Parallel Cerebrospinal Fluid and Serum Temporal Profile Assessment of Axonal Injury Biomarkers Neurofilament-Light Chain and Phosphorylated Neurofilament-Heavy Chain: Associations With Patient Outcome in Moderate-Severe Traumatic Brain Injury.

Neurofilament-light chain (NF-L) and phosphorylated neurofilament-heavy chain (pNF-H) are axonal proteins that have been reported as potential diagnostic and prognostic biomarkers in traumatic brain injury (TBI). However, detailed temporal profiles for these proteins in blood, and interrelationships in the acute and chronic time periods post-TBI have not been established. Our objectives were: 1) to characterize acute-to-chronic serum NF-L and pNF-H profiles after moderate-severe TBI, as well as acute cerebrospinal fluid (CSF) levels; 2) to evaluate CSF and serum NF-L and pNF-H associations with each other; and 3) to assess biomarker associations with global patient outcome using both the Glasgow Outcome Scale-Extended (GOS-E) and Disability Rating Scale (DRS). In this multi-cohort study, we measured serum and CSF NF-L and pNF-H levels in samples collected from two clinical cohorts (University of Pittsburgh [UPITT] and Baylor College of Medicine [BCM]) of individuals with moderate-severe TBI. The UPITT cohort includes 279 subjects from an observational cohort study; we obtained serum (n = 277 unique subjects) and CSF (n = 95 unique subjects) daily for 1 week, and serum every 2 weeks for 6 months. The BCM cohort included 103 subjects from a previous randomized clinical trial of erythropoietin and blood transfusion threshold after severe TBI, which showed no effect on neurological outcome between treatment arms; serum (n = 99 unique subjects) and CSF (n = 54 unique subjects) NF-L and pNF-H levels were measured at least daily during Days (D) 0-10 post-injury. GOS-E and DRS were assessed at 6 months (both cohorts) and 12 months (UPITT cohort only). Results show serum NF-L and pNF-H gradually rise during the first 10 days and peak at D20-30 post-injury. In the UPITT cohort, acute (D0-6) NF-L and pNF-H levels correlate within CSF and serum (Spearman r = 0.44-0.48; p < 0.05). In the UPITT cohort, acute NF-L CSF and serum levels, as well as chronic (Months [M]2-6) serum NF-L levels, were higher among individuals with unfavorable GOS-E and worse DRS at 12 months (p < 0.05, all comparisons). In the BCM cohort, higher acute serum NF-L levels were also associated with unfavorable GOS-E. Higher pNF-H serum concentrations (D0-6 and M2-6), but not CSF pNF-H, were associated with unfavorable GOS-E and worse DRS (p < 0.05, all comparisons) in the UPITT cohort. Relationships between biomarker levels and favorable outcome persisted after controlling for age, sex, and Glasgow Coma Scale. This study shows for the first time that serum levels of NF-L and pNF-H peak at D20-30 post-TBI. Serum NF-L levels, and to a lesser extent pNF-H levels, are robustly associated with global patient outcomes and disability after moderate-severe TBI. Further studies on clinical utility as prognosis and treatment-response indicators are needed.

Aptamer Technologies in Neuroscience, Neuro-Diagnostics and Neuro-Medicine Development.

Aptamers developed using in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology are single-stranded nucleic acids 10-100 nucleotides in length. Their targets, often with specificity and high affinity, range from ions and small molecules to proteins and other biological molecules as well as larger systems, including cells, tissues, and animals. Aptamers often rival conventional antibodies with improved performance, due to aptamers' unique biophysical and biochemical properties, including small size, synthetic accessibility, facile modification, low production cost, and low immunogenicity. Therefore, there is sustained interest in engineering and adapting aptamers for many applications, including diagnostics and therapeutics. Recently, aptamers have shown promise as early diagnostic biomarkers and in precision medicine for neurodegenerative and neurological diseases. Here, we critically review neuro-targeting aptamers and their potential applications in neuroscience research, neuro-diagnostics, and neuro-medicine. We also discuss challenges that must be overcome, including delivery across the blood-brain barrier, increased affinity, and improved in vivo stability and in vivo pharmacokinetic properties.

Isolated Traumatic Subarachnoid Hemorrhage on Head Computed Tomography Scan May Not Be Isolated: A TRACK-TBI Study.

Isolated traumatic subarachnoid hemorrhage (tSAH) after traumatic brain injury (TBI) on head computed tomography (CT) scan is often regarded as a "mild" injury, with reduced need for additional workup. However, tSAH is also a predictor of incomplete recovery and unfavorable outcome. This study aimed to evaluate the characteristics of CT-occult intracranial injuries on brain magnetic resonance imaging (MRI) scan in TBI patients with emergency department (ED) arrival Glasgow Coma Scale (GCS) score 13-15 and isolated tSAH on CT. The prospective, 18-center Transforming Research and Clinical Knowledge in Traumatic Brain Injury Study (TRACK-TBI; enrollment years 2014-2019) enrolled participants who presented to the ED and received a clinically-indicated head CT within 24 hours (h) of TBI. A subset of TRACK-TBI participants underwent venipuncture within 24h for plasma glial fibrillary acidic protein (GFAP) analysis, and research MRI at 2-weeks post-injury. In the current study, TRACK-TBI participants aged ≥17 years with ED arrival GCS 13-15, isolated tSAH on initial head CT, plasma GFAP level, and 2-week MRI data were analyzed. In 57 participants, median age was 46.0 years [quartile 1 to 3 (Q1-Q3): 34-57] and 52.6% were male. At ED disposition, 12.3% were discharged home, 61.4% were admitted to hospital ward, and 26.3% to intensive care unit. MRI identified CT-occult traumatic intracranial lesions in 45.6% (26 of 57 participants; 1 additional lesion type: 31.6%; 2 additional lesion types: 14.0%); of these 26 participants with CT-occult intracranial lesions, 65.4% had axonal injury, 42.3% had subdural hematoma, and 23.1% had intracerebral contusion. GFAP levels were higher in participants with CT-occult MRI lesions compared to without (median: 630.6 pg/ml, Q1-Q3: [172.4-941.2] vs. 226.4 [105.8-436.1], p=0.049), and were associated with axonal injury (no: median 226.7 pg/ml [109.6-435.1], yes: 828.6 pg/ml [204.0-1194.3], p=0.009). Our results indicate that isolated tSAH on head CT is often not the sole intracranial traumatic injury in GCS 13-15 TBI. Forty-six percent of patients in our cohort (26 of 57 participants) had additional CT-occult traumatic lesions on MRI. Plasma GFAP may be an important biomarker for the identification of additional CT-occult injuries, including axonal injury. These findings should be interpreted cautiously given our modest sample size and await validation from larger studies.

Diagnostic Utility of Glial Fibrillary Acidic Protein Beyond 12 Hours After Traumatic Brain Injury: A TRACK-TBI Study.

Blood levels of glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) within 12h of suspected traumatic brain injury (TBI) have been approved by the Food and Drug administration to aid in determining the need for a brain computed tomography (CT) scan. The current study aimed to determine whether this context of use can be expanded beyond 12h post-TBI in patients presenting with Glasgow Coma Scale (GCS) 13-15. The prospective, 18-center Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) study enrolled TBI participants aged ≥17 years who presented to a United States Level 1 trauma center and received a clinically indicated brain CT scan within 24h post-injury, a blood draw within 24h and at 14 days for biomarker analysis. Data from participants with emergency department arrival GCS 13-15 and biomarker values at days 1 and 14 were extracted for the primary analysis. A subgroup of hospitalized participants with serial biomarkers at days 1, 3, 5, and 14 were analyzed, including plasma GFAP and UCH-L1, and serum neuron-specific enolase (NSE) and S100 calcium-binding protein B (S100B). The primary analysis compared biomarker values dichotomized by head CT results (CT+/CT-). Area under receiver-operating characteristic curve (AUC) was used to determine diagnostic accuracy. The overall cohort included 1142 participants with initial GCS 13-15, with mean age 39.8 years, 65% male, and 73% Caucasian. The GFAP provided good discrimination in the overall cohort at days 1 (AUC = 0.82) and 14 (AUC = 0.72), and in the hospitalized subgroup at days 1 (AUC = 0.84), 3 (AUC = 0.88), 5 (AUC = 0.82), and 14 (AUC = 0.74). The UCH-L1, NSE, and S100B did not perform well (AUC = 0.51-0.57 across time points). This study demonstrates the utility of GFAP to aid in decision-making for diagnostic brain CT imaging beyond the 12h time frame in patients with TBI who have a GCS 13-15.

Functional connectivity, tissue microstructure and T2 at 11.1 Tesla distinguishes neuroadaptive differences in two traumatic brain injury models in rats: A Translational Outcomes Project in NeuroTrauma (TOP-NT) UG3 phase study.

The damage caused by contusive traumatic brain injuries (TBIs) is thought to involve breakdown in neuronal communication through focal and diffuse axonal injury along with alterations to the neuronal chemical environment, which adversely affects neuronal networks beyond the injury epicenter(s). In the present study, functional connectivity along with brain tissue microstructure coupled with T2 relaxometry were assessed in two experimental TBI models in rat, controlled cortical impact (CCI) and lateral fluid percussive injury (LFPI). Rats were scanned on an 11.1 Tesla scanner on days 2 and 30 following either CCI or LFPI. Naive controls were scanned once and used as a baseline comparison for both TBI groups. Scanning included functional magnetic resonance imaging (fMRI), diffusion weighted images (DWI), and multi-echo T2 images. fMRI scans were analyzed for functional connectivity across laterally and medially located region of interests (ROIs) across the cortical mantle, hippocampus, and dorsal striatum. DWI scans were processed to generate maps of fractional anisotropy, mean, axial, and radial diffusivities (FA, MD, AD, RD). The analyses focused on cortical and white matter (WM) regions at or near the TBI epicenter. Our results indicate that rats exposed to CCI and LFPI had significantly increased contralateral intra-cortical connectivity at 2 days post-injury. This was observed across similar areas of the cortex in both groups. The increased contralateral connectivity was still observed by day 30 in CCI, but not LFPI rats. Although both CCI and LFPI had changes in WM and cortical FA and diffusivities, WM changes were most predominant in CCI and cortical changes in LFPI. Our results provide support for the use of multimodal MR imaging for different types of contusive and skull-penetrating injury.

Lifelong Chronic Sleep Disruption in a Mouse Model of Traumatic Brain Injury.

Chronic sleep/wake disturbances (SWDs) are strongly associated with traumatic brain injury (TBI) in patients and are being increasingly recognized. However, the underlying mechanisms are largely understudied and there is an urgent need for animal models of lifelong SWDs. The objective of this study was to develop a chronic TBI rodent model and investigate the lifelong chronic effect of TBI on sleep/wake behavior. We performed repetitive midline fluid percussion injury (rmFPI) in 4-month-old mice and monitored their sleep/wake behavior using the non-invasive PiezoSleep system. Sleep/wake states were recorded before injury (baseline) and then monthly thereafter. We found that TBI mice displayed a significant decrease in sleep duration in both the light and dark phases, beginning at 3 months post-TBI and continuing throughout the study. Consistent with the sleep phenotype, these TBI mice showed circadian locomotor activity phenotypes and exhibited reduced anxiety-like behavior. TBI mice also gained less weight, and had less lean mass and total body water content, compared to sham controls. Further, TBI mice showed extensive brain tissue loss and increased glial fibrillary acidic protein and ionized calcium-binding adaptor molecule 1 levels in the hypothalamus and vicinity of the injury, indicative of chronic neuropathology. In summary, our study identified a critical time window of TBI pathology and associated circadian and sleep/wake phenotypes. Future studies should leverage this mouse model to investigate the molecular mechanisms underlying the chronic sleep/wake phenotypes post-TBI early in life.

Serial Measurements of Serum Glial Fibrillary Acidic Protein in Moderate-Severe Traumatic Brain Injury: Potential Utility in Providing Insights into Secondary Insults and Long-Term Outcome.

In patients with traumatic brain injury (TBI), serum biomarkers may have utility in assessing the evolution of secondary brain injury. A panel of nine brain-injury- associated biomarkers was measured in archived serum samples over 10 days post-injury from 100 patients with moderate-severe TBI. Among the biomarkers evaluated, serum glial fibrillary acidic protein (GFAP) had the strongest associations with summary measures of acute pathophysiology, including intracranial pressure (ICP), cerebral perfusion pressure (CPP), and brain tissue pO2 (PbtO2). Group based trajectory (TRAJ) analysis was used to identify three distinct GFAP subgroups. The low TRAJ group (n = 23) had peak levels of 9.4 + 1.2 ng/mL that declined rapidly. The middle TRAJ group (n = 48) had higher peak values (31.5 + 5.0 ng/mL) and a slower decline over time. The high TRAJ group (n = 26) had very high, sustained peak values (59.6 + 12.5 ng/mL) that even rose among some patients over 10 days. Patients in the high TRAJ group had significantly higher mortality rate than patients in low and middle TRAJ groups (26.9% vs. 7.0%, p = 0.028). The frequency of poor neurological outcome (Glasgow Outcome Score Extended [GOS-E] 1-4) was 88.5% in the high TRAJ group, 54.2% in the middle TRAJ group, and 30.4% in the low TRAJ group (p < 0.001). ICP was highest in the high TRAJ group (median 17.6 mm Hg), compared with 14.4 mmHg in the low and 15.9 mm Hg in middle TRAJ groups (p = 0.002). High TRAJ patients spent the longest time with ICP >25 mm Hg, median 23 h, compared with 2 and 6 h in the low and middle TRAJ groups (p = 0.006), and the longest time with ICP >30 mm Hg, median 5 h, compared with 0 and 1 h in the low and middle TRAJ groups, respectively (p = 0.013). High TRAJ group patients more commonly required tier 2 or 3 treatment to control ICP. The high TRAJ group had the longest duration when CPP was <50 mm Hg (p = 0.007), and PbtO2 was <10 mm Hg (p = 0.002). Logistical regression was used to study the relationship between temporal serum GFAP patterns and 6-month GOS-E. Here, the low and middle TRAJ groups were combined to form a low-risk group, and the high TRAJ group was designated the high-risk group. High TRAJ group patients had a greater chance of a poor 6-month GOS-E (p < 0.0001). When adjusting for baseline injury characteristics, GFAP TRAJ group membership remained associated with GOS-E (p = 0.003). When an intensive care unit (ICU) injury burden score, developed to quantify physiological derangements, was added to the model, GFAP TRAJ group membership remained associated with GOS-E (p = 0.014). Mediation analysis suggested that ICU burden scores were in the causal pathway between TRAJ group and 6-month mortality or GOS-E. Our results suggest that GFAP may be useful to monitor serially in moderate-severe TBI patients. Future studies in larger cohorts are needed to confirm these results.

Temporal Profiles of P-Tau, T-Tau, and P-Tau:Tau Ratios in Cerebrospinal Fluid and Blood from Moderate-Severe Traumatic Brain Injury Patients and Relationship to 6-12 Month Global Outcomes.

Traumatic brain injury (TBI) can initiate progressive injury responses, which are linked to increased risk of neurodegenerative diseases known as "tauopathies." Increased post-TBI tau hyperphosphorylation has been reported in brain tissue and biofluids. Acute-to-chronic TBI total (T)-tau and phosphorylated (P)-tau temporal profiles in the cerebrospinal fluid (CSF) and serum and their relationship to global outcome is unknown. Our multi-site longitudinal study examines these concurrent profiles acutely (CSF and serum) and also characterizes the acute- to-chronic serum patterns. Serial serum and CSF samples from individuals with moderate-to-severe TBI were obtained from two cohorts (acute, subacute, and chronic samples from University of Pittsburgh [UPitt] [n = 286 unique subjects] and acute samples from Baylor College of Medicine [BCM] [n = 114 unique subjects]) and assayed for T-tau and P-tau using the Rolling Circle Amplification-Surround Optical Fiber ImmunoAssay platform. Biokinetic analyses described serum T-tau and P-tau temporal patterns. T-tau and P-tau levels are compared with those in healthy controls (n = 89 for both CSF and serum), and univariate/multivariable associations are made with global outcome, including the Disability Rating Scale (DRS) and the Glasgow Outcome Scale-Extended (GOS-E) scores at 3 and 6 months post-TBI (BCM cohort) and at 6 and 12 months post-TBI (UPitt cohort). For both the UPitt and BCM cohorts, temporal increases in median serum and CSF T-tau and P-tau levels occurred over the first 5 days post-injury, while the initial increases of P-tau:T-tau ratio plateaued by day 4 post-injury (UPitt: n = 99, BCM: n = 48). Biokinetic analyses with UPitt data showed novel findings that T-tau (n = 74) and P-tau (n = 87) reached delayed maximum levels at 4.5 and 5.1 days, while exhibiting long serum half-lives (152 and 123 days), respectively. The post-TBI rise in acute (days 2-6) serum P-tau (up to 276-fold) far outpaced that of T-tau (7.3-fold), leading to a P-tau:T-tau increase of up to 267-fold, suggesting a shift toward tau hyperphosphorylation. BCM analyses showed that days 0-6 mean CSF T-tau and P-tau levels and P-tau:T-tau ratios were associated with greater disability (DRS) (n = 48) and worse global outcome (GOS-E) (n = 48) 6 months post-injury. Days 0-6 mean serum T-tau, P-tau, and P-tau:T-tau ratio were not associated with outcome in either cohort (UPitt: n = 145 [DRS], n = 154 [GOS-E], BCM: n = 99 [DRS and GOS-E]). UPitt multivariate models showed that higher chronic (months 1-6) mean P-tau levels and P-tau:T-tau ratio, but not T-tau levels, are associated with greater disability (DRS: n = 119) and worse global outcomes (GOS-E: n = 117) 12 months post-injury. This work shows the potential importance of monitoring post-TBI T-tau and P-tau levels over time. This multi-site longitudinal study features concurrent acute TBI T-tau and P-tau profiles in CSF and serum, and also characterizes acute-to-chronic serum profiles. Longitudinal profiles, along with no temporal concordance between trajectory groups over time, imply a sustained post-TBI shift in tau phosphorylation dynamics that may favor tauopathy development chronically.

Screening Performance of S100 Calcium-Binding Protein B, Glial Fibrillary Acidic Protein, and Ubiquitin C-Terminal Hydrolase L1 for Intracranial Injury Within Six Hours of Injury and Beyond.

The Scandinavian NeuroTrauma Committee (SNC) guidelines recommend S100 calcium-binding protein B (S100B) as a screening tool for early detection of Traumatic brain injury (TBI) in patients presenting with an initial Glasgow Coma Scale (GCS) of 14-15. The objective of the current study was to compare S100B's diagnostic performance within the recommended 6-h window after injury, compared with glial fibrillary acidic protein (GFAP) and UCH-L1. The secondary outcome of interest was the ability of these biomarkers in detecting traumatic intracranial pathology beyond the 6-h mark. The Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) core database (2014-2017) was queried for data pertaining to all TBI patients with an initial GCS of 14-15 who had a blood sample taken within 6 h of injury in which the levels of S100B, GFAP, and UCH-L1 were measured. As a subgroup analysis, data involving patients with blood samples taken within 6-9 h and 9-12 h were analyzed separately for diagnostic ability. The diagnostic ability of these biomarkers for detecting any intracranial injury was evaluated based on the area under the receiver operating characteristic curve (AUC). Each biomarker's sensitivity, specificity, and accuracy were also reported at the cutoff that maximized Youden's index. A total of 531 TBI patients with GCS 14-15 on admission had a blood sample taken within 6 h, of whom 24.9% (n = 132) had radiologically confirmed intracranial injury. The AUCs of GFAP (0.86, 95% confidence interval [CI]: 0.82-0.90) and UCH-L1 (0.81, 95% CI: 0.76-0.85) were statistically significantly higher than that of S100B (0.74, 95% CI: 0.69-0.79) during this time. There was no statistically significant difference in the predictive ability of S100B when sampled within 6 h, 6-9 h, and 9-12 h of injury, as the p values were >0.05 when comparing the AUCs. Overlapping AUC 95% CI suggests no benefit of a combined GFAP and UCH-L1 screening tool over GFAP during the time periods studied [0.87 (0.83-0.90) vs. 0.86 (0.82-0.90) when sampled within 6 h of injury, 0.83 (0.78-0.88) vs. 0.83 (0.78-0.89) within 6 to 9 h and 0.81 (0.73-0.88) vs. 0.79 (0.72-0.87) within 9-12 h]. Targeted analysis of the CENTER-TBI core database, with focus on the patient category for which biomarker testing is recommended by the SNC guidelines, revealed that GFAP and UCH-L1 perform superior to S100B in predicting CT-positive intracranial lesions within 6 h of injury. GFAP continued to exhibit superior predictive ability to S100B during the time periods studied. S100B displayed relatively unaltered screening performance beyond the diagnostic timeline provided by SNC guidelines. These findings suggest the need for a reevaluation of the current SNC TBI guidelines.

Sleep Disruption in a Mouse Model of Chronic Traumatic Brain Injury.

Chronic sleep/wake disturbances are strongly associated with traumatic brain injury (TBI) in patients and are being increasingly recognized. However, the underlying mechanisms are largely understudied and there is an urgent need for animal models of lifelong sleep/wake disturbances. The objective of this study was to develop a chronic TBI rodent model and investigate the lifelong chronic effect of TBI on sleep/wake behavior. We performed repetitive midline fluid percussion injury (rmFPI) in four months old mice and monitored their sleep/wake behavior using the non-invasive PiezoSleep system. The sleep/wake states were recorded before injury (baseline) and then monthly thereafter. We found that TBI mice displayed a significant decrease in sleep duration in both the light and dark phases, beginning at three months post-TBI and continuing throughout the study. Consistent with the sleep phenotype, these TBI mice showed circadian locomotor activity phenotypes and exhibited reduced anxiety-like behavior. TBI mice also gained less weight, and had less lean mass and total body water content, compared to sham controls. Furthermore, TBI mice showed extensive brain tissue loss and increased GFAP and IBA1 levels in the hypothalamus and the vicinity of the injury, indicative of chronic neuropathology. In summary, our study identified a critical time window of TBI pathology and associated circadian and sleep/wake phenotypes. Future studies should leverage this mouse model to investigate the molecular mechanisms underlying the chronic sleep/wake phenotypes following TBI early in life.

Alzheimer's pathology is associated with altered cognition, brain volume, and plasma biomarker patterns in traumatic encephalopathy syndrome.

BACKGROUND: Traumatic encephalopathy syndrome (TES) is a clinical phenotype sensitive but non-specific to underlying chronic traumatic encephalopathy (CTE) neuropathology. However, cognitive symptoms of TES overlap with Alzheimer's disease (AD), and features of AD pathology like beta-amyloid (Aß) plaques often co-occur with CTE, making clinical-to-pathological conclusions of TES diagnoses challenging. We investigated how Alzheimer's neuropathological changes associated with cognition, brain volume, and plasma biomarkers in patients with repetitive head impacts (RHI)/TES, clinical AD, or typically aging controls. METHODS: We studied 154 participants including 33 with RHI/TES (age 61.5 ± 11.5, 100% male, 11/33 Aß[ +]), 62 with AD and no known prior RHI (age 67.1 ± 10.2, 48% male, 62/62 Aß[ +]), and 59 healthy controls without RHI (HC; age 73.0 ± 6.2, 40% male, 0/59 Aß[ +]). Patients completed neuropsychological testing (memory, executive functioning, language, visuospatial) and structural MRI (voxel-based morphometry analysis), and provided plasma samples analyzed for GFAP, NfL, IL-6, IFN-γ, and YKL-40. For cognition and plasma biomarkers, patients with RHI/TES were stratified as Aß[ +] or Aß[ -] and compared to each other plus the AD and HC groups (ANCOVA adjusting for age and sex). Differences with at least a medium effect size (Cohen's d > 0.50) were interpreted as potentially meaningful. RESULTS: Cognitively, within the TES group, Aß[ +] RHI/TES performed worse than Aß[-] RHI/TES on visuospatial (p = .04, d = 0.86) and memory testing (p = .07, d = 0.74). Comparing voxel-wise brain volume, both Aß[ +] and Aß[ -] RHI/TES had lower medial and anterior temporal lobe volume than HC and did not significantly differ from AD. Comparing plasma biomarkers, Aß[ +] RHI/TES had higher plasma GFAP than HC (p = .01, d = 0.88) and did not significantly differ from AD. Conversely, Aß[ -] RHI/TES had higher NfL than HC (p = .004, d = 0.93) and higher IL-6 than all other groups (p's ≤ .004, d's > 1.0). CONCLUSIONS: Presence of Alzheimer's pathology in patients with RHI/TES is associated with altered cognitive and biomarker profiles. Patients with RHI/TES and positive Aß-PET have cognitive and plasma biomarker changes that are more like patients with AD than patients with Aß[ -] RHI/TES. Measuring well-validated Alzheimer's biomarkers in patients with RHI/TES could improve interpretation of research findings and heighten precision in clinical management.

Associations of Microvascular Injury-Related Biomarkers With Traumatic Brain Injury Severity and Outcomes: A Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) Pilot Study.

Traumatic brain injury (TBI) is characterized by heterogeneity in terms of injury severity, mechanism, outcome, and pathophysiology. A single biomarker alone is unlikely to capture the heterogeneity of even one injury subtype, necessitating the use of panels of biomarkers. Herein, we focus on traumatic cerebrovascular injury and investigate associations of a panel of 16 vascular injury-related biomarkers with indices of TBI severity and outcomes using data from 159 participants in the Transforming Research and Clinical Knowledge in TBI (TRACK-TBI) Pilot Study. Associations of individual biomarkers and clusters of biomarkers identified using non-linear principal components analysis with TBI severity and outcomes were assessed using logistic regression models and Spearman's correlations. As individual biomarkers, higher levels of thrombomodulin, angiopoietin (Ang)-2, von Willebrand factor, and P-selectin were associated with more severe injury; higher levels of Ang-1, Tie2, vascular endothelial growth factor (VEGF)-C, and basic fibroblast growth factor (bFGF) were associated with less severe injury (all p < 0.05 in age-adjusted models). After false discovery rate correction for multiple comparisons, higher levels of Ang-2 remained associated with more severe injury and higher levels of Ang-1, Tie2, and bFGF remained associated with less severe injury at a p < 0.05 level. In principal components analysis, principal component (PC)1, comprised of Ang1, bFGF, P-selectin, VEGF-C, VEGF-A, and Tie2, was associated with less severe injury (age-adjusted odds ratio [OR]: 0.63, 95% confidence interval [CI]: 0.44-0.88 for head computer tomography [CT] positive vs. negative) and PC2 (Ang-2, E-selectin, Flt-1, placental growth factor, thrombomodulin, and vascular cell adhesion protein 1) was associated with greater injury severity (age-adjusted OR: 2.29, 95% CI: 1.49-3.69 for Glasgow Coma Scale [GCS] 3-12 vs. 13-15 and age-adjusted OR 1.59, 95% CI: 1.11-2.32 for head CT positive vs. negative). Neither individual biomarkers nor PCs were associated with outcomes in adjusted models (all p > 0.05). In conclusion, in this trauma-center based population of acute TBI patients, biomarkers of microvascular injury were associated with TBI severity.

Neuroinflammatory Biomarkers for Traumatic Brain Injury Diagnosis and Prognosis: A TRACK-TBI Pilot Study.

The relationship between systemic inflammation and secondary injury in traumatic brain injury (TBI) is complex. We investigated associations between inflammatory markers and clinical confirmation of TBI diagnosis and prognosis. The prospective TRACK-TBI Pilot (Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot) study enrolled TBI patients triaged to head computed tomography (CT) and received blood draw within 24 h of injury. Healthy controls (HCs) and orthopedic controls (OCs) were included. Thirty-one inflammatory markers were analyzed from plasma. Area under the receiver operating characteristic curve (AUC) was used to evaluate discriminatory ability. AUC >0.7 was considered acceptable. Criteria included: TBI diagnosis (vs. OC/HC); moderate/severe vs. mild TBI (Glasgow Coma Scale; GCS); radiographic TBI (CT positive vs. CT negative); 3- and 6-month Glasgow Outcome Scale-Extended (GOSE) dichotomized to death/greater relative disability versus less relative disability (GOSE 1-4/5-8); and incomplete versus full recovery (GOSE <8/ = 8). One-hundred sixty TBI subjects, 28 OCs, and 18 HCs were included. Markers discriminating TBI/OC: HMGB-1 (AUC = 0.835), IL-1b (0.795), IL-16 (0.784), IL-7 (0.742), and TARC (0.731). Markers discriminating GCS 3-12/13-15: IL-6 (AUC = 0.747), CRP (0.726), IL-15 (0.720), and SAA (0.716). Markers discriminating CT positive/CT negative: SAA (AUC = 0.767), IL-6 (0.757), CRP (0.733), and IL-15 (0.724). At 3 months, IL-15 (AUC = 0.738) and IL-2 (0.705) discriminated GOSE 5-8/1-4. At 6 months, IL-15 discriminated GOSE 1-4/5-8 (AUC = 0.704) and GOSE <8/ = 8 (0.711); SAA discriminated GOSE 1-4/5-8 (0.704). We identified a profile of acute circulating inflammatory proteins with potential relevance for TBI diagnosis, severity differentiation, and prognosis. IL-15 and serum amyloid A are priority markers with acceptable discrimination across multiple diagnostic and outcome categories. Validation in larger prospective cohorts is needed. ClinicalTrials.gov Registration: NCT01565551.

Neurobiochemical, Peptidomic, and Bioinformatic Approaches to Characterize Tauopathy Peptidome Biomarker Candidates in Experimental Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a multidimensional damage, and currently, no FDA-approved medicine is available. Multiple pathways in the cell are triggered through a head injury (e.g., calpain and caspase activation), which truncate tau and generate variable fragment sizes (MW 400-45,000 K). In this study, we used an open-head TBI mouse model generated by controlled cortical impact (CCI) and collected ipsilateral (IC) and contralateral (CC) mice htau brain cortices at one (D1) three (D3), and seven (D7) days post-injury. We implemented immunological (antibody-based detection) and peptidomic approaches (nano-reversed-phase liquid chromatography/tandem mass spectrometry) to investigate proteolytic tau peptidome (low molecular weight (LMW) < 10 K)) and pathological phosphorylation sites (high-molecular-weight (HMW); > 10 K) derived from CCI-TBI animal models. Our immunoblotting analysis verified tau hyperphosphorylation, HMW, and HMW breakdown products (HMW-BDP) formation of tau (e.g., pSer202, pThr181, pThr231, pSer396, and pSer404), following CCI-TBI. Peptidomic data revealed unique sequences of injury-dependent proteolytic peptides generated from human tau protein. Among the N-terminal tau peptides, EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA (a.a. 96-125) and AQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARM (a.a. 91-127). Examples of tau C-terminal peptides identified include NVSSTGSIDMVDSPQLATLADEVSASLAKQGL (a.a. 410-441) and QLATLADEVSASLAKQGL (a.a. 424-441). Our peptidomic bioinformatic tools showed the association of proteases, such as CAPN1, CAPN2, and CTSL; CASP1, MMP7, and MMP9; and ELANE, GZMA, and MEP1A, in CCI-TBI tau peptidome. In clinical trials for novel TBI treatments, it might be useful to monitor a subset of tau peptidome as targets for biomarker utility and use them for a "theranostic" approach.

Effects of age and time since injury on traumatic brain injury blood biomarkers: a TRACK-TBI study.

Older adults have the highest incidence of traumatic brain injury globally. Accurate blood-based biomarkers are needed to assist with diagnosis of patients across the spectrum of age and time post-injury. Several reports have suggested lower accuracy for blood-based biomarkers in older adults, and there is a paucity of data beyond day-1 post-injury. Our aims were to investigate age-related differences in diagnostic accuracy and 2-week evolution of four leading candidate blood-based traumatic brain injury biomarkers-plasma glial fibrillary acidic protein, ubiquitin carboxy-terminal hydrolase L1, S100 calcium binding protein B and neuron-specific enolase-among participants in the 18-site prospective cohort study Transforming Research And Clinical Knowledge in Traumatic Brain Injury. Day-1 biomarker data were available for 2602 participants including 2151 patients with traumatic brain injury, 242 orthopedic trauma controls and 209 healthy controls. Participants were stratified into 3 age categories (young: 17-39 years, middle-aged: 40-64 years, older: 65-90 years). We investigated age-stratified biomarker levels and biomarker discriminative abilities across three diagnostic groups: head CT-positive/negative; traumatic brain injury/orthopedic controls; and traumatic brain injury/healthy controls. The difference in day-1 glial fibrillary acidic protein, ubiquitin carboxy-terminal hydrolase L1 and neuron-specific enolase levels across most diagnostic groups was significantly smaller for older versus younger adults, resulting in a narrower range within which a traumatic brain injury diagnosis may be discriminated in older adults. Despite this, day-1 glial fibrillary acidic protein had good to excellent performance across all age-categories for discriminating all three diagnostic groups (area under the curve 0.84-0.96; lower limit of 95% confidence intervals all >0.78). Day-1 S100 calcium-binding protein B and ubiquitin carboxy-terminal hydrolase L1 showed good discrimination of CT-positive versus negative only among adults under age 40 years within 6 hours of injury. Longitudinal blood-based biomarker data were available for 522 hospitalized patients with traumatic brain injury and 24 hospitalized orthopaedic controls. Glial fibrillary acidic protein levels maintained good to excellent discrimination across diagnostic groups until day 3 post-injury irrespective of age, until day 5 post-injury among middle-aged or younger patients and until week 2 post-injury among young patients only. In conclusion, the blood-based glial fibrillary acidic protein assay tested here has good to excellent performance across all age-categories for discriminating key traumatic brain injury diagnostic groups to at least 3 days post-injury in this trauma centre cohort. The addition of a blood-based diagnostic to the evaluation of traumatic brain injury, including geriatric traumatic brain injury, has potential to streamline diagnosis.

Characterization of Calpain and Caspase-6-Generated Glial Fibrillary Acidic Protein Breakdown Products Following Traumatic Brain Injury and Astroglial Cell Injury.

Glial fibrillary acidic protein (GFAP) is the major intermediate filament III protein of astroglia cells which is upregulated in traumatic brain injury (TBI). Here we reported that GFAP is truncated at both the C- and N-terminals by cytosolic protease calpain to GFAP breakdown products (GBDP) of 46-40K then 38K following pro-necrotic (A23187) and pro-apoptotic (staurosporine) challenges to primary cultured astroglia or neuron-glia mixed cells. In addition, with another pro-apoptotic challenge (EDTA) where caspases are activated but not calpain, GFAP was fragmented internally, generating a C-terminal GBDP of 20 kDa. Following controlled cortical impact in mice, GBDP of 46-40K and 38K were formed from day 3 to 28 post-injury. Purified GFAP protein treated with calpain-1 and -2 generates (i) major N-terminal cleavage sites at A-56*A-61 and (ii) major C-terminal cleavage sites at T-383*Q-388, producing a limit fragment of 38K. Caspase-6 treated GFAP was cleaved at D-78/R-79 and D-225/A-226, where GFAP was relatively resistant to caspase-3. We also derived a GBDP-38K N-terminal-specific antibody which only labels injured astroglia cell body in both cultured astroglia and mouse cortex and hippocampus after TBI. As a clinical translation, we observed that CSF samples collected from severe human TBI have elevated levels of GBDP-38K as well as two C-terminally released GFAP peptides (DGEVIKES and DGEVIKE). Thus, in addition to intact GFAP, both the GBDP-38K as well as unique GFAP released C-terminal proteolytic peptides species might have the potential in tracking brain injury progression.

Incremental prognostic value of acute serum biomarkers for functional outcome after traumatic brain injury (CENTER-TBI): an observational cohort study.

BACKGROUND: Several studies have reported an association between serum biomarker values and functional outcome following traumatic brain injury. We aimed to examine the incremental (added) prognostic value of serum biomarkers over demographic, clinical, and radiological characteristics and over established prognostic models, such as IMPACT and CRASH, for prediction of functional outcome. METHODS: We used data from the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) core study. We included patients aged 14 years or older who had blood sampling within 24 h of injury, results from a CT scan, and outcome assessment according to the Glasgow Outcome Scale-Extended (GOSE) at 6 months. Amounts in serum of six biomarkers (S100 calcium-binding protein B, neuron-specific enolase, glial fibrillary acidic protein, ubiquitin C-terminal hydrolase L1 [UCH-L1], neurofilament protein-light, and total tau) were measured. The incremental prognostic value of these biomarkers was determined separately and in combination. The primary outcome was the GOSE 6 months after injury. Incremental prognostic value, using proportional odds and a dichotomised analysis, was assessed by delta C-statistic and delta R2 between models with and without serum biomarkers, corrected for optimism with a bootstrapping procedure. FINDINGS: Serum biomarker values and 6-month GOSE were available for 2283 of 4509 patients. Higher biomarker levels were associated with worse outcome. Adding biomarkers improved the C-statistic by 0·014 (95% CI 0·009-0·020) and R2 by 4·9% (3·6-6·5) for predicting GOSE compared with demographic, clinical, and radiological characteristics. UCH-L1 had the greatest incremental prognostic value. Adding biomarkers to established prognostic models resulted in a relative increase in R2 of 48-65% for IMPACT and 30-34% for CRASH prognostic models. INTERPRETATION: Serum biomarkers have incremental prognostic value for functional outcome after traumatic brain injury. Our findings support integration of biomarkers-particularly UCH-L1-in established prognostic models. FUNDING: European Union's Seventh Framework Programme, Hannelore Kohl Stiftung, OneMind, Integra LifeSciences, and NeuroTrauma Sciences.

Prognostic value of day-of-injury plasma GFAP and UCH-L1 concentrations for predicting functional recovery after traumatic brain injury in patients from the US TRACK-TBI cohort: an observational cohort study.

BACKGROUND: The prognostic value of glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1) as day-of-injury predictors of functional outcome after traumatic brain injury is not well understood. GFAP is a protein found in glial cells and UCH-L1 is found in neurons, and these biomarkers have been cleared to aid in decision making regarding whether brain CT should be performed after traumatic brain injury. We aimed to quantify their prognostic accuracy and investigate whether these biomarkers contribute novel prognostic information to existing clinical models. METHODS: We enrolled patients from the Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) observational cohort study. TRACK-TBI includes patients 17 years and older who are evaluated for TBI at 18 US level 1 trauma centres. All patients receive head CT at evaluation, have adequate visual acuity and hearing preinjury, and are fluent in either English or Spanish. In our analysis, we included participants aged 17-90 years who had day-of-injury plasma samples for measurement of GFAP and UCH-L1 and completed 6-month assessments for outcome due to traumatic brain injury with the Glasgow Outcome Scale-Extended (GOSE-TBI). Biomarkers were analysed as continuous variables and in quintiles. This study is registered with ClinicalTrials.gov, NCT02119182. FINDINGS: We enrolled 2552 patients from Feb 26, 2014, to Aug 8, 2018. Of the 1696 participants with brain injury and data available at baseline and at 6 months who were included in the analysis, 120 (7·1%) died (GOSE-TBI=1), 235 (13·9%) had an unfavourable outcome (ie, GOSE-TBI ≤4), 1135 (66·9%) had incomplete recovery (ie, GOSE-TBI <8), and 561 (33·1%) recovered fully (ie, GOSE-TBI=8). The area under the curve (AUC) of GFAP for predicting death at 6 months in all patients was 0·87 (95% CI 0·83-0·91), for unfavourable outcome was 0·86 (0·83-0·89), and for incomplete recovery was 0·62 (0·59-0·64). The corresponding AUCs for UCH-L1 were 0·89 (95% CI 0·86-0·92) for predicting death, 0·86 (0·84-0·89) for unfavourable outcome, and 0·61 (0·59-0·64) for incomplete recovery at 6 months. AUCs were higher for participants with traumatic brain injury and Glasgow Coma Scale (GCS) score of 3-12 than for those with GCS score of 13-15. Among participants with GCS score of 3-12 (n=353), adding GFAP and UCH-L1 (alone or combined) to each of the three International Mission for Prognosis and Analysis of Clinical Trials in traumatic brain injury models significantly increased their AUCs for predicting death (AUC range 0·90-0·94) and unfavourable outcome (AUC range 0·83-0·89). However, among participants with GCS score of 13-15 (n=1297), adding GFAP and UCH-L1 to the UPFRONT study model modestly increased the AUC for predicting incomplete recovery (AUC range 0·69-0·69, p=0·025). INTERPRETATION: In addition to their known diagnostic value, day-of-injury GFAP and UCH-L1 plasma concentrations have good to excellent prognostic value for predicting death and unfavourable outcome, but not for predicting incomplete recovery at 6 months. These biomarkers contribute the most prognostic information for participants presenting with a GCS score of 3-12. FUNDING: US National Institutes of Health, National Institute of Neurologic Disorders and Stroke, US Department of Defense, One Mind, US Army Medical Research and Development Command.