Abstracts from The 29 th Annual National Neurotrauma Symposium July 10–13,2011 Hollywood Beach,Florida

OC1 The Role of Fas/Fasl Mediated Apoptosis and Inflammation after Spinal Cord Injury: Evidence from Human Tissue and Mouse Models

The Fas/FasL system plays an important role in apoptosis, the inflammatory response and in gliosis in a variety of neurologic disorders. A better understanding of these mechanisms could lead to effective therapeutic strategies following SCI. We explored these mechanisms by examining molecular changes in post-mortem human spinal cord tissue from acute and chronic SCI. Complementary studies were conducted using the in vivo Fejota^™ clip compression model of SCI in Fas-deficient (lpr) and wild-type (Wt) mice to test Fas-mediated apoptosis, inflammation, gliosis and axonal degeneration by immunohistochemistry, western blotting, gelatin zymograph and ELISA. We report novel evidence that shows that Fas-mediated apoptosis of neurons and oligodendrocytes, which was associated with infiltration by CD68-positive microglia/macrophages, occurred in the injury epicenter in all cases of acute and subacute SCI and not in the setting of chronic SCI or in control cases. We also found significantly reduced apoptosis, glial scar formation, NF-kB, and iba1 expression and an increased number of CD4 positive T cells and MMP2 expression in lpr mice when compared with Wt mice after SCI. We observed a significant increase of cytokines and chemokines, which act as inflammatory mediators in SCI. In the Wt mice, we observed an increase in the cytokines IL-1alpha, IL-12p40, IL-7, IL-15, IL-7, IL-13, and the chemokines MIP-1ą/CCL4, MCP-1/CCL2, RANTES/CCL5, MIG/CXCL9, M-CSF, Exotion, IP10/CXCL10. We furthermore found a significant reduction in the amount of IL-1alpha, IL-7, IL-15, IFN gamma, MCP-1/CCL2 and Eotaxin expression at 14 days in lpr mice when compared to Wt mice after SCI. In conclusion, we provide multiple lines of evidence that Fas/FasL activation plays an important role in apoptosis, the inflammatory response, glial scar formation and inhibiting degeneration after SCI. This work provides a compelling rationale for therapeutically targeting Fas in the setting of human SCI.

OC2 Stem Cell-Based Transplantation Strategies for Sub-Cortical Remyelination

HYPOTHESIS/AIM: This study investigates the capacity for both adult and pluripotent stem cell-derived NSPCs to functionally remyelinate the sub-cortical white matter, prior to neuronal cell loss, as a treatment strategy for Cerebral Palsy.

BACKGROUND: Cerebral Palsy (CP) is the most common neurodevelopmental disorder, affecting 2.5/1000 live births. Demyelination, followed by motoneuron cell death and subsequent neuromotor impairment, is characteristic of CP. Neural stem and precursor cells (NSPCs) show great potential for cellular replacement and remyelination in models of sub-cortical injury and dysmyelination. It is, however, also imperative to delineate whether transplantation is effective at the most clinically relevant disease timepoints - which commonly follow age-dependent immune privilege.

METHODS: NSPCs derived from the sub-ventricular zone of yellow fluorescent protein transgenic animals, as well as other pluripotent sources, were injected bilaterally into the anterior and posterior aspects of the anlagen of the corpus callosum and also into the anlagen of the cerebellar peduncle. Animals were injected, using a pulled glass needle, on P0, P7 (equivalent to human birth) and P21 (equivalent to ∼2 years human age) in the shiverer mouse model, which genetically lacks compacted myelin. They survived for 4 days, 14 days or 45 days, with or without immune ablation.

RESULTS AND CONCLUSIONS: Cells integrated both anatomically and functionally into the local white matter, as well as the periventricular areas, and preferentially became electrophysiologically-competent MBP + oligodendrocytes, as measured using 2-D and 3-D confocal histologic imaging, electron microscopy to visualize extruded myelin and by two-point recordings of electrophysiological conductance. This provides the first evidence that these cells can functionally incorporate into the disordered CNS and shows their promise for future extension into disease models.

OC5 Reducing Neuropathic Pain in the Chronic Phase of Spinal Cord Injury by Targeting Inflammation

Little is known about the role of inflammatory signaling in the maintenance of chronic neuropathic pain following spinal cord injury (SCI). We have detected high levels of pro-inflammatory cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) products in the spinal cord 9 months post-SCI. We hypothesized that COX and 5-LOX activity might contribute to the maintenance of chronic SCI-associated pain. Thus, we investigated the effects of systemic treatment with the dual COX/5-LOX inhibitor Licofelone on neuropathic pain in chronic SCI rats. Licofelone has passed phase III clinical trials for the treatment of osteoarthritis, thus establishing safety and efficacy in human subjects.

In order to investigate the effects of Licofelone treatment on chronic SCI, we performed a moderate T10 spinal contusion injury on female Sprague-Dawley rats. 9 months post-injury, animals were treated with Licofelone, or vehicle, once daily for 1 month via the clinically-relevant oral route. Prior to and following treatment, locomotor activity was measured using a Photobeam Activity System, and mechanical allodynia was measured using von Frey hair testing.

Mechanical withdrawal thresholds of all four limbs were decreased in SCI rats compared to uninjured controls. However, thresholds of Licofelone-treated rats were significantly increased over vehicle-treated rats. Additionally, vehicle-treated rats spent significantly more time resting than uninjured controls, consistent with increased levels of pain in SCI rats. Strikingly, this effect was reversed in Licofelone-treated rats. Together, this data indicates that neuropathic pain in animals with chronic SCI is attenuated by systemic Licofelone treatment.

Finally, we performed a metabolomic analysis of spinal cord tissue from these animals. Between Licofelone- and vehicle-treated groups, we detected differential expression of components of anti-inflammatory and anti-oxidant biochemical pathways. Thus, we propose that in addition to the anti-inflammatory action of direct COX/5-LOX inhibition, Licofelone may also act by upregulating endogenous anti-inflammatory pathways which antagonize the molecular mechanisms causing neuropathic pain.

OC12 Inhibition of the JNK Pathway Improves the Long-Term Functional Recovery after Juvenile Traumatic Brain Injury

OBJECTIVES: Traumatic brain injury (TBI) in the juvenile population is associated with a high risk of mortality and long-term disability. Young victims' brains are particularly vulnerable to TBI due to their unique developmental features and are often associated with long-term disabilities. The c-Jun-N-terminal-kinase (JNK) pathway has been recognized as playing a critical role in mediating post-injury cascade related to glial and neuronal survival. Our objective was to determine the therapeutic effectiveness of D-JNKI, global JNK-inhibiting peptide, in improving behavioral and neuroimaging outcomes.

METHODS: A moderate controlled cortical impact (CCI) injury to the right parietal cortex (2.7 mm-diameter impactor tip, 1.5 mm depth) was induced in 17-day-old rats followed by D-JNKI (11 mg/kg) or saline administered intraperitoneally at 3 hours. Lesion volumes post-injury were quantified using T2-weighted MRI until at 60 days. Behavioral outcomes were measured from 1 to 60 days post-injury using beam balance and foot-fault, rotarod, open field, turn bias, Morris water maze, and zero maze tests.

RESULTS: D-JNKI-treated animals show significant improvement in foot-fault and rotarod tests over the first 60 dpi. Similarly, D-JNKI1-treated animals compared to the non-treated in cognitive behavioral tests such as MWM test showed a significant improvement in the strategy to find the hidden platform at 30 and 60 days (p < 0.01). Turn bias revealed asymmetry of motor function (p = 0.05) in the saline group that resolved after D-JNKI-treatment. These functional improvements were associated with MRI-derived lesion volumes that showed a 50%-reduction in lesion volume in the DJNKI1-treated animals.

CONCLUSIONS: Administration of D-JNKI1 at 3 hours post-TBI in juvenile rats reduces lesion volume and improves behavioral outcomes. This lends credence to the idea that D-JNKI1 might be beneficial for potential clinical application for jTBI patients.

Funded By NIH 1RO1HD061946-01

OC15 tPA Variant TPA-S481A Prevents Impairment of Cerebral Autoregulation During Hypotension and Histopathology After TBI.

INTRODUCTION: Tissue plasminogen activator (tPA) can enhance excitotoxic neuronal death through interactions with NMDA receptors. TBI is associated with cerebral hypoperfusion and impaired responsiveness to cerebrovascular dilator stimuli, including loss of autoregulation during hypotension. Impaired cerebral hemodynamics contribute to neuronal necrosis. tPA upregulation post TBI contributes to impaired cerebral hemodynamics, histopathology, and aggravation of NMDA-receptor mediated impairment of cerebral hemodynamics. We hypothesize that generation of a mutant tPA that competes with wild type tPA for binding to NMDA receptors and protects it from cleavage/activation by wild type tPA will improve outcome after TBI. tPA-S481A is a catalytically inactive tPA variant with single mutation in the active site that maintains its docking site and capacity to bind to the NMDA receptor, but cannot cleave/activate the receptor. This study investigated the ability of tPA-S481A to prevent vascular dysregulation and histopathology after TBI.

METHODS: Pial artery diameter was determined during normotension and hypotension before and after fluid percussion brain injury (FPI) in piglets given vehicle or tPA-S481A (1 mg/kg iv) 30 min post injury. H + E staining was used to assess histopathology.

RESULTS: FPI produced pial artery vasoconstriction, which was blocked by tPA-S481A. Pial artery dilation in response to hypotension was blunted after FPI, while responses to papaverine were unchanged. In contrast, tPA-S481A prevented impairment of autoregulation during hypotension, but had no further effect on papaverine induced vasodilation. FPI was associated with marked neuronal cell loss in CA3 hippocampus, which was prevented by tPA-S481A.

CONCLUSIONS: These data indicate that tPA-S481A prevents impairment of cerebral autoregulation and histopathology after FPI via its ability to bind but not activate the NMDA receptor. Use of this tPA variant is a novel approach towards limiting toxicity of NMDA receptor activation associated with the robust increase in tPA and glutamate within the brain following TBI.

P001 Combinational Drug Therapies as Treatment for Traumatic Brain Injury

Traumatic Brain Injury (TBI) initiates a cascade of secondary biological events which cause cell death over the course of several days. This delay presents a therapeutic window for treatment. Because the post-injury cascade is dynamic and complex, it is likely that several therapeutic agents will be necessary for an effective treatment. In this study we used a combination of memantine and 17β-estradiol (E2) to reduce cell death in rat organotypic hippocampal slice cultures (OHSC) subjected to mechanical injury. OHSC were cultured from Sprague-Dawley rat pups (P8-P10). Briefly, the hippocampus was dissected and cut in to 400μm thick sections that were cultured in custom silicone membrane wells in Neurobasal media with B27 (1X), L-glutamine (2 μM), and D-glucose (4.5 mg/mL). After 14 days in vitro (DIV), cultures were subjected to a moderate injury (20% biaxial strain, 20/s strain rate) using a custom, feedback-controlled injury device. Tissue biomechanics were verified by image analysis of high speed video of the dynamic deformation. Cell death was measured by propidium iodide (PI) staining and quantified as percent area above a threshold. Cultures were treated with either memantine (1 uM) or estradiol (1 nM) or both beginning one hour after injury. Post-injury treatment with either memantine (n = 15) or E2 (n = 14) alone did not significantly reduce cell death compared to vehicle-treated (n = 13) cultures. In contrast, the combination treatment of E2 and memantine (n = 11) significantly (p < 0.05) reduced cell death at 1, 2, 3, and 4 d post-injury compared to vehicle treated controls. In conclusion, memantine and E2 individually did not reduce cell death after mechanical injury in our model of TBI. However, in combination, they significantly decreased cell death. Our results suggest that using a combination of drugs to target multiple injury cascades may be a promising therapeutic strategy which warrants additional studies in vivo.

P003 NIM811, a Mitochondrial Permeability Transition Inhibitor, Reduces NMDA Excitotoxicity by Enhancing Network Activity

Excitotoxicity is a common mechanism of cell death in many neurologic disorders. Recently, there has been much interest in mitochondria-targeted agents for reducing excitotoxic cell death. However, it is unclear if this approach is beneficial for network dynamics and behavior because we do not fully understand how the “rescued” neurons integrate into the existing neural network to either maintain the same level of function, or alternatively, result in a dysfunctional circuit. The aims of this study were 1) to study the short- and long-term effects of N-Methyl-D-Aspartate (NMDA) stimulation, of varying magnitude, on the activity patterns of cortical neural network and 2) to investigate whether pretreatment with NIM811, a mitochondrial permeability transition inhibitor, improves network activity and/or decreases cell death. We used high-speed calcium imaging of a population of dissociated cortical neurons to monitor changes in network activity. We found that low levels of NMDA receptor activation led to an immediate increase in synchronized network-wide oscillations and an increase in functional connectivity that persisted for up to 6-hours. Blocking all activity with tetrodotoxin abolished the neuroprotective effect of short-duration NMDA stimulus. Longer stimulus led to an immediate suppression of spontaneous activity, which later returned in a subpopulation of neurons. There was a significant decrease in functional connectivity and neurons that did not reestablish integration with the rest of the network eventually died of apoptosis (caspase-3 positive). Pretreatment with NIM811 improved functional integration of the injured neurons into the network and decreased cell death relative to untreated cultures. We believe that improved calcium regulation with NIM811 treatment promotes synaptic transmission and reduces network impairment.

P004 Acute Bisperoxovanadium Therapy Stimulates AKT and MTOR Activity, Reduces Autophagy, and Promotes Neuroprotection and Functional Recovery Following Cervical Contusive Spinal Cord Injury

Following primary spinal cord injury (SCI), secondary events follow, extending pathology into adjacent areas of the cord initially spared by the primary insult. At the injury epicenter, the acute inflammatory response promotes cellular necrosis, while programmed cell death of neurons and glia follow in the surrounding injury penumbra. Recently, the PI3K/Akt/mTOR signaling cascade has received much attention due to its survival and regenerative functions following central nervous system (CNS) injury. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) negatively regulates this pathway, promoting programmed cell death, and prohibiting regeneration and repair. Bisperoxovanadium (bpV) compounds, known inhibitors of PTEN activity, have promoted neuroprotection following SCI, though no signaling mechanism has been linked to these effects in this injury type. However, other CNS injury models have linked bpV-mediated benefits to the PI3K/Akt/mTOR pathway. This study investigated the role of PI3K/Akt/mTOR pathway modulation on neuroprotection and functional recovery by bpV(pic) in a clinically-relevant cervical hemi-contusion SCI model. One animal group received twice-daily bpV(pic) treatments for 1 week following SCI, and underwent behavioral testing for 6 weeks. The tissue was used to assess long-term neuroprotection mediated by the treatment. Another group of injured animals were treated for 24 hours, and sacrificed to assess acute intracellular changes in signal transduction and physiological/pathological processes in order to link a mechanism to use of this drug in vivo following SCI. Our results show that acute administration of bpV(pic) promoted neuroprotection and functional recovery in our SCI model. Additionally, Akt and mTOR activity increased while autophagic activity decreased following treatment, suggesting this pathway plays a potential mechanistic role in cell survival processes post-injury, and that acute pharmacological intervention with bpV compounds may be a viable therapy for promoting neuroprotection and recovery following SCI.

P006 Intraspinal Transplantation of Recombinat Neuroprogenitor Cells in a Spinal Cord Injury Model of Central Neuropathic Pain

Chronic pain significantly reduces the quality of life of spinal cord injured patients and interferes with rehabilitative strategies. With low efficacy of conventional therapy, chronic pain presents a serious clinical problem. Therefore, there is a need for an alterative pain relief strategies. Hypothesized mechanisms underlying chronic neuropathic pain following injury to the nervous system include increased hyperexcitability of spinal dorsal horn neurons due to loss or dysfunction of inhibitory γ-aminobutyric acid (GABA)-ergic interneurons, and enhanced excitatory glutamate signaling through NMDA receptors. Intraspinal transplantation of engineered neuronal progenitor cells (NPCs) releasing GABA and/or glutamate receptor inhibitor- serine histogranin (SHG)- could restore inhibitory tone and reduce pain symptoms. Spinal clip compression injury was used to induce central neuropathic pain. Treatment with predifferentiated GABAergic NPCs injected intraspinally 2 weeks post injury reduced cold allodynia in injured rats and partially suppressed tactile allodynia. The addition of intrathecally injected SHG further improved the effects of NPC treatment in reducing neuropathic pain symptoms. Therefore, in order to enhance cell therapy, SHG cDNA was introduced into GABAergic progenitors. One week after intraspinal injection of engineered cells, over 60% reduction in cold allodynia was observed in treated animals. This effect was sustained for several weeks and was reduced by intrathecal injection of SHG antibody. Tactile allodynia was partially reduced by treatment as well. These findings support the potential for transplantation of genetically enhanced cells in pain therapy for spinal cord injured patients.

Supported by NS51667

P007 Neural Precursor Cells Express Trophins Following Transplantation But Remyelination is the Key Mechanism by Which they Augment Functional Recovery Following Rodent Spinal Cord Injury

BACKGROUND: Though myelination has been demonstrated following neural precursor cell (NPC) transplantation, the mechanism(s) by which NPCs enhance functional recovery from spinal cord injury (SCI) is unclear.

METHODS: Two weeks following clip compression SCI at the T7 bony level, rats underwent cellular or control transplantation. Cellular transplants included wild-type mouse NPCs and NPCs derived from shiverer mice unable to produce central myelin. In most experimental groups animals received minocycline, cyclosporin and peri-lesional infusion of trophins. Hind-leg motor function was assessed according to the BBB scale. qPCR for trophin expression was performed on NPCs and host cord isolated by FACS 1w post-transplant. H&E/LFB staining was used to assess grey and white matter, cyst and lesional tissue. Confocal microscopy was used to assess the relationship between transplanted cells and axons.

RESULTS: Animals transplanted with wild-type NPCs showed significantly greater functional recovery than any other group. Animals transplanted with shiverer NPCs performed worse on functional testing than even injured untreated animals. Both wild-type and shiverer NPCs ensheathed axons and but the latter did not produce MBP. NPCs isolated post-transplant expressed all four neurotrophins, CNTF, EGF, and bFGF at higher levels than host tissue. Pharmacotherapy and trophin infusion was associated with grey, white matter and oligodendrocyte sparing. Trophin infusion was, however, also associated with increased cyst and lesional tissue volume as well as reduced functional recovery.

DISCUSSION: The mere presence of transplanted cells did not lead to a functional benefit suggesting that the trophins they produce are without a functional effect. Shiverer NPCs may prevent remyelination by endogenous cells capable of myelin formation.

CONCLUSIONS: These results suggest that remyelination by NPCs is their most important contributor to functional recovery following SCI. Though these findings may be protocol-specific, they may lead to strategies to augment functional recovery associated with cellular transplantation for SCI.

P009 Electrophysiological Abnormalities after Minimal Traumatic Brain Injury

INTRODUCTION: Mild traumatic brain injury (mTBI) accounts for more than 90% of all cases of head trauma. Despite lasting cognitive, emotional, and behavioral deficits seen after mTBI, there are frequently no overt morphological defects, suggesting that the changes result from alterations in the activity and interaction of surviving neurons rather than direct damage to cells or tracts. We investigated hippocampal neural activity during task performance in rats to determine the effect of mTBI on physiological activity.

METHODS: Three male Sprague-Dawley rats (300-350 g) underwent mTBI via lateral fluid percussion (1.5 Atm) and were compared with three sham-operated rats. All six underwent stereotactic implantation of electrodes into the CA1 and CA3 hippocampal subfields ipsilateral to injury and were trained to a delayed non-match-to-sample swim T-maze. Single-neuron and local field potential (LFP) activity were analyzed during task performance 28 days after trauma.

RESULTS: There were no histological differences between control and mTBI rats. Rats subjected to mTBI demonstrated significantly worse memory with increasing delay. Examination of single-neuron spiking activity revealed fewer cells that change firing rate predictably during the task. Examination of local field potentials revealed delayed suppression of low-frequency LFP activity with task onset after mTBI despite no difference in overall theta power. Pyramidal cells were less entrained to the theta frequency, and coherence between spikes and LFP oscillations was significantly lower after mTBI.

CONCLUSIONS: Memory deficits after mTBI are correlated with disorganized patterns of hippocampal neural activity. Since this is seen in the absence of clear morphological defects, these data suggest that functional impairment after mTBI may result from stereotypic alterations in activity of individual neurons and may therefore be amenable to therapeutic neuromodulation.

P011 Minocycline Improves Olfactory Integrity and Olfactory Behaviour After Traumatic Brain Injury in Mice

INTRODUCTION: Olfactory dysfunction can often arise after traumatic brain injury (TBI). Olfactory bulb lesions result from the immediate loss of olfactory neurons due to the site and severity of the mechanical insult. Moreover, there is increasing awareness that diseases involving persistent neuroinflammation demonstrate deterioration of the olfactory bulbs. Since TBI triggers multiple neuroinflammatory cascades, we examined the effects of an acute anti-inflammatory treatment with minocycline on post-TBI olfactory behavior and olfactory bulb integrity.

METHODS: The mouse model of TBI by mechanical percussion was applied on anesthetized Swiss mice. The treatment protocol included three injections of minocycline (i.p.) at 5 min (90 mg/kg), 3 h and 9 h (45 mg/kg) post-TBI. An olfactory avoidance test was run from 1 up to 12 weeks post-TBI. At 12 weeks post-TBI, the digital images of the whole brains were taken and the surface of olfactory bulbs was measured.

RESULTS: TBI induced an olfactory avoidance behavior deficit that was significant from 2 weeks (P < 0.05) to at least 12 weeks (P < 0.001) post-TBI. Additionally, substantial olfactory bulb atrophy was observed at 12 weeks post-TBI (P < 0.001) that was strongly correlated with the olfactory impairment (n = 33; r = − 0.62; P = 0.001). The acute post-TBI treatment with minocycline was able to attenuate both the behavioral dysfunction (P < 0.01) and olfactory lesions (P < 0.01) in the long-term.

CONCLUSION: Inhibition of the acute phase of post-TBI neuroinflammation by minocycline is associated with the sparing of olfactory bulb tissue and improvement of olfactory behavior. These results emphasize the potential role of minocycline as a promising neuroprotective strategy for the prevention of olfactory lesions and deficits following TBI.

P013 Medial Septal Stimulation Following Traumatic Brain Injury Increases Exploration

RATIONALE: Traumatic brain injury (TBI) often results in cognitive deficits, including inattention and memory loss. Hippocampal theta rhythms are important in learning and spatial navigation, and we have shown that brain injured animals have significantly lower hippocampal theta power compared to uninjured animals. Since the medial septal projections to the hippocampus play an important role in the generation and maintenance of theta activity, we determined if continuous electrical stimulation of the rat medial septum would alter behavior in the metric spatial processing task.

METHODS: 26 male Sprague-Dawley rats (300-350 grams) underwent a sham (n = 5) or lateral fluid percussion injury (n = 21, 2.12-2.15 atm). Immediately after injury, we stereotactically implanted a medial septal stimulating electrode and hippocampal recording electrode ipsilateral to the injury site. On post-injury day 7, rats were tested with or without stimulation in the metric task (exploration of two novel objects before and after moving the objects closer together). Sham injured rats (n = 5) did not undergo stimulation, while injured rats received either no stimulation (n = 5) or continuous medial septal stimulation at 20μA (n = 5), 80μA (n = 6) or 200μA (n = 5) at a frequency of 7.7 Hz. Time spent exploring two novel objects was analyzed.

RESULTS: TBI animals without stimulation spent less time exploring objects than sham animals (14.2 ± 2.6 vs. 59.8 ± 7.7 seconds, p < 0.001). TBI animals with stimulation at 80μA and 200μA spent longer times exploring (75.1 ± 4.7 and 63.6 ± 5.1 seconds, respectively, p < 0.05) than TBI animals without stimulation (14.2 ± 2.6 seconds), while TBI animals stimulated at 20μA (28.3 ± 4.8 seconds, p > 0.05) were not different from unstimulated TBI animals. Mean exploration time did not differ between sham animals and TBI animals stimulated with either 80μA or 200μA (p > 0.05); however, only sham animals demonstrated habituation prior to moving the objects.

CONCLUSION: Medial septal stimulation in the theta frequency range results in current-dependent increases in exploration of novel objects.

P014 Repeat TBI: Duration of Vulnerability is Related to Duration of Decreased Cerebral Glucose Metabolism in the Juvenile Rat

Transient decreases in cerebral metabolism of glucose (CMRglc) are seen after brain injury and reflect a compromised state of the brain. We hypothesize that the duration of decrease CMRglc marks the duration of vulnerability of the brain to a 2^nd insult. Postnatal day 35 rats were given sham (n = 4),single (n = 3), repeat TBI (RTBI) at 24hr interval (n = 4) or RTBI at 120hr interval (n = 3)with a closed head injury. At 1 or 3 days after the last injury animals were prepared for ¹⁴C-2DG autoradiography. In the ipsilateral cortex after a single TBI, CMRglc decreased by 18.7% at 1d and 11% at 3d relative to sham CMRglc. When the 2^nd injury was introduced during this depression (24hr interval), the CMRglc decreased by 32% at 1d and 21%at 3d. When the 2^nd insult was introduced after the depression of the 1^st injury (120hr), CMRglc in the cortex decreased by 9.2% at1d and 10.6% at 3d. In the ipsilateral CA1, CMRglc decreased by 28.5% at 1d and 20% at 3d after a single injury. When the 2^nd injury was during the depression, CMRglc decreased further to 40.6% at 1d and 10.7% at 3d. When the 2^nd injury was delivered after the depression, CMRglc did not change significantly. Similar patterns were seen in the thalamus and perirhinal cortex. Introduction of a 2^nd injury during the depression of the 1^st injury caused significant changes in both the magnitude and duration of the subsequent CMRglc depression in the cortex, thalamus, hippocampus and perirhinal cortex. However, when the 2^nd injury occurred after CMRglc from the 1^st injury recovered, the consequence changes in CMRglc were truncated in magnitude and duration. These findings support the idea that CMRglc depression may be a biomarker for the window of vulnerability after a mild TBI.

Supported by NS058489, UCLA BIRC and NFL Charities Grant

P016 Expression of Neuroprotective Globin Genes Following Traumatic Brain Injury in Adult Mice

Improved TBI outcomes may be achieved through treatments that target the globin gene family (e.g., hemoglobin, myoglobin, neuroglobin, and cytoglobin). Globins are vital for binding oxygen. Neuroglobin has a high affinity for oxygen within the brain and is neuroprotective following hypoxia and ischemia. Cytoglobin is an efficient nitric oxide (NO) scavenger, and protects cells from reactive oxygen species (ROS). We examined expression of neuroglobin and cytoglobin post-TBI, and in relation to behavioral outcomes of adult mice. Controlled cortical impact (CCI) was induced in adult (5-6 month) C57/BL6 wild-type mice, and transgenic mice (The Jackson Laboratory, ME) expressing neuroglobin and enhanced green fluorescent protein (CAG-NGB, EGFP) via the chicken beta actin promoter coupled with the cytomegalovirus distal enhancer. Neuroglobin and cytoglobin mRNA was assessed (in wild-type mice only) prior to injury, and at 1, 3, 7, and 14 days post-TBI in samples of the injured cortex using quantitative real time polymerase chain reaction (qRT-PCR). The time couse study demonstrated late upregulation of neuroglobin and cytoglobin post-TBI. Significant differences in adult mice were observed for neuroglobin at 7 days post-TBI, and for cytoglobin at 14 days post-TBI when compared to pre-injury mRNA expression levels. The gridwalk was used for sensorimotor testing of adult (5-6 month) wild-type and CAG-NGB, EGFP mice, prior to injury, and at 2, 3, 7, and 14 days post-TBI. CAG-NGB, EGFP mice displayed significant reductions in the average number of foot faults per minute during gridwalk testing at 2, 3, and 7 days post-TBI when compared to wild type mice at each time point. Our gridwalk data show that overexpression of neuroglobin reduces sensorimotor deficits following TBI in adult mice. Based on the qRT-PCR and gridwalk data presented in this study we suggest that recovery from TBI may be improved by targeting neuroglobin and cytoglobin through novel therapeutic interventions.

P017 Neuroprotective Effects of EPHRINB3 Following Traumatic Brain Injury

Membrane bound ephrin ligands and their cognate Eph receptors are important developmental proteins that have recently been shown to regulate anti-apoptotic events during adult neurogenesis.

PURPOSE: To investigate whether EphB3/ephrinB3 signaling plays a role in blocking traumatic brain injury (TBI) induced damage in the adult cortex and hippocampus.

METHODS: Wild type, EphB3 (EphB3^-/-) and ephrinB3 (ephrinB3^-/-) knockout mice were subjected to controlled cortical impact (CCI) injury then administered pre-clustered soluble ephrinB3-Fc molecules (eB3-Fc) using mini-osmotic pumps. Cell death was quantified using terminal deoxynucleotidyl transferase dUTP nick end labeled (TUNEL) staining. Histological data was then compared to functional behavioral outcomes using rotarod motor assessment.

RESULTS: Here, we show that ephrinB3 is minimally expressed in the adult cortex, is down-regulated following CCI injury and is neuroprotective when infused in the adult murine forebrain. Application of eB3-Fc into the contralateral ventricle of wild type and ephrinB3^-/- mice for 3 days after CCI injury reduced cortical infarct volume which correlated with a reduced number of TUNEL-positive neurons in the cortex, dentate gyrus and CA3 hippocampus compared to Fc-control mice. This improvement in histopathology coincided with reduced motor deficits at 3, 7 and 14 days following CCI injury. Interestingly, similar neuroprotective events were also observed in the absence of EphB3 receptor (EphB3^-/-). Finally, stimulation of EphB3 using eB3-Fc infusion increased bcl-2 anti-apoptotic protein expression in the cortex at 3 days post-CCI injury compared to Fc-control infused mice.

CONCLUSION: EphB3/ephrinB3 signaling exerts neuroprotective effects via bcl-2 induction to prevent TBI-induced neuronal loss. These studies also suggest that EphB3 acts as a dependence receptor to mediate cell death in the injured adult cortex and may be a novel therapeutic target for reducing the deleterious effects following TBI.

P018 Pericontusional Transplantation of Neural Progenitor Cells Releasing a Multineurotrophin Reverses Creb Signaling Deficits After Traumatic Brain Injury in the Rat

Although traumatic brain injury (TBI) remains a leading cause of mortality and debilitation in the U.S. and elsewhere, there are no proven treatments currently available to attenuate disability. In addition to the gross histopathological consequences after brain injury, a series of signaling pathways are affected including extracellular signal-regulated kinase (ERK) and cAMP response element-binding (CREB) cascades. There is pronounced deficiency in the activation of these pathways, which are critical for hippocampal-dependent memory formation. The purpose of this investigation is to determine whether a novel combinatorial therapeutic approach resulting from transplantation of immunologically-privileged neural progenitor cells (NPCs) that have been transduced to secrete a prosurvival multineurotrophin (MNTS1) reverses ERK and CREB activation deficits and improves functional outcome. We generated a synthetic neurotrophin with multiple neurotrophic specificities through the exchange of seven key amino acids. This construct binds all prosurvival tyrosine kinase (Trk) receptors and seems to support neuronal survival more so than any one neurotrophin individually. To verify that MNTS1 recapitulates the biological activity of multiple neurotrophins, we assayed survival of dorsal root ganglion (DRG) cells and found MNTS1 generates extensive neurite outgrowth on whole DRG explants. For the in vivo study, male Sprague-Dawley rats received either sham surgery or parasagittal fluid-percussion brain injury. One week after trauma, animals were injected with NPCs transduced to release MNTS1/GFP or express GFP alone. One week post-injection, hippocampal slices from each group were generated and biochemically stimulated with glutamate or KCl. Our preliminary findings indicate a recovery of activation in CREB signaling after pericontusional transplantation of NPCs releasing MNTS1 in our clinically-relevant animal model of TBI. Using a novel combinatorial approach to reverse signaling deficits and attenuate cognitive impairment after TBI may be more effective in improving functional outcome than one-dimensional therapies alone.

Supported by NS030291.

P019 Variants of SLC6A4 and BDNF in Depression Risk and Onset Following Severe TBI

Depression following traumatic brain injury (TBI), or post-traumatic depression (PTD), presents a complex challenge in treatment and recovery following injury. In idiopathic depression, the interaction of serotonin and brain-derived neurotrophic factor (BDNF) signaling appears to play a role in etiology. However, it is still not clear if these interactions remain unchanged in PTD. In this study, genetic variants of the polymorphic region 5-HTTLPR of the serotonin transporter, including a single nucleotide polymorphism (SNP) RS25531 and a length variation, as well as the variable number of tandem repeats (VNTR) in Intron 2 were evaluated. These variants, along with the functional variant RS6265 and the tagging SNP RS7124442 in the BDNF gene were studied in relation to PTD incidence, onset, and duration in 78 persons with moderate to severe TBI. All patients were genotyped, and depressive symptoms were assessed with the Patient Health Questionnaire (PHQ-9) at 6 and 12 months post-injury. Of these participants, 21 (30.9%) were depressed at 6 months and 15 (25.4%) were depressed at 12 months, an increased incidence compared to idiopathic rates. Results at 6 months indicate an early protective role for the S allele, contrary to idiopathic depression literature. At 12 months, the LG allele was protective. The S and 12 allele of the VNTR seem to be protective against all subsets of PTD. Half of LG carriers never became depressed while the other 50% experienced transient depression with recovery by 12 months. Contrary to idiopathic depression, initial analysis of BDNF variants showed no association to PTD, but it may have implications in other outcome measures. Our study is unique in examining depression across recovery, differentiating between transient, persistent and late-onset depression. The implications of this study warrant further investigation into the role of genetic variation in post-TBI depression, allowing for more specialized interventions.

P020 Severe, Brief, Pressure-Controlled Hemorrhagic Shock After Traumatic Brain Injury Exacerbates Long-Term Neuropathological Damage and Functional Deficits in Mice

BACKGROUND: Hypotension after traumatic brain injury (TBI) critically worsens outcome. Mouse models of TBI plus hemorrhagic shock (HS) are limited. We published the first report of TBI + HS in mice and noted increased hippocampal neuronal death at 7d post-injury. HYPOTHESIS: TBI + HS in mice will exacerbate long-term neuropathological damage and cognitive dysfunction vs TBI or HS alone.

METHODS: C57BL6 male mice (n = 50) were randomized into four groups (n = 15/group): sham, TBI, HS, TBI + HS. TBI was induced using mild controlled cortical impact. A severe pressure controlled HS targeted a mean arterial pressure (MAP) of 25-27 mmHg for 35min. We included a HS phase, followed by a 90min “pre-hospital” resuscitation where lactated Ringer's was given to maintain MAP > 70 mmHg and then a “hospital phase” where the shed blood was re-infused. MAP and temperature were monitored. Arterial blood gases, pH, and lactate were serially assessed. On d14-20 mice were tested in the Morris water maze (MWM, n = 10/group) using a spatial memory acquisition paradigm (latency to find hidden platform). On d21, lesion volume (n = 10/group) or hippocampal cell counts (n = 5/group) were assessed.

RESULTS: Groups that underwent HS required fluid resuscitation (∼150-200 mL/kg). Lactate was increased in HS and TBI + HS groups, with TBI + HS having a larger increase than HS (p < 0.05). MWM latency was increased vs sham only in TBI + HS (p < 0.05). The TBI + HS group had a 33.0% increase in lesion volume vs TBI (7.33 ± 0.64 vs 5.51 ± 0.36, p < 0.05). Hemispheric volume loss was increased 33.3% in TBI + HS vs TBI (17.32 ± 1.14 vs 12.99 ± 1.14, p < 0.05). MWM latencies correlated with percent hemispheric volume loss (p < 0.005). CA1 cell counts were reduced 28.9% in TBI + HS vs sham (p < 0.05) but not in HS or TBI.

CONCLUSIONS: We characterized a clinically relevant model of TBI + HS in mice. The long-term outcome data confirm our initial 7d studies and suggest that CA1 hippocampus is a key target for testing novel therapies after TBI + HS.

P024 Additive Effects of Multiple Mild Traumatic Brain Injuries in Hippocampal Slice Cultures

The majority of traumatic brain injury (TBI) occurrences are mild TBI (mTBI), which result in increased likelihood for repeated mTBI.¹ To quantify the synergistic effect of two sub-threshold mTBI events as compared to one, hippocampal organotypic cultures (OTC) were prepared using published methods.² In brief, hippocampii of 8-10 day old Sprague-Dawley rats were sectioned and plated on coated silicone membranes. OTC were cultured for 14-16 days at 37°C and 5% CO₂ prior to delivery of either one or two 10% biaxial stretch-injuries 24 hours apart. The injury device and loading mechanism have been described previously and characterized in detail.² Control cultures were clamped on the device, but received no injury. Prior to and four days following the first injury, cell death was quantified by propidium iodide (PI) staining as the percent area above an intensity threshold. A single, sub-threshold injury did not significantly increase cell death four days after injury in any region of the hippocampus. In contrast, cell death in OTC receiving two identical injuries 24 hours apart was significantly increased. These results suggest there is a synergistic cell death response within the hippocampus for two mild injuries delivered 24 hours apart as compared one injury, quantifiable four days post-injury. An in vitro model of repeated mTBI may help elucidate cellular mechanisms responsible for this increased susceptibility.

P025 Extracellular Galectin-3 Confers Neuroprotection Against Excitotoxic Injury Through Dual Activity at Glutamate Transporters and Glycosylated Neuronal Cell Surface Proteins

Levels of the beta-galactoside binding lectin galectin-3 (gal-3) are elevated in brain or spinal cord after traumatic or ischemic injury, as well as in neurodegenerative disease, such as ALS. In all these conditions, gal-3 is primarily expressed by activated microglia. However, the precise role of gal-3 in secondary injury processes and/or inflammation is unclear. As excitotoxic injury is a shared mechanism of neuronal death in both acute and chronic neurodegeneration, we evaluated whether gal-3 may influence cell death induced by glutamate. In an organotypic model of slow excitotoxicity, gal-3 dose-dependently reduced cell death, and this neuroprotection was coincident with increased excitatory amino acid uptake, primarily through non-GLT1 glutamate transporters. Further, gal-3 also enhanced glutamate uptake in pure cultures of activated primary astrocytes, and the increase in glutamate uptake was associated with increased expression of GLAST and GLT-1 glutamate transporters. Gal-3 also reduced glutamate-induced injury in pure NSC-34 motor neuron cultures that contained no glia. Such neuroprotection was reduced in the presence of lactose, a competitive inhibitor of the gal-3 carbohydrate recognition domain. Moreover, prior incubation with a selective O-glycosylation inhibitor had no effect on the neuroprotective activity of gal-3 against glutamate, whereas a selective N-glycosylation inhibitor prevented gal-3 neuroprotection, though this drug was also toxic, with or without glutamate-induced injury, even at low micromolar concentrations. Collectively, these data suggest that expression and release of gal-3 by activated microglia after injury may support neurons in several ways. Gal-3 may indirectly protect neurons by enhancing glutamate uptake through increased expression of specific glutamate transporters on astrocytes. In addition, gal-3 may be directly neuroprotective, potentially through binding to N-glycan containing glycosylated proteins on the neuronal cell surface.

Supported by NIH NCMRR/NINDS 2R24HD050846-06 and CNMC RAC.

P027 The Extracellular Environment May Influence the Neuronal Response to Mechanical Forces in Mild Traumatic Brain Injury

The high incidence of blast-induced Traumatic Brain Injury in the conflicts in Afghanistan and Iraq has highlighted the challenges associated with treating mild Traumatic Brain Injury (mTBI). Previous studies aimed at understanding the mechanisms which initiate neuronal injury have primarily focused on the magnitude of forces and strains required to elicit a response, but have generally overlooked how these forces or strains are transduced by the cell. Previous research in non-neuronal cell types has shown that cells actively respond to extracellular mechanical stimuli transmitted through focal adhesion proteins such as integrins. Therefore, we hypothesized that force transmission through extracellular matrix proteins linked to integrins may be an important factor in neuronal injury initiation in mTBI. To study this, we built an in vitro high speed stretcher device which allowed us to modify the extracellular matrix substrate through which injurious forces could be delivered to a population of neurons. Cortical neurons from neonatal Sprague-Dawley rats were seeded on stretchable membranes coated with poly-l-lysine, fibronectin, or laminin and cultured for 5 days. A single, high speed stretch (1% per ms) was administered to mimic blast wave propagation through the brain. Injured neurons were identified by widespread focal swelling of the neurites, similar to the hallmark injury morphology of diffuse axonal injury in vivo. We found that the density of focal adhesions formed between neurites and the substrate depended upon substrate coating, indicating that the extracellular environment may influence how forces are transmitted to neurons. Furthermore, substrate coating affected the percentage of injured neurons for a given strain magnitude, suggesting that injury sensitivity is dependent upon the extracellular environment. An understanding of how extracellular force transmission through mechanically sensitive cellular components such as integrins contributes to the initiation of neuronal injury may lead to the identification of novel treatment strategies for mTBI.

P029 Development of an In Vitro Model to Study Shear Injury in Brain Slice Cultures

Blast induced Traumatic brain injury is a significant risk in warfare. The blast pressure wave passes through the brain and triggers closed-head injury, which is partly due to shear forces that stretch axons (primary injury). Further shear injury may occur following the blast when the soldier's head is exposed to rapid acceleration or deceleration in a vehicle (tertiary injury). We have developed a model to create shear injury in cultured hippocampal brain slices, a tractable in vitro model in which physiologic synaptic circuitry is preserved. Such a model allows study of the earliest modifications in neuronal morphology and physiology, which contribute to the later pathologies observed in animal models and in humans. Our model is a modification of an earlier model reported by Bottlang et al [J. Neurotrauma 24:1068 (2007)]. It consists of a small air tank, which is pressurized from a regular lab air outlet. Rapid release of the compressed air drives a plunger, which accelerates a sled holding up to 6 Millicell membranes with slices growing on top. The sled travels down a track, impacts a damper at the end, and decelerates. This acceleration and deceleration cause rapid changes in g forces (up to 280 g) over the duration tens of milliseconds. We are currently developing and calibrating biological readouts of the injury. Our aim is to visualize the earliest changes in synaptic microstructure and circuitry by combining biolistic transfection of fluorescent proteins and 2-photon microscopic analysis of injured brain slices. Further, we are testing whether the biolistic transfection pressure wave itself (produced by the Gene-gun) can induce a controlled injury in the same slice culture model.

P031 Protective Effects of Phenelzine, a Scavenger of Lipid Peroxidation-Derived 4-Hydroxynonenal, on Cerebral Mitochondria: An in Vitro Study

Previous studies in our laboratory have demonstrated that free radical-induced lipid peroxidation (LP) and its toxic aldehydic byproduct 4-hydroxy-2-nonenal (4-HNE) are associated with posttraumatic mitochondrial respiratory dysfunction in the injured mouse brain (Singh et al., J.Cereb. Blood Flow & Metab. 26:1407-1418, 2006), and that inhibitors of post-TBI LP such as U-83836E can preserve brain mitochondrial function (Mustafa et al., J. Neurochem. 114: 271-280, 2010). More recently, we have confirmed that direct application of 4-HNE to isolated healthy mouse brain mitochondria mimics the effects of TBI (Vaishnav et al., J Neurotrauma. 27:1311-20, 2010). In the current study, we investigated the possible mitochondrial protective effects of phenelzine, an older MAO inhibitory antidepressant drug, against 4-HNE-induced mitochondrial respiratory dysfunction. Phenelzine has been shown to be a chemical scavenger of LP-derived reactive aldehydes by virtue of its containing a hydrazine functional group which can covalently react with 4-HNE. Bioenergetics in healthy, ficoll gradient-isolated mouse cortical mitochondria were measured using the Seahorse Bioscience XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Complex I (pyruvate + malate substrate) and complex II (succinate substrate)-driven oxygen consumption rates (OCR, pmoles O₂/min) were assessed after exposure to 4-HNE which inhibited both in a dose-dependent manner. On the other hand, pretreatment of mitochondria with equimolar concentrations of phenelzine significantly antagonized the respiratory depressant effects of 4-HNE consistent with its ability to act as a 4-HNE scavenger. For instance, 30 μM 4-HNE attenuated complex I function by 37% whereas phenelzine pretreatment reduced this to 17% (p < 0.05 vs. 4-HNE alone). Complex II was inhibited by 24% which was completely prevented by phenelzine. Ongoing studies are evaluating the mitochondrial protective effects of phenelzine against the toxic effects of SIN-1 (3-morpholinosydnonimine), a peroxynitrite donor, and its ability to preserve cerebral cortical mitochondrial function in vivo after experimental TBI.

P032 The Interaction Between Caspase-Dependent and -Independent Pathways in In Vitro Models of Neuronal Cell Death

Neuronal loss is a key feature of both acute central nervous system (CNS) injury and chronic neurodegeneration. Understanding the mechanisms of neuronal cell death will facilitate the development of novel therapeutic strategies for CNS injury. Caspase-dependent and apoptosis-inducing factor (AIF)-dependent (caspase-independent) cell death are two of the most important cell death pathways following CNS injury and in this study we investigated their roles and interdependencies using in vitro models.

We used primary cortical neurons to examine the relative role of caspase-dependent and -independent cell death pathways using inducers such as etoposide and N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG). Cell death was evaluated by release of lactate dehydrogenase ad calcein fluorescence; caspase-3 activity was determined by Ac-DEVD-AMC fluorescence and detection of active caspase fragments; NAD and ATP measurements were performed to determine bioenergetic status in neurons.

Our data indicate that etoposide predominantly induces caspase-dependent cell death characterized by intense caspase activity. Caspase inhibitors were highly neuroprotective in the etoposide paradigm. In contrast, MNNG predominantly induces caspase-independent cell death that features activation of the poly(ADP-ribose) polymerase (PARP)-AIF pathway. Treatments with PARP inhibitors such as PJ34 were neuroprotective in this cell death paradigm while no improvement was observed with caspase inhibitors. Unlike etoposide-induced neuronal death, the MNNG cell death paradigm is characterized by compromised bioenergetic status with rapidly decreasing NAD and ATP levels that are reversed by PJ34 treatment. Interestingly, neurons exposed to MNNG and treated with PJ34 showed increased caspase activity suggesting that following PARP inhibition and/or bioenergetic recovery the neuronal death processes attempt to redirect to alternative pathways including caspase mechanisms that require a more preserved bioenergetic status.

We conclude that multi-potential interventions targeting several cell death pathways will show consistent and sustained neuroprotection and combination therapies for both neuronal cell death pathways should be evaluated in in vivo CNS injury models.

P033 Pro-Inflammatory Cytokine Regulation of Cyclic-Amp-Phosphodiesterase-4 Signaling in Microglia

Cyclic AMP levels are critical in suppressing immunoactivation and pro-inflammatory cytokine production as demonstrated by adenylyl cyclase activators, cAMP analogs and phosphodiesterase (PDE4) inhibitors. The positive feedback loop of pro-inflammatory cytokine production and immunoactivation implies that pro-inflammatory cytokines may not only be regulated by cAMP but conversely regulate cAMP signaling. In this study we investigated how the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) may differentially alter cyclic AMP signaling in microglia with a particular emphasis on the PDE4 family of enzymes, the main PDEs responsible for cyclic AMP hydrolysis in the CNS. Exposure of cultured microglia to either TNF-α or IL-1β produced a profound reduction (>90%) in cAMP within 30min that then recovered by 1h after IL-1β but remained persistently suppressed with TNF-α through 48h. Accompanying TNF-α-induced cAMP reductions, but not IL-1β, was a matching increase in cAMP-PDE activity; acute IL-1β-induced cAMP changes may be related to altered adenylyl cyclase activity. The role of PDE4 activity in TNF-α-induced cAMP reductions was confirmed by simultaneous addition of the PDE4 inhibitor Rolipram, which abrogated these effects. Examination of pde4 mRNA by RT-PCR revealed an immediate, persistent increase in pde4b with TNF-α; IL-1β increased all pde4 mRNAs (a, b and d). Immunoblot quantification of PDE4 showed that both cytokines increased PDE4A1, but only TNF-α also increased PDE4B2. PDE4 immunocytochemistry revealed nuclear translocation of PDE4A with IL-1β but not TNF-α, though TNF-α induced PDE4B nuclear translocation. These studies show that TNF-α and IL-1β differentially affect cAMP-PDE signaling in microglia and that specific targeting of PDE4B2 may be a putative therapeutic direction for reducing microglial activation in CNS injury and neurodegenerative diseases.

P034 Astrocytic Modulation of Neuronal Networks Through Glutamate Gliotransmission

Activation of post-synaptic NMDA and AMPA receptors from astrocytic glutamate release has been shown to be a potentially significant mode of synaptic modulation and excitotoxic damage. Translating studies in gliotransmission at the synaptic level into a larger understanding of neuronal network activity may provide the key to accurately model post-injury excitotoxicity, physiologic slow-inward currents (SICs), and restoration of large-scale function to damaged networks.

In this study we generated simple network models of a single astrocyte coupled into a small population of neurons to see the effect of astrocytic modulation on the overall activity of the model neurons. We determine the response of post-synaptic receptor populations to an astrocytic glutamate release at one dendritic spine then generate an idealized geometry for a group of spines that can be embedded in a more physiologic topology. A modeled astrocyte surface covers each spine and provides the source of the vesicular release. By simulating this calcium wave (Ca-wave) in a model astrocyte, we are able to track the responses of a set of adjacent neurons through this topology and determine how signalling through Ca-waves and gliotransmission can influence activity of connected neurons through post-synaptic receptors preferentially activated by astrocytic vesicles.

Our model demonstrates large, sustained SICs that resemble in vitro observations, as well as distributed activity that leads to further questions about the role of astrocytes in maintaining the integrity of the neuronal network. Extending the model by incorporating neuron- > astrocyte communication and threshold-based neuron damage can further provide us with a more realistic view capable of fully characterizing the role of gliotransmission in post-injury damage to networks of neurons, giving us a powerful tool in our attempts to minimize damage, restore function and understand distributed pathological signalling in the brain.

P035 Traumatic Brain Injury's Impact on MRNA Translational Mechanisms

Developing effective therapies for TBI-impaired learning and memory is a difficult challenge because the underlying pathological mechanisms are not known. Neuronal apoptosis and necrosis are clear mechanisms for dysfunction, but some studies indicate cognitive deficiencies without neuronal cell death. Studies reporting changes in receptor expression suggest the existence of an injury induced pathological plasticity that alters the function of the surviving cells. A key calcium-dependent, mechanism implicated in plasticity is local dendritic mRNA translation. Previous fluid percussion injury studies by others (Atkins et al., JCBFM, 2006 and Chen et. al, JCBFM, 2007) found CaMKII, CPEB and mTor pathways to be activated in the cortical and hippocampal tissue of injured rats. This study measures the impact of the injury-induced changes in all three pathways which are key regulators for both dendritic and global mRNA translation. In this in vitro study, we find the mTor, CPEB, and CaMKII pathways to be dynamically modulated by stretch injury in an NMDAR dependent manner. The impact of stretch injury on mRNA translation was also directly measured using an in vitro imaging assay; changes in translation of dendritically localized proteins were found within minutes of stretch injury. The results of this study suggest that mechanical injury can alter the expression of dendritic proteins via modulation of translational pathways.

P036 Temporal Course of Mitochondrial Damage in Primary Cortical Neurons After Mechanical Stretch Injury: Focus on Mitophagy

Traumatic brain injury (TBI) can lead to both direct mechanical damage and functional disturbance in mitochondria. Mitochondrial dysfunction is a key contributor to neuronal death after TBI via release of pro-apoptotic factors. Thus, timely elimination of impaired mitochondria via selective mitochondrial autophagy (mitophagy) could be an effective strategy for prevention of neuronal death. In order to investigate TBI-induced mitophagy, we evaluated the temporal course of mitochondrial damage with a focus on mitophagy. Because cardiolipin (CL), a mitochondria-specific phospholipid, plays an important role in the maintenance of mitochondrial functions, we also explored possible effects of manipulation of CL content on the mitophagy after mechanical stretch in primary cortical neurons. Mitophagy was assessed by LC 3 shift, electron microscopy (EM), and degradation of mitochondrial markers (Cytochrome c oxidase (COX IV) and TOM 40). Mitophagy was seen as early as 1h and continued for 24h after injury. However, neuronal cell death (assessed by increased caspase 3/7 activities and phosphatidylserine externalization) did not occur until 6-12 h after injury. Manipulation of neuronal CL levels by knocking down CL synthase (CLS, the rate limiting enzyme in the synthesis of CL) using siRNA technology did not change neuronal mitochondrial morphology and function (assessed by EM, COX IV and TOM 40 expressions, and cellular ATP levels). Although administration of CLS siRNA did not change CL molecular speciation (assessed by electro-spray ionization mass spectrometry), it produced a 15 and 46% decrease in CL content at 72 and at 96h, respectively. Moreover, CLS deficiency in neurons markedly inhibited mechanical stretch induced mitophagy early after stretch. Taken together, our data indicate that mechanical stretch can induce neuronal mitophagy—a process that requires CLS early after injury. Further studies are required to evaluate whether elimination of damaged mitochondria is beneficial after TBI.

P037 Studies on Blast-Induced Traumatic Brain Injury Using an in Vitro Model System with Shock Tube

The mechanism of blast-induced traumatic brain injury (bTBI) and subsequent neurobehavioral deficits are still not completely understood which impede the development of effective therapeutics for pre/post-exposure treatment strategies to protect military and civilian population. We have developed an in vitro model system for studying the mechanism of bTBI and identify effective pre/post-exposure therapeutics using a compressed air-driven shock tube and utilizing SH-SY5Y human neuroblastoma cells as well as NG108-15, a rat neuroblastoma/mouse glioblastoma cell line in 96 well tissue culture plates (Arun et. al, NeuroReport 2011). By using this model, we tested the effect of repeated blast exposures on the neurobiological effects in SH-SY5Y cells and screened potential therapeutics for protection. The results indicate highest cellular injury following the first exposure and reduced injury following repeated blast exposures indicating that repeated blast exposures induce the production/release of cellular factors involved in protection. Proteomic analysis of cell homogenate after repeated blast exposures showed significant reduction in the level of cyclophilin A (CyP-A), which is reported to be involved in cellular protection after secreting in to the extracellular matrix. Western blot analysis confirmed the decreased intracellular levels of CyP-A and detectable amount in the culture medium after repeated blast exposures suggesting that CyP-A secreted in to the culture medium is likely involved in providing protection against repeated blast exposures. Therapeutic screening showed that histone deacetylase inhibitors and drugs having antioxidant and anti-inflammatory properties can provide significant protection against bTBI. Studying the mechanism of protection by secreted CyP-A may lead to the development of effective therapeutics against bTBI. The in vitro model system using shock tube can be successfully used for screening therapeutics against bTBI before testing in vivo.

P038 Brain Death in a Muslim Population: A Ten Year Experience in a Tertiary Care Hospital in Saudi Arabia

INTRODUCTION: In Muslim patients, end of life care in brain dead patients is not defined. We conducted this study to look at the outcome of Muslim brain dead patients in our non-trauma tertiary care hospital.

METHODS: We retrospectively collected clinical data for all adult patients who were declared brain dead between January 2001 and March 2011.

RESULTS: A total of forty patients were found. The average age was 45 years, 22 male and 18 female. Twenty patients had intra-cranial hemorrhage, five had cerebral neoplasm and rest of the patients had miscellaneous etiologies like anoxic brain injury. The average time from admission to diagnosis of brain death was 7.6 days. Per hospital policy, two attending physicians are required to confirm brain death. Five patients expired before completion of the protocol. In rest of the patients, it was completed by an Intensivist and the second physician was either Neurologist (67%) or Neurosurgeon (33%). Electroencephalogram was used as a confirmatory test in all patients, except five who had an additional perfusion scan done. 25 patients were “Full Code” before brain death confirmation. However, after brain death three patients remained “Full Code”, nineteen had “Do Not Resuscitate (DNR)” orders and eighteen had DNR with limitations on vasopressors. Ventilator withdrawal occurred only in eleven patients. Average time to death from confirmation of brain death was two days. 24 patients were eligible for organ donation, however only two had organs harvested. There was no involvement of social worker or chaplain in the end of life discussions.

CONCLUSION: Our experience shows that even though the diagnosis of brain death follows international standards, the care afterwards is yet to be defined in Muslim patients. The concept of terminal withdrawal and organ donation is not accepted by most of the families. Future studies should focus to improve these issues.

P039 Stent Angioplasty is an Effective and Safe Method for Carotid Artery Stenosis

OBJECTIVE: Stent angioplasty of carotid artery stenosis was known as an alternative to surgical endarterectomy. The research was to observe the efficacy and safety of stent angioplasty for symptomatic carotid artery stenosis.

METHODS: a total of 72 patients were selected into treatment group after carotid artery detection of color-coded Doppler ultrasonography. The mean age of the patients was 62.7 years. The most common indications for the operation were symptomatic carotid stenosis with a level of stenosis of over 70%. DSA was performed to diagnose the position and severity of carotid artery stenosis. And then, all cases were treated by carotid artery stent angioplasty.

RESULTS: DSA showed that the mean pre-procedural carotid stenosis degree is 81.7%. A level of residual stenosis less than 20% was achieved in all 72 cases after stent angioplasty. A week later, all patients showed profound clinical improvement and great changes in neurological function. 23 cases (31.9%) with heart rate slow down. The blood pressure declined in 15 cases (20.8%). However, 8 patients (11.1%) had complications associated with the procedure, of which 5 patients (6.9%) were found of carotid vasospasm during stents implantation. 1 case (1.4%) with cerebral embolization. 2 cases (2.8%) were clinical presentations of a re-perfusion syndrome. During a mean clinical follow-up time of 12 months. None had additional transient ischemic attacks or stroke. No sign of de novo in-stent stenosis was detected through Doppler ultrasonography.

CONCLUSION: The research showed the stent angioplasty is a safe and effective method for symptomatic carotid artery stenosis. However, the procedures do carry a risk of complications such as thromboembolization, arterial injury, and high perfusion syndrome. Perfect operation skills and performed by appropriately trained specialists can make the risk minimum. Accordingly, we propose the Stent angioplasty can be an ideal treatment option of carotid artery stenosis.

P041 Role of RyR2 in Spinal Cord Hypoxic/Reperfusion Injury

PURPOSE: Calcium influx into cells is responsible for initiating the cell death in neuronal tissue after hypoxic injury. Changes in intracellular calcium with subsequent increased expression of RyR2 are hypothesized to cause cell death after hypoxic injury.

METHODS: In this study we used western blotting, RT-PCR, Immunofluorescence, Immunohistochemistry and biochemical assays to examine changes in protein and gene expression of RyR2 after spinal cord injury (SCI) in the rat.

RESULTS: Quantitative immunoblotting showed increase in the expression of RyR2 at 4 hours during hypoxic/reperfusion injury of dorsal column. Moreover, RT-PCR showed 36 fold increases in mRNA of RyR2 after 4 hours of hypoxic injury of white matter. With histochemistry we found RyR2 localized in axons and astrocytes in the white matter of the spinal cord. After treatment with KN-62; (inhibitor of CaMKII) and SP600125 (inhibitor of JNK), there is a significant reduction in the expression of RyR2, indicating the role of these molecules in RyR2 regulation. BAPTA-AM, an intracellular chelator of Ca⁺⁺ significantly reduced the expression of RyR2 and phosphorylation of CaMKII. Finally, bioassay with quantitative analysis showed that treatment with KN-62 and SP600125 significantly reduced the cellular oxidative stress suggesting that increased expression of RyR2 is one of the factors responsible for increased cellular oxidative load during spinal cord injury.

CONCLUSION: We conclude that RyR2 gene and protein expression in astrocytes and axons is markedly increased after hypoxic injury. Further CaMKII/JNK pathway upregulates RyR2 expression after hypoxic injury. Increased RyR2 expression leads to increase oxidative stress. Therefore we propose that inhibitors of CaMKII/JNK pathway would reduce the cellular oxidative load and thereby have a neuroprotective role.

P042 Upregulation of Inositol 1, 4, 5-Triphosphate Receptor, Type 1 (IP3R1) in Spinal Cord Hypoxic Injury

PURPOSE: In the present study we have seen the regulation of IP3R1 isoform in hypoxic/reperfusion injury of spinal cord dorsal column in vitro. Deregulation of IP3Rs has pathological consequences.

METHODS: Dorsal columns were isolated from the spinal cord of adult rats and injured by exposure to hypoxic condition for 1 hour. After injury, reperfusion was carried out for 0, 2, 4 and 8 hour. In treatment group, inhibitor was perfused both during hypoxia and during reperfusion, which was carried out for different time interval. Tissues were collected and processed for western blotting, Immunohistochemistry and real time PCR.

RESULTS: We found increased expression of IP3R1 after hypoxic/reperfusion injury of spinal cord dorsal column in vitro. Maximum expression of IP3R1 was seen at 4 hr after hypoxia. Immunohistological studies show the localization of IP3R1 in axons and glial cells. Further identifying the signaling pathway involved in the regulation, we found CaMKII inhibitor KN-62 and JNK inhibitor SP600125 reduced the expression of IP3R1 suggesting the role of CaMKII and JNK in the regulation of IP3R1 expression. We further checked the effect of KN-62 and SP600125 on release of cytochrome c and caspase 3 and found these inhibitors reduced the release of cytochrome c and caspase 3 cleavages.

CONCLUSION: Our study concludes that IP3R1 expression is increased in hypoxic/ reperfusion injury of spinal cord and it is regulated by CaMKII/JNK pathway. This pathway also inhibit the release of cytochrome c and cleavage of caspase 3 probably by down regulating expression of Ca⁺⁺ channels.

P043 Optoacoustic Monitoring of Oxygen Saturation in the Superior Sagittal Sinus of Neonates

Continuous, noninvasive monitoring of cerebral blood flow (CBF) and cerebral oxygen consumption (CMRO₂) in neonates provides information to assess cerebral ischemia with or without associated intraventricular hemorrhage (IVH). Measuring cerebral vascular function of preterm neonates with low birth weights (lbw) and very low birth weights (vlbw) is particularly useful, since normal mean arterial pressure (MAP) is poorly defined and small changes in MAP in these groups may cause large changes in cerebral oxygen delivery. Monitoring of oxygen saturation (SO₂) in the superior sagittal sinus (SSS) reflects the ability of CBF to meet the demands of CMRO₂. SO₂ in the SSS (SsssO₂) varies directly with CBF and inversely with CMRO₂. An optoacoustic monitor was designed and built to noninvasively assess neonatal SsssO₂ through both the anterior and posterior fontanels. The optoacoustic monitor uses a pulsed laser source to generate an ultrasound signal that is directly transmitted from the SSS to a custom-designed acoustic detector. Continuous measurements were obtained for up to 10 minutes in 7 neonates. SsssO₂ in the individual neonates was 72.0% ± 1.7%, 71.3% ± 2.0%, 74.2 ± 2.0%, 66.6% ± 4.0%, 69.8% ± 1.9%, 75.7% ± 0.9%, and 77.2 ± 3.4%, respectively. Mean SsssO₂ for the 7 neonates was 72.4% ± 2.3%. Although there is no gold standard for comparison, the measurements are consistent with expected values, such as those obtained from jugular venous bulb measurements in children and adults. These measurements show promise for noninvasive research into the function of the cerebral circulation in lbw and vlbw infants and for clinical monitoring of these high-risk infants.

P044 The Effect of Poly(3-Hydroxybutyrate-CO-3-Hydroxyhexanoate)(PHBHHX) and Human Mesenchymal Stem Cell (hMSC) on Axonal Regeneration in Experimental Sciatic Nerve Damage

OBJECTIVES: To evaluate the treatment effect of PHBHHX and hMSC on axonal regeneration in experimental rat sciatic nerve damage, and also to compare the results of this modality with autologus nerve graft which is known to be the gold standart for pheripheral nerve repair.

MATERIALS AND METHODS: In this research, an experimental 10 mm long nerve gap was created in Spraque-Dawley albino rats. Three groups, 9 rats in each, were constituted. The gaps were repaired with autologous nerve graft, PHBHHX nerve graft tube with oriented nanofiber three dimensional surface alone and PHBHHX nerve graft tube with oriented nanofiber three dimensional surface filled with hMSCs inside, respectively. The rats were evaluated for functional recovery at the end of fourth and eighth weeks with sciatic functional index. At the end of eighth week, electromyographic evaluation was performed using compound muscle action potantials. Rats then were sacrificed, histopathologic evaluation with ligth microscopy for axonal regeneration, and transmission electron microscopy for myelin formation was carried out.

RESULTS: Autograft and the PHBHHX with hMSC groups showed functional improvement with time, whereas PHBHHX alone group did not, in sciatic functional index. Electromyographic evaluation showed better results in autograft and PHBHHX with hMSC groups, when compared with PHBHHX alone group. There were no statistical difference between autograft and PHBHHX with hMSC groups. Histopathological examination showed regenerated and myelinated axons in each group. Autograft group was better than the others, and PHBHHX with hMSC group was better than PHBHHX alone group by means of axonal regeneration and myelin formation.

CONCLUSIONS: Study showed that the nerve grafts prepared from PHBHHX with oriented nanofiber three dimensional surface aided to nerve regeneration either used alone or with hMSC. PHBHHX, when used with mesenchymal stem cells, provided better nerve regeneration than alone, and reached same statistical treatment effect in functional and electromyographic evaluation with autografting.

P045 Effects of (-)-Epigallocatechin-3-Gallate (EGCG) on Functional and Sensory Recovery in Sciatic Nerve Crush Model

Recent studies revealed the neuroprotective effects of green tea (GT) epigallocatechin-3-gallate (EGCG) on a variety of neuronal injury. We have shown that GT consumption improves both reflexes and sensation which are often affected in the course of peripheral neuropathy in unilateral chronic constriction injury to the sciatic nerve. The purpose of this study was to determine the effects of EGCG on the tissue protection and behavioral improvement in the sciatic nerve crush model. Wistar male rats (n = 8) were randomly assigned to three groups as follows: sham, crush treated with saline (i.p.) and experimental treated with 50mg/ml EGCG (i.p.) (1-2 hours, 1 day and 2 day post injury). The duration and magnitude of functional, thermal and behavioral hyperalgesia recovery were monitored. EGCG-treated axonotmized group showed significant improvement in the toe spread and foot positioning analysis. EGCG treatment resulted in an earlier and significant gain of hindlimb extension force as evaluated by the EPT and derived percentage motor deficit (P < 0.01). Likewise, the proprioceptive and motor function expressed by the hopping response was fully restored 2-3 weeks earlier. EGCG significantly reduced the axonotmesis-induced thermal and mechanical hyperalgesia as well as mechanical allodynia compared to saline treated animals. Also, treated rats showed significant recovery measured by paw withdrawal reflex (P < 0.0001) and latency (P < 0.005) to nociceptive mechanical pressure. Tail flick thresholds showed that the EGCG treatment significantly (P < 0.003) accelerated the recovery. Photographs obtained from light and electron microscopy and morphometric analysis of the sciatic nerves showed significant (P < 0.0001) increase in the number of myelinated fibers and a significant decrease (P < 0.0001) in the number of myelinated axonal fibers. Our results demonstrate that EGCG enhances functional and sensory recovery of peripheral nerve injuries in rat and thus improves nerve regeneration.

P047 Survival, Proliferation, Migration, and Myelination of GFP-Expressing Schwann Cells Transplanted into the Injured Rat Spinal Cord

Schwann cells (SCs) have been considered one of the best cell types for transplantation to treat spinal cord injury (SCI) due to their unique growth-promoting properties. Despite the extensive use as donor cells for transplantation in SCI models, the fate of SCs following transplantation remains controversial and needs to be fully elucidated. The controversy is due in part to the lack of a reliable marker for tracing the grafted SCs. To precisely assess the fate and temporal profile of transplanted SCs, we isolated purified SCs from sciatic nerves of adult transgenic rats overexpressing GFP (SCs-GFP). SCs were directly transplanted into the epicenter of a moderate contusive SCI at the mid-thoracic level at 1 week post-SCI. SCs-GFP or SCs-GFP labeled with Brdu were quantified at 5min, 1d, 7d, 14d, 28d, and 90d following grafting. BBB, footfall error, and thermal withdrawal latency were measured weekly after SCs-GFP transplantation. We found a remarkable survival of grafted SCs up to 90 days post-grafting. The proliferation rate of SCs-GFP (Brdu⁺) increased on day 1 (5.1%) and day 7 (8.0%), peaked on day 14 (22%), decreased on day 28 (4.7%), and stopped at day 90 (≈ 0) post-grafting. Immediately after transplantation, SCs-GFP quickly filled the lesion cavity and migrated to the surrounding host tissue as early as 1 day post-SCI. SCs-GFP migrated up to 5.0 mm along the central canal at 28 days post-grafing. While transplanted SCs-GFP myelinated regenerated axons within the graft region and expressed P0 and MBP at 90 days post-grafting, we did not observe a significant BBB score, thermal withdrawal latency, or footfall error rate change compared with control animals that received no SCs-GFP. We conclude that while SCs-GFP can survive, proliferate, migrate, and myelinate regenerated axons within the lesion site, combinatorial strategies are essential to achieve a meaningful functional regeneration after SCI.

P048 Development of a Novel Forelimb Assessment of Skilled Coordinated Object Manipulation Following Cervical Spinal Cord Injury

After spinal cord injury (SCI), a variety of sensorimotor deficits can be induced depending on the severity and location of injury. The most common forms of SCI are compression/contusion injuries at the cervical level, primarily caused by automobile accidents and falls. One common outcome following such injury is forelimb paresis and paralysis. Methods to gauge recovery of various functions of the forelimb in experimental animal models are many, though such procedures often require extensive training of the animal to perform the task, rely on the motivation of the animal, and ultimately, vary considerably in the sensitivity to the parameter being measured. As such, assessing behaviors innate to the animal, such as food manipulation, should be most beneficial in limiting such disadvantages. Given the severity of effects on overall forelimb motor function following cervical SCI, we have designed a novel treat manipulation test utilizing flavored cereal rings which is highly sensitive, requires no training, and can be repeated for multiple trials per animal. We observed the sensitivity of our forelimb test over 6 weeks in a study employing bisperoxovanadium (bpV) for investigation of PI3K/Akt pathway modulation and neuroprotection following cervical contusive SCI. The sensitivity of our forelimb assessment is based on our evaluation parameters, including treat grasp ability (open or partially closed palm), number of times the treat is dropped during eating, and treat manipulation. Also reinforcing the test's validity is its strong correlation (R²  = 0.88) with forelimb articulation scores derived from an established forelimb locomotion test. Importantly, improvements in functional forelimb recovery corresponded to higher scores on our test, as well as reduced spinal cord lesion volume and increased motor neuron survival.

P058 Tissue Type Plasminogen Activator (tPA) Promotes Functional Recovery After Compressive Spinal Cord Injury in Rats

The establishment of glial scar in response to central nervous system (CNS) injuries creates a dense physical barrier around the injury as well as a chemical barrier due to the release by reactive astrocytes of chondroitin sulfate proteoglycans (CSPG) that are growth inhibitory molecules. This inhibitory environment mainly contributes to the failure of injured axons to regenerate leading to persistent functional impairments. Our results show that compressive spinal cord injury (SCI) in rats causes an acute activation of astrocytes within the spinal cord leading to a persistent glial scar 3 weeks after the injury. Reactive astrocytes synthesized and released the main inhibitor of tPA (tissue Plasminogen activator), PAI-1 (type 1 Plasminogen Activator Inhibitor), into the extracellular space, where it inhibits the proteolytic activity of tPA. We expect that the tPA/PAI-1 axis may have an influence on secondary damages occurring after SCI, in particular on axonal regeneration and glial scar. So, we have further investigated the role of tPA in vitro in primary mixed cultures of medullary neurons and astrocytes. Our results have evidenced that tPA decreased glial reactivity which could in turn promote neuritse growth. We determine the molecular and cellular mechanisms by which tPA could favour neurites growth. We have evidenced in vitro and in vivo that tPA is regulating a protease X able to destroy neurocan, a potent CSPG of the CNS. The degradation of neurocan promotes axonal regeneration which improves functional recovery after SCI. To conclude, we are the first to demonstrate the relevance of tPA as new original therapeutic strategy for SCI.

P059 Spinal Cord Injury Harms the Brain and Spinal Cord and these Effects are Reduced by Exercise

Besides the main deficits in motor and sensory functions, cognitive and emotional disorders are common features in patients suffering SCI. We have initiated studies to determine how SCI can compromise molecular systems important for plasticity and function of neural circuits in the hippocampus and spinal cord. Adult C57BL6 mice (n = 46) were either exposed to cages provided with running wheel or regular cages (sedentary). A common observation is that the outcome of SCI patients is heavily dependent on pre-injury conditions associated with lifestyle. Accordingly, we have attempted to understand how the level of experience incurred before the onset of the injury can affect healing mechanisms. We evaluated how exercise prior to the injury influences the process of post-injury recovery by assessing molecular systems important for synaptic plasticity and function. After 21 days of voluntary exercise, a complete spinal cord transection (T6-T8) was performed in sedentary (Sed/SCI) and exercise (Exc/SCI) animals, and sacrificed two days after. Protein levels were measured in the hippocampus, cervical (SCC) and lumbar (SCL) spinal cord enlargement regions. BDNF levels were significantly reduced in Sed/SCI animals as compared to Sed/Int in the hippocampus, cervical and lumbar regions. For the three regions, exercise prevented the BDNF reduction, such that the BDNF levels of Exc/SCI group remained same as Sed/Int group in all measured regions. In addition, SCI reduced the levels of phospho-synapsin I phospho-CREB and phospho-CaMK II in Sed/SCI group compared to Sed/Int, while exercise prevented these reductions (Exc/SCI). Exercise prevented the reduction in p-synapsin, p-CREB, p-CaMKII. These results indicate that the effects of SCI go beyond than the local SC environment and can reach brain centers. Interestingly, exercise provided before the injury onset had a protective effect in the brain and SC from the effects of the injury.

Funding Support: R01 NS056413; CRAIG H. NEILSEN FOUNDATION

P060 Dietary Therapy to Promote Neural Repair in Chronic Spinal Cord Injury

Cervical spondylotic myelopathy (CSM), a commonly encountered form of chronic spinal cord injury (SCI), is related to primary mechanical and secondary neurodegeneration caused by disc disease. We explore a novel, non-invasive method of promoting neural repair in CSM by using the capacity of omega − 3 fatty acids to support membrane structure and improve neurotransmission, and curcumin to minimize oxidative cellular injury. We have developed an animal model of CSM using a nonresorbable expandable polymer placed in the thoracic epidural space that causes delayed myelopathy. Animals that underwent placement of the expandable polymer were exposed to a diet rich in DHA (1.2, Nordic Naturals, CA %) and curcumin (DHA/Cur) (500 ppm), or to a standard Western diet (WD). Animals (n = 28) underwent serial gait testing, and protein measurements were performed after 7 week study period. The Noldus Catwalk program quantified the stepping patterns of rats under a number of gait parameters; in multiple parameters, the neurological behavior was significantly worse in the WD than the control group. In contrast, the gait parameters in the DHA/Cur group remained consistent with those found in the control group. We measured levels of BDNF, syntaxin 3, and the BDNF receptor trkB in the thoracic region affected by the compression and the lumbar enlargement. Results showed BDNF levels in the DHA/cur group were not significantly different than intact animals, and significantly greater than the WD group. Higher spinal cord BDNF levels were significantly associated with better gait performance. Animals that received DHA/curcumin also had significantly higher levels of syntaxin-3 and the BDNF signaling receptor TrkB than those receiving the standard diet. This study has demonstrated that DHA and Curcumin can promote spinal cord neural repair and neutralize the clinical and biochemical effects of myelopathy.

Funding Support: NIH RO1 NS056413; CRAIG H. NEILSEN FOUNDATION

P065 Diffusion Weighted MRI of white Matter Injury Produced by Ethidium Bromide and Cervical Contusion Spinal Cord Injuries

INTRODUCTION: Literature suggests that diffusion-weighted-MRI (DW-MRI) can distinguish between demyelination of spinal cord white matter (elevated radial diffusion) and axonal injury (decreased longitudinal diffusion/diffusivity). We hypothesized that ethidium bromide (EB) demyelination would result in preserved longitudinal diffusion and isolated increase in radial diffusion. In addition, we sought to compare diffusion parameters produced by EB demyelination to those produced by contusion spinal cord injury (SCI).

MATERIALS AND METHODS: 18 adult female Long-Evans rats were used. Animals (n = 12) received EB microinjections into the right lateral funiculus at C4-C5 and saline into the left lateral funiculus, or were subjected to unilateral SCI (100kdyn) at C5 using the IH impactor (n = 4). 2 served as normal controls. MRI was performed using a 7T 300 MHz Horizontal Bore Varian MR System 7-8 days after injury. Anatomical sequences included T1 and T2 weighted imaging, and DW-MRI included anisotropy maps (AM) and longitudinal (LDW) and radial diffusion weighted (RDW) images.

RESULTS: To our knowledge, this is the first in vivo DW-MRI study of a focal demyelinating SCI. In EB demyelinating lesions, we found a significant increase in radial diffusion. In addition we found a decrease in longitudinal diffusion, despite utilizing a lesion with relative sparing of axons. In SCI, we found evidence of spinal cord contusion including hemorrhage and edema. In these injuries DW-MRI patterns were similar as in the EB lesions.

DISCUSSION: Both increased radial and decreased longitudinal diffusion/diffusivity is common to most spinal cord white matter injury models studied, including cuprizone, axotomy, hemisection, lysolesithin, ischemic, experimental autoimmune encephalomyelitis, and contusion lesions, and now, EB lesions. This data collectively suggest that directional diffusion values/diffusivities may be sensitive but are quite nonspecific in vivo markers of white matter injury.

Support: NIH: 5 T32 EB001631-07 (JT); AG032518 (MB); NS038079 (JB/MB); Margulis Society (JT); C.H. Neilsen Foundation (JB/YN).

P072 Cellular Localization of Calpastatin Splice Variants

Shortly following traumatic brain and spinal cord injury, widespread necrotic cell death is observed around the injury site. This is thought to be due to the overactivation of the calcium-activated Calpain proteolytic system. The Calpain family is comprised of 15 calpain isoforms and a single endogenous inhibitor Calpastatin (CAST). Currently four N-terminal splice variants of CAST have been identified; however their intracellular localization is unclear. In silico analysis suggested that the N-terminus of type I CAST may function as a mitochondrial targeting sequence. We therefore examined the intracellular localization of the three N-terminal CAST splice variants: Type I (XL domain), Type II (exons 1xb, 1y), and Type III (L domain). Type IV is testis-specific and was not examined. SH-SY-5Y neuroblastoma cells were transfected with Rat CAST types I, II, or III with C-terminus GFP or FLAG tags. Immunocytochemical (ICC) results indicated Type I was present in both the cytosol, mitochondria and also associated with cell membranes. Type II was associated with vimentin filaments as shown by ICC and pulldown assays. Type III was diffusely distributed throughout the cell. We also examined mitochondrial localization of Type I using mitochondrial enriched fractions collected 48 hours post-transfection of the XL domain-GFP construct. Immunoreactivity was detected in mitochondrial fractions, but at lower molecular weights than cell homogenates, suggesting cleavage of the XL domain. Taken together these results demonstrate that the three N-terminal CAST splice variants have distinct intracellular localizations, with a portion of Type I being in the mitochondria, Type II associated with vimentin and possibly other intermediate filaments, and Type III predominately cytosolic.

Research was supported by NIH grant P01 NS058484.

P073 Biosynthesis of Chondroitin Sulfate Proteoglycans in HEK293T Cells

The role of chondroitin sulfate proteoglycans (CSPGs; e.g. aggrecan) in the inhibition of neurite outgrowth is an important factor in CNS injury and developmental neuroscience. In spinal cord injury (SCI), the development of a glial scar and reactive astrocytosis results in increased CSPG expression and inhibition of neuronal regeneration. The sulfation pattern of CSPG-associated glycosaminoglycans (GAGs) plays a pivotal role in neurite inhibition. In the present study, we developed a model to examine the biosynthesis of CSPGs with varying sulfation patterns. Human Embryonic Kidney (HEK) 293T cells were transiently transfected with the gene for bovine aggrecan (pBAGG71-28). Through G50-Sephadex column chromatography and DEAE column elution, several peaks were detected using a DMMB assay. The presence of aggrecan within these peaks was verified by dot blot with an antibody to aggrecan. The resulting differences in retention times for aggrecan likely represent varying sulfation patterns of the GAGs. This novel pattern of aggrecan sulfation implicates HEK293T cells as a promising model for the continuing development of our novel “Designer PGs” (Snow et al., 2007) to study the inhibitory influence of sulfation on neurite outgrowth. By targeting individual chondroitin sulfotransferases with siRNA, “Designer PGs” with varied sulfation patterns can be synthesized and then analyzed for their inhibitory effect on neurite outgrowth. The model is not only promising for the development of CSPG constructs to study mechanisms of inhibition, but targeting genes of human origin is a clinically relevant approach as well, and could lead to the development of novel gene therapies.

[Support: R01 NS053470 and grant #10-11A from the Kentucky Spinal Cord and Head Injury Research Trust].

P074 Mitochondrial Dysfunction: A Crictial Target for Treatment of Acute Spinal Cord Injury

We have reported that targeting mitochondrial dysfunction following T10 contusion spinal cord injury (SCI) with acetyl-l-carnitine (ALC), which can acts as an antioxidant and alternative bio-fuel, significantly improves mitochondrial bioenegetics. Also, daily ALC treatment increases spinal cord tissue sparing versus vehicle after one-week, however, the identical 7-day regimen did not improve hind limb function (BBB scores) versus vehicle at 5-weeks post-injury (n = 6/group). Therefore, we changed our contusion injury site to upper lumber (L1/L2) spinal level to target gray matter thought to be critical for rhythmic hind-limb movements in rats. Four weeks after L1/L2 SCI, prolonged daily treatment with ALC versus vehicle (n = 14-17/group) indeed rendered significant increases in the BBB scores and both spared gray and white matter; showing significant correlations between BBB and spared tissues. We then carried out a comparative study employing 1) ALC, 2) N-acetyl cysteine amide (NACA), a glutathione precursor, 3) a combination of ALC + NACA or 4) vehicle administration at 15 min post-injury, followed by 6 hr boosters. After 24 hrs, both ALC and NACA alone significantly improved mitochondrial bioenergetics post-SCI; however, ALC + NACA where found to be antagonistic, approximating values with vehicle treatment. In a subsequent long-term behavioral study, we similarly administered ALC, NACA, ALC + NACA or saline at 15 min post injury followed by 6 hr booster. We also inserted subcutaneous osmotic pumps containing these four reagents to continually deliver for 7 days post-injury (n = 5/group). After 6 weeks of testing, treatment with ALC or NACA alone each rendered significant increases in the BBB scores (∼11-walking) compared to vehicle treatment (∼8-dragging limbs). However, similar to the bioenergetic profiles, the combined ALC + NACA treatment did not improve functional recovery. Overall, both ALC and NACA alone, but not in combination, promotes neuroprotection by targeting mitochondrial dysfunction that is directly correlated with improved hind-limb function over weeks post-injury.

P075 Single Filopodial Contact with Inhibitory Chondroitin Sulfate Proteoglycans Induces Behavioral Changes in Sensory Neurons In Vitro

Chondroitin sulfate proteoglycans (CSPGs) are up-regulated in response to spinal cord injury (SCI), and consequently inhibit axonal regeneration. The majority of CSPGs produced after injury stem from reactive astrocytes of the glial scar. Axons attempting to regrow “evaluate” inhibitory molecules and make “decisions” about whether to continue to advance, stop, or turn. To grow beyond the glial scar and toward appropriate targets, neurons must overcome the CSPG-induced inhibitory milieu. The current study examined behavioral changes in the leading edge of regenerating neurons, the growth cone, as they come into first contact with CSPGs in vitro, thus modeling primary interactions with the glial scar. Using analyses of time-lapse video images, growth cone properties such as morphology, filopodial length and number, approach velocity (to substratum adsorbed CSPGs), and approach angle were compared before and after first contact with CSPGs. Using this methodology, we report that growth cone velocity was significantly reduced following first contact by a single filopodia. We recently reported that single filopodial contact with CSPGs resulted in regulation of growth cone area as well, and the two characteristics may be interrelated. Collectively, these data are the first demonstrations of significant behavioral changes in growth cone behavior resulting from a single filopodial contact with CSPGs. This intriguing result represents a potential therapeutic target for regeneration and recovery of function following SCI.

[Support provided by NIH/NINDS (NS053470); the Kentucky Spinal Cord and Brain Injury Research Center (#10-11A); and a grant from the Department of Defense].

P076 Wheelchair Immobilization and Activity-Based Rehabilitation After Contusive SCI: Anatomical Correlates of Gain and Loss of Function

It has been shown that both the type and amount of afferent input can effect spinal cord injury rehabilitation, presumably via plasticity occurring in one or more of the three functional spinal locomotor compartments; primary afferents, spinal interneurons, and/or motoneurons. We have recently developed a model of gain and loss of function using activity-based retraining or wheelchair immobilization after contusive spinal cord injury (cSCI). We hypothesize that hindlimb activity post-cSCI will lead to altered expression of plasticity and inflammatory related molecules, giving rise to anatomical correlates for locomotor changes and functional recovery. Adult female Sprague-Dawley rats receiving moderate cSCI at the thoracic 3/9 (T3 or T9) level were randomly assigned to one of three experimental groups; swim trained starting at 7 days post-injury, wheelchair immobilized starting at day 4, or normally housed 2 per cage. Following sacrifice at 21 days post-injury, RNA was isolated from lumbar levels 1-3 (L1-L3) for RT-PCR-based microarray analysis of 172 mRNAs coding for selected ion channels, neurotransmitters and receptors. Robust transcriptional changes for genes encoding several classes of receptors, including NK1 (Taclr) and solute carrier family 5 choline transporter (Cht1), occurred differentially based on the amount/type of hindlimb activity. Immunohistochemical analysis of relative expression levels for regions of interest (ROI) in L1-L5 segments is presented. L1-L5 dorsal root ganglion (DRG) are positive for the presence of activating transcription factor 3 (ATF3) indicating a peripheral tissue inflammatory mediated process exists in our model. Morphological changes in motoneurons in DRGs and L1-L5 segments are assessed using cholera toxin B horseradish peroxidase conjugate (BHRP) tracing of ankle extensors. Our studies begin to establish expression profiles of distinct morphological and molecular signatures of our gain and loss models so that pharmocological and neuromodulatory targets for combination therapies can emerge.

P077

Withdrawn

Wu Junfang 1 Stoica Bogdan 1 Dinizo Michael 1 Pajoohesh-Ganji Ahdeah 2 Faden Alan 1 1 University of Maryland School of Medicine, Batimore, MD, USA 2 George Washington University Medical School, Washington DC, USA P078

Role of Cell Cycle in Experimental Spinal Cord Injury: Delayed Treatment with a Pan-CDK Inhibitior is Protective

Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction through both mechanical damage and secondary biochemical and physiological responses. We have previously shown the role of cell cycle pathway in secondary injury following rat contusion SCI by examining effects a cell cycle inhibitor or gene knockout. In our prior study the non-selective CDK inhibitor flavopiridol was administered centrally beginning 30 min after trauma. In the present study, we investigate cell cycle pathway changes over time following SCI as well as the functional effects of cell cycle inhibition in rat contusion SCI model using delayed systemic administration of flavopiridol. Expression of cell cycle proteins was assessed at various time point post-injury using immunoblot analysis. Behavioral assessments including the BBB scale and the combined behavioural score (CBS) were performed, lesion volume and spared white matter were assessed using histological analysis; stereological assessment of microglia/macrophages, oligodendrocytes, and neuron were also performed. Immunoblot analysis shows a marked up-regulation of cell cycle-related proteins, including CDK4, E2F5, PCNA, cyclin D1, pRb at various time points after SCI, whereas the endogenous CDK inhibitor p27 is down-regulated. Treatment with flavopiridol reduced cell cycle protein induction and increased p27 expression in injured spinal cord. Functional recovery was significantly improved from 7 days through 4 weeks after SCI. Treatment significantly reduced lesion volume and numbers of Iba-1⁺ microglia in the preserved tissue, while, increased the area of spared white matter, as well as numbers of CC1⁺ oligodendrocytes. Furthermore, flavopiridol markedly inhibited proliferation of astrocytes and microglia, attenuating the production of factors associated with microglial activation and astrocytic reactivity in both in vivo and in vitro. Our results support a role for cell cycle activation in the pathophysiology of SCI, and provide further support for the therapeutic potential of cell cycle inhibitors for the treatment of clinical SCI.

Noga Brian Xie Songtao Sanchez Francisco University of Miami, Miami, FL, USA

P079 Noradrenergic NAα_2B Receptor Involvement in Spinal Locomotor Processes

PURPOSE: Spinal noradrenergic receptors critically affect locomotion processes following spinal cord injury. Recent work has demonstrated that locomotor-activated neurons within the intermediate zone of the thoraco-lumbar spinal cord are innervated by descending noradrenergic pathways and express NAα_2B receptors (Noga et al. 2011). Here we examine the functional relationship between NAα_2B receptors and locomotor activity.

METHODS: Experiments were performed on decerebrate cats, paralyzed with a muscle relaxant, in which locomotion occurred spontaneously or was induced by electrical stimulation of the mesencephalic locomotor region (MLR). Pharmacological antagonism of the NAα_2B receptor was accomplished by superfusion of the lumbar (L3-L7) spinal cord with imiloxan hydrochloride, a moderately potent and highly selective NAα_2B receptor antagonist.

RESULTS: In mesencephalic preparations (decerebrated at precollicular-postmamillary level), low-dose (5 mM) imiloxan blocked locomotion evoked by unilateral but not bilateral MLR stimulation. In thalamic preparations (decerebrated at precollicular-premamillary level), this low dose did not block locomotion that was spontaneous or evoked unilaterally from MLR. An intermediate dose (10 mM) blocked locomotion evoked by bilateral MLR stimulation in all cats but not spontaneous locomotion in thalamic cats. The highest dose (20 mM) abolished both spontaneous and MLR-evoked locomotion in thalamic preparations. Before complete blocking, stepping frequency and burst amplitudes were reduced and stepping patterns became irregular. All effects were reversible. Imiloxan concentrations in the mid-dorsal horn, voltammetrically measured at microelectrodes, were ∼1/1000^th of the superfused concentration and within the intermediate zone were undetectable (<1-5 μM). Effects on locomotor activity did not correspond with the appearance of imiloxan within the dorsal horn, but rather with the modeled temporal profile of diffusion into the deeper levels of the spinal cord.

CONCLUSION: These results indicate that noradrenergic initiation/modulation of locomotion likely involves NAα_2B receptors and identifies the NAα_2B receptor as a therapeutic target for locomotion facilitation following spinal injury.

Supported by NIHR01-NS046404

Noor Natassya 1 Truettner Jessie 2 Steer David 3 Dziegielewska Kate 1 Dietrich W Dalton 2 Ek C Joakim 1 Richardson Samantha 4 Wheaton Benjamin 1 Saunders Norman 1

1 University of Melbourne, Parkville, Victoria, Australia 2 University of Miami Miller School of Medicine, Miami, Florida, USA 3 Monash University, Clayton, Victoria, Australia 4 School of Medical Sciences and Health Innovations Research Institute, Bundoora, Victoria, Australia P080 Age-Dependent Changes in Protein Expression Following Complete Spinal Cord Transection in Postnatal Monodelphis Domestica

The ability of mature spinal cords to recover after complete transection is very limited. However, the immature cord shows a remarkable capacity for repair, particularly in early development. We used the gray, short tailed opossum, Monodelphis domestica, to investigate changes in the proteome following complete spinal cord transection.

Immature pups at P7 or P28 were subjected to complete transection at thoracic level T10 and cord sections caudal to the lesion were collected at 24h or 7d after injury along with age matched uninjured controls. Proteins were separated based on isoelectric point and subunit molecular weight and those that changed in response to injury were identified by densitometry and analyzed by mass spectrometry. Another group of animals was prepared for quantitative RT-PCR. Primers for genes encoding proteins that changed in the proteomics study were designed based on the published sequence (Esembl).

Fifty-seven proteins were identified as being differentially regulated in response to spinal transection. More than 50% were cytoplasmic and 70% belonged to families of proteins with binding properties. More proteins were down-regulated at P7 and more were up-regulated at P28. More changes were observed at 1d than 7d post-injury. Several of these proteins such as ubiquitin were validated using western blotting and immunocytochemistry. To determine if proteins that were most changed had a transcriptional element to their regulation, quantitative RT-PCR was performed on ubiquitin, 14.3.3 gamma/epsilon, cofilin, α-enolase, and heart and brain fatty acid binding proteins 3 and 7. There was good concordance between the results. For example, ubiquitin was downregulated at P7, but was elevated at P28. 14.3.3 epsilon protein and mRNA were lower in P28 1d animals.

This study showed that changes in the proteome following spinal cord injury are age dependent and may contribute to the differences in the ability of spinal cords to repair.

de Rivero Vaccari Juan Pablo 1 Minkiewicz Julia 1 Wang Xiaoliang 2 German Ramong 1 Marcillo Alex 1 Dietrich W. Dalton 1 Keane Robert W. 1

1 University of Miami Miller School of Medicine, Miami, Florida, USA 2 Nanjing University, Nanjing, China P081 Rig-Like Receptor Signaling Modulates Innate Immunity and Astrocyte Activation Following Spinal Cord Injury

Injury to the central nervous system (CNS) induces a glial response, in which astrocytes become activated and produce inflammatory mediators. However, the molecular basis for glial activation and the signaling pathways regulating glial/innate immune responses remains poorly understood. Here, we examine the activation of retinoic acid inducible gene-like (RIG) receptors (RLRs) and the involvement of RLR signaling in the regulation of type I IFNs following cervical (C5) spinal cord injury (SCI). Immunohistochemical analysis followed by confocal microscopy procedures reveals that astrocytes express two intracellular RLRs, RIG-I and melanoma-associated gene 5 (MDA5). Immunoblotting of spinal cord protein lysates after SCI and stretch injury of culture astrocytes indicates that injury to the CNS activates RLR signaling as determined by phosphorylation of IRF3 leading to production of type I IFNs. Stimulation of RIG-I and MDA5 with synthetic RNA (poly(I:C)) results in RLR signaling activation, phosphorylation of IRF3, and increased expression of glial fibrillary acidic protein and the intermediate filament protein vimentin, two hallmarks of reactive astrocytes. To the best of our knowledge this is the first time that the RLR signaling pathway is described as being involved in regulating astrocyte activation and modulation of the innate immune response after SCI.

Supported by NS059836, Craig Nielsen Foundation and The Miami Project to Cure Paralysis.

Minkiewicz Julia de Rivero Vaccari Juan Pablo Keane Robert W. University of Miami, Miami, FL, USA

P082 The NLRP2 Inflammasome in Astrocytes Contributes to Innate Immunity Following CNS Injury

Injury to the central nervous system (CNS) induces an innate inflammatory response involving activation of astrocytes. The initial sensing of danger signals after injury is mediated by innate pattern recognition receptors (PRRs), which include the NOD-like receptors. However, the PRR receptors and signaling cascades regulating innate glial responses to CNS injury remain largely undefined. Here, primary human astrocytes were evaluated for expression of inflammasome proteins. We show that human astrocytes express NLRP2 inflammasome components, including NLRP2, Apoptosis-associated speck-like protein with CARD domain (ASC), caspase-1, pannexin 1, P2X7 and X-linked inhibitor of apoptosis protein (XIAP). Treatment of astrocytes with ATP resulted in a dose-dependent activation of the NLRP2 inflammasome resulting in caspase-1 cleavage and IL-1 beta production. ATP induction of the NLRP2 inflammasome was blocked by the pannexin-1 inhibitor, probenecid and by treatment with the P2X7 receptor antagonist, brilliant blue G. Our findings suggest that the NLRP2 inflammasome in astrocytes is an important component of the CNS inflammatory response and may be an important therapeutic target to inhibit inflammation induced by CNS injury.

Supported by NINDS grant 1RO1NS59836 to RWK

Santamaria Andrea Benavides Francsico Guest James The Miami Project to Cure Paralysis, Miami, FL, USA

P083 Assessment of CAM Kinase II Alpha Antibody Labeling of the Corticospinal Tract After Spinal Cord Injury in Rodents, Pigs and Non Human Primates

INTRODUCTION: Quantification of corticospinal tract axons (CST) in spinal cord injury (SCI) models is important. Due to lack of denominator values indicating total axons in the tract, neuroanatomic tracing methods are limited for quantification. Although anti-neurofilament or silver staining may be used to identify axons, these methods are non-specific. CaM-kinase II alpha (CaMKIIα) immunostaining has been reported to label the CST. We report a comparison of the CST identified by CaMKIIα in perfused tissue of three species used in SCI research: primates, pigs and rodents, after either paraffin embedding or cryopreservation.

METHODS: Brainstem and spinal cord tissues of Macaca fascicularis, Yucatan minipigs and Sprague dawley rats were immunostained using HRP chromagen precipitation. Animals were fixated with 4% paraformaldehyde. Frozen sections were cut axially at 30 μm. Paraffin embedded sections were cut at 10 μm and antigen retrieval performed. Primary antibody; mouse IgG1 CaMKIIα (1:200) was incubated overnight. Secondary antibody, biotinylated goat anti-mouse IgG (Vector laboratories) was applied, washed, and then incubated with avidin-biotin-peroxidase. The reaction was initiated with 3,3′-diaminobenzidine and H2O2 and stopped by rinsing in acetate imidazole buffer, sections where then dehydrated with a graded ethanol series, cleared with xylene and coverslipped. Axon size and numbers were recorded using image analysis techniques. In the pyramid, the number of CaMKIIα positive axons was compared to the number in adjacent silver stained sections.

RESULTS: CaMKIIα labeled CST in all three species. The major and ventral CST was detected. Fine detail could be observed including emergence of collateral branches. However, CaMKIIα also labeled axons of the superficial dorsal horn in rodents, and neuronal soma of the substantia gelatinosa in primates.

CONCLUSIONS: CaMKIIα labels myelinated and unmyelinated axons of the CST in uninjured and injured animals at both acute and chronic time points and is a useful tool for studies of the CST.

Hulsebosch Claire E. Xu Guo-Ying Johnson Kathia M. Rea Harriett C. Unabia Geda Nesic Olivera Perez-Polo J. Regino University of Texas Medical Branch at Galveston, Galveston, Texas, USA

P084 DNA Decoy Treatment Improves Sensory and Motor Recovery After Spinal Cord Injury in Rats By Neuroprotection and Microglial Inhibition

Spinal cord injury (SCI) results in a number of deficits, triggering primary and secondary injury signaling cascades characterized by early and prolonged inflammatory responses. Early after SCI, IL-1B increases trigger increased activation of the transcription factor Nuclear Factor-kB (NF-kB) that mediates secondary injuries via synthesis of proteins that maintain inflammation. There are different NF-kB subunits. Subunit-specific inhibition can be accomplished with synthetic double stranded “decoy” deoxyoligonucleotides containing selective NF-kB protein dimer binding consensus sequences. Spinal cord contusion injury (IH Impactor, 150 kdynes, 1 sec dwell) was given to male Sprague-Dawely (235 to 245 gm) rats and divided randomly into two groups: 1) decoy treated (N = 10) or 2) vehicle treated (N = 9). Treatment was delivered stereotaxically with a Hamilton syringe (28 gage needle) into the spinal lesion site in an injection regimen of four treatments of 2ul/10min at 15 minutes, 7, 14 and 21 days after injury. Locomotor scores were recorded daily for 21 days and then weekly using the BBB rank score system. At presurgical times and 42 days or longer following SCI, cutaneous mechanical thresholds, deep pressure thresholds (Randall and Selitto, 1957) and thermal thresholds were measured for paw withdrawal accompanied by supraspinal measures in forepaws and separately for hindpaws (Hulsebosch et al., 2000). Levels of COX-2 and iNOS were measured in both groups. In this study, DNA “decoys” that target the COX-2 gene promoter NF-kB binding site, attenuated the SCI-induced increases in COX-2 and iNOS protein levels, improved locomotor function, improved sensory, decreased neuronal death and attenuated microglial activation.These experiments demonstrate the efficacy of novel interventions in the inflammatory cascade triggered by SCI as a strategy for treatment of SCI-induced physiological functional impairments. The approach is innovative because of the new technology (DNA promoter decoys) used to selectively block signaling mechanisms.

Mission Connect/TIRR Foundation, M.D.AndersonFoundation, TheLiddellandTheDunnFoundations, NS11255.

Hulsebosch Claire Bhupendra Kaphalia Nauduri Dhananjaya Hassler Shayne UTMB, Galveston, Texas, USA

P085 Measures of Lipid Peroxidation Formation After Chronic Spinal Cord Injury

The effects of injury on the nervous system can be seen long after the initial injury has already healed. We have previously shown that lipid peroxidation products are highly up regulated after spinal cord injury (SCI). In the current experiment we investigate lipid peroxidation in chronic SCI. Lipid peroxidation products such as 4-hydroxynonenol (4-HNE) and malondialdehyde (MDA) have been shown to have toxic effects in nervous tissue and persist much longer than the reactive oxygen species that generate lipid peroxidation products. We hypothesize that in chronic SCI, spinal tissue from a SCI animals will contain greater amounts of lipid peroxidation products than sham injured animals and that when given apocynin, a super-oxide inhibitor, a reduction in lipid peroxidation products will occur in chronic SCI animals. We measure lipid peroxidation (LP) products as hydroxy alkenals in the spinal cord after chronic spinal cord injury. Spinal cord tissue is taken from animals that undergo an impact mediated spinal cord contusion injury at spinal segment T10 (IH device, 150 kdynes, 1 sec dwell). Mass spectrometry is used to quantify and analyze LP products in spinal cord tissue, and is compared to tissue from the sham and treatment animals. The measures of lipid peroxidation products in the chronic spinal cord injury model suggest that the LP products caused by the initial up regulation of reactive oxygen species after injury are still present long after the injury is healed. LP products, which have been shown to be toxic and cause pathology in other injury or disease models, may be responsible for symptoms seen in chronically injured spinal cord or nervous tissue patients, specifically neuropathic pain. Therefore, further studies on the toxicity of identified LP products need to be conducted to understand their role in injury to nervous tissue.

Wanchoo Sheshali Durham-Lee Julieann Wu Yewen Nesic Olivera UTMB, Galveston, TX, USA

P086 Tamoxifen Decreases Spinal Cord Injury Pain

In the present study we examined the analgesic effects of tamoxifen, not previously tested. Tamoxifen is an FDA-approved drug for the treatment of breast cancer patients. However, it is also neuroprotective and affects many pathways that are implicated in the pathophysiology of SCI pain, such as estrogen receptors, protein kinase C (PKC) or microglial activation. It might therefore be an effective intervention for SCI pain, especially because tamoxifen crosses the blood brain barrier and its safety has been thoroughly evaluated in the past decades. We used a clinically relevant rat model of contusion SCI, in which the majority of SCI rats develop hypersensitivity to mechanical stimuli applied to their hind limbs with VonFrey filaments. We found the mechanical thresholds in SCI rats at 28d after SCI decreased by ∼60% compared to pre-SCI values. Tamoxifen (1mg/day) was delivered from 42 to 56d after SCI via subcutaneously inserted tamoxifen-containing timed-release pellets. Our data showed that delayed, 2 weeks-long continuous tamoxifen administration significantly decreased mechanical hypersensitivity of SCI rats, but did not affect motor recovery of hind limbs. The analgesic effect of tamoxifen on SCI pain lasted longer than the tamoxifen delivery, indicating that tamoxifen treatment of SCI pain may not have to be chronic. In sum, our data strongly suggest that tamoxifen may be a novel therapeutic intervention for SCI pain, and that further study of the analgesic effects of tamoxifen is warranted.

Gwak Young Hulsebosch Claire University of Texas Medical Branch, Galveston, USA

P087 The Role of Reactive Oxygen Species on Below-Level Neuropathic Pain Following Spinal Contusion Injury

Spinal cord injuries (SCI) result in neuronal hyperexcitability in the spinal dorsal horn that cause chronic central neuropathic pain. Our previous data suggested that SCI produced overproduction of reactive oxygen species (ROS) that contributed to chronic below-level neuropathic pain following SCI. In this study, we examined the roles of ROS on below-level neuropathic pain in rat SCI models. SCI was produced by T10 contusion (150 kdyne, 1 sec dwell time, Infinite Horizon Impactor) injury in male SD rats (230-250 g). In immunohistochemical studies, the SCI group showed significantly increased Dhet (Dihydroethidium, red autoimmunofluorescence ROS indicator) intensity in the lumbar (L4/5) dorsal horn on post operation day (POD) 40 compared to sham controls which was attenuated by early treatment with the ROS scavenger, PBN (phenyl-N-tert-butylnitrone, free radical scavenger, i.p., 100 mg/kg, 7 days consecutive treatments following SCI). In electrophysiological studies, the SCI group showed significantly increased neuronal activity to non-noxious and noxious mechanical stimuli, respectively and attenuated by single intrathecal treatment of PBN (i.t., 3 mg/kg). To investigate ROS-mediated intracellular events, changes of phosphorylated calcium/calmodulin-dependent protein kinase II (pCamKII) were studied. The SCI group showed significant increases of pCamKII expression in dorsal horn neurons. However, neither astrocytes nor microglia showed pCamKII expressions. The changes in pCamKII expression were confirmed by Western Blot analyses. The neuronal hyperexcitability on POD 40 following SCI was attenuated by a single intrathecal treatment of KN-93 (i.t., 100 μM), an inhibitor of pCamKII. In addition, early treatment with PBN significantly attenuated both neuronal hyperexcitability and pCamKII expression on POD 40. In addition, a single i.t. treatment of t-BOOH (ROS donor, i.t., 10 mg) showed significant increases in neuronal activity to mechanical stimuli. The present study suggests that SCI produces below-level neuropathic pain via ROS-neuronal pCamKII pathways.

Fan Xin Yan Susan 1 Mothe Andrea 2 Tator Charles 1

1 University of Toronto, Toronto, Ontario, Canada 2 Toronto Western Research Institute, Toronto, Ontario, Canada P088 The Effect of EPHRIN-B3 on the Survival of Transplanted Neural Stem/Progenitor Cells (NSPC) in an Adult Rat Spinal Cord Injury (SCI) Model

PURPOSE: Ephrin-B3 was shown to reduce endogenous neural stem/progenitor (NSPC) death by inhibiting EphA4 receptors in the mouse brain. This project aims to determine the effect of ephrin-B3 on NSPC survival after spinal cord injury (SCI).

METHODS: (1) In cultures of rat spinal cord NSPC and in rat spinal cord sections, we detected the expression of EphA4 receptors. (2) In cultures of NSPC, we tested cytotoxicity of recombinant ephrin-B3-Fc and viability of NSPC after 3 days of ligand infusion. Fc fragments and PBS were used as controls. (3) In 26g clip compression SCI rats, we examined the survival of transplanted NSPC. Ephrin-B3-Fc was delivered intrathecally starting at the time of transplantation for 3 or 7 days. Fc fragments and PBS were used as controls.

RESULTS: (1) EphA4 receptors were expressed in cultured NSPC and in healthy and injured rat spinal cord, although expression is lower in the healthy spinal cord compared to the injured spinal cord. (2) In vitro, ephrin-B3-Fc did not affect NSPC survival except at the highest concentration tested, where survival was significantly reduced. Fc significantly reduced NSPC survival. (3) 3 days after transplantation, all groups contained many living transplanted NSPC. In contrast, 14 days after transplantation, there were no surviving transplanted cells in the ephrin-B3-Fc or Fc groups. At 3 and 14 days, transplanted NSPC and degraded remnants were localized to the sites of injection, and some were in macrophages.

CONCLUSIONS: Cultured NSPC express EphA4 receptors and can respond to ephrin-B3. However, ephrin-B3 and Fc are toxic to NSPC in vitro and after transplantation in SCI rats.

Gonzalez-Lara Laura Chen Yuhua Brown Arthur Foster Paula The University of Western Ontario, London, ON, Canada

P089 In Vivo Magnetic Resonance Imaging of Acute Inflammation After Spinal Cord Injury in the Rat

The inflammatory response after an initial traumatic spinal cord injury (SCI) further damages the neural pathways, exacerbating motor and sensory deficits. This ‘secondary’ injury may include: hemorrhage, edema, and cyst formation. These pathologies can be clinically observed through Magnetic Resonance Imaging (MRI). Current research targets modulation of leukocyte influx in order to reduce oxidative damage, decrease the size of the lesion and improve neurological outcomes. The purpose of this study was to observe macrophage infiltration in vivo using MRI in a traumatic SCI model.

METHODS: A clip compression SCI was induced in 16 female Wistar rats at the 4th thoracic cord segment. Rats were scanned in a 3T clinical MRI system, using custom-built RF and gradient coils and a 3D balanced steady-state free precession imaging pulse sequence with 200x200x200μm resolution 3 days after SCI. Molday ION^™, an iron oxide-based superparamagnetic blood pool agent, was then injected at 3 different doses: 5mg/kg (n = 3), 15mg/kg (n = 5), and 30mg/kg (n = 3); 5 control rats were not injected. 24 hours later the rats were imaged again.

RESULTS: Areas of signal heterogeneity at the epicenter with diffuse areas of signal loss indicating hemorrhage were identified in all animals on day 3 and on day 4 on controls. In the animals that received Molday ION^™, additional areas appeared hypo-intense on day 4 where the signal loss was more pronounced. Histology showed intracellular iron in these animals, which indicates that the increased signal loss observed after the injection is from macrophages that phagocytized the Molday ION^™ agent.

CONCLUSION: We are the first to have used cellular MRI to study macrophage infiltration in vivo in a rat model of SCI. Being able to follow this infiltration over time may contribute to understand changes in timing, severity and patterns as a response to potential anti-inflammatory treatments.

Tu Tsang-Wei 1 Bayly Philip 1 Song Sheng-Kwei 2

1 Department of Mechanical Engineering, Washington University, St. Louis, MO, USA 2 Department of Radiology, Washington University, St. Louis, MO, USA P090 Understanding the Coexisting Distal Vascular and Proximal Axonal Injuries in Contusion Spinal Cord Injury

PURPOSE: In contusion SCI, vascular disruption has been observed proximally and distally resulting in intra-parenchymal hemorrhage to exacerbate the secondary injury. Scarce evidence has been reported to explain such injury patterns. In present study, in vivo diffusion- and T2*-weighted MRI, combined with FE biomechanical simulations, provide evidence that explains the causes of concomitant proximal and distal vascular injury with focal axonal injury.

METHOD: T9-contusion was generated on 14-week-old C57BL/6 mice by a modified-OSU impactor with mild, moderate and severe injuries (n = 3 each group). The injured animals were imaged by T2*-weighted and diffusion MRI hyper-acutely (within 3hrs) to evaluate the injury patterns. ABAQUS was used to create a FE model based on MRI. The constitutive model, material properties, and boundary conditions were adapted from literatures and adjusted according to MRI results.

RESULTS: Distal hemorrhage increased with injury severities and axonal injuries extended evenly along tracts. Both hemorrhage and axonal injuries extended more in the rostral than caudal direction. The impact produced a focal stress and stretch in epicenter resulting proximal hemorrhage and axonal injuries. During impactor retraction, the stress propagated, extending rostrally, consistent with more severe rostral hemorrhage away from epicenter. The stretch was confined focally at epicenter resulting in the even injury distribution.

CONCLUSION: Our results suggest that the vascular disruption is caused by the focal stress accumulation while the axonal injury is more sensitive to the diffuse strain field. Structures, such as nerve roots, narrower rostral area, and the size of laminectomy may contribute to the stress accumulation. Our study indicates that the unexpected, distally-dominated, vascular injury could significantly affect the characteristics of the injury animal model via hemorrhage-induced secondary injury. Injury severity should be carefully characterized first, using, for example, in vivo MRI, to provide a consistent and reproducible platform on trials of preclinical therapeutic intervention.

Nott Melissa 1 Jones Andrew 1 Heriseanu Roxana 2 Boyle Claire 1 Weber Gerard 2 Meares Sue 3 Middleton James 1 Batchelor Jenny 3 Baguley Ian 1

1 University of Sydney, NSW, Australia 2 Royal Rehabilitation Centre, Sydney, NSW, Australia 3 Macquarie University, NSW, Australia P091 The Impact of Dual Diagnosis: Spinal Cord Injury and Traumatic Brain Injury

PURPOSE OF STUDY: People who have a traumatic spinal cord injury (SCI) are at risk of concomitant traumatic brain injury (TBI). The incidence and consequences for such ‘dual diagnosis’ (DD) patients are under-investigated in the literature. The project aimed to identify: 1) the incidence of DD in SCI patients; 2) the nature and severity of DD injuries; 3) the accuracy of TBI diagnosis in SCI patients; and 4) the medical, vocational, functional and psychological outcomes of DD patients.

METHODS: Consecutive traumatic SCI admissions to a tertiary metropolitan spinal rehabilitation service in Sydney, Australia, were eligible for the study. DD was classified where evidence of a TBI was found in the acute hospital medical records or the rehabilitation service's database. Consenting DD patients were assessed via a battery of validated questionnaires covering the domains of medical, functional, and psychosocial function.

RESULTS: Of 199 traumatic SCI admissions, 74 (37%) had concomitant TBI. Of these, only half had been identified in the rehabilitation service's database. SCIs were cervical in 35%, thoracic in 53% and lumbar in 12%, with 50% complete (ASIA A). 68% of TBIs were severe to extremely severe. 34 DD participants were interviewed between 1 and 6 years post-injury. Common medical complaints included bowel/bladder dysfunction, fatigue, sexual dysfunction and chronic pain. 30% reported “often” experiencing cognitive difficulties such as memory and attention impairment. Around one quarter endorsed depressive or anxiety-related symptoms. Most were in paid work or study (68%), while 36% required personal care/support.

CONCLUSION: DD was very common and often undiagnosed on entering spinal rehabilitation. Medical, cognitive and psychosocial issues were common. Future research should compare outcomes between DD patients and their single diagnosis TBI/SCI peers to determine whether acquiring combined SCI/TBI incurs additional personal disability.

Chen Liang 1 Gao Xiang 2 Chen Jinhui 2

1 Department of Neurobiology, Guangzhou, China 2 Stark Neuroscience Research Institute, Indianapolis, IN, USA P092 Traumatic Brain Injury Promotes Neural Stem Cell Proliferation VIA MTOR Signaling

Traumatic brain injury (TBI) is one of the most serious injuries that humans can suffer. Currently, there is no effective treatment available to reduce the neurological dysfunction following TBI. The identification of neural stem/progenitor cells (NSCs) in the adult brain gives hope for repair of the damaged brain following TBI. By using a transgenic reporter mouse line, Nestin-GFP, in which green-fluorescent protein (GFP) is driven by nestin promoter to express in the NSCs, NSCs are easily visualized. Different subtypes of NSCs, quienscent neural progenitors (QNPs) and active neural progenitors (ANPs), are distinguishable using this model. We took advantage of this transgenic mouse to quantify the proliferation of different subtypes of NSCs following TBI. We found that TBI transiently promotes QNP proliferation, while ANP proliferation is not significantly changed. The proliferation of NSCs was correlated with activation of the mammalian target of the rapamycin (mTOR) signaling pathway. Blocking this signaling pathway with rapamycin attenuated TBI-enhanced NSCs proliferation. These results suggest that TBI promotes neural stem cell proliferation, and the mTOR signaling pathway is required for NSCs activation. NSCs activation by TBI may reflect the induction of innate repair and plasticity mechanisms by the injured brain. Thus, the mTOR signaling pathway may serve as a novel target for augmenting neurogenesis in the adult brain in order to promote post-traumatic functional recovery.

Prima Victor 1 Glushakova Olena 1 Svetlov Archie 1 Sherman Alexandra 1 Kirk Daniel 2 Gutierrez Hector 2 Curley Kenneth 3 Hayes Ronald 1 Wang Kevin 1 Svetlov Stanislav 1

1 Center of Innovative Research, Banyan Biomarkers, Alachua, FL, USA 2 Florida Institute of Technology, Melbourne, FL, USA 3 US Army Medical Research and Material Command, Fort Detrick, MD, USA P093 Responses to Primary Vs. “Composite” Blast Overpressure in the Mirror of Protein Biomarkers in Rat Models

Pathophysiological processes following brain trauma are accompanied by the release of characteristic proteins and protein derivatives into circulation. Thorough analysis of dynamic byproducts spectrum provides valuable diagnostic information about the pathological foci and identifies clinically relevant biomarkers of traumatic brain injury (TBI).

Our previous studies characterized positional interaction of head and whole body with blast wave in a rat model of overpressure-induced TBI. Here, we compared the effects of body/head exposure to a moderate primary overpressure with brain injury by a severe composite blast. Assessment of previously characterized and novel TBI biomarkers was done by immunohistochemistry, ELISA, antibody microarrays and Western blot.

High speed imaging revealed a negligible degree of acceleration at rat positioning “off-axis” toward shock tube (primary blast) compared to “on-axis” experimental setup (strong head/cervical acceleration). We examined brain expression of glial and neural markers including GFAP, CNPase, and revealed strong glyosis after exposures to blast. GFAP and neuronal markers UCH-L1 and NSE were also detectable in plasma/serum after blast exposures. Serum levels of IL-1 and IL-10 were significantly elevated, predominantly after primary blast reflecting systemic body responses. Brain up-regulation of cell adhesion molecules L-selectin and ICAM-1; metalloproteinases 2, 8 and 13, nerve growth factor beta-NGF and neuronal receptor Neuropilin-2 was also detected in a time-dependent fashion.

In summary, a specific dynamics of serum biomarkers, reflecting crucial pathogenic components of neural and systemic responses was established and characterized. For all biomarkers, the detected levels raised at all the setups studied, nevertheless most significant and persistent serum changes were observed when the total animal body was subjected to blast wave compared to setups where only animal head was targeted. We suggest that the mechanisms underlying blast brain injuries, particularly mild and moderate, may be triggered by systemic, cerebrovascular and neuro-glia responses as consecutive but overlapping events.

Zoltewicz Susie 1 Yang Boxuan 1 Yang Changping 2 Lu Xi-Chun May 2 Dave Jitendra R. 2 Shear Deborah A. 2 Schmid Kara 2 Tortella Frank C. 2 Hayes Ronald L. 1 Zhang Zhiqun 1 Wang Kevin K.W. 1

1 Banyan Biomarkers, Inc., Alachua, FL, USA 2 Walter Reed Army Institute of Research, Silver Spring, MD, USA P094 Biomarkers Track Injury Severity in a Rat Model of Penetrating Ballistic Brain Injury

The goal of this project is to determine whether the severity of brain injuries resulting from bullets, shrapnel, or other penetrating objects can be tracked or diagnosed by measuring biomarker levels. We have used a rat model of penetrating ballistic-like brain injury (PBBI) for these studies. To assess the relationship between injury magnitude and biomarker levels, rats were subjected to one of three discreet PBBI severity levels operationally defined by the magnitude of the ballistic component of the injury (calibrated to equal 5%, 10%, or 12.5% of total rat brain volume; n = 5 per group). Brain tissue and cerebrospinal fluid (CSF) were collected from these rats at two time points (1 and 7 days post injury), and levels of two biomarkers (SBDP150 and GFAP; markers of axonal injury and gliosis, respectively) were measured using quantitative immunoblotting and ELISAs. We found that SBDP150 levels were significantly elevated 1 day after PBBI in the brain tissue compared to controls, but not at 7 days. SBDP150 levels in brain at 1 day showed clear stepwise increases as injury severity increased across all three injury magnitudes, although differences did not reach statistical significance at n = 5. In contrast, GFAP levels were observed to increase significantly over controls after 7 days in brain, but not at 1 day. However the increase in GFAP in brain 7 days post-PBBI was not correlated with injury magnitude. Regarding CSF, levels of both SBDP150 and GFAP were significantly elevated 1 day after injury compared to controls, and the level of GFAP correlated to the magnitude of brain injury. Therefore we have two promising biomarkers for early detection of PBBI which show promising changes associated with injury magnitude. Further studies are warranted to increase sample sizes.

Glushakova Olena Jeromin Andreas Johnson Danny Zhang Zhiqun Hayes Ronald L Wang Kevin K.W. Banyan Biomarkers, Inc., Alachua, FL, USA

P095 Increased Levels of Brain Injury Biomarkers: UCH-L1, GFAP and α-II Spectrin Breakdown Products in Rat Model of Kainic Acid-Mediated Neurotoxicity

BACKGROUND: Glutamate induced excitotoxicity has been long implicated in traumatic brain injury (TBI) as well as many other neurological disorders, as one of the major contributory factors in triggering axonal and dendritic degeneration. The neuropathological consequences of TBI are mediated through sustained glutamate induced increase in cytosolic Ca²⁺ leading to activation of proteases such as calpains and caspases causing fragmentation of certain proteins and selected release of brain proteins/fragments into biofluids (e.g. CSF, serum), which are considered as biomarkers of brain injury. Kainic acid (KA) is a known excitotoxin that mimics the effect of glutamate. Thus, we used rat KA excitotoxicity model to examine the distribution, relationship and localization of several biomarkers of TBI: ubiquitin C-terminal hydrolase 1 (UCHL 1), glial fibrillary acidic protein (GFAP), αll-spectrin break down products (SBDP150, SBDP145 and SBDP120).

METHODS: Neurotoxicity in adult rats was induced by subcutaneous injections of KA (9 mg/kg). Brain tissues and CSF were collected at 6, 24, 48 and 72 h after KA injection. The levels and cellular localization TBI biomarkers were examined using ELISA, Western blot and immunohistochemical analysis on paraffin-embedded 6 μm brain sections.

RESULTS: Experiments were performed in two groups of animals: control (saline injection) and treatment (KA injection). Western Blot analysis indicated the decrease in intact αll-spectrin level and appearance of is breakdown fragments produced by both calpain (SBDP150 and SBDP145) and caspase-3 (SBDP120) in hippocampus and to a lesser extent in cortex in KA group. Immunohistochemical analysis showed increased level of GFAP, SBDP150, SBDP145 and SBDP120 in hippocampus and cortex of the KA treated animals, but not in the animals from control group. ELISA analysis indicated the increase in expression of UCHL 1, GFAP and buildup of αll-spectrin break down products (SBDP150, SBDP145 and SBDP120) in CSF of the KA groups, as compared with control group.

Chawla Aarti Fang Lihuan Wang Kevin Banyan Biomarkers,Inc., Alachua, FL, USA

P096 TBI Biomarker UCH-L1 Protein Monomer and Oligomers Overexpressed in E.Coli as Recombinant Soluable Protein Calibrator for Human GFAP Biomarker Assay

Traumatic brain injury (TBI) affects 1.7 million patients each year in the USA. Approximately 170,000 of these injuries are classified as moderate-severe traumatic brain injury. While some patients suffering from moderate-severe TBI make good recoveries, most have severe, lifelong cognitive deficits. Ubiquitin C-terminal hydrolase 1 (UCHL1) was identified as a potential cerebrospinal fluid (CSF) and serum biomarker of CNS damage and disease states. Overexpression of the de-ubiquitinating enzyme UCH-L1 leads to inclusion formation in response to proteasome impairment. We observed some degree of UCH-L1 protein oligomerization during expression of UCH-L1 protein in E. coli and purification of the UCH-L1 protein using HisPur cobalt column. Apparently possible dimers and trimers in the molecular weight of monomer UCH-L1 (about 27 kDa) were detected by size exclusion chromatography with Superose-12 column, and reverse phase (C-18) HPLC separation then analysis by TOF-TOF mass spectrometry. A single peak was detected by A241 from reverse phase C-18 HPLC. From fractions of the C-18 column, three mass peaks were detected at 26324, 52984 and 79220, which might indicate monomer, dimer and trimer of UCH-L1 protein. This finding might shed light on TBI pathology a high concentration of UCH-L1 protein oligomer might form protein aggregate in CNS following TBI, which in turn might cause release of high levels of UCH-L1 into CSF and serum. Finally we demonstrated that the monomer fraction of UCH-L1 can be used to serve as calibrator for our sandwich ELISA for the quantification of UCH-L1 from CSF and serum of TBS patients as a diagnostic tool.

Zheng Wenrong Kobeissy Firas Liu Ming Cheng Hayes Ronald L Wang Kevin KW Banyan Biomarkers,Inc., Alachua, FL, USA

P097 Degradation of Microtubule-Associated Protein 2 (Map-2) in Rat Brain After Traumatic Brain Injury

A large body of research has focused on the pathological significance of mechanical and chemical insults (eg, calcium homeostasis disturbances) to the brain following traumatic brain injury (TBI). While traumatic axonal injury (TAI) is recognized as an important pathological component of TBI, the exact mechanism of of TAI is not well-characterized. In our previously published work (Liu et al. 2011), axonally specific microtubule-associated protein tau, component of neurofibrillary tangles, was shown to be degraded following TBI and vulnerable to both calpain and caspase-3 proteolysis. In this work, we describe the calpain-induced degradation of microtubule-associated protein 2 (MAP-2), a protein found predominantly in the somatodendritic environment. Here we report that the 350 kDa MAP2a/b were extensively degraded into smaller fragments (130 kDa and 80 kDa) in the ipsilateral hippocampus and cortex within 2 hours after controlled cortical impact (a rat model of TBI) up to 14 days. In contrast, in the contralateral counterparts, MAP 2a/b was not degraded. Also, degradation of MAP 2a/b was not observed in the naive and sham groups. MAP 2a/b is preferentially enriched in the dendritic regions which are associated with axonal/dendritic disruption leading to the proteolysis of MAP 2a/b leading to its release in the CSF and subsequently to the blood after mechanical injury or brain injury models of ischemic stroke. The presence and monitoring of MAP2a/b breakdown products (130 and 80 kDa) cannot be overstressed as they can be potential dendritic specific markers following mechanical or chemical insults.

Samuelson Rodney Kalani Yashar Nakaji Peter Spetzler Robert Barrow Neurological Institute, Phoenix, AZ, USA

P098 Preserving the Under-FASCIA of the Temporalis Muscle Reduces Postoperative Edema Following Decompressive Hemicraniectomy

PURPOSE: Decompressive hemicraniectomy for trauma frequently results in excessive swelling and mass effect from the temporalis muscle. The dissection is often done in an emergent fashion with the periosteal elevator or bovie coagulator in a cranial to caudal direction. We explored the potential benefit of the surgical technique for temporalis fascia preservation originally described by Dr. Yasargil for vascular neurosurgery. In this technique, the temporalis muscle is carefully stripped from the temporal bone with a Penfield one dissector, or similar instrument, in a caudad to cranial direction. This preserves the soft pink fascia on the undersurface of the muscle, as well as the neurovascular structures supplying the temporalis muscle.

METHODS: Decompressive hemicraniectomies were performed with the temporalis sparing technique. The resulting size of the temporalis muscle was compared to the contralateral side, as well as matched controls which underwent hemicraniectomy with the standard (cranial to caudal) method of temporalis dissection.

RESULTS: The temporalis fascia preserving method of dissection resulted in temporalis muscle volume measuring 98% of the contralateral side. Postoperative hematoma formation was reduced. The standard method of temporalis dissection resulted in temporalis muscle volume measuring 213% of the contralateral side. Postoperative hematoma volume was increased.

CONCLUSIONS: Hemicraniectomy using Yasargil's temporalis fascia preserving technique substantially limits post operative edema and mass effect. It preserves the neurovascular supply to the temporalis muscle. This results in greater space available to the swollen brain following the decompressive hemicraniectomy procedure. Prospective research is warranted to determine if this effects postoperative ICP reduction and clinical outcome.

McCauley Stephen 1 Pedroza Claudia 2 Barnes Amanda 1 Frisby Melisa 1 Garza Hector 1 Varghese Reni 1 Wilde Elisabeth 1

1 Baylor College of Medicine, Houston, TX, USA 2 Center for Clinical Research and Evidence-Based Medicine at the University of Texas Medical School at Houston, Houston, TX, USA P099 Neuropsychological Recovery Over the First Week Following Mild Traumatic Brain Injury

PURPOSE: This study was designed to investigate patterns of neuropsychological recovery measured at multiple intervals over the first week postinjury following mild traumatic brain injury (mTBI).

METHODS: Patients (age 12-40 years) with mTBI and negative CT scan (n = 12) were recruited and assessed within 24 hours of injury (baseline) and on alternate days during the first week postinjury (4 total occasions) with a 3-month follow-up. Orthopedically-injured controls (OI n = 4) were assessed at baseline and 3 months.

RESULTS: Growth curve analyses were performed. Between-group differences in slope adjusted for baseline performance were calculated corresponding to Cohen's d effect sizes (ES). Large ES were found for age-adjusted measures of verbal learning (Hopkins Verbal Learning Test-Revised; 1.43) and delayed recall (0.83); small ES were found for simple (0.21) and complex (0.48) reaction time. There was no significant change in slope for postconcussion symptom severity (Rivermead Post Concussion Symptom Questionnaire) between groups. In analyses within the TBI group comparing those sustaining motor-vehicle accidents (n = 7) versus all other injury mechanisms (falls, sports-related, n = 5), medium effect sizes were found for verbal learning (0.76) and delayed recall (0.51), and postconcussion symptom severity (0.67). Small ES were found for simple (0.23) and complex (0.26) reaction time.

CONCLUSIONS: Substantial recovery was observed in the first week following mTBI in verbal learning and recall with modest changes in reaction time. Postconcussion symptoms remained elevated in the mTBI group with no difference in slopes compared to OI. Recovery trajectories within mTBI differed based on injury mechanism with substantially different slopes for verbal memory and postconcussion symptom severity. These results demonstrate the importance of early multiple assessments to improve current understanding of neuropsychological recovery from civilian mTBI, the critical role of injury mechanism on early recovery, and the dynamic nature of recovery within the first week.

Levin Harvey 1 Hanten Gerri 1 Li Xiaoqi 1 Wilde Elisabeth 1 McCauley Stephen 1 Barnes Amanda 1 Smith Douglas 2

1 Baylor College of Medicine, Houston, TX, USA 2 Univ Pennsylvania, Philadelphia, PA, USA P100 Early Effects of Mild TBI on Cognition are Age and Test-Dependent

In a prospective, longitudinal study 58 patients ranging from 12 to 30 years (mean age = 19.01, SD = 5.47) underwent cognitive assessment within 96 hours (mean post-injury interval = 2.93 days, SD = 1.09) after sustaining a mild TBI (mTBI), i.e., a Glasgow Coma Scale score 13-15 with loss or alteration of consciousness and normal computed tomographic (CT) scan. Fifty-two patients who sustained orthopedic injury (OI) (mean age = 19.24 years, SD = 5.41) were studied for comparison (mean post-injury interval = 3.20 days, SD = 0.98). Motor vehicle crashes (MVCs) predominated in mTBI patients (37.9% vs 11.5% of OI group) whereas sports injuries were more frequent in the OI group (46.2% vs 22.4% of mTBI patients), = 12.24, p = .0023. The Automated Neuropsychological Assessment Metrics (ANAM), a series of seven cognitive tests, disclosed that a between-group difference in performance depended on age and the specific ANAM test, F(6,106) = 2.21, p = 0.048. An interaction of age with group was significant only on the CodeSub subtest (F(1,104) = 6.89, p = 0.01), indicating that older mTBI patients had slower RTs, but this was not the case in OI patients. Therefore, the group difference became larger for older patients. Validity of the CodeSub test in measuring cognitive processing speed was supported by its Pearson correlation with performance on the well-validated Digit Symbol Modality Test which is not part of the ANAM, (r = −0.55, p < .0001 in mTBI; p = −0.63, p < .0001 in OI). Pending confirmation as our patient accrual increases, one interpretation of these data is that traumatic forces in mTBI sustained by adults may produce more severe cognitive sequelae relative to OI whereas this difference is reduced in adolescents. However, at this point in the study, we cannot differentiate the effects of age from those related to mechanism of mTBI. These results also encourage caution in extrapolating findings from sports concussion to mTBI resulting from MVCs.

McCauley Stephen 1 Wilde Elisabeth 1 Pedroza Claudia 2 Barnes Amanda 1 Ibarra Alyssa 1 Levin Harvey 1

1 Baylor College of Medicine, Houston, TX, USA 2 Center for Clinical Research and Evidence-Based Medicine at the University of Texas Medical School at Houston, Hosuton, TX, USA P101 Patterns of Early Emotional and Neuropsychological Sequelae Following Mild Traumatic Brain Injury

PURPOSE: This prospective, longitudinal study was designed to investigate patterns of emotional and neuropsychological performance following mild traumatic brain injury (mTBI) over the first three months postinjury. We report baseline data (within 96 hours postinjury).

METHODS: Participants (age 12-30 years) with mTBI and negative CT scan (n = 68; mean age = 19.2 + 5.4) or orthopedic injuries (OI; n = 49; mean age = 19.3 + 5.5) were compared. Motor vehicle accidents (MVAs) were more frequent in mTBI (37.3% versus 6.1% of OIs) compared to sports-related injuries in OI (51.0% versus 20.9% of mTBI). Participants were assessed on measures of emotional distress (Brief Symptom Inventory), postconcussion symptoms (Rivermead), posttraumatic stress (PTSD-Checklist), processing speed (Symbol-Digit Modalities Test), memory (Verbal Selective Reminding, Brief Visuospatial Memory Test-Revised), and executive functioning (Color-Word Interference Test, Verbal Fluency).

RESULTS: Participants with mTBI reported significantly greater emotional distress (F = 18.06, p < .0001) and postconcussion symptom severity (F = 46.33, p < .0001). Significant interactions included PTSD symptom severity X age (F = 10.6, p < .002) such that severity increased with increasing age in mTBI, and postconcussion symptoms X time postinjury of baseline assessment (F = 9.03, p < .004) such that symptoms worsened in mTBI while diminishing in OI. Significant differences were found for reduced processing speed (p < .008) and a positive trend for verbal learning (p = .06) in mTBI, but not delayed verbal recall, visual learning and recall, or executive functions.

CONCLUSIONS: These preliminary data suggest that emotional and postconcussional sequelae are more prominent in the acute stage following mTBI than reduced neuropsychological functioning and that these sequelae vary with age and the time postinjury when assessed in the first 96 hours.

Cruz Jovany Ponce Lucido Pillai Shibu Cherian Leela Robertson Claudia Baylor College of Medicine, Houston, Texas, USA

P102 Role of eNOS in the Vascular Effects of Erythropoietin After Experimental Traumatic Brain Injury

Erythropoietin (Epo) has been shown to improve pressure autoregulation, to increase post-traumatic cerebral blood flow (CBF), and to enhance vascular reactivity to L-arginine. The purpose of this study was to examine the role of endothelial nitric oxide synthase (eNOS) on the cerebral hemodynamic effects of Epo using eNOS-deficient mice. Using a Laser Doppler Flow imaging device, CBF was monitored in wild-type (WT) and eNOS-deficient mice that underwent a 3 m/sec controlled cortical impact (CCI) followed by administration of either Epo 5000 mg/kg, or normal saline. Mean arterial pressure (MAP) was monitored throughout the experiment. Changes in means of variables between groups were estimated using a mixed modeling procedure (SAS software system). CBF decreased in all groups post-injury, with the greatest reductions occurring at the impact site. Significant genotype and treatment differences were found in the CBF in peri-contusional tissue. Despite maintaining a higher MAP; eNOS-deficient mice had significantly lower CBF in peri-contusional areas by 2 hrs after injury compared to WT mice (52.5 ± 2.37 % and 61.6 ± 2.37 of baseline, respectively; p < 0.05). Epo administration resulted in significantly higher CBF in the peri-contusional sites after injury, but only in the WT mice (70.2 ± 3.35 % in Epo-treated compared to 53 ± 3.3 % of baseline in saline-treated mice; p < .0001). A trend for better CBF recovery contralateral to the injury was observed over time in WT mice groups. No significant CBF differences were found over time or by genotype at the core impact site (CBF dropped to 20-25% of baseline in all treatment groups; p > 0.05). These differences in response of the cerebral vasculature to Epo between eNOS-deficient and wild-type mice suggest that Epo may improve CBF recovery in the contused neural tissue after TBI by enhancing the activity of eNOS.

Bitner Brittany 1 Cherian Leela 1 Fabian Roderic 1 Tour James 2 Kent Thomas 1 Robertson Claudia 1

1 Baylor College of Medicine, Houston TX, USA 2 Rice University, Houston TX, USA P103 The Cerebrovascular Effect of Hydrophilic Carbon Clusters in Mild Traumatic Brain Injury Model Complicated by Hemorrhagic Shock in Rats

Traumatic brain injury (TBI) is associated with oxidative damage which may impair cerebral blood flow (CBF). Both superoxide radicals and hydroxyl radicals are increased in experimental TBI models, suggesting that antioxidants may be effective in decreasing the oxidative damage. Polyethylene glycolated hydophilic carbon clusters (PEG-HCCs), which are highly modified nano-structures derived from single wall carbon nanotubes, have potent antioxidant properties in in vitro models of oxidative stress. The present study was to determine the beneficial effects of PEG-HCCs in a mild TBI model complicated by hemorrhagic hypotension.

Mild cortical impact injury was induced at the right parietal cortex followed by first phase of hypotension (by withdrawing blood ) lasting for 50 minutes, then second phase of “prehospital care” (on air and infusion of Ringer's lactate) for 30 minutes followed by third phase of “definitive hospital care (oxygenation and infusion of shed blood) for 30 minutes. A total of 14 Long Evans rats were given phosphate buffered saline or PEG-HCCs just prior to the definitive hospital care phase and the animals were monitored for 6 hrs post-TBI by measuring mean arterial pressure, intracranial pressure and CBF. The CBF was assessed at the injury site, penumbra and contralateral brain. The CBF was expressed as % of baseline.

Treatment with PEG-HCCs significantly increased CBF at the center of the impact compared to PBS treated animals post-TBI (RM ANOVA: time effect p < .0001, treatment effect p = 0.0059, Bonferroni post test). CBF was restored to pre-injury levels in the perilesional area of the brains of rats treated with PEG-HCCs. The duration of effect was 200 minutes, consistent with the half life in the vasculature of the PEG-HCC's.

Treatment with PEG-HCCs can increase CBF after TBI complicated by hemorrhagic shock. This effect may be due to the antioxidant properties of the PEG-HCCs.

Goodman J. Clay Saucedo Hector Pillau Shibu Van Mai Robertson Claudia Baylor College of Medicine, Houston, TX, USA

P104 Hypoxia Inducible Factor 1 (HIF1), Vascular Endothelial Growth Factor (VEGF), Erythropoietin (EPO) and Erythropoietin Receptor (EPOR) Expression After Controlled Cortical Impact Injury (CCI) in Mice

PURPOSE OF STUDY: Numerous studies have shown a neuroprotective effect of EPO treatment in ischemia and trauma. In ischemia models, this neuroprotection may be mediated by HIF-1 activating its target genes EPO, EPOR and VEGF. We studied the magnitude and time course of these proteins in controlled cortical impact. Since EPO induces nitric oxide synthase (NOS) resulting in increased nitric oxide (NO) leading to increased cerebral blood flow, we also measured serum NO 2 hours after injury.

METHODS USED: Moderate CCI was induced in C57BL6 wild-type and endothelial NOS knock-out mice HIF-1, EPO, EPOR and VEGF were measured at 6 hr, 24 hr, 48 hr, 72 hr, 120 hr and 14 days. In addition, serum NO levels were examined following CCI.

SUMMARY OF RESULTS: During the first 24 hours after injury HIF-1 expression is two times higher than the non-injured side and then decreases over time. The expression of HIF-1α on the non-injured side also increases but after 48 hours. Epo and VEGF expression is higher in the injured side of the brain but not as dramatic as the HIF-1α. There was no change in EPOR expression. There was no difference between the wild-type and NOS knockout mice.

NO serum levels were, however, elevated in the wild-type mice while there was no change in the NO levels in the knockout mice.

CONCLUSION: Previous studies have shown neuroprotection in CCI by EPO as well as arginine induced elevations of NO. The cellular hypoxia response is activated by CCI leading ultimately to increased EPO and NO. This signaling pathway provides several potential targets that may provide neuroprotection including HIF-1, EPO and NOS. If NOS is not present, the neuroprotective effect is abrogated.

(Supported by NIH, NINDS PO1 NS38660-01)

Nelson Neta Paluy Vadim MacAllister Thomas BHR Pharma, LLC, Herndon, VA, USA

P105 Synapse: Study BHR-100-301: A Randomized, Double-Blind, Placebo-Controlled Phase 3 Study to Investigate the Efficacy and Safety of Progesterone in Patients with Severe Traumatic Brain Injury (TBI)

PURPOSE: Preclinical studies have demonstrated that progesterone has beneficial effects in TBI through multiple mechanisms. In a pilot and two Phase 2 studies conducted in moderate-to-severe TBI patients (Xiao et al., 2007; Wright et al., 2007; Xiao et al., 2008), progesterone showed promising efficacy results in functional outcomes and mortality, with an acceptable safety profile. BHR Pharma, LLC is conducting the SyNAPSe^® Phase 3 trial to evaluate BHR-100 progesterone infusion in 1180 severe TBI patients (Glasgow Coma Scale 4-8).

STUDY DESIGN: Subjects are entered into the SyNAPSe^® study following proxy consent, then centrally randomized in a 1:1 ratio of BHR-100 or placebo. Subjects are treated with a loading dose of 0.71 mg/kg/hr of BHR-100 or placebo i.v. for the first hour followed by a continuous maintenance infusion of 0.5 mg/kg/hr for a total of 120 hours/5 days. The infusion must be initiated within 8 hours of brain injury. The primary endpoint is the Glasgow Outcome Scale (GOS) assessed at 6-months post-injury. Secondary endpoints include the GOS-Extended, mortality, and the SF-36.

SUMMARY: The study has received approval in 17 countries, and 99 sites in the U.S., Argentina, Europe, and Asia have received Investigational Review Board/Ethics Committee approval to conduct the trial. Most of the 150 sites will be approved by May 2011. The study is under review in the Russian Federation and the People's Republic of China. An interim analysis is planned once 400 subjects (200 in each arm) complete the 6-month assessment.

CONCLUSION: BHR-100 is a promising candidate as a neuroprotectant for severe TBI patients. Successful completion of the SyNAPSe Phase 3 study may lead to the approval and registration of BHR-100 as the first such drug to have a positive outcome in the acute treatment of these patients.

Goldstein Lee 1 Fisher Andrew 1 Tagge Chad 1 Wojnarowicz Mark 1 Casey Noel 1 Moncaster Juliet 1 Wolozin Benjamin 1 Blusztajn Jan 1 Ikezu Tsuneya 1 Stanton Patric 2 Kowall Neil 3 McKee Ann 4

1 Boston University School of Medicine, Boston, MA, USA 2 New York Medical College, Valhalla, NY, USA 3 VA Boston Healthcare System, Jamaica Plain, MA, USA 4 New England VA Healthcare System, Bedford, MA, USA P106 Blast Exposure Induces Microvascular Disruption, Neuroinflammation, TAU Neuropathology, and Hippocampal Electrophysiology Deficits in a Murine Model of Chronic Traumatic Encephalopathy

Exposure to blasts from improvised explosive devices (IEDs) is a leading cause of traumatic brain injury (TBI) in US troops and raises concern about later development of chronic traumatic encephalopathy (CTE). We hypothesize that blast shock waves induce shearing forces that disrupt microvascular integrity, damage fiber tracts, and induce neuroinflammation leading to CTE. We developed a murine blast neurotrauma model system to investigate blast-induced cerebrovascular and blood-brain barrier disruption, neuroinflammation, hippocampal electrophysiology, and CTE neuropathology in C57/B6 mice.

METHODS: Our murine blast neurotrauma system utilizes a 27-foot-long 12-inch diameter air-driven shock tube to deliver sublethal shock waves scaled to military IED blasts. Brain microvascular integrity and elemental homeostasis (Ca, Cu, Fe, Zn) were assessed by metallomic imaging mass spectrometry (MIMS, Center for Biometals & Metallomics). Focal tauopathy and neuroinflammation were evaluated by neuropathological analysis and immunophenotyping. Hippocampal long-term synaptic plasticity and conduction velocity were assessed in organotypic slice culture.

RESULTS: Our murine blast neurotrauma system delivered reproducible graded blast shock waves in accordance with Friedlander wave theory. Maximal sublethal blast overpressure was achieved at 17.0 + 0.9 psi (10 ms, 88 psi-ms, Mach 1.29) and induced persistent focal microvascular disruption, neuroinflammation, cytoskeletal reorganization, and tauopathy. Persistent electrophysiology deficits, including accelerated LTP decay and decreased conduction velocity, were detected in blast-exposed mice. Blast-induced neuropathological findings reproduced the major neuropathological hallmarks of human CTE. Neurotrauma is induced by linear and angular acceleration of the head.

CONCLUSIONS: We have developed a murine blast neurotrauma model system that reproduces blast intensities and shock wave dynamics comparable to IED explosions experienced by soldiers. Single blast exposure induces persistent focal microvascular disruption, neuroinflammation, cytoskeletal reorganization, tauopathy, and electrophysiology deficits that replicate major features of human CTE.

Acknowledgments: Boston University Alzheimer's Disease Center, Army Research Laboratory, Boston University Photonics Center, Boston University School of Medicine.

Edlow Brian 1 Takahashi Emi 2 Cummings Peter 3 Wu Ona 2 Klein Joshua 1 Bu Lihong 4 Greer David 5 Greenberg Steven 6 Kinney Hannah 4 Folkerth Rebecca 7

1 Brigham and Women's Hospital, Department of Neurology, Boston, MA, USA 2 Massachusetts General Hospital, Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, MA, USA 3 Office of the Chief Medical Examiner, Boston, MA, USA 4 Children's Hospital of Boston, Department of Pathology, Boston, MA, USA 5 Yale-New Haven Hospital, Department of Neurology, New Haven, CT, USA 6 Massachusetts General Hospital, Department of Neurology, Boston, MA, USA 7 Brigham and Women's Hospital, Department of Pathology, Boston, MA, USA P107 Diffusion Tractography in Traumatic Brain Injury: A New Paradigm for Investigating the Neuroanatomic Basis of Altered Consciousness

PURPOSE: Diffuse axonal injury (DAI) causes altered consciousness in traumatic brain injury (TBI) by disrupting brainstem arousal networks, hemispheric awareness networks, or both. Yet, there is a critical lack of diagnostic tools for identifying neuroanatomic networks disrupted by DAI, particularly in the brainstem, where conventional imaging is insufficient. In this study, we used a magnetic resonance technique, high angular resolution diffusion imaging (HARDI), to investigate the neuroanatomic connectivity of arousal and awareness networks susceptible to DAI. We hypothesized that HARDI tractography would reveal the neuroanatomic basis of altered consciousness.

METHODS: High-field HARDI scans (3T and 4.7T) were performed on two post-mortem brains: from a 53-year-old woman who died of non-neurological causes, and from a 62-year-old woman who died three days after severe TBI causing coma. The normal specimen was scanned as a dissected block of the brainstem, hypothalamus, and thalamus. The trauma specimen was scanned as a whole brain and as a dissected block. HARDI tractography was used to investigate brainstem connectivity with the hypothalamus and thalamus, as well as cortical connectivity with the thalamus and corpus callosum. Upon completion of imaging, the specimens were sectioned and stained for correlative histopathologic analysis.

RESULTS: HARDI tractography revealed that brainstem arousal networks were severely disrupted by DAI in the trauma specimen. These disruptions were consistent with histopathologically confirmed tissue tears and axonal swellings. By contrast, HARDI tractography and histopathologic analysis both demonstrated that hemispheric awareness networks were relatively preserved, suggesting that DAI of brainstem arousal networks caused the patient's coma.

CONCLUSION: This study suggests that HARDI tractography can delineate the neuroanatomic basis of altered consciousness in TBI. Clinical implementation of this new imaging tool has the potential to revolutionize TBI care, as disruptions in specific neuroanatomic networks can be uniquely defined in each patient, allowing individualized prognostication and targeted therapy.

Zakharova Natalia Potapov Alexander Kornienko Valeriy Pronin Igor Arutyunov Nikita Fadeeva Ludmila Gavrilov Anton Oshorov Andrey Zaytsev Oleg Sychov Alexander Burdenko Neurosurgery Institute, Moscow, Russian Federation

P108 Identification of Degeneration and Possible Regeneration of White Matter Tracts by Dynamic DT MRI

INTRODUCTION: Diffusion-tensor MRI allows us to perform qualitative and quantitative evaluation of damage of white matter tracts. Dynamics of structural changes in corpus callosum (CC) and corticospinal tracts (CST) at various periods after severe TBI is not studied enough.

AIM: To study dynamic changes of the CC and CST at various periods after severe DAI by Diffusion-Tensor (DT) MRI.

PATIENTS AND METHODS: Dynamic 1.5T MRI studies were primarily performed in 21 patients (pts) aged 9-72 years (average age 27) with severe TBI (GCS ≤ 8) within 2 - 21 days after injury, and was repeated in 19 of 21 pts in 1-8 months, in 2 pts - 3 years after injury; in 6 of 21 pts MRI was repeated 3-5 times within 3 months-4 years.

RESULTS: In pts primarily examined 10-17 days after injury the values of fractional anisotropy (FA) in the CC and CST significantly correlated with outcome. A significant decrease of FA and thinning of CST fibers were observed in pts with unfavourable outcome and motor deficit. The data analysis of the second DTMRI studies in 21 pts in 1-3 months after injury showed different degrees of CC degeneration in 19 of them. Only 2 of these 19 pts showed some signs of partial recovery of CC fibers and favourable outcome in 6-7 months.

CONCLUSION: DT MRI in pts with DAI identifies dynamics of the degenerative changes in the white matter tracts and their possible recovery.

Alexandrova Evgeniya Tenedieva Valeriya Sychev Alexander Tenedieva Nina Zaitsev Oleg Voronov Vladimir Zakharova Nataliya Gavrilov Anton Okhlopkov Vladimir Kravchuk Alexander Potapov Alexander Burdenko Neurosurgery Institute, Moscow, Russian Federation

P109 Gender Dimorphism and Oxidative Stress in Patients with Severe Traumatic Brain Injury

BACKGROUND: Recent published studies gender dimorphism in stroke models, showed different cell death pathways in males and females. But the role of gender difference for TBI course and outcome remains unclear.

GOAL: To estimate hormonal, cytokine changes and clinical manifestations in TBI patients according to their gender.

MATERIALS AND METHODS: Sex hormones (progesterone-PROG, estradiol-E2, testosterone-TEST, LH, FSH), cytokines (IL-6, sIL-2R, EPO), ferritin (oxide stress marker) were monitored in 85 males and 27 females (GCS < 8 at admission) by immunochemiluminescent analysis. Besides, hemodynamic (HD) patterns were evaluated by transpulmonary thermodilution (PiCCO) method.

RESULTS: 1) Levels of IL-6, sIL-2R, ferritin (<0.0001) were higher in male vs female. 2) Intracranial pressure-ICP, stroke volume index, cardiac function index, intrathoracic blood volume and global end-diastolic volume indexes were also prevailed in male. 3) Extravascular lung water index, proBNP and EPO levels predominated in female. Sex hormone levels in both genders were very low during the observation period.

CONCLUSION: We found less pronounced inflammatory responses in female and more severe HD disturbances in male. Significant increase of ferritin levels in male may be related to different pathways of cell apoptosis that is NO/PARP/AIF mitochondrial and cytochrome C/caspase pathways for male and female respectively. Perhaps our data would be useful for developing more effective sex-specific neuroprotective therapy protocols for severe TBI.

Deng-Bryant Ying Chen Zhiyong van der Merwe Christopher Liao Zhilin Lu Xi-Chun May Dave Jitendra Shear Deborah A. Tortella Frank C. Center for Military Psychiatry and Neuroscience, Silver Spring, MD, USA

P110 Neuroprotective Effects of Amnion-Derived Cellular Cytokine Suspension in an Experimental Model of Penetrating Ballistic-Like Brain Injury

Previous work has shown that amnion-derived multipotent progenitor (AMP) cells are neuroprotective in a rat model of penetrating ballistic-like brain injury (PBBI). To some extent, this neuroprotection may be mediated by the sustained secretion of AMP cell-derived neurotrophic factors which are abundant in the amnion-derived cellular cytokine suspension (ACCS) medium. To test this hypothesis, we investigated 1) the bioactive stability and neuroprotective capacity of ACCS on cultured embryonic cortical neurons treated with staurosporine (an apoptosis-inducer) and 2) the effects of chronic ACCS delivery (via Alzet osmotic pumps) on PBBI-induced motor and cognitive deficits. Results confirmed that ACCS is thermally stable for at least one week at 37°C and demonstrated that co-treatment with ACCS facilitated neurite outgrowth (i.e. neuroplasticity) in an in vitro model of apoptotic cell death. Chronic delivery of ACCS or control medium (1μl/hr or 5μl/hr) into the cerebral ventricles was initiated within 30 min following PBBI. Rotarod motor function assessments and Morris water maze (MWM) spatial learning tests were performed at 1, 2, 3, or 4 weeks for each respective group. The high dose (5μl/hr) of ACCS improved rotarod performance (10, 15 and 20 rpm) at 1 and 2 weeks post injury with a significant effect at 2 weeks (p < 0.05). However, this infusion rate resulted in some mortality possibly due to the buildup of cerebral spinal fluid. The low dose (1μl/hr) of ACCS (which exhibited no signs of toxicity) produced extended therapeutic effects evident out to 3 weeks post-injury on the rotarod test (p < 0.05) with the optimal effect observed again at 2 weeks post-injury. Chronic ACCS infusion (at either dose) failed to produce significant improvement on MWM performance at any time point tested. Collectively, our results support the hypothesis that the neuroprotective effects of AMP cells may be partially mediated through a sustained delivery of neurotrophic factors.

Kreber Lisa Johnson Sarah Ashley Jessica Ashley Mark Centre for Neuro Skills (CNS), Bakersfield, CA, USA

P111 Growth Hormone Deficiency Following Brain Injury May Negatively Impact Activities of Daily Living

Approximately 20% of people with brain injury (BI) also suffer from post-traumatic growth hormone deficiency (GHD). Individuals with GHD experience fatigue, increased abdominal adiposity, reduced exercise capacity, memory impairments, inability to concentrate, anxiety and depression. These symptoms overlap considerably with deficits commonly observed in patients with BI. The objective of this study was to determine if patents with BI who were also GHD actualized less recovery in rehabilitation than individuals who had a BI and were not GHD. Upon admission into the rehabilitation facility, basal hormone levels were assessed. Blood levels of thyroid (TSH, T3 and T4), follicle stimulating hormone (FSH), luteinizing hormone (LH), cortisol (AM level), testosterone, estradiol and insulin-like growth factor-1 (IGF-1) were measured. It has been documented that low levels of IGF-1 increase the likelihood of GHD. For patients whose IGF-1 level was less than 200 ng/mL, a provocative glucagon stimulation test was conducted to investigate GH levels.

Glucagon stimulation tests were subsequently completed in 41 individuals with BI. Of these, 21 patients had deficient levels of GH. These 21 patients did not receive GH replacement prior to discharge from the rehabilitation facility. Preliminary results indicate patients who were GHD, did not actualize as much change per day on the Independent Living Scale (ILS), a measure of functional activities of daily living, as patients who had normal GH levels (p < 0.05). There were no differences in level of disability or ILS scores at admission between the GHD and GH normal patients. Patients who were GHD did have a slightly longer length of stay than the GH normal group, although this difference did not reach statistical significance. These results support previous findings that concomitant GHD can negatively influence recovery from brain injury.

Ashley Jessica Ashley Mark Seneca Philip Kreber Lisa Centre for Neuro Skills, Bakersfield, CA, USA

P112 Rehabilitation Length of Stay Following Traumatic Brain Injury: Are Patient's Getting the Full Dose?

Studies have provided reasonable evidence to suggest that the intensity of treatment and duration of treatment both contribute to greater reduction of disability. The goal of this study was to conceptualize the time required for a patient to transition to a lower disability level during inpatient rehabilitation. This retrospective analysis included data obtained during the years 1990-2009 from the Traumatic Brain Injury Model Systems (TBIMS) national dataset. Survival analysis using Disability Rating Scale (DRS) admission disability categories and scores from admission and discharge was conducted to determine the length of time required to change disability level to a lower specified level. Separate analysis was done based upon injury severity upon admission. The first survival analysis addressed those patients admitted with a moderate disability rating at admission (score of 3.5-6.0). The terminal event was defined as achieving a rating of partial disability (DRS score of 3 or below). The median length of stay for the moderate category of patients to reach the terminal event of DRS rating was 18 days. A second survival analysis included those patients admitted to inpatient rehabilitation with a DRS category of moderately severe and above (score of 6.5-29.0). The terminal event for this subset of the sample was defined as achieving a rating of moderate disability (DRS score of 6 or below). The median length of stay to reach the terminal event was 17 days for moderately severe, 30 days for severe, 58 days for extremely severe, and 94 days for vegetative patients. The data suggests time to reach the terminal DRS score varies with level of disability at admission. Further, the data allows a more objective prognostication about the likelihood of a specific patient attaining a particular level of improvement during inpatient rehabilitation and may provide for an objective determination of required rehabilitation treatment.

Alves Óscar L Bullock Ross Centro Hospitalar de Gaia, Porto, Portugal

P113 Pharmacokinetic and Pharmakodynamic Studies on Human TBI

INTRODUCTION: Virtually all neuroprotective trials failed to shown any encouraging results in the human injured brain despite positive outcomes in laboratory studies using different animal models. We hypothesize that poor injured brain pharmacokinetics is a putative cause for this failure.

MATERIAL AND METHODS: We have done pioneer work using brain microdialysis to quantify the amount of unbound drugs, such as topiramate, Cyclosporin A, and cephazolin in the brain extracellular fluid of TBI patients. Dialysate and CSF samples were collected during five days and the drugs measured in the brain dialysate using HPLC.

RESULTS: An important inter- and intra-individual variability was observed among patients receiving the same drug dosage of cephazolin, topiramate and CsA. Only 1/11 patients showed minimal inhibitory concentrations for S. aureus when receiving conventional dosage of cephazolin. Topiramate and CsA dialysate levels are significantly different from drug CSF levels (p < 0.001). Brain levels are higher for CsA than CSF levels, but lower for topiramate. A lower dialysate/plasma ratio was found when topiramate dosage was doubled (0.15 versus 0.25) suggesting an active transport mechanism for topiramate CNS delivery that can be saturated. Using equivalent dosage, brain CsA levels are different in humans than in rats (p < 0.0001). in the group 10 patients receiving 11.4 mg/Kg of topiramate, in the first two days, the decrease in glutamate levels is even more marked denoting a better effect upon glutamate release.

CONCLUSIONS: Microdialysis provides continuous information on unbound drug concentration-time profiles in the brain ECF and CNS drug delivery. It may be possible to quantify in vivo drug influx and efflux process across BBB. This will have a major impact on drug design in order to manipulate drug penetration in CNS for which a central effect is (un)desired. Better knowledge of injured human brain drug pharmacokinetics will increase the probability of positive neuroprotective trials.

See Jill Elkind Jaclynn Horyn Oksana Nissim Ilana Nissim Itzhak Cohen Akiva Children's Hospital of Philadelphia, Philadelphia, PA, USA

P114 Alterations in Neuronal-Glial Metabolic Coupling Caused by Lateral Fluid Percussion Injury

Traumatic brain injury (TBI) is a major public health issue, affecting over 1.4 million people annually in the USA. TBI often causes enduring disabilities including emotional alterations, cognitive impairment and memory dysfunction. These functional deficits result from changes in hippocampal network excitability that are precipitated by regional imbalances between excitatory and inhibitory synaptic activity, including decreased network excitability in CA1 and increased excitability in the dentate gyrus. Furthermore, both neuronal and astrocyte metabolism following injury are altered, including increased accumulation of lactate and an increase in intracellular glutamate, but the interaction between astrocyte-derived metabolites and neuronal metabolism following injury is unclear. Thus, the purpose of this study is to investigate the changes in metabolic interactions between glia and neurons following injury. We have investigated the effects of fluid percussion injury (FPI) on cellular metabolism in hippocampus by treating hippocampal slices from injured and sham mice with 13C-labeled acetate, a glial specific substrate. Slices were incubated 13C acetate and samples were analyzed to determine whether injury alters neurotransmitter production, amino acid metabolism or energy production. We have determined that total neutotransmitter and amino acid concentrations are unchanged by injury. In addition, 13C glutamate enrichment also remains unchanged. Interestingly, 13C GABA enrichment is increased, suggesting that neuronal metabolism from glial-derived metabolites is augmented following injury. In addition, we have investigated whether injury alters the expression of metabolic transporters integral in the metabolic coupling of glia and neurons. Preliminary data suggests that SNAT3, which releases glutamine from the astrocyte, is decreased following injury, while SNAT1, which takes up glutamine into the neuron, remains unchanged. These experiments suggest that changes in coupling of neuronal and glial metabolism may underlie and contribute to changes in excitability following TBI.

Supported by NIH-NINDS- R01NS069629, NICHD- R01HD059288, and NINDS- 5T32NS007413-13

Salonia Rosanne 1 Empey Philip 3 Miller Tricia 3 Bell Michael 4 6 Adelson P. David 5 Kochanek Patrick 2 Poloyac Samuel 3

1 Children's Hospital of Pittsburgh of UPMC; Dept. of Pediatric Critical Care Medicine, Pittsburgh, PA, USA 2 University of Pittsburgh School of Medicine; Safar Center for Resuscitation Research, Pittsburgh, PA, USA 3 University of Pittsburgh School of Medicine; School of Pharmacy, Pittsburgh, PA, USA 4 University of Pittsburgh School of Medicine; Department of Neurological Surgery, Pittsburgh, PA, USA 5 Phoenix Children's Hospital; Department of Neurological Surgery, Phoenix, AZ, USA 6 University of Pittsburgh School of Medicine; Department of Critical Care Medicine, Pittsburgh, PA, USA P115 Identification of Arachidonic Acid (AA) Metabolites in Cerebrospinal Fluid of Infants and Children After Traumatic Brain Injury

PURPOSE: The preservation of cerebral blood flow (CBF) to match metabolic demands after TBI is vital to preserve neurologic function. AA metabolites are powerful mediators with cerebral vasoactive and anti-inflammatory effects. 20- hydroxyeicosatetraenoic acid (20-HETE) is a potent mediator of cerebral vasoconstriction and has been identified in CSF of adults after subarachnoid hemorrhage (Poloyac, 2004). 12-HETE is a pro-inflammatory mediator with vasodilatory effects. Quantification of AA metabolites has not been explored in children.

HYPOTHESIS: We hypothesize that AA metabolites are able to be quantified by Ultra triple quadruple mass spectrometer (UPLC-MS/MS) and will be present in CSF of children after TBI.

METHODS: CSF (n = 134) was collected via external ventricular device (EVD) in children (n = 18, 8d) after severe TBI. AA metabolites were extracted via a solid phase extraction procedure (Waters Oasis) and separated by Water's Acquity UPLC with identification by TSQ Quantum MS/MS. The assay was validated with an intraday and interday variability of < 20%.

RESULTS: 20-HETE and 12-HETE were quatified in the CSF of subjects. The concentration of 20-HETE ranged from 0.18 to 17.87 ng/ml (mean 1.696 ng/ml; SEM 0.696 ng/ml) and was present at multiple time points in 13 of 18 subjects. The concentration of 12-HETE ranged from 0.21 to 135.74 ng/ml (mean 9.4 ng/ml; SEM 3.44 ng/ml) and was present at multiple time points in 17 of 18 subjects.

CONCLUSIONS: We conclude that we were able to identify AA metabolites in the CSF of children after severe TBI, they were quantified by UPLC-MS/MS and were present at physiologically relevant concentrations. The temporal profile of these metabolites suggests a prolonged effect at the vascular level post-TBI. Further study is warranted to pursue the role of these metabolites and may offer insight into mechanisms of secondary injury after TBI that may contribute to changes in CBF.

Grant Support: NHLBI T32HL007820; NIH5U01NS052478

Ferguson Nikki Miller Kochanek Patrick M. Clark Robert S. B. Tyler-Kabara Elizabeth Bell Michael J. Childrens Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA

P116 Intracranial Hypertension in Children with Severe Traumatic Brain Injury (sTBI): Quantifying the Secondary Insult and its Effect on Outcome

sTBI (GCS ≤ 8) is the leading killer of children aged 1 - 18 yet published guidelines are inadequate in many aspects. Intracranial pressure (ICP) monitoring is recommended but interpretation of ICP-generated data is subject to wide interpretation. The impact of number of intracranial hypertension events and their duration on outcome has not been well studied.

A prospectively-collected database was interrogated for the first 7 d. Hourly ICP recordings (in mm Hg) were collated and the number above thresholds (>15, > 20 and > 30) was quantified. Intracranial hypertension index (IHI, [# h ICP > 20/# h of monitoring] x 100) was calculated. Glasgow outcome scale (GOS) scores at 6 months were determined and analyzed categorically and dichotomously (GOS 3 - 5 unfavorable). Data were compared using Mann-Whitney Rank Sum and linear regression, all data is expressed as mean ± SEM.

Children (n = 85, 9 deaths) were enrolled with initial GCS 6.4 ± 0.2. Children with unfavorable outcome (n = 34, 40%) had more hourly readings of ICP > 20 (20.1 ± 4.6 vs. 7.6 ± 1.4; p = 0.02), greater mean ICP (14.6 ± 1.7 vs. 10.8 ± 0.6; p = 0.049) and greater IHI (17.1 ± 4.0 vs. 6.0 ± 1.1; p = 0.014). Moreover, children with unfavorable outcome trended toward more hourly readings of ICP > 15 and > 30 (47.0 ± 6.0 vs. 32.6 ± 3.9, p = 0.06; 8.7 ± 3.5 vs. 1.0 ± 0.3, p = 0.07). Regression analysis demonstrates a strong correlation between GOS and ICP > 30, ICP > 20, mean ICP and IHI (p < 0.001 for all).

Increased ICP is strongly associated with outcome in children after sTBI. As a variable that is observed at the bedside, the optimal manner to describe ICP-related events after TBI remains to be determined.

Hue Christopher 1 Panzer Matthew 2 Bass Cameron 2 Meaney David 3 Morrison Barclay III 1

1 Columbia University, New York, NY, USA 2 Duke University, Durham, NC, USA 3 University of Pennsylvania, Philadelphia, PA, USA P117 Blast Overpressure Induces Disruption of Brain Endothelial Monolayer Integrity

PURPOSE: While blast injury is recognized as a major cause of brain injuries, the pathobiology of blast-induced traumatic brain injury (bTBI) remains poorly understood, and there is a scarcity of information available for its consequences on the blood-brain barrier (BBB). This work investigates blast-induced disruption of the integrity of an endothelial monolayer - a major component of the BBB - by measuring time-course changes in transendothelial electrical resistance (TEER).

METHODS: Using an endothelial monoculture BBB model (bEnd.3 mouse brain cell line), cells were seeded on poly-L-lysine coated Transwell inserts, grown in DMEM supplemented with 10% newborn calf serum and 4 mM glutamine, and cultured for 8 days prior to blast exposure. A blast injury condition of approximately 240 kPa peak incident overpressure of 0.8 ms duration was generated using a 76 mm diameter shock tube with a helium-pressurized 25 mm driver section. Changes in TEER were quantified using an EVOMX Epithelial Voltohmmeter. Uninjured controls were processed identically to injured cultures but not exposed to blast.

RESULTS: After blast overpressure of approximately 240 kPa/0.8 ms, TEER of bEnd.3 monolayers significantly (p < 0.05) decreased acutely at day 0 to 8 ± 1.1 Ω cm² compared to 18 ± 0.9 Ω cm² in uninjured controls. TEER of blast-exposed cultures was significantly depressed compared to age-matched uninjured control cultures for 3 days after injury and remained depressed at 6 and 8 days post-exposure.

CONCLUSION: These data demonstrate that a shock wave is capable of disrupting the integrity of an endothelial monoculture BBB model through direct interaction with a primary blast wave. TEER values monitored for 8 days following injury partially recovered, suggesting an intrinsic ability of the BBB to repair itself. A fundamental outcome envisioned from this research is elucidating pathobiological implications and functional recovery time for the blast-injured BBB.

Kang Woo Hyeun Yu Zhe Morrison Barclay III Columbia University, New York, NY, USA

P118 Changes in Electrophysiological Function After Controlled Mechanical Deformation of Hippocampal Slice Cultures

In their current form, finite element models only predict mechanical responses and not biological responses to TBI. To address this deficiency, we are developing tolerance criteria based on alterations in neuronal network function in brain tissue in response to controlled mechanical stimuli. We have utilized an in vitro tissue culture approach to generate this criterion to relate changes in electrophysiological function to tissue-level, biomechanical measures of injury, i.e. tissue strain and strain rate. Organotypic hippocampal slice cultures were subjected to a single, biaxial deformation with our well-characterized in vitro model of TBI at specific combinations of strain and strain rate. At 4-6 days post-injury, electrophysiological activity was quantified throughout the hippocampus with a 60-electrode microelectrode array. Stimulus-response curves were generated using mossy fiber and Schaffer collateral stimulation to determine the maximum evoked response (R_max) and the stimulus intensity necessary to generate a half-maximal response (I₅₀). 0.05 and 0.10 strain injuries decreased R_max in the CA1, CA3 and DG hippocampal regions. However, moderate injury (0.20 strain) increased R_max. I₅₀ was not altered at 0.05 strain, increased at 0.10 strain, but decreased at 0.20 strain. Our results suggest that after mild injury (0.05, 0.10 strain) synaptic function is reduced as indicated by R_max, while moderate injury paradoxically increased synaptic transmission. These seemingly paradoxical results may suggest an impairment of inhibitory interneurons and neuronal network inhibition leading to a relative increase in excitatory drive, as indicated by I₅₀, and therefore, increased responses and hyperexcitability, as indicated by R_max, at 0.20 strain. Our ongoing study suggests that alterations in the hippocampus neuronal network may be related to injury severity in a more complex fashion than previously understood, possibly due to the difference in biomechanical tolerance of inhibitory and excitatory neurons.

Finan John D. Elkin Benjamin S. Morrison Barclay III Columbia University, New York, USA

P119 Viscoelastic Properties of the Rat Brain Depend on Age, Loading Direction and Anatomical Structure

Modern finite element analysis methods can describe the mechanical heterogeneity of the brain explicitly, potentially leading to more accurate brain injury metrics, but these models require accurate experimental measurements of brain mechanical properties. In this study, we used microindentation to measure the viscoelastic properties of the juvenile (P17/18) and adult rat brain in the sagital direction. Specific anatomical regions of the brain were indented with a cylindrical punch. A closed-form solution for indentation of a linearly viscoelastic material incorporating a hereditary integral was fit numerically to the force displacement data to determine regional relaxation functions. The rat brain was highly heterogeneous and viscoelastic in the sagital plane. The hippocampus was the stiffest structure, followed by the cortex and then the white matter (i.e., alveus and corpus callosum) while the cerebellum was the softest structure. This heterogeneity was more pronounced in the adult than in the juvenile rat because the hippocampus stiffened relative to the rest of the brain as the animal matured. There was also mechanical heterogeneity within the hippocampus, which was age-dependent. The properties of the juvenile brain differed from those of the adult brain in magnitude and in their heterogeneous distribution. These differences imply that the strain field, and hence the pattern of injury, is different in the juvenile animal. In a similar study in the coronal plane, the corpus callosum was found to be softer than the alveus in adult rats but as stiff as the alveus in juvenile animals. Here, this temporal pattern is reversed, suggesting that the rat corpus callosum is anisotropic, consistent with its highly directed microstructure and previous work on porcine corpus callosum. By incorporating the effect of age, direction and anatomy on stiffness into finite element models, we can better address hypotheses about how these factors affect the anatomical distribution of injury.

Taylor Sabrina 1 Smith Colin 4 Harris Brent 3 Costine Beth 2 Duhaime Ann-Christine 2

1 Dartmouth College, Hanover, NH, USA 2 Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA 3 Georgetown University Medical Center, Washington, DC, USA 4 University of Edinburgh, Edinburgh, Scotland, United Kingdom P120 Neurogenesis After Traumatic Brain Injury During Immaturity: Translational Considerations

Traumatic brain injury (TBI) is the leading cause of acquired disability in children, yet repair mechanisms are incompletely understood. The observation of numerous ectopic neurons in white matter resected for intractable epilepsy 10 years after severe unilateral TBI in a child led us to question whether injury-induced migration of immature neurons in young children might occur, similar to what has been seen in animal models.

Previously we utilized a piglet model of focal cortical injury to study trauma-related neurogenesis and neuronal migration. Doublecortin positive (DCX⁺) migrating neuroblasts in the white matter of the ipsilateral and contralateral hemispheres in injured (N = 3) and uninjured (N = 3) 1 week old subjects were counted using stereology. A greater number of DCX⁺ neuroblasts were found in the white matter of the ipsilateral compared to contralateral hemispheres (p < 0.05). To learn whether such events might occur in children, we used immunohistochemistry for doublecortin (DCX) in autopsy specimens from patients aged 1 month, 5 months, and 3 years who died of various non-traumatic conditions. Elongated DCX-positive (DCX⁺) cells with 1-2 processes were considered to represent migrating immature neurons.

Analysis demonstrated an observable decrease in DCX expression throughout maturation in human children. The 1 month old patient had the greatest expression of DCX⁺ cells migrating from the subventricular zone to the white matter, with progressively fewer seen in the 5 month old and the 3 year old. The youngest patient also had an area of left frontal leukoencephalomalacia, likely reflecting a pre-morbid hypoxic-ischemic insult, in which DCX⁺ cells congregated in large numbers, suggesting that they migrated to that area as a possible mechanism for repair. Quantitative analysis of DCX in the brains of 8 additional children with TBI and two additional non-injured children of different ages is currently underway.

Wang Yushan 1 Chavko Mikulas 2 Weiss Tracy 1 Adeeb Saleena 2 McCarron Richard 2 Yu Simon 1

1 Defence R&D Canada Suffield, Medicine Hat, Alberta, Canada 2 Naval Medical Research Center, Silver Spring, MD, USA P121 Changes in Glutamate Receptor Trafficking in Blast-Induced Traumatic Brain Injury in the Rat

Glutamate receptor trafficking has been implicated in various brain diseases and injuries, including traumatic brain injury. Cell surface expression of two types of ionic glutamate receptors (AMPA and NMDA) was investigated in the rat brain after exposure to blast. Animals were subjected to a 120 kPa blast overpressure at a side-on orientation in a pneumatic-pressure driven shock tube and allowed to recover for 3h, 24 h, 1 week and 3 weeks following blast. At the end of each period, animals were euthanized and their brains harvested. Whole rat brains were fast frozen and kept at − 80⁰C until further analysis. Frontal cortices and hippocampi of both sides (contralateral and ipsilateral to blast exposure) of the brain were processed to separate synaptic membranes (synaptoneurosomes, SN). The expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits in SNs and in total homogenates from both sides of the brain were analyzed using Western blot analysis. Results showed that synaptic expression of AMPA receptor subunits, GluR1 and GluR2, were dramatically reduced 24 h on both sides of the brain. These changes became less significant 1 week after blast. However, both GluR1 and GluR2 were significantly increased 3 weeks after blast. Expression of GluR1 and GluR2 in the homogenates remain unchanged after blast injury. The expression of NR2A on SNs or homogenates did not show any change after blast while the expression of NR2B was dramatically increased, especially in homogenates, resulting a net decrease in the ratio of NA2A/NA2B. Changes in NA2A/NA2B ratio has been implicated in various brain diseases including traumatic brain injury. Results from these experiments indicate that the blast overpressure produces changes in glutamate receptor trafficking. These changes may contribute to neurodegenerative and neurobehavioral changes observed after blast.

Seidl Stacey 1 Lance Steven 1 Chishti Athar 2 Kozlowski Dorothy 1

1 DePaul University, Chicago, Illinois, USA 2 Tufts University School of Medicine, Boston, MA, USA P122 Contusion Size is Significantly Decreased in Calpain-1 Knockout Mice Following a Controlled Cortical Impact (CCI)

An effective pharmacological treatment for individuals suffering from a traumatic brain injury (TBI) has remained elusive. One challenge has been the characterization of numerous proteins that are integral components in the death of neurons following a TBI. Recent research has indicated that calcium-activated neutral proteases, otherwise known as calpains, are essential mediators of cell death in numerous chronic and acute neurodegenerative disorders. Following TBI, calpain activation has been demonstrated in both dendrites and axons of the cortex and hippocampus. Additionally, calpain activation has been linked to TBI-induced pathology. General inhibition of calpain activity following TBI resulted in cytoskeletal protection and behavioral improvement. Two isoforms of calpain exist in the brain, m-calpain (calpain-2) and μ-calpain (calpain-1), yet the specific roles of each enzyme following TBI are not well understood. Using calpain-1 knockout mice, the current study examined the role of calpain-1 in TBI-induced neural degeneration following the controlled cortical impact (CCI) rodent model of TBI. Calpain-1 knockout mice were generated using mice with a pure C57BL/6 genetic background by first producing heterozygotes that contained the mutant calpain-1 allele. Through PCR based analysis, mice containing the mutant calpain-1 allele were identified and mated with one another to produce the homozygote calpain-1 knockout mice (Cpn1-/-). Both Cpn1-/- and wild type mice (n = 6-8) received a CCI unilaterally over the forelimb sensorimotor cortex. Mice were sacrificed three days post-CCI and their brains were sliced coronally and stained with Nissl. Contusion size was examined with an analysis of remaining cortical volume utilizing computerized microscopy (NeuroLucida). Our results demonstrate that the calpain-1 knockout mice exhibit a significantly smaller contusion when compared to the wild type mice (p < 0.02). This finding suggests that calpain-1 is a mediator of cell death and the down-regulation of this enzyme following a TBI may result in neuroprotection in vivo.

Tenovuo Olli 1 Menon David 2 Katila Ari 1 Coles Jonathan 2 Frantzén Janek 1 Outtrim Joanna 2

1 Turku University Central Hospital, Turku, Finland 2 University of Cambridge, Cambridge, United Kingdom P123 TBIcare – A New Way to Use Traumatic Brain Injury Biomarkers

BACKGROUND: TBIcare aims to produce individual evidence-based diagnostic and treatment solutions by combining various TBI biomarkers in their wide sense.

MATERIALS AND METHODS: This EU-funded collaborative project analyses large clinical retrospective and prospective TBI databases containing detailed clinical, imaging, and laboratory biomarkers, and outcome data. The information provided by these biomarkers will be analyzed and combined using statistical and system simulation modeling. The available data includes clinical databases from Cambridge, UK and Turku, Finland + the IMPACT database, forming thus a source of tens of thousands subjects with TBI.

RESULTS: This three-year project will create a software tool where individual parameters can be compared to a large modeling database to yield best available assessments about the nature of the TBI, the treatment options available, and the recommended diagnostic markers and tests to improve the reliability of the model for an individual.

CONCLUSION: TBIs are an area where randomized controlled trials are extremely hard to accomplish due to the vast variability of the injuries and injured subjects, and where almost all currently available treatments lack strong scientific evidence. TBIcare is a major and innovative effort to produce evidence-based solutions to an individual level in TBIs, in order to assist clinical decision-making and improve outcome.

TBIcare participants: VTT Technical Research Centre of Finland (coordinator), University of Cambridge, Turku University Central Hospital, Imperial College London, GE Healthcare (UK + Fin), Kaunas University of Technology, Complexio S.a.r.L.

This project is partially funded by the European Commission under the 7^th Framework Programme (FP7-270259 TBI care).

Cope Elise 1 Morris Deborah 1 Vanlandingham Jacob 1 Scrimgeour Angus 2 Levenson Cathy 1

1 Florida Statue University, Tallahassee, FL, USA 2 US Army Reserach Insitute for Environmental Medicince, Natick, MA, USA P124 Zinc Supplementation Improves Resliency to TBI by Reducing Depression and Enhancing Spatial Learning and Memory in a Rat Model

In addition to cognitive deficits, a significant number of TBI patients suffer from depression and depression-related disorders such as anxiety. Given the well-known inverse relationship between serum zinc levels and the severity of depression in uninjured populations, we hypothesized that zinc supplementation could be used to prevent depression-like behaviors in a rat model of TBI. Using controlled cortical impact (CCI) we induced a moderately-severe TBI in adult male Sprague-Dawley rats. We have previously shown that this injury induces both depression- and anxiety-like behaviors. Four weeks of dietary zinc supplementation (180 ppm) prior to the TBI resulted in trends toward reduced anxiety accompanied by significant reductions in post-injury adrenal weights (p < 0.05). Furthermore, zinc supplemented diets significantly reduced anhedonia (p < 0.05) a depression-like behavior, measured by the 2-bottle saccharin preference test, and increased cognitive resilience in the Morris Water Maze test of spatial learning and memory (p < 0.05). A second study was performed to examine the possible efficacy of providing supplemental zinc only after injury. In this work we show that while use of zinc as a treatment for TBI did improve spatial learning and memory (p < 0.05), it was not effective in treating depression and did not improve adrenal weights. These data suggest that chronic zinc supplementation may be a novel and effective strategy for improving cognitive and behavioral resiliency in populations at risk for traumatic brain injury.

Suber John Phipps Helen Levenson Cathy VanLandingham Jacob Florida State University College of Medicine, Tallahassee, FL, USA

P125 Development of a Repetitive Mild Traumatic Brain Injury Model to Test the Treatment Effects of Progesterone

Repetitive mild traumatic brain injury (mTBI) can lead to long term disability. The purpose of this study was to develop an animal model of repetitive mTBI and subsequently test both the prophylactic and acute post-treatment effects of the neurosteroid, progesterone. Male Sprague Dawley rats were placed in one of four groups (n = 6); injury-vehicle, injury-pre-treated progesterone (PROG, 16 mg/kg), injury-post-treated PROG, sham. All injured groups received 3 mTBI, 30 min apart. Pre-treatments were administered 15 min prior to the first impact and post-treatments at 15 min following the third impact. Briefly, the animals were anesthetized and stabilized in prone with a strap across the thoracic spine. A scalp incision was made followed by placement of a 1mm thick rubber disk over the right frontal plate. A metal impactor was activated at a 15° angle, perpendicular to the disk, at a velocity of 6.6 m/s. The impact induced a downward rotation of the head to the left. Behavioral analysis using Morris Water Maze (MWM) testing was then performed on all animals. 3 animals for the injury-vehicle group and shams were evaluated using diffusion tensor imaging (DTI) software with a 21 T magnet at 24h post-injury. At 6 and 24h, animals treated with either pre-or post-PROG had a significant reduction in latency to find the MWM platform on the first and second trial compared to the injury-vehicle group (p < 0.05). These same findings were evident when comparing thigmotactic behavior. DTI analysis showed that our repetitive model of mild TBI causes diffuse axonal injury (DAI). Our findings show that both prophylactic and acute post-injury treatment with PROG reduces learning and memory deficits associated with repetitive mild TBI. Furthermore, this model of mild TBI induces DAI that can be used in future studies to determine the effects of treatments such as PROG on brain morphology.

Washington Patricia M. Parsadanian Maia Hoe Hyang-Sook Turner R. Scott Burns Mark P. Laboratory for Brain Injury and Dementia, Department of Neuroscience, Georgetown University, Washington, DC, USA

P126 Accumulation of β-Amyloid Monomers, Oligomers and Fibrils After TBI in an Alzheimer's Disease Mouse Model

Increased levels of amyloid-β (Aβ) after traumatic brain injury (TBI) have been reported in both humans and animal models of brain trauma. Amyloid plaque formation occurs in approximately 30% of all fatal TBI cases in humans, including a 20% incidence rate in young adults under the age of forty. Plaque formation is reported to happen extremely quickly and studies on surgically excised human TBI brain show that deposition can occur within 24 hours of injury. In the present study we examine the production and accumulation of Aβ40 and Aβ42 after TBI in a mouse model of Alzheimer's disease. Using a range of biochemical and immunohistochemical techniques we measure shifts in Aβ solubility and aggregation, and identify the regions where Aβ is accumulating after TBI. We demonstrate the accumulation of soluble and insoluble Aβ species 24 hours after TBI, and for the first time characterize the presence of the highly-toxic oligomeric-Aβ species after controlled cortical impact in eight-week old transgenic mice. Although these transgenic mice do not usually deposit Aβ plaques until eighteen months of age, we find an increase in both soluble and insoluble Aβ species, with a shift in distribution towards insoluble species, and identify the early deposition of amyloid plaque. Furthermore, using neoepitope specific antibodies, we find that the most intense Aβ40 and Aβ42 immunoreactivity occurs in the ipsilateral corpus callosum between the cortex and hippocampus in areas of diffuse axonal injury. We believe the rapid accumulation, oligomerization and fibrillization of Aβ contributes to the secondary injury cascade after TBI.

Ramesh Sangeetha Sukumari Kimbler Donald Vender John Dhandapani Krishnan Georgia Health Sciences University, Augusta, GA, USA

P127 Suberoylanilide Hydroxamic Acid (SAHA) Attenuates Cerebral Edema Following Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) remains a leading cause of death and disability, posing a significant challenge to health care systems. Although there have been many advances in the care for patients with severe TBI, mortality rates remain at 25-35% with eighty percent of these deaths due to high intracranial pressure. Brain edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP), brain herniation, and a poor clinical prognosis following a TBI. The present study addresses the role of suberoylanilide hydroxamic acid (SAHA), a clinically well tolerated, pan-histone deacetylase inhibitor (HDACi), in the modulation of cerebral edema after TBI. To test this question, adult male CD-1 mice were treated with SAHA and then subjected to controlled cortical impact, which reproducibly induces a moderate brain injury. SAHA (75 mg/kg and 100 mg/kg) significantly attenuated cerebral edema, as determined by the wet-dry method of brain water content, after TBI, as compared to placebo treated TBI mice. In contrast, 50 mg/kg SAHA did not reduce cerebral edema. TBI induced hypoacetylation within the peri-contusional cortex and was normalized to levels observed in sham-operated mice following treatment with SAHA. The reduction in edema was paralleled by a concomitant increase in acetylation status within the peri-contusional cortex. Together, these data suggest HDACi may represent a novel class of drugs to reduce cerebral edema following TBI.

Saez Pedro 1 Wright Matthew 1 McArthur David 2 Alger Jeff 3 Hovda David 2 Vespa Paul 2

1 Psychology Division, Department of Psychiatry, Harbor-University of California, Los Angeles Medical Center, Torrance, CA, USA 2 Department of Neurosurgery, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA 3 Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA P128 Early Metabolic Crisis-Related Brain Atrophy and Neuropsychological Functioning

Recent work by our group has shown that acute metabolic crisis (AMC) following traumatic brain injury (TBI) is associated with greater frontal and temporal lobe atrophy that is associated with poorer neuropsychological outcomes. In the current study we employed positron emission tomography, magnetic resonance imaging, and neuropsychological assessments to determine if this ‘frontal-temporal’ pattern of AMC atrophy confers greater neuropsychological deficits than other patterns of AMC brain atrophy. We compared the neuropsychological performances of 10 TBI participants with AMC-related frontal-temporal atrophy (FTA) to 9 TBI participants with other patterns of AMC-related brain atrophy (OA). The FTA and OA groups were similar in age, education, and injury severity, as indexed by the Glasgow Coma Scale. Nonparametric analyses showed that FTA participants demonstrated greater impairments in attention, executive ability, and psychomotor function at 6 and 12 months post-injury in comparison to OA participants. Furthermore, FTA participants exhibited more clinically significant neuropsychological deficits relative to OA participants. The current study suggests that AMC related brain atrophy in the frontal and temporal lobes may greatly impact outcomes for TBI survivors. Interventions that reduce AMC may lead to improved functional outcomes for TBI survivors.

Wu Hongtao Mahmood Asim Chopp Micheal Henry Ford Hospital, Detroit, MI, USA

P129 mTOR/GSK3β/APC Pathway Mediates the Effect of Simvastatin on Axon Regeneration after Traumatic Brain Injury

OBJECT: The beneficial effects of simvastatin on functional recovery after traumatic brain injury (TBI) have been shown in previous studies. In this study, we investigate the effect of simvastatin on traumatic axonal injury (TAI) and axon regeneration after TBI as well as the underlying mechanism.

METHODS: Adult Wistar rats were subjected to controlled cortical impact (CCI) or sham surgery. Saline or simvastatin (1 mg/kg) was orally administered to rats starting 1 day after TBI and then daily for consecutive 14 days. A modified neurological severity score (mNSS) was performed to evaluate functional recovery. Rats were sacrificed and the brain tissues were harvested at 14 days after TBI. Immunohistochemistry were performed to examine the axonal injury after TBI. Primary cortical neurons (PCNs) were cultured and subjected to oxygen glucose deprivation (OGD), followed by various treatments. Neurite outgrowth length was calculated to evaluate the effect of simvastatin on axon regeneration. Western blot analysis was utilized to investigate the protein expression after simvastatin treatment.

RESULT: Simvastatin significantly decreased the density of APP-positive profiles, while increased the density of Neurofilament H and Bielshowsky silver immunoreactive axons compared to the saline-treated controls. Simvastatin enhanced the functional recovery, which is correlated with the increase of axonal density. In vitro study demonstrated that simvastatin stimulated the neurite outgrowth of PCNs after OGD. This effect was attenuated by LY294002, enhanced by lithium chloride (LiCl) and not affected by cholesterol supplement. Western blot analysis showed that simvastatin activated Akt and mTOR, inactivated GSK3β and promoted the release of APC.

CONCLUSIONS: Our data suggest that simvastatin reduces axonal injury, enhances axon regeneration and promotes neurological functional recovery after TBI. The beneficial effects of simvastatin on axon regeneration may be mediated through manipulation of PI-3K/mTOR/GSK3β/APC pathway in the hypoxic neurons.

Ferreira Luiz Carlos Brasiliano 2 Regner Andrea 1 Simon Daniel 1 Ikuta Nilo 1 da Silva Carvalho Fabiana 3 Mioto Karen Dal Lago 3 Vargas Andréia Escosteguy 4 Ghies José Artur 5

1 Programa de Pós-Graduação em Diagnostico Genético e Molecular,Universidade Luterana do Brasil, Canoas, Rio Grande Do Sul, Brazil 2 Hospital Municipal De Pronto Socorro Dep Nelson Marchezan, Canoas, Rio Grande Do Sul, Brazil 3 Curso De Medicina da Ulbra, Canoas,Rio Grande Do Sul, Brazil 4 Fundação Universitária de Cardiologia FUC, Porto Alegre,Rio Grande Do SUL, Brazil 5 Departamento de Genética UFRGS, Porto Alegre,Rio Grande do Sul, Brazil P130 Increased Levels of Interleukin-6, − 8 and − 10 are Associated to Fatal Outcome in Men with Severe Traumatic Brain Injury

Traumatic brain injury (TBI) is the major cause of death among individuals between 1-45 years-old. The outcome of the TBI patients may be related to both the severity of the primary lesion and the extent of the secondary brain damage. Secondary brain damage is associated with neuroinflammatory phenomena, characterized by activation of microglia and astrocytes, damage to the blood-brain barrier and production of cytokines. Although increased cytokine production and cellular inflammation are clearly involved on the pathophysiology of TBI, no clear relationships have been established between measurements of cytokines and clinical outcome following TBI. The present study was designed to examine which changes in cytokine levels (of IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α) are associated with primary outcome (death or survival) and clinical measures following severe TBI in men. The study group consisted of 24 male patients, victims of severe TBI. Venous blood samples were taken in the Intensive Care Unit (ICU) (study entry), 24 and 48 hours later. The plasma cytokine levels were assayed by flow cytometry. Severe TBI was associated with a 42% mortality rate. TBI patients had significant increase in the levels of all cytokines measured, except for IL-1β, compared to controls. Statistically significant increases in the IL-10, − 8 and − 6 levels were observed in non-survivors TBI patients compared to survivors subgroup measured in the first sample (study entry) and in the subsequent sample (24 hours latter). There were no significant differences in IL-1β, TNF-α and IL-12p70 levels between survivors and non-survivors in any time investigated. Our findings indicate that increased IL-10, − 8 and − 6 levels may constitute an early predictor of unfavorable outcome in severe TBI patients.

Keywords: traumatic brain injury, cytokines, biomarkers, prognosis

McCann Kathleen 1 Walsh Mark 2 Castellino Frank 3 Yount Robert 2 Thomas Scott 2 Evans Edward 2 Howard Jan 2 Ploplis Victoria 3 Donahue Deborah 3 Navari Rudolf 1

1 Indiana University School of Medicine, South Bend, IN, USA 2 Memorial Hospital of South Bend, South Bend, IN, USA 3 W.M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IN, USA P131 Expanding Role of Platelet Mapping in Traumatic Brain Injury

Introduction: Platelet dysfunction, a critical factor in acute coagulopathy in traumatic brain injury (TBI), is often not quantified in routine serum based trauma labs. We purport the expanded use of thromboelastography (TEG©) with Platelet Mapping^™ to study and manage platelet dysfunction in TBI. Here we discuss early findings of our ongoing study, and compare to previously published series.

Methods: We analyzed data collected on all patients admitted to a Level II Trauma Center with TBI since January 2010. Specific parameters considered include (1) TEG results: values for R, K, α angle, MA, and Ly30 (2) platelet mapping results: percentages of arachidonic acid (AA) and ADP inhibition; inhibition of >30% of either was considered abnormal. Overall clinical outcome was measured using Glasgow Outcome Score (GOS); a score of ≤3 was judged “poor”.

Results: TEG studies were normal, and failed to correlate with coagulopathy or poor clinical outcomes in 81% of cases. Platelet mapping results, however, correlated with clinical outcomes in 93%; this variable was independent of GCS, age, co-morbidities, or injury. Platelet mapping was more sensitive than TEG for detecting coagulation abnormalities secondary to anticoagulants. In contrast to previously published series, we found no significant correlation between clinical course and whether a patient had AA inhibition, ADP inhibition, or both. AA inhibition had no correlation to ADP inhibition. The degree of inhibition of AA or ADP was not significant; inhibition of >90% invariably indicated anticoagulant use.

Discussion/Conclusion: The underlying multifactorial pathogenesis of platelet dysfunction in TBI, as measured here by AA and ADP inhibition, is poorly understood. We demonstrate that Platelet Mapping^™ has an expanding role in TBI research as well as management of coagulopathy. It may also have prognostic predictive value. It may have potential to rapidly identify the presence of anticoagulants in acute neurotrauma patients who present with no medical history.

Deng Ping Xu Zao Indiana University School of Medicine, Indianapolis, IN, USA

P132 I _h Contributes to Neuroprotection in the Hippocampus After Controlled Cortical Injury

Traumatic brain injury (TBI) causes selective neuronal damage in the hippocampus. However, the underlying mechanisms are not well understood. Hyperpolarization-activated cation current (I _h) is critical to maintaining neuronal excitability. Posttraumatic alterations of I _h may play important roles in excitotoxicity, and thus contributes to TBI-induced neuronal injury. The present study examined the posttraumatic changes of I _h in hippocampal neurons, and to explore whether I _h is involved in TBI-induced neuronal injury.

Controlled cortical injury (CCI) was performed to produce moderate TBI in male adult SD rats. Whole-cell recordings on hippocampal slices were performed to detect the changes of I _h 24 h after TBI. In some experiments, intraventricular injection was performed 10 min after CCI to deliver drugs to either block or increase I _h. Histological assessment of neuronal injury was performed 7 days after TBI.

Moderate TBI produced neuronal death of CA3 neurons and mossy cells in the hilus, but not CA1 pyramidal cells. The damage of CA3 neurons and mossy cells could be significantly ameliorated with intraventricular injection of lamotrigine, a I_h activator. In contrast, administration of I _h channel blocker (ZD7288) caused obvious cell death in CA1 region after TBI. Whole-cell recordings revealed that, different from CA3 neurons, CA1 pyramidal cells expressed larger I _h and exhibited a posttraumatic increase of I _h amplitude 24 h after TBI. Moreover, blocking I _h led to an increase of input resistance and thus neuronal excitability, with greater effects in posttraumatic CA1 pyramidal cells than that in CA3 neurons. In addition, the I _h in mossy cells was dramatically inhibited early (24 h) after TBI.

TBI causes differential alteration of I _h in hippocampal neurons, which may be one of the mechanisms of selective cell death. Enhancement of functional I _h may protect hippocampal neurons against TBI.

Carre Emilie 1 Duchossoy Yann 2 Lieutaud Thomas 3 Carrillon Romain 3 Ogier Michael 1 Labaune Emmanuelle 3 Tremeau Sebastien 3 Bourdon Lionel 1 Risso Jean-Jacques 1 Bezin Laurent 3

1 Institut de Recherche Biomedicale des Armees, Bretigny-sur-Orge, France 2 MAPREG SAS, Le Kremlin-Bicêtre, France 3 Lyon Neuroscience Center, TIGER team, UMR5292 and U1028, UCB Lyon I, Lyon, France P133 Anxiety Behavior is Differentially Affected in Distinct Models of Brain Trauma

PURPOSE: The present study was aimed at investigating anxiety behavior at mid and longterm in different models of brain trauma in adult rats.

METHODS: After trauma, rats received a daily subcutaneous administration of sesame oil for six days, to model care provided to injured animals and subjected to pharmacological treatments with lipophilic molecules. In the lateral fluid percussion (LFP) (1.8-2.2 atm) and the controlled cortical impact (CCI) models, craniotomy was performed over the hippocampus, at stereotaxic coordinates: − 3.9 from Bregma, + 3.0 from midline (Paxinos and Watson, 1998). Controlled cortical impact (CHI) was induced by dropping a 250 g weight from 200 cm. Sham-operated rats served as controls. During surgery and brain trauma procedures, rats were anesthetized with isoflurane. Anxiety was measured using the Water Exploration Test, 7 and 27 days after injury. Finally, T2 MRI was performed at 28 days to assess brain edema in the different models.

RESULTS: In injured rats treated with sesame oil, anxiety behavior differed according to the model used. In the LFP group, anxiety behavior was reduced at 27 days, with no alteration observed at 7 days. By contrast, after CCI, anxiety behavior transiently increased 7 days post-trauma, control values being recovered by day 27. After CHI, data obtained at 7 days did not differ from controls. MRI data obtained at 28 days will allow us to determine whether the extent of brain edema is associated with anxiety behavior alterations at 27 days.

CONCLUSIONS: In this study, CCI is the only model associated with increased anxiety behavior in the midterm. Intriguingly, anxiety behavior was not increased after LFP, contrasting with prior studies showing a transient increase (days 1 to 9 post-trauma) in non-injected rats. Therefore, the care provided to injured animals may interfere with the pathophysiological mechanisms leading to increased anxiety following brain trauma.

Zhang Feng 1 Feng Mengling 1 Pan Jialin Sinno 1 Loy Liang Yu 1 Guo Wenyuan 1 Zhang Zhuo 1 Guan Cuntai 1 King Nicolas Kon Kam 2 Ang Beng Ti 2

1 Institute for Infocomm Research, Singapore, Singapore 2 National Neuroscience Institute, Singapore, Singapore P134 Short-Term Trend Forecast of Neurophysiological Signals for Medical Decision Support

PURPOSE: Timely treatment is crucial to mitigate the secondary injury caused by intracranial pressure (ICP) elevation to patients with traumatic brain injury. In the current approach, treatment is often applied only after sustained and significant ICP elevation is observed. It is difficult and troublesome for clinicians to conduct timely treatment for patients without information of the future trends in neurophysiological signals. Therefore, we propose an autoregressive moving average based mean forecast algorithm (ARMA-MFA) to predict the short-term future trends in the neurophysiological signals.

METHODS: In our proposed ARMA-MFA algorithm, in order to obtain the features for forecast, the data of a neurophysiological signal is divided into windows. Each past window is further segmented into several finer sub-windows with varying size. Nearer data has a higher resolution than remote ones, because future trends are more likely to be related to the more recent neurophysiological conditions. The ARMA-MFA algorithm predicts the future mean of each sub-window in a future window and then obtains the mean of that future window based on the weighted average of those of the sub-windows.

RESULTS: ARMA-MFA algorithm achieved satisfactory results in forecasting the future mean of ICP, mean arterial pressure, central venous pressure and heart rate. 94%, 89% and 82% of the forecasted ICP mean values are within one sigma interval of the actual values for 5 minutes, 15 minutes and 30 minutes forecast ranges, respectively. The shorter the prediction horizon is, the easier to catch the trends of signals, and therefore the more accurate the forecast is.

CONCLUSION: Our proposed short-term trend forecast method assists clinicians in choosing the optimal medical treatment option. By providing alerts for possible future life threatening conditions identified, it also helps clinicians get better prepared for critical interventions or surgeries, so as to optimize the resource allocation and outcome of patients.

Feng Mengling 1 Loy Liang Yu 1 Zhang Feng 1 Htun Ja Lu 1 Myo Min Tun 1 Lam Jiongle 1 de Rozario Andrew 1 King Nicolas Kon Kam 2 Ang Beng Ti 2

1 Institute for Infocomm Research, Singapore, Singapore 2 National Neuroscience Institute, Singapore, Singapore P135 Artifact Removal from Intracranial Pressure Monitoring Signals: A Solution Based on Robus Statistics & Empirical Mode Decompsition

BACKGROUND & MOTIVATION: Intracranial Pressure (ICP) monitoring signal collected in Neuro Intensive Care Units (NICU) are often contaminated by large amount of artifacts. The artifacts not only directly lead to false alarms in automatic Intracranial Hypertension (IH) alert systems, and they also severely contaminate the characteristics of the underlying signal, which makes accuracy forecasting of impending IH impossible. Therefore, in this paper, we propose a novel solution to effectively remove artifacts from ICP monitoring signals.

MATERIALS & METHOD: ICP monitoring signals of 53 patients, who were admitted between January 2009 to December 2010, are used for the study. ICP levels are invasively measured with a fibre-optic intraparenchymal gauge (Codman and Shurtleff, Taynham, MA). Measured ICP levels were sampled and recorded every 10 seconds with a bedside computer system.

The proposed method effectively detects artifacts by decomposing the ICP monitoring signal with Empirical Mode Decomposition (EMD) method. An iterative filtering method is also proposed to extract artifacts from the decomposed components of ICP signals. The proposed filter is “robust”, i.e. instead of classical statistical tools, parameters of the iterative filter are estimated with “robust statistics”. This ensures the performance of the proposed filter will not be unduly affected by artifacts. The detected artifacts are then imputed based on the Auto-Regressive Moving Average (ARMA) model to preserve the characteristics of the ICP signal.

RESULTS & CONCLUSION: On average, the proposed method achieves 100% precision and 80.2% recall (F-score = 0.88) in identifying the positions of artifacts. In terms of estimating the exact number of artifact data points, the proposed method, on average, achieves 83.2% precision and 97.3% recall (F-score = 0.89). In short, the proposed method is empirically demonstrated to be an effective solution to remove artifacts in ICP monitoring signals.

Lee Eui Jung 1 Shin Sang Do 2 Song Kyoung Jung 3 Park Chang Bae 2 Kim Yu Jin 4

1 Department of Emergency Medicine, JeJu National Univerisity Hospital, Jeju, Republic of Korea 2 Department of Emergency Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea 3 Department of Emergency Medicine, Seoul National University, Boramae Medical Center, Seoul, Republic of Korea 4 Department of Emergency Medicine, Seoul National University, Bundang Hospital, Seongnam, Republic of Korea P136 Sex Differences in Mortality After Moderate to Severe Traumatic Brain Injury

PURPOSE OF STUDY: The objective of this study was to evaluate the association of sex with mortality after moderate to severe traumatic brain injury(TBI).

DESIGN: We collected moderate-severe traumatic brain injury (3 < = GCS ≤ 13) patients aged from a 7 ED based Traumatic Brain Injury Network (2008–2010). Primary outcome was mortality in ED or hospital stay. Logistic regression analysis was used to identify the association between sex and mortalit controlling for age, GCS, brain CT finding, presence of shock (systolic blood pressure < 90mmHg).

RESULTS: Of 20867 TBI patients, 459 patients including severe (GCS ≤ 8, n = 216, 47.1%), and moderate (9 ≤ GCS ≤ 13, n = 243, 52.9%) TBI met inclusion criteria. Among 459 patients, 117(25.5%) were female and 342(74.5%) were male. The mortalities were 57(12.4%). The adjusted ORs of mortality were 1.41 (female, 95% CI 0.71-2.82), 4.79 (severe TBI, 95% CI 2.26-10.11), age (increasing 10 years, 95% CI 1.01-1.05), 9.04 (presence of shock, 95% CI 4.21-19.41), 3.82 (abnormal brain CT, 95% CI 1.42-10.24), respectively.

CONCLUSION: Female sex was not associated with significant higher odds of mortality after moderate to severe TBI.

Duckworth Josh L. 1 Qin Qin 2 Van Zijl Peter 2 Barker Peter 2 Zheng Wei 1 Brawner Terri 2 Burman Hillary 1 Campbell-Malone Regina 1 German Rebecca Z. 1 Stevens Robert D. 1

1 The Johns Hopkins University, Baltimore, MD, USA 2 Kennedy Krieger Institute, F.M. Kirby Center for Functional Brain Imaging, Baltimore, MD, USA P137 Yucatan Miniature Swine Imaging Model of Non-Impact Blast Traumatic Brain Injury (PreVENT II)

INTRODUCTION: The incidence of blast induced traumatic brain injury (bTBI) has increased in both military and civilian populations underscoring the need for models to enhance understanding of the pathophysiology associated with primary blast. Limitations of rodent-based bTBI models may be in part overcome in larger animal models. Here, we demonstrate feasibility of combined high resolution, multi-parametric, magnetic resonance imaging (MRI), and magnetic resonance spectroscopy imaging (MRSI) in a Yucatan miniature swine model of non-impact bTBI.

METHODS: 6 month old, 30-35kg, male, Yucatan miniature swine were allocated to sham and blast exposed groups. They were then imaged using a Philips Actieva, 7-Tesla, MRI research system at both pre-blast and at prescribed intervals following blast exposure. Multi-modal MRI and MRSI protocols were implemented to evaluate parenchymal ischemic or hemorrhagic changes, alterations in perfusion, and alterations in neurochemistry.

All blast exposures, MRI scans, transport, and perfusions were conducted under general anesthesia (isoflurane). Animals were intubated and continuous monitoring of blood pressure, heart rate, respiratory rate, pulse oximetry, end-tidal CO2 was performed throughout to minimize effects of secondary injury, such as hypoxia and hypotension.

RESULTS: T1, T2, FLAIR, DWI/ADC, SWI, DTI, MRA, MRSI, and MR perfusion data for 4 control and 2 blast exposed animals have been obtained. This data allows for evaluation of blood brain barrier disruption, qualitative changes in cerebral blood flow, parenchymal lesions and disruption of white matter fiber tracts, evidence of vascular injury, and changes in neuro-metabolite concentrations.

CONCLUSION: We demonstrate feasibility of serial high resolution multi-parametric MRI and MRSI imaging in a juvenile Yucatan miniature swine model of bTBI. As part of the multi-center PreVENT II study, MR data will be integrated with neurocognitive/behavioral testing (see abstract by Campbell-Malone et al.), CSF and blood proteomic changes, and neuropathology.

Robertson Courtney 1 Scafidi Susanna 2 Saraswati Manda 1 McKenna Mary 2

1 Johns Hopkins School of Meicine, Baltimore, MD, USA 2 University of Maryland School of Medicine, Baltimore, MD, USA P138 Determination of Metabolic Alterations After Traumatic Brain Injury in Immature Rats Using Proton Spectrocscopy

INTRODUCTION: Studies have demonstrated alterations in cerebral metabolism after traumatic brain injury (TBI). However, information on the timeline of alterations in the developing brain after TBI is limited. Using ex-vivo proton (¹H) nuclear magnetic resonance (NMR), we previously reported reductions in N-acetylaspartate (NAA) and increases in lactate that began early after injury (4h) in a pediatric rat model. We have investigated other key metabolites from 4h to 1 week after TBI.

METHODS: Immature rats (PND 16-17, n = 5-8/group) underwent controlled cortical impact (CCI) to the left parietal cortex. Sham rats (n = 6/group) underwent cranietomy only. Brains were removed at 4h, 24h and 7d after CCI, separated into ipsilateral (injured) and contralateral sides, and frozen. Samples were prepared with perchloric acid extraction, using TMSP as an internal standard. Spectra were obtained using a Varian Inova 500 MHz spectrometer. The total concentrations of NAA, lactate, alanine, acetate, GABA, glutamate, succinate, glutamine, aspartate, creatine, choline, taurine and myoinositol were determined.

RESULTS: In sham rats there was no difference between sides in any metabolite at any time. At 4 hrs after CCI, alanine was increased and creatine was decreased ipsilaterally (p < 0.05 vs. contra, paired t-test). At 4h and 24h after CCI there was a trend towards decreased NAA ipsilaterally (p = 0.06). There were no inter-hemispheric differences in lactate, but lactate was higher bilaterally in CCI (∼150 vs. ∼115 nmol/mg in sham). At 7d, NAA, glutamate and taurine were decreased ipsilaterally, whereas glutamine and choline were increased (p < 0.05 vs. contra).

CONCLUSION: Reductions in NAA and glutamate suggest decreased neuronal metabolism and/or cell loss, whereas increased glutamine reflects increased glial metabolism. Increased choline can reflect increased glial metabolism and/or gliosis. The use of ¹H spectra to evaluate the characteristics of TBI-mediated alterations could target metabolic rescue following TBI in children.

Support NIH K08NS42805 (CLR) and NIH P01HD016596 (MCM)

Campbell-Malone Regina 1 Zheng Wei 2 Duckworth Josh L. 2 Qin Qin 3 Burman Hillary 2 German Rebecca Z. 1 Stevens Robert D. 2

1 Johns Hopkins School of Medicine, Department of Physical Medicine and Rehabilitation, Baltimore, MD, USA 2 Johns Hopkins School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, MD, USA 3 Kennedy Krieger Institute, F.M. Kirby Center for Functional Brain Imaging, Baltimore, MD, USA P139 Assessing the Neurobehavioral and Cognitive Effects of Traumatic Brain Injury in a Juvenile Mini-PIG Model System

INTRODUCTION: Small animal models of traumatic brain injury (TBI) have provided limited evidence of correlation between post-injury neurological deficits and spatially-localized neuropathology. We have developed a thirteen-point neurobehavioral assessment tool to determine how the behavior of blast-exposed Yucatan miniature swine compares to that of control animals.

METHODS: We constructed quantitative ethograms based on continuous digital video and audio recordings of animal behavior at prescribed intervals. Data taken every minute in a 15 minute recording included measures of (1) motility, (2) body position, (3) body posture, (4) head position, (5) activity (6) respiration and (7) vocalization. Additionally, the tool included continuous counts of (8) vertical exploration behavior and (9) the number and duration of drinking events at an ad libitum water source, as well as cognitive assessments including, (10) distractibility, (11) awareness, and (12) spontaneous object recognition (non-spatial memory assessment). The final measurement was (13) the presence or absence of the head shake reflex.

RESULTS: This protocol has been evaluated in 2 test specimens and preliminary data have been collected for 2 control and 2 experimental pigs. This protocol will permit the evaluation of the neurobehavioral and cognitive effects of moderate-to-severe TBI.

CONCLUSION: This assessment tool will allow us to link neurological deficits to neuropathology when used in conjunction with magnetic resonance imaging and magnetic resonance spectroscopic imaging of the same subjects (see abstract by Duckworth et al.). Such correlations in animal models will provide powerful insights into brain-behavior relationships of moderate-to-severe traumatic brain injury in human subjects.

Plowman R. Scooter 1 Harris Janna L. 1 Craciunas Sorin C. 2 Reintjes Stephen 1 Brooks William M. 1

1 Hoglund Brain Imaging Center, Kansas University Medical Center, Kansas City, Kansas, USA 2 Department of Neurosurgery, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania P140 Durotomy: The Effects of Breaching the Dura on Behavioral Recovery, Lesion Volume, and Cellular Morphology Following Acute TBI

Traumatic brain injury is a leading cause of death and disability among people ages 15–40 and the primary cause of coma. Since elevated intra-cranial pressure (ICP) is generally considered to contribute to morbidity and mortality, decompressive craniectomy (DC) is often regarded as a neurosurgical option to reduce secondary injury. However, although the question of whether to rupture the dura is still open, few preclinical studies have investigated the effects of durotomy following craniectomy on TBI recovery. We examined whether a breached dura in a rat model of controlled cortical injury (CCI) altered behavioral recovery, lesion volume using MRI, and neuronal survival using histology. We compared the outcome in male Fisher rats with breached (n = 9) vs. intact (n = 9) dura following a unilateral CCI to the sensorimotor cortex. The CCI (tip = 5mm, velocity = 3.5m/s, depth = 2mm, contact time = 300ms) was performed following a 6mm craniectomy. The bone flap was not replaced in either group. Animals were placed into two groups based on whether or not incidental dural rupture occurred during CCI as observed by the surgeon immediately following the injury. Prior to the injury the animals underwent behavioral training and a baseline MRI scan. Rotarod (p < 0.01) and beamwalk (p < 0.05) performance at 1, 3, 7, and 14 days post-injury demonstrated less sensorimotor function loss and improved recovery in breached rats. Lesion volumes measured from hyperintensity on T₂-weighted MRI at 14 and 28 days post-injury were not different between groups (p > 0.05). Neurodegeneration assessed by thionin and H&E staining indicated greater perilesional neuronal survival in the breached cohort. These results suggest that decompressive durotomy following CCI elicits improved functional recovery and neuronal preservation in rats. However, lesion volume does not appear to be altered as a result of dural breach. Likely explanations include lower ICP and improved intracranial circulation, as well as increased neuronal plasticity and less axonal degeneration.

Nekludov Michael Mobarrez Fariborz Bellander Bo-Michael Wallen Håkan Karolinska Institute, Stockholm, Sweden

P141 Formation of Microparticles in Traumatic Brain Injury

PURPOSE: Cells may release microparticles (MPs) upon cellular activation and cell-death. MPs may be involved in coagulopathy, a common finding and risk factor for adverse events in traumatic brain injury (TBI). We assessed MPs in 15 patients with severe isolated TBI.

METHODS: Blood was collected repeatedly from an arterial line and from a jugular bulb catheter at admittance, 6h, 12h, 24h, 48h and 72h after the injury. Microparticles (MPs) were measured by flow cytometry, and platelet function by whole blood impedance aggregometry (Multiplate^™) or modified thromboelastography (TEG-PM). MPs were identified by size and binding of fluorescently labelled lactadherin (phosphatidylserine expression) and labelled antibodies towards CD144 (endothelial-MPs) or CD42a (platelets-MPs).

RESULTS: Plasma concentration of endothelial-MPs (CD144+), and platelet-MPs (CD42a+) were higher in TBI patients, compared to healthy controls (3.5x10⁶ vs 0.6x10⁶ /L, and 13.5x10⁶ vs 9.9x10⁶/L, respectively. p < 0.001 for both). All MPs co-varied with brain damage marker S100b (r = 0.48, p < 0.01 for lactadherin + MPs; r = 0.65, p < 0.01 for CD42a + and r = 0.28, p = 0.05 for CD144+). Comparing arterial and cerebrovenous samples we found a significant transcranial gradient for lactadherin + MPs and CD42a + MPs (p < 0.05) in early samples, whereas for endothelial MPs (CD144+) gradient was only marginally significant (p = 0.06). There were no differences in platelet function in arterial vs cerebrovenous samples, as measured by either TEG-PM or Multiplate^™.

CONCLUSIONS: Early after TBI there is an increased formation of circulating platelet- and endothelial-derived microparticles over the brain. The relationship between microparticles and S100b suggest that this is due to damage of brain tissue. Later on, the number of MPs declines in cerebrovenous blood, but remains unchanged or increases slightly in arterial samples. Sepsis or SIRS may be a cause of increased MP formation later on in the course of trauma.

Thelin Eric 1 Johannesson Louise 2 Bellander Bo-Michael 1

1 Karolinska Institute, Department of Clinical Neuroscience, Stockholm, Sweden 2 Stockholm University, Department of Economics, Stockholm, Sweden P142 The Sensitivity and Role of Protein S100B in Detecting Secondary Injuries After Traumatic Brain Injury in Humans

BACKGROUND: Patients suffering from traumatic brain injury (TBI) are often treated in specialized neuro-intensive care units (NICU) using multi-modal monitoring to detect harmful secondary insults such as increased intra cranial pressure. In addition to different monitoring devices, serum biomarkers have been shown to provide additional important information regarding the patient. Elevated serum levels of S100B have been detected following TBI, cerebral ischemia, spontaneous intra-cerebral hematomas and edema formation. S100B is known to correlate to outcome. Severe secondary cerebral injuries have been shown to correlate to secondary peaks in serum levels of S100B. Increases of more than 0.1μg/L are considered pathological.

METHOD: 267 patients treated in the NICU for TBI with S100B samples obtained every 12 hours during a minimum of a 96-hour time period were included. Secondary increases of S100B after 48 hours following trauma were noted. All patients had at least 2 CT-scans and/or MRI scans performed.

RESULTS: 67 secondary injuries during the NICU stay were detected using MRI or CT-scans. The most common lesions were diffuse ischemic injuries (29), edema (13), cerebral infarctions (11) and intra-cerebral hematoma (5). Looking at secondary peaks of S100B in detecting radiological verifiable cerebral lesions during the NICU stay, a cut-off level of more than 0.5μg/L the specificity was 100% and sensitivity 8.9%. Decreasing the cut-off level to 0.1μg/L a specificity of 95.9% and sensitivity of 64.2% was obtained, while a cut-off level of 0.05μg/L presented a specificity of 92.7% and sensitivity of 92.5%.

CONCLUSIONS: S100B is a sensitive marker for secondary cerebral injuries occurring in the NICU after TBI. A low cut-off point for the secondary peak of S100B (0.05 μg/L) increases sensitivity without any major deficit of specificity in detecting secondary injuries during the NICU stay.

Shikuev Alex 1 Bagumyan Arthur 2 Danilenko Uliana 2 Dambinova Svetlana 1

1 Kennesaw State University, Kennesaw, GA, USA 2 GRACE Laboratories, LLC, Decatur, GA, USA P143 Ampar Peptide Assay Differentiates Mild TBI from TIA

BACKGROUND & PURPOSE: A large majority of mild TBI patients, both symptomatic and asymptomatic have normal CT and MRI scans. A variety of potential biomarkers of brain damage from TBI have been investigated. However, no single brain-specific biomarker has yet been unanimously established for traumatic brain injury in the routine clinical practice. We aimed to assess AMPA receptor (AMPAR) peptide biomarker in patients with mild TBI and healthy controls.

METHODS: Venous blood samples were collected from patients with symptoms suggestive of mTBI (n = 61) from 9 hours to 5 days from injury, age-/gender matched group of patients with TIA (n = 11), and healthy volunteers (n = 20). AMPAR peptide was measured in plasma samples using MP-ELISA assay. Neurological examination and CT scans were performed for all patients with mild TBI.

RESULTS: All patients with mild TBI had normal CT scans and diagnosis was based on clinical symptoms, history of head injury, and Glasgow Coma Scale (GCS). AMPAR peptide levels (range 1.3-1.8 ng/ml) were significantly higher in mTBI group (p < 0.01) than in patients with TIA and control group (range 0.10-0.25 ng/ml). In plasma samples from mTBI patients (GCS 13-15) drastically elevated AMPAR peptide concentrations were detected within 24 h of injury gradually declining to control level by 72 h.

CONCLUSION: In conjunction with neurologic assessment and neuropsychological testing AMPAR peptide assay has the potential to assess mTBI patients vs TIA and healthy controls. The highest levels of AMPAR peptide within 24 h of brain injury have been registered.

Yutthakasemsunt Surakrant 2 Kittiwattanagul Warawut 1 Piyavechvirat Parnumas 1 Phuenpathom Nakornchai 3 Thinkhamrop Bandit 2 Lumbiganon Pisake 2

1 Khon Kaen Regional Hospital, Khon Kaen, Thailand 2 Khon Kaen University, Khon Kaen, Thailand 3 Prince of Songkla University, Songkla, Thailand P144 Tranexamic Acid for Patients with Traumatic Brain Injury: A Randomized, Double-Blinded, Placebo-Controlled Trial

BACKGROUND: Traumatic brain injury (TBI) is commonly accompanied by intracranial bleeding which can worsen after hospital admission. Tranexamic acid (TXA) has been shown to reduce bleeding in elective surgery and TXA could reduce re-bleeding in spontaneous intracranial haemorrhage. We aimed to determine the effectiveness and safety of TXA in preventing progressive intracranial haemorrhage in patients with TBI.

METHODS: We enrolled 238 patients older than 16 years with moderate to severe TBI (post-resuscitation Glasgow Coma Scale (GCS) 4 to 12) who had a Computerized Tomography (CT) brain scan within eight hours of injury and there was no immediate indication for surgery. We excluded patients if they had a coagulopathy or a serum creatinine over 176•8 micromole/litre. The treatment was a single dose of 2 grams of TXA in addition to other standard treatments. The primary outcome was progressive intracranial hemorrhage (PIH) which was defined as an intracranial hemorrhage seen on the second CT scan that was not seen on the first CT scan, or an intracranial hemorrhage seen on the first scan that had expanded by 25% or more on any dimension (height, length, or width) on the second scan.

FINDINGS: PIH was present in 16 (14%) of 115 patients allocated to TXA and in 28 (25%) of 114 patients allocated to placebo [Relative risk (RR) = 0•57 (95%CI 0•32 to 0•99)]. The RR of death from all causes and the RR for the unfavourable Glasgow Outcome Scale allocated to TXA compared with placebo were 0•69 (95% CI 0•35 to 1•39) and 0•76 (95% CI 0•46 to 1•27) respectively. There was no evidence of increased risk of thromboembolic events in those allocated to TXA.

INTERPRETATION: TXA may reduce PIH in patients with TBI. Large clinical trials are needed to assess the effect of TXA on death and disability after TBI.

Kokiko-Cochran Olga Bhaskar Kiran Lee Yu-Shang Lamb Bruce Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

P145 Traumatic Brain Injury Induces TAU Phosphorylation in a Mouse Model of Alzheimer's Disease

Epidemiological evidence suggests that traumatic brain injury (TBI) is a major risk factor for many neurodegenerative diseases, including Alzheimer's disease (AD). Many patients suffering from moderate head trauma display several neuropathological features of AD, including: extracellular deposition of beta-amyloid peptide in senile plaques, intracellular phosphorylation and aggregation of microtubule associated protein tau in neurofibrillary tangles, and marked activation of astrocytes and microglia. We hypothesize that TBI induces activation of brain microglia and factors secreted from these activated microglia in combination with the amyloidogenic processing of amyloid precursor protein subsequently induce tau phosphorylation and aggregation and neuronal dysfunction. To test this hypothesis, two month old R1.40 and C57BL/6J mice were subjected to moderate lateral fluid percussion or sham brain injury followed by biochemical and neuropathological analysis at three days postinjury. Histological analysis revealed a focal cortical contusion in brain injured mice. Activated microglia and astrocytes were observed around the injury cavity and in subcortical structures including the hippocampus and thalamus. Western blot analysis revealed that TBI induced tau hyperphosphorylation at the AT8 epitope (Ser202) in the ipsilateral cortex of brain injured mice. Tau hyperphosphorylation was enhanced in brain injured R1.40 mice compared to control mice. Neuropathological analysis confirmed there was marked elevation of AT8 immunoreactivity at the site of injury in brain injured mice. Notably, a more detailed analysis revealed an increase in AT8 + dystrophic neurites and cellular AT8 immunoreactivity in a subset of cortical neurons in brain injured R1.40 mice compared to controls. These studies show that TBI facilitates microglial activation and induces acute tau phosphorylation in a transgenic mouse model of AD. Our results suggest that one mechanism which TBI may contribute to AD is via the induction of tau hyperphosphorylation and is consistent with the recent observation that TBI can lead to traumatic encephalopathy, where tau pathology predominates.

Olson Brenda Bartnik 1 Kroh Julia 2 Ropacki Susan 2 Tong Karen 1 Ashwal Stephen 3 Wong Valarie 4 Holshouser Barbara 1

1 Department of Radiology, Loma Linda University, Loma Linda, CA, USA 2 Department of Psychology, Loma Linda University, Loma Linda, CA, USA 3 Department of Pediatric Neurology, Loma Linda University, Loma Linda, CA, USA 4 Redlands Pediatric and Adult Medicine, Redlands, CA, USA P146 Adolescents with Post-Concussive Symptoms Following Mild TBI Show Lower Subcortical NAA/CR if Studied Within 30 Days of Injury

Recent studies have shown metabolite changes in subjects with adult mild TBI. This study was designed to study regional cerebral metabolite changes in a pediatric population after mild TBI who have post concussive symptoms. Fourteen adolescent mTBI subjects (13.9 ± 2.9 yrs) and 9 normal control subjects (14.3 ± 2.9 yrs) were studied. mTBI subjects had MRI (T2, T1, FLAIR, SWI) and 3D MRSI (PRESS TR/TE = 1700/144 ms) imaging 6 - 515 days post-injury, using a Siemens Tim Trio 3T scanner. LCmodel was used to obtain semi-quantitative metabolite ratios. In-house custom designed software was used to obtain tissue composition and metabolic information from the same anatomical position. Voxels were grouped into gray matter (GM), white matter (WM) or subcortical nuclei (basal ganglia and thalami; SCN) regions. Statistical differences were determined using an independent samples Kruskal-Wallis test where p < 0.05 was considered significant. NAA/Cr was lower (1.83 ± 0.07) in the SCN of mTBI subjects studied ≤ 30 days of injury (n = 4; p = 0.02), compared to controls (2.09 ± 0.20), with 73% of subjects having > 20% of voxels with decreased NAA/Cr. There were no differences in GM or WM metabolite ratios between the mTBI group imaged within 30 days of imaging and mTBI subjects imaged > 30 days post-injury or controls. These preliminary findings show lower NAA in the SCN of adolescent mild TBI subjects suggesting an early alteration of neuronal metabolism since these changes were detected only in subjects who were studied within 30 days of injury. It is possible that these early alterations in neuronal metabolism may play a role in persistent memory and attention deficits; however, further studies are needed.

Coats Jacqueline Castello Michael Obenaus Andre Huang Lei Loma Linda University, Loma Linda, CA, USA

P147 Cumulative White Matter Injury After Repeated Mild TBI

PURPOSE: Disruption of white matter integrity has been associated with latent cognitive disturbances after mild traumatic brain injury (mTBI). Experimental and clinical evidence shows that repeated mTBI worsens cognitive outcomes. In this study we modeled repeated mTBI in adult rats and evaluated the changes in the corpus callosum (CC) using diffusion tensor imaging (DTI).

METHODS: Mild TBI was induced via controlled cortical impact (CCI) (speed of 6.0m/s, depth of 0.5mm) in adult male rats. Animals in the repeated mTBI group had a second CCI at the same site 1, 3 or 7 days after the initial CCI. Shams had no CCI. DTI data were acquired 14 days after the first mTBI. Axial, radial and mean diffusivity and relative anisotropy data were extracted from the CC at the level of the CCI. DTI measurements were not statistically different between the 1, 3 or 7 day repeated mTBI groups, so data were pooled into one repeated mTBI group.

RESULTS: A single mTBI resulted in significantly higher axial and mean diffusivity compared to Shams. Conversely, axial and mean diffusivities were significantly reduced in the repeated mTBI group compared to Shams. Relative anisotropy and radial diffusivity in both the Single and Repeated mTBI groups were not statistically different from Shams.

CONCLUSIONS: DTI allows quantification of white matter neuropathology associated with mTBI. The increased axial and mean diffusivity in the single mTBI group is consistent with chronic axonal damage. The reductions in axial and mean diffusivity in the repeated mTBI group represent acutely injured white matter. Fourteen days after the first mTBI myelin appears preserved in both the single and repeated mTBI groups. These results suggest that a second mTBI extends the acute injury phase compared to a single mTBI.

Huang Lei 1 Coats Jacqueline 1 Mohd-Yusof Alena 2 Obenaus Andre 3

1 Department of Biophysics and Bioengineering, Loma Linda University, Loma Linda, CA, USA 2 California State University, San Bernardino, CA, USA 3 Department of Biophysics and Bioengineering, Radiation Medicine, Radiology, Loma Linda, Loma Linda, CA, USA P148 Hyperbaric Oxygen Intervention Improves the Neuroimaging Outcomes in a Rat Model of Repetitive Mild Traumatic Brain Injury

PURPOSE: Repetitive mild traumatic brain injury (rmTBI) is an important public health concern as subsequent injuries are thought to exacerbate existing neuropathology. The pathophysiological processes underlying mTBI likely involve cellular metabolic perturbations in the injured brain and a neuroprotective approach favoring cerebral aerobic metabolism could be beneficial. Hyperbaric oxygen (HBO) fulfills this requirement and has been explored as a novel therapeutic approach for management of TBI. In this study, we investigated both prophylactic and therapeutic HBO strategies in a rat model of rmTBI.

METHODS: Sprague Dawley adult male rats were randomized into six groups: Sham, 3d and 7d rmTBI with or without HBO. A mild controlled cortical impact (0.5mm depth) was delivered to the parietal cortex. For animals receiving rmTBI, a second CCI was delivered at the same location 3 or 7 days later. Shams had no CCI. HBO (100% oxygen at 2 ATA) was given 1hr daily for 3 consecutive days either prior to or 24 hrs post the initial TBI. T2 weighted imaging (T2WI) and susceptibility weighted imaging (SWI) were acquired and lesion and hemorrhage volumes were quantified..

RESULTS: Both HBO pre-treatment and post-treatment improved neuroimaging outcomes following rmTBI in contrast to those seen in tissues without HBO intervention. There were significant reductions in the T2WI-dervided lesion and SWI-identified hemorrhage volumes at 24 hrs post rmTBI. The most dramatic HBO treatment effects were observed in animals receiving rmTBI 3d apart where there was a 3-fold decrease hemorrhage volumes back to Sham levels.

CONCLUSIONS: Both prophylactic and therapeutic HBO provides a potential neuroprotective strategy that can be applied to victims at high risk for repetitive mTBI. MRI is a sensitive neuroimaging biomarker for monitoring experimental and clinical treatment effects within the setting of rmTBI.

Thompson Floyd 1 2 3 Nelson Rachel 1 Hou Jiamei 2 Parmer Ron 2 Williams Kelli 2 Sennhauser Susie 2 Nissim Nicole 2 Jaiswal Poonam 2 Keener Jonathon 2 Bose Prodip 1 2 4

1 North Florida/South Georgia Veterans Health System, Gainesville, FL, USA 2 Department of Physiological Sciences at the University of Florida, Gainesville, FL, USA 3 Department of Neuroscience at the University of Florida, Gainesville, FL, USA 4 Department of Neurology the University of Florida, Gainesville, FL, USA P149 Experimental Model of Closed Head TBI (CH-TBI) to Study the Neurobiology and Pathophysiology of Long Term Disabilities: Spasticity, Cognitive, Balance, Anxiety, and Pain Related Behaviors

CH-TBI is a common form of head injury in every day life and particularly on the military battlefield. CH-TBI results in a broad range of life-long disabilities. In addition to cognitive recovery, motor (spasticity, and gait), pain and anxiety disorders are high incident disabilities that produce lingering barriers for re-entry of a TBI patient into active service or the work place. Currently, there is no clear understanding of the neurobiology of spasticity, pain, and anxiety disabilities induced by TBI, and effective treatments are awaiting this information. We report findings in a rat model of mild CH-TBI (450g × 1.25m, Marmarou Model) performed to assess alterations in stretch reflex (spasticity) and other comorbidities: velocity dependent ankle torque & ankle extensor muscle EMGs (for spasticity), 3D kinematic & footprints (for gait), rotorod (for balance performance), Morris water maze (MWM) spatial learning (for cognition), elevated plus maze (for anxiety), thermal test (for pain) tested before and after injury. The TBI animals developed an enduring spasticity (a significant elevated ankle torques and corresponding EMGs) and revealed impaired gait parameters (e.g. deterioration of step sequences and increased joint angles in 3-D footprints and kinematics, respectively). The TBI animals also showed significant disorder of cognitive and balance performance, and showed a significant reduction in motor evoked potential amplitude, hyperesthetic responses to thermal stimuli (number of leak/guard of the hind-limb plantar surfaces), decreased rearing, and decreased open arm exploration in an elevated plus maze. These findings were correlated with immunohistological changes in Locus Coeruleus cells and projection fibers to key neural substrates of motor, cognitive, anxiety (amygdala) and somesthesis (spinal cord). Collectively, these studies represent the first model of TBI-induced multiple morbidity in which the neurobiology and pathophysiology of these long term disabilities can be studied.

Supported by Veterans Affairs RR&D Merit Review B5037R and B6570R.

Garber Kent Zhu Xiaoxia Park Juyeon Qiu Jianhua Whalen Michael Massachusetts General Hospital, Boston, MA, USA

P150 Plasmalemma Permeability and Regulation of Monocyte Inflammation by CX3CR1 After Intracerebral Hemorrhage

PURPOSE: Intracerebral hemorrhage induces apoptotic cell death programs as well as influx of blood monocytes into injured brain, however the contribution of necrosis to cell type-specific injury and death after ICH remains unknown. We tested the hypothesis that necrotic-like cell death occurs in neurons and other cell types after collagenase intracerebral hemorrhage in mice.

METHODS : In vivo propidium iodide (PI) was used to assess plasmalemma damage. Ex vivo caspase substrates, TUNEL, and electron microscopy (EM) were used to assess the relationship between plasmalemma permeability and mode of cell death. Cell types were determined by immunohistochemistry. CX3CR1 heterozygous and knockout (KO) mice expressing GFP in monocytes/microglia were used to examine trafficking of blood monocytes and brain microglia.

RESULTS : Plasmalemma permeability peaked at 24-48 h (p < 0.05 ANOVA). At 6 and 24 h, permeable cells were neurons and IBA-1-positive or GFP + cells (in CX3CR1 + /- mice) with morphological features of monocytes but not resident microglia and astrocytes. Monocyte influx into hemorrhagic brain was markedly reduced in CX3CR1-/- mice. At 48 h, greater than half of injured cells had a pure necrotic phenotype (PI + /DEVD- or PI + /TUNEL-) and less than 5% had a pure apoptotic phenotype (PI-/TUNEL + or PI-/DEVD+)(p < 0.05 ANOVA). Ultrastructural features of necrosis were also confirmed by EM. Permeabilized cells remained in brain for at least 24 h, with less than 10% undergoing spontaneous resealing. CX3CR1 -/- mice had decreased Fluoro-jade B stained cells at 48 h, but increased motor deficits vs. wild type after ICH (p < 0.05).

CONCLUSIONS: Delayed necrosis with concomitant activation of apoptotic programs contributes to cell demise after ICH. CX3CR1 recruits monocytes into hemorrhagic brain and contributes to neuronal cell death and motor recovery after ICH. Monocytes are a main source of permeabilized cells early after ICH whereas resident microglia and astrocytes are resistant. Programmed necrosis and plasmalemma damage may represent novel therapeutic targets to prevent cell death or rescue injured cells after ICH.

Dapul Heda Park Juyeon Whalen Michael Massachusetts General Hospital, Boston, MA, USA

P151 Development of a Combined Concussion/Contusion Model of Traumatic Brain Injury in Mice

PURPOSE: TNF/Fas pathways are activated in brain after controlled cortical impact (CCI) and closed head (concussive) injury (CHI, a non-cell death model) in mice. However, genetic inhibition of TNF/Fas prior to CCI improves postinjury cognitive function whereas inhibition of either of these pathways in CHI leads to worse cognitive outcome (p < 0.05 for group, Morris water maze performance, TNF/Fas knockout mice vs. WT in CCI and CHI, RM ANOVA). Thus, therapies based on TNF/Fas pathways may improve or exacerbate cognitive deficits in patients with mixed injury types depending on whether contusion or concussion injury predominates. Using a new model of combined CCI/CHI, we tested the hypothesis that TNF/Fas knockout mice would have worse functional outcome vs. wild type.

METHODS: Adult C57/BL6 mice were subjected to CHI, CCI, CHI + CCI, or combined sham injuries. Postinjury motor function was assessed by wire grip test, cognitive function by the Morris water maze (MWM), and lesion volume by image analysis. Data were analyzed by ANOVA (lesion size) or RM ANOVA (behavioral data) followed by the appropriate post hoc tests.

RESULTS: Compared to CCI or CHI alone, combined injury produced increased motor deficits on postinjury day 1, a tendency toward increased MWM deficits, and significantly greater lesion volume (p < 0.05 ANOVA). In wild type mice, MWM deficits occurred similarly whether CCI preceded or followed CHI. Mice deficient in TNF/Fas had MWM performance in the combined model similar to that in CCI, not CHI.

CONCLUSIONS: Concussive and contusion TBI may activate similar pathways with opposite effects on functional outcome. Development of a combined injury mouse TBI model may help predict effects of novel therapeutics in human TBI in which concussive and contusion brain injuries co-exist. Effects of TNF/Fas inhibition in the combined injury model may be predicted by the CCI model alone.

Zhang Jimmy Zhu Xiaoxia Park Juyeon Qiu Jianhua Massachusetts General Hospital, Boston, MA, USA

P152 Divergent Function of AKT and mTOR Signaling in Contusion Versus Concussion Models of Traumatic Brain Injury in Mice

PURPOSE: Necrostatin-1 is a specific inhibitor of RIPK1, a mediator of programmed necrosis. Akt and mTOR mediate protective responses to cellular stress and are activated after traumatic brain injury (TBI). We tested the hypothesis that dual inhibition of Akt/mTOR would exacerbate postinjury cell death and cognitive function in two TBI models.

METHODS: Adult C57/BL6 mice were subjected to controlled cortical impact (CCI) or closed head concussive injury (CHI). Protein phosphorylation was assessed by Western blot, cell death by in vivo propidium iodide, and functional outcome by wire grip test and Morris water maze (MWM). Data were analyzed by rank sum or t-test (PI + cells, densitometry) or RM ANOVA (behavioral data).

RESULTS: Necrostatin-1 improved MWM performance after CCI and CHI (p < 0.05 for each). Phosphorylation of proteins downstream of Akt and mTOR was increased in cortex/hippocampus in both models, albeit with different kinetics (p < 0.05 vs. sham for all). Akt and mTOR activity was regulated by TNF/Fas, RIPK3, and RIPK1 in CHI. Administration of Akt inhibitor and rapamycin (mTOR inhibitor) prior to TBI reduced phosphorylation of downstream proteins in both models (p < 0.05 vs. vehicle), reduced PI + cells in CA1 and CA3 after CCI (p < 0.01 vs. vehicle) and improved motor and MWM performance in CCI (p < 0.05) but worsened MWM performance in CHI (p < 0.05). Necrostatin-1 improved MWM performance in both models (p < 0.05) and increased phosphorylation of Akt-473 and S6RP in CHI- these effects of necrostatin-1 in CHI were abolished by co-administration of Akt inhibitor or rapamycin.

CONCLUSIONS: Necrostatin-1 improves cognitive outcome after CHI via Akt/mTOR signaling. Akt/mTOR is beneficial in CHI but detrimental in CCI. The data suggest that inhibition of RIPK1 is broadly protective but that inhibition of Akt/mTOR signaling is a rational therapy for contusion but not concussion TBI.

Aygok Gunes 1 Marmarou Anthony 1 Ward John 1 Bullock Ross 2

1 Medical College of Virginia, Richmond, VA, USA 2 University of Miami, Miami, FL, USA P153 A Multicenter Observational Study to Evaluate Dynamics of Endothelin in Cerebrospinal Fluid and Plasma in Moderate and Severe Human Head Injury

INTRODUCTION: The need for a pharmaceutical agent to blunt the cascade of neurotoxins released with TBI is critically important. The purpose of this observational study was to document the levels of one of these neurotoxins, endothelin in cerebrospinal fluid (CSF) and plasma of TBI patients.

METHODS: Subjects who had severe or moderate TBI together with lesions on CT scans sufficient to warrant placement of ventriculostomy catheter were eligible. The study was implemented among 12 centers, 8 in the USA, 1 in Canada and 3 in Europe. Aliquots of CSF and blood were obtained at 8 hour intervals as long as a ventriculostomy was required for routine management. The samples were analyzed for endothelin-1 (ET-1), big-endothelin-1 (big ET-1), atrial (ANP) and brain natriuretic peptides (BNP) and cGMP over a 14 day period. Data was collected and coordinated by the American Brain Injury Consortium.

RESULTS: Total of 46 patients and 5 controls had been enrolled in this study. Increased plasma concentrations of big ET-1 and ET-1 were observed in the first few days post-TBI, followed by significant reductions in levels thereafter. CSF concentrations of big ET-1 and ET-1 were below the detection point in most cases through day 1 to 14. There was no temporal pattern for CSF concentrations of ANP and BNP.

DISCUSSION: We found contradictory results compared with the literature. Sample collection error seemed to be unlikely because same technique was utilized for controls and patients. The concentrations obtained from controls were all above the detection point. While working with low concentrations, many authors preferred to report estimated values which were less precise. Our aim was to obtain the most accurate concentrations therefore, we used validated values. Any value below lower limit of validated concentration was counted as an undetectable and this was the case in most of our samples.

Shah Alok 1 Stemper Brian 1 Yoganandan Narayan 1 Shender Barry 1 2

1 Medical College of Wisconsin, Milwaukee, WI, USA 2 Naval Air Warfare Center Aircraft Division, Patuxent River, MD, USA P154 Pressure Profiles and Attenuation Characteristics of Shockwaves using Post Mortem Human Subject Model for Blast-Induced Mild Traumatic Brain Injury

PURPOSE: A better understanding of mTBI mechanisms due to open field blast is essential for establishing injury thresholds and design of safety devices. Blast-induced shockwaves travel through the composite medium of skull and brain. Transmission-attenuation characteristics of the shockwave through the human skull have not been well defined. This is the purpose of the study.

METHODS: Intracranial contents were removed from post mortem human subject (PMHS) heads, filled with Sylgard gel, and pressure transducers were mounted on the skull (frontal, bilateral) and inserted intracranially (ipsilateral, contralateral, temporal, frontal). Preparations were attached to a surrogate human neck and placed at different distances and orientations from the shock tube, consisting of a hollow-metal cylinder (2.83-m driven, 0.35-m driver sections). Shockwaves were generated by accumulating driver section pressure with nitrogen gas behind a Mylar diaphragm. Varying diaphragm thicknesses controlled bursting and peak shockwave pressures. Shockwave data were recorded at 10 MHz.

RESULTS: Shockwaves applied from the lateral side, outside of the blast wind, had peak pressures of 88 and 114 kPa on the skull surface. Ipsilateral internal and lateral skull surface profiles were typical, overpressure followed by underpressure. However, the pattern was reversed internally on contralateral side. Peak internal contralateral pressures were approximately 60% of peak ipsilateral external pressures, albeit not time matched. The lag increased from ipsilateral to contralateral sites by approximately 300 microseconds.

CONCLUSION: These preliminary tests show that shockwaves attenuate and change profile as it passes through human skull and simulated brain medium. Megahertz recordings of data coupled with PMHS models allow improved analysis of shockwave transmission within the human skull-brain system, and these data can be used to advance computational models for understanding the mechanisms of blast-induced mTBI and safety devices.

Stemper Brian D. 1 Baumgartner Daniel 2 Yoganandan Narayan 1 Lamy Michael 2 Willinger Remy 2

1 Medical College of Wisconsin, Milwaukee, WI, USA 2 University of Strasbourg, Strasbourg, France P155 Tissue-Level Stresses Within the Brain During Rotationally-Induced MTBI: A Three-Dimensional Finite Element Model of the Rat Coupled with Experiments

PURPOSE: To quantify anatomical region-dependent tissue-level stresses during head rotational acceleration sufficient to induce mTBI.

METHODS: A unique hybrid experimental-computational approach was employed. The experimental study subjected thirty-one adult Sprague Dawley rats to mTBI using the MCW Rotational Injury Device. Rats were divided into five experimental groups with different rotational acceleration pulses that independently increased rotational acceleration magnitude to three levels while holding duration constant and increased duration to three levels while holding magnitude constant. Following injury, unconscious time was measured from administration of anesthesia reversal agent to return of the corneal reflex and histological evidence of injury was measured using GFAP. The FEM was developed using high-resolution CT and MR images of rats. The mesh consisted of six anatomical structures, with cerebrum subdivided into 18 anatomical regions. Anatomical region-based peak stresses and an integrated stress-time metric from the FEM were compared to experimental unconscious times and histological evidence of injury.

RESULTS: Greater experimental unconscious times (i.e., injury severities) were associated with increases in rotational acceleration magnitude and duration. Maximum FEM Von Mises stresses, maximum FEM stress-time magnitudes, and greatest histological evidence of injury were obtained in the hippocampus. Peak stresses increased with increasing acceleration magnitude, which agreed with unconscious times and histological evidence of injury. However, while unconscious times and evidence of injury increased for increasing durations, maximum FEM stresses did not. Conversely, the stress-time metric matched increases in unconscious time and evidence of injury for increasing acceleration magnitude and duration.

CONCLUSIONS: Results of this unique hybrid analysis indicate that the integrated stress-time variable may be more suited to explain variation of mTBI severity than pure peak metrics (e.g., Von Mises stress). These findings highlight the independent roles that head rotational acceleration magnitude and duration play in modulating injury type and severity, and region-specific biomechanical correlates for mTBI.

Stemper Brian D. 1 Yoganandan Narayan 1 Budde Matthew D. 1 Pintar Frank A. 2

1 Medical College of Wisconsin, Milwaukee, WI, USA 2 Veterans Affairs Medical Center, Milwaukee, WI, USA P156 Behavioral Alterations in Rats Exposed to Repeated Mild Traumatic Brain Injury: Changes in Open Field Test and Elevated Plus Maze Performance

PURPOSE: To identify differences in behavioral deficits between rats exposed to single mTBI and rats exposed to repeated mTBI using a novel head rotational acceleration injury model.

METHODS: Under general anesthesia, adult female Sprague Dawley rats sustained mTBI through rapid head rotation using the MCW Rotational Injury Device. The first group sustained a single injury. The second group sustained a single injury, followed by a second injury seven days later. A third group of controls was subjected to a sham procedure that involved placement in the injury device without head rotational acceleration. All rats were exposed to five minutes in the Elevated Plus Maze (EPM) on the sixth post-injury day and five minutes in the Open Field Test (OFT) on the seventh post-injury day.

RESULTS: Rats with repeated injury spent significantly less time in the EPM closed arms (p < 0.05), along with a greater number of rearing events per 60 sec in the closed arms and a greater number of head dips. These metrics were not markedly different between controls and rats with single injury. In the OFT, rats with repeated injury had a significantly higher number of rearing events (p < 0.05), along with 20% greater ambulatory time/distance and a greater number of center entries. These metrics were not markedly different between controls and rats with single injury.

CONCLUSIONS: Although rats with single injury did not demonstrate remarkable differences from controls, rats receiving repeated injury of equal magnitude demonstrated decreased defensive along with hyperactive locomotion behaviors that have previously been reported as an indicator of depression in olfactory bulbectomized rats. Behavioral deficits resulting from repeated exposure to previously non-injurious head accelerations highlight increased susceptibility for second injury following initial concussion event. Continued research along this line will focus on defining relationships between magnitude and duration of increased susceptibility following ignition concussive injury.

Khan Mushfiquddin 1 Sakakima Harutoshi 1 Im Yeong-Bin 1 Shunmugavel Anandakumar 1 Dhammu Tajinder Singh 1 Singh Avtar 2 Singh Inderjit 1

1 Medical University of South Carolina, Charleston, SC, USA 2 Ralph H Johnson VA Medical Center, Charleston, SC, USA P157 S-Nitrosoglutathione Reduces Secondary Injury and Stimulates Neurorepair Following Traumatic Brain Injury in Rats

BACKGROUND: Traumatic brain injury (TBI) induces primary and secondary damage in the neurovascular unit. While neurons die quickly by necrosis, a vicious cycle of secondary injury in endothelial cells exacerbates the initial injury following TBI. In activated endothelial cells, excessive superoxide reacts with nitric oxide (NO) to form peroxynitrite. Peroxynitrite has been implicated in blood brain barrier (BBB) leakage and neurobehavioral impairment. S-nitrosoglutathione (GSNO), a signaling molecule, was reported not only to reduce brain levels of peroxynitrite but also to ameliorate TBI and stroke. Therefore, we investigated whether GSNO promotes the neurorepair by decreasing the levels of peroxynitrite and reducing the degree of oxidative injury.

METHODS: TBI was induced by controlled cortical impact (CCI) in adult male rats. GSNO or 3-Morpholino-sydnonimine (SIN-1) (50 μg/kg body weight) was administered orally at two hours after CCI. The same dose was repeated daily until endpoints. GSNO-treated (GSNO group) or SIN-1-treated (SIN-1 group) injured animals were compared with injured (TBI group) and sham animals in terms of peroxynitrite, BBB leakage, edema, axon/myelin integrity, glutathione, and neurotrophic factors.

RESULTS: SIN-1 treatment of TBI increased but GSNO treatment decreased peroxynitrite, lipid peroxides/aldehydes, BBB leakage, and edema in a short-term treatment. GSNO also reduced brain infarction and restored axonal integrity and levels of both glutathione and myelin. In a long-term treatment, GSNO promoted synaptic plasticity and enhanced the expression of neurotrophic factors.

CONCLUSION: Our findings indicate the participation of peroxynitrite in TBI. GSNO treatment of TBI not only reduces peroxynitrite but also protects the neurovascular unit, indicating that GSNO blunts the deleterious effects of peroxynitrite. A long term treatment of TBI with GSNO promotes synaptic plasticity and enhances the expression of neurotrophic factors. The results support that GSNO reduces the levels of oxidative metabolites, protects the neurovascular unit, and promotes neurorepair mechanisms in TBI.

Dams-O'Connor Kristen 1 Kulubya Edwin 1 Gordon Wayne 1 Yue John 2 Puccio Ava 3 Okonkwo David 3 Valadka Alex 4 Manley Geoff 2

1 Mount Sinai School of Medicine, Department of Rehabilitation Medicine, New York, NY, USA 2 University of California San Francisco, Brain and Spinal Injury Center at SF General Hospital, San Francisco, CA, USA 3 University of Pittsburgh Medical Center, Brain Trauma Research Center, Pittsburgh, PA, USA 4 University Medical Center, Brackenridge-Austin, Austin, TX, USA P158 Predicting TBI Outcomes in the Transforming Research and Clinical Knowledge in Traumatic Brain Injury (Track-TBI) Study

PURPOSE: The current study explores the utility of the Extended Glasgow Outcome Scale (GOS-E) in accounting for objective and subjective outcomes beyond the acute phase of injury. The GOS-E is one of the most widely used methods for quantifying overall outcome after head injury, yet its prognostic value in explaining post-acute outcomes has not been thoroughly investigated.

METHODS: A sample of 214 individuals with mild to severe TBI were recruited from Emergency Departments and inpatient brain injury rehabilitation units at four USA hospitals. Participants completed the GOS-E via telephone 3 months post-injury, and then completed a NIH/NINDS common data elements consensus-based battery of neurocognitive assessments 6 months post-injury. In addition to the GOS-E, participants completed a set of self-reported measures (Satisfaction With Life Scale (SWLS), the Rivermead Postconcussion Symptoms Questionnaire (RPQ), and the Brief Symptom Inventory 18 (BSI 18)), an objective measure of functional independence (Craig Handicap Assessment and Reporting Technique - Short Form (CHART-SF)), and a battery of administered, timed neurocognitive measures (California Verbal Learning Test-II (CVLT-II), Processing Speed Index of the Wechsler Adult Intelligence Scales-IV (WAIS-IV), and the Trail Making Test (TMT)).

RESULTS: The GOS-E administered 3 months post-injury is not significantly associated with timed measures of neurocognitive function at 6-months post TBI in this cohort (F(1,165) = 0.16-2.7; p > .05). Cross-sectional analyses of data collected at 6-month follow up reveals that GOS-E is significantly correlated with self-report indices of functioning (i.e., RPQ, SWLS, BSI; p < .01) and select CHART-SF subscales, but is not associated with timed measures of neurocognitive function (i.e., TMT, WAIS-IV; p > .05).

CONCLUSION: Cognitive impairment is a major cause of long-term disability after brain injury. The lack of association between GOS-E and common indices of neurocognitive functioning may limit the usefulness of the GOS-E, when used in isolation, as an predictor or indicator of TBI outcomes.

Turtzo l. Christy 2 Budde Matthew 1 Gold Eric 2 Lewis Bobbi 1 Janes Lindsay 2 Watson William 2 Frank Joseph 1

1 NIH, Bethesda, Maryland, USA 2 Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA P159 The Evolution of Traumatic Brain Injury in a Rat Model: Implications for MRI Cell Tracking

Serial MRI facilitates in vivo evolutionary analysis of traumatic brain injury (TBI) lesions. MRI has been used to track the delivery of superparamagnetic iron oxide (SPIO) labeled cells in TBI models. Despite the availability of MRI, the natural history of experimental TBI lesions in the CCI model is not well described in the literature. The motor cortex of anesthetized female Wistar rats (n = 34) underwent CCI with an impactor tip driven by an electromagnetic piston. In vivo MRI was performed at 7T on days 2, 9, and 30 post-CCI using a T2w sequence. Cortical and lesion volumes were determined using MEDx software. The appearance and volume of CCI-induced lesions at days 2, 9, and 30 was variable and different than previously reported. There was little correlation between the percent change of CCI side cortex volume to contralateral side cortex volume on days 2 to subsequent exams on 9 and 30 (mean ± STD: Day 2 = 24.4 ± 8.6%; Day 9 = − 5.9 ± 11.0%; Day 30 = −11.6 ± 12.0%). Hemorrhagic conversion within the CCI lesion occurred in 45% of rats between days 2 and 9. Some early variation in CCI lesions can be attributed to differences in surgical technique. The further divergence of similar lesions between days 2 and 30 demonstrates the inherent biological variability of the CCI rat model. The possibility of hemorrhagic evolution between days 2 and 9 raises caution in using SPIO-labeled cells to track their delivery to CCI lesions, as hemorrhage appears identical on MRI and Prussian blue staining. Investigators utilizing the CCI model in the rat should be aware of the higher degree of variability and hemorrhage in results obtained with this method of inducing TBI.

King Nicolas KK Neilson Sam J Lee Kah Keow Wang Ernest Ng Ivan National Neuroscience Institute, Singapore, Singapore

P160 Validation of the Crash Prognostic Score in Severe Head Injury in a South East Asian Population

PURPOSE OF STUDY: Traumatic brain injury (TBI) represents a significant cause of mortality and morbidity. An accurate model able to predict outcome in terms of survival and disability would be very beneficial to those involved in patient care to make informed decisions regarding patient management and to communicate these effectively with the patient's family. Clinical prediction models must be validated both internally and externally before they are to be considered widely applicable. In this study, we validated the prognostic model developed by the Corticosteroid Randomisation After Significant Head injury (CRASH) trial investigators which was developed using one of the largest number of patients.

METHODS: Data from 300 consecutive patients admitted to the National Neuroscience Institute, Singapore with severe head injury were analyzed (median GCS5). The individual factors used to calculate the prognosis were extracted. The CRASH model estimates the probability of 14-day mortality and 6-month dichotomized GOS using two different models. The base model has presentation clinical factors alone, and does not include CT brain imaging data. The estimates were compared with the observed outcomes.

RESULTS: The overall observed 14-day mortality was 47.7% and the observed 6-month poor outcome was 71.3%. Using the CRASH prognostic score with the CT model, the predicted mortality was 46.7% with a predicted poor outcome of 75.0%. Similarly, using the base model, mortality was 38.2% and poor outcome was 67.7%. Calibration was judged to be best for 14-day mortality using the CT model. The GOS outcome was over-estimated using the model. The base model had a poorer fit.

CONCLUSION: The CRASH model has been further externally validated using our cohort of patients and found that the CRASH prognostic score using the combination of base clinical information combined with additional information provided by CT (CT model) satisfactorily predicted outcome in our patients with severe TBI.

Pang Boon Chuan 1 Yin Hongli 2 Chan Siew-Pang 3 Khan Sanaullah 1 Lee Kah Leow 1 Ang Beng Ti 1 Goh Jia Jun 1 Tze Yun Leong 2 Ng Ivan 1

1 Department of Neurosurgery, National Neuroscience Institute, Singapore, Singapore 2 Medical Computing, Department of Computer Science, School of Computing, Yong Loo Lin School, Singpaore, Singapore 3 Clinical Research Unit, Tan Tock Seng Hospital, Singapore, Singapore P161 Analysis of Clinical Criteria in Predicting Clinical Deterioration Following Minor Head Injury

Canadian CT head rule (CCHR) and New Orleans criteria (NOC) were developed and validated to guide cranial computed tomography (CT) use following minor head injury. The role of these rules in identifying neurological deterioration is not known. Using predictors lacking in specificity will lead to oversubscription of admission beds whereas using predictors lacking in sensitivity will result in missing possible late clinical deterioration due to inappropriate allocation of resources.

Data from 1805 consecutive patients with minor head injury (GCS 13-15) were prospectively collected from 2003 to 2005 in a tertiary neurosurgical unit. All patients seen in the emergency department who warrant a cranial CT would be admitted to the neurosurgical unit for the scan and a minimum of one-day hospital stay for observation. Clinical parameters and radiological parameters including those outlined in the CCHR and NOC were collected. Attempts to predict neurological deterioration was carried out using logistic regression, neural network, support machine learning, decision tree and bayesian approach. A comparison of CCHR, NOC and a proposed 10 parameter criteria in predicting clinical deterioration (drop of admission GCS to < 9) were made. Glasgow Outcome Score (GOS) at six months was collected.

29 (1.6%) patients deteriorated following admission. This study reported the following results. Firstly, a Bayesian Network approach provided the best model in outcome prediction. Secondly, CCHR and NOC had very poor specificity, despite a sensitivity of 100%. A 10 variable criteria was proposed which achieved specificity of ∼90% and sensitivity ∼50%. Thirdly, clinical deterioration occurred within 24 hours of injury.

The results above will be used to validate prospectively the cohort of patients with minor head injury in the consecutive years from our database and to further refine our prediction model so as to strengthen its generalisability in the Asian population.

Shaughness Michael 1 Maudlin-Jeronimo Eric 1 Hall Aaron 1 Shear Deborah 2 McCarron Richard 1 Stone James 3 Ahlers Stephen 1

1 Naval Medical Research Center, Silver Spring, MD, USA 2 Walter Reed Army Institute of Research, Silver Spring, MD, USA 3 University of Virginia School of Medicine, Charlottesville, VA, USA P162 Evaluation of Corticosterone After Repeated Exposure to Low Level Blast Overpressure in Rats

There is a high co-morbidity of mild traumatic brain injury (mTBI) and post traumatic stress disorder (PTSD) in Warfighters. It is generally assumed that the manifestation of mTBI symptoms result from exposure to IEDs and PTSD symptoms from prolonged stress on the battlefield. We recently demonstrated that rats administered 12 exposures to 5.6 psi blast overpressure (BOP) showed an impairment of acquisition on a Morris Water Maze when training occurred 24 hours after the last BOP exposure. We subsequently observed this impairment at 1, 2, and 4 weeks after BOP exposure indicating that repeated exposures to BOP can result in the manifestation of long-lasting, post-concussive-like, symptoms (PCS). While we continue to characterize the performance decrements following repeated BOP exposure to determine how long cognitive impairment endures, we are interested to assess how, and if, changes in the stress response might accompany, and possibly contribute to, the protracted cognitive impairment resulting from BOP exposure. Accordingly, we assessed the stress response by evaluating serum corticosterone levels in blasted and sham exposed rats where we observed cognitive impairment in the conditions described above. Corticosterone was assayed in rats after they were repeatedly exposed (under anesthesia) to 5.6 psi BOP or a sham control condition, once per day for 12 days and assessed at 24 hours, 1, 2, 4, and 8 weeks post BOP exposure. Corticosterone levels were higher than controls at all time points except at 24 hours where corticosterone in BOP exposed animals were lower than controls. While changes in the stress response do not appear to explain the impairment of cognitive performance observed with repeated exposure to BOP, they do suggest that there are alterations in the stress response following repeated BOP exposure which may contribute to the manifestation of symptoms after exposure to IEDs.

Subject Category: Blast-induced mTBI

Siriwardane Mevan 1 Skop Nolan 1 Levison Steve 2 Calderon Frances 2 Jiang Yuhui 2 Gandhi Chirag 3

1 University of Medicine & Dentistry, New Jersey Institute of Technology, Department of Biomedical Engineering, Newark, NJ, USA 2 University of Medicine & Dentistry, Department of Neurology & Neurosciences, Newark, NJ, USA 3 University of Medicine & Dentistry, Department of Neurological Surgery, Newark, NJ, USA P163 Characterization of Controlled Cortical Impact Trauma and Evaluation of Acute and Sub-Acute Neural Stem Cell Delivery

INTRODUCTION: Cell transplantation for treating traumatic brain injury (TBI) can improve regeneration and repair of the central nervous system. Timing of cell delivery remains to be optimized to ensure survival of transplanted cells within a pro-inflammatory microenvironment.

MATERIALS AND METHODS: TBI was performed on the adult rat neocortex using controlled cortical impact (CCI) injury model. The viability, location, and differentiation of transplanted neural stem and progenitor cells (NPs) following CCI at acute (24 hours) and sub-acute (7 days) intervals was evaluated. NPs were harvested from the subventricular zone (SVZ) of GFP transgenic postnatal rat brains. Morphological differences and differentiation were assessed by immuno-labeling tissue sections after 3 and 7 days survival. Control sections of CCI without cell transplants were immunostained at 24, 48, and 72 hours post-trauma. Neuronal and oligodendrocyte cell loss and the level of gliosis were analyzed at chronic stages.

RESULTS: Engrafted GFP-cells in brain tissue sections revealed differences in morphology and location of cells in host tissue at both time points. Furthermore, only few cells entered the lesion cavity. Unlike cells grafted acutely, cells engrafted subacutely were diffusely located surrounding the lesion site. At both time-points, most cells were negative for common neuronal and glial markers. Immunohistochemistry analysis of control CCI brains at 24 hours and 7 days survival with neuronal and oligodendrocyte markers showed considerable cell loss.

CONCLUSION: Acute and subacute delivery of NPs led to survival at 3 and 7 days post-transplantation. However, cells injected at both time points did not express mature neural markers suggesting that postnatal SVZ-derived NPs may have a limited potential for neural differentiation. More primitive embryonic ventricular zone-derived NPs and biomaterial-based carriers are now being studied.

Hilz Max J. 1 Marthol Harald 2 Anders Stefan 2 Aurnhammer Felix 2 Schroeder Tim 2 Roßmeißl Anja 2 Lang Christoph J. 2 Flanagan Steven 3 DeFina Philip A. 4 Schwab Stefan 2

1 Depts. of Neurology, Medicine, Psychiatry, New York University, New York, NY, USA 2 Dept. of Neurology, University of Erlangen-Nuremberg, Erlangen, Germany 3 Dept. of Rehabilitation Medicine, New York University, New York, NY, USA 4 International Brain Research Foundation Inc., IBRF, Edison, NJ, USA P164 Frequency Analysis Reveals Subtle Cardiac Autonomic Dysfunction in Patients with Mild Traumatic Brain Injury

BACKGROUND: After mild traumatic brain injury (mTBI), there is a long-lasting increased risk of mortality that might be due to trauma-induced dysfunction of the central autonomic network with subsequent cardiovascular dysregulation.

OBJECTIVE: To determine whether there is cardiovascular-autonomic dysregulation after mTBI.

METHODS: 5-43months after mTBI, 20 patients (37 ± 13years; GCS-scores 13-15, assessed 30minutes after injury), and 20 age-matched healthy controls (26 ± 9years) underwent autonomic testing at supine rest and during active standing. We monitored respiratory frequency, electrocardiographic RR-intervals (RRI) and systolic blood pressure (SBP). We calculated the root mean square successive RRI differences (RMSSD) reflecting cardiovagal modulation, and the ratio of maximal and minimal RRIs around the 30^th and 15^th RRI upon standing-up (30:15-ratio) reflecting baroreflex-sensitivity (BRS). Using trigonometric-regressive-analysis, we determined spectral powers of mainly sympathetic low (LF: 0.04-0.15Hz) and parasympathetic high (HF: 0.15-0.5Hz) frequency RRI-fluctuations, sympathetically mediated LF-powers of SBP, RRI-LF/HF-ratios reflecting sympathovagal balance, and the gain between BP- and RRI-oscillations as additional BRS-index (BRS_gain).

We calculated statistics by repeated measurement analysis of variance, comparing supine and standing parameters of patients and controls; significance: p < 0.05.

RESULTS: While supine, patients had lower RRIs (874.2 ± 157.8 vs. 1024.3 ± 165.4ms), RMSSDs (30.1 ± 23.6 vs. 56.3 ± 31.4ms), RRI-HF-powers (298.1 ± 309.8 vs. 1507.2 ± 1591.4ms²), BRS_gain (8.1 ± 4.4 vs. 12.5 ± 8.1ms · mmHg⁻¹), but higher RRI-LF/HF-ratios (3.0 ± 1.9 vs. 1.2 ± 0.7) than controls. Upon standing, patients had lower RRI-30:15-ratios (1.3 ± 0.3 vs. 1.6 ± 0.3) and RRI-LF-powers (2450.0 ± 2110.3 vs. 4805.9 ± 3453.5ms²) than controls; RMSSDs and RRI-HF-powers decreased significantly in controls, but not in patients.

CONCLUSION: While supine, cardiovagal modulation was reduced in mTBI-patients. During orthostasis, patients did not adequately increase sympathetic outflow, nor withdraw parasympathetic modulation. Additionally, BRS was reduced while supine and during active standing compared to values of healthy controls. In mTBI-patients, the impairment of autonomic modulation might contribute to cardiovascular complications.

Acknowledgement: The study was partially funded by the International Brain Research Foundation Inc., Edison, NJ, USA.

Hilz Max J. 1 Aurnhammer Felix 2 Anders Stefan 2 Koehn Julia 2 Marthol Harald 2 Blaszczynska Paulina 1 Schroeder Tim 2 Roßmeißl Anja 2 Flanagan Steven R. 3 De Fina Philip A. 4

1 Depts. of Neurology, Medicine, Psychiatry, New York University, New York, NY, USA 2 Dept. of Neurology, University of Erlangen-Nuremberg, Erlangen, Germany 3 Dept. of Rehabilitation Medicine, New York University, New York, NY, USA 4 International Brain Research Foundation Inc., IBRF, Edison, NJ, USA P165 Ocular Pressure Shows Altered Cardiovascular Responses in Mild Traumatic Brain Injury Patients

BACKGROUND: Patients with a history of mild traumatic brain injury (mTBI) have a significantly increased long-term risk of mortality that might be related to central autonomic dysregulation. Ocular-pressure-testing (OPT), a procedure increasing central parasympathetic activity and thus decreasing heart rate and blood pressure (BP), might be suited to unveil central autonomic dysfunction in mTBI-patients.

OBJECTIVE: To evaluate cardiovascular and BP responses to OPT in post-mTBI-patients.

METHODS: 5-86months after mTBI, 24 patients (34 ± 12years; GCS-scores 13-15, assessed 30minutes after injury), and 27 age-matched healthy controls (30 ± 11years) underwent OPT. Before and during 2minutes of 30mmHg right-sided OP (Oculosionspelotte^®, Bausch & Lomb, Germany), we monitored respiratory frequency (RESP), electrocardiographic RR-intervals (RRI), systolic and diastolic BP (SBP, DBP).

We determined spectral powers of mainly sympathetic low (LF: 0.04-0.15Hz) and parasympathetic high (HF: 0.15-0.5Hz) frequency RRI-fluctuations as well as sympathetically mediated LF-powers of SBP. Statistics were calculated by repeated measurement analysis of variance with t-test post-hoc analysis (significance: p < 0.05).

RESULTS: Before OPT, parameters were similar between patients and controls. During OPT, SBP (121.8 ± 14.9 vs. 128.5 ± 13.8mmHg) and DBP (64.1 ± 8.4 vs. 67.0 ± 9.4mmHg) increased in mTBI-patients only. In controls but not in patients, LF-powers of RRI (1001.5 ± 1213.6 vs. 570.3 ± 468.3ms²) and of SBP (14.4 ± 10.2 vs. 10.1 ± 6.4mmHg²) decreased, while normalized HF-powers of RRI (48.1 ± 24.4 vs. 55.9 ± 22.0) increased with OPT.

CONCLUSION: In healthy controls, OPT induced normal oculocardiac reflex responses, with decreases in LF-powers of RRI and of SBP and an increase in normalized HF-powers of RRI, showing a sympatho-vagal shift towards parasympathetic tone. In contrast, the OPT-induced increase in SBP and DBP in mTBI-patients indicates a higher vascular tonicity with a slight shift towards increased sympathetic activation. In mTBI-patients, this subtle autonomic dysfunction might contribute to cardiovascular complications and further the risk of unexplained death.

Acknowledgement: The study was partially funded by the IBRF Inc., Edison, NJ, USA.

Tado Masahiro Mori Tatsuro Maeda Takeshi Tahara Junichi Fukushima Masamichi Katayama Yoichi Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan

P166 Etiology of Sport-Related Repetitive Head Injury; Prevention Against the Catastrophic Brain Damages

INTRODUCTION: Sport-related concussion is a common injury that occurs in a variety and the repeated concussion occurring within a short period occasionally lead to fetal outcome. In the present study we investigated severe brain damages due to repetitive head injury in sports and discussed the pathophysiology of sport-related repetitive injury.

METHODS: We reviewed the literature of severe sport-related repeated head injury described in detail. Total 19 cases included our cases were analyzed as age, gender, the kind of sports, symptoms before second injury and diagnoses of brain CT scan.

RESULTS: The majority of these cases involved male adolescents or young adults who sustained a second head injury before symptoms from the first head injury resolved. Eleven of 19 cases did not have a loss of consciousness at the insult. Eight cases were confirmed brain CT scan after second injury and all 8 cases showed brain swelling associated with a thin subdural hematoma.

CONCLUSIONS: Second impact syndrome is thought to occur because of a loss of autoregulation of the cerebral blood flow, which leads to vascular engorgement, increased intracranial pressure, and eventual herniation. The present study suggests that the existent of subdural hematoma is one of the major causes of brain swelling after sport-related repetitive head injury. It is important that coaches, as well as players and trainers, understand the medical issues involved in concussion. Physical evaluation is need for decide when athlete can return to competition especially contact sports such as boxing and American football.

Papa Linda 1 Demery Jason 2 Dixit Neha 3 Braga Carolina 1 Zhang Zhiqun 5 Brophy Gretchen 6 Burks Stephen 7 Ilic Sanja 5 Streeter Jackson 5 Tortella Frank 4 Hayes Ronald 5 Wang Kevin K.W. 1

1 Orlando Regional Medical Center, Orlando, Florida, USA 2 University of Florida, Gainesville, Florida, USA 3 NF/SG Veteran's Health System, Gainesville, Florida, USA 4 Walter Reed Army Institute of Research, Silver Spring, Maryland, USA 5 Banyan Biomarkers Inc., Alachua, Florida, USA 6 Virginia Commonwealth University, Richmond, Virgina, USA 7 University of Miami, Miami, Florida, USA P167 Early Serum Levels of Glial Fibrillary Acidic Protein Breakdown Product (GFAP-BDP) are Associted with Global Outcome at One Month Post Injury in Mild and Moderate Traumatic Brain Injury

OBJECTIVE: Glial Fibrillary Acidic Protein (GFAP) is found glial cells and is specific to the central nervous system. This study compared levels of GFAP-BDP from patients with mild and moderate TBI (MMTBI) with good and poor outcome at one month post-injury.

METHODS: This prospective cohort study enrolled adult patients presenting to the Emergency Department (ED) of a tertiary care Level 1 Trauma Center following blunt head trauma with a GCS 9-15. Blood samples were obtained in all patients within 4 hours of injury and measured by ELISA for GFAP-BDP (ng/ml + /-SEM). Patients were assessed in person at one month post-injury. The main outcome was measured using the traditional dichotomization of GOS at one-month post-injury. Poor outcome was defined as death, vegetative state or severe disability and good outcome was moderate disability or good recovery.

RESULTS: There were 35 MMTBI patients included in the analysis: 33 with a GCS 13-15, and 2 with a GCS 9-12. The mean age of TBI patients was 39 years (range 19-68) with 63% males. Ten patients (29%) had poor outcome at one month post-injury. Mean serum GFAP-BDP levels obtained within 4 hours of injury were 0.071 (0.260) in those with good outcome and 2.162 (0.777) in those with poor outcome (P = 0.039). The AUC for distinguishing good versus poor outcome was 0.76.

CONCLUSIONS: Early levels of GFAP-BDP measured in the emergency department were able to distinguish good versus poor outcome at one month post-injury. These results need further study in a larger cohort.

Papa Linda 1 Robicsek Steve 3 Robertson Claudia S. 2 Brophy Gretchen 4 Gabrielli Andrea 3 Tepas Joseph III 7 Heaton Shelley 3 Hannay H. Julia 6 Hayes Ronald L. 5 Wang Kevin K.W. 5

1 Orlando Regional Medical Center, Orlando, Florida, USA 2 Baylor College of Medicine, Houston, Texas, USA 3 University of Florida, Gainesville, Florida, USA 4 Virginia Commonwealth University, Richmond, Virginia, USA 5 Banyan Biomarkers Inc., Alachua, Florida, USA 6 University of Houston, Houston, Texas, USA 7 University of Florida, Jacksonville, Florida, USA P168 Early CSF Levels of UCH-L1 are Associated with Injury Severity in Patients with Severe TBI

OBJECTIVE: Ubiquitin C-terminal hydrolase (UCH-L1), also called neuronal-specific protein gene product (PGP 9.3), is highly abundant in neurons. This study assessed whether early levels of UCH-L1 in CSF were associated with acute and long-term measures of injury severity in severe TBI patients.

METHODS: This study was designed as prospective observational study that enrolled adults with severe TBI presenting to 3 Level I Trauma Centers. Patients were included if they had a blunt head injury with a GCS score of ≤ 8 and required intraventricular intracranial pressure monitoring. Ventricular CSF was sampled from each patient at enrollment, 6, 12, 18, 24, 48, 72, 96, 120, 144, 168, 192, 216 and 240 hours following TBI and analyzed for UCH-L1 (ng/ml) using ELISA. Injury severity was assessed by the best 24-hour GCS score, pupillary reactivity and the 6-month dichotomized GOS score.

RESULTS: There were 51 patients included in the analysis. Mean age of patients was 38 years and 75% were male. There was a very early rise in UCH-L1 in the CSF. Mean UCH-L1 levels at sequential time-points were 94.9, 113.1, 80.4, 69.6, 50.5, 14.4, 9.0, 4.5, 4.8, 2.6, 3.8, 4.1, 13.0, and 7.9 respectively. Early levels of UCH-L1, drawn within 24 hours of injury, were significantly higher in patients with a best GCS score 3-5 compared to those with a best GCS score 6-8 (P = 0.004). Patients with at least one unreactive pupil also had significantly higher initial UCH-L1 levels than those with bilaterally reactive pupils (P = 0.026). Additionally, UCH-L1 was markedly elevated in those with a poor 6-month global outcome compared to those with a good outcome (p = 0.016).

CONCLUSION: These data suggest that early levels of this novel biomarker are associated with both early and late measures of TBI severity. Ongoing studies are being conducted to validate these findings.

Beziaud Tiphaine Chen Xiao Ru Shafey Nelly El Frechou Magalie Teng Fei Beray-Berthat Virginie Palmier Bruno Plotkine Michel Marchand-Leroux Catherine Besson Valérie Paris Descartes University, Department of Diagnostic and Therapeutic Innovation, Pharmacology of Cerebral Circulation, Paris, France

P169 Anti-Edematous Effect by Simvastatin in Traumatic Brain Injury: Evidence and Putative Mechanisms

OBJECTIVES: Traumatic brain injury (TBI) leads to a deleterious cerebral edema which may originate from endothelial cell junction protein degradation, polymorphonuclear neutrophil (PMN) infiltration and matrix metalloproteases (MMP) activation, all contributing to blood-brain barrier (BBB) loss of integrity. As we previously showed that simvastatin, an HMG-CoA reductase inhibitor, reduced neurological deficit and brain edema in a rat model of TBI, we evaluated whether anti-edematous effect is associated with modulation of claudin-5, a tight junction protein, ICAM-1 expression, PMN infiltration, MMP2 and 9 activation and thus BBB permeability.

METHODS: TBI was induced by lateral fluid percussion on rats. Simvastatin was administered p.o. 1 and 6h after brain injury. Brain edema was evaluated by measuring brain water content (BWC). Claudin-5 expression was determined by western-blot. ICAM-1 expression was done by immunohistochemistry, PMN infiltration by measuring MPO activity and MMP2 and 9 activities by zymography. BBB permeability was assessed by extravasation of two fluorescent dyes, Evans blue (EB) and Fluorescein sodium salt (NaFl). PMN infiltration and BBB permeability were measured on temporoparietal cortex (contusion), frontal and occipital cortices (pericontusional areas) in order to evaluate more precisely areas affected. All other measures were done in the contusion.

RESULTS: TBI induced brain edema (P < 0.001) that was reduced by simvastatin (P < 0.01), showing an anti-edematous effect. Simvastatin suppressed post-TBI decrease of claudin-5 expression (P < 0.05). TBI led to ICAM-1 staining and PMN infiltration in the contusion and pericontusional areas, which were decreased by simvastatin (P < 0.05). At 24h post-TBI, permeability was extended to the 3 cortical areas (for EB, P < 0.01). Even if simvastatin failed to reduce post-TBI MMP2 and 9 activities, it decreased EB extravasation in the pericontusional zones (P < 0.05).

CONCLUSION: Simvastatin is able to prevent post-TBI tight junction impairment, PMN parenchyma infiltration, and thus reduces BBB loss of integrity. These effects could contribute to its anti-edematous effect.

Siopi Eleni Dabén Gemma Llufriu Fanucchi Francesca Plotkine Michel Marchand-Leroux Catherine Jafarian-Tehrani Mehrnaz Paris Descartes University, Lab of Pharmacology, Paris, France

P170 Acute Minocycline Treatment Improves Memory Function Following Traumatic Brain Injury in Mice

INTRODUCTION: Comorbidity of cognitive and stress disorders is a common clinical sequel of traumatic brain injury (TBI). It is determined by the site and severity of the insult, but also by the extent of the ensuing neuroinflammatory response. The present study examined the effects of closed-head TBI on memory function and anxiety in the mouse, in order to examine the efficacy of an acute treatment with minocycline.

METHODS: The model of closed-head injury by mechanical percussion was applied on anesthetized Swiss mice. This model is known to induce moderate to severe TBI with a pronounced neuroinflammatory response. The treatment protocol included three injections of minocycline (i.p.) at 5 min (90 mg/kg), 3 h and 9 h (45 mg/kg) post-TBI. A novel object recognition test was run from 4 to 12 weeks post-TBI, to evaluate memory function. The Plus Elevated Maze test (PEM) and the Zero Elevated Maze test (ZEM) were run at 3 and 7 weeks post-TBI respectively, to assess the level of post-TBI anxiety.

RESULTS: Our results revealed a significant post-TBI memory deficit at 4 weeks (P < 0.001), 9 weeks (P < 0.001) up to at least 12 weeks post-TBI (P < 0.05). Minocycline was able to efficiently attenuate the memory deficit at all three time points of the test (P < 0.01; P = 0.06; P < 0.05 vs vehicle). However, neither PEM nor ZEM revealed any alteration in post-TBI anxiety levels.

CONCLUSION: Severe closed-head TBI in the mouse induces memory impairment without any alteration in stress behavior. Minocycline is able to attenuate memory dysfunction in an effective and lasting manner, highlighting its therapeutic potential in TBI-induced memory deficits.

Siopi Eleni Cho Angelo H Homsi Shadi Croci Nicole Plotkine Michel Marchand-Leroux Catherine Jafarian-Tehrani Mehrnaz Paris Descartes University, Lab of Pharmacology, Paris, France

P171 Reduction of the Long-Term Consequences of Traumatic Axonal Injury by Minocycline Following Closed Head Injury in Mice: The Potential Role of sAPPalpha

INTRODUCTION: Traumatic axonal injury (TAI) is a detrimental consequence of traumatic brain injury (TBI) that refers to extensive lesions in white matter tracts and for which no treatment is available. It has been recently reported that an endogenous neuroprotector, the soluble form of the amyloid precursor protein (sAPPalpha) is able to reduce TAI. However, the emergent post-traumatic neuroinflammatory environment compromises sAPPalpha production and may promote TAI development and subsequent white matter atrophy. Hence, the aim of this study was to examine the effects of an anti-inflammatory drug, minocycline, on post-TBI sAPPalpha production and on the long-term TAI consequences.

METHODS: The mouse model of TBI by mechanical percussion was applied on anesthetized Swiss mice. Minocycline was injected (i.p.) at 5 min (90 mg/kg), 3 h and 9 h (45 mg/kg) post-TBI. The ELISA and the cresyl violet staining were used to measure sAPPalpha production and TAI-induced atrophies respectively. Immunohistochemistry of GFAP and CD11b was performed to measure gliar scar formation.

RESULTS: A decrease of sAPPalpha levels was observed at 24h post-TBI (P < 0.001) that was significantly attenuated by minocycline (P < 0.01). Corpus callosum atrophy (P < 0.05), striatal atrophy (P < 0.001) and ventriculomegaly (P < 0.01), accompanied by an increased GFAP and CD11b immunolabeling (P < 0.01) were observed at 3 months post-TBI. All the three delayed consequences were significantly reduced by minocycline.

CONCLUSION: Inhibition of the acute phase of post-TBI neuroinflammation by minocycline is associated with the sparing of sAPPalpha and the protection of the white matter tracts in the long-term, emphasizing the potential role of minocycline as a promising neuroprotective treatment in TBI and proposing sAPPalpha as potential therapeutic target.

Buttram Sandra Garcia-Filion Pamela Youssfi Mostafa Brown Danielle Dalton Heidi Adelson P. David Phoenix Children's Hospital, Phoenix, AZ, USA

P172 CT Versus MRI for Identifying Lesions in Pediatric Traumatic Brain Injury

PURPOSE: Pediatric traumatic brain injury (TBI) is a leading cause of morbidity and mortality with potential for undetected lesions. While computed tomography (CT) is an effective screening tool for cranial injury, the purpose of this study was to compare lesion frequency detected by CT and magnetic resonance (MR) imaging following pediatric TBI.

METHODS: Retrospective study of children with TBI admitted to a pediatric trauma center between 2006 and 2010 with a head CT on day of and a brain MR within two weeks of injury. Two pediatric radiologists, blinded to clinical information, reviewed the CT and MR images to identify presence of abnormalities (extra-axial lesions, basal cisterns, midline shift, traumatic axonal injury (TAI), contusion, and ischemia). Agreement in the detection of abnormalities by CT and MR were determined by the kappa statistic, and stratified on TBI severity.

RESULTS: We studied 105 children (age range: < 1–204 months) with TBI. Of these, 78% had mild, 10% moderate, and 12% severe TBI. The MR was obtained an average 1.8 days post initial CT. As expected, they showed significant agreement identifying extra-axial lesions (84.8%; κ = 0.61, p < 0.001), basal cistern compression (92%) and midline shift (94.3%; κ = 0.64, p < 0.001). There was moderate agreement for contusions (82.9%; κ = 0.48, p < 0.001) and ischemia (95.2%; κ = 0.43, p < 0.001) with MR detecting lesions not visualized on CT. When stratified by TBI severity, there was moderate to substantial agreement in identification of extra-axial lesions (mild κ = 0.61, moderate κ = 0.55, severe κ = 0.68) and contusions (mild κ = 0.44, moderate κ = 0.74, severe κ = 0.45). Interestingly, MR identified TAI in 7 subjects not seen on CT. MR detected intraparenchymal lesions in 42 cases (40%) compared to only 16 (15%) identified with CT.

CONCLUSION: MR was more effective in identifying lesions than CT. Future study of early MR following TBI should be done to evaluate the relationship between intraparenchymal lesions and prognosis.

Adelson P. David 1 2 Perez Roxanna E. 2 Salevitz Nicole 2 Treiman Lucy J. 2

1 Children's Neuroscience Institute, Phoenix Children's Hospital, Phoenix, AZ, USA 2 Barrow Neurological Institute, Phoenix, AZ, USA P173 Controlled Cortical Impact Lowers the Threshold for Pentylenetetrazole-Induced Seizures in Mice

RATIONALE: There are few existing animal models of post-traumatic epilepsy and none are both clinically relevant and highly reproducible. Our goal was to begin to examine the effects of seizure threshold and susceptibility following experimental traumatic brain injury using controlled cortical impact (CCI) in mice and the standardized pentylenetetrazole (PTZ) seizure inducibility protocol.

METHODS: A 4mm right parietal area craniotomy was performed in Kv1.1 mice, a knockout mouse model lacking one allele of the Kcna1 gene encoding a potassium channel (Kv1.1) gene and CCI (3mm tip; velocity = 3.5m/s; depth = 1.2 mm) injury induced; following which the bone was replaced using dental acrylic. Bilateral parasagittal epidural recording electrodes were implanted following a 7-day recovery period. The animals were then observed for 4 months after injury, assessed monthly with continuous EEG recording (for 1 week/ month) for spontaneous recurrent seizures. After 4 months, the rats were tested in a standardized PTZ seizure susceptibility procedure with PTZ infused into the tail vein while awake and freely moving. Times to first twitch and onset of clonus were recorded, as was the amount of PTZ required for effects. PTZ was stopped at the onset of clonus to allow for later histology.

RESULTS: CCI lowered the threshold for PTZ-induction of seizures. Mean time to first twitch was lower in CCI vs. controls (38.4 vs. 49.7 sec; p < 0.001) and to onset of clonus (53.6 sec vs. 67.4 sec, p < 0.001), respectively. The amount of PTZ necessary to induce these effects also differed: for first twitch (18.9 mg/kg vs. 23.3 mg/kg; p < 0.001) and for onset of clonus (26.4 mg/kg vs. 32.7 mg/kg (p < 0.001).

CONCLUSION: CCI lowers the threshold for PTZ-induced seizures in an experimental mouse model. The tail vein infusion PTZ test has potential for the study of TBI-induced epileptogenesis.

VanLaeken Amanda 1 Ardito Tara 1 Hockey Kevin 2 Rzigalinski Beverly 2 Whiting Mark 1

1 Radford University, Radford, VA, USA 2 Virginia College of Osteopathic Medicine, Blacksburg, VA, USA P174 (-)-Epigallocatechin Gallate Improves Outcome Following in Vitro and in Vivo Traumatic Brain Injury

INTRODUCTION: Flavonoids such as (-)-epigallocatechin gallate (EGCG), the primary catechin found in green tea, may have beneficial effects following nervous system injury. The present studies investigated the application of EGCG in both in vitro (stretch injury) and in vivo (fluid percussion) models of traumatic brain injury (TBI).

METHODS: In our in vitro studies, mixed organotypic neuronal cultures were pretreated with EGCG or vehicle followed by moderate or severe stretch injury. Neurons were assessed for cell damage, lipid peroxidation, and response to glutamate stimulation. In our in vivo studies, adult male and female rats were administered moderate fluid-percussion TBI and treated with 50 or 100mg/kg EGCG 15min or 3hr post-injury. Behavioural assessment included vestibulomotor testing (beam balance) and assessment of cognitive function (water maze).

RESULTS: Our in vitro studies suggest that EGCG treatment reduced the number of injured cells as measured by propidium iodide uptake, reduced lipid peroxidation by 50%, and attenuated the abnormal response to glutamate stimulation typically seen in neurons following stretch injury. Our in vivo studies suggest that post-injury EGCG treatment improved both behavioural and histological outcome when EGCG was administered 15min post-injury. Benefits of EGCG were reduced when treatment was delayed 3hr post-injury, and the observed benefits were significantly diminished in females.

CONCLUSION: Our in vitro and in vivo studies provide support for EGCG as a neuro- and behaviourally-protective agent for treating TBI. The narrow therapeutic window observed in this study, combined with the results of our in vitro studies, suggests that the benefits observed following EGCG treatment may be due to protection from early-stage post-injury pathophysiological events, such as intracellular free calcium deregulation and lipid peroxidation. Treatment beyond the early post-injury stage will likely require extended, multiple doses in order to realize the benefits of EGCG therapy.

P175

Withdrawn

Crawford Fiona 1 2 Crynen Gogce 1 Reed Jon 1 2 Mouzon Benoit 1 2 Bishop Alex 1 Katz Benjamin 1 Ferguson Scott 1 2 Phillips John 1 Roses Allen 3 Mathura Venkatarajon 1 2 Mullan Michael 1 2 1 Roskamp Institute, Sarasota, FL, USA 2 James A. Haley VA Hospital, Tampa, FL, USA 3 R. David Thomas Executive Training Center, Durham, NC, USA P176

Proteomic Identification of Biomarkers and Target Therapeutic Pathways in Traumatic Brain Injury

Traumatic brain injury (TBI) is a significant cause of mortality and acute chronic morbidity and is cited to be the leading cause of death and disability in more physically active populations. Currently, there are no effective therapies to treat TBI, especially the multiple long-term consequences which can persist and progress for years. By gaining a more complete understanding of the neurodegenerative and neuroreparative mechanisms triggered in the brain in response to injury, it will be possible to identify or design more effective treatments. Moreover, the current lack of diagnostic and prognostic biomarkers for TBI confounds management of patients is of increasing concern as the TBI population grows. We have used a TBI mouse model and a quantitative proteomics approach to generate plasma and brain proteomic profiles and thus identify potential peripheral biomarkers of injury and cellular mechanisms triggered in response to TBI. We have carried out these studies in mice transgenic for different isoforms of human APOE to discriminate between the cellular mechanisms associated with a favorable (APOE3) versus unfavorable (APOE4) outcome after TBI. Our data identify significant changes in expression of many proteins in the mouse plasma, hippocampi and cortices at 24hrs, 1 month and 3 months after TBI, including proteins that were significantly differently modulated in APOE3 compared to APOE4 mice. The plasma protein changes reveal both Injury-dependent (diagnostic) and Genotype*Injury interaction-dependent (prognostic) biomarkers even as late as 3-months after injury. The datasets of brain proteins showing significant changes in expression have been mapped onto known molecular relationships to determine the functional significance of the observed changes. These extensive datasets reveal molecular pathways that are dependent upon TBI, TBI severity, time-post-TBI, brain region and APOE genotype, many of which may represent targets for therapeutic intervention.

Mouzon Benoit 1 Chaytow Helena 1 Bachmeier Corbin 1 Stewart William 2 Crawford Fiona 1

1 Roskamp Institute, Sarasota, Florida, USA 2 Southern General Hospital, Glasgow, Scotland, United Kingdom P177 Pathological and Behavioral Outcome in a Mouse Model of Single and Multiple Concussions

Concussion or mild traumatic brain injury (mTBI), is the most common type of TBI. Despite its prevalence, mTBI has only recently become accepted as a major health issue since the intense media attention regarding the high incidence of TBI in military conflicts and in professional athletes. The purpose of this study is to develop and characterize a novel non invasive mTBI model in rodents that replicates the pathological components or phases of human clinical mTBI.

To evaluate the structural and functional changes associated with mTBI, 48 male C57BL/6J mice were divided into groups of receiving a single hit or multiple hits, with appropriate sham mice to control for the effects of repeated anesthesia. Compared to sham-injured mice, brain-injured mice exhibited significant deficits in short term memory measured using the Barnes maze over the last three days of acquisition (p < 0.05). Sensorimotor functions following mTBI were evaluated with the Rotarod test. Mice receiving multiple concussions were not able to recover their baseline performance and did significantly worse than their sham counterparts at day 7 (p < 0.05). Brain pathology was also examined with various putative biomarkers. Among those, glial fibrillary acid protein immunohistochemistry was found to be increased in the multiple injury group (p < 0.05).

These data validate our model of mTBI within a temporal window of vulnerability to repetitive mTBI resulting in behavioral dysfunction. This model has several advantages over currently available rodent models of TBI like controlled cortical impact, weight drop, or the fluid percussion model. It is of particular interest to investigate military or sports related concussions, where the soldier/athletes usually receive multiple hits over a relatively short period of their lifetime. Furthermore, the location of the hit on the midline suture allows the whole brain to be used for biochemistry and pathology analyses.

Ferguson Scott 1 2 Mouzon Benoit 1 2 Phillips John 1 Crynen Gogce 1 2 Chaytow Helena 1 Bishop Alex 1 Abdullah Laila 1 Mullan Myles 1 Mullan Michael 1 2 Crawford Fiona 1 2

1 The Roskamp Institute, Sarasota, FL, USA 2 James A Haley Veterans' Hospital, Tampa, FL, USA P178 CD40 Ligand Deficiency Improves Rate of Functional Recovery Following Traumatic Brain Injury

PURPOSE: Traumatic brain injury (TBI) has been diagnosed in 178,876 service members from 2000 to the first quarter of 2010. The neurobehavioral sequelae of TBI persist long after the injury, which consists of both a primary insult to the brain as well as a secondary injury that occurs in the hours and days thereafter. In order to find targets for therapeutic intervention we have analyzed the proteomic profile of the response to injury by Apolipoprotein E (APOE) transgenic mice. Polymorphisms in APOE are known to impact the outcome after TBI, with the APOE4 allele (and concomitant ApoE4 expressed protein) associated with worse outcome than APOE3 following TBI.

Proteomic analysis of the response to injury by APOE transgenic mice revealed multiple pathways differentially regulated by genotype following injury, suggesting a potential role of those pathways in modulating the outcome from injury. One of these pathways involved CD40-related molecules. CD40 and its ligand have been shown to be upregulated in patients following cerebral ischemia (Garlichs et al, 2003). Given that ischemic conditions are known to occur in the brain following TBI, we used CD40 ligand (CD40L) knockout mice to study the effect of CD40 signaling in a controlled cortical impact (CCI) model of TBI.

METHODS: 24 CD40L knockout and 24 wild type mice were given a CCI or a craniotomy-only as sham controls. Rotarod testing was used to evaluate neuromotor performance and the Barnes maze was employed to test spatial learning and memory.

RESULTS: Our results showed that CD40L knockout mice showed an improved rate of motor recovery following TBI compared to wild type controls, as well as improved performance on both the Rotarod and Barnes maze tasks overall.

References: Garlichs, C. D. et al, 2003. Upregulation of CD40-CD40 ligand (CD154) in patients with acute cerebral ischemia. Stroke. 34, 1412-1418.

Mitchell Brian Byrne Richard Rush Medical College, Chicago, IL, USA

P179 Temporal Lobectomy: A Human Lesion Study into the Mechanisms of Sleep, Dreams, and Memory Consolidation

Temporal Lobe Epilepsy (TLE) is characterized by the occurrence of focal seizures resulting in global modification of neural functioning. TLE is associated with progressive deficits in three aspects of long-term memory function: formation, consolidation, and retrieval. The progression of these deficits is directly correlated with the progression of the disease. Patients with TLE may experience seizures during sleep. These commonly occur during non-REM (NREM) sleep and are associated with a disruption of the transition between NREM and REM stages of sleep. Dreaming occurs during REM sleep. The alternation between dreaming (REM) and non-dreaming (NREM) states during sleep is implicated as being primarily responsible for the introduction of relative synaptic strength and memory salience, that is, memory consolidation during sleep. The hippocampus is essential to each of these three processes and is completely or partially removed during temporal lobectomy.

A retrospective review of the effects of temporal lobectomy on memory consolidation, sleep, and dreams will be performed through the compilation and analysis of the post-operative, mail in surveys from 200 patients. All the data received from the surveys will then be combined with Rush University's extensive Surgical Epilepsy Database. These results will be compared with data collected from two control groups: patients who underwent epilepsy and brain tumor surgery not involving the temporal lobe.

The connection between sleeping, dreaming, memory consolidation, and the hippocampus has never been explored before using a lesion study. Patients with TLE are excellent models to study such a relationship because the induction of a temporal lobe (and hippocampal) lesion is an excellent treatment option for patients with chronic TLE. In effect, the performance of this study involves the treatment (and possible cure) of hundreds of cases of chronic TLE, while allowing this team to study a poorly understood relationship between anatomical and cognitive substrates.

Yost Ann-Marie 1 Seibert Pennie 1 2 Schommer Julie 1 Connley Madeline 1

1 Saint Alphonsus Regional Medical Center, Boise, ID, USA 2 Boise State University, Boise, ID, USA P180 The Treatment and Evaluation of Clinically Symptomatic Vasospasm in a Case of Traumatic Brain Injury

INTRODUCTION: Vasospasm occurs in an estimated 30-40% of traumatic brain injury (TBI) cases, with the occurrence of clinically symptomatic vasospasm uncommon. The exact pathophysiology of vasospasm remains unknown despite continued research, which has focused primarily on radiographic vasospasm. Moreover, the relationship among TBI, angiographic vasospasm and symptomatic vasospasm is ambiguous, and the varying nature of TBIs complicates analysis of the pathophysiology of clinically symptomatic vasospasm.

METHODS: We detail the unusual case of a 44 year-old female who sustained a TBI and subsequently experienced clinically symptomatic vasospasm. CT revealed hemorrhagic contusion in the anterior left temporal lobe and subarachnoid blood within the left sylvian fissure and left cerebral convexity. Shortly following the injury she was found to have abnormal flexion to painful stimuli, no verbal response, and her eyes opened to verbal command. She had a history of ethanol abuse and mental illnesses with subsequent ethanol withdrawal complicating evaluation for clinical signs of vasospasm.

RESULTS: A CT angiogram seven days post-trauma revealed significant vasospasm of the left M1. Hypertensive hemodilutional hyperdynamic (HHH) therapy was initiated but then tapered after six days due to clinical improvement. The following day the vasospasm worsened and she began having neurological decline. Angioplasty was conducted of the left ICA, left M1, and left M2, resulting in clinical improvement during subsequent evaluations. All radiographic evidence of vasospasm was resolved by day 20. Three months post-trauma she showed improvement in symptoms although neurological deficits continued.

DISCUSSION: This case demonstrates an unusual onset of clinically symptomatic vasospasm following TBI. Additionally, it provides an exemplar for radiographic evaluation and treatment of clinically symptomatic vasospasm when clinical evaluation is complicated by substance abuse and mental illnesses. Further research regarding clinically symptomatic vasospasm is needed to establish reports in the literature to ultimately improve evaluation and treatment of vasospasm.

Bunton Amanda Clayton Elyse Hoane Michael Southern Illinois University, Carbondale, IL, USA

P180 The Functional Recovery Effects of Nicotinimide Treatment Prior to Traumatic Brain Injury

Nicotinimide (vitamin B₃) has been shown to have neuroprotective effects when administered post-injury. Additionally, several other B-group vitamins have been shown to improve function after traumatic brain injury (TBI). In the present study, the pretreatment of rats with nicotinimide was examined in order to determine if this treatment regimen would improve behavioral recovery or cortical sparing after a controlled cortical impact (CCI) injury. Thirty Sprague Dawley rats received five intraperitoneal injections of nicotinamide (75 mg/kg) or saline beginning 48 hours prior to CCI. The last injection was administered 30 minutes immediately prior to CCI. The rats were then given either a unilateral CCI over the hippocampus or sham procedure. Beginning on Day 2 post CCI, the rats were tested on several behavioral tasks: the bilateral tactile removal task, the locomotor placing task, Rotor-Rod, and the Morris water maze (MWM). Following behavioral testing, the rats were perfused and the brains were removed for histological analysis. They were sliced on a frozen microtome, mounted on slides for staining, and analyzed for lesion comparison. In general it appears that the pretreatment regimen of nicotinimide greatly enhanced behavioral recovery on the sensorimotor and motor tasks. However, only marginal effects were observed in the MWM. There also appeared to be only a marginal reduction in tissue loss in the nicotinimide-treated group compared to the vehicle. Although, some beneficial effects were observed in the present study it appears that this current pretreatment regimen does may not be as efficacious when compared to previous studies with post-injury nicotinimide administrations.

Bae Kwang Soo 1 Park So Young 1 Kim Yu Jin 1 Lee Eui Jung 2

1 Seoul National University, Bundang Hospital, Sungnam-si, Gyeonggi-do, Republic of Korea, ²Jeju National University College of Medicine, Jeju, Republic of Korea P181 Value of Repeat Brain CT in Head Injured Children Visiting ED Without Traumatic Brain Injury in Initial CT

PURPOSE: Unnecessary CT imaging is avoided in children due to risks of radiation hazard. Our aim was to assess the value of a repeat brain CT in children with minor head injury (MHI) without traumatic brain injury confirmed initial CT in ED.

METHODS: A retrospective study performed within a single emergency department between May 2003 and April 2010. Enrolled patients younger than 15 years old, presensting blunt head trauma with GCS 13-15 who met our criteria of TBI (Intracranial haemorrhage or contusion, Diffuse axonal injury, Midline shift or brain herniation, Diastasis of the skull, Pneumocephalus, Skull fracture depressed by at least the width of the table of the skull) confirmed in initial CT. Outcome data included newly diagnosed TBI in repeated CT, neurosurgical intervention, and neurologic outcome on discharge.

RESULTS: A total of 3527 children were assessed whose mean age was 5.61 ± 4.16 years. 1154(32.7%) were female. 3520(99.8%) were included in GCS 15. Cases of skull fracture without TBI in initial CT were 128(3.7%). Patients with repeated CT were 57(1.6%) (average 2.08 per person). Cases of skull fracture in initial CT were 12(21.1%) with average 2.33 per person. There were 45(1.3%) repeated scanned patients without TBI and skull fracture in initial CT scan (average 2.02 per person). Only 2 cases were newly developed TBI in repeated CT. The positive and negative predictive values, sensitivity and specificity were 3.5%, 100%, 100%, and 98.38%, respectively. All of 2 patients had no skull fracture and recovered without neurosurgical intervention.

CONCLUSION: Repeated brain CT, in children with a MHI without TBI evidence in initial CT scan, resulted in newly developed TBI rarely(3.5%), and neurosurgical intervention were not indicated. Especially those who had skull fracture, had no TBI in repeated brain CT regardless of high rate of CT repetition.

Park Gwan Jin 1 Shin Sang Do 1 Song Kyoung Jun 2 Kwak Young Ho 1 Park Chang Bae 1

1 Department of Emergency Medicine, Seoul National University College of Medicine, Seoul, Korea, Republic of 2 Department of Emergency Medicine, Seoul National University, Boramae Medical Center, Seoul, Republic of Korea P182 A Risk Prediction Model for Determining Early Mortality Following Traumatic Brain Injury

We developed and validated practical prognostic models for short term mortality (death at day 1 and 3) after traumatic brain injury. We identified moderate-severe traumatic brain injury (GCS ≤ 13) patients aged over 18 years from a 7 ED based Traumatic Brain Injury Network (2008-2010). We excluded cases with unknown hospital outcomes. Candidate predictors were gender, age, pupil reflex, motor GCS, presence of shock (systolic blood pressure < 90mmHg), abnormal brain CT findings, and injury mechanism. Outcome was defined as early mortality at day 1 and 3. Multivariable logistic regression was used to select variables associated with mortality. We measured the area under the curve (AUC) to assess the performance of the model. Among a 2-year adult TBI patients of 52,160 cases, a total of 742 patients including severe (GCS ≤ 8, n = 373, 50.3%), and moderate (9 ≤ GCS ≤ 13, n = 369, 40.7%) TBI met inclusion criteria. The mortality at day 1 and 3 were 14.6% (n = 108) and 17.5% (n = 130), respectively. This model included 5 outcome predictors: age, pupil reactivity, motor GCS, shock, and the presence of brain CT lesion. The adjusted ORs of 1 day mortality were 3.17(no pupil reactivity, 95% CI 1.41-7.11), 1.67 (motor GCS, 95% CI 1.34-2.08), 9.11 (presence of shock, 95% CI 4.79-17.3), 2.12 (abnormal brain CT, 95% CI 0.72-6.21), respectively. The measured area under the ROC curve was 0.92 (95% CI 0.89-0.95). The adjusted ORs of 3 day mortality were 1.58 (age, 95% CI 1.24-2.02), 1.55 (motor GCS, 95% CI 1.29-1.88), 3.52 (no pupil reactivity, 95% CI 1.7-7.31), 8.31 (shock, 95% CI 4.48-15.43), 3.37 (abnormal brain CT, 95% CI 1.17-9.76), respectively. The measured area under the ROC curve was 0.92 (95% CI 0.90-0.94). Age, pupil reactivity, motor GCS, the presence of shock, and abnormal brain CT lesion were the predictors of early mortality of moderate to severe TBI patients.

Gao Xiang Ding Xinchun Chen Jinhui SNRI, Indianapolis, IN, USA

P183 Notch Signaling Regulates Dendrite Development of Adult-Born Neurons Through Mtor Pathway

Although traumatic brain injury (TBI) causes significant cell death in the cortex and hippocampus, most neurons survive the initial insult. Our recent study revealed that a significant number of spared neurons exhibited dendritic degeneration and synaptic elimination following TBI. The number of neurons that experience dendrite degeneration is hundreds of times greater than the number of neurons lost in the hippocampus following moderate TBI. Since dendrites provide enormous surface area for spine formation and determine the range and scope of synaptic inputs, dendritic degeneration following TBI could cause significant disruption in synaptic transmission between neurons, in turn, contributing to neurological disorders. Understanding molecular mechanisms that regulate dendrite outgrowth in the postnatal brain may lead to novel approaches to enhance dendrite regeneration following TBI. Recently, we found that conditional knockout of Notch1 in postnatal born neurons attenuated mTOR activity and reduced dendrite arborization. In contrast, conditional activation of Notch signaling increased dendrite arborization. These results suggest that Notch signaling regulated the activity of the mammalian target of rapamycin (mTOR) pathway and played novel roles in enhancing dendrite arborization of neurons in the postnatal brain. The importance of Notch in dendrite arborization may open a potential for repairing the damaged brain through regrowth of degenerated dendrites even in the adult, a concept previously thought restricted to the developing brain.

Gao Xiang Chen Jinhui SNRI, Indianapolis, IN, USA

P184 Moderate Traumatic Brain Injury Promotes Cell Proliferation Without Increasing Neurogenesis in the Adult Hippocampus

It has been shown that traumatic brain injury (TBI) promotes neural stem/progenitor cell (NPC) proliferation in the adult hippocampus. However, whether TBI increases neurogenesis in the adult hippocampus is still not conclusive. By tracing the fate of newly divided cells one month following TBI with Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) labeling, we found that the number of survived BrdU-positive cells increased in the hippocampus of TBI injured mice compared to the sham control; however, further studied using double immunostaining to distinguish their cell types showed that most of the survived BrdU-positive cells in the hippocampus were glia. Only a small subpopulation of them differentiated into granular neurons and there was no significant difference of neurogenesis in the adult hippocampus between injured and control mice. These results indicated that TBI promoted cell proliferation, including astrocyte activation and NPC proliferation. Nevertheless, majority of those proliferated and survived BrdU-positive cells are astrocytes, and neurogenesis in the adult hippocampus did not significantly increased following TBI. These data suggested that an innate repair and/or plasticity mechanisms in the brain is activated by TBI through promoting NPC proliferation, however, additional intervention is required to increase neurogenesis for successfully repairing the damaged brain following TBI.

Gao Xiang Chen Jinhui SNRI, Indianapolis, IN, USA

P185 Extensive Neural Degeneration: A Neuropathological Basis for Neurological Disorders Following Mild TBI

Although mild traumatic brain injury (mTBI) leads to long-term cognitive and emotional difficulties and behavioral disturbances, its diagnosis and treatment have been histologically hampered by a lack of evidence-based substantiations of these implications. Unlike moderate and severe TBI, mTBI does not show significant tissue lesions or cavity in the cortex, and neuroimaging by magnetic resonance imaging (MRI) or computed tomography (CT) is usually negative, suggesting that the damage is beyond the resolution of structure-based scanning technologies. Therefore, we investigated at high resolution and in great detail the morphologies of spared neurons in the cortex following mTBI using a controlled cortical impact (CCI) injury model. Our results showed that although mTBI caused only mild cell death, it led to extensive dendrite degeneration and synapse reduction. These data elucidate the pathological basis for the impaired cognitive function following mTBI in animal model. This experimental study sheds light on the neuropathology of mild TBI in humans, and suggests that neurodegeneration may be a novel target for developing diagnostic methods and therapeutic approaches for mTBI in the future.

Swan Alicia Hoane Michael Southern Illinois University, Carbondale, USA

P186 Never Too Late to Learn: Cognitive Training Improves Functional Recovery of Cognitive Behaviors Following Traumatic Brain Injury in Aged Animals

Age at the time of injury is a consistent predictor of poor outcome following traumatic brain injury (TBI). Although aged individuals are more likely to endure a TBI and have a higher rate of hospitalization, institutionalization, and death following injury, few preclinical studies examine altered injury sequelae or potential therapies following injury. Research indicates that environmental enrichment may have a particularly robust effect for aged individuals, although investigations of the model for this population have been limited. This study was conducted to investigate the relationship of cognitive ability prior to injury and the potential benefit of a novel cognitive rehabilitative training procedure following injury in 22-month old Fischer 344 rats. Prior to TBI, animals were evaluated in a Morris water maze (MWM) reference memory paradigm to establish cognitive status as impaired or unimpaired. Animals were then assigned to either a controlled cortical impact (CCI) centered over the hippocampus or sham condition. The rats were also assigned to receive either cognitive rehabilitation or control condition. Animals in the cognitive rehabilitation condition were trained on an odor discrimination digging task that required them to dig for a reinforcer buried in scented sand. After four weeks of rehabilitative training, animals were re-evaluated in the MWM reference memory task. The findings indicate that the precharacterization showed that some rats were cognitively impaired (∼ 750 cm), while others were cognitively unimpaired (∼ 225 cm). Post-CCI, animals classified as cognitively impaired showed that the rehabilitation training significantly improved cognitive performance. Furthermore, in injured animals, the cognitive performance of the impaired cognitively rehabbed animals became indistinguishable from that of their intact counterparts later in testing, while the impaired non-rehabbed group continued to perform significantly worse. These findings indicate that cognitive rehabilitation can ameliorate cognitive impairment following TBI, even in the most aged, impaired, and vulnerable individuals.

Martens K.M. Haar Cole Vonder Hoane Michael Southern Illinois University, Carbondale, IL, USA

P187 Additional Options for Behavioral Testing in Rodent Models of TBI: A Simple Discrimination Task Used as a Novel Method of Testing Decision-Making Behavior

TBIs result in a multitude of deficits following injury. Some of the most pervasive in humans are the drastic personality changes that affect decision making. The assessment of decision-making behavior in rodents has been extensively tested in the field of behavior analysis. However, due to the narrow therapeutic window following TBI, time intensive operant paradigms are rarely incorporated into the battery of tests traditionally used; the majority of which assess motor and sensory functioning. The cognitive measures that are used are frequently limited to memory and do not account for changes in decision-making behavior. The purpose of the present study was to develop a simplified discrimination task that can be implemented into any existing battery in order to assess deficits in decision-making behavior in rodents. For the task, rats were trained on a scent discrimination task with cocoa-scented vs. unscented sand in which rats were required to dig in cocoa-scented sand for a Froot Loop reinforcer. Rats were given 12 sessions per day until a criterion level of 80% accuracy for three days straight was reached. Once criterion was reached, cortical contusion injuries were induced (frontal, parietal, or sham). Following a recovery period, rats were re-tested on cocoa vs. unscented. Upon reaching criterion, reversal discrimination was evaluated in which the reinforcer was placed in unscented sand. Finally, a novel scent discrimination (basil vs. coffee with basil reinforced) and a reversal (coffee) were evaluated. The results indicated that there was a significant difference in the cumulative percentage correct between the three groups. Specifically, differences existed between parietal vs. frontal (unscented, basil, coffee), parietal vs. sham (unscented) and frontal vs. sham (cocoa, basil). The general findings from this study suggest that the dig task is a simple experimental preparation that can be used to assess deficits in decision-making behavior following TBI.

Bunton Amanda Clayton Elyse Hoane Michael Southern Illinois University, United States

P188 The Functional Recovery Effects of Nicotinimide Treatment Prior to Traumatic Brain Injury

Nicotinimide (vitamin B₃) has been shown to have neuroprotective effects when administered post-injury. Additionally, several other B-group vitamins have been shown to improve function after traumatic brain injury (TBI). In the present study, the pretreatment of rats with nicotinimide was examined in order to determine if this treatment regimen would improve behavioral recovery or cortical sparing after a controlled cortical impact (CCI) injury. Thirty Sprague Dawley rats received five intraperitoneal injections of nicotinamide (75 mg/kg) or saline beginning 48 hours prior to CCI. The last injection was administered 30 minutes immediately prior to CCI. The rats were then given either a unilateral CCI over the hippocampus or sham procedure. Beginning on Day 2 post CCI, the rats were tested on several behavioral tasks: the bilateral tactile removal task, the locomotor placing task, Rotor-Rod, and the Morris water maze (MWM). Following behavioral testing, the rats were perfused and the brains were removed for histological analysis. They were sliced on a frozen microtome, mounted on slides for staining, and analyzed for lesion comparison. In general it appears that the pretreatment regimen of nicotinimide greatly enhanced behavioral recovery on the sensorimotor and motor tasks. However, only marginal effects were observed in the MWM. There also appeared to be only a marginal reduction in tissue loss in the nicotinimide-treated group compared to the vehicle. Although, some beneficial effects were observed in the present study it appears that this current pretreatment regimen does may not be as efficacious when compared to previous studies with post-injury nicotinimide administrations.

Peterson Todd 1 Ward Jacob 1 Logue Mahala 1 Anderson Gale 2 Hoane Michael 1

1 Southern Illinois University, Carbondale, IL, USA 2 University of Washington, Seattle, WA, USA P189 An Evaluation of the Neuroprotective Effects of Progesterone and Nicotinamide on Functional Recovery Following Cortical Contusion Injury in the Rat

The primary goal of this study was to compare clinically relevant doses of progesterone and nicotinamide (NAM; vitamin B3) within the same injury model. Progesterone has been shown to promote remyelination and regeneration of axons in the periphery, reduce edema and inflammation, and improve functional outcomes following injury. Additionally, NAM has been shown to be an effective neuroprotective treatment following ischemia, fluid percussion, and cortical contusion injuries (CCI). Numerous studies have shown that the neuroprotective effects of these two compounds are dependent, not only on the route and time of administration, but also the dose. In the current study, NAM was administered beginning 4 hours post-CCI with a loading dose (75 mg/kg, i.p.) combined with continuous infusion (12 mg/hr/kg, s.c.) for 72 hours post-injury. Progesterone was administered beginning 4 hours post-CCI at a dose of 10 or 20 mg/kg, i.p. every twelve hours for 72 hours. This resulted in the following groups: Injured-NAM treated, Injured-Progesterone-10 treated, Injured-Progesterone-20 treated, Injured-vehicle treated, and Sham. Functional recovery was assessed with a battery of behavioral tests including motor coordination (Rotor-Rod) and two spatial memory tasks in the Morris water maze. It was found that both progesterone (10 and 20 mg/kg) and NAM improved motor functioning on the Rotor-Rod test in comparison to vehicle-treated animals. Additionally, progesterone (10 mg/kg) and NAM both improved reference and working memory in the Morris water maze compared to the vehicle treatment. The beneficial effects of progesterone appear to be dose dependent with the lower 10 mg/kg dose producing stronger effects. Direct comparison between NAM and low dose progesterone appear to suggest that both are equally effective. The general findings of this study suggest that both NAM and progesterone produce significant improvements in recovery of function following CCI.

Haar Cole Vonder Emery Michael Hoane Michael Southern Illinois University-Carbondale, Carbondale, IL, USA

P190 Chronic Folic Acid Administration Does not Improve Recovery of Function in Either a Low or a High Dose Following Unilateral Controlled Cortical Impact Injury

Recent research has shown that folic acid (vitamin B₉) increases behavioral recovery and axonal regrowth in a spinal cord injury model. In addition, several other B-group vitamins have been shown to be effective in improving function after traumatic brain injury (TBI). The current study investigated whether or not folic acid administration improved behavioral recovery or provided any cortical sparing after a controlled cortical impact (CCI) injury. Thirty-seven male Sprague-Dawley rats were given a unilateral CCI over the sensorimotor cortex or sham surgery. Thirty minutes following CCI and every 24 hours after for two weeks, rats were given injections of either folic acid (80μg/kg or 800μg/kg, i.p.) or vehicle (1ml/kg saline, i.p.). Starting on day two post-CCI and continuing till day 19, the four groups (B₉-High, B₉-Low, Vehicle, Sham) were tested on various behavioral tasks, including the bilateral tactile adhesive removal, Rotor-Rod and the reference memory and working memory paradigms of the Morris water maze. Following behavioral testing, rats were perfused and brains were removed for histological analysis. Brains were sliced on a frozen microtome at 40μm and a series was mounted to slides, then stained with cresyl-violet. Lesion volumes were compared across groups to assess the effect of folic acid on cortical sparing. There were no improvements in any of the behavioral measures examined or in cortical tissue loss for either the low-dose or high-dose folic acid groups. The findings from this study suggest that folic acid may not be as efficacious of a treatment for TBI as it has been shown to be in spinal cord injury. However, variations on the dosing regimen beyond high and low dose may need to be explored. Potential follow-ups could include pre-treatment with folic acid or restricting dosing to the acute post-CCI period.

Torres Roland 1 Kouzminova Natalia Kouzminova 2 Sherck John 2

1 Dept. of Neurosurgery, Stanford univ. Medical School, Stanford, CA, USA 2 Dept. of Surgery Santa Clara Valley Medical Center, San Jopse, CA, USA P191 The Role of Hemodilution Coagulopathy in Bleedingprogression After Isolated Blunt Head Injury

INTRODUCTION: Coagulopathy after traumatic brain injury (TBI) is conventionally attributed to massive release of tromboplastin proportional to the severity of trauma and consumption of coagulation factors, but acute mild coagulopathy in patients with head trauma is not always directly related to severity of injury. We hypothesized that mild coagulopathy may be due to hemodilution, and investigated factors associated with coagulopathy in relation to clinical outcome.

METHODS: We reviewed records of 153 consecutive patients with isolated moderate to severe blunt head injuries (Glasgow Coma Scale score, 12; head abbreviated injury score, 2; extracranial abbreviated injury score sum, 2) without preexisting coagulopathies or previous anticoagulant therapy. Coagulopathy was coded “mild” if the international normalized ratio (INR) was between upper laboratory norm (ULN) and 1.5, “moderate” when the INR was between 1.5 and 2, and “severe” if the INR was more than 2.

RESULTS: Twenty-one of the 153 patients (14%) had coagulopathy on admission, and 48/153 patients (31.4%) had coagulopathy by third day of hospitalization. Spearman's test demonstrated significant correlation between drop in hematocrit and elevation of INR. In our study group, 91.6% of patients with moderate or severe coagulopathy and 48% of patients with mild coagulopathy had progression of intracranial bleeding compared with only 12.5% in patients without confirmed coagulopathy (Kruskal-Wallis test, The median time from arrival at the hospital to start of plasma transfusion in the patients with coagulopathy on admission who survived was shorter than in those who died 95 minutes v.s 150 minutes.

CONCLUSION: We demonstrated that hematocrit, reflecting the amount of blood loss and hemodilution, strongly correlates with INR in the patients with isolated TBI. Even mild coagulopathy was associatedwith intracranial bleeding progression. Earlier diagnosis of coagulopathyand faster coagulation factors replacement may lead to improved outcome in patients with post-traumatic intracranial bleeding with goal, to arrest bleeding and orrect coagulopathy early.

Ping Xingjie 1 Lee Seung Young 2 Cheng Ji-Xin 2 Jin Xiaoming 1

1 Stark Neuroscience Research Institute, Indiana University, Indianapolis, IN, USA 2 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA P192 MPEG-PDLLA Micelles Improve the Function of Cortical Unmyelinated Axons After Traumatic Brain Injury

Axonal injury is a common feature of traumatic brain injury (TBI), and is one of the most important causes of morbidity in TBI. Axonal injury begins with membrane failure and loss of ionic homeostasis, with rapid progression to neuronal cell death via necrosis and apoptosis. Here we used monomethoxy poly (ethylene glycol) - poly (D, L - Lactic acid) di-block copolymer micelles (mPEG-PDLLA) as a membrane sealant to repair the injured cortical axons following TBI. Mice between postnatal day 30-35 were subjected to severe TBI via controlled cortical impact (CCI). Mice in the treatment group received mPEG-PDLLA injection via tail vein either immediately (0 hour) or 4 hours after injury. To evaluate the function of the cortical axons, field potential recordings of evoked compound action potentials (CAPs) were made from the corpus callosum in coronal cortical slices prepared 48 hours after injury. The CAPs waveform components generated by myelinated axons (N₁ wave) and by unmyelinated axons (N₂ wave) were separately quantified in peak amplitude and covered area. CCI caused a significant decrease in average CAP amplitude and area of N₂ wave. Treatment with mPEG-PDLLA at 0 or 4 hours after CCI resulted in a significant larger amplitude and area in N₂ wave, almost reaching the levels of that in the uninjured sham group. There were no significant differences in amplitudes and areas of N₁ waves among sham, injured, and injured with treatment groups. These data indicate that intravenous administration of mPEG-PDLLA within 4 hours after TBI can effectively repair the function of unmyelinated axons.

Rom Slava 1 Elliot Melanie 2 Tang Waixing 1 Ramirez Servio 1

1 Temple University School of Medicine, Philadelphia, PA, USA 2 Thomas Jefferson University, Philadelphia, PA, USA P193 Tight Junction Complex Modification at the Blood-Brain Barrier in TBI

The pathophysiological progression in TBI is significantly compounded by cerebral vascular damage which induces permeability of the Blood-Brain Barrier (BBB) and leaves neurons vulnerable to injury. The physical composition of the BBB is characterized by the presence of specialized tight-junctions (TJ) between endothelial cells. Although BBB dysfunction has long been known in TBI, no studies have addressed the biochemical changes that occur at the TJ complexes after a TBI event. Furthermore, detection of shed TJ proteins caused by the trauma or vascular remodeling thereafter offers a novel approach for biomarker discovery. The goals of this study were to 1) characterize the degree of TJ disruption and 2) whether TJ elements could be detected in blood plasma. Using the CCI mouse model of TBI, mice were exposed to increased severity of CCI-TBI. At seven days post-injury, BBB permeability was assessed by multiphoton intravital microscopy. Also, blood was collected and used for developing a TJ specific ELISA while brain tissue was collected for histological analysis. The pathophysiology of CCI-TBI was confirmed by the increased BBB permeability, increased neuroinflammation (activated microglia) and the presence of neurodegeneration. Assessment of the TJ integrity by ZO-1, occludin and claudin-5 immunostaining revealed significant morphological disruptions. The disruptions to the TJs appeared discontinuous, punctuated, and even absent when compared to the sham control. Our results also showed the presence of phosphorylated forms (known to contribute to permeability) of the TJ proteins in vessels proximal and distal from the site of injury. Blood samples tested on a developed ELISA panel showed the presence of TJ proteins in the blood plasma of injured animals and correlated with injury severity. In sum, our study demonstrates that a significant level of TJ modification exists in TBI and that protein members of these complexes are detectable in the blood following injury.

Tharakan Binu Sawant Devendra Reilly John Hunter Felicia Childs Ed Texas A&M Health Science Centre College of Medicine and Scott and White Memorial Hospital, Temple, Texas, USA

P194 Regulation of Blood Brain Barrier Endothelial Cell Hyperpermeability by Matrix Metalloproteinases

Traumatic brain injury (TBI) is associated with blood brain barrier (BBB) disruption and brain edema. The BBB consist of tight junctions between the adjacent endothelial cells that constitute tight junction proteins (TJPs). Recent studies suggest that matrix metalloproteinases (MMPs) contribute to acute edema and lesion formation following traumatic and ischemic brain injuries. We hypothesized that the activation of MMPs following various traumatic injuries such as TBI would lead to proteolytic cleavage of the TJPs leading to hyperpermeability and brain edema. Our objective was to determine if BBB endothelial cell hyperpermeability is MMP dependent. Rat brain microvascular endothelial cell (RBMEC) monolayers grown on Transwell inserts or chamber slides were treated with activated MMP-9 in presence or absence of MMP inhibitors (doxycycline or MMP-9 inhibitor-1). The change in monolayer permeability was studied using FITC-dextran. Tight junction integrity was studied using immunofluorescence of TJPs, ZO-1, claudin-5 and occludin. The change in f-actin cytoskeletal assembly was studied using rhodamine phalloidin staining. Activated MMP-9 significantly increased RBMEC permeability compared to untreated control monolayers (p < 0.05), and this effect was prevented by doxycycline as well as MMP-9 inhibitor-1 (p < 0.05). Activated MMP-9 disrupted tight junction proteins and this effect was prevented by doxycycline as well as MMP-9 inhibitor-1. Doxycycline as well as MMP-9 inhibitor-1 prevented MMP-9-induced increase in actin stress fiber formation. These results suggest that MMP-9-mediated BBB disruption may be an important mechanism that regulates BBB endothelial cell hyperpermeability associated with TBI and related injuries.

Friess Stuart 1 Smith Colin 3 Kilbaugh Todd 1 Ralston Jill 2 Helfaer Mark 1 Margulies Susan 2

1 The Children's Hospital of Philadelphia, Philadelphia, PA, USA 2 University of Pennsylvania, Philadelphia, PA, USA 3 Western General Hospital, Edinburgh, Scotland, United Kingdom P195 Aggressive Cerebral Perfusion Pressure Augmentation with Phenylephrine Early After Traumatic Brain Injury Reduces Secondary Injury in a Pediatric Swine Model

INTRODUCTION: Cerebral hypoperfusion is an early and common phenomenon in pediatric traumatic brain injury (TBI). Cerebral perfusion pressure (CPP) less than 40 mm Hg following TBI has been associated with increased mortality independent of age, and current guidelines recommend maintaining CPP between 40-60 mm Hg. We hypothesize that targeting a CPP of 70 with the use of phenylephrine early after injury in young children will reduce secondary injury.

METHODS: After IACUC approval, 21 four week old pigs (equivalent to toddler aged child) were randomly assigned to injury with CPP = 70 mm Hg, (N = 10) or CPP = 40 mm Hg (N = 11). Diffuse TBI was produced by a single rapid rotation of the head in the axial plane. Cerebral microdialysis, brain tissue oxygen, intracranial pressure, and cerebral blood flow (CBF) were measured 20 min - 6 h post-injury. One hour after injury, CPP was manipulated with the vasoconstrictor phenylephrine. Animals were euthanized 6 h post-TBI, brains fixed, and stained with H&E or beta-APP to assess regions of cell injury (CI) and axonal dysfunction (AD).

RESULTS: Augmentation of CPP to 70 resulted in no change in AD, but significantly smaller CI volumes at 6 hours post-injury compared to CPP 40 (1.1% vs 7.4%, p < 0.05). Lactate/pyruvate ratios from microdialysis samples were improved at CPP = 70 compared to CPP = 40 (40.1 vs 24.7, p < 0.05). CBF was higher in the CPP = 70 group but did not reach statistical significance (45.7 vs 40.1 mL/100g/min). Phenylephrine was well tolerated and there were no observed increases in arterial blood lactate levels, or intracranial pressure in either group.

CONCLUSION: Early CPP augmentation with vasopressors to levels higher than currently recommended after pediatric TBI may have the potential to reduce secondary injury.

Elliott Melanie 1 Amenta Peter 1 Tuma Ronald 2 Browne Karena 1 Jallo Jack 1

1 Thomas Jefferson University, Philadelphia, PA, USA 2 Temple University School of Medicine, Philadelphia, PA, USA P196 Selective Cannabinoid-2 Receptor Activation in a Mouse Model of Traumatic Brain injury

The cannabinoid-2 receptor (CB₂R), expressed by microglial cells, astrocytes, and neurons, has been shown to possess immunomodulatory and anti-inflammatory properties in the setting of central nervous system pathology. Using a murine model of controlled cortical impact (CCI), we evaluated the effects of CB₂R stimulation on the cellular, biochemical, and behavioral outcomes at multiple time points post traumatic brain injury (TBI). Male C57BL/6 mice underwent CCI and were administered 0-1966, a CB₂R agonist, or vehicle (i.p.) at two, 24, 48, and 72 hours post-injury. Pre- and post-injury motor function and exploratory behavior were evaluated via the rota-rod, forelimb cylinder, and open-field tests. Cerebral edema was quantified using the wet-dry weight technique. Neurogenic inflammation, macrophage/microglial activation, and neuronal degeneration were evaluated with substance P (SP) and Iba-1 protein immunofluorescence and fluorojade C (FJC) labeling. Treatment with 0-1966 significantly improved motor function and exploratory activity compared to vehicle-treated mice. Cerebral edema and perivascular SP immunoreactivity were significantly reduced in treated animals. Perilesional tissue in 0-1966-treated mice demonstrated a significant decrease in the number of macrophages/ microglia when compared to vehicle-treated mice. Additionally, morphologic analysis showed these cells to more often exhibit a resting phenotype following the administration of 0-1966, while macrophages/ microglia in vehicle-treated animals more commonly displayed an activated morphology. 0-1966 treated mice had a significant reduction in the number of FJC labeled degenerating neurons compared to vehicle-treated animals. Our findings show that the immunomodulatory effects of 0-1966, on a cellular and biochemical level, are associated with significant improvements in behavioral testing in the wake of TBI. This study provides evidence of the neuroprotective effects of selective CB₂R activation that are mediated, in part, by early attenuation of neurogenic inflammation and cerebral edema following TBI. Ultimately, selective CB₂R activation may represent a novel pharmacologic target for the treatment of TBI.

Elliott Melanie Amenta Peter Awe Olatilewa Browne Karena Jallo Jack Thomas Jefferson University, Philadelphia, PA, USA

P197 Persistent Trigeminal Sensitization in a Murine Model of Mild Controlled Cortical Impact

Headache is a common neurological disorder in patients with traumatic brain injury or patients undergoing craniotomy. Neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP) are well-known for their roles in mediating headache. In our previous study using a murine model of controlled cortical impact (CCI), we reported early (48 hr) changes in SP in the injured cerebral cortex and spinal trigeminal tract. In the present study, we characterized biochemical and behavioral markers for abnormal activation of the trigeminovascular system in mice undergoing mild CCI and craniotomy only. Male C57BL/6 mice received craniotomy or CCI followed by periorbital mechanical sensory testing with von Frey monofilaments on postoperative days seven to 28. CGRP levels in the brain stem were evaluated using ELISA. SP and glial activation (Iba1⁺ microglia and GFAP⁺ astrocytes) were evaluated using immunohistochemistry. Our results show CCI induces persistent bilateral periorbital mechanical hypersensitivity that intensifies over time compared to craniotomy and naïve mice. CCI induces increases in CGRP and SP in brain stem compared to craniotomy and naïve mice. CGRP levels continued to increase while SP declines over time. Increases in glial activation in the somatosensory cortex persist up to 14 days and resolves by 28 days. Mice with craniotomy showed periorbital mechanical hypersensitivity, increased CGRP and SP along with glial activation for up to post-operative day 14, however, changes were significantly less when compared to CCI. Our study shows CCI injury results in chronic periorbital mechanical hypersensitivity accompanied by persistent CGRP and SP increases in the trigeminal system. Findings provide evidence of abnormal trigeminal activation in mice with CCI and craniotomy. In conclusion, CCI injury and craniotomy represent a novel means by which to study post-traumatic headache and post-craniotomy headache.

Fan Weijia Huang Huiling Huanhu Hospital, Tianjin, China

P198 Effect of Taurine on Enzymes of Mitochondrion Following Traumatic Brain Injury in Rats

OBJECTIVE: Study the effect of taurine on respiration chain enzymes activity of brain mitochondria in rats after 24h following with severe traumatic brain injury (TBI).

METHODS: 56 rats were randomly divided into 7 groups: sham, TBI, taurine (TAU), prevented (pre-TAU), beta-Alanine (Ala), Vitamin C (Vc), folic acid (FA)group. All animals were subjected to lateral fluid percussion brain injury except sham group. Taurine, Vc, beta-Alanine, folic acid (200mg/kg) were injected through caudal vein after traumatic brain injury, while normal saline was given in sham and TBI group. Mitochondria were extracted immediately from brain tissue after 24 hours by using mitochondrial extract kit. Then SDH, COX, NADH, ATPase activity of mitochondrial were detected by analyzed kits.

RESULTS: The activity of SDH, COX, NADH, ATPase were significant lower in TBI group compared those in sham group (1.65 ± 0.17 vs 2.06 ± 0.25; 0.231 ± 0.01 vs 0.241 ± 0.01; 28.54 ± 1.49 vs 36.24 ± 1.72; 3.51 ± 0.15 vs 3.83 ± 0.45) and in TAU group (1.65 ± 0.17 vs 2.00 ± 0.115; 0.231 ± 0.01 vs 0.246 ± 0.03; 28.54 ± 1.49 vs 39.43 ± 6.25; 3.51 ± 0.15 vs 3.89 ± 0.37); COX, NADH in pre-TAU group (0.231 ± 0.01 vs 0.244 ± 0.03; 28.54 ± 1.49 vs 38.49 ± 5.95), SDH in Vc group (1.65 ± 0.17 vs 1.94 ± 0.26), NADH in FA group (28.54 ± 1.49 vs 39.33 ± 7.96) were increased and had statistic significance compared TBI group.

CONCLUSION: The mitochondrial enzymes activity of brain tissue was impaired after traumatic brain injury. Treatment of taurine could improve the state of brain mitochondrial respiration chain enzymes activity and protect mitochondria from impairment.

Huang Huiling Cai Ying Zhang Xuebin Chang Xiaoli Fan Weijia Tianjin Neruosurgical Institute, Tianjin, China

P199 Protection of Taurine on the Brain Edema and Expression of AQP4,SCN5A,SCA7A After Traumatic Brian Injury in Rats

OBJECT: The goal of this study was to investigate the therapeutic effects of taurine on Traumatic brain injury (TBI) by reducing the brain edema and modifying the expressing of AQP4, SCN5A and SCN7A.

METHODS: Forty Sprague-Dawley rats were randomly divided into four groups: sham group, TBI group, Tau-L group(low taurine dosage, 100mg/Kg) and Tau-H group(high taurine dosage, 200mg/Kg). All animals were subjected to lateral fluid percussion brain injury except sham group; taurine was injected intravenously (tail vein) right after TBI, while normal saline was given in sham and TBI group. After 24 hours, Brain water content was determined. AQP-4 mRNA and protein expression, SCN5A, SCN7A mRNA expression were analyzed by real-time PCR and western blotting technique respectively.

RESULTS: The brain water content was significantly higher in the TBI group than that of surgical controls (83.74 ± 1.29 vs 78.95 ± 0.56, p < 0.01) and was decreased in Tau-H and Tau-L group compared TBI group (80.57 ± 1.42 and 82.09 ± 1.38 vs 83.74 ± 1.29, p < 0.01), and there was much difference in the two inventional groups. Compared with the Sham group, the mRNA level and protein expression of AQP4 in TBI group were upregulated significantly. Taurine could recovery it (1.05 and 0.98 fold than sham group). brain water content had positive correlation with AQP4 mRNA level (r = 0.499, p = 0.001) and protein expression (r = 0.459, p = 0.024). The mRNA level of SCN5A in TBI group were upregulated significantly compared with the Sham group, (13.35 ± 5.67 vs 2.35 ± 0.86, p < 0.01). Taurine could recovery it (Tau-H group: 1.64 ± 0.64 vs 2.35 ± 0.86, Tau-L group: 1.59 ± 0.49 vs 2.35 ± 0.86). SCN7A mRNA level was decreased significantly in two interventional groups compared with the TBI group. (Tau-H group: 47.93 ± 16.97 vs 16.93 ± 7.38, p < 0.01; Tau-L group: 47.93 ± 16.97 vs 19.23 ± 7.40 p < 0.01).

CONCLUSIONS: Taurine reduced significantly brain edema in rats and inhibited upregulation of mRNA and protein expression of AQP4, SCN5A, SCN7A after TBI. So taurine showed a well therapeutic effect and may be a potential protective agent for TBI.

Villapol Sonia 1 Yaszemski Alexandra 1 Sanchez-Lemus Enrique 2 Saavedra Juan M 2 Symes Aviva 1

1 Center for Neuroscience and Regenerative Medicine, and Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA 2 Section on Pharmacology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA P200 The Angiotensin Receptor Blocker, Candesartan, Improves Recovery After Traumatic Brain Injury in Mice

PURPOSE OF THE STUDY: Traumatic brain injury (TBI) results in complex pathological reactions. Damage from the initial lesion is worsened by secondary inflammation and edema. Angiotensin II is produced in the brain and is known to be vasoconstrictive and pro-inflammatory. Drugs that block angiotensin II receptor function – angiotensin II receptor blockers (ARBs) are neuroprotective in models of stroke. However, little is known about the response of the Renin Angiotensin System (RAS) after TBI.

METHODS USED: We performed moderate controlled cortical impact (CCI) injury on 8-week-old male C57BL/6 mice (3.6 m/s, 2 mm depth, 100 msec dwell time), and sacrificed mice at 3 or 28 days post injury (dpi). We then sought to determine if the angiotensin receptor blocker (ARB), candesartan, would mitigate the effects of the injury. Mice were implanted with an osmotic minipump containing candesartan (1mg/kg/day) or vehicle, 5 hours before CCI injury. Rotarod test and Morris water maze test were performed at different time points after injury.

SUMMARY OF THE RESULTS: We found angiotensin receptor type 1 (AT1R) expression upregulated in the damaged cortex and hippocampus after 3dpi. Candesartan treatment reduced the lesion volume after CCI injury by approximately 50%. The number of dying neurons labeled by TUNEL staining was also significantly reduced. Behavioral assays indicated that CCI injured mice given candesartan demonstrated significantly longer latencies on the rotarod, and show a trend towards improved performance in the Morris Water Maze.

CONCLUSION REACHED: Treatment with the ARB, candesartan reduces lesion size and neuronal cell death, and also improves motor recovery after TBI mice model. Thus, candesartan, and potentially other ARBs, may have efficacy for treating TBI.

Selwyn Reed Hockenbury Nicole Jaiswal Shalini Mathur Sanjeev Byrnes Kimberly Uniformed Services University, Bethesda, MD, USA

P201 Mild Traumatic Brain Injury Results in Sustained Hypoglycolysis: An ¹⁸FDG Pet Study

Moderate to severe traumatic brain injury (TBI) in humans and rats induces measurable metabolic changes, including a sustained depression in cerebral glucose metabolism. In rodent studies using a moderate lateral fluid percussion (LFP) injury, glucose metabolism as measured by positron emission tomography (PET) remained depressed for up to 10 days. However, the effect of a mild TBI on brain glucose metabolism is currently unknown. Therefore, this study aimed to determine the glucose metabolism pattern in the brain after a mild TBI. Briefly, adult male rats were subjected to LFP (1.2 + /− 0.1 atm) or a closed head impact (2mm deformation, 5m/s impact speed, without skull damage). PET imaging with ¹⁸FDG was performed prior to injury and at 3, 8, 24, and 48 hours and 5, 9 and 16 days post-injury. In addition, locomotor function was assessed prior to injury and 1, 2, 7, 14 and 21 days after injury using modified beam walk tasks. PET imaging revealed that, contrary to a moderate or severe TBI, mild LFP-induced TBI does not result in a significant peak in glucose metabolism over sham-injured controls within a few hours after injury. However, from 24 hours to 9 days post-injury, glucose metabolism is depressed in comparison to sham-injured and naïve controls. Investigation of ipsilateral versus contralateral metabolism demonstrated that this depression was initially localized to the ipsilateral cortex, but had spread to the contralateral cortex by 48 hours post-injury. In contrast, closed head injury did not result in a measurable depression in glucose metabolism. Analysis of function revealed a negative correlation between beam walk performance and brain metabolism at 24 hours post-injury. In conclusion, we now show that mild TBI results in an observable depression in brain glucose metabolism that correlates with functional deficits during early time points post-injury.

Leonessa Fabio 1 Kitces Eleanor 1 Prieto DaRue 2 Chan King 2 Issaq Haleem 2 Veenstra Timothy 2 Kovacs Krisztian 1 Murphy Erin 1 Ecklund James 3

1 Uniformed Services University of the Health Sciences, Bethesda, MD, USA 2 SAIC-Frederick, Frederick, MD, USA 3 Inova Fairfax Hospital, Falls Church, VA, USA P202 Identification of Potential Novel Plasma Biomarkers of Closed Head and Penetrating Ballistic Traumatic Brain Injury by Comparative Count of Unique Peptides

OBJECTIVE: To identify novel potential candidate biomarkers of closed head and ballistic penetrating traumatic brain injury (TBI).

METHODS: Plasma samples were collected transcardially at euthanasia from one hundred four Sprague-Dawley 15 min, and1, 6, 24 and 72 hours following lateral fluid percussion, penetrating ballistic brain injury or the respective control treatments (sham, anesthesia only and naïve). Each experimental group, as defined by injury/equivalent and survival time included 5-7 rats. Following abundant protein depletion, samples were digested with sequencing grade modified trypsin. Strong cation exchange fractions of the protein plasma digests were then analyzed by liquid chromatography-tandem mass spectrometry. Proteins were identified by at least two unique peptide sequences. Potential candidate biomarkers of TBI were identified by comparing the number of unique peptides per protein across different experimental groups. Potential candidates were prioritized by statistical significance of differences and biological significance.

RESULTS: 10,217 proteins were identified by at least two unique sequences across 104 plasma samples; 1500-2500 proteins per sample. Based on the results, we have identified 24 nervous tissue proteins as potential novel candidate biomarkers of closed head and/or penetrating ballistic TBI.

CONCLUSIONS: As an alternative for the preliminary identification of candidate biomarkers, a comparative count of unique peptide sequences detected per protein has the potential advantages of allowing for the comparative screen of a wider number of proteins, and of an intrinsic bias against more abundant proteins. Ongoing evaluations with antibody-based assays are aimed to obtain proof of concept for the validity of this identification approach, as well to verify some of the identified proteins as novel candidate biomarkers of TBI.

Support: Defense Advanced Research Project Agency

Kovacs S. Krisztian 1 Murphy Erin 1 Kitces Eleanor 1 Magnuson John 1 Parks Steve 2 Swauger Peter 2 Ecklund James 3 Leonessa Fabio 1

1 Uniformed Services University Of The Health Sciences, Bethesda, MD, USA 2 Ora Inc., Fredericksburg, VA, USA 3 Inova Fairfax Hospital, Falls Church, VA, USA P203 Evidence of Brain Injury in Rats Exposed to the Blast Wave Generated by the Detonation of High Explosive

OBJECTIVE: Characterizing the brain neuropathological and neurobehavioral outcome of exposure to high explosive-driven blast overpressure and to identify related thresholds in a rat model.

METHODS: Anesthetized Sprague-Dawley rats were exposed to a range of primary blast intensities (incident pressure peaks: 18-52 p.s.i., pulse duration 5-10msec) as generated by the detonation of different charges of sensitized liquid nitromethane within a cylindrical blast wave generator (test section: 50′x 6′). Animals were exposed with back facing the blast source. With the exclusion of head and neck, the rat body was protected within a lead-lined cylindrical aluminum holder to decrease morbidity and lethality by lung injury. Animals were euthanized and perfused-fixed 24 hours after blast exposure. Brains, spinal cords and lungs were collected and post-fixed. Frozen sections were stained for H&E, and immunostained for albumin, amyloid precursor protein and OX-42.

RESULTS: No significant behavioral changes, except for reversible episodes of apnea during recovery from anesthesia in two rats exposed to 37 p.s.i blasts. A direct correlation has been observed between blast overpressure levels and lung, but not brain injury. Pathological results include subdural hemorrhages and discoloration of the brain surface. Histological results show: albumin staining of the microvessel walls, mild to diffuse subarachnoid, intraventricular and intracerebral (micro) hemorrhages; but only sporadic evidence of breach of the blood brain barrier, microglia activation and axonal injury.

CONCLUSIONS: Available results show a certain inconsistency of results, and, more surprisingly an apparent lack of correlation between blast overpressure levels and brain injury even when this is evident for lung injury. This may suggest the involvement of physical mechanisms other than blast overpressure as cause of the observed pathology. Ongoing evaluations are focusing on additional pathological and neurobehavioral endpoints through a range of survival times.

Support: DARPA; USUHS Center for Neuroscience and Regenerative Medicine

Moratz Chantal 1 Burton Ellen 2 Morgan Amy 2 Schrot John 3 McCarron Richard 3 Liu Yunbo 4 Myers Matthew 4 McCabe Joseph 5

1 Department of Medicine, Uniform Services University of the Health Sciences, Bethesda, USA 2 The Center for Neuroscience and Regenerative Medicine, USUHS, Bethesda, USA 3 Naval Medical Research Center, Silver Spring, USA 4 Center for Devices and Radiological Health, Food and Drug Administration, White Oak, USA 5 Department of Anatomy, Physiology and Genetics, USUHS, Bethesda, USA P204 Rat Blood-Brain Barrier Disruption and Neuro-Immune Alterations from Non-Impact Pressure Alterations by High-Intensity Focused Ultrasound and Blast Overpressure Shock Tube Exposure

Traumatic brain injury (TBI), often from improvised explosive device blasts, has become the “signature injury” of our military personnel. The blast overpressure (BOP) shock tube is a well-established model of whole-body-related TBI. An alternative approach, allowing exposure to a restricted region of the body, is from high-intensity focused ultrasound (HIFU). The goal of this project is to determine the biological effects of mild TBI from non-impact mechanical injury. To evaluate alterations of the integrity of the blood-brain barrier, male Sprague-Dawley rats were injected with dextran dyes of various molecular weights (4kD, 10kD, 40kD, or 70kD) prior to a single, or after three exposures (1/day) of HIFU (400mV amplitude) or BOP (70kPa). Brain tissue was harvested 2 or 24 hours after injury, and examined for leakage of the dyes, histopathological changes, and altered expression of neuroimmune inflammation markers. Routine physical characteristics, such as body weight, were also assessed daily. Histological evaluation of brain sections indicated there was no evidence of intracerebral hemorrhage or observable damage to the blood-brain barrier architecture. However, within two hours after a single exposure to a 70kPa BOP or a 100kPa HIFU there was infiltration of small molecular weight (4kD) dextran into the brain parenchyma and adhesion of IgG to the cerebral vasculature. Results from the single and multiple exposures provide baseline data for BOP and HIFU model comparisons.

McCabe Joseph 1 Burton Ellen 2 Morgan Amy 2 Budinich Craig 1 Woodard Geoffrey 1 Liu Yunbo 3 Myers Matthew 3 Moratz Chantal 1

1 Uniformed Services University of the Health Sciences, Bethesda, MD, USA 2 The Center for Neuroscience and Regenerative Medicine, Bethesda, MD, USA 3 Food and Drug Administration, White Oak, MD, USA P205 Mouse Blood-Brain Barrier Integrity and Neuro-Immune Sequelae From Non-Impact Pressure Alterations by High-Intensity Focused Ultrasound

Blast-related traumatic brain injuries are a significant challenge for military medicine. Non-impact pressure alterations by high intensity focused ultrasound (HIFU) are a means of inducing mechanical injury, to mimic mild TBI, since the blast can be localized to the head region and elicits few overt signs of neurological dysfunction. A HIFU wave generator was used to modulate the amplitude of a 1 MHz frequency train to produce an envelope that was characteristic of a blast wave. The positive portion of the wave was ∼10 MPa max and the negative portion ∼3 MPa, and the wave form was 1 msec in duration. Pressure measurements made inside the mouse skull indicated the final maximal pressure was ∼100kPa. Using male C57/Bl6 mice, we observed the infiltration of small molecular weight dextran-coupled dyes into brain parenchyma, but not high molecular weight (70kDa) dextran, two hours after a single HIFU blast exposure. Alterations in blood-brain barrier function were seen with the adherence of IgG to cerebral vascular walls, but not extravasation into the brain parenchyma. In addition, western blotting indicated HIFU exposure resulted in complement activation, where levels of C3b were elevated 2 and 24 h after a single exposure, and this was seen throughout the neuroaxis. Other signs of brain injury, however, were not evident. For example, no areas showed signs of activated astrocytes (GFAP staining changes) or neuronal death (FluoroJadeC staining. Shock waves produced by HIFU can transfer energy pressure through the skull, and can be a useful animal model to study blast-related neuro-immune injuries and impairments of cerebral vasculature integrity. These findings may have relevance to known perturbations associated with mild, mechanically-induced TBI.

Day Nicole D'Alessandro Tracy Floyd Candace University of Alabama at Birmingham, Birmingham, AL, USA

P206 A Combination of 17β-Estradiol and Memantine is Neuroprotective Following Traumatic Brain Injury in the Rat

Traumatic brain injury (TBI) is a significant public health problem, annually affecting over 1.7 million people in the USA. A main component of the pathophysiology of TBI is the complex secondary injury cascade. To date there are no approved drug therapies for TBI that reduce secondary damage. We hypothesize that a polytherapeutic approach may be necessary due to the complexity of secondary injury and that by combining promising drugs, several injury processes could be targeted simultaneously. Previous research has demonstrated that 17β-estradiol (E2) is effective in attenuating apoptosis, inflammation and reactive gliosis following spinal cord injury and TBI. In addition, it has been reported that memantine (a non-competitive NMDA receptor antagonist) reduces neuronal death, edema and lipid peroxidation, and restores blood brain barrier function in brain injury models. Therefore, we hypothesized that a combination of E2 and memantine would be neuroprotective when administered acutely following TBI. Adult male Sprague-Dawley rats were subjected to moderate-severe lateral fluid percussion TBI and given a bolus venous infusion of either E2 (1mg/kg), memantine (5mg/kg), a combination of E2 and memantine (1mg/kg; 5mg/kg), or vehicle 1 hour post-TBI. We observed a significant reduction in numbers of Fluoro-Jade B-positive neurons in both ipsilateral cortex and the CA2/3 region of the hippocampus of animals that received E2 + memantine compared to animals that received vehicle, E2, or memantine alone. In addition, a reduction in Caspase 3 immunoreactivity was observed in the ipsilateral cortex, CA2/3 region of the hippocampus and hilus of the dentate gyrus in animals that received the E2 + memantine combination compared to animals that were administered vehicle, E2, or memantine alone. Taken together, these data suggest that a combination of E2 and memantine, when administered acutely post-TBI, confers neuroprotection and may represent a promising new polytherapy for treatment of TBI.

Supported by W81XWH-08-2-0153.

Niedzielko Tracy Floyd Candace University of Alabama at Birmingham, Birmingham, AL, USA

P207 Comparision of Blast- and Impact-Induced Mild Traumatic Brain Injury in Adult Male Mice

Approximately 1.7 million people acquire a traumatic brain injury (TBI) every year in the US with approximately 75% classified as mild traumatic brain injury (mTBI). Mild TBI can be caused from a large array of events including falls, car accidents, acts of violence, and recently of interest, exposure to blast waves due to explosions. However, it remains unclear if the pathophysiology of blast-induced mTBI differs from that of impact-induced mTBI. Consequently, the goal of this study was to compare the behavioral effects incurred in a blast model of mTBI with those of an impact acceleration model of mTBI. Adult, C57BL/6 mice were randomly assigned one of the following groups 1) sham uninjured controls 2) blast-induced mTBI or 3) impact-induced mTBI. Motor function, learning and memory, anxiety, depression/despair, and ethological behaviors were assessed using the following behavior tasks: Rotorod, Morris water maze, elevated plus maze, forced swim, and the Laboras bioactivity chamber. No significant differences between groups were observed on motor function or learning and memory tasks. Blast-induced mTBI increased anxiety and depression/ despair behaviors over both sham and impact-induced groups. Blast-induced mTBI animals also exhibited an increase in overgrooming in the light and dark cycles, which is an indicator of obsessive compulsive- like behavior. Also, mice in the blast-induced mTBI group exhibited an increase in locomotion during the dark cycle as compared to both sham and impact-induced mTBI groups. These data indicate that while blast may not cause impairment in locomotor function and learning and memory, it may induce changes in anxiety, depression, and ethological behaviors indicative of obsessive compulsive disorder and insomnia.

Supported by W81XWH-08-1-0289.

Van Ken 1 Wang Tao 1 Russell Michael 2 Lyeth Bruce 1

1 UC Davis, Davis, CA, USA 2 Aaken Labs, Davis, CA, USA P208 Cognitive Effects of Repeated Mild Traumatic Brain Injury in Adult Rats

There are increasing reports that repetitive concussive or mild traumatic brain injury (mTBI) in athletes can result in brain damage and long-term cognitive deficits. Preliminary data shows that a single lateral fluid percussion mTBI (1.25 ATMs) does not produce significant cognitive deficits in the Morris water maze (MWM). We hypothesized that multiple mTBIs would cause cumulative brain damage resulting in significant cognitive deficits. Three groups of rats were subjected to a varied number of repeated lateral fluid percussion mTBIs either unilaterally or bilaterally as well as at different inter-injury intervals: Group 1 (2X-unilateral, 24 hrs apart, n = 2); Group 2 (2X-bilateral, 24 hrs apart, n = 3); Group 3 (2X-bilateral, 1 min apart followed 6 hrs later by 2X-bilateral, 1 min apart, n = 5). There was no mortality associated with any of the mild injury groups, in contrast to a ∼20% mortality rate found with our moderate TBI model (∼ 2.15 ATMs). All groups were tested in the MWM for 5 consecutive days (12-16 post-TBI) for acquisition of cognitive performance. Compared to sham-injured animals, two unilateral mTBIs (Group 1) had deficits only on test day 2; two bilateral mTBIs (Group 2) had deficits on test days 2 and 3; four bilateral mTBIs (Group 3) had deficits on test days 2-5. The MWM deficits in Group 3 were greater than our historical data for moderate TBI. Body weight loss after TBI was similar for Groups 1,2, 3 and was less than for a single moderate TBI. Hence, we demonstrated that cognitive performance declines with multiple mTBI and also that cognitive deficits are greater after bilateral mTBI. In separate, ongoing experiments, Fluoro-Jade B staining is being compared between the three injury groups. Our paradigm of repeated mTBI may be a useful model for investigating the effects of repeated concussions in athletes.

Gurkoff Gene 1 Van Ken 1 Izadi Ali 1 Feng Jun-feng 1 Lowe David 2 Lyeth Bruce 1

1 University of California, Davis, Davis, CA, USA 2 PsychoGenics Inc., Tarrytown, NY, USA P209 Post-Treatment with the NAAG Peptidase Inhibitor PGI-02776 Improves Functional Outcome Following Moderate Lateral Fluid Percussion with Secondary Hypoxia

N-acetyl-aspartyl glutamate (NAAG), a prevalent neuropeptide in the CNS, is released into the synapse with glutamate and selectively activates presynaptic mGluR3 (group II metabotropic glutamate receptor) and attenuates further release of glutamate. Synaptic NAAG is broken down by the catalytic enzyme glutamate carboxypeptidase II and converted into NAA and glutamate. Previously we have demonstrated that the NAAG peptidase inhibitor ZJ-43 reduced accumulation of glutamate, and reduced cell death when administered immediately prior to a moderate lateral fluid percussion injury (LFP). In the current study we combine LFP with a clinically relevant model of secondary hypoxia. Immediately following moderate LFP FiO₂ was reduced to 11% for 30 minutes which caused a significant drop in mean arterial blood pressure (MABP; p < 0.05). When returned to normoxia, MABP rapidly recovered to baseline. In a second group of animals, we injected the NAAG peptidase inhibitor PGI-02776 i.p. 30-minutes following LFP to determine whether treatment would improve motor and cognitive function. Motor performance was assessed on the beam walk, rotorod and inclined plane on post-injury days (PID) 1, 4, 7, 11 and 15. Post-treatment with PGI-02776 led to a statistically significant improvement in performance on the beam walk (p < 0.05) in the first 15 days following injury. While not significant there was a trend toward improvement on the rotorod in the first 7 days post-injury. Additionally, we demonstrate that following LFP with hypoxia there was a significant deficit in MWM performance on PID 11-15 (p < 0.05). There was no significant difference between sham animals and LFP with hypoxia animals that received PGI-02776 30-minutes following injury. These data demonstrate that the NAAG peptidase inhibitor PGI-02776 has therapeutic potential to reduce both motor and cognitive deficits when administered systemically 30-minutes following a clinically relevant model of TBI with hypoxia.

Valino Hannah McArthur David Yudovin Sue Giza Christopher UCLA, Los Angeles, CA, USA

P210 Sports-Related Brain Injuries in a Pediatric TBI Clinic

Sports-related traumatic brain injuries (TBI) comprise a large segment of all TBI. This prospective study utilized data from children presenting to a pediatric TBI clinic to compare and contrast sports-related TBI from other TBI in this clinic cohort. Children admitted to UCLA or referred to UCLA's pediatric neurology clinic for TBI since 2006 were eligible for consent and inclusion. Sports activities included both team (football, soccer, etc.) and individual sports (skiing, skateboarding, etc.). Only patients with follow-up data within 6 months of their injury were included in this analysis. Data were analyzed using R version 2.12.2 and WinPepi. 188 patients were enrolled (125 males, 63 females); 27.1% of those endured a sports-related TBI (34 mild, 14 moderate, 3 severe). Sports-related TBI occurred more frequently among older children (binomial logistic regression, p < 0.0001), were less severe (Fisher Exact Test, p < 0.0001), and often blunt injuries or falls (Fisher Exact Test, p < 0.0001). The odds of sports-related TBI in patients with a history of TBI was 9.57 times that of patients without a history of TBI (CI_.95 = 3.56, 27.28). Contact-sport TBI was the most common sports injury. Wheeled-sport TBI was associated with increased severity (Fisher Exact Test, p < 0.0001). Headaches (OR = 3.90, CI_.95 = 1.66, 9.18) and cognitive impairment (OR = 3.41, CI_.95 = 1.34, 8.64) were more likely after sports-related TBI than other TBI. A history of prior TBI greatly increased the chance of another TBI. Pediatric sports-related TBI were associated with age and more likely to result in headaches and cognitive impairments than other TBI in this cohort. Further research can assess the effect of a specialized pediatric TBI clinic on sports-related TBI cases and their rate of recovery.

Supported by UCLA Brain Injury Research Center, NS05720, NS027544, Winokur Family Foundation, Today's and Tomorrow's Children Fund, Thrasher Research Fund, UCLA Faculty Grants Program.

Wanner Ina B. 1 Kwon Eunice 1 Fernandez Ana B. 1 Czerwieniec Gregg 2 Sondej Melissa A. 2 Paez Pablo M. 1 Bullock Ross M. 3 Dietrich Dalton W. 3 Glenn Thomas C. 4 Hovda David A. 4 Loo Joseph A. 2 Sofroniew Michael V. 5

1 Semel Institute for Neuroscience and Human Behavior, UCLA, Los Angeles, CA, USA 2 Dept. Chemistry and Biochemistry, Molecular Instrumentation Center, MIC, UCLA, Los Angeles, CA, USA 3 The Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA 4 Dept. of Neurosurgery, Brain Injury Research Center, BIRC, UCLA, Los Angeles, CA, USA 5 Dept. of Neurobiology, UCLA, Los Angeles, CA, USA P211 Mining the Astrocyte Traumatome for Biomarkers of CNS Injury

There is an unmet need for biomarkers to diagnose traumatic brain injury (TBI) without visible wounds. The goal of this study is to define the protein release profile of astrocytes mechanically traumatized in vitro in order to identify new potential neurotrauma biomarkers that can be screened for in cerebrospinal fluid (CSF) samples from TBI patients. Astrocytes are the most abundant cell type in the human brain and react sensitively to CNS trauma; hence the CSF protein composition from TBI patients is predicted to carry a measurable astrocyte signature. For translational relevance we used rodent and established human astrocyte cultures that were matured in vitro. Cultured rodent and human astrocytes expressed prototypical proteins of mature astrocytes including glutamate transporters, tenascin, aquaporin4, S100 and GFAP. Astrocytes grown on deformable membranes received a brief pressure pulse creating widespread shear strains that caused reactivity, membrane damage and death. Mechanical stretching caused widespread stellation and increased GFAP. Time-lapse imaging showed up to 70 kD large dyes being taken up during stretching, suggesting considerable membrane microporation due to the mechanical trauma. Cross-species comparison revealed large differences in cell death rates, with mouse astrocytes being most vulnerable followed by rat and human astrocytes being most robust when exposed to mechanical stretch injury. Proteomic analysis of conditioned medium from traumatized astrocytes defined a list of released, 15-120 kD sized proteins including metabolic enzymes, cytoskeleton and signaling proteins. Western blot studies comparing cell lysates and media between species, revealed striking release differences of selected trauma markers reflecting the severity of the trauma. Several of the released astrocytic proteins could be detected in CSF samples from TBI patients using western blotting and an antibody-independent mass spectrometry approach called multiple reaction monitoring (MRM). MRM is used to detect and quantify candidate astrocyte trauma markers in patient CSF for future diagnostic use.

Sutton Richard L. Moro Nobuhiro Ghavim Sima Hovda David A. Harris Neil G. David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

P212 Neuroprotection and Improved Brain Metabolism Results from Ethyl Pyruvate Treatment After Cortical Contusion Injury

We examined the effects of ethyl pyruvate (EP) on neuronal injury and cerebral metabolic rates of glucose (CMRG) 24 h after unilateral cortical contusion injury (CCI). Adult male rats with sham injury (n = 8-10/group) or left CCI (n = 9-10/group) were injected once (1X; immediate treatment) or four times (4X; at 0, 1, 3 and 6 h) with EP (40 mg/kg; IP) or saline (Sal) post-surgery. 14C-2-deoxyglucose studies at 24 h assessed CMRG (μmol/100g/min) in left (L) and right (R) cortical and subcortical regions; the magnitude of ipsilateral CMRG depression was assessed via regional asymmetry scores ((L-R)/L + R)*100. FJB-positive (Fluoro-Jade B) neurons in the ipsilateral hippocampus (Hilus and CA3) and midline peri-contusional cortex were quantified (n = 8/group).

Sham controls with 1X or 4X Sal or EP treatments did not differ on any CMRG asymmetry scores, and no FJB-positive cells were detected. All CCI groups showed significant (p < 0.001) reductions in CMRG asymmetry scores compared to sham controls, reflecting widespread injury-induced metabolic depression in the ipsilateral hemisphere (cortex, caudate, hippocampus, amygdala and thalamus). The 1X CCI-EP group had significantly fewer FJB-positive neurons in the left midline cortex and hippocampus compared to 1X CCI-Sal (p < 0.05). 1X EP administration after CCI significantly reduced the magnitude of CMRG asymmetry scores in 6 cortical and 3 thalamic regions compared to 1X CCI-Sal (p < 0.05). FJB-positive neurons in ipsilateral midline cortex and hippocampus were significantly decreased in the 4X CCI-EP group compared to 4X CCI-Sal (p < 0.05), and CMRG asymmetry was significantly improved in 1 thalamic and 3 cortical regions of the 4X CCI-EP group compared to 4X CCI-Sal (p < 0.05).

These results demonstrate that a single treatment of EP immediately after CCI is neuroprotective and improves CMRG ipsilateral to injury. Four EP treatments within 6 h of CCI, while also beneficial, did not improve on the outcomes produced by the single post-injury treatment.

Gomez-Pinilla Fernando Ying Zhe Wu Aiguo Dept. Neurosurgery, Dept. Integrative Biology and Physiology, UCLA, Los Angeles, CA, USA

P213 Separate DHA or Combination with Curcumin Protects Against Behavior Deficits After Traumatic Brain Injury

We have previously demonstrated that TBI results in impaired cognitive function, and that the curry spice curcumin or DHA supplemented in the diet before TBI protect the brain against the effects of TBI. In the current study, we investigated the effects of DHA in combination with curcumin supplemented in the diet after TBI. We supplemented curcumin (500ppm), DHA (1.2%), or a combination of curcumin + DHA (low dose: curcumin, 250ppm; DHA, 0.6% or High dose: curcumin, 500ppm; DHA, 1.2%) to the diets after moderate fluid percussion injury (FPI). Rats were maintained on control diet or special diets for 21 days after FPI. Barnes Maze testing showed that FPI reduced learning ability, while the supplementation of DHA, curcumin, or the combination of both (low or higher dose) improved learning ability. Elevated plus maze (EPM) testing showed that FPI increased anxiety-like behavior (i.e. the time spent in open arms: 16 ± 5s in FPI rats vs 30 ± 14s in sham rats-control diet), which was reduced by supplementation of DHA, curcumin, or combination of both (low or higher dose). In addition, TBI reduced BDNF (69% of control), TrkB (59% of control), and p-TrkB (51% of control) in the hippocampus. TBI increased anxiety-related glucocorticoid receptor (GR) (124% of control). These alterations were counteracted by supplementation of curcumin, DHA, or combination of both (low or higher dose). The combination of curcumin and DHA (half dose of each) has similar and/or even greater beneficial effects on behavior and cognitive function in TBI animals. These findings suggest that a combination of curcumin and DHA may produce collaborative action in reducing damaging effects of TBI on cognition and mood.

Funding support: NIH R01 NS050465; NIH R01 NS045804;NIH RC1 NS068473

Xu Kathleen Prins Mayumi UCLA, Los Angeles, CA, USA

P214 Determining Recovery Time After Single and Repeat Mild Traumatic Brain Injury

Previous research has shown that after mild traumatic brain injury (mTBI), juvenile rats showed increased cognitive deficits proportional to the number of head injuries induced. Both single and repeat TBI (RTBI) animals showed deficits at 1 day post injury. We hypothesize that the recovery time for mTBI in juvenile rats also increases with the number of head injuries. Postnatal day 35 (PND35) rats were divided into sham (n = 9), single (n = 6) and repeat TBI (n = 5) groups, subjected to 0, 1 or 2 closed head injuries, respectively. Novel object recognition task (NOR) was performed on post injury day 3 to test cognitive and behavioural deficits between the groups. All rats experienced habituation (10 min/rat) in the open chamber (52x52x9 cm). On post-injury day 3 rats were exposed to two similar objects (5 min/rat) and on day 4 were exposure to one novel object (5 min/rat). Objects of age-appropriate size and with a recognizable difference between novel and familiar objects were kept constant throughout experimentation. Data of total number of interactions and total duration of interaction with each object was recorded using timer and video analysis. Based on number of interactions per object, the sham and single mTBI groups on average showed significant novel object recognition after a 24 hr delay, at 61.3 ± 3.7% and 57.54 ± 3.0% novel interaction, respectively. However, the repeat mTBI group, at 51.09 ± 4.6% novel interaction, showed no significance in object recognition since 50% is recognition by chance. Upon introduction to the new object 98% of sham, 83% single and 60% of RTBI animals explored the new object first. Our data suggests that repeat mTBI rats show slower recovery with cognitive deficits remaining at 3 days post-injury relative to single or sham animals.

Supported by: NS058489, NFL Charities Grant and the UCLA Brain Injury Research Center.

Ponnaluri Aditya Maria Naomi Santa Hovda David Giza Christopher University of California, Los Angeles, Los Angeles, CA, USA

P215 Decreased Fractional Anisotropy in Corpus Callosum Following Lateral Fluid Percussion Injury in the Immature Rat

In normal white matter, axonal integrity and myelination can be correlated to water diffusion along axons; Fractional Anisotropy (FA) measures the extent of unidirectional water diffusion using Diffusion Tensor Imaging (DTI). Previous studies have shown that following traumatic brain injury (TBI) in mature brains diffuse axonal injury occurs, characterized by axonal swelling and myelin damage. Concurrently FA values in the both white and gray matter are reduced post-injury. In the developing brain, myelin also undergoes a developmental profile, which may pose a challenge to tracking FA in the young brain. Here we demonstrate that FA values in the immature rat brain can be obtained following a diffuse model of TBI, lateral fluid percussion injury (LFPI). We hypothesized that after LFPI, young rats will show reduced FA values in hippocampus and corpus callosum (CC) acutely (post-injury day (PID) 3), recovering at a chronic time point (PID14). Sprague Dawley rats underwent sham and LFPI surgeries on post natal day 19. Using a Bruker 7Tesla scanner, DTI was performed on PID3 (Sham N = 5, LFPI N = 8) and PID14 (Sham N = 3, LFPI N = 4). In CC, LFPI pups showed significantly lowered FA values on PID3 (ANOVA, p = 0.035) ipsilateral to injury (FA = 0.30 ± 0.03) compared to Shams (FA = 0.40 ± 0.03). Contralaterally, LFPI did not significantly affect FA values (Sham: FA = 0.41 ± 0.05; LFPI: FA = 0.38 ± 0.02). By PID14, LFPI rats still showed trends for reduced FA in ipsilateral CC (FA = 0.33 ± 0.02) compared to shams (FA = 0.38 ± 0.00) (ANOVA, p = 0.062). No effect of injury was observed in acute and chronic hippocampal FA in both sides. We found that CC undergoes persisting changes that may underlie lasting dysfunction. FA can be used as biomarker for the assessment of injury severity and to target a window for therapeutic intervention.

Supported by UCLA BIRC, NS27544, NS057420, NS058489, UCLA FGP, Winokur Family Foundation/Child Neurology Foundation, Today's and Tomorrow's Children Fund, NFL Charities.

Rodriguez Alexandra Prins Mayumi UCLA, Los Angeles, CA, USA

P216 The Age Dependent Effects of Ketones on Oxidative Damage Following Controlled Cortical Impact

Traumatic brain injury (TBI) has been shown to induce oxidative stress, leading to the formation of free radicals, lipid peroxidation, protein nitration and eventual cell death. Ketone bodies have been shown to increase glutathione peroxidase activity (Ziegler et al., 2003), increase mitochondrial uncoupling (Sullivan et al., 2004) and protect cells from glutamate and hypoglycemia (Noh et al., 2006; Hace et al., 2008). Previous work from our laboratory has shown ketone metabolism to be neuroprotective after controlled cortical impact injury (CCI). We hypothesized that reduction of oxidative damage may be the mechanism for age dependent neuroprotection. Postnatal day (PND) 35, PND70 rats where placed on either a standard or ketogenic (KG) diet after CCI injury. 3NT western blots detected 15kD and 25kD proteins for PND35 animals; only a 25kD protein was detected for PND70 animals. 4HNE western blots for both age groups detected a 25kD protein. PND35 KG-fed animals showed a statistically significant ____% reduction in protein nitration on the 15kD protein (p < 0.05) relative to standard fed animals, but no significant difference on 25kD protein. No significant changes in lipid peroxidation levels were observed. The KG diet had no effect on PND70 protein nitration (p > 0.05) relative to standard-fed animals and an increased in lipid peroxidation (p < 0.05) relative to standard-fed animals. Contralateral and hippocampal regions for both age groups did not show a detectable amount of oxidative damage.

Ketone metabolism may confer oxidative protection to specific cellular compartments in the juvenile brain. Supported by: NS058489, NFL Charities Grant and the UCLA Brain Injury Research Center.

Tio Delia Nair Shyama Taylor Anna Hovda David Griesbach Grace UCLA, Los Angeles, CA, USA

P217 Differential Effects of Voluntary and Forced Exercise After Mild Traumatic Brain Injury on BDNF and Stress Responses

Exercise increases levels of brain derived neurotrophic factor (BDNF) after traumatic brain injury (TBI) when it occurs at a delayed time window. Increases in glucocorticoids suppress levels of BDNF. We set out to determine if a heightened stress response is observed with acute exercise. We also explored if the type of exercise (forced versus voluntary running-wheel) will have differential effects on the stress and BDNF responses.

Adult rats were implanted with telemetry devices to monitor changes in heart rate, body temperature and non-exercise activity. Mild lateral fluid-percussion injury (FPI), sham injury and anesthesia controls were assigned to a sedentary (Sed), voluntary running (vRW), or forced running (fRW) condition on the day of the injury. Rats were exercised for 10 days. Blood was collected at post-injury days 0, 4, 7 and 11 to determine corticosterone (CORT) and adrenocorticotropic hormone (ACTH). Rats were sacrificed at PID 11 and BDNF levels were determined.

In all the experimental groups, fRW resulted in higher levels of both CORT and ACTH compared to vRW (p < 0.0005). CORT increases were more pronounced after FPI in both the fRW and vRW conditions compared to the control-fRW (p < 0.05) and control-vRW (p < 0.005) groups. A sham effect was observed on the day of the injury, in that levels of ACTH were lower in the sham-fRW and FPI-fRW compared to anesthesia controls (p < 0.05). The FPI group was the only group that showed a significant increase in ACTH as a response to vRW (p < 0.05). As previously reported, vRW did not increase BDNF in the FPI group (when immediately after injury). BDNF was increased in the control-vRW(p < 0.05). Forced exercise did not increase BDNF in any group. This suggests that a mild FPI produces a heightened stress response to exercise that contributes to the inability of acute exercise to increase BDNF.

Support: NS06190, UCLA BIRC

Thomsen Gretchen 1 Le Belle Janel 2 Harnisch Jessi 1 Hovda David 1 Kornblum Harley 2 Harris Neil 1

1 Dept. Neurosurgery, UCLA, Los Angeles, CA, USA 2 NPI-Semel Inst. of Neuroscience & Human Behav. & Dept. Psychiatry & Biobehavioral Sci., Los Angeles, CA, USA P218 Non-Proliferative Expansion of SVZ Astrocyte and Activated Stem Cell Populations Occurs as EGFR⁺ Cells Become Less Proliferative After Brain Injury

It is known that subventricular (SVZ) neurogenesis is enhanced after brain injury and that some SVZ neuroblasts are directed toward the injury site. However the early response of specific SVZ cell phenotypes is not well understood. It is unknown which cell types are directly responsible for these injury-induced changes, and whether cell division is stimulated in populations of normally quiescent GFAP + resident stem cells, or in fast dividing, more committed progenitors such as Mash1 + transit-amplifying cells and/or DCX + neuroblasts. Resolution of this is important for guiding future, potential therapeutic avenues of research.

We studied the effect of controlled cortical impact injury on SVZ neurogenesis in wild-type and Gfap-HSV-TK transgenic mice at 1, 3 and 7days post-injury using confocal and epifluorescent immunohistochemistry and stereology, as well as in vitro neurosphere clonal analysis. We found that Mash1 + transit-amplifying cells are the primary SVZ cell type that is stimulated to divide following brain injury, and that they account for the majority of the proliferative response. In contrast, a transit-amplifying EGFR + cell population becomes significantly less proliferative after injury, and more likely to express GFAP. While Mash1 + cells derive from a population of slowly dividing GFAP + SVZ stem cells, GFAP + cells themselves are surprisingly not stimulated to divide more after injury. Paradoxically, there is a non-proliferative increase in total numbers of GFAP + cells, and therefore an increase in the potential stem cell pool, that can be numerically attributed to increased GFAP + /EGFR + activated stem cells. This can occur by either disruption to the normal lineage progression or by phenotypic reversion. Together with the known effect of EGF on SVZ maturation and proliferation, and the trauma-induced increase in EGFR-ligands, these data indicate that the EGFR pathway is an important determinant of SVZ function after injury. This might be manipulated to potentiate the response and enhance recovery.

Nogueira Marcia Verley Derek Hovda David Sutton Richard Harris Neil Dept. Neurosurgery, UCLA, Los Angeles, CA, USA

P219 Enhanced Brain Activation Following Removal of Axon-Growth Inhibition in the Brain Injured Rat

Removal of the axon-growth inhibitory chondroitin-sulphate proteoglycans (CSPGs) around the glial scar after cortical brain injury enhances neuronal sprouting and leads to some behavioral improvements. As part of a longer term goal to determine whether functional improvements specifically arise from regions of increased axon sprouting, we assessed whether post-injury interventions to reduce CSPGs results in improved brain activation, as assessed indirectly by immunostaining for the immediate-early-gene c-FOS.

Following controlled cortical impact injury over the left, cortical forelimb region in adult, male rats, chondroitinase-ABC (1.5μl, 48U/μl) or vehicle (n = 6,3) was infused immediately and at 3 days post-injury into the injury site through an indwelling cannula. At 7-days, all rats, including additional groups of non-injected-injured (n = 4) and naïve rats (n = 3) received right (affected) forepaw, electrical stimulation (3.5mA) for 60mins under medetomidine sedation (0.1mg/kg/hr). Rats were perfused-fixed 120mins after the end of the stimulation period and processed for c-FOS immunohistochemistry. A final injured group were not stimulated (n = 2). Cortical grey (GM) and corpus callosum white matter (WM) c-FOS + cell-density was determined bilaterally on 3-sections/rat, 600μm apart, within the sensory-motor cortex.

FOS + cell-density was low throughout the entire cortical GM/WM in injured-non-stimulated rats (2 − 19cells/mm²). There was no effect of vehicle-infusion in injured-stimulated rats and so data were pooled. As expected, FOS-activation in injured-stimulated rats was higher in ipsilateral-GM (28 ± 9.1cells/mm²) compared to contralateral (8 ± 3.0cells/mm²,P < 0.05), but also compared to naïve, ipsilateral-GM (8 ± 2.4cells/mm², P < 0.05). However, WM FOS + density was similar bilaterally, and between both groups.

Chondroitinase infusions to reduce cortical CSPGs was associated with significantly increased c-FOS + density in ipsilateral-GM (128 ± 27.3cells/mm², P < 0.01) and bilaterally in WM matter compared to vehicle or non-injected injured-stimulated rats (P < 0.05). These data indicate that reduction of pericontusional CSPGs has a significant effect on brain function, and this might underlie functional recovery in this model.

Verley Derek Hovda David Sutton Richard Harris Neil UCLA, Los Angeles, CA, USA

P220 Time-Course of Sensorimotor Brain Activation After Traumatic Brain Injury in the Rat

There is continued debate over whether the contralesional cortex, the cortex contralateral to an injury, participates in recovery of function. While clinical functional imaging evidence is accumulating in support of the importance of contralaesional activation in functional recovery, solid experimental evidence is lacking for the mechanism(s) of plasticity that underlie such recovery. As a first step towards investigating this, we set out to determine how sensory-evoked brain activation change after cortical brain injury, and whether there is evidence of contralesional, or “wrong-side” activation in response to fore-limb stimulation paradigm.

Functional brain activation data were acquired from medetomidine-sedated rats (n = 7) before, and at weekly intervals to 28 days, after mild-moderate controlled cortical contusion injury over the left forelimb cortex. A multi-slice, gradient-echo, single shot echo-planar sequence was used to acquire BOLD data using a 7 Tesla Bruker spectrometer. A paradigm of both affected- or unaffected fore-limb, electrically evoked stimulations (2mA) was used to activate the brain using a classic “on-off” block design. Group-wise statistical analysis was performed following registration and warping to a common brain template.

As expected, significant activation of the sensorimotor cortices was observed in the cortex opposite the stimulated limb at baseline. Brain activation levels were generally low following injury, particularly when the affected limb was stimulated. We therefore report data from uncorrected images (p < 0.05). Paired, two-sample t-test between baseline and 7 days post- injury after stimulation of the affected limb showed “wrong side” activation in the uninjured, contralesional cortex (p < 0.05). At 28 days post-injury, this activation had shifted back to the injured cortex in the peri-contusional region (p < 0.05). The activation was especially prominent in cortex immediately caudal and rostral to the contusion. We interpret these findings to indicate that contralesional activation after injury is important and requires further study to determine any role in recovery of function.

Donovan Virginia 1 Coats Jacqueline 2 Badaut Jerome 2 Wilson Sean 2 Huang Lei 2 Obenaus Andre 2

1 University of California, Riverside, Riverside, CA, USA 2 Loma Linda University, Loma Linda, CA, USA P221 Long-Term White Matter Changes in a Bilateral Model of Repetitive Mild Traumatic Brain Injury

INTRODUCTION: Repetitive mTBIs (multiple concussions) are an important health concern as subsequent impacts can exacerbate existing neuropathology leading to long-term neurological deficits. In this study we focused on the sub-acute and chronic white matter changes that occurred as a result of bilateral repetitive mTBI, using a model in which cortical impacts were induced on opposite sides of the brain.

METHODS: Adult male rats were grouped into sham controls (no mTBI), single mTBI controls, and repetitive mTBI (rmTBI) animals. Initial mTBIs were induced to the right cortex via a controlled cortical impact (CCI) with a second CCI delivered to rmTBI animals 7 days later on the left cortex. Animals were sacrificed at 14, 21, and 60 days to evaluate white matter injury using Diffusion Tensor Imaging (DTI). Regions of interest (ROI) included the corpus callosum on brain slices anterior, posterior, and at the site of impact. Radial, axial, relative anisotropy (RA), and trace diffusivity measurements were extracted.

RESULTS: Anterior regions of the day 21 rmTBI group revealed increased axial diffusivity compared to single animals in both hemispheres, which waned by 60 days. Day 60 rmTBI animals demonstrated a 20-30% increase in radial diffusivity throughout the brain, which was not seen in any other group. Single mTBI animals had similar trace values within the anterior regions at 14 and 60 days, while the rmTBI groups progressively increased over time. RA values of day 21 and 60 rmTBI groups progressively increased anterior-to-posterior in the right hemisphere and decreased in the left hemisphere.

CONCLUSIONS: Our findings suggest that white matter remains vulnerable to a second mTBI. Further, our results indicate that injury appears to develop first as axonal damage, which recovers by 60 days, followed by demyelination.

Funding provided by DCMRP # DR080470 (AO), NSF # DGE 0903667(VMD), NSF # MRI - DBI 0923559 (SW).

Lopez Nicole Krzyzaniak Mike Ortiz-Pomales Yan Hageny Ann-Marie Putnam James Eliceiri Brian Coimbra Raul Bansal Vishal UCSD, San Diego, CA, USA

P222 Early Treatment With Ghrelin Prevents Disruption of Blood Brain Barrier and Apoptosis After Traumatic Brain Injury

PURPOSE: Significant effort has been focused on reducing neuronal damage from post-traumatic brain injury (TBI) inflammation and blood brain barrier (BBB) mediated edema. The orexigenic hormone, ghrelin, has been shown to be anti-inflammatory and neuroprotective in other models of neurologic injury. These models use repeated dosing of ghrelin. We hypothesize that treatment with ghrelin, within the first hour alone, will prevent breakdown of the BBB and apoptosis 24 hours following TBI.

METHODS: A weight drop model was used to create severe TBI in Balb/c mice. Animals were divided into 3 groups. Group TBI: TBI only; Group TBI/ghrelin: animals were treated with 10 μg of intraperitoneal ghrelin immediately prior to, and following TBI; Group Sham: no TBI or ghrelin treatment. Brain vascular permeability to injected 70kDa FITC-Dextran was measured 24 hours following injury and quantified in arbitrary integrated fluorescence (afu). Coronal sections of brain tissue were subjected to TUNEL staining and TUNEL positive cells were quantified as cells per high-powered field (hpf).

RESULTS: TBI increased cerebral vascular permeability by three-fold compared to sham as measured by arbitrary fluorescence (132 ± 41.7 vs. 43.4 ± 1.52 afu; p < 0.05). Ghrelin treatment restored vascular permeability to the level of shams (61.1 ± 17.5 afu; p < 0.05). TUNEL staining revealed significant apoptosis in TBI compared to sham as detected per high power field (hpf). Again, ghrelin treatment significantly decreased apoptosis compared to the TBI.

CONCLUSIONS: In a mouse TBI model, early ghrelin treatment prevents TBI BBB disruption as measured by permeability to FITC-Dextran. Ghrelin also prevents TBI mediated neuronal apoptosis 24 hours following injury. These preliminary results demonstrate the neuroprotective potential of ghrelin. Future studies will need to be conducted to determine the precise mechanism of ghrelin neuroprotection.

Cheong Maxwell 1 Sinha Tuhin 1 Yuh Esther 1 Cooper Shelly 2 Mukherjee Pratik 1 2 Yue John 2 Okonkwo David 3 Valadka Alex 4 Gordon Wayne 5 Manley Geoffrey 2

1 University of California, San Francisco, Quantitative Image Processing Center (UCSF), San Francisco CA, USA 2 University of California, San Francisco, Brain and Spinal Injury Center at SF General Hospital (UCSF), San Francisco CA, USA 3 University of Pittsburgh Medical Center, Brain Trauma Research Center (UPMC), Pittsburgh PA, USA 4 University Medical Center, Brackenridge-Austin (UMCB), Austin TX, USA 5 Mount Sinai Medical Center, Dept. of Rehabilitation Medicine (MSMC), New York NY, USA P223 Transforming Research and Clinical Knowledge in Traumatic Brain Injury: A Comprehensive Imaging Data Management System for a Multicenter Observational Study of Acute Traumatic Brain Injury

PURPOSE: NINDS, NIDRR, DVBIC, and DCOE have jointly supported an effort to develop consensus-based common data elements for traumatic brain injury and psychological health (TBI-CDE, www.commondataelements.ninds.nih.gov/TBI.aspx). One key aspect of the TBI-CDEs is descriptors of intracranial pathology as identified on head CT and brain MRI studies. Imaging studies, unlike most other clinical information, are high-volume data that require special techniques for anonymization, transmission, storage, and visualization by multiple users. We sought to develop a comprehensive imaging data management system for TRACK-TBI, a multicenter observational study of acute TBI that is evaluating the utility of the TBI-CDEs as well as comprehensively phenotyping a cohort of TBI patients across the injury spectrum from concussion to coma.

METHODS: We implemented a research PACS (rPACS) with DCM4chee, Osirix and ClearCanvas, all open-source DICOM software, that allows controlled remote access for multiple users. To address HIPAA requirements for de-identification and secure transfer of imaging studies from multiple remote sites, we developed a multiplatform tool for simultaneous anonymization and transmission of DICOM images, based on open-source Java-based software available through the RSNA Medical Imaging Resource Center (www.rsna.org/mirc).

RESULTS: Noncontrast head CT studies for 604 patients and 3T MRI studies for 235 patients from 4 sites across the USA are being transferred to this rPACS. The system addresses key requirements including 1) avoiding disruption of the clinical PACS by large numbers of research studies, 2) simultaneous anonymization and secured file transfer of de-identified images from multiple remote sites, 3) remote access by researchers to images without protected health information, 4) reliable organization, storage and backup of images, and 5) control of access to formal radiological clinical reports.

CONCLUSION: We have implemented a comprehensive imaging data management system for anonymization, transmission, storage and remote multiuser access to imaging studies in a multicenter observational study of acute TBI.

Investigators Track-Tbi 1 Yue John 1 Puccio Ava 2 Schnyer David 3 Dams-O'Connor Kristen 4 Yuh Esther 1 Mukherjee Pratik 1 Okonkwo David 2 Valadka Alex 3 Gordon Wayne 4 Maas Andrew 5 Manley Geoffrey 1

1 University of California, San Francisco, Brain and Spinal Injury Center at SF General Hospital (UCSF), San Francisco, CA, USA 2 University of Pittsburgh Medical Center, Brain Trauma Research Center (UPMC), Pittsburgh, PA, USA 3 University Medical Center, Brackenridge-Austin (UMCB), Austin, TX, USA 4 Mount Sinai Medical Center, Department of Rehabilitation Medicine, NY (MSMC), New York, NY, USA 5 University Hospital Antwerp, Department of Neurosurgery, Antwerp, Belgium P224 Transforming Research and Clinical Knowledge in Traumatic Brain Injury: Multicenter Implementation of the TBI Common Data Elements

PURPOSE: NINDS, NIDRR, DVBIC, and DCOE have jointly supported an effort to develop consensus-based common data elements for traumatic brain injury and psychological health (TBI-CDE, www.commondataelements.ninds.nih.gov/TBI.aspx). The goal of the TRACK-TBI study is to evaluate the feasibility and utility of the TBI-CDEs and to extensively phenotype a cohort of TBI patients across the injury spectrum from concussion to coma.

METHODS: Phase I focused on developing infrastructure, establishing collaborations and pilot testing of data collection and outcome measures. For the three acute care centers, patients were enrolled that presented to the emergency department within 24 hours of head injury and required CT. For the rehabilitation center, referrals from acute hospitals were enrolled. Patients were consented to participate in components: clinical profile; blood draws for measurement of proteomic and genomic markers; 3T MRI within 2 weeks; three-month Glasgow Outcome Scale-Extended (GOS-E); and six-month TBI-CDE Core outcome assessments.

SUMMARY: A web-enabled database, imaging repository, and biospecimen bank was developed using the TBI-CDE recommendations. A total of 605 patients were enrolled. Of these subjects, 88% had a GCS 13-15, 5% had a GCS 9-12, and 7% had a GCS of 8 or less. Three-month GOS-E's were obtained for 78% of the patients. Comprehensive 6-month outcome measures, including PTSD assessment, are ongoing until September 2011. TBI-CDE outcome measures could not be applied to non-English speakers and children. Blood specimens were collected from 450 patients. Initial CTs for 605 patients and 235 patients with 3T MRI studies were transferred to an imaging repository.

CONCLUSION: Phase I of the TRACK-TBI study was completed within an accelerated timeframe of 10 months. We demonstrate that it is feasible to develop the infrastructure, collaboration, and tools needed to implement TBI-CDEs for multicenter clinical research across the spectrum of TBI. Analyses of neuroimaging, genomic, proteomic, and outcome data are underway.

Pawlowicz Artur 1 Braga Carolina 2 Ponce Michelle 2 McVay-Foti Sally 2 Papa Linda 1

1 University of Central Florida College of Medicine, Orlando, FL, USA 2 Orlando Regional Medical Center, Department of Emergency Medicine, Orlando, FL, USA P225 Abnormal Levels of End- Tidal Carbon Dioxide (ETCO2) are Associated with Severity of Injury in Mild and Moderate Traumatic Brain Injury (MMTBI)

Capnography is a fast, non-invasive technique that is easily administered and accurately measures exhaled ETCO2 concentration. ETCO2 levels respond to changes in ventilation, perfusion, and metabolic state, all of which may be altered following TBI.

OBJECTIVE: This study examined the relationship between ETCO2 levels and severity of TBI as measured by clinical indicators including Glasgow Coma Scale (GCS) score, Computerized Tomography (CT) findings, requirement of neurosurgical intervention, and levels of a serum biomarker.

METHODS: This prospective cohort study enrolled adult patients presenting to a Level 1 trauma center following a MMTBI defined by blunt head trauma followed by loss of consciousness, amnesia, or disorientation and a GCS 9-15. ETCO2 measurements were recorded from the prehospital and emergency department records and compared to indicators of TBI severity.

RESULTS: Of the 46 patients enrolled, 21 (46%) had a normal ETCO2 level and 25 (54%) had an abnormal ETCO2 level. The mean age of enrolled patients was 40 (range 19-70) and 32 (70%) were male. Mechanisms of injury included motor vehicle collision in 19 (41%), motorcycle collision in 9 (20%), fall in 8 (17%), bicycle/pedestrian struck in 8 (17%) and other in 2 (4%). Eight (17%) patients had a GCS 9-12 and 38 (83%) had a GCS 13-15. Of the 11 (24%) patients with intracranial lesions on CT, 10 (91%) had an abnormal ETCO2 level (p = 0.006). Of the 5 (11%) patients who required a neurosurgical intervention, 100% had an abnormal ETCO2 level (p = 0.05). Levels of a biomarker indicative of astrogliosis were significantly higher in those with abnormal ETCO2 compared to those with a normal ETCO2 (p = 0.026).

CONCLUSION: Abnormal levels of ETCO2 were significantly associated with clinical measures of brain injury severity. Further research with a larger sample of MMTBI patients will be required to better understand and validate these findings.

Pitkanen Asla Sierra Alessandra Laitinen Teemu Grohn Olli University of Eastern Finland, Kuopio, Finland

P226 Diffusion Tensor MRI of Plasticity During Epileptogenesis in the Rat Hippocampus

PURPOSE: Our objective was to investigate whether diffusion tensor imaging (DTI) reveals epileptogenesis-related plasticity in the hippocampus after traumatic brain injury (TBI), and compare the changes to that after status epilepticus (SE).

METHODS: TBI was induced with lateral fluid-percussion brain injury and SE with pilocarpine in adult rats. Ex vivo DTI was performed at 7 months after TBI and 6-12 months after SE. Maps of fractional anisotropy (FA), axial (D _|| ) and radial (D _^ ) diffusivities were obtained. After imaging, brain sections were stained with Timm, Nissl, or gold chloride protocols.

RESULTS: After both injuries, DTI parameters were remarkably changed in different hippocampal subfields. Changes in FA, D _|| and D _^ were mainly found in the CA3 and dentate gyrus. Changes in orientation of the principal eigenvector were more pronounced in the CA3 and stratum lacunosum-moleculare of CA1. As expected based on histology, DTI changes after SE were more robust and widespread than after TBI. Moreover, in TBI the changes were the most clearly in the distal CA3 where the most severe principal cell degeneration occurs.

CONCLUSION: DTI parameters of each hippocampal subfield can provide additional information about the dynamics of ongoing plasticity in injured hippocampus. The detection of differences in hippocampal plasticity between TBI and SE creates a scenario for the use of DTI to understand the etiology-specific circuitry reorganization during epileptogenesis.

Mondello Stefania 1 Gabrielli Andrea 1 Catani Sheila 3 D'Ippolito Mariagrazia 3 Schmid Kara 4 Tortella Frank 4 Wang Kevin 2 Hayes Ronald 2 Formisano Rita 3

1 University of Florida, Gainesville, USA 2 Banyan Biomarkers, Inc., Alachua, FL, USA 3 Santa Lucia Foundation, Rome, Italy 4 Walter Reed Army Institute of Research, Silver Spring, MD, USA P227 MAP-2 Concentration in Serum of Patients After Traumatic Brain Injury: Insight into the Pathophysiological Mechanisms of the Chronic Phase

OBJECTIVES: Severe traumatic brain (TBI) injuries are often associated with long-term and disabling consequences. Prognosis and chronic treatment planning following severe TBI remain challenging. The discovery of specific brain biomarkers could create new opportunities for more accurate clinical assessments identifying groups that may experience better outcomes when exposed to an intervention. The objective of the present study was to evaluate Microtubule-associated proteins (MAP-2), a marker of dendritic damage, in serum of survivors after severe TBI and examine the association with long-term outcome.

METHODS: In 16 patients with severe TBI (Glasgow Coma Scale score [GCS] ≤ 8), serum, taken at 6 months post-injury was analyzed for MAP-2. Physical and cognitive outcomes were assessed, respectively using the Glasgow Outcome scale Extended (GOSE) and Levels of Cognitive Functioning Scale (LCFS).

RESULTS: TBI patients had significantly higher serum MAP-2 concentrations than normal controls (p < 0.0001) at 6 months post-injury. Serum MAP-2 values separated subjects with previous TBI well from normal controls with no history of TBI (p < 0.0001; area under the curve [AUC] = 0.96, sensitivity 91%, specificity 96%). MAP-2 levels correlated with the GOSE (R = 0.58, P = 0.02), and LCFS (R = 0.65, P = 0.007) at month 6.

CONCLUSION: Severe TBI results in a sustained chronic abnormal release of MAP-2 into the peripheral circulation, suggesting ongoing processes after several months from TBI. This study demonstrated that MAP-2 might be of value in refining the prediction of long-term global outcome. The future use of biomarkers will substantially enhance our understanding of chronic conditions following brain injury.

Mondello Stefania 1 Jeromin Andreas 2 Bullock Ross 3 Mo Sweaney Jixiang 2 Streeter Jackson 2 Schmid Kara 4 Tortella Frank 4 Hayes Ronald 2 Wang Kevin 2

1 University of Florida, Gainesville, FL, USA 2 Banyan Biomarkers, Inc., Alachua, FL, USA 3 University of Miami, Miami, FL, USA 4 Walter Reed Army Institute of Research, Silver Spring, MD, USA P228 In Vivo Monitoring of Cytokines and Brain Biomarker Damage Following Severe Traumatic Brain Injury: A Microdialysis Study

OBJECTIVES: Microdialysis has become a routine method for biochemical surveillance of patients with severe traumatic brain injury (TBI). This technique can evaluate patterns of protein expression with high a spatial and temporal resolution than samples from cerebrospinal fluid (CSF) and blood. The aim of this pilot study was to evaluate levels of cytokine and neuronal damage biomarkers, Ubiquitin Carboxyl-terminal Hydrolase-L1 (UCHL1) and Microtubule Associated Protein-2 (MAP-2) and to study their temporal profiles and relationships using microdialysis in patients with severe traumatic brain injury.

METHODS: Six patients were included in the study. 24-h MD samples were collected to analyse cytokines and biomarkers after TBI, including IL-1β, IL-6, TNF-α, INF-γ, UCH-L1 and MAP-2 from 24h up to 4 days post-injury. The concentrations of cytokines were detected using a multiplexed electrochemiluminescence assay, while biomarkers were determined by absorbance-based ELISA assay.

RESULTS: There was a substantial interindividual variability between cytokine and biomarker values. The highest concentrations for UCH-L1 were measured during the first 24 hrs followed by a gradual decline and secondary increases, while MAP-2 showed a sustained elevation that persisted for at least 4 days post-injury. IL-6 showed high initial values that then decrease, in contrast IL-1β, TNF-α and INF-γ showed later elevations. Surprisingly, UCH-L1 levels negatively correlated (p < 0.05) with IL-1β, widely used biomarker of inflammation.

CONCLUSIONS: This study demonstrates the feasibility of analyzing concurrently biomarkers of neuronal injury and inflammatory mediators in microdialysate. Serial measurements of biomarkers and cytokines confirm distinct time patterns. Microdialysis might be used to monitor different temporal characteristics of biomarkers and inflammatory mediators. Our results support a close relationship between brain damage and neuroinflammation following human TBI. Nevertheless, interpretation of microdialysis data is difficult because of the variations in recovery rates and the nonuniformity of patient groups as well as the absence of controls. Further studies are needed.

Bose Prodip 1 2 3 Nelson Rachel 1 Hou Jiamei 2 Parmer Ron 2 Williams Kelli 2 Nissim Nicole 2 Sennhauser Susie 2 Keener Jonathon 2 Wacnik Paul 4 Thompson Floyd 1 2

1 North Florida/South Georgia Veterans Health System, Gainesville, FL, USA 2 Department of Physiological Sciences at the University of Florida, Gainesville, FL, USA 3 Department of Neurology at the University of Florida, Gainesville, FL, USA 4 Neuromodulation Research, Medtronic Inc., Minneapolis, Minnesota, USA P229 Intrathecal Baclofen (ITB) Initiaed in Acute Traumatic Brain Injury (TBI) More Effectively Reduced Spasticity than When Initiated in the Subchronic Setting

Spasticity is a major health problem for patients with TBI. Currently, application of early anti-spastic medication, such as ITB baclofen, a GABA_b agonist, is confounded by insufficient information on interactions with cognitive, balance, and motor recovery, and, insufficient data on the effectiveness of early intervention strategies. Therefore, the purpose of this study was to provide a comprehensive evaluation of safety, feasibility, and efficacy of acute (post TBI 1 week) and subchronic (post TBI 1 month) continuous ITB treatments (Alzet pumps; 0.8μg/hr for 4 weeks). Following closed head TBI in a laboratory rat model (Marmarou model; 450 g/1.25m weight drop), velocity dependent ankle torque, ankle extensor muscle EMGs, 3D footprints (gait), and rotorod balance performance were recorded before injury, and at post-treatment weeks 2 and 4. Serial learning was tested using the Morris water maze (MWM). In the acute treatment group, two, weeks of ITB-treatment completely blocked spasticity (elevated ankle torques and corresponding EMGs). By the fourth treatment week, ankle extensor spasticity was still reduced by 63% compared to untreated injured controls. By contrast, in the subchronic group, treatment produced 65% and 42% attenuation of spasticity following 2 and 4 weeks of ITB-treatments respectively compared to untreated injured controls. All ITB treated animals showed improved scores (trends) for serial learning (probe trail), and improved gait performance compare to untreated injured controls, and no difference in balance performance was observed between the treated and untreated animals. The data indicate that one month ITB treatment in the acute setting fully blocked the early onset spasticity and partially attenuated late onset spasticity with no adverse effects on cognitive and balance performance. Accordingly, acute ITB treatment strategy appeared to be more effective in controlling spasticity than initiating ITB in the subchronic treatment setting.

Supported by Medtronic Inc. and VA Merit Review B6570R.

Russell Kristin Berman Nancy Levant Beth University of Kansas Medical Center, Kansas City, KS, USA

P230 Dietary N-3 Fatty Acid Content Influences Sensorimotor Outcomes After TBI in Juvenile Rats

Children 0-4 years old are at high risk for sustaining traumatic brain injuries (TBI). N-3 polyunsaturated fatty acids, a major component of neural membranes, have neuroprotective and anti-inflammatory activities. Low dietary n-3 content results in reduced accumulation of these fatty acids in the developing brain. This study examined the effects of dietary n-3 content on sensorimotor outcomes after TBI in a juvenile rat model. Male rats (n = 11-12/group), raised from conception on diets containing adequate n-3s (N-3) or low in n-3s (Low N-3), received a unilateral controlled cortical impact injury to the sensorimotor cortex or sham surgery on postnatal day 17. Sensorimotor function was evaluated 1, 7, 14, 21, and 28 days post-surgery using tests previously identified as appropriate for juvenile rats: spontaneous forelimb elevation test, beam walk, and assessment of motor function using the force-plate actometer. All rats with TBI had greater forelimb laterality and hindlimb foot slips as measured with the spontaneous forelimb elevation test and beam walk, respectively. TBI rats also had fewer bouts of low mobility and decreased low mobility distance on day 1. Low N-3 diet treatment alone caused increased total distance traveled and decreased bouts of low mobility in sham rats on day 1. No other diet-induced sensorimotor changes were detected in sham rats. In contrast, TBI Low N-3 rats exhibited greater forelimb deficits than TBI N-3 rats. TBI Low N-3 rats also had fewer bouts of low mobility and decreased low mobility distance on the day after injury compared to TBI N-3 rats, suggesting decreased grooming. TBI Low N-3 rats also exhibited increased total distance traveled on day 1, an effect not observed in TBI N-3 rats. These findings suggest that higher dietary n-3 content may foster improved sensorimotor outcomes after TBI in toddlers.

Supported by HD059939, HD02528, RR016475, and ES007079.

Lee Phil Kim Jieun Gregory Eugene Brooks William Berman Nancy University of Kansas Medical Center, Kansas City, KS, USA

P231 T1 Mapping Reveals Greater Blood Brain Barrier Opening in Aged than Adult Mice After Controlled Cortical Impact

Outcomes after traumatic brain injury are worse in the elderly, but mechanisms underlying this difference are poorly understood. Previously, we (Onyszchuk et al., 2008) demonstrated that blood brain barrier (BBB) opening is greater in aged (22 months) than adult (5 months) mice after controlled cortical impact (CCI). The present study reexamined this issue using longitudinal noninvasive quantitative MRI. T₂-weighted MRI was used to delineate lesions and quantitative T₁ mapping before and after a administering a contrast agent (Gd-DTPA) was used to assess BBB permeability changes. Permeability indices were calculated as the difference between T₁ relaxation rate (R₁) after and before Gd-DTPA administration obtained from the CCI core, peri-CCI cortex, and contralateral cortex 1, 3 and 7 days after CCI in adult and aged mice. In addition, the area under the curve (AUC) of the BBB permeability index over three time points was calculated to characterize the overall impact of the BBB opening. Both the CCI core and peri-CCI cortex showed large initial BBB opening and gradual closing over time, with almost complete recovery at Day 14. No BBB opening was observed in contralateral cortex. The pattern of BBB opening at Day 3 was consistent with our previous report. T1 mapping demonstrated a 1.6 times greater (p = 0.036) BBB opening in the CCI core and 2 times greater (p = 0.002) BBB opening in the peri-CCI cortex (AUC) in aged mice than in adult mice. BBB permeability was also assessed using immunohistochemical analysis of mouse IgG staining of the peri-CCI region. Aged mice had significantly more IgG staining in the peri-CCI region at day 3 (p < 0.001) and day 7 after injury (p < 0.05) than adult mice. Thus, both T1 mapping and IgG staining demonstrate increased blood brain barrier disruption after CCI in aged mice.

Support:R01AG31140.

Sandhir Rajat Anderson Joshua Gregory Eugene Berman Nancy University of Kansas Medical Center, Kansas City, USA

P232 Differential Response of Microrna-21 and its Targets is Associated with Poor Outcome After Traumatic Brain Injury in Aging Mice

The elderly have higher rates of mortality and worse functional outcomes than middle aged or younger adults following traumatic brain injury (TBI). The mechanisms involved in poor outcomes in the elderly are not well understood. We have previously reported exacerbated inflammatory responses, increased cell death, impaired neuroprotective responses, and increased blood brain barrier disruption after TBI in the aged brain. All of these changes are likely to result from altered gene and protein expression patterns after injury. One mechanism that could produce changes in many downstream events is an alteration in microRNA (miR) expression following CNS trauma. The present study investigated the role of miR-21, which confers survivability, in response to traumatic brain injury in the adult and aged brain. Controlled cortical impact injury was induced in adult (5-6 months) and aged (22-24 months) C57/BL6 mice. miR-21 and its targets (PDCD4, TIMP3, RECK, PTEN) were analyzed at 1, 3, 7 days post injury in samples of injured cortex using real-time PCR analysis. Basal mIR-21 expression was higher in the aged brain than in the adult brain. In the adult brain, miR-21 expression increased in response to injury, with the maximum increase 24 hours after injury followed by a gradual decrease, returning to baseline 7 days post-injury. In contrast, in aged mice, mR21 showed no injury response, and expression of miR-21 target genes (PDCD4, TIMP3, RECK, PTEN) was up-regulated at all post injury time points, with a maximal increase at 24 hours post injury. Based on these results, we conclude that the diminished miR21 injury response in the aged brain leads to up-regulation of its targets, contributing to the poor prognosis following TBI in aging brain, and that strategies aimed at up-regulation of miR-21 might be useful in improving outcomes in the elderly following TBI.

Support: R01 AG31140.

Harris Janna Choi In-Young Lee Phil Yeh Hung-Wen Brooks William University of Kansas Medical Center, Kansas City, KS, USA

P233 Imaging Biomarkers of Neuroprotective and ANTI-Oxidative Effects of Citicoline in Traumatic Brain Injury

Citicoline (CDP-choline) is an endogenous compound that has received recent attention as a promising new therapy for traumatic brain injury (TBI). Treatment with exogenous citicoline may provide neuroprotection through several mechanisms, including inhibition of apoptosis and attenuation of oxidative stress. In the present study, we sought to evaluate the effects of post-injury citicoline treatment on neuroimaging biomarkers visible with proton magnetic resonance spectroscopy (¹H-MRS) at 9.4T. Adult male rats received unilateral controlled cortical impact to the sensorimotor cortex and citicoline (200 mg/kg i.p.) or an equivalent volume of saline vehicle at 5 minutes and 4 hours after impact. ¹H-MRS was acquired at 1 hour, 1 day (D1), 3 days (D3), and 7 days (D7) after TBI. Behavioral outcomes were assessed with the Rotorod and Beam Walk tests. An acute post-injury decline in NAA was attenuated in the citicoline treated animals compared to saline (93.7% vs 86.0% of pre-injury levels at 1h) suggesting neuroprotection. However NAA levels in the two groups continued to fall, reaching the same lowest point by D3 (86.8% vs 86.7%). Glutathione (GSH) levels were depleted in both groups at D1, but subsequent recovery of GSH was greater in the citicoline treated animals (98.4% vs 86.3% at D3; 104% vs 87.3% at D7), suggesting the effective antioxidant action of the drug. Citicoline treatment also improved performance on the Rotorod at all time points tested, but did not improve Beam Walk scores. The present results are consistent with the known neuroprotective and antioxidant effects of citicoline, and further support the utility of non-invasive ¹H-MRS for elucidating the mechanisms of novel TBI therapies. This takes on particular significance since ¹H-MRS biomarkers can be measured in the clinical setting with existing MR scanners and techniques.

Singh Ranjana 1 2 Benson Adam 2 Saatman Kathryn 2 3 Geddes James 1 2

1 Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY, USA 2 Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky, Lexington, KY, USA 3 Department of Physiology, University of Kentucky, Lexington, KY, USA P234 Calpain 5 is Expressed in Neurons and May Contribute to Neurodegeneration Following Traumatic Brain Injury

Following traumatic brain injury (TBI), calpain activation is thought to contribute to the neurodegeneration. Several previous studies examined the possible roles of the typical, ubiquitous calpains 1 and 2 in TBI. Typical calpains have a distinct large catalytic subunit and a common small regulatory subunit. Calpain 5 (Capn5) is an additional isoform which lacks the small subunit and the C-terminal EF hand domain of the typical calpains, but has significant homology with the large subunit, non-EF hand calcium binding regions and has a conserved catalytic region. Capn5 is present in the CNS of human, rat and mouse. In C. elegans, silencing of the Capn5 homologue TRA-3 protects from necrotic cell death, indicating that Capn5 may play an important role in cell death. To investigate its role in TBI, we obtained Capn5 deficient mice (B6.129P2-Capn5^tm1Dgen /J) from Jackson labs, originally created by Deltagene. These mice have an IRES-lacZ-Neo555G insert in the calpain 5 gene. Homozygote Capn5 knockout is embryonically lethal, while heterozygotes (Capn5 + /-) develop normally. Using lacZ staining and immunohistochemistry against catalytic domain of Capn5 in the mouse brain sections, we observed that Capn5 was predominantly expressed in neurons. Capn5 immunostaining was not evident in oligodendrocytes and GFAP expressing astrocytes. This contrasts with the more widespread distribution of typical calpains. Capn5 was localized to the cytoplasm of SHSY5Y neuroblastoma cells. To investigate the role of Capn5 in TBI, Capn5 + /- and wild-type (Capn5 + /+) mice received a severe (0.9 mm depth) controlled cortical impact brain Injury. Capn5 + /- (n = 6) mice showed significantly smaller cortical contusion volumes 24hrs post injury, as compared to Capn5 + / + mice (n = 5) (p = 0.03), with no difference between sham groups. These results suggest the possible involvement of the atypical Calpain 5 in neurodegeneration following TBI and identify Calpain 5 as a possible new therapeutic target.

Miller Darren 1 2 Buchanan Ashley 1 Wang Juan 1 Hall Edward 1 2

1 SCoBIRC, University of Kentucky College of Medicine, Lexington, KY, USA 2 Anatomy&Neurobiology, University of Kentucky College of Medicine, Lexington, KY, USA P235 Temporal and Spatial Dynamics of NRF2-Are Mediated Gene Targets in Cortex and Hippocampus Following Controlled Cortical Impact Traumatic Brain Injury in Mice

The pathophysiological importance of oxidative damage after traumatic brain injury (TBI) has been extensively demonstrated. The transcription factor Nrf2 mediates transcription of antioxidant/cytoprotective genes by binding to the antioxidant response element (ARE) within DNA. Upregulation of these genes constitutes a pleiotropic cytoprotective-defense pathway. We investigated the time-course of Nrf2-ARE-mediated expression in cortex and hippocampus using a unilateral controlled cortical impact (CCI) model. Young-adult male CF-1 mice underwent sham surgery (craniotomy only) or severe CCI (1.0mm). Ipsilateral hippocampus and cortex were collected for Western-blot protein analysis (n = 6/group) or quantitative-RT-PCR of mRNA (n = 3/group) at 3, 6, 12, 24, 48, 72 hours or 1-week post-injury. HO-1 mRNA and protein increased at 24, 72 hours in cortex and 24, 48 hours in hippocampus (p < .05). SOD1 mRNA decreased in hippocampus at 3, 6, 48, 72 hours (p < .05) but no changes occurred in cortex. GFAP mRNA increased at 24, 48, 72 hours and 1-week (p < .05) in both regions. GR mRNA increased in hippocampus at 24 hours (p < .05) but without change in cortex or GR protein expression. NQO1 mRNA increased at 72 hours and 1-week (p < .05) in cortex but not in hippocampus. Catalase mRNA increased at 72 hours in hippocampus and at 48, 72 hours in cortex (p < .05). GST-mu5 mRNA decreased at 6, 24, 48 hours in hippocampus and 24, 48 hours in cortex (p < .05). GPx-3 mRNA decreased at 3, 6, 24 hours in cortex but increased at 72 hours and 1-week in hippocampus (p < .05). Nrf2 mRNA increased at 48, 72 hours in cortex and at 48, 72 hours and 1-week in hippocampus (p < .05) demonstrating the first evidence of such changes post-TBI. Interestingly, increased Nrf2-ARE-mediated expression was not observed until ∼24 hours, whereas prior work showed oxidative damage occurring 1-12 hours post-TBI. These results underscore the need to discover pharmacological agents to enhance Nrf2-ARE-mediated expression early post-TBI.

Miller Darren 1 2 Wang Juan 1 Carrico Kimberly 1 Hall Edward 1 2

1 SCoBIRC, University of Kentucky College of Medicine, Lexington, KY, USA 2 Anatomy & Neurobiology, University of Kentucky College of Medicine, Lexington, KY, USA P236 Time-Course of Nitric Oxide Synthase Expression in Cortex and Hippocampus in a Controlled Cortical Impact Mouse Model of Traumatic Brain Injury

We previously demonstrated the pathophysiological importance of peroxynitrite (PN), formed by reaction of nitric oxide (•NO) and superoxide radical (O₂ ^•-), in traumatic brain injury (TBI) (Deng et al., Exp. Neurol. 205: 154-165, 2007). Since •NO is produced by multiple isoforms of nitric oxide synthase (NOS): endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS), we compared the time course of NOS expression post-TBI to PN-mediated protein nitration (3-nitrotyrosine, 3-NT) in cortex (CTX) and hippocampus (HIPP) in a unilateral controlled cortical impact (CCI) TBI mouse model. Young adult male CF-1 mice underwent sham surgery (craniotomy only) or severe CCI (1.0mm). Ipsilateral HIPP and CTX was collected for Western blot analysis of protein (n = 6/group) or quantitative RT-PCR of mRNA (n = 3/group) at 3, 6, 12, 24, 48, 72 hours or 1 week post-injury. CTX eNOS mRNA levels increased at 24, 48 hours post-injury (p < .05), but not in HIPP. eNOS protein increased at 48 and 72 hours in CTX, and at 72 hours and 1 week in HIPP (p < .05). CTX nNOS mRNA decreased at 3 hours and at all times in HIPP (p < .05). CTX nNOS protein increased at 72 hours and 1 week (p < .05). iNOS mRNA increased at 24, 48, 72 hours (p < .05) in both CTX and HIPP. No change was observed in CTX iNOS protein but it was increased at 3 hours (p < .05) in HIPP. Interestingly, increased eNOS and iNOS expression was not observed until 24 hours post-TBI, in contrast to our prior work which showed increased 3-NT from 1-12 hours. Thus, NOS induction may be temporally unrelated to early PN-mediated nitration where formation of PN may be provided in the first post-TBI hours by activation of existing NOS protein. The role of the > 24 hr eNOS/iNOS expression in delayed secondary injury or possibly repair mechanisms remains to be elucidated.

Rowe Rachel 1 3 Striz Martin 1 4 O'Hara Bruce 1 4 Lifshitz Jonathan 1 2 4 5

1 University of Kentucky, Lexington, KY, USA 2 Spinal Cord and Brain Injury Research Center, Lexington, KY, USA 3 Dept. Anatomy and Neurobiology, Lexington, KY, USA 4 Dept. of Biology, Lexington, KY, USA 5 Physical Medicine and Rehabilitation, Lexington, KY, USA 6 College of Medicine, Lexington, KY, USA P237 Effects of Diffuse Traumatic Brain Injury on Sleep in the Mouse

Sleep is a naturally recurring state characterized by reduced or absent consciousness and inactivity of nearly all voluntary muscles. The purpose of sleep is largely unknown; however, current hypotheses indicate a regenerative function. Immediately following traumatic brain injury (TBI), the current dogma is for brain-injured individuals to avoid sleep or be frequently awoken; this practice is not supported by biomedical literature. If sleep is regenerative, then post-traumatic sleep may contribute to recovery of function following injury. We contend that post-traumatic sleep results from injury-induced inflammatory processes, for which some inflammatory mediating cytokines (e.g. IL-1) have dual roles as sleep regulatory substances. Here, we test the hypothesis that diffuse TBI increases quantitative measures of sleep in the mouse. Diffuse TBI was induced by midline fluid percussion injury in young adult, male C57Bl/6J mice. Groups of mice were sham (n = 6), mild (n = 9; 0.8 atm; 1-3 min righting reflex time) or moderate (n = 9; 1.4 atm; 4-6 min righting reflex time) brain-injured one hour after dark onset to evaluate injury-induced sleep behavior when mice are typically active. After brain injury, mice were returned to individual sleep monitoring cages. Sleep profiles (quantitative aspects of sleep/wake activity) were measured non-invasively using a system that utilizes piezoelectric materials configured as highly-sensitive pressure detectors. The pressure detectors are incorporated into the bottom of the animal cage to detect sleep. The sleep profiles of the animals were measured for seven days post-injury. Results pending further analysis expected to be completed by May 2011.

Support: KSCHIRT 10-5A, NIH R21 NS072611, NIH ROI NS-065052.

Marti Francesc 1 Bachstetter Adam 2 3 Rowe Rachel 3 4 Ellis Gavin 1 Van Eldik Linda 2 3 4 Lifshitz Jonathan 3 4 5

1 Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, USA 2 Sanders-Brown Center on Aging, University of Kentucky College of Medicine, Lexington, KY, USA 3 Anatomy & Neurobiology, University of Kentucky College of Medicine, Lexington, KY, USA 4 Spinal Cord & Brain Injury Research Center, University of Kentucky College of Medicine, Lexington, KY, USA 5 Physical Medicine & Rehabilitation, University of Kentucky College of Medicine, Lexington, KY, USA P238 Systemic Loss of Peripheral Regulatory T-Cell Differentiation After Diffuse Brain Injury in the Mouse

Inflammation follows injury, whether sustained by the body, peripheral nervous system or central nervous system (CNS). Typically, inflammatory signaling begins at the injury site shortly after injury, remains elevated during cellular repair and then subsides over a protracted time course. However, inflammatory signaling in the presence of blood-brain barrier disruption could transfer immunogenicity between systemic and CNS compartments. Here we test the hypothesis that diffuse brain injury initiates a broad CNS inflammatory program, from which delayed deficits in systemic immune regulation emerge. In particular, we evaluate the potential for T-cells to transition into regulatory T-cells (T_reg) after CNS injury. Diffuse brain injury was induced by moderate fluid percussion injury (1.4 atm; 4-6 min righting reflex time) in young adult male C57Bl/6 mice. At eight post-injury time points, cortical biopsies were dissected from sham and brain-injured mice (n = 3 per time point). The inflammatory cytokine response was determined by ELISA for IL-1β, IL-6, CXCL1, TNFα, IL-10, IFN-γ and IL-12p70. Inflammatory cytokines (IL-1β, IL-6, CXCL1, TNFα) were elevated within 1 hour post-injury compared to sham, with significant peak responses occurring within 9 hours post-injury. By 7 days post-injury, cytokine levels in the cortex were at or below sham levels. From separate mice at 7 days post-injury, T-cells were cultured from the pooled collection of lymph nodes of sham and brain-injured mice. The harvested T-cell population was indistinguishable between sham and brain-injured mice. Then, T-cells underwent a protocol to induce T_reg differentiation in response to Retinoic Acid (RA). Results revealed that T_reg were generated from uninjured sham T cells. Conversely, RA-mediated T_reg differentiation was completely abrogated in T cells from brain-injured mice. Thus, acute CNS inflammation, in the presence of blood-brain barrier disruption, affects the dynamic response of the systemic immune system to subsequent challenge.

Support: UK College of Medicine; KSCHIRT 10-5A; NIH NS072611

Cao Tuoxin 1 2 Lisembee Amanda 2 Lifshitz Jonathan 2 3 4

1 Department of Biology, College of Arts and Sciences 2 Spinal Cord and Brain Injury Research Center 3 Department of Anatomy and Neurobiology 4 Department of Physical Medicine and Rehabilitation, College of Medicine, University of Kentucky, Lexington, KY, USA P239 Microglia in the Somatosensory Barrel Cortex Align Perpendicular to the Dural Surface After Diffuse Brain Injury in Rats

Morbidities experienced by traumatic brain injury (TBI) survivors are thought to develop from secondary molecular and cellular events that compound primary neuronal, glial and vascular injuries. In experimental diffuse TBI, we observed consistent axonal injury, neuronal atrophy and neuroplasticity in the primary somatosensory barrel fields (S1BF), which correlated with increased sensory sensitivity to whisker stimulation. To expand upon these studies, here we investigated temporal changes in microglial morphology in S1BF after diffuse TBI in the rat.

Adult male, Sprague-Dawley rats were subjected to a single midline fluid percussion injury (FPI) of moderate severity (1.9 atm; 6-10 min righting reflex time)or sham injury. Using Iba-1 immunolabeling, microglia morphology was compared between sham and brain-injured animals. Microglia in S1BF of brain-injured rats showed swollen cell bodies and reduced microglial processes compared to sham animals, indicating microglial activation. Furthermore, these microglia showed dynamic alignment such that cells appeared to be elongated and extending their processes in alignment with neuronal dendrites. The alignment of microglia was observed starting 2 days post-injury, becoming most prominent at 7 days post-injury. By 28 days post-injury, microglia activation subsided, as indicated by a return of ramification and decreased cell body swelling compared to 7days post-injury. Additional studies to be presented will double-label uninjured and brain-injured tissue with Iba-1 and neurofilament (NF-M), microtubule associated protein 2 (MAP-2), myelin associated glycoprotein (MAG) or synaptophysin to determine the relationship between microglia and axons, dendrites, oligodendrocytes and synaptic terminals, respectively.

Thus, diffuse brain injury induces a sub-acute morphological change in microglia aligned with neuronal processes in S1BF. We postulate that these microglia ‘rod-cells’ are involved in synaptic stripping associated with whisker circuit disruption.

Supported, in part, by University of Kentucky College of Medicine, NIH NINDS R01 NS065052, Kentucky Spinal Cord and Head Injury Research Trust (KSCHIRT) 7-11 and NIH NINDS P30 NS051220.

Learoyd Annastazia 1 2 Lifshitz Jonathan 2 3 4

1 Department of Biology and Biochemistry, University of Bath, Bath, UK 2 Spinal Cord & Brain Injury Research Center 3 Department of Anatomy & Neurobiology 4 Department of Physical Medicine & Rehabilitation, University of Kentucky College of Medicine, Lexington, KY, USA P240 Comparison of Rat Sensory Behavioral Tests to Detect Somatosensory Morbidity After Diffuse Brain Injury

Brain injury disrupts neuronal circuits, which can impact neurological function. To pursue therapeutic interventions and explore anatomical underpinnings of neurological dysfunction, selective and sensitive behavioral tests are necessary to evaluate the injury and recovery processes. Previously, we reported that the whisker nuisance task (WNT), where whiskers are manually stimulated in an open field, shows delayed-onset sensory sensitivity in diffuse brain-injured rats. In the present communication, we evaluate a battery of tests to explore this somatosensory deficit. In addition to the WNT, we selected the gap cross test, a novel angle entrance test and the whisker guided exploration test, because they require sensory input from the whiskers without pre-requisite training or learning. Brain-injured (n = 11) and sham (n = 8) rats were tested prior to, one and four weeks after midline fluid percussion brain injury (moderate: 2.0 ATM). For the WNT, we confirmed that brain-injured rats develop significant sensory sensitivity to whisker stimulation over 28 days. In the gap cross test, where rats cross progressively larger elevated gaps, we found that animals were inconsistent in the crossable distance regardless of injury. In the angle entrance task, where rats enter corners of 30°, 40°, 50° or 80°, rats entered equally far into the corner regardless of injury. In the whisker guided exploration test, where rats voluntarily explore an oval circuit, we identified significant decreases in the expression of certain behavioral traits (number of rears and reversals) and differences in the predominant location (injured rats spend more time in the inside turn compared to the outside turn) after injury and increased thigmotaxis after sham and brain-injury. Both the WNT and whisker guided exploration tasks show injury-induced somatosensory behavioral morbidity; however, the WNT is superior in the detection of neurological dysfunction, possibly due to the repeated manual stimulation while other tasks were voluntary behaviors.

Reneer Dexter 1 Hisel Richard 4 Hoffman Joshua 2 Kryscio Richard 3 Lusk Braden 2 Geddes James 1

1 University of Kentucky Spinal Cord and Brain Injury Research Center, Lexington, KY, USA 2 University of Kentucky Department of Mining Engineering, Lexington, KY, USA 3 University of Kentucky Department of Biostatistics, Lexington, KY, USA 4 GLR Enterprises, LLC., Nicholasville, KY, USA P241 Blast-Induced Brain Injury: Influence of Shockwave Components

Blast-induced mild traumatic brain injury (bTBI) has become increasingly common in current military conflicts. Additionally, civilians are also at risk due to terrorism and industrial accidents. The mechanisms by which non-impact blast exposure results in bTBI are not completely understood. Current small animal bTBI models utilize mostly compressed gas-driven membrane rupture as their blast wave source, while large animal models use chemical explosives. The pressure-time signature of each blast mode is unique, making it difficult to evaluate the contributions of the different components of the blast wave to bTBI when using a single blast source. We utilized a multi-mode shock tube, the McMillan blast device, capable of utilizing compressed air- and compressed helium-driven membrane rupture, and the explosives oxyhydrogen and cyclotrimethylenetrinitramine (RDX, the primary component of C-4 plastic explosives) as the driving source. At equal maximal peak overpressures, the positive pressure phase of compressed air-driven blasts was longer, and the positive impulse was greater, than those observed for shockwaves produced by other driving sources. Helium-driven shockwaves more closely resembled RDX blasts, but by displacing air created a hypoxic environment within the shock tube. Pressure-time traces from oxyhydrogen-driven shockwaves were very similar to those produced by RDX, although they resulted in elevated carbon monoxide levels due to combustion of the polyethylene bag used to contain the explosive gases prior to detonation. Rats exposed to compressed air-driven blasts had more pronounced vascular damage than those exposed to oxyhydrogen-driven blasts of the same peak overpressure, suggesting that variations in blast wave components other than peak overpressure may alter the extent of bTBI. Use of this multi-mode shock tube in small animal models will enable comparison of the extent of brain injury with the pressure-time signature produced using each blast mode, facilitating evaluation of the blast wave components contributing to bTBI.

Madathil Sindhu Kizhakke Brelsfoard Jennifer Saatman Kathryn University of Kentucky, Lexington,KY, USA

P242 Astrocyte-Specific Overexpression of Insulin-Like Growth Factor-1 Protects Hippocampal Neurons Following Traumatic Brain Injury in Mice

Cognitive impairment is one of the chronic effects of traumatic brain injury (TBI). Previously, systemic administration of insulin-like growth factor-1 (IGF-1) has been shown to attenuate behavioral dysfunction caused by TBI in rodents. Our group also found improved behavioral function in IGF-1 overexpressing (IGF-1Tg) mice following TBI. However, less is known regarding the neuroprotective efficacy of IGF-1 in TBI. Here we evaluated the ability of IGF-1 to reduce hippocampal pathology following moderate or severe controlled cortical impact (CCI) injury. IGF-1 was targeted to the injured hippocampus by using transgenic mice with astrocyte-specific IGF-1overexpression. Adult mice were subjected to 0.5mm or 1.0mm CCI injury or sham surgery. At 1d or 3d after CCI, subsets of animals were used for histological (n = 6-8 injured/genotype/severity, n = 4 shams/genotype) and western blot (n = 5-8 injured/genotype/severity, n = 4 shams/genotype) analyses. A separate cohort of mice (n = 13 injured/genotype, n = 8 shams/genotype) received severe CCI and was euthanized 10d post-injury to analyze sustained neuroprotection. Hippocampal IGF-1 overexpression was verified using IGF-1 immunohistochemistry and ELISA. TBI induced an increase in astrocytic IGF-1 expression in IGF-1Tg mice. Compared to wild type (WT) mice, astrocytic hypertrophy was more prominent in IGF-1Tg mice. Western blot analysis confirmed higher hippocampal GFAP levels in injured IGF-1Tg mice. Akt phosphorylation was increased in IGF-1Tg compared to WT mice, suggesting a role for the PI3K/Akt pathway in mediating neuroprotection. IGF-1 overexpression attenuated early neurodegeneration in the CA-3 and dentate gyrus subregions of the hippocampus when quantified using Fluorojade-C. Qualitative assessment of NeuN and Nissl staining suggests neuroprotection sustained up to 10d after CCI. Stereological quantification of neuronal survival is ongoing. Our results indicate that IGF-1 protects against hippocampal neuronal loss following brain trauma. This hippocampal protection may contribute to improved cognitive outcomes previously observed in IGF-1Tg or IGF-1 treated rodents.

Supported by: NS058484, NS051220, and KSCHIRT 7-20.

Carlson Shaun Brelsfoard Jennifer Saatman Kathryn University of Kentucky, Lexington, KY, USA

P243 Evaluation of Systemic and Central Infusion of Insulin-Like Growth Factor-1 Following Controlled Cortical Impact

Traumatic brain injury (TBI) produces neuronal dysfunction and loss, which can culminate in lasting motor and cognitive impairment. Insulin-like growth factor-1 (IGF-1) is a potent neurotrophic factor capable of mediating both neuroprotective and neuroreparative mechanisms. While systemic administration of human IGF-1 (hIGF-1) improves motor and cognitive function in brain-injured rats, little is known regarding the ability of IGF-1 to promote cell survival in TBI. We hypothesized that elevating brain levels of hIGF-1 would reduce cell loss and attenuate behavioral dysfunction after severe controlled cortical impact (CCI) brain injury. To this end, C57BL/6 mice were subjected to 0.8mm CCI and subcutaneously infused with either 4mg/kg/d hIGF-1 or vehicle for 3 days beginning at 15 minutes post-injury (n = 3 sham and n = 5 injured/ treatment). IGF-1 treated animals exhibited no improvement in motor function or overt neuroprotection as assessed by histological measures. To evaluate the effectiveness of extending the treatment duration, a second cohort (n = 5 injured/treatment) received a 7 day infusion, using an identical daily dose and route of administration. Compared to mice receiving vehicle, those treated with IGF-1 demonstrated a slight improvement in motor function and significant preservation of neurofilament protein in cortical homogenates, consistent with neuroprotection. Despite increased cortical levels of phosphorylated Akt, a signaling protein downstream of IGF-1, hIGF-1 was not detectable by ELISA. To try to achieve higher brain levels of IGF-1, we next administered 0.33mg/kg/d hIGF-1 (n = 8) or vehicle (n = 4) for 3 days by intracerebroventricular infusion starting 15 minutes after 0.8mm CCI injury. hIGF-1 infusion increased cortical Akt activation and hIGF-1 was detected in the cortex and hippocampus of treated animals. Future experiments will verify if central infusion is neuroprotective and attenuates behavioral deficits associated with severe brain injury.

Supported by: NIH P01 NS058484, KSCHIRT 7-20, NIH P30 NS051220, and T32 DA022738

Schoch Kathleen M. 1 von Reyn Catherine R. 2 Telling Glenn C. 1 Meaney David F. 2 Saatman Kathryn E. 1

1 University of Kentucky, Lexington, KY, USA 2 University of Pennsylvania, Philadelphia, PA, USA P244 Reduction in Posttraumatic Calpain-Mediated Cleavage of Spectrin and Sodium Channel Protein with Calpastatin Overexpression

Calpains, calcium-activated neutral proteases, are activated early after traumatic brain injury (TBI), with subsequent proteolysis of substrates including cytoskeletal components and membrane receptors. The prolonged activity of calpains after trauma suggests that the action or levels of its endogenous inhibitor, calpastatin, may be insufficient to fully inhibit the proteolytic activity of calpains. Therefore, we hypothesize that calpastatin overexpression via the prion promoter (PrP-hCAST) will reduce calpain activity and calpain-mediated substrate proteolysis after TBI. Previously, we showed that cortical protease activity via fluorogenic assay was lower in homogenates obtained from transgenic mice (n = 6) compared to wildtype (WT) mice (n = 5; p < 0.001). Here we demonstrate that hCAST overexpression results in reduced posttraumatic cytoskeletal and membrane channel cleavage. Contusion brain injury was created with a controlled cortical impact (CCI) device, transiently indenting the exposed cortex to a 1.0mm depth. Immunoblots for alpha-spectrin revealed that PrP-hCAST mice (n = 6) exhibited significantly less cortical and hippocampal calpain-mediated proteolysis than WT (n = 5-7) at 6 hours (p < 0.0.01) and 24 hours (p < 0.005) post-injury. Brain injury resulted in a reduction in cortical full-length voltage-gated sodium channel (NaCh) protein, coupled with the appearance of fragments detected by pan, Nav1.2 I-II loop and C-terminus-specific antibodies. PrP-hCAST mice (n = 4) exhibited a significant reduction in NaCh breakdown products detected by pan, Nav1.2 I-II loop, and Nav1.2 C-terminal antibodies compared to WT (n = 4) at 6 h. At 24 h post-CCI, an 85 kDa Nav1.2 I-II loop fragment was also attenuated in PrP-hCAST mice (n = 4) compared to WT (n = 5); however, other sodium channel fragments were equivalent. Considering the structural and functional importance of spectrin and NaCh dynamics in neurons, preservation of these proteins after injury may contribute to a reduction in neuronal damage or dysfunction. Future studies will continue to investigate sparing of calpain substrates with hCAST overexpression, supporting the therapeutic potential for augmenting endogenous calpastatin levels.

Readnower Ryan Geddes James Sullivan Patrick University of Kentucky-Spinal Cord and Brain Injury Research Center, Lexington, KY, USA

P245 Genetic Analysis of the Role of Cyclophilin D in Traumatic Brain Injury Pathology

Cyclophilin D (CypD) dependent cell death mechanisms, including Ca²⁺ dyshomeostasis and increased reactive oxygen species production, have been implicated in traumatic brain injury (TBI). Pharmacological inhibition of CypD using cyclosporin A (CsA) and N-methyl-4-isoleucine-cyclosporin (NIM811) following TBI has been shown to result in neuroprotection. However, interpretation of the protective mechanisms of CsA and NIM811 are confounded by potential off-target effects of both compounds. In order to directly assess the contribution of CypD in TBI pathology, wild-type (WT) mice (n = 5) and mice lacking the Cyp-D enconding gene Ppif (n = 4) were subjected to a controlled cortical impact model of TBI. At 18 days post-injury cortical tissue sparing assessment showed that CypD knockout (KO) mice had significantly more spared cortical tissue than WT mice. Injury in WT mice resulted in a significant decrease in neurons in hippocampal regions; dentate gyrus, CA1, and CA3. Importantly, CypD KO prevented the cell loss in area CA3. Initial reports indicate that CypD KO mice (n = 4) have improved mitochondrial complex I function compared to WT mice (n = 3) following TBI. This is the first study to report the effects of Cyp-D knockout in a model of traumatic brain injury and we showed that the lack of Cyp-D significantly decreased the damage associated with TBI. Taken together, Cyp-D dependent cell death is critically involved in TBI, supporting the hypothesis that Cyp-D is a valid target for neuroprotection following TBI.

Bains Mona Gilmer Lesley Hall Edward University of Kentucky Spinal Cord & Brain Injury Research Center, Lexington, KY, USA

P246 Inhibition of Cytoskeletal Degradation with Post-Injury Administration of the Calpain Inhibitor SNJ-1945 in Severe Mouse Traumatic Brain Injury

Calpain, a calcium-dependent cysteine protease, is activated in response to rapid increases in intracellular calcium levels following traumatic brain injury (TBI). Calpain, in turn degrades vital cytoskeletal and receptor proteins and signal transduction enzymes, which contributes to the secondary injury response and ultimately neuronal cell death. Although targeted calpain inhibition holds promise as a potential therapeutic for TBI, the available calpain inhibitors are limited by their low membrane permeability, solubility and metabolic stability. We have previously shown that the calpain inhibitor MDL-28170 decreases 24-hour cytoskeletal degradation in a mouse model of TBI, but this required repeated dosing over a period of 5 hours (Thompson et al., J. Neurotrauma 27: 2233-2243, 2010). In the present study, we examined a newer calpain inhibitor, SNJ-1945, which has been described as having improved membrane permeability, using the controlled cortical impact TBI model in male CF-1 mice. We found that compared to MDL-28170, an early (15 min), single post-injury dose of SNJ-1945 effectively decreased cytoskeletal degradation at 24 hours post-injury. Dose response studies (3.125 – 100 mg/kg, i.p.) demonstrated a U-shaped curve with 12.5 and 25 mg/kg, i.p. producing the maximal effect of SNJ-1945 on calpain-mediated degradation of the neuronal cytoskeletal protein α-spectrin. SNJ-1945 equally reduced calpain-nonspecific (150-kDa) and calpain-specific (145-kDa) α-spectrin breakdown products in both the ipsilateral cortex and hippocampus at 24 hours post TBI. Both 12.5 and 25 mg/kg significantly reduced the calpain specific (145-kDa) spectrin breakdown product by 55% in cortex and 33% in hippocampus compared to vehicle. Ongoing experiments include a comparative efficacy study of MDL-28170 versus SNJ-1945 with a repeated dosing regimen as well as a therapeutic window analysis on calpain-mediated α-spectrin degradation in cortical and hippocampal tissue post-injury.

These studies were supported by 2P30 NS051220 and the Kentucky Spinal Cord & Head Injury Research Trust.

Thomas Theresa Currier 1 Hinzman Jason 1 2 4 Gerhardt Greg 2 4 5 Lifshitz Jonathan 1 2 3

1 Spinal Cord and Brain Injury Research Center, Lexington, KY, USA 2 Anatomy and Neurobiology, Lexington, KY, USA 3 Physical Medicine & Rehabilitation, Lexington, KY, USA 4 Center for Microelectrode Technology, Lexington, KY, USA 5 Morris K. Udall Parkinson's Disease Research Center of Excellence, University of Kentucky College of Medicine, Lexington, KY, USA P247 Diffuse Brain Injury-Induced Increases in Glutamate Neurotransmission Parallel the Development of Late-Onset Behavioral Morbidity in Rats

Midline fluid percussion injury (mFPI) in rodents causes diffuse axonal injury and deafferentation. After mFPI, brain-injured rats demonstrate robust sensory sensitivity in response to manual whisker stimulation by 28 days post-injury. Whisker somatosensation relies on intact thalamocortical glutamatergic relays through the ventral posterior medial (VPM) thalamic nucleus. These experiments were designed to elucidate functional, pathological, and structural consequences of diffuse brain injury in the functionally-relevant VPM. Using glutamate-selective microelectrodes with amperometric detection in anesthetized rats, we measured a 110% increase in tonic glutamate levels and a 100% increase in potassium-evoked glutamate release, with no change in glutamate clearance by 28d post-injury. Increased glutamate signaling paralleled the temporal development of post-traumatic sensory sensitivity. A 50% greater reduction in evoked glutamate release after Ω-conotoxin was observed in brain-injured animals indicating that the increased evoked glutamate release in the VPM primarily originated from presynaptic terminals. Coronal tissue sections were assessed for gross cellular histology, neurodegeneration and neuronal morphology in the VPM at 1, 7, and 28 days post-injury. Giemsa staining confirmed no overt changes in neuronal morphology or regional cytoarchitecture. Silver staining showed marked neurodegeneration at 1d post-injury (10.1% of the VPM stained) that increased through 7d post-injury (16.8%), and persisted at 28d post-injury (13.6%) as compared to uninjured brain. In sham animals, Golgi staining revealed individual axons and dendrites with long and evenly distributed branches between neurons. At 1d post-injury, remains of neuronal processes appeared severely blunted and/or fractured. By 7 and 28d post-injury, neuronal processes re-appeared, but were truncated, disorganized and clustered compared to sham. Quantitative neuronal morphology in VPM of brain-injured rats is ongoing. Compromised structural integrity of surviving circuits following diffuse brain injury may contribute to changes in neurotransmission and maladaptive plasticity underlying behavioral morbidity.

Support: KSCHIRT #7-11, P30 NS051220-01, NIH R01 NS-065052, T32 AG000242, UK College of Medicine

Hinzman Jason Thomas Theresa Quintero Jorge Gerhardt Greg Lifshitz Jonathan University of Kentucky, Lexington, KY, USA

P248 Diffuse Brain Injury Disrupts Glutamate Regulation in the Rat Striatum

Central nervous system function requires strict regulation of extracellular glutamate. Disrupted glutamate release and glutamate uptake can increase extracellular glutamate to excitotoxic levels, propagating secondary injury cascades and promoting neurological dysfunction. Clinically, increased extracellular glutamate levels have correlated with poor outcomes following TBI. Recent development of enzyme-based microelectrode arrays (MEAs) have permitted unprecedented insight into extracellular glutamate regulation near synapses. We hypothesize that diffuse TBI produces post-traumatic disruptions in glutamate regulation. Using MEAs, we examined glutamate regulation in male Sprague Dawley rats two days after sham or moderate midline fluid percussion injury (FPI). Previously, we showed injury-dependent increases in tonic glutamate levels and glutamate release. Here, we confirm the original finding detecting a significant increase in tonic glutamate after FPI (4.07 ± 0.61 μM) compared to sham (2.15 ± 0.37 μM). Next, we pharmacologically manipulated specific components of glutamate regulation to determine the source of increased glutamate levels after TBI. To examine calcium-mediated vesicular glutamate release on tonic glutamate levels, we blocked N-type calcium channels with Ω-conotoxin. Tonic glutamate levels were largely calcium-independent after FPI, being reduced by only (0.36 ± 0.7 μM) compared to (2.6 ± 0.8 μM) after sham. Next, we examined glutamate uptake by blocking the excitatory amino acid transporters (EAATs) with a competitive inhibitor (TBOA). Inhibition of glutamate uptake produced significantly larger increases in tonic glutamate after FPI (8.5 ± 0.8 μM), compared to sham (4.4 ± 0.9 μM). To further examine glutamate uptake kinetics, we locally applied glutamate and examined the signal decay time (T₈₀). Glutamate clearance was significantly slower after FPI with a T₈₀ of (4.48 ± 0.57 s) after FPI compared to (3.19 ± 0.17 s) after sham. Here, we report specific mechanisms responsible for disrupting glutamate regulation after diffuse brain injury. Future work will examine if targeted therapeutics to improve glutamate regulation leads to improved outcomes after TBI.

Support: NIH F31 NS067899, KSCHIRT 7–11.

Sour Chandler Janowich Jacqueline Betz Joshua Stein Deborah Aarabi Bizhan Shanmuganathan Kathirkamanthan Gullapalli Rao University of Maryland School of Medicine, Baltimore, MD, USA

P249 Default Mode Network Changes Following Mild TBI - Preliminary Findings from Magnets Study

PURPOSE: The default mode network (DMN) has been shown to be disrupted in patient populations with cognitive deficits such as ADHD, schizophrenia, and Alzheimer's disease. Due to the cognitive deficits and diffuse axonal injury associated with mild TBI (mTBI), we hypothesized that the DMN would be disrupted following mTBI.

METHODS: Resting state data was obtained on patients suffering from mTBI within ten days of initial injury (N = 32) and again one month following initial injury (N = 20) and compared with normal control subjects (N = 14). All resting state images were obtained on a 3T scanner using a T2*-weighted EPI sequence covering the entire brain over 6 min 42 s yielding 171 volumes. Time series from a 5mm ROI from the posterior cingulate cortex (PCC) was correlated with every voxel in the brain to create an individual whole brain resting state correlation map. The strength of functional connectivity was obtained from average correlation coefficients between a ROI from the PCC and ROI in the bilateral MTL, frontal, thalamus, parietal and medial ACC.

RESULTS: Results suggest that the strength of the functional connectivity within the DMN is decreased in the bilateral Medial Temporal Lobe (MTL), bilateral thalamus and left frontal in the acute stages, but only the bilateral MTL and L frontal in the sub-acute stages suggesting recovery within the DMN in the thalamus.

CONCLUSION: We propose that this recovery is unlikely due to recovery of the integrity axonal tracts, but a reorganization through recruitment and strengthening of alternative neural connections. Further support for this explanation comes from preliminary data from cognitive testing using the ANAM in a subset of these patients which suggests that mTBI patients have comparable accuracy to controls but increased reaction times. We believe that this is evidence for a less efficient neural network due to reorganization following mTBI.

Zhuo Jiachen Xu Su Mullins Jake Hazelton Julie Fiskum Gary Gullapalli Rao University of Maryland School of Medicine, Baltimore, MD, USA

P250 Direct Detection of Astrogliosis in Vivo Following Traumatic Brain Injury

PURPOSE: The goal of this study was to determine whether diffusion kurtosis imaging (DKI) was sensitive in detecting microstructural changes due to reactive astrogliosis following controlled cortical impact injury (CCI) in several gray and white matter regions of the brain.

METHODS: Adult male Sprague-Dawley rats were subjected to left parietal CCI injury and imaged using a 7T. For DKI, diffusion weighted images were acquired with single shot spin-echo EPI sequence. Following 5 images acquired using b = 0 s/mm², two separate b-values (1000, 2000 s/mm²) were acquired for 30 direction (δ/Δ = 4/23 ms). Imaging was performed at 2 hours and 7 days following injury induction and afer obtaining baseline images. Animals were perfused after imaging at 7 days and stained for GFAP.

RESULTS: Both fractional anisotropy and mean diffusivity normalized by seven days. However, mean kurtosis (MK) was significantly elevated at 7 days in the ipsilateral regions of the brain. Further, at 7 days abnormal MK was observed in the contralateral regions while no changes were observed with MD and FA. GFAP staining demonstrated reactive astrogliosis in regions of elevated MK.

CONCLUSION: Our study suggests that DKI is sensitive to microstructural changes resulting from increased astrocytotic activity that may be missed by standard diffusion tensor imaging parameters. Monitoring changes in MK allows the investigation of molecular and morphological changes due to reactive astrogliosis and may complement information available from standard DTI parameters. To date the use of diffusion tensor imaging has been limited to study changes in white matter integrity following traumatic insults. Given the sensitivity of DKI in detecting microstructural changes in vivo even in the gray matter extends the use of DTI techniques to understand patho-morphological changes following a traumatic insult.

Hazelton Julie 1 Gullapalli Rao 1 Fourney William 2 Fiskum Gary 1

1 University of Maryland, School of Medicine, Baltimore, USA 2 University of Maryland, School of Engineering, College Park, USA P251 Mild Brain Injury in a Rat Model of Underbody Blast-Induced Hyper-Acceleration

PURPOSE: Many victims of blast TBI are occupants in vehicles subjected to underbody blasts and are exposed to extremely high G-forces. We studied the effects of underbody blast-induced acceleration on rats to determine if G force alone, in the absence of blast overpressure, causes brain injury and to characterize the nature of this injury.

METHODS: An explosive charge is placed under the center of a heavy metal plate at various stand-off distances that generate peak G-forces of from 20 − 50 Gs. The explosion causes the plate with anesthetized rats secured on top to accelerate vertically several inches, reaching maximum acceleration within 5 msec. Pressure sensors indicate blast exposures of less than 5 psi. At 30 min to 30 days after the blast, 40 rats were perfusion-fixed and their brains analyzed for evidence of neuronal degeneration (FJB), astrocyte and microglial activation (Iba-1 and GFAP), and axonal fiber injury (aminocupric silver staining). MRI and MRS were used to compare brain water diffusivity and levels of different neurochemicals present prior to and at several periods after the blast.

RESULTS: Widespread silver staining indicative of degenerating axonal fibers was evident primarily in the internal capsule, corpus callosum, and cerebellum at 24 hrs and 7 days post-injury but was markedly reduced by 30 days. Perivascular microglial activation was evident, as was astrocyte accumulation near ventral cortical surfaces. FJB-stained, degenerating neurons were found mainly at the base of the pituitary stalk. Diffusion tensor imaging and MRS focusing on the hippocampus provided evidence for edema and reduced glutathione levels within 4 hr post-blast.

CONCLUSIONS: 1. Underbody blast-induced Gs of 20 - 50 produce region-selective axonopathy. 2. Blast-induced hyperacceleration is followed by chronic brain inflammation. 3. Brain edema and oxidative stress occur within a few hr after the blast.

Support: Office of Naval Research

Zhao Zaorui Loane David Murray Michael Faden Alan Department of Anesthesiology and the Center for Shock, Trauma and Anesthesiology Research (STAR), University of Maryland School of Medicine, Baltimore, MD, USA

P252 Comparing the Predictive Value of Multiple Cognitive and Motor Tasks After Rodent TBI

PURPOSE: Controlled cortical impact injury (CCI) is a widely used, clinically relevant model of traumatic brain injury (TBI). Although functional outcomes have been used for years in this model, little work has been done to compare the predictive value of various cognitive and sensorimotor assessment tests, singly or in combination. Such information would be particularly useful for assessing mechanisms of injury or therapeutic intervention.

METHODS: Following anesthesia, C57BL/6 mice were subjected to sham, mild (5.0 m/sec), moderate (6.0 m/sec), or severe (7.5 m/sec) CCI. A battery of behavioral tests were evaluated and compared.

RESULTS: The beam walk task - performed at 0, 1, 3, 7, 14, 21, and 28 days post-injury (PID), showed good discrimination across severities of injury. The classical Morris water maze and reversal Morris water maze (rMWM) tests (PID14-23), novel object recognition (PID24, 25), passive avoidance test (PID 27, 28), and tail-suspension test (PID24) effectively discriminated spatial, non-spatial, fear-related emotional memory, and depression status, respectively, across injury severities. Notably, the rMWM showed the greatest separation between mild and moderate/severe injury. Search strategy utilized by mice to locate the hidden platform was able to better discriminate behavioral outcomes across injury severities, especially in rMWM. A composite behavioral score was also developed based on the multiple behavioral tests. We are currently comparing specific functional changes with cell loss across anatomical regions.

CONCLUSIONS: This study shows the feasibility of using a larger number of complementary functional outcome behavioral tests than traditionally employed to follow post-traumatic recovery for rodent TBI and should be helpful in screening new neuroprotective agents or in investigating injury mechanism.

Stein Deborah 1 Lindell Allison 2 Murdock Karen 2 Kufera Joseph 2 Menaker Jay 1 Bochicchio Grant 1 Aarabi Bizhan 1 Scalea Thomas 1

1 R Adams Cowley Shock Trauma Center, University of Maryland School of Medicine, Baltimore, MD, USA 2 Shock Trauma and Anesthesiology Organized Research Center (STAR-ORC), University of Maryland School of Medicine, Baltimore, MD, USA P253 Use of Serum Biomarkers to Predict Cerebral Hypoperfusion Following Severe Traumatic Brain Injury

PURPOSE: The management of severe traumatic brain injury (sTBI) focuses on prevention and treatment of secondary insults, such as cerebral hypoxia (CH). Predicting which patients will develop these secondary insults is currently not possible. This study evaluates the association between serum biomarkers and CH as measured by brain tissue oxygen partial pressure (Pb02) in patients with sTBI.

METHODS: Patients with sTBI (head AIS > 3, age > 14, “isolated” TBI, need for intracranial pressure monitor) were prospectively enrolled. Serum was collected within 24 hours of injury and twice daily for 7 days. Pressure times time (PTD; mmHg*h), measuring the depth and duration of CH, was calculated for 12-hour periods for episodes of moderate and severe CH (PbO2 < 20 and < 15mmHg) and compared to serum levels of neuron-specific enolase (NSE), S100-beta (S100b), and glial fibrillary acidic protein (GFAP) drawn prior to periods of monitoring. Specimens were analyzed by ELISA. Adjusted mixed models accounted for longitudinal correlations within patients and analysis was conducted of area under the curve (AUC) obtained from Receiver Operating Characteristics (ROC) curves.

RESULTS: Of 68 patients enrolled, 24 had PbO2 monitoring. 130 serum samples were matched with 12-hour periods of monitoring. Significant correlations were found in adjusted analysis between serum levels of NSE, S100b, and GFAP and PTD of moderate (p < 0.01) and severe (p < 0.01) CH. AUCs for classification of moderate and severe CH ranged from 0.55-0.71 for NSE and S100-b, indicating fair to good accuracy, with specificities between 76 and 90%.

CONCLUSIONS: NSE, S100-b, and GFAP demonstrate promise as candidate serum markers of impending CH. These data suggest that we may be able to ‘predict’ imminent events following TBI prior to clinical manifestations. Given the morbidity of CH, early intervention and prevention may have a significant impact on outcome and help guide decisions about timing of interventions.

Skop Nolan 1 Siriwardane Mevan 1 Calderon Frances 2 Jiang Yuhui 2 Cho Cheul 4 Levison Steve 2 Gandhi Chirag 3

1 University of Medicine & Dentistry, New Jersey Institute of Technology, Department of Biomedical Engineering, Newark, NJ, USA 2 University of Medicine & Dentistry, Department of Neurology & Neurosciences, Newark, NJ, USA 3 University of Medicine & Dentistry, Department of Neurological Surgery, Newark, NJ, USA 4 New Jersey Institute of Technology, Department of Biomedical Engineering, Newark, NJ, USA P254 Engineering a Microscaffold to Improve Neural Stem Cell Transplantation for Traumatic Brain Injury Repair

INTRODUCTION: Regenerating the central nervous system has become more feasible with the growing field of stem cell therapies. However, stem cells injected into a cystic cavity are limited in regenerative potential due to the lack of appropriate substrate and growth factor support. The goal of this study is to fabricate chitosan-based microspheres to create an ideal biomaterial scaffold suitable for transplantation.

METHODS: Differentiation of newborn subventricular zone (SVZ) cells, embryonic ventricular zone (VZ) cells, and the radial glia cell line (RG3.6) were evaluated for their regenerative potential, in vitro. Laminin, fibronectin, gelatin, collagen, and poly-lysine were adsorbed to chitosan to determine their effects on RG3.6. Surface chemistry may increase cell adhesion and survival while maintaining the “stem-like” properties of cells through integrin receptor signaling pathways. Chitosan microspheres were generated by ionic coagulation (100-300μm) and electrospraying (1-50μm). Fibroblast growth factor-2 (FGF2) was covalently crosslinked to the chitosan spheres using heparin and genipin.

RESULTS: VZ cells formed greater percentages of neurons than SVZ cells (30% neurons from VZ cells and 10% neurons from SVZ cells) while many RG3.6 cells remained undifferentiated. RG3.6 cells grown on chitosan adsorbed with fibronectin possessed longer processes than when grown on poly-l-lysine, gelatin, laminin or collagen. Cells grown on fibronectin also had fewer processes, thus morphologically more similar to radial glial cells. RG3.6 cells were successfully grown on modified chitosan microspheres and formed all three neural cell types upon differentiation.

CONCLUSION: Results from parallel studies (Siriwardane et al. 2011) demonstrate that cells transplanted adjacent to cystic cavities have limited regenerative potential. Data presented here demonstrate that radial glial-like precursors can be grown on modified chitosan microspheres whereupon they can be delivered directly into the lesion cavity to improve engraftment, survival and ultimately long-term cell replacement.

Supported by a grant from the NJ Commission on Brain Injury Research.

Diaz Julio Sanchez Jose Bullock Ross University of Miami, Miami, FL, USA

P255 Changes in Brain and Ventricle Volumes After Decompressive Craniectomy in Patients with Severe Traumatic Brain Injuries

Fluctuations in brain volume are apparent in patients with severe traumatic brain injuries (TBI) who have undergone decompressive craniectomy (DC). Here we describe the changes in brain and ventricular volumes that result from DC during the acute phase of the injury. Whole-brain CT scans were compiled for decompressed patients with severe non-penetrating TBI (Glasgow Comma Scale < 8, n = 9, female = 1, mean age = 36.7). Brain and ventricular volumes were quantified for week one post-DC using the software Analyze 10.0. Preliminary data do not reveal significant variations in brain volume during the first post-operatory week, and indicate a significant decrease in ventricular volume at the second post-operatory day (p = 0.004, paired samples t-test). No significant correlations were found between changes in brain and ventricular volumes (r = -0.139, n = 36, p = 0.420, Pearson's correlation test). Further evaluations will reassess the present results in a larger patient pool, and will examine the contributions of edema, hemorrhage, and extra-ventricular cerebrospinal fluid to brain herniation.

O'Phelan Kristine 1 Sanchez-Chavez Jose 1 Nemeth Zsuzsanna 1 Morrison Christopher 1 Jaggid Jonathan 1 Hartings Jed 2 Olvey Stephen 1 Bullock M. Ross 1

1 University of Miami, Miller School of Medicine, Miami, Florida, USA 2 University of Cincinnati College of Medicine, Cincinnati, Ohio, USA P256 Evidence of Metabolic Stress Associated with Cortical Spreading Depression

BACKGROUND: Many factors contribute to secondary injury after traumatic brain injury (TBI). Cortical spreading depression (CSD) -a slow depolarization occuring after injury, has been associated with increased tissue damage after experimental TBI. Electrocorticography (ECoG) can detect CSD post TBI. Cerebral microdialysis can demonstrate ongoing cellular stress. Elevations in the lactate-pyruvate ratio (LPR), and increased glutamate have been associated with ischemia, failure of oxidative phosphorylation and necrosis.

HYPOTHESIS: CSD is associated with microdialysis markers of cellular stress; decreased extracellular glucose and increased LPR.

METHODS: Inclusion criteria: TBI, GCS < 9, enrolled in the COSBID study, cerebral microdialysis monitor placed. ECoG strips were placed at craniotomy. Microdialysis was performed using a 10mm CMA 70 probe and artificial CSF at 0.3 ml/min. Dialysate glucose, lactate and pyruvate were measured.

RESULTS: 10 episodes of CSD occurred during microdialysis monitoring in three patients, one patient had no CSD events. CSD duration 5-24 minutes. 5/10 events were associated with a decrease in extracellular glucose concentration. 7/9 events were associated with an elevation in LPR. Individual subjects: SubA- five events, mean preSCD glucose 0.13mM/L post SCD glucose 0.06mM/L, mean pre SCD LPR 54, mean post SCD LPR 62. SubB- four SCD events, mean preSCD glucose 1.6 mM/L, mean postSCD glucose 1.7mM/L, mean preSCD LPR 23, mean postSCD LPR 24. SubC- one SCD PreSCD glucose 3.48mM/L post SCD glucose 3.46 mM/L. SubD- no SCD mean glu 2.5 mM/L, mean LPR 26.

DISCUSSION: The metabolic response to SCD is variable. LPR is altered more consistently than glucose concentration. Individual differences exist between subjects' tissue vulnerability to stress. Limitations include variability in probe distance to the ECoG strip, CSD duration and length of time between CSD and microdialysis vial collection. Future studies examining differences in tissue vulnerability and metabolic reserve in individual patients may be helpful.

Bidot Carlos J Sanchez Jose Ponce Erick Nemeth Zsuzsanne Morrison Christopher O'Phelan Kristine Jagid Jonathan Olvey Stephen Hong Lee Fong Bullock Ross University of Miami, Miami, Florida, USA

P257 Microdialysis in Severe Traumatic Brain Injury (TBI): Cerebral Chemistry Vs Glasgow Outcome Scale

INTRODUCTION: Detection of secondary events remains a major target in severe TBI. Microdialysis monitoring (MD) of cerebral extracellular chemistry has the potential for early detection of metabolic derangements associated with such events.

OBJECTIVE: To determine the relationship between biochemical markers and neurological outcome in severe TBI.

MATERIALS AND METHODS: MD data from 12 TBI patients was analyzed. MD markers included: glucose, lactate, pyruvate, and lactate/pyruvate ratio (LPR). Glasgow Outcome Scale (GOS) was assessed at the time of discharge and patients were grouped as Bad Outcome (GOS < 3) and Good Outcome (GOS > 4). MD was done from D1 to D7. Statistic tests were performed to determine correlation of the different parameters.

RESULTS: Of 12 patients, 10 /2 (male/female). Median age was 38 (18 - 70). 58.4% had BO at discharge. Positive correlation between lactate vs pyruvate and glucose vs pyruvate (p < 0.01) was found. In contradistinction, negative correlation was found between glucose and LPR. Glucose, lactate, pyruvate and LPR were significantly higher in BO group (p < 0.001, p < 0.01, p < 0.05) vs GO during the first 72 h. A remarkable decrease in glucose levels was observed in Day 4 in BO group.

CONCLUSION: Results suggest that extracellular metabolic markers are associated with outcome and could help with pronostication. High levels of LPR and falling glucose predic poor outcome. Although our findings were significant in the first 72 h, sample size needs to be greater to confirm and better understand the physiological significance of biochemical markers.

Sakurai Atsushi Titus David J Kang Yuan Furones Concepcion Jergova Stanislava Atkins Coleen M University of Miami Miller School of Medicine, Miami, FL, USA

P258 Rolipram Rescues Cognitive Impairments Resulting from Traumatic Brain Injury

Traumatic brain injury (TBI) is a devastating condition that severely affects the CNS and results in several physiological changes. The hippocampus is highly vulnerable to TBI and insult to this region affects cognitive performance and in particular memory formation. Our previous study suggests that the cAMP-PKA signaling cascade is downregulated after TBI, and that treatment with a phosphodiesterase (PDE) IV inhibitor rescues this decrease in cAMP levels. Given that hippocampal long-term memory formation is dependent on activation of the cAMP-PKA signaling pathway, we hypothesized that preventing the degradation of cAMP with a PDE inhibitor (rolipram) may rescue these memory deficits. Accordingly, in the present study, we examined the effect of rolipram on TBI-induced cognitive impairments. Adult male Sprague Dawley rats were subjected to either sham surgery or moderate parasagittal fluid-percussion brain injury (TBI). At 2 weeks after recovery, the sham and TBI animals received vehicle (0.15% DMSO in saline) or rolipram (0.03mg/kg, i.p.) 30 minutes prior to training on a contextual and cue fear conditioning paradigm. 24 hours after training, the animals were tested for cue and contextual fear conditioning. Deficits in contextual and cue fear conditioning were observed in vehicle-treated TBI animals as compared to vehicle-treated sham animals and these deficits were rescued with rolipram treatment. To further understand the underlying mechanisms of these memory impairments, hippocampal long-term potentiation (LTP) was investigated in the Schaffer collateral pathway of the CA1 region at 2 weeks after TBI or sham surgery. TBI animals showed a 40% reduction in the expression of LTP as compared to sham surgery animals (p < 0.01). We are currently analyzing the effect of rolipram in the restoration of hippocampal LTP. Thus, these results indicate that administration of a rolipram rescues cognitive impairments after TBI and suggests a potential therapeutic strategy to treat TBI-induced memory dysfunction.

Supported by NS069721, NS056072.

Tomura Satoshi de Rivero Vaccari Juan Pablo Keane Robert W. Alonso Ofelia Bramlett Helen Dietrich W. Dalton University of Miami Miller School of Medicine, Miami, Florida, USA

P259 The Effect of Therapeutic Hypothermia on the NLRP1 Inflammasome Signaling After Traumatic Brain Injury

Traumatic brain injury (TBI) elicits acute inflammation that in turn exacerbates primary brain damage. In previous reports, we demonstrated that the nucleotide-binding, leucine-rich repeat pyrin domain containing protein 1 (NLRP1) inflammasome consisting of NLRP1, caspase-1, caspase-11, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), the X-linked inhibitor of apoptosis protein and pannexin 1 constitutes an important component of the innate CNS inflammatory response after TBI. Therapeutic hypothermia has been reported to improve outcomes in several animal models of brain injury and has been successfully translated to specific patient populations. In this study, we investigated the influence of therapeutic hypothermia on NLRP1 inflammasome signaling after TBI. Adult male Sprague-Dawley rats (300 - 400 g) were subjected to parasaggital fluid percussion brain injury at a moderate severity (1.7 - 2.2 atm). Temperature manipulation (33°C) was initiated 30 min after TBI and maintained using cooled air and heating lamps for a period of 4 h. The normothermia groups were maintained at 37°C throughout the procedure. At the end of the temperature manipulation or at 24 h after TBI, rats were sacrificed and the samples of brain tissue were immediately excised from the traumatized cortex and prepared for Western blot analysis. In the normothermic groups, caspase-1 and caspase-11 tended to increase overtime. In contrast, the hypothermic groups demonstrated an increase at 4 h after TBI, and then decreased at 24 h after trauma. Therapeutic hypothermia seems to have an effect on inflammasome signaling and reduces the innate immune response to TBI.

Supported by NS042133 and NS030291.

Adamczak Stephanie 2 de Rivero Vaccari Juan Pablo 1 Dietrich W. Dalton 1 Keane Robert W. 2

1 Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA 2 Dept. of Physiology and Biophysics, University of Miami, Miami, FL, USA P260 dsDNA Induces an Innate Immune Response in Cortical Neurons

The AIM2 inflammasome is activated by dsDNA and has been characterized in macrophages, predominantly in the context of viral infection. However, the AIM2 inflammasome also responds to self-DNA and induces pyroptosis, a recently-characterized, inflammatory cell death program. My central hypothesis is that AIM2 forms an inflammasome in neurons, is activated by endogenous dsDNA released from necrotic cells following TBI, and contributes to caspase-1 activation, inflammatory cytokine production, pyroptotic cell death, and injury pathology. Here, we cultured cortical neurons from E18-E19 rat embryos and stimulated them with dsDNA to activate the AIM2 inflammasome. Immunocytochemistry and western blotting show that the AIM2 inflammasome is expressed in neurons and mediates an innate immune response to dsDNA. Neurons exposed to dsDNA show a 2.5-fold increase in propidium iodide (PI) uptake and form oligomers of Apoptosis-associated Speck-like protein Containing a CARD (ASC), a reliable indication of pyroptosis. The data suggest that non-microbial dsDNA activates an innate immune response and induces an inflammatory cell death program in neurons. Self-dsDNA released from necrotic cells is present in the plasma and cerebrospinal fluid of TBI patients and may contribute to the deleterious effects of the inflammatory response. Therefore, AIM2 may be useful therapeutic target to reduce secondary injury mechanisms and limit the chronic, progressive loss of neurons following TBI.

Sanchez Juliana Truettner Jessie Moreno W. Javier Bramlett Helen University of Miami Miller School of Medicine, Miami, Florida, USA

P261 Alterations in Adhesion Molecules Following Polytrauma

Traumatic brain injury (TBI) combined with secondary insults such as respiratory distress and organ trauma is referred to as polytrauma. Our laboratory is currently modeling this type of injury using moderate fluid percussion (FP) brain injury combined with secondary hypoxia and a systemic injection of interleukin-1β. This complicated model of polytrauma has been reported to further exacerbate the amount of damage seen after TBI alone. The current study is designed to determine the degree of changes in adhesion molecules due to vascular dysfunction/damage after polytrauma. Male Sprague-Dawley rats underwent moderate FP injury followed by 30 minutes of either normoxic (TBI-NO, n = 6-8/time point) or hypoxic (TBI-HY, n = 6-8/time point) gas levels. After returning to normoxic gas levels, an intraperitoneal injection of IL-1β (40mg/kg) or saline vehicle was injected in all rats. Sham operated animals underwent all surgical procedures plus normoxia and vehicle administration (Sham-NO + Veh, n = 4-6/time point). Animals were sacrificed at 3 and 24 hrs for RT-PCR and western blotting assessment of adhesion molecules (P- and E-selectin, ICAM-1 (intercellular adhesion molecule), VCAM-1 (vascular cell adhesion molecule)). We observed changes in several mRNA adhesion molecules in all groups. However, the most robust cortical changes were seen in the TBI plus normoxia and IL-1β group. Structural differences appear to correlate with the an increased upregulation of adhesion molecules occurring in the hippocampus following polytrauma compared to cortical mRNA levels. Although, adhesion molecule protein levels did not consistently follow the pattern of mRNA readings, the immunohistochemistry for these adhesion molecules demonstrate increases in all groups. It appears that adhesion molecule upregulation following polytrauma is a contributing factor to the deleterious effects of this clinically relevant model of complicated TBI.

Supported by W81WXH-08-1-0146.

Yokobori Shoji 1 Bullock Ross 1 Gajavelli Shyam 1 Mondello Stefania 2 Mo Jixiang 2 Scharf Dancia 2 Wang Kevin 2 Hayes Ronald 2 Sakurai Atsushi 1 Tomura Satoshi 1 Bramlett Helen 1 Dietrich Dalton 1

1 Department of Neurosurgery, University of Miami Miller School of Medicine, Miami, FL, USA 2 Center of Innovate Research, Banyan Biomarkers, Inc., Alachua, FL, USA P262 Preoperative-Induced Mild Hypothermia Attenuates Brain Biomarkers in a Rat Subdural Hematoma Model

Posttraumatic hypothermia has been shown in previous studies to improve histopathological and behavioral consequences of traumatic brain injury using various experimental models. However, therapeutic hypothermia has not shown efficacy in clinical multi-center trials. Based on the data from latest hypothermia trial (NABISH:II), we hypothesized that hypothermia may be beneficial in acute subdural hematoma (ASDH), by blunting the effects of reperfusion injury and excitotoxicity, at the time of craniotomy. 20 S-D rats had induced ASDH and were allocated into one of four groups (5 rats each). Their temperatures were manipulated as below: Group1 (Normothermia); maintained at 37°C throughout. Group 2 (Early hypothermia); brain temp. (BT) reduced to 33°C at 30 min. prior to craniotomy and ASDH removal, continued for three hours. Group 3 (Late hypothermia); BT lowered to 33°C at 30 min. after decompression and maintained 3 hours. Group 4 (Sham); normothermia without ASDH. For estimation of glial and neuronal cell damage, we analyzed serum concentrations of two protein biomarkers: astroglia injury marker, Glial Fibrillary Acidic Protein (GFAP) and neural injury marker, Ubiquitin Carboxyl-Terminal Hydrolase -L1 (UCH-L1) in these groups. We also applied microdialysis techniques and compared the concentrations of GFAP and UCH-L1 in the extracellular space among these. Furthermore, we made TTC (2,3,5-triphenyltetrazolium chloride) -stained sections in several rats, and compared injury volumes. This was the first study to compare concentrations of GFAP and UCH-L1 in the extracellular fluid (ECF) space and plasma in a head injury model. On TTC staining, injury volumes were smallest in the early hypothermia group in which induced hypothermia was done before craniotomy and cerebral reperfusion. Early mild hypothermia might reduce permanent brain damage after ASDH in the rat model. ECF GFAP and UCH-L1 concentrations may be more useful for estimation of injury severity.

Spurlock Markus Hosein Khadil Bomberger Christine Diaz Daniel Zeidan Michelle Calabrese Jordan Al Sulaimani Sara Gajavelli Shyam Yokobori Shoji Bullock Ross Bramlett Helen Dietrich Dalton Miami Project to Cure Paralysis,Department of Neurosurgery, University of Miami Miller School of Medicine., Miami/FL, USA

P263 Histological Evaluation of the Neuroprotective Effects of Preoperative Mild Therapeutic Hypothermia in Rat Subdural Hematoma Model

PURPOSE OF THE STUDY: Posttraumatic hypothermia has been shown in previous studies to improve histopathological and behavioral consequences of traumatic brain injury using various experimental models. However, therapeutic hypothermia has not shown efficacy in clinical multi-center trials. Based on the data from the latest hypothermia trial (NABISH: II), we hypothesized that hypothermia may be beneficial in acute subdural hematoma ( ASDH ) by blunting the effects of reperfusion injury and excitotoxicity at the time of craniotomy.

MATERIALS AND METHODS: Acute subdural hematoma was induced in Sprague-Dawly rats (n = 20). The rats were allocated into one of four groups (5 rats each). Their temperatures were manipulated as follows: Group1 (Normothermia) maintained at 37°C throughout. Group 2 (Early hypothermia) brain temperature (BT) reduced to 33°C at 30 min prior to craniotomy and ASDH removal then continued for three hours. Group 3 (Late hypothermia) BT lowered to 33°C at 30 min after decompression and maintained for 3 hours. Group 4 (Sham) normothermia without ASDH. The animals were sacrificed after 24 hour. Cryopreserved brains were sectioned for histological analyses. To estimate neuronal sparing we looked at the numbers of neuronal nuclei (NeuN) immunoreactive cells in the subcortical region and hippocampus. Similarly, to estimate neuronal degeneration we assessed the numbers of Flurojade B positive cells. Hematoxylin and eosin staining was performed to assess tissue architecture.

RESULTS: The numbers if NeuN + cells did not significantly differ between groups. However the distribution of FJB positive cells varied between groups. The FJB labeling was predominantly in the subcortical region and absent in the hippocampus. Compared to the normothermia group there were more FJB positive cells in the cortices (both contra and ipsilateral) in the late hypothermia group.

CONCLUSION: Early mild hypothermia may reduce permanent brain damage by reducing the number of degenerating neurons following ASDH in rats.

Atkins Coleen Kang Yuan Furones Concepcion Bramlett Helen Dietrich W. Dalton University of Miami Miller School of Medicine, Miami, FL, USA

P264 Traumatic Brain Injury Induces Increased Phosphodiesterase Expression

Traumatic brain injury (TBI) results in significant memory impairments that impede recovery. Understanding the molecular changes that occur after TBI is an important step to develop therapies to improve memory performance. Previously we have demonstrated that cAMP, a molecule required for memory formation, is downregulated after traumatic brain injury (TBI). cAMP is synthesized from ATP via adenylyl cyclases and degraded by phosphodiesterases (PDEs). To determine the mechanism by which cAMP is downregulated after TBI, we determined whether TBI induces changes in PDE expression levels. Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury (FPI) or sham injury, and the ipsilateral, parietal cortex and hippocampus were analyzed by western blotting. We chose to study changes in PDE1, 3, 4, 8, and 10 because these specific isoforms are found in the brain or in cells that could potentially infiltrate the brain after injury. In the ipsilateral parietal cortex, expression of PDE1A, PDE4B2, and PDE4D2 significantly increased from 30 min to 24 hr post-injury. Phosphorylation of PDE4A also significantly increased in the ipsilateral parietal cortex from 6 hr to 7 days post-injury. In contrast, PDE1B, PD4A5, and PDE4A8 were significantly decreased in the ipsilateral parietal cortex after TBI. No changes were observed with PDE1C, PDE3A, PDE4B1, PDE4B3, PDE4D3, PDE4D4, PDE8A, PDE8B, or PDE10A. Regulation of PDE in the ipsilateral hippocampus was similar to that observed in the cortex with a few exceptions. Levels of PDE1A, PDE4B2, PDE4D2, PDE4D3, PDE4D4, and phosphorylation of PDE4A were significantly increased in the ipsilateral hippocampus after TBI, whereas PDE4B1 and PDE4B3 were significantly decreased. No changes were observed with PDE1B, PDE1C, PDE3A, PDE4A5, PDE4A8, PDE8A, PDE8B, and PDE10A. These findings suggest that therapies to improve memory impairments after TBI could be facilitated with targeted therapies, in particular for PDE1A, PDE4B2 and PDE4D2.

Supported by NS069721, NS030291, NS056072.

Sakurai Atsushi Atkins Coleen Alonso Ofelia Bramlett Helen Dietrich W. Dalton University of Miami Miller School of Medicine, Miami, Florida, USA

P265 The Effects of Hyperthermia on Mild Traumatic Brain Injury

The highest incident rate of brain trauma occurs following mild traumatic brain injury (mTBI). One potential confounding variable of mTBI is the presence of elevated body temperature resulting in hyperthermia. Heightened temperature levels are a potential risk factor in the civilian or military population prior to mTBI, either due to excessive activity and/or elevated ambient temperatures. The goal of this study was to determine whether pre- or post-traumatic hyperthermia exacerbates histopathological outcome after mTBI. Adult male Sprague-Dawley rats were randomized into 3 groups: pre/post-traumatic hyperthermia (pre/post-hyper), post-traumatic hyperthermia (post-only hyper) and normothermia. The pre/post-hyper group was treated with whole body hyperthermia (40°C) starting 15 min before mild parasaggittal fluid-percussion brain injury (1.4-1.6 atm). Both pre/post-hyper and post-only hyper groups underwent hyperthermia for 2 h after mTBI. The normothermic group was maintained at normothermia (37°C) for an equivalent duration after mTBI. At 72 h after mTBI, contusion area and volume were quantified. Contusion area measurements at bregma levels − 3.3, − 4.3, and − 5.8 mm were larger in the pre/post-hyper group as compared to the post-only hyper and normothermic groups. These findings extended as well to the contusion volume assessment with the pre/post-hyper group exhibiting a 38% increase in contusion volume as compared to the normothermic group. In contrast to normothermic conditions where only minimal structural damage was observed, mild hyperthermic mTBI produced consistent histopathological abnormalities including neuronal cell loss and a well defined contusion. These novel findings demonstrate that pre- and post-traumatic hyperthermia exacerbates histopathological damage after mTBI. Importantly, individuals exhibiting mild hyperthermic temperatures prior to or immediately after mTBI may be predisposed to aggravated brain damage and subsequent neurological impairments.

Supported by NS042133 and NS030291.

Sanchez Jose Bidot Carlos O'Phelan Kristine Jagid Jonathan Ponce Eric Diaz Julio Nemeth Zsuzsanna Rhagnanan-Kramer Violet Olvey Stephen Bullock Ross Neurointensive Care Unit Jackson Memorial Hospital, Miami, Florida, USA

P266 Changes in Brain Dialysate Analytes After Craniotomy for High Intracranial Pressure (ICP) in Severe Trauma Brain Injury (TBI)

BACKGROUND: Microdialysis (MD) permits the measurement of metabolic changes that occur in damaged brain tissue that is at risk to progress to permanent cell loss (Penumbra).

HYPOTHESIZE: The values of brain metabolyte analytes are related with the severity of the TBI, the type of lesion and the outcome of the tissue in which the MD probe is embedded.

METHODS: In 1,260 MD samples from 12 consecutive patients with focal severe TBI who required craniotomy for high ICP, dialysate glucose, lactate and pyruvate were measured, for up to seven days. Analytes were correlated with the Glasgow Coma Scale (GCS) before surgery, the type of focal lesion and with the Glasgow Outcome Scale (GOS) at the time of discharge.

RESULTS: Brain Dialysate analytes changed significantly, over time after the craniotomy (p < 0.001). Glucose levels were frequently low while Lactate and Lactate/Pyruvate ratio (LPR) were high. Pyruvate and Lactate/glucose ratio (LGR) were commonly within the normal range.

GCS correlation with Dialysate analytes: A statistically significant negative correlation was found between glucose, pyruvate and LGR with GCS. The glucose showed a sustained relationship with GCS during the whole seven days of monitoring (p < 0.05), lactate only during the first 72 hours (p < 0.01), pyruvate from day three to seven and the LGR from day one to five. Meanwhile the LPR showed a positive correlation (p < 0.01) from day three to five.

GOS correlation with brain dialysate analytes: Glucose showed a significant negative correlation (p < 0.01), while lactate, pyruvate, LPR and LGR showed a significant positive correlation (p < 0.05).

CONCLUSION: After craniotomy extracellular glucose appears to be a good “biomarker” for monitoring brain tissue integrity. We speculate that high extracellular glucose was an indicator of more severe neurological damage and thus poor outcome, due to impaired glycolitic metabolism.

Odland Rick 1 Johnson Jami 2 Horn Molly 2

1 University of Minnesota, Minneapolis, MN, USA 2 Bethel University, St. Paul, MN, USA P267 Three-Dimensional Analysis of Intracranial Compliance

PURPOSE: Compliance measurements have been performed using a variety of devices in living tissue. While the importance of tissue compliance is well accepted and change in tissue compliance is well known with ischemia or trauma, clinically meaningful measurement and interpretation of compliance changes has been elusive. The compliance or pressure-volume curve is typically a positive exponential relationship, but the true pressure volume curve (Boyle's law) is an inverse relationship. Volume of tissue can be defined as the “container” (or the skull), and as the “mass” or amount of material within the container. Both variables must be examined for an understanding of the state of brain compliance.

METHODS: A bench model with three variables was constructed to examine the effect of changes in material compliance, volume of container, and mass of material within container.

It was observed that, with a lower initial pressure, the compliance of the system increased more rapidly with increasing mass and volume than when the system began at a higher initial pressure. For an initial pressure of 10 mmHg, compliance increased from 0.33 mL/mmHg to 2.37 mL/mmHg over a 71.14 mL volume difference. With an initial pressure of 100 mmHg, compliance increased from 0.22 mL/mmHg to 0.73 m L/mmHg over a 50.87 mL volume difference.

RESULTS: A three dimensional plot of pressure versus mass versus volume was created. The model can be used to examine clinical findings of “u-shaped” compliance curves determined clinically, and the effects of edema and hemicraniectomy on tissue pressure.

A linear relationship between pressure and the addition volume was observed similar to findings in Spiegelberg intracranial pressure model by Yun-Hom Yau, et. al.

CONCLUSION: Tissue compliance is a complicated topic and further understanding the effect of all three variables may lead to clinical application. Compliance changes may have great importance in foretelling changes in edema and perfusion.

Hyson Morton University of Nevada, Las Vegas, NV, USA

P268 Anticephalgic Photoprotective Premedicated Mask: A Report of a Successful Study of a Treatment for Migraine and/or Tension Headaches

OBJECTIVES: This study was performed to determine the efficacy of an anticephalgic photoprotective mask in conjunction with a topical medication containing bryonia and rhus toxicodendron in the treatment of migraine and/or tension headache.

METHODS: Thirty-three patients were given masks and tubes of topical medication containing the bryonia and rhus toxicodendron. They were instructed to apply the medication to their frontalis and/or temporalis regions in the event they should suffer a headache and apply a photoprotective mask. Furthermore, they were instructed to take their usual oral or parenteral medications if required for the relief of the headache. They subsequently filled out forms rating the degree of relief which they attributed to the topical medication and the mask using a 0-10 scale. At the interview following the completion of their participation in the study, the patients were also simply asked if this form of treatment helped or not.

RESULTS: Thirty out of 33 patients stated the medication and the mask were effective over and above the normal degree of relief they were receiving from their oral and/or parenteral medications. This study demonstrated a significant efficacy rate (91%) in the treatment of migraine and/or tension headache with the anticephalgic mask in conjunction with a topical cream containing bryonia and rhus toxicodendron.

INTERPRETATIONS: This study demonstrated a significant efficacy rate in the treatment of migraine and/or tension headache with the anticephalgic mask in conjunction with a topical cream containing bryonia and rhus toxicodendron

Holguin Elizabeth Stippler Martina Yonas Howard University of New Mexico, Albuquerque, NM, USA

P269 Teleradiology and Online Consultation: A Solution of Care for Traumatic Brain Injury in Rural New Mexico

PURPOSE: The main purpose of our study is to demonstrate that teleradiology consultation with a neurosurgeon has been demonstrated to be a safe way to manage mild traumatic brain injured (mTBI) patients locally without transfer to a higher level of care. We will also make clinicians aware of the potential cost savings to their facilities and to the patients that they serve by utilizing the technology and software system in addition to phone consultation. An additional component of the study will allow us to identify the opportunity for staff education regarding the treatment and care of the mTBI patient within the Indian Health Service (IHS) system. The data obtained will allow for projection of future benefits and impact by implementing teleradiology and reforming current traumatic brain injury care policies.

METHODS: In an effort to promote safe management and quality care of the mTBI patient at a local level, a teleradiology system has been implemented through collaboration between IHS and the University of New Mexico Hospital (UNMH). Data on outcome and utilization of teleradiology from January to November 2010 were retrospectively reviewed.

RESULTS: Between January 2010 and November 2010, 70 head CT scans were reviewed with a teleradiology software program. Thirty six patients had a diagnosis pertaining to TBI. Ten head CT scans were negative. The remaining 26 patients had pathology that included: Epidural (n = 1) and subdural (n = 11) hematomas, traumatic subarachnoid hemorrhage (n = 12), contusion (n = 5), intraparenchymal (n = 3) and intraventricular bleeds (n = 1). Of the 26 scans reviewed, 42% (n = 11) patients were treated locally and 58% (n = 15) were transferred to UNMH or elsewhere.

CONCLUSION: Teleradiology is safe and feasible in rural areas to facilitate triage of patients with mTBI but is currently underutilized.

Kraynik Jessica Stippler Martina Yonas Howard Nemoto Edwin University of New Mexico, Albuquerque, NM, USA

P270 Cerebral Perfusion Pressure (CPP) and Brain Tissue O₂ (PBTO₂) Correlation After Brain Injury

PURPOSE: Underlying patho-physiological mechanisms of brain injury are very heterogeneous and can complicate treatment decisions. Heterogeneity of the regional brain circulation and metabolism is often postulated for the post insult time after severe traumatic brain injury (TBI) and aneurysmal subarachnoid hemorrhage (SAH). Our aim was to understand the heterogeneity in this patho-physiological processes occurring after TBI and SAH. We hypothesized that intact cerebral blood flow (CBF) autoregulation is consistent with a lack of correlation between CPP and PbtO₂ (>20mmHg) whereas good correlation indicates loss of CBF autoregulation.

METHODS: We used a multimodal neuromonitoring system, the CNS monitor (Moberg, Inc, Philadelphia, PA.) to collect minute to minute multimodal neuromonitoring data including cerbral blood flow, ICP, MAP, CCP and PbtO2. We studied the dependence of PbtO₂ on CPP in ten patients for up to seven days post injury. Correlations between CPP and PbtO₂ in ten patients (3 SAH and 7 severe TBI) from day 0 to day 7 post injury were examined by linear regression analysis.

SUMMARY OF THE RESULTS: Of a total of 39 days of CPP vs PbtO₂ correlations in the ten patients, 69% or 27 days showed significant correlations (R values > .25 and P < 0.002). The frequency of significant correlations between CPP and PbtO₂ appeared to occur earlier rather than later post injury. Days with significant correlations between CPP and PbtO₂ were followed by days of no correlation indicating day to day alteration in CBF autoregulation.

CONCLUSIONS: The day to day variability in the correlation between CPP and PbtO₂ may indicate variable CBF autoregulation post injury. CPP-PbtO2 correlation could be an important parameter for clinicians in setting the optimal CPP during the first post injury days. Our findings suggest that the optimal CPP for brain oxygenation may differ for each patient from day to day.

Bragin Denis Bush Rachel Nemoto Edwin Deptartment of Neurosurgery University of New Mexico SOM, Albuquerque, NM, USA

P271 Cerebral Perfusion Pressure Elevation at High ICP Raises the Transition Threshold From Capillary to Non-Nutritive Microvascular Shunt Flow

PURPOSE: Recently we showed that decreasing cerebral perfusion pressure (CPP) by increasing intracranial pressure (ICP) in rats is associated with tissue hypoxia and brain edema due to a flow transition from capillary (CAP) to microvascular shunt flow (MSF), which did not occur when CPP was reduced by lowering arterial pressure (AP). We now ask the question, does raising CPP at intracranial hypertension affect the threshold for this transition?

METHODS: In rats, ICP was progressively increased from 10 to 30, 40 and 60 mmHg by raising an artificial cerebrospinal fluid reservoir connected to an ICP catheter, while arterial pressure (AP) was progressively elevated by i.v. dopamine infusion to maintain CPP at 50, 60, 70 or 80 mmHg (n = 3 per group). Using in vivo 2-photon laser scanning microscopy through a cranial window over the parietal cortex, we measured microvascular red blood cells flow velocity (fluorescein dextran) and NADH autofluorescence (tissue oxygenation). ICP, AP, rectal and cranial temperatures, Doppler flow and arterial blood gases were also monitored.

RESULTS: Increasing ICP at constant CPP led to redistribution of flow from CAP to MSF, BBB dye extravasation and reduced tissue oxygenation (NADH increase). At an ICP of 30 mmHg, the threshold for the transition to MSF, reflected by the ratio of CAP/MSF, increased significantly from 0.6 ± 0.1, 0.9 ± 0.1, 1.4 ± 0.1 to 1.9 ± 0.1 with increasing CPP of 50, 60, 70 and 80, respectively (mean ± SEM, P < 0.05). BBB opening and the rise in NADH occurred at similarly (P < 0.05) higher ICP as CPP was increased. Doppler flow increased with ICP elevation reflecting MSF.

CONCLUSIONS: CPP elevation at intracranial hypertension increases the ICP at which the transition from CAP to non-nutritive MSF, BBB opening and tissue hypoxia occurs. Our data suggest that in the management of patients with raised ICP, raising CPP above 70 mmHg may be beneficial.

Malik Saafan Park Soojin Maloney Eileen Friedman Bess Robinson Yolanda Frangos Suzanne Sitterly TT Bernal Olivia Watson Deborah University of Pennsylvania, Philadelphia, PA, USA

P272 The Effect of Pre-Injury Fitness Level on Prognosis and Clinical Outcome After a Single Moderate-to-Severe TBI: A Cohort Study

Clinical experience suggests that people with active lifestyles tend to recover better from TBI than more sedentary people. Surprisingly, however, there are no published studies about the effect of pre-injury fitness level on prognosis or recovery in humans after TBI. Our study aims to evaluate how the pre-injury physical fitness level affects the clinical outcome after a single moderate-to-severe traumatic brain injury. The enrollment of the subjects will be done through the UPHS trauma and clinical databases for consecutive patients who suffered a single moderate-to-severe TBI over the course of the study and or during the past one year. Several data points, including GCS, APACHE, Marshall Score, FIM on discharge, Modified Rankin Scale, OT/PT assessment will be extracted from the databases for pre-injury fitness and post-injury outcome assessment. In addition to the retrospective chart review the patients and or the caregivers will be contacted telephonically and or via postal mail and asked a standard set of questions (Physical activity questionnaire and AMPAC) to determine pre-injury physical fitness and post-injury outcome. Informed consent will be obtained at the time of enrollment either in person or through their caregivers during their initial hospital stay or via telephone if subject was identified from the database established in the past year. The results of this study will help clinicians advise their patients about prognosis, investigate the role (if any) of pre-injury fitness level on the recovery from TBI, and suggest molecular and biochemical pathways for future laboratory investigations and translational research.

Chen YungChia Wierzbicki Brittainy Meaney David Unniversity of Pennsylvania, Philadelphia, PA, USA

P273 The Role of Signalling Attenuation in Astrocytes in Traumatic Brain Injury in Vivo

Traumatic brain injury (TBI) affects an estimated 1.7 million Americans each year, generating an annual cost of 60 billion dollars. These statistics show how important it is to find a viable cure for a condition that currently has limited treatments, most of which involve rehabilitation. There is an increasing body of evidence that suggests targeting astrocytes, a major glial cell type, could be a potential therapeutic strategy. This study seeks to determine what role astrocytic signaling plays in brain injury recovery. Both the acute and delayed astrocytic response has been shown to affect neuronal survival. The initial injury response can induce astrocytes to release neurotransmitters that in excess become toxic to neurons. This acute response can also potentially lead to delayed, long term calcium dysregulation and induce further neuronal death. Impact injury induces in astrocytes an acute calcium wave that is propagated due to purinergic receptor activation and ATP release. The initial and subsequent ATP release can also activate other P2 receptors (P2R) leading to calcium dependent and independent release of glutamate, furthering excitotoxicity. Past in vitro stretch injury results have shown that inhibiting this immediate astrocytic response is neuroprotective. The in vitro findings led to this in vivo study using two transgenic mouse lines, Venus-tagged IP3 5-phosphatase (VIPP) and dominant negative SNARE (dnSNARE), to determine whether inhibiting astrocytic calcium signaling after a TBI resulted in a neuroprotective or neurodegenerative effect. The VIPP transgenic mouse line attenuates IP3 dependent calcium signaling; dnSNARE mouse line uses an over expression of the dominant negative SNARE domain to attenuate SNARE dependent gliotransmission. The mice are subjected to a severe CCI (1mm depth, 6m/s impact speed). Their recovery from CCI is determined by a neurological severity score (NSS) as well as their performance on the Morris water maze (MWM).

Wolf John 1 Johnson Brian 2 Mietus Constance 1 Browne Kevin 1 Smith Douglas 1 Grady M. Sean 1 Cohen Akiva 2 Cullen D. Kacy 1

1 University of Pennsylvania, Philadelphia, PA, USA 2 Children's Hospital of Philadelphia, Philadelphia, PA, USA P274 Diffuse Brain Injury Causes Alterations in Hippocampal Excitability in Swine

The rodent hippocampus has been shown to be preferentially vulnerable to fluid-percussion brain injury, including shifts in excitability in hippocampal subregions. Diffuse brain injury (DBI) induced by inertial loading has been extensively studied using non-impact head rotational acceleration in swine, and demonstrated loss of cells in hippocampal area CA1. To date, however, there has been no direct demonstration that rotational injury leads to changes in hippocampal function. We hypothesized that excitability in the hippocampus would also be altered by non-impact head rotational DBI in swine. Therefore we examined limbic function using hippocampal slices from DBI or sham pigs with extracellular recording techniques. Either eight hours or seven days post rotational injury, pigs were transcardially perfused with aCSF, one of the hemispheres was extracted, and the hippocampus isolated. The contralateral side was perfused with paraformaldehyde and prepared for histological examination. Transverse slices of the live hippocampal tissue (350μM) were prepared and extracellular recordings were performed in an interface chamber while slices were bathed in oxygenated aCSF. Input-output curves were generated and paired-pulse paradigms were utilized to examine changes in excitability and transmitter release probabilities in slices from injured versus sham animals. Experiments were carried out in hippocampal dentate gyrus and area CA1. Our data suggest that area CA1 becomes more excitable seven days post injury. In addition, there was a decrease in paired pulse potentiation in area CA1 and dentate gyrus lateral perforant path, potentially due to changes in release probability in these areas. These data indicate that non-impact inertial DBI may also lead to dysfunction in various aspects of hippocampal circuitry and suggests that this may be a useful pre-clinical model of human traumatic brain injury.

Mietus Constance 1 Browne Kevin 1 Meng Xu 2 Wolf John 1 Smith Douglas 1 Adeeb Saleena 3 Kawoos Usmah 3 Rosen Arye 2 Chavko Mikulas 3 Cullen D. Kacy 1

1 Center for Brain Injury and Repair, Dept of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA 2 2School of Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, PA, USA 3 3Dept of Trauma and Resuscitative Medicine, Naval Medical Research Center, Silver Spring, MD, USA P275 Neuroanatomical Patterns of Biophysical Responses and Neuropathology Based on Orientation to Blast Wave

Current military conflicts have highlighted the importance of understanding blast-induced traumatic brain injury (bTBI). However, potentially unique mechanism(s) by which blast exposure detrimentally affects neural cells/tissue remains unknown. We have demonstrated that blast exposure in rats directly elicits biophysical plasmalemmal disruptions in specific neural populations, in patterns that differ markedly from fluid percussion injury. The objective of the current study was to determine neuroanatomical patterns of acute neural cellular damage based on animal orientation to the blast wave. Varying orientation may permit segregation of general blast-sensitive neural populations versus those responding to directional-based biomechanical parameters. Lucifer yellow (LY), a small cell-impermeant dye, was delivered via intracerebroventricular injection and permitted to diffuse throughout the interstitial tissue. Rats were then exposed to blast overpressure at 120 kPa using an air-driven shock tube in lateral, front, or back orientations, or sham conditions. At either < 5 min (n = 20) or 3 hrs (n = 8; lateral only) post-blast, animals were euthanized and the brains processed for routine histology and intracellular uptake of LY. Following bTBI for each orientation, diffuse patterns of LY + cells were observed, demonstrating immediate blast-induced plasmalemmal disruption. Substantial LY uptake was observed in the hippocampus, cerebral cortex, and the cerebellum. In the hippocampus, the density of LY + cells was quantified, revealing that acute cell permeability varied based on exposure direction (p < 0.01) and based on sub-regions (p < 0.05). Specifically, the hilar region of the dentate gyrus was distinctly vulnerable in acute bTBI and up to 3 hrs following lateral blast. We found that virtually all LY + cells were neurons, and we are currently investigating preferential susceptibility based on specific neuronal phenotypes. Moreover, we are expanding our neuroanatomical map of acutely vulnerable populations, tracking their potential dysfunctional or degenerative fates, and regionally correlating with astrogliosis and inflammatory responses to further define a “signature” of bTBI neuropathology.

Johnson Victoria E. 1 Stewart William 2 Trojanowski John Q. 1 Smith Douglas H. 1

1 University of Pennsylvania, Philadelphia, PA, USA 2 University of Glasgow, Glasgow, United Kingdom P276 TDP-43 Immunohistochemistry Following Varying Survival from a Single Traumatic Brain Injury in Humans

Evidence suggests an association between traumatic brain injury (TBI) and the later development of syndromes of cognitive impairment, such as Alzheimer's disease (AD). In addition, we recently reported that a single TBI is associated with increased neurofibrillary tangle (NFTs) and amyloid beta (Aβ) plaque pathologies many years after injury, similar to those observed in AD. Although not previously examined following a single TBI, the pathologic phosphorylation and sub-cellular translocation of TAR-DNA binding protein (TDP-43) in neurons has emerged as a shared feature of a proportion of AD cases and the few, selected cases of repetitive TBI thus far reported.

Using antibodies specific for pathologic (hyper-phosphorylated) and physiological (non-phosphorylated) TDP-43, we examined human postmortem tissue from both acute (n = 23; 10hrs-14days survival) and long-term (n = 39; 1-47 years survival) survivors of a single TBI and compared them to uninjured age-matched controls (n = 47). Regions examined included the hippocampus and medial temporal lobe, cingulate gyrus, insular cortex, midbrain, pons and medulla.

No association was found between a history of single TBI and the development of pathologic TDP-43. Specifically, just 3 of 62 TBI cases displayed hyper-phosphorylated TDP-43 pathology versus 2 of 47 controls. However, in material from acute post-TBI cases, abnormal neuronal cytoplasmic immunoreactivity to physiological TDP-43 was commonly noted.

This observation of increased cytoplasmic immunoreactivity for physiological TDP-43 acutely following TBI may suggest a role for TDP-43 in injury or repair. Moreover, the absence of pathologic TDP-43 in this material at all survival intervals suggests a fundamental difference in the pathology of a single TBI versus repetitive TBI. Further elucidation of the role of TDP-43 in the injury state may be important in comprehending its evolution into aggregated inclusions and why multiple insults may be required for this process.

This work was supported by NIH grants NS038104, NS056202, AG10124 and AG17546.

Malik Saafan Motamedi Shahab Bernal Olivia Sitterly TT Paul Mohona Watson Deborah University of Pennsylvania, Philadelphia, PA, USA

P277 A Method to Monitor Neurotrophin Mediated Plasticity After a Unilateral TBI

We have demonstrated that trkB ligand neurotrophin (NT4/5) administered to an LFP lesion is highly neuroprotective for the most vulnerable hippocampal neurons in layer CA3. After TBI, neurotrophin support is lost at the injury cavity and could contribute to axonal dieback of trkB-expressing axons. By administering an infusion of NT4/5 at the injury site we will be able to see how it affects the axonal sprouting and regeneration. Many traumatic brain injury (TBI) patients increasingly recover function during the next few years after their injury, suggesting that plasticity of neuronal circuits likely plays a significant role in recovery. We have developed a method to label specific group of neurons and their axons wth GFP to study the regrowth and reconnectivity of axonal terminals in the hippocampus that occur following traumatic brain injury. In an initial experiment using normal rats, Sprague Dawley rats (250-275g) were injected with 2 ul of AAV2/9.CMV.eGFP.bGH into the CA3 region of ipsilateral hippocampus. Four weeks later, GFP labeling was observed in the ipsilateral CA3 cell bodies, in the crossing axons and in the terminals contralaterally. The main tracts highlighted by the transduced cells included the mossy fiber pathway ipsilaterally and the dorsal commisure and ventral commisure tracts contralaterally across the midline. In a second experiment, we labeled CA3 neurons one week before a contralateral application of LFP-injury and an infusion of NT4/5 via mini-osmotic pump. The brains were studied four weeks later, corresponding to five weeks post-AAV injection. By labeling axons with GFP prior to the injury, we are able to monitor post-injury distribution of the terminal fields from specific populations of neurons. We hypothesize that axons rescued by neurotrophin infusion will sprout into the lesion at intermediate times (4w-2mo) post-injury, and begin to redistribute around the lesion cavity at chronic time points (4mo).

Miller Megan Carter Michelle Scanlon Joelle Niyonkuru Christian Ferrell Robert Conley Yvette Arenth Patricia Wagner Amy University of Pittsburgh, Pittsburgh, PA, USA

P278 COMT Gene Variant Associations with Depression, Cognition and Recovery After Severe TBI

Posttraumatic depression (PTD) is one of the most common psychological disorders resulting from TBI and can adversely impact cognition and hinder TBI recovery. Several studies have linked genetic influences to idiopathic depression, however little is known of the genetic correlations with PTD. This study aimed to look at genetic associations between COMT genes and susceptibility to PTD. Both tagging single nucleotide polymorphisms (tSNPs) and functional variants were assessed. Overall, the population included 81 moderately to severely injured Caucasian patients. Individuals with pre-morbid depression were excluded from analysis. PTD was measured at 6 and 12 months using the Patient Health Questionnaire (PHQ-9) through both a categorical and numeric approach. The Functional Independence Measure Cognitive Subscales were also assessed At 6 months 32.2% of the population was categorized as depressed, while at 12 months the rate dropped to 24.6%. Out of the entire population 27 patients were never designated as depressed, and 5 were categorized as depressed at both time points. RS174696 was associated with depression status at 12 months post-injury. Univariate analysis shows that RS4680 were associated with PHQ-9 scores at 6 months while RS9332977, RS174696, and RS933271 were associated with 12 month depression and/or PHQ-9 scores. RS1654774, RS9332977, RS 740603 and RS5993883 were associated with FIM-COG scores at 6 and 12 months. RS9332977 associations with both cognition and depression may suggest that variability in the COMT gene is one factor by which depression is linked to cognition after TBI. Further studies, including multivariate and mediational analyses are required.

Hricik Allison 1 Puccio Ava 1 Yue John 2 Schnyer David 3 Pacheco Jenni 3 Valadka Alex 3 Okonkwo David 1 Manley, on behalf of the TRACK_TBI Investigators Geoffrey 2

1 University of Pittsburgh Medical Center (UPMC), Neurotrauma Clinical Trials Center, Pittsburgh, PA, USA 2 Univ Calif, San Francisco, Brain and Spinal Injury Center at SF General Hospital (UCSF), San Francisco, CA, USA 3 University Medical Center, Brackenridge-Austin (UMCB), Austin, TX, USA P279 Occurrence of Post-Concussive Symptoms in Mild TBI: Results from the Transforming Research and Clinical Knowledge in Traumatic Brain Injury (Track-TBI) Study

PURPOSE: Mild traumatic brain injury (mTBI) symptom sequelae is often thought to resolve over time by the acute care team. The purpose of this study was to prospectively examine the occurrence of the cluster of symptoms classified as post-concussive syndrome (PCS) by the World Health Organization at 3 post mTBI.

METHODS: This population-based cohort study performed through the Transforming Research And Clinical Knowledge in TBI (TRACK-TBI) study included all mTBI patients ≥ 18 years of age evaluated at an acute Level 1 trauma center (University of California, San Francisco, UCSF; University of Pittsburgh Medical Center, UPMC; and University Medical Center, Brackenridge-Austin, Austin Texas, UMCB) within 24 hours of injury between April 2010 and February 2011. Self-reported PCS symptoms were prospectively collected at 3 months post-injury and classified within 4 domains (physical, sleep, cognitive and emotional disturbances). These symptoms were compared to discharge status and functional outcome (Glasgow Outcome Scale-Extended (GOS-E) score and return to work/school). Symptoms were also compared to the Rivermead Post Concussion Symptom Questionnaire (RPCSQ) and Brief Symptom Inventory (BSI) Scale scores.

RESULTS: 427 mTBI patients (mean ± SD age, 43.7 ± 17.6; 28% female, 72% male) were consented with a site IRB approved protocol, 283 at UCSF, 82 at UPMC, and 62 at UMCB, with completed follow up at 3 months at 83%; 85%; and 67%, respectively. Overall, 77% experienced at least one PCS symptom at 3 months and was consistent across centers. A diagnosis of PCS was confirmed in 48% of subjects followed at 3 months post-mTBI. Self-reported symptoms were confirmed by the objective RPCSQ and BSI scores.

CONCLUSION: mTBI results in prolonged PCS symptomology, is commonly unreported, and is under appreciated by medicine providers. Mild TBI is often anything but mild.

Bales James W. 1 Garringer Julie 1 Conley Yvette P. 1 Puccio Ava M. 1 Okonkwo David O. 1 Dixon C. Edward 1 2

1 University of Pittsburgh, Pittsburgh, PA, USA 2 VA Pittsburgh Healthcare System, Pittsburgh, PA, USA P280 Association Between the PPP3CC Gene, Coding for the Calcineurin Gamma Catalytic Subunit, and Severity of Traumatic Brain Injury in Humans

PURPOSE: Calcineurin activity is upregulated post injury in animal models of traumatic brain injury (TBI). Inhibition of calcineurin following a TBI is associated with improved outcomes. Genetic variation in the calcineurin catalytic isoform A-gamma (CnAγ) has been associated with increased risk of schizophrenia and bipolar disorder. No evaluation has examined the association of calcineurin polymorphisms to outcomes after TBI in human tissue. We examined the CnAγ gene (PPP3CC) and clinical data for 277 subjects with TBI.

METHODS: Tagging single nucleotide polymorphisms (SNPs) rs10108011, rs2443504, rs2461491, and rs2469749 were genotyped from DNA extracted from blood or CSF, and variant associations with Glasgow Coma Scale (GCS) Score and Glasgow Coma Outcome Scale (GOS) score were determined using multivariate analysis with ordinal regression. For analysis GCS was split into GCS < 6 and GCS ≥ 6 and GOS was split into 3 categories of severe (GOS1,2), moderate (3), and good (4,5). Age and gender were controlled.

RESULTS: Carrying the ‘AA’ genotype of the rs2443504 polymorphism was associated with initial GCS scores < 6 and more severe GOS scores. Risk genotype analysis showed that subjects with the ‘AA’ genotype had a 54.2% chance of initial GCS < 6, a 59.3% chance of 3 month severe GOS, a 53.1% chance of 6 month severe GOS, and a 56.2% chance of 12 month severe GOS. In contrast subjects with the ‘GG’ genotype had a 26.6% chance of initial GCS < 6, and 31.3%, 32.4%, and 33.3% chance of severe GOS at 3, 6, and 12 months respectively.

CONCLUSIONS: These results suggest that polymorphisms within calcineurin catalytic subunits, the PPP3CC gene in particular, might be important predictors of susceptibility and recovery in TBI. Furthermore these results provide rationale for further examination of variable responses to calcineurin inhibitors in the context of underlying genetic differences within TBI.

Support: NIH P50NS030318, VA RR&D#B6761R, NIH R01NR008424

Hatz Laura 1 Scanlon Joelle 1 Miller Megan 1 Niyonkuru Christian 1 Conley Yvette 2 Ferrell Robert 3 Ricker Joseph 1 Wagner Amy 1

1 Department of Physical Medicine & Rehabilitation, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA 2 Health Promotion & Development, University of Pittsburgh, School of Nursing, Pittsburgh, PA, USA 3 Department of Human Genetics, University of Pittsburgh, Graduate School of Public Health, Pittsburgh, PA, USA P281 Kibra Polymorphism Associations with Episodic Memory in a TBI Population

Recent studies have implicated SNP rs17070145, a common T → C polymorphism on the KIBRA gene, in mediating differences in episodic memory. In healthy adult populations, carriers of the T allele (CT/TT) performed better than non-carriers (CC) on measures of episodic memory. However, there is evidence suggesting that this allelic association may be reversed in adults with subjective memory complaints and populations vulnerable to memory deficits, a problem common in traumatic brain injury (TBI). This study assessed the relationship between this variation in the KIBRA gene and cognitive function in 111 individuals with severe TBI (mean age 31.8 ± 12.8 years, mean GCS 6.21 ± 1.4). We predicted that non-carriers of the T allele would perform better than carriers in TBI populations on measures specific for episodic memory. Neuropsychological tests, including the Buschke Selective Reminding Test (SRT), were administered at 6 and 12 months post-injury. When grouped by the presence or absence of the T allele, Mann-Whitney tests demonstrated that non-carriers of the T allele performed better than carriers on the delayed cued (p = .002) and delayed free (p = .034) recall tasks at 6 months post-injury as well as on Long Term Retrieval (LTR; p = .033) and Long Term Storage (LTS; p = .032) at 12 months post-injury. A multivariate analysis controlling for age supported the association observed at 6 months post-injury for delayed cued recall. No associations were found between this KIBRA variant and other measures of executive function or functional outcome. The results of the present study are consistent with literature suggesting that the effects of the rs17070145 T allele are specific to episodic memory. The results also support the hypothesis that the relationship between rs17070145 variation and memory outcomes are disparate between healthy and impaired populations. Future research in diverse TBI populations is necessary to validate and generalize the relationship between KIBRA rs17070145 variation and episodic memory.

Puccio Ava Scruggs Bobby Panczykowski David Hricik Allison Bauer Joshua Beers Sue Okonkwo David University of Pittsburgh Medical Center, Pittsburgh, PA, USA

P282 Prospective Independent Validation of ‘Crash’ Modeling as a Prognostic Tool in Severe TBI

PURPOSE: Mortality and outcome prognosis of severe TBI patients at initial presentation has inherent decision-making difficulties for both medical planning and clinical trial inclusion. Medical Research Council (MRC) CRASH (Corticosteroid Randomisation After Significant Head Injury) prognostic model has been purposed as a means of risk adjustment and outcome prediction for use in trial design and analysis. The purpose of this study was to evaluate the CRASH model in predicting 14-day mortality and 6-month functional outcome using prospectively collected data at a large, Level 1 neurotrauma center.

METHODS: This population-based cohort study included all TBI patients (≥14 years of age, GCS ≤ 8) admitted to the University of Pittsburgh Medical Center between 1994 and 2009. Clinical data was linked to 14-day mortality and 6-month functional outcome, measured by the Glasgow Coma Scale (GOS). The prognostic CRASH models were composed of admission characteristics including country (US), age, Glasgow Coma Scale (GCS) score, and pupillary reactivity (core model); as well as results of computed tomography (core + CT). The discriminatory power and calibration of the CRASH models were assessed using multiple regression analyses and indicated by the area under the receiver operating characteristic curve (AUC).

RESULTS: A sample of 503 patients was available for analysis [mean age 37.8 ± 17 and median GCS 6 (IQR 3)]. The 14-day mortality was 29% while the median 6-month GOS was 3 (IQR 3). Both core and core + CT models displayed good prediction ability for unfavorable outcome and mortality (14-day mortality AUC = 0.74 & 0.73; 6-month unfavorable outcome AUC = 0.80 & 0.70, respectively). All model iterations displayed adequate calibration for predicting mortality and unfavorable outcome.

CONCLUSION: Prospective, independent validation supports the CRASH prognostic model's prediction of 14-day mortality and 6-month outcome after severe TBI. The CRASH prognostic model is an effective instrument to assist TBI study design and analysis.

Parey Phillip Bauer Joshua Puccio Ava Panczkowski David Okonkwo David University of Pittsburgh Medical Center, Pittsburgh, PA, USA

P283 Validation of VerifyNow in Detecting Platelet Dysfunction Caused by Antithrombotic Agents within the TBI Population

PURPOSE: Traumatic brain injury (TBI) has increased in the elderly population, many of whom have pre-existing comorbidities requiring antithrombotic medications. The impact of these agents has been shown to worsen outcome resulting in a conservative treatment approach of platelet transfusion with evidence of an intracranial bleed and history of antithrombotic agent. The VerifyNow assay detects the effect of Aspirin and P2Y12 inhibitors (Plavix) in the cardiac population. Validation in TBI is unreported, and could possibly decrease unnecessary transfusions.

METHODS: Between 2010 and 2011, we conducted a prospective comparative cohort study of patients presenting with a positive head CT and a reported history of antithrombotic use (Aspirin and P2Y12 inhibitors). VerifyNow assay was performed upon admission and 6 hours post-transfusion, with the primary endpoint detection of disinhibition given platelet transfusion.

RESULTS: 44 patients were available for analysis [mean age 78 ± 9 years, 67% male (IQR 22), median GCS 14 (IQR 1) and Marshall score 2 (IQR 0)], with 51%(17) taking Aspirin, 15%(5) taking Plavix, and 33%(11) taking both Aspirin and Plavix. The average baseline platelet count was 224 ± 61. According to the VerifyNow assay not all patients were inhibited (therapeutic) at admission; Aspirin only, Plavix and Aspirin: 6% (1), and 64% (11), respectively. After transfusing with platelets, not all patients were disinhibited (non-therapeutic); Aspirin only, Plavix and Aspirin: 24% (4), and 18% (2), respectively.

CONCLUSION: This study provides a means for detecting patients reported to be on antithrombotic agents whom are not at therapeutic levels and receive unnecessary transfusions following a TBI. In addition, a single platelet transfusion may not be sufficient to reverse the platelet inhibitory effects in the subgroup of patients who are inhibited. The VerifyNow assay is sensitive in the TBI population and needs to be validated in a larger cohort sample with control groups.

Bauer Joshua 2 Monaco Edward 1 Wang Li 2 Puccio Ava 2 Okonkwo David 1

1 University of Pittsburgh Medical Center, Pittsburgh, PA, USA 2 University of Pittsburgh School of Medicine, Pittsburgh, PA, USA P284 Acute Phase Diagnostic Neurophysiology Testing as a Predictive Measure of Outcome and Mortality in Severe TBI

INTRODUCTION: Current prognostic measures for severe traumatic brain injury (TBI) mortality and outcome are Glasgow Coma Scale (GCS) score, pupillary response, and computerized tomography results; however, their predictive value is limited by confounding variables contributing to outcome and poor inter-rater reliability. Use of diagnostic neurophysiology testing in the acute phase may provide a reliable objective mechanism for predicting patient prognosis.

METHODS: Under an IRB-approved protocol, clinical data and neurological outcome were prospectively collected from severe TBI patients (initial Glasgow Coma Scale (GCS) score ≤ 8, admitted ages 18-75) admitted to the University of Pittsburgh Medical Center between 2006-2009. All patients were treated with a standard ICU protocol, focusing on ICP management. Neurophysiology diagnostic brainstem auditory evoked responses (BAER) and somatosensory evoked potentials (SSEP) were performed as a standard-of-care by a trained neurophysiology technician on post-injury day four, and analyzed by a neurophysiologist. Outcome assessment included mortality (30 days and 1 year) and neurological outcome [1 year Glasgow Outcome Scale (GOS) score]. Logistic regression was utilized controlling for age and initial GCS.

RESULTS: A total of 90 patients with complete outcome testing were available for analysis [median GCS 6 (IQR 2), Marshall score 3 (IQR 2), Injury Severity Score 32 (IQR 16), and mean age 42 (36.67,47.6)]. BAER and SSEP results were coded as normal or abnormal (46%, 40%, respectively abnormal). Diagnostic BAER was a significant predictor of thirty day mortality (p = 0.001), and 1 year mortality (p < 0.001). Diagnostic SSEP was a significant predictor of 1 year outcome (p = 0.004), and 30 day mortality (p = 0.016); with a trend toward significance for 1 year mortality (p = 0.066).

CONCLUSION: Diagnostic neurophysiology testing (BAER and SSEP) is shown to be a significant objective means in prognosis of 30-day and 1 year mortality, with SSEP additionally being significant in neurological outcome prognosis.

Hurwitz Max Brayer Samuel Carter Michelle Zou Huichao Arenth Patricia Skidmore Elizabeth Thiels Edda Wagner Amy University of Pittsburgh, Pittsburgh, PA, USA

P285 Using the Morris Water Maze to Dissociate Implicit and Explicit Learning in the CCI Model of TBI

Separate networks exist for implicit and explicit learning and memory, and clinical research suggests that implicit networks are largely preserved following TBI. The Morris water maze (MWM) is used prolifically to study learning/memory deficits. However, the spatial learning paradigm does not fully differentiate between explicit/implicit learning and memory ability. Non-spatial training in the MWM can provide the necessary behavioral components to complete the task while limiting the ability of the animal to generate spatial maps. Our study utilized non-spatial pre-training (NSPT) prior to controlled cortical impact (CCI) injury as a construct for assessing interactions between implicit memory (search strategy) and explicit learning (place learning) deficits in the MWM task. 76 adult male Sprague-Dawley rats [sham N = 32, injured (CCI 2.8 mm; 4m/s, applied to the parietal cortex) N = 44] were divided into eight groups where pre-trained and non-pre-trained CCI and sham rats were subjected to place learning and retention trials, both with/without the use of extra-maze spatial cues. Prior to surgery, NSPT was performed across four days with a rotating hidden platform, extra-maze cues covered, and a static entry point. On d14-21 MWM testing was performed with rats divided between a traditional spatial place learning paradigm using extra-maze cues and a non-spatial version without extra maze cues. NSPT completely eliminated deficits in hidden platform latencies for CCI rats relative to non-pretrained shams in the cued condition. After pre-training, the injury deficits (compared to pre-trained shams) for place learning in the extra-maze cued condition likely represents the true spatial learning deficits in our CCI model. Acquisition trial performance in the non-cued condition showed sham and CCI rats with pre-training perform similarly, suggesting that implicit memory is largely intact two weeks after CCI. These results have significant implications for pre-clinical trial behavioral testing and methods for cognitive rehabilitation in the clinical population with TBI.

Phelps Thomas I McAloon Rose L Garcia Alexandra N Shah Mansi A Cheng Jeffrey P Kline Anthony E Physical Medicine & Rehabilitation, Psychology, Center for Neuroscience, and Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA

P286 The Therapeutic Efficacy of Aripiprazole After Experimental Traumatic Brain Injury

Antipsychotic drugs (APDs) are routinely administered after TBI to reduce agitation and aggression. However, APDs with D₂ receptor antagonist properties (e.g., haloperidol and risperidone) produce deleterious effects on behavioral recovery after TBI. Hence, the evaluation of APDs with different mechanisms of action is warranted. Aripiprazole (ARIP) exhibits 5-HT_1A and D₂ agonist activity, but not D₂ antagonism. Studies have shown that pharmacotherapies with these properties enhance behavioral outcome after TBI. Thus, the aim of the current study was to test the hypothesis that ARIP will enhance motor and cognitive performance after TBI. Adult male rats were anesthetized and subjected to either a cortical impact or sham injury and then randomly assigned to TBI + ARIP (0.1, 0.5, or 1.0 mg/kg), TBI + VEH (1.0 mL/kg, saline VEHicle), Sham + ARIP (1.0 mg/kg) or Sham + VEH (1.0 mL/kg) groups (n = 7-8 per condition). Treatments were administered intraperitoneally once daily for 19 days. Motor (beam-walk/beam-balance) and cognitive (Morris water maze) performance was assessed on post-operative days 1-5 and 14-19, respectively. The Sham groups did not differ from one another and thus were pooled. No motor differences were revealed among the TBI + VEH and TBI + ARIP groups [p > 0.05]. No cognitive differences were revealed among the TBI + VEH and TBI + ARIP (0.5 and 1.0 mg/kg) groups [p = 0.94 and p = 0.43, respectively]. In contrast, the TBI + ARIP (0.1 mg/kg) group performed better in the water maze vs. the TBI + VEH group [p = 0.0036], but did not differ from the SHAM controls [p > 0.05]. No deleterious effects on motor or cognitive function were observed following chronic administration of ARIP after TBI. Furthermore, the lower dose of ARIP (0.1 mg/kg) facilitated the acquisition of spatial learning relative to the vehicle group and did not differ from SHAMs. The findings support our hypothesis and present ARIP as a safer APD for alleviating behavioral disturbances in TBI patients and perhaps also improving cognitive function.

Harun Rashed 1 Zhou Huichao 1 Wagner Amy 1 Leak Rehana 2

1 University of Pittsburgh, Pittsburgh, USA 2 Duquesne University, Pittsburgh, USA P287 An Examination of Striatal C-FOS Activation Following Controlled Cortical Impact in Rats

Traumatic brain injury (TBI) often results in impairments in concentration, learning, and mental flexibility. Striatal dysfunction may underlie these TBI-related cognitive deficits. Our lab has previously shown deficits in a key striatal neurotransmitter, dopamine (DA), following a controlled cortical impact (CCI) model of TBI it rats. Daily treatment with the commonly prescribed psychostimulant methylphenidate (MPH) (5mg/kg i.p.) reverses some of the observed DAergic deficits. This study attempts to localize distinct subregions of the striatum that are altered in the context of CCI and to characterize the effects of chronic MPH treatment on a molecular level using c-Fos immunohistochemistry. c-Fos is a marker of cellular activation that is downstream of various signaling pathways, and it forms part of the AP-1 transcription factor complex. We examined striatal c-Fos expression profiles in CCI (2.7mm deformation at 4m/s applied over R parietal lobe) and naïve rats treated daily with MPH (5mg/kg) or saline (1mL/kg) for 2 weeks. Striatal sections were then examined with immunohistochemical labeling of c-Fos along the rostrocaudal extent (AP: + 1, − 1, − 2mm relative to bregma). Our results show over a two-fold increase in c-Fos immunopositivity in the striatum for CCI rats compared to naïve, with the most pronounced effects in the medial and dorsomedial striatum (208% and 294%, respectively). Furthermore, chronic MPH treatment reversed the injury-induced increase in c-Fos positivity to a level that is comparable to naïve animals. These findings are similar in nature to the observed restorative effects of MPH treatment on injury-induced DA neurotransmission deficits. These results suggest that medial regions of the striatum that are involved in cognitive and executive functions may be more susceptible to injury-induced changes, and MPH may act to restore these alterations.

Abrahamson Eric 1 Foley Lesley 2 Hitchens Kevin 2 Ho Chien 2 Paljug William 1 Melick John 1 DeKosky Steven 3 Kochanek Patrick 1 Ikonomovic Milos 1

1 University of Pittsburgh, Pittsburgh, PA, USA 2 Carnegie Mellon University, Pittsburgh, PA, USA 3 University of Virginia, Charlottesville, VA, USA P288 Simvastatin Improves Cerebral Blood Flow and Reduces Brain Tissue Loss After Brain Injury in Mice Expressing Human Beta-Amyloid Peptide

Traumatic brain injury (TBI) is a risk factor for developing Alzheimer's disease (AD), possibly due to altered amyloid-β (Aβ) metabolism. Brain hemodynamics are also altered after TBI, however it is unknown whether post-injury deficits in cerebral blood flow (CBF) can be ameliorated using therapies that prevent TBI induced increases in brain Aβ concentration. We recently demonstrated that daily treatment with simvastatin prevented post-injury increases in brain Aβ concentration in APP^NLh/NLh/C57BL/6 mice that express human Aβ. The current study examined whether simvastatin treatment also prevents CBF impairments and reduces brain tissue loss at 3 weeks after controlled cortical impact (CCI) injury in APP^NLh/NLh/C57BL/6 mice. Regional CBF was quantified using arterial spin-labeling magnetic resonance imaging (MRI) and brain tissue loss was measured using high resolution MRI as well as ex vivo histology analyses. Compared to injured C57BL/6 wild types, injured APP^NLh/NLh/C57BL/6 mice had greater CBF deficits in the ipsilateral cortex and lack of hyperemia response on the contralateral (non-injured) side, particularly in the hippocampus and thalamus. Compared to vehicle treated APP^NLh/NLh/C57BL/6 mice, daily oral administration of 3 mg/kg simvastatin for 3 weeks resulted in higher CBF in the thalamus (p = 0.02) and in a trend for higher CBF in the hippocampus ipsilateral to CCI. Simvastatin treatment also improved CBF in the cortical contusion area (p = 0.04) with a trend for improvement in the thalamus (p = 0.05) and amygdala (p = 0.07) on the contralateral side. This improvement in post-injury CBF was associated with less tissue loss. Our results demonstrate that post-injury treatment with simvastatin 1) ameliorates impairments in brain hemodynamics which are associated with increased concentration of human Aβ and 2) reduces brain tissue loss after CCI injury. We conclude that simvastatin therapy may improve outcome and reduce risk for developing long-term hypoperfusion and AD pathology in brains of human TBI survivors.

Zou Huichao Hurwitz Max Lorraine Fowler Wagner Amy University of Pittsburgh, Pittsburgh, USA

P289 Reversed Downregulation of Glutamate Transporter Expression and Inhibition of Neuroinflammation Followed by Levetiracetam Treatment in TBI Rats

Levetiracetam (LEV, Keppra) is a relatively new anti-epileptic drug that is used clinically to prevent post-traumatic seizures. However, little is known about its molecular effects on the injured brain. Our previous finding shows that acute LEV treatment improved motor performance in the controlled cortical impact (CCI) model of TBI without affecting cognition. We used young adult male Sprague-Dawley rats to explore regional changes in Glutamate transporter (GLT-1), reactive astrocytosis (GFAP), and inflammation (IL-1 β) expression 12-36 hours after CCI (2.8mm; 4m/s) or sham surgery and treatment with either LEV (i.p., 50mg/kg) or vehicle (VEH). We found enhanced pericontusional IL-1 β immunoreactivity and increased GFAP immunoreactive astrocytes in vehicle treated TBI animals that was effectively reduced by acute LEV treatment. LEV treatment also minimized post-TBI reductions in GLT-1 expression in the ipsilateral frontal cortex and striatum. Our findings suggest that the neuroprotective effects of LEV treatment on motor function may, in part, be due to an attenuation of TBI-induced glutamate transporter loss associated with reduced regional gliosis and neuroinflammation.

Yan Hong Q. 1 Dixon C. Edward 1 2

1 University of Pittsburgh, Pittsburgh, PA, USA 2 VA Pittsburgh Healthcare System, Pittsburgh, PA, USA P290 Effects of Two Craniotomy Sizes on Motor and Cognitive Function in the Rat

PURPOSE: A recent study by Cole, et al., (2011, J. Neurotrauma) demonstrated that the craniotomies performed in sham rats that do not undergo experimental traumatic brain injury (TBI), can result in substantial pro-inflammatory responses, morphological damage, and behavioral derangements. Also, intracranial neurosurgical procedures can have complications associated with craniotomies, and minimally invasive techniques using smaller craniotomies have been developed to reduce damage. This study examined the effects of two sizes of craniotomies on motor and cognitive function in the rat.

METHODS: There were four experimental groups of Sprague-Dawley rats (n = 10/group): Naïve control, anesthesia-alone control; anesthesia + 3 mm craniotomy; and anesthesia + 6 mm craniotomy. All craniotomies were performed over the right parietal cortex using an air-powered drill. Rats were assessed for motor function (beam balance and beam walking) prior to surgery and at one day post-surgery. Cognitive testing was performed on days 2-6 post-surgery using the Morris water maze.

RESULTS: None of the manipulations altered beam balance performance. For beam walking, the 6mm craniotomy was significantly worse 1 day after surgery. Anesthesia alone has no effect on either tests of motor performance the next day after surgery. In the water maze, the 3mm craniotomy group was significantly worse than anesthesia-alone and 6mm craniotomy groups.

CONCLUSIONS: The results confirm the findings by Cole, et al. that craniotomies alone in rats can produce detectable brain dysfunction. The present proposal extends these findings by studying two different sizes of craniotomies, and included an anesthesia-alone control. The size of the craniotomy appears to have differential effects on motor and cognitive function.

Supported by NIH grant 1F30NS030315, VA RR&D #B6761R, and the Copeland Fund of the Pittsburgh Foundation.

Liu Ying Zou Huichao Michael Adrian Wagner Amy University of Pittsburgh, Pittsburgh, PA, USA

P291 Chronic Methylphenidate Treatment Restored Spontaneous Dopamine Transients in The Striatum of Freely Moving Rats with Experimental Traumatic Brain Injury

The neurostimulant, methylphenidate (MPH), is clinically effective in treating cognitive deficits after traumatic brain injury (TBI). We previously used fast scan cycle voltammetry (FSCV) to show that a single dose of MPH (5mg/kg) challenge increases electrically evoked dopamine (EO DA) in the striatum of anesthetized naïve rats, however, controlled cortical impact (CCI) results in reduced levels of striatal EO DA and DA clearance. We have also shown that deficits in stiatal DA neurotransmission in anesthetized rats can be reversed by daily treatment with MPH (5mg/kg) for 2 weeks after CCI. In contrast to anesthetized preparations, the goal of this study was to evaluate: 1) How a single dose challenge of MPH induces changes in spontaneous striatal DA neurotransmission in naïve versus CCI rats; and 2) how two weeks daily MPH treatment (5 mg/kg) induces changes in spontaneous DA transients in the striatum of CCI rats. Striatal levels (μM) and frequency (per minute) of spontaneous DA transients were assessed in freely moving naïve rats, CCI rats without daily treatment, and CCI rats with daily MPH treatment both before and after a single dose challenge of MPH (2mg/kg). Spontaneous striatal DA transients were not detected in CCI rats baseline or after a single MPH challenge. DA transient levels and frequency increased after MPH challenge in both naïve (level: 32%; frequency: 83% increase) and CCI rats with daily MPH treatment (level: 39%; frequency: 36% increase). DA transient levels from CCI rats with daily MPH treatment was significantly higher compared to naïve rats both before (253% of naive) and after (272% of naive) MPH challenge. There was no significant difference in DA transient frequency between naïve and CCI rats with daily MPH treatment. The findings in freely moving rats provide further support the concept that daily MPH therapy restores striatal DA neurotransmission after CCI.

Shin Samuel S. 1 Bray Eric R. 1 Dixon C. Edward 1 2

1 University of Pittsburgh, Pittsburgh, PA, USA 2 VA Pittsburgh Healthcare System, Pittsburgh, PA, USA P292 Nicotine Improves Striatal Dopamine Release and Tyrosine Hydroxylase Activity Following Traumatic Brain Injury in Rats

PURPOSE: Striatal dopamine (DA) neurotransmission and signaling deficits resulting from traumatic brain injury (TBI) are important therapeutic targets. In our recent studies, we demonstrated decreased DA release, decreased activity of tyrosine hydroxylase (TH), and decreased phosphorylation of dopamine- and cAMP-regulated phosphoprotein at threonine 34 (pDARPP-32-T34) in the striatum of rats after TBI. While nicotine has been shown to be beneficial in TBI, its effects on striatal DA signaling are unknown. The purpose of this study was to assess enhancement of striatal DA signaling following TBI.

METHODS: Anesthetized rats (n = 5-7/group) were injured by controlled cortical impact and treated with saline or 2mg/kg dose nicotine for 7 days. For sham animals, only craniectomy was performed. Microdialysis measurement of potassium stimulated DA release, in-vivo TH activity assay, and Western blots were used on day 7.

RESULTS: In microdialysis experiments, potassium stimulated DA release showed significant between-group differences (F_3,17 = 4.372; p ≤ 0.05) in peak DA levels. Injured animals treated with saline showed statistically significant decrease from shams treated with saline (p ≤ 0.05). In addition, injured animals treated with nicotine showed no difference from sham, demonstrating recovery of DA release. TH activity assay showed statistically significant difference among the groups (F_3,23 = 14.360, p ≤ 0.05). Injured animals treated with saline showed significantly reduced TH activity compared to shams treated with saline, whereas injured animals treated with nicotine did not (p ≤ 0.05). There was statistically significant between-group differences with injured animals treated with saline and injured animals treated with nicotine showing significantly lower levels than shams treated with saline (p ≤ 0.05).

CONCLUSIONS: Although nicotine treatment was able to induce recovery of TH activity and DA release after TBI, no postsynaptic signaling deficit was reversed. Additional research is needed to better understand the relationship between DA release deficits and postsynaptic signaling changes after TBI.

Support: NIH/1F30NS067731-01, NIH/5R01NS060672-02, VA/RR&D #B6761R, and the Copeland Foundation.

Blyth Brian Bazarian Jeffrey University of Rochester, Rochester, NY, USA

P293 Serum Apolipoprotein A-I Concentrations are an Accurate Surrogate for the Clinical Diagnosis of MTBI

BACKGROUND: We recently observed that apoliprotein A-I (apoA-I) is elevated in the serum of patients suffering from mTBI relative to uninjured controls. This observation suggested the possibility that serum apoA-I concentrations would provide an accurate and objective surrogate for the clinical diagnosis of mTBI that was superior to extant protein biomarkers.

OBJECTIVE: To assess the accuracy of serum apoA-I concentrations for the diagnosis of mTBI.

METHODS: Sera from uninjured control subjects (n = 19) or from subjects meeting a clinical definition of mTBI (n = 100) were collected and assayed for apoA-1 concentration by commercially available ELISA. Sera from all mTBI subjects were collected within 6 hours of injury. Biomarker values between control and injured subjects were compared by the non-parametric Mann-Whitney test. For each observed apoA-I concentration estimates of the corresponding sensitivity and specificity were calculated and plotted on a ROC curves. In a separate but similar cohort, S100B was assayed by ELISA in mTBI (n = 438) and uninjured (n = 395) subjects. With these data, an ROC analysis analogous to that with apoA-I was performed.

RESULTS: ApoA-I concentrations were significantly elevated in mTBI subjects (2.39 mg/ml, SD 1.37) relative to controls (0.73 mg/ml, SD 0.25), p < 0.0001. For apoA-I, the AUC and associated 95% confidence interval was 0.94 (0.90-0.98, p < 0.0001). S100B concentrations were significantly elevated in mTBI subjects (0.29 mg/ml, SD 0.48) relative to controls (0.13 mg/ml, SD 0.22), p < 0.0001. For S100B, the AUC and associated 95% confidence interval was 0.72 (0.68-0.75, p < 0.0001).

CONCLUSIONS: Serum apoA-I concentrations are an accurate surrogate for the clinical diagnosis of mTBI. The respective performance of apoA-I and S100B in separate but similar cohorts suggest that apoA-I is likely to have greater accuracy for the clinical diagnosis of mTBI. A direct comparison of both biomarkers in a single cohort (n = 1250) is underway.

Kis David 1 Mencser Zoltan 1 Czigner Andrea 2 Kincses Tamas 3 Babos Magor 5 Toth Zoltan 5 Voros Erika 4 Barzo Pal 1

1 Department of Neurosurgery, University of Szeged, Szeged, Hungary 2 Department of Anatomy, University of Szeged, Szeged, Hungary 3 Department of Neurology, University of Szeged, Szeged, Hungary 4 Department of Radiology, University of Szeged, Szeged, Hungary 5 Euromedic Diagnostics Szeged Ltd., Szeged, Hungary P294 Prediction of Long Term Outcome in Severe Traumatic Brain Injury using Diffusion Tensor Imaging and Probabilistic Tractography

Traumatic brain injury (TBI) is among the leading causes of permanent disability. Techniques which reliably identify irreversibly injured brain areas would allow us to predict the long term outcome of severe TBI patients, thus indicate the need for aggressive treatment. Diffusion tensor imaging (DTI) and probabilistic tractography are powerful tools to quantitatively examine white matter integrity and create probabilistic connectivity (PC) maps at the individual level. Our aim was to identify structural abnormalities as biomarkers which can predict the long term outcome of the individual and are easy to use in the clinical practice. Magnetic resonance imaging (MRI) scans were performed in the subacute phase of the trauma using the following sequences: high resolution T1, fluid attenuation inversion recovery (FLAIR), susceptibility weighted imaging (SWI), T2 and DTI. From the DTI images fractional anisotropy (FA) and PC (connection to the medial and sensory thalamus, primary motor cortex) maps of the brain stem were reconstructed. In healthy controls (n = 15) there was a high correlation between the FA and PC maps of the brain stem and the anatomical structure. Ten TBI patients were tested who had similar initial clinical and CT scores indicating same clinical outcomes. There was no significant correlation between the outcome and T1, T2, FLAIR and SWI abnormalities. In patients who remained in unconscious state (n = 4) we observed disorganization of the FA and PC maps in the upper pons. In those who regained consciousness (n = 6) the brain stem structure showed no difference compared to the controls. In conclusion the FA and PC maps highly correlated with the clinical state at the individual level. According to our results DTI and probabilistic tractography may be clinically useful methods to predict long term outcome of severe TBI patients.

Combs Hannah 1 Jones Theresa 1 Kozlowski Dorothy 2 Adkins DeAnna 1

1 The University of Texas, Austin, TX, USA 2 DePaul University, Chicago, IL, USA P295 Skill Training, Exercise and Constraint-Like Therapy Together Promote Major Functional Reorganization of Remaining Motor Cortex After Controlled Cortical Impact Injury in Rats

There is a great deal of research on the neural mechanisms underlying motor rehabilitation after stroke, but not after traumatic brain injury (TBI). This is problematic given growing evidence that rehabilitative training regimes effective after stroke may be ineffective after TBI, even when injuries are similar. Previous studies in our lab demonstrated that, unlike rats with similarly placed stroke-like lesions, rats with controlled cortical impact (CCI) injury to the sensorimotor cortex did not show improved motor behavior following rehabilitative reach training alone. However, the combination of reach training, exercise, and forced forelimb use effectively improved motor function. We examined the effects of this behavioral treatment combination after CCI on the functional reorganization of forelimb representations in motor cortex, and its relationship with behavioral function. Adult male rats that were proficient in skilled reaching, received a CCI and three days later began rehabilitation (n = 13) or yoked control procedures (n = 13). The rehabilitation group participated in daily skilled reach training and exercise over 42 days. During days 10 through 20, rats wore vests that constrained the intact forelimb to force reliance on the impaired limb. Yoked controls were exposed to reaching chambers, locked running wheels, and were placed in non-limb restricting vests. The combination treatment significantly improved reaching success and normalized reaching strategies. Further, the rehabilitation increased wrist representations in the motor cortex, as established using intracortical microstimulation mapping of the injured hemisphere, indicating an expansion or reinstatement of this area compared with controls. These findings indicate that sufficient rehabilitative training can greatly improve motor function and improve the functional integrity of remaining motor cortex. When compared with findings from stroke models, they also suggest that more intense rehabilitation may be needed to improve motor function and remodel the injured cortex after TBI.

Grill Raymond Redell John Moore Anthony Zhao Jing Dash Pramod University of Texas Health Science Center at Houston, Houston, Texas, USA

P296 Comparison of Early Inflammatory Responses in Two Rodent Models of Mild Traumatic Brain Injury

RATIONALE: More than 30% of combat casualty survivors are reported to suffer from a traumatic brain injury (TBI). Many survivors received what can be called a “mild” TBI that, in many instances, permitted a rapid return to the battlefield. It has become apparent, however, that many of those who experienced a mild TBI exhibit long-term cognitive and emotional deficits.

OBJECTIVE: Several animal models of TBI are currently employed to both mimic the pathophysiological mechanisms of TBI and serve as “test beds” for the development of therapies. Our goal is to identify and compare early responses occurring in the rodent brain in two mild models of TBI: mild controlled cortical impact (mCCI; 0.8 mm depth) and mild fluid percussion injury (mFPI; 1 atm).

DESIGN: Adult, male, Sprague-Dawley rats received either a unilateral mCCI or mFPI. Subjects were sacrificed at 3 and 24 hours post-insult. Cortices ipsilateral to the lesion were assessed for changes in inflammatory cytokine mRNA and protein expression using Illumina Bead- and Luminex Protein Arrays, respectively. Additional subjects were euthanized at each time point to assess the immunohistochemical profile of inflammatory protein changes.

RESULTS: Animals receiving a mCCI or mFPI exhibited increased expression of a range of pro-inflammatory cytokines, both in terms of mRNA and protein as early as 3 hrs post-insult. Upregulation of the pro-inflammatory cytokine interleukin-1b could be detected in the ipsilateral cortex following both mCCI and mFPI by3 hours, with increased expression detected at 24 hours. The Inter-Cellular-Adhesion-Molecule-1 (ICAM-1), utilized by peripheral immune cells to access the CNS, was also dramatically upregulated in the ipsilateral cortex by 3 hours post-injury.

CONCLUSIONS: mCCI and mFPI both elicit pronounced inflammatory responses that are similar between the two models. Our goal is to better understand the consequences of these responses in order to better design strategies to promote recovery.

Ruppert Katherine Parsley Margaret Zeng Yaping Prough Donald DeWitt Douglas University of Texas Medical Branch, Galveston, TX, USA

P297 Blast Induced Neurotrauma Causes Decreases in Cerebral Blood Flow, Mean Arterial Blood Pressure and Myogenic Vasodilatory Responses of Pressurized Middle Cerebral Arteries in a Rodent Model

PURPOSE: Blast induced neurotrauma (BINT) has been called the signature injury of Operations Iraqi Freedom and Enduring Freedom. Many combatants exposed to BINT exhibit cognitive deficits, sleeplessness and behavior changes are all symptoms of BINT. While there are numerous investigations of the neurological and behavioral consequences of BINT, the vascular effects of blast exposure have not been described thoroughly.

METHODS: Using a gun powder-driven blast injury device, isoflurane-anesthetized, adult, male Sprague-Dawley rats were exposed to moderate blast injury. Laser Doppler cerebral blood flow (CBF) and mean arterial pressure (MAP) were recorded 5, 15, 30, 45 and 60 minutes after blast injury. We also examined myogenic responses to intraluminal pressure changes in isolated, pressurized middle cerebral arterial segments (MCAs) harvested from rats exposed to mild, moderate or severe BINT in vivo.

RESULTS: In rats exposed to moderate BINT (righting reflex suppression for 10-14 min), MAP decreased to 45% of baseline within 50 seconds of BINT then returned to baseline levels within 15 min. LDF decreased to 50% of baseline values within 5 min and remained at these levels for the entire 60 minute monitoring period. The MCAs harvested from rats subjected to mild, moderate and severe BINT decreased in diameter as intraluminal pressure was reduced from 100mmHg to 20mmHg.

CONCLUSION: The observed CBF reductions in the presence of normal MAP, suggest that BINT is associated with significant increases in cerebral vascular resistance. Additionally, the reductions in MCA diameters during progressive reductions in intravascular pressure suggest that autoregulation is impaired by BINT. Our observations of blast-induced increases in cerebral vascular and impaired autoregulation suggest that blast exposure may be associated with significant cerebral hypoperfusion.

Supported by award W81XWH-08-2-0132 from the Department of Defense and the Moody Center for Traumatic Brain & Spinal Cord Injury Research/Mission Connect.

Boone Deborah Kumar Aswathi Micci Adelaide Cowart Jeremy Parsley Margaret Prough Donald DeWitt Douglas Hellmich Helen University of Texas Medical Branch, Galveston, Galveston, Texas, USA

P298 AAV-Mediated Gene Knockdown Influences Functional Outcome After Traumatic Brain Injury

The lack of effective treatments for traumatic brain injury (TBI) patients is a great impediment to reducing the twin burdens of long-term disability, i.e. cognitive problems associated with hippocampal damage and enormous health care costs. Therapeutic gene silencing of injury-induced genes has the potential to improve functional outcome in TBI patients but this potential has not yet been demonstrated in pre-clinical studies of TBI. The goal of this study is to examine the functional consequences of knocking down two TBI-induced genes, neuronal nitric oxide synthase (nNOS) and glutathione peroxidase-1 (GPX-1), both shown to mediate neuronal injury/death and neuroprotection, respectively, after TBI. Three siRNA sequences for each gene, previously shown to knockdown gene expression in cultured hippocampal neurons, were cloned into the Adeno-Associated Virus (AAV) vector expressing the green fluorescent protein (GFP) reporter gene. High titer recombinant AAV viruses were produced for stereotactic injection into the rat hippocampus. Rats received a severe fluid-percussion injury and were injected with AAV virus constructs one hour post injury. Hippocampal-dependent working memory deficits were assessed using the Morris water maze on days 11-15 post injury. On day 15, brains were removed from euthanized rats, sectioned, and total RNA was isolated from laser capture microdissected neurons expressing GFP. Gene knockdown was confirmed by quantitative real time PCR for all nNOS and GPX-1 constructs. The control scramble virus had no effect on gene knockdown and functional outcome was indistinguishable from TBI alone. Knockdown of nNOS with one of the AAV constructs improved working memory deficits after TBI and knockdown of GPx-1 with one of the viral constructs had the opposite effect, supporting the hypothesized roles of these genes in neurodegeneration or recovery after TBI. Immunohistochemical analysis showed that the siRNA viruses did not elicit an immune response in the brain which could have confounded the behavioral results.

Micci Maria-Adelaide Parsley Margaret Kennedy Debbie Wei Jingna Patrikeev Igor DeWitt Douglas Hellmich Helen UTMB, Galveston, Texas, USA

P299 Development of a Novel Method for Non-Invasive In Vivo Imaging of Viable Grafted Stem Cells in the Brain

INTRODUCTION: Stem cells represent a promising therapeutic approach for several neurological disorders of the central nervous system (CNS) as well as for traumatic brain injury (TBI). The successful development of stem cells therapy and its translation to the clinical setting is currently hampered by the lack of a reliable and safe method to accurately monitor the location, migration and phenotypical differentiation of transplanted cells. The aim of our work is to develop a novel non-invasive in vivo imaging approach for monitoring viable grafted cells in the brain repeatedly over time. Here we present proof of principle data that demonstrate the feasibility of using the human sodium-iodide symporter (hNIS) as a reporter gene for tracking neural stem cell (NSC) after transplantation in the brain using SPECT/CT imaging.

METHODS: NSC were isolated from the hippocampus of adult rats (Hipp-NSC) and transfected with a lentiviral vector containing the hNIS gene. Uptake of pertechnetate (^99mTc) into the cells via the hNIS was measured with a gamma counter. Immunofluorescence analysis using specific antibodies against neuronal and glial markers was used to study the differentiation potentials of Hipp-NSC in vitro. hNIS-expressing Hipp-NSC were transplanted into the brain of adult male rats and the cells imaged in vivo using a small rodent SPECT/CT apparatus.

RESULTS: The expression of the hNIS in Hipp-NSC results in the functional uptake ^99mTc and does not affect their ability to generate neuronal and glial cells in vitro. After transplantation in the rat brain, Hipp-NSC expressing the hNIS can be visualized non-invasively using SPECT-CT imaging.

CONCLUSIONS: These data support the use of the hNIS as a reporter gene for non-invasive imaging of NSC in the brain. The repeated, non-invasive tracking of implanted cells will accelerate the development of effective stem cell therapies for TBI and other types of central nervous system injury.

Sell Stacy Paulucci-Hothauzen Adriana Leavitt Sheri Prough Donald DeWitt Douglas University of Texas Medical Branch, Galveston, TX, USA

P300 Estradiol Effects on GAP Junction Communication in the Cerebral Vasculature After Traumatic Brain Injury

17ß-estradiol (E₂) may be protective after traumatic brain injury (TBI) through effects on the cerebral vasculature such as increasing gap junction (GJ) coupling. Gap junction intercellular communication contributes to propagated vasoconstriction, vasodilation, myogenic tone and other aspects of cerebral vascular reactivity such as autoregulation. We tested the hypothesis that the administration of E₂ after TBI improves GJ communication between cerebrovascular smooth muscle cells. Fluorescence recovery after photobleaching (FRAP) was used to measure GJ coupling between vascular smooth muscle cells in middle cerebral artery (MCA) segments harvested from rats subjected to parasagittal fluid percussion injury (FPI). Under isoflurane anesthesia, ovariectomized female rats received moderate FPI (2 atm) or sham FPI followed by treatment with E₂ (0 - 66 μg/kg, s.c.) or vehicle 15 minutes post-injury. Middle cerebral arteries were harvested 15 minutes after injection and mounted on a confocal microscope to measure FRAP. The diffusion-uncoupled FRAP curve was fit to a mathematical model and quantitatively analyzed to calculate the mobile fraction, percentage of recovery related to baseline, and the rate of fluorescence recovery (T_1/2). While the mobile fraction and percent recovery was not different between treatment groups, the T_1/2 was greater after TBI (3.8 ± 1.3 sec) compared to sham-injured rats (1.7 ± 0.3). Treatment with E₂ reduced T_1/2 to levels that were similar to sham-injured rats (1.7 ± 0.4 for the 33 μg/kg dose). These observations that E₂ increases the rate of GJ communication after TBI and our previous results that E₂ improved vasodilation responses in MCAs after TBI (Sell, et al., JNT 27:A89, 2010), suggest that E₂ improves cerebral vascular reactivity after TBI by enhancing GJ communication. Thus, E₂ may be protective after TBI in part by improving intercellular communication and cerebral vascular reactivity, thereby reducing the deleterious effects of posttraumatic insults such as hemorrhagic hypotension or hypoxemia.

Perez-Polo J. Regino 1 Rea Harriet C. 1 Johnson Kathia M. 1 DeWitt Douglas S. 1 Grill Raymond J. 2 Parsley Margaret A. 1 White Mellodee M. 1 Unabia Gedda C. 1 Hulsebosch Claire E. 1

1 UT Med Br at Galveston, Galveston, TX, USA 2 UT Med School at Houston, Houston, TX, USA P301 Inflammatory Response to MTBI in Rat

Mild traumatic brain injury (mTBI) has long term cognitive and behavioral deficits. There is consensus that “inflammation” is a component of mTBI response. The time course of “inflammatory” responses beyond the acute stage, low level chronic events taking place after mTBI or the specifics of the “inflammatory” response outcomes in several components of physiological function are not fully understood. The behavioral outcomes, the “mild” nature of the injury has made assessments that are significant in the clinical context difficult to evaluate. We focused on inflammatory outcomes of mTBI after 1 atm. lateral fluid percussion (mLFP) in the rat, amodel that causes a combination of focal cortical contusion and diffuse injury of subcortical brain areas, akin to human brain neuropathology after TBI. We characterized inflammatory cytokine responses and found that there is a robust early expression of IL-1β by microglia as a result of an earlier breakdown in blood brain barrier and activation of astrocytes. We have also shown that there is significant early impairment of cognitive function based on measures of working memory. In the mTBI rats microglial cells were immunolabeled with antibody against IBA-1 (ionized calcium binding adaptor) a biomarker for microglia colocalizing with the pro-inflammatory cytokine Interleukin-1β (IL-1β). As early as 3 hours after mLFP there was an increase in activated astrocytes (GFAP) and deterioration of the blood brain barrier. Evidence for the involvement of impaired vascular function after mTBI which would be consistent with chronic low level inflammatory events which because of cross talk with other systems may explain some of the persistance of behavioral outcomes. We chose working memory assays as the most relevant indicators of cognitive impairment. The most robust deficit was evident at 11 days post mLFP. Supported in part by DOD grant T074693 and the Mission Connect Mild TBI Translational Research Consortium.

Zeng Yaping Prough Donald S. Dewitt Douglas S. UTMB, Galveston, TX, USA

P302 Moderate Traumatic Brain Injury Plus Hemorrhagic Hypotension Reduces Myogenic Responses in Pressurized Rat Middle Cerebral Arteries

INTRODUCTION: TBI reduces cerebral vascular compensatory responses to changes in arterial blood pressure in patients and experimental animals. To determine whether hemorrhagic hypotension (HH) after TBI also alters cerebral vascular reactivity, the responses of cerebral arterial to reduced intravascular pressure were measured in middle cerebral arterial (MCA) segments harvested from rats after moderate fluid percussion TBI followed by a clinically relevant HH and fluid resuscitation protocol.

METHOD: Rats were anesthetized isoflurane in a mixture of air and oxygen and prepared for moderate fluid percussion TBI. Rats were then randomized to one of four groups: Sham injury, TBI only, HH only, TBI + HH. HH was produced by withdrawing sufficient blood to reduce MAP to 40 mmHg for 30 min. In the second “prehospital care” phase, saline or lactated Ringer's was infused in 1 mL boluses to increase MAP to 50 mmHg for 30 minutes. At the end of the period of hemorrhage, shed blood was returned and the rats were allowed to survive for 24 hrs. At the end of the survival period, rats were anesthetized with isoflurane, decapitated and segments of the MCA were harvested. The MCA segments were mounted on an arteriograph and pressurized and MCA diameters were measured as transmural pressure was sequentially reduced from 100 mmHg to 20 mmHg in 20 mmHg increments.

RESULTS: TBI alone and TBI + HH produced similar impairment in vasodilatory responses to controlled reductions in intravascular pressure in isolated, pressurized MCAs. Although differences were not significant, vasoconstriction at intravascular pressures of 20 mmHg was slightly greater after TBI + HH than after TBI alone.

DISCUSSION: Both TBI alone and TBI + HH reduced vasodilatory responses to reduced intravascular pressure in rodent MCAs.

Oldenkamp Catherine 1 Spence Jeffrey 2 Warner Matthew 3 Harper Caryn 1 Moore Carol 1 Madden Christopher 4 Sarode Ravi 5 Diaz-Arrastia Ramon 1

1 Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX, USA 2 Department of Internal Medicine and Clinical Science, University of Texas Southwestern Medical Center, Dallas, TX, USA 3 Mayo Medical School, Rochester, MN, USA 4 Department of Neurological Surgery, University of Texas Southwestern Medical Center, Parkland Memorial Hospital, Dallas, TX, USA 5 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA P303 Effects of Platelet and Plasma Transfusions on Long-Term Functional Outcome After Traumatic Brain Injury

BACKGROUND & PURPOSE: Previous research suggests red blood cell (RBC) transfusions in traumatic brain injury (TBI) patients are associated with poor hospital outcomes and increased mortality. The preferred transfusion thresholds for fresh frozen plasma (FFP) and platelets have not been studied. This study evaluates transfusion's relationship with long-term functional outcome and survival in TBI patients with moderate platelet and coagulation factor abnormalities.

METHODS: This study consists of a retrospective review of prospectively collected data from 480 TBI inpatients hospitalized at a single center between 2005 and 2009. Inpatient information includes initial Glasgow Coma Score (GCS), Abbreviated Injury Score (AIS), length of hospital stay, volumes of all transfusions received, and 11 laboratory parameters. Moderate anemia was defined as HCT between 21-30%, moderate coagulopathy as INR between 1.4-1.9, and moderate thrombocytopenia as platelet count 50-107 x103/μL. Outcome measures recorded at 6 months post-injury include Glasgow Outcome Score Extended (GOS-E), Functional Status Exam (FSE), and survival. Two sample independent t-tests, univariate correlations, and general linear models were used to detect differences in outcome while accounting for confounding differences between transfused and nontransfused groups.

RESULTS: Univariate analyses showed significant correlations between poor outcome scores and transfusion volume of pRBCs, FFP, and platelets in all 3 moderate deficiency groups. Several measures of initial injury and lab abnormalities also correlated significantly with poor long-term outcome. In simple linear regression, poor outcome scores were significantly associated with RBC transfusion in the moderately anemic group (F = 6.0, p = 0.02) and with FFP and platelet transfusion in the moderately coagulopathic group (F = 3.9, p = 0.05; F = 5.0, p = 0.03, respectively).

CONCLUSION: Blood product transfusions may contribute to poor long-term functional outcome in TBI patients with moderate anemia or coagulopathy. More conservative transfusion strategies should be considered in this population.

Oldenkamp Catherine 1 Paliotta Christopher 1 Peng Lifang 1 De La Plata Carlos Marquez 2 Moore Carol 1 Harper Caryn 1 Diaz-Arrastia Ramon 1

1 Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX, USA 2 Center for BrainHealth, University of Texas at Dallas, Dallas, TX, USA P304 Magnetic Resonance Imaging (MRI) Methods for Detection of Traumatic Axonal Injury in the Acute Phase After Traumatic Brain Injury (TBI)

BACKGROUND: Traumatic Axonal Injury (TAI) results from decelerating or impacting the brain leading to shearing of axons. TAI is not well imaged by conventional computerized tomography (CT) scanning. Fluid Attenuated Inversion Recovery (FLAIR) identifies white matter lesions associated with post-TBI cerebral atrophy and functional recovery. Diffusion Tensor Imaging (DTI) is an advanced MRI technique thought to be more sensitive than FLAIR for detecting white matter pathology. It is unknown which modality is more useful in the acute phase following TBI. This study compares FLAIR and DTI by assessing the relationship between identified lesions and measures of outcome.

METHODS: DTI and FLAIR images were obtained from 17 uninjured controls and 29 patients in the acute phase following TBI (median 2 days after injury) and again during the chronic phase (median 7.8 months after injury). FLAIR lesion volume was measured using a semi-automated thresholding algorithm. Statistical Parametric Mapping was used to identify voxels where Fractional Anisotropy (FA) and Mean Anisotropy (MD) were significantly different in patients (FDR = 0.05, cluster size 20). Brain volumes were assessed using Freesurfer software. Functional outcome was assessed using the Glasgow Outcome Score-Extended (GOS-E) at the time of the second scan.

RESULTS: There was no correlation between white matter pathology identified by FLAIR with that identified by DTI FA or MD. Whole brain atrophy correlated with acute and chronic FLAIR white matter lesion indices (r = -0.433, p = 0.044; r = -0.439, p = 0.041, respectively), but not DTI measures. GOS-E correlated with acute and chronic FLAIR lesion volume (rho = -0.514, p = 0.004; rho = -0.593, p = 0.001, respectively). Chronic DTI FA and MD lesion volumes correlated with GOS-E, while acute DTI measures did not.

CONCLUSIONS: FLAIR and DTI measure different aspects of white matter injury. FLAIR may be the more useful imaging sequence in the acute stage, as its lesions are associated with subsequent global atrophy and functional outcome.

Bosworth Christopher 2 Culver Carlee 1 Krishnan Kamini 2 Arenivas Ana 2 Shokri-Kojori Ehsan 1 Diaz-Arrastia Ramon 2 de la Plata Carlos Marquez 1

1 University of Texas at Dallas Center for Brain Health, Dallas, TX, USA 2 University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA P305 Hippocampal Interhemispheric Functional Connectivity in Traumatic Axonal Injury

PURPOSE OF THE STUDY: Research suggests hippocampal interhemispheric connectivity (IHC) is positively correlated with memory performance in healthy individuals. However, white matter damage, like that which occurs during traumatic axonal injury (TAI), compromises connectivity between hippocampi, and this potentially impacts memory. We examined whether hippocampal IHC is compromised among a sample of moderately to severely injured patients with TAI at both the acute and chronic stages, and whether the degree of hippocampal IHC is associated with memory.

METHOD: Resting-state functional connectivity data were acquired using 3T MRI. Images from 22 acute stage (2-7 days post-injury) and 25 chronic stage patients (6-11 months post-injury) were analyzable. Seventeen uninjured controls were scanned for comparison. Neurocognitive outcomes were assessed on the day of the chronic scan. The hippocampi were divided into head, body, and tail for analysis. Diffusion tensor tractography data for the corpus callosum (CC) were analyzed as measures of structural connectivity.

RESULTS: The structural integrity of the CC was compromised for both acute and chronic stages. Decreased IHC was found in the head of the hippocampus among individuals with acute and chronic TAI compared to controls. However, no group differences in IHC were found between the body and tail of the hippocampi. Chronic stage patients performed in the average range on a memory task. Anterior hippocampal IHC in the acute stage was negatively correlated with performance on memory tests at the chronic stage; however, anterior hippocampal IHC at the chronic stage was not correlated with memory.

CONCLUSIONS: Decreased hippocampal IHC at both acute and chronic stages compared to controls suggests the limbic system is not operating normally, possibly due to compromise in interhemispheric white matter. The relationship between hippocampal IHC and memory at the acute phase may reflect a need for functional reorganization, and evidence of reorganization at the chronic stage.

Bedi Supinder 1 Walker Peter 1 Shah Shinil 1 Jimenez Fernando 1 Smith Philippa 1 Hamilton Jason 2 Mays Robert 2 Pati Shibani 1 Dash Pramod 1 Cox Charles 1

1 University of Texas, Health Science Center at Houston, Houston, Texas, USA 2 Department of Regenerative Medicine, Athersys Inc., Cleveland, OH, USA P306 Multipotent Adult Progenitor Cell Therapy for Traumatic Brain Injury: Modulation of Microglia

Traumatic brain injury (TBI) results in inflammation and cell death. As a result, TBI can cause cognitive, physical and behavioral deficits that are dependent on the severity and location of the injury. Immune responses are key regulators of TBI-induced alterations in the central nervous system. One key component of the immune response is microglia. In these experiments, we are investigating interactions of multipotent adult progenitor cells [MAPC (Athersys, Inc)] and splenocytes to attenuate the immune response of microglia (pro to anti-inflammatory: M1 to M2) after TBI. Specifically, antibodies to CD86 and CD206 were used to assess the ratio of M1 and M2 phenotypes respectively using flow cytometry. For the in vivo experiments, MAPC treatment was administered 2 and 24 hr after injury. Microglia harvested 48 and 120 hr after injury showed a significant increase in the M2:M1 phenotype when compared to microglia from untreated animals. For the in vitro experiments, isolated microglia were stimulated with Lipopolysaccharide (LPS). They were then incubated with supernatant derived from MAPC in direct contact with stimulated splenocytes for 72 hr. There was a significant increase in the M2:M1 phenotype of MAPC media cultured microglia when compared with LPS stimulated microglia alone. This suggests that there is a secreted soluble factor (s) due to the interactions between the spleen and MAPC that modulates the microglia phenotype from pro to anti-inflammmatory. MAPC, a primitive form of bone marrow derived progenitor cells modulate the systemic inflammatory response [via the spleen (Walker 2011)] that significantly alters the resident microglia/macrophage populations from M1 to M2. Delineating the mechanisms that modulate the pro to anti-inflammatory state of microglia with MAPC treatments is vital in attenuating the harmful effects of TBI. MAPC therapy, one day may be administered to patients immediately after TBI to help aid in the recovery process.

Coats Brittany 1 Binenbaum Gil 3 Peiffer Robert L. 5 Sullivan Sarah 2 Ralston Jill 2 Akella Trupti 2 Smith Colin 4 Duhaime Ann-Christine 6 Margulies Susan S. 2

1 University of Utah, Salt Lake City, UT, USA 2 University of Pennsylvania, Philadelphia, PA, USA 3 Children's Hospital of Philadelphia, Philadelphia, PA, USA 4 University of Edinburgh, Edinburgh, Scotland, USA 5 Merck & Co, Inc, Whitehouse Station, NJ, USA 6 Massachusetts General Hospital, Boston, MA, USA P307 Ocular and Neuropathology from Cyclic, Low-Velocity Head Rotations in Immature Pigs

The objective of this study was to investigate ocular and neuropathological changes in immature pigs undergoing cyclic, non-impact head rotations. Immature piglets (n = 41, 3-5 days old) underwent cyclic (3hz), low-velocity (28.1 ± 3.2 rad/s) head rotations for 10 seconds, 30 seconds, or five 6 second intervals. A subset of these animals (n = 8) experienced a second cyclic head rotation 24 hrs after the first rotation. An additional group of animals (n = 5) underwent a single head rotation at a velocity similar to that of the cyclic group (31.8 ± 6.6 rad/s). Sham animals (n = 5) experienced similar procedures without rotation. Brains and eyes were perfusion fixed and harvested at 6 hrs (n = 20), 24 hrs (n = 21), or 6 days (n = 5) post-insult. Sections were stained with H&E and βAPP to quantify cerebral and ocular pathology, including axonal injury (AI). At 6 hrs post injury, no βAPP staining or extra-axial hemorrhage (EAH) was seen in animals undergoing either cyclic or single, low velocity head rotations. At 24 hrs post injury, the 10 and 30 second cyclic head rotations resulted in a variable amount of βAPP-positive AI (0-0.35% of total brain area stained with βAPP) and EAH (0-15% of visible brain surface covered by blood). At 6 days post injury, more animals had evidence of AI (80%), but the range of values for AI (0.05-0.2%) and EAH (0-9%) were similar to 24 hrs. The subset of animals that received 2 cyclic insults 24 hrs apart were survived an additional 24 hrs and had 0-0.49% AI and 0-6% EAH. No ocular pathology was seen in any animal. Axonal injury and extra-axial hemorrhage was greater at 24 hrs and 6 days post-insult compared to 6 hrs. This suggests that cyclic, low-velocity head rotations may be associated with evolving injury, showing different temporal and pathological characteristics compared to single, high-velocity rotational events.

Yeoh Stewart Bell David Monson Kenneth University of Utah, Salt Lake City, UT, USA

P308 Thresholds of Cerebrovascular Trauma in Mild Primary Blast Injury

Mechanisms and thresholds of primary blast traumatic brain injury have not been well defined. Cerebral vessel injury is a common sequela of severe blast exposure, but little is known about how these injuries occur and whether minor vascular dysfunction may contribute to mild traumatic brain injury. In order to better understand thresholds of cerebrovascular injury in primary blast, rats protected with a Kevlar vest were exposed to a blast wave expanding out the end of a rifle barrel. The explosion was produced by firing the primer in an otherwise empty casing. Animals were placed off-axis to ensure exposure to primary blast alone, characterized by peak pressures between 125 and 280 kPa and positive phase durations ranging from 0.028 to 0.016 ms. Brains were sectioned and stained to detect IgG and GFAP via fluorescent microscopy. In general, IgG was detected in multiple distinct locations throughout the brain, often at regions of tissue transition such as the gray-white matter interface. GFAP staining clearly identified astrocyte structure throughout the brain except at lesion locations, corresponding directly with locations of extravascular IgG. As expected, more and larger focal regions of injury were generally present in animals exposed to higher levels of pressure. Injury was present in some, but not all, of the animals exposed at the 125 kPa level, while most animals exposed to 150 kPa suffered identifiable injury, suggesting that the injury threshold is somewhere near these levels of peak pressure. These results add to the growing set of data defining thresholds of blast injury in rats. Other groups have reported significant vascular injury at lower levels of peak pressure but with much longer durations, consistent with expectations that injury would be dependent on duration.

Tustison Nicholas 1 Avants Brian 2 Cook Philip 2 Kim Junghoon 3 Whyte John 3 Gee James 2 Ahlers Stephen 4 Stone James 1

1 University of Virginia, Charlottesville, VA, USA 2 University of Pennsylvania, Philadelphia, PA, USA 3 Moss Rehabilitation Research Institute, Philadelphia, PA, USA 4 Naval Medical Research Center, Silver Spring, MD, USA P309 Multivariate Analysis of Diffusion Tensor Imaging and Cortical Thickness Maps in a Traumatic Brain Injury (TBI) Cohort using Advanced Normalization Tools (ANTS)

TBI is a complex disease process involving mechanical disruption of neurological tissues and activation of secondary injury cascades, culminating in loss of function. Multiple neuroimaging approaches have recently been developed to detect white matter (WM) and gray matter injury following TBI. Diffusion tensor imaging (DTI) affords quantification of microstructural white matter injury. High-resolution T1-weighted sequences allow for computation of cortical thickness maps. It is hypothesized that a multivariate analysis of these two imaging modalities may provide insight into the alteration of cortical and WM circuits following TBI. To address this hypothesis, we employ a novel multivariate technique, sparse canonical correlation analysis (SCCA), to quantify traumatically induced disruption of WM and cortical networks. This approach increases detection power over traditional univariate models. Our cohort includes 17 controls and 16 patients with TBI (age and gender matched). Each patient had a history of non-penetrating TBI of at least moderate severity. T1-weighted MP-RAGE and 30-direction diffusion tensor images (DTI) were collected. We measure white matter integrity via the DTI's fractional anisotropy (FA) and, separately, quantify gray matter integrity from probabilistic segmentation of the T1-weighted imaging. We factor out variation in brain shape across subjects by diffeomorphically mapping these data into a population-specific template space. These image processing steps rely on the Camino and ANTs (Advanced Normalization Tools) neuroimage analysis open source toolkits. SCCA demonstrates significant differences between the control and patient groups in both the FA (p < 0.002) and gray matter (p < 0.01) that are widespread but largely focus on thalamocortical networks related to the limbic system. Specific regional differences included the medial thalamic nuclei, hypothalamus, amygdala, hippocampus, anterior cingulate cortex, orbitofrontal cortex and fornix. Using these SCCA-identified regions, we demonstrate a strong correlation of the degree of injury in WM and GM within the patient group.

Chesnut Randall 1 Celix Juanita 1 Chaddock Kelley 1 Dikmen Sureyya 1 Lujan Sylvia 2 Petroni Gustavo 2 Pridgeon James 1 Rondina Carlos 2 Temkin Nancy 1 Barber Jason 1 Machamer Joan 1 Videtta Walter 3

1 University of Washington, Seattle, WA, USA 2 Fundación ALAS, Rosario, Santa Fe, Argentina 3 Latin American Brain Injury Consortium (LABIC), Buenos Aires, Buenos Aires, Argentina P310 Outcome From Severe Traumatic Brain in Latin America: Results from the Latin American Pilot Traumatic Coma Databank

PURPOSE: The outcome from severe Traumatic Brain Injury (TBI) in Latin America has not been previously described using a prospective multicentre, multinational study approach.

METHODS: We have constructed a pilot Traumatic Coma Data Bank (TCDB) from eight centres in five countries in South America in order to better understand this population and as a model of low- and middle-income countries (LMICs). Between 2008 and 2010, we prospectively collected screening admission data on 748 patients and complete datasets from prehospital through 6 month Extended Glasgow Outcome Scale (GOS-E) on 404 patients presenting with a Glasgow Coma Scale (GCS) scale score ≤ 8 or deteriorating to that level within 48 hours.

RESULTS: Early (14 day) mortality is remarkably similar to modern outcomes in high-income countries (HICs), despite resource limitations including very rare use of ICP monitoring. However, unique to this population, there is a “delayed” mortality, representing ca. 30% of the deaths, that brings the six-month figure (31%) to approximately those of the middle to late 1980's in the US TCDB. There was a high proportion of diffuse injury (DI) patterns on admission CT (30% DI III, 6% DI IV), despite that 36% of total injuries were largely unhelmeted motorcyclists. Outcome was distinctly bimodal, with a 31% mortality and 46% favourable (GOS-E 5-8) outcomes. We also discovered a previously undescribed but common (22%) group of sTBI patients whose ICU admission is delayed 24 + hours or eliminated due to ICU bed unavailability.

CONCLUSIONS: Outcome from sTBI in LMICs appears sufficiently dissimilar to that in HICs to render questionable any extrapolation of epidemiological or treatment studies from HICs to LMICs.

Supported by RO1NS058302.

Petroni Gustavo 1 Perel P 1 Rondina Carlos 2 Videtta Walter 1 Lujan Silvia 1 Temkin Nancy 3 Dikmen Sureyya 3 Pridgeon Jim 3 Celix Juanita 3 Barber Jason 3 Chaddock Kelley 3 Chesnut Randall 3

1 Latin American Brain Injury Consortium, Buenos Aires, Buenos Aires, Argentina 2 Fundacian ALAS, Rosario, Santa Fe, Argentina 3 Universit of Washington, Seattle, WA, USA P311 Prognosis in Severe Traumatic Brain Injury Cross Validation of Predictive Models using a Latin American Data Base

PURPOSE: South America and the Caribbean region have some of the highest rates of traumatic brain injury (TBI) in the world. Unfortunately, little data is available about the burden of these injuries. Although early, accurate prognosis is crucial to the patient and family, there is no widely accepted and used predictive model worldwide, largely due to the lack of external validation.

The objective of this work is to test the performance of two previously-published prognostic models developed with patients from low- and middle-income countries (LMIC) using a current prospective cohort of severe traumatic brain injured (sTBI) patients in five Latin-American countries.

METHODS: We assessed the external validity of 8 published models generated using a single center Argentinean TBI database and the CRASH sample in a new group of 404 subjects from Latin America. Both models included age, admission GCS, pupillary response and CT findings. The Argentinean model also listed presence of hypotension, and the CRASH model presence of major extra-cranial injury. Fourteen day and 6 month mortality and 6 month unfavorable outcome (GOS 1-3) were the dependent variables.

RESULTS: R² shows the strength association of the prediction and the outcome. The C-statistic (area under the ROC curve) is presented for each model to assess predictability and the Hosmer-Lemeshow test and curves are presented to evaluate calibration. The applicability of the available LMIC sTBI prognostic models to the Latin American population is discussed.

CONCLUSION: This is the first work that assesses the external validity of the previously developed models to predict outcome from TBI in LMIC.

Supported by RO1NS058302.

Celix Juanita 1 Dikmen Sureyya 1 Lujan Silvia 2 Petroni Gustavo 2 Pridgeon Jim 1 Rondina Carlos 3 Temkin Nancy 1 Barber Jason 1 Machamer Joan 1 Chaddock Kelley 1 Videtta Walter 2 Chesnut Randall 1

1 University of Washington, Seattle, WA, USA 2 Latin American Brain Injury Consortium, Buenos Aires, Buenos Aires, Argentina 3 Fundacion ALAS, Rosario, Santa Fe, Argentina P312 The Association Between ICP Monitoring and Resource Level and Outcome from Severe Traumatic Brain Injury in Latin America

PURPOSE: In many high-income countries (HICs), the use of invasive intracranial pressure (ICP) monitoring is common in severe traumatic brain injury (sTBI) patients. In most low- and middle-income countries (LMICs) there is unequal access to acute medical care and variable availability and utilization of medical resources to treat TBI, unlike HICs. TBI management guidelines developed in resource-rich settings may not be applicable in resource-limited settings. This study evaluates the association between resource availability and outcomes from sTBI in LMICs in South America.

METHODS: We conducted a prospective observational study of 228 patients with severe non-penetrating TBI admitted to one of four metropolitan hospitals in Argentina, Brazil, Ecuador, and Colombia from August 2008 through December 2009. Study hospitals were dichotomized to low (2) or high (2) resource based on the availability of invasive ICP monitoring. Demographic, injury, clinical, and outcome data were compared.

RESULTS: Transportation to the hospital via ambulance occurred more frequently at high- than low-resource hospitals (95% vs. 64%). Between the two high resource hospitals, there were significant differences in the use of ICP monitors (74% vs. 14%). The lack of availability of an intensive care unit (ICU) bed was the primary determinant preventing ICP monitoring in the low utilization hospital. Based on this finding, outcomes were compared in patients with and without ICP monitoring as well as by level of resources. Functional outcome assessed by the 6 month Extended Glasgow Outcome Scale (GOS-E) differed by both ICP monitoring and by resource level.

CONCLUSION: There are associations between outcomes from sTBI in Latin America and both the use of ICP monitoring and level of resources; however, one must be concerned about likely confounding. We are optimistic that the ongoing ICP randomized controlled trial in Latin America will help resolve such confounding.

Supported by RO1NS058302.

Videtta Walter 2 Rondina Carlos 3 Lujan Silvia 2 Petroni Gustavo 2 Celix Juanita 1 Dkimen Sureyya 1 Barber Jason 1 Machamer Joan 1 Pridgeon James 1 Chaddock Kelley 1 Chesnut Randall 1

1 University of Washington, Seattle, WA, USA 2 Latin American Injury Brain Consortium (LAIBIC), Buenos Aires, Buenos Aires, Argentina 3 Fundacion ALAS, Rosario, Santa Fe, Argentina P313 Patterns of “ICP” Management in Severe Traumatic Brain Injury Patients in the Absence of ICP Monitoring in Latin America

PURPOSE: Management of severe Traumatic Brain Injury (sTBI) patients in Latin America appears to involve intracranial pressure (ICP) monitoring in only a small subset of patients. Preliminary analysis of our observational study in five counties in South America suggests that early (14 day; “ICU”) mortality from sTBI in Latin America is not markedly different from that published from recent studies in high-income countries (HICs). Methods of management of patients with sTBI without ICP monitoring have not been described in the modern era.

METHODS: The Latin American Pilot Traumatic Coma Data Bank (LAPTCDB) prospectively collected information on 228 non-penetrating sTBIs in teenagers and adults at 8 centers in 5 low- and middle-income countries (LMICs) in South America. In parallel with the hourly collection of general ICU data, the Therapeutic Intensity Level (TIL) was measured, including any treatments directed at intracranial hypertension based on CT data and the neurologic exam (in the absence of monitoring). An experienced Neurointensivist evaluated the data on the management of individual patients in terms of the use of individual agents, their patterns, and interactions.

RESULTS: Despite the lack of protocols and the involvement of separate centres and numerous individual physicians, there appear to be identifiable patterns of management of “ICP” in the absence of formal monitoring. We will present and discuss these patterns in the context of the management of sTBI as likely occurs in the majourity of LMICs.

CONCLUSIONS: This is the first description of what appear to be general management approaches currently used for managing “ICP” in sTBI patients in the absence of ICP monitoring.

Supported by RO1NS058302.

Temkin Nancy 1 Lujan Silvia 2 Petroni Gustavo 2 Rondina Carlos 2 Videtta Walter 3 Dikmen Sureyya 1 Barber Jason 1 Machamer Joan 1 Celix Juanita 1 Pridgeon James 1 Chaddock Kelley 1 Chesnut Randall 1

1 University of Washington, Seattle, WA, USA 2 Fundación ALAS, Rosario, Santa Fe, Argentina 3 LABIC, Buenos Aires, BA, Argentina P314 Latin American Pilot Traumatic Coma Data Bank: Baseline Characteristics

PURPOSE: The South American and Caribbean region has one of the highest rates of traumatic brain injuries (TBIs) in the world. Despite this, there is little data about the characteristics of these injuries and the people injured.

METHODS: The Latin American Pilot Traumatic Coma Data Bank (LAPTCDB) prospectively collected information on 748 non-penetrating TBIs in teenagers and adults at 8 centers in 5 low- and middle-income countries (LMICs) in South America. To be included, patients had to arrive at the study hospital within 24 hours of injury and have a Glasgow Coma Scale score (GCS) of 3-8 at admission or deteriorate to that level within 48 hours. GCS motor had to be 5 or less if the patient was intubated.

RESULTS: Patients were entered between 2008 and 2010. Half of the participants were in their teens or 20s; beyond the 20s, the number of cases decreased with each decade. Less than 10% were over 60. Unlike in high income countries, motorcycle crashes were the most common cause of injury; assaults were rare. For patients arriving directly, half arrived at the study hospital within an hour; 2/3 arrived by ambulance. Information about pre-hospital treatment was rarely available. Most patients arrived without intubation or sedation. Hypotension on admission was documented in only about 25% of cases, although this underestimates the overall occurrence of this secondary insult as pre-hospital blood pressure was rarely reported and blood pressure was recorded hourly or less in the emergency department (ED). About one third of subjects had no assessment of hypoxia through the time of resuscitation. Over 1/3 of participants had CT with Marshall classification DI-III.

CONLUSIONS: TBI in LMICs has many different characteristics from that in high income countries and deserves further study.

Supported by NIH grant R01NS058302.

Anderson Gail 1 Farin Fred 1 Bammler Theo 1 Beyer Richard 1 Kantor Eric 1 Hoane Michael 2

1 University of Washington, Seattle, WA, USA 2 Southern Illinois University, Carbondale, IL, USA P315 Gene Expression in Brain and Liver After Nicotinamide Treatment Following Traumatic Brain Injury

OBJECTIVE: To examine the effects of nicotinamide (NAM) administration (75 mg/kg s.c. bolus plus 12 mg/kg/day infusions) for 72 hours starting at 4 hr post-TBI on brain and liver gene expression.

METHOD: 30 rats received moderate Cortical Contusion injury (CCI), with another 5 rats serving as intact sham controls. Fresh brain samples from the injured cortex and liver samples were harvested in 5 animals in each treatment arm at 24 hr, 72 hr and 7 days post-CCI. Samples were removed with a tissue punch, were snap frozen and stored at − 80°C. RNA was isolated from the tissue punches. RNA samples passed stringent quality control were processed and submitted to microarray analysis using Affymetrix GeneChip Rat Gene 1.0 ST arrays. Microarray data analysis was performed with the Bioconductor packages limma and topGO.

RESULTS AND CONCLUSION: Serum NAM concentrations obtained from tail snips during NAM treatment were 55 ± 11 μg/mL (mean ± sd). In brain, the number of differentially expressed genes (>1.5-fold up or down, p < 0.05) for NAM compared to saline at 24 hr, 72 hr and 7 days post-CCI were 150, 119 and 5, respectively. In the liver, the number of differentially expressed genes for the NAM compared to saline at 24 hr, 72 hr and 7 days post-CCI were 1318, 1683 and 269, respectively. NAM resulted in a statistically significant (p < 0.05) enrichment of a large variety of Biological Process Gene Ontology (GO) categories. In brain, GO categories included pathways involving immune and inflammatory response, oxygen transport, ion transport, GABA and glutamate signaling pathways. In liver, the GO categories included inflammation and immune pathways, apoptosis, oxygen and reactive oxygen species metabolism, lipid oxidation and others. The extent and duration of effect on gene expression was significantly greater in the liver than in the brain.

Marchand-Leroux Catherine Girgis Haymen Pamier Bruno Croci Nicole Soustrat Mathieu Michel Plotkine Unuversité Paris Descartes, Department of Diagnostic and Therapeutic Lab Pharmacology, Paris, France

P316 Indomethacin Promotes Neurological Recovery Independently of Cerebral ŒDema After Closed Head Injury in Mice

The role of cyclooxygenase (COX) family of enzymes in traumatic brain injury (TBI) is still controversial. In rat models of TBI, the pharmacological inhibition of the inflammation-induced isoform, the COX-2, seems to be neuroprotective. However, the genetic disruption of either COX-2 or COX-1,the constitutive isoform, has no effect in mice.Therefore, we investigated the effect of different COX inhibitors on the TBI-induced neurological deficit and brain œdema. Closed head injury model was realised in anaesthetised Swiss male mice using a weight-drop device. COX-2 protein expression was evaluated by Western Blot and brain 6-keto prostaglandin F1alpha (6-Keto PGF_1α), was determined by Enzyme immunoassay. A single dose of a preferential COX-2 inhibitor, meloxicam (2mg/kg) was administered intraperitoneally 10minutes after TBI. Another group received 3 doses of a non selective COX inhibitor, indomethacin (5 mg/kg) at 10 minutes, 6 and 12 hours after trauma. Neurological deficit was evaluated by an 8-point score at 6 and 24 hours. Brain œdema was calculated by wet weight/dry weight technique at the same time-points. COX-2 protein was significantly increased at 6 and 12 hours after injury. This was associated with an increased production of 6-Keto PGF1α which was partially or completely suppressed by meloxicam and indomethacin respectively. While meloxicam had no effect on neurological deficit, indomethacin significantly promoted neurological recovery at 6 and 24 hours. However, no COX inhibitor modified brain œdema. Our results show that COX-2 is not implicated in post-traumatic consequences. On the other hand, we demonstrate for the first time in a mouse model of TBI that non selective COX inhibition promotes neurological recovery independently of an eventual anti-œdematous effect.

Brophy Gretchen 1 Mondello Stefania 2 Papa Linda 3 Robicsek Steve 2 Gabrielli Andrea 2 Tepas Joseph III 4 Buki Andras 5 Schmid Kara 7 Robertson Claudia 6 Tortella Frank 7 Hayes Ron 8 Wang Kevin 8

1 Virginia Commonwealth University, Richmond, VA, USA 2 University of Florida, Gainesville, FL, USA 3 Orlando Regional Medical Center, Orlanado, FL, USA 4 University of Florida, Jacksonville, FL, USA 5 University of Pécs, Pécs, Hungary 6 Baylor College of Medicine, Houston, TX, USA 7 Walter Reed Army Institute of Research, Silver Springs, MD, USA 8 Banyan Biomarkers Inc., Alachua, FL, USA P317 Quantitative Cerebrospinal Fluid Biokinetic Parameters of Alpha II-Spectrin Breakdown Products in Severe Traumatic Brain Injury Patients

BACKGROUND: This study describes the biokinetic characteristics of alpha II-spectrin breakdown products (SBDP145 produced by calpain, and SBDP120 produced by caspase-3) in cerebrospinal fluid (CSF) in severe traumatic brain injury (TBI) patients.

MATERIALS AND METHODS: This study included 33 adult patients with severe TBI (Glasgow Coma Scale [GCS] score ≤ 8) who had CSF SBDP145/120 concentrations and clinical data available for analysis. Patients requiring CSF drainage for other medical reasons served as controls. CSF samples from time points every 6 h after injury for a maximum of 7 days were assessed using novel quantitative fragment-specific ELISAs for SBDPs. Biokinetic parameters evaluated include area under the curve (AUC), maximum concentrations (Cmax), time to maximum concentration (Tmax), mean residence time (MRT) and half-life (t ½ ).

RESULTS: The biokinetic profiles for CSF SBDP145 and SBDP120 are described, respectively: AUC 1243 vs 660 ng/ml*hr; Cmax 26 vs 12 ng/ml; Tmax 34 vs 94 hrs; and MRT 58 vs 83 hrs; and half -life 9.6 vs 12 hrs. The median concentration of SBDP145 and SBDP120 in control patients (n = 20) was 0 ng/ml, and this was significantly different than the Cmax for these biomarkers in the CSF (p < 0.001). These trends were similar to our previous study looking at SBDP145 and SBDP120 that were analyzed by Western Blot.

CONCLUSION: These quantitative biokinetic parameters suggest that SBDP145, a potential TBI biomarker associated with calpain activity, is more predominant in the acute period after TBI as compared to SBDP120, associated with caspase-3 activity. The maximum concentrations of these products of proteolysis were significantly different than control patients. These data confirm our previous biokinetic studies of alpha II-spectrin breakdown products analyzed by Western Blot.

Doperalski Adele Ottens Andrew Phillips Linda Virginia Commonwealth University, Richmond, VA, USA

P318 Glial and Extracellular Matrix Response in the Internal Capsule Following Traumatic Brain Injury

Axonal damage is a major feature of traumatic brain injury (TBI). Injured axons interact with reactive glia and the extracellular matrix (ECM) to achieve recovery. The principle cellular mediators of recovery are astrocytes, oligodendrocytes and microglia, secreting repair promoting molecules and inhibitory proteins, such as chondroitin sulfated proteoglycans (CSPGs), into the ECM. Prior TBI studies of corpus callosum document time dependent alterations in reactive glia and their expression of ECM molecules critical to recovery. Differential myelinated and unmyelinated fiber recovery was correlated with distinct glial and matrix change. Because TBI affects multiple fiber tracts throughout the brain, it is important to understand glial and ECM response in pathways with different axon composition. The internal capsule is a tract composed principally of large caliber, myelinated fibers with multiple targets in the brain, brainstem, and spinal cord. Using the central fluid percussion model of diffuse TBI, we examined glial cell response in the internal capsule at 6 hrs, 1 and 3 days post-injury using confocal immunohistochemistry with antibodies specific for astrocytes, oligodendrocytes, or microglia. To explore ECM mediation of axonal recovery, we assessed CSPG co-localization within each glial type. Altered microglial and astrocyte morphology was observed at 3 d. Microglia were reactive, with elongated somas and short, thick processes. Hypertrophic astrocytic processes had increased filament staining. In contrast, oligodendrocyte response appeared at 1 d, with cell body redistribution along axon bundles. CSPGs co-localized within reactive astrocytes. Preliminary Western blot analysis of internal capsule αII spectrin lysis revealed increase of calpain derived fragments at 3d. Together, these data support complex glial reactivity in the internal capsule within 3 d after TBI, a response which correlates with significant αII spectrin breakdown. Further, CSPG localization in reactive astrocytes links ECM cues with the process of axonal recovery.

Supported by: NIH-NS4437, NS57758, NS55102

Reeves Thomas Colley Beverly Smith Terry Lee Nancy Phillips Linda Virginia Commonwealth University, Richmond, VA, USA

P319 Regional Variations in Axonal Populations Influence Post-Traumatic Functional Deficits

Recent findings suggest that axonal pathology, following traumatic brain injury (TBI), is not uniformly expressed among all axons. Instead, TBI induces distinct functional and morphological changes in subpopulations of axons. For example, myelination status strongly determines the course of posttraumatic fiber pathology. Prior results from our laboratories indicate that unmyelinated axons respond to TBI, and to neuroprotective drugs, in a manner distinct from myelinated fibers. Unmyelinated axons are the most numerous fiber type in the mammalian brain, however, the ratio of unmyelinated to myelinated axons is variable among white matter regions. To explore how regional variations in the density of these fiber subtypes determines axonal pathophysiology, we have examined ultrastructural and electrophysiological changes at three loci within the corpus callosum after TBI. Adult rats underwent moderate midline fluid percussion TBI (N = 19), or a sham injury (N = 21). At 1 day postinjury, ultrastructural stereology was used to quantify the density and cross-sectional area of axons at three callosal subregions (genu, midportion, splenium) and, in subgroups of rats, evoked compound action potentials (CAPs) were acquired at the same callosal subregions. In sham rats the proportion of unmyelinated axons varied in a rostrocaudal gradient, from 73% of total genu fibers to 85% of total splenium fibers. TBI decreased cross-sectional area of unmyelinated axons (-12%), but not myelinated. TBI did not significantly alter density of either fiber type. TBI-induced changes in CAPs were strongly determined by subregion. With the genu, deficits were confined to suppression (-30%) of unmyelinated CAPs. Mid-callosal recordings showed abnormalities in both fiber types, whereas splenium dysfunction was confined mainly to myelinated CAPs (-55%). These results suggest an inverse relationship between subregional density of fiber subtype and the severity of post-TBI electrophysiological deficits. Moreover, they underscore the importance of distinct fiber populations in models of axonal pathology.

Supported by: NIH NS56247, NS57758.

Greer John E Povlishock John T Jacobs Kimberle M Virginia Commonwealth University, Richmond, VA, USA

P320 Altered Physiology of Axotomized and Non-Axotomized, Intact Pyramidal Neurons in a Model of Diffuse Axonal Injury

PURPOSE: Mild traumatic brain injury (TBI) induced in Thy-1-YFP-H mice results in diffuse axonal injury in which axotomized layer V pyramidal neurons are easily distinguished from adjacent intact neurons by the presence of axonal swellings (Greer et al 2011). We hypothesized that the associated structural changes would produce functional alterations in axotomized neurons. Unknown in models of TBI is whether the adjacent non-axotomized intact neurons are functionally affected.

METHODS: We have used standard methods for in vitro slice preparation and whole cell patch clamp (George 2011) to analyze the intrinsic membrane properties of YFP-labeled layer V pyramidal neurons 1 day after mild central fluid percussion injury in adult mice. The response to a series of current steps was measured under current clamp. Only the responses of the regular spiking neuronal type were analyzed here.

SUMMARY: Measures of resting potential, membrane time constant and input resistance in neurons from injured brains were not different from controls, suggesting that injured neurons survive with some normal functions. In comparison to the controls, axotomized neurons had an increased action potential (AP) amplitude, a decreased duration of the after hyperpolarization (ahp), and a decrease in the late adaptation that occurred during trains of 6 or more APs. Intact neurons from injured brains manifested a significantly lowered rheobase, as well as a slower decaying AP and a decreased duration of the ahp.

CONCLUSION: These results demonstrate that both axotomized and intact neurons within the injured brain have altered intrinsic properties. The increase in AP amplitude likely reflects a change in K⁺ rather than Na⁺ channels since the rate of AP rise was unaffected. Together, these alterations may be due to multiple potassium channelopathies, increasing neuronal excitability of both the axotomized and intact neurons.

Supported by: HD055813.

Wang Jiaqiong Povlishock John Virginia Commonwealth University, Richmond, VA, USA

P321 Axonal Dieback Following Traumatic Brain Injury Results from Local Glial and Macrophage Activation Rather than Retinal Ganglion Cell Death

PURPOSE: Traumatic brain injury (TBI) is a major health problem with most of its morbidity associated with traumatic axonal injury (TAI). Previously, we have developed a model of TAI in the optic nerve, wherein the TAI evolved rapidly with dramatic anterograde and retrograde axonal dieback. Given this dramatic damage and its rapid progression, we questioned whether this was the result of local inflammation and/or concomitant cell death within the retina.

METHODS: YFP-16 transgenic mice were subjected to mild central fluid percussion injury at 1.40 ± 0.05atm and allowed to survive from 24h to 28d post TBI. Optic nerve frozen sections were reacted with antibodies targeting astrocytes (GFAP) and microglia/macrophages (Iba1). Retina frozen sections were immunolabeled with antibodies to the cleaved caspase-3 as well as TUNEL approaches interfaced with electron microscopy.

RESULTS: Both reactive astrocytes and numerous activated microglia/macrophages were recognized in the optic nerve at 24h and 48h post TBI, with many of the activated microglia/macrophage lying adjacent to swollen axonal segments in the process of dieback. YFP positive cells in the retinal ganglion cell layer revealed no overt loss at 2d, 7d and 14d post TBI. Further, they were not labeled by the cleaved caspase-3 antibody. Overall, the optic nerve showed improved structural detail and reorganization at 28d post TBI in comparison to that seen at 24h and 48h post TBI.

CONCLUSION: Collectively, our results suggest that astrocyte and microglia/macrophage activation are associated with the axonal dieback identified in the pathogenesis of TAI within the optic nerve. The absence of concomitant retinal degeneration and/or ganglion cell death argues that they are not directly involved within the dieback process. The implications of these findings for our understanding of TAI will be discussed, together with a consideration of their relevance to other optic nerve injury models.

(Supported by HD055813 and NS047463)

Chan Julie Phillips Linda Virginia Commonwealth University, Richmond, VA, USA

P322 Osteopontin: An Acute Inflammatory Mediator of Successful Synaptic Recovery Following Traumatic Brain Injury

Osteopontin (OPN) is a pleiotropic inflammatory cytokine documented to modulate CNS growth and plasticity. Following traumatic brain injury (TBI), it is likely a player during reactive synaptogenesis as a substrate of acutely activated matrix metalloproteinases, and a recruiter of resident microglia for debris clearance. Our previous studies following unilateral entorhinal cortex lesion (UEC) showed elevated OPN transcript and protein at 2-7d post-injury. Questions regarding OPN's role during acute TBI inflammation and its contribution to successful synaptogenesis are addressed here at more acute (1d) post-injury intervals. Further, we tested OPN response following maladaptive synaptic plasticity in combined midline fluid percussion injury and bilateral entorhinal cortex lesion (TBI + BEC). By contrasting OPN profile during acute inflammation, we can identify alterations in expression which might correlate with effective synaptic recovery. Rats subjected to UEC, TBI + BEC, or sham injury were evaluated for OPN at 1 or 2d post-injury via Western blot or confocal immunohistochemistry utilizing antibodies to OPN and either microglia or astroglia. At 1d post-UEC, we observed a robust OPN increase matching the response at 2d, suggesting rapid and intense OPN activation during the acute inflammatory response. Following maladaptive TBI + BEC, OPN increased at 1 and 2d, however expression was significantly reduced relative to adaptive recovery. Finally, confocal imaging at 1d showed OPN localization in reactive microglia and astrocytes, and that microglia were diffusely distributed within the deafferented zone relative to distinct laminar organization at 2d. Together, these results support a significant OPN role in acute inflammatory response to TBI. Further, differences in OPN induction between adaptive and maladaptive recovery suggest immune-related regulation of successful synaptic repair. This process may involve OPN effects on microglial activation and migration, facilitating efficient debris clearance to promote axonal sprouting. Additional studies manipulating the immune response are underway to confirm potential OPN roles during synaptic recovery.

Support: NIH-NS4437, NS57758.

Lafrenaye Audrey D McGinn Melissa J Henderson Scott C Povlishock John T Virginia Commonwealth University, Richmond, VA, USA

P323 Increased Inracranial Pressure is Associated with Neuronal Somatic Membrane Perturbation Following Diffuse Traumatic Brain Injury in the Rat

Increased intracranial pressure (ICP) associated with traumatic brain injury (TBI) is linked to long-term morbidity and negative outcomes. Although progress has been made in understanding the complex pathobiology associated with TBI, questions remain regarding the specific damaging consequences of elevated ICP independent of its impact on cerebral perfusion. The current study examined the effect of increased ICP on the magnitude of neuronal somatic membrane perturbation following diffuse TBI. To evaluate membrane perturbation in a temporal fashion different fluorescently labeled dextrans were infused intraventricularly allowing dissemination into the extracellular space prior to and four hours following moderate central fluid percussion injury in Fischer rats. The number of NeuN positive neurons displaying mechanoporation demonstrated by the uptake of either dextran was evaluated six hours following injury. Intraventricular measurements of ICP both pre and post injury revealed the existence of two distinct responses to injury. One population revealed a transient increase in ICP following injury that returned to baseline levels acutely. In contrast, approximately half of the animals maintained a consistently elevated ICP (≥20mmHg). Rigorous systemic physiological assessments were conducted, including evaluation of cerebral perfusion pressure, and strict inclusion criteria were implemented to limit variability between the two animal groups showing differences in ICP. In animals exhibiting consistently elevated ICP following injury, the number of cortical neurons displaying membrane perturbation was increased compared to that observed for animals exhibiting only transient increases in ICP. Strikingly, the number of neurons exhibiting delayed membrane perturbation, assessed via the uptake of the post injury administered dextran, doubled in animals with persistently elevated ICP. These findings indicate that a persistent increase in ICP post TBI directly exacerbates neuronal plasmalemmal perturbation independent of attenuated cerebral perfusion pressure, with the caveat that this membrane perturbation could precipitate neuronal impairment and/or death.

This work was supported by NIH grant NS045824.

Fujita Motoki Wei Enoch Povlishock John Department of Anatomy & Neurobiology, Virginia Commonwealth University, Richmond, VA, USA

P324 Effects of Hypothermia on Cerebral Autoregulation in Two Rodent Models of Traumatic Brain Injury

BACKGROUND: It is well known that cerebral autoregulation is frequently impaired following traumatic brain injury (TBI). To explore the potential beneficial effects of hypothermia on cerebral autoregulation, we examined using cranial windows, the autoregulatory response in the pial arteriolar bed in rats subjected to either impact acceleration injury (IAI) or lateral fluid percussion injury (LFPI).

METHODS: Male adult Sprague-Dawley rats were randomly assigned to 5 groups; sham, IAI with normothermia, IAI with hypothermia, LFPI with normothermia, and LFPI with hypothermia. IAI was produced by a weight of 450 g dropped from a height of 2 m on the metal helmet. LFPI was generated at 2.0 atm pressure pulse. Hypothermia was initiated one hour post injury and maintained at 33°C for 60 minutes, followed by rewarming over a span of 90 minutes. Pial arteriolar diameter was measured 4 hours post injury following the sequential reduction of mean arterial pressure (MAP) to 100, 80, 60, and 40 mmHg.

RESULTS: Pial arteriolar diameter in sham rats dilated against progressive reductions of MAP. In animals subjected to IAI, vascular dilation to hypotension was reduced by approximately 50%. However, this reduced reactivity to hypotension was fully restored with delayed hypothermia. In contrast, LFPI completely blocked the pial vascular response to hypotension, even in the delayed hypothermia group.

CONCLUSIONS: TBI impaired the ability of cerebral arterioles to dilate in the face of decreasing blood pressure following both IAI and LFPI models. Further, these results suggest that LFPI generated more severe brain vascular damage than that seen with IAI. The impaired autoregulatory response in the IAI model could be restored with delayed hypothermia. In contrast, the severity of LFPI insult most likely overshadowed the beneficial effect of hypothermia.

Churn Severn Campbell John Virginia Commonwealth University, Richmond, VA, USA

P325 Inhibition of Post-Traumatic Epileptogenesis by an Acute Administration of Tacrolimus

Every 16 minutes in the USA, someone suffers a traumatic brain injury which leads to lifelong recurrent seizures, an acquired form of epilepsy known as post-traumatic epilepsy (PTE). PTE is typically resistant to anti-seizure medications, and no therapy has yet been shown to prevent PTE. Designing such a therapy will require a better understanding of the neurobiological changes which lead to epilepsy. Previous research has shown that brain injury may lead to epilepsy through pathological activation of a calcium-sensitive phosphatase, calcineurin (Campbell et al., J Neurotrauma, 2011a,b). Therefore, the present study characterized the effect of acute, post-injury inhibition of calcineurin on a rat model of PTE. Adult male Sprague-Dawley rats (n = 17) received a moderate traumatic brain injury by lateral fluid percussion (fluid percussion pressure, 2.6-2.8atm), or were kept as age-matched, uninjured controls (n = 4). One hour after TBI, rats were treated with a calcineurin inhibitor, Tacrolimus (5mg/kg; i.p.; n = 8 rats), or vehicle (70% ethanol; n = 2 rats), or received no injection (n = 7 rats). Six months later, injured rats and uninjured controls were monitored with video-electrocorticography (video-ECoG; > 50 hours of recording per rat, over at least 5 days). Video-ECoG monitoring revealed spontaneous, recurrent seizures in all post-TBI rats, consistent with previous reports by other labs. However, no seizures were observed in age-matched, uninjured controls. In rats subjected to TBI but no drug, seizures occurred frequently (6.42 ± 1.36 seizures/hour), had an average duration of 13 ± 0.4 seconds (range, 3 - 51 sec), and coincided with behavioral arrest, consistent with complex-partial type seizures. In contrast, in rats subjected to TBI followed by Tacrolimus, preliminary data indicate a significant reduction in both seizure frequency (1.42 seizures/hour) and duration (5 ± 0.5 sec). To our knowledge, this is the first demonstration of an acute treatment which ameliorates post-traumatic epilepsy in an animal model.

Cunningham Tracy L. Shear Deborah A. Lu Xi-Chun May Mountney Andrea Long Melissa van der Merwe Christopher Tortella Frank Walter Reed Army Institute of Research, Silver Spring, Maryland, USA

P326 The Temporal Gradient Profile of Blood-Brain Barrier Disruption Following Penetrating Ballistic-Like Brain Injury using Large and Small Molecule Tracers

Breakdown of the blood-brain barrier (BBB) is a hallmark of severe traumatic brain injury (TBI) and is associated with infiltration of peripheral fluid and leukocytes into the central nervous system. Previously, we reported that leukocyte infiltration peaks at 24h - 72h post injury following penetrating ballistic-like brain injury (PBBI) in rats. More recently, we have identified a biphasic pattern of BBB disruption in this brain injury model (Phase I: < 24h, Phase II: 48h - 72h) using Evan's blue (EB; 65 kDA) extravasation. However, the temporal profile of BBB disruption following PBBI needs to be more fully elucidated in order to better understand how BBB breakdown and secondary neuroinflammatory events interact. The present study was designed to further investigate the temporal gradient of BBB permeability following PBBI using small and large molecule tracers, which may provide a foundation for the subsequent study of associated neuroinflammation. For this purpose, adult male rats were anesthetized and received a 10%, right frontal PBBI injury. Horseradish peroxidase (HRP; 44 kDA) or biotin-dextran amine (BDA; 3 kDA) were injected intravenously and allowed to circulate for 0.5h prior to perfusion at 4h, 24h, 48h, 72h, and 7 days post-PBBI. Experimental groups included PBBI + HRP and PBBI + BDA, with appropriate injured and non-injured control groups (n = 6/group/time-point). Significant BDA and HRP extravasation, indicative of increased BBB permeability, was evident at 4h, 24h, 48h, and 72h post-PBBI. Compared to previous EB extravasation results, which suggested that BBB disruption resolved by 24h post-injury but then re-appeared from 48h – 72h, these results suggest that for smaller molecules the BBB opens and closes in a gradient fashion following PBBI. The next phase of studies will be aimed at evaluating the spatial-temporal relationship between injury-induced BBB disruption and secondary neuroinflammatory events (i.e. peripheral leukocyte infiltration and native immune cell activation) using the same time frame.

Wang Ying Wei Yanling Oguntayo Samuel Arun Peethambaran Biggemann Lionel Valiyaveettil Manojkumar Song Jian Nambiar Madhusoodana Walter Reed Army Institute of Research, Silver Spring, MD, USA

P327 Repeated Blast-Induced Neurobiological Effects in Mice

OBJECTIVE: The clear spectrum of biochemical and molecular mechanisms involved in blast induced traumatic brain injury (bTBI) and the subsequent pathological processes and behavioral abnormalities are not well understood. We developed a pre-clinical model of repeated bTBI in mice to unravel the molecular mechanisms involved in the pathophysiology of blast-induced brain injury.

METHODS: Mouse models of single and repeated blast exposures with 10-30 min intervals were developed using a shock tube. Isoflurane anesthetized restrained C57BL/6J mice were exposed to different levels of blast overpressure (BOP) and the animals were allowed to recover for multiple time points. Biochemical and molecular changes in the brain of blast exposed animals were quantified by using histopathology, immunostaining, Western blotting and gene expression analysis. Rota-rod was used for behavioral study.

RESULTS: A significant decrease in body weight was observed in mice exposed to single or repeated blast and was BOP dependent. Animals exposed to repeated blast showed significant increase in brain edema. Cerebellum and cortex showed significant axonal degeneration in repeated BOP mice. Increased neuronal death was observed in dorsal piriform cortex of repeated blast exposed mice. Significant neurobiological effects with decreased adenosine triphosphate levels and increased reactive oxygen species formation in the cortex were observed after repeated BOP. Glial fibrillary acidic protein expression showed a biphasic change after repeated blast exposure. Expression of beta amyloid precursor protein and protein kinase Akt3 was up-regulated in the cortex of blast exposed animals. Repeated blast exposures also resulted in reduced behavioral performance in rota-rod test.

CONCLUSION: These data suggest that repeated BOP exposures leads to more severe neurobiological effects compared to single blast exposure. Biochemical and molecular analysis revealed significant changes in the brain after repeated blast exposure. The repeated blast exposure model is valuable to evaluate the protective efficacy of potential therapeutics for blast induced neurotrauma.

Chen Zhiyong Liao Zhilin Leung LaiYee Mountney Andrea Yao Changping Yang Weihong Lu Xi-Chun May Dave Jitendra Wei Guo Deng-Bryant Ying Schmid Kara Shear Deborah Tortella Frank Walter Reed Army Institute of Research, Silver Spring, MD, USA

P328 The Wrair Model of Projectile Concussive Impact (PCI) Injury: II. In Vivo Modeling Acros Injury Severities

We have recently developed a device capable of producing a non-invasive projectile concussive impact (PCI) head injury in rodents. The primary aim of this study was to evaluate the PCI across a range of injury severity. In this model anesthetized rats are placed in a supine position on a raised platform located above the projectile such that the surface of the rat's head can be impacted at different distances and/or angles. A tightly sealed microcentrifuge tube packed with dry ice is placed beneath the dorsal surface of a rat's head. Upon heating, rapid sublimation of dry ice produces an eruptive force launching an intact ‘projectile’ (i.e. tube cap) at the rat's head. Using energy forces capable of inducing a severe injury causes intracranial hemorrhage, neural progenitor cell activation, GFAP activation, activation of heat shock protein-27, axonal degeneration (silver staining), and increases in blood-brain barrier permeability (albumin staining). However, decreasing the force of projectile impact (by decreasing the energy or using a protective helmet), induces concussive events ranging from complicated mTBI (with no global cellular damage except at the foci of impact) to mTBI without any evidence of intracranial hemorrhage or gross morphological damage. Importantly, we have detected protein changes and behavioral abnormalities following mild PCI-induced injury where 1) we have measured GFAP upregulation in cortical tissue (bilateral PCI) and in hippocampal regions (lateral PCI), and 2) detected elevated levels of ubiquitin carboxyl-terminal hydrolase L1 in cortical tissue and plasma. Critically, we have also measured subtle, yet significant, motor/postural abnormalities during the acute post-injury period (1h - 24h) in PCI-injured animals sustaining a mTBI event. Overall, these results indicate that our newly developed PCI model is capable of inducing closed-head concussive impact injury across a spectrum of injury severity. Further studies are underway to characterize the full extent of the injury.

Leung Lai Yee Chen Zhiyong Liao Zhilin Lu Xi-Chun May Dave Jitendra Wei Guo Yang Weihong Schmid Kara Shear Deborah Tortella Frank Walter Reed Army Institute of Research, Silver Spring, MD, USA

P329 The Wrair Model of Projectile Concussive Impact (PCI): I. Device Development

To study clinically relevant experimental concussion we are developing a simple, non-invasive rodent model of closed-head impact TBI triggered by a pressurized projectile. Materials required for this model include a microcentrifuge tube, a heating device and dry ice. For these studies, fixed amounts of dry ice are packed into a tube sealed with a screw cap. Upon heating, rapid sublimation of dry ice produces a build-up of compressed CO₂ that triggers an eruptive force which causes the cap to burst off the tube and launch as an intact ‘projectile’. The velocity of the projectile is 47.2 ± 2.3 m/s. To study the projectile concussive impact (PCI) injury, anesthetized rats are placed supine on a platform positioned above the projectile such that the surface of the rat's head can be impacted at different distances and/or angles by the projectile. The tube is then rapidly heated to eruption, causing a vertical release of the cap resulting in a targeted PCI injury to the rat's head. In order to control for the effects of the pressure wave generated by the energy released from the tube, a highly perforated metal plate was engineered to be positioned between the rat's head and the projectile. By varying the amount of dry ice (0.1 to 0.8 g) and the distance between the projectile and the rat skull (5.1 to 12.7 cm), the impact force (3.9 to 30.8 N) and pressure magnitude (103.4 to 310.3 kPa) can be titrated to produce concussive TBI's ranging from mild to severe. A stainless steel helmet (oval: 4.5 x 3.0 x 0.7 cm) was developed to protect the head from bruising, yet allow the brain to sustain a true PCI event. Compared to other concussion/mTBI models, this model is simple, compact, very inexpensive and suitable for high throughput rodent research.

Mountney Andrea Leung Lai Yee Chen Zhiyong Lu Xi-Chun May Liao Zhilin Yang Weihong Schmid Kara E. Shear Deborah A. Tortella Frank C. Walter Reed Army Institute of Research, Silver Spring, MD, USA

P330 The Wrair Model of Projectile Concussive Impact (PCI): III. Acute Behavioral Analysis in a Mild Brain Injury

Mild traumatic brain injury (mTBI) is a significant concern for the civilian and military populations. To date, there are few research models to examine and understand the extent of mTBI. We have recently developed a device capable of producing a non-invasive projectile concussive impact (PCI) which can be used to model mild to severe close-head concussive injury in rats. The purpose of this study was to evaluate the acute motor dysfunction following mTBI induced by closed-head PCI using a computer-assisted gait analysis system (CatWalk system, Noldus) which is able to detect subtle and dynamic changes in sensorimotor function. Anesthetized rats (n = 9/group) protected with a stainless helmet were subjected to a lateral PCI induced by a projectile ejected from rapid sublimation of dry ice at a projectile distance of 5.1cm. Sensorimotor function was assessed on the CatWalk device at 1h, 4h, and 24h post-PCI. PCI-injured rats showed neither gross brain morphology nor overt gait disturbances measured by run speed, run duration, stride length, and gait pattern. However, parameters of individual paw performance, such as intensity, contact area, interpaw support, and interpaw coordination, were significantly affected following mTBI. Overall, PCI-injured mTBI rats displayed increased surface contact area and pressure of both hind and forepaws while crossing the walkway at 1h post-injury that remained evident at 4 and 24h (p < .05) compared to sham rats. PCI-injured rats also showed an abnormal pattern of interpaw support at 1h and 4h post-PCI (p < .05) but not at 24h post. Collectively, this study describes a single PCI injury capable of producing mTBI with subtle, yet significant, sensorimotor abnormalities that can be detected during the acute post-injury period (≤24h) using the CatWalk system.

Wei Guo Yang Xiaofang Lu XI-Chun M Shear Deborah A Tortella Frank C Walter Reed Army Institue of Research, Silver Spring, MD, USA

P331 Selective Brain Cooling Attenuates the Axonal Damage Following Penetrating Ballistic-Like Brain Injury in Rats

Induced hypothermia has been reported to provide neuroprotection against traumatic brain injury (TBI). We previously reported that selective brain cooling (SBC) significantly protects against the acute injury-induced neuropathophysiology measured in a rodent model of penetrating ballistic-like brain injury (PBBI). The current study was designed to (1) characterize the spatiotemporal distribution of acute PBBI-induced axonal damage using ß-amyloid precursor protein (APP) immunostaining and (2) evaluate the effect of SBC on acute injury-induced APP pathology. APP histopathology was assessed in PBBI and sham animals at 6h, 24h, 72h, 7 and 21 days post-surgically. No APP-positive staining was detected in the brains of sham-operated control animals at any time-point. However, significant APP-positive staining was evident in PBBI-injured brains (primarily in regions surrounding the core injury site) by 6h post-PBBI. The presence of APP-positive immunostaining peaked between 24h and 72h post-injury, but was greatly diminished by 7 days post-PBBI. SBC (∼34°C for 4h), initiated immediately following PBBI in anesthetized rats, reduced the extent of injury-induced APP positive staining by 30% compared to PBBI control animals (p < .05) at 72h post-injury. In summary, compared to our previous studies where silver staining for axonal injury was not evident until approximately 7 days post-PBBI, but could be seen for several weeks post-injury, this study has identified an acute post-injury profile of axonal transport damage detected using APP immunostaining. Critically, the current results also demonstrate that SBC protects against PBBI-induced axonal damage during the acute post-injury phase thereby providing further support for SBC as a promising therapy for ameliorating the effects of severe TBI.

Dave Jitendra 1 Yao Changping 1 Ahlers Stephen 2 Stone James 3 Shaughness Michael 2 Hall Aaron 2 McCarron Richard 2 Schmid Kara 1 Tortella Frank 1

1 Walter Reed Army Institute of Research, Silver Spring, MD, USA 2 Naval Medical Research Center, Silver Spring, MD, USA 3 University of Virginia School of Medicine, Charlottesville, VA, USA P332 Serum GFAP: A Potential Biomarker of Mild Traumatic Brain Injury Produced by Repeated Exposure to Low Level Blast Overpressure

From the shear number of reported cases that have emerged since 2002, mild traumatic brain injury (mTBI) resulting from exposure to improvised explosive devices (IEDs) represents a major medical issue for deployed military personnel. This has created an urgent need to identify potential serum/blood biomarkers of brain injury resulting from exposure to blast overpressure (BOP), one component of IEDs, which may play a role in the long-term manifestation of cognitive and other impairments. We have previously reported cognitive impairment after a single exposure to 75 and 120 kPa BOP and a long lasting cognitive deficit in rats given 12 exposures to 40 kPa. Since 1) our WRAIR TBI/biomarker group has demonstrated serum biomarker changes in a rodent model of penetrating brain injury that 2) have also been reported to be sensitive indicators of mTBI diagnosed in human patients, the objective of the present study was to determine if repeated, once daily exposures to 75 KPa for 3 days or 40 kPa for 3 days produced any changes in targeted serum biomarkers of brain injury. Anesthetized adult male rats exposed to BOP were assessed for serum levels of GFAP, Nestin, UCH-L1 and SBDP-145 (spectrin breakdown product) by Western blot analysis. Although the results of Nestin, UCH-L1 and SBDP-145 measurements in plasma were inconclusive, a significant increase in serum GFAP was observed that was also dependent on the severity of BOP. Further studies are under progress to determine alterations in these biomarkers in brain tissue using immunocytochemical analysis. These preliminary studies suggest that serum GFAP level may be a potential candidate biomarker of low-level blast overpressure-mediated mTBI.

Khatri Vivek Shear Deborah A. Pedersen Rebecca C. Sun Justin A. Long Melissa Lu Xi-Chun May Tortella Frank C. Walter Reed Army Institute for Research, Silver Spring, MD, USA

P333 Progesterone Dose-Response Profile After Penetrating Ballistic-Like Brain Injury in Rodents

The neuroprotective efficacy of progesterone is well-established in animal models of traumatic brain injury (TBI). Here, we examined the therapeutic benefits of intravenously (i.v.) delivered progesterone in a military-relevant model of penetrating ballistic-like brain injury (PBBI) in rodents. Unilateral frontal PBBI was produced in the right hemisphere of anesthetized rats (10% injury severity level). Progesterone (5, 10, or 25 mg/kg) dissolved in 22.5% 2-hydroxopropyl-β-cyclodextrin was administered 30m, 6h, 24h, 72h, 4 days, and 5 days post-injury. Neuroprotective efficacy was evaluated in two tasks: (1) on post-injury day 7 using the rotarod to measure motor coordination and balance at speeds of 10, 15, and 20 rpm and (2) on post-injury days 13 through 17 using the Morris water maze (MWM) to measure spatial learning performance. Results showed that progesterone provided dose-dependent protection against PBBI-induced motor abnormalities on the rotarod task, with the lowest dose (5 mg/kg) being the most effective (p < 0.05 compared to vehicle). The mean rotarod latency across speeds was 2.5-fold higher in the 5 mg/kg treated group relative to vehicle-treated animals (means: 38.2 versus 15.2 sec). However, progesterone (at any dose) did not protect against PBBI-induced cognitive impairment in the MWM task. To our knowledge, this is the first study investigating the dose-response effects of i.v. administration of progesterone on neurobehavioral outcome in an animal model of brain injury. Overall, these results suggest that relatively small dosages of progesterone may be required with i.v. administration compared to other systemic routes of administration. Further work is warranted to determine the lowest effective dose of progesterone, and to investigate whether a gradual reduction in progesterone over the same dosing period (particularly for the higher doses) might improve neurofunctional outcome in the PBBI model.

Leung Lai Yee Wei Guo Khatri Vivek Shear Deborah Tortella Frank Walter Reed Army Institute Of Research, Silver Spring, MD, USA

P334 Development of a Military-Relevant Polytrauma Model: Combined Effects of Penetrating Ballistic-Like Brain Injury and Hemorrhagic Hypotension in Rats

In a military setting, traumatic brain injury often occurs in conjunction with additional trauma resulting in secondary complications such as hypotension (HT) due to blood loss. This study investigated the combined effects of 10% unilateral penetrating ballistic-like brain injury (PBBI) and hemorrhagic HT (30-min duration; mean arterial blood pressure of 40 to 45 mmHg) on physiological parameters including acute changes in cerebral blood flow (CBF), brain tissue oxygen tension (PbtO₂), and cortical spreading depolarizations (CSDs). CBF and PbtO₂ were monitored using laser Doppler flowmetry and Licox oxygen probe, respectively. The occurrence of CSDs was measured using direct current recordings from both ipsilateral (injured) and contralateral cortices in anesthetized rats using silver/silver chloride electrodes. All recordings were initiated prior to injury (PBBI, HT, or both) and maintained for 2.5 hours. Results showed that the combined effects of PBBI + HT produced a sustained impairment of ipsilateral CBF, which was significantly lower (2-fold, p < .05) than the sham or HT alone groups. Temporary reductions in CBF were also observed in rats subjected to PBBI alone, but returned to 85% of baseline by 2.5h post-PBBI. Notably, contralateral CBF increased after PBBI or PBBI + HT, suggestive of a compensatory response. PBBI + HT also caused significant and sustained reductions in PbtO₂ (50% of sham PbtO₂ levels) in the injured hemisphere. PBBI alone produced similar, albeit smaller, reductions in PbtO₂ levels (80% of sham PbtO₂.levels) but this effect was transient. Critically, a significant (p < .05) 4-fold increase in the occurrence of CSDs was observed in PBBI + HT group compared to PBBI or HT alone, and this increase was detected bilaterally. Our data suggests that the combined effects of PBBI + HT cause persistent impairment of CBF and brain tissue oxygen, increasing the probability of CSDs that likely contribute to secondary neuropathology and compromise neurological recovery.

Cartagena Casandra M. Schmid Kara Williams Garry Harris Jessie Tortella Frank Dave Jitendra WRAIR, Silver Spring, MD, USA

P335 Effects of NNZ-2566 on Transcription Regulation of IL6 Family Members

To better understand the mechanism of action of the clinical stage neuroprotection drug NNZ-2566, we examined its effect on transcription regulation of genes which are altered following penetrating ballistic-like brain injury (PBBI). We first examined mRNA levels of transcription and growth factors by PCR array using pooled RNA from sham or PBBI rats collected 4 hr, 24 hr, and 3 days post-injury. We found 1) that PBBI upregulated ATF3, a transcription factor which negatively regulates IL6 cytokine production, at all time points and 2) that CLCF1, an IL6 family member, was upregulated 24 hr post-PBBI. Previous studies showed that NNZ-2566 treatment following PBBI significantly decreased injury-induced upregulation of IL-6 mRNA levels. Although IL6 family members have a variety of functions, they may have similar mechanisms of transcription regulation. To confirm injury-induced changes in ATF3 and CLCF1 mRNA and to determine the effects of NNZ-2566 treatment, mRNA from individual animals (n = 6/ group: sham, PBBI, and PBBI/ NNZ-2566) was evaluated by realtime PCR at 4, 12, and 24 hr and 3 days post-PBBI. As seen earlier, ATF3 mRNA levels were upregulated by PBBI at all time points. At 12 hr post-PBBI, NNZ-2566 further increased ATF3 levels by > 4 fold in comparison to PBBI alone (p < 0.05). CLCF1 mRNA levels were also upregulated by PBBI at 12 hr and 24 hr. However, here NNZ-2566 significantly decreased this upregulation at the 24 hr time point by > 5 fold (p < 0.001), suggesting increased ATF3 levels have downstream effects on IL6 family member transcription. In addition, we evaluated 6 other IL6 family members and found that two genes (IL11 and CTF1) were affected by injury. NNZ mitigated the injury induced affects on both these genes, again at 24 hr post-injury. These effects on ATF3 and downstream IL6 family members demonstrate a possible mechanism of action for NNZ-2566.

Shear Deborah A. 1 Chen Zhiyong 1 Schmid Kara E. 1 Caggiano Anthony O. 2 Iaci Jennifer F. 2 Tortella Frank C. 1 Dave Jitendra R. 1

1 Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD, USA 2 ACORDA Therapeutics, Inc, Hawthorne, NY, USA P336 Acute Neuroprotective Effects of Glial Growth Factor 2 in a Model of Penetrating Ballistic-Like Brain Injury

Glial growth factor 2 (GGF2; ACORDA Therapeutics) is a member of the neuregulin family of growth factors that is best known for its ability to promote the survival and proliferation of oligodendrocytes and stimulate remylenation in preclinical models of multiple sclerosis. More recently, the GGF2 molecule was demonstrated to have a range of other effects in neural protection and repair including providing trophic support and exerting potent anti-inflammatory and antioxidant effects in the brain. In addition, neuregulins have been shown to play a role in synaptic plasticity, which is important for memory. Thus GGF2 shows promise as a potential neuroprotective therapy for the treatment of traumatic brain injury (TBI). The current study was designed to test the potential neuroprotective effects of GGF2 in our model of penetrating ballistic-like brain injury (PBBI). GGF2 (100 or 500 μg/kg) or vehicle was administered via i.v. infusion at 30 min post-PBBI and given once/day until the experimental endpoint. Animals were sacrificed at 24h, 72h, and 7 days post-injury (n = 6/grp/time) and the tissue was processed for histopathological analysis. GGF2 (either dose) treatment did not exhibit any significant neuroprotective effects on lesion size, apoptosis (Bax/BCL2), neutrophil infiltration (MPO), or axonal injury (silver) following PBBI. However, GGF2 dose-dependently reduced the extent of GFAP reactivity in the cortex of PBBI rats at 72h post-PBBI. Critically, no toxic side-effects were observed during acute administration of GGF2, even after multiple treatment days. This observation, combined with our current observations of a GGF2-mediated reduction in GFAP reactivity, provide support for a dose-response study that would combine modified dosing regimens with extended treatment duration.

Shear Deborah A. Mountney Andrea Pedersen Rebecca Rosen Elliot Sun Justin Long Melissa Lu X.C. May Tortella Frank C. Brain Trauma Neuroprotection and Neurorestoration Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD, USA

P337 Neuroprotective Dose-Response Profile of Dextromethorphan in a Rodent Model of Penetrating Ballistic-Like Brain Injury

Dextromethorphan (DM) has been well characterized as a neuroprotective agent in experimental models of CNS injury. We recently reported that DM provides significant protection against acute (≤7 days) injury-induced neuropathology and behavioral abnormalities in our penetrating ballistic-like brain injury (PBBI) model. The goal of this study was to identify the optimal dose-response of DM in the PBBI model as a potential candidate for combination therapy studies. DM (1, 5, 10 or 20 mg/kg) was administered intravenously (i.v.) at 0.5h, 2h, 4h, and 6h post-injury and continued once/day out to 72h post-PBBI. Neuroprotective efficacy was evaluated on: (1) the fixed-speed rotarod task to measure motor coordination and balance at speeds of 10, 15, and 20 rpm and (2) the Morris water maze (MWM) to measure spatial learning performance. DM improved motor performance on the rotarod task with the 5-10 mg/kg doses being the most effective. The mean rotarod latency across speeds was approximately 2-fold higher in both the 5 mg/kg and 10 mg/kg treated groups relative to vehicle-treated PBBI animals (means: PBBI + vehicle = 17.95 ± 4s; PBBI + 5mg/kg = 34.02 ± 5s; PBBI + 10mg/kg = 37.04 ± 5s). DM also provided dose-dependent protection against cognitive deficits as measured by mean latency to locate the hidden platform over the last 2 days of MWM testing. However, only the 10 mg/kg dose of DM resulted in significant improvements in performance on this task (mean latencies: PBBI + vehicle = 53.33 ± 8s; PBBI + 5mg/kg = 54.05 ± 8s; PBBI + 10mg/kg = 29.68 ± 4s). In order to determine whether extending the treatment duration would produce added benefits, DM (10 mg/kg) administration was carried out to 5 days post-injury in an additional group. However the 5-day treatment duration did not produce any additional therapeutic benefits on the rotarod (mean = 34.69 ± 6s) or MWM tasks (latency = 32.75 ± 5s).

Overall, these results confirm the neuroprotective efficacy of DM in the PBBI model and identified the lowest effective dose and treatment duration for potential use in combination with other neuroprotective agents.

Dave Jitendra R. 1 Yao Changping 1 Cartagena Casandra M. 1 Shear Deborah A. 1 Chen Zhiyong 1 Schmid Kara E. 1 Caggiano Anthony O. 2 Iaci Jennifer F. 2 Tortella Frank C. 1

1 1Brain Trauma Neuroprotection and Neurorestoration, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD, USA 2 2Acorda Therapeutics, Inc, Hawthorne, NY, USA P338 Acute Neuroprotective Effects of Glial Growth Factor 2 on Protein Changes Following Penetrating Ballistic-Like Brain Injury

Traumatic brain injury (TBI) is a significant cause of morbidity and mortality for the civilian and military populations. To date there are no approved drug treatments for TBI. Glial growth factor 2 (GGF2; ACORDA Therapeutics) is a member of the endogenous protein family known as neuregulins. Neuregulins are required for normal development of the heart and brain and have recently demonstrated neuroprotective and neurorestorative effects in stroke injury models. Therefore, GGF2 was examined for possible acute neuroprotective effects in our model of penetrating ballistic-like brain injury (PBBI). Animals (n = 6/group) were dosed via i.v. infusion at 30 min post-injury with GGF2 (100 or 500 μg/kg) or vehicle control. Changes in α-2 spectrin breakdown product-145 (SBDP-145), and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) were examined in brain and plasma at 24 hr post injury using western blot analysis. PBBI injury caused a significant increase in both of these proteins in brain tissue and in plasma (p < 0.05). GGF2 (both doses) significantly reduced PBBI-induced increases in UCH-L1 levels in brain tissue and (high dose only) significantly reduced SBDP-145 levels in plasma (p < 0.05). In a separate experiment, GGF2 (both doses) significantly attenuated the PBBI-mediated up-regulation of caspase-3 activity in brain tissue. Overall, the current results indicate that a single, post-injury infusion of GGF2 provides significant protection against acute (≤24 hr) PBBI-induced protein changes. Since recent studies have suggested that GGF2 may have a stronger restorative function than a protection function, optimal dosing regiments of GGF2 should be examined during the sub-acute or chronic periods in the PBBI model.

Lu May Chen Zhiyong Wei Guo Cao Ying Leung Laiyee Cunningham Tracy Khatri Vivek Mountney Andrea Shear Deborah Tortella Frank Walter Reed Army Institute of Research, Silver Spring, MD, USA

P339 Dose Response Effects of Phenytoin on Attenuation of Nonconvulsive Seizures Caused by Penetrating Ballistic-Like Brain Injury in Rats

Acute post-traumatic nonconvulsive seizures (NCS) require prompt intervention. As NCS, they are more difficult to diagnose and more refractory to antiepileptic drug treatments. Phenytoin (PHT) is widely considered as the first choice for seizure treatment in the neurointensive care unit. In this study we evaluated the dose-response effects of PHT to attenuate acute nonconvulsive seizures caused by a penetrating ballistic-like brain injury (PBBI) in rats. Continuous EEG recordings were collected for 72h following PBBI from awake animals. Four doses of PHT (PHT-10/5, PHT-20/10, PHT-40/20, and PHT-60/30, N = 15/dose) were tested. Each dose consisted of a loading dose (10, 20, 40, 60 mg/kg i.v. injection) initiated at 30 min after injury followed by a maintenance dose (5, 10, 20, 30 mg/kg, respectively) given at 8h after the loading dose on Day 1 and then twice per day (at 8h intervals) on days 2-3. Overall, 73% of vehicle-treated animals exhibited an average of 7.4 NCS episodes/rat yielding a total NCS duration of 303 ± xx sec/rat during the 72h post-injury period. The onset latency was @27h post-injury. PHT treatments, except for the lowest dose, significantly reduced NCS incidence to 33-40%. In all PHT treated groups, the NCS episodes and durations were also dose-dependently mitigated, resulting in an average of 6.2-1.8 episodes/rat (267-66 sec/rat) measured from the highest to the lowest doses, with the significant reductions being afforded by the two highest dose regimens. Transient (5-30 min) sedation was also correlated to the escalating doses of PHT, i.e. the higher the dose, the longer the sedation. Since PHT treatment was initiated before NCS occurred, these results indicate that PHT is effective in ameliorating post-traumatic NCS as prophylactic treatment and the PHT-40/20 dose has a better therapeutic index than PHT-60/30 dose because of its similar efficacy but lesser degree of sedation.

Clayton Erik Bayly Philip Washington University in St. Louis, Saint Louis, MO, USA

P340 The Frequency Response of the Human Brain to Extracranial Pressure Excitation in Vivo

Spatiotemporal patterns of tissue deformation during blast-induced traumatic brain injury (TBI) are poorly understood. While numerical simulations are available, experimental data are needed for model validation and to better understand injury mechanism(s).

Magnetic resonance elastography (MRE) was used to record spatiotemporal images of brain deformation as the cranium was exposed to acoustic frequency pressure loading. Three healthy male human subjects, 21-30 years-old (mean: 23.7 yr) were exposed to extracranial pressure oscillations at frequencies of 45, 60, and 80 Hz. Phase contrast images, with contrast proportional to displacement, were collected using a 1.5T MAGNETOM Avanto (Siemens) MRI scanner with a phased-array head coil. Imaging parameters: TR/TE: 138/27.5 ms, flip angle: 25°, NEX: 1, resolution: 3 mm cubic. Tensorial strains were estimated from displacement components in x-, y- and z-directions. Shear strains were normalized by pressure amplitude for comparison across subjects and frequencies. Pressure-normalized shear-strains were evaluated in two ways: (i) globally - across all brain regions, and (ii) regionally - in three concentric annular zones.

Symmetric acoustic pressure excitation of the human skull leads to propagation of shear waves in the brain. Mean RMS shear strains are significantly higher (173 μɛ/Pa) at 45 Hz than at 60 Hz (68 μɛ/Pa) and 80 Hz (31 μɛ/Pa). At 45 Hz, the mean RMS strain amplitude is 217 μɛ/Pa in the outermost region; RMS strain amplitude is 43% lower in the brain interior. At 60 and 80 Hz, similar spatial attenuation characteristics are observed, however the attenuation in RMS strain amplitude is nearly double that observed at 45 Hz.

The magnitude and phase of shear deformation, relative to known pressure excitation, can be used to validate computer models of TBI. These data offer new insight into the effects of brain mechanical parameters, brain-skull attachments, and skull transmissibility on the brain's response to external pressure.

Kou Zhifeng Hanks Robin Gattu Ramtilak Yang Jie Zheng Weili Mika Valerie Welch Robert Benson Randall Millis Scott Haacke E Mark Wayne State University, Detroit, MI, USA

P341 Quantifying Changes of Hemorrhage Over Time in Traumatic Brain Injury by Using MR Susceptibility Mapping

OBJECTIVE: Hemorrhagic blood has been considered a radiological biomarker for the diagnosis of traumatic brain injury (TBI). After brain injury, the blood itself may undergo a complicated biochemical process along with patient's recovery. Tracking and quantifying the changes of hemorrhage longitudinally may help reveal the recovery process of the brain after injury. Susceptibility weighted imaging (SWI) has been reported as a sensitive means of detecting hemorrhagic blood but short of quantifying the signal of irons in blood. The next generation technique, referred as susceptibility weighted imaging and mapping (SWIM), could efficiently overcome this technical difficulty from both image reconstruction and post processing perspectives. It has the potential of quantitatively mapping the susceptibility signal, which is in proportion of irons in blood. The objective of this study is to use SWIM as a means to quantify the iron concentrations contained in the blood over time after TBI.

MATERIALS AND METHOD: Five mild to severe TBI patients were imaged at the acute or subacute stage and further followed up at up to 1-year period. SWIM images were collected on 3T Siemens Verio magnet and further processed to quantify the irons by using our in-house software SPIN. SWIM images at two time points have been co-registered to overcome image acquisition difference. The patients' clinical and neurological data have been collected and neuropsychological assessment has been done at follow up.

RESULTS AND DISCUSSION: Over 1-year period after injury, we found most significant decrease of SWIM signal at the extra-axial space and moderate decease of bleed at brain parenchyma. This is associated with the recovery of the patients. It also manifests that the blood has been absorbed by the brain during recovery. SWIM has the potential to quantify hemorrhage transformations over time in association of brain recovery.

Sajja Sujith 1 Perrine Shane 2 Ghoddoussi Farhad 3 Galloway Matthew 3 VandeVord Pamela 4

1 Biomedical Engineering,Wayne State University, Detroit, Michigan, USA 2 Psychiatry and Behavioral Neurosciences, Wayne State University, Detroit, Michigan, USA 3 Psychiatry and Behavioral Neurosciences, and Anesthesiology, Wayne State University, Detroit, Michigan, USA 4 Biomedical Engineering,Wayne State University, and John D. Dingell Veteran's Administration Medical Center, Detroit, Michigan, USA P342 Imparied Working Memory is Associated with MYO-lNOSITOL in the Frontal Cortex of Rats Exposed to Blast Overpressure

Working memory, which is dependent on higher-order executive function in the prefrontal cortex, is often disrupted in clinical cases of exposure to blast overpressure. In this study, we evaluated working memory impairment associated with blast overpressure along with neurochemical assessment in a rat model of blast. Animals (adult male SD rats) anesthetized with 3% isoflurane were exposed to calibrated blast overpressure (17 psi or 117 KPa) while sham animals received only anesthesia. Animals were sacrificed at 1, 2 and 7 days post blast (n = 10/group). To test working memory, animals underwent novel object recognition (NOR) test 7 days after overpressure. After behavioral assessment, animals were sacrificed and neurochemical profiles were determined with magic angle spinning proton magnetic resonance spectroscopy (¹ H-MRS) ex vivo obtained from frontal cortex.

Early effects included a significant decrease in betaine and increase in GABA at day 1 and significant increases in glycerophosphorylcholine, phosphorylethanolamine, also glutamate/creatine and lactate/creatine ratios on day 2. Seven days after blast, only myo-inositol levels were altered (15% increase). In behavioral tests, NOR revealed significantly impaired working memory (23%) in blast group compared to shams. Interestingly, working memory in control animals was negatively correlated with myo-inositol levels (r = − .759, p < 0.05), an association that was absent in blast exposed animals.

The major finding of our report is that 7 days after a single exposure to blast overpressure, rats have a deficit in working memory and an increase in prefrontal cortex levels of myo-inositol. Early observations are consistent with glutamate dysregulation, increased inflammation, and disrupted equipoise of energy status. Although higher-order cognitive aspects of working memory are dependent on recruitment of the prefrontal cortex, blast-induced damage to the hippocampus may also contribute to the memory deficit. Increased myo-inositol may represent tardive glial scarring and contribute to the impaired working memory.

Li Yan Zhang Liying Kallakuri Srinivasu Zhou Runzhou Cavanaugh John Wayne State University, Detroit, MI, USA

P343 Injury Predictors of Traumatic Axonal Injury in Corpus Callosum and Pyramidal Tract Following Impact Acceleration Induced Traumatic Brain Injury in Rodent

A modified Marmarou impact acceleration model was developed to study the kinematics of the rat head, to quantify traumatic axonal injury (TAI) in corpus callosum (CC) and pyramidal tracts (Py), and to determine injury predictors and establish injury thresholds for severe TAI.

Thirty-one anesthetized male Sprague-Dawley rats (375-425 grams) were impacted using impact device from 2.25m and 1.25m heights. Linear and angular responses of the head were measured in vivo with attached accelerometer and angular rate sensor. Impact was captured by a high-speed video camera at 10,000 fps. Latency to surface right was assessed post-TBI. β-amyloid precursor protein immunocytochemistry was used to assess and quantify axonal injury and to develop injury maps of TAI in CC and Py. Injury predictors were evaluated using logistic regression analysis. Receiver operator characteristic (ROC) curves were used to assess the predictive power of individual and paired injury predictors.

Over 600 injury maps in CC and Py were constructed in the 31 impacted rats. TAI distribution along the rostro-caudal direction, as well as across left-right hemispheres in CC and Py were determined. Biomechanical data showed that average linear acceleration, peak angular velocity, average angular acceleration, Power Index, HIC and time to surface right were significantly different between the two impact heights. Logistic regression suggested that the occurrence of severe TAI in CC was best predicted by average linear acceleration, followed by the Power Index and time to surface right. In Py, severe TAI was best predicted by the time to surface right, followed by the peak angular velocity and average angular velocity. The combination of average linear acceleration plus time to surface right in CC, and peak angular velocity plus time to surface right in Py achieved area under the ROC curve of 0.949 and 0.898, respectively; better than results predicted by single predictors. NIHR01EB006508

Kallakuri Srinivasu Li Yan Zakaria Nisrine Zhou Runzhou Zhang Liying Cavanaugh John Wayne State University, Detroit, MI, USA

P344 Quantification and Comparison of Axonal Pathology in a Rodent Traumatic Brain Injury Model

Traumatic axonal injury (TAI) involves focal impairment of axonal transport (IAT) and neurofilament side arm alterations leading to neurofilament-compaction (NFC). Although IAT and NFC are found in corpus callosum (CC), studies on their quantification in CC and comparison to that in pyramidal tract (Py) are lacking and thus form the purpose of this study.

TBI (modified Marmaoru impact acceleration device) was induced in anesthetized male rats by dropping a 450 gram mass on to helmeted skull from height of 2.25m (n = 16) or 1.25m (n = 15). 24 hours post-TBI rats were perfused. IAT and NFC were assessed in two sets of coronal (CC) and sagittal sections (Py) by β-APP and RMO14 immunocytochemistry. For CC, every twelfth section was analyzed (a total of 13-15 sections). For Py, 5 sagittal sections (0-1000um) were used. β-APP and RMO14 reactive CC and Py regions were photographed (×10) and panoramic images were digitally stitched. Then, a 200 × 200 mm grid system was digitally superimposed on each panoramic image. The number of damaged axons in each grid was counted by ImageJ cell counter function.

At 2.25m, Py APP count (784 ± 182) was significantly higher than that in CC (179 ± 55). (p < 0.05). At 2.25m, average IAT (combined CC and Py APP) was significantly higher than NFC (combined CC and Py RMO14) and also significantly higher than corresponding IAT and NFC at 1.25m (p < 0.05). Average combined TAI counts at 2.25m were significantly higher than that at 1.25m (p < 0.05). A correlation between total TAI versus surface righting time was also observed.

This is the first study comparing TAI counts in CC and Py in the Marmarou model. TAI count was greater at 2.25m than at 1.25m with higher TAI counts in Py than in CC. Furthermore, axonal counts indicated that IAT was more prominent than NFC in this study. NIHR01EB006508

Zhang Liying Yang King King Albert Wayne State University, Detroit, MI, USA

P345 Characterization of Mechanical Properties and Recovery Rate From Cyclic Loading for a Low Density Foam used in Marmarou's Rodent Model of TAI

Marmarou's impact acceleration model is a widely used TBI model capable of producing a substantial degree of axonal injury in rodents. The mechanical characteristics of the low-density polyurethane (PU) foam, an essential piece of energy management used in the model, have not been fully characterized. The goals were to 1) determine the mechanical properties of the foam in compression at various loading rates, 2) ascertain the rate of recovery under cyclic loading, and 3) validate a finite element (FE) foam model in simulating rate-dependent responses.

The foam samples (N = 21) were tested in uniaxial compression to 80% strain at seven strain-rates ranging from 0.014 to 42.86s⁻¹. Cyclic loading was conducted at every 5-minutes for the first five cycles followed by cyclic loading at one-hour (6^th), one-day (7^th), three-days (8^th) and three-weeks (9^th cycle). The predicted force-deflection responses at various strain-rates from a FE foam model were compared to test results.

The stiffness and Young's modulus ranged from 3100 to 5600N/m and from 44.75 to 78.08kPa, respectively. The derivatives of the engineering stress-strain rate curve fitted by power functions indicated that the foam properties were strongly related to the strain-rate up to 15s⁻¹. 30% stress softening was observed at the 2^nd cycle in comparison to the stress from the initial cycle. Further reduction in stress (5%) was found in the 3^rd, 4^th and 5^th cycles. The stress results from 6^th to 9^th cycles were similar to the 2^nd cycle.

The foam is strain-rate dependent and is reusable for multiple impacts. However, the stress resistance of a used foam is about 70% of a new foam. Allowing one-hour recovery time between the subsequent tests after 2^nd cycle is recommended to assure the consistency of mechanical responses. The material model of the FE solver is adequate to simulate this rate-sensitive foam. NIH R01EB006508

Zhang Liying Zhou Runzhou Li Yan Kallakuri Srinivasu Cavanaugh John Wayne State University, Detroit, MI, USA

P346 Local Brain Strain Predicts Traumatic Axonal Pathology in a Rodent Model of Head Impact Acceleration Induced Brain Injury

Finite element (FE) modeling of traumatic brain injury (TBI) is an effective approach to quantify the local mechanical responses and to define injury thresholds. A combined in-vivo experimental and computer modeling approach was used to establish the relationships between tissue level strain and axonal pathology in a rodent impact-acceleration model of TBI.

The FE model of a SD rat head consists of the scalp, skull, dura, arachnoid-pia, cerebral gray and white matter, ventricles, corpus callosum(cc), brainstem with pyramidal tract(py), medial lemniscus, cervical spine and facial tissues. The head model had over 990,000 3D elements. A FE rat body was recently added to mimic spinal-brainstem stretching during impact. 13 different material properties were used to represent the regional heterogeneities and the anisotropies of white matter tracts. The validated rat model was applied to simulate 2.25m and 1.25m impact-acceleration experiments conducted on anesthetized rats. The tissue strain calculated in cc and py was related to axonal pathology quantified by beta-APP and RMO14 immunochemistry.

The average principal strain was 0.15 in the entire cc and 0.19 in the entire py from 2.25m head impact. The average strain of 0.08 in the cc and 0.10 in the py in 1.25m was significantly lower (p < 0.01) than from 2.25m impact. The average strain predicted at two heights agreed with the injury density (average axonal counts/200um³) being significantly higher at 1.25m (2.1/200um³ in cc, 9.85/200um³ in py) than 1.25m (0.3/200um³ in cc, 2.0/200um³ in py) in the live rats. The model also predicted high strain in medial lemniscus and pyramidal decussation.

An anatomically detailed FE model of rat head/body enabled analysis of biomechanical responses at tissue/cellular levels in an in-vivo TBI model. The correlation of tissue strain with histopathology in white matter tracts may lead to the development of thresholds for traumatic axonal injury.

Mao Haojie 1 Zhu Feng 1 Jin Xin 1 Clausen Fredrik 2 Colgan Niall 3 Gilchrist Michael 3 Yang King 1

1 Wayne State University, Detroit, MI, USA 2 Uppsala University Hospital, Uppsala, Sweden 3 University College Dublin, Belfield, Dublin, Ireland P347 Biomechanical Analysis of Fluid Percussion Model of Brain Injury

INTRODUCTION: Fluid percussion (FP) is a widely used experimental model for studying traumatic brain injury (TBI). The severities of FP-induced TBI (mild, moderate, or severe) are normally characterized by extracranial pressure, or physiological, morphological, and behavioral responses without considering local mechanical strain/pressure applied on brain tissues. The purposes of this study were to develop a numerical rat brain model based on fluid-solid interaction technique to simulate FP experiments, validate the numerical model-predicted ventricular pressure against experimental data, and analyze regional tensile strain and intracranial pressure during FP.

METHODS: A previously developed finite element (FE) rat brain model was improved by incorporating a layer of cerebrospinal fluid determined from MRI scans of four S-D adult rats. A Gruneisen equation was adopted to simulate the pressurized saline with peak values of 2.6 or 2.9 atm (Clausen and Hillered, 2005) to load the rat FE model. Fluid-solid interaction was implemented between the saline and brain using a commercially available software LS-DYNA.

RESULTS: The FE model-predicted ventricular pressures in the ipsilateral and contralateral sides were about 5% to 9% lower than those measured experimentally. Peak intracranial pressures ranged between 140 and 165 kPa and distributed into both hemispheres. Regions with maximum principal (tensile) strains greater than 0.1 were located in the ipsilateral cortex, with volumes of 2.5 or 2.7 mm³ for two FP loading conditions. The low strain result explained why brain contusions induced in FP were fewer compared to controlled cortical impact in which cortical strains were much higher (Mao et al., 2010).

CONCLUSIONS: A numerical model of FP was developed and validated. Numerical analysis indicated FP was a complex experimental TBI model with high strain concentrated in the ipsilateral cortex and intracranial pressure distributed into both hemispheres. More investigations regarding the combined effect of strain, pressure, duration, and incident rate are needed.

Mao Haojie Skelton Paul Yang King Wayne State University, Detroit, MI, USA

P348 Computational Simulation of Controlled Cortical Impact on C57BL/6 Mouse Brain

INTRODUCTION: The effects of external settings of rat controlled cortical impact (CCI) on intracranial responses have been mechanically characterized using a numerical rat brain model. However, mechanical characterizations of mouse CCI are still limited. Additionally, the use of transgenic mice, whose intracranial structures were different from those of wild mice, results in different intracranial mechanical pathways. The purposes of this study were to: explore a method to generate a detailed mouse-specific brain model, and analyze the effect of different CCI parameters on C57BL/6 mouse brain tissue.

METHODS: ANSYS ICEM CFD/HEXA was used to create a mouse brain “block” system which could be associated to specific mouse brain geometries. For baseline mouse CCI simulation, a flat 3.0 mm diameter impactor was driven to compress the exposed dura to 1 mm depth at 3.5 m/s impact velocity. Five additional mouse CCI cases were simulated by adjusting: impact depth, impactor shape, impactor diameter, impact velocity, and craniotomy pattern.

RESULTS: The mouse brain “block” system was created and used to develop an all-hexahedral C57BL/6 mouse brain model. Generally, the intracranial mechanical responses in simulated mouse CCI were similar to those of rat CCI. The numerical model-predicted high tensile strains beneath the impactor matched experimentally observed ipsilateral brain contusions. The flat impactor induced highest strain concentrations along the impactor rim while there is no such “edge effect” for hemispherical impactor, which is consistent with recent findings (Pleasant et al., 2011). Only with bilateral craniotomy, high strains were distributed to the contralateral side. Impact depth was a leading factor affecting tissue strains surpassing impactor diameter and impact velocity.

CONCLUSION: A mouse brain “block” system was developed which can be used to create transgenic mouse-specific brain models. Also, numerical simulation conducted before experimentation can be used to provide clear descriptions of regional brain injury intensity.

Lowing Jennifer L. 1 Susick Laura L. 1 Provenzano Anthony M. 1 Raghupathi Ramesh 2 Conti Alana C. 1

1 John D. Dingell VA Medical Center; Departments of Neurosurgery and Pediatric Neurosurgery, Wayne State University, Detroit, MI, USA 2 Department of Neurobiology & Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA P349 Traumatic Brain Injury Increases Ethanol Sensitivity in Mice

Traumatic brain injury (TBI) victims commonly develop long-lasting behavioral disturbances, such as susceptibility to alcohol use disorders. Our overall objective is to elucidate the mechanisms by which TBI increases ethanol sensitivity and promotes alcohol addiction.

Here we present data characterizing a mouse model of non-contusive closed-head TBI and an associated increase in alcohol sensitivity. Using an electromagnetically-driven controlled cortical impact device, we demonstrate graded injury severities as evaluated by latency to right after injury, motor deficits, diffuse axonal injury and reactive astrogliosis. Ethanol sensitivity was measured using a loss of righting reflex (LORR) assay.

Wild type (WT) C57/BL6 mice (6-8 weeks of age) were subjected to sham or moderate TBI. Moderate TBI resulted in significantly longer latency to right after injury compared to sham controls. Using an accelerating rotarod assay, TBI mice were significantly impaired for 48 h after injury compared to controls, recovering to sham levels by 7 d post-injury. Histological assessment revealed accumulation of APP bilaterally in white matter structures at 6, 24 and 72 hrs after injury compared to sham controls. Subsequent to traumatic axonal injury, GFAP immunoreactivity was observed bilaterally in the cortex by 7 d following injury, largely observed under the site of injury. Little evidence of GFAP immunoreactivity was detected in sham controls.

At 14 d after injury, mice were tested for LORR following acute administration of ethanol. Sham and injured mice received ethanol (4 g/kg, i.p.) and were placed on their backs after losing the ability to right themselves. Mice subjected to TBI exhibited a significant increase in sedation time compared to sham controls.

These data demonstrate that experimental TBI increases sensitivity to ethanol sedation. Characterizing preclinical models of non-contusive TBI and examining the effects of TBI on ethanol sensitivity may be beneficial to both civilian and military TBI populations.

Peduzzi Jean 1 Kumaran Saravanan 1 Cavanaugh John 1 Kreipke Christian 1 Lima Carlos 2 Meythaler Jay 1

1 Wayne State University, Detroit, MI, USA 2 Hospital Egas Moniz, Lisbon, Portugal P350 Functional Improvement in TBI Using Syngeneic Olfactory Mucosa Stem Cells in Inbred Rat

INTRODUCTION: The safest and most effective treatment for traumatic brain injury (TBI) may be autologous stem cells delivered into the cerebrospinal fluid (CSF). CSF-injected stem cells home to the injured area which would be critical in diffuse axonal injury that damages several areas. These CSF-injected stem cells penetrate the brain and induce recovery. Olfactory mucosa (OM) stem cells can be obtained from the nose and their normal fate is neurons, making them safer to use.

METHODS: Adult inbred Lewis rats (345-395g) received a sham surgery or severe TBI using the Marmarou model (450g weight dropped 2 meters). Two weeks later, 3 groups (6/group) received CSF injections of saline or iron-oxide-labeled OM stem cells. Blinded functional testing (1-7 weeks post-injury) included the Beam Balance, Inclined Plane, 8-Arm Radial Maze and Barnes Table tests.

RESULTS/DISCUSSION: In the Beam Balance at one week post-treatment, the stem cell-injected injured group was similar to the sham group while significantly different from the vehicle-injected injured group (p = 0.017). At later times, performance was similar. For the Inclined Plane, Radial Arm Maze and Barnes Table tests, the stem cell-injected injured group performed similarly to the sham group and consistently performed better than the saline-injected injured group at 3 and 4 weeks after treatment. In the Radial Arm Maze, this difference reached significance level at week 4 after treatment (p = 0.041). In Barnes Table test, the sham was significantly different from the vehicle-treated injured group at week 4 after injury (p = 0.047).

CONCLUSIONS: OM stem cells caused no adverse effects and resulted in functional improvement when evaluated in experimental TBI producing diffuse axonal injury. Clinical translation is facilitated by the fact that OM stem cells can be obtained and delivered in a minimally-invasive manner.

Traumatic Brain Injury Research Program at Wayne State University & R01 NS064976(CK), VA Merit RX000224(CK)

Turner Ryan 1 Mills James 1 Bailes Julian 1 Dodson Sean 1 Sakai Jun 1 Maroon Joseph 1

1 West Virginia University, Morgantown, WV, USA 2 University of Pittsburgh, Pittsburgh, PA, USA P351 Anabolic Steroids and Traumatic Brain Injury: Do Steroids Play a Role in Chronic Traumatic Encephalopathy?

BACKGROUND: The syndrome of chronic traumatic encephalopathy (CTE) has received increasing attention from the medical community, general population, and media following evidence of CTE in numerous former athletes. The suggestion has been made that perhaps the neurological and pathological changes seen in CTE may be due to exogenous anabolic steroid (AS) use rather than TBI.

OBJECTIVE: The purpose of this study was to determine if the administration of AS alters the pathophysiology of traumatic brain injury.

METHODS: Sixty adult male Sprague-Dawley rats were used. A standard linear acceleration-deceleration model of TBI was used. Experimental groups were: 1) pre-injury AS, 2) pre-injury placebo carrier, 3) AS without injury, 4) no AS and no injury, 5) post-injury placebo carrier, 6) post-injury AS. Following a 30-day recovery, rats were sacrificed and brainstem white matter tracts underwent fluorescent immunohistochemical processing and labeling of beta amyloid precursor protein (APP), a marker of axonal injury. Digital imaging and statistical analyses were used to determine if AS administration resulted in a significant change in the number of injured axons.

RESULTS: There was no statistically significant difference in number of APP-positive axons by immunohistochemical analysis between respective AS and placebo groups.

CONCLUSION: Using a standard model of mild TBI (MTBI), we have shown successful visualization of traumatically injured axons with antibody staining of APP. Our results indicate no statistically significant effect of AS on the number of APP positive axons. Using this model and within its limitations, we see no adverse effect or causative role of AS administration on the brain following MTBI using APP counts as a marker for axonal injury. As such, it seems unlikely that AS use contributes to development of CTE, regardless of whether they were used prior to or following brain injury.

Bailes Julian 1 Smith David 1 Fisher Joseph 2 Robles Javier 1 Turner Ryan 1 Mills James 1

1 West Virginia University, Morgantown, WV, USA 2 University of Toronto, Toronto, ON, Canada P352 Internal Jugular Vein Compression Mitigates Traumatic Axonal Injury in Rat Model by Reducing Intracranial Slosh Effect

BACKGROUND: Traumatic brain injury (TBI) remains a devastating condition for which traditionally extra-cranial protection has been utilized in the form of helmets. Helmets are effective in preventing penetrating injury but often fail to prevent concussions.

OBJECTIVE: The purpose of this study was to determine if pathologic outcome after TBI can be improved by reduction of intracranial Slosh forces via modulating intracranial volume.

METHODS: Two groups of rats were subjected to impact acceleration traumatic brain injury. Prior to injury, the experimental group had a cervical collar with two beads located over the IJVs applied. Intracranial pressure (ICP) and intraocular pressure (IOP) were measured before and after IJV compression to assess collar performance. All rats were sacrificed after a 7-day recovery period, and brainstem white matter tracts underwent fluorescent immunohistochemical processing and labeling of beta amyloid precursor protein (APP), a marker of axonal injury. Digital imaging and statistical analyses were used to determine if IJV compression resulted in a diminished number of injured axons.

RESULTS: IJV compression resulted in an immediate and reversible elevation of ICP and IOP. Most notably, quantitative analysis showed 13,540 APP positive axons in the experimental group versus 77,474 in the control group (P < 0.01), a marked reduction of greater than 80%.

CONCLUSION: Using a standard acceleration-deceleration impact laboratory model of mild TBI, we showed successful and marked reduction of axonal injury following IJV compression as indicated by immunohistochemical staining of APP. We argue IJV compression reduces Slosh-mediated brain injury by increasing intracranial blood volume and reducing compliance and potential for brain movement within confines of the skull. The potential for such technique to mitigate linear and rotational brain injury in humans by “internal protection” requires further investigation.

He Xiaosheng Gao Wei Xu Hongyu Liang Ming Fei Zhou Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China

P353 The Association of Hippocampal Glucocorticoid Receptor Expression and Cognitive Function in a Rat Model of Traumatic Brain Injury Due to Lateral Head Acceleration

OBJECTIVE: Learning and memory dysfunction after traumatic brain injury (TBI) is one of the important cognitive sequela, and seriously impacts on the patient's capability in living and working. The glucocorticoid receptors (GRs) had been observed to be widely expressed in the animal hippocampus which was highly associated with their cognitive functions. The relation between the hippocampal GRs changes and learning and memory deficits was investigated in this present study.

METHODS: A TBI rat model by lateral head rotational acceleration device was produced. The hippocampal GRs was tested by Western blotting analysis, and the rat cognitive function was examined with Morris water maze.

RESULTS: GRs-positive staining was observed microscopically to be distributed in the area of hippocampal CA1, GD sector among the sections from the sham and TBI rats. The OD values obtained in the sections expressing GRs immunoreactivity of the hippocampus in TBI rats revealed a peak at 4d survival and an obviously-declined trend over the following time-points significantly. The OD value, reflecting GRs protein- and mRNA- expression, obtained in the electro-phoresis bands from the hippocampus tissue among the sham and TBI rats, also showed a same change across various survivals after TBI. The control group and TBI group were trained with a spatial learning task at the same time. The spatial memory of rats was tested in MWM, which tests rats' ability for spatial learning and memory by measuring a rat's ability to navigate and exploration. According to the data of experiment, latency was shortened during the 4-day acquisition period. But latency of TBI group was long than the sham. AT 7day after TBI, the number of TBI group cross-platform was more than sham.

CONCLUSION: There must be an association between reduced hippocampal GRs and learning and memory dysfunction. The mechanism needs to be further investigated.

Koizumi Hiroyasu 1 Fujisawa Hirosuke 1 Suehiro Eiichi 1 Iwanaga Hideyuki 2 Nakagawara Jyoji 3 Suzuki Michiyasu 1

1 Department of Neurosurgery, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan 2 Department of Radiology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan 3 Department of Neurosurgery and Nuclear Medicine, Nakamura Memorial Hospital, Sapporo, Hokkaido, Japan P354 Recovered Neuronal Viability Disclosed by Iodine-123-Iomazenil Spect Following Traumatic Brain Injury

PURPOSE: Iodine-123-iomazenil (IMZ) is a specific radioligand for the central benzodiazepine receptor developed as a tracer for single photon emission computed tomography (SPECT). We evaluated cortical damages following traumatic brain injury (TBI) in the acute phase with IMZ SPECT.

METHODS: Twelve patients with cerebral contusion following TBI were employed in this study. The distribution of benzodiazepine receptors in the cortical regions was evaluated by IMZ SPECT within 1 week of injury. To investigate the changes in IMZ distribution in the cortex in the chronic phase, after conventional treatment, patients underwent IMZ SPECT again. Additionally, in vivo microdialysis was used to examine the focal metabolism by measuring the extracellular concentration of amino acids in the cortex.

RESULTS: In all patients, a reduction of IMZ uptake in the location of the cerebral contusions was observed in the TBI acute phase. In 9 of 12 patients, images of IMZ SPECT obtained in the TBI chronic phase showed that areas with decreased IMZ distribution were remarkably reduced compared to those obtained in the acute phase. In one case, the patient underwent placement of in vivo microdialysis probes. These were placed in the left frontal and temporal cortex where a decreased IMZ distribution was shown in the acute phase. The extracellular levels of glutamate were increased during the 168-hour monitoring period. In the chronic phase, IMZ uptake in the region with the microdialysis probes was recovered.

CONCLUSION: In brain injury including cerebral ischemia, reduced binding potential of central benzodiazepine receptor ligands indicates irreversible brain damage. The results suggest that this reduction is not a sign of irreversible tissue damage in TBI. The recovery of IMZ uptake in the cortical regions could be associated with changes in brain metabolism.

Jefferson Stephanie 1 Donlan Nicole 1 Kozlowski Dorothy 2 Jones Theresa 1 Adkins DeAnna 1

1 University of Texas at Austin, Austin, TX, USA 2 DePaul University, Chicago, IL, USA P355 Effects of Cortical Electrical Stimulation Combined with Motor Training on Anatomical Plasticity and Inhibitory Substrates in a Focal Model of Traumatic Brain Injury (TBI) in Rats

Cortical electrical stimulation (CS) coupled with motor rehabilitative training (RT) improves forelimb (FL) motor function and enhances functional and structural plasticity in the motor cortex of rats and non-human primates following stroke (Adkins et al., 2008; Kleim et al., 2003; Teskey et al., 2003; Plautz et al., 2003). Recently, we have demonstrated that, after moderate to large unilateral controlled cortical impact (CCI) to the FL sensorimotor cortex (FL-SMC), CS + RT significantly improves motor function and increases the FL motor cortical representation in the injured hemisphere, especially anterior to the injury, compared to RT alone (Adkins et al., 2009 SFN abstract). The goal of the present study is to investigate whether the behavioral improvements seen with CS + RT are correlated with anatomical plasticity and the reduction of growth inhibitory molecules in the peri-injury motor cortex as well as in remaining motor cortex anterior to the CCI following focal traumatic brain injury. In this study, adult male Long Evans hooded rats received 100HZ epidural stimulation at 50% of movement threshold over remaining peri-injury SMC during reach training with the impaired FL (4 days per week for 9 weeks). CS + RT animals have a significantly greater surface density of the dendritic marker microtubule associated protein-2 (MAP-2) anterior to the injury in layer V, congruent with motor map changes(p = 0.034), and significantly less MAP-2 in remaining motor cortex medial to the contusion in layer II/III (p = 0.036) in peri-injury cortex compared to RT alone. CS + RT also tends to decrease the density of cells expressing the neurite growth inhibitory protein Nogo-A, in layer V of the remaining motor cortex (p = 0.10) compared to RT alone. Analysis of axonal growth changes through assessments of the surface density of the phosphorylated axonal form of neurofilament heavy chain (pNF-H) in the anterior dorsolateral striatum, showed no significant difference across groups, however further investigation is currently being conducted in other cortical regions. These findings suggest that the behavioral improvements and motor map alterations following CS + RT may be in part due to enhancements in dendritic plasticity as well as diminishment of inhibitory molecules in remaining ipsilateral motor cortex in this focal model of TBI.

Abstract Author Index

Aarabi, Bizhan, P249, P253

Abdullah, Laila, P178

Abrahamson, Eric, P288

Adamczak, Stephanie, P260

Adeeb, Saleena, P121, P275

Adelson, P David, P115, P172, P173

Adkins, Deanna, P295, P355

Agin, Veronique, P058

Agrawal, Sandeep, P041, P042

Ahlers, Stephen, P162, P309, P332

Ajao, David, OC12

Akella, Trupti, P307

Al-Allaf, Faisal, P057

Alexander, Daya, P014

Alexandrova, Evgeniya, P109

Alger, Jeff, P128

Ali, Carine, P058

Alonso, Ofelia, P259, P265

Al Sulaimani, Sara, P263

Alves, Óscar L, P113

Amenta, Peter, P196, P197

Amoscato, Andrew, P036

Anagli, John, P022

Anderson, Gail, P315

Anderson, Gale, P189

Anderson, Iain, P030

Anderson, Joshua, P232

Anders, Stefan, P164, P165

Ang, Beng Ti, P134, P135, P16

Ardito, Tara, P174

Arenivas, Ana, P305

Arenth, Patricia, P278, P285

Armonda, Rocco, OC9

Armstead, William, OC15

Arun, Peethambaran, P037, P327

Arutyunov, Nikita, P108

Ashley, Jessica, P111, P112

Ashley, Mark, P111, P112

Ashwal, Stephen, OC12, P146

Atkins, Coleen, P018, P258, P264, P265

Atkins, Kristal D, OC14

Aurnhammer, Felix, P164, P165

Austin, James, P056

Avants, Brian, P309

Awe, Olatilewa, P197

Aygok, Gunes, P153

Ayoub, Kareem, OC8

Babos, Magor, P294

Bachmeier, Corbin, P177

Bachstetter, Adam, P238

Badaut, Jerome, OC12, P221

Bae, Kwang Soo, P181

Baguley, Ian, P091

Bagumyan, Arthur, P143

Bailes, Julian, P351, P352

Bains, Mona, P246

Bales, James W, P280

Baligand, Celine, P069

Bammler, Theo, P315

Bansal, Vishal, P222

Barber, Jason, P310, P311, P312, P313, P314

Barker, Peter, P137

Barnes, Amanda, P099, P100, P101

Bartley, Stephanie, P006

Bartnik Olson, Brenda, P146

Barzo, Pal, P294

Bass, Cameron, P117

Batchelor, Jenny, P091

Bauer, Joshua, P282, P283, P284

Baumgartner, Daniel, P155

Bayir, Hülya, P020, P036

Bayly, Philip, P090, P340

Bayon, Yves, P054

Bazan, Nicolas G, OC14

Bazarian, Jeffrey, P293

Beattie, Michael, P061, P062, P064, P065, P066, P067, P068

Bedi, Supinder, P306

Beers, Sue, P282

Belayev, Ludmila, OC14

Bellander, Bo-Michael, P141, P142

Bell, David, P308

Beller, Justin, P075

Beller, Justin A, P073

Bell, Michael, P115

Bell, Michael J, P116

Bell, Randy, OC9

Benavides, Francisco, P050

Benavides, Francsico, P083

Benson, Adam, P234

Benson, Randall, P341

Benton, Richard, P076, P077

Beray-Berthat, Virginie, P169

Berman, Nancy, P016, P230, P231, P232

Berman, Robert, P013

Bernal, Olivia, P272, P277

Besson, Valérie, P169

Betz, Joshua, P249

Beyer, Richard, P315

Beziaud, Tiphaine, P169

Bezin, Laurent, P133

Bhaskar, Kiran, P145

Bhatia, Harsharan, P060

Bhupendra, Kaphalia, P085

Bidot, Carlos, P266

Bidot, Carlos J, P257

Biggemann, Lionel, P037, P327

Bigler, Erin, OC8

Binenbaum, Gil, P307

Bishop, Alex, P176, P178

Bitner, Brittany, P103

Blaskiewicz, Donald, P060

Blaszczynska, Paulina, P165

Blusztajn, Jan, P106

Blyth, Brian, P293

Bochicchio, Grant, P253

Bohn, Martha, P010

Bomberger, Christine, P263

Bondada, Vimala, P072

Boone, Deborah, P298

Bose, Prodip, P069, P070, P149, P229

Bosworth, Christopher, P305

Bourdon, Lionel, P133

Boyle, Claire, P091

Bozkurt, Gokhan, P044

Braga, Carolina, P167, P225

Bragin, Denis, P271

Bramlett, Helen, P018, P028, P259, P261, P262, P263, P264, P265

Brasiliano Ferreira, Luiz C, P130

Brawner, Terri, P137

Bray, Eric R, P292

Brayer, Samuel, P285

Brelsfoard, Jennifer, P242, P243

Bresnahan, Jacqueline, P061, P062, P064, P065, P066, P067, P068

Brock, John, P061

Brooks, William, P140, P231, P233

Brophy, Gretchen, OC10, P167, P168, P317

Brown, Arthur, P089

Brown, Danielle, P172

Brown, Edward, P076

Browne, Karena, P196, P197

Browne, Kevin, P274, P275

Buchanan, Ashley, P235

Buckley, Jennifer, P064

Budde, Matthew, P156, P159

Budinich, Craig, P205

Buki, Andras, P317

Bu, Lihong, P107

Bullock, Ross, OC6, P113, P153, P211, P228, P255, P256, P257, P262, P263, P266

Bunton, Amanda, P188

Burke, Darlene, P076

Burke, Darlene A, P066

Burkhardt, Josh, P019

Burks, Stephen, P167

Burman, Hillary, P137, P139

Burns, Mark P, P126

Burton, Ellen, P204, P205

Bush, Rachel, P271

Buttram, Sandra, P172

Byrne, Richard, P179

Byrnes, Kimberly, P201

Caggiano, Anthony O, P336, P338

Cai, Ying, P199

Calabrese, Jordan, P263

Calabria, Silvia, P011

Calderon, Frances, P163, P254

Calulot, Christopher M, P075

Cameron, Andrew, P012

Campbell, John, P325

Campbell-Malone, Regina, P137, P139

Cannon, Steve, OC3

Cao, Tuoxin, P239

Cao, Xudong, OC4

Cao, Ying, P339

Cardenas, Diana, OC7

Carlson, Shaun, P243

Carre, Emilie, P133

Carrico, Kimberly, P236

Carrillon, Romain, P133

Cartagena, Casandra M, P335, P338

Carter, Michelle, P019, P278, P285

Casey, Noel, P106

Castellino, Frank, P131

Castello, Michael, P147

Catani, Sheila, P227

Caudle, Krista, P076

Cavanaugh, John, P343, P344, P346, P350

Cebak, John E, P031

Celix, Juanita, P310, P311, P312, P313, P314

Çetinkaya, Duygu, P044

Chaddock, Kelley, P310, P311, P312, P313, P314

Chang, Xiaoli, P199

Chan, Julie, P322

Chan, King, P202

Chan, Siew-Pang, P161

Chavko, Mikulas, P121, P275

Chawla, Aarti, P096

Chaytow, Helena, P177, P178

Cheng, Jeffrey P, P286

Cheng, Ji-Xin, P192

Chen, Jinhui, P092, P183, P184, P185

Chen, Liang, P092

Chen, Xiao Ru, P169

Chen, Yuhua, P089

Chen, Yungchia, P273

Chen, Zhiyong, P110, P328, P329, P330, P336, P338, P33

Cheong, Maxwell, P223

Cherian, Leela, P102, P103

Chesnut, Randall, P310, P311, P312, P313, P314

Chew, Derek, P007

Childs, Ed, P194

Chishti, Athar, P122

Cho, Angelo H, P171

Cho, Cheul, P254

Choi, In-Young, P233

Choi, Nicole, P023

Cho, Newton, P056

Chopp, Micheal, P129

Christensen, Randolph N, P062

Churn, Severn, P325

Chu, Zili, OC8

Cines, Douglas, OC15

Clark, Daniel, P010

Clark, Robert S B, P020, P036, P116

Clausen, Fredrik, P347

Clayton, Elyse, P188

Clayton, Erik, P340

Coats, Brittany, P307

Coats, Jacqueline, P147, P148, P221

Cohen, Akiva, P114, P274

Coimbra, Raul, P222

Coles, Jonathan, P123

Colgan, Niall, P347

Collante, David, P006

Colley, Beverly, P319

Combs, Hannah, P295

Conley, Yvette, P019, P278, P280, P281

Connley, Madeline, P180

Conti, Alana C, P349

Cook, Philip, P309

Cooper, Shelly, P015, P223

Cope, Elise, P124

Costine, Beth, P120

Cowart, Jeremy, P298

Cox, Charles, P306

Coyle, Matthew, OC4

Craciunas, Sorin C, P140

Crawford, Fiona, P176, P177, P178

Creasey, Graham, P067

Croci, Nicole, P171, P316

Cron, Courtney, P076

Cruz-Almeida, Yenisel, OC7, P005

Cruz, Jovany, P102

Crynen, Gogce, P176, P178

Cullen, Dkacy, P274, P275

Culver, Carlee, P305

Cummings, Peter, P107

Cunningham, Tracy, P326, P339

Curley, Kenneth, P093

Czerwieniec, Gregg, P211

Czigner, Andrea, P294

Dabiri, Borna, P027

Dahl, Ellen, P062, P064, P068

D'Alessandro, Tracy, P206

Dal Lago Mioto, Karen, P130

Dalton, Heidi, P172

Dambinova, Svetlana, P143

Dams-O'connor, Kristen, P158

Dams-O'connor, Kristin, P224

Danilenko, Uliana, P143

Dapul, Heda, P151

Dash, Pramod, P296, P306

Da Silva Carvalho, Fabiana, P130

Dave, Jitendra, P094, P110, P328, P329, P332, P335, P336, P338

Davidoff, Allen W, OC14

Day, Nicole, P206

Dechickera, Sonali, P052

Defina, Philip A, P164, P165

Dekaban, Gregory, P052

Dekosky, Steven, P288

Demery, Jason, OC10, P167

Deng-Bryant, Ying, P110, P328

Deng, Ping, P132

Denkbas, Emir Baki, P044

De Rivero Vaccari, Juan Pablo, P081, P082, P259, P260

Dewitt, Douglas, P297, P298, P299, P300, P301, P302

Dhammu, Tajinder Singh, P157

Dhandapani, Krishnan, P127

Diaz-Arrastia, Ramon, P303, P304, P305

Diaz, Daniel, P263

Diaz, Julio, P255, P266

Dietrich, Dalton, P033, P211, P262, P263

Dietrich, W Dalton, P018, P040, P080, P081, P259, P260, P264, P265

Dikmen, Sureyya, P310, P311, P312, P314

Ding, Xinchun, P183

Dinizo, Michael, P078

D'ippolito, Mariagrazia, P227

Dixit, Neha, P167

Dixon, C Edward, P020, P280, P280, P290, P290, P292, P292

Dkimen, Sureyya, P313

Dodson, Sean, P351

Donahue, Deborah, P131

Donaldson, Katherine, P076

Donlan, Nicole, P355

Donohue, Kevin, P071

Donovan, Virginia, P221

Doperalski, Adele, P318

Dore-Duffy, Paula, P040

Dragunow, Mike, P030

Dreier, Jens, OC6

Duchossoy, Yann, P133

Duckworth, Josh L, P137, P139

Duhaime, Ann-Christine, P120, P307

Du, Lina, P036

Dulin, Jennifer, OC5

Durham-Lee, Julieann, P086

Dziegielewska, Kate, P080

Eady, Tiffany N, OC14

Eakin, Katharine, P009

Eberwine, James, P035

Ecker, Robert, OC9

Ecklund, James, P055, P202, P203

Edgerton, Reggie, P061

Edlow, Brian, P107

Effgen, Gwen, P001, P024

Ek, C Joakim, P080

Eldahan, Khalid, P071

Eliceiri, Brian, P222

Elkin, Benjamin, P001, P119

Elkind, Jaclynn, P114

Elliot, Melanie, P193, P196, P197

Ellis, Gavin, P238

El Shafey, Nelly, P169

Emery, Evelyne, P058

Emery, Michael, P190

Empey, Philip, P115

Endo, Takashi, P051

Escosteguy Vargas, Andréia, P130

Esenaliev, Rinat, P043

Evans, Edward, P131

Fabian, Roderic, P103

Fabricius, Martin, OC6

Fadeeva, Ludmila, P108

Faden, Alan, P032, P078, P252

Falk, Jay, OC10

Fang, Lihuan, P096

Fanucchi, Francesca, P170

Fan, Weijia, P198

Fan, Weijia, P199

Fan, Xin Yan Susan, P088

Farin, Fred, P315

Fehlings, Michael, OC1, OC2, P007, P008, P054, P056

Fei, Zhou, P353

Felix, Elizabeth, OC7, P005

Feng, Cameron, P060

Feng, Jun-Feng, P209

Feng, Mengling, P134, P135

Ferguson, Adam, P061, P062, P064, P067, P067, P068

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Fernandez, Ana B, P211

Ferrell, Robert, P019, P278, P281

Figley, Sarah, P008

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Finger, Isaac, P021, P022

Fischer, Jason, P064

Fisher, Andrew, P106

Fisher, Daniel, OC3

Fisher, Joseph, P352

Fiskum, Gary, P250, P251

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Floyd, Thompson, P069

Foley, Lesley, P288

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Fong Hong, Lee, P257

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Foster, Paula, P052, P089

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Frangos, Suzanne, P272

Frank, Joseph, P159

Frantzén, Janek, P123

Frechou, Magalie, P169

Friedman, Bess, P272

Friess, Stuart, P195

Frisby, Melisa, P099

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Gao, Wei, P353

Gao, Xiang, P092, P183, P184, P185

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Garcia-Canet, Cesar, P050

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Garcia-Filion, Pamela, P172

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Gerhardt, Greg, P247, P248

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Ghavim, Sima, P212

Ghies, José Artur, P130

Ghoddoussi, Farhad, P342

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Gilmer, Lesley, P246

Girgis, Haymen, P316

Giza, Christopher, P210, P215

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Glavaski-Joksimovic, A, P010

Glenn, Thomas C, P211

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Goldstein, Lee, P106

Golshani, Roozbeh, P033

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Gonzalez-Lara, Laura, P089

Goodman, J Clay, P104

Gordon, Wayne, P158, P223, P224

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Grady, Msean, P274

Grau, James, P068

Greenberg, Steven, P107

Greer, David, P107

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Gregory, Eugene, P231, P232

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Grill, Raymond J, P301

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Guan, Cuntai, P134

Gu, Chengwu, P039

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Guo, Wenyuan, P134

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Hanks, Robin, P341

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Hanscom, Marie, P032

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Harper, Caryn, P303, P304

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Harris, Janna, P233

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Harris, Jessie, P335

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Hartman, Richard, OC12

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Hassler, Shayne, P085

Hatz, Laura, P281

Hawkins, James, P065

Hawryluk, Gregory, P007

Hayakawa, Kentaro, P051

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Hebert, Marie, P058

Heffernan, Ann, P015

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Hemphill, Matthew, P027

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Hochheimer, Sven, OC9

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Hockenbury, Nicole, P201

Hockey, Kevin, P174

Hodge, Jessica, P023

Hoe, Hyang-Sook, P126

Hoffer, Barry, P009

Hoffman, Eric, P025

Hoffman, Joshua, P241

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Hricik, Allison, P279, P282

Htun, Ja Lu, P135

Huang, Huiling, P198, P199

Huang, Lei, P147, P148, P221

Hue, Christopher, P117

Huie, J Russell, P062, P068

Hulsebosch, Claire, P084, P085, P087, P301

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Hunter, Jill, OC8

Hurwitz, Max, P285, P289

Hyson, Morton, P268

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Ikezu, Tsuneya, P106

Ikonomovic, Milos, P288

Ikuta, Nilo, P130

Ilic, Sanja, P167

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Jaiswal, Poonam, P149

Jaiswal, Shalini, P201

Jallo, Jack, P196, P197

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Janes, Lindsay, P159

Janowich, Jacqueline, P249

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Jergova, Stanislava, P006, P258

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Jiang, Shijie, P039

Jiang, Yuhui, P163, P254

Ji, Jing, P036

Jimenez, Fernando, P306

Jin, Xiaoming, P192

Jin, Xin, P347

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Johnson, Jami, P267

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Johns, Roger, P049

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Kalani, Yashar, P098

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Kang, Eugene, P012

Kang, Woo Hyeun, P118

Kang, Yuan, P258, P264

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Katyshev, Vladimir, P040

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Khalid, Tabindeh, P038

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Kim, Junghoon, P309

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Russell, Michael, P208

Rzigalinski, Beverly, P174

Saatman, Kathryn, P234, P242, P243, P244

Saavedra, Juan M, P200

Saez, Pedro, P128

Sagen, Jacqueline, P006

Sajja, Sujith, P342

Sakai, Jun, P351

Sakakima, Harutoshi, P157

Sakar, Mustafa, P044

Sakowitz, Oliver, OC6

Sakurai, Atsushi, P258, P262, P265

Salevitz, Nicole, P173

Salonia, Rosanne, P115

Sampat, Mehul, P015

Samuelson, Rodney, P098

Sanchez-Chavez, Jose, P256

Sanchez, Francisco, P079

Sanchez, Jose, P255, P257, P266

Sanchez, Juliana, P261

Sanchez-Lemus, Enrique, P200

Sanchez-Molina, Juliana, P028

Sandhir, Rajat, P232

Santamaria, Andrea, P083

Santamaria, Jimena, P050

Santa Maria, Naomi, P215

Saraswati, Manda, P138

Sarode, Ravi, P303

Satkunedrarajah, Kajana, P056

Saucedo, Hector, P104

Saunders, Norman, P080

Sawant, Devendra, P194

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Scanlon, Joelle, P019, P278, P281

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Schell, Michael, P029

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Schnyer, David, P224, P279

Schoch, Kathleen M, P244

Schommer, Julie, P180

Schroeder, Tim, P164, P165

Schrot, John, P204

Schwab, Stefan, P164

Schwartzkroin, Philip, P013

Scrimgeour, Angus, P124

Scruggs, Bobby, P282

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Sehli, Deema, P038

Seibert, Pennie, P180

Seidl, Stacey, P010, P122

Sell, Stacy, P300

Selwyn, Reed, P201

Seneca, Philip, P112

Sennhauser, Susie, P070, P149, P229

Shabashvili, Arseniy, P021

Shah, Alok, P154

Shahlaie, Kiarash, P013

Shah, Mansi A, P286

Shah, Shinil, P306

Shanawani, Hasan, P038

Shanmugalingam, U, OC4

Shanmuganathan, K, P249

Shaughness, Michael, P162, P332

Shear, Deborah, P094, P110, P162, P326, P328, P329, P330, P331, P333, P334, P336, P337, P338, P339

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Sheng, Morgan, OC3

Sherck, John, P191

Sherman, Alexandra, P093

Shikuev, Alex, P143

Shin, Samuel S, P292

Shin, Sang Do, P136, P182

Shoemaker, James, P026

Shokri-Kojori, Ehsan, P305

Shum-Siu, Alice, P076

Shunmugavel, A, P157

Shutter, Lori, OC6

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Singh, Avtar, P157

Singh, Inderjit, P157

Singh, Indrapal N, P031

Singh, Pallab, P034

Singh, Ranjana, P234

Sinha, Tuhin, P223

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Stewart, William, P177, P276

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Tremeau, Sebastien, P133

Trojanowski, John Q, P276

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Zhao, Zaorui, P252

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Zheng, Wenrong, P097

Zhou, Huichao, P287

Zhou, Runzhou, P343, P344, P346

Zhuang, Yumei, P059

Zhu, Feng, P347

Zhuo, Jiachen, P250

Zhu, Xiaoxia, P150, P152

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Zou, Huichao, P285, P289, P291

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