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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.

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.

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.