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IntroductionSequelae from traumatic brain injuries (TBIs) and post-traumatic stress disorder (PTSD) are major career-limiting factors for combat soldiers. Overlap between TBI and PTSD symptoms alongside other common comorbidities complicate the diagnosis and treatment. Systems-level and high-throughput approaches are key in understanding the underlying biomolecular mechanisms and differentiating these conditions.MethodsThe present study identifies dietary factors and proposes mechanisms behind psychological stress and TBI, using established preclinical animal models and a multi-omics approach. Here, we used microbiome characterizations of rats exposed to simulations of blast-induced TBI and underwater trauma (UWT)-induced psychological stress. We further studied the effect of dietary omega-6 versus omega-3 polyunsaturated fatty acid (n-6, n-3 PUFA) enrichment on the insult responses. The use of excess n-6 PUFA was chosen due to its high prevalence in the Western diet and pro-inflammatory nature. Prior to TBI or UWT, animals were maintained for 6 weeks and continued thereafter on either a standard diet or two customized chows imbalanced and diminished in omega-3 content. Corresponding shams were carried out for all groups. Fecal bacterial microbiome populations were assessed using 16S rRNA gene sequencing.ResultsPhysiologic outcome modeling identified that dietary status affected post-TBI lactate dehydrogenase (LDH) and triglyceride levels, with n-3 PUFA having a large attenuating influence. The UWT model showed similar trends, with diet significantly altering LDH, terminal corticosterone (14 days post-exposure), and a fear behavior susceptibility. Fecal microbiome alpha diversity was significantly reduced by high levels of n-3 PUFA. Likewise, beta diversity of the microbiome was significantly affected by both diet and time but not exposure to TBI or UWT. Compositionally, temporal effects on the microbiome were more likely to be observed with the diets. The most affected features fell within the Proteobacteria phyla, in which n-3 PUFA enrichment significantly reduced Alphaproteobacteria in the TBI model and increased Gammaproteobacteria in the UWT group.DiscussionAll these observations can influence the vulnerability or resilience of the warfighter to blast-induced TBI and acute psychological stress. The microbiome mechanisms facilitate and provide a knowledge-driven unbiased panel of signatures to discriminate between the two insults and is an essential tool for designing precise care management.

Blast-related traumatic brain injury (bTBI) is a major cause of neurological disorders in the U.S. military that can adversely impact some civilian populations as well and can lead to lifelong deficits and diminished quality of life. Among these types of injuries, the long-term sequelae are poorly understood because of variability in intensity and number of the blast exposure, as well as the range of subsequent symptoms that can overlap with those resulting from other traumatic events (e.g., post-traumatic stress disorder). Despite the valuable insights that rodent models have provided, there is a growing interest in using injury models using species with neuroanatomical features that more closely resemble the human brain. With this purpose, we established a gyrencephalic model of blast injury in ferrets, which underwent blast exposure applying conditions that closely mimic those associated with primary blast injuries to warfighters. In this study, we evaluated brain biochemical, microstructural, and behavioral profiles after blast exposure using in vivo longitudinal magnetic resonance imaging, histology, and behavioral assessments. In ferrets subjected to blast, the following alterations were found: 1) heightened impulsivity in decision making associated with pre-frontal cortex/amygdalar axis dysfunction; 2) transiently increased glutamate levels that are consistent with earlier findings during subacute stages post-TBI and may be involved in concomitant behavioral deficits; 3) abnormally high brain N-acetylaspartate levels that potentially reveal disrupted lipid synthesis and/or energy metabolism; and 4) dysfunction of pre-frontal cortex/auditory cortex signaling cascades that may reflect similar perturbations underlying secondary psychiatric disorders observed in warfighters after blast exposure.

Blast-induced traumatic brain injury (bTBI) frequently results in sleep and circadian rhythm disturbances. We have investigated whether dysregulation of circadian rhythm after bTBI is mediated by dysregulation of clock genes in the hypothalamus and pineal gland of rats at acute (24?h) and chronic (1 month) time points post-blast. Expression of core circadian genes (Bmal1, Clock, Per1, Per2, Cry1, and Cry2) in the hypothalamus and pineal gland were quantified using quantitative real-time polymerase chain reaction. Hypocretin (Hcrt) and hypocretin receptor (Hcrtr1 and Hcrtr2) expression in the hypothalamus were also quantified along with plasma corticosterone levels. Blast-exposed rats showed a statistically significant increase in Bmal1 and decreases in Per1, Per2, and Cry2 in the pineal gland at 24?h post-blast in rats euthanized at night. In the hypothalamus, increases in Bmal1, Cry1, and Cry2 were noted along with decreases in Per1 and Per2 gene expression at 24?h post-blast in rats euthanized at night. Except for Bmal1 in the hypothalamus, no statistically significant changes in expression of any of the clock genes were detected in the hypothalamus or pineal gland samples collected during daylight post-blast. In the hypothalamus, a decrease in Hcrt associated with increases in Hcrtr1 and Hcrtr2 were noted at 24?h post-blast in rats euthanized during daytime and nighttime. Increased plasma corticosterone was noted at 24?h post-blast in samples collected at night. No statistically significant changes in any of the core circadian genes?hypocretin, hypocretin receptors, or plasma corticosterone?were observed in the samples collected at 1 month post-blast injury. Blast exposure causes differential expression of core circadian genes in the hypothalamus and pineal gland during nighttime, along with dysregulation of hypocretin and its receptors, which might play a key role in the sleep disruptions associated with bTBI.

IntroductionMild traumatic brain injury (mTBI) caused by repetitive low-intensity blast overpressure (relBOP) in military personnel exposed to breaching and heavy weapons is often unrecognized and is understudied. Exposure to relBOP poses the risk of developing abnormal behavioral and psychological changes such as altered cognitive function, anxiety, and depression, all of which can severely compromise the quality of the life of the affected individual. Due to the structural and anatomical heterogeneity of the brain, understanding the potentially varied effects of relBOP in different regions of the brain could lend insights into the risks from exposures.MethodsIn this study, using a rodent model of relBOP and western blotting for protein expression we showed the differential expression of various neuropathological proteins like TDP-43, tight junction proteins (claudin-5, occludin, and glial fibrillary acidic protein (GFAP)) and a mechanosensitive protein (piezo-2) in different regions of the brain at different intensities and frequency of blast.ResultsOur key results include (i) significant increase in claudin-5 after 1x blast of 6.5 psi in all three regions and no definitive pattern with higher number of blasts, (ii) significant increase in piezo-2 at 1x followed by significant decrease after multiple blasts in the cortex, (iii) significant increase in piezo-2 with increasing number of blasts in frontal cortex and mixed pattern of expression in hippocampus and (iv) mixed pattern of TDP-3 and GFAP expression in all the regions of brain.DiscussionThese results suggest that there are not definitive patterns of changes in these marker proteins with increase in intensity and/or frequency of blast exposure in any particular region; the changes in expression of these proteins are different among the regions. We also found that the orientation of blast exposure (e.g. front vs. side exposure) affects the altered expression of these proteins.

Abstract Animal studies provide valuable insights on how the interaction of blast waves with the head may injure the brain. However, there is no acceptable methodology to scale the findings from animals to humans. Here, we propose an experimental/computational approach to project observed blast-induced molecular changes in the rat brain to the human brain. Using a shock tube, we exposed rats to a range of blast overpressures (BOPs) and used a high-fidelity computational model of a rat head to correlate predicted biomechanical responses with measured changes in glial fibrillary acidic protein (GFAP) in rat brain tissues. Our analyses revealed correlates between model-predicted strain rate and measured GFAP changes in three brain regions. Using these correlates and a high-fidelity computational model of a human head, we determined the equivalent BOPs in rats and in humans that induced similar strain rates across the two species. We used the equivalent BOPs to project the measured GFAP changes in the rat brain to the human. Our results suggest that, relative to the rat, the human requires an exposure to a blast wave of a higher magnitude to elicit similar brain-tissue responses. Our proposed methodology could assist in the development of safety guidelines for blast exposure.

Abstract The interaction of explosion-induced blast waves with the head (i.e., a direct mechanism) or with the torso (i.e., an indirect mechanism) presumably causes traumatic brain injury. However, the understanding of the potential role of each mechanism in causing this injury is still limited. To address this knowledge gap, we characterized the changes in the brain tissue of rats resulting from the direct and indirect mechanisms at 24 h following blast exposure. To this end, we conducted separate blast-wave exposures on rats in a shock tube at an incident overpressure of 130 kPa, while using whole-body, head-only, and torso-only configurations to delineate each mechanism. Then, we performed histopathological (silver staining) and immunohistochemical (GFAP, Iba-1, and NeuN staining) analyses to evaluate brain-tissue changes resulting from each mechanism. Compared to controls, our results showed no significant changes in torso-only-exposed rats. In contrast, we observed significant changes in whole-body-exposed (GFAP and silver staining) and head-only-exposed rats (silver staining). In addition, our analyses showed that a head-only exposure causes changes similar to those observed for a whole-body exposure, provided the exposure conditions are similar. In conclusion, our results suggest that the direct mechanism is the major contributor to blast-induced changes in brain tissues.

Previous findings have indicated that pain relieving medications such as opioids and non-steroidal anti-inflammatory drugs (NSAIDs) may be neuroprotective after traumatic brain injury in rodents, but only limited studies have been performed in a blast-induced traumatic brain injury (bTBI) model. In addition, many pre-clinical TBI studies performed in rodents did not use analgesics due to the possibility of neuroprotection or other changes in cognitive, behavioral, and pathology outcomes. To examine this in a pre-clinical setting, we examined the neurobehavioral changes in rats given a single pre-blast dose of meloxicam, buprenorphine, or no pain relieving medication and exposed to tightly-coupled repeated blasts in an advanced blast simulator and evaluated neurobehavioral functions up to 28 days post-blast. A 16.7% mortality rate was recorded in the rats treated with buprenorphine, which might be attributed to the physiologically depressive side effects of buprenorphine in combination with isoflurane anesthesia and acute brain injury. Rats given buprenorphine, but not meloxicam, took more time to recover from the isoflurane anesthesia given just before blast. We found that treatment with meloxicam protected repeated blast-exposed rats from vestibulomotor dysfunctions up to day 14, but by day 28 the protective effects had receded. Both pain relieving medications seemed to promote short-term memory deficits in blast-exposed animals, whereas vehicle-treated blast-exposed animals showed only a non-significant trend toward worsening short-term memory by day 27. Open field exploratory behavior results showed that blast exposed rats treated with meloxicam engaged in significantly more locomotor activities and possibly a lesser degree of responses thought to reflect anxiety and depressive-like behaviors than any of the other groups. Rats treated with analgesics to alleviate possible pain from the blast ate more than their counterparts that were not treated with analgesics, which supports that both analgesics were effective in alleviating some of the discomfort that these rats potentially experienced post-blast injury. These results suggest that meloxicam and, to a lesser extent buprenorphine alter a variety of neurobehavioral functions in a rat bTBI model and, because of their impact on these neurobehavioral changes, may be less than ideal analgesic agents for pre-clinical studies evaluating these neurobehavioral responses after TBI.

Blast-induced traumatic brain injury (bTBI) frequently results in sleep-wake disturbances. However, limited studies have investigated the molecular signaling mechanisms underlying these sleep disturbances, and potentially efficacious therapies are lacking. We investigated the levels of melatonin and genes involved in melatonin synthesis pathway in the pineal glands of Sprague Dawley rats exposed to single and tightly coupled repeated blasts during the night and daytime. Rats were exposed to single and tightly coupled repeated blasts using an advanced blast simulator. The plasma, cerebrospinal fluid (CSF), and pineal gland were collected at 6 h, 24 h, or 1 month postblast at two different time points: one during the day (1000 h) and one at night (2200 h). Differential expressions of genes involved in pineal melatonin synthesis were quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Plasma and CSF melatonin levels were assessed using a commercial melatonin ELISA kit. The plasma and CSF melatonin levels showed statistically significant decreases at 6 h and 24 h in the blast-exposed rats euthanized in the night (in dim light), with no significant alterations noted in rats euthanized in the morning (daylight) at all three-time points. Blast-exposed rats showed statistically significant decreases in Tph1, Aanat, Asmt, and Mtnr1b mRNA levels, along with increased Tph2 mRNA, in the pineal gland samples collected at night at 6 h and 24 h. No significant changes in the mRNA levels of these genes were noted at 1 month. These findings imply that the melatonin circadian rhythm is disrupted following blast exposure, which may be a factor in the sleep disturbances that blast victims frequently experience.

According to the US Defense and Veterans Brain Injury Center (DVBIC) and Centers for Disease Control and Prevention (CDC), mild traumatic brain injury (mTBI) is a common form of head injury. Medical imaging data provides clinical insight into tissue damage/injury and injury severity, and helps medical diagnosis. Computational modeling and simulation can predict the biomechanical characteristics of such injury, and are useful for development of protective equipment. Integration of techniques from computational biomechanics with medical data assessment modalities (e.g., magnetic resonance imaging or MRI) has not yet been used to predict injury, support early medical diagnosis, or assess effectiveness of personal protective equipment. This paper presents a methodology to map computational simulations with clinical data for interpreting blunt impact TBI utilizing two clinically different head injury case studies. MRI modalities, such as T1, T2, diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC), were used for simulation comparisons. The two clinical cases have been reconstructed using finite element analysis to predict head biomechanics based on medical reports documented by a clinician. The findings are mapped to simulation results using image-based clinical analyses of head impact injuries, and modalities that could capture simulation results have been identified. In case 1, the MRI results showed lesions in the brain with skull indentation, while case 2 had lesions in both coup and contrecoup sides with no skull deformation. Simulation data analyses show that different biomechanical measures and thresholds are needed to explain different blunt impact injury modalities; specifically, strain rate threshold corresponds well with brain injury with skull indentation, while minimum pressure threshold corresponds well with coup–contrecoup injury; and DWI has been found to be the most appropriate modality for MRI data interpretation. As the findings from these two cases are substantiated with additional clinical studies, this methodology can be broadly applied as a tool to support injury assessment in head trauma events and to improve countermeasures (e.g., diagnostics and protective equipment design) to mitigate these injuries.

Blast-induced auditory dysfunctions including tinnitus are the most prevalent disabilities in service members returning from recent combat operations. Most of the previous studies were focused on the effect of blast exposure on the peripheral auditory system and not much on the central auditory signal-processing regions in the brain. In the current study, we have exposed rats to single and tightly coupled repeated blasts and examined the degeneration of neuronal cytoskeletal elements using silver staining in the central auditory signal-processing regions in the brain at 24 h, 14 days, 1 month, 6 months, and 1 year. The brain regions evaluated include cochlear nucleus, lateral lemniscus, inferior colliculus, medial geniculate nucleus, and auditory cortex. The results obtained indicated that a significant increase in degeneration of neuronal cytoskeletal elements was observed only in the left and right cochlear nucleus. A significant increase in degeneration of neuronal cytoskeletal elements was observed in the cochlear nucleus at 24 h and persisted through 1 year, suggesting acute and chronic neuronal degeneration after blast exposure. No statistically significant differences were observed between single and repeated blasts. The localized degeneration of neuronal cytoskeletal elements in the cochlear nucleus suggests that the damage could be caused by transmission of blast shockwaves/noise through the ear canal and that use of suitable ear protection devices can protect against acute and chronic central auditory signal processing defects including tinnitus after blast exposure.

Exposure to the repeated low-level blast overpressure (BOP) periodically experienced by military personnel in operational and training environments can lead to deficits in behavior and cognition. While these low-intensity blasts do not cause overt changes acutely, repeated exposures may lead to cumulative effects in the brain that include acute inflammation, vascular disruption, and other molecular changes, which may eventually contribute to neurodegenerative processes. To identify these acute changes in the brain following repeated BOP, an advanced blast simulator was used to expose rats to 8.5 or 10 psi BOP once per day for 14 days. At 24 h after the final BOP, brain tissue was collected and analyzed for inflammatory markers, astrogliosis (GFAP), tight junction proteins (claudin-5 and occludin), and neurodegeneration-related proteins (Aβ40/42, pTau, TDP-43). After repeated exposure to 8.5 psi BOP, the change in cytokine profile was relatively modest compared to the changes observed following 10 psi BOP, which included a significant reduction in several inflammatory markers. Reduction in the tight junction protein occludin was observed in both groups when compared to controls, suggesting cerebrovascular disruption. While repeated exposure to 8.5 psi BOP led to a reduction in the Alzheimer’s disease (AD)-related proteins amyloid-β (Aβ)40 and Aβ42, these changes were not observed in the 10 psi group, which had a significant reduction in phosphorylated tau. Finally, repeated 10 psi BOP exposures led to an increase in GFAP, indicating alterations in astrocytes, and an increase in the mechanosensitive ion channel receptor protein, Piezo2, which may increase brain sensitivity to injury from pressure changes from BOP exposure. Overall, cumulative effects of repeated low-level BOP may increase the vulnerability to injury of the brain by disrupting neurovascular architecture, which may lead to downstream deleterious effects on behavior and cognition.

Exposure to blast overpressure waves is implicated as the major cause of ocular injuries and resultant visual dysfunction in veterans involved in recent combat operations. No effective therapeutic strategies have been developed so far for blast-induced ocular dysfunction. Lysophosphatidic acid (LPA) is a bioactive phospholipid generated by activated platelets, astrocytes, choroidal plexus cells, and microglia and is reported to play major roles in stimulating inflammatory processes. The levels of LPA in the cerebrospinal fluid have been reported to increase acutely in patients with traumatic brain injury (TBI) as well as in a controlled cortical impact (CCI) TBI model in mice. In the present study, we have evaluated the efficacy of a single intravenous administration of a monoclonal LPA antibody (25 mg/kg) given at 1 h post-blast for protection against injuries to the retina and associated ocular dysfunctions. Our results show that a single 19 psi blast exposure significantly increased the levels of several species of LPA in blood plasma at 1 and 4 h post-blast. The anti-LPA antibody treatment significantly decreased glial cell activation and preserved neuronal cell morphology in the retina on day 8 after blast exposure. Optokinetic measurements indicated that anti-LPA antibody treatment significantly improved visual acuity in both eyes on days 2 and 6 post-blast exposure. Anti-LPA antibody treatment significantly increased rod photoreceptor and bipolar neuronal cell signaling in both eyes on day 7 post-blast exposure. These results suggest that blast exposure triggers release of LPAs, which play a major role promoting blast-induced ocular injuries, and that a single early administration of anti-LPA antibodies provides significant protection.

Blast-induced traumatic brain injury (bTBI) is one of the major causes of persistent disabilities in Service Members, and a history of bTBI has been identified as a primary risk factor for developing age-associated neurodegenerative diseases. Clinical observations of several military blast casualties have revealed a rapid age-related loss of white matter integrity in the brain. In the present study, we have tested the effect of single and tightly coupled repeated blasts on cellular senescence in the rat brain. Isoflurane-anesthetized rats were exposed to either a single or 2 closely coupled blasts in an advanced blast simulator. Rats were euthanized and brains were collected at 24 h, 1 month and 1 year post-blast to determine senescence-associated-β-galactosidase (SA-β-gal) activity in the cells using senescence marker stain. Single and repeated blast exposures resulted in significantly increased senescence marker staining in several neuroanatomical structures, including cortex, auditory cortex, dorsal lateral thalamic nucleus, geniculate nucleus, superior colliculus, ventral thalamic nucleus and hippocampus. In general, the increases in SA-β-gal activity were more pronounced at 1 month than at 24 h or 1 year post-blast and were also greater after repeated than single blast exposures. Real-time quantitative RT-PCR analysis revealed decreased levels of mRNA for senescence marker protein-30 (SMP-30) and increased mRNA levels for p21 (cyclin dependent kinase inhibitor 1A, CDKN1A), two other related protein markers of cellular senescence. The increased senescence observed in some of these affected brain structures may be implicated in several long-term sequelae after exposure to blast, including memory disruptions and impairments in movement, auditory and ocular functions.

A history of mild traumatic brain injury (mTBI), particularly repeated mTBI (rmTBI), has been identified as a risk factor for late-onset neurodegenerative conditions. The mild and transient nature of early symptoms often impedes diagnosis in young adults who are disproportionately affected by mTBIs. A proportion of the affected population will incur long-term behavioral and cognitive consequences but the underlying pathomechanism is currently unknown. Diffusion tensor imaging (DTI) provides sensitive and quantitative assessment of TBI-induced structural changes, including white matter injury, and may be used to predict long-term outcome. We used DTI in an animal model of blast rmTBI (rmbTBI) to quantify blast-induced structural changes at 7 and 90 days post-injury, and their evolution between the two time points. Young adult male rats (~P65 at injury) were exposed to repeated mild blast overpressure, or anesthetized as shams, and their fixed brains were imaged using high-field (7 T) MRI. We found that whole brain volumes similarly increased in injured and sham rats from 7 to 90 days. However, we detected localized volume increases in blast-exposed animals 7 days post-injury, mainly ipsilateral to incident blast waves. Affected regions included gray matter of the frontal association, cingulate, and motor cortex, thalamus, substantia nigra, and raphe nuclei (median and dorsal), as well as white matter of the internal capsule and cerebral peduncle. Conversely, we measured volume reductions in these and other regions, including the hippocampus and cerebellum, at 90 days post-injury. DTI also detected both transient and persistent microstructural changes following injury, with some changes showing distinct ipsilateral versus contralateral side differences relative to blast impact. Early changes in fractional anisotropy (FA) were subtle, becoming more prominent at 90 days in the cerebral and inferior cerebellar peduncles, and cerebellar white matter. Widespread increases in radial diffusivity (RD) and axial diffusivity (primary eigenvalue or E1) at 7 days post-injury largely subsided by 90 days, although RD was more sensitive than E1 at detecting white matter changes. E1 effects in gray and white matter, which paralleled increases in apparent diffusion, were likely more indicative of dysregulated water homeostasis than pathologic structural changes. Importantly, we found evidence for a different developmental trajectory following rmbTBI, as indicated by significant injury x age interactions on volume. Our findings demonstrate that rmbTBI initiates dynamic pathobiological processes that may negatively alter the course of late-stage neurodevelopment and adversely affect long-term cognitive and behavioral outcomes. Keywords: White matter injury, Neurodegeneration, Traumatic brain injury

Blast overpressure exposure has been linked to transient, but measurably deteriorated performance and symptomatologies in law enforcement and military personnel. Overlapping sub-concussive symptomatology associated with the very low level blast overpressures (vLLB) but high sound pressure (

Recent evidence linking repeated low-level blast overpressure exposure in operational and training environments with neurocognitive decline, neuroinflammation, and neurodegenerative processes has prompted concern over the cumulative deleterious effects of repeated blast exposure on the brains of service members. Repetitive exposure to low-level primary blast may cause symptoms (subclinical) similar to those seen in mild traumatic brain injury (TBI), with progressive vascular and cellular changes, which could contribute to neurodegeneration. At the cellular level, the mechanical force associated with blast exposure can cause cellular perturbations in the brain, leading to secondary injury. To examine the cumulative effects of repetitive blast on the brain, an advanced blast simulator (ABS) was used to closely mimic “free-field” blast. Rats were exposed to 1–4 daily blasts (one blast per day, separated by 24 h) at 13, 16, or 19 psi peak incident pressures with a positive duration of 4–5 ms, either in a transverse or longitudinal orientation. Blood-brain barrier (BBB) markers (vascular endothelial growth factor (VEGF), occludin, and claudin-5), transactive response DNA binding protein (TDP-43), and the mechanosensitive channel Piezo2 were measured following blast exposure. Changes in expression of VEGF, occludin, and claudin-5 after repeated blast exposure indicate alterations in the BBB, which has been shown to be disrupted following TBI. TDP-43 is very tightly regulated in the brain and altered expression of TDP-43 is found in clinically-diagnosed TBI patients. TDP-43 levels were differentially affected by the number and magnitude of blast exposures, decreasing after 2 exposures, but increasing following a greater number of exposures at various intensities. Lastly, Piezo2 has been shown to be dysregulated following blast exposure and was here observed to increase after multiple blasts of moderate magnitude, indicating that blast may cause a change in sensitivity to mechanical stimuli in the brain and may contribute to cellular injury. These findings reveal that cumulative effects of repeated exposures to blast can lead to pathophysiological changes in the brain, demonstrating a possible link between blast injury and neurodegenerative disease, which is an important first step in understanding how to prevent these diseases in soldiers exposed to blast.

Due largely to the use of improvised explosive devices (IEDs) and other explosives in recent military conflicts, blast-related TBI has emerged as a prominent injury sustained by warfighters. In the recent wars in Iraq and Afghanistan, traumatic brain injury (TBI) has been one of the most common types of injury sustained by soldiers and military personnel; of the ~380,000 TBIs reported in service members from 2000 to 2017, 82.3% were classified as mild (mTBI). While mTBI is associated with normal structural imaging, brief or no loss of consciousness, and rapid recovery of mental state, mTBI can nevertheless lead to persistent behavioral and cognitive effects. As in other cases of mTBI, exposure to low-level blast often does not cause immediate overt neurological effects, but may similarly lead to persistent behavioral and cognitive deficits. These effects are likely to be compounded when multiple exposures to blast and/or impact are sustained, since there is increasing evidence that multiple mTBIs can lead to chronic neurodegeneration. One common form of this deleterious outcome is frontotemporal lobar degeneration (FTLD), which is a progressive neurodegenerative process marked by atrophy of the frontal and temporal lobes, leading to frontotemporal dementia, a common form of dementia affecting behavior, cognition and language. About half of all cases of FTLD are marked by TAR-DNA binding protein (TDP-43)-positive protein inclusions. TDP-43, a DNA/RNA binding protein, controls the expression of thousands of genes and is associated with several neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, and chronic traumatic encephalopathy. TDP-43 abnormalities have also been associated with traumatic brain injury in both pre-clinical and clinical studies. The role of TDP-43 in the manifestation of FTLD pathology in military TBI cases is currently unclear, and to date there has been only a limited number of pre-clinical studies addressing the effects of repeated blast-related mild TBI (rbTBI) in relation to FTLD and TDP-43. This review will summarize some of these findings and address the concerns and critical knowledge gaps associated with FTLD manifestation with military populations, as well as clinical findings on other forms of mTBI.

Repeated mild blast-induced traumatic brain injury (rmbTBI), caused by recurrent exposure to low levels of explosive blast, is a significant concern for military health systems. However, the pathobiology of rmbTBI is currently poorly understood. Animal models are important tools to identify the molecular changes of rmbTBI, but comparisons across different models can present their own challenges. In this study, we compared two well-established rodent models of mbTBI, the “KI model” and the “USU/WRAIR model.” These two models create different pulse forms, in terms of peak pressure and duration. Following single and double exposures to mild levels of blast, we used in situ hybridization (ISH) to assess changes in mRNA levels of tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH2), and galanin in the locus coeruleus (LC) and dorsal raphe nucleus (DRN). These systems and their transmitters are known to mediate responses to stress and anxiety. We found increased mRNA levels of TH, TPH2 and galanin in the LC and DRN of single-exposed rats relative to sham rats in the KI but not the USU/WRAIR model. Sham mRNA values measured in the USU/WRAIR model were substantially higher than their KI counterparts. Double exposure caused similarly significant increases in mRNA values in the KI model but not the USU/WRAIR model, except TPH2 and galanin levels in the DRN. We detected no cumulative effect of injury in either model at the used inter-injury interval (30 min), and there were no detectable neuropathological changes in any experimental group at 1 day post-injury. The apparent lack of early response to injury as compared to sham in the USU/WRAIR model is likely caused by stressors (e.g., transportation and noise), associated with the experimental execution, that were absent in the KI model. This study is the first to directly compare two established rodent models of rmbTBI, and to highlight the challenges of comparing findings from different animal models. Additional studies are needed to understand the role of stress, dissect the effects of psychological and physical injuries and to identify the window of increased cerebral vulnerability, i.e., the inter-injury interval that results in a cumulative effect following repeated blast exposure.

Psychological stress and traumatic brain injury (TBI) can both result in lasting neurobehavioral abnormalities. Post-traumatic stress disorder (PTSD) and blast induced TBI (bTBI) have become the most significant health issues in current military conflicts. Importantly, military bTBI virtually never occurs without stress. In this experiment, we assessed anxiety and spatial memory of rats at different time points after repeated exposure to stress alone or in combination with a single mild blast. At 2 months after injury or sham we analyzed the serum, prefrontal cortex (PFC), and hippocampus (HC) of all animals by proteomics and immunohistochemistry. Stressed sham animals showed an early increase in anxiety but no memory impairment at any measured time point. They had elevated levels of serum corticosterone (CORT) and hippocampal IL-6 but no other cellular or protein changes. Stressed injured animals had increased anxiety that returned to normal at 2 months and significant spatial memory impairment that lasted up to 2 months. They had elevated serum levels of CORT, CK-BB, NF-H, NSE, GFAP, and VEGF. Moreover, all of the measured protein markers were elevated in the HC and the PFC; rats had an increased number of TUNEL+ cells in the HC and elevated GFAP and Iba1 immunoreactivity in the HC and the PFC. Our findings suggest that exposure to repeated stress alone causes a transient increase in anxiety and no significant memory impairment or cellular and molecular changes. In contrast, repeated stress and blast results in lasting behavioral, molecular, and cellular abnormalities characterized by memory impairment, neuronal and glial cell loss, inflammation, and gliosis. These findings may have implications in the development of diagnostic and therapeutic measures for conditions caused by stress or a combination of stress and bTBI.

Mild blast induced traumatic brain injury (mbTBI) and its associated memory impairment and anxiety elevation currently represent one of the major military health challenges. Our earlier work using a rodent model of mbTBI indicated that the pathology underlying the observed neurobehavioral abnormalities includes neuroinflammation. The aim of our present study was to determine the effect of acute treatment with minocycline, an FDA approved non-steroidal anti-inflammatory drug, on the functional and molecular outcomes of mbTBI. Beginning four hours after a single exposure to mild blast overpressure, animals received a daily dose of minocycline (50 mg/kg) or physiological saline intraperitoneally (i.p.) for 4 days. Physiological parameters (arterial blood O2 saturation, heart and breath rates, and pulse distension) and neurobehavior (locomotor activity, anxiety, and spatial memory) were monitored at multiple time points. At the termination of the experiment (51 days post-injury), we analyzed sera and select brain regions for changes in protein markers of inflammation as well as vascular, neuronal, and glial integrity. We found that acute treatment with minocycline completely prevented memory impairment and anxiety development by ameliorating the inflammatory response to injury and substantially reducing neuronal and glial cell loss. Based on our findings, we urge testing the effect of minocycline treatment in human mbTBI.

Explosive blast results in multiple organ injury and polytrauma, the intensity of which varies with the nature of the exposure, orientation, environment and individual resilience. Blast overpressure alone may not precisely indicate the level of body or brain injury after blast exposure. Assessment of the extent of body injury after blast exposure is important, since polytrauma and systemic factors significantly contribute to blast-induced traumatic brain injury. We evaluated the activity of plasma enzymes including aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and creatine kinase (CK) at different time points after blast exposure using a mouse model of single and repeated blast exposures to assess the severity of injury. Our data show that activities of all the enzymes in the plasma were significantly increased as early as 1 h after blast exposure. The elevated enzyme activity remained up to 6 h in an overpressure dose-dependent manner and returned close to normal levels at 24 h. Head-only blast exposure with body protection showed no increase in the enzyme activities suggesting that brain injury alone does not contribute to the systemic increase. In contrast to plasma increase, AST, ALT and LDH activity in the liver and CK in the skeletal muscle showed drastic decrease at 6 h after blast exposures. Histopathology showed mild necrosis at 6 h and severe necrosis at 24 h after blast exposures in liver and no changes in the skeletal muscle suggesting that the enzyme release from the tissue to plasma is probably triggered by transient cell membrane disruption from shockwave and not due to necrosis. Overpressure dependent transient release of tissue enzymes and elevation in the plasma after blast exposure suggest that elevated enzyme activities in the blood can be potentially used as a biological dosimeter to assess the severity of blast injury.

De novo hippocampal neurogenesis contributes to functional recovery following traumatic brain injury (TBI). Enriched environment (EEN) can improve the outcome of TBI by positively affecting neurogenesis. Blast induced traumatic brain injury (bTBI) characterized by memory impairment and increased anxiety levels, is a leading cause of chronic disability among soldiers. Using a rodent model of bTBI we asked: a) whether long-term exposure to EEN after injury can ameliorate behavioral abnormalities and b) what the effects of EEN are at the molecular and cellular levels and on de novo neurogenesis. We found that housing injured animals in EEN resulted in significantly improved spatial memory while animals in normal housing (NH) showed persistent memory impairment. VEGF and Tau-protein but not IL-6 levels were normalized in the dorsal hippocampus (DHC) of EEN rats while all three markers remained elevated in NH rats. Interestingly, after peaking at 6 weeks post-injury, anxiety returned to normal levels at 2 months independent of housing conditions. Housing animals in EEN had no significant effect on VEGF and Tau-protein levels in the ventral hippocampus (VHC) and the amygdala (AD). We also found that EEN reduced IL-6 and INF levels in the VHC; these markers remained elevated following NH. We observed an increase in GFAP and DCX immunoreactivities in the VHC of NH animals at 2 months post-injury. Conversely, injured animals housed in EEN showed no increase in GFAP or DCX immunoreactivity in their VHC. In summary, long-term exposure of injured animals to EEN appears to play a positive role in the restoration of memory functions but not on anxiety, which returned to normal levels after a significant period of time. Cellular and molecular changes in response to EEN appear to be a part of neurogenesis-independent as well as dependent recovery processes triggered by bTBI.