki Taninishi, Motomu Kobayashi, Kiyoshi Morita
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan
Background: In rat focal ischemia, the permanently depolarizing ischemic core has been suggested to result in infarction, and it is well known that NADH concentration is increased by ischemia.
In the present study, we examined changes in cortical NADH fluorescence and DCpotential to determine whether intraischemic hypothermia results in repolarization of the area showing persistent depolarization.
Methods: Three male spontaneously hypertensive rats weighing 300±20 g were used in this study. Anesthesia was maintained with 1% halothane in a mixture of 30% O2 and 70% N2O.
Focal ischemia was initiated by occlusion of the left common carotid artery and middle cerebral artery (MCA).
Hypothermia was initiated at 15 minutes after occlusion of the MCA using a nasopharyngeal cooling method described by Hagioka et al. (1) Brain temperature decreased to 25°C within 15 minutes and was maintained at that temperature for 2 hours. Rectal temperature was maintained at over 34°C during hypothermia.
A large cranial window was made on the left parietal-temporal bone, and two glass microelectrodes (for monitoring DCpotential) were placed in this window. One was placed 3 mm posterior to the bregma and 3 mm lateral from the sagittal line, and the other was placed 5 mm posterior to the bregma and 3 mm lateral from the sagittal line.
We observed changes in DCpotential and cortical NADH fluorescence using an in vivo fluorescence imaging technique described by Higuchi et al. (2)
Results: At the DC recording sites, persistent depolarization was observed for the first 15 minutes in all rats. After starting hypothermia, recovery of DC potentials was observed in two of the three rats.
In the two rats in which recovery of DC potentials was observed, the number of pixels showing more than 120% NADH fluorescence, which we call the “ischemic core”, had decreased from 5000 to 4000 and 4800 to 3800, respectively, at 10 minutes after starting hypothermia.
Conclusion: Hypothermia induced 15 minues after focal brain ischemia by nasopharyngeal cooling repolarized the area showing persistent depolarization and reduced the ischemic core in NADH fluorescence images.
Our results suggest that hypothermia induced by nasopharyngeal cooling repolarizes neural cells by changing the mitochondrial NADH redox state and improving the energy metabolism of neural cells.
THE MEASUREMENT OF CEREBRAL METABOLIC RATE OF OXYGEN WITH CYTOCHROME OXIDASE
Owen D. Price, Alexander B. Rowley, Stephen John Payne
Department of Engineering Science, University of Oxford, Oxford, UK
Background
Quantifying cerebral metabolic activity accurately is well-known to be difficult. A number of different measurement techniques exist and much recent work has been devoted to using measurement techniques to measure the cerebral metabolic rate of oxygen (CMRO2). In particular, Near Infra-Red Spectroscopy (NIRS) provides much information about haemoglobin transport. Although a recent study, which combined a mathematical model with NIRS data, showed that it was not possible to measure CMRO2 without further information, the potential use of cytochrome oxidase (CtOx) information has not been explored. There are also potentially significant confounding effects due to other physiological variability, such as fluctuations in Arterial Blood Pressure (ABP): these have not been investigated fully, although they have recently been shown to have a large potential confounding factor.
Methods
A joint mathematical model has been developed that predicts the response of the cerebral vasculature to changes in ABP and neural activity (coupling together the models of Wilson et al., Archives of Biochemistry and Biophysics, 182, 749-762, 1977, and Payne, Mathematical Biosciences, 204, 260-281, 2006), in terms of both haemoglobin transport and CtOx. Using experimental data for ABP obtained from volunteers and simulated neural activity patterns, a range of measurable outputs have been simulated, including CBF, CtOx and both oxyhaemoglobin and deoxyhaemoglobin. The resulting model outputs have been analysed using Independent Component Analysis (ICA) to investigate whether the fluctuations in the simulated measurable outputs can be decomposed into those caused by ABP and those due to neural activity. ICA is used as this provides maximal separation between independent signals.
Results and conclusions
It is found that there are significant components of the pressure fluctuations in the CtOx signal, but that some degree of separation of the pressure and neural components can be achieved using ICA on simulated CBF and CtOx. However, there is still some cross-contamination of the signals, preventing complete separation. Further work will need to investigate whether including more signals (such as oxyhaemoglobin and deoxyhaemoglobin) or including dynamical information (ICA treating the signals as independent samples from an underlying distribution) will aid in separating these signals. This will then help in determining whether CMRO2 can be accurately measured from multiple experimental recordings. It is hoped that this analysis will aid in quantifying metabolic activity on both a global and regional basis, which will be valuable in interpreting the metabolic pathways in both normal subjects and diseased states such as stroke and dementia.
EFFECTS OF MITOCHONDRIAL ATP-SENSITIVE K+ CHANNEL OPENER ON MITOCHONDRIAL POTENTIAL IN VIVO
Yoshimasa Takeda, Motomu Kobayashi, Hideki Taninishi, Toshihiro Sasaki, Minako Arai, Kiyoshi Morita
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan
Introduction
It has been reported that activation of the mitochondrial K channel attenuates mitochondrial potential without affecting ATP production in vitro. To elucidate the effects of diazoxide on mitochondrial potential and ATP production in vivo, mitochondrial potential, NADH fluorescence, DC potential and onset time of ischemic depolarization were examined in the rat cortex.
Methods
Male Wistar rats (n=5) were anesthetized with 1% halothane and were artificially ventilated. Two cranial windows were made on the left parietal cortex. For the measurement of mitochondrial potential, a potentiometric dye, JC-1 (2 micro l), was injected into the parietal cortex through the two cranial windows. After a 30 minute equalization period, DC electrodes were inserted into the cortex, where JC-1 was injected. Then a mitochondrial ATP-sensitive K+ channel opener, diazoxide (1mmol/l in 1.5 mmol/l NaOH, 150 mmol/l NaCl), and its carrier (1.5 mmol/l NaOH, 150 mmol/l NaCl) were continuously infused at the rate of 0.5 micro l/min through the DC electrodes. Five minutes after the onset of infusion, 4vessel occlusion was performed for 10 minutes to observe the onset time of ischemic depolarization.
Mitochondrial potential
The cortex was illuminated with excitation light (485 nm). The intensity of red fluorescence (590 nm) and that of green fluorescence (530 nm) in an area adjacent to the DC electrode were measured every 20 seconds. The ratio of red:green fluorescence, indicative of mitochondrial potential, was normalized to that of the control value of the red:green ratio.
NADH fluorescence
The cortex was illuminated with excitation light (360 nm). The intensity of fluorescence (460 nm) in an area adjacent to the DC electrode was measured every 20 seconds. The intensity of fluorescence, indicative of aerobic glycolysis in mitochondria, was normalized to that of the control value.
Results
Mitochondrial potential was decreased to 71 · 16 % of the control level with continuous infusion of diazoxide but was unchanged with infusion of the carrier (99 · 5%, p=0.006). There were no statistical differences in DC potential (diazoxide, −0.6 · 0.2 mV; carrier, −1.1 · 0.8 mV, p=0.18) and in intensity of NADH fluorescence (diazoxide, 101 · 1 %; carrier, 99 · 3 %, p=0.26) with infusion of diazoxide and its carrier. Onset times of DC depolarization after the initiation of 4-vessel occlusion were 3.3 · 1.3 min at the site of diazoxide infusion and 2.8 · 1.0 min (p=0.5) at the site of the carrier infusion.
Conclusions
In the present experiment, mitochondrial potential was decreased by 29% with activation of the mitochondrial ATP-sensitive K+ channel without affecting DC potential, intensity of NADH fluorescence and onset time of DC depolarization. This observation suggests that K+ gradient is an important factor making mitochondrial potential but is independent of energy production in mitochondria in vivo.
EARLY DISTURBANCE OF CEREBRAL METABOLIC FUNCTIONS AFTER THE ONSET OF STROKE IN SPONTANEOUSLY HYPERTENSIVE RATS (SHR)
Takashi Temma1, Y. Kuge1, K. Sano1, N. Obokata1, J. Kamihashi1, H. Kawashima2, Y. Magata3, H. Saji1
1Department of Patho-Functional Bioanalysis, Kyoto Univeristy Graduate School of Pharmaceutical Sciences, Kyoto, Japan, 2Department of Nuclear Medicine and Diagnostic Imaging, Kyoto University Graduate School of Medicine, Kyoto, Japan, 3Laboratory of Genome Bio-Photonics, Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan
Objectives: Although hypertension is widely regarded as the most stringent risk factor leading to stroke, the effects on cerebral metabolic functions after the onset of stroke have not been evaluated. Thus, we measured cerebral metabolic rate for oxygen (CMRO2) and cerebral metabolic rate for glucose (CMRglc) at 1 hour after the onset of stroke in SHR. Methods: Male SHR (n=12, 13-17 week-old) and Wistar Kyoto rats (WKY, n=12, 14-17 week-old) were used for an ischemic model and divided into two groups, PET and ARG groups. The right middle cerebral artery (MCA) was intraluminally occluded. Cerebral blood flow (CBF), oxygen extraction fraction (OEF) and CMRO2 were measured by two serial PET scans (SHR-7700L, Hamamatsu Photonics, Hamamatsu, Japan) using 15O-H2O and radiolabelled 15O-O2 blood (injectable 15O-O2; ref. J Cereb Blood Flow Metab 26:1577:2006) at 1 hour after the occlusion. CMRglc was obtained by the ARG method using 18F-FDG in other experiments. Results & Discussion: SHR showed a significant CBF decrease in the left hemisphere compared with that of WKY (55%) besides a consequent CBF decrease in the right hemisphere (47%), indicating the disturbance of autoregulation mechanism in SHR. However, CMRO2 in each hemisphere was comparable between SHR and WKY, probably owing to the compensatory mechanism in SHR. Furthermore, CMRO2 exhibited a significant difference between hemispheres in SHR and WKY while CMRglc showed that only in SHR (78%), suggesting that the anaerobic glycolysis functioned in the ischemic boundary region in WKY. Since CMRO2 inherently reflects the energy production in mitochondria, different from CMRglc calculated by the tracer uptake, the combination of CMRO2 and CMRglc can provide further information on ischemic progression. Conclusion: The results showed early disturbance of cerebral metabolic functions at 1 hour after the occlusion in SHR compared with WKY, indicating that hypertension deteriorates the metabolic functions and prognosis after the onset of stroke.
VESICULAR GABA TRANSPORTER (VGAT) IS CLEAVED, BY CALPAINS, IN EXCITOTOXIC CONDITIONS AND IN-VIVO ISCHEMIA
Joao Gomes1, Bruno Manadas1, Carlos Melo1, Tamas Atlasz2, Dora Reglodi3, Francisco Amado4, Pedro Domingues4, Robert Gabriel2, Carlos Duarte1
1Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 2Department of Experimental Zoology and Neurobiology, University of Pecs, Pecs, Hungary, 3Department of Anatomy, Medical School, University of Pecs, Pecs, Hungary, 4Department of Chemistry, University of Aveiro, Aveiro, Portugal
Although GABA is the major inhibitory neurotransmitter in CNS, and may be neuroprotective under excitotoxic conditions, it has been suggested that activation of GABA A receptors may contribute to cell damage due to Cloride ion entry and consequent neuronal swelling. Recent findings showed that vesicular transporter expression directly regulates the extent of neurotransmitter release, controlling the quantal efficacy of a synaptic transmission. Therefore, putative changes in VGAT protein levels during ischemia may affect GABAergic transmission and cell death. In this work we investigated the fate of VGAT in cultured hippocampal neurons, under excitotoxic conditions. We found that VGAT (58KDa) is cleaved upon glutamate stimulation of hippocampal neurons, in a biphasic manner, giving rise to a product of about 46KDa. About 40% of VGAT is cleaved within the first hour after glutamate stimulation, and 35% of the protein is cleaved 2-7h later. Truncated VGAT is stable for more than 24h, and de novo protein synthesis slowly allows recovering full VGAT protein levels. VGAT cleavage was also observed in the cerebral cortex following Middle Cerebral Artery Occlusion (MCAO), a cerebral ischemia model. Calpain I inhibition with ALLN or MDL28170 prevented the early cleavage of VGAT in hippocampal neurons subjected to excitotoxicity, but had no effect on the late truncation. Inhibition of Caspases with Z-VAD-FMK did not affect glutamate-induced VGAT cleavage. In-vitro studies, using cerebrocortical synaptic vesicles and recombinant calpain I, also showed the cleavage of VGAT to the same product of about 46KDa. Immunoblot experiments using different antibodies against VGAT showed that the calpain cleavage site is located in the N-terminal region of the protein. Accordingly, calpain also cleaved a fusion protein of GST with the N-terminal region of VGAT, after Glu80, as determined by mass spectrometry MALDI-TOF/TOF. Cleavage of VGAT may affect the intracellular traffic of the protein since the VGAT immunoreactivity in neuritis changes following excitotoxic stimulation with glutamate. Taken together, our results show that glutamate toxicity causes a down-regulation of VGAT, which may affect GABAergic neurotransmission and cell death, during ischemia. (Supported by FCT and FEDER)
DIFFERENTIAL EXPRESSION OF THE MONOCARBOXYLATE TRANSPORTERS MCT1 AND MCT2 INDUCED BY FOCAL BRAIN ISCHEMIA IN NON-DIABETIC AND DIABETIC RATS
Tiago Prazeres Moreira1, Karin Pierre1, Aleta Cebere2, Claes-Goran Ostenson3, Suad Efendic3,
Sture Liljequist2, Luc Pellerin1
1Department of Physiology, University of Lausanne, Lausanne, Switzerland, 2Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden, 3Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden
Background and aims. The combination of diabetes and brain ischemia has been reported to worsen the clinical outcome of stroke. Similarly, larger infarct sizes and neurodegeneration have been observed in several models of brain injury in rats rendered diabetic with pharmacologic treatment but, to our knowledge, few studies of recovery from brain ischemia have been performed in constitutively diabetic rat strains such as the Goto-Kakizaki (GK) strain. The GK rat is a type 2 diabetic rat model generated by selective breeding of Wistar rats with hyperglycemia in the oral glucose tolerance test. Contrary to other in vivo models of type 2 diabetes, the GK rat has the advantage of showing mild-to-moderate hyperglycemia, deficient insulin secretion and late-occurring insulin resistance, and of avoiding the contribution of confounding variables such as obesity, ketosis, hyperlipidemia and the need for exogenous insulin treatment.
During recovery of brain ischemia, supplementary energy is required for the removal of necrotic tissue, elimination of apoptotic cells and restoration of function in surviving cells. To date, little is known about how energy substrates are used during recovery from brain ischemia. Monocarboxylate transporters (MCTs) facilitate the distribution of energy substrates such as lactate, known to be overproduced during ischemia, from astrocytes to neurons for oxidation. We thus hypothesized that MCT expression could be modulated in brain cells to supply energy following ischemia.
Methods. Gene expression of MCT1 and MCT2 was investigated using real time reverse transcriptase polymerase chain reaction (RT-PCR) at baseline and at 48h of reperfusion from focal brain ischemia induced by cortical compression. The regional distribution of MCT1/MCT2 protein expression was investigated by immunohistochemistry at 2 and 7 days of reperfusion. To assess if MCT expression would be affected by blood glucose levels, control Wistar and diabetic GK rats were compared.
Results. Higher baseline MCT2 mRNA expression levels were detected in the cortex of diabetic rats. Cortical ischemia induced hyperglycemia in diabetic GK but not in control Wistar rats. In the injured cortex of both strains, MCT1 expression was upregulated in astrocytes whereas MCT2 expression was detected in surviving neurons. MCT1 mRNA levels increased in the cortex and hippocampus of Wistar rats while MCT2 mRNA significantly decreased in the cortex of diabetic rats. At 7 days post-reperfusion, only GK rats showed MCT1-labelled astrocytes in the hippocampus, bilaterally.
Conclusions. Expression of MCTs in astrocytes and neurons suggests that these transporters may be used to supply energy to neurons recovering from ischemia. Differential changes in gene expression of MCTs between strains, both at baseline and at 48 h of reperfusion, indicate that the involvement of MCTs during recovery is affected by blood glucose levels. Overall, these observations are in line with a beneficial action of MCTs during recovery from focal brain ischemia and provide support for targeting the modulation of MCT expression on blood vessels and brain parenchyma as a promising therapeutic approach.
DYNAMIC CHANGES OF CORTICAL NADH FLUORESCENCE IN RAT FOCAL ISCHEMIA: NORMOTHERMIA VS. HYPOTHERMIA
Toshihiro Sasaki, Yoshimasa Takeda, Minako Arai, Hideki Taninishi, Motomu Kobayashi, Kiyoshi Morita
Department of Anesthesiology and Resuscitology/Okayama University Medical School, Okayama City, Okayama, Japan
[INTRODUCTION]
Preischemic hypothermia has been suggested to reduce infarct volume. However, the dynamics of the growth of infarct volume during hypothermia has not been observed.
In this study, we examined differences in expansion of the ischemic core in normothemic and hypothermic groups using a cortical NADH fluorescence imaging technique described by Higuchi et al. (1)
[METHODS]
Nine male spontaneous hypertensive rats weighing 300±20 g were used in this study. Permanent occlusion of the left common carotid and middle cerebral arteries was performed.
In the hypothermic group (n=4), hypothermia was induced before ischemia, and then the animals were kept at 30~31°C for two hours and rewarmed to 36.5°C over a period of 60 minutes.
In the normothermic group (n=5), the animals were kept at 37.0±0.5%°C.
A large cranial window was made on the left parietal-temporal bone, and cortical NADH fluorescence in this window was examined.
The infarct volumes were measured 24 hours after onset of ischemia.
[RESULTS]
The areas of the ischemic core in the normothermic and hypothermic groups had increased to a maximal level at about 30 minutes and 60 minutes respectively.
The areas of the ischemic core at 15, 30, 60 and 120 minutes after onset of ischemia and at the end of rewarming were significantly reduced in the hypothermic groups compared to those in the normothermic group.(see figure)
The infarct volumes were also significantly reduced in the hypothermic group (P<0.05).
[CONCLUSION]
We could elucidate differences between growth of the ischemic core in the normothermic group and that in the hypothermic group using cortical NADH fluorescence images.
The results of this study suggested that preischemic hypothermia not only reduced area of the ischemic core but also delayed expansion of the ischemic core, resulting in reduction of infarct volume.
Cortical NADH fluorescence imaging is useful for real-time monitoring of growth of the ischemic core in focal ischemic rat models.
REGULATION OF IL-1BETA-INDUCED PROINFLAMMATORY GENE EXPRESSION IN ASTROCYTES AND MICROGLIA BY RHO KINASE INHIBITOR
Abdullah Md Sheikh, Atsushi Nagai, Junichi Masuda
Department of Laboratory Medicine, Shimane University School of Medicine, Izumo, Shimane, Japan
Purpose and objective: The expression of IL-1? is increased in the plaque area of Alzheimer's disease by glial cells, which plays an important role in the progression of disease pathology; such as increased expression of amyloid precursor protein, further glial cell activation which is frequently found in the lesion area. Fasudil, a Rho kinase inhibitor, is known to have anti-inflammatory properties. We have investigated the functions of IL-1? on cytokine gene expression, and the role of fasudil on that gene expression in glial cells. Methods and results: (1) The mRNA expression of proinflammatory cytokines in primary astrocytes and a human microglial cell line HMO6 was investigated by quantitative real time PCR, and the results showed that (a) IL-1? increased the mRNA expression of TNF?, IL-1?, IL-6 and MCP-1 in both primary astrocytes and HMO6 time dependently. (b) Fasudil inhibited the IL-1?-induced mRNA expression of IL-1? and IL-6 in both astrocytes and HMO6. (c) IL-1?-induced TNF? mRNA expression was transiently inhibited by fasudil in astrocytes. Conversely, fasudil increased IL-1?-induced TNF? mRNA expression in HMO6. (2) The secretion of TNF? into the culture supernatant was investigated by ELISA, and the results showed that (a) fasudil inhibited IL-1?-induced TNF? secretion into the culture supernatant in astrocytes, but (b) increased that secretion in HMO6. To elucidate this differential regulation of TNF? expression in astrocytes and HMO6 by fasudil, (3) we analyzed the promoter activity of TNF? gene using a reporter construct containing a human TNF? promoter, which showed that (a) IL-1? did not increase the TNF? promoter activity in HMO6. (b) The TNF? promoter activity was increased by 40% in HMO6 after stimulation with IL-1? and fasudil. (4) The mRNA stability assay of TNF? using ActinomycinD showed that (a) IL-1? increased the TNF? mRNA stability in HMO6. (b) Fasudil did not change IL-1?-induced TNF? mRNA stability in astrocytes or microglia. Conclusion: Thus our results showed that IL-1? increased the mRNA expression of TNF?, IL-1?, IL-6 and MCP-1, and TNF? secretion in astrocytes and microglia cell line HMO6, and fasudil inhibited the mRNA expression of IL-1? and IL-6 in astrocytes and HMO6. But IL-1?-induced TNF? expression was increased in HMO6 and decreased in astrocytes by fasudil indicating its differential regulation on TNF? expression in astorcytes and HMO6. Through these regulations of proinflammatory gene expression by fasudil, it might be possible to modulate the glial inflammatory reaction in neurodegenerative diseases, such as Alzheimer's disease.
A NEW MODEL OF LACUNAR INFARCTION IN THE MINIAURE PIG: COMPREHENSIVE EVALUATION FOR THE PURE WHITE MATTER ISCHEMIA
Yukitaka Tanaka1, Hideaki Imai1, Kenjiro Konno2, Takaaki Miyagishima1, Chisato Kubota1, Sandra Sandra1, Takeo Aoki3, Hidekazu Hata2, Yuhei Yoshimoto1, Nobuhito Saito4
1Department of Neurosurgery, Gunma University Graduate School of Medicine, Maebashi, Japan, 2Institute of Experimental Animal Research, Gunma University Graduate School of Medicine, Gunma, Japan, 3Department of Anatomy and Cell Biology, Gunma University Graduate School of Medicine, Gunma, Japan, 4Department of Neurosurgery, University of Tokyo Graduate School of Medicine, Tokyo, Japan
Background and Purpose: There has been no previous animal model of lacunar infarction relevant to the lacunar infarction of human so far. In this study, we aimed to develop a new model of lacunar infarction in gyrencephalic brain as a result of in situ small perforating arterial occlusion of internal carotid artery, anterior choroidal artery (AchA) occlusion. This paper provides information on precise neurophysiologic and histopathological findings accompanied with the assessment of neurological deficit from the acute to chronic phase in lacunar infarction.
Methods: Mexican hairless miniature pigs, weighing 19-42 kg, were undergone to AchA occlusion, using a modification of the permanent MCA occlusion method (1). Animals were allowed to recover for 24 hours (n=7), 2 days (n=5), 1 week (n=7), 4 weeks (n=3) and other days (n=13). Sham animals were included (n=5). Muscle Motor evoked potentials (MMEP) were used physiologically to monitor corticospinal tract function during the procedure (n=7). Comprehensive assessments such as neurological, electrophysiological, histopathological (immunohistochemical and electronmicroscopical) methods and MRI were used.
Results: The present protocol provided a 91.4% rate in successful production of the internal capsule. For MMEP results, internal capsule tissues that were exposed to ischemia between the first 6-15 minutes of AchA occlusion were considered penumbra zone. Lacunar infarction animal group usually displayed signs of neurological deficit after stroke onset. The neurological deficit spontaneously recovered to became nearly same as to the control animals at 12 day after ischemia. On the other hand, histopathological study revealed that the lesion of internal capsule expands gradually as time dependent manner which is particularly attributed to significant expansion of the ischemic lesion during the first week after ischemia (P<0.05). Ultrastructural analysis unveiled the characteristics in terms of the mechanism of expansion in white matter ischemia. At peri-infarct zone the axon was initially damaged to be swollen accompanied with the edema formation. Another notable finding in this study is that small vessels are highly resistant to ischemic stress. The small vessels amount at internal capsule remains constant before and after ischemia, even in the chronic phase. This is probably attributed to the difference in cellular susceptibility. The environment seems to be favorable in terms of the repair and/or axonal regeneration, because the system for blood supply in the core of lacunar infarction even at the chronic phase was kept stable.
Conclusion
A new model in the miniature pig to study white matter ischemia would be extremely useful to investigate the specific cellular mechanisms of white matter damage and to provide robust insight (evidence) about penumbra in white matter. Our study suggests that peri-infarct zone even in the small subcortical white matter must be a dynamic and malleable area for long time after stroke allowing that we could contribute to salvage it administrating an adequate treatment. Also, this model can be widely used for translational research such as subclinical trial for axonal regeneration in the way to conform an advanced stroke therapy.
CALCIUM REGULATION OF GLUTAMATE ASPARTATE TRANSPORTER GLAST IN ASTROCYTES
Yu-Peng Liu, Shun-Fen Tzeng
Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan-R.O.C.
During acute traumatic CNS injury, highly accumulation of extracellular glutamate induces neuronal excitotoxicity by calcium ion influx through their glutamate receptors and causes a secondary CNS tissue damage. Astrocytes, a major glial cell type in the central nervous system (CNS), have capability to reduce excess extracellular glutamate via uptaking glutamate molecules primarily through astrocytic glutamate transporter 1 (GLT-1)/EAAT-2 and glutamate aspartate transporter (GLAST). Thus, malfunction and abnormal expression of glutamate transporters in astrocytes could cause the accumulation of the toxic concentrations of glutamate to augment neuronal damage, which leads to the progressive neurodegeneration. However, little is known about the regulation of the astrocytic glutamate transporter expression by injury-induced signaling. In this study, using an in vitro model of cadmium (Cd)-induced cellular stress we found that glutamate uptake and GLAST transcription in astrocytes were reduced and calcium ion influx into astrocytes was increased. Inhibition of Cd-induced calcium influx by the extracellular calcium chelator EGTA or the intracellular calcium chelator BAPTA-AM, efficiently restored GLAST expression and glutamate uptake activity in astrocytes, suggesting that the reduction of GLAST expression in astrocytes was calcium-dependent. This was confirmed by the finding that GLAST mRNA expression was reduced in astrocytes treated with the calcium ionophore A23187. The GLAST promoter analysis and the DNA binding analysis of calcium-sensitive transcription factors (AP-1 and CREB) also demonstrated that AP-1 and CREB could be the downstream components of the calcium-dependent signaling and might mediate the inhibition of astrocytic GLAST transcription. Together, injury-induced calcium ion influx into astrocytes can cause the downregulation of astrocytic GLAST, and reduce astrocytic glutamate uptake function, which in turn may enhance the secondary damage after CNS injury (supported by National Science Council, Taiwan; NSC 95-2311-B-006 −0040).
UPREGULATION OF BCL-XS BY NF-??B CONTRIBUTES TO NEURONAL DEATH IN ISCHEMIA
Wanlu Du, Yizheng Wang
Institute of Neuroscienc, SIBS, CAS, Shanghai, China
It has been shown that NF-κB plays a role in neuronal cell death after focal ischemia. However, the mechanism by which NF-κB affects neuronal survival is controversial. Here, we report that up-regulation of Bcl-Xs via an NF-κB-dependent pathway is important for neuronal death induced by ischemia.
The rats were subjected to transient focal ischemia and followed by reperfusion for different times. Western blot analysis revealed that the level of nuclear p65, the main subunit of NF-κB, was rapidly increased and maintained at a high level during reperfusion periods. Immunostaining showed the nuclear accumulation of p65. The protein level of Bcl-Xs, a protein regulated by NF-κB, was also elevated in the ischemic side compared to the non-ischemic side, whereas that of Bcl-Xl was not affected. In cultured neurons down-regulation of p65 or BCL-Xs protected neurons from death induced by glutamate. These results suggest that NF-κB might promtoe neuronal death in ischemia possibly by up-regulating the pro-apoptotic proteins.
PROTECTIVE EFFECT OF 12/15-LIPOXYGENASE DEFICIENCY OR INHIBITION ON CEREBRAL EDEMA AFTER TRANSIENT FOCAL ISCHEMIA
Guang Jin, Ken Arai, Yoshihiro Murata, Eng H. Lo, Klaus van Leyen
Neuroprotection Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA
Background and Purpose
Cerebral ischemic stress can cause blood-brain barrier (BBB) disruption and increase cerebral vascular permeability, leading to the formation of brain edema. Lipoxygenases are lipid-oxidizing enzymes involved in a variety of cellular processes, including membrane remodeling and cell death. Our previous studies have shown that 12/15-lipoxygenase knock-out mice were protected against ischemic brain injury by reducing neuronal cell death. However, the function of 12/15-lipoxygenase in cerebral edema after stroke has not been clarified. The present study was designed to test if the inhibition or genetic knockout of 12/15-lipoxygenase reduces the cerebral edema related to blood-brain barrier injury in transient focal ischemia.
Methods
Transformed human brain endothelial cells (THBEC) were subjected to H2O2 oxidative stress in the presence or absence of baicalein, which is a specific inhibitor of 12/15-lipoxygenase. The release of LDH was detected for the evaluation of the cells death. The presence of 12/15-lipoxygenase in the mouse brain was detected by immunohistochemistry. Brain edema was evaluated in a mouse model of transient middle cerebral artery occlusion with and without baicalein treatment, and in wild-type and 12/15-lipoxygenese knock-out mice, respectively.
Results
In H2O2-treated THBEC cells, the cells death was greatly reduced by treatment with baicalein (17.2 ± 5.19 %, p < 00.1) and AA-861 (39.6 ± 1.62 %, p < 0.01), compared to the control group (66.2 ± 10.78 %). In ischemic mouse brain, significant 12/15-lipoxygenase expression was shown in the peri-infarct area at 24 hours after ischemia, and the expression was colocalized with neuronal marker NeuN and endothelial marker CD31. Administration of Baicalein significantly reduced edema at 24 hours (water content 79.7 ± 1.66 % vs. 82.1 ± 1.77 %, p < 0.01, n=9). Likewise, 12/15-lipoxygenase knockout mice had significantly reduced edema compared to wildtype mice (80.6 ± 2.02 % vs. 82.5 ± 1.24 %, p < 0.05, n=8).
Conclusion
These results demonstrate that inhibition or deficiency of 12/15-lipoxygenase reduces the formation of edema in transient cerebral ischemia, associated with the protection of endothelial cells and reduced ischemic degradation of BBB.
Grant support: supported by NIH grant R01NS049430, and a SDG from the American Heart Association.
HYPERBARIC OXYGEN PRECONDITIONING ATTENUATES BRAIN EDEMA AFTER EXPERIMENTAL INTRACEREBRAL HEMORRHAGE
Zhiyong Qin1, Shuijiang Song1, Guohua Xi1, Robert Silbergleit2, Richard Keep1, Julian Hoff1, Ya Hua1
1Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA, 2Department of Emergency Medicine, University of Michigan, Ann Arbor, MI, USA
Background and Aims: Hyperbaric oxygen preconditioning (HBOP) induces ischemic tolerance and reduces ischemic brain damage. Activation of p44/42 mitogen-activated protein kinases (p44/42 MAPKs) has been associated with preconditioning-induced brain tolerance. This study investigated whether or not HBOP can induce brain tolerance against brain edema formation after intracerebral hemorrhage (ICH) and the role of p44/42 MAPKs in HBOP-induced protection.
Methods: There were three parts of the studies. In the first part, male Sprague-Dawley rats received 2, 3 or 5 consecutive sessions of HBOP (3 ATA, 100% oxygen, one hour daily for 2, 3 or 5 days). Control animals received normobaric room air. Twenty-four hours after HBOP, the rats received an intracaudate injection of autologous whole blood (100-µl) and were killed one or three days later for brain edema measurement. In the second part, rats received either 5 sessions of HBOP or control pretreatment and were killed for Western blot analysis and immunohistochemistry of p44/42 MAPKs 24h after the final HBOP. In the last part, rats received an intracaudate injection of an inhibitor of p44/42 MAPK activation, PD 098095 (5 nmol) or vehicle (4% dimethyl sulfoxide) before the first session of HBOP, then underwent 5 sessions of HBOP. Twenty-four hours after the final HBOP, rats received an intracaudate injection of 100-µl autologous whole blood. Brain water content was measured 24 hours after blood injection.
Results: Fewer than five sessions of HBOP did not significantly attenuate brain edema 24 hours after ICH (p>0.05). Five sessions of HBOP significantly reduced water content in the ipsilateral basal ganglia 24 hours (79.5±0.9% vs. 81.2±1.1% in the control group, n=9-10, p<0.01) and 72 hours (81.3 ± 1.4% vs. 82.8 ± 1.1% in the control group, n=6, p<0.05) after ICH. Western blot analysis showed that five sessions of HBOP significantly activated p44/42 MAPKs in the basal ganglia (activated p44 MAPK: 3116±972 vs. 15 ± 4 pixels in the control, p<0.01; activated p42 MAPK: 10750±2192 vs. 173±33 pixels in the control, p<0.01). Strong p44/42 MAPK immunoreactivity was found in the basal ganglia 24 hours after five sessions of HBOP, whereas few p44/42 MAPK positive cells were detected in the basal ganglia in the control rats. Intracaudate infusion of PD 098059 abolished HBOP-induced brain tolerance against brain edema formation 24 hours after ICH. Edema was 80.7± 1.1% in rats treated with HBOP plus PD098059, not significantly different from rats without preconditioning, while HBOP plus vehicle was identical to HBOP alone, 79.5 ± 0.5% (p<0.05).
Conclusions: Five sessions of HBOP induces brain tolerance against edema formation following ICH, and activation of p44/42 MAPK pathway contributes to this HBOP-induced brain tolerance.
CHOLINERGIC IMPAIRMENTS FOLLOWING EXPERIMENTAL TRAUMATIC BRAIN INJURY
Cornelius L. Donat1, Cornelia Voigt2, Martin U. Schuhmann2, Winnie Deuther-Conrad1, Karen Nieber3, Reinhard Schliebs4, Peter Brust1
1Institute of Interdisciplinary Isotope Research, Radiopharmacy, Leipzig, Germany, 2Department of Neurosurgery, University of Leipzig, Leipzig, Germany, 3Institute of Pharmacy, University of Leipzig, Leipzig, Germany, 4Paul Flechsig Institute for Brain Research, Department of Neurochemistry, University of Leipzig, Leipzig, Germany
Background and Aims
Traumatic brain injury (TBI) is one of the leading causes of death and disability mainly among young people and often results in significant cognitive impairments. Working memory deficits shown in animal models are believed to be connected with deficits of the cholinergic system, especially of the acetylcholine receptors (AChR). Findings obtained from different animal models have shown declines in AChR receptor densities and changes in associated enzyme activities. The present study was conducted to evaluate these findings in a special animal model considering the time-course of posttraumatic events and the critical brain regions. The identification of sensitive targets within the cholinergic system could be a precondition for diagnostic neuroimaging in TBI-patients with Positron-Emission-Tomography (PET).
Methods
Male Sprague-Dawley rats selected in three groups (post-TBI survival time: 2 h, 24 h and 72 h) were anaesthetized and subjected to sham injury (control, n = 8) or controlled-cortical-impact injury (CCI) (n = 8) with 2 mm depth of impact at a velocity of 4 m/sec. Following surgery, animals were antagonized and returned to their homecage with free access to water and food. Following the designated time after injury, the rats were decapitated, brains were quickly removed and frozen in ?35°C 2-methylbutan.
To quantify the receptor density, 12 µm cryostat brain sections were incubated with 2 nM[3H]-epibatidine, a ligand for nicotinic acetylcholine receptors (nAChR), for 90 min or 8 nM [3H]-QNB, specific for muscarinic acetylcholine receptors (mAChR), for 120 minutes. Non-specific binding was determined in the presence of 300 µM nicotine or 1 µM atropine, respectively. All slides were preincubated with assay buffer (50 mM TRIS-HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2) for 15 minutes and experiments were carried out at room temperature. Additionally acetylcholinesterase (AchE) staining was performed using acetylcholiniodide.
Results
A significant decline in the receptor density of the nAChR in thalamus (72 h: TBI 16.2±2.1 vs. sham 18.1±1.8 fmol/mg protein) and motor cortex (72h: TBI 7.92±1.05 vs. sham 9.81±1.02 fmol/mg protein) was observed after TBI.
The mAChR showed a similar, time-dependent reduction in these regions (thalamus 72 h: TBI 90.6±4.9 vs. sham 100.7±13.1 fmol/mg protein) and additionally in the corpus callosum (72h: TBI 33.2±3.5 vs. sham 37.3±4.1 fmol/mg protein) and basal forebrain (72h: TBI 268±29. vs. sham 305±42 fmol/mg protein).
Histochemical staining indicated an increase in AChE activity (in terms of optical density) in the basal forebrain 2 h after TBI (120% increase of optical density compared to control activity). A decreased enzyme activity was found in the hippocampus (85% of control activity).
Conclusions
Cholinergic receptor densities are significantly impaired after experimental TBI, especially in regions associated with attention. Considering the role of the AChR for cognition in the brain, it seems that the decline in receptor density contributes to attention and memory deficits as found in behavioural experiments.
The changes in the AChE found in our experimental TBI model could be related to short-term impairments after traumatic brain injury.
FOCAL HYPERPERFUSION AFTER SUPERFICIAL TEMPORAL ARTERY - MIDDLE CEREBRAL ARTERY (STA-MCA) ANASTOMOSIS IN THE ISCHEMIC CEREBROVASCULAR DISEASE
Hideaki Watanabe, Yoshiaki Kumon, Shiro Oue, Takanori Onishi
Department of Neurosurgery, Ehime University, Toon-City, Ehime, Japan
Background and aims
In the recent report, hyperperfusion syndrome has been recognized after superficial temporal artery ? middle cerebral artery (STA-MCA) anastomosis and careful postoperative management has been required. In this study, we examine the relationship between STA-MCA anastomosis and the incidence of hyperperfusion syndrome in the ischemic cerebrovascular diseases.
Subject and method
STA-MCA anastomosis was performed to 18 sides of 17 cases during last 3 years. Eight sides of 7 cases were adult onset ischemic Moyamoya disease (Moya group) and 10 sides of 10 cases were atherosclerotic occlusive disease (ASO group; IC occlusion and MCA occlusion). The detail of subjects are shown in Fig. 1. The diffusion weighted image (DWI) by MRI was performed within 3 days after surgery to evaluate newly developed cerebral infarction during surgery. PAO-SPECT was performed within 1week after surgery for hyperperfusion evaluation. Xe-SPECT was performed pre and post 1 month after surgery for evaluation of cerebral blood flow (CBF) and cerebral vascular response (CVR).
Results
In 12 sides (67%), postoperative focal hyperperfusion were shown. The incidence sides were 6 sides in each group, however, the incidence ratio in Moya group (75%) was relatively higher than in the ASO group (60%). The mean degree of CBF increase was 16% in comparison with a contra lateral side. No significant difference of the CBF increase was shown between Moya group (17% increases) and ASO group (14% increase). In all cases, no DWI positive lesion was detected by MRI and no CBF decrease lesion was detected by PAO SPECT, we could not recognized ischemic lesion after surgery. Temporary postoperative neurological deterioration (motor paresis, aphasia and convulsion) occurred in 7 sides of 6 cases (39%). The Moya group was 5 sides of 4 cases (63%) and ASO group was 2 sides (20%), the incidence ratio in Moya group was significantly higher. In these symptomatic sides, 6 sides of 5 cases (28%) were hyperperfusion syndrome. The changing in CBF measured by Xe-SPECT was no significant difference between pre and post 1 month in the both group. Even in the hyperperfusion sides were also no difference, postoperative hyperperfusion was temporary phenomenon. The CVR was significantly improved in the both group, especially in the Moya group, the CVR reached to the normal range (3.4% ? 24.8%).
Conclusion
Temporary focal hyperperfusion after STA-MCA anastomosis occurs in quit often case and a precise diagnosis and postoperative management are required.
BED-SIDE MONITORING OF CEREBRAL PERFUSION IN PATIENTS WITH ANEURYSMAL SUBARACHNOID HEMORRHAGE BY NEAR INFRARED TIME - RESOLVED SPECTROSCOPY
Noriaki Yokose, Takayuki Awano, Tatsuya Hoshino, Shin Nakamura, Norio Fujiwara, Yoshihiro Murata, Tsuneo Kano, Kaoru Sakatani, Yoichi Katayama
Department of Neurological Surgery, Nihon University School of Medicine, Itabashi-Ku, Tokyo, Japan
Introduction: Early detection of cerebral vasospasm is essential for the treatment of delayed ischemic neurological deficits in patients with subarachnoid hemorrhage (SAH). Transcranial Doppler sonography (TCD) has been used to detect vasospasm after SAH, but its sensitivity is not high. In addition, TCD does not provide information about the cerebral circulation and oxygenation in the cortex. In contrast, near infrared time-resolved spectroscopy (TRS), an optical diagnostic technique, allows assessments of cortical blood oxygenation and hemodynamics. In this study, we examined whether TRS can detect cerebral ischemia caused by vasospasm in aneurysmal SAH. Initially, we measured normal values for the hemoglobin (Hb) concentrations and oxygen saturation (SaO2) in normal adults. We then repeatedly measured the SaO2 and Hb concentrations in patients after SAH, and these data were compared with the results of TCD. In addition, we evaluated whether TRS measurements on day 1 after SAH can be useful for predicting the occurrence of vasospasm and clinical outcome.
Methods: We investigated eight normal adults and seven SAH patients with poor clinical conditions (WFNS grade 5) who had undergone embolization of their aneurysms using Guglielmi detachable coils. Employed TRS (TRS-10, Hamamatsu Photonics K.K, Japan), we measured SaO2 and baseline concentrations of oxygenated Hb (Oxy-Hb), deoxyhemoglobin (Deoxy-Hb) and total Hb in the cortices. Measurement of TRS and TCD were performed repeatedly for 14 days after SAH. Angiography was performed day 7 after SAH or when TRS or TCD indicated occurrence of vasospasm.
Results: In normal adults, TRS demonstrated relatively constant values of SaO2 (about 70%) regardless of the cortical regions, although the hemoglobin concentrations varied depending on the recording regions. In four of the seven patients, the SaO2 and hemoglobin concentrations measured in the middle cerebral artery territory remained stable after SAH. However, in three patients, TRS revealed abrupt decreases in SaO2 and total hemoglobin between five and nine days after SAH. Cerebral angiography performed on the same day revealed severe vasospasms in these patients. Although TCD detected the vasospasm in two of three cases, it failed to do so in one case. In addition, TRS performed on day 1 after SAH revealed significantly higher SaO2 in patients with vasospasms or with severe disability (n=5) as compared to the normal controls, although there was no significant difference in total Hb.
Conclusions: TRS could detect vasospasms after SAH by evaluating the cortical blood oxygenation and may be more sensitive than TCD. The high SaO2 in patients with vasospasms or with severe disability suggests a reduction of cerebral oxidative metabolism in the patients rather than hyperperfusion, since there was no difference in total Hb (≒cerebral blood volume) between the patients and controls. Several studies have reported that initial aneurysmal rupture could cause brain damage resulting in a reduction of cerebral oxidative metabolism. These findings suggest that it may be possible to predict the occurrence of vasospasms and prognosis of SAH patients based on the cortical blood oxygenation measured by TRS on day 1 after SAH.
VALIDATION OF A NOVEL SUBARACHNOIDAL HEMORRHAGE MODEL IN THE MOUSE
Sergej Feiler2, Serge Thal2, Nikolaus Plesnila1,2
1Laboratory of Experimental Neurosurgery, Department of Neurosurgery, Munich, Germany, 2Institute for Surgical Research, University of Munich Medical Center ? Groshadern, Munich, Germany
Introduction: Subarachnoid hemorrhage (SAH) has a high mortality and morbidity. The pathophysiology of SAH, especially on the molecular level, is poorly understood. The aim of the current study was to characterize a model of SAH in mice, which allows to investigate SAH in transgenic animals.
Materials and methods: C57/Bl6 mice were anesthetized (medetomidine, fentanyl, midazolam i.p.), intubated and ventilated under control of endtidal pCO2. A blunted 5/0 monofilament nylon suture was introduced over the external carotid artery into the internal carotid artery (ICA). Under monitoring of cerebral blood flow and intracranial pressure (ICP) the filament was advanced until it perforated the vessel wall near the bifurcation of the middle and anterior cerebral artery thereby causing SAH. In sham operated mice the filament was advanced into the ICA without perforation of the vessel. 24 hours and 3 days after SAH the ICP and brain edema were measured. 7 days after SAH the animals were sacrificed and neuronal cell loss was quantified in the CA1, CA2, and CA3 regions of the hippocampus.
Results: Under baseline conditions ICP was 4.0 +/− 0.3 mmHg (MW +/− SEM). After SAH ICP increased to 56 +/− 10 mmHg and leveled off at ~20 mmHg thereafter. Cerebral blood flow decreased to 15 +/− 3% just after SAH and returned to subnormal values within 15 min. 24 hours after SAH the average ICP in SAH mice was 10 +/− 8 mmHg as compared to 4 +/− 2 mmHg in sham operated animals. After 3 days ICP returned to almost baseline values (SAH: 5 +/− 3 mmHg; sham: 3+/− 1 mmHg). Brain edema formation quantified by determination of brain water content was increased by 1% and 1.5% 24 and 72 h after SAH, respectively.
The number of surviving neurons in the CA1 region of the ipsilateral hippocampus decreased significantly by 37% after SAH (56 +/− 41 vs. 90 +/− 9 in sham operated animals; p<0.04). No significant decrease was found in the CA2 and CA3 regions.
Three out of 10 mice died within the first 3 days after SAH.
Conclusion: The SAH filament perforation model in mice is technically feasible, can be performed in a standardized manner, and has a good reproducibility. Additionally, it resembles many features of SAH in man, e.g. the post hemorrhagic ICP increase and CBF decrease and the high mortality within the first few days. Therefore our findings suggest that the present SAH model is well suited for investigations on the molecular pathophysiology of SAH, e.g. by using transgenic mouse models or other molecular tools only available for mice.
PROMINENT DECREASE OF BRAIN TISSUE K+, [K+]BR, IN THE PERIPHERAL REGIONS OF ISCHEMIC CORE EVALUATED BY QUANTITATIVE HISTOLOGICAL POTASSIUM STAINING
Alexander Kharlamov1, Victor E. Yushmanov1, Stephen C. Jones1,2
1Department of Anesthesiology, Allegheny-Singer Research Institute, Pittsburgh, PA, USA, 2Department of Neurology, Allegheny-Singer Research Institute, Pittsburgh, PA, USA
Background and Aims
Maximum changes of sodium (1), MAP2 (2), aquaporin (3), water and K+ (4) occur in the border regions of ischemic core (BRIC) after ischemia. This phenomenon may be related to relatively more residual blood flow supply to the peripheral areas of the ischemic core. We propose that the decrease of [K+]br over time after ischemia is more prominent in BRIC areas at early times after ischemic onset.
Methods
Experimental focal ischemia was induced by middle cerebral artery transection and bilateral common carotid artery occlusion in six Sprague-Dawley rats anesthetized with isoflurane and N2O with arterial pressure and blood gas monitoring for 2.5–5. h. Physiological parameters were stable and within normal limits. Brains were alternatively sectioned coronally (40 µm, −14 ?C) for K+ histochemistry and punched (2 mm × 0.5 mm diameter) using reflective change (5) to identify ischemic regions for flame photometry analysis of [K+]br. Sections stained for K+ using sodium cobaltinitrite (6) were digitized and aligned with the punch positions (MCID system, InterFocus Imaging Ltd.). Flame photometry-determined [K+]br values in these punch positions were used as standards to calibrate the OD in histological sections for the evaluation of regional [K+]br in ischemic cortex.
Results
Results demonstrated that [K+]br (mean±sem) in the ischemic core was decreased from 69±2 to 33±1 mM from 2.5 to 5 h (see Fig.). At each time point the [K+]br in the BRIC areas was significantly (p=0.016, n=6, paired t-test) lower than in the core. In seven of twelve BRIC regions [K+]br was decreased at least 20% compared to the ischemic core [K+]br. Five of those seven regions were located in the ventral BRIC.
Conclusions
Differences between [K+]br in ischemic core and BRIC at each time-point could be due to different rates of K+ removal via trickle blood flow or differences in edema formation. We conclude that at early times after ischemia the maximum [K+]br decrease occurs in the peripheral regions of ischemic core suggesting that the K+ decrease is associated with dynamic changes of edema formation and infarct expansion.
Support: NIH R01-NS30839
EXTRACELLULAR BRAIN PH IS A MARKER OF PROFOUND METABOLIC DERANGEMENT DURING HYPOXIA AFTER TRAUMATIC BRAIN INJURY
Ivan Timofeev1, Jurgens Nortje2, Philippa Al-Rawi1, Peter Hutchinson1, Arun Gupta2
1Academic Neurosurgery Unit, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK, 2Division of Anaesthesia, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK
Background and aims
The aim of this study was to evaluate the contribution of acidosis to changes in cerebral chemistry in the presence of low (<1.5kPa) or normal (?1.5kPa) brain tissue oxygen (PbO2) in patients with severe traumatic brain injury (TBI).
Methods
Prospective observational data from 41 patients with TBI, who underwent monitoring with microdialysis and Neurotrend? sensors, were analysed.
Results
Brain pH (pHb) was negatively correlated with lactate/pyruvate rato (LP), a marker of anaerobic metabolism (r=-.22 p<0.001, Spearman rho). No correlation was observed between PbO2 and LP at pHb above 7.1. In the presence of low pHb (<7.1) negative correlation between PbO2 and LP became significant (r=-.35 p<0.001, Spearman). Monitoring periods when both pHb and PbO2 were reduced exhibited more profound derangement of metabolism, consistent with ischaemia (low glucose (Mean±SD 0.64±2.2 µmol/L) high lactate (4±1.6 µmol/L) and high LP (38±1.4)), as compared to periods with low PbO2 alone or normal PbO2 (ANOVA p=0.0001–0..02, Tamhane or Dunnet correction). Periods with low pHb, but normal PbO2 were also metabolically abnormal (lactate = 3.55±1.7 and LP = 29.5±1.4) and differed from normal pHb and PbO2 states (lactate 2.5±1.5 and LP 24±1.4 p<0.001)
Conclusions
Decreasing brain pH is associated with deteriorating energy metabolism. Measuring PbO2 together with pHb may be more robust to detect critical metabolic derangements
RESCUEICP ? RANDOMISED MULTICENTRE STUDY OF DECOMPRESSIVE CRANIECTOMY IN TRAUMATIC BRAIN INJURY
Ivan Timofeev1, The RESCUEicp Investigators2
1Academic Neurosurgery Unit, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK, 2International Study Centres
Background and aims
To provide Class I randomised evidence as to whether decompressive craniectomy is effective for the management of patients with raised and refractory intracranial pressure (ICP) following traumatic brain injury.
Methods
A multi-centre randomised trial comparing decompressive craniectomy with medical management. Patients (n=50 for the pilot phase, n=500 for the main study) with traumatic brain injury and raised ICP (>25mmHg) refractory to initial treatment measures are eligible for the study. Patients are randomised to one of two arms: continuation of optimal medical management (including barbiturates) versus surgery (decompressive craniectomy). The inclusion criteria are: traumatic brain injury, age 10-65 years, abnormal CT scan and the exclusion criteria: bilateral fixed and dilated pupils, bleeding diathesis, devastating injury, patient not expected to survive 24 hours. Outcome is assessed using the extended Glasgow Outcome Score and SF-36 quality of life questionnaire at 6 months with additional surrogate endpoints (ICP control, length of stay in ITU, length of stay in Neurosurgical Unit).
Results
The pilot phase of the study has been completed. The study is on-going. The background, protocol, progress of the study will be presented. Information on how to join the study and eligibility criteria for interested new centres will be provided.
Conclusions
Randomising patients with traumatic brain injury to decompressive craniectomy versus optimal medical management is feasible. Whether this operation is effective and safe remains to be seen.
CHARACTERIZATION OF PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE (PACAP) INDUCED PIAL ARTERIOLAR DILATION IN PIGLETS
Ferenc Bari1, Laura Lenti1, David Kis1, Ferenc Domoki1,2, Gabor Toth3, David W. Busija2
1Department of Physiology, Faculty of Medicine, University of Szeged, Szeged, Hungary, 2Dept Physiol & Pharmacol, Wake Forest Univ Health Sciences, Winston-Salem, NC, USA, 3Dept of Medical Chemistry, University of Szeged, Szeged, Hungary
PACAP has been found neuroprotective in numerous models of cerebral ischemia. PACAP-induced neuroprotection in vivo is partially based on its vasorelaxant effect. Cerebral ischemia occurring during the perinatal period often result in neurological sequelae. One possible contributor to the neurological damage is the cerebrovascular dysfunction, e.g. hypercapnia-induced arteriolar vasodilation is severely attenuated after ischemia reperfusion (I/R). The mechanism of PACAP induced cerebrovascular changes in newborns is unexplored.
We sought to characterize the vascular reactivity to PACAP-27 and −38 (naturally existing forms), as well as to shorter PACAP sequences in a piglet model that correlates well with the vascular physiology of newborn babies. Using the non selective cyclooxygenase (COX) inhibitor indomethacin (5 mg/kg, iv), the selective COX-1 inhibitor SC 560 (1 mg/kg, iv) and the selective COX-2 inhibitor NS 398 (1 mg/kg, iv), we investigated if COX-derived metabolites play a role in the cerebrovascular effects of PACAP. We also determined the effect of the nitric oxide synthase (NOS) inhibition by N-nitro-l-arginine methyl ester (L NAME, 15 mg/kg, iv). We examined the influence of short PACAP fragments (10-5 M topically) on PACAP-27 and-38 induced vasodilation. Further, we tested whether application of PACAP preserves hypercapnia-induced cerebrovascular dilator responses after I/R. Anesthetized (Na thiopenthal 40 mg/kg, ip, followed by α chloralose 40 mg/kg, iv), ventilated piglets (1 day old, 1 2 kg, n=104) were equipped with closed cranial windows. Pial arteriolar diameters were determined via intravital microscopy.
Topical PACAP-27 and −38 elicited similar, repeatable, dose dependent pial arteriolar dilations. Percent changes in diameters to 10-8, 10-7, and 10-6 M PACAP-38 and-27 were 6±1, 16±2, and 40±4, and 9±2, 19±3, 36±4 (mean±SEM), respectively. In contrast, the shorter segments of the peptides (6-27, 6-38, 6-15, 20-31, and 1-15) did not display any vasoactivity. Arteriolar dilations to PACAP-38 were abolished 20 min after indomethacin and SC-560 (to 10-6 M 35±4% vs. −1±1%*, and 36±6% vs. 1±1*, *p<0.05), but the responses were unaffected by NS-398 (to 10-6 M 39±4% and 45±8%, before and after the treatment, respectively). L NAME was also ineffective (34±5% vs. 26±5%). Co-application with PACAP 6-27 and 6-38 (putative PACAP receptor antagonists) reduced the PACAP induced caliber changes efficiently (e.g. changes to 10-6 M PACAP-38 were 46±10% vs. 7±3%* without and with PACAP 6-27), however other short segments had no impact on this response. Graded hypercapnia resulted in large, concentration-dependent, reversible increases in pial arteriolar diameters. Pretreatment with PACAP (10-8 M, topically for 30 minutes) preserved the hypercapnia-induced vasodilation after I/R (62±5% and 56±3% to 10% CO2 before and after I/R).
In summary, PACAP is a potent vasodilator in the neonatal cerebral circulation, and this vascular reaction depends critically on COX-1 derived prostanoids. The role of NO seems to be ancillary. Preservation of arteriolar dilator responsiveness to hypercapnia by PACAP may contribute to neuroprotection.
Supported by the Hungarian Research grants: OTKA-T046531, ETT-2006 and NKTH.
VEGF STIMULATES THE ERK1/2 SIGNALING PATHWAY AND APOPTOSIS IN CEREBRAL ENDOTHELIAL CELLS AFTER OXYGEN-GLUCOSE DEPRIVATIION
Purnima Narasimhan, Jing Liu, Yun-Seon Song, Pak Chan
Departments of Neurosurgery, Neurology and Neurological Sciences and Program in Neurosciences, Stanford University School of Medicine, Stanford, CA, USA
Background and aims: Cerebral endothelial cells play an important role in blood brain barrier (BBB) injury and contribute to postischemic secondary injury with increased vascular permeability and vasogenic brain edema. Although VEGF is potent in angiogenesis, it can have detrimental effects, such as an increase in endothelial permeability, leading to vasogenic edema. Cerebral endothelial cells are injured by hypoxia-induced reperfusion, leading to BBB breakdown and exacerbation of ischemic injury. We investigated the mode of cerebral endothelial cell death induced by oxygen-glucose deprivation (OGD).
Materials and Methods: Using primary cultures of mouse cerebral endothelial cells we examined cell death after 8 h of OGD. Western analysis of different proteins was carried out. VEGF was blocked using a neutralizing VEGF antibody and siVEGF to study the role of ERK activation. U0126 (10mM) was used to inhibit ERK to study its role in OGD. To examine the role of oxidative stress in the induction of VEGF and p-ERK, endothelial cells from SOD1 Tg mice were used and TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay and DNA fragmentation were performed.
Results: Caspase-3 cleavage was observed after OGD, indicating apoptotic cell death. HIF-1? increased transiently as early as 5 min after OGD, and VEGF protein expression showed a 2-fold induction over the control. Flk-1, the VEGF receptor through which VEGF exerts its effects, was also stimulated by 6 h and remained high until 24 h. There was an upregulation of phosphorylation of ERK1/2 protein levels. Using neutralizing VEGF antibody (10 µg/ml) and siVEGF, we show that p-ERK expression decreased after OGD in endothelial cells when VEGF was blocked. Furthermore, U0126 (10 µM), an ERK1/2 inhibitor, decreased the phosphorylation of ERK1/2 and significantly reduced cell death from 30% to 17%. Thus, ERK1/2 activation is important in OGD injury of cerebral endothelial cells. Blocking VEGF in the endothelial cells did not alter phosphorylated JNK or p38 expression levels. To examine the role of reactive oxygen species (ROS) in OGD we used SOD1-overexpressing endothelial cells. There was a reduction in phosphorylated ERK1/2 expression after OGD in these SOD1-overexpressing endothelial cells, suggesting that activation of ERK occurs in the presence of superoxide radicals or ROS produced after OGD, and that ROS and oxidative stress are critical in ERK activation. Apoptosis measured by TUNEL assay and DNA fragmentation assay showed a significant decrease in TUNEL-positive SOD1-overexpressing endothelial cells compared with wild-type cells after OGD.
Conclusion: Our data suggest that OGD induces VEGF signaling via its receptor, Flk-1, and activates ERK via oxidative-stress-dependent mechanisms. ERK1/2 phosphorylation has a crucial role in cell injury after OGD.
A NEW RAT MODEL FOR INVESTIGATION OF CHRONIC VENOUS INTRACRANIAL HYPERTENSION
Frederick H. Daher1, Axel Heimann2, Torben Scholz1, Carsten Stuer3, Michael Stoffel3, Bernhard Meyer3, Oliver Kempski2, Carlo Schaller1
1Department of Neurosurgery, University of Bonn Medical Center, Bonn, Germany, 2Institute for Neurosurgical Pathophysiology, University of Mainz, Mainz, Germany, 3Department of Neurosurgery, Technical University Munich, Munich, Germany
Objective: Reversal of a preexisting intracranial arteriovenous shunt, e.g. by removal of an arteriovenous malformation (AVM) may have deleterious consequences due to so called hyperperfusion injury. We have hypothesized that chronic venous hypertension (CVHT) causes a reduction of cerebral blood flow plus weakening of the blood brain barrier (BBB), thus facilitating perfusion pressure breakthrough following AV-shunt occlusion. The current experiment shall deliver proof of this concept by application of a new AV-fistula model, which restricts CVHT to the intracranial compartment.
Methods: In five male Sprague Dawley rats a fistula was created between the right common carotid artery (CCA) and the exit vein from the right transverse sinus (TS). All facial and pterygopalatinal branches were ligated for creation of direct inflow from the CCA via the TS. After 12 weeks, transdural assessment of cortical capillary oxygen saturation (%SO2) and of regional cerebral blood flow (rCBF) by Laser Doppler scanning was performed in the fistula rats and in four controls. This was followed by perfusion-fixation and hematoxylin-eosin staining of the brains in all animals.
Results: Cortical oxygenation was significantly lower in the fistula group (58,2±19,1 %SO2) than in controls (72,8±8,3 %SO2). CBF values were not significantly different with 85,1±56,4 LDU in the fistula group and 83,3±52,4 LDU in controls. Hippocampal histological slices showed no signs of ischemia, but cortical thickness was reduced to app. 60% of the controls. Furthermore, an increase in diameter of arterioles and venules was observed in the fistula group.
Conclusion: CVHT results in a significant global drop in cortical oxygen saturation without parallel decreases in CBF. This decrease in %SO2 may result from an increase in energy expenditure or from remodelling of vascular channels. The effect of this model leads to a substantial increase in the diameter of cerebral arterioles and venules. Further investigations regarding weakening of the BBB by Evans-blue staining, and of the role of vascular endothelial growth factors are warranted.
Key words: cerebral blood flow- venous hypertension -arteriovenous fistula - Laser Doppler - cortical oxygen saturation - blood brain barrier
DIFFERENTIAL DIAGNOSIS BETWEEN RADIATION NECROSIS AND GLIOMA PROGRESSION USING SEQUENTIAL PROTON MAGNETIC RESONANCE SPECTROSCOPY AND METHIONINE POSITRON EMISSION TOMOGRAPHY
Takeshi Nakajima1, Toshihiro Kumabe1, Masayuki Kanamori1, Ryuta Saito1, Manabu Tashiro2, Mika Watanabe3, Teiji Tominaga1
1Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai, Japan, 2Division of Nuclear Medicine, Tohoku University Cyclotron and Radioisotope Center, Sendai, Japan, 3Department of Pathology, Tohoku University Hospital, Sendai, Japan
Background and aims: Differential diagnosis between the radiation necrosis and the recurrent tumor is exceedingly important in clinical management of malignant glioma after the radiation therapy. In this study, the authors verified the clinical usefulness of multi-modality exploration including proton magnetic resonance spectroscopy (1H-MRS) and L-[methyl-11C] methionine (MET) - positron emission tomography (PET) in patients with irradiated glioma to develop the reliable protocol for distinguishing these two pathological statuses.
Methods: Eighteen patients with irradiated glioma underwent sequential 1H-MRS and MET-PET. The values of metabolites including choline-containing compounds (Cho), creatine phosphate (Cre), and lactate (Lac) evaluated by 1H-MRS were subsequently calculated as a ratio of Cho to Cre (Cho/Cre) and Lac to Cho (Lac/Cho), respectively. The uptake of MET was determined as a ratio of the lesion to contralateral reference region (L/R). The validity of both 1H-MRS and MET-PET was evaluated by correlating these calculated values with final diagnosis determined by histological examination and/or long-term follow-up clinical course and MR images of the patients.
Results: The Lac/Cho was 0.63 +/− 0.25 (average +/− SD) in recurrent group (7 cases) and 2.25 +/− 1.89 in necrosis group (11 cases). There was significant difference between the recurrence and necrosis in Lac/Cho ratio (P <0.01). Consecutive investigation of 1H-MRS revealed temporary elevation of Cho peak in 3 of 10 cases with radiation necrosis, which could be identified as false positive finding for recurrence. Even including those situations, MET-PET demonstrated statistically significant difference between the two groups (2.18 +/− 0.42 vs. 1.49 +/− 0.35, P <0.01). According to a 2 × 2 factorial table analysis, the borderline of Lac/Cho and L/R values those could differentiate the tumor recurrence from necrosis were 1.05 and 2.00, respectively.
Conclusions: The sequential evaluation using 1H-MRS is actually reliable and accessible technique for differentiating between the tumor recurrence and the radiation necrosis. But the temporary elevation of Cho in the course of radiation necrosis should be recognized. Additional application of MET-PET can draw a conclusion of the diagnosis.
CEREBRAL ISCHEMIA INDUCED CELL MIGRATION OF IMMUNOCYTES AND MESENCHYMAL STEM CELLS
Shu-Yun Cheng, Chun-Hung Chang, Chi-Mei Hsueh
Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan-R.O.C.
Brain injury including cerebral ischemia can often break the immune privilege and lead to significant damage of brain. The primary goal of the study was to verify the brain infiltration capability of immunocytes and mesenchymal stem cells (MSCs) from periphery. In the study, a focal cerebral ischemia model was developed in S.D. rats by 90 min bilateral common carotid artery occlusion plus unilateral middle cerebral artery occlusion (CCAO/MCAO) followed by reperfusion. Brain infarct size and blood brain barrier (BBB) permeability were determined at various reperfusion times (0, 1, 8, 24, 48 h) by TTC staining and evans blue extraction, respectively. The number of immnuocytes (neutrophils and CD45+ cells) in the ischemic brain was also determined at the same time by Hematoxylin-Eosin (HE) and immunohistochemistry staining. In a different set of experiments, the migration of PKH26 labeled MSCs was further analyzed in the ischemic brain at various reperfusion times using the fluorescence microscope. The current results showed that significant brain infarct did not appear until 24 h after reperfusion, whereas disruption of BBB began at 1 h after reperfusion and exacerbated with time till 48 h. Along with the BBB disruption, neutrophil-like cells were observed in the injured brain at 24 h after reperfusion. The number of activated immunocytes (CD45+) was also increased significantly at the infarct core area (cortex) at 24 h followed by a subsequent increase at the peri-infarct area (striatum) at 48 h. In addition, significant brain infiltration of the PKH26 (+) MSCs was also observed at 48 h after reperfusion. These results indicate that cerebral ischemia induced brain injury and BBB disruption allow significant entrance of immunocytes and MSCs from the periphery into the injured brain. Immunocyte movement within the ischemic brain may further expand the brain infarct into the surrounding area (penumbra). Details regarding the pathophysiological roles of the immigrant immunocytes and MSCs currently are still under the investigation in our laboratory. Results from the study will provide valuable information for cellular treatment of cerebral ischemia.
NO VASOSPASM OCCURRED IN SMALL ARTERIES ON BRAIN STEM IN CANINE SUBARACHNOID HEMORRHAGE MODEL
Masataka Takahashi1, R. Loch Macdonald2, Kazuo Mizoi1
1Division of Neurosurgery, Department of Neuro-Locomotor Science, Akita University School of Medicine, Akita, Akita, Japan, 2Section of Neurosurgery, Department of Surgery and Pritzker School of Medicine, University of Chicago, Chicago, IL, USA
Introduction
Delayed cerebral vasospasm due to subarachnoid hemorrhage is usually detected in relatively large intracranial arteries such as internal, anterior and middle cerebral artery. There are, however, only several reports which examine question about whether vasospasm would occur in much smaller arteries and the issue still is shelved. To elucidate the matter we tested subarachnoidal perforating artery on brain stem surface and basilar main trunk in canine vasospasm model using pressurized artery technique.
Method
SAH was created by double injection of autologus blood. Main basilar arteries and subarachnoid portion of perforating arteries were removed from control and day4, day7, day21 after subarachnoid hemorrhage. The vessels were cannulated and tested at luminal pressure in normal blood pressure range of canine. To clarify strength of vasoconstriction potassium dose response were also investigated.
Results
SAH produced significant vasospasm of the basilar main trunk on day 7 after subarachnoid hemorrhage (mean value±SE inµm, Control 1143.5±49.4, day 4 1085.5±31.8, day 7 843.2±34.8, day 21 922±33.8). Basilar arteries were also narrow significantly on day 4, day7, day21 after subarachnoid hemorrhage at intraluminal pressure of 25 ? 100 mmHg (P<0.05, vs control). On the other hand there were no vasospasm in perforating small arteries (Control 103.4±10.1, day 4 118.9±9.8, day 7 111.7±12.9, day 21 118.7±11.0 at 0mmHg). From the results of KCl dose response experiment, contractility of basilar main trunk to potassium chloride by maximal % contraction, calculated as follow equation: [(D0-Da)/D0] × 100, where D0 is the baseline diameter at 0mmHg and Da is the smallest diameter measured at certain concentration of KCl, decreased significantly on day 4 at 50mmHg, day 7 at 0, 25, 50, 75 mmHg, day21 at 25, 50 mmHg (P<0.05). Contractility of perforating small arteries in internal diameter were increased on day 7 and day 21 after subarachnoid hemorrhage at higher pressure (Control 29.7±10.9%, day 4 45±6.5%, day 7 59.6±7.5%, day 21 66.3±4.0%, % contraction at 50mmHg, P<0.05).
Conclusion
Delayed cerebral vasospasm of small perforating arteries in intraarachnoidal space was not observed differently from basilar main trunk in canine vasospasm model.
Small arteries were not influenced by subarachnoid hemorrhage altogether considering that contractility to potassium was elevated at higher pressure in contrast to decreased response on basilar main trunk. This result suggests that small arteries were remodeled and may compensate function of larger artery impaired by subarachnoid hemorrhage to maintain appropriate blood supply for brain.
THRESHOLDS FOR CEREBRAL ISCHEMIA ON BALLOON TEST OCCLUSION USING REGIONAL CEREBRAL OXYGEN SATURATION
Hideyuki Kamii1, Kiyotaka Sato1, Hiroaki Shimizu2, Yasushi Matsumoto3, Satoru Fujiwara2, Teiji Tominaga4
1Department of Neuroanesthesia, Kohnan Hospital, Sendai, Japan, 2Department of Neurosurgery, Kohnan Hospital, Sendai, Japan, 3Departmetn of Neuroendovascular Therapy, Kohnan Hospital, Sendai, Japan, 4Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai, Japan
Background and aims: Balloon test occlusion (BTO) of the internal carotid artery (ICA) has been an accepted procedure for preoperative evaluation of patients with ICA large aneurysms. We investigated thresholds for cerebral ischemia on BTO using regional cerebral oxygen saturation (rSO2).
Methods: In 19 patients with an ICA large aneurysm, BTO was performed under rSO2 monitoring. Then, in a room for single-photon emission computed tomography (SPECT), cerebral blood flow (CBF) was evaluated during the ICA occlusion. The sensors for rSO2 were bilaterally attached on the forehead, and regions of interest in the SPECT were defined in the area below the rSO2 sensor of each hemisphere.
Results: The rSO2 significantly decreased during BTO (73.5 to 64.2%, p<0.001). The individual decreases in rSO2 correlated with decreases of CBF from SPECT (r=0.954, p<0.001). Moreover, 13 patients with rSO2 decrease less than 12 points had no ischemic symptoms, while 4 patients with rSO2 decrease more than 14 points had some symptoms.
Conclusion: The threshold for cerebral ischemia may be 12 points of rSO2 decrease on BTO. It might be important to prevent rSO2 decrease more than 12 points, when ICA occlusion is inevitable during some intravascular techniques, especially carotid artery stenting.
RAPID ELEVATION OF INTRACRANIAL PRESSURE AND HYPONATREMIA FOLLOWING SUBARACHNOID HEMORRHAGE IN RATS
Tatsuro Mori, Takahiro Igarashi, Nobuhiro Moro, Takahiro Matsuzaki, Jyun Kojima, Tatsuro Kawamata, Yoichi Katayama
Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan
Background: Hyponatremia is common complication in patients with aneurysmal subarachnoid hemorrhage (SAH). These patients demonstrate excessive natriuresis, which decreases total blood volume through osmotic diuresis and increases the risk of symptomatic cerebral vasospasm. However, precise mechanisms underlying SAH-induced hyponatremia remain unclear. In the present study, in order to establish an experimental model of hyponatremia following SAH, we induced SAH in rats, and serum sodium (Na) levels, Na excretion and physiological parameters were determined. Methods: Male Wistar rats were used (n=18). SAH was induced by an endovascular puncture method. The mean arterial blood pressure (MABP), intracranial pressure (ICP), and cerebral blood flow (CBF) were continuously monitored. The urine was cumulatively collected for 12 hours post SAH, and the urine Na concentration was determined by a spectrophotometer. The serum Na levels and body weight were measured at 12 hrs, 2 and 4 days following the SAH induction. Results: The baseline ICP level was 3.5±2.6 mmHg, and was increased to 67.4±17.6 mmHg immediately following the SAH induction. The CBF decreased rapidly and gradually recovered up to 70–80% of baseline. The urine volume and the total Na excretion significantly increased as comparison to the sham (P<0.05). The serum Na level was significantly decreased at 4 days following SAH (P<0.05). Conclusions: The endovascular puncture model is suitable for the study of the hyponatremia concomitant with natriuresis and diuresis after SAH.
INCREASE IN GLUTAMATE AS A SENSITIVE INDICATOR OF EXTRACELLUALR MATRIX INTEGRITY IN PERITUMORAL EDEMA: 1H-MRS STUDY ON A 3.0T SYSTEM
Teruo Kimura1,2, Masaki Ohkubo1,2, Hironaka Igarashi1, Ingrid L. Kwee2, Tsutomu Nakada1,3
1Cener for Integrated Human Brain Science, Brain Resaerch Institute, University of Niigata, Niigata, Japan, 2Department of Radiological Technology, School of Health Sciences Faculty of Medicine, University of Niigata, Niigata, Japan, 3Department of Neurology, University of California Davis, Davis, CA, USA
Object: Previous studies based on diffusion tensor imaging (DTI) indicate that there are two types of peritumoral edema, namely, edema with preserved structural integrity of the glial matrix and edema with compromised glial matrix. We hypothesize that functionality of the glutamate-glutamine shuttle, a vital neuron-glia interaction, may be differentially affected by peritumoral edema. We tested this hypothesis using proton magnetic resonance spectroscopy (1H-MRS) on a 3.0T system which is capable of quantifying glutamate without need of editing.
Methods: Twenty-three patients who have a single brain tumor mass and peritumoral edema (nine high-grade gliomas, eight metastatic brain tumors, six meningiomas), and nine normal subjects participated in this study. Single voxel 1H-MRS targeted on the region of peritumoral edema was performed using a 3.0T magnetic resonance imaging (MRI) system.
Conclusion: Glutamate in peritumoral edema of non-glial tumors was significantly elevated (p<0.01) compared to edema associated with glial tumors or normal white matter. The finding confirmed that periedema in non-glial tumors is distinct from that of glial tumors as previously indicated by DTI studies. The former condition represents a compensatory increase in activities of the glutamate-glutamine shuttle brought about by simple expansion of the extracellular space due to edema, whereas the latter condition represents serious disruption of the structural integrity of the glial matrix. Assessment of glutamate concentration in peritumoral edema using 1H-MRS on a 3.0T system can provide another objective index of the structural integrity of the glial matrix, an indirect marker of tumor histology.
WHEN CEREBRAL VASOSPASM AFTER SUBARACHNOID HEMORRHAGE BEGINS AND ENDS?: DEFINITION OF VASOSPASM USING ANGIOGRAPHY
Shigeki Ono, Keisuke Onoda, Koji Tokunaga, Kenji Sugiu, Isao Date
Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistory and Pharmaceutical Sciences, Okayama, Japan
Cerebral vasospasm (VS) after subarachnoid hemorrhage (SAH) is one of the fatal strokes causing severe brain ischemia. So far, many types of treatments for VS have been applied, such as triple H therapy, clot fibrinolysis, spinal drainage, endovascular treatment, or administration of vasodilatory agents, but its pathophysiology has been still unknown, and absolute therapeutic strategies have not been established yet. One of the reasons why the strategies of VS treatment has not been confirmed is that clear definition of VS has not given until now. For instance, there are many cases which have no symptoms while these patients have obvious angiographic VS, or it is difficult to distinguish between real VS symptoms and neurological deterioration due to fever, abnormal blood levels of electrolytes, primary brain damages from SAH. Therefore, incidence of VS and the timing of its treatment are sometimes different among each facility. From 2000, we define VS as angiographic VS in our institute. When symptomatic VS is suspected clinically, emergency angiography is immediately performed and confirm VS. And then if necessary, endovascular therapy is given. Even if there are no VS symptoms during spasm periods, we routinely perform angiography on around day 7 as shown in the following protocol (see table). Under this protocol, angiographic VS was seen in 59.7% of the 57 patients, while, symptomatic VS was seen in 37.1%. In this study, we discuss the advantages of this definition of VS by angiography.
Table:
A. Treatment after surgery
1) Drains(always inserted)
Ventricular drainage (VD) + cisternal (CD) or lumber drainage (LD)
2) Fibrinolysis
UK 30,000IU/day, injected through VD Repeated if necessary by Day 4
3) General management(under strict control)
Normo~hypertension, normovolemia (≧120mmHg, CVP≒8~10)
Hct(≦9.0mg/dl), ICP(≧20mmH2O), and electrolytes(≧130meq/L)
Ozagrel Na (i.v.) Fasudil (i.v.)
4) Intravascular treatment (if severe angiographic VS was detected.)
Fasudil (i.a.)
Percutaneous transluminal angioplasty (PTA)
B. Detection of VS
1) TCD monitor
2) Neurological changes
3) Angiography
When TCD or neurological changes are detected On day 7 (routine exam.)
4) INVOS5100R(Somanetics, USA): 2005~
Near infrared spectroscopy, measuring rSO2
INTRA-CISTERNAL INFUSION OF MAGNESIUM SOLUTION REVERSES THE REDUCED CEREBRAL BLOOD FLOW INDUCED BY EXPERIMENTAL SUBARACHNOID HEMORRHAGE IN THE RAT
Kentaro Mori1, Masahiro Miyazaki2, Junko Iwata2
1Department of Neurosurgery, Juntendo University, Shizuoka Hospital, Shizuoka, Japan, 2Juntendo Casualty Center, Shizuoka, Japan
Background and aims: Magnesium may act as a neuroprotectant and antivasospastic agent in the presence of ischemic brain injury. Recent randomized clinical studies of intravenous infusion of magnesium in patients with SAH have failed to show any prevention of angiographic vasospasm. The present study investigated the effect of intra-cisternal administration of magnesium on cerebral vasospasm in the experimental SAH rat model.
Methods: The rat double SAH model (0.2 ml autologous blood injected twice into the cisterna magna) was used. Normal saline (SAH group: N=8) or 10 mmol/L magnesium sulfate in normal saline (SAH-MG group: N=8) was infused into the cisterna magna at 1.5 µL/min for 30 minutes on day 5. Control rats without SAH also received intra-cisternal infusion of normal saline (Control group: N=6). Local CBF (24 locations and weighted average) were quantitatively measured by the autoradiographic technique using [14C]iodoantipyrine after infusion.
Results: The weighted average CBF in the SAH group (0.78±0.16 mL/g/min) was significantly reduced compared to the Control group (1.0±0.15) and was significantly improved in the SAH-MG group (0.98±0.18). Local CBF was significantly reduced in 16 locations in the SAH group and significantly improved in 12 locations in the SAH-MG group.
Conclusions: Intra-cisternal infusion of magnesium sulfate significantly improved the reduced CBF induced by experimental SAH in the rat.
METALLOTHIONEIN AND BRAIN INJURY AFTER INTRACEREBRAL HEMORRHAGE
S. Yamashita, M. Okauchi, R. Keep, Y. Hua, J. Hoff, Guohua Xi
Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA
Background and Aims: Metallothioneins (MT) are metal-binding proteins, which can be upregulated in the brain after injury and are associated with neuroprotection. A recent genomics study has shown that brain MT-1 and ?2 mRNA levels are upregulated following intracerebral hemorrhage (ICH) in rats. The current study examined whether or not brain MT-1 and ?2 protein levels are increased after ICH. We also investigated the effect of exogenous MT-1 in perihematomal edema formation in vivo and iron-induced cell death in vitro.
Methods: There were three sets of experiments in this study. In the first set, male Sprague-Dawley rats received an intracaudate injection of 100-µl autologous whole blood. Sham animals had a needle insertion. The rats were killed at 1, 3 and 7 days later and the brains were sampled for Western blot analysis and immunohistochemistry. The antibody used detects both MT-1 and MT-2. In the second set, rats receive an intracaudate infusion of 100-µl blood with 10-µl MT-1 (0.1 nmol or 0.2 nmol) or saline. The rats were euthanized 3 days later for brain water content measurement. In the last set, cultured primary astrocytes were treated with MT-1 (5, 10, 50 and 100 nM) or vehicle for one hour. The astrocytes were then exposed to ferrous chloride (500 µM). Culture medium was collected for lactate dehydrogenase (LDH) measurement 48 hours following treatment.
Results: MT-1/?2 immunoreactivity was very low in normal brain tissue. MT-1/−2 immunoreactivity in the ipsilateral basal ganglia was significantly increased after ICH. MT-1/−2 positive cells were found in the ipsilateral basal ganglia after ICH and most of those positive cells appeared to be glia-like. Western blot analysis, showed a marked increase in MT-1/−2 content at day 1 after ICH, but it was still detectable at day 7. At day 3 after ICH, MT-1/−2 levels in the ipsilateral basal ganglia were 3327±1523 vs. 338±270 pixels in the sham control, p<0.01.
Exogenous MT-1 attenuated edema formation following ICH in vivo. ICH-induced brain edema at day 3 was reduced by co-injection with MT-1 at dose of 0.2 nmol (80.7±1.4% vs. 82.2±0.9% in the vehicle-treated group, p<0.05) but not at dose of 0.1 nmol (p>0.05). MT-1 also protected against iron-induced cell death in cultured astrocytes. The LDH release induced by ferrous chloride was significantly reduced by MT-1 pretreatment at all doses (e.g. MT-1 at dose of 50 nM: 141±62 vs. 313±71 mU/ml in the vehicle-treated group, p<0.05).
Conclusion: Brain MT levels were upregulated after ICH and MT-1 reduced ICH-induced brain edema in vivo and iron-induced cell death in vitro suggesting MT could be a therapeutic target of ICH.
ISCHEMIC NEUROPROTECTION BY GLYCINE TRANSPORTER INHIBITION AND NMDA RECEPTOR ANTAGONISM: THE ROLE OF CEREBRAL METABOLISM
Laszlo Szabo
Neuroscience Discovery Research, Abbott GmbH, Ludwigshafen, Germany
Background and aims? The off-label use of atypical antipsychotic drugs in elderly patients has been linked to an increased risk of stroke, but the underlying mechanism of this phenomenon is not known. Enhancement of NMDA receptor activity by blockade of the glycine transporter 1 (GlyT1) is a novel experimental approach for the treatment of schizophrenia. Since NMDA receptor activation might exacerbate cerebral ischemic damage, we previously investigated the effect of several GlyT1 inhibitors in a rat stroke model and found that pre-ischemic treatment with 30 mg/kg SSR504734 i.p. significantly reduced the extent of ischemic infarction while the dose of 10 mg/kg was ineffective. The present study evaluated the neuroprotective property of SSR504734 in a post-occlusion treatment paradigm and assessed its effect on brain metabolism in direct comparison with the NMDA receptor antagonist MK-801.
Methods?Focal cerebral ischemia was induced by permanent distal occlusion of the middle cerebral artery (MCA) in Long Evans rats under sevoflurane anesthesia. The animals were treated with a single intraperitoneal injection of either 30 mg/kg SSR504734 or 0.5 mg/kg MK-801 administered 1.5 hours after MCA occlusion. Cortical infarct volume was quantified by TTC staining and image analysis 24 hours later. The compounds' effect on brain metabolism was evaluated in additional experiments by determining the cerebral metabolic rate of glucose (CMRG) in 13 predefined regions of the cerebral cortex with the aid of the [14C]deoxyglucose technique. The tracer was administered intravenously to freely moving animals 80 minutes after application of the test drugs; the experiments were terminated 45 minutes later. The statistical significance of the findings was assessed by analysis of variance followed by the Holm-Sidak post-hoc procedure for multiple comparisons. Differences with a P value of less than 0.05 were considered statistically significant.
Results?Treatment with both SSR504734 and MK-801 led to a virtually identical reduction in infarct volume by approximately 26% (P < 0.05). Administration of 30 mg/kg SSR504734 i.p. reduced local CMRG in each cerebrocortical region by 20% to 30% in a statistically significant manner whereas the dose of 10 mg/kg was without effect on glucose metabolism. In contrast, the administration of 0.5 mg/kg MK-801 i.p. resulted either in a statistically significant increase in cortical CMRG by up to 50% (in seven out of 13 investigated regions) or in no significant change.
Conclusions?The GlyT1 inhibitor SSR504734 maintains its neuroprotective effect even if it is administered with a significant delay after the onset of ischemia. In this therapeutically relevant setting, GlyT1 inhibition proved to be as effective as NMDA receptor antagonism. The data on brain metabolism suggest that the neuroprotection brought about by GlyT1 inhibition could be explained by the reduced metabolic demand of the tissue at risk; this effect is likely to be mediated by inhibitory glycine receptors. However, the selective activation of synaptic, as opposed to extrasynaptic, NMDA receptors may represent an alternative mechanism of glycine-mediated prevention of neuronal death. The nature of MK-801-induced cerebral metabolic changes indicates that a reduction in brain metabolism is not a necessary prerequisite of ischemic neuroprotection.
EFFECT OF STATINS ON PLASMA LEVELS OF ASYMMETRIC DIMETHYLARGININE IN PATIENTS WITH NONCARDIOGENIC ISCHEMIC STROKE
Yasuhiro Nishiyama1, Yuichi Komaba1, Masayuki Ueda1, Tatsushi Kamiya2, Nobuo Kamiya1, Yasuo Katayama1
1Division of Neurology, Nephrology and Rheumatology, Nippon Medical School, Tokyo, Japan, 2Department of Neurology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
Introduction: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, has reportedly been involved in the pathogenesis of atherosclerosis (1). Elevated plasma ADMA levels have been observed in atherosclerotic patients with various risk factors (2). Recent studies have reported that treatment of hypercholesterolemia with hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) might lower plasma ADMA levels, contributing to the restoration of endothelial dysfunction. Although ADMA is related to atherosclerotic diseases, the association between ADMA and ischemic stroke has rarely been reported. The present study examined effect of statins for treatment of hypercholesterolemia on plasma ADMA levels and differences in the effectiveness among statins in patients with ischemic stroke.
Methods: Consecutive 132 outpatients suffering from noncardiogenic ischemic stroke with written informed consents were included in the study. All patients received aspirin at a daily dosage of 100mg for anti-platelet medication. The subjects were divided into high cholesterol (HC) group (LDL 140 mg/dl or higher) and normal cholesterol (NC) group (LDL<140 mg/dl). Statins, including 10mg of pravastatin (n=20), 20mg of fluvastatin (n=14), 1mg of pitavastatin (n=12) and 10mg atorvastatin (n=2), were administered in 48 HC patients, and total 103 patients were followed-up for 3months. Baseline clinical characteristics of the participants are shown in Table 1.
Results: Treatment with statins for 3 months in HC group led to a significant reduction in plasma ADMA concentrations, compared with NC group (p=0.007). Multiple backward linear regression analysis revealed that treatment with statins was an independent determinant (p=0.0038) of delta ADMA (difference between pre- and post-values). No significant differences in the effectiveness were observed among the type of statins (p=0.94).
Conclusion: Treatment with statins in HC patients with noncardiogenic ischemic stroke leads to significant reduction in plasma ADMA levels, suggesting an improvement in endothelial function by statins.
PREVENTION OF CEREBROVASCULAR K+ CHANNELS DYSFUNCTION BY KMUP-1 AFTER ACUTE SUBARACHNOID HEMORRHAGE IN RATS
Bin-Nan Wu1, Yi-Lin Tsai1, Yi-Ren Hong2, Shen-Long Howng3, Ing-Jun Chen1
1Department and Graduate Institute of Pharmacology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan-R.O.C., 2Graduate Institute of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan-R.O.C., 3Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan-R.O.C.
Background and aims: Sustained narrowing of cerebral vessels remains a major complication after aneurysmal subarachnoid hemorrhage (SAH). The large conductance Ca2+-activated K+ (BKCa) channel plays a negative feedback role to prevent cerebral vasospasm. The absence of an adequate therapy for cerebral vasospasm continues to motivate preclinical and clinical studies. KMUP-1, a xanthine-based derivative, has been demonstrated to activate K+ channels resulting in the relaxation of aortic and tracheal smooth muscles. In this study, we tried to investigate whether SAH-induced vasospasm could be a defect in the function of vascular BKCa channels. We also attempted to explore the protective activity of KMUP-1 in SAH rats.
Methods: Animals were divided into four groups including control, SAH, KMUP-1-treated and pinacidil-treated rats. Rats were subjected to experimental SAH by injecting autologous blood into the cisterna magna, and then KMUP-1 (1 mg/kg) and pinacidil (1 mg/kg) were administered intraperitoneally 1 h after SAH. All rats were sacrificed 24 h after SAH. Cerebrovascular smooth muscle cells (CVSMCs) were enzymatically isolated from rat basilar arteries.
Results: Using whole-cell patch-clamp techniques, iberiotoxin (IbTX)-sensitive BKCa currents attenuated in SAH rats compared with the control. Interestingly, pretreatment with KMUP-1 or pinacidil, the IbTX-sensitive BKCa current was restored. In inside-out patches, the unitary conductance and voltage sensitivity of SAH, KMUP-1 and pinacidil groups showed effects similar to those of control groups. However, the BKCa channel open probability (NPo) and calcium sensitivity were decreased in SAH rats compared with the control.
Conclusions: We suggest that the down-regulation of the BKCa channel activity could be due to the modification of the BKCa channel's calcium sensitivity. The results indicated that KMUP-1 and pinacidil could protect against the SAH-induced vasospasm.
C-FOS EXPRESSION IN EXOFOCAL REGIONS FOLLOWING EXPERIMENTAL MIDDLE CEREBRAL ARTERY OCCLUSION MODELS
Tomoya Omae1, Taku Sugawara1, Masaya Oda1, Suguru Yamaguchi1, Kazuo Mizoi1, Hiroyuki Kinouchi2
1Division of Neurosurgery, Department of Neuro and Locomotor Science, Akita University School of Medicine, Akita, Japan, 2Department of Neurosurgery, University of Yamanashi Faculty of Medicine, Yamanashi, Japan
Introduction: Experimental permanent middle cerebral artery (MCA) occlusion in rodents results in the cerebral infarction in MCA territory within hours after ischemia. Delayed neuronal degeneration is also known to occur in the areas remote from the infarct, such as substantia nigra and thalamus. Immediate early gene c-fos, a regulator of neuronal excitability and survival, was reported to be induced in these exofocal regions after ischemia in the previous studies. These studies were performed using intraluminal suture-inserting MCA occlusion model (Suture model), in which blood flow in the exofocal regions could also be reduced. Therefore, the mechanism of c-fos induction in these regions has been obscure. To eliminate the effects of blood flow reduction in these regions, we utilized selective MCA coagulation model (Tamura model). Methods: The rats were subjected to permanent MCA occlusion by Tamura model and c-fos immunohistochemistry and Western blot were performed 1, 6 and 24 hours (h) after ischemia. Another group of rats was also subjected to permanent MCA occlusion by Suture model and c-fos expression was examined 6 h after ischemia. Results: In Tamura model, c-fos was expressed at 1 h, peaked at 6 h and declined at 24 h in substantia nigra reticulata and was also detected at 6 h in the thalamus. Western blot confirmed the changes in c-fos on immunohistochemistry in Tamura model. Temporal and spatial expression pattern in these areas was similar to that in Suture model. Comments: The results suggest that the c-fos induction in substantia nigra reticulata and thalamus is not the consequence of blood flow reduction. Considering the neural connection between MCA territory and these exofocal regions, c-fos induction is presumably due to transsynaptic excitation.
THE ADMINISTRATION OF CEREBROLYSIN IN THERAPY OF VIRAL NEUROINFECTIONS
Andrey Kulchikov2, Alexander N. Makarenko1, Irina Vasileva3
1Orlov State University, Orel, Russia, 2Orlov Regional Clinical Hospital, Orel, Russia, 3Institute of Pharmacology and Toxicology of AMS of Ukraine, Kiev, Ukraine
Background and aims: the study of curative effect of Cerebrolysin in complex therapy of acute viral encephalitis.
Methods: the patients with a clinical picture of acute viral encephalitis were included in the study; therapy with Cerebrolysin was started in the first day from the onset of the disease. The assessment of preparation's action was made by means of: 5-score rating scale with standardized assessment criteria of intensity of each symptom, study of cerebrospinal fluid, immunologic indexes, CT/MRI of brain, ECG, EEG, glucose level, white blood count, C-reactive protein, and blood sedimentation rate. Statistical analysis was carried out as well.
Results: before therapy both groups (receiving Cerebrolysin and baseline therapy and group receiving only baseline therapy) were comparable on the severity of clinical symptomatology and laboratory indexes. To the 10th day of the disease (termination of carried out therapy) in the group receiving Cerebrolysin, the accelerated restoration of neurologic dysfunctions, normalization of body temperature, respiration rate and heart rate, normalization of blood values (leucocytes, C-reactive protein, blood sedimentation rate), cerebrospinal fluid sanation, normalization of immune system indexes (p<0,01) were marked compared to the control group. On the 28th day of the disease in the group receiving Cerebrolysin, full recovery is marked. In the control group residual effects were marked: cerebrospinal fluid cytosis, headache, general weakness, lack of energy, meningial symptoms, focal neurological symptoms and mental disorders.
Conclusions: 1. Cerebrolysin is the effective drug in the treatment of viral neuroinfections. 2. Therapy with Cerebrolysin promotes faster and full recovery of patients with viral encephalitis, allows the accelerating of cerebrospinal fluid sanation and normalization of laboratory indexes. 3. The administration of Cerebrolysin allows the achieving of absence of residual effects after viral neuroinfections. 4. Cerebrolysin normalizes immune system in patients with vital neuroinfections.
THE EFFECT OF FLUVOXAMINE MALEATE ON POSTSTROKE DEPRESSION
Eiko Sunami, Kazuhiro Usuda, Yuichi Komaba, Yasuo Katayama
Divisions of Neurology, Nephrology and Rheumatology, Department of Internal Medicine, Nippon Medical School, Tokyo, Japan
Background and aims: The patients after ischemic stroke frequently have depression and insomnia. The purpose of this study is to evaluate the effect of fluvoxamine maleate on poststroke depression (PSD).
Methods: We gave fluvoxamine maleate (75mg/day) to the depressive patients who had more than 40 on self-rating depression scale (SDS) score three weeks after onset of cerebral infarction among 30 ischemic stroke inpatients. Before and two weeks after medication of fluvoxamine maleate depression and insomnia were quantified by SDS and Pittsburgh Sleep Quality Index (PSQI).
Results: Administration of fluvoxamine maleate to one who agreed to participate in this study out of 10 patients with more than 40 on SDS score improved the score on SDS and PSQI from 50 to 24, from 27 to 25, respectively.
Conclusions: This study shows that depression and insomnia ameliorated after medication of fluvoxamine maleate in a case of PSD. It has been reported that administration of fluvoxamine maleate increases melatonin, N-acetyl-5-methoxytryptamine, which is said to have circadian rhythm and be related to sleep. At present we are increasing the number of the cases and are investigating the influence on blood melatonin level and the effect on symptoms of depression and insomnia by treatment of fluvoxamine maleate.
SPATIAL, TEMPORAL PROTEOMIC ASSAY OF CORTEX IN ISCHEMIC BRAIN INJURY
Cheng-Fu Chang, Y.H. Chen, H.A. Shui, Y.H. Chiang
Department of Neurological Surgery, National Defense Medical Center and The Tri-Service General Hospital, Taipei, Taiwan-R.O.C.
Recently, array technology has allowed the simultaneous study of hundreds to thousands of genes in single samples from animal models of brain disease. In experimental brain ischemia, some studies have reported to address the patterns of gene expression in various models of permanent and transient ischemia by focusing on the infarct area and the surrounding penumbra. So far, only one study addressed gene expression in remote non-ischemic cortex, but examined only one time point in the late sub-acute stage of 10 days after ischemia and did not compare responses in non-ischemic vs. ischemic brain areas. Alterations in protein synthesis seem to account for the outcome of central neurons following hypoxia. In the rat nucleus tractus solitarii, c-fos protein expression was induced by relatively mild hypoxia (10% O2) and nNOS expression was increased by cycles of episodic hypoxia. However, there are no extensive assessments of protein expression differences in the cortical regions, and their response patterns to acute ischemic injury. In this study, we reported the application of proteomic techniques to this important issue could provide us with insights into potential mechanisms underlying the neurocognitive morbidity and contribute to the identification of critical proteins implicated in CNS vulnerability.
GABA RELEASED FROM SPECIFIC SUBTYPES OF ACTIVATED CORTICAL INTERNEURONS CONTRIBUTES TO THE HEMODYNAMIC RESPONSE TO BASAL FOREBRAIN STIMULATION
Ara Kocharyan1, Priscilla Fernandes1, Xin-Kang Tong1, Elvire Vaucher2, Edith Hamel1
1Laboratory of Cerebrovascular Research, Montreal Neurological Institute, McGill University, Montreal, QC, Canada, 2Ecole D'optometrie De L'Universite De Montreal, Montreal, QC, Canada
Introduction: Recently, we found that the increased cortical cerebral blood flow (CBF) induced by basal forebrain (BF) stimulation occurred concomitantly with activation of specific subsets of cortical GABA interneurons that co-localize somatostatin (SOM) and/or neuropeptide Y (NPY). In the present study, we assessed the contribution of BF and cortical neuronal mediators, as well as that of glial factors, in this hemodynamic response.
Methods: The increase in cortical CBF induced by electrical BF stimulation (80µA, 100Hz, 1sec on/1sec off, 20sec) was measured bilaterally by laser Doppler flowmetry before (baseline) and after intracisternal (i.c., 3µl of a pH7.4 buffered solution) or intraperitoneal (i.p.) injection of drug vehicles, antagonists of muscarinic (scopolamine, 10-4M), GABA-A (picrotoxin, 10-4M), NPY-Y1 (BIBP 3226, 10-6M), or SOM (cyclo-somatostatin/C-SOM, 10-4M) receptors, or respective inhibitors (isoniazid, 350mg/kg i.p and miconazole, 30nmol in 5µl, i.c.) of glutamate decarboxylase (GAD, GABA synthesizing enzyme) and P-450 epoxygenase (epoxyeicosatrienoic acids (EETs) synthesizing enzyme). CBF changes were expressed as percent changes from baseline, and compared by paired Student's t-test between drugs and corresponding vehicle. Selective cholinergic denervation of the neocortex was achieved with the specific cholinergic immunotoxin 192 IgG-saporin (4.1µg/2µl saline, n=3, control rats received saline) administered into the left lateral ventricle. Selectivity of lesion was confirmed by immunostaining of choline acetyltransferase (ChAT), GAD67, or neuronal nitric oxide synthase (NOS).
Results: Vehicles did not affect the BF-evoked CBF response when compared to baseline (Fig). Inhibition of GABA synthesis with isoniazid and antagonism of GABA-A receptor with picrotoxin decreased (−50 and −30%, p<0.05 and 0.01, respectively) the induced CBF response (Fig). Scopolamine, in accord with previous investigations, significantly reduced the hemodynamic response (−56.2%, p<0.001), and a combined injection of scopolamine and isoniazid demonstrated no additive inhibitory effect (−56%, p<0.05, Fig). In contrast, antagonism of SOM receptors with C-SOM, NPY-Y1 receptors with BIBP 3226 or inhibition of EETs synthesis with myconazole slightly but not significantly altered the perfusion response (Fig). In rats with selective cortical cholinergic denervation, the BF-induced CBF response was virtually abolished (−85%), despite preserved GAD67, NOS and ChAT BF neurons.
Conclusions: Together with previous findings, we conclude that ACh neurons from the BF drive the CBF response and that GABA, released from SOM- and/or NPY-containing cortical GABA interneurons, major targets of BF cholinergic afferents, mediate part of this response. Preliminary data also suggest a minor contribution of the EET pathway in this neurovascular coupling response. Supported by CIHR grant (MOP-53334, EH).
HETEROGENEITY ACROSS LAYERS IN RAT SOMATOSENSORY CORTEX
Peter Herman1,2, Basavaraju Sanganahalli1, Fahmeed Hyder1,3
1Department of Diagnostic Radiology, Yale University, New Haven, CT, USA, 2Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary, 3Department of Biomedical Engineering, Yale University, New Haven, CT, USA
INTRODUCTION
To date, layer specific comparison of neuronal activity and neurometabolic-neurovascular responses are lacking possibly due to method limitations. We have measured neurometabolic-neurovascular responses (BOLD, CBF, pO2) and electrical activity (MUA or AP, LFP) in rat during 30 s forepaw stimulation, as a function of cortical depth.
METHODS
Artificially ventilated rats (n = 43) were anesthetized with α-chloralose (40 mg/kg/hr). All fMRI experiments were conducted at 11.7T. Gradient-echo EPI data (TE=15ms) were acquired with TR of 1 s. Data were collected in 120 s windows: 30 s before and 60 s after the 30 s forepaw stimulation. Electrical activities (MUA, LFP) were measured with high impedance microelectrodes (2-4 MΩ) simultaneously with laser Doppler flowmetry and pO2 responses with optical methods. Responses were measured in during 30 s forepaw stimulation (3 Hz, 2 mA, 0.3 ms) at different cortical depths.
RESULTS
Laminar-specific averaged responses of MUA, LFP, CBF, and pO2 during a 30 s stimulation across all layers generally show some agreement, but there were some differences (Figs. 1,2). For BOLD data, only voxels surpassing the statistical threshold of P<0.05 were used for the analysis, with decreasing intensities from top to bottom layers. The MUA and LFP signals in the middle layers showed a slight depressed activity lasting ~5s immediately following stimulation offset. The LFP signal in all layers showed a rapid rise and then decay to a plateau within ~5s immediately following stimulation onset, which was different from the laminar responses of the MUA signal. The CBF changes were uniform across most layers, whereas the pO2 signal was significantly delayed compared to the other signals in top and bottom layers. Although the dynamics of the different multi-modal signals were generally correlated, these results have significant implications for dynamic modeling of the BOLD signal.
ACKNOWLEDGEMENTS
Supported by NIH (DC-003710, MH-067528) and OTKA (T34122) grants.
PIAL ARTERIOLAR RESPONSES AND TISSUE OXYGENATION STATE IN CEREBRAL ISCHEMIA AND ISCHEMIC TOLERANCE IN GERBILS
Ji-Yao Li1, Hirokazu Ueda1,3, Akitoshi Seiyama2, Junji Seki4, Kuni Konaka1, Toshio Yanagida2, Saburo Sakoda1, Takehiko Yanagihara1
1Department of Neurology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan, 2Department of Physiology and Biosignaling, Osaka University Graduate School of Medicine, Suita, Osaka, Japan, 3Department of Neurology, Osaka Kosei-Nenkin Hospital, Osaka, Japan, 4Department of Biomedical Engineering, National Cardiovascular Center Research Institute, Suita, Osaka, Japan
Background and aims
The pathophysiological mechanism of ischemic tolerance is still speculative. Our previous study with bilateral carotid artery occlusion (BCO) in gerbils [1] indicated that improvement of the tissue oxygenation state during ischemia might contribute to ischemic tolerance. In the present study, we investigated the pial arteriolar constriction in response to energy failure [2], intra-arteriolar red blood cell velocity (Vrbc) and tissue oxygenation state, with and without ischemic preconditioning.
Methods
Under pentobarbital anesthesia (50mg/kg), adult male Mongolian gerbils with sham operation (S-group, n=5) and those with pretreatment consisting of BCO for 2 minutes, twice at 3 days and 2 days earlier (T-group, n=5), were again subjected to BCO for 5 minutes. Using video microscopy and microspectroscopy, the diameter change of the pial arteriole, whose preischemic diameter was about 50µm, and the redox state of cytochrome oxidase (cyt.aa3; ?A590nm-A605nm) in the underlying cerebral cortex were monitored through a cranial window as described [2], and intra-arteriolar Vrbc was simultaneously monitored with a fiber-optic laser-Doppler anemometer [3]. Seven days after the analyses, tissue damage was assessed immunohistochemically using antibodies against microtubule-associated proteins.
Results
In the S-group, a slight reduction of the diameter of pial arterioles (immediate vasoconstriction) developed within the initial 1 min of ischemia and extensive vasoconstriction (late vasospasm) occurred later [2] with the maximum constriction at 1.50 ± 0.40 min. In the T-group, immediate vasoconstriction persisted longer with the ischemic time for maximal vasospasm being significantly (P<0.05) delayed at 3.3 ± 0.53 min. The minimum diameter was not significantly different between the two groups (57.1 ± 0.26 % of the preischemic value in the S-group and 52.8 ± 0.20 % in the T-group, respectively). The reduction pattern of cyt.aa3 in the neighboring cerebral cortex was similar between the two groups with initial quick reduction, subsequent transient improvement and final rapid reduction down to complete reduction [2]. However, the time for initiation of final rapid reduction and the time for complete reduction were significantly delayed in the T-group (1.12 ± 0.07 and 1.90 ± 0.90 min, respectively) compared with those in the S-group (0.97 ± 0.10 and 1.48 ± 0.23 min, respectively)(p<0.05, each). Vrbc (mm/sec) was not significantly different between the two groups (30.2 ± 2.5 in the S-group and 31.0 ± 3.3 in the T-group, respectively) before ischemia, and decreased to about 0.10 mm/sec soon after ischemia in both groups. Neuronal damage developed in the cerebral cortex immunohistochemically in the S-group, but not in the T-group.
Conclusions
The present investigation demonstrated that ischemic tolerance was associated with improvement of the tissue oxygen metabolism and alleviation of the ischemic vasoconstriction without changes in the intra-arteriolar red blood cell velocity, indicating the induction of adaptive tissue metabolic suppression without changes in oxygen supply.
LINEAR CORRELATION BETWEEN BOLD SIGNAL AND SOMATOSENSORY EVOKED POTENTIAL IN RAT CORTEX AS A FUNCTION OF STIMULUS FREQUENCY AND CURRENT
Ikuhiro Kida1, Toru Yamamoto2
1Graduate School of Dental Medicine, Hokkaido University, Sapporo, Hokkaido, Japan, 2School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan
Introduction: The neurovascular coupling plays an important role in the interpretation of the functional brain imaging signal. Although the local cerebral blood flow (CBF) is certainly coupled to the neuronal activity, the correlation of BOLD fMRI signals related to CBF response with the neuronal activity is still under investigation. The aim of the present study is to investigate the relationship between BOLD signals and somatosensory evoked potentials (SEP) in rat cortex evoked by forepaw sitmulation as a function of stimulus current and frequency. Methods: Male Sprague-Dawley rats (n=11) were used in this study. An femoral arterial and vein catheters were placed for blood sampling and blood pressure monitoring, and for immobilization by pancuronium bromide, respectively, throughout the experiment. The silver ball electrodes were placed on the surface of somatosensory area for the SEP measurement. After the surgery, isoflurane was discontinued and alpha-chloralose anesthesia was given. A pair of needle electrodes was inserted beneath the skin of a forepaw. Forepaw electrical stimulation was applied at a current of 0.5–2..0 mA with fixed frequency and at a frequency of 1-10 Hz with a fixed current for 4 second duration. All fMRI experiments were carried out on a horizontal 7 T/11cm magnet interfaced to a VarianINOVA console (Palo Alto, CA) with a single turn 10 mm transmit/receive surface coil. FMRI data were acquired using a single-shot spin echo EPI sequence with scan parameters as follows: TR/TE = 1000/40 ms, slice thickness = 2 mm, FOV=20×20 mm2, matrix size=32×32. Results: Figure 1 shows the summed SEP amplitudes within entire stimulation period with all stimulus conditions. The summed SEP amplitudes increased with an increase in the stimulus intensity for all stimulus frequency and has a peak at 3–5 Hz. Figure 2 shows the correlation between the maximum BOLD signals and the summed SEP amplitudes for all stimulus conditions. The maximum value of the BOLD signal increases linearly with the summed SEP amplitude over all stimulus conditions in the present study and a least-squares fit of a power-law to the data gave a slop = 8.3 and section = 2.3 (R = 0.96). Conclusion: In the rat forepaw model, the BOLD response measured by spin echo sequence at high magnetic field is linear with neuronal activity. Thus, the BOLD response measured by spin echo sequence at high magnetic field may be used to derive the neural activity as a function of stimulus current and frequency. Supported by The Akiyama Foundation, Takeda Science Foundation and The Mitsubishi Foundation (IK).
NONLINEAR LOCAL ELECTROVASCULAR COUPLING
Jorge Riera1, Juan Carlos Jimenez2, Tohru Ozaki3, Ryuta Kawashima1, Xiaohong Wan4
1The Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan, 2The Institute of Cybernetics, Mathematics and Physics, Havana, Cuba, 3The Institute of Statistics Mathematics, Tokyo, Japan, 4RIKEN Brain Science Institute, Wako, Japan
We will present a biophysical model of how electrical/hemodynamic brain signals are generated within a basic cortical unit (Riera et al. 2006a). The model has three components. The first is the neural mass model of three neuronal sub-populations that responds to incoming excitatory synaptic currents. The second and third components model the generation of measured electrical and hemodynamic meso-states, respectively. We establish the face validity of the model by using simulations of visually evoked responses under different luminous contrasts and flicker frequencies. We reformulate the model in the context of the continuous-discrete state space theory in order to make inferences about the unknown parameters and the unobservable states (i.e. membrane potentials, extra-cellular ionic current, NO concentration, flow-inducing signal, CBF, CBV, dHb) from the local linearization filter and the innovation approach (Riera et al. 2006b). Inference on this model is performed from concurrent EEG/fMRI signals obtained during a visual experiment in which a subject observes passively during 4s a radial white/black checkerboard with pattern reversal at 4Hz, follows by 21s of resting condition. Figure summarizes our results:
the spatial-distributions and temporal-dynamics of the EEG/fMRI meso-states (left)
the dynamics of the unobservable states and the incoming synaptic currents at V1 (right)
PRIMARY INJURY OF RAT NEOCORTICAL VASCULATURE: EFFECTS OF ALBUMIN ADMINISTRATION ON LOCAL VASCULAR DYNAMICS
Anitha Nimmagadda, Hee-Pyoung Park, Ricardo Prado, Myron Ginsberg
Department of Neurology, University of Miami, Miami, FL, USA
BACKGROUND AND AIMS: Regional blood-flow dynamics play an important role in thrombus formation and dissolution. Local intravascular conditions in the environment surrounding a thrombus contribute to its composition and susceptibility to lysis. Currently, approved treatment methods for acute ischemic stroke attempt to limit tissue injury by thrombolytic recanalization of the occluded vessel. However, vascular patency is achieved in only 30% of patients. Thus, there is a need for ancillary measures to enhance the efficacy of thrombolytic therapy. Human albumin therapy is highly neuroprotective in rodent models of focal cerebral ischemia and reperfusion. The results of a recently completed pilot-phase clinical trial suggest that albumin therapy appeared to be particularly beneficial in stroke patients who also underwent thrombolysis. To study further the role of albumin on microvascular hemodynamics in thrombotic stroke, we employed two-photon laser scanning microscopy to assess laser-induced thrombosis of superficial neocortical arterioles in the rat.
METHODS: Male Sprague-Dawley rats were anesthetized with isoflurane/nitrous oxide, intubated, and mechanically ventilated. Arterial blood pressure and acid-base status were maintained within normal limits. A cranial window was created over the fronto-parietal cortex; the dura was kept intact. The animal was then transferred to a specially constructed stereotaxic frame attached to the stage of a two-photon microscope. The cranial defect was filled with artificial CSF and the skull defect sealed within a circulating chamber that maintained cranial temperature at 36.5oC. Arterioles of interest were identified at 20x magnification via z-scans performed at 1 ·m intervals after plasma labeling with fluorescein isothiocyanate-dextran (~0.7 mg/kg i.v). Baseline blood flow velocities and diameters were measured in the selected microvessels by the method of Kleinfeld et al. In brief, RBC flow velocity was determined by repeated line-scans along a vessel's longitudinal (y) axis. Computer stacking of these scans revealed oblique shadows, from whose slopes with respect to time (dy/dt) flow velocity was computed. To produce vascular injury, a segment of an arteriole of interest was selected at the maximal optical zoom setting for laser-irradiation at the two-photon wavelength of 840 nm. With the laser shutter in the off position, irradiation intensity (W/cm2) was set according to the vessel diameter. The vessel was then irradiated for ~5 min. Animals were allocated to albumin or isotonic saline treatment groups by coin toss. Treatment was carried out 25 min following vascular injury, and flow velocities determined over a 90 min period.
RESULTS: Average baseline blood flow velocity was 24.5 ± 12.5 mm/s (n=6). Following injury, flow velocity decreased to 9.2 ± 2.8 mm/s (p< 0.05 by ANOVA vs. baseline). After saline infusion, blood flow velocity remained unchanged at 9.9 ± 2.4 mm/s. By contrast, after albumin infusion, blood flow velocity increased to 20.7 ± 4.0 mm/s (p< 0.05 by ANOVA vs. post-injury flow).
CONCLUSIONS: Albumin administration following laser-induced microvascular thrombosis improves local hemodynamics; this may be an important component of its therapeutic effect.
EVALUATION OF MEAN TRANSIT TIME AS AN INDEX OF CEREBRAL PERFUSION PRESSURE IN RABBITS WITH GRADED SYSTEMIC HYPOTENSION
Matthew Murphy1,2,3, Ting-Yim Lee1,2,3, Xiaogang Chen2
1Department of Medical Biophysics, University of Western Ontario, London, ON, Canada, 2Imaging Research Laboratories, Robarts Research Institute, London, ON, Canada, 3Imaging Division, Lawson Health Research Institute, London, ON, Canada
BACKGROUND: A reliable indicator of local cerebral perfusion pressure (CPP) is needed to better understand the pathophysiology of cerebrovascular diseases. Mean arterial pressure (MAP) is not a good indicator of CPP without knowledge of intracranial pressure and it does not assess local vascular territories at risk. A better indicator is cerebral vasoreactivity (CVR), defined as the ratio of cerebral blood flow (CBF) post stimulus to its basal value, and it can be measured locally by non-invasive imaging methods; but it requires administration of a vasodilatory drug and two separate CBF measurements. We propose that MTT, or cerebral blood volume (CBV):CBF, is a surrogate index of local CPP in the brain without the need to evaluate its changes before and after vasodilation and can be measured by CT scanning.
METHOD: 8 New Zealand White rabbits had a cannula stereotaxically implanted into the right lateral ventricle of the brain as an access port for ICP measurement. To achieve a range of CPP values all rabbits were studied at a normo- and hypo-tensive state in random order with a week interval between studies. For each study, baseline CBF was measured twice with CT, then 20mg/kg of acetazolamide (ACZ) was given and CBF was measured again at 5, 15, 25, 40 and 60 minutes post ACZ induced vasodilation. For the hypotensive state induced with Prostaglandin II (3µg/kg/min), once a stable MAP was reached, a baseline hypotensive CBF measurement was made before continuing with the ACZ portion of the protocol as in the normotensive study. MAP and ICP were measured throughout while CVR was measured at all time points post ACZ for both the normo- and hypotensive state.
RESULTS: Prostaglandin II lowered MAP significantly (p<0.05) compared with basal MAP (24.1 ± 6.8 vs. 41.2 ± 8.9 mmHg), resulting in a significant decrease in CPP (52.5%) from the baseline measurement that lasted the duration of the normo- and hypo-tensive study. CVR was statistically similar for both MAP states at all time points following ACZ. Over the range of MAP studied, a Pearson correlation demonstrated that CPP was significantly (p<0.01) correlated with both CVR and MTT with R=0.377 and −0.580.
CONCLUSIONS: The results indicate that both CVR and MTT are significantly correlated with CPP. However, MTT may be a more sensitive indicator of CPP by accounting for changes in both CBV and CBF that occur as the lower limit on the pressure-autoregulation curves “plateau” is approached. The said relationship may suggest that it may be possible to identify areas at risk in cerebrovascular disease with a single measurement of MTT without the need to assess CVR which requires measuring CBF before and after a vasodilatory stimulus.
IMPORTANCE OF BASELINE CBF FOR DETECTION OF CRITICAL HEMODYNAMIC STATUS IN CEREBROVASCULAR DISEASE: COMPARISON WITH ACETAZOLAMIDE TEST USING PET
Hidehiko Okazawa1, Tetsuya Tsujikawa1, Yoshikazu Arai2, Masato Kobayash1, Makoto Isozaki2, Yasuhisa Fujibayashi1
1Biomedical Imaging Research Center, University of Fukui, Eiheiji-Cho, Fukui, Japan, 2Department of Neurosurgery, University of Fukui, Eiheiji-Cho, Fukui, Japan
Background and aims: Autoregulatory mechanism to keep cerebral blood flow (CBF) may be impaired in patients with occlusive cerebrovascular disease (CVD). To investigate the critical hemodynamic status in the impaired cerebral circulation, baseline regional CBF (rCBF) and cerebral vascular reactivity (CVR) determined by changes in rCBF induced by the acetazolamide (ACZ) challenge were measured in CVD, as well as conventional hemodynamic parameters such as cerebral blood volume (CBV), oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2).
Methods: Fifty-six patients (mean age = 64.3±8.2 y) with unilateral major cerebral arterial occlusive disease underwent O-15 water PET at baseline and 10 min after ACZ administration. All patients also underwent O-15 gas PET study before the water PET scans. CBF, CVR determined by %change in CBF after ACZ administration, OEF, and other hemodynamic parameters in territories of bilateral middle cerebral arteries were analyzed. A significant elevation of OEF greater than mean+2SD of healthy controls (n=14, 28 cerebral hemispheres) was defined as misery perfusion. Diagnostic accuracy in detection of misery perfusion using the criteria determined by baseline rCBF alone, both by CVR and baseline rCBF, and by CVR alone was evaluated. Significant decreases for these parameters were defined by <mean-2SD of control data.
Results: Eleven of 19 patients with misery perfusion showed a significant CVR decrease. Using criteria determined by significant decrease in baseline rCBF alone, misery perfusion was detected with sensitivity of 63.2% and specificity of 89.2% in all patients. Using criteria determined by both decreases in CVR and baseline rCBF, misery perfusion was detected with sensitivity of 52.6% and specificity of 86.5%. The criteria using baseline rCBF alone provided greater diagnostic accuracy than the criteria using CVR and baseline rCBF (Table).
Conclusion: Significant decrease in baseline rCBF is more accurately detect misery perfusion than reduced CVR alone or reductions of CVR and baseline rCBF in the ACZ challenge. Quantitative measurement of baseline rCBF would be essential for evaluation of CVD hemodynamic status and can detect misery perfusion with higher accuracy.
A NOVEL APPROACH USING HIGHER ORDER CROSSINGS TO ASSESS DYNAMIC CEREBRAL AUTOREGULATION IN DIABETICS WITH AUTONOMIC NEUROPATHY
Chuang-Chien Chiu1, Ben-Yi Liau1, Shoou-Jeng Yeh2
1Graduate Institute of Electrical and Communications Engineering, Feng Chia University, Taichung, Taiwan-R.O.C., 2Section of Neurology and Neurophysiology, Cheng-Ching General Hospital, Taichung, Taiwan-R.O.C.
The cerebral autoregulation (CA) mechanism refers to the cerebral blood flow (CBF) tendency to maintain relatively constant in the brain. In this study, a novel approach using higher order crossings (HOC) analysis was applied to evaluate the relationship between blood pressure and cerebral blood flow velocity (CBFV) signals acquiring from normal subjects and diabetics with autonomic neuropathy both in supine and tilting positions. There were 2 groups of subjects being recruited in this study, i.e., 66 diabetics (14 diabetics without autonomic neuropathy, 25 diabetics with mild autonomic neuropathy and 17 diabetics with severe autonomic neuropathy) and 11 age-matched healthy adults for normal subjects. All subjects were informed consent prior to entry to the study and it had been approved by the Research Ethics Committee of Cheng-Ching General Hospital. The subjects were included only if they had no history of cardiovascular disease, heart problems, hypertension, migraine, epilepsy, cerebral aneurysm, intracerebral bleeding or other pre-existing neurological conditions. None of the subjects were receiving any medication during the period of the study. Continuous cerebral blood flow velocity (CBFV) was measured in the middle cerebral artery using TCD (Transcranial Doppler ultrasound, EME TC2020) in conjunction with a 5-MHz transducer fixed over the temporal bones by an elastic headband. Continuous arterial blood pressure (ABP) recordings were obtained through the Finapres (Ohmeda 2300) device with the cuff attached to the middle finger of the right hand. Subjects were investigated on a tilt-table that enabled a motor-driven change from a supine to an upright position 75° within 4 seconds. Data acquisition was started after a 10-min relaxation in supine position. Spontaneous ABP and CBFV were recorded simultaneously to PC for off-line analysis. The acquisition periods were approximately 5 minutes in both the supine and 75°tilt-up positions by a self-developed data acquisition system. The approach counts the number of zero-crossings to evaluate the degree of oscillating in time series. The results revealed that the zero-crossings values of both MABP and MCBFV in all normal subjects converge to a constant. However, the zero-crossings values do not converge to a constant in diabetics. The values of zero-crossings between normal subjects and diabetics with severe autonomic neuropathy were significantly different (Mann-Whiteny test, p<0.001). It might indicate that the CA mechanisms in normal subjects are better than that in diabetics. Therefore, the HOC can be a useful tool to evaluate dynamic cerebral autoregulation in clinical practice.
ESTROGEN PRETREATMENT MIMICS ISCHEMIC PRECONDITIONING IN THE BRAIN
Ami Raval1, Kunjan Dave1, Ricardo Prado1, Isa Saul1, Raul Busto1, Helen Bramlett2, Miguel A. Perez-Pinzon1
1Department of Neurology and Neuroscience, Cerebral Vascular Disease Center, University of Miami School of Medicine, Miami, FL, USA, 2Department of Neurological Surgery, Miami Project To Cure Paralysis, University of Miami School of Medicine, Miami, FL, USA
Background and aims: Estrogen has proven beneficial actions on neuronal survival against cerebral ischemia (1), (2). Because of promising preclinical data on neuroprotection, the Women's Estrogen for Stroke Trial was initiated. The outcomes from this trial were, however, unsuccessful and questions emerged about the safety of chronic estrogen treatment in women. In contrast to the chronic estrogen treatment strategy, our recent study demonstrated an estrogen bolus 48 h prior to ischemia induces neuroprotection in the hippocampal CA1 region in an in vitro model of organotypic slice cultures (3). Furthermore, the degree of neuroprotection induced by estrogen is similar to that achieved by ischemic preconditioning. Prompted from our in vitro study, we hypothesized that a bolus of estrogen provides pharmacological neuroprotection against ischemia in a rat model.
Method: Normal cycling female rats were ovariectomized (OvX; performed at diestrus stage of estrous cycle) and 7 days later 17s-estradiol (5 or 50 µg/Kg; i.p) or vehicle (oil) was administered. Cerebral ischemia was induced in 17s-estradiol/vehicle treated rats, 48h later. Cerebral ischemia was produced by 10 min of bilateral carotid occlusion and systemic hypotension (50mmHg). Seven days after ischemia rats were fixed for histopathological assessment. Hippocampal sections at the level of 3.8 mm posterior to bregma were examined for normal neurons. Results were expressed, as mean ± SEM. Statistical significance was determined with an ANOVA test followed by a Bonferroni's post-hoc test.
Results: The number of normal neurons per slice in the CA1 hippocampal region in control rats (without an ischemic episode) was 1067 ± 64 (n = 3). The ischemic insult in intact or OvX rats decreased the number of normal neurons by 87% (136 ± 9, n = 4, p < 0.05) and 88% (150 ± 10, n = 3), respectively, as compared to the control group. Interestingly, an estrogen bolus to ovariectomized rats 48h prior to cerebral ischemia increased the number of normal neurons by 39% (562 ± 116, n = 3, dose 5 µg/Kg) and 28% (450 ± 42, n = 3, dose 50 µg/Kg) as compared to ovariectomized (OvX) group (p < 0.05). Vehicle treatment did not show any significant difference in the number of normal neurons as compared to OvX groups. We also carried out a control experiment by exposing female rats to a procedure of sham-OvX. Ischemia was induced 7 days later in sham-operated females. The number of normal neurons in CA1 hippocampal region in sham-ovariectomized group did not vary from that in the OvX group, suggesting no neuroprotective effect.
Conclusion: Our results demonstrate that an estrogen bolus protects the hippocampus against cerebral ischemia in a preconditioning manner. Future investigations in estrogen preconditioning may suggest new estrogen regimens that avoid potential side effects of chronic estrogen treatment for stroke patients.
Grant support: Supported by PHS grants NS34773, NS045676, NS054147, and AHA-SDG-National Center #0730089N
NEURONAL PRECONDITIONING WITH THE ANTIANGINAL DRUG, BEPRIDIL
Tamas Gaspar1, Bela Kis1, James A. Snipes1, Gabor Lenzser1,2, Keita Mayanagi1, Ferenc Bari3,
David W. Busija1
1Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston Salem, NC, USA, 2Institute of Clinical Experimental Research and Human Physiology, Semmelweis University, Budapest, Hungary, 3Department of Physiology, Faculty of Medicine, University of Szeged, Szeged, Hungary
Background and aims. Bepridil (BEP) is an antianginal drug with a large variety of cardiovascular effects. While a recent study has demonstrated that BEP activates mitochondrial ATP sensitive potassium (mitoKATP) channels and protects the heart against ischemia, the effects of this drug on the central nervous system are not entirely known. The aim of our in vitro study was to investigate whether BEP can protect rat cortical neuronal cultures against toxic stimuli. We also examined the effect of BEP on reactive oxygen species (ROS) generation, plasma membrane and mitochondrial membrane potentials, mitoKATP channels, and on the expression and activity of cytoprotective proteins.
Methods. Cortical neurons of 18-day old Sprague-Dawley rat fetuses were cultured in B27 supplemented Neurobasal medium. Preconditioning was carried out by treating the cells with different doses of BEP for 3 days, once a day. Twenty-four hours after the last treatment, neuronal cultures were exposed to either 180 min of oxygen and glucose deprivation (OGD) or 60 min of glutamate excitotoxicity (200 µM). Cell viability was evaluated with CellTiter 96 AQueous One Solution assay. ROS production was monitored with a fluorescent microplate reader using hydroethidine, plasma membrane and mitochondrial membrane potentials with confocal microscopy using di-8-ANEPPS and tetramethylrhodamine ethyl ester, respectively. The expression of phosphorylated protein kinase C (PKC), manganese dependent superoxide dismutase (MnSOD), glutathione peroxidase (GPx), catalase, and Bcl2 was examined using Western blotting. Enzyme activity of antioxidants was assessed with a microplate reader using commercially available kits.
Results. As an immediate effect, application of BEP did not change plasma membrane potential, induced an increase in ROS generation, and dose-dependently depolarized mitochondria, but no acute protection could be found. Three-day treatment with BEP, however, provided dose-dependent neuroprotection against both OGD (cell viability: untreated, 54.7±0.67%; BEP 1µM, 66.4±1.45%*; BEP 2.5 µM, 77.3±1.19%*; BEP 5 µM, 97.8±0.94%*; BEP 10 µM, 84.7±1.67%*; p<0.05 vs. untreated) and glutamate excitotoxicity (cell viability: untreated, 54.1±0.69%; BEP 1 µM, 61.2±1.19%*; BEP 2.5 µM, 78.1±1.67%*; BEP 5 µM, 91.2±1.20%*; BEP 10 µM, 94.6±2.12%*; p<0.05 vs. untreated), and inhibited the ROS surge upon exposure to glutamate. The neuroprotective effect of BEP could be antagonized with the SOD mimetic M40401 (cell viability after OGD for 180 min: untreated, 36.7±0.59%; BEP 5 µM, 82.0±1.23%*; BEP 5 µM + M40401 50 µM, 20.5±2.70%*#; *p<0.05 vs. untreated, #p<0.05 vs. BEP 5 µM), but not with catalase, reduced glutathione, or the putative mitoKATP channel blocker, 5-hydroxydecanoate. BEP treatment resulted in elevated protein levels of PKC, MnSOD, GPx, catalase, and Bcl-2 and increased enzymatic activity of MnSOD and GPx.
Conclusion. Our results indicate that BEP induces late preconditioning in isolated rat neuronal cultures conferring neuroprotection which is dependent on superoxide generation and a sequential activation of PKC and cytoprotective proteins but perhaps not on direct mitoKATP activation.
HYPOTHERMIA INHIBITS THE INFLAMMATORY RESPONSE ASSOCIATED WITH GLOBAL CEREBRAL ISCHEMIA AT THE TRANSCRIPTIONAL LEVEL
Carla M. Webster1,2, Maya A. Koike1,2, Valarie Chock2, Stephen Kelly3, Ronna G. Giffard2,3, Midori A. Yenari1
Department of Neurology, UCSF VAMC, San Francisco, CA, USA, Department of Anesthesiology, Stanford University, Palo Alto, CA, USA, Department of Neurosurgery, Stanford University, Palo Alto, CA, USA
Mild hypothermia is clinically and experimentally neuroprotective in response to brain injury due to global cerebral ischemia. Previously, we showed that hypothermia attenuates the inflammatory response in models of focal cerebral ischemia (FCI) at the level of nuclear factor-kappa B (NFκB) regulation by inhibiting its activating kinases IKKγ and IKKβ. This study aims to establish a connection between the effects of mild hypothermia on inflammation in models of global cerebral ischemia (GCI), aseptic meningitis (LPS stimulation) and the NFκB pathway.
Sprague-Dawley rats or C57/BL6 mice were subjected to GCI by occluding the common carotid arteries for 8 (rat) or 12min (mice) followed by reperfusion. In the rat, systemic hypotension was also applied. As a positive control, a separate group of mice were injected with LPS (5 mg/kg LPS IP) to cause brain inflammation without brain cell death. In all in vivo models, hypothermia was induced by total body cooling to 30°C (33°C brain) for 2h immediately after the onset of ischemia or LPS injection. Primary microglial cultures were prepared from neonatal mouse brains, and were subjected to oxygen glucose deprivation for 24h followed by 1h reperfusion (OGD) or stimulated with LPS (10 µg/ml).
The inflammatory response in both GCI models was accompanied by microglial activation, but no neutrophil infiltration, or blood brain barrier disruption, in contrast to FCI. Mild hypothermia reduced the amount of CA1 damage 72h after GCI (n=4-6 per group, p<0.01) and decreased microglial staining/activation. In vitro, hypothermia protected primary microglial cultures from cell death following OGD compared to normothermic groups (P<0.01), and reduced inflammatory cytokine secretion upon LPS exposure (P<0.05 for TNF-α, P<0.01 for IL1-β). Hypothermia resulted in less nuclear NFκB staining following both GCI and LPS treatment. An NFκB ELISA activation assay revealed that nuclear extracts from LPS and GCI animals showed a robust decrease in NFκB activation in the hypothermic groups (p< 0.05 GCI 33°C, p< 0.01LPS 33°C).
NFκB regulatory proteins, IκB-α (and phosphorylated IκB-α, p-IκB-α), and IKKγ, were examined via Western blot. In contrast to our previous observations in FCI showing decreases in p-IκB-α and IKKγ, hypothermia did not alter expression of any of these proteins. Thus, the effect of reduced NFκB nuclear translocation and activity under hypothermic conditions is probably not related to changes in its regulatory protein expression, but could represent temperature dependent effects of NFκB itself.
The inflammatory response following GCI is different from that observed in FCI, where inflammation appears to be mediated only by endogenous brain elements in the former. Nevertheless, hypothermia shows a robust decrease in many elements of the inflammatory response, including reductions in microglial activation, inflammatory cytokine secretion, and NFκB activity, Although different from that observed in FCI, this data demonstrates the importance of the inflammatory response in GCI, and its attenuation by mild hypothermia.
NOVEL NEUROPROTECTIVE APPROACHES IN EXPERIMENTAL MODEL OF CEREBRAL VENOUS INFARCTION
Hiroyuki Nakase1, Kentaro Tamura1, Ryo Tamaki1, Toshikazu Nishioka1, Ryota Kimura1, Toshisuke Sakaki1, Axel Heimann2, Oliver Kempski2
1Department of Neurosurgery, Nara Medical University, Nara, Japan, 2Institute for Neurosurgical Pathophysiology, Johannes Gutenberg-University, Mainz, Germany
Background and Purpose: Although brain ischemia by arterial occlusion has been in the focus of attention for decades, currently there is considerable interest in the study of cerebral injury following cerebral venous occlusion. Cerebral venous disorders are potentially good outcome if diagnosed and treated promptly. In the paper, novel neuroprotective approaches in experimental model of cerebral venous infarction (CVI) are described.
Method and Results: We used the rat two cortical veins occlusion (2-VO) model. <Experiment-1> Male Wistar rats were divided into two groups: the control and the treated group with Vascular endothelial growth factor (VEGF) antagonist. They were evaluated by magnetic resonance imaging (MRI) 24 hours after the operation. VEGF expression was observed exclusively in the area of vasogenic edema which was shown as a high intensity area in apparent diffusion coefficient of water (ADCw) map. The both volumes of the infarction and the vasogenic edema in MRI were significantly smaller in the treated group than the control (P<0.05). <Experiment-2> Caspase-inhibitor z-VAD.fmk was applied intraventricularly 3, 24, 48, or 96 hours after 2-VO. Vehicle was injected 3 (n=6) or 24 hours (n=7) after 2-VO. Inhibition of caspases by z-VAD.fmk given 3 and even 24 hours after 2-VO significantly reduced infarct volume (P<0.05).
Conclusions: The result in experiment-1 provides an evidence that the inhibition of VEGF attenuates the brain edema formation and also reduces the infarction at the acute stage of CVI, and the experiment-2 shows that 2-VO induces delayed neuronal death in which caspase-induced cell death is involved. On the basis of these data, we concluded that the inhibition of VEGF and apotosis might be new therapy against CVI. Therapeutic window in time and space is much wider in cerebral venous infarct than arterial stroke. Therefore, neuroprotective candidates with the success obtained with animal models are more promising in human trial in CVI.
A LONG-TERM EFFECT OF EDARAVONE GIVEN AT ISCHEMIA - REPERFUSION ON ISCHEMIC STROKE IN MICE
Hisakazu Uno1, Akihiko Taguchi2, Takayuki Nakagomi1, Orie Saino1, Masatoshi Fujikawa1, Tomohiro Matsuyama1
1Institute for Adavanced Medical Sciences, Hyogo Medical College, Nishinomiya, Hyogo, Japan, 2Department of Cerebrovascular Disease, National Cardiovascular Center, Suita, Japan
Background and aims: Several previous reports have suggested that free radicals play an important role in the development of ischemia-reperfusion injury following resuscitation of brain circulation by treatment with tPA. Free radical scavenger, edaravone, has been used widely for treating ischemic stroke patients in Japan, but its long-term effects are still under investigation. In the present study, to clarify the neuroprotective properties of edaravone, we examined a long-term effect of edaravone by using a newly developed murine model of focal cerebral ischemia which is highly reproducible and can survive for long period.
Methods: To assess the effect of edaravone on stroke, we developed a murine stroke model applying our previous method (Taguchi et al., 2004) to adult CB17 mice (20-30g). Transient focal ischemia was produced by occlusion of the M1 distal portion of the left MCA for 60 minutes. Regional cerebral blood flow (rCBF) in the MCA area was measured by laser Doppler flowmetry before, during, and after ischemia. Mice were treated with infusion of edarabone via tail vein (3mg/kg), immediately before and 60 minutes after reperfusion (edaravone group, n=10). They were followed by treating with 10mg/kg of edaravone twice a day and continuing for 3 days. As controls, the same volume of saline was administered (control group, n=10). Some of each group underwent an examination of brain MRI to evaluate the change of ischemic lesion from 1 day to 4 weeks after stroke. Subsequently behavioral study (open field, water maze, elevated plus-maze) was performed. Finally the infarct volume was caluculated as the relative infarct volume using the brain sections.
Results: All the mice operated showed ~75% decreased rCBF immediately after the procedure, and developed cerebral infarct localized in left MCA area of the cerebral cortex 1 month after the insult. The size of infarct is almost same as that of permanent MCA occlusion when the period of ischemia was 60 minutes. There were no significant differences in rCBF between the edaravone and control groups during the course of ischemia and reperfusion. Also no significant differences were noted in infarct volume measured by MRI at 1 day. One month later, however, infarct volume was significantly smaller in edaravone group than control animals (18±2% vs 24±4%, p=0.011). Behavioral testing on day 30 after stroke (n=10, for each group) displayed significantly improved behavior according to the elevated plus maze test.
Conclusions: The present study clearly demonstrated that edaravone protected a brain ischemia-reperfusion injury which can develop for long time after the insult. Resuscitation of brain circulation is essential for treating cerebral thrombosis. It is suggested that administration of edaravone on period of recirculation has a mild protective effect with respect to brain atrophy and results in beneficial functional outcome, even if the infarct size was not different compared with the non-treated group.
NEUROPROTECTIVE EFFECTS OF ETHYL PYRUVATE ON ENERGY METABOLISM OF RAT BRAIN AFTER ISCHEMIA: A 31P-NMR STUDY
Osamu Tokumaru1, Chihiro Kuroki1, Hidehiro Takei2, Noriko Yoshimura1, Naoko Nisimaru1, Takaaki Kitano3, Isao Yokoi1
1Department of Physiology, Oita University Faculty of Medicine, Yufu, Oita, Japan, 2Department of Pathology, The Methodist Hospital, Houston, TX, USA, 3Medical Education Center, Oita University Faculty of Medicine, Yufu, Oita, Japan
Background and aims: Pyruvate is not only an intermediary metabolite of energy metabolism but also a neuro-protectant as a potent antioxidant and free radical scavenger or an inhibitor of zinc excitotoxicity (Fink, 2003; Gonzalez-Falcon et al., 2003; Yu et al., 2005). It was reported that administration of pyruvate improved cerebral metabolic and functional status during hemorrhagic shock and reduced neuronal death after cerebral ischemia. Neuroprotective effects of ethyl pyruvate (EP), a stable and lipophilic derivative of pyruvate, on energy metabolism of rat brain exposed to ischemia were investigated by phosphorous nuclear magnetic resonance (31P-NMR) spectroscopy.
Methods: Brain slices (400 µm-thick) prepared from a male Wistar rat (6 week-old) were incubated in standard artificial cerebrospinal fluid (ACSF) with 2mM EP or ACSF only (control) at 25°C. The brain slices were exposed to ischemia by halting the perfusion for 1 hr. Recovery of the levels of high-energy phosphates, phosphocreatine (PCr) and γ-ATP, after reperfusion were measured by 31P-NMR (AMX300wb, BRUKER).
Results: Recovery of the level of PCr after ischemia was significantly greater when EP was present in ACSF than in its absence (78.2 ± 3.3% vs 60.8 ± 1.9%, respectively; p = 0.001). EP was less effective when it was administered only during the recovery period (71.9 ± 4.6%; p = 0.059). When the brain slices were treated with 100µM fluorocitrate (FC), a selective glial poison, PCr level was decreased to 92.0 ± 3.3% of the untreated level. In neuron-rich brain slices pretreated with FC, difference in recovery of the PCr level after ischemia was not detected (69.8 ± 6.4% vs 70.0 ± 4.5%; p = 0.66).
Conclusions: These results indicated that EP is neuroprotective against ischemia when administered before the ischemic exposure. The importance of timing of administration of EP in clinical use was suggested. Our results also showed that EP did not show significant neuroprotective effects in neuron-rich slices pretreated with FC. It was suggested that the administration of EP before ischemia and the presence of glial cells are required for EP to exert neuroprotective effects. It is reported that astrocytes protect neurons from oxidative stress (Desagher et al., 1996). Thus damage to astrocytes by FC may remove protective factors and allow death pathways to be activated in neurons (Zhao et al., 2000). Our results might indicate that EP assists neuroprotective effects of astrocyte against reactive oxygen species or zinc excitotoxicity after ischemic stress. Recently a hypothesis on energy metabolism in brain has been proposed that uptake of glucose takes place predominantly in astrocytes and that neurons are supplied with lactate produced in astrocytes by glycolysis (Pellerin & Magistretti, 1994; Magistretti et al., 1999; Hyder et al., 2006). It was also reported that glial cells are the main source of lactate utilized for the recovery of synaptic function posthypoxia (Schurr et al., 1997). It might also be possible that EP has some effects either on glycolytic process in astrocytes or monocarboxylate transport system from astrocytes to neurons when brain slices are exposed to ischemia.
NEUROPROTECTIVE EFFECT OF ERYTHROPOIETINS WITH TRANSIENT FOCAL CEREBRAL ISCHEMIA IN RATS
Ken-Ichiro Katsura, Kouichi Utsumi, Megumi Watanabe, Makoto Sakurazawa, Yasuo Katayama
Department of Internal Medicine, Division of Neurology, Nippon Medical School, Tokyo, Japan
Introduction: For more than a decade, recombinant human erythropoietin has been used to treat anemia associated with chronic renal failure, chemotherapy for cancer patients etc. Epoetin alpha (ESPO) and epoetin beta are recombinant forms of endogenous erythropoietin, while darbepoetin alpha (AranespR) is a modified erythropoiesis-sitmulating protein that contains higher sialic acid carbohydrate content than endogenous erythropoietin, which extends its serum half-life. Stimulation of red blood cell formation was considered to be the sole physiological function of Erythropoietin (EPO), however, studies which report novel neuroprotective and neurotrophic functions of EPO were appeared recently. In this study, we aimed to explore if ESPO and Aranesp would be neuroprotective with a single bolus shot of intravenous administration and compare the two erythropoietins.
Methods: Male Sprague Dawley rats (250-300 g) were anesthetized with halothane in nitrous oxide/oxygen. The animals underwent MCA occlusion for 90 min and then reperfused. The drug (ESPO 5000 U/kg, Aranesp 5000U/kg) or vehicle was administered intravenously at 30 min before MCA occlusion. Three time points were studied: 24 h after ischemia, 7 days after, and 14 days after. At those time points, after evaluating neurological score and motor functions (grip strength, rotametric assessments), brains were taken out and infarct volumes were calculated. In the separate groups at 24h, 3 days and 7 days, rats were perfusionfixed with paraformaldehyde and imunohistochemical assessments of Bcl-2, Bax, and Tunnel staining were performed on the peri-infarct area.
Results: Body temperature and weights were preserved with EPO treated groups (ESPO or Aranesp). The volume of cerebral infarction were not significantly different at 24h after ischemia, however, at 7 and 14 days, EPO treated groups showed significant reduction(Figure). Neurological score and motor function showed significant improvement in EPO treated groups at 24h and thereafter. Bcl-2 expressions were significantly increased and Bax expressions decreased with EPO treated groups at 24h. TUNEL positive cells were significantly decreased with EPO treated groups at 24h, and 72h.
Discussion: EPO showed significant improvement of neurological score and motor function without reducing cerebral infarct volumes at 24h after ischemia. At 72h, increases of infarct volume were inhibited with EPO treatment probably because of increased Bcl-2 expression and decreased Bax expression around the infarct area as shown at 24h. Protective mechanisms of EPO may involve the stimulation of surviving signals through Bcl-2 and inhibition of death signal through Bax around the area of infarction.
THE AROMATASE INHIBITOR LETROZOLE DOES NOT INFLUENCE OUTCOME IN A MOUSE MODEL OF PERMANENT FOCAL ISCHAEMIA
Tracy Deanne Farr1, I. Mhairi Macrae1, Barry Condon2, Lindsay Gallagher1, Jim Mullin1, Hilary V.O. Carswell1
1Wellcome Surgical Institute and 7T Experimental MRI Facility, Division of Clinical Neuroscience, University of Glasgow, Glasgow, Scotland, UK, 2Department of Clinical Physics and Bioengineering, Southern General Hospital, Glasgow, Scotland, UK
Pre-clinical research has highlighted the potential of oestrogen as a neuroprotectant in experimental stroke. Aromatase, the enzyme responsible for producing oestrogen, may represent a therapeutic target. Indeed, inhibition of aromatase has been shown to exacerbate neuronal damage after a neurotoxic insult and transient ischaemia (Azcoitia et al. 2001 J Neurobiol. 47, 318; McCullough, et al., 2003. J Neurosci. 23: 8701). The present study investigated the effects of the aromatase inhibitor letrozole (FemaraR, Novartis) in a mouse model of distal diathermy middle cerebral artery occlusion (MCAO). Male 129SvJ mice were randomly subcutaneously injected daily with vehicle (0.3% hydroxypropylmethylcellulose n=7) or 10ug/mouse/day of letrozole (n=5) and experimenter and analyser were blinded to treatment. Animals were trained in the ladder rung walking test (Farr et al, 2006. J Neurosci Methods 153(1), 104) for sensorimotor function pre-MCAO. Forty-eight hours post-MCAO magnetic resonance imaging (MRI) was performed for oedema corrected infarct volume determination using a RARE T2 weighted scan (TR/TE of 5086/73msec, 600µm slice thickness) on a 7T/30 Bruker BioSpec. Animals were tested for sensorimotor impairments at 2 and 8 days post-MCAO. The results indicated that there was no significant difference in hemispheric lesion volume between vehicle (17.2 ± 8%) and letrozole (17.4 ± 4.6%) treated animals (% of hemisphere, mean ± standard deviation, unpaired t-test, Figure 1). Furthermore, all animals exhibited significantly lower scores on the ladder test following the MCAO, but there was no significant difference between groups. In conclusion, pretreatment with 10ug daily dose of letrozole did not significantly influence infarct volume or sensorimotor function in mice following MCAO. A higher daily dose of 100ug letrozole is currently being tested.
Acknowledgements
This work was supported by a Research Fellowship Grant from Research into Ageing to HVC, a research development grant from SHEFC, and a University of Glasgow postgraduate studentship, ORS, and Canadian Stroke Network postgraduate studentship to TDF. The authors are grateful to Novartis Pharma AG, Basel, Switzerland for the gift of letrozole.
Figure 1. Percent hemispheric lesion volume (± standard deviation of the mean) calculated from the MRI images for the two groups (vehicle (n=7) and letrozole 10µg/day (n=5)).
INTRANASAL NAD+ ADMINISTRATION FOR TREATING BRAIN ISCHEMIA
Weihai Ying, Guangwei Wei, Dongming Wang, Peng Zhang, Huafei Lu
Department of Neurology, University of California at San Francisco and San Francisco VA Medical Center, San Francisco, CA, USA
Background and aims: Excessive poly(ADP-ribose) polymerase-1 (PARP-1) activation plays a significant role in ischemic brain damage. Based on our in vitro finding that NAD+ treatment can abolish PARP-1-mediated cell death, we hypothesized that NAD+ administration may decrease ischemic brain injury. The major goal of this study was to test this hypothesis.
Methods: A rat model of 2-hr transient focal ischemia was used. NAD+ was administered by intranasal delivery approach at 2 hrs after ischemic onset. At 24 or 72 hrs after ischemia, both brain infarct formation and neurological deficits were assessed.
Results: We observed that intranasal NAD+ delivery significantly increased NAD+ contents in the brains. Intranasal delivery with 10 mg / kg NAD+ at 2 hours after ischemic onset profoundly decreased infarct formation when assessed either at 24 or 72 hours after ischemia. The NAD+ administration also significantly attenuated ischemia-induced neurological deficits. In contrast, intranasal administration with 10 mg / kg nicotinamide did not decrease ischemic brain damage.
Conclusions: These results provide the first in vivo evidence that NAD+ metabolism is a new target for treating brain ischemia, and that NAD+ administration may be a novel strategy for decreasing brain damage in cerebral ischemia and possibly other PARP-1-associated neurological diseases.
ASSOCIATION BETWEEN NICOTINE DEPENDENCE AND STRIATAL DOPAMINE RELEASE FOLLOWING NICOTINE ADMINISTRATION
Hidehiko Takahashi, Yota Fujimura, Harumasa Takano, Hiroshi Ito, Tetsuya Suhara
Department of Molecular Neuroimaging, National Institute of Radiological Sciences, Chiba, Japan
Background and aims
It has been suggested that mesolimbic dopamine pathway mediate the rewarding effect of nicotine. [11C]raclopride PET method has been used for the indirect measurement of changes in synaptic dopamine concentration in response to addictive drugs like cocaine and amphetamine. Since synaptic dopamine competes with [11C]raclopride for D2 receptor binding, dopamine release is thought to decrease [11C]raclopride binding potential (BP). Decreases in [11C]raclopride BP in the ventral striatum have also been demonstrated following tobacco smoke in smokers. However there is no PET study comparing dopamine release following nicotine administration in nonsmokers with those in smokers. In this study, we used nicotine gum aiming to administer nicotine in nonsmokers as in smokers.
Methods
Twelve healthy male subjects (6 smokers, mean age 25.8 years and 6 nonsmokers mean age 23.7 years) participated in the study. The Fagerstrom test for nicotine dependence (FTND) was used for assessing nicotine dependence. Smokers were instructed not to smoke for 24 hours before scanning. The study was a double-blind, randomized, placebo-controlled cross-over design. Each subject participated in two PET measurements separated by at least 4 weeks. One hour before each PET measurement, subjects received two pieces of either nicotine or taste-matched placebo gum. Plasma nicotine concentration was measured just before gum administration, 60, 75, 90, 105, 120min after gum administration. Dynamic PET scans were performed for 60 minutes immediately after the injection of a bolus of [11C]raclopride. BP values were quantified using the simplified reference tissue model for regions-of-interest (ROI), those were the dorsal caudate, dorsal putamen, ventral caudate and ventral putamen. The cerebellum was used as reference region. BP values of each ROI following nicotine and placebo administration were compared by paired t-tests. Pearson correlation coefficients between changes in BP values of each ROI and nicotine dependence assessed with FTND were calculated.
Results
Plasma concentrations of nicotine during PET scan using this procedure were similar to those achieved by smoking a typical cigarette (6-16 ng/ml). Although smokers and nonsmokers did not differ significantly in BP of each ROI in both nicotine and placebo conditions, smokers showed significant decreases in [11C]raclopride BP in the ventral caudate [2.75±0.24 (placebo), 2.46±0.21 (nicotine)] and ventral putamen [3.59±0.31(placebo), 3.14±0.33(nicotine)] in response to nicotine, but nonsmokers did not. Reduction of BP in the ventral striatum was correlated with the degree of nicotine dependence.
Conclusions
Decrease in [11C]raclopride BP is thought to reflect dopamine release following nicotine administration in smokers. Enhanced dopamine release in smokers may underlie reinforced rewarding effect of nicotine in dependence after repeated exposure.
EFFECT OF REPEATED ADMINISTRATION OF AN ANTIHISTAMINE, OLOPATADINE, ON CNS HISTAMINE H1 RECEPTOR MEASURED WITH PET: VISUALIZATION OF DOWN REGULATION
Michio Senda1, Nobuo Kubo2, Kazuhiko Adachi3, Keiichi Matsumoto1, Keiji Shimizu1, Miho Suzuki1, Hideyuki Tominaga1, Shusaku Tazawa1, Jun Nakakuma3, Masaru Takaishi4, Jun-ichi Nagase4
1Division of Molecular Imaging, Institute of Biomedical Research and Innovation, Kobe, Japan, 2Department of Otorhinolaryngology, Kansai Medical University, Yawata, Kyoto, Japan, 3Department of Mechanical Engineering, Kobe University, Kobe, Japan, 4MICRON Inc., Kobe, Japan
Background and aims: Antihistamines are widely used as a medication for common allergic disorders such as seasonal pollinosis, chronic rhinitis and urticaria. Some antihistamine drugs have a sedative side effect as they enter the brain and block histamine H1 receptor, which may cause traffic accidents and other serious events, but is difficult to evaluate due to large variation in neuropsychological measures. Measurement of cerebral histamine receptor occupancy (RO) using PET with C-11 doxepin under a single administration of antihistamines has been effectively used for evaluation of the sedative effect of the drugs to classify them into sedative and less- or non-sedative groups. Olopatadine has been reported as a less sedative antihistamine as measured with PET, but the sedative effect after repeated administration has been shown to wear off in neuropsychological tests. The purpose of the present study is to explore the mechanism by measuring the H1 receptor following repeated administration.
Methods: A total of 17 young male subjects (age 20-24) with perennial nasal allergy but without current antihistamine medication underwent PET measurement of H1 receptor three times in the single blinded manner using ECAT HR+ PET camera: baseline (placebo), 75 minutes after the initial 5mg single dose administration of olopatadine, and 75 minutes after a 5mg dose following repeated administration of 10mg/day for 4 weeks. They were also interviewed for the sleepiness at each PET measurement. The time course of the brain radioactivity was measured with PET for 90 minutes starting the injection of 769+/−41MBq of C-11 doxepin (22+/−4MBq/nmol) together with three-point venous blood sampling, from which the tracer distribution volume was computed with Logan plots using the standard input function and metabolite analysis data. Using the cerebellum as the reference tissue, the H1 receptor binding potential (BP) in the frontal cortex was obtained.
Results: The BP under the initial single dose of olopatadine was slightly lower than the baseline BP (0.347 +/− 0.066 vs. 0.395 +/− 0.123) (p<0.1). The RO by the initial single dose was estimated to be 11.1 +/− 14.0 %, although one subject presented a much higher RO (58.3%) than the others. The sleepiness scale at the PET scan showed mild sedative effect by the initial single dose.
The BP under another dose following 4-week medication of olopatadine showed marked decrease (0.188 +/− 0.093) as compared with BP at the initial single dose (p<0.0001). Assuming that the repeated administration had no effect on the BBB permeability or receptor affinity or occupancy, the amount of receptor (maximum BP) was estimated to have decreased to an average of 54.2% after repeated dose, suggesting down regulation of H1 receptor possibly due to increased endogenous histamine level. The sleepiness scale at the PET scan following repeated administration was variable.
Conclusions: The results indicate that it is possible and plausible to evaluate the effect of repeated administration of an antihistamine drug on the H1 receptor, which may be useful for drug development. It also suggests that the reduced sleepiness after repeated administration may be caused by the H1 receptor down regulation.
SHIFT IN PROPRIOCEPTIVE ACTIVITY IN RECOVERY FROM POST-STROKE HEMIPARESIS
Alexander Thiel2, Beatrice Aleksic2, Johannes C. Klein2, Jobst Rudolf2, Wolf-Dieter Heiss1
1Max Planck Institute for Neurological Research, Cologne, Germany, 2Department of Neurology, University of Cologne, Cologne, Germany
Objective
Imaging studies of the motor system employing active motor tasks have been applied extensively in patients after ischemic stroke (1)–(3), but have not yielded reliable measures for predicting recovery. This might be in part due to the complexity and flexibility of the motor system to cope with lesions, in part to the variability of the lesion site causing motoric deficits. Little is known about the role of the proprioceptive system for recovery after stroke despite rehabilitation strategies utilize proprioceptive facilitation (4) and despite the role of proprioceptors for spasticity. This study was designed to investigate the activity of the proprioceptive system during early recovery after pure motor stroke.
Methods
Ten patients with unilateral infarction of the posterior limb of the internal capsule were investigated using O-15-water PET during passive extension of the index finger of the paretic and non-paretic hands. PET scanning and assessment of motor recovery were performed early (mean 4.7 days) and after rehabilitation with 30 minutes of physiotherapy daily for an average of 17.6 days. Changes in brain activation patterns induced by passive finger movement were analyzed using SPM99.
Results
All patients showed significant improvement of motor-function of the paretic limb after rehabilitation. During passive finger movement of the non-paretic index-finger, significant CBF-increases were observed in the SI (BA3) and SII (BA 40 and 39) areas of the non-infarcted hemisphere, as previously reported for normal subjects. Additionally, significant activation of ipsilateral SII in the infarcted hemisphere was observed. After rehabilitation ipsilateral SII activation vanished and the normal activation pattern was restored. During passive movement of the paretic index-finger opposite changes of activation patterns were observed. Initially only SI and SII of the infarcted hemisphere were activated, like in normal subjects. During rehabilitation additional recruitment of SII in the non-infarcted hemisphere occurred.
Conclusion
Recovery from internal capsule infarction is accompanied by substantial changes in activity of proprioceptive systems not only of the paretic but also of the non-paretic limb. These changes are evident in ipsilateral activity of SII during the acute phase of stroke, which vanishes for stimulation of the non-paretic limb after recovery, but newly occurs after rehabilitation of the paretic limb. The consecutive alternating activations of SII are observed during non-complex, non-sequential passive movements. These changes in the activation patterns cannot be easily explained by plasticity of the proprioceptive system, since the time span is too short for adaptation. It might in part be due to a reduction of transcallosal inhibition of the affected onto the intact hemisphere; it may also reflect an interhemispheric shift of attention to proprioceptive stimuli associated with recovery.
ABSOLUTE QUANTITATION OF REGIONAL CEREBRAL BLOOD FLOW IN MOUSE USING 123I-IODOAMPHETAMINE AND PINHOLE SPECT
Tsutomu Zeniya1, Hiroshi Watabe1, Takayuki Ose1, Takuya Hayashi1, Noboru Teramoto1, Kazunori Myojin2, Akihiko Taguchi2, Hiroshi Sato3, Akihide Yamamoto1, Antti Sohlberg1, Toru Inomata1, Hidehiro Iida1
1Department of Investigative Radiology, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Suita, Osaka, Japan, 2Department of Cardiovascular Dynamics, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Suita, Osaka, Japan, 3Laboratory for Diagnostic Solution, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Suita, Osaka, Japan
Background and aims: Pre-clinical study using small animals is essential for evaluation of new therapy. Regional cerebral blood flow (rCBF) as physiological parameter is one of indexes to evaluate the effect of therapy for cerebral infarction. For imaging distribution of radioisotope in mouse, imaging modality with high-spatial resolution (<1 mm) is required. Unlike autoradiography (ARG), pinhole SPECT allows to obtain 3D distribution of radioisotope without cutting specimen into thin slices. This study was aimed at evaluating feasibility of absolute quantitation of rCBF in mouse using 123I-iodoamphetamine (IMP) and our pinhole SPECT system.
Methods: The pinhole SPECT system consists of an object rotating stage, a conventional gamma camera (Toshiba GCA-7200A) and a pinhole collimator. The pinhole of 1 mm was used. The spatial resolution was 0.9 mm. This study was performed on six mice of cerebral infarction model. The MCA-infarction was created on 9 days before SPECT experiments. The anesthetized mice were sacrificed 13-23 min after administration of IMP (0.44–0..47 mCi), and then brains were extracted and placed on the rotating stage with holder. Immediately before sacrifice, arterial blood in heart chamber was sampled to calibrate population-based input function which was determined separately on five mice by frequent blood sampling. Each brain was scanned for one hour. The images were reconstructed by pinhole 3D-OSEM method and CBF values were calculated using a two-compartment model (2CM) with distribution volume of 45 ml/ml as proposed previously (Iida et al. JNM 1994). CBF images were generated on a pixel-by-pixel basis. T2 weighted images of brain in all mice were obtained by MRI (GE Signa 3T) scans the day before the SPECT study, which has been used for identification of region-of-interest (ROI). SPECT images were superimposed on MR images through X-ray CT images. Two ROIs were set on infarction region including penumbra and the symmetrical region in unaffected side.
Results: SPECT images could be obtained and successfully superimposed on MRI images (Fig. 1). CBF images showed significant defect in infarction regions, and demonstrated significantly decreased CBF values in infarct side as compared with unaffected side, namely 0.68+/−0.20 versus 0.88+/−0.25 ml/g/min, respectively. The CBF values in normal regions were within a range measured previously by 14C-iodoantipyrine and autoradiography (Maeda et al. JCBFM 2000).
Conclusions: This result suggested feasibility of absolute quantitation of rCBF in mouse using IMP and our pinhole SEPCT system.
A FLOW SENSITIVE ALTERNATING INVERSION RECOVERY (FAIR) MRI PROTOCOL TO MEASURE CEREBRAL BLOOD FLOW IN A MOUSE STROKE MODEL
Christoph Leithner1, Karen Gertz1, Helmut Schroeck2, Josef Priller3, Konstantin Prass1, Jens Steinbrink4, Arno Villringer4, Matthias Endres1, Ute Lindauer1, Ulrich Dirnagl1, Georg Royl1
1Department of Experimental Neurology, Center for Stroke Research, Charite Universitaetsmedizin Berlin, Berlin, Germany, 2Department of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany, 3Laboratory of Molecular Psychiatry and Department of Experimental Neurology, Charite Universitaetsmedizin, Berlin, Germany, 4Berlin Neuroimaging Center and Department of Neurology, Charite Universitaetsmedizin and Center for Stroke Research Berlin, Berlin, Germany
Background and Purpose: In vivo determination of cerebral blood flow is an important tool in cerebrovascular research. Mice as experimental animals are of special interest because of the potential of genetic engineering. MRI provides three-dimensional non-invasive quantitative methods of CBF (cerebral blood flow) imaging, but MRI techniques of CBF determination have not yet been validated for mice. We compared CBF imaging using flow sensitive alternating inversion recovery (FAIR) MRI at 7T and 14C-iodoantipyrine (IAP)-autoradiography in a mouse model of acute stroke.
Materials and Methods: Twenty-nine male 129S6/SvEv mice were subjected to filamentous left middle cerebral artery occlusion (MCAo). CBF imaging was performed using 14C-IAP autoradiography and FAIR-MRI under two different anaesthesia protocols, either intravenous infusion of etomidate or inhalation of isoflurane, which differentially affect cerebral perfusion. Physiological parameters, such as arterial blood gas concentrations, body temperature and mean arterial pressure were carefully monitored and kept within the physiological range for the mouse.
Results: Using 14C-IAP autoradiography, the average hemispheric CBF was 160 ± 34 (isoflurane, n=5) and 59 ± 21 ml/(100g*min) (etomidate, n=7) in the intact hemisphere and 43 ± 12 (isoflurane, n=5) and 36 ± 12 ml/(100g*min) (etomidate, n=7) in the MCAo hemisphere. Using FAIR-MRI the corresponding average CBF were 208 ± 56 (isoflurane, intact hemisphere, n=7), 84 ± 9.3 (etomidate, intact hemisphere, n=7), 72 ± 22 (isoflurane, MCAo hemisphere, n=7) and 48 ± 13 ml/(100g*min) (etomidate, MCAo hemisphere, n=7).
Regression analysis showed a strong linear correlation of CBF measured with FAIR-MRI and 14C-IAP autoradiography with FAIR-MRI overestimating CBF by approximately 30% compared to autoradiography.
Conclusion: FAIR-MRI provides repetitive quantitative measurements of hemispheric CBF in a mouse model of stroke.
IMPACT OF CHRONIC ALCOHOL ADMINISTRATION ON THE BRAIN ACETATE METABOLISM OF RAT
Ya-Ju Hsieh1, R.S. Liu2, Y.C. Lin3, K.H. Su1, C.C. Ke3, J.W. Kuo3, L.C. Wu4, J.C. Chen1, T.P. Su5
1Institute of Radiological Sciences, National Yang-Ming University, Taipei, Taiwan-R.O.C., 2Department of Nuclear Medicine, Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan-R.O.C., 3Molecular and Genetic Imaging Core, National Yang-Ming University, Taipei, Taiwan-R.O.C., 4National PET and Cyclotron Center, Taipei Veterans General Hospital, Taipei, Taiwan-R.O.C., 5Department of Psychiatry, Taipei Veterans General Hospital, Taipei, Taiwan-R.O.C.
Objectives: The aim of this study is to assess the effect of chronic alcohol administration on the acetate metabolism of rat brain. Methods: Ten male Spraque-Dawley (SD) rats were studied. 1-[C-11]-acetate (ACE) microPET imaging was carried out for each animal before and after ingestion of a dose of 2.4%, 4.8%, 7.2% alcohol for 3, 3, 15 days, respectively. 0.2 mL of saline solution containing about 40 MBq of ACE was injected into the tail vein. A 30 min dynamic imaging acquisition in 3D list mode started immediately after bolus injection of ACE by a Concorde R4 microPET system. ROIs are defined for the cerebral hemispheres, cerebellum and cardiac chamber. A decay-corrected tissue time-activity curve was obtained from the dynamic data. After completion of the base-line study, rats were fed with liquid diet containing various concentrations of alcohol for 3 weeks. The image-derived input (k1) and output (k2) functions were extracted from the dynamic brain microPET imaging using independent component analysis (ICA). Results: The k1 and k2 of ACE showed no significant difference between cerebrum and cerebellum. The values of k1 and k2 in Cerebrum were 0.085±0.012 and 0.104±0.015 to 0.089±0.013 and 0.105±0.014 before and after ingestion of alcohol, respectively. Cerebellum also revealed the similar results. k1 and k2 were 0.069±0.018 and 0.104±0.015 to 0.074±0.014 and 0.095±0.014. No significant changes of k1 and k2 were observed in rats with and without chronic alcoholism. Both the time-activity curves before and after administration of alcohol showed that the input of ACE reached the peak between 400 to 600 sec, followed by a flat curve. After alcohol ingestion, the uptake curve of ACE is lower than the baseline study, but the curve shapes were similar. Conclusion: Chronic alcohol administration does not affect the input and output function of acetate in cerebrum as well as cerebellum, but may destroy some brain cells so that the input of ACE decreases significantly. Quantification method using ICA is promising for the study of the metabolic consequence in the central nervous system caused by alcohol.
RELATIONSHIP BETWEEN MDR-1 POLYMORPHISM AND BRAIN PENETRATION OF FEXOFENADINE, AN ANTIHISTAMINE, EVALUATED BY PET WITH [11C]DOXEPIN
Manabu Tashiro1, Masayuki Tsujimoto3, Motohisa Kato2, Hisakazu Ohtani4, Masatoshi Itoh1, Yasufumi Sawada4, Kazuhiko Yanai2
1Division of Nuclear Medicine, Tohoku University Cyclotron and Radioisotope Center, Sendai, Miyagi, Japan, 2Department of Pharmacology, Graduate School of Medicine, Tohoku University, Sendai, Miyagi, Japan, 3Department of Hospital Pharmacy, Kyoto Pharmaceutial University, Kyoto, Japan, 4Laboratory of Drug Informatics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
Background and aims
Histamine H1 receptor (H1R) antagonists, or antihistamines, mainly act on the peripheral tissues but can induce sedation. This undesirable central side effect is caused by blockade of nerve transmission in the histaminergic neuron system. Variation in sedative properties of antihistamines is due to difference in their blood-brain barrier (BBB) permeability, partly defined by activity of P-glycoprotein (P-gp), an efflux pump located in the membrane of endothelial cells in brain capillaries. The aim of the present study is to evaluate the effects of single nucleotide polymorphisms (SNP) of the P-gp encoding “multi-drug resistance gene (MDR)-1” on BBB permeability of a non-sedative antihistamine, fexofenadine, that is a substrate of P-gp, using positron emission tomography (PET) with [11C]doxepin.
Methods
Healthy young male volunteers (n=39, ranging 19-36 y.o.) were recruited and examined for genotyping of the common SNP in exon 26 (C3435T). Twenty-two out of the 39 subjects were studied by PET with [11C]doxepin following oral administration of placebo or fexofenadine 120 mg, in a single blind cross-over design. PET scan started 90 min following administration of each drug and the dynamic data, obtained taking 90 min, were analyzed based on Logan reference method to yield binding potential in various cortical structures. Based on the biding potential data of each subject in two drug conditions, histamine H1 receptor occupancy (H1RO) due to fexofenadine was calculated for each subject and the individual H1RO values were compared between wild-type (CC: n=6), heterozygous (CT: n=13) and homozygous (TT: n=3) subgroups.
Results
Overall mean H1RO due to fexofenadine in the 22 subjects was calculated as −5%. The mean H1ROs of the 3 subgroups, CC, CT and TT, were −13.4, −3.7 and −1.4%, respectively, although the difference was not significant.
Conclusions
The reason that H1RO of fexofenadine was calculated as lower than zero is not known. The PET results might suggest the presence of slight decrease in efflux function of P-glycoprotein in BBB among holders of C3435T mutation in comparison to wild-type in human. Further investigation is needed to draw a definitive conclusion with larger sample size, including studies on other mutations and other influx and efflux transporters. Functional and molecular imaging will be a strong tool for establishment of “individualized medicine”.
VESSEL ISCHENIC SIGN ON T2*-WEIGHTED IMAGES OF ACUTE CEREBRAL INFARCTION USING 3-TESLA MRI
Masaaki Uno1, Naomi Morita2, Masafumi Harada2, Shinji Nagahiro1
1Department of Neurosurgery, The University of Tokushima, Tokushima, Japan, 2Department of Radiology, The University of Tokushima, Tokushima, Japan
Purpose: We report new ischemic findings on gradient echo-type T2*-weighted images (T2*-WI) of acute ischemic stroke by 3T-MRI, and we also evaluated the reliability of the new ischemic signs compared with a cerebral perfusion study by the flow-sensitive alternating inversion recovery (FAIR) methods.
Material and methods: We initially diagnosed the acute stroke patients by 3T-MRI since April 2004. Twenty-nine patients with middle cerebral artery (n=21) or internal carotid artery occlusion (n=8) (16 men and 13 women; mean age 68 year) were examined by emergency 3T-MRI within 24 hours of symptom onset. New ischemic findings on T2*-WI were compared with diffusion-weighted images (DWI) and perfusion-weighted images (PWI). Detection of artery susceptibility sign was also evaluated.
Results: Twenty-two patients (75.9%) showed an artery susceptibility sign at occluded artery on T2*-WI. All 29 patients showed new ischemic findings such as hypointensity of the cortical vessels (vessel enlargement sign) and the deep white mater vessels (brush sign) on T2*-WI. In 13 patients the area showing the ischemic vessel signs was almost same as the hypoperfusion area shown by PWI, and in 6 patients this area was smaller than hypoperfusion area shown by PWI. In all patients, hyperintenity areas on DWI were smaller than those showing the ischemic vessel sign on T2*-WI.
Conclusion: In the diagnosis of acute ischemic stroke by 3T-MRI, these new ischemic signs will be useful in the detection of ischemic areas suggesting the necessity for an additional perfusion study or acute thrombotytic therapy.
REPERFUSION TIME WINDOW BASED ON ISCHEMIC AND HEMORRHAGIC CBF THRESHOLDS MEASURED BY XENON-ENHANCED CT IN HYPERACUTE ISCHEMIC STROKE
Shigeru Nogawa1, T. Dembo2, K. Ohki2, N. Suzuki2
1Tokyo Dental College, Ichikawa General Hospital, ‘Ichikawa, Japan, 2Department of Neurology, Keio University, Tokyo, Japan
Background and Aims: Although the newly emerged thrombolytic treatment for stroke is effective, its therapeutic time window is extremely narrow, and it may trigger intracranial hemorrhage. Therefore, it would be beneficial, if we could apply some clinical techniques that are not time-consuming and useful for selecting appropriate candidates for treatment. It is well established in animal models that the viability of ischemic brain tissue depends on its residual cerebral blood flow (CBF) and the duration of the ischemic period. Moreover, lower CBF in the affected brain has been implicated in increased incidence of hemorrhagic transformation in humans. However, substantial clinical data are still lacking, especially in the hyperacute period. We therefore tried to determine the ischemic and hemorrhagic CBF thresholds at each ischemic time point in patients with recanalization after an ischemic insult.
Methods: Forty-three sequential patients (≦85 year, mean age: 64.7+12.8, NIHSS: 17.1+9.3) with sudden-onset cortical ischemic stroke who had presented to our hospital within 2 hours (44.8+34.5 min) after the onset were studied. In the case series, local CBF was measured by xenon-enhanced CT (Xe-CT) (124.0+49.8 min). In 6 selected patients, intra-arterial thrombolytic treatment with urokinase was performed according to the procedural protocol of PROACT II. In other 5 patients, the neurological deficit abruptly disappeared, strongly suggesting spontaneous recanalization. We estimated the duration of the ischemic period in these 11 patients from the time point of intervention or symptomatic recovery. The infarction and hemorrhagic areas were determined from CT and MRI images which were obtained more than 72 hours after the onset. Mean CBF values in hemorrhagic infarction, non-hemorrhagic infarction, intact area on the ischemic side, and the symmetrical area to the infarction on the contralateral side were calculated by setting ROI on the CT images in a blind manner.
Results: At 3.3 hours after the onset, an area with a low CBF of 9 (ml/100 g/min) could escape from infarction. However, the CBF thresholds for the non-hemorrhagic infarction in gray and white matter reached a plateau (19 and 12, respectively) after 3.3 hours. The CBF threshold for hemorrhagic infarction was initially found to be lower (5-9) than that for non-hemorrhagic infarction at 4.0–4..7 hours. However, the thresholds for hemorrhagic infarction in gray and white matter reached comparable levels (17 and 9, respectively) to those for non-hemorrhagic infarction at 5.5 hours.
Conclusions: Although our cases were limited, these data suggested that the duration of the ischemic period, as well as residual CBF, is closely related to tissue viability and the risk of hemorrhage in patients with reperfusion. These data also support that thrombolytic therapy is reasonable within 3 hours, while it might trigger hemorrhagic transformation more frequently if it is performed after 6 hours. Xe-CT may be a useful decision-making tool in selecting candidates for thrombolytic therapy in the hyperacute phase, providing residual CBF values which are associated with the outcome.
TIME COURSE OF CT DENSITY CHANGES AFTER ACUTE STROKE IN MONKEYS
Edwin Nemoto1, Howard Yonas2, Oscar Menendez3, Andrew Firlik5, Kevin Stevenson5, Mary Kerr4, Tudor Jovin3
1Department of Radiology, University of Pittsburgh, Presbyterian University Hospital, Pittsburgh, PA, USA, 2Department of Neurosurgery, University of New Mexico, Albuquerque, NM, USA, 3Department of Neurology, University of Pittsburgh, Presbyterian University Hospital, Pittsburgh, PA, USA, 4Department of Nursing Research, University of Pittsburgh, Presbyterian University Hospital, Pittsburgh, PA, USA, 5Department of Neurosurgery, University of Pittsburgh, Presbyterian University Hospital, Pittsburgh, PA, USA
Background and Aims: The utility of CT imaging and xenon-enhanced CT (Xe/CT) CBF is well-established in stroke. CT density changes indicative of irreversible ischemic injury signals caution in the administration of tissue plasminogen activator (tPA) or other methods of revascularization. However, the time required for visualization of CT density changes with respect to onset of focal ischemia has not been clearly defined. Methods: Eight M. mulatta monkeys of either sex greater than 5 kg body weight were subjected to transorbital permanent focal cerebral ischemia of the middle cerebral artery (MCA) territory. The ipsilateral posterior cerebral artery (P2) was clipped prior to baseline CBF. Clipping of the ipsilateral MCA and internal carotid artery (ICA) initiated onset of the stroke. Subsequent Xe/CT CBF measurements were made at 30 m and hourly for up to six hours thereafter. End-tidal CO2, arterial blood pressure and rectal temperature were continuously monitored and kept within normal limits. Arterial blood gases and pH were obtained hourly. Group means of hemispheric CBF, CT density and stroke volume (CBF values < 10 ml/100g/min) were analyzed. In addition, individual animal hemispheric CBF and CT density changes were analyzed with time after stroke onset to determine when CT density changes were first observable.
Results: This model of focal cerebral ischemia produced a large focal ischemic insult 30–40% of hemispheric volume early after stroke onset with little growth with time based upon the CBF values obtained within the core volume of CBF <10 ml/100g/min. CBF in the ischemic focus fell almost immediately to 10 ml/100g/min where it remained over the six hours except for a transient increase at 2 hrs. CT density (Fig. 1B) changes showed that a significant decrease in CT density occurred at two hours after stroke and continued to decline for up to three hours of ischemia where it reached a plateau for up to six hours. Analysis of CT density changes between the ischemic and contralateral hemisphere in individual animals showed that visible CT density changes could be observed one hour after onset of ischemia.
Conclusions: CT density changes in the core (CBF < 10 ml/100g/min) are visible between one and two hours after onset of severe ischemia with CBF between 0 and 10 ml/100g/min.
Fig. 1. (B) CT density (Hounsfield Units) changes after MCA occlusion in the ischemic core of the stroke compared to an equal volume in the contralateral hemisphere. CT density was significantly lower (P<0.05) in the stroked hemisphere at 2 hours after occlusion. N=8.
ACUTE EMBOLIC STROKE MODEL IN CYNOMOLGUS MONKEYS USING AUTOLOGOUS BLOOD CLOT
Rishu Piao, Takuya Hayashi, Noboru Teramoto, Nobuyuki Kudomi, Youichirou Ohta, Hiroshi Sato, Hiroshi Watabe, Hidehiro Iida
Department of Investigative Radiology, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Osaka, Japan
Introduction and Purpose: For efficient development of novel therapeutics in stroke, it is expected to use a non-human primate stroke model that has a similarity in pathophysiology, as well as species to human. However, conventional techniques of MCA occlusion model often need enucleation and occlusion of M1 portion by a transorbital approach, so that it is difficult to evaluate long-term neurological deficits. Here, we show preliminary results which tried to develop a selective embolization model of MCA using cynomolgus monkeys combined with serial PET evaluations of hemodynamics.
Subjects and Methods: We used four cynomolgus monkeys and performed experiments under general anesthesia. To produce whole MCA territory or localized branch territory infarction, we placed a thin catheter at Rt M1 or M2 portion under angiography and thereafter occluded the MCA branch by injecting autologous blood clots. The dual-tracer single scan ARG (DARG) method PET was performed to measure CBF, CMRO2 and OEF at pre embolization, and 5min, 3 and 6hours post embolization repeatedly. We defined penumbra regions as regions with elevated OEF (mean value+2SD), thereafter divided the ROI into two subtypes (reversible region: RR or irreversible region: IR) by the final tissue outcome defined by the T2-MRI at 24hours after embolization. For control, we made ROI in normal regions. Neurological symptoms were scored by a monkey stroke scale.
Results: All animals showed left hemiparesis, among which two had unilateral spatial neglect, and the other two showed homonymous hemianopia. The infarction volume and neurological score were 9.4±2.4cm3 (account for 26 % of whole brain) and 25±0 in M1 occluded animals (N=2); and 2.8±3.9cm3 (account for 7% of whole brain) and 14±11 in M2 or M3 occluded model (N=1 and 1), however, animals with M1 occlusion both died of cerebral hernia 1day after embolization;. The neurological score was correlated with infarct volume (R=0.869).
Even though OEF values did not significantly differ between IR and RR (120% vs. 118%), the IR areas that finally became infarcted, showed lower CBF (70% in RR vs. 55% in IR) and CMRO2 (88% in RR vs. 65% in IR) than those in RR areas that did not. Areas of IR showed variable changes in their time course, whereas RR areas tended to keep lowered in their CBF and CMRO2.
Discussions and Conclusion: We presented a less invasive embolization technique in which M1, M2, or M3 portion of MCA was selectively catheterized in cynomolgus monkeys. We confirmed sites of occlusion in MCA branch after each embolization, and suggest that embolization of M1 portion results in large hemispheric infarction so that the animal may hardly survive in later days, whereas embolization of M2 or M3 portion does not affect animal's survival but cause significant neurological deficits that are needed for long-term behavioral evaluation. In addition, we showed time-dependent changes in CBF and CMRO2, especially in a mildly ischemic part within penumbra, suggesting that frequent and serial PET evaluations may be useful to estimate risk of infarction and correlate with the final outcome.
MEASUREMENT OF CEREBRAL ASTROCYTIC FUNCTION USING N-13 AMMONIA PET
Naoya Hattori1, Hisashi Nagashima2, Kazuo Kitazawa2, Hiroyoshi Hara3, Kazuhiro Oguchi4
1Advanced Clinical Research Institute, Aizawa Hospital, Matsumoto, Nagano, Japan, 2Department of Neurosurgery, Aizawa Hospital, Matsumoto, Nagano, Japan, 3Rehabilitation Center, Aizawa Hospital, Matsumoto, Nagano, Japan, 4Positron Imaging Center, Aizawa Hospital, Matsumoto, Nagano, Japan
Background and aims
N-13 ammonia (NH3) diffuses into cerebral tissue and is rapidly trapped in amino acid pools via glutamine synthetase. Anatomical localization of the enzyme (exclusively in astrocytes) and slow turnover of the amino acids pool allow measurement of cerebral astrocytic function as a retention of NH3. The purpose of this study was to investigate astrocytic function using NH3 PET in patients with stenotic lesion(s) in internal carotid arteries.
Methods
Ten patients (2 women, 8 men, mean age 71 ± 3 years old) underwent F-18 FDG and N-13 NH3 PET. All had stenotic lesion(s) in one of the internal carotid arteries. Four of them underwent percutaneous intervention immediately after the PET scan, and repeated a PET study after a 3 months period to investigate the effect of the intervention. Retention of NH3 was investigated using a parametric image of standard uptake vale (SUV) anatomically coregistered to his/her MRI and FDG-PET images. Regions of interest were placed on the normal grey matter territories of middle cerebral arteries on right and left sides at the slice of centrum semiovale. SUV between pathologic and normal sides were compared using a t-test.
Results
In normal grey and white matters, retention of NH3 was visually in parallel with FDG uptake except for hippocampus, pons and pituitary gland. These 3 regions showed increased retention of NH3 (compared to FDG) in all patients. Statistical analysis showed significantly lower retention of NH3 in apparently normal grey matter of the pathologic side (2.35±0.69) than that of normal side (2.53±0.6, p < 0.05). Percutaneous intervention increased regional NH3 SUV in 3/4 of the patients. Among 5 patients with subacute cortical infarction, 2 had infarcted cortices showing complete defect of FDG, while the remaining 3 had infarcted cortices showing preserved (normal to reduced) FDG. The former cortices showed minimal retention of NH3 (SUV: 0.18 and 0.58) while the latter cortices showed higher retention of NH3 (SUV: 2.45, 2.71 and 3.93, respectively) than that of the normal side (SUV: 2.23, 2.01 and 2.36, respectively) in each patient (Figure).
Conclusions
Reduced retention of NH3 in the pathologic side may suggest impaired astrocytic function in apparently normal grey matter. Percutaneous intervention increased regional retention of NH3 in 3/4 patients, suggesting the improved astrocytic function. In conclusion, retention of NH3 can be a clinical marker of cerebral astrocytic function.
EFFECTS OF CILNIDIPINE ON BP, CBF AND AUTONOMIC NERVES IN CHRONIC CEREBRAL INFARCTION
Tatsuo Otori1, sizuki Sakamoto1, Sunao Mizumura2, Seiji Okubo1, Megumi Watanabe1, Toshiya Katsumata1, Yutaka Nishiyama1, Yuichi Komaba1, Yasuo Katayama1
1Department of Neurology, Nippon Medical School, Tokyo, Japan, 2Department of Radiology, Nippon Medical School, Tokyo, Japan
In addition to both L- and N-type calcium channel inhibition, cilnidipine also blocks sympathetic hyperactivity induced by blood pressure decrease. Thereby it is estimated to provide benefits for patients with chronic cerebral infarction. To clarify these favorable actions, this study investigated 24-hour heart rate (HR), blood pressure (BP), and cerebral blood flow in a number of patients.
Subjects were 11 male and female patients with cerebral infarction who had been untreated for more than one month since onset and had blood pressure over 150/90 mmHg. All were over 20 years of age. Written consent was obtained from them (according to the requirements of the Ethics Committee). Cilnidipine dosage of 10-20 mg was administered once every morning with the decreased BP target of less than 170-150/95 mmHg. Changes in BP and autonomic nervous activity over 24 hours were measured before treatment and 3 months after treatment using a portable automatic BP/HR meter (A&D: TM2425/2025). Cerebral blood flow was determined by the head 123I-MP SPECT ARG method. SPECT images were converted to standard brain model, in which 55 regions of interest were set. The following sympathetic and parasympathetic nervous indicators were measured: LF (low frequency): sympathicotonus + parasympathicotonus; HF (high frequency): parasympathicotonus; and LF/HF: sympathicotonus.
The mean 24-hour BP (mean ± SD) was found to drop significantly from 146 ± 15/84 ± 15 mmHg before treatment to 138 ± 14/77 ± 10 mmHg after treatment. HR remained unchanged at 71 ± 4/min and 70 ± 9/min, respectively. Power spectrum analysis of HR fluctuation calculated and compared LF, HF, and LF/HF, but no significant treatment-associated changes were seen. The mean cerebral blood flow volume increased significantly from 31.8 ± 5.9 ml/min/100 g before treatment to 34.8 ± 6.5 ml/min/100 g after treatment (p <0.05).
As cilnidipine does not cause sympathetic hyperactivity induced by decreased BP and increases cerebral blood flow, it is considered a favorable antihypertensive agent in patients with chronic cerebral infarction.
OLMESARTAN INCREASES CEREBRAL BLOOD FLOW
Yasuo Iwasaki, Kiyokazu Kawabe, Riya Nagata, Ken Ikeda
Department of Neurology, Toho Univerasity Omori Hospital, Tokyo, Japan
Background and aims: Olmesartan is a long-acting angiotensin II type1(AT1) receptor blocker with a high affinity for the AT1 receptor. Olmesartan is a widely used for patients with hypertension. It is not known whether Olmesartan increases cerebral blood flow(CBF). We evaluated whether CBF is increased after administration of Olmesartan in hypertensive patients.
Methds: Ten adult hyertensive patients in whom they have no additional diseases participated in this study. Doses of Olmesartan was initially started 10mg per a day, and increased up to 20mg per a day. CBF was evaluated before and 6 months after administration of Olmasartan using 99mTc-EDC. Changes of CBF in the 13 parts of brain lesions was determined.
Results: Increases of CBF in the whole brain lesion was seen after administration of Olmasartan, but statistically no significant. However, there was a statistically significant increased CBF in the both hippocampus after administration of Olmasartan.
Conclusions: Increased CBF in the hippocampus after administration of Olmasartan suggest Olmasartan may have improvement in cognitive function.
FUNCTIONAL P-GLYCOPROTEIN IN THE BLOOD-BRAIN BARRIER OF THE POST STATUS EPILEPTICUS RAT : MEASUREMENTS USING (R)-[C-11]VERAPAMIL AND PET
Gert Luurtsema1, Mark Lubberink1, Robert Schuit1, Rob Voskuyl2,3, Harry Hendrikse1, Carla Molthoff1, Adriaan Lammertsma1, Elizabeth de Lange2
1Department of Nuclear Medicine and PET Research, VU University Medical Centre, Amsterdam, The Netherlands, 2Department of Pharmacology, University of Leiden, Leiden, The Netherlands, 3Epilepsy Institute of The Netherlands (SEIN), Heemstede, The Netherlands
Background
About one third of epilepsy patients cannot be treated satisfactorily with standard medication, illustrating that pharmacoresistance is a major problem in the treatment of epilepsy. In vitro studies have shown that the multidrug transporter P-glycoprotein (P-gp) is overexpressed in the endothelial cells of the blood-brain barrier (BBB). It has been hypothesized that this overexpression of P-gp could be an important mechanism contributing to pharmacoresistance in epilepsy patients. As many anti-epileptic drugs are substrates for P-gp, overexpression of P-gp could result in brain concentrations of these drugs that are too low to be effective.
Aim
The purpose of the present study was to investigate in vivo whether functional P-gp in the BBB was increased in an animal model of epilepsy.
Methods
Spontaneous epilepsy was achieved in three Sprague Dawley rats by induction of Status Epilepticus (SE) using i.v. injection of kainic acid. Three months after SE induction, presence of spontaneous epileptic activity was confirmed by EEG measurements. (R)-[C-11]verapamil was synthesized according to a standard procedure as described previously. Specific activity of (R)-[C-11]verapamil ranged from 10 to 40 GBq/µmol, radiochemical purity was > 98%. After a 6 min transmission scan, anesthetized (5% isoflurane) rats were injected with 20 MBq (R)-[C-11]verapamil via the tail vein. A 60 min list mode PET scan was acquired and histogrammed into 20 frames of increasing duration. Data were corrected for randoms, scatter and dead time and reconstructed using attenuation and normalisation weighted OSEM. Arterial blood samples were taken at set times to estimate plasma to whole blood ratios and metabolite fractions, used to convert the left ventricle time-activity curve into a plasma input function. Whole brain Vd over a time interval of 30 to 60 minutes was obtained using Logan analysis.
Results
Whole brain Vd 30-60 minutes was in the SE rats was 0.41 ± 0.07 and in control group 0.60 ± 0.11 (p =0.034, two tailed Mann-Whitney U test). Metabolite fractions of (R)-[C-11]verapamil in plasma were different in the SE rats compared with the control group. However, these differences in plasma concentration of the tracer were taken into account in the analysis and therefore the differences in Vd could not be explained by the differences in plasma kinetics.
Conclusion
A significant decrease in Vd of (R)-[C-11]verapamil of around 30% was measured in the SE rat relative to control. This indicates increased P-gp function at the BBB, which in turn might explain increased brain elimination of anti-epileptic drugs. Using similar studies in patients, this hypothesis now needs to be tested in patients with and without pharmacoresistant epilepsy.
TC-99M-MIBI IMAGING FOR PREDICTION OF THERAPEUTIC EFFECTS OF SECOND-GENERATION MDR1 INHIBITORS IN MALIGNANT BRAIN TUMORS
Toshio Sasajima, N. Shimada, Y. Naitoh, Y. Hu, M. Takahashi, T. Satoh, K. Mizoi
Department of Neurosurgery, Akita University School of Medicine, Akita, Japan
Introduction: Overexpression of P-glycoprotein (Pgp), encoded by multidrug-resistance 1 (MDR1) gene, has been associated with treatment failure in malignant tumors. Tc-99m-methoxyisobutylisonitrile (Tc-99m-MIBI) is a transport substrate recognized by Pgp and has been identified as sensitive probe of Pgp function. The aim of this study is to explore whether Tc-99m-MIBI is suitable to elucidate multidrug resistance and prediction of potentiation of antitumor agents by second-generation MDR1 inhibitors (PSC833, MS209) in malignant brain tumors.
Methods: In vitro experiment: Malignant tumor cells (RG2 and C6 gliomas, Walker 256 carcinoma: W256) were incubated with low dose vincristine (VCR) to induce multidrug resistance. MTT assay demonstrated significant increase of surviving fractions in all VCR-resistant sublines (RG2R, C6R, W256R) compared with those of drug-naive cell lines. The effects of the second-generation MDR1 inhibitors on proliferation activities and Tc-99m-MIBI accumulation of tumor cells were investigated using double-label accumulation assay (C-14 thymidine (TdR), Tc-99m-MIBI or Tc-99m-DTPA). Uptake values of tracers, which were corrected for extracellular fluid and damaged cells using Tc-99m-DTPA, were quantified as corrected cell-to-medium concentration ratio. Tc-99m-MIBI accumulation profiles are fitted to a one compartment model and parameter for volume distribution (Vd) was determined. And the steady state accumulation rate (Ki) of TdR was also calculated. Expression levels of MDR1 mRNA of drug-naive cells and VCR-resistant cells were assessed by RT-PCR. VCR-resistant cells were also incubated with second-generation MDR1 inhibitors (1µM) one hour before double-label accumulation assay and RT-PCR.
In vivo experiment: C6 and C6R cells were stereotactically inoculated in the right and left basal ganglia of Sprague-Dawley rats, respectively. Autoradiography using Tc-99m-MIBI was performed 10 days after tumor implantation. The second-generation MDR1 inhibitors (100 mg/kg) were orally given one hour before intravenous injection of Tc-99m-MIBI. The therapeutic effects of VCR (2 mg/kg, i.v.) with or without the MDR1 inhibitors (100 mg/kg) were also evaluated by autoradiography using C-14 methyl-L-methionine (Met) and MIB-5 index indicating the growth fraction of tumors.
Results: In vitro experiment: RT-PCR revealed higher expression of MDR1 mRNA in VCR-resistant cells than that of drug-naive cells in each line. Vd of Tc-99m-MIBI is negatively correlated with MDR1 mRNA expression among drug-naive and VCR-resistant cells (r=0.962, p<0.01). MDR1 inhibitors decreased surviving fractions and increased Vd of Tc-99m-MIBI significantly in VCR-resistant sublines, whereas MDR1 mRNA expression was unchanged. These findings indicated that Tc-99m-MIBI efflux was functionally suppressed by MDR1 inhibitors. MDR1 inhibitors had no significant effects on cell proliferation as measured by TdR accumulation rate (Ki).
In vivo experiment: Autoradiographic images of Tc-99m-MIBI revealed higher uptake in drug-naive cells at the right basal ganglia compared with VCR-resistant cells on the opposite side of tumor-bearing rat brain. Oral administration of the second-generation MDR1 inhibitors significantly increased Tc-99m-MIBI accumulation of both tumors. Met uptake and MIB-5 index of both tumors treated with VCR following the MDR 1 inhibitor treatment significantly decreased than those of tumors treated with VCR alone.
Conclusions: Tc-99m-MIBI SPECT should be informative for detecting MDR1-mediated drug resistance and for monitoring therapeutic effects of MDR1 inhibitors visually and non-invasively in patients with malignant brain tumors.
WHITE MATTER CHANGES AFTER STROKE IN RATS AS STUDIED BY SERIAL DIFFUSION TENSOR IMAGING AND MANGANESE-ENHANCED MRI
Jet P. van der Zijden, Annette van der Toorn, Rick M. Dijkhuizen
Image Sciences Institute, University Medical Center Utrecht, Utrecht, The Netherlands
Background and Aims
Restoration of lost function after stroke is often associated with alterations in neuronal circuitry in the brain. However, the exact pattern of neural reorganization after stroke has not been fully characterized. Changes in neuronal connectivity may be assessed in vivo with diffusion tensor imaging (DTI)1,2 or manganese-enhanced MRI (MEMRI).3 The aim of this study was to characterize the temporal pattern of white matter changes with DTI and MEMRI after stroke in rats.
Methods
Transient focal cerebral ischemia was induced in four male Wistar rats by 30-minutes intraluminal occlusion of the right middle cerebral artery.4 Repetitive T2 mapping and DTI were performed at 3 hours, 3 days, 3 weeks and 9 weeks after ischemia induction. T2 maps were used for lesion assessment. From DTI data we calculated mean diffusivity (ADCav) and fractional anisotropy (FA) maps. MEMRI was performed at 10 weeks after stroke, four days after injection of 0.2 ml 1M MnCl2 into spared ipsilesional sensorimotor cortex. Manganese enhancement was calculated from changes in R1 (1/T1).5
To assess temporal white matter changes after stroke, we selected three white matter structures as regions-of-interest (ROIs) for data analysis: ipsilesional internal capsule (ICipsi); ipsilesional external capsule (ECipsi); and contralateral internal capsule (ICcontra).
Results and Conclusions
In ICcontra, no significant changes in T2, ADCav and FA were observed over time. ICipsi and ECipsi were both part of the T2-defined lesion area at 3 days after stroke. However, while T2 in ECipsi remained prolonged, in ICipsi T2 was normalized after 3 and 9 weeks. As compared to ICcontra (0.44 ± 0.04), FA was significantly decreased in ICipsi (0.22 ± 0.02, P < 0.05) and ECipsi (0.21 ± 0.02, P < 0.05) at 3 days after stroke. After 9 weeks, FA in ECipsi remained significantly reduced as compared to ICcontra (0.32 ± 0.04 vs. 0.44 ± 0.03, P < 0.05). FA in ICipsi, however, was significantly increased (0.58 ± 0.03, P < 0.05) at this time-point. In addition, at 10 weeks after stroke, there was significant manganese-induced R1 increase in ICipsi, suggestive of intact connectivity with the ipsilesional sensorimotor cortex.
This study demonstrates that early ischemia-induced changes in white matter structures that are reflective of tissue degeneration (i.e., increased T2 and decreased FA), may recover at chronic time-points. The observed T2 normalization, elevated FA, and significant manganese enhancement in ICipsi at 9 weeks after stroke, may indicate reorganization of white matter including axonal sprouting and/or remyelination.
In conclusion, combining DTI and MEMRI can provide unique in vivo information on changes in tissue architecture and connectivity, and may give important insights in mechanisms underlying functional recovery after stroke.
FLAVOPROTEIN AUTOFLUORESCENCE AND SOMATOSENSORY EVOKED POTENTIAL DURING TRANSIENT CEREBRAL ISCHEMIA
Takahiro Igarashi, Noriaki Yokose, Tatsuya Hoshino, Norio Fujiwara, Yoshihiro Murata, Tuneo Kano, Jun Kojima, Kaoru Sakatani, Yoichi Katayama
Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan
Introduction: Neural activity causes a transient increase of energy metabolism in the brain, which evokes autofluorescence of mitochondrial flavoproteins. The evoked-changes in flavoprotein fluorescence (FPF) have been applied for functional brain imaging; however, the effect of cerebral ischemic period on FPF is not yet clear. In this study, we compared change in FPF and somatosensory evoked potential (SEP) during transient cerebral ischemia.
Methods: Initially, we investigated stimulus frequency responses and intensity responses of FPF and SEP in F344 rats under non-ischemic conditions; the left median nerve was stimulated at various frequencies (0.5–30.Hz) and intensities (1-10mA). Then, transient forebrain ischemia was induced in the rat by occlusion of the bilateral common carotid arteries at 45min model (n=5) or 60min model (n=10). The cortical blood flow in the somatosensory cortex was measured continuously by Laser-Doppler flowmetry. FPF and SEP were measured simultaneously via a cranial window made on the right sensory-motor cortex; FPF images were obtained by a CCD camera system (AQUACOSMOS, Hamamatsu Photonics). Supra-maximal stimulation (8mA) at 50 Hz and 0.5Hz were applied to the left median nerve for recordings of FPF and SEP, respectively.
Results: 1) SEP amplitude decreased with increases of the stimulus frequency, and the maximal SEP amplitude was observed at 0.5Hz. In contrast, FPF intensity increased with increases of the stimulus frequency, and reached the maximal intensity at 50Hz. Both of FPF intensity and SEP amplitude reached plateau levels at a stimulus intensity of over 4mA. 2) The cerebral blood flow decreased rapidly after clamping of the bilateral common carotid arteries and recovered rapidly after reperfusion. FPF and SEP responses decreased simultaneously during occlusion of the arteries, and disappeared at 30 min after the occlusion. In the 45min-ischemia model, both of FPF and SEP responses rapidly recovered after reperfusion, and reached to the control level; there were no significant differences in the recovery rate between FPF and SEP responses. In contrast, in the 60min-ischemia model, the recovery rate of FPF tended to be higher than that of SEP after reperfusion. There were significant differences in the recovery rate between SEP and FPF responses at 15 and 30min after reperfusion (SEP amplitude=11.9±17.2% versus FPF=42.1±36.1%, p<0.05 (15 min); SEP amplitude=9.0±7.4% versus FPF=61.6±48.9%, p<0.01 (30 min)).
Conclusions: The present study demonstrated a difference in recovery rates between FPF and SEP after reperfusion. The difference might be caused by a difference in the cortical layer responsible for signal generations between FPF and SEP: SEP is generated in the fourth layer of the cortex which receives specific fibers from thalamus, while FPF is generated in the second and third layers of the cortex which receive non-specific fibers. A possible difference in ischemic damages between the layers may cause the difference in recovery rates between FPF and SEP.
HYPEROXIA- AND HYPERCAPNIA-INDUCED CHANGES OF CEREBRAL BLOOD FLOW AND METABOLIC RATE OF OXYGEN IN ISCHEMIC BRAIN TISSUE
Mahmoud Ashkanian1,2, Per Borghammer2, Grethe Andersen3, Albert Gjedde1,2, Leif Ostergaard1, Manouchehr Vafaee1,2
1CFIN, Aarhus University Hospital, Bygn. 30, Aarhus, Denmark, 2PET Center, Aarhus University Hospital, Aarhus, Denmark, 3Department of Neurology, Aarhus University Hospital, Aarhus, Denmark
Background: Ischemic penumbra is defined as a hypoperfused brain tissue with preserved membrane potential and ion homestasis. Study of neurometabolic and neurovascular behavior of penumbral cells is essential to shed some light on the pathophysiology of stroke. Patients with stroke or occlusion of carotid artery demonstrate local or global decreases of oxygen tension. The ischemic brain tissue caused by the decline in oxygen tension undergoes certain metabolic changes which can result in apoptotic cell death. Improvement of tissue oxygenation in ischemic penumbra will therefore inhibit the degenerative process seen in the early stage of stroke.
Objectives: The aim of the current study is to investigate whether whole brain cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow (CBF) could be manipulated by inhalation of oxygen and carbon dioxide which in turn could lead to a therapeutic means for ischemic tissue.
Methods: We used positron emission tomography (PET) to measure CMRO2 and CBF in two groups of subjects i.e. patients with occlusion of carotid artery (n=6) and healthy controls (n=11). During the four 3-min CBF-PET scans the subjects inhaled a semirandom sequence of 100% O2 (hyperoxia), 5% CO2 (mild hypercapnia), carbogen (95% O2 + 5% CO2) or atmospheric air (baseline). The sequence was repeated for the measurements of CMRO2. Parametric subtraction maps (gas minus baseline) of CBF and CMRO2 for healthy group were calculated following co-registration of PET images to the corresponding anatomical MRI images (patient data is under completion).
Results: Significant increases of CBF in the whole brain (compared to baseline) were observed when the subjects were given CO2 and carbogen (paired t-test; p-values: 0.0018 and 0.0042 accordingly) while a significant decrease in CBF was observed during the inhalation of pure oxygen (p-value: 0.0055). However, no significant changes in the global CMRO2 were observed following the inhalation of any of the three gases. The results also were confirmed by statistical cluster analysis. Arterial blood oxygen saturation (SaO2) significantly rose from baseline (97.2%) when oxygen and carbogen were applied (99.89% & 99.80% respectively). However, there was no significant difference between oxygen and carbogen potency in increasing SaO2.
Discussion: Animal experiments have shown that either oxygen therapy or mild hypercapnia could reduce the final infarct volume (1). The present data demonstrate that inhalation of carbogen and CO2 increases CBF significantly in healthy brain tissue. Moreover, only carbogen was able to enhance the vital physiological factor such as SaO2. This implies that inhalation of carbogen is capable of optimizing oxygenation of brain tissue, a vital factor for tissue survival. Patients with stroke could be treated by thrombolysis during a 3-hour window. Improvement of brain tissue oxygenation during the pre-admission time has potential of reducing the tissue damage. The completion of our patient data will reveal whether it is possible to accomplish the same effect in ischemic tissue. If so, this will slow down the tissue degeneration which will eventually enable us to extend the 3-hour therapeutic window.
CEREBRAL HYPOMETABOLISM IN POSTVIRAL ANOSMIA; AN FDG-PET STUDY
Yu Kyeong Kim1, Jeong-Whun Kim2, Sang Eun Kim1
1Department of Nuclear Medicine, Seoul National University College of Medicineand Seoul National University Bundang Hospital, Seoul, South Korea, 2Department of Otolaryngology, Head and Neck Surgery, Seoul National University College of Medicine and Seoul National University Bundang Hospital, Seoul, South Korea
Backgroud and aims : Postviral smell loss associated with an upper respiratory track infection (URTI) is relatively severe and prolonged after subsiding of the URTI symptoms. The exact location and nature of the damage in olfactory dysfunction after URTI are not fully understood, yet. Even though a direct damage of the olfactory epithelium is very likely in postviral anosmia, the damage of central pathway has also been suspected as the possible mechanism of the disease. In this study, we investigated whether the changes in the cerebral activity occurs in patients with postviral olfactory disorder using FDG PET.
Methods : Nine patients (mean age: 57 ± 9 yr, 8 females) who suffering from disability of smelling were enrolled in this study. All patients had neither apparent sinusitis nor rhinitis, however, all recalled causative URTI and temporal connection with development of the symptom. Olfactory function tests using butanol threshold test (BTT) and odor identification task (OI) confirmed that all subjects were anosmic or severely impaired in olfaction. FDG PET studies during a rest state were acquired after initial evaluation, and the cerebral metabolic abnormality was tested using voxel-wised comparison with those from age and gender matched healthy controls.
Results : As comparison with healthy controls, patients showed the significant hypometabolism in the right ventral frontal cortex and bilateral amygdala and parahippocampal area where the olfactory neurons primarily project. Furthermore, hypometabolism was also demonstrated the bilateral insular cortices, medial and lateral temporal cortex where the olfactory information is integrated to produce the sensation. Furthermore, correlation between subsequent olfactory improvement and the severity of hypometabolism was also demonstrated in the ventral frontal cortex as well as medial/lateral temporal cortices.
Conclusions : This study clearly demonstrated that the postviral anosmia is typically associated with metabolic suppression in the brain regions where the olfactory information is received and integrated.
SPECT ANALYSIS IS USEFUL IN DIFFERENTIATION OF PURE AKINESIA FROM EARLY PROGRESSIVE SUPRANUCLEAR PALSY
Tomoko Kurata1, Takeshi Hayashi1, Tetsuro Murakami1, Kazunori Miyazaki1, Nobutoshi Morimoto1, Yasuyuki Ota1, Yasushi Takehisa1, Makiko Nagai1, Yoshiki Takao2, Taisei Ota3, Yasuo Harigaya4, Yasuhiro Manabe5, Tatsushi Kamiya1, Mikio Shoji6, Koji Abe1
1Department of Neurology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan, 2Department of Neurology, Kurashiki Heisei Hospital, Kurashiki, Japan, 3Department of Neurology, Ota Memorial Hospital, Fukuyama, Japan, 4Department of Neurology, Maebashi Red Cross Hospital, Maebashi, Japan, 5Department of Neurology, National Hospital Organization Okayama Medical Center, Okayama, Japan, 6Department of Neurology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
Background: Progressive supranuclear palsy (PSP) was first described by Steele, Richardson, and Olszewski as a clinical entity that showed supranuclear ophthalmoplegia, dystonia, rigidity of the neck and upper trunk, pseudobulbar palsy, and dementia. On the other hand, pure akinesia (PA) was described as a 3,4-dihydroxyphenylalanine (L-DOPA) unresponsive neurodegenerative disorder that causes acceleration and freezing of repeated movement without rigidity and tremor. Because the clinical features of PSP and PA patients resemble, it is still controversial whether these two disorders belong to distinct entities. Methods: We investigated 15 PA and 41 PSP patients clinical and radiological features including head MRI, ethyl cysteinate dimmer-single photon emission-computed tomography (ECD-SPECT) and iodine-123 meta-iodobenzyl guanidine (123I-MIBG) myocardial scintigraphy. In the ECD-SPECT study, the cerebral blood flow (CBF) reduction was quantitatively expressed as Z-score. The largest Z-score in the frontal lobe, as well as that in the left area 8 (frontal eye field) was measured (Fig. 1-A, B), because gaze disturbance may be associated with CBF reduction in this area.
Results: Many PSP patients claimed falls as the initial symptom but no PA patients did. Eye movement, as well as opticokinetic nystagmus elicitation, was significantly more frequently disturbed in PSP. Dementia, dysarthria and rigidity were also more frequently in PSP than in PA. By head MRI, midbrain tegmentum atrophy was more frequently observed in PSP. CBF in the frontal lobe, especially in the frontal eye field, was significantly lower in PSP than in PA. The largest Z-score in the frontal lobe of PSP tended to be larger than that of PA, but was not statistically significant; however, that in the left area 8 (frontal eye field) was significantly larger in PSP (Fig.1-C, D, Fig.2). MIBG myocardial scintigraphy showed no difference between these two groups. Conclusions: PA and PSP show distinct symptoms from the early stage, indicating that they are distinct disorders. The occurrence of falls and eye movement disturbance, as well as CBF reduction at the frontal eye field, is very important for distinguishing these disorders.
SPECT AND PET EVALUATION IN PATIENTS WITH LIMBIC ENCEPHALITIS
Kyungah Chun1, Ihnho Cho1, Eunjung Kong1, Jungsang Hah2, Meeyoung Park2, Sejin Lee2, Jun Lee2
1Department of Nuclear Medicine, Yeungnam University Hospital, Daegu, South Korea, 2Department of Neurology, Yeungnam University Hospital, Daegu, South Korea
Case Report: A 48-year-old man underwent psychiatric evaluation and treatment due to psychotic symptom for several months. But there was no clinical improvement, even more worse and mental change. He had no previous history of stroke or meningitis. MRI shows tumor-like lesion on left hippocampus area. He underwent brain SPECT with Tc-99m ECD. SPECT shows increased uptake in both medial temporal lobe, especially in left side. But, we missed at initial scan. We suspect limbic encephalitis at the follow up scan. We also evaluated with F-18 FDG PET. PET also showed increased uptake in medial temporal lobe, more evident than SPECT images. Previous report showed that FDG-PET was more sensitive in showing the initial bitemporal involvement. After treatment, SPECT findings were improved. Our study shows SPECT is useful for diagnosis and evaluation of treatment response in encephalitis.
MEASUREMENT OF CEREBRAL ACTIVATION DURING DIFFERENT MOTOR TASKS OF A LOWER LIMB USING NEAR INFRARED SPECTROSCOPY
Junichiroh Kaneko1,2, Kotaro Takeda1,2, Taizo Shiomi1,2, Misao Ogano1,2, Ituki Imai3, Hitoshi Maruyama1, Hiroyuki Kato1,2
1International University of Health and Welfare, Ohtawara, Nasushiobara, Japan, 2CREST, Japan Science and Technology Agency, Kawaguchi, Japan, 3Department of Rehabilitation, Nasu Neurosurgical Center, Nasushiobara, Japan
Objectives: The aim of this study is to assess the effect of chronic alcohol administration on the acetate metabolism of rat brain. Methods: Ten male Spraque-Dawley (SD) rats were studied. 1-[C-11]-acetate (ACE) microPET imaging was carried out for each animal before and after ingestion of a dose of 2.4%, 4.8%, 7.2% alcohol for 3, 3, 15 days, respectively. 0.2 mL of saline solution containing about 40 MBq of ACE was injected into the tail vein. A 30 min dynamic imaging acquisition in 3D list mode started immediately after bolus injection of ACE by a Concorde R4 microPET system. ROIs are defined for the cerebral hemispheres, cerebellum and cardiac chamber. A decay-corrected tissue time-activity curve was obtained from the dynamic data. After completion of the base-line study, rats were fed with liquid diet containing various concentrations of alcohol for 3 weeks. The image-derived input (k1) and output (k2) functions were extracted from the dynamic brain microPET imaging using independent component analysis (ICA). Results: The k1 and k2 of ACE showed no significant difference between cerebrum and cerebellum. The values of k1 and k2 in Cerebrum were 0.085±0.012 and 0.104±0.015 to 0.089±0.013 and 0.105±0.014 before and after ingestion of alcohol, respectively. Cerebellum also revealed the similar results. k1 and k2 were 0.069±0.018 and 0.104±0.015 to 0.074±0.014 and 0.095±0.014. No significant changes of k1 and k2 were observed in rats with and without chronic alcoholism. Both the time-activity curves before and after administration of alcohol showed that the input of ACE reached the peak between 400 to 600 sec, followed by a flat curve. After alcohol ingestion, the uptake curve of ACE is lower than the baseline study, but the curve shapes were similar. Conclusion: Chronic alcohol administration does not affect the input and output function of acetate in cerebrum as well as cerebellum, but may destroy some brain cells so that the input of ACE decreases significantly. Quantification method using ICA is promising for the study of the metabolic consequence in the central nervous system caused by alcohol.
APOLIPOPROTEIN HAS A MARKED EFFECT ON STEM CELL SURVIVAL, MIGRATION AND DIFFERENTIATION IN A MODEL OF NEURONAL INJURY
Karen Horsburgh1, Andrew Wong1, Helen Hodges2
1Centre for Neuroscience Research, University of Edinburgh, Edinburgh, Scotlanf, UK, 2Institute of Psychiatry, Kings College London, London, UK
Background/aims
Stem cell grafts may be beneficial in promoting repair and recovery in different neurodegenerative diseases such as Alzheimer's disease or ischaemic stroke. We previously reported the ability of MHP36 stem cells to survive and migrate, up to 4 weeks posttransplantation, and reduce the extent of neuronal damage in a mouse model of cerebral ischaemia (Wong et al. 2005). However, the mechanisms by which stem cells promote repair are unclear. Apolipoprotein E, (APOE) is a lipid transport protein that has a critical role in repair mechanisms. The present study sought to investigate whether endogenous APOE influences MHP36 stem cell graft integrity after neuronal injury induced by transient global cerebral ischaemia.
Methods
In wild-type (WT, mice with endogenous APOE, n=7) and in APOE deficient mice (APOE KO, n=9), transient global cerebral ischaemia was induced by temporary bilateral carotid artery occlusion. The duration of ischemia was titrated to produce similar levels of neuronal damage in the WT and APOE KO mice. In this model, ischaemia is associated with selective neuronal damage in the striatum which is maximal at 3 days post-ischaemia. Two weeks post-ischaemia, mice received a graft of fluorescently labelled MHP36 cells (0.5ul, 25,000 cells/ul) into the striatum. Mice were immunosuppressed by injections of cyclosporin A (10mg/kg, s.c.). Mice survived for 4 weeks and the brains sectioned. Stem cell survival and migration were assessed by imaging fluorescently labelled cells in defined grids within the striatum. The number of cells and the distance the cells migrated were then compared between the groups. Adjacent sections were labelled with markers of mature neurons (Neu-N) and astrocytes (GFAP) and co-localisation of stem cells with these markers quantified and compared between the groups. Statistical differences were assessed using Mann-Whitney.
Results
The results of this study indicated significantly greater numbers of MHP36 cells present in the striatum of WT mice as compared to APOE KO mice (6.4 +/− 3.2 cells/mm2 vs. 0.9 +/− 0.5 cells/mm2; p < 0.001). Migration of MHP36 cells was also significantly increased in WT mice as compared to APOE KO mice (0.7 +/− 0.2mm vs. 0.3 +/− 0.1mm, p < 0.01). Co-localisation of MHP36 cells with immunohistochemical markers indicated that MHP36 cells differentiated at 4 weeks post-transplantation into mature neurons and astrocytes. A significantly higher percentage of MHP36 cells differentiate into a neuronal phenotype in WT compared to APOE KO mice (79.8 +/− 6.9% vs. 25.0 +/− 17.1%, p <0.001). In contrast, there is reduced astrocytic differentiation in WT mice as compared to APOE KO mice (8.3 +/− 6.9% vs. 19.1 +/− 10.6%, p <0.05).
Conclusions
The study clearly demonstrates that endogenous APOE has a beneficial effect on stem cell survival, migration and differentiation. Modulation of APOE levels may have utility in future stem cell transplantations.
CONTINUOUS DEEP BRAIN STIMULATION HAS A REGENERATIVE EFFECT ON THE RAT MIDDLE CEREBRAL ARTERY OCCLUSION MODEL IN THE CHRONIC STAGE
Takamasa Morimoto, Tetsuro Shingo, Masahiro Kameda, Satoshi Kuramoto, Naoki Tajiri, Takashi Uozumi, Tomohito Kadota, Takao Yasuhara, Yasuyiki Miyoshi, Isao Date
Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Okayama, Japan
[Purpose]Continuous deep brain stimulation (DBS) has been used to treat a variety of neurological disorders such as Parkinson's disease. DBS is considered to modulate pathological neuronal network. Our recent study showed that DBS had a neuroprotective effect on the ischemic brain. Here we examined the effects of DBS on the rat middle cerebral artery occlusion (MCAO) model in the chronic stage.
[Methods]In this experiment, the brain was assessed with magnetic resonance imaging (MRI) and we used MCAO model rats. One month after the lesion, the stimulation electrode was implanted into the ischemic border zone, and low frequency and low voltage stimulation were delivered to the electrode for a week. The electrode was removed after DBS, and infarct volume was reassessed with MRI at two months after the lesion. The animals were sacrificed and the brain tissue was analyzed by using immunohistochemistry and western blot.
[Result]The rats with DBS showed significantly better behavioral recovery in the spontaneous activity or limb placement test than those without DBS at two months after the lesion. The infarct volume significantly reduced in the lesioned striatum with DBS compared to that of pre-DBS or without DBS. In immunohistochemistry, the proliferated cells (astrocytes or neural progenitor cells) accumulated around the DBS electrode and the newly differentiated neurons (Doublecortin or NeuN/BrdU positive cells) were found around the ischemic core and ischemic border zone in the DBS group. These neurons are considered to be generated in the subventricular zone (SVZ) by the pattern of migration. Western blot analysis for VEGF showed that DBS induced a significant change in VEGF in MCAO model in the chronic stage.
[Conclusion]These results suggest that continuous DBS enhanced neurogenesis in SVZ and the migration of immature neurons into the ischemic region. DBS may control the survival of neurons around the ischemia and play a role as an attractanct to induce neurogenesis
NEURAL CELL TRANSPLANTATION IMPROVES BLOOD FLOW RESPONSE TO SOMATOSENSORY STIMULATION IN CORTICAL INJURED MICE
Yasushi Kondoh1, Kazuhiro Nakamura2, Noboru Suzuki3, David Wright1, Shigenori Mizusawa4, Ken Nagata1, Iwao Kanno5
1Department of Neurology, Akita Research Institute of Brain and Blood Vessels, Akita, Japan, 2Department of Radiology and Nuclear Medicine, Akita Research Institute of Brain and Blood Vessels, Akita, Japan, 3Department of Immunology and Regenerative Medicine, St. Marianna University, Kawasaki, Japan, 4Department of Internal Medicine, Akita Research Institute of Brain and Blood Vessels, Akita, Japan, 5Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan
Background and aims: Recent results have demonstrated that transplantation of various types of cells improves neurological deficits in animals with stroke or head injury [1]. We have also shown that transplantation of neural cells derived from mouse embryonic stem (ES) cells regenerates the neural network and improves motor function in hemiplegic mice [2]. Since the neuronal responses to somatosensory stimulation correlate with the stimulus-induced increase of CBF in the cerebral cortex, to evaluate the effect of transplantation of neural cells we measured cerebral perfusion under electric stimulation in mice with cortical injury by using 4.7- tesla experimental MRI system.
Methods: Neural cells were obtained from mouse ES cells by transfection of MASH1, a neuron-specific differentiation factor gene. Photochemical induced thrombo-embolic lesion was made in the left somatosensory cortex. Seven days later, neural cells (1 × 105 cells) were administered into the cisterna magna (MASH1 group). Animals injected with undifferentiated ES cells were served as control. A continuous arterial spin labeling technique was used for perfusion weighted MRI during electric stimulation (5Hz, 2mA) of right hind-paw 7 or 8 days, 14 days and 28 or 30 days after transplantation. The mean perfusion ratio (stimulation/rest) and SD were calculated in the healthy hemisphere, and the voxels having higher ratio than this mean + 2SD in the lesion side were considered as significant perfusion increased areas by stimulation.
Results: Histological examination revealed that the neural cells injected into cisterna magna accumulated in the lesion as early as 2 days after transplantation. The perfusion ratio in MASH1 group (n=6) was significantly higher than control group (n=4) by ANOVA with repeated measurements, but no significant difference was observed among the time after transplantation [Fig.1].
Conclusions: This result indicates an improved blood flow response to somatosensory stimulation after neural cell transplantation. As blood flow reflects synaptic transmission and postsynaptic processing, the results also suggest functional recovery of neuronal activity.
HEMATOPOIETIC STEM CELLS PREVENT HAIR CELL DEATH AFTER TRANSIENT COCHLEAR ISCHEMIA THROUGH PARACRINE EFFECTS
Tadashi Yoshida1, Ryuji Hata2, Nobuhiro Hakuba1, Fang Cao2, Pengxiang Zhu2, Masahiro Sakanaka2, Kiyofumi Gyo1
1Department of Otolaryngology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan, 2Department of Functional Histology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
Background and aims
Idiopathic sudden hearing loss (ISHL) is usually unilateral and can be anything within a range of a slight impairment of hearing to virtual deafness. One of the most common etiologies for ISHL is circulatory disturbance (most often vertebrobasilar ischemia). Vertebrobasilar ischemia (VBI) causes deafness because most of the auditory system including the inner ears is supplied from the vertebrobasilar system. ISHL of vascular cause is important for neurologists to recognize, because it sometimes appears as a warning sign of impending vertebrobasilar ischemic stroke. Previously, we produced cochlear ischemia by occluding both vertebral arteries in gerbils and established an animal model of ISHL [1]. We also showed progressive sensory hair cell loss after cochlear ischemia [1]. Sensory hair cell loss results in the sensorineural hearing loss (SNHL) and the most common type of hearing loss in adults is SNHL. Recent advances in stem cell biology have gained interest in the hope that stem cell therapy will come closer to regenerating sensory hair cells. Transplantation of hematopoietic stem cells (HSC) is regarded to be a potential approach for promoting repair of damaged organs. Here, we investigated the influence of hematopoietic stem cells on progressive hair cell degeneration after transient cochlear ischemia in gerbils.
Methods & Results
Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra [2]. Intrascalar injection of HSC ameliorated hearing impairment (** indicates p< 0.01, n=6 in each group)(Fig. A) and prevented ischemia-induced inner hair cell (IHC) degeneration while there were no apparent outer hair cell (OHC) degeneration after ischemia (** indicates p< 0.01, n=6 in each group)(Fig. B). We also showed that the protein level of glial cell line-derived neurotrophic factor (GDNF) in the organ of Corti was upregulated after cochlear ischemia and that treatment with HSC augmented this ischemia-induced upregulation of GDNF. A tracking study revealed that HSC injected into the cochlea were retained in the perilymphatic space of the cochlea, although they neither transdifferentiated into cochlear cell types nor fused with the injured hair cells after ischemia.
Conclusions
Our results suggest that HSC may have therapeutic potential possibly through paracrine effects. Thus, we propose HSC as a potential new therapeutic strategy for hearing loss.
THE SURVIVAL AND DIFFERENTIATION OF THE MESENCHYMAL STEM CELL AFTER THE TRANSPLANTATION IN MIDDLE CEREBRAL ARTERY OCCLUSION RAT MODEL
Kengo Kato1,2, Kuniko Shimazaki1, Satoshi Suda1,3, Masayuki Ueda3, Tatsushi Kamiya3, Toshiki Inaba3, Hidenori Yokota4, Keiji Oguro4, Eiju Watanabe4, Masaru Okabe5, Yasuo Katayama3, Tatsuhiko Kanno2
1Department of Physiology, Jichi Medical University, Tochigi, Japan, 2Musashi Ranzan Hospital, Saitama, Japan, 3Second Department of Internal Medicine, Nippon Medical School, Tokyo, Japan, 4Department of Surgical Neurology, Jichi Medical University, Tochigi, Japan, 5Department of Experimental Genome Research, Genome Information Research Center, Osaka University, Osaka, Japan
Background and aims
It was reported that the transplantation of the mesenchymal stem cell (MSC) protects against the stroke in the acute phase. It has also been suggested that they might be able to differentiate into other phenotypes as well, include neuronal cells. MSC transplantation therapy may have the potential to promote brain repair and recovery after stroke. To compare how environment affects the survival and differentiation by different area, we examined cerebral cortex and striatum after MSC plantation in stroke rats by intravenous routes.
Methods
The enhanced green fluorescent protein (EGFP) expressing MSC were transplanted in adult rats 3 h after the transient 90 min the middle cerebral artery occlusion. The rats were allowed to survive for from 1 day to 2 months after stroke and the migration and survival of transplanted MSC were examined with fluorescence microscopy.
Results
The transplanted MSC were migrated in penumbra and a few cells had engrafted at 2 months without using the immunosuppressant and growth factor.
Conclusions
These results suggest that MSC have relevant therapeutic potential in chronic phase after stroke.
ROLE OF TOLL-LIKE RECEPTOR 4-MYELOID DIFERENTIATION FACTOR 88 PATHWAY IN NEONATAL HYPOXIC-ISCHEMIC BRAIN INJURY
Xiaoyang Wang1, Chunxia Nie1, Kristina Eriksson2, Henrik Hagberg1,3, Carina Mallard1
1Perinatal Center, Department of Physiology, Goteborg University, Goteborg, Sweden, 2Department of Medical Microbiology and Immunology, Goteborg University, Goteborg, Sweden, 3Perinatal Center, Department of Obstetrics and Gynecology, Sahlgrenska Academy, Goteborg, Sweden
BACKGROUND AND AIMS Perinatal complications, which are often associated with infection, frequently result in brain injury with subsequent neurological deficits in children such as cerebral palsy. The innate immune system recognizes a broad spectrum of pathogens via specific receptors on immune cells, so called toll-like receptors (TLRs). Bacterial endotoxin lipopolysaccharide (LPS) activates the innate immune system through interaction with TLR-4 and mice deficient in TLR-4 has attenuated cell death following neonatal LPS-induced injury (Lehnardt et al, PNAS 2003; Lehnardt et al, J Neurosci. 2002) Recent advances in the understanding of TLRs, suggest that in addition to their function as receptors for infectious pathogens, they may also play a role in the response to endogenous ligands, including necrotic cells in the CNS. Activation of TLR4 may trigger several different down-stream pathways, including activation of interleukin 1 receptor-associated kinases (IRAK), tumor necrosis factor-alpha (TNF-α), c-Jun N-terminal kinases (JNK), nuclear factor kappa B (NF-κB) via either the myeloid differentiation factor-88 (MyD88)-dependent pathway, or alternatively, MyD88-independent pathway.
The aim of this project was to examine the effects of TLR4 and MyD88 on neonatal HI brain injury by using TLR4 (C3H/HeJ) and MyD88 deficient mice.
METHODS 1) C3H/HeJ (TLR4 gene deficient) and C3H/HeN (controls) mice were bred under the same conditions. At postnatal day (PND) 9, C3H/HeJ and C3H/HeN mice were subjected to left carotid artery ligation and 10% O2 for 40 minutes in the same chamber. Mice were sacrificed at 3 days after HI on PND12. 2) Littermates of mixed genotype (MyD88 deficient (MyD88 KO), heterozygotes (Het) and wild type (WT)) were subjected to HI on PND9 and sacrificed at 3 days after HI on PND12 as described above. Brain sections were stained with microtubule protein-2 (MAP-2) and the infarct volume and the total tissue loss was calculated by measuring the MAP-2 stained negative and positive areas.
RESULTS 1) TLR4 gene deficient mice did not demonstrate any differences in the brain injury compared with control mice. The brain injury score was 1.33 ± 0.30 (n=21) vs 1.47 ± 0.29 (n=19) in C3H/HeJ vs C3H/HeN mice (p=0.74). 2) MyD88 deficiency did not result in significant overall differences in brain injury compared with the WT mice after HI. The total tissue loss was 15.14 ± 1.03 mm3 in MyD88 KO mice (n=19) vs 17.64 ± 1.14 mm3 in WT mice (n=25) (p=0.14). However, a more detailed analysis of the brain damage revealed a gender dependent difference in injury in MyD88 deficient mice. Female mice lacking the MyD88 gene showed significantly less brain injury compared with female wild type mice The total tissue loss was 15.28 ± 0.92 mm3 (n=6) in MyD88 KO mice vs 20.07 ± 1.32 mm3 (n=14) in WT mice (p=0.037).
CONCLUSIONS The TLR4-MyD88 dependent pathway does not play a critical role in HI induced brain injury in neonatal mice. However, the role of MyD88 in HI brain injury in female mice, need to be further investigated.
THE NEUROPLASTICITY POTENTIAL OF MELATONIN FOLLOWING TRANSIENT FOCAL CEREBRAL ISCHEMIA IN RATS
Yi-Hua Wang1, E-Jian Lee2, Jian-Ping Wang3, Shur-Tzu Chen1
1Department of Cell Biology and Anatomy, National Cheng Kung University Medical School, Tainan, Taiwan-R.O.C., 2Neurophysiology Laboratory, Neurosurgical Service, Department of Surgery, National Cheng Kung University Medical Center and Medical School, Tainan, Taiwan-R.O.C., 3Department of Life Sciences, National Cheng Kung University School, Tainan, Taiwan-R.O.C.
Introduction: Melatonin (N-aceyl-5-methoxytryptamine) is a well-known, potent free radical scavenger and an antioxidant (1)–(2). It is known that melatonin reduces infarct volumes and enhances neurobehavioral and electrophysiological recoveries following transient middle cerebral artery (MCA) occlusion and reperfusion in rats (3). In this study, we examined the melatonin's potential for neuroplasticity at 1, 7 and 28 days after reperfusion in rats subjected to MCA occlusion for 1 hour.
Methods: Adult male Sprague-Dawley rats, weighing 200-260 gm, were subjected to intraluminal MCA occlusion for 60 minutes. Local cerebral blood perfusion and physiological parameters were measured. Melatonin (5 mg/kg) or vehicle was given intraperitoneally at the commencement of reperfusion. Brain was sectioned and stained using the Golgi-Cox procedure (4), and spines density was quantified in the second-, third-order basilar dendrites of the pyramidal neurons in the ischemic core (layer II-III), penumbra (layer III-IV), and layer V-VI of forepaw and hindpaw areas in the ischemic hemisphere, as well as in the layer V-VI of the contralateral intact brain.
Results: At 7 days of reperfusion, our data indicate that melatonin did not affect core temperature, local cerebral blood flow or other physiological parameters. Melatonin treatment, however, significantly enhanced dendritic spines density in the second-, third-order basilar dendrites of the pyramidal cells. As compared to the vehicle-treated controls, in the ischemic core of the ispilateral (25±20% vs. 2±0% and 32±23% vs. 2±0%, P<0.05, respectively); in the penumbra of the ispilateral (41±6% vs. 21±5% and 44±8% vs. 25±10%, P<0.001, respectively); in the layer V-VI of the ispilateral (59±5% vs. 28±8% and 74±14% vs. 37±11%, P<0.001, respectively); in the layer V-VI of the contalateral (50±6% vs. 43±6% and 91±3% vs. 84±4%, P<0.05, respectively). In addition, melatonin improves neurobehavioral outcome at various time points measured following transient focal cerebral ischemia.
Conclusion: Melatonin has the potential to improve neurobehavioral outcome after stroke via upregulating the dendritic spine density in the residual cortical neurons in the ischemic hemisphere as well as in the non-injured, intact brain. The findings support melatonin's potential for decreasing ischemic brain injury in the territory at risk of ischemia and also suggest that it may have a beneficial role of neuroplasticity for the contralateral, intact brain in the filed of ischemic stroke.
CYCLOPHILIN A PARTICIPATES IN THE NUCLEAR TRANSLOCATION OF APOPTOSIS INDUCING FACTOR IN NEURONS AFTER CEREBRAL HYPOXIA ISCHEMIA
Changlian Zhu1,2, Xiaoyang Wang2,3, Zhiheng Huang1,2, Jianfeng Gao1,2, Martha Neagu4, Michael Nilsson1, Peter Eriksson1, Henrik Hagberg3, Jeremy Luban4, Guido Kroemer5, Klas Blomgren1,6
1Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, Goteborg University, Goteborg, Sweden, 2Department of Pediatrics, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China, 3Perinatal Center, Institute of Neuroscience and Physiology, Goteborg University, Goteborg, Sweden, 4Institute for Research in Biomedicine, Bellinzona, Switzerland, 5INSERM, Unit ≪ Apoptosis, Cancer and Immunity ≫, Institut Gustave Roussy, Villejuif, France, 6Department of Pediatric Oncology, The Queen Silvia Children's Hospital, Goteborg, Sweden
Cyclophilin A (CypA) has earlier been demonstrated to cooperate in vitro with apoptosis-inducing factor (AIF) in apoptosis-associated chromatinolysis. Nine-day-old wild type (WT), CypA+/− or CypA-/− mice were subjected to unilateral cerebral hypoxia-ischemia (HI). The infarct volume after HI was reduced by 46.7% (p=0.0089) in CypA-/− mice compared with their WT littermates. Nuclear translocation of AIF in ischemic neurons was impaired in the absence of CypA. Conversely, nuclear translocation of CypA was prevented in AIF-deficient harlequin (Hq) mice. Immunoprecipitation of AIF revealed co-precipitation of CypA only in the injured, ischemic tissue. This indicates that nuclear translocation of AIF and subsequent chromatinolysis requires binding of CypA, providing an attractive model where two proteins normally residing in separate cellular compartments, upon the appropriate stimulus, can bind to each other and execute cell death mechanisms they would not be capable of doing on their own. This is the first in vivo study to our knowledge demonstrating that CypA deficiency affords neuroprotection in vivo.
PS1 M233L AND H146R COMMONLY PRESENTING SEVERE DEMENTIA, PSYCHIATRIC SYMPTOMS AND MOTOR DEFICITS BUT DIVERSE PROGRESSION OF DETERIORATION
Masaki Ikeda1, Kimie Yonemura2, Etsuro Matsubara3, Tamao Taukie4, Koichi Ishiguro5, Noboru Oriuchi6, Ryozou Kuwano4, Yasuo Harigaya7, Koichi Okamoto1
1Department of Neurology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan, 2Department of Psychiatry, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan, 3National Institute for Longevity Science, Obu, Aichi, Japan, 4Genome Science Branch, Center for Bioresource-Based Researches, Brain Research Institute, Niigata University, Niigata, Niigata, Japan, 5Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan, 6Departments of Diagnostic Radiology and Nuclear Medicine, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan, 7Department of Neurology, Maebashi Red Cross Hospital, Maebashi, Gunma, Japan
Background: Presenilin-1 (PS1) is most frequent causative gene in familial Alzheimer's disease (FAD). We examined possible causative genes of two Japanese FAD families that have four diseased individuals from three generations respectively.
Objectives: We disclose causative gene mutations of this family, and elucidate the clinical and radiological features.
Methods: We analyzed gene sequences of PS1, tau and ?-synuclein as causative genes of independent two FAD families (S Family and O Family). We also analyzed the correlation between temporal profiles of neurological symptoms and radiological findings of MRI/CT and SPECT.
Results: Two siblings in S family had common clinical phenotype characterized by several neurological symptoms (dementia, apraxia, agnosia) and psychiatric symptom (apathy), with severe motor deficits since onset of the disease. PS1 M233L mutation was confirmed in these two cases. A proband of O family presented dementia and apathy, with rigid-bradykinetic parkinsonism with abnormal posture like forward flexion and dropped head. PS1 H163R was confirmed in this case. MRI of two siblings in S family showed that the temporo-parietal lobes and hippocampus were atrophied at the initial stage, then atrophies of the frontal lobe and basal ganglia were followed. SPECT showed low CBF in right parietal lobe. Phosphorylated tau199 in CSF increased slightly, while Abeta42 in CSF was very low. MRI of the proband of O family showed severe temporal atrophy, and fronto-temporal atrophy, corresponding to low CBF in SPECT.
Conclusions: Three FAD patients with PS1 mutation of M233L and H163R showed common clinical phenotype of dementia and apathy corresponded to the frontal and temporal lobe atrophies. However, divergences of progression processes in deterioration of neuropsychiatric symptoms exist between two FAD families. This suggested that PS1 mutation might possess some pathological functions to neurodegeneration in certain areas of FAD brains in addition to A? accumulation and NFT appearance.
FOCAL METABOLIC IMPAIRMENT AND GLOBAL ATROPHY IN MILD ALZHEIMER'S DISEASE
Stephen Francis Carter1, D.J. Coope1, M. Lambon Ralph2, A. Burns3, K. Herholz1
1Wolfson Molecular Imaging Centre, The University of Manchester, Manchester, UK, 2The School of Psychological Sciences, The University of Manchester, Manchester, UK, 3Division of Psychiatry, The University of Manchester, Manchester, UK
Background
Brain atrophy, especially of the medial temporal lobe, is a common feature of Alzheimer's disease (AD). Reduced glucose metabolism, as measured by FDG PET, is usually most pronounced in neocortical association areas. The relation between atrophic and metabolic changes is unclear at present, although it is commonly thought that metabolic impairment precedes atrophy. The high-resolution research tomograph (HRRT, Siemens/CTI) provides a spatial resolution of 2.5 mm FWHM which allows the assessment of cortical glucose metabolism with much less partial volume effects than standard PET scanners. We therefore used this instrument to address the issue of the relation between atrophic and metabolic changes in normal controls and AD.
Subjects and methods
We measured FDG uptake (20-60 min after i.v. injection of 250-370 MBq FDG) in seven healthy age matched controls (mean age 69 ± 9 yrs, 4/3 male/female, MMSE ? 29) and one AD patient with generalised brain atrophy (age 78, male, MMSE 28) under resting conditions with eyes closed. All subjects underwent a detailed neuropsychological assessment to determine cognitive status. T1-weighted MR and 18F-FDG PET images were acquired on 3T and HRRT scanners respectively. FDG scans were normalised to same global average. MR and PET images were co-registered and an old age MR template was created by spatially normalising all the T1 MR scans to the ICBM single subject template and averaging the scans. The individual scans were then coregistered to the new template and these transformations were reapplied to the individual PET images, thus creating a FDG PET reference data set and template. In a case study design ROIs were placed within the hippocampus, posterior cingulate cortex (PCC) and primary motor cortex (PMC) to compare the whole brain mean normalised FDG uptake in the controls to the same regions in the AD patient to investigate differences using t-statistics. In addition a t-map was created comparing the patient to the reference image data set.
Results
The AD patient had a significantly reduced FDG uptake in the left hippocampus (t(6) = 7.27, p = .001) and PCC (t(6) = 3.10 p = .05) and there was a relative increase in the PMC which was used as a control region. In the hippocampus atrophy corresponded with metabolic impairment. Both, PCC and PMC were affected similarly by global atrophy, but metabolic impairment was found in PCC only.
Conclusion
This pilot data demonstrates that when using combined high resolution modalities significant AD related changes can be detected at an early stage. In spite of widespread global atrophy metabolic impairment may appear focally only.
DIFFERENTIAL DIAGNOSIS BETWEEN IDIOPATHIC PARKINSON'S DISEASE AND MULTIPLE SYSTEM ATROPHY USING SPM
Kyung Hoon Hwang1, Nam-Bum Kim1, Chanmo Goo1, Wonsick Choe1, Uhn Lee2
1Department of Nuclear Medicine, Gachon University Gil Medical Center, Incheon, South Korea, 2Department of Neurosurgery, Gachon University Gil Medical Center, Incheon, South Korea
Background
A differential diagnosis between Idiopathic Parkinson's Disease (IPD) and Multiple System Atrophy (MSA) is clinically difficult. We analyzed the difference in glucose metabolism between IPD and MSA using SPM (statistical parametric mapping).
Methods
This study included 7 IPD patients (age range: 47-77 yr), 3 MSA patients (age range: 57-65 yr) and 11 healthy controls (age range: 33?68 yr) who underwent F-18 FDG PET.
Using SPM2 (Statistical parametric Mapping 2, Wellcome Department of Cognitive Neurology, London, UK) software, all the images were spatially normalized into the standard template, smoothed with 6 mm FWHM isotropic Gaussian kernel. The count of each voxel was normalized versus the total count for the brain (proportional scaling in SPM) to remove global cerebral blood flow differences between the individuals.
Images of IPD were compared with those of healthy controls and MSA patients in a voxel wise manner using SPM2 (uncorrected p < 0.01).
Results
The IPD patients were found to have a significant glucose hypermetabolism in comparison with the healthy controls in both basal ganglia, thalami, cerebellar areas and decreased metabolism in motor cortical areas. The MSA patients demonstrated diffusely decreased cerebral glucose metabolism in both both cerebellar areas compared to the IPD patients.
Conclusion
Although the number of patients studied is small, SPM analysis may be useful in differential diagnosis of patients with Idiopathic Parkinson's Disease and Multi-System Atrophy. Further studies including more patients may be warranted
SERIAL CHANGES ON DWI IN PATIENTS WITH TRANSIENT GLOBAL AMNESIA
Hiroki Ueno1, Hiromitsu Naka2, Tomohiko Ohshita1, Yukari Imon1, Shinichi Wakabayashi3, Masayasu Matsumoto1
1Department of Clinical Neuroscience and Therapeutics, Hiroshima University, Graduate School of Biomedical Sciences, Hiroshima, Japan, 2Department of Neurology, Suiseikai Kajikawa Hospital, Hiroshima, Japan, 3Department of Neurosurgery, Suiseikai Kajikawa Hospital, Hiroshima, Japan
Background and aims: The etiology of transient global amnesia (TGA) is not well understood. MR studies, including studies using diffusion-weighted imaging (DWI), have been used to investigate the pathophysiology of TGA, and focal hippocampal lesions have been detected in some studies. The aim of this study was to investigate serial changes in MR images from patients with TGA.
Methods: In seven TGA patients, serial MRI scans (from the day to several days of the onset of symptoms) using a 1.5-T MR unit were prospectively evaluated.
Results: In four patients, the duration of TGA was over 12 hours. Three of those patients, showed small punctate hippocampal hypersensitivity with decreased ADC values on DW images (right side, n = 2; left side, n = 1). These lesions were detected in the postacute phase (a time window of 24?48 hours after the onset of symptoms). In follow-up studies performed several days after the onset of symptoms, DWI lesions had disappeared in the chronic phase (7-10 days after the TGA episode).
Conclusion: The delayed hippocampal lesion on DW images with 1.5-T MRI in patients with TGA appears to be associated longer duration of symptoms, to persist for a several days and to disappear in the chronic phase.
PEDUNCULAR HALLUCINOSIS
Takahito Miyazawa
Department of Neurosurgery, National Defense Medical College, Tokorozawa, Saitama, Japan
?Background and aims]
The purpose of this paper is to clarify the clinical characteristics and pathomechanism of Peduncular Hallucinosis (PH).
?Methods]
I analyzed 38 previously reported cases of PH in the literature, in which the lesions responsible for the PH were clarified. Representative cases of PH will be presented at the meeting.
?Results]
The causal pathology of PH was classified into brain tumors (tumor; 12), brainstem infarction (BI; 7), subarachnoid hemorrhage (SAH; 6), and brainstem hemorrhage (BH; 13). The localization in 32 cases, the exceptions being those of SAH was in the thalamus (6.2%), midbrain (31.2%), pons (37.5%) and midbrain-pons (2.5%). PH was not necessarily confined to elderly patients. The sex ratio (man/woman) was 20/18. The most common time for PH seems to be the evening. The cause of PH does not necessarily have a close relationship with the timing of withdrawal of the hemorrhage and edema. In 10 cases of tumor in which PH appeared preoperatively, the hallucination disappeared after removal of the tumor in all. For SAH, the duration of PH was from 3 to 12 days, which is interestingly consistent with that of symptomatic vasospasms. For BI and BH, although the hallucination usually disappeared in parallel with withdrawal of the causal pathology, PH continued even at the chronic stage in some cases. There was no particular difference in hallucination contents with the causal pathology of PH. The most frequent hallucinations involved people or figures, i.e., approximately 80%. Then, animals (dogs, cats, snakes, etc.), insects, and fishes were also frequently involved. Sleep abnormality was observed in 27 cases (71%). Agitation was observed in 18 cases (47.3%), in all of which it was accompanied by sleep abnormality. Increased intracranial pressure was observed in 8 (21%). Hydrocephalus was observed in 4 (10.5%). Prior visual impairment was recorded in 7 (18.4%).
?Conclusioins]
On the analysis of 38 cases with PH, it became clear that PH occurs not only in focal brainstem intrinsic stroke lesions, but also in extrinsic lesions like subarachnoid hemorrhage and brain tumor compression. Reciprocal connections between the serotonergic and cholinergic systems, especially between the raphe and the pedunculopontine tegmental nucleus, may be impaired in the reticular formation. Surprisingly, these lesions do not directly involve the retino-geniculo-cortical system, which is generally considered to be required for the appreciation of complex forms and color. The association between sleep-awake disruption and PH may occur because lesions affecting the raphe nucleus cause a transient increase in ponto-geniculate-occipital spikes, which may be related to reorganization and plasticity. Although increased intracranial pressure and hydrocephalus were considered as insults to the “hallucination-suppression mechanism?, they do not seem to be significant as a cause of PH. The clinical manifestations of PH are often mistaken for those of postoperative “altered sensorium? or “psychosis?. Medical doctors who manage organic brain lesions should take PH into consideration.
OBESITY DETERIORATES THE OUTCOME OF THROMBOTIC ISCHEMIC STROKE
Nobuo Nagai, Roger H. Lijnen
Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
Background and Aims: The objective of this study was to test the hypothesis that obesity, a major risk factor for development of cardiovascular disease, may affect the expansion of ischemic stroke.
Methods: Ischemic stroke and associated intracerebral hemorrhage (ICH) were studied in mice with nutritionally induced or genetically determined (leptin deficient) obesity and their lean counterparts. The ischemic stroke was induced by photochemical middle cerebral artery (MCA) occlusion, and the occlusion time after was measured by direct observation under the microscope. The infarct size and the hemorrhagic volume were quantified 24 hours after MCA occlusion.
Results: In a first study, five weeks old male C57Bl/6 mice were kept on standard fat diet (caloric value of 10.9 kJ/g) or on high fat diet (20.1 kJ/g) for 15 weeks, resulting in significantly higher (p<0.05) total body weights for the obese (43 ± 3.2 g; mean ± SD n = 13) as compared to the lean mice (29 ± 3.6 g; n = 12). High fat diet feeding also resulted in significantly elevated cholesterol levels (270 ± 64 mg/dl versus 100 ± 16 mg/dl; p<0.05). The MCA occlusion time was significantly shorter in the obese as compared to the lean mice (160 ± 34s versus 200 ± 16 s; p < 0.05), and the infarct size was significantly larger (32 ± 6.4 mm3 versus 24 ± 2.8 mm3; p < 0.05). Expansion of the damaged zone was observed both in the cortical and subcortical regions. Furthermore, ICH was enhanced in obese as compared to lean mice (4.9 ± 2.8 mm3 versus 2.7 ± 1.1 mm3; p < 0.05).
In a second study, five weeks old male leptin deficient (ob/ob) and corresponding wild-type (WT) mice (genetic background 100 % C57Bl/6) were kept on standard fat diet for 15 weeks, resulting in significantly higher (p<0.05) total body weights for the genetically obese ob/ob (53 ± 5.6 g; n =9) as compared to the WT mice (29 ± 2.3 g; n = 12). The MCA occlusion time was significantly shorter in the ob/ob as compared to the WT mice (170 ± 34 s versus 210 ± 19 s; p<0.05), and the infarct size was significantly larger (30 ± 8.6 mm3 versus 23 ± 5.2 mm3; p < 0.05), mainly as a result of expansion in the cortical region. ICH was also enhanced in ob/ob as compared to WT mice (5.0 ± 2.8 mm3 versus 2.7 ± 1.2 mm3; p < 0.05).
Conclusions: In mice with either nutritionally induced or genetically determined obesity, MCA occlusion causes more extensive ischemic damage and ICH than in the corresponding lean mice. These data suggest that obesity related prothrombotic tendency may deteriorate the outcome of thrombotic ischemic stroke.
VOXEL-WISE CORRELATION OF BINDING POTENTIAL OF PITTSBURGH COMPOUND B TO CBF IN OLDER SUBJECTS AS REVEALED BY JOINT FREQUENCY HISTOGRAMS
Hiroto Kuwabara1, Jitka Sojkova1,2, Anil Kumar1, James Brasic1, Mohab Alexander1, Daniel Holt1, Robert Dannals1, Dean F. Wong1, Susan M. Resnick2
1Department of Radiology, Johns Hopkins University, Baltimore, MD, USA, 2National Institute On Aging, Baltimore, MD, USA
Background and Aims: Although amyloid deposition is negatively correlated with regional glucose metabolism in patients with dementia, little is known about associations between amyloid deposition and functional activity in pre-morbid stages. We hypothesized that amyloid accumulation is proportional to functional demand in normal aging until exceeding a threshold. Accordingly, we investigated the correlation between binding potential (BP) of PIB, an estimate of amyloid deposition, and resting CBF, an estimate of functional demand, using joint frequency histogram analysis of gray matter voxels.
Methods: Thirty-two participants of the Baltimore Longitudinal Study of Aging (age: 80.3 +/−6.2) had one [11C]PIB PET scan and one static [15O]water scan. Participants had normal cognition (n=26) or mild cognitive impairment by clinical dementia rating of 0.5 (n=6). BP maps of PIB scans were constructed by the bolus-plus-infusion transformation (BPIT; Kuwabara et al., 2002). CBF images were normalized (nCBF) to the cerebellum value (50 ml/mg/min). BP and CBF maps were coregistered by SPM2 and smoothed with a Gaussian kernel (8 mm FWHM). Gray voxels defined on MRI were transferred to PET space by SPM2. PIB-BP (cutoff: −0.7 and 1.5; 100 bins; x-axis) versus CBF (cutoff: 30 and 80 ml/mg/min; 100 bins; y-axis) joint frequency histograms were constructed (voxel value = number of BP-CBF voxels within the histogram voxel limits).
Results: The figure shows typical joint histograms. On visual inspection, one group of subjects displayed one long cluster only (Type T for ‘tight’ correlation; n=20) while another showed a broader distribution (Type D for ‘diffuse’ correlation; n=12). Some Type T subjects displayed a small ‘hub’ of negative correlation at the highest end of PIB-BP (Type T1; n=12) while others did not (Type T0; n=8). Voxel-wise regression analysis confirmed positive slopes in all cases (range 9.5 ? 42.3; F>8120; p<0.001). Individuals with mild cognitive impairment showed a significant tendency toward the diffuse pattern of correlation (p < 0.05).
Conclusion: We demonstrate a positive voxel-wise correlation of PIB-BP with resting CBF in normal and mildly impaired older subjects except for a small ‘hub’ of negative correlation in some individuals. The positive correlation suggests the possibility that amyloid accumulation is greater in functionally more active brain tissue, reflecting a compensatory increase in CBF proportional to amyloid deposition. The tendency for cognitively impaired individuals to show more diffuse associations suggests a failure of these compensatory processes. The clinical significance of these patterns of association will be determined through continued longitudinal follow-up.
Grant support: Intramural Research Program, NIA, NIH; N01-AG-3-2124; K24 DA00412 (DFW)
COMPARISON OF INVERSED Z-SCORE IMAGES OF INTERICTAL PREOPERATIVE FDG-PET AND POSTOPERATIVE MRI IN NEOCORTICAL EPILEPSY PATIENTS
Etsuko Imabayashi, Ichiei Kuji, Hiroshi Matsuda
Department of Nuclear Medicine, Saitama Medical University Hospital, Saitama, Japan
Background and aims: Usefulness of Z-score images of interictal FDG-PET for detecting eileptogenic foci has been already reported. The Z-score images are spatially normalized to a standardized stereotactic space based on the Talirach and Tournoux atlas. However, malformation which is frequently seen in epileptic brain is not suitable for spatial normalization. In this study, using inversed parameter, we re-warped Z-score images to the original brain shape and precisely compared the operated areas with glucose metabolism decrease precisely.
Methods: 14 neocortical epilepsy patients were studied, 9M, 5F (mean age 18, range 2-35) yrs. 13 had focal cortical dysplasia and 1 had astrocytoma. Prognosis of patients was Engel I (Free of disabling seizure) or II (Rare disabling seizures) after 2-year follow up after operation. FDG-PET scan were obtained and nomalized using SPM2. Then Z-score images are obtained from this normalized patient's image and normal data base consisted from 31 normal control subfjects using a soft ware program of eZIS (easy Z-score imaging system). Then using SPM2, the inverse parameter was calcureted from normalising parameter and it was applied to the obtained Z-score image to rewarp to the original brain shape. This inversed Z-score image was corregistered to each patient's MRI which had been previously corregistered to FDG-PET image. Then this inversed Z-score images was compared with operated area on the MRI obtaied after operation.
Results: In 11 patients, operated areas corresponded to abnormal Z-score areas. Among them, in 4 patients, abnormal Z score areas exist in peripheral areas of operated site. In the other 3 patients, abnormal Z-score areas are more extensive than operated areas. They are the 1 oldest (35 yrs) and the 2 yongest (2 and 3 yrs) patients and all of them underwent corticectomy.
Conclusions: Inversed Z-score images are useful and necessary for evaluation of epileptogenic foci on functional images. They make it possible to inspect images anatomically at gyrus level for each individual. Prospective studies are needed to assure their anatomical accuracy and relationship between electrical discharge and propagation and metabolic changes.
THERAPEUTIC BENEFIT OF INTRATHECAL INJECTION OF INSULIN-LIKE GROWTH FACTOR-1 IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Kazunori Miyazaki, Isao Nagano, Hristelina Ilieva, Mito Shiote, Tetsuro Murakami, Masataka Yokoyama, Mikio Shoji, Koji Abe
Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan
Background. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by selective motor neuron loss and death within 3–5 yaers after onset. Although most of the cases occur sporadically, 10% of these are familial. 10–20% of familial cases are associated with missense mutation or small deletions in the Cu/Zn superoxide disumutase (SOD1) gene, one of the main free radical scavenging enzymes that protects cells against oxidative stress. There are many hypothesis about the cause of this disease. However, the mechanism were still obscure and therapeutic strategies are required. Insulin-like growth factor (IGF)-1 has been shown to have a protective effect on motor neurons both in vitro and in vivo, but has limited efficacy in patients with ALS when given subcutaneously. We hypothesized that direct infusion of IGF-1 into the intrathecal space could improve the drug delivery and may strengthen its efficacy against motor neuron death in ALS.
Material and method. To examine the possible effectiveness of IGF-1 in a mouse model of familial ALS, transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) with a G93A mutation were treated by continuous IGF-1 delivery into the intrathecal space of the lumbar spinal cord. Dissolved IGF-1 was give 1 mg/kg body weight/day as a high-dose treatment (n=6) or 100 µg/kg body weight/day as a low-dose treatment (n=6). Five mice were treated with vehicle (aCSF) as controls. Continuous intrathecal infusion into the lumbar spinal cord region was continued at 140 days of age to the end stage of the disease. Rotarod test and wheel-running test was performed for behavioral studies. For histological studies, the region of the spinal cord spanning L4?L5 was segmented and stained for motor neuron counting. For immunohistochemistry, frozen transverse sections were treated with polyclonal antibodies against p-AKT, p-ERK and p-bcl-2. Immunoblot analysis was performed to detect p-AKT and anti-pERK.
Result. We found that the intrathecal administration of IGF-1 improved motor performance, delayed the onset of clinical disease, and extended survival in the G93A transgenic mice. Furthermore, it increased the expression of phosphorylated Akt and ERK in spinal motor neurons, and partially prevented motor neuron loss in these mice.
Discussion and conclusion. Taken together, the results suggest that direct administration of IGF-1 into the intrathecal space may have a therapeutic benefit for ALS. However, it is uncertain whether this treatment is beneficial in human ALS, since the therapy was started before the Tg mice developed the disease. In addition, it is possible that Tg animal models may not reflect the sporadic form of human ALS. Nevertheless, this treatment with IGF-1 could be a new therapeutic strategy for the treatment of human ALS, although the optimal dose and timing for humans remain to be determined in the future.
1H MRS IDENTIFIES LACTATE RISE IN THE STRIATUM OF MPTP-TREATED C57BL/6 MICE
Hisashi Kitagawa1, Atsushi Mori1, Keiko Koga2, Satoshi Ohashi1, Naoki Kurihara1, Makoto Ishikawa2, Yasuhide Mitsumoto3, Masami Nakai1
1Research Unit for Neurological Diseases, Second Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan, 2Laboratory of Bioenergetics Research, Tokushima Research Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan, 3Department of Alternative Medicine and Experimental Therapeutics, Faculty of Pharmaceutical Sciences, Hokuriku University, Tokushima, Japan
Mitochondrial dysfunction has been implicated in the death of nigrostriatal dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated experimental models of Parkinson's disease (PD). Here we utilized proton magnetic resonance spectroscopy (1H MRS) to identify changes in energy metabolism in the striatum of MPTP-treated C57BL ? 6 mice. Remarkable increases in lactate ? creatine (Lac ? Cr) ratio were observed at 2 h and then quickly returned to about the basal level by 7 h after injection of MPTP. Neurochemical and Western blot analyses revealed that dopamine contents and protein levels of tyrosine hydroxylase and dopamine transporter in the striatum were profoundly decreased at 3 days after MPTP treatment. Pretreatment with deprenyl, a monoamine oxidase B inhibitor, or GBR-12909, a dopamine uptake inhibitor, almost completely attenuated both the increases in striatal Lac ? Cr ratio and the subsequent loss of dopaminergic nerve terminals in MPTP-treated mice. The present study indicates that 1H MRS is a sensitive measure of biochemical alterations of the brain in a mouse model of PD, and further shows that the increases in striatal Lac ? Cr ratio induced by MPTP may be associated with mitochondrial energy crisis, followed by dopaminergic neurotoxicity.
DIFFERENT REGIONAL CEREBRAL BLOOD FLOWS IN ALZHEIMER'S DISEASE WITH/WITHOUT VISUAL HALLUCINATION
Junghee Kim1,3, Ho-Chun Song1, Seong-Min Choi2, Byeong-Il Lee1, Byung Chae Kim2, Ki Hyun Cho2, Taejin Park3
1Department of Nuclear Medicine, Chonnam National University Medical School, Gwangju, South Korea, 2Department of Neurology, Chonnam National University Medical School, Gwangju, South Korea, 3Department of Psychology, Chonnam National University, Gwangju, South Korea
Background: Visual hallucination is a common behavioral symptom in Alzheimer's disease (AD) and associated with institutionalization and mortality. To clarify which neural substrate participate in visual hallucination in AD, we evaluated the differences of regional cerebral blood flow (rCBF) between AD with and without visual hallucination (as determined by the Neuropsychiatric Inventory).
Methods: Three AD patients with visual hallucination (AD+VH group, 1 males, 2 females; mean age 75.0±6.2 years) and 9 AD patients without visual hallucination (AD-VH group, 3 males, 6 females; mean age 70.3±7.2 years) were studied. Mini-Mental State Examination (MMSE), Global Deterioration Scale scores and Clinical Dementia Rating were evaluated in all AD patients. Brain perfusion was measured by 99mTc-ECD brain perfusion SPECT. All images of two AD subgroups were processed using spatial normalization and smoothing functions implemented in statistical parametric mapping (SPM) operating under Matlab 6.1 and identified brain regions with hypoperfusion compared with 11 healthy normal control subjects.
Results: There were no significant differences in age, sex, Mini-Mental State Examination, or Global Deterioration Scale scores between two groups. Clinical Dementia Ratings of all AD patients were stage 2. Compared with the healthy control subjects, AD-VH group had significantly lower rCBF in the left superior and bilateral medial frontal regions. AD+VH group had significantly decreased rCBF in the left amydgala, bilateral cuneus, left lingual gyrus, and right caudate head (with exception of same area of hypoperfusion; uncorrected p<0.01).
Conclusions: This preliminary study showed significant differences in rCBF between healthy control subjects and AD with or without visual hallucination patients. Visual hallucination in Alzheimer's disease has been associated with hypoperfusion in the amygdala and cuneus. Because these regions are important for emotion and visual perception, visual hallucination in AD might be related to altered emotion and decreased visual perception.
ANALYSIS OF AMYLOID ANGIOPATHY IN TG2576 TRANSGENIC MOUSE MODEL OF ALZHEIMER'S DISEASE
Takeshi Kawarabayashi1, Yusuke Seino1, Yasuhito Wakasaya1, Tamaki Kimura1, Chieko Suzuki1, Masahiko Tomiyama1, Etsuro Matsubara2, Mikio Shoji1
1Department of Neurology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki, Aomori, Japan, 2Department of Alzheimer's Disease Research, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan
Background and aims: Cerebral amyloid angiopathy (CAA) affects about 30% of the normal elderly and over 90% of those with Alzheimer's disease (AD). The most frequent clinical manifestation is lobar cerebral hemorrhage, but CAA can also cause cerebral infarction, ischemic leucoencephalopathy and CAA-related inflammation. Though many therapies for AD are tested, no therapy for CAA has been developed. By immunotherapy against amyloid beta protein (Abeta), CAA gets worse while parenchymal Abeta decreases. CAA was evaluated in the Tg2576 mouse model of AD if it reproduces features of CAA.
Methods: Tg2576 mice(n=117, 2-33 month) was examined by immunostaining, electronmicroscopy (EM) and immunoEM.
Results: The all mice older than 10 month-old developed CAA in both meningeal and parenchymal blood vessels in the brain while core plaques were detected at 7–8 months. The Abeta in blood vessels was preferentially labeled by the BA-27 antibody to Abeta40. In EM and immunoEM, deposition of amyloid fibrils was detected in the media of vascular wall. Very old 33 month-old mouse showed the pathology that has close similarities to the CAA-related inflammation; infiltration of mononuclear cells around vessels with CAA, microhemorrhage, and racefaction.
Conclusions: The Tg2576 mouse can reproduce many features of CAA, and can be a model to examine CAA.
VARIABLE CLINICAL PHENOTYPES OF CEREBRAL AMYLOID ANGIOPATHY AND THEIR TREATMENTS
Shu-Ichi Ikeda
Department of Medicine, Neurology and Rheumatology, Shinshu University School of Medicine, Matsumoto, Japan
Background and aims: Cerebral amyloid angiopathy (CAA) is characterized by amyloid deposition within the walls of leptomeningeal and cortical blood vessels in the brains of elderly individuals. Amyloid fibril protein in this disorder consists of amyloid β protein (Aβ). CAA is known to cause two major clinical manifestations: intracerebral hemorrhage and diffuse white matter degeneration. However, the pathogenesis of the latter is still incompletely understood and any effective treatments have not been established for the CAA-related disorders.
Methods: We present 3 cases who had biopsy-proven Aβ type CAA and suffered from diverse clinical phenotypes, adding that two of them were successfully treated by large dose of corticosteroid.
Results: Case 1 was a 65-year-old woman who experienced intracerebral hemorrhages 4 times during 23 days but these serious strokes did not recur after the use of steroid pulse therapy and the following oral administration of predonisolone. Case 2 was a 69-year-old man who had developed dizziness, dementia and generalized seizure during the preceding 4 years. An initial CT and MRI showed bilateral symmetrical periventricular white matter lesions closely resembling those of Binswanger's disease. Subsequently, the lesions expanded slowing, involving a large area of the right cerebral hemisphere with an obvious mass effect. Brain biopsy revealed the presence of angiitis in the cortical vessels in addition to CAA and the former seemed to be responsible for his extensive white matter lesions. Case 3 was a 69-year-old woman who developed subacute onset encephalopathy. CT and MRI revealed diffuse edematous changes and her CSF disclosed xanthochromia. Her condition was temporarily relieved using large doses of dexamethazone but finally fell into decerebrate state, which was caused by communicating hydrocephalus. She underwent a V-P shunt operation and was again treated by dexamethazone. Her condition has been gradually improving.
Conclusions: Clinical phenotypes of CAA-related disorders are more heterogeneous than previously recognized. Corticosteroid seems to be effective for these conditions.
GENDER AND AGE ASSOCIATED DIFFERENCES AND CHANGES OF CEREBRAL GLUCOSE METABOLISM IN NORMAL HEALTHY POPULATIONS
Sang Heon Song1, Ihm Soo Kwak1, Seong-Jang Kim2, Yong-Ki Kim2, In-Ju Kim2
1Department of Internal Medicine Pusan National University Hospital, Busan, South Korea, 2Department of Nuclear Medicine, Pusan National University Hospital, Busan, South Korea
The present study used F-18 FDG brain PET and statistical parametric mapping analysis to examine the effects of gender and sex related changes in the cerebral glucose metabolism in normal healthy populations. In male, negative correlation was found in right temporopolar area, right orbitofrontal, both frontopolar area, both dorsolateral frontal gyri, left insula, both precentral gyri, right ventral posterior cingulate gyrus. Positive relationship was found in left claustrum, left thalamus, left paracentral lobule, right postcentral gyrus, left visual association cortex, right dorsal posterior cingulate cortex. In female, negative changes were found in left caudate body, both temporopolar areas, right dorsolateral prefrontal cortex, both orbitofrontal gyri, right caudate head, right superior frontal gyrus, right dorsolateral prefrontal cortex, right frontopolar area. Positive association was found in left subthalamic nucleus, superior temporal gyri, right superior frontal gyrus, right inferior temporal gyrus. We found regions of age and gender associated distinctive differences and changes of cerebral glucose metabolism that had not previously been identified any other functional neuroimaging methods.
ROSUVASTATIN SELECTIVELY PROTECTS CULTURED RAT CORTICAL NEURONS AGAINST OXYGEN-GLUCOSE DEPRIVATION
Ferenc Domoki1,2, Bela Kis1, Tamas Gaspar1, Ferenc Bari2, David W. Busija1
1Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC, USA, 2Department of Physiology, University of Szeged, Szeged, Hungary
Background and Aims: Protective effects of statins in different compartments of the neurovascular unit have not been examined completely. We determined whether rosuvastatin (RST) protects against oxygen-glucose deprivation (OGD) in primary rat cortical neuronal, astrocytic, and endothelial cultures.
Methods: Primary rat cortical neuronal, astrocytic, and endothelial cell cultures were obtained from E18 fetuses, 1-day and 2-week old pups, respectively. Cultures were treated with RST (10nM-5µM, treatment for 1–5 days) before exposure to OGD. OGD duration was chosen to result in ~50% cell death assessed with MTT/formasan and/or LDH release viability assay 24h after OGD. After RST and/or OGD treatments, cellular ATP content was determined with luciferase luminescence, and reactive oxygen species (ROS) production was detected with hydroethidine (HET, 5µM) fluorescence.
Results: RST did not prevent OGD-induced cell death in cultured astrocytes and endothelial cells. In contrast, RST (?2 days treatment) exerted dose-dependent, potent neuroprotection against OGD (Fig A, mean±SEM, n=8-32 for each data in Fig). RST (5 µM, 3d) reduced ATP to 59±2% of baseline levels, and resulted in unchanged baseline but reduced post-OGD ROS production (Fig B). RST-induced neuroprotection was dose-dependently antagonized by mevalonate (mev), geranyl-geranyl pyrophosphate (GGPP), but not by cholesterol (chol, Fig C, 3d RST-drug 5-10 µM coapplication, respectively). RST-induced ATP reduction was also reversed by both mevalonate and GGPP.
Conclusions: RST elicits metabolic stress selectively in neurons resulting in an OGD-resistant phenotype. The protection afforded by RST is indeed mediated by inhibition of HMG-CoA reductase but appears to be independent of cholesterol depletion. Instead, altered geranylgeranylation of neuron-specific proteins may play a role. The reduced ATP levels and ROS response to OGD after RST treatment suggest the involvement of mitochondria in the effector mechanism of protection against OGD.
Support: HL-50587, HL-65380, HL-77731, and OTKA T046531.
NUROPROTECTIVE EFFECTS OF OVER-EXPRESSING TISSUE INHIBITOR OF METALLOPROTEINASE TIMP1: CELL CULTURE AND IN VIVO STUDIES OF BRAIN TRAUMA AND ISCHEMIA
Emiri Tejima1, Shu-Zhen Guo1, Yoshihiro Murata1, Ken Arai1, Klaus van Leyen1, Michael J. Whalen2, Xiaoying Wang1, Yoichi Katayama3, Eng H. Lo1
1Neuroprotection Research Laboratory, Departments of Neurology and Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, 2Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, 3Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan
INTRODUCTION: Accumulating data suggest that matrix metalloproteinases (MMPs) are important mediators in the pathophysiology of acute brain injury. By degrading neurovascular matrix, MMPs may induce blood-brain barrier damage, edema and hemorrhage. By disrupting cell-matrix homeostasis, MMPs can induce anoikis-like cell death pathways. Here, we test the hypothesis that genetic over-expression of the endogenous tissue inhibitor of metalloproteinase TIMP1 is neuroprotective in vitro and in vivo. METHODS: For in vitro studies, primary cortical neuronal cultures were exposed to cellular hypoxia and treated with recombinant TIMP1. For in vivo studies, two brain injury models were used. Brain trauma was induced using controlled cortical impact. Transient and permanent focal cerebral ischemia was induced using intraluminal MCA occlusions. RESULTS: Neuronal cultures exposed to 6hours hypoxia and 18hours reoxygenation suffered 45% neurotoxicity. Addition of recombinant human TIMP1 (500ng/ml) significantly reduced neuronal death (22% neurotoxicity). In vivo studies confirmed these cell culture results. Over-expression of TIMP1 reduced MMP-9 activation after brain trauma and ischemia. At 7 days after brain trauma, lesion volumes were significantly decreased by TIMP1 over-expression (5.80±0.5 mm3 in transgenic mice vs 6.92±0.9 mm3 in wildtype mice. Neuroprotection was also observed in our models of focal cerebral ischemia. 24 h ischemic lesion volumes were significantly reduced in transgenic mice after transient (45.9±17.1 mm3) and permanent (108.1±17.5 mm3) ischemia, compared with wildtype mice (116.2±16.1 mm3 transient ischemia, 147.8±10.3 mm3 permanent ischemia). Correspondingly, blood-brain barrier injury as measured by Evans' blue leakage after transient ischemic was ameliorated in TIMP1 over-expressing transgenics (2227±1207 ng/hemisphere) compared with wildtype mice (4452±1136 ng/hemisphere). CONCLUSIONS: Our results demonstrate that genetic over-expression of the endogenous MMP inhibitor TIMP1 is neuroprotective against acute brain traumatic injury and focal cerebral ischemia. These results further confirm that MMPs are deleterious in brain pathology and may serve as valid therapeutic targets in brain injury and neurodegeneration. Supported in part by NIH grants R01-NS37074, R01-NS48422, R01-NS56548, and P50-NS10828.
SOLUBLE EPOXIDE HYDROLASE: SEX DIFFERENCES AND ROLE IN ESTROGEN-MEDIATED NEUROPROTECTION
Wenri Zhang, Jeffrey J. Iliff, Yazan K. Alhadid, Ardi Ardeshiri, Ines P. Koerner, Patricia D. Hurn, Nabil J. Alkayed
Department of Anesthesiology and Peri-Operative Medicine, Oregon Health and Science University, Portland, OR, USA
INTRODUCTION: In experimental models of stroke, females sustain smaller brain damage than males, in part due to the neuroprotective effects of estrogen. The precise mechanism of estrogen-mediated neuroprotection remains unknown. We previously identified P450 eicosanoids termed eopoxyeicosatrienoic acid (EETs) as important mediators of ischemic preconditioning and tolerance. We subsequently demonstrated that inhibition or gene deletion of EETs-metabolizing enzyme soluble epoxide hydrolase (sEH) reduces infarct size after middle cerebral artery occlusion (MCAO) in mice. In peripheral tissue, sEH expression is sexually dimorphic and regulated by sex hormones. Therefore, we tested the hypothesis that the protective effect of sEH inactivation is sex-specific and modulated by estrogen.
METHODS: Adult male and female C57/Bl6 wild type (WT) and sEH knockout (sEHKO) mice were subjected to 2-hour MCAO, and infarct size was measured at 22 hours of reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC). Male and female WT mice were also treated with sEH inhibitor 12-(3-adamantan-1-yl-ureido)- dodecanoic acid n-butyl ester (AUDA-BE, 10 mg/kg i.p.) or vehicle at reperfusion. Body temperature was kept within normal range, and cortical laser-Doppler perfusion was monitored to confirm vascular occlusion and reperfusion. The level of sEH expression was determined by Western blotting, and sEH activity was determined by measuring EETs metabolite 14,15-dihydroxyeicosatrienoic acids (14,15-DHETs), specifically produced by sEH, in plasma using ELISA. To determine the effect of estrogen on sEH expression, ovariectomized female rats (OVX) were implanted subcutaneously with pellets containing 17beta-estradiol (E2, 25 µg, 21-day release) for 7 days.
RESULTS: The level of sEH immunoreactivity (IR) in brain was higher in WT male compared to female mice (0.45·0.02 vs 0.34·0.03 relative to beta-actin, n=4 each, p=0.007), and was undetectable in male and female sEHKO mice (n=4 each). Strong sEH-IR was consistently detected in brains from OVX rats. Replacement with E2 reduced sEH protein by 38% (n=4 each, p=0.029). Plasma 14,15-DHET was higher in male vs. female mice [3908·458 (n=14) vs. 710·257 (n=11) pg/ml, p<0.001], and was reduced by AUDA-BE (n=10) in male mice by approximately 35% (p=0.04). The level of 14,15-DHET in male sEHKO mice was significantly lower than WT males [115·73 (n=10) vs 4179·482 (n=12), p<0.001].
Female WT mice sustained smaller infarcts after MCAO compared to male mice (23.9·7.9%, n=5 vs. 35.9·2.5%, n=5, p<0.05). The sex difference, however, was absent in sEHKO mice and mice treated with AUDA-BE. Infarct size was significantly smaller in sEHKO compared to WT male (16.0·6.4% vs. 35.9·2.5%, n=5 each, p<0.05), but not female mice (17.0·7.7% compared to 23.9·7.9, n=5 each, p>0.05). Infarct size was also smaller in AUDA- compared to vehicle-treated male (15.7·5.6% vs. 33.1·2.3%, n=5 each, p<0.05), but not female mice (28.1·6.0 % vs. 25.4·5.8 %, n=5 each, p>0.05).
CONCLUSION: We conclude that sEH is expressed at a lower level in young adult female vs male brain, which serves a beneficial role due to reduced breakdown of protective 14,15-EET, and may contribute to the protection against cerebral ischemia observed in females, and to the neuroprotective effect of estrogen.
POLYUNSATURATED FATTY ACIDS ARE CEREBRAL VASODILATORS VIA THE TREK-1 POTASSIUM CHANNEL
Nicolas Blondeau, Olivier Petrault, Valerie Giordanengo, Michel Lazdunski, Catherine Heurteaux
IPMC, CNRS/UNSA UMR6097, Valbonne, France
Vessel occlusion is the most frequent cause for impairment of local blood flow within the brain resulting in neuronal damage and is a leading cause of disability and death worldwide. Polyunsaturated fatty acids and especially a-linolenic acid (ALA) have been previously shown to considerably improve brain resistance against cerebral ischemia. The purpose of the present study was to evaluate the effects of polyunsaturated fatty acids and particularly ALA and docosahexanoic acid (DHA) on the cerebral blood flow and on the tone of vessels that regulate brain perfusion.
Our results show that ALA and DHA injections increased cerebral blood flow and induced vasodilation of the basilar artery but not of the carotid artery. The saturated fatty acid palmitic acid did not produce vasodilation. This suggested that the target of the polyunsaturated fatty acids effect could be the TREK-1 potassium channel. Indeed, we demonstrate the presence of this channel in basilar but not in carotid arteries and show that the basilar artery vasodilation and CBF increase produced by the polyunsaturated fatty acids and particularly ALA and DHA is abolished in TREK-1-/− mice.
Altogether our data indicate that TREK-1 activation elicits a robust dilation that probably accounts for the increase of cerebral blood flow induced by polyunsaturated fatty acids such as a-linolenic acid or docosahexanoic acid and that the selective expression and activation of TREK-1 in brain collaterals could play an important role in the protective mechanisms of polyunsaturated fatty acids against stroke providing residual circulation during ischemia.
PROTECTIVE MECHANISMS OF ANGIOTENSIN II AT1 RECEPTOR BLOCKER, CANDESARTAN, AGAINST CEREBRAL ISCHEMIA; IN VIVO AND IN VITRO STUDIES
Keiko Kitazato, Hao Liu, Masaaki Uno, Yasuhisa Kanematsu, Tetsuya Tamura, Kenji Yagi, Yoshiteru Tada, Atsuhiko Suzue, Shinji Nagahiro
Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
Backgroun and aims; to elucidate the mechanisms underlying the protective effects of candesartan against cerebral ischemia, we performed in vivo and in vitro studies
Methods; Normotensive Wistar rats were treated with 0.5 and 1.0 mg/kg candesartan for 2weeks and then subjected to 2-hr middle cerebral artery occlusion-reperfusion (MCAO-R). Human umbilical endothelial cells (HUVEC) in our in vitro experiments were stimulated with candesartan and angiotensin II (Ang II).
Results; compared to the control rats, blood pressure was significantly decreased in 1.0 mg/kg candesartan-treated rats (p<0.05). The cortical infarct volume at 0.5 and 1.0 mg/kg candesartan was decreased in a dose-dependent manner (p<0.05, p<0.01vs control, respectively) and the improved hypoxic status reflected by HIF-1alpha, the protection from oxidative damage indicated by 8-OHdG and the augmented expression of repair- and growth-associated proteins in their cortical penumbra were observed. Candesartan also increased eNOS mRNA level of MCA. In human umbilical endothelial cells, candesartan and angiotensin II (Ang II) increased eNOS protein buat affected NO availability differently. Ang II increased intracellular ROS and NO production. Candesartan inhibited these increase induced by Ang II and promoted the extracellular release of NO, suggesting that the increased NO by candesartan was available to restore endothelial function, while Ang II led to eNOS “uncoupling” to form superoxide.
Conclusions; Ours is the documentation that candesartan may exert protection against cerebral ischemia by several blood pressure-dependent and -independent mechanisms. Candesartan-induced restoration of endothelial function may represent an attractive therapeutic goal against cerebral ischemia.
WHY IS TISSUE PLASMINOGEN ACTIVATOR NOT WIDELY USED FOR ACUTE ISCHEMIC STROKE ?
Shigeharu Takagi, Shunya Takizawa, Wakoh Takahashi, Sari Okamoto, Youichi Ohnuki, Hiroyuki Mandokoro, Yuko Morita, Takashi Ohtomo, Tatsuya Kumazawa, Tsuyoshi Uesugi, Takashi Yasuda, Satoshi Kontani, Reiko Nagano, Kazunari Honma
Department of Neurology, Tokai University School of Medicine, Isehara, Kanagawa, Japan
Background : Intravenous injection of tissue plasminogen activator (t-PA) has been used to treat acute ischemic stroke for more than 10 years. The ratio of treated/total number of acute stroke patients, however, is less than 2 percent. In October 2005 intravenous t-PA treatment was started in Japan.
Purpose : The purpose of this presentation is to clarify the factors which prevent the use of t-PA for acute ischemic stroke.
Subjects and Methods : Seventy-two subjects with acute cerebral infarction due to cardiogenic embolism were admitted to Neurological and Cerebrovascular Center of Tokai University Hospital from October 2005 to September 2006. Eleven patients (15.3%) were treated with t-PA. We studied 61 subjects with acute cerebral embolism, not treated with t-PA. There were 36 males and 25 females, with age of 72 ± 11 years. We evaluated medical records and CT, MRI images of these subjects.
Results : (1) patients arrived within 3 hours after onset: This group include 26 subjects. The reason not to use t-PA was too mild symptom or significant improvement before treatment in 8, contraindication such as early ischemic CT sign of hemorrhage in 5, too severe neurological symptom in 7. Since there was an overlap, these 19 out of 26 patients were considered not to be the candidate for t-PA treatment. In the remaining 7 subjecs, the reason not to use t-PA was age older than 75, intra-hospital delay, inability to contact with family to give informed consent. These 7 patients might benefit from t-PA treatment. (2) patients arrived after 3 hours: This group includes 20 patients. Four patients initially refuse to come to hospital, and 9 patients did not come immediately after onset. Since there were other contraindication in these patients, we calculated that 9 subjects could be treated if they came to our hospital earlier. (3) patients with unknown time of onset: These 14 patients can not be treated by t-PA right now.
Discussion and Conclusion : In 16 of 61 patients t-PA could be indicated. In order to increase the number of candidates of t-PA treatment, appropriate information to intra- and outside the hospital is necessary. Indication to subjects older than 75 should be discussed. By these improvement, the ration of t-PA-treated/total patients could be increased up to 30 % of more (27/72 in this study).
THE EFFECT OF SIMVASTATIN ON PLASMA ASYMMETRIC DIMETHYLARGININE (ADMA) IN CHRONIC INPATIENT
Makoto Sakurazawa1,2, Yasuhiro Nishiyama1, Yuichi Komaba1, Toshiya Katsumata1, Ken-Ichiro Katsura1, Yasuo Katayama1
1Second Department of Internal Medicine, Nippon Medical School, Tokyo, Japan, 2Department of Internal Medicine, Tajirigaoka Hospital, Ibaraki, Japan
Background: Recently it has been reported that ADMA (asymmetric dimethylarginine) reduces NO production by eNOS inhibition, and research attentions are focused on its relation to atherosclerosis.
Purpose: The aim of this study was to evaluate the relation between risk factor of atherosclerosis and ADMA, and to evaluate the effect of simvastatin on plasma ADMA level.
Method: Fifty-four chronic stage inpatients were recruited (exclusion criteria: CRE>2.0mg/dl, CRP>3.0mg/dl, patients who cannot intake intraoral nutrition). Twenty patients who had plasma total cholesterol≧180mg/dl were administered Simvastatin (5mg/day) for 1 month. We assessed 1) correlation between atherosclerosis risk factor (age, gender, history of hyperlipidemia, hypertension, diabetes mellitus, smoking, cerebrovascular diseases) and ADMA, 2) correlation between lipid level and ADMA, and 3) changes of ADMA level after 5mg/day simvastatin administration for 1 month.
Results: There was a correlation between history of hyperlipidemia and ADMA. Although there were no correlation between T-CHO, LDL, MDA-LDL, TG, FBS level and ADMA, there was a correlation between HDL level and ADMA (figure). Plasma ADMA level was not changed by administration of 5mg/day simvastatin for 1 month. There were correlation between ADMA and LDL, HDL, TG after administration of simvastatin.
Conclusion: It was suggested there might be a relationship between plasma ADMA level and the progression of atherosclerosis. Although we could not find significant relationship between ADMA and LDL, or TG before simvastatin treatment, the correlation turned to be clear and significant after 1 month of simvastatin treatment. Simvastatin treatment might have reduced the daily changing values of LDL or TG to the individual intrinsic value, thereby revealing significant relationship to plasma ADMA levels.
LONG-TERM PROGNOSIS IN TYPE 3 ISCHEMIA OF OCCLUSIVE CAROTID ARTERY DISEASES
Masaaki Hokari1, Satoshi Kuroda1, Tohru Shiga2, Chietsugu Katoh2, Naoki Nakayama1, Tetsuya Ishikawa1, Hiroshi Yasuda3, Hisatoshi Saitoh3, Yoshinobu Iwasaki1
1Department of Neurosurgery, Hokkaido University, Sapporo, Japan, 2Department of Nuclear Medicine, Hokkaido University, Sapporo, Japan, 3Sapporo Asabu Neurosugical Hospital, Sapporo, Japan
Background and Purpose: Reduced cerebral blood flow (CBF) and cerebrovascular reactivity (CVR) to acetazolamide has been proven as an alternative predictor for subsequent ischemic stroke in patients with carotid occlusive diseases. However, recent studies have shown that type 3 ischemia (reduced CBF and CVR to acetazolamide) may not always represent elevated OEF and may be divided into 2 pathophysiologically different subgroups: type 3 with elevated OEF and type 3 but normal OEF. This study was aimed to clarify the prognosis and time course of cerebral hemodynamic and metabolic changes in the patients with Type 3 ischemia who were medically treated with symptomatic occlusive carotid diseases.
Methods: This study included 20 patients with Type 3 ischemia (reduced CBF and CVR on 123I-IMP SPECT) who were medically treated with symptomatic occlusive carotid diseases. Hemodynamic and metabolic parameters were initially estimated in all patients by 15O-gas PET. All patients were classified into elevated OEF group and normal OEF group and were followed up with average follow-up period of 45.6 months.
Results: According to PET study, twenty patients with type 3 ischemia were classified into elevated OEF in 9 patients, and normal OEF in 11 patients. There was no significant difference in these risk factors such as age, sex, clinical diagnosis, and initial PET parameters variables between two groups, except for OEF value. In nine patients with elevated OEF, three patients suffered from ipsilateral strokes. In the other six patents with elevated OEF, we observed improvement of their hemodynamic and metabolism parameter in three patients, no change of them in two patients, and transition into matched metabolism in one. In the eleven patients with normal OEF, no strokes occurred.
Conclusion: This study indicates that the patients with Type 3 ischemia (reduced CBF and CVR) and normal OEF are stable in cerebral homodynamics without stroke recurrence. However, the patients with type 3 ischemia and elevated OEF have high risk in stroke recurrence. Further study would be necessary to confirm the speculation in a larger number of patients.
OXYGEN RADICAL PRODUCTION IN RAT ISCHEMIC BRAIN MEASURED BY INTRAVITAL FLUORESCENCE IMAGING
Yong Wang, Seiji Yamamoto, Atsuo Miyakawa, Takashi Sakurai, Kyoko Ibaraki, Susumu Terakawa
Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan
INTRODUCTION: Several studies using electron-spin resonance showed increased oxygen free radical formation during ischemia. However, the results remain controversial. In the present study, to analyze the controversial issue, we tried to establish a new semi-quantitative analysis system that could spatiotemporally detect the cortical intracellular oxygen radical changes of adult rat brain, and sought to determine how the production of oxygen radicals could change in ischemia-reperfusion.
METHODS: Adult male SD rats (300 g) were anesthetized and spontaneously ventilated. After instrumentation, bilateral vertebral arteries were coagulated, and transient (10 min) forebrain ischemia was induced by occluding bilateral common carotid arteries with balloon occluders. The regional cerebral blood flow (rCBF) was measured by laser-Doppler flowmetry. To observe the oxygen radical production, cranial window was made over the frontal cortex, and a fluorescent dye (2.5 µl) was microinjected to the cortical region. To measure superoxide radical (?O2-) production, MitoSOX Red (5µM, Molecular Probes), a fluorescent dye for mitochondrial ?O2-, was used, and to measure hydroxyl radical (?OH) production, Hydroxyphenyl Fluorescein (20 µM, Daiichi Pure Chemicals, Japan), a fluorescent ?OH indicator, was used. After microinjection, closed cranial window was made over the cortex, and the fluorescence images were taken under a microscope (BX50WI, Olympus) equipped with a confocal scanner of a microlens-attached Nipkow-disk (CSU-21, Yokogawa, Japan) every 30 s before and after 10-min ischemia. The free radical scavenger, edaravon (3-methyl-1-phenyl-2-pyrazolin-5-one, Mitsubishi Pharma Corporation, Japan), which has been developed as a neuroprotective agent, is approved for the treatment of acute cerebral infarction in Japan. To test the feasibility of our intravital imaging system, edaravon (3.0 mg/kg) was intravenously injected 5 min before ischemia, and measured ?O2- and ?OH.
RESULTS: Mean arterial pressure and blood gases were within normal limits for anesthetized rats, and did not differ among the groups. In the control animals without ischemia, ?O2- and ?OH did not significantly increase (n=3/each). During 10-min ischemia, the rCBF (mean±SD) dropped to 29±7% (n=6). The ?O2- production (n=5) began to significantly increase at the 5 min after the onset of ischemia, reached to 160±13% at the end of ischemia, and persistently increased up to 186±20% during 20-min reperfusion. The ?OH production (n=5) started to increase at the 5 min after the onset of ischemia, elevated to 131±12% at the end of ischemia, and persistently increased up to 183±10% during reperfusion. The treatment with edaravon completely scavenged ?OH to the control level (p<0.01, n=3), but did not do so in ?O2- generation (n=5).
COMMENTS: In the present study, the intravital imaging indicated that ?O2- and ?OH production increased in the ischemia and reperfusion in rats. Whereas ischemia (ischemic rCBF=30%) produced oxygen radicals, reperfusion strongly enhanced the production. Especially, during reperfusion, production of ?OH, toxic reactive oxygen species, was more than ?O2-. Our system allows us to observe real-time production of oxygen radicals, which is useful for investigating the pathological roles of free radical in the brain.
Supported by the grant #16390407 (SY) from Japan Society for the Promotion of Science.
NEUROPROTECTION OF RAT HIPPOCAMPAL SLICES EXPOSED TO OXYGEN-GLUCOSE DEPRIVATION BY ENRICHMENT WITH DOCOSAHEXAENOIC ACID AND BY INHIBITION OF I-PLA2
Mikhail Strokin1, Klaus Reymann2, Georg Reiser1
1Institut Fuer Neurobiochemie, Medizinische Fakultaet, Otto-Von-Guericke Universitaet, Magdeburg, Germany, 2Leibniz Institute for Neurobiology, Magdeburg, Germany
Background and aims: Polyunsaturated fatty acids play an important role in the development of pathological states in brain after hypoxia/ischemia. Here, we investigated the role of docosahexaenoic acid (22:6n-3, DHA) in brain phospholipids for neuronal survival.
Methods: We used organotypic cultures of rat brain hippocampal slices exposed to 40 min of oxygen-glucose deprivation (OGD), to study the consequences of experimental ischemia. In [14C]DHA-labeled cultures, OGD induced significant release of radioactive DHA. This release could be blocked by the selective inhibitor of the Ca2+-independent phospholipase A2, BEL (10 µM), when it was added 30 min prior to OGD.
Results: Addition of BEL at 30 min prior to OGD markedly decreased the neuronal damage induced by OGD. The protective effect was substantially higher in dentate gyrus (DG) than in CA1 and CA3 areas. Enrichment of the hippocampal tissue with DHA by incubation with 10 µM DHA for 24 h exerted the same neuroprotective effect, which was observed after treatment with BEL. In contrast to the 24 h-preincubation, simultaneous addition of DHA with the onset of OGD had no protective effect. This suggests that incorporation of DHA into phospholipids is required for the protective effect observed. Then the possible involvement of arachidonic acid (AA) metabolism in DHA-induced neuroprotection was tested. Inhibition of prostaglandin (PG) production by ibuprofen produced no change in neuroprotection after 24-h incubation of the hippocampal slices with DHA. Simultaneous inhibition of Ca2+-independent and Ca2+-dependent phospholipases A2 by treatment with the general PLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP; 3 µM, 30 min prior to OGD) resulted in significant enhancement of the neuroprotective effect in the DG, but not in the CA1 and CA3 areas.
Conclusions: The results reported here indicate that DHA and DHA-containing phospholipids provide potent protection against neurodegeneration after hypoxia/hypoglycemia. Furthermore, our data suggest that iPLA2, the isoform, which has been largely ignored so far, is a possible target for treatment of ischemia-related pathologies in brain.
ANTIOXIDANT PROPERTY OF NEUROPEPTIDE CART
Peizhong Mao, Philippe Thuillier
Department of Public Health and Preventive Medicine, Cancer Institute, Oregon Health and Science University, Portland, OR, USA
BACKGROUND and HYPOTHESIS: The neuropeptide CART, Cocaine and Amphetamine Regulated Transcript, was first found in 1980, its cDNA was identified in 1995. Recently CART has been implicated in a variety of brain functions, including energy metabolism, appetite control, drug addiction and neuroprotection, indicating its potential use for some human diseases, such as stroke and diabetes. We have previously shown that CART interacts with succinate dehydrogenase (SDH) and stimulates SDH activity in primary cultured rat neuron cells at neuroscience and brain meetings. High concentrations of CART peptides in the hypothalamus, pituitary, adrenal medulla and blood are consistent with a wider role for CART in body homeostasis or cellular defense system. Thus, we tested the hypothesis that CART may protect human cells from oxidative stress using cultured human cells.
METHODS and RESULTS: First, in order to increase CART delivery into cells, we expressed a CART fusion protein with TAT-EGFP vector. The plasmid was confirmed by sequencing and purified TATEGFP-CART was recognized by CART antibody. Second, we used hydrogen peroxide (H2O2) to induce cellular oxidative stress. Human embryonic kidney (HEK293) cells were exposed to H2O2 for 1hr. DNA was isolated and PCR amplification of mitochondrial DNA (mtDNA) was performed using mtDNA specific primers. The 16kb mitochondrial fragment was amplified in each reaction. However, H2O2 treatment of the cells dramatically reduced the mtDNA amplification and pretreatment with the CART protein (0.005mg/ml) inhibited the damaging effect of H2O2, while no protective effect was seen with TATEGFP. Interestingly, a similar observation was obtained in human lymphocyte cells, suggesting that the effect was not cell specific. Third, to measure oxidative stress at the protein level, HEK293 cells were exposed to 0.2mM H2O2 for 2hrs. Total cellular proteins were isolated and oxidized proteins were determined by Western blot. The results showed that H2O2 increased protein oxidation. However, pretreatment of the cells with TATEGFP-CART or pure CART peptide remarkably reduced oxidation. Finally, using confocal microscopy we observed that TATEGFP-CART was more stably localized to cellular mitochondria than TATEGFP in live HEK293 cells.
CONCLUSIONS: Reactive oxygen species (ROS) can damage cellular protein and DNA, especially mtDNA. For the first time we report that CART localizes to the mitochondria and significantly reduces H2O2 induced damage of both mtDNA and proteins, indicating that CART is a potential endogenous antioxidant. Our study may provide a new possibility for treatment of some human diseases associated with oxidative stress and mitochondrial disorder.
RESVERATROL PRETREATMENT PROTECTS BRAIN FROM CEREBRAL ISCHEMIC DAMAGE
David Della Morte, Kunjan R. Dave, Miguel A. Perez-Pinzon
Cerebral Department of Neurology and Neuroscience, Vascular Disease Research Center, University of Miami School of Medicine, Miami, FL, USA
Beckground and aims: Ischemic preconditioning (IPC) is a phenomenon whereby an organ's adaptive transient resistance to a lethal insult occurs by preconditioning this organ with a sub-lethal/mild insult of short duration. Recently, using an in vitro model of cerebral ischemia we demonstrated that resveratrol mimics IPC via the SIRT1 (a member of Sirtuin proteins family) pathway [1]. In the present study we tested the hypothesis that tolerance for ischemia can be induced in brain by resveratrol pre-treatment.
Methods: We compared efficacy of resveratrol preconditioning with IPC in protecting CA1 hippocampal neurons in a rat model of asphyxial cardiac arrest (CA). IPC was induced by tightening the carotid ligatures bilaterally following hypotension (50 mmHg) for 2 min [2]. Resveratrol preconditioning was induced by injecting resveratrol (i.p.) at 10, 50 and 100 mg/kg dose. Eight minutes of CA was induced 48 h after IPC or resveratrol injection as described earlier [3]. At the end of 7 days of resuscitation, the brain sections were examined for histopathological changes. The numbers of normal neurons were counted in CA1 hippocampus at 3.8 mm posterior to bregma. Results were expressed, as mean ± SD. Statistical significance was determined with an ANOVA test followed by a Bonferroni's post-hoc test.
Results: The number of normal neurons in sham operated rats was 1328±23 (n = 9) and 70% lower in cardiac arrest/vehicle rats (405±78 (n = 5), p<0.001 sham vs. CA). IPC was able to provide tolerance against CA since the number of normal neurons were higher by 88% (760±120, n = 5; P<0.01 IPC vs. CA) as compared with the CA group. The number of normal neurons in 10 and 50 mg/kg resveratrol groups were higher by 73% (702±54, n = 7, p<0.05 10 mg vs. CA) and 11% (449±57, n = 7) as compared to CA group, respectively. No significant protection was observed at higher resveratrol dosages (10 mg/kg, 292±15, n = 6). To determine if IPC and resveratrol preconditioning resulted in increased SIRT1 activity, we measured SIRT1 activity following IPC and resveratrol treatments. IPC and resveratrol preconditioning were able to stimulate SIRT-1 activity by 29 % (n = 4, p< 0.02) and 36 % (n = 4, p< 0.01) as compared to control rats (n = 4), respectively.
Conclusion: We conclude that preconditioning can be emulated by pre-treating rats with low dosages of resveratrol. Our data suggests that resveratrol and IPC may exert its neuroprotection via activation of the SIRT1 pathway.
Grant support: PHS grants NS34773, NS05820, NS045676 and NS054147.
ERYTHROPOIETIN PREVENTS SPINAL CORD DAMAGE BY ACTIVATING SERINE-THREONIN KINASE AFTER TRANSIENT SPINAL CORD ISCHEMIA IN RABBITS
Hiroaki Kokubo1, Masahiro Sakurai4, Yasuto Itoyama2, Koji Abe3, Koichi Tabayashi1
1Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan, 2Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan, 3Department of Neurology, Okayama University Graduate School of Medicine, Okayama, Japan, 4Department of Cardiovascular Surgery, National Hospital Organization Sendai Medical Center, Sendai, Japan
Background and aims : The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. We have examined whether recombinant human erythropoietin (rhEPO), which is reported to have potent neuroprotective activity against a variety of potential brain injuries such as ischemia and reperfusion, can protect against ischemic spinal cord damage.
Methods : After induction of ischemia, rhEPO(800U/kg) or vehicle was immediately injected intravenously. Cell damage was analyzed by counting the number of motor neurons. To investigate the mechanism, by which rhEPO prevents ischemic spinal cord damage, we observed the immunoreactivity of Erythropoietin receptor (EPO R), and antiapoptic protein of its downstream, phosphorylated serine-threonin kinase (pAkt).
Results : The neurological function and the survived cell number 7 days after induction of ischemia of rhEPO administration group (group E) was greater than that of ischemic group (group I). The immunoactivity of EPO R was activated in both group I and group E. The immunoactivity of pAkt was much activated in group E than in group I.
Conclusion : These results suggest that rhEPO can protect motor neurons from ischemic spinal injury by up-regulating EPO R and activating anti-apoptotic pathway that involve pAkt. RhEPO could be a potent candidate for a use as a therapeutic agent in the treatment of ischemic spinal cord injury.
WOX1 PARTICIPATES NEURONAL CELL DEATH DURING HYPOXIA
Chen-Peng Lo1, Ming-Shu Tsai2, Jiann-Pyng Wang3, Shur-Tzu Chen1
1Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan-R.O.C., 2Department of Health, Executive Yuan, Putz General Hospital, Chiayi, Taiwan-R.O.C., 3Department of Life Sciences, National Cheng Kung Univeristy, Tainan, Taiwan-R.O.C.
Introduction
WOX1 is a stress-induced factor (such as hyaluronidase, UV irradiation or toxin), which may increase certain cancer cell sensitivity to tumor necrosis factor (TNF) cytotoxicity. Human WW domain-containing oxidoreductase WOX1 (WOX1) gene is regarded as a tumor suppressor gene, located at the chromosome region 16q23.3–24..1, a commonly fragile area. A high frequency of loss of heterozygosity (LOH) of this gene has been shown in prostate, lung, breast and other cancers. It becomes phosphorylated at Tyr33, further complex with p53 or c-Jun N-terminal protein kinase 1 (JNK1). Thus WOX1 regulates apoptosis through an unknown pathway. Stroke is a serious factor in causing death. It is caused by cerebral arteries bleeding or occlusion. Ischemia or hypoxia in local area makes neurons undergoing apoptosis. Hypoxia-induced factor-1 alpha (HIF-1α) regulates many transcripts under hypoxic conditioning. It protects p53 from degradation mediated by Mdm2, and leads to activation of p53-mediated transcription in cells. HIF-1α coordinates with p53 and promotes delayed neuronal cell death after focal ischemia.
Materials and Methods
Human neuroblastoma NT2era cells and SK-N-SH cells were grown with DMEM containing 10% fetal bovine serum. Cells were then incubated in a humid incubator containing 1% oxygen and 5 % CO2, or treated with CoCl2 at various concentrations for serial time points. Protein and DNA were collected for applying Western blotting and DNA fragmentation analysis.
Results
Cells exhibited obviously morphological change after CoCl2 treatment for 24 hours. DNA fragmentation was detected in the cells treated with CoCl2 for 24 hours, or incubated with 1% oxygen for 72 hours. Cell viability was decreased after CoCl2 treatment for 24 hours. Under hypoxic conditioning, the phosphorylation level of WOX1, c-Jun and extracellular signalregulated kinase (ERK) was increased. The expression of activation transcription factor 3 (ATF3) was over-expressed only after 24 hours or high dosage of CoCl2 treatment. Increased HIF-1α was regarded as a hypoxic indicator. Dramatically, the phosphorylation level of WOX1 exhibited a CoCl2 dose dependent manner. By using immunostaining, phosphor-WOX1 translocated into cell nuclei after hypoxic treatment.
Conclusion
Over-expression and nuclear translocation of WOX1 suggested WOX1 involved in hypoxic damage. Up-regulation of MAP kinase and other transcription factors indicated the possible mechanism of WOX1-mediated neuronal cell death during hypoxia.
FOS EXPRESSION IN NORADRENERGIC CELLS IN THE RAT BRAIN AFTER HYPERBARIC OXYGEN SEIZURE
Minako Arai, Yoshimasa Takeda, Naomi Aoe, Motomu Kobayashi, Hideki Taninishi, Toshihiro Sasaki
Department of Anestheology, Okayama University Hospital, Okayama, Japan
[Background] The mechanisms of seizures induced by hyperbaric oxygen (HBO) have been studied extensively. Various biochemical changes occur in the brain. Among these, changes in the brain concentration of noradrenaline has been suggested to have an important role in the origin and propagation of HBO-induced seizures. Noradrenergic cells in the rat brain are distributed through A1~A7 areas in the brainstem. These areas are divided into two pathways, the locus coeruleus noradrenergic pathway (A6, A4) and the lateral tegmentum noradrenergic pathway (A1, A2, A3, A5, A7). The locus coeruleus noradrenergic pathway differs from the other pathway in that it projects to the whole cortex area. Fos, a typical immediate early gene, has been widely used as a marker for neuronal excitation and has been to reported to be expressed in some seizure models.
We examined Fos expression using an immunohistochemical technique in noradrenergic cells in the rat brain after HBO-induced seizure to determine the involvement of noradrenergic cells in the brain in HBO-induced seizure and whether there is a difference between the two noradrenergic pathways, locus coeruleus pathway and lateral tegmentum pathway.
[Materials and methods] Six male Wistar rats were pressurized at five atmospheres absolute with 100% oxygen for 120 minutes (pressurized group), and another three male Wistar rats were put into a chamber at one atmosphere absolute with room air for 120 minutes (control group). All animals were sacrificed and perfused with 4% paraformaldehyde solution for immunohistochemistry two hours after the experiments. We distinguished noradrenergic cell groups A1 to A7 using double immunofluorescence with TH and Fos and the anatomical location and then evaluated Fos immunoreactivity.
[Results] Seizure was observed in all of the rats in the pressurized group. All of the noradrenergic cell groups A1 to A7 in the pressurized group had Fos expression, while the control group had no Fos immunoreactivity (Table 1).
[Table 1] The ratio of Fos expression to TH positive cells in
[Conclusion] Our results suggested that noradrenergic cells in the rat brain affected the origin and propagation of HBO-induced seizure, and there was no statistical difference between the two noradrenergic pathways, locus coeruleus noradrenergic pathway and lateral tegmentum noradrenergic pathway.
ESTROGEN INHIBITS FAS-MEDIATED APOPTOSIS IN EXPERIMENTAL STROKE
Jia Jia1,2, Zhenjun Yang1,2, Lihui Wang1, Zichun Hua2, Patricia D. Hurn3, Nabil J. Alkayed3, Yun Xu1,2
1Department of Neurology, The Affiliated Drum Tower (Gulou) Hospital of Nanjing University, Nanjing, China, 2State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University, Nanjing, China, 3Department of Anesthesiology and Peri-Operative Medicine at Oregon Health and Science University, Portland, OR, USA
INTRODUCTION. It is well known that 17 beta-estradiol protects against brain injury in experimental stroke, and the mechanism (s) is likely multi-factorial. However, neuronal apoptosis following stroke plays a pivotal role in the pathological processes of brain ischemia and reperfusion[1]. Increasing evidence showed that the Fas/FADD signaling pathway is integral to apoptotic cell death [2]. Recent data show that increased expression of both Fas and Fas ligand in the ischemic rat brain and that infarction and measures of apoptosis are reduced in mice with a loss-of-function mutation in Fas [3]. The aim of our experiments was to determine whether brain injury after middle cerebral occlusion (MCAO) involves Fas/FADD pathway and if estradiol acts as an anti-apoptotic agent via this pathway.
METHODS. Ovariectomy in mice is performed 1–2 weeks before the experiment, and estradiol is replaced by implanting a silastic capsule containing 35 ul of 180 ug/ml 17 beta-estradiol (E2, 7 days), as previously described [4]. MCAO was produced using the intraluminal suture technique [5]. Ischemic or contralateral cortex was collected at 3, 6 or 12 hr post-MCAO and analyzed for Fas and FADD mRNA and protein expression using RT-PCR and Western blot, and activation of caspases-8 and caspase-3 was determined fluorometrically. Primary cultured cortical neurons were pretreated with 17 beta-estradiol [6] in the presence and absence of monoclonal anti-Fas antibody for 48 hrs, and apoptotic cells were quantified using fluorescence activated cell sorting (FACS).
RESULTS. Fas and FADD mRNA and protein were elevated in ischemic cortex relative to contralateral cortex at 3, 6 and 12 hours post-MCAO (p<0.05), with peak levels at the 6h time point. Estradiol suppressed Fas/FADD mRNA and protein expression at all time points (p<0.05). Cortical caspase-8 and caspase-3 activities were increased at 3, 6 and 12 hours after MCAO (p<0.05) as part of the downstream mechanism of injury. Caspase activity was significantly reduced in estradiol-treated animals. In cultured cells, application of Fas-antibody induced neuronal apoptosis, which was attenuated by estradiol.
CONCLUSIONS. Our results indicate that estradiol mitigates Fas/FADD signaling after focal cerebral ischemia and Fas-induced neural apoptosis. We speculate that this mechanism may be an important part of estradiol's anti-ischemic effects in the female brain.
APOPTOSIS INDUCING FACTOR (AIF) IS ESSENTIAL FOR NEURONAL CELL DEATH FOLLOWING GLOBAL CEREBRAL ISCHEMIA
Simone Thal, Serge Thal, Nikolaus Plesnila
Laboratory of Experimental Neurosurgery, Department of Neurosurgery and Institute for Surgical Research, University of Munich Medical Center, Grosshadern, Munich, Germany
Introduction: Delayed neuronal cell death is a hallmark of cardiac arrest induced brain damage and a primary target for neuroprotective strategies. Apoptosis-inducing factor (AIF), an oxidoreductase located in the mitochondrial intermembrane space promotes caspase-independent apoptosis upon release from mitochondria and translocation to the nucleus. In the present study we studied the role of AIF for neuronal cell death after global cerebral ischemia (GI).
Methods: Harlequin mutant (HQ-) mice, which express AIF at low levels (~20 % of wild type mice), and their wild type litter mates (HQ+) were anesthetized with isoflurane, intubated and mechanically ventilated. The basilar artery was exposed and permanently occluded by coagulation and dissection. After a 2 week recovery period bilateral occlusion of the common carotid artery was performed under control of cerebral blood flow by laser Doppler fluxmetry for 10 min. Neuronal damage in the hippocampus was quantified 5 days after GI on Nissl-stained sections.
Results: All sham operated animals survived the observation period of 5 days. The mortality rate in animals subjected to GI was 40% and there was no difference between HQ- and HQ+ animals. HQ- and HQ+ mice and their wild type littermates mates not subjected to GI had similar neuronal cell counts in the hippocampus (CA1; sham: 82±3; HQ+: 80±4, sham HQ-: 75±3). HQ- mice, however, displayed significantly less cell death in the hippocampus (−50 %, p<0.05) as compared to their wild type littermates (CA1: HQ+: 43±3, HQ-: 66±6; CA2: HQ+: 40±2, HQ- 64±7, n=6 each).
Conclusion: These results provide evidence for the primary involvement of AIF in neuronal cell death after global cerebral ischemia and suggest that therapies targeting caspase-independent cell death signaling may provide protection in cerebrovascular disease or other pathological conditions where programmed cell death is prominent.
CHRONIC MILD ISCHEMIA RECOVERS CBF AUTOREGULATION CURVE AND REDUCES INFARCT SIZE AFTER MCA OCCLUSION
Emi Omura-Matsuoka, Kazuo Kitagawa, Tsutomu Sasaki, Yasukazu Terasaki, Naoki Ohyama, Kenichi Todo, Yoshiki Yagita, Masatsugu Hori
Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
Background and Purpose: We previously demonstrated that occlusion of common carotid artery (CCA) for two weeks preserves cortical perfusion and reduces infarct size after occlusion of middle cerebral artery (MCA) in mice. The purpose of this study is to (1) show the same phenomenon in rat MCA occlusion model and (2) clarify the change in cerebral blood flow (CBF) autoregulation curve after CCA occlusion.
Methods: 1. MCA occlusion subsequent to CCA occlusion in rats: Adult male wistar rats aged 7 to 9 weeks were used in this study. After attachment of a probe, the left CCA was occluded. The left MCA was subsequently occluded permanently 14 days later (n=13). The change in cortical perfusion during MCA occlusion was recorded by laser-Doppler flowmetry. A sham group rats received only exposure of the CCA and MCA occlusion 14 days later (n=15). Infarct size and neurological deficit were determined 2 days after MCA occlusion. Motor function was serially evaluated by the rotarod test after MCA occlusion. 2. Change in CBF Autoregulation Curve after CCA occlusion: We measured the CBF at rest and during hemorrhagic hypotension, using laser-Doppler flowmetry. A femoral artery was cannulated with PE10 tube and blood pressure (BP) was monitored continuously. There were three groups of rats; (1) Control group (n=5), (2) CCAO 30 min group (n=5), the rats with hypotension 30 minutes after the left CCA occlusion and (3) CCAO 2 wks group (n=5), the rats with hypotension 2 weeks after the left CCA occlusion. Blood pH, PaO2 and PaCO2 were determined three times, at rest and under hypotension. The lower limit of CBF autoregulation was defined as mean BP at which CBF decreased by 10% of the baseline value.
Results: Cortical perfusion after MCA occlusion was significantly preserved in CCA occlusion group (51.8 ± 10.8 %) compared with sham group (37.8 ± 8.5 %). Infarct size was also significantly attenuated in CCA occlusion group (126.5 ± 111.0 mm3) compared with sham group (328.8 ± 81.9 mm3). CCA occlusion group also showed better recovery in neurological findings and motor function than sham group. In the control group, the lower limit of CBF autoregulation was 63.0 ± 6.7 mmHg. In the CCA 30 min group, it was shifted to a higher level, 77.6 ± 7.4 mmHg (P<0.01). However, in the CCAO 2 wks group, the lower limit of CBF autoregulation returned to a lower level, 63.2 ± 6.3 mmHg.
Conclusion: Chronic CCA occlusion preserved cortical perfusion and attenuated infarct size after MCA occlusion in rats. Improvement of CBF autoregulation curve during 2 weeks after CCA occlusion could be involved in preservation of cortical perfusion after MCA occlusion.
NO-INDUCED DOWN-REGULATION OF HSP10 AND HSP60 EXPRESSION IN THE POSTISCHEMIC BRAIN
Seung-Woo Kim1, Joo-Hyun Kim1, In-Sun Baek2, Pyung-Lim Han2, Ja-Kyeong Lee1
1Department of Anatomy, Center for Advanced Medical Education (BK21 Project), Inha University School of Medicine, Inchon, South Korea, 2Division of Nano Sciences, Department of Life Science, Ewha Womans University, Seoul, South Korea
Background and aims: Heat shock protein 60 (HSP60) and HSP10 are mitochondrial chaperonin proteins, and are responsible for the folding of newly imported proteins and of 13 polypeptides encoded by mitochondrial DNA. Expressions of HSP60 and HSP10 are simultaneously regulated because HSP60 and HSP10 genes are localized head to head on chromosome, separated by a bidirectional promoter, which harbors heat shock element (HSE), CHOP, STAT3 and SP1 binding sites. In the present study, we showed down-regulation of the HSP10 and HSP60 by NO in the postischemic brain and identified the promoter element responsible for that regulation.
Methods: We examined the expressions of HSP10 and HSP60 in the brain after 1 hour middle cerebral artery occlusion (MCAO). The down-regulation of the HSP10 and HSP60 by NO was confirmed in C6 astroglioma cells. To identify the promoter element, reporter gene analysis combined with deletion and mutation studies and elctrophoretic mobility shift assay were employed.
Results and conclusions: The expressions of HSP10 and HSP60 in the brain after middle cerebral artery occlusion (MCAO) were significantly induced. Interestingly, the expressions of HSP10 and HSP60 were further up-regulated by the administration of the aminoguanidine (AG), an inhibitor of the inducible nitric oxide synthesis (iNOS), which has been known to reduce ischemic damage in the animal model after MCAO. The results suggest that HSP10 and HSP60 are induced in the postischemic brain, yet they are under the down-regulation by NO generated beginning 12 hours after the MCAO/reperfusion. Down-regulation of the HSP10 and HSP60 by NO was confirmed in in vitro experiments, wherein HSP10 and SHP60 expressions were increased by LPS/IFN gamma-treatment in C6 astroglioma cells and it was further up-regulated by NMMA, another iNOS inhibitor. Previously we have identified a STAT3 binding site localized in the bidirectional promoter which mediates IFN gamma-induced up-regulations of HSP60 and HSP10 in C6 astroglioma cells. Reporter gene analysis combined with deletion and mutation studies revealed that STAT3 binding site in the bidirectional promoter is responsible for the LPS/IFN gamma-induced up-regulation and also for the down-regulation by NO. Together the results indicated that NO suppresses HSP10 and HSP60 inductions in the postischemic brain by suppressing STAT3 binding to its recognition site.
NITRIC OXIDE PRODUCTION DURING CEREBRAL ISCHEMIA AND REPERFUSION IN VMAT2 KNOCKOUT MICE
Yuji Kato, Nobuo Araki, Takeshi Ohkubo, Yoshio Asano, Daisuke Furuya, Tomokazu Shimazu, Harumitsu Nagoya, Yasuo Ito, Kaori Itokawa, Kunio Shimazu
Department of Neurology, Saitama Medical University, Saitama, Japan
Background and aims: Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging monoamines into synaptic vesicles and thereby plays an essential role in neurotransmission. We investigated the dynamics of cerebral NO production in VMAT2 knockout mice during cerebral ischemia and reperfusion for the purpose of analyzing the relationship between NO metabolism and monoamine system in the brain.
Methods: Male VMAT2 knockout mice (n = 5) and wild type mice (control group) (n = 7) were anesthetized by halothane. NO production was continuously monitored by in vivo microdialysis. A microdialysis probe was inserted into the left striatum and perfused with Ringer's solution at a constant rate 2 µl/min. A laser Doppler probe was placed on the right skull surface. After 2 hours equilibrium period, fractions were collected every 10 minutes. Forebrain ischemia was produced by occlusion of both common carotid arteries for 10 minutes. Levels of nitric oxide metabolites, nitrite (NO2-) and nitrate (NO3-), in the dialysate were determined using the Griess reaction.
Results: (1) Blood Pressure: There were no significant differences between VMAT2 knockout mice and wild type. Only in 10 minutes after reperfusion, VMAT2 knockout mice (70.7±4.9 mmHg) showed significantly lower blood pressure than those of wild type (84.4±9.6) (p<0.05).
(2) Cerebral Blood Flow (CBF): During cerebral ischemia, CBF in VMAT2 knockout mice (52.6±30.8%; % of the baseline level) was significantly higher than that of wild type (11.6±11.7%). CBF in VMAT2 knockout mice was significantly higher than that of wild type during reperfusion.
(3) Nitric oxide: (a) NO2-: The levels of NO2- in VMAT2 knockout mice were significantly lower than those of wild type before and in 20, 60, 90 minutes after reperfusion.
(b) NO3-: The levels of NO3- in VMAT2 knockout mice were significantly higher than those of wild type in 10, 20 minutes after reperfusion.
(c) NO2-/NO3- ratio: The ratio in VMAT2 knockout mice was significantly lower than those of wild type before and during ischemia and just after reperfusion [Fig.1].
Conclusion: Although ischemia in VMAT2 knockout mice was not so severe as the wild type mice, there were significant differences in NO2-/NO3- ratio. These data suggest that VMAT2 knockout mice may be more easily oxidized than wild type mice.
EXPRESSION OF CYCLOPHILIN C-ASSOCIATED PROTEIN IN FOCAL CEREBRAL ISCHEMIA IN RAT
Rei Yamaguchi1, Hideaki Imai1, Masahiro Hosaka2, Toshiyuki Takeuchi2, Yuhei Yoshimoto1, Nobuhito Saito3
1Department of Neurosurgery, Gunma University Graduate School of Medicine, Maebashi, Japan, 2Institute of Molecular and Cellular Regulation, Gunma University, Maebashi, Japan, 3Department of Neurosurgery, University of Tokyo Graduate School of Medicine, Tokyo, Japan
Background and Purpose: Immunophilin ligands, such as cyclosporine A, are know to have neuro-protective efficacy in experimental stroke models, although the precise mechanism is unclear. Cyclophilin C-associated protein (CyCAP) is a natural cellular ligand for the immunophilin, cyclophilin C. It has an inflammation modifying factor intrinsically and shows a protective efficacy against endotoxins by down-modulating the proinflammatory response. Previously we have reported mRNAs of CyCAP and cyclophilin C are up-regulated in penumbral neurons and microglia after focal cerebral ischemia in the rat (1). There was no specific antibody to CyCAP which is available for immunohistochemistry and in vitro function analysis so far. The first aim of this study is to generate the specific antibody for CyCAP. The second is to investigate the temporal and spatial profile of the protein expression of CyCAP within the ischemic hemisphere using the antibody for CyCAP after focal cerebral ischemia.
Methods: The antibody to CyCAP was raised in rabbits by injecting the synthetic oligopeptide covering Rat MAMA (98% homology of Mouse CyCAP) amino acid sequence 77-96. The specificity of the antibody was examined by Western blot analysis using the organ lysate of the rats. Permanent middle cerebral artery occlusion model was employed in the adult Sprague Dawley rats (300-330g, n= 30). Rats were decapitated at 2 hours, 24 hours, 3 days, 7 days and 14 days after ischemia. Paraffin-embedded sections were stained with haematoxylin & eosin for identification of ischemic damage with adjacent sections taken for CyCAP immunohistochemistry. Fluorescence double labeling was employed to specify the CyCAP immunopositive cells to use Neu-N, GFAP, and ED-1 antibodies. For Western blot analysis, proteins were isolated from dissected region such as ipsilateral and contralateral hemisphere.
Results: This antibody reacted with proteins of approximate 77 kDa from cell extracts by western blotting. Also we have confirmed that GFP tagged CyCAP bind with FLAG tagged Cyclophilin-C in vitro by using immunoprecipitation. Immunohistochemical study revealed that CyCAP were ubiquitously distributed in the neurons of the normal brain at minimal level. Expression increased in predominately in the neurons of the peri-infarct zone at 2hours after MCA occlusion. CyCAP were predominantly expressed in microglia/macrophage of the ischemic core at 7 days and 14days. The quantification of CyCAP by Western blot analysis showed CyCAP expression once increased at 2hr after MCAO, then gradually decreased and re-increased at 7days through 14 days after ischemia.
Conclusions: The newly developed polyclonal antibody reacted with a protein which is supposed to be CyCAP of the rat. The observed changes in CyCAP expression in response to permanent focal cerebral ischemia strongly suggest that this protein have at least two independent functions; to participate in intrinsic cellular response against the ischemic damage at acute phase and activation of microglia/macrophage at subacute to chronic phase. Further both in vitro and in vivo study are needed to elucidate the function of CyCAP preciously.
TARGETED DISRUPTION OF ORGANIC CATION TRANSPORTER 3 IN MICE REDUCES ISCHEMIC BRAIN INJURY THROUGH DOWN-REGULATION OF IL-6 AND MCP-1
Pengxiang Zhu1, Ryuji Hata1, Masahito Ogasawara2, Fang Cao1, Kazutaka Maeyama2, Masahiro Sakanaka1
1Department of Functional Histology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan, 2Department of Pharmacology, Ehime University Graduate School of Medicine, Toon, Ehime, Japan
Background and aims
Organic cation transporter 3 (OCT3) participates in the control of histamine and pro-Th2 cytokine synthesis by modulating intracellular histamine levels [1]. Once it has attained a critical concentration in the cytosol, histamine is ready to exert its negative feedback control; this alleviates its deleterious effect during allergic reactions, and hampers the development of Th2 immune response [2]. Since inflammatory reactions play pivotal roles in ischemic insult, we investigated the effect of OCT3 on cerebral ischemia.
Methods & Results
Transient focal ischemia for 1 hour was induced by occlusion of the middle cerebral artery in homozygous oct3 knockout mice (n=6) and their wild-type littermates (n=6). One day after reperfusion, infarct volume was assessed by TTC staining and oct3 ablation significantly decreased infarct size (Figs. A and B). To gain an insight into the mechanisms underlying targeted disruption of oct3, animals were sacrificed after deep anesthesia at predetermined time periods (0h, 3h, 6h, 1d and 3d after reperfusion). Their brains were removed and divided into four areas of ischemic side (cortex and caudoputamen) and contra-lateral side (cortex and caudoputamen). Samples were homogenized in lysis buffer and the levels of Monocyte chemoattractant protein-1 (MCP-1) and Interleukin-6 (IL-6) in were determined by ELISA. As shown in Figs. C and D, and oct3 ablation significantly decreased the levels of MCP-1 and IL-6 in the ischemic cortex at one day after reperfusion.
Conclusions
Our data suggest that the targeted disruption of oct3 in mice modulates inflammatory responses after ischemia and reduces ischemic brain injury through down-regulation of IL-6 and MCP-1
ACTIVATION OF RHO-KINASE IS INVOLVED IN INFARCT EXPANSION AFTER FOCAL CEREBRAL ISCHEMIA
Yoshiki Yagita, Kazuo Kitagawa, Tsutomu Sasaki, Naoki Oyama, Yasukazu Terasaki, Kenichi Todo, Emi Omura-Matsuoka, Masatsugu Hori
Stroke Division, Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita City, Osaka, Japan
Background and Purpose: Microcirculatory disturbances contribute to the expansion of infarct lesions after focal cerebral ischemia. Because cerebral infarction gradually develops even more than 24 hours after ischemia, therapeutic intervention in endothelial cell dysfunction may inhibit the progression of ischemia peripherally in the early phase after ischemia. Recently, it was shown that Rho-kinase attenuates function of endothelial nitric oxide synthase in a rodent stroke model. However, the contribution of Rho-kinase activation to microcirculatory disturbances has not been determined. In this study, we investigated the profiles of Rho-kinase activation and the effect of the Rho-kinase inhibition on microcirculatory disturbances induced by endothelial cell dysfunction in the ischemic brain.
Methods: Male Wistar rats were subjected to focal cerebral ischemia with the use of a 4–0 nylon monofilament. Rho-kinase activation was evaluated by analyzing the phosphorylation of adducin, a direct substrate of Rho-kinase by immunohistochemistry. Microcirculation was assessed by the density of labeled microvessels after dichlorotriazinyl amino fluorescein-conjugated plasma injection at 6 hour following induction of ischemia. Immunohistochemistry of von Willebrand factor (vWF) was carried out to detect abnormal increase of vWF immunoreactivity in endothelial cells with functional perturbation. We assessed the effect of post-ischemic treatment with fasudil (10mg/kg, 5min after ischemia), a Rho-kinase inhibitor, on microcirculation, endothelial cell dysfunction after focal cerebral ischemia. Additionally, rats were treated with fasudil (10mg/kg) 5 min and 24 hours after transient or permanent cerebral ischemia. To evaluate infarct size, brains were removed 48 hours after ischemia and were subjected to 2,3,5-triphenyltetrazolium chloride staining.
Results: Signals of phosphorylated adducin was increased in microvessels in the ischemic area 6 hours after induction of ischemia. Microcirculatory disturbances and increased endothelial cell staining for vWF were observed in the same area. Post-ischemic treatment with fasudil preserved microcirculation and ameliorated vWF staining up-regulation in peripheral ischemic areas. Labeled-microvessel densities compared to non-ischemic cortex were 18.6 % (vehicle-treated group) and 32.9 % (fasudil-treated group, P<0.05). Microvessel densities with increased vWF signals were 7.6-fold (vehicle-treated group) and 3.8-fold (fasudil-treated group, P<0.05) increased compared to non-ischemic cortex. Inhibition of infarct expansion and improvement of neurological score were observed in fasudil-treated group.
Conclusions: These findings indicate that Rho-kinase is activated in the endothelial cells after cerebral ischemia and it contributes to microcirculatory disturbances in the peripheral ischemic area and infarct expansion. Activation of Rho-kinase in the brain endothelial cells may be a therapeutic target in the acute phase of ischemic stroke.
CHANGES IN EXTRACELLULAR CALCIUM AGAINST CBF REDUCTION AND ISCHEMIC DEPOLARIZATION DURING WHITE MATTER ISHCEMIA
Eiji Kumura1, Dohmen Christian2, Rudolf Graf2, Toshiki Yoshimine3, Wolf-Dieter Heiss2
1Department of Neurosurgery, Kobe Ekisaikai Hospital, Hyogo, Japan, 2Max Planck Institute for Neurological Research, Cologne, Germany, 3Department of Neurosurgery, Osaka University Graduate School of Medicine, Osaka, Japan
Introduction: Calcium influx is the most deleterious factor of ischemic brain injury. In gray matter (GM), membrane depolarization due to blood flow abruption produces bulk influx of Ca2+. In white matter (WM), the relationship among regional blood flow (rCBF), membrane depolarization and Ca2+ influx is not elucidated. To clarify the relationship of these factors in ischemic white matter, we measured extracellular calcium concentrations ([Ca2+]o), direct current (DC) potentials and rCBF during middle cerebral artery (MCA) occlusion in GM and WM regions of the cat brain. Methods: In 25 alpha-chloralose anesthetized cats, left MCA was occluded for 60 min. Two double-barrel ion-selective electrodes measured [Ca2+]o and direct current (DC) potentials in ectosylvian gyrus (GM) and corona radiata (WM). Platinum electrodes measured rCBF using hydrogen clearance that inserted together with each Ca2+-selective electrode. Regional CBF of GM and WM was measured repeatedly before, and at 10, 40 min after MCA occlusion. Regional [Ca2+]o and DC potentials were measured continuously and evaluated before, and at 10, 40min after MCA occlusion. Results: Pre-ischemic values of rCBF of GM were 50±3 ml/100g.min, and those of WM were 23±3 ml/100g.min. Nineteen animals showed significant rCBF reduction in both GM (3±2ml/100g.min) and WM (2±2ml/100g.min) at 10 min after MCA occlusion with persistent negative shifts of DC potentials. At 40 min after MCA occlusion, rCBF was recovered in 5 animals, and the rests remained severe rCBF reduction. In the remaining 6 animals, MCA occlusion elicited mild decrement of rCBF with minimal DC negative shifts. Fig.1 shows the relationship between [Ca2+]o and rCBF, and DC potentials at 10 min after MCA occlusion in 25 animals. In GM, [Ca2+]o (preischemic value, 1.2±0.1mM) was decreased significantly (0.5±0.3mM, p<0.01) when rCBF fell below 10 ml which produced profound negative shifts of DC potentials (−17±4mV, p<0.01). Negative shifts of DC potentials were proportional to [Ca2+]o (r=0.89, p<0.01, Fig.1 right). In WM, [Ca2+]o (preischemic value, 1.2±0.1mM) was increased significantly reaching 1.4±0.3mM (p<0.01, Fig.1 left) when rCBF fell below 10 ml. DC potentials showed negative shifts (−11±5mV, p<0.01), which were indifferent to [Ca2+]o (r=-0.27, Fig.1 right). At 40 min after MCA occlusion, [Ca2+]o were 0.2±0.2mM (p<0.01) and DC potentials were −18±4mV in GM of 14 animals with persistent severe rCBF reduction (4±3ml/100g.min). In WM, [Ca2+]o was 1.0±0.3mM (p<0.01) and DC potentials were −18±9mV in the same animals. Conclusion: The present study demonstrated that ischemic changes of [Ca2+]o in white matter were indifferent to the decrease in CBF or negative shifts of membrane potential. During early ischemia, [Ca2+]o of white matter even increased which may reflect significant shrinkage of extracellular space of the region. Lack of voltage-dependent transmitter gated channels and lack of spreading depression that is regularly seen in gray matter ischemia are responsible for the dynamics of extracellular Ca2+ during white matter ischemia.
THERAPEUTIC WINDOW OF BRADYKININ B2 RECEPTOR INHIBITION AFTER
Benjamin Klaesner2, David Lumenta2, Didier Pruneau3, Stefan Zausinger1,2, Nikolaus Plesnila1,2
1Laboratory of Experimental Neurosurgery, Department of Neurosurgery & Institute for Surgical Research, University of Munich Medical Center ? Grosshadern, Munich, Germany, 2Institute for Surgical Research, University of Munich Medical Center, Grosshadern, Munich, Germany, 3Groupe De Pharmacochimie Des Recepteurs, Centre De Recherche, Fournier Pharma, Daix, France
Backround and aims
Following cerebral ischemia bradykinin/kinin B2 receptors mediate inflammatory responses resulting in edema formation and secondary brain damage. However, the therapeutic window for B2 receptor inhibition determining its potential clinical use has not been investigated so far. The aim of the current study was therefore to investigate the effect of delayed B2 receptor inhibition on morphological and functional outcome following experimental stroke.
Methods
Rats were subjected to 90 min of middle cerebral artery occlusion (MCAo) by an intraluminal filament. Animals received 0.9% NaCl or 1.0 mg/kg/day Anatibant (LF 16-0687 Ms), a selective bradykinin B2 receptor antagonist, for 3 days beginning at different time points after MCAo: 1, 2.5, 4.5, or 6.5 h (n = 10 per group). Neurological recovery was examined daily, infarct volume on day 7 after MCAo.
Results
Animal physiology was not influenced by B2 receptor inhibition. Significant improvement of functional outcome was observed when treatment was delayed up to 4.5 h after ischemia (p < 0.05 versus vehicle). Inhibition of B2 receptors during ischemia, i.e. when the inhibitor was given 1 h after MCAo, reduced infarct volume in the basal ganglia and in the cortex by 49% (p < 0.05) and 26% (p < 0.05), respectively. Inhibition of B2 receptors at later time points (2.5, 4.5, or 6.5 after MCAo) reduced penumbral damage, i.e. cortical infarction, by 19?26% (p < 0.05).
Conclusions
In conclusion, the current study shows that the therapeutic window of B2 receptor inhibition extends for up to 6.5 h after MCAo. Our data therefore suggest that inhibition of kinin B2 receptors represents a treatment strategy for ischemic stroke which may warrant clinical validation.
EFFECT OF INTRAPERITONEAL ADMINISTRATION OF STAPHYLOCOCCAL ENTEROTOXIN-B ON THE PHOSPHORYLATION OF EXTRACELLULAR-REGULATED KINASE 1/2 IN THE HYPOTHALAMUS OF THE MICE
Fang Kuang, Bairen WAng, Zhenghua Zhu, Gong Ju
Institute of Neurosciences, Fourth Military Medical University, Xi'an, Shaanxi, China
Background and aims: The bacterial superantigen staphylococcal enterotoxin B (SEB) can trigger T-cell producing cytokines and activate the hypothalamic-pituitary-adrenal (HPA) axis to increase corticosterone secretion in vivo. Present study aimed to figure out the relationship between the phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in the hypothalamus and peripheral immune system response to SEB.
Methods: We injected SEB intraperitoneally to the adult BALB/c mice who were treated (intracerebralventricular administration) with an ERK pathway inhibitor, PD98059, or the solution control in advance. Six hours later, the blood sample was taken for detection of tumor necrosis factor-(TNF) alpha and corticosterone levels. Then the animals were sacrificed by perfusion, and the brain was sectioned to investigate the p-ERK1/2 by immunohistochemistry. The co-location of p-ERK1/2 and the neuron marker, NeuN was also observed with confocal microscopy.
Results: We found that p-ERK1/2 immunoreactive staining in hypothalamic paraventricular nucleus and supraotic nucleus was dramatically increased in BALB/c mice treated with SEB. The increased p-ERK1/2 by SEB stimulation was coincident with the elevation of TNF-alpha cytokine and corticosterone level in serum. Microscopically, the p-ERK1/2 was co-localized with NeuN, indicating the neuronal event. Furthermore, intracerebroventricular administration of PD98059 attenuated the elevation of serum TNF-alpha level responded to SEB stimulation, while the level of serum corticosterone was not affected significantly in the same time course.
Conclusions: These results provide evidences that ERK signaling in hypothalamus is affected by T cell-dependent immune challenge with superanitgen, or vice versa, and suggest that ERK1/2 pathway play an important role in brain modulation to SEB-induced immune activity. In addition, the results imply that the modulation of hypothalamus to the peripheral immune activity might not all dependent on the common route of HPA axis.
GLIAL GAP-JUNCTIONS IN THE RAT BTRAIN: ARE THEY ESSENTIAL FOR BRAIN FUNCTION DURING BRAIN ISCHEMIA?
Kentaro Tamura1, Ryo Tamaki1, Hiroyuki Nakase1, Toshisuke Sakaki1, Beat Alessandri2, Axel Heimann2, Oliver Kempski2
1Department of Neurosurgery, Nara Medical University, Kashihara, Japan, 2Institute for Neurosurgical Pathophysiology, Johanes-Gutenberg University, Mainz, Germany
Background: Gap junctions are reported to be important to maintain the physiological function in brain, e.g. spatial buffering of potassium. And they form gap junction intercellular communications (GIJC). But, it is still unclear which role gap junctions play in pathophysiological processes. We investigated 1) effect of gap junction blockade under physiological conditions with the gap junction blocker Carbenoxolone (CBX). 2) effect of gap junction blockade under ischemic conditions with CBX using our rat 2 vein occlusion model, which has rather wide penumbra area compared to infarct core size.
Method: 1) Male Wistar rats were used. After anesthetizing with chloral hydrate, lt. temporal craniotomy was made. CBX was injected into the rt. ventricle before spreading depression induction (25, 50, 250µg, saline, 10µg each). Spreading depression was induced with KCl injection into the cortex (150mM, 5µl, every 10 minutes, 7 times). Impedance and cerebral blood flow (CBF) were monitored.
2) After craniotomy, 2 adjacent cortical veins were occluded photochemically. CBX was injected into the rt. ventricle (250µg or saline, 10µl). Spreading depressions were induced with KCl injection (150mM, 5µl, every 7 minutes,10 times). Histological analysis was done 7 days after the operation (HE stain).
Results:1) After injection of CBX, CBF was elevated in the 250µg,50µg treated group compared to 25µg or saline treated group. Velocity of spreading depression was accelerated with 250µg CBX injection compared to saline treated group.2) Ischemic damage was aggravated with 250µg CBX injection compared to saline treated group(P<0.05).
Conclusion: Disturbed function of gap junctions can aggravate outcome from venous ischemia.
THE EFFECT OF OREXIN-A ON CORTICAL SPREADING DEPRESSION
Junko Yonekura, Junichi Hamada, Kenzo Koizumi, Michinari Fukuda, Shigeyoshi Maruyama, Fumihiko Sakai
Department of Neurology, Kitasato University School of Medicine, Kanagawa, Japan
Background and aims
Cortical spreading depression(CSD) is a short-lasting depolarization wave which moves across the cortex accompanied by changes in cellular activity and in cerebral blood flow(CBF). CSD has been discussed as a possible mechanism for the aura phase of migraine. Recently, orexin (hypocretin) was found as a regulator of sleep and awake cycle. We have recently observed that orexin-A induces an increase of cerebral blood flow in rats(unpublished observation). In the present study, we investigated the possible role of orexin-A in the CSD.
Methods
Eight male Sprague Dawley rats (350-450g) were anesthetized with α-chloralose(50 mg/kg) and urethane(500 mg/kg), intubated, and ventilated mechanically. The right femoral artery was cannulated for measurement of blood pressure, and other physiological parameters were measured. CBF was continuously monitored by laser-Doppler flowmetry. The cortical DC-potential was measured by extracellular platinum electrode. The stainless tube with polyethylene resin was placed in the right lateral ventricle to administer orexin-A. First, CSD was induced by dropping 1M KCl on rat brain surface and DC-potential and CBF were observed. Then, 0.3nmol orexin-A was injected in right lateral ventricle, and CSD was evaluated again. The mean value of CBF change was compared between pre and post injection. The average amplitude change of DC-potential was also evaluated in the same condition. For statistical analysis, we used paired t-test.
Results
After the administration of orexin-A, the mean value of CBF was decreased to 88.5± 6.3%(mean±SEM)(p=0.07) of pre injection. The amplitude of DC potential was suppressed to 82.6± 9.1%(mean±SEM)(p=0.06)after orexin-A. There was a tendency for suppression of CSD by orexin-A administration. There was no difference in physiological parameters between pre and post administration of orexin-A.
Conclusions
Our study evaluated CBF and DC potential during CSD and suggested that orexin-A may inhibit CSD. CSD is known to occur during migraine aura. We have previously shown that orexin-A has some role in the regulation of CBF. In the present study, it was suggested that orexin-A may play a role in the pathogenesis of migraine aura. It is a possibility that orexin-A prevent the aura of migraine. This is the first report that evaluated the relationship of orexin-A and CSD.
PREVENTING HYPOXIC BRAIN DAMAGE: EFFECTS OF DIRECT AND INDIRECT STEMCELL ADMINISTRATION IN A NEW CELL CULTURE MODEL OF THE PENUMBRA
Doreen Melanie Reich1, Susann Hau1, Manja Kamprad2, Markus Scholz3, Frank Emmrich1, Johannes Boltze1
1Department of Cell Therapy and Immunology, Fraunhofer Institute, Leipzig, Saxony, Germany, 2Institute Clinical Immunology and Transfusion Medicine, Leipzig University, Leipzig, Saxony, Germany, 3Institute for Medical Informatics, Statistics and Epidemiology, Leipzig University, Leipzig, Saxony, Germany
Pathophysiological models developed from animal studies form the basis of our understanding of the acute phase of stroke. In vivo data display a perfusion-releated dependency of neuronal cell damage. Nearly total loss of cerebral blood perfusion leads to necrotic cell death in the ischemic core while residual energy supply in the surrounding penumbra induces apoptosis whose early phases are reversible. Consequently, rescue of the penumbra is the basis of stroke therapy and the main determinant of clinical recovery. In this context, administration of external cell fractions recently demonstrated clear therapeutic benefit in vivo. However, complexity of in vivo models have hindered understanding of the mechanisms involved in functional recovery so far.
We established an well-defined in vitro model of the penumbra that can facilitate to address specific pathophysiological processes underlying ischemic damage and cell mediated neuroprotection. Our model is based on fully matured SH-SY5Y neuroblastoma cells that were cultivated in an oxygen reduced atmosphere for 48 hours to simulate apoptotic cell death upon hypoxia in the penumbra. Apoptosis level was detected via AnnexinV / PI assay and by quantification of apoptosis specific proteins.
We employed the system for direct as well as for indirect co-culturing with various fractions of stem cell containing mononuclear cells (MNC) from umbilical cord blood.
Direct co-culturing with MNC as well as with pure haematopietic stem cells derived from MNC provided significant protection from neuronal apoptosis over the course of 3 days, suggesting that transplanted cells may act in a paracrine manner with apoptotic neuronal cells to influence their activity and survival. Direct cell-cell-contacts did not only influence apoptotic ratio in neuronal cultures but also triggert retaining morphologial neuronal characteristics such as formig networks through multiple branched neurites.
Potential neuroprotective effects of soluble factors were investigated by affixing MNC and stem-cell-depleted-MNC-fractions in impermeable cell culture inserts. In this version of co-culturing external cell fractions prevented apoptosis in neuronal cells significantly as well, but not in the extent seen in direct co-cultures.
PREVENTION OF DELAYED INFARCT GROWTH BY NATURAL REGULATORY T-LYMPHOCYTES IS MEDIATED BY INTERLEUKIN-10
Arthur Liesz1, Elisabeth Suri-Payer2, Clemens Sommer3, Claudia Veltkamp4, Henrike Doerr3, Thomas Giese5, Roland Veltkamp1
1Department of Neurology, University Heidelberg, Heidelberg, Germany, 2Department of Immunogenetics, DKFZ Heidelberg, Heidelberg, Germany, 3Department of Neuropathology, University Mainz, Mainz, Germany, 4Department of Gastroenterology, University Heidelberg, Heidelberg, Germany, 5Department of Immunology, University Heidelberg, Heidelberg, Germany
Background and Aims: Inflammatory mechanisms are involved in brain damage and protection. Tregs are master anti-inflammatory modulators. We have recently shown that thymus derived CD4+CD25+Foxp3+ regulatory T-Lymphocytes are capable of preventing secondary infarct progression in a murine stroke model. In the present study, we tested the hypothesis that this natural adaptive Tcell response is mediated by Interleukin-10 (IL-10). We also examined the effect of Tregs on systemic cytokine levels after ischemia.
Methods: Permanent focal cerebral ischemia was induced by transtemporal middle cerebral artery occlusion (MCAO). In the first set of experiments, Tregs were eliminated by preischemic depletion with a monoclonal antibody (clone PC61) in C57Bl/6 mice. Mice were either treated with intraventricular IL-10 (200 µg) or with bovine serum albumin (control). Secondly, infarct size was compared in lymphocyte deficient rag2-/− mice after adoptive transfer of either CD4+CD25- plus CD4+CD25+ (both derived from wild type mice) or CD4+CD25- plus CD4+CD25+ (the latter derived from IL-10 -/− mutant mice). Plasma cytokine levels (TNF-alpha, IFN-gamma, IL-10, TGF-beta) were measured at various time points after MCAO using ELISA.
Results: Antibody-induced preischemic depletion of Tregs resulted in a significant increase of infarct size 7 d after MCAO (control: 7.4mm3; antibody-treated mice: 12.1mm3). Intraventricular injection of IL-10 reversed this effect almost completely (8.3 m3) to levels of mice with normal Treg cell counts. To determine whether Treg derived IL-10 is of particular importance, we performed adoptive cell transfer experiments with CD4+CD25+ cells derived from IL-10 -/− mice into rag -/−. Similar to mice lacking Tregs after antibody depletion, these animals had significantly larger infarcts 7 d after MCAO. Proinflammatory cytokines were significantly elevated in Treg-depleted mice
Conclusion: Thymic derived Tregs appear to be important counterregulators of inflammation-induced secondary infarct progression. This effect is largely mediated by IL-10 which is primarily derived from Tregs. In addition to affecting cerebral cytokine expression, Tregs also modify the systemic levels of key inflammatory cytokines.
CILOSTAZOL PROVIDES PROTECTION AGAINST CEREBRAL HYPOPERFUSION-INDUCED COGNITIVE IMPAIRMENT AND WHITE MATTER DAMAGE IN RAT
Takao Urabe, Terubumi Watanabe, Ning Zhang, Nobukazu Miyamoto, Yuji Ueno, Ryota Tanaka, Nobutaka Hattori
Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
Background and Aims: The number of patients afflicted with cerebral infarction is on the increase at present and stroke is a leading cause of disability worldwide, with no effective clinical treatment that enhances recovery. Post-stroke cognitive impairment and depressive disorders are recognized complications in such patients. The present study was designed to assess the neuroprotective mechanisms of cilostazol, a potent inhibitor of type III phosphodiesterase (PDE III), through signaling pathways, which lead to activation of transcription factor cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB) phosphorylation using rat chronic cerebral hypoperfusion model.
Methods: Adult male Wister rats (8-week-old) weighing 250-270 g underwent bilateral common carotid artery ligation. They were divided into the cilostazol group (n=80) and the vehicle (control) group (n=80). Performance at the Morris water maze task and immunohistochemistry for 4-hydroxy-2-nonenal (HNE), GST-pi, Iba-1, phosphorylated CREB (p-CREB), Bcl-2, and cyclooxygenase (COX)-2 were analyzed at baseline and at days 3, 7, 14, 21, and 28 days after hypoperfusion.
Results: Cilostazol significantly improved spatial learning memory (6.8±2.3 sec; P<0.05) at 7 days after hypoperfusion. Cilostazol markedly suppressed accumulation of HNE-modified protein and loss of GST-pi-positive oligodendrocytes in the cerebral white matter during the early period after hypoperfusion (P<0.05). Cilostazol up-regulated p-CREB and Bcl-2 (P<0.05), increased COX-2 expression, and reduced microglial activation in the early period of hypoperfusion.
Conclusions: We have shown that cilostazol provides protection against cerebral hypoperfusion-induced cognitive impairment and white matter damage in a rat bilateral carotid artery occlusion model. The results suggest that cilostazol has potential therapeutic and brain protective effects based on multi-target mechanism through cell signaling pathway of CREB phosphorylation, and may be helpful in the treatment of patients suffering from post-stroke complications.
VALPROIC ACID EFFECT ON THE REGULATION OF PURINERGIC RECEPTOR EXPRESSION AND PHAGOCYTOTIC ACTIVITY IN MICROGLIA
Wen-Hsin Lu, Kuan Ming Fang, Tsung-I. Lee, Shun-Fen Tzeng
Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan-R.O.C.
Microglia, the immune cells of the central nervous system (CNS), are responsible for the removal of cell debris after CNS injury, and play a constructive role in tissue remodeling and repair. Valproic acid (VPA) which is a common anti-seizure drug clinically used for the treatment of bipolar disorder has recently been found to have neuroprotective and anti-inflammatory activity by the regulation of glial response to inflamed environment. In our previous study, treatment of BV2 cells, an immortalized murine microglial cell line, with VPA caused their intracellular calcium rise and enhanced their phagocytotic activity without the involvement of inducible nitric oxide synthase. The present study showed that the expression of P2X4, a subtype of ATP receptors, was downregulated in VPA-treated BV2 cells by quantitative RT-PCR analysis, while VPA had no effect on P2X7 mRNA in BV2 cells. Contradictorily, upon treatment with a selective P2X agonist 2-methylthioATP (2-meSATP), a slight increase in phagocytotic activity was observed along with an increase in the intracellular calcium levels of VPA-treated BV2 cells. The results provide the view that P2X4 may not be the major player for BV2 phagocytosis. Since immortalized BV2 cells may not retain the nature of primary microglia, we continued to examine the effect of VPA on P2X4/P2X7 expression in primary rat microglia. In contrast to the observation from VPA-treated BV2 cells, the mRNA expression of P2X7, but not P2X4, was inhibited by VPA application to primary microglial culture. The phagocytotic activity in VPA-treated microglia was also significantly suppressed in response to 2-meSATP, indicating the possible link of P2X7 role to microglial phagocytotic activity. Together, our findings demonstrate that VPA may modulate puringeric signaling via the regulation of P2X receptor expression to mediate microglial phagocytotic function.
THE KINETICS OF OREXIN-A/HYPOCRETIN-1 IN STROKE - CLINICAL AND BASIC INVESTIGATIONS-
Kenji Dohi1, Beth Ripley3, Nobuhiro Fujiki3, Hirokazu Ohtaki2, Tomoya Nakamachi2, Seiji Shioda2, Tohru Aruga1, Seiji Nishino3
1Department of Emergency and Critical Care Medicine, Showa University, Tokyo, Japan, 2Department of Anatomy, Showa University, Tokyo, Japan, 3Stanford University Center for Narcolepsy, Palo Alto, CA, USA
Background and Methods
Two novel hypothalamic neuropeptides, orexin-A (hypocretin-1) and orexin-B (hypocretin-2), have recently been identified. Subsequent studies discovered that a chronic human sleep disorder, namely narcolepsy, is specifically associated with a reduced production of the hypocretin/orexin peptides in the hypothalamus. This orexin/hypocretin deficiency in the brain of narcoleptics has been clinically detected to demonstrate either reduced or undetectably low orexin-A/hypocretin-1 levels in the CSF. Orexins/hypocretins were initially characterized as potent stimulants of food intake, but they have also been shown to be involved in various hypothalamic functions, including energy homeostasis, neuroendocrine and autonomic nervous system functions, pain perception and vigilance control. Orexin/hypocretin may also be involved in various neurological conditions associated with hypothalamic dysfunctions, and reduced CSF orexin-A/hypocretin-1 levels have been reported to occur in subsets of patients with brain tumors, neurotrauma, vascular diseases, infections, Parkinson's disease and Guillain? Barre' syndrome, as well as symptomatic cases of narcolepsy due to various underlying causes. The aim of this study was to examine the role of the hypothalamic orexin-A/hypocretin-1 system in patients with hemorrhagic stroke (SAH and ICH) patients.
(Results and Conclusions)
CSF orexin-A/hypocretin-1 levels were low both in SAH and ICH patients. In SAH patients, CSF hypocretin-1/orexin-A levels were lower in patients with symptomatic vasospasms complications than in those who did not develop. In ICH patients, the CSF orexin-A/hypocretin-1 levels were lower in the thalamic hemorrhage patients than those in other patients.
These data suggest that a reduction in hypocretin/orexin production in hemorrhagic stroke patients is associated with alterations in brain hypocretin/orexin signaling in response to ischemia and hypothalamic dysfunction.
In the current study, we also refer to the time-dependent changes in cerebrospinal fluid (CSF) orexin-A concentration and the expression of the orexin-1 receptor (OX1R) in the rat hippocampus after global ischemia-reperfusion (5 min cardiopulmonary arrest).
FEBRILE NEUTROPENIA IN PATIENTS WITH MULTIPLE SCLEROSIS (MS) AND HASHIMOTO THYROIDITIS (HT)
Dimitrios Kountouris, Kon. Koutsobelis, Agg. Bougioukou, Kon. Karachristou
Neurological Diagnostic Center, Athens, Greece
BACKGROUND AND AIMS
Relapsing infections are usual and severe complications in patients genetically susceptible for neutropenia or suffering from periodical neutropenia in the context of autoimmune disorders. As a result, regular biochemical control is necessary for the prevention of febrile neutropenia.
METHODS
We examined three patients (two male and one female) diagnosed with definite Multiple Sclerosis (MS), according to EDSS Kurtzke scale and subclinical Hashimoto Thyreoiditis (HT), with raised titles of antithyroid antibodies. The patient periodically suffered from febrile neutropenia accompanying relapsing infections, not responding to antibiotics. We observed that these infectious relapses occurred after stressful events or in the context of MS deterioration. During periods of febrile neutropenia, the patients undertook detailed neurological, neurophysiological and biochemical control.
RESULTS
According to our findings, neutropenia co-existed with significantly increased levels of thyroid antibodies along with neurophysiological and radiological evidence for MS relapse. Furthermore, febrile neutropenia subsided only after treatment with intravenous immunosuppressive therapy.
We observe that febrile neutropenia indisputably originates from an autoimmune aetiology, given that it follows HT and MS outbreaks. We should also notice that autoimmune disorders regression commnoly coincides with stressful events.
CONCLUSION
In patients suffering from autoimmune disorders, regular complete control is preventive both for MS or HT relapse, but also for the manifestation of febrile neutropenia.
PUMA IS INVOLVED IN ACUTE BRAIN INJURY AFTER SUBARACHNOID HEMORRHAGE IN RATS
Kuniyasu Niizuma1, Hidenori Endo1,2, Miki Fujimura2, Teiji Tominaga2, Pak H. Chan1
1Departments of Neurosurgery, Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Stanford, CA, USA, 2Department of Neurosurgery, Tohoku University Graduate School of Medicine, Sendai, Japan
Background and aims: Subarachnoid hemorrhage (SAH) results in a high mortality rate, despite sophisticated medical management and neurosurgical techniques. Recent studies have emphasized the importance of acute brain injury after SAH, in which apoptosis is involved as one of the major mechanisms. We have reported the involvement of mitochondrial cytochrome c release in apoptotic cell death after SAH. However, the molecular mechanism of this cytochrome c release remains unresolved. PUMA (p53-upregulated modulator of apoptosis), which is one of the BH3-only proteins, was shown to localize to mitochondria and to function to induce cytochrome c release in several recent in vitro studies. These reports inspired us to investigate the role of PUMA in the mechanisms of acute brain injury after SAH.
Methods: To examine the relationship between acute brain injury and the PUMA pathway following SAH, we used a perforation SAH model in rats. One, 6 and 24 hours after SAH, samples were taken from the cerebral cortex and used for Western blot, coimmunoprecipitation or immunohistochemistry. Protein extraction of the mitochondrial fraction was performed using a multiple centrifugation method for Western blot and coimmunoprecipitation. To investigate apoptotic cell death after SAH, DNA fragmentation was analyzed with a commercial enzyme immunoassay. Finally, we intravenously administered an inhibitor against p53, pifithrin-α (PFT), to examine the role of PUMA as a downstream target of p53 and the relationship between PUMA inhibition and acute brain injury.
Results: Western blot analysis showed that the expression of PUMA significantly increased in the mitochondrial fraction 6 hours after SAH. Double immunofluorescence for PUMA and COX demonstrated that PUMA colocalized with COX (which was used as a mitochondrial marker) in the cerebral cortex 6 hours after SAH. PUMA expression precipitated by Bcl-XL in the mitochondrial fraction increased time dependently and a significant increase was observed at 1 and 6 hours. Western blot analysis showed a significant increase in cytosolic cytochrome c 24 hours after SAH compared with sham-operated brains. Double immunofluorescence showed that some cortical cells with strong PUMA immunoreactivity colocalized with cytochrome c-positive cells at 24 hours. Western blot analysis showed that mitochondrial localization of PUMA, which was transcriptionally controlled by p53, and cytosolic cytochrome c release were significantly inhibited by PFT 6 hours after SAH. Moreover, DNA fragmentation at 24 hours was significantly decreased in the PFT-treated animals compared with vehicle-treated animals.
Conclusions: We found that PUMA is upregulated in mitochondria and directly binds to Bcl-XL after SAH. Mitochondrial localization of PUMA and protein interaction between PUMA and Bcl-XL might induce release of cytochrome c to the cytosol, resulting in acute brain injury following SAH.
INDUCTION OF PLATELET DERIVED-ENDOTHELIAL CELL GROWTH FACTOR IN THE BRAIN AFTER ISCHEMIA
Xiquan Wang, Takeshi Hayashi, Hanzhe Zhang, Kentaro Deguchi, Shoko Nagotani, Yoshihide Sehara, Atsushi Tsushiya, Toru Yamashita, Violeta Lukic, Tatsushi Kamiya, Koji Abe
Department of Neurology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
Backgrounds: Platelet derived-endothelial cell growth factor (PD-ECGF) is a highly potent angiogenic factor. Although angiogenesis plays active role in pathophysiology of stroke, the expression pattern of this molecule in ischemic brain has not been investigated. In the present study, therefore, we investigated change in PD-ECGF expression in the brain after ischemia.
Methods: With use of male Wistar rats (250-289 g), the right middle cerebral artery (MCA) was occluded by a nylon thread for 90 min under anesthesia with isoflurane. The animals were decapitated at 3 h, 1, 4, and 10 d after the reperfusion, and frozen sections were prepared. We then performed immunohistochemistry for PD-ECGF, and counted the positively stained cells in the cerebral cortex at the MCA boundary area. In addition, we carried out fluorescent double staining for PD-ECGF and NeuN or glial fibrillary acidic protein (GFAP), in order to identify the cell phenotype which strongly expressed PD-ECGF.
Results: In the sham-operated brain, only small numbers of cells slightly expressed PD-ECGF. The number of positively stained cells increased at the peri-ischemic area from 3 h of reperfusion. Not only small-sized cells but also large-sized cells became stained. The number of stained cells further increased, and peaked at 4 d for large-sized cells and at 10 d as to small-sized cells. Fluorescent double staining revealed that both large-sized and small-sized cells which were stained for PD-ECGF expressed NeuN (Fig. A) but not GFAP (Fig. B), indicating that neurons are the main source of PD-ECGF production in the ischemic brain.
Conclusion: PD-ECGF has a strong angiogenic property without vascular permeability increasing effect. This molecule was useful for reducing ischemic heart injury. This molecule may have therapeutic potential also for ischemic stroke treatment.
CLINICAL EXPERIENCE WITH USE OF ICM+ SOFTWARE FOR CONTINUOUS ANALYSIS OF CEREBROVASCULAR DYNAMICS
Peter Smielewski, Marek Czosnyka, Ivan Timofeev, Andrea Lavinio, John Pickard
Department of Clinical Neurosciences, University of Cambridge, UK
Background and aims: The ICM+ software encapsulates 20 years of experience of brain monitoring gained in multiple neurosurgical and intensive care centres. It collects data from bedside monitors and produces time trends of parameters defined using configurable signal processing formulas. We summarized our experience from the first 3 years of use of the software.
Methods: 122 severely head injured patients were managed in the Neuro Critical Care Unit. Intracranial pressure (ICP) was monitored using Codman intraparenchymal probes and arterial blood pressure (ABP) was measured invasively from the radial artery. Signals were monitored with ICM+ software, calculating mean values of ICP, ABP, cerebral perfusion pressure (CPP) and various indices describing pressure reactivity, compensation and vascular waveforms of ICP.
Results: Mean ICP was 18 mm Hg, mean CPP was 72 mm Hg. Nine patients showed permanent disturbance of cerebral autoregulation (mean pressure reactivity index above 0.3). Pressure reactivity index demonstrated significant U-shape relationship with CPP, suggesting loss of pressure reactivity at low (CPP<55 mmHg) and high CPPs (CPP>95 mm Hg). The valley of the relationship CPP (termed ‘optimal CPP’) differed from patient to patient but also during patient stay in the unit.
Conclusion: ICM+ software proved to be useful clinically in the Neuro Critical Care Unit. It allows continuous monitoring of pressure reactivity and its ‘optimal CPP’ which could redefine the meaning of ‘CPP’ orientated therapy.
SUPPRESSION OF AQUAPORINS EXPRESSION IN BRAIN EDEMA AFTER TRANSIENT FOCAL CEREBRAL ISCHEMIA BY AGMATINE ADMINISTRATION
Jae Hwan Kim1,2, Yong Woo Lee1,2, Kyung Ah Park1, Won Taek Lee1, Jong Eun Lee1,2
1Department of Anatomy, Yonsei University College of Medicine, Seoul, South Korea, 2Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea
Background and aims: Edema is frequently observed in brain ischemia. Brain edema, defined as an abnormal increase in brain water content, which leads to an expansion of brain volume, has a crucial impact on morbidity and mortality after stroke (Klatzo, 1985). Aquaporins (AQPs) are a family of water channel proteins that facilitate the diffusion of water through the plasma membrane (Agre, 2002). In the rodent brain, three aquaporins have been clearly identified, AQP-1, AQP-4, and AQP-9 (Badaut, 2002; Agre, 2004). AQP-1 has been detected in epithelial cells of the choroid plexus (Nielsen, 1993), AQP-4 in astrocytes with a polarization on astrocyte endfeet (Nielsen, 1997), and AQP-9 in astrocytes of the white matter and in catecholaminergic neurons (Badaut, 2001; 2004). AQP-1 and AQP-4 are permeable only to water and are presumed to be involved in cerebrospinal fluid formation and brain water homeostasis (Amiry-Moghaddam, 2003). These channels may be implicated in water movements occurring during the formation and resolution of cerebral edema after ischemia. Agmatine was previously reported to be neuroprotective on cerebral ischemia (Kim, 2004). In this study, the change of aquaporins expression by agmatine administration was investigated in brain edema after ischemia.
Methods: Using a well established model of focal cerebral ischemia, male ICR mice (36±2 g) were subjected to 2 hours middle cerebral artery occlusion (MCAO). Agmatine was treated at the end of occlusion (100mg/kg, IP). Water content was analyzed in 24 hours after ischemia and the expression of aquaporins was analyzed using immunohistochemistry and immunoblotting.
Results: The water content in ischemic injured brains at 22 hours reperfusion is shown in Figure 1. Ischemia led to a significant increase in water content in the ipsilateral hemispheres (88.23 ± 0.59 %, P < 0.01). However, in agmatine treatment group, water content was significantly decreased in the ipsilateral hemispheres (80.61 ± 1.33 %, P < 0.01). The expression of aquaporin-1 and −4, correlated with brain as water channels, was plainly decreased by agmatine treatment (Figure 2).
Conclusions: These results suggest that agmatine may be a novel therapeutic material for brain edema through the control of water contents and the expression of aquaporins.
SPM AND SPAM ANALYSES OF BASAL/ACETAZOLAMIDE BRAIN SPECT FOR EFFICACY ASSESSMENT OF ENDOVASCULAR STENT PLACEMENT OF MIDDLE CEREBRAL ARTERY STENOSIS
Tae-Hong Lee1,2, Seong-Jang Kim2,3, In-Ju Kim2,3, Yong-Ki Kim2,3, Kyung Pil Park2,4
1Department of Radiology, PNUH, Busan, South Korea, 2Medical Research Institute, PNUH, Busan, South Korea, 3Department of Nuclear Medicine, PNUH, Busan, South Korea, 4Department of Neurology, PNUH, Busan, South Korea
Purpose: Statistical parametric mapping (SPM) and Statistical probabilistic anatomical mapping (SPAM) were applied to basal/acetazolamide Tc-99m ECD brain perfusion SPECT images in patients with middle cerebral artery (MCA) stenosis to assess efficacy of endovascular stent of MCA.
Material & methods: Eleven patients (8 men and 3 women, the mean age of 54.2±6.2 year) who were undergone endovascular stent placement for MCA stenosis were enrolled. Using SPM and SPAM analyses, we compared the significant number of voxels and cerebral counts between pre-and post-stent basal/acetazolamide SPECT, and assessed the perfusion changes and cerebral vascular reserve index (CVRI).
Results: The numbers of hypoperfusion voxels in basal and acetazolamide SPECT were decreased from 10,083 ± 8,326 to 4,531 ± 5,091 in basal images (p=0.0317) and from 13,398±14,222 to 7,699±10,199 in acetazolamide images (p=0.0142) after MCA stenting. With SPAM analysis, the increases in cerebral counts were significant in the acetazolamide images (90.9±2.2 to 93.5±2.3, p=0.0098) but not in basal images (91±2.7 to 92±2.6, p=0.1602). The CVRI showed also statistically significant increase from pre-stent (median 0.32; 95% CI, −2.19–2..37) to post-stent (median 1.59; 95% CI, −0.85–4..16) (p=0.0068).
Conclusion: This study revealed the usefulness of voxel based analysis of basal/acetazolamide brain perfusion SPECT after MCA stent placement. From this study, SPM and SPAM analyses of basal/acetazolamide Tc-99m brain SPECT could evaluate the short term hemodynamic efficacy of successful MCA stent placement.
THE INFLUENCE OF BLOOD AND VOLUME ON BRAIN SWELLING, HISTOLOGICAL DAMAGE AND FUNCTIONAL OUTCOME AFTER ACUTE SUBDURAL HEMATOMA IN RATS
Oliver Kempski1, Heidrun Baechli2, Melika Behzad1, Swetlana Ens1, Chrysostomos Papaioannou1, Beat Alessandri1
1Institute for Neurosurgical Pathophysiology, Johannes Gutenberg-University, Mainz, Germany, 2Neurosurgical Clinic, University Hospital and Children's University Hospital Basel, Basel, Switzerland
Bachground and Aim: Acute subdural hematoma (ASDH) is a devastating injury following traumatic brain injury. The evacuation of the extravasated blood mass early after hemorrhage is able to reduce mortality significantly. Still mortality remains high which leads to the suggestion that blood itself affects lesion development also. Therefore, we compared the effect of autologous blood with paraffin oil as sheer volume on acute pathophysiological parameters, histological and functional outcome in a model of ASDH in rats.
Method: We used an established model of ASDH and infused either 300 µL autologous blood or paraffin oil (50 µL/min). Sprague-Dawley rats were monitored for arterial blood pressure (MAP), intracranial pressure (ICP), brain tissue oxygen (ptiO2) and local cerebral blood flow (CBF) for up to 2 hours after infusion. Water content was measured 2 and 24 hours after injury and histological damage was assessed at 2 hours, 1, 4 and 12 days post-injury. In addition, animals were tested for neurological deficits for 11 days after trauma.
Results: Sham-operated animals had stable MAP(78.2±2.5 mmHg), ICP (4.4±0.5 mmHg) ptiO2 (30.6±1.4 mmHg) and CBF values (38.4±1.7 LDU) throughout the acute observation period and ipsilateral water content was 78.79±0.12%. There was no histological and functional deficit. Blood infusion increased ICP to 45.3±3.0 mmHg and decreased ptiO2 to 11.2±1.9 mmHg as well as CBF to 7.5±1.4 LDU measured at 10 min post-trauma which was not significantly different to the values after paraffin oil infusion (ICP: 40.5±3.1 mmHg, ptiO2: 15.2±3.0 mmHg; CBF: 9.6±1.5 LDU) (n=5-9/group, p??s>0.25). Within 30-40 minutes ICP dropped in both groups to 20 mmHg, but CBF remained very low. Both infusion groups had no elevated water content at 2h after trauma, but differed significantly at 24h (blood: 80.5±0.33 %; paraffin: 79.3±0.17 %; n=10/group, p<0.01). Contrarily, histological damage was already visible at 2h(11.6±3.6 mm3 vs 2.4±0.7 mm3) and increased within 24 hours to 35.7±9.0 mm3 (blood) and 4.2±2.4 mm3 (paraffin) reaching 34.7±8.7 mm3 and 15.8±2.6 mm3 (n=10/group, p<0.05) at day 11 after trauma. This was accompanied by an impaired neurological outcome score at day 11 (blood: 10±2.0; paraffin 2.0±1.3, n=10/group, p<0.001).
Conclusion: The results indicate that a subdural volume causes a repeatable pattern of acute pathophysiological changes during and after infusion. Although there is no difference in acute swelling at 2h between blood and paraffin oil infusion, blood causes significantly more swelling at later time-points. A difference in histological damage, however, starts to appear already hours after injury and persists, as also seen for the difference in functional deficits, throughouts the experiment between the two groups. This suggests that factors released from extravasated blood early after subdural hemorrhage potentiate the volume-induced damage.
The study is supported by a grant of ??Deutsche Forschungsgesellschaft?? (KE-338/6-2).
PHOSPHOLIPASE C FUNCTION IN VASCULAR CONTRACTION BY ENDOTHELIN-1
Yoshifumi Kawanabe1, Nobuo Hashimoto2
1Department of Neurosurgery, Takatsuki Red Cross Hospital, Osaka, Japan, 2Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
Endothelin-1 (ET-1) is a major cause of cerebral vasospasm after subarachnoid hemorrhage (SAH). In addition, extracellular Ca2+ influx plays an essential role for ET-1-induced vasospasm. We recently demonstrate that ET-1 activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in rabbit basilar artery vascular smooth muscle cells (VSMCs). In this study, we investigate the effects of phospholipase C (PLC) on ET-1-induced activation of these Ca2+ channels and basilar artery contraction by using PLC inhibitor, U73122. To determine which Ca2+ channels are activated via PLC-dependent pathway, monitoring of intracellular free Ca2+ concentration ([Ca2+]i) is performed. Role of PLC in ET-1-induced vascular contraction is examined by tension study using rabbit basilar artery rings. U73122 inhibits ET-1-induced transient increase in [Ca2+]i, which results from mobilization of Ca2+ from the intracellular store in VSMCs. PLC also inhibits ET-1-induced extracellular Ca2+ influx through SOCC and NSCC-2, but not NSCC-1. U73122 inhibits the ET-1-induced contraction of rabbit BA rings that depends on extracellular Ca2+ influx through SOCC and NSCC-2. These results indicate that (1) SOCC and NSCC-2 are stimulated by ET-1 via PLC-dependent cascade, whereas NSCC-1 is stimulated via PLC-independent cascade; (2) PLC is involved in the ET-1-induced contraction of rabbit basilar artery rings that depends on extracellular Ca2+ influx through SOCC and NSCC-2.
CONTINUOUS EVALUATION OF REGIONAL OXYGEN SATURATION USING INVOSR FOR MONITORING OF CEREBRAL VASOSPASM AFTER SUBARACHNOID HEMORRHAGE
Shigeki Ono, Keisuke Onoda, Koji Tokunaga, Kenji Sugiu, Isao Date
Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistory, and Pharmaceutical Scinences, Okayama, Japan
Objective
There are several tools for detection of cerebral vasospasm (VS) after subarachnoid hemorrhage (SAH), however, transcranial Doppler (TCD) is not always accurate for detecting VS, and microdialysis is, somehow, an invasive method. Although angiography is a gold-standard method to make a diagnosis of VS, it is not continuous and is not a bed-side examination for detecting VS. INVOSR monitoring which measures the regional oxygen saturation (rSO2) by using near inflated spectroscopy has been used for detecting cerebral ischemia during operations as reported elsewhere. We here use this noninvasive monitoring system for detecting cortical ischemia due to VS by measuring the continuous rSO2 after SAH, and determine whether it is useful for detecting VS and beneficial for treatment of VS.
Methods
Six patients who suffered from SAH were nominated in this study. The probes of INVOSR (INVOS 5100R, Somanetics Corporation, MI, USA) were attached to the scalp in the areas anticipated to show VS considering the thickness of clots, from day 3 to the end of VS. After clipping or coiling, angiography was routinely performed around day 7 after SAH, or, at the timing if rSO2 continuously decreased by 10% compared to that of the contralateral side, or when the patients showed additional neurological deficits.
If VS was detected, interventional treatment was performed using superselective intraarterial injection of Fasudil hydrochloride by microcatheters or percutaneous transluminal angioplasty if possible. This performance was once in a day at a maximum, and if the patients deteriorated the next day, the interventional therapy was repeatedly performed until the neurological deficits recovered.
Results
The locations of ruptured aneurysms were 2 distal anterior cerebral artery, and 2 internal carotid-posterior communicating artery, one middle cerebral artery, and one basilar artery-superior cerebral artery. Angiographic VS was seen in 4 of 6 patients. Symptomatic VS was seen in 2 of the patients having angiographic VS. Generally, INVOS value was around 70-65 in the both side in the early phase of the hemorrhagic attack, and then, gradually stabilized around the value of 60 from around the day 4 to 6. When INVOS value became less than 55, angiographic or symptomatic VS tended to occur. Recovery from ischemia seen on angiography or single photon emission CT also correlated with reincrease of rSO2 by INVOSR.
Discussion
INVOSR is handy and noninvasive monitor for detecting VS after SAH. And it also evaluates chronological real-time rSO2 in the region of VS. This monitoring system may be superior to the conventional monitoring systems such as TCD or single photon emission CT.
NEW TREATMENT PROTOCOL OF METASTATIC BRAIN TUMOR FOR REDUCING RADIATION-INDUCED COGNITIVE DYSFUNCTION
Shinya Sato, Wataru Mouri, Kaori Sakurada, Makoto Saino, Takamasa Kayama
Department of Neurosurgery, Yamagata University Faculty of Medicine, Yamagata, Japan
(Introduction) Cognitive function is increasingly regarded as an important outcome measure in patients with metastatic brain tumor. Radiotherapy is the mainstay in the treatment of patients with brain metastases of extracranial tumors. The risk of developing a devastating late-delayed encephalopathy caused by whole-brain radiation therapy has been estimated to range between 1.9% and 18.4%. In particular, the use of high daily fractions (over 2Gy) is thought to be deteimental. The role of whole-brain therapy after surgery or stereotactic radiosurgery of one or few brain metastasis is therefore the subject of debate. Herein, we report the ongoing randomized phase III trial of prospective whole brain radiation(WBRT) therapy compared with salvage stereotactic radiosurgery(SRS) in patients with one to four brain metastases: Japan Clinical Oncology Group Study (JCOG0504) for reducing cognitive function damage caused by irradiation.
(Objectives) The aim of this study is to evaluate non-inferiority of salvage SRS in the patients who received surgical resection for brain metastases in comparison with postoperative WBRT.
(Assessment) Primary outcome: overall survival, Secondary outcomes: Propotion of performance status preservation, proportion of mini-mental status examination preservation, and adversive events.
(Treatment Arms) A: Whole brain radiation therapy arm: Patients receive WBRT in daily 2.5Gy fractions to a total 37.5 Gy over 3 weeks. No concomitant systemic therapy is given in WBRT period. B: Salvage stereotactic radiosurgery arm: If patients have residual brain metastases after surgical resection, they receive SRS for residual metastases. If patients have no residual brain metastasis, they observed without radiotherapy for brain. When the recurrence or progression of residual brain metastasis is observed, patients receive salvage SRS. SRS is restricted up to 8 lesions.
(Eligibility Criteria) (1) one to four brain metastases with a maximum diameter of 3 cm or more for the largest lesion, (2) all brain metastases localized within cerebrum or cerebellum, (3) before surgical resection for brain metastasis, PS is 0-2, or 3 by neurological deficits, (4) surgical resection for the largest brain metastases has achieved, (5) after surgical resection, four or fewer (0-4) residual lesions with maximum diameter under 3 cm, (6) histologically proven non-small cell carcinoma, breast cancer or colorectal cancer, (7) primary lesion and the other metastases (i.e. lung, liver, bone metastases except for brain) is consider to be controlled, (8) an age of 20-79 years, (9) no prior surgery or irradiation for brain.
(Trial registration) umin.ac.jp/ctr identifier: C000000307.
EVALUATION OF CALCIFIED FOCUS FOR SURGERY OF CAROTID STENOSIS
Hiroyuki Katano, Masao Nakatsuka, Yotaro Takeuchi, Atsuo Masago, Noritaka Aihara, Atsushi Umemura, Mitsuhito Mase, Kazuo Yamada
Department Of Neurosurgery And Restorative Neurosciences, Nagoya City University Graduate School Of Medical Sciences, Nagoya Aichi, Japan
Background and aims: We previously investigated carotid plaques in focus of calcification using MD (multi-detector row) CT and the specimens removed during carotid endarterectomy (CEA) were pathologically investigated for comparison. We reported calcifications were microscopically classified into granular, lump and laminar types, and lesions depicted as similar calcification in MDCT may include relatively soft calcifications of granular type. We pointed out that the fact should influence the result of carotid angioplasty and stenting (CAS), because hard calcification might sometimes prevent expansion of stent. The aim of the study was to analyze calcification of carotid plaques in quality (hardness) and quantity (volume) of each calcified focus on MDCT and discussed whether Agatston calcium score could also apply as a useful tool for evaluating plaques with calcification as in coronary artery diseases, especially for deciding indications of options for surgical treatment for carotid stenosis, CEA or CAS.
Methods: A total of 124 carotid bifurcations of consecutive 62 patients (mean age 70.6 ± 6.4 years, male : female = 53 : 9) were examined with 16-row multidetector CT (IDT-16, Philips, Netherland) before and after surgical interventions. Seventy carotid arteries which at least two pixels (0.67mm3) of calcification were detected on volume rendering (VR) or maximum intensity projection (MIP) images of MDCT were chosen for further calcium analysis. Among all carotid bifurcations, surgical interventions were performed on 65 carotid arteries, 44 for CEA and 21 for CAS including three restenoses. Calcification of the carotid plaque was quantified using the specialized software implemented in the workstation (Aquarious®). Calcium scores were determined according to the method described by Agatston et al.
Results: Agatston calcium scores ranged from 3.7 − 4274.2 (mean 540.5). Volume scores ranged from 2.5 − 3205.7mm3 (mean 405.8 mm3), while averaged Hounsfield Units ranged from 255.9 − 871.7 (mean 595.2) and peaked HU 389 − 2075 (mean 1025.4). Mean and peak Hounsfield Units in order of volume scores demonstrated that calcification with larger volume score tended to have bigger difference between mean and peak Hounsfield Units in a single case. Of all calcified plaques, 44.7 % (38/85) had the 80% harder portion compared to the area with mean hardness. The calcified lesions having this feature existed only 35.3% (24/68) of those with volume score under 500 mm3, while volume score is higher than 500, 600 and 700, the percentage rose up to 82.4 (14/17, Fig. 4), 90.9 (10/11) and 100% (8/8), respectively.
Conclusions: The present study indicated that Agatston calcium score is useful in consideration of indication for CAS, when calcium in carotid plaque being small to medium size, but in larger ones, individual inspection for Hounsfield units and volumes should be recommended in certain cases.
A NEW PRIMATE MODEL OF FOCAL STROKE: INTRALUMINAL OCCLUSION OF THE MIDDLE CEREBRAL ARTERY IN THE COMMON MARMOSET
Thomas Freret1,2, Valentine Bouet1, Jerome Toutain1, Romaric Saulnier1, Palma Pro-Sistiaga1, Eric MacKenzie1, Simon Roussel1, Pascale Schumann-Bard1, Omar Touzani1
1University of Caen, CNRS-UMR 6185, Cyceron, Caen, France, 2University of Caen, Laboratory of Pharmacology-Physiology, CERMN-EA 3915, Caen, France
Background and aims: The failure of many clinical trials of neuroprotection is often attributed, at least in part, to poor animal models of human stroke. In the present study, we sought to develop a stroke model in non-human primates by an intraluminal approach to occlude the middle cerebral artery (MCA). This technically simple procedure allows both transient and permanent ischemia with minimal morbidity.
Methods: Ten common marmosets underwent either transient (3 h) or permanent ischemia by the insertion of a nylon filament through the external carotid artery up to the origin of the MCA. Cortical cerebral blood flow (CBF) was monitored by the laser-Doppler flowmetry technique. Sensorimotor functions were regularly evaluated by a battery of tests previously described by Marchall et al., (1996). Histological and MRI analyses were performed 8 days following the induction of ischemia. The data are mean ±SD.
Results: MRI-derived angiography shows that the internal carotid artery of the marmoset, presents a distinct carotid siphon before the emergence of the MCA. Despite this intracranial curvature, the filament, with a distal cylinder of 3 mm length and 0.54 mm diameter, was introduced straightforwardly through the ECA up to the origin of the MCA. Not post-operative mortality or overt cerebral hemorrhage was seen after the surgery. During the three hours following the occlusion, no difference in decreased CBF was observed between the two groups (two-way ANOVA, p=0.08). In transiently occluded marmosets, withdrawal of the filament resulted in a hyperperfusion that lasted at least 30 min following reperfusion. In all animals, ischemic damage was observed in the caudate, the putamen, the internal capsule as well as the cortical areas surrounding the lateral sulcus. The volume of cortical infarction was not different between reperfused and non-reperfused animals (164±117 and 172±108mm3, respectively). However, the former displayed less damage in sub-cortical structures, (73±34 and 186±61mm3, respectively p<0.05 t-test). In all the behavioral tests used, reperfused marmosets exhibited less neurological and functional impairments compared to permanently occluded ones.
Conclusion: We show the feasibility of the induction of permanent or transient focal cerebral ischemia in the marmoset by the intraluminal approach with minimal invasion. This model could be suitable as an advanced screening for potential stroke therapies in which behavioral, imaging and histological analyses can be compared.
THE GENETIC RESPONSE TO ISCHEMIC TOLERANCE IN RAT HIPPOCAMPUS
Hemal Patel1, Zheng Feng1, Daniel Davis1, Roman Sasik1, Brian Head1, Yasuo Tsutsumi1, David Roth1,2, John Drummond1,2, Piyush Patel1,2
1University of California, San Diego, San Diego, CA, USA, 2VA San Diego Healthcare Systems, San Diego, CA, USA
Ischemic tolerance (IT) is a phenomenon whereby a sublethal ischemic insult (SLI) provides robust protection against subsequent lethal ischemia. Numerous studies have outlined the genetic program initiated by a preconditioning stimulus; however, limited studies have considered how a preconditioning stimulus modifies gene response during lethal ischemia. Thousands of genes can be simultaneously screened by microarray analysis; however, difficulty remains in identifying candidate genes from mere epiphenomenon. Activation of N-methyl-D-aspartate (NMDA) receptors is essential for IT induced by SLI. We therefore administered MK801, an NMDA antagonist, to define a specific subset of genes that mediate IT in rat hippocampus. Animals subjected to SLI underwent 3 min of bilateral carotid artery occlusion with simultaneous hypotension (BCAO). Seventy-two hours later, the rats underwent 6 min BCAO. Brain tissue was harvested immediately after lethal ischemia or 1h, 4h and 24h post ischemia (n=5/group/time point). Extracted mRNA were pooled and hybridized to Affymetrix arrays. Genes were sorted according to the extent of differences between the vehicle and MK801 or SLI over time. Independent verification of a subset of gene expression via real-time PCR showed high correlation to gene array results. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene ontology (GO) analysis were used to identify statistically significant functionally related groups of genes, while hierarchical cluster analysis was used to visualize the expression level changes within these groups. Pathways with statistically significant expression included, MAP kinase signaling pathway, TGF-β signaling pathways, Wnt signaling pathways, Toll receptor pathway, ribosome, cell adhesion molecules, VEGF signaling, and oxidative phosphorylation. However, the overall response initiated by SLI is different than that of NMDA stimulation suggesting that a discrete gene program is initiated by NMDA stimulation to produce IT. Our results identify pathways/transcripts that may play a crucial role in the acquisition of IT and identify genes that may constitute novel targets as therapeutics for stroke.
NEUROPROTECTIVE EFFECTS OF GIF0173, A DERIVATIVE OF CYCLOPENTENONE PROSTAGLANDIN IN A RAT STROKE MODEL
Kazuya Hokamura1, Min Thura1, Seiji Yamamoto2, Masahide Maeda3, Kyoji Furuta3, Masaaki
Suzuki3, Kazuo Umemura1
1Deparment of Pharmacology, Hamamatsu University School of Medicine, Shizuoka, Japan, 2Photo Medical Research Center, Hamamatsu University School of Medicine, Shizuoka, Japan, 3Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu, Japan
[Background and aims]
It is known that certain cyclopentenone prostaglandin derivatives have biological activities of promoting neurite outgrowth. In line with this understanding, GIF 0173, a derivative of cyclopentenone prostaglandin with cross conjugated dienone at C (7) - C (11) position, prevents neuronal death by inhibiting apototic processes and by promoting antiapototic process. However, it is not know if GIF 0173 can protect cerebral damage in the acute phase of cerebral ischaemia. This study was to investigate the neuroprotective effects of GIF 0173 in an in vivo setting, using the rat photothrombotic middle cerebral artery occlusion (MCAo) model and in in vitro setting by using the rat primary cultured cortical neurons.
[Methods]
In vivo experiments
Male Sprague-Dawley rats were anaesthetized with halothane. A catheter for the administration of either the drug or Rose Bengal was inserted into the femoral vein. Subtemporal craniotomy was performed with a dental drill, under an operating microscope and a 3 mm diameter oval bony window was prepared. The window was irradiated with green light (wavelength 540 nm) from a xenon lamp. Photoillumination was continued for 10 min after an intravenous injection of Rose Bengal (20 mg/kg) and then the MCA was thrombotically occluded. Neurological scores were examined at 24hr after MCAo and then the brain was quickly excised under pentobarbital sodium anaesthesia. The brain was coronally sectioned on a brain matrix starting from the frontal lobe. Six consecutive slices were stained with 1% triphenyltetrazolium chloride. GIF 0173 (0.01, 0.03, and 0.1 mg/kg) was administered intravenously as a bolus injection just after and 1, 2 and 3hr after MCAo.
In vitro experiments
Effects of GIF 0173 were investigated on morphological changes, acute cellular swellings, neuronal death and intracellular calcium influx in the rat primary cortical neuronal cultures using respective imaging unit.
[Results]
In vivo experiments
When GIF 0173 was administered just after the MCAo, neurological scores showed a significant improvement compared to control and the infarct size was reduced in a dose-dependent manner. GIF 0173 at a dose of 0.1 mg/kg reduced the infarct size by 40 % compared to control. Likewise, GIF 0173, administered at 1hr after the MCAo, it tended to reduce the infarct size, but starting 2 or 3hr after MCAo, the infarct size was unaffected.
In vitro experiments
GIF 0173 inhibited acute cellular swelling in a concentration-dependent manner and at 0.3µM completely inhibited the cellular swelling. Similarly, GIF 0173 inhibited the neuronal death up to 6hr and at 0.3µM suppressed the neuronal death by 39.7%, compared to control (P<0.05). Additionally, intracellular calcium influx induced by glutamate was inhibited at the late phase by GIF 0173.
[Conclusion]
GIF 0173 appears to have pharmacological effects that reduce infarct size and improve neurological scores as observed in the MCAo model in in vivo experiments. In in vitro experiments, acute cellular swelling, cell death and intracellular calcium influx stimulated by glutamate were suppressed by GIF 0173. These findings suggest that potentially, GIF 0173 might become an effective medication against acute cerebral infarction.
RETINAL ISCHEMIA INDUCED BY INTRALUMINAL INTERNAL CAROTID ARTERY OCCLUSION IN MICE
Ernest Steele, Qingmin Guo, Shobu Namura
Department of Anatomy and Neurobiology, Neuroscience Institute, Morehouse School of Medicine, Atlanta, GA, USA
Background and aims: Middle cerebral artery occlusion (MCAO) by insertion of nylon filament into the internal carotid artery is one of the most frequently used focal cerebral ischemia models in mice and rats. Since the ophthalmic artery arises from the internal carotid artery very close to the origin of middle cerebral artery, the intraluminal MCAO is likely to induce retinal ischemia. Unlike the most commonly used models induced by increased intraoclular pressure or optic sheath ligation, the current method does not cause any direct insult to ocular structures. The effect of retinal ischemia induced by this technique is poorly studied [1], [2] and no published study utilized mice, to the best of our knowledge. Here, we report the first findings of retinal ischemic damage in mice using intraluminal MCAO.
Methods: Male SV129EV mice were subjected to surgery under isoflurane anesthesia. Silicon/resin coated nylon filament (8-0) was inserted through the external carotid artery and advanced into the internal carotid artery as described previously [3]. Regional cerebral blood flow in the ischemic hemisphere was monitored by either laser Doppler flowmetry or laser speckle imaging. Retinal perfusion was evaluated by fluorescence microsphere beads (1µm diameter) injection through the femoral vein. Retinal injury was histologically examined by cresyl violet staining and fluorescent TUNEL assay.
Results: Fluorescence microsphere beads injection at 60 min after filament insertion demonstrated filling in the contralateral retina but the ipsilateral retina was devoid of beads. Twenty four hours after reperfusion following 60 min occlusion, TUNEL positive cells were detected in the ganglion cell layer (30% of total cells) and inner nuclear layer (25% of total cells). This was consistent with the observation of pyknotic nuclei in these layers with cresyl violet staining.
Conclusions: Intraluminal internal carotid artery occlusion represents a useful technique for simultaneously studying ischemia/reperfusion injury in the retina and brain of the same mice. Although functional significance needs to be investigated, our data support additional consideration of visual dysfunction when neurological outcome is analyzed in this model.
Support Contributed By: National Institute of Neurological Disorders and Stroke Grant NS048532, NS034194.
“CHESTNUT” (AMAGURI) METHOD TO VISUALIZE VESSELS IN THE DURA AND SKULL BONE IN MICE
Haruki Toriumi1, M. Tomita1, Y. Tomita1,2, T. Osada1, M. Unekawa1, T. Shimizu1, N. Suzuki1
1Department of Neurology, Keio University School of Medicine, Tokyo, Japan, 2Department of Neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Gunma, Japan
Pain-sensitive apparati (nociceptors including presumably chemo-, mechano-, osmo-, and thermo-sensors) in the brain appear to be gathered in dural vessels and sinuses densely innervated with sympathetic and trigeminal sensory nerves. On the other hand, the middle meningeal and calvarial vessels are an obstacle to noninvasive measurement of cerebral blood flow and observation on changes in arterial vessels through the thin skull bone. However, detailed descriptions of such the vessels in mice are lacking in the literature. This paper presents a new method to visualize the meningeal and calvarial vessels. Ten C57BL/6J mice of both sexes, (either 5 weeks or 18 weeks after birth) were used under halothane anesthesia. Immediately after injection of FITC dextran (0.4 ml of 0.25% FITC dextran) into the tail vein, the mouse was guillotined. After removal of the scalp, the skull bone was cut with a pair of scissors to open the cavity at the horizontal plane of an orbito-foramen magnum line. The brain was easily removed from the skull, leaving the dura on the inner surface of the calvarium. This procedure was quite simple, resembling removal of a chestnut (amaguri) from the skin, leaving the astringent membrane. The calvarium was placed upside down on the stage of a confocal fluorescence (λ=488 nm) microscope and vessels in the dura adhering to the skull bone were observed. The vessels in the dura and calvarium were clearly stained with bright green fluorescence. It took only 10 min for this procedure. When the dura was peeled off from the skull, two categories of vessels were separated (Fig. 1, a hand drawing picture). We found that the middle meningeal artery occupied the temporoparietal area. It arborized to small, poorly developed capillaries and plexuses connecting to the superior saggital sinus which lay between the cranial layer and meningeal layer. The calvarial vessels were localized mainly within the skull bone in the nasal area and temporo-occipital area. Specific points were: 1) The vessels formed an independent vascular tree in 5 week mice but became communicated in 18 week mice; 2) The microvessels were characterized by tortuosity, multiple ramifications with arborized loops, apical enlargement of ‘capillaries' (megacapillaries or giant capillaries) and ectasia of microvessel efferent branches, and venous plexuses in the diploe. 3) The hairpin heads of vascular loops were specifically well stained with FITC dextran. 4) The vascular trees appeared to continue developing with age even after 5 weeks. This method is expected to be useful for studying the mechanisms of vascular headache.
INHIBITION OF L-TYPE CALCIUM CHANNELS BY A CYCLIC GMP-DEPENDENT RHO KINASE INHIBITOR KMUP-1 IN RAT BASILAR ARTERY MYOCYTES
Ing-Jun Chen, Min-Chi Jiang, Bin-Nan Wu
Department and Graduate Institute of Pharmacology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan-R.O.C.
Background and aims: KMUP-1, a chemically synthetic xanthine-based derivative, has been demonstrated not only increase of cyclic nucleotides and inhibition of phosphodiesterases, but also activation of K+ channels resulting in relaxation in rat aortic smooth muscle (SM), and rabbit corpus cavernosum, which is also sensitive to Rho kinase inhibitor Y27632. However, comparison of calcium current inhibition by KMUP-1 and Y27632 has not yet been documented in basilar artery myocytes. This study is to examine the effects of KMUP-1 on L-type calcium currents (ICa,L), targeting the applicability of KMUP-1 for cerebral artery vasodilatation.
Methods: We used the conventional whole cell patch-clamp technique to investigate Ba2+ currents (IBa) through L-type Ca2+ channels in rat basilar artery myocytes.
Results Under voltage-clamp conditions, KMUP-1 inhibited the IBa in a concentration-dependent manner without any change in current-voltage relationship of IBa. Additionally, KMUP-1 inhibited the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA, 1 uM), induced IBa. Pretreatment with the PKC inhibitor chelerythrine (5 uM) enhances the inhibition of IBa by KMUP-1. However, a Rho kinase inhibitor Y-27632 (30 uM) failed to affect the inhibition of IBa by KMUP-1. In fura-2-loaded rat basilar arteries, KMUP-1 inhibited the Ca2+ signal evoked by 100 mM KCl and 1 uM PMA. In addition, KMUP-1 also inhibited the Ca2+ signal evoked by 10 uM thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase.
Conclusions: In light of these results, we suggest that KMUP-1 inhibits the L-type calcium channels in concentration- and voltage-dependent manners in rat basilar artery myocytes and the effects may partially related to the PKC pathway. KMUP-1 is more potent than Y27632 to inhibit L-type calcium channels.
ANALYSIS OF GENE EXPRESSION RELATED TO ENRICH ENVIRONMENT AFTER TRANSIENT FOCAL BRAIN ISCHEMIA IN RATS
Yuji Shono1, Yuji Kuge2, Chiaki Yokota1, Shinsuke Kido2, Koichi Kokame3, Hiroyasu Inoue4, Mariko Hotta4, Kazuo Minematsu1, Hideo Saji2
1Cerebrovascular Division, Department of Medicine, National Cardiovascular Center, Osaka, Japan, 2Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan, 3National Cardiovascular Center Research Institute, Osaka, Japan, 4Department of Food Science and Nutrition, Nara Women's University, Nara, Japan
Background and Purpose: Rehabilitation is essential for functional recovery after stroke. Several studies showed that animals housed in an enriched environment after an experimental stroke obtained better functional outcome as compared with those housed in a standard cage1]~3]. The aim of this study is to examine genes associated with functional recovery in an enriched environment after ischemic stroke.
Methods: Male Sprague-Dawley rats (300-350g) were used in this study. Transient focal brain ischemia was produced by occlusion of the right middle cerebral artery (tMCAO) intraluminally with a nylon monofilament for 60 minutes. After tMCAO, rats were housed in an enrich environment or standard cages. Examination of neurological severity score (NSS) and behavioral tests such as inclined plane test were done at baseline, 2 and 4 weeks after tMCAO. Soon after the final examinations of both NSS and behavioral test, rats were sacrificed. Immunoreactivity to microtubule-associated protein-2 (MAP-2) was detected in frozen sections. Microarray analysis of peri-infarct area in the ischemic brain was conducted.
Results: Infarct expansion evaluated by immunoreactivity to MAP-2 in an enrich environment was as the same as that in standard cages. Although there was no significant difference in NSS between the two groups, results of behavioral tests in an enrich environment were better as compared with those in standard cages. In microarray analysis, a decrease in the expression of brain derived neurotrophic factor (BDNF) as well as increase in the expression of nectin-3 gene was demonstrated in rats housed in an enrich environment.
Conclusion : Decrease in BDNF and increase in nectin-3 gene were associated with improvement of behavioral function in an enrich environment after transient focal brain ischemia.
INFLUENCE OF BRAIN TRAUMA ON THE EXPRESSION OF CONTROL GENES IN QUANTITATIVE REAL-TIME PCR
Serge Thal1, Sebastian Wyschkon1, Raimund Trabold2, Nikolaus Plesnila2, Kristin Engelhard1, Christian Werner1
1Department of Anaesthesiology, Johannes Gutenberg University, Mainz, Germany, 2Laboratory of Experimental Neurosurgery, Department of Neurosurgery & Institute for Surgical Research, University of Munich Medical Center, Grosshadern, Munich, Germany
Introduction: Quantitative measurements of gene expression require the correction of data for differences in tissue sample size, RNA extraction variations, RNA quantity/quality, and reverse transcription efficiency.
It is, therefore, essential to characterize housekeeping genes which are not changed by the experimental setting. However, in mouse brain trauma the expression profile of commonly used housekeeping genes has not been determined. We, therefore, analysed the time course of absolute expression of four commonly used control genes after brain trauma.
Methods: Male C57Bl/6 mice were anaesthetized with isoflurane (0.8%) and a pneumatic brain trauma was induced on the right parietal cortex (controlled cortical impact (CCI), 8 m/s, 1 mm depth). Brain tissue was removed 15 min, 3, 6, 12 and 24 hr (each n=6) after CCI and from naive animals (n=7). The absolute beta-2-microglobin (B2M), cyclophylin A (PPIA), s-actin, and glyceraldehyd-3-phosphate dehydrogenase (GAPDH) amount in 1 µg RNA (determined by fluorescent labelling, QubitR) was determined by real-time RT-PCR (LightcyclerR) in the traumatic focus and health tissue. Statistics: ANOVA on RANKS, p<0.05.
Results: The expression of B2M was reduced 15 min to 12 hr after CCI and increased at 24 hr (B2M: 15 min: −7 %, 3 hr: −11 %, 6 hr: −20 %, 12 hr:
2 %, 24 hr: 15% vs. naive). The expression of PPIA was stable 15 min and 24 hr after CCI, but decreased at 3?12 hr (PPIA: 15 min: 4 %, 3 hr:
36 %, 6 hr: −34 %, 12 hr: −37 %, 24 hr: −6 % vs. naive). s-actin and GAPDH were strongly up regulated after 6 hr post CCI (s-Actin: 15 min:
3 %, 3 hr: 15 %, 6 hr: 186 %, 12 hr: 150 %, 24 hr: 417 % vs. naive;
GAPDH: 15 min: 11 %, 3 hr: −3 %, 6 hr: 162 %, 12 hr: 16 %, 24 hr: 254 % vs. naive)
Conclusion: The expression of all studied genes was changed in the time course after CCI. The expression of the four genes was rather stable until 3 hr after CCI. At later time points the expression of s-actin and GAPDH increased. These genes, therefore, are inadequate to determine mRNA levels at time points later than 3 hr post CCI. Despite minor regulatory response of PPIA and B2M, the genes expression exhibited sustained stability. These results suggest that the knowledge of the time course of the house keeping genes after CCI is essential to properly interpret the results of mRNA expression analysis.
Footnotes
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Bauer et al. Pediatr Res (2004) 56, 639-46.
