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.

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.

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.

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.

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.

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.

Proteomic analysis and brain-specific systems biology in a rodent model of penetrating ballistic-like brain injury.

Proteomics and systems biology have significantly contributed to biomarker discovery in the field of brain injury. This study utilized 2D-DIGE-PMF-MS as a preliminary screen to detect biomarkers in a rat model of penetrating ballistic-like brain injury (PBBI). Brain-specific systems biology analysis of brain tissue identified 386 proteins having a fold change of more than 2, of which 321 proteins were increased and 65 were decreased 24 h after PBBI compared to sham controls. The majority of upregulated proteins were cytoskeletal (10.5%), nucleic acid binding (9.3%), or kinases (8.9%). Most proteins were involved in protein metabolism (22.7%), signal transduction (20.4%), and development (9.6%). Pathway analysis indicated that these proteins were involved in neurite outgrowth and cell differentiation. Semiquantitative Western blotting of 6, 24, 48, and 72 h after PBBI indicated ubiquitin carboxyl-terminal hydrolase isozyme L1 (a proposed traumatic brain injury biomarker in human clinical trials), tyrosine hydroxylase, and syntaxin-6 were found to be consistently elevated in brain tissue and cerebral spinal fluid after PBBI compared to sham controls. Combining proteomics and brain-specific systems biology can define underlying mechanisms of traumatic brain injury and provide valuable information in biomarker discovery that, in turn, may lead to novel therapeutic targets.

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.

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.

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.

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.

Acute and delayed neuroinflammatory response following experimental penetrating ballistic brain injury in the rat.

BACKGROUND: Neuroinflammation following acute brain trauma is considered to play a prominent role in both the pathological and reconstructive response of the brain to injury. Here we characterize and contrast both an acute and delayed phase of inflammation following experimental penetrating ballistic brain injury (PBBI) in rats out to 7 days post-injury. METHODS: Quantitative real time PCR (QRT-PCR) was used to evaluate changes in inflammatory gene expression from the brain tissue of rats exposed to a unilateral frontal PBBI. Brain histopathology was assessed using hematoxylin and eosin (H&E), silver staining, and immunoreactivity for astrocytes (GFAP), microglia (OX-18) and the inflammatory proteins IL-1beta and ICAM-1. RESULTS: Time course analysis of gene expression levels using QRT-PCR indicated a peak increase during the acute phase of the injury between 3-6 h for the cytokines TNF-alpha (8-11 fold), IL-1beta (11-13 fold), and IL-6 (40-74 fold) as well as the cellular adhesion molecules VCAM (2-3 fold), ICAM-1 (7-15 fold), and E-selectin (11-13 fold). Consistent with the upregulation of pro-inflammatory genes, peripheral blood cell infiltration was a prominent post-injury event with peak levels of infiltrating neutrophils (24 h) and macrophages (72 h) observed throughout the core lesion. In regions of the forebrain immediately surrounding the lesion, strong immunoreactivity for activated astrocytes (GFAP) was observed as early as 6 h post-injury followed by prominent microglial reactivity (OX-18) at 72 h and resolution of both cell types in cortical brain regions by day 7. Delayed thalamic inflammation (remote from the primary lesion) was also observed as indicated by both microglial and astrocyte reactivity (72 h to 7 days) concomitant with the presence of fiber degeneration (silver staining). CONCLUSION: In summary, PBBI induces both an acute and delayed neuroinflammatory response occurring in distinct brain regions, which may provide useful diagnostic information for the treatment of this type of brain injury.

Effect of penetrating brain injury on aquaporin-4 expression using a rat model.

Cerebral edema (CE) is a frequent and potentially lethal consequence of various neurotraumas, including penetrating brain injury (PBI). Aquaporin-4 (AQP4) water channel is predominantly expressed by astrocytes and plays an important role in regulating water balance in the normal and injured brain. Using a rat model of PBI, we show that AQP4 immunoreactivity was substantially increased in the peri-injury area at both 24 and 72 h after PBI. The increase in AQP4 expression was paralleled by increased GFAP expression. The two proteins were co-expressed by peri-vascular astrocytes, whereas reactive astroglia identified by their stellar morphology did not express AQP4 at either time points after injury. Western analysis confirmed the increase in AQP4 immunoreactivity observed in the injured tissue. The apparent increase in AQP4 immunoreactivity was likely due to de novo AQP4 protein synthesis, as most of the increased AQP4 immunoreactivity was found in the soluble (cytosolic) fraction. Our results demonstrate dynamic spatial and temporal changes in AQP4 expression that contribute to the molecular pathophysiology of PBI.

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.

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.

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.

Alpha-1-antichymotrypsin promotes beta-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease.

Alpha(1)-antichymotrypsin (ACT), an acute-phase inflammatory protein, is an integral component of the amyloid deposits in Alzheimer's disease (AD) and has been shown to catalyze amyloid beta-peptide polymerization in vitro. We have investigated the impact of ACT on amyloid deposition in vivo by generating transgenic GFAP-ACT-expressing mice and crossing them with the PDGF-hAPP/V717F mice, which deposit amyloid in an age-dependent manner. The number of amyloid deposits measured by Congo Red birefringence was increased in the double ACT/amyloid precursor protein (APP) transgenic mice compared with transgenic mice that only expressed APP, particularly in the hippocampus where ACT expression was highest, and the increase was preceded by elevated total amyloid beta-peptide levels at an early age. Our data demonstrate that ACT promotes amyloid deposition and provide a specific mechanism by which inflammation and the subsequent upregulation of astrocytic ACT expression in AD brain contributes to AD pathogenesis.

Regulation of neuregulin expression in the injured rat brain and cultured astrocytes.

In this report, we investigated whether reactive astrocytes produce neuregulins (glial growth factor 2/heregulin/acetylcholine receptor-inducing activity or neu differentiation factor) and its putative receptors, ErbB2 and ErbB3 tyrosine kinases, in the injured CNS in vivo. Significant immunoreactivities with anti-neuregulin, anti-ErbB2, and anti-ErbB3 antibodies were detected on astrocytes at the injured site 4 d after injury to the adult rat cerebral cortex. To elucidate the mechanisms for the upregulation of neuregulin expression in astrocytes, primary cultured astrocytes were treated with certain reagents, including forskolin, that are known to elevate the intracellular level of cAMP and induce marked morphological changes in astrocytes. Western blot analysis showed that the expression of a 52 kDa membrane-spanning form of a neuregulin protein was enhanced in cultured astrocytes after administration of forskolin. The upregulation of glial fibrillary acidic protein was also observed in astrocytes treated with forskolin. In contrast, inactivation of protein kinase C because of chronic treatment with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate downregulated the expression of the 52 kDa isoform, although other splice variants with apparent molecular sizes of 65 and 60 kDa were upregulated. These results suggest that the enhancement of neuregulin expression at injured sites is induced, at least in part, by elevation in intracellular cAMP levels and/or a protein kinase C signaling pathway. The neuregulin expressed on reactive astrocytes may stimulate their proliferation and support the survival of neurons surrounding cortical brain wounds in vivo.