Abstract
AANS/CNS Sessions
TREATMENT OF THE DIFFICULT THORACOLUMBAR FRACTURES
The thoracolumbar junction, from T11 to L2, is the most commonly injured portion of the spinal column. Over 15,000 major thoracolumbar spine injuries occur per year in the United States, with over 5,000 resulting in neurological deficits. Identifying these injuries is paramount to prevent secondary neurologic injury and assist in clinical decision-making. Several classification schemes are in use to describe these fractures, determine their overall stability, and help predict which fractures will require operative versus non-operative management. Some types of fractures are conventionally treated with or without surgery, but there still remains controversy regarding how to best manage certain fractures.
During this session, there will be a brief overview of the different fracture types which occur in the thoracolumbar spine and the classifications systems in use today. The management for each of these fractures will be discussed with respect to their fracture morphology, neurologic injury, and spinal stability. It will conclude with a review of the current literature regarding treatment of the difficult and controversial fractures.
Keywords: thoracolumbar fracture, spinal stability, management
BRAIN TISSUE OXYGENATION–WHAT HAVE WE LEARNED FROM BOOST-2?
ICU management of patients with severe TBI has focused on intracranial pressure (ICP) and cerebral perfusion pressure (CPP), but this approach was never validated in a randomized clinical trial, and recent studies have questioned the benefit of ICP-directed therapies. These studies indicate that a strategy focused on ICP may be overly simplistic. Some have suggested that monitoring the partial pressure of oxygen in brain tissue (PbtO2) provides a useful marker of tissue metabolism, and that interventions based on PbtO2 may improve patient's outcome. However, existing studies using non-randomized designs have conflicting results. We recently completed a Phase II randomized clinical trial of Brain Oxygen Optimization in Severe TBI (BOOST-2). The primary hypothesis was that a neurocritical care management strategy informed by PbtO2 values as well as ICP values would reduce the total burden on hypoxia. Secondary hypotheses related to safety, feasibility, and non-futility.
122 participants were enrolled in the study. There was a substantial and highly statistically significant reduction in total hypoxia burden (p < 0.0001). No patients in the PbtO2 + ICP treatment group experience a hypoxia burden >400 hrs*mm Hg, while more than 20% of ICP only treated patients experienced a similar hypoxia burden. There were no significant differences in the rate of serious adverse events (SAEs) between the two groups. Functional outcome was assessed using the GOS-E 6 months after injury. There was a trend towards reduced mortality (GOS-E 1) and increased good outcome (GOS-E 7–8) in the PbtO2 + ICP treatment group, compared to the ICP-only treatment group. The mean (SD) GOS-E was 3.4 (2.2) in the ICP-only group and 4.2 (2.5) in the PbtO2 + ICP group (p = 0.111). In the pre-specified analysis dichotomizing the GOS-E at 4, the Odds Ratio favoring good recovery in the PbtO2 + ICP treatment group was 1.8 (p = 0.221, Fisher's exact test). These findings were supportive of the pre-determined non-futility hypothesis. A Phase III study designed to obtain definitive evidence of clinical benefit is currently under review.
Keywords: PbtO2, randomized clinical trial, Glasgow Outcome Scale-Extended
Massachusetts General Hospital, Neuroradiology, Boston, USA
Keywords: low-level light therapy, advanced MRI imaging, diffusion tensor imaging, resting-state fMRI, hypercapnia, cerbrovascular reserve
Spinal cord injury (SCI) is a significant public health problem with approximately 12,000 new cases each year. More than 50% SCI occur in the cervical spine (i.e., tetraplegia), resulting in some loss of arm and/or hand function. Hand function is consistently rated as the most desired function for persons with tetraplegia above bowel and bladder function, sexual function, standing and pain control.
Nerve transfers to treat brachial plexus and peripheral nerve injuries have gained significant momentum over last decade. The central principle of nerve transfers is the conversion of a high level injury, to a low injury, placing regenerating axons in close proximity to the target end-organ. Increased experience with nerve transfer procedures has markedly improved the functional outcomes obtained in comparison with traditional graft techniques for peripheral nerve injuries and changed the treatment algorithm in patients with nerve injuries. While tendon transfers have an established role in the management of patients with tetraplegia, only recently have nerve transfers been considered as a potential treatment option.
Utilizing donor nerves above the SCI, nerve transfers can be done either acutely into the zone of the injury (upper and lower motor neuron dysfunction) or in a delayed fashion below the zone of injury. Motor neurons in the zone of injury are subject to lower motor degeneration, with a similar degeneration pattern seen in peripheral nerve injuries. Injuries in the zone of injury should be treated aggressively, to prevent progressive motor endplate fibrosis and contractures. Motor neurons below the level of injury are still in continuity with distal motor endplates, these nerves do not undergo typical Wallerian degeneration as observed in the zone with injury. This provides two distinct windows of opportunity for acute treatment (<6 months) after injury and chronic treatment (years) after injury.
We have treated several tetraplegic patients with nerve transfers following a cervical spinal cord injury. Nerve transfers can reliably provide improved function below the level of injury reanimating muscles 1–3 myotomes below the level of injury, this treatment can be considered in the long-term management of cervical SCI patients.
Keywords: spinal cord injury, Nerve transfer, tetraplegia, hand function
PAIRED MAGNETIC AND ELECTRICAL STIMULATION FOR CERVICAL SCI
James J. Peters VA Medical Center, Neurology, Spinal Cord Injury, Bronx, USA
Keywords: non-invasive brain stimulation, TMS, cervical electrical stimulation, spike timing-dependent plasticity
This presentation will review the current status of cellular therapeutics for spinal cord injury. Spinal cord injury currently has no effective therapies and cell transplantation therapy has been suggested to be of benefit. There are 4 major categories of cells being transplanted: schwann cells, olfactory ensheathing cells, mesenchymal stromal cells and related cells, and neural stem/progenitor cells, each with different proposed mechanisms of action. Review of the website “clinicaltrials.gov” reveals 31 studies currently active around the world. Of these listed trials, there are 4 relevant groups currently active in the United States. These four groups and their trials will be discussed. Three of these groups are utilizing allogeneic neural stem cells or their progenitors. It has been suggested that autologous cells would provide a superior source due to concerns of immune rejection. We have developed an efficient, reproducible, clinically compatible protocol for generating neural stem cells and their oligodendrocytic and neuronal progenitors from an autologous source. We believe that this type of personalized medicine represents the future in this field.
Keywords: Cell Transplantation for Spinal Cord Injury
ARE WE EVER GOING TO HAVE A TBI CLINICAL TRIAL THAT SUCCEEDS?
Eurotherm 3235 enrolled 387 out of a planned 600 patients, prior to early termination for identification of worse outcomes in the test group. PROTECT III enrolled 882 out of a planned 1140 patients before early termination for futility. SYNAPSE enrolled 1195 patients. ROC was terminated after enrolling 1331 of a planned 2500 patients. And these are only the latest of more than 33 failed consecutive trials demonstrating lack of efficacy for treatment of traumatic brain injury. All of these massively funded, well conducted multicenter prospective randomized trials were built on a foundation of preclinical evidence supporting efficacy. For progesterone alone, more than 200 papers demonstrated potential utility for treatment of TBI. Clearly, the failure of these trials suggests fundamental problems in our understanding of brain injury, and the limitations of translating animal research to humans. Consider this: would a hospital conduct a clinical trial of a treatment (eg anticoagulation) for “chest pain” in the emergency department, classifying patients on the basis of severity (mild/moderate/severe), and using an 8-point outcome measure (ranging from normal to disabled)? Given that chest pain in the emergency department has more than 20 common myriad causes, some of which might improve with the treatment (myocardial infarction, pulmonary embolism) and others of which might be exacerbated (hemothorax, cardiac tamponade), the idea is preposterous. Yet we attempt to treat brain injury in clinical trials without first classifying its pathophysiology. Improved multimodal classification of brain injury, based on eye tracking, serum biomarkers and imaging modalities is a prerequisite for testing and development of effective therapeutics. Given the complex and heterogeneous nature of both brain function and injury, better outcome measures that accurately capture treatment effects are also needed. Until these goals are obtained, effective therapeutics for brain injury will remain elusive.
Keywords: brain injury, clinical trials
CAN TECHNOLOGY SAVE US? THE BIOMECHANICS OF CONCUSSION PREVENTION
Children's Hospital of Philadelphia, Center for Injury Research and Prevention, Philadelphia, USA
Nearly 90% of pediatric traumatic brain injuries (TBIs) are considered “mild” (mTBI and concussion), and they can lead to poor neurological outcomes and functional disabilities that adversely affect academic, behavioral and emotional aspects of quality of life, and produce deficits in multiple domains (memory, concentration, sleep, processing speed, sleep, and eye and motor function). The best strategy for mitigating these long term consequences is to prevent the injury from occurring. Development of effective prevention approaches requires consideration of two fundamental questions: what is the clinical entity that is mTBI and what is the mechanical event that leads to the injury occurring? Current research is advancing on both of these fronts including 1) the development of objective, age-specific metrics for diagnosis and monitoring of recovery and 2) understanding of injury mechanisms and injury risk curves to guide the development of preventative equipment and policies. Animal studies and human studies are both being utilized to advance the fundamental knowledge. In both of these efforts, the promise of technology is great – technology for prevention, technology for injury monitoring and technology for diagnosis. Science and evidence should guide the utilization of all types of technology. Four fundamental questions should be asked: 1) what is the technology designed to do, 2) does it measure what it intends to measure and does it do that accurately, 3) is there high quality evidence of its effectiveness and reliability, and 4) for what scenarios (i.e. age, sport, stage of recovery) does it apply? By understanding the answers to these questions, we gain insight into the role that technology can play in mitigating the long-term consequences of mTBI on children and adolescents.
Keywords: prevention, technology, protective equipment
NNS 2016 Trainee Competition Finalists
Barrow Neurological Institute at Phoenix Children's Hospital, Phoenix, USA
Development and aging are influenced by external factors with potential to impact health throughout the lifespan. While there is ongoing research of experimental pediatric and adult TBI, few studies have incorporated animal models of pediatric, adolescent, and adult TBI to understand the role of age-at-injury across the lifespan. Our aim was to follow neurological morbidities across the rodent lifespan, with respect to age-at-injury. A single cohort of rats (n = 69) was received at post-natal day (PND)10. Subgroups (n = 11–12/group) were subjected to midline fluid percussion injury at age PND17, PND35, 2 mo, 4 mo, or 6 mo or were naïve (n = 12). Rats were assessed for motor function, anxiety-like behavior, cognitive performance and depression-like behavior across their lifespan. TBI resulted in deficits on a beam walk. Brain-injured rats committed significantly more foot-faults compared to naive at 1.5 mo, 3 mo, and 5 mo. TBI resulted in anxiety-like-behaviors at 8 mo assessed by the open field task. Rats injured at 2 mo, 4 mo, and 6 mo spent significantly less time in the center of the arena compared to naïve. TBI also resulted in memory deficits assessed using novel location task. There was an overall effect on average duration of visits to the novel location at 8 mo and 9 mo and rats injured at PND17 and PND35 had significantly shorter visits to the novel location compared to naïve. Lastly, TBI did not result in depressive-like behavior at 9 mo assessed by forced swim task measured as the amount of time spent trying to escape or total number of escape attempts. These data support TBI can negatively impact neurological function during discrete stages of development and aging. The interplay of age-at-injury and aging with an injury are translationally important factors that influence behavioral performance as a quality of life metric. More complete understanding of these factors can direct rehabilitative efforts and personalized medicine for TBI survivors.
Support:Science-Foundation-Arizona, Arizona-Alzheimer-Consortium
Keywords: age-at-injury, aging, development, TBI, behavior
Mammalian target of rapamycin (mTOR) – a signaling molecule involved in protein synthesis, cell growth, and cell proliferation – is linked to neural plasticity and regeneration in the CNS. The aim of this study is twofold: to determine whether mTOR signaling is a necessary component of axonal regeneration in the injured spinal cord and to determine whether mTOR signaling is involved in the increase in regeneration that occurs with forced exercise. This is accomplished using rapamycin, which blocks mTOR signaling. Axons damaged during spinal cord transection will regenerate through a peripheral nerve graft if apposed to the lesion. Neurons whose axon regenerated through the graft can then be labeled, and subsequently quantified, by applying the retrograde fluorescent tracer True Blue to the distal end of the graft. A complete T12 spinal cord transection was performed on adult Sprague-Dawley rats and a tibial nerve graft was apposed to the injured spinal cord. The rats were divided into four groups: no exercise and no rapamycin; no exercise plus rapamycin; exercise and no rapamycin; exercise plus rapamycin. Five weeks after grafting, True Blue was applied to the distal end of the graft. One week later, the number of neurons that regenerated an axon through the graft was quantified and compared between the four groups. Among the non-exercised animals, those not given rapamycin demonstrated an almost three-fold (p < 0.05) increase in number of neurons that regenerate over those that received rapamycin injections. Among the exercised animals, the same result was found, with the rats not given rapamycin showing three times (p > 0.05) the regeneration as those given rapamycin. Our results demonstrate that blocking mTOR signaling with rapamycin significantly reduces the regenerative potential of the injured spinal cord. The pro-regenerative effects of exercise are impaired by blocking mTOR signaling, potentially pointing to the mTOR pathway as a mechanism by which exercise enhances regeneration. Supported by the Alpha Omega Alpha Carolyn Kuckein Student Research Fellowship.
Keywords: SCI, peripheral nerve graft, rapamycin
EPIGENETIC, TRANSCRIPTOMIC, AND SLEEP ALTERATIONS FOLLOWING PENETRATING BALLISTIC BRAIN INJURY (PBBI)
Icahn School of Medicine at Mount Sinai, Neuroscience/Neurology, New York, USA
Traumatic brain injury (TBI) affects 1.7 million Americans each year according to the Centers for Disease Control and Prevention. Long-term consequences of TBI significantly impact quality of life and one of the most commonly reported symptoms is sleep disturbance. Reduction in the quality and amount of sleep can affect a wide range of behavioral and physiological symptoms, such as impaired cognition, mood and anxiety disorders, and cardiovascular effects. In an established model of PBBI, we investigated the epigenetic and transcriptional alterations associated with sleep changes following PBBI. Rats were randomly assigned to injury cohorts, implanted with bilateral electrodes, and subjected to unilateral frontal PBBI. Continuous electroencepholographic recordings were collected from baseline through 7 days post-injury and assessed for changes in sleep-wake architecture. PBBI-injured animals showed significant deviations in sleep-wake architecture compared to baseline recordings. Animals demonstrated irregular sleep patterns with increased daytime and nighttime sleepiness, reduced REM sleep, and greater time to REM onset. The most salient effects were detected within 24 h post-injury. To examine epigenetic alterations associated with these sleep disturbances, we performed DNA methylation profiling via expanded reduced representation bisulfite sequencing on genomic DNA extracted from brain cortical tissue and leukocytes. To determine whether PBBI related methylation alterations confer changes in gene expression, we performed genome-wide gene expression profiling of whole brain tissue and blood samples using RNA sequencing on the Illumina HiSeq2500. Data showed DNA methylation and transcriptional perturbations associated with sleep disturbances following PBBI. These findings highlight the importance of the role of sleep in TBI symptomatology and may identify genetic targets for future translational studies of TBI and associated sleep disturbance.
Keywords: epigenetic, methylation, gene expression, blast, sleep
Traumatic brain injury (TBI) is the leading cause of death and disability amongst children and adolescents. Moreover, mild/moderate TBI (mTBI) is frequently under-diagnosed, with patients being soon discharged from the hospital. However, growing evidence shows that behavioral impairments endure or even arise months after injury, without a full knowledge of their pathophysiology. We aimed to characterize these deficits and identify the cellular mechanisms underlying juvenile mTBI (jmTBI).
We developed a new model of closed-head jmTBI on postnatal day 17 mice using an electromagnetic impactor on non-fixed head, allowing rotation, with 2 severity grades with no skull fracture: mild (1 mm impact depth, 2 m/s speed) and moderate (3 mm impact depth, 3 m/s speed). Mice were evaluated with a battery of behavioral tests followed by morpho-functional neurovascular unit assessment.
Increasing severity caused a significant increase in the time to resume exploratory behavior after impact. Over 60% of mice from the moderate group and none in the mild group exhibited subdural/ intraparenchymal hemorrhages 24 h after injury. An impairment in vascular reactivity (ex vivo contraction of cortical vessels in response to thromboxane agonist applied to acute brain slices) was observed in the moderate group 24 h post-jmTBI. Both grades of jmTBI induced significant blood-brain barrier dysfunction (assessed byIgG infiltration) and significant astrogliosis (assessed by GFAP staining) in the ipsilateral hemisphere 3 days after injury. IgG infiltration and astrogliosis were also observed in the contralateral corpus callosum, suggesting that fibers were especially sensitive to rotation. As in clinical mild TBI, there was no major neurological deficit at 1 and 3 days post-jmTBI. However, our longitudinal analysis up to 1 month post-jmTBI showed significant anxiety (open field test) and a trend for cognitive decline (novel object location test) in the moderate group 1 month post-jmTBI.
In summary, early significant vascular and astrocytic changes may contribute to longterm behavioral impairments. Our results strongly suggest that the neurovascular unit represents a promising target for pediatric mTBI patients.
Keywords: concussion / mTBI, pediatric / juvenile, neurovascular unit, long-term damage, behavior
DOUBLE DIFFUSION ENCODING FOR RAPID IN VIVO EVALUATION OF RAT SPINAL CORD INJURY
Medical College of Wisconsin, Neurosurgery, Milwaukee, USA
Noninvasive assessment of spinal cord injury (SCI) severity and functional outcome are important challenges in acute SCI care. A promising magnetic resonance imaging (MRI) technique known as Diffusion Tensor Imaging (DTI) has been extensively studied for SCI evaluation by quantifying microstructural tissue changes following injury. However, in-vivo DTI application is complicated as its specificity to the important functional status marker of axonal integrity is reduced by confounding diffusion changes from edema. Furthermore, technical difficulties and involved post-processing requirements reduce clinical feasibility. To overcome these challenges, we developed a double diffusion encoding (DDE) MRI application to improve specificity to axonal integrity, reduce scan time, and minimize data processing. This is accomplished with a strong diffusion “filter” that attenuates edema and non-neuronal tissue signal before preferentially sampling axonal diffusion in a spectroscopic acquisition. In-vivo testing and comparison to DTI was performed in a rat contusion SCI model with severe, moderate, mild, and sham severities at the T10 vertebral level. At 48 hours post-injury, diffusion MRI data were collected at the lesion epicenter with acquisition times of 3 minutes for DDE and 65 minutes for DTI. Standard DTI analysis used manual region of interest drawing for whole-cord measurements while DDE signal analysis was automated. Regression of diffusion measurements against spinal cord compression during injury showed significance with multiple DDE parameters, including fraction of restricted diffusion (R2 = 0.67, p < 0.001), while only fractional anisotropy from DTI showed significance (R2 = 0.31, p = 0.02). Similar results were seen for regression against functional scores at 24 hours and 30 days post-injury, indicating significant association with functional status and potential prognostic value. Thus, DDE provides a sensitive measurement of injury severity and functional status while significantly reducing acquisition and analysis demands. These improvements over DTI show promise for DDE in future preclinical and clinical applications for rapid, sensitive SCI assessment.
Keywords: spinal cord injury, double diffusion encoding, diffusion tensor imaging, magnetic resonance imaging
Naval Medical Research Center/ Henry Jackson Foundation, Neurotrauma, Silver Spring, USA
The incidence of traumatic brain injury (TBI) resulting from exposure to blast overpressure from improvised explosive devices (IEDs) has been on the rise in combat troops. Cerebral vasospasm is a prominent and potentially life-threatening complication experienced by casualties exposed to IED's. The underlying mechanisms of the pathology in blast-induced TBI patients are still unclear; however, we have observed vascular injury in rats exposed to low intensity blast overpressure (BOP). The purpose of this study is to evaluate the effects of BOP on functional responses of the cerebral microcirculation. Specifically, cerebrovascular responses of pial microvessels were assessed in control and blast-exposed rats after a single 75 kPa blast 2 hours and 1,3, and 14 days after BOP exposure. Pial microcirculation was accessed through a cranial window in the parietal bone of anesthetized male Long Evans rats. The responsiveness of pial arterioles was probed using hypercapnia (7% CO2), topical application of barium chloride (BaCl2), and serotonin (5HT); arteriolar diameters were measured before and after treatment. At 2 h after blast, cerebrovascular responses were not different from those of control animals. However, one day after BOP, BaCl2 had a statistically significant (P < 0.0001) vasoconstrictive effect on pial arterioles, while 5HT and CO2 had a dilatory effect. The effects of BaCl2 and 5HT (but not CO2) were different from control. A similar trend was observed 3 days post-BOP. By 14 days days post-BOP, vascular responses to BaCl2 and 5HT diminished significantly compared to 1 and 3 days post-blast. Collectively, the data show that while pial microvessels remain reactive to vasoactive mediators after blast, their responses are different from those of control non-blast animals. These results suggest functional changes in cerebrovascular responses after blast that occur in a time-dependent manner. Further research into the structural changes occurring in the endothelium and the perivascular space are indicated.
Keywords: blast, cerebral microcirculation, vascular function, time-course of dysfunction
Ohio State University, Department of Neuroscience, Columbus, USA
Traumatic brain injury (TBI) elicits immediate neuroinflammation that contributes to acute cognitive, motor, and affective disturbances. Although acute impairments resolve after mild to moderate TBI, inflammatory processes persist and may underlie neuropsychiatric and cognitive complications that arise long after injury. Microglia, the innate immune cells resident to the brain, are key mediators of acute and chronic inflammation. We have previously reported that methylene blue (MB), an antioxidant and anti-inflammatory agent, reduces microglia-mediated neuroinflammation after midline fluid percussion injury, a murine model of diffuse TBI. This was associated with improved recovery of motor coordination 7 days post-injury (dpi). In examining microglia morphology 7 dpi, we found that MB intervention prevented TBI-induced formation of rod microglia in the cortex. Although the formation of rod microglia after FPI has been observed, their origin and role in pathophysiology is unclear. Here we show novel data that rod microglia are in close proximity to axotomized (ATF-3+) neurons 7 dpi and form trains along nearby blood vessels. Furthermore, both rod microglia and nearby activated microglia upregulate CD45. We confirmed that CD45+ rod microglia originate from resident CNS cells (unlabeled in GFP bone marrow chimeras), but not through proliferation (unlabeled by BrdU). Overall, these findings suggest that TBI drives resident microglia to take on a rod morphology, align with blood vessels proximal to damaged cortical neurons, and that this process can be prevented by anti-inflammatory intervention. One possible interpretation is that the rod morphology facilitates movement of microglia along blood vessels toward sites of neuronal injury.
Keywords: neuroimmunology, traumatic brain injury
Neurogenic bowel following spinal cord injury (SCI) is recognized as a lifelong physical and psychological challenge. Despite the enormity of the problem, the mechanisms leading to colonic dysmotility after SCI have yet to be fully understood or effectively treated. Smooth muscle cells, interstitial cells of Cajal (ICC), and PDGFRα+ cells form a functional syncytium which induces slow-wave propagation and modulates contractility along much of the gastrointestinal (GI) tract, including the colon. Cholecystokinin (CCK), a potent post-prandial regulator of GI function, facilitates giant migratory contractions (GMCs) in the colon and anecdotal evidence from the SCI population suggests that bowel programs are facilitated post-prandially. We hypothesized that acute spinal cord injury results in colonic inflammation and elevated reactive oxygen species (ROS) ultimately disrupting colonic GMCs. Adult male Wistar rats received a 300 kdyn T3-SCI. Three days or three weeks post-injury, both injured and age-matched controls underwent in vivo experimentation followed by tissue harvest for histological evaluation. In vivo GMCs were measured via two micro-pressure transducers following injection of heparinized saline or CCK. Colonic cross-sections from SCI and control animals were processed with DHE to visualize oxidative status and c-Kit immunofluorescence was performed to evaluate colonic ICC populations. Baseline GMCs were significantly reduced following acute SCI. CCK dose-dependently invoked responses in both control and SCI colons; however, responses were greatly reduced in injured animals. DHE staining revealed an increase in ROS within the myenteric plexus of the SCI colon vs. surgical control. Immunofluorescent c-Kit labeling displayed intact ICC networks throughout the myenteric plexus sham colon. Conversely, 3-day and 3-week SCI samples displayed incomplete ICC networks that became progressively disorganized and fragmented. Our data suggest that post-SCI inflammation and oxidative stress may be prolonged and provoke diminished colonic motility, possibly through the disruption of intrinsic neuromuscular control of the colon. Diminished CCK-mediated facilitation of GMCs may reflect a general lack of sensitivity to GI peptides following SCI.
Support: NINDS #049177
Keywords: spinal cord injury, colonic dysmostiltiy, reactive oxygen species
MORPHINE UNDERMINES RECOVERY FOLLOWING SCI: OPIOID-IMMUNE INTERACTIONS
Texas A&M Health Science Center, Neuroscience & Experimental Therapeutics, Bryan, USA
Opioids are one of few effective analgesics for the treatment of pain following spinal cord injury (SCI). Unfortunately, we have shown that morphine administered in the acute phase of SCI undermines locomotor recovery, increases mortality and pain reactivity, and decreases signs of general health in a rodent contusion model. Our studies suggest that morphine produces these adverse effects by exacerbating the inflammatory response innate to SCI. Indeed, a single administration of morphine significantly increases IL-1β at the lesion site at 30 min and 24 h post-treatment. Pre-treatment with minocycline also blocks the adverse effects of morphine on locomotor recovery. In the current study, we used flow cytometry to characterize morphine-induced changes in resident and infiltrating immune cell populations. Sham and contused (T12) subjects were implanted with an intrathecal cannula. Twenty-four hours following surgery, subjects were treated with morphine or vehicle. At 24 hours post-treatment (48 h post-surgery), 1 cm of spinal tissue encompassing the injury was collected and dissociated. The resulting cell suspension was plated and stained with markers for CD45, CD11b, IBA1, CD86, and CD68. Cells were quantified using a FACSFortessa flow cytometer, and data were analyzed using FlowJo software. We found both a significant main effect of surgery and drug treatment on total immune cells present at the injury site, with contused-morphine subjects showing significantly higher numbers than their contused-vehicle counterparts. Importantly, we also found a significant main effect of morphine on total and M1-polarized microglia. Irrespective of surgery, morphine increased the number of inflammatory microglia present at the injury site. Our results support the hypothesis that opioid-immune interactions underlie the adverse effects on recovery observed in our model. Morphine may bind to opioid receptors on microglia, synergistically increasing the release of pro-inflammatory factors after SCI, and producing neurotoxicity. Research support: Grant DA031197 to M.A. Hook and the NIDA Drug Supply Program.
Keywords: spinal cord injury, opioids, flow cytometry, rodent, morphine
University of Kentucky, Physiology, Lexington, USA
Traumatic spinal cord injury (SCI) results in excitotoxicity, excessive reactive oxygen and nitrogen species (ROS/RNS) production, and necrotic cell death. This contributes to development of secondary pathophysiological cascades which results in widespread tissue damage. Many branches of these cascades such as ROS/RNS production, glutamate-induced excitotoxicity, calcium dysregulation, decreased ATP production, compromised bioenergetics, and apoptotic cell death can stem from mitochondrial dysfunction. Thus, a single therapy targeting mitochondrial dysfunction after SCI can be far reaching in maintaining cellular bioenergetics to foster functional neuroprotection. We hypothesized that mitochondria transplanted after SCI can be taken up in vivo and replace damaged endogenous mitochondria, providing a multi-mechanistic approach to restore cellular bioenergetics. Methods were refined for transgenically-labeling mitochondria in cell culture, isolating and microinjecting them into naïve and injured rat spinal cords, and assessing bioenergetic integrity after transplantation. Results indicate that mitochondrial transplantation increased overall respiration in the acutely contused cord in a dose-dependent manner. Specifically, oxygen consumption rates of injured tissues transplanted with a total of 50 ug mitochondria showed significantly increased respiration compared to vehicle-treated injured or contusion injury alone after 24 hr. We also visually identified transplanted mitochondria in the injured cords and found them co-localized within a variety of host cell types by 24 hr. Ongoing studies are characterizing cellular uptake of exogenous mitochondrial over time and, based on our previous reports correlating increased bioenergetics with neuroprotection, we are further examining the effects of acute mitochondrial transplantation on tissue sparing and long-term functional recovery.
Support:
NIH T32 Training Grant 5T32 NS077889(JLV)
SCoBIRC Chair Endowment (AGR)
Conquer Paralysis Now (AGR)
NIH/NINDS 2P30NS051220
Keywords: mitochondria, spinal cord injury, respiration
University of Melbourne (Royal Melbourne Hospital), Department of Medicine, Parkville, Australia
Chronic social behavior problems after pediatric traumatic brain injury (pTBI) significantly contribute to poor quality of life for survivors, however, the mechanisms underlying such deficits have not been elucidated. We hypothesized that interconnected brain regions comprising the 'social brain network' undergo aberrant neuroplasticity changes during development following pTBI, to influence social functioning at adulthood. We therefore asked whether pTBI influences neuronal morphology in the medial prefrontal cortex (mPFC), a region involved in social cognition and behavior, prior to the development of social problems. Littermate male and female C57Bl/6 mice were subjected to a unilateral controlled cortical impact or sham-operation at postnatal day 21, approximating TBI during early childhood. One cohort were euthanized at 3 weeks post-surgery for Golgi-Cox staining (n = 5/group); a second cohort were maintained until 8 weeks post-surgery for the evaluation of psychosocial and neurocognitive function (n = 8–10/group). Morphological analysis of layer III pyramidal neurons in the ipsilateral mPFC revealed a reduction in dendritic complexity at adolescence after pTBI in male mice compared to sham controls, including fewer branch nodes and ends, as well as reduced basal dendritic length. By adulthood, consistent with previous studies, male pTBI mice showed deficits in social and sociosexual behaviors. In contrast, mPFC neuroanatomy was unaffected by pTBI in female mice, which also showed a more limited profile of social dysfunction. pTBI mice exhibited robust hyperactivity across multiple paradigms, to a greater extent in males compared to females. Together, our findings demonstrate changes in neuronal morphology, remote from the injury site, several weeks after pTBI in male mice, and associated with the subsequent emergence of social behavior deficits. Sex is a determinant of both regional neuroplasticity and social outcomes after pTBI. It remains unclear whether these changes are an indirect, stress-related consequence of pTBI, or a direct result of aberrant connectivity of the social brain network.
Keywords: social behavior, neuronal morphology, sex, prefrontal cortex
University of Miami Miller School of Medicine, Neurological Surgery, Miami, USA
Traumatic brain injury (TBI) initiates a deleterious inflammatory response that exacerbates pathology and worsens outcome. The anti-inflammatory benefits of inhibiting a cAMP-hydrolyzing enzyme, phosphodiesterase 4 (PDE4), are well established in models of CNS injury. Studies of PDE4 knockout mice have found that the PDE4B subfamily is predominantly responsible for the anti-inflammatory effects of pan-PDE4 inhibitors by regulating tumor necrosis factor (TNF) levels and neutrophil recruitment. Furthermore, PDE4 inhibitors shift the activation state of microglia from a pro-inflammatory (M1) to an anti-inflammatory (M2) phenotype. However, whether selective PDE4B inhibition reduces inflammation and pathology after TBI is unknown. To address this question, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury (2 ± 0.2 atm) or sham surgery. To determine whether PDE4B inhibition reduces inflammation after TBI, animals received either vehicle (5% DMSO in saline) or a PDE4B inhibitor, A33 (2-(4-{[2-(5-chlorothiophen-2-yl)-5-ethyl-6-methylpyrimidin-4-yl]amino}phenyl)acetic acid) at 0.3 mg/kg (i.p.) at 30 min and 5 hrs post-surgery. PDE4B inhibition significantly decreased TNF levels at 6 hrs post-injury and neutrophil infiltration at 24 hrs after TBI. Using flow cytometry to differentiate between microglia expressing a classical M1 marker (inducible nitric oxide synthase, iNOS) versus an M2 marker (arginase 1, Arg1), we found that PDE4B inhibition also increased Arg1+ microglia at 3 hrs post-injury. Lastly, to determine whether PDE4B inhibition reduces pathology after TBI, animals received vehicle or A33 at 30 min and once daily for 3 days post-surgery. Cortical contusion volume was evaluated in serial brain sections stained with hematoxylin and eosin. PDE4B inhibition significantly reduced cortical contusion volume at 3 days post-injury. Overall, these results suggest that PDE4B inhibition may be a viable treatment to reduce inflammation and pathology after TBI.
Supported by The Miami Project to Cure Paralysis and NIH/NINDS NS056072 and NS089351.
Keywords: TBI, PDE4, inflammation, microglia
University of Pennsylvania, Center for Brain Injury & Repair, Neurosurgery, Philadelphia, USA
Neural tissue engineering is an attractive therapeutic strategy following neurotrauma, with the potential to replace neural cells and circuitry in order to improve neurological function. We are building on our previously reported micro-tissue engineered neural networks (micro-TENNs), which are miniature constructs grown in vitro that consist of discrete neuronal populations spanned by long axonal tracts within a protective tubular micro-column. Micro-TENNs are the first transplantable neural networks that mimic gray-white matter architecture, and therefore may be useful for the targeted neurosurgical reconstruction of vulnerable neural populations and long-distance axonal pathways following trauma. In the current study, we advanced our micro-tissue engineering techniques be seeding micro-columns with engineered neuronal aggregates rather than traditional methodology of seeding with dissociated neurons lacking organization. Here, micro-columns with an agarose outer-shell (345–701 μm) and collagen-laminin inner-core (180–350 μm) were seeded with aggregated or dissociated cerebral cortical neurons. Neuronal aggregates exhibited rapid neurite extension within the micro-columns, attaining outgrowth rates of >500 μm/day versus <100 μm/day from dissociated neurons. Aggregate micro-TENNs demonstrated improved neuronal architecture and more robust axonal tracts. Micro-TENNs built using aggregate or dissociated neurons were then stereotaxically microinjected to mimic cortical-thalamic pathways in rats. At 1 week and 1 month post-implant, neuronal network survival and host inflammatory/gliotic responses were assessed via immunohistochemistry and microscopy. Implanted micro-TENN neurons survived and maintained their axonal architecture. A modest astrogliotic response was observed at the micro-TENN–host tissue interface, and this response waned as the hydrogel encasement was gradually resorbed. Neural network survival, neurite integration, and host astrogliotic responses are currently being quantified based on the neuronal architecture and micro-column dimensions. Micro-TENNs may represent a novel platform to treat a range of neurological disorders by simultaneously replacing neurons as well as their long-distance axonal connections. Funding provided by the NIH and NSF.
Keywords: axonal reconstruction, micro-column, micro-tissue engineered neural networks, tissue-engineering, neuroengineering
University of Pittsburgh, Physical Medicine and Rehabilitation, Pittsburgh, USA
Distinct regulatory signaling mechanisms exist between cortisol and brain derived neurotrophic factor (BDNF), and both acute cerebral spinal fluid (CSF) cortisol and BDNF can predict TBI outcome. We investigated concurrent CSF BDNF relationships with CSF cortisol in 92 patients week-1 after severe traumatic brain injury (sTBI). BDNF genetics and age were assessed for relationships with 6-month mortality. We hypothesized cortisol would mediate BDNF effects on 6-month mortality when also considering age and BDNF genetics. We also hypothesized BDNF genetics would moderate relationships between CSF BDNF and cortisol levels. BDNF variants, rs6265 and rs7124442, were used to create a gene risk score (GRS) in reference to “younger patients” (< 48 years old). GRS = 0 was hypothesized as the no-risk group (Val/Val, T/T), GRS = 1 included carriers for one risk-allele (Val/Val, C-carrier or T/T, Met-carrier), and GRS = 2 included carriers of both risk-alleles (Met-carrier, C-carrier). Group based trajectory analysis was used to create patient cortisol groupings, and spline analysis was used to identify the 80th percentile as an appropriate cut-point for high vs. low BDNF levels. CSF BDNF predicted patient cortisol trajectory grouping (p = 0.041) when controlling for age and injury severity. Additionally, cortisol grouping predicted 6-month mortality (p = 0.020), when controlling for age, injury severity and genetics. In mediation analysis, the direct effect of BDNF predicting mortality, when controlling for cortisol trajectory grouping, was not significant. However, the total BDNF effect predicting mortality, with cortisol acting as the mediator, was significant (p = 0.040), yielding a mediation percentage of 20.30%. Also, BDNF was associated with cortisol trajectory grouping among patients with a BDNF GRS = 1 or GRS = 2 (p = 0.026). A BDNF*GRS interaction predicted mortality in younger subjects when controlling for cortisol trajectory grouping, GCS, and GRS (p = 0.018). Thus, we conclude that 6-month mortality after sTBI can be predicted through a mediation model with CSF cortisol and BDNF while also considering age and BDNF genetics.
Support: NIDILRR-90DP0041; DoD-W81XWH-071-0701; NIH-R01NR008424; NIH- R01HD048162
Keywords: cortisol, BDNF, genetic variation, rehabilomics
University of Pittsburgh, Neurosurgery, Pittsburgh, USA
Experimental models of traumatic brain injury (TBI) reproduce cognitive impairments and secondary injury sequela observed in TBI patients. Our previous work suggests that reductions in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, the machinery facilitating vesicular fusion, contribute to impaired neurotransmission. TBI significantly reduces cysteine-string protein α (CSPα), an important chaperone protein that facilitates SNARE complex formation. Lithium treatment in naïve rats has been shown to increase expression of CSPα. Lithium-treated TBI-injured rats exhibit improved outcome, but the mechanisms mediating the improvement have not been elucidated. The objective of this study was to evaluate the effect of lithium on SNARE protein abundance, evoked striatal neurotransmission, cortical contusion volume, and neurobehavioral function after controlled cortical impact (CCI). Sprague-Dawley rats received CCI (2.7 mm) or sham injury, and injected daily (i.p.) with vehicle or 1.0 mmol/kg/ml lithium chloride for up to 14 d, beginning 5 minutes post-injury. The brains were dissected at 1, 3, 7, 14 d post-injury and processed as whole cell or synaptosomal lysates for immunoblotting (n = 6/group). Evoked-dopamine release was evaluated using microdialysis at 7 d post-injury (n = 6–7/group). Lesion volume was assessed at 14 d post-injury (n = 6/group). Cognitive function was assessed at 9–14 d post-injury (n = 14–16/group). Lithium significantly attenuated CCI-induced reductions in CSPα and SNARE complexes at multiple time points. Assessment of contusion volume revealed that lithium did not reduce cortical cell loss. At 7 d post-injury, lithium significantly improved striatal high-potassium evoked dopamine release. Lithium significantly improved Morris water Maze acquisition and probe trial performance. Taken together, lithium improves SNARE protein abundance, neurotransmission, and cognitive performance following TBI. The observation that lithium did not reduce cortical cell loss, suggests that the beneficial effects of lithium may not be mediated via neuroprotection. These findings highlight that lithium increased the abundance of important synaptic proteins that facilitate neurotransmission and promoted functional recovery after TBI. Acknowledgement: 1-F32NS090748, The Pittsburgh Foundation, NIH-NS40125, NIH-NS060672, VAI01RX001127.
Keywords: CCI, lithium, SNARE
University of Toronto, Toronto, Canada
Keywords: MRI, DTI, SCI, prognosis
University of Toronto, Neurosurgery, Toronto, Canada
Cervical spinal cord injuries (cSCI) have devastating long-term consequences for patients' physical, social, and vocational wellbeing. Induced pluripotent stem cell-derived neural stem cell (iPS-NSC) transplants are an exciting therapeutic strategy to remyelinate denuded axons, replace neural circuits, and provide trophic support. However, the dense chondroitin sulfate proteoglycan (CSPG) scar in chronic injuries poses a significant challenge to effective cell migration and neurite outgrowth. Chondroitinase ABC (ChABC) is an enzyme capable of rapidly degrading CSPGs to enhance recovery. We investigated a novel combinatorial approach delivering intrathecal ChABC for 7 days followed by intraparenchymal murine iPS-NSC transplant in a clinically-relevant murine chronic cSCI model. We compared the (1) combinatorial treatment with (2) ChABC alone, (3) iPS-NSC transplant alone, (4) vehicle-treatment alone, and (5) sham injury (laminectomy alone). Behavioural assessments were completed weekly for 9 weeks post-transplant. We found that ChABC pre-treatment dramatically reduced CSPG scarring and resulted in greater subsequent iPSC-NSC survival (7.8% vs 2.4%), particularly at the lesion epicenter. Combinatorial treatment also resulted in greater preservation of cholinergic neurons (ChAT+, NeuN+; 7516 ± 832) compared to iPS-NSC (4904 ± 1243) or ChABC (5890 ± 552) treatments alone. Functionally, only animals receiving the combined therapy demonstrated consistent recovery of forelimb grip strength compared to vehicle-treated controls. This study provides key evidence for the synergistic effects of ChABC pre-treatment with iPS-NSC transplant and opens a new avenue of research in developing an effective therapy for patients with cSCI. This work was generously supported by the Canadian Institutes of Health Research and the Krembil Foundation.
Keywords: chondroitinase, neural stem cell, chronic injury, induced pluripotent stem cell, spinal cord injury, proteoglycans
University of Toronto, Institute of Medical Science, Toronto, Canada
Spinal cord injury (SCI) is a life-threatening condition with multi-faceted complications and limited treatment options. In SCI, the initial physical trauma is closely followed by a series of secondary events, including inflammation and blood spinal cord barrier (BSCB) disruption, which further exacerbate injury. This secondary pathology is partially mediated by the systemic immune response to trauma, where cytokine production leads to the recruitment/activation of inflammatory cells. Since early intravenous delivery of mesenchymal stromal cells (MSCs) has been shown to mitigate inflammation in various models of neurologic disease, this study aimed to assess these effects in a rat model of SCI (C7-T1, 35 gram clip compression) using human brain-derived stromal cells. qPCR for a human specific DNA sequence was used to assess cell biodistribution/clearance and confirmed that only a small proportion (approximately 0.001–0.002%) of cells are delivered to the spinal cord, with the majority residing in the lung, liver, and spleen. Intriguingly, while cell populations drastically declined in all aforementioned organs, there remained a persistent population in the spleen at 7 days. Further, the cell infusion significantly increased splenic and circulating levels of IL-10–a potent anti-inflammatory cytokine. Through this suppression of the systemic inflammatory response, the cells also reduced acute spinal cord BSCB permeability, hemorrhage and lesion volume. These early effects further translated into enhanced functional recovery and tissue sparing 10 weeks post-SCI. Taken together, this work demonstrates an exciting therapeutic approach whereby a minimally-invasive cell transplantation procedure can effectively reduce secondary damage post-SCI through systemic immunomodulation.
Keywords: mesenchymal stem cells, interleukin-10, spleen, spinal cord injury
Two mechanisms have been proposed for the progressive tauopathy in neural tissue responsible for chronic traumatic encephalopathy (CTE): 1) a mechanical shearing of the tissue that disrupts neurons and surrounding structures, and 2) an immune or pathological response that is triggered by or independent of the mechanical disruption. Proving some insight into the possible mechanism, recent clinical studies have shown the unique presentation of injured neurons and tau protein aggregates in perivascular domains. The prevailing hypothesis for this distinctive CTE neuropathology is that inclusions such as vessels lead to localized and elevated stresses in tissues surrounding the vessels during brain deformation. We hypothesize that mechanical property differences at this vessel-tissue interface causes these stresses. This study aims to quantify this mechanism in vascularized tissues using high-resolution, micro-scale finite element (FE) models.A 3D representative volume of vascularized brain tissue was developed to investigate the potential stress concentrations and elevated shear strain in the perivascular tissue. Simple shear deformations were applied to the vascularized tissue model parallel and perpendicular to the vessel, and the distribution of stress was observed. Stresses at the vessel-tissue interface were 40–60% higher than stresses in tissue without a vessel, with deformation perpendicular to the vessel having the highest stress concentrations. A subsequent sensitivity analysis identified transitions in material properties at the interface of the vessels and the neural tissue as the cause of these stress concentrations. The vascularized tissue model was then simulated using deformations predicted by a whole brain FE model subjected to impacts caused by helmeted football impacts and automotive impact. Deformations from four locations within the brain were selected for the micro-scale analysis, and a stochastic model of stress concentration was developed for a random vessel orientation. Preliminary results indicate these stress concentrations also occur in complex, dynamic loading scenarios, ranging 30–60% above what is predicted without vasculature. Given the prevalence of tau protein build-up in perivascular tissues, these results further support the mechanical disruption hypothesis of CTE, and can be used to improve FE brain models for more sensitive injury prediction and analysis.
Keywords: finite element analysis, chronic traumatic encephalopathy
PERSISTENT EPIGENETIC CHANGES IN HIPPOCAMPAL NEURAL STEM CELLS FOLLOWING TRAUMATIC BRAIN INJURY
The hippocampus is one of the two regions in the adult brain where new neurons are constantly generated from neural stem cells (NSC) located in the dentate gyrus (DG) (a process known as neurogenesis). We have previously reported that, following traumatic brain injury (TBI), although a significant increase of neural progenitor cells is observed in the hippocampus DG, the maturation and integration of newly formed neurons is significantly reduced. The aim of this work was to study epigenetic regulation of neurogenesis in the DG in a rat model of TBI.
Support: These studies were completed as part of an interdisciplinary research team funded by The Moody Project for Translational Traumatic Brain Injury Research.
Keywords: microRNA, hippocampus, laser capture microdissection, fluid percussion injury
MILD TRAUMATIC BRAIN INJURY (MTBI) ALTERS NEOCORTICAL GABAERGIC INTERNEURON STRUCTURE AND FUNCTION VIA AXONAL INJURY
A major pathology following mTBI is diffuse axonal injury (DAI). Historically, DAI has been studied in white matter, where long-distance projections are susceptible to trauma-induced forces. Recent clinical and experimental evidence however, suggests the involvement of locally projecting axons within neocortical gray matter. Previously, we have demonstrated structural vulnerability of the pyramidal neuron's perisomatic axonal domain, with electrophysiological studies suggesting involvement of GABAergic interneurons. This investigation sought to determine if mTBI alters locally projecting GABAergic interneurons within neocortical gray matter. We evaluated parvalbumin (PV)-expressing GABAergic interneurons, which control pyramidal neuron output and regulate neocortical excitatory-inhibitory (E-I) balance. Cre/lox mice were used to genetically label PV-expressing interneurons with tdTomato, a red fluorescent protein variant, allowing detailed morphological and electrophysiological analysis. PV-cre; tdTomato mice subjected to sham or mild central fluid percussion injury were sacrificed after 3 or 24 h and prepared for confocal and ultrastructural analysis. By 3 h post-mTBI, the occurrence of DAI was reflected in tdTomato+ axonal swellings observed throughout the somatosensory cortex. Colocalization of APP+/PV+ immunoreactivity with tdTomato+ swellings confirmed the presence of DAI. At 24 h post-mTBI, widespread tdTomato+ pathology consistent with axonal degeneration was readily observed. Ultrastructural analysis corroborated these findings demonstrating intra-axonal disruption within tdTomato+ swellings. Quantitative analysis yielded ∼10 tdTomato+/APP+ swellings/mm2, with ∼80% of swellings occurring near their somatic origin. Accordingly, we estimate 5–10% of tdTomato+ cells undergo DAI. Parallel electrophysiological studies were performed in layer 5 PV-interneurons, identified via tdTomato expression and fast-spiking behavior. Whole-cell current and voltage clamp were applied to record intrinsic and synaptic properties 24 h after sham-injury or mTBI. Fast-spiking/tdTomato+ cells intrinsic properties did not change, consistent with their overall health. However, both frequency and amplitude of spontaneous inhibitory post-synaptic currents were reduced significantly. Since PV-interneurons are highly interconnected, this suggests the loss of PV-mediated inhibition as a consequence of DAI and its attendant axonal degeneration/deafferentation. Together, these observations reveal for the first time that locally projecting GABAergic interneurons are susceptible to DAI with major implications for overall neocortical E-I balance.
Keywords: mild traumatic brain injury, GABAergic interneurons, axonal injury, circuit disruption, gray matter, neocortical excitation- inhibition
DEVELOPMENT OF METHODS TO PERFORM RADIOLOGICAL-PATHOLOGICAL CORRELATIONS IN CHRONIC TRAUMATIC ENCEPHALOPATHY
Washington University in St. Louis, St. Louis, USA
Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative condition that occurs following repetitive mild traumatic brain injury, and is distinguishable by neuropathological features such as phosphorylated tau tangles and axonal degeneration. Because these microstructural changes in brain tissue cannot be detected using standard imaging methods, CTE remains a post-mortem diagnosis. Advanced diffusion MRI methods are currently being explored as a more sensitive means to detect CTE, but the relationship between the diffusion signal and the neuropathological features of the disease is unclear. We use robust image registration to perform highly sensitive quantitative correlations between histological markers of CTE-related pathology and diffusion MRI parameters.
Human ex vivo cortical tissue was scanned using an 11.7T Varian MRI scanner, 202 diffusion weighted gradient directions, and a voxel size of 250 × 250 × 500 μm. Diffusion based directionality was calculated using a generalized q-space model. The tissue was serially sectioned into 50 μm slices and stained for myelinated axons using Black Gold II. Registration of histological data to the diffusion data was performed by placing landmarks on both data sets and applying a local weighted mean transform to the histological image. Two dimensional Fourier transforms of each equivalent voxel of the Black Gold II stained image were used to quantify histological fiber direction and randomness of fiber orientation.
We found a strong correlation between the histologically and diffusion derived fiber directionality on a voxel-wise level. Co-registration between imaging and histology was found to have >93% accuracy when comparing voxels classified as gray or white matter, along with high inter and intra-rater reliability of landmark placement. Furthermore, we were able to obtain histological voxel-based measures of white matter fiber disruption using a blinded ROI-based analysis of cortical sulci. These findings demonstrate that application of robust radiological-pathological correlations can determine whether advanced diffusion MRI methods are likely to sensitively and specifically detect CTE-related pathology.
Keywords: histology-MRI co-registration, diffusion MRI, quantitative histology, white matter directionality
Poster Presentations - Group A
The Roskamp Institute, Neuroscience, Sarasota, USA
Repetitive mild traumatic brain injury (r-mTBI) is a risk factor for development of Chronic Traumatic Encephalopathy (CTE), a disease characterized by Tau pathology throughout the cortices, and often co-presenting with conditions such as Alzheimer Disease (AD). It has been well documented that mild to severe TBI can result in transient reductions in Cerebral Blood Flow (CBF), with severe injuries often accompanied by presence of varying degrees of vascular pathology post-mortem. Aberrant CBF readings precede gross Amyloid pathology in AD patients, suggesting that hypo-perfusion is key in the pathogenesis of conditions such as CTE and AD, for which r-mTBI is a pre-disposing factor. We have herein expanded on our previous animal model of r-mTBI, showing robust neuroinflammation and pronounced spatial memory deficit in wild type mice as late as 18 months post-injury. We now show this pathology and concomitant behavioral phenotype to be emulated in a separate animal model of r-mTBI, described herein, and accompanied by chronic impairment of global CBF, and altered expression of cerebrovascular markers. These results are the first to demonstrate chronic cerebrovascular dysfunction in the pathogenesis and evolution of r-mTBI-induced illness, and validate this model for investigation of CTE.
Keywords: repetitive mild traumatic brain injury, cerebrovasculature, cerebral blood flow, inflammation
There is an increasing incidence of spinal cord injury (SCI) in aged individuals. Previously, we demonstrated that aged mice exhibit worse functional deficits associated with altered macrophage activation following SCI. Reactive oxygen species (ROS)-mediated damage following neurotrauma contributes to the secondary injury. It has been suggested that NADPH oxidase (NOX) plays an essential role in microglia/macrophage activation and subsequent inflammatory responses. We hypothesize that age increases oxidative damage in the injured spinal cord via increased NOX activity. In order to understand the mechanisms behind age-related differences in recovery, we compared oxidative stress and macrophage activation in 4-month-old (4 MO) and 14 MO mice after contusion SCI. By tracking oxidized dihydroethidine (ox-DHE), a superoxide marker, we identified that overall superoxide generation is significantly higher in 14 vs. 4 MO SCI mice at 3 days post injury (dpi). Notably, the expression of NOX2, which we primarily detected in ROS-producing macrophages, is significantly increased in 14 MO mice. This suggests that macrophages and NADPH oxidase are the major cellular and subcellular sources of oxidative stress and may potentiate secondary injury in older animals. We detected increased activation of neurotoxic M1 macrophages (CD16/32-positive) in 14 MO SCI mice. Interestingly, while the number of M2 macrophages (arginase-1-positive) was similar between ages, we detected a larger percentage of ROS-producing, ARG-1-positive SCI macrophages in 14 vs. 4 MO mice. These data indicate that age plays an important role in macrophage polarization with normally protective M2 macrophages potentially contributing to secondary injury through ROS generation after SCI. Understanding the differences in the inflammatory and oxidative stress responses after SCI is important to determine how age at time of injury might affect endogenous repair processes, pathology, and clinical therapies.
Acknowledgements: this work is supported by Craig H. Neilsen Foundation and NIH NINDS R01NS091582 and P30 NS051220.
Keywords: NADPH oxidase, reactive oxygen species, macrophage polarization, arginase-1, dihydroethidium
ACUTE SEVERITY PREDICTION USING NEW ASTROGLIAL MARKERS AFTER RECOVERABLE SWINE SPINAL CORD CONTUSION INJURY
UCLA, Semel Institute for Neuroscience and Human Behavior, Los Angeles, USA
Evaluating injury severity of acute spinal cord injury (SCI) immediately after an accident or combat trauma is challenging. Accurate patient assessment can minimize exacerbation during transportation and guide urgent care decisions. Fast-released, sensitive and specific neurotrauma biomarkers are unmet needs. We have recently identified a novel neurotrauma marker panel in a trauma-release proteome of mechanically injured and dying astrocytes (Levine et al., 2015; Halford et al., submitted). This project aims to determine acute severity after recoverable swine contusion SCI using new astroglial biomarkers, quantitative histopathology and behavioral outcome. Immunoblotting densitometry and peptide mass spectrometry measured biomarker amounts. GFAP, S100b and new astroglial markers aldolase C (ALDOC), brain lipid binding protein (BLBP), and glutamine synthetase (GS) were robustly elevated 20–30 min hour post-SCI in cerebrospinal fluid compared to same animal baselines. Temporal trajectories for GFAP and S100b decreased drastically 2–7 days post-injury, while ALDOC increased by 7 days post-injury. Blood testing is feasible, as ALDOC was detected in serum after 20–30 min and increased by 7 days post-SCI, suggesting early blood-brain barrier passage and robust blood stability. Hemorrhage was documented by post-SCI magnetic resonance imaging and was quantitated using interstitial immunoglobulin expansion from the contusion. Blood presence in CSF was quantified using hemoglobin mass spectrometry, providing a proteomic fingerprint of bleeding. Diffuse white matter astroglial injury was documented using GFAP-stains showing glial fiber beading (clasmatodendrosis). Fiber interruption progressed rostro- caudally with increasing injury severity. Newly generated peri-lesional bundle-forming astrocytes segregated distinct zones of astroglial injury/demise versus scar-forming astrogliosis. Histopathology associated with acute post-injury biomarker levels with high accuracy and is being correlated with locomotor recovery. In conclusion, these neurotrauma biomarkers monitor severity of astroglial injury validating them for early neurotrauma patient risk stratification.
Support: CDMRP/USAMRAA W81XWH-13-2-0047
Keywords: proteomic, contusion spinal cord injury, swine
North Carolina State University, College of Veterinary Medicine/Department of Clinical Sciences, Raleigh, USA
Acute thoracolumbar spinal cord injury (TLSCI) is common in dogs secondary to disc herniation and trauma. Severe injuries frequently result in permanent motor, sensory and autonomic impairment making dogs a viable model to study chronic SCI. 4-Aminopyridine (4-AP) improves hind limb function in these dogs, however, the response is variable. We hypothesized that dogs with electrophysiologically detectable connections across the site of injury would show a greater response to 4AP. Our objective was to investigate differences in baseline clinical and electrophysiologic parameters between dogs that do and do not respond to 4-AP. Twenty dogs that failed to recover by 3 months after clinically complete, acute TLSCI were enrolled. Gait was quantified using a 0–12 open field gait scale (OFS). Electrophysiologic testing included transcranial magnetic motor evoked potentials (TMMEPs) and somatosensory spinal evoked potentials (SSEPs). All testing was performed at baseline and post-4-AP. Baseline gait scores were compared between responders (defined as 1+ OFS change) and non-responders, and association between response and presence of TMMEP or SSEP was evaluated. Four dogs had recordable TMMEPs, while none had recordable SSEPs. Seven dogs responded to 4AP with a mean baseline OFS of 1.6 (SD = 2.1) and change of 1.57 (1–3) compared to non-responders (mean OFS 3.25 (SD = 3.5), change 0). No responders had detectable baseline TMMEPs. No factors investigated were associated with response to 4-AP. Presence of intact conduction across the lesion was not predictive of a response to 4AP; 4AP response was more common in dogs with lower baseline gait scores.
Keywords: canine, model, chronic spinal cord injury, 4-aminopyridine
UCSF, Neurosurgery, San Francisco, USA
Diffuse axonal injury (DAI) causes considerable disruption to neuronal integrity and cerebral metabolism following traumatic brain injury (TBI). Improved characterization is needed for outcomes after isolated DAI. TBI patients from the TRACK-TBI Pilot study with isolated DAI (DAI+) or no intracranial pathology on computed tomography (CT-) were extracted for cohort analysis. Patients with complete 3-month Glasgow Outcome Scale-Extended (GOSE, ≤6 = moderate/severe disability) and postconcussional assessments were included. Univariate analyses are described by cohort and multivariable regression adjusted for age, education, psychiatric history, Glasgow Coma Scale (GCS), and loss of consciousness (LOC). Overall, 249 patients were included (16 DAI+, 233 CT-), aged 42.3 ± 17.1-years, 65.9%-male and 76.3%-Caucasian. Falls accounted for 45.8% of injuries, followed by motor vehicle-28.5%, pedestrian/cyclist-15.7%, assault-13.3%, other-4.0%. Patients were predominantly mild TBI (GCS 13–15: 96.4%) and +LOC (63.5%). All DAI+ patients (56.3%-floor, 43.7%-ICU) and 49.4% of CT- patients (41.7%-floor, 7.7%-ICU) were admitted to hospital. At 3-months, 50.0% of DAI+ patients reported moderate/severe disability (vs. CT-, 26.6%), associating with a multivariable OR of 3.30 (95% CI [1.09–9.99]; p = 0.034). DAI+ patients showed marginally higher odds of emotional lability (62.5%-vs.-37.8%; multivariable OR 2.52 [0.84–7.50]; p = 0.097). Of patients employed at baseline, 61.5% of DAI+ were not back-to-work compared to 28.5% of CT- (multivariable OR 4.53 [1.32–15.49]; p = 0.016). Patients with isolated DAI demonstrate increased risk of impaired functional outcome and failure to return to work at 3-months following TBI compared to those without intracranial pathology. Heightened surveillance and post-discharge resource allocation are recommended to optimize recovery.
Keywords: traumatic brain injury, diffuse axonal injury, outcomes, functional recovery, human studies
Severe TBI (sTBI) often results in long-term cognitive deficits such as reduced processing speed and attention. The intraparietal sulcus (IPS) is a neocortical structure that plays a crucial role in the deeply interrelated processes of multisensory processing and top down attention. Therefore, we hypothesized that disruptions in the functional and structural connections of the IPS may play a role in the development of such deficits. To examine these connections we used resting state fMRI (rsfMRI) and diffusion kurtosis imaging (DKI) in a cohort of 27 sTBI patients (29.3 ± 8.9 yrs) and 32 control participants (33.5 ± 13.4 yrs). Participants were prospectively recruited and received rsfMRI and neuropsychological assessments including the Automated Neuropsychological Assessment Metrics (ANAM) at greater than 6 months post injury. Results suggest that sTBI patients performed worse than controls on multiple subtests of the ANAM suggesting reduced cognitive performance. Reduced resting state functional connectivity between the IPS and the cortical regions associated with multisensory processing and the dorsal attention network was observed in the sTBI patients. The patients also showed reduced structural integrity of the Superior Longitudinal Fasciculus (SLF), a key white matter tract connecting the IPS to anterior frontal areas, as measured by reduced mean kurtosis (MK) and fractional anisotropy (FA) and increased mean diffusivity (MD). Furthermore, this reduced structural integrity of the SLF was associated with a reduction in overall cognitive performance. These findings suggest that disruptions in the structural and functional connectivity of the IPS may contribute to chronic cognitive deficits experienced by these patients.
Keywords: multisensory, severe TBI, resting state, diffusion kurtosis imaging, intra-parietal sulcus, cognitive deficits
MYELIN PLASTICITY SUPPORTS RECOVERY OF NERVE CONDUCTION VELOCITY AFTER EXPERIMENTAL TBI
USUHS, Anatomy, Physiology & Genetics, Bethesda, MD, USA
Patients with mild TBI (mTBI) often exhibit slow information processing speed which may indicate demyelination in white matter tracts. We used a mouse model of mTBI with traumatic axonal injury in the corpus callosum to examine myelin pathology and plasticity after mTBI. Functional integrity of myelinated axons in the corpus callosum was examined using electrophysiological recording in brain slice preparations from C57BL/6 mice after mTBI. Conduction velocity of the N1 myelinated axon waveform was significantly decreased at 3 days and 2 weeks post-TBI, consistent with demyelination. By 6 weeks, the N1 conduction velocity recovered while the refractory period was delayed, suggesting remyelination with incomplete recovery of conduction efficiency. To monitor active remyelination in vivo after mTBI, we generated fluorescent myelin reporter mice. Conditional expression of Cre recombinase was driven in mature oligodendrocytes using PLPCreERT2 mice or in oligodendrocyte progenitor cells using NG2CreERT2 mice. Each driver line was crossed to mTmG reporter mice so that tamoxifen administration induced green fluorescent protein (GFP) labeling of membranes. Analysis of naïve PLPCreERT2:mTmG mice confirmed GFP labeled mature oligodendrocytes and myelin throughout the corpus callosum within 7 days of tamoxifen administration. NG2CreERT2:mTmG mice were administered tamoxifen on days 2 and 3 post-mTBI to assess the progenitor response. At 7 days post-mTBI, GFP labeled progenitors extended multiple short processes that were morphologically distinct from the myelin sheath labeling in PLPCreERT2:mTmG mice. By 1 month post-mTBI, NG2 fate-labeled cells had matured into oligodendrocytes and elaborated myelin along axons. The corpus callosum area labeled with GFP myelin membrane was significantly greater in mTBI mice, relative to sham NG2CreERT2 mice, demonstrating remyelination after mTBI. Overall, mTBI produced demyelination resulting in functional deficits followed by remyelination and recovery that was still incomplete at 6 weeks after mTBI. This study was funded by the Department of Defense within the Center for Neuroscience and Regenerative Medicine.
Keywords: demyelination, remyelination, oligodendrocyte, myelin, conduction velocity, progenitor
Blast related TBI (bTBI) resulting from improvised explosive devices is the hallmark injury of recent wars affecting many returning veterans who have been exposed either to a direct or indirect exposure. Many of these exposed veterans suffer from long term neuro-cognitive symptoms. However, there is very little evidence to show whether blast-induced acceleration alone, in the absence of secondary impacts, can cause mild TBI. In this study, we examine the effect of under-vehicle blast-induced hyperacceleration (uBIH) on the biochemical and microstrucutral changes using diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS). Two groups of adult male Sprague-Dawley (SD) rats were subjected to asham procedure and uBIH respectively. Axonal and neurochemical alterations were assessed using in vivo diffusion DTI and MRS at 2 hours, 24 hours and 7 days after uBIH. Significant reduction in mean diffusivity, axial diffusivity and radial diffusivity was observed in the hippocampus, thalamus, internal capsule and corpus callosum as early as 2 hours and sustained up to 7 days post uBIH. Total creatine (Cr) and glutamine (Gln)were reduced in the internal capsule at 24 hours post uBIH. The reductions in DTI parameters, Cr and Glnin vivo suggests significant upregulation of astrocytes and diffuse axonal injury following a single underbody blast confirming previous histology reports.
Keywords: underbody hyperacceleration, MR spectroscopy, diffusion tensor imaging, corpus callosum, insula
REGIONAL ACUTE AND SUBACUTE ALTERATIONS IN GFAP, SPECTRIN, AND BREAKDOWN PRODUCTS FOLLOWING PENETRATING BALLISTIC-LIKE TBI IN RATS
Acute alterations of glial fibrillary acidic protein (GFAP), GFAP breakdown products (GFAP-BDPs), spectrin, and spectrin breakdown products (SBDPs) have been well studied following TBI but their longitudinal trajectory has not been well described. Here, we assess these pathology markers out to 1 month post injury following a penetrating ballistic-like brain injury (PBBI). Unilateral frontal PBBI was induced by inserting a probe through the right frontal cortex (FCx) and striatum (St) followed by a rapid balloon inflation to create a temporary cavity. Additional animals were exposed to probe insertion without balloon inflation or sham surgery (craniotomy only). At 3d, 7d, or 1m following injury, the ipsilateral FCx, St, hippocampus (HC), and residual midbrain (RMb) were dissected for analysis by western blot. Percent change from sham was determined at each time point. PBBI produced acute increases in GFAP at 3d (FCx, St, and HC) and 7d (FCx, St, HC and RMb) that remained evident at 1m post-injury in FCx, St and RMb. Concomitantly, GFAP-BDPs were increased in all regions at 3d and 7d and remained elevated at 1m in FCx, St, and RMb. Probe insertion also resulted in significant regional specific increases in GFAP and GFAP-BDPs that were mostly resolved by 1m post-injury. At 3d post-PBBI, spectrin decreased in the FCx whereas SBDPs increased in FCx, St, and RMb. At 7d, spectrin decreased in HC while SBDPs increased in FCx, St, and RMb. At 1m, spectrin decreased and SBDPs increased in St. Probe insertion resulted in increased SBDPs at 3d in FCx and decreased spectrin at 1m. Correlation analyses between these pathology metrics and acute neuroscore were performed and significant correlations were obtained, even one month following injury. These data demonstrate that TBI pathology remains evident into the subacute period and is not limited to the area of the injury tract. This highlights the need to consider long-term interventions when assessing TBI therapeutics.
Keywords: GFAP, GFAP break down products, spectrin, spectrin break down products, PBBI
LONGITUDINAL CHANGES IN BAX AND BCL-2 IN KEY REGIONS OF INTEREST FOLLOWING PENETRATING TRAUMATIC BRAIN INJURY IN RATS
Acute alterations of the pro- and anti- apoptotic factors BAX and Bcl-2 have been well established following severe penetrating traumatic brain injury (TBI). However, changes into the subacute period have not been well described. Here, we examine longitudinal alterations of these two apoptotic factors up to one month following penetrating ballistic-like brain injury (PBBI) in specific regions of interest. Unilateral frontal PBBI was induced by inserting a probe through the right frontal cortex (FCx) and striatum (St) followed by rapid balloon inflation to create a temporary cavity. Additional animals were exposed to probe insertion without balloon inflation or sham surgery (craniotomy only). Acute motor deficits were assessed 24 hr following injury. At 24 hr, 3d, 7d, or 1m post-injury, the ipsilateral FCx, St, hippocampus (HC), and residual midbrain (RMb) were dissected for analysis of BAX and Bcl-2 expression levels by western blot. Percent change from sham was determined for PBBI and probe only animals at each time point. PBBI resulted in significant elevations of BAX at 24 hr (FCx, St, and RMb) and at 3d and 7d (FCx and St), while probe resulted in more limited elevations in BAX at 24 hr (FCx), 3d (FCx and St), and 7d (FCx). Reduced expression of Bcl-2 was observed at 7d (FCx) in both the PBBI and probe groups. Significant correlations were detected between the expression of apoptotic factors and acute motor deficits at one week post-injury. These results indicate that expression of pro- and anti- apoptotic factors remains altered one week following severe penetrating TBI. For the most part, the observed alterations in BAX and Bcl2 expression appear to be limited to areas surrounding the injury trajectory. This work establishes a timeline against which these pathological markers can be used to evaluate the efficacy of anti-apoptotic pharmacotherapies and emphasizes the need to design drug therapy treatment regimens that extend beyond the acute post-injury phase.
Keywords: BAX, Bcl-2, apoptotic factors, PBBI
MINO PLUS NAC SYNERGISTICALLY TREATS TBI WITHIN A CLINICALLY RELEVANT THERAPEUTIC WINDOW
Currently there are no effective treatments for TBI. We have previously shown that the combination of FDA-approved drugs minocycline (MINO) and N-acetylcysteine when dosed 1-hour after injury synergistically improves both cognition and memory, modulates inflammation, limits grey matter injury and induces remyelination. The dosing window of MINO plus NAC needs to be explored to determine its utility as a therapeutic. In a rat controlled cortical impact model (CCI) of TBI. We report that MINO and MINO plus NAC improved cognition and memory when dosed 24-hours after injury. This therapeutic window was assessed using two behavioral tasks. In the active place avoidance task, a task with high cognitive demand. MINO plus NAC, improved cognition in the rat CCI model when dosed 12-hours after injury. The 12-hour therapeutic window of MINO plus NAC was increased to 24-hours using Barnes maze, a behavioral task that likely has a lower cognitive demand than active place avoidance. When dosed at 1-hour MINO plus NAC increased myelin content in white matter regions that lost axons, supporting the hypothesis that the drug combination of MINO plus NAC induces remyelination, this was seen with luxol fast blue staining. It is anticipated that MINO plus NAC will modulate inflammation, limit grey matter injury and induce remyelination when dosed beyond 1-hour window. These data suggest that MINO plus NAC limits brain injury within a clinically useful therapeutic window. These preclinical studies provide further evidence that that MINO plus NAC has sufficient potency and safety to be tested against clinical TBI.
Keywords: TBI, CCI, therapeutic window
USING A CERVICAL DORSAL HEMISECTION INJURY MODEL, THE AGGRECAN-DEGRADING ENZYME, ADAMTS-4, ENHANCES FUNCTIONAL RECOVERY
Univ. of Kentucky, Spinal Cord and Brain Injury Research Center, Lexington, USA
Following spinal cord injury (SCI), expression of chondroitin sulfate proteoglycans (CSPGs) in the resulting glial scar is elevated. Growing neurites are unable to penetrate the glial scar, in part due to CSPGs, deterring regeneration and preventing restoration of function. We have been investigating the potential of an enzyme that specifically degrades the CSPG, aggrecan, called A Disintegrin And Metalloproteinase with ThromboSpondin motifs, or ADAMTS-4, for promoting axonal outgrowth across the glial scar, and specifically, restoring forepaw function. Following a rodent cervical dorsal hemisection spinal cord injury (SCI), recombinant ADAMTS-4 was infused via mini-pump at the site of injury for 14 days. For some animals, tissue was processed for immunocytochemistry using antibodies to aggrecan, endogenous and recombinant aggrecan, axons, and astrocytes. For other animals, functional assessment was done using two behavioral paradigms, specifically, a Sticker Sensory Task and a Staircase Pellet Retrieval Task. Immunocytochemistry showed localization of ADAMTS-4 to the injury site and identifies axon and glial behavior within and beyond the injury region. While results using the Staircase test were inconclusive, results for the Sticker test indicated an improvement in sensory function related to ADAMTS-4 treatment. Tract tracing is in progress to determine the affects of ADAMTS-4 on dorsal column neurons in vivo. Although preliminary, the data suggest that ADAMTS-4 treatment, either alone or in combination with other successful treatments, such as chondroitinase, may be a promising avenue for enhancing regeneration and promoting functional recovery following SCI.
Keywords: sticker sensory test, dorsal hemisection, aggrecan, proteoglycans
University of Louisville, KSCIRC, Neurological Surgery, Anatomical Sciences & Neurobiology and Bioengineering, Louisville, USA
During walking, the nervous system receives continuous sensory feedback from both external and internal conditions. During Locomotor Training (LT), therapists use different techniques to provide sensory information similar to that which occurs during normal walking to activate and re-organize spinal circuitry responsible for basic stepping. The LT process, however, is slow with incomplete motor recovery in individuals with spinal cord injuries (SCIs). Despite the concentration of sensory receptors on the foot's sole, little attention has been paid to this area for facilitation of stepping-related movements. We are using a variety of surfaces to examine the influence of different stimuli on standing and walking activities following SCI. Initially, surfaces are tested in controls and rat pups with low thoracic SCIs on post-natal day 2 (P2). Those with injuries show limited hindlimb movement at P19 (17 days post-SCI) on smooth craft paper. In contrast, surfaces with hard punctate-type stimuli enhance hindlimb activity and weight support. When rearing activity is assessed during a cylinder task at P20, SCI rats show an abnormally smaller base of support compared to controls. By P51, the bases of support become more similar. Based upon findings in rats, surfaces are tested in children. A surface with hard punctate stimuli enhanced leg movements and muscle activity (EMG) in a non-ambulatory 4 year old with a severe SCI sustained shortly after birth. These preliminary findings suggest changes in walking surface characteristics may be beneficial in enhancing motor output. Supported by: Helmsley Foundation, Rebecca F. Hammond Endowment, VA-RR&D-RCS-B9249-S, Kentucky Spinal-Cord and Head-Injury Research Trust. Presentation content does not represent the views of the DVA or US-government.
Keywords: sensory input, locomotor training, mechanoreceptors, SCI
Traumatic brain injury (TBI) affects 2 million individuals in the United States each year, and many survivors endure long-lasting cognitive impairments associated with frontal lobe disturbances, while also being highly vulnerable to neuropsychiatric disorders. Clinical and preclinical research has highlighted the importance of chronic stress, particularly when presented in an unpredictable fashion, as a major risk factor for many psychopathological conditions. In the current study, we are beginning to assess clinically-relevant cognitive-behavioral and anxiety-like dimensions sensitive to both TBI and chronic unpredictable stress (CUS). We hypothesized that moderate TBI produced by controlled cortical impact (CCI) injury, as well as CUS exposure will render cognitive impairments in male rats in an attentional set-shifting test (AST), reduced sucrose preference and open field exploration, as well as blunted weight gain. Thirty isoflurane-anesthetized adult male rats were subjected to a CCI (2.8 mm cortical tissue deformation at 4 m/sec) or sham injury over the right parietal cortex. Following surgery, rats were randomly assigned to receive CUS (21 days starting 5 days post-surgery) or 30 sec of handling (CTRL). Upon cessation of stress, rats were tested for perceived state of anxiety (open field test) and anhedonia (reduced preference of 1% sucrose-water versus regular water overnight). At 4 weeks post-surgery, rats were then tested on the AST, which involves a series of increasingly difficult discriminative tasks to obtain food reward. Preliminary results demonstrate that CUS exposure leads to a 5–10% weight gain reduction compared to CTRL group. We anticipate that TBI will lead to cognitive deficits, as previously reported (Bondi et al., 2014, J Neurotrauma), which will be augmented by CUS exposure. This ongoing project will provide novel outcomes pertaining to cognitive capability, as well as anxiety- and depressive-like symptoms following overlapping chronic stress exposure and the recovery phase of TBI. Supported by UPP/UPMC (CB) and NS060005, HD069620 and NS084967 (AEK).
Keywords: chronic unpredictable stress, executive function, anhedonia, open field test
DOSE-RESPONSE EVALUATION OF AMANTADINE IN THE CONTROLLED CORTICAL IMPACT MODEL OF TRAUMATIC BRAIN INJURY: AN OBTT CONSORTIUM STUDY
University of Pittsburgh, Neurosurgery, Pittsburgh, USA
Amantadine (AMA) is a compound with several potential mechanisms useful in treating traumatic brain injury (TBI). Amantadine reportedly augments dopaminergic neurotransmission, acts as a partial NMDA antagonist and inhibits microglial activation. Amantadine was chosen as the ninth drug to be tested by the multicenter consortium Operation Brain Trauma Therapy. The University of Pittsburgh site tested Amantadine in the controlled cortical impact (CCI) model. Male Sprague-Dawley rats were anesthetized and underwent CCI (4 m/sec, 2.8 mm deformation) or sham surgery. Rats were randomized into four groups and administered Amantadine (10 mg or 45 mg/kg IP) or vehicle daily beginning 1 day after TBI. Groups were TBI-AMA-Low (n = 10), TBI-AMA-High (n = 10), TBI-Veh (n = 10) or Sham (n = 10). Functional outcomes were tested via beam balance Task, beam walking test (days 1–5), Morris water maze (MWM) acquisition (days 14–18) and probe trial (day 18). Rats were sacrificed on day 21 for histology. A repeated-measures ANOVA revealed a significant group main effects for beam balance and beam walk latencies (p < 0.01). None of the injured groups differed from each other. All injury groups performed significantly worse after TBI compared to the sham group. A significant group main effect (p = 0.0001) was found for the MWM test. Swim latencies across days did not differ between the injured groups regardless of treatment. Swim latencies for all the injured groups differed significantly from shams. There were no group differences in the probe trial. We conclude that treatment with either dosage of Amantadine after CCI did not improve sensorimotor or cognitive function. Although histology and biomarker data are still pending, behavioral findings of Amantadine treatment in the CCI model in rats do not support its further testing in OBTT. Support: US Army W81XWH-10-1-0623.
Keywords: traumatic brain injury, neuroprotection, behavior, OBTT, controlled cortical impact
Emory University School of Medicine, Pediatrics, Atlanta, USA
Keywords: proteomics, biomarker, traumatic brain injury, hypoxia-ischemia, osteopontin
Henry M Jackson Foundation/ Uniformed Services University of Health Sciences, Department of Pathology, Bethesda, USA
MicroRNAs (MiRNAs) are small endogenous RNA molecules and have emerged as novel serum diagnostic biomarkers for several diseases due to their stability and detection at minute quantities. In this study, we have identified a novel serum miRNA signature in human serum samples of mild and severe TBI, which can be used for diagnosis of mild TBI (MTBI). Human serum samples of MTBI, severe TBI (STBI), orthopedic injury and healthy controls were used and miRNA profiling was done using taqman real time PCR platform. The real time PCR data for the MTBI, STBI and orthopedic injury was normalized to the control samples which showed upregulation of 39, 37 and 33 miRNAs in mild, severe and orthopedic injury groups respectively. TBI groups were compared to orthopedic injury group which showed up-regulation of 13 and 17 miRNAs in MTBI and STBI groups. Among these, a signature of 10 miRNAs was found to be present in both MTBI and STBI groups. These 10 miRNAs were also validated in cerebrospinal fluid (CSF) from clinical samples of STBI. In conclusion, we identified a subset of 10 unique miRNAs which can be used for diagnosis of MTBI and STBI.
The opinions expressed herein are those of authors and are not necessarily representative of those of the Uniformed Services University of the Health Sciences, Department of Defense or, the United States Army, Navy, or Air Force and DMRDP.
*Dr Radha K Maheshwari has recently deceased. He has not seen this version of the abstract but he was the principal investigator on this project and has previously approved similar abstract.
Keywords: microRNA, TBI, biomarker
Withdrawn
Uniformed Services University of Health Sciences, Neurology, Bethesda, USA
Keywords: Von Willebrand factor, cellular fibronectin, phosphorylated neurofilament H, mild TBI
University of California Los Angeles, UCLA BIRC, Dept. Neurosurgery, Los Angeles, USA
The complexity of the pathology of TBI involving multiple components poses challenges for elaboration of effective diagnosis and therapeutics. We carried out a systems biology study using next generation sequencing and integrative genomics analyses to determine how TBI affects networks of genes that could characterize main events in the pathology, and how these signatures could serve as biomarkers of mild TBI (mTBI). We found that mTBI promoted reprogramming of important aspects of gene regulation (DNA methylation, transcript abundance, alternative splicing, and organization of genes in networks), which have the potential to alter the course of brain homeostasis and disease. We also analyzed leukocytes to trace homologies between central and peripheral events, and found homology between hippocampus and blood at gene-, pathway-, and network levels. Transcriptomic signatures in the hippocampus overlap with those in leukocytes, and that these signatures correspond to curated functional categories related to vascularity, cell integrity, and immune response. mTBI also elicited select methylomic changes in hippocampus and leukocytes that colocalized with the respective transcriptomic signature genes. We also found that mTBI promotes specific changes in the organization of genes in networks, under the regulatory control of key driver genes such as Anxa2 and Ogn. Gene networks identified in our rodent model of mTBI overlapped with candidate causal genes in human gene-wide association studies (GWAS) for several neurodegenerative diseases such as Alzheimer's disease, bipolar disorder, autism, cognitive performance, PTSD, and psychiatric disorders, which are proposed long-term sequel of mTBI. The overall results support the potential of our novel unbiased strategy to diagnose and to treat mTBI. In addition, our approach has the promise to get insightful information to elucidate one of the most intriguing aspects of mTBI, which is why many patients become vulnerable to a range of neurological disorders such as CTE, Alzheimer's, and psychiatric (NIH R01NS50465).
Keywords: epigenetic, genomic, system biology, GWAS
Turku University Hospital, Rehabilitation and Brain Trauma, Turku, Finland
Keywords: GFAP, UCH-L1, orthopedic injury
USUHS, Neurology, Bethesda, USA
Keywords: cerebrovascular reactivity, traumatic vascular injury, biomarker, phosphodiesterase 5 (PDE5) inhibitor, sildenafil
Washington Univ. School of Med., Div. of Critical Care Medicine, St. Louis, USA
Care for severe traumatic brain injury (sTBI) centers around avoidance of intracranial hypertension (ICH) and other secondary insults. Biomarkers predicting the development of ICH and ICH trajectory may help guide care. Using FDA compliant assays we evaluated the utility of cytoplasmic ubiquitin C-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein breakdown products (GFAP-BDP's) as candidate biomarkers to predict the development of ICH and ICH trajectories in children with sTBI. Using a threshold of 20 mmHg to define ICH we calculated pressure-time area under the curve values (AUC-ICH) to quantify ICH trajectories. When measured 6 h post-injury, both biomarkers predicted development of ICH in the first 12 h post-injury (C-statistic = 0.99 and 0.98 respectively). For ICH trajectories, UCH-L1 measured 6 h post injury predicted ICH-AUC (coefficient of 1.57; p < 0.01), and GFAP-SBDP-s was a reliable predictor at 24 h (coefficient of 2.65; p < 0.01). We evaluated the utility of combining both biomarkers into the neuronal-astrocytic injury index (NAII = sample quantile of UCHL-1 × sample quantile of GFAP-SBDP). When measured 24 h post-injury the NAII predicted total 72 h cumulative ICH exposure (1 NAII unit increase = 1,490 mmHg·h increase in total ICH-AUC; p < 0.001) and the patient's ICH-AUC on day 3 post injury (1 NAII unit increase = ICH-AUC on day 3 increases by 405.15 mmHg·h; p = 0.009). Both linear regression models included intensity of ICH therapy in the first 24 h and patient characteristics that may affect outcome (age, gender and race). The NAII correlated with outcome at 24 and 48 h post injury (r = −0.5, t = −3.4 and; r = −0.34, t = −2.17 respectively).
Keywords: UCH-L1, GFAP, serum, severe TBI
Medical College of Wisconsin, Neurosurgery, Milwaukee, USA
Repetitive subconcussive head impact (RSCHI) exposure in sports has important clinical implications, is thought to decrease injury tolerance, and was associated with cognitive decline even in the absence of concussion. Development of a preclinical model capable of recreating physics of RSCHI using a repeatable and noninvasive injury mechanism can provide valuable information to understand these phenomena. Sprague-Dawley rats were exposed to 25 subconcussive head rotational accelerations (58% or concussive acceleration magnitude from our previous studies) over five days using the MCW Rotational Injury Model, and cognitive deficits and changes in emotionality were assessed using Morris water (MWM) and elevated plus (EPM) mazes. Subconcussive level of exposure was confirmed by lack of any significant differences in MWM or EPM metrics following single head acceleration exposure compared to shams. Following 25 subconcussive accelerations, RSCHI rats demonstrated significantly increased (p < 0.05) EPM arm changes and open arm entries during the first week post-exposure, indicating greater activity and a lack of inhibition consistent with findings from concussive exposures delivered using this model. Although not statistically significant, rats exposed to RSCHI also demonstrated 38% increased EPM open arm time and 45% longer acquisition latencies during the third MWM session. Behavioral changes resolved by 30 days post-injury, although RSCHI rats maintained 32% greater EPM open area time compared to shams. Due to its noninvasive nature, scalable head acceleration exposures, and incorporation of an injury mechanism that matches the physics of human exposures, the present model is ideal to study effects of subconcussive exposures. This preliminary study identified significant acute changes in emotionality following 25 exposures, along with non-significant trends demonstrating possible cognitive deficits. Continued work using this model will define effects of prior subconcussive exposure on the magnitude of insult required to produce concussion and the correlation between number/severity of subconcussive exposures with behavioral changes in the acute and chronic phases.
Keywords: biomechanics, subconcussive, repetitive exposure, sports concussion
Injury from blunt impact is a multi-scale problem where impact forces transmit from the skull to the cellular level. We hypothesize the type and degree of trauma is related to mechanical loading and deformation patterns throughout the brain tissue where each trauma is unique to other types of head trauma. The span and degree of tissue deformation (corresponding to the level of injury) varies across different regions of the brain and is determined by the biomechanical parameters of the blunt injury event (velocity, momentum, direction) and the anatomical boundary conditions of the human head, brain and neck. In this study we developed a full scale skull-brain-neck model with a simulated brain to map the spatial and temporal distribution of deformation in various regions in the brain under blunt impact conditions. A skull filled with a ballistic gelatin was be fixed to a biofidelic Hybrid 3 neck and exposed to controlled blunt impact on a Cadex droptower system. Markers spaced >5 mm apart were painted onto the surface of a simulate brain to measure deformations patterns throughout the impact using a high speed 3D video imaging system operating at 1000 fps. A 5 kg impactor was dropped from heights for impact velocities of 3&5 mph to the forehead and crown. Models were constructed to examine the brain deformation at various depths using a half, & and full skull. Captured video of the motion of the brain markers was used to calculate the tissue level strains and rates of strains using ProAnalyst Software and a custom algorithm. While rotations of the head (not linear motions) are generally agreed to produce brain deformation, strains were found through the cranium under blunt impacts with primarily linear motion of the skull. Maximum strains of 15% were produced from the 5 mph impact within the hippocampal region. Deformation maps show the measured motion extending to the brain skull interface. The strain values and location for the produced strain maps provide an important insight into the deformation pattern of the brain under blunt impact that will be useful in linking brain deformation to the type and degree of neuronal damage.
Keywords: physical model, brain model, brain deformation, biomechanics, blunt injury
COMPRESSION IMPAIRS FUNCTIONAL AND ANATOMICAL RECOVERY AT VARIABLE IMPACT FORCES IN CONTUSIVE SPINAL CORD INJURY
Spinal cord injury (SCI) causes victims life-long hardships, largely due to limited treatment options following injury. Effective and accurate models are pivotal in treatment development, but an obstacle to translating findings from basic science models to clinical application is heterogeneity of human SCI. Two important elements of SCI, contusion and compression, are commonly utilized in SCI models; however, little is known about the effects of compression at variable impact forces or the effects of the compression on specific aspects of secondary cascades following injury. Using an Infinite Horizons impactor, our study characterizes the anatomical and functional recovery in C57/Bl6 mice after SCI with or without a 20s compressive element at two contusion impact forces (50 or 75 kdyn). We hypothesized that compression will increase tissue damage and impair functional recovery regardless of impact force. Indeed, 28 days post injury (dpi), cross sectional tissue sparing at the lesion epicenter was significantly reduced in animals that received compressive SCI, regardless of impact force (50 kdyn >20s50 kdyn and 75 kdyn >20s75 kdyn). Lesion length also increased with compression, but less drastically than tissue sparing (50 kdyn <20s50 kdyn and 75 kdyn <20s75 kdyn), indicating a greater effect of compression on lesion epicenter circumference than rostral-caudal spread. Functional recovery, measured by BMS, closely resembles cross sectional sparing with significant decreases due to compression (50 kdyn >20s50 kdyn and 75 kdyn >20s75 kdyn). Interestingly, compression groups cease recovery at 14 dpi while non-compression groups continue improving until 28 dpi; the earlier recovery plateau may indicate differences in secondary recovery cascades due to compression in the primary injury. These data further justify our investigation into effects of compression on specific aspects of secondary cascades following SCI. This study will improve the understanding of the effects of compression on secondary cascades and outcomes of SCI, and will aid in development of SCI therapies.
Supported by NIH NINDS R01NS091582.
Keywords: compression, contusion
CHARACTERIZATION OF A NOVEL CLOSED HEAD MODEL OF DIFFUSE TRAUMATIC BRAIN INJURY
Over 2.5 million people are admitted to an emergency department for traumatic brain injury (TBI) every year in the United States. An additional 3–5 million people are living with the long-term effects of TBI at an estimated annual cost of $60 billion. Moderate-severe TBI is the most debilitating subtype. Representative models of TBI are required to devise therapies for such patients. Though many small animal models exist to study brain trauma, current methods require substantial surgical intervention, or have suffered from experimental variability. Surgery may confound important effects of neuroinflammation, cerebral edema, and alterations of the blood-brain barrier or cerebral blood flow from the primary injury. We therefore developed a non-surgical, tunable model of mouse moderate-severe TBI focused on reproducibility and based off of the CHIMERA platform. This platform delivers precise, monitored, closed-skull impact via a pneumatically-driven piston, and allows rotation of the body post-impact. Two primary modifications of the CHIMERA model were required to produce moderate-severe TBI: a padded backboard to limit hyperflexion injury of the spine, and a semi-rigid helmet to diffuse the piston impact, preventing skull fracture. Animals undergoing this procedure exhibit diffuse traumatic axonal injury as demonstrated by bAPP staining. Axonal injury is apparent in the corpus callosum, anterior commissure, hippocampal commissure, and fimbria bilaterally. Neuroinflammation, revealed by staining against microglial markers, is prominent. Injured animals demonstrate multidomain neurobehavioral deficits that remain evident weeks after injury. Importantly, the procedure does not generate a focal cavitary lesion as occurs in many moderate-severe TBI models. The absence of a dominant focal lesion permits analysis of brain injury without gross cortical or white matter degeneration. We believe that this moderate-severe TBI model provides an important tool for the analysis of post-traumatic brain injury, and will form the basis of future therapeutic trials targeting the behavioral impairments resulting from such pathophysiology.
Keywords: chimera, diffuse, amyloid, moderate, severe, brain
CONTROLLED LOW-PRESSURE BLAST-WAVE EXPOSURE CAUSES DISTINCT BEHAVIORAL AND MORPHOLOGICAL RESPONSES MODELLING MTBI AND PTSD
Ben-Gurion University of the Negev, Faculty of Health Sciences, Anxiety and Stress Research Unit, Beer-sheva, Israel
The intense focus in the clinical literature on the mental and neuro-cognitive sequelae of explosive blast-wave exposure, especially when they co-morbid with post-traumatic stress-related disorders (PTSD) is justified and warrants the design of translationally valid animal studies to provide valid complementary basic data.
We employed a controlled experimental blast-wave paradigm in which non-anesthetized animals were exposed to visual, auditory, olfactory, and tactile effects of an explosive blast-wave. By combining cognitive-behavioral paradigms and ex-vivo brain MRI to assess mild traumatic brain injury phenotype with a validated behavioral model for PTSD, complemented by morphological assessments, this study sought to examine our ability to evaluate the bio-behavioral effects of low-intensity blast overpressure on rats, in a translationally valid manner.
There were no significant differences between blast and sham exposed rats on motor coordination and strength, or sensory function. Whereas almost half of the male rats exposed to the blast-wave displayed normal behavioral and cognitive responses, 38.5% of the rats displayed a significant, retardation of spatial learning acquisition, fulfilling criteria for mTBI-like responses. In addition, 6.0% of the blast-exposed animals displayed an extreme response in the behavioral tasks used to define PTSD-like criteria, whereas 6.8% of the rats developed both long-lasting and progressively worsening behavioral and cognitive “symptoms”, suggesting comorbid PTSD-mTBI-like behavioral and cognitive response patterns. Neither groups displayed changes on MRI.
This experimental animal model can be a useful tool to elucidate neurobiological mechanisms underlying the effects of blast wave-induced mTBI and PTSD and comorbid mTBI-PTSD.
Keywords: blast wave, post-traumatic stress disorder, mild traumatic brain injury, animal model
Naval Medical Research Center, NeuroTrauma, SilverSpring, USA
Grant#603115HP.2380.001.A1304.
Keywords: stress, en-route care, coagulation, animal model
Naval Medical Research Center, Neurotrauma, Silver Spring, USA
Traumatic brain injury (TBI) following blast may be associated with changes in the cerebral vasculature. A prominent complication associated with severe TBI is significant cerebral vasospasm. It has been observed that blast-exposed rats demonstrate vascular weakening, reduction of endothelial glycocalyx structure, and blunting of induced vasoconstriction in cerebral arteries. Despite clinical indications of vascular insult and supporting experimental data in animals, there is little information on structural and functional changes in the cerebral vasculature that occur after blast exposure. Established rodent models were used to assess effects of exposures to overpressures associated with mild to moderate TBI. Three blast intensities were chosen, 37, 75, and 140 kPa, to represent that range. Vessels were observed through a cranial window at two and twenty-four hours after injury. Vessel reactivity was tested by elevating pCO2, application of barium chloride, and application of serotonin. Between treatments the pial surface was washed with artificial cerebrospinal fluid (CSF). There was little change in vessel reactivity for all blast levels compared to control at two hours after injury. After 24 hours there was significant change in the three blasted groups compared with control animals (p < 0.01, ANOVA and post-hoc Tukey HSD): the responsiveness of vessels to treatment with barium chloride was increased after 24 hours. Additionally, both 37 kPa and 75 kPa were significantly different from control when treated with serotonin (p < 0.05 and 0.01 respectively). At these time points there was no significant effect of the different blast ‘doses.’ It may be that there are differences that this experiment cannot detect. It may also be that in the blood vessels, the physiological changes following blast occur regardless of blast intensity, so that even a mild exposure has a significant effect on vessel responsiveness. A better understanding of short term changes in the cerebral vasculature following blast TBI is critical to understanding the long term effects of blast on the brain.
Keywords: blast, vascular, mTBI, vasoconstriction, vasospasm
New Jersey Institute of Technology, Center for Injury Biomechanics Materials and Medicine, Newark, USA
Blast-induced Traumatic Brain Injury (bTBI) has been a major health hazard to warfighters in the recent conflicts in Iraq and Afghanistan. When an object (e.g. human or animal models) is placed in the path of a blast wave, the shock overpressure is felt unabated whenever there is a flow path for the shock wave to travel from outside to the surface of the body. The magnitude of overpressure is determined by the direct (transmitted) as well as indirect loading associated with how the gaps are filled. This knowledge is critical in the design of protective gear for the humans and protective shield for experimental animal models in shock tubes. Our goal was to design a shield protecting the head of an animal, which allows loading of the torso. The shell of the head cover is made of plastic (PLA, UPET) or metal (steel, aluminum) while soft and hard foam, and ballistic gel served as gap fillers between the specimen and the shield. It was found that the pressure profile inside the animal head cavity resembled closely external overpressure profile when the filler was not present. The foam use resulted in only marginal reduction in pressure and moderately affected the pressure rise slope. The only way to eliminate pressure surge in the cavity is to completely block the rear side using metal; the presence of ballistic gel or foams results in increased overpressure in the cavity. These observations lead to the conclusion that in order to fully isolate the effect of external shock wave, complete isolation of the region of interest with solid surfaces is necessary. This is the key finding to properly design protective equipment for humans and to design experiments based on animal models.
Work funded by US Army under contract W81XWH-15-1-0303.
Keywords: BINT, attenuation/propagation of shock wave, head protection, mitigation of blast wave.
Blast-induced traumatic brain injury (bTBI) is the signature wound of the military conflicts in Iraq and Afghanistan. Understanding the physical mechanism(s) of injury is critical for the development of preventative measures and therapeutic treatments for affected soldiers and civilians. Direct shock transmission, thoracic surge, skull flexure, rotational acceleration/deceleration and cavitation are hypothesized to be the biomechanical mechanisms causing bTBI. In this study, we investigate cavitation formation as a possible response to a shock wave in a pseudo-biomimetic surrogate head-brain model system. The deleterious effect associated with cavitation is the formation of bubbles followed by subsequent implosion causing pressure jet release. This sequence of events has been previously proposed based on studies using computational models and to date no experimental validation have been attempted. A fluid-filled, polycarbonate cylinder instrumented with pressure sensors was exposed to blast waves with peak overpressure in the 70–180 kPa range in a compressed gas-driven shock tube and bubble formation was observed using high speed video (5000 fps). We were able to vary four key parameters and discern the effects of each on cavitation formation (blast intensity, boundary conditions of head, skull geometry, and viscoelastic properties of brain matter and CSF). Experiments comparing constrained vs unconstrained boundary conditions revealed the role of bending on the head-brain system. Shell thickness is inversely related with cavitation formation, caused by the flexure of the polycarbonate cylinder. We can conclude that boundary conditions of our head-brain system significantly affect pressure measurements and bubble formation, while increasing shock intensity and reducing shell thickness increases propensity for cavitation in response to a blast wave. Our results also show that as blast overpressure increases, the number of bubbles visually observed also increases
This work was supported by N00014-14-1-2637, Office of Naval Research (Project monitor: Timothy Bentley)
Keywords: blast, TBI, cavitation
PSYCHOSOCIAL LEARNING DEFICITS AFTER MILD BLAST INJURY ARE INDUCED BY INTRACRANIAL DEFORMATION AND OXIDATIVE STRESS
Purdue University, Biomedical Engineering, West Lafayette, IN, USA
Mild blast-induced traumatic brain injury (mild bTBI) has been labeled the ‘signature injury’ of modern conflict and has been reported to account for over half of battlefield-related TBIs. The aim of this investigation was to explore physical and biological mechanisms linking mild bTBI to development of post-blast neuropsychiatric abnormalities common among military veterans. Rats were exposed to mild bTBI and evaluated in behavioral metrics under normal and anxiogenic conditions. Post-mortem brain tissue was assessed via immunohistochemistry (IHC) and immunoblotting (IB), and daily post-injury urine samples were analyzed for changes in an oxidative stress metabolite. In addition, wireless deformation sensors were implanted to measure brain deformation in skull-brain phantoms and in vivo during blast exposure. Mild bTBI rats demonstrated psychosocial safety learning deficits from 9–14 days post-injury despite a lack of other motor or cognitive impairments. The severity of psychosocial dysfunction correlated with 24 hr post-injury urine metabolite elevations of a toxic post-trauma neurotoxin, acrolein, which was elevated for up to three days in urine and five days in brain tissue. Further, IB investigation of acrolein revealed significant elevations in regions associated with the psychosocial safety learning paradigm at 24 hours post-injury. Most other brain regions did not demonstrate significant elevation, suggesting intrinsic mechanical or biological susceptibility of these areas to our mild bTBI model. Additionally, IHC revealed blood-brain-barrier disruption and inflammatory activity in and around brain regions where acrolein was elevated. Significant brain deformation is suspected in these regions at the time of injury based on our phantom and in-vivo deformation recordings. Taken together, shock wave exposure can physically disrupt and biochemically dysregulate the brain in key regions involved in psychosocial learning. This study has significant implications suggesting that damage from mild bTBI can, if left unabated, directly lead to long-term neuropsychiatric consequences and reduced quality of life.
Keywords: psychosocial learning, oxidative stress, blood-brain barrier, neuroinflammation
Univ. of Maryland, Baltimore, Anesthesiology, Baltimore, USA
Many victims of blast-TBI are in vehicles targeted by explosions from land mines. This study used a rat model of underbody blast TBI to test the hypothesis that at induced accelerations greater than 1000G, there is dose-dependent brain injury, based on behavior, histology, and neurochemistry. We also tested the hypothesis that hull designs which reduce the acceleration experienced by vehicle occupants can reduce brain injury and improve survival at high G forces. Male rats were restrained on top of a platform that was accelerated vertically at 1200, 2400, or 2800G, in response to the detonation of a small explosive positioned under a second, bottom platform separated from the top by different structures. Blast and sham animals were subjected to behavioral assays to assess working memory and anxiety-like behavior using Y maze and Plus maze tests, respectively, for up to 28 days post-injury. Rats were euthanized at 24 hr or 30 days post-blast and their brains used for either histopathology or neurochemical measurements. 67% of rats exposed to 2800G blasts died almost immediately. All rats exposed to 1200 or 2400G blasts survived and exhibited the following dose-dependent changes: Increased number of cleaved caspase-3 immunoreactive or TUNEL positive cells neurons in the hippocampus, amygdala and cerebellum. Increased perivascular F4/80 positive activated macrophages/microglia. Increased cortical GFAP-stained glial scars. Reduced cortical levels of synaptophysin and synaptophilin. Impaired performance on the Y maze and particularly the Plus maze. The presence of polyurea-coated cylinders between the top and bottom platforms (hulls), reduced the G force on the rats from 2800 to 500G, resulting in complete survival and reduced TBI. The acceleration experienced by occupants of vehicles targeted by underbody blasts is sufficient to cause brain injury and mortality. Double hull vehicle designs that dampen the acceleration experienced by occupants can mitigate brain injury and death in a combat-relevant paradigm. Supported by US Army W81XWH-13-1-0016.
Keywords: acceleration, apoptosis, depression, memory, oxidative stress
Virginia Tech, Biomedical Engineering, Blacksburg, USA
Clinical manifestations of blast induced neurotrauma (BINT) include a diverse array of cognitive and behavioral symptoms driven by persistent inflammation at the cellular level. Epigenetic regulation serves as an important mediator of gene expression and cellular function which may underlie the chronic inflammation likely resulting in neurodegeneration. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Animals were subjected to blast overpressure (10 or 17 psi) within an Advanced Blast Simulator. Sham animals underwent same procedure without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at two and seven days post-injury. Sham animals showed in-tact memory at both time points. Both injury groups showed dramatically decreased performance between time points. This is indicative the onset of memory impairment. Tissues were collected at seven days for ex vivo assessment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure at 10 and 17 psi. No changes were observed for Ionized Calcium-Binding Adapter molecule 1 (IBA-1), a marker for microglia. Analysis of histone protein extract, showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups. Colocalization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments focused on the anterior cingulate cortex (ACC) within the PFC and showed a similar decrease in H3 acetylation in astrocytes exposed to a 17 psi blast but not the 10 psi blast. Such changes in astrocytes may play a role in the chronic activation associated with BINT and may lead to potential therapeutic targets.
Keywords: epigenetic, cognitive impairment, astrocyte, chronic
The cyclin-dependent kinase inhibitor 1 (p21Cip1) was initially characterized as a key inhibitor of the complexes of CDK2 and CDK1. The protein is encoded by the cdkn1a gene. Recent evidence suggests that an abnormal reactivation of the cell cycle may precede and cause the hyperphosphorylation and filament formation of tau protein in Alzheimer's disease and other tauopathies. Although significant increases in tau and phosphorylated tau (p-Tau) proteins have been widely described in multiple brain regions following blast-induced traumatic brain injury (bTBI) in rodents, the mechanism(s) underlying these changes is still undefined. To explore possible involvement of proteins involved in cell-cycle progression in these mechanisms, in the present study we investigated expression of these proteins in the rat cerebral cortex following exposure to the combination of blast with blunt brain injury. Anesthetized SD rats (male, 350 g) were exposed to blast overpressure in an air-driven shock tube (19 psi total peak pressure) followed immediately by weight-drop (500 g, 125 cm). When compared to sham controls, cdkn1a gene expression was upregulated at 6 hrs post-injury and p21 Cip1 was significantly increased at both 1 day and 1 month after the traumatic insults. Although the expression of the cdkn1a gene is tightly controlled by the tumor suppressor protein p53 in response to a variety of stress stimuli, changes in the expression of p53 gene or protein were not found. The association between p21 and p-Tau was further assessed in an in vitro experiment in which p21 CRISPR/dCas9 Lentiviral activation particles were transfected to primary cultures of cortical neurons. Overexpression of p21Cip1caused a significant increase in Tau and p-Tau expressions in these neurons. These combined results indicate that p21Cip1 may play significant roles in cytoskeletal dynamics and apoptosis in addition to cell cycle regulation. In particular, TBI-induced overexpression of p21Cip1 might contribute to tauopathy and might therefore serve as a potential therapeutic target for CTE mitigation.
Keywords: blast neurotrauma, p21(Cip1), p-Tau, tauopathy
INVESTIGATION OF NANOPARTICLE ACCUMULATION AFTER EXPERIMENTAL BRAIN INJURY
Arizona State University, School of Biological and Health Systems Engineering, Tempe, USA
Nanoparticles (NP) are an important area of research for various disease diagnosis and therapy because of their ability to deliver a wide range of particles/drugs to varying areas of the body for sustained periods of time. In the case of traumatic brain injury (TBI), that leads to a transient dysfunction in the blood-brain barrier (BBB), it provides a “window of opportunity” to deliver NPs, which otherwise maybe impermeable. However, there is a critical gap in understanding the influence of NP size for effective delivery to completely utilize this opportunity. In this study, our objective was to evaluate the effect of NP size after experimental brain injury in both focal and diffuse injury models. In this direction, a cocktail of four different sized PEGylated NPs (20, 40, 100 and 500 nm) was injected intravenously in mouse at various time points after brain injury (either controlled cortical impact, CCI, or midline fluid percussion, FPI). For the CCI model, peak accumulation at 1 h post-injury for all sized NPs near the injury penumbra was observed. Moreover, the 20 and 40 nm NPs displayed prolonged (13 h post-injury) accumulation as compared to 100 and 500 nm (6 h post-injury). Furthermore, NP displayed preferential accumulation within the cortex where the primary motor and somatosensory areas demonstrated higher accumulation as compared to the parietal association and visual area. For the FPI model (midline), our preliminary results indicate higher accumulation of NPs (20, 40, 100 and 500 nm) specifically near the injured cortex (motor/sensory areas) at 3 h post-injury, as compared to the sham. Taken together, we have shown the potential for accumulation of up to 500 nm sized NP acutely after both focal and diffuse brain injury. Results from our study can be used to strategically design efficient NPs for TBI diagnostics and/or therapeutics.
Keywords: nanoparticles, blood brain barrier breakdown, intravascular delivery, theranostics
Although cytosolic phospholipid A2 (cPLA2) has long been associated with inflammation and neuronal injury, its profile and specific role in traumatic brain injury (TBI) remain unclear. In this study, we investigated expressional change of cPLA2 and, if so, its possible role in TBI. A controlled cortical impact (CCI) injury was produced at an impact depth of 2.0 mm for SD rats or an impact depth of 1.0 mm for C57BL/6 cPLA2 knockout mouse using an electromagnetic impactor (Impactor OneTM, MyNeuro Lab; tip diameter: 3 mm; speed: 3 m/s; dwell: 50 ms). Western blot analysis showed that the cPLA2 expression level in the rat cortex significantly increased and peaked at 3 (3.18-fold; p < 0.05) and 7 days (2.88-fold; p < 0.05), compared to the sham-operated group, and returned to the basal level at 14 days after CCI. Immunohistochemical examination revealed that cPLA2 immunoreactivity (IR) was weak in the rat cortex contralateral to the injury. In contrast, cPLA2 IR was markedly increased in the injured cortex particularly at the lesion penumbra. Immunofluorescence double labeling further showed that cPLA2 expressed in neurons, oligodendrocytes, astrocytes, and macrophages following TBI. In cortical neuronal cultures, exogenous delivery of PLA2 or melittin, an activator of endogenous PLA2, induced cortical neuronal death in a dose-dependent manner, which was substantially reversed by annexin A1, a PLA2 inhibitor. Importantly, blocking cPLA2 pharmacologically at 30 minutes post-injury in rats or deleting cPLA2 genetically in cPLA2 knockout mice inhibited caspase-3 apoptotic cascade and reduced neuronal death. These findings collectively suggest that cPLA2 may play an important role in the pathogenesis of TBI and as such could be an attractive target for TBI treatment.
Keywords: traumatic brain injury, cPLA2, secondary injury, apoptosis
CARDIOLIPIN REMODELING RESPONSE TO TRAUMATIC BRAIN INJURY IN THE IMMATURE BRAIN
Traumatic brain injury (TBI) triggers a series of metabolic responses beginning with the oxidation of a mitochondria specific phospholipid, cardiolipin (CL). The oxygenated CL species formed, undergo phospholipase A2γ (iPLA2γ)-catalyzed hydrolysis generating two products: monolyso-CL and free oxygenated fatty acids whereby the former can undergo remodeling to CL by monolysocardiolipin acyltransferase (mCL-AT) and tafazzin (TAZ). We hypothesized that CL remodeling pathway is induced after TBI in a time and region specific manner. Post-natal day (PND) 17 male rats underwent CCI (4 ± 0.2 m/sec velocity, 2.5 mm depth, 50 msec duration). Rats were sacrificed at 1 h, 4 h or 24 h after injury. Levels of Tim23 (translocase of inner membrane), β-actin, mCL-AT, TAZ and iPLA2g were evaluated by western blot in ipsilateral cortex, hippocampus and striatum. Liquid-chromatography mass spectrometry-based lipidomics analysis was performed in the contusional cortex. Consistent with the high diversity of CLs, the levels of TAZ and mCL-AT were lower in the normal cortex compared to hippocampus and thalamus. Tim23 levels (normalized to actin) decreased in all three regions at 1 h and 4 h after CCI suggesting increased mitophagy after the injury returning to the normal levels at 24 h. CCI caused oxidation of CL which peaked at 1 h post injury. Concomitantly, an increase in the cortical iPLA2γ level (normalized to Tim23) was observed which was accompanied by the increased monolyso-CL content (2-fold at 1 h and 3-fold at 4 h and 24 h after injury). These changes were followed by the enhanced expression of remodeling enzymes in the cortex: mCL-AT at 1 h and 4 h and TAZ at 24 h after CCI. Similar CCI induced temporal changes in iPLA2γ, mCL-AT and TAZ levels were observed in the hippocampus. However mCL-AT protein levels did not increase at any time point in thalamus despite an increase in iPLA2γ at 1 h and 4 h after the injury. These results indicate that CL oxidation and hydrolysis are associated with time- and region-specific activation of remodeling pathways after TBI.
Support: AI068021, NS061817, NS076511
Keywords: cardiolipin remodeling, traumatic brain injury, liquid-chromatography mass spectrometry, tafazzin
LYSOSOMAL DAMAGE AFTER SPINAL CORD INJURY IS ASSOCIATED WITH NECROPTOSIS
University of Maryland, Baltimore, Anesthesiology & Shock, Trauma and Anesthesiology Research (STAR) Center, Baltimore, USA
Necroptosis is a caspase-independent programmed cell death mediated by the receptor-interacting protein kinase 1 (RIPK1). Necroptosis is induced following spinal cord injury (SCI) and is thought to pathologically contribute to the injury. However, the mechanisms leading to activation of necroptosis after SCI remain unclear. Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner. Current literature suggests that inhibition of the autophagy-lysosomal system can facilitate necroptosis by providing a platform for assembly of necrosomes on autophagosomal and autolysosomal membranes. Using a rat model of contusive SCI, we observed accumulation of LC3-II positive autophagosomes starting at post-trauma day 1. This was accompanied by a pronounced accumulation of autophagy substrate protein p62, indicating that early elevation of autophagy markers reflects disrupted autophagosome degradation. Levels of lysosomal protease cathepsin D (CTSD), numbers of CTSD-positive lysosomes and CTSD enzymatic activity were also decreased at this time, suggesting that lysosomal damage is the likely cause of autophagy flux inhibition. Interestingly, motor neurons showing disrupted autophagy co-expressed RIPK1, suggesting defects in lysosomal function and disrupted autophagy after SCI are associated with induction of necroptosis. High-resolution imaging revealed that RIPK1 accumulated in intracellular punctate structures, which co-localized with CTSD positive lysosomes. Therefore, lysosomal damage may contribute to the observed RIPK1 accumulation in motor neurons. In agreement with lysosomal inhibition contributing to RIPK1 accumulation, in vitro treatment of PC12 cells and cortical rat neurons with lysosomal inhibitors also lead to increased expression of RIPK1 protein. RIPK1 localized to lysosomes in both untreated and lysosomal inhibitor treated cells, suggesting that lysosome-dependent degradation may contribute to normal RIPK1 homeostasis. Therefore, lysosomal dysfunction and inhibition of autophagy after SCI may contribute to non-apoptotic neuronal cell death by promoting RIPK1 accumulation and facilitating necrosome assembly.
Keywords: autophagy, necroptosis, lysosome, RIPK1
MJC Veterans Affairs Medical Center, Philadelphia, USA
Diffuse traumatic brain injury (TBI) typically results from rapid rotational acceleration-deceleration of the head. The resulting cellular damage is often heterogeneous, as certain individual neurons are more susceptible to acute biophysical responses, such as plasmalemmal disruption, and subsequent physiological aberrations post-injury. Using an established model of closed-head rotational acceleration in swine, we have previously shown that various neuronal populations become flooded by the normally cell-impermeant dye Lucifer Yellow (LY) within 15 minutes, indicating that membrane disruptions may occur rapidly in diffuse TBI. In the current study, we extend these findings to determine whether acutely permeabilized neurons exhibited markers of apoptosis following a single injury or repetitive injuries. To assess acute apoptosis, we performed immunohistochemistry using an antibody recognizing caspase-cleaved α-spectrin, a protein product that denotes an irreversible step in the apoptosis pathway. Confirmation of apoptosis was performed using a standard TUNEL assay. Sham injured animals exhibited a paucity of permeabilized (LY+) neurons, and were devoid of caspase cleaved α-spectrin immunoreactivity. Following single injury, approximately 25% of acutely permeabilized neurons in the cerebral cortex exhibited characteristics of apoptosis. Not surprisingly, there was a significant increase in the density of LY+ neurons in the cerebral cortex after repetitive TBI. TUNEL-positive staining and caspase-cleaved α-spectrin immunoreactivity was also significantly elevated following repetitive TBI. Moreover, we found a 3-fold increase in the percentage of LY+ neurons that were also positive for apoptotic markers compared to single injury. This demonstrated a significantly elevated density of apoptotic neurons strongly correlating with plasmalemmal disruptions in repetitive, but not single, head rotational injury. Our results shed light on the pathophysiological progression and ultimate fate of acutely injured neurons following repetitive TBI in a clinically relevant large animal model of closed-head diffuse brain injury. Financial support provided by NIH and Department of Veterans Affairs.
Keywords: apoptosis, TBI
University of New Mexico, Neurosurgery, Albuquerque, USA
Support: NIH-NINDS 8P30GM103400.
Keywords: TBI, hemorrhagic shock, resuscitation, hemorheology, drag reducing polymers
University of Pennsylvania, Dept. of Anesthesia & Critical Care, Philadelphia, USA
Traumatic brain injury (TBI) is the leading cause of injury related death in children, with boys and children under 4 years having particularly poor outcomes. Cerebral autoregulation is often impaired after TBI, contributing to cerebral ischemia and poor outcome. Previous studies showed that cerebral autoregulation is more impaired in male compared to female newborn pigs after TBI, which parallels the clinical experience. Cerebral perfusion pressure and CBF are often augmented by use of vasoactive agents such as epinephrine (EPI). Yet, cerebral effects of this clinically commonly used vasoactive agent are not known. Some vasoactive agents (Phenyleprhine) improve brain blood flow and protect autoregulation in female newborn piglets but make these hemodynamic indices of outcome worse in male newborn piglets after TBI, which mirrors what is seen clinically. The c-Jun-terminal kinase (JNK) isoform of mitogen activated protein kinase produces hemodynamic impairment after TBI, but less is known about its role in histopathology. We investigated whether EPI age and sex dependently protected cerebral autoregulation and limited histopathology after TBI and the role of JNK in that outcome. Results show that EPI protects autoregulation, prevents histopathology, and blocks JNK upregulation in newborn males and females and juvenile females but not juvenile males after TBI. These data indicate that EPI protects cerebral autoregulation and limits histopathology after TBI through blockade of JNK in an age and sex dependent manner.
Supported By NIH RO1NS090998.
Keywords: cerebral autoregulation, CPP, signaling, neurovascular unit, histopathology
University of Pittsburgh, Neurosurgery, Pittsburgh, USA
Keywords: progesterone, SyNAPSE trial, CSF vs serum, severe TBI
University of Louisville, Neurosurgery, Louisville, USA
Current spinal immobilization technologies are inadequate for treating spinal injuries because they are designed to have a degree of extension. Forced extension is counterproductive in injuries that require optimal volume area in the cervical spinal canal and space at the neural foramina. Furthermore, fracture alignment is not adequete nor maintained with the current generation of immobolization. Current limitations of existing cervical collars were identified including pain and pressure; noncompliance; poor immobilization of axial spine; and muscle atrophy. Patient characteristics that limit a collar's effectiveness were identified: cervical deformity; obese or long thin necks; pediatric population; atrophy; and incisions. A novel three-piece design was proposed for the shell with custom fixation points based off of a patient's MRI or CT scan and a inner insert that alings the spine so that the maximum space is available for the cord or ideal fracture alignment. Mimics software was used for additive manufacturing and 3D printing. Custom design using negative space to protect healing incsions and pressure points and low-cost reprints of the inner insert to accomodate for atrophy over the course of wearing the collar are also available. 3D models of patient's spines and soft tissues could reliably be created and ideal alignment achieved on software with a formula built upon space available for the cord or fracture alignment to determining the amount of flexion or extension the patient should be placed in. A case of a 26 yo male with unilateral facet fracture who failed conservative management with standard cervical orthotic showed only 40% limitation of movement in sagittal plane, low comfort score, and noncompliance required surgical fixation. A custom printed collar based upon the patient's 1 mm computerized tomography scan aligning his facets showed 90% reduction of movement with greater comfort score. Our cervical collar design allows the spine to be more effectively stabilized than the current standard of external cervical orthotics. Initial analysis also suggests improved cost effectiveness.
Keywords: computer modeling, 3D printing/additive manufacturing, cervical orthosis and immobilization, cervical spine trauma
Mount Sinai, Neurology, New York, USA
Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder associated with traumatic brain injury (TBI) characterized by the presence of hyperphosphorylated tau into neurofibrillary deposits. The diagnosis of CTE is established postmortem. However, a new molecular ligand for positron emission tomography (PET), [18F]-T807, may provide the antemortem detection of pathological neurofibrillary aggregates. Our goal in the present study was to examine [18F]-T807 uptake in athletes with mild traumatic brain injury (mTBI) resulting from multiple concussions. Here we report a 39-year-old retired National Football League player with progressive cognitive decline who suffered at least 22 concussions. Serial neuropsychological exams revealed decline in executive functioning, processing speed, and fine motor skills. Naming was below average but performance in other cognitive tests was preserved. Longitudinal structural analysis revealed cortical thickness loss in left frontal and lateral temporal areas, and volume loss in the basal ganglia. PET with [18F]florbetapir was negative for amyloidosis showing a standard uptake value ratio (SUVr) of 0.929. The [18F]T807 tau radioligand showed multifocal retention at the cortical grey matter-white matter junction, a distribution similar to the tauopathy of CTE defined postmortem. [18F]T807 SUVr analysis showed increased uptake (SUVr ≥1.1) in bilateral cingulate, occipital, and orbitofrontal cortices, and several temporal areas. This case study provides a window into the cognitive and structural progression of CTE. While identification of the neuropathological underpinnings of [18F]T807 retention requires postmortem correlation, our data suggest that [18F]T807 tau imaging is a promising tool to detect and diagnosis of CTE-related neuropathology in living subjects.
Keywords: CTE, tauopathy, neurofibrillary tangles, tau PET imaging
Lerner research institute at Cleveland clinic, Neurosciences, Cleveland, USA
Alzheimer's disease (AD) is the most common neurodegenerative disease with no cure or way to stop or slow down the progression of the disease. One of the postulated environmental risk factors for AD is traumatic brain injury (TBI). Key characteristics of both TBI and AD include an increase of neuroinflammation, which is characterized by enhanced microglial activation, increased cytokine production, and recruitment of peripheral macrophages. Recently, heterozygous mutations in
Keywords: macrophages, TREM2, neuroinflammation, immunohistochemistry
Previous studies have shown that Azithromycin (AZM), a macrolide antibiotic, has the ability to induce alternative macrophage polarization, and improve behavioral/ biochemical outcomes in animal models of spinal cord injury (Zhang et. Al 2015; Amantea et al., 2016). The present study determined AZM whether improves cognitive outcomes and reduces brain inflammation in a rat model of diffuse TBI. Adult male Sprague-Dawley rats were administered AZM (40 mg/kg) via oral gavage, once-daily for three days prior to medial fluid percussion injury (MFPI; 1.9 atm), with an additional treatment 15 minutes post-MFPI. Animals were allowed to recover for 3 days then underwent spatial memory assessment using the Morris Water Maze (MWM). All animals had 4 daily training trials across 4 days of testing in the MWM. MFPI animals treated with vehicle had significantly impaired performance in the MWM, in terms of latency/distance to goal, compared to sham animals. AZM treated MFPI animals exhibited improved performance in the MWM compared to sham animals. Following behavioral testing (7 days post MFPI), brain sections were prepared for [3H]-PK11195 autoradiography to assess expression of the Translocator Protein 18 KDa, a marker of neuroinflammation. Image J was used to perform a thresholding analysis of [3H]-PK11195 binding in the somatosensory cortex, hippocampus and thalamus. Preliminary analysis of a subset of animals revealed no significant attenuation of brain inflammation by AZM as assessed by this measure in any brain region evaluated. These studies support the idea that behavioral outcomes following neurotrauma can be mitigated by AZM administration, but the specific biological underpinnings of this beneficial action remain to be determined.
These studies were supported by Grant # 13-8A from the Kentucky Spinal Cord and Brain Injury Research Trust
Keywords: neuroinflammation, neurotrauma, translocator protein 18 KDa, Morris Water Maze
CHRONIC VS INTERMITTENT ADMINISTRATION OF THE ANTIPSYCHOTIC DRUGS, HALOPERIDOL OR RISPERIDONE AFTER EXPERIMENTAL TRAUMATIC BRAIN I
Preclinical studies have shown that chronic administration of the antipsychotic drugs (APDs), haloperidol (HAL) or risperidone (RISP), impairs neurobehavioral recovery after TBI. However, APDs are often administered intermittently rather than continuously to TBI patients as restlessness and agitation occur irregularly during the recovery phase. The goal of this study was to test the hypothesis that intermittent (1 or 3 times per week) administration of HAL and RISP would produce less deleterious effects on motor and cognitive recovery compared to chronic (daily) administration. Anesthetized adult male rats received a controlled cortical impact (2.8 mm tissue deformation at 4 m/s) or sham injury and then were provided HAL (0.5 mg/kg; i.p.), RISP (0.45 mg/kg; i.p.) or vehicle (VEH; 1 mL/kg; i.p.), with administration starting at 24 h after surgery and then either once daily (chronic), or 1 or 3 times weekly for 19 days. Beam-balance/walk and Morris water maze performance were assessed on post-injury days 1–5 and 14–19, respectively. The data revealed that the TBI groups administered HAL or RISP intermittently did not significantly differ from the VEH+TBI group in both motor and cognitive outcome, which is opposite to the chronic (once daily) treatment paradigm, which performed worse in all behavioral measures compared to the VEH+TBI group (p < 0.05). These findings replicate previous reports that chronic administration of HAL or RISP negatively impact motor and cognitive recovery after cortical impact. Additionally, the data expands the research by demonstrating that intermittent administration of HAL or RISP, regardless of whether once or three times a week, did not impair recovery as those groups did not differ from the TBI+VEH-treated controls. The potential clinical implications of these findings suggest that intermittent administration of the APDs HAL or RISP may be safely used to control agitation in TBI patients, without negatively impacting subsequent recovery.
Keywords: environmental enrichment, antipsychotics
PREDICTING BRAIN INJURY USING HEAD KINEMATICS
Effective brain injury risk models can improve how we predict, diagnose, and prevent brain injury in many applications, from assessing head impact severity with wearable helmet sensors, to evaluating efficacy of automobile restraint systems during an automobile crash. Kinematic-based metrics relate the severity of a head impact to a mathematical function of the velocity and/or acceleration components of translational and/or rotational head motion. Currently, no single injury criterion is universally accepted for a diverse range of head impact conditions. In this study, fifteen kinematic-based metrics were compared to finite element (FE) model-predicted brain deformations from simulations using a broad range of head kinematics including data from football impacts and various automotive crash test modes. Correlations between brain strain and angular velocity-based metrics were highest among those evaluated, while metrics based on linear acceleration were least correlative. BrIC and RVCI, two rotationally-based metrics, had the highest overall correlation with FE strains; however both metrics had limitations. These results suggest that rotational metrics are the most important kinematic parameters for predicting brain injury. Based on our correlation assessment, a parametric study was performed using a FE head model to elucidate the physics of brain deformation caused by rotational head motion. Brain deformation in short-duration impacts (<30 ms) was proportional only to maximum angular velocity, while in long-duration impacts (>60 ms), deformation was proportional only to maximum angular acceleration. For impacts with moderate duration, a combination of velocity, acceleration, and duration was needed to determine maximum brain deformation. These results explain some limitations associated with existing brain injury models. Finally, existing brain injury criteria were evaluated for their ability to correctly predict non-injurious impact. Several criteria, including ones proposed for automotive safety assessment and helmet protection ratings, substantially overpredict the likelihood of concussion and diffuse axonal injury when applied to head kinematic data from a series of well-instrumented human volunteer impact studies. This work highlights the need for further development of a universal brain injury criterion.
Keywords: injury criteria, finite element modeling, head kinematics, automotive, football
CHRONIC TRAUMATIC ENCEPHALOPATHY IN ATHLETES IN THE SUBACUTE PERIOD AFTER CONCUSSIVE IMPACT AND A MOUSE MODEL OF IMPACT CONCUSSION
Boston university School of Medicine, Boston, USA
The mechanisms by which head injury induces acute concussion and chronic sequelae are not known. We examined postmortem brains from young athletes in the subacute period after closed-head concussive impact injury and found parenchymal contusion, myelinated axonopathy, microvasculopathy, neuroinflammation, neurodegeneration, and phosphorylated tauopathy consistent with early chronic traumatic encephalopathy (CTE). We developed a biofidelic mouse model of closed-head impact injury that induces non-skull-deforming head acceleration and transient signs of concussion in non-anesthetized C57BL/6 mice. Mice subjected to lateral head impact exhibited contralateral limb and trunk weakness, impaired gait and balance, and locomotor abnormalities that recapitulate neurological signs and temporal course of acute concussion in humans. Neurological function rapidly returned to baseline; however, CTE-linked pathology, phosphorylated tau proteinopathy, and functional sequelae persisted long after recovery. We used an albumin-binding gadolinium contrast agent to demonstrate in vivo blood-brain barrier disruption that upon histological analysis colocalized to brain regions exhibiting neuroinflammation and CTE-linked tau proteinopathy. Notably, concussion did not correlate with markers of CTE pathology or functional sequelae.
Keywords: modeling, preclinical animal model, neuroinflammation, imaging
A subconcussive impact is a milder form of brain injury that often goes undetected due to a lack of immediate concussion-like symptoms. However, multiple subconcussive impacts can produce behavioral deficits and cellular dysfunction. Subconcussive impacts have not been extensively examined in the TBI field. Specifically, the mechanisms of subconcussive impacts and their relationship to behavioral dysfunction are not fully understood. In order to further investigate these underlying mechanisms, we developed a clinically relevant closed head animal model of a subconcussive impact in adult rats using a Leica controlled cortical impact (CCI) device. Animals received either a single subconcussive injury or three subconcussive injuries administered 48 hours apart. Motor coordination (foot fault test) was tested at baseline, post-injury day (PID) 3, 7, 11, 21, and 27. No significant motor deficits were observed in either the single or repeat group at either time point. Object recognition was examined using the Novel Object task at PID 7 and 27. No significant memory deficits were observed in either group at either time point. No apparent gross pathology was observed on the surface of the brains for either group. In conclusion, we have developed a closed head injury model where one to three subconcussive impacts were not enough to produce any memory or motor deficits. Future studies will investigate a higher number of impacts to determine the threshold for behavioral symptoms. We will also perform histological examination of the tissue for markers of pathology. We would like to thank the Kozlowski Lab members and the RFUMS collaborators. This study is funded by DePaul University Research Council.
Keywords: Subconcussive impact, Animal model, Closed head injury, Stereotaxic surgery
LONGITUDINAL ANALYSIS OF ARTERIAL SPIN LABELING PERFUSION IN MILD TRAUMATIC BRAIN INJURY
GE Healthcare, Waukesha, USA
Keywords: mild traumatic brain injury, arterial spin labeling, linear mixed effects model, cerebral blood flow
Johns Hopkins University School of Medicine, Baltimore, USA
Funding: ImmunArray, Inc.
Keywords: substance abuse, outcome, recovery, traumatic brain injury
Loma Linda University, Radiology, Loma Linda, USA
Approximately 14% of school age children with sports-related concussions (SRC) remain symptomatic 3 months after injury. We and others have previously shown regions of white matter diffusivity changes and hypoperfusion in symptomatic patients in the chronic phase of mild TBI; however data in the pediatric population remains limited. Using a whole-brain spatial mapping and a voxel-wise statistical approach, we investigated the anatomical distribution of white matter diffusivity and hypoperfusion in chronic symptomatic pediatric SRC subjects. Twenty two adolescents (15 ± 3 years) who sustained a SRC (3–24 months before imaging) and 15 controls (15 ± 3 years) were enrolled in the study. Conventional 3D T1 weighted (T1w), 30 direction diffusion tensor imaging (DTI; b = 0 and 1000 mm/s2), and dynamic susceptibility contrast (DSC) perfusion weighted imaging (PWI) were acquired at 3.0T (Siemens Tim Trio) using a 12 channel receive-only head coil. Relative CBF maps were generated using Olea Sphere (Olea Medical, Cambridge, USA) using a Bayesian probabilistic estimation algorithm with automatic arterial input function selection. Maps of fractional anisotropy (FA), mean diffusivity (MD), axial and radial diffusivity (AD, RD) were calculated using Camino (University College London, United Kingdom). Intrasubject image registration, warping to a T1w template and segmentation was performed using ANTs. Voxel-wise analysis was performed using the Randomise tool in FSL, where highlighted clusters represent significant perfusion differences at p < 0.05. Cluster voxel-wise analysis of the DTI maps showed clusters of elevated AD in the right frontal white matter (p < 0.05) and elevated MD in the right frontal and parietal white matter (p < 0.09) suggesting injury to the axons. Clusters of hypoperfusion were seen in the temporal lobe (p < 0.05) and thalamus (p < 0.09) and likely reflect regions of reduced metabolism or neuronal loss. These findings reflect chronic neuronal injury following SRC which likely contributes to long term cognitive and behavior deficits and posttraumatic headaches in these student athletes.
Keywords: pediatric, sports concussion, diffusion tensor imaging, cerebral blood flow
Mind Research Network, Albuquerque, USA
History of mild traumatic brain injury (mTBI) and repetitive subconcussive head blows (rSHB) have separately been associated with increased symptomatology and long-term white matter damage. However, few studies have examined both factors in well-powered samples. A total of 216 collegiate athletes (133 males) underwent a multishell, high angular resolution diffusion imaging sequence. Participants were stratified into high (football or soccer; N = 108) or low (spirit squad, baseball, basketball or volleyball; N = 108) rSHB risk groups. A subset of participants completed a modified version of the Rivermead Postconcussion Symptoms Questionnaire (RPSQ) and Cogstate battery. Eighty-two athletes self-reported at least one mTBI (mTBI+), with 115 athletes indicating no history (mTBI-). Preliminary results indicate that frequency of mTBI+ was similar across both high (48/106; 45.2%) and low (34/91; 37.4%) risk groups (X2 = 1.26, p = 0.261); however, a statistical trend (X2 = 3.19, p = 0.074) was observed for higher number of repetitive injuries in the high (17/106; 16.0%) compared to low (7/91; 7.7%) risk group. There were no differences (p > 0.10) between high and low risk groups based on age of first mTBI or average symptom burden/degree of unresolved symptomatology (RPSQ_3 or RPSQ_13). There were no significant differences between either grouping variable (high vs. low risk rSHB; mTBI+ vs. mTBI-) for age, history of migraines or ADHD, or handedness (p > 0.10). mTBI+ athletes more frequently endorsed a learning disorder (Fisher's Exact p = 0.044). Finally, no differences were observed between either grouping variable on Cogstate performance (accuracy or reaction time). Current results therefore suggest minimal cognitive or post-concussive symptom differences for either exposure or mTBI history in collegiate athletes on brief measures. Analyses comparing traditional diffusion metrics (fractional anisotropy) and geometrically-derived measures of intracellular and isotropic water fractions are ongoing.
Keywords: subconcussive head blows, mTBI history, cogstate, RPSQ
Naval Medical Research Center, Operational and Undersea Medicine/ Neurotrauma Department, Silver Spring, USA
A growing body of literature indicates high rates of post-traumatic stress disorder (PTSD) in military personnel experiencing a blast related mild TBI (mTBI), however it is unclear if the direct effects of blast exposure predispose an individual to PTSD. If blast exposure does predispose an individual to PTSD, it follows that brain regions involved in fear should be impacted by a blast event. Two areas that may be affected are the amygdala and the medial prefrontal cortex (mPFC). The amygdala plays a key role in the brain's “fear circuitry” interacting with the mPFC to mediate fear responses. Evidence indicates that exposure to repeated blast overpressure (BOP) may damage or causes alteration to the brain's fear circuitry, but it is unknown how and if the amygdala is damaged. Evidence indicates the fear-related protein Stathmin-1 is elevated in the amygdala of an mTBI animal model. To assess fear circuitry damage, and molecular correlates of repeated blast exposure, animals were exposed to BOP (75 kPa) once daily over 3 consecutive days. Following repeated BOP exposure animals were behaviorally tested to examine locomotion, anxiety, and fear. Animals were euthanized 9 days following the last BOP exposure. Brain tissue and serum were collected for molecular assays. No mortality and no abnormal locomotor activity were observed following repeated BOP exposure. Stathmin-1 protein and corticosterone levels were measured using western blot and ELISA respectively. Rats exposed to repeated BOP exhibited similar freezing behavior to controls during acquisition and lower freezing during extinction compared to controls. Additionally, Stathmin-1 protein levels were elevated in the amygdala of BOP exposed rats. Finally, there was a trend for corticosterone levels to be higher in the BOP exposed animals. Overall, the behavioral phenotype of our animal model reflected no elevated freezing during acquisition and reduced freezing during extinction. However, the elevated Stathmin-1 levels are in line with previous research in a repeated BOP model examined at a later time point (6–8 months) compared to the current study, indicating repeated BOP does affect this particular fear-related protein (Elder 2012). Further research is necessary to develop our repeated BOP animal model and understand the time course of the relationship between the behavior and the molecular correlates.
Keywords: blast, traumatic brain injury, animal model, PTSD, amygdala
Neurostructural Research Labs, Neurotrauma Studies Group, Tampa, USA
TBI from multiple concussions is strongly associated with contact sports. The relationship between repetitive concussions and neuronal damage is poorly understood. We assessed the effects of multiple concussions on dendritic branching of cortical, hippocampal, and striatal neurons in the adult mouse. Using a closed head injury paradigm, a 30 gram weight was dropped on the right temporal-parietal region of anesthetized mice (N = 3 per group). There were 5 concussions, each with a 24 hour interval (5xTBI). After a recovery period of 30 days, the mice were sacrificed. Sham controls were handled identically. For coded slides, brains were stained using the Rapid Golgi method. For dendritic branching analysis, camera lucida drawings were prepared and analyzed for amount, distribution, and complexity of the dendritic arbor.
Keywords: dendrites, golgi staining, multiple concussions, cortex, hippocampus, striatum
Traumatic brain injury (TBI) is closely and bidirectionally linked with alcohol use, as by some estimates intoxication is the direct or indirect cause of one-third to one-half of all TBI cases. Alcohol use following injury can reduce the efficacy of rehabilitation and increase the chances for additional injury. Finally, TBI itself may be a risk factor for the development of alcohol use disorders. Children who suffer TBIs have poorer life outcomes and more risk of substance abuse. We used a standardized closed head injury to model mild traumatic brain injuries. We found that mice injured as juveniles but not during adulthood exhibited much greater alcohol self-administration in adulthood. Further, this phenomenon was limited to female mice. Using behavioral testing, including conditioned place preference assays, we showed that early injuries increase the rewarding properties of alcohol. Environmental enrichment administered after injury reduced axonal degeneration and prevented the increase in drinking behavior. Additionally, brain derived neurotrophic factor gene expression, which was reduced by TBI, was normalized by environmental enrichment. In a follow up experiment, we have shown that a single intraperitoneal dose of alcohol induces neuroinflammatory responses that are of greater magnitude in mice injured as juveniles. Finally, administering binge drinking-like levels of ethanol to mice induces neurodegeneration and inflammation which is exacerbated by prior juvenile injury. Together these results suggest a novel model of alterations in reward circuitry, inflammatory responses and behavior following trauma during development.
Keywords: mTBI, alcohol, inflammation, juvenile development, reward
EPIGENOMIC APPROACH TO THERAPEUTIC TARGET IDENTIFICATION, VALIDATION AND DRUG DISCOVERY FOR TRAUMATIC BRAIN INJURY
Roskamp Institute, Sarasota, USA
Traumatic brain injury (TBI) is the main cause of disability in various population groups including military/veterans and athletes, and has long been known as a risk factor for neurodegenerative disorders such as Alzheimer's disease (AD). Growing evidence now links a distinct neurodegenerative pathology termed chronic traumatic encephalopathy (CTE) to exposure to repetitive mild TBI (r-mTBI) or to single, moderate or severe TBI. However, the neuropathological processes that occur years following these traumas are acknowledged as being complex, mixed, and poorly understood. Investigation of clinically relevant laboratory models is critical to understanding the neuropathology of brain injuries and to reveal potential therapeutic targets, enabling future translation into human practice. Truly longitudinal studies have been missing from the research field, and will make substantial contribution to understanding TBI progression. Our mouse model of r-mTBI (5 hits/48 hours interval) elicited neurodegenerative outcomes persisting up to 24 months after injury. Proteomic analysis of r-mTBI mice at a range of different time-points post injury showed significant changes in molecular profiles at chronic (3,6,9,12 months) compared to early time-points (24 hours). For example, our data revealed time-dependent dysregulation of histone H1A which showed opposite activation patterns at 9 and 12 months compared to 3 months after TBI. Histone H1A regulates glycoprotein M6A which is responsible for neuroplasticity and was shown to be downregulated in patients who suffered from stress and those who committed suicide. Moreover, we observed changes in transcriptional factor EIF2AK4 activation in cortex and mR-122 dysregulation in hippocampus. In order to further investigate chronic epigenomic changes in r-mTBI we are exploring methylation profiles in our models. Epigenetic responses to TBI could explain the persistence of pathogenic processes long after the injury event(s), and reveal novel pathways and proteins as targets for therapeutic intervention.
Keywords: epigenomics, mild TBI, animal models, proteomics
Stanford University, Mechanical Engineering, Stanford, USA
In this study, we identified a mechanistic relationship between head acceleration and brain strain that may explain sports-related mild traumatic brain injury (mTBI) symptoms. Animal, computational, and physical models suggest the brain has direction-, structure-, and rate-dependent tolerance to strain. However, human data is limited and prior laboratory findings have not been combined to form a biomechanical injury mechanism with sequential consistency. We analyzed acceleration measurements of a diagnosed mTBI and hypothesized that the preceding impact caused cellular damage in the corpus callosum through coronal head rotation, lateral motion of the falx cerebri, and fast-rate fiber strain. Coronal rotational acceleration (12890 rad/s2) was most atypical among all six rotation and translation directions, a standard deviation above the next highest of 108 non-injury impacts. In finite element simulations, we found strong correlation between peak coronal rotation and lateral falx displacement (r > 0.76), largest (7.5 mm) in the mTBI impact. Lateral falx displacement was most correlated with fiber tract strain in the corpus callosum (r > 0.75) and thalamus (r > 0.66), periventricular brain structures just below the longitudinal fissure and strongest (lowest deviance) strain predictors of diagnosed mTBI among all structures. Removing both the fissure and falx in the mTBI simulation doubled peak cortical tract strain but halved peak strain in the corpus callosum. mTBI corpus callosum strain and strain rate were large enough to exceed proposed thresholds of microtubule rupture and cell death. Our results suggest the corpus callosum may be sensitive to coronal rotation because it drives deep lateral motion of a structural weakness, the longitudinal fissure, in the direction of commissural fibers. Since corpus callosum damage may produce mTBI symptoms due to impaired interhemispheric communication, further investigation of this mechanistic relationship is prudent.
We thank NIH, NSF, David and Lucile Packard Foundation, Stanford Child Health Research Institute, and Ford Foundation for financial support.
Keywords: sports concussion, finite element method, wearable sensors, injury mechanism, blunt head trauma, corpus callosum
Keywords: concussion, mild traumatic brain injury (mTBI), head impact dose, intelligent mouthguard, American football, boxing
LINEAR HEAD KINEMATICS PREDICTS DURATION OF UNCONSCIOUSNESS AND SEVERITY OF AXONAL INJURY IN THE CHIMERA MOUSE MODEL OF CONCUSSION
CHIMERA (Closed Head Injury Model of Engineered Rotational Acceleration) is a rodent traumatic brain injury (TBI) platform that integrates head kinematics with functional and neuropathological outcomes. We previously showed that mice subjected to two mild, closed-head TBIs with an impact energy of 0.5 J using CHIMERA faithfully replicate several features of human TBI including repeatable human-like head kinematics (Namjoshi et al 2015). The present study was conducted to address two aims: 1) to assess head kinematics and acute neurological and histopathological outcomes following single TBI with increasing impact energy dose and 2) to determine the relationship between impact energy dose-dependent changes in head kinematics and post-TBI outcomes. Male C57Bl/6 mice at 4 months of age were subjected to a single TBI using impact energies of either 0 (sham) or 0.1, 0.3, 0.4, 0.5, 0.6 or 0.7 J. Head kinematics were assessed with high-speed (9000 fps) videography. Duration of loss of righting reflex (LRR) was recorded immediately after TBI. Axonal injury was assessed with silver stain at 2 d post-TBI. Mice subjected to single TBI showed an impact energy dose-dependent increase in several parameters of linear head kinematics (displacement, velocity and acceleration), LRR duration and silver uptake in white matter regions. Further analyses revealed a significant positive correlation of the all three linear head kinematic parameters with LRR duration (R2 > 0.41 and p < 0.02) as well as the degree of silver uptake in the optic tracts (R2 > 0.46 and p < 0.02). Our present data indicates that linear head kinematics may be able to predict the duration of unconsciousness and severity of axonal injury following single closed-head TBI in mice using CHIMERA.
Keywords: Animal model, Head Kinematics, Loss of consciousness, Axonal injury
CANNABINOID RECEPTOR TYPE-2 MODULATES NOCICEPTIVE SIGNALING MOLECULES AND PAIN BEHAVIOR IN A MODEL OF POST-CONCUSSION HEADACHE
Headache, a common symptom of post-concussion syndrome, persists in a substantial portion of patients. The cannabinoid receptor type-2 (CB2R) is an ideal analgesic target for post-traumatic headache as it's devoid of psychotropic properties, modulates nociception, and regulates inflammation. The goal was to determine the role of the CB2R in the trigeminal pain pathway in a model of post-concussion headache. Sprague Dawley rats were randomized to receive either a single mild closed head injury (CHI), repeated mild CHI, or served as incision controls. Von Frey for trigeminal allodynia was performed at baseline, up to 4, and at 24 hours post-treatment with either saline or CB2R agonist (JWH133). Changes in CGRP, iNOS, nNOS, and Iba-1 for microglia were assessed in the trigeminal ganglia and/or trigeminal nucleus caudalis (TNC) using immunohistochemistry, western blot or ELISA. An in-vitro brain slice study was performed using 300 μm slices from TNC and cerebrum regions incubated with capsaicin, JWH133, or media control for 24 hours, followed by an assessment of CGRP and PGE2 released using ELISA. Repeated CHI increases the levels of CGRP in the caudal brainstem compared to incision, p < 0.001. There was no change in CGRP in these regions found in single CHI. Repeated CHI show altered IBA1 and iNOS immunoreactivity in the TNC compared to control. Repeated CHI increases CGRP in capsaicin-stimulated TNC slices compared control, p < 0.01. JWH-133 blocked capsaicin-induced increases in CGRP and PGE2 in the TNC and cerebrum slices, p < 0.001. Repeated CHI induced increases in capsaicin-triggered PGE2 in the cerebrum slices, p < 0.01, but not in TNC slices indicating other pain mediators are important in this pain region. There was a graded effect on trigeminal allodynia, in which JWH-133 increased trigeminal thresholds after repeated CHI to baseline levels, effects that were comparable with the NSAID, Ketorolac. Findings show the CB2R contribute to trigeminal pain in a model of concussion, although the mechanisms eliciting analgesia warrant a more in-depth investigation. DODW81XWH-14-1-0594; DODW81WH-12-1-0326
Keywords: post-traumatic headache, post-concussion headache, cannabinoid receptor type-2, nociceptive signaling
POSIPHEN REVERSES BEHAVIORAL DEFICITS AND NEUROPATHOLOGY IN A RAT MODEL OF MILD TRAUMATIC BRAIN INJURY
UCLA, Neurology, Los Angeles, USA
We tested Posiphen in the lateral fluid percussion (LFP) model of mild TBI to investigate its ability to prevent the neurological damage secondary to TBI. In adult rats, LFP produces cognitive impairment in the Morris water maze (MWM) and leads to progressive damage to dopaminergic neurons in the substantia nigra, a characteristic of Parkinson's disease.
Keywords: TBI, lateral fluid percussion, learning and memory, neuroinflammation, neurodegeneration
University of Arizona College of Medicine Phoenix, Translational NeuroTrauma Research Program, Phoenix, USA
Traumatic brain injury (TBI) in children can result in cognitive, emotional, and somatic neurological impairments. Hypo-pituitarism can impact these impairments, likely due to mechanical damage, which are rarely reported in children. Post-traumatic endocrinopathies may leave them with lifelong disabilities that impair quality of life. To address this issue, we identified patients who experienced a TBI and subsequently developed a new-onset hypothalamically-regulated endocrine disorder. We retrospectively analyzed patients from 2008–2016 seen at a nationally-recognized level-1 Trauma Center in Arizona. We identified 121 patients who were diagnosed with both a TBI and an endocrine disorder. Of these patients, 62 were excluded because their endocrine disorder diagnosis predated their TBI diagnosis; 46 more were excluded because of pre-existing medical conditions that influenced their endocrine function. We report on 16 patients who have one or more of 40 head injury ICD-9 codes and subsequently diagnosed with one or more of 116 new-onset endocrinopathy ICD-9 codes. The patients age of TBI onset ranged from 3 months to 14 years, their endocrinopathies developed between 3 days and 16 years after their TBI, Glascow Coma Scale Score at time of injury ranged from 4 to 15, and 56% (9/16) were male. 70.6% (12/16) of patients developed hypothyroidism, 11% (2/16) delayed puberty, 5.6% (1/16) diabetes insipidus, 5.6% (1/16) growth hormone insufficiency, and 5.6% (1/16) short stature. TSH values ranged from 0.02–584.2 mIu/mL; Free T4 from 0.2–5.4 ng.dL; and IGF-1 from 16–238 ng/mL. Radiological series including CT and MRI showed subdural hematomas variable in size and location, without localization to endocrine structures. Children who experience TBIs have the potential to develop hypo-pituitarism, though this relationship needs further investigation with larger-scale epidemiological studies.
Keywords: hypothalamic, endocrinopathy, idiopathic endocrine disorder
San Francisco General Hospital, Brain and Spinal Injury Center, San Francisco, USA
Traumatic brain injury (TBI) produces a complex disorder that is traditionally stratified based on clinical signs and symptoms. Recent imaging and molecular biomarker innovations provide unprecedented opportunities for improved TBI precision medicine, incorporating patho-anatomical and molecular mechanisms. Complete integration of diverse data for TBI diagnosis and patient stratification remains an unmet challenge. The TRACK-TBI Pilot multicenter study enrolled 599 acute TBI patients and collected common data elements (CDEs) across all patients, including imaging, genetics, and clinical outcomes. Topological data analysis (TDA) algorithms were applied to these CDE data to identify natural subgroups of TBI patients to organize and map them in multidimensional space, identifying a subset of mild TBI patients with a specific multidimensional phenotype associated with impaired recovery at 6 month outcome. Further analyses revealed that this patient subset had high rates of post-traumatic stress disorder (PTSD), and enrichment in several distinct genetic polymorphisms in striatal dopamine processing (ANKK1, COMT, DRD2), and a gene associated with cell death (PARP1). The findings identify a unique diagnostic subgroup of patients with unfavorable outcome after mild TBI. Biomarker screening for this unique subgroup may help direct improved therapeutic targeting in an at-risk population of mild TBI patients (ClinicalTrials.gov Identifier NCT01565551).
Funding: DoD grant W81XWH-13-1-0441 (GTM), NIH grants NS067092 (ARF), NS069409 (GTM) and NS069409-02S1 (GTM), Craig H. Neilsen Foundation grant 224308 (ARF) and Wings for Life Foundation (ARF).
Keywords: traumatic brain injury, post-traumatic stress disorder, topological data analysis, genetic polymorphisms
UCSF, Neurosurgery, San Francisco, USA
Over 20% of mild traumatic brain injury (mTBI) patients experience persistent impairment at 6-months, which may be complicated by patterns of excessive alcohol use. Blunt mTBI patients without acute intracranial pathology from the TRACK-TBI Pilot study with emergency department (ED) Glasgow Coma Scale (GCS) 13–15 and recorded blood alcohol level (BAL) were extracted (100-mg/dl as proxy for excessive use (>U.S. legal limit 80-mg/dl)). Multivariable regression was performed for patients with 6-month Glasgow Outcome Scale-Extended (GOSE, n = 67) and Wechsler Adult Intelligence Scale-Processing Speed Index Composite Score (WAIS-PSI, n = 58), adjusting for age, education, psychiatric history, GCS, loss-of- consciousness (LOC). Overall, 107 patients aged 42.7 ± 16.8-years, 67.3%-male, and 80.4%-Caucasian were included; 65.4% had zero BAL, 5.6% BAL <100-mg/dl, and 29.0% BAL ≥100-mg/dl (range 100–440). Injury mechanisms included falls-47.7%, motor vehicle-24.3%, pedestrian/cyclist-16.8%, and assault-9.3%. Most patients experienced LOC (49.5%-yes, 26.2%-unknown) and amnesia (65.1%-yes, 17.8%-unknown); 65.5% had GCS = 15. BAL differed across LOC (none: [median 0/IQR 0–0], <30 min: [0/0–43], ≥30 min: [224/50–269], unknown: [108/0–232]; p = 0.002) but not amnesia (p = 0.062). GCS <15 associated with higher BAL ([19/0–204]-vs.-[0/0–20]; p = 0.013). BAL did not differ for psychiatric history or admission destination. On univariate analysis, BAL ≥100-mg/dl associated with less-than-full-recovery (GOSE ≤7; 38.1%-vs.-11.5%; p = 0.025) and lower WAIS-PSI (92.4 ± 12.7/30th-percentile; 105.1 ± 11.7/63rd-percentile; p < 0.001). Multivariable regression confirmed BAL ≥100-mg/dl associated with odds ratio of 8.05 [1.35–47.92] for GOSE ≤7 (p = 0.022), and mean decrease of 8.88 [0.67–17.09] points on WAIS-PSI (p = 0.035). Excessive alcohol consumption diminishes acute consciousness and may be a biomarker for impaired 6-month nonverbal processing speed and recovery. Further research is needed to delineate the prognostic value of BAL for maladaptive coping and/or decreased resilience after mTBI.
Keywords: traumatic brain injury, blood alcohol level, concussion, cognition, outcomes, human studies
Upwards of 3 million traumatic brain injuries occur every year in the United States with 75–80% classified as mild. We previously reported that closed head injury (CHI) repeated five times at 24 h intervals in mice caused acute regional microgliosis and cell death. When the time interval was extended to 48 h, much of the histological damage was attenuated to sham levels. To determine whether our model of repeated CHI was associated with behavioral dysfunction, mice received five CHI at 24 h intervals (n = 9), five CHI at 48 h intervals (n = 9), or five sham injuries at 24 h intervals (n = 9) and were evaluated across a 10 week period. Animals with repeated CHI exhibited motor coordination (beam walking) and memory (novel object recognition) deficits which persisted across the 10 wk period. Lengthening the inter-injury interval from 24 h to 48 h did not effectively reduce these behavioral deficits. Ongoing degeneration and microglial activation was noted in the visual system pathway, which was comparable for the two injured groups. To examine the longevity of these functional deficits and to determine whether progranulin deficiency, a condition associated with increased neuroinflammation and age-dependent TDP43 pathology, was associated with worsened behavioral dysfunction, progranulin knockout (GRN KO) and wildtype (WT) mice were tested intermittently for 6 months after five repeated CHI at 24 h intervals (n = 15 GRN KO; n = 18 WT) or sham injury (n = 8 GRN KO; n = 10 WT). Cognitive dysfunction, assessed with novel object recognition, persisted out to 6 months following injury. Beam walking deficits were induced in both WT and GRN KO mice by repeated CHI with recovery of function occurring by 8 wks after injury. These results demonstrate that repeated mild CHI induces long-term cognitive dysfunction and transient impairment in coordinated motor function and that behavioral impairment is not accentuated by increased microgliosis associated with progranulin deficiency.
Keywords: closed head injury (CHI), behavioral dysfunction, progranulin, microgliosis
NETWORK ANALYSIS OF EVENT-RELATED POTENTIALS AFTER MILD BRAIN INJURY
With the prevalence of concussion occurring in children in the U.S., there is profound need for involuntary objective methods for traumatic brain injury (TBI) diagnosis. We present the novel study of functional Event-Related Potentials (ERPs) networks after mild TBI in our established large animal model for TBI. Our objective was to compare longitudinal ERPs network behavior after rapid head rotations. Our previous studies found widespread white matter injury after single sagittal (SAG) head rotations, but not coronal (COR). These direction-specific responses are attributable to the alignment of tissue strains and white matter tracts. Using customized 32-channel, low impedance electroencephalography net, 300 ms auditory ERPs were recorded in awake piglets at 1 day before and 1, 4 and 7 days after rapid SAG or COR head rotations (131 rad/s, n = 4/group). Two auditory paradigms were employed: a uniform sequence of white-noise clicks (STD) and an oddball (OB) sequence composed of 70% STD clicks interspersed with 30% random target clicks. We evaluated weighted network edges (EdgeWt), defined as the maximum absolute cross-correlations of the ERP responses between all pairs of electrodes. At the global level, we observed global similarities between STD and OB paradigms, with significant (p < 0.05) increases in EdgeWt 1 week after SAG, and hypo-connectivity after COR. Regional topographical alterations in STD EdgeWt were observed, that were absent in OB. As expected, network metrics based on OB paradigm often better discriminate deficits than STD, because of the elevated cognitive burden of the task. We found that in COR, OB-standard click EdgeWt decreased significantly on all post-injury days, while OB-target click EdgeWt decreased only at 7 days. In contrast, after SAG, both OB-Standard and OB-target click EdgeWt increased significantly at both 4 and 7 days post-injury. We speculate that increased auditory information flow and computation between various brain regions after SAG may reflect a demanding compensation for damaged connections, while the brain may adapt to hearing clicks after COR. In conclusion, ERPs may offer promise for detecting mild TBI. Support: NIH U01 NS069545, CHOP, and Fontaine.
Keywords: event related potentials, auditory, network, TBI, pediatric
TEAM-TBI: A MONITORED MULTIPLE INTERVENTIONAL TBI RESEARCH TRIAL
University of Pittsburgh, Neurological Surgery, Pittsburgh, USA
Keywords: clinical trial, multiple interventional
UTMB, Neuroscience and Cell Biology, Galveston, USA
Traumatic brain injury (TBI) is an important public health problem. 42–57 million mild TBIs occur worldwide annually, with 1.4–3.8 million people in the US alone, representing 75–87% of all TBIs. The main mechanisms of brain damage in TBI are direct contact (impact) and acceleration/deceleration injuries. Acceleration/deceleration have been associated with diffuse injury. The features of blunt impact and acceleration/deceleration components make the closed-skull weight-drop injury model developed by Kane et al. clinically relevant. However, variability is a concern. This project addresses the concern with the addition of a sensor that allows us to accurately calculate the kinetic energy of the weight immediately before impact, to quantify the variability in the model. Two-month old C57BL/6 male mice were randomly divided into 3 groups: sham, TBI with a 95 g and TBI with 150 g weight. Mice received mild TBIs using either a 95 g or 150 g weight from a height of one meter. Velocity of the dropping weight was measured using sensors in the distal portion of guiding tube. Locomotor activity was evaluated by photo beam activity system after TBI. Brain tissues were collected at 5 days post injury for western blot and immunohistochemical analysis. We found a direct relationship among the weight, its kinetic energy, and righting reflex. Particularly, the kinetic energy of the weight dropped was inversely correlated to the locomotor activity of the injured animals. This study is our first attempt to optimize the closed-skull weight-drop model by implementing quantifiable measures to monitor and consequently reduce injury variations.
Support: Studies were completed as part of a team funded by The Moody Project for Translational Traumatic Brain Injury Research, supported by Mission Connect a project of TIRR Foundation, Coalition for Brain Injury Research, CONACYT-COPOCYT and Fundación Marrón Cajiga.
Keywords: animal model, weight drop, sensors, kinetic energy
USUHS/CNRM, Center for Neuroscience and Regenerative Medicine, Bethesda, MD, USA
Non-invasive detection of mild traumatic brain injury (mTBI) is important for evaluation of acute through chronic effects of head injuries, particularly after repetitive impacts. To better detect abnormalities from mTBI, we performed longitudinal studies of magnetic resonance diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI) in adult mice after single mTBI (s-mTBI) and repetitive mTBI (r-mTBI; daily × 5), or respective sham procedures. The s-mTBI impact was reduced for r-mTBI to differentiate whether repetition lowered the impact threshold for pathology. In the corpus callosum, s-mTBI reduced DTI fractional anisotropy (FA) at all post-injury time points (3, 6, 42 days). Conversely, cortical regions were not altered after s-mTBI but cortical axial diffusivity (AD) was reduced after r-mTBI at all time points with a corresponding increase in axial kurtosis (Ka) at 6 days post injury. Post-imaging histology revealed differential pathology in the corpus callosum versus cortex. Microhemorrhages were not visible in any regions. The corpus callosum exhibited microglial activation, astrogliosis, and demyelination after s-mTBI, with much less robust changes after r-mTBI. However, microglial activation was increased in the cortex after r-mTBI. Additional r-mTBI studies inThy1-YFP-16 mice, which express yellow fluorescent protein in a high density of neurons, detected pathology in corpus callosum axons as well as beading and retraction of neuron processes in the cortex in both models. Finally, r-mTBI, but not s-mTBI resulted in social deficits consistent with the function of this anterior cingulate region of cortex. Overall, DTI and DKI revealed a vulnerability of cortical regions to mild repetitive injury, with underlying differences of microglial activation and behavioral deficits found in r-mTBI but not s-mTBI. This study was funded by the Department of Defense within the Center for Neuroscience and Regenerative Medicine.
Keywords: axonal injury, myelin, diffusion tensor imaging, diffusion kurtosis imaging, Thy1-YFP mice, repetitive TBI
Walter Reed Army Institute of Research, Silver Spring, USA
Changes in FDG-PET imaging can be observed following clinical TBI cases across the injury severity spectrum. Here, we utilize FDG-PET imaging studies in a rat mild concussion model to characterize longitudinal alterations post-injury. Injuries were induced using the Projectile Concussive Impact (PCI) model, in which a projectile impacts the helmet covered head in the right temporoparietal region. Righting reflex, gait, and FDG uptake changes were assessed following single-sham (sSham), repeat-sham (rSham), single-PCI (sPCI), or repeat-PCI (rPCI). Injured rats showed significant increases in righting reflex and acute gait dysfunction. At 2 h post-injury, animals showed decreased cadence, increased stance, and decreased swing speeds compared to controls. Deficits were equally distributed between limbs and more severe in rPCI animals. FDG-PET neuroimaging studies were performed 24 hr, 3d, 7d, 1m, 3m, and 6m post-injury. Distinct regions of interest were evaluated for changes in FDG uptake. After sPCI, uptake increased bilaterally in the olfactory bulbs (24 hr) and in the ipsilateral hypothalamus (7d). Following rPCI, uptake increased in the ipsilateral olfactory bulb (24 hr) and the contralateral cortex (3m) but decreased in the ipsilateral thalamus (3d and 3m) and white matter (3m). Further sub-region analysis revealed uptake changes in the entorhinal, primary somatosensory, and primary motor cortical areas. Significant correlations of altered FDG-PET regions with clinical mTBI assessors revealed the strongest relationships between righting reflex and speed related gait abnormalities with alterations in the olfactory bulb, an area vulnerable to coup counter-coup injury, and cortical areas controlling motor function. Overall, these data indicate that mild concussive injuries result in longitudinal glucose metabolism alterations as detected by FDG-PET imaging that correlate with currently established clinical assessors of concussion.
Keywords: projectile concussive impact, FDG-PET, gait analysis, single injury, repeat injury
Wayne State University, Biomedical Engineering, Detroit, USA
While resting state functional magnetic resonance imaging (rsfMRI) studies in mild traumatic brain injury (mTBI) report alterations in brain function, the heterogeneity of mTBI remains a major obstacle to characterizing these alterations. Each patient is unique in terms injury location and biomechanical profile, making generalization of group-level findings to the individual level difficult. However, we hypothesize that mTBI patients follow similar patterns of alterations in higher-order brain functions despite the heterogeneity. We investigated the relationship between group-level and individual-level alterations in the default mode network (DMN) after mTBI in seventeen mTBI patients at the acute stage and 17 healthy controls using the connectivity domain, a new innovative approach recently developed by the authors. rsfMRI data was transformed into the connectivity domain using predefined seed networks. With a nonparametric statistical comparison of the connectivity matrices that represents the relationship between the seed networks and intrinsic connectivity networks, we observed decreased activity within the DMN after mTBI at the group level, similar to previous findings. The connectivity strength between the DMN and seed networks associated with the DMN was decreased, as was connectivity strength between the DMN and seed networks inversely linked to the DMN. A significant correlation was observed between neuroimaging findings and neurocognitive performance. A subject-level analysis shows that, in general, the same pattern was observed at the subject level as at the group level; 56 out of 60 statistically significant subject-level differences reveal a decrease in connectivity strengths. In conclusion, despite a high level of heterogeneity and inter-individual variability, mTBI patients do share commons patterns of alterations in the DMN. This finding serves to validate previous group-level findings despite current doubt in the field about their relevance.
Keywords: resting state fMRI (rsfMRI), connectivity domain, subject-level analysis, default mode network (DMN), brain networks, intrinsic connectivity networks (ICNs)
New Jersey Medical School - Rutgers Biomedical and Health Sciences, Pharmachology, Physiology, and Neurosciences, Newark, NJ, USA
Mild traumatic brain injury (mTBI) and depression have long been linked in civilians, are also becoming a health concern in military personnel and veterans of the recent conflicts. However, it has not been clear whether depressed mood develops secondary to neurocognitive and somatic symptoms or is a primary outcome of mTBI-induced neurochemical dysfunction. Coping with stress was assessed in a lever-press escape/avoidance (E/A) paradigm in rats after mTBI, by comparing lateral fluid percussion injury (LFPI) and repeated blast injury (rBI) models. The acoustic startle reflex (ASR) was also assessed before and after the E/A training. In E/A training, rats learn to lever-press to limit shock exposure (escape), and to prevent shock exposure all together (avoidance). Efficient stress coping is expressed as a balance of escape and avoidance responding. Consistent with the literature, LFPI induced a profound attenuation of ASRs without a sign of recovery until post-injury day (PID) 42. In contrast, attenuated ASRs were only evident on PID 1 in rBI rats. Both LFPI and rBI rats exhibited poor avoidance expression with unimpaired escape responding. However, the effect of rBI on avoidance appears to be bi-modal. Using a criteria of attaining greater than 50% avoidance during any of the last three sessions, 7/9 LFPI-SHAMs, 0/9 LFPI, 8/10 rBI-SHAMS, and 5/12 rBI rats attained criterion. Although a direct comparison of the two models is difficult without a common biomarker, poor coping after mTBI in rats supports a neuro-organic basis for mTBI-induced depression.
Keywords: depression, fluid percussion, blast
Brain injuries have long been presumed to result from trauma, where the imminent deficits in the brain structure or the individual's behavior can be detected by conventional clinical measures such as magnetic resonance imaging (MRI) or computed tomography (CT). Recent reports argued that initial effects of most brain injuries, particularly the mild cases, can be too subtle to detect during diagnosis. In this preliminary work, we developed a highly sensitive electrophysiological measure to monitor progression of mild injuries in rats that were exposed to a single shock wave. Our approach utilized electrocorticography (ECoG) in the rats implanted with multi-electrode arrays (MEAs), which allowed the cerebellar signals to be monitored during an on-going state of injury. The cerebellum, which is a major brain site in motor related functions, has been viewed as a secondary brain region affected in the TBI context. However, recent reports from both human and animal studies showed that the cerebellum is highly susceptible to brain insults. We evaluated the cerebellar signals by characterizing the local field potentials (LFPs) with sensory evoked potentials (EPs) and current state oscillations in the anesthetized as well as awake animals. The EPs that contained the mossy and climbing fiber responses showed nearly 3 fold decline in amplitude at the end of the survival period. Interestingly, we found critical changes on days 1 and 3 post-injury where signals were deteriorated by 62% and 38%, respectively, compared to the preceding day measurements. We also performed the spectral analysis of LFP oscillations for commonly defined frequency bands; theta (5–8 Hz), beta (12–20), low gamma (30–80 Hz) and high gamma (80–150 Hz). While oscillations in the theta band showed an increased mean power (about +5dB) and correlation r = 0.79 ± 0.1 (0.77 ± 0.09), high gamma oscillations indicated significant drops in the mean power (about −8dB) as well as in correlation r = 0.61 ± 0.065 (0.8128 ± 0.018, p = 0.001) across channels. These results demonstrate the potential utility of continuously monitoring the electrophysiological response as a tool to pinpoint acute and delayed phases of the post-injury period.
This study was supported by individual research grant to M.S. from New Jersey Commission on Brain Injury Research (NJCBIR-CBIR15IRG022).
Keywords: electrophysiology, animal models, cerebellum, blast injury
ABCC8 SINGLE NUCLEOTIDE POLYMORPHISMS ARE ASSOCIATED WITH CEREBRAL EDEMA IN SEVERE TRAUMATIC BRAIN INJURY
Keywords: Abcc8, TBI, single nucleotide polymorphism, cerebral edema
NETWORK LEVEL ANALYSIS OF NEURAL OSCILLATIONS FOLLOWING FLUID PERCUSSION INJURY IN THE RAT
UC Davis, Neurological Surgery, Davis, USA
Traumatic brain injury (TBI) is associated with behavioral and cognitive deficits that are accompanied by changes in brain activity. More specifically, TBI rats perform poorly in spatial memory tasks and have altered electroencephalography (EEG) as observed by a reduction in hippocampal theta oscillatory activity. Interestingly, it has been known for some time that neural oscillations in general, and theta in particular, play a role in information processing. As such we have previously demonstrated that stimulating the medial septal nucleus (MSN) in the theta frequency range following fluid percussion improves behavioral performance. In order to better understand the deficits in network function as they pertain to specific behaviors we recorded local field potentials not only during passive context exposure but also during task specific stages of spatial and non-spatial tasks. We characterized the septohippocampal system; including different hippocampal subfields and MSN, as well as the anterior ventral thalamus (AV) and anterior cingulate cortex. Initial analysis of the behavioral data indicates different learning curves in the Barnes maze and T-maze, with the TBI animals lagging behind the shams 2–4 weeks post-surgery. Consistent with the behavioral data we detected a general decrease expression of theta in the septohippocampal system of TBI rats. This trend was present when rats were in the start box prior to the Barnes maze trial as well as during active exploration. In addition, coherence between several regions (e.g. MSN-AV) tended to be reduced in TBI animals in the start box, arguing against a strictly movement driven effect on theta. Behavioral performance in the Barnes maze was also linked to changes in theta in both the CA1 subfield and AV. These data demonstrate that TBI alters theta oscillations across a broad network and supports our hypothesis that restoring normal oscillatory activity can improve outcome in brain-injured rats.
Keywords: hippocampus, neurostimulation, deep brain stimulation, oscillations, theta, EEG
Sleep disturbances are common following traumatic brain injury (TBI) and often exacerbate symptoms, impeding rehabilitation. This study provides a comprehensive profile of sleep architecture changes following severe penetrating ballistic-like brain injury (PBBI). Animals were randomly assigned to cohorts, implanted with bilateral electrodes and subjected to PBBI or sham. Electroencepholographic (EEG) recordings were scored into vigilance states and spectral analysis quantified relative power and frequency shifts during periods of wakefulness, slow-wave-activity (SWA), and rapid eye movement (REM) sleep. PBBI induced significant changes in bilateral sleep architecture. Sham animals displayed light-dependent rhythmic changes which oscillated between sleep-wake states, whereas, brain-injured rats showed irregular sleep patterns which deviated from baseline recordings. Following PBBI, rats showed rapid reductions in wakefulness (63%) and rapid eye movement (REM) sleep (67%) with concomitant increases in SWA (25%). Rats showed delayed REM onset, fewer sleep-stage transitions, and increased sleep disruptions. PBBI induced bioelectrical discordance—changes in REM and SWA were more prominent and persistent in the ipsilateral hemisphere. To discriminate SWA periods from TBI-induced delta slowing, SWA was further resolved into SWA-1 and SWA-2 according to the percentage of delta activity. PBBI-injured rats showed significant reductions in ipsilateral SWA-1 with increases in high-delta SWA-2. These rats showed rapid increases in delta activity (200% compared to baseline) and decreases in theta, alpha, beta, and gamma frequencies. Compared to baseline, post-injury sleep tracings showed acute peak frequency shifts and attenuation in EEG amplitude during SWS and REM. Overall, our results indicate that TBI produces significant effects on sleep architecture: altering wake, SWA, and REM periods compared to respective controls and indicate a decrease in restorative and quality sleep. Reduced REM sleep has been linked to decreases in memory consolidation, reduced reaction reflexes, increased risk of cardiovascular disease, and increased occurrence of mental health comorbidities. Furthermore, we report that slow-wave activity can be classified power into periods of slow-wave sleep and slow-wave delta activity which accompanies TBI.
Keywords: PBBI, REM, qEEG
ESTRADIOL TO ANDROSTENEDIONE RATIOS MODERATE THE RELATIONSHIP BETWEEN INJURY SEVERITY AND MORTALITY RISK AFTER SEVERE TBI
Early onset of pituitary dysfunction, combined with elevated sex steroid production, suggests increased aromatization occurs with traumatic brain injury (TBI). Our previous work shows that increased serum estradiol (E2), the product of increased extra-gonadal aromatization within the steroidogenesis pathway, serves as a potent mortality biomarker among individuals with severe TBI. A well-characterized model incorporating the aromatization precursor, Androstenedione (Andro), and its relationship to E2 and mortality has not yet been explored. Serum Andro and E2 levels over the first week post-TBI were evaluated for 82 patients with severe injuries. Daily values for both hormones were calculated and E2:Andro ratios were then generated. E2:Andro ratios were then averaged to produce 3-day mean values. After data distribution inspection, E2:Andro values were further categorized into two groups, above and below the 50th percentile. Demographic and clinical variables were used in multivariate analysis to predict 6-month mortality. An initial bivariate model (n = 82) with E2:Andro ratios show a significant association with 6-month mortality (p = 0.0236), where higher ratios were associated with increased mortality. A multivariate model (n = 79) including, Glasgow Coma Scale (GCS), Neurological Burden Score (NBS), and E2:Andro ratios resulted in a significant interaction between GCS*E2:Andro (p = 0.0188) when predicting 6-month mortality; the area under the curve (AUC) for this model was 86.7%. The relationship between injury severity and mortality varied by E2:Andro group. Individuals in the higher E2:Andro group had a similar GCS, regardless of survival status, while non-survivors in the lower E2:Andro group have more severe injuries than survivors in the same E2:Andro group, indicating that GCS predicts mortality among those with lower levels of aromatization. This work needs to be replicated in larger cohorts, and at earlier time-points post-TBI; however, the work has potential implications for understanding which individuals may benefit from early treatment interventions, including progesterone therapy, which is a molecular substrate in the steroidogenesis pathway for both androstenedione and E2.
Support: CDC-R49CCR323155, DOD-W81XWH-071-0701, NIDILRR-90DP0041-02-01.
Keywords: aromatization, hormone physiology, rehabilomics, injury severity
DEVELOPMENT AND CHARACTERIZATION OF A NEW MODEL OF POSTTRAUMATIC EPILEPSY
Traumatic brain injury (TBI) is a major cause of morbidity and mortality in the United States and is a primary public health concern. Injury to the brain may result in serious behavioral and neurological deficits. A consequence of TBI is the development of posttraumatic epilepsy (PTE) where recurrent spontaneous seizures occurs after a brain injury. The pathophysiology in which trauma to the brain leads to spontaneous seizures is unknown and clinically relevant models of PTE are key to understanding the molecular and cellular mechanisms underlying the development of PTE. Current models of PTE have focused on using pentylenetetrazole (PTZ) for testing seizure susceptibility. For example, injured animals injected with a subdose of PTZ were more susceptible to generalized seizures and displayed a decrease in latency to the first spike of an epileptiform discharge as compared to control animals. Diffusion MRI have also associated hippocampal damage to decreased seizure susceptibility in animals that have undergone a TBI, however, a correlation between cortical damage and seizure susceptibility was not observed. In our study, we utilized optical coherence tomography (OCT) to detect changes in tissue dynamics in a controlled cortical impact (CCI) injury mouse model of severe TBI (sTBI). Additionally, animals underwent in vivo intrahippocampal electrical stimulation for the assessment of electrographic seizure threshold (EST) and electrographic seizure duration (ESD). OCT imaging revealed structural changes within the cortex after sTBI and injured animals had a lower EST compared to sham controls. Altogether, our data suggest that EST and ESD can be quantitatively determined and OCT can be a potentially powerful imaging tool for optical biomarker detection in our model of PTE.
Keywords: posttraumatic epilepsy, optical coherence tomography, electrographic seizure threshold
DOSE RESPONSE EFFECTS OF SIMVASTATIN ON ATTENUATION OF NONCONVULSIVE SEIZURES INDUCED BY PENETRATING BALLISTIC-LIKE BRAIN INJURY
Simvastatin (SIM) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor commonly used to reduce serum cholesterol. Recently the neuroprotective and anti-seizure effects of SIM have emerged from animals studies. For example, we have demonstrated that intravenous treatment of SIM mitigated cognitive deficits following penetrating ballistic-like brain injury (PBBI). Preclinical studies by others have shown that SIM reduced kainic acid or picrotoxin-induced seizures. A clinical study has also shown that patients treated with statins, including simvastatin, within 3 days after a stroke had a reduced risk of developing post-stroke seizures. In this study, we evaluated the anti-seizure effects of SIM against PBBI induced nonconvulsive seizures (NCS), which were detected by 72 h continuous EEG recordings immediately following the injury. Four doses of SIM (0.001, 0.01, 0.1, or 1.0 mg/kg) were tested intravenously, twice/day for three days, initiated 30 min post-injury. Control animals received matching vehicle treatments. The results showed that 73% of vehicle-treated animals exhibited an average of 14.5 NCS episodes/rat, yielding an average accumulative duration of 386.6 sec/rat during the 72 h post-injury period. The onset latency was 26 h post-injury. Compared to the vehicle treatment, SIM manifested anti-seizure activities in a dose-dependent fashion by significantly decreasing NCS frequency to 6.0 – 9.7 NCS/rat (59%-33% reduction) and shortening total NCS duration to 173–282 sec/rat (55%-27% reduction) at the two does tested. However, the effects of SIM treatment on NCS incidence and onset latency were non-significant. In summary, the findings of this study provide further support on the anti-seizure properties of SIM in a clinically relevant animal model of post-traumatic seizures. Considering the clinical evidence of beneficial effects of SIM on post-stroke seizures, our results are highly encouraging, warranting further support for more advanced preclinical and clinical evaluations of SIM as a potential seizure prophylaxis in patients having suffered severe brain trauma.
Keywords: post-traumatic nonconvulsive seizures, simvastatin, penetrating brain injury
CAN SURFER'S MYELOPATHY OCCUR IN NON-SURFERS? A CASE OF ACUTE ONSET PARAPLEGIA IN A GYMNAST
Keywords: spinal cord injury, spinal cord infarct, surfer's myelopathy
IDENTIFICATION OF SECONDARY INJURY PATHWAYS USING IN VIVO STABLE ISOTOPE LABELING PROTEOMICS AFTER EXPERIMENTAL SPINAL CORD TRAUMA
George Washington Univ/CNMC, Center for Genetic Medicine, Washington DC, USA
Quantitative proteomics utilizing Stable Isotope Labeling of Amino Acids in Culture (SILAC) has tremendously improved the accuracy of proteomic profiling. Recently, the method was adapted to in vivo studies using heavy Stable Isotope Labeling of Amino Acids in Mammals (SILAM). We used this approach to enhance detection of protein changes that may be associated with secondary injury pathways after experimental spinal cord trauma induced by a weight-drop method in the mouse. Proteins were measured at 30 min, 7 days and 28 days after injury. A mouse SILAM repository quantitative reference standard was created by feeding a wild-type C57BL6 mouse cohort with standard chow containing “heavy” L-lysine (13C6) to the F2-generation. Spinal cord homogenates from these mice were spiked (1:1 ratio) into equivalent protein homogenates from the impact site of unlabeled/”light” C57BL6 injured or sham-injured mice. Samples were then digested with trypsin, fractionated, and analyzed by data dependent LC-MS/MS on a Q exactive mass spectrometer, and the proteins identified and quantified with IP2 software. This approach detected a relatively high number of differentially expressed proteins (58 at 30 min, 270 at 7 days and 136 at 28 days). At 30 min after injury, protein changes were primarily associated with stress-response and inflammation (eg ATG12, IFN-gamma, CEBP-gamma, others). At 7 and 28 days, different proteins related to the inflammatory response were represented (GFAP, peroxiredoxins, stress-induced phosphoprotein 1, astrocytic phosphoprotein PEA 15, cathepsins, HMGB-B1, others). Also at 7 and 28 days, proteins functionally related to synaptic plasticity and neuropathic pain were altered (synapsins-1 − 2, MAP-2, DDAHs-1, −2, GABA and glycine transporters, neuronal membrane glycoprotein M6, others). To our knowledge, this is the first study to utilize SILAM to measure proteins after CNS trauma. These extended time course data support an ongoing reorganization process concurrent with persistent inflammation at the lesion site, weeks after the initial impact. Support:R24HD050846
Keywords: proteomics, profiling
University of Pittsburgh, Critical Care Medicine, Pittsburgh, USA
The pro-death protein RNA binding motif 5 (RBM5) modulates alternative splicing of apoptotic genes like caspase-2 in cancer cells. RBM5 promotes exon-9 exclusion from caspase-2 mRNA yielding a larger pro-apoptotic protein (caspase-2L). Conversely, RBM5 inhibition increases exon-9 inclusion yielding a smaller pro-survival protein (caspase-2s). RBM5 -dependent regulation of gene expression/splicing is thought to be tissue specific. Only a few mRNA targets have been identified to date, and the majority in cancer cells. Identification of neuron-specific RBM5 gene targets has not been explored previously but may help to elucidate its role after brain injury. Here we performed exploratory investigations to identify novel RBM5 regulated genes in neurons. Primary rat cortical neurons were harvested from embryonic brains. High titer lentivirus was made by transfecting HEK293 cells with Origene expression and packaging plasmids. Viral particles were concentrated by ultracentrifugation and titered by ELISA. Neurons were transduced at a multiplicity of infection (MOI) 60 or 30 with vectors to deliver RBM5 siRNAs (knockdown) or alternatively recombinant DDK-tagged RBM5 (overexpression), respectively. At day in vitro 6 (DIV6) neurons were harvested by trizole extraction and high-purity mRNA collected. A subset of neurons were harvested for Western blot analysis to confirm RBM5 manipulations (i.e. knockdown determined to be ∼80–90% and overexpression ∼40–50% above control levels). RNA samples (n = 3–4/group) were converted to cDNA then analyzed on Rat Gene 1.0 ST Affymetrix Arrays by the University of Pittsburgh Genomics Research Core. Data were processed in Affymetrix Expression Console then analyzed in Transcription Analysis console (TAC). 115 coding genes were differentially expressed after RBM5 knockdown; 70 genes were upregulated and 45 downregulated in RBM5 knockdown vs. control neurons. Trends were found for individual gene differences including Rab4a (ANOVA = p = 0.000002; FDR-corrected = 0.069), Sec23 (ANOVA = p = 0.000003; FDR-corrected = 0.069), and RBM5 as expected (ANOVA = p = 0.000002; FDR-corrected = 0.069). Our study suggests that RBM5 regulates expression of neuronal genes which have not been previously identified. This work was supported by NIH grant R21NS088145.
Keywords: microarray, RBM5, RNA splicing, primary cortical neurons, apoptosis
University of Pittsburgh, Physical Medicine & Rehabilitation, Pittsburgh, USA
Previous work has linked the Val158Met polymorphism within the COMT gene to behavioral dysfunction among those dealing with depression following traumatic brain injury (TBI). In TBI and other populations, Met-homozygotes, (i.e. having higher synaptic DA levels), have more behavioral dysfunction. Since the biological rationale for this relationship is unclear, further efforts are needed to elucidate how Val158Met may influence behavior and depression. The literature suggests a link between Val158Met status and the hypothalamic- pituitary-adrenal (HPA) axis, proposing that Val158Met status may confer a differential response to stress sensitivity, wherein carrying the Met-allele increases sensitivity to environmental and/or behavioral stressors, increasing the HPA axis response. To examine this relationship within TBI, cortisol measurements were made using ELISA with samples collected from individuals post-injury who also had COMT genetic data and depression status available for analysis. To examine temporal relationships between cortisol levels by Val158Met group, we first examined general trends within our entire cortisol study population. Overall, serum cortisol levels were significantly elevated for individuals with TBI (n = 251) versus controls (n = 17) initially after injury (220.01+/− 97.81 ng/ml vs. 154.12 +/− 54.44 n/mL; p = 0.0065), but cortisol trended downward significantly over time for all subjects (n = 50) by 3 months (p = 0.001) to conrol levels. At 6-months post-TBI (n = 60), individuals who were Met-homozygotes (n = 17) had significantly higher cortisol levels (169.11+/− 66.16 ng/ml) versus Val/Met-heterozygotes (123.08 +/− 63.02 ng/ml) (n = 29; p = 0.018) or Val-homozygotes (111.21 +/− 31.72 ng/ml) (n = 17; p = 0.008). Suggesting increased sensitivity for Met-homozygotes to post-acute stressors. These findings may help to elucidate previously observed relationships between Val158Met and behavior through the lens of HPA axis response.
Support: NIDILRR-90DP0041; DoD-W81XWH-071-0701; NIH- R01HD048162
Keywords: dopamine, cortisol, COMT, chronic stress, depression, Val158Met
Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) are major neurotrophic factors that promote cell survival and brain plasticity. However, little is known about the endogenous response of these molecules following penetrating ballistic-like brain injury (PBBI). The aim of this study was to identify regional and temporal alterations in BDNF and IGF-1 expression following PBBI. Rats received either sham (n = 6/time point) or PBBI (10% injury, n = 12/time point), and were euthanized at different time points (1 h, 6 h, 24 h, 48 h, 72 h, 7 and 14 days) post-injury. One cohort was used for immunohistochemical staining and another for ELISA. Immunohistochemical analyses revealed temporal and regional changes in BDNF and IGF-1 expression. Immunoreactivity of neurotrophins was increased in the ipsilateral cortex at 1 h compared to sham staining. At 6 h, although both the neurotrophins showed sparse reduction in immuno staining, the staining intensity was increased in selective neuronal populations. The transient loss of staining at 6 h was mostly restored by 24 h except in the injury core. At 72 h, while BDNF staining intensity was similar to sham, IGF-1 immunostaining showed an increase in the ipsilateral cortex and subjacent subcortical white matter. Hippocampal BDNF and IGF-1 immunostaining showed an increase only at early time points (1–24 h) and then returned back to basal levels. An interesting observation was the change in cellular phenotype of neurotrophin expressions as a result of PBBI. More specifically, the basal expression of BDNF and IGF-1 was mainly restricted to neurons. However, following PBBI, both neurotrophins were expressed in astrocyte-like cells throughout the hippocampus at 6 h-24 h and in the cortex at 48 h-72 h time point. Corroborating the immunohistochemical observation, BDNF levels analyzed by ELISA showed an early (1 h-72 h) increase (p < 0.05) in the hippocampus and cortical homogenates. Our findings indicate that PBBI results in a brief upregulation of neurotrophins during the acute post-traumatic period, providing critical information for targeted therapeutic interventions aim at enhancing neuronal survival and brain plasticity for better functional outcome.
Keywords: BDNF, IGF-1, neurotrophins, neurorepair
TRAUMATIC RETROCLIVAL EPIDURAL HEMATOMA: REPORT OF 2 CASES AND REVIEW
A 16 year old boy fell backwards from the height of a jump during a basketball game and struck his occiput on the floor. There was no loss of consciousness but the patient was amnestic to the event. CT and MR scans of the patient's brain and cervical spine revealed a retroclival epidural hematoma. CT and MR angiography were normal. He complained of double vision, and neurologic exam showed a bilateral CN VI palsy. He was discharged home in a cervical collar. On followup exam two weeks later, he was neurologically intact and the hematoma had resolved.
Only 2 reported patients underwent surgery for hematoma evacuation. There were 16 reported instances of CN VI palsy, 6 of CN IX palsy, and 9 of CN XII palsy, all transient.
Mechanisms for formation of retroclival hematomas are controversial. Tectorial membrane disruption may be an important factor. Bleeding may arise from the basilar venous plexus, the odontoid arterial arch system, or the anastomotic arterial network involving the neuromeningeal trunk, ascending pharyngeal artery, meningohypophyseal trunk, and inferolateral trunk.
Keywords: retroclival epidural hematoma
FIBRINOLYSIS PREDICTS PROGRESSIVE HEMORRHAGIC INJURY FOLLOWING TRAUMATIC BRAIN INJURY
The University of Texas Health Science Center at Houston, Department of Surgery, Center for Translational Injury Research, Houston, USA
Progressive hemorrhagic injury (PHI) occurs in 50% of patients with traumatic brain injury (TBI) and is associated with worse outcomes including mortality. PHI pathophysiology remains poorly understood and is difficult to predict. We hypothesize that fibrinolysis is associated with PHI and fibrinolytic markers may predict PHI. A retrospective review of a cohort of highest-level activation adult polytrauma patients with prospectively collected data was performed. TBI was defined by a Head Acute Injury Severity Score ≥3 and intracranial hemorrhage (ICH) on initial CT. In patients with TBI, stable hemorrhage (SH) and PHI was determined based on the absence or presence of ICH expansion on repeat CT within 6 hours, respectively. Demographic and clinical parameters were also collected. Coagulation factor levels, specifically fibrinolytic markers- tissue plasminogen activator (tPA), plasmin-inhibitor (PI), and d-dimer (DD), were determined from blood samples collected at 0, 2, 4, and 6 hours after admission. Twenty-five patients met inclusion criteria and were dichotomized into SH (n = 6, 24%) and PHI (n = 19, 76%) groups. There were no group differences in regards to demographics, injury severity scores, rapid thromboelastography parameters, or outcomes. Generalized estimating equation models determined that collectively across time, tPA is positively associated while PI is negatively associated with developing PHI (both p < 0.05). Moreover, DD exhibits strong time-interactions, and higher levels were associated with higher likelihood of developing PHI. Receiver operating curve analysis for admission DD levels provided an area under the curve of 0.7222 (“fair-accuracy”) and a level of 12.82 μg/mL to differentiate SH from PHI patients. Fibrinolysis, evidenced by DD time-interactions, is associated with PHI. DD may represent a clinically available biomarker for early prediction of PHI in TBI patients.
Keywords: traumatic brain injury, fibrinolysis, progressive hemorrhagic injury, d-dimer
University of Pittsburgh, Neurological Surgery, Pittsburgh, USA
Keywords: acute subdural hematoma, subdural drainage system, post-surgical management, traumatic brain injury
Keywords: stroke, behavior, vitamin D deficiency
DETECTING CONSCIOUSNESS AND PREDICTING RECOVERY IN ACUTE SEVERE TRAUMATIC BRAIN INJURY
Massachusetts General Hospital (Harvard University), Neurology, Boston, USA
For patients with acute severe traumatic brain injury (TBI), current clinical tools may fail to detect consciousness and predict long-term recovery. Functional MRI (fMRI) and electroencephalography (EEG) may detect cognitive-behavioral dissociation (CBD) in the chronic stage of recovery from TBI, but the ability of these techniques to detect CBD acutely is unknown. In this prospective longitudinal study, we tested two hypotheses: 1) fMRI and EEG can detect CBD in patients with acute severe TBI; and 2) acute fMRI and EEG predict 6-month outcomes. Sixteen consecutive patients admitted to the ICU for acute severe TBI underwent fMRI and EEG during language, music, and motor imagery stimuli. Behavioral examination with the Coma Recovery Scale-Revised (CRS-R), fMRI and EEG were performed within 24 hours of one another. The criteria for CBD were defined as stimulus-induced fMRI activation of association cortex and EEG cortical reactivity determined by support vector machine, and absent or minimal behavioral signs of consciousness. Functional outcomes were assessed at 6 months using the Glasgow Outcome Scale-Extended. fMRI and EEG were performed 9.2+/−5.0 and 9.8+/−4.6 days post-injury, respectively. CRS-R indicated coma (n = 2), vegetative state (VS; n = 3), minimally conscious state without language (MCS-; n = 3), minimally conscious state with language (n = 4) or post-traumatic confusional state (n = 4). We detected CBD in 3 patients: 1 whose exam suggested VS and 2 MCS-. All three demonstrated modulation of brain activity in response to language and music, and one (MCS-) had fMRI evidence of command-following on the motor imagery task. fMRI and EEG responses did not correlate with functional outcomes, but all 3 patients with CBD recovered behavioral evidence of consciousness at 6-months. These results provide initial evidence that MRI and EEG may improve detection of conscious awareness in patients with acute severe TBI.
Keywords: coma, consciousness
There is increasing evidence that traumatic brain injury (TBI) elicits a rapid and robust neuroinflammatory response within the brain. This innate immune response to injury involves prominent activation and proliferation of microglia, which can engage in a plethora of neurotoxic or neuroregenerative effector responses. Although immunohistochemical methods can be used to characterize the innate immune response to TBI in post-mortem samples, few methods are available to visualize microglial activation in vivo in human subjects. Because activated microglia express TSPO, PET tracers binding to TSPO have been considered putative markers of the innate immune response to neurological damage (or microglial activation) in various neurological disorders. However, the literature investigating the characteristics and significance of TSPO PET tracer binding in TBI remains limited. In order to investigate the potential utility of second-generation TSPO PET tracer [(11)C]-PBR28 for visualizing microglial activation secondary to TBI, a cohort of adult patients who had sustained a moderate to severe TBI in the past 6 months underwent [(11)C]-PBR28 PET dynamic scanning on a Siemens Biograph mCT scanner, in addition to concurrent structural MRI and TSPO genotyping (rs6971). As a preliminary exploratory step, static SUV images depicting tracer uptake for select patients were reviewed and compared to similar images from healthy controls matched for TSPO genotype. Visual inspection suggested plausible relationships between [(11)C]-PBR28 and known focal pathology secondary to TBI. In particular, notable asymmetries and focal areas of reduced or increased uptake were compatible with acute and concurrent abnormalities on structural neuroimaging. For example, significant hemorrhagic contusions eventually demonstrated reduced binding as they progressed to encephalomalacia. The findings suggest that further investigation is warranted to elucidate the relationship of [(11)C]-PBR28 binding to focal neuropathology in TBI, and to clarify the potential utility of this tracer for clinical and research purposes relating to TBI.
Keywords: traumatic brain injury, microglia, neuroinflammation, neuroimaging, PBR28, TSPO PET
DOUBLE DIFFUSION ENCODING SPECTROSCOPY TO DETECT SPINAL CORD INJURY
Medical College of Wisconsin, Neurosurgery, Milwaukee, USA
Diffusion weighted imaging is a sensitive marker of microscopic nervous system injury, but simplistic models such as diffusion tensor imaging (DTI) have inherent limitations. Likewise, imaging the spinal cord is technically challenging and quantifying diffusion parameters often requires extensive post-processing. To overcome these limitations and provide a rapid estimate of tissue microstructure, a diffusion weighted spectroscopy sequence was implemented with double diffusion encoding (DDE) to probe novel DWI measure of tissue microstructure. MRI experiments were performed on Sprague-Dawley rats subjected to a spinal cord injury using a weight-drop. A spectroscopy (PRESS) sequence included two pairs of diffusion sensitizing gradients adjustable in their duration (δ), separation (Δ), strength (b-value), and direction. A single voxel (10x10x6 mm) was placed in the spinal cord injury epicenter, and diffusion weighting was performed under 3 conditions:
Keywords: double diffusion encoding, diffusion weighted imaging, spinal cord injury, magnetic resonance imaging, microscopic anisotropy
The Amino Cupric Silver (ACS) stain of de Olmos reveals neurons in the degenerative (disintegrating) state in the brain and spinal cord. These neurons stain intensely black, while normal neurons do not take up the stain. To assess the degree or the amount of degeneration, a numbered score (0,1,2,3,4, with 4 being highest level of degeneration) is usually assigned. Here we describe a more quantitative means for this type of evaluation that can be achieved by applying a second stain “over” the ACS stain. The antibody NeuN reveals a marker expressed in all normal neurons over 2 weeks old. When combining the ACS stain with NeuN IHC on the same tissue, degenerative neurons stained with the ACS protocol can be counted against “live” neurons that stain positive for NeuN.
Using imaging software (FIJI or “Fiji Is Just ImageJ”) the numbers of degenerating (black) neuron and NeuN positive (brown) neurons can be determined for any given region of interest. Using “batch processing” methods, captured images are pre-processed using Adobe Photoshop to make them more amenable for image analysis. Further batch processing using brightness, contrast, selection and thresholding tools, black degenerating neurons and brown “live” neurons are isolated for quantification using the “particle analysis” tool in FIJI. Although this method does not replace quantitative determinations provided by the more time consuming and expensive stereologic analysis, it does provide a numeric index of observables rather than a subjective assessment.
Keywords: analysis
IN VIVO VASCULAR IMAGING OF TRAUMATIC BRAIN INJURY IN THE SECOND NEAR-INFRARED WINDOW
Stanford University, Stanford, USA
Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Disruption of vasculature is a primary event in TBI that can lead to a cascade of secondary injuries. Investigation of cerebrovascular responses to TBI with high spatial and temporal resolution is therefore important to understanding disease pathogenesis at the cellular level. Here, we designed a bright, renal-excreted and biocompatible near infrared II (NIR-II, fluorescence in 1,000–1,700 nm range) molecular fluorophore for in vivo imaging the dynamics of cerebrovascular responses to TBI in mice. Video-rate dynamic imaging of cerebral blood perfusion was performed by detecting the emission of circulating fluorophore above 1,300 nm through the intact mouse skull and scalp at sub-centimeter imaging depths. We observed a transient hypoperfusion in the injured cerebral region, followed by fluorophore leakage and accumulation associated with blood vessel damage, blood-brain barrier (BBB) disruption and subdural hematoma. Time and spatially resolved imaging revealed ultra-slow blood perfusion at the single vessel level. The results suggest that deep tissue fluorescence imaging with biocompatible NIR-II fluorophores could provide a non-invasive, high resolution and real-time assessment of BBB permeability and cerebrovascular responses to TBI. These authors (X.Zhang, H.Wang, A.L.Antaris) contributed equally to this work. Correspondence should be addressed to H.Dai (hdai@stanford.edu), J.Luo (jianl@stanford.edu) and Y.Liang (liangyy@sustc.edu.cn).
Keywords: traumatic brain injury, vascular imaging, near infrared II, blood-brain barrier, hypoperfusion, molecular fluorophore
Thomas Jefferson University, University College of Health Professions, Phildelphia, USA
Keywords: diffusion tensor imaging, pediatrics
UCLA, Neurosurgery, BIRC, Los Angeles, USA
While current clinical rehabilitation strategies used after brain injury promote significant behavioral benefits, it is possible that therapies might be more optimally employed using more detailed feedback from comparison of brain functional connectivity (fc) before and after intervention. We hypothesized that resting state fMRI (rsfMRI) might be an ideal technique to monitor whole brain function in response to rehabilitation. We have recently shown that fc is significantly impacted by experimental brain trauma in the rat, and that brain circuitry adjacent and contralateral to the injury mounts a response to offset the loss in connectivity. However, it is unknown whether the beneficial effects of rehabilitation can be followed by monitoring brain circuity through changes in fc. We therefore followed fc in control-cortical-impact-injured and sham-injured adult rats that were housed immediately after injury for 4 weeks in either standard (STD) or environmental enriched (EE) conditions (n = 5–6/group). We obtained rsFMRI data at 1 and 4 wks post-injury, and then at 4 wks following resumption of STD conditions for all groups (8 wks post-injury) and analyzed data for network-based connectivity over 96 brain regions. The data confirm and extend prior published work showing significant injury effects in STD conditions compared to sham-STD at all time-points (P < 0.05). There was no global effect of EE vs STD on any network parameter at 1 wk post-injury, although there was a trend of reduced modularity. However, 4 wks of EE normalized many of the injury+STD effects of reduced shortest path and increased mean local connectivity to sham-STD / sham-EE levels. Modularity was significantly reduced at 4 wks after injury+EE (P < 0.05) compared to: injury-STD, sham-EE and sham-STD, all of which were not different, indicating a potential biomarker of reorganization due to EE. Importantly, these effects persisted until 4 wks after removal of EE conditions (8 wks post-injury). These global effects were underpinned by significant regional effects of normalized connection strength in injured-EE vs injured-STD, bilaterally in S1 cortex, thalamus and caudate, and in contralateral hippocampus and M1 cortex (P < 0.05).
Keywords: environmental enrichment, traumatic brain injury, reorganization, rsfMRI, functional connectivity
UCSF, Neurosurgery, San Francisco, USA
The National Institute of Neurological Disorders and Stroke (NINDS) traumatic brain injury (TBI) Common Data Elements (CDEs) for neuroimaging are designed to minimize inter-rater ambiguity through strict definition of intracranial lesion types. Fifty subjects were selected randomly from the prospective multicenter Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot (TRACK-TBI Pilot) study. Board-certified neuroradiologists blinded to clinical and outcome data except age independently reviewed brain computed tomography (CT) and magnetic resonance imaging (MRI) using Osirix (64-bit version, Pixmeo, Geneva, Switzerland). During interpretation of the studies, discrepancies were discovered that were attributable to variations in individual radiologist interpretation of CDE lesion definitions, with preliminary Fleiss kappa statistics ranging from 0.7 to 1.0 for most lesions, with a few falling below 0.7. Given these discrepancies, we constructed an atlas demonstrating specific lesion types with pictorial examples based on NINDS TBI-CDE definitions. The objective was to create a comprehensive visual tool that would accompany the TBI-CDE classification system in order to improve inter-rater reliability among all neuroimaging readers. Examples of each CDE-defined lesion include axial, coronal, and sagittal views from two different MRI series as well as at least one CT image. Both typical and atypical examples of each lesion were included. The utility of this comprehensive brain atlas, including enhancements to lesion identification accuracy and inter-rater reliability when used in conjunction with NINDS TBI-CDE definitions, is currently under evaluation using the larger TRACK-TBI dataset and will be the subject of future reports.
Keywords: traumatic brain injury, common data elements, neuroimaging, interrater reliability, diagnostics, human studies
University of Toronto, Toronto, Canada
Keywords: MRI, DTI, SCI, prognosis
Macrophages are mature phagocytic leukocytes that have roles in both physiological and pathological processes. They can be activated through numerous stimuli, such as injury, infection, and inflammation. The activation state of macrophages is often defined as either classically activated M1 (pro-inflammatory), or alternatively activated M2 (anti-inflammatory). While this dichotomy can prove useful in a general context, current research points to the fact that there are many states between the two extremes.
Spinal cord injury (SCI) results in massive axon death which produces large amounts of myelin debris. There is a large degree of bone marrow derived macrophage infiltration at the primary injury site. Upon engulfment of myelin debris, macrophages take on an M1-like phenotype which contributes to the secondary injury and prevents functional recovery. Little is known about the transcript level changes that myelin debris phagocytosis induces in macrophages, or how this contributes to their pathological role in the injured spinal cord. We have implemented mRNA microarray analysis of murine bone marrow derived macrophage transcripts. Using this method we have identified several related gene clusters that are correlated with known phenotypic changes. Among the clusters with known correlation are immune response, taxis, and G-protein coupled signaling. Additionally, mapping to previously unreported epigenetic methylation pathways was identified using this method.
Keywords: microarray, bioinformatics, myelin, macrophage
THE EFFECTS OF INTERLEUKIN-1 RECEPTOR ANTAGONIST TREATMENT IN AN ANIMAL MODEL OF MULTITRAUMA
La Trobe University, Department of Physiology, Anatomy and Microbiology, Bundoora, Australia
Keywords: multitrauma, bone fracture, interleukin-1
The Ohio State University, School of Health and Rehabilitation Sciences, Columbus, USA
Spinal cord injury (SCI) produces a toxic inflammatory microenvironment that negatively affects plasticity and recovery. Recently we showed that glial activation and infiltration of bone-marrow derived myeloid cells extends past the epicenter and through remote areas of the lumbar cord after thoracic SCI. Recruitment of monocytes into intact regions of the lumbar cord may further impede locomotor rehabilitation depending on their activation profile. Therefore, our objective was to compare the inflammatory profile of resident microglia and peripheral myeloid cells after SCI. GFP+ bone marrow chimeras received a moderate mid-thoracic contusion injury and trafficking of myeloid cells was determined 1–7 days later. FAC sorting showed that both neutrophils and macrophages infiltrated the epicenter and lumbar cord in a similar time frame. Through 7 days, the number of neutrophils was attenuated while the number of macrophages remained. These data suggest that macrophages play an important long-term role in influencing the microenvironment while neutrophils act acutely. Nanostring gene array identified a strong pro-inflammatory profile of infiltrating macrophages relative to microglia at both epicenter and lumbar sites. For instance, macrophages had elevated expression of inflammatory cytokines (IL-1b, IFNg), chemokines (CCL2, CXCL2), mediators (COX-1, MMP-9), and receptors (CCR2, Ly6C), and decreased expression of growth promoting genes (GDNF, BDNF). Such a high inflammatory potential of infiltrating macrophages within the central pattern generators for locomotion in the lumbar cord is likely to impede activity-dependent recovery. Importantly, macrophages recruited to the lumbar cord had elevated expression of genes associated with active trafficking (CCR2, L-selectin, MMP-9) compared to macrophages at the epicenter. Therefore, limiting active trafficking of macrophages into the lumbar cord identifies a novel target for SCI therapies to improve locomotion.
Keywords: nanostring gene array, neutrophils, macrophage, microglia, inflammatory cytokines, spinal cord injury
UCSF, Brain and Spinal Injury Center, San Francisco, USA
Recent work has shown that the aged brain may be predisposed to altered inflammatory responses following neurotrauma due to maladaptive signaling mechanisms. However, little is known concerning how these maladaptive responses affect the activation and recruitment of peripheral macrophages to the injured brain. Multiple approaches were used to define these responses in both young (3m) and aged (23m) WT and CX3CR1GFP/+CCR2RFP/+ reporter mice. Herein, we used unsupervised hierarchical gene clustering and pathway analysis to define the effect of age upon injury-induced inflammatory sequelae. Our data show that age predisposes the injured brain to an exaggerated inflammatory response involving the enrichment of chemotactic mediators associated with the recruitment and activation of peripheral monocytes. Specifically, our data show that aged TBI animals have an exacerbated response in the production of ligands that bind to both CCR2 and CCR5 at a critical time point associated with migration of peripheral monocytes/macrophages to the injured brain parenchyma. This molecular response was paralleled by an increased recruitment of CCR2+ macrophages to the injured brain of aged mice. Given the exacerbated CCR2/5 cellular and molecular signatures, we used a novel small molecule antagonist for CCR2/5 (Cenicriviroc; CVC) to abrogate this phenotype. Using a clinically relevant pharmacological approach, our data show that the age-related phenotype in response to injury is significantly mitigated in treated animals. These data implicate a new important role for the accumulation of monocyte/macrophages in the maintenance of neuroinflammatory sequelae following neurotrauma in the aged brain.
Keywords: aging, CCR2, macrophage, antagonist
Neurodegeneration following spinal cord injury (SCI) is due, in part, to inflammation and immune responses. Activation of B lymphocytes and production of autoantibodies contributes to neurodegeneration subsequent to traumatic SCI in humans and in animal models. Flubendazole is a benzimidazole anthelmintic used to attack parasitic worm infections, and approved for human use. It binds to nematode tubulin and impairs microtubule-dependent mechanisms. However, Flubendazole can also interact with mammalian tubulin and impair proliferation of rapidly dividing cells such as B lymphocytes. The aim of this research was to determine whether post-SCI treatment with Flubendazole decreases neurodegeneration and improves functional outcomes when administered post-SCI in a rat model. Flubendazole was administered via intraperitoneal injection, 10 mg/kg/day to Sprague-Dawley rats for 2 weeks, beginning 3 hrs post-SCI (180 kdyn at T10), n = 10. This resulted in improved locomotor function (BBB scores) 7 weeks after contusion SCI compared to vehicle-treated controls, n = 9. Flubendazole treatment also improved total tissue sparing, white matter sparing, and gray matter sparing at 7 weeks after contusive SCI. Flubendazole reduced the splenic population of CD45RA-positive B cells and suppressed IgG immunoreactivity at the lesion site 4 weeks post-injury. In conclusion, our results suggest that Flubendazole targets the pathogenic B cell pathway and improves functional recovery after SCI.
Keywords: B cells, autoimmune, tubulin
COMPRESSION ALTERS MACROPHAGE ACTIVATION IN CONTUSIVE SPINAL CORD INJURY
University of Kentucky, ScoBIRC, Lexington, USA
Initial tissue damage after SCI is exacerbated by secondary mechanisms that drive cell death for days to weeks following injury. One key mechanism in this secondary response is inflammation. Activation of pro-inflammatory cells, specifically cytotoxic (M1) macrophages, drives cell death and increases the severity of SCI. Conversely, activation of neuroprotective (M2) macrophages can decrease cell death and overall SCI severity. Therefore, there is increased focus on the development and identification of immunomodulatory therapies to treat SCI. It is currently not known, however, if differences in the mode of the initial injury alter how macrophages are activated. Specifically, there is a gap in our understanding if compression and force of injury alter macrophage polarization states. We have previously shown, in a mouse model of SCI, that altering the initial impact force and compression changes the amount of tissue damage. With longer impact duration, we observe more tissue damage and worse functional recovery. This damage is not evident directly after injury but becomes evident one week later suggesting distinct activation of secondary mechanisms. Based upon these observations, we hypothesize that longer impact duration increases activation of pro-inflammatory macrophages at the injury site. To determine if macrophage activation is altered by the initial mode of impact, we quantify macrophage subtypes using immunohistochemistry and compare four impact injuries: 50 kdyn, 75 kdyn, 50 kdyn with 20 second dwell and 75 kdyn with 20 second dwell. We find that the classic M1/M2 markers, CD86 and Arg1 respectively, are not significantly different among treatment groups. However, we do see significant changes in the number of macrophages expressing phagocytic proteins suggesting a possible shift in macrophage activity depending upon duration of initial compression. These results highlight the need to consider mode of injury in the design and implementation of immunomodulatory therapies for SCI.
NIH NINDS R01NS091582 and The University of Kentucky Postdoctoral Research Fellowship
Keywords: macrophage, compression injury
University of Kentucky, Sanders Brown Center on Aging, Lexington, USA
Many disorders of the central nervous system (CNS) involve neuroinflammation, an inflammatory/immune response in the brain. Following traumatic brain injury (TBI), dysregulated neuroinflammatory responses are thought to contribute to neurological damage and cognitive deficits in part through an increased production of proinflammatory cytokines such as interleukin-1 beta (IL-1β). Addressing these damaging proinflammatory processes without affecting endogenous recovery responses has therefore yielded a potential intervention target. This study investigated the use of MW151, a CNS-penetrant, small molecule experimental therapeutic that restores overproduction of proinflammatory cytokines towards homeostasis without general immunosuppression when administered post-injury in multiple TBI models. In a midline fluid percussion model of diffuse brain injury in mice administration of a low dose (0.5–5.0 mg/kg) of MW151 suppressed IL-1β levels in the cortex. Reactive astrocyte and microglia morphological responses were not affected. In an in vitro study the BV-2 microglial cell line was used to demonstrate that phagocytosis, proliferation, and migration of microglial cells was not affected by treatment with MW151. These results demonstrate that the use of selective therapeutic modulation is a feasible tool to target the increase of the proinflammatory cytokine IL-1β without affecting physiological responses of glial cells.
Keywords: interleukin-1 beta, MW151, small molecule therapeutic, neuroinflammation, mouse model, diffuse TBI
University of Texas Medical Branch, Anesthesiology, Galveston, USA
Keywords: chronic activation of microglia, fluid percussion injury in rat, CD68, Iba1
USUHS, Neuroscience, Bethesda, USA
Microglia are the macrophages of the central nervous system (CNS), which function to monitor and maintain homeostasis. Microglia activation occurs after CNS injury, infection or disease. Prolonged microglia activation is detrimental to the CNS as they produce nitric oxide (NO), reactive oxygen species (ROS) and pro-inflammatory cytokines, resulting in neuronal cell dysfunction and death. Microglia activation is implicated in the neurological deficits following traumatic brain injury (TBI) and Alzheimer's disease. Intranasal insulin administration is a promising treatment of Alzheimer's disease and TBI. Previous work in our lab showed a significant reduction in Iba1, a marker of microglia and macrophage, in the hippocampus of rats treated with intranasal insulin after controlled cortical impact injury. This reduction in Iba1 corresponded with improved performance in Morris water maze. Therefore, the goal of this study was to specifically examine the effect of insulin administration on activated microglia.
BV2 microglia were exposed to a pro-inflammatory stimulus, lipopolysaccharide (LPS), and then treated with insulin. Outcome measures were conducted at 24 hours after treatment. In vitro assays quantified NO and ROS production. Western blot and immunocytochemistry further examined the effect of insulin on microglia polarization markers. Insulin treatment significantly reduced NO production but had no effect on ROS production following LPS activation. Insulin treatment had no significant effect on any M1 or M2 macrophage polarization markers examined. Insulin treatment also resulted in a reduction in two chemoattractants, JE/monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1A. These data suggest that insulin acts on microglia to reduce NO production and chemoattractant production. Therefore, administration of insulin to the CNS is a promising anti-inflammatory treatment.
Keywords: insulin, microglia, inflammation, nitric oxide, cytokines
Hofstra Northwell School of Medicine, Department of Neurosurgery, Manhasset, NY, USA
Keywords: external ventricular drain, intraparenchymal ICP monitor, ICP measurement
Montreal Neurological Institute, Neurosurgery, Montreal, Canada
External ventricular drain (EVD) placement is one of the most frequently performed neurosurgical procedures. Inaccuracies in the drain positioning and the need for multiple passes using the classic freehand insertion technique are increasingly reported in the literature. The problem is seen most frequently in the severe traumatic brain injury (TBI) population. Many proposed methods were discussed to improve the placement accuracy and none gained enough support to be implemented in EVD placement. The purpose of this study is to evaluate the use of electromagnetic neuronavigation guidance to aid EVD insertion to improve the accuracy and minimize the number of passes in severe TBI patients. The navigation was applied prospectively for all new severe TBI patients who required ventricular catheter placement over a year period, and this was compared to a retrospective cohort of severe TBI patients who had EVD inserted freehand in the preceding year. Fifty-four cases were recruited, 35 (64.8%) had their EVD placed using the freehand technique and 19 (35.2%) using navigation guidance. In the navigation group, the placement accuracy was as follows: 94.7% (18/19) achieved a grade 1 and 5.3% (1/19) a grade 2, while none were in grade 3. In comparison, freehand placement was associated with misplacement (grade 2 and 3) in 42.9% of the cases (P-Value = 0.009). The number of passes was significantly lower in the navigation group with a mean of 1.16 ± 0.38 {P-Value = 0.018, 95% CI (-0.86, −0.09)}, compared to the freehand group with a mean of 1.63 ± 0.88. This suggests that using the navigation to guide EVD placement was associated with better accuracy and lower number of passes in challenging cases of severe TBI, which means less associated morbidities.
Keywords: external ventricular drain, severe TBI, neuronavigation
The fate of the bone flap is a significant decision during surgical treatment of acute subdural hematoma (SDH). A general guideline revolves around the surgeon's concern for brain swelling. The objective of this study was to assess factors that contribute to perceived perioperative brain swelling. From 2012 to 2015, 38 patients who underwent decompressive craniectomy (no replacement of bone flap) for traumatic acute SDH were reviewed. Clinical data were extracted [age, gender, initial (Glasgow coma scale) GCS, antiplatelet / anticoagulation status, sodium level, hematocrit, and intraoperative blood loss]. CT imaging was loaded into OsiriX MD. From the preoperative scan, SDH volume, midline shift (MLS), and volume within the skull (to estimate baseline brain volume) were measured. From the postoperative scan, MLS and brain volume (including any herniating regions) were measured. Volume was obtained by a semi-automated protocol to select the region of interest. The extent of brain swelling was defined as Δ%, the percentage change in postoperative brain volume compared to preoperative volume. Other parameters [presence of contralateral injury, contusions, or intraventricular hemorrhage (IVH)] were noted. Overall, fifteen patients (39%) demonstrated negative Δ%. Univariate analysis found significant correlations between Δ% and the following: preoperative MLS, initial GCS, IVH, and contralateral injury (all p < 0.05). A multiple linear regression for Δ% that combined preoperative MLS, initial GCS, and presence of IVH elicited a significant model [F (3,34) = 17.387), p < 0.01)] with R2 0.605, where Δ% = 16.197–1.246*GCS-0.986*MLS +3.292*IVH (with 0 = no IVH, 1 = presence of IVH). In conclusion, a high proportion can exhibit negative Δ%, or relative brain compression, after decompression of SDH. For these patients, replacement of the bone flap may be reasonable to avoid obligatory interval cranioplasty. Preoperative MLS, initial GCS, and IVH may help predict whether overall brain volume will swell or compress within the normal confines of the cranium. This can guide the decision to retain or remove the bone flap.
Keywords: decompressive craniectomy, brain swelling, acute subdural hematoma
RTBI INDUCED CEREBRAL METABOLIC CRISIS UPREGULATES THE AMYLOIDOGENIC PATHWAY
Traumatic brain injury (TBI) is an identified risk factor for Alzheimer's disease (AD). It is unknown what cellular mechanisms facilitate AD pathogenesis in the adolescent brain following repeat mild TBI (RTBI). In order to understand how RTBI may act as an AD “ trigger”, P35 male transgenic AD rats were given 4 closed head injuries with 24 and 72 hr intervals. Hippocampal amyloid plaque (Aβ) load was determined at 12 months of age. Injury intervals were based on previous data that shows injury interval significantly affects the duration of cerebral metabolic depression. Two injuries within 24 hrs have a cumulative effect and significantly extend metabolic recovery. 4RTBI-24 hr animals had significantly more Aβ deposits relative to sham and 4RTBI-72 hr groups. Beta amyloid precursor protein processing is divided into two pathways; non-amyloidogenic and amyloidogenic and is cleaved by alpha- (ADAM10) and beta-secretases (BACE1), respectively. The activities of ADAM10 and BACE1 exist in a ratio and their cleavage products inhibit each other, maintaining this balance. ADAM10 activity is regulated by PKC and is an ATP dependent process. We hypothesize that sustained decreases in ATP in 4RTBI-24 h animals decreases ADAM10 activity, disrupting inhibitory regulation of BACE1 resulting in increased BACE1 activity and Aβ production. P35 male wildtype rats were given 4 closed head injuries with 24 hr intervals. At 24 and 48 hrs post-injury BACE1, ADAM10 and PKC activity and ATP levels were quantified. RTBI caused significant decreases in PKC activity at 24 hr along with significant decreases in ATP levels and ADAM10 activity at 24 and 48 hrs post-injury. In contrast, BACE activity significantly increased at 24 and 48 hrs post-injury. These data identify a mechanism where prolonged disruptions in cerebral metabolism disrupt the balance of βAPP production in favor of amyloidogenesis and are the first evidence of a link between TBI, cerebral glucose metabolism, injury interval and pathogenesis of AD. This work suggests acute restoring enzymatic balance following RTBI may prevent AD pathogenesis.
Keywords: repeat traumatic brain injury, metabolism, concussion, adolescent, Alzheimer's Disease, neurodegeneration
GLOBAL METABOLOMICS ANALYSIS IN RATS FOLLOWING PENETRATING BALLISTIC-LIKE BRAIN INJURY
There is increasing interest in metabolic management in the treatment of traumatic brain injury (TBI) patients. However, clinical studies have yielded mixed results, in part due to the complex pathological responses of the neurometabolic network. This study was designed to profile the metabolomes in rat brain tissue, cerebrospinal fluid (CSF), and serum following penetrating ballistic-like brain injury (PBBI). Rats received either a sham craniotomy or a PBBI. Ipsilateral frontal cortices, CSF, and serum were collected at 30 min, 6 h, 24 h, 72 h, and 7d post-injury (n = 6/group/time-point). High throughput mass spectrometry-based metabolomics was performed to determine the entire metabolome. Metabolomics profiling detected a total of 535, 448, and 629 biochemicals in the brain tissue, CSF, and serum respectively. Immediately following injury at 30 min, the percentage of biochemicals significantly altered compared to sham in the three biological compartments were 23.2% (brain), 4.7% (CSF), and 9.1% (serum) of total biochemicals (p < 0.05); this quickly elevated to 51.0–65.0% (brain), 8.5–21.0% (CSF), and 13.4–28.5% (serum) starting at 6 h and lasting out to 7d post-injury (p < 0.05). Importantly, biochemical pathway analysis showed a significant and complex metabolic response of the brain to severe TBI that includes osmotic stress, imbalance in multiple neurotransmitters, increase in glycolytic metabolism, depletion of creatine stores, and enhanced complex lipid hydrolysis. Additionally, principal component analysis (PCA) revealed a distinct separation between sham and PBBI samples starting from 6 h post-injury in brain tissues and serum samples, indicating a PBBI metabolic profile that is different from the sham in these two compartments. Interestingly, this distinct PBBI metabolic profile was only observed at 6 h post-injury in the CSF, and was absent at other time points. Overall, the comprehensive metabolomics analysis of the traumatically injured brain tissue, and the comparison of its compositions in the CSF and peripheral circulation have defined a pathological metabolic profile that is different from a normal physiological state, which provides crucial information for the advancement of translating metabolomics to the clinical setting. US Army Combat Casualty Care Research Program H_026_2014_WRAIR.
Keywords: metabolomics, penetrating ballistic-like brain injury
STRESS WORSENS GLOBAL ISCHEMIA OUTCOME BY EXACERBATING INFLAMMATORY RESPONSE: IMMUNOMODULATORY EFFECTS OF PROGESTERONE
Emory University, Emergency Medicine, Atlanta, USA
Keywords: microglia, global ischemia, stress, progesterone, neuroinflammation, hippocampus
Uniformed Services University, Neuroscience Program, Bethesda, USA
Microglial polarization is primarily pro-inflammatory after traumatic brain injury (TBI). Numerous stimuli, such as damage associated molecular patterns (DAMPS), induce M1 polarization within hours after TBI, and can persist for weeks or even potentially years after the primary injury. Currently, the mechanisms to explain this chronic pro-inflammatory state are not fully elucidated. We suspect iron may play an important role within these mechanisms. Despite iron's ubiquitous presence among cells within the neural parenchyma, excessive levels of iron seem to contribute to the pathology of similar chronic pro-inflammatory neurodegenerative diseases. We therefore hypothesized that excessive iron alone can overwhelm cellular antioxidant mechanisms and induce a pro-inflammatory polarization among microglia. To test this hypothesis, we exposed immortalized BV2 microglia to various concentrations of ferrous sulfate (FeSO4), a Fe2+ donor, with and without lipopolysaccharide (LPS) for 24 hours. The cells were evaluated utilizing cellular activity measures, western blotting, flow cytometry (FACS), and cytokine profiling to characterize and determine polarization alterations. We detected a significant increase in reactive oxygen species (ROS) synthesis among cells exposed to FeSO4 in a concentration dependent manner, with and without LPS co-stimulation. Control experiments determined equipotent doses of sodium sulfate did not induce similar ROS production, while administration of deferoxamine (DFO), an iron chelator, completely reversed the effect of FeSO4. In addition, we detected the presence of intracellular iron through significantly elevated ferritin (FER) concentrations after FeSO4 exposure. Next, we detected increased expression of catalase (CAT), peroxiredoxin 6 (PRDx6), and superoxide dismutase 1. Interestingly, glutathione peroxidase (GPx) concentrations did not increase in any group. Multiple polarization markers were used to determine microglia phenotypes utilizing western blotting and FACS. M1 marker (CD86) expression increased incrementally after iron exposure while M2 marker (CD206) concentrations decreased in a dose response manner. These findings potentially indict iron as significant contributor and mediator of the inflammatory response among microglia.
Keywords: iron, polarization alterations, reactive oxygen species, antioxidant
Baylor College of Medicine, Neurosurgery, Houston, USA
Keywords: traumatic brain injury, multiple organ dysfunction syndrome, cytokines, critical care, prognosis, sepsis
Barrow Neurological Institute and MEDITECH Foundation, Neurosurgery, Phoenix, USA
Effectiveness of treatment for TBI are dependent of institutional resources. Lack of infrastructure and limitations of intensive care are just few examples. The aim of this study is to compare the management of severe pediatric TBI in two university hospitals; one in USA and one in Colombia. We analyze etiology, management and outcomes, describing differences between institutions. We review medical records of pediatric patients, with severe TBI between 2010 and 2015. Mean age was 6.7 ± 4 years in Colombia vs 7.3 ± 5 years in USA, 74% male patients in Colombia vs 51.5% in USA. MVA was the main cause in Colombia and falls were the main cause in USA. 85.2% of the patients in Colombia were transferred from another hospital and 62.1% of the patients in USA come from the scene. The initial GCS was similar in both groups and the initial CT shows more frequently a Subdural Hematoma in USA and a Diffuse Axonal Injury in Colombia. In Colombia 14.8% received surgical management and 40.9% in the USA. 11.1% of Colombian patients receive ICP monitoring and 71.2% in the USA. GOS discharge of 5 (51.9%) was the most frequent in Colombia and in USA 3 and 5 were the most common (27.3% & 27.3%). When comparing Pediatric TBI characteristics of two university hospitals in USA and Colombia including demographics, causes of injury and management, differences emerge. MVA, transfer of patients and low neuromonitoring was more frequent in Colombia. Falls, primary transport from the field and advanced neuromonitoring was more frequent in USA. Subdural hematoma was more frequent in USA and axonal injury in Colombia. GOS at discharge was different probably due to the differences in type of primary injury.
Keywords: neurotrauma, neurosurgery, traumatic brain injury, low and middle income country
Keywords: systematic review, clinical guideline, traumatic spinal cord injury, anticoagulation
GUIDELINES FOR THE MANAGEMENT OF PATIENTS WITH SPINAL CORD INJURY: THE TYPE AND TIMING OF REHABILITATION
Keywords: systematic review, clinical guidelines, spinal cord injury, anticoagulation
SAFETY OF A RESTRICTIVE TRANSFUSION PROTOCOL IN PATIENTS WITH TRAUMATIC BRAIN INJURY
University of California San Francisco, Neurosurgery, SanFrancisco, USA
The safety of a lower transfusion threshold in critical care patients has been demonstrated. Although such policies are often embraced in trauma patients, patients with traumatic brain injury (TBI) are often excluded. Transfusing TBI patients at a restrictive transfusion threshold of hemoglobin (Hgb) <7 versus Hgb <10, was evaluated at a Level I trauma center before and after a hospital-wide protocol change. Patients admitted to the ICU with a diagnosis of TBI in isolation or as part of a polytrauma were included, and differences between transfusion protocol groups were evaluated by Chi-Square or t-test as appropriate. Among a total subject population of 1565, there were few differences in demographic and admission characteristics between groups. However, fewer patients received a transfusion of packed red blood cells (PRBCs) with the restrictive protocol (24% vs. 32%, p = 0.001). Among the 451 patients that received a transfusion, the restrictive protocol group was maintained at a significantly lower mean Hgb during their hospital stay (9.6 [SD 1.3] vs. 10.3 [SD 1.1]; p < 0.001). There were no differences between groups in total hospital days or mortality at discharge (p = 0.277, p = 0.748, respectively). The units of PRBCs transfused per patient also did not differ among protocols (p = 0.442). Therefore, a restrictive transfusion protocol is safe in patients with TBI, and the overall decrease in the number of patients receiving a transfusion of PRBCs represents a decrease in allocation of hospital resources.
Keywords: blood transfusion, hemoglobin, traumatic brain injury, hospital outcomes
Spinal cord injury (SCI) generally results from a trauma or tumor that fosters the interruption of sensory and motor function below the site of injury. Given the CNS has little capacity for true regeneration, SCI often produces permanent changes in the strength, sensation, and body function below the injury site, in most cases leading to paralysis. NINDS estimates 12,000 SCIs to occur every year in the United States alone, and more than a quarter of a million Americans to be currently living with SCIs. Managing the care of SCI patients currently costs the health industry approximately $3 billion each year. Sadly, despite numerous research endeavors, an effective clinical therapy following SCI remains elusive.
SCI triggers a rapid endoplasmic reticulum (ER) stress response (ERSR), resulting in upregulation of components of the unfolded protein response (UPR). The UPR is a cell defense mechanism that serves to restore ER homeostasis in response to cell stress. Three signaling branches, PERK, IRE-1, and ATF6, each of which is rapidly initiated following SCI, mediate the UPR. More specifically, expression of phosphorylated eif2α, an immediate downstream target of PERK, has been shown to be upregulated following SCI. Here we show PERK itself to be activated in the spinal cord lesion epicenter following a T9 injury. We also show cell type specificity with respect to PERK activation. We hypothesize pharmacologic PERK inhibition following SCI to reduce lesion cavity expansion by preventing apoptotic cell death, thereby increasing white matter sparing and functional motor recovery. Our data support exploring PERK inhibition as a potential clinical therapy for SCI patients.
Keywords: UPR, PERK
PREDICTION OF SEDATION SCORE USING WIRELESS ACCELEROMETERS
Maintenance of sedation during a patient stay in the hospital has been an important area of investigation following results suggesting a direct correlation between level of sedation and mortality in the ICU setting. Inappropriate sedation not only extends the stay of patients in the ICU but is also associated with increased mortality rates. Specifically, it has been shown that heavier sedation results in a higher risk of mortality at 30 days. Wireless accelerometers measure motion in three-dimensional while requiring miniscule power. We hypothesized accurate measurements of movements would correlate well with nursing assessment scores for patients requiring mild to moderate sedation. Methods: Patients requiring mechanical ventilation in the neuroICU at the University of Minnesota were screened and enrolled for placement of wrist-mounted, bluetooth wireless accelerometers under IRB approval. Richmond Agitation Sedation Scale (RASS) scores were collected from hourly nursing assessments. An algorithm was developed with multiple times window scales to predict RASS scores for each hour and compared with the nursing assessments. Results: Ten patients in the neuroICU were enrolled in the study. Variation in windowed movement data significantly explained variation in RASS scores observed by nurses with greater than 90% correlation. The data was used to create an algorithm for predicting RASS score based on accelerometer data. Conclusion: In ventilated patients who require mild-to-moderate sedation in the neuroICU, wireless accelerometers perform similarly to nursing assessments while providing substantial improvement in time resolution. Improved resolution in sedation scoring will help further delineate a patient's level of sedation and allow for optimization of sedatives for maximization of mortality.
Keywords: sedation, accelerometer, neurocritical care, monitoring, informatics
INCIDENCE AND RISK FACTORS FOR DEVELOPMENT OF PULMONARY EMBOLI IN SEVERE TRAUMATIC BRAIN INJURY
UTHSCSA, Neurosurgery, San Antonio, USA
Keywords: pulmonary embolism, severe traumatic brain injury, DVT prophylaxis, intracranial pressure monitoring
University of Pennsylvania, Neurosurgery, Philadelphia, USA
Neurotrauma may result in significant neurodegeneration, potentially resulting in permanent functional deficits due to limited capacity of the central nervous system (CNS) to reconstitute lost neuronal populations. In the brain, specific astrocyte populations form longitudinally oriented micro-structures called “glial tubes” that direct migrating neuronal progenitors in the rostral migratory stream (RMS) from a stem cell niche in the subventricular zone. Our objective was to apply micro-tissue engineering techniques to create “living scaffolds” designed to mimic features of in vivo migratory pathways in order to recruit endogenous neural stem cells to CNS lesions. We devised a biomaterial encasement scheme using tubular hydrogel-collagen micro-columns that facilitated the self-assembly of seeded astrocytes into 3-D living scaffolds consisting of long, cable-like aligned astrocytic networks. We systematically tested the effects of the micro-column biomaterial scheme and astrocyte seeding parameters to achieve optimal 3-D aligned astrocyte network formation. These studies revealed that micro-column physical cues such as the radius of curvature dictated astrocyte morphology and extent of alignment; whereas seeding density determined the compaction of the resulting aligned networks. Indeed, high density seeding of small diameter (< 350 μm) micro-columns led to the formation of ultra-dense 3-D “bundles” of aligned bi-polar astrocytes measuring less than 150 μm in diameter yet extending to a remarkable length of over 2.5 cm. Importantly, co-seeded neurons migrated and extended neurites directly on aligned astrocytic bundles, demonstrating permissive cues for neuronal adhesion and neurite outgrowth. These miniature transplantable cable-like astrocytic networks structurally mimic the glial tube that guides neuroblast migration along the RMS. We are currently assessing the ability of these aligned astrocytic networks to orchestrate neural progenitor migration to repopulate sites of widespread neurodegeneration following CNS injury. Financial support provided by the Penn Medicine Neuroscience Center and NIH.
Keywords: astrocyte, tissue engineering, regeneration, neuronal migration
Hippocampal neurogenesis results in the generation of new neurons which have distinct neurophysiological properties essential for proper functioning and plasticity of the network. While there is evidence for an early increase in neurogenesis after traumatic brain injury (TBI), long-term changes in neurogenesis after TBI remain largely unexplored. We examined whether the early post-injury abnormal increase in neurogenesis leads to subsequent decrease in neural precursor cells (NPCs) and exhaustion of the proliferative capacity. Young adult male rats (25 day) were subject to moderate (2 atm) lateral fluid percussion injury (FPI). Histological studies were conducted 3, 7, 30 and 90 days after FPI and in age-matched sham controls. Doublecortin labeling for immature granule cells (IGC) revealed that compared to age-matched controls, FPI rats showed an early increase in IGCs 3 and 7 days post-injury followed by a decrease at 30 and 90 days. BrdU labeling revealed an increase in Prox-1 positive cells co-labeled with BrdU 45 days after injury indicating that a majority of proliferating cells survive and mature into granule cells. Co-labeling with BLBP a neural progenitor cell (NPCs) marker and Ki67 which labels proliferating cells demonstrated the NPCs and proliferation rates were increased 3 days after FPI and decrease at 90 days. In vivo post-injury antagonism of VEGF receptor 2 limited the TBI-induced increase in neurogenesis at 3 days and reversed the long-term reduction in neurogenesis 90 days after injury. VEGFR2 treatment also rescued the post-injury reduction in neurogenic capacity quantified as the increase in neurogenesis following a challenge with sub-convulsive dose of kainic acid administered one month after injury. Thus, in contrast to the prevailing view that post-traumatic increase in neurogenesis is beneficial, we find that early increase in neurogenesis after brain injury may adversely affect long-term outcomes.
Support: NJCBIR CBIR14IRG024 to VS
Keywords: dentate gyrus, epilepsy, neurogenesis, neurogenic potential
RIT MEDIATED SOX2 ACTIVATION INDUCES NEUROGENESIS IN HIPPOCAMPUS: STRATEGY FOR EFFECTIVE REGENERATION FOLLOWING NEUROTRAUMA
Traumatic brain injury leads to the compromised survival of newly born hippocampal neurons and hence limits the prospect for effective regeneration. Therefore strategies that either increase the survival and/or increase the generation of newly born hippocampal neurons may improve the quality of life of traumatic injury subjects. We expressed the constitutively active form of the Ras related small GTPase Rit (RitQ79L) using a doxycycline (Dox) controlled Tet off system in hippocampus and investigated the generation of DCX+ newborn neuroblasts. After 3 weeks of Dox removal the RitQ79L mice displayed a robust increase in DCX+ neuroblasts without affecting the basal NPC pool. In order to understand the mechanism of Rit-mediated expansion of the DCX+ neuroblast pool, we performed a transcription factor screen which identified upregulation of the Sox2 transcription. Rit-mediated Sox2 activation leads to NPC expansion (including upregulation of Neurod1+, Neurog1+ and Tbr2+) and subsequent neuronal differentiation. Luciferase gene reporter assays in isolated hippocampal neuronal precursor cells (hNPcs) confirmed RitQ79L-mediated Sox2 activation. Moreover, active Rit stimulates Akt-dependent phosphorylation of Sox2 at T118, leading to its stabilization and transcriptional activation. Our results demonstrate a strategy to increase the hippocampal neurogenesis by driving the Sox2 based transcription. Therefore, therapeutic Rit activation may provide a targeted approach towards increasing newborn neuroblasts and limit the neuropsychiatric burden of traumatic brain injury subjects.
Keywords: neurogenesis, Sox2, Rit, dentate gyrus
MECHANISMS OF CHRONIC COGNITIVE DEFICITS AFTER TBI
University of Cincinnati, Psychiatry and Behavioral Neuroscience, Cincinnati, USA
The biological mechanisms leading to persistent cognitive and behavioral symptoms after traumatic brain injury (TBI) are not well characterized. In healthy brain, microglia have a central role in neuroplasticity. Microglia are thought to mediate spine turnover and other neuroplastic events via engulfment of spines and processes, and release of paracrine factors that help maintain synaptic fidelity. Developmental synaptic pruning depends on presynaptic complement 3 (C3) and microglial complement 3 receptor (C3R) interactions. Aberrant complement activity is linked with psychiatric syndromes and neurodegenerative disorders with cognitive symptoms such as schizophrenia, Alzheimer's disease, and multiple sclerosis. Accumulating evidence implicates pathological activation of the complement system early after TBI; complement proteins C1q and C3 increase over at least 6 days, spine density is reduced for at least 1 week, and acute acceleration of complement turnover with exogenous complement regulatory factors improves short-term outcome. However the role of complement activation in chronic TBI is unknown. Our initial studies suggest ongoing microglial activation in frontal cortex 8 weeks after lateral fluid percussion. Additionally, expression of complement regulatory factors is decreased in frontal cortex in rats with chronic cognitive impairment. We propose that cognitive symptoms of TBI are linked to altered maintenance and turnover of synapses which is mediated by microglia and dependent on complement activation.
Keywords: rat, chronic, complement regulation, fluid percussion, frontal cortex
CNRM/HJF/NIH, NINDS/SB, Bethesda, USA
Traumatic microbleeds visible on T2* MRI of TBI patients, which appear as linear or branching structures, suggest accumulation of iron along the track of a vessel. Traditional 2D pathology demonstrates iron-laden macrophages in perivascular space surrounding large index vessels with associated damage to the adjacent parenchyma. Iron surrounding small vessels is also seen distant to injury, but is not visualized as discrete microbleeds on MRI. We sought to investigate the morphology of injured vasculature using 3D histological reconstruction. A whole human brain was procured from a patient who expired 7 months post TBI. Specimen was formalin-fixed and imaged at 7T. Brain was sliced into parallel slabs. Sections from the left frontal lobe were imaged in small-bore animal 7T MRI and used for analysis in correlation with histology. Myelin, Nissl, and Prussian blue staining were interleaved to create a 3D brain construction of histology. Minimum intensity projection (MIP) image was created to co-register to MRI for visualization of histology in tri-planar view. On T2* images microbleeds appear as hypointensities that track from gray to white matter. These MRI findings co-localized on histology to areas of Prussian blue staining where iron-laden macrophages are observed in the perivascular space surrounding a 50 micron vessel. Tracing some of these Prussian blue positive vessels leads to regions of cortex containing a collection of iron in cortex extending beyond the immediate vasculature. Nissl and myelin stained sections, in comparison, reveal a lack of cell bodies and reduced myelin in surrounding areas, signifying neuronal cell loss and demyelination. 3D MIP of iron demonstrates regions of connected iron that branch and extend over millimeters. Interleaved serial section of traumatic microbleeds revealed a vascular tree beyond what is seen on MRI, suggesting T2* imaging may only identify the “tip-of-the-iceberg” of vascular injury.
Keywords: microbleed, histology
La Trobe University, Physiology, Anatomy and Microbiology, Bundoora, Australia
Concomitant traumatic brain injury (TBI) and long bone fracture are routinely observed in multitrauma as a consequence of high energy impacts resulting from motor vehicle collisions, falls and warzone injuries. While there is growing evidence that extracranial injuries such as bone fractures may influence TBI outcomes, few studies have investigated the effect of TBI on fracture healing. Therefore in order to further explore the effect of TBI on fracture healing, here we developed a novel mouse model that involved a closed-skull weight drop and concomitant tibial fracture. Adult male mice were randomly assigned into either fracture + sham TBI (FX) or fracture + TBI (MULTI) injury groups. Mice were killed at 21 and 35 days post-injury and fractures analyzed via micro computed tomography (μCT) and histomorphometry. μCT analysis revealed calluses from MULTI mice had a greater bone and tissue volume, and a higher mean polar moment of inertia when compared to calluses from FX mice at 21 days post-injury. Histological results complemented the μCT findings, demonstrating an increased amount of newly formed trabecular bone in MULTI calluses at 21 days post-injury. By 35 days post-injury there were no differences in callus structural parameters between MULTI and FX mice indicating MULTI calluses remodelled to the size of their FX counterparts. Taken together, these findings indicate that closed-skull TBI results in calluses that are larger in size and have an increased bone volume, which is consistent with the notion that TBI induces a more robust callus formation response.
Keywords: multitrauma, bone fracture, fracture healing
R. Adams Cowley Shock Trauma Center, University of Maryland School of Medicine, Neurocritical Care, Baltimore, USA
Keywords: histology, MRI, microvascular, extracellular matrix, ischemia, human
The need to determine reliable markers of neuronal injury following a traumatic spinal cord injury (SCI) is of critical importance to both the clinical diagnosis of injury severity and to preclinical models for the study of medical interventions to treat a SCI. Neurodegeneration, as a result of traumatically induced neuronal damage, is believed to play a role in the secondary injuries that occur post-SCI. The current study proposes to determine a novel marker of neurodegeneration by examining the hyperphosphorylation of the protein tau, a post-traumatic modification which has been examined as a marker of neurodegeneration to a high degree in studies of traumatic brain injury but has not yet been examined in SCI. The characterization of tau hyperphosphorylation will be done by examining the extent of hyperphosphorylated tau accumulation in the cell bodies of neurons affected by a traumatic SCI. Female Wistar rats (N = 60) will be used in the current study. Rats will be subjected to a severe traumatic spinal cord clip compression injury and sacrificed at three time points post-injury: 4 hours, 24 hours, and 1 week. The spinal cord will be dissected after perfusion with fixative, and segments rostral and caudal to the injury site will be sectioned. This will allow a serial analysis to be done on the degree of hyperphosphorylated tau accumulation in cell bodies in spinal cord segments progressively farther from the injury site. The three time points will be used to determine how early and to what extent hyperphosphorylated tau accumulation occurs post-SCI.
Acknowledgments: Spinal Cord Injury Ontario
Keywords: tau, hyperphosphorylation, compression, spinal cord, preclinical
RANDOMIZED, PLACEBO-CONTROLLED, DOUBLE-BLINDED TRIAL OF GRANULOCYTE COLONY STIMULATING FACTOR-MEDIATED NEUROPROTECTION FOR SCI
Chiba University Graduate School of Medicine, Dept of Orthopedic Surgery, Chiba, Japan
Next we moved to early phase of clinical trials. In a phase 1/2a trial, no adverse events were observed. Next, we conducted a non- randomized, non-blinded, comparative trial, which suggested the efficacy of G-CSF for promoting neurological recovery. We are now performing a phase 3 trial to confirm G-CSF treatment efficacy for SCI.
Our primary endpoint is changes in ASIA motor scores from baseline to 3 months. Each group includes 44 patients (88 total patients). Our protocol was approved by the Pharmaceuticals and Medical Device Agency and this trial is funded by the Center for Clinical Trials, Japan Medical Association.
The current trial is now underway at the 18 sites in Japan. At present, 16 cases were enrolled to this trial.
Keywords: G-CSF, clinical trial, neuroprotection, drug approval
Safar Center, Univ. of Pittsburgh, Critical Care Medicine, Pittsburgh, USA
The eighth drug to be tested by the multicenter consortium Operation Brain Trauma Therapy was AER-271, a drug developed by Aeromics to target brain edema and reduce ICP. We tested an intravenous infusion of AER-271 on neurobehavioral and histopathological outcomes in the CCI model. Thirty adult male Sprague Dawley rats were prepared for CCI (4 m/sec, 2.8 mm deformation) or sham. Rats were randomized into three groups: CCI+vehicle (vehicle bolus IV beginning at 15 min after injury and administered over 30 min, followed immediately by a continuous IV infusion of vehicle for 48 h), CCI+AER-271 (2.5 mg/kg bolus IV beginning 15 min after injury and administered over 30 min, followed by continuous IV infusion of 1 mg/kg/h for 48h), and Sham. Functional outcomes were tested via Beam Balance Task (BBT) and Beam Walking Test (BWT)(days 3–5), Morris water maze acquisition (days 14–18) and probe trial (day 18). Rats were sacrificed on day 21 for lesion volume analysis. Groups differed on BBT and BWT performance (p > 0.0001); post hoc testing revealed sham animals balanced longer than vehicle and AER animals. For both tests there were no significant differences between the two CCI groups. MWM latency differed by group (p > 0.001); post hoc testing revealed longer latencies (vs. sham) in the CCI+vehicle (p > 0.001) and CCI+AER group (p = 0.0001). There were no significant differences between the two CCI groups. Probe trial was significantly poorer for both CCI groups vs. Sham, but no differences between the CCI groups. There was a non-significant trend towards AER-271 attenuating lesion volume (18.8 vs 15.1 mm3) and hemispheric volume loss (21.1 vs 17.2 mm3). AER-271 was not beneficial in the CCI model within the rigors of our OBTT assessment paradigm. Support:US Army(W81XWH-10-1-0623).
Keywords: TBI, controlled cortical impact, rat
Traumatic brain injury (TBI) is one of the leading causes of mortality in the United States. Functional deficits sustained from TBI persist throughout the life of the patient due to a lack of effective clinical treatments. Phenelzine (PZ) is an effective therapy for anxiety and atypical depression and has been shown to express neuroprotective effects in models of TBI. Additionally, research from our lab has demonstrated that rats raised in enriched environments (EE) perform better on behavioral tasks than rats housed in traditional or standard laboratory environments (SE). The purpose of the current study was to examine the effect of PZ on recovery of function in rats reared in an EE or purchased as adults and housed in a SE. Twenty-five day old male Long-Evans rat pups were reared in an EE. After ninety days in the EE, MFC contusion injuries were administered to EE-housed rats. Three month old male rats were purchased and placed into SE upon arrival. After seven days, eighteen of the SE-housed rats received an MFC contusion injury. After each injury, at fifteen minutes post-injury, half of the animals received a subcutaneous (10 mg/kg) injection of phenelzine, and the other half received an injection of an equal volume of the vehicle. Behavioral analysis began one week post-injury and included multiple behavioral tasks. Upon completion of behavioral testing, the animals were euthanized and perfused, and their brains extracted. Tissue underwent histological preparations and stereological analysis. Overall, injured animals continued to express significant functional deficits, regardless of their pre-injury housing condition. The data suggest that PZ-treated animals housed in SE performed better on tasks than vehicle-treated/SE-housed animals. However, there were no significant differences between PZ-treated and vehicle-treated animals that were housed in the EE. These findings suggest that housing environment may be an important variable in recovery of function following brain injury.
Keywords: phenelzine, enriched environment
INTRA-ARTERIAL PHARMACOTHERAPY ADMINISTRATION FOLLOWING EXPERIMENTAL STROKE IMPROVES NEURONAL SURVIVAL AND FUNCTIONAL OUTCOME
The disconnect between laboratory stroke models and the human clinical condition has long been recognized as a limitation to translating preclinical neuroprotective research into therapeutics. Experimental neuroprotective stroke therapies may fail, in part, due to their inability to reach stroke-affected brain tissue if the blood clot has not been removed prior to treatment. To that end, we proposed to use the MCAO mouse stroke model in combination with post-reperfusion selective intra-arterial (IA) neuroprotectant administration to accurately mimic large vessel occlusion and the IA techniques used during surgical thrombectomy in patients. Given the complex pathways involved in stroke, it is also unlikely that monotherapy would yield highly significant benefits in the human condition. Therefore, we proposed to study synergistic effects of two neuroprotective agents administered selectively after stroke reperfusion. We administered two distinct neuroprotective agents, a calcium channel blocker and an experimental NMDA receptor modulator. Selective administration of either agent significantly reduced mean brain infarct volumes and improved functional outcomes without any systemic side-effects. Selective IA administration of these agents following successful recanalization proved safe under physiological measurements of heart rate and blood pressure when compared to IA control (saline). We next looked at blood flow through the MCA following drug administration and found no difference between IA administration of combinational therapy and IA control. To determine the potential effects of IA therapy on post-stroke motor function, we exposed the mice to both rotor rod and open field motor assessment at baseline (before stroke), post-stroke day (PSD) 1 and 7. We noted a significant increase in functional recovery from PSD 1 to PSD 7 in both tests. Lastly we examined infarct volume and found a significant reduction (75%) when comparing IA combination versus IA control. We conclude that the selective IA administration of potential neuroprotective agents can be successfully modeled in the laboratory to mimic contemporary human large vessel acute stroke management and may result in the successful translation of experimental stroke treatments.
Keywords: translational, pharmacotherapy, intra-arterial, combinational therapy
PROTECTION AGAINST TRAUMATIC BRAIN INJURY AFFORDED BY RIN GTPASE DEFICIENCY
University of Kentucky, Biochemistry, Lexington, USA
Traumatic brain injury (TBI) affects approximately 1.4 million people in the United States annually and remains a leading cause of death and permanent disability. TBI results in diverse manifestations of damage throughout the brain, including cell death, inflammation, excitotoxicity and axonal degeneration, which ultimately results in a long-term loss of cellular function and cognition. Given the heterogeneity of the injury and downstream effects, the search for potent neuroprotective pharmaceuticals to combat multiple secondary effects of TBI remains an active, however, enigmatic area of research. Ras family proteins act as GDP/GTP regulated molecular switches governing diverse cellular processes by allowing for the relay of extracellular stimuli into cellular signaling pathways. Rin was identified as a small G-protein with expression limited to the central nervous system. Through the generation of a Rin knockout mouse, our lab has elucidated a model in which the loss of Rin signaling aids in neuroprotection following brain injury. Our data suggests the absence of Rin is neuroprotective following a controlled cortical impact (CCI) model of TBI, decreasing the amount of neurodegeneration (FJC+) in the dentate gryrus and CA3 region of the hippocampus. Although sparing was seen within the dentate gryrus of Rin-/- mice, no alterations were observed in immature (Dcx+) neuronal survival or proliferation (Ki67+) was seen at 2d post-injury when compared to wild-type controls. Furthermore, recovery of immature neurons 10d following TBI was also unchanged. Together, these data led to the hypothesis that Rin loss spared mature neurons from injury related cell death. Stereological counts of mature neurons within the CA3 10d following trauma indicate that indeed Rin loss increased mature neuronal survival compared to WT littermates. Additionally, Rin loss proved to enhance cognitive function after trauma in a novel object recognition paradigm. Taken together, these data suggest that the loss of Rin is protective against TBI.
Keywords: signal transduction
Following traumatic brain injury (TBI), mitochondria play an essential role in maintaining cellular homeostasis and survival. Therefore, mitochondria are promising therapeutic targets for prevention of cellular death and dysfunction following TBI. Mitochondria are heterogeneous, consisting of both synaptic and non-synaptic populations, which have distinct properties. It is essential mitochondria targeted pharmacotherapies protect both synaptic and non-synaptic populations to be considered optimally effective. One of the most promising mitochondrial targeted TBI therapies is the mitochondrial permeability transition pore (mPTP) inhibitor, cyclosporine A (CsA). However, previous studies have focused on CsA protection of total mitochondria. This study is the first to examine the effects of acute CsA administration on isolated synaptic and non-synaptic mitochondria following TBI, as well as the first to study the effects of CsA on mitochondrial bioenergetics 24 h following TBI in adult rodents. Our results indicate that synaptic mitochondria sustain more damage 24 h following severe controlled cortical impact injury (CCI) than non-synaptic mitochondria, and that administration of CsA (20 mg/kg, i.p.) 15 min following injury is able to significantly attenuate mitochondrial respiratory dysfunction in both populations. However, following injury CsA treatment results in synaptic mitochondria that are still significantly impaired compared to non-synaptic mitochondria. Further characterization of both synaptic and non-synaptic mitochondria in TBI, as well as their responses to pharmacotherapy following injury is essential in order to better understand the contribution each population makes to TBI pathology, and to the successful development of mitochondria directed therapeutic strategies.
Keywords: mitochondria, synaptic, non-synaptic, lipid peroxidation, cyclosporine A
DOSE-RESPONSE EVALUATION OF AMANTADINE IN THE MIAMI FLUID PERCUSSION MODEL OF TRAUMATIC BRAIN INJURY: AN OBTT CONSORTIUM STUDY
Amantadine (AMA) is a compound with several potential mechanisms useful in treating traumatic brain injury (TBI). Published reports have shown that Amantadine augments dopaminergic neurotransmission, acts as a partial NMDA antagonist and inhibits microglial activation. Amantadine was chosen as the ninth drug for testing by the multicenter consortium Operation Brain Trauma Therapy. The University of Miami site tested Amantadine in our model of fluid percussion TBI. Male Sprague-Dawley rats were anesthetized and underwent moderate fluid percussion (FP; 1.8–2.1 atm) TBI or sham surgery. Rats were randomized into three treatment groups and administered Amantadine (10 mg or 45 mg/kg IP) or vehicle daily beginning 1 day after TBI. Animal groups were TBI-AMA-Low (n = 15), TBI-AMA-High (n = 13), TBI-Veh (n = 15) or Sham (n = 15). Rats were tested on day 7 post-injury for sensorimotor function (gridwalk, cylinder task). On days 13–21, rats were assessed for cognitive function utilizing the simple place task, probe trial and working memory task. On day 21, brain tissue was processed for histology. One-way ANOVA was not significant for the cylinder or gridwalk tasks. For the hidden platform task, two-way repeated measures ANOVA for latency was significant for group (p < 0.05) but not for group × day. Neither dosage improved function on this task vs. TBI-Veh. Surprisingly, the high dose group exhibited increases on both path length and latency. There was no significant difference between groups for the probe trial. Repeated measures ANOVA for working memory latency was significant for trial and group (p < 0.05) and again, worse in the high dose group. We conclude that treatment with either dosage of Amantadine after FP did not improve sensorimotor or cognitive function, with paradoxical deleterious effects in the high dose group. At this time, behavioral findings of Amantadine treatment in the FP model in rats do not support its further testing in OBTT. Support: US Army W81XWH-10-1-0623.
Keywords: traumatic brain injury, neuroprotection, behavior, OBTT, fluid percussion
INTRANASAL GDNF DELIVERY TO TREAT TRAUMATIC BRAIN INJURY
University of Texas Medical Branch, Neuroscience & Cell Biology, Galveston, USA
Traumatic brain injury (TBI) causes mortality and morbidity in adolescent and young adults who are involved in sports, automobile accidents and military services. Currently there are no effective treatments to reduce brain damage and prevent TBI-induced acute and chronic disabilities. Our long-term goal is to develop novel therapeutic strategies to treat TBI patients by enhancing neural repair. Here we examined the pharmacokinetics and efficacy of acute intranasal administration of glial cell line-derived neurotrophic factor (GDNF), a non-invasive, simple and clinically relevant approach to treat TBI. Our preliminary data showed that intranasal GDNF induced significant increases of GDNF in cerebrospinal fluid and critical brain regions such as hippocampus and cortex from both rats and pigs. Intranasal GDNF treatment protected electrophysiological function of hippocampus and improved cognitive function in rats. GDNF also reduced the pathological changes of rat brains after TBI. Furthermore, through an academic and industrial collaboration, we found that GDNF retained its biological function after packaging into Mystic blisters, a novel technology developed by the Mystic Pharmaceuticals that ensures accurate drug administration. Our results indicate the potential of intranasal GDNF treatment as a novel strategy to treat TBI.
Support: Studies were completed as part of a team funded by The Moody Project for Translational Traumatic Brain Injury Research, and supported by Mission Connect a project of TIRR Foundation, Coalition for Brain Injury Research, Gilson Longenbaugh Foundation, J.S. Dunn Foundation and Mystic Pharmaceuticals.
Keywords: intranasal delivery, glial cell line-derived neurotrophic factor, traumatic brain injury
Vanderbilt University, Vanderbilt Eye Institute, Nashville, USA
Erythropoietin (EPO) is a promising neuroprotective agent and is currently in Phase III clinical trials for the treatment of traumatic brain injury. The goal of this study was to determine if EPO is also protective to the retina after traumatic eye injury. The left eyes of anesthetized DBA/2J or Balb/c mice were exposed to a single 26 psi over-pressure air-wave while the rest of the body was shielded. DBA/2J mice were given intraperitoneal injections of EPO or buffer and analyses were performed at 3 or 7-days post-blast. Balb/c mice were given intramuscular injections of rAAV.EpoR76E or rAAV.eGFP either pre-or post-blast and analyses were performed at 1-month post-blast. EPO had a bi-modal effect on cell death, glial reactivity, and oxidative stress. All measures were increased at 3-days post-blast and decreased at 7-days post-blast. Increased retinal ferritin and NADPH oxygenases were detected in retinas from EPO-treated mice. Gene delivery of EPO.R76E protected against axon degeneration, cell death and oxidative stress when given after blast, but not before. Systemic EPO.R76E protects the retina after trauma even when initiation of treatment is delayed by up to 3-weeks. Systemic treatment with EPO or EPO-R76E beginning before or soon after trauma may exacerbate protective effects of EPO within the retina as a result of increased iron and oxygen levels from erythropoiesis and thus, increased oxidative stress within the retina. This is likely overcome with time as a result of an EPO-induced increase in gene expression of antioxidant enzymes. Either intraocular delivery of EPO or treatment with non-erythropoietic forms of EPO may be more efficacious. Funded by Department of Defense W81XW-10-1-0528 (TSR), W81XWH-13-1-0048 (TSR), W81XWH-15-1-0096 (TSR), NIH R01 EY022349 (TSR) and P30 EY008126 (D. Calkins), Fight for Sight (LDS) and Research to Prevent Blindness (P. Sternberg, Jr.).
Keywords: erythropoietin, eye trauma, gene therapy, retina, optic nerve
AER-271 is a proprietary prodrug from Aeromics and is the 8th drug to be tested by OBTT. It is rapidly converted to AER-270, an aquaporin 4 antagonist and has been shown to decrease edema following MCAO in mice. This drug was chosen by the consortium as part of our endeavor to test novel compounds as neuroprotective and neurorestorive agents against brain injury. The Walter Reed Army Institute of Research site evaluated the effectiveness of AER-271 on neurobehavioral and neuropathological outcomes using the penetrating ballistic-like brain injury (PBBI) model. PBBI (10%) was performed unilaterally in the right hemisphere of anesthetized rats. Rats were randomized into three groups and administered AER-271 (2.5 mg/kg IV bolus followed by continuous 48 h infusion) or vehicle 15 min post-TBI. Dosing was based on previous PK studies. Animal groups were PBBI+AER-271 (n = 16), PBBI+Veh (n = 15) or Sham (n = 14). Rats were tested on days 7 and 10 post-injury for motor function on the rotarod task. On days 13–21, rats were assessed for spatial learning and memory in the Morris water maze (MWM) task. On day 21, brain tissue was processed for histology. No significant differences were detected between AER-271 and vehicle-treated rats on motor performance. MWM results revealed significant deficits in both injury groups with the average latency to locate the hidden platform increased by 61% (vehicle) and 60% (AER-271) vs. sham. Similarly, thigmotaxic (wall-hugging) behavior was increased by 63% (vehicle) and 77% (AER-271) vs. sham (p < .05). The probe (missing platform) trial failed to reveal significant between-group differences although a trend towards worsened behavior was observed in the AER-treated group. Lesion volume did not differ between AER-271 and vehicle-treated rats. Overall, this study indicates that AER-271 is not effective in improving neurobehavioral function or histopathology following PBBI when given in the current dosing regimen. Supported by U.S. Army Grant W81XWH-10-1-0623.
Keywords: Neuroprotection, Aquaporin 4, Therapeutics, Operation Brain Trauma Therapy Consortium
INTRATHECAL NOGGIN ADMINISTRATION IN RATS TEMPORALLY AMELIORATES MECHANICAL ALLODYNIA INDUCED BY A CHRONIC CONSTRICTION INJURY
Chiba Medical Center, Orthopaedic Surgery, Chiba, Japan
Chronic intractable neuropathic pain after central or peripheral nervous system injury remains refractory to therapeutic intervention. Using microarray and RT-qPCR methods, we found that Noggin mRNA is downregulated in the lumbar enlargement 2 weeks after chronic constriction injury (CCI) in rats.
Eight-week-old female Sprague Dawley rats were used for the CCI model. Two weeks after CCI, rats underwent a laminectomy at L5 under halothane anesthesia, and a silicone tube connected to an osmotic mini pump was inserted intrathecally for 14 days. Rats were administered Noggin ranging from 10 ng/ml to 10 mg/ml. Phosphate buffered saline (PBS) was used as a control. The time course of mechanical allodynia was assessed for 5 weeks using von Frey filaments. An ANOVA showed that rats administered Noggin at 2 mg/ml had significantly less mechanical allodynia compared with controls.
We next compared the effect of intrathecal administration (14 days) of Noggin (2 mg/ml), bone morphogenetic protein 4 (BMP4; 2 mg/ml), or BMP4 (2 mg/ml)+Noggin (2 mg/ml) with controls. Only Noggin administration significantly reduced mechanical allodynia in the CCI model.
Fluorescence immunohistochemistry indicated that Noggin administration decreased astrocyte accumulation in the dorsal horn compared with PBS after administration for one week. BMP4-driven conversion of oligodendrocyte progenitor cells (OPCs) to type 2 astrocytes is inhibited by Noggin (Hampton et al., 2007). We speculated that Noggin administration inhibits the conversion of OPCs to astrocytes, and decreases glial fibrillar acidic protein expression. This histological condition could decrease neuropathic pain.
Keywords: chronic constriction injury (CCI), noggin, microarray, allodynia
Clinical studies indicate that traumatic brain injury (TBI) patients frequently experience chronic post-traumatic pain, particularly vascular-type headache. A recent prospective study provides compelling evidence that such pain is unrelated to injury severity. Although headache descriptions predominate, patients may also experience allodynia, hyperalgesia/hyperesthesia, or spontaneous pain. Periorbital and extra-cephalic (paw) mechanical allodynia have been reported in rodent models of TBI, which may persist for weeks after injury. However, there has been little research devoted to understanding the pathobiology to such hyperesthesia. The present study characterized post-TBI sensory changes in mice with mild, moderate or severe controlled cortical impact injury (CCI) by testing mechanical/thermal allodynia and presence of spontaneously face pain (MGS). Microglia/astrocytes-mediated inflammation was examined in brain regions as well as spinal dorsal horns after CCI. The von Frey hair force was significantly decreased on the left and right hindpaws of mice subjected to moderate/severe TBI when compared to sham mice. The threshold for hot plate temperature was decreased in a severity-dependent manner. The threshold for cold plate was significantly increased in all grades of TBI severity at early time points but returned to baseline level at 4 weeks post-injury. The MGS based on ear position, orbital tightening, and nose bulge was transiently increased at day 1 for all groups. Sham and mild TBI group returns to the baseline level at week 1. However, moderate/severe TBI groups showed extended increases of MGS. Quantitative IHC analysis revealed that chronic inflammation occurs in pain regulatory areas such as brainstem, thalamus, and spinal dorsal horns. The present study characterizes the time course of hyperesthesia after TBI of varying severity as well as microglia/astrocytes-mediated inflammation. These observations indicate that more generalized hyperesthesia and pain, as well as vascular-like headaches, may occur after TBI, and associated with chronic inflammation specifically in pain regulatory areas.
Keywords: TBI, pain, hyperesthesia, spontaneous face pain, inflammation, mice
DEFICITS IN DECISION-MAKING SKILLS AFTER POST-NATAL DAY 28 BUT NOT DAY 17 FRONTAL CONTROLLED CORTICAL IMPACT
Currently, there are still questions as to how the young brain copes with traumatic brain injury (TBI). The developmental time window in which brain injury can best be rehabilitated remains unclear. Further investigation of developmental time windows can help to figure out when TBIs do the most damage and how these injuries impact neurocognitive abilities later in life. The current study sought to investigate the differences in impulsive decision-making skills between rats that received a TBI at post-natal day 17 and those that received one on post-natal day 28. In order to assess these differences, male rats (PND17 & PND28) were anesthetized and given either a bilateral frontal controlled cortical impact (CCI) or a sham surgery. When the subjects reached early adulthood (i.e. PND 60) they were tested on the Dig Task in order to test for decision-making deficits. Assessment in the Dig Task included three discrimination paradigms and has been previously found to be sensitive to frontal injury occurring in adulthood. Results showed that those receiving a CCI on PND28 performed significantly worse on the task than those that received one on PND17. Specifically, those that received an injury on PND28 had a significantly lower cumulative percentage of correct choices in the discriminations than the PND17 group and the shams and took significantly longer than the shams to complete each discrimination. Moreover, the PND28-injured rats had significantly fewer correct first choice percentages than the other two groups for the first two discrimination paradigms, as well as fewer correct first choice percentages in the third paradigm than those in the PND17-injured group. The PND28 groups also showed significantly fewer correct last choice percentages in the first discrimination paradigm than the other groups. Taken together, these findings indicate that frontal injuries occurring early in development may not lead to increased impulsivity and deficits in decision-making; however, the same injury at a later developmental period results in severe impairments later in life.
Keywords: developmental, frontal, immature, decision making, impulsivity, controlled cortical impact
EVALUATION OF TRUNK AND RESPIRATORY MOTOR CONTROL IN CHILDREN WITH SPINAL CORD INJURY
University of Louisville, Physiology, Louisville, USA
Keywords: respiratory motor control, trunk control, neurodevelopment, surface EMG, SCI injury in children
Gettysburg College, Health Sciences, Gettysburg, USA
Gastrointestinal reflexes from the esophagus to the proximal colon are dominated by parasympathetic control within the vagus nerve. For example, gastrointestinal vagal afferent neurotransmission occurs in response to mechanical (e.g., stroking) and chemosensitive (e.g., gut peptide) stimuli. These vago-vagal circuits remain anatomically intact following SCI. Our previous qRT-PCR and neurophysiological data showed an increased expression of gastrointestinal inflammatory markers as well as nodose ganglion CCK and CCK-1 receptor (CCK-1r) accompanied by diminished vagal afferent nerve sensitivity to systemic administration of CCK in 3-day T3-SCI rats. In other animal model systems, TRPV1 is upregulated in gastrointestinal inflammation and mediates a heightened sensitivity of vago-vagal reflexes. The functional effects of TRPV1 activation of vagal afferents and the time course of CCK-1r and TRPV1 upregulation following SCI remained unresolved. We hypothesized that acute T3-SCI leads to gastric dysfunction, in part, due to prolonged neuroplasticity of vagal afferent transmission. Male Wistar rats received a contusion injury at spinal T3 or laminectomy. In vivo neurophysiological recordings of gastric vagal afferent activity following I.V. infusion of CCK-8s and the TRPV1-agonist, capsaicin. The left gastric vagus nerve was isolated sub-diaphragmatically in anesthetized control vs T3-SCI rats. The expression of nodose CCK-1r and TRPV1 was not significant 3-weeks following T3-SCI. Our 3-week neurophysiological data extended our previous 3-day observations of a diminished gastric vagal afferent nerve response to I.V. infusion of CCK-8s in T3-SCI rats. This response was blocked by the CCK-1r antagonist, lorglumide. In response to I.V. infusion of capsaicin, T3-SCI rats demonstrated a significant increase in sensitivity of gastric vagal afferent response that was attenuated, but not fully absent. T3-SCI neurophysiological recordings reveal a differential sensitivity to CCK-8s and capsaicin. Specifically, the experimental data indicate that: 1) T3-SCI provokes a significant decrease in the sensitivity of gastric vagal afferents to CCK-8s; and 2) T3-SCI provokes a significant increase in the sensitivity of gastric vagal afferents to the TRPV1 agonist, capsaicin. Support: NS 49177, NS 87834
Keywords: SCI, TRPV1, vago-vagal, capsaicin, Neurophysiology
Texas A&M Health Science Center, Neuroscience and Experimental Therapeutics, Bryan, USA
Opioids are commonly used to treat pain in the acute phase of spinal cord injury (SCI). Our data, however, indicate that early administration of morphine undermines locomotor recovery and increases pain reactivity in a rodent contusion model. These adverse effects appear to depend on kappa opioid receptor (KOR) activation. We have shown that activation of the KOR is sufficient to attenuate recovery after SCI, and that blocking KOR activation prevents morphine's adverse effects. Our experiments also implicate glia in the morphine-induced deficit. This study explored the interaction between KORs and glia. Evidence suggests that KORs are expressed on immune cells. We hypothesized that an increase in KOR-expressing immune cells following injury mediates the adverse effects of morphine. To test this, sham and contused (T12) subjects were treated with intrathecal morphine or vehicle 24 h following surgery. At 48 h post-surgery, spinal tissue encompassing the lesion site was collected and dissociated. The cell suspension was plated and stained with markers for CD45, CD11b, IBA1, and KOR. Cells were quantified using a FACSFortessa flow cytometer. We found that SCI significantly increased the number of CD45+ cells at the injury site, relative to shams. There was also a significant effect of surgery on the number of CD45+ cells expressing KORs. Differentiating between resident and infiltrating immune cells, SCI per se did not increase the number of microglia at the injury site at 48 h. Morphine, however, significantly increased the number of microglia expressing the KOR. SCI also produced a 6–8 fold increase in the number of KOR-expressing macrophages, but there was no effect of morphine on this immune cell population. These data indicate that both morphine and SCI modulate the loci of spinal KORs, increasing the expression on immune cells. Activation of KOR receptors on these immune cells may dramatically alter the consequences of opioids in the SCI model.
Keywords: spinal cord injury, rodent, kappa opioid receptor, morphine, glia
University of Pittsburgh, Nursing, Pittsburgh, USA
Keywords: traumatic brain injury, melatonergic receptors, melatonin, pre-clinical, controlled cortical impact
While cortical contusions are among the most common type of traumatic intracranial lesions in humans, most of our knowledge of post-TBI neurogenesis is from work in the rodent hippocampus. In humans, brain injury typically involves both gray and white matter. We utilize our immature piglet model, which has similar proportions of gray and white matter as humans, to study neuroblasts post-TBI. Previously, we demonstrated that scaled cortical impact caused an age-dependent increase the area of the subventricular zone, and an increased density of neuroblasts in gray matter injury. Here, we investigated the white matter component of the cortical impact injury. One-week old piglets received cortical impact to the rostral gyrus or underwent sham surgery. Brains were collected 1 week after injury and the density of doublecortin+ neuroblasts in the white matter were compared to sham piglets. The resulting injury was heterogeneous with half of piglets exhibiting a cavity into the depths of the gyral white matter and half lacking cavitation. The maximum density of neuroblasts in gyral white matter tended to be greater in injured vs. sham piglets (p = 0.09). In piglets with lesion cavitation, neuroblasts were densely arranged in the white matter at the cavity edge. We observed a dense meshwork of calretinin+ neurons intermixed with phenotypically differentiating doublecortin+ neuroblasts, and rarely, co-labeled neurons in the injured white matter. Quantification of calretinin+ neurons in the white matter of injured vs. sham piglets is underway. Future work will aim to determine if this focal aggregation of neurons in the white matter is transient, aiding in repair recapitulating development as interstitial neurons, becomes a glial scar, or persists as ectopic gray matter with the potential to create epileptic foci. In summary, while others have shown that neuroblasts create a dense meshwork around gray matter injury in rodent models of TBI, we demonstrate a similar phenomenon in an immature gyrencephalic species but in the white matter, which may contribute to post-TBI sequelae.
Keywords: cortical impact, gyrencephalic, doublecortin, white matter
MODULATION OF THE MAMMALIAN TARGET OF RAPAMYCIN TO ALTER MALADAPTIVE PLASTICITY ASSOCIATED WITH AUTONOMIC DYSREFLEXIA
Mounting evidence shows that genetic enhancement of mammalian target of rapamycin (mTOR) cortical activity in spinal cord injury (SCI) models promotes corticospinal tract sprouting and improves locomotor recovery. Alternatively, we have shown that the development of autonomic dysreflexia (AD) after complete high thoracic spinal cord transection correlates with maladaptive plasticity of both primary afferent fibers and ascending propriospinal pathways. Therefore, while others have used genetic models to increase mTOR activity, we are testing the hypothesis that inhibiting mTOR with the FDA-approved drug rapamycin (RAP) can mitigate aberrant plasticity associated with AD. Preliminary protein expression data from naïve and injured spinal cord tissues showed an approximately two-fold elevation in phosphorylated mTOR at 10 days post-injury (DPI), with further increases induced by prolonged, intermittent colorectal distension (CRD) at 21 DPI. Similarly, phosphorylation of the downstream ribosomal protein S6 was increased at 10 and 21 DPI, with further expression following CRD at 21 DPI. Critically, RAP treatment (6 mg/kg i.p., every other day) significantly reduced pS6 at 10 and 21 DPI, and prevented CRD-induced increases in pS6. Furthermore, while CRD elicited a five-fold increase in c-FOS expression compared to naive, RAP treatment significantly abated this effect. Preliminary immunohistochemical and behavioral analyses revealed that RAP treatment for 3 weeks attenuated CGRP+ c-fiber sprouting into the lumbosacral dorsal horns and reduced hindlimb spasticity during CRD, the latter a surrogate indicator of AD severity. To fully establish whether mTOR inhibition can prevent aberrant reorganization of spinal circuitry underlying AD, we are employing hemodynamic monitoring to assess the effects of chronic RAP administration on basal cardiovascular parameters and the incidence and severity of AD to inform whether RAP treatment is viable as a prophylactic intervention to prevent the development of AD altogether.
Funding: KSCHIRT #10-10; SCoBIRC Chair Endowment (AGR); NIH/NINDS 2P30NS051220
Keywords: autonomic dysreflexia, sprouting, cell signaling, cardiovascular, pharmacology
CRANIOPLASTY COMPLICATIONS AND REHABILITATION- WILL A CRANIOPLASTY DISRUPT REHABILITATION STAYS?
Cranioplasty (CP) may improve neurocognitive and physical functioning fairly immediately, and may contribute to a greater progress during inpatient rehabilitation. Conversely, periprocedural complications following CP may interrupt the rehabilitation stay. Therefore, the risk and benefit of CP during the acute rehabilitation phase should be carefully weighed. The aim of this study was to evaluate timing of surgery, timing and nature of complications, and risk factors for complications for CP performed in the acute phase.
Keywords: cranioplasty, complication cranioplasty, cranioplasty risk, cranioplasty duration
IMPROVEMENTS IN BLADDER OUTCOMES FOLLOWING TASK-SPECIFIC TRAINING IN HUMAN SPINAL CORD INJURY
University of Louisville, Kentucky Spinal Cord Injury Research Center, Louisville, USA
Following spinal cord injury (SCI), urological complications account for the vast majority of morbidities affecting well-being and quality of life. Life-long bladder management is just one of many adaptive challenges individuals encounter post-injury. The urological deficits manifest as a neurogenic bladder and include bladder overactivity, dyssynergic contractions between the detrusor and sphincter, decreased compliance and incontinence. While standard pharmacological therapy aims to manage bladder dysfunction, therapies targeting functional bladder recovery are lacking. However, locomotor training (LT) effective for improving post-SCI motor outcomes has also been shown to have a beneficial impact on responses from the cardiovascular and respiratory systems. Our recent animal studies and a few clinical cases have further reported improvements in bladder function through the use of LT. Given the overlap of neural networks controlling bladder and locomotor function in the lumbosacral cord, we hypothesized that a vesico-somatic relationship is influenced by LT resulting in improved bladder-sphincter integrity and function. In this study, eight subjects who sustained a SCI received 80 daily 1-hr sessions of LT on a treadmill, using body-weight support, or 1-hr of LT and stand training (on alternate days). Urodynamic assessments were performed at pre-and post-training time points, revealing significant increases in bladder capacity and detrusor contraction time as well as a significant decrease in voiding pressure post-training. These results suggest an appropriate level of sensory information provided to the spinal cord, generated through task-specific stepping and/or loading, appears to influence the neural circuitry controlling bladder function, facilitating an improvement in overall compliance.
Keywords: bladder, human spinal cord injury, locomotor training, activity-dependent plasticity
The antipsychotic drug (APD) haloperidol (HAL), a D2 antagonist, impedes recovery after traumatic brain injury (TBI), and these effects persist up to 3 months. Also, HAL reduces the effectiveness of environmental enrichment (EE), a pre-clinical model of neurorehabilitation. Patients often experience agitation post-TBI and, thus, will be administered APDs so they can be safely treated. Because ARIP provides its antipsychotic properties via a different mechanism than HAL (D2 partial agonist vs. D2 antagonist), it is hypothesized that ARIP will not impair recovery or reduce the effectiveness of EE regardless of whether administered every day (i.e., chronic agitation) or every other day (occasional agitation). Anesthetized adult male rats received a CCI of moderate severity or sham injury and were then randomly assigned to EE or standard (STD) housing. Treatments with ARIP (0.1 mg/kg; i.p.) or vehicle (VEH; 1.0 mL/kg; i.p.) began 24 hr after injury and continued once daily for 19 days, or once every other day for the same period. Motor and cognitive outcome were assessed after surgery on days 1–5 and 14–19, respectively. As expected, the TBI+EE alone group was significantly better in both motor and cognitive function compared to the TBI+STD alone group. Moreover, the administration of ARIP, regardless of dosing regimen, performed significantly better on all endpoints relative to the TBI+VEH controls, but did not differ from one another. The data replicate previous work from our laboratory showing the EE improves motor and cognition after TBI. Furthermore, ARIP, unlike others APDs such as haloperidol or risperidone, did not impair recovery or reduce the efficacy of EE, which supports the hypothesis. The significance of the findings is that ARIP is beneficial on its own and does not negate the benefits of rehabilitation (i.e., EE) and thus can be used safely to control TBI-induced agitation and aggression without compromising recovery.
Supported by the NIH grants R01HD069620, R01NS084967 (AEK)
Keywords: experimental traumatic brain injury, antipsychotic drug
NETWORK MODULARITY PREDICTS BRAIN RESPONSE TO REASONING TRAINING IN CHRONIC TBI
University of Texas at Dallas, School of Behavioral and Brain Sciences, Dallas, USA
Previously, we demonstrated changes in cortical thickness of individuals with chronic Traumatic Brain Injury (TBI) following Strategic Memory Advanced Reasoning Training (SMART). While the effort to characterize training-related brain changes is informative, identifying biomarkers for training-related responses prior to training could be more valuable due to the heterogeneity of TBI and need for individualized interventions. Previous studies exhibited disrupted modular organization in TBI and the potential for modularity (i.e., a graph theoretic measure for the balance between brain network integration and segregation) to predict training outcomes. Here, we assessed the baseline modularity of individuals with TBI whose resting-state functional Magnetic Resonance Imaging scans were acquired and who completed the training (N = 13) to determine whether baseline modularity predicts changes in cortical thickness following the SMART program. We obtained average cortical thickness changes over six regions (left dorsolateral prefrontal and orbital frontal cortices; right dorsal prefrontal and anterior medial prefrontal cortices, postcentral gyrus, and middle temporal complex) where full sample of the SMART group (N = 28) showed greater cortical thickness changes relative to active controls (N = 26). Then, we linearly regressed the cortical thickness changes on the modularity with an age covariate. The regression analysis (R-squared = 0.58) revealed that higher baseline modularity predicted greater change in cortical thickness over time (p = 0.02). There were also statistically significant effects of age on the amount of changes in cortical thickness following training (p = 0.01). Younger participants showed greater changes in cortical thickness over time. These results indicate that the functional network modularity may be a potentially effective neuroimaging biomarker for predicting brain response to cognitive intervention for individuals with chronic TBI.
Keywords: cortical thickness, modularity, rehabilitation, TBI
Naval Medical Research Center, Neurotrauma, OUMD, Silver Spring, USA
Keywords: PbtO2, oxygen therapeutics, phosphorescence quenching method, PFC
Keywords: diffuse axonal injury, secondary injury, microRNA, target gene
SECONDARY INJURY EVENTS ASSOCIATED WITH TRAUMATIC BRAIN INJURY ALTER EXPRESSION OF MITOCHONDRIA ASSOCIATED MICRORNA
University of Kentucky, Sanders Brown Center on Aging, Lexington, USA
Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. There is clear evidence of compromised mitochondrial function following traumatic brain injury (TBI), however, the pathological consequences are not well known. MiRNA are small non-coding RNA molecules that regulate post-transcriptional gene expression, and serve as important mediators of neuronal development, synaptic plasticity, and neurodegeneration. Our recent studies suggest that mitochondria may serve as regulators of cellular miRNA activity following TBI. Here, we extend our initial observations by reporting the effect of TBI related secondary injury events on mitochondria associated miRNA expression. Synaptic and non-synaptic mitochondria were isolated from naïve adult rat cortex and exposed to different concentrations of Ca2+ that inhibit State III mitochondrial respiration by 15–30%. RT-qPCR analysis demonstrated that Ca2+ treatment significantly decreased miR-150 and miR-146a levels in non-synaptic, but not synaptic mitochondria. This observation is particularly intriguing for two reasons. First, non- synaptic mitochondria are known to be more sensitive to Ca2+ treatment compared to synaptic mitochondria. Second, the levels of miR-150 and miR-146a have been shown to decrease in mitochondria isolated from the hippocampal formation following TBI. In a separate set of experiments, rat primary cortical astrocytes were treated with various concentrations of glutamate followed by mitochondrial isolation and RT-qPCR measurement of miRNAs. These studies revealed that mitochondrial levels of selected miRNAs, including miR-223 are altered in a dose-dependent fashion. Collectively, these findings suggest that individual TBI-related secondary events, such as high Ca2+ and exposure to excitotoxic levels of glutamate, trigger different miRNA pathways that may directly impact their downstream activities.
Keywords: microRNA, mitochondria, non-coding RNA, synaptic mitochondria, miR-146a, miR-150
Barrow Neurological Institute at Phoenix Children's Hospital, Phoenix, USA
Chronic neurological impairments can manifest from repetitive traumatic brain injury (rTBI), particularly when subsequent injuries occur before a previous injury has resolved. For sports-related TBI, return-to-play guidelines are trending towards a personalized approach, rather than a time-dependent prescription, based on symptomology. The opportunity exists to define quantitative metrics–biomarkers–that represent risk for worse outcomes following rTBI. We have pioneered research on post-traumatic-sleep identifying injury-induced sleep lasting 6 hrs in brain-injured mice as a bio-indicator of unregulated inflammation. Here, we apply post-traumatic-sleep as a physiological biomarker of vulnerability to hypothesize that a second TBI occurring during the period of post-traumatic-sleep worsens functional and histological outcome compared to mice receiving one TBI or a second TBI after post-traumatic-sleep resolves. Mice were subjected to sham or midline fluid percussion injury (FPI). Brain-injured mice received one FPI, or two FPIs (3 hr or 9 hr interval). As expected, FPI groups showed significantly more post-traumatic-sleep (non-invasive monitoring cages) over 24 hrs. For mice with rTBIs within 3 hrs, functional assessments showed significantly lower latencies on rotarod and increased Neurological Severity Scores. Anxiety-like behaviors in the open field task were significantly increased for mice with rTBIs at 3 hrs. Neuropathology in mice from both rTBI groups was significantly greater at 28 days post-injury (DPI) compared to sham and single-FPI, measured by pixel density of silver accumulation. Neuroinflammation in mice receiving rTBI at 3 hrs was significantly increased at 7, 14, and 28DPI compared to sham, as measured by Iba-1 positive, activated microglia morphology distributions. Orexin-positive neurons regulate wakefulness and contribute to chronic sleep disturbances after TBI. Here, orexin-A positive cells in the lateral hypothalamus did not differ across groups, indicating loss of wake-promoting neurons did not contribute to post-traumatic-sleep. Thus, post-traumatic sleep served as a physiological biomarker defining a period of vulnerability to a second TBI. Monitoring individual post-traumatic sleep is a potential clinical tool for personalized TBI management, where regular sleep patterns may inform rehabilitative strategies and return-to-play guidelines.
Support: Science-Foundation-Arizona, Diane-and-Bruce-Halle-Foundation
Keywords: TBI, sleep, biomarker, inflammation, repetitive TBI
City of Hope National Medical Center, Developmental and Stem Cell Biology, Duarte, USA
Preclinical studies indicate that neural stem cells (NSCs) can limit or reverse central nervous system (CNS) damage through direct cell replacement, promotion of regeneration, or delivery of therapeutic agents. Immortalized NSC lines are in growing demand due to the inherent limitations of adult patient-derived NSCs, including availability, expandability, potential for genetic modifications, and costs. Here, we describe the generation and characterization of a new human fetal NSC line, immortalized by transduction with L-Myc (LM-NSC008) that in vitro displays both self-renewal and multipotent differentiation into neurons, oligodendrocytes and astrocytes. These L-myc.hNSCs were non-tumorigenic in vivo, and migrated to orthotopic glioma xenografts in immunodeficient mice. When administered intranasally, L-myc.hNSCs distributed specifically to sites of traumatic brain injury (TBI). These data support the therapeutic development of immortalized L-myc.hNSC cells for allogeneic use in TBI and other CNS diseases
Keywords: neural stem cell, TBI, distribution, TBI specific migration, human stem cells
University of Arizona College of Medicine-Phoenix, Phoenix, USA
Approximately 2.4 million people sustain a traumatic brain injury (TBI) annually, many of whom suffer long-term morbidities such as auditory and visual sensitivity. After experimental TBI, rodents develop late-onset sensory sensitivity to manual whisker stimulation that corresponds to hypersensitive glutamate release and regional hyper-activation, suggesting altered synaptic connections. During the first week post-injury, there are dynamic changes in glial activation, neuropathology and neuron morphology within the whisker circuit, likely contributing to this sensory sensitivity. The FDA approved drug, riluzole, has been shown to reduce inflammation, apoptosis and glutamate-mediated excitotoxicity while promoting neuronal outgrowth and branching after CNS insult.
We predict that early treatment with riluzole after diffuse TBI will preserve the number of synapses in whisker circuit relays.
Riluzole (8 mg/kg, i.p.) or vehicle was administered to adult male, Sprague–Dawley rats at 15 minutes, 6 hours and 24 hours following a single moderate midline fluid percussion injury or sham surgery. Brains were collected at 3 or 7 days post-injury. Brain sections were immunohistochemically double-labeled for pre- and post-synaptic markers, VGLUT1/VGLUT2 and PSD95, respectively. Image stacks were captured at 63X magnification in the cortical and thalamic relays of the whisker circuit. Synapses were quantified by colocalization of VGLUT and PSD95 using ImageJ Puncta Analyzer.
We have optimized data collection through the depth of tissue and stack size. The optimal method to minimize within-animal variability requires six 2 μm z-stacks (0.2 μm intervals) per animal. This refined image collection protocol permits unbiased assessment of synapse number.
We are now poised to investigate synapse number in cortical and thalamic relays of the whisker circuit and the response to riluzole in order to understand the role of circuit reorganization in TBI.
Support: ADHS14-00003606, NIH-R03-NS-077098, NIH-R01-NS-065052, PCH Mission Support
Keywords: traumatic brain injury, synapses, riluzole, synapse quantification
University of Pittsburgh, Neurosurgery, Pittsburgh, USA
Docosahexaenoic acid (DHA), the main omega-3 fatty acid, is an essential polyunsaturated fatty acid primarily found in marine fish. It is an integral component of neuronal cell membranes and synaptic terminals. DHA has been reported to be neuroprotection is several neurotrauma models, yet its mechanism is unknown. Synucleins (Syn), a family of synaptic proteins, includes alpha-synuclein (α-Syn), which is associated with Parkinson's disease and related neurodegenerative diseases. The native function of α-synuclein is not completely understood, but is thought to involve regulation of synaptic vesicle trafficking. While the pathological forms of α-syn are considered to be the primary targets of TBI-associated neurodegeneration, disruption of the native function of α-Syn may contribute to pathology by diminishing synaptic function. Thus, the goal of the project is to examine the effects of post injury intraperitoneal (i.p.) treatment of DHA on wild-type α-Syn expression at 1 day post injury. Male Sprague-Dawley rats were anesthetized and surgically prepared for CCI injury (4 m/sec, 2.8 mm) or sham surgery. Male Sprague-Dawley rats were randomly assigned and treated with either DHA (16 mg/kg, N = 6) or vehicle (DMSO, N = 6) administered 5 minutes after TBI or sham surgery. Semiquantitative measurements of the hippocampal tissues from rats sacrificed at 1 day after surgery that were assessed by Western blot analysis show that expression of α-Syn are decreased ipsilaterally in the hippocampus (P < 0.05). However, such a decreased expression of α-Syn can be attenuated by the acute post-injury DHA treatment (P < 0.05). This study suggests that a single acute post-injury DHA treatment attenuates TBI-induced decreased α-Syn expression. However, additional work is required to examine the mechanisms and determine the optimal DHA dose regimens after TBI. Support: Veterans Administration, The Pittsburgh Foundation, NIH-NS40125, NIH-NS060672.
Keywords: traumatic brain injury, alpha-synuclein, Western blot, docosahexaenoic acid (DHA)
American Life Science Pharmaceuticals, La Jolla, USA
Gene knockout data validate the cysteine proteases cathepsin B and calpain 1 as traumatic brain injury (TBI) drug targets but cathepsin B/calpain inhibitors have not been developed for clinical use in TBI. In the 1980s, the small molecule compound E64d (2S, 3S-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, EST), was developed and completed Phase 3 trials in pediatric muscular dystrophy patients without a significant adverse event but did not advance due to insufficient efficacy for that indication. Nonetheless, extensive clinical and preclinical data on E64d, including pharmacokinetics, biodistribution, toxicology, and mutagenicity were published. E64d is a prodrug, which is orally bioavailable and converted to its acid form E64c, which irreversibly inhibits cathepsin B and calpain. Subsequently, E64d/E64c were found to produce benefical outcomes in TBI and TBI-related animal models. Oral E64d administration post-trauma improved neuromotor deficits and reduced brain lesion volume, hippocampal cell death and cell death protein Bax in a controlled cortical impact TBI mouse model. Similarly, E64d administration in a spinal cord injury rat model reduced Bax levels. Intravenous (IV) E64c treatment post-insult prevented neuronal hippocampal cell death in an ischemic monkey model. Intraperitoneal (ip) prophylactic E64d treatment prevented hippocampal mossy fiber pathology in an epilepsy rat model. Further, feeding E64d to transgenic Alzheimer's disease mice reduced memory deficits and neurotoxic amyloid beta. Finally, ip E64d treatment in a rat rheumatoid arthritis model dramatically reduced inflammation. In summary, E64d/E64c have been safely used in man and inhibit cathepsin B/calpain and can be orally or IV administered to improve outcomes in many TBI and TBI related animal models. These data provide strong support for using E64d/E64c as a scaffolds upon which to develop cathepin B/calpain inhibitors as TBI clinical therapeutics.
Keywords: cysteine proteases, E64d, E64c
Contusive spinal cord injury (SCI) produces cellular necrosis and secondary tissue loss culminating in a fluid-filled cystic cavity. Limited endogenous repair results in focal cellular trabeculae composed of Schwann cells, fibroblasts, astrocytes, pericytes, macrophages, collagen, and sprouting axons. We hypothesized that implantation of a biodegradable, biomaterial scaffold into the injured spinal cord could serve as a locus for appositional healing and tissue remodeling that would enhance endogenous repair. We evaluated the effect of implantation of scaffolds composed of the block copolymer poly(lactic-co-glycolic acid)-poly(L-lysine) (PLGA-PLL) on tissue remodeling in a contusion model of SCI. A T10 contusion injury was created in female Sprague-Dawley rats (IH Impactor 220 kDyn). Cylindrical scaffolds (1.0 mm diameter, 2.0 mm length) were surgically implanted at the lesion site 24 to 72 hours later. Remodeled tissue at the injury epicenter was evaluated at 12 weeks by histology and immunolabeling of paraformaldehyde-fixed frozen sections. Rats in the non-treated control group developed large cavities surrounded by a rim of spared tissue. In contrast, in rats treated with scaffold implantation, cavity volume decreased by 86%, and spared white matter width increased by 44%. Although scaffolds were fully resorbed by 12 weeks after implantation, the amount of remodeled tissue at the implantation site in the lesion epicenter increased by 111%. Remodeled tissue contained laminin and sparse collagen. Schwann cells (myelin protein zero positive) were present in significant numbers in the remodeled tissue of the lesion epicenter and the perilesional white matter. Sprouting axons (β3-tubulin and neurofilament positive fibers) indicated neural regeneration within the remodeled tissue. These results demonstrate that PLGA-PLL scaffold implantation in the acutely injured spinal cord can act through appositional healing to reduce cavitation, promote tissue sparing, and support neural regeneration. Schwann cells may contribute to both neural regeneration and remyelination of focally demyelinated white matter axons. Scaffold implantation may augment endogenous cellular recovery processes to contribute to functional neurological improvement.
Keywords: scaffold, contusion, schwann cells, cavitation, PLGA-PLL, regeneration
LIDOCAINE BLOCKS THE DETRIMENTAL EFFECTS OF C-FIBER ACTIVATION ON RECOVERY AFTER SPINAL CORD INJURY
The majority of spinal cord injuries are the consequence of car accidents or falls that result in injury to other parts of the body and the activation of pain (C-fibers). Research in rats has shown that pain signals after the spinal cord injury leads to impaired behavioral recovery and increased pro-inflammatory cytokines and markers of apoptosis. Here we examined how blocking these signals with epidural lidocaine impacts recovery and cellular pathways. Twenty-four hours after rats received a moderate lower thoracic (T12) spinal cord contusion, BBB locomotor scoring was assessed to provide a baseline and to balance groups. Subjects were lightly anesthetized before receiving an epidural injection of either lidocaine or vehicle. Thirty-minutes after the injection, subjects were restrained in Plexiglas tubes before receiving six minutes of intermittent uncontrollable electrical stimulation (shock) to the tail to activate C-fibers. Controls remained unshocked. In the acute study, rats were sacrificed three hours after testing for cellular assays. For long-term recovery, subjects were assessed for the next six weeks using the BBB locomotor scale. In addition, weights were assessed daily throughout the recovery period. At the end of the recovery period, measures of pain reactivity and coordination/balance were examined. Acutely, lidocaine treatment reduced the expression of pro-inflammatory cytokines in subjects receiving shock. Analysis of locomotor recovery revealed a significant impairment in vehicle-shock subjects across time. Epidural lidocaine completely blocked the detrimental effect of electrical stimulation (lidocaine-shock) on locomotor recovery. Lidocaine also blocked the exaggerated weight-loss seen in vehicle-shock subjects. There were no differences between lidocaine-shock, lidocaine-unshocked, and vehicle-unshocked subjects in locomotor performance, weight-loss, or thermal and tactile reactivity. However, there was a significant increase in at-level pain (Girdle test) in all subjects receiving lidocaine. Overall, lidocaine treatment improved locomotor function in subjects receiving C-fiber activation. The results suggest that epidural lidocaine can attenuate the adverse effect noxious stimulation has on spinal function after injury. Supported by the Neilsen Foundation and NIH (NS091723).
Keywords: contusion, lidocaine, inflammation, locomotion, electrical stimulation, recovery
CATHEPSIN B IS A VALIDATED DRUG TARGET FOR TRAUMATIC BRAIN INJURY AND RELATED DISORDERS, FOR IMPROVING BEHAVIOR AND NEUROPATHOLOGY
Univ. of California, San Diego, Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, USA
There currently are no or limited drug treatments for traumatic brain injury (TBI) and the related conditions of Alzheimer's disease (AD), ischemia, epilepsy, chronic pain, and multiple sclerosis (MS). There is a critical need to discover new drug targets from which effective therapeutics may emerge. Data presented here illustrates the beneficial effects of cathepsin B gene knockout for improving TBI and TBI-related behavioral deficits and neuropathology. Specifically, cathepsin B deficient animals that suffer TBI have significantly improved neuromotor dysfunction, reduced brain tissue loss, and reduced neuronal cell death than controls. In a mouse model of AD, cathepsin B gene knockout improves memory deficits and reduces brain amyloid-b plaques. In an ischemia model, cathepsin B knockout reduces brain damage and decreases cytokine production compared to wild-type (wt) animals. In an epilepsy model, animals lacking cathepsin B display reduced brain neuronal apoptosis than those expressing cathepsin B. In an inflammatory chronic pain model, cathepsin B knockout mice display reduced chronic pain with reduced cytokine production than wt mice. In a multiple sclerosis (MS) model, deletion of cathepsin B and another cysteine protease, cathepsin S, results in improved MS clinical scores, delayed onset, and reduced lymphocyte infiltration into the spinal cord compared to wt mice. Clinical data shows that plasma cathepsin B is elevated in polytrauma patients which correlates with the severity of trauma. In AD patients, plasma cathepsin B is elevated and correlates with reduced cognition. These data validate cathepsin B as a new drug target for TBI and related brain disorders, indicating that drug inhibitors of cathepsin B are likely to provide effective treatment for TBI and TBI-related conditions.
Keywords: traumatic brain injury, cathepsin B, protease, drug target
Sustained increases in non-physiological concentration of glutamate contribute to neuronal dysfunction after traumatic brain injury (TBI). In people at risk for Alzheimer's disease (AD), brain accumulation of amyloid-β (Aβ) could exacerbate glutamate-related neuropathology after TBI. We measured human Aβ concentration, histological, and behavioral outcomes in human Aβ (hAβ) knock-in mice subjected to controlled cortical impact (CCI) injury and treated with memantine hydrochloride (memantine), a clinically well-tolerated moderate- affinity noncompetitive glutamate receptor antagonist proven beneficial in excitotoxicity models, currently in use for symptomatic treatment of AD. Adult hAβ mice received memantine (2.5, 5, 10 mg/kg) or vehicle by intraperitoneal injection daily for 3 weeks starting 1 hr after CCI (depth = 1.6 mm; velocity = 6 m/s). CCI impaired vestibulomotor function on days 1–5 after CCI, in both memantine and vehicle groups. CCI also impaired performance on the novel object recognition test on days 9 and 10 after CCI, which was moderately attenuated by all doses of memantine. CCI-induced spatial memory impairments on the Morris water maze acquisition and probe tests on days 14–20 after CCI were significantly ameliorated by the 5 mg/kg dose (both tests p < 0.05) but not the 2.5 or 10 mg/kg doses. Brain concentrations of Aβ1-42 and Aβ1-40 were elevated in CCI/vehicle group, while 5 mg/kg memantine significantly reduced Aβ1-42 concentration in cerebral cortex ipsilateral to injury (p < 0.01). Cortical lesion size was also reduced in mice receiving 5, but not 2.5 or 10 mg/kg dose of memantine. CCI-induced reductions in hippocampal synaptic densities (regions CA1 and CA3) were moderately attenuated with the 2.5 and 5 mg/kg doses. These results demonstrate protective effects of daily treatment with 5 mg/kg memantine over three weeks after TBI. The observed attenuation of pathological Aβ accumulation indicates that this treatment could be effective in breaking the link between TBI and AD.
Keywords: amyloid, alzheimer, memantine, severe TBI
RHO INHIBITOR VX-210 IN ACUTE TRAUMATIC CERVICAL SPINAL CORD INJURY: DESIGN OF A PHASE 2B/3 STUDY
University of Toronto, Division of Neurosurgery and Spine Program, Toronto, Canada
Recovery following spinal cord injury (SCI) is limited by several intermediate signaling proteins that prevent axonal repair and regrowth. These growth inhibitory proteins overactivate the neuronal intracellular molecule Rho, which plays an integral role in inhibiting axonal regeneration. Rho inhibition therefore may provide a therapeutic strategy for acute SCI.
VX-210 is a derivative of the bacterial enzyme C3 transferase that inhibits Rho activity. It is administered topically in a fibrin sealant to the dural surface of the spinal cord. In rodent models, VX-210 stimulated axon regeneration and plasticity and promoted functional recovery after SCI. A Phase 1/2a study in 48 patients with acute, complete thoracic/cervical SCI demonstrated good safety and tolerability of VX-210 at doses from 0.3 mg to 9 mg. In that study, motor strength in patients with cervical SCI suggested improvement when compared to patients in natural history studies.
Vertex Pharmaceuticals has initiated a Phase 2b/3, randomized, double-blind, placebo-controlled study of VX-210 [NCT02669849]. The study will enroll ≈150 patients, 14 through 75 years of age, across ≈35 sites in the United States and Canada. All patients will have acute traumatic SCI with motor levels of C4 to C6 on each side and American Spinal Injury Association Impairment Scale (AIS) grades of A or B. Patients will be randomized (1:1:1) to receive a single 3-mg dose of VX-210, single 9-mg dose of VX-210, or placebo, applied directly to the site of injury during decompression/stabilization commencing within 72 hours of injury. Patients will be followed for 1 year, with 6-week, 3-month, 6-month, and 12-month follow-up assessments. The primary endpoint is the change from baseline in upper extremity motor score (UEMS) at 6 months. This study tests the hypothesis that extradurally–applied VX-210 promotes improvement of upper extremity motor strength versus placebo, possibly by enhancing axonal regeneration.
Sponsored by Vertex Pharmaceuticals Incorporated
Keywords: Rho inhibitor, VX-210
Impaired breathing is a devastating consequence of cervical spinal cord injury (SCI) that increases morbidity and the risk of mortality. Injuries at high-to-mid cervical levels (C1-4) result in the most severe deficits as the phrenic motor circuitry – controlling the diaphragm – is directly compromised, typically resulting in dependence on assisted-ventilation. While there is mounting evidence for spontaneous respiratory improvement, the extent of recovery – or functional plasticity – remains limited. Thus, there is a need to develop therapeutic strategies for enhancing repair of respiratory pathways. The ongoing research in our lab aims to elucidate neuroanatomical changes that may influence respiration post-SCI, and assess whether treatments can harness ongoing neuroplasticity to improve function post-injury. These studies have identified that spinal interneurons represent a potential therapeutic target for enhancing plasticity and recovery of phrenic motor function. With a particular focus on the phrenic motor system, the goal of the present research is to assess whether transplantation of neural precursor cells (NPCs) can facilitate repair of the injured adult rat cervical spinal cord and promote lasting, functional recovery.
Adult, female Sprague-Dawley rats (∼250 g) received lateralized C3/4 contusions (200 kilodynes, Infinite Horizons Pneumatic Impactor). One week post-injury, NPCs derived from developing rat spinal cord (E13.5 Sprague-Dawley or Fisher-UBC-EGFP) were injected directly into the injury cavity (∼1 million cells). Transplant recipients were immunosuppressed (1 mg/kg cyclosporine i.p. daily) as required. Transplanted animals are compared against injured, vehicle treated animals. Four weeks or one year later, a transynaptic retrograde tracer (pseudorabies virus, PRV) was delivered to the ipsilateral hemidiaphragm or directly into the transplant. Tracing revealed synaptic integration between donor neurons and host phrenic circuitry. Phrenic function assessed with terminal electrophysiology revealed enhanced phrenic and diaphragm recovery in those animals that received NPC transplants following injury. These ongoing studies are providing insight into the therapeutic potential for NPC therapy in the injured spinal cord.
Keywords: respiration, interneuron, phrenic, neural progenitor, contusion
ADULT NEURAL STEM CELL ICV TRANSPLANTATION IN EXPERIMENTAL TBI: SONIC HEDGEHOG MEDIATED REGENERATIVE RESPONSE AND IMMUNOMODULATION
USUHS, Anatomy, Physiology & Genetics, Bethesda, MD, USA
Neural stem cells (NSCs) hold promise to promote brain repair by replacing lost cells and/or interacting with host cells to modulate the immune response and stimulate endogenous regenerative capacity. Transplantation strategies are challenging for traumatic brain injury (TBI) due to diffuse rather than focal lesions. Therefore, an intracerebroventricular delivery route may be advantageous in TBI patients, and designed via external ventricular drains implanted to control intracranial pressure. To test this concept, NSCs were isolated from adult mouse subventricular zone (SVZ) and transplanted into the lateral ventricle of adult mice at two weeks post-TBI (subacute stage) followed by analysis at four weeks post-TBI. TBI produced reactive astrogliosis and microglial activation in the corpus callosum (CC) that was significantly reduced by NSC transplantation. We then examined in vivo activation of the Sonic hedgehog (Shh) pathway. Shh has important regenerative roles and Gli1 transcription is an effective readout for canonical Shh signaling. Mouse reporter lines were generated for induced genetic fluorescent labeling of cells actively transcribing Shh or Gli1 in vivo. Transplanted NSCs from ShhCreERT2:RosamTmG mice showed rare Shh expression in vivo. In host ShhCreERT2:RosaTdTomato mice, Shh was primarily expressed in neurons, including CC axons. TBI did not induce Shh expression in reactive astrocytes or microglia. Gli1CreERT2:RosaTdTomato host mice demonstrated only rare Shh activation in CC cells, even after TBI or NSC transplantation. Additionally, NSC transplantation did not activate Shh signaling in host SVZ cells. Therefore, intracerebroventricular NSC transplantation in subacute stage TBI did not stimulate Shh-mediated regeneration yet did significantly suppress neuroinflammation. Funded by the DoD in the Center for Neuroscience and Regenerative Medicine (CNRM).
Keywords: sonic hedgehog, microglia, astrocyte, reporter mice
Mild traumatic brain injury (TBI) is one of the most common neurological disorders often leading to devastating health problems. The harmful effects of TBI occur during primary injury, which is induced by a mechanical force resulting in compression and physical damage of vessels and neurons, and after secondary complications (e.g. inflammation), that may follow hours or days after the injury and involves blood-brain barrier deterioration. Impaired memory, particularly short-term memory, is one of the frequent problems of people with head injury. We tested the hypothesis that TBI-induced an increase in cerebrovascular permeability leads to extravasation of fibrinogen (Fg), which is one of the inflammatory markers, and its deposition in vasculo-astrocyte interface causing neuronal degeneration and the resultant reduction in short-term memory. Pial venular permeability to proteins was studied in pericontusional area after mild cortical contusion injury (CCI) in C57BL/6J (wild-type, WT) mice using an intravital fluorescence microscopy. Immunohistochemistry of brain cryosections was used to assess deposition of Fg in vasculo-astrocyte interface. While astrocytes were identified with glial fibrillary acidic protein (GFAP), neuronal degeneration was assessed using a neuronal specific nuclear protein (NeuN). Short-term memory was evaluated by a novel object recognition and Y maze (spontaneous alternation and two trial recognition) tests. We found that protein transcytosis and Fg deposition in vasculo-astrocyte interface were increased after TBI. These effects were accompanied with increased association of deposited Fg with cellular prion protein (PrPC), which is known to have a strong effect on loss of memory. Neuronal degeneration was also greater in mice with CCI compared to that in control animals. Thus, an increased blood content of Fg that occurs after TBI can lead to an increased formation of Fg-PrPC complex in vasculo-astrocyte interface causing their uncoupling and the resultant neuronal degeneration leading to reduction in short-term memory.
Supported in part by NIH grants P30 GM-103507, NS-084823, HL-108621, HL-74185.
Keywords: cerebrovascular permeability, fibrinogen, cellular prion protein, vasculo-astrocyte interface, short-term memory
ASSESSMENT OF WHITE MATTER TRACTS IN ACUTE HEAD TRAUMA USING ANATOM-E SOFTWARE
Massachusetts General Hospital, Neuroradiology, Boston, USA
Keywords: acute head trauma, white matter tracts systems, software with deformable anatomic template
University of Louisville, Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, Louisville, USA
Myelination of the central nervous system (CNS) is a tightly regulated process. Oligodendrocyte precursor cells (OPCs) proliferate and migrate to target axons, where they mature to myelinating oligodendrocytes (OL), the sole providers of CNS myelin. Many factors have been identified that regulate OL differentiation and myelination. However, few factors have been found that act specifically in myelin formation, and the mechanisms regulating myelin sheath wrapping and compaction are poorly understood. Here, we present evidence that autophagy, the targeted isolation of cytoplasm and organelles by the double-membrane autophagosome for lysosomal degradation, regulates myelin formation in mature OLs. Autophagy is critical for cell homeostasis, but recent evidence has shown that autophagy regulates OL survival and differentiation and morphogenesis of neurons and astrocytes in the brain. Our data show: 1) increased autophagosome formation, trafficking, and autophagic flux, especially in OL processes, during late stages of OL differentiation and myelin wrapping, 2) autophagy regulatory proteins co-localization with markers of the OL lineage during myelin development, 3) detection of key autophagy regulatory proteins in enriched myelin fractions from corpus callosum (CC) at the peak of myelination and immunostaining of DRG co-cultures, 4) time-lapse imaging demonstrates autophagosome movement from myelin to the OL soma in DRG co-cultures, 5) pharmacological autophagy inhibition blocked and autophagy stimulation enhanced myelination, 6) genetic deletion of the essential autophagy gene Atg5 leads to near complete loss of CNS OPCs, failed myelin compaction, a rapid tremor, and neonatal death at PD12. These results provide insight into novel functions of autophagy in OL and myelin development and identify autophagy as an attractive target to both promote OL survival and subsequent myelin repair after injury. Supported by NS073584, GM103507, and the Kentucky Spinal Cord and Head Injury Research Trust.
Keywords: autophagy, myelin, oligodendrocyte
The objective of this study was to evaluate a potential scoring system to determine if it correlates with the perceived need for craniotomy in traumatic brain injury (TBI) patients. The Glasgow Coma Scale (GCS) has been effective in describing the severity of TBI but is inadequate for predicting the potential need for surgical intervention. Using trauma databases, we have described a scoring system in both children and adults which correlates well with the perceived need for surgical decompression following TBI. In children, we have used a single pediatric trauma center registry (1/1/2010 and 1/1/2014). For the adult population, we utilized the patient database for the Progesterone for the Treatment of Traumatic Brain Injury III Trial (PROTECT III). The SITI score was calculated using presenting GCS; presence of a unilateral enlarged pupil; and radiographic findings of midline shift, temporal pathology, and presence of an epidural hematoma. For the pediatric arm of this retrospective study, there were 915 children in the trauma registry during the observation period. Of those, 880 were managed non-operatively and 35 required operative intervention. The mean SITI scores for these groups were 0.47 ± 0.03 and 4.74 ± 0.36, respectively (p < 0.0001). For the adult arm of the study, 871 patients were reviewed, 159 underwent craniotomy, and 712 were treated non-operatively. The mean SITI score was 5.1 for operative patients and 2.5 for non-operative patients (p < 0.0001). The SITI scale was designed to be a simple, objective system for communication between clinical services regarding the potential need for surgical decompression for TBI. Application of the SITI scale retrospectively to databases of adult and pediatric patients demonstrated that the SITI score does correlate well with a perceived need for craniotomy. These results further demonstrate the potential utility of the SITI scale in clinical practice and perhaps justify pursuit of a prospective study.
Keywords: traumatic brain injury, surgical decompression, Triage, communication
Poster Presentations - Group B
AGING AND SPINAL CORD INJURY ALTER INFLAMMATORY GENE EXPRESSION
Uniformed Services University of the Health Sciences, Anatomy, Physiology, and Genetics, Bethesda, USA
There has been a steady rise in the number of spinal cord injuries (SCI) among the aging population since the 1980's. Aging tissue shows increases in oxidative stress and reactive oxygen species (ROS), which are suggested to increase the activation of glial cells. The NADPH oxidase (NOX) 2 enzyme is a primary producer of ROS in glial cells. This study aimed to determine age related changes to gene expression of NOX2 components, microglial polarization, and inflammatory cytokines both basally and after SCI. Briefly, spinal cord tissue was obtained from groups of naïve or spinal cord injured young (3 months old) and aged (12 months old) male Sprague Dawley rats. Naïve or 30 day post-injury (dpi) tissue was processed for quantitative PCR analysis using the Trizol RNA isolation method to determine basal and post injury gene expression. The studies demonstrate that aged naïve rats showed significantly increased gene expression of the pro-inflammatory microglial marker CD86 (p = 0.0050) and of the NOX2 component p47PHOX compared to young rats (p < 0.0001). At 30 dpi, 12 month aged rats showed significantly increased gene expression of both of these markers (CD86, p = 0.0008; p47PHOX, p = 0.0404) as well as significantly increased gene expression of the NOX2 membrane component p22PHOX (p = 0.0192), the anti-inflammatory microglial marker CD206 (p = 0.0045), and inflammatory cytokines TNFα (p = 0.0405) and NFκB (p = 0.0156) compared to 3 month aged rats. These results demonstrate that, with aging, pro-inflammatory microglial polarization markers are increased both before and 30 dpi, while anti-inflammatory microglial polarization is increased only at 30 dpi. Further, both NOX2 components and inflammatory cytokine gene expression are increased in 12 month old compared to 3 month old rats. These findings suggest that NOX2 and pro-inflammatory microglia gene expression are increased with age and may influence the inflammatory response after injury, resulting in altered microglial polarization and a more pro- inflammatory environment.
Keywords: spinal cord injury, oxidative stress, rat injury model, inflammatory cytokine expression, glial polarization
Thrombin is elevated at sites of neurotrauma and well known for its ability to generate fibrin monomers supporting hemostasis. In addition, thrombin is a high affinity agonist for Protease Activated Receptor 1 (PAR1), also known as the thrombin receptor. PAR1 is a seven transmembrane G-protein-coupled receptor that is activated by proteolysis within its extracellular N-terminus. Signaling at PAR1 is emerging as an important translator of the extracellular proteolytic microenvironment into cellular responses that contribute to tissue injury, remodeling and regeneration. In this study, we critically evaluated the role and mechanism of action of PAR1 in spinal cord injury (SCI) by determining the impact of PAR1 gene deletion on functional recovery and cellular and molecular signs of pathogenesis in experimental murine contusion-compression SCI. SCI in PAR1 knockout mice was associated with greater improvements in motor coordination and strength compared to wild type littermates. Molecular profiling of the injury site demonstrated PAR1-/- mice had significantly attenuated elevations in pro-inflammatory cytokines (IL-6, TNF and IL-1β) and in key hallmarks of astrogliosis (GFAP, vimentin, neurocan). SCI in PAR1-/- mice was also accompanied by improved preservation of PKC-ϒ- positive corticospinal axons, and reductions in BIM expression and STAT3 signaling. The mechanistic link between PAR1, STAT3 and astrogliosis was investigated in primary astrocyte cultures revealing that thrombin promotes IL-6 secretion in a PAR1-dependent manner. A model is proposed where PAR1-elicited IL-6 secretion drives expression of GFAP and vimentin through canonical STAT3 signaling. Since IL-6 also markedly increased the expression of PAR1 and thrombin by astrocytes, these data collectively point to an IL-6-driven PAR1 signaling circuit that works hand-in-hand with STAT3 to promote inflammatory-astrogliosis. Given the superior neuromotor recovery observed in PAR1 knockout mice, we suggest that targeting PAR1 to limit inflammation and astrogliosis represents a promising drug target to reduce axon degeneration and improve motor outcomes after SCI. Supported by NIH R01 NS052741, NMSS RG4958 and the Mayo Clinic Center for Regenerative Medicine.
Keywords: inflammation, STAT3, astrogliosis, interleukin 6, GFAP
RE-EXPRESSION OF THROMBOSPONDINS IS CONCOMITANT WITH INCREASED SYNAPTIC MARKERS AFTER DIFFUSE TRAUMATIC BRAIN INJURY
University of Arizona, Child Health/Surgery, Phoenix, USA
Damaged circuits after traumatic brain injury (TBI) are repaired by the formation of new synaptic connections (synaptogenesis). For some TBI survivors, aberrant synaptogenesis misguides reparative processes leading to the development of persistent post-concussive symptoms. Synaptogenic regulation post-injury is unknown, and if misguided in the hippocampus (HC) could contribute to persistent emotional and cognitive impairment. Developmental synaptogenesis involves thrombospondin (TSP) interactions with the α2δ-1 subunit on voltage-gated calcium channels (α2δ-1), however, TSP is down-regulated in the adult CNS. In models of epilepsy and stroke, TSP re-expression regulates post-insult synaptogenesis, such that TSP-α2δ-1 antagonism modulates post-insult symptomatology.
Support: ADHS14-00003606, NIH R03 NS-077098, NIH R01 NS-065052, Diane&Bruce Halle Foundation, and PCH Mission Support Funds.
Keywords: astrocyte, TBI, thrombospondin, synaptogenesis, hippocampus
The functional loss associated with traumatic spinal cord injury (SCI) is a primary consequence of axonal degeneration and demyelination. It is known that damage to myelin induces a sequel of events including breakdown of paranodal axoglial junctions, myelin retraction, and exposure of underlying juxtaparanodal K+ channels which subsequently disrupts conduction. It is well established that axonal conduction can be partially restored by potassium channel blockers such as 4-aminopyridine (4-AP). While 4-AP significantly restores functions in animal studies, its effectiveness in human is hampered by its low safe clinical dosage. In light of this, we have examined 4-aminopyridine-3-methanol (4-AP-3-MeOH) as an alternative for improving axonal conduction and motor function recovery post-SCI. The current study draws direct comparisons between the effects of 4-AP and 4-AP-3-MeOH for restoring axonal conduction and motor and sensory function using both ex vivo and in vivo models of SCI. In particular, using isolated spinal cord segments and a double sucrose gap recording apparatus, we observed a significant increase in CAP amplitude with 100 μM 4-AP-3-MeOH treatment (30.1 ± 2.9%), which is more significant than the restoration rendered by 4-AP with same dosage (22.4 ± 2.0%). Both compounds show no preference in axonal conduction enhancement based on axonal caliber. Furthermore, while 4-AP-recruited axons displayed both longer absolute and relative refractory periods, 4-AP-3-MeOH-recruited axons appear to conduct in a manner similar to uninjured axons with relatively normal refractory periods. Similar differences were also observed between 4-AP and 4-AP-3-MeOH when axonal conduction responded to rapid train stimulation. In addition, both 4-AP-3-MeOH and 4-AP also improved motor and sensory dysfunction following in vivo systemic application in a rat contusion SCI model. These findings demonstrate the potential benefits of 4-AP-3-MeOH as a viable alternative medication to 4-AP for restoring axonal conduction and functional recovery in SCI.
Keywords: demyelination, potassium channel blockers
PROGRANULIN DEFICIENCY EXACERBATES MICROGLIOSIS BUT NOT AXONAL INJURY IN THE VISUAL PATHWAY AFTER MILD TBI
Athletes and military personnel are at risk to receive multiple mild head injuries during their careers. Repeated mild traumatic brain injury (mTBI) induced using closed head impacts (CHI) at 24 h intervals in mice produces visual system axonal degeneration and gliosis. Progranulins are the precursors of granulins, growth factors involved in inflammation, neuron survival and outgrowth, and protein homeostasis. Progranulin deficiency induces age-related increases in inflammation while mutation can lead to fronto-temporal dementia. Progranulin deficient (GRNKO) mice exhibit increased microgliosis compared to wildtype (WT) mice after a stab wound to the brain. Microglial activation is an established feature of diffuse axonal injury and has been postulated to mediate ongoing axonal degeneration. We examined the effect of progranulin deficiency on white matter pathology following mTBI in mice. GRNKO and WT mice received five CHI or sham injuries at 24 h intervals and survived either 48 h or 7d after the final injury (N = 4–5 sham, 9–10 injured /genotype/time point). The superior colliculus (SC), optic tract (OT) and optic nerve (ON) were analyzed for axonal injury and microgliosis. Repeated mTBI produced significant ON axonal damage at 48 h and 7d, as detected by accumulation of dephosphorylated neurofilaments; axonal damage was comparable across genotypes. Silver stain showed axonal degeneration in the OT and SC at 7d in both GRNKO and WT mice. Injured WT mice exhibited delayed microgliosis (CD68-positive microglia) in the OT, while the onset of microgliosis was earlier in injured GRNKO mice. Microgliosis in the SC and ON was significantly greater at both 48 h and 7d after injury in GRNKO compared to WT mice. These results demonstrate that progranulin deficiency augments posttraumatic microgliosis but does not increase the severity of axonal injury in the first week after injury. Therapies targeting microgliosis in diffuse brain injury may need to be combined with those targeting axonal pathology directly. Supported in part by NS087878-02.
Keywords: Optic Nerve, Progranulin, Visual Pathway, Optic tract, Superior Colliculus
LOCALIZED ECTOPIC CALCIUM SPARKS MAY PLAY A ROLE IN NETWORK CALCIUM DESYNCHRONIZATION FOLLOWING TRAUMATIC AXONAL INJURY
Traumatic axonal injury (TAI), a common consequence of traumatic brain injury (TBI), results in an influx of calcium into the axon even after mild injury, resulting in a dysregulation of intra-axonal calcium concentrations. Here, we examined temporal changes in calcium concentration in axon networks following TAI using a model of dynamic mechanical stretch of isolated cortical axons in vitro. Briefly, primary rat cortical neurons were grown on micropatterned deformable membranes, whereby a series of parallel 2 mm-long lanes containing only axons spanned two populations of neuronal soma. The axon only region was rapidly stretched via mechanical parameters based on clinical TBI. Temporal changes in intra-axonal and somal calcium following TAI was monitored using Fluo-4AM along with continuous high-speed video microscopy. Pre-injury, individual axons display distinct, uniform and synchronized calcium bursts throughout the network at a regular frequency, However, immediately following injury, calcium network activity frequency is dampened and highly desynchronized in contrast to control cultures, with fluctuating increases in intra-axonal calcium levels that persist for up to 24 hrs post-injury. Following injury, localized “ectopic” calcium sparks are observed that are initiated at distinct positions along the axon, and are in direct contrast to those initiated by the cell soma or synapse. Over 24 hours, while calcium intensity remained elevated in surviving axons, synchronicity of calcium bursts gradually re-emerged. These data demonstrate that calcium bursts and general increases in intracellular calcium concentrations were associated with network-wide desynchronization. Although the mechanisms of these changes are as yet unknown, the localized ectopic calcium sparks may be contributing to this network desynchronization. These findings may have a clinical correlate of network slowing, such as is observed with reduced processing speed following mild TBI. This work was supported by DOD grant, PT110785 and NIH grants NS056202 and NS038104.
Keywords: calcium, network activity, diffuse axonal injury, axonal injury
EPIGENETIC AND TRANSCRIPTIONAL SIGNATURES OF BLAST EXPOSURE
Icahn School of Medicine at Mount Sinai, Neuroscience, New York, USA
Injuries from exposure to explosive blasts rose dramatically during Operations Iraqi Freedom and Enduring Freedom, motivating investigations of blast-related neurotrauma. In this effort, we have undertaken human studies involving “Breachers,” military and law enforcement personnel who are exposed to repeated blasts as part of their occupational duty. Breachers have reported a range of physical, emotional, and cognitive symptoms: headache, sleep issues, anxiety, and lower cognitive performance. The goal of this study is to identify biomarkers associated with blast exposure. We investigated DNA methylation, a highly stable epigenetic mark associated with gene repression and gene expression patterns, in blast-exposed subjects to identify potential epigenetic and coordinated gene expression abnormalities. The data presented involved 32 individuals participating in a 2-week data collection cycle at U.S. Army explosive entry training sites, where blood samples were obtained pre- and post-training for epigenetic and gene expression studies. We performed genome-scale DNA methylation profiling using the Illumina Infinium HumanMethylation450K BeadChip platform covering all coding genes and whole genome transcription profiling via RNA-seq. For the Breacher samples assayed, we found DNA methylation and transcriptional changes associated with a history of blast exposure and lifetime history of mild TBI at baseline (prior to training), as well as pre- vs. post-training. We identified changes associated with genes involved in mood disorder, inflammatory response in low vs. high breaching, and inflammatory response, neurodegenerative disorders, intra-individually following the two-week training course. This is the first human study that has systematically investigated coordinated epigenetic and gene expression changes associated with repeated blast exposure. These data show the power of genomic approaches to identify dynamic chromatin and transcription perturbations associated with blast.
Keywords: epigenetics, methylation, blast
Caffeine (1, 3, 7-trimethyxanthanine) is one of the most widely used psychoactive drugs to combat fatigue and sleep loss. As a non-specific adenosine receptor antagonist, caffeine has recently been shown to exert neuroprotective effects against brain injury in animal models of Parkinson's disease (PD) and stroke. However, studies of the neuroprotective effects of caffeine on traumatic brain injury (TBI) are limited. This study assessed the potential therapeutic effect of caffeine on neurobehavioral recovery in the WRAIR penetrating ballistic-like brain injury (PBBI) model. Unilateral frontal PBBI was produced in the right hemisphere of anesthetized rats (10% injury severity level). Animals were randomly assigned to pretreatment groups: (1) acute caffeine (25 mg/kg CAF) or vehicle (gavage, 1 h prior to PBBI); (2) chronic caffeine (0.25 g/L CAF) or water (water bottle, 3 weeks prior to PBBI). Blood samples (0.7 mL) were collected at 1 h, 4 h, 24 h, and the terminal endpoints to measure plasma levels of the glial biomarker GFAP and inflammatory cytokines. Motor function was evaluated on the rotarod (7 and 10 days post) at fixed-speed increments of 10, 15, and 20 rpm. Cognitive performance was evaluated on the Morris water maze (MWM). In pilot studies, cognitive performance in the MWM revealed deficits in all injury groups with the average latency to find the hidden platform (across all testing days) increased by 140% (PBBI) and 130% (25 mg/kg CAF) vs. respective sham (p < .05). Results of the probe trial indicated that caffeine-treated rats showed reduced thigmotaxic behavior and spent more time in the target annulus zone; however, these results were not statistically significant. The effects of chronic caffeine treatment and plasma GFAP and inflammatory cytokine levels are currently ongoing. Overall, the results of this study indicate that acute caffeine does not promote significant neurofunctional recovery when delivered via oral gavage in the 10% PBBI model. Future studies will evaluate additional doses and injury severity levels.
Keywords: TBI, caffeine, brain injury, rat, behavior
BRAIN CATHEPSIN B PROTEIN LEVELS AND CYSTEINE PROTEASE ACTIVITY ARE AFFECTED BY SUBACUTE CRANIOTOMY AND PENETRATING BRAIN TRAUMA
Walter Reed Army Institute of Research, Brain Trauma Neuroprotection and Neurorestoration Branch, Silver Spring, MD, USA
Comprehensive analysis of key mediators involved in subacute traumatic brain injury (TBI) is tantamount to understanding, and mitigating, mechanisms of injury progression. Cathepsin B is a cysteine protease implicated in neurodegeneration and TBI. The purpose of this preliminary study was to determine the extent of cathepsin B up-regulation, and enzymatic activity within proximal and distal brain regions affected by subacute craniotomy or penetrating ballistic-like brain injury (PBBI). Rat ipsilateral frontal cortex (FC) and hippocampal (HC) brain tissues were collected 7 days after craniotomy or PBBI. Mature (25–26 kDa) cathepsin B protein levels were determined by western blotting. Enzymatic activity was determined by substrate cleavage to generate a fluorescent product. Craniotomy or PBBI values are expressed as a fold change (x) compared to naïve controls. To determine if activity was specific to cathepsin B, assays were repeated with the addition of CA-074. Changes in activity are expressed as percent (%) of values without inhibitors. PBBI led cathepsin B protein levels to increase by 6–7x in the FC and by 1.3–1.6x in the HC compared to naïve and craniotomy cohorts. FC enzyme activity increased by 1.4x and 2.3x after craniotomy or PBBI, respectfully. In FC extracts, CA-074 nearly ablated enzymatic activity caused by craniotomy, and suppressed the PBBI-induced activity by 60%. Within the HC, craniotomy decreased enzymatic activity to 0.8x of naive levels, while PBBI had no effect. CA-074 suppressed activity in naïve, craniotomy, and PBBI by 40–50%. PBBI led to expected upregulation of cathepsin B protein levels the FC and HC. Craniotomy-induced FC enzyme activity is largely due to cathepsin B. In contrast, PBBI likely leads to activation of several proteases. Protease protein levels and activity may be crucial to the elucidating differential mechanisms within lesioned and non-lesioned brain tissues affected by subacute TBI of varying severities.
Keywords: cathepsin B, cysteine protease activity, craniotomy
Acute renal dysfunction has been observed in patients with severe traumatic brain injury (TBI) or polytrauma. Yet, such systemic effects are scarcely investigated in preclinical models of TBI/polytrauma. This study examined the changes of creatinine levels, a major marker of kidney function, in a rat model of penetrating ballistic-like brain injury (PBBI) with concomitant hypoxemia and hemorrhagic shock. Rats were randomly assigned into four groups: (1) sham (2) hypoxemia and hemorrhagic shock (HH) (3) PBBI only (4) PBBI combined with HH (PHH). In HH or PHH group, hypoxemia (PaO2<40 mmHg) was initiated 5 minutes following PBBI or sham procedures and maintained for 30 minutes. After restoring normoxia, hemorrhagic shock (mean arterial pressure ∼40 mmHg) was initiated and maintained for 30 minutes. Prior to injury and at the specified end points (1–28 days), urine output, food and water intake were recorded in metabolic cages. Urine and blood serum were analyzed for creatinine levels using colorimetric assays. While urine creatinine levels in the PBBI or PHH groups were significantly increased at day 1, 7 and 14 (p < 0.05), their serum creatinine levels remained comparable to the sham and HH groups. Although peak creatinine excretion occurred at day 1 post-injury, food and water consumption at 1–2 days were found to be reduced significantly in PBBI and PHH groups compared to the sham or HH groups. As expected, low water consumption led to significant reduction in urine output at 1–2 days following PBBI or PHH. Overall, these results suggest that PBBI may augment renal clearance of creatinine while maintaining a normal serum creatinine level. Critically, hypoxemic and hypotensive insults following PBBI did not result in additional maladaptive effects on the kidney function. Apart from the augmented renal clearance, the higher levels of urine creatinine following PBBI may be due to an increased production/utilization of adenosine triphosphate from phosphocreatine, and subsequent conversion of creatine/phosphocreatine to more creatinine.
Keywords: polytrauma, hypoxemia, creatinine, hemorrhagic shock
DIETARY RESTRICTION (DR) TREATMENT REVERSES TBI-INDUCED COGNITIVE IMPAIRMENT IN MICE
Traumatic brain injury (TBI), a brain dysfunction for which there is no present effective treatment, is often caused by a concussive impact to the head. The impact of diet on brain function and susceptibility to neuropsychiatric and neurodegenerative disorders is increasingly appreciated. In addition, rodents maintained on dietary restriction (DR) perform better on learning tasks than do rodents fed ad libitum (AL). Here, we demonstrate a neuroprotective effect of DR in a mouse model of induced mild TBI (mTBI). Mice were divided into two main groups following a weight drop (30, 50 and 70 gr). AL group was fed normally while the DR group was maintained on an alternate day feeding regimen. Cognitive and behavioral performance was assessed 30 days post injury using the Novel Object Recognition (NOR) and the Elevated Plus Maze (EPM) tasks. Results taken from the NOR task show that all groups of mTBI mice (30, 50 and 70 gr) maintained on normal diet were significantly impaired post injury compared with the no- mTBI (p < 0.03, p < 0.001 and p < 0.001 respectively) and the mTBI+DR groups (p < 0.01, p < 0.001 and p < 0.007). EPM analysis shows no difference between all groups, indicating that injury failed to cause any anxiety like behavior. Currently we are examining the role of Sirtuin1 in this neuroprotective mechanism, as it is known to have a key role in the biological effect of DR. These results bolster accumulating evidence that DR may be an effective approach for increasing the resistance of the brain to damage.
Keywords: TBI, dietary restriction, neuroprotective, mice
GETTING A GRIP: WALKING SURFACE INFLUENCES ON STEPPING PHENOTYPE WHEN LONG ASCENDING PROPRIOSPINAL NEURONS ARE SILENCED
U of L, Interdisciplinary Program in Translational Neuroscience, Louisville, USA
Central pattern generators (CPGs) are neuronal networks that generate coordinated muscle activities associated with behaviors like stepping and swimming. Despite >100 years since the recognition of spinal CPGs, many functional details of those circuits, including the effects of afferent sensory input, are unknown. In the mammalian spinal cord, long ascending propriospinal neurons (LAPNs) are part of an “inter-enlargement” population of neurons that likely provide direct connections between the hindlimb and forelimb CPGs in the spinal cord. Previously, we reversibly blocked synaptic transmission of LAPNs using a doxycycline (DOX)-mediated two-viral vector system. Results showed that LAPNs play a critical role in right-left alternation of both the hindlimbs and forelimbs during over ground stepping. We hypothesized that afferent feedback from the stepping surface contributes to the locomotor gait changes seen during LAPN silencing. To test this hypothesis, we examined kinematic and gait changes in locomotion on different surfaces when LAPNs were silenced. We utilized a smooth surface (slippery) and a Silguard-coated surface (grippy), and assessed locomotor function. Disruption of right-left alternation after silencing was more severe on the grippy surface versus the slippery surface. Furthermore, alternation appears to remain intact during swimming, a task that involves alternating limbs but no cutaneous contact with a surface. These data demonstrate that afferent input from a slippery walking surface results in more consistent right-left alternation when LAPNs are silenced, implying a supraspinal influence on CPG and inter-enlargement circuitry. Thus, sensory input appears capable of modulating the balance between supraspinal and spinal circuitry during overground stepping. Supported by GM103507, NS089324, The Kentucky Spinal Cord and Head Injury Research Trust, Norton Healthcare, and the Commonwealth of Kentucky Challenge for Excellence.
Keywords: silencing, viral vectors, locomotor CPG, sensory feedback
Medicinal Cannabis Reduces Agitation In Acquired Brain Injury: Case Study
UNM Center for Brain Recovery and Repair
Keywords: Cannabis, Neurobehavioral
University of Pittsburgh, Neurosurgery, Pittsburgh, USA
Acknowledgements: NIH-NS40125, NIH-NS060672, VAI01 RX001127, 1-F32NS090748
Keywords: alpha synuclein, transgenic, CCI, function
Human mild traumatic brain injury (mTBI) displays long-term behavioral dysfunctions yet lacks anatomical alterations on magnetic resonance imaging (MRI). Majority of the animal models that replicate many of the features observed following mTBI involve either craniotomy or generate anatomical changes detected using MRI. These characteristics reflect an injury that goes beyond the mild classification. The objective of the current study is to examine the longitudinal behavioral and microstructural white matter changes in brain following experimental mTBI. The current study uses the Maryland closed head TBI model, which causes anterior-posterior plus sagittal rotational acceleration of the brain and is frequently observed in motor vehicle and sports related TBI injuries. The injury reflects a concussive injury model without skull fracture. We examined the exploratory behavior using computerized activity monitoring and performed MRI at 72 hours, 3 and 4 weeks post-injury. MRI imaging consists of high-resolution anatomical imaging and diffusion tensor imaging (DTI). DTI provides information about microstructural alterations and neurodegeneration following injury compared to baseline (pre-injury) measures. We assessed changes in the fractional anisotropy, mean, longitudinal, and radial diffusivities compared to baseline measures. Our results indicated significant decrease in exploratory behaviors such as ambulatory distance, ambulatory counts, stereotypic counts, vertical (rearing) counts, and ambulatory episodes that persist into the chronic phase of injury. Our DTI results indicated microstructural changes. Histological examination to detect contributions of astrocytes (GFAP), oligodendrocytes (MBP), neurons (neurofilament), and microglia (Iba-1) were correlated with the DTI measures.
Keywords: diffusion tensor imaging, myelin degeneration, inflammation, axonal plasticity
UNTARGETED LIPIDOMICS FOR THE DISCOVERY OF NOVEL LIPID BIOMARKERS OF TRAUMATIC BRAIN INJURY
Biomarkers offer the potential to predict disease states by measuring endogenous species concentrations. Through the use of a data-driven, untargeted approach, potential biomarker candidates of traumatic brain injury (TBI) were identified in the serum lipidome of adult male Sprague Dawley rats (n = 34). A predictive model for the classification of samples into two groups (injury and control) was developed, with a cross-validated accuracy of 85.3%. Animals were injured by controlled cortical impact (CCI), and serum samples were collected pre-injury, as well as 3 and 7 days post-injury. Both naïve and sham controls were included in the study. Following the precipitation of proteins, serum samples were analyzed in both positive and negative ion mode by Ultra Performance Liquid Chromatography Mass Spectrometry (UPLC-MS) using a Waters Xevo QTOF mass spectrometer. Feature selection for the predictive model was conducted using omniclassifier software. The optimized predictive model identified 26 unique dysregulated species following TBI belonging to a variety of lipid classes, 17 of which were statistically significant as individual compounds. Polyunsaturated fatty acids (PUFAs) and diacylglycerols containing esterified PUFAs were found to be upregulated in injured samples, while changes in oxidized phospholipids, sphingolipids, and membrane phospholipids were also observed, many of which confirm the results of previous TBI biomarker studies. Overall, the predictive model may serve as a starting point for the development of an objective biomarker panel to aid in the diagnosis of TBI and identify a variety of metabolic pathways that may aid in further understanding of TBI pathophysiology.
We acknowledge the Parker H. Petit Institute for Bioengineering & Bioscience, the Center for Chemical Evolution; Vasser-Wooley Professorship (FF), NIH (5T32EB006343-05 (SH) and NS079739 (MAV, ML, RB).
Keywords: lipidomics, TBI, biomarker, diagnostics
A PANEL OF THREE SERUM BIOMARKERS ACCURATELY IDENTIFIES BRAIN INJURED PATIENTS
ImmunArray, Program of Neurological Diseases, Richmond, USA
Keywords: biomarker panel, concussion, mTBI
The University of New Mexico Health Sciences Center, The Center for Brain Recovery and Repair, Albuquerque, USA
Keywords: auditory attention, magnetoencephalography, inhibition of return (IOR), orienting response, cognitive control
University Hospital of Erlangen-Nürnberg, Germany, Department of Neurosurgery, Erlangen, Germany
Keywords: S100B, semi-automated MR-volumetry, neuropsychological outcome, subarachnoid hemorrhage
University of Messina, Messina, Italy
This work, part of the Operation Brain Trauma Therapy (OBTT) a multi-center pre-clinical drug screening consortium, investigated the temporal course of circulating brain damage biomarkers and their associations with behavioral and histo-pathological outcomes across multiple traumatic brain injury (TBI) models.
Levels of ubiquitin C-terminal hydrolase (UCH-L1) and glial fibrillary acid protein (GFAP) were serially measured 1 hr, 6 hr, and 24 hr following injury in rats subjected to controlled cortical impact (CCI), fluid percussion (FPI), or penetrating ballistic-like brain injury (PBBI). Sham rats underwent all manipulations except trauma.
Both biomarkers were significantly increased as early as 1 hr after injury compared to shams with the exception of UCH-L1 in CCI. Higher UCH-L1 levels were consistently observed in sham and injured CCI groups when compared to the other models, albeit the levels remained unchanged over study duration across the groups. Conversely, GFAP concentration changed over time following injury, with magnitude and temporal profile differing significantly by group (interaction P < 0.001). Specifically, contrasting the acute elevation following FPI and PBBI, a delayed peak in GFAP at 6 hr was observed after CCI. UCH-L1 and GFAP at 1 hr correlated with latency to find the platform across models, and with the performance in the probe trial in FPI and PBBI. UCH-L1 at 1 hr also correlated with lesion volume and tissue loss in CCI.
This study provides direct evidence of differential release kinetics of GFAP across distinct injury models supporting the adoption of an adjusted diagnostic time window for detection of TBI based on the injury type. Furthermore, these data support the acute measurement of UCH-L1 and GFAP as potential surrogates for behavioral outcomes.
Support:W81XWH-10-1-0623/WH81XWH-14-2-0018.
Keywords: UCH-L1, GFAP, animal models, CCI, FPI, PBBI
Turku University Hospital, Rehabilitation and Brain Trauma, Turku, Finland
Keywords: metabolomics, outcome, traumatic brain injury
Severe traumatic brain injury (TBI) is a risk factor for tauopathies. The degree to which TBI contributes to tau pathology, and the role of tau or its phosphorylation state as a TBI biomarker remains debated. This preliminary study sought to determine the effect upon total and phosphorylated tau in a rat model of penetrating ballistic-like brain injury (PBBI). Ipsilateral frontal cortex (FC), cerebral spinal fluid (CSF), and serum were collected 1, 2, and 4 weeks (wks) after craniotomy or PBBI. Total and phosphorylated (threonine 231) tau (pTau) were measured using electrochemilluminescent ELISAs. All values are displayed as the fold change (x) vs. craniotomy per time-point. The p-tau to tau ratio (p-tau: tau) is also described. In the FC, tau increased in a stepwise manner after PBBI. Here, tau rose to 1.67x at 2 wks, then to1.89x at 4 wks. P-tau increased by 1.85x 2 wks after PBBI, yet decreased to 0.22x of craniotomy after 4-wks. Total tau was 12x higher in CSF collected 1wk post-PBBI, yet fell to near craniotomy levels at 2–4 wks. CSF p-tau mirrored this trend, and was increased by 3.17x 1wk after injury. Serum tau was suppressed by 0.17x after 1 wk, normalized to craniotomy levels at 2 wks, but was again suppressed to 0.048x 4 wks post-PBBI. Within the same cohorts, the serum p-tau peaked 1 wk after PBBI to 4.21x. In the FC, the p-tau:tau ratio decreased to 0.77x at 1 wk, increased by 1.12x after 2 wks, then fell to 0.047x at 4 wks. The ratio in CSF rose from 0.093x at 1 wk, to 0.22x at 2 wks, then to 0.33x at 4 wks post-PBBI. Tau, pTau, and the p-tau:tau ratio, may be indicators of tauopathies in brain tissues driven by TBI. Further, these analytes remain detectable throughout subacute injury in both serum (2 wks) and CSF (4 wks). Tau, p-tau and the ratio may be useful as diagnostic and theranostic indicators relevant to the long-term effects of TBI.
Keywords: Severe TBI, tau, phosphorylated tau, serum
BIOMECHANICS OF MOTORCYCLE HELMET PROTECTION
Motorcycle accident victims account for more than 340,000 fatalities annually, with the Unites States ranking 8th highest worldwide in number of motorcycle accident deaths. Seventy-five percent of all fatal motorcycle accidents involve head and brain injury, with rotational forces acting on the brain the primary cause of mortality. Current motorcycle helmets are reasonably effective at reducing head injuries associated with blunt impact. However, the mechanism of traumatic brain injury is biomechanically very different from that associated with head injury. This biomechanics study was conducted to evaluate the effectiveness of current motorcycle helmets at reducing the risk of traumatic brain injuries, including hemorrhages and concussion. A variety of motorcycle helmet designs, including full-face, three-quarter, half-helmets and novelty (non DOT) helmets were evaluated at impact speeds up to 25 mph using a validated test apparatus outfitted with a crash test dummy head and neck. Sensors installed at the center of gravity of the headform and on the helmet enabled high-speed data acquisition of linear and angular head kinematics associated with impact. Variables depicting the impact characteristics and protective properties of the helmet were computed using Matlab
Keywords: motorcycle, helmet, accident, TBI
PROJECT HEAD TO HEAD II: YEAR ONE
Approximately 300,000 sports-related concussions occur annually in the United States. Amateur football players reported higher rates of concussions compared to other contact sports. Subconcussive impacts through routine participation in sports were theorized to contribute towards increased concussion susceptibility. Therefore, it is important to characterize head impact exposure data. 197 participants were recruited from four NCAA Division III college and 36 participants were recruited from two varsity high school football teams during the 2015 season. Each athlete received a Riddell Speed helmet (Des Plaines, IL) outfitted with the Head Impact Telemetry System (Simbex, Lebanon, NH), which recorded impact data when one of six single-axis accelerometers exceeded a predefined threshold of 10 g. Data were collected during games and practices, each referred to as a session. College athletes sustained 13.13 ± 8.9 and high school athletes sustained 14.05 ± 8.1 impacts per session, which varied significantly (p < 0.05) between positions, with linemen sustaining the highest frequency. Median Peak Linear Acceleration (PLA) was 20.2 g and 20.7 g for college and high school, respectively. Median Peak Rotational Acceleration (PRA) was 941.86 rad/s2 for college and 949.82 rad/s2 for high school. A total of 12 concussions were recorded during the season (9-college, 3-high school). The average PLA for concussive impacts was 99.05 ± 29.12 g (college- 107.57 ± 28.48 g, high school- 73.5 ± 10.56 g) and PRA was 4506.28 ± 1673.77 rad/s2 (college - 4840.05 ± 1444.52 rad/s2, high school - 3504.99 ± 2248.65 rad/s2). There were no significant differences between mean PLA of concussed athletes (26.8 g) and non-concussed athletes (25.1 g). Similarly, mean PRA showed no significant difference between concussed (1182.45 rad/s2) and non-concussed (1108 rad/s2) athletes. However, distribution of head impacts showed that concussed players sustained slightly more impacts and higher magnitudes for PLA and PRA. This descriptive analysis from a relatively large sample provides additional data to understand head impact exposure in amateur football. The focus of the ongoing aspects of the study will be to understand correlations between head impact exposure, onset of concussion, and clinical/imaging aspects of the injury.
Keywords: concussion, football, subconcussive, HITS
A HIGH CONTENT, HIGH THROUGHPUT, HUMAN, IN VITRO MODEL OF NEURONAL STRETCH INJURY
NorthShore University HealthSystem, Neurosurgery, Evanston, USA
Neuronal stretch injury is an important component of traumatic brain injury (TBI) pathology. TBI is a major cause of mortality and morbidity with limited therapeutic options. Drug development for TBI is resource-intensive and low yield because existing pre-clinical models are low throughput and non-primate. In an effort to address these challenges, a high throughput, human, in vitro model of stretch injury was created using a 96 well format and human neurons derived from induced, pluripotent stem cells. Silicone membranes were covalently bonded to bottomless, 96 well plates to create stretchable culture substrates. The external geometry of the plate was maintained so that it remained compatible with robotic devices typically employed in drug screening. A custom-built device driven by an electromagnetic voice coil was designed and validated to apply a range of rapid, highly repeatable, biofidelic strains and strain rates to these plates. The device stretches the membrane by indenting it with a rigid post to create a spatially homogenous, equibiaxial strain field. A high content approach was used to measure injury in a hypothesis-free manner compatible with drug screening experiments. Fluorescent images of intact cells in culture were analyzed to determine cell viability along with several measures of altered neurite morphology. These measurements are shown to provide a sensitive, dose- dependent, multi-modal description of the response of human neurons to mechanical insult. Human neurons transition from a healthy phenotype to an injured phenotype at approximately 35% Lagrangian strain. The injury phenotype had three components: cell death, shortening of neurites, and changes in neurite shape. Morphological metrics integrate the effects of multiple biochemical processes into a single outcome, which is particularly useful in a multi-modal disorder such as TBI. This model creates novel opportunities for drug discovery and exploration of the role of human genotype in TBI pathology.
Keywords: human induced pluripotent stem cell derived neuron, high throughput, high content
In abusive head trauma, subdural and subarachnoid hemorrhage (SDH/SAH) is attributed to rupture of fine parasagittal bridging veins (BVs) tethering the brain to the superior sagittal sinus, which elongate as the brain and skull move past each other during vigorous shaking. We examined the longitudinal mechanical properties and behavior of BVs from N = 12 adult pigs, N = 9 infant pigs, and N = 7 infant humans. BVs from each subject experienced high rate or low rate elongation to failure (13.91 ± 2.84 s−1 and 1.36 ± 0.26 s−1 respectively) and cyclic loading (1.2 stretch ratio, at 3 Hz, for 30 s) followed by elongation to failure (2.56 ± 0.32 s−1). BVs fatigued during cyclic loading, such that peak stresses decayed exponentially with increasing cycle number, with no significant influence of age or species. ANOVA and post-hoc Tukey analyses of BV yield stress, yield stretch, ultimate stress, ultimate stretch, and Young's modulus during elongations to failure revealed no age-dependence, but human infant tissue had higher values than porcine for all metrics except Young's modulus. Yield stress and ultimate stress were higher in high rate tests than low rate and post-cyclic. Young's modulus was larger in high rate tests than low rate tests (post-cyclic Young's modulus was indistinguishable from both high and low rate). Finally, yield stretch was greater in post-cyclic tests than high rate and low rate. In all cases, the shape of the stress-stretch curve for post-cyclic failure pulls was also qualitatively different than those for high rate and low rate tests, with a longer low-stress regime (toe region). We conclude that 1) repeated loading before failure softens BVs; 2) BV tissue behavior is rate-dependent; and 3) human infant BVs are stronger than porcine. Future computational studies will utilize these BV mechanical properties to identify BV failure in falls and abusive TBI scenarios. Funding: American Heart Association Predoctoral Fellowship and NIH R21HD078842.
Keywords: pediatric TBI, biomechanics, computational model
DYSREGULATIONS IN THE BRAIN CAUSED BY MILD BLAST EXPOSURE AND NEUROPROTECTIVE TREATMENT WITH A NRF2 ACTIVATOR
Bay Pines VA Healthcare System, Research & Development, Bay Pines, USA
Approximately 1/200 individuals suffer a traumatic brain injury worldwide each year and the rate is 30 fold higher among deployed service personnel. The most common injuries are mild yet they do result in significant deficits in brain function and cognitive performance. We have identified certain transcription factor pathways, involved in inflammatory responses, that are important to the health of neurons after traumatic brain injury. The explosive blast injury system is an extension of our closed head injury model. The sharp pressure wave produced by a TNT detonation produces memory deficits in the exposed mice. The degree of injury is titrated by distance from the calibrated blast in a controlled field environment. The advantages of the explosion model are that it most accurately reproduces the environment surrounding explosive devices and the resultant microsecond pressure wave. We found that the blast-injured brain displayed altered levels of regulators of plasticity and transcription factor pathways, e.g., Inhibitor of DNA binding 2, Id2. Protein and mRNA levels for the transcription factor, Nrf2 were measured after mild TBI exposures at different distances and times from the blast. We found that the injury exposure itself induced an upregulation in Nrf2 levels and this may indicate that Nrf2 partially responds to this form of TBI insult. Post-injury treatment involved administration of tert-butylhydroquinone, an activator of the transcription factor Nrf2. Several different proteins were evaluated that could affect neuronal health and we concentrated on one of the most sensitive brain regions to this kind of injury, the hippocampus. In summary, the examination of altered expression levels and signaling pathways should help advance the identification of therapeutic targets that will benefit Veterans and others suffering traumatic brain injuries.
Keywords: transcription factor, Nrf2, mice, hippocampus
Columbia University in the City of New York, Department of Biomedical Engineering, New York, USA
Blast-induced traumatic brain injury (TBI) has been described as the signature wound of the conflicts in Iraq and Afghanistan. Currently, there is no FDA-approved therapeutic for TBI. Therapeutic candidates that have demonstrated experimental efficacy for treatment of TBI include phosphodiesterase-4 (PDE4) inhibitors. This study investigated the therapeutic potential of roflumilast, an FDA-approved PDE4 inhibitor for chronic obstructive pulmonary disorder, on deficits in long-term potentiation (LTP) caused by primary blast in rat organotypic hippocampal slice cultures. Mild primary blast exposure (336 kPa/0.84 ms/87 kPa·ms) was applied with a compressed-gas driven shock tube. Roflumilast (1 μM) was delivered immediately following injury, and its effects were compared to vehicle (0.07% DMSO) treatment. Electrophysiological recordings were acquired at 24 hours following injury using 60-channel microelectrode arrays. Long-term potentiation was induced in CA1, via the Schaffer collateral pathway, using 100 Hz stimuli. LTP induction was calculated as percent potentiation above baseline based off of the last 10 minutes of pre- and post-induction recordings. Compared to vehicle (28 ± 11%), roflumilast treatment (69 ± 14%) prevented blast-induced LTP deficits measured at 24 hours (Avg ± SEM). Roflumilast treatment did not enhance LTP in sham exposed cultures (78 ± 11%) over vehicle treatment (79 ± 13%). The effects of injury and drug treatment were analyzed statistically with analysis of variance, where significance was considered p < 0.05 (N = 6 for all groups). We conclude that roflumilast may have therapeutic potential to prevent memory deficits following primary blast exposure. LTP deficits may be the source of memory loss commonly observed in TBI patients. Future research will confirm the cellular mechanisms responsible for this therapeutic effect following primary blast injury.
Keywords: in vitro, hippocampus, cell death, LTP
Naval Medical Research Center, Neurotrauma, Silver Spring, USA
Traumatic brain injury (TBI) occurs in high frequency in returning military service members from the recent military conflicts of Operation Enduring Freedom/Operation Iraqi Freedom (OEF/OIF). A common mechanism of injury in OEF/OIF are blast-related mild TBI (mTBI) caused by exposure to blast- overpressure (BOP; shock) waves from incendiary devices, such as improvised explosive devices (IEDs). Additionally, recent literature has indicated high rates of posttraumatic stress disorder (PTSD), as well as postconcussive symptoms in military personnel following blast-related mTBI. It is currently unclear whether BOP exposure predisposes an individual to PTSD. However, if repeated BOP does predispose an individual with PTSD, then there should be some behavioral disruption similar to PTSD symptomology, such as elevated anxiety, stress, and enhanced fear. One such behavioral assessment used to assess fear and anxiety is a cued-fear conditioning paradigm. In the current study, rats were exposed to repeated BOP of 75 kPa once daily over 3 consecutive days. Following the final BOP exposure, animals were assessed in general ambulation, via a motor activity test; fear and anxiety via cued-fear conditioning. Results revealed no abnormal locomotor activity following BOP exposure. Rats exposed to BOP exhibited similar freezing behavior to controls during acquisition trials and relativity lower freezing during extinction trials compared to controls in the fear conditioning paradigm. However, animals appear to adopt differing strategies during the intertrial intervals (ITIs), with BOP animals tending to show reduced freezing during ITIs compared to controls in both acquisition and extinction. In order to assess stress levels, blood serum was collected and corticosterone levels were analyzed using a commercially available ELISA kit. Results revealed a nonsignificant trend for elevated corticosterone levels in the repeated BOP exposed animals compared to controls. Overall, the current results suggest animals exposed to repeated BOP demonstrate reduced freezing following fear conditioning trials whereas control animals tend to demonstrate relatively higher fear response throughout the fear conditioning paradigm. The trend for elevated corticosterone in BOP animals may be related to the differences in freezing behavior in the fear conditioning paradigm.
Keywords: blast-overpressure, rodent model, blast-related mTBI, behavioral assessment
Neurostructural Research Labs, Neurotrauma Studies Group, Tampa, USA
Blast-related traumatic brain injury (bTBI) from improvised explosive devices (IEDs) has been the signature injury of the war against terror for both combatants and civilians. TBI is regarded as a major risk factor for the development of Post-Traumatic Stress Disorder (PTSD). The amygdala plays a vital role in fear conditioning and emotion; human studies have shown that the amygdala demonstrates hyperactivity in PTSD patients. As part of a larger study characterizing neuronal damage in a mouse model of blast-related TBI (bTBI) we evaluated the neuropathological impact of a mild bTBI, e.g., the effects on dendritic branching and spines of stellate neurons from the basolateral amygdalar. Anesthetized 7 week-old mice were exposed to a single explosion which generated a pressure wave of 5.5 psi (lbs/sq in) and sacrificed 72 hrs later. Using the Golgi impregnation technique, from coded slides, the dendritic branching and spines of basolateral amydgalar neurons were quantified (N = 6 subjects/group, n = 5 neurons/subject). The Sholl analysis was used to evaluate the extent and distribution of the dendritic arbor. Dendritic spines were evaluated in terms of total spine density and spine configurations.
Keywords: amygdalar, dendritic branching, dendritic spines, golgi staining
Blast traumatic brain injury (bTBI) is recognized as the signature injury of the wars in Iraq and Afghanistan. Brain injury is not only determined by the blast loading environment (magnitude and duration of the blast overpressure) but also by the orientation of the animal to the oncoming shockwave. This will correspond to a soldier encountering blast waves from different direction. In order to study the effect of various body orientations, we subjected 10-week old male Sprague Dawley rats to mild (blast overpressure equal to 130 kPa) and moderate (180 kPa) brain injury conditions using a field validated compressed gas driven shock tube. Rats were aligned parallel to shock wave propagation direction in either the prone or supine position. 24 hours after blast exposure the rats were sacrificed and brain and plasma samples were collected. In this study we investigated the effect of the subject's body orientation on the levels of neuroinflammation (IL-1β, TNF-α, nf-kB), oxidative/ nitrosative stress (NOX1, iNOS, 4HNE, 3NT), microglial activation (Iba1), neuronal cell death/ necrosis/ apoptosis, (caspase-3, Neurofilaments, neuron specific enolase (NSE), alpha II-spectrin), and blood brain barrier breakdown (Matrix metalloproteinases 2 and 9). These biomarkers were analyzed using immunohistochemistry, western blotting, and enzyme linked immunosorbent assay. Our results show that in both prone and supine positions, there was a significant upregulation of all analyzed biomarker compared to the control. Further these levels were higher for moderate TBI than mild TBI in most cases. Rats exposed in the prone position showed higher levels of all the biomarkers comparted to that of supine position. From this study we can conclude that prone position has higher levels of biochemical activities compared supine. The study indicates that one need to take the orientation into account while determining the injury severity.
This work was funded by US Army Materials and Medical Command. Award number: X81XWH-15-1-0303
Keywords: neuroinflammation, oxidation stress, apoptosis, blood brain barrier breakdown
PARKINSON'S DISEASE-LIKE NEUROPATHOLOGY FOLLOWING A MILD-BLAST TRAUMATIC BRAIN INJURY
Purdue University, Basic Medical Sciences, West Lafayette, USA
The increasing prevalence of blast-induced traumatic brain injury (bTBI) has contributed significantly to the rising incidence of long-term neurological deficits among military TBI victims. Notably, survivors of bTBI have three-fold higher susceptibility to Parkinson's disease (PD), which is characterized by α-synuclein protein-rich inclusions and the progressive degeneration of nigrostriatal dopaminergic neurons. However, preclinical studies on the pathophysiological changes in the brain post-bTBI, particularly for the signature mild injuries common in the military, have not been investigated in detail. The goal of this study is to evaluate biochemical changes in the brain post-mild bTBI that are known to be related to the pathologies of PD, aiming to understand key pathways leading to augmented vulnerability of PD post bTBI. Specifically, we have focused on the expression levels of α-synuclein, regulation of tyrosine hydroxylase (TH), both of which contribute to PD pathology, and acrolein, a marker of oxidative stress with widespread effects in neurotrauma and neurodegeneration, aiming to explore the synergistic mechanisms of bTBI and PD. We have shown that acrolein, a highly reactive aldehyde species and pro-inflammatory compound, persists up to a week following mild bTBI. In addition, we have also noted that acrolein may be a major contributing factor in the aberrant expression of α-synuclein and dysregulation of TH post-m-bTBI. Our data suggest acrolein, and more broadly oxidative stress, is likely a point of convergence between mild blast TBI and PD, and plays a critical role in the higher incidence of PD in bTBI victims. These results are expected to advance our understanding of the long-term consequences of blast-related injuries leading to the development of PD. Such efforts could eventually lead to the establishment of biomarkers for earlier diagnosis as well as strategies for prevention and treatment to curtail the elevating incidence of post-bTBI PD. Ultimately, we hope to significantly improve the quality of life for the military men and women who suffer daily from the long-lasting effects of bTBI.
Keywords: mild blast, parkinson's disease, alpha synuclein, tyrosine hydroxylase
Uniformed Services University of the Health Sciences, Neurology, Bethesda, USA
Most of preclinical studies carried out so far on blast-induced traumatic brain injury have focused on blast's “primary” mechanism of injury, namely, transmission of shock waves across the brain. The objective of this study is to develop a new in vivo model to explore how blast-induced acceleration may affect primary blast-induced neurotrauma. We have designed a new holding system allowing the open-field exposure of rodents under conditions permissive for explosive-driven blast-induced full body acceleration. Anesthetized rodents are exposed in a prone position to the blast wave generated by detonation of an explosive charge placed quasi-vertically above the targets. Acceleration-permissive conditions are obtained by laying the rodent on a sheet of paper stretched over the upper opening of an acceleration-capture box. At shock impact, the support paper is quasi-instantaneously removed from under the rodent allowing its unrestrained fall/acceleration. A high-speed video camera is used to obtain records for the subsequent analysis of acceleration/velocity parameters. Initial evaluations have been carried out on rat dummies and live Sprague-Dawley rats, with exposures to blasts with overpressures up to 30 psi and 2–2.5 msec positive phase duration (ppd). At equal overpressures, acceleration increases with ppd. As opposed to brains of rats exposed to 30 psi × 8–10 msec primary blasts in an explosive-driven blast wave generator, we observed no 7-day FD Neurosilver evidence of axonal injury or of related astrocyte and microglia reaction. Increased levels of calpain-specific alpha II-spectrin breakdown were detected in the hippocampus. Higher blast impulses, at minimally lung damaging overpressures, may be needed to obtain more significant acceleration-induced brain injury in the rat, though tested blast exposure conditions may be sufficient in a mouse model. Independent of acceleration, open field exposures under conditions of minimum restrain appear to be less injurious than those reported for less intense exposures in other preclinical models, reinforcing concerns about the relative role of artificial mechanisms of injury in these models.
Keywords: blast, acceleration, TBI, model
Traumatic brain injury (TBI) is often associated with reduced cerebral perfusion and impaired cerebrovascular function. However, little is known about cerebral vascular effects of blast induced TBI (bTBI). We examined relative cerebral perfusion, mean arterial pressure (MAP), cerebral arterial responses to reduced intravascular pressures, neuronal injury and cognitive function in rats subjected to bTBI using a compressed air-driven shock tube. Adult male Sprague-Dawley rats were surgically prepared for bTBI and randomly assigned to receive bTBI (17–22 psi) or sham-bTBI. After bTBI, righting reflexes (RR) were measured. Vascular reactivity was assessed in collected middle cerebral arteries (MCA) through measurements of dilator responses to reduced intravascular pressure. MAP and cerebral perfusion were measured using laser Doppler flowmetry before and for two hours after bTBI or sham-bTBI. Neuronal injury was assessed using FluoroJade-C while cognitive function was assessed through beam walk, beam balance and Morris water maze (MWM). The effects of peroxynitrite scavenger penicillamine on cerebral vascular function after bTBI were also determined. MCA dilator responses to reduced intravascular pressure and cerebral perfusion were significantly reduced while cerebral vascular resistance and duration of RR suppression were significantly increased by bTBI. bTBI also resulted in significant neuronal injury, impaired beam balance performance and an increase in MWM latencies. Reductions in cerebral perfusion and elevation of cerebral vascular resistance at levels of bTBI that resulted in small (<20%) increases in RR suppression that did not reduce MAP suggest that blast exposure insufficient to produce a sustained loss of consciousness may be associated with significant cerebral arterial dysfunction. Impaired cerebral vascular reactivity may contribute to impaired behavioral and cognitive function and increased acute neuronal injury. Blast-induced impairment of cerebral dilator responses to reduced intravascular pressure might contribute to further reductions in cerebral perfusion in the presence of arterial hypotension, a common occurrence in combat situations when blast exposure is accompanied by hemorrhage. Studies were completed as part of a team funded by The Moody Project for Translational Traumatic Brain Injury Research and award W81XWH-08-2-0132 from the Department of Defense.
Keywords: blast induced neurotrauma, primary blast injury, cerebrovascular circulation, neuropathology, behavior
CHARACTERIZATION OF INFLAMMATION PROCESSES IN THE VISUAL SYSTEM OF RATS INDUCED BY EXPOSURE TO PRIMARY BLAST WAVES
Walter Reed Army Institute of Research, Center for Military Psychiatry and Neuroscience Research, Silver Spring, USA
Blast injury is arguably the greatest threat to Warfighters in current campaigns, and is a leading cause of vision loss due to closed injuries to the eyes (retina) or brain from blast shock waves. Thus, there is an urgent need to carry out advanced studies in a well-established rodent model of blast, which allows evaluations of neuronal injures to the retina and brain. Our hypothesis is that immune cells play a primary role in exacerbating neurodegeneration following blast. Our objective is to monitor the nature and timing of neuroinflammation processes so as to discern potential drug targets and therapeutic windows. We are also examining the impact of nutrients known to be anti-inflammatory, e.g. omega-3 polyunsaturated fatty acids, on blast vulnerability. Adult male rats were fed for one month an omega-3 fatty acid deficient versus enriched (fish oil supplemented) diet. Blast injury was produced in anesthetized animals secured in a compressed air driven shock tube and then exposed twice (1 min interval) to blast over pressure waves (20 psi, 8 msec). At 3 to 28 days post-blast, retina and brain injuries were followed by electroretinography, visual acuity assessment, magnetic resonance imaging, histopathology, and cytokine level outcome measures. Our findings reveal that vision impairments occur early post-blast (i.e., within 7 days), and are accompanied by neurodegeneration in the retina and brain along with macrophage accretion, activated microglia and astrocytes, and increased cytokines. Dietary omega-3 fatty acids have so far shown slight, if any, ability to alleviate these acute injury events. Overall, our mission is to discover new drug treatments for blast-induced neurotrauma sustained by military personnel.
Supported by DoD grants from MOMRP and USAMRMC / CDMRP, #: W81XWH-14-2-0178.
Keywords: blast wave, rat, neuroinflammation, retina, brain, omega-3 polyunsaturated fatty acid
Blast-induced traumatic brain injury (bTBI) is one of the major disabilities in Service Members returning from recent military operations. The neurobiological underpinnings of bTBI, which are associated with acute and chronic neuropathological and neurobehavioral deficits, are uncertain. Increased oxidative stress in the brain is reported to play a significant role promoting neuronal damage associated with both brain injury and neurodegenerative disorders. In this study, brain regions of rats exposed to repeated blasts in a shock tube underwent untargeted profiling of primary metabolism by automatic linear exchange/cold injection GC-TOF mass spectrometry and revealed acute and chronic disruptions in the metabolism of amino acids and antioxidants. Closely coupled repeated blast exposures (19 psi peak total pressure, 8 msec duration) affected the metabolism of the essential amino acids tryptophan, phenylalanine and methionine. Tryptophan levels decreased on day 1 whereas phenylalanine showed a significant increase in the brain at 28 day after blast exposure. Methionine sulfoxide, the oxidized metabolite of methionine, showed a sustained increase in the brain after blast exposure which was associated with a significant decrease in cysteine, the amino acid derived from methionine. Glutathione, the antioxidant synthesized from cysteine, similarly decreased as also did the antioxidant ascorbic acid. Reductions in ascorbic acid were accompanied by increased levels of its oxidized metabolite, dehydroascorbic acid and other metabolites such as threonic acid, isothreonic acid, glycolic acid and oxalic acid. In view of the fundamental importance of glutathione in the brain as an antioxidant, including its role in the reduction of dehydroascorbic acid to ascorbic acid, the disruptions in methionine metabolism elicited by blast might prominently contribute to neuronal injury by promoting increased and sustained oxidative stress. Increasing the levels of cysteine in the brain by dietary supplementation of cysteine or administration of N-acetyl cysteine could be a potential therapeutic strategy against bTBI.
Keywords: metabolites, amino acids, antioxidants, oxidative stress
EFFICACY OF SELECTIVE INHIBITORS OF NUCLEAR EXPORT IN TRAUMATIC BRAIN INJURY CORRELATES WITH BLOOD BRAIN BARRIER PENETRATION
Karyopharm Therapeutics, Newton, USA
Exportin 1 (XPO1) – a nuclear export protein that regulates the trafficking of over 200 cargoes, including many important neuroprotective and anti-inflammatory factors – has surfaced as an attractive target for the treatment of traumatic brain injury (TBI). A new class of molecules inhibiting this protein, termed Selective Inhibitor of Nuclear Export (SINE) compounds, has demonstrated therapeutic activity and tolerability in preclinical and clinical trials. Previous studies in models of TBI demonstrated that SINE compounds act to improve neurologic outcomes via multiple pathways, including protection of the blood brain barrier (BBB). In the present study, three SINE compounds with different pharmacokinetic properties were used to assess the relationship between BBB penetration and post-TBI functional outcomes.
We investigated the effects of KPT-350, KPT-335 and KPT-8602 – SINE compounds with low, moderate and high brain bioavailability, respectively – in a rat controlled cortical impact (CCI) model of TBI. CCI rats received SINE compound or vehicle twice per week for three weeks, starting 2 hours post-injury. Following the dosing period, behavioral tests and histological assays were performed.
The efficacy of SINE compounds correlated directly with the compounds' relative brain bioavailability. Animals treated with KPT-350 and KPT-335, compounds that readily penetrate the blood brain barrier, showed significant improvements in the Rotarod and tail hang tests of behavior and coordination compared to vehicle. Functional outcomes in the KPT-8602 treatment group, however, were markedly worse than in the control group, suggesting that brain bioavailability is important for SINE compound efficacy. Histological assessments revealed that TBI-induced lesion size was directly related to BBB penetrance, with KPT-350 affording the largest reduction in impact area.
These findings demonstrate that SINE compound efficacy in TBI is predicated upon BBB permeability and highlight the therapeutic potential of KPT-350.
Keywords: blood brain barrier, nuclear export inhibition, neuroprotection, anti-inflammation
Medical College of Wisconsin, Department of Neurosurgery, Milwaukee, USA
Traumatic brain injury (TBI) with its devastating outcomes remains an unmet medical need. For the development of novel and more effective therapies for TBI, it is essential to better understand mechanisms that lead to the primary and secondary cell death following the injury. The aim of the present study was to characterize acute cellular effects of weight-drop injury in rat organotypic hippocampal slice cultures (OHCs). Hippocampi obtained from neonatal Sprague Dawley rats (P7-10) were cut into 400 μm thick slices and grown using the membrane interface method. At 7 days in the culture, OHCs were injured using the NYU weight-drop impactor with the custom made rod. The rod was positioned above the cornu ammonis 1 (CA1) region of the hippocampus and dropped from a 6.25 mm height at the impact velocity of 0.35 m/s. Resultant cell death at 2 h post-injury was analyzed by propidium iodide (PI) uptake assay. Live microglia imaging in OHCs following injury was preformed with isolecitin B4 (IB4) conjugated to FITC. Additionally, effects of injury on neurons and astrocytes were assessed by immunostaining against neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP), respectively. A drop of the impactor rod resulted with the tissue damage below the point of impact and PI fluorescent dead cells were observed already at 2 h post-injury. In addition, cells that were co-labeled with PI and IB4, GFAP, or NeuN were identified. Astrocytic clasmatodendrosis characterized by cytoplasmic swelling, beading and dissolution of distal processes was also detected. Our data suggest that both glial cells and neurons are affected in the acute phase of mechanical injury. Future studies will elucidate mechanism involved in the acute cell death following the weight-drop injury. Supported by Department of Neurosurgery, MCW and VA Research
Keywords: In vitro model, organotypic hippocampal slice cultures, brain injury, acute cell death
Cellular senescence refers to irreversible growth arrest that occurs when cells experience stress and damage from exogenous and endogenous sources. Senescent cells secrete various factors that can contribute to tissue dysfunction. We hypothesized that senescent cells appear in the brain as a response to traumatic brain injury (TBI). We performed histochemical analysis in the brain for senescence-associated β-galactosidase (SA-βgal), a marker of senescent cells, at 1, 4, 7 and 14 days after TBI in a controlled cortical impact (CCI) mouse model. On comparing the data from control and TBI animal groups, we found that SA-βgal was preferentially expressed in the cortex ipsilateral to the site of the injury on 4, 7, and 14 days after TBI. Immunohistochemical analyses also showed that macrophages and microglial cells expressed SA-βgal at the site of cortical contusion. These findings suggest that senescent cells are associated with TBI. Because senescent cells are deleterious to brain tissue, the appearance of senescent cells may contribute to the progression of post-TBI brain damage.
Keywords: cellular senescence, senescent cells, senescence-associated β-galactosidase, controlled cortical impact
CPLA2 MEDIATED LYSOSOMAL DAMAGE LEADS TO IMPAIRMENT OF AUTOPHAGY AFTER TBI
University of Maryland, Baltimore, Anesthesiology & Shock, Trauma and Anesthesiology Research (STAR) Center, Baltimore, USA
Autophagy is a lysosome-dependent intracellular degradation process compromised in many neurodegenerative diseases. Recently we demonstrated impairment of autophagy after controlled cortical impact (CCI) traumatic brain injury (TBI) in mice. One day after TBI autophagosome degradation was inhibited mainly within neurons and contributed to neuronal cell death. In the current study we investigated the mechanisms leading to impairment of autophagy after TBI. Our data demonstrate that it is due to lysosomal dysfunction, evidenced by lower protein levels and enzymatic activity of the lysosomal enzyme, cathepsin D (CTSD). Unlike the typical vesicular localization in the sham mice, CTSD immunostaining in the injured cortex appeared diffuse, suggesting leakage into the cytosol. Consistently, CTSD enzyme activity was lower in the lysosomal but not cytosolic fractions from injured cortex as compared to sham. Therefore, TBI causes lysosomal membrane damage and leakage of lysosomal contents into the cytosol leading to lysosomal impairment and inhibition of autophagy. Our data further indicate that lysosomal damage after TBI is mediated by the cytosolic phospholipase A2 (cPLA2). cPLA2 was activated in the injured cortex, its levels were increased within neurons and co-localized with markers of blocked autophagy. Activation of cPLA2 by ceramide-1-phosphate (C1P) in vitro in human H4 cells and rat cortical neurons lead to block of autophagosome degradation. This was due to lysosomal damage evidenced by lower lysotracker fluorescence as compared to controls. Knockdown of the PLA2G2A gene or pretreatment with cPLA2 inhibitor AACOCF3 prevented autophagosome accumulation, demonstrating dependence on cPLA2 function. In vivo, pretreatment of mice with AACOCF3 significantly decreased accumulation of LC3-II and p62 after TBI, indicating attenuation of autophagy defect. Together, our data demonstrate that lysosomal function and integrity is compromised after TBI, and implicate cPLA2 as an important mediator of damage to the lysosomal membrane and inhibition of autophagy flux.
Support: R01NS091218
Keywords: autopahgy, lysosome, membrane damage, cytosolic phospholipase A2, autopahgy flux
UMASS Medical School, Neurology, Worcester, USA
Keywords: mouse, closed head injury, outcome, laser doppler, neurological severity score
Support: NIH-NIGMS P20GM109089-01A1, Rio Grande Neurosciences.
Keywords: transcranial direct current stimulation (tDCS), TBI, rehabilitation, animal model
CHANGES IN CEREBRAL BLOOD FLOW AND DEFAULT MODE NETWORK CONNECTIVITY FOLLOWING MTBI OBSERVED WITH PULSED ARTERIAL SPIN LABELING
Wayne State University, Department of Psychiatry and Behavioral Neurosciences, Detroit, USA
Mild traumatic brain injury (mTBI) affects more than 1 million Americans each year, accompanied by short-term and sometimes long-term changes in cognitive functioning. However, the etiology of these changes still requires some elucidation. Investigation of cerebral blood flow (CBF) and brain functional connectivity together, as biomarkers of brain metabolism and activity, may provide some insight into how brain function changes following mTBI. A single MRI sequence, PASL, can provide us a unique opportunity to simultaneously assess both CBF and intrinsic connectivity networks (ICNs) due to its sensitivity to both static and dynamic CBF. We performed PASL in 30 healthy individuals and 22 mTBI patients at the acute stage, generated individual subject CBF maps and extracted the default mode network (DMN) from the PASL time series using independent component analysis (ICA). A voxel-wise nonparametric t-test with family-wise error (FWE) correction showed increases in CBF in mTBI patients, which mainly overlap with the default mode network (DMN). The ICA showed reductions in connectivity within the DMN, similar to our previous findings using rsfMRI; however, this did not survive FWE correction. In conclusion, identification of similar networks from PASL as from rsfMRI suggests that ICNs can be identified from PASL data. Moreover, because the PASL signal is mainly derived from blood flow, PASL ICNs may be more representative of brain metabolism then blood oxygen level dependent (BOLD)-based ICNs, since BOLD is also affected by other physiological parameters, while also providing extra information from the PASL data at essentially no cost. The combination of decreased connectivity within the DMN and increased CBF in some regions of the DMN may represent an acute brain response to injury. We plan to further investigate this using a method more specific to oxygen consumption.
Keywords: arterial spin labeling (ASL), connectivity, default mode network (DMN), mild traumatic brain injury (mTBI)
Medical College of Wisconsin, Neurosurgery, Milwaukee, USA
Large-scale network analysis based on the characterization of the brain as a complex network of nodes and edges allows for the evaluation of functional connectivity patterns. The utility of graph-based techniques has been proven by an increasing number of resting-state functional MRI (rs-fMRI) studies in normal and diseased brain. However, to our knowledge, graph theory has not been used to study the reorganization pattern of resting-state brain networks in patients with complete spinal cord injury (SCI). In the present analysis, we applied a graph-theoretical approach to explore changes to global brain network architecture as a result of SCI. Fifteen subjects with chronic (> 2 years) complete (ASIA A) cervical SCI as well as 15 neurologically intact controls were scanned. The data was preprocessed followed by parcellation of the brain into 116 nodes. The average time series was extracted and correlation analysis was performed between every pair of nodes. A functional connectivity matrix for each subject was then generated. Subsequently, the matrices were averaged across groups and network changes were evaluated between groups using network based statistic (NBS) method. Our results revealed a decrease in connectivity in the whole brain network in SCI compared to control subjects. Upon further examination, we observed an increase in connectivity in SCI compared to controls in the network comprised of sensorimotor cortex and cerebellum. In conclusion, we have demonstrated whole brain changes in resting-state functional connectivity in SCI using large-scale network analysis. Our findings not only emphasis the applicability of large-scale network analysis in SCI, but also provide important insight into the reorganization of brain networks, which could have potential prognostic and therapeutic implications.
Keywords: spinal cord injury, resting-state functional connectivity, large-scale network analysis, whole brain network
University of South Dakota Sanford School of Medicine, Sioux Falls, USA
Cervical spondyloptosis is a very rare neurological entity in which there is total anterior or posterior dislocation of the superior vertebral body with respect to the inferior vertebral body. We report a case of a post-traumatic C5-6 spondyloptosis in a 49 year-old woman following rollover motor vehicle accident. The patient presented with quadriplegia and absent sensation distal to the C5 dermatome. CT of the cervical spine demonstrated C5-6 spondyloptosis, lamina fractures on the right side of C3-4, and widening of the facet joint on the right side of C6-7. The patient underwent cervical traction in the operating room at the beginning of surgery and then received anterior stabilization with a C5-6, C6-7 anterior cervical discectomy and fusion. Six months post-operatively, the patient's neurological status remained stable with complete loss of function below C5. A literature search was conducted for the key terms spondyloptosis and grade 5 spondylolithesis. Each of the articles were reviewed and analyzed tabulating the details for the following fields: age, mechanism of injury, and types of treatment among other categories. Of the 33 total cases identified 10 (30.30%) underwent anterior fusion only, 7 (21.21%) underwent posterior fusion only, and 14 (42.42%) underwent 360 degree fusion. Surgical management of post-traumatic cervical spondyloptosis varies from anterior only, to posterior only, to 360° fusion in a single surgery or staged. There is no consensus on the best treatment strategy and each patient is managed on an individual basis. Merits of these three surgical options have been discussed since the early 1980s with few subsequent reports in literature. Here we provide a comprehensive analysis of the literature and report a C5-6 spondyloptosis that was successfully treated by an anterior-only approach with unicortical, variable-angle screws on a fixed plate. This helps support the validity of this relatively conservative treatment option for appropriately selected patients.
Keywords: spondyloptosis, cervical spondyloptosis, post-traumatic cervical spondyloptosis, spondylolithesis
UPMC Presbyterian, Dept of Neurosurgery, Pittsburgh, USA
Keywords: PET, tau, chronic TBI, neurodegeneration
Long-term effects of traumatic brain injury are not well described in animal models of brain injury. The novel-object recognition (NOR) test is one way to evaluate short-term memory in rodents. We evaluated the effects of fluid-percussion injury (FPI) on the ability of rats to recognize a novel versus a familiar object six months after injury. Twenty-four, male, Sprague-Dawley rats (300–370 grams) were handled, trained and baseline measurements were taken for reflexes, balance and coordination. Rats were anesthetized (isoflurane) and randomly assigned to receive right-sided FPI (2.21–2.36 atm; n = 12) or sham injury (n = 12). Neurological and vestibulomotor functions were tested 1–3 days and memory was assessed 6 months post-TBI. For NOR assessment, rats were first habituated to the chamber for 15 min followed the next day by 5 min exposure to matching objects. After a 30 min inter-trial interval, rats were replaced in the chamber with a pair of objects (one familiar and one new) for a 5 min test session. More time spent with the novel object than the familiar object indicates that the rat remembers the familiar object. Injured rats showed behavioral deficits on tests of neurological function, balance and coordination on days 1 – 3 after injury. However, in the NOR test, there were no differences between novel and familiar objects in the number of entries or the time spent in the object zones for either group of rats. While both groups of rats spent more time exploring the chamber than the objects; injured rats spent less time exploring objects than uninjured rats, possibly indicating reduced activity. Thus, at six months after injury injured rats were less active than uninjured rats, however deficits in short-term memory were not detected using the current NOR paradigm. Studies were completed as part of a team funded by The Moody Project for Translational Traumatic Brain Injury Research.
Keywords: novel-object recognition, short-term memory, behavior, rat, fluid-percusion injury, chronic injury
SELECTIVE ALLOSTERIC MODULATION OF PDE4D REVERSES CHRONIC MEMORY DEFICITS FOLLOWING TRAUMATIC BRAIN INJURY
University of Miami Miller School of Medicine, Neurological Surgery, Miami, USA
Learning and memory impairments significantly affect a majority of traumatic brain injury (TBI) survivors. However, there are no effective therapeutic strategies to improve cognitive functioning in these individuals. Our previous studies demonstrated that a pan-phosphodiesterase 4 (PDE4) inhibitor rolipram improves cognitive function and synaptic plasticity after TBI. However, unwanted side effects such as nausea and emesis prevent clinical development of rolipram. A novel allosteric modulator of PDE4D, D159687, has been shown to have reduced emetic potential while maintaining efficacy in cellular and in vivo models. Therefore, in the present study, we hypothesized that treating animals with a PDE4D allosteric modulator could reverse the cognitive deficits caused by TBI. To test this hypothesis, adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury. After 3 months recovery, animals were treated with D159687 (0.3 mg/kg, intraperitoneal) 30 min prior to cue and contextual fear conditioning, acquisition in the water maze or working memory training. Treatment with D159687 reversed deficits in TBI animals in cue and contextual fear conditioning, water maze performance and also improved working memory. To further explore the underlying cellular mechanisms, changes in basal synaptic transmission and long-term potentiation (LTP) at the CA3-CA1 synapse were studied at 3 months post-injury. Hippocampal slices from TBI animals showed a significant reduction in basal synaptic transmission and impairment in the expression of LTP as compared to sham surgery animals. The deficits in LTP were rescued with D159687 treatment. These results indicate that a novel allosteric modulator of PDE4D may be a potential cognitive enhancer to improve chronic cognitive dysfunction following TBI.
Keywords: traumatic brain injury, learning and memory, syanptic plasticity, long-term potentiation, fear conditioning
Animal models are used extensively to study blast-induced neurotrauma (BINT). Primary blast injuries are caused by shock waves comprised of sharp rising overpressure followed by exponential decay lasting a few-to-tens of milliseconds with no other pressure surges or violent high velocity winds. We established that to mimic field-validated blast profiles that specimen should be tested inside the shock tube. However, the end effects or structural reflections can induce unwanted pressure pulses and hence inappropriate loading. Using a shock tube equipped with an end-plate we demonstrate both numerically and experimentally that we can generate a single, isolated shock wave characterized by Friedlander waveform, without deleterious secondary pressure surges. When the shock tube is fully closed, the high-pressure compressive waves are reflected from the end, but when it is fully open, tensile waves are created by exiting original shock wave. These secondary waves traveling back into the tube can lead to additional types of injuries in an animal model, and thus are unwanted from the perspective of correct injury loading conditions. Moreover, it is highly unlikely that these secondary artifacts are encountered in the field, and hence should not be included as a part of laboratory model. We conducted a series of experiments with five different end plate configurations for three peak overpressures 130 kPa, 200 kPa and 290 kPa (corresponding to mild, moderate, and severe BINT conditions). Judicious choice of end opening results in elimination of artifacts and in each of those cases, we can obtain a pure Friedlander waveform. Finite Element numerical simulations are also carried out to elucidate the physics of the problem.
This work was funded by US Army Materials and Medical Command. Award number: W81XWH-15-1-0303: “Primary Blast Injury Criteria for Animal/Human TBI Models using Field Validated Shock Tubes.”
Keywords: shock tube, animal model, Friedlander waveform, end plate
MEMANTINE IMPROVES FUNCTIONAL AND HISTOPATHOLOGICAL SEQUELAE AFTER REPETITIVE TRAUMATIC BRAIN INJURY
Boston Children's Hospital, Harvard Medical School, Emergency Medicine, Boston, USA
Repetitive mild traumatic brain injury (rmTBI; eg, sports concussions) is a common injury that has been shown to result in significant cognitive impairment and yet, current targeted therapies for rmTBI are lacking. There is a paucity of preclinical and clinical data regarding NMDAR antagonist efficacy in the rmTBI setting. Evidence from other injury models indicates that strategies mitigating N-methyl-d-aspartate (NMDA) receptor (NMDAR)-mediated excitotoxicity might mitigate rmTBI-induced neurologic deficits. This is particularly attractive as NMDAR antagonists are widely-available and already in clinical use. Thus, we investigated whether a posttraumatic NMDAR blockade protects against adverse functional and histopathological sequelae in an established mouse rmTBI model.
Immediately after the last rmTBI injury (5 injuries in 5 days), mice were randomized to receive memantine treatment or vehicle. Functional outcomes were assessed by motor, anxiety/impulsivity and mnemonic behavioral tests. At the synaptic level, NMDAR-dependent long-term potentiation (LTP) was assessed in isolated neocortical slices. At the molecular level, the magnitude of microgliosis and tau hyper-phosphorylation was tested by immunostaining, while NMDAR subunit expression was evaluated by western blot and polymerase chain reaction (PCR).
Compared to injured vehicle treated mice, injured memantine treated mice had partially mitigated APP upregulation and reductions in tau phosphorylation. Memantine also ameliorated deficits in LTP and a decline in NMDARs. Furthermore, treatment with memantine in injured mice reduced moicrogliosis after rmTBI. Finally, Open Field testing showed that injured, memantine-treated mice spent less time in the wall zone compared to injured vehicle treated mice. Elevated Plus Maze also revealed that injured, memantine-treated mice performed more like sham mice compared to injured, vehicle-treated mice. No improvement in learning and memory was observed in the injured, memantine-treated group.
Our results indicate that early treatment of memantine ameliorates TBI-mediated histopathological changes at the molecular and electrophysiological levels. Furthermore, memantine improves certain behavioral outcomes after rmTBI. This data raises the prospects for therapeutic, post-concussive NMDAR antagonism. Further evaluation of the therapeutic window and dosing for treatment is warranted.
Keywords: closed head injury, concussion, traumatic brain injury, behavior, gliosis
CNRM / NIH / NINDS, Stroke Branch, Bethesda, USA
Keywords: chart review, diagnosis
French Armed Forces Biomedical Research Institute, Bretigny-sur-Orge, France
The influence that recurrent stress exposure before injury exerts on outcome after traumatic brain injury (TBI) has been little studied, although prevalence of TBI is high in both civilian and military populations repeatedly exposed to life-threatening stressful situations. Here, using a rat model, we aimed at determining if early post-TBI modulation of the expression of brain-derived neurotrophic factor (BDNF) and of its high-affinity receptor TrkB, which are known to promote neuroprotection after brain injury, is influenced by pre- exposure to life-threatening stress. Adult male Sprague-Dawley rats were repeatedly exposed to predator odor 2-4-5-trimethyl-3-thiazoline (TMT), which elicits fear response in rodents, or were exposed to water instead of TMT for 2 weeks, and then were subjected to either bilateral mild-to-moderate fluid percussion brain injury (LFP) or Sham surgery. We measured the expression of BDNF and TrkB at 8 hours post-TBI/surgery in the neocortex, hippocampus and ventral limbic region (VLR) using RT-qPCR. The most robust alteration of BDNF expression compared to Sham condition was observed when rats were pre-exposed to TMT before LFP, although it was not specific to a given brain area. TMT, LFP and combination of both all influenced TrkB expression with regional specificities. Overall, TBI alone had weak effects on TrkB expression, contrasting with the significant alterations induced by TMT exposure alone. The combination of TMT exposure and LFP produced effects similarly to stress alone. Thus, the reactivity of TrkB expression to LFP alone was different from that triggered by the TMT-LFP combination. Hence, these data suggest that repeated pre-exposure to life-threatening stimulus modifies the reactivity of the BDNF-TrkB system, and may therefore influence outcome after TBI. Acknowledgements of support: This work was funded by the Direction Générale de l'Armement (French Ministry of Defense).
Keywords: TBI, stress, preclinical, brain-derived neurotrophic factor, TrkB, gene expression
Georgia Institute of Technology/ Emory University, Atlanta, USA
Keywords: concussion, mild traumatic brain injury, symptoms, vertigo, balance
Kansas University Medical Center, Neurosurgery, Kansas City, USA
Keywords: NHP, controlled cortical impact, model, sustained focal deficit
Medical College of Wisconsin, Milwaukee, USA
Growing evidence suggests that repeated concussions and sub-concussive head injuries may have long-term neurological consequences. We have previously demonstrated smaller hippocampal (HPC) volumes in football players with (foot-w) and without (foot-wo) a previous concussion relative to healthy controls (HC), as well as thinner orbital frontal (OFC), pregenual anterior cingulate (pg-ACC), and motor cortices in foot-w relative to HC and foot-wo. Moreover, smaller HPC volume was associated with plasma levels of neurotoxic kynurenine metabolites that have been implicated in structural and functional changes in a variety of neurodegenerative diseases. Here, we tested the hypothesis that football players have abnormal resting state functional connectivity of the HPC, OFC, pgACC, and motor cortex relative to HC, and that connectivity is associated with plasma levels of kynurenine metabolites in football players. Seed-based functional connectivity was calculated for HPC, pgACC, OFC, and motor cortex based on our previous evidence of structural differences in these regions in 25 foot-w, 24 foot-wo, and 27 HC. FMRI analyses were conducted using AFNI; kynurenine metabolites were quantified in plasma using high performance liquid chromatography with tandem mass spectrometry. Both football groups had significantly increased connectivity of the HPC and OFC to regions in the executive control and default mode networks relative to HC. Foot-w had greater connectivity between the motor cortex and supplementary motor area relative to foot-wo and HC. Secondary analyses demonstrated that the relationship between neurotoxic kynurenine metabolites and connectivity of the pgACC and HPC differed as a function of football experience and concussion history. These results demonstrate that football is associated with changes in intrinsic functional connectivity and are the first evidence of a relationship between intrinsic brain connectivity and neuroactive kynurenine metabolites in any disorder.
Keywords: resting state functional connectivity, kynurenine, blood biomarker, football
Mind Research Network, Albuquerque, USA
Substance abuse and trauma are highly comorbid, with approximately 30–50% of new mild traumatic brain injuries (mTBI) occurring under the influence of alcohol. mTBI is often associated with damage to prefrontal and limbic circuitry, regions also implicated in the development and maintenance of alcohol use disorders (AUD). However, few studies have examined the consequences of mTBI on subsequent drinking-related behaviors and neuronal abnormalities. The current study therefore investigated the long-term consequences of self- reported mTBI on maladaptive drinking behaviors and behavioral disinhibition in individuals with AUD (N = 176). A subset of participants also underwent functional magnetic resonance imaging (fMRI) during a cue reactivity task to directly probe disturbances in addiction circuitry. Preliminary results indicated that 107/176 AUD (60.8%) reported a positive lifetime history of mTBI (AUD+mTBI group), approximately 1.6 times the rate of self-reported mTBI (36.4%) in the general population. Behavioral results from a delay discounting task indicated significantly (t106 = 2.93; p < 0.005; Cohen's d = 0.57) increased discounting rates in AUD+mTBI (N = 63; log(k) = -3.35 ± 1.18) relative to AUD alone (N = 45; log(k) = -4.01 ± 1.10). Although no significant group differences on measures of alcohol craving (d = 0.23), average number of drinks per drinking day (d = 0.15), drinking days (d = 0.07), the maximum reported numbers of drinks in an episode (d = 0.20) or failure to control drinking behavior (d = 0.28) were observed, all effects were in the expected direction (AUD+mTBI > AUD). The range of these measures was potentially limited by severity of alcohol use in the sample. fMRI results indicated that AUD+mTBI (N = 61) exhibited increased cue reactivity (alcohol > juice) within prefrontal and limbic circuitry (key regions d = 0.44–0.63) relative to the AUD only group (N = 41) following appropriate correction for false positives (p < 0.05). In summary, current results provide some of the first evidence of the behavioral and neuronal consequences of combined AUD+mTBI relative to AUD alone.
Keywords: mTBI, alcoholism, prefrontal circuitry, drinking behaviors
Heightened awareness of mild traumatic brain injuries (mTBI) and the millions of cases that occur worldwide each year highlight the need for better biomarkers which enable diagnosis and monitoring of the progression of injury. Cerebral hemodynamics have traditionally been used for severe TBI management; however, recent literature supports cerebral hemodynamic dysfunction as a primary indicator in mTBI as well. A number of technologies including fMRI, ASL and transcranial Doppler (TCD) have shown promise by measuring alterations in cerebral blood flow (CBF) and CBF regulation following mTBI. We performed a study investigating cerebrovascular reactivity (CVR) in high school athletes following mTBI to better understand the progression of cerebral hemodynamic recovery. Pre-injury baseline controls (n = 96 16.45 ± 1.25 years) and post injury (concussion) patients (n = 151 16.35 ± 0.91 years) were measured with TCD. The participants completed a CVR protocol which included both rest (normal breathing) and periods of hypercapnia (25 seconds of breath holding) in addition to the standard clinical exam. Concussions were diagnosed by physicians. TCD features included mean velocity, Pulsatility Index (PI) and CVR change were analyzed at (0 to 1 n = 9), (2 to 3 n = 10), (4 to 5 n = 18), (6 to 7 n = 23), (8 to 9 n = 16), (10 to 11 n = 22), (12 to 13 n = 16), (14 to 18 n = 16) and (19 to 30 n = 21) days following the initial injury. Cerebral hemodynamics of athletes with mTBI deviated significantly from controls, peaking between days 2 and 3 post injury (mean CVR of 55.7%), and returning to control levels on average 10 days following injury (mean CVR of 41.0%). This work shows that CBF and CBF regulation measured using TCD is a low cost, portable, and timely option as a biomarker for the management of mTBI recovery.
Keywords: mTBI, transcranial doppler, cerebral blood flow, longitudinal study
CHARACTERIZATION OF CUMULATIVE SUBCONCUSSIVE EXPOSURES OF BLUNT AND BLAST INJURY
New Jersey Inst. of Technology, Dept. of Biomedical Engineering, Newark, USA
Soldiers in training and operational environments are routinely exposed to either one or more low-level blunt or blast pressure wave impacts that might not prompt immediate medical attention. The effects of repeated exposure to mild blunt or blast related traumatic brain injury is not clearly known but has been identified to worsen the effects of single impact injuries in terms of neuronal damage and cognitive deficits. Furthermore, the outcome of repetitive subconcussive impacts or a combination of blunt and blast impact has not been extensively examined. The paucity of information on cumulative subconcussive impacts has led to little guidance in management of repetitive subconcussive TBI (scTBI) within a single day, a common occurrence among young soldiers. We present here a model of scTBI defined by the absence of acute behavioral markers and neuronal degeneration and applied repetitive paradigm. Wistar rats (21–25 day old) were subjected to repetitive lateral fluid percussion injury (FPI) at peak pressures 7.237 ± 0.38 or sham injury spaced at 5-minute intervals. Behavioral measures including apnea, righting time, toe-pinch reflex and seizures were assessed immediately after injury. Righting times did not differ between groups with repetitive injury peak pressure and sham (independent-samples t-test, t(20) = .737, p = .420) ScTBI group 1502.74 ± 21.33 and Sham, 1479.9 ± 22.25 n = 11 for each. However, 5 out of the 11 repetitive scTBI revealed qualitative Fluoro-Jade staining in the granule cell layer, corpus callosum and cortex. Although there was no hemorrhage or dentate hilar neuronal degeneration after single injury, rats with repetitive insults showed evidence of hemorrhage in the hippocampal fissure despite the absence of acute behavioral signs, while degenerating neurons marked by Fluoro-Jade were observed in 45% of the repetitive scTBI subjects. Thus, we have a model of scTBI in young rats and demonstrated that repetitive scTBI can lead to the brain hemorrhage and degenerating neurons. Future directions of our lab are to identify neurological correlates of combined low-level primary blast exposure and subconcussive blunt impacts. Soldiers in training and operational environments are routinely exposed to either one or more low-level blunt or blast pressure wave impacts that might not prompt immediate medical attention. The effects of repeated exposure to mild blunt or blast related traumatic brain injury is not clearly known but has been identified to worsen the effects of single impact injuries in terms of neuronal damage and cognitive deficits. Furthermore, the outcome of repetitive subconcussive impacts or a combination of blunt and blast impact has not been extensively examined. The paucity of information on cumulative subconcussive impacts has led to little guidance in management of repetitive subconcussive TBI (scTBI) within a single day, a common occurrence among young soldiers. We present here a model of scTBI defined by the absence of acute behavioral markers and neuronal degeneration and applied repetitive paradigm. Wistar rats (21–25 day old) were subjected to repetitive lateral fluid percussion injury (FPI) at peak pressures 7.237 ± 0.38 or sham injury spaced at 5-minute intervals. Behavioral measures including apnea, righting time, toe-pinch reflex and seizures were assessed immediately after injury. Righting times did not differ between groups with repetitive injury peak pressure and sham (independent-samples t-test, t(20) = .737, p = .420) ScTBI group 1502.74 ± 21.33 and Sham, 1479.9 ± 22.25 n = 11 for each. However, 5 out of the 11 repetitive scTBI revealed qualitative Fluoro-Jade staining in the granule cell layer, corpus callosum and cortex. Although there was no hemorrhage or dentate hilar neuronal degeneration after single injury, rats with repetitive insults showed evidence of hemorrhage in the hippocampal fissure despite the absence of acute behavioral signs, while degenerating neurons marked by Fluoro-Jade were observed in 45% of the repetitive scTBI subjects. Thus, we have a model of scTBI in young rats and demonstrated that repetitive scTBI can lead to the brain hemorrhage and degenerating neurons. Future directions of our lab are to identify neurological correlates of combined low-level primary blast exposure and subconcussive blunt impacts.
Keywords: fluid percussion injury, repetitive injury, mTBI, concussion
Traumatic brain injury (TBI) is a complex emerging epidemic. Concussion (or “mild” TBI) cases are extremely heterogenous and the mechanism of injury appears to be based on context. Many aspects of recovery remain unpredictable and accurate prognostic methods remain elusive. Here we present a systems-level dynamic model describing the pathophysiological mechanisms of concussion over multiple scales.
Keywords: concussion, mTBI, modeling, system dynamics, systems science
IMMATURE FRONTAL CONTROLLED CORTICAL IMPACT INJURY RESULTS IN SELECT DEFICITS IN NEUROCOGNITIVE ABILITIES
Traumatic brain injury (TBI) is the leading cause of death and disability in children. Currently, it is unknown how the brain handles injury before the frontal lobe is fully developed. While there has been investigation into the effects that frontal TBI has on neurocognitive abilities in the adult brain, the neurocognitive deficits resulting from injury to the juvenile brain are relatively unexplored. Further, little is known about what impact early frontal injury has upon the development of cognitive behaviors later in life. The current study was designed to investigate the effects of early frontal TBI on motor and cognitive abilities. Additionally, this study examined potential sex differences following early injury. At post-natal day (PND) 17, male and female juvenile rats received a bilateral frontal controlled cortical impact (CCI) or a sham surgery. The week following injury, motor behavior was assessed using the foot fault task. While injured males did not demonstrate significant impairments in motor behavior, injured females showed a significant increase in the number of foot faults. Female rats with CCIs had more foot faults than sham females. Cognitive abilities were tested using the Morris Water Maze (MWM; PND 30) and the Dig Task (PND 60+). Significance was found in the MWM for males and females, such that those that received frontal CCI demonstrated impaired learning and cognitive flexibility. For the Dig Task, results show that early TBIs can lead to cognitive deficits with respect to the time taken to make a decision, but not in the ability to make the correct discrimination. There was a significant difference between the males and females overall in the number of days taken to correctly discriminate in the new learning paradigm, but not in the cumulative proportion correct within that discrimination. From this study, it can be concluded that neurocognitive deficits occur later in life following TBI in the pediatric model. This includes impairments in tasks requiring spatial memory, and, to a lesser degree, those that require the use of impulsive decision-making strategies.
Keywords: juvenile, impulsivity, development, gender
VESTIBULAR AND VISUAL DYSFUNCTION FOLLOWING PEDIATRIC CONCUSSION (64)
The Children's Hospital of Philadelphia, Pediatrics/Orthopedic Surgery, Philadelphia, USA
Keywords: vestibular, oculomotor, vision, balance
Keywords: symptoms, symptom burden, adult, outcomes
BEHAVIORAL, BLOOD, AND ADVANCED MAGNETIC RESONANCE IMAGING BIOMARKERS OF EXPERIMENTAL CONCUSSION
The University of Melbourne, Parkville, Australia
Repetitive concussions may result in cumulative and chronic neurological consequences, and these effects may be due to the subsequent concussions occurring while the brain is in a period of increased vulnerability after the initial insult. Thus, the identification of markers that indicate when the brain is no longer in a state of increased vulnerability might allow them to be used to guide medical decisions, so as to reduce the effects of repeated concussion. The current management of concussion is based on assessing for the resolution of symptoms, which can be subjective and may not accurately indicate when the brain has recovered. Advanced MRI and blood-based biomarkers represent objective methods that may be more sensitive to the pathophysiological changes that occur in the concussed brain. Here we assessed the ability of multi-modal MRI, blood proteomics, and behavioral outcomes to detect changes and estimate recovery after an experimental concussion. Adult male rats were given either a sham injury or a mild fluid percussion injury (mFPI), and serial MRI, blood collection, and behavioral testing was performed at 1, 3, 5, 7, and 30 days post-injury. Plasma was analyzed using reverse phase protein arrays to assess markers sensitive to neuronal and glial cell loss, metabolic abnormalities, neuroinflammation, and axonal injury. MRI data was acquired using a 4.7T Bruker pre-clinical scanner, and analyses included structural, diffusion tensor imaging (DTI), tractography, and magnetic resonance spectroscopy (MRS). Behavioral analyses involved measures of cognition, sensorimotor function, anxiety, and depression. A mFPI induced transient cognitive abnormalities that persisted for 3 days post-injury, and sensorimotor impairments that persisted for 1 day post-injury. MRI (i.e., DTI, tractography and MRS) and blood biomarkers detected abnormalities that remained after the resolution of behavioral symptoms, some of which were still present ay day 30 post-injury. These findings indicate that MRI, blood, and behavior can detect changes induced by a mFPI. However, MRI and blood biomarkers may be more sensitive.
Keywords: MRI, DTI, MRS, behavior, proteomics
USAMRMC, Fort Detrick, USA
In this study we evaluated a number of neurotrauma biomarkers in serum as predictive aids for the diagnosis of concussion in a combat zone. This was an observational study conducted in Regional Command Southwest, Afghanistan with 233 U.S. military service member participants stratified into three cohorts: Concussed, Uninjured Control, and Injured (non-concussed) Control. There were two peripheral blood draws: one within 8 hours of injury (or upon enrollment for Controls) and a second approximately 24 hours later. Provider clinical diagnosis was supplemented by research assessment with the Sport Concussion Assessment Tool 2 (symptomology, postural stability, and cognitive performance). Results showed that within 8 hours following injury serum concentrations of Ubiquitin C-terminal Hydrolase-L1 (UCH-L1) were elevated for the Concussed cohort relative to Uninjured Control cohort [t(149) = 4.18, p < .001] and also to concentrations 24 hours following injury [t(70) = 5.51, p < .001]. Among other markers tested, none showed reliable elevation following injury. However, total tau and Interleukin 6 (IL-6) showed decline 24 hours following injury for the Concussed cohort [t(19) = 3.05, p = .007; t(45) = 2.65, p < .011; respectively] and no decline for Uninjured Controls, mirroring the post-injury decline observed for UCH-L1. Also, β-amyloid (Aβ)-42 showed lower serum concentrations for the Concussed cohort relative to Uninjured Control cohort within 8 hours of injury [t(112) = 2.18, p < .032] and remained low 24 hours following injury. Interestingly, the elevation of UCH-L1 within 8 hours of injury did not correlate with symptom severity (r = .10, p = .475) whereas concentrations of total tau and Aβ did correlate with symptom severity (r = .45, p < .001; r = -.24, p = .022; respectively). Concentrations of total tau also correlated with delayed recall performance (r = -.31, p = .018). These results are consistent with clinical studies in civilian settings. Because a combat zone can present unique conditions, such as injury from explosive blast, studies of this type are required to evaluate the comparability of findings reported in civilian medical research to military medicine.
Keywords: military, acute, serum, UCH-L1
University of Arizona, Department of Child Health, Phoenix, USA
Traumatic brain injury (TBI) is not a transient event from which all people recover; the resulting damage can evolve into neurological disease. In rats, TBI disrupts circuits, expressed as cognitive impairments and sensitivity to whisker stimulation. The current study sought to establish a rehabilitation task targeting somatosensory and cognitive functions. The task takes place in a box with a peg board floor that allows for 3” plastic pegs to be inserted at 1 inch intervals anywhere across the board. The configuration of pegs promotes whisker stimulation as rats explore the environment. To test feasibility of this approach, a pilot study was conducted by exposing rats to navigate through increasingly denser peg configurations as rehabilitation for TBI. Rats received sham or midline fluid percussion injury. Rats explored the peg-filled arena (or served as caged controls) and tested for whisker sensitivity and working memory. Brain-injured rats that received rehabilitation showed sensory sensitivity similar to shams, while injured rats without rehabilitation showed elevated sensitivity. When tested for working memory, injured controls showed significant memory impairments compared to shams (t(3) = 5.22, p < 0.05). Injured rats receiving rehabilitation performed similarly to shams and improved performance compared to injured controls (t(3) = 2.56, p = 0.051). This indicates that following a TBI, rehabilitation through a forest of pegs reduced whisker sensitivity and cognitive impairment. A power analysis on the pilot data indicated group sizes of 6 would have >90% power for detecting an effect size (0.79) with 0.05 significance criteria. Guided by this, a study is underway to determine the benefits of self-guided rehabilitation compared to open field exploration in sham and injured rats. Efficacy is evaluated on a battery of behavioral tests, with primary endpoints on cognitive performance evaluated in object recognition tasks. We approach rehabilitative treatments for TBI as amenable to clinical translation to improve quality of life. Supported, in part, by-the-Diane-and-Bruce-Halle-Foundation
Keywords: traumatic brain injury, Peg Forest, somatosensory sensitivity, object recognition, translational neurotrauma
University of British Columbia, Pathology & Laboratory Medicine, Vancouver, Canada
Keywords: APP/PS1 mouse model, aging, inflammation, white matter pathology
University of California San Francisco, Neurosurgery, San Francisco, USA
Patients that experience mild traumatic brain injury (mTBI) often report problems with concentration, learning, and memory. Cognitive problems can negatively affect the ability of mTBI patients to recover. Currently, streamlined identification of this at-risk population is limited. To better define subjective cognitive complaints (SCC) in mTBI, and to identify prognostic factors and relationship to objective cognitive performance, subjects were assessed at 6 months post-injury as part of the Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) Pilot study. SCC was defined as an affirmative response to any of 4 cognitive questions from a symptoms questionnaire. At 6 months post-injury, 60% of a total 284 subjects reported SCC. Multivariable logistic regression modeling demonstrated that the variables associated with increased risk of SCC at 6 months included pre-existing headache (RR 1.3, 95% CI [1.0–1.6]; p = 0.02), pre-existing psychiatric disease (RR 1.3, 95% CI [1.0–1.5]; p = 0.01), and TBI due to assault (RR 1.4, CI [1.2–1.7]; p < 0.01). Univariate regression models adjusted for age, gender, race, and years of education showed that subjects with SCC at 6 months performed worse on the trail making test and the California Verbal Learning Test than controls. SCC are common after mTBI and represent objective cognitive deficits. Use of a simple 4-question screen may identify patients at risk for poor cognitive recovery and direct treatment goals.
Keywords: traumatic brain injury, subjective complaints, cognitive outcomes, cognitive complaints
The majority of the estimated three million traumatic brain injuries (TBI) that occur each year are classified as “mild” and do not require surgical intervention. However, many people are subject to multiple head injuries that can lead to chronic pathological and behavioral consequences. We previously reported that repeated closed head injury (CHI) using a pneumatically controlled device with a silicone tip delivering a diffuse, midline impact directly onto the mouse skull caused acute regional microgliosis and cell death. At ten weeks after repeated injury, microgliosis and neurodegeneration were observed in the visual pathway. It is presumed that chronic microgliosis observed after TBI and in a number of neurodegenerative diseases contributes to the cascade of neurodegeneration. We hypothesized that the extent of mild TBI-related neuropathology would be proportional to the amount of microgliosis. An increased microglial response was previously reported in progranulin deficient mice (Grn-/-) after a brain-stab wound. To test our hypothesis Grn-/- and C57BL/6 mice were injured with five repeated CHI or sham injury at 24 h inter-injury intervals. Immediately following recovery from the first CHI, half of the Grn-/- cohort was provided rodent chow with 375 ppm ibuprofen for two weeks to mitigate injury-induced microgliosis. Histological analyses were performed on n = 4–5 sham/group and n = 9–10 injured/group to assess microgliosis, neurodegeneration, and axon loss 7 months after injury. Microgliosis was not increased in C57BL/6 mice 7 mo after injury. However, increased microgliosis was observed in the visual pathway of injured Grn-/- mice with and without acute ibuprofen treatment. Despite substantial differences in repeated CHI-induced microgliosis between C57BL/6 and Grn-/- mice, all groups showed similar amounts of repeated CHI-induced axonal injury and ongoing neurodegeneration in these same regions. These data suggest that microgliosis may not play a pivotal role in chronic neurodegeneration. Supported in part by NS087878-02.
Keywords: Progranulin, Ibuprofen
DELETERIOUS EFFECT OF HYPERHOMOCYSTEINEMIA ON SHORT-TERM MEMORY AFTER TRAUMATIC BRAIN INJURY
Traumatic brain injury (TBI) is a devastating public health problem worldwide accompanied with inflammation. Elevated blood level of Homocysteine (Hcy), called hyperhomocysteinemia (HHcy) is considered to be an independent and high risk factor for many cerebrovascular disorders. One of the important problems after TBI is a memory impairment, particularly loss of short-term memory. A greater role of cellular prion protein (PrPC) in cognition is well known. We demonstrated that PrPC deposition is increased after TBI along with enhanced formation of Fg-PrPC complex. We hypothesize that HHcy exacerbates the TBI-induced macromolecular protein leakage resulting in enhanced Fg-PrPC complex formation leading to the short-term memory reduction. Permeability of pial venules in pericontusional area formed after mild cortical contusion injury (CCI) was studied in wild-type (WT, C57BL/6J) and in mouse model of HHcy, cystathionine- β-synthase heterozygote (CBS+/−) mice. Venular permeability was assessed by measuring the extravascular leakage of Alexa-flour 647-labeled bovine serum albumin (647-BSA) in sham-operated or CCI mice using an intravital fluorescence microscopy. Immunohistochemistry on brain cryosections was used to assess deposition of Fg and Fg-PrPC complex formation. Short-term memory changes were evaluated by novel object recognition and Y maze (spontaneous alternation and two trial recognition) tests. Pial venular permeability to 647-BSA was greater in animals with CCI compared to that in sham-operated mice in all experimental groups. However, in CBS+/− mice protein leakage was greater than that in WT animals. Deposition of Fg was increased after TBI, and formation of Fg-PrPC complex was greater in CBS+/− mice compared to that in WT animals. The cognitive deficiency was developed in WT mice after TBI. In CBS+/− mice the short-term memory was also reduced. Our study reveals a novel effect of HHcy in TBI-induced memory impairment, which can be therapeutically targeted in future.
Supported in part by NIH grants P30 GM-103507, NS-084823, HL-108621, HL-74185.
Keywords: cerebrovascular permeability, hyperhomocysteinemia, fibrinogen, cellular prion protein, short-term memory
SPORT CONCUSSION ASSESSMENT TOOL-3RD EDITION: UTILITY TO DIAGNOSE CONCUSSION IN GENERAL POPULATION
University of Minnesota, Neurosurgery, Minneapolis, USA
Mild head injuries are a common cause of presentation to the emergency department (ED), where a standardized symptom assessment would streamline their care. Previous research has used SCAT-3 with an athlete population; however a pre-injury baseline for each individual is required. Here we present baseline normative data, and demonstrate the utility of the SCAT-3 in the evaluation of concussion in a non-athlete population. The SCAT3 was administered to 98 non-athlete controls, 46 non-head injured patients, 87 head-injured patients with negative CT, and 31 head-injured patients with a positive CT who presented to the ED. A classifier function was built to assess the utility of SCAT3 in discriminating head-injured versus controls. The control population had a mean of 2.30 (S.D. = 3.62) symptoms, 4.38 (S.D. = 8.73) symptom severity score (SSS), and 26.02 (S.D. = 2.52) standardized assessment of concussion score (SAC). There was no correlation with age or gender in the control population. Number of symptoms, SSS and SAC did differ significantly when compared across they four subgroups (Kruskal-Wallis test; p-values <0.001). The logistic regression based model achieved an AUC = 0.945. Patients were more likely to be diagnosed with a concussion if the SSS >7; or SSS <7 but with an SAC ≤22, (sensitivity of 96% and specificity of 77%). The SCAT-3 is a reasonable tool for screening a non-athlete population for concussions given its high sensitivity. Population data can be used as a baseline for trauma patients presenting to the ED after TBI. Patients with a SSS >7, or <7 but with a SAC ≤22 should undergo additional testing and follow-up.
Keywords: standardized assessment, SCAT-3, brain trauma, acute trauma
University of Pennsylvania, Neurosurgery, Philadelphia, USA
Each year in the United States, over 1.5 million people experience mild traumatic brain injury (mTBI) and recent studies suggest that mTBI is associated with long term physical and mental health problems. Functional and structural changes induced by mTBI may occur in the hippocampus, a structure crucial for learning and memory formation, with the hilar region showing particular vulnerability in preclinical models of TBI. Mossy cells, one of the predominant cells in the hilus, have been a focus of attention due to their potential role in post-TBI hyperexcitability and epileptogenesis. The objective of the current study was to assess mossy cell pathology and microglial changes after mTBI injury using an established model of closed-head rotational acceleration induced TBI in swine. Swine were subjected to sham conditions or rapid head rotation in the coronal plane, with some specimens undergoing in vivo electrophysiological recordings post-injury. Neuropathological changes in the hilar region of the hippocampus were assessed by labeling whole coronal sections of tissue using antibodies for MAP2, Synapsin, and IBA1. Using confocal microscopy, we measured the mossy cell somal area via MAP2, and visualized synaptic and microglial locations using Synapsin and IBA1 respectively. We observed microglial processes engulfing mossy cells in injured specimens, suggesting that microglia are recruited post-injury to remove disrupted synaptic components around mossy cells. Additionally, quantitative analysis of mossy cell somal area demonstrated an increase in somal area at 7 days post-injury in specimens with and without electrophysiological recordings compared to sham specimens with and without electrophysiological recordings. This alteration of mossy cell area paired with the increase of microglia processes around the cell demonstrates the vulnerability of hilar mossy cells after mTBI. Moreover, this hilar mossy cell pathology may play a role in aberrant hippocampal function post-TBI, potentially affecting dentate granule cells along the entire dorsoventral axis and enhancing perforant path inputs.
Keywords: large animal models, mossy cell, diffuse brain injury, synaptic changes
University of Rochester Medical Center, Emergency Medicine, Rochester, USA
Brain protein biomarkers like tau have been suggested as possible means to diagnose concussion and predict outcome. Prior research suggests that many proteins in the peripheral circulation are detectable in sweat, but this fluid has not been examined for brain proteins in the setting of sport-related concussion (SRC). The objective of this study was to pilot a novel method to collect, identify, and measure tau in sweat of athletes following SRC. NCAA contact-sport athletes were prospectively monitored for a SRC. Eight had a SRC and underwent serial dermal sweat patch sampling; patches were applied within 6-hours post-SRC and remained in place for 3-days, removed and replaced. This process was repeated for a maximum of 24-days. Eight non-concussed athletes served as controls and also underwent sampling. Sweat was extracted and total tau was measured using an ultrasensitive single-molecule ELISA. Tau was detected in all sweat samples. Concentrations ranged from 0.037–0.964 and 0.020–0.428 pg/mL in SRC and control athletes, respectively. None of the controls had a sweat tau value exceeding 0.428 pg/mL, whereas 4 of the SRC-athletes exceeded this threshold. Mean (SD) tau concentrations during the first post-SRC interval were nearly identical in the two groups (0.163 ± 0.058 vs. 0.148 ± 0.064 pg/mL; p = 0.80). By the second post-SRC interval, levels were over two times higher in SRC-athletes compared to controls (0.468 ± 0.301 vs. 0.221 ± 0.126 pg/mL; p = 0.23); the increase persisted into the third interval (0.419 ± 0.188 vs. 0.211 ± 0.149 pg/mL; p = 0.07). Comparing longitudinal patterns of sweat tau between the two groups over all time points revealed that concentrations were significantly higher among the concussed than control athletes (0.302 vs. 0.193 pg/mL; p = 0.03). Peripheral tau concentrations are detectible in sweat. That SRC-athletes, in this pilot study, seem to have higher levels than non-concussed athletes suggests that sweat tau might be a concussion biomarker worthy of future research efforts.
Keywords: tau, sports, sport related concussion, sweat
University of Washington, Neurological Surgery, Seattle, USA
Previous reports have identified high percentages of service members with poor outcome defined as moderate to severe disability on the GOS-E who sustain concussive TBI in combat. The current study explores the GOS-E subdomains and other clinical data collected at a 6–12 month evaluation post-injury in order to ascertain factors influencing this poor outcome in this mild brain injury population (n = 170 blast TBI). These patients are compared to combat deployed controls (n = 103) who screened negative for any history of major head injury at the time of enrollment and to blast exposed controls (n = 45) who screened negative for TBI history but endorsed prior blast exposure. Compared to both control groups, concussive TBI service members exhibited worse global disability, neurobehavioral impairment, post-traumatic stress disorder (PTSD) severity and depression severity (all p < 0.0001). Interestingly, even the blast exposed controls faired significantly worse on all the measures in comparison to their non-blast-exposed counterparts. Best fit logistic regression model of dichotomized GOS-E disability (7–8 good recovery, 6 or less poor outcome) revealed that poor outcome was largely driven by TBI diagnosis, evacuation status, depression, and PTSD severity, but not by neuropsychological performance, age, education, self-reported sleep deprivation, or injury mechanism (area under the curve = 0.8351). Poor disability outcome on the GOS-E was influenced primarily by psychological health symptoms, not cognitive function in combat concussive TBI. This finding has important implications for rehabilitative strategies in this service member population.
Keywords: clinical outcomes, combat concussion
Negative emotional states, or negative affect, resulting from concussion is of increasing concern in both civilian and military populations. Evidence of repeated concussion resulting in depression and anxiety is mounting. Studies currently used to test negative affect following concussion in animals, such as the elevated plus maze and Morris water maze, have yielded conflicting results. In the current study, we developed a model to investigate negative affect following multiple concussions induced by the projectile concussive injury (PCI) model. 22 kHz ultrasonic vocalizations are associated with negative affective stimuli in rats. Forty-eight hours post-injury, negative affect was examined using the air-puff induced negative vocalization test. Animals were placed into a clean acrylic box. A 5 min baseline recording was followed by 15 air puffs (55 psi) spaced 30 sec apart aimed at the upper back and neck. Recording continued until vocalization was terminated for 1 min. Total 22 kHz vocalizations, duration of vocalization, and threshold (number of puffs needed to elicit response) were significantly different between injured animals and shams (p < 0.05). Injured animals produced on average 153.5 ± 55.13 more vocalizations. Additionally, injured animals vocalized to fewer air-puffs, exhibiting a 1.5 fold lower threshold for the expression of negative affect. They also vocalized on average 4 min longer than shams, demonstrating that the same stimulus induced a more negative state in injured animals. Negative vocalizations are an indication of general negative affect and do not alone denote a specific type of negative state (e.g. sadness, anxiety, fear). However, it is likely that injured animals in the current study show greater fear and anxiety and perhaps have a lower threshold for negative environmental stimuli. Further studies are underway to examine these possibilities. Here we show that the air-puff vocalization test is an innovative, non-invasive, straightforward approach to assessing negative affect resulting from mild brain injury that can be utilized to evaluate novel therapies for the treatment of negative affect following concussive injury.
Keywords: ultrasonic vocalization, affective neuroscience
TIME-COURSE PROFILE OF EEG ABNORMALITY FOLLOWING REPEATED CONCUSSIVE BRAIN INJURY DETECTED BY QEEG POWER SPECTRAL ANALYSIS
Previously we demonstrated moderate EEG slowing following a single concussive brain injury in rats detected by quantitative EEG (qEEG) power spectral analysis. In this study we examined the time-course of EEG power shifts following repeated concussive injury. Rats received sham surgery or repeated projectile concussive impacts (rPCI), spaced apart by 1 h intervals, targeting the right frontal dorsal quadrant of the brain. Skull EEG electrodes were implanted immediately after the last PCI for continuous EEG recording for 14-days. qEEG power spectral analysis (60 sec epoch) was performed at 12, 24, 48, 72 h, and 7 and 14 days post-injury. The EEG relative power of delta (0–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), and beta (12–30 Hz) frequency bands was calculated for the left and right hemispheres at each time point for each rat, and expressed as the percent of the total power of the global frequency band (0–30 Hz). The qEEG power spectrum analysis revealed that rPCI injury caused moderate but significant EEG slowing within 24 h post-injury in the ipsilateral hemisphere, evidenced by increases in EEG delta power at 12 and 24 h post-injury (p < 0.05 vs. sham). A trend of EEG slowing was also measured between 48 and 72 h post rPCI. EEG slowing during the acute post-injury phase following rPCI appeared similar to what was previously observed following a single PCI. However, a trend showing increased alpha and beta activities was observed between 7th and 14th day after rPCI, which was not apparent after single impacts. In summary, although multiple concussive brain injury replicated previously reported early EEG slowing caused by single concussive brain injuries, the subtle delayed increase in EEG activities in the high frequency bands detected only following repeated concussions indicated the potential diagnostic value of qEEG in discriminating brain injury severity dynamics.
Keywords: EEG power spectrum analysis, repeated concussive brain injury
COMPARISON OF EMOTIONALITY BEHAVIORS IN THE ELEVATED PLUS MAZE: SENSITIVITY AND INDEPENDENCE
Medical College of Wisconsin, Neurosurgery, Milwaukee, USA
The focus of TBI research has moved from severe to mild injuries with the realization that repetitive low-level injuries contribute to chronic disability. Physiological changes and cognitive deficits are not as pronounced following mild TBI, where injury outcomes include changes in emotionality such as anxiety. Rodent models often assess emotionality changes using number of entries into or amount of time spent in open arms of the elevated plus maze (EPM). However, open arm entries are linked to locomotor activity (i.e., arm changes). A metric such as grooming duration may be an independent measure, more specific to emotionality changes. Grooming is a natural maintenance behavior indicative of stress dearousal. The MCW Rotational Acceleration Injury model produced mTBI in rats, with acute behavior assessed using the EPM. Compared to shams, rats receiving mTBI demonstrated increased (p < 0.05) total arm changes, open arm entries, and open arm duration. Injured rats also demonstrated decreased (p < 0.005) grooming behavior. As expected, total arm changes were strongly correlated to open arm entries (p < 0.05, R-squared = 0.73). Grooming behavior also demonstrated a negative correlation with total arm changes (p < 0.05), albeit with lower correlation (R-squared = 0.48). Interestingly, open arm time had lower correlations with total arm changes (p < 0.05, R-squared = 0.28) and open arm entries (p < 0.05, R-squared = 0.34), suggesting this anxiety metric was less influenced by locomotor activity. Of the EPM anxiety metrics, we found that open arm and grooming duration had the lowest association with overall locomotor activity. However, due to the large effect size, grooming behavior (Hedges' g = 2.33) may be more sensitive to emotionality changes than open arm duration (Hedges' g = 1.56). These results indicate that mTBI-treated rats had higher EPM locomotor response accompanied by increased open arm duration and reduced grooming behavior, indicating reduced anxiety-like behavior and/or behavioral disinhibition.
Keywords: rotational acceleration, elevated plus maze, sports concussion, anxiety
Texas A&M Health Science Center, Neuroscience and Experimental Therapeutics, Bryan, USA
Previously, we have shown that, following spinal cord injury (SCI), a subset of rats exhibit depression-like behaviors. These subjects display decreased sucrose preference, open field activity, and social exploration, and increased immobility on the forced swim test. These behaviors are thought to reflect depressive symptoms (i.e. decreased interest or ability to experience pleasure, psychomotor retardation, fatigue) analogous to those of human patients. In this experiment, we extend these findings and examine the relationship between depression-like behavior and physiological function. Subjects received a moderate contusion or sham injury and were assessed for depression-like behavior on days 9–10, 19–20, and 29–30 post SCI, with a battery of established behavioral tests (sucrose preference, social exploration, open field activity, burrowing activity, and appetite deviation). Using implantable telemetry devices, home cage activity, body temperature, heart rate and heart rate variability were collected for five-minute intervals every hour throughout the duration of the experiment. Locomotor performance (BBB scale) and measures of pain reactivity (girdle, tactile, and tail flick tests) were also assessed throughout the recovery period. Using principal components and hierarchical cluster analyses, subjects were classified into two groups: exhibiting behaviors indicative of depression or no depression. The depressed and not-depressed groups displayed significant differences in both behavior and physiological function. Subjects characterized as depressed had significantly higher heart rates, and decreased heart rate variability. Body temperature and home cage activity did not differ between depressed and not-depressed SCI subjects, although both groups displayed lower activity levels than the intact controls. Here we replicate our previous study, showing depression-like behavior in a subset of rodents after SCI. Moreover in addition to behavior, depressed and not-depressed subjects differ on measures of physiological function that are also associated with depression in humans. These physiological differences further validate the rodent model of depression after SCI. Research support: Grant DA031197 to M.A. Hook.
Keywords: spinal cord injury, depression, behavior, telemetry, rodent
Columbia University, Biomedical Engineering Department, New York, USA
We propose a new conceptual framework for understanding the etiology of edema formation after traumatic brain injury (TBI) and a novel therapeutic approach for reducing it. The “Donnan effect” describes the tendency of a porous and negatively-charged matrix to generate an osmotic gradient that attracts positive ions, and water, into the matrix—causing the matrix to swell. In the brain, hyaluronan and glycosaminoglycans (GAGs) are large, fixed, negatively-charged matrix molecules that contribute to the fixed charge density (FCD) of the tissue. We hypothesized that hyaluronidase, a hyaluronan-degrading enzyme, should reduce the FCD and reduce edema formation after TBI. First, using an in vitro swelling assay, we determined whether hyaluronidase could prevent swelling of cortical explant tissue. Compared to 24 h incubation in Gey's solution, incubation in 1 U/ml hyaluronidase reduced water content by 1.5% (p < 0.05) as measured by the wet-weight/dry-weight method. Enzyme activity was confirmed using the dimethylmethylene blue (DMMB) assay to measure GAG content, which was reduced in hyaluronidase-treated explants compared to control (p < 0.05). Next, to test if hyaluronidase could reduce edema after TBI, we exposed mice to controlled cortical impact (CCI) or sham injury and administered hyaluronidase (1 U/10 μl) or sterile PBS via intracerebroventricular injection on the contralateral side 2–4 min after injury. CCI increased water content in the ipsilateral hippocampus of vehicle-treated mice at 24 h by 4% (80.4%; p < 0.001 vs. sham veh; 77.4%), but hyaluronidase reduced this by 2.8% (78.2%; p < 0.01 vs. TBI veh) so that hyaluronidase-treated mice were similar to shams. Reduction of the FCD was indicated by reduced GAG content in hyaluronidase-treated vs. non-treated CCI brains (p < 0.05). Hyaluronidase did not alter blood brain barrier integrity in sham or CCI mice as measured by Evan's blue extravasation. We provide evidence that the Donnan effect helps explain edema formation after TBI and demonstrate that reducing the FCD with hyaluronidase reduces edema after TBI in vivo.
Keywords: edema, controlled cortical impact, biomechanics, extracellular matrix, hyaluronan, glycosaminoglycans
Massachusetts General Hospital, Neurology, Boston, USA
Recovery of consciousness after traumatic coma is associated with increased alpha and reduced delta frequencies on electroencephalogram (EEG). However, the cortical network properties that underlie these changes are unknown. In this prospective longitudinal study, we focused on small-worldness - the ratio of modular clustering to long-range connections within a network - as small-worldness is a known property of cortical networks in conscious humans. We hypothesized that patients who recover consciousness after traumatic coma show an increase in cortical network small-worldness, and that this correlates with improvement on the Coma Recovery Scale-Revised (CRS-R) score at follow-up. We enrolled 7 patients with traumatic coma (3F; mean age 24 ± 4 years) admitted to the intensive care unit. Patients underwent EEG and CRS-R evaluation at 9 ± 5 days post-injury and follow-up EEG/CRS-R at median day 235 [range 174–656]. For each time point, we calculated maximal cross-correlation between all electrode pairs for each second during five minutes of rest and averaged the resulting networks in order to measure clustering coefficient C, characteristic path length L, and small-worldness (SW = C/L). We used a Wilcoxon signed-rank test to compare network properties between acute and follow-up EEG and a Spearman test to estimate correlations between longitudinal changes in network small-worldness and CRS-R score. Small-worldness significantly increased between acute and follow-up EEG (p = 0.03). Both network properties that comprise small-worldness (C and L) also increased significantly (p = 0.02 and p = 0.03, respectively). At follow-up, all subjects attained a maximum score of 23 on the CRS-R (mean acute-to-chronic increase in CRS-R = 11 ± 10). We did not observe a correlation between longitudinal changes in network measures and CRS-R improvement. Our results suggest that increased cortical small-worldness is associated with recovery of consciousness after traumatic coma. Based on these results, EEG-based network measures may provide quantitative biomarkers of cortical activity, and hence recovery of consciousness, after traumatic coma.
Keywords: consciousness, coma, EEG, networks
Keywords: electroencephalography, microdialysis, percent alpha, depth EEG, cerebral metabolic crisis
ROLE OF VASOPRESSIN IN THE DEVELOPMENT OF POLYURIA AFTER ACUTE SPINAL CORD INJURY
University of Louisville, Department of Anatomical Sciences and Neurobiology, Louisville, USA
Spinal cord injury (SCI) often leads to bladder dysfunction which severely impacts patient morbidity and quality of life. Improvements in bladder function is a top priority for SCI individuals, however research in this area is very limited. Polyuria (the increase production of and/or passage of urine) is often seen following SCI and leads to more frequent bladder catheterizations and disruptions in sleep and daily activities. The mechanisms that underlie the development and maintenance of polyuria after SCI are unknown. One of the major hormones responsible for the regulation of water balance is vasopressin, with a decrease in vasopressin levels leading to increased urine production. Two weeks after a T8 spinal contusion injury (215 kd; adult male Wistar rats), 24 hour urine output significantly increased compared to preinjury baseline levels. Urinalysis revealed that the specific gravity and creatinine levels were significantly decreased at two weeks post-SCI, indicating that the urine was less concentrated. At this same time point, basal levels of vasopressin also decreased significantly. Despite this decrease in vasopressin levels, blood osmolarity (the strongest stimulus that controls circulating vasopressin levels) did not change. To determine how SCI could lead to a decrease in vasopressin, corticosterone levels were measured as an increase in corticosterone is known to suppress vasopressin release. At two weeks following SCI, corticosterone levels increased significantly. Thus it appears that several factors contribute to the development of polyuria after SCI. Having identified that vasopressin levels decrease after SCI and a mechanism that can lead to this decrease, we can now use these targets to develop interventions that may decrease polyuria within the SCI community and thereby improve the quality of life for SCI individuals.
Keywords: bladder, urogenital system
Excess cortisol is associated with cognitive dysfunction, possibly via hippocampal and frontal lobe neurotoxicity; however the relationship between cortisol levels and cognitive outcomes after traumatic brain injury (TBI) has not been well-described. The objective of this study was to test the hypothesis that acute serum cortisol levels (days 0–6) after moderate-to-severe TBI in adults correlate with cognitive testing measures at 6 and 12 months post-injury. Outcome measures at 6 and 12 months assessed attention (Trail Making Test A and Digit Span sub-test of Wechsler Adult Intelligence Scale-R), language fluency (Controlled Oral Word Association test and Delis-Kaplan Executive Function Systems Verbal Fluency test), memory (Rey-Osterreith Complex Figure Task and California Verbal Learning Test), and executive function (Trail Making Test B and Stroop Color/Word Test). Individual test scores were aggregated to produce one Cognitive Composite score for each individual. The Functional Independence Measure was also assessed, and the cognition-centered subscales were used as a measure of functional cognition. We included 66 individuals with 6 month outcome data; 51 individuals had 12 month outcome data. At 6 months post-TBI, mean acute cortisol serum levels negatively correlated with Cognitive Composite score (r = -0.360, p = 0.003), including significant correlations with language fluency, memory, and executive function performance (p < 0.05 all comparisons). Functional cognition also negatively correlated with mean acute cortisol levels (r = -0.259, p = 0.036). At 12 months, mean acute cortisol levels were significantly associated with language fluency (r = -0.301, p = 0.032) but not Cognitive Composite score or other cognitive domains assessed. Our data suggest that elevated acute cortisol serum levels are associated with functionally significant cognitive deficits during the first 6 months post-injury. However, this association is attenuated by 12 months post-injury, suggesting a non-cortisol dependent recovery process in this timeframe. Acute serum cortisol levels appear to play a role in and are a marker of cognitive function through the first 6 to 12 months post-TBI.
Supported by NIDILRR 90DP0041; DoD W81XWH-071-0701.
Keywords: cortisol, cognition, rehabilomics
SYNERGISTIC EFFECTS OF PHENYTOIN AND LEVETIRACETAM COMBINATION THERAPY ON ATTENUATION POST-TRAUMATIC NONCONVULSIVE SEIZURES
Walter Reed Army Institute of Research, Silver Spring, USA
Post-traumatic seizures during the emergent state of traumatic brain injury (TBI) are deemed detrimental to the recovery of TBI patients. Phenytoin (PHT) and levetiracetam (LEV) are the commonly prescribed drugs as prophylaxis. Although LEV has become more favorable than PHT owing to its improved safety profile, many patients are still intractable to the monotherapy of either drug. In this study we applied the isobolographic approach to evaluate PHT+LEV combination therapy against penetrating ballistic-like brain injury (PBBI) induced nonconvulsive seizures (NCS) in rats, aimed at achieving synergism with properly paired dose ratios of the two drugs. NCS were induced by PBBI and detected by EEG recording for 72 h. PHT and LEV were tested at the following dose ratios (PHT/LEV): 1.8/6.3, 3.6/12.7, 7.2/25.4, or 14.5/50.7 mg/kg, which were derived from previous monotherapy results of each drug. Treatments were given intravenously twice/day for three days. Control animals received matching vehicle treatments. The results showed that except for the lowest dose ratio, PHT+LEV combination therapy significantly reduced NCS frequency and total seizure duration by 61%–84% across the three high dose ratios (as compared to vehicle treatments). The highest dose ratio also significantly reduced NCS incidence from 65% (Vehicle) to 25% and delayed NCS latency from 39 h (vehicle) to 57 h post-injury. The dose equivalent analysis of the isobolographic design indicated an achievement of synergism because the observed effects across all dose ratios exceeded the expected additive effects. Compared to our previous results of a PHT or LEV monotherapy derived from the identical TBI, the combined treatments of PHT+LEV out-performed the monotherapy of each individual drug by achieving statistically defined synergism. Critically, the doses of each drug used in combination were 2–3 fold lower than the monotherapy doses required of each drug, which may alleviate the safety concerns of these drugs used in TBI patients.
Keywords: post-traumatic nonconvulsive seizures, phenytoin, levetiracetam, combination therapy, isobolographic analysis, synergism
The post-TBI metabolic depression impairs the ability of neuronal circuits to comply with activity demands, limiting the success of rehabilitative strategies, particularly during the acute period of TBI, which is critical for athletes to return to play, or patients to engage in rehabilitation programs. Although most neurons survive mild or moderate TBI, they cannot operate efficiently, and this severely compromises brain function. Our paradigm is based on the concept that TBI disrupts brain functional connectivity (fc), and that synaptic function and cell metabolism are underlying substrates of fc. We capitalize on the enhancing action of flavonoid derivative, 7,8-dihydroxyflavone (7,8-DHF, BDNF analog) on cell metabolism and synaptic plasticity to restore fc. We performed moderate fluid percussion injury (FPI) and 7,8-DHF (5 mg/kg, ip) was administered in animals receiving FPI with or without exposure to voluntary exercise. TBI resulted in disturbances in energy homeostasis markers (PGC-1α, COX II) and plasticity/growth (GAP-43, synaptophysin), concurrent with reductions in memory in Barnes maze. Treatment with 7,8-DHF or exercise ameliorated impairments in cognitive function and energy homeostasis, and enhanced the activation of TrkB BDNF receptor signaling. The combined action of exercise and 7,8-DHF resulted in elevated values in most of the variables. Resting state functional MRI (rsfMRI) data acquired from the same groups to monitor circuit-level changes associated with functional brain reorganization showed that the action of 7,8-DHF improved brain circuit connectivity (nodal connection strength) compared to FPI alone, where it is was reduced compared to sham animals. Exercise and 7,8-DHF also normalized connectivity. These data show that 7,8-DHF can render as a prototype molecule that links metabolism and synaptic plasticity, with the potential to normalize functional brain connectivity crucial to enhance the effects of training on TBI pathology. (supported by NIH R01NS50465).
Keywords: metabolism, flavonoid, connectome, synaptic plasticity
KNOCKOUT OF THE NOX2 ENZYME IMPROVES FUNCTION AND REDUCES ACUTE INFLAMMATION AFTER SPINAL CORD INJURY
Spinal cord injury (SCI) affects approximately 12,000 people each year in the United States; there currently is no approved therapy to improve functional loss after injury. Reactive oxygen species (ROS) have been implicated as a contributing factor of this impaired function and elevated histopathology. The NADPH oxidase (NOX) enzyme is a key producer of ROS. Previously, we showed that a temporary inhibition of NOX2 by an intrathecal administration of gp91ds-tat immediately after impact improved recovery in a mouse SCI model. However, chronic inflammation and oxidative stress were not affected by this single acute inhibition of NOX2. Therefore, we aimed to explore the effect of constant inhibition of NOX2 by investigating recovery after SCI in a NOX2 knockout (KO) mouse model. A moderate SCI contusion injury was performed in 3 month old KO and wild-type (WT) mice. Outcomes measured included locomotor function scored using the Basso Mouse Scale (BMS) and assessment of inflammation, polarization and oxidative stress markers using immunohistochemistry and western blotting. NOX2 KO mice demonstrated significantly improved BMS scores at 7, 14, and 28 days post injury (DPI) in comparison to WT mice. ROS, as measured by oxidative stress damage to spinal cord tissue, was significantly reduced at both sub-acute and chronic periods in KO mice in comparison to WT mice. Furthermore, a shift in microglial polarization toward the neuroprotective M2 phenotype was observed in KO mice at 7 days post injury. However, by 28 days post-injury, no significant difference in inflammation or microglial polarization was noted between groups. Thus, we now show that NOX2 KO enables a sub-acute neuroprotective shift in microglial phenotype that may contribute to improved long-term recovery but does not affect long-term inflammatory responses in the injured spinal cord.
Keywords: reactive oxygen species, NADPH oxidase, polarization, NOX2
LONGITUDINAL GENE EXPRESSION ANALYSIS REVEALS SYSTEMS-LEVEL PERSPECTIVE OF TBI
University of California, Davis, Department of Neurological Surgery, Davis, USA
TBI results in multiple concurring pathologies including neuroinflammation, apoptosis, and altered neurotransmission. We propose that measurement of global transcriptional changes can provide a comprehensive assessment of processes post-TBI, and importantly, evaluate the effects of interventions to improve TBI outcome across multiple systems. In the following study, ipsilateral rat hippocampus was isolated at 1 and 14 days following lateral fluid percussion or sham injury and processed for RNA sequencing. After alignment and transcript abundance estimation, differentially expressed genes were identified in injured brains relative to sham, at a false discovery rate <0.05. Dynamic expression changes were observed, with 1071 genes significantly different at 1 day-post injury, 1275 genes at 14 days post-injury, and 391 common to both time points. Gene co-expression modeling and functional enrichment analysis identified perturbed signaling pathways and molecular signatures, in which representative candidates were confirmed by immunohistology. Data included inflammatory genes (e.g. IL-1beta), apoptotic genes (e.g. caspase-8) and metabolic genes (e.g. hexokinase 3). While many biological networks were implicated across time points, unique to 14 days post-injury was a widespread decrease in transcript abundance of genes related to neurotransmitter receptors and ion channels, an effect larger than expected by cell loss alone. Interestingly, many of these receptors (e.g. nAch receptor α7; serotonin receptor 7; histamine receptor 3) are also key for learning and memory, processes highly relevant to the hippocampus and well documented in spatial learning deficits in injured rodents. Moreover, there was increased transcription of genes involved in coagulation cascades as well as a reduction in genes responsible for steroid synthesis, perhaps reflecting reparative processes at this later stage. These data demonstrate how genomics approaches can be used to assay changes across cellular and pathological systems following TBI. Future work includes applying this systems-level approach to further characterize TBI pathology and the global effects of specific interventions.
Keywords: RNA sequencing, hippocampus, differential expression
Support: These studies were completed as part of an interdisciplinary research team funded by the Moody Project for Translational Traumatic Brain Injury
Keywords: chronic TBI, genomic
YING-YANG OF BDNF: KCC2 EXPRESSIONAL CHANGE REGULATE THE EFFECT OF BDNF BEFORE AND AFTER SPINAL CORD INJURY (SCI)
Texas A&M University, Neuroscience, College Station, USA
In spinally transected rats, prior work has shown that intrathecal (i.t.) administration of BDNF has a beneficial effect on spinal cord plasticity that promotes adaptive learning and blocks the learning impairment and enhanced mechanical reactivity (EMR) observed after shock or peripheral capsaicin treatment. In contrast, in intact rats BDNF is thought to enhance the development of spinally-mediated central sensitization, which would enhance capsaicin-induced EMR. To evaluate the differential effects of BDNF within the spinal cord, we compared the effects of BDNF (i.t.) treatment in intact rats and spinal injured rats. Subjects received a complete spinal transection at T2 or sham operation. All subjects then had an i.t. cannula placed along the dorsal surface above the lumbar enlargement for drug delivery. Twenty-four hours later, subjects received either saline or BDNF followed by peripheral treatment with capsaicin. In intact subjects, BDNF enhanced capsaicin-induced EMR. In contrast, BDNF blocked capsaicin-induced EMR in transected rats. BDNF treatment also had opposite effects on a cellular marker (ERK phosphorylation) of central sensitization. These findings show that SCI alters how BDNF affects nociceptive processing within the spinal cord.
BDNF is known to affect KCC2 expression, a Cl- transporter, which regulates GABAergic function. We hypothesized that SCI could alter how BDNF affects membrane-bound KCC2 expression. To examine this possibility, we examined KCC2 expression in BDNF treated intact and spinally transected rats. We found that BDNF up-regulated KCC2 expression in transected rats, but down-regulated it in intact rats. We also found that BDNF had opposing effects on the inhibitory action of muscimol, a GABAA antagonist; in uninjured rats, BDNF reduced muscimol's inhibiting effect, whereas it enhanced inhibition in transected rats. These results suggest that SCI alters how BDNF affects nociceptive processing within the spinal cord and that this is due in part to how it affects GABA function.
Supported by the Neilsen Foundation and NIH (NS091723)
Keywords: BDNF, GABA, KCC2, SCI, spinal cord injury, allodynia
Massachusetts General Hospital, Neuroradiology, Boston, USA
Hematoma expansion within the early hours after intracranial hemorrhage (ICH) is among the most important predictors of neurological deterioration. The “spot sign,” presence of active leakage of iodinated contrast from the vessels, has been shown to be an indicator of ongoing bleeding, and thus, an accurate and powerful predictor of hematoma expansion. In this pilot study, we sought to develop and validate a new methodology using Dual Energy CT (DECT) to predict ICH expansion. We reviewed all consecutive head DECT angiograms performed in our emergency department since October 2014, and included those cases that had at least one DECTA with a delayed phase acquisition for iodine measurement and a follow-up head CT to assess hematoma expansion (N = 16). We later developed an automated algorithm to identify the hematoma ROI on virtual non-contrast image series, to replicate the same ROI to iodine image series, to statistically identify spot signs, and to assess relative iodine concentration in the hematoma and in spot signs to determine extravasations. Follow-up CT scans showed hematoma expansion in 4/16 patients. Hematoma volume was significantly larger in expanders (60 ± 17 vs 23 ± 13 cm3; p = 0.03). Our automated algorithm correctly identified all 4/4 patients with and 9/12 without hematoma expansion (100% sensitivity, 75% specificity). The volume of the false positive “spot signs” were substantially smaller compared to true positives (2 ± 3 vs 96 ± 43 mm3; p < 0.01), and their relative iodine content was markedly lower (143 ± 25 vs 48 ± 44 in the spot sign; p = 0.06). Thus, DECT of the head can detect early extravasation of iodine contrast, and our automated detection algorithm for spot signs as a surrogate marker can provide an accurate predictor of hematoma expansion in ICH.
Keywords: intracranial hemorrhage, dual energy computed tomography, hematoma expansion, spot sign
Traumatic spinal cord injuries are often accompanied by secondary injuries that complicate the recovery process. Further, nociceptive stimulation appears to increase the amount of red blood cells at the lesion site, which may indicate compromised vasculature. Progressive hemorrhagic necrosis (PHN), a phenomenon originating from the inflammatory environment induced by the initial injury, leads to the death of endothelial cells at the lesion site and hemorrhage. The present study investigated the impact of nociceptive stimulation on the amount of lesion-site hemorrhage, a hallmark of PHN. Adult rats received a laminectomy and a moderate contusion injury at the T12 vertebra. After a 24-hour recovery period, half of the subjects received noxious input in the form of electrical shock to the tail. Three hours post-shock, subjects were sacrificed and a 1-cm section of tissue around the lesion site was collected and sectioned. Following H&E staining, the amount of hemorrhage was quantified as a percentage of the total section area. Subjects that received shock had more hemorrhage at and around the lesion site, compared to the unshocked group. The amount of hemorrhage peaked at the lesion epicenter and decreased with distance in both groups. Additionally, the greatest difference in hemorrhage between the two groups was found rostral to the lesion. Based on these findings, PHN may play a role in the detrimental effect of noxious input on recovery after SCI. Future work includes examination of the effect of noxious input on capillary segmentation, another hallmark of PHN, and further investigation into the cellular mechanisms behind the effect.
Keywords: progressive hemorrhagic necrosis, nociception, contusion, immunohistochemistry
AGE-DEPENDENT EFFECTS OF HAPTOGLOBIN DELETION IN NEUROBEHAVIORAL AND ANATOMICAL OUTCOMES FOLLOWING TRAUMATIC BRAIN INJURY
University of Florida, Anesthesiology, Gainesville, USA
Cerebral hemorrhages are common features of TBI and their presence are associated with chronic disabilities. Recent clinical and experimental evidenced suggest that haptoglobin (Hp), an endogenous hemoglobin-binding protein most abundant in blood plasma, would be involved in the intrinsic molecular defensive mechanism; though, its role in TBI is poorly understood. The aim of this study was to investigate the effects of haptoglobin deletion on the anatomical and behavioral outcomes in controlled cortical impact model using wildtype (WT) C57BL/6 mice and genetically modified mice lacking Hp gene (Hp-/-) in two age cohorts [(2–4 mo old (young adult) and 7–8 mo old (older adult)]. The data obtained suggest age-dependent significant effects on the behavioral and anatomical TBI outcomes and recovery from the injury. Moreover, in the adult cohort, neurological deficits of Hp-/- mice at 24 h were significantly improved as compared to WT; whereas, there were no significant differences in brain pathology between these genotypes. In contrast, in the older adult cohort, Hp-/- mice had significantly larger lesion volumes compared to WT, but neurological deficits were not significantly different. Immunohistochemistry for ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) did not reveal significant differences in microglial and astrocytic reactivity between Hp-/- and WT mice in both age cohorts. In conclusion, the data obtained in the study provide clarification on the age-dependent aspects of the intrinsic defensive mechanisms involving Hp that might be involved in complex pathways differentially affecting acute brain trauma outcomes.
Keywords: controlled cortical impact, hemorrhage, Iba1, GFAP, gliosis, hemoglobin
Heme Oxygenase-1 (HO-1) is rapidly induced following traumatic brain injury (TBI), mediating breakdown of erythrocyte released heme and generating potentially cytotoxic molecules CO, Biliverdin/ Bilirubin, and Fe. Since rise in HO-1 expression is time dependent relative to the extent of heme accumulation and is driven by oxidant induced transcription factor Nrf2 (known to upregulate cytotoxic buffering proteins) we hypothesized that acute postinjury activation of Nrf2 could preemptively drive HO-1 elevation for more rapid clearance of heme, add protective buffering and improve behavioral outcome post-TBI. Using the male rat central fluid percussion model of TBI, we delivered Nrf2 inducer sulforaphane or hemin (FDA approved compounds) activating Nrf2 gene transcription during early postinjury intervals, testing for effects on cortical HO-1 expression, heme processing and motor behavior. Results show both compounds elevate cortical Nrf2 in a dose dependent manner. Further, acute post-injury delivery of either sulforaphane or hemin reduces the time dependent HO-1 peak expression within the cortex of 3d survivors approximately 70% relative to untreated cases. Notably, histological analysis showed advanced loss of heme pigment within injured areas of treated animals, with evidence of fully processed heme (identified as pale yellow bilirubin) as early as 1 day post injury. This facilitated cortical heme processing and reduced accumulation correlated with significant improvement in rotarod motor behavioral performance 1–3d post-injury relative to untreated injured cases. Paired confocal analysis after sulforaphane/hemin treatment shows altered patterns of cellular HO-1 expression after TBI. HO-1 and LCN2 (an iron sequestering protein) expression within reactive glia was reduced surrounding identifiable hemorrhagic/necrotic sites, suggesting a narrowing of the injury penumbral field. Together, these results suggest Nrf2 manipulation of HO-1 and antioxidant gene transcription may provide a means to proactively clear heme-related toxins post-TBI and improve both structural and functional outcome. While current motor behavioral results are associated with brain regions known to experience hemorrhage in our TBI model, we propose that our parallel observations of HO-1 activation in the hippocampus indicate that post-injury Nrf2 gene transcription modulation may also improve recovery in a non-hemorrhagic tissue.
NIH NS056247 & NS057758
Keywords: traumatic brain injury, heme oxygenase 1, Nrf2, hemin, sulforaphane
EXAMINING ANIMAL SURVIVABILITY FOLLOWING STRESS, ACUTE LOW LEVEL BLAST EXPOSURE, AND HYPOBARIC CONDITIONS
In combat, service members undergo stress and physical injury such as from blast overpressure. Therefore, service members are often medically evacuated via airplane to receive medical care. During medical evacuation, there is a risk of exposure to hypobaric conditions that may have a detrimental effect on cardiovascular physiology, which may also be impaired from battlefield stress and injuries from pressure blast exposure. Therefore, there is a need to assess which physiological measures are at greatest risk for impairment and survivability after a blast injury and during hypobaric conditions. To examine these effects, rats were exposure to 45-minutes of restraint stress and then a blood draw was taken from a femoral artery catheter to assess changes in cardiovascular (i.e., mean atrial pressure) and physiological parameters (i.e., electrolytes, coagulation factors). Then, using a shock tube, rats were exposed to a 75 KPa blast followed by a 35% blood volume hemorrhage. Fifteen minutes after hemorrhage, rats received saline as resuscitation fluid. Finally, animals underwent a simulated flight (within a hypobaric chamber) for 3 hours and were then euthanized. Blood draws from the femoral artery were again taken after blast exposure and after flight to assess changes in cardiovascular and physiological parameters. Pressure blast hemorrhagic shock injury and restraint stress were significant predictors of survival, whereas the hypobaric flight condition was not. Physiological parameters such as hematology cell counts (i.e., hematocrit), and the blood chemistry measures (i.e., creatin kinease) were all significantly associated with stress and injury. These results suggest that hypobaric conditions are not as much of concern for survival, but rather physiological impairments of injuries involving blood loss combined with stress may pose greater risk to warfighters.
Keywords: mild TBI, stress, hypobaric, survival
DTI ABNORMALITIES IN THE CLOSED HEAD INJURY MODEL OF ENGINEERED ROTATIONAL ACCELERATION (CHIMERA) MOUSE MODEL
Henry M. Jackson Foundation for the Advancement of Military Medicine, CNRM, Rockville, USA
Acceleration and rotation causing deformation and contusion of the brain within the skull accompanies most traumatic brain injury (TBI) incidents. These forces cause diffuse axonal injury (DAI), a hallmark TBI pathology. Recently, the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA) was developed to provide an ecologically relevant injury that generates white matter damage closely resembling human DAI. Diffusion MRI has the capability to detect abnormalities that may be invisible to anatomical MRI. In particular, diffusion tensor imaging (DTI) can probe the microstructure of the brain by measuring the magnitude and shape of water diffusion. DTI metrics Trace and Fractional Anisotropy (FA) provide tools to identify axonal abnormalities. Further, non-Gaussian diffusion MRI approaches including mean apparent propagator (MAP)-MRI offer new parameters that may be more specific for DAI pathomechanisms such as axonal damage, demyelination and glial reactivity. Ex vivo imaging of CHIMERA mouse brains were directly compared to histopathology, using silver stain to identify degenerating tracts, and immunohistochemistry with antibodies to glial fibrillary acid protein (GFAP), Iba-1, Beta-amyloid precursor protein (B-APP), myelin basic protein (MBP), and neurofilament (NF). Optic tract, anterior corpus callosum, and the brachium of the superior colliculus were among the regions that showed both DTI and histopathologic abnormalities. Regions of reduced FA showed abundant silver stain, increased Iba-1 and GFAP immunoreactivity, and accumulations of NF indicative of axonal damage. Regions positive for neurodegeneration and increased neuroinflammation were carefully investigated using MAP-MRI. This study elucidates metrics of DTI that correspond with regions of DAI pathophysiology.
Keywords: DTI, animal model, CHIMERA, diffuse axonal injury, diffusion MRI
Keywords: MRI, CT, neuroimaging, management, emergency department, intensive care unit
SENSITIVITY COMPARISON BETWEEN GLUCOCEST, FDG-PET AND 2DG-AUTORADIOGRAPHY IN MEASURING GLUCOSE METABOLIC DISORDER IN MILD TBI
Keywords: glucoCEST, FDG-PET, 2DG Autoradiography, diffuse axonal injury, diffusion tensor imaging
ISCHEMIA AND LATE BLOOD BRAIN BARRIER DISRUPTION DURING EVOLUTION OF WHITE MATTER MICROVASCULAR INJURY
NIH/NINDS, Bethesda, USA
The presence of post-traumatic microbleeds in white matter on MRI has traditionally been considered a marker of diffuse axonal injury. These microbleeds may have a linear and/or branching appearance, suggesting vascular injury. We present an index case from our observational study demonstrating the evolution of microbleed-associated injury from hyperacute (< 6 hours) to early chronic (90 day) phases. The subject was a 39-year-old woman presenting with head injury and lacerations from a motor vehicle accident, with brief loss of consciousness, a negative head CT, and a small hypointense lesion in frontal white matter on T2* MRI at 5 hours post-injury. Juxtaposed with the smaller microbleed was a subtle hyperintense lesion on diffusion weighted imaging with decreased ADC values, and absent changes on T2-FLAIR and post-contrast T1; together suggesting acute cytotoxic edema and ischemia. Dural enhancement was observed on post-contrast FLAIR. Repeat CT obtained at 24 hours remained negative. By 30 days post-injury, the subject had developed a conspicuous linear microbleed. The adjacent tissue had elevated ADC values, associated hyperintensity on FLAIR, evidence of cavitation on 3DT1, and enhancement on post-contrast T1; together suggesting progression to infarction. Dural enhancement had resolved. By 90 days post-injury, the linear microbleed remained well-defined. The temporal pattern suggests a) the initial trauma involved microvascular injury with subsequent development of microhemorrrhage; b) microbleeds can be associated with acute ischemia followed by late, secondary injury, such as blood-brain barrier disruption, focal infarction, and vasogenic edema and/or gliosis; and c) the observation of microbleeds in white matter post-trauma is not solely indicative of diffuse axonal injury. Most TBI patients do not have initial MRIs obtained as early after injury as this patient did. Additional study of TBI subjects able to undergo hyperacute scanning will help define prevalence of this pathological process in a broader sample of the population.
Keywords: traumatic microvascular injury
Thomas Jefferson University, College of Heath Professions, Philadelphia, USA
Support: Craig H. Neilsen Foundation Grant #260637 (Krisa, PI), Shriners Hospitals for Children Grant #8509 (Krisa, PI), and Thomas Jefferson University Department of Physical Therapy.
Keywords: pediatrics, resting state, fMRI
Despite published reports of widespread axonal injury in the controlled cortical impact (CCI) model of TBI, many still refer to CCI as a focal injury and/or question its clinical utility. To further investigate this issue we acquired in vivo diffusion tensor imaging (DTI) data before and at 1 and 4 wks post-CCI in adult rats (n = 17). Unbiased, whole brain, voxel-wise statistical analysis of white matter skeleton was used to compare DTI scalar measures pre-versus-post-injury using contusion volume (injury severity) as a covariate. Whole brain deterministic tractography was used to compute fiber tract density (FTD) and length (FTL) and for network analysis using graph theory. All data reported at P < 0.05, multiple comparison corrected, compared to pre-injury. The data show widespread ipsilateral regions of significantly reduced FA at 1 wk post-injury, driven by temporally changing values of axial, radial and mean diffusivity (AD, RD, MD) that persist to 4 wks. Analysis of AD, RD, MD unconstrained by FA, revealed bilateral reductions at 4 wks, implying FA alone underestimates damage. However, bilateral regions show a strong dependence between FA and injury severity at 4 wks (P < 0.001), indicating the importance of assessing injury severity. These data were confirmed by retrograde tract tracing studies. Significant FA increases occurred in subcortical (thalamus, caudate) and corticospinal tract (CST) regions at 1 and 4 wks that were spatially distinct from regions of FA decrease. These data were not confounded by gliosis or by tract degeneration as indicated by tensor mode changes. We found significantly increased and decreased FTD in regions of high and low FA, respectively, possibly indicating reorganization in high FA regions. In support of this, FTL significantly increased in high FA regions, especially in the contralateral CST, with significantly lower FTL in the ipsilateral CST. Network analysis of tractography data support these widespread changes in structural connectivity, and show that contrary to current dogma, the CCI model is not limited to focal damage and is clinically relevant.
Acknowledgements: UCLA BIRC; NS091222; Center-for-Neuroskills, Bakersfield, California
Keywords: reorganization, connectivity, network analysis
MRI OF BRAIN PATHOLOGY IN A MODEL OF REPEATED MILD TBI
University at Buffalo, Neurosurgery, Buffalo, USA
The risk of epileptogenesis increases with the severity of TBI. As mild TBI (mTBI) represents 75% of all TBI, the number of subjects who develop epilepsy after mTBI may outweigh those with epilepsy after severe TBI. Nothing is known about epileptogenesis after mTBI or whether the risk of epileptogenesis increases with repeated mTBI (rmTBI). We hypothesize that epileptogenic pathology, particularly microvascular changes, neuroinflammation, and white matter damage increase with the number of mild impacts.
We induced mTBI in rats by lateral fluid-percussion injury as a single or repeated injuries. Neurological Severity Score assessments at 24 hours post injury confirmed negligible functional impairment. MRI imaging was performed at 4–5 weeks after the first injury with a Bruker 9.4T MRI using diffusion tensor imaging (DTI) and quantitative susceptibility mapping (QSM).
FA and DTI image analysis suggested abnormal white mater tracts in the fimbria and external capsule ipsilateral to the injury following rmTBI. QSM showed hemhorrages at gray-white matter junctions and suggested focal calcification in the thalamus. Corresponding to MRI, analysis of thionin-stained sections of animals revealed neuronal loss in the deep layers of the ipsilateral cortex, thalamus and dentate hilus. White matter damage with gliosis was apparent in the ipsilateral external capsule, corpus callosum, and internal capsule. Iron deposits were most clear in the external capsule, and calcifications became apparent in the ipsilateral thalamus. Robust immunoreactivity to total tau was also observed in the lesioned cortex and external capsule following rmTBI.
The lateral FPI mTBI model replicates clinically relevant pathology that can be detected by MRI techniques and rmTBI produces a measured increase in pathology.
Keywords: MRI, quantitative susceptibility mapping, Tau, calcification
Mild traumatic brain injury (mTBI) patients account for more than 70% of all TBI. Although CT and conventional MRI are negative, a significant portion of the patients experience persistent post concussive symptoms more than 3 months following the initial injury. In this study we examined regional volumetric changes in mTBI patients and their association with post concussive symptoms(PCS) at 6 months following the injury.
Volumetric T1-weighted MP-RAGE images were obtained on 48 mTBI patients (age: 40.92 ± 2.30, M/F: 34/14) and 33 healthy controls (age: 38.55 ± 3.10, M/F: 18/15) were obtained on a 3T system within 10 days of the injury and these patients were re-imaged at the chronic ∼6 months following injury. FreeSurfer was used for subcortical segmentation of brain regions, from which the regional volumes were extracted.
Overall the patient symptoms recovered over the six months with patients reporting lower number and levels of symptoms. However, significant volume atrophy was observed in mTBI population compared to the control population in the thalamus, hippocampus and the amygdala (p < 0.05) at 6 months. Longitudinal volume atrophy from acute to chronic stages was observed in the thalamus and hippocampus in mTBI patients (p < 0.05). Thalamus volume atrophy was found to significantly correlate with patient symptom ratings at chronic such as: vertigo (p = 0.024), loss of balance (p = 0.029), difficult making decisions (p = 0.024). Hippocampus volume atrophy was found to significantly correlate with the nausea symptom (p = 0.036).
Overall the study indicates that the thalamus and the hippocampus may be particularly vulnerable to the long-term consequences of PCS. The thalamus may be particularly vulnerable as it is involved in relaying sensory and motor signals and may bear the brunt of disruptions in the signaling pathways from various cortical areas leading to thalamic atrophy.
Keywords: atrophy, thalamus, hippocampus, behavior, mild TBI
NEUROIMAGING OF INFLAMMATION IN EXPERIMENTAL TRAUMATIC BRAIN INJURY (TBI) UTILIZING THE 18F-GE180 TRANSLOCATOR PROTEIN (TSPO) LIGA
University of Virginia, Radiology and Medical Imaging, Charlottesville, VA, USA
Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in individuals under the age of 35. One of the most significant challenges in detecting TBI is the limited nature of current neuroimaging tools for diagnosing this condition. In recent years, effort has been devoted to the creation of imaging agents that accumulate at sites of interest and emit a signal that can be detected by positron emission tomography (PET). 18F-GE180 is a tricyclic, second-generation radiopharmaceutical that binds to the translocator protein (TSPO). TSPO is an 18 kDa protein that modulates leukocyte oxidative activity and is a biomarker for brain inflammation. The current investigation explores the utility of 18F-GE180 as a PET imaging agent for detection of TBI. Sprague Dawley rats underwent controlled cortical impact TBI and were designated for brain slice autoradiography or microPET analyses. Both groups received 18F-GE180 injections through a tail vein route at 3 or 7 days post-injury. Animals designated for autoradiography received tail vein injections one hour prior to euthanasia, after which brains were removed, sectioned, placed on radio-sensitive screens, and then analyzed utilizing a digital phosphor imager. Animals designated for microPET received 18F-GE180 injections and were then immediately scanned for a total of 90 minutes following tracer injection. SHAM controls were utilized at each time point within both groups. Significant differences were seen within the cortex and subcortical white matter ipsilateral to injury that were not seen in contralateral hemispheres or in SHAM controls. These data provide initial insight into the potential utility of molecular imaging probes targeting inflammation for the diagnosis and characterization of TBI.
Keywords: TSPO, positron emission tomography, molecular imaging, controlled cortical impact
Barrow Neurological Institute at Phoenix Children's Hospital, Phoenix, USA
Central and peripheral inflammation, secondary injury processes initiated by traumatic brain injury (TBI), have been implicated in the potentiation of somatic nociceptive pain. We hypothesized that the robust inflammatory response to diffuse TBI potentiates persistent pain through central-peripheral immune crosstalk. To test this, mice were subjected to midline fluid percussion injury or sham. One cohort of mice was analyzed for inflammation-related cytokine levels along an acute time course. TBI increased both central (cortex) and peripheral (serum) levels of IL-6 and CXCL1 indicating central-peripheral immune crosstalk. In a second cohort, peripheral inflammation was induced 7 days after surgery with an intraplantar injection of carrageenan. Mechanical pain thresholds, measured by Von Frey's filament test, were decreased in response to peripheral inflammatory challenge in brain-injured mice compared to uninjured shams. To test if this potentiated hyperalgesia may be attributable to peripheral immune dysregulation we used flow cytometric analysis of T-cell proliferation and differentiation. Mesenteric lymph node (MLN) T-cells from brain-injured mice displayed a distinct deficiency in the ability to proliferate and differentiate into inflammation-suppressing regulatory T-cells (Tregs). MLN T-cells from brain-injured mice cultured with all-trans retinoic acid (ATRA), a Treg inducing factor, showed less than 1% Treg differentiation compared to 28.6% differentiation from sham MLN T-cells. A proliferation assay indicated T-cells from brain-injured mice showed a diminished capacity for proliferation when cultured with DMSO (64.9%) or ATRA (81.1%) compared to uninjured shams, DMSO (97.6%) or ATRA (96.7%). We conclude the reduced ability of T-cells to proliferate and differentiate into Tregs after TBI may contribute to increased inflammatory pain. Further, we identify Tregs as a target for therapeutic rebalancing of peripheral immune homeostasis to improve functional outcome and decrease the incidence of somatic inflammatory pain following the double insult of TBI and cutaneous tissue injury.
Support:Science-Foundation-Arizona; Diane-and-Bruce-Halle-Foundation; NIH-F31NS090921
Keywords: traumatic brain injury, immunology, regulatory T cells, hyperalgesia, pain, inflammation
Indiana University, Stark Neuroscience Institute, Indianapolis, USA
Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Substantial epidemiological evidence implicates brain injury as a major risk factor for multiple neurodegenerative diseases. TBI has been shown to induce widespread neuroinflammation as well as accumulation of microtubule associated protein tau (MAPT) into insoluble deposits, two key pathological features of tauopathies. This study sought to characterize the macrophage response to TBI in genomic-based MAPT transgenic mice in a Mapt knockout background (called hTau), which produce only wild-type human MAPT isoforms, develop age-related MAPT hyperphosphorylation and aggregation and neurodegeneration. Two-month-old hTau and age-matched control mice received a single lateral fluid percussion TBI or sham injury. Separate groups of mice were aged to an acute (3 days post-injury [DPI]) or chronic (120 DPI) time point. Notably, the macrophage response to TBI was enhanced at 3 DPI in hTau mice compared to control mice. Although TBI mice demonstrated region specific MAPT phosphorylation at 3 DPI, no differences were observed between genotypes. By 120 DPI, a sizeable reduction in the macrophage response occurred in all brain injured mice; however, hTau mice displayed a heightened presence of infiltrating macrophages regardless of injury group. TBI-induced MAPT pathology declined by 120 DPI and was notable in hTau mice regardless of injury group. Compared to control mice, hTau mice displayed a preferential use of non-spatial strategies to complete a water maze memory test. Non-spatial strategies were most prevalent in hTau TBI mice at 120 DPI. Collectively, these data indicate that the presence of wild-type human tau alters the macrophage response to a single TBI and subsequent cognitive recovery. These results highlight the potential significance of communication between MAPT and macrophages following TBI and emphasize the role of neuroinflammation in mediating recovery following brain injury.
Keywords: macrophage, tauopathy, Alzheimer's disease, cognitive recovery
Medical College of Wisconsin, Neurosurgery, Milwaukee, USA
Spinal cord injury (SCI) is a frequent and severe condition, and strategies that improve the outcome after SCI can have far-reaching effects on health and quality of life. The primary tissue damage after SCI occurs from the trauma itself, secondary damage is caused by subsequent events, including hemorrhage and inflammation. These events contribute significantly to the pathology and thereby to the severity of the functional deficits. Inflammation after SCI is exacerbated and prolonged. Activated microglia and blood-derived macrophages are among the main immune cell types present after SCI. Previous work has shown that macrophages which phagocytose red blood cells (RBCs) develop a pro-inflammatory phenotype. RBCs are present at the site of SCI due to trauma-induced hemorrhage. One of the proinflammatory cytokines upregulated in macrophages by RBC phagocytosis is TNF. In TNF knockout mice, locomotor recovery after SCI is significantly improved. However, this effect is partial, suggesting that modification of other pro-inflammatory mediators could also reduce the secondary damage after SCI. Interestingly, Il12b, which is coding for the shared p40 subunit of the pro-inflammatory cytokines IL-12 and IL-23, is strongly upregulated by RBC phagocytosis. Both IL-12 and IL-23 are expressed by a variety of cell types and can induce the production of other proinflammatory cytokines. This project is aimed at investigating and dissecting the roles of IL-12 and IL-23 after SCI in female C57BL/6 mice, using a model of moderate contusion injury. Preliminary results suggest that IL-12 is upregulated one day after SCI. Furthermore, IL-12 and CD11b, a macrophage/microglia marker, are co-expressed in the injured tissue 3 days after injury. We are also investigating the time course of expression and the cell types expressing these cytokines and assessing their roles in vitro. Our preliminary data suggest a potential role of IL-12 and/ or IL-23 in SCI.
Keywords: cytokine, macrophage, proinflammatory, red blood cell
UCSF, Neurological Surgery, San Francisco, USA
Spinal cord injury (SCI) results in extensive infiltration of pro-inflammatory leukocytes that promote secondary damage through the release of neurotoxic substances. L-selectin, a lectin-like receptor broadly expressed on the surface of leukocytes, plays an important role in the recruitment of leukocytes to areas of inflammation. Our previous genetic and pharmacologic studies have revealed that L-selectin contributes to oxidative stress and degradation of myelin in the acutely injured spinal cord and long-term neurological deficits. However, the specific leukocyte subtypes involved in this early pathogenic role of L-selectin remain unknown. As a starting point, we examined the expression of L-selectin (CD62L) in leukocyte subsets from the peripheral blood of uninjured mice by flow cytometry. We found high L-selectin expression in inflammatory monocytes (Ly6Chi/Ly6G-) and neutrophils (Ly6Clow/Ly6G+), while expression in patrolling monocytes (Ly6Clow/Ly6G-) was low. We have previously found that diclofenac (DFA), an FDA-approved non-steroidal anti-inflammatory drug that has been shown to activate the cleavage (shedding) of L-selectin by cell surface metalloproteases, improves long-term neurological recovery after SCI. To determine which of the leukocyte subsets are susceptible to L-selectin cleavage, we systematically administered a single bolus injection of DFA or vehicle and performed flow cytometry at 2, 8, and 24 hours post-injection to assess the onset and duration of L-selectin shedding. There were no differences in the expression of L-selectin on patrolling or inflammatory monocytes between DFA- and vehicle-treated mice at 2, 8, or 24 hours post-injection. While DFA resulted in a modest yet significant increase in L-selectin on neutrophils at 2 hours post-injection, we observed a robust shedding of L-selectin on neutrophils at 8 and 24 hours post-injection. These preliminary findings reveal the preferential shedding of L-selectin on neutrophils and suggest that L-selectin expressed on neutrophils is a determinant of acute secondary pathogenesis after SCI.
Keywords: L-selectin, diclofenac
UCSF, Brain and Spinal Injury Center, San Francisco, USA
Traumatic brain injury (TBI) initiates a robust innate immune response, which has been shown to persist for years following the initial event, ultimately affecting cognitive function. Surviving patients of TBI have increased cerebrospinal levels of the chemokine CCL2, suggesting a brain-peripheral dialogue involving CCR2 competent cells. We have recently shown that TBI induces the recruitment of peripheral CCR2+ macrophages to the brain at discreet time points following injury. However, the molecular signaling contribution of recruited macrophages to the TBI-damaged brain remains largely unknown. Herein, we used parabiontes consisting of a wildtype B.6 mouse paired to a CX3CR1GFP/+ CCR2RFP/+ mouse to accurately delineate the accumulation of three subpopulations of monocytes/macrophages; CX3CR1+CCR2- CX3CR1+ CCR2+, and CX3CR1-CCR2+ at multiple time points following injury. This approach allowed us to unambiguously track these donor (GFP+ or RFP+) monocyte/macrophage subpopulations in the injured WT animal. Data indicate that following TBI across multiple time points, there is not a robust recruitment of peripheral CX3CR1+CCR2- monocytes/macrophages to the injured parenchyma. Next, we followed these experiments using FACS in the CX3CR1GFP/+CCR2RFP/+ mouse at 24 hours post injury, which represented the largest influx of peripheral macrophages to the injured brain. FACS enriched microglia (CX3CR1+CCR2-), and two peripheral macrophage populations (CX3CR1+CCR2+ and CX3CR1-CCR2+) were analyzed for gene expression by RNA sequencing. RNAseq revealed unique heterogeneous signatures of these three enriched subpopulations. Importantly, among these three subpopulations of microglia/macrophages there was not a clear delineation of the M1/M2 polarization phenotypes, as each displayed simultaneous expression profiles. We next examined how deletion of CX3CR1 and CCR2 chemokine receptors altered these molecular signatures in tissue 24 hours after TBI. Lastly, we examined how age at the time of TBI as well as Cenicivoric, a novel small molecule antagonist of CCR2 affected these molecular signatures. Cumulatively, these data point toward a unique molecular contribution of peripheral-derived macrophages in the etiology of TBI-induced neuroinflammatory sequelae.
Keywords: macrophage, CCR2, RNAseq, aging
Keywords: experimental traumatic brain injury, inflammation, IL-1
THERAPEUTIC APPROACHES TO TARGET INFLAMMATION FOLLOWING TBI THROUGH INHIBITION OF THE P38ALPHA MAPK
University of Kentucky, Spinal Cord and Brain Injury, Lexington, USA
Closed head traumatic brain injury (TBI) triggers an acute inflammation response that involves resident glia and other immune cells. Neurologic outcome is dependent on the essential balance between restoration of tissue homeostasis after the initial injury and resolution of the injury-induced innate immune response. Natural resolution of the injury-induced proinflammatory cytokine response such that neurologic sequelae are attenuated is generally not successful. Therefore, therapeutic interventions during critical dosing time windows are needed in order to reduce the dysregulated inflammation that is causally linked to the neuropathologic sequelae. Previous work has generated a causal link between the p38α mitogen-activated protein kinase (MAPK) mediated intracellular signaling pathway and the injurious proinflammatory cytokine response in neurodegenerative animal models of disease. The recent availability of highly specific in vivo molecular probes for p38α inhibition allow a more refined in vivo analysis of this intracellular signaling pathway and its link to dysregulated glia function and neuroinflammation in TBI with its more rapid pathology progression kinetics. We have recently explored these processes in TBI through the combined use of these in vivo p38α dynamic molecular probes and genetics based in vivo tools, such as targeted knockdown of p38α in specific inflammatory cell types. We found that genetic suppression of p38α in myeloid cells resulted in less TBI induced deficits in a running wheel behavioral task and cognitive deficits as measured by the radial arm water maze. Suppression of p38α activity through selective pharmacological action or through reduction of p38α protein levels generated reduction of injury injury induced cytokine levels in the brain. The congruence of outcomes from genetic and pharmacological approaches provides a unique battery of outcomes consistent with p38α as a potential therapeutic target in TBI.
Keywords: inflammation, drug discovery, cytokines, microglia
University of Pittsburgh, Physical Medicine and Rehabilitation, Pittsburgh, USA
Our previous work used principal components analysis (PCA) to characterize cerebrospinal fluid (CSF) inflammatory profiles following severe Traumatic Brain Injury (sTBI). However, systemic immunity is distinct from the brain's inflammatory response to TBI, as systemic cytokine production is coordinated via a sympathetic nervous system mediated acute phase response (APR) in the liver. APR contributes to multi-organ dysfunction, amplified aromatization, and increased mortality risk, supporting the rationale to study variability in peripheral inflammation after sTBI. The objective of this study is to leverage PCA to identify principal components (PCs) that make up serum inflammatory profiles after sTBI, and assess potential PC associations with 6 mo mortality. Serum samples (n = 615) were collected 0–5d post-injury in 121 adults with sTBI and analyzed for the following biomarkers: interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, TNFα, sVCAM-1, sICAM-1, and sFAS. PCA is a dimension-reducing procedure for correlated continuous variables by identifying sources of variation within data. A weekly average for each marker was calculated and z-score standardized. There were two significant PCs identified, PC1 and PC2, accounting for 20.2% and 15.0% of the variance, respectively. Relevant PCs were entered into a multivariate logistic regression model for 6-month mortality. Significant markers positively loading to PC1 were: TNFα, IL-1β, IL-8, IL-10, IL-12, and sFAS. Significant markers that positively loaded for PC2 were: sICAM-1, IL-6 and sVCAM-1, and negatively were: IL-12 and IL-4. Upon covariate adjustment, PC1 scores were significantly associated with mortality (OR = 2.61, p = 0.023). PC2 scores were non-significant (p = 0.991). PC1 scores 1) capture the greatest variance for peripheral inflammation; and 2) increases in scores (higher levels) are a significant risk factor for mortality after sTBI. TNFα is the strongest loading PC1 marker, and due to its role as a pro- inflammatory agent that also facilitates estradiol production, there is a biological plausibility that it is a critical peripheral marker of inflammation and aromatization associated mortality.
Support: CDC: R49-CCR323155, DOD: W81XWH-071-0701, NIDILRR 90DP0041-02-01.
Keywords: principal components analysis, statistical modeling, post-traumatic inflammation, peripheral inflammation
Krembil Research Institute (University Health Network), Genetics and Development, Toronto, Canada
Spinal cord injury (SCI) is a debilitating condition where damage caused by physical trauma on the spinal cord is enhanced by neuroinflammation. Neutrophils and resident microglia, which are the earliest immune cells activated after trauma, are detrimental after SCI by propagating neuroinflammation and expanding the susceptible perilesional region. Immunomodulatory therapies involving biomolecules with high availability and good safety profiles represent an attractive approach to halt this damage. Currently, Immunoglobulin G (IgG) treats various inflammatory diseases in central nervous system. However, immunomodulatory effects of IgG on neuroinflammation after SCI remain elusive. We hypothesize that early infusion of IgG after SCI confers neuroprotection by creating an anti-inflammatory environment in the injured spinal cord and modulating local and systemic leukocyte populations. Female adult Wistar rats were injured at C7-T1 level. At 15 minutes post-SCI, IgG (0.2, 0.4, 1 and 2 g/kg), methylprednisolone and their respective vehicle controls (IgG-free serum, saline) was injected intravenously. Blood and spinal cord were analyzed for various inflammatory readouts at 24 hours post-SCI. Relative to IgG-free serum, in the spinal cord, IgG (2 g/kg) reduced number of activated neutrophils (p < 0.05), increased expression of IL-10 and fractalkine (p < 0.05) and decreased expression of activated resident microglia/macrophage marker (Iba-1) (p < 0.001). Peripherally, IgG (2 g/kg) reduced circulating neutrophils relative to IgG (0.2 g/kg) (p < 0.05). On all readouts, no difference was seen between saline and IgG-free serum. This study presents the first evidence showing that IgG has immunomodulatory effects on various leukocyte populations after SCI through IL-10 and fractalkine. Given the current use of IgG in the clinic, our work to elucidate how IgG promotes functional recovery through immunomodulation has potentially significant therapeutic value.
Acknowledgement of support: This work is supported by grants from Baxter Pharmaceuticals, Krembil Family Foundation and CIHR (J.C.).
Keywords: neutrophils, intravenous immunoglobulin G, rat model, fractalkine, interleukin-10, microglia/macrophages
Virginia Commonwealth University, Department of Neurosurgery, Richmond, USA
TBI is a risk factor for chronic degenerative disorders, including chronic traumatic encephalopathy (CTE) and Alzheimer's and Parkinson's diseases. Our recent preclinical studies have shown that acute TBI triggers reciprocally interconnected secondary molecular and cellular pathways, resulting in chronic pathological consequences that include white matter degeneration, delayed microvascular pathology, microbleeds, punctate blood-brain barrier breakdown, caspase-induced apoptosis and progressive inflammatory responses. The goal of this study was to investigate the association of progression of white mater alterations with these pathophysiological pathways in the cerebral cortex and hippocampus.
Keywords: chronic TBI, caspase-3, inflammation, blood-brain barrier
Univrsity of Miami, Miami Project for Cure Paralysis, Miami, USA
The lack of reproducibility in many areas of science, including spinal cord injury (SCI) research, is due in part to the lack of common reporting standards. Over the past four years an ad hoc consortium of scientists has developed a minimal information reporting standard for SCI experiment, called MIASCI (PMID: 24870067). We have developed a web-based annotation tool, called MIASCI ONLINE, which eases the collection of metadata and data about SCI studies. An important and innovative feature of MIASCI ONLINE is that it establishes formal relationships between different metadata concepts and experimental data. The experimental information is then provided in a form that can be stored and interpreted by computer based knowledge systems, and is ready for deposit in databases, such as RegenBase. MIASCI ONLINE also provides a PDF document that can be submitted as a supplementary file for a SCI manuscript, documenting compliance with MIASCI. MIASCI ONLINE improves transparency in the description of experiments, enabling the use of uniform reporting standards for experimental design parameters. Therefore, this web-based annotation tool encourages the use of “best practices” in the SCI community.
Acknowledgments: NINDS NS080145 and NICHD HD057632
Keywords: axon regeneration, ontology, knowledgebase, MIASCI, reporting standard, kinase
Traumatic brain injury (TBI) resulting from exposure to blast overpressure (BOP) is associated with several behavioral and cognitive impairments. Although the underlying mechanism(s) of blast-induced TBI is unclear, it appears to involve elevated intracranial pressure (ICP) and impairment of the blood brain-barrier (BBB). This study examined the effects of treatment with the antioxidant N-acetylcysteine amide (NACA) after blast exposure on ICP and BBB integrity assessed directly via telemetry and intravital microscopy (IVM), respectively. Anesthetized male Sprague- Dawley rats were exposed to three blasts separated by 0.5 h each at 110 kPa. Rats were injected i.p. with NACA (500 mg/kg) or placebo (PBS) 2 h after the first BOP exposure. After a pre-blast baseline equilibration, ICP was recorded continuously for six days post blast. For the IVM study, BBB integrity was examined in vivo in rats equipped with a cranial window exposing pial microcirculation. Tetramethylrhodamine isothiocyanate Dextran (TRITC-Dextran, MW = 40 kDa) was injected intravenously (25 mg/kg) 2.5 h after the first blast and the leakage of marker from pial micovessels into the brain parenchyma was assessed over 5 minutes. Rats were randomized into 4 groups: BOP no treatment, sham no treatment, BOP followed by NACA injection, and sham with NACA injection groups. Data analyses show a significant increase in ICP after blast in placebo treated animals. Treatment with NACA resulted in a significant reduction of the blast-induced ICP elevation. The rate of TRITC-Dextran leakage through the pial vessels was higher (60%) after exposure to blast than the control uninjured group (36%). NACA treatment reduced the rate of leakage from ∼60% to 18% in BOP-exposed rats. Collectively, these data suggest that NACA treatment at the single dose may significantly protect against ICP increase and BBB breakdown after exposure to blast.
Keywords: blast, TBI, ICP, BBB
REPEATED TRAUMATIC BRAIN INJURIES INDUCE PERSISTENT CENTRAL INSULIN RESISTANCE
Traumatic brain injury (TBI)-induced central metabolic dysfunction (i.e. changes in glucose utilization) corresponds to an impaired capacity for tissue repair and a persistence of cognitive deficits. In general, tissue repair and associated deficits improve when the energetic crisis resolves, but the capacity for central metabolic processes to normalize is greatly reduced after severe or repeated TBI. Increasing evidence of both direct and indirect roles of insulin in the regulation of brain metabolism, coupled with the known neuroprotective effects of insulin following recovery from CNS insults, led us to investigate the effect of TBI on brain insulin sensitivity. To determine a time course of TBI-induced changes in brain insulin signaling, male C57/Bl6 mice underwent single, repeated, or sham mild TBI (mTBI) and fresh brain slices were treated with insulin and assessed via Western blotting for a marker of insulin resistance at various time points. In order to assess the effect of pre-existing insulin resistance on TBI outcome, a separate cohort was fed either a low fat or a high fat diet (HFD) prior to undergoing the same procedure. Brain pathology (silver staining), neuroinflammation (IBA1+ microglia), and behavioral outcomes were also assessed. Our data reveal that a single mTBI produces insulin resistance which recovers within 7 days, while repeated TBI-induced insulin resistance persists up to 28 days after injury. Pre-existing insulin resistance (via HFD) altered TBI-induced insulin signaling, increased cortical microglial activation, and exacerbated anxiety-like behavior. Taken together, our data reveal a significant TBI-induced disruption of insulin signaling, which is further exacerbated by repeated TBI and corresponds to greater inflammation and behavioral deficits.
Keywords: insulin resistance, repeated TBI, obesity, neuroinflammation
DIFFERING TIME COURSES OF SYNAPTIC & NON- SYNAPTIC MITOCHONDRIAL DYSFUNCTION & OXIDATIVE DAMAGE FOLLOWING TBI IN YOUNG ADULT MALES
Traumatic brain injury (TBI) results in secondary injury caused by the reactive oxygen species peroxynitrite (PN), whose decomposition into highly reactive free radicals that initiate lipid peroxidation (LP) and the production of reactive aldehydes such as 4-hydroxynonenal (4-HNE). Our laboratory previously characterized the temporal profile of LP-mediated oxidative damage and mitochondrial dysfunction following TBI. While these experiments demonstrated the progressive decline in total mitochondrial function and increased levels of 4-HNE, the question remained: was one mitochondrial population, synaptic (i.e. neuronal terminals) or non-synaptic (i.e. neuronal soma and axons, endothelial, glial and infiltrating inflammatory cells), more susceptible than the other to LP-mediated damage and dysfunction following TBI. Male Sprague-Dawley rats received a severe (2.2 mm) controlled cortical impact (CCI)-TBI. Cortical mitochondria were isolated and evaluated using a Clarke-type electrode at from 3 hrs out to 5 days post injury using a Ficoll gradient and burst in a nitrogen cell disruption bomb. Mitochondrial samples were assessed for 4-HNE as a marker of LP by Western immunoblot. We observed a more rapid and severe decline in synaptic mitochondrial function compared to the non-synaptic population. Within 3 hrs post-TBI, synaptic mitochondrial respiratory function was significantly reduced and continued to decline. Between 24 and 72 hrs, synaptic mitochondrial function continued to decline, but not as dramatically as within the first 24 hrs. The non-synaptic population did not show significant decreases in respiratory function until 2–3 days post-injury. In this study, we have thus established a temporal profile of post-traumatic synaptic and non-synaptic mitochondrial bioenergetics and identified the peak and duration of dysfunction for these two populations. These findings, therefore, suggest that mitochondrial-targeted neuroprotective therapies may have differing therapeutic windows for protecting synaptic vs. non-synaptic mitochondrial populations following CCI-TBI.
Acknowledgments: Supported by NIH/NINDS R01 NS083405 and R01 NS084857
Keywords: traumatic brain injury, synaptic mitochondria, lipid peroxidation, 4-HNE
IDENTIFICATION OF PREFERRED SUBSTRATES OF MITOCHONDRIAL ENERGY METABOLISM FOLLOWING PENETRATING BALLISTIC-LIKE BRAIN INJURY
Mitochondria constitute a central role in brain energy metabolism, and play a pivotal role in the development of secondary pathophysiology and subsequent neuronal cell death following traumatic brain injury (TBI). Under normal circumstances, brain consumes glucose as the preferred energy source for adenosine triphosphate (ATP) production over ketones. However following TBI, the ATP synthesis rates of glucose intermediate metabolites (i.e. pyruvate, glutamate and succinate) and ketones (i.e. β-hydroxybutyrate) have not been individually compared. We hypothesize that TBI leads to an overall decline or metabolic shift in the utilization of these substrates for the ATP production. Adult male rats were subjected to either 10% unilateral penetrating ballistic-like brain injury (PBBI) or Sham craniotomy (n = 5 per group). At 24 hours post-injury, mitochondria were isolated from pooled brain regions (frontal cortex and striatum) of the ipsilateral hemisphere. Mitochondrial ATP synthesis was measured using Clark-type oxygen electrode in the presence of metabolic substrates pyruvate+malate (PM), glutamate+malate (GM), succinate (Succ) and β-hydroxybutyrate+malate (BHBM). As compared to Sham group, the PBBI group showed a reduction in ATP synthesis with PM (-43%, p < 0.05); GM (-42%, non-significant); Succ (-50%, p < 0.05) and BHBM (-44%, p < 0.05). Next, we compared the ATP synthesis rates of the four substrates within the experimental group. In the Sham group, the BHBM showed significantly less ATP synthesis than the other substrates (PM = GM = Succ > BHBM, p < 0.05). In the PBBI group, the ATP synthesis of PM, Succ and BHBM were significantly distinct amongst each other (PM > Succ > BHBM, p < 0.05). Additionally, the ATP synthesis of PM and GM were identical, and were the highest amongst the four substrates tested. Collectively, PBBI leads to an overall reduction and metabolic shift in the intermediate substrates utilization for the ATP production. These results provide a strong basis for the use of neutraceuticals as “alternative biofuels” for higher energy production following brain trauma. Support: US Army Combat Casualty Care Research Program H_026_2014_WRAIR.
Keywords: mitochondrial bioenergetics, preferred metabolic substrates, penetrating ballistic-like brain injury, alternative biofuels
MICROGLIAL ACTIVATION AND THE IMMUNOINFLAMMATORY RESPONSE AFTER A MODERATE TRAUMATIC BRAIN INJURY
Traumatic brain injury (TBI) is a common result of head injury in both sports and military personnel, accounting for over 1.7 million injury-related deaths in the United States alone. A large percentage of the clinical deficits found in TBI are caused by secondary neural degeneration that occurs days to months after the original injury. Long-term glial accumulation, heightened immunoinflammatory markers, and dysregulation of calcium and NMDA channels are hypothesized to contribute to secondary neural cell loss. Immunofluorescent labeling was used to characterize the changes in microglial and neuronal cellular markers following a controlled cortical impact that was moderate in severity. The progression of microglial proliferations and activation was measured at 4-hours, 72-hours, and 1-week post injury. In the dorsal thalamus ipsilateral to the site of injury, significant increases in microglia were found to occur at 72-hours and 1-week, but not after 4-hours. Additionally, in the medial and lateral portions of the cortex ipsilateral to the site of injury, significant increases in microglia were found after 72-hours and 1-week post injury. Relative distance to the injury site may influence the increases in glial levels. Regions that are closer to the site of injury may have higher microglia concentrations than those located more distal. Additionally, disruption of the blood-brain barrier, allowing for peripheral glia to enter into the brain, may account for increased glial levels. The findings of this study are also not consistent with the hypothesis that microglia peak at 72 hours then begin to drop. Glial levels in the dorsal thalamus peaked at 72 but then were sustained up to a week after injury. Finally, no significance was found in either the dorsal thalamus or cortical regions ipsilateral to the site of injury for mature neurons at any time points. The timeline of glial activation is critical in determining when to administer treatments. Narrowing the window for administering treatments can greatly impact the extent of recovery after injury.
Keywords: inflammation, activation, neuron, mice
RAPID MICROGLIAL ACTIVATION NEAR INJURED NEURONS FOLLOWING DIFFUSE BRAIN INJURY IN SWINE
Veterans Affairs Medical Center, PHILADELPHIA, USA
It is recognized that disruption of neuronal circuitry from traumatic brain injury (TBI) contributes to acute and persistent symptomology. However, it is not well understood how microglia, the inflammatory modulators of the CNS, contribute to acute neuronal health and homeostasis after TBI. To better elucidate the influence of microglia on injury progression, we investigated acute microglial reactivity around injured neurons immediately following single and repetitive TBI. Using a previously characterized porcine closed-head, rotational acceleration injury model, we induced mild or severe TBI and labeled mechanically permeabilized neurons using Lucifer yellow (LY). Acutely after injury (15 minutes), animals were sacrificed and microglia were analyzed using a novel cell body to cell size ratio to investigate distribution and morphology as a function of distance from neurons exhibiting trauma-induced plasmalemmal disruptions (LY+). In regions without acutely permeabilized neurons, there were no differences in microglia reactivity in either injured or sham animals. However, activated microglia were frequently observed adjacent to permeabilized neurons post-injury. Following both mild and severe injuries, microglia density increased and morphology became more amoeboid in the immediate vicinity of LY+ neurons in a distance-dependent manner. Interestingly, the extent of localized reactivity was similar in both TBI severities, suggesting that microglia may contain intrinsic limitations in acute reactivity. Animals subjected to repetitive injuries exhibited marginally heightened microglia reactivity relative to single TBI. This is the first report of rapid microglial activation around damaged neurons following closed-head TBI. Further investigation into the influence of microglia on acutely injured neurons could inform the development of immunomodulatory therapeutics. Financial support provided by the NIH, NSF, and Department of Veterans Affairs.
Keywords: diffuse brain injury, plasmalemmal disruption, glial reactivity
The Brain and Spinal Injury Center (BASIC; UCSF-SFGH) a level-one trauma center with both basic and clinical investigators, is uniquely positioned to study early critical care variables. SCI patients arrive within 20 min (mean) of injury (due to excellent EMS, small catchment area, dense population). We showed (Nout et al, 2012) that BP immediately after a T3 MASCIS-SCI in rats was correlated with locomotor outcome; animals with MAP of ∼80 mmHg or less had poorer function as compared to subjects in the normal range (∼100–110 mmHg). A retrospective analysis of recorded MAP after SCI in the ICU at BASIC/SFGH (Hawryluk et al, 2015) also suggested that epochs of low-BP acutely after human SCI were associated with poorer outcome at discharge (AIS-grade). An independent data-mining study using the VISION-SCI repository and topological data analysis (Nielson et al, 2015) identified high BP during T9-10 MASCIS injury surgery as being associated with poorer locomotor function. A new experimental study in rats controlled BP over 4 hrs after T3-SCI (250KD IH), at below 80 mmHg (n = 9), at ∼100–110 mmHg (n = 12), or at ∼130 mmHg (n = 11), and showed that both high and low BP groups performed more poorly over the early weeks after injury than normotensive rats. We have retrospectively identified 85 SCI patients' OR records from decompression surgery, and will evaluate the relationship between their cardiovascular parameters and neurological outcomes. From early retrospective clinical data, BP was identified as an important predictor of outcome, and laboratory results confirmed this. We have now initiated a prospective study, TRACK-SCI, to systematically gather data on a wide variety of critical care and outcome variables to identify those predicting better function for use in guiding SCI patient care. (Support: DoD W81XWH-13-1-0297, CH Neilsen)
Keywords: blood pressure, predictors of outcome, translational, bench to bedside
AUTOMATED CONTROL OF BLOOD PRESSURE IN HEMORRHAGED SWINE WITH TRAUMATIC BRAIN INJURY
UTMB, Anes., Galveston, USA
Studies were completed as part of a team funded by The Moody Project for Translational Traumatic Brain Injury Research and the Office of Naval Research.
Keywords: automated, swine, hypotension, resuscitation, TBI
Keywords: clinical guideline, spinal cord injury, timing of surgery, systematic review
PROGRESSIVELY DECREASING GLYCEMIC VARIABILITY AFTER TBI ASSOCIATED WITH IMPROVED OUTCOMES
Landmark studies of therapeutic glycemic control produced somewhat discrepant results on the benefits of intensive insulin therapy. How we control blood glucose impacts critically ill TBI patients in addition to what range we target. High glycemic variability has been implicated in negative long-term outcomes following TBI. This study's objective was to investigate whether acute glycemic variability predicts 6-month outcomes for severely injured human TBI patients, hypothesizing that daily glycemic variability is higher in patients with poor outcomes. Point-of-care (POC) glucose was extracted from the UCLA BIRC electronic database for forty-two consented adults (8 Female, 39 yo [IQR 26–57], GCS 5 [IQR 3–7]). Glycemic variability was quantified by daily mean amplitude glycemic excursions (MAGE) between post-injury day (PID) 1–10. Clinical outcomes were Glasgow Outcome Scale extended (GOSe) scores obtained 6 months post- injury, dichotomized as good (5–8) versus poor outcomes (1–4). Our mixed effects model included daily mean POC glucose, PID, outcome, and the interaction between PID and outcome; subject ID was the random effect. Daily mean POC glucose had the strongest effect on MAGE; increasing mean POC glucose 10 mg/dL was associated with increasing MAGE 1 mg/dL (p < 0.001). There was a decrease in MAGE over time in the good outcome group, averaging 17 mg/dL on PID1 and decreasing 6 mg/dL by PID10 (p = 0.002). MAGE on PID1 were not significantly different between good and bad outcomes (p = 0.20) but MAGE did not decrease over time in the poor outcome group (p = 0.67). MAGE on PID10 were 4 mg/dL higher in the poor outcome group (p = 0.060). The clinical implications of this study are the evolution of MAGE over time, rather than glucose averages or MAGE alone, distinguished good outcomes and rapid correction of low or high glucose may negatively impact acute recovery.
Keywords: glycemic control, glucose, insulin, glycemic variability
CIRCADIAN VARIABILITY OF THE INITIAL GLASGOW COMA SCALE IN TRAUMATIC BRAIN INJURY PATIENTS
There is scant literature describing the potential influence of circadian rhythms and/or emergency department (ED) admission hour on the Glasgow Coma Scale (GCS) after traumatic brain injury (TBI). Retrospective cohort analysis of blunt adult TBI was performed using the National Sample Program of the National Trauma Data Bank (2003–2006). ED admission GCS was characterized by midday (10am-4pm) and midnight (12am-6am) cohorts (N = 24,548). Multivariable regression was performed to assess associations between arrival hour and GCS. Statistical significance assessed at p < 0.05. Patients were 43.5 ± 19.9 years old and 69.5% male. GCS was 12.63 ± 4.20 (median 15, IQR 13–15; 77.2% mild, 4.2% moderate, and 17.6% severe TBI). Overall, 85.7% were admitted (33.5% ICU). ISS was 15.65 ± 11.22 and did not differ between day/night. Nighttime admissions were associated with decreased medical comorbidities (p < 0.001). GCS demonstrated a circadian pattern with peak at 12pm (13.03 ± 0.08 (SE)) and nadir at 4am (12.12 ± 0.12 (SE)). Midnight patients demonstrated significantly lower GCS (12am-6am: 12.23 ± 0.04, 10am-4pm: 12.95 ± 0.03, p < 0.001). Multivariable regression adjusted for age, mechanism, comorbidities, hypotension and ISS confirmed that midnight-hours were independently associated with decreased GCS (B = -0.29 [−0.40, −0.19]). In patients who did not die in the ED or go directly to surgery (N = 21,862), multivariable regression demonstrated midnight-hours (OR 1.73 [1.30–2.31]) associated with increased likelihood of ICU admission, while increasing GCS (per-unit OR 0.82 [0.80–0.83]) associated with decreased odds. Notably, the interaction factor GCS*ED arrival hour independently demonstrated an OR of 0.96 [0.94–0.98], suggesting that the influence of GCS on ICU admission odds is less important at night than during the day. Nighttime TBI patients present with decreased GCS and are admitted to ICU at higher rates, yet have fewer prior comorbidities and similar systemic injuries. The interaction between nighttime hours and decreased GCS on ICU admissions has important implications for clinical assessment/triage.
Keywords: Glasgow Come Scale, circadian rhythm, traumatic brain injury, emergency department
AIM2 INFLAMMASOME SIGNALING IN TRAUMATIC BRAIN INJURY-INDUCED ACUTE LUNG INJURY
University of Miami, Neurological Surgery, Miami, USA
Pulmonary dysfunction often occurs in patients with severe Traumatic Brain Injury (TBI). Acute lung injury (ALI) is one of the most common respiratory complications after TBI, which is seen in 20–25% of patients with TBI. The DNA binding protein, high mobility group box protein 1 (HMGB1) is a damage-associated molecular pattern (DAMP) that is immediately released after TBI and has been implicated in TBI-induced pulmonary dysfunction. However, the precise underlying molecular mechanisms remain unclear. HMGB1 is also known to activate the DNA sensing inflammasome, absent in melanoma (AIM2) and may be involved in the pathomechanisms of TBI-induced ALI. We hypothesized that the AIM2 inflammasome plays a central role in innate immune signaling after TBI, thus resulting in the development of ALI. C57/BL-6 mice were injured at 6m/s using a Controlled Cortical Impact (CCI) injury and were sacrificed at 4 and 24 hours post-injury. Mouse lung and brain tissue lysates were collected for inflammasome protein expression using Western Blot analysis. Mouse lungs were perfused intratracheally for immunohistochemical analysis. Lungs were sectioned and stained with hematoxylin and eosin (H&E) for evidence of alveolar edema and inflammatory cell infiltration. At 4 and 24 hours after TBI there was a significant increase in the levels of inflammasome proteins caspase-1, apoptosis speck like protein containing a caspase recruiting domain (ASC), Interleukin-18, AIM2, and HMGB1 in mouse brain and lung lysates. Lungs showed changes in alveolar morphology changes at 4 and 24 hours, which were more pronounced at 4 hours. In addition, lung sections showed co-localization of caspase-1 and ASC with surfactant protein C (SPC), a type II alveolar epithelial cell marker, at 4 and 24 hours post-TBI. Our results support the hypothesis that inflammasome signaling plays a role in the pathophysiology of ALI after TBI, and may identify novel targets for therapeutic intervention in TBI-induced ALI and pulmonary dysfunction.
Keywords: inflammasome, traumatic brain injury, acute lung injury, extracellular vesicles
University of Utah, Neurosurgery, Salt Lake City, USA
Keywords: guidelines, compliance, intracranial pressure monitor, survey
Traumatic brain injury (TBI) affects an average of 1.4 million Americans each year, with at least 50,000 reported injuries resulting in death. Long-term effects of TBI include headaches, cognitive decline, seizures, mood swings, motor skill impairment, increased fatigue, and sleep disturbances. TBI is also a prominent risk factor for neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD). There are currently no treatments for TBI. We recently found that PERK, an endoplasmic reticulum stress kinase that mediates the adaptive branch of the unfolded protein response, was chronically activated in brains following TBI. We show here that PERK is activated in a time dependent and region specific manner following injury with the controlled cortical impact (CCI) model of TBI. We also show inhibition of PERK following traumatic brain injury in 6-week-old mice modulated overall protein synthesis, broad neuronal function, and the inflammatory response. Our data provide novel insight into the physiological mechanisms of TBI, and suggest that PERK plays an important role in both injury progression and neurological recovery. These data also support the exploration of PERK inhibition as a therapeutic option for traumatic brain injury.
Keywords: UPR, PERK, ER stress
PLASMALEMMAL PERMEABILITY & INCREASED α- SYNUCLEIN EXPRESSION IN SWINE SUBSTANTIA NIGRA NEURONS FOLLOWING DIFFUSE BRAIN INJURY
MJC Veterans Affairs Medical Center, Philadelphia, PA, USA
Experimental models of traumatic brain injury (TBI) have suggested that the plasma membrane of neural cells may become disrupted immediately following mechanical insults and lead to delayed cellular injury. However, the specific consequences of this transient membrane permeability are unknown. Recent evidence suggests that this acute response may play a role in pathological protein aggregations commonly associated with neurodegenerative diseases such as Parkinson's disease (PD), in which α-synuclein accumulates in substantia nigra (SN) neurons. We utilized an established porcine model of closed-head rotational acceleration to investigate related neuronal changes following mild TBI. As the anatomy of the SN has not been detailed in the Yucatan minipig, as a first step we characterized the structure of the SN pars compacta (SNc), SN pars reticulata (SNr), and surrounding nuclei in naïve animals. Next, swine were subjected to sham conditions or single/repetitive rotational head acceleration (100–300 radians/second) using a HYGE pneumatic actuator. Two hours prior to final injury, the normally cell-impermeant dye Lucifer Yellow was injected into the interstitial space to assess plasmalemmal permeability. Animals were sacrificed 15 minutes after final injury for immunohistological analysis. Acutely permeabilized neurons were observed in both divisions of the SN and related nuclei, and increased α-synuclein expression was observed in a subpopulation of these cells. As many permeabilized cells did not express dopaminergic markers, ongoing studies are assessing the susceptibility of inhibitory neurons in the SNr, as well as the emergence of pathological isoforms of α-synuclein in the SN overall. These results suggest that diffuse brain injury induces complex, multi-faceted pathophysiology in the SN, which has important implications for linking physical injury at the cellular level to chronic neurodegenerative pathways like those found in PD. Financial support provided by NIH and Department of Veterans Affairs.
Keywords: traumatic brain injury, alpha-synuclein, cell permeability, substantia nigra
Safar Center for Resuscitation Research, University of Pittsburgh, Neurorehabilitation, Pittsburgh, U.S.A
Environmental enrichment (EE), a preclinical model of rehabilitation, increases basal neurogenesis and promotes cognitive recovery after experimental traumatic brain injury (TBI) in rats. However, the effect of EE on learning-induced neuroplasticity has not been studied. In the present work we evaluated the effect of two different models of EE on dentate gyrus neurogenesis after training in a spatial learning task and examined any potential region-specific effects within the hippocampal subfields along the rostro-caudal axis. Anesthetized adult male rats received a controlled cortical impact (2.8 mm depth at 4 m/sec) and were randomly assigned to either standard housing (TBI+STD), continuous EE (TBI+EE), or delayed and abbreviated EE (TBI+EE, 3 day delay, 6 hr day). Rats were trained in a Morris water maze on post-operative days 14–18 and a single probe trial was performed on day 19 to measure memory retention. Rats were sacrificed, the brains were cut on a freezing microtome at 40 μm and immunostaining for DCX was performed. We estimated the volume of the subgranular layer (SGL) of the dentate gyrus and the number and density of DCX+ cells by stereology. Our results showed continuous EE increased DCX+ cell number and mean density in TBI rats without causing any effect on SHAMs. Delayed-and-abbreviated EE decreased DCX+ cell number and volume in SHAM animals which resulted in no differences in density without affecting TBI group. A rostro-caudal analysis revealed that EE effect was selective to the rostral part of the dorsal hippocampus. A significant increase of DCX+ cell density was also present in the more rostral part of delayed and abbreviated EE + TBI group. In conclusion, EE selectively increases training-induced neuroplasticity in the rostral hippocampus after TBI.ter your own text!
Keywords: hippocampus, plasticity, controlled cortical impact, stereology
University of Texas Medical Branch, Department of Anesthesiology, Galveston, USA
Support: These studies were completed as part of an interdisciplinary research team funded by The Moody Project for Translational Traumatic Brain Injury Research.
Keywords: near infrared light, ultrasounds, neuroprotection, hippocampus, brain injury
Baylor College of Medicine, Pathology & Immunology (Neuropathology), Houston, USA
This is a case report of an unusual wooden spinal foreign body that became symptomatic two decades after the initial injury.
A 30-year-old man presented with lower back pain for 8 months with the pain worsening accompanied by urinary retention, left leg numbness, saddle anesthesia and impaired ambulation in the 2 weeks prior to admission. He had a T10 sensory level, weakness of both lower extremities, hyper-reflexia with plantar extensor responses and diminished rectal tone. MRI demonstrate a T10 extradural and intradural enhancing mass with severe spinal cord compression.
The patient underwent T9-11 laminectomy and intra-operatively extradural and intradural inflammatory tissue with a 2 cm tapered intramedullary wooden foreign body – a thorn – was encountered. Histological examination confirmed that the foreign body was of plant origin with cellulose cells and brown pigmentation. The inflammatory pseudo-tumor had granulomatous and chronic lymphoplasmacytic inflammation as well as foci of acute inflammation. In retrospect, the patient recalled that as a child he fell off a horse onto a bush, resulting in several thorns penetrating his back. After the incident he had some trouble ambulating for several days. The patient's postoperative course uncomplicated with improvement of sensation and strength. Postoperative MRI showed good decompression of the T10 mass.
Spinal wooden foreign bodies are rare and usually result from falls into plants with penetration of the spine. Presentation is usually acute but occasional latent presentation has occurred. The present case is very unusual because of the almost 25-year interval between the penetration of the spine and presentation due to an inflammatory pseudo-tumor. It is possible that the thorn may have migrated recently inciting the inflammatory reaction. The thorn was not seen on pre-operative imaging and as numerous observers have noted, wooden foreign bodies are not well visualized by conventional imaging techniques.
Keywords: foreign body, spinal cord, inflammatory pseudotumor, thorn, plant matter
CSHL, Cold Spring Harbor, USA
Traumatic brain injury through mechanical forces acting on the parenchyma is suspected to cause direct injury to the vasculature. Blood is extravasated into the perivascular space and surrounding parenchyma, and secondary injury such as ischemia and inflammation can occur. As these vessels are surrounded by iron-laden macrophages, visualizing this pathology digitally may push patient and neuroanatomical data into an era of quantitative and unbiased analysis of cellular, vascular, and network changes induced by injury. We are interested in defining tissue injury as a result of the vascular injury, while also defining potential metrics and the correlation related to axonal injury. Given the rapid growth in whole-slide imaging, it is desirable to obtain high quality histological materials co-registered to MRI data sets for pathology and analysis. Fixed blocked brain tissue of a TBI subject imaged immediately post-injury and postmortem at 3T and 7T were processed using a high-throughput neuroanatomy pipeline. Brain tissue was sectioned at 20 μm using a custom tape-transfer system; mounted sections were stained for myelin, Nissl, and Iron-Prussian blue. Slides imaged using the NanoZoomer 2.0-HT system at 0.46 μm/pixel produced ∼550 serial JPEG2000 images per sample. The resulting digital datasets each of 3TB are displayed on a Djatoka image server allowing for dynamic dissemination. We create a volumetric brain stack by aligning images within a reference frame via 2D rigid transformations. The stack is registered to a reference MRI by optimizing correspondences between the volumes through a nonlinear transformation. The aligned 3D-stack is further processed to yield reconstructed sagittal, axial, and maximum intensity projections in each plane. Development for processing half and whole hemispheres are on-going and will permit the unbiased study of tissue without the previous constraint to small regions.
Keywords: histology, digital, pathology, whole-slide imaging, TBI
Support: NINDS #049177
Keywords: spinal cord injury, pancreas, vagus
DYSREGULATION OF MITOCHONDRIAL DYNAMICS AFTER TBI: A POSSIBLE ROLE FOR DYNAMIN-RELATED PROTEIN 1
University of Texas Health Science Center at Houston, Neurobiology and Anatomy, Houston, USA
Mitochondrial function is intimately linked to cellular survival, growth, and death. Mitochondria not only generate ATP from oxidative phosphorylation, but also mediate intracellular calcium buffering, generation of reactive oxygen species (ROS), and apoptosis. Electron leakage from the electron transport chain, especially from damaged or depolarized mitochondria, can generate excess free radicals that damage cellular proteins, DNA, and lipids. Furthermore, mitochondrial damage releases pro-apoptotic factors to initiate cell death. Previous studies have reported that traumatic brain injury (TBI) reduces mitochondrial respiration, enhances production of ROS, and triggers apoptotic cell death, suggesting a prominent role of mitochondria in TBI pathophysiology. Mitochondria maintain cellular energy homeostasis and health via balanced processes of fusion and fission, continuously dividing and fusing to form an interconnected network throughout the cell. An imbalance of these processes, particularly an excess of fission, can be detrimental to mitochondrial function, causing decreased respiration, ROS production, and apoptosis. Mitochondrial fission is regulated by the cytosolic GTPase, dynamin-related protein 1 (Drp1), which translocates to the mitochondrial outer membrane to initiate fission. Aberrant Drp1 activity has been linked to excessive mitochondrial fission and neurodegeneration. Measurement of Drp1 levels in purified hippocampal mitochondria showed an increase in TBI animals as compared to sham controls. Analysis of cryo-electron micrographs of these mitochondria also showed that TBI caused an initial increase in the length of hippocampal mitochondria at 24 hours post-injury, followed by a significant decrease in length at 72 hours. Post-TBI administration of Mdivi-1, a pharmacological inhibitor of Drp1, prevented this decrease in mitochondria length. Mdivi-1 treatment also reduced the loss of newborn neurons in the hippocampus and improved novel object recognition memory and context-specific fear memory. Taken together, our results show that TBI increases mitochondrial fission and that inhibition of fission improves hippocampal-dependent learning and memory, suggesting that strategies to reduce fission may have translational value after injury.
Keywords: mitochondrial dynamics, Drp1, neurogenesis, TBI
Elevated intracranial pressure (ICP) following traumatic brain injury (TBI) is associated with increased morbidity. Previously we found a correlation between ICP elevation and increased sub-acute neuronal membrane poration diffusely distributed throughout the lateral neocortex 6 h following TBI. While membrane porated neurons demonstrate both the potential to reseal their membranes or to remain perturbed and potentially progress to death, the time-course over which these processes are initiated is unknown. Therefore, in the current study, we investigated the time course of neuronal membrane poration sub-acutely following diffuse brain injury. We utilized a moderate central fluid percussion injury model in adult male rats paired with intraventricular infusions of cell-impermeable fluorescent tracers prior to and/or at various time points (6 h, 1d, 2d, 3d, 7d) following injury. Uptake of these tracers, indicative of membrane poration/perturbation, was assessed via confocal microscopic analysis at 1–7d post-injury. Cell loss 1w post-injury was quantified via hematoxylin and eosin analysis. To investigate the ongoing subcellular alterations occurring in sub-acutely membrane porated neurons, electron micrographs, labeled to detect the infused tracers, were assessed throughout the 1w sub-acute window. Through these approaches we observed ongoing neuronal membrane poration in layers V and VI of the lateral neocortex at all time points assessed following diffuse moderate TBI, however no correlative cell loss was observed at 1w post-injury. Neurons sustaining acute membrane poration following injury were readily apparent 7d post-injury, with indications of progressive membrane resealing throughout the sub-acute time course. While some neurons displayed subcellular changes consistent with perturbation or cell death, the majority of neurons sustaining active membrane poration exhibited few ultrastructural alterations indicative of degenerative change. These findings are in contrast to previous studies indicating that membrane poration within pericontusional and/or ischemic lesions primarily progress to cell death within the first week following injury, suggesting that diffuse neuronal membrane poration has a different pathogenesis following TBI. Understanding the pathological progression of diffuse neuronal membrane poration could lead to development of novel therapeutics for the treatment of TBI patients, in particular, those suffering from secondary ICP elevation.
Keywords: traumatic brain injury, membrane poration, central fluid percussion injury, sub-acute pathology, rat model
DIMERCAPROL PROVIDES NEUROPROTECTION IN ACROLEIN-EXPOSED PC-12 CELLS AND RATS CONTUSIVE SPINAL CORD INJURY
Purdue University, Weldon School of Biomedical Engineering, West Lafayette, IN, USA
Oxidative stress has been demonstrated to be involved in secondary injury in central nervous system trauma. Oxidative stress causes damages to key biomacromolecules, depletion of antioxidant defenses, dysfunction of mitochondria, elevation of neuroinflammation, and incites neuronal hyperexcitability, all of which are known contributory factors to neuronal death and myelin degradation. Acrolein, both a product and a catalyst of oxidative stress, due to its higher reactivity and longer existence in the body than better known reactive oxygen species, plays an important role in perpetuating oxidative stress. Decreasing acrolein levels post-spinal cord injury (SCI) using known acrolein scavengers, such as hydralazine and phenelzine, appears to be a promising therapy to halt secondary injury and neural degeneration by providing neuroprotection and enhancing motor and sensory functional recovery. Aiming to offer viable alternative acrolein scavengers to overcome potential side effects associated with the existing pharmacophores, we have identified dimercaprol, a FDA approved drug for metal poisoning, as a novel acrolein scavenger. Dimercaprol possesses two thiol functional groups capable of binding and neutralizing acrolein. In this study, we confirmed the reaction between acrolein and dimercaprol in an abiotic condition using Nuclear Magnetic Resonance (NMR) spectra. We have also revealed that dimercaprol at non-toxic concentrations could protect PC-12 cells from acrolein-mediated cell death in a dose dependent manner. Furthermore, we have discovered that dimercaprol at a safe dose reduced acrolein levels and associated tissue damage, promoted locomotor behavioral recovery, and mitigated neuropathic pain in a rat contusive SCI model. Taken together, this study suggests that dimercaprol may be an effective acrolein scavenger, which could offer neuroprotection by enhancing post-SCI motor function and mitigation of neuropathic pain post-SCI.
Keywords: acrolein, oxidative stress, dimercaprol, spinal cord injury, neuropathic pain
In the United States 1.7 million people receive a traumatic brain injury (TBI) annually and it is estimated there are 5.3 million people in the U.S. living with long term disabilities. Currently, there are no clinical treatments that are effective in alleviating the functional deficits of TBI. Progesterone (PROG), a neurosteroid with pleiotropic effects has been shown to be beneficial in multiple brain injury models. The purpose of this study was to investigate the neurologically protective effect of progesterone following traumatic brain injury in animals reared in Enriched Environments (EE). The current study used 27 male Long-Evans rats purchased at post-natal day 25 and reared to maturity in EE. After 91 days, 18 subjects received a bilateral controlled cortical impact (CCI) placed over the medial frontal cortex to produce a severe injury. Rats were administered intraperitoneal injection of either 10 mg/kg PROG or vehicle injections (peanut oil) 4 h post-injury and every 12 h for 72 h following the initial injection. Seven days post-injury the rats were tested on several behavioral tasks including the open field test, Barnes maze, Morris water maze, rotor-rod task, elevated plus maze, and forced swim task. Results from behavioral testing suggest that overall the intact animals performed significantly better than injured animals. Contrary to the established literature we found an intermediate effect of PROG when animals were tested on the MWM and rotor rod tasks, where intact animals performed significantly better than untreated group. An interesting additional finding using Elevated plus maze (EPM) to assess anxiety-like behaviors, overall intact animals spent significantly greater time in the closed arms than the injured groups. Analysis of the behavioral data from this study has shown that enriched housing before injury can impact behavior tasks in intact animals as well as functional recovery in injured animals when in combination with PROG. Future research should explore potential mechanisms related to pre-enriched housing that may influence recovery from TBI.
Keywords: progesterone, enriched environment, functional recovery
PIOGLITAZONE MAINTAINS ACUTE MITOCHONDRIAL INTEGRITY AND IMPROVES LONG-TERM FUNCTIONAL NEUROPROTECTION AFTER SPINAL CORD INJURY
University of Kentucky, Spinal Cord and Brain Injury Research Center, Lexington, USA
Emerging evidence suggests that pharmacological interventions which preserve mitochondrial integrity acutely following neurotrauma promote significant neuroprotection and functional recovery. The current study assessed the protective efficacy of pioglitazone on acute mitochondrial respiration and long-term functional neuroprotection following spinal cord injury (SCI). For acute mitochondrial assessments, adult male C57BL/6 mice (n = 31) were divided into 4 treatment groups: 1) Sham, 2) SCI + Vehicle 15 min, 3) SCI + Pio 15 min, and 4) SCI + Pio 3 hr. Mice received either T9 laminectomy (n = 7) or contusion SCI (75 kdyn, IH Impactor; n = 7–9/group). Injured mice received vehicle (DMSO, i.p., 15 min and 24 hr post-injury) or pioglitazone (10 mg/kg) either at 15 min or 3 hr post-injury, and their respective booster injections at 24 hr. At 25 hr, isolated mitochondria from sham and injured spinal cords were assessed for mitochondrial respiration. For long-term behavioral testing, injured mice (n = 22) were divided in two treatment groups 1) SCI + Vehicle and 2) SCI + Pio (n = 10–12/group) and received DMSO vehicle or pioglitazone (10 mg/kg) administered at 15 min post-injury and once daily for 5 days. Mice were assessed weekly for hindlimb function using the Basso Mouse Scale (BMS) for 4 weeks followed by terminal gridwalk analysis. The mice were then perfused with paraformaldehyde and coronal spinal cord sections were processed for histological assessments. Results showed that compromised mitochondrial respiration following acute SCI was significantly attenuated by pioglitazone. Moreover, behavioral assessments showed that pioglitazone significantly improved BMS scores over time, as well as terminal BMS subscores and gridwalk performance. Pioglitazone also significantly increased chronic grey and white matter sparing, suggesting that maintaining mitochondrial bioenergetics limited secondary tissue damage which promoted significantly improved functional recovery. Funding: NIH/NINDS 2P30NS051220; NIH/NINDS (R01 NS069633); SCoBIRC Chair Endowment (AGR)
Keywords: neuroprotection, therapeutics, bioenergetics, behavioral function
Traumatic brain injury (TBI) results in the production of peroxynitrite (PN), leading to oxidative damage of lipids and protein. PN-mediated lipid peroxidation (LP) results in production of 4-hydroxy-2-trans-nonenal (4-HNE). The goal of these studies was to explore the hypothesis that interrupting secondary oxidative damage following a TBI via acute pharmacological inhibition of LP by phenelzine (PZ), a 4-HNE scavenger, would protect against LP-mediated mitochondrial and neuronal damage. Male Sprague-Dawley rats received a severe (2.2 mm) controlled cortical impact (CCI)-TBI. PZ was administered subcutaneously (s.c.) at 15 min (10 mg/kg) and 12 hrs (5 mg/kg) post-injury. Cortical mitochondria were isolated at 24 and 72 hrs post-injury. Mitochondrial respiration was measured using a Clarke-type electrode and Ca2+ buffering was monitored using a spectrofluorometer. Protein samples were assessed for α-spectrin breakdown as an indicator of axonal cytoskeletal degradation and 4-HNE as a marker of LP by western immunoblot. Administration of PZ significantly improved mitochondrial respiration at 24 hrs compared to vehicle-treated animals and PZ-treated animals were never significantly lower than Sham. At 72 hours, injured animals had significantly lower respiration compared to Sham and PZ administration significantly improved mitochondrial respiration compared with vehicle-treated animals. These results demonstrate that PZ administration preserves mitochondrial bioenergetics at 24 hrs and that this protection is maintained out to 72 hrs post-injury. PZ administration also improved mitochondrial Ca2+ buffering capacity and mitochondrial membrane potential parameters compared to vehicle-treated animals at 24 hrs. The amount of α-spectrin breakdown in cortical tissue was also significantly reduced at both 24 hrs post-injury compared to vehicle-treated animals following administration of PZ. These results indicate that acute PZ treatment successfully attenuates LP-mediated oxidative damage eliciting multiple neuroprotective effects following TBI.
Acknowledgments: This work was supported by NIH/NINDS R01 NS083405 and R01 NS084857
Keywords: traumatic brain injury, mitochondria, lipid peroxidation, 4-HNE, α-spectrin, Ca2+ buffering
EVALUATION OF AER-271 IN THE MIAMI FLUID PERCUSSION MODEL OF TRAUMATIC BRAIN INJURY: AN OBTT CONSORTIUM STUDY
AER-271 is a drug developed by Aeromics to target brain edema and reduce ICP. This drug has previously been shown to reduce brain edema in a water intoxication model and after MCAO. AER-271 was chosen as the eighth drug to be tested by the multicenter consortium Operation Brain Trauma Therapy. The University of Miami site tested AER-271 in our model of fluid percussion TBI. Male Sprague-Dawley rats were anesthetized and underwent moderate fluid percussion (FP; 1.8–2.1 atm) TBI. Rats were randomized into three groups and administered AER-271 (2.5 mg/kg IV bolus followed by 48 h infusion) or vehicle 15 min post-TBI. Dosing was based on PK studies carried out in separate rats. Animal groups were TBI AER-271 (n = 15), TBI-Veh (n = 15) or Sham (n = 15). Rats were tested on day 7 post-injury for sensorimotor function (gridwalk, cylinder task). On days 13–21, rats were assessed for cognitive function utilizing the simple place task, probe trial and working memory task. On day 21, brain tissue was processed for histology. One-way ANOVA was not significant for the cylinder or the gridwalk tasks. For the hidden platform task, two-way repeated measures ANOVA for latency was not significant for group × day. TBI AER-271 treated animals treated groups exhibited similar latencies to TBI-Veh. There was no significant difference between groups for the probe trial. Repeated measures ANOVA for working memory latency was significant for trial (p < 0.001), but not group or group × trial. Lesion volume or cortical volume loss was not significant between the TBI groups. We conclude that sustained treatment with AER-271 after FP did not improve motor or cognitive function or decrease histological damage. Although this drug potentially targets several pathomechanisms important after TBI, the current study reports no benefit for AER-271 treatment in the rat FPI model. Support: US Army W81XWH-10-1-0623.
Keywords: traumatic brain injury, neuroprotection, behavior, OBTT, fluid percussion
NEUROPROTECTIVE EFFECTS OF CLOVANEMAGNOLOL IN AN IN VITRO MODEL OF RAPID STRETCH INJURY
University of Texas Medical Branch, Department of Anesthesiology, Galveston, USA
An in vitro injury model is an invaluable way to rapidly screen drug treatments in a high throughput manner without the use of a costly and labor intensive animal model of brain injury. Previously defined in vitro models have not used hippocampal cell types, which is an important feature for identifying neuroprotective drugs that may impact hippocampal dependent learning and memory. Our goal was to use a hippocampal cell line to establish a model for screening novel neuroprotective natural compounds. Here we test a compound derived from the magnolia tree, Clovanemagnolol, which has been shown to be neuroprotective in C. elegans and embryonic mouse hippocampal neuron cultures. H19-7 fibroblasts, an immortalized hippocampal cell line, were plated at a density of 150,000 cells per well in 0.01% poly-L-lysine coated collagen I BioFlex plate. Cells were cultured for 48 hours before receiving a moderate or severe rapid stretch injury (RSI) using the Cell Injury Controller II. Moderate and severe injury levels were established using Propidium Iodine and Fluoro-Jade C. Stretched cells were immediately treated with Clovanemagnolol (5–100 nanomolar). After 24 hours, we assessed cellular damage using a lactate dehydrogenase (LDH) assay. We chose to define moderate cell injury as 20–30% dying cells and severe cell injury as 50–60% dying cells. Treatment with Clovanemagnolol reduced cellular injury, which was demonstrated by a reduction in LDH levels after rapid stretch injury compared to cells that only received RSI. These findings show that our in vitro model of hippocampal injury can be a valuable tool for screening neuroprotective drugs such as Clovanemagnolol. We are currently testing other potentially neuroprotective compounds using this in vitro injury model.These studies were completed as part of an interdisciplinary research team funded by the Moody Project for Translational Traumatic Brain Injury.
Keywords: hippocampal injury, rapid stretch injury, in vitro model
UTHSCSA, Physiology, San Antonio, USA
Traumatic brain injuries (TBIs) are common amongst military personnel and in civilian populations, and there are currently no FDA-approved treatments. Over 6% of all epilepsies are due to TBIs. Throughout the CNS, voltage-gated “M-type” K+ ion channels play dominant roles in controlling neuronal firing and excitability; consequently, M-channel “openers” such as retigabine, have emerged as novel anti-convulsants. Astrocytes also play major roles in reducing abnormal neuronal electrical activity by activating energy-dependent transport mechanisms. TBI-induced seizures are accompanied by pronounced cytotoxic and vasogenic edema. Our research relies on identifying novel molecular targets to reduce acquired epilepsies and associated co-morbidities that develop after TBIs. Mice were subjected to controlled closed-cortical impacts (CCCI), with administration of retigabine (i.p. 1 mg/kg), the P2Y1-receptor agonist (MRS2365, (i.p. .85 mg/kg) both drugs or only vehicle, within 30 minutes of TBI, or a smaller cohort of sham TBI. After 24 hours, mice were implanted with EEG electrodes and seizure activity recorded the following day. Six days post-TBI, mice were given the chemoconvulstant, pilocarpine (i.p. 75 mg/kg) at 30-minute intervals to induce subsequent seizures as a test for alterations in seizure susceptibility. Methylscopolamine (1 mg/kg) was administered i.p. 30 minutes before application of pilocarpine to minimize peripheral cholinergic effects and mortality. Seizure susceptibility was assayed 24 hours after 3 injections of pilocarpine. Seizures were monitored by behavioral video and EEG recording. Our preliminary data show that administration of both retigabine and MRS2365 in combination to mice within 30 min of TBI decreases subsequent post-TBI seizures as well as seizure susceptibility. Thus, no spontaneous post-TBI seizures were detected after TBI in retigabine treated animals and the percentage of mice displaying post-TBI seizures was decreased in both combinational and MRS2365 groups vs. vehicle controls. This approach has the potential to reduce the incidence of seizures after a TBI. We will present further preliminary data on mice undergoing CCCI-induced TBIs and likely also blast-TBIs, for this meeting.
Supported by DOD#CDM-RPW81XWH-15-1-0284
Keywords: potassium channel, traumatic brain injury, seizures, epilepsy
Walter Reed Army Institute of Research, BTNN, Silver Spring, USA
Amantadine hydrochloride was the 9th drug selected for testing by the Operation Brain Trauma Therapy Consortium. Amantadine is a non-competitive NMDA antagonist shown to inhibit microglial activation and improve cognitive recovery in pre-clinical TBI models. Clinically, Amantadine has produced beneficial effects on cognition, motor performance, and/or wakefulness in TBI patients. The WRAIR site evaluated the effectiveness of Amantadine in the penetrating ballistic-like brain injury (PBBI) model. PBBI (10%) was performed unilaterally in the right hemisphere of anesthetized rats. Rats were randomized into three groups. Amantadine (AMA; 10 mg or 45 mg/kg IP) or vehicle administered once daily for 18 days, beginning 1 day post-PBBI. Animal groups were PBBI-AMA-Low (n = 13), PBBI-AMA-High (n = 12), PBBI-Vehicle (n = 14), or sham (n = 15). Rats were tested on days 7 and 10 post-injury for motor function on rotarod task and on days 13–21, for cognitive function in Morris water maze (MWM) task. On day 21, brain tissue was processed for histology. Both PBBI-Vehicle and PBBI-AMA-Low groups displayed significant motor impairment versus sham (p < .05). However, no significant motor impairment was detected in PBBI-AMA-High. Mean overall Motor scores were reduced by *30% (PBBI-Vehicle), *26% (PBBI-AMA-Low), and 9% (PBBI-AMA-High) (*p < .05 vs. sham). MWM results showed significant deficits in all injury groups with mean latencies to the hidden platform increased by 108% (PBBI-Vehicle), 88% (PBBI-AMA-Low), and 108% (PBBI-AMA-High) versus sham (p < .05). A trend towards improved cognitive performance was observed in PBBI-AMA-Low, but not significant. Notably, AMA reduced lesion volume (both doses) and hemispheric/cortical volume loss (high dose) versus PBBI-vehicle (p < .05). Overall despite not performing well in other OBTT models, Amantadine has shown the most benefit of any OBTT drug tested in the PBBI model and, thus, may warrant further exploration in this model with a special potential for penetrating TBI. Supported by U.S. Army Grant W81XWH-10-1-0623.
Keywords: traumatic brain injury, OBTT, behavior
Levetiracetam (LEV) is a US Food and Drug Administration (FDA) approved, second generation anti-epileptic drug that has demonstrated reproducible neuroprotective effects in multiple preclinical traumatic brain injury (TBI) models using a limited range of dose concentrations. We have recently reported that an extended dosing regimen out to at least 10 days post-injury is required to confer neuroprotection in our model of severe penetrating TBI. The goal of the current study was to evaluate the comprehensive dose-response profile of LEV on motor and cognitive measures using our established treatment regimen in the penetrating ballistic-like brain injury (PBBI) model. Unilateral frontal PBBI was produced in the right hemisphere of isoflurane anesthetized rats (10% injury severity level). LEV (Keppra, 5 mL sterile ampule 100 mg/ml; Sun Pharmaceuticals; 25, 50, 100 or 200 mg/kg) was administered as bolus injections via indwelling jugular vein catheters at 30 min and 8 hrs post-injury, and twice daily thereafter (0900 and 1700 hrs) for 10 consecutive days. Motor function was evaluated on the rotarod (7 and 10 days post-injury) using fixed-speed increments of 10, 15, and 20 rpm. Cognitive performance was evaluated on the Morris water maze (MWM) on days 13–17 post-injury (4 trials/day). Pharmacokinetic analysis of blood plasma and brain tissue (50 mg/kg dose only) was completed for acute time points of 2 and 8 hrs post-injury. Histopathological analysis included lesion volume and total hemispheric tissue loss at terminal end point of 22 days post-injury. Motor and cognitive testing revealed significant deficits in all injury groups versus sham and a dose-dependent profile of recovery as compared to vehicle controls. Given its broad therapeutic index, nearly 100% bioavailability and potent anti-seizure and neuroprotective effects, LEV is emerging as an exciting candidate for future combination drug therapy studies for TBI. This study was funded by the Army Combat Casualty Care Research Program.
Keywords: Sprague Dawley rat, drug therapy, penetrating TBI, Morris water maze, rotarod
DELETION OF T1 ISOFORM OF TYROSINE-KINASE B ELIMINATES NEUROPATHIC PAIN AFTER SCI THROUGH REGULATION OF ASTROCYTES
In addition to causing sensorimotor deficits, spinal cord injury (SCI) also results in post-traumatic neuropathic pain in a majority of patients, which is often resistant to conventional pain therapy. Identifying better interventions to manage SCI-Pain requires improved understanding of the pathophysiological mechanisms involved. We previously demonstrated that up-regulation of TrkB.T1, a truncated isoform of the Brain-derived Neurotrophic Factor (BDNF) receptor, contributes to the pathobiology of SCI and SCI-Pain through modulation of cell cycle pathways. Astrocytes express only the TrkB.T1 isoform, and therefore provide a model for exploring the role of TrkB.T1 in SCI-Pain. We examined spatially and temporally cell-specific TrkB.T1 changes following SCI. We also investigated effects of TrkB.T1 in cultured astrocytes derived from neonatal TrkB.T1+/+ and TrkB.T1-/- mice, as well as in SCI-Pain using astrocytic TrkB.T1 knock-out (KO) mice. Immunohistochemistry showed that astrocytes expressing TrkB.T1 were significantly up-regulated in both the lesion area and lumbar spinal dorsal horn after SCI. TrkB.T1 was also highly expressed by neurons in the gray matter and by microglia/macrophages, most significantly in the white matter nearest to the traumatic lesion. In vitro, TrkB.T1-/- astrocytes showed a slower proliferation in response to 10% FBS than that of TrkB.T1+/+ astrocytes. BDNF stimulated proliferation of astrocytes derived from TrkB.T1+/+ but not TrkB.T1-/- mice. Following SCI, astrocytic TrkB.T1 KO mice showed reduced hyperpathic responses compared to WT controls in von Frey mechanical stimulation and in pain-related measurements on the CatWalk, as well as improved recovery of motor function and coordination. Together, our data suggest that the presence of TrkB.T1 in astrocytes may play an important role in the modulation of neuropathic pain and functional recovery following SCI. Thus, TrkB.T1 may provide a potential therapeutic target for treatment of SCI.
Keywords: spinal cord injury, neuropathic pain, TrkB.T1, brain-derived neurotrophic factor
ISG15 PHOSPHORYLATES GLUT1 REGULATING GLUCOSE TRANSPORT ACROSS BBB IN A PEDIATRIC TBI EXPERIMENTAL MODEL
LSUHSC, Department of Pediatrics, New Orleans, USA
Protein expression
BBB Disruption
Keywords: pediatrics, traumatic brain injury, ISG15, glucose
UCSF, Neurosurgery, San Francisco, USA
Neutrophils are the “first responders” to sites of infection. However, in the injured developing brain, activated neutrophils are a source of proteolytic enzymes and reactive oxygen species that contribute to early secondary pathogenesis and may influence long-term recovery. We have previously shown that traumatic injury to the developing murine brain results in persistent cognitive deficits in adulthood and that these adverse outcomes are partially rescued in mice genetically deficient in neutrophil elastase, a protease released by activated neutrophils. We propose that the activated neutrophils direct early pathogenesis and are determinants of long-term cognitive injury. As neutrophils are activated via the downstream signaling pathway dependent on spleen tyrosine kinase (Syk), we evaluated brain-injured conditional knockouts of Syk (sykf/fMRP8-cre +) along with congenic littermates (sykf/f ). Flow cytometry revealed not only prolonged recruitment of neutrophils but also provided the first evidence of infiltration of distinct leukocyte subsets across both genotypes. While the magnitude of recruitment did not differ across genotypes, barrier disruption to fluorescently tagged dextrans, corresponding to the molecular weights of albumin (MW 70 kD) and fibrinogen (MW 500 kD), was significantly reduced in the sykf/fMRP8-cre + mice. We next examined the long-term behavioral and cognitive consequences after injury. Injury-induced hyperactivity and risk taking behavior was limited to sykf/f mice. Assessment of cognitive function in adulthood revealed robust, significant improvements in brain-injured sykf/f MRP8-cre+ mice in task learning and short and long-term spatial memory retention. Together, these findings confirm the extended recruitment of neutrophils, and identify different subsets of infiltrating leukocytes to the injured developing brain. Furthermore, there was a profound disruption of the barrier to a broad range of molecular tracers that is in part attributed to activated neutrophils. This study establishes the first mechanistic link between activated neutrophils and long-term cognitive injury.
Keywords: traumatic brain injury, neutrophils, pediatric, signaling, cognitive behavior
University of New Mexico, Pediatrics & Neurosciences, Albuquerque, USA
Traumatic brain injury (TBI) is a leading cause of death and severe morbidity for infants born healthy at term. Despite the high prevalence and incidence of lifelong deficits from infant TBI, no targeted treatment currently exists to actively promote repair. Using a translatable preclinical model of infant TBI, we hypothesized that magnetic resonance imaging (MRI) and touchscreen cognitive testing could be used to quantify outcomes and potential efficacy of the neuro-reparative agent erythropoietin (EPO). Accordingly, controlled cortical impact (CCI) was performed on postnatal day 12 (P12) rats of both sexes. On post-injury day (pid) 1, rats were randomized to intraperitoneal EPO (3000 U/kg/dose) or vehicle (sterile saline) over pid 1–8, and coded. Beginning at P35, rats were trained on a touchscreen operant platform by blinded investigators. Following successful training, rats performed visual discrimination (VD) and reversal tasks to assess executive function and cognitive flexibility (n = 6–8/group). Ex vivo MRI was performed on a 4.7T at P15 and P45. Sham, CCI-veh, and CCI-EPO rats were compared using two-way ANOVA with Bonferroni's correction. Results show diffusion tensor imaging (DTI) of ipsilateral and contralateral regions in CCI-veh P45 rats have widespread bilateral injury and significant abnormalities of functional anisotropy (FA), mean diffusivity (MD), axial (AD) and radial diffusivity (RD). Treatment with EPO reversed changes in MD, AD and RD. EPO treatment also ameliorated deficits in cognitive flexibility on a touchscreen platform, including error reduction and perseveration (all p < 0.05). In conclusion, in our model of moderate-severe infantile TBI, EPO, administered in an extended dosing paradigm congruent with its mechanisms of action, proved efficacious in reversing microstructural and functional impairment in developing rats. To our knowledge, this is the first demonstration using the highly translatable, touchscreen platform of cognitive assessment for TBI. These data support the use of age-appropriate preclinical models with human clinical trial-compatible imaging biomarkers and outcome measures.
Keywords: infant tbi, erythropoietin, executive function, diffusion tensor imaging
Keywords: sonic hedgehog signaling, transgenic mice, spinal cord development/injury, expression profile
INSULIN-LIKE GROWTH FACTOR-1 MEDIATES REGIONAL ALTERATIONS TO THE MTOR SIGNALING PATHWAY IN THE HIPPOCAMPUS FOLLOWING TBI
University of Kentucky, SCoBIRC/Physiology, Lexington, USA
Insulin-like Growth factor 1 (IGF1), an endogenous growth factor that promotes neuronal survival and plasticity, has significant potential as a therapeutic candidate for TBI. In the nervous system PI3-K/Akt signaling predominates in mediating many functions of IGF1, including precursor proliferation and differentiation and neuronal survival. However, the complex signaling pathways through which this growth factor modulates brain plasticity in the setting of TBI remain unclear. In a transgenic mouse model with conditional IGF1 overexpression restricted to astrocytes, we previously showed that increased IGF1 levels in the hippocampus by means of injury-induced astrogliosis leads to increased activation of Akt. Akt activation results in the phosphorylation of multiple downstream signaling molecules including mammalian target of rapamycin (mTOR). Following brain injury, mTOR is transiently activated in the hippocampus. We hypothesized that increased brain levels of IGF1 would potentiate posttraumatic activation of the mTOR signaling pathway, a pathway associated with growth and differentiation. To this end, astrocyte-specific IGF1 overexpressing (IGF1-TG) mice and wild-type (WT) mice received controlled cortical impact (CCI, n = 8/genotype/injury level/time point) or sham (n = 2–3/genotype/time point) injuries. To evaluate if the effects of IGF1 on mTOR activity were dependent on injury severity, moderate and severe CCI were compared. At 24 hrs and 72 hrs following injury, immunohistochemical labeling of pS6, a well characterized downstream effector of mTOR, was quantified in the granule cell layer, molecular layer, and the hilus of the dentate gyrus. Analysis of pS6 at the injury epicenter suggests that IGF1 stimulates activity of the mTOR pathway following TBI in a manner dependent upon the severity of injury.
Funding Sources: Kentucky Spinal Cord and Head Injury Research Trust (KSCHIRT) 14-12A and NIH awards: R01 NS072302-02S1, R01 NS0072302, T32 NS077889, P30 NS051220.
Keywords: mTOR, IGF1
BioAxone BioSciences Inc., Cambridge, USA
PTEN (phosphatase and tensin homologue) is a novel target to promote axon regeneration in the central nervous system (CNS) that has been validated by experimental techniques that delete PTEN expression. Therapeutic drugs that target PTEN selectively to down-regulate its expression have not been developed. We created self-delivering interfering RNA (sdRNA) against PTEN sequences conserved in the rat and human to evaluate PTEN as a target to promote axon regeneration in the CNS and to create a potential therapeutic to treat CNS trauma. Self-deliverable RNAs are double stranded siRNA-like molecules that are modified to be cell permeable and reduce the risk of off-target effects. Protein reduction by sdRNA molecules of similar design have been translated to Phase 2 clinical trials. Unlike siRNA, sdRNA is extensively modified and many candidates must be screened to select highly potent drugs. We screened multiple in silico-predicted lead sequences with a luciferase reporter assay and the most efficient sequences were further screened by qPCR of PTEN mRNA. The best sequence, called BA-434 was further tested to confirm PTEN protein knockdown in both PC12 cells and primary neurons. In primary neurons, sdRNA knockdown was maximal 3 days after transfection, and maintained for over 5 days, even after sdRNA was removed from the culture medium. We tested efficacy in vivo in rat retinal ganglion cells (RGCs) after intraocular injection of 30–60 μg sdRNA. BA-434 knocked down PTEN expression in RGCs, as observed by both immunocytochemistry in retinal whole mounts and by Western blot. Experiments to investigate the ability of PTEN knockdown by sdRNA to promote RGC regeneration in the rat optic nerve after injury are in progress.
Keywords: PTEN, regeneration, RNAi
Texas Womans University, Biology, Denton, USA
Traumatic spinal cord injury leads to neuronal damage and results in varying levels of functional impairment. Nanomaterial-based drug delivery systems provide potential for axon regeneration from specific neurons by crossing blood-brain barrier. In our work, we analyzed whether magnetic nanoparticles (MNPs) detrimentally affect outgrowth processes in primary dorsal root ganglion (DRG) neurons from embryonic chick. The DRG neurons were extracted from E9-12 chick embryos and dissociated. The dissociated DRG was treated with different concentrations of MNPs for 72 hours. Images were obtained and analyzed using a Nikon A1 confocal microscope system. Our study showed that the highest dose of MNPs applied was minimally toxic for DRG cells treated for 72 hours. We found that 72 hours of MNP treatment did not affect the number of neurites, branches and neurite length in DRG cells. We did not see any effect on the morphology of neurons after MNP treatment. Also, we used rat primary neurons to study the uptake efficiency of surface functionalized nanoparticles (SFNPs) by different cell types in mixed cortical culture. Primary rat cortical cells were extracted from P0-P2 rats and cultured on PDL coated plates. The cultured cells were treated with -NH2 and -COOH surface functionalized nanocarriers for 30 mins. After immunocytochemistry, images were obtained using a Nikon A1 confocal microscopy and analyzed. Uptake of a portion of our nanoparticles was seen in cortical neurons. Together, these results show the feasibility of nanocarriers for targeted drug delivery to encourage axon regeneration following nervous system damage.
Keywords: DRG, Nanomaterial, uptake efficiency, mixed cortical culture
John D. Dingell VA Medical Center, Detroit, USA
Traumatic brain injury (TBI) increases susceptibility to a variety of neurobehavioral disorders, such as mood disorders, learning and memory impairments, and substance use disorders, including alcohol use disorders (AUDs). AUD is the second most common comorbid Axis I diagnosis resulting from TBI, yet the mechanisms involved are not fully understood. AUD is thought to be influenced by changes in striatal regions, including the nucleus accumbens (NAC), potentially due impaired neuroplasticity. Previously, we have observed no structural alterations in dendrites of medium spiny neurons of the NAC following TBI, suggesting that NAC-mediated neurobehavioral disruptions may be subserved by subcellular/molecular mechanisms that are independent of structural modifications. Here we examine effects of mild and moderate TBI on protein markers of synaptic plasticity: GAP-43 (known to be concentrated in growth cones of axons during development and neuroregeneration), and PSD-95 (a post-synaptic scaffolding protein found in dendritic spines), in NAC in the sub-acute and delayed post-injury periods.
Anesthetized male C57BL/6 mice (6–8 wks) were given a mild or moderate, midline impact over the intact skull, or sham surgery. At 14 or 30 d post-injury, brains were harvested and frozen. For Western blot analysis, protein lysates were made from 1.5 mm diameter NAC punches taken from 2 mm thick coronal slices.
Moderate TBI decreased GAP-43 protein levels in the NAC, while mild TBI increased PSD-95 protein levels at 14 d post-injury. Decreases in GAP-43 may represent a loss of axonal migration (less regeneration); while increased PSD-95 suggests changes in subcellular structural elements or glutamate receptor clustering, that may affect synaptic transmission and plasticity. These data demonstrate that severity differentially influences the synaptic response profile to injury in the NAC. Understanding the subcellular mechanisms by which TBI alters striatal plasticity in the NAC is crucial in clarifying the relationship between TBI and striatal-mediated behaviors related to AUDs.
Keywords: TBI, alcohol, striatum, synaptic plasticity
University of Louisville, Physiology and Biophysics, Louisville, USA
Support: This study was funded by the DOD (SC110169).
Keywords: locomotor function, stretching, Nociceptive afferents, muscle
University of Louisville, KSCIRC, Louisville, USA
Disruption of autonomic pathways following spinal cord injury (SCI) leads to altered cardiovascular homeostasis. Cervical and high thoracic lesions disrupt supraspinal control of sympathetic preganglionic neurons of the heart and upper body vasculature, leading to a state in which episodic hypertensive crises known as autonomic dysreflexia (AD) can develop. Rodent studies utilizing thoracic transection have shown that appropriately-timed rehabilitation has the ability to reduce AD severity. To date, no study has examined the impact of acute rehabilitation on CV function in a more clinically relevant contusive model.
Keywords: cardiovascular, blood pressure regulation
University of Pittsburgh, Safar Center for Resuscitation Research, Pittsburgh, USA
Environmental enrichment (EE) has been shown to facilitate motor recovery and hasten spatial learning and memory when provided after traumatic brain injury (TBI). These effects are observed in both male and female rats as well as adult and pediatric populations. Typical explanations for the EE-mediated benefits are reductions in hippocampal cell loss and increased neurogenesis. The goal of this study was to assess other pathological mechanisms that are prevalent after TBI. Anesthetized male rats received a controlled cortical impact or sham injury, then were housed in EE or standard (STD) conditions, and subsequently evaluated for motor (beam-walk/balance) and cognitive (Morris water maze) performance as well as inflammation via microglial activation (Iba1) and oxidative stress (3-NT). EE improved both motor and cognitive performance relative to STD (p < 0.05). Moreover, EE downregulated TBI-induced Iba1 expression levels in both hemispheres (p < 0.05) and also reduced 3-NT immunostaining in the ipsilateral hemisphere (p < 0.05). These data suggest that in addition to neurogenesis, EE may mediate benefits after TBI by attenuating inflammation and oxidative stress.
Supported by: NIH, R01HD069620 and R01NS084967 (AEK)
Keywords: environmental enrichment, oxidative stress
Naval Medical Research Center, NeuroTrauma Department, Silver Spring, USA
Perfluorocarbons (PFCs) can transport 50 times more oxygen than human plasma which may be advantageous in treating patients with TBI. We hypothesized that a prehospital intravenous dose of the PFC emulsion NVX-108 (NuvOx Pharmaceuticals, Tucson, AZ), could improve brain tissue oxygenation and other physiological parameters in a swine model of Fluid Percussion Traumatic Brain Injury (FP-TBI). Anesthetized and instrumented Yorkshire swine were assigned to four experimental groups: 1) TBI-PFC:FP-TBI at time 0 (T0) and NVX-108 (1 ml/kg) at time 15 minutes (T15); 2) TBI-Controls: FP-TBI at T0 and no NVX-108 at T15; 3) Sham-PFC: No FP-TBI at T0 and NVX-108 (1 ml/kg) at T15; and 4) Sham-Controls: No FP-TBI at T0 and No NVX-108 at T15. Along with other physiological parameters, partial pressure of brain tissue oxygen (PbtO2) was measured with LICOX® probe for 6 hours after FP-TBI, followed by euthanasia. Compared to controls, NVX-108 did not improve PbtO2 irrespective of whether the group had TBI or not (p > 0.05). Mean arterial pressure (MAP) was higher (10 mm of Hg) only in the Sham-PFC group compared to Sham-Controls (p < 0.001). In groups that received NVX-108, there was a transient increase in mean pulmonary artery pressure immediately after infusion compared to controls (p < 0.001). Furthermore, there was a significant increase in cardiac output (CO) and intracranial pressure (ICP) and a significant decrease in cerebral perfusion pressure (CPP) following TBI compared to controls (p < 0.001). CPP was also higher only in Sham-PFC group compared to Sham-Controls (p < 0.001). In Conclusion, NVX-108 did not improve PbtO2 compared to controls. Furthermore, NVX-108 increased MAP and CPP only in animals without TBI. These results differ from previous promising results in a rodent model. Additional studies would be needed to explain these differences.
Keywords: traumatic brain injury, perfluorocarbon, brain tissue oxygenation
After sustaining a traumatic brain injury (TBI), a person's ability to make daily decisions can be affected. Simple tasks such as, deciding what to wear are no longer effortless choices, but are instead difficult decisions. Incorporating behavioral assays that address decision-making skills after TBI can help a pharmacological treatment become easily translatable, as it is specifically assessing a certain aspect of cognitive functioning. Magnesium is a multimodal treatment that can decrease apoptosis, decrease breakdown of the blood brain barrier, and lessen brain edema after a TBI, which can affect the recovery of a patient. A discrimination task was used in conjunction with a magnesium treatment in order to examine how decision-making is affected after TBI and if the treatment helps to attenuate cognitive and motor deficits. Thirty-one male Sprague-Dawley rats (Harlan, Indianapolis, IN) were used and separated into MAG/TBI, VEH/TBI, or VEH/Sham groups. Before induction of a bilateral frontal injury, rats were shaped to learn to dig in the sand for a reinforcer and then pre-trained on the dig task. After surgery, rats received either an i.p. injection of 2 mmol/kg magnesium chloride or 0.1% phosphate buffer solution (PBS). Magnesium injections occurred 4 hours post-surgery, then at 24 hours and at 72 hours. Dig task testing began 7 days post-injury, lasting for 4 weeks. The discriminations included two scent pairings; basil (baited) versus coffee then the reversal and then cocoa (baited) versus cumin then the reversal. The locomotor placing task was conducted in order to assess for the recovery of motor function after TBI. The results of the study showed that MAG/TBI animals performed just as well as the sham animals on the initial discrimination and the reversal, as well as the reversal of the second discrimination. The magnesium treatment was also successful at attenuating motor deficits and lessening the lesion effect after the TBI. Discrimination testing and a magnesium treatment both have the potential to positively impact the millions of people suffering from a TBI.
Keywords: discrimination tasks, TBI, magnesium, the dig task
EFFECT OF POLYTRAUMA VERSUS ISOLATED TRAUMATIC BRAIN INJURY ON 3-MONTH OUTCOME
UCSF, Neurosurgery, San Francisco, USA
Traumatic brain injury (TBI) is a leading cause of death and disability in the United States. The Glasgow Outcome Scale Extended (GOS-E) is the most widely used measure to assess TBI recovery, however its accuracy in evaluating and tracking outcome is limited because peripheral injuries from polytrauma may confound its measurement. The Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot dataset was utilized to investigate the potential effects of polytrauma on 3-month GOS-E. Polytrauma was defined as an Abbreviated Injury Score (AIS) >2 in any extracranial region. Univariate statistics are reported for TBI+Polytrauma vs. isolated TBI cohorts, and a binary logistic regression tested association of AIS with failure to reach good recovery (GOSE <7) at 3-months adjusting for demographics, history of psychiatric disorder, Glasgow Coma Scale, loss of consciousness, and intracranial pathology on CT. 422 patients (median age, 44 years; IQR 27–58) were included in the analysis. The majority (83%) had mild TBI (GCS 13–15), and 18.4% of patients had TBI with polytrauma (TBI+polytrauma). On univariate analysis, TBI+polytrauma showed a greater risk of disability at 3-months (58% vs. 37%, p = 0.001). Findings were confirmed on multivariable analysis as TBI+polytrauma demonstrated an odds ratio (OR) of 2.04 ([95% CI 1.15–3.61]; p = .014) for failure to achieve good recovery at 3-months post injury (GOSE <7). Thus the poorer outcome of TBI+polytrauma subjects was associated with the secondary trauma rather than the TBI per se. These results highlight the need to closely examine the characteristics of those with poorer outcome. Further, methodological improvements on the utility of the GOS-E to assess disability attributable specifically to TBI versus other systemic injuries will be beneficial in qualifying a standardized assessment tool for clinical care.
Keywords: traumatic brain injury, polytrauma, outcome, GOSE
This study examined the temporal change in cardiac baroreflex response following severe traumatic brain injury (TBI) with or without additional hypotensive/hypoxemic insults. Rats were randomly assigned into three groups – sham craniotomy only, 10% PBBI only, and PBBI combined with hypoxemia/hemorrhagic shock (PHH). The PHH group received 30-min hypoxemia (fraction of inspired oxygen = 0.1) and then 30-min hemorrhagic hypotension (mean arterial pressure = 40 mmHg) following PBBI. To assess the baroreflex function, phenylephrine was injected via femoral vein catheter with a sequential dosing regimen of 2, 1, 0.5, 5, 10 μg/kg. Systolic blood pressure (SBP) and electrocardiogram were recorded. Twenty minutes after the last dose of phenylephrine, the dosing regimen was repeated. This phenylephrine pressor test was performed on the injury day (day 0), and again on 1 and 5 days post-injury. A linear regression line of heart period (R-R interval from the electrocardiogram) against SBP was plotted for each test, in which the slope of the line was taken as an index of baroreflex sensitivity (BRS). Additionally, heart rate was derived from the electrocardiogram recordings. In PBBI and PHH group, BRS was higher than that in the sham group at all time points. While BRS remained steady in the sham control group (ranged 0.26 ± 0.08 – 0.36 ± 0.11 ms/mmHg) throughout the experiment, it exhibited a gradual increase in the PBBI group with the peak (0.65 ± 0.15 ms/mmHg) occurring at day 5 post-injury. In contrast, a decreasing trend was observed in the PHH group, in which an acute increase in BRS (0.59 ± 0.1 ms/mmHg) was detected on day 0. The differences in heart rate were not significant between groups, yet PHH trended lower acutely following injury and at 5 days post-injury, indicating bradycardia. Persistent disturbance of cardiovascular responses induced by TBI and the additional insults may potentially result in reduced perfusion of the vital organs and consequently worsen the clinical outcomes. (Funding: USAMRMC CCCRP H_024_2014_ WRAIR)
Keywords: polytrauma, cardiac response, baroreflex sensitivity, hypoxemia, hemorrhagic shock
CAFFEINE PRE-TREATMENT ALTERS TBI-INDUCED SLEEP DISRUPTIONS
Sleep disturbances are commonly reported during military deployment and can have lasting effects which impede restorative processes, impair cognition, disrupt sensorimotor function and promote a pro-inflammatory state. Service members often rely on stimulants (caffeine) to counteract the effects of sleep-loss. The impact of these drugs on physiological function and TBI recovery is unknown. We tested the hypothesis that caffeine-induced alterations in pre-injury sleep patterns modulate sleep homeostasis following TBI. Animals were randomly assigned to treatment cohorts of caffeine (25 mg/kg) or vehicle and subjected to penetrating ballistic-like brain injury (PBBI) or sham procedure. Caffeine or vehicle was administered gavage 1 h prior to the light phase and electrocorticographic recordings were objectively scored and analyzed for changes in sleep homeostasis. In uninjured animals, caffeine dose-dependently altered sleep parameters; rats showed significant decreases in total sleep time with concomitant increases in sleep. We found a time-dependent effect of caffeine on percentage of time spent in SWS, REM, and WAKE in the first 4 hours as well as a significant decrease in the number of SWS-REM transitions. Following PBBI, sleep recordings in vehicle-treated rats revealed reductions in total wakefulness (45%) and REM (55%) sleep with concomitant increases in SWS (17%) compared to sham. Caffeine appeared to mitigate these effects—changes in total REM and SWS periods in caffeine pre-treated PBBI rats were not significantly different from sham animals. Caffeine pre-treatment also increased the number of SWS-REM sleep transitions and reduced systemic levels of the inflammatory marker, C-Reactive Protein (12%) compared to vehicle. Studies determining the effect of chronic caffeine pre-treatment on TBI sleep architecture are currently ongoing. Overall, our results suggest that pretreatment with caffeine prior to TBI may reduce TBI-induced sleep alterations in a vulnerable population with pre-disposed sleep disorders.
Keywords: caffeine, sleep, EEG, REM
GLUTAMATE INCREASES SURVIVAL, PROLIFERATION AND ATTENUATES OXIDATIVE STRESS-INDUCED CELL DEATH IN ADULT SPINAL CORD-DERIVED NSPCS
Krembil Neuroscience Centre, Toronto Western Hospital, University Health Network, Toronto, Canada
Traumatic spinal cord injury (SCI) leads to a cascade of secondary chemical insults including oxidative stress and glutamate excitotoxicity that damage host neurons and glia. Transplantation of neural stem/progenitor cells (NSPCs) has shown promise in enhancing regeneration after SCI although survival of transplanted cells remains poor. Understanding the response of NSPCs to the chemical mediators of secondary injury is essential in finding therapies to enhance survival. We therefore examined the in vitro effects of glutamate on adult rat spinal cord-derived NSPCs both alone and in the setting of oxidative stress. NSPCs isolated from the periventricular region of the adult rat spinal cord were exposed to glutamate (5–1000 μM) in vitro. Oxidative stress was induced using 500 μM hydrogen peroxide (H2O2). Glutamate receptor-specific antagonists and agonists were used to elucidate the mechanisms by which glutamate acts on NSPCs. Glutamate treatment (500 μM for 96h) significantly increased cell viability and proliferation but did not alter phenotype of NSPCs. Concurrent glutamate treatment in the setting of H2O2 exposure increased cell survival compared to H2O2 exposure alone. The effects of glutamate on NSPCs were blocked by the AMPA/kainate receptor antagonist GYKI-52466 but not by the NMDA receptor antagonists MK-801 or DL-AP5, or the mGluR3 antagonist LY-341495. Furthermore, treatment of NSPCs with AMPA/kainate receptor agonists (AMPA, kainic acid or ATPA) mimicked the responses seen with glutamate both alone and in the setting of oxidative stress. Together, these results show that glutamate increases survival and proliferation and attenuates oxidative stress-induced cell death in adult NSPCs via AMPA/kainate receptors. These findings offer important insights into potential mechanisms to enhance NSPC survival and suggest that glutamate may have a role in promoting NSPC survival and proliferation after traumatic SCI.
Keywords: neural stem cells, glutamate, oxidative stress, AMPA receptors, kainate receptors
University of Pittsburgh, Neurosurgery, Pittsburgh, USA
Traumatic brain injury (TBI) impairs neuronal function which can culminate in lasting cognitive dysfunction. Previous work from our lab implicates impaired neurotransmission as a contributor to neurobehavioral dysfunction after TBI, but little is known about the mechanisms underlying this pathology. Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is an essential step in vesicle fusion and neurotransmitter release at the synapse. We recently showed that severe TBI significantly reduces SNARE complex formation; however, it is unknown if these deficits persist in models of milder injury with persistent cognitive deficits. We hypothesized lateral fluid percussion injury decreases SNARE complex formation. To this end, male Sprague Dawley rats were subjected to sham or 2 atm lateral fluid percussion injury (FPI) and euthanized at either 1 (n = 6/group) or 3 weeks post-injury (n = 8 sham, n = 13 FPI). Assessment of motor and cognitive performance was completed in the 3 week cohort. Immunoblotting was completed to quantify changes in protein abundance in hippocampal synaptosomal lysates. At 1 week post-injury, FPI resulted in a significant reduction in multiple SNARE proteins and reduced SNARE complex formation. Assessment of beam balance and beam walking revealed transient impairments in vestibulomotor function at 1 day post-injury. Assessment of cognitive abilities by the Morris water maze revealed significant deficits in learning latencies and probe trial scores at 3 weeks post-injury, consistent with previously published literature. Future directions will evaluate the effect of FPI on regional changes in SNARE proteins and to evaluate potential deficits in evoked neurotransmitter release. These data provide novel insight into the effect of TBI on SNARE protein abundance and synaptic function.
Acknowledgement: 1-F32NS090748, The Pittsburgh Foundation, NIH-NS40125, NIH-NS060672, VAI01RX001127
Keywords: FPI, neurotransmission
Traumatic brain injury (TBI), of which mild TBI (mTBI) accounts for more than 80%, significantly increases the risk of developing alcohol use disorders (AUDs) over a patient's lifetime. Experimental mTBI has been shown to impair ethanol-induced behavioral sensitization and increase chronic and binge ethanol consumption and preference in the delayed post-TBI period. With its known relationship to ethanol consumption and the physiological effects of ethanol, the endocannabinoid system has become of interest in AUDs secondary to TBI. Specifically, ethanol vapor and self-administration decreased striatal expression of cannabinoid receptor 1 (CB1) and altered ethanol preference has been observed following CB1 antagonism in rodent models. In this study, we investigated changes in cannabinoid receptor (CB1 and CB2) proteins in the anterior striatum (ASTR) and nucleus accumbens (NAC) at 8 and 25 d post-mTBI. Anesthetized male C57BL/6 mice (8–10 wks) were given a mild, midline impact over the intact skull or sham surgery. At 8 and 25 d post-injury, brains were harvested and frozen. For Western blot analysis, protein lysates were made from 1.5 mm diameter tissue punches of striatal subregions taken from 2 mm thick coronal slices. At 8 d post-injury, there was a trending decrease in CB1 in ASTR, which became significantly reduced at 25 d post-injury. The NAC showed an initial increase in CB1 at 8 d following mTBI, but was decreased at 25 d. There were no changes in CB2 protein levels at either time point. The observed time- and region-dependent alterations in CB1 expression in the ASTR and NAC may underlie the progression of AUDs that develop in the post-TBI period.
Keywords: alcohol, plasticity, cannabinoid, striatum
EFFECTS OF CUTAMESINE ON RECOVERY FROM SPINAL CORD INJURY IN RATS
Chiba Rehabilitation Center, Chiba, Japan
Keywords: cutamesine, motor functional recovery, rehabilitative training, combination therapy
Safar Center/Univ. of Pittsburgh, Crit Care Med, Pittsburgh, USA
OBTT is a multi-center pre-clinical drug and biomarker screening consortium supported by the U.S. DoD. It evaluates therapies across three severe TBI models (parasagittal fluid percussion injury [FPI], controlled cortical impact [CCI] and penetrating ballistic-like brain injury (PBBI) in rats). FPI is the mildest model while PBBI is the most severe. OBTT's goals are 1) to define therapies showing efficacy across models which should have the best chance for clinical translation, and/or 2) to define model-dependent therapeutic effects to guide precision medicine–based clinical trials. The results of the first 5 therapies tested by OBTT (nicotinamide, erythropoietin, cyclosporine [CsA], simvastatin, and levetiracetam) were published in J Neurotrauma. OBTT has assessed 4 additional therapies (glibenclamide, kollidon-VA64, AER 271, and amantadine). Only levetiracetam has shown benefit in multiple models, including benefit in at least one model on cognitive outcome, histology, and serum biomarker levels. The second most successful drug, glibenclamide demonstrated improved motor function and reduced contusion volume, but benefit was largely restricted to CCI. Several other therapies showed model dependent effects (tissue sparing by nicotinamide in CCI, some benefit from CsA in FPI but toxicity in PBBI, and benefit from amantadine on behavior in PBBI but deleterious effects in FPI). Glial fibrillary acidic protein (GFAP) has performed well as a serum biomarker showing correlations with histology and theranostic utility. Levetiracetam merits additional pre-clinical and clinical investigations. However, it failed to show benefit in our micropig model. Glibenclamide also merits additional investigations in contusion. Amantadine may require testing in penetrating TBI. Finally, our work suggests utility of GFAP in pre-clinical screening. Support:W81XWH-10-1-0623/WH81XWH-14-2-0018.
Keywords: multi-center, consortium, reproducibility, clinical translation
Our objective was to define the chronically altered gene expression signature of traumatic brain injury (TBI-sig), and apply it to discover novel treatments in order to reverse pathologic gene expression or reinforce the expression of recovery-related genes. TBI was induced with lateral fluid-percussion injury in adult male rats. Genome-wide RNA-seq of the perilesional cortex, ipsilateral thalamus and ipsilateral hippocampus was performed at 3 months post-TBI. Data highlighted chronic transcriptional changes, particularly, in the perilesional cortex (4 964 regulated genes) and thalamus (1 966)(FDR <0.05). Gene set enrichment analysis (GSEA) indicated down-regulation of ion channel and mitochondrial gene sets and upregulation of inflammatory gene sets (GSEA q-value <0.01). Transcriptomics data were used to design the TBI-sig which was used in LINCS analysis. We identified 18 of 1064 compounds which regulated gene expression in three cell lines with strong connectivity score with TBI-sig. Eleven of these 18 compounds showed a strong connectivity with TBI-sig in neuronal cell lines. Two of 11 compounds (celecobix, sirolimus) were recently shown to have a disease-modifying effect in in vivo animal models of epilepsy. The nine other compounds revealed by the analysis were (BRD-K91844626; BRD-A11009626; NO-ASA; BRD-K55260239; SDZ-NKT-343; STK-661558; BRD-K75971499; ionomycin; desmethylclomipramine). Transcriptomics signatures of the 11 compounds were compared in more details with TBI-sig. IPA analysis of overlapping genes revealed the effects of these compounds on tubulins (Tubb2a, Tubb3, Tubb4b), Nfe2l2, S100a4, Cd44, and Nfkb2, which have been linked to TBI-relevant outcomes, including epileptogenesis and tissue repair. From all compounds revealed by the LINCS analysis, desmetylclomipramine, an active metabolite of antidepressant clomipramine, modulated most of the gene targets considered favorable for the TBI outcome. Our data demonstrate long-lasting transcriptomics changes after TBI in the perilesional cortex and thalamus, suggesting a long-lasting therapeutic window. LINCS analysis predicted that these changes can be modulated by various compounds, some of which are already in clinical use.
Keywords: transcriptomics, bioinformatics, pharmacotherapy, fluid percussion
DEVELOPING AZITHROMYCIN DERIVATIVES FOR ALTERING MACROPHAGE PHENOTYPE
University of Kentucky, Physiology- Spinal Cord and Brain Injury Research Center, Lexington, USA
Azithromycin (AZM) is a macrolide antibiotic with significant anti-inflammatory actions in rodent models of lung infection, skin inflammation, and sepsis. We have previously shown that AZM improves tissue sparing and locomotor recover in a mouse model of contusion spinal cord injury (SCI). Further, we have shown that in-vitro application of AZM to pro-inflammatory M1 bone marrow derived macrophages (BMDMs) dampens the release of pro-inflammatory cytokines, increases M2 associated anti-inflammatory cytokines and reduces BMDM supernatant neurotoxicity. While promising, the therapeutic applications for AZM remain limited. AZM is one of the primary antibiotics used to treat respiratory infections such as pneumonia in SCI patients so the risk of developing antibiotic resistance would discourage its use. Thus, we have obtained a library of non-antibiotic AZM derivatives with a variety of chemical modifications from the University of Kentucky College of Pharmacy. Initial in-vitro screening of these compounds have identified several non-antibiotic candidates that retain immunomodulatory activity. With continued screening we aim to find a non-antibiotic compound that retains or exceeds AZM's characteristics. While the initial focus of these studies is to elucidate the molecular pathways through which we are able to modulate macrophage biology, we ultimately aim to obtain a new drug for the treatment of SCI and other neuroinflammatory conditions.
Supported by the University of Kentucky Ignite Program and NIH R01NS091582-01A1
Keywords: azithromycin, macrophage, macrolide antibiotics, phenotype, cell proliferation, microglia
Keywords: Tau oligomers, fluid percussion injury, behavior, vascular
CERIUM OXIDE NANOPARTICLES REDUCE OXIDATIVE STRESS AND PRESERVE COGNITIVE FUNCTION FOLLOWING MILD TRAUMATIC BRAIN INJURY
Virginia College of Osteopathic Medicine, Pharmacology, Blacksburg, USA
Despite considerable research efforts at improving outcome after mild traumatic brain injury (mTBI), treatment remains limited. Cerium oxide nanoparticles (CeONP) are regenerative antioxidants and mitochondrial protectants, with demonstrated efficacy in treatment of neurodegenerative disorders. Here, we test the hypothesis that administration of CeONP reduces oxidative stress and improves functional outcome after mTBI. A mild lateral fluid percussion injury was delivered to Sprague-Dawley rats. CeONP were administered in 2 injection paradigms; acute (1, 3, and 15 min following injury) or chronic (additional injections at 24 and 48 hrs after injury). Two drug concentrations were tested (0.05 and 0.5 μ/g). Controls received injury, without drug intervention and shams underwent surgery only. Biochemical assays were conducted on ipsilateral and contralateral cortices at the injury site. Endogenous antioxidant levels, including SOD, catalase, and reduced/oxidized glutathione (GSH/GSSG) were measured. Free radical damage to macromolecules was assessed by measuring 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), markers for lipid peroxidation and tyrosine nitration. A separate group of animals were used for behavioral assessments including novel object recognition (NOR) and open field task (OF), two weeks following injury. mTBI produced significant depletion of SOD and catalase, and a decline in GSH/GSSG. These changes correlated with significant increases in 4-HNE and 3-NT. Both CeONP injection paradigms significantly increased SOD, catalase, and GSH/GSSG and reduced levels of of 4-HNE and 3-NT. Using the high (0.5 μ/g) dose in the chronic injection paradigm, antioxidant enzymes, 4-HNE, and 3-NT were restored to sham levels. No significant differences were observed during the OF test, and each group showed similar anxiety and activity levels. However short term memory deficits observed in injured rats using the NOR task, were improved by CeONP. Our results demonstrate that CeONP treatment following mTBI reduces oxidative stress and mitigates pathological memory impairments. Supported by NIH-NINDS-NS072873.
Keywords: cerium oxide nanoparticles, nanomedicine, nanotechnology
More than half of all spinal cord injuries (SCIs) occur at the cervical level resulting in persistent, life-threatening respiratory deficits. Despite this challenge, studies have demonstrated an intrinsic capacity of spinal interneurons to alter activity post-SCI and undergo short distance sprouting that can contribute to spontaneous neuroplasticity via the formation of novel functional neuronal relays. With a primary focus on spinal interneurons, the present study tests whether transplantation of interneuronal precursors can provide a source of cells that contributes to functional neuronal relays capable of improving respiratory function. The present work documents the development of cultured and non-cultured (freshly-dissociated) neural precursor cells (NPCs) after transplantation into a lateral, C3/4 contusion injury, which often results in diaphragm paresis and persistent respiratory deficits. Anatomical connectivity of grafted cells is assessed using a retrograde, transynaptic tracing technique while the functional contribution of grafted cells is analyzed via electrophysiological methods (diaphragm electromyograms and phrenic nerve neurograms). Despite donor cell survival, differentiation and integration with the injured host phrenic circuitry, electrophysiological analysis did not reveal significant functional recovery for the cultured NPC grafts. In contrast, transplantation of freshly-dissociated developing spinal cord showed some improvement in phrenic motor function, albeit variable. These results suggest that donor cell survival, differentiation and integration are not sufficient to result in recovery of respiratory function following cervical SCI. We propose that specific (excitatory) interneuronal phenotypes are necessary for the consistent enhancement of motor function and recovery. Ongoing experiments are now exploring the contribution of donor excitatory and inhibitory interneurons to anatomical and functional relays that are capable of improving phrenic motor function. We are grateful to NIH, NINDS (R01-NS081112) and Craig H. Neilsen (338432) for funding this work.
Keywords: spinal cord injury, cell transplantation, neural plasticity, respiration, neurotrauma
ACTIVITY-DEPENDENT CELLULAR CHANGES IN RAT VASCULATURE AND SPINAL CORD AFTER HIGH THORACIC SCI
University of Louisville, Bioengineering, Louisville, USA
Cardiovascular dysfunction is one of the leading causes of death in the SCI population. High thoracic spinal cord injury (SCI) damages sympathetic neurons, which disrupts neural regulation of various systems at and below the level of injury, including the cardiovascular system. Our preliminary work has shown increased sensitivity to physiologic acetylcholine concentration (parasympathetic vasodilator) in rat femoral arteries after T2 and T3 25 g/cm injuries compared to controls. Chronic endoplasmic reticulum (ER) stress induces autophagic and apoptotic pathways in cells. It has been demonstrated that exercise decreases ER stress in neural tissue. In this study, activity-dependent changes in several vasomotor proteins and ER stress markers are evaluated. Rats were divided into three groups: uninjured controls (n = 5), small cage (n = 6), and large cage (n = 6) housing with T2 25 g/cm injuries. The small cage was designed to limit the physical activity of the rats by reducing the ground area of the cages. The activity levels of the injured rats were determined by weekly overnight recording of the distance traveled by the rats in the small/large cages. The rats were then sacrificed 4 weeks post-SCI, the spinal cord, femoral and brachial arteries were harvested. Various parasympathetic and sympathetic mediated vasomotor proteins were analyzed using western blotting. ER stress markers were evaluated with qPCR at multiple sites in the cord. The proteins and genetic markers assessed in this study are involved in cellular homeostatic pathways. The results from this study could elucidate potential cellular mechanisms for the cardiovascular dysfunction seen after high thoracic SCI.
Keywords: vasomotor, spinal cord injury, ER stress, activity
Keywords: TBI myogenic response, middle cerebral arteries, rifampicin
REMYELINATION REVERSES COGNITIVE DEFICITS IN MICE FOLLOWING CHEMICAL DEMYELINATION OR CLOSED HEAD INJURY
State University of New York-Downstate Medical Center, Physiology and Pharmacology, New York, USA
Mild traumatic brain injury selectively damages white matter. Much is to be learned about the behavioral consequences of this white matter injury. We examined the behavioral consequences of myelin loss in two experimental mouse models, closed head injury and stereotaxic injections of lysolecithin (LPC). Closed head injury induces a long-lasting demyelination that does not remyelinate. Lysolecithin induces demyelination that spontaneously remyelinates. Myelin loss was significantly limited when two FDA-approved drugs, minocycline and N-acetylcysteine, were dosed 12 h after closed head injury. In contrast, myelin was lost when minocycline and N-acetylcysteine were dosed at 24 h after injury. Mice receiving 12 h dosing acquired Barnes maze and active place avoidance. Mice receiving 24 h dosing acquired Barnes maze, but not active place avoidance. Saline-treated injured mice acquired neither of these tasks. To address whether demyelination contributed to these behavioral impairments, LPC was stereotaxically injected into the corpus callosum. LPC injections locally demyelinated a region of corpus callosum at 7 DPI that spontaneously remyelinated by 23 DPI. At times of demyelination, mice acquired Barnes maze, but not active place avoidance. At times of remyelination, LPC-injected mice acquired both tasks. Stereotaxic saline injections did not alter myelin content and saline-injected mice acquired both tasks at both time points. These data suggest that tests of cognition and memory differ in their sensitivity to white matter injury, with active place avoidance more sensitive than Barnes maze to demyelination of corpus callosum. Treatment with minocycline and N-acetylcysteine at the clinically relevant time period of 12 hours improved cognition by preserving myelin integrity. Remyelination may be needed to optimally improve clinical outcomes following TBI.
This study was supported by the grants USAMRCC 09127004 and NIH RO1070512 to P.J.B.
Keywords: remyelination, drug combination
Univ. of Kentucky, Spinal Cord and Brain Injury Research Center, Lexington, USA
Reactive astrocytes of the glial scar produce inhibitory chondroitin sulfate proteoglycans (CSPGs) that act as barriers to axonal outgrowth and regeneration. Numerous studies, in vitro and in vivo, have focused on developing methods to overcome CSPG-induced inhibition. Here, we observed a peculiar phenomenon – the inhibitory influence of the CSPG, aggrecan (150 μg/ml), was dependent upon the density of dorsal root ganglion (DRG) sensory neurons present surrounding aggrecan. Thus, DRG neurons can “self attract”, seemingly to overcome inhibitory influences, such as following spinal cord injury. To test this, DRG neurons were grown in a choice assay either 1) placed on one side of a stripe of aggrecan (control), or 2) placed on each side of a stripe of aggrecan, aligned horizontally. Neurons were cultured for 48 hrs, and fixed. Using immunofluorescence and image analysis, a region of interest including the DRG explant(s) and the CSPG stripe was established, and neurite outgrowth was measured. Results of this quantitative analysis showed that one-sided DRG explants were fully inhibited, as shown previously by our lab and others. However, when DRG explants were grown on each side of the CSPG stripe, directly across from one another in the horizontal plane, they exhibited outgrowth toward the adjacent DRG, even so much as to grow into the region adsorbed with aggrecan, which is typically inhibitory. These data suggest that DRG explants may influence the outgrowth of one another, and under certain conditions, can overcome a barrier of inhibitory CSPG. These data are consistent with the interpretation that DRG neurons in sufficient density (or other cells within the DRG explants) may secrete a factor(s) that promotes elongation and/or blocks the inhibitory effects of CSPGs. Studies are on-going to identify the promoting factor(s) to test their efficiency in ameliorating inhibition of sensory neuron outgrowth and their potential to promote regeneration in vivo.
Keywords: proteoglycans, extracellular matrix, cell culture, dorsal root ganglion neurons, aggrecan
Keywords: spinal fracture, transverse process fracture
TRANSLATIONAL RELEVANCE OF NEUROPROTECTIVE GENES AND PUTATIVE DRUG TARGETS
Predicting that results will translate from preclinical animal models to the clinic remains difficult, in particular with regard to the central nervous system. In well studied brain regions, reports of cellular and anatomical similarity between model species abound yet lack gene expression data necessary for drug targeting. Comparing greater gene coverage and additional brain regions would increase confidence earlier in the discovery process. Further, assessing brain regions elaborated in primates relative to rodents (e.g. prefrontal cortex) may also aid in anticipating possible side effects. In rodent models of brain injury, relevant baseline gene expression patterns and levels across brain regions are often unverified in other species, such as non-human primates and humans.
To examine such translational assumptions, we utilized public gene expression maps to compare the relative expression of the ∼40 genes that comprise the ontological category “negative regulation of apoptotic process” (GO:0043066; applying 3 filters primates/direct assay evidence/neuron). We found a general congruence in gene expression, yet with some interesting exceptions. For example, CRLF1 (Cytokine receptor-like factor 1), encodes a protein which can promote neuron survival. In the adult mouse, highly selective expression levels were observed (almost exclusive to the hippocampus), whereas in human and non-human primate brains, expression levels are more uniformly expressed across many brain regions and at lower levels. These descriptive findings of variability both between brain regions, and between species, suggest that potential neuroprotective mechanisms and drug targets may differ depending on the model species. Future studies could extend these findings with gene regulatory mechanisms, variation across developmental stages, and validation with protein differences (e.g. IHC) for more effective drug target selection.
Keywords: Neuroprotection, Gene expression, Drug Discovery, Informatics, Translational