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.

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.

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.

Serum Protein Biomarker Findings Reflective of Oxidative Stress and Vascular Abnormalities in Male, but Not Female, Collision Sport Athletes.

Studies have indicated that concussive and sub-concussive brain injuries that are frequent during collision sports may lead to long-term neurological abnormalities, however there is a knowledge gap on how biological sex modifies outcomes. Blood-based biomarkers can help to identify the molecular pathology induced by brain injuries and to better understand how biological sex affects the molecular changes. We therefore analyzed serum protein biomarkers in male (n = 50) and female (n = 33) amateur Australian rules footballers (i.e., Australia's most participated collision sport), both with a history of concussion (HoC) and without a history of concussion (NoHoC). These profiles were compared to those of age-matched control male (n = 24) and female (n = 20) athletes with no history of neurotrauma or participation in collision sports. Serum levels of protein markers indicative of neuronal, axonal and glial injury (UCH-L1, NfL, tau, p-tau, GFAP, BLBP, PEA15), metabolic (4-HNE) and vascular changes (VEGF-A, vWF, CLDN5), and inflammation (HMGB1) were assessed using reverse phase protein microarrays. Male, but not female, footballers had increased serum levels of VEGF-A compared to controls regardless of concussion history. In addition, only male footballers who had HoC had increased serum levels of 4-HNE. These findings being restricted to males may be related to shorter collision sport career lengths for females compared to males. In summary, these findings show that male Australian rules footballers have elevated levels of serum biomarkers indicative of vascular abnormalities (VEGF-A) and oxidative stress (4-HNE) in comparison to non-collision control athletes. While future studies are required to determine how these findings relate to neurological function, serum levels of VEGF-A and 4-HNE may be useful to monitor subclinical neurological injury in males participating in collision sports.

Comparisons between the ON- and OFF-edge motion pathways in the Drosophila brain.

Understanding the circuit mechanisms behind motion detection is a long-standing question in visual neuroscience. In Drosophila melanogaster, recently discovered synapse-level connectomes in the optic lobe, particularly in ON-pathway (T4) receptive-field circuits, in concert with physiological studies, suggest a motion model that is increasingly intricate when compared with the ubiquitous Hassenstein-Reichardt model. By contrast, our knowledge of OFF-pathway (T5) has been incomplete. Here, we present a conclusive and comprehensive connectome that, for the first time, integrates detailed connectivity information for inputs to both the T4 and T5 pathways in a single EM dataset covering the entire optic lobe. With novel reconstruction methods using automated synapse prediction suited to such a large connectome, we successfully corroborate previous findings in the T4 pathway and comprehensively identify inputs and receptive fields for T5. Although the two pathways are probably evolutionarily linked and exhibit many similarities, we uncover interesting differences and interactions that may underlie their distinct functional properties.

Harmonization of pipeline for preclinical multicenter plasma protein and miRNA biomarker discovery in a rat model of post-traumatic epileptogenesis.

The Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is an international, multicenter, multidisciplinary study aimed at preventing epileptogenesis (EpiBioS4Rx: https://epibios.loni.usc.edu/). One of the study's major objectives is the discovery of diagnostic, prognostic, and predictive plasma protein and microRNA (miRNA) biomarkers that are sensitive, specific, and translatable to the human condition. Epilepsy due to structural brain abnormalities, secondary to neurological insults such as traumatic brain injury (TBI), currently represents ∼50% of all epilepsy cases. In the preclinical EpiBioS4Rx study, TBI was induced in adult male Sprague Dawley rats using a standardized protocol for lateral fluid-percussion injury. Whole blood was collected from the tail vein at baseline and 2, 9 and 30 days post-injury and processed for plasma separation. Biomaterial properties, sample preparation and integrity, and choice of analysis platform can significantly impact measured marker levels and, in turn, interpretation with respect to injury and/or other variables. We present here the results of procedural harmonization for the first 320 rats included in the EpiBioS4Rx study study, from three international research centers, and preliminary proteomic and miRNA analyses. We also discuss experimental considerations for establishing rigorous quality controls with the goal of harmonizing operating procedures across study sites, and delivering high-quality specimens for preclinical biomarker discovery in a rat model of post-traumatic epilepsy (PTE).

A connectome of a learning and memory center in the adult Drosophila brain.

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB's α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall.

Synaptic circuits and their variations within different columns in the visual system of Drosophila.

We reconstructed the synaptic circuits of seven columns in the second neuropil or medulla behind the fly's compound eye. These neurons embody some of the most stereotyped circuits in one of the most miniaturized of animal brains. The reconstructions allow us, for the first time to our knowledge, to study variations between circuits in the medulla's neighboring columns. This variation in the number of synapses and the types of their synaptic partners has previously been little addressed because methods that visualize multiple circuits have not resolved detailed connections, and existing connectomic studies, which can see such connections, have not so far examined multiple reconstructions of the same circuit. Here, we address the omission by comparing the circuits common to all seven columns to assess variation in their connection strengths and the resultant rates of several different and distinct types of connection error. Error rates reveal that, overall, <1% of contacts are not part of a consensus circuit, and we classify those contacts that supplement (E+) or are missing from it (E-). Autapses, in which the same cell is both presynaptic and postsynaptic at the same synapse, are occasionally seen; two cells in particular, Dm9 and Mi1, form ≥ 20-fold more autapses than do other neurons. These results delimit the accuracy of developmental events that establish and normally maintain synaptic circuits with such precision, and thereby address the operation of such circuits. They also establish a precedent for error rates that will be required in the new science of connectomics.