Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET.

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

Novel mitoNEET ligand NL-1 improves therapeutic outcomes in an aged rat model of cerebral ischemia/reperfusion injury.

Cerebral ischemic stroke is a leading cause of mortality and disability worldwide. Currently, there are a lack of drugs capable of reducing neuronal cell loss due to ischemia/reperfusion-injury after stroke. Previously, we identified mitoNEET, a [2Fe-2S] redox mitochondrial protein, as a putative drug target for ischemic stroke. In this study, we tested NL-1, a novel mitoNEET ligand, in a preclinical model of ischemic stroke with reperfusion using aged female rats. Using a transient middle cerebral artery occlusion (tMCAO), we induced a 2 h ischemic injury and then evaluated the effects of NL-1 treatment on ischemic/reperfusion brain injury at 24 and 72 h. Test compounds were administered at time of reperfusion via intravenous dosing. Results of the study demonstrated that NL-1 (10 mg/kg) treatment markedly improved survival and reduced infarct volume and hemispheric swelling in the brain as compared aged rats treated with vehicle or a lower dose of NL-1 (0.25 mg/kg). Interestingly, the protective effect of NL-1 was significantly improved when encapsulated in PLGA nanoparticles, where a 40-fold lesser dose (0.25 mg/kg) of NL-1 produced an equivalent effect as the 10 mg/kg dose. Evaluation of changes in blood-brain barrier permeability and lipid peroxidation corroborated the protective actions of NL-1 (10 mg/kg) or NL-1 NP treatment demonstrated a reduced accumulation of parenchymal IgG, decreased levels of 4-hydroxynonenal (4-HNE) and a decreased TUNEL positive cells in the brains of aged female rats at 72 h after tMCAO with reperfusion. Our studies indicate that targeting mitoNEET following ischemia/reperfusion-injury is a novel drug target pathway that warrants further investigation.

The Mitochondrial mitoNEET Ligand NL-1 Is Protective in a Murine Model of Transient Cerebral Ischemic Stroke.

PURPOSE: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here on a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. METHOD: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. RESULTS: NL-1 decreased hydrogen peroxide production with an IC50 of 5.95 µM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1 h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. CONCLUSION: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents.

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of medical visits in the United States. There are currently no FDA-approved treatments available for TBI. The increased incidence of military-related, blast-induced TBI further accentuates the urgent need for effective TBI treatments. Therefore, new preclinical TBI animal models that recapitulate aspects of human blast-related TBI will greatly advance the research efforts into the neurobiological and pathophysiological processes underlying mild to moderate TBI as well as the development of novel therapeutic strategies for TBI. Here we present a reliable, reproducible model for the investigation of the molecular, cellular, and behavioral effects of mild to moderate blast-induced TBI. We describe a step-by-step protocol for closed-head, blast-induced mild TBI in rodents using a bench-top setup consisting of a gas-driven shock tube equipped with piezoelectric pressure sensors to ensure consistent test conditions. The benefits of the setup that we have established are its relative low-cost, ease of installation, ease of use and high-throughput capacity. Further advantages of this non-invasive TBI model include the scalability of the blast peak overpressure and the generation of controlled reproducible outcomes. The reproducibility and relevance of this TBI model has been evaluated in a number of downstream applications, including neurobiological, neuropathological, neurophysiological and behavioral analyses, supporting the use of this model for the characterization of processes underlying the etiology of mild to moderate TBI.

Exposure to mild blast forces induces neuropathological effects, neurophysiological deficits and biochemical changes.

Direct or indirect exposure to an explosion can induce traumatic brain injury (TBI) of various severity levels. Primary TBI from blast exposure is commonly characterized by internal injuries, such as vascular damage, neuronal injury, and contusion, without external injuries. Current animal models of blast-induced TBI (bTBI) have helped to understand the deleterious effects of moderate to severe blast forces. However, the neurological effects of mild blast forces remain poorly characterized. Here, we investigated the effects caused by mild blast forces combining neuropathological, histological, biochemical and neurophysiological analysis. For this purpose, we employed a rodent blast TBI model with blast forces below the level that causes macroscopic neuropathological changes. We found that mild blast forces induced neuroinflammation in cerebral cortex, striatum and hippocampus. Moreover, mild blast triggered microvascular damage and axonal injury. Furthermore, mild blast caused deficits in hippocampal short-term plasticity and synaptic excitability, but no impairments in long-term potentiation. Finally, mild blast exposure induced proteolytic cleavage of spectrin and the cyclin-dependent kinase 5 activator, p35 in hippocampus. Together, these findings show that mild blast forces can cause aberrant neurological changes that critically impact neuronal functions. These results are consistent with the idea that mild blast forces may induce subclinical pathophysiological changes that may contribute to neurological and psychiatric disorders.

A mouse Model of Focal Vascular Injury Induces Astrocyte Reactivity, Tau Oligomers, and Aberrant Behavior.

Neuropsychiatric symptom development has become more prevalent with 270,000 blast exposures occurring in the past 10 years in the United States. How blast injury leads to neuropsychiatric symptomology is currently unknown. Preclinical models of blast-induced traumatic brain injury have been used to demonstrate blood-brain barrier disruption, degenerative pathophysiology, and behavioral deficits. Vascular injury is a primary effect of neurotrauma that can trigger secondary injury cascades and neurodegeneration. Here we present data from a novel scaled and clinically relevant mouse blast model that was specifically developed to assess the outcome of vascular injury. We look at the biochemical effects and behavioral changes associated with blast injury in young-adult male BALB/c mice. We report that blast exposure causes focal vascular injury in the Somatosensory Barrel Field cortex, which leads to perivascular astrocyte reactivity, as well as acute aberrant behavior. Biochemical analysis revealed that mild blast exposure also invokes tauopathy, neuroinflammation, and oxidative stress. Overall, we propose our model to be used to evaluate focal blood-brain barrier disruption and to discover novel therapies for human neuropsychiatric symptoms.

Single low-dose lipopolysaccharide preconditioning: neuroprotective against axonal injury and modulates glial cells.

AIM: Over 7 million traumatic brain injuries (TBI) are reported each year in the United States. However, treatments and neuroprotection following TBI are limited because secondary injury cascades are poorly understood. Lipopolysaccharide (LPS) administration before controlled cortical impact can contribute to neuroprotection. However, the underlying mechanisms and whether LPS preconditioning confers neuroprotection against closed-head injuries remains unclear. METHODS: The authors hypothesized that preconditioning with a low dose of LPS (0.2 mg/kg) would regulate glial reactivity and protect against diffuse axonal injury induced by weight drop. LPS was administered 7 days prior to TBI. LPS administration reduced locomotion, which recovered completely by time of injury. RESULTS: LPS preconditioning significantly reduced the post-injury gliosis response near the corpus callosum, possibly by downregulating the oncostatin M receptor. These novel findings demonstrate a protective role of LPS preconditioning against diffuse axonal injury. LPS preconditioning successfully prevented neurodegeneration near the corpus callosum, as measured by fluorojade B. CONCLUSION: Further work is required to elucidate whether LPS preconditioning confers long-term protection against behavioral deficits and to elucidate the biochemical mechanisms responsible for LPS-induced neuroprotective effects.

Endoplasmic Reticulum Stress Modulation as a Target for Ameliorating Effects of Blast Induced Traumatic Brain Injury.

Blast traumatic brain injury (bTBI) has been shown to contribute to progressive neurodegenerative disease. Recent evidence suggests that endoplasmic reticulum (ER) stress is a mechanistic link between acute neurotrauma and progressive tauopathy. We propose that ER stress contributes to extensive behavioral changes associated with a chronic traumatic encephalopathy (CTE)-like phenotype. Targeting ER stress is a promising option for the treatment of neurotrauma-related neurodegeneration, which warrants investigation. Utilizing our validated and clinically relevant Sprague-Dawley blast model, we investigated a time course of mechanistic changes that occur following bTBI (50 psi) including: ER stress activation, iron-mediated toxicity, and tauopathy via Western blot and immunohistochemistry. These changes were associated with behavioral alterations measured by the Elevated Plus Maze (EPM), Forced Swim Test (FST), and Morris Water Maze (MWM). Following characterization, salubrinal, an ER stress modulator, was given at a concentration of 1 mg/kg post-blast, and its mechanism of action was determined in vitro. bTBI significantly increased markers of injury in the cortex of the left hemisphere: p-PERK and p-eIF2α at 30 min, p-T205 tau at 6 h, and iron at 24 h. bTBI animals spent more time immobile on the FST at 72 h and more time in the open arm of the EPM at 7 days. Further, bTBI caused a significant learning disruption measured with MWM at 21 days post-blast, with persistent tau changes. Salubrinal successfully reduced ER stress markers in vivo and in vitro while significantly improving performance on the EPM. bTBI causes robust biochemical changes that contribute to neurodegeneration, but these changes may be targeted with ER stress modulators.

Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.

Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10-30 min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system.

Elucidating the role of compression waves and impact duration for generating mild traumatic brain injury in rats.

BACKGROUND: In total, 3.8 million concussions occur each year in the US leading to acute functional deficits, but the underlying histopathologic changes that occur are relatively unknown. In order to improve understanding of acute injury mechanisms, appropriately designed pre-clinical models must be utilized. METHODS: The clinical relevance of compression wave injury models revolves around the ability to produce consistent histopathologic deficits. Mild traumatic brain injuries activate similar neuroinflammatory cascades, cell death markers and increases in amyloid precursor protein in both humans and rodents. Humans, however, infrequently succumb to mild traumatic brain injuries and, therefore, the intensity and magnitude of impacts must be inferred. Understanding compression wave properties and mechanical loading could help link the histopathologic deficits seen in rodents to what might be happening in human brains following concussions. RESULTS: While the concept of linking duration and intensity of impact to subsequent histopathologic deficits makes sense, numerical modelling of compression waves has not been performed in this context. In this interdisciplinary work, numerical simulations were performed to study the creation of compression waves in an experimental model. CONCLUSION: This work was conducted in conjunction with a repetitive compression wave injury paradigm in rats in order to better understand how the wave generation correlates with histopathologic deficits.

Behavioral and biochemical effects of ketamine and dextromethorphan relative to its antidepressant-like effects in Swiss Webster mice.

Ketamine has been shown to produce rapid and robust antidepressant effects in depressed individuals; however, its abuse potential and adverse psychotomimetic effects limit its widespread use. Dextromethorphan (DM) may serve as a safer alternative on the basis of pharmacodynamic similarities to ketamine. In this proof-of-concept study, behavioral and biochemical analyses were carried out to evaluate the potential involvement of brain-derived neurotrophic factor (BDNF) in the antidepressant-like effects of DM in mice, with comparisons to ketamine and imipramine. Male Swiss, Webster mice were injected with DM, ketamine, or imipramine and their behaviors were evaluated in the forced-swim test and the open-field test. Western blots were used to measure BDNF and its precursor, pro-BDNF, protein expression in the hippocampus and the frontal cortex of these mice. Our results show that both DM and imipramine reduced immobility time in the forced-swim test without affecting locomotor activity, whereas ketamine reduced immobility time and increased locomotor activity. Ketamine also rapidly (within 40 min) increased pro-BDNF expression in an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-dependent manner in the hippocampus, whereas DM and imipramine did not alter pro-BDNF or BDNF levels in either the hippocampus or the frontal cortex within this timeframe. These data show that DM shares some features with both ketamine and imipramine. Additional studies examining DM may aid in the development of more rapid, safe, and efficacious antidepressant treatments.

Salubrinal reduces oxidative stress, neuroinflammation and impulsive-like behavior in a rodent model of traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of trauma related morbidity in the developed world. TBI has been shown to trigger secondary injury cascades including endoplasmic reticulum (ER) stress, oxidative stress, and neuroinflammation. The link between secondary injury cascades and behavioral outcome following TBI is poorly understood warranting further investigation. Using our validated rodent blast TBI model, we examined the interaction of secondary injury cascades following single injury and how these interactions may contribute to impulsive-like behavior after a clinically relevant repetitive TBI paradigm. We targeted these secondary pathways acutely following single injury with the cellular stress modulator, salubrinal (SAL). We examined the neuroprotective effects of SAL administration on significantly reducing ER stress: janus-N-terminal kinase (JNK) phosphorylation and C/EBP homology protein (CHOP), oxidative stress: superoxide and carbonyls, and neuroinflammation: nuclear factor kappa beta (NFκB) activity, inducible nitric oxide synthase (iNOS) protein expression, and pro-inflammatory cytokines at 24h post-TBI. We then used the more clinically relevant repeat injury paradigm and observed elevated NFκB and iNOS activity. These injury cascades were associated with impulsive-like behavior measured on the elevated plus maze. SAL administration attenuated secondary iNOS activity at 72h following repetitive TBI, and most importantly prevented impulsive-like behavior. Overall, these results suggest a link between secondary injury cascades and impulsive-like behavior that can be modulated by SAL administration.

Aneurysmal Subarachnoid Hemorrhage and Neuroinflammation: A Comprehensive Review.

Aneurysmal subarachnoid hemorrhage (SAH) can lead to devastating outcomes including vasospasm, cognitive decline, and even death. Currently, treatment options are limited for this potentially life threatening injury. Recent evidence suggests that neuroinflammation plays a critical role in injury expansion and brain damage. Red blood cell breakdown products can lead to the release of inflammatory cytokines that trigger vasospasm and tissue injury. Preclinical models have been used successfully to improve understanding about neuroinflammation following aneurysmal rupture. The focus of this review is to provide an overview of how neuroinflammation relates to secondary outcomes such as vasospasm after aneurysmal rupture and to critically discuss pharmaceutical agents that warrant further investigation for the treatment of subarachnoid hemorrhage. We provide a concise overview of the neuroinflammatory pathways that are upregulated following aneurysmal rupture and how these pathways correlate to long-term outcomes. Treatment of aneurysm rupture is limited and few pharmaceutical drugs are available. Through improved understanding of biochemical mechanisms of injury, novel treatment solutions are being developed that target neuroinflammation. In the final sections of this review, we highlight a few of these novel treatment approaches and emphasize why targeting neuroinflammation following aneurysmal subarachnoid hemorrhage may improve patient care. We encourage ongoing research into the pathophysiology of aneurysmal subarachnoid hemorrhage, especially in regards to neuroinflammatory cascades and the translation to randomized clinical trials.

Endoplasmic reticulum stress implicated in chronic traumatic encephalopathy.

OBJECTIVE: Chronic traumatic encephalopathy is a progressive neurodegenerative disease characterized by neurofibrillary tau tangles following repetitive neurotrauma. The underlying mechanism linking traumatic brain injury to chronic traumatic encephalopathy has not been elucidated. The authors investigate the role of endoplasmic reticulum stress as a link between acute neurotrauma and chronic neurodegeneration. METHODS: The authors used pharmacological, biochemical, and behavioral tools to assess the role of endoplasmic reticulum stress in linking acute repetitive traumatic brain injury to the development of chronic neurodegeneration. Data from the authors' clinically relevant and validated rodent blast model were compared with those obtained from postmortem human chronic traumatic encephalopathy specimens from a National Football League player and World Wrestling Entertainment wrestler. RESULTS: The results demonstrated strong correlation of endoplasmic reticulum stress activation with subsequent tau hyperphosphorylation. Various endoplasmic reticulum stress markers were increased in human chronic traumatic encephalopathy specimens, and the endoplasmic reticulum stress response was associated with an increase in the tau kinase, glycogen synthase kinase-3ß. Docosahexaenoic acid, an endoplasmic reticulum stress inhibitor, improved cognitive performance in the rat model 3 weeks after repetitive blast exposure. The data showed that docosahexaenoic acid administration substantially reduced tau hyperphosphorylation (t = 4.111, p < 0.05), improved cognition (t = 6.532, p < 0.001), and inhibited C/EBP homology protein activation (t = 5.631, p < 0.01). Additionally the data showed, for the first time, that endoplasmic reticulum stress is involved in the pathophysiology of chronic traumatic encephalopathy. CONCLUSIONS: Docosahexaenoic acid therefore warrants further investigation as a potential therapeutic agent for the prevention of chronic traumatic encephalopathy.

The role for infarct volume as a surrogate measure of functional outcome following ischemic stroke.

The failed translation of proposed therapeutic agents for ischemic stroke from preclinical to clinical studies has led to increased scrutiny of preclinical studies, namely the model and outcome measures utilized. Preclinical studies routinely use infarct volume as an experimental endpoint or measure in studies employing young-adult, healthy male animals despite the fact that clinically, ischemic stroke is a disease of the elderly and improvements in functional outcome from pre- to post-intervention remains the most widely utilized assessment. The validity of infarct volume as a surrogate measure for functional outcome remains unclear in clinical studies as well as preclinical studies, particularly those utilizing a more clinically relevant aged thromboembolic model. In this work, we will address the relationship between acute and chronic functional outcome and infarct volume using a variety of functional assessments ranging from more simplistic, subjective measurements such as the modified Neurologic Severity Score (mNSS), to more complex, objective measurements such as grip strength and inclined plane.

Modeling Chronic Traumatic Encephalopathy: The Way Forward for Future Discovery.

Despite the extensive media coverage associated with the diagnosis of chronic traumatic encephalopathy (CTE), our fundamental understanding of the disease pathophysiology remains in its infancy. Only recently have scientific laboratories and personnel begun to explore CTE pathophysiology through the use of preclinical models of neurotrauma. Some studies have shown the ability to recapitulate some aspects of CTE in rodent models, through the use of various neuropathological, biochemical, and/or behavioral assays. Many questions related to CTE development, however, remain unanswered. These include the role of impact severity, the time interval between impacts, the age at which impacts occur, and the total number of impacts sustained. Other important variables such as the location of impacts, character of impacts, and effect of environment/lifestyle and genetics also warrant further study. In this work, we attempt to address some of these questions by exploring work previously completed using single- and repetitive-injury paradigms. Despite some models producing some deficits similar to CTE symptoms, it is clear that further studies are required to understand the development of neuropathological and neurobehavioral features consistent with CTE-like features in rodents. Specifically, acute and chronic studies are needed that characterize the development of tau-based pathology.

Traumatic brain injury and epilepsy: Underlying mechanisms leading to seizure.

Post-traumatic epilepsy continues to be a major concern for those experiencing traumatic brain injury. Post-traumatic epilepsy accounts for 10-20% of epilepsy cases in the general population. While seizure prophylaxis can prevent early onset seizures, no available treatments effectively prevent late-onset seizure. Little is known about the progression of neural injury over time and how this injury progression contributes to late onset seizure development. In this comprehensive review, we discuss the epidemiology and risk factors for post-traumatic epilepsy and the current pharmacologic agents used for treatment. We highlight limitations with the current approach and offer suggestions for remedying the knowledge gap. Critical to this pursuit is the design of pre-clinical models to investigate important mechanistic factors responsible for post-traumatic epilepsy development. We discuss what the current models have provided in terms of understanding acute injury and what is needed to advance understanding regarding late onset seizure. New model designs will be used to investigate novel pathways linking acute injury to chronic changes within the brain. Important components of this transition are likely mediated by toll-like receptors, neuroinflammation, and tauopathy. In the final section, we highlight current experimental therapies that may prove promising in preventing and treating post-traumatic epilepsy. By increasing understanding about post-traumatic epilepsy and injury expansion over time, it will be possible to design better treatments with specific molecular targets to prevent late-onset seizure occurrence following traumatic brain injury.

The Quest to Model Chronic Traumatic Encephalopathy: A Multiple Model and Injury Paradigm Experience.

Chronic neurodegeneration following a history of neurotrauma is frequently associated with neuropsychiatric and cognitive symptoms. In order to enhance understanding about the underlying pathophysiology linking neurotrauma to neurodegeneration, a multi-model preclinical approach must be established to account for the different injury paradigms and pathophysiologic mechanisms. We investigated the development of tau pathology and behavioral changes using a multi-model and multi-institutional approach, comparing the preclinical results to tauopathy patterns seen in post-mortem human samples from athletes diagnosed with chronic traumatic encephalopathy (CTE). We utilized a scaled and validated blast-induced traumatic brain injury model in rats and a modified pneumatic closed-head impact model in mice. Tau hyperphosphorylation was evaluated by western blot and immunohistochemistry. Elevated-plus maze and Morris water maze were employed to measure impulsive-like behavior and cognitive deficits respectively. Animals exposed to single blast (~50 PSI reflected peak overpressure) exhibited elevated AT8 immunoreactivity in the contralateral hippocampus at 1 month compared to controls (q = 3.96, p < 0.05). Animals exposed to repeat blast (six blasts over 2 weeks) had increased AT8 (q = 8.12, p < 0.001) and AT270 (q = 4.03, p < 0.05) in the contralateral hippocampus at 1 month post-injury compared to controls. In the modified controlled closed-head impact mouse model, no significant difference in AT8 was seen at 7 days, however a significant elevation was detected at 1 month following injury in the ipsilateral hippocampus compared to control (q = 4.34, p < 0.05). Elevated-plus maze data revealed that rats exposed to single blast (q = 3.53, p < 0.05) and repeat blast (q = 4.21, p < 0.05) spent more time in seconds exploring the open arms compared to controls. Morris water maze testing revealed a significant difference between groups in acquisition times on days 22-27. During the probe trial, single blast (t = 6.44, p < 0.05) and repeat blast (t = 8.00, p < 0.05) rats spent less time in seconds exploring where the platform had been located compared to controls. This study provides a multi-model example of replicating tau and behavioral changes in animals and provides a foundation for future investigation of CTE disease pathophysiology and therapeutic development.

Amelioration of nicotinamide adenine dinucleotide phosphate-oxidase mediated stress reduces cell death after blast-induced traumatic brain injury.

A total of 1.7 million traumatic brain injuries (TBIs) occur each year in the United States, but available pharmacologic options for the treatment of acute neurotrauma are limited. Oxidative stress is an important secondary mechanism of injury that can lead to neuronal apoptosis and subsequent behavioral changes. Using a clinically relevant and validated rodent blast model, we investigated how nicotinamide adenine dinucleotide phosphate oxidase (Nox) expression and associated oxidative stress contribute to cellular apoptosis after single and repeat blast injuries. Nox4 forms a complex with p22phox after injury, forming free radicals at neuronal membranes. Using immunohistochemical-staining methods, we found a visible increase in Nox4 after single blast injury in Sprague Dawley rats. Interestingly, Nox4 was also increased in postmortem human samples obtained from athletes diagnosed with chronic traumatic encephalopathy. Nox4 activity correlated with an increase in superoxide formation. Alpha-lipoic acid, an oxidative stress inhibitor, prevented the development of superoxide acutely and increased antiapoptotic markers B-cell lymphoma 2 (t = 3.079, P < 0.05) and heme oxygenase 1 (t = 8.169, P < 0.001) after single blast. Subacutely, alpha-lipoic acid treatment reduced proapoptotic markers Bax (t = 4.483, P < 0.05), caspase 12 (t = 6.157, P < 0.001), and caspase 3 (t = 4.573, P < 0.01) after repetitive blast, and reduced tau hyperphosphorylation indicated by decreased CP-13 and paired helical filament staining. Alpha-lipoic acid ameliorated impulsive-like behavior 7 days after repetitive blast injury (t = 3.573, P < 0.05) compared with blast exposed animals without treatment. TBI can cause debilitating symptoms and psychiatric disorders. Oxidative stress is an ideal target for neuropharmacologic intervention, and alpha-lipoic acid warrants further investigation as a therapeutic for prevention of chronic neurodegeneration.