Structural and functional connectivity of the whole brain and subnetworks in individuals with mild traumatic brain injury:predictors of patient prognosis

Patients with mild traumatic brain injury have a diverse clinical presentation,and the underlying pathophysiology remains poorly understood.Magnetic resonance imaging is a non-invasive technique that has been widely utilized to investigate neuro biological markers after mild traumatic brain injury.This approach has emerged as a promising tool for investigating the pathogenesis of mild traumatic brain injury.G raph theory is a quantitative method of analyzing complex networks that has been widely used to study changes in brain structure and function.However,most previous mild traumatic brain injury studies using graph theory have focused on specific populations,with limited exploration of simultaneous abnormalities in structural and functional connectivity.Given that mild traumatic brain injury is the most common type of traumatic brain injury encounte red in clinical practice,further investigation of the patient characteristics and evolution of structural and functional connectivity is critical.In the present study,we explored whether abnormal structural and functional connectivity in the acute phase could serve as indicators of longitudinal changes in imaging data and cognitive function in patients with mild traumatic brain injury.In this longitudinal study,we enrolled 46 patients with mild traumatic brain injury who were assessed within 2 wee ks of injury,as well as 36 healthy controls.Resting-state functional magnetic resonance imaging and diffusion-weighted imaging data were acquired for graph theoretical network analysis.In the acute phase,patients with mild traumatic brain injury demonstrated reduced structural connectivity in the dorsal attention network.More than 3 months of followup data revealed signs of recovery in structural and functional connectivity,as well as cognitive function,in 22 out of the 46 patients.Furthermore,better cognitive function was associated with more efficient networks.Finally,our data indicated that small-worldness in the acute stage could serve as a predictor of longitudinal changes in connectivity in patients with mild traumatic brain injury.These findings highlight the importance of integrating structural and functional connectivity in unde rstanding the occurrence and evolution of mild traumatic brain injury.Additionally,exploratory analysis based on subnetworks could serve a predictive function in the prognosis of patients with mild traumatic brain injury.

Exosomes derived from microglia overexpressing miR-124-3p alleviate neuronal endoplasmic reticulum stress damage after repetitive mild traumatic brain injury

We previously reported that miR-124-3p is markedly upregulated in microglia-derived exosomes following repetitive mild traumatic brain injury.However,its impact on neuronal endoplasmic reticulum stress following repetitive mild traumatic brain injury remains unclear.In this study,we first used an HT22 scratch injury model to mimic traumatic brain injury,then co-cultured the HT22 cells with BV2 microglia expressing high levels of miR-124-3p.We found that exosomes containing high levels of miR-124-3p attenuated apoptosis and endoplasmic reticulum stress.Furthermore,luciferase reporter assay analysis confirmed that miR-124-3p bound specifically to the endoplasmic reticulum stress-related protein IRE1α,while an IRE1αfunctional salvage experiment confirmed that miR-124-3p targeted IRE1αand reduced its expression,thereby inhibiting endoplasmic reticulum stress in injured neurons.Finally,we delivered microglia-derived exosomes containing miR-124-3p intranasally to a mouse model of repetitive mild traumatic brain injury and found that endoplasmic reticulum stress and apoptosis levels in hippocampal neurons were significantly reduced.These findings suggest that,after repetitive mild traumatic brain injury,miR-124-3 can be transferred from microglia-derived exosomes to injured neurons,where it exerts a neuroprotective effect by inhibiting endoplasmic reticulum stress.Therefore,microglia-derived exosomes containing miR-124-3p may represent a novel therapeutic strategy for repetitive mild traumatic brain injury.

Quantitative proteomic and phosphoproteomic analyses of the hippocampus reveal the involvement of NMDAR1 signaling in repetitive mild traumatic brain injury

The cumulative damage caused by repetitive mild traumatic brain injury can cause long-term neurodegeneration leading to cognitive impairment.This cognitive impairment is thought to result specifically from damage to the hippocampus.In this study,we detected cognitive impairment in mice 6 weeks after repetitive mild traumatic brain injury using the novel object recognition test and the Morris water maze test.Immunofluorescence staining showed that p-tau expression was increased in the hippocampus after repetitive mild traumatic brain injury.Golgi staining showed a significant decrease in the total density of neuronal dendritic spines in the hippocampus,as well as in the density of mature dendritic spines.To investigate the specific molecular mechanisms underlying cognitive impairment due to hippocampal damage,we performed proteomic and phosphoproteomic analyses of the hippocampus with and without repetitive mild traumatic brain injury.The differentially expressed proteins were mainly enriched in inflammation,immunity,and coagulation,suggesting that non-neuronal cells are involved in the pathological changes that occur in the hippocampus in the chronic stage after repetitive mild traumatic brain injury.In contrast,differentially expressed phosphorylated proteins were mainly enriched in pathways related to neuronal function and structure,which is more consistent with neurodegeneration.We identified N-methyl-D-aspartate receptor 1 as a hub molecule involved in the response to repetitive mild traumatic brain injury,and western blotting showed that,while N-methyl-D-aspartate receptor 1 expression was not altered in the hippocampus after repetitive mild traumatic brain injury,its phosphorylation level was significantly increased,which is consistent with the omics results.Administration of GRP78608,an N-methyl-D-aspartate receptor 1 antagonist,to the hippocampus markedly improved repetitive mild traumatic brain injury-induced cognitive impairment.In conclusion,our findings suggest that N-methyl-D-aspartate receptor 1 signaling in the hippocampus is involved in cognitive impairment in the chronic stage after repetitive mild traumatic brain injury and may be a potential target for intervention and treatment.

Severe bilateral anterior cingulum injury in patients with mild traumatic brain injury

The cingulum,the neural tract connecting the orbitofrontal cortex with the medial temporal lobe,plays an important role in cognition（Bush et al.,2000）.It is also important in memory because it provides cholinergic innervations to the cerebral cortex after obtaining innervation from the medial septal nucleus,the vertical nucleus of the diagonal band, and the nucleus basalis of Meynert via the medial cholinergic pathway （Nieuwenhuys et al., 2008; Naidich and Duvernoy, 2009; Hong and Jang, 2010a）.

Pulsed arterial spin labeling effectively and dynamically observes changes in cerebral blood flow after mild traumatic brain injury

Cerebral blood flow is strongly associated with brain function, and is the main symptom and diagnostic basis for a variety of encephalopathies. However, changes in cerebral blood flow after mild traumatic brain injury remain poorly understood. This study sought to observe changes in cerebral blood flow in different regions after mild traumatic brain injury using pulsed arterial spin labeling. Our results demonstrate maximal cerebral blood flow in gray matter and minimal in the white matter of patients with mild traumatic brain injury. At the acute and subacute stages, cerebral blood flow was reduced in the occipital lobe, parietal lobe, central region, subcutaneous region, and frontal lobe. Cerebral blood flow was restored at the chronic stage. At the acute, subacute, and chronic stages, changes in cerebral blood flow were not apparent in the insula. Cerebral blood flow in the temporal lobe and limbic lobe diminished at the acute and subacute stages, but was restored at the chronic stage. These findings suggest that pulsed arterial spin labeling can precisely measure cerebral blood flow in various brain regions, and may play a reference role in evaluating a patient＇s condition and judging prognosis after traumatic brain injury.

Diffusion tensor tractography characteristics of axonal injury in concussion/mild traumatic brain injury

The main advantage of diffusion tensor tractography is that it allows the entire neural tract to be evaluated.In addition,configurational analysis of reconstructed neural tracts can indicate abnormalities such as tearing,narrowing,or discontinuations,which have been used to identify axonal injury of neural tracts in concussion patients.This review focuses on the characteristic features of axonal injury in concussion or mild traumatic brain injury(m TBI)patients through the use of diffusion tensor tractography.Axonal injury in concussion(m TBI)patients is characterized by their occurrence in long neural tracts and multiple injuries,and these characteristics are common in patients with diffuse axonal injury and in concussion(m TBI)patients with axonal injury.However,the discontinuation of the corticospinal tract is mostly observed in diffuse axonal injury,and partial tearing and narrowing in the subcortical white matter are frequently observed in concussion(m TBI)patients with axonal injury.This difference appears to be attributed to the observation that axonal injury in concussion(m TBI)patients is the result of weaker forces than those producing diffuse axonal injuries.In addition,regarding the fornix,in diffuse axonal injury,discontinuation of the fornical crus has been frequently reported,but in concussion(m TBI)patients,many collateral branches form in the fornix in addition to these findings in many case studies.It is presumed that the impact on the brain in TBI is relatively weaker than that in diffuse axonal injury,and that the formation of collateral branches occurs during the fornix recovery process.Although the occurrence of axonal injury in multiple areas of the brain is an important feature of diffuse axonal injury,case studies in concussion(m TBI)have shown that axonal injury occurs in multiple neural tracts.Because axonal injury lesions in m TBI patients may persist for approximately 10 years after injury onset,the characteristics of axonal injury in concussion(m TBI)patients,which are reviewed and categorized in this review,are expected to serve as useful supplementary information in the diagnosis of axonal injury in concussion(m TBI)patients.

Positron emission tomography imaging for the assessment of mild traumatic brain injury and chronic traumatic encephalopathy:recent advances in radiotracers

A chronic phase following repetitive mild traumatic brain injury can present as chronic traumatic encephalopathy in some cases,which requires a neuropathological examination to make a definitive diagnosis.Positron emission tomography(PET)is a molecular imaging modality that has high sensitivity for detecting even very small molecular changes,and can be used to quantitatively measure a range of molecular biological processes in the brain using different radioactive tracers.Functional changes have also been reported in patients with different forms of traumatic brain injury,especially mild traumatic brain injury and subsequent chronic traumatic encephalopathy.Thus,PET provides a novel approach for the further evaluation of mild traumatic brain injury at molecular levels.In this review,we discuss the recent advances in PET imaging with different radiotracers,including radioligands for PET imaging of glucose metabolism,tau,amyloid-beta,γ-aminobutyric acid type A receptors,and neuroinflammation,in the identification of altered neurological function.These novel radiolabeled ligands are likely to have widespread clinical application,and may be helpful for the treatment of mild traumatic brain injury.Moreover,PET functional imaging with different ligands can be used in the future to perform largescale and sequential studies exploring the time-dependent changes that occur in mild traumatic brain injury.

Experimental repetitive mild traumatic brain injury induces deficits in trabecular bone microarchitecture and strength in mice

To evaluate the long-term consequence of repetitive mild traumatic brain injury （mTBI） on bone, mTBI was induced in 10-week-old female C57BL/6J mice using a weight drop model, once per day for 4 consecutive days at different drop heights （0.5, 1 and 1.5 m） and the skeletal phenotype was evaluated at different time points after the impact. In vivo micro-CT （μ-CT） analysis of the tibial metaphysis at 2, 8 and 12 weeks after the impact revealed a 5%-32% reduction in trabecular bone mass. Histomorphometric analyses showed a reduced bone formation rate in the secondary spongiosa ofl.5 m impacted mice at 12 weeks post impact. Apparent modulus （bone strength）, was reduced by 30% （P 〈 0.05） at the proximal tibial metaphysis in the 1.5 m drop height group at 2 and 8 weeks post impact. Ex vivo μ-CT analysis of the fifth lumbar vertebra revealed a significant reduction in trabecular bone mass at 12 weeks of age in all three drop height groups. Serum levels of osteocalcin were decreased by 22%, 15%, and 19% in the 0.5, 1.0 and 1.5 m drop height groups, respectively, at 2 weeks post impact. Serum IGF-I levels were reduced by 18%-32% in mTBI mice compared to control mice at 2 weeks post impact. Serum osteocalcin and IGF-I levels correlated with trabecular BV/TV （r2 = 0.14 and 0.16, P 〈 0.05）. In conclusion, repetitive mTBI exerts significant negative effects on the trabecular bone microarchitecture and bone mechanical properties by influencing osteoblast function via reduced endocrine IGF-I actions.

Quetiapine augmentation of prolonged exposure therapy in veterans with PTSD and a history of mild traumatic brain injury: design and methodology of a pilot study

Background: Selective serotonergic reuptake inhibitors(SSRIs) are first-line pharmacologic treatments for patients with posttraumatic stress disorder(PTSD), but must be given over extended period of time before the onset of action. The use of SSRIs in PTSD patients with mild traumatic brain injury(m TBI) is problematic since SSRIs could exacerbate post-concussion syndrome(PCS) symptoms. VA/DOD guidelines identify trauma-focused psychotherapy as the best evidence-based treatment for PTSD, but overall effectiveness is limited by reduced levels of patient engagement and retention. A previous study from this research group suggested that quetiapine monotherapy, but not risperidone or valproate, could increase engagement in trauma-focused psychotherapy.Methods: We report the study protocol of a pilot study funded under the South-Central Mental Illness Research, Education, and Clinical Center pilot study program from the U.S. Department of Veterans Affairs. This randomized, open-label study was designed to evaluate the feasibility of completing a randomized trial of quetiapine vs. treatment as usual to promote patient engagement in PTSD patients with a history of m TBI.Discussion: We expect that the success of this ongoing study should provide us with the preliminary data necessary to design a full-scale randomized trial. Positive efficacy results in a full-scale trial should inform new VA guidelines for clinical practice by showing that quetiapine-related improvements in patient engagement and retention may be the most effective approach to assure that VA resources achieve the best possible outcome for veterans.Trial registration: NCT04280965.

Weak phonation due to unknown injury of the corticobulbar tract in a patient with mild traumatic brain injury: a diffusion tensor tractography study

In this study,we report on a patient who showed weak phonation following mild traumatic brain injury（TBI）,which was demonstrated by diffusion tensor tractography（DTT）.

The value of neurocognitive testing for acute outcomes after mild traumatic brain injury

Background: Traditionally, neurocognitive testing is performed weeks to months after head injury and is mostly performed on patients who continue to have symptoms or difficulties. In this study, we sought to determine whether these tests, when administered acutely, could assist in predicting short-term outcomes after acute traumatic brain injury(TBI).Methods: This is an IRB-approved prospective study of adult patients who came to the emergency department of our Level-1 trauma center with TBI. Patients were enrolled prospectively after providing written informed consent and underwent three separate neurocognitive tests: the Galveston Orientation Amnesia Test(GOAT), the Rivermead PostConcussion Survey Questionnaire(RPCSQ), and the Mini Mental Status Examination(MMSE).Results: A lower GOAT score was significantly associated with hospitalization(P=0.0212) and the development of post-concussion syndrome(PCS) at late follow-up(P=0.0081). A higher RPCSQ score was significantly associated with hospital admission(P=0.0098), re-admission within 30 days of discharge(P=0.0431) and evidence of PCS at early follow-up(P=0.0004). A higher MMSE score was significantly associated with not being admitted to the hospital(P=0.0002) and not returning to the emergency department(ED) within 72 hours of discharge(P=0.0078). Lower MMSE was also significantly associated with bleeding or a fracture on the brain CT(P=0.0431).Conclusions: While neurocognitive testing is not commonly performed in the ED in the setting of acute head injury, it is both feasible and appears to have value in predicting hospital admission and PCS. These data are especially important in terms of helping patients understand what to expect, thus, aiding in their recovery.

Animal model of repetitive mild traumatic brain injury for human traumatic axonal injury and chronic traumatic encephalopathy

Chronic traumatic encephalopathy（CTE）is a chronic neurodegenerative disease featured with tauopathy.CTE is tightly related with repetitive mild traumatic brain injury（m TBI）,which is interchangeably known as concussion（Mc Kee et al.,2009,2013）.This disease is differentiated by neuropathological features from other neurological diseases that involve tau protein aggregation and tangle formation abnormalities like Alzheimer＇s disease （AD）, frontotemporal dementia, and Parkinson- ism linked to chromosome 17 （FTDP-17）.

Post-Concussion Syndrome after a Mild Traumatic Brain Injury: A Minefield for Clinical Practice

In this clinical practice review, the controversies and difficulties managing post concussion symptoms following mild traumatic brain injury are discussed. Based on considerable clinical experience in a designated Concussion Clinic, the authors (a neuropsychologist, a psychiatrist, and a neurologist) review relevant literature and issues for clinical practice, particularly with respect to understanding risk factors for and vulnerability to, development of chronic post-concussion symptoms. We contend it is not just the kind of head that matters but also the kind of complications, the kind of outcomes and the kind of management that can influence injury recovery. Given these complexities, a bio-psychosocial conceptualization of chronic post-concussion syndrome is appropriate. Though understanding is still elusive, management should not be biased by physiogenic or psychogenic aetiological theories for management needs to address patient reported outcomes regardless of underpinning aetiology.

Application of diffusion kurtosis imaging technology in evaluating early mild traumatic brain injury

Objective To explore the value of magnetic resonance diffusion kurtosis imaging in diagnosing early tiny changes of brain tissue after mild traumatic brain injury.Methods A total of 22 patients with mild traumatic brain injury(study group)and 20 healthy subjects(control group)were enrolled in this study,and diffusion kurtosis imaging magnetic resonance scanning was employed in all subjects.fractional anisotropy,fractional anisotropy of kurtosis,mean kurtosis,axial kurtosis and radial kurtosis of diffusion kurtosis imaging parameters in the genu of corpus callosum,splenium corporis callosi,internal capsule,thalamus,putamen,cortex of frontal lobe,temporal lobe and parietal lobe at control group,the injured side and the mirror regions were measured,and the results were compared between the two groups.The receiver operating characteristic curve was used to evaluate the ability of different parameters in diagnosing mild traumatic brain injury.Results Compared with the control group,in the study group fractional anisotropy values of bilateral genu of corpus callosum,splenium corporis callosi,internal capsule and thalamus were significantly reduced,and fractional anisotropy of kurtosis values of bilateral thalamus and putamen were significantly reduced,and the differences were statistically significant(P<0.05).Compared with the control group,in the study group mean kurtosis and axial kurtosis values of bilateral genu of corpus callosum,posterior limb of Internal capsule,thalamus,putamen and cortex of temporal lobe were significantly reduced,while radial kurtosis values in the genu of corpus callosum,thalamus,cortex of frontal lobe,temporal lobe at the injured side were increased,and the differences were statistically significant(P<0.05).Conclusion DKI techniques can sensitively detect the early tiny pathologic changes of cerebral tissue in mild traumatic brain injury patients,which provide more imaging evidence for the clinical early diagnosis treatment and prognosis.

Electroacupuncture improves cognitive function in a rat model of mild traumatic brain injury by regulating the SIRT-1/PGC-1α/mitochondrial pathway

Background:Mild traumatic brain injury(mTBI)is a common neurological trauma that can lead to cognitive impairment.The sirtuin-1(SIRT-1)/peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)pathway has been reported to have neuroprotective effects in rats with craniocerebral injury.We evaluated potential mechanisms underlying electroacupuncture-mediated recovery of cognitive function after mTBI,focusing on the SIRT-1/PGC-1α/mitochondrial pathway.Methods:We included forty 6-week-old male Sprague-Dawley rats in this study.Rats were randomly divided into four groups:controlled cortical impactor(CCI,n=10),sham operation(sham,n=10),electroacupuncture-treated CCI(CCI+EA,n=10),and electroacupuncture-treated sham(sham+EA,n=10)group.Randomization was performed by assigning a random number to each rat and using a random number table.The mTBI rat model was established using a controllable cortical impactor.Electroacupuncture therapy was performed on the back of rats,by inserting acupuncture needles to the specific acupoints and setting appropriate parameters for treatment.We evaluated spatial learning and memory functions with the Morris water maze test.We performed quantitative real-time polymerase chain reaction(qRT-PCR),western blotting,adenosine triphosphate(ATP)determination,and mitochondrial respiratory chain complex I(MRCC I)determination on rat hippocampal tissue.We analyzed SIRT-1/PGC-1α expression levels and the results of mitochondrial function assays,and compared differences between groups using bilateral Student’s t-tests.Results:Compared with the sham group,SIRT-1/PGC-1α expression was downregulated in the hippocampus of CCI group(P<0.01).Although this expression was upregulated following electroacupuncture,it did not reach the levels observed in the sham group(P<0.05).Compared with the sham group,MRCC I and ATP levels in the CCI group were significantly reduced,and increased after electroacupuncture(P<0.01).In the Morris water maze,electroacupuncture reduced the incubation period of rats and increased average speed and number of crossing platforms(P<0.05).Conclusion:Electroacupuncture may improve cognitive function in the mTBI rat model by regulating the SIRT-1/PGC-1α/mitochondrial pathway.

Exploring cerebral structural and functional abnormalities in a mouse model of post-traumatic headache induced by mild traumatic brain injury

Mild traumatic brain injury(mTBI)-induced post-traumatic headache(PTH)is a pressing public health concern and leading cause of disability worldwide.Although PTH is often accompanied by neurological disorders,the exact underlying mechanism remains largely unknown.Identifying potential biomarkers may prompt the diagnosis and development of effective treatments for mTBI-induced PTH.In this study,a mouse model of mTBI-induced PTH was established to investigate its effects on cerebral structure and function during short-term recovery.Results indicated that mice with mTBI-induced PTH exhibited balance deficits during the early post-injury stage.Metabolic kinetics revealed that variations in neurotransmitters were most prominent in the cerebellum,temporal lobe/cortex,and hippocampal regions during the early stages of PTH.Additionally,variations in brain functional activities and connectivity were further detected in the early stage of PTH,particularly in the cerebellum and temporal cortex,suggesting that these regions play central roles in the mechanism underlying PTH.Moreover,our results suggested that GABA and glutamate may serve as potential diagnostic or prognostic biomarkers for PTH.Future studies should explore the specific neural circuits involved in the regulation of PTH by the cerebellum and temporal cortex,with these two regions potentially utilized as targets for non-invasive stimulation in future clinical treatment.

White Matter Changes in Posttraumatic Stress Disorder Following Mild Traumatic Brain Injury： A Prospective Longitudinal Diffusion Tensor Imaging Study

Background： The ability to predict posttraumatic stress disorder （PTSD） is a critical issue in the management of patients with mild traumatic brain injury （mTBI）, as early medical and rehabilitative interventions may reduce the risks of long-term cognitive changes. The aim of the present study was to investigate how diffusion tensor imaging （DTI） metrics changed in the transition from acute to chronic phases in patients with mTBI and whether the alteration relates to the development of PTSD. Methods： Forty-three patients with mTBI and 22 healthy volunteers were investigated. The patients were divided into two groups： successful recovery （SR, n = 22） and poor recovery （PR, n = 21）, based on neurocognitive evaluation at 1 or 6 months after injury. All patients underwent magnetic resonance imaging investigation at acute （within 3 days）, subacute （10-20 days）, and chronic （1-6 months） phases after injury. Group differences of fractional anisotropy （FA） and mean diffusivity （MD） were analyzed using tract-based spatial statistics （TBSS）. The accuracy of DTI metrics for classifying PTSD was estimated using Bayesian discrimination analysis. Results： TBSS showed white matter （WM） abnormalities in various brain regions. In the acute phase, FA values were higher for PR and SR patients than controls （all P 〈 0.05）. In subacute phase, PR patients have higher mean MD than SR and controls （all P 〈 0.05）. In the chronic phase, lower FA and higher MD were observed in PR compared with both SR and control groups （all P 〈 0.05）. PR and SR groups could be discriminated with a sensitivity of 73%, specificity of 78%, and accuracy of 75.56%, in terms of MD value in subacute phase. Conclusions： Patients with mTBI have multiple abnormalities in various WM regions. DTI metrics change over time and provide a potential indicator at subacute stage for PTSD following mTBI.

Nanometer ultrastructural brain damage following low intensity primary blast wave exposure

Blast-induced mild traumatic brain injury（m TBI） is of particular concern among military personnel due to exposure to blast energy during military training and combat.The impact of primary low-intensity blast mediated pathophysiology upon later neurobehavioral disorders has been controversial.Developing a military preclinical blast model to simulate the pathophysiology of human blast injury is an important first step.This article provides an overview of primary blast effects and perspectives of our recent studies demonstrating ultrastructural changes in the brain and behavioral disorders resulting from open-field blast exposures up to 46.6 k Pa using a murine model.The model is scalable and permits exposure to varying magnitudes of primary blast injuries by placing animals at different distances from the blast center or by changing the amount of C4 charge.We here review the implications and future applications and directions of using this animal model to uncover the underlying mechanisms related to primary blast injury.Overall,these studies offer the prospect of enhanced understanding of the pathogenesis of primary low-intensity blast-induced TBI and insights for prevention,diagnosis and treatment of blast induced TBI,particularly m TBI/concussion related to current combat exposures.

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

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