TRAUMATIC BRAIN INJURIES (TBI) are the signature injury sustained by military personnel in recent combat operations with an estimated 25% of combat veterans sustaining a TBI since 2001.1 Given enduring operations in global conflicts, Special Operations Forces (SOF) combat soldiers are subject to sustained blast and blunt exposures known to increase TBI risk. Between 2000 and 2018, the Defense and Veterans Brain Injury Center reported 383 947 first-time TBI diagnoses; 82.3% were classified as mild TBI (mTBI).2 Despite this classification, both the acute and cumulative effects of mTBI can substantially affect soldier readiness and increase the risk of long-term neurological impairments.3,4 Multiple mTBIs have been associated with increased chronic behavioral and neurological symptom rates in Gulf War veterans.5 Measuring the effects of cumulative mTBI exposure is challenging because symptom presentation is heterogenous and diagnosis is reliant on self-reporting. Impairments following mTBI may not be captured by subjective clinical examinations.6 Assessment is further complicated by reports that neurophysiological recovery may not coincide with clinical recovery.7–9 Clinicians have sought more objective assessment tools to aid in determining mTBI etiology. However, no objective blood biomarkers or advanced neuroimaging techniques have demonstrated clinical utility beyond discriminating mTBI from intracranial injuries.10,11
Identifying objective blood biomarkers indicating cumulative neurotrauma or ongoing neuroinflammation is necessary to detect those at risk for cognitive deficits and long-term neurological sequelae. Numerous protein biomarkers originating from neuronal, axonal, and glial injury have been proposed to reflect the underlying mTBI pathophysiology. Clinically useful biomarkers should be readily measurable in biofluids, elevated in accordance with mTBI severity, and progressively decreased throughout recovery. However, mTBI pathophysiology involves both acute injury and progressive secondary neurodegenerative processes caused by multiple biological reactions and neurometabolic events.12,13 Hence, it is unlikely that a single blood biomarker will reflect the spectrum of changes at the tissue and cellular levels. Thus, blood biomarker panels should match the different cascading processes that occur following mTBI.
In light of the aforementioned information, it is clear that a single blood biomarker has failed to emerge as a prognostic indicator for chronic neurodegeneration in those with an mTBI history. Based upon prior research, we propose that tissue-derived and neuroinflammatory blood biomarkers are worth studying in SOF combat soldiers and they are as follows: (1) neuron-specific enolase (NSE), (2) S100 calcium-binding protein B (S100B), (3) neurofilament light chain (NfL), and (4) interleukin-6 (IL-6). Neuron-specific enolase is a glycolytic enzyme found in neuronal and neuroendocrine cell cytoplasm14,15 and is secreted from the cytoplasm following traumatic biomechanical forces.16 Neuron-specific enolase elevation following mTBI has been documented and thought to reflect neuronal injury severity.17,18 S100B is predominantly expressed by astrocytes and is the predominant mTBI biomarker studied and reported in the literature.19 Increases in S100B following mTBI are attributed to astrogliosis and increased blood-brain barrier permeability.20 Primary and secondary neurodegenerative axonal injury may lead to cytoskeletal destabilization, calcium influx, and protease activity causing neurofilament protein breakdown.21 The NfL is an abundantly expressed cytoskeletal component of large caliber myelinated subcortical axons.22 Peripheral increases in NfL concentrations may indicate ongoing axonal disruption or chronic neurodegeneration within white matter.23,24 Finally, in addition to many processes affecting IL-6, IL-6 is a neurotrophic cytokine reported to modulate neuroinflammation and is expressed in response to mTBI.25,26 Both proinflammatory and neuroprotective properties have been attributed to IL-6 increases.27 Therefore, the study purpose was to determine the effects of mTBI history, lifetime mTBI incidence, and mTBI recency on NSE, S100B, NfL, and IL-6 concentrations in healthy, asymptomatic SOF combat soldiers.
This cross-sectional study utilized data collected over 4 years (2015-2019) at The University of North Carolina at Chapel Hill. Our final study sample included 104 male SOF combat soldiers (mean age = 33.3 ± 3.5 y) for which all data we required (eg, blood biomarker concentrations and mTBI history information) were collected and available for analyses. All participants completed verbal consent and study procedures were approved by the office of human research ethics at our institution. We asked our participants to self-report mTBI history as a dichotomized response: 49 (47.1%) SOF combat soldiers (mean age = 32.8 ± 3.3 y) self-reported no mTBI history, while 55 (52.9%) SOF combat soldiers (mean age = 33.7 ± 3.7 y) self-reported an mTBI history. We also asked them to self-report lifetime mTBI incidence (“None,” “1,” “2,” “≥3”; median [(IQR) interquartile range] = 3 [2.5]) and mTBI recency (“past month,” “past year,” or “year+”).
Fasted venous blood samples were collected in a laboratory setting via antecubital venipuncture into 2 collection tubes (in order): (1) 10.0-mL K2-ethylenediaminetetraacetic acid (EDTA) used to separate plasma, and (2) 10.0-mL serum separation tube (SST) used to separate serum (Becton-Dickinson Company, Plymouth, United Kingdom). Both the EDTA and SST collection tubes were then inverted 8 to 10 times. The EDTA collection tubes were centrifuged within 30 minutes of collection and the SST tubes were spun after a period of 30 minutes. Both were spun at 4°C at 4000 revolutions per minute for 10 minutes. Supernatants of both separated plasma and serum were pipetted into 1.0-mL cryovials and stored at −80°C until analysis to ensure a single freeze/thaw cycle per aliquot. Blinded sample analyses were conducted by an independent laboratory at our institution. Laboratory personnel were blinded to participant mTBI history. Prior to assay completion, samples were individually verified to confirm the consistency between vial labeling and our inventory documentation. Then samples were thawed and vortexed 10 seconds and centrifuged at 3000 revolutions per minute for 15 minutes. Supernatants were taken and assays were completed following the reagent kit manufacturer's protocol. For all blood biomarkers, reruns were conducted when concentrations were too high (out of the standard ranges), or when the assay coefficient of variation was higher than 15%. All samples were analyzed in duplicate and averaged for our analytic purposes. Information related to specific enzyme-linked immunosorbent assay, standard detection ranges, and assay sensitivity for all blood biomarkers is provided in the Supplemental Digital Content Table, available at: http://links.lww.com/JHTR/A354.
Means (and SDs) and medians (and IQRs) were used to characterize continuous variables, and frequency distributions (and percentages) were used to characterize discrete variables. Correlations were assessed using the Spearman rank test between age, NSE, NfL, S100-B, and IL-6. The Mann-Whitney U test was used to compare differences in biomarker concentrations across mTBI history. Kruskal-Wallis tests were used to examine group differences in biomarker concentrations between mTBI recency and number of reported mTBI. For significant findings observed following the Kruskal-Wallis test, post hoc pairwise Mann-Whitney U tests with Dwass-Steel-Critchlow-Fligner correction were applied to provide family-wise error rate protection. All statistical tests were 2-tailed with an a priori α level of P ≤ .05. Statistical analyses were conducted using SAS 9.4 (SAS Institute Inc, Cary, North Carolina) and figures were generated in R Studio (RStudio Inc, Boston, Massachusetts).
Blood biomarker concentration values and group-level sample sizes for all analyses are presented in Table 1.
TABLE 1 -
Blood biomarker concentrations presented as median (IQR) for overall sample and across the 3 grouping variables of interest: mTBI history, lifetime incidence, and mTBI recency
|Overall (n = 104)
|Yes (n = 55)
|No (n = 49)
|0 (n = 49)
|1 (n = 14)
|2 (n = 13)
|3+ (n = 28)
|Past month (n = 8)
|Past year (n = 8)
|Year+ (n = 39)
Abbreviations: IL-6, interleukin-6; mTBI, mild traumatic brain injury; NfL, neurofilament light chain; NSE, neuron-specific enolase; S100B, S100 calcium-binding protein B.
aNSE concentrations significantly higher in combat soldiers with mTBI history.
bNSE concentrations significantly higher in combat soldiers with 3+ mTBI versus 0 mTBI.
cS100B concentrations significantly higher in combat soldiers with 2 mTBI versus 1 mTBI.
dNfL concentrations significantly higher in combat soldiers sustaining mTBI in past year versus year+.
Neuron-specific enolase concentrations were significantly higher (z = −2.60, P = .01) for combat soldiers who self-reported mTBI history (median = 3.22 ng/mL, IQR = 2.61) than for those without self-reported mTBI history (median = 2.57 ng/mL, IQR = 1.54). We did not observe any differences for NfL, S100B, and IL-6 between those with and without mTBI history.
Lifetime mTBI incidence
There was a significant difference in NSE concentrations observed across lifetime mTBI incidence (0, 1, 2, and 3+ mTBI) (χ2(3) = 9.52, P = .02). These differences were driven largely by combat soldiers who have sustained 3+ mTBI (median = 3.64 ng/mL, IQR = 2.87) compared with combat soldiers with no mTBI history (median = 2.57 ng/mL, IQR = 1.54) (z = −2.75, P = .03). No pairwise differences in NSE concentrations were observed for the other lifetime mTBI incidence categories. We also observed a significant effect of lifetime mTBI incidence on S100B concentrations (χ2(3) = 8.21, P = .04), driven largely by the difference between combat soldiers self-reporting 1 mTBI and 2 lifetime mTBIs (z = −2.60, P = .04). The biomarker concentrations across lifetime mTBI incidence are illustrated in Figure 1.
Mild TBI recency
There was a main effect of mTBI recency on NfL concentrations (χ2(2) = 6.02, P = .049). Specifically, NfL concentrations were higher in those who sustained an mTBI within the past year than in those who last sustained an mTBI beyond 1 year ago (z = 2.42, P = .02). There were no significant differences in NSE, S100B, or IL-6 concentrations across mTBI recency levels. A nonsignificant elevation in median S100B concentration was observed in combat soldiers reporting more recent mTBI (χ2(2) = 4.81, P = .09). The biomarker concentrations across mTBI recency are illustrated in Figure 2.
This study aimed to describe blood biomarkers associated with mTBI in a sample of asymptomatic, healthy SOF combat soldiers. We evaluated differences in NSE, NfL, S100B, and IL-6 concentrations in healthy SOF combat soldiers based on mTBI history, lifetime mTBI incidence, and mTBI recency. Neuron-specific enolase was the only biomarker in this panel that was significantly higher in soldiers with mTBI history. This finding was complemented by an observation such that elevations in NSE were observed in combat soldiers with 3+ mTBI compared with those with no mTBI history. In the meta-analysis by Cheng et al,28 increased NSE obtained from patients reporting to the emergency department following severe TBI was associated with mortality and poor neurological outcomes. It is important to note that these injuries were caused by macrotrauma events (eg, motor vehicle crashes), and many other systemic injuries were likely concurrently responsible for these poor outcomes. Future studies should refine the prognostic properties offered by NSE in these environments and, as it relates to our particular study, in forward military operations. Despite the obvious population differences and incomplete neurological development, NSE concentrations following a single acute mTBI are not different from controls in pediatric populations.29 Neuron-specific enolase concentrations were not different between preseason baseline and postconcussion (ie, post-mTBI) concentrations in ice hockey players.30 Interestingly, NSE levels were significantly elevated in boxers compared with controls following a 2-month rest after a 1-week national boxing camp.31 The boxing data suggest a prolonged release of NSE due to neuronal decay following exposure to frequent repetitive head trauma, especially given the NSE half-life is 24 to 48 hours.31–33 These data support our findings that NSE remains elevated in combat soldiers who are exposed to more than 3 mTBIs. We validated elevations in NSE with self-reported mTBI frequency (ie, 3+ mTBIs). Therefore, NSE in a military setting may be a useful biomarker for sustained neuronal damage following exposure to both diagnosed mTBI and repetitive subclinical blast and blunt neurotrauma in cases in which soldiers are unable or unwilling to accurately self-report mTBI history.
Although we observed a significant difference in S100B between those who sustained 1 versus 2 mTBIs, no differences were observed when comparing other lifetime mTBI incidence groups. Our data point to a trend (P = .09) suggesting that S100B may be an indicator of mTBI recency, given its linear decrease as recency increased (see Figure 2, mTBI recency). While studies have shown increases in S100B concentrations following mTBI,30,34 elevated levels have also been reported following regular physical activity including football and rugby game-play, weight lifting, and treadmill running.35,36 It is possible that these observed S100B levels could be attributed to regular military training the combat soldiers were engaged in preceding their visit. Longitudinal sampling of S100B following mTBI would be useful to discern the true effects mTBI recency has on S100B levels in combat soldiers.
We observed differences in NfL concentration in SOF combat soldiers based on mTBI recency. Our overall findings demonstrate that SOF combat soldiers exhibited NfL concentrations similar to those reported for civilian patients with more severe brain injury and neurodegenerative disease.21 Disanto et al37 estimated the 99th percentile for NfL in 35-year-old, healthy controls to be 41.5 (35.8-49.4) pg/mL. The sample median NfL concentration in this SOF combat soldier cohort (461.50 [198.70] pg/mL) was similar to case studies of patients who sustained diffuse axonal injuries.23 Our observed NfL concentrations were also greater than a sample of boxers measured 7 to 10 days after repetitive head impacts.38 Unlike S100B and NSE, NfL concentrations appear unaffected by physical activity.24 Studying how NfL is influenced by other exposure types is warranted. For example, chronic occupational blast exposure may contribute to axonal degenaration.39 The science behind this is in its infancy and requires further study. Evaluating the relationship between white matter microstructure and NfL concentrations may elucidate potential reasons for these elevated values relative to civilians and athletes with brain injuries.
Interleukin-6 concentrations did not differ between any of our grouping variables: mTBI history, lifetime mTBI incidence, and mTBI recency. Previous studies have shown that IL-6 is acutely elevated following severe TBI, with the concentration dropping between 24 and 72 hours postinjury.40 Higher concentrations are associated with worse outcomes, and high blood levels are a predictor of mortality within 24 hours of severe TBI.40,41 The IL-6 inflammatory response following brain injury may appear only in the acute phase and differences between groups with and without mTBI resolve over time. However, previously reported significant differences in IL-6 concentrations were observed between active duty military personnel with (3.01 ± 2.10 pg/mL) and without (1.62 ± 0.56 pg/mL) general TBI history.42 These average IL-6 concentrations were higher than the median (IQR) IL-6 values we report in our SOF combat soldiers (mTBI history: 1.01 [1.16] pg/mL; no mTBI history: 1.18 [0.72] pg/mL). Our study may have missed the initial window where neuroinflammation could be observed where there is no longer a need for cell death and new neuronal growth, or IL-6 may not be a sensitive physiological mTBI measure in SOF combat soldiers. Nevertheless, further investigation is needed to better understand the physiological effects mTBI has in this population. This is especially true acutely following injury.
Our study is not without its limitations. Our analysis relied on SOF combat soldiers self-reporting mTBI history, lifetime mTBI incidence, and mTBI recency. This approach, though commonly employed in the mTBI literature, may introduce inaccuracies or underreporting due to the lack of definitive diagnostic criteria. Despite this, mTBI nondisclosure is prevalent in both military and civilian populations. Several factors contribute to nondisclosure in both populations. These include fear of missing game/practice time,43 service career repercussions,44 or simply not knowing that it was a concussion.43 Nondisclosure would ultimately lead to inaccurate medical records. This limitation is not restricted to our study but, rather, to all those in our field. We additionally also could not account for subclinical head impact exposures resulting from blast and blunt exposures sustained during training and missions that did not result in a diagnosed mTBI. In addition, a distinction between blast and blunt mechanisms is worth investigating in future work, as these mechanisms may result in different neurobiological processes. The biomarkers we evaluated can be affected by confounding factors including physical activity that we did not control for in this initial study. Each biomarker was treated as an independent analysis; therefore, corrections for multiple comparisons were applied only to the ensuing post hoc tests within each biomarker analysis.
These findings suggest several avenues for future investigation. Despite our noted differences, our cross-sectional design revealed substantial overlap between groups for all biomarkers. This suggests that these concentrations alone may not be discriminatory and longitudinal sampling would be more useful in a clinical setting. Future prospective studies and clinical trials should consider these biomarkers' naturally occurring variability. These distributions should be strongly considered when powering such trials with sufficient sample sizes and in interpreting the positive and negative predictive values derived for clinical application in this population. Furthermore, these biomarkers maybe sensitive to neurotrauma below thresholds, which would cause symptoms. Expanding this current work to relate elevated biomarker concentrations with neuroimaging findings, blast or impact sensor data, and other objective physiological measures could clarify clinically meaningful differences in neurological outcomes in SOF combat soldier populations.
Monitoring NSE concentration in combat soldiers exposed to multiple mTBI could provide evidence of ongoing neuroinflammation following repetitive head trauma, and our data suggest a dose-response relationship between NSE concentration and lifetime mTBI incidence. Currently, the clinical significance of chronically elevated NSE levels is unknown; however, this important discovery provides the framework for future investigations to correlate these findings with relevant health outcomes. Serial sampling of S100B following mTBI may reflect a longitudinal decrease following more recent mTBI; however, further research in this population is needed.
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