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.

A Simplified Workflow for Protein Quantitation of Rat Brain Tissues Using Label-Free Proteomics and Spectral Counting.

Mass spectrometry-based proteomics is an increasingly valuable tool for determining relative or quantitative protein abundance in brain tissues. A plethora of technical and analytical methods are available, but straightforward and practical approaches are often needed to facilitate reproducibility. This aspect is particularly important as an increasing number of studies focus on models of traumatic brain injury or brain trauma, for which brain tissue proteomes have not yet been fully described. This text provides suggested techniques for robust identification and quantitation of brain proteins by using molecular weight fractionation prior to mass spectrometry-based proteomics. Detailed sample preparation and generalized protocols for chromatography, mass spectrometry, spectral counting, and normalization are described. The rat cerebral cortex isolated from a model of blast-overpressure was used as an exemplary source of brain tissue. However, these techniques may be adapted for lysates generated from several types of cells or tissues and adapted by the end user.

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.

A systems biology strategy to identify molecular mechanisms of action and protein indicators of traumatic brain injury.

The multifactorial nature of traumatic brain injury (TBI), especially the complex secondary tissue injury involving intertwined networks of molecular pathways that mediate cellular behavior, has confounded attempts to elucidate the pathology underlying the progression of TBI. Here, systems biology strategies are exploited to identify novel molecular mechanisms and protein indicators of brain injury. To this end, we performed a meta-analysis of four distinct high-throughput gene expression studies involving different animal models of TBI. By using canonical pathways and a large human protein-interaction network as a scaffold, we separately overlaid the gene expression data from each study to identify molecular signatures that were conserved across the different studies. At 24 hr after injury, the significantly activated molecular signatures were nonspecific to TBI, whereas the significantly suppressed molecular signatures were specific to the nervous system. In particular, we identified a suppressed subnetwork consisting of 58 highly interacting, coregulated proteins associated with synaptic function. We selected three proteins from this subnetwork, postsynaptic density protein 95, nitric oxide synthase 1, and disrupted in schizophrenia 1, and hypothesized that their abundance would be significantly reduced after TBI. In a penetrating ballistic-like brain injury rat model of severe TBI, Western blot analysis confirmed our hypothesis. In addition, our analysis recovered 12 previously identified protein biomarkers of TBI. The results suggest that systems biology may provide an efficient, high-yield approach to generate testable hypotheses that can be experimentally validated to identify novel mechanisms of action and molecular indicators of TBI.

Temporal alterations in aquaporin and transcription factor HIF1α expression following penetrating ballistic-like brain injury (PBBI).

OBJECTIVES: Brain edema is a primary factor in the morbidity and mortality of traumatic brain injury (TBI). The various isoforms of aquaporin 4 (AQP4) and aquaporin 9 (AQP9) are important factors influencing edema following TBI. Others have reported that these AQPs are regulated by the transcription factor hypoxia inducible factor (HIF) 1α. Therefore, we examined the temporal alterations in the multiple isoforms of AQP4 and AQP9, and its possible upstream regulation by HIF1α, and evaluated whether different severities of penetrating injury influence these mechanisms. METHODS: In the penetrating ballistic-like brain injury (PBBI) model, a temporary cavity and resultant injury was formed by the rapid inflation/deflation (i.e. <40ms) of an elastic balloon attached to the end of the custom probe, injuring 10% of total rat brain volume. Tissue from the ipsilateral core and perilesional injury zones was collected. Total RNA was isolated at 4, 12, and 24h, 3 and 7days post-injury (sham and PBBI, n=6 per group). cDNA was synthesized using oligodT primers. Quantitative real time PCR was performed using Taqman expression assays for aqp4 (recognizing all isoforms), aqp9, and hif1α. Using separate animals, tissue lysate was collected at 4 and 24h, 3 and 7days post-injury and analyzed by immunoblot for protein expression of multiple isoforms of AQP4, the single known isoform of AQP9 and for expression of transcription factor HIF1α (sham, probe only control, and PBBI, n=8-10 per group). RESULTS: Global aqp4 mRNA was decreased at 24h (p<0.01) with PBBI. Three of the four known protein isoforms of AQP4 were detected, M1 (34kDa), M23 (32kDa) and isoform 3 (30kDa). AQP4 M1 decreased at 3 and 7days post-injury (p<0.001; p<0.01). AQP4 M23 levels were highly variable with no significant changes. AQP4 isoform 3 levels were decreased 3days post-PBBI (p<0.05). From 4, 12, and 24h aqp9 mRNA levels were decreased with injury (p<0.01, p<0.05, p<0.01) while AQP9 levels were decreased at 3 and 7days after PBBI (p<0.001, p<0.01). At 12 and 24h post-PBBI hif1α mRNA levels increased (p<0.05, p<0.01) but at 3 and 7days mRNA levels decreased (p<0.05, p<0.01). From 24h and 3 and 7days HIF1α protein levels were decreased (p<0.0001, p<0.0001, p<0.0001). In comparison to probe control, PBBI led to greater decreases in protein for AQP4 M1 (trend), AQP4 isoform 3 (trend), AQP9 (p<0.05) and HIF1α (p<0.05). CONCLUSION: PBBI is characterized by a loss of AQP4 M1, AQP4 isoform 3 and AQP9 at delayed time-points. The severity of the injury (PBBI versus probe control) increased these effects. Therefore, AQP9 and the AQP4 M1 isoform may be regulated by HIF1α, but not AQP4 isoform 3. This delayed loss of aquaporins may markedly reduce the ability of the brain to efflux water, contributing to the protracted edema that is a characteristic following severe penetrating TBI. Factors contributing to edema differ with different types and severities of TBI. For example, cellular based edema is more prominent in diffuse non-penetrating TBI whereas vasogenic edema is more prevalent with TBI involving hemorrhage. Molecular regulation leading to edema will likely also differ, such that treatments which have been suggested for non-hemorrhagic moderate TBI, such as the suppression of aquaporins, may be detrimental in more severe forms of TBI.

Human traumatic brain injury induces autoantibody response against glial fibrillary acidic protein and its breakdown products.

The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38-50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0-1 days) to late (7-10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.

Correlations between blood-brain barrier disruption and neuroinflammation in an experimental model of penetrating ballistic-like brain injury.

Abstract Blood-brain barrier (BBB) disruption is a pathological hallmark of severe traumatic brain injury (TBI) and is associated with neuroinflammatory events contributing to brain edema and cell death. The goal of this study was to elucidate the profile of BBB disruption after penetrating ballistic-like brain injury (PBBI) in conjunction with changes in neuroinflammatory markers. Brain uptake of biotin-dextran amine (BDA; 3 kDa) and horseradish peroxidase (HRP; 44 kDa) was evaluated in rats at 4 h, 24 h, 48 h, 72 h, and 7 days post-PBBI and compared with the histopathologic and molecular profiles for inflammatory markers. BDA and HRP both displayed a uniphasic profile of extravasation, greatest at 24 h post-injury and which remained evident out to 48 h for HRP and 7 days for BDA. This profile was most closely associated with markers for adhesion (mRNA for intercellular adhesion molecule-1) and infiltration of peripheral granulocytes (mRNA for matrix metalloproteinase-9 [MMP-9] and myeloperoxidase staining). Improvement of BBB dysfunction coincided with increased expression of markers implicated in tissue remodeling and repair. The results of this study reveal a uniphasic and gradient opening of the BBB after PBBI and suggest MMP-9 and resident inflammatory cell activation as candidates for future neurotherapeutic intervention after PBBI.

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.

Nefiracetam attenuates post-ischemic nonconvulsive seizures in rats and protects neuronal cell death induced by veratridine and glutamate.

AIMS: Stroke patients are at a high risk of developing post-ischemic seizures and cognitive impairment. Nefiracetam (NEF), a pyrrolidone derivative, has been shown to possess both anti-epileptic and cognitive-enhancing properties. In this study the anti-seizure effects of NEF were evaluated in a rat model of post-ischemic nonconvulsive seizures (NCSs). Its potential mechanisms were investigated in neuronal cell culture assays of neurotoxicity associated with ischemic brain injury and epileptogenesis. MAIN METHODS: In the in vivo study, rats received 24h permanent middle cerebral artery occlusion. NEF was administered intravenously either at 15 min post-injury but prior to the first NCS event (30 mg/kg, pre-NCS treatment) or immediately after the first NCS occurred (30 or 60 mg/kg, post-NCS treatment). In the in vitro study, neuronal cell cultures were exposed to veratridine or glutamate and treated with NEF (1-500 nM). KEY FINDINGS: The NEF pre-NCS treatment significantly reduced the NCS frequency and duration, whereas the higher NEF dose (60 mg/kg) was required to achieve similar effects when given after NCS occurred. The NEF treatment also dose-dependently (5-500 nM) protected against neuronal cell death induced by veratridine as measured by MTT cell viability assay, but higher doses (250-500 nM) were required against glutamate toxicity. SIGNIFICANCE: The anti-seizure property of NEF was demonstrated in a clinically relevant rat model of post-ischemic NCS. The preferential effects of NEF against in vitro veratridine toxicity suggest the involvement of its modulation of sodium channel malfunction. Future studies are warranted to study the mechanisms of NEF against ischemic brain injury and post-ischemic seizures.

Systems biology approaches for discovering biomarkers for traumatic brain injury.

The rate of traumatic brain injury (TBI) in service members with wartime injuries has risen rapidly in recent years, and complex, variable links have emerged between TBI and long-term neurological disorders. The multifactorial nature of TBI secondary cellular response has confounded attempts to find cellular biomarkers for its diagnosis and prognosis or for guiding therapy for brain injury. One possibility is to apply emerging systems biology strategies to holistically probe and analyze the complex interweaving molecular pathways and networks that mediate the secondary cellular response through computational models that integrate these diverse data sets. Here, we review available systems biology strategies, databases, and tools. In addition, we describe opportunities for applying this methodology to existing TBI data sets to identify new biomarker candidates and gain insights about the underlying molecular mechanisms of TBI response. As an exemplar, we apply network and pathway analysis to a manually compiled list of 32 protein biomarker candidates from the literature, recover known TBI-related mechanisms, and generate hypothetical new biomarker candidates.

Biomarkers track damage after graded injury severity in a rat model of penetrating brain injury.

The goal of this project was to determine whether biochemical markers of brain damage can be used to diagnose and assess the severity of injury in a rat model of penetrating ballistic-like brain injury (PBBI). To determine the relationship between injury magnitude and biomarker levels, rats underwent three discrete PBBI severity levels defined by the magnitude of the ballistic component of the injury, calibrated to equal 5%, 10%, or 12.5% of total rat brain volume. Cortex, cerebrospinal fluid (CSF), and blood were collected at multiple time points. Levels of three biomarkers (αII-spectrin breakdown product [SBDP150], glial fibrillary acidic protein [GFAP], and ubiquitin C-terminal hydrolase-L1 [UCH-L1]), were measured using quantitative immunoblotting and/or enzyme-linked immunosorbent assays. In injured cortex, SBDP150 and GFAP levels were increased significantly over controls. Cortical SBDP150 was elevated at 1 day but not 7 days, and GFAP at 7 days but not 1 day. At their respective time points, mean levels of SBDP150 and GFAP biomarkers in the cortex rose stepwise as injury magnitude increased. In the CSF, increasing severity of PBBI was associated with increasing concentrations of both neuronal and glial biomarkers acutely at 1 day after injury, but no trends were observed at 7 days. In plasma, SBDP150 was elevated at 5 min after 10% PBBI and at 6 h after 12.5% PBBI. UCH-L1 levels in plasma were elevated acutely at 5 min post-injury reflecting injury severity and rapidly decreased within 2 h. Overall, our results support the conclusion that biomarkers are effective indicators of brain damage after PBBI and may also aid in the assessment of injury magnitude.

Changes in apoptotic mechanisms following penetrating ballistic-like brain injury.

We investigated apoptotic pathways in a model of severe traumatic brain injury, penetrating ballistic-like brain injury (PBBI). TUNEL staining identified increasing apoptosis within 24 h. From targeted arrays, 11 genes were identified for temporal mRNA evaluation. In addition, mRNA levels and enzyme activity for caspases 3, 8, and 9 were examined. In the death receptor-mediated apoptosis pathway, the relative quantities (RQs) of mRNA for tnfr1, fas, and tnf were upregulated while trail mRNA was downregulated. In the anti-apoptotic TNF-R2 pathway, tnfr2 and flip were upregulated while xiap was downregulated. These findings indicate that increases in tnf levels following injury are not only pro-apoptotic but may also signal competing anti-apoptotic mechanisms. For the mitochondria-mediated apoptosis pathway, RQs of anti-apoptotic factors bcl2a1d and birc3 were upregulated while both bcl2 and bax were downregulated. RQs for casp 3 and casp 8 increased while casp9 decreased. Enzymatic activity increased for caspases 3, 8, and 9. While multiple mechanisms promoting and inhibiting apoptosis are at play during the first week after a PBBI, the cumulative result remains increased apoptosis. The ability to understand and dissect these events will assist in the development and evaluation of treatments targeting apoptosis following severe brain injury.

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.

Long-term administration of amnion-derived cellular cytokine suspension promotes functional recovery in a model of penetrating ballistic-like brain injury.

BACKGROUND: Previous work has shown that human amnion-derived progenitor (AMP) cell therapy is neuroprotective in a penetrating ballistic-like brain injury (PBBI) model. However, the neuroprotective capacity of AMP cells seemed to be mediated by the sustained secretion of AMP cell-derived neurotrophic factors, which are abundant in the amnion-derived cellular cytokine suspension (ACCS). To test this theory, the current study assessed the neuroprotective efficacy of long-term ACCS delivery in the PBBI model. METHODS: Experiment 1 assessed the bioactive stability and neuroprotective capacity of ACCS in an in vitro model of neurodegeneration. Experiment 2 evaluated the therapeutic effects of ACCS delivery initiated 15 minutes after PBBI and continued for 2 weeks after injury. Experiment 3 was designed to identify the therapeutic window for long-term ACCS delivery in the PBBI model. Outcome metrics included neurobehavioral assessments and neuropathologic measures of neuroinflammation and axonal/neuronal degeneration. RESULTS: Experiment 1 demonstrated that ACCS is thermally stable for 1 week at 37°C and that ACCS treatment protected neurite against staurosporine toxicity. Experiment 2 identified the optimal infusion rate of ACCS (1 µL/h) and demonstrated that long-term infusion of ACCS was capable of promoting significant protection against PBBI-induced neuropathology and motor abnormalities, but was not sufficient for reducing cognitive deficits. Finally, the results of Experiment 3 showed that ACCS is effective in promoting significant neuroprotection even when onset of treatment is delayed out to 24 hours (but not 48 hours) after PBBI. CONCLUSIONS: Collectively, our results support the hypothesis that the neuroprotective effects of AMP cells are mediated through a sustained delivery of ACCS, which implicates ACCS as a promising neuroprotection agent for clinical study.

A novel animal model of closed-head concussive-induced mild traumatic brain injury: development, implementation, and characterization.

Closed-head concussive injury is one of the most common causes of traumatic brain injury (TBI). While single concussions result in short-term neurologic dysfunction, multiple concussions can result in cumulative damage and increased risk for neurodegenerative disease. Despite the prevalence of concussion, knowledge about what occurs in the brain following this injury is limited, in part due to the limited number of appropriate animal research models. To study clinically relevant concussion we recently developed a simple, non-invasive rodent model of closed-head projectile concussive impact (PCI) TBI. For this purpose, anesthetized rats were placed on a platform positioned above a torque-sealed microcentrifuge tube packed with fixed amounts of dry ice. Upon heating, rapid sublimation of the dry ice produced a build-up of compressed CO(2) that triggered an eruptive force causing the cap to launch as an intact projectile, resulting in a targeted PCI head injury. A stainless steel helmet was implemented to protect the head from bruising, yet allowing the brain to sustain a mild PCI event. Depending on the injury location and the application of the helmet, PCI-induced injuries ranged from severe (i.e., head injury with subdural hematomas, intracranial hemorrhage, and brain tissue damage), to mild (no head injury, intracranial hemorrhage, or gross morphological pathology). Although no gross pathology was evident in mild PCI-induced injury, the following protein changes and behavioral abnormalities were detected between 1 and 24 h after PCI injury: (1) upregulation of glial fibrillary acidic protein (GFAP) in hippocampal regions; (2) upregulation of ubiquitin carboxyl-terminal hydrolase L1 (UCHL-1) in cortical tissue; and (3) significant sensorimotor abnormalities. Overall, these results indicated that this PCI model was capable of replicating salient pathologies of a clinical concussion, and could generate reproducible and quantifiable outcome measures.

Severity profile of penetrating ballistic-like brain injury on neurofunctional outcome, blood-brain barrier permeability, and brain edema formation.

This study evaluated the injury severity profile of unilateral, frontal penetrating ballistic-like brain injury (PBBI) on neurofunctional outcome, blood-brain barrier (BBB) permeability, and brain edema formation. The degree of injury severity was determined by the delivery of a water-pressure pulse designed to produce a temporary cavity by rapid (<40 ms) expansion of the probe's elastic balloon calibrated to equal 5%, 10%, 12.5%, or 15% of total rat brain volume (control groups consisted of sham surgery or insertion of the probe only). Neurofunctional assessments revealed motor and cognitive deficits related to the degree of injury severity, with the most clear-cut profile of PBBI injury severity depicted by the Morris water maze (MWM) results. A biphasic pattern of BBB leakage was detected in the injured hemisphere at all injury severity levels at 4 h post-injury, and again at 48-72 h post-injury, which remained evident out to 7 days post-PBBI in the 10% and 12.5% PBBI groups. Likewise, significant brain edema was detected in the injured hemisphere by 4 h post-injury and remained elevated out to 7 days post-injury in the 10% and 12.5% PBBI groups. However, following 5% PBBI, significant levels of edema were only detected from 24 h to 48h post-injury. These results identify an injury severity profile of BBB permeability, brain edema, and neurofunctional impairment that provides sensitive and clinically relevant outcome metrics for studying potential therapeutics.

Selective brain cooling in rats ameliorates intracerebral hemorrhage and edema caused by penetrating brain injury: possible involvement of heme oxygenase-1 expression.

Brain edema formation associated with trauma-induced intracerebral hemorrhage (ICH) is a clinical complication with high mortality. Studies have shown that heme oxygenase-1 (HO-1) plays an important role in ICH-induced brain edema. In order to understand the role of HO-1 in the protective effect of selective brain cooling (SBC), we investigated the time course of HO-1 changes following penetrating ballistic-like brain injury (PBBI) in rats. Samples were collected from injured and control animals at 6, 24, 48, and 72 h, and 7 days post-injury to evaluate HO-1 expression, heme concentration, brain water content, and immunohistochemistry (IHC). Following a 10% frontal PBBI, HO-1 mRNA and protein was increased at all time points studied, reaching maximum expression levels at 24-48 h post-injury. An increase in the heme concentration and the development of brain edema coincided with the upregulation of HO-1 mRNA and protein during the 7-day post-injury period. SBC significantly decreased PBBI-induced heme concentration, attenuated HO-1 upregulation, and concomitantly reduced brain water content. These results suggest that the neuroprotective effects of SBC may be partially mediated by reducing the heme accumulation, which reduced injury-mediated upregulation of HO-1, and in turn ameliorated edema formation. Collectively, these results suggest a potential value of HO-1 as a diagnostic and/or therapeutic biomarker in hemorrhagic brain injury.

Synergism of human amnion-derived multipotent progenitor (AMP) cells and a collagen scaffold in promoting brain wound recovery: pre-clinical studies in an experimental model of penetrating ballistic-like brain injury.

One of the histopathological consequences of a penetrating ballistic brain injury is the formation of a permanent cavity. In a previous study using the penetrating ballistic-like brain injury (PBBI) model, engrafted human amnion-derived multipotent progenitor (AMP) cells failed to survive when injected directly in the injury tract, suggesting that the cell survival requires a supportive matrix. In this study, we seated AMP cells in a collagen-based scaffold, injected into the injury core, and investigated cell survival and neuroprotection following PBBI. AMP cells suspended in AMP cell conditioned medium (ACCS) or in a liquefied collagen matrix were injected immediately after a PBBI along the penetrating injury tract. Injured control rats received only liquefied collagen matrix. All animals were allowed to survive two weeks. Consistent with our previous results, AMP cells suspended in ACCS failed to survive; likewise, no collagen was identified at the injury site when injected alone. In contrast, both AMP cells and the collagen were preserved in the injury cavity when injected together. In addition, AMP cells/collagen treatment preserved some apparent brain tissue in the injury cavity, and there was measurable infiltration of endogenous neural progenitor cells and astrocytes into the preserved brain tissue. AMP cells were also found to have migrated into the subventricular zone and the corpus callosum. Moreover, the AMP cell/collagen treatment significantly attenuated the PBBI-induced axonal degeneration in the corpus callosum and ipsilateral thalamus and improved motor impairment on rotarod performance. Overall, collagen-based scaffold provided a supportive matrix for AMP cell survival, migration, and neuroprotection.

Acute and chronic Chlamydia pneumoniae infection in pregnancy complicated with preeclampsia.

OBJECTIVE: To investigate whether Chlamydia pneumoniae (Cp) infection is more common in women whose current pregnancy is complicated with preeclampsia (PE) as compared to pregnant women without PE. METHODS: Thirty pregnant women with PE and 30 pregnant women without PE were studied between 29 and 30 weeks of gestation. The presence of an acute or chronic Cp infection was determined by the estimations of serum IgG, IgM, and IgA Cp antibodies. RESULTS: None of the women were diagnosed as having acute Cp infection. Prevalence of chronic Cp infection was 53 and 66% in the PE and control groups, respectively (X(2), p = 0.068). CONCLUSION: Chronic Cp infection is not more common in women whose pregnancy is complicated with PE as compared to pregnant women without PE. Therefore, no association between Cp infection and PE can be established.

Neuroproteomics: a biochemical means to discriminate the extent and modality of brain injury.

Diagnosis and treatment of stroke and traumatic brain injury remain significant health care challenges to society. Patient care stands to benefit from an improved understanding of the interactive biochemistry underlying neurotrauma pathobiology. In this study, we assessed the power of neuroproteomics to contrast biochemical responses following ischemic and traumatic brain injuries in the rat. A middle cerebral artery occlusion (MCAO) model was employed in groups of 30-min and 2-h focal neocortical ischemia with reperfusion. Neuroproteomes were assessed via tandem cation-anion exchange chromatography-gel electrophoresis, followed by reversed-phase liquid chromatography-tandem mass spectrometry. MCAO results were compared with those from a previous study of focal contusional brain injury employing the same methodology to characterize homologous neocortical tissues at 2 days post-injury. The 30-min MCAO neuroproteome depicted abridged energy production involving pentose phosphate, modulated synaptic function and plasticity, and increased chaperone activity and cell survival factors. The 2-h MCAO data indicated near complete loss of ATP production, synaptic dysfunction with degraded cytoarchitecture, more conservative chaperone activity, and additional cell survival factors than those seen in the 30-min MCAO model. The TBI group exhibited disrupted metabolism, but with retained malate shuttle functionality. Synaptic dysfunction and cytoarchitectural degradation resembled the 2-h MCAO group; however, chaperone and cell survival factors were more depressed following TBI. These results underscore the utility of neuroproteomics for characterizing interactive biochemistry for profiling and contrasting the molecular aspects underlying the pathobiological differences between types of brain injuries.