Circulating GFAP and Iba-1 levels are associated with pathophysiological sequelae in the thalamus in a pig model of mild TBI.

Serum biomarkers are promising tools for evaluating patients following traumatic brain injury (TBI). However, their relationship with diffuse histopathology remains unclear. Additionally, translatability is a focus of neurotrauma research, however, studies using translational animal models are limited. Here, we evaluated associations between circulating biomarkers and acute thalamic histopathology in a translational micro pig model of mTBI. Serum samples were collected pre-injury, and 1 min-6 h following mTBI. Markers of neuronal injury (Ubiquitin Carboxy-terminal Hydrolase L1 [UCH-L1]), microglial/macrophage activation (Ionized calcium binding adaptor molecule-1 [Iba-1]) and interleukin-6 [IL-6]) and astrogliosis/astrocyte damage (glial fibrillary acidic protein [GFAP]) were measured. Axonal injury and histological features of neurons and glia were also investigated using immunofluorescent labeling and correlated to serum levels of the associated biomarkers. Consistent with prior experimental and human studies, GFAP, was highest at 6 h post-injury, while no substantial changes were observed in UCH-L1, Iba-1 or IL-6 over 6 h. This study also found promising associations between thalamic glial histological signatures and ensuing release of Iba-1 and GFAP into the circulation. Our findings suggest that in diffuse injury, monitoring serum Iba-1 and GFAP levels can provide clinically relevant insight into the underlying acute pathophysiology and biomarker release kinetics following mTBI, providing previously underappreciated diagnostic capability.

Serum-Based Phospho-Neurofilament-Heavy Protein as Theranostic Biomarker in Three Models of Traumatic Brain Injury: An Operation Brain Trauma Therapy Study.

Glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase (UCH-L1), markers of glial and neuronal cell body injury, respectively, have been previously selected by the Operation Brain Trauma Therapy (OBTT) pre-clinical therapy and biomarker screening consortium as drug development tools. However, traumatic axonal injury (TAI) also represents a major consequence and determinant of adverse outcomes after traumatic brain injury (TBI). Thus, biomarkers capable of assessing TAI are much needed. Neurofilaments (NFs) are found exclusively in axons. Here, we evaluated phospho-neurofilament-H (pNF-H) protein as a possible new TAI marker in serum and cerebrospinal fluid (CSF) across three rat TBI models in studies carried out by the OBTT consortium, namely, controlled cortical impact (CCI), parasagittal fluid percussion (FPI), and penetrating ballistics-like brain injury (PBBI). We indeed found that CSF and serum pNF-H levels are robustly elevated by 24 h post-injury in all three models. Further, in previous studies by OBTT, levetiracetam showed the most promising benefits, whereas nicotinamide showed limited benefit only at high dose (500 mg/kg). Thus, serum samples from the same repository collected by OBTT were evaluated. Treatment with 54 mg/kg intravenously of levetiracetam in the CCI model and 170 mg/kg in the PBBI model significantly attenuated pNF-H levels at 24 h post-injury as compared to respective vehicle groups. In contrast, nicotinamide (50 or 500 mg/kg) showed no reduction of pNF-H levels in CCI or PBBI models. Our current study suggests that pNF-H is a useful theranostic blood-based biomarker for TAI across different rodent TBI models. In addition, our data support levetiracetam as the most promising TBI drug candidate screened by OBTT to date.

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.

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.

Synthesis of Findings, Current Investigations, and Future Directions: Operation Brain Trauma Therapy.

Operation Brain Trauma Therapy (OBTT) is a fully operational, rigorous, and productive multicenter, pre-clinical drug and circulating biomarker screening consortium for the field of traumatic brain injury (TBI). In this article, we synthesize the findings from the first five therapies tested by OBTT and discuss both the current work that is ongoing and potential future directions. Based on the results generated from the first five therapies tested within the exacting approach used by OBTT, four (nicotinamide, erythropoietin, cyclosporine A, and simvastatin) performed below or well below what was expected based on the published literature. OBTT has identified, however, the early post-TBI administration of levetiracetam as a promising agent and has advanced it to a gyrencephalic large animal model--fluid percussion injury in micropigs. The sixth and seventh therapies have just completed testing (glibenclamide and Kollidon VA 64), and an eighth drug (AER 271) is in testing. Incorporation of circulating brain injury biomarker assessments into these pre-clinical studies suggests considerable potential for diagnostic and theranostic utility of glial fibrillary acidic protein in pre-clinical studies. Given the failures in clinical translation of therapies in TBI, rigorous multicenter, pre-clinical approaches to therapeutic screening such as OBTT may be important for the ultimate translation of therapies to the human condition.

Traumatic axonal injury in the perisomatic domain triggers ultrarapid secondary axotomy and Wallerian degeneration.

Traumatic axonal injury (TAI) arising from diffuse brain injury (DBI) results in focally impaired axonal transport with progressive swelling and delayed disconnection over several hours within brainstem axons. Neocortical DBI-mediated perisomatic axotomy does not result in neuronal death, suggesting that a comparably delayed axotomy progression was responsible for this unanticipated response. To evaluate delayed perisomatic axotomy, the current study was initiated. Rats received intracerebroventricular 10-kDa dextran followed by moderate midline/central fluid percussion injury (FPI) or FPI alone. At 15, 30, 60, and 180 min post-injury, light and transmission electron microscopy identified impaired axonal transport via antibodies targeting amyloid precursor protein (APP), while double-label fluorescent microscopy explored concomitant focal axolemmal alterations via dextran-APP co-localization. At 15 min post-injury, perisomatic TAI was identified with LM within dorsolateral and ventral posterior thalamic nuclei. Using TEM, many sustaining somata and related proximal/distal axonal segments revealed normal ultrastructural detail that was continuous with focal axonal swellings characterized by cytoskeletal and organelle pathology. In other cases, axotomy was confirmed by loss of axonal continuity distal to the swelling. By 30 min post-injury, perisomatic axotomy predominated. By 60-180 min, somatic, proximal axonal segment, and swelling ultrastructure were comparable to earlier time points although swelling diameter increased. Distal axonal segment ultrastructure now revealed the initial stages of Wallerian degeneration. The site of perisomatic axotomy did not internalize dextran, suggesting that its pathogenesis occurred independent of altered axolemmal permeability. Collectively, this DBI-mediated ultrarapid perisomatic axotomy and its sequelae further illustrate the varied axonal responses to trauma.