Microglial CD300f immune receptor contributes to the maintenance of neuron viability in vitro and after a penetrating brain injury.

Emerging evidences suggest that immune receptors participate in diverse microglial and macrophage functions by regulating their immunometabolism, inflammatory phenotype and phagocytosis. CD300f, a TREM2-like lipid sensing immune receptor, that integrates activating and inhibitory cell-signalling pathways, modulates inflammation, efferocytosis and microglial metabolic fitness. In particular, CD300f overexpression was described to be neuroprotective after an acute brain injury, suggesting a role for this immune receptor in neurotrophic interactions. Thus, we hypothesised that CD300f modulates neuronal survival through neuron-microglial interactions. In order to study its biological function, we used in vitro and in vivo approaches, CD300f-/- animals and rCD300f-Fc, a fusion protein that interrupts the endogen interaction between CD300f receptor-ligands. In hippocampal cocultures containing neurons and mixed glia, we observed that rCD300f-Fc, but not control IgGs induced neuronal death. In accordance, in vivo studies performed by injecting rCD300f-Fc or control IgGs into rat or WT or CD300 KO mice neocortex, showed an increased lesioned area after a penetrating brain injury. Interestingly, this neuronal death was dependent on glia, and the neurotoxic mechanism did not involve the increase of proinflammatory cytokines, the participation of NMDA receptors or ATP release. However, exogenous addition of glial cell line-derived neurotrophic factor (GDNF) prevented this process. Taken together, our results suggest that CD300f modulates neuronal survival in vitro and after a penetrating brain injury in vivo and that CD300f inhibition alters microglial phenotype generating a neurotoxic microenvironment.

Time-Course Evaluation of Brain Regional Mitochondrial Bioenergetics in a Pre-Clinical Model of Severe Penetrating Traumatic Brain Injury.

Mitochondrial dysfunction is a pivotal target for neuroprotection strategies for traumatic brain injury (TBI). However, comprehensive time-course evaluations of mitochondrial dysfunction are lacking in the pre-clinical penetrating TBI (PTBI) model. The current study was designed to characterize temporal responses of mitochondrial dysfunction from 30 min to 2 weeks post-injury after PTBI. Anesthetized adult male rats were subjected to either PTBI or sham craniectomy (n = 6 animals per group × 7 time points). Animals were euthanized at 30 min, 3 h, 6 h, 24 h, 3 days, 7 days, and 14 days post-PTBI, and mitochondria were isolated from the ipsilateral hemisphere of brain regions near the injury core (i.e., frontal cortex [FC] and striatum [ST]) and a more distant region from the injury core (i.e., hippocampus [HIP]). Mitochondrial bioenergetics parameters were measured in real time using the high-throughput procedures of the Seahorse Flux Analyzer (Agilent Technologies, Santa Clara, CA). The post-injury time course of FC + ST showed a biphasic mitochondrial bioenergetics dysfunction response, indicative of reduced adenosine triphosphate synthesis rate and maximal respiratory capacity after PTBI. An initial phase of energy crisis was detected at 30 min (-42%; p < 0.05 vs. sham), which resolved to baseline levels between 3 and 6 h (non-significant vs. sham). This was followed by a second and more robust phase of bioenergetics dysregulation detected at 24 h that remained unresolved out to 14 days post-injury (-55% to -90%; p < 0.05 vs. sham). In contrast, HIP mitochondria showed a delayed onset of mitochondrial dysfunction at 7 days (-74%; p < 0.05 vs. sham) that remained evident out to 14 days (-51%; p < 0.05 vs. sham) post-PTBI. Collectively, PTBI-induced mitochondrial dysfunction responses were time and region specific, evident differentially at the injury core and distant region of PTBI. The current results provide the basis that mitochondrial dysfunction may be targeted differentially based on region specificity post-PTBI. Even more important, these results suggest that therapeutic interventions targeting mitochondrial dysfunction may require extended dosing regimens to achieve clinical efficacy after TBI.

Penetrating Traumatic Brain Injury Triggers Dysregulation of Cathepsin B Protein Levels Independent of Cysteine Protease Activity in Brain and Cerebral Spinal Fluid.

Cathepsin B (CatB), a lysosomal cysteine protease, is important to brain function and may have dual utility as a peripheral biomarker of moderate-severe traumatic brain injury (TBI). The present study determined levels of pro- and mature (mat) CatB protein as well as cysteine protease activity within the frontal cortex (FC; proximal injury site), hippocampus (HC; distal injury site), and cerebral spinal fluid (CSF) collected 1-7 days after craniotomy and penetrating ballistic-like brain injury (PBBI) in rats. Values were compared with naïve controls. Further, the utility of CatB protein as a translational biomarker was determined in CSF derived from patients with severe TBI. Craniotomy increased matCatB levels in the FC and HC, and led to elevation of HC activity at day 7. PBBI caused an even greater elevation in matCatB within the FC and HC within 3-7 days. After PBBI, cysteine protease activity peaked at 3 days in the FC and was elevated at 1 day and 7 days, but not 3 days, in the HC. In rat CSF, proCatB, matCatB, and cysteine protease activity peaked at 3 days after craniotomy and PBBI. Addition of CA-074, a CatB-specific inhibitor, confirmed that protease activity was due to active matCatB in rat brain tissues and CSF at all time-points. In patients, CatB protein was detectable from 6 h through 10 days after TBI. Notably, CatB levels were significantly higher in CSF collected within 3 days after TBI compared with non-TBI controls. Collectively, this work indicates that CatB and its cysteine protease activity may serve as collective molecular signatures of TBI progression that differentially vary within both proximal and distal brain regions. CatB and its protease activity may have utility as a surrogate, translational biomarker of acute-subacute TBI.

Alterations in Peripheral Organs following Combined Hypoxemia and Hemorrhagic Shock in a Rat Model of Penetrating Ballistic-Like Brain Injury.

Polytrauma, with combined traumatic brain injury (TBI) and systemic damage are common among military and civilians. However, the pathophysiology of peripheral organs following polytrauma is poorly understood. Using a rat model of TBI combined with hypoxemia and hemorrhagic shock, we studied the status of peripheral redox systems, liver glycogen content, creatinine clearance, and systemic inflammation. Male Sprague-Dawley rats were subjected to hypoxemia and hemorrhagic shock insults (HH), penetrating ballistic-like brain injury (PBBI) alone, or PBBI followed by hypoxemia and hemorrhagic shock (PHH). Sham rats received craniotomy only. Biofluids and liver, kidney, and heart tissues were collected at 1 day, 2 days, 7 days, 14 days, and 28 days post-injury (DPI). Creatinine levels were measured in both serum and urine. Glutathione levels, glycogen content, and superoxide dismutase (SOD) and cytochrome C oxidase enzyme activities were quantified in the peripheral organs. Acute inflammation marker serum amyloid A-1 (SAA-1) level was quantified using western blot analysis. Urine to serum creatinine ratio in PHH group was significantly elevated on 7-28 DPI. Polytrauma induced a delayed disruption of the hepatic GSH/GSSG ratio, which resolved within 2 weeks post-injury. A modest decrease in kidney SOD activity was observed at 2 weeks after polytrauma. However, neither PBBI alone nor polytrauma changed the mitochondrial cytochrome C oxidase activity. Hepatic glycogen levels were reduced acutely following polytrauma. Acute inflammation marker SAA-1 showed a significant increase at early time-points following both systemic and brain injury. Overall, our findings demonstrate temporal cytological/tissue level damage to the peripheral organs due to combined PBBI and systemic injury.

Spatiotemporal dynamics of PDGFRß expression in pericytes and glial scar formation in penetrating brain injuries in adults.

AIMS: Understanding the spatiotemporal dynamics of reactive cell types following brain injury is important for future therapeutic interventions. We have previously used penetrating cortical injuries following intracranial recordings as a brain repair model to study scar-forming nestin-expressing cells. We now explore the relationship between nestin-expressing cells, PDGFRß+ pericytes and Olig2+ glia, including their proliferation and functional maturation. METHODS: In 32 cases, ranging from 3 to 461 days post injury (dpi), immunohistochemistry for PDGFRß, nestin, GFAP, Olig2, MCM2, Aquaporin 4 (Aq4), Glutamine Synthetase (GS) and Connexin 43 (Cx43) was quantified for cell densities, labelling index (LI) and cellular co-expression at the injury site compared to control regions. RESULTS: PDGFRß labelling highlighted both pericytes and multipolar parenchymal cells. PDGFRß LI and PDGFRß+ /MCM2+ cells significantly increased in injury Zones at 10-13 dpi with migration of pericytes away from vessels with increased co-localization of PDGRFß with nestin compared to control regions (P < 0.005). Olig2+ /MCM2+ cell populations peaked at 13 dpi with significantly higher cell densities at injury sites than in control regions (P < 0.01) and decreasing with dpi (P < 0.05). Cx43 LI was reduced in acute injuries but increased with dpi (P < 0.05) showing significant cellular co-localization with nestin and GFAP (P < 0.005 and P < 0.0001) but not PDGFRß. CONCLUSIONS: These findings indicate that PDGFRß+ and Olig2+ cells contribute to the proliferative fraction following penetrating brain injuries, with evidence of pericyte migration. Dynamic changes in Cx43 in glial cell types with dpi suggest functional alterations during temporal stages of brain repair.

Sonic hedgehog signaling is negatively regulated in reactive astrocytes after forebrain stab injury.

Following injury to the central nervous system, astrocytes perform critical and complex functions that both promote and antagonize neural repair. Understanding the molecular signaling pathways that coordinate their diverse functional properties is key to developing effective therapeutic strategies. In the healthy, adult CNS, Sonic hedgehog (Shh) signaling is active in mature, differentiated astrocytes. Shh has been shown to undergo injury-induced upregulation and promote neural repair. Here, we investigated whether Shh signaling mediates astrocyte response to injury. Surprisingly, we found that following an acute, focal injury, reactive astrocytes exhibit a pronounced reduction in Shh activity in a spatiotemporally-defined manner. Shh signaling is lost in reactive astrocytes at the lesion site, but persists in mild to moderately reactive astrocytes in distal tissues. Nevertheless, local pharmacological activation of the Shh pathway in astrocytes mitigates inflammation, consistent with a neuroprotective role for Shh signaling after injury. Interestingly, we find that Shh signaling is restored to baseline levels two weeks after injury, a time during which acute inflammation has largely subsided and lesions have matured. Taken together, these data suggest that endogenous Shh signaling in astrocytes is dynamically regulated in a context dependent manner. In addition, exogenous activation of the Shh pathway promotes neuroprotection mediated by reactive astrocytes.

Crosstalk between Estrogen Withdrawal and NFκB Signaling following Penetrating Brain Injury.

OBJECTIVES: Characterized by neuroinflammation, traumatic brain injury (TBI) induces neuropathological changes and cognitive deficits. Estrogens are neuroprotective by increasing cell survival and this increase is mediated by a decrease in neuroinflammation. To further explore the relationship between estrogens, brain injury, and neuroinflammation, we examined the expression of the IKK/NFκB complex. The IKK/NFκB complex is a pleiotropic regulator of many cellular signaling pathways linked to inflammation, as well as three major cytokines (IL-1ß, IL-6, and TNF-α). We hypothesized that NFκB expression would be upregulated following injury and that this increase would be exacerbated when circulating estrogens were decreased with fadrozole (aromatase inhibitor). METHODS: Using adult zebra finches, we first determined the expression of major components of the NFκB complex (NFκB, IκB-α, and IκB-ß) following injury using qPCR. Next, male and female finches were collected at 2 time points (2 or 24 h after injury) and brain tissue was analyzed to determine whether NFκB expression was differentially expressed in males and females at either time point. Finally, we examined how the expression of NFκB changed when estrogen levels were decreased immediately after injury. RESULTS: Our study documented an increase in the expression of the major components of the NFκB complex (NFκB, IκB-α, and IκB-ß) following injury. Decreasing estrogen levels resulted in a surprising decrease in the NFκB complex studied here. DISCUSSION: These data further expand the model of how estrogens and other steroid hormones interact with the inflammatory pathways following injury and may prove beneficial when developing therapies for treatment of TBI.

Exogenous progesterone is neuroprotective following injury to the male zebra finch brain.

The use of progesterone following brain injury has a controversial history. On one hand, some lab-based models have showed progesterone as being neuroprotective, but on the other, clinical trials have showed quite the opposite. One of many complaints that arose from this discrepancy was the lack of a diverse pool of animal models and paradigms employed during the preclinical phase. However, over the past decade, the zebra finch has emerged as an optimal organism for the study of steroid-mediated neuroprotection. Following an injury, steroid hormones and receptors are upregulated, serving to decrease neuroinflammation and overall damage to the brain. As compared to other vertebrate models, zebra finches can upregulate expression of both estrogens and androgens at a faster and more robust response, suggesting that vertebrates differ in their neuroprotective mechanisms and timing following injury. Therefore, to expand the types organisms studied in pre-clinical trials, we chose to use zebra finches. While the majority of work in the zebra finch brain has focused on estrogens and androgens, we sought to clarify the role of progesterone following injury. Adult male zebra finches were given daily injections of progesterone following a penetrating injury and then were assessed for the size of injury and expression of various genes associated with neuroinflammation and cell survival. Treatment with progesterone decreased the injury size in zebra finches over controls and increased expression of various genes associated with cell survival and neuroinflammation. These data suggest that progesterone does mediate neuroprotection, most likely through the alteration of neuroinflammatory and cell survival pathways.

Alterations in brain-derived neurotrophic factor and insulin-like growth factor-1 protein levels after penetrating ballistic-like brain injury in rats.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) are essential for neuroplasticity and neuronal survival. Despite the importance of these endogenous factors in mediating posttraumatic recovery, little is known about their response after penetrating type traumatic brain injury. The objective of this study was to quantify the expression levels BDNF and IGF-1, two well-known neuroplasticity mediators, after penetrating ballistic-like brain injury (PBBI). METHODS: Rats were randomly assigned to receive unilateral sham or PBBI injuries. Using enzyme-linked immunosorbent assay and immunohistochemistry, we performed a comprehensive evaluation of BDNF and IGF-1 expression at acute (1 hour, 6 hours, 1 day) and subacute (2, 3, 7, and 14 days) timepoints after injury. RESULTS: BDNF and IGF-1 expression was transiently upregulated in both cortex and hippocampus after PBBI. Although BDNF levels increased at acute timepoints, IGF-1 expression peaked at 3 days in cortical homogenates. Although there was loss of staining in cells bordering the cavity, increased BDNF and IGF-1 immunoreactivity was observed in scattered neurons away from the contusion site. Glial upregulation of both growth factors was observed at early timepoints in the hippocampus. CONCLUSION: Our findings demonstrate that PBBI results in a brief upregulation of BDNF and IGF-1 during early posttraumatic period, providing critical information for interventions aiming to enhance neuronal survival and brain plasticity.

Acute and subacute microRNA dysregulation is associated with cytokine responses in the rodent model of penetrating ballistic-like brain injury.

BACKGROUND: MicroRNAs (miRNAs) are small stable RNAs that regulate translational degradation or repression of genes involved in brain trauma-mediated inflammation. More recently, miRNAs have emerged as potential novel TBI biomarkers. The aim of this study was to determine if a select set of miRNAs (miR-21, Let-7i, miR-124a, miR-146a, miR-107) that were previously associated with TBI models and clinical studies would be dysregulated and correlated to inflammatory cytokine abundance in the rat penetrating ballistic-like brain injury (PBBI) model. METHODS: Adult male Sprague-Dawley rats received a unilateral frontal 10% PBBI, which produces a temporary cavity. Sham animals received a craniotomy only. Ipsilateral brain tissue and serum were collected 4 hours to 7 days post-injury. Quantitation of miR-21, Let-7i, miR-124a, miR-146a, or miR-107 levels was conducted using Taqman PCR assays normalized to the endogenous reference, U6 snRNA. Brain tissue derived from matching cohorts was used to determine 1L-1beta and IL-6 levels by enzyme-linked immunosorbent assay. RESULTS: Brain tissue Let-7i and miR-21 increased at 4 hours and 1 day, whereas miR-124a and miR-107 were enhanced only 1 day post-injury. MiR-146a displayed a biphasic response and increased 1 day and 7 days, whereas elevation of miR-21 was sustained 1 day to 7 days after PBBI. Pathway analysis indicated that miRNAs were linked to inflammatory proteins, IL-6 and IL-1beta. Confirmation by enzyme-linked immunosorbent assay indicated that both cytokines were increased and peaked at 1 day, but fell at 3 days through 7 days after PBBI, indicating an inverse relationship with miRNA abundance. Serum Let-7i, alone, was differentially abundant 7 days after PBBI. CONCLUSION: Brain tissue-derived miRNAs linked to increased cytokine levels demonstrates a plausible therapeutic target of TBI-induced inflammation. Suppression of serum derived Let-7i may have utility as a biomarker of subacute injury progression or therapeutic responses.

Neurochemical changes following combined hypoxemia and hemorrhagic shock in a rat model of penetrating ballistic-like brain injury: A microdialysis study.

BACKGROUND: Energy metabolic dysfunction is a key determinant of cellular damage following traumatic brain injury and may be worsened by additional insults. This study evaluated the acute/subacute effects of combined hypoxemia (HX) and hemorrhagic shock (HS) on cerebral interstitial levels of glucose, lactate, and pyruvate in a rat model of penetrating ballistic-like brain injury (PBBI). METHODS: Rats were randomly assigned into the sham control, PBBI, and combined injury (P + HH) groups. The P + HH group received PBBI followed by 30-minute HX and 30 minute HS. Samples were collected from striatum (perilesional region) using intracerebral microdialysis at 1 to 3 hours after injury and then at 1 to 3, 7, and 14 days after injury. Glucose, lactate, and pyruvate were measured in the dialysate samples. RESULTS: Glucose levels dropped significantly up to 24 hours following injury in both PBBI and P + HH groups (p < 0.05). A reduction in pyruvate was observed in the PBBI group from 24 to 72 hours after injury (vs. sham). In the P + HH group, the pyruvate was significantly reduced from 2 to 24 hours after injury (p < 0.05 vs. PBBI). This prominent reduction persisted for 14 days after injury. In contrast, lactate levels were significantly increased in the PBBI group during the first 24 hours after injury and remained elevated out to 7 days. The P + HH group exhibited a similar trend of lactate increase as did the PBBI group. Critically, P + HH further increased the lactate-to-pyruvate ratio by more than twofold (vs. PBBI) during the first 24 hours. The ratio reached a peak at 2 hours and then gradually decreased, but the level remained significantly higher than that in the sham control from 2 to 14 days after injury (p < 0.05). CONCLUSION: This study identified the temporal profile of energy-related neurochemical dysregulation induced by PBBI and combined injury in the perilesional region. Furthermore, combined HX and HS further reduced the pyruvate level and increased the lactate-to-pyruvate ratio following PBBI, indicating the exacerbation of posttraumatic metabolic perturbation.

Subacute Changes in Cleavage Processing of Amyloid Precursor Protein and Tau following Penetrating Traumatic Brain Injury.

Traumatic brain injury (TBI) is an established risk factor for the development of Alzheimer's disease (AD). Here the effects of severe penetrating TBI on APP and tau cleavage processing were investigated in a rodent model of penetrating ballistic-like brain injury (PBBI). PBBI was induced by stereotactically inserting a perforated steel probe through the right frontal cortex of the anesthetized rat and rapidly inflating/deflating the probe's elastic tubing into an elliptical shaped balloon to 10% of total rat brain volume causing temporary cavitation injury. Separate animals underwent probe injury (PrI) alone without balloon inflation. Shams underwent craniectomy. Brain tissue was collected acutely (4h, 24h, 3d) and subacutely (7d) post-injury and analyzed by immunoblot for full length APP (APP-FL) and APP beta c-terminal fragments (ßCTFs), full length tau (tau-FL) and tau truncation fragments and at 7d for cytotoxic Beta amyloid (Aß) peptides Aß40 and Aß42 analysis. APP-FL was significantly decreased at 3d and 7d following PBBI whereas APP ßCTFs were significantly elevated by 4h post-injury and remained elevated through 7d post-injury. Effects on ßCTFs were mirrored with PrI, albeit to a lesser extent. Aß40 and Aß42 were significantly elevated at 7d following PBBI and PrI. Tau-FL decreased substantially 3d and 7d post-PBBI and PrI. Importantly, a 22 kDa tau fragment (tau22), similar to that found in AD, was significantly elevated by 4h and remained elevated through 7d post-injury. Thus both APP and tau cleavage was dramatically altered in the acute and subacute periods post-injury. As cleavage of these proteins has also been implicated in AD, TBI pathology shown here may set the stage for the later development of AD or other tauopathies.

Serum Glial Fibrillary Acidic Protein Predicts Tissue Glial Fibrillary Acidic Protein Break-Down Products and Therapeutic Efficacy after Penetrating Ballistic-Like Brain Injury.

Acute traumatic brain injury (TBI) is associated with neurological dysfunction, changes in brain proteins, and increased serum biomarkers. However, the relationship between these brain proteins and serum biomarkers, and the ability of these serum biomarkers to indicate a neuroprotective/therapeutic response, remains elusive. Penetrating ballistic-like brain injury (PBBI) was used to systematically analyze several key TBI biomarkers, glial fibrillary acidic protein (GFAP) and its break-down products (BDPs)-ubiquitin C-terminal hydrolase-L1 (UCH-L1), α-II spectrin, and α-II spectrin BDPs (SBDPs)-in brain tissues and serum during an extended acute-subacute time-frame. In addition, neurological improvement and serum GFAP theranostic value was evaluated after neuroprotective treatment. In brain tissues, total GFAP increased more than three-fold 2 to 7 d after PBBI. However, this change was primarily due to GFAP-BDPs which increased to 2.7-4.8 arbitrary units (AU). Alpha-II spectrin was nearly ablated 3 d after PBBI, but somewhat recovered after 7 d. In conjunction with α-II spectrin loss, SBDP-145/150 increased approximately three-fold 2 to 7 d after PBBI (vs. sham, p<0.05). UCH-L1 protein levels were slightly decreased 7 d after PBBI but otherwise were unaffected. Serum GFAP was elevated by 3.2- to 8.8-fold at 2 to 4 h (vs. sham; p<0.05) and the 4 h increase was strongly correlated to 3 d GFAP-BDP abundance (r=0.66; p<0.05). Serum GFAP showed such a strong injury effect that it also was evaluated after therapeutic intervention with cyclosporin A (CsA). Administration of 2.5 mg/kg CsA significantly reduced serum GFAP elevation by 22.4-fold 2 h after PBBI (vs. PBBI+vehicle; p<0.05) and improved neurological function 1 d post-injury. Serum biomarkers, particularly GFAP, may be correlative tools of brain protein changes and feasible theranostic markers of TBI progression and recovery.

Chapter 3 animal models of traumatic brain injury: is there an optimal model that parallels human brain injury?

Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in the younger population worldwide. Survivors of TBI often experience long-term disability in the form of cognitive, sensorimotor, and affective impairments. Despite the high prevalence in, and cost of TBI to, both individuals and society, some of its underlying pathophysiology is not completely understood. Animal models have been developed over the past few decades to closely replicate the different facets of TBI in humans to better understand the underlying pathophysiology and behavioral impairments and assess potential therapies that can promote neuroprotection. However, no effective treatment for TBI has been established to date in the clinical setting, despite promising results generated in preclinical studies in the use of neuroprotective strategies. The failure to translate results from preclinical studies to the clinical setting underscores a compelling need to revisit the current state of knowledge in the use of animal models in TBI.

Glucose and oxygen metabolism after penetrating ballistic-like brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability in all age groups. Among TBI, penetrating traumatic brain injuries (PTBI) have the worst prognosis and represent the leading cause of TBI-related morbidity and death. However, there are no specific drugs/interventions due to unclear pathophysiology. To gain insights we looked at cerebral metabolism in a PTBI rat model: penetrating ballistic-like brain injury (PBBI). Early after injury, regional cerebral oxygen tension and consumption significantly decreased in the ipsilateral cortex in the PBBI group compared with the control group. At the same time point, glucose uptake was significantly reduced globally in the PBBI group compared with the control group. Examination of Fluorojade B-stained brain sections at 24 hours after PBBI revealed an incomplete overlap of metabolic impairment and neurodegeneration. As expected, the injury core had the most severe metabolic impairment and highest neurodegeneration. However, in the peri-lesional area, despite similar metabolic impairment, there was lesser neurodegeneration. Given our findings, the data suggest the presence of two distinct zones of primary injury, of which only one recovers. We anticipate the peri-lesional area encompassing the PBBI ischemic penumbra, could be salvaged by acute therapies.

Common biochemical defects linkage between post-traumatic stress disorders, mild traumatic brain injury (TBI) and penetrating TBI.

Post-traumatic stress disorder (PTSD) is a complex mental disorder with psychological and emotional components, caused by exposure to single or repeated extreme traumatic events found in war, terrorist attacks, natural or man-caused disasters, and by violent personal assaults and accidents. Mild traumatic brain injury (TBI) occurs when the brain is violently rocked back and forth within the skull following a blow to the head or neck as in contact sports, or when in close proximity to a blast pressure wave following detonation of explosives in the battlefield. Penetrating TBI occurs when an object penetrates the skull and damages the brain, and is caused by vehicle crashes, gunshot wound to the head, and exposure to solid fragments in the proximity of explosions, and other combat-related head injuries. Despite clinical studies and improved understanding of the mechanisms of cellular damage, prevention and treatment strategies for patients with PTSD and TBI remain unsatisfactory. To develop an improved plan for treating and impeding progression of PTSD and TBI, it is important to identify underlying biochemical changes that may play key role in the initiation and progression of these disorders. This review identifies three common biochemical events, namely oxidative stress, chronic inflammation and excitotoxicity that participate in the initiation and progression of these conditions. While these features are separately discussed, in many instances, they overlap. This review also addresses the goal of developing novel treatments and drug regimens, aimed at combating this triad of events common to, and underlying, injury to the brain.

COX-2 regulation and TUNEL-positive cell death differ between genders in the secondary inflammatory response following experimental penetrating focal brain injury in rats.

INTRODUCTION: Traumatic brain injury is followed by secondary neuronal degeneration, largely dependent on an inflammatory response. This response is probably gender specific, since females are better protected than males in experimental models. The reasons are not fully known. We examined aspects of the inflammatory response following experimental TBI in male and female rats to explore possible gender differences at 24 h and 72 h after trauma, times of peak histological inflammation and neuronal degeneration. METHODS: A penetrating brain injury model was used to produce penetrating focal TBI in 20 Sprague-Dawley rats, 5 males and 5 females for each time point. After 24 and 72 h the brains were removed and subjected to in situ hybridization and immunohistochemical analyses for COX-2, iNOS, osteopontin, glial fibrillary acidic protein, 3-nitrotyrosine, TUNEL and Fluoro-Jade. RESULTS: COX-2 mRNA and protein levels were increased in the perilesional area compared to the uninjured contralateral side and significantly higher in males at 24 h and 72 h (p < 0.05). iNOS mRNA was significantly increased in females at 24 h (p < 0.05) although protein was not. TUNEL was increased in male rats after 24 h (p < 0.05). Glial fibrillary acidic protein, osteopontin, 3-nitrotyrosine and Fluoro-Jade stained degenerating neurons were increased in the perilesional area, showing no difference between genders. CONCLUSIONS: COX-2 regulation differed between genders after TBI. The increased COX-2 expression in male rats correlated with increased apoptotic cell death detected by increased TUNEL staining at 24 h, but not with neuronal necrosis measured by Flouro-Jade. Astrogliosis and microgliosis did not differ, confirming a comparable level of trauma. The gender-specific trait of the secondary inflammatory response may be connected to prostaglandin regulation, which may partially explain gender variances in outcome after TBI.

A molecular method to correlate bloodstains with wound site for crime scene reconstruction.

Bloodstain pattern analysis to determine the wound-of-origin of bloodstains is problematic with nonspecific patterns. In this proof-of-concept study, the authors examined a molecular approach to correlate bloodstains with injuries using the rat as a model. Specifically, investigations were conducted on the rat brain marker, rno-miR-124-3p, with the QIAGEN miScript System and real-time PCR analysis. Rno-miR-124-3p was detected in brain homogenates diluted 100,000 times; in 3-week-old, room temperature stored, simulated brain-blood stains; and in bloodstains from head gunshot wounds collected with swabs and subsequently frozen for 9-18 months; however, rno-miR-124-3p was not detected in whole blood. Proof-of-principle was demonstrated by the ability to distinguish bloodstains from a gunshot wound to the head versus bloodstains from a gunshot wound to the chest, by the testing of otherwise identical bloodstains from the two patterns for the presence of the marker. The results suggest a viable approach to a longstanding problem in casework.

Mechanism of action for NNZ-2566 anti-inflammatory effects following PBBI involves upregulation of immunomodulator ATF3.

The tripeptide glycine-proline-glutamate analogue NNZ-2566 (Neuren Pharmaceuticals) demonstrates neuroprotective efficacy in models of traumatic brain injury. In penetrating ballistic-like brain injury (PBBI), it significantly decreases injury-induced upregulation of inflammatory cytokines including TNF-α, IFN-γ, and IL-6. However, the mechanism by which NNZ-2566 acts has yet to be determined. The activating transcription factor-3 (ATF3) is known to repress expression of these inflammatory cytokines and was increased at the mRNA and protein level 24-h post-PBBI. This study investigated whether 12 h of NNZ-2566 treatment following PBBI alters atf3 expression. PBBI alone significantly increased atf3 mRNA levels by 13-fold at 12 h and these levels were increased by an additional fourfold with NNZ-2566 treatment. To confirm that changes in mRNA translated to changes in protein expression, ATF3 expression levels were determined in vivo in microglia/macrophages, T cells, natural killer cells (NKCs), astrocytes, and neurons. PBBI alone significantly increased ATF3 in microglia/macrophages (820%), NKCs (58%), and astrocytes (51%), but decreased levels in T cells (48%). NNZ-2566 treatment further increased ATF3 protein expression in microglia/macrophages (102%), NKCs (308%), and astrocytes (13%), while reversing ATF3 decreases in T cells. Finally, PBBI increased ATF3 levels by 55% in neurons and NNZ-2566 treatment further increased these levels an additional 33%. Since increased ATF3 may be an innate protective mechanism to limit inflammation following injury, these results demonstrating that the anti-inflammatory and neuroprotective drug NNZ-2566 increase both mRNA and protein levels of ATF3 in multiple cell types provide a cellular mechanism for NNZ-2566 modulation of neuroinflammation following PBBI.

The contribution of ciliary neurotrophic factor receptors to adult motor neuron survival in vivo is specific to insult type and distinct from that for embryonic motor neurons.

Exogenous ciliary neurotrophic factor (CNTF) promotes motor neuron (MN) survival following trauma and in genetic models of MN disease. Unconditional disruption of the mouse CNTF receptor α (CNTFRα) gene leads to MN loss, demonstrating a developmental role for endogenous CNTF receptor signaling. These data also suggest that CNTF receptors may promote adult MN survival and that appropriately manipulating the receptors could effectively treat adult MN disorders. This effort would greatly benefit from a better understanding of the roles played by CNTF receptors in adult MNs. We have previously found that adult onset disruption of CNTFRα in facial MNs of "floxed CNTFRα" mice by AAV-Cre vector injection leads to significantly more MN loss than in identically treated controls. While indicating that CNTF receptors can promote adult MN survival, the data did not distinguish between potential roles in MN maintenance versus roles in protecting MNs from the injection associated trauma or the toxicity of the chronic Cre recombinase (Cre) produced by the AAV-Cre. Here we used an inducible Cre gene construct to produce adult-onset CNTFRα disruption in facial MNs without the traumatic and toxic effects of the AAV-Cre procedure. The MNs survive without CNTFRα, even when challenged by facial nerve crush or the injection-associated trauma, thereby suggesting, in conjunction with our previous study, that endogenous CNTF receptor signaling can protect MNs against toxic insult, such as that produced by chronic Cre. The data also indicate that in vivo CNTF receptors play very different roles in adult and embryonic MNs.