Spatial lipidomics maps brain alterations associated with mild traumatic brain injury.

Traumatic brain injury (TBI) is a global public health problem with 50-60 million incidents per year, most of which are considered mild (mTBI) and many of these repetitive (rmTBI). Despite their massive implications, the pathologies of mTBI and rmTBI are not fully understood, with a paucity of information on brain lipid dysregulation following mild injury event(s). To gain more insight on mTBI and rmTBI pathology, a non-targeted spatial lipidomics workflow utilizing high resolution mass spectrometry imaging was developed to map brain region-specific lipid alterations in rats following injury. Discriminant multivariate models were created for regions of interest including the hippocampus, cortex, and corpus callosum to pinpoint lipid species that differentiated between injured and sham animals. A multivariate model focused on the hippocampus region differentiated injured brain tissues with an area under the curve of 0.99 using only four lipid species. Lipid classes that were consistently discriminant included polyunsaturated fatty acid-containing phosphatidylcholines (PC), lysophosphatidylcholines (LPC), LPC-plasmalogens (LPC-P) and PC potassium adducts. Many of the polyunsaturated fatty acid-containing PC and LPC-P selected have never been previously reported as altered in mTBI. The observed lipid alterations indicate that neuroinflammation and oxidative stress are important pathologies that could serve to explain cognitive deficits associated with rmTBI. Therapeutics which target or attenuate these pathologies may be beneficial to limit persistent damage following a mild brain injury event.

Integrative Analysis of Cytokine and Lipidomics Datasets Following Mild Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is a significant source of disability in the United States and around the world and may lead to long-lasting cognitive deficits and a decreased quality of life for patients across injury severities. Following the primary injury phase, TBI is characterized by complex secondary cascades that involve altered homeostasis and metabolism, faulty signaling, neuroinflammation, and lipid dysfunction. The objectives of the present study were to (1) assess potential correlations between lipidome and cytokine changes after closed-head mild TBI (mTBI), and (2) examine the reproducibility of our acute lipidomic profiles following TBI. Cortices from 54 Sprague Dawley male and female rats were analyzed by ultra-high-performance liquid chromatography mass spectrometry (LC-MS) in both positive and negative ionization modes and multiplex cytokine analysis after single (smTBI) or repetitive (rmTBI) closed-head impacts, or sham conditions. Tissue age was a variable, given that two cohorts (n = 26 and n = 28) were initially run a year-and-a-half apart, creating inter-batch variations. We annotated the lipidome datasets using an in-house data dictionary based on exact masses of precursor and fragment ions and removed features with statistically significant differences between sham control batches. Our results indicate that lipids with high-fold change between injury groups moderately correlate with the cytokines eotaxin, IP-10, and TNF-α. Additionally, we show a significant decrease in the pro-inflammatory markers IL-1ß and IP-10, TNF-α, and RANTES in the rmTBI samples relative to the sham control. We discuss the major challenges in correlating high dimensional lipidomic data with functional cytokine profiles and the implications for understanding the biological significance of two related but disparate analysis modes in the study of TBI, an inherently heterogeneous neurological disorder.

Spatial Lipidomics Maps Brain Alterations Associated with Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) is a global public health problem with 50-60 million incidents per year, most of which are considered mild (mTBI) and many of these repetitive (rmTBI). Despite their massive implications, the pathologies of mTBI and rmTBI are not fully understood, with a paucity of information on brain lipid dysregulation following mild injury event(s). To gain more insight on mTBI and rmTBI pathology, a non-targeted spatial lipidomics workflow utilizing ultrahigh resolution mass spectrometry imaging was developed to map brain region-specific lipid alterations in rats following injury. Discriminant multivariate models were created for regions of interest including the hippocampus, cortex, and corpus callosum to pinpoint lipid species that differentiated between injured and sham animals. A multivariate model focused on the hippocampus region differentiated injured brain tissues with an area under the curve of 0.994 using only four lipid species. Lipid classes that were consistently discriminant included polyunsaturated fatty acid-containing phosphatidylcholines (PC), lysophosphatidylcholines (LPC), LPC-plasmalogens (LPC-P) and PC potassium adducts. Many of the polyunsaturated fatty acid-containing PC and LPC-P selected have never been previously reported as altered in mTBI. The observed lipid alterations indicate that neuroinflammation, oxidative stress and disrupted sodium-potassium pumps are important pathologies that could serve to explain cognitive deficits associated with rmTBI. Therapeutics which target or attenuate these pathologies may be beneficial to limit persistent damage following a mild brain injury event.

Comparing Brain and Blood Lipidome Changes following Single and Repetitive Mild Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is a major health concern in the United States and globally, contributing to disability and long-term neurological problems. Lipid dysregulation after TBI is underexplored, and a better understanding of lipid turnover and degradation could point to novel biomarker candidates and therapeutic targets. Here, we investigated overlapping lipidome changes in the brain and blood using a data-driven discovery approach to understand lipid alterations in the brain and serum compartments acutely following mild TBI (mTBI) and the potential efflux of brain lipids to peripheral blood. The cortices and sera from male and female Sprague-Dawley rats were analyzed via ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) in both positive and negative ion modes following single and repetitive closed head impacts. The overlapping lipids in the data sets were identified with an in-house data dictionary for investigating lipid class changes. MS-based lipid profiling revealed overall increased changes in the serum compartment, while the brain lipids primarily showed decreased changes. Interestingly, there were prominent alterations in the sphingolipid class in the brain and blood compartments after single and repetitive injury, which may suggest efflux of brain sphingolipids into the blood after TBI. Genetic algorithms were used for predictive panel selection to classify injured and control samples with high sensitivity and specificity. These overlapping lipid panels primarily mapped to the glycerophospholipid metabolism pathway with Benjamini-Hochberg adjusted q-values less than 0.05. Collectively, these results detail overlapping lipidome changes following mTBI in the brain and blood compartments, increasing our understanding of TBI-related lipid dysregulation while identifying novel biomarker candidates.

An Automated Virtual Reality Program Accurately Diagnoses HIV-Associated Neurocognitive Disorders in Older People With HIV.

Background: HIV-associated neurocognitive disorders (HANDs) remain prevalent despite antiretroviral therapy, particularly among older people with HIV (PWH). However, the diagnosis of HAND is labor intensive and requires expertise to administer neuropsychological tests. Our prior pilot work established the feasibility and accuracy of a computerized self-administered virtual reality program (DETECT; Display Enhanced Testing for Cognitive Impairment and Traumatic Brain Injury) to measure cognition in younger PWH. The present study expands this to a larger sample of older PWH. Methods: We enrolled PWH who were ≥60 years old, were undergoing antiretroviral therapy, had undetectable plasma viral loads, and were without significant neuropsychological confounds. HAND status was determined via Frascati criteria. Regression models that controlled for demographic differences (age, sex, education, race/ethnicity) examined the association between DETECT's cognition module and both HAND status and Global Deficit Score (GDS) derived via traditional neuropsychological tests. Results: Seventy-nine PWH (mean age, 66 years; 28% women) completed a comprehensive neuropsychological battery and DETECT's cognition module. Twenty-five (32%) had HAND based on the comprehensive battery. A significant correlation was found between the DETECT cognition module and the neuropsychological battery (r = 0.45, P < .001). Furthermore, in two separate regression models, HAND status (b = -0.79, P < .001) and GDS impairment status (b = -0.83, P < .001) significantly predicted DETECT performance. Areas under the curve for DETECT were 0.78 for differentiating participants by HAND status (HAND vs no HAND) and 0.85 for detecting GDS impairment. Conclusions: The DETECT cognition module provides a novel means to identify cognitive impairment in older PWH. As DETECT is fully immersive and self-administered, this virtual reality tool holds promise as a scalable cognitive screening battery.

Lipidome Alterations following Mild Traumatic Brain Injury in the Rat.

Traumatic brain injury (TBI) poses a major health challenge, with tens of millions of new cases reported globally every year. Brain damage resulting from TBI can vary significantly due to factors including injury severity, injury mechanism and exposure to repeated injury events. Therefore, there is need for robust blood biomarkers. Serum from Sprague Dawley rats was collected at several timepoints within 24 h of mild single or repeat closed head impacts. Serum samples were analyzed via ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) in positive and negative ion modes. Known lipid species were identified through matching to in-house tandem MS databases. Lipid biomarkers have a unique potential to serve as objective molecular measures of injury response as they may be liberated to circulation more readily than larger protein markers. Machine learning and feature selection approaches were used to construct lipid panels capable of distinguishing serum from injured and uninjured rats. The best multivariate lipid panels had over 90% cross-validated sensitivity, selectivity, and accuracy. These mapped onto sphingolipid signaling, autophagy, necroptosis and glycerophospholipid metabolism pathways, with Benjamini adjusted p-values less than 0.05. The novel lipid biomarker candidates identified provide insight into the metabolic pathways altered within 24 h of mild TBI.

A Novel Neuropsychological Tool for Immersive Assessment of Concussion and Correlation with Subclinical Head Impacts.

Mild traumatic brain injury (mTBI) remains a diagnostic challenge and therefore strategies for objective assessment of neurological function are key to limiting long-term sequelae. Current assessment methods are not optimal in austere environments such as athletic fields; therefore, we developed an immersive tool, the Display Enhanced Testing for Cognitive Impairment and mTBI (DETECT) platform, for rapid objective neuropsychological (NP) testing. The objectives of this study were to assess the ability of DETECT to accurately identify neurocognitive deficits associated with concussion and evaluate the relationship between neurocognitive measures and subconcussive head impacts. DETECT was used over a single season of two high school and two college football teams. Study participants were instrumented with Riddell Head Impact Telemetry (HIT) sensors and a subset tested with DETECT immediately after confirmed impacts for different combinations of linear and rotational acceleration. A total of 123 athletes were enrolled and completed baseline testing. Twenty-one players were pulled from play for suspected concussion and tested with DETECT. DETECT was 86.7% sensitive (95% confidence interval [CI]: 59.5%, 98.3%) and 66.7% specific (95% CI: 22.3%, 95.7%) in correctly identifying athletes with concussions (15 of 21). Weak but significant correlations were found between complex choice response time (processing speed and divided attention) and both linear (Spearman rank correlation coefficient 0.262, p = 0.02) and rotational (Spearman coefficient 0.254, p = 0.03) acceleration on a subset of 76 players (113 DETECT tests) with no concussion symptoms. This study demonstrates that DETECT confers moderate to high sensitivity in identifying acute cognitive impairment and suggests that football impacts that do not result in concussion may negatively affect cognitive performance immediately following an impact. Specificity, however, was not optimal and points to the need for additional studies across multiple neurological domains. Given the need for more objective concussion screening in triage situations, DETECT may provide a solution for mTBI assessment.

Immuno-suppressive hydrogels enhance allogeneic MSC survival after transplantation in the injured brain.

Traumatic brain injury (TBI) triggers multiple biochemical and cellular processes that exacerbate brain tissue damage through a secondary injury. Therapies that prevent or limit the evolution of secondary injury could significantly reduce the neurological deficits associated with TBI. Mesenchymal stem cell (MSC) transplantation after TBI can ameliorate neurological deficits by modulating inflammation and enhancing the expression of neurotrophic factors. However, transplanted MSCs can be actively rejected by host immune responses, such as those mediated by cytotoxic CD8+ T cells, thereby limiting their therapeutic efficacy. Here, we designed an agarose hydrogel that releases Fas ligand (FasL), a protein that can induce apoptosis of cytotoxic CD8+ T cells. We studied the immunosuppressive effect of this hydrogel near the allogeneic MSC transplantation site and its impact on the survival of transplanted MSCs in the injured brain. Agarose-FasL hydrogels locally reduced the host cytotoxic CD8+ T cell population and enhanced the survival of allogeneic MSCs transplanted near the injury site. Furthermore, the expression of crucial neurotrophic factors was elevated in the injury penumbra, suggesting an enhanced therapeutic effect of MSCs. These results suggest that the development of immunosuppressive hydrogels for stem cell delivery can enhance the benefits of stem cell therapy for TBI.

Pre-Clinical Common Data Elements for Traumatic Brain Injury Research: Progress and Use Cases.

Traumatic brain injury (TBI) is an extremely complex condition due to heterogeneity in injury mechanism, underlying conditions, and secondary injury. Pre-clinical and clinical researchers face challenges with reproducibility that negatively impact translation and therapeutic development for improved TBI patient outcomes. To address this challenge, TBI Pre-clinical Working Groups expanded upon previous efforts and developed common data elements (CDEs) to describe the most frequently used experimental parameters. The working groups created 913 CDEs to describe study metadata, animal characteristics, animal history, injury models, and behavioral tests. Use cases applied a set of commonly used CDEs to address and evaluate the degree of missing data resulting from combining legacy data from different laboratories for two different outcome measures (Morris water maze [MWM]; RotorRod/Rotarod). Data were cleaned and harmonized to Form Structures containing the relevant CDEs and subjected to missing value analysis. For the MWM dataset (358 animals from five studies, 44 CDEs), 50% of the CDEs contained at least one missing value, while for the Rotarod dataset (97 animals from three studies, 48 CDEs), over 60% of CDEs contained at least one missing value. Overall, 35% of values were missing across the MWM dataset, and 33% of values were missing for the Rotarod dataset, demonstrating both the feasibility and the challenge of combining legacy datasets using CDEs. The CDEs and the associated forms created here are available to the broader pre-clinical research community to promote consistent and comprehensive data acquisition, as well as to facilitate data sharing and formation of data repositories. In addition to addressing the challenge of standardization in TBI pre-clinical studies, this effort is intended to bring attention to the discrepancies in assessment and outcome metrics among pre-clinical laboratories and ultimately accelerate translation to clinical research.

3-D multi-electrode arrays detect early spontaneous electrophysiological activity in 3-D neuronal-astrocytic co-cultures.

Three-dimensional (3-D) neural cultures represent a promising platform for studying disease and drug screening. Tools and methodologies for measuring the electrophysiological function in these cultures are needed. Therefore, the purpose of this work was primarily to develop a methodology to interface engineered 3-D dissociated neural cultures with commercially available 3-D multi-electrode arrays (MEAs) reliably over 3 weeks to enable the recording of their electrophysiological activity. We further compared the functional output of these cultures to their structural and synaptic network development over time. We reliably interfaced a primary rodent neuron-astrocyte (2:1) 3-D co-culture (2500 cells/mm3 plating cell density) in Matrigel™ (7.5 mg/mL) that was up to 750 µm thick (30-40 cell-layers) with spiked 3-D MEAs while maintaining high viability. Using these MEAs we successfully recorded the spontaneous development of neural network-level electrophysiological activity and measured the development of putative synapses and neuronal maturation in these co-cultures using immunocytochemistry over 3 weeks in vitro. Planar (2-D) MEAs interfaced with these cultures served as recording controls. Neurons within this interfaced 3-D culture-MEA system exhibited considerable neurite outgrowth, networking, neuronal maturation, synaptogenesis, and culture-wide spontaneous firing of synchronized spikes and bursts of action potentials. Network-wide spikes and synchronized bursts increased rapidly (first detected at 2 days) during the first week in culture, plateaued during the second week, and reduced slightly in the third week, while maintaining high viability throughout the 3-week culturing period. Early electrophysiology activity occurred prior to neuronal process maturation and significant synaptic density increases in the second week. We successfully interfaced 3-D neural co-cultures with 3-D MEAs and recorded the electrophysiological activity of these cultures over 3 weeks. The initial period of rapid increase in electrophysiological activity, followed by a period of neuronal maturation and high-level of synapse formation in these cultures suggests a developmental homeostatic process. This methodology can enable future applications both in fundamental investigations of neural network behavior and in translational studies involving drug testing and neural interfacing.

Neuronal Plasma Membrane Integrity is Transiently Disturbed by Traumatic Loading.

The acute response of neurons subjected to traumatic loading involves plasma membrane disruption, yet the mechanical tolerance for membrane compromise, time course, and mechanisms for resealing are not well understood. We have used an in vitro traumatic neuronal injury model to investigate plasma membrane integrity immediately following a high-rate shear injury. Cell-impermeant fluorescent molecules were added to cortical neuronal cultures prior to insult to assess membrane integrity. The percentage of cells containing the permeability marker was dependent on the molecular size of the marker, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were positively correlated with insult loading rate. Membrane disruption was transient, evidenced by a membrane resealing within the first minute after the insult. In addition, chelation of either extracellular Ca2+ or intracellular Ca2+ limited membrane resealing. However, injury following chelation of both extracellular and intracellular Ca2+ caused diminished permeability as well as a greater resealing ability compared to chelation of extracellular or intracellular Ca2+ alone. Treatment of neuronal cultures with jasplakinolide, which stabilizes filamentous actin, reduced permeability increases, while latrunculin-B, an actin depolymerizing agent, both reduced the increase in plasma membrane permeability and promoted resealing. This study gives insight into the dynamics of neuronal membrane disruption and subsequent resealing, which was found to be calcium dependent and involve actin in a role that differs from non-neuronal cells. Taken together, these data will lead to a better understanding of the acute neuronal response to traumatic loading.

Molecular dynamics simulations showing 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) membrane mechanoporation damage under different strain paths.

Continuum finite element material models used for traumatic brain injury lack local injury parameters necessitating nanoscale mechanical injury mechanisms be incorporated. One such mechanism is membrane mechanoporation, which can occur during physical insults and can be devastating to cells, depending on the level of disruption. The current study investigates the strain state dependence of phospholipid bilayer mechanoporation and failure. Using molecular dynamics, a simplified membrane, consisting of 72 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) phospholipids, was subjected to equibiaxial, 2:1 non-equibiaxial, 4:1 non-equibiaxial, strip biaxial, and uniaxial tensile deformations at a von Mises strain rate of 5.45 × 108 s-1, resulting in velocities in the range of 1 to 4.6 m·s-1. A water bridge forming through both phospholipid bilayer leaflets was used to determine structural failure. The stress magnitude, failure strain, headgroup clustering, and damage responses were found to be strain state-dependent. The strain state order of detrimentality in descending order was equibiaxial, 2:1 non-equibiaxial, 4:1 non-equibiaxial, strip biaxial, and uniaxial. The phospholipid bilayer failed at von Mises strains of .46, .47, .53, .77, and 1.67 during these respective strain path simulations. Additionally, a Membrane Failure Limit Diagram (MFLD) was created using the pore nucleation, growth, and failure strains to demonstrate safe and unsafe membrane deformation regions. This MFLD allowed representative equations to be derived to predict membrane failure from in-plane strains. These results provide the basis to implement a more accurate mechano-physiological internal state variable continuum model that captures lower length scale damage and will aid in developing higher fidelity injury models.

Mechanoporation is a potential indicator of tissue strain and subsequent degeneration following experimental traumatic brain injury.

BACKGROUND: An increases in plasma membrane permeability is part of the acute pathology of traumatic brain injury and may be a function of excessive membrane force. This membrane damage, or mechanoporation, allows non-specific flux of ions and other molecules across the plasma membrane, and may ultimately lead to cell death. The relationships among tissue stress and strain, membrane permeability, and subsequent cell degeneration, however, are not fully understood. METHODS: Fluorescent molecules of different sizes were introduced to the cerebrospinal fluid space prior to injury and animals were sacrificed at either 10 min or 24 h after injury. We compared the spatial distribution of plasma membrane damage following controlled cortical impact in the rat to the stress and strain tissue patterns in a 3-D finite element simulation of the injury parameters. FINDINGS: Permeable cells were located primarily in the ipsilateral cortex and hippocampus of injured rats at 10 min post-injury; however by 24 h there was also a significant increase in the number of permeable cells. Analysis of colocalization of permeability marker uptake and Fluorojade staining revealed a subset of permeable cells with signs of degeneration at 24 h, but plasma membrane damage was evident in the vast majority of degenerating cells. The regional and subregional distribution patterns of the maximum principal strain and shear stress estimated by the finite element model were comparable to the cell membrane damage profiles following a compressive impact. INTERPRETATION: These results indicate that acute membrane permeability is prominent following traumatic brain injury in areas that experience high shear or tensile stress and strain due to differential mechanical properties of the cell and tissue organization, and that this mechanoporation may play a role in the initiation of secondary injury, contributing to cell death.

Pre-Clinical Testing of Therapies for Traumatic Brain Injury.

Despite the large number of promising neuroprotective agents identified in experimental traumatic brain injury (TBI) studies, none has yet shown meaningful improvements in long-term outcome in clinical trials. To develop recommendations and guidelines for pre-clinical testing of pharmacological or biological therapies for TBI, the Moody Project for Translational Traumatic Brain Injury Research hosted a symposium attended by investigators with extensive experience in pre-clinical TBI testing. The symposium participants discussed issues related to pre-clinical TBI testing including experimental models, therapy and outcome selection, study design, data analysis, and dissemination. Consensus recommendations included the creation of a manual of standard operating procedures with sufficiently detailed descriptions of modeling and outcome measurement procedures to permit replication. The importance of the selection of clinically relevant outcome variables, especially related to behavior testing, was noted. Considering the heterogeneous nature of human TBI, evidence of therapeutic efficacy in multiple, diverse (e.g., diffuse vs. focused) rodent models and a species with a gyrencephalic brain prior to clinical testing was encouraged. Basing drug doses, times, and routes of administration on pharmacokinetic and pharmacodynamic data in the test species was recommended. Symposium participants agreed that the publication of negative results would reduce costly and unnecessary duplication of unsuccessful experiments. Although some of the recommendations are more relevant to multi-center, multi-investigator collaborations, most are applicable to pre-clinical therapy testing in general. The goal of these consensus guidelines is to increase the likelihood that therapies that improve outcomes in pre-clinical studies will also improve outcomes in TBI patients.

Discovery of Lipidome Alterations Following Traumatic Brain Injury via High-Resolution Metabolomics.

Traumatic brain injury (TBI) can occur across wide segments of the population, presenting in a heterogeneous manner that makes diagnosis inconsistent and management challenging. Biomarkers offer the potential to objectively identify injury status, severity, and phenotype by measuring the relative concentrations of endogenous molecules in readily accessible biofluids. Through a data-driven, discovery approach, novel biomarker candidates for TBI were identified in the serum lipidome of adult male Sprague-Dawley rats in the first week following moderate controlled cortical impact (CCI). Serum samples were analyzed in positive and negative modes by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). A predictive panel for the classification of injured and uninjured sera samples, consisting of 26 dysregulated species belonging to a variety of lipid classes, was developed with a cross-validated accuracy of 85.3% using omniClassifier software to optimize feature selection. Polyunsaturated fatty acids (PUFAs) and PUFA-containing diacylglycerols were found to be upregulated in sera from injured rats, while changes in sphingolipids and other membrane phospholipids were also observed, many of which map to known secondary injury pathways. Overall, the identified biomarker panel offers viable molecular candidates representing lipids that may readily cross the blood-brain barrier (BBB) and aid in the understanding of TBI pathophysiology.

Mining standardized neurological signs and symptoms data for concussion identification.

The Centers for Disease Control estimate that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Studies have shown that concussions increase the risk of future injuries and mild cognitive disorders. Despite extensive research on sports related concussion risk factors, the factors which are most predictive of concussion outcome and recovery time course remain unknown. In order to overcome the issue of physician bias and to identify the factors which can best predict concussion diagnosis, we propose a multi-variate logistic regression based analysis. We demonstrate our results on a dataset with 126 subjects (ages 12-31). Our results indicate that among 322 features, our model selected 27-29 features which included a history of playing sports, history of a previous concussion, drowsiness, nausea, trouble focusing as measured by a common symptom list, and oculomotor function. The features picked using our model were found to be highly predictive of concussions and gave a prediction performance accuracy greater than 90%, Matthews correlation coefficient greater than 0.8 and the area under the curve greater than 0.95.

Bilateral gene interaction hierarchy analysis of the cell death gene response emphasizes the significance of cell cycle genes following unilateral traumatic brain injury.

BACKGROUND: Delayed or secondary cell death that is caused by a cascade of cellular and molecular processes initiated by traumatic brain injury (TBI) may be reduced or prevented if an effective neuroprotective strategy is employed. Microarray and subsequent bioinformatic analyses were used to determine which genes, pathways and networks were significantly altered 24 h after unilateral TBI in the rat. Ipsilateral hemi-brain, the corresponding contralateral hemi-brain, and naïve (control) brain tissue were used for microarray analysis. RESULTS: Ingenuity Pathway Analysis showed cell death and survival (CD) to be a top molecular and cellular function associated with TBI on both sides of the brain. One major finding was that the overall gene expression pattern suggested an increase in CD genes in ipsilateral brain tissue and suppression of CD genes contralateral to the injury which may indicate an endogenous protective mechanism. We created networks of genes of interest (GOI) and ranked the genes by the number of direct connections each had in the GOI networks, creating gene interaction hierarchies (GIHs). Cell cycle was determined from the resultant GIHs to be a significant molecular and cellular function in post-TBI CD gene response. CONCLUSIONS: Cell cycle and apoptosis signalling genes that were highly ranked in the GIHs and exhibited either the inverse ipsilateral/contralateral expression pattern or contralateral suppression were identified and included STAT3, CCND1, CCND2, and BAX. Additional exploration into the remote suppression of CD genes may provide insight into neuroprotective mechanisms that could be used to develop therapies to prevent cell death following TBI.

Randomized, Placebo-Controlled, Double-Blind Pilot Study of D-Cycloserine in Chronic Stroke.

Stroke is a leading cause of death and disability in the USA. Up to 60% of patients do not fully recover despite intensive physical therapy treatment. N-Methyl-D-aspartate receptors (NMDA-R) have been shown to play a role in synaptic plasticity when activated. D-Cycloserine promotes NMDA receptor function by binding to receptors with unoccupied glycine sites. These receptors are involved in learning and memory. We hypothesized that D-cycloserine, when combined with robotic-assisted physiotherapy (RAP), would result in greater gains compared with placebo + RAP in stroke survivors. Participants (n = 14) were randomized to D-cycloserine plus RAP or placebo plus RAP. Functional, cognitive, and quality-of-life measures were used to assess recovery. There was significant improvement in grip strength of the affected hand within both groups from baseline to 3 weeks (95% confidence interval for mean change, 3.95 ± 2.96 to 4.90 ± 3.56 N for D-cycloserine and 5.72 ± 3.98 to 8.44 ± 4.90 N for control). SIS mood domain showed improvement for both groups (95% confidence interval for mean change, 72.6 ± 16.3 to 82.9 ± 10.9 for D-cycloserine and 82.9 ± 13.5 to 90.3 ± 9.9 for control). This preliminary study does not provide evidence that D-cycloserine can provide greater gains in learning compared with placebo for stroke survivors.

A three-dimensional image processing program for accurate, rapid, and semi-automated segmentation of neuronal somata with dense neurite outgrowth.

Three-dimensional (3-D) image analysis techniques provide a powerful means to rapidly and accurately assess complex morphological and functional interactions between neural cells. Current software-based identification methods of neural cells generally fall into two applications: (1) segmentation of cell nuclei in high-density constructs or (2) tracing of cell neurites in single cell investigations. We have developed novel methodologies to permit the systematic identification of populations of neuronal somata possessing rich morphological detail and dense neurite arborization throughout thick tissue or 3-D in vitro constructs. The image analysis incorporates several novel automated features for the discrimination of neurites and somata by initially classifying features in 2-D and merging these classifications into 3-D objects; the 3-D reconstructions automatically identify and adjust for over and under segmentation errors. Additionally, the platform provides for software-assisted error corrections to further minimize error. These features attain very accurate cell boundary identifications to handle a wide range of morphological complexities. We validated these tools using confocal z-stacks from thick 3-D neural constructs where neuronal somata had varying degrees of neurite arborization and complexity, achieving an accuracy of ≥95%. We demonstrated the robustness of these algorithms in a more complex arena through the automated segmentation of neural cells in ex vivo brain slices. These novel methods surpass previous techniques by improving the robustness and accuracy by: (1) the ability to process neurites and somata, (2) bidirectional segmentation correction, and (3) validation via software-assisted user input. This 3-D image analysis platform provides valuable tools for the unbiased analysis of neural tissue or tissue surrogates within a 3-D context, appropriate for the study of multi-dimensional cell-cell and cell-extracellular matrix interactions.