Biofluid,Imaging,Physiological,and Functional Biomarkers of Mild Traumatic Brain Injury and Subconcussive Head Impacts

Abstract

Post-concussive symptoms are frequently reported by individuals who sustain mild traumatic brain injuries (mTBIs) and subconcussive head impacts, even when evidence of intracranial pathology is lacking. Current strategies used to evaluate head injuries, which primarily rely on self-report, have a limited ability to predict the incidence, severity, and duration of post-concussive symptoms that will develop in an individual patient. In addition, these self-report measures have little association with the underlying mechanisms of pathology that may contribute to persisting symptoms, impeding advancement in precision treatment for TBI. Emerging evidence suggests that biofluid, imaging, physiological, and functional biomarkers associated with mTBI and subconcussive head impacts may address these shortcomings by providing more objective measures of injury severity and underlying pathology. Interest in the use of biomarker data has rapidly accelerated, which is reflected by the recent efforts of organizations such as the National Institute of Neurological Disorders and Stroke and the National Academies of Sciences, Engineering, and Medicine to prioritize the collection of biomarker data during TBI characterization in acute-care settings. Thus, this review aims to describe recent progress in the identification and development of biomarkers of mTBI and subconcussive head impacts and to discuss important considerations for the implementation of these biomarkers in clinical practice.

Introduction

Mild traumatic brain injuries (mTBIs) were long considered to induce only transient impairments, but it is now known that these injuries can have long-term effects on patient quality of life even when macroscale brain damage is undetectable. TBIs account for nearly 2.5 million emergency department visits, hospitalizations, and deaths in the United States alone each year and are particularly prevalent in the military and athlete populations.¹ Over 80% of TBIs are classified as mild,¹ which is currently defined in the military as a Glasgow Coma Scale (GCS) score of 13–15, confused or disoriented state lasting less than 24 h, loss of consciousness for up to 30 min, and/or memory loss lasting less than 24 h²; similar criteria have been defined by the American Congress of Rehabilitation Medicine (ACRM) and World Health Organization Collaborating Centre Task Force on Mild Traumatic Brain Injury.³ The long-term effects of subconcussive head impacts, which are currently defined as head impacts that do not result in immediate symptoms, are also under increasing study.^4
–6 Despite the often subtle nature of mTBI and subconcussive head impacts, some symptoms, including fatigue and headache, have been shown to persist for years after injury in a considerable proportion of mTBI patients^5,7; in addition, there is emerging evidence suggesting a link between cumulative exposure to subconcussive head impacts, such as those sustained by military personnel and athletes, and neurological and neurobehavioral symptoms in some individuals.^4,8

The clinical evaluation of individuals with a suspected TBI currently relies heavily on subjective techniques involving self-reports, as well as cognitive and neurological assessments of injury severity, such as the GCS, which lack the quantitative power necessary to gain an objective view of the pathophysiological mechanisms that underlie injury and inform mTBI treatment and management.^5,9 Efforts have been made to develop more sensitive and objective measures to aid TBI assessment, including various neurocognitive assessment tools, and some of these tools, such as the Automated Neuropsychological Assessment Metrics and the Immediate Post-Concussion Assessment and Cognitive Testing, have been cleared by the U.S. Food and Drug Administration (FDA) to aid in the evaluation of patients with suspected head injury.^10,11 While such tools are promising measures of cognitive alterations following a head impact,¹² such as the GCS, they do not provide information on the specific pathologies that may contribute to these changes and could be targeted therapeutically. The heterogeneous nature of mTBI pathology and outcomes further complicates efforts to develop effective treatment regimens and predict patient prognosis.

To address the limitations of current strategies for evaluating TBI, many investigators have aimed to identify biomarkers that could be used to aid TBI diagnosis, outcome prediction, long-term monitoring, pharmacodynamic analysis, and assessments of treatment safety.¹³ Generally, biomarkers are defined as indicators of normal biological processes, pathogenic processes, or responses to an exposure or intervention¹⁴; importantly, changes in biomarker characteristics should accurately reflect the underlying processes involved in the pathology of a disease and should be reproducible.¹⁵ In the context of mTBI, these processes include cytoskeletal damage, metabolic perturbations, and inflammation among others,¹⁶ and the most promising biomarker candidates that reflect these processes are molecules isolated from bodily fluids, metrics obtained from brain imaging modalities, physiological indicators, and measures of brain or cardiovascular function.

Many studies have been conducted to determine the optimal context for the use of potential candidates based on their kinetic properties, sensitivity, cost, or invasiveness. Some may be better suited to act as continuous measures for tracking cumulative cellular damage, recovery, or individual response to treatment over time. Others may enable the establishment of universal threshold values to standardize diagnosis and identify TBI endophenotypes at the time of injury.¹⁷ Because multiple pathological processes that could contribute to TBI-associated outcomes may be activated depending on TBI severity, the mechanism of head injury, and patient characteristics among other factors, emerging biomarkers have been evaluated both in isolation and in different combinations to enable a more comprehensive and personalized assessment of TBI pathology. Thus, this review describes evidence on the use of biofluid, imaging, physiological, and functional biomarkers associated with mTBI and subconcussive head impacts and important considerations for implementing these analyses in clinical settings. Collectively, recent evidence suggests that many biomarker candidates in these categories have the potential to be used with clinical data and current TBI assessment tools to improve patient management.

Search Method

PubMed was utilized to conduct the literature search for this narrative review. The search used terms related to biomarkers (“PET,” “EEG,” “MEG,” “tau,” “phosphorylated tau,” “GFAP,” “BLBP,” “small non-coding RNAs,” “microRNA,” “CT,” “DTI,” “MRI,” “SWI,” “MRS,” “NODDIQSM,” “FDG,” “NSE,” “UCHL1,” “amyloid beta,” “APP,” “IL-10,” “IL-6,” “TNF-alpha”), terms related to evaluation (“diagnostic,” “prognostic,” “clinical,” “nonclinical”), FDA status (“FDA cleared,” “FDA noncleared”), terms related to military populations (“military,” “veterans”), other related terms (“neuroimaging,” “evaluate,” “diagnosis,” “assess,” “etiology,” “comorbid,” “treatment,” “risk,” “risk factor,” “prevalence,” “protective factors”), and terms related to TBI (“head injuries,” “closed,” “brain injuries,” “traumatic,” “brain concussion,” “post-concussion,” “traumatic brain injury,” “TBI,” “mTBI,” “concuss,” “brain injuries”). Articles were retrieved and evaluated for relevance to the subject. References within the identified articles were also searched, and relevant articles were retrieved.

Fluid Biomarkers

The identification of fluid biomarkers of neurotrauma, including molecules isolated from bodily fluids such as blood, cerebrospinal fluid (CSF), urine, and saliva, has been a key objective of recent mTBI research. Interest in assays that can be performed using whole, unprocessed blood has increased, as this approach would bypass the need for centrifugation and processing into plasma or serum (i.e., the supernatant containing blood proteins) and thus supports the use of blood-based assays in more remote or austere environments. Markers of neuronal or astrocyte injury, inflammation, and blood–brain barrier disruption isolated from blood and saliva have received much attention in the context of mTBI as they have the potential to provide a minimally invasive evaluation of TBI pathology in various settings.¹⁸ These processes may also occur after subconcussive impacts, allowing the dual use of some fluid biomarkers for the assessment of both subconcussive impacts and mTBI.¹⁹

Biomarkers of neuronal injury and astrocyte activation

To date, neuronal and astrocyte proteins are the most studied biofluid markers of TBI (Table 1). Of note, while several novel neuronal and astrocyte-derived biomarkers have been identified as promising candidates worthy of further study, this review focuses only on the most studied.

Table 1.

Summary of Neuronal and Glial Biomarkers of mTBI and Subconcussive Head Impacts

Biomarker	Fluid tested	Time course after injury	FDA status and indication	Type of evaluation assisted by biomarker	Injury characteristic related to biomarker	Primary function (independent of injury)
Glial markers
Brain lipid binding protein (BLBP; also known as aldolase-C)	CSF, blood (serum)	Levels peak at 1–8 h after injury, decline by 24 h	Not cleared	Diagnostic	Astrocyte injury; gliosis	Astrocyte marker involved in fatty acid uptake, transport, metabolism
Glial fibrillary acid protein (GFAP)*	CSF, blood (plasma, serum)	Levels peak at 20 h after injury, decline across 7 days	Plasma test is FDA-cleared to aid in determining whether CT scans are needed within 12 h of injury	Diagnostic	Astrocyte injury; gliosis	Astrocyte marker (structure, function)
S100 calcium binding protein β (S100β)	Blood (serum)	Levels peak within 6 h of injury	Not FDA-cleared in the United States; used for clinical evaluation in Sweden	Diagnostic	Glial and blood–brain barrier dysfunction	Astrocyte marker involved in the cell cycle, and glial proliferation
Neuronal markers
Neuron-specific enolase (NSE)	CSF, blood (serum)	Levels increase during the first 12 h and decrease within hours or days; half-life is 24 h	Not cleared	Prognostic	Neuronal injury	Neuronal glycolytic enzyme
Ubiquitin C-terminal hydrolase-L1 (UCHL1)*	CSF, blood (plasma, serum)	Levels peak 6–24 h following injury and decline over 48 h	Plasma test is FDA-cleared to aid in determining whether CT scans are needed within 12 h of injury	Diagnostic	Neuronal injury	Neuronal enzyme that hydrolyzes the C-terminal adducts of ubiquitin
Axonal markers
Neurofilament light (NFL)	CSF, blood (serum, plasma)	Levels begin to increase within the first 48 h of injury; levels increase over time after injury	Not cleared	Diagnostic, prognostic	Axonal injury	Maintenance/support of the cytoskeleton
Phosphorylated/ Total Tau	CSF, blood (plasma, serum)	Elevations in tau levels can be detected 4–10 days after injury	Not cleared	Diagnostic, prognostic	Axonal injury	Microtubule-associated protein that stabilizes neuronal microtubules
Neurodegeneration markers
Amyloid precursor protein (APP)	CSF, blood (plasma, serum)	Accumulation begins 2–3 h after injury	Not cleared	Diagnostic, prognostic	Amyloidosis; neuronal injury	Integral membrane protein and regulator of synapse formation
Amyloid beta(Aβ−40, Aβ−42)	CSF, blood (plasma, serum)	Peaks 24 h post-injury and declines between 3–14 days	Not cleared	Diagnostic, prognostic	Amyloidosis; neuronal injury	Neuronal growth and repair

Asterisks indicate biomarkers that are cleared by the FDA for uses related to the assessment of head injury. CSF, cerebrospinal fluid; CT, computed tomography; FDA, U.S. Food and Drug Administration.

Astrocyte-derived biomarkers

S100 calcium binding protein β

S100β was one of the first blood-based biomarkers investigated for its utility in TBI assessment, and S100β analysis is now considered most appropriate for use during the first 24 h after injury.^20,21 S100β levels have been shown to be elevated after mTBI²² and in football players who sustained repetitive subconcussive head impacts but did not receive an mTBI diagnosis.^23,24 The most promising use of S100β has been in the detection of intracranial pathology on head computed tomography (CT) within 24–48 h after TBI,^25
–27 and these findings led to the inclusion of S100β measurements in guidelines for the routine clinical evaluation of mTBI in Scandinavia. While the addition of S100β analysis has resulted in a one-third reduction in the number of unnecessary CT scans and has reduced the cost of care per patient,²² its lack of specificity has prevented its widespread use in other countries. Although primarily expressed in astrocytes, S100β is also expressed in skeletal muscle and adipocytes; thus, elevated S100β levels have been reported in association with exercise and non-central nervous system (CNS) trauma.^21,25,28

Glial fibrillary acidic protein

Glial fibrillary acidic protein (GFAP) is an intermediate filament that is also expressed in astrocytes that is considered a marker of structural brain damage. Elevated levels of GFAP in blood have been observed at 24 h,^29
–31 2 days,^29,32 and 13 days³² after injury, but typically not at time-points beyond this period.^20,33,34 Elevated GFAP levels have also been measured in football players after a season of exposure to repetitive subconcussive head impacts.²⁴ Many studies have demonstrated that GFAP levels within 24 h post-injury can be used to discriminate mTBI patients from controls,³¹ CT-positive patients from CT-negative patients,^35

–39 and CT-negative, magnetic resonance imaging (MRI)-positive patients from CT- and MRI-negative patients.⁴⁰ GFAP levels could also accurately predict prolonged recovery from sports-related mTBI⁴¹ and patient mortality in a meta-analysis.³⁶ Another study reported elevated GFAP levels in military personnel exposed to moderate or repeated low-level blast overpressures on days 6 and 7 of a blast training program, suggesting the potential utility of GFAP for evaluating individuals exposed to subconcussive head impacts.⁴²

Brain lipid-binding protein

Recently, a proteomic study performed using CSF from severe TBI patients revealed the potential value of the brain lipid-binding protein (BLBP) (also known as aldolase-C), another astrocyte-derived protein, as a novel biomarker of astrocyte pathology that may address the specificity limitations of S100β and GFAP.⁴³ The levels of BLBP in blood within 8 h post-injury were shown to be more accurate than S100β levels in discriminating athletes who sustained an mTBI and those who did not.⁴⁴ Another study reported significantly higher BLBP levels in mTBI patients with positive CT scan findings than in those with negative CT scan findings using blood samples collected within 3 h of injury.⁴⁵ However, further study is needed to confirm these findings and determine other possible applications of BLBP analysis in the context of mTBI, and studies on changes in BLBP levels after subconcussive head impacts are warranted. Additional proteomic studies should also be conducted to identify novel protein biomarker candidates that could be well suited to predict patient outcome or inform treatment.

Neuron-derived biomarkers

Neuron-specific enolase

Neuron-specific enolase (NSE), also known as enolase 2, is a glycolytic enzyme expressed in the cell bodies of neurons that is released upon injury. NSE has been most studied as a potential biomarker of moderate-to-severe injury, in which extensive neuronal damage is more likely,⁴⁶ and has been shown to accurately predict mortality and unfavorable outcome across multiple studies.⁴⁷ While elevated levels of NSE in blood have also been observed in individuals who have sustained an mTBI,^48,49 findings on the ability of NSE levels to predict outcomes associated with mTBI have been mixed. One study showed that changes in serum NSE levels detected within 1 week after mTBI could be used to predict reductions in default mode network efficiency and centrality 3 months post-injury,¹⁶ but a recent meta-analysis failed to identify an association between early NSE levels (assessed within 2 weeks post-injury) and mTBI symptoms.⁵⁰

Ubiquitin C-terminal hydrolase-L1

Similar to NSE, ubiquitin C-terminal hydrolase-L1 (UCHL1) is a cytosolic protein expressed in neuronal cell bodies and is considered a marker of neuronal injury. Some studies have reported increased UCHL1 levels in blood collected within 24 h of mTBI^29,51 and an association between these elevations and intracranial pathology detectable on CT.²⁰ Notably, UCHL1 levels were also shown to be higher in mTBI patients who developed an unfavorable neurological outcome within 24 h of injury⁵² and in football players who sustained multiple subconcussive head impacts.⁵³ Another study reported incremental increases in blood UCHL1 levels with increasing injury severity from subconcussive head impacts to concussion using samples obtained within 4 h of injury.³⁰ However, most studies indicate that UCHL1 levels peak within the first 2 days of injury, with some showing peak UCHL1 levels as early as within 6 h of TBI, indicating that this marker may be most appropriate for aiding mTBI diagnosis and assessment during this period.^20,33,54

Neurofilaments

Neurofilament proteins are expressed in the axonal cytoskeleton, and neurofilament light (NFL) has shown mounting promise as a marker of axonal injury for use within days to years after TBI. A recent study reported that elevated serum NFL levels in concussed athletes persisted for 6 and 13 days post-injury and could be used to accurately discriminate athletes with and without concussion at the 2-, 6-, and 13-day post-injury time-points.³² Other studies have reported increased plasma NFL levels persisting for as long as 3 months after mTBI^31,55 and associations between elevated NFL levels and intracranial MRI findings,^31,56 persistent mTBI symptoms,⁵⁵ and an unfavorable 6–12-month outcome.^56,57 In a study of mild, moderate, and severe TBI patients, serum NFL levels remained elevated 5 years post-injury, and the 30-day levels could accurately distinguish TBI severity groups, as well as TBI patients from healthy controls.³⁴ Increased NFL levels were also associated with increased frequency and magnitude of subconcussive head impacts in a study of sports-related concussion.⁵⁸

Amyloid-beta isoforms and amyloid precursor protein

Since TBI and prolonged exposure to repetitive head trauma have been reported to increase the risk of developing neurodegenerative diseases,⁵⁹ neuronal proteins involved in these pathologies, including tau and amyloid-beta (Aβ) isoforms, have also been investigated as potential mTBI biomarkers.⁶⁰ The levels of tau, a microtubule protein expressed in axons, as well as tau fragments and phosphorylated tau have been shown to increase within days after mTBI,^31,61

–64 correlate with the duration of mTBI symptoms⁶² and the presence of intracranial pathology,³¹ accurately discriminate mTBI patients from trauma and healthy controls,^31,32,63 and predict prolonged recovery in concussed athletes.⁴¹ Elevated levels of Aβ−42, NFL, and tau were also observed in mTBI patients who exhibited poor sleep quality and lower executive function,⁶⁵ and a recent study of military service members reported a significant correlation between tau levels, but not the levels of Aβ isoforms, and symptom severity.⁶⁶ Other studies have reported alterations in blood Aβ−40, Aβ−42, and tau levels in military personnel at the 24-, 48-, and 72-h time-points after subconcussive, low-level blast exposure,^67
–69 whereas one study observed altered peripheral amyloid precursor protein levels after moderate blast exposure.⁷⁰

Blood-based biomarker panels

To leverage the different types of information provided by astrocyte- and neuron-derived biomarkers, many researchers have begun to evaluate the use of multiplexed biomarker panels for mTBI assessment. The most promising results in this area have involved the use of blood-based assays for GFAP and UCHL1. One of the largest studies of GFAP and UCHL1 to date, which assessed 1959 patients with mild-to-moderate TBI, demonstrated that serum GFAP and UCHL1 levels within 12 h post-injury had high sensitivity (0.973) and a negative predictive value (0.995), but low specificity (0.367), for the detection of intracranial injury on head CT in individuals with GCS scores of 14–15.³⁸ These findings were used to support the FDA clearance of a biomarker panel comprising GFAP and UCHL1, known as the Banyan Brain Trauma Indicator, to aid in the evaluation of patients with a suspected mTBI and assist in determining the need for a CT scan in 2018.⁷¹ Subsequently, Abbott Diagnostics adopted the Banyan assay and adapted it for use with their blood analyzer capability-i-STAT (i-STAT Alinity) TBI cartridge, a handheld device that was FDA-cleared in 2021 to aid in mTBI evaluation and rule out the need for a head CT.^71,72 The i-STAT TBI Cartridge also received FDA clearance for use in whole blood in 2024, which may support the use of this assay in more remote or austere settings.⁷³ Similarly, the Biomerieux VIDAS TBI blood test, which also measures GFAP and UCHL1 levels, obtained CE marking for use in Europe in 2023.⁷¹

However, some studies have reported that the performance of biomarker panels containing GFAP and the performance of GFAP alone for predicting CT positivity are similar,^33,54 suggesting the particular importance of GFAP in these panels. In addition, a recent study noted that there was no significant difference in the specificity of S100β and the i-STAT GFAP/UCHL1 panel in identifying intracranial lesions, indicating that either approach could be used for this indication.⁷⁴ Other panels have been developed for measuring brain-derived protein levels; these include the Neurology 4 Plex, which measures GFAP, UCHL1, NFL, and tau levels using the Single Molecule Array platform developed by Quanterix Corp. In a recent study of U.S. military cadets that utilized this assay, combined analysis of GFAP, UCHL1, NFL, and tau levels within 6 h of TBI accurately discriminated mTBI patients from controls, but the panel did not offer a significant benefit over the Sport Concussion Assessment Tool–Third Edition (SCAT-3) in diagnosing mBTI.²⁹

While markers of neuronal and astrocyte pathology have shown considerable promise and have been the most widely studied biofluid markers of mTBI in recent years, several challenges are worth considering. First, since the pathological processes triggered by TBI occur at different time-points after the initial injury,^16,75 the timing of biomarker measurement is critical. In addition, the cutoff values used for protein biomarkers vary widely among studies, which may challenge the development of reliable assays; for example, the thresholds for GFAP levels that have been tested for the prediction of intracranial pathology on CT range from 0.6 ng/mL to 190 pg/mL across different studies.^35

–39 Consensus regarding the optimal time-points, cutoff values, and applications for the analysis of neuronal and astrocyte markers should be established to support the routine clinical analysis of these biomarkers.

Inflammatory biomarkers

Biomarkers of inflammatory processes associated with TBI have also received increasing attention for their potential value in monitoring mTBI progression and identifying pathways that could be targeted therapeutically (Table 2). Elevated levels of the inflammatory cytokines IL-6 and IL-2 and various chemokines have been observed within 24 h after mTBI,^76,77 and the levels of IL-2, IL-8, tumor necrosis factor (TNF)-α, and IL-10, an anti-inflammatory cytokine, at this time-point were reported to be associated with more severe neuropsychological symptoms at both the 1-week and 6-month time-points post-mTBI.⁷⁷ Calcitonin gene-related peptide (CGRP), which plays a role in the pathophysiology of headache, has been investigated as a marker of post-TBI sequelae; in one recent study, CGRP levels were found to be higher in individuals reporting persisting post-concussive symptoms than in healthy controls.⁷⁸ Similar to GFAP, IL-10 levels have been reported to accurately differentiate CT-negative and CT-positive mTBI patients; in addition, the specificity of this assay depended on the timing of sample collection and patient age.⁷⁹ In another study, the levels of IL-8, IL-9, TNF, IL-17a, and monocyte chemoattractant protein-1 at the time of admission were associated with persistent post-concussive symptoms at 3 months post-injury.⁸⁰ Notably, the levels of brain-derived markers, including GFAP, NFL, and tau, were not associated with persistent post-concussive symptoms in this study, suggesting that inflammatory markers may be more useful for predicting outcome after TBI.⁸⁰

Table 2.

Summary of Inflammatory and RNA Biomarkers of mTBI and Subconcussive Head Impacts

Biomarker	Fluid tested	Time course after Injury	FDA status and indication	Type of evaluation assisted by biomarker	Injury characteristic related to biomarker	Primary function (independent of injury)
Inflammatory markers
Interleukin-6 (IL-6)	CSF, blood (plasma, serum)	Levels increase within 6 h of injury	Not cleared	Prognostic	Neuroinflammation	Chronic inflammation; autoimmunity
Interleukin-10 (IL-10)	CSF, blood (plasma, serum)	Levels increase within 24 h of injury and remain elevated up to 18 months post-injury	Not cleared	Diagnostic, prognostic	Neuroinflammation	Anti-inflammatory
Interleukin-12 (IL-12)	CSF, blood (plasma, serum)	Levels increase within the first 5 days of injury	Not cleared	Diagnostic	Neuroinflammation	Proinflammatory; regulates T-cells/NK cells
Tumor necrosis factor alpha (TNF- α)	CSF, blood (plasma, serum)	Levels increase within the first 5 days of injury	Not cleared	Diagnostic, prognostic	Neuroinflammation	Proinflammatory protein
RNAs
Salivary miRNAs (e.g., miR-26b-3p, miR-29c-3p)	Saliva	Levels change within the first 5 days of injury	Not cleared	Diagnostic, prognostic	Unknown	Autophagy, senescence, cell cycle
Exosomal miR-139-5p and hsa-miR-18a-5p	Blood (plasma)	Expression changes associated with remote history of TBI and repetitive TBI	Not cleared	Diagnostic, prognostic	Unknown	Inflammatory regulation, neurological disease, cell development

These markers have not been FDA-cleared to aid in the evaluation of mTBI. CSF, cerebrospinal fluid; FDA, U.S. Food and Drug Administration; mTBI, mild traumatic brain injury; NK, natural killer.

The profiles of inflammatory molecules in individuals who have sustained subconcussive head impacts have been less characterized, but recent evidence suggests that these markers may be less informative in this context. For example, one study of military personnel revealed significant elevations in IL-6 and TNF-α levels within 24 h after moderate, but not low-level, blast exposure,⁸¹ and another showed no changes in chemokine levels in soccer players within 24 h of exposure to repetitive subconcussive head impacts.⁸² In an effort to move beyond measuring changes in the levels of cytokines and chemokines, which often have redundant or pleiotropic roles, one recent study investigated the use of whole-blood stimulation as a measure of altered immune function in sports-related concussion.⁸³ This study showed higher immune reactivity to the inflammatory molecules lipopolysaccharide and resiquimod in athletes with a history of sports-related concussion than in healthy athletes,⁸³ and this approach could likely be applied to regularly monitor changes in immune function in individuals exposed to repetitive subconcussive head impacts.

The production of brain-targeting autoantibodies has been well documented in the context of some neurological diseases, such as Alzheimer’s disease,⁸⁴ but autoimmunity in TBI has been relatively less studied and has primarily focused on common antigens, such as S100β and GFAP. In an early study on autoantibodies and mTBI, anti-S100β antibody levels in blood were elevated in football players who sustained repeated subconcussive hits and correlated with persistent MRI abnormalities.²³ Another study reported elevated blood anti-GFAP levels within 24 h of injury only in TBI patients who experienced loss of consciousness, and most of these patients (82%) sustained mTBIs.⁸⁵ More recently, the levels of immunoglobulin-A autoantibodies to various protein fragments were shown to be elevated in biobanked saliva samples from athletes who incurred concussive and subconcussive head impacts.⁸⁶

Despite these promising findings, many factors beyond CNS injury, including age, infection, and other conditions, can influence cytokine and chemokine levels. Thus, the analysis of inflammatory molecules will likely be more useful as one component of a multi-dimensional TBI assessment than as a stand-alone technique. Additional studies evaluating the benefit of inflammatory markers in mTBI assessment in conjunction with other biomarkers or clinical data are warranted before their routine use can be recommended.

Extracellular vesicles

Extracellular vesicles (EVs) are emerging biomarkers of mTBI that play important roles in the cell–cell communication processes that underlie CNS pathology. These membranous vesicles are released by astrocytes, microglia, and neurons into bodily fluids and enclose various proteins, microRNAs (miRNA), metabolites, and other molecules as cargo.⁸⁷ The EV double membrane protects this cargo from degradation, and after EVs are released, they can be taken up by recipient cells where their cargo can exert effects that range from promoting inflammatory processes to stimulating excitatory transmission.⁸⁸ Exosomes, which are nanoscale EVs produced using the endocytic machinery of the cell, are of particular research interest as novel biomarkers for TBI diagnosis and monitoring.⁸⁹

Several of the promising protein biomarker candidates described above have been detected within exosomes, and changes in the exosomal levels of these proteins have been linked to mTBI in different populations. In studies of veterans, elevations in exosomal tau, phosphorylated tau, and NFL levels were observed in individuals who sustained 3 or more mTBIs, and these elevations were correlated with the occurrence of post-concussive symptoms and post-traumatic stress disorder (PTSD).^90,91 A study of military service members revealed elevated levels of Aβ−42, IL-10, and tau in neuron-derived exosomes in those who sustained an mTBI, and exosomal tau and IL-10 levels correlated with post-concussive symptoms and PTSD, respectively.⁹² In another study of military service members, participants who sustained three or more TBIs in the Chronic Effect of Neurotrauma Consortium longitudinal study were reported to exhibit dysregulated expression of several exosomal miRNAs involved in apoptosis, neuroinflammation, neurodegeneration, and other cell signaling pathways; the levels of one of these miRNAs also correlated with symptom severity.⁹³

In civilians, exosomal biomarkers have been increasingly investigated in the context of sports-related concussion, and in these studies, miRNAs and other non-coding RNAs isolated from saliva have received much attention (Table 2).⁹⁴ Changes in the levels of salivary miRNAs have been shown to relate to mTBI diagnosis,^95

–99 number of head impacts,^96
–98 symptom severity and persistence,^100,101 and neurocognitive and balance impairments.^{95,98,100,101} The levels of miRNAs from plasma-isolated EVs were also shown to be increased with repetitive head impact exposure¹⁰² and to be associated with mTBI-related neurocognitive changes.¹⁰³ In another study of TBI patients admitted to the emergency department and concussion clinic, the levels of several genes related to Alzheimer’s disease located within salivary EVs were shown to be higher than control levels.¹⁰⁴

While EVs and their cargo are promising biomarkers for TBI, several limitations should be addressed before they can be used clinically. First, EVs are difficult and time-consuming to isolate using traditional methods; without substantial modifications to how these biomarkers are isolated, analyzing them in point-of-care or remote settings will remain challenging.⁸⁹ Techniques enabling the enrichment of EVs directly from CNS cells are also needed to ensure the specificity of EV-based biomarkers, because EVs are released constitutively by nearly all cells of the body. While some CNS-specific EV assays have been developed,¹⁰⁵ most have been tested primarily in pre-clinical settings or in human studies with small sample sizes. Finally, the biological significance of EV-derived miRNAs remains particularly unclear, and further studies are needed to determine which EV miRNA candidates are relevant to TBI pathology.

Considerations regarding the clinical implementation of fluid biomarkers

Several factors, including storage and processing requirements, degradation of the marker through metabolic processes, TBI specificity, and feasibility of clinical use, should be considered in studies of biofluid markers.^106,107 In addition, there are a wide variety of platforms used for the analysis of biofluid markers, each with differing limits of detection and quantification, which has been shown to influence findings on biomarker levels.¹⁰⁸ The requirement for specialized equipment or advanced technological training may also limit the utility of some biomarker assays, particularly in remote or point-of-care settings. Some clinicians also express concerns with forgoing brain imaging even when biomarker data indicate a low probability of intracranial pathology. Identifying other indications for the use of blood-based biomarkers may promote more enthusiasm for their use; while there is some evidence that elevated GFAP and UCHL1 levels may be associated with outcomes such as cognitive impairment after TBI,¹⁰⁹ additional studies are needed to confirm this relationship and identify other possible indications for the use of common biofluid biomarkers. Finally, although markers such as Aβ−40, Aβ−42, and tau have shown promise as biofluid markers of subconcussive head trauma,^67
–69 additional studies are needed to determine whether assays for these and other commonly studied candidates are sensitive enough to detect pathologies that may be associated with subconcussive head impacts.

The nature of the sample and biomarker should also be considered during biomarker studies. While TBI biobanks have been established to support large-scale, multi-center studies,^110,111 archived samples may be more suitable for the analysis of protein than RNA or EVs. The levels of protein biomarkers were found to remain stable over 3 days in blood samples stored at 4–5°C,¹¹² and biomarker levels in blood samples even 10 years old were found to be similar to those in more recently collected samples from TBI patients when stored at −80°C.¹¹³ However, RNA transcripts are less stable than proteins in blood and are affected by the method of sample collection and storage.¹¹⁴ A recent study showed that long-term −80°C storage and repeated freeze–thaw cycles also significantly reduced the concentration of EVs in biospecimens in a time-dependent manner,¹¹⁵ indicating that fresh, non-archival samples should be used for EV analysis whenever possible. Although a TBI working group did help to establish guidelines for the processing and storage of biospecimens for biomarker studies in 2010,¹¹⁶ these guidelines primarily focus on protein or genomic biomarkers, and updated guidelines that describe the optimal handling of specimens for the analysis of novel biomarker candidates are warranted.

Imaging Biomarkers

In addition to causing considerable intracranial pathology in some patients, mTBI and subconcussive head impacts have been shown to involve microstructural damage, prompting research efforts to identify potential biomarkers related to patient outcome through specialized imaging modalities, such as MRI and positron emission tomography (PET) (Table 3).

Table 3.

Summary of CT, MRI, and PET Imaging Biomarkers of mTBI and Subconcussive Head Impacts

Biomarker	Modality	Optimal timing of use after injury	Clinical use	Type of evaluation assisted by biomarker	Injury characteristic detected by method	Measures related to injury
Computed tomography (CT)	Computerized X-ray	Recommended for use from the time of injury to 7 days post-injury	Indicated for patients with loss of consciousness, post-traumatic amnesia, focal neurological deficit, GCS <15 or other symptoms to identify potential intracranial pathology	Diagnostic	Intracranial lesion	Positive results indicate intracranial lesions or abnormalities
Diffusion tensor imaging (DTI)	MRI	Can be used from 0–7 days post-injury for diagnosis and from 8 days to 89 days for prognosis	In early stages of being incorporated into routine clinical practice to identify patients with mild TBI	Diagnostic, prognostic	Axonal injury	Changes in fractional anisotropy, mean diffusivity, and/or radial diffusivity allow the identification of microstructural (white matter) abnormalities
Susceptibility weighted imaging (SWI)	MRI	Can be used from 0–7 days post-injury for diagnosis, and from 8 days to 89 days for prognosis	In early stages of being incorporated into routine clinical practice to identify patients with mild TBI	Diagnostic, prognostic	Cortical contusions, cytotoxic edema, microhemorrhage (diffuse vascular injury often occurring with diffuse axonal injury)	Changes in susceptibilities allow the identification of microstructural (i.e., white matter or microvascular) abnormalities
T1, T2, FLAIR, magnetic resonance imaging (MRI)	MRI	Can be used from 0–7 days post-injury for diagnosis, and from 8 days to 89 days for prognosis	Can be applied to patients displaying symptoms associated with mild TBI (with or without negative CT results) to identify intracranial abnormalities	Diagnostic, prognostic	Intracranial lesions	Positive results are indicative intracranial lesions or abnormalities
Blood oxygenation level-Dependent functional MRI (BOLD-fMRI)	MRI	Can be used from 0–7 days post-injury for diagnosis, and from 8 days to 89d for prognosis	Not used clinically to diagnose head injury; restricted to research settings	Diagnostic, prognostic	Functional connectivity	Changes in BOLD signal between regions of interest signify altered functional connectivity
Brain sodium MRI (Na MRI)	MRI	Can be used from 0–7 days post-injury for diagnosis, and from 8 days to 89 days for prognosis	Not used clinically to diagnose head injury; restricted to research settings	Diagnostic, prognostic	Ionic homeostasis, axonal injury	Changes in volume weighted average of intra- and extracellular sodium concentrations signify ionic imbalance
Magnetic resonance spectroscopy (MRS)	MRI	Can be used from 0–7 days post-injury for diagnosis, and from 8 days to 89 days for prognosis	Not used clinically to diagnose head injury; restricted to research settings	Diagnostic, prognostic, Predictive	Neurochemical changes, neurotransmitter changes	Changes in the peaks in the spectra indicate in vivo alterations in chemicals in the brain (i.e., creatine or neurotransmitters)
Multi-dimensional MRI	MRI	Can be used from 0–7 days post-injury for diagnosis	Not used clinically to diagnose head injury; restricted to research settings	Diagnostic	Axonal injury	Changes in T1, T2 and diffusion could be indicative of abnormal white matter
Neurite orientation dispersion and density imaging (NODDI)	MRI	Can be used from 0–7 days post-injury for diagnosis, and from 8 days to 89 days for prognosis	Not used clinically to diagnose head injury; restricted to research settings	Diagnostic, Prognostic	Axonal injury	Changes in metrics obtained using this technique may indicate white matter microstructural damage
Quantitative susceptibility maps (QSM)	MRI	Can be used from 0–7 days post-injury for diagnosis, and from 8 days to 89 days for prognosis	Not used clinically to diagnose head injury; restricted to research settings	Prognostic	Axonal injury, edema, cerebral blood flow	Changes in results from this analysis, which is applied to SWI, indicate dysfunction in myelin, water content, and cerebral blood flow
Fluorodeoxyglucose (FDG)	PET	Can be used from 8 days to 89 days for prognosis, or beyond 90 days to better understand sequelae	Not used clinically to diagnose head injury; restricted to research settings	Diagnostic, prognostic	Glucose metabolism	Changes revealed by this technique indicate altered glucose metabolism in discrete brain regions
[F-18] FDDNP; 18F-T807 (18F-AV-1451)	PET	Can be used from 8 days to 89 days for prognosis, or beyond 90 days to better understand sequelae	Not used clinically to diagnose head injury; restricted to research settings	Diagnostic	Protein dysregulation	Radiotracers for measuring tau and amyloid beta (Tau/Aβ); changes revealed by this technique indicate in vivo protein dysregulation following injury

GCS, Glasgow Coma Scale; PET, positron emission tomography.

Structural and functional MRI markers

Recent evidence has shown that mTBI patients with MRI lesions can exhibit poorer outcome months after TBI.¹¹⁷ Specifically, MRI studies have revealed the presence of diffuse punctate lesions often located in subcortical brain regions after mTBI, which can be attributed to either axonal or vascular injury.^118,119 Various diffusion tensor imaging (DTI) metrics that are associated with this pathology, including changes in fractional anisotropy, have also been shown to correlate with the incidence and persistence of post-concussive symptoms.^120,121 Changes in DTI metrics have been found in multiple white matter tracts, including the corpus callosum, corona radiate, and superior longitudinal fasciculus, and correlate with functional impairments.^122,123 Longitudinal changes in DTI metrics have also been observed in studies of sports-related subconcussive head impacts, and these changes were associated with the magnitude and frequency of head impacts.¹²⁴ DTI studies performed at multiple time-points indicate that these metrics may serve as prognostic markers that could enable tracking of the recovery process.¹²⁵

However, DTI findings exhibit variability across different studies. The directionality of the changes in DTI metrics varies, which may be due to the heterogeneity of TBI pathology across patients or differences in imaging acquisition or analysis techniques.¹²⁶ The timing of DTI studies of TBI ranges from 1 day to multiple years after injury, which challenges robust conclusions on the dynamics of intracranial lesions detectable on DTI. The changes in DTI metrics found to be associated with subconcussive head impacts have also been inconsistent. For example, some studies of low-level blast exposure in military personnel showed no differences in DTI measurements,^127,128 while another reported alterations in DTI metrics with repetitive low-level blast exposure.¹²⁹ Additional studies conducted using larger samples are necessary to standardize methodologies for assessing DTI findings and understand their clinical implications.

Other MRI techniques can also provide valuable information on mTBI pathology. Standard structural MRI has revealed regional gray matter volume changes following mTBI that persisted up to 6 months and were correlated with various outcome scores.¹³⁰ Quantitative susceptibility maps are extensions that can be applied to susceptibility-weighted imaging to derive information on myelination, water content, and cerebral blood flow (CBF), and one study found abnormalities in both gray matter and white matter in mTBI patients within 48 h of injury using this approach.¹³¹ One small study using multi-dimensional MRI, which incorporates T1, T2, and diffusion imaging, found that normal and abnormal white matter could be distinguished, as mild and severe lesions contained larger T1 and T2 components and a significantly larger abnormal diffusion-T2 component.¹³² Brain sodium MRI calculates a volume-weighted average of intra- and extracellular sodium concentrations to derive the total sodium concentration and identify changes in sodium ion homeostasis, which is related to resting membrane potential, fluid balance, and the initiation of action potentials. One study found that sodium concentrations in gray and white matter were lower in mTBI patients than in controls and correlated with scores on cognitive tests.¹³³ Another study showed that hybrid diffusion MRI coupled with the neurite orientation dispersion and density imaging method could also identify changes in white matter that were associated with symptoms that persisted for at least 3 months after mTBI.¹³⁴

Functional MRI (fMRI) also shows promise for the assessment of mTBI and subconcussive head trauma. Functional MRI can be used to identify changes in brain activity by measuring the blood oxygenation level-dependent (BOLD) signal. Differences in BOLD signal between regions of interest indicate altered functional connectivity after mTBI.^135,136 Changes in BOLD signals have also been observed following low-level blast exposure¹³⁷ and subconcussive head impacts.^138,139 When paired with structural MRI, fMRI can be used to determine the functional ramifications of structural connectivity changes. In one study that combined DTI and fMRI, the decoupling of structural and functional connectivity networks was observed following mTBI.¹⁴⁰ The use of fMRI analyses alone to measure functional connectivity between brain regions may also provide biomarkers for mTBI. One study showed that differences in small-world global network connection were correlated with mTBI, prolonged return-to-play, and outcomes 1-year after return-to-play.¹⁴¹ Another study reported functional connectivity changes associated with deployment-related mTBI, noting higher connectivity between the right caudate and lateral occipital regions in these individuals than in the TBI-negative group and those who sustained non-deployment mTBIs.¹⁴² Resting-state functional connectivity in the frontoparietal network, which is considered to be involved in cognitive flexibility, was recently shown to differ between mTBI patients and orthopedic controls in a pilot study; longitudinal functional connectivity changes in a network involved in executive control were also observed in the mTBI group.¹⁴³

Magnetic resonance spectroscopy (MRS) enables an MRI-based analysis of chemicals in the brain. These chemicals are identified based on a spectrum of peaks characteristic of each molecule.¹⁴⁴ Common MRS findings in patients with sports-related concussion include significant reductions in the levels of N-acetyl aspartate (a neuronal marker), creatine (which is important for adenosine triphosphate [ATP] synthesis), and glutamate (an excitatory neurotransmitter).¹⁴⁵ These changes can persist even after clinical symptoms resolve.^146,147 Lower creatine levels are also directly correlated with a higher number of repetitive head impacts, suggesting MRS can aid in quantifying the frequency of mTBI exposures.¹⁴⁸ Subconcussive head impacts can also produce persistent alterations in glutamate levels.¹⁴⁹

PET markers

PET is an imaging method that tracks radioactive ligands to visualize metabolic processes in the brain.¹⁵⁰ Studies using PET ligands show that neuroinflammatory changes can be observed after mTBI.^151,152 Glucose hypometabolism is also commonly observed after mTBI and can be measured in discrete brain regions using the fluorodeoxyglucose PET ligand.^153,154 Ligands used to visualize proteins that are commonly dysregulated after injury, such as tau and Aβ, are also being studied for efficacy and reliability.^155,156 PET analyses have been shown to track aberrant changes in these proteins following injury in specific brain regions (i.e., thalamic and midbrain regions) in vivo.¹⁵² Additional research is required to identify optimal PET radiotracers with high affinities for proteins, such as key isoforms of tau, involved in pathological processes initiated by mTBI.¹⁵⁷

Single positron emission computed tomography (SPECT) is another imaging technique that provides information on metabolic processes in the brain by using gamma ray radiotracers to map blood flow.¹⁵⁸ One study that combined quantitative electroencephalography (qEEG) and SPECT analyses concluded that SPECT could be used to identify cerebral changes in mTBI patients who showed persisting symptoms for at least 3 months after injury and exhibited positive qEEG readings for structural injury and locate areas within the brain that had undergone functional changes and that were associated with behavioral changes; they also observed that all patients displayed abnormalities in central perfusion on SPECT that were undetectable by conventional CT or MRI.¹⁵⁹ Although SPECT appears to be a promising tool for mTBI assessment, further studies are required to confirm its utility.

Considerations regarding the clinical implementation of imaging biomarkers

Among the various advanced imaging techniques that have been investigated for use during mTBI evaluation, to date, MRI has received the most widespread attention in TBI research and has shown the most clinical utility; thus, MRI has been the first to become incorporated into clinical recommendations and guidelines for acute TBI care.^160,161 However, the clinical use of imaging biomarkers currently faces several challenges. While specialized imaging techniques are a standard component of the assessment of some neurological disorders, there is no consensus on the use of these methods for mTBI evaluation. In the Military Health System, the Department of Defense (DoD) Clinical Recommendation on the use of neuroimaging after mTBI indicates that CT scans should be performed within the first 7 days after a suspected mTBI to rule out severe pathology, but specialized imaging is indicated only in patients who exhibit persistent symptoms beyond this period and is performed at the discretion of the clinician.¹⁶⁰ To date, most specialized imaging techniques are used primarily for research purposes as the precise therapeutic implications of the microstructural lesions or functional pathologies detected using these methods are unclear. In addition, most specialized imaging techniques are more expensive and time-consuming than standard CT imaging and are less accessible since they require specialized equipment, reagents, or data analysis techniques.¹⁶² Notably, since functional imaging techniques, such as PET and SPECT, do not provide detailed anatomical information, these modalities are likely more useful for assessing patient prognosis and guiding therapy than for mTBI evaluation as stand-alone imaging methods.¹⁶² Finally, emerging studies have indicated changes in the BOLD signal and MRS as promising tools for assessing subconcussive head trauma,^{138,139,148,149} although further study is needed to confirm their sensitivity in this context.

Physiological Biomarkers

Physiological markers of mTBI, including impairments in balance and vision, are commonly assessed through clinical mTBI evaluation tools, offering easily accessible, non-invasive data that could inform patient management and mTBI progression. The use of physiological markers will likely become more widespread with the further development of devices and tools for assessing these metrics.

Balance/vestibular markers

Deficits in balance, which is coordinated by the combined efforts of specific cortical and subcortical structures, may occur following mTBI.¹⁶³ These impairments can occur immediately after injury and persist for days after injury,¹⁶⁴ and subconcussive head impacts and low-level blast have also been shown to elicit transient changes in balance.^165,166 Balance impairments have been thought to indicate dysfunction in one or multiple vestibular, somatosensory, and visual brain regions that could persist after other neurological symptoms have resolved.^167,168 To measure these impairments, devices that deliver stochastic stimulation of the lower limbs can be used to provide data on changes in progressive vestibular dysfunction resulting from repetitive head impacts.¹⁶⁹ Balance assessments, such as the Clinical Test of Sensory Integration in Balance, can also be used to provide information regarding sensory coordination to promote postural stability that can distinguish symptomatic patients with mTBI from controls.¹⁷⁰ Thus, device and non-device paradigms can be used to track changes in balance during recovery.

Markers of visual dysfunction

Visual dysfunction is frequently associated with mTBI. Aspects of eye movement, in particular, are affected by mTBI and are measurable through eye-tracking technologies.¹⁷¹ Patients who sustain blast-related mTBI often experience deficits in saccades, smooth pursuits, and convergence movements.¹⁷² Sports-related concussion can also elicit similar deficits.¹⁷³ In addition, deviations in the visual field and pattern standard and high spatial frequency contrast sensitivity can be observed in veterans during the first 1 to 5 years following mTBI.¹⁷⁴ Alterations in visual and vestibular integration can also be measured through eye-tracking tests. The optokinetic after nystagmus (OKAN) reaction reflects the response of visual and vestibular self-motion signals and can be measured via eye-tracking and video head impulse test paradigms. Patients with concussion have been shown to display an abnormally prolonged OKAN reaction.¹⁷⁵ Vision and oculomotor functions indicate changes in cognitive processes, such as attention, which is often impaired after a concussion.¹⁷⁶ Similarly, changes in reaction time elicited by injury and related to cognitive dysregulation can also be detected using common eye-tracking assessments.¹⁷⁷

Several diagnostic devices on the market use eye-tracking as an indicator of mTBI, as well as for monitoring recovery and guiding treatment approaches. Of these, the EyeBOX and Eye-SYNC systems have been FDA-cleared to aid in the evaluation of patients with suspected brain injury. The EyeBOX measures eye movements and gaze-related functions during a 4-min visual task, and EyeBOX measures have been shown to accurately distinguish mTBI patients from controls.¹⁷⁸ The Eye-SYNC utilizes a virtual-reality-based assessment to record, view, and analyze eye movements to aid mTBI diagnosis. However, a recent DoD-funded study showed age and sex differences in the performance of this assessment,¹⁷⁹ and other studies have reported that features of the task protocol can also influence diagnostic ability.¹⁸⁰ Additional studies on these devices are warranted in larger populations to confirm their sensitivity and specificity and assess their relationship to patient outcome.

Considerations regarding the clinical implementation of physiological biomarkers

Vestibular and oculomotor symptoms are commonly reported during the initial diagnosis of mTBI, and recent studies also indicate their association with exposure to subconcussive head impacts.^165,181,182 The Vestibular Ocular Motor Screening (VOMS) tool can be used to evaluate impairments in subdomains of the vestibular and oculomotor systems quickly and reliably.¹⁸³ A recent systematic analysis of 18 primary articles reported that positive VOMS findings obtained at the time of the injury or first clinical encounter consistently predicted a longer recovery time than negative VOMS results.¹⁸⁴ Due to this improved understanding of the importance of vestibular and oculomotor symptoms after mTBI, efforts should be made to promote the routine use of VOMS during mTBI evaluation and assessment of subconcussive head trauma. To this end, for example, the Military Acute Concussion Evaluation was revised in 2021 to incorporate VOMS assessment for military personnel with suspected head trauma.¹⁸⁵

Although the changes in balance and visual function that occur after mTBI may provide objective metrics of injury and recovery, several factors limit the identification of gold standard tools for measuring these domains in mTBI patients. The tools used to measure these functions vary widely regarding the instrumentation, protocol, and thresholds or strategies used to define impairment, which may influence the results.¹⁸⁶ Device paradigms used to assess these functions can also be difficult to use or access and may require extensive training to administer and interpret, limiting their widespread use. The subtle or transient nature of visual and balance deficits in some patients may also pose a challenge to establishing robust criteria for impairment that can be used broadly among clinicians, which emphasizes the need for innovative and multi-dimensional approaches, particularly in the context of predicting the potential for long-term impairment following repeated subconcussive head trauma.¹⁸⁷ Finally, these impairments can occur in the context of a variety of other neurological disorders of mTBI^188
–190; thus, the assessment of visual and vestibular symptoms should be performed alongside other methods of mTBI evaluation.

Functional Biomarkers

Many recent studies have reported changes in electrophysiological, cerebrovascular, and peripheral autonomic function associated with mTBI, providing additional information that could be used for routine TBI assessment and monitoring (Table 4).

Table 4.

Summary of Physiological Biomarkers of mTBI and Subconcussive Head Impacts

Biomarker	Modality	Optimal timing of use after Injury	Clinical use	Type of evaluation assisted by biomarker	Injury characteristic related to indicator	Measures related to injury
Electroencephalography (EEG)	EEG	Can be used from 0–7 days post-injury for diagnosis, from 8 days to 89 days for prognosis, or beyond 90 days to better understand sequelae	Not used clinically to diagnose head injury	Diagnostic, prognostic	Electrophysiological abnormality	Changes in electrophysiological activity in the brain may indicate injury
Event-related potentials (ERPs)	EEG	Can be used from 0–7 days post-injury for diagnosis, from 8 days to 89 days for prognosis, or beyond 90 days to better understand sequelae	Not used clinically to diagnose head injury	Diagnostic, prognostic	Electrophysiological abnormality	Changes in EEG signals time-locked to the onset of a stimulus can reveal impairments in a brain region’s electrical response to stimuli
Magnetoencephalography (MEG)	MEG	Can be used from 0–7 days post-injury for diagnosis, from 8 days to 89 days for prognosis, or beyond 90 days to better understand sequelae	Not used clinically to diagnose head injury	Diagnostic, prognostic	Electrophysiological abnormality	Changes in electrophysiological activity in the brain can indicate injury

Electrophysiological markers

The axonal stretching and white matter abnormalities caused by mTBI can ultimately disrupt neuronal communication between key brain regions.^191,192 This damage can interrupt the oscillations in brain activity generated by normal, coordinated networks. Abnormalities in these oscillations are detected using event-related potentials derived from electroencephalography (EEG) recordings.^193,194 Changes in specific oscillation frequencies have been found to be related to post-concussive symptoms and chronic pain.¹⁹⁵ A study on service members with a remote history of mTBI found diminished synchrony in the alpha, beta, and theta frequency bands, and disrupted synchrony at the low-gamma frequency was significantly correlated with reduced white matter integrity detected via DTI.¹⁹⁴ One study of individuals who sustained two or more mTBIs showed abnormalities in alpha, beta, and theta oscillations during a test used to evaluate task-switching, indicating that mTBI-associated changes in neural communication may relate to global changes in task performance.¹⁹⁶ In one study of ice hockey players who sustained a concussion, a portable framework for assessing electrocardiogram (ECG) potentials detected impairments in signals related to auditory sensation, attention, and cognitive processing within 24 h after concussion and at return-to-play.¹⁹⁷ Using this approach, deficits in the signal related to cognitive processing were also observed in players who sustained subconcussive head impacts.¹⁹⁷ Due to consistent findings on impaired brain activity in TBI patients, the FDA cleared the use of BrainScope, a portable qEEG device, to aid in the evaluation of TBI.¹⁹⁸

Magnetoencephalography (MEG) is an imaging technique that also provides information about electrophysiological activity in the brain following mTBI. One study using this technique reported reduced regional power and impairments in functional connectivity across several brain regions; importantly, changes in beta functional connectivity more accurately classified mTBI diagnosis than regional power or the SCAT2.¹⁹⁹ In a study of combat-related mTBI, the combined algorithmic analysis of delta-theta, alpha, beta, and gamma frequency bands outperformed the analysis of individual bands at discriminating mTBI patients and controls, showing high sensitivity and specificity.²⁰⁰ This study also revealed that delta-theta and gamma activity in the mTBI group correlated with poorer neuropsychological test performance.²⁰⁰ Collectively, these findings indicate that identifying electrophysiological abnormalities using ECG and MEG holds promise for the diagnosis and monitoring of mTBI and subconcussive head impacts.

Cerebrovascular and peripheral autonomic markers

CBF may be altered after mTBI and could be used to track recovery.²⁰¹ CBF can be measured through MRI-based methods, such as arterial spin labeling and dynamic susceptibility contrast, as well as through PET and transcranial Doppler ultrasound.²⁰² Regional increases in CBF have been shown to occur in mTBI patients within 10 days after injury, indicating increased cerebral metabolic demand following mTBI.²⁰³ CBF changes also occur after repetitive subconcussive head impacts in atheletes.^204,205 In one study of sports-related concussion, CBF was shown to decrease during the period from 24 h post-injury to 8 days post-injury, while cognitive and clinical symptoms resolved during the same period.²⁰⁶ Another study reported that decreased CBF at 1 month after mTBI was observed in football players who showed slower recovery of post-concussive symptoms, suggesting the possible use of CBF as a prognostic marker in mTBI.²⁰⁷ However, additional studies conducted in larger samples are needed to establish a reliable trajectory of CBF changes post-injury.

In addition to functional changes measured within the CNS, peripheral changes may occur following mTBI, including transient autonomic dysfunction, which can be detected by measuring heart rate variability (HRV). One study observed decreases in an indirect measure of stroke volume in concussed athletes one week after concussion and differences in arterial stiffness in athletes who took longer to return to play.²⁰⁸ Altered HRV has been shown to persist even after the resolution of clinical symptoms.²⁰⁹ These cardiovascular changes are detectable via electrocardiogram recordings, spectral analyses of the heart rate, and/or systolic blood pressure.^210,211 However, HRV changes after mTBI are not consistent, especially at months-to-years following injury.²¹²

Considerations regarding the clinical implementation of functional biomarkers

Changes in brain activity and cardiovascular function can be measured using cost-effective, non-invasive tools and may be useful for predicting prognosis, and studies have shown the utility of these tools for assessing both mTBI and subconcussive head trauma. However, there are several barriers to the standardized implementation of functional assessments of mTBI and subconcussive head impacts in clinical practice. Similar to the physiological impairments observed among mTBI patients, functional abnormalities are not specific to mTBI²¹³ and are measured through a variety of techniques, indicating the need for identifying gold standards for their detection. In blast TBI, the blast force itself can contribute to arrhythmias, ischemia, and myocardial infarction, even in the absence of cerebral concussion.²¹⁴ Accordingly, HRV may not be a reliable biomarker for mTBI or subconcussive head trauma in these scenarios.²¹⁴ These heterogeneous findings indicate that further investigation of HRV is warranted to determine its reliability and clinical utility. Although EEG is a more established method for evaluating neurological function, it can be difficult to reliably perform in remote settings, even with the use of BrainScope.²¹⁵ In addition, as with other biomarker categories, it will be important to evaluate whether tools that assess functional changes are sufficiently sensitive to detect pathologies associated with subconcussive head trauma.

Discussion

This review describes progress in the use of fluid, imaging, physiological, and functional biomarkers of mTBI and subconcussive head impacts and discusses the potential barriers to their implementation in clinical settings. Several tools for assessing mTBI-associated biomarkers, including biofluid platforms, eye-tracking devices, and a portable EEG device, have been FDA-cleared to aid in the clinical evaluation of mTBI, but all are currently considered auxiliary assessments that should not be used as stand-alone methods to diagnose mTBI. While blood-based biomarkers are minimally invasive and can be cost-effective, many studies highlight variability in the levels of promising biomarker candidates in mTBI patients and individuals exposed to subconcussive head impacts, indicating that their reliability, validity, and overall utility are currently unknown. Imaging metrics can provide important information on the microstructural pathology associated with mTBI and subconcussive head impacts, but the cost and requirement for advanced technological expertise associated with imaging may limit its use to research and high-resource acute-care settings. Although physiological and functional changes are not specific to mTBI, their inclusion in mTBI analysis may enable improved prediction of mTBI-associated outcomes. Further investigation of biomarker candidates and their applications in large, multi-center prospective clinical trials is necessary to confirm their utility in clinical settings.

Based on currently available data, several organizations, including the National Academies of Sciences, Engineering, and Medicine (NASEM); the ACRM; and the National Institute of Neurological Disorders and Stroke (NINDS), have begun to advocate for the prioritization of biomarker analyses during mTBI evaluation. The NASEM report on Accelerating Progress in TBI describes the utility of blood-based and neuroimaging assessments primarily in hospital settings,¹⁶¹ and the ACRM added neuroimaging and laboratory findings (e.g., blood-based biomarker data) to the recently updated diagnostic algorithm for mTBI.²¹⁶ Similarly, the NINDS recently held a workshop to discuss evidence on the use of biomarker data and other clinical features to improve the characterization of TBI, concluding that the available evidence supports the use of blood-based and neuroimaging during the initial evaluation of individuals with suspected TBI.²¹⁷ These efforts will likely accelerate the use and understanding of multi-modal biomarker assessments for TBI, which could allow precision medicine for TBI by promoting an individualized approach to identifying the specific symptoms and pathologies associated with injury in each patient. These advancements may also shift how mTBIs, as well as subconcussive head trauma, are clinically defined and understood in the future as evidence of their long-term effects continues to emerge.

Conclusion

Increased understanding of the dynamic outcomes associated with mTBI and subconcussive head impacts has sparked fervent efforts to improve TBI evaluation through the identification of reliable biomarkers. It is now well known that TBI is not a singular event, but a trigger for a dynamic, chronic disease process with pathological features that evolve over time as the brain attempts to recover.²¹⁸ The comprehensive study and validation of biomarker candidates could drastically alter the classification and management of mTBI by enabling the identification of the specific injury features that could be targeted therapeutically in individual patients as they arise during disease progression. The blood biomarkers UCHL1, GFAP, and S100β have already received widespread acceptance as tools for identifying mTBI patients likely to exhibit intracranial pathology on head CT, and their inclusion in mTBI evaluation guidelines will likely promote the collection of more data on these markers for future studies on their other possible uses. With further study, other promising biomarkers will likely also become components of routine mTBI assessment and monitoring in the future.

The combined analysis of multiple biomarker types at specific time-points and in appropriate patient populations following mTBI could allow determination of optimal windows for treatment to prevent the progression of mTBI symptoms.²¹⁹ This process will involve establishing clear and robust guidelines on the biomarkers most indicative of specific mTBI endophenotypes and outcomes.²²⁰ In addition, if certain biomarkers are consistently associated with a specific TBI severity, a threshold may be established. A universal threshold for subconcussive head impacts, for example, could be used to warn athletic trainers, military leaders, or medical providers that an individual is approaching a grievous level of exposure, and this information could be used to identify preventative measures and appropriate treatment as warranted. Collectively, biomarker studies suggest that combined analysis of biofluid, imaging, physiological, and functional features of mTBI could help achieve great progress in mTBI management.

Authors’ Contributions

All authors made substantial contributions to the conceptualization, literature search, interpretation, writing, and approval of the article.

Transparency, Rigor, and Reproducibility Summary

This review does not report primary data.

Footnotes

Author Disclosure Statement

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding Information

This work was supported by the Traumatic Brain Injury Center of Excellence. The views expressed in this article are those of the authors and do not necessarily represent the official policy or position of the Defense Health Agency, Department of Defense, or any other U.S. government agency. This work was prepared under Contract HT0014-22-C-0016 with DHA Contracting Office (NM-CD) HT0014 and, therefore, is defined as U.S. Government work under Title 17 U.S.C.§101. Per Title 17 U.S.C.§105, copyright protection is not available for any work of the U.S. Government. For more information, please contact dha.TBICOEinfo@health.mil. UNCLASSIFIED

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