Feasibility and acceptability for LION, a fully remote, randomized clinical trial within the VA for light therapy to improve sleep in Veterans with and without TBI: An MTBI2 sponsored protocol: LION: A remote RCT protocol within VA.

Sleep-wake disturbances frequently present in Veterans with mild traumatic brain injury (mTBI). These TBI-related sleep impairments confer significant burden and commonly exacerbate other functional impairments. Therapies to improve sleep following mTBI are limited and studies in Veterans are even more scarce. In our previous pilot work, morning bright light therapy (MBLT) was found to be a feasible behavioral sleep intervention in Veterans with a history of mTBI; however, this was single-arm, open-label, and non-randomized, and therefore was not intended to establish efficacy. The present study, LION (light vs ion therapy) extends this preliminary work as a fully powered, sham-controlled, participant-masked randomized controlled trial (NCT03968874), implemented as fully remote within the VA (target n=120 complete). Randomization at 2:1 allocation ratio to: 1) active: MBLT (n=80), and 2) sham: deactivated negative ion generator (n=40); each with identical engagement parameters (60-min duration; within 2-hrs of waking; daily over 28-day duration). Participant masking via deception balanced expectancy assumptions across arms. Outcome measures were assessed following a 14-day baseline (pre-intervention), following 28-days of device engagement (post-intervention), and 28-days after the post-intervention assessment (follow-up). Primary outcomes were sleep measures, including continuous wrist-based actigraphy, self-report, and daily sleep dairy entries. Secondary/exploratory outcomes included cognition, mood, quality of life, circadian rhythm via dim light melatonin onset, and biofluid-based biomarkers. Participant drop out occurred in <10% of those enrolled, incomplete/missing data was present in <15% of key outcome variables, and overall fidelity adherence to the intervention was >85%, collectively establishing feasibility and acceptability for MBLT in Veterans with mTBI.

Research Letter: Relationship of Blood Biomarkers of Inflammation With Acute Concussion Symptoms and Recovery in the CARE Consortium.

OBJECTIVE: Determine the association of inflammatory biomarkers with clinical measures and recovery in participants with concussion. SETTING: Multicenter study in National Collegiate Athletic Association member institutions including military service academies. PARTICIPANTS: Four hundred twenty-two participants with acute concussion. DESIGN: Clinical visits and blood draws were completed preinjury and at multiple visits postconcussion (0-12 hours, 12-36 hours, and 36-60 hours postinjury). Clinical measures included Sport Concussion Assessment Tool (SCAT) symptom severity, Balance Error Scoring System, Standardized Assessment of Concussion (SAC), Brief Symptom Inventory-18 (BSI-18) scores, time to initiation of graduated return-to-play (RTP) protocol, and time to RTP. Interleukin (IL)-6, IL-10, IL-8, IL-1 receptor antagonist (RA), tumor necrosis factor (TNF), c-reactive protein, and vascular endothelial growth factor (VEGF) were measured in serum. Prespecified analyses focused on IL-6 and IL-1RA at 0 to 12 hours; exploratory analyses were conducted with false discovery rate correction. RESULTS: For prespecified analyses, IL-1RA at 0 to 12 hours in female participants was positively associated with more errors on the SAC (B(standard error, SE) = 0.58(0.27), P < .05) and worse SCAT symptom severity (B(SE) = 0.96(0.44), P < .05). For exploratory analyses, higher levels of IL-1RA at 12 to 36 hours were associated with higher global (B(SE) = 0.55(0.14), q < 0.01), depression (B(SE) = 0.45(0.10), q < 0.005), and somatization scores on the BSI (B(SE) = 0.46(0.12), q < 0.01) in participants with concussion; Higher TNF at 12 to 36 hours was associated with fewer errors on the SAC (B(SE) = - 0.46(0.14), q < 0.05). Subanalyses showed similar results for male participants and participants who were athletes. No associations were discovered in nonathlete cadets. Higher IL-8 at 0 to 12 hours was associated with slower RTP in female participants (OR = 14.47; 95% confidence interval, 2.96-70.66, q < 0.05); no other associations with recovery were observed. CONCLUSIONS: Peripheral inflammatory markers are associated with clinical symptoms following concussion and potentially represent one mechanism for psychological symptoms observed postinjury. Current results do not provide strong support for a potential prognostic role for these markers.

A Pilot Study Investigating the Use of Serum Glial Fibrillary Acidic Protein to Monitor Changes in Brain White Matter Integrity After Repetitive Head Hits During a Single Collegiate Football Game.

Repetitive head hits (RHHs) in sports and military settings are increasingly recognized as a risk factor for adverse neurological outcomes, but they are not currently tracked. Blood-based biomarkers of concussion have recently been shown to increase after nonconcussive RHHs during a single sporting contest, raising the possibility that they could be used in real time to monitor the brain's early response to repeated asymptomatic head hits. To test this hypothesis, we measured GFAP in serum immediately before (T0), immediately after (T1) and 45 min (T2) after a single collegiate football game in 30 athletes. Glial fibrillary acidic protein (GFAP) changes were correlated with three measures of head impact exposure (number of hits, total linear acceleration, and total rotational acceleration captured by helmet impact sensors) and to changes in brain white matter (WM) integrity, estimated by regional changes in fractional anisotropy (FA) and mean diffusivity (MD) on diffusion tensor imaging from 24 h before (T1) to 48 h after (T3) the game. To account for the potentially confounding effects of physical exertion on GFAP, correlations were adjusted for kilocalories of energy expended during the game measured by wearable body sensors. All 30 participants were male with a mean age of 19.5 ± 1.2 years. No participant had a concussion during the index game. We observed a significant increase in GFAP from T0 to T1 (mean 79.69 vs. 91.95 pg/mL, p = 0.008) and from T0 to T2 (mean 79.69 vs. 99.21 pg/mL, p < 0.001). WM integrity decreased in multiple WM regions but was statistically significant in the right fornix (mean % FA change -1.43, 95% confidence interval [CI]: -2.20, -0.66). T0 to T2 increases in GFAP correlated with reduced FA in the left fornix, right fornix, and right medical meniscus and with increased MD in the right fornix (r-values ranged from 0.59 to 0.61). Adjustment for exertion had minimal effect on these correlations. GFAP changes did not correlate to head hit exposure, but after adjustment for exertion, T0 to T2 increases correlated with all three hit metrics (r-values ranged from 0.69 to 0.74). Thus, acute elevations in GFAP after a single collegiate football game of RHHs correlated with in-game head hit exposure and with reduced WM integrity 2 days later. These results suggest that GFAP may be a biologically relevant indicator of the brain's early response to RHHs during a single sporting event. Developing tools to measure the neurological response to RHHs on an individual level has the potential to provide insight into the heterogeneity in adverse outcomes after RHH exposure and for developing effective and personalized countermeasures. Owing to the small sample size, these findings should be considered preliminary; validation in a larger, independent cohort is necessary.

Association of Blood Biomarkers of Inflammation With Acute Concussion in Collegiate Athletes and Military Service Academy Cadets.

BACKGROUND AND OBJECTIVES: The objective was to characterize the acute effects of concussion (a subset of mild traumatic brain injury) on serum interleukin (IL)-6 and IL-1 receptor antagonist (RA) and 5 additional inflammatory markers in athletes and military service academy members from the Concussion Assessment, Research, and Education Consortium and to determine whether these markers aid in discrimination of concussed participants from controls. METHODS: Athletes and cadets with concussion and matched controls provided blood at baseline and postinjury visits between January 2015 and March 2020. Linear models investigated changes in inflammatory markers measured using Meso Scale Discovery assays across time points (baseline and 0-12, 12-36, 36-60 hours). Subanalyses were conducted in participants split by sex and injury population. Logistic regression analyses tested whether acute levels of IL-6 and IL-1RA improved discrimination of concussed participants relative to brain injury markers (glial fibrillary acidic protein, tau, neurofilament light, ubiquitin c-terminal hydrolase-L1) or clinical data (Sport Concussion Assessment Tool-Third Edition, Standardized Assessment of Concussion, Balance Error Scoring System). RESULTS: Participants with concussion (total, N = 422) had elevated IL-6 and IL-1RA at 0-12 hours vs controls (n = 345; IL-6: mean difference [MD] (standard error) = 0.701 (0.091), p < 0.0001; IL-1RA: MD = 0.283 (0.042), p < 0.0001) and relative to baseline (IL-6: MD = 0.656 (0.078), p < 0.0001; IL-1RA: MD = 0.242 (0.038), p < 0.0001), 12-36 hours (IL-6: MD = 0.609 (0.086), p < 0.0001; IL-1RA: MD = 0.322 (0.041), p < 0.0001), and 36-60 hours (IL-6: MD = 0.818 (0.084), p < 0.0001; IL-1RA: MD = 0.317 (0.040), p < 0.0001). IL-6 and IL-1RA were elevated in participants with sport (IL-6: MD = 0.748 (0.115), p < 0.0001; IL-1RA: MD = 0.304 (0.055), p < 0.0001) and combative-related concussions (IL-6: MD = 0.583 (0.178), p = 0.001; IL-1RA: MD = 0.312 (0.081), p = 0.0001). IL-6 was elevated in male (MD = 0.734 (0.105), p < 0.0001) and female participants (MD = 0.600 (0.177), p = 0.0008); IL-1RA was only elevated in male participants (MD = 0.356 (0.047), p < 0.0001). Logistic regression showed the inclusion of IL-6 and IL-1RA at 0-12 hours improved the discrimination of participants with concussion from controls relative to brain injury markers (χ2(2) = 17.855, p = 0.0001; area under the receiver operating characteristic curve [AUC] 0.73 [0.66-0.80] to 0.78 [0.71-0.84]), objective clinical measures (balance and cognition; χ2(2) = 40.661, p < 0.0001; AUC 0.81 [0.76-0.86] to 0.87 [0.83-0.91]), and objective and subjective measures combined (χ2(2) = 13.456, p = 0.001; AUC 0.97 [0.95-0.99] to 0.98 [0.96-0.99]), although improvement in AUC was only significantly relative to objective clinical measures. DISCUSSION: IL-6 and IL-1RA (male participants only) are elevated in the early-acute window postconcussion and may aid in diagnostic decisions beyond traditional blood markers and common clinical measures. IL-1RA results highlight sex differences in the immune response to concussion which should be considered in future biomarker work.

Plasma phosphorylated tau181 as a biomarker of mild traumatic brain injury: findings from THINC and NCAA-DoD CARE Consortium prospective cohorts.

Objective: The 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. Methods: This 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. Results: Concentrations 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). Conclusion: These 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.

Elevated Serum Tau and UCHL-1 Concentrations Within 12 Months of Injury Predict Neurobehavioral Functioning 2 or More Years Following Traumatic Brain Injury: A Longitudinal Study.

OBJECTIVE: Blood-based biomarkers have received considerable attention for their diagnostic and prognostic value in the acute and postacute period following traumatic brain injury (TBI). The purpose of this study was to examine whether blood-based biomarker concentrations within the first 12 months of TBI can predict neurobehavioral outcome in the chronic phase of the recovery trajectory. SETTING: Inpatient and outpatient wards from 3 military medical treatment facilities. PARTICIPANTS: A total of 161 service members and veterans classified into 3 groups: (a) uncomplicated mild TBI (MTBI; n = 37), (b) complicated mild, moderate, severe, penetrating TBI combined (STBI; n = 46), and (c) controls (CTRL; n = 78). DESIGN: Prospective longitudinal. MAIN MEASURES: Participants completed 6 scales from the Traumatic Brain Injury Quality of Life (ie, Anger, Anxiety, Depression, Fatigue, Headaches, and Cognitive Concerns) within 12 months (baseline) and at 2 or more years (follow-up) post-injury. Serum concentrations of tau, neurofilament light, glial fibrillary acidic protein, and UCHL-1 at baseline were measured using SIMOA. RESULTS: Baseline tau was associated with worse anger, anxiety, and depression in the STBI group at follow-up (R2 = 0.101-0.127), and worse anxiety in the MTBI group (R2 = 0.210). Baseline ubiquitin carboxyl-terminal hydrolase L1 (UCHL-1) was associated with worse anxiety and depression at follow-up in both the MTBI and STBI groups (R2Δ = 0.143-0.207), and worse cognitive concerns in the MTBI group (R2Δ = 0.223). CONCLUSIONS: A blood-based panel including these biomarkers could be a useful tool for identifying individuals at risk of poor outcome following TBI.

Longitudinal Associations Between Blood Biomarkers and White Matter MRI in Sport-Related Concussion: A Study of the NCAA-DoD CARE Consortium.

BACKGROUND AND OBJECTIVES: To study longitudinal associations between blood-based neural biomarkers (including total tau, neurofilament light [NfL], glial fibrillary acidic protein [GFAP], and ubiquitin C-terminal hydrolase-L1) and white matter neuroimaging biomarkers in collegiate athletes with sport-related concussion (SRC) from 24 hours postinjury to 1 week after return to play. METHODS: We analyzed clinical and imaging data of concussed collegiate athletes in the Concussion Assessment, Research, and Education (CARE) Consortium. The CARE participants completed same-day clinical assessments, blood draws, and diffusion tensor imaging (DTI) at 3 time points: 24-48 hours postinjury, point of becoming asymptomatic, and 7 days after return to play. DTI probabilistic tractography was performed for each participant at each time point to render 27 participant-specific major white matter tracts. The microstructural organization of these tracts was characterized by 4 DTI metrics. Mixed-effects models with random intercepts were applied to test whether white matter microstructural abnormalities are associated with the blood-based biomarkers at the same time point. An interaction model was used to test whether the association varies across time points. A lagged model was used to test whether early blood-based biomarkers predict later microstructural changes. RESULTS: Data from 77 collegiate athletes were included in the following analyses. Among the 4 blood-based biomarkers, total tau had significant associations with the DTI metrics across the 3 time points. In particular, high tau level was associated with high radial diffusivity (RD) in the right corticospinal tract (ß = 0.25, SE = 0.07, p FDR-adjusted = 0.016) and superior thalamic radiation (ß = 0.21, SE = 0.07, p FDR-adjusted = 0.042). NfL and GFAP had time-dependent associations with the DTI metrics. NfL showed significant associations only at the asymptomatic time point (|ß|s > 0.12, SEs <0.09, psFDR-adjusted < 0.05) and GFAP showed a significant association only at 7 days after return to play (ßs > 0.14, SEs <0.06, psFDR-adjusted < 0.05). The p values for the associations of early tau and later RD were not significant after multiple comparison adjustment, but were less than 0.1 in 7 white matter tracts. DISCUSSION: This prospective study using data from the CARE Consortium demonstrated that in the early phase of SRC, white matter microstructural integrity detected by DTI neuroimaging was associated with elevated levels of blood-based biomarkers of traumatic brain injury. Total tau in the blood showed the strongest association with white matter microstructural changes.

Are EPB41 and alpha-synuclein diagnostic biomarkers of sport-related concussion? Findings from the NCAA and Department of Defense CARE Consortium.

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.

Effects of Physical Exertion on Early Changes in Blood-Based Brain Biomarkers: Implications for the Acute Point of Care Diagnosis of Concussion.

Blood-based brain biomarkers (BBM) such as glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have potential to aid in the diagnosis of concussion. Recently developed point-of-care test devices would enable BBMs to be measured in field settings such military and sport environments within minutes of a suspicious head hit. However, head hits in these environments typically occur in the setting of vigorous physical exertion, which can itself increase BBMs levels. Thus, efforts to develop BBMs as acute concussion aids in field settings need to account for the effects of physical exertion. To determine the acute effects of physical exertion on the BBMs, we measured GFAP, UCH-L1, tau, and neurofilament light chain (NF-L) immediately before, immediately after, and 45 min after a single workout session consisting of aerobic and resistance exercises in 30 collegiate football players. Subjects wore body sensors measuring several aspects of exertion and underwent diffusion tensor imaging 24 h before and 48 h after exertion. All subjects were male with a mean age of 19.5 ± 1.2 years. The mean duration of activity during the workout session was 94 ± 31 min. There was a significant decrease in serum GFAP immediately after (median decrease of 27.76%, p < 0.0001) and a significant increase in serum UCH-L1 45 min after (median increase of 37.11%, p = 0.016) exertion, compared with pre-exertion baseline. No significant changes in tau or NF-L were identified. The duration of exertion had a significant independent linear correlation to the increase in serum UCHL1 from pre-exertion to 45 min after exertion (r = 0.68, p = 0.004). There were no significant pre- to post-exertional changes in any of the 39 examined brain white matter regions, and biomarker changes did not correlate to variation in white matter integrity in any of these regions. Thus, exertion appeared to be associated with immediate decreases in serum GFAP and very acute (45 min) increases in UCH-L1. These changes were related to the duration of exertion, but not to changes in brain white matter integrity. Our results have important implications for how these BBMs might be used to aid in the on-scene diagnosis of concussion occurring in the setting of physical exertion.

Serum GFAP, NfL, and tau concentrations are associated with worse neurobehavioral functioning following mild, moderate, and severe TBI: a cross-sectional multiple-cohort study.

Introduction: The 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 methods: Participants 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. Results: Significant associations were found between biomarkers and MMPI-2-RF scales (all p < 0.05; R2Δ > 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. Discussion: Results 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.

Cytokine Profiles Differentiate Symptomatic from Asymptomatic PTSD in Service Members and Veterans with Chronic Traumatic Brain Injury.

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 <25 (asymptomatic). Cytokines IL6, IL8, TNFα, and IL10 were significantly elevated (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.

Proteomic Profiling of Plasma Biomarkers Associated With Return to Sport Following Concussion: Findings From the NCAA and Department of Defense CARE Consortium.

Objective: To investigate the plasma proteomic profiling in identifying biomarkers related to return to sport (RTS) following a sport-related concussion (SRC). Methods: This 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 <14-days (n = 99) and (2) recovery ≥14-days (n = 41). We applied a highly multiplexed proteomic technique that uses DNA aptamers assay to target 1,305 proteins in plasma samples from concussed athletes with <14-days and ≥14-days. Results: We identified 87 plasma proteins significantly dysregulated (32 upregulated and 55 downregulated) in concussed athletes with recovery ≥14-days relative to recovery <14-days groups. The significantly dysregulated proteins were uploaded to Ingenuity Pathway Analysis (IPA) software for analysis. Pathway analysis showed that significantly dysregulated proteins were associated with STAT3 pathway, regulation of the epithelial mesenchymal transition by growth factors pathway, and acute phase response signaling. Conclusion: Our data showed the feasibility of large-scale plasma proteomic profiling in concussed athletes with a <14-days and ≥ 14-days recovery. These findings provide a possible understanding of the pathophysiological mechanism in neurobiological recovery. Further study is required to determine whether these proteins can aid clinicians in RTS decisions.

Dietary Supplementation With Branched Chain Amino Acids to Improve Sleep in Veterans With Traumatic Brain Injury: A Randomized Double-Blind Placebo-Controlled Pilot and Feasibility Trial.

Study Objectives: Traumatic 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. Methods: Thirty-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. Results: Six 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. Conclusion: Dietary 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].

Elevations in Tumor Necrosis Factor Alpha and Interleukin 6 From Neuronal-Derived Extracellular Vesicles in Repeated Low-Level Blast Exposed Personnel.

Objective: The 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. Methods: Participants 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. Results: Serum 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). Conclusions: These 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.

ErbB Signaling Pathway Genes Are Differentially Expressed in Monozygotic Twins Discordant for Sports-Related Concussion.

Transcriptional changes involved in neuronal recovery after sports-related concussion (SRC) may be obscured by inter-individual variation in mRNA expression and nonspecific changes related to physical exertion. Using a co-twin study, the objective of this study was to identify important differences in mRNA expression among a single pair of monozygotic (MZ) twins discordant for concussion. A pair of MZ twins were enrolled as part of a larger study of concussion biomarkers among collegiate athletes. During the study, Twin A sustained SRC, allowing comparison of mRNA expression to the nonconcussed Twin B. Twin A clinically recovered by Day 7. mRNA expression was measured pre-injury and at 6 h and 7 days postinjury using Affymetrix HG-U133 Plus 2.0 microarray. Changes in mRNA expression from pre-injury to each postinjury time point were compared between the twins; differences >1.5-fold were considered important. Kyoto Encyclopedia of Genes and Genomes identified biologic networks associated with important transcripts. Among 38,000 analyzed genes, important changes were identified in 153 genes. The ErbB (epidermal growth factor receptor) signaling pathway was identified as the top transcriptional network from pre-injury to 7 days postinjury. Genes in this pathway with important transcriptional changes included epidermal growth factor (2.41), epiregulin (1.73), neuregulin 1 (1.54) and mechanistic target of rapamycin (1.51). In conclusion, the ErbB signaling pathway was identified as a potential regulator of clinical recovery in a MZ twin pair discordant for SRC. A co-twin study design may be a useful method for identifying important gene pathways associated with concussion recovery.

Elevated Axonal Protein Markers Following Repetitive Blast Exposure in Military Personnel.

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.

Feasibility and preliminary efficacy for morning bright light therapy to improve sleep and plasma biomarkers in US Veterans with TBI. A prospective, open-label, single-arm trial.

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.

Extracellular vesicle neurofilament light is elevated within the first 12-months following traumatic brain injury in a U.S military population.

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 < 0.001) and mTBI (AUC:76.1%, p < 0.001) groups. No other group differences were observed for NfL or GFAP at either timepoint. NfL correlated with post-concussion symptoms (rs = - 0.38, p = 0.04) in the mTBI group, and with PTSD symptoms in mTBI (rs = - 0.43, p = 0.021) and smcTBI groups (rs = - 0.40, p = 0.024) within one year after injury, which was not confirmed in regression models. Our results suggest the potential of NfL, a protein previously linked to axonal damage, as a diagnostic biomarker that distinguishes TBI severity within the first year after injury.

Remote blast-related mild traumatic brain injury is associated with differential expression of exosomal microRNAs identified in neurodegenerative and immunological processes.

BACKGROUND: Blast traumatic brain injury (TBI) and subconcussive blast exposure have been associated, pathologically, with chronic traumatic encephalopathy (CTE) and, clinically, with cognitive and affective symptoms, but the underlying pathomechanisms of these associations are not well understood. We hypothesized that exosomal microRNA (miRNA) expression, and their relation to neurobehavioral outcomes among Veterans with blunt or blast mild TBI (mTBI) may provide insight into possible mechanisms for these associations and therapeutic targets. METHODS: This is a subanalysis of a larger Chronic Effects of Neurotrauma Consortium Biomarker Discovery Project. Participants (n = 152) were divided into three groups: Controls (n = 35); Blunt mTBI only (n = 54); and Blast/blast+blunt mTBI (n = 63). Postconcussive and post-traumatic stress symptoms were evaluated using the NSI and PCL-5, respectively. Exosomal levels of 798 miRNA expression were measured. RESULTS: In the blast mTBI group, 23 differentially regulated miRNAs were observed compared to the blunt mTBI group and 23 compared to controls. From the pathway analysis, significantly dysregulated miRNAs in the blast exposure group correlated with inflammatory, neurodegenerative, and androgen receptor pathways. DISCUSSION: Our findings suggest that chronic neurobehavioral symptoms after blast TBI may pathomechanistically relate to dysregulated cellular pathways involved with neurodegeneration, inflammation, and central hormonal regulation.

A Pilot Study of Whole-Blood Transcriptomic Analysis to Identify Genes Associated with Repetitive Low-Level Blast Exposure in Career Breachers.

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 <0.05: One upregulated gene, 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.