Fluid-Based Protein Biomarkers in Traumatic Brain Injury: The View from the Bedside.

There has been an explosion of research into biofluid (blood, cerebrospinal fluid, CSF)-based protein biomarkers in traumatic brain injury (TBI) over the past decade. The availability of very large datasets, such as CENTRE-TBI and TRACK-TBI, allows for correlation of blood- and CSF-based molecular (protein), radiological (structural) and clinical (physiological) marker data to adverse clinical outcomes. The quality of a given biomarker has often been framed in relation to the predictive power on the outcome quantified from the area under the Receiver Operating Characteristic (ROC) curve. However, this does not in itself provide clinical utility but reflects a statistical association in any given population between one or more variables and clinical outcome. It is not currently established how to incorporate and integrate biofluid-based biomarker data into patient management because there is no standardized role for such data in clinical decision making. We review the current status of biomarker research and discuss how we can integrate existing markers into current clinical practice and what additional biomarkers do we need to improve diagnoses and to guide therapy and to assess treatment efficacy. Furthermore, we argue for employing machine learning (ML) capabilities to integrate the protein biomarker data with other established, routinely used clinical diagnostic tools, to provide the clinician with actionable information to guide medical intervention.

Protein biomarkers of epileptogenicity after traumatic brain injury.

Traumatic brain injury (TBI) is a major risk factor for acquired epilepsy. Post-traumatic epilepsy (PTE) develops over time in up to 50% of patients with severe TBI. PTE is mostly unresponsive to traditional anti-seizure treatments suggesting distinct, injury-induced pathomechanisms in the development of this condition. Moderate and severe TBIs cause significant tissue damage, bleeding, neuron and glia death, as well as axonal, vascular, and metabolic abnormalities. These changes trigger a complex biological response aimed at curtailing the physical damage and restoring homeostasis and functionality. Although a positive correlation exists between the type and severity of TBI and PTE, there is only an incomplete understanding of the time-dependent sequelae of TBI pathobiologies and their role in epileptogenesis. Determining the temporal profile of protein biomarkers in the blood (serum or plasma) and cerebrospinal fluid (CSF) can help to identify pathobiologies underlying the development of PTE, high-risk individuals, and disease modifying therapies. Here we review the pathobiological sequelae of TBI in the context of blood- and CSF-based protein biomarkers, their potential role in epileptogenesis, and discuss future directions aimed at improving the diagnosis and treatment of PTE.

Modelling traumatic brain injury and posttraumatic epilepsy in rodents.

Posttraumatic epilepsy (PTE) is one of the most debilitating and understudied consequences of traumatic brain injury (TBI). It is challenging to study the effects, underlying pathophysiology, biomarkers, and treatment of TBI and PTE purely in human patients for a number of reasons. Rodent models can complement human PTE studies as they allow for the rigorous investigation into the causal relationship between TBI and PTE, the pathophysiological mechanisms of PTE, the validation and implementation of PTE biomarkers, and the assessment of PTE treatments, in a tightly controlled, time- and cost-efficient manner in experimental subjects known to be experiencing epileptogenic processes. This article will review several common rodent models of TBI and/or PTE, including their use in previous studies and discuss their relative strengths, limitations, and avenues for future research to advance our understanding and treatment of PTE.

The epilepsy bioinformatics study for anti-epileptogenic therapy (EpiBioS4Rx) clinical biomarker: Study design and protocol.

The Epilepsy Bioinformatics Study for Anti-epileptogenic Therapy (EpiBioS4Rx) is a longitudinal prospective observational study funded by the National Institute of Health (NIH) to discover and validate observational biomarkers of epileptogenesis after traumatic brain injury (TBI). A multidisciplinary approach has been incorporated to investigate acute electrical, neuroanatomical, and blood biomarkers after TBI that may predict the development of post-traumatic epilepsy (PTE). We plan to enroll 300 moderate-severe TBI patients with a frontal and/or temporal lobe hemorrhagic contusion. Acute evaluation with blood, imaging and electroencephalographic monitoring will be performed and then patients will be tracked for 2 years to determine the incidence of PTE. Validation of selected biomarkers that are discovered in planned animal models will be a principal feature of this work. Specific hypotheses regarding the discovery of biomarkers have been set forth in this study. An international cohort of 13 centers spanning 2 continents will be developed to facilitate this study, and for future interventional studies.

Biofluid biomarkers of traumatic brain injury.

PRIMARY OBJECTIVE: The purpose of this paper is to review the clinical and research utility and applications of blood, cerebrospinal fluid (CSF), and cerebral microdialysis biomarkers in traumatic brain injury (TBI). RESEARCH DESIGN: Not applicable. METHODS AND PROCEDURES: A selective review was performed on these biofluid biomarkers in TBI. MAIN OUTCOME AND RESULTS: Neurofilament heavy chain protein (NF-H), glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCHL1), neuron-specific enolase (NSE), myelin basic protein (MBP), tau, and s100ß blood biomarkers are elevated during the acute phase of severe head trauma but have key limitations in their research and clinical applications to mild TBI (mTBI). CSF biomarkers currently provide the best reflection of the central nervous system (CNS) pathobiological processes in TBI. Both animal and human studies of TBI have demonstrated the importance of serial sampling of biofluids and suggest that CSF biomarkers may be better equipped to characterize both TBI severity and temporal profiles. CONCLUSIONS: The identification of biofluid biomarkers could play a vital role in identifying, diagnosing, and treating the underlying individual pathobiological changes of TBI. CNS-derived exosomes analyzed by ultra-high sensitivity detection methods have the potential to identify blood biomarkers for the range of TBI severity and time course.

The effect of concomitant peripheral injury on traumatic brain injury pathobiology and outcome.

BACKGROUND: Traumatic injuries are physical insults to the body that are prevalent worldwide. Many individuals involved in accidents suffer injuries affecting a number of extremities and organs, otherwise known as multitrauma or polytrauma. Traumatic brain injury is one of the most serious forms of the trauma-induced injuries and is a leading cause of death and long-term disability. Despite over dozens of phase III clinical trials, there are currently no specific treatments known to improve traumatic brain injury outcomes. These failures are in part due to our still poor understanding of the heterogeneous and evolving pathophysiology of traumatic brain injury and how factors such as concomitant extracranial injuries can impact these processes. MAIN BODY: Here, we review the available clinical and pre-clinical studies that have investigated the possible impact of concomitant injuries on traumatic brain injury pathobiology and outcomes. We then list the pathophysiological processes that may interact and affect outcomes and discuss promising areas for future research. Taken together, many of the clinical multitrauma/polytrauma studies discussed in this review suggest that concomitant peripheral injuries may increase the risk of mortality and functional deficits following traumatic brain injury, particularly when severe extracranial injuries are combined with mild to moderate brain injury. In addition, recent animal studies have provided strong evidence that concomitant injuries may increase both peripheral and central inflammatory responses and that structural and functional deficits associated with traumatic brain injury may be exacerbated in multiply injured animals. CONCLUSIONS: The findings of this review suggest that concomitant extracranial injuries are capable of modifying the outcomes and pathobiology of traumatic brain injury, in particular neuroinflammation. Though additional studies are needed to further identify the factors and mechanisms involved in central and peripheral injury interactions following multitrauma and polytrauma, concomitant injuries should be recognized and accounted for in future pre-clinical and clinical traumatic brain injury studies.

Traumatic Brain Injury in the Long-COVID Era.

Major determinants of the biological background or reserve, such as age, biological sex, comorbidities (diabetes, hypertension, obesity, etc.), and medications (e.g., anticoagulants), are known to affect outcome after traumatic brain injury (TBI). With the unparalleled data richness of coronavirus disease 2019 (COVID-19; ∼375,000 and counting!) as well as the chronic form, long-COVID, also called post-acute sequelae SARS-CoV-2 infection (PASC), publications (∼30,000 and counting) covering virtually every aspect of the diseases, pathomechanisms, biomarkers, disease phases, symptomatology, etc., have provided a unique opportunity to better understand and appreciate the holistic nature of diseases, interconnectivity between organ systems, and importance of biological background in modifying disease trajectories and affecting outcomes. Such a holistic approach is badly needed to better understand TBI-induced conditions in their totality. Here, I briefly review what is known about long-COVID/PASC, its underlying-suspected-pathologies, the pathobiological changes induced by TBI, in other words, the TBI endophenotypes, discuss the intersection of long-COVID/PASC and TBI-induced pathobiologies, and how by considering some of the known factors affecting the person's biological background and the inclusion of mechanistic molecular biomarkers can help to improve the clinical management of TBI patients.

Long-term consequences of single and multiple mild blast exposure on select physiological parameters and blood-based biomarkers.

Mild traumatic brain injury (mTBI), especially when it is repeated (rmTBI), can lead to progressive degenerative diseases and lasting neuropsychiatric abnormalities. To better understand the long-term pathobiological changes in mTBI and rmTBI, we exposed rats to single or repeated (5 total; administered on consecutive days) mild blast overpressure, monitored changes in physiological parameters, and determined the plasma levels of select biomarkers at 42 days post injury by proteomics. We unexpectedly found comparable changes in arterial oxygen saturation levels and heart rates of single-injured (SI) and multiple-injured (MI) rats throughout the observation period. Our analyses indicated lasting oxidative stress, vascular abnormalities, and neuronal and glial cell loss in both injured groups. However, MI rats exhibited a relatively more pronounced increase in the plasma levels of most of the tested markers-particularly those associated with inflammation-albeit the differences between the two injured groups were not statistically significant. Our findings indicate that the frequency of blast exposures is an important determinant of the resulting cumulative damage in rmTBI.

Evaluating Targeted Therapeutic Response With Predictive Blood-Based Biomarkers in Patients With Chronic Mild Traumatic Brain Injury.

Chronic consequences of mild traumatic brain injury (mTBI) are heterogeneous, but may be treatable with targeted medical and rehabilitation interventions. A biological signature for the likelihood of response to therapy (i.e., "predictive" biomarkers) would empower personalized medicine post-mTBI. The purpose of this study was to correlate pre-intervention blood biomarker levels and the likelihood of response to targeted interventions for patients with chronic issues attributable to mTBI. Patients with chronic symptoms and/or disorders secondary to mTBI >3 months previous (104 days to 15 years; n = 74) were enrolled. Participants completed pre-intervention assessments of symptom burden, comprehensive clinical evaluation, and blood-based biomarker measurements. Multi-domain targeted interventions for specific symptoms and impairments across a 6-month treatment period were prescribed. Participants completed a follow-up testing after the treatment period. An all-possible model's backward logistic regression was built to identify predictors of improvement in relation to blood biomarker levels before intervention. The minimum clinically important difference (MCID) of the change score (post-intervention subtracted from pre-intervention) for the Post-Concussion Symptom Scale (PCSS) to identify treatment responders from non-responders was the primary outcome. The MCID for total PCSS score was 10. The model to predict change in PCSS score over the 6-month intervention was significant (R2 = 0.09; p = 0.01) and identified ubiquitin C-terminal hydrolase L1 (odds ratio [OR] = 2.53; 95% confidence interval [CI], 1.18-5.46; p = 0.02) and hyperphosphorylated tau (p-tau; OR = 0.70; 95% CI, 0.51-0.96; p = 0.03) as significant predictors of symptom improvement beyond the PCSS MCID. In this cohort of chronic TBI subjects, blood biomarkers before rehabilitation intervention predicted the likelihood of response to targeted therapy for chronic disorders post-TBI.

Time-Dependent Changes in the Biofluid Levels of Neural Injury Markers in Severe Traumatic Brain Injury Patients-Cerebrospinal Fluid and Cerebral Microdialysates: A Longitudinal Prospective Pilot Study.

Monitoring protein biomarker levels in the cerebrospinal fluid (CSF) can help assess injury severity and outcome after traumatic brain injury (TBI). Determining injury-induced changes in the proteome of brain extracellular fluid (bECF) can more closely reflect changes in the brain parenchyma, but bECF is not routinely available. The aim of this pilot study was to compare time-dependent changes of S100 calcium-binding protein B (S100B), neuron-specific enolase (NSE), total Tau, and phosphorylated Tau (p-Tau) levels in matching CSF and bECF samples collected at 1, 3, and 5 days post-injury from severe TBI patients (n = 7; GCS 3-8) using microcapillary-based western analysis. We found that time-dependent changes in CSF and bECF levels were most pronounced for S100B and NSE, but there was substantial patient-to-patient variability. Importantly, the temporal pattern of biomarker changes in CSF and bECF samples showed similar trends. We also detected two different immunoreactive forms of S100B in both CSF and bECF samples, but the contribution of the different immunoreactive forms to total immunoreactivity varied from patient to patient and time point to time point. Our study is limited, but it illustrates the value of both quantitative and qualitative analysis of protein biomarkers and the importance of serial sampling for biofluid analysis after severe TBI.

Association of Plasma Biomarkers with Sleep Outcomes and Treatment Response After Mild Traumatic Brain Injury.

Sleep disturbances occur in up to 70% of patients with mild traumatic brain injury (mTBI). Modern mTBI management recommends targeted treatment for the patient's unique clinical manifestations (i.e., obstructive sleep apnea, insomnia). The purpose of this study was to evaluate the association of plasma biomarkers with symptom reports, overnight sleep evaluations, and response to treatment for sleep disturbances secondary to mTBI. This study is a secondary analysis of a prospective multiple interventional trial of patients with chronic issues related to mTBI. Pre- and post-intervention assessments were conducted, including overnight sleep apnea evaluation, the Pittsburgh Sleep Quality Index (PSQI), and blinded analysis of blood biomarkers. Bivariate Spearman correlations were conducted for pre-intervention plasma biomarker concentrations and 1) PSQI change scores and 2) pre-intervention sleep apnea outcomes (i.e., oxygen saturation measures). A backward logistic regression model was built to evaluate the association of pre-intervention plasma biomarkers with improvement in PSQI over the treatment period (p < 0.05). Participants were 36.3 ± 8.6 years old and 6.1 ± 3.8 years from their index mTBI. Participants reported subjective improvements (PSQI = -3.7 ± 3.8), whereas 39.3% (n = 11) had improved PSQI scores beyond the minimum clinically important difference (MCID). PSQI change scores correlated with von Willebrand factor (vWF; ρ = -0.50; p = 0.02) and tau (ρ = -0.53; p = 0.01). Hyperphosphorylated tau correlated with average saturation (ρ = -0.29; p = 0.03), lowest desaturation (ρ = -0.27; p = 0.048), and baseline saturation (ρ = -0.31; p = 0.02). The multi-variate model (R 2 = 0.33; p = 0.001) retained only pre-intervention vWF as a predictor (odds ratio = 3.41; 95% confidence interval, 1.44-8.08; p = 0.005) of improving PSQI scores beyond the MCID. vWF had good discrimination (area under the curve = 0.83; p = 0.01), with an overall accuracy of 77%, sensitivity of 46.2%, and specificity of 90.0%. Validation of vWF as a potential predictive biomarker of sleep improvement post-mTBI could optimize personalized management and healthcare utilization.

Early preclinical plasma protein biomarkers of brain trauma are influenced by early seizures and levetiracetam.

OBJECTIVE: We used the lateral fluid percussion injury (LFPI) model of moderate-to-severe traumatic brain injury (TBI) to identify early plasma biomarkers predicting injury, early post-traumatic seizures or neuromotor functional recovery (neuroscores), considering the effect of levetiracetam, which is commonly given after severe TBI. METHODS: Adult male Sprague-Dawley rats underwent left parietal LFPI, received levetiracetam (200 mg/kg bolus, 200 mg/kg/day subcutaneously for 7 days [7d]) or vehicle post-LFPI, and were continuously video-EEG recorded (n = 14/group). Sham (craniotomy only, n = 6), and naïve controls (n = 10) were also used. Neuroscores and plasma collection were done at 2d or 7d post-LFPI or equivalent timepoints in sham/naïve. Plasma protein biomarker levels were determined by reverse phase protein microarray and classified according to injury severity (LFPI vs. sham/control), levetiracetam treatment, early seizures, and 2d-to-7d neuroscore recovery, using machine learning. RESULTS: Low 2d plasma levels of Thr231 -phosphorylated tau protein (pTAU-Thr231 ) and S100B combined (ROC AUC = 0.7790) predicted prior craniotomy surgery (diagnostic biomarker). Levetiracetam-treated LFPI rats were differentiated from vehicle treated by the 2d-HMGB1, 2d-pTAU-Thr231 , and 2d-UCHL1 plasma levels combined (ROC AUC = 0.9394) (pharmacodynamic biomarker). Levetiracetam prevented the seizure effects on two biomarkers that predicted early seizures only among vehicle-treated LFPI rats: pTAU-Thr231 (ROC AUC = 1) and UCHL1 (ROC AUC = 0.8333) (prognostic biomarker of early seizures among vehicle-treated LFPI rats). Levetiracetam-resistant early seizures were predicted by high 2d-IFNγ plasma levels (ROC AUC = 0.8750) (response biomarker). 2d-to-7d neuroscore recovery was best predicted by higher 2d-S100B, lower 2d-HMGB1, and 2d-to-7d increase in HMGB1 or decrease in TNF (P < 0.05) (prognostic biomarkers). SIGNIFICANCE: Antiseizure medications and early seizures need to be considered in the interpretation of early post-traumatic biomarkers.

Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex.

Pyramidal neurons of the neocortex can be subdivided into two major groups: deep- (DL) and upper-layer (UL) neurons. Here we report that the expression of the AT-rich DNA-binding protein Satb2 defines two subclasses of UL neurons: UL1 (Satb2 positive) and UL2 (Satb2 negative). In the absence of Satb2, UL1 neurons lose their identity and activate DL- and UL2-specific genetic programs. UL1 neurons in Satb2 mutants fail to migrate to superficial layers and do not contribute to the corpus callosum but to the corticospinal tract, which is normally populated by DL axons. Ctip2, a gene required for the formation of the corticospinal tract, is ectopically expressed in all UL1 neurons in the absence of Satb2. Satb2 protein interacts with the Ctip2 genomic region and controls chromatin remodeling at this locus. Satb2 therefore is required for the initiation of the UL1-specific genetic program and for the inactivation of DL- and UL2-specific genes.

Time-dependent changes of protein biomarker levels in the cerebrospinal fluid after blast traumatic brain injury.

Time-dependent changes of protein biomarkers in the cerebrospinal fluid (CSF) can be used to identify the pathological processes in traumatic brain injury (TBI) as well as to follow the progression of the disease. We obtained CSF from a large animal model (swine) of blast-induced traumatic brain injury prior to and at 6, 24, 72 h, and 2 wk after a single exposure to blast overpressure, and determined changes in the CSF levels of neurofilament-heavy chain, neuron-specific enolase, brain-specific creatine kinase, glial fibrillary acidic protein, calcium-binding protein ß (S100ß), Claudin-5, vascular endothelial growth factor, and von Willebrand factor using reverse phase protein microarray. We detected biphasic temporal patterns in the CSF concentrations of all tested protein markers except S100ß. The CSF levels of all markers were significantly increased 6 h after the injury compared to preinjury levels. Values were then decreased at 24 h, prior to a second increase in all markers but S100ß at 72 h. At 2 wk postinjury, the CSF concentrations of all biomarkers were decreased once again; brain-specific creatine kinase, Claudin-5, von Willebrand factor, and S100ß levels were no longer significantly higher than their preinjury values while neurofilament-heavy chain, neuron-specific enolase, vascular endothelial growth factor, and glial fibrillary acidic protein levels remained significantly elevated compared to baseline. Our findings implicate neuronal and glial cell damage, compromised vascular permeability, and inflammation in blast-induced traumatic brain injury, as well as demonstrate the value of determining the temporal pattern of biomarker changes that may be of diagnostic value.

Neurobehavioral, cellular, and molecular consequences of single and multiple mild blast exposure.

Mild traumatic brain injury, caused by the exposure to single or repeated blast overpressure, is a principal concern due to its pathological complexity and neurobehavioral similarities with posttraumatic stress disorder. In this study, we exposed rats to a single or multiple (five total; administered on consecutive days) mild blasts, assessed their behavior at 1 and 16 days postinjury) and performed histological and protein analyses of brains and plasma at an early (2 h) and a late (22 days) termination time point. One day postinjury, multiple-injured (MI) rats showed the least general locomotion and the most depression- and anxiety-related behaviors among the experimental groups; there were no such differences at 16 days. However, at the later time point, both injured groups displayed elevated levels of select protein biomarkers. Histology showed significantly increased numbers of TUNEL+ (terminal-deoxy-transferase-mediated dUTP nick-end labeling)-positive cells in the dorsal and ventral hippocampus (DHC and VHC) of both injured groups as early as 2 h after injury. At 22 days, the increase was limited to the VHC of MI animals. Our findings suggest that the exposure to mild blast overpressure triggers early hippocampal cell death as well as neuronal, glial, and vascular damage that likely contribute to significant, albeit transient increases in depression- and anxiety-related behaviors. However, the severity of the observed pathological changes in MI rats failed to support the hypothesized cumulative effect of repeated injury. We infer that at this blast frequency, a potential conditioning phenomenon counteracts with and reduces the extent of subsequent damage in MI rats.

Incorporating Blood Flow in Nerve Injury and Regeneration Assessment.

Peripheral nerve injury is a significant public health challenge, with limited treatment options and potential lifelong impact on function. More than just an intrinsic part of nerve anatomy, the vascular network of nerves impact regeneration, including perfusion for metabolic demands, appropriate signaling and growth factors, and structural scaffolding for Schwann cell and axonal migration. However, the established nerve injury classification paradigm proposed by Sydney Sunderland in 1951 is based solely on hierarchical disruption to gross anatomical nerve structures and lacks further information regarding the state of cellular, metabolic, or inflammatory processes that are critical in determining regenerative outcomes. This review covers the anatomical structure of nerve-associated vasculature, and describes the biological processes that makes these vessels critical to successful end-organ reinnervation after severe nerve injuries. We then propose a theoretical framework that incorporates measurements of blood vessel perfusion and inflammation to unify perspectives on all mechanisms of nerve injury.

Targeting the Cerebrovascular System: Next-Generation Biomarkers and Treatment for Mild Traumatic Brain Injury.

The diagnosis, prognosis, and treatment of mild traumatic brain injuries (mTBIs), such as concussions, are significant unmet medical issues. The kinetic forces that occur in mTBI adversely affect the cerebral vasculature, making cerebrovascular injury (CVI) a pathophysiological hallmark of mTBI. Given the importance of a healthy cerebrovascular system in overall brain function, CVI is likely to contribute to neurological dysfunction after mTBI. As such, CVI and related pathomechanisms may provide objective biomarkers and therapeutic targets to improve the clinical management and outcomes of mTBI. Despite this potential, until recently, few studies have focused on the cerebral vasculature in this context. This article will begin by providing a brief overview of the cerebrovascular system followed by a review of the literature regarding how mTBI can affect the integrity and function of the cerebrovascular system, and how this may ultimately contribute to neurological dysfunction and neurodegenerative conditions. We then discuss promising avenues of research related to mTBI biomarkers and interventions that target CVI, and conclude that a clinical approach that takes CVI into account could result in substantial improvements in the care and outcomes of patients with mTBI.

Blood-Based Biomarkers of Repetitive, Subconcussive Blast Overpressure Exposure in the Training Environment: A Pilot Study.

Because of their unknown long-term effects, repeated mild traumatic brain injuries (TBIs), including the low, subconcussive ones, represent a specific challenge to healthcare systems. It has been hypothesized that they can have a cumulative effect, and they may cause molecular changes that can lead to chronic degenerative processes. Military personnel are especially vulnerable to consequences of subconcussive TBIs because their training involves repeated exposures to mild explosive blasts. In this pilot study, we collected blood samples at baseline, 6 h, 24 h, 72 h, 2 weeks, and 3 months after heavy weapons training from students and instructors who were exposed to repeated subconcussive blasts. Samples were analyzed using the reverse and forward phase protein microarray platforms. We detected elevated serum levels of glial fibrillary acidic protein, ubiquitin C-terminal hydrolase L1 (UCH-L1), nicotinic alpha 7 subunit (CHRNA7), occludin (OCLN), claudin-5 (CLDN5), matrix metalloprotease 9 (MMP9), and intereukin-6 (IL-6). Importantly, serum levels of most of the tested protein biomarkers were the highest at 3 months after exposures. We also detected elevated autoantibody titers of proteins related to vascular and neuroglia-specific proteins at 3 months after exposures as compared to baseline levels. These findings suggest that repeated exposures to subconcussive blasts can induce molecular changes indicating not only neuron and glia damage, but also vascular changes and inflammation that are detectable for at least 3 months after exposures whereas elevated titers of autoantibodies against vascular and neuroglia-specific proteins can indicate an autoimmune process.

Serum Protein Biomarkers of Inflammation, Oxidative Stress, and Cerebrovascular and Glial Injury in Concussed Australian Football Players.

Clinical decisions related to sports-related concussion (SRC) are challenging, because of the heterogenous nature of SRC symptoms coupled with the current reliance on subjective self-reported symptom measures. Sensitive and objective methods that can diagnose SRC and determine recovery would aid clinical management, and there is evidence that SRC induces changes in circulating protein biomarkers, indicative of neuroaxonal injury. However, potential blood biomarkers related to other pathobiological responses linked to SRC are still poorly understood. Therefore, here we analyzed blood samples from concussed (male = 30; female = 9) and non-concussed (male = 74; female = 27) amateur Australian rules football players collected during the pre-season (i.e., baseline), and at 2, 6, and 13 days post-SRC to determine time-dependent changes in serum levels of biomarkers related to glial (i.e., brain lipid-binding protein [BLBP]; phosphoprotein enriched in astrocytes 15) and cerebrovascular injury (i.e., von Willebrand factor, claudin-5), inflammation (i.e., fibrinogen, high mobility group box protein 1), and oxidative stress (i.e., 4-hydroxynoneal). In females, BLBP levels were significantly decreased at 2 days post-SRC compared with their pre-season baseline; however, area under the receiver operating characteristic curve (AUROC) analysis found that BLBP was unable to distinguish between SRC and controls. In males, AUROC analysis revealed a statistically significant change at 2 days post-SRC in the serum levels of 4-hydroxynoneal, however the associated AUROC value (0.6373) indicated little clinical utility for this biomarker in distinguishing SRC from controls. There were no other statistically significant findings. These results indicate that the serum biomarkers tested in this study hold little clinical value in the management of SRC at 2, 6, and 13 days post-injury.

The Known Unknowns: An Overview of the State of Blood-Based Protein Biomarkers of Mild Traumatic Brain Injury.

Blood-based protein biomarkers have revolutionized several fields of medicine by enabling molecular level diagnosis, as well as monitoring disease progression and treatment efficacy. Traumatic brain injury (TBI) so far has benefitted only moderately from using protein biomarkers to improve injury outcome. Because of its complexity and dynamic nature, TBI, especially its most prevalent mild form (mild TBI; mTBI), presents unique challenges toward protein biomarker discovery and validation given that blood is frequently obtained and processed outside of the clinical laboratory (e.g., athletic fields, battlefield) under variable conditions. As it stands, the field of mTBI blood biomarkers faces a number of outstanding questions. Do elevated blood levels of currently used biomarkers-ubiquitin carboxy-terminal hydrolase L1, glial fibrillary acidic protein, neurofilament light chain, and tau/p-tau-truly mirror the extent of parenchymal damage? Do these different proteins represent distinct injury mechanisms? Is the blood-brain barrier a "brick wall"? What is the relationship between intra- versus extracranial values? Does prolonged elevation of blood levels reflect de novo release or extended protein half-lives? Does biological sex affect the pathobiological responses after mTBI and thus blood levels of protein biomarkers? At the practical level, it is unknown how pre-analytical variables-sample collection, preparation, handling, and stability-affect the quality and reliability of biomarker data. The ever-increasing sensitivity of assay systems and lack of quality control of samples, combined with the almost complete reliance on antibody-based assay platforms, represent important unsolved issues given that false-negative results can lead to false clinical decision making and adverse outcomes. This article serves as a commentary on the state of mTBI biomarkers and the landscape of significant challenges. We highlight and discusses several biological and methodological "known unknowns" and close with some practical recommendations.