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.

Minor and repetitive head injury.

Traumatic brain injury (TBI) is the leading cause of death and disability in the young, active population and expected to be the third leading cause of death in the whole world until 2020. The disease is frequently referred to as the silent epidemic, and many authors highlight the "unmet medical need" associated with TBI.The term traumatically evoked brain injury covers a heterogeneous group ranging from mild/minor/minimal to severe/non-salvageable damages. Severe TBI has long been recognized to be a major socioeconomical health-care issue as saving young lives and sometimes entirely restituting health with a timely intervention can indeed be extremely cost efficient.Recently it has been recognized that mild or minor TBI should be considered similarly important because of the magnitude of the patient population affected. Other reasons behind this recognition are the association of mild head injury with transient cognitive disturbances as well as long-term sequelae primarily linked to repeat (sport-related) injuries.The incidence of TBI in developed countries can be as high as 2-300/100,000 inhabitants; however, if we consider the injury pyramid, it turns out that severe and moderate TBI represents only 25-30 % of all cases, while the overwhelming majority of TBI cases consists of mild head injury. On top of that, or at the base of the pyramid, are the cases that never show up at the ER - the unreported injuries.Special attention is turned to mild TBI as in recent military conflicts it is recognized as "signature injury."This chapter aims to summarize the most important features of mild and repetitive traumatic brain injury providing definitions, stratifications, and triage options while also focusing on contemporary knowledge gathered by imaging and biomarker research.Mild traumatic brain injury is an enigmatic lesion; the classification, significance, and its consequences are all far less defined and explored than in more severe forms of brain injury.Understanding the pathobiology and pathomechanisms may aid a more targeted approach in triage as well as selection of cases with possible late complications while also identifying the target patient population where preventive measures and therapeutic tools should be applied in an attempt to avoid secondary brain injury and late complications.

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.

The challenge of mild traumatic brain injury: role of biochemical markers in diagnosis of brain damage.

During the past decade there has been an increasing recognition of the incidence of mild traumatic brain injury (mTBI) and a better understanding of the subtle neurological and cognitive deficits that may result from it. A substantial, albeit suboptimal, effort has been made to define diagnostic criteria for mTBI and improve diagnostic accuracy. Thus, biomarkers that can accurately and objectively detect brain injury after mTBI and, ideally, aid in clinical management are needed. In this review, we discuss the current research on serum biomarkers for mTBI including their rationale and diagnostic performances. Sensitive and specific biomarkers reflecting brain injury can provide important information regarding TBI pathophysiology and serve as candidate markers for predicting abnormal computed tomography findings and/or the development of residual deficits in patients who sustain an mTBI. We also outline the roles of biomarkers in settings of specific interest including pediatric TBI, sports concussions and military injuries, and provide perspectives on the validation of such markers for use in the clinic. Finally, emerging proteomics-based strategies for identifying novel markers will be discussed.

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.

Elevated levels of serum glial fibrillary acidic protein breakdown products in mild and moderate traumatic brain injury are associated with intracranial lesions and neurosurgical intervention.

STUDY OBJECTIVE: This study examines whether serum levels of glial fibrillary acidic protein breakdown products (GFAP-BDP) are elevated in patients with mild and moderate traumatic brain injury compared with controls and whether they are associated with traumatic intracranial lesions on computed tomography (CT) scan (positive CT result) and with having a neurosurgical intervention. METHODS: This prospective cohort study enrolled adult patients presenting to 3 Level I trauma centers after blunt head trauma with loss of consciousness, amnesia, or disorientation and a Glasgow Coma Scale (GCS) score of 9 to 15. Control groups included normal uninjured controls and trauma controls presenting to the emergency department with orthopedic injuries or a motor vehicle crash without traumatic brain injury. Blood samples were obtained in all patients within 4 hours of injury and measured by enzyme-linked immunosorbent assay for GFAP-BDP (nanograms/milliliter). RESULTS: Of the 307 patients enrolled, 108 were patients with traumatic brain injury (97 with GCS score 13 to 15 and 11 with GCS score 9 to 12) and 199 were controls (176 normal controls and 16 motor vehicle crash controls and 7 orthopedic controls). Receiver operating characteristic curves demonstrated that early GFAP-BDP levels were able to distinguish patients with traumatic brain injury from uninjured controls with an area under the curve of 0.90 (95% confidence interval [CI] 0.86 to 0.94) and differentiated traumatic brain injury with a GCS score of 15 with an area under the curve of 0.88 (95% CI 0.82 to 0.93). Thirty-two patients with traumatic brain injury (30%) had lesions on CT. The area under these curves for discriminating patients with CT lesions versus those without CT lesions was 0.79 (95% CI 0.69 to 0.89). Moreover, the receiver operating characteristic curve for distinguishing neurosurgical intervention from no neurosurgical intervention yielded an area under the curve of 0.87 (95% CI 0.77 to 0.96). CONCLUSION: GFAP-BDP is detectable in serum within an hour of injury and is associated with measures of injury severity, including the GCS score, CT lesions, and neurosurgical intervention. Further study is required to validate these findings before clinical application.

Increased levels of serum MAP-2 at 6-months correlate with improved outcome in survivors of severe traumatic brain injury.

OBJECTIVE: To evaluate microtubule-associated proteins (MAP-2), a dendritic marker of both acute damage and chronic neuronal regeneration after injury, in serum of survivors after severe TBI and examine the association with long-term outcome. METHODS: Serum concentrations of MAP-2 were evaluated in 16 patients with severe TBI (Glasgow Coma Scale score [GCS] ≤ 8) 6 months post-injury and in 16 controls. Physical and cognitive outcomes were assessed, using the Glasgow Outcome Scale Extended (GOSE) and Levels of Cognitive Functioning Scale (LCFS), respectively. RESULTS: Severe TBI patients had significantly higher serum MAP-2 concentrations than normal controls with no history of TBI (p = 0.008) at 6 months post-injury. MAP-2 levels correlated with the GOSE (r = 0.58, p = 0.02) and LCFS (r = 0.65, p = 0.007) at month 6. Significantly lower serum levels of MAP-2 were observed in patients in a vegetative state (VS) compared to non-VS patients (p < 0.05). A trend tracking the level of consciousness was observed. CONCLUSIONS: Severe TBI results in a chronic release of MAP-2 into the peripheral circulation in patients with higher levels of consciousness, suggesting that remodelling of synaptic junctions and neuroplasticity processes occur several months after injury. The data indicate MAP-2 as a potential marker for emergence to higher levels of cognitive function.

Extensive antibody cross-reactivity among infectious gram-negative bacteria revealed by proteome microarray analysis.

Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising approximately 70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms. We first examined rabbit antibodies produced against proteomes extracted from Y. pestis, Burkholderia mallei, Burkholderia cepecia, Burkholderia pseudomallei, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies enabled detection of shared cross-reactive proteins, fingerprint proteins common for two or more bacteria, and signature proteins specific to each pathogen. Recognition by rabbit and non-human primate antibodies involved less than 100 of the thousands of proteins present within the Y. pestis proteome. Further antigen binding patterns were revealed that could distinguish plague from anthrax, caused by the Gram-positive bacterium Bacillus anthracis, using sera from acutely infected or convalescent primates. Thus, our results demonstrate potential biomarkers that are either specific to one strain or common to several species of pathogenic bacteria.

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.

Conversion of a mouse Fab into a whole humanized IgG antibody for detecting botulinum toxin.

Antibodies serve as the gold standard in most immunodiagnostic assays. Recent advances in recombinant DNA technology have offered the production of antibody fragments or Fabs as promising alternatives. However, the lack of the Fc region of the antibody can be difficult in many standard diagnostic platforms. Therefore we sought to convert a murine Fab into a whole humanized IgG. The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated conversion to human IgG with no traces of mouse Fab as determined by Western blot analysis. In addition, the humanized IgG performed better as both a detection and capture reagent in an ELISA format and detected the botulinum toxoid at a lower concentration than the parental murine Fab. This technique offers the ability to convert various species of antibodies or antibody fragments into humanized antibodies with improved characteristics in immunodiagnostic assays, for use as human controls in serological assays, or for possible therapeutic benefit.

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.

Approach to Modeling, Therapy Evaluation, Drug Selection, and Biomarker Assessments for a Multicenter Pre-Clinical Drug Screening Consortium for Acute Therapies in Severe Traumatic Brain Injury: Operation Brain Trauma Therapy.

Traumatic brain injury (TBI) was the signature injury in both the Iraq and Afghan wars and the magnitude of its importance in the civilian setting is finally being recognized. Given the scope of the problem, new therapies are needed across the continuum of care. Few therapies have been shown to be successful. In severe TBI, current guidelines-based acute therapies are focused on the reduction of intracranial hypertension and optimization of cerebral perfusion. One factor considered important to the failure of drug development and translation in TBI relates to the recognition that TBI is extremely heterogeneous and presents with multiple phenotypes even within the category of severe injury. To address this possibility and attempt to bring the most promising therapies to clinical trials, we developed Operation Brain Trauma Therapy (OBTT), a multicenter, pre-clinical drug screening consortium for acute therapies in severe TBI. OBTT was developed to include a spectrum of established TBI models at experienced centers and assess the effect of promising therapies on both conventional outcomes and serum biomarker levels. In this review, we outline the approach to TBI modeling, evaluation of therapies, drug selection, and biomarker assessments for OBTT, and provide a framework for reports in this issue on the first five therapies evaluated by the consortium.

Erythropoietin Treatment in Traumatic Brain Injury: Operation Brain Trauma Therapy.

Experimental studies targeting traumatic brain injury (TBI) have reported that erythropoietin (EPO) is an endogenous neuroprotectant in multiple models. In addition to its neuroprotective effects, it has also been shown to enhance reparative processes including angiogenesis and neurogenesis. Based on compelling pre-clinical data, EPO was tested by the Operation Brain Trauma Therapy (OBTT) consortium to evaluate therapeutic potential in multiple TBI models along with biomarker assessments. Based on the pre-clinical TBI literature, two doses of EPO (5000 and 10,000 IU/kg) were tested given at 15 min after moderate fluid percussion brain injury (FPI), controlled cortical impact (CCI), or penetrating ballistic-like brain injury (PBBI) with subsequent behavioral, histopathological, and biomarker outcome assessments. There was a significant benefit on beam walk with the 5000 IU dose in CCI, but no benefit on any other motor task across models in OBTT. Also, no benefit of EPO treatment across the three TBI models was noted using the Morris water maze to assess cognitive deficits. Lesion volume analysis showed no treatment effects after either FPI or CCI; however, with the 5000 IU/kg dose of EPO, a paradoxical increase in lesion volume and percent hemispheric tissue loss was seen after PBBI. Biomarker assessments included measurements of glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) in blood at 4 or 24 h after injury. No treatment effects were seen on biomarker levels after FPI, whereas treatment at either dose exacerbated the increase in GFAP at 24 h in PBBI but attenuated 24-4 h delta UCH-L1 levels at high dose in CCI. Our data indicate a surprising lack of efficacy of EPO across three established TBI models in terms of behavioral, histopathological, and biomarker assessments. Although we cannot rule out the possibility that other doses or more prolonged treatment could show different effects, the lack of efficacy of EPO reduced enthusiasm for its further investigation in OBTT.

Nicotinamide Treatment in Traumatic Brain Injury: Operation Brain Trauma Therapy.

Nicotinamide (vitamin B3) was the first drug selected for cross-model testing by the Operation Brain Trauma Therapy (OBTT) consortium based on a compelling record of positive results in pre-clinical models of traumatic brain injury (TBI). Adult male Sprague-Dawley rats were exposed to either moderate fluid percussion injury (FPI), controlled cortical impact injury (CCI), or penetrating ballistic-like brain injury (PBBI). Nicotinamide (50 or 500 mg/kg) was delivered intravenously at 15 min and 24 h after injury with subsequent behavioral, biomarker, and histopathological outcome assessments. There was an intermediate effect on balance beam performance with the high (500 mg/kg) dose in the CCI model, but no significant therapeutic benefit was detected on any other motor task across the OBTT TBI models. There was an intermediate benefit on working memory with the high dose in the FPI model. A negative effect of the low (50 mg/kg) dose, however, was observed on cognitive outcome in the CCI model, and no cognitive improvement was observed in the PBBI model. Lesion volume analysis showed no treatment effects after either FPI or PBBI, but the high dose of nicotinamide resulted in significant tissue sparing in the CCI model. Biomarker assessments included measurements of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase-1 (UCH-L1) in blood at 4 or 24 h after injury. Negative effects (both doses) were detected on biomarker levels of GFAP after FPI and on biomarker levels of UCH-L1 after PBBI. The high dose of nicotinamide, however, reduced GFAP levels after both PBBI and CCI. Overall, our results showed a surprising lack of benefit from the low dose nicotinamide. In contrast, and partly in keeping with the literature, some benefit was achieved with the high dose. The marginal benefits achieved with nicotinamide, however, which appeared sporadically across the TBI models, has reduced enthusiasm for further investigation by the OBTT Consortium.

Ability of Serum Glial Fibrillary Acidic Protein, Ubiquitin C-Terminal Hydrolase-L1, and S100B To Differentiate Normal and Abnormal Head Computed Tomography Findings in Patients with Suspected Mild or Moderate Traumatic Brain Injury.

Head computed tomography (CT) imaging is still a commonly obtained diagnostic test for patients with minor head injury despite availability of clinical decision rules to guide imaging use and recommendations to reduce radiation exposure resulting from unnecessary imaging. This prospective multicenter observational study of 251 patients with suspected mild to moderate traumatic brain injury (TBI) evaluated three serum biomarkers' (glial fibrillary acidic protein [GFAP], ubiquitin C-terminal hydrolase-L1 [UCH-L1] and S100B measured within 6 h of injury) ability to differentiate CT negative and CT positive findings. Of the 251 patients, 60.2% were male and 225 (89.6%) had a presenting Glasgow Coma Scale score of 15. A positive head CT (intracranial injury) was found in 36 (14.3%). UCH-L1 was 100% sensitive and 39% specific at a cutoff value >40 pg/mL. To retain 100% sensitivity, GFAP was 0% specific (cutoff value 0 pg/mL) and S100B had a specificity of only 2% (cutoff value 30 pg/mL). All three biomarkers had similar values for areas under the receiver operator characteristic curve: 0.79 (95% confidence interval; 0.70-0.88) for GFAP, 0.80 (0.71-0.89) for UCH-L1, and 0.75 (0.65-0.85) for S100B. Neither GFAP nor UCH-L1 curve values differed significantly from S100B (p = 0.21 and p = 0.77, respectively). In our patient cohort, UCH-L1 outperformed GFAP and S100B when the goal was to reduce CT use without sacrificing sensitivity. UCH-L1 values <40 pg/mL could potentially have aided in eliminating 83 of the 215 negative CT scans. These results require replication in other studies before the test is used in actual clinical practice.

Levetiracetam Treatment in Traumatic Brain Injury: Operation Brain Trauma Therapy.

Levetiracetam (LEV) is an antiepileptic agent targeting novel pathways. Coupled with a favorable safety profile and increasing empirical clinical use, it was the fifth drug tested by Operation Brain Trauma Therapy (OBTT). We assessed the efficacy of a single 15 min post-injury intravenous (IV) dose (54 or 170 mg/kg) on behavioral, histopathological, and biomarker outcomes after parasagittal fluid percussion brain injury (FPI), controlled cortical impact (CCI), and penetrating ballistic-like brain injury (PBBI) in rats. In FPI, there was no benefit on motor function, but on Morris water maze (MWM), both doses improved latencies and path lengths versus vehicle (p < 0.05). On probe trial, the vehicle group was impaired versus sham, but both LEV treated groups did not differ versus sham, and the 54 mg/kg group was improved versus vehicle (p < 0.05). No histological benefit was seen. In CCI, there was a benefit on beam balance at 170 mg/kg (p < 0.05 vs. vehicle). On MWM, the 54 mg/kg dose was improved and not different from sham. Probe trial did not differ between groups for either dose. There was a reduction in hemispheric tissue loss (p < 0.05 vs. vehicle) with 170 mg/kg. In PBBI, there was no motor, cognitive, or histological benefit from either dose. Regarding biomarkers, in CCI, 24 h glial fibrillary acidic protein (GFAP) blood levels were lower in the 170 mg/kg group versus vehicle (p < 0.05). In PBBI, GFAP blood levels were increased in vehicle and 170 mg/kg groups versus sham (p < 0.05) but not in the 54 mg/kg group. No treatment effects were seen for ubiquitin C-terminal hydrolase-L1 across models. Early single IV LEV produced multiple benefits in CCI and FPI and reduced GFAP levels in PBBI. LEV achieved 10 points at each dose, is the most promising drug tested thus far by OBTT, and the only drug to improve cognitive outcome in any model. LEV has been advanced to testing in the micropig model in OBTT.

Insight into Pre-Clinical Models of Traumatic Brain Injury Using Circulating Brain Damage Biomarkers: Operation Brain Trauma Therapy.

Operation Brain Trauma Therapy (OBTT) is a multicenter pre-clinical drug screening consortium testing promising therapies for traumatic brain injury (TBI) in three well-established models of TBI in rats--namely, parasagittal fluid percussion injury (FPI), controlled cortical impact (CCI), and penetrating ballistic-like brain injury (PBBI). This article presents unique characterization of these models using histological and behavioral outcomes and novel candidate biomarkers from the first three treatment trials of OBTT. Adult rats underwent CCI, FPI, or PBBI and were treated with vehicle (VEH). Shams underwent all manipulations except trauma. The glial marker glial fibrillary acidic protein (GFAP) and the neuronal marker ubiquitin C-terminal hydrolase (UCH-L1) were measured by enzyme-linked immunosorbent assay in blood at 4 and 24 h, and their delta 24-4 h was calculated for each marker. Comparing sham groups across experiments, no differences were found in the same model. Similarly, comparing TBI + VEH groups across experiments, no differences were found in the same model. GFAP was acutely increased in injured rats in each model, with significant differences in levels and temporal patterns mirrored by significant differences in delta 24-4 h GFAP levels and neuropathological and behavioral outcomes. Circulating GFAP levels at 4 and 24 h were powerful predictors of 21 day contusion volume and tissue loss. UCH-L1 showed similar tendencies, albeit with less robust differences between sham and injury groups. Significant differences were also found comparing shams across the models. Our findings (1) demonstrate that TBI models display specific biomarker profiles, functional deficits, and pathological consequence; (2) support the concept that there are different cellular, molecular, and pathophysiological responses to TBI in each model; and (3) advance our understanding of TBI, providing opportunities for a successful translation and holding promise for theranostic applications. Based on our findings, additional studies in pre-clinical models should pursue assessment of GFAP as a surrogate histological and/or theranostic end-point.

The WRAIR projectile concussive impact model of mild traumatic brain injury: re-design, testing and preclinical validation.

The WRAIR projectile concussive impact (PCI) model was developed for preclinical study of concussion. It represents a truly non-invasive closed-head injury caused by a blunt impact. The original design, however, has several drawbacks that limit the manipulation of injury parameters. The present study describes engineering advancements made to the PCI injury model including helmet material testing, projectile impact energy/head kinematics and impact location. Material testing indicated that among the tested materials, 'fiber-glass/carbon' had the lowest elastic modulus and yield stress for providing an relative high percentage of load transfer from the projectile impact, resulting in significant hippocampal astrocyte activation. Impact energy testing of small projectiles, ranging in shape and size, showed the steel sphere produced the highest impact energy and the most consistent impact characteristics. Additional tests confirmed the steel sphere produced linear and rotational motions on the rat's head while remaining within a range that meets the criteria for mTBI. Finally, impact location testing results showed that PCI targeted at the temporoparietal surface of the rat head produced the most prominent gait abnormalities. Using the parameters defined above, pilot studies were conducted to provide initial validation of the PCI model demonstrating quantifiable and significant increases in righting reflex recovery time, axonal damage and astrocyte activation following single and multiple concussions.

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.