Association of Blast Exposure in Military Breaching with Intestinal Permeability Blood Biomarkers Associated with Leaky Gut.

Injuries and subclinical effects from exposure to blasts are of significant concern in military operational settings, including tactical training, and are associated with self-reported concussion-like symptomology and physiological changes such as increased intestinal permeability (IP), which was investigated in this study. Time-series gene expression and IP biomarker data were generated from "breachers" exposed to controlled, low-level explosive blast during training. Samples from 30 male participants at pre-, post-, and follow-up blast exposure the next day were assayed via RNA-seq and ELISA. A battery of symptom data was also collected at each of these time points that acutely showed elevated symptom reporting related to headache, concentration, dizziness, and taking longer to think, dissipating ~16 h following blast exposure. Evidence for bacterial translocation into circulation following blast exposure was detected by significant stepwise increase in microbial diversity (measured via alpha-diversity p = 0.049). Alterations in levels of IP protein biomarkers (i.e., Zonulin, LBP, Claudin-3, I-FABP) assessed in a subset of these participants (n = 23) further evidenced blast exposure associates with IP. The observed symptom profile was consistent with mild traumatic brain injury and was further associated with changes in bacterial translocation and intestinal permeability, suggesting that IP may be linked to a decrease in cognitive functioning. These preliminary findings show for the first time within real-world military operational settings that exposures to blast can contribute to IP.

Neurotrauma Biomarker Levels and Adverse Symptoms Among Military and Law Enforcement Personnel Exposed to Occupational Overpressure Without Diagnosed Traumatic Brain Injury.

Importance: There is a scientific and operational need to define objective measures of exposure to low-level overpressure (LLOP) and concussion-like symptoms among persons with specialized occupations. Objective: To evaluate serum levels of neurotrauma biomarkers and their association with concussion-like symptoms reported by LLOP-exposed military and law enforcement personnel who are outwardly healthy and cleared to perform duties. Design, Setting, and Participants: This retrospective cohort study, conducted from January 23, 2017, to October 21, 2019, used serum samples and survey data collected from healthy, male, active-duty military and law enforcement personnel assigned to operational training at 4 US Department of Defense and civilian law enforcement training sites. Personnel aged 18 years or older with prior LLOP exposure but no diagnosed traumatic brain injury or with acute blast exposure during sampling participated in the study. Serum samples from 30 control individuals were obtained from a commercial vendor. Main Outcomes and Measures: Serum levels of glial fibrillary acidic protein, ubiquitin carboxyl hydrolase (UCH)-L1, neurofilament light chain, tau, amyloid ß (Aß)-40, and Aß-42 from a random sample (30 participants) of the LLOP-exposed cohort were compared with those of 30 age-matched controls. Associations between biomarker levels and self-reported symptoms or operational demographics in the remainder of the study cohort (76 participants) were assessed using generalized linear modeling or Spearman correlations with age as a covariate. Results: Among the 30 randomly sampled participants (mean [SD] age, 32 [7.75] years), serum levels of UCH-L1 (mean difference, 4.92; 95% CI, 0.71-9.14), tau (mean difference, 0.16; 95% CI, -0.06 to 0.39), Aß-40 (mean difference, 138.44; 95% CI, 116.32-160.56), and Aß-42 (mean difference, 4.97; 95% CI, 4.10-5.83) were elevated compared with those in controls. Among the remaining cohort of 76 participants (mean [SD] age, 34 [7.43] years), ear ringing was reported by 44 (58%) and memory or sleep problems were reported by 24 (32%) and 20 (26%), respectively. A total of 26 participants (34%) reported prior concussion. Amyloid ß-42 levels were associated with ear ringing (F1,72 = 7.40; P = .008) and memory problems (F1,72 = 9.20; P = .003). Conclusions and Relevance: The findings suggest that long-term LLOP exposure acquired during occupational training may be associated with serum levels of neurotrauma biomarkers. Assessment of biomarkers and concussion-like symptoms among personnel considered healthy at the time of sampling may be useful for military occupational medicine risk management.

DNA Methylation Patterns of Chronic Explosive Breaching in U.S. Military Warfighters.

Background: Injuries from exposure to explosions rose dramatically during the Iraq and Afghanistan wars, which motivated investigation of blast-related neurotrauma. We have undertaken human studies involving military "breachers" -exposed to controlled, low-level blast during a 3-days explosive breaching course. Methods: We screened epigenetic profiles in peripheral blood samples from 59 subjects (in two separate U.S. Military training sessions) using Infinium MethylationEPIC BeadChips. Participants had varying numbers of exposures to blast over their military careers (empirically defined as high ≥ 40, and conversely, low < 39 breaching exposures). Daily self-reported physiological symptoms were recorded. Tinnitus, memory problems, headaches, and sleep disturbances are most frequently reported. Results: We identified 14 significantly differentially methylated regions (DMRs) within genes associated with cumulative blast exposure in participants with high relative to low cumulative blast exposure. Notably, NTSR1 and SPON1 were significantly differentially methylated in high relative to low blast exposed groups, suggesting that sleep dysregulation may be altered in response to chronic cumulative blast exposure. In comparing lifetime blast exposure at baseline (prior to exposure in current training), and top associated symptoms, we identified significant DMRs associated with tinnitus, sleep difficulties, and headache. Notably, we identified KCNN3, SOD3, MUC4, GALR1, and WDR45B, which are implicated in auditory function, as differentially methylated associated with self-reported tinnitus. These findings suggest neurobiological mechanisms behind auditory injuries in our military warfighters and are particularly relevant given tinnitus is not only a primary disability among veterans, but has also been demonstrated in active duty medical records for populations exposed to blast in training. Additionally, we found that differentially methylated regions associated with the genes CCDC68 and COMT track with sleep difficulties, and those within FMOD and TNXB track with pain and headache. Conclusion: Sleep disturbances, as well as tinnitus and chronic pain, are widely reported in U.S. military service members and veterans. As we have previously demonstrated, DNA methylation encapsulates lifetime exposure to blast. The current data support previous findings and recapitulate transcriptional regulatory alterations in genes involved in sleep, auditory function, and pain. These data uncovered novel epigenetic and transcriptional regulatory mechanism underlying the etiological basis of these symptoms.

Overpressure Exposure From .50-Caliber Rifle Training Is Associated With Increased Amyloid Beta Peptides in Serum.

Background: Overpressure (OP) is an increase in air pressure above normal atmospheric levels. Military personnel are repeatedly exposed to low levels of OP caused by various weapon systems. Repeated OP may increase risk of neurological disease or psychological disorder diagnoses. A means to detect early phase effects that may be relevant to brain trauma remain elusive. Therefore, development of quantitative and objective OP-mediated effects during acute timeframes would vastly augment point-of-care or field-based decisions. This pilot study evaluated the amplitude of traumatic brain injury (TBI)-associated biomarkers in serum as a consequence of repeated OP exposure from .50-caliber rifle use over training multiple days. Objective: To determine the acute temporal profile of TBI-associated serum biomarkers and their relationship with neurocognitive decrements or self-reported symptoms among participants exposed to low-level, repeated OP from weapons used in a training environment. Methods: Study participants were enrolled in .50-caliber sniper rifle training and exposed to mild OP (peak pressure 3.8-4.5 psi, impulse 19.27-42.22 psi-ms per day) for three consecutive days (D1-D3). Defense automated neurobehavioral assessment (DANA) neurocognitive testing, symptom reporting, and blood collection were conducted 2-3 h before (pre-) and again 0.45-3 h after (post-) OP exposure. The TBI-associated serum biomarkers, glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), neurofilament light (Nf-L), tau, and amyloid beta peptides (Aß-40 and Aß-42) were measured using digital ELISAs. Results: Serum GFAP decreased on D1 and D3 but not D2 after OP exposure. Nf-L was suppressed on D3 alone. Aß-40 was elevated on D2 alone while Aß-42 was elevated each day after OP exposure. Suppression of GFAP and elevation of Aß-42 correlated to OP-mediated impulse levels measured on D3. Conclusions: Acute measurement of Aß-peptides may have utility as biomarkers of subconcussive OP caused by rifle fire. Fluctuation of GFAP, Nf-L, and particularly Aß peptide levels may have utility as acute, systemic responders of subconcussive OP exposure caused by rifle fire even in the absence of extreme operational deficits or clinically defined concussion.

Brain-related proteins as serum biomarkers of acute, subconcussive blast overpressure exposure: A cohort study of military personnel.

Repeated exposure to blast overpressure remains a major cause of adverse health for military personnel who, as a consequence, are at a higher risk for neurodegenerative disease and suicide. Acute, early tracking of blast related effects holds the promise of rapid health assessment prior to onset of chronic problems. Current techniques used to determine blast-related effects rely upon reporting of symptomology similar to that of concussion and neurocognitive assessment relevant to operational decrement. Here, we describe the results of a cross sectional study with pared observations. The concentration of multiple TBI-related proteins was tested in serum collected within one hour of blast exposure as a quantitative and minimally invasive strategy to augment assessment of blast-exposure effects that are associated with concussion-like symptomology and reaction time decrements. We determined that median simple reaction time (SRT) was slowed in accordance with serum Nf-L, tau, Aß-40, and Aß-42 elevation after overpressure exposure. In contrast, median levels of serum GFAP decreased. Individual, inter-subject analysis revealed positive correlations between changes in Nf-L and GFAP, and in Aß-40 compared to Aß-42. The change in Nf-L was negatively associated with tau, Aß-40, and Aß-42. Participants reported experiencing headaches, dizziness and taking longer to think. Dizziness was associated with reaction time decrements, GFAP or NfL suppression, as well as Aß peptide elevation. UCH-L1 elevation had a weak association with mTBI/concussion history. Multiplexed serum biomarker quantitation, coupled with reaction time assessment and symptomology determined before and after blast exposure, may serve as a platform for tracking adverse effects in the absence of a head wound or diagnosed concussion. We propose further evaluation of serum biomarkers, which are often associated with TBI, in the context of acute operational blast exposures.