Repeated Blast Exposures Research Articles

MicroRNA Profile Changes in Brain, Cerebrospinal Fluid, and Blood Following Low-Level Repeated Blast Exposure in a Rat Model.

It is well documented that service members are exposed to repeated low-level blast overpressure during training with heavy weapons such as artillery, mortars and explosive breaching. Often, acute symptoms associated with these exposures are transient but cumulative effect of low-level repeated blast exposures (RBEs) can include persistent deficits in cognitive and behavioral health. Thus far, reliable diagnostic biomarkers which can guide countermeasure strategies have not been identified. In this study, rats were exposed to multiple field-relevant blast waves with 8.5 and 10 psi peak positive overpressures, applying one exposure per day for 14 consecutive days. micro-RNAs that can potentially be used as biomarkers for RBEs were assessed in blood, brain, and cerebrospinal fluid (CSF). RBE caused a differential pattern of changes in various miRNAs in blood, brain and CSF in an overpressure-dependent manner. Our key outcomes were decrease of mir-6215 and let-7 family miRNAs and increase of mir-6321 and mir-222-5p in brain, blood, and CSF. Expression pattern of these miRNAs is in concurrence with various neurological conditions such as upregulation of mir-6321 in focal ischemic injury and downregulation of mir-6215 in nerve injury model. Contrarily, Let-7 family miRNAs have neuroprotective role and their downregulation suggests progression of blast induced traumatic brain injury (bTBI) with RBE at 14× -8.5 psi. Repeated blast caused alterations in miRNAs that are likely involved in vascular integrity, inflammation, and cell death. These results indicate that miRNAs are differentially dysregulated in response to blast injuries and may represent better prognostic and diagnostic biomarkers than traditional molecules to identify blast-specific brain injury.

Five-Year Serial Brain MRI Analysis of Military Members Exposed to Chronic Sub-Concussive Overpressures.

The Canadian Special Operations Forces Command conducts explosives operations and training which exposes members to explosive charges at close proximity. This 5-year longitudinal trial was conducted in follow-up to our initial trial which examined military breachers with MRI and EEG pre and post blast exposure. To examine brain MRI findings in military personnel exposed to multiple repeated blast exposures. Five-year longitudinal prospective trial. Ninety-two males aged 23-42 with an average of 9.4 years of blast exposure. 3 T brain MRI/T1-weighted 3D with reconstruction in three planes, T2-weighted, T2-weighted fluid attenuated inversion recovery (FLAIR) 3D with reconstruction in three planes, T2-weighted gradient spin echo (GRE), saturation weighted images, DWI and ADC maps, diffusion tensor imaging. All MRI scans were interpreted by the two neuroradiologists and one neuroradiology Fellow in a blinded fashion using a customized neuroradiology reporting form. Matching parametric statistics represented the number of participants whose brain parameters improved or deteriorated over time. Odds ratio (OR) and 95% confidence intervals (CI) were computed using log regression modeling to determine volume loss, white matter lesions, hemosiderosis, gliosis, cystic changes and enlarged Virchow Robin (VR) spaces. A Kappa (κ) statistic with a 95% CI was calculated to determine rater variability between readers. A significant deterioration was observed in volume loss (OR = 1.083, 95% CI 0.678-1.731, permutation test), white matter changes (OR: 0.754, 95% CI 0.442-1.284, permutation test), and enlargement of VR spaces (OR: 0.775, 95% CI 0.513-1.171). Interrater reliability was low: κ = 0.283, 0.156, and 0.557 for volume loss, white matter changes, and enlargement of VR spaces, respectively. There were significant changes in brain volume, white matter lesions, and enlargement of VR spaces. 2 TECHNICAL EFFICACY: Stage 2.

Neurological Effects of Repeated Blast Exposure in Special Operations Personnel.

Exposure to blast overpressure has been a pervasive feature of combat-related injuries. Studies exploring the neurological correlates of repeated low-level blast exposure in career "breachers" demonstrated higher levels of tumor necrosis factor alpha (TNFα) and interleukin (IL)-6 and decreases in IL-10 within brain-derived extracellular vesicles (BDEVs). The current pilot study was initiated in partnership with the U.S. Special Operations Command (USSOCOM) to explore whether neuroinflammation is seen within special operators with prior blast exposure. Data were analyzed from 18 service members (SMs), inclusive of 9 blast-exposed special operators with an extensive career history of repeated blast exposures and 9 controls matched by age and duration of service. Neuroinflammation was assessed utilizing positron emission tomography (PET) imaging with [18F]DPA-714. Serum was acquired to assess inflammatory biomarkers within whole serum and BDEVs. The Blast Exposure Threshold Survey (BETS) was acquired to determine blast history. Both self-report and neurocognitive measures were acquired to assess cognition. Similarity-driven Multi-view Linear Reconstruction (SiMLR) was used for joint analysis of acquired data. Analysis of BDEVs indicated significant positive associations with a generalized blast exposure value (GBEV) derived from the BETS. SiMLR-based analyses of neuroimaging demonstrated exposure-related relationships between GBEV, PET-neuroinflammation, cortical thickness, and volume loss within special operators. Affected brain networks included regions associated with memory retrieval and executive functioning, as well as visual and heteromodal processing. Post hoc assessments of cognitive measures failed to demonstrate significant associations with GBEV. This emerging evidence suggests neuroinflammation may be a key feature of the brain response to blast exposure over a career in operational personnel. The common thread of neuroinflammation observed in blast-exposed populations requires further study.

Mitigation ofHearing Damage With Liraglutide Treatment inChinchillas After Repeated Blast Exposures at Mild-TBI.

Although hearing protection devices (HPDs) have been widely used during training and combat, over one million veterans experience service-connected hearing loss. Hearing damage has been reported to be associated with blast-induced mild traumatic brain injury (mTBI) and there is a lack of understanding and treatment. Liraglutide is a glucagon-like peptide-1 receptor agonist and a potential treatment for TBI-induced memory deficits. This study aims to investigate the function of the liraglutide to prevent damage and facilitate hearing restoration in chinchillas exposed to multiple high-intensity, mTBI-level blasts. Chinchillas were divided into three treatment groups: blast control, pre-blast drug treatment, and post-blast drug treatment. On day 1, the chinchilla ears were protected by HPDs and exposed to three blasts with peak pressure levels of 15-25 psi. The auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and middle latency response (MLR) were recorded pre- and post-blast on day 1 and on days 4, 7, 14, and 28. Substantial acute damage was observed and progressively recovered in chinchillas after the blast exposures. The pre-blast treatment group exhibited the lowest elevation of the ABR threshold and reduction of the wave I amplitude on day 1 after blasts. The liraglutide treatment insignificantly facilitated the recovery of the DPOAE levels and ABR thresholds on days 14 and 28. The pre-blast treatment chinchillas showed reduced MLR amplitudes on days 4 and 7. This study indicated that the pre-blast liraglutide administration provided damage protection against blasts in addition to the HPDs. Current evidence suggests that the effect of liraglutide is more prominent in the early phase of the experiment.

Neuroinflammation Profiling of Brain Cytokines Following Repeated Blast Exposure.

Due to use of explosive devices and heavy weapons systems in modern conflicts, the effect of BW on the brain and body is of increasing concern. These exposures have been commonly linked with neurodegenerative diseases and psychiatric disorders in veteran populations. A likely neurobiological link between exposure to blasts and the development of neurobehavioral disorders, such as depression and PTSD, could be neuroinflammation triggered by the blast wave. In this study, we exposed rats to single or repeated BW (up to four exposures-one per day) at varied intensities (13, 16, and 19 psi) to mimic the types of blast exposures that service members may experience in training and combat. We then measured a panel of neuroinflammatory markers in the brain tissue with a multiplex cytokine/chemokine assay to understand the pathophysiological process(es) associated with single and repeated blast exposures. We found that single and repeated blast exposures promoted neuroinflammatory changes in the brain that are similar to those characterized in several neurological disorders; these effects were most robust after 13 and 16 psi single and repeated blast exposures, and they exceeded those recorded after 19 psi repeated blast exposures. Tumor necrosis factor-alpha and IL-10 were changed by 13 and 16 psi single and repeated blast exposures. In conclusion, based upon the growing prominence of negative psychological health outcomes in veterans and soldiers with a history of blast exposures, identifying the molecular etiology of these disorders, such as blast-induced neuroinflammation, is necessary for rationally establishing countermeasures and treatment regimens.

Double Blast Wave Primary Effect on Synaptic, Glymphatic, Myelin, Neuronal and Neurovascular Markers

Explosive blasts are associated with neurological consequences as a result of blast waves impact on the brain. Yet, the neuropathologic and molecular consequences due to blast waves vs. blunt-TBI are not fully understood. An explosive-driven blast-generating system was used to reproduce blast wave exposure and examine pathological and molecular changes generated by primary wave effects of blast exposure. We assessed if pre- and post-synaptic (synaptophysin, PSD-95, spinophilin, GAP-43), neuronal (NF-L), glymphatic (LYVE1, podoplanin), myelin (MBP), neurovascular (AQP4, S100β, PDGF) and genomic (DNA polymerase-β, RNA polymerase II) markers could be altered across different brain regions of double blast vs. sham animals. Twelve male rats exposed to two consecutive blasts were compared to 12 control/sham rats. Western blot, ELISA, and immunofluorescence analyses were performed across the frontal cortex, hippocampus, cerebellum, and brainstem. The results showed altered levels of AQP4, S100β, DNA-polymerase-β, PDGF, synaptophysin and PSD-95 in double blast vs. sham animals in most of the examined regions. These data indicate that blast-generated changes are preferentially associated with neurovascular, glymphatic, and DNA repair markers, especially in the brainstem. Moreover, these changes were not accompanied by behavioral changes and corroborate the hypothesis for which an asymptomatic altered status is caused by repeated blast exposures.

Mitigation of Hearing Damage After Repeated Blast Exposures in Animal Model of Chinchilla.

High-intensity sound or blast-induced hearing impairment is a common injury for Service members. Epidemiology studies revealed that the blast-induced hearing loss is associated with the traumatic brain injury (TBI), but the mechanisms of the formation and prevention of auditory injuries require further investigation. Liraglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, has been reported as a potential treatment strategy for TBI-caused memory deficits; however, there is no study on therapeutics of GLP-1R for blast-induced hearing damage. This paper reports our current study on progressive hearing damage after repeated exposures to low-level blasts in the animal model of chinchilla and the mitigation of hearing damage using liraglutide. Chinchillas were divided into three groups (N = 7 each): blast control, pre-blast treatment, and post-blast treatment. All animals were exposed to six consecutive blasts at the level of 3-5 psi (21-35kPa) on Day 1. The auditory brainstem response (ABR) was measured on Day 1 (pre- and post-blast) and Days 4, 7, and 14 after blast exposure. Upon the completion of the experiment on Day 14, the brain tissues of animals were harvested for immunofluorescence studies. Significant damage was revealed in blast-exposed chinchillas by increased ABR thresholds, decreased ABR wave I amplitudes, and cell apoptosis in the inferior colliculus in the blast control chinchillas. Treatment with liraglutide appeared to reduce the severity of blast-induced hearing injuries as observed from the drug-treated chinchillas comparing to the blast controls. This study bridges the gap between TBI and hearing impairment and suggests a possible intervention for blast-induced hearing loss for Service members.

Transfer Function for Relative Blast Overpressure Through Porcine and Human Skulls In Situ.

The overarching objective of the Office of Naval Research sponsored Blast Load Assessment Sense and Test (BLAST) program was to quantify neurofunctional risk from repeated blast exposure. However, human studies have limitations in data collection that can only be addressed by animal models. To utilize a large animal model in this work, researchers developed an approach for scaling blast exposure data from animal to human-equivalent loading. For this study, energy interacting with the brain tissue was selected as a translation metric because of the hypothesized association between observed neurological changes and energy transmitted through the skull. This article describes the methodology used to derive an energy-based transfer function capable of serving as a global correspondence rule for primary blast injury exposure, allowing researchers to derive human-appropriate thresholds from animal data. To generate data for the development of the transfer functions, three disarticulated cadaveric Yucatan minipigs and three postmortem human surrogate heads were exposed to blast overpressure using a large bore, compressed-gas shock tube. Pressure gauges in the free field, on the skull surface, and pressure probes within the brain cavity filled with Sylgard silicone gel recorded the pressure propagation through the skull of each specimen. The frequency components of the freefield and brain cavity measurements from the pig and human surrogates were interrogated in the frequency domain. Doing so quantifies the differences in the amount of energy, in each frequency band, transmitted through both the porcine and the human skull, and the transfer function was calculated to quantify those differences. Nonlinear energy transmission was observed for both the porcine and human skulls, indicating that linear scaling would not be appropriate for developing porcine to human transfer functions. This study demonstrated similar responses between species with little to no attenuation at frequencies below 30 Hz. The phase of the pressure transmission to the brain is also similar for both species up to approximately 10 kHz. There were two notable differences between the porcine and human surrogates. First, in the 40-100 Hz range, human subjects have approximately 8 dB more pressure transmitted through the skull relative to porcine subjects. Second, in the 1-10 kHz range, human subjects have up to 10 dB more pressure transmitted into the brain (10 dB more attenuation) relative to the porcine subjects. The fundamental goal of this study was to develop pig-to-human transfer functions to allow researchers to interpret data collected from large animal studies and aid in deriving risk functions for repeated blast exposures. Similarities in porcine and human brain physiology make the minipig experimental model an excellent candidate for blast research. However, differences in the skull geometry have historically made the interpretation of animal data difficult for the purposes of characterizing potential neurological risk in humans. Human equivalent loading conditions are critical so that the thresholds are not over- or underpredicted due to differences in porcine skull geometry. This research provides a solution to this challenge, providing a robust methodology for interpreting animal data for blast research.

Phosphorylated Neurofilament Heavy Chain in the Cerebrospinal Fluid Is a Suitable Biomarker of Acute and Chronic Blast-Induced Traumatic Brain Injury.

Blast-induced traumatic brain injury (bTBI) has been documented as a significant concern for both military and civilian populations in response to the increased use of improvised explosive devices. Identifying biomarkers that could aid in the proper diagnosis and assessment of both acute and chronic bTBI is in urgent need since little progress has been made towards this goal. Addressing this knowledge gap is especially important in military veterans who are receiving assessment and care often years after their last blast exposure. Neuron-specific phosphorylated neurofilament heavy chain protein (pNFH) has been successfully evaluated as a reliable biomarker of different neurological disorders, as well as brain trauma resulting from contact sports and acute stages of brain injury of different origin. In the present study, we have evaluated the utility of pNFH levels measured in the cerebrospinal fluid (CSF) as an acute and chronic biomarker of brain injury resulting from single and tightly coupled repeated blast exposures using experimental rats. The pNFH levels increased at 24 h, returned to normal levels at 1 month, but increased again at 6 months and 1 year post-blast exposures. No significant changes were observed between single and repeated blast-exposed groups. To determine whether the observed increase of pNFH in CSF corresponded with its levels in the brain, we performed fluorescence immunohistochemistry in different brain regions at the four time-points evaluated. We observed decreased pNFH levels in those brain areas at 24 h, 6 months, and 1 year. The results suggest that blast exposure causes axonal degeneration at acute and chronic stages resulting in the release of pNFH, the abundant neuronal cytoskeletal protein. Moreover, the changes in pNFH levels in the CSF negatively correlated with the neurobehavioral functions in the rats, reinforcing suggestions that CSF levels of pNFH can be a suitable biomarker of bTBI.

Prevention of Blast-induced Auditory Injury Using 3D Printed Helmet and Hearing Protection Device – A Preliminary Study on Biomechanical Modeling and Animal

Repeated blast exposures result in structural damage to the peripheral auditory system (PAS) and the central auditory system (CAS). However, it is difficult to differentiate injuries between two distinct pathways: the mechanical damage in the PAS caused by blast pressure waves transmitted through the ear and the damage in the CAS caused by blast wave impacts on the head or traumatic brain injury. This article reports a preliminary study using a 3D printed chinchilla "helmet" as a head protection device associated with the hearing protection devices (e.g., earplugs) to isolate the CAS damage from the PAS injuries under repeated blast exposures. A finite element (FE) model of the chinchilla helmet was created based on micro-computed tomography images of a chinchilla skull and inputted into ANSYS for FE analysis on the helmet's protection against blast over pressure. The helmet was then 3D printed and used for animal experiments. Chinchillas were divided into four cases (ears open, with earplug only, with both earplug and helmet, and with helmet only) and exposed to three blasts at blast over pressure of 15 to 20 psi. Hearing function tests (e.g., auditory brainstem response) were performed before and after blast on Day 1 and Days 4 and 7 after blasts. The FE model simulation showed a significant reduction in intracranial stress with the helmet, and the animal results indicated that both earplug and helmet reduced the severity of blast-induced auditory injuries by approximately 20 dB but with different mechanisms. The biomechanical modeling and animal experiments demonstrated that this four-case study in chinchillas with helmet and hearing protection devices provides a novel methodology to investigate the blast-induced damage in the PAS and CAS.

Optimizing Helmet Pad Placement Using Computational Predicted Injury Pattern to Reduce Mild Traumatic Brain Injury

This effort, motivated and guided by prior simulated injury results of the unprotected head, is to assess and compare helmet pad configurations on the head for the effective mitigation of blast pressure transmission in the brain in multiple blast exposure environments. A finite element model of blast loading on the head with six different helmet pad configurations was used to generate brain model biomechanical responses. The blast pressure attenuation performance of each pad configuration was evaluated by using the calculated pressure exposure fraction in the brain model. Monte Carlo simulations generated repetitive blast cumulative exposures. Significant improvement of a 6-Pad Modified configuration compared to a 6-Pad Baseline configuration indicates the importance of providing protection against the side blast. Both 12-Pad configurations are very effective in mitigating pressure in the brain. Repetitive blast exposure statistics for operational exposures shows that pad configurations with a larger number of pads and smaller gaps between pads perform better than the configurations with a smaller number of pads and larger gaps between pads. Optimizing helmet pad size and/or placement could provide an improved protection by minimizing the side blast orientation effects and mitigating high-pressure fields in the brain from repeated blast exposures.

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.

Blast Exposure Leads to Accelerated Cellular Senescence in the Rat Brain.

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.

Repeated blast model of mild traumatic brain injury alters oxycodone self-administration and drug seeking.

Each year, traumatic brain injuries (TBI) affect millions worldwide. Mild TBIs (mTBI) are the most prevalent and can lead to a range of neurobehavioral problems, including substance abuse. A single blast exposure, inducing mTBI alters the medial prefrontal cortex, an area implicated in addiction, for at least 30days post injury in rats. Repeated blast exposures result in greater physiological and behavioral dysfunction than single exposure; however, the impact of repeated mTBI on addiction is unknown. In this study, the effect of mTBI on various stages of oxycodone use was examined. Male Sprague Dawley rats were exposed to a blast model of mTBI once per day for 3days. Rats were trained to self-administer oxycodone during short (2h) and long (6h) access sessions. Following abstinence, rats underwent extinction and two cued reinstatement sessions. Sham and rbTBI rats had similar oxycodone intake, extinction responding and cued reinstatement of drug seeking. A second group of rats were trained to self-administer oxycodone with varying reinforcement schedules (fixed ratio (FR)-2 and FR-4). Under an FR-2 schedule, rbTBI-exposed rats earned fewer reinforcers than sham-exposed rats. During 10 extinction sessions, the rbTBI-exposed rats exhibited significantly more seeking for oxycodone than the sham-injured rats. There was a positive correlation between total oxycodone intake and day 1 extinction drug seeking in sham, but not in rbTBI-exposed rats. Together, this suggests that rbTBI-exposed rats are more sensitive to oxycodone-associated cues during reinstatement than sham-exposed rats and that rbTBI may disrupt the relationship between oxycodone intake and seeking.

Defective methionine metabolism in the brain after repeated blast exposures might contribute to increased oxidative stress

Blast-induced traumatic brain injury (bTBI) is one of the major disabilities in Service Members returning from recent military operations. The neurobiological underpinnings of bTBI, which are associated with acute and chronic neuropathological and neurobehavioral deficits, are uncertain. Increased oxidative stress in the brain is reported to play a significant role promoting neuronal damage associated with both brain injury and neurodegenerative disorders. In this study, brains of rats exposed to repeated blasts in a shock tube underwent untargeted profiling of primary metabolism by automatic linear exchange/cold injection GC-TOF mass spectrometry and revealed acute and sub-acute disruptions in the metabolism of the essential amino acid methionine and associated antioxidants. Methionine sulfoxide, the oxidized metabolite of methionine, showed a sustained increase in the brain after blast exposure which was associated with a significant decrease in cysteine, the amino acid derived from methionine. Glutathione, the antioxidant synthesized from cysteine, also concomitantly decreased as did the antioxidant ascorbic acid. Reductions in ascorbic acid were accompanied by increased levels of its oxidized metabolite, dehydroascorbic acid and other metabolites such as threonic acid, isothreonic acid, glycolic acid and oxalic acid. Fluorometric analysis of the brains showed acute and sub-acute increase in total reactive oxygen species. In view of the fundamental importance of glutathione in the brain as an antioxidant, including its role in the reduction of dehydroascorbic acid to ascorbic acid, the disruptions in methionine metabolism elicited by blast exposure might prominently contribute to neuronal injury by promoting increased and sustained oxidative stress.

A critical literature review on primary blast thorax injury and their outcomes.

Since World War II, researchers have been interested in exploring the injury mechanisms involved in primary blast on the thorax by using animal model surrogates. These studies were mostly concerned with the finding of the lung injury threshold, the relationship between the physical components of the air blast wave, and the biological response. Studies have also been conducted to investigate the effect of repeated blast exposures on the injury outcome threshold. This has led to several injury criteria, such as the Bowen curves based on pressure history's characteristics or the Axelsson Chest Wall Velocity Predictor that used measurement from the mammals' chest wall. This article aims at doing a critical literature review of this specific topic.

The Vestibular Effects of Repeated Low-Level Blasts.

The objective of this study was to use a prospective cohort of United States Marine Corps (USMC) instructors to identify any acute or long-term vestibular dysfunction following repeated blast exposures during explosive breaching training. They were assessed in clinic and on location during training at the USMC Methods of Entry School, Quantico, VA. Subjects received comprehensive baseline vestibular assessments and these were repeated in order to identify longitudinal changes. They also received shorter assessments immediately following blast exposure in order to identify acute findings. The main outcome measures were the Neurobehavioral Symptom Inventory, vestibular Visual Analog Scale (VAS) of subjective vestibular function, videonystagmography (VNG), vestibular evoked myogenic potentials (VEMP), rotary chair (including the unilateral centrifugation test), computerized dynamic posturography, and computerized dynamic visual acuity. A total of 11 breachers and 4 engineers were followed for up to 17 months. No acute effects or longitudinal deteriorations were identified, but there were some interesting baseline group differences. Upbeat positional nystagmus was common, and correlated (p<0.005) with a history of mild traumatic brain injury (mTBI). Several instructors had abnormally short low-frequency phase leads on rotary chair testing. This study evaluated breaching instructors over a longer test period than any other study, and the results suggest that this population appears to be safe from a vestibular standpoint at the current exposure levels. Upbeat positional nystagmus correlated with a history of mTBI in this population, and this has not been described elsewhere. The data trends also suggest that this nystagmus could be an acute blast effect. However, the reasons for the abnormally short phase leads seen in rotary chair testing are unclear at this time. Further investigation seems warranted.

Repeated Blast Exposures Cause Brain DNA Fragmentation in Mice

The pathophysiology of blast-induced traumatic brain injury (TBI) and subsequent behavioral deficits are not well understood. Unraveling the mechanisms of injury is critical to derive effective countermeasures against this form of neurotrauma. Preservation of the integrity of cellular DNA is crucial for the function and survival of cells. We evaluated the effect of repeated blast exposures on the integrity of brain DNA and tested the utility of cell-free DNA (CFD) in plasma as a biomarker for the diagnosis and prognosis of blast-induced polytrauma. The results revealed time-dependent breakdown in cellular DNA in different brain regions, with the maximum damage at 24 h post-blast exposures. CFD levels in plasma showed a significant transient increase, which was largely independent of the timing and severity of brain DNA damage; maximum levels were recorded at 2 h after repeated blast exposure and returned to baseline at 24 h. A positive correlation was observed between the righting reflex time and CFD level in plasma at 2 h after blast exposure. Brain DNA damage subsequent to repeated blast was associated with decreased mitochondrial membrane potential, increased release of cytochrome C, and up-regulation of caspase-3, all of which are indicative of cellular apoptosis. Shock-wave-induced DNA damage and initiation of mitochondrial-driven cellular apoptosis in the brain after repeated blast exposures indicate that therapeutic strategies directed toward inhibition of DNA damage or instigation of DNA repair may be effective countermeasures.

Cytoskeletal protein α–II spectrin degradation in the brain of repeated blast exposed mice

Repeated blast exposures commonly induce traumatic brain injury (TBI) characterized by diffuse axonal injury (DAI). We hypothesized that degradation of cytoskeletal proteins in the brain can lead to DAI, and evaluated α-II spectrin degradation in the pathophysiology of blast-induced TBI using the tightly-coupled three repetitive blast exposure mice model with a 1–30min window in between exposures. Degradation of α-II spectrin and the expression profiles of caspase-3 and calpain-2, the major enzymes involved in the degradation were analyzed in the frontal cortex and cerebellum using Western blotting with specific antibodies. DAI at different brain regions was evaluated by neuropathology with silver staining. Repeated blast exposures resulted in significant increases in the α-II spectrin degradation products in the frontal cortex and cerebellum compared to sham controls. Expression of active caspase-3, which degrades α–II spectrin, showed significant increase in the frontal cortex after blast exposure at all the time points studied, while cerebellum showed an acute increase which was normalized over time. The expression of another α–II spectrin degrading enzyme, calpain-2, showed a rapid increase in the frontal cortex after blast exposure and it was significantly higher in the cerebellum at later time points. Neuropathological analysis showed significant levels of DAI at the frontal cortex and cerebellum at multiple time points after repeated blast injury. In summary, repeated blast exposure results in specific degradation of α-II spectrin in the brain along with differential expression of caspase-3/calpain-2 suggesting cytoskeletal breakdown as a possible contributor of DAI after repeated blast exposure.

Acute Mitochondrial Dysfunction after Blast Exposure: Potential Role of Mitochondrial Glutamate Oxaloacetate Transaminase

Use of improvised explosive devices has significantly increased the incidence of traumatic brain injury (TBI) and associated neuropsychiatric deficits in the recent wars in Iraq and Afghanistan. Acute deleterious effects of single and repeated blast exposure can lead to long-term neurobiological effects and neuropsychiatric deficits. Using in vitro and in vivo shock tube models of blast-induced TBI, we studied changes in mitochondrial energy metabolism after blast exposure. Single and repeated blast exposures in vitro resulted in significant decreases in neuronal adenosine triphosphate (ATP) levels at 6 h post-blast that returned towards normal levels by 24 h. Similar changes in ATP also were observed in the cerebral cortices of mice subjected to single and repeated blast exposures. In neurons, mitochondrial glutamate oxaloacetate transaminase (GOT2) plays a critical role in metabolism and energy production. Proteomic analysis of brain cortices showed a significant decrease in GOT2 levels 6 h after repeated blast exposures, which was further confirmed by Western blotting. Western blot analysis of GOT2 and pyruvate dehydrogenase in the cortex showed direct correlation only between GOT2 and ATP levels. Activity of GOT2 in the isolated cortical mitochondria also showed significant decrease at 6 h supporting the results of proteomic and Western blot analyses. Knowing the significant role of GOT2 in the neuronal mitochondrial energy metabolism, it is quite likely that the down regulation of GOT2 after blast exposure is playing a significant role in mitochondrial dysfunction after blast exposure.