Journal of Cerebral Blood Flow & Metabolism

It has been suggested that reactive oxygen species (ROS) play a role in the pathophysiology of brain damage. A number of therapeutic approaches, based on scavenging these radicals, have been attempted both in experimental models and in the clinical setting. In an experimental rat and mouse model of closed-head injury (CHI), we have studied the total tissue nonenzymatic antioxidant capacity to combat ROS. A major mechanism for neutralizing ROS uses endogenous low-molecular weight antioxidants (LMWA). This review deals with the source and nature of ROS in the brain, along with the endogenous defense mechanisms that fight ROS. Special emphasis is placed on LMWA such as ascorbate, urate, tocopherol, lipoic acid, and histidine-related compounds. A novel electrochemical method, using cyclic voltammetry for the determination of total tissue LMWA, is described. The temporal changes in brain LMWA after CHI, as part of the response of the tissue to high ROS levels, and the correlation between the ability of the brain to elevate LMWA and clinical outcome are addressed. We relate to the beneficial effects observed in heat-acclimated rats and the detrimental effects of injury found in apolipoprotein E-deficient mice. Finally, we summarize the effects of cerebroprotective pharmacological agents including the iron chelator desferal, superoxide dismutase, a stable radical from the nitroxide family, and HU-211, a nonpsychotoropic cannabinoid with antioxidant properties. We conclude that ROS play a key role in the pathophysiology of brain injury, and that their neutralization by endogenous or exogenous antioxidants has a protective effect. It is suggested, therefore, that the brain responds to ROS by increasing LMWA, and that the degree of this response is correlated with clinical recovery. The greater the response, the more favorable the outcome.

Resting- and acetazolamide (Acz)-activated-regional cerebral blood flow (rCBF) measurements were performed by consecutive single-photon emission computed tomography (SPECT) studies before and after Acz administration using equal-volume-split technetium-99m-L,L-ethyl cysteinate dimer. Quantitative rCBF images were converted from qualitative axial SPECT images by the application of Patlak plot graphical analysis with radionuclide angiography and Lassen's linearization correction. Total time span required for this study was 53 minutes. The unaffected side of 37 studies with unilateral vascular lesions and 45 studies without apparent vascular lesions showed 132 ± 17% and 140 ± 15% increase of mean CBF (mCBF), respectively, under Acz administration. Comparing these values, the Acz-activated rCBF increases of less-affected and affected hemispheres of 23 studies with bilateral vascular lesions (116 ± 13% and 113 ± 12%, respectively) was lower with high statistical significance (P < 0.001). For the other 20 cases, physiologic saline was administered instead of Acz. This group showed no changes in mCBF under placebo administration (after placebo/baseline; 100 ± 6%). Acetazolamide-activated rCBF increase was recognized clearly and easily using quantitative images. This noninvasive method is easy to perform and may be helpful to detect regional abnormalities of hemodynamic reserve in cerebrovascular diseases.

We adapted and implemented a permutation test (Holmes 1994) to single-subject positron emission tomography (PET) activation studies with multiple replications of conditions. That test determines the experimentwise α error as well as location and extent of focal activations in each individual. Its performance was assessed in five normal volunteers, using ¹⁵O-H2O-PET data acquired on a high-resolution scanner, with septa retracted (3D mode), during functional activation by repeating words versus resting (four replications each). Calculated α errors decreased and the size of activated tissue volumes (voxels with P ≤ 0.05) increased with increasing filter kernel size applied to the difference images. At a filter kernel of 12 mm Gaussian full width at half maximum, significant focal activations were seen bilaterally in superior temporal cortex, including Brodmann's areas 41 and 42, in all five subjects. Additional foci were detected in the precentral gyrus, left inferior frontal gyrus, supplementary motor area, and cerebellum of several subjects. The average CBF increase in activated voxels ranged from 17.6% to 28.7%. Activated volumes were smaller than those detected with a standard parametric test procedure. We conclude that the permutation test is a less sensitive procedure, having the advantage of not depending on unproven distributional assumptions, that detects strong activation foci in individual subjects with high reproducibility.

In the α-chloralose-anesthetized rat during single forepaw stimulation, a spatially localized ¹H[¹³C] nuclear magnetic resonance spectroscopic method was used to measure the rate of cerebral [C4]-glutamate isotopic turnover from infused [1,6-¹³C]glucose. The glutamate turnover data were analyzed using a mathematical model of cerebral glucose metabolism to evaluate the tricarboxylic acid (TCA) cycle flux (V_TCA). During stimulation the value of V_TCA in the sensorimotor region increased from 0.47 ± 0.06 (at rest) to 1.44 ± 0.41 μmol·g⁻¹ min⁻¹ (P < 0.01) in the contralateral hemispheric compartment (24 mm³) and to 0.65 ± 0.10 μmol·g⁻¹min⁻¹ (P < 0.03) in the ipsilateral side. Each V_TCA value was converted to the cerebral metabolic rates of glucose oxidation (oxidative-CMR_glC) and oxygen consumption (CMR_O2). These rates were corrected for partial-volume based on activation maps obtained by blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI). The percent increase and the absolute value of oxidative-CMR_glc in the activated regions are similar to values reported previously for total-CMR_glc using the same activation paradigm. This indicates that the large majority of energy required for brain activation, in going from the resting to an activated state, is supplied by glucose oxidation. The level of activity during stimulation is relevant to awake animals because the oxidative-CMR_glc (1.05 ± 0.28 μmol·g⁻¹·min⁻¹; current study) is in the range of total-CMR_glc previously reported for awake rats undergoing physiologic activation (0.7–1.4 μmol·g⁻¹ min⁻¹). It is concluded that oxidative glycolysis is the main source of energy for increased brain activity and a positive BOLD fMRI signal-change occurs in conjunction with a large increase in CMR_O2.

During reperfusion after ischemia, deleterious biochemical processes can be triggered that may antagonize the beneficial effects of reperfusion. Research into the understanding and treatment of reperfusion injury (RI) is an important objective in the new era of reperfusion therapy for stroke. To investigate RI, permanent and reversible unilateral middle cerebral artery/common carotid artery (MCA/CCA) occlusion (monitored by laser Doppler) of variable duration in Long-Evans (LE) and spontaneously hypertensive (SH) rats and unilateral MCA and bilateral CCA occlusion in selected LE rats was induced. In LE rats, infarct volume after 24 hours of permanent unilateral MCA/CCA occlusion was 31.1 ± 34.6 mm³ and was only 28% of the infarct volume after 120 to 300 minutes of reversible occlusion plus 24 hours of reperfusion, indicating that 72% of the damage of ischemia/reperfusion is produced by RI. When reversible ischemia was prolonged to 480 and 1080 minutes, infarct volume was 39.6 mm³ and 16.6 mm³, respectively, being indistinguishable from the damage produced by permanent ischemia and significantly smaller than damage after 120 to 300 minutes of ischemia. Reperfusion injury was not seen in SH rats or with bilateral CCA occlusion in LE rats, in which perfusion is reduced more profoundly. Reperfusion injury was ameliorated by the protein synthesis inhibitor cycloheximide or spin-trap agent N-tert-butyl-alpha-phenylnitrone pretreatment.

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [³H]nimodipine and [¹⁴C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 ± 0.11 in normoglycemic rats (n = 12) and 6.57 ± 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group (P < 0.001). In contrast the ratio of [³H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 ± 0.45) than hyperglycemic (1.40 ± 0.50) rats (P < 0.05). Within this region of ischemic cortex CBF was 31 ± 22 mL/100 g in normoglycemic rats (n = 8) and 33 ± 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic rats.

In this study we explored if the secondary bioenergetic failure, which occurs a few hours after recirculation, following transient middle cerebral artery occlusion (MCAO) in rats, is caused by a compromised reflow. We induced 2 hours of MCAO and measured CBF at the end of the ischemia, as well as 15 minutes, 1, 2, and 4 hours after the start of recirculation, using autoradiographic or tissue sampling ¹⁴C-iodoantipyrine techniques. After 2 hours of MCAO, the autoradiographically measured CBF in the ischemic core areas was reduced to 3 to 5% of contralateral values. The reduction in CBF was less in neighboring, penumbral areas. After recirculation, flow already normalized in core tissues after 15 minutes, and remained close to normal for the 4 hours recirculation period studied. However, in penumbral tissues, recovery CBF values were usually below normal. The results show that tissues that are heavily compromised by the 2-hour period of ischemia and are destined to incur infarction, show a “relative hyperemia” during recirculation. In fact, some areas of the previously densely ischemic tissue showed overt hyperperfusion. This finding raises the question whether the relative or absolute hyperemia reflects events that are pathogenetically important. Because drugs that clearly ameliorate the final damage incurred fail to alter the relative hyperperfusion of previously ischemic tissues, it is concluded that vascular events in the reperfusion period do not play a major role in causing the final damage.

We investigated the l-arginine-induced, regional cerebral blood flow (rCBF) enhancement after different durations of transient focal cerebral ischemia in the rat to determine if l-arginine increases rCBF after transient focal cerebral ischemia. Focal ischemia (5 minutes and 20 minutes) followed by 90 minutes of reperfusion was induced in a normotensive rat suture-model. Regional cerebral blood flow in both hemispheres was measured by laser-Doppler-flowmetry. Reactivity of rCBF to l-arginine (300 mg/kg) was measured 45 minutes after reperfusion, and hypercapnia 90 minutes after reperfusion. The effect of d-arginine and pretreatment with the nitric oxide (NO) synthase inhibitor N^ω-nitro-l-arginine (l-NA) (10 mg/kg) was examined in additional groups. Hypercapnia and l-arginine increased rCBF in sham operated controls and on the nonischemic hemispheres. d-arginine did not. Twenty-minute long ischemia significantly reduced the response to l-arginine (control side: 115 ± 5.9%; ischemic side: 107 ± 6.1%, n = 7) and hypercapnia, 5 minutes of ischemia did not. N^ω-nitro-l-arginine pretreatment partly restored the l-arginine-induced rCBF increase. Thus, rCBF increase caused by l-arginine in the reperfusion period was unaffected by 5 minutes of ischemia, but reduced by 20 minutes of ischemia. The restoration after pretreatment with l-NA may be caused by attenuated production of cytotoxic substances, e.g., NO and related compounds.

We developed a mouse model of embolic focal cerebral ischemia, in which a fibrin-rich clot was placed at the origin of the middle cerebral artery (MCA) in C57BL/6J mice (n = 31) and B6C3 mice (n = 10). An additional three non-embolized C57BL/6J mice were used as a control. Embolus induction, cerebral vascular perfusion deficit, and consequent ischemic cell damage were confirmed by histopathology, immunohistochemistry, laser confocal microscopy, and regional cerebral blood flow (rCBF) measurements. Reduction in rCBF and cerebral infarct were not detected in the control animals. An embolus was found in all C57BL/6J and B6C3 mice at 24 hours after injection of a clot. Regional CBF in the ipsilateral parietal cortex decreased to 23% (P < 0.05) and 17% (P < 0.05) of preembolization levels immediately and persisted for at least 1 hour in C57BL/6J mice (n = 6) and in B6C3 mice (n = 3), respectively. A significant decrease of rCBF was accompanied by a corresponding reduction of plasma perfusion in the ipsilateral MCA territory. Neurons exhibited marked reduction in microtubule-associated protein-2 immunostaining coincident with the area of perfusion deficit. The percent infarct volume was 30.3% ± 13.4% for C57BL/6J mice (n = 17), and 38.3% ± 15.3% for B6C3 mice (n = 7) at 24 hours after embolization. This model of embolic ischemia is relevant to thromboembolic stroke in humans and may be useful to investigate embolic cerebral ischemia in the genetically altered mouse and for evaluation of antiembolic therapies.

We tested the hypothesis that nitric oxide (NO) plays a role in CBF autoregulation in the brain stem during hypotension. In anesthetized rats, local CBF to the brain stem was determined with laser-Doppler flowmetry, and diameters of the basilar artery and its branches were measured through an open cranial window during stepwise hemorrhagic hypotension. During topical application of 10⁻⁵ mol/L and 10⁻⁴ mol/L N^ω-nitro-L-arginine (L-NNA), a nonselective inhibitor of nitric oxide synthase (NOS), CBF started to decrease at higher steps of mean arterial blood pressure in proportion to the concentration of L-NNA in stepwise hypotension (45 to 60 mm Hg in the 10⁻⁵ mol/L and 60 to 75 mm Hg in the 10⁻⁴ mol/L L-NNA group versus 30 to 45 mm Hg in the control group). Dilator response of the basilar artery to severe hypotension was significantly attenuated by topical application of L-NNA (maximum dilatation at 30 mm Hg: 16 ± 8% in the 10⁻⁵ mol/L and 12 ± 5% in the 10⁻⁴ mol/L L-NNA group versus 34 ± 4% in the control group), but that of the branches was similar between the control and L-NNA groups. Topical application of 10⁻⁵ mol/L 7-nitro indazole, a selective inhibitor of neuronal NOS, did not affect changes in CBF or vessel diameter through the entire pressure range. Thus, endothelial but not neuronal NO seems to take part in the regulation of CBF to the the brain stem during hypotension around the lower limits of CBF autoregulation. The role of NO in mediating dilatation in response to hypotension appears to be greater in large arteries than in small ones.

Our previous studies demonstrated coordinate expression of platelet-derived growth factor (PDGF) -B chain and β-receptor in neurons at risk in the rat brain with focal ischemia. To clarify a role of the -B chain in the brain further, we examined whether PDGF-A or -B chain protects CA1 pyramidal neurons from delayed neuronal death after forebrain ischemia in rats. Pretreatment with PDGF-BB, but not -AA, at 120 ng/d for 2 days until forebrain ischemia was performed markedly ameliorated delayed neuronal death in CA1 pyramidal neurons on day 7 after ischemia. This neuroprotective effect of PDGF-BB was dose-dependent, and pretreatment with PDGF-BB at 240 ng/d showed almost complete inhibition of delayed neuronal death. In contrast, posttreatment with PDGF-BB at 120 ng/d starting 20 minutes after ischemia demonstrated no significant neuroprotective effect. The current study established marked neuroprotective actions of PDGF-BB in ischemic neuronal damage.

The mRNA expression of the proinflammatory cytokine interleukin-1β (IL-1β) has been shown to be induced in neural elements during ischemia. It is not clear which cells generate the IL-1β mRNA and eventually synthesize IL-1 protein and which cells respond to this signaling by producing IL-1 receptors during ischemia. To clarify this question, rats were subjected to global ischemia by bilateral carotid occlusion and hypotension for 20 minutes, followed by reperfusion for 2 hours (n = 7), 8 hours (n = 7), or 24 hours (n = 7). Cryostat sections were hybridized using antisense oligonucleotide probes (30 dimer). Multiple cell markers were used in immunohistochemical staining to identify the cells expressing IL-1β and IL-1R protein. The sham animals (n = 5) showed no or only a weak expression of IL-1R or IL-1β mRNA. The number of IL-1β mRNA-expressing cells was significantly increased by 2 hours of reperfusion in several brain areas including cortex (12-fold compared with sham) and caudate-putamen (14-fold), and was maximally increased in most hippocampal regions by 8 hours of reperfusion (mean ± SD of positive cells/field versus sham equivalent being 37.9 ± 12.3 versus 4.0 ± 3.3; 30.6 ± 9.0 versus 3.1 ± 2.3; 41.3 ± 17.5 versus 2.9 ± 1.9; in CA1; CA2; CA3/CA4 regions of the hippocampus, respectively). IL-1β mRNA signal was also intensified in the white matter areas. Changes in IL-1R mRNA were seen in the hippocampus (after 2 hours CA1: 16-fold; CA2: 17-fold; DG: 24-fold increase; and CA3/CA4: 10-fold increase after 8 hours), and the expression was prolonged especially in CA1 and CA2 regions up to 24 hours of reperfusion. The major cellular source of IL-1β protein was glia (astrocytes, oligodendrocytes, microglia, and scattered perivascular macrophages/monocytes), while neurons and sporadic microvascular endothelia showed IL-1R immunoreactivity. The data suggest that neurons in discrete areas vulnerable for selective neuronal death, and possibly the vascular endothelium, are target cells for ischemia-induced glial IL-1β production.

We have studied the possible mechanisms underlying the decrease of excitatory transmission induced by glucose deprivation by using electrophysiological recordings in corticostriatal slices. Extracellular field potentials were recorded in the striatum after cortical stimulation; these potentials were progressively reduced by glucose deprivation. The reduction started 5 minutes after the onset of aglycemia. The field potential was fully suppressed after 40 minutes of glucose deprivation. After the washout of the aglycemic solution only a partial recovery was observed. Aglycemia also induced a delayed inward current during single-microelectrode voltage-clamp recordings from spiny neurons. This inward current was coupled with an increased membrane conductance. The A1 adenosine receptor antagonists, 8-cyclopentyl-1,3-dimethylxanthine (CPT, 1 μmol/L) and 1,3-dipropyl-8-cyclopentylxanthine (CPX, 300 nmol/L), significantly reduced the aglycemia-induced decrease of field potential amplitude. Moreover, in the presence of CPT and CPX, a full recovery of the field potential amplitude after the interruption of the aglycemic solution was observed. Conversely, these antagonists affected neither the inward current nor the underlying conductance increase produced by glucose deprivation. The ATP-sensitive potassium channel blockers glibenclamide (10 μmol/L) and glipizide (100 nmol/L) had no effect on the aglycemia-induced decrease of the field potential amplitude. We suggest that endogenous adenosine, but not ATP-dependent potassium channels, plays a significant role in the aglycemia-induced depression of excitatory transmission at corticostriatal synapses probably through a presynaptic mechanism. Moreover, adenosine is not involved in the postsynaptic changes induced by glucose deprivation in spiny striatal neurons.

The nonproportional relationship between instantaneous arterial blood pressure (BP) and cerebral blood flow velocity (CBFv) is well explained by the concept of critical closing pressure (CCP). We aimed to determine the frequency response of the neonatal cerebrovascular system, and to establish the exact mathematical relationship between cerebrovascular impedance and CCP under physiologic conditions. In 10 preterm neonates (gestational age, 25–32 weeks; birth weight, 685–1,730 g; age 1–7 days) we Doppler-traced CBFv of the internal carotid artery. Blood pressure was traced simultaneously. Critical closing pressure was graphically determined. Cerebrovascular impedance was calculated as the square root of the ratio of the corresponding peaks in the power spectra of BP and CBFv at zero frequency, and at heart rate (H) and harmonics (xH). Uniformly, the impedance between H and 3H (2 to 6 Hz) was reduced about fivefold, compared with the impedance at zero frequency. The cerebrovascular system behaves like a high-pass filter, leading to a reduction of the DC (direct current) component of CBFv (analogous to current) relative to that of the driving force BP (analogous to voltage). The frequency response of cerebrovascular impedance reflects the ratio of CCP and DC BP. A mathematical derivation of this relationship is given matching the observed results. Thus, both the CCP and the impedance approach are valid.

Sixteen of 24 Sprague-Dawley rats with permanent middle cerebral artery occlusion for 24 hours were subjected to immediate or 8-hour delayed 2,3,5-triphenyltetrazolium chloride (TTC) staining (n = 8 at each time point); the other 8 animals were subjected to immediate or 8-hour delayed measurement of succinate dehydrogenase activity (n = 4 at each time point). The TTC staining was of good quality good in all animals, and the infarcted region could be distinguished easily from normal tissue. There was no significant difference in corrected infarct volume between the two groups (263.8 ± 43.1 versus 264.4 ± 54.8 mm³ [mean ± standard deviation]). The activity of succinate dehydrogenase was not significantly different when normal or infarcted tissue was measured immediately after death or with an 8 hour delay, although less activity was detected at both time points in the infarcted tissue. These results demonstrate that an 8-hour delay of TTC staining is reliable for evaluating brain infarct volume in a rat stroke model and this probably is attributable to the slow deterioration of mitochondrial enzyme activity in nonischemic brain over this time period.