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Abstract A reduction in the thickness and volume of the brain entorhinal cortex (EC), together with changes in blood arachidonic acid (AA) and docosahexaenoic acid (DHA), are associated with Alzheimer’s disease (AD) among apolipoprotein E ε4 carriers. Magnetic Resonance Imaging (n = 631) and plasma lipidomics (n = 181) were performed using the LIMBIC/CENC cohort to examine the influence of ε4 on AA- and DHA-lipids and EC thickness and volume in relation to mild traumatic brain injury (mTBI). Results showed that left EC thickness was higher among ε4 carriers with mTBI. Repeated mTBI (r-mTBI) was associated with reduced right EC thickness after controlling for ε4, age and sex. Age, plus mTBI chronicity were linked to increased EC White Matter Volume (WMV). After controlling for age and sex, the advancing age of ε4 carriers with blast mTBI was associated with reduced right EC Grey Matter Volume (GMV) and thickness. Among ε4 carriers, plasma tau and Aβ40 were associated with mTBI and blast mTBI, respectively. Chronic mTBI, ε4 and AA to DHA ratios in phosphatidylcholine, ethanolamides, and phosphatidylethanolamine were associated with decreased left EC GMV and WMV. Further research is needed to explore these as biomarkers for detecting AD pathology following mTBI.

IntroductionThe purpose of this study was to examine whether blood-based biomarkers associate with neurobehavioral functioning at three time points following traumatic brain injury (TBI).Materials and methodsParticipants were 328 United States service members and veterans (SMVs) prospectively enrolled in the Defense and Veterans Brain Injury Center-Traumatic Brain Injury Center of Excellence (DVBIC-TBICoE) 15-Year Longitudinal TBI Study, recruited into three groups: uncomplicated mild TBI (MTBI, n = 155); complicated mild, moderate, severe TBI combined (STBI, n = 97); non-injured controls (NIC, n = 76). Participants were further divided into three cohorts based on time since injury (≤12 months, 3–5 years, and 8–10 years). Participants completed the Minnesota Multiphasic Personality Inventory-2-Restructured Format (MMPI-2-RF) and underwent blood draw to measure serum concentrations of glial fibrillary acidic protein (GFAP), neurofilament light (NfL), and tau. A total of 11 MMPI-2-RF scales were examined (e.g., depression, anxiety, anger, somatic, cognitive symptoms). Stepwise hierarchical regression models were conducted within each group.ResultsSignificant associations were found between biomarkers and MMPI-2-RF scales (all p 0.10). GFAP was inversely related to (a) neurological complaints in the MTBI group at ≤12 months, (b) demoralization, anger proneness in the STBI group at ≤12 months, and (c) head pain complaints in the STBI group at 8–10 years. NfL was (a) related to low positive emotions in the NIC group; and inversely related to (b) demoralization, somatic complaints, neurological complaints, cognitive complaints in the MTBI group at ≤12 months, (c) demoralization in the STBI group at ≤12 months, and (d) demoralization, head pain complaints, stress/worry in the STBI group at 3–5 years. In the STBI group, there were meaningful findings (R2Δ > 0.10) for tau, NFL, and GFAP that did not reach statistical significance.DiscussionResults indicate worse scores on some MMPI-2-RF scales (e.g., depression, stress/worry, neurological and head pain complaints) were associated with lower concentrations of serum GFAP, NfL, and tau in the sub-acute and chronic phase of the recovery trajectory up to 5 years post-injury, with a reverse trend observed at 8–10 years. Longitudinal studies are needed to help elucidate any patterns of association between blood-based biomarkers and neurobehavioral outcome over the recovery trajectory following TBI.

Background: Current protein biomarkers are only moderately predictive at identifying individuals with mild traumatic brain injury or concussion. Therefore, more accurate diagnostic markers are needed for sport-related concussion. Methods: This was a multicenter, prospective, case-control study of athletes who provided blood samples and were diagnosed with a concussion or were a matched non-concussed control within the National Collegiate Athletic Association–Department of Defense Concussion Assessment, Research, and Education Consortium conducted between 2015 and 2019. The blood was collected within 48 h of injury to identify protein abnormalities at the acute and subacute timepoints. Athletes with concussion were divided into 6 h post-injury (0–6 h post-injury) and after 6 h post-injury (7–48 h post-injury) groups. We applied a highly multiplexed proteomic technique that used a DNA aptamers assay to target 1305 proteins in plasma samples from athletes with and without sport-related concussion. Results: A total of 140 athletes with concussion (79.3% males; aged 18.71 ± 1.10 years, mean ± SD) and 21 non-concussed athletes (76.2% males; 19.14 ± 1.10 years) were included in this study. We identified 338 plasma proteins that significantly differed in abundance (319 upregulated and 19 downregulated) in concussed athletes compared to non-concussed athletes. The top 20 most differentially abundant proteins discriminated concussed athletes from non-concussed athletes with an area under the curve (AUC) of 0.954 (95% confidence interval: 0.922‒0.986). Specifically, after 6 h of injury, the individual AUC of plasma erythrocyte membrane protein band 4.1 (EPB41) and alpha-synuclein (SNCA) were 0.956 and 0.875, respectively. The combination of EPB41 and SNCA provided the best AUC (1.000), which suggests this combination of candidate plasma biomarkers is the best for diagnosing concussion in athletes after 6 h of injury. Conclusion: Our data suggest that proteomic profiling may provide novel diagnostic protein markers and that a combination of EPB41 and SNCA is the most predictive biomarker of concussion after 6 h of injury.

ObjectiveThe aim of this study was to investigate phosphorylated tau (p-tau181) protein in plasma in a cohort of mild traumatic brain injury (mTBI) patients and a cohort of concussed athletes.MethodsThis pilot study comprised two independent cohorts. The first cohort—part of a Traumatic Head Injury Neuroimaging Classification (THINC) study—with a mean age of 46 years was composed of uninjured controls (UIC, n = 30) and mTBI patients (n = 288) recruited from the emergency department with clinical computed tomography (CT) and research magnetic resonance imaging (MRI) findings. The second cohort—with a mean age of 19 years—comprised 133 collegiate athletes with (n = 112) and without (n = 21) concussions. The participants enrolled in the second cohort were a part of a multicenter, prospective, case-control study conducted by the NCAA-DoD Concussion Assessment, Research and Education (CARE) Consortium at six CARE Advanced Research Core (ARC) sites between 2015 and 2019. Blood was collected within 48 h of injury for both cohorts. Plasma concentration (pg/ml) of p-tau181 was measured using the Single Molecule Array ultrasensitive assay.ResultsConcentrations of plasma p-tau181 in both cohorts were significantly elevated compared to controls within 48 h of injury, with the highest concentrations of p-tau181 within 18 h of injury, with an area under the curve (AUC) of 0.690–0.748, respectively, in distinguishing mTBI patients and concussed athletes from controls. Among the mTBI patients, the levels of plasma p-tau181 were significantly higher in patients with positive neuroimaging (either CT+/MRI+, n = 74 or CT−/MRI+, n = 89) compared to mTBI patients with negative neuroimaging (CT−/MRI−, n = 111) findings and UIC (P-values < 0.05).ConclusionThese findings indicate that plasma p-tau181 concentrations likely relate to brain injury, with the highest levels in patients with neuroimaging evidence of injury. Future research is needed to replicate and validate this protein assay's performance as a possible early diagnostic biomarker for mTBI/concussions.

Abstract Traumatic brain injury (TBI) can be associated with long-term neurobehavioral symptoms. Here, we examined levels of neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in extracellular vesicles isolated from blood, and their relationship with TBI severity and neurobehavioral symptom reporting. Participants were 218 service members and veterans who sustained uncomplicated mild TBIs (mTBI, n = 107); complicated mild, moderate, or severe TBIs (smcTBI, n = 66); or Injured controls (IC, orthopedic injury without TBI, n = 45). Within one year after injury, but not after, NfL was higher in the smcTBI group than mTBI (p = 0.001, d = 0.66) and IC (p = 0.001, d = 0.35) groups, which remained after controlling for demographics and injury characteristics. NfL also discriminated the smcTBI group from IC (AUC:77.5%, p

Abstract Background Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) represent leading causes of trauma-induced mortality, especially when co-occurring in pre-hospital settings where standard therapies are not readily available. The primary objective of this study was to determine if 17α-ethinyl estradiol-3-sulfate (EE-3-SO4) increases survival, promotes more rapid cardiovascular recovery, or confers neuroprotection relative to Placebo following TBI + HS. Methods All methods were approved by required regulatory agencies prior to study initiation. In this fully randomized, blinded preclinical study, eighty (50% females) sexually mature (190.64 ± 21.04 days old; 28.18 ± 2.72 kg) Yucatan swine were used. Sixty-eight animals received a closed-head, accelerative TBI followed by removal of approximately 40% of circulating blood volume. Animals were then intravenously administered EE-3-SO4 formulated in the vehicle at 5.0 mg/mL (dosed at 0.2 mL/kg) or Placebo (0.45% sodium chloride solution) via a continuous pump (0.2 mL/kg over 5 min). Twelve swine were included as uninjured Shams to further characterize model pathology and replicate previous findings. All animals were monitored for up to 5 h in the absence of any other life-saving measures (e.g., mechanical ventilation, fluid resuscitation). Results A comparison of Placebo-treated relative to Sham animals indicated evidence of acidosis, decreased arterial pressure, increased heart rate, diffuse axonal injury and blood–brain barrier breach. The percentage of animals surviving to 295 min post-injury was significantly higher for the EE-3-SO4 (28/31; 90.3%) relative to Placebo (24/33; 72.7%) cohort. EE-3-SO4 also restored pulse pressure more rapidly post-drug administration, but did not confer any benefits in terms of shock index. Primary blood-based measurements of neuroinflammation and blood brain breach were also null, whereas secondary measurements of diffuse axonal injury suggested a more rapid return to baseline for the EE-3-SO4 group. Survival status was associated with biological sex (female > male), as well as evidence of increased acidosis and neurotrauma independent of EE-3-SO4 or Placebo administration. Conclusions EE-3-SO4 is efficacious in promoting survival and more rapidly restoring cardiovascular homeostasis following polytraumatic injuries in pre-hospital environments (rural and military) in the absence of standard therapies. Poly-therapeutic approaches targeting additional mechanisms (increased hemostasis, oxygen-carrying capacity, etc.) should be considered in future studies.

ObjectiveTo investigate the plasma proteomic profiling in identifying biomarkers related to return to sport (RTS) following a sport-related concussion (SRC).MethodsThis multicenter, prospective, case-control study was part of a larger cohort study conducted by the NCAA-DoD Concussion Assessment, Research, and Education (CARE) Consortium, athletes (n = 140) with blood collected within 48 h of injury and reported day to asymptomatic were included in this study, divided into two groups: (1) recovery

Study ObjectivesTraumatic brain injury (TBI) is associated with chronic sleep disturbances and cognitive impairment. Our prior preclinical work demonstrated dietary supplementation with branched chain amino acids (BCAA: leucine, isoleucine, and valine), precursors to de novo glutamate production, restored impairments in glutamate, orexin/hypocretin neurons, sleep, and memory in rodent models of TBI. This pilot study assessed the feasibility and preliminary efficacy of dietary supplementation with BCAA on sleep and cognition in Veterans with TBI.MethodsThirty-two Veterans with TBI were prospectively enrolled in a randomized, double-blinded, placebo-controlled trial comparing BCAA (30 g, b.i.d. for 21-days) with one of two placebo arms (microcrystalline cellulose or rice protein, both 30 g, b.i.d. for 21-days). Pre- and post-intervention outcomes included sleep measures (questionnaires, daily sleep/study diaries, and wrist actigraphy), neuropsychological testing, and blood-based biomarkers related to BCAA consumption.ResultsSix subjects withdrew from the study (2/group), leaving 26 remaining subjects who were highly adherent to the protocol (BCAA, 93%; rice protein, 96%; microcrystalline, 95%; actigraphy 87%). BCAA were well-tolerated with few side effects and no adverse events. BCAA significantly improved subjective insomnia symptoms and objective sleep latency and wake after sleep onset on actigraphy.ConclusionDietary supplementation with BCAA is a mechanism-based, promising intervention that shows feasibility, acceptability, and preliminary efficacy to treat insomnia and objective sleep disruption in Veterans with TBI. A larger scale randomized clinical trial is warranted to further evaluate the efficacy, dosing, and duration of BCAA effects on sleep and other related outcome measures in individuals with TBI.Clinical Trial Registration[http://clinicaltrials.gov/], identifier [NCT03990909].

ObjectiveThe purpose of this pilot study was to determine if military service members with histories of hundreds to thousands of low-level blast exposures (i. e., experienced breachers) had different levels of serum and neuronal-derived extracellular vesicle (EV) concentrations of interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNFα), compared to matched controls, and if these biomarkers related to neurobehavioral symptoms.MethodsParticipants were experienced breachers (n = 20) and matched controls without blast exposures (n = 14). Neuronal-derived EVs were isolated from serum and identified with mouse anti-human CD171. Serum and neuronal-derived EVs were analyzed for IL-6, IL-10, and TNFα using an ultra-sensitive assay.ResultsSerum TNFα concentrations were decreased in breachers when compared to control concentrations (p < 0.01). There were no differences in serum concentrations of IL-6, IL-10, or the IL-6/IL-10 ratio between breachers and controls (p's > 0.01). In neuronal-derived EVs, TNFα and IL-6 levels were increased in breachers compared to controls (p's < 0.01), and IL-10 levels were decreased in the breacher group compared to controls (p < 0.01). In breachers the IL-6/IL-10 ratio in neuronal-derived EVs was higher compared to controls, which correlated with higher total Rivermead Post-concussion Questionnaire (RPQ) scores (p's < 0.05).ConclusionsThese findings suggest that exposure of personnel to high numbers of low-level blast over a career may result in enduring central inflammation that is associated with chronic neurological symptoms. The data also suggest that peripheral markers of inflammation are not necessarily adequate surrogates for central neuroinflammation.

Blast exposures that occur during training are common in military personnel; however, the biomarkers that relate to these subtle injuries is not well understood. Therefore, the purpose of this study is to identify the acute biomarkers related to blast injury in a cohort of military personnel exposure to blast-related training. Thirty-four military personnel who participated in the training program were included in this study. Blood samples were collected before and after repetitive blast-related training on days 2 (n = 19) and days 7 (n = 15). Serum concentration (pg/mL) of tau, glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), and phosphorylated tau181 (p-tau181) were measured using an ultrasensitive immunoassay platform. We observed that serum p-tau181 concentrations were elevated after exposed to repetitive blast on days 2 (z = −2.983, p = 0.003) and days 7 (z = −2.158, p = 0.031). Serum tau (z = −2.272, p = 0.023) and NfL (z = −2.158, p = 0.031) levels were significantly elevated after exposure to repetitive blasts on days 7. Our findings indicate that blast exposure affects serum biomarkers indicating axonal injury.

Mild traumatic brain injury (TBI) is associated with persistent sleep-wake dysfunction, including insomnia and circadian rhythm disruption, which can exacerbate functional outcomes including mood, pain, and quality of life. Present therapies to treat sleep-wake disturbances in those with TBI (e.g., cognitive behavioral therapy for insomnia) are limited by marginal efficacy, poor patient acceptability, and/or high patient/provider burden. Thus, this study aimed to assess the feasibility and preliminary efficacy of morning bright light therapy, to improve sleep in Veterans with TBI (NCT03578003). Thirty-three Veterans with history of TBI were prospectively enrolled in a single-arm, open-label intervention using a lightbox (~10,000 lux at the eye) for 60-minutes every morning for 4-weeks. Pre- and post-intervention outcomes included questionnaires related to sleep, mood, TBI, post-traumatic stress disorder (PTSD), and pain; wrist actigraphy as a proxy for objective sleep; and blood-based biomarkers related to TBI/sleep. The protocol was rated favorably by ~75% of participants, with adherence to the lightbox and actigraphy being ~87% and 97%, respectively. Post-intervention improvements were observed in self-reported symptoms related to insomnia, mood, and pain; actigraphy-derived measures of sleep; and blood-based biomarkers related to peripheral inflammatory balance. The severity of comorbid PTSD was a significant positive predictor of response to treatment. Morning bright light therapy is a feasible and acceptable intervention that shows preliminary efficacy to treat disrupted sleep in Veterans with TBI. A full-scale randomized, placebo-controlled study with longitudinal follow-up is warranted to assess the efficacy of morning bright light therapy to improve sleep, biomarkers, and other TBI related symptoms.

Background: Elevations of inflammatory cytokine levels occur immediately after mild traumatic brain injury (mTBI) and can persist for years. These elevations have been associated with neuropsychological outcomes, including depression and PTSD symptoms. Sleep disorders, another common sequelae of mTBI, are independently associated with inflammation in otherwise healthy individuals. However, whether sleep and inflammation are linked in chronic mTBI has not been reported.Methods: A retrospective cross-sectional cohort of warfighters was used to investigate the hypothesis that inflammation may be linked to sleep quality in chronic mTBI. Clinical history, peripheral blood samples, and sleep quality scores were collected from 182 warfighters (n = 138 mTBI; n = 44 controls) during enrollment in the Chronic Effects of Neurotrauma Consortium study. Biomarkers of inflammation (IL-6, IL-10, TNFα cytokines) from plasma and plasma-derived extracellular vesicles (EVs) were quantified using single molecule array. Relationships between sleep quality and cytokine levels were assessed, controlling for age, sex, and BMI. Using clinical cutoff scores for sleep quality, mTBI patients were then divided into “good” and “poor” sleepers and cytokine levels compared between groups.Results: In mTBI participants, sleep quality was significantly associated with EV levels of IL-10 [ß (SE) = 0.11 (0.04), p = 0.01] and TNFα [ß (SE) = 0.07 (0.03), p < 0.01]. When divided according to “good” versus “poor” sleepers, those reporting poor sleep had significantly elevated EV IL-10 compared to those reporting good sleep [ß (SE) = 0.12 (0.04), p < 0.01]. Plasma-derived associations were not significant. No associations were found between sleep quality and cytokine levels in controls.Conclusion: These results suggest a significant relationship between sleep quality and chronic inflammation in mTBI patients. Clinically, mTBI patients with a high likelihood of sleep disorders demonstrate elevated levels of inflammatory cytokines. Signal from EVs, though smaller in magnitude, may have stronger clinical associations than from plasma. Sleep-focused interventions may also serve to regulate chronic inflammatory processes in these patients. Larger prospective studies are needed to investigate the mechanisms and therapeutic implications of the likely bi-directional relationship between sleep and inflammation following mTBI.

Traumatic brain injuries (TBI) and posttraumatic stress disorder (PTSD) are commonly observed comorbid occurrences among military service members and veterans (SMVs). In this cross-sectional study, SMVs with a history of TBI were stratified into symptomatic and asymptomatic PTSD groups based on posttraumatic stress checklist-civilian (PCL-C) total scores. Blood-based biomarkers were assessed, and significant differential markers were associated with scores from multiple neurobehavioral self-report assessments. PCL-C cutoffs were total scores >50 (PTSD symptomatic) and p < 0.05–0.001) in the TBI+/PTSD symptomatic group compared to the TBI+/asymptomatic group. Cytokine levels of IL8, TNFα, and IL10 were strongly associated with PCL-C scores (0.356 < r > 0.624 for all, p < 0.01 for all), while TNFα and IL10 were additionally associated with NSI totals (r = 0.285 and r = 0.270, p < 0.05, respectively). This is the first study focused on PTSD symptom severity to report levels of circulating pro-inflammatory IL8, specifically in SMVs with TBI. These data suggest that within the military TBI population, there are unique cytokine profiles that relate to neurobehavioral outcomes associated with TBI and PTSD.

Repetitive low-level blast exposure is one of the major occupational health concerns among US military service members and law enforcement. This study seeks to identify gene expression using microRNA and RNA sequencing in whole-blood samples from experienced breachers and unexposed controls. We performed experimental RNA sequencing using Illumina’s HiSeq 2500 Sequencing System, and microRNA analysis using NanoString Technology nCounter miRNA expression panel in whole-blood total RNA samples from 15 experienced breachers and 14 age-, sex-, and race-matched unexposed controls. We identified 10 significantly dysregulated genes between experienced breachers and unexposed controls, with FDR corrected LINC00996 (long intergenic non-protein coding RNA 996); and nine downregulated genes, IGLV3-16 (immunoglobulin lambda variable 3-16), CD200 (CD200 molecule), LILRB5 (leukocyte immunoglobulin-like receptor B5), ZNF667-AS1 (ZNF667 antisense RNA 1), LMOD1 (leiomodin 1), CNTNAP2 (contactin-associated protein 2), EVPL (envoplakin), DPF3 (double PHD fingers 3), and IGHV4-34 (immunoglobulin heavy variable 4-34). The dysregulated gene expressions reported here have been associated with chronic inflammation and immune response, suggesting that these pathways may relate to the risk of lasting neurological symptoms following high exposures to blast over a career.

Chronic mild traumatic brain injury (mTBI) has long-term consequences, such as neurological disability, but its pathophysiological mechanism is unknown. Exosomal microRNAs (exomiRNAs) may be important mediators of molecular and cellular changes involved in persistent symptoms after mTBI. We profiled exosomal microRNAs (exomiRNAs) in plasma from young adults with or without a chronic mTBI to decipher the underlying mechanisms of its long-lasting symptoms after mTBI. We identified 25 significantly dysregulated exomiRNAs in the chronic mTBI group (n = 29, with 4.48 mean years since the last injury) compared to controls (n = 11). These miRNAs are associated with pathways of neurological disease, organismal injury and abnormalities, and psychological disease. Dysregulation of these plasma exomiRNAs in chronic mTBI may indicate that neuronal inflammation can last long after the injury and result in enduring and persistent post-injury symptoms. These findings are useful for diagnosing and treating chronic mTBIs.