Applying Proteomics and Computational Approaches to Identify Novel Targets in Blast-Associated Post-Traumatic Epilepsy.

Traumatic brain injury (TBI) can lead to post-traumatic epilepsy (PTE). Blast TBI (bTBI) found in Veterans presents with several complications, including cognitive and behavioral disturbances and PTE; however, the underlying mechanisms that drive the long-term sequelae are not well understood. Using an unbiased proteomics approach in a mouse model of repeated bTBI (rbTBI), this study addresses this gap in the knowledge. After rbTBI, mice were monitored using continuous, uninterrupted video-EEG for up to four months. Following this period, we collected cortex and hippocampus tissues from three groups of mice: those with post-traumatic epilepsy (PTE+), those without epilepsy (PTE-), and the control group (sham). Hundreds of differentially expressed proteins were identified in the cortex and hippocampus of PTE+ and PTE- relative to sham. Focusing on protein pathways unique to PTE+, pathways related to mitochondrial function, post-translational modifications, and transport were disrupted. Computational metabolic modeling using dysregulated protein expression predicted mitochondrial proton pump dysregulation, suggesting electron transport chain dysregulation in the epileptic tissue relative to PTE-. Finally, data mining enabled the identification of several novel and previously validated TBI and epilepsy biomarkers in our data set, many of which were found to already be targeted by drugs in various phases of clinical testing. These findings highlight novel proteins and protein pathways that may drive the chronic PTE sequelae following rbTBI.

Blast neurotrauma impairs working memory and disrupts prefrontal myo-inositol levels in rats.

Working memory, which is dependent on higher-order executive function in the prefrontal cortex, is often disrupted in patients exposed to blast overpressure. In this study, we evaluated working memory and medial prefrontal neurochemical status in a rat model of blast neurotrauma. Adult male Sprague-Dawley rats were anesthetized with 3% isoflurane and exposed to calibrated blast overpressure (17 psi, 117 kPa) while sham animals received only anesthesia. Early neurochemical effects in the prefrontal cortex included a significant decrease in betaine (trimethylglycine) and an increase in GABA at 24 h, and significant increases in glycerophosphorylcholine, phosphorylethanolamine, as well as glutamate/creatine and lactate/creatine ratios at 48 h. Seven days after blast, only myo-inositol levels were altered showing a 15% increase. Compared to controls, short-term memory in the novel object recognition task was significantly impaired in animals exposed to blast overpressure. Working memory in control animals was negatively correlated with myo-inositol levels (r=-.759, p<0.05), an association that was absent in blast exposed animals. Increased myo-inositol may represent tardive glial scarring in the prefrontal cortex, a notion supported by GFAP changes in this region after blast overexposure as well as clinical reports of increased myo-inositol in disorders of memory.

Traumatic white matter injury and glial activation: from basic science to clinics.

An improved understanding and characterization of glial activation and its relationship with white matter injury will likely serve as a novel treatment target to curb post injury inflammation and promote axonal remyelination after brain trauma. Traumatic brain injury (TBI) is a significant public healthcare burden and a leading cause of death and disability in the United States. Particularly, traumatic white matter (WM) injury or traumatic axonal injury has been reported as being associated with patients' poor outcomes. However, there is very limited data reporting the importance of glial activation after TBI and its interaction with WM injury. This article presents a systematic review of traumatic WM injury and the associated glial activation, from basic science to clinical diagnosis and prognosis, from advanced neuroimaging perspective. It concludes that there is a disconnection between WM injury research and the essential role of glia which serve to restore a healthy environment for axonal regeneration following WM injury. Particularly, there is a significant lack of non-invasive means to characterize the complex pathophysiology of WM injury and glial activation in both animal models and in humans. An improved understanding and characterization of the relationship between glia and WM injury will likely serve as a novel treatment target to curb post injury inflammation and promote axonal remyelination.

Quantifying acute changes in neurometabolism following blast-induced traumatic brain injury.

Brain health is largely dependent on the metabolic regulation of amino acids. Brain injuries, diseases, and disorders can be detected through alterations in free amino acid (FAA) concentrations; and thus, mapping the changes has high diagnostic potential. Common methods focus on optimizing neurotransmitter quantification; however, recent focus has expanded to investigate the roles of molecular precursors in brain metabolism. An isocratic method using high performance liquid chromatography with electrochemical cell detection was developed to quantify a wide range of molecular precursors and neurotransmitters: alanine, arginine, aspartate, serine, taurine, threonine, tyrosine, glycine, glutamate, glutamine, and γ-Aminobutyric acid (GABA) following traumatic brain injury. First, baseline concentrations were determined in the serum, cerebrospinal fluid, hippocampus, cortex, and cerebellum of naïve male Sprague Dawley rats. A subsequent study was performed investigating acute changes in FAA concentrations following blast-induced traumatic brain injury (bTBI). Molecular precursor associated FAAs decreased in concentration at 4 h after injury in both the cortex and hippocampus while those serving as neurotransmitters remained unchanged. In particular, the influence of oxidative stress on the observed changes within alanine and arginine pathways following bTBI should be further investigated to elucidate the full therapeutic potential of these molecular precursors at acute time points.

Spatial Intracranial Pressure Fields Driven by Blast Overpressure in Rats.

Free-field blast exposure imparts a complex, dynamic response within brain tissue that can trigger a cascade of lasting neurological deficits. Full body mechanical and physiological factors are known to influence the body's adaptation to this seemingly instantaneous insult, making it difficult to accurately pinpoint the brain injury mechanisms. This study examined the intracranial pressure (ICP) profile characteristics in a rat model as a function of blast overpressure magnitude and brain location. Metrics such as peak rate of change of pressure, peak pressure, rise time, and ICP frequency response were found to vary spatially throughout the brain, independent of blast magnitude, emphasizing unique spatial pressure fields as a primary biomechanical component to blast injury. This work discusses the ICP characteristics and considerations for finite element models, in vitro models, and translational in vivo models to improve understanding of biomechanics during primary blast exposure.

Comparative assessment of iridium oxide and platinum alloy wires using an in vitro glial scar assay.

The long-term effect of chronically implanted electrodes is the formation of a glial scar. Therefore, it is imperative to assess the biocompatibility of materials before employing them in neural electrode fabrication. Platinum alloy and iridium oxide have been identified as good candidates as neural electrode biomaterials due to their mechanical and electrical properties, however, effect of glial scar formation for these two materials is lacking. In this study, we applied a glial scarring assay to observe the cellular reactivity to platinum alloy and iridium oxide wires in order to assess the biocompatibility based on previously defined characteristics. Through real-time PCR, immunostaining and imaging techniques, we will advance the understanding of the biocompatibility of these materials. Results of this study demonstrate iridium oxide wires exhibited a more significant reactive response as compared to platinum alloy wires. Cells cultured with platinum alloy wires had less GFAP gene expression, lower average GFAP intensity, and smaller glial scar thickness. Collectively, these results indicated that platinum alloy wires were more biocompatible than the iridium oxide wires.

Regional variances depict a unique glial-specific inflammatory response following closed-head injury.

Mild traumatic brain injuries (mTBI) constitute a significant health concern with clinical symptoms ranging from headaches to cognitive deficits. Despite the myriad of symptoms commonly reported following this injury, there is still a lack of knowledge on the various pathophysiological changes that occur. Preclinical studies are at the forefront of discovery delineating the changes that occur within this heterogeneous injury, with the emergence of translational models such as closed-head impact models allowing for further exploration of this injury mechanism. In the current study, male rats were subjected to a closed-head controlled cortical impact (cCCI), producing a concussion (mTBI). The pathological effects of this injury were then evaluated using immunoflourescence seven days following. The results exhibited a unique glial-specific inflammatory response, with both the ipsilateral and contralateral sides of the cortex and hippocampus showing pathological changes following impact. Overall these findings are consistent with glial changes reported following concussions and may contribute to subsequent symptoms.

The Imbalance of Astrocytic Mitochondrial Dynamics Following Blast-Induced Traumatic Brain Injury.

Mild blast-induced traumatic brain injury (bTBI) is a modality of injury that has been of major concern considering a large number of military personnel exposed to explosive blast waves. bTBI results from the propagation of high-pressure static blast forces and their subsequent energy transmission within brain tissue. Exposure to this overpressure energy causes a diffuse injury that leads to acute cell damage and, if chronic, leads to detrimental long-term cognitive deficits. The literature presents a neuro-centric approach to the role of mitochondria dynamics dysfunction in bTBI, and changes in astrocyte-specific mitochondrial dynamics have not been characterized. The balance between fission and fusion events is known as mitochondrial dynamics. As a result of fission and fusion, the mitochondrial structure is constantly altering its shape to respond to physiological stimuli or stress, which in turn affects mitochondrial function. Astrocytic mitochondria are recognized to play an essential role in overall brain metabolism, synaptic transmission, and neuron protection. Mitochondria are vulnerable to injury insults, leading to the increase in mitochondrial fission, a mechanism controlled by the GTPase dynamin-related protein (Drp1) and the phosphorylation of Drp1 at serine 616 (p-Drp1s616). This site is critical to mediate the Drp1 translocation to mitochondria to promote fission events and consequently leads to fragmentation. An increase in mitochondrial fragmentation could have negative consequences, such as promoting an excessive generation of reactive oxygen species or triggering cytochrome c release. The aim of the present study was to characterize the unique pattern of astrocytic mitochondrial dynamics by exploring the role of DRP1 with a combination of in vitro and in vivo bTBI models. Differential remodeling of the astrocytic mitochondrial network was observed, corresponding with increases in p-Drp1S616 four hours and seven days post-injury. Further, results showed a time-dependent reactive astrocyte phenotype transition in the rat hippocampus. This discovery can lead to innovative therapeutics targets to help prevent the secondary injury cascade after blast injury that involves mitochondria dysfunction.

An automated rat grimace scale for the assessment of pain.

Pain is a complex neuro-psychosocial experience that is internal and private, making it difficult to assess in both humans and animals. In pain research, animal models are prominently used, with rats among the most commonly studied. The rat grimace scale (RGS) measures four facial action units to quantify the pain behaviors of rats. However, manual recording of RGS scores is a time-consuming process that requires training. While computer vision models have been developed and utilized for various grimace scales, there are currently no models for RGS. To address this gap, this study worked to develop an automated RGS system which can detect facial action units in rat images and predict RGS scores. The automated system achieved an action unit detection precision and recall of 97%. Furthermore, the action unit RGS classifiers achieved a weighted accuracy of 81-93%. The system's performance was evaluated using a blast traumatic brain injury study, where it was compared to trained human graders. The results showed an intraclass correlation coefficient of 0.82 for the total RGS score, indicating that the system was comparable to human graders. The automated tool could enhance pain research by providing a standardized and efficient method for the assessment of RGS.

CNS4 causes subtype-specific changes in agonist efficacy and reversal potential of permeant cations in NMDA receptors.

The NMDA subtype of glutamate receptor serves as an attractive drug target for the treatment of disorders evolving from hyper- or hypoglutamatergic conditions. Compounds that optimize the function of NMDA receptors are of great clinical significance. Here, we present the pharmacological characterization of a biased allosteric modulator, CNS4. Results indicate that CNS4 sensitizes ambient levels of agonists and reduces higher-concentration glycine & glutamate efficacy in 1/2AB receptors, but minimally alters these parameters in diheteromeric 1/2A or 1/2B receptors. Glycine efficacy is increased in both 1/2C and 1/2D, while glutamate efficacy is decreased in 1/2C and unaltered in 1/2D. CNS4 does not affect the activity of competitive antagonist binding at glycine (DCKA) and glutamate (DL-AP5) sites; however, it decreases memantine potency in 1/2A receptors but not in 1/2D receptors. Current-voltage (I-V) relationship studies indicate that CNS4 potentiates 1/2A inward currents, a phenomenon that was reversed in the absence of permeable Na+ ions. In 1/2D receptors, CNS4 blocks inward currents based on extracellular Ca2+ concentration. Further, CNS4 positively modulates glutamate potency on E781A_1/2A mutant receptors, indicating its role at the distal end of the 1/2A agonist binding domain interface. Together, these findings reveal that CNS4 sensitizes ambient agonists and allosterically modulates agonist efficacy by altering Na+ permeability based on the GluN2 subunit composition. Overall, the pharmacology of CNS4 aligns with the need for drug candidates to treat hypoglutamatergic neuropsychiatric conditions such as loss function GRIN disorders and anti-NMDA receptor encephalitis.

Atypical Neurogenesis, Astrogliosis, and Excessive Hilar Interneuron Loss Are Associated with the Development of Post-Traumatic Epilepsy.

BACKGROUND: Traumatic brain injury (TBI) remains a significant risk factor for post-traumatic epilepsy (PTE). The pathophysiological mechanisms underlying the injury-induced epileptogenesis are under investigation. The dentate gyrus-a structure that is highly susceptible to injury-has been implicated in the evolution of seizure development. METHODS: Utilizing the murine unilateral focal control cortical impact (CCI) injury, we evaluated seizure onset using 24/7 EEG video analysis at 2-4 months post-injury. Cellular changes in the dentate gyrus and hilus of the hippocampus were quantified by unbiased stereology and Imaris image analysis to evaluate Prox1-positive cell migration, astrocyte branching, and morphology, as well as neuronal loss at four months post-injury. Isolation of region-specific astrocytes and RNA-Seq were performed to determine differential gene expression in animals that developed post-traumatic epilepsy (PTE+) vs. those animals that did not (PTE-), which may be associated with epileptogenesis. RESULTS: CCI injury resulted in 37% PTE incidence, which increased with injury severity and hippocampal damage. Histological assessments uncovered a significant loss of hilar interneurons that coincided with aberrant migration of Prox1-positive granule cells and reduced astroglial branching in PTE+ compared to PTE- mice. We uniquely identified Cst3 as a PTE+-specific gene signature in astrocytes across all brain regions, which showed increased astroglial expression in the PTE+ hilus. CONCLUSIONS: These findings suggest that epileptogenesis may emerge following TBI due to distinct aberrant cellular remodeling events and key molecular changes in the dentate gyrus of the hippocampus.

Blast-induced neurotrauma leads to neurochemical changes and neuronal degeneration in the rat hippocampus.

Blast-induced neurotrauma is a major concern because of the complex expression of neuropsychiatric disorders after exposure. Disruptions in neuronal function, proximal in time to blast exposure, may eventually contribute to the late emergence of clinical deficits. Using magic angle spinning ¹H MRS and a rodent model of blast-induced neurotrauma, we found acute (24-48 h) decreases in succinate, glutathione, glutamate, phosphorylethanolamine and γ-aminobutyric acid, no change in N-acetylaspartate and increased glycerophosphorylcholine, alterations consistent with mitochondrial distress, altered neurochemical transmission and increased membrane turnover. Increased levels of the apoptotic markers Bax and caspase-3 suggested active cell death, consistent with increased FluoroJade B staining in the hippocampus. Elevated levels of glial fibrillary acidic protein suggested ongoing inflammation without diffuse axonal injury measured by no change in ß-amyloid precursor protein. In conclusion, blast-induced neurotrauma induces a metabolic cascade associated with neuronal loss in the hippocampus in the acute period following exposure.

Chronic Anxiety- and Depression-Like Behaviors Are Associated With Glial-Driven Pathology Following Repeated Blast Induced Neurotrauma.

Long-term neuropsychiatric impairments have become a growing concern following blast-related traumatic brain injury (bTBI) in active military personnel and Veterans. Neuropsychiatric impairments such as anxiety and depression are common comorbidities that Veterans report months, even years following injury. To understand these chronic behavioral outcomes following blast injury, there is a need to study the link between anxiety, depression, and neuropathology. The hippocampus and motor cortex (MC) have been regions of interest when studying cognitive deficits following blast exposure, but clinical studies of mood disorders such as major depressive disorder (MDD) report that these two regions also play a role in the manifestation of anxiety and depression. With anxiety and depression being common long-term outcomes following bTBI, it is imperative to study how chronic pathological changes within the hippocampus and/or MC due to blast contribute to the development of these psychiatric impairments. In this study, we exposed male rats to a repeated blast overpressure (~17 psi) and evaluated the chronic behavioral and pathological effects on the hippocampus and MC. Results demonstrated that the repeated blast exposure led to depression-like behaviors 36 weeks following injury, and anxiety-like behaviors 2-, and 52-weeks following injury. These behaviors were also correlated with astrocyte pathology (glial-fibrillary acid protein, GFAP) and dendritic alterations (Microtubule-Associated Proteins, MAP2) within the hippocampus and MC regions at 52 weeks. Overall, these findings support the premise that chronic glial pathological changes within the brain contribute to neuropsychiatric impairments following blast exposure.

Web Conferencing Facilitation Within Problem-Based Learning Biomedical Engineering Courses.

Problem-based learning (PBL) has been effectively used within BME education, though there are several challenges in its implementation within courses with larger enrollments. Furthermore, the sudden transition to online learning from the COVID-19 pandemic introduced additional challenges in creating a similar PBL experience in an online environment. Online constrained PBL was implemented through asynchronous modules and synchronous web conferencing with rotating facilitators. Overall, facilitators perceived web conferencing facilitation to be similar to in-person, but noted that students were more easily "hidden" or distracted. Students did not comment on web conferencing facilitation specifically, but indicated the transition to online PBL was smooth. Course instructors identified that a fully synchronous delivery as well as modifications of Group Meeting Minutes assignments as potential modifications for future offerings. Future work will aim to address the perceptions and effectiveness of web conferencing facilitation for PBL courses within an undergraduate BME curriculum, as web conferencing could prove to be another significant breakthrough in addressing challenges of problem-based learning courses.

A 3-D Finite-Element Minipig Model to Assess Brain Biomechanical Responses to Blast Exposure.

Despite years of research, it is still unknown whether the interaction of explosion-induced blast waves with the head causes injury to the human brain. One way to fill this gap is to use animal models to establish "scaling laws" that project observed brain injuries in animals to humans. This requires laboratory experiments and high-fidelity mathematical models of the animal head to establish correlates between experimentally observed blast-induced brain injuries and model-predicted biomechanical responses. To this end, we performed laboratory experiments on Göttingen minipigs to develop and validate a three-dimensional (3-D) high-fidelity finite-element (FE) model of the minipig head. First, we performed laboratory experiments on Göttingen minipigs to obtain the geometry of the cerebral vasculature network and to characterize brain-tissue and vasculature material properties in response to high strain rates typical of blast exposures. Next, we used the detailed cerebral vasculature information and species-specific brain tissue and vasculature material properties to develop the 3-D high-fidelity FE model of the minipig head. Then, to validate the model predictions, we performed laboratory shock-tube experiments, where we exposed Göttingen minipigs to a blast overpressure of 210 kPa in a laboratory shock tube and compared brain pressures at two locations. We observed a good agreement between the model-predicted pressures and the experimental measurements, with differences in maximum pressure of less than 6%. Finally, to evaluate the influence of the cerebral vascular network on the biomechanical predictions, we performed simulations where we compared results of FE models with and without the vasculature. As expected, incorporation of the vasculature decreased brain strain but did not affect the predictions of brain pressure. However, we observed that inclusion of the cerebral vasculature in the model changed the strain distribution by as much as 100% in regions near the interface between the vasculature and the brain tissue, suggesting that the vasculature does not merely decrease the strain but causes drastic redistributions. This work will help establish correlates between observed brain injuries and predicted biomechanical responses in minipigs and facilitate the creation of scaling laws to infer potential injuries in the human brain due to exposure to blast waves.

Mechano-stimulation initiated by extracellular adhesion and cationic conductance pathways influence astrocyte activation.

Traumatic brain injury (TBI) represents a major cause of long-term disability worldwide. Primary damage to brain tissue leads to complex secondary injury mechanisms involving inflammation, oxidative stress and cellular activation/reactivity. The molecular pathways that exacerbate brain cell dysfunction after injury are not well understood and provide challenges to developing TBI therapeutics. This study aimed to delineate mechanisms of astrocyte activation induced by mechano-stimulation, specifically involving extracellular adhesion and cationic transduction. An in vitro model was employed to investigate 2D and 3D cultures of primary astrocytes, in which cells were exposed to a single high-rate overpressure known to cause upregulation of structural and proliferative markers within 72 h of exposure. An inhibitor of focal adhesion kinase (FAK) phosphorylation, TAE226, was used to demonstrate a relationship between extracellular adhesion perturbations and structural reactivity in the novel 3D model. TAE226 mitigated upregulation of glial fibrillary acidic protein in 3D cultures by 72 h post-exposure. Alternatively, incubation with gadolinium (a cationic channel blocker) during overpressure, demonstrated a role for cationic transduction in reducing the increased levels of proliferating cell nuclear antigen that occur at 24 h post-stimulation. Furthermore, early changes in mitochondrial polarization at 15 min and in endogenous ATP levels at 4-6 h occur post-overpressure and may be linked to later changes in cell phenotype. By 24 h, there was evidence of increased amine metabolism and increased nicotinamide adenine dinucleotide phosphate oxidase (NOX4) production. The overproduction of NOX4 was counteracted by gadolinium during overpressure exposure. Altogether, the results of this study indicated that both extracellular adhesion (via FAK activation) and cationic conductance (via ion channels) contribute to early patterns of astrocyte activation following overpressure stimulation. Mechano-stimulation pathways are linked to bioenergetic and metabolic disruptions in astrocytes that influence downstream oxidative stress, aberrant proliferative capacity and structural reactivity.

Glial Activation in the Thalamus Contributes to Vestibulomotor Deficits Following Blast-Induced Neurotrauma.

Vestibular impairment has become a frequent consequence following blast-related traumatic brain injury (bTBI) in military personnel and Veterans. Behavioral outcomes such as depression, fear and anxiety are also common comorbidities of bTBI. To accelerate pre-clinical research and therapy developments, there is a need to study the link between behavioral patterns and neuropathology. The transmission of neurosensory information often involves a pathway from the cerebral cortex to the thalamus, and the thalamus serves crucial integrative functions within vestibular processing. Pathways from the thalamus also connect with the amygdala, suggesting thalamic and amygdalar contributions to anxiolytic behavior. Here we used behavioral assays and immunohistochemistry to determine the sub-acute and early chronic effects of repeated blast exposure on the thalamic and amygdala nuclei. Behavioral results indicated vestibulomotor deficits at 1 and 3 weeks following repeated blast events. Anxiety-like behavior assessments depicted trending increases in the blast group. Astrogliosis and microglia activation were observed upon post-mortem pathological examination in the thalamic region, along with a limited glia response in the amygdala at 4 weeks. These findings are consistent with a diffuse glia response associated with bTBI and support the premise that dysfunction within the thalamic nuclei following repeated blast exposures contribute to vestibulomotor impairment.

A Pilot Study Assessing the Impact of rs174537 on Circulating Polyunsaturated Fatty Acids and the Inflammatory Response in Patients with Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading cause of death and disability in persons under age 45. The hallmark secondary injury profile after TBI involves dynamic interactions between inflammatory and metabolic pathways including fatty acids. Omega-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) have been shown to provide neuroprotective benefits by minimizing neuroinflammation in rodents. These effects have been less conclusive in humans, however. We postulate genetic variants influencing PUFA metabolism in humans could contribute to these disparate findings. Therefore, we sought to (1) characterize the circulating PUFA response and (2) evaluate the impact of rs174537 on inflammation after TBI. A prospective, single-center, observational pilot study was conducted to collect blood samples from Level-1 trauma patients (N = 130) on admission and 24 h post-admission. Plasma was used to quantify PUFA levels and inflammatory cytokines. Deoxyribonucleic acid was extracted and genotyped at rs174537. Associations between PUFAs and inflammatory cytokines were analyzed for all trauma cases and stratified by race (Caucasians only), TBI (TBI: N = 47; non-TBI = 83) and rs174537 genotype (GG: N = 33, GT/TT: N = 44). Patients with TBI had higher plasma DHA levels compared with non-TBI at 24 h post-injury (p = 0.013). The SNP rs174537 was associated with both PUFA levels and inflammatory cytokines (p < 0.05). Specifically, TBI patients with GG genotype exhibited the highest plasma levels of DHA (1.33%) and interleukin-8 (121.5 ± 43.3 pg/mL), which were in turn associated with poorer outcomes. These data illustrate the impact of rs174537 on the post-TBI response. Further work is needed to ascertain how this genetic variant directly influences inflammation after trauma.

Cerium Oxide Nanoparticles Improve Outcome after In Vitro and In Vivo Mild Traumatic Brain Injury.

Mild traumatic brain injury results in aberrant free radical generation, which is associated with oxidative stress, secondary injury signaling cascades, mitochondrial dysfunction, and poor functional outcome. Pharmacological targeting of free radicals with antioxidants has been examined as an approach to treatment, but has met with limited success in clinical trials. Conventional antioxidants that are currently available scavenge a single free radical before they are destroyed in the process. Here, we report for the first time that a novel regenerative cerium oxide nanoparticle antioxidant reduces neuronal death and calcium dysregulation after in vitro trauma. Further, using an in vivo model of mild lateral fluid percussion brain injury in the rat, we report that cerium oxide nanoparticles also preserve endogenous antioxidant systems, decrease macromolecular free radical damage, and improve cognitive function. Taken together, our results demonstrate that cerium oxide nanoparticles are a novel nanopharmaceutical with potential for mitigating neuropathological effects of mild traumatic brain injury and modifying the course of recovery.

Astrocyte Mechano-Activation by High-Rate Overpressure Involves Alterations in Structural and Junctional Proteins.

Primary blast neurotrauma represents a unique injury paradigm characterized by high-rate overpressure effects on brain tissue. One major hallmark of blast neurotrauma is glial reactivity, notably prolonged astrocyte activation. This cellular response has been mainly defined in primary blast neurotrauma by increased intermediate filament expression. Because the intermediate filament networks physically interface with transmembrane proteins for junctional support, it was hypothesized that cell junction regulation is altered in the reactive phenotype as well. This would have implications for downstream transcriptional regulation via signal transduction pathways like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Therefore, a custom high-rate overpressure simulator was built for in vitro testing using mechanical conditions based on intracranial pressure measurements in a rat model of blast neurotrauma. Primary rat astrocytes were exposed to isolated high-rate mechanical stimulation to study cell junction dynamics in relation to their mechano-activation. First, a time course for "classical" features of reactivity was devised by evaluation of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression. This was followed by gene and protein expression for both gap junction (connexins) and anchoring junction proteins (integrins and cadherins). Signal transduction analysis was carried out by nuclear localization of two molecules, NF-κB p65 and mitogen-activated protein kinase (MAPK) p38. Results indicated significant increases in connexin-43 expression and PCNA first at 24 h post-overpressure (p < 0.05), followed by structural reactivity (via increased GFAP, p < 0.05) corresponding to increased anchoring junction dynamics at 48 h post-overpressure (p < 0.05). Moreover, increased phosphorylation of focal adhesion kinase (FAK) was observed in addition to increased nuclear localization of both p65 and p38 (p < 0.05) during the period of structural reactivity. To evaluate the transcriptional activity of p65 in the nucleus, electrophoretic mobility shift assay was conducted for a binding site on the promoter region for intracellular adhesion molecule-1 (ICAM-1), an antagonist of tight junctions. A significant increase in the interaction of nuclear proteins with the NF-κB site on the ICAM-1 corresponded to increased gene and protein expression of ICAM-1 (p < 0.05). Altogether, these results indicate multiple targets and corresponding signaling pathways which involve cell junction dynamics in the mechano-activation of astrocytes following high-rate overpressure.