Effects of pregabalin on neurobehavior in an adult male rat model of PTSD.

Posttraumatic stress disorder (PTSD) can be a very debilitating condition. Effective approaches to prevent and treat PTSD are important areas of basic science research. Pregabalin (PGB), a gabapentinoid derivative of γ-aminobutyric acid, possesses the potential to positively affect neurobehavioral changes associated with PTSD. Using a rodent model of PTSD, the aims of this study were to determine the effects of PGB as a possible prevention for the development of PTSD-like symptoms and its use as a possible treatment. A prospective, experimental, between groups design was used in conjunction with a three-day restraint/shock PTSD stress model. Sixty rats were randomly assigned between two groups, non-stressed and stressed (PTSD). Each of the main two groups was then randomly assigned into six experimental groups: control vehicle, control PGB, control naïve, PTSD vehicle, PTSD Pre-PGB (prophylactic), PTSD Post-PGB (non-prophylactic). The neurobehavioral components of PTSD were evaluated using the elevated plus maze (EPM), Morris water maze (MWM), and forced swim test (FST). Pregabalin administered 24 hours before the initial PTSD event or for 10 days following the last PTSD stress event did not statistically improve mean open arm exploration on the EPM, spatial memory, and learning in the MWM or behavioral despair measured by the FST (p > 0.05).

Protein Citrullination: A Proposed Mechanism for Pathology in Traumatic Brain Injury.

Protein citrullination is a calcium-driven post-translational modification proposed to play a causative role in the neurodegenerative disorders of Alzheimer's disease, multiple sclerosis (MS), and prion disease. Citrullination can result in the formation of antigenic epitopes that underlie pathogenic autoimmune responses. This phenomenon, which is best understood in rheumatoid arthritis, may play a role in the chronic dysfunction following traumatic brain injury (TBI). Despite substantial evidence of aberrations in calcium signaling following TBI, there is little understanding of how TBI alters citrullination in the brain. The present investigation addressed this gap by examining the effects of TBI on the distribution of protein citrullination and on the specific cell types involved. Immunofluorescence revealed that controlled cortical impact in rats profoundly up--regulated protein citrullination in the cerebral cortex, external capsule, and hippocampus. This response was exclusively seen in astrocytes; no such effects were observed on the status of protein citrullination in neurons, oligodendrocytes or microglia. Further, proteomic analyses demonstrated that the effects of TBI on citrullination were confined to a relatively small subset of neural proteins. Proteins most notably affected were those also reported to be citrullinated in other disorders, including prion disease and MS. In vivo findings were extended in an in vitro model of simulated TBI employing normal human astrocytes. Pharmacologically induced calcium excitotoxicity was shown to activate the citrullination and breakdown of glial fibrillary acidic protein, producing a novel candidate TBI biomarker and potential target for autoimmune recognition. In summary, these findings demonstrate that the effects of TBI on protein citrullination are selective with respect to brain region, cell type, and proteins modified, and may contribute to a role for autoimmune dysfunction in chronic pathology following TBI.

Blood Biomarkers in Moderate-To-Severe Traumatic Brain Injury: Potential Utility of a Multi-Marker Approach in Characterizing Outcome.

BACKGROUND: Blood biomarkers are valuable tools for elucidating complex cellular and molecular mechanisms underlying traumatic brain injury (TBI). Profiling distinct classes of biomarkers could aid in the identification and characterization of initial injury and secondary pathological processes. This study characterized the prognostic performance of a recently developed multi-marker panel of circulating biomarkers that reflect specific pathogenic mechanisms including neuroinflammation, oxidative damage, and neuroregeneration, in moderate-to-severe TBI patients. MATERIALS AND METHODS: Peripheral blood was drawn from 85 isolated TBI patients (n = 60 severe, n = 25 moderate) at hospital admission, 6-, 12-, and 24-h post-injury. Mortality and neurological outcome were assessed using the extended Glasgow Outcome Scale. A multiplex platform was designed on MULTI-SPOT(®) plates to simultaneously analyze human plasma levels of s100 calcium binding protein beta (s100B), glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), brain-derived neurotrophic factor (BDNF), monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-5, and peroxiredoxin (PRDX)-6. Multivariable logistic regression and area under the receiver-operating characteristic curve (AUC) were used to evaluate both individual and combined predictive abilities of these markers for 6-month neurological outcome and mortality after TBI. RESULTS: Unfavorable neurological outcome was associated with elevations in s100B, GFAP, and MCP-1. Mortality was related to differences in six of the seven markers analyzed. Combined admission concentrations of s100B, GFAP, and MCP-1 were able to discriminate favorable versus unfavorable outcome (AUC = 0.83), and survival versus death (AUC = 0.87), although not significantly better than s100B alone (AUC = 0.82 and 0.86, respectively). CONCLUSION: The multi-marker panel of TBI-related biomarkers performed well in discriminating unfavorable and favorable outcomes in the acute period after moderate-to-severe TBI. However, the combination of these biomarkers did not outperform s100B alone.

Autoimmune Profiling Reveals Peroxiredoxin 6 as a Candidate Traumatic Brain Injury Biomarker.

Autoimmune profiling in rats revealed the antioxidant enzyme, peroxiredoxin 6 (PRDX6), as a target for autoantibodies evoked in response to traumatic brain injury (TBI). Consistent with this proposal, immunohistochemical analysis of rat cerebral cortex demonstrated that PRDX6 is highly expressed in the perivascular space, presumably contained within astrocytic foot processes. Accordingly, an immunosorbent electrochemiluminescence assay was developed for investigating PRDX6 in human samples. PRDX6 was found to be measurable in human blood and highly expressed in human cerebral cortex and platelets. Circulating levels of PRDX6 were elevated fourfold over control values 4 to 24 h following mild-to-moderate TBI. These findings suggest that PRDX6 may serve as a biomarker for TBI and that autoimmune profiling is a viable strategy for the discovery of novel TBI biomarkers.

Multivariate analysis of traumatic brain injury: development of an assessment score.

Important challenges for the diagnosis and monitoring of mild traumatic brain injury (mTBI) include the development of plasma biomarkers for assessing neurologic injury, monitoring pathogenesis, and predicting vulnerability for the development of untoward neurologic outcomes. While several biomarker proteins have shown promise in this regard, used individually, these candidates lack adequate sensitivity and/or specificity for making a definitive diagnosis or identifying those at risk of subsequent pathology. The objective for this study was to evaluate a panel of six recognized and novel biomarker candidates for the assessment of TBI in adult patients. The biomarkers studied were selected on the basis of their relative brain-specificities and potentials to reflect distinct features of TBI mechanisms including (1) neuronal damage assessed by neuron-specific enolase (NSE) and brain derived neurotrophic factor (BDNF); (2) oxidative stress assessed by peroxiredoxin 6 (PRDX6); (3) glial damage and gliosis assessed by glial fibrillary acidic protein and S100 calcium binding protein beta (S100b); (4) immune activation assessed by monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (MCP1/CCL2); and (5) disruption of the intercellular adhesion apparatus assessed by intercellular adhesion protein-5 (ICAM-5). The combined fold-changes in plasma levels of PRDX6, S100b, MCP1, NSE, and BDNF resulted in the formulation of a TBI assessment score that identified mTBI with a receiver operating characteristic (ROC) area under the curve of 0.97, when compared to healthy controls. This research demonstrates that a profile of biomarker responses can be used to formulate a diagnostic score that is sensitive for the detection of mTBI. Ideally, this multivariate assessment strategy will be refined with additional biomarkers that can effectively assess the spectrum of TBI and identify those at particular risk for developing neuropathologies as consequence of a mTBI event.

Protein carbonylation after traumatic brain injury: cell specificity, regional susceptibility, and gender differences.

Protein carbonylation is a well-documented and quantifiable consequence of oxidative stress in several neuropathologies, including multiple sclerosis, Alzheimer׳s disease, and Parkinson׳s disease. Although oxidative stress is a hallmark of traumatic brain injury (TBI), little work has explored the specific neural regions and cell types in which protein carbonylation occurs. Furthermore, the effect of gender on protein carbonylation after TBI has not been studied. The present investigation was designed to determine the regional and cell specificity of TBI-induced protein carbonylation and how this response to injury is affected by gender. Immunohistochemistry was used to visualize protein carbonylation in the brains of adult male and female Sprague-Dawley rats subjected to controlled cortical impact (CCI) as an injury model of TBI. Cell-specific markers were used to colocalize the presence of carbonylated proteins in specific cell types, including astrocytes, neurons, microglia, and oligodendrocytes. Results also indicated that the injury lesion site, ventral portion of the dorsal third ventricle, and ventricular lining above the median eminence showed dramatic increases in protein carbonylation after injury. Specifically, astrocytes and limited regions of ependymal cells adjacent to the dorsal third ventricle and the median eminence were most susceptible to postinjury protein carbonylation. However, these patterns of differential susceptibility to protein carbonylation were gender dependent, with males showing significantly greater protein carbonylation at sites distant from the lesion. Proteomic analyses were also conducted and determined that the proteins most affected by carbonylation in response to TBI include glial fibrillary acidic protein, dihydropyrimidase-related protein 2, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A. Many other proteins, however, were not carbonylated by CCI. These findings indicate that there is both regional and protein specificity in protein carbonylation after TBI. The marked increase in carbonylation seen in ependymal layers distant from the lesion suggests a mechanism involving the transmission of a cerebral spinal fluid-borne factor to these sites. Furthermore, this process is affected by gender, suggesting that hormonal mechanisms may serve a protective role against oxidative stress.

Impaired cortical mitochondrial function following TBI precedes behavioral changes.

Traumatic brain injury (TBI) pathophysiology can be attributed to either the immediate, primary physical injury, or the delayed, secondary injury which begins minutes to hours after the initial injury and can persist for several months or longer. Because these secondary cascades are delayed and last for a significant time period post-TBI, they are primary research targets for new therapeutics. To investigate changes in mitochondrial function after a brain injury, both the cortical impact site and ipsilateral hippocampus of adult male rats 7 and 17 days after a controlled cortical impact (CCI) injury were examined. State 3, state 4, and uncoupler-stimulated rates of oxygen consumption, respiratory control ratios (RCRs) were measured and membrane potential quantified, and all were significantly decreased in 7 day post-TBI cortical mitochondria. By contrast, hippocampal mitochondria at 7 days showed only non-significant decreases in rates of oxygen consumption and membrane potential. NADH oxidase activities measured in disrupted mitochondria were normal in both injured cortex and hippocampus at 7 days post-CCI. Respiratory and phosphorylation capacities at 17 days post-CCI were comparable to naïve animals for both cortical and hippocampus mitochondria. However, unlike oxidative phosphorylation, membrane potential of mitochondria in the cortical lining of the impact site did not recover at 17 days, suggesting that while diminished cortical membrane potential at 17 days does not adversely affect mitochondrial capacity to synthesize ATP, it may negatively impact other membrane potential-sensitive mitochondrial functions. Memory status, as assessed by a passive avoidance paradigm, was not significantly impaired until 17 days after injury. These results indicate pronounced disturbances in cortical mitochondrial function 7 days after CCI which precede the behavioral impairment observed at 17 days.