Sports concussion is a major problem affecting thousands of people in North America every year. Despite negative neuroimaging findings many athletes display neurophysiological alterations and post-concussion symptoms such as headaches and sensitivity to light and noise. It is suspected that neurometabolic changes may underlie these changes. The present study thus investigated the effects of sports concussion on brain metabolism using 1H-MR Spectroscopy by comparing a group of 12 non-concussed athletes with a group of 12 concussed athletes of the same age (mean: 22.5) and education (mean: 16 yrs). All athletes were scanned 24-36 hours post-concussion in a 3T Siemens MRI as well as administered a symptom scale to evaluate post-concussion symptomatology. Participants also completed a neuropsychological test battery to assess verbal memory, visual memory, information processing speed, and reaction time where no group differences were detected relative to controls. Concussed athletes showed a higher level of symptoms than non-concussed athletes and they also showed a significant decrease in glutamate in primary motor cortex as well as significant decrease in N-acetyl aspartate in prefrontal and primary motor cortices. No changes were observed in the hippocampus. Furthermore, the metabolic changes in M1 correlated with self-reported symptom severity despite equivalent neuropsychological performance. These results confirm cortical vulnerability in the acute postconcusion phase and demonstrate for the first time a correlation between subjective self-reported symptoms and objective physical changes, namely cortical metabolic anomalies.

Increases of synaptically released zinc and intracellular accumulation of zinc in hippocampal neurons after traumatic or ischemic brain injury is neurotoxic and chelation of zinc has been shown to reduce neurodegeneration. Although our previous studies showed that zinc chelation in traumatically brain-injured rats correlated with an increase in whole-brain expression of several neuroprotective genes and reduced numbers of apoptotic neurons, the effect on functional outcome has not been determined, and the question of whether this treatment may actually be clinically relevant has not been answered. In the present study, we show that treatment of TBI rats with the zinc chelator calcium EDTA reduces the numbers of injured, Fluoro-Jade-positive neurons in the rat hippocampus 24h after injury but does not improve neurobehavioral outcome (spatial memory deficits) 2 weeks post-injury. Our data suggest that zinc chelation, despite providing short-term histological neuroprotection, fails to improve long-term functional outcome, perhaps because long-term disruptions in homeostatic levels of zinc adversely influence hippocampus-dependent spatial memory.

Memory impairment is one of the most significant residual deficits following traumatic brain injury (TBI) and is among the most frequent complaints heard from patients and their relatives. It has been reported that the hippocampus is particularly vulnerable to TBI, which results in hippocampus-dependent cognitive impairment. There are different regions in the hippocampus, and each region is composed of different cell types, which might respond differently to TBI. However, regional and cell type-specific neuronal death following TBI is not well described. Here, we examined the distribution of degenerating neurons in the hippocampus of the mouse brain following controlled cortical impact (CCI) and found that the majority of degenerating neurons observed were in the dentate gyrus after moderate (0.5 mm cortical deformation) CCI-TBI. In contrast, there were only a few degenerating neurons observed in the hilus, and we did not observe any degenerating neurons in the CA3 or CA1 regions. Among those degenerating cells in the dentate gyrus, about 80% of them were found in the inner granular neuron layer. Analysis with cell type-specific markers showed that most of the degenerating neurons in the inner granular neuron layer are newborn immature neurons. Further quantitative analysis shows that the number of newborn immature neurons in the dentate gyrus is dramatically decreased in the ipsilateral hemisphere compared with the contralateral side. Collectively, our data demonstrate the selective death of newborn immature neurons in the hippocampal dentate gyrus following moderate injury with CCI in mice. This selective vulnerability of newborn immature dentate neurons may contribute to the persistent impairment of learning and memory post-TBI and provide an innovative target for neuroprotective treatment strategies.(c) 2008 Wiley-Liss, Inc.

OBJECTIVES: Our objective was to characterize the heat shock response (HSR) in a model of traumatic brain injury (TBI) and to determine the association of HSR to cell death. METHODS: We used immunofluorescent detection of HSP-70 to characterize HSR and TUNEL labeling to determine the pattern of cell death. RESULTS: HSP-70 immunofluorescence revealed a steady increase from 4 to 48 hours following TBI, culminating in a ubiquitous expression with the capillary bed 48 hours post-TBI. TUNEL labeling revealed a small subset of endothelial cell death and a most robust staining of putative pericyte cell death. DISCUSSION: Our results show that while injury causes a detectable stress response, cell death is not a direct consequence of the HSR.

Studies involving animal models of acute central nervous system (CNS) stroke and trauma strongly indicate that sex and/or hormonal status are important determinants of outcome after brain injury. The present study was undertaken to examine the ability of estradiol to protect hippocampal neurons from lateral fluid percussion brain injury. Sprague-Dawley female rats (211-285 g; n = 119) were ovariectomized, and a subset (n = 66) were implanted with 17beta-estradiol pellets to provide near physiological levels of estradiol. Animals were subjected to lateral fluid percussion brain injury or sham injury 1 week later. Activation of caspase-3 (n = 26) and TUNEL staining (n = 21) were assessed at 3 and 12 h after injury, respectively, in surviving control and estradiol-treated animals. Memory retention was examined using a Morris water maze test in a separate subset of animals (n = 43) at 8 days after injury. Activated caspase-3 and TUNEL staining were observed in the dentate hilus, granule cell layer, and CA3 regions in all injured rats, indicative of selective hippocampal cell apoptosis in the acute posttraumatic period. Estradiol did not significantly alter the number of hippocampal neurons exhibiting caspase-3 activity or TUNEL staining. Brain injury impaired cognitive ability, assessed at 1 week post-injury (p < 0.001). However, estradiol at physiological levels did not significantly alter injury-induced loss of memory. These data indicate that estradiol at physiological levels does not ameliorate trauma-induced hippocampal injury or cognitive deficits in ovariectomized female rats.

Two mechanisms of brain cell death coexist, necrosis and apoptosis. We investigated the correlation between the apoptotic index and the expression of apoptosis modulators and stress response in an ultraviolet-induced cortical microinfarct. Adult rat neocortex was exposed to an ultraviolet beam and brains removed at different intervals after injury were paraffin-embedded and processed for TUNEL assay and immunohistochemistry against apoptotic modulators Bax and Bcl-2, and stress protein HSP70. During the 12-72h postirradiation period, apoptotic nuclei decreased from 11% to 4% in the infarcted area whereas only 1.2% of such nuclei was seen in the perilesional area. While Bcl-2 was always negative in the lesion focus, Bax was positive at all survival times, mainly in glial cells. HSP70 was expressed over a broad area of the ipsilateral hemisphere from 3h after brain injury, firstly in neurons and progressively in glial cells and finally in endothelium. At longer survival times, positive cells could be also seen in the contralateral hemisphere. Apoptosis seems to play only a quantitatively modest role in the progression of brain damage in penumbra areas despite the wide expression of pro-apoptotic factors. On the other hand HSP70 appears to be one of the main protective responses to injury stress.

Target ablation (removal of the olfactory bulb, OBX) induces apoptotic death of olfactory sensory neurons (OSNs) and an immune response in which activation and recruitment of macrophages (mphis) into the olfactory epithelium (OE) occupies a central role. Mphis phagocytose apoptotic neurons and secrete cytokines/growth factors that regulate subsequent progenitor cell proliferation and neurogenesis. Scavenger receptor A (SR-A) is a pattern recognition receptor that mediates binding of mphis to apoptotic cells and other relevant immune response functions. The aim of this study was to determine the impact of the absence of SR-A on the immune response to OBX. The immune response to OBX was evaluated in mice in which functional expression of the mphi scavenger receptor (MSR) was eliminated by gene disruption (MSR(-/-)) and wild type (wt) mice of the same genetic background. OBX induced significant apoptotic death of mature OSNs in the 2 strains. However, subsequent mphi infiltration and activation and progenitor cell proliferation were significantly reduced in MSR(-/-) vs. wt mice. Gene expression profiling at short intervals after OBX demonstrated significant differences in temporal patterns of expression of several gene categories, including immune response genes. Many immune response genes that showed different temporal patterns of expression are related to mphi function, including cytokine and chemokine secretion, phagocytosis, and mphimaturation and activation. These studies suggest that impairment of the immune response to OBX in the OE of MSR(-/-) mice most likely resulted from decreased mphi adhesion and subsequent reduced infiltration and activation, with a resultant decrease in neurogenesis.

Lateral fluid percussion injury (LFP), a model of mild-moderate concussion, leads to the temporary loss of the capacity for experience-dependent plasticity in developing rats. To determine if this injury-induced loss in capacity for plasticity is due to cell death, we conducted stereological measurements within the cerebral cortex and CA3 of the hippocampus 2 weeks following mild, moderate or severe LFP in the post-natal day 19 (P19) rat. Results indicated that there was no significant change in the absolute number of neurons, regardless of injury severity, in either the ipsilateral cortex (sham = 10.6 +/- 1.7, mild = 11.5 +/- 2.1, moderate = 10.0 +/- 1.0, severe = 10.9 +/- 1.3 million neurons) or CA3 region of the hippocampus (sham = 251 +/- 38, mild = 289 +/- 2, moderate = 245 +/- 48, severe = 255 +/- 62 thousand neurons). Even though there was no evidence of a significant degree of injury-induced cell death, animals exhibited cognitive deficits as revealed in a Morris water maze task (MWM). The MWM results indicated that regardless of injury severity, P19-injured rats exhibited a significant increase in escape latency compared to age-matched shams (injury by day; P < 0.001) and a significant increase in the number of trials needed to reach criterion (P < 0.05). Analysis of a probe trial one week post-MWM training, however, indicated that there was no deficit in storage or recall of the learned behavior as analyzed by platform hits (sham = 2.9 +/- 0.37, mild = 2.0 +/- 0.40, moderate = 1 +/- 0, severe = 2.8 +/- 0.62) or percent time spent in, or immediately surrounding, the platform area (sham = 13.5 +/- 1.71, mild = 10.8 +/- 2.32, moderate = 12.7 +/- 0, severe = 13.5 +/- 1.69). Taken together, these results indicate that while LFP in P19-injured animals does not lead to significant cell death, it does generate acute, mild deficits in MWM performance.

Drug development for CNS disorders faces the same formidable hurdles as other therapeutic areas: escalating development costs; novel drug targets with unproven therapeutic potential; and health care systems and regulatory agencies demanding more compelling demonstrations of the value of new drug products. Extensive clinical testing remains the core of registration of new compounds; however, traditional clinical trial methods are falling short in overcoming these development hurdles. The most common CNS disorders targeted for drug treatment are chronic, slowly vitiating processes manifested by highly subjective and context dependent signs and symptoms. With the exception of a few rare familial degenerative disorders, they have ill-defined or undefined pathophysiology. Samples selected for treatment trials using clinical criteria are inevitably heterogeneous, and dependence on traditional endpoints results in early proof-of-concept trials being long and large, with very poor signal to noise. It is no wonder that pharmaceutical and biotechnology companies are looking to biomarkers as an integral part of decision-making process supported by new technologies such as genetics, genomics, proteomics, and imaging as a mean of rationalizing CNS drug development. The present review represent an effort to illustrate the integration of such technologies in drug development supporting the path of individualized medicine.

Increases of synaptically released zinc and intracellular accumulation of zinc in hippocampal neurons after traumatic or ischemic brain injury is neurotoxic and chelation of zinc has been shown to reduce neurodegeneration. Although our previous studies showed that zinc chelation in traumatically brain-injured rats correlated with an increase in whole-brain expression of several neuroprotective genes and reduced numbers of apoptotic neurons, the effect on functional outcome has not been determined, and the question of whether this treatment may actually be clinically relevant has not been answered. In the present study, we show that treatment of TBI rats with the zinc chelator calcium EDTA reduces the numbers of injured, Fluoro-Jade-positive neurons in the rat hippocampus 24h after injury but does not improve neurobehavioral outcome (spatial memory deficits) 2 weeks post-injury. Our data suggest that zinc chelation, despite providing short-term histological neuroprotection, fails to improve long-term functional outcome, perhaps because long-term disruptions in homeostatic levels of zinc adversely influence hippocampus-dependent spatial memory.

BACKGROUND: After traumatic brain injury, memory dysfunction is due in part to damage to the hippocampus. To study the molecular mechanisms of this selective vulnerability, the authors used laser capture microdissection of neurons stained with Fluoro-Jade to directly compare gene expression in injured (Fluoro-Jade-positive) and adjacent uninjured (Fluoro-Jade-negative) rat hippocampal neurons after traumatic brain injury and traumatic brain injury plus hemorrhagic hypotension. METHODS: Twelve isoflurane-anesthetized Sprague-Dawley rats underwent moderate (2.0 atm) fluid percussion traumatic brain injury followed by either normotension or hemorrhagic hypotension. Animals were killed 24 h after injury. Frozen brain sections were double stained with 1% cresyl violet and 0.001% Fluoro-Jade. RNA from 10 Fluoro-Jade-positive neurons and 10 Fluoro-Jade-negative neurons, obtained from the hippocampal CA1, CA3, and dentate gyrus subfields using laser capture microdissection, was linearly amplified and analyzed by quantitative ribonuclease protection assay for nine neuroprotective and apoptosis-related genes. RESULTS: In injured CA3 neurons, expression of the neuroprotective genes glutathione peroxidase 1, heme oxygenase 1, and brain-derived neurotrophic factor was significantly decreased compared with that of adjacent uninjured neurons. Superimposition of hemorrhagic hypotension was associated with down-regulation of neuroprotective genes in both injured and uninjured neurons of all subregions. Expression of apoptosis-related genes did not vary between injured and uninjured neurons, with or without superimposed hemorrhage. CONCLUSIONS: The authors show, in the first direct comparison of messenger RNA levels in injured and uninjured hippocampal neurons, that injured neurons express lower levels of neuroprotective genes than adjacent uninjured neurons.

Hippocampal damage contributes to cognitive dysfunction after traumatic brain injury (TBI). We previously showed that Fluoro-Jade, a fluorescent stain that labels injured, degenerating brain neurons, quantifies the extent of hippocampal injury after experimental fluid percussion TBI in rats. Coincidentally, we observed that injured neurons in the rat hippocampus also stained with Newport Green, a fluorescent dye specific for free ionic zinc. Here, we show that, regardless of injury severity or therapeutic intervention, the post-TBI population of injured neurons in rat hippocampal subfields CA1, CA3 and dentate gyrus is indistinguishable, both in numbers and anatomical distribution, from the population of neurons containing high levels of zinc. Treatment with lamotrigine, which inhibits presynaptic release of glutamate and presumably zinc that is co-localized with glutamate, reduced numbers of Fluoro-Jade-positive and Newport Green-positive neurons equally as did treatment with nicardipine, which blocks voltage-gated calcium channels through which zinc enters neurons. To confirm using molecular techniques that Fluoro-Jade and Newport Green-positive neurons are equivalent populations, we isolated total RNA from 25 Fluoro-Jade-positive and 25 Newport Green-positive pyramidal neurons obtained by laser capture microdissection (LCM) from the CA3 subfield, linearly amplified the mRNA and used quantitative ribonuclease protection analysis to demonstrate similar expression of mRNA for selected TBI-induced genes. Our data suggest that therapeutic interventions aimed at reducing neurotoxic zinc levels after TBI may reduce hippocampal neuronal injury.

In head-injured patients and experimental traumatic brain injury (TBI), important cerebrovascular abnormalities include decreases in cerebral blood flow (CBF) and impairment of cerebral pressure autoregulation. We evaluated CBF and pressure autoregulation after fluid percussion injury (FPI) and hypothermia in rats with the hypothesis that hypothermia would ameliorate changes in posttraumatic CBF. Male Sprague-Dawley rats, intubated and mechanically ventilated, were prepared for parasagittal FPI (1.8 atm) and laser Doppler CBF flow (LDF) measurement. The abdominal aorta was cannulated for rapid removal and reinfusion of blood. Baseline autoregulatory testing in all groups consisted of LDF measurements at normothermia and a mean arterial pressure (MAP) of 100 mm Hg, followed by randomly ordered changes of MAP to 80, 60, and 40 mm Hg. Animals were then randomized to one of five groups: normothermic control without FPI; normothermia with FPI; hypothermic control (32 degrees C) without FPI; hypothermia initiated before FPI; and hypothermia initiated immediately after FPI injury. For each group, a complete, randomly ordered autoregulatory sequence was performed at 30 and 60 min after FPI or sham TBI. In a second study, rats were prepared identically, maintained at normothermic temperatures and autoregulation was tested before and after TBI using a set of randomly ordered levels of hypotension or using progressive reductions in MAP (i.e., 80, 60, 40 mm Hg) with the hypothesis that the technical manner and timing of decreasing of the blood pressure would effect CBF after TBI. Due to high acute mortality, the group in which hypothermia was induced before FPI was excluded from the analysis. At baseline, autoregulation was similar in all groups. There was no change in CBF or autoregulation in the normothermic control group at 30 and 60 min. In the other groups at 30 and 60 min, there was a similar, statistically significant decrease in absolute CBF (i.e., a decrease of 27-57% of baseline values), but pressure autoregulation was intact except at the lowest blood pressure tested at 60 min, where there was a slight improvement in the hypothermic group. Thus, in these experiments, absolute CBF decreased with hypothermia and FPI, while neither hypothermia nor FPI significantly altered autoregulation. In the second study, autoregulatory function was not different before TBI when comparing random and sequential blood pressure changes, but, when comparing the groups after TBI at the 60 mm Hg blood pressure level, CBF was significantly lower in the sequential group than in the random order group. This suggests that the mechanism of creating hypotension, whether random or sequential, significantly affects the measurement of CBF and autoregulation after TBI in rats.

Chelation of excessive neuronal zinc ameliorates zinc neurotoxicity and reduces subsequent neuronal injury. To clarify the molecular mechanisms of this neuroprotective effect, we used a focused cDNA array of stress-response genes with zinc chelation (calcium EDTA) in our rat model of fluid percussion brain injury at 2 h, 24 h, and 7 days after injury. In parallel experiments, we compared neuronal cell death in TUNEL-stained brain sections in traumatized rats with and without calcium EDTA treatment. Zinc chelation induced the expression of several neuroprotective genes; neuroprotective gene expression correlated with substantially decreased numbers of TUNEL-positive cells.

Peroxynitrite is a powerful oxidant capable of nitrating phenolic moieties, such as tyrosine or tyrosine residues in proteins and increases after traumatic brain injury (TBI). First, we tested the hypothesis that TBI increases nitrotyrosine (NT) immunoreactivity in the brain by measuring the number of NT-immunoreactive neurons in the cerebral cortex and hippocampus of rats subjected to parasagittal fluid-percussion TBI. Second, we tested the hypothesis that treatment with L-arginine, a substrate for nitric oxide synthase, further increases NT immunoreactivity over TBI alone. Rats were anesthetized with isoflurane and subjected to TBI, sham TBI, or TBI followed by treatment with L-arginine (100 mg/kg). Twelve, 24, or 72 h after TBI, brains were harvested. Coronal sections (10 mum) were incubated overnight with rabbit polyclonal anti-NT antibody, rinsed, and incubated with a biotinylated secondary antibody. The antigen-antibody complex was visualized using a peroxidase-conjugated system with diaminobenzidine as the chromagen. The number of NT-positive cortical and hippocampal neurons increased significantly in both ipsilateral and contralateral hemispheres up to 72 h after TBI compared with the sham-injured group. Remarkably, treatment with L-arginine reduced the number of NT-positive neurons after TBI in both cortex and hippocampus. Our results indicate that L-arginine actually prevents TBI-induced increases in NT immunoreactivity.Journal of Cerebral Blood Flow & Metabolism advance online publication, 9 July 2008; doi:10.1038/jcbfm.2008.66.

ABSTRACT Explosive munitions account for more than 50% of all wounds sustained in military combat, and the proportion of civilian casualties due to explosives is increasing as well. But there has been only limited research on the pathophysiology of blast-induced brain injury, and the contributions of alterations in cerebral blood flow (CBF) or cerebral vascular reactivity to blast-induced brain injury have not been investigated. Although secondary hypotension and hypoxemia are associated with increased mortality and morbidity after closed head injury, the effects of secondary insults on outcome after blast injury are unknown. Hemorrhage accounted for approximately 50% of combat deaths, and the lungs are one of the primary organs damaged by blast overpressure. Thus, it is likely that blast-induced lung injury and/or hemorrhage leads to hypotensive and hypoxemic secondary injury in a significant number of combatants exposed to blast overpressure injury. Although the effects of blast injury on CBF and cerebral vascular reactivity are unknown, blast injury may be associated with impaired cerebral vascular function. Reactive oxygen species (ROS) such as the superoxide anion radical and other ROS, likely major contributors to traumatic cerebral vascular injury, are produced by traumatic brain injury (TBI). Superoxide radicals combine with nitric oxide (NO), another ROS produced by blast injury as well as other types of TBI, to form peroxynitrite, a powerful oxidant that impairs cerebral vascular responses to reduced intravascular pressure and other cerebral vascular responses. While current research suggests that blast injury impairs cerebral vascular compensatory responses, thereby leaving the brain vulnerable to secondary insults, the effects of blast injury on the cerebral vascular reactivity have not been investigated. It is clear that further research is necessary to address these critical concerns.

BACKGROUND:: Resuscitation with hypertonic saline or hypertonic saline plus l-arginine acutely improves cerebral blood flow after traumatic brain injury (TBI) followed by hemorrhagic hypotension. The authors investigated whether hypertonic saline or hypertonic l-arginine would improve long-term neuronal survival and behavioral outcomes 15 days after TBI and hemorrhagic hypotension. METHODS:: Mean arterial pressure, arterial blood gases, pH, plasma glucose, hematocrit, and hemoglobin were measured in male Sprague-Dawley rats before and after moderate (2.0 atm) fluid percussion TBI. Rats were assigned to one of six groups:(1) sham TBI,(2) hemorrhage only,(3) TBI only,(4) TBI plus hemorrhage and resuscitation with 0.9% saline,(5) TBI plus hemorrhage and resuscitation with hypertonic saline (7.5%), or (6) TBI plus hemorrhage and resuscitation with l-arginine (100 mg/kg) in hypertonic saline. On postinjury days 1-5, vestibulomotor function was assessed using beam balance and beam walking tasks. On postinjury days 11-15, spatial memory function was assessed using the Morris water maze. After behavioral testing, neuronal counting was performed bilaterally on specific hippocampal regions. RESULTS:: Groups receiving hypertonic saline (P < 0.05, day 15 vs. day 11) or hypertonic l-arginine (P < 0.05, days 13-15 vs. day 11) showed improved performance over time on the Morris water maze, as well as significantly improved neuronal survival in the contralateral hippocampus (P < 0.05, hypertonic saline or hypertonic l-arginine vs. normal saline) compared with untreated TBI or normal saline-treated TBI plus hemorrhage groups. CONCLUSIONS:: Hypertonic saline and hypertonic l-arginine were both effective at promoting long-term neuronal survival and behavioral recovery. The slightly earlier improvement in Morris water maze performance in the hypertonic l-arginine group warrants further studies to determine whether higher doses of l-arginine provide additional improvement. This study supports the therapeutic benefits of hypertonic resuscitation after TBI plus hemorrhagic hypotension.

Aged traumatic brain injury (TBI) patients suffer higher rates of mortality and disability than younger patients. Cognitive problems common to TBI patients are associated with damage to the hippocampus, a central locus of learning and memory. To investigate the molecular mechanisms of age-related vulnerability to brain injury in a mouse model of TBI, we studied the effects of TBI on hippocampal gene expression in young and aged mice. Young and aged male C57Bl/6 mice were subjected to sham injury or TBI and sacrificed 24h post-injury. We used laser capture microdissection to obtain pure populations of neurons from the CA1, CA3, and dentate gyrus subfields of the hippocampus. We compared injury-induced gene expression in hippocampal neurons of young and aged mice using quantitative ribonuclease protection assay analysis of linearly amplified mRNA from laser captured neurons. Both increased age and TBI were associated with increased expression of neuroprotective (brain-derived neurotrophic factor), pro-inflammatory (interleukin-1beta), and proapoptotic (caspase-3) genes in mouse hippocampal neurons. Our data support previous reports that suggested the CA3 subregion is highly susceptible to fluid percussion TBI and that age-related changes in gene expression are one potential mechanism of increased vulnerability of the aged brain to TBI.

The goal of the present study was to determine in vivo whether peroxynitrite, at the concentration and duration produced by SCI, contributes to membrane lipid peroxidation (MLP) after traumatic spinal cord injury (SCI) and the capability of a broad spectrum scavenger of reactive species, Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), to reduce MLP. This was accomplished by administering a peroxynitrite donor 3-morpholinosydnonimine (SIN-1) into the gray matter of an uninjured rat spinal cord through a microdialysis fiber to generate ONOO() at the SCI-elevated levels. The resulting MLP was characterized by measuring the productions of extracellular malondialdehyde and of intracellular 4-hydroxynonenal. We demonstrated that extracellular SIN- 1 administration significantly increased the concentration of malondialdehyde (p < 0.001) and the numbers of hydroxynonenal-positive cells (p < 0.001) as compared to a control group in which ACSF was administered. Simultaneous administration of MnTBAP through a second microdialysis fiber significantly reduced SIN-1-induced malondialdehyde production (p < 0.001) and the numbers of HNE-positive cells (p < 0.001). There was no significant difference between MnTBAP-treated and ACSF-controls (p = 0.3). These results demonstrate in vivo that (1) SCI-produced levels of peroxynitrite sufficient to cause MLP, and therefore that peroxynitrite is an agent of secondary damage after acute SCI;(2) MnTBAP can efficiently reduce SIN-1-induced MLP.

OBJECTIVES: Given after traumatic brain injury (TBI), Dexamethasone (DXM) reduces cerebral edema but impairs retrograde memory. This study was designed to test the hypothesis that administration of DXM to rats with TBI promotes learning deficits that are correlated with the morphological changes of hippocampal pyramidal neurons. METHODS: Adult male Wistar rats were subjected to fluid percussion injury (FPI), received DXM (5 and 10 mg/kg), and then trained for spatial acquisition. Brain sections were examined by H.E. and Golgi impregnation to quantitatively measure the morphological changes of hippocampal pyramidal neurons. RESULTS: The latency and path length were significantly higher in rats with FPI than those in control groups, particularly in rats receiving post-trauma high-dose DXM. At the same time, Golgi impregnation revealed a significant decrease in the number of apical and basal dendrites of pyramidal neurons of ipsilateral hippocampus in rats after injury, but the decrease was greatest of CA3 pyramidal neurons of ipsilateral hippocampus in injured rats that also received high-dose DXM. DISCUSSION: These findings indicate that the administration of high-dose DXM after TBI could worsen the dendritic atrophy of hippocampal CA3 pyramidal neurons and, as a result, exacerbate spatial acquisition deficits.

The integrity of the arterial baroreflex is central to cardiovascular homeostasis. There is evidence of altered cardiovascular regulation following acute traumatic brain injury (TBI). We hypothesised that arterial baroreflex is modified by acute TBI. An experimental study using 18 terminally anaesthetized male Wistar rats weighing 240-260g was undertaken at a University Laboratory setting. Brain injury was induced using the lateral fluid percussion (LFP) brain injury model. The fluid percussion device delivered an applied cortical pressure of 1.2atm and 1.8atm, producing mild and moderate TBI respectively. Control animals underwent identical surgical procedures but no applied cortical pressure. Arterial baroreflex was assessed by determining the relationship between heart period (R-R interval) and systolic blood pressure using the modified phenylephrine pressor test adapted for the rat. The arterial baroreflex was tested before (Tcon), post TBI, at 10 mins (T10) and 30 mins (T30). Analysis of baroreflex function following moderate TBI using repeated measures ANOVA revealed significant differences in baroreflex sensitivity (BRS) at T10 and T30 (F-ratio=10.18, df =2,15, p=0.005) compared to pre-TBI (Tcon: 0.39+/-0.00ms.mmHg; T10: 0.85+/-0.01ms.mmHg; T30: 0.81+/-0.01ms.mmHg, weighted mean +/- SD). The changes in BRS were not significant following mild TBI (p = 0.152). Repeated measures ANOVA comparing trends between the three groups indicated significant differences between the control and moderate TBI groups only (F ratio = 6.26, df =2,15, p =0.01). Acute TBI of moderate severity is associated with an early significant modification in arterial baroreflex sensitivity. This is a key component of cardiovascular homeostasis the clinical implications of this observation require further investigation.

The temporal and regional expression profiles of matrix metalloproteinases-9 (MMP-9), after moderate or severe traumatic brain injury (TBI) were measured to investigate the effects of posttraumatic hypothermia (33 degrees C) or hyperthermia (39 degrees C). In the first phase of this study, adult male Sprague-Dawley rats were randomly assigned to the groups of moderate TBI (1.8-2.2 atmospheres), severe TBI (2.4-2.7 atmospheres), and sham injured control. Rats were killed at 4, 6, 12, 24, 48, 72 hours or 1 week after TBI, for relative mRNA and protein analysis. In the second phase, rats underwent moderate fluid-percussion brain injury, followed immediately by 4 hours of posttraumatic normothermia (37 degrees C), hyperthermia (39 degrees C), or hypothermia (33 degrees C), rats were killed at 12 and 48 hours after TBI for mRNA expression analyses. Other groups were killed at 24, 72 hours after TBI for protein expression analyses. MMP-9 levels in both ipsilateral and contralateral hemispheres were significantly increased after TBI compared with those of sham injured (p < 0.01). Two expression peaks of MMP-9 were observed in ipsilateral cortex and hippocampus. An Increase in injury severity was associated with an increase in the respective mRNA (12 and 48 hours) and protein (24 and 72 hours) levels of MMP-9. Posttraumatic hypothermia attenuated the increase in both the mRNA and protein levels of MMP-9, compared with normothermia and hypothermia (P<0.01). In contrast, hyperthermia had no significant effect on mRNA (12 hours) and protein levels (24 hours) of MMP-9, compared with normothermic values (P>0.05), but resulted in the significant increase in the levels of MMP-9 mRNA and protein at 24 or 72 hours respectively (P<0.01). Injury severity determines the elevation degree of MMP-9 mRNA and protein levels after TBI. The effects of posttraumatic hypothermia on the expression of MMP-9 may partially explain the established effects of posttraumatic temperature on secondary injury after TBI.

To investigate the effect of hypothermia on the expression of apoptosis-regulating protein TIMP-3 after fluid percussion traumatic brain injury (TBI) in rats. 210 adult male Sprague Dawley rats were randomly assigned to the groups of TBI with hypothermia treatment (32 degrees C), TBI with normothermia (37 degrees C), and sham injured control. TBI model was induced by fluid percussion TBI device. Mild hypothermia (32 degrees C) was achieved by partial immersion in a water bath (0 degrees C) under general anesthesia for 4 hours. All the rats were killed at 4, 6, 12, 24, 48, 72h and 1w after TBI. The mRNA and protein level of TIMP-3 in both injured and uninjured hemispheres of each group were measured using RT-PCR and western blot techniques. In the normothermic group, TIMP-3 levels in both injured and uninjured hemispheres were significantly increased after TBI compared with those of sham injured animals (p < 0.01). In contrast, post-traumatic hypothermia significantly attenuated such an increase. According to the RT-PCR and western blot analysis, the maximum mRNA levels of TIMP-3 were reduced to 60.60%+/-2.30, 55.83%+/-1.80, 66.03%+/-2.10 and 64.51%+/-1.50 of the corresponding values in the normothermic group in injured and uninjured hemispheres (cortex and hippocampus) by hypothermia treatment, respectively (p < 0.01), while the respective maximum protein levels of TIMP-3 were reduced to 57.50%+/-1.50, 52.67%+/-2.20, 60.31%+/-2.50and 54.76%+/-1.40 (p < 0.01). Our data suggests that moderate F-P brain injury would significantly upregulate TIMP-3 expression, while such an increase could be efficiently suppressed by hypothermia treatment.

Abstract In this investigation, we evaluated the effect of post-traumatic mild hypothermia on cell death in the hippocampus after fluid percussion traumatic brain injury (TBI) in rats. Adult male Sprague-Dawley rats were randomly divided into three groups (n = 40/group): TBI with hypothermia treatment (32 degrees C), TBI with normothermia (37 degrees C), and sham injury. The TBI model was induced by a fluid percussion TBI device. Mild hypothermia (32 degrees C) was achieved by partial immersion in a water bath (0 degrees C) under general anesthesia for 4 h. All rats were killed at 24 or 72 h after TBI. The ipsilateral hippocampal CA1 in all rats were analyzed by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL), and 4',6-diamidino-2-phenylindole (DAPI) staining for determining cell death. Caspase-3 expression was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. At 24 h, based on TUNEL and DAPI results, the cell death index was 28.80 +/- 2.60% and 32.10 +/- 1.40% in the normothermia TBI group, while reaching only 14.30 +/- 2.70% and 18.40 +/- 2.10% in the hypothermic TBI group (p < 0.01). Based on RT-PCR and Western blotting results, the expression of caspase-3 was 210.20 +/- 5.30% and 170.30 +/- 4.80% in the normothermic TBI group, while reaching only 165.10 +/- 3.70% and 130.60 +/- 4.10% in the hypothermic TBI group (p < 0.05). At 72 h, based on TUNEL and DAPI results, the cell death index was 20.80 +/- 2.50% and 25.50 +/- 1.80% in the normothermic TBI group, while reaching only 10.20 +/- 2.60% and 15.50 +/- 2.10% in the hypothermic TBI group (p < 0.01). Based on RT-PCR and Western blotting results, the expression of caspase-3 was 186.20 +/- 6.20% and 142.30 +/- 5.10% in the normothermic TBI group, versus only 152.10 +/- 3.60% and 120.60 +/- 3.90% in the hypothermic TBI group (p < 0.05). Based on our findings, we conclude that post-traumatic hypothermia significantly attenuates cell death within the hippocampus following fluid percussion injury. Taken together with other studies, these observations support the premise that post-traumatic mild hypothermia can provide cerebral protection for patients with TBI.

Abstract The expression of the neutrophil chemokine macrophage inflammatory protein-2 (MIP-2/CXCL2) and the monocyte chemokine monocyte chemotactic protein-1 (MCP-1/CCL2) have been described in glial cells in vitro but their origin following TBI has not been established. Furthermore, little is known of the modulation of these chemokines. Chemokine expression was investigated in male Sprague-Dawley rats following moderate lateral fluid percussion injury (LFPI). At 0, 4, 8, 12, and 24 h after injury, brains were harvested and MIP-2/CXCL2 and MCP-1/CCL2 levels measured by ELISA. To investigate the inhibition of chemokine expression a second cohort of animals received dexamethasone (1-15 mg/kg), FK506 (1 mg/kg), or vehicle, systemically, immediately after injury. These animals were sacrificed at the time of peak chemokine expression. A third cohort of animals was also sacrificed at the time of peak chemokine expression and immunohistochemistry performed for MIP-2/CXCL2 and MCP-1/CCL2. Following LFPI, chemokines were increased in the ipsilateral hemisphere, MIP-2/CXCL2 peaking at 4 h and MCP-1/CCL2 peaking at 8-12 h post-injury. Dexamethasone significantly reduced cortical MCP-1/CCL2, but not MIP-2/CXCL2 concentrations. FK506 did not inhibit chemokine expression. In undamaged brain, chemokine expression was localized to cells with a neuronal morphology. For MIP-2/CXCL2 this was supported by double staining for the neuronal antigen NeuN. In contused tissue, increased MIP-2/CXCL2 and MCP-1/CCL2 staining was visible in cells with the morphology of degenerating neurons. MIP-2/CXCL2 and MCP-1/CCL2 are increased after injury, and neurons appear to be the source of this expression. Chemokine expression was selectively inhibited by dexamethasone. The implications of this are discussed.

Hippocampal damage contributes to cognitive dysfunction after traumatic brain injury (TBI). We previously showed that Fluoro-Jade, a fluorescent stain that labels injured, degenerating brain neurons, quantifies the extent of hippocampal injury after experimental fluid percussion TBI in rats. Coincidentally, we observed that injured neurons in the rat hippocampus also stained with Newport Green, a fluorescent dye specific for free ionic zinc. Here, we show that, regardless of injury severity or therapeutic intervention, the post-TBI population of injured neurons in rat hippocampal subfields CA1, CA3 and dentate gyrus is indistinguishable, both in numbers and anatomical distribution, from the population of neurons containing high levels of zinc. Treatment with lamotrigine, which inhibits presynaptic release of glutamate and presumably zinc that is co-localized with glutamate, reduced numbers of Fluoro-Jade-positive and Newport Green-positive neurons equally as did treatment with nicardipine, which blocks voltage-gated calcium channels through which zinc enters neurons. To confirm using molecular techniques that Fluoro-Jade and Newport Green-positive neurons are equivalent populations, we isolated total RNA from 25 Fluoro-Jade-positive and 25 Newport Green-positive pyramidal neurons obtained by laser capture microdissection (LCM) from the CA3 subfield, linearly amplified the mRNA and used quantitative ribonuclease protection analysis to demonstrate similar expression of mRNA for selected TBI-induced genes. Our data suggest that therapeutic interventions aimed at reducing neurotoxic zinc levels after TBI may reduce hippocampal neuronal injury.

Aged traumatic brain injury (TBI) patients suffer higher rates of mortality and disability than younger patients. Cognitive problems common to TBI patients are associated with damage to the hippocampus, a central locus of learning and memory. To investigate the molecular mechanisms of age-related vulnerability to brain injury in a mouse model of TBI, we studied the effects of TBI on hippocampal gene expression in young and aged mice. Young and aged male C57Bl/6 mice were subjected to sham injury or TBI and sacrificed 24h post-injury. We used laser capture microdissection to obtain pure populations of neurons from the CA1, CA3, and dentate gyrus subfields of the hippocampus. We compared injury-induced gene expression in hippocampal neurons of young and aged mice using quantitative ribonuclease protection assay analysis of linearly amplified mRNA from laser captured neurons. Both increased age and TBI were associated with increased expression of neuroprotective (brain-derived neurotrophic factor), pro-inflammatory (interleukin-1beta), and proapoptotic (caspase-3) genes in mouse hippocampal neurons. Our data support previous reports that suggested the CA3 subregion is highly susceptible to fluid percussion TBI and that age-related changes in gene expression are one potential mechanism of increased vulnerability of the aged brain to TBI.

Traumatic brain injury is a leading cause of death and disability in the United States. Pathological examinations of humans and animal models after brain injury demonstrate hippocampal neuronal damage, which may contribute to cognitive impairments. Data from our laboratories have shown that, at 1 week after brain injury, mice possess significantly fewer neurons in all ipsilateral hippocampal subregions and a cognitive impairment. Since cognitive function is distributed across both cerebral hemispheres, the present paper explores the morphological and physiological response of the contralateral hippocampus to lateral brain injury. We analyzed the contralateral hippocampus using design-based stereology, Fluoro-Jade (FJ) histochemistry, and extracellular field recordings in mice at 7 and 30 days after lateral fluid percussion injury (FPI). At 7 days, all contralateral hippocampal subregions possess significantly fewer healthy neurons compared to sham-injured animals and demonstrate FJ-positive neuronal damage, but not at 30 days. Both the ipsilateral and contralateral dentate gyri demonstrate significantly increased excitability at 7 days post-injury, but only ipsilateral dentate gyrus hyperexcitability persists at 30 days compared to sham. In the contralateral hippocampus, the transient decrease in the number of healthy neurons, concomitant with FJ damage, and electrophysiological alterations establish a stunned period of cellular and circuit dysfunction. The return of healthy neuron number, absence of FJ damage, and sham level of excitability in the contralateral hippocampus suggest recovery of structure and function by 30 days after injury. The cognitive recovery observed after human traumatic brain injury may stem from a differential injury exposure and time course of recovery between homologous regions of the two hemispheres.

Abstract Traumatic brain injury (TBI) causes selective hippocampal cell death, which is believed to be associated with cognitive impairment observed both in clinical and experimental settings. Although neurotrophin administration has been tested as a strategy to prevent cell death following TBI, the potential neuroprotective role of neurotrophin-4/5 (NT-4/5) in TBI remains unknown. We hypothesized that NT-4/5 would offer neuroprotection for selectively vulnerable hippocampal neurons following TBI. Measurements of NT-4/5 in rats subjected to lateral fluid percussion (LFP) TBI revealed two-threefold increases in the injured cortex and hippocampus in the acute period (1-3 days) following brain injury. Subsequently, the response of NT-4/5 knockout (NT-4/5(-/-)) mice to controlled-cortical impact TBI was investigated. NT-4/5(-/-) mice were more susceptible to selective pyramidal cell loss in Ahmon's corn (CA) subfields of the hippocampus following TBI, and showed impaired motor recovery when compared with their brain-injured wild-type controls (NT-4/5(wt)). Additionally, we show that acute, prolonged administration of recombinant NT-4/5 (5 microg/kg/day) prevented up to 50% of the hippocampal CA pyramidal cell death following LFP TBI in rats. These results suggest that post-traumatic increases in endogenous NT-4/5 may be part of an adaptive neuroprotective response in the injured brain, and that administration of this neurotrophic factor may be useful as a therapeutic strategy following TBI.