Abstract
Trainee Competition Finalists
SMART HUMAN NEURAL STEM CELLS TO MODIFY SCAR AND OPTIMIZE REGENERATION AFTER TRAUMATIC CERVICAL SPINAL CORD INJURY
University of Toronto, Neurosurgery, Toronto, Canada
Human induced pluripotent stem cell-derived neural stem cells (hiPS-NSCs) represent an exciting therapeutic strategy for traumatic spinal cord injury (SCI) as they can replace lost neural circuits, remyelinate denuded axons and provide local trophic support. Unfortunately, most patients are in the chronic phase of their injuries where dense chondroitin sulfate proteoglycan (CSPG) scarring significantly impairs neurite outgrowth and regenerative cell migration. Several scar-modifying enzymes have been shown to synergistically enhance NSC-mediated recovery, however, nonspecific intrathecal administration can produce off-target effects. We aimed to generate a genetically-engineered line of hiPS-NSCs, termed Spinal Microenvironment Modifying and Regenerative Therapeutic (SMaRT) cells, uniquely capable of expressing a scar-modifying enzyme within the host environment to enhance functional recovery. A proprietary enzyme was non-virally integrated into hiPS-NSCs and a monocloncal line of SMaRT cells was generated and extensively characterized. The expressed enzyme rapidly degrades CSPGs on biochemical assays and allows neurons to extend into CSPG-rich regions in vitro. Furthermore, unlike wild-type hiPS-NSC media, conditioned SMaRT cell media can degrade post-injury rodent CSPGs in ex vivo injured cord cryosections. T-cell deficient rats (N = 60) with translationally-relevant chronic C6-7 clip-contusion injuries have been randomized to receive: (1) vehicle, (2) hiPS-NSCs, (3) SMaRT cells, or (4) sham surgery (laminectomy). While blinded sensorimotor behavioural assessments and rehabilitation are ongoing with a long-term 32-week endpoint, interim histologic analysis shows that grafted human cells are extending remarkably long (≥ 20,000 μm) axons along host white matter tracts in the rostral and caudal directions. This work provides exciting proof-of-concept data that genetically-engineered SMaRT cells can degrade CSPGs in vitro and that human NSC transplants can grow long axons in chronic cervical SCI to potentially form a bridge for sensorimotor signal transmission. This work is generously supported by the Canadian Institutes of Health Research, Phillip and Peggy DeZwirek, OIRM, and the Krembil Foundation.
Keywords: Chronic, Glial scar, Neural precursor cell, Rehabilitation
Understanding the mechanical response of the human brain during impact is crucial to predicting injury severity and elucidating injury mechanisms. Currently, finite element models of the human brain are the state-of-the-art technique for assessing brain injury risk, investigating potential TBI mechanisms, and developing preventative mechanisms. However, they have been developed using limited experimental data quantifying relative brain-skull motion during head impacts. The objective of this study was to develop a methodology to quantify the 3D deformation of cadaveric brain specimens in situ during high-rate rotational head motion. An array of neutrally-dense sonomicrometry crystals were implanted into the brain of a single cadaveric head specimen (male, 53 years, 116 kg, 173 cm). These crystals are capable of transmitting and receiving ultrasonic pulses where the ultrasonic time-of-flight is used to determine the distance between each crystal pair. Eight transmitting crystals were affixed to the inner skull, and 24 receiving crystals were implanted in the brain. Dynamic 3D spatial time-history data for each receiving crystal was calculated using trilateration and reported as brain tissue motion relative to the skull. Four purely rotational motion conditions, ranging from a peak angular velocity of 20–40 rad/s with a duration of 30–60 ms, were applied to the same cadaveric head/brain specimen in the three anatomic planes. This is the first study to quantify 3D brain deformation in one specimen in response to varying severity rotations in all three anatomical planes using sonomicrometry. Sonomicrometry provided highly repeatable 3D displacement data of the dynamic motion of the brain. Brain deformation response was dependent on peak angular velocity, duration, and loading direction – axial rotation resulted in the greatest deformation. Peak-to-peak displacement reached as high as 23 mm in the most severe case. The transient response of the brain lasted between 100–200 ms. The natural frequency of the brain markers was found to be 12–20 Hz. Funding was provided under NHTSA contract number DTNH221500022/0002.
Keywords: mTBI/Concussion, Brain Deformation, Sonomicrometry, Finite Element Validation
EARLY SURGICAL INTERVENTION AMONG PATIENTS WITH ACUTE CENTRAL CORD SYNDROME IS NOT ASSOCIATED WITH HIGHER MORTALITY
Barrow Neurological Institute, Department of Neurosurgery, Phoenix, USA
Keywords: central cord syndrome, national trauma data bank, mortality
Formal clinical testing has become widespread in the assessment and management of athletes with sport-related concussion (SRC). A variety of assessments tools are available to assess the symptoms, cognitive, postural stability, and other impairments that can arise after injury. Yet there are limited data directly comparing the performance of differing assessment tools from which clinicians can identify the most sensitive measures for clinical use. The aims of this study were to compare the sensitivity of an array of clinical assessment tools to SRC and, secondarily, to explore patterns of heterogeneity across athletes in clinical domains affected by SRC in order to inform recommendations about the multidimensional clinical assessment of SRC. Male football players (N = 917) enrolled in Project Head-to-Head II at preseason examinations, where they completed assessments of symptoms, cognitive performance, oculomotor functioning, and postural stability. Participants who sustained SRCs (n = 60) and matched non-injured controls (n = 64) underwent repeat assessments at 24–48 hours and at days 8, 15, and 45 post-injury. Measures of oculomotor and vestibular functioning were the most sensitive performance metrics to SRC (King-Devick Test and Balance Error Scoring System area under the ROC curve, AUCs, at 24–48 hours = .75 and .73, respectively, p's <.01). Additionally, memory performance as measured by the computerized Immediate Post-Concussion and Cognitive Testing (ImPACT) battery was sensitive to SRC to a lesser degree (AUC = .61, p = .036), while computerized and paper-and-pencil measures of attention, processing speed, and executive functioning (e.g., Standardized Assessment of Concussion, Trail Making Test, Wechsler Adult Intelligence Scale-IV Processing Speed Index) did not significantly distinguish SRC from control subjects. Evaluation of patterns of impairment among subjects across clinical domains revealed significant heterogeneity in the clinical manifestations of SRC. The data help to identify the assessment tools with the strongest overall sensitivity to SRC while also supporting the multidimensional assessment of athletes with SRC.
Keywords: Sport-related Concussion, Neuropsychological Testing, Multidimensional Assessment, Oculomotor Functioning
IN VIVO CALCIUM IMAGING OF HIPPOCAMPAL CA1 NEURONS REVEALS A FUNCTIONAL INJURY SIGNATURE OF PRIMARY BLAST NEUROTRAUMA
The extent of neuronal dysfunction following mild traumatic brain injury (mTBI) is largely unknown, and is a major limitation to the development of treatment strategies. A key unknown is how the neural circuits in brain areas important for cognition change after mTBI, and how these changes affect the neural computations during behavior. Recent reports indicate that the blast overpressure component of an explosion alone can produce some pathology and behavioral deficits common amongst other forms of TBI. We used a miniaturized endoscope implanted in awake behaving mice, in combination with a genetically encoded calcium indicator in the hippocampus, to (1) identify a functional neural circuit ‘signature’ of blast induced TBI (bTBI) and (2) to track the progression of this circuit signature over time after blast exposure to elucidate the trajectory of recovery at the single cell/network level. We transduced CA1 Hippocampal neurons with GCaMP6f, a genetically encoded calcium indicator, and utilized a miniaturized, head-mounted microscope to detect transients of intracellular calcium – serving as a proxy of action potential events - in awake, unconstrained mice. We measured changes in calcium transient morphology from multiple mice (N = 6) and utilized a regression model to identify factors that best distinguished injured and sham groups. We found that changes in variation in magnitude and duration were statistically significant in predicting injured and sham cells. We then scored each cell immediately post blast and tracked its progression at 1 and 5 days. Cells with higher initial scores exhibited larger deviations in Calcium signals at day 5 compared to cells with lower scores, indicating a predictive capacity to identify cells that exhibit sustained responses. These results indicate that primary blast overpressure bTBI alters Calcium signaling at the level of single neurons, with sustained effects of at least 5 days. This platform for tracking the response of individual cells in vivo in time will facilitate the future interrogation of functional recovery and the effects of therapeutic strategies on its trajectory. (Funding Source: MURI W911MF-10-1-0526).
Keywords: Blast Induced TBI, in vivo Imaging, Functional Injury, Hippocampus CA1, Calcium Imaging, Mild TBI
INSIGHTS INTO THE PBTO2 TREATMENT THRESHOLD: EXPLORING A TREATMENT WINDOW SUGGESTED BY ‘BIG DATA’
University of Utah, Neurosurgery, Salt Lake City, USA
Brain tissue hypoxia is common after traumatic brain injury (TBI). Technology exists to detect hypoxia and guide corrective therapy. Current guidelines for the management of severe TBI recommend maintaining PbtO2 > 15–20 mmHg, however uncertainty persists as to the optimal treatment threshold. PbtO2 measures were prospectively and automatically collected every minute from consecutive patients admitted to the San Francisco General Hospital (SFGH) intensive care unit over a 6-year period. We analyzed mean PbtO2 values in TBI patients and the proportion of PbtO2 values below each of 75 thresholds between 0 mmHg to 75 mmHg over various epochs up to 30 days from time of admission. Patient outcomes were calculated using the Glasgow Outcome Scale. We explored putative treatment thresholds by generating 675 separate receiver operator curves (ROC) and 675 generalized linear models (GLM) to examine each 1 mmHg threshold for various epochs. A total of 1,380,841 PbtO2 values were recorded from 190 patients recovering from TBI. A high proportion of PbtO2 measures were below 20 mmHg irrespective of examined epoch. Time below treatment thresholds was more strongly associated with outcome than mean PbtO2. A treatment window was suggested; a threshold of 19 mmHg most robustly distinguished patients by outcome, especially from days 3–5. Benefit to maintaining values at least as high as 33 mmHg was suggested, however. Our “big data” analysis substantially informs the relationship between PbtO2 values and outcome. We were able to discern a therapeutic window for PbtO2 in TBI patients along with minimum and preferred PbtO2 treatment thresholds. Traditional treatment thresholds which have the strongest association with outcome may not be optimal.
Keywords: Brain oxygenation, PbtO2, Outcome, Threshold, Treatment window
Miami Project to Cure Paralysis, Department of Neurological Surgery, Miami, USA
Penetrating traumatic brain injury (PTBI) remains a significant cause of death and disability in the United States without effective therapies. A rodent PTBI model, penetrating ballistic-like brain injury (PBBI) has uncovered several secondary pathophysiological mechanisms such as reduced glucose uptake, neurodegeneration, inflammation, and apoptosis that magnify the primary injury. Targeting components of these mechanisms may help improve PTBI outcomes. Activators of innate immunity contribute to secondary injury mechanisms following traumatic brain injury (TBI). Inflammasomes are the key regulators of Il-1β mediated inflammation after TBI and present as clinically relevant targets for therapy. The role of inflammasomes in PBBI pathophysiology has yet to be determined. Towards this, adult male Sprague-Dawley rats were subjected to unilateral sham or PBBI surgery and sacrificed at various time points. Tissues were assessed for expression of cytokines IL-1β, IL-18 and components of the inflammasome, ASC (apoptosis-associated speck like protein containing a caspase activation and recruitment domain), caspase-1, NLRP3 (NOD-like receptor protein 3) and GSDMD (gasdermin-D) by immunoblot analysis and assessed for ASC cell-type expression by immunohistochemistry. Cortical IL-1β and IL-18 expression increased 4 h–48 h and 48 h–72 h, respectively after injury. PBBI also increased caspase-1, ASC, NLRP3, and GSDMD expression from 24 h–48 h. Compared to sham and contralateral cortex, microglial numbers significantly increased 48 h post-injury in the ipsilateral cortex. ASC expression was predominantly increased in activated microglia, permeating the enlarged cell bodies and into the processes. Taken together, this is the first report of inflammasome activation after PBBI and suggests that these activators of inflammation lead to an exacerbation of the pro-inflammatory state post-injury which could underlie the long-term sequlae of PBBI. Inhibition of the inflammasome in PBBI will evaluate its therapeutic potential for PTBI. Supported by NIH NINDS award R01NS089443.
Keywords: PBBI, Penetrating Ballistic-like Brain Injury, Inflammasome, Microglia, Inflammation
Veterans Affairs Puget Sound Health Care System, Geriatric Research Education and Clinical Center, Seattle, USA
A number of groups, including ours, indicate that mild blast-induced traumatic brain injury (mTBI) can cause transient blood-brain barrier (BBB) disruption. Whether short-lived BBB repair processes afford prolonged neuroprotection to mTBI, or can subsequently promote chronic pathologic and functional impairment is not well understood. To begin addressing this issue we mapped the temporal and anatomical profile of BBB disruption following blast exposure. C57/BL6 mice received two consecutive blasts that recapitulate detonation of high explosives in the open field. To assess blast-induced BBB disruption, we co-injected radiolabeled [14C]-sucrose and [99mTc]-albumin, which do not readily cross the intact BBB. We quantified [14C]-sucrose and [99mTc]-albumin in ten brain regions at 0.25, 3, 24, 72, and 168 hours post-blast. A significant increase in [14C]-sucrose was observed in all brain regions, except striatum, at 0.25 h post-mTBI (p < 0.01) compared to sham controls. A similar effect was also observed at 3, 24 and 72 h post-mTBI (p < 0.05). Results suggest an immediate, and sustained BBB opening after mTBI. A significant increase in [99mTc]-albumin was also observed at 0.25 h post-mTBI (p < 0.01), but only in frontal cortex and brain stem. No significant differences in [99mTc]-albumin were found among any brain region at 3 and 24 h post-mTBI. However, a significant increase in BBB disruption was measured in the frontal cortex and brain stem at 72 h post-mTBI (p < 0.05). These results argue that large openings of the BBB are restored within 24 h post-mTBI, followed by a secondary BBB disruption at 72 h. We hypothesize that the secondary BBB opening could be targeted to potentially reduce the chronic pathophysiology associated with neurotrauma.
Funding: T32AG05235401 (A.F.L.)
Keywords: Blast injury, Blood-brain barrier, Radioactivity, Neuropathology
Keywords: Blood Brain Barrier, Apolipoprotein-E4, Neurovascular Unit, Pericyte signaling, BBB permeability and stabilization
THE STEALTH PATHOLOGIES OF MILD TBI AND DEMONSTRATION THAT ADVANCED NEUROIMAGING MAY NOT BE AS SENSITIVE AS ADVERTISED
Keywords: Mild TBI, Concussion, Neuroimaging, Diffuse Axonal Injury
CANNABIDIOL ADMINISTRATION AFTER SPINAL CORD INJURY REDUCES ALLODYNIA IN MALE AND FEMALE RATS, WITH MOST ROBUST EFFECTS IN FEMALES
University of Alabama at Birmingham, Physical Medicine and Rehabilitation, Birmingham, USA
Over 60% of persons with spinal cord injury (SCI) experience neuropathic pain (SCI-NP), which frequently is listed as a top concern due to few treatment options. Clinically, cannabidiol (CBD), a non-psychotropic constituent of Cannabis sativa, has shown efficacy in treating some chronic pain disorders. However, effectiveness of CBD in treating SCI-NP has not been examined. The central hypothesis of this study is that acute post-SCI administration of CBD will ameliorate SCI-NP development and that these effects are sexually dimorphic. We recorded baseline motor and sensory function measures from adult male and female Sprague Dawley rats. Rats then received either laminectomy or SCI with the SCI groups receiving a unilateral 300 kdyne impact at the 5th cervical level. SCI groups where randomized to receive vehicle or CBD (100 mg/kg) beginning 30 minutes post-SCI, then once daily for 7 consecutive days. Weekly assessments of motor and sensory function were taken for 6 weeks. We found SCI reduced motor function in both sexes and CBD treatment conferred modest improvement in motor function in females. Regarding sensory tests, SCI induced an increase in sensitivity to mechanical stimuli in males and CBD reduced this tactile allodynia in males. Robust cold allodynia was observed in both sexes after SCI and CBD reduced development of cold allodynia in both males and females, with more pronounced reduction in females. Additionally, over-grooming of the affected hindpaw was observed in both sexes following SCI and the percentage of rats exhibiting over-grooming was reduced in CBD treated males and females. In conclusion, CBD administration post-SCI conferred modest reduction in motor deficits in females and significant effects in ameliorating cold allodynia and over-grooming in both sexes, with more robust effects in females. Support: UAB TJ Atchinson SCI Research Program and Conquer Paralysis Now Grant (CF, SM).
Keywords: spinal cord injury, neuropathic pain, cannabidiol
We have previously shown that the injured brain is both functionally disconnected as well as hyperconnected, and in a region-dependent manner after controlled cortical impact (CCI) injury in the adult rat. Our recent data shows that remote, homotopic cortical regions become hyperexcitable with time post-injury, and we hypothesized that this results in increased trans-hemispheric cortical inhibition, preventing reorganization of the primary injured hemisphere. In support of this, other data from our lab show that temporary silencing the contralesional sensorimotor cortex at 1wk after CCI injury normalizes affected forelimb use, but not when conducted at 4 wks. To determine whether this occurs due to restoration of the afferent pathway connectivity, or to reorganization of brain-centric circuits, we acquired forelimb-evoked-fMRI and resting state fMRI data at 1 and 4 wks post-injury after contralateral silencing with intraparenchymal injection of muscimol, and compared data to a vehicle-injected-injured (n = 9 vs 3) as well as sham rats (n = 3). Data were analyzed by statistical parametric mapping for the forelimb-evoked cortical map, and by graph theory for network-based functional connectivity (fc). As predicted, contralesional silencing at 1wk and 4 wks post-injury decimated the unaffected-limb-evoked contralesional cortical map, compared to vehicle-injected (P < 0.05, z = 1.7 cluster-corrected), but this did not restore the affected-limb-evoked cortical map to the ipsilesional hemisphere at either post-injury time-points. However, contrary to this, while fc analysis also demonstrated that silencing led to the expected contralateral cortical disconnection (reduction in strength of connections, P < 0.05, FDR-corrected q = 0.05) at both time-points, it also showed that silencing significantly lessened the injury-induced ipsilateral disconnection at 1wk that was present in vehicle-injected-injured rats, so that connection strength was more similar to sham rats. This effect occurred coincident with a normalization of hyperconnectivity in remote regions, likely indicating a causal effect and in agreement with prior data showing behavioral gains due to silencing at this time post-injury. Crucially, this cortical silencing-induced balancing of cortical circuits was much reduced or absent in the same rats at 28 days, also in agreement with the absence of behavioral gains in our prior data.
Keywords: Traumatic Brain Injury, Reorganization, contralesional cortex, cortical silencing, resting state fMRI, functional connectivity, forelimb stimulation-induced fMRI
INFLAMMATORY RESPONSE TO PERIPHERAL PAIN INPUT AFTER SCI IS BLOCKED BY A SPINAL TRANSECTION
When communication with the brain is disrupted by means of a spinal transection, noxious stimulation applied below the injury induces a state of over-excitation (central sensitization) that impairs adaptive plasticity. When this same stimulation is applied after a contusion injury, it increases the extent of secondary injury and impairs long-term recovery (reviewed in Grau et al., J Neurotrauma, 2017). The present study examined how spared fibers regulate the effect of nociceptive stimulation after a contusion injury. Sprague-Dawley rats (∼350 g) received a spinal contusion at T12. Eighteen hours later, subjects were scored on the BBB locomotor scale before receiving either a spinal transection or a sham surgery at T2. Six hours following the second surgery, subjects were exposed to 6 min of nociceptive stimulation (intermittent shock applied via tail electrodes). As previously reported, nociceptive stimulation increased the area of secondary injury and hemorrhage. Western blotting revealed that this effect of stimulation was correlated with increased expression of IL-18, IL-1b, TNF, Caspase 1, Caspase 3, Caspase 8, IGG, and Hemoglobin. These effects were most evident at the contusion site. All of these effects were eliminated when communication with the brain was blocked by means of a spinal transection. To explore the generality of these effects, we have also examined how the application of a peripheral irritant (capsaicin) affects tissue sparing after a contusion injury. Here too, nociceptive input increased the extent of hemorrhage and this effect was eliminated by a spinal transection. These findings suggest that the adverse effect nociceptive stimulation has at the site of injury depends upon communication with rostral neural systems. We are currently exploring the processes that underlie this interaction.
Supported by the Craig H Neilsen Foundation and the National Institute of Health award number R21465891-00001 to JW
Keywords: SCI, Hemorrhage, C-fibers, Pyroptosis
ENRICHED ENVIRONMENT REARING DIFFERENTIALLY AFFECTS DENTATE SPINES AFTER REPEAT CONCUSSIONS IN ADOLESCENT RAT
The greatest incidence of concussive injuries occurs during adolescence and young adulthood, during the dynamic maturational period of ongoing myelination and synaptic pruning. A single mild TBI during adolescence has been shown to interfere with normal pruning (Mychasiuk 2015), behavioral impairments and transient metabolic alterations (Prins). Repeat mTBI at short intervals has been shown to exacerbate outcomes in adolescent rats (Prins 2013) and it is hypothesized that it will decrease synaptic density, contributing to behavioral impairments. It is also hypothesized that enriched environment(EE)-induced plasticity can improve anatomy and function. Postnatal day 35 rats (n = 3/group) were given sham or 2 closed head concussion (24 h interval) and immediately placed in single or EE housing for 30days, followed by Novel object recognition testing. Golgi-Cox staining was analyzed for spine density and type in the dentate granule cells of the hippocampus. Three neurons per animal were selected and both the proximal and distal segments were isolated for synaptic density and synaptic type analysis with RECONSTRUCT. Sham single housed and EE housed rats interacted with the novel object 67% and 84% of the time. rTBI single housed showed decrease in novel object recognition (53%), which improved to 61% among rTBI EE animals. Sham EE reared showed 20–24% greater spine density on segments proximal and distal to granule cell bodies, with greatest increases in long-thin, filopodia and thin synapses, reflecting newer synapses. Following rTBI in single housing there was a 15–19% decrease in synaptic density at proximal and distal segments, with thin and mushroom synapses predominant lost. rTBI EE animals showed significant decrease in synaptic density proximally with thin and mushroom synapses lost, but distally segments did not show loss of mushroom synapses with EE. RTBI during development affects EE-induced plasticity as measured by reductions in working memory and decreases in hippocampal synaptic connections.
Marilyn & Austin Anderson, UCLA BIRC, NFL Charities, UCLA Easton Neurotrauma Laboratories, UCLA Steve Tisch BrainSPORT program
Keywords: adolescent, repeat concussion, environment enrichment, synapse spines
RECORDINGS OF THE HIPPOCAMPUS IN BEHAVING RODENTS AFTER TRAUMATIC BRAIN INJURY REVEAL FIELD AND NEURONAL CODING DISRUPTIONS
University of Pennsylvania, Department of Neurosurgery, Philadelphia, USA
Persistent memory deficits are a major sequela of TBI, yet network level mechanisms of memory impairment are poorly understood. Studies using the fluid percussion injury (FPI) model have reported reduced theta power in the hippocampus (HC), but how this change influences memory-relevant HC networks is unknown. We have previously examined hippocampal circuitry post-injury acutely, demonstrating robust changes in CA1 neuronal entrainment to HC theta. To overcome limitations due to anesthesia, we chronically implanted electrodes in rodents, allowing examination of HC networks engaged in relevant awake behavior after injury. Experimental TBI was induced in male Sprague Dawley rats by FPI at mild to moderate (1.6–1.8 atm) severity. Implantation of multi-shank silicon probes with 32–64 electrodes on a high-resolution microdrive allowed for high-quality recordings of single units and simultaneous laminar field potentials. These recordings were also obtained while the animal was freely moving in an open field and a radial arm maze using 64-channel wireless telemetry. We demonstrate robust extracellular field potentials and stable unit recordings post-implant following FPI out to 14 days post-injury. Preliminary results from acute and chronic recordings indicate that CA1 neurons remain entrained to theta in non-injured and injured animals. However, distribution of preferred phase in injured CA1 neurons revealed two sharp peaks compared to a wide distribution with one peak in non-injured animals. These results indicate abnormal theta entrainment which could cause failure to coordinate neuronal ensembles that represent place during memory tasks. Distribution of axonal injury (revealed with amyloid precursor protein) suggests that loss of inputs from entorhinal cortex and medial septum may contribute to these changes, and reduction in performance on the Morris Water maze. Ongoing work to characterize memory deficits in the radial arm maze combined with awake recordings during this task may reveal mechanisms of trauma-induced network dysfunction.
Keywords: electrophysiology, behavior, learning and memory, hippocampus
Very little is known about the role of N-Methyl-D-aspartate receptors (NMDAR) and downstream pro-plasticity and pro-death molecular pathways following developmental TBI. Recent evidence indicates beneficial effects of D-Cycloserine (DCS), a partial NMDAR agonist, to treat cognitive and memory deficits following TBI, and this may occur partly by alteration of neuroplasticity-associated molecular pathways. We tested the hypothesis that sub-acute treatment with DCS following injury results in an enhancement of hippocampal pro-plasticity pathways and prevents upregulation of pro-death pathways after lateral fluid percussion injury (LFPI). 32 postnatal day 19 male Sprague Dawley rats were randomized into 4 groups (sham-saline, sham-DCS, LFPI-saline, LFPI-DCS, 8/group). All received 5 i.p. injections of 30 mg/kg DCS or saline every 12 hours until post-injury day (PID) 3. Ipsilateral hippocampal tissues were harvested on PID14 and processed for immunoblotting of pro-plasticity (pAkt, pCamKII, NR1, and NR2B) and pro-death markers (nNOS and p38). Results indicate that after injury there was a significant effect of DCS treatment on pro-plasticity and pro-death pathways compared to saline-treated injured rats in ipsilateral hippocampus: an upregulation of pAkt (8.8%, P < 0.05), pCaMKII (29.18%, P < 0.05), NR1 (10.79%, P < 0.05), NR2B (10.81%, P < 0.05), and a significant decrease in nNOS (11.29%, P < 0.05) and p38 (3.8%, trend). These findings support the hypothesis that NMDAR agonist administration enhances neuroplasticity-related molecular pathways following developmental TBI. Mechanism-based treatments have particular promise for translation to improved TBI recovery. Supported by: R01NS27544, R01NS091222 UCLA Easton Labs for Brain Injury, UCLA Steve Tisch BrainSPORT program, UCLA BIRC.
Keywords: NMDAR agonist, Chronic treatment, plasticity, developmental TBI, D-cycloserine
CHRONIC CORTISOL TRAJECTORIES MEDIATE SIL6R EFFECTS ON GLOBAL OUTCOME AFTER SEVERE TBI
University of Pittsburgh, Department of Physical Medicine and Rehabilitation, Pittsburgh, USA
Severe traumatic brain injury (TBI) elicits a stress response due to similation of the hypothalamic-pituitary adrenal (HPA) axis that is associated with poor recovery. Distinct inflammatory signaling mechanisms associated with autonomic injury underlie HPA axis stimulation. With brief exposure, cortisol can have anti-inflammatory properties. However, prolonged elevated cortisol levels perpetuate pathological immune responses. Interleukin 6 (IL-6) is an important pro-inflammatory mediator associated with 6-month outcome, that when bound to its soluble receptor (sIL-6R), stimulates the HPA-axis into a state of sustained stress. Relationships between post-acute serum cortisol and inflammatory cytokines have not been extensively studied in TBI. Thus, we investigated a possible regulatory role for cortisol in mediating IL6R effects on 6-month outcome for N = 89 individuals with severe TBI. Individual mean serum cortisol and sIL-6R levels from samples collected over the first 3 months post-injury were assessed, and sIL-6R levels for the cohort were quartiled. Group based trajectory analysis (TRAJ) on cortisol levels measured in the same samples identified two subgroups–a stable-high group (N = 50) and a decliner (N = 39) group. 6-month Glasgow Outcome Scale (GOS) scores were dichotomized as poor (GOS = 2/3) or good (GOS = 4/5) outcome. Logistic regression showed sIL-6R quartiles predicted 6-month GOS (OR = 1.82, p = 0.014), where higher quartiles were associated with poor outcome. Cortisol TRAJ also predicted GOS (OR = 5.53, p = 0.0004), where the stable-high group had greater odds for poor outcome. sIL-6R quartiles also predicted cortisol TRAJ group membership (OR = 0.639, p = 0.024). Mediation analysis considering cortisol TRAJ as the mediator variable showed that cortisol TRAJ attenuated the relationship of sIL6R to GOS (OR = 1.5, p = 0.087). These results indicate IL-6 signaling through the sIL6R is influenced by cortisol and may represent an important post-acute contribution to global outcome post-injury. Bidirectional communication between the HPA axis and IL-6 family proteins provides novel insight onto the propagation of chronic inflammation and the long-term stress response post-TBI. Support: DODW81XWH-071-0701, NIDILRR-90DP0041.
Keywords: Chronic cortisol, Chronic inflammation, Soluble interleukin 6 receptor, Traumatic brain injury
ICORD-University of British Columbia (UBC), Vancouver, Canada
Axons in the central nervous system (CNS) typically fail to regenerate after SCI. Mechanistically, this failure to regenerate is due to an inhibitory environment as well as a diminished intrinsic capacity of CNS neurons for axon growth including a disruption of the cytoskeleton. The cytoskeleton, and in particular microtubules (MT), plays a critical role in axonal transport and axon growth during development. In this regard, members of the kinesin superfamily of proteins (KIFs) have been demonstrated to regulate the extension of axons and growth of collateral branches. Some KIFs have been shown to promote axonal regeneration while in contrast the kinesin KIF2A has been found to negatively control axonal and collateral branches growth by depolymerizing microtubules. Using dorsolateral funiculus crush (DLF) and contusion (Infinite Horizon (IH) impactor) models to induce SCI in adult rats, we examined the temporal and cellular expression of KIF2A in the injured spinal cord. By Western blot analysis we observed an increase of KIF2A expression with a maximal level at 10 days to 8 weeks post injury. KIF2A immunoreactivity was localized in cultured cortical neurons, as well as in tissue sections of injured spinal cords in neurons and mature oligodendrocytes at the vicinities of the injury. Importantly, we observed increased KIF2A at the injured axonal tips as well as in Calbindin and Parvalbumin positive propriospinal neurons. We also show the inhibition of KIF2A in vitro with siRNA promoting neurite extension and therefore propose that the expression in the injured axons may contribute to neurite outgrowth inhibition after injury. It remains to be shown whether the expression of KIF2A in Calbindin and Parvalbumin positive neurons might contribute to a prevent random neurite outgrowth and play a role in neuropathic pain after SCI. Further studies will determine whether the KIF2A becomes a viable target for the treatment of SCI.
Keywords: cytoskeleton, kinesin, spinal cord, neurophatic pain, axonal regeneration
IL-1 is a central regulator of the immune response to acute brain injury. IL-1α and IL-1β are the best-characterized cytokines of this family and signal through IL-1 receptor (IL-1RI). An understanding of the contribution of individual IL-1 pathway molecules following TBI is lacking and is of vital importance in developing potential anti-IL-1 therapies. We use lateral fluid percussion injury adapted to mice targeting moderate-severe TBI. C57BL/6J and global IL-1 KO mice (on C57BL/6J background) were used. Gene expression was evaluated by qPCR. Barnes maze testing was used to evaluate cognitive function 2 weeks post-TBI. FPI resulted in increased inflammatory cytokine expression (IL-1α, IL-1β, TNF and IL-6) in both focal (left parietal cortex, hippocampus) and remote regions (brainstem, cerebellum). At 6 hours, IL-1RI ablation decreased the spread of IL-1β and IL-6 expression to remote regions (brainstem, cerebellum). At 24hrs, IL-1RI ablation improved resolution of IL-1β and IL-6 in both focal (parietal cortex) and remote (brainstem) regions. Compared to WT littermates, IL-1RI KO mice performed better in the Barnes maze probe trial whereas IL-1α and IL-1β KO mice showed no significant improvement.
Support: K12 HD27748-11
Keywords: Interleukin-1, fluid percussion injury, cytokine
PERSISTENT DECREASE OF CEREBRAL BLOOD FLOW IN TRAUMATIC BRAIN INJURY AND SPORTS-RELATED CONCUSSION
Uppsala university, Institution of neurosciencies, Dep of neurosurgery, Uppsala, Sweden
We hypothesized that persisting cerebrovascular abnormalities contribute to chronic cognitive problems commonly observed in traumatic brain injury (TBI) and sports-related concussions (SRC). To test this hypothesis, we used arterial spin labeling (ASL) magnetic resonance imaging (MRI) to evaluate cerebral blood flow (CBF) in patients with moderate to severe TBI and in symptomatic athletes with SRCs.
Seven healthy controls (age 29.7 ± 6.4) with no prior history of concussion/TBI, 8 athletes (age 27.6 ± 6.8) with previous SRC, fulfilling the criteria for post concussive syndrome, sustained in median 23 (6-132) month previously and 6 patients (age 28.8 ± 7.5) with a moderate-severe TBI sustained in median 18 (10–29) months prior to the investigations were included. ASL and structural imaging was performed on a 3.0 T integrated PET/MR-system. Neuropsychological evaluation using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) cognitive screening tool was performed at time of MRI evaluation.
A globally decreased CBF was observed in grey and white matter, in TBI patients (55 ± 13 ml/100g/min and 41 ±-8 ml/100g/min, respectively) and in SRC 59 ± 5 ml/100g/min and 43 ± 3 ml/100g/min, respectively, compared to controls 72 ± 12 ml/100g/min and 54 ± 11 ml/100g/min respectively. There were no CBF differences between the TBI and SRC groups. The RBANS test showed that both TBI patients (75 ± 24) and SRC athletes (76 ± 12) performed worse than controls (102 ± 16).
Our results suggest that ASL using 3T MRI is a suitable method for evaluating CBF following TBI and SRC and that cerebrovascular abnormalities persist chronically following both moderate-severe TBI and in symptomatic athletes with SRC. It is plausible that CBF decrease contributes to persisting cognitive and behavioral deficits observed in both post-concussive syndrome and TBI.
Keywords: Cerebral perfusion, Concussion, ASL, athletes
Toronto Western Research Institute and Spine Program, Krembil Neuroscience Center, University Health Network, Genetics & Development, Toronto, Canada
Degenerative cervical myelopathy (DCM) is caused by age-related degeneration of the cervical spine, leading to chronic compression of the spinal cord. The current treatment consists of surgical decompression. Our laboratory has shown that after decompression, increased neuroinflammation and spinal cord reperfusion contribute to the development of a secondary injury. Methylprednisolone (MPSS) has been used to moderate inflammation in the spinal cord following spinal cord injury, but has been shown to compromise the peripheral immune system. The primary objective of this study was to assess the effectiveness of MPSS in enhancing neurological recovery following decompression and evaluate whether it compromises the composition of circulating immune cells. Gradual compression of the spinal cord was induced in C57B/L mice by inserting a biomaterial underneath the C5-6 lamina. After 12 weeks of DCM, animals received one dose of MPSS (30mg/kg) or vehicle 30 minutes before decompression and two weeks after decompression. Composition of circulating white blood cells was analyzed by flow cytometry and locomotor recovery was assessed with the Catwalk system. One single dose of MPSS before surgical decompression significantly enhanced the beneficial effects of surgical decompression for two weeks, by further improving locomotion (*p < 0.05). In addition, MPSS treatment reduced IL-1b production without inducing changes in the composition of circulating white blood cells compared to the vehicle-treated group. Our data suggest that MPSS has an early protective effect in improving locomotion following decompression for degenerative cervical myelopathy without compromising the composition of circulating immune cells.
Keywords: decompression, degenerative cervical myelopathy, gait assessment
IUPUI, Neurological Surgery, Indinanapolis, USA
The corticospinal tract (CST), the major tract for voluntary control of dexterous movement, fails to regenerate in adult mammals after spinal cord injury (SCI). Furthermore, after an incomplete SCI, spared CST axons lack sufficient plasticity to induce meaningful functional improvement. Previous studies have shown that augmenting the activity of cortical neurons produces some effects after electrical or chemical stimulations. However, the lack of spatial and temporal resolutions hinders an understanding of the mechanisms of therapeutic effects, which is important for developing activity-dependent therapies for SCI. In the present study, we hypothesized that augmentation of CST neural activity would be sufficient to promote its plasticity, and subsequently, functional recovery. We used an optogenetic stimulation approach to selectively stimulate the motor cortex in which axons were spared in the CST contralateral to a cervical 5 (C5) right-sided hemisection, thus mimicking human Brown-Séquard syndrome. Our results showed that optogenetic stimulation for 1 week promoted robust sprouting of spared CST axons across the midline to innervate the denervated side of the gray matter. Such a “cross the midline” phenomenon was not found in the non-stimulated group. Notably, the optogenetic-mediated CST plasticity correlated well with improved functional recoveries, measured by pellet retrieval, grid-walking, cylinder, and rotarod tests. Brain mapping further revealed that optogenetic stimulation enhanced the spot number and amplitude of electrophysiological recordings, as compared to non-stimulation. Finally, optogenetic stimulation significantly increased the production of brain-derived neurotrophic factor (BDNF), a critical factor for synaptic plasticity, in both motor cortex and spinal cord. In conclusion, selective augmentation of the pyramidal neurons in the motor cortex using optogenetic stimulation is sufficient to initiate CST axonal sprouting in the spinal cord, leading to forelimb functional improvement after a cervical spinal hemisection.
Keywords: Spinal cord injury, optogenetics, CST, Plasticity, locomotor recovery
The average age at the time of spinal cord injury (SCI) has steadily increased from the average age of 29 years old in the 1970's to the current age of 42 years old. Recent findings from our group and others demonstrate that middle-aged mice exhibit worse functional deficits and exacerbated tissue damage after SCI. This is associated with age-dependent increases of reactive oxygen species (ROS) production, NADPH oxidase (NOX) activity and pro-inflammatory macrophage activation. Despite these age-specific differences, clinical therapies are being examined in individuals regardless of age and are based upon preclinical data generated almost exclusively using young animals. The purpose of the current study is to test the extent to which age affects the efficacy of SCI treatment. Specifically, we hypothesize that the effectiveness of apocynin, a NOX inhibitor for SCI, is age-specific. We applied mild-to-moderate contusion injury at the thoracic level (T9 laminectomy, 50 kdyn Infinite Horizons) to 4-month-old (4 MO) and 14 MO mice. We treated mice with apocynin (5 mg/kg, intraperitoneal injection) or vehicle (1% DMSO) at 1 and 6 hours post injury, then daily for 1 week. We examined the effect of apocynin treatment on functional and anatomical recovery from SCI. Our results show that apocynin effectively improves functional recovery and decreases lesion volume in 14 MO but not in 4 MO SCI mice. This suggests that apocynin may exhibit age-dependent neuroprotection by blocking excessive NOX-mediated ROS production. To the best of our knowledge, our data is the first to identify age as a critical regulator for SCI treatment efficacy. Age therefore needs to be considered as an important clinical variable to tailor therapeutic interventions and best serve the diverse SCI community.
Keywords: Aging, Apocynin, Functional recovery, Reactive oxygen species
PRIMARY TRAUMATIC AXONOPATHY AFTER IMPACT ACCELERATION: CHARACTERIZATION OF PATHOLOGY WITH 3D HIGH-RESOLUTION METHODS
Diffuse axonal injury (DAI), a primary axonopathy encountered in traumatic brain injury (TBI) of various types and degrees of severity, is a common neuropathological entity with mechanisms that are not well understood and no effective treatments. In experimental settings, it is often grouped together with axonal degeneration secondary to perikaryal injury. Animal models of primary traumatic axonopathy that replicate the conditions of human DAI are very important for the understanding of the basic cellular and molecular mechanisms of this condition. In these experiments we used the impact acceleration (IA) model of diffuse axonal injury that we modified for use in the mouse and explored the advantages of novel, high-resolution neuroanatomical methodologies. To characterize axonal pathology, we used the transgenic mouse line Thy1-eYFP-H expressing YFP in distinct populations of neurons, including layer V pyramidal neurons. Using CLARITY to render the whole mouse brain transparent and two-photon microscopy coupled with high-working-distance objective with submicron resolution, we visualized in 3D the whole corticospinal tract (CST) from the cerebral peduncles to pyramidal decussation in single-axon resolution. Individual axons and classical axonal abnormalities were identified in exceptionally high detail and quantified at 3 h and 24 h post injury. CLARITY-based immunohistochemistry for amyloid precursor protein (APP) was used to compare the sensitivity of conventional markers of axonal injury with that of high-resolution methods. We found that CLARITY is extraordinarily revealing of the location, type, and magnitude of axonal abnormalities that appear to cluster in lower pyramids and pyramidal decussation. In parallel, we found that the cell bodies of CTB-filled, layer V pyramidal neurons whose axons were injured in the CST (based on perikaryal p-c-JUN immunoreactivity) atrophied, but did not die. In addition, in experiments using extravasation of IgG from LEA-labeled endothelial cells as a marker of blood-brain-barrier disruption, we found positive signal in brain stem very early post injury. Furthermore, we established the role of endogenous and blood-borne inflammatory response involving activated or transformed microglia and blood macrophages. Together, we present a high-resolution multivariate model that can be used for molecular interrogation of specific mechanisms and serve as potential platform for the development of novel therapeutics.
Keywords: Diffuse Axonal Injury, Impact Acceleration, CLARITY, 2-Photon Imaging, Corticospinal Tract, 3D Neuropathology
Poster Abstracts
University of Kansas Medical Center, Anatomy and Cell Biology, Kansas City, USA
Adults over the age of 65 have a higher incidence of, and worse outcomes from, traumatic brain injury (TBI) than younger adults. Developing effective treatments to improve outcomes in this vulnerable population will require preclinical studies in aged animal models to better understand the effects of aging on TBI pathophysiology. In this study we sought to characterize functional and tissue effects in adult (3 month) and aged (20 month) male F344 rats that had received identical controlled cortical impact (CCI) injuries targeting the sensorimotor cortex. Sensorimotor outcomes were assessed with beam walk and adhesive removal tests from day 1–14 after TBI. On day 14, rats were scanned with T2-weighted MRI at 9.4 Tesla to assess lesion volumes. We found that acute functional impairment after TBI was worse in the aged rats than in younger adults, and that subsequent spontaneous recovery of function was diminished. However, brain lesion volumes measured at day 14 did not differ across age groups (21.8 ± 8.0 mm3 aged vs. 21.9 ± 8.1 mm3 adult), similar to our previous findings after CCI in mice (Onyszchuk et al. 2008). These findings confirm that the effects of older age on TBI outcomes in humans can be reproduced in the CCI model in aged rats. The similar lesion size in adult and aged rats suggests that age-related differences in the plasticity or function of spared brain tissue may be more important than lesion size in determining functional outcomes after TBI. Future studies to understand aging effects on the metabolic function of spared perilesional brain areas after TBI are warranted.
Keywords: TBI, Aging, Behavioral outcome, Animal models, Magnetic resonance imaging
North Shore University Hospital, Department of Neurosurgery, Manhasset, USA
Keywords: coagulation, platelets, Aspirin
University of California, San Francisco, Neurosurgery, San Francisco, USA
The incidence of traumatic brain injury (TBI)-related hospitalizations among older adults is four to 12 times that of younger age groups. Yet, older adults are underrepresented in TBI clinical research, thus limiting evidence-based management.
To elucidate this age-related disparity in research participation, we compared rates of exclusion criteria between older (≥ 65y) vs. younger (< 65y) adults screened 04/2014-03/2016 at the Zuckerberg San Francisco General Hospital (ZSFG) for participation in the ongoing 18-site Transforming Research and Clinical Knowledge in Traumatic Brain Injury (TRACK-TBI) study. TRACK-TBI aims to enroll 3,000 patients with acute TBI across the spectrum of age and TBI-severity.
Of 9471 screened patients, 2656 (28%) were older adults. 88% of older adults vs. 78% of younger adults met ≥1 exclusion criteria (p < 0.001). Overall, the highest number of patients were excluded due to non-English speaking (18%), substance abuse (14%), homelessness (14%), significant past medical history (PMH; 10%), neurological disorder (8%), polytrauma (5%), psychiatric disorder (4%), in custody (4%), unsuitable home location (3%), and psychiatric hold (1%). Exclusion criteria with highest age-related discrepancy (older vs. younger, p < 0.001) included: non-English speaking (27%-vs.14%), significant PMH (16%-vs.-8%), neurological disorder (15%-vs.5%), substance abuse (6%-vs.-18%), homelessness (4%-vs.-17%), psychiatric disorder (3%-vs.-5%), and in custody (0.4%-vs.-6%).
In this single center study, older adults with TBI were more likely than younger adults to screen out of TBI research due to lack of English ability or pre-existing medical or neurological disorders. These findings suggest that exclusion criteria pertaining to English ability and pre-existing conditions serve to preferentially exclude older adults from TBI clinical research, effectively functioning as age cut-offs. As the global population ages and grows and numbers of older adults with TBI continues to rise, it will become increasingly important to consider broadening standard inclusion/exclusion criteria in TBI clinical research studies in order to ensure generalizability of results.
Keywords: Traumatic Brain Injury, Aging, Recruitment
Barrow Neurological Institute at Phoenix Children's Hospital, Child Health, Phoenix, USA
Traumatic brain injury (TBI) is more than a singular event, such that a disease state with evolving pathophysiology ensues over an extended timeframe following the initial insult. Effective recovery from injury and enduring/remaining pathology may depend on many parameters, including the biological age at the time of injury. Throughout “normal” aging, neuroinflammatory status is in constant flux and homeostatic processes oscillate. TBI activates inflammatory processes, such that the sustained inflammatory state disrupts neurovascular units, including microglia, that can contribute to injury-induced behavioral morbidities. Given the comparable inflammatory profiles of the aged and the injured brain, we hypothesize that TBI accelerates pathological hallmarks of aging in the brain. A single cohort of rats was received at post-natal day (PND)10 and subgroups of this cohort (n = 11–12/group) were subjected to a moderate midline fluid percussion injury at PND17, PND35, 2 mo, 4 mo, or 6 mo or were naïve. Rats were assessed for motor function, anxiety-like behavior, cognitive performance, and depression-like behavior across their lifespan. These published data indicated TBI can negatively impact neurological function during discrete stages of development and aging. Tissue was collected at 10 mo for histopathology. A skeleton analysis method was used to quantify microglial ramification in Iba-1 stained cortical tissue. There was no overall difference in number of microglia endpoints per cell or process length per cell among treatment groups at 10 mo of age. For the first time, rod microglia, an activated morphology identified in brain tissue following diffuse brain injury, Alzheimer's disease, and aging, were identified in rats injured at PND17, PND35, and 6 mo. The interplay of age-at-injury and aging with an injury are translationally important factors and continued investigation of injury-induced neuropathology, including astrocytes and neurons are ongoing. More complete understanding of these factors can direct therapeutic interventions and personalized medicine for TBI survivors. Funding:AZ-Alzheimer-Consortium;WiNTR-VISA
Keywords: pediatric, age-at-injury, concussion, neurovascular unit
Phoenix Children's Hospital, Child Health, Phoenix, USA
Alzheimer's disease (AD) affects over 5.3 million Americans and cost the US $226 billion dollars in the year 2015 alone (Alzheimer's Association). Traumatic brain injury (TBI) can increase the risk of developing multiple age-related neurological disorders and neurodegenerative diseases, including AD. However, chronic consequences of a single diffuse TBI remain poorly understood. Many patients that suffer from TBI exhibit similar neurological pathologies to AD patients including deposition of amyloid plaques, formation of neurofibrillary tau tangles, and increased neuroinflammation. However, it is unknown how biological age at time of injury and severity of TBI contribute to these pathologies. The aim of this study is to determine whether a chronic diffuse TBI exacerbates AD-like pathology, neuroinflammation, and cognitive deficits and if this effect is dependent on age-at-injury. For this study we used 3 × Tg (B6;129-Psen1 Tg[APPSwe, tauP301L]1Lfa) mice, a commonly used model of AD, and wild-type controls. Mice received a midline fluid percussion injury (mFPI) or sham injury at 2 mo of age or 6 mo of age and assessed for cognitive function at 10 mo of age. Behavior analyses included Morris water maze and radial arm maze to evaluate reference memory; novel object recognition to evaluate recognition memory; and contextual fear conditioning to evaluate associative learning. Sensorimotor function was assessed using rotarod. At 10 mo of age (4–8 mos post-injury), brains were collected for histopathological analyses. F4/80 and Iba1 immunostaining determined the persisting inflammatory burden in chronic stages of TBI. Ongoing analyses correlating inflammation in chronic stages of TBI with behavioral deficits will allow us to determine effects of age-at-injury on TBI outcomes. Data from this study will provide much needed evidence for who is at greater risk for developing AD-like pathologies and cognitive deficits after a diffuse TBI.
Funding: Arizona Alzheimer's Consortium
Keywords: Diffuse injury, 3 × Tg mouse model
Keywords: Traumatic SCI, Frailty, Outcomes
GERIATRIC TRAUMATIC BRAIN INJURY: UNIQUE CONSIDERATIONS FOR PRESENTATION AND OUTCOME
UCSF, Neurosurgery, San Francisco, USA
Older adults have the highest rates of traumatic brain injury (TBI)-related emergency department visits, hospitalizations, and deaths. Few studies have investigated age-related differences in presentation and neurobehavioral recovery following acute TBI. We compared injury characteristics and clinical outcomes between young (18–39 years), middle-aged (40–59) and older (≥60) adults suffering acute TBI from the prospective multicenter Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot (TRACK-TBI Pilot) study. Outcomes included Glasgow Outcome Scale-Extended (GOS-E), Rivermead Postconcussional Symptoms Questionnaire (RPQ), Post-Traumatic Stress Disorder (PTSD) Checklist-Civilian Version (PCL-C) scored using the Diagnostic and Statistical Manual, Fourth Edition (DSM-IV) criteria. Categorical variables were compared across age groups using the Pearson's chi-squared test, and continuous variables were compared using analysis of variance for continuous variables followed by Tukey's post hoc test. In 572 patients, (older-21%/middle-aged-35%/young-44%) falls predominated in older compared to young and middle-aged adults (71%-vs.-47%/34%; p < 0.001). TBI severity was worse among older adults by Abbreviated Injury Scale-Head (72%-vs.-50%/44%; p < 0.001) but not by Glasgow Coma Scale (p = 0.238). Compared to middle-aged and young-adults, older adults had lower rates of loss of consciousness (64%-vs.-83%/80%; p = 0.001) and post-traumatic amnesia (64%-vs.-68%/77%; p = 0.023), and higher rates of intracranial pathology on computed tomography scan (64%-vs.-41%/39%; p < 0.001) intensive care unit admissions (51%-vs.-29%/36%; p < 0.001), and six-month mortality (18%-vs.-7%/1%). Fewer older and middle-aged adults achieved good functional recovery compared to young-adults (GOSE ≥7: 53%/51%-vs.-66%; p = 0.017). Older adults reported less postconcussional symptoms (RPQ: 9.6 ± 10.9-points-vs.-15.0 ± 13.8-points/9.7 ± 11.1; p < 0.001) and had lower rates of screening positive for PTSD (12%-vs.-31%/23%; p = 0.016). Older TBI patients presented with lower rates of loss of consciousness and amnesia, but suffered increased intracranial pathology, intensive care admissions, and overall mortality compared to younger patients. At six-months, however, greater than half of older adults had good outcome per GOSE and were less likely to have PCS and PTSD compared to middle-aged patients. Critical avenues for future research will be to determine predictors and mechanisms of favorable versus unfavorable recovery in geriatric TBI.
Keywords: Geriatric, Traumatic brain injury, Postconcussional symptoms, Glasgow outcome scale-extended
Aging spinal cord tissue shows increased inflammatory activity and oxidative stress, resulting in reduced neuronal viability and contributing to worsened outcome following spinal cord injury (SCI). Previously, we have demonstrated using positron emission tomography (PET)-based measurements of glucose uptake that 12 month (middle-aged) rats show an altered glucose uptake profile compared to 3 month (young adult) rats. To explore the potential effects and mechanism behind these findings, we aimed to determine how age affects glucose transporter (GLUT) gene expression and neuronal health. Briefly, spinal cord tissue was obtained from groups of naïve or injured (1 or 30 days post-injury (dpi)) young adult and middle-aged male Sprague Dawley rats. Tissue was processed for quantitative-PCR analysis, protein quantification or immunostaining of NeuN, a neuronal specific marker of nuclear proteins. In naïve tissue, middle-aged rats showed significant increases in GLUT3 and GLUT4 gene expression compared to young adults. After SCI, neuron specific GLUT3 was significantly decreased and non-specific GLUT4 significantly increased at all time-points post injury in young adult rats, likely due to decreased neuronal viability and increased inflammatory activity. In middle-aged rats, however, while GLUT3 was significantly decreased at all time points, GLUT4 was significantly increased only at 1 dpi; the reduction in GLUT3 was significantly greater than that observed in young adult rats at 30 dpi. Further, in middle-aged rats, GLUT1 (expressed by endothelial cells) was significantly increased at 1 dpi and significantly decreased by 30 dpi. Although protein quantification of NeuN revealed no significant difference between young adult and middle-aged rats, immunostaining revealed an altered staining pattern with NeuN localized outside of the neuronal nucleus, suggesting compromised neuronal function. Overall, these findings show an inverse relationship between GLUT gene expression and glucose uptake patterns, as middle-aged rats show significantly reduced basal glucose uptake that was increased at chronic post-injury time-points compared to young rats. Our findings suggest that alterations in glucose uptake with age, resulting from compromised neuronal viability and increased inflammatory activity, may influence GLUT gene expression profiles.
Keywords: glucose uptake, glucose transporters, neuronal viability, contusion injury, rodent models
Astrocytes are the most abundant glial cell population in the brain which are critical to maintenance of central nervous system. Astrocytes mediate the inflammatory cascade following traumatic brain injury (TBI). MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that regulate gene expression. Exosomes are extracellular membrane vesicles secreted by mammalian cells containing proteins, lipids and miRNAs. The cargo of exosomes may reflect the physiological state of the cells and hence, they have been studied as mediators of cell signaling and biomarkers.
In this study, we evaluated the exosomal miRNAs released from astrocytes under inflammatory conditions and studied their potential role in mediating inflammation and potential for an inflammatory biomarker for TBI. Human primary fetal astrocytes were treated with interleukin 1beta (IL-1b). The exosomes from the astrocytes were pelleted from the cell free supernatant by ultracentrifugation. MiRNA profiling was performed on the total RNA extracted from the exosomes. Ingenuity pathway analysis (IPA) was used to identify the role of the secretory miRNAs.
Treatment with Il-1b induced an increased expression of GFAP. The presence of exosomes was confirmed by immunoblotting of tetraspanin membrane markers (Alix and CD9). MiRNA profiling of the exosomal RNA identified 5 miRNAs which were exclusively released only by activated astrocytes whereas, 23 miRNAs were differentially secreted on IL-1ß stimulation. Furthermore, IPA analysis indicated that most of these miRNAs are involved in the regulation of the neuroinflammatory pathways. Moreover, some of these exosomal miRNAs were also reported as biomarkers of human TBI. Therefore, we conclude that inflammation lead to a release of astrocytic miRNAs which may be involved in cellular crosstalk for mediating inflammation during TBI. The miRNAs can be used as astrocyte injury biomarker and can help in differentiating neuronal injury from inflammation during TBI.
The opinions expressed herein are those of authors and are not necessarily representative of those of the Uniformed Services University, Department of Defense or, the United States Army, Navy, or Air Force and DMRDP.
Keywords: Inflammation, Astrocyte, Exosome, Biomarker, TBI
ENHANCING INTEGRATION OF SCHWANN CELL TRANSPLANTS FOLLOWING SPINAL CORD INJURY
Miller School of Medicine, University of Miami, Miami Project to Cure Paralysis, Miami, USA
Schwann cell (SC) transplantation has been extensively studied for spinal cord injury (SCI) repair. SCs are neuroprotective and promote axon regeneration and myelination in the lesion site. However, glial scar tissue surrounding the lesion site is inhibitory for both SC integration and axon growth into the host cord. Astrocytes contribute to this inhibition by secreting chondroitin sulfate proteoglycans (CSPGs), expressing ephrins, and forming an intricate network of astrocytic processes. The goal of this study was to investigate a drug that can transiently suppress astrocyte function to enable SCs to integrate and axons to grow across the implant-host spinal cord interface. 6-Aminonicotinamide (6-AN) disrupts the metabolic pathway in glial cells, and in the proper concentration can be used to transiently decrease their inhibitory actions following injury. Our in vitro data shows that 6-AN acts in a time and dose-dependent manner, causing retraction of astrocyte processes and decreasing their metabolic rates. The metabolic rate in SCs is only marginally decreased by 6-AN. Importantly, using an in vitro SC/astrocyte confrontation assay, we found that 6-AN treatment caused SC migration into the astrocyte terrain. In a rat model of spinal cord contusion, extensive migration of SCs into the adjacent spinal cord tissue was seen 3 weeks after SC transplantation and 6-AN treatment. No additional damage was observed in the injection site, suggesting that the dose used was not harmful. We suggest that 6-AN treatment is a tool that can transiently alter the glial scar, resulting in improved SC-astrocyte integration. Future studies will investigate axon growth, motor function, and immune cell responses in response to 6-AN treatment.
Keywords: Glial Scar
Virginia Tech, Biomedical Engineering and Mechanics, Blacksburg, USA
Astrocytes contribute to brain function through both active and homeostatic support to neurons. They play an equally integral role in pathological sequelae associated with traumatic brain injury and other CNS insults. Much evidence exists to suggest that astrocyte response to injury is governed by impulse mechanics, however, mechanisms of their mechanosensitivity remain largely uncharacterized. This study aimed to understand mechanobiological roles in reactive astrocytosis in response to high rate mechanical impulses. We have previously shown that environmental factors influence astrocyte reactivity to shock wave. Together, these evidences lend to the hypothesis that astrocyte reactivity is at least in part governed by mechanical disturbances and associated mechanotransduction. In this study, primary rat astrocytes were exposed to a single high rate overpressure using an underwater shock wave generator. Structural reactivity was assessed by quantifying alterations in gene and protein expression of intermediate filament proteins up to 48 hours post-insult. Inflammatory potential was evaluated through alterations in cytokine expression. In order to determine potential correlative mechanotransduction pathway activation, key transcription factors in the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and mitogen-activated protein kinase (MAPK) pathways were quantified for cells exposed to mild severity overpressure at the same time points following insult. Results indicate that reactivity occurs within the 48 hour timeframe, as marked by gene and protein upregulation of glial fibrillary acidic protein. Fluctuations in gene expression of several cytokines, including interleukin-1β and interleukin-6, were also observed when compared to sham. Moreover, differential activation of NFκB and MAPK pathways occurred parallel to these phenotypic changes. Specifically, phosphorylated p65 and p38 were significantly altered as compared to sham. Increased levels of phosphorylated focal adhesion kinase suggests that these changes may be the direct result of integrin activation following mechanical stimulation. Further characterization of integrin clustering and mechanotransduction signaling as a result of shock wave exposure will be necessary to establish definitive links in the mechanisms associated with astrocyte reactivity to isolated mechanical stimulus.
Keywords: in-vitro injury, mechanotransduction, reactivity, shock wave
Traumatic brain injury (TBI) is a leading cause of death worldwide. It can also lead to chronic impairment of function and causes a significant socioeconomic burden. Current therapies are limited and no intervention can alter the underlying pathological process. Over the past two decades a number of studies have investigated the potential for neural stem cell (NSC) transplantation in TBI models.
Keywords: traumatic brain injury, neural stem cells, stem cells, functional neurons, systematic review, metanalysis
ATTENUATION OF AXONAL DEGENERATION RELATIVE TO MYELIN PATHOLOGY AFTER MILD TRAUMATIC BRAIN INJURY IN MICE LACKING SARM1
USUHS, Anatomy, Physiology & Genetics, Bethesda, USA
Traumatic brain injury (TBI) results in traumatic axonal injury (TAI) in white matter tracts. Myelin pathology may contribute to deficits experienced after TBI. Myelin pathology may be due to secondary effects along degenerating axons or may occur as primary pathology of white matter injury. The current studies investigate the relationship between axonal degeneration and myelin pathology. Sarm1 is essential for execution of the conserved axon death pathway in injured axons. Sarm1-/- mice exhibit suppressed Wallerian degeneration and long-term survival of injured axons. Using a mild TBI (mTBI) model that results in TAI in the corpus callosum, we assessed axon degeneration and myelin pathology in Sarm1-/- and wild type mice. The corpus callosum was sampled using electron microscopy 3 days post-mTBI or sham procedure. In wild type mice, TBI increased axon degeneration and produced two forms of myelin pathology - demyelination of intact axons and excessive myelin extending out from axons. In Sarm1-/- mice, axon degeneration and demyelination were both suppressed after TBI. However, formation of excessive myelin figures after mTBI was similar in wild type and Sarm1-/- mice. We generated myelin reporter mice to label myelin with green fluorescent protein (GFP) to visualize these excessive myelin figures by confocal microscopy. PLPcreERT2 ; mTmG mice were given a single low-dose of tamoxifen prior to mTBI/sham procedure, to induce recombination in sparse oligodendrocytes. At 3 days post-injury, aberrant myelin formations were observed along GFP-labeled internodes in mTBI animals as compared to sham controls. These myelin abnormalities may represent dynamic myelin remodeling in a compromised axon-myelin unit. These results indicate that Sarm1 may be an important therapeutic target to interrupt processes that produce axon degeneration and demyelination after mTBI. In addition, mTBI also produces excessive myelin that was not mitigated by suppressing axon degeneration. Funded by the Center for Neuroscience and Regenerative Medicine.
Keywords: myelin, demyelination, neuropathology, plasticity
Diffuse axonal injury (DAI) has emerged as a primary pathological substrate of mild traumatic brain injury (mTBI) or concussion. DAI includes swollen axonal profiles resulting from mechanical and chemical damage to the cytoskeleton with interruption of axonal transport. In addition, dysregulation of axonal sodium channels with substantial influx of sodium has been observed in an in vitro model of axon trauma. In turn, these high sodium levels trigger increases in intra-axonal calcium levels in a feed-forward pathway of protease activation and axon degeneration. Although there is indirect evidence that this same process occurs in TBI in vivo, little is known about specific changes to axonal sodium channels in the injured brain.
Here, we used a model of head rotational acceleration in swine that mimics the biomechanics of concussion in humans to examine potential post-injury changes in specific subtypes of voltage gated sodium channels. Seventy-two hours after injury the brain was fixed and immunohistochemical analyses were performed on brain sections that included the corpus callosum. For sham animals, typical NaV1.6 positive nodal clusters were found throughout the white matter region. However, for injured animals, NaV1.6 labeling appeared to be split into heminodal-like profiles in APP co-labelled axonal swellings. In addition, patchy loss of NaV1.2 was observed in the white matter, suggesting proteolysis of these channels as is seen in vitro after trauma. These data suggest that the two main sodium channel subtypes found on both myelinated and unmyelinated axons in the brain are modified after trauma. These findings support examination of potential therapies targeting sodium channels to combat progressing DAI. This work was supported by DOD grant, PT110785 and NIH grants NS092389, NS038104 and EB021293.
Keywords: Sodium Channels, Diffuse Axonal Injury, APP, Neurodegeneration
DTI VOXEL-WISE ANALYSIS OF MILD TBI IN NEONATAL PIGS FOLLOWING NON-IMPACT HEAD ROTATION
University of Utah, Mechanical Engineering, SALT LAKE CITY, USA
The objective of this study was to develop a robust voxel-wise image analysis routine to detect subtle changes in axonal diffusion metrics following repetitive head trauma in a neonatal pig model. To achieve this objective, diffusion weighted images of anesthetized piglets (3–5 days old) were acquired before and after a repetitive, non-impact inertial head rotation. Animals were divided into three groups. Group 1 experienced cyclic head rotation and was imaged 24 hours post-injury. Groups 2 and 3 experienced thoracic compressions designed to increase cerebral blood volume (CBV) and ICP to levels reported for intense infant crying, and then experienced the cyclic head rotation. Group 2 was imaged immediately post-injury (acute) and Group 3 was imaged 24 hours post-injury. Fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were computed. A weighted integral measure (WIM) for each metric was created to account for the cluster size and magnitude of the statistical differences. Significant differences from baseline imaging were detected in all groups. WIM levels in Group 1 were lower than in Groups 2 and 3, suggesting there is overall less injury in Group 1 compared to Groups 2 and 3. Significant clusters in Group 1 were observed primarily in the internal capsule and corona radiata, correlating well with locations of positive βAPP staining. Groups 2 and 3, which had the same injury induced, had different responses depending on the timing of imaging. Acute imaging (Group 2) resulted in significant increases in MD and decreases in AD, while longer term imaging (24 hours in Group 3) resulted in significant decreases in FA and increases in RD. These data suggest increased CBV and ICP prior to mechanical loading can increase axonal injury. Further, DTI biomarkers of axonal injury will be highly dependent on the timing of the imaging.
Keywords: Diffusion Tensor Imaging, Repetitive Head Trauma, Pediatric, Voxel-based imaging
Traumatic axonal injury (TAI), a common consequence of traumatic brain injury (TBI), results in both axonal degeneration and cell death in regions far removed from the site of injury. Here, we examined health and calcium network activity changes in axons and cell soma networks adjacent and far away from axonal tracts after exposure to media taken from axons experiencing TAI. Briefly, primary rat cortical neurons were grown on micropatterned deformable membranes, whereby a series of parallel 2 mm-long lanes containing only axons span two populations of neuronal soma. The axon-only region was rapidly stretched via mechanical parameters based on clinical TBI. Thirty minutes following injury, media from injured axons was removed and either placed on uninjured cultures or separated into <3KDa or >3KDa and then placed on uninjured cultures. Calcium levels and network activity was assessed at 24, 48 and 72 hrs later using the genetically encoded calcium indicator GCaMP6. Significant axonal degeneration was observed within 24 hrs following addition of injured media where numerous calcium accumulations along the length of the axons is observed which progressively worsens over 72 hrs. Cells adjacent to the axon-only region start to degenerate within 24 hrs exposure and are all dead within 72 hrs. Delayed degeneration in cells far away from the axon-only region starts around 48 hrs, with a few surviving cells observed at 72 hrs. By 72 hrs, there is no associated calcium network activity observed. When media was separated into <>3KDa, >3kDa injured media showed increased cell death and decreased calcium activity compared to <3KDa injured media. These findings may shed some light on potential molecules responsible for cell death far removed from regions of traumatic axonal injury. This work was supported by DOD grant, PT110785 and NIH grants NS056202 and NS038104.
Keywords: Traumatic axonal injury, Cell death, Calcium activity, Secondary injury
LINEAR DISCRIMINANT ANALYSIS FOR HIGH CONTENT SCREENING IN NEUROTRAUMA
NorthShore University HealthSystem, Neurosurgery, Evanston, USA
The molecular pathology of neurotrauma is multi-modal so target-based drug screening is difficult. Therefore, phenotypic screening is attractive. Phenotypic screening requires an in vitro model and a phenotypic parameter with a high z’, indicating low signal-to-noise ratio. Conventionally, z’>0 is required and z’>0.5 is preferred. High content image analysis generates many phenotypic parameters. Typically, the parameter with the highest z’ is used for screening. This study hypothesized that performance could be further improved by combining several injury parameters into a composite parameter using linear discriminant analysis (LDA). 19 quantitative measures of cell viability and neurite morphology were calculated from images of an in vitro neurotrauma experiment using MetaXpress software. In the model, circular silicone cell culture membranes were indented from underneath with cylindrical posts to induce trauma. Indentations 1, 2, 3, 4 and 5 mm deep were used. The midpoint of the dose response was 3 mm. 4mm was defined as negative control. 2 mm was defined as positive control. The highest z’ for any single parameter was 0.121. A LDA transform was trained across all parameters to optimally discriminate injured and uninjured neurons using the 1mm and 5 mm data. This transform was then applied to the 2 mm and 4 mm data (that were not included in the training of LDA) and the z’ was 0.261. In addition to image parameters (i.e. parameters with a single value for each image), image analysis returned several cell parameters (i.e. parameters with a single value for each cell). For a given image, each of these is characterized by its density distribution. Specifically, we used a 10-bin histogram for each individual cell parameter. This process expanded the number of image parameters to 160. LDA on this dataset produced z’ = 0.582, which renders a significant improvement. Therefore, a high content neurotrauma screen was elevated from marginally viable to high performing using LDA. Supported by NIH R21NS098129.
Keywords: high content screening, computer vision, multi-parametric methods, drug discovery
University of Pennsylvania, Neurology, Philadelphia, USA
Traumatic Brain Injury (TBI) results in diffuse axonal injury (DAI) and diffuse vascular injury (DVI). Both DAI and DVI result from inertial shearing forces, and the two terms are often used interchangeably, the spatial relationships between DAI and DVI have not been carefully studied. Multimodal magnetic resonance imaging (MRI) can help to distinguish these injury mencahisms: diffusion tensor imaging (DTI) provides information about axonal integrity, while arterial spin labeling (ASL) and functional Blood Oxygen Level Dependent imaging (BOLD) with hypercapnia challenge, reflect cerebral blood flow (CBF) and cerebrovascular reactivity (CVR) respectively. Chronic TBI participants (n = 27) and age- and education-matched healthy controls (n = 15) underwent multimodality MRI. The Freesurfer image analysis suite (MGH, Harvard, MA) was used to segment each MP-RAGE image into regions of interest (ROIs). Mean values of mean diffusivity (MD), fractional anisotropy (FA), CBF, and CVR were extracted for each ROI. Additionally, maps were normalized into a common space (MNI Atlas) and z-score maps were generated based on a pool of healthy controls. Normality of an ROI/voxel was determined based on z-score (abnormal MD: z-score >2.5; abnormal FA, CBF, and CVR: z-score < −2.5). Abnormal ROIs in one modality were not predictive of abnormalities in another modality. Approximately 8–10% of abnormal voxels for CVR and CBF also show an abnormal voxel value for MD, while only 1% of abnormal CVR and CBF voxels show a concomitant abnormal FA value. These data indicate that chronic TBI patients display two distinct endophenotypes: microstructural tissue/axonal injury and vascular injury that are spatially independent.
Keywords: Diffusion Tensor Imaging, Cerebral Vascular Reactivity, Chronic TBI, MRI
Potentially due to their unique ultrastructure and anisotropic arrangement in fascicles, axons are particularly vulnerable to damage during mechanical loading from traumatic brain injury. This has been extensively demonstrated via in vitro models of traumatic axonal injury, where mechanical disruption of the axon cytoskeleton has been shown to trigger pathophysiological cascades. However, since most of the in-vitro studies used rodent neurons, corroboration using human neurons is important to determine the clinical relevance. To use human iPSC in an established in vitro model to examine the effects of dynamic stretch injury of micropatterened human axon fascicles in comparison to injured rat axon fascicles. Micropatterning of human iPSC neurons on deformable membranes created two populations of neuron soma spanned by parallel lanes of axon fascicles. Cultures were placed in a pressure-controlled device that injects a controlled air pulse into the pressure chamber. This cause dynamic selective stretch limited to the axon region. Three different pressures were used to cause injury. Over three days post-injury the cultures were examined for morphological and physiological changes and viability. After stretch injury, undulations were observed in a periodic arrangement along axons, which slowly relaxed back to the pre-stretch straight orientation. This reflects immediate mechanical damage to axonal microtubules that then buckle causing delayed relaxation of the axon, previously been characterized with rat axons. Varicose swellings began to form along the injured axons and by three days post injury, some axons had degenerated and disappeared. The extent of this damage and degeneration was greatly reduced compared to the sequence of rat axon degeneration at the same levels of injury. These data demonstrate that dynamic stretch injury in human iPSC axons induces mechanical microtubule damage leading to transport interruption and degeneration, similar to rat axons. The human axons appeared more resilient to the same level of injury in rat axons. This may be due to a larger diameter and more microtubules in human axons, which could provide more protection against mechanical disruption.
Keywords: Axonal injured, iPSC derived neuron, Calcium influx, neurodegeneration
RBM5 INCREASES NEURONAL DAMAGE IN A MODEL OF MECHANICAL STRETCH-INJURY
RNA binding motif 5 (RBM5) induces apoptosis in cancer cells. Little is known concerning its pro-death function(s) in the CNS. We reported that lentivirus mediated RBM5 knockdown (KO) decreases the activation of caspases following staurosporine (STS) injury in human neuronal SHSY5Y cells. Others have shown that RBM5 inhibition prevents caspase-3 activation, and the upregulation of pro-death p53 expression, in rat PC12 cells after being subjected to hydrogen peroxide injury. Taken together the evidence supports our hypothesis that RBM5 is toxic to CNS cells. However, RBM5 mediated neurotoxicity has not been established directly in primary neurons. Here we tested if lentivirus mediated RBM5 overexpression augments damage to primary cortical rat neurons after a mechanical stretch-injury. Cortical tissue was harvested from E16 rat embryonic brains. Cells were cultured on Silastic membrane plates (BioFlex). Day in vitro (DIV) 9 neurons were subjected to a gas-induced deformation using the cell injury controller (CIC-II) device (50 ms; 54% bilateral stretch). The following day (24 h post-injury), media was collected to assay lactate dehydrogenase (LDH) levels for quantification of cell death, and subsequently cultures were harvested for protein analysis. Mechanical stretch-injury significantly increased LDH release at 24 h post-injury compared to uninjured controls. Stretch-injured neurons overexpressing RBM5 had significantly higher LDH levels vs. empty-vector controls. In non-viral characterization studies, spectrin breakdown products (SBDPs) were also higher at 24 h post-injury in neurons subjected to stretch. Stretch induced SBDP levels were further augmented by RBM5 overexpression. Finally, an auxiliary study was done to test if RBM5 overexpression exacerbates neuronal injury by other insult mechanisms. RBM5 overexpressing neurons had significantly lower 24 h viability (measured by CellTiter-Blue®) in response to 100 nM STS. Results confirm that RBM5 promotes damage in primary neurons, and in a TBI-relevant injury model. This work was supported by NIH grant R21NS088145. Key words: stretch-injury, neuron, TBI, RNA binding motif 5.
Keywords: Mechanical stretch-injury, RNA binding motif 5 (RBM5), Lentivirus
DECREASED EFFECTIVENESS OF THE AXON INITIAL SEGMENT IN GENERATING AN ACTION POTENTIAL AFTER MILD CENTRAL FLUID PERCUSSION INJURY
Recently there has been an increased recognition that structural and physiological alterations within the neocortical gray matter likely contribute to long term morbidity after mild traumatic brain injury (mTBI). We have previously shown that within 2 days after mild central fluid percussion injury, layer V neocortical pyramidal neurons (LVpyr) have abnormal intrinsic properties. Here we have refined our previous approach, with a second derivative (2ndDeriv) analysis of the action potential (AP), separating the peak current amplitude at the axon initial segment (AIS) from that at the soma. Using whole cell patch clamp, a series of depolarizing pulses were injected to produce APs. The AP was smoothed (7 point boxcar filter, repeated 3 times) prior to calculation of the 2nd Deriv. In shams, this always produced two peaks, the first corresponding to the AP generated at the AIS and the second to that initiated at the soma. At 1 and 2 days after mTBI fewer than 40% of axotomized LVpyr neurons (AX) showed the AIS peak. Surprisingly, the intact LVpyr neurons (InT) also showed a loss of the AIS peak 1 day after injury (<30% with 2 peaks). This recovered quickly, however with all InT neurons having two peaks by 2 days after injury. In addition, the somal 2ndDeriv peak was significantly decreased for all AX and for the 1day InT, perhaps reflecting a reduced density of sodium channels. When this study was repeated in Cyclophilin-D (CypD) knock-out mice, all groups had two 2ndDeriv peaks, suggesting that the AIS was capable of producing APs in these mice. The reduced somal peak was partially ameliorated in CypD knock-out mice. Together these results suggest that the AIS is functionally affected by mTBI, even for structurally intact neurons, and that mitochondria protection is important for maintenance of AIS AP firing capabilities. Supported by NIH grant NS077675. CypDKO mouse strain originally supplied by Michael Forte, Vollum Institute, OHSU.
Keywords: Axon Initial Segment, Cyclophilin-D, Axotomized, Neocortex, Intact Neurons
Recently, interest has increased in the damaging consequences of repetitive traumatic brain injury (rTBI). We and others have confirmed that the injury severity, together with the time between insults are important variables, with the suggestion that rTBI can exacerbate both vascular and axonal injury (AI). In assessing AI, most have relied on the visualization of amyloid precursor protein (APP), although this typically underestimates the total burden of AI. We previously reported the increased sensitivity and utility of antibodies to phosphorylated c-Jun (p-c-Jun) in detecting both acute and long term AI. Because of this, we now reevaluate the burden of AI following rTBI using the quantitative assessment of neuronal p-c-Jun. Adult mice were subjected to central fluid percussion injury (CFPI) at an intensity of 1.40 atm 2x with a 3-hour interval between injuries. Controls employed an initial CFPI followed by a sham injury 3 h later. All animals were euthanized 24 hour after second insult and were perfused with 4% paraformaldehyde. Brain regions incident to the craniotomy were sectioned at 40 μm followed by immunofluorescent labeling for NeuN and p-c-Jun. Images of layer V in neocortical gray matter were captured using Zeiss LSM710 confocal microscope and quantitatively analyzed using ImageJ software. Statistical analyses were performed by R software. No animal showed any evidence of contusion or hematoma formation. All animals revealed significantly longer and consistent righting reflex times after injury compared to the sham injury alone. Among the layer V pyramidal neuron population, neither NeuN+ cells nor p-c-Jun+ cells revealed significant differences in number. The proportion of those neurons revealing p-c-Jun positive immunoreactivity was approximately 4%. These analyses suggest that the rTBI did not cause a significant increase in the number of axotomized neurons nor did it impact on the recovery of righting reflex times. This unanticipated finding may be related to the low intensity injury used. Thus, this issue mandates reevaluation with repetitive injuries of increased intensity. This study was funded by NIH grant NS077675.
Keywords: fluid percussion injury, c-Jun, repetitive TBI
OPTOGENETICALLY ENGINEERED HUMAN NEURONAL PRECURSORS FOR TRANSPLANTATION IN THE NORMAL AND INJURED RODENT BRAINS
JHMI SOM, Neuropathology, Baltimore, USA
Keywords: Optogenetics, TBI, Stem cells, Transplantation, Electrophysiology
Keywords: Traumatic Fracture, Nerve Laceration, Nerve Transection, Nerve Injury, Nerve Palsy
Biofluid based biomarkers hold great potential for tracking severe TBI progression. Increased CSF levels of glial fibrillary acidic protein (GFAP), alpha-II-spectrin, and their break-down products (BDPs) have been described acutely after TBI. However, their use as subacute-chronic biomarkers is unclear. This work conducted longitudinal analyses of GFAP, alpha-II-spectrin, and their BDPs following severe TBI. Penetrating ballistic-like brain injury (PBBI) was induced by inserting a probe through the right frontal pole followed by rapid balloon inflation to create a temporary cavity. Additional rats sustained a less severe probe insertion without balloon inflation (probe) or were exposed to craniotomy alone (sham). CSF was collected 24 h, 3 d, 7 d, 1 m, or 3 m after injury and concentrated with centrifugal spin-filters. GFAP, alpha-II-spectrin, and BDPs were assessed by Western blot and/or ELISA. Based on western blots, alpha-II-spectrin and its BDPs were not elevated following probe injury but PBBI led to widespread elevation compared to sham. Full length protein (280kDa) was significantly elevated at 24 h and 3 d following PBBI. SBDP145-150 were robustly increased at 24 h-1 m. SBDP-120 was detected at 3 d. ELISAs indicated that GFAP levels were not significantly upregulated at 24 h or 3 d. At 7 d-3 m, GFAP was not detected in the majority of samples. To confirm these results, western blots were performed to determine if full length GFAP or its BDPs could be ascertained. These data indicate that GFAP-BDPs were significantly increased at 24 h following PBBI. In accordance with ELISA quantitation, GFAP and its BDP were not detectable in most samples collected at 7 d-3 m. However, nearly half of PBBI rats at 1m and 10% of sham rats at 3 m did display the characteristic banding of GFAP-BDPs. These data indicate that elevation in injury-induced biomarkers remain detectable in CSF as late as 1m following PBBI. Alpha-II-spectrin and its SBDP145-150, as opposed to GFAP, may have greater utility as acute-chronic CSF biomarkers.
Keywords: GFAP, alpha-II-spectrin, chronic, rat
INITIAL OBSERVATIONS ON A BALLISTIC MODEL OF BLUNT INJURY IN THE GYRENCEPHALIC BRAIN: BEHIND HELMET BLUNT TRAUMA IN PIGS
Johns Hopkins University School of Medicine, Department of Pathology/ Division of Neuropathology, Baltimore, USA
Our goal here was to explore the neuropathology of blunt TBI when high-velocity impactors strike the head. Such impactors include baseball bats or bullets when the head is helmeted. In the latter case, the helmet slows down or stops the bullet while its material elastically deforms, pushing down on the cranium at high velocity. This type of injury is called “Behind Helmet Blunt Trauma”, and is the model of blunt injury used here. Yucatan miniature swine, anesthetized with Propofol, were subjected to ballistic (gunshot) trauma to the head with rifle while wearing a combat helmet. Bullet impact velocities averaged 1800 ft/s. Vital signs were monitored throughout the injury and the post-injury survival. Subjects with stable vitals were euthanized at 4 hours with transcardial perfusion with 4% neutral-buffered paraformaldehyde. If vitals decompensated to critical levels, subjects were euthanized without further delay using the same procedures. Cranial CT was performed before and after the gunshot. Crania were carefully inspected for evidence of fracture. Brain tissues were embedded in paraffin and then sectioned (10 μm) or processed as floating sections (40 μm). Serial sections were processed for: general anatomy using Nissl stain (cresyl violet); vascular anatomy/pathology (Masson's trichrome); and axonal pathology (amyloid precursor protein immunohistochemistry). The initial characterization of lesions in 7 subjects is summarized as follows: In general, there appeared to be a relationship between bullet velocity, severity of cranial fractures, and vital sign outcome. In turn, there was an association between fracture and lobe “bursting”, i.e. severe contusion underneath. Coup sites were associated with subarachnoid blood, and multiple superficial linear or round hemorrhagic foci more so than large contusions. There were some contrecoup contusions in olfactory sites. Subarachnoid hemorrhage was ubiquitous. In some cases, there was a more diffuse pattern of small hemorrhages over several gyri, at the white-gray matter border. Multiple fronts of classical APP (+) axonal abnormalities were seen in prefrontal white matter, medial parietal, occipital white matter, next to the roof of frontal horn, splenium, and brain stem, were remarkably similar in distributions to the ones observed with inertia acceleration. There was no obvious association between hemorrhagic foci and axonal abnormalities. These observations suggest that high-velocity blunt trauma is associated with subarachnoid bleeding and diffuse lesions, both vascular and axonal, and axonal pathologies resemble the ones seen with acceleration injuries.
Keywords: Ballistic Injury, Axonal Injury, Hemorrhage, Behind Helmet Blunt Trauma, 3D Brain Mapping
University of Miami, The Miami Project to Cure Paralysis, Miami, USA
We previously reported amelioration of PBBI-induced cognitive deficits following engraftment of NSI-566 human neural stem cells (hNSC). However, the mechanism of such amelioration is unknown. To gain insights we hypothesized that transplant induced behavior modification will depend on transplant location, extent of tissue spared, numbers of cells engrafted derived processes. To test the hypothesis, one million green fluorescent protein (GFP) expressing hNSCs transplanted one week following injury either in the core of injured tissue (intralesional) or in undamaged tissue surrounding the core (perilesional). Same dose of cells were transplanted into an uninjured sham group to uncover hNSCs effects. The animals were tested after 12 weeks (by this time hNSC have been shown to undergo neuronal differentiation) in a standard Morris Water Maze for acquisition (latency on day 5 after 4 days of training) and performance following spatial reversal (day 12). Linear regression to explore relationship between transplant location, lesion volume, cell numbers and latencies was assessed. Surprisingly there was no relationship between transplant location, extent of engraftment and latency to find platform. Similar analysis on additional parameters such as extent of GFP processes in corpus callosum and internal capsule will be presented to gain insights to the mechanism. These results are similar to those with bone marrow stem cell therapies in stroke patients.
Keywords: human neural stem cell, PBBI
This study examined the changes in blood coagulation profiles in rats exposed to penetrating ballistic-like brain injury (PBBI) combined with hemorrhagic shock (HS), and compared the effect of resuscitation with Hextend, Lactated Ringer's solution (LR) or whole blood (WB) on post-injury coagulation. Rats were assigned into three groups: Hextend, LR, and WB. All animals received a frontal PBBI followed by a 35-minute of HS (mean arterial pressure < 45 mmHg). In the Hextend and LR groups, resuscitation fluid was infused followed by shed blood reinfusion. In WB group, shed blood was re-infused 15 minutes after the hypotensive period. Blood samples were analyzed using thromboelastography (TEG) and blood gas analyzer at four time points: Pre-injury baseline, post-PBBI, post-HS, and after the infusion of Hextend, LR or WB. TEG parameters included Reaction Time (R), Kinetics Time (K), Angle (α) and Maximum Amplitude (MA). R was significantly reduced vs. baseline by both PBBI and HS. K was significantly shorter and α was significantly greater immediately following PBBI and HS vs. baseline. MA was significantly greater following PBBI vs. either baseline or post-HS. Hextend, LR and WB produced different coagulation profiles after PBBI and HS insults. Hextend resulted in a significantly smaller α (p < .05 vs. WB) and MA (p < .05 vs. Baseline and WB), indicating that Hextend infusion leads to a slower coagulation rate and reduced clot strength. In contrast, the parameters in LR and WB groups were comparable to the baseline values. Blood gas analysis revealed a significant reduction in hemoglobin and hematocrit following HS and PBBI, which were restored to baseline levels by WB resuscitation. On the contrary, a significant decrease (p < .05 vs. baseline or WB) in ctHb and Hct as well as an increased lactate level were detected following LR or Hextend resuscitation. The current results show that PBBI altered the coagulation parameters, which returned to the baseline levels after LR or WB resuscitation whereas Hextend resuscitation after PBBI/HS resulted in hypocoagulation.
Keywords: Coagulation, Resuscitation, Hemorrhagic Shock, Thromboelastography
PENETRATING GUNSHOT HEAD INJURIES WITH PROLONGED SURVIVAL: A POTENTIAL RISK FACTOR FOR CHRONIC TRAUMATIC ENCEPHALOPATHY
Post-traumatic neurodegeneration in the form of chronic traumatic encephalopathy (CTE) is associated with a history of traumatic brain injury in susceptible individuals, although the types and number of insults required to initiate neurodegeneration remain incompletely defined. Penetrating gunshot head trauma has not previously been considered a risk factor for neurodegeneration despite being a major cause of traumatic brain injury in many urban centers in the United States. Penetrating gunshot head wounds do not perforate the head and kinetic energy possessed by the projectile is entirely absorbed by the head (including brain). We hypothesized that in penetrating gunshot head wounds with a survival interval, the crushing and stretch injury of brain tissue by the bullet and the contusion of cortical surfaces from rapid, brief expansion of skull contents, might act as substrates for aggregation of hyperphosphorylated tau. In New York City, gunshot lethalities are investigated by the Office of the Chief Medical Examiner (OCME). Over a 6-month period we prospectively examined brains of all gunshot head wounds referred to the forensic neuropathology consultation service at the NYC OCME. Of these, 4 had retained bullet(s) or fragments, and a survival period after injury; these underwent full neurodegenerative disease workup including Bielschowsky silver preparation and immunohistochemistry for tau, beta-amyloid, alpha-synuclein and ubiquitin. All 4 cases were male. Age range at injury was 14 to 29 years and at death was 19 to 50 years. Survival periods ranged from 1 month to 30 years. All victims had neurologic deficits following injury and two had post-traumatic seizure disorder. Neuropathologic evaluation revealed tau deposition in 2: one with rare threads in the bank of the middle frontal gyrus (survival, 1 month), the other with rare threads around cortical vessels in the wound cavity and rare tangles in the locus ceruleus and thalamus (survival, 30 years). No other abnormal protein expression was detected. Detection of tau at autopsy suggests penetrating gunshot trauma represents an independent risk factor for CTE among injured survivors.
Keywords: Gunshot trauma, forensic pathology
PREDICTING SURVIVAL AFTER ACUTE CIVILIAN PENETRATING BRAIN INJURIES: THE SPIN SCORE
University of Massachusetts Medical School (UMMS), Neurology (Neurocritical Care), Worcester, MA, USA
Keywords: Penetrating Brain Injury, Gun Shot Wound, Prognostication, Outcome Prediction
Mitochondria play a pivotal role in the secondary pathophysiological sequelae following traumatic brain injury (TBI) and thus are leading targets for therapeutic interventions. The current study is designed to characterize the temporal and regional post-injury responses of mitochondrial energy crisis up to 14 days following penetrating ballistic-like brain injury (PBBI) in rats. Adult male rats were subjected to either 10% unilateral PBBI or Sham craniotomy (n = 6/group). At 30 min, 3 h, 6 h, 24 h, 3 d, 7 d and 14 d post-PBBI, mitochondria were isolated from the ipsilateral hemisphere of two brain regions: the injury core, i.e. frontal cortex + striatum (FC+ST) and a region distant from the injury core, i.e. hippocampus (HIP). Mitochondrial bioenergetics parameters were measured using a high-throughput procedure of the Seahorse Flux Analyzer. The time-course of FC+ST mitochondria showed a biphasic energy dysfunction response, indicated by a decline in ATP synthesis and maximum respiratory capacity. The first phase of energy crisis started immediately at 30 min (−42%; p < 0.05 vs. Sham) and attained baseline levels between 3 h to 6 h (nonsignificant vs. Sham), followed by a second phase of more robust energy crisis observed between 24 h to 14 d post-injury (−55% to −90%; p < 0.05 vs. Sham). In contrast, the HIP mitochondria showed a significantly delayed decline in mitochondrial bioenergetics parameters at 7 d (−74%; p < 0.05 vs. Sham) and 14 d (−51%; p < 0.05 vs. Sham) post-PBBI. Collectively, PBBI produced temporal and region-specific alterations in mitochondrial bioenergetics that are unique to the penetrating, temporary cavity mechanism. More importantly, the results underscore an extended therapeutic window (between 3 h to 24 h post-injury) for mitochondria targeted intervention following PBBI.
Support: US_Army_CCCRP_H_026_2014_WRAIR.
Keywords: Traumatic Brain Injury, Penetrating Ballistic like Brain Injury, Brain Mitochondrial Energy Metabolism, Time Course of Secondary Injury, Seahorse Flux Analyzer, Mitochondria Targeted Therapeutics
A MOUSE MODEL OF FOCAL VASCULAR INJURY INDUCES ASTROCYTE REACTIVITY, TAU OLIGOMERS, AND ABERRANT BEHAVIOUR
Keywords: Ballistic injury, focal vascular injury, Astrocyte reactivity, Behavioral outcome
DIFFERENTIAL EFFECTS OF ACUTE AND CHRONIC CAFFEINE PRE-EXPOSURE IN THE WRAIR PENETRATING BALLISTIC-LIKE BRAIN INJURY MODEL
Sleep disturbance is a commonly reported behavioral health diagnosis in the military, and Soldiers increasingly rely on pharmacological intervention, such as caffeine, to counteract the effects of sleep-loss. This study assessed the potential therapeutic effect of caffeine on neurobehavioral recovery in the WRAIR PBBI model. Unilateral frontal PBBI was produced in the right hemisphere of anesthetized rats at moderate (7%-PBBI) or severe (10%-PBBI) injury levels. Animals were randomly assigned to pretreatment groups: (1) acute caffeine (25mg/kg CAF) or vehicle (gavage, 1h prior to PBBI), (2) chronic caffeine (0.25g/L CAF) or water (30 days prior to PBBI). Motor function was evaluated on the rotarod (7 and 10 days post). Cognitive performance was evaluated on the Morris water maze (MWM). A single bolus dose of 25 mg/kg caffeine (p.o.) one hour prior to PBBI had no effect on motor outcome. Cognitive deficits were observed in all injury groups with average latency to find hidden platform increased by 130% (PBBI) and 140% (25mg/kg CAF) vs. sham (p < 0.05). Rats exposed acute caffeine prior to sustaining 10% PBBI displayed a significantly higher thigmotaxic response compared to injured rats not exposed to caffeine, which may indicate that pre-injury exposure to caffeine exacerbates post-injury anxiety/attention decrements. Results of the chronic caffeine study revealed a significant improvement in motor outcome at 7 and 10 days post-injury in the 7%-PBBI group that had received chronic, pre-injury dosing with caffeine (p < 0.05). However, chronic caffeine exposure prior to PBBI significantly increased the latency to locate the platform in the MWM task compared to vehicle at both 7% and 10% injury levels (p < 0.05), indicating that chronic pre-injury caffeine exposure may worsen cognitive outcome. Overall, the results of this study indicate that chronic caffeine consumption prior to injury may provide moderate beneficial effects to motor recovery, but may worsen the neurocognitive outcome following TBI.
Keywords: traumatic brain injury, caffeine
PERILESIONAL NEURAL STEM CELL TRANSPLANTATION MITIGATES LESION VOLUME FOLLOWING PENETRATING BALLISTIC-LIKE BRAIN INJURY (PBBI)
University of Miami, Neurosurgery, Miami, USA
We previously confirmed engraftment of NSI566 human neural stem cells (hNSC) in PBBI. In this study, we aimed to determine the optimal transplant location relative to PBBI lesion. To do so, we transplanted one million hNSC into each animal one week following injury either in the core of injured tissue (intralesional) or in undamaged tissue immediately surrounding the core (perilesional). We also transplanted the same dose of cells into an uninjured sham group. In a final vehicle group, we transplanted only media with no cells one week following injury. After 12 weeks, we assessed the survival of the transplanted cells and the overall volume of the lesion. Although the survivability of the cells was not significantly different between intra and perilesional groups, there was greater cell survival in the inujred groups compared to non injured control. This suggests that engraftment of cells was not adversely by the PBBI lesion milleu. The injury environment after one week may be conducive to transplanted stem cells in terms of survivability and/or proliferation. PBBI-injured animals that received perilesional hNSC engraftments showed a significant reduction in lesion volume compared to animals that received intralesional hNSC transplants [Unijured + hNSC = 2.64 ± 0.68 mm3; PBBI + Vehicle = 64.35 ± 4.48 mm3; PBBI + Perilesional hNSC = 27.57 ± 3.78 mm3; PBBI + Intralesional hNSC = 55.74 ± 4.77 mm3]. The lesion volume was significantly less in the perilesional transplant group when compared to the vehicle group (p < .05 vs. PBBI + Vehicle). Both transplant groups had reduced lesion volume compared to vehicle group.
Keywords: human neural stem cell, PBBI
Firearm injuries cause an estimated 33,000 deaths annually in the United States. Management algorithms following gunshot wounds to the head (GSWH) have been static for two decades and outcomes remain in need of systematic characterization. Adult GSWH patients in the National Sample Program (NSP) of the National Trauma Data Bank (NTDB), years 2003–2012, were extracted. Multivariable regression identified predictors for hospital length of stay (HLOS), intensive care unit length of stay (ICU LOS), medical complications, mortality, and discharge disposition. Odds ratios (OR) and 95% confidence intervals (CI) were reported. Statistical significance was assessed at p < 0.001. In total, 8,148 patients were included extrapolating to 32,439 national incidents. Age was 36.6 ± 16.4 years. Mean HLOS was 13.9 ± 17.9 days and ICU LOS was 8.6 ± 17.4 days. The mortality was 54.6%. Age, injury intent, Glasgow Coma Scale (GCS) score, Injury Severity Score (ISS), and hypotension were significant predictors as previously reported (p < 0.001). Compared to privately insured patients, Medicare/Medicaid patients had longer HLOS (mean increase 4.4 days, 95% CI [2.6-6.3 days], p < 0.001). Compared to the Midwest, the South had longer HLOS (mean increase 3.7 days [2.0-5.4 days], p < 0.001) and more complications (OR 1.7 [1.4-2.0], p < 0.001); the West had less complications (OR 0.6 [0.5-0.7], p < 0.001). Compared to handgun, injury from shotgun and hunting rifle had lower odds of death (OR 0.3 [0.2-0.4], p < 0.001; OR 0.5 [0.4-0.8], p < 0.001). Suicide had lower odds of discharge to home compared to accident (OR 0.5 [0.3-0.7], p < 0.001). Relative to Level I trauma centers, Level II trauma centers had lower odds of discharge to home (OR 0.7 [0.5-0.8], p < 0.001). In conclusion, firearm type, injury intent, insurance type, and geographic region are important prognosticators for acute recovery and outcome following GSWH, in addition to older age, hypotension, and injury severity. Improved understanding of civilian GSWH is critical to increasing awareness and reducing injury burden from firearm injuries.
Keywords: firearm injury, mortality, penetrating brain injury, traumatic brain injury, epidemiology
Uniformed Services University of the Health Sciences, Pathology, Bethesda, USA
Traumatic brain injury (TBI) is considered as one of the major causes of disability and death. Currently, there is no FDA approved diagnostic test for diagnosis of TBI. The usefulness of Glasgow comma scale (GCS) and imaging (CT and MRI scans) is limited in the diagnosis of mild TBI. MicroRNAs (miRNAs) are small non-coding RNA molecules which regulate gene expression and have been reported as biomarkers of several diseases due to their stability and ease of detection. We have previously reported a panel of 10 miRNAs as potential acute TBI biomarkers using real-time PCR. In this study, we have used a droplet digital PCR (ddPCR) platform to quantitate the absolute miRNA concentration in a larger cohort of acute and sub-acute TBI cases. Human severe TBI serum and cerebrospinal fluid samples were collected from the patients at 48 hr and 192 hr post injury. Total miRNA from injury and healthy controls groups was isolated and reverse transcription was performed using primers for the following: miR-195, miR-20a, miR-30d, miR-328, miR-451, miR- 362-3p, miR-486, miR-505*and miR-92a. MiRNAs were quantitated (copies /μl) in serum from severe TBI cases (n = 30) at 48 hr post injury and 192 hr post injury and compared to the healthy controls. Data from ddPCR showed significantly higher miRNA concentration of all miRNAs in the 48 hr serum samples in comparison to the healthy controls and their concentrations remained elevated until 192 hr post injury. The results of this study showed that this panel of miRNA biomarkers is promising in detecting acute and sub-acute TBI. Further, ddPCR platform enabled a precise absolute miRNA expression in comparison to real-time PCR.
The opinions expressed herein are those of authors and are not necessarily representative of those of the Uniformed Services University, Department of Defense or, the United States Army, Navy, or Air Force and DMRDP.
Keywords: Biomarker, traumatic brain injury, microRNAs, diagnostics
University of Texas Medical Branch, Anesthesiology, Galveston, USA
Sensitive and accurate assessment of genomic changes plays a major role in personalized medicine. We hypothesized that two broad types of TBI, focal or diffuse injury, can be defined and distinguished using unique signatures of circulating miRNAs. Distinct combinations of circulating miRNAs have been shown to serve as diagnostic and prognostic biomarkers in other neurodegenerative disorders. As a first step in correlating neuroimaging data with serum miRNA levels, CT scan data were used to distinguish two groups of TBI patients (focal or diffuse injury). The RNA isolated from TBI patient serum was assessed for integrity, reverse transcribed, pre-amplified, and run on the Human Serum and Plasma miScript PCR array panels; these panels consist of 84 well characterized miRNAs expressed in biofluids. Global mean analysis was performed in Global Gene software to determine the average ΔCT value for each miRNA, and fold changes were calculated by comparing each TBI sample to controls. One-way ANOVA was used to compare control, focal, and diffuse injury groups, and was followed by the Benjamin-Hochberg test and Tukey's test for multiple comparisons. PCR array data were validated using Droplet Digital PCR analysis of differentially expressed miRNAs. Preliminary analysis shows several miRNAs are differently expressed in human serum samples between control and injury groups, and one differently expressed miRNA distinguishes between focal and diffuse injury. Ingenuity pathway analysis suggests these miRNAs regulate multiple pro-death and pro-survival cell signaling pathways affected by TBI. The development of serum miRNA signatures as prediction tools will help us determine if changes in a small, discrete panel of miRNAs could serve as a surrogate of changes in miRNA expression in different brain areas of TBI patients diagnosed with focal or diffuse brain injury.
Keywords: Brain injury, microRNA expression, Biomarker, Human serum
Walter Reed Army Institute of Research, Brain Trauma Neuroprotection Branch, Silver Spring, USA
Cathepsin B is a lysosomal cysteine protease that may be an important therapeutic target and biomarker for traumatic brain injury (TBI). The current study was designed to evaluate the regional and temporal profiles of mature cathepsin B (mCatB) protein levels and cysteine protease activity in the penetrating ballistic-like brain injury (PBBI) model. Rat ipsilateral frontal cortex (FC), hippocampus (HC), and cerebellum (CB) were collected 1–7 d after PBBI or craniotomy alone, compared to unoperated sham controls (baseline for fold change comparison). MCatB increased after 7 d after craniotomy (2.3-fold) and PBBI (11.9-fold) in the FC. HC levels of mCatB were elevated at 3 d (1.6-fold) and 7d (2.5-fold) after PBBI. CB mCatB was suppressed 3 d after craniotomy (0.37-fold) and 1-3 d after PBBI (range: 0.43–0.55-fold) vs. sham. However, at 7d post-injury CB mCatB levels in PBBI animals exceeded that of sham (1.2-fold). Craniotomy did not have a significant effect on cysteine protease activity in the FC. PBBI resulted in increased FC cysteine protease activity at 3 d (3.4-fold) and 7 d (2.13-fold) post-injury. In the HC, activity increased 7d after craniotomy (1.8-fold). Non-linear dysregulation of mCatB protein levels infer that proteolytic processes within proximal and distal brain regions vary with injury severity and time over the course of TBI progression. Differential increases in cysteine protease activity suggest that TBI elicits a complex proteolytic response, inclusive of cathepsin B, thereby incentivizing the need for spatio-temporally defined therapeutic strategies.
Keywords: Cathepsin B, Cysteine Proteases, Craniotomy, Penetrating Ballistic-like Brain Injury, Spatiotemporal Resolution
University of Toronto, Institute of Medical Science, Toronto, Canada
Clinical trials within the field of traumatic spinal cord injury (SCI) have been largely unsuccessful due to the lack of reliable and consistently accurate measures of patient injury severity. Specifically, the field of SCI lacks biofluid-based biomarkers capable of objectively assessing the severity of a patient's injury. Tau, a microtubule-associated protein, is primarily located within the axonal compartment of central nervous system (CNS) axons. Following traumatic brain injury, tau becomes hyperphosphorylated, and research in this field has proven the utility of hyperphosphorylated tau (p-tau) as a histological and serological biomarker of axonal injury; however, no study to date has examined the utility of p-tau as a biomarker post-SCI. Therefore, for the current study, we utilized the clip-compression SCI rat model as well as a full spinal cord transection to assess the histological profile of tau hyperphosphorylation; sham-controls consisted of a laminectomy-only procedure. Tau hyperphosphorylation was assessed using antibodies specific for tau when phosphorylated at the serine 396 and threonine 205 amino acid positions; antibody specificity was assessed by using a P301L transgenic Alzheimer's disease mouse and tau knockout mouse. Current results demonstrate that tau becomes hyperphosphorylated within injured white matter axons as early as 4 hours post-clip-compression SCI and persists as late as 7 days post-injury, to a much lesser extent, indicated by significantly reduced p-tau white matter axonal immunoreactivity. Additionally, p-tau white matter axonal immunoreactivity was demonstrated in animals subjected to a full spinal cord transection. Sham-controls displayed no p-tau axonal immunoreactivity. Therefore, similarly to traumatic brain injury, the tau protein becomes hyperphosphorylated within injured spinal cord axons, and antibodies specific for phosphorylated tau amino acid residues are capable of histologically assessing the extent of axonal injury post-SCI. Further experiments are currently being conducted to assess the serological profile of p-tau in animals subjected to both a clip-compression and full transection SCI.
Keywords: Tau, Spinal Cord Injury, Phosphorylation, Biomarker
University of Pennsylvania, Bioengineering, Philadelphia, USA
We investigated the plasma concentration of amino acids (AA) as the potential biomarkers of focal and diffuse traumatic brain injury (TBI). Twenty-five female, 4-week-old piglets received a diffuse TBI via rapid non-impact head rotation (RNR, n = 13) or a focal TBI via controlled cortical impact (CCI, n = 12), and blood samples were obtained pre-injury, and either at 24-hours or 4-days post-TBI. The concentrations of seventeen AAs were determined via HPLC (Agilent 1260 Infinity). Only Glycine, Serine, Taurine and Alanine concentrations were significantly decreased at 24-hours post-injury compared with pre-injury (Mann–Whitney test, P < 0.05). Glycine levels continued to remain depressed significantly at 4-days post-TBI while Serine, Taurine and Alanine returned to pre-injury levels at 4-days. In addition, we observed significant increases in Arginine and Lysine levels and significant decrease in Isoleucine at 4-days post-TBI compared with pre-injury. To find a robust TBI biomarker while taking into consideration possible interactions between AA levels, we combined RNR and CCI injuries and used multivariate logistic regression analysis, coupled with the best-subset selection technique and k-fold cross-validation method, to perform a thorough search of all possible subsets of AAs, and evaluated the ability of the subset to correctly identify serum from injured or uninjured animals. Our goal was to determine the optimal multivariate AA biomarker model with the highest TBI prediction capability, assessed using the receiver operating characteristics curve analysis. Selection criteria, such as Akaike's and Bayesian information criteria, adjusted R-squared, and maximum log-likelihood, were used to determine the best subset of AAs for groups of 1-to-17 AAs. Instead of any single AA, the logistic regression combining Glycine, Taurine, and Ornithine was optimal for TBI diagnosis, with 80% sensitivity and 86% overall prediction rate. This optimal model was validated on a separate dataset (4 uninjured and 5 RNR piglets), yielding excellent TBI diagnostic performance, with 100% sensitivity and 78% overall prediction rate. We propose a new multivariate AA serum biomarker to detect mild-to-moderate. Funded by NINDS U01NS069545.
Keywords: TBI Biomarker, Amino Acids, Multivariate Analysis, TBI Diagnosis, Diffuse and Focal TBI
University of Minnesota, Neurosurgery, Minneapolis, USA
The objective of this study was to evaluate blood levels of biomarkers such as GFAP (glial fibrillary acidic protein) and UCH-L1 (ubiquitin carboxyl-terminal hydrolase L1 enzyme) in predicting computed tomography (CT)-positive TBI. As part of preliminary analyses from early enrollees in the CLASSIFY TBI trial, blood samples were collected from 78 trauma subjects within 6 hours of injury who underwent a head CT scan. Biomarker concentrations of subjects with positive (Marshall classification ≥ 2; n = 34) or negative (Marshall classification = 1; n = 44) CT scans were compared. In addition, biomarker concentrations of subjects in the trauma group were compared with 35 nontrauma control subjects. Median levels of UCH-L1 for control versus trauma groups were 0.074 and 0.780 ng/mL. Corresponding median levels of GFAP were 0.009 and 0.109 ng/mL. Blood concentrations of both biomarkers were significantly different between the groups (both P < 0.001). In trauma subjects, GFAP (P < 0.001) and UCH-L1 (P = 0.008) were higher in subjects with positive CT scans compared to those with negative CT scans. These findings indicate that concentrations of UCH-L1 and GFAP are increased in patients with TBI. Furthermore, these preliminary data suggest higher levels of GFAP and UCH-L1 predict a positive CT scan with high sensitivity, suggesting that these biomarkers may have clinical utility in the management of brain injury.
Keywords: Positive Head CT, Biomarker, Traumatic brain injury
Keywords: electroencephalogram, neurophysiology, frequency, coherence
CIRCULATING GFAP LEVELS TO MONITOR THERAPEUTIC RESPONSE TO GLIBENCLAMIDE IN CONTROLLED CORTICAL IMPACT: FINDINGS FROM OBTT
Messina University, Messina, Italy
Glibenclamide (GLI) is a sulfonylurea receptor antagonist that has shown promise as a therapy to prevent secondary injury following traumatic brain injury (TBI). The present work, part of the Operation Brain Trauma Therapy (OBTT) multi-center pre-clinical drug screening consortium, investigated the effects of GLI treatment on the levels of circulating glial fibrillary acidic protein (GFAP) and the relationships with histopathological and behavioral outcomes after controlled cortical impact (CCI). OBTT demonstrated that GLI treatment reduced contusion volume in CCI; thus, we sought to determine whether circulating GFAP in the initial 24 h could inform theranostically on lesion volume at 21 d post-injury. Adult male rats subjected to CCI received a bolus (10μg/kg IP) 15 min after-injury, followed by a continuous SQ infusion (0.2μg/h) via osmotic pumps throughout 7 d of GLI or vehicle. GFAP levels in blood were measured at 1, 4 and 24 h after CCI. Vehicle-treated rats displayed distinct temporal profiles for GFAP vs. GLI-treated rats. GFAP in vehicle-treated animals demonstrated a sustained increase after-injury vs. shams (p < 0.0001) peaking at 4 h. Conversely, rats treated with GLI initially had high GFAP levels similar to vehicle. However, by 24 h GFAP in GLI-treated rats did not differ from sham and were lower (p < 0.05) than vehicle. Overall, GLI-treated rats had significantly lower GFAP release (area under the curve) throughout the study vs. vehicle. GFAP at 24h also strongly correlated with contusion volume at 21d (r = 0.88, p < 0.0001), hemispheric tissue loss (r = 0.86, p < 0.0001) and MWM latency (r = 0.68, p = 0.0001). Our findings support a role for GFAP in predicting tissue damage and as a marker of therapeutic response corroborating advantageous effects of GLI in CCI. Circulating GFAP may be useful for high-throughput screening of drugs in pre-clinical investigations. Support: USArmyW81XWH-10-1-0623.
Keywords: Biomarkers, GFAP, TBI, controlled cortical impact, rat, neuroprotection
Univ. of Eastern Finland, Neurobiology, Kuopio Fin, Finland
Keywords: Exosome, microRNA, Plasma
Keywords: chronic TBI, amyloid B, tau, neuropsychological outcome
SGP130 MODERATES THE RELATIONSHIP BETWEEN CHRONIC IL-6/SIL-6R COMPLEX IN DIFFERENTIATING OUTCOME AFTER SEVERE TBI
University of Pittsburgh, Department of Physical Medicine and Rehabilitation, Pittsburgh, USA
The secondary injury response after traumatic brain injury (TBI) is characterized by an acute innate immune response. While research has focused on acute neuroinflammatory markers, our previous work suggests sub-acute and chronic peripheral inflammatory markers influences long-term global outcomes. Specifically, pro-inflammatory mediators, including interleukin 6 (IL-6), 2 weeks to 3 months post-injury, were associated with worse global outcomes at 6 and 12 months. Further, the relationship between IL-6 and its soluble receptor, sIL-6R, facilitates a signaling cascade predisposing individuals to a chronic inflammatory state; in contrast soluble gp130 (sgp130), a potent IL-6 inhibitory transmembrane protein, moderates this relationship. To date, no clinical or experimental TBI study has examined the association between IL-6/sIL-6R and sgp130. The objective of this study was to evaluate relationships between serum IL-6, sIL-6R, and sgp130 in the subacute period post-injury for N = 100 individuals with severe TBI. Monthly ratios were produced for IL-6:sIL-6R and sgp130:sIL-6R, and IL-6 levels were quartiled using levels from samples collected up to 3 months post-injury. Six-month GOS scores were dichotomized to reflect poor (GOS = 2/3) vs. good (GOS = 4/5) outcome. Bivariate analysis showed significant differences in sIL-6R by GOS group (p = <0.0001), where higher sIL-6R levels were associated with poor outcome. sgp130:sIL-6R ratios also significantly differed by GOS group (p = 0.0034), where lower ratios were associated with poor outcome. A multivariate logistic regression model including age, IL-6, sgp130:sIL-6R, and a sgp130:sIL-6R*IL-6 resulted in a significant interaction (OR = 5.527, p = 0.0047) in predicting 6-month outcome. The interaction suggests sgp130:sIL-6R ratios influence global outcome by attenuating the IL-6/sIL-6R complex, resulting in higher IL-6 levels. These results suggest the preferential binding of sgp130 to sIL-6R selectively blocks progression of inflammation through the inhibition of IL-6 signaling by the sIL-6R after severe TBI. This work has novel implications for understanding how sgp130 potentially serves as a modifiable target for prevention and/or resolution of chronic inflammation post-TBI. Support: DoD-W81XWH-071-0701; NIDILRR-90DP0041; R49-CCR323155.
Keywords: chronic inflammation, traumatic brain injury, innate immunity, interleukin 6, sgp130
UCLA, Neurosurgery, Los Angeles, USA
Hippocampal lateral fluid percussion injury (LFPI) has been previously correlated with memory and recognition deficits in developing rats and dysfunction of glutamatergic neurotransmission via the N-methyl-D-aspartate receptor (NMDAR), is a plausible factor. Protein expression studies have confirmed a downregulation of NMDAR protein after experimental traumatic brain injury (TBI). To study the dynamics of NMDARs in vivo, and their role in memory-related network dysfunction after developmental TBI, we acquired pilot pharmacological magnetic resonance imaging (phMRI) data at day 4 following injury (n = 6) or sham (n = 4) of post-natal day-19 rats. This included a 5 minute baseline Cerebral Blood Volume (CBV) enhanced imaging (7T Bruker spectrometer using a single-shot, gradient-echo sequence, echo/repetition time: 20/1000ms, 300 repetitions, 128 × 128 matrix, 30 × 30mm field-of-view and 1mm slice-thickness) followed by systemic injection of 30 mg/kg DCS/ Saline in a 1 × 4 experimental design. Image acquisition continued 15 minutes post-injection. After typical preprocessing of time-series data and standard space registration, Region of Interest (ROI) based rCBV response analysis (pre vs. post drug challenge) was then performed for five brain regions identified in a prior study as the memory and recognition circuit, Prefrontal Cortex (PFC); Hippocampus (Hip); Thalamus (Tha); Perirhinal (PRh) and Entorhinal (Ent) cortex. No significant changes in regional CBV signal were observed in control animals following the saline injection. Regional CBV percent signal changes showed DCS-modulated network activation (PFC: +2.29; Ent: +2.64; Hip: +3.46; Prh: +3.05; Tha: +2.99) in sham control rats. Injured brain responses to the DCS challenge were in agreement with our hypothesis of injury induced hippocampal neural network irregularities (PFC: +0.85; Ent: +0.20; Hip: 0.90; Prh: +1.12; Tha: +1.34). Perceived NMDAR-modulated responses were then fed into a multivariate Granger causality analysis which also confirmed the existence of significant (p < 0.01) changes in the DCS-mediated hippocampal neural network between injury and sham.
Funding: R01NS27544, R01NS091222, UCLA Easton Labs for Brain Injury, UCLA Steve Tisch BrainSPORT program, UCLA BIRC.
Keywords: Pharmacological MRI, Developmental TBI, NMDA receptor, D-Cycloserine, Bain Connectivity Network
Yamaguchi University Graduate School of Medicine, Department of Neurosurgery, Ube, Japan
Keywords: traumatic brain injury, coagulopathy, biomarker
ImmunArray, Program of Neurological Diseases, Richmond, VA, USA
Diagnostic monitoring of mild traumatic brain injury (mTBI) requires robust biomarkers for precise, sensitive, objective assessment. We recently identified Aldolase C (ALDOC) as a protein released from acutely traumatized astrocytes in vitro (Levine et al., 2016). GFAP release and proteolysis was associated with delayed cell death, dependent on injury severity (Halford et al., accepted). The current study examined ALDOC and GFAP levels in clinical samples after TBI. Healthy controls, mTBI, severe TBI and athletic cohorts were studied in replicate electrochemiluminescence-based ELISAs and compared with detectable protein bands in standardized immunoblotting assays. Rank Sum, permutation and paired T-tests were used to measure significant differences between cohorts. Studies demonstrated the C-isoform specificity of the antibodies and showed CNS-specific expression. Both assays confirmed ALDOC elevation after TBI compared to healthy controls (all reported comparisons were significant at α = 0.05). A cohort of Italian mTBI patients showed serum ALDOC elevation irrespective of CT-status, while GFAP levels rose only in CT+ mTBI. In HeadSMART mTBI patients, ALDOC was significantly elevated versus healthy controls. ALDOC levels rose significantly in concussed NCAA athletes after injury, versus controls and non-concussed players. In Israeli football players, ALDOC elevation, but not GFAP, was detected in concussed players versus controls. ELISA testing and immunoblotting for GFAP breakdown-products found no GFAP elevation in either athlete study despite elevation in severe TBI and some mTBI patients. Overall, multiple mTBI cohorts of different age, athletic groups, and geographical location support ALDOC as a robust biomarker for mTBI.
Keywords: Biomarker, GFAP, ALDOC, Athlete, mild TBI
University of Pittsburgh, Department of Physical Medicine and Rehabilitation, Pittsburgh, USA
Traumatic brain injury (TBI) is associated with long-term complications, including persistent hypogonadotropic hypogonadism (PHH), which our studies suggest a link to autoimmunity. Autoantibodies (AAb) to the pituitary (APA) and hypothalamus (AHA) are present up to one-year following TBI and reduced IgM AAb increases PHH-risk among men with TBI. Adaptive immunity, including interleukin 7 (IL-7) production, may promote brain tissue-specific AAb production, which we hypothesize is neuro-reparative and reduces neuroendocrine dysfunction. Serum IL-7, IgM/IgG APA and AHA levels were evaluated 2-weeks to 12-months post-injury for N = 129 individuals with severe TBI. Mean IL-7 levels for 2 wk-3 mos, 4–6 mos, and 7-12mos were assessed. Age was dichotomized (median = 32). Group-based trajectory analyses for APA/AHA IgM and IgG levels identified high, medium, and low trajectory (TRAJ) AAb profiles. Ordinal logistic regression modeled IL-7, dichotomized age, and age*IL-7 interaction effects on AAb TRAJ. At 2wk-3mo, IL-7 was associated with APA IgM TRAJ (β = -0.023, p = < 0.001); though age was not a moderator. At 4–6 mos, older age moderated the IL-7 relationship to IgM APA levels, such that at a given age, increases in IL-7 were associated with lower APA IgM TRAJ (β = -0.022, p = 0.04). A similar trend was noted at 7–12 mos for the age*IL-7 interaction (β = -0.026, p = 0.08). IL-7 did not influence IgM AHA production as strongly, with only trends (p < 0.08) noted 4–12 mos post-injury. IL-7 was associated with IgG APA across the entire sampling period (p < 0.04 all comparisons), while IL-7 was associated with IgG AHA 7–12 mos post-injury (p ≤ 0.015). These data implicate IL-7 as relevant to the autoimmunity to pituitary proteins and as relevant to ongoing neuroendocrine dysfunction after TBI. Further work should replicate findings and explore how aging and immunomodulation might affect adaptive immunity post-TBI. Support: UPP-Foundation, NIDILRR 90DP0041, DODW81XWH-071-0701, R21-NS085455-01-(KKW).
Keywords: Adaptive Immunity, Autoantibodies, Interleukin 7, Aging
UCLA, Semel Institute for Neuroscience and Human Behavior, Los Angeles, USA
Spinal cord injury (SCI) is heterogeneous, hampering treatment optimization and clinical trials. SCI-patient management lacks non-invasive tools evaluating severity and predicting outcome. This project established multimodal tools using novel
Support: USAMRMC:W81XWH-13-2-0047
Keywords: functional recovery, swine, mass spectrometry, kinetics, multimodal analyses, transport
University of Texas Medical Branch, Department of Anesthesiology, Galveston, USA
Minimally invasive biomarkers can distinguish focal or diffuse TBI injury types and may prove clinically useful. Circulating microRNA (miRNA) expression was examined after fluid percussion injury (FPI) and controlled cortical impact (CCI), which produce diffuse and focal injuries, respectively. We hypothesized that there would be unique differential regulation of miRNA in various brain regions dependent on injury type and that these changes would be reflected in serum. Brain regions examined in this study have previously been shown to be functionally affected after TBI. Our long-term goal is to define a panel of miRNAs that can be used to supplement conventional imaging techniques, which currently lack specificity for injury classification. In this study, rats were anesthetized and subjected to either severe FPI or CCI or remained uninjured. Serum was isolated from whole blood in all animals and rats were sacrificed 4 h after TBI. Brains were prepared for laser capture microdissection (LCM) of TBI-affected brain regions. Serum and tissue miRNA were isolated, cDNA was synthesized, and pre-amplification was performed. RT-qPCR was performed using arrays populated with 84 common disease associated miRNAs. Data was analyzed using Geneglobe Data Analysis and Ingenuity Pathway Analysis software (Qiagen). Our analysis shows there are distinct miRNAs which are differentially regulated, in a brain region dependent manner, between either focal or diffuse injuries. Pathway analysis of these tissue-expressed miRNA suggests their involvement in the pathophysiologic response after TBI. Serum miRNA expression revealed significant differences among FPI and CCI comparisons and to naïve controls. In a long term perspective, understanding regulation of tissue miRNAs in TBI injury subtypes and measuring respective changes in serum will improve clinical diagnosis by supplementing imaging modalities. These studies were supported in part by the Moody Project for Translational TBI Research.
Keywords: microRNA, serum, diffuse, focal
University of British Columbia, Orthopaedics, Vancouver, Canada
Dislocation is the most common clinically observed spinal cord injury (SCI) mechanism, occurring in roughly 45% of SCI cases, however, there are few preclinical models. Despite demonstrating different injury outcomes compared to more common contusion models, the dislocation models exhibit greater variability. Since dislocation injury models involve clamps which grip and displace the spine, a potential cause of variability may be slipping at this interface. The objectives of this study were to: i) design new injury clamps, ii) measure intervertebral kinematics during a high-speed dislocation injury in an in vivo rat model; and iii) quantify relative motion at the vertebrae-clamp interface to determine which clamps provide the most rigid connection.
New clamps were designed to pivot, and self-align when tightened, ensuring each clamp holds both intended vertebrae. The dislocation injury was performed using both the existing and redesigned clamps, where 400 μm radiopaque tantalum beads were fixed to C3-C6 and the clamps to track relative motion. The injury was performed at C4/C5 on anesthetized rats (n = 17 per group), to a displacement of 2.3 mm at a velocity of 700 mm/s. A high-speed x-ray system recorded the tests at 8000 frames per second, and motion of the vertebrae with respect to the clamps was tracked.
The relative motion of C5 with respect to the caudal clamp was significantly less for both translation and rotation for the redesigned clamps, and the maximum observed relative motion was four times less for the redesigned clamps.
This is the first time measurement of clamp-vertebral motion in a high-speed dislocation model. This study demonstrated that relative motion was occasionally present between the existing dislocation clamps and the vertebrae. By redesigning the clamps, relative motion was reduced, and produced more repeatable kinematics during injury. These improvements address a potential source of variability and progress toward a more repeatable rat dislocation model, better suited for investigating SCI interventions, and further shed light on the importance of injury mechanism.
Keywords: biomechanics, spinal cord injury, dislocation, animal model
University of British Columbia, Orthopaedics, Vancouver, Canada
Finite Element (FE) models have been used to determine the relationship between strain and tissue damage in traumatic spinal cord injury. Previous FE studies have used mechanical properties determined from ex vivo loading of spinal cord tissue, despite the variation in results from such tests, and evidence that suggests spinal cord tissue degrades rapidly after death. The current study aims to develop a method with which to determine the relative mechanical properties of the two primary constituent tissues in the spinal cord; the grey and white matter.
The current study used a quasi-static, 3D FE model to recreate mild contusion-type spinal cord injuries imposed on Sprague-Dawley rats (n = 6) by Bhatnagar et al previously. MR images of the undeformed and deformed spinal cords were used to guide the initial mesh generation and the gross cord deformation, respectively. The resulting predicted grey matter deformations were compared with experimental results. The ratio of elastic moduli between the grey and white matter (W/G) was varied and the resulting morphologies of the spinal cords were observed. The morphological similarity was quantified using Dice Similarity Coefficient (DSC). The grey and white matter in each spinal cord were modeled as homogeneous, isotropic, linear elastic solids. W/G values of 0.5, 1.0 and 2.0 were tested with Poisson's ratio held at 0.42.
For all cords, the DSC values ranged between 72% and 87%. The best qualitative agreement was seen for contusions that were central on the spinal cord, for all values of W/G. For the asymmetric contusions, the model accurately predicts deformation at the lateral edges of the grey matter, but predicts greater grey matter deformation at the mediolateral center of the cord than seen in the experimental deformations for all W/G values. These results suggest possible heterogeneity of the spinal cord white matter.
Keywords: biomechanics, spinal cord injury, finite element modelling, material properties
University of Virginia, Center for Applied Biomechanics, Charlottesville, USA
Current football concussion risk assessments were developed based on head kinematics measured using sensors that were worn by players to determine accelerations associated with injury. While head motion is associated with concussion risk, tissue-level strains are the primary mechanism of brain injury. Finite element (FE) head models are used to obtain brain strains for potentially developing more reliable concussion tolerances through simulated head impact. In this study, six-degree-of-freedom head kinematics obtained from laboratory reconstructions of 55 on-field football head impacts (11 concussed cases) were simulated using a FE human brain model. Strain-based metrics including maximum principal (MPS) and cumulative brain strain (CSDM) were calculated in various brain regions for each impact. Estimates of maximum axonal-tract strain (MAS) in the corpus callosum were also considered. Strain-based predictors were evaluated relative to injury outcomes from the players, and used to identify potential tissue-level tolerances for concussion. For all cases, maximum brain strains were highest in cortical grey matter tissue, and cerebral white matter and cerebellum tended to have the lowest strains. Brain strains were higher in concussed players (p < 0.01); median MPS was 22% for concussed cases and 14% for non-concussed, CSDM values were overall low (< 0.05) but slightly larger in the concussed group. MAS in the corpus callosum were also higher in concussed cases (5% vs 8% median value, p < 0.05). The MPS metric had the highest ROC AUC value (0.822), followed by CSDM (0.815) and MAS (0.711). Additionally, rotational head motion response had the highest correlation with brain strains in all regions, Although FE brain models are not validated for regional strain prediction, these results highlight the efficacy of developing concussion risk models using tissue-level strains. These methods can be used to improve helmet design to ultimately reduce the incidence of concussion in football. The authors acknowledge the UVa-VTC Neuroscience Seed Fund Program for their support of this research.
Keywords: Brain strain, Concussion, Finite element modeling, Football, Head kinematics
Here we present a high frame-rate (6200 images/second) ultrasound imaging method that can accurately (<1 μm) measure the internal brain motion during the rapid transient events associated with a mild impact in an ex vivo porcine brain. Attempts to measure the in situ nonlinear brain mechanics with imaging methods (MRI, CT) have lacked the penetration, frame rate, or motion detection accuracy to capture the rapid and nonlinear shear wave motion during traumatic injury. The substantial increases in frame-rate and motion detection accuracy of our ultrasound method allow us to investigate previously unexplored biomechanical regimes. We present the first experimental observation of shear shock wave generation in the brain. The highly localized increases in acceleration at the shock front suggests that shear shocks are a previously unappreciated primary mechanism for traumatic injuries.
Our method relies on two main advancements 1) A high frame-rate ultrasound sequence which increases the penetration and improves the image quality compared to conventional high frame-rate methods 2) An adaptive tracking algorithm that can accurately detect the fine shock structure within the brain. To the best of our knowledge no other imaging method has been able to achieve this combination of speed, accuracy, and penetration in the brain.
By imaging brain motion directly we discovered that shear shock waves form within the brain. The measured shock waves have a specific odd harmonic signature predicted by theory describing a cubically nonlinear elastic soft solid. Measurements of the frequency dependent attenuation and dispersion were used to fit this nonlinear theoretical model to our data. This yielded the first estimates of the cubic nonlinear parameter for brain tissue. This previously unobserved shear shock wave phenomenology dramatically amplifies the acceleration at the shock front, deep in the brain, compared with the acceleration imposed at the brain surface (up to a factor 8.5). A 30 g acceleration at the brain surface therefore develops into a 255 g shock wave deep inside the brain. Strain and strain rate are also amplified at the shock front.
NIHR0106052014
Keywords: Shear shock wave, Ultrasound imaging
BIOMECHANICS OF A WEIGHT-DROP MODEL OF SPORTS CONCUSSION
Emory University, Biomedical Engineering, Atlanta, USA
There is a critical need to understand the biomechanical factors influencing mTBI severity in order to guide preventative measures and novel therapies. Numerous existing pre-clinical models of concussion may help address this need, but it is essential that the animal models yield realistic impacts that are comparable to those seen in humans. The “Harvard weight-drop model” claims clinical relevance to sports concussion in that (1) it features blunt impact followed by rotational acceleration and (2) repetitive insults lead to significant cognitive deficits without overt structural brain injury. However, the biomechanical forces of this model have yet to be quantified. Herein, we assess the linear and rotational accelerations incurred in this murine model, and we compare the scaled values to literature reports of human sports concussion. This model involves impact of the intact skull/scalp using a 55 g bolt dropped through a guide tube from 48” followed by rotation in the anterior-posterior plane. To quantify, N = 12 slow motion videos (480 fps) of impact were taken on adult mice. Particle tracking software was used to quantify the position of center of mass of the bolt across 3 frames before/after impact. Using the momentum principle, we related the change in velocity of the bolt pre/post-impact to the linear and the rotational accelerations of the mouse head. Mean (SD) linear acceleration was 83 (27) g and rotational acceleration was 253,000 (75,600) rad/s. Assuming that equal stress is required to reproduce similar injury mechanism in both mice and humans, we scaled our values using the ratio of mice/human brain masses. These accelerations translate to roughly 5.4 (1.8) g and 1,100 (324) rad/s in humans. The scaled linear accelerations are significantly lower than reported thresholds for human mTBI; however, the scaled rotational accelerations obtained with this model are on the same order of magnitude as human mTBI thresholds. In conclusion, the Harvard weight-drop mouse model yields rotational accelerations that realistically scale to corresponding human sports concussion biomechanics.
Keywords: animal model, rotational acceleration, linear acceleration, scaling
University of British Columbia, Orthopaedics and Mechanical Engineering, Vancouver, Canada
The effect of timing of decompression following traumatic SCI remains unclear, as clinical and preclinical studies have demonstrated varying results. Some studies suggest that certain sub-groups of SCI could see greater benefits than others from early decompression. Dislocation is the most commonly seen injury mechanism clinically, but it has never been investigated with respect to residual compression in an animal model. The objective of this study was to develop a model for evaluating the effect of velocity and timing of residual compression in a rodent dislocation SCI model. Based on a previous dislocation model using custom vertebral injury clamps, a novel method for holding residual compression immediately following SCI was developed. This method involves use of an electromagnetic actuator to perform the injuries at both high and low velocity and a custom stereotaxic frame which rigidly holds the clamps during injury and in residual compression after insult. After residual compression, while still held securely within the stereotaxic frame, the clamps are realigned via linear translation stage where they are rigidly fixed together. The clamps were modified to also function as surgical fixation implants, ensuring spinal stability during all phases of the study. This model was used for a study involving 4 groups of SD rats (n = 12) plus shams: 2 timings of decompression (24 minutes, 240 minutes) and 2 velocities (5 mm/s, 500 mm/s). All injuries involve dislocation between the C5/C6 vertebrae in an anterior-posterior direction to 1.45 mm and residual compression of 0.8mm. Animals were evaluated for motor function using the Martinez open field, grip strength, and grooming tests for 6 weeks post-injury. This design is able to precisely create dislocation injuries with immediate residual compression afterwards and represents a novel model of SCI, relevant to the question of timing of decompression.
Keywords: Residual Compression, Dislocation, Animal Model, Decompression, Design, Acute
The Emmes Corporation, Bethesda, USA
Keywords: Common Data Elements, Devices, Sensors
There is an urgent need to elucidate the injury mechanisms of traumatic brain injury. Blunt impact to the head is hypothesized to generate negative intracranial pressure transients sufficient to cause CSF cavitation. Upon bubble collapse, jet streaming and immense localized pressures may occur, potentially damaging surrounding tissue. The objective for this study was to investigate cavitation bubble formation and collapse as an injury mechanism using a biofidelic head model under impact. A simplified head surrogate with brain phantom was created consisting of a skull (a rigid Lucite container), cerebrospinal fluid (degassed distilled water of 2 mm thickness), and different brain tissue simulants (Perma-Gel, ballistic gelatin composite, Sylgard gel). Brain phantoms were varied in order to investigate cavitation limits from the range of bulk modulus material properties. The head was subject to linear blunt impact loading from a weighted pendulum, with resulting peak linear accelerations ranging from from 500–1500 m/s2 as measured by a rigidly mounted accelerometer. Hydrophones were externally adhered to the vessel and placed at the coup and contrecoup sites to measure sonoacoustic emissions from bubble formation and collapse. A high speed camera recording at 40,000 fps directly captured fluid behavior. Cavitation was directly observed in the CSF layer and confirmed by acoustic data. The occurrence of cavitation was dependent on the peak acceleration of the system as generated by the impact. Cavitation occurred in all tests above Head Impact Criterion values of 1000, a value for which the loading scenario would create a high risk for injury. Changing brain phantom material and their associated bulk modulus properties altered cavitation thresholds. Bubble formation and collapse elapsed approximately 0.15 to 0.4 msec. Power spectrum analysis of sonoacoustic data showed dominant frequencies ranging from 10–15 kHz during cavitation bubble collapse. This data supports the hypothesis that fluid cavitation is a potential injury mechanism for TBI. Furthermore, the methodology detailed here provides the framework to investigate cavitation in live animal TBI models, as currently there are no empirical studies demonstrating cavitation in vivo.
Keywords: Mechanisms of Injury, Cavitation
DEVELOPMENT OF CRASH INDUCED INJURY CRITERIA FOR PREDICTING BRAIN INJURIES USING A HUMAN HEAD COMPUTER MODEL
Finite element (FE) modelling can serve as a powerful tool to study biomechanical process of head and brain injuries that is difficult to investigate experimentally on living human subjects. Recently, a detailed human head model, GHBMC (Global Human Body Modelling Consortium) M50, representing a 50th percentile male adult head has been developed, validated and used to develop tissue level injury criteria for TBI. The objective of this study was to validate a detailed GHBMC 5th percentile female (F05) head model which accounts for gender related size, geometrical and anatomical differences in order to properly predict injury risk sustained by this population. A number of Crash Induced Injury (CII) criteria for injuries to the skull, face, and brain of various regions were developed to enable the prediction of the injury risk by the model. Thirty-one sets of published cadaveric head impact experimental data were simulated to validate the biomechanical response of the head model in terms of force-deflection for various facial and cranial bones, intracranial pressure and brain/skull relative displacement for brain of various regions. Then, Forty-four sets of head impact experiments with injurious and non-injurious conditions were simulated to develop CII values for skull fracture, facial fracture, acute subdural hematoma (ASDH), cerebral contusion, and diffuse axonal injury (DAI) at various white matter structures/regions and their associated severities. Model predicted biomechanical responses correlated well to the experimentally measured results with objective ratings greater than 0.7 (1 being perfect). The current GHBMC F05 head model has been rigorously validated against all exiting data head impact experiments and responses. The current model is capable of predicting six different head injury types affecting nine regions/locations with reasonable predictive capability. With further improvement and exercises, the human head model can enable assessment of possible real-world injury scenarios to understand the injury mechanisms and allow for engineering improvements to help prevent potential head/brain injury from traumatic events.
Keywords: Biomechanical Response, Finite Element Human Head Model, Crash Induced Injury Criteria for Head, Diffuse Axonal Injury, Bridging Vein Rupture, Coup and Contrecoup Contusion
The C statistics for risk for death of the CORE model was 0.687 and for the CORE+CBF was 0.754 which was statistically significant different (p = 0.0007). The C statistics for death of the CORE+CT was 0.735 and of CORE+CT+CBF was 0.787 which was statistically significant different (p = 0.0015).
The C statistics for risk for poor outcome of the CORE model was 0.752 and of the CORE+CBF was 0.787. The C statistics for risk for poor outcome of the CORE+CT was 0.772 and for CORE+CT+CBF was 0.801. Both were statistically significant different p = 0.0116 and p = 0.0038 respectively.
Keywords: Prognosis Prediction, Severe Traumatic Brain injury, Impact Calculator, TBI outcome
CHRONIC CEREBROVASCULAR ABNORMALITIES IN A MOUSE MODEL OF REPETITIVE MILD TRAUMATIC BRAIN INJURY
The Roskamp Institute, Neuroscience, Sarasota, USA
Keywords: Repetitive Traumatic Brain Injury, Cerebrovascular, Cerebral Blood Flow, Animal Model
Clinical Center, NIH, Radiology and Imaging Sciences, Bethesda, USA
Pre- and post-contrast FLAIR was used to assess for TME, identified as high signal along the dura seen on post contrast FLAIR without extension into the subarachnoid space. High signal on precontrast FLAIR in similar distribution along the dura was considered evidence of subdural hematoma (SDH).
BFFE was used to assess for subdural effusion/hygroma (SDEH) manifest as a separation of the subarachnoid membranes from the inner table of the skull. Such visualization is possible using BFFE due to the combination of high spatial resolution (∼500 μm isotropic scans in 3–4 minutes) and high image contrast between CSF (high signal) and arachnoid membranes (low signal).
Keywords: mTBI, MRI, acute
University of Minnesota, Neurosurgery, Minneapolis, USA
Significant discrepancies between continuous and intermittent pressure monitoring of intraventricular catheters have been found at our institution using the Integra® Camino® Flex Ventricular Catheter. These discrepancies generated clinical uncertainty about the status of cerebral autoregulation of patients suffering severe traumatic brain injury in accordance with Segal's law. Our efforts endeavored to fully characterize the two signals during simultaneous recordings to understand the source of the discrepancy. After IRB approval, simultaneous recordings were obtained from four patients when the external ventricular drain was clamped resulting in more than 40 million data points. Data was sampled at 124 Hz, resulting in 90 hours for comparison. Power spectra were calculated, and mean squared cross coherence was compared. Significant error was found in the low frequency component. Low frequency data less than 0.3 Hz was found to have a correlation coefficient of 0.3 while data above 0.3 Hz was found to be 0.84. False positive readings, when intermittent ICP read above 20 mmHg while continuous ICP remained below, constituted more than 10% of the data, while false negative data constituted less than 0.8%. By examining the low frequency components of the relationship of the two sources of pressure, it was found that the most significant source of discrepancy remained changes in the zero level for the intermittent external ventricular drain readings, corresponding to head position changes or nursing leveling. Continuous ICP monitoring from an intraventricular pressure sensor consistently reads lower values than an external ventricular pressure sensor while maintaining consistency.
Keywords: Sensor, continuous ICP, flex cathetar, Intraventricular
Chiba University Graduate School of Medicine, Dept. of Orthopaedic Surgery, Chiba, Japan
Keywords: surgical decompression, sub-acute phase, chronic phase, clinical outcome, cervical myelopathy, spinal cord injury
University of Toronto, Institute of Medical Science, Toronto, Canada
Traumatic spinal cord injury (SCI) is a devastating neurological condition that occurs primarily at the cervical (cSCI, >65%) and thoracic (tSCI) levels. Despite the marked neurovascular distinctions of the two levels and strikingly positive response of cSCI to trial drugs such as cethrin compared to tSCI, the mechanisms driving level-specific heterogeneity between their respective milieu remains elusive. We posit that the increased vascularity and grey-white ratio of the cervical cord—relative to the thoracic—results in greater susceptibility to neurovascular disruption, ultimately manifesting a secondary injury of earlier onset, severity, and chronicity. A rat model of moderate clip compression injury was used to induce SCI at the C6-7 and T6-7 levels, with laminectomy-only animals serving as surgical controls. Following sacrifices at 3, 7, 14, and 56 days, samples were subject to RNA-seq, protein work, imaging, and immunohistochemistry. Results of RNA-sequencing revealed striking differences in the onset and temporal profile of astrocytic and pericytic neurovascular processes with canonic stat3-dependent gliotic markers—lcn2, gfap and serpina3n—being upregulated in the cervical cord across time. Further, 3D ultrasound and immunostaining revealed rapid tissue loss and hemorrhage starting as early as 3 days post-cSCI with increased gfap and cspg4 staining in the cord. Finally, Western blotting confirmed an increase in stat3-dependent gliotic markers accompanied by a loss of key blood-brain-barrier proteins tjp1 and ocln in cSCI across time. Taken together, this data demonstrates—for the first time—the level-specific heterogeneity of SCI, with cSCI having a quicker onset and chronicity compared to tSCI. Further, these results reconcile the potential reasons behind why preliminary tSCI-derived trial paradigms may not be suited–in both strategy and timing—to cSCI, and hopes to engage clinicians and scientists in the design and study of level-specific therapeutics.
Keywords: RNASeq, Neurovascular unit, Secondary injury, Pericyte
Chiba University Graduate School of Medicine, Orthopedics, Chiba, Japan
Keywords: cervical compressive myelopathy, rat model
MetroHealth Medical Center, Neurosurgery and Surgery, Cleveland, USA
The effect of regionalized trauma care (RT) on traumatic spine injury (TSI) outcomes in the United States is unknown. The Northern Ohio Trauma System was organized in 2010 as a multi-institution trauma system across northeastern Ohio. We hypothesized that RT would be associated with earlier time to spine surgery and decreased length of stay (LOS). All patients >14 years with a TSI were identified from the RT database using ICD-9-CM diagnostic codes. Data from 2008 through 2012 were analyzed before and after RT in 2010. A total of 4,072 patients were identified; 1904 (47%) pre-RT and 2168 (53%) post-RT. TSI admissions to the regional level 1 center post-RT increased from 67% to 74% (p < 0.0001). TSIs in the post-RT group were older (median age: 52 vs. 50, p = 0.02). Injury severity scores, Spine Abbreviated Injury Scale scores, and the percentage of TSIs with spinal cord injury were similar between time periods. Post-RT TSIs demonstrated a lower median ICU LOS (0 day vs. 1 days; p < 0.0001), underwent spine surgery more frequently (13% vs. 11%; p = 0.01), and had a higher rate of spine surgery performed within 24 hours of admission (65% vs. 55%; p = 0.02). In patients with tSCI post-RT, ICU length of stay was decreased (1 day vs. 2 days; p < 0.0001) and ventilator days were reduced (average days: 2 vs. 3; p = 0.006). The post-RT time period was an independent predictor for spine surgery performed in less than 24 hours for all TSIs (OR 1.51, 95% CI: 1.03–2.22, C-stat = 0.66). Multivariate linear regression analysis demonstrated an independent effect on reduced ICU LOS post-RT for both TSIs (OR −0.6; 95% CI: −1.04--.181; R2 = 0.11) and tSCIs (OR −1.01, 95% CI: −1.6--0.40; R2 = 0.11). Regionalization of trauma is associated with increased surgical rates, earlier time to surgery, and decreased ICU LOS for patients with TSI.
Keywords: Spine trauma, Spine surgery, Trauma systems, Clinical outcomes
Keywords: HALO vest immobilization, cervical trauma, cervical instability
University of Texas Medical Branch, Neuroscience and Cell Biology, Galveston, USA
Mild traumatic brain injury (mTBI) represents 75–87% of the reported 1.4–3.8 million cases of TBI per year. Furthermore, athletes often face the risk of repetitive mTBI and its consequences. However, the model of mTBI for research remains less optimal. In order to obtain a better understanding of mTBI-related disorders, we modified the weight drop model developed by Kane et al. in 2012 by adding a velocity sensor to calculate the force of the injury and monitoring impact variability. Fifteen female C57BL/6 mice (about 2.5 months of age) were randomly assigned into 5 groups: Sham, single mTBI with 95- or 120-gram weight (95 × 1 and 120 × 1), or repetitive mTBI with 95- or 120-gram weights (95 × 5 and 120 × 5). Weight was dropped from one meter height. For repetitive injuries, animals were hit once daily for 5 consecutive days. Kinetic energy (joules) were calculated: Sham = 0, 95 × 1 = 0.83 ± 0.007J, 95 × 5 = 0.82 ± 0.31J, 120 × 0 = 1.06 ± 0J, and 120 × 5 = 1.05 ± 0.31J. Elevated plus maze (EPM) test was conducted 3 days post injury and brains were collected on day 4 for immunohistochemical analyses. We observed a significant increases in righting reflex following repetive mTBI. There was a trend towards increased time spent in the open arm of the EPM that also appeared to be dose-dependent. Finally, we observed alterations in immunoreactivity of GFAP, APP and αSMA in multiple areas of the mouse brains. In conclusion, the 120-gram weight at one meter height is the most consistent and optimal injury for modeling mTBI in our hands.
Support: Studies were completed as part of a team funded by The Moody Project for Translational Traumatic Brain Injury Research, Steve Dunn Foundation, Coalition for Brain Injury Research, CONACYT-COPOCYT and Fundación Marrón Cajiga.
Keywords: Sensor, Kinetic Energy
University of British Columbia, Pathology and Laboratory Medicine, Vancouver, Canada
CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration) is a recently described animal model of traumatic brain injury (TBI) that primarily produces diffuse axonal injury (DAI) characterized by white matter inflammation and axonal damage. CHIMERA was specifically designed to reliably generate a variety of TBI severities using precise and quantifiable biomechanical inputs in a nonsurgical user-friendly platform. The objective of this study was to define the lower limit of single impact mild TBI (mTBI) using CHIMERA by characterizing the dose-response relationship between biomechanical input and neurological, behavioral, neuropathological and biochemical outcomes. Wild-type male mice aged 4–5 months were subjected to a single CHIMERA TBI using six impact energies ranging from 0.1 to 0.7 J, and post-TBI neurological, behavioral, neuropathological and biochemical outcomes were assessed at 6 h, 1 d, 2 d, 7 d, and 14 d time-points. We report that single TBI using CHIMERA induces injury dose- and time-dependent changes in behavioral and neurological deficits, axonal damage, white matter tract microgliosis and astrogliosis. Impact energies of 0.4 J or below produced no significant phenotype (sub-threshold), 0.5 J led to significant changes for one or more phenotypes (threshold), and 0.6 and 0.7 J resulted in significant changes in all outcomes assessed (mTBI). We further show that linear head kinematics are the most robust predictors of duration of unconsciousness, severity of neurological deficits, white matter injury, and microgliosis following single TBI. Our data extend the validation of CHIMERA as a biofidelic animal model of DAI and establish working parameters to guide future investigations of the mechanisms underlying axonal pathology and inflammation induced by mechanical trauma.
Keywords: CHIMERA, Traumatic brain injury, Animal model of TBI, Behavior, Biomechanics, Neuropathology
University of British Columbia, Department of Pathology and Laboratory Medicine, Vancouver, Canada
Incidence of traumatic brain injury (TBI) peaks in young and old individuals, and older age is associated with worse outcome and poorer recovery. TBI is also an established risk factor for neurodegenerative conditions such as dementia and chronic traumatic encephalopathy (CTE), and much remains to be learned about how age at injury affects susceptibility to neurodegeneration. We aimed to delineate how TBI, age at injury, and genetic predisposition to amyloid deposition interact. Repetitive mild TBI was induced in APP/PS1 and wildtype (WT) mice using the CHIMERA model at 6 or 13 months of age, and acute behavioural, histological and biochemical changes were assessed up to 14 days post-injury. We observed impaired post-TBI spatial learning in old but not young mice regardless of genotype, and age-dependent post-TBI changes in Ab deposition in APP/PS1 mice. Age at injury and genotype showed a complex interaction with respect to cytokine and microglial responses, where the neuroinflammatory response is exacerbated in young APP/PS1 but blunted in old APP/PS1 mice, whereas in WT mice the response is sensitized by older age. In conclusion, both age at injury and APP/PS1 genotype modify TBI outcomes, with chronological age at injury being the more robust modifier.
Keywords: Alzheimer Disease, CHIMERA, Aging, Traumatic Brain Injury, Neurodegeneration
The Roskamp Institute, −, Sarasota, USA
Keywords: Traumatic Brain Injury, Biomarkers, Tau, Mouse Models
Worcester Polytechnic Institute, Biomedical Engineering, Worcester, USA
Robust and objective prediction of traumatic brain injury (TBI) is important for its clinical management. Previous efforts have largely relied on univariate logistic regression and a single “training” injury dataset to evaluate performance. Recently, we developed a deep learning approach to formulate TBI prediction as a supervised binary classification. In this study, we evaluate the injury classifier performance using a two-step cross-validation procedure and compare with four popular injury metrics: Brain Injury Criterion, BrIC), cumulative strain damage measure for the whole-brain (CSDM-WB) and corpus callosum (CSDM-CC), and peak fiber strain in the corpus callosum (Peak-CC). Reconstructed NFL head impacts (25 concussions and 33 non-injuries) were simulated. Peak white matter fiber strains were generated on diffusion tensor imaging voxels to create a 128 × 128 × 65 strain-encoded image. The injury dataset was repeatedly (N = 20) and randomly split into non-overlapping training (N = 39) and cross-validation (N = 19) datasets. For each random trial, a fully-connected, five-layer deep learning network was trained in a supervised manner. The same training dataset was used to fit a logistic regression for other four competing metrics. The trained network and regression models were used to predict TBI using the cross-validation dataset, as well as a separate, independent injury dataset (7 injuries and 1 non-injury; reconstructed from automobile crashes and football) for further cross-validation. Using the first validation dataset, the five injury classifier/metrics had largely comparable accuracy, sensitivity and specificity. However, deep learning had a significantly better average accuracy (0.7829 vs. 0.6062, 0.7375, 0.6125, and 0.675 for BrIC, CSDM-WB, CSDM-CC and Peak-CC, respectively) and sensitivity (0.7519 vs. 0.55, 0.7, 0.5571, and 0.6929 for the other four metrics, respectively), statistically, when using the second independent cross-validation dataset. Deep learning has not been used in TBI biomechanics studies before. Our results suggest that this technique may have an improved injury prediction performance than other state-of-the-art injury metrics. They also highlight the need for independent cross-validation using fresh/unmet injury datasets for more rigorous comparison of injury prediction performances.
Keywords: concussion, injury prediction, traumatic brain injury, deep learning, cross-validation, fiber strain
UCLA, Neurology, Los Angeles, USA
The lack of an objective, physiological biomarker to detect concussion has resulted in the injury remaining a clinical diagnosis that relies on subjective symptom-based assessments. Historically, concussion grading scales have classified injury without acknowledgement of diagnostic uncertainty and have not resulted in prognostic value. Clinical certainty based scales for diagnosis are currently used in other neurological disorders such as multiple sclerosis and Alzheimer's disease but no such scale is currently used for concussion. A novel classification system was introduced to the literature in 2014 by Kutcher and Giza, which incorporated degree of clinical certainty into the concussion diagnosis. The objectives of the current study are to (1) adapt and refine this concussion classification system and (2) apply it retrospectively to a cohort of mild TBI patients at the UCLA BrainSPORT concussion clinic to determine if it can predict outcome. The classification system categorizes concussions as definite, probable, possible, or not a concussion based on the following clinical criteria: (a) witnessed mechanism of injury, (b) typical symptoms, (c) time of symptom onset, (d) improving time course, and (e) presence/absence of alternative diagnosis. This classification system was used to retrospectively diagnose 160 patients at the UCLA BrainSPORT concussion clinic from June 2015 to October 2016. Post injury symptom scores were assessed with the Sport Concussion Assessment Tool 3 (SCAT-3) graded symptom checklist scores as well as an “Overall rating score” from 0–100% at the initial clinic appointment. Patients were then contacted at a second time point between 6–12 months post injury at which time the patient's SCAT-3 symptom score and overall rating score were reassessed. Concussions classified as “Definite” had a higher likelihood of symptom resolution (<9 symptom score, or >95% overall rating) at 6–12 months post-injury when compared to probable or possible concussions. These findings suggest that a concussion classification system based on degree of certainty can provide value for clinical management by increasing our diagnostic specificity and assisting in prognostication.
Keywords: Concussion, Diagnosis, Prognosis
Keywords: TBI, MEG, somatization, depression, anxiety, Post Traumatic Stress
CORRELATION BETWEEN SYMPTOMS AND MEG-DERIVED BRAIN PATTERNS IN CHRONIC TBI
TEAM-TBI, n = 62: somatization, r = 0.38; p = 0.009; depression, r = 0.46; p = 0.007
HIV-risk, n = 54: somatization, r = 0.33; p = 0.014; depression, r = 0.49; p = 0.018
Combined, n = 116: somatization, r = 0.2; p = 0.029, depression, r = 0.33; p = 0.002, anxiety, r = 0.29; p = 0.006
Keywords: somatization, anxiety, Post traumatic stress, TBI, MEG, depression
EXPRESSION OF TRPM4 INDUCES ASTROCYTE SWELLING BUT NOT CELL DEATH FOLLOWING DIFFUSE MODERATE TRAUMATIC BRAIN INJURY IN THE RAT
Virginia Commonwealth University, Anatomy and Neurobiology, Richmond, USA
Traumatic brain injury (TBI) is a highly prevalent disease that has significant costs. Although progress has been made in understanding the complex pathobiological consequences of contusive TBI, questions still remain regarding the diffuse responses to injury. Recently expression of transient receptor potential melastatin 4 (Trpm4), as a component of the sulfonylurea receptor 1- transient receptor potential melastatin 4 (Sur1-Trpm4) channel, has been linked to cell death during hemorrhagic contusion expansion. However, very little is known about the expression patterns of the Sur1-Trpm4 channel following diffuse TBI, in which hemorrhagic contusions are absent. To explore the expression pattern of the Sur1-Trpm4 channel in diffuse TBI adult male Sprauge Dawley rats were subjected to a moderate (2.05 ± 0.05atm) central fluid percussion injury and survived for 6 h-8 w. Immunolabeling of the Trpm4 component of the Sur1-Trpm4 channel was assessed over time following injury. Hemotoxylin and eosin labeling was undergone to evaluate cell damage/death correlating to Trpm4 expression following injury. To investigate the cell type expressing Trpm4 following diffuse TBI, immunohistochemistry against the common astrocyte marker, GFAP, and the common microglial marker, Iba-1, was done. Finally, ultrastructural assessments were also performed to evaluate the integrity of Trpm4+ cells and potential for cellular swelling over time following diffuse TBI. Acutely following TBI Trpm4 expression was restricted to GFAP+ astrocytes within the hippocampus. By 1 w following diffuse TBI Trpm4+ astrocytes encompassed the hippocampus, fimbra and aspects of the subcortical white mater. This expression pattern, was observed as chronically as 8 w post-injury. Correlative assessments of cell damage/death demonstrated little evidence of hippocampal or subcortical white matter damage (< 0.0004 damaged cells/100μm2 of hippocampus at all time points), suggesting that expression of Trpm4 by astrocytes does not precipitate cell death in this model of TBI. Additionally, ultrastructural assessments showed Trpm4+ astrocytes have double the area of somatic cytoplasm (21.46 ± 9.70μm2) as compared to Trpm4- astrocytes (10.26 ± 4.60μm2) at 4weeks post-TBI, suggesting cellular swelling with Trpm4 expression. This work was supported in part by NINDS grants 1R01NS096143 and 5P30NS047463 and the Commonwealth fund.
Keywords: TRPM4, central fluid percussion injury, hippocampus, Confocal microscopy, Electron microscopy
Georgia Tech, Atlanta, USA
The potential long-term consequences of repetitive head impacts in football and other collision sports highlight a need for improved concussion mitigation strategies. The purpose of this study was to analyze biomechanical impact variables across two high school football seasons to assess whether the use of a helmet add-on contributes to the reduction of head impact magnitudes. We collected helmet impact data using the Riddell Head Impact Telemetry (HIT) system during games and practices on 72 players, n = 42 in season 1 and n = 30 in season 2. During season 2 practices, players wore Guardian helmet pads. A total of 33,023 impacts were recorded; with 16,106 (383.5/player) impacts during practices and 6,178 (147.1/player) impacts during games in season 1, and with 3,884 (129.5/player) impacts during practices and 6,855 (228.5/player) impacts during games in the 2nd season. The total number of impacts decreased significantly for practice sessions from season 1 to season 2, reflecting national recommendations for reduced contact practice time. In season 2 practices there was a 13% reduction in linear acceleration (p < 0.0001) and a 3.3% reduction in rotational acceleration (p < 0.01). For games, there was an 8.5% reduction in linear acceleration (p < 0.0001). Interestingly, between skilled players from two different schools in season 2 (i.e. same rules changes) there was no difference in linear or rotational acceleration with or without Guardian cap. However, in linemen, the Guardian-wearing school had significant reductions in both linear and rotational acceleration. Concussion incidence did not significantly change from season to season. A combination of concussion mitigating strategies (rule changes and use of a helmet add-on) can reduce helmet impact frequency and acceleration, yet more research is needed to assess the clinical significance of lower impact magnitudes. Supported by NFL/GE/UnderArmour (Head Health Challenge II) and USMRMC W81XWH12C0203.
Keywords: Concussion, Linear acceleration, Rotational acceleration
The Children's Hospital of Philadelphia, Surgery, Philadelphia, USA
Functional near-infrared spectroscopy (fNIRS) is a noninvasive and portable neuroimaging modality that detects changes in blood oxygenation related to human brain function. It is a promising objective biomarker for identifying pathophysiologic changes associated with concussion. Te objective of this study was to determine the utility of fNIRS to detect and differentiate cortical brain activity between concussed and healthy subjects when performing oculomotor and visual tasks which has not been previously described. A prospective study of 38 concussed subjects and 9 healthy controls was undertaken where all participants completed standard oculomotor and visual tasks, including the King-Devick test (K-D), while wearing an fNIRS headband that recorded anterior prefrontal cortex oxygenation changes with 4 optodes and12 channels at 4Hz sampling rate. Linear mixed model analysis was performed to compare oxygenation changes in the two cohorts. Among this pilot cohort, healthy controls showed significantly greater oxygenation changes upon initiation of the King-Devick test compared to concussed subjects, but had decreased oxygenation changes over each successive test card (F1,172 = 4.06, p < 0.05) compared to injured subjects, indicating a habituation to the task over time in healthy controls that did not occur in concussed subjects. There were also significant differences in spatial patterns of oxygenation changes between the left and right prefrontal cortex between concussion subjects and healthy controls with monocular accommodation assessment (F1,66 = 12.05, p < 0.001) indicating recruitment of different cortical areas for the task in concussed subkects versus healthy controls. Our preliminary experimental results suggest that fNIRS has utility in detecting differences in cerebral blood oxygenation change between concussed and healthy subjects with excellent temporal and spatial resolution based on the given oculomotor or visual task. Further investigation into this neuroimaging modality for quantifying changes in cognitive workload after injury and over the course of recovery is warranted. fNIRS may be useful as an objective measure of injury and recovery, supplementing the clinical assessment and management of concussion.
Keywords: functional near infrared spectroscopy, vision, oculomotor
The Children's Hospital of Philadelphia, Surgery, Philadelphia, USA
Vision and vestibular deficits are common following concussion. It is unknown, however, which of these findings, may be associated with prolonged recovery. The objective of this study was to determine which vision or vestibular deficits are associated with prolonged recovery in children and to identify interrelationships between such deficits. A retrospective cohort study of pediatric patients with concussion was conducted in a subspecialty concussion program within a large pediatric care network. 432 random patient records were abstracted for data. The presence of vision or vestibular deficits upon initial presentation for clinical care at a subspecialty concussion program was determined and the main outcome measure of interest was time to functional clinical recovery. Study subjects ranged from 5–18 years (median = 14). 378 of 432 (88%) patients presented with one of the following vision or vestibular deficits: saccades (82%), balance (68%), smooth pursuits (66%), vestibulo-oculomotor reflex (VOR) (66%), near point of convergence (35%) or accommodative amplitude (22%). A prior history of motion sickness was associated with vision and vestibular dysfunction. Physical examination deficits associated within distinct vision and vestibular clusters. Abnormal accommodative amplitude predicted prolonged recovery time, as did abnormal balance, VOR, and smooth pursuits. Abnormal accommodative amplitude is associated with prolonged concussion recovery in children, as are deficits in balance, VOR and smooth pursuit function. Vision and vestibular deficits associate within specific clusters. A prior history of motion sickness was associated with vision and vestibular deficits following concussion. Vision assessments in concussion must move beyond visual acuity.
Keywords: vision, oculomotor, vestibular, balance
This study's objective was to investigate the performance of three standard criteria sets for diagnosing postconcussion syndrome/disorder (PCS/PCD).
Comparability of studies of patients with postconcussional syndrome (PCS) are frequently hampered by nonuniformity of symptoms used to diagnose the disorder. Although the International Classification of Diseases, 10th Edition (ICD-10) has clinical and research criteria for PCS, and the Diagnostic and Statistical Manual, 4th Edition (DSM-4) included provisional criteria for postconcussion disorder (PCD), few if any studies strictly observe these criteria. Consequently, little is known about how these criteria sets perform and compare. To explore this issue, 76 participants with mild traumatic brain injury (mTBI) were recruited from consecutive admissions to two Level 1 trauma centers in Houston, TX. Participants ranged in age from 18–50 years (mean age = 29.8 ± 9.1). Measures included the SF-12 and the Rivermead Postconcussion Symptoms Questionnaire (RPSQ) in addition to specific items addressing all ICD-10 and DSM-4 criteria. Because PCD can only be diagnosed at three months postinjury, data from this end point was analyzed for comparability between all criteria sets (mean = 94.5 ± 10.6 days). Incidence rates varied by criteria set used (ICD-10 clinical = 27.6%; ICD-10 research = 21.1%, and DSM-4 = 11.8%). Analyses between those meeting criteria versus not meeting criteria were conducted and significant differences were found using all three criteria sets for SF-12 Mental Health (p < .002 to < .0001) and Physical Health (p < .02 to < .0007) Summary scores. Significant differences were found for the total RPSQ score (p < .0001) for all three criteria sets.
Although the incidence rates varied widely, all three criteria sets appear to identify a subgroup of patients with mTBI who demonstrate significant symptoms at three months postinjury. There was no evidence supporting the use of one criteria set over the others. This may be elucidated employing different outcome measures.
This work was supported by the Congressionally Directed Medical Research Program by the Department of Defense (W81XWH-08-2-0133, PT074693P2).
Keywords: Post concussion Syndrome, Traumatic Brain Injury, Diagnostic criteria, Quality of Life
SYSTEMIC AND CEREBRAL HEMODYNAMICS IN MILD AND MODERATE TRAUMATIC BRAIN INJURY PATIENTS WITH PERSISTENT SYMPTOMS
UT Southwestern Medical Center at Dallas, Dallas, USA
Keywords: cardiac baroreceptor reflex, dynamic cerebral autoregulation, blood pressure, hemodynamics, heart rate, cerebrovascular circulation
Traumatic brain injury (TBI) is a leading cause of death and disability that is often studied using animal surrogates. Many closed head injury animal models of TBI place the animal on a foam bed during injury; however, the characteristics of these foams are rarely reported and may contribute to variation in TBI severity between laboratories. In this study, adult male rats were anesthetized, placed on one of two types of foam (n = 7 each foam; sham, n = 6) without head fixation, and given a mild TBI using a controlled cortical impact (CCI) device (3 m/s, 5 mm displacement, 16 mm diameter padded impactor). The two foams were chosen for their divergent properties: one was soft open-pore foam and the other was firmer and closed-pore. Toe pinch response and righting reflex time were recorded immediately following injury, and brains were sectioned 24 hours post-injury and immunostained for glial fibrillary acidic protein (GFAP) expression. Results indicate that GFAP was more highly elevated in brains injured on the firm foam than in those injured on the soft foam, suggesting a difference in TBI severity based on foam stiffness. There was no GFAP expression observed in sham animals. There was no significant difference in toe pinch or righting reflex time between groups. These results demonstrate that the type of foam used in closed head impact models can affect degree of injury for the same impact conditions; there is therefore a need to report and more fully characterize the foams used. We are currently investigating the injury response using other types of foam and additional outcome measures. Supported by R21 NS091832.
Keywords: animal model, foam, GFAP, TBI, concussion, cortical impact
DIAGNOSING THE GOSE: STRUCTURAL AND PSYCHOMETRIC PROPERTIES USING ITEM RESPONSE THEORY
Medical College of Wisconsin, Psychiatry, Milwaukee, WI, USA
The Glasgow Outcome Scale – Extended (GOSE) was designed to assess global functional outcome following traumatic brain injury (TBI). Since its introduction, several empirically founded criticisms of the GOSE have been raised, including poor reliability; an insensitivity to small but potentially meaningful changes; a tendency to produce ceiling effects; inconsistent associations with neurocognitive, psychological, and quality of life measures; and an inability to assess the multidimensional nature of TBI outcome. To distill the structural elements and psychometric properties of the GOSE that may contribute to reported shortcomings, the current study used an Item Response Theory (IRT) approach including one-parameter (1PL, or Rasch) logistic modeling. Data were from the TRACK-TBI Pilot Study, a large (N = 586), prospective (3M, 6M, 12M), multisite project that included recommended core common data elements (CDEs) and TBI cases of all injury severity levels. To assess the level of latent functional “impairment” captured by facets of the GOSE independent of its conventional 1–8 rating system, GOSE item responses were dichotomized (1 = impairment, 0 = no or non-debilitating amount of impairment) and combined into composites representing the seven domains (Consciousness, Independence at Home, Independence Outside of the Home, Return to Work, Social and Leisure Activities, Family and Friendships, and Return to Normal Life). CDEs were then used to predict each of the domains at each time point. Results of 1PL analyses indicated that, overall, the GOSE's items captured impairment across a broad disability spectrum at 3, 6, and 12 months. However, there was evidence of item redundancy (multiple items capturing similar levels of impairment), and a deficiency of items that detect lower levels of impairment to possibly explain ceiling effects. Findings also indicated that different CDEs predict different GOSE domains within and across time. The findings illustrate the value of IRT to illuminate the strengths and weaknesses of clinical outcome assessment measures and provides a framework for future measure refinement.
Keywords: Glasgow Outcome Scale Extended, traumatic brain injury, item response theory, Rasch modeling, common data elements, functional outcome
UPMC Presbyterian, Dept of Neurosurgery, Pittsburgh, USA
Keywords: chronic TBI, brain injury, psychological health
UPMC Presbyterian, Dept of Neurosurgery, Pittsburgh, USA
Keywords: chronic TBI, brain injury, symptoms, neurological outcome
Generally, closed head injury models of TBI result in fewer measurable behavioral and other functional deficits. Repetitive injuries, in addition to potential modeling of injuries in military personnel or athletes, often result in a more significant phenotype. Here, we describe a novel model utilizing repeated impacts on a closed head in male mice that produces substantial behavioral deficits and pathology. In the repeat-injury group, four moderate impacts to the head were performed, with a 24 hour interval between each impact. The single injury group only received one such injury. Repeat-injured mice exhibited hyperactivity and disinhibition in the open field test, social anhedonia in the social interaction maze, and abnormal nesting behavior. No major motor deficits were noted in either model. Behavioral changes were measurable for more than three weeks after injury. Different cohorts were sacrificed at 7, 28, or 56 days post-injury (dpi). Glymphatic function was measured by the distribution of fluorescent tracer injected into the cisterna magna 30 minutes before sacrifice. The distribution of astrocytic aquaporin-4 in the perivascular endfeet was also examined. We found that in injured animals, the tracer had restricted distribution within the brain parenchyma in comparison to that in sham injured animals. Immunohistochemistry revealed primary foci of injury directly under the impact as well as a few secondary discreet injury sites in the single impact model and more widespread pathology in the repeat injury model. Glial pathology from the single injury was largely resolved by 56 dpi however astrocytes and microglia were still activated in the repeat injured animals at 56 dpi. Fluoro-Jade C staining showed significant neuronal degeneration at 7dpi. Our data show that the repeat closed head injury results in significant behavioral deficits that are present several weeks after injury, together with reduced glymphatic movement of tracer, differential aquaporin 4 expression, increased neuronal degeneration and enhanced glial reactivity. Measurement of glymphatic system function, and nesting behavior are somewhat novel functional indicators after TBI that show deficits in this injury model. This model may therefore be useful for understanding interventions to treat closed head injury.
Keywords: glymphatic, nesting, repeat, repetitive, closed-head, CHI
EVALUATION OF ENVIRONMENTAL SENSORS IN MILITARY TRAINING ENVIRONMENTS: PRELIMINARY RELATIONSHIPS BETWEEN EXPOSURE AND OUTCOME
Given the prevalence of and attention to TBI in both military combat and training environments, the DoD has prioritized identifying and/or developing sensory technologies to detect and quantify head impact exposures. Subsequently, the Environmental Sensors in Training (ESiT) program was developed to evaluate an environmental sensor's (ES) utility to identify potentially concussive events occurring in military-training environments. A portion of the program evaluated DoD and commercially developed ES in the Basic Airborne Course (BAC). The primary objective is to examine the relationships between sensor measurements and changes in performance on neurocognitive, non-postural dynamic balance/sensory integration, reaction time, and vestibular/oculomotor tasks immediately and at delayed time points following head impacts. Participants were instrumented with multiple ES to record head impact events exceeding specified thresholds. Participants were videotaped during the drills to visually identify head impact events. Participants completed a series of tasks administered on a handheld device comprised of an enhanced virtual reality display, noise-reducing headphones, and a handheld controller. The device effectively provides an immersive environment for multimodal neurologic assessment in remote, noisy, or distracting environments. Additionally, participants completed the MACE, the PCL-M (military version), and a concussion history questionnaire. Testing occurred at in-processing to obtain a baseline and the end of each training week (progressively greater risk for head impact) for a total of four testing sessions. Complete datasets were available for 15 of 20 volunteers (1 female and 14 male). The majority of the participants indicated no prior history of concussion (90 percent), and all participants scored below the accepted cut-off criterion for the PCL-M, suggesting a healthy sample relative to PTSD and concussion. Results of the MACE tests suggest no changes in performance over the course of the training despite the occurrence of head impacts during drills. If results of the functional performance tests show sensitivity to head impacts, given the different time points for assessment, the tests may prove more advantageous for use in the training environment.
Keywords: concussion, wearable sensor, sensor, biomechanics
EVALUATING THE ABILITY OF HEAD INJURY CRITERIA TO PREDICT CONCUSSION THROUGH RECONSTRUCTIONS OF COLLEGIATE FOOTBALL HEAD IMPACTS
Head injury criteria are used in automotive and sports protective equipment safety standards. The standards currently in use are based on linear head acceleration during impact, and have greatly reduced the number of fatal head injuries in automobile accidents and contact sports like football. However, a large body of research suggests that both linear and rotational acceleration contribute to brain injury mechanisms, and a combination of kinematic parameters may better predict injury risk than a single parameter alone. In order to compare the predictive capabilities of different head injury criteria, we reconstructed head impacts in collegiate football players in a laboratory setting. A total of 11 impacts resulting in concussion and 44 not resulting in concussion were reconstructed. The field impacts were collected with helmet-mounted accelerometers, which compute impact location, peak linear acceleration, and peak rotational acceleration. The laboratory reconstructions were performed to determine the 6-degree-of-freedom linear and rotational kinematics needed to calculate all head injury criteria of interest. The impacts were reconstructed with a pneumatic ram striking an instrumented head and neck assembly fitted with a matched helmet for the field impact. Head kinematics from the reconstructions were used to calculate 10 different head injury criteria. These criteria used either linear kinematics, rotational kinematics, or a combination of both. Receiver operating characteristic (ROC) curves were used to evaluate the predictive capability of all criteria. For all criteria evaluated, the area under the curve (AUC) was significantly better than random guessing (p < 0.0007). Additionally, criteria that included both linear and rotational kinematics performed better than those that only included one or the other. The results of this study have applications toward evaluation of protective equipment to better predict concussion risk and further reduce those injuries.
Keywords: Acceleration, Linear, Rotational, Risk
INTERNAL JUGULAR VEIN COMPRESSION: THINKING OUTSIDE OF THE BOX FOR CONCUSSION, BLAST TBI, AND BLAST INDUCED HEARING INJURY
A traumatic brain injury, TBI, is due to acceleration of the head leading to relative motion of the brain parenchyma within the skull causing both macroscopic (hemorrhage) and microscopic injury (diffuse axonal injury). A novel approach to reducing this movement has been proposed through internal jugular vein compression (IJVC). The focus of this presentation will be to present the mechanism and theory behind IJVC and its resultant positive benefits in blunt, blast, and sports related TBI. A historical perspective reviewing the previously performed preclinical rodent models where IJVC demonstrated a reduction in APP, microscopic injury, post blunt injury in a rodent drop weight model (Smith et al., 2012; Turner et al. 2012) and parenchymal hemorrhage, macroscopic injury, in a swine cortical impact model (Sindelar et al. 2016). We will then discuss the two clinical studies demonstrating a reduction in axonal injury seen on DTI in subconcussed high school hockey and football athletes (Myer et al 2016).
Beyond the use in blunt TBI, IJVC has been suggested for use also in blast induced TBI. Due to the compensatory properties of the intracranial space, IJVC results in mild increases in intracranial pressure, but its resultant influence on other structures that communicate with this space, like the cochlea, has only begun to be researched. The discussion will then conclude with the recently completed study demonstrating the significant effects of IJVC specifically in audiological injury following exposure to a blast wind. The author exposed 20 Sprague Dawley rats to a 16.8 ± 0.3 PSI (195.3 dB SPL) right sided shock wave in which 10 had application of a custom IJVC collar in place at the time of injury. IJVC was shown to reduce the incidence of tympanic membrane rupture, initial temporary and permanent threshold shifts on otoacoustic emissions (OAE), and also a significant reduction in hair cell loss at the base of the cochlea secondary to mechanical trauma from the blast wind.
Keywords: internal jugular vein compression
AFFECTIVE PROFILING FOR ANXIETY-LIKE BEHAVIOR
Naval Medical Research Center, Silver Spring, USA
Traumatic brain injury (TBI) is a frequent injury in military conflicts, often caused by exposure to overpressure (shock) waves from devices such as improvised explosive devices. Studies indicate high rates of co-morbid mTBI and posttraumatic stress disorder (PTSD). Specifically, Ahlers et al. demonstrated blast-exposed rats display PTSD-like behavior, indicating possible relationships between blast and PTSD development. However, not every blast-exposed subject will display this behavior and may display phenotypes similar to controls. Therefore, including animals from experimental groups that do not appear anxious may obscure results that would demonstrate differences due to blast. To account for this, normal and abnormal behaviors can be determined from the control group, and the experimental group can be split into those displaying normal behaviors (“unaffected”) and anxiety-like behaviors (“affected”). These subgroups can then be compared separately to controls for a better understanding of changes that blast exposure may cause in behavior and molecular variables.
Data sets consist of molecular variables (e.g., blood or protein biomarkers) and behavioral data that identify the “affected” group. Using an affective profiling approach introduced by Richter-Levin and his colleagues, control animal behavioral data was analyzed to determine boundary values for normal behavioral results. These boundary values were then applied to the experimental group's behavioral data, and animals whose behavior fell outside this range were separated into an “affected” experimental group; remaining animals were labeled “blast-unaffected”. Using these procedures, data for molecular variables were then compared.
When this method was used on test data containing behavioral data from an elevated zero maze and stathmin and corticosterone levels, clear differences in the average values between “affected” and “unaffected” groups were present. This method shows promise as another method to study effects of blast, while accounting for animals whose data may obscure results due to similarities to control animals.
Keywords: behavior, animal model, PTSD, biomarkers
University of Michigan, Kinesiology, NeuroTrauma Research Laboratory, Ann Arbor, USA
Keywords: Concussion/ mTBI, Biomarker, Accelerometry, Pediatric
Few experimental models attempt to recapitulate the progressive brain atrophy and tau pathology that defines human chronic traumatic encephalopathy (CTE). The absence of stringent diagnostic tools together with variable disease progression limit the understanding of this disease. Here, we use a recently described novel rat model of CTE to examine temporal changes in cortical neuron loss, inflammation, and hyper-phosphorylated tau (P-tau) following repeat concussion.
Keywords: rat model, inflammation, neuronal loss, corticospinal motor neurons
PRE-INJURY SOMATIC COMPLAINTS AND NEGATIVE EMOTIONALITY PREDICT SYMPTOM RECOVERY AFTER SPORT-RELATED CONCUSSION
It is increasingly being recognized that athletes vary in their recoveries from sport-related concussion (SRC) and that pre-premorbid individual patient factors play a role in their risk for persistent post-concussive symptoms. We previously published evidence that higher pre-injury somatic symptoms prolong symptom recovery via their influence on acute post-concussive symptom burden. This study aimed to replicate this previous finding and additionally examine the degree to which broad personality traits, shown to predict other health outcomes, contribute to symptom presentation and recovery after SRC. High school and collegiate football athletes (N = 917) completed preseason evaluations. Athletes who sustained a concussion during the season (n = 62) completed serial post-injury assessments (<6 and 24–48 hours; 8, 15 and 45 days). Cox proportional hazard modeling was conducted to predict symptom duration (in days) from various pre-injury and acute (<48-hour) post-injury demographic, injury, and clinical assessment variables. The best overall predictors of symptom duration were neurologic complaints (Minnesota Multiphasic Personality Inventory-2-Restructured Form, MMPI-2-RF, NUC scale score), somatic complaints (MMPI-2-RF RC1 scale score), and post-concussive symptom burden (Sport Concussion Assessment Tool-3 symptom severity) assessed within 48 hours of injury (p's < .009). Additionally, path analyses indicated that both pre-injury somatic complaints and trait negative emotionality (Multidimensional Personality Questionnaire, MPQ) contribute to prediction of symptom duration by exacerbating acute post-injury head pain and neurologic symptoms. The findings replicate previous reports that pre-injury somatic symptoms affect clinical recovery from SRC and point to negative emotionality as an additional individual difference construct with relevance to concussion recovery. These data align with a broad literature pointing to the predictive value of negative emotionality for diverse health outcomes. Clinicians and athletic staff could use these findings to identify and focus treatment efforts on concussed athletes who are at higher risk of prolonged recoveries. Future work should investigate associations between these pre-injury characteristics and neurobiological outcomes and develop early interventions to maximize the recovery of athletes at high risk of persistent symptoms.
Keywords: High School and Collegiate Football, Sport related concussion, Personality, Somatic Complaints, Recovery
PHASES OF HEMODYNAMIC RECOVERY AFTER MILD TRAUMATIC BRAIN INJURY
Neural Analytics, Los Angeles, USA
The lack of biomarkers which measure specific pathophysiologic mechanisms has hindered the development of improved therapeutic and diagnostic tools for managing traumatic brain injuries (TBI). Recent research has identified abnormalities in cerebral blood flow and it is clear that the microvasculature is prominently affected after TBI, including mild TBI (mTBI). Detailed understanding of hemodynamic alterations following mTBI has the potential for providing an objective diagnostic aid as well informing the design of therapeutic trials aimed at cerebrovascular dysfunction. In this study, we assessed cerebral hemodynamic changes in high school sports participants 14–19 years old after suffering clinically diagnosed mTBI using transcranial Doppler (TCD). Cerebral blood flow velocity of mTBI subjects (n = 109, age 16.2 years (1.16)) were compared to age matched controls (n = 70, age 16.1 years (1.56)). We identified two phases of hemodynamic change after concussion not previously reported. During the first 48 hours, Pulsatility Index (PI), Resistivity Index, and peak ratio (P2R), showed a significant difference from the controls (U = −3.10; P < 0.01, U = −2.86; P < 0.01, and U = 2.62; P < 0.01, respectively). In addition, PI and P2R were not correlated (rp = −0.36; P = 0.23). After 48 hours post-injury, differences in pulsatile features were no longer observable. However, breath holding index (BHI), a measure of cerebral vascular reactivity, was significantly increased after concussion. BHI was measured 2–3, 4–5, and 6–7 days post-injury with U = 2.72; P < 0.01, U = 2.46; P = 0.014, and U = 2.38; P = 0.018, respectively. Measurements were repeated for 30 days following injury and longitudinal results will be presented. Understanding of the multiple phases of hemodynamic recovery after mTBI may provide further insight into the initial mechanical injury and subsequent recovery.
Keywords: transcranial Doppler
Boston university school of medicine, Boston, USA
The mechanisms underpinning impact concussion, traumatic brain injury (TBI), and chronic traumatic encephalopathy (CTE), and the relationships between these disorders, are poorly understood. We examined postmortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation and phosphorylated tau pathology. We developed a mouse model of impact concussion that uses momentum transfer to induce traumatic head acceleration in non-anesthetized C57BL/6 and Ccr2RFP/Cx3cr1GFP mice. Experimental head injury recapitulated brain pathologies associated with mild TBI and early CTE in humans. Unilateral closed-head injury resulted in ipsilateral neuronal dropout, neuroinflammation, and traumatic microvascular injury with serum albumin extravasation that was detectable in living mice by dynamic contrast-enhanced MRI. Delayed microgliosis and infiltrating myeloid cells were detected by flow cytometry and confirmed by fluorescence microscopy in Ccr2RFP/Cx3cr1GFP mice. Phosphorylated tauopathy was detected in ipsilateral cortex by 24 hours post-injury and progressed to both hemispheres where tau pathology persisted for at least 5.5 months post-injury. These pathologies were accompanied by persistent bilateral impairments in hippocampal axonal conduction and defective long-term potentiation (LTP) in the prefrontal cortex. TBI and CTE brain pathologies and neurophysiological defects did not correlate with neurobehavioral measures of acute concussion. Closed-head impact injury, independent of concussion signs, can induce mild TBI and trigger early brain pathologies and functional sequelae associated with CTE.
Keywords: Animal Modeling, Tau Protein, Microvascular Injury, Neuroinflammation
Emory University, Emergency Neurosciences, Atlanta, USA
The goal of this study was to describe current concussion screening, diagnosis, and management patterns in the emergency department (ED) of a large urban county hospital. This pilot was part of a larger initiative, Screen-Inform-Prevent, which was developed by a national multidisciplinary workgroup to improve concussion identification in the ED setting.
We implemented a prospective screening tool embedded within the electronic medical record and then examined the number of patients who received a concussion diagnosis, corresponding ICD-10 code, and discharge instructions. Clinicians were not alerted to the results of the initial screen. Chart review was used to document specific elements in the physician note and the ICD-10 codes assigned. Injury mechanism and complex (i.e. isolated head injury vs. multiple injuries) predicted whether or not a patient received a diagnosis of concussion. Over half of the population that screened positive for concussion did not have any elements of a concussion exam documented.
Of the 98 patients who screened positive, concussion was included in the differential diagnosis for 45 patients and in the final diagnosis for 36 patients; 15 patients received discharge instructions, and 6 patients received a concussion ICD-10 code. Patients with and without a concussion diagnosis were primarily distinguished by their injury complex (i.e. isolated head injury vs. multiple injuries), whether a head CT was ordered, and whether concussion was included in the differential diagnosis. Similarly, patients who received discharge instructions were distinguished by their injury complex (i.e. isolated head injury vs. multiple injuries).
Our study provides additional support that ED patients with likely concussion do not receive an examination necessary to identify a concussion, and of those diagnosed, half did not receive concussion-specific discharge instructions. Improved recognition and patient education has the potential to prevent and reduce concussion morbidity. More work is needed to improve concussion screening and evaluation in the ED setting.
Keywords: Screen, Inform, Prevent, Concussion
Stanford University, Bioengineering, Stanford, USA
Keywords: Concussion Diagnostic, Injury Criteria, Reduced Order Modeling
Johns Hopkins University, Pathology/Division of Neuropathology, Baltimore, USA
We have recently established a model of primary multifocal axonopathy in the mouse after diffuse TBI using impact acceleration (IA) (Xu et al, 2016). Among the various CNS tracts involved, the visual system was greatly prominent, with IA dose -dependent retinal ganglion cell (RGC) axonal and perikaryal degeneration and with neuropathy-related neuroinflammation. This model can serve not only to replicate the specific conditions of traumatic optic neuropathy but also to more broadly model the problem of traumatic axonal injury because of the well-defined origin, course, and termination of the optic tract and the accessibility of RGCs to experimental manipulations. Here we delve further into molecular mechanisms operating in traumatic optic axonopathy after IA, with focus on the dual leucine zipper kinase (DLK)-jun terminal kinase (JNK) axis. Using relatively mild IA settings, we performed a time course of changes in expression of key members of the DLK-JNK axis and we then began to delete specific members of the axis and explore outcomes. By immunohistochemistry and Western, we found that phosphorylation of c-jun in RGCs increased significantly at day 1 and 3 and started to drop at day 7; similar trends were observed with other members of the DLK-JNK axis. Using gamma-synuclein as marker of RGCs, we found a significant RGC loss and optic nerve degeneration at 14 and 28 days after injury (19% and 37% cell loss, respectively). A CTB pulse in retina was blocked in the distal third of the optic nerve when chased at day 2 but appeared to have recovered after day 14. We are presently exploring the effect of deleting dlk expression with Cre-lox recombination strategies on c-jun phosphorylation on day 3 post-injury. We are also exploring the effect of deleting both dlk and lzk on the survival of RGCs 8 weeks post-injury. We propose that traumatic optic neuropathy after IA is a powerful model for traumatic axonopathy and, because of the accessibility of the retina to genetic and pharmacological manipulations, it is especially appealing for exploring molecular mechanisms of traumatic degeneration, including the role of mixed-lineage kinases.
Keywords: concussive injury, traumatic optic neuropathy, apoptosis, axonal injury
University of Michigan, Department of Computational Medicine and Bioinformatics, Ann Arbor, USA
Traumatic brain injury (TBI) is a massive public health problem worldwide. Approximately 1.5 to 2 million Americans sustain a traumatic brain injury each year with the lifetime and total costs associated with these injuries estimated at $60 billion. Accurate and fast automatic detection of hematoma in brain are important for TBI diagnosis, which can efficiently decrease the medical cost, reduce the error from visual examination and provide guidance for appropriate medical treatment. The evaluation of hematoma regions is also important for epidemiologic studies and agent efficacy estimation. In this study, we developed an automated system to detect and segment hematoma regions in head Computed Tomography (CT) images of patients with acute TBI. Our proposed method has three main stages as follows. 1) Pre-processing, 2) Feature extraction and 3) Training a learning model. We first process head CT images to extract regions of interest and decrease noises and variances among patients. Then, we extract clinically relevant features from CT images as well as statistical, texture, and geometrical features to capture characteristics of hematoma regions. After that, we combine uncertainty-based active learning strategy to adaptively select the most samples to train a SVM classifier. In this study, we used CT images of 40 patients from Treatment of Traumatic Brain Injury Experimental Clinical Treatment Trial (ProTECT) and our final SVM model achieved the area under curve (AUC), specificity and sensitivity of 0.909, 0.674, and 0.985, respectively. Our results show that the proposed automatic hematoma detection system can overcome the challenge of the limited, highly unbalanced and varied dataset.
Keywords: acute TBI, image processing, segmentation, active learning
Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
Traumatic brain injury (TBI) is one of the major public health concerns in the U.S. that could result in permanent disability. However, the impact of the injury could be limited/decreased by its early management and treatment. TBI can cause subdural hematoma (SDH), diffuse axonal injury, lacerations and other injuries. The focus of our study is on developing a decision support system for SDH detection as it is one of the most common consequences of TBI and its detection through visual examination could be difficult. In this study, we propose a fully automated image processing and machine learning based approach for detecting convexity subdural hematomas and measuring the volume of SDH quantitatively. We analyze head Computed Tomography (CT) images and extract textural, statistical and geometrical features of sample points from the intracranial region to train a classifier. We implement a RUSBoost classifier to classify each image pixel as hematoma or no-hematoma. We apply our method on 55 CT image sets captured from patients with SDH at the University of Michigan Hospital. The total number of CT images used in our analysis is 1281 and our method could yield sensitivity and specificity values of 75.98% and 87.60% respectively.
UCSF, Neurosurgery, San Francisco, USA
Over 70% of traumatic brain injuries are classified as mild (mTBI), which present heterogeneously, leading to challenges in management and prognosis. Improved characterization of baseline medical comorbidities may delineate their status as risk factors for unfavorable recovery. Patients from the TRACK-TBI Pilot study with Glasgow Coma Scale 13–15, completing three- and six-month outcomes (Glasgow Outcome Scale-Extended (GOSE); Acute Concussion Evaluation (ACE): physical/cognitive/sleep/emotional subdomains) were extracted. Presence/absence of 12 preinjury medical comorbidities and intracranial pathology on acute computed tomography (CT) were considered. Statistical significance was assessed at p < 0.001 correcting for multiple comparisons. Two-hundred sixty mTBI patients were aged 44.0 ± 18.7-years, 70.4%-male, 78.6%-Caucasian; 36.2% were CT-positive. Comorbidities of highest incidence were psychiatric-30.0%, cardiac-20.4%, gastrointestinal-15.8%, asthma-13.1%, headache/migraine-11.5%, seizures-8.5%. Overall three- and six-month GOSE were 6.83 ± 1.14, 6.92 ± 1.13 respectively. ACE symptomatology did not differ significantly between CT-positive/negative groups. In CT-negative patients, psychiatric-history associated with decreased three- and six-month GOSE (p = 0.001); seizure-history associated with decreased six-month GOSE (p = 0.001). At three-months, increased ACE-physical symptomatology associated marginally with gastrointestinal-history (p = 0.004), increased ACE-cognitive symptomatology associated with seizure-history (2.53-vs.-1.12-symptoms; p<0.001), ACE-sleep symptomatology associated with gastrointestinal-history (1.56-vs.-0.77-symptoms;p = 0.001) and headache/migraine-history (1.58-vs.-0.76-symptoms;p<0.001), ACE-emotional symptomatology associated with headache/migraine-history (2.04-vs.-0.91-symptoms;p<0.001) and marginally with seizure-history (p = 0.003). At six-months, ACE-physical associated with headache/migraine-history (5.08-vs.-2.71-symptoms; p < 0.001) and marginally with seizure-history (p = 0.003), ACE-sleep associated marginally with hepatic-history (p = 0.005), and ACE-emotional associated with seizure-history (2.79-vs.-1.37-symptoms;p<0.001). Psychiatric-history associated with increased symptomatology across all ACE subdomains at three- and six-months (range: 0.71-to-2.36 mean increase in symptom number; p<0.001). In CT-positive patients, thyroid-history associated marginally with ACE-physical (p = 0.003), and hepatic-history associated with ACE-emotional (3.67-vs.-0.91-symptoms; p = 0.001). The contribution of baseline comorbidities to evolving symptomatology within six-months post-mTBI deserves increased scrutiny. In CT-negative patients, comorbidities associating with postconcussional symptoms across three-and six-month timepoints include psychiatric-history, and to a less conserved extent, headache/migraine- and seizure-history. Gastrointestinal-history may increase symptoms burden prior to three-months.
Keywords: medical comorbidities, mild traumatic brain injury, outcome measures, postconcussional symptoms, prognosis
UCSF, Neurosurgery, San Francisco, USA
The status of facial bones as a protective cushion for cranial contents, versus risk factor for cranial injury, during traumatic brain injury (TBI) is unclear. The contribution of acute facial fractures on long-term functional disability following uncomplicated mild TBI (mTBI) remains to be investigated. Adult subjects suffering acute TBI requiring clinically-indicated brain computed tomography (CT) scan <24-hours of injury from the prospective Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot (TRACK-TBI Pilot), with Glasgow Coma Scale (GCS) score 13-15, without intracranial CT pathology nor history of stroke, brain tumor, and/or learning/developmental disability, completing six-month disability measures [Glasgow Outcome Scale-Extended (GOSE), Craig Handicap Assessment and Reporting Scale-Short Form (CHARTSF) subscales) were extracted. Outcomes were analyzed by CT evidence of facial fracture. Multivariable regressions controlling for age, education, employment, psychiatric and seizure history, GCS, loss-of-consciousness, and polytrauma were performed for univariate analyses with p < 0.10. One-hundred sixty-five uncomplicated mTBI subjects were included, aged 41 ± 16-years, 67%-male, and 75%-Caucasian. Facial fracture (n = 20) was associated with hospital admission (70%-vs.-39%;p = 0.018). Most patients achieved good recovery at six-months (GOSE ≥7; facial fracture = 61%, no fracture = 70%). On univariate analysis, facial fracture was associated with lower CHARTSF-Social Integration scores (82.2 ± 31.5 vs. 92.9 ± 16.3;p = 0.017) and marginally lower CHARTSF-Economic Self-Sufficiency scores (64.0 ± 39.4 vs. 77.6 ± 32.9;p = 0.092). On multivariable analysis, the association between facial fracture and decreased CHARTSF-Social Integration persisted (adjusted-mean 75.7 ± 5.0 vs. 84.7 ± 3.4; mean difference = -9.1, 95% confidence interval [0.5–17.7]; p = 0.039). Adjusted Social Integration scores for fracture/non-fracture groups were below/above the 50th percentile CHARTSF normative data for all-severity TBI, respectively. Subjects suffering facial fractures in the setting of acute uncomplicated mTBI may be at risk for impaired return to social participation independent of other injury factors, and may constitute a subpopulation benefitting from heightened surveillance and/or therapeutic intervention. Confirmation in larger datasets is however warranted.
Keywords: CHART-SF, disability, facial fracture, mild traumatic brain injury, social integration
Traumatic brain injury (TBI) is a leading cause of death and permanent disability worldwide, affecting an estimated 10 million people annually. The prevalence of Axis I and/or Axis II disorders following TBI is 20-50%. Although there is a great need for evidence-based treatment for psychiatric impairments after TBI, such research is scant. Disorders of mood and cognition may remain even after recovery of neurologic function is achieved. One mechanism by which this occurs is disruption of brainstem neurons during TBI, which effectively disrupts pontine and medullary serotonergic projections, resulting in impaired metabolism of serotonin and associated deficits in mood regulation, executive control and homeostasis. Selective serotonin reuptake inhibitors (SSRI) are highly specific antidepressant agents modulating arousal, emotion, and working memory. They constitute first-line treatment for a variety of neurocognitive and neuropsychiatric disorders. This review investigates the utility of SSRIs in treating post-TBI sequelae. Twenty-seven unique reports were consolidated from the Cochrane Central Register for Controlled Trials and National Library of Medicine PubMed databases (seven randomized controlled trials, nine open-label studies, 11 case reports). SSRIs are associated with improvement of depressive symptoms but not cognitive symptoms; pooled results using the Hamilton Depression Scale (HAM-D) demonstrate a significant mean decrease of depression severity following sertraline administration compared to placebo. A number of case reports and small open-label studies cite mood improvement following SSRI administration, while effects on cognitive and functional recovery were largely absent or negative. No studies specifically focused on SSRI treatment effects for post-TBI post-traumatic stress disorder (PTSD). Discernment of SSRI efficacy for TBI-induced PTSD represents an important direction of future research; in addition, placebo-controlled studies with extended follow-up periods and concurrent biomarker, neuroimaging and behavioral data are necessary to accurately delineate the attributable pharmacological effect of SSRIs in the TBI population.
Keywords: cognition, depression, postconcussive disorder, selective serotonin reuptake inhibitor, traumatic brain injury, posttraumatic stress disorder
Traumatic brain injuries (TBI) carry a risk for the development of post traumatic epilepsy (PTE). Currently, animal models that produce clinically relevant spontaneous seizures are lacking. The aim of the study was to characterize PTE following severe penetrating TBI (pTBI) and severe closed-head TBI (cTBI) in rats. Severe pTBI was induced by stereotactically inserting a perforated steel probe into the brain of an anesthetized rat and rapidly inflating the probe's elastic tubing into an elliptical shaped balloon. Two trajectories (frontal and lateral) and two severity levels (10% and 12.5%) were used. Severe cTBI was induced by launching a steel ball bearing (10.5 mm in diameter) aimed at the dorsal-lateral skull surface of anesthetized rats. Investigation of spontaneous seizure activity was carried out to 1 or 6 months post-injury. At 1 and 6 months post-injury, seizure susceptibility was tested in all animals by pentylenetetrazole (PTZ) injection (30 mg/kg). Eight pTBI groups (2 trajectories × 2 severities × 2 time points) and 2 cTBI groups (2 time points) were included in the study. Among 80 pTBI rats and 20 cTBI rats included in the study, no spontaneous convulsive behavior was detected out to 6 months post-injury. Spontaneous and daily recurrent nonconvulsive seizures were detected in only one cTBI rat via EEG recording at 6 months post-injury. Increased seizure susceptibility in the PTZ test was observed in 50–80% of all injured animals with higher seizure scores and shorter seizure latencies detected in pTBI injured animals. In the cTBI model, seizure susceptibility to the PTZ challenge was moderately increased 1 month post-injury and even to a lesser degree at 6 months post-injury. Overall, while results of the current study showed lowered threshold to the PTZ challenge in both brain injury models, it failed to demonstrate consistent, reproducible evidence of spontaneous PTE in either model, in the absence of the PTZ challenge.
Keywords: Post-traumatic seizures, Post-traumatic epilepsy, model development, Ballistic-like brain injury
ASTROGLIAL GLUTAMATE TRANSPORTER GENETIC ASSOCIATIONS WITH POST-TRAUMATIC SEIZURES
University of Pittsburgh, Physical Medicine and Rehabilitation, Pittsburgh, USA
Individuals with TBI have an estimated 50-times increased relative risk for unprovoked seizure versus healthy age-matched controls. Published studies have identified factors that increase risk for post-traumatic seizure (PTS), like subdural hematoma and craniectomy; however, there remains significant unexplained variance in clinical prognostic models. Variation in genes involved in both secondary injury after TBI and seizure pathology (e.g. glutamate transport/excitotoxicity), may help address this knowledge gap. SLC1A3, which encodes the glial glutamate transporter excitatory amino acid transporter-1, clears extracellular glutamate from synaptic clefts across astroglial membranes, impacting local bioenergetics, ammonia homeostasis and GABA-glutamine production, which may each lead to increased PTS risk. The study objective was to assess the association between genetic variation in SLC1A3 and time until first seizure over the first 3 years post-injury in N = 267 self-reported White adults with severe TBI and no pre-injury history of seizure disorder. Eighteen tagging single nucleotide polymorphism (SNPs) were examined for independent associations between minor allele variant carriers and PTS. Adjustment for multiple comparisons was made using the minimum number of effective tests (Meff) method. Rs4869682 (GG vs. T-carriers) was associated with time until first seizure (log-rank p = 0.003). PTS incidence for GG homozygotes was 29.7% versus 16.9% for T-carriers. After adjustment for subdural hematoma, rs4869682 T-carriers had a 53% reduced PTS risk versus GG-homozygotes (aHR = 0.47, 95% CI: 0.25, 0.89, p = 0.021). No other SNPs were associated with PTS after multiple comparisons adjustment. Our data suggest variation in the glial transporter gene, SLC1A3, is associated with epileptogenesis, such that T-minor allele carriers for rs4869682 are protected against PTS. These study results should be validated in independent samples; though, the relevance of this glial transporter gene in PTS pathophysiology makes it a candidate to use for 1) PTS risk stratification and 2) as a potential treatment target. Supported by NIH-R01HD048162; NIH-R01NR013342; DoD-W81XWH-071-0701, CURE Epilepsy Award; NIDILRR-90DP0041.
Keywords: Post-traumatic seizure, Astroglia, Candidate gene study
We previously demonstrated synergistic effects of combination therapy using phenytoin paired with levetiracetam (PHT+LEV) or ethosuximide (PHT+EXM) for mitigating acute post-traumatic nonconvulsive seizures (NCS) induced by penetrating ballistic like brain injury (PBBI) in rats. We also observed sub-additive effects of LEV paired with gabapentin (LEV+GBP). In this study we evaluated two different pairs of combinations of these drugs: EXM+LEV and PHT+GBP, in the PBBI model. All rats received a frontal PBBI and skull EEG electrodes implantation. EXM+LEV were tested at four dose ratios: EXM/LEV: 5.6/6.3, 11.1/12.7, 22.2/25.4, or 44.5/50.7 mg/kg. PHT+GBP were tested at five dose ratios: PHT/GBP: 1.8/0.6, 3.6/1.3, 7.2/2.5, 14.5/5.0, or 28.9/10.0 mg/kg. Treatments were given intravenously twice/day for three days following the injury. The NCS were detected by continuous EEG recording for 72 h. Similar to previous studies, approximately 65% of PBBI-vehicle treated animals showed evidence of post-traumatic NCS within the first 72h following injury. PBBI animals treated with EXM+LEV showed reduced NCS incidence to 33–44% across all dose ratios tested and dose-dependently decreased NCS frequency and duration by 10–84% (p < 0.05 at 2 highest dose ratios) and 2–88% (p < 0.05 at 2 highest dose ratios), respectively. However, for PHT+GBP treatments, only the highest dose ratio reduced NCS incidence to 41% and significantly reduced seizure frequency by 78% (p < 0.05) and seizure duration by 82% (p < 0.05). Isobolographic analysis further indicated that EXM+LEV combination therapy achieved synergism because the observed effects exceeded the expected additive effects, whereas PHT+GBP had sub-additive effects. These results extended our previous findings on AED combinations involving PHT, EXM, and LEV and indicated their favourable interactions when any two pairs were combined at lower dose constituents than their monotherapy doses. On the other hand, similar to the lack of beneficial effects of LEV+GBP combination therapy, PHT+GBP also failed to improve their anti-seizure activities, suggesting that GBP may not be a good candidate for combination therapy against post-traumatic NCS.
Keywords: post-traumatic nonconvulsive seizures, penetrating brain injury, anti-epileptic drugs, combination therapy, EEG, rats
LOWER DOES OF PROPHYLACTIC LEVETIRACETAM IN THE ELDERLY FOR SUBACUTE AND CHRONIC SUBDURAL HEMATOMAS AFTER SURGICAL EVACUATION
Keywords: Subdural hematoma, Prophylactic, Optimal dosage, Seizures
ABNORMAL CA2+ SIGNALING IN REACTIVE ASTROCYTES CONTRIBUTES TO SPONTANEOUS SEIZURES IN A NEW MOUSE MODEL OF POST-TRAUMATIC EPILEPSY
Traumatic brain injury (TBI) is the most common cause of acquired epilepsy. Currently available anti-epileptic therapy is ineffective for post-traumatic seizures prevention pointing to differences in the pathogenesis. To date nearly all research has focused on neuronal dysfunction. Yet, the field has identified other potential underlying causes that individually lead to generation of epileptiform activities, including reactive astrogliosis and calcium[Ca2+]-dependent gliotransmission. These mechanisms are actively induced by TBI. The role of astrocytes and their Ca2+ transients in epilepsy remains largely unknown due to uncertainty of how they affect seizures. Even less is known how these phenomena contribute to post-traumatic epilepsy (PTE). Clinically, even after mild TBI, the risk for PTE is increased. Today, spontaneous seizures were only confirmed in two PTE models, where TBI is induced by moderate-to-severe penetrating trauma, PTE incidence is low and profound tissue loss following brain injury interferes with the emergence of a concise picture detailing disease mechanisms and biomarkers. We developed a new model of PTE induced by repetitive mild non-penetrating TBI. The TBI was induced (100 g weight, 50cm height, 3 impacts, 45min inter-injury interval) in a small cohort of mice. After the final righting time was assessed, mice were fitted with electrodes for electroencephalographic (EEG) recordings. EEG monitoring was started 4 days post-injury. All recorded animals had at least two seizures within 6 weeks post-injury. The first seizure was captured on day 21’ post-injury. Seizures recurred in a subset of mice and were accompanied by behavioral abnormalities including freezing, facial automatisms, tail extension, rearing, and falling. Using 2-photon imaging we determined abnormal astrocyte Ca2+ signaling acutely and within first 4 days post-injury. We provide proof-of-concept evidence that our model of PTE with recurrent unprovoked spontaneous electrographic and behavioral seizures in the settings of repetitive diffuse mild TBI is a promising animal model for studying the pathogenesis of PTE and will help in finding new potential therapeutic targets. Early post-traumatic abnormalities in astrocyte Ca2+ and downstream mechanisms are promising targets to explore for their role in post-traumatic epileptogenesis.
Keywords: Calcium Oscillations, Reactive Astrocytes, Spontaneous Seizures, Post-Traumatic Epilepsy
OPTICAL AND ELECTROGRAPHIC CHANGES IN THE BRAIN AFTER TRAUMA
University of California, Riverside, of Biomedical Sciences, Division Riverside, USA
Traumatic brain injury (TBI) is a major cause of morbidity and mortality in persons under the age of 45 and is a primary public health concern in both the civilian and military populations. Posttraumatic epilepsy (PTE) is a long-term negative consequence of TBI in which one or more unprovoked seizures occur at least one week after the initial trauma and accounts for up to 20% of all symptomatic epilepsy. Severity of injury and early posttraumatic seizures are risk factors associated with PTE, however, predictors of PTE still remain highly variable. Current studies of PTE have focused on pharmacologically testing seizure threshold using pentylenetetrazole and imaging studies, while limited, have not identified a correlation between trauma and seizure susceptibility. In our study, we utilized optical coherence tomography (OCT) to detect functional and structural changes in the brain in a controlled cortical impact (CCI) injury mouse model of TBI. Additionally, animals underwent long-term video-electroencephalography (vEEG) recording to identify spontaneous seizures after TBI and intrahippocampal electrical stimulation to quantitatively define electrographic seizure threshold (EST) and duration (ESD). OCT imaging detected alterations in optical attenuation, a measure of both scattering and absorption of the biological sample, with and without vascular contribution at different time points after TBI. Additionally, injured animals exhibited altered EST and ESD compared to naïve and sham controls. Altogether, our data suggest that seizure threshold can be quantitatively measured and OCT imaging can potentially offer useful information to identify predictors of PTE in our model.
Keywords: Optical coherence tomography, Seizure threshold, EEG
Experimental models of dissociated neuronal cultures have offered insight into the single-cell pathophysiology of neuronal injury, but fail to incorporate the modular architecture inherent to the brain. Without this modular architecture, it is difficult to directly evaluate the role of architecture in driving neural activity, particularly across different scales of brain organization. In this study, we explored how network size affected neuronal activity during recovery after injury in vitro. Primary rat cortical cultures were patterned as multi-neuron isolated clusters of neurons (islands) ranging in size from 750 microns (μm) to 1.4 mm. Selective injury of 20–30% randomly chosen neurons in islands was performed using a microinjector device, at a level of injury that does not cause cell death. A genetically-encoded calcium indicator (GCaMP6f) was used to estimate neural activity at baseline, immediately after, and 6–20 hours following injury at 13–15 days in vitro (DIV). We computed single-cell Ca2+ transient rates, and estimated functional connectivity among neurons using previously published methods. Neuronal islands progressed from non-synchronous patterns of activity to full network bursts as they matured. Ten days following plating, we observed slow-paced, full-system bursts (0.8 +/- 0.33 events/minute) in smaller islands, with faster rates of non-synchronous activity appearing in larger islands (11.3+/-7 events/minute, p < 0.01 relative to smaller islands). At the single cell level, two characteristic neuronal populations appeared: (1) fast-pacing neurons characterized by periodic transients with a peak at 0.33 Hz in the frequency domain, and (2) regular pacing neurons with activity in the 0.03–0.1 Hz range. Following injury, activity is perturbed in islands with non-synchronous activity, but maintained in cultures with bursting pre-injury patterns. Additionally, there is a transient increase in the proportion of fast-pacing neurons 6 hours after injury, suggesting that they are more resilient to injury or potentially serve as re-initiators of activity. Taken together, these data suggest that activity state, as determined by network size, plays a role in the response to and recovery after targeted injury.
Keywords: in vitro model, controlled neuronal networks, targeted injury, Calcium imaging, functional connectivity
FACTOR ANALYSIS TO DETERMINE WHITE MATTER INJURY PATTERNS UNDERLYING COGNITIVE CHANGES FOLLOWING PEDIATRIC TRAUMATIC BRAIN INJURY
Loma Linda University, Radiology, Loma Linda, USA
Traumatic brain injury (TBI) is associated with widespread disruption in white matter (WM) integrity. Diffusion tensor imaging (DTI) shows promise as a tool to detect changes underlying neurocognitive outcome. Factor analysis can condense a large number of variables into relatively few new variables that best characterize the data. These factors can be used to predict outcomes of interest. The purpose of our study was to use factor analysis to identify patterns of WM injury that predict neurocognitive outcome in pediatric TBI patients. 30 children (ages 4–18) with moderate or severe TBI were enrolled. DTI was acquired at 3T using 30 directions and b = 0 and 1000 s/mm2. Patients were imaged 6–17 days post injury. Cognitive testing was done 1 year after injury and included the Wechsler Abbreviated Scale of Intelligence (FSIQ), Performance Intelligence Quotient (PIQ), and the Test of Everyday Attention- Children (TEA-CH G). TBSS was used to create FA, ADC, AD and RD maps. Individual cortical, combined regions and whole brain DTI measures were subjected to factor analysis. Principal components of individual variables were used in a logistic regression to determine the factors ability to predict neurocognitive outcome. Three factors were identified, accounting for 69.8% of the variance in the original data set. Factor 1 and 2 included ADC, AD, and RD measurements from individual cortical, combined, and total WM regions. Factor 3 included the mean FA of individual cortical, combined, and total WM regions. All factors predicted impairments in FSIQ and TEA-CH G with 82.5% and 87.5% accuracy. Factor 3 was the best predictor of PIQ at 86.2%. Our findings show that factor analysis of DTI in pediatric patients after a TBI can successfully identify patterns of WM injury that predict impairments in cognitive function. Supported by NIH/NINDS R01-NS054001
Keywords: diffusion tensor imaging, pediatric, factor analysis, cognition
USC Keck School of Medicine, IGC, SNII, Los Angeles, USA
Many studies over the last decade have used dMRI (diffusion magnetic resonance imaging) to investigate TBI, due to its sensitivity to white matter (WM) disruptions post-injury. However, while dMRI can identify areas of WM disruption post-injury, it is limited in its abilities to interpret those regions of disruption. Lower FA can indicate demyelination, but it can also indicate axonal loss, inflammation, and changes to axonal packing. We collected whole brain magnetic resonance spectroscopy (MRS) to combine with dMRI and give more detail about areas of WM disruption. We scanned 29 TBI patients 2–5 months post-injury and again 13-19 months post-injury, along with 38 well-matched controls. Rarlier analyses identified 2 subgroups based on interhemispheric transfer time (IHTT) collected using ERP (event-related potentials), with differences in cognitive function and WM structural organization. To combine dMRI and MRS, we co-registered the modalities and extracted summaries of 6 measures (FA – fractional anisotropy, MD – mean diffusivity, RD – radial diffusivity, AD – axial diffusivity, NAA – N-acetylaspartate, Cho - choline) within 19 WM bundles. Group differences in NAA corresponded to group differences in dMRI measures, while there was no correspondence between dMRI measures and differences in Cho. Using a partial-F test, we found that a model including MRS measures explained significantly more variance in cognitive outcome than a reduced model including only demographic, dMRI, and IHTT variables. Our results suggest demyelination plays an important role in TBI-related WM disruption, and that multi-modal data improves outcome prediction.
Keywords: MRS, dMRI, tractography, longitudinal
University of Utah, School of Medicine, Salt Lake City, USA
Traumatic Brain Injury (TBI) is an important public health concern. Computerized Tomography (CT) of the head is the gold standard for initially evaluating moderate to severe TBI patients. Adequate and timely evaluation of head CT in TBI is important for immediate management. Current ATLS guidelines for CT evaluation in TBI do not incorporate Brain Trauma Foundation (BTF) guidelines. We aim to investigate a novel method of evaluating trauma head CT. The objective of this study is to compare a new standardized method (ABCDE) of initially assessing head CT with the Advanced Trauma Life Support (ATLS) recommendations in patients with TBI. This is a cross-sectional survey study to assess the performance of a novel imaging interpretation method. Eight CT scans of patients were sent to 18 participants, randomized and blinded to 2 reading methods: the novel method (ABCDE) and ATLS guidelines (ATLS 9th edition). Participants included residents in surgery, emergency medicine, neurology, neurosurgery and medical students. 144 total readings were collected and compared to a gold standard (attending neurosurgeon). Both ABCDE and ATLS methods showed equivalence or near equivalence in identifying abnormal CT (ABCDE sensitivity 85%, ATLS 83%), blood on CT imaging (ABCDE sensitivity 81%, ATLS 83%), presence of basal cistern compression (ABCDE sensitivity 77%, ATLS 76%), correctly identifying the underlying CNS pathology (ABCDE 74%, ATLS 79%) and identifying the need for surgical decompression (ABCDE sensitivity 60%, ATLS 73%). ABCDE was superior in correctly identifying the grade of basal cistern compression (ABCDE 50%, ATLS 37.5%). Limitations include the small sample size and heterogeneity and level of expertise between the two groups. Both methods showed similarity in identifying the need for surgical decompression. Method ABCDE was superior in identifying the grade of basal cistern compression. Being equal, utilization of ABCDE, which incorporates BTF guidelines in CT evaluation, is recommended. Increasing compliance with BTF guidelines is an important determinant of quality measures.
Keywords: Computed Tomography
Oregon Health and Science University, Neurosurgery, Portland, USA
Keywords: Rotterdam Score, Neurologic Deterioration
University of Southern California, Leonard Davis School of Gerontology, Los Angeles, USA
Cerebral micro-bleeds (CMBs) are frequent acute neuroimaging findings in patients with mild traumatic brain injury (mTBI), particularly in older ones. Nevertheless, the clinical significance and potential interpretation of TBI-related CMBs remains controversial partly because their long-term effects are poorly understood. Here we report the development of a neuroimaging-based framework for the longitudinal quantification of CMB effects upon the white matter (WM) of aging mTBI victims. The multimodal neuroimaging approach involves T1 - and T2 -weighted structural magnetic resonance imaging (MRI), fluid-attenuated inversion recovery (FLAIR), diffusion weighted and diffusion tensor imaging (DWI and DTI, respectively) and susceptibility weighted imaging (SWI). Co-registration of neuroimaging volumes is first accomplished four-dimensionally (across space and time) and across modalities. CMBs are then identified at each acquisition time point based on SWI, which is particularly amenable to CMB detection. WM fiber bundle reconstruction is performed via DTI-based streamline interpolation using a novel algorithm for topologically-consistent spatiotemporal mapping of WM connections. WM bundles passing through the vicinity of CMBs (i.e. through umbral and/or penumbral regions) are identified automatically and their properties [fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), etc.] are calculated. The framework is (A) amenable to an arbitrary number of time points at which neuroimaging is performed, and (B) scalable to include an arbitrary number of imaging modalities. The workflow was validated based on a neuroimaging dataset acquired from (A) 10 healthy volunteers aged 22 to 69 who were scanned repeatedly over a 6-week period and (B) 10 mTBI patients aged 21 to 67 scanned acutely and 6 months post-injury. Validation results indicate minimal differences across scans pertaining to WM fiber bundle properties [mean differences across subjects and scans: 1.2 ± 0.3% (FA); 0.8 ± 0.5% (MD); 2.1 ± 0.6% (AD)]. Thus, our analytic approach is sufficiently robst for enabling the study of CMB effects upon the aging mTBI brain.
Keywords: blood-brain barrier, magnetic resonance imaging, micro-hemorrhage, penumbra
University of Southern California, Leonard Davis School of Gerontology, Los Angeles, USA
It has long been known that older adults exhibit increased permeability of the blood-brain barrier, defective neuroinflammatory responses to traumatic brain injury (TBI) and abnormal microglial activation subsequent to the onset of TBI-related cerebral micro-bleeds (CMBs). Nevertheless, the nature of the association between CMBs and the long-term trajectories of grey matter (GM) and white matter (WM) structure and volumetrics remains poorly understood, particularly throughout senescence. Here we report the results of a longitudinal analysis of this association based on neuroimaging scans acquired from 36 aging mTBI victims (age range: 51–78 years; μ = 66.4 years; σ = 5.2 years). Volunteers were scanned with IRB approval both acutely and 6 months post-injury using a protocol involving T1 - and T2 -weighted magnetic resonance imaging (MRI), fluid attenuated inversion recovery (FLAIR), diffusion tensor imaging (DTI) and susceptibility weighted imaging (SWI). In all volunteers, the only acute neuroimaging findings included the presence of CMBs (as obviated by SWI) and CMB-related penumbral edema (as obviated by FLAIR). At follow-up, all volunteers were found to have suffered brain atrophy (mean brain volume change: −2.4 ± 1.2%) and GM loss (mean GM volume loss: −1.6 ± 0.9%). Additionally, cortical thickness was found to have decreased in various regions, including parahippocampal gyrus (mean cortical thickness change: −12.8 ± 4.2%), prefrontal sulcus (−3.3 ± 2.4%), anterior cingulate gyrus (−4.2 ± 2.5%), middle temporal gyrus (−8.3 ± 2.8%) and orbital gyrus (−4.5 ± 1.7%). When interpreted together, our preliminary results suggest that, in older adults, the presence of acute CMBs is correlated with detectable neuroanatomical changes, including decreases in total brain volume, total GM volume as well as cortical thinning across a variety of brain regions. Future research in a larger sample should aim to determine the relationship between age at injury and the significance of the association between acute CMB occurrence and GM loss, particularly in older adults.
Keywords: blood-brain barrier, cerebral micro-bleed, grey matter, susceptibility weighted imaging
Chiba University Graduate School of Medicine, Orthopedic Surgery, Chiba, Japan
Keywords: Diffusion Tensor Imaging, prediction, cervical myelopathy
North Carolina State University, College of Veterinary Medicine/Department of Clinical Sciences, Raleigh, USA
Lesion heterogeneity amongst chronically paralyzed dogs after acute, complete thoracolumbar spinal cord injury (TLSCI) is poorly described. We hypothesized that Diffusion Tensor Imaging (DTI) would define and quantify the continuum of lesion severity in this population. Our objective was to use fractional anisotropy (FA), mean diffusivity (MD) and tractography to quantify lesion severity and investigate associations with hind limb motor function. Dogs were recruited that had suffered clinically complete TLSCI and failed to recover hind limb sensation. They underwent thoracolumbar MRI with DTI and gait analysis. FA and MD were calculated on regions of interest at the lesion epicenter and cranial and caudal to the visible extent of the lesion. Tractography was performed to detect trans-lesional fibers. Gait was quantified using an ordinal scale (OFS) ranging from 0-12 with scores ≥4 indicating weight bearing stepping. Relationships between FA, MD, presence of trans-lesional fibers and OFS were investigated using logistic regression or Wilcoxon rank sum. Twenty-two dogs were enrolled with median OFS of 2 (0–6); 5 had an OFS ≥4. Median values were FA: 0.188 (0.107–0.320), MD: 2.06 × 10−3 (1.33–2.96 × 10−3) at the lesion epicenter, FA: 0.420 (0.391–0.516), MD: 1.52 × 10−3 (1.03–1.87 × 10−3) cranial, and FA: 0.369 (0.265–0.513), MD: 1.31 × 10−3 (0.82–2.08 × 10−3) caudal to the lesion. Four dogs had no trans-lesional fibers. Injury duration (median: 18 months, 3-64) was associated with MD (p < 0.05) but not FA values. FA at the lesion epicenter and presence of trans-lesional fibers were associated with OFS (p ≤ 0.03). These findings suggest DTI quantifies injury severity amongst chronically paralyzed dogs after severe TLSCI.
Keywords: diffusion tensor imaging, canine, chronic paralysis
University of Pennsylvania, Neurology, Philadelphia, USA
Magnetic resonance imaging (MRI) is a powerful tool for visualizing traumatic brain injury-related pathology. Trauma-induced encephalomalacia is frequently identified by Fluid-attenuated inversion recovery (FLAIR) MRI. Often thought to represent gliosis, the underlying microstructural tissue and vascular injury within FLAIR hyperintensities are not well understood. The current study utilized a multimodal MRI protocol to assess mean diffusivity (MD), fractional anisotropy (FA), and vascular alterations within FLAIR normal and abnormal regions in chronic TBI subjects. MRI was performed on TBI subjects (subjects with FLAIR abnormalities, n = 13; subjects without FLAIR abnormalities, n = 8) and healthy controls (n = 15). Subjects received MPRAGE-T1, FLAIR, Diffusion Tensor Imaging (DTI), Arterial Spin Labeling (ASL), and Blood-oxygen level dependent imaging (BOLD) with hypercapnia challenge to assess cerebral vascular reactivity (CVR). MD, FA, cerebral blood flow (CBF), and CVR within regions of FLAIR hyperintensities were compared to normal appearing tissue. All values were converted to z-scores using the pool of healthy controls from the study. There was a significant reduction in FA, CBF, and CVR with a complementary increase in MD within regions of FLAIR abnormalities (p < 0.05 for all comparisons). Grey matter regions without FLAIR hyperintensities did not show a reduction in CBF (p < 0.05). However, normal appearing grey matter in TBI patients with and without FLAIR hyperintensities exhibited significantly reduced CVR relative to controls (p < 0.05). A standardized symptoms questionnaire (RPQ-13) did not reveal differences between TBI subjects with FLAIR hyperintensities versus TBI subjects without FLAIR abnormalities (p > 0.05), consistent with prior studies indicating that FLAIR- abnormalities are poorly associated with clinical outcome. These findings lend insight into the neurobiology underlying FLAIR abnormal regions in chronic TBI, and indicate that CVR is a more sensitive measure of microstructural integrity.
Keywords: MRI/FLAIR, Cerebral Vascular Reactivity, Chronic TBI, Diffusion Tensor Imaging
Boston University School of Medicine, Molecular Aging & Development Laboratory, Boston, USA
Despite growing awareness of links between concussion, traumatic brain injury (TBI), and long-term effects of neurotrauma, the underlying mechanisms in the brain and possible changes in the retina are unknown. In this study, we used a new mouse model of concussion and a state-of-the-art retinal eye scanner to investigate post-traumatic inflammatory responses in the brain and retina. We utilized Ccr2RFP/Cx3cr1GFP mice (Jackson Laboratory) to enable immune cell visualization by class (monocyte, microglia), origin (peripheral, brain, retina), morphology, and location. We used a new closed-head impact injury mouse model (Tagge et al., submitted) that recapitulates key features of human concussion. We used a multimodal adaptive optics small-animal imager (MAOSI) (Physical Sciences, Inc.) for in vivo retinal studies. MAOSI has several channels: adaptive optics optical coherence tomography (AO-OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO) with reflectance and fluorescence channels. Before injury, Ccr2RFP/Cx3cr1GFP mice showed normal cellular distribution and morphological phenotype of microglia in retina and brain. We observed significant increase in microglia in retina and brain post-injury. Moreover, microgliosis in both retina and brain was notable for reactive cellular phenotype transformation with overlapping ramification fields. Closed-head impact injury is associated with similar reactive inflammatory responses and sequelae in both retina and brain. The retina can be used as a “brain proxy” for noninvasive diagnosis, prognosis, and monitoring of neuroinflammation after closed-head injuries.
Keywords: Impact Concussion, Mouse Model, Retinal Imaging, Neuroinflammation
University of California, Irvine, Pediatrics, Irvine, USA
Keywords: gray matter, diffusion tensor imaging, juvenile, water mobility
Keywords: susceptibility weighted imaging, diffusion tensor imaging, microhemorrhage, fractional anisotropy
ULTRA HIGH RESOLUTION SUSCEPTIBILITY WEIGHTED IMAGING AT 3 TESLA
Keywords: susceptibility weighted imaging, high resolution, microhemorrhage, motion correction
CRANIOTOMY ALONE RESULTS IN DEFAULT MODE NETWORK DYSFUNCTION IN THE IMMATURE RAT
University of California Los Angeles, Neurosurgery, Los Angeles, USA
It remains controversial whether rodents with a craniotomy-only are required, or even appropriate to serve as a sham group to control for the effect of surgery after experimental TBI. Published data show significant molecular and behavioral changes that occur due to craniotomy compared to naïve controls, indicating that craniotomy alone likely constitutes a brain insult. We hypothesized that these confounding effects of craniotomy are also accompanied with alterations in neural circuit dysfunction. We tested this by acquiring resting state functional-MRI data from male, 23 day-old Sprague Dawley rat pups at day 4 post-craniotomy (3mm diameter, −3mm, +4mm left-lateral; intact dura) as well as from age-matched, naïve controls with no craniotomy but with time-matched exposure to isoflurane anesthesia (n = 5/group). Imaging data were acquired on a 7 T Bruker spectrometer using a single-shot, gradient-echo sequence, echo/repetition time: 20/1000ms, 300 repetitions, 128 × 128 matrix, 30 × 30mm field-of-view and 1mm slice-thickness). After typical preprocessing of the time-series data, voxel-wise functional connectivity analysis was then performed by calculating Pearson correlation coefficients between all brain voxels. The Root Mean Square of the correlation values for each voxel were calculated as an index of global functional connectivity (fc), clusterized for the presence of 30 voxels ore more. Large scale, significant (p < 0.01) differences in fc were found between the two groups following group ANOVA. Center of mass for the peaks of the clusters that survived statistical correction for multi voxel comparison were located predominantly in regions previously assigned to the rodent default mode network: bilaterally in auditory, temporal association, and primary visual cortex, and in right retrosplenial cortex and hippocampus. These network alterations provide additional evidence to support the idea that craniotomy-alone constitutes a brain injury, and that it might not always serve as an appropriate control. Funding: R01NS27544, R01NS091222, UCLA Easton Labs for Brain Injury, UCLA Steve Tisch BrainSPORT program, UCLA BIRC.
Keywords: Craniotomy, Default mode network, Developing Brain, MRI, Experimental model of TBI
University of Pennsylvania, Neurology, Philadelphia, PA
Traumatic Cerebral Vascular Injury (TCVI) is a universal feature of traumatic brain injury (TBI) and likely contributes to TBI-related disability. Prior studies have shown a significant decrease in whole brain, gray and white matter cerebrovascular reactivity (CVR) in TBI subjects. The relationship between regional CVR measures and brain parenchyma integrity is unknown. To investigate whether TCVI correlates with brain parenchymal volume loss, analysis was performed on chronic TBI subjects and healthy controls. TCVI was measured via Blood Oxygen Level Dependent magnetic resonance imaging (MRI-BOLD) with hypercapnia challenge to generate CVR maps, as well as arterial spin labeling (ASL) to generate cerebral blood flow (CBF) maps. Cerebral volumes were calculated with the Freesurfer image analysis suite. The segmentation maps were imported into MATLAB and superimposed onto CBF and CVR maps to calculate mean values for each region of interest (ROI). Volume measurements were compared to CBF and CVR values within each ROI. CVR was significantly decreased in multiple subcortical ROIs in the chronic TBI subjects relative to healthy controls (p < 0.001), while CBF was not. TBI subjects exhibited significantly more ROIs with reduced volume and cortical thickness (z-score<-2.5, p < 0.05). Group level analysis did not reveal significant volume changes in any particular ROI between the TBI and healthy control groups. Regional volumetric changes, CBF, and CVR were not significantly correlated over multiple ROIs. Cortical gyri with a substantial reduction in thickness (z-score<-2.5) in TBI subjects did not show a concurrent abnormal CBF or CVR reduction (z-score>-2.5). This suggests that regional deficits in CBF and CVR in chronic TBI reflect direct vascular injury and dysfunction, and are not a consequence of neural injury. Vascular and neuronal injuries represent distinct TBI endophenotypes, which warrant independent study to further understand their contributions to TBI.
Keywords: MRI, Cerebral Vascular Reactivity, Chronic TBI, MRI-BOLD, Arterial Spin Labeling
Keywords: External ventricular drain, grading system, ventriculostomy
ANALYSIS OF OUTCOMES OF BARBITURATE THERAPY FOR REFRACTORY INTRACRANIAL HYPERTENSION AFTER TBI
Recent clinical trials have shown inconsistent clinical benefit for decompressive craniectomy or hypothermia to treat intracranial hypertension in TBI. Barbiturate therapy continues to be used as a third-tier option for refractory intracranial hypertension. The aim of this study was to determine if ICP control with barbiturate therapy is associated with improved clinical outcomes in a series of TBI patients treated with barbiturate therapy for refractory intracranial hypertension. Patients with closed head injury who received pentobarbital for control of refractory intracranial hypertension from 1986–2012 were reviewed. Average ICP prior to and during barbiturate coma was analyzed. Outcomes were recorded at 6 months after injury using the Glasgow Outcome Scale. Primary outcomes of this study included ICP control during barbiturate coma and mortality at 6 months after injury. Univariate and multivariate regression analysis for primary outcomes were performed for clinical and imaging parameters. A total of 176 patients were included in this study. ICP was adequately controlled during barbiturate therapy in 47 of 146 patients (32.2%), who received pentobarbital for a minimum of 24 hours. Diffuse injury on initial CT scan (p = 0.04) and higher motor GCS score (p = 0.046) was associated with higher likelihood of ICP control. ICP control was associated with lower rates of mortality (86.5% vs 13.5%, p < 0.001) and higher rates of severe disability in patients with ICP control (67.9% vs 32.1%, p < 0.001). There was no significant difference for the rates of good recovery (p = 0.12), moderate disability (p = 0.21) or persistent vegetative state (p = 0.65) based on ICP control. Uncontrolled ICP during barbiturate coma was significantly associated with 6-month mortality (OR 9.7 95% CI 3.8-24.9, p < 0.001). ICP control with barbiturate therapy was superior in patients with diffuse injury on initial CT scan and higher admission motor GCS scores. Although ICP control with barbiturate therapy in TBI was associated with significant survival advantage, higher rates of severe disability were observed among survivors.
Keywords: barbiturate therapy, pentobarbital coma, glasgow outcome scale
Keywords: multimodal brain monitoring, Cerebral Microdialysis, informatics, Pupillometer
MULTIMODALITY MONITORING IN SEVERE TRAUMATIC BRAIN INJURY: CLINICAL FEASIBILITY AND RELIABILITY
The cornerstone of management of severe traumatic brain injury (sTBI) patients involves the identification and treatment of increased intracranial pressure (ICP). However, the detection and prevention of secondary brain injury requires a multimodality monitoring (MMM) approach that provides data capture of not only ICP, but also brain tissue oxygen (PbtO2), cerebral blood flow (rCBF), and electrocorticography (dEEG). We present our institutional experience with MMM using a four-lumen bolt to facilitate simultaneous measurement of multiple intracranial parameters through a single bur hole. Data was retrospectively collected from consecutive adult sTBI patients admitted to our Neuroscience Intensive Care Unit (NSICU) between April 2015 and March 2017 who underwent MMM. Demographics, injury characteristics, duration of monitoring for each probe, device related complications, and length of stay (LOS) were recorded. Forty patients were included (mean age = 43.0 ± 17.3 SD). Bolt and probe placement occurred a median of 17.7 hours (interquartile range [IQR]: 8.3–16.9 hours) from time of injury. Insertion related minor complications (e.g. small tract hemorrhage, intracranial bone chips, or pneumocephalus) occurred in 37.5% of cases, but none were clinically significant or required intervention. Median total monitoring time was 103.5 hours (IQR 48.8–133.6 hours). Probe reliability analysis showed that ICP data was the most reliable, with data available a mean of 94.8% of the total monitoring time with 1.2% error time. PbtO2 and dEEG data were reliable for >90% of total monitoring time with less than 2% error time. The least reliable measure was rCBF, with 77% of total monitoring time and 17.9% error time. Average NSICU and hospital LOS was 12.2 and 15.2 days, respectively. We conclude that MMM in sTBI patients can be done through a single bur hole without clinically significant complications. Our experience with this alternative to solitary ICP monitoring yields reliable patient data that can support individualized patient care.
Keywords: multimodality monitoring, severe traumatic brain injury, brain tissue oxygen, electrocorticography
University of Louisville School of Medicine, Louisville, USA
Trauma accounts for 4.7 million deaths each year, with an estimated 90% of these occurring in low- and middle-incomes countries (LMICs). Approximately half of trauma-related deaths are due to central nervous system injury, most often in the form of traumatic brain injury (TBI). Odds of mortality following TBI are twice as high in LMICs as in high-income countries. An understanding of TBI in LMICs is essential to mitigate mortality; however, there is a paucity of data on TBI in these settings. This retrospective review of prospectively collected data from Kamuzu Central Hospital, a tertiary care center serving 6 million people in central Malawi, includes all patients admitted from October 2016 through February 2017 with a history of head trauma plus altered consciousness and/or radiographic evidence of TBI. 202 patients met inclusion criteria. 159 (70.1%) were male, with mean age 28.0±16.2. Mean GCS was 11.1±3.8. Road traffic crashes constituted the most common injury mechanism (125, 62.9%). 105, or 52.0%, of patients received a CT scan, with the most common findings being contusions (49, 24.4%), skull fractures (44, 21.9%), and subdural hematomas (15, 7.5%). 61 (30.1%) patients had severe TBI, defined as a GCS of ≤ 8, of whom 10 (16.4%) were intubated, while 5 (8.2%) received tracheostomies. Overall mortality was 26.6%. Of patients who survived, 81.5% made a good recovery, 10.3% were moderately disabled, 6.2% were severely disabled, and 1.4% were in a vegetative state. An adjusted survival time analysis, with failure defined as mortality or poor functional outcome, was done. Female sex was found to be protective. On Cox analysis, deterioration of GCS in the first 24 hours of hospitalization was significantly predictive (HR = 1.25, 95% CI: 1.15, 1.37). Implementation of proven in-hospital interventions for these patients, aimed at preventing secondary brain damage and detecting neurological deterioration in a timely manner, is critical to attenuate TBI-related morbidity and mortality in resource-poor settings.
Keywords: global neurosurgery, low-resource settings, sub-Saharan Africa, epidemiology, low- and middle-income countries, global health
University of Miami, Neurological Surgery, Miami, USA
Acute Lung Injury (ALI) is a common systematic complication of severe Traumatic Brain Injury (TBI). However, the pathophysiologic mechanisms of TBI-induced ALI are not well known. We have previously shown that the inflammasome plays a critical role in TBI-induced secondary pathophysiology and that inflammasome proteins are released in Extracellular Vesicles (EV) after TBI. Here we investigate whether EV-mediated inflammasome signaling contributes to the etiology of TBI-induced ALI. In this study, C57/BL6 mice were subjected to Controlled Cortical Impact Injury (CCI) and brains and lungs were examined for inflammasome activation, pyroptosome formation, and ALI. Also, an adoptive transfer experiment was performed by injecting serum-derived EV from sham and TBI injured mice into healthy mice and lungs were analyzed for inflammasome protein expression and ALI. Our findings indicate that brain and lungs of CCI-injured mice showed a significant increase in the expression of AIM2, IL-1b, caspase-1, IL-18 and HMGB1 acutely after TBI. Moreover, injured lungs also showed evidence of pyroptosis. Lungs of CCI-injured animals demonstrated evidence of ALI as determined by thickening of the alveolar septum, alveolar edema and inflammation, thus resulting in a higher ALI score. Adoptive transfer of serum-derived EV from TBI mice, but not sham mice, into healthy mice induced higher expression of inflammasome proteins in lungs and higher ALI scores. Interruption of this axis by administration of Enoxaparin or an anti-ASC significantly inhibited inflammasome activation and improved ALI scores. In conclusion, our data show that inflammasome activation plays a major role in TBI-induced ALI. These data provide strong evidence for activation of a Neural-Respiratory Inflammasome Axis, and demonstrate that targeting this axis with Enoxaparin or anti-ASC antibody may provide a novel therapeutic approach for neurotrauma-induced ALI.
Funding: Miami Project to Cure Paralysis, NIH grants: R42NS086274, F31 HL132425-01A1
Keywords: Traumatic Brain Injury, Acute Lung Injury
Keywords: hyperbaric oxygen, Severe traumatic brain injury, clinical trial, adaptive design
LOCAL BRAIN TISSUE WATER CONTENT IN TRAUMATIC BRAIN INJURY PATIENTS
Brain edema predicts poor neurological outcomes in traumatic brain injury (TBI) patients, however, it is indirectly assessed by intracranial pressure (ICP) monitoring. Intraparenchymal thermal conductivity-based probes measure local cerebral blood flow but more recently have been used to derive a measure of tissue water (TW) content in the brain (i.e. local cerebral edema). Hypertonic saline boluses lower the TW content measure in neurosurgical patients. However, how this measure responds over time through a patient's ICU course has yet to be established. We investigated whether TW content responds to hyperosmolar and hypothermic therapy administered over time in neurosurgical patients. TBI patients with ICP monitoring placed due to poor neurological status were reviewed. A Camino Bolt (Integra Life Sciences Inc) monitoring system or an extraventricular drain was used. Patients were chemically paralyzed and cooled to a goal temperature of 34–35 degrees Celsius. A QFlow500 probe (Hemedex Inc.) was placed in the hemisphere containing more intact brain tissue according to current guidelines. Patient were monitored for several days. Correlation between TW and ICP, TW and serum sodium, and TW and temperature were calculated. N = 15 patients were included in this study. Results showed that there is a positive correlation between TW and ICP over time (mean r = 0.17, ps <0.001). An ICP of 17–20 mmHg is related to 75–76% TW content. Overall, there is a negative relationship between TW and serum sodium, though this seems to depend on the prior state of the brain, i.e. time from injury. Bolusing with hypertonic saline was related to lower TW content. Finally, there is a positive correlation between TW and body temperature over time for the patients whom were cooled (n = 3). Local TW content is a novel measure that can be used as part of current multimodal neuromonitoring systems to allow for both global and local, targeted treatment of brain injury.
Keywords: Hemedex
WHAT PROGNOSTIC INFORMATION DO TBI FAMILIES NEED AND PHYSICIANS DELIVER? RESULTS FROM A MULTI-CENTER QUALITATIVE STUDY
University of Massachusetts Medical School (UMMS), Neurology, Worcester, USA
Keywords: Shared decision-making, Prognostication, Family-centered care, critical care
Western Regional Center for Brain and Spine Surgery, Las Vegas, USA
Keywords: Thoracolumbar spine burst fractures, acute treatment, minimally invasive lateral corpectomy, ASIA scores, outcomes, complications
Hypernatremia is independently associated with increased mortality in critically ill patients; however, there is limited data on the effect of hypernatremia on TBI patients. The aim of the present study was to evaluate the impact of hypernatremia in patients with severe TBI treated in a neurocritical care unit. Patients with severe TBI treated in a single neurocritical care unit from 1986-2012 were reviewed. Patients with a minimum of three recorded serum sodium values were selected. Patients with a diagnosis of diabetes insipidus or hypernatremia on admission were excluded. Hypernatremia was classified based on the highest serum sodium, and was classified as no hyponatremia (≤ 150 mEq/L), mild (151–155 mEq/L), moderate (156-160 mEq/L) and severe (> 160 mEq/L). The independent effect of hypernatremia on mortality was assessed using a multivariate cox regression analysis. A total of 588 severe TBI patients were studied. Initial GCS was 3/15 in 220 patients (37.4%). The median number of serum sodium measurements for patients in this study was 17 (range 3–190). Mannitol was used in 109 patients (18.5%) and no patient received hypertonic saline. No hypernatremia was seen in 371 patients (63.1%), mild hypernatremia in 77 patients (13.1%), moderate hypernatremia in 50 patients (8.5%) and severe hypernatremia in 90 patients (15.3%). At discharge, 148 patients (25.2%) died. Mild (hazard ratio 3.4, 95% CI 1.94–5.97, p < 0.001), moderate (hazard ratio 4.4, 95% CI 2.47–8.02, p < 0.001), and severe (hazard ratio 8.4, 95% CI 5.13–13.86, p < 0.001) hypernatremia were significant independent predictors of mortality. Significantly lower survival rates were observed for TBI patients with greater degrees of hypernatremia (log rank test, p < 0.001). In this study, hypernatremia after admission was observed in 36.9% of severe TBI patients treated in a neurocritical care unit. In addition to severe hypernatremia, mild and moderate hypernatremia was associated with early mortality. These results highlight additional mortality risk associated with hypernatremia after severe TBI.
Keywords: hypernatremia, severe traumatic brain injury
PHYSICIAN COMMUNICATION STRATEGIES IN EMERGENT BRAIN TRAUMA
Keywords: traumatic brain injury, physician communication, patient decision making
CONTINUOUS, NONINVASIVE OPTOACOUSTIC MONITORING OF CEREBRAL VENOUS BLOOD OXYGENATION IN PATIENTS WITH TRAUMATIC BRAIN INJURY
Universtiy of Texas Medical Branch, Anesthesiology, Galveston, USA
Keywords: optoacoustic O2 saturation, superior sagittal sinus (SSS), jugular bulb O2 saturation, cerebral venous blood oxygenation
Keywords: Polytrauma, hypobaria, oxygen, lung injury, Aeromedical evacuation
University of Glasgow, Queen Elizabeth University Hospital, Glasgow, United Kingdom
Keywords: traumatic brain injury, amyloid, cerebral amyloid angiopathy, chronic traumatic encephalopathy
Minocycline (MIN) has been studied in various TBI models which have been positive. This may be due to the drugs pleiotropic effects targeting microglial activation, reduced cellular apoptosis and inflammation. Minocycline was chosen as the tenth drug for testing by the multicenter consortium Operation Brain Trauma Therapy. The University of Miami site tested Minocycline in our model of fluid percussion TBI. Male Sprague-Dawley rats were anesthetized and underwent moderate fluid percussion (FP; 1.8-2.1atm) TBI or sham surgery. Rats were randomized and administered a bolus (30 mg/kg, IV) of Minocycline followed by 72 h infusion (2 mg/kg/h) or vehicle beginning 15 min after TBI. This protocol produced steady state blood levels mimicking those seen in the successful phase II clinical trial in human SCI. Animal groups were TBI-MIN (n = 15), TBI-Veh (n = 15) or Sham (n = 15). Rats were tested on day 7 post-injury for sensorimotor function. On days 13–21, rats were assessed for cognition 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 sensorimotor 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. MIN appears to provide some improvement on latency with a trend towards TBI-MIN better than TBI-Veh (p = 0.062). There was no significant difference between groups for the probe trial. Repeated measures ANOVA for working memory latency was not significant for group (p < 0.05). We conclude that treatment with Minocycline after FP did not improve sensorimotor but did slightly improve cognitive function. At this time, behavioral findings of Minocycline treatment in the FP model in rats do not support its further testing across models in OBTT, but may merit additional testing in combination therapy, particularly in mild TBI models. DoD W81XWH-10-1-0623 W81XWH-14-2-0018.
Keywords: traumatic brain injury, OBTT, fluid percussion
EVALUATION OF MINOCYCLINE IN THE CONTROLLED CORTICAL IMPACT MODEL OF TRAUMATIC BRAIN INJURY: AN OBTT CONSORTIUM STUDY
University of Pittsburgh, Safar Center for Resuscitation Research, Pittsburgh, USA
Several studies have reported positive effects with minocycline therapy in experimental traumatic brain injury via multiple mechanisms. Minocycline was chosen as the tenth drug tested by the multicenter consortium Operation Brain Trauma Therapy. The University of Pittsburgh site tested minocycline 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 three groups (n = 10/group) and administered a minocycline regimen that mimics steady state blood levels seen in a successful phase II clinical trial in spinal cord injury (single IV bolus 30 mg/kg 15 min after CCI followed by infusion [2 mg/kg/h] for 72hr), CCI followed by vehicle treatments, or sham surgery. Functional outcomes were tested via beam balance and beam walking tests (days 3–5), 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 no significant group effects for beam balance and a significant group effect for beam walk latencies (p < 0.016). None of the injured groups differed from each other. In the beam walking test, only the CCI+Mino group differed from the sham group. A significant group effect (p = 0.0001) was found for the MWM test. While swim latencies for the injured groups differed significantly from shams, they did not differ from each other. There were no group differences in the probe trial. There were no treatment effects on lesion volume or hemispheric tissue loss. We conclude that treatment with minocycline after CCI did not improve function or histopathology. Although biomarker data are pending, these findings do not support further testing of minocycline monotherapy in the CCI model in OBTT. Support: US DoD W81XWH-10-1-0623 W81XWH-14-2-0018.
Keywords: OBTT, Neurobehavioral Function, Controlled cortical impact, Traumatic brain injury
Levetiracetam (LEV) is an antiepileptic drug and Simvastatin (SIM) is a drug used to reduce serum cholesterol. Both drugs have shown neuroprotective effects when tested individually in several pre-clinical models of traumatic brain injury (TBI). Our previous monotherapy studies of these drugs confirmed their neuroprotective efficacy in a model of penetrating ballistic-like brain injury (PBBI) in rats. Based on the monotherapy dose-response profile of each drug, isobolographic analysis was used to construct fixed-dose ratios for a combination therapy of LEV and SIM to identify potential additive or synergistic effects in the rat model of PBBI. Unilateral frontal PBBI (10%) was induced in the right hemisphere of isoflurane anesthetized rats. LEV and SIM were tested with the following dose ratios (LEV/SIM, mg/kg): 6.25/0.01, 12.5/0.02, 25/0.04, 50/0.06, and 100/0.08. Both drugs were administered intravenously (LEV by bolus injection; SIM by infusion) at 30 min and 6 hours post-PBBI. Additional treatments were given daily (LEV 2x/day; SIM 1x/day) for 10 days post-PBBI. The order of administration was counter-balanced. The rotarod task was used to evaluate motor function 7 and 10 days post-PBBI. The Morris water maze was used to evaluate cognitive performance 13–17 days post-PBBI. Brains were collected 22 days post-PBBI for histopathological analysis. There were significant motor and cognitive deficits in all injury groups compared to sham. Isobolic analysis of the results identified sub-additive effects between Levetiracetam and Simvastatin in the PBBI model indicating this drug combination may not be useful for neuroprotective therapy.
Keywords: Levetiracetam, Simvastatin, TBI, Penetrating Ballistic-Like Brain Injury, Rat, Behavior
NEUROPROTECTIVE EFFECTS OF NANO-PULSED LASER THERAPY (NPLT) IN THE RAT FLUID PERCUSSION INJURY MODEL
University of Texas Medical Branch, Anesthesiology, Galveston, USA
Keywords: Fluid percussion injury, Nano-Pulsed Laser Therapy, Fluoro Jade C, Active caspase-3, Sprague-Dawley rats
Despite higher rates of hospitalization and mortality following traumatic brain injury (TBI) in patients over 65 years old, this age group is highly underrepresented in drug development and treatment studies. Worse outcomes in elderly individuals compared to younger adults could be attributed to exacerbated injury mechanisms including oxidative stress, inflammation, blood-brain barrier disruption, and bioenergetic dysfunction. Taurine, an endogenous amino acid and nutritional supplement that functions as an antioxidant, anti-inflammatory, cellular osmolyte, and neuromodulator is neuroprotective in adult rats with TBI. However, its effects on the aged brain have not been explored. Therefore, the objective of our study was to determine whether taurine is neuroprotective in an aged rat model of TBI. Aged male F344 rats (21 months old) were anesthetized and subjected to unilateral controlled cortical impact injury to the sensorimotor cortex (5 m/s, 2.5 mm depth). Rats were then randomized into four groups (n = 9–10 per group) and administered taurine (200 mg/kg, 50 mg/kg, or 25 mg/kg i.p.) or saline 20 minutes post-injury and then daily for 7 days. Sensorimotor functional outcomes were assessed using beam walk and bilateral adhesive removal test at 1, 3, 7, 10 and 14 days post-TBI. Magnetic resonance images were obtained at day 14 post-TBI and analyzed using ImageJ to estimate lesion volumes. Experimenters were blinded to the treatment group for the duration of the study. MRI analysis showed a dose-dependent decrease in TBI lesion volume in taurine-treated groups compared to saline. However, taurine did not significantly improve sensorimotor outcomes. This suggests that while taurine promotes tissue sparing in aged rats following TBI, this may not be sufficient to rescue gross function of the remaining tissue.
Supported by: Funds from NIH (R21 NS091920) and a University of Kansas Lied Basic Science grant to Dr. Harris. The Hoglund Brain Imaging Center is supported by a generous gift from Forrest and Sally Hoglund.
Keywords: Taurine, Neuroimaging, Sensorimotor function
PHENELZINE ADMINISTRATION FOLLOWING TRAUMATIC BRAIN INJURY IMPROVES SYNAPTIC AND NON-SYNAPTIC MITOCHONDRIAL RESPIRATORY FUNCTION
Keywords: Traumatic Brain Injury, Synaptic and Non-Synaptic Mitochondria, LP-Mediated Oxidative Damage, 4-HNE, Phenelzine
PHARMACOLOGICAL INHIBITION OF OXIDATIVE DAMAGE BY THE LAZAROID U-74389G IMPROVES CORTICAL MITOCHONDRIAL FUNCTION FOLLOWING TBI
Traumatic brain injury (TBI) results in hemoprotein breakdown leading to cellular accumulation of free iron. Iron (Fe) can react with hydrogen peroxide (H2O2) and/or lipids resulting in post-traumatic Fe-mediated oxidative damage. The lazaroid U-74389G (desmethylated Tirilazad; TZ), an inhibitor of Fe-dependent lipid peroxidation (LP), was used to protect mitochondrial function following TBI in young adult male rats. Male SD rats received a severe (2.2 mm) controlled cortical impact-TBI. TZ was administered intravenous (iv) at 15 min and 2 hrs post injury (hpi) followed by an intraperitoneal (ip) dose at 8 hpi at the following doses (mg/kg): 0.3 (iv) +1 (ip), 1 + 3, 3 + 10, 10 + 30. Total cortical mitochondria were isolated at 72 hpi and respiratory rates were measured using a Clarke-type electrode. Mitochondrial 4-HNE and acrolein were evaluated as indicators of LP-mediated oxidative damage by quantitative Western immunoblot. At 72 hrs post-TBI injured animals had significantly lower mitochondrial respiration rates compared to Sham. Administration of TZ at the 1 mg/kg dosing paradigm significantly improved mitochondrial respiration rates for State II, State III, Complex II driven State V and RCR compared to vehicle-treated animals. At 72 hrs post-TBI injured animals had significantly higher levels of mitochondrial 4-HNE and acrolein compared to Sham and administration of TZ reduced reactive aldehydes levels compared to vehicle-treated animals. The aim of this study was to explore the hypothesis that interrupting secondary oxidative damage via acute pharmacological inhibition of Fe-mediated LP by TZ following a CCI-TBI would provide mitochondrial neuroprotective effects in a dose-dependent manner. We found that acute administration of TZ to injured rats resulted in improved mitochondrial function and lowered the levels of reactive aldehydes in the mitochondria. These results establish not only the most effective dose of TZ treatment to attenuate LP-mediated oxidative damage, but also set the foundation for further studies to explore additional neuroprotective effects following TBI.
This work supported by NIH/NINDS R01 NS083405.
Keywords: Traumatic brain injury, Mitochondria, Iron-mediated lipid peroxidation, 4-HNE, Acrolein
EXPRESSION PATTERNS OF HEAT SHOCK PROTEIN 27, 70 AND 90 DEPENDING ON TIME COURSE IN CONTUSIVE SPINAL CORD INJURED RATS
Korea Univ. College of Health Science, Dept. of Health Science, Seoul, Korea South
Traumatic spinal cord injury (SCI) causes sensory and motor dysfunction below the injured level. A poor intervention in the primary damage period may result in permanent handicap. Heat Shock Proteins (Hsp), proteins families of the molecular chaperone, are induced by cell stressful conditions. Recently, Hsp70 has been found to play an important role for neuroprotection relevance to cell death after SCI. However, the underlying mechanisms of the contribution Hsp70 on neuroprotection after SCI remain unclear. Moreover there is a lack of study about the role of Hsp27, and 90 following the damage of spinal cord. Thus, we investigated the changes of Hsp27, 70, and 90 expression patterns in spinal cord after injury. The contusive SCI was made by New York University (NYU) impactor at the T 10 level from 12.5 mm height under anesthesia. To assess the changes of Hsp27, 70, and 90 expressions was performed immuno-blotting in rostral, epicenter, caudal and remote site at 4 h, 8 h, 12 h, 24 h, 3 d, and 7 d after injury. Hsp27 expression was slightly increased in all spinal segment from 4 h to 7 d after SCI. A significant change was shown in 7 days after damage. Hsp70 was increased immediately after damage in the epicenter but it was returning back around basal level. In contrast to Hsp27 and 70, the expression of Hsp90 was decreased to less than basal level until 7 days after SCI. Our findings suggest that in studying mechanisms of Hsp, a critical time point for neuroprotection is different depending on target Hsp families.
This study is supported by NRF funded by the Ministry of Science, ICT, and Future planning Grant (2014M3C1B2048632)
Keywords: Heat shock protein, HSP, Spinal cord injury
There are currently no FDA-approved pharmacotherapies able to prevent the devastating neurological deficits caused by traumatic brain injury (TBI). Due to complex secondary injury mechanisms which occur following TBI, combinational therapies should enhance neuroprotection above single agents alone. Two pivotal secondary injury mechanisms, lipid peroxidation (LP) and mitochondrial dysfunction, peak 72 h following injury. Therefore, continuous infusion of neuroprotective agents over the first 72 h following injury may also be required for optimal neuroprotection. Mitochondria are essential mediators of the TBI secondary injury cascade, including lipid peroxidation. As such, mitochondria are promising therapeutic targets for the prevention of cellular death and dysfunction following TBI. Phenelzine (PZ), an anti-depressant capable of scavenging LP-derived neurotoxic aldehydes, and cyclosporine A (CsA), an immunosuppressant capable of inhibiting the mitochondrial permeability transition pore (mPTP), have previously been shown to be partially neuroprotective, including protection of mitochondria, following TBI. This is the first study to evaluate the effects of a continuous 72 h infusion by subcutaneous osmotic pump of PZ, CSA, or PZ + CsA on mitochondria 72 h following severe controlled cortical impact injury in rats. Although neither PZ, CsA, nor PZ + CsA were able to improve mitochondrial respiratory function in the first dosing paradigm evaluated (PZ: 10mg/kg s.c. 15min post-injury +10mg/kg/day/72 h; CsA: 20mg/kg i.p. 15 min post-injury +10 mg/kg/day/72 h), individually PZ and CsA, but not the combination PZ+CSA, were able to attenuate modification of mitochondrial proteins by LP-derived neurotoxic aldehydes. However, it is premature to conclude that combining aldehyde scavenging with inhibition of mPTP is not neuroprotective following TBI. Therefore, kinetics studies, assessment of additional outcome measures, and evaluation of additional PZ mechanisms of action, such as monoamine-oxidase inhibition, will be pursued in order to optimize dosing paradigms.
This research is funded by NIH-NINDS 5R01 NS083405 and 5R01 NS084857. JR Kulbe is currently supported by NINDS F30 NS096876
Keywords: Mitochondria, Combinational Therapy, Neurotoxic Aldehydes, Lipid Peroxidation, Phenelzine, Cyclosporine A
EVALUATION OF MINOCYCLINE IN THE WRAIR PBBI MODEL: STUDIES FROM THE OPERATION BRAIN TRAUMA THERAPY (OBTT) CONSORTIUM
Minocycline was the 10th drug selected for testing by the OBTT consortium. Minocycline is a broad-spectrum tetracycline antibiotic shown to be neuroprotective in neurodegenerative diseases and experimental models of stroke, spinal cord injury (SCI), and brain trauma. It has been reported to inhibit microglial activation, prevent oligodendrocyte and/or neuronal apoptosis, decrease oxidative damage, and reduce inflammation. A phase II clinical trial for acute SCI found Minocycline safe and tended to improve several motor recovery outcomes. The WRAIR site evaluated the effectiveness of Minocycline in the penetrating ballistic-like brain injury (PBBI) model. PBBI (10%) was performed unilaterally in the right hemisphere of anesthetized rats. A 30 mg/kg Minocycline loading bolus was administered intravenously 15 mins post-PBBI followed by continuous 72-hour infusion (2 mg/kg/hr). This protocol was shown in PK studies by our group to produce steady state blood levels ranging between 5–10 mcg/mL, mimicking those in the successful phase II clinical trial. Groups consisted of TBI-MIN (n = 14), TBI-Veh (n = 19) or Sham (n = 16). Motor and cognitive performance were assessed using rotarod (days 7 and 10 post-PBBI) and Morris water maze (MWM, days 13–17 post-PBBI). On day 21, brain tissue was processed for histology. Motor testing showed significant injury-induced deficits versus sham (p < .05). Overall rotarod latencies were reduced by 51.8 ± 5.5% (TBI-Veh) and 47.4 ± 8% (TBI-MIN) vs. sham. No significant therapeutic effects were detected on the rotarod task. MWM results revealed significant injury-induced deficits with latencies to locate the submerged platform increased by 56.8 ± 14% (TBI-Veh) and 52.9 ± 14.8% (TBI-MIN) versus sham. Positive trends towards improved memory retention (probe trial) and reduced lesion size were observed, but not significant. Current findings do not support further testing of Minocycline using this dosing regimen. However, additional testing may be warranted using higher doses and/or extended treatment durations in order to overcome peak microglial and inflammatory responses in the PBBI model. Supported by U.S. Army Grant W81XWH-10-1-0623.
Keywords: traumatic brain injury, OBTT
MITOCHONDRIAL TRANSPLANTATION FOLLOWING CONTUSION SPINAL CORD INJURY
University of Kentucky, Spinal Cord & Brain Injury Research Center, Dept of Physiology, Lexington, USA
The current study tested whether transplantation of exogenous mitochondria into the contused rat spinal cord results in 1) their incorporation into various host cell types, 2) improving overall bioenergetics of host cells, and 3) increasing long-term functional neuroprotection. For visualization of transplanted mitochondria, we used transgenically modified PC12 cells in which mitochondria were labeled with tGFP, and for clinical relevance, we also used mitochondria isolated from rat soleus muscle. Freshly isolated tGFP (50, 100 or 150 μg/cord) or muscle (50 or 100 μg/cord) mitochondria were microinjected into the penumbra of severely contused spinal cords within 1 hr after spinal cord injury (SCI) at L1/L2 (250 kdyn using IH Impactor) in adult female Sprague-Dawley rats. Depending on outcome measures, they survived 24 hr, 48 hr, 7 days or up to 6 weeks. Results showed that transplantation of either tGFP or muscle mitochondria significantly maintained bioenergetics of injured spinal cord tissues 24 hr after injury, with maximum effects at the 100 ug dosage. Confocal imaging showed prominent rostro-caudal spread of exogenous tGFP mitochondria from injection sites after 24–48 hr that dissipated by 7 days. At the earlier time points, tGFP mitochondria co-localized conspicuously with microglia/macrophages and endothelial cells, with less incidences in astrocytes and oligodendrocytes, and none in neurons. Assessments of hindlimb functional recovery (BBB-LRS) and paw withdrawal latencies (Von Frey hair) over 6 weeks after transplanting 100ug tGFP or muscle mitochondria showed no significant differences in over-ground locomotion or mechanical hypersensitivity compared to vehicle-injected injured groups. Morphometric analyses further showed no differences in grey or white matter tissue sparing. In summary, intraspinal injections of mitochondria after contusion SCI improved cellular bioenergetics acutely, but that such maintenance of respiration did not translate into improved long-term functional neuroprotection. Supported by NIH/NINDS R21NS096670 (AGR), NRSA F31NS093904 (JLG).
Keywords: Respiration, Enzyme activities, Hindlimb locomotor recovery, Von Frey hair
Bay Pines VA Healthcare System, R&D, Bay Pines, USA
In the US, approximately 1.7 million people sustain a traumatic brain injury (TBI) each year. In our military and athletes, TBI remains a major cause of morbidity and mortality. The majority of TBIs are mild, but can result in deleterious cognitive effects for which there is no effective treatment. tBHQ is an activator of the transcription factor, Nrf2, and has been shown to induce production of several neuroprotective factors. Pifithrin-α compounds, which inhibit p53, demonstrated neuroprotective effects in an in vitro excitotoxicity model and an in vivo head injury model. To evaluate the effects of a repetitive physical injury on a neuronal cell line and characterize the potential protective effects of treatment, we used an in vitro “injury in a dish” model. tBHQ plus the sulfur containing inhibitor, PFT-α, produced significantly improved neuronal function over single treatments measured with an MTS assay. We investigated treatment with tBHQ, oxygen containing inhibitor, PFT-α (O), and a combination to determine whether activation of Nrf2 and inhibition of p53 would provide a synergistic neuroprotective effect. Observations indicated that treatment with tBHQ plus PFT-α (O) improved overall neuronal survival and neurite health, when compared to untreated cells or those that received only one treatment. In addition, qRT-PCR array data has identified differentially regulated genes in response to combination treatment. Anti-inflammatory heme oxygenase 1 mRNA was increased, which may account for some neuroprotective effects. This suggests that simultaneous activation of Nrf2 and inhibition of p53 produces enhanced neuroprotective effects in response to neuronal injury and could represent a novel treatment in response to head injury.
Keywords: transcription factors, in vitro injury, Nrf2, p53, synergistic treatment
Following TBI, the brain undergoes serious pathological changes, which compromise neuronal survival and plasticity, and contributes to numerous functional deficits. Thus far, published studies have found that two natural products, curcumin (a dietary product) and melatonin (the major pineal hormone), have beneficial effect for TBI recovery. Recently, we have developed a novel hybrid compound derived from curcumin and melatonin, ZCM-I-1. In a mouse transgenic model of Alzheimer's disease, this novel compound showed significant effect in reduction of oxidative stress and microglial activation, enhancement of mitochondrial function and synaptic plasticity. Based on this observation, we explored the effect of ZCM-I-1 for TBI treatment. In this study, adult male Sprague-Dawley rats were subjected to a moderate cortical impact injury. At 30 minutes, 1 day, and 2 days following injury, 50 mg/kg of ZCM-I-1 was administrated i.p. for a total of 3 doses. Sensorimotor functions were tested using beam walking, rotarod, and neurological score testing methods. Cognitive functions were assessed using Morris Water Maze latency and probe trial tests. Animals were sacrificed at 2 or 28 days post-injury in order to assess acute and chronic response to treatment, respectively. Brain tissues were processed for histological examination to assess injury-induced degenerative neurons with FJB staining. Cortical lesion volume and neuronal cell survival were also assessed. We demonstrated that following TBI, ZCM-I-1 administration significantly reduce the number of degenerative neurons in the injured cortex and hippocampal neurons. Injured animals received ZCM-I-1 treatment also had significant improvement in both motor and cognitive functions. Our data suggest that our novel compound has neuroprotective effects for TBI. Further study examining the effect of ZCM-I-1treatment on post-TBI neuroplasticity is ongoing.
Keywords: traumatic brain injury, neuroprotection, cell death, cognitive function, neuroplasticity
NEUROPROTECTIVE AND COGNITIVE BENEFITS OF ACUTE PHOSPHODIESTERASE 4B INHIBITION AFTER TRAUMATIC BRAIN INJURY
University of Miami Miller School of Medicine, Neurological Surgery, Miami, USA
Finding molecular targets to attenuate traumatic brain injury (TBI)-induced pathologies and cognitive deficits is of critical importance for the estimated 3–5.3 million people currently living with TBI-related disabilities in the United States. Previously, we reported that acute inhibition of the cAMP-hydrolyzing enzyme, phosphodiesterase 4B (PDE4B), reduced inflammation and cortical contusion volume after TBI. However, it was unknown whether the therapeutic benefits of acute PDE4B inhibition led to reduced behavioral deficits, neuronal loss and atrophy after TBI. To determine whether acute PDE4B inhibition attenuated TBI-induced behavioral deficits and pathology, adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury (2 ± 0.2 atm) and were then treated with a PDE4B-selective inhibitor, A33, or vehicle for up to 3 days post-surgery. Animals were assessed from 1 to 6 weeks post-surgery for forelimb placement asymmetry, contextual fear conditioning, water maze performance and spatial working memory. We found that A33 treatment significantly improved contextual fear conditioning and water maze retention at 24 hrs post-training. However, this treatment did not rescue sensorimotor or working memory deficits. At 2 months after surgery, atrophy and neuronal loss were assessed. A33 treatment significantly reduced neuronal loss in the pericontusional cortex and hippocampal CA3 region. This treatment paradigm also reduced cortical, but not hippocampal, atrophy. These results suggest that acute PDE4B inhibition may be a viable treatment to reduce pathology and memory deficits after TBI. Altogether, the multifactorial and beneficial effects observed with A33 treatment support the use of PDE4B inhibitors as an anti-inflammatory, and possibly neuroprotective, treatment strategy for TBI.
This work was supported by NINDS F31 NS089351 and R01 NS056072, NIMH R44 MH091791 and The Miami Project to Cure Paralysis.
Keywords: PDE4B, cAMP
Indiana University School of Medicine, Dept. Neurological Surgery, Indianapolis, USA
Our previous studies showed that ferulic acid-glycol chitosan (FA-GC), a nanoparticle, or bisperoxovanadium (bpV), an inhibitor of phosphatase and tensin homolog (PTEN), promoted neuroprotection and functional recovery after a contusive spinal cord injury (SCI) in adult rats. Here we investigated whether a combination of the two would maximize the effect on neuroprotection. A moderate contusive SCI was performed at the 10th thoracic level using the Louisville Injury System Apparatus (LISA, n = 61). FA-GC (20 mg/kg), bpV (400 ug/kg), or their combination was injected through the jugular vein after SCI. The bpV was injected intraperitoneally daily for an additional 6 days in the bpV and combination groups. Rats received Basso-Bresnahan-Beattie (BBB) locomotor rating scale, transcranial magnetic motor evoked potential (tcMMEP), and metabolic cage assessments for 6 weeks post-injury. All rats were sacrificed at 7 weeks post-injury. Our BBB results showed significantly improved locomotor function in the combination group compared with the vehicle group, and moderately better motor function compared with either of the single treatments. The tcMMEP showed the highest response rate in the combination group (95%) compared with the FA-GC (70%), bpV (69%), or vehicle group (60%). The metabolic cage study showed significantly decreased void volume in the combination, FA-GC, and bpV groups, and significantly increased void frequency in the combination and FA-GC groups compared with the vehicle group. Bladder size was significantly reduced by 24%, 21%, and 13% in the combination, FA-GC, and bpV groups, respectively. The combination also significantly reduced the lesion area and increased the white matter sparing. All these data suggest that the combination of FA-GC and bpV promotes greater motor and bladder recoveries than either treatment alone following a moderate contusive SCI in adult rats.
Supported by DOD W81XWH-12-1-0562 and ISDH13679 (XW).
Keywords: Spinal cord injury, contusion, FA-GC, bpV
Despite great research efforts, there is still no established effective treatment to improve recovery following spinal cord injury (SCI). In part, this reflects incomplete understanding of the complex secondary pathobiological mechanisms involved and the historical emphasis on targeting single injury mechanism. There are many processes that contribute to the pathophysiology of SCI, such as neuronal cell death, axonal damage, vascular disruption, neuroinflammation, among others. It is increasingly clear that effective clinical strategies will require multi-functional drugs. MicroRNAs (miRs) are small (20–23 nucleotide) non-protein-coding RNAs that negatively regulate target gene expression at the posttranscriptional level by binding to their mRNAs and inducing its degradation and/or inhibiting translation. As single miRs can simultaneously modulate the expression of various proteins in multiple pathways, miRs are attractive candidates as upstream regulators of the secondary SCI progression. Here we examined miRs expression changes in the injured spinal cord using a moderate contusion SCI model in mice. We observed rapid and persistent up-regulation (approximately 10-fold) of miR-711, in response to specific post-traumatic transcriptional activation. This was associated with down-regulation of the pro-survival protein Akt, a target of miR-711, with sequential activation of GSK3α/β and pro-apoptotic BH3-only molecules PUMA. Moreover, inhibition of miR-711 attenuated the injury-induced down-regulation of Akt and sequential activation of GSK3ß and pro-apoptotic Bcl2 family members. Furthermore, Angiopoietin-1, a predicted target of miR-711, is decreased in the injured spinal cord and elevated by inhibition of miR711. Importantly, miR711 inhibitors reduced neuronal loss and axonal damage, rescued epicenter blood vessels, and improved recovery of motor function and coordination. Together, our data indicate that miR-711 elevation after SCI serves to several endogenous neuroprotective pathways, leading to neuronal cell death and axonal and blood vessel damage. Thus, miR-711 represents a potential therapeutic target that may lead to novel, effective clinical therapeutic strategies.
Keywords: MicroRNA 711, spinal cord injury, neuroprotection, locomotor function
TESTING THE EFFECT OF PROTEIN KINASE INHIBITORS ON TRAUMATIC BRAIN INJURY-RELEVANT CELL-BASED MODEL OF TAU HYPERPHOSPHORYLATION
Traumatic brain injury (TBI) increases the subsequent risk of chronic neurodegenerative disorders, including Alzheimer disease and chronic traumatic encephalopathy (CTE). Tauopathies are neurodegenerative disorders that are related to the abnormal aggregation of hyperphosphorylated tau protein into oligomers. It was found in both human and animal model of TBI, Tau is also hyperphosphorylated post-injury. However, the biological mechanism through which tau get hyperphosphorylated need further elucidation. In this study, okadiac acid (OA, a protein phosphatase PP1/PP2A inhibitor) was used to induce Tau hyperphosphorylation and oligomerization as model for TBI. This model was used to set up a screening method for studying various protein kinase inhibitors on tau hyperphosphorylation using rat primary cortical culture (CTX) and mouse neuroblastoma (N2A) cells. The inhibitors used were pan kinase inhibitors (k252a and STS), GSK3b inhibitors (LiCl, AR-A014418 and A-1070722), CDK5 inhibitor (Roscovitine), Calcineurin inhibitor (Cyclosporin), Calmodulin antagonist (Calmidazolium), Casein kinase II inhibitor (tTBB), fyn/src kinase inhibitor (Saracatinib) or calcium chelators (EGTA and EDTA). The phosphorylation and oligomerization of Tau protein was studied by western blotting. Identification of tau oligomers was achieved by antibodies that detects total tau at 102–140 (DA9). Phospho-specific epitopes studied were pSer202/pThr205, pThr181, pSer202, pSer396/pSer404, pThr231. Treatment of OA for 24 hours achieved tau hyperphosphorylation at multiple TBI-related phospho-epitopes. In N2A cells, treatment with OA lead to the formation of tau oligomers observed at ∼170 kDa, which were completely reversed when cells were pre-treated with tTBB. The hyperphosphorylation was partially blocked by EDTA, EGTA and k252a and completely inhibited by tTBB and cyclosporine. As for CTX cells, LiCl, AR-A014418 and A-1070722) were the most potent inhibitors of Tau hyperphosphorylation followed by tTBB and roscovitine. Identification of protein kinases that inhibit tau hyperphosphorylation in TBI can form the basis for screening of compounds and could provide potential therapeutic targets for the treatment of TBI-induced tauopathy-related neurodegenerative diseases.
Keywords: Tauopathy, Tau hyperphosphorylation, TBI, Kinase Inhibitors, Cell culture
TARGETING KCNQ CHANNELS TO TREAT MOTOR AND SENSORY DYSFUNCTION AFTER SPINAL CORD INJURY
Traumatic injury to the spinal cord results in acute necrosis and delayed secondary expansion of neurological damage, and leads to a lifetime of paralysis, sensory dysfunction, and chronic pain. Excessive excitation is a primary source of neural injury evoked by various insults, causing neuronal cytotoxicity, and slowly expanding lesions. The proposed studies attempt to reduce persistent hyperexcitability of neurons during acute phases of spinal cord injury (SCI), thereby preserving neurological function that would be lost during the chronic stage. KCNQ/Kv7 channels are abundant in spinal neurons and axons, controlling their excitability. Retigabine, an FDA-approved drug for anti-epilepsy that specifically opens KCNQ/Kv7 channels, could be a plausible treatment to reduce SCI-induced pathology. We produced contusive SCI at T10 in adult, male rats, which then received 10 consecutive days' treatment with retigabine or vehicle 3 hours or 3 days after contusion. Two different concentrations and two different delivery methods were used. With pumps (250 mg/kg/hr) delivery 3 hours after contusion, retigabine promoted recovery of locomotor function, not intraperitoneal delivery. Remarkably, retigabine delivery in both method significantly attenuated the development of mechanical stimuli-induced hyperreflexia (hindlimb and torso) although there were no significant difference in thermal threshold. Retigabine delivered by pump 3 days after contusion only significantly attenuated the development of mechanical hypersentivity, no effect on locomotor function. Finally, we found early application of retigabine protect gray matter, not the white matter after SCI. Our result indicates that very early opening of KCNQ/Kv7 channels promotes locomotor function recovery and preemptively mitigates development of neuropathic pain following SCI.
Keywords: KCNQ/Kv7 channels, locomotor function, chronic pain, spinal cord injury, T10 contusion
ESTROGEN REDUCES INTRACELLULAR CALCIUM AFTER MILD RAPID STRETCH INJURY IN ADULT RAT PRIMARY CORTICAL NEURONS IN VITRO
Traumatic brain injury (TBI) initiates numerous processes that can lead to neuronal injury, behavioral/cognitive dysfunction, seizures and neurodegenerative diseases (e.g. Alzheimer's, Parkinson's) Estradiol has neuroprotective effects in models of experimental traumatic and/or ischemic brain injury. In the present study, we examined the effects of 17-beta estradiol (E2) on intracellular calcium (Ca2+) and neuron viability in adult rat brain-derived primary cortical neuron or hippocampal cells subjected to one of three levels of rapid stretch injury (RSI), an in vitro model that replicates many features of TBI in vivo using a model 94A cell injury controller II. Rat hippocampus and cerebral cortex were sliced at 0.5 mm and digested with papain at 30°C for 30 min. and triturated to release cells. The cells were plated onto poly-l-lysine-coated 2-mm-thick Silastic bottom 25-mm-diameter Flex Plate wells. Rat neuron cells in Neurobasal _A/B27+ bFGF medium were cultured to confluency in 95% air, 5% CO2 at 37°C and then were subjected to mild RSI (30, 40 psi for 50 ms) and treated with vehicle or E2 (80nM) for 30 min immediately post-injury. 1,3,5 and 24 hours post-RSI, Intracellular Ca2+ levels were measured with Fura-4/AM and the neuron viability was also assessed with P.I and Hoechst. Mild RSI increased intracellular Ca2+ levels and reduced neuron survival. Post-injury E2 treatment significantly reduced stretch-induced increases in Ca2+ influx and neuronal death assessed 1 hr after 30 psi RSI. Our results suggest the E2 improved of neuronal cell survival by reducing intracellular Ca2+ after mild stretch injury.
Supported by the Moody Project for Translational Traumatic Brain Injury Research
Keywords: Adult rat brain primary cortical neuron, Rapid stretch cell injury, Intracellular calcium, Estrogen, Neuron cell survival
The benefit of early enteral nutrition in the care of critically injured patients is well established. Further benefits are derived by administering immune-enhancing formulas including glutamine, arginine, nucleotides, and omega-3-fatty acids. Despite this recognized standard of care following trauma, the application of clinical nutrition for individuals with spinal cord injury (SCI) remains controversial and poorly defined. Adult male Wistar rats received a 300kdyn T3-SCI or surgical control. Three days post-injury, both groups underwent in vivo experimentation to quantify mesenteric and duodenal blood flow in response to enteral delivery of low (10 mM) or high (100 mM) doses of arginine or glutamine. In accordance with previous data, mean arterial pressure (MAP) and superior mesenteric artery (SMA) irrigation of the viscera were significantly lower in T3-SCI rats. In T3-SCI rats, 30 or 60 minutes of enteral supplementation with either 10mM or 100mM arginine provoked a reduction in local duodenal blood flow while SMA flow and MAP remained unchanged. This reduction in local duodenal blood flow was significantly lower than control animals. Conversely, enteral delivery of 10 mM glutamine provoked a significant elevation in local duodenal blood flow at 30 min while SMA flow and MAP remained unchanged. Control animals did not demonstrate a significant elevation in local duodenal blood flow until 60 min. The 100mM dose of glutamine provoked a reduction in local duodenal blood flow in T3-SCI rats while remaining unchanged in control rats. The reduction in local duodenal blood flow of T3-SCI rats was significantly lower than controls. Our data suggest that unlike glutamine supplementation, the microvascular benefits derived from enteral arginine supplementation are not realized in the SCI individual. Therefore, accepted standards of care for maintaining GI health such as administering pharmaconutrients, such as arginine, that require active transport but do not provide a local energy substrate for the gut may be detrimental to GI integrity in the SCI population. Support: CH Neilsen #295319
Keywords: Mesenteric blood flow, Gastrointestinal, Postprandial hyperemia, Enteral nutrition
Roskamp Institute, Sarasota, USA
Keywords: PTSD, mTBI, Fear Memory, Depression, Mouse model, Closed Head Injury
UCLA, Neurosurgery, Los Angeles, USA
Cognitive impairments and emotional liability are common long-term consequences of Traumatic Brain Injury (TBI). Increasing prevalence of comorbid TBI and Post-Traumatic Stress Disorder (PTSD) emphasizes an urgency for a better understanding of how injury affects interactions between sensory, cognitive, and emotional systems that may underlie maladaptive responding. We have previously shown changes in auditory-emotional network activity and enhanced contextual fear learning following white noise fear conditioning early after TBI, prior to the resolution of typical physical symptoms. In the current study, we asked whether TBI would have chronic effects on auditory fear learning and responses to novel stimuli. Four weeks following either mild-moderate lateral fluid percussion injury (FPI) or sham surgery, adult male rats were fear conditioned to either white noise or unsignaled shocks. All groups were tested for contextual fear memory and context extinction then subsequently tested for fear responses to either pure tones or white noise auditory stimulus trials in a new context. While FPI did not impact freezing across acquisition trials, FPI led to increased shock reactivity if white noise preceded the shock. As expected, unsignaled conditioned groups had greater contextual fear relative to noise-shock groups. However, FPI noise-shock animals froze more to the context than respective shams, consistent with our previous findings. During white noise cue testing, FPI noise-shock rats had increased freezing on the first trial of white noise cue testing compared to respective shams. Interestingly, when presented with novel pure tones FPI noise-shock animals displayed robust fear to the novel, untrained auditory stimulus compared to noise-shock shams. These data indicate an injury-induced increase in auditory stimulus generalization, and a unique phenotype chronically following diffuse TBI. This novel finding illustrates both a cognitive impairment and increased fear in the chronic phase after TBI, where stimulus generalization may underlie maladaptive fear and hyperarousal common to comorbid TBI-PTSD.
Keywords: amygdala, learning and memory, fear, PTSD, fear generalization
VA Boston Healthcare System/Boston Univ Sch Medicine, Psychiatry and Pharmacology, Boston, USA
The high rates of comorbid TBI and PTSD diagnosed in veterans in recent years have resulted in increased research investigation into these conditions and putative treatments. In this study, mild traumatic brain injury (mTBI) and posttraumatic stress disorder (PTSD) were modeled in male C57BL/6 mice in order to determine the comorbid effects of mTBI and PTSD on cognition, behavior and neuroinflammation within 3 weeks of injury. Mice with mTBI induced by fluid percussion injury (FPI) at one of two intensity levels (0.7 and 1.7 atmospheres), and also sham FPI mice, were subsequently fear conditioned (FC). FC was used to model aspects of PTSD and fear cues and contexts produced near-complete freezing behavior. Mice were then assessed for novel object recognition and fear retention/extinction. Novel object recognition was impaired in FPI groups compared to sham FPI and naive controls. Re-exposure to the fear cue and context several days after fear acquisition showed the retention of freezing behavior in FPI/FC and sham FPI/FC groups. Following behavioral testing, brains were harvested to for immunohistochemical (IHC) studies. IHC staining of activated macrophage/microglia marker CD68, and pro-inflammatory marker TNF-α produced evidence of neuroinflammation in cortical, hippocampal and subcortical brain regions as a consequence of FPI/FC (vs. controls). IHC staining of beta-amyloid precursor protein was used as a measure of diffuse axonal injury and this protein was induced in several regions in FPI/FC groups. This FPI/FC mouse model shows translational efficacy as a model for mTBI/PTSD and produces subacute cognitive impairment and fear retention accompanied by neuroinflammation and axonal injury in cortical, subcortical and hippocampal regions. This mouse model can serve as a platform to examine longer term behavioral and neural effects and potential therapeutics. Supported by a VA Merit Award to GBK and Gordon Project/C.S.C./U.W.S.F. fellowships to LW and TG.
Keywords: fluid percussion injury, fear conditioning, neuroinflammation, comorbidity
the First Affiliated Hospital of Nanjing Medical University, Neurosurgery, Nanjing, china
Extra-cranial organ injury is one of severe complications in patients with traumatic brain injury (TBI) and is a predictor of poor clinical outcome, involving in increased apoptosis, enhanced inflammatory response, and excessive oxidative stress in extracranial organs. Autophagy is known as a primary homeostatic process that can promote cell survival under stress. Thus, we hypothesized that autophagy could be an effective strategy for prevention of cell death in TBI-induced stress damage to extracranial organs. TBI can lead to lung and intestine injury, and induce autophagy in lung tissue and mitophagy in the intestine tissue in our model. Because extracellular signal-regulated kinase 1/2 (ERK1/2) is known to play an important role in regulating autophagy, we investigate the effect of the ERK1/2 signaling pathway on autophagy in extracranial organs after TBI by using PD98059 (ERK inhibitor). Autophagy or mitophagy was assessed by electron microscopy (EM) and biochemical evidences(western blot, immunobiochemical staining). Rats were preconditioned with autophagy promoter rapamycin or inhibitor 3-methyladenine before they were challenged with TBI. We found that autophagy can suppress apoptosis, inflammation and oxidative stress, which were activated after TBI. Moreover, autophagy was mediated by the ERK1/2/mTOR/Stat3 signaling pathway in the lung and mitophagy was mediated by the ERK1/2/Nrf2/HO-1 signaling pathway in the intestine after TBI, which may serve to reduce the stress injury and improve intestinal or pulmonary function. It appears that autophagy plays a protective role in TBI-induced stress damage to extracranial organ and this effect may be enhanced by moderately improving autophagy level. Meanwhile, modulating ERK1/2 signaling pathway may be important for the prevention and treatment of of symptoms associated with TBI.
Keywords: TBI, Extra-cranial organ injury, Autophagy, oxidative stress, inflammatory response, apoptosis
Support: NIH-NIGMS P20GM109089.
Keywords: Transcranial Direct Current Stimulation, Metabolism, Vascular Tone, Cerebral Microcirculation
A COMBINATION OF ENVIRONMENTAL ENRICHMENT AND NICOTINAMIDE AFTER TBI IN JUVENILE RATS LEADS TO TRANSFER EFFECTS IN COGNITIVE TASKS
Few effective therapeutic interventions have demonstrated the ability to mediate behavioral deficits following damage in juvenile populations. Even fewer have shown long-term beneficial effects. Most treatment regimens consist of single treatments, whereas combinational therapies may serve to augment rehabilitation. Utilization of a combined treatment may not only maximize recovery, but also provide a sustained effect in the absence of the therapy. The current study used a combination of continuous environmental enrichment (EE) and a 500mg/kg dose of nicotinamide (NAM) given three times following a bilateral controlled cortical impact (CCI) or sham surgery on post-natal day 28 rats. After surgery, animals were divided into 5 groups: CCI+EE, CCI+NAM, CCI+NAM+EE, CCI+noEE+noNAM, and sham no treatment. Rats were tested on motor (Foot Fault and Beam Walk) and cognitive tasks (Morris Water Maze and Radial Arm Maze). Results showed that the combinational therapy group performed similar to the sham group in both motor tasks. There was also significance improvement in spatial memory performance of the combined group in the water maze, as the latencies were comparable to those of the sham group. After cessation of enrichment, the positive effects of the combination group were transferred to a different spatial memory task (Radial Arm Maze). Specifically, the combinational therapy made significantly fewer reference and working memory errors relative to the other injured groups. Overall, these findings point to the benefits of combining environmental enrichment with pharmacological treatments when compared to individual treatments alone in treating cognitive dysfunction following juvenile brain trauma. When administered together combinational therapy may be more efficacious with respect to long-term functional recovery.
Keywords: polytherapy, nicotinamide, development, transfer
OUTCOME AFTER POST-ACUTE REHABILITATION IN TRAUMATIC BRAIN INJURY PATIENTS
Keywords: TBI, rehabilitation, outcome, follow-up, longitudinal
IMPACT OF COMPLETE SPINAL CORD INJURY ON NEOVASCULARIZATION AND TISSUE GRANULATION IN MOUSE MODEL OF SKIN PRESSURE ULCERS
Rutgers, The State University of New Jersey, Biomedical Engineering, Piscataway, USA
Pressure ulcers or sores (PUs) are a very common debilitating secondary complication of traumatic spinal cord injury (SCI) beside sensory and motor functional deficit. It tends to occur in soft tissues located around bony prominences. There is however little known about the impact of SCI on wound healing due to the lack of suitable animal model to perform relevant studies in controlled experimental settings. Herein we describe a reproducible and clinically relevant mouse model of PUs in the context of complete SCI. Adult male (BALB/c) mice were subjected to thoracic (T9-T10) complete SCI. Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnet discs held in place for 12 hours, thus creating an ischemic area that developed into a pressure ulcer over the following days. The wounded areas demonstrated tissue edema and epidermis disappearance by day 3 after magnets removed. The PUs spontaneously healed slowly (p < 0.001) in SCI mice compared to Control mice (35 versus 21 days). Histology data showed that there was slower migration of epidermal cells over the granulation tissue in SCI group compared to Control group. The SCI group also showed smaller thickness of epidermis and dermis, lower blood vessel density, decreased numbers of proliferating cells, and decreased expression alpha-smooth muscle actin compared to Control group on their wound closing day. Taken together, these data suggest that SCI significantly slows down the dynamics of healing process in experimental pressure wounds in mice. This work is supported by grant of New Jersey Commissions on Spinal Cord Research.
Keywords: Spinal cord injury, Pressure ulcers, Neovascularization, Granulation, Proliferation, Alpha-smooth muscle actin
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 memory circuits, expressed as impaired cognitive performance. Experimental rehabilitation strategies, such as enriched environment and exercise, have limited success in alleviating symptoms. The persistent symptoms of TBI are heterogeneous and therefore require focused therapies to alleviate neurological symptoms. In our hands, diffuse brain injury by midline fluid percussion leads to cognitive impairments at 1 month post-injury. Therefore, we hypothesize that rehabilitation targeting spatial and contextual memory circuit will prevent the onset of injury-induced memory impairments. Rehabilitation occurs in a box with a peg board floor (24” × 24”) that allows for 3” plastic pegs to be inserted at 1” intervals in designated layouts; termed Peg Forest Rehabilitation. Uninjured and brain-injured rats were exposed to the rehabilitation task (15 min/day), allowing free navigation through random layouts of the peg-filled arena for 10 days over 2 weeks. Controls were exposed to an open-field arena (15 min/day) or served as caged-controls for 10 days over 2 weeks. One week post-rehabilitation (1 month post-injury), cognitive performance was tested for short-term memory (novel object recognition), long-term memory (novel location recognition), and working memory (temporal object recognition). Brain-injured animals exposed to Peg Forest rehabilitation showed similar levels of object discrimination to uninjured rats on all three object recognition tasks. Whereas, brain-injured open field and caged-control rats performed significantly worse than shams on all three object recognition tasks, demonstrating injury-induced cognitive impairment without benefit of rehabilitation. Sham animals had no cognitive benefit from Peg Forest rehabilitation. Thus, passive, intermittent rehabilitation targeting specific neurological impairments can prevent cognitive symptomatology. The Peg Forest is a viable rehabilitation strategy to explore cellular and molecular mechanisms underlying preservation of neurological function. Supported by the Diane and Bruce Halle Foundation
Keywords: rehabilitation, cognition, diffuse axonal injury, memory preservation
Southern Illinois University-Carbondale, Psychology, Carbondale, USA
Environmental enrichment (EE) is a versatile tool that affords animals the opportunity to engage in social interactions, explore novel stimuli, as well as freely explore a larger living space. The effects of EE have been shown to promote neural plasticity after traumatic brain injury and is an acceptable form of rehabilitation. The use of abbreviated EE, which is defined as access to an enriched environment for a set amount of time each day, is comparable to rehabilitation programs performed in the clinic. Furthermore, proteins associated with neural plasticity peak during the day when the rats are less active and are at their lowest during the night, when the rats tend to be in a more active state. These proteins are important for the maintenance and consolidation of long-term memories. The objective of the current study was to investigate how the time at which EE was administered during the light cycle impacts performance in the Morris Water Maze (MWM). Adult male Sprague-Dawley rats either received a bilateral controlled cortical impact (CCI) or sham surgery. Animals received 6 hours of EE per day, starting 1 week post injury and began MWM training 2 weeks post injury. Testing began after the 6 hours of EE during the light phase or dark phase of the daily cycle. We hypothesized that rats given EE during their light phase will experience recovery in the MWM when compared to rats tested in the dark phase possibly due to peak levels of neural plasticity proteins. The results concluded that receiving EE and MWM training during the dark phase negates the beneficial effects of abbreviated EE. Further research is needed to study the cyclical effects of proteins such as glycogen synthase kinase-3 Beta (GSK3β) following treatment and the role it plays in the modulation of neural plasticity proteins associated with EE treatment.
Keywords: Environmental Enrichment, Chronotherapeutics, Circadian Rhythm, Neural Plasticity Proteins
In juveniles under the age of 15, traumatic brain injury (TBI) is the leading cause of death and disability. While prior research has demonstrated that a bilateral craniectomy increases axonal strain and leads to cell death in the contralesional hemisphere; unilateral craniectomy models are commonly used. However, it remains to been seen what differences exist when it comes to induced recovery between these two models. Through utilizing both bilateral and unilateral models in this study, we aimed to assess mechanisms underlying recovery of function in the juvenile rat model. The animals were given a controlled cortical impact (CCI) or sham surgery on postnatal day 28 (PND 28). CCI subjects were given either a unilateral or a bilateral craniectomy, with the other opening occurring over the identical region in the opposite hemisphere. In both models, an impact was delivered to the right parietal cortex at a velocity of 5.5 m/s with a 2-mm deformation of the cortex. To facilitate recovery of function post-injury, we utilized a 24-hour environmental enrichment (EE) protocol starting one day post-injury, where EE subjects remained in EE throughout the entirety of the experiment. The recovery of both EE-treated animals was compared to animals the remained in a standard housing environment. While we found no behavioral differences (as assessed with the Morris water maze (MWM)) between the two injury models; we did observe significant differences with respect to EE treatment. EE-treated subjects with a unilateral craniectomy demonstrated significant improvement in the MWM when compared to subjects with bilateral craniectomies. Future research will look to evaluate how EE treatment impacts the histological differences between the two injury models with respect to axonal strain. Through examining differences between these two models we can better understand the mechanisms surrounding functional recovery after treatment and better improve rehabilitative strategies for injury models with varying degrees of injury severity improving recovery of function post-injury.
Keywords: Enriched Environment, Juvenile Injury, Recovery of Function, Neural Plasticity
EFFECT OF NEUROREHABILITATION ON BRAIN CIRCUITS AFTER MILD TBI: AN EXPERIMENTAL FUNCTIONAL RSFMRI STUDY
UCLA, Neurosurgery, Brain Injury Research Center, Los Angeles, USA
We present an update of our previous pilot work to determine the effects of environmental enrichment on brain circuitry after controlled cortical impact injury (CCI) in the adult male rat. We tested whether resting state fMRI (rsfMRI) functional connectivity can be used to monitor brain circuitry in response to rehabilitation in expanded cohorts of injured and anesthetic-sham rats that were housed immediately after injury for 4 weeks in either standard (STD) or environmental enriched (EE) conditions (n = 9-11/group). We acquired rsfMRI data at 1 and 4 wks post-injury, and then 4 wks later following resumption of STD conditions for all groups (8 wks post-injury). We analyzed data for network-based connectivity differences over 96 brain regions using graph theory at both global and regional levels. There was no effect of time on sham-group network parameters over the 8 wk study indicating good reproducibility. There was no effect of EE on sham-group brain circuitry compared to sham-STD for any global network parameter at any time-point apart from small-worldness (SW), which was reduced immediately following 4 wks of EE (P < .05), but this did not endure after resumption of STD conditions. Compared to our published work in moderate/severe TBI, global network parameters after mild CCI in STD conditions were relatively unaffected at both 1 and 4 wks post-injury, apart from temporary reduced modularity and SW (P < .05), indicating potential circuit reorganization. By 8 wks however, most network parameters trended towards alterations similar to 4 wks post-injury in moderate TBI. This was highlighted by the regional development of hyperconnectivity (increased nodal P < .05 and edge strength, P < .005) in multiple brain regions compared to sham-STD rats at 8 wks. There was no effect of EE on CCI for any global network parameter compared to sham+EE at any time-point except for reduced modularity and SW at 4 wks (P < .05), possibly indicating EE-induced reorganization. This did not persist into STD conditions at 8wks. CCI+EE resulted in only sparse areas of hyperconnectivity compared to sham-EE (bilateral caudate, hippocampus, P < .05), so that compared to CCI+STD, EE resulted in reductions in bilateral retrosplenial and sensory cortex (P < .05).
Keywords: Environmental Enrichment, Traumatic Brain Injury, Reorganization, rsfMRI, Functional Connectivity
Chiba University Graduate school of Medicine, Department of Orthopedic Surgery, Chiba, Japan
The neural contorl of rhythmical hind-limb locomotion is known to be organized by a central pattern generator(CPG) in the lumbar enlargement. In some previous reports, epidural spinal cord stimulation(SCS) for CPG enabled to elicit full weight-bearing standing and stepping in patients with motor complete spinal cord injury. However, the precise mechanism of improvement by SCS is still unclear. The purpose of this study was to elucidate efficacy and molecular mechanism of SCS with body weight support treadmill training. Ten female Sprague-Dawley rats were used in this study. All rats were performed spinal cord transection at the T8-T9 level under general anesthesia. Two weeks after the spinal cord transection, rats were divided into SCS group and control group. The rats in SCS group were performed laminectomy at L3-4 level followed by implantation of a SCS electrode at L2 level. SCS and body weight support treadmill training were performed for 30 minutes per day, 5 days per weeks, for 4 weeks. The rats in control group were performed laminectomy without putting an electrode. Rats in control group were set to do treadmill training without stimulation during the same periods of SCS group. We evaluated hind-limb locomotion function by using Basso-Beattie-Bresnahan(BBB) score. We also assessed force for full extension of hind-limb as evaluation of spasticity before treadmill training, two and four weeks after treadmill training. For histological examination, we performed immunohistostaining for glutamic acid decarboxylase(GAD)65 of lumbar enlargement after behavioral assessment. There was no siginificant difference of BBB score between two groups, but spasticity of hind-limb in SCS group was significantly reduced. Histological assessment revealed that expression of GAD65 tended to increase in the SCS group. SCS ameliorated hind-limb spasticity through up-regulation.
Keywords: epidural spinal cord stimulation
Univ. of Alabama at Birmingham, Dept of Physical Med & Rehab., Birmingham, USA
The United States Centers for Disease Control and Prevention indicate that the incidence rate for traumatic brain injury (TBI) has increased over the last decade. Unfortunately, protective treatment options remain limited. We hypothesize that antagonists of AMPA receptors may confer acute protection after TBI. The goal of the current study is to evaluate the effects of post-injury administration of a non-competitive AMPA antagonist, perampanel (FYCOMPA®), on cognitive behavioral outcomes in adult male rats after moderate TBI. Perampanel is clinically approved as an antiepileptic. Following moderate lateral fluid percussion TBI (3.0ATM) or sham uninjured control procedure, rats were randomized to receive 0, 0.2, 1.0, or 2.0 mg/kg of perampanel (i.p.) starting at 1 hour post-TBI and then once per day for 7 days. Anxiety-like behaviors were assessed on post-TBI day 4 via the elevated plus maze. Learning and memory were evaluated using the Morris Water Maze on post-TBI days 7 − 11. Motivation was measured via the female encounter test on post-TBI day 25. We found that TBI induced deficits in learning and memory as compared to uninjured controls and that post-injury administration of perampanel ameliorated these impairments, regardless of dose. Additionally, rats in the TBI group spent less time with the female in the female encounter test than uninjured controls and the 1.0 mg/kg dose of perampanel reduced this effect. No significant effects of TBI or perampanel were observed in the elevated plus maze. Taken together, these data suggest that acute administration of perampanel after TBI reduces injury-induced deficits in learning and memory as well as motivation. Thus, acute administration of perampanel following TBI may be an effective protective agent. Supported by a Research Contract from Eisai, Inc. to JPS.
Keywords: Moderate TBI, Perampanel (FYCOMPA®), Lateral Fluid Percussion Model, Cognitive Deficits
University of Miami Miller School of Medicine, Miami Project to Cure Paralysis, Miami, USA
Spinal cord injury (SCI) leads to hypokinesis and skeletal muscle atrophy. The association of muscle deficit with metabolic dysfunction makes preservation of lean mass a clinical priority. Myostatin, a member of the TGFb family of growth factors, is a key negative regulator of skeletal muscle mass and is associated with muscle pathophysiology in many degenerative processes. The objective of this study was to determine the effect of anti-myostatin pharmacotherapy on sub-lesional skeletal muscle following experimental SCI. Myostatin signaling was blocked by systemic delivery of a highly selective monoclonal antibody, muSRK-015P, that blocks release of active growth factor from the latent form of myostatin. muSRK-015P is a parental counterpart of Scholar Rock's lead compound, SRK-015, expressed on a mouse IgG1 framework. SCI was induced by subjecting mice to severe contusion (65 kDyne) at thoracic level T9. Mice were administered test articles, muSRK-015P or controls (either vehicle or IgG), immediately following injury. A sham group was subjected to laminectomy only. Test articles were re-administered 1-week post-SCI. Animals were sacrificed at 2-weeks post-SCI. Treatment control groups had significant sub-lesional muscle atrophy in soleus and gastrocnemius (p < 0.05 compared to sham), while muSRK-015P treatment significantly improved maintenance of mass in both muscles (p < 0.05 compared to controls; not significant compared to sham). Oil Red O staining showed significant lipid infiltration into control muscles, which was absent in sham (p < 0.05) and significantly reduced in muscles from the muSRK-015P group (p < 0.05 compared to controls). These impovements in muscle health were associated with improved grip strength (p < 0.05) and BMS functional scores (p < 0.05) at 1- and 2-weeks post SCI in the muSRK-015P group compared to controls. These results indicate that pharmacological inhibition of myostatin signaling with muSRK-015P reduces atrophy and fatty infiltration, and improves muscle function following SCI.
Keywords: Musculoskeletal, Metabolism, Treatment, Rehabilitation
University of Texas - Medical Branch, Anesthesiology, Galveston, USA
Traumatic brain injury (TBI) can cause brain region-specific changes in mRNA and miRNA expression. We hypothesized that an immunotherapy that targets removal of tau oligomers given one hour post-TBI would result in decreased expression of neurodegenerative genes or increased expression of neuroprotective genes in the hippocampus. Adult male Sprague Dawley rats received either a single fluid percussion or sham injury before assessing the duration of suppression of the righting reflex. An intracerebroventricular injection of anti-tau oligomer-specific monoclonal antibody (TOMA) or nonspecific Immunoglobulin G control was administered one hour post-TBI. Animals underwent cognitive behavior testing and were euthanized fifteen days later for tissue collection. Based on our previous studies and a literature review, we created a custom Qiagen PCR plate array that targets 86 TBI-related genes and five housekeeping genes; the array also includes five wells that control for genomic contamination, RNA, PCR and RT quality. Normalized gene expression was analyzed by ANOVA for each gene, and differences among treatment groups were assessed by Tukey-adjusted contrasts followed by Benjamini-Hochberg control of false discovery rate. Our results show that up-regulation of neuroprotective genes in the TOMA treated animals varied with brain region, suggesting TBI may initiate brain region-specific injury and/or recovery processes. Studies including additional time intervals post-TBI are warranted. These studies were funded by The Moody Project for Translational TBI Research, Darrell K Royal Research Fund for Alzheimer's Disease, Mission Connect (program of the TIRR Foundation), and the UTMB Technology and Commercialization Program.
Keywords: Immunoglobulin G, PCR Array, Monoclonal Antibody, Tau Oligomers, Tau
University of Texas Health Science Center McGovern Medical School, Neurosurgery, Houston, USA
Keywords: traumatic brain injury, bioinformatics, pathway analysis
University of Texas Medical Branch (UTMB), Moody Project for Translational Traumatic Brain Injury Research, Department of Anesthesiology, Galveston, USA
Considerable interest exists to translate novel therapies into human clinical trials for the treatment of traumatic brain injury (TBI). We present key findings from the Moody TBI Symposium held in Galveston in which invited experts in clinical and preclinical TBI provided their opinions on preclinical study design. The purpose of this initiative was to seek perspectives on a number of translational issues that included: 1) model and therapy selection, 2) outcomes and data collection procedures, 3) analysis of results and reproducibility of studies, 4) data management and sharing plans, and 5) communications between labs during large scale multi-site projects. It is anticipated that the knowledge generated from this initiative will help to guide future scientific discussions and expert guidelines towards translation of novel therapies to improve TBI outcomes. The Moody TBI Symposium was generously supported by The Moody Project for Translational Traumatic Brain Injury Research and Mission Connect, a program of the TIRR Foundation.
Keywords: consensus, roadmap, preclinical
Children's Hospital of Philadelphia, Philadelphia, USA
Alterations in mitochondrial function is a critical component of the secondary injury cascade initiated by traumatic brain injury. We hypothesize that improving mitochondrial function will improve outcomes. Ciclosporin A (CsA) primarily exerts its neuroprotective effect through inhibiting Cyclophilin D-mediated activation of the mitochondrial transition pore. NeuroSTAT® is a novel lipid emulsion containing CsA. Our objective was to evaluate the pharmacokinetics and efficacy of NeuroSTAT using a large animal model of focal controlled cortical impact injury (CCI). Four-week-old (7–9 kg) piglets were studied. A mild-to-moderate injury was induced using a spring-loaded CCI device. One hour post-CCI, a loading dose of CsA was administered followed by a continuous infusion for either 24 h or 5 days. In a 24-hour bioequivalence study, piglets were randomized to 20 mg/kg/day (IV) of either NeuroSTAT or Sandimmune formulation of CsA (N = 3/group). In a pharmacokinetic dose escalation study: 5, 10, 20 or 40 mg/kg/day of NeuroSTAT (N = 3/group) was administered over 24 h, and CsA levels were measured in brain tissue to determine optimal dosing. Finally, in a randomized blinded placebo controlled study, animals were treated for 5 days with continuous infusion (20 mg/kg/d NeuroSTAT®, N = 10) or placebo (N = 13), and multiple efficacy outcome metrics were obtained: blood and CSF biomarkers, microdialysis, mitochondrial respiration and reactive oxygen species generation, and advanced neuroimaging. Mean brain tissue concentrations of Sandimmune and NeuroSTAT (436 ± 187 ng/g vs. 566 ± 80 ng/g, P = 0.3538), indicated equivalent penetration. NeuroSTAT brain concentrations significantly increased with each escalation from 10 to 40 mg/kg/day. Finally, animals treated with NeuroSTAT (20 mg/kg/day) for 5 days displayed a trend towards higher maximal oxidative phosphorylation (OXPHOS) (P = 0.252) and a decrease in reactive oxygen species generation (P = 0.122) compared to placebo. Analysis of the additional outcome metrics are ongoing.
Keywords: Mitochondria, Ciclosporin A, Focal controlled cortical impact injury
University of Pittsburgh, Safar Center, Pittsburgh, USA
Operation brain trauma therapy (OBTT) is a multi-center pre-clinical drug and biomarker screening consortium supported by the US DoD. OBTT screens therapies across three models in rats (parasagittal fluid percussion injury [FPI], controlled cortical impact [CCI] and penetrating ballistic-like brain injury [PBBI]) reflecting a range of phenotypes. Our goals include 1) to define therapies efficacious across models with the best chance for clinical translation, and 2) to define model-dependent effects to guide trials in targeted pathologies. The results of the first 5 therapies tested (nicotinamide, erythropoietin [EPO], cyclosporine [CsA], simvastatin, and levetiracetam) were published. OBTT has now screened 10 therapies, with ongoing testing of drugs 11–12. Drugs 6–12 include glibenclamide, kollidon-VA64, AER-271, amantadine, minocycline, E-64d and P7C3-A20. Levetiracetam showed benefit in multiple models. The second most successful drug, glibenclamide showed multiple benefits in CCI. Other therapies showed model dependent effects: tissue sparing by nicotinamide in CCI, benefit from CsA in FPI but toxicity in PBBI, benefit from amantadine in PBBI. Serum glial fibrillary acidic protein (GFAP) and Ubiquitin carboxy-terminal hydrolase-L1 were assessed at 4 and 24 h. GFAP predicted 21 d histology and drug effects. Levetiracetam merits additional testing and is being evaluated in our large animal model. Glibenclamide and Amantadine merit additional testing in contusion and penetrating brain injury, respectively. Our data support theranostic use of serum GFAP. Support: W81XWH-10-1-0623; WH81XWH-14-2-0018.
Keywords: consortium, rat, reproducibility, rigor, pre-clinical, therapy
There remains a need for more effective therapeutics in the treatment of traumatic brain injury (TBI). In this study, we tested whether an effective TBI intervention could be developed around a post-translational target occurring within an opportune treatment window as guided by temporal proteomics. Of interest were delayed-onset processes best managed after patient stabilization on the intensive care unit, focusing on events initiated a day or more after insult, out to two weeks following controlled cortical impact injury. We identified a subset of protein changes with the temporal profile of interest utilizing a self-organizing map approach. Enriched in this map were post-translational processes tied with ionic dysregulation, among which was a highly dynamic processing of neuron-specific K+-Cl- cotransporter 2 (KCC2), an essential component for maintaining chloride homeostasis that is critical to inhibitory neurotransmission. We identified a potential therapeutic window of opportunity starting on day 1 preceding unique acetylation, phosphorylation and ubiquitination events guiding the functional loss of KCC2. To test this window, we administered the KCC2-targeting compound CLP290 daily (50 mg/kg, p.o.) before, at, and after the identified 1-day point of KCC2 post-translational processing. The therapy was most effective at 1-day, preserving plasmalemmal KCC2 within perilesional somatosensory neocortex needed to maintain chloride homeostasis and effective inhibitory neurotransmission. Furthermore, TBI-impacted sensorimotor integration improved significantly with the 1-day intervention on rotarod and whisker adhesive removal task assessments. Together, our findings demonstrate the therapeutic targeting of post-translational processes revealed with temporal proteomics to effectively preserve KCC2-mediated chloride homeostasis with improved functional recovery. Furthermore, the approach defines an effective administration window that can be extended for the intervention of other post-translational events. Lastly, KCC2-targeted therapy may be extended to other neurological insults known to involve chloride dysregulation such as epilepsy and stroke.
Supported by NINDS NIH 1F31NS100322-01, Department of Anatomy and Neurobiology, and VCU Neuroscience PhD Program.
Keywords: KCC2, Proteomics, Inhibition, CCI, Chloride, Post-translational
USE OF BEHAVIORAL MEASURES TO ASSESS THE THERAPEUTIC VALUE OF NANO-PULSED LASER THERAPY AFTER FLUID-PERCUSSION INJURY
Univ. Texas Medical Branch, Anesthesiology, Galveston, USA
Traumatic brain injury (TBI) is considered a chronic condition due to the fact that it can both cause and accelerate disease; thus, the search for non-invasive, non-pharmaceutical therapies continues. Here, we assessed a novel, non-invasive nano-pulsed laser therapy (NPLT) system that combines near-infrared laser light (808 nm) and laser-generated, low-energy optoacoustic waves using fluid-percussion injury (FPI) in rats. Adult, male Sprague-Dawley rats were divided into four groups (SHAM, FPI, SHAM+NPLT, FPI+NPLT). All rats experienced two days of training followed by baseline assessment using the Neuroscore, Beam-Balance, and Beam-Walk tests prior to surgical preparation. Rats were anesthetized, intubated and a craniotomy was trephined lateral-right of the sagittal suture. Half of the rats received a moderate (2.0–2.2 atm) FPI and half received sham surgery. NPLT was applied for five minutes at one hour post-surgery. Neurological and vestibulomotor function were assessed using Neuroscore, Beam-Balance, and Beam-Walk for five consecutive days post-surgery. Cognitive function was assessed using a working memory version of the Morris water maze on post-injury days 11–15. Data were analyzed using a mixed model ANOVA to test for differences between treatments, days, trials, and in the case of the working memory test, the trials delta (Trial 1 – Trial 2). Results indicated that NPLT restored the performance of injured rats to that of sham-injured rats treated with NPLT, providing evidence of the neuroprotective effect of NPLT and supporting the continued investigation of NPLT as a therapeutic intervention after TBI. Funded by The Moody Project for Translational TBI Research
Keywords: nano-pulsed laser therapy, working memory, fluid-percussion injury, rodent behavior
Arizona State University, Biomedical Engineering, Tempe, USA
Traumatic brain injury (TBI) is the leading cause of injury related death in America. Current clinical treatment modalities for TBI focus on minimizing the secondary symptoms and complications associated with TBI; however, no clinical treatments currently exist to address the underlying neuropathology for any level of TBI severity ranging from mild to severe. The long-term goal of this project is to develop novel intervention strategies that directly tackle neurodegenerative cues present in the extracellular matrix to shift the balance toward regeneration. The primary objective of this study was to identify unique targeting moieties of the neural injury microenvironment in order to mask neurodegenerative cues in injured neural tissue. We employed the molecular biology screening technique, phage display, to identify nanobodies that bind to markers that are more abundant in injured neural tissue versus healthy neural tissue. Here, we focus on the chondroitin sulfate proteoglycan neurocan. We successful completed biopanning screens against neurocan, confirmed via high throughput ELISAs. Subsequent concentration dependent ELISAs with four top clones demonstrated a superior nanobody clone displaying preferential binding to neurocan compared to non-specific targets. Recombinant protein expression/production of this nanobody is underway where we are conducting additional binding affinity assays to determine the robustness of its targeting capabilities. Ultimately, this approach will contribute significant advances to improved understanding of how the extracellular microenvironment impacts neural regeneration after brain injury.
Keywords: Glial scar, nanobody, neurocan
Harvard Medical School, PM&R and Neurosurgery, Charlestown, USA
Inflammation plays major roles in the pathogenesis and recovery after acute spinal cord injury (SCI). Carbon monoxide (CO) confers anti-inflammatory and pro-healing effects in rodent models and clinical cases of lung lesions when applied in low concentrations. The goal of this project was to investigate the impacts of low-dose CO ventilatory exposure on the inflammatory events and spinal cord neuronal survival following T9-10 injury of 35 g × 5 min compression in adult female S-D rats. Three-four hours after SCI, 4 groups of rats were randomly housed inside whole body exposure chambers that were maintained with room air or 100, 250 or 500 ppm CO (n = 7/group), respectively. The rats underwent 1 hour CO exposure/24 hours for 12 consecutive days. In addition to hindlimb locomotion, lesion volume, white matter sparing and neuronal survival were evaluated up to 6 weeks post T9-10 compression. Anti-inflammation and neural protection parameters were assessed by immunohistochemical, histopathological and stereological quantifications. We found that CO treatment demonstrated a dose-dependent therapeutic effect for neural repair. The 500 ppm CO-treated rats showed the highest level of hindlimb functional improvement, lesion volume reduction, white matter sparing and neuronal preservation, compared to the control group; furthermore, 500 ppm CO inhalation significantly decreased pro-inflammatory cytokine (e.g., IL-1β and TNF-α) expression, macrophage/microglia activation (e.g., CD68+), reactive gliosis, and neuronal protein nitration. CO ventilation therapy directed and sustained macrophage polarization towards an arginase 1+ M2 phenotype to promote neural recovery. Importantly, the total number of interneurons and the numbers of GAD67+ inhibitory and ChAT+ excitatory interneurons were significantly higher in the 500 ppm CO-treated spinal cords. The results of 500 ppm ventilation were independently replicated. Taken together, the studies demonstrate that low-dose CO inhalation may mitigate inflammatory damage and support functional recovery by enhancing interneuron and neurite survival, suggesting a potential treatment for clinical neurotrauma.
Keywords: Spinal cord injury, Carbon monoxide, Inflammation, Macrophage, Interneuron, Recovery Neurobiology
Traumatic SCI significantly causes mitotic cell proliferation of B cells and astrogcytes, which leads to inflammation, astroglial scar formation, autoimmunity, locomotor deficits and pain. However, no approved anti-B cell or anti-astroglial products are available for SCI indications. Flubendazole (FluBZ) is a novel tubulin-binding antimitotic agent that has been widely used in the treatment of intestinal and neural parasites in human and can be safely administered long term up to two years in human without side effects. The objective is to evaluate whether FluBZ anti-mitotic therapy reduces proliferating signaling pathways of B cells and astrocytes and demonstrates potent pain relief and improved locomotor function after SCI. Intraperitoneal (IP) injection with 5 and 10 mg/kg/day (n = 10/group) of FluBZ to Sprague-Dawley rats for 2 or 4 weeks started at 3 or 5 hrs post-SCI (180 kdyn) at T10. These FluBZ treatments resulted in improved locomotor function (BBB scores and kinematic analysis) 7 weeks after SCI and reduced pain behaviors 4 weeks after excitotoxic SCI compared to vehicle-treated controls (n = 9/group). FluBZ IP treatment also improved total tissue sparing, white matter sparing, and gray matter sparing at 7 weeks after SCI. Mechanistic studies revealed that FluBZ reduced splenic population of CD45RA-positive B cells and spinal GFAP-positive astrocytes at 4 or 7 weeks post-injury and suppressed production of antibody IgG at lesion site post-injury. Moreover, we found that FluBZ inhibited mitotic signaling activation of mitogen-activated protein kinase pERK1/2 and mitotic cell cycle protein activator cyclin B1 at lesion site 4 weeks post-injury. In conclusion, our results demonstrate that FluBZ antimitotic therapy inhibiting proliferation of B cells and astrocytes as well as mitotic signaling is a valid therapeutic strategy for treating paralysis and pain following SCI.
Keywords: flubendazole, B cells, astrocytes, spinal cord injury, neuropathic pain, paralysis
University of Toronto, Institute of Medical Science, Toronto, Canada
There are currently no therapeutic treatments for traumatic brain injury (TBI). Primary injury from the direct impact to the skull followed by secondary injury mechanisms consisting of delayed cellular and molecular pathways can contribute to breakdown of the blood brain barrier (BBB) among other events. Following TBI, vascular disruption leads to edema formation that is associated with increased intra-cranial pressure adversely affecting outcome. Human umbilical cord derived perivascular cells (HUCPVCs) have been shown to express mesenchymal, neurotrophic and vascular factors, making them a potential therapeutic strategy. A TBI was modeled using the rat fluid percussion injury (FPI) device. Rats were systemically infused with 1.5 × 106 cells either 1 hour pre-injury or 1.5 hours post injury, and survived for 24 h, 48 h or 7 days. Brains were assessed for Evan's blue vascular leakage and edema formation. Immunohistochemistry for RECA-1 at 24 h and 48 h was performed to assess microvascular density. Western blot was used to examine the expression of tight junction proteins after trauma. At 24 h and 48 h Evan's blue vascular leakage was 6.4 μg/g and 15.5 μg/g, respectively and 1.7 μg/g in sham rats. Pre-injury treatment values were 4.0 μg and 5.6 μg at 24 and 48 hours, respectively. Post-injury leakage values were 5.5 μg/g and 3.3 μg/g at 24 and 48 hours, respectively. At 7 days post-injury, FPI rats demonstrated leakage values similar to sham rats. Edema formation was not significantly different across treatment groups. At 24 h and 48 h, vascular density, expressed as fluorescence/area was 0.056 and 0.064 respectively compared to 0.081 for sham, and 0.076 and 0.082 for HUCPVC treated animals at 24 h and 48 h respectively. Preliminary results indicate some therapeutic capacity of HUCPVCs in mitigating acute BBB pathophysiology after TBI.
Keywords: Mesenchymal stromal cells, Fluid percussion injury, Umbilical cord derived stem cells, Tight junctions
UTHealth Medical School at Houston, Diagnostic and Interventional Imaging, Houston, USA
Gastrointestinal (GI) dysfunction is observed clinically after spinal cord injury (SCI) and contributes to the diminished long-term quality of life after SCI. GI vascular permeability changes can results in undiagnosed abdominal complications after SCI. Our study examined the acute and chronic GI vascular changes that occurs following spinal cord injury. We demonstrated that the GI vascular tract in mice becomes compromised during the acute phase of injury and persists into the chronic phase of injury. The contusion injury was delivered using the Infinite Horizon Impactor (60 kDynes with a 1 second dwell time) at thoracic level 8. The study examines GI vasculature permeability was measured using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) at 48 hours, 2 and 4 weeks following injury. Our hypothesis was that the observed vascular permeability could be attenuated by an intravenous (IV) administration of angiopoietin-1 (Ang-1). Ang-1 is a vascular stabilizing protein expressed constitutively by endothelial cells, pericytes, astrocytes, smooth muscle cells, and fibroblasts. Our results indicated that a single administration of Ang-1 reduced vascular permeability at 48 hours but the effect was only transient, as there were no significant difference between treated and the vehicle control treated group at 2 and 4 weeks. When the treatment paradigm was changed from a single dose to multiple doses of Ang-1 following contusion injury. Ang-1 (150μg, IV for 7 days), our DCE-MRI data indicated a significant decrease in GI vascular permeability 4 weeks after injury compared to vehicle control treated animals. We also demonstrated that Ang-1 reduced the expression of sICAM-1 in the ileum compared to the vehicle treated group. Together this data indicates that the GI vasculature is compromised into the chronic phase of injury and that multiple administrations Ang-1 can attenuate GI vascular permeability.
Keywords: gastrointestinal, vascular permeability, dynamic contrast enhanced imaging, angiopoietin-1
Loma Linda University, Basic Science, Loma Linda, USA
Traumatic brain injuries (TBI) occur in 1.7 million people each year in the USA. Little is known about how the cerebrovasculature is altered after TBI. We previously reported that TBI elicits acute decrements in cerebral vessels near the injury site in rats followed by revascularization over the subsequent 2 weeks. Sexual dimorphism of the brain is well documented and different hormonal levels in males and females differentially modify the recovery process after brain injury. However, little is known about the effects of biological sex on the temporal evolution of revascularization following moderate TBI. Using a model of controlled cortical impact in male and female mice, we set out to determine if the injury and the repair process are affected by sex. Lesion volume was assessed using MRI T2-weighted imaging at 1 and 7 days post-injury (dpi). To evaluate the vascular network, we used a new “vessel painting” technique that uses a fluorescent dye (DiI) to stain blood vessels. Vascular parameters such as vessel numbers and complexity were analyzed using Angiotool and Fraclac, ImageJ plugins. Blood-brain barrier (BBB) alteration, neurodegeneration, inflammation and endothelial activation were assessed through immunohistochemistry. We found no sex differences in lesion volume, BBB alteration or neurodegeneration. However at 1 dpi, females exhibited more astrocytic hypertrophy, whereas males presented with increased endothelial activation and expression of beta-catenin, which has been shown to be involved in angiogenesis. At 7 dpi, we observed an increase in the number of vessels and an enhancement in vessel complexity in injured cortex of males compared to females. Our results imply that the cerebrovasculature recovers differently after TBI, suggesting sex should be considered when patients are being treated. Further studies are needed to determine if these sex differences are beneficial or deleterious.
Keywords: biological sex, beta-catenin
Cerebral vessels commonly tear and bleed as a consequence of traumatic brain injury (TBI). It follows that neighboring vessels experience deformations that are also significant, though not severe enough to produce bleeding. Although these vessels remain intact, little is known about potential changes to their integrity and their prognosis as functioning vessels. Preliminary work from our laboratory shows that both the mechanical properties and contractility of cerebral arteries are altered by deformation. We hypothesized that these observed changes would correlate with damage to elastin and collagen in the extracellular matrix (ECM) and thus aimed to characterize ECM changes following large deformations. Middle cerebral arteries were dissected from euthanized sheep, attached to an isolated vessel testing device, and subjected to overstretch in either the axial or circumferential direction. The tested vessels were incubated in fluorescently-tagged collagen hybridizing peptize (CHP), a newly discovered marker of tropocollagen disruption, and evaluated via confocal microscopy. Elastin was assessed using autofluorescence. Extent of damage was correlated with magnitude and direction of applied overstretch. Damaged collagen was found following subfailure overstretch in both directions, but it was located primarily in the media after circumferential loading and in the adventitia following axial overstretch, consistent with known dominant fiber directions in the arteries. Extent of collagen damage, defined by the intensity of CHP fluorescence, increased linearly with overstretch, but only above thresholds of 1.15 and 1.23 times the in vivo diameter and length for the circumferential and axial directions, respectively. We also commonly observed disruption of the internal elastic lamina prior to complete rupture of the vessel, occurring, on average, at 1.31 times the in vivo length, compared to 1.57 for failure. It was common for multiple disruptions to occur in a single vessel prior to failure. Findings show that vessel ECM is damaged in cerebral arteries experiencing large deformations below the threshold of complete vessel disruption. To our knowledge, such damage has not yet been observed in vivo, but these changes could explain the observed changes in function and structural properties and may be relevant to TBI.
Keywords: extracellular matrix, collagen, internal elastic lamina, overstretch
TRAUMATIC BRAIN INJURY RESULTS IN ACUTE RAREFICATION OF THE VASCULAR NETWORK
University of California, Irvine, Pediatrics, Irvine, USA
Support: NIH NINDS 1P01NS082184, Project 3
Keywords: vessel painting, vascular repair, fractal, network
Indiana University School of Medicine, Goodman Campbell Brain and Spine, Indianapolis, USA
Keywords: Blunt cerebrovascular injury, treatment protocol, antiplatelet medication, anticoagulation
University of Pennsylvania, Neurology, Philadelphia, USA
Traumatic cerebrovascular injury (TCVI) is common after TBI and is responsible for a significant portion of TBI-related disability. Development of therapies targeting TCVI will require reliable and inexpensive biomarkers to measure vascular dysfunction and document target engagement. Cerebrovascular reactivity (CVR) is impaired following TBI and methods which reliably and non-invasively measure CVR are available, making CVR an attractive candidate predictive and pharmacodynamic biomarker for TCVI-directed therapies. This study was designed to assess CVR longitudinally after TBI in humans using functional Near Infrared Spectroscopy (fNIRS) from the acute to the subacute and chronic stages. We also studied the effect of treatment with a phosphodiesterase 5 inhibitor, sildenafil citrate, in order to assess the utility of fNIRS as a pharmacodynamic biomarker in future clinical trials. 7 participants with complicated mild TBI were studied in the acute (within 72 after injury) and subacute (14 days after injury) stages, in addition to 4 age- matched healthy controls (HC), who were studied once. CVR was assessed by measuring the changes in oxygenated hemoglobin (ΔHbO) and deoxygenated hemoglobin (ΔHbR) concentration produced by mild hypercapnia (5% CO2). The change in CVR before and one hour after the administration of single dose of sildenafil citrate (60 mg orally) was also assessed. Mean (± SEM) CVR was lower in TBI patients compared to HC (HC: 0.100 ± 0.028%/mmHg; and TBI: CVR 0.057 ± 0.011%/mmHg, p = 0.22). Sildenafil administration did not result in an increase in CVR in HC (t = 0.1463 df = 3, p = .89) whereas TBI patients show a clear potentiation of the CVR response (p < .08). These findings support the hypothesis that vascular injury represents a distinct endophenotype following TBI and PDE5 inhibition as a potential therapy for TCVI.
Keywords: FNIRS
More than 10 million people worldwide sustain a traumatic brain injury (TBI) each year. Many survivors suffer long-lasting cognitive impairments associated with frontal lobe disturbances, as well as psychopathological consequences. Previously, we demonstrated that a controlled cortical impact (CCI) injury over the parietal cortex produced deficits in executive function in the attentional set-shifting test (AST) in rats, a complex cognitive paradigm analogous to the Wisconsin Card Sorting Test, which measures strategy-switching deficits in patients with frontal lobe damage, TBI, and psychiatric disorders. Considering that a large percentage of TBIs occur via direct impact to the frontal part of the skull (e.g., windshield during a car accident), this study aimed to investigate complex cognitive deficits in rats subjected to frontal lobe CCI. Isoflurane-anesthetized adult male rats were subjected to CCI (2.0, 2.2, and 2.4 mm cortical tissue deformation at 4 m/sec) or sham injury over the prefrontal cortex region in the right hemisphere. Rats were tested on the AST at four weeks post-surgery. The test involves a series of increasingly difficult discriminative stages to obtain food reward, including simple and compound discriminations, stimulus reversals, and intra- and extradimensional (ED) shifts. Frontal CCI produced significant deficits in attentional performance on the ED stage and stimulus reversals of AST at four weeks post-injury, seen as increased total trials to reach criterion and significantly higher total errors compared to SHAM rats (p < 0.05 for Injury, n = 7–8/group). These effects were particularly robust in the two more severe injury groups, namely 2.2 and 2.4 mm cortical deformation depth (p < 0.05). These results suggest that frontal lobe injury negatively impacts complex cognitive functioning. Ongoing and future studies will focus on further disentangling brain constructs and neurotransmitter alterations responsible for such attentional deficits following brain trauma, as well as identifying necessary pharmacotherapies for cognitive performance and advance rehabilitation research.
Supported by NIH grants NS094950, NS099683, UPP/UPMC Academic Foundation and UPMC Rehabilitation Institute (COB); HD069620, HD069620-S1, NS060005, and NS084967 (AEK).
Keywords: Controlled cortical impact, Attentional set-shifting, Frontal injury, Executive function
DELAYED ADMINISTRATION OF INTRANASAL INSULIN PROVIDES LIMITED COGNITIVE IMPROVEMENT AFTER MODERATE CCI
Uniformed Services University, Anatomy, Physiology and Genetics, Bethesda, USA
Following traumatic brain injury (TBI), there is an acute period of elevated glucose demand followed by a period of cerebral hypometabolism, which directly affects long term patient outcome and is associated with cognitive and physical deficits. Our previous research demonstrated that intranasal insulin administration within 4 hours after injury significantly increased glucose uptake in the hippocampus and improved cognitive function after a moderate controlled cortical impact (CCI) in rats. In order to more fully understand the therapeutic potential of intranasal insulin administration for TBI, this study aimed to evaluate the effect of delaying treatment. Male adult Sprague Dawley rats were exposed to a moderate CCI injury followed by intranasal insulin or saline treatment beginning 24 hours post-injury and continuing with daily administration for 14 days. Glucose uptake was assessed by measuring 18Fluorodeoxyglucose (FDG) uptake in positron emission tomography (PET) scans at 2 and 10 days post-injury. Motor and cognitive testing were performed through 60 days post-injury. Significant differences in FDG uptake were noted in the cortex and hippocampus at 2 and 10 days post-injury, respectively, in control and insulin-treated groups. In addition, analysis of the novel object recognition task showed that insulin treated rats demonstrated significant improvement at 2 days post-injury in comparison to rats that received saline. However, injured animals treated with intranasal insulin demonstrated no significant difference in cognitive performance on the MWM or motor function in the beam walk task, unlike our previous study. These data suggest that more acute treatment with intranasal insulin is optimal for observing functional effects of the therapy, and may suggest that the acute hyperglycolysis and elevated glucose demand are the primary target of insulin administration. However, delaying treatment to 24 hours may still offer some benefit.
Keywords: insulin, glucose, intranasal, PET
Antipsychotic drugs, such as haloperidol (HAL), are prescribed in the clinic to manage traumatic brain injury (TBI)-induced agitation. While preclinical studies have consistently shown that HAL hinders functional recovery after TBI in male rats, its effects in females is unknown. Hence, the objective of this study was to directly compare neurobehavioral and histological outcomes in both sexes to determine whether the reported deleterious effects of HAL extend to females. Anesthetized adult female and male rats received either a controlled cortical impact (CCI) or sham injury and then were randomly assigned to a dosing regimen of HAL (0.5 mg/kg, i.p.) or vehicle (VEH; 1 mL/kg, i.p.) that was initiated 24 hours after injury and continued once per day for 19 consecutive days. Motor function was tested using established beam-balance/walk protocols on post-operative days 1–5 and acquisition of spatial learning was assessed with a well-validated Morris water maze task on days 14-19. Cortical lesion volume was quantified at 19 days. No statistical differences were revealed between the HAL and VEH-treated sham groups and thus they were pooled for each sex. HAL impaired motor recovery in males, but not females. However, HAL significantly diminished spatial learning in both sexes. Taken together, the data show that daily HAL does not prohibit motor recovery in females, but does negatively impact cognition. The deleterious effects of HAL on cognition in females replicate that observed in males and suggests that other treatments need to be explored to combat agitation after TBI.
Supported, in part, by NIH grants HD069620, HD069620-S1, NS060005, NS084967 (AEK), NS094950, NS099683 (COB), and the University of Pittsburgh Physicians /UPMC Academic Foundation (COB).
Keywords: Controlled cortical impact, Water maze, Antipsychotics
LONG-TERM EFFECTS OF ADOLESCENT CHRONIC STRESS ON TBI COGNITIVE AND EMOTIONAL IMPAIRMENTS IN ADULT MALE RATS
Exposure to early life stress has lasting effects on behavior and brain function due to dynamic plasticity occurring in the developing adolescent brain. However, it is yet to be determined how stress exposure in this developmental period influences functional recovery post traumatic brain injury (TBI) later in life. Thus, the goal of this study was to test the hypothesis that stress in adolescence would confer deleterious effects on behavioral impairments post TBI in adulthood. Adolescent male Sprague-Dawley rats (n = 40) were exposed to 4 weeks (postnatal day, PND, 30–60) of chronic unpredictable stressors (CUS) or no stress, and after a 1-month resting period (PND 60-90), were anesthetized and received a cortical impact of moderate severity (2.8 mm tissue deformation at 4 m/s) or sham injury. After one week of recovery, anxiety-like behavior in the open field test (OFT) and elevated plus maze (EPM), and cognitive performance in the novel object recognition NOR) task and, Morris water maze (MWM) were measured, and the brains collected 25 days post TBI for histological analysis. Preliminary results show increased time spent in the anxiogenic zones of the OFT and EPM, and improved NOR memory after a 24 h delay, and reduced time to reach the platform in the MWM for CUS groups compared to no-stress groups, although TBI rats remained significantly more anxious and cognitively impaired compared to sham controls. These results suggest that aversive environmental conditions in adolescence induces adaptive behavioral responses in TBI rats, albeit, without leading to full functional recovery.
Keywords: Early-Life Stress, Anxiety-Like Behavior, Cognitive Behavior, Controlled Cortical Impact (CCI)
VISUAL DYSFUNCTION OF THE OPTOMOTOR RESPONSE IN APOE TRANSGENIC MICE AFTER TBI
Keywords: APOE, TBI, Vision, Optomotor
EXPLORING SPATIAL MAP VERSUS COMPENSATORY STRATEGY FORMULATION FOR PLACE-LEARNING IN THE MORRIS WATER MAZE AFTER EXPERIMENTAL TBI
University of Pittsburgh, Physical Medicine and Rehabilitation, Pittsburgh, USA
Explicit learning and memory deficits after traumatic brain injury (TBI) have implications for skill learning, reacquisition, retention, and cognitive rehabilitation approaches that leverage implicit-learning networks, which remain relatively intact, as compensatory mechanisms. The Morris water maze (MWM) assesses learning/memory deficits through metrics like latency and target zone time allocation (TZTA). MWM protocols include extra-maze cues to formulate spatial maps (explicit-learning) representing the escape platform's location (place-learning). However, latency improvements can be achieved via alternative strategies without clear spatial map formulation. We hypothesized MWM place-learning post-experimental TBI results primarily from acquiring non-spatial (Implicit-learning) strategies, and post-injury cognitive training (CT) on the MWM's implicit components facilitates further compensatory reductions (via implicit-learning networks) in platform latencies, and supports increased spatial strategy selection. Adult male Sprague-Dawley rats (n = 50) underwent controlled cortical impact (CCI) injury or sham surgery. Beginning D8 post-surgery, CCI/Shams completed 6 days of non-spatial CT or no-training (NT). Acquisition trials [AC (D14-D18, D20-D24)], and periodic short/long-term retention probe trials (PT) were performed. Platform latencies, peripheral zone time allocation (PZTA), TZTA, and search strategies were assessed. CCI-CT and CCI-NT groups improved latency and TZTA by D18 (p < 0.001). PZTA improved by D24 (p < 0.01). Shams improved latency, PZTA, and TZTA by D18 (p < 0.05, all comparisons) and further reduced PZTA by D24 (p < 0.001). Compared to CCI-NT animals, CCI-CT had lower AC latencies (p < 0.001), higher TZTA (p < 0.01), and lower PZTA (p < 0.01) than CCI-NT on long-term retention PTs, where CT-Shams also had lower PZTA than NT-Shams (p < 0.01). Frequency analysis showed CT groups used more spatial strategies than NT counterparts with long-term retention PTs (D19:CCI-CT = 25%, CCI-NT = 0%, Sham-CT = 66.67%, Sham-NT = 44.4%; D25:CCI-CT = 40%, CCI-NT = 18.18%, Sham-CT = 90%, Sham-NT = 33.3%). These findings suggest MWM improvements can be achieved via new learning involving implicit networks, and CT on the MWM's implicit components improves place-learning and facilitates spatial map retention post-TBI. Support: NIH-1R21HD071728.
Keywords: Morris Water Maze, cognitive rehabilitation, implicit learning networks, Spatial Map Formation, Experimental TBI
The majority of behavioral assessment studies are conducted during the day, which is not when rats are most active. This discrepancy may preclude optimal performance. Hence, the goal of this study was to determine if differences in neurobehavior exist in traumatic brain injured (TBI) rats when assessed during the day vs. night. The hypothesis was that the night group would perform better than the day group in all behavioral tasks. Isoflurane-anesthetized adult male rats received a controlled cortical impact (2.8 mm depth at 4 m/sec) or sham injury and were randomly assigned to either day (1:00 - 3:00 p.m.) or night (07:30 – 09:30 p.m.) testing. Motor function (beam-balance and beam-walk) was conducted on post-operative days 1–5 and cognitive performance (acquisition of spatial learning) was assessed on days 14–18. CORT levels were quantified at 24 hr and 21 days after TBI. No significant differences were revealed between the TBI rats tested during the day vs. night for beam-balance, beam-walk, or water maze (p's < 0.05). CORT levels were higher in the TBI and sham groups tested at night at 24 hr (p < 0.05), but returned to baseline and were no longer different by day 21 (p > 0.05), suggesting an initial, but transient stress response, that did not affect neurobehavioral outcome. These data suggest that the time rats are tested has no impact on their performance, which does not support the hypothesis. The finding is important because it validates the interpretations from numerous studies conducted when rats were tested during the day vs. their biologically active period.
Supported by NIH grants HD069620, HD069620-S1, NS060005, NS084967 (AEK), NS094950, NS099683 (COB), and the University of Pittsburgh Physicians /UPMC Academic Foundation (COB)
Keywords: Controlled cortical impact, day vs night, spatial learning, Morris water maze, motor
EFFECT OF GALANTAMINE ON ATTENTIONAL SET-SHIFTING PERFORMANCE AFTER EXPERIMENTAL BRAIN TRAUMA
Traumatic brain injuries (TBI) impact millions of Americans, with older patients being more likely to have a co-occurring condition, particularly dementia. Galantamine (GAL), a first-line drug used to treat dementia, acts primarily as an acetylcholinesterase inhibitor and has been reported to positively impact cognitive function in older adults. Previously, we demonstrated that a controlled cortical impact (CCI) injury produced significant impairments in executive function in the attentional set-shifting test (AST), a complex cognitive paradigm analogous to the Wisconsin Card Sorting Test, which is used to measure strategy-switching deficits in patients with frontal lobe damage, TBI, and psychiatric disorders. In the current study, we predicted that daily GAL injections would normalize AST performance after a parietal lobe TBI in rats. Isoflurane-anesthetized adult male rats were randomly assigned to either a CCI or sham group. Surgery was administered following a previously established CCI protocol. Following surgery, rats were randomly distributed into three treatment groups: saline or GAL (1 or 2 mg/kg/day), until the test day 4 weeks later. AST results indicated that TBI significantly impairs performance on the first reversal stage, deficits attenuated by both GAL chronic doses (p < 0.05). In particular, GAL (2 mg/kg/day) also significantly reduced TBI-induced cortical lesion volumes (p < 0.05). In summary, chronic GAL administration provides an efficacious treatment for higher-order cognitive recovery following TBI. Further studies will investigate whether these results are maintained when using aged Sprague-Dawley rats in order to mirror the elderly segment of adults typically treated with GAL in the clinic, as well as assess protein expression of brain cholinergic markers involved in the GAL mechanism of action for restoring executive function after TBI.
Keywords: Controlled Cortical Impact, Attentional Set-Shifting, Executive Function, Acetylcholine, Galantamine
INTERACTION OF BRAIN TRAUMA AND CHRONIC STRESS ON COGNITION, ANXIETY, AND MARKERS OF NEUROTRANSMISSION AND NEUROINFLAMMATION
Traumatic brain injury survivors endure cognitive impairments associated with frontal lobe disturbances, and are vulnerable to neuropsychiatric disorders. Research has highlighted chronic unpredictable stress (CUS) as a major risk factor for many psychopathological conditions. Herein, we began to assess clinically-relevant cognitive- and anxiety-like dimensions sensitive to both TBI and CUS. We hypothesized that moderate TBI produced by controlled cortical impact (CCI) injury, as well as CUS exposure will render cognitive impairments in rats in an attentional set-shifting test (AST), reduced sucrose preference, open field exploration, blunted weight gain, as well as increased inflammatory markers and altered brain neurotransmission markers. Adult male rats were subjected to a CCI (2.8 mm deformation, 4 m/s) or sham injury over the right parietal cortex. Following surgery, rats were assigned to receive CUS (21 days) or handling (CTRL). Upon cessation of stress, rats were tested for anxiety (open field test) and anhedonia (reduced preference of 1% sucrose-water). At 4 weeks post-surgery, rats were tested on the AST, which involves a series of increasingly difficult discriminative tasks. Results demonstrate that CUS leads to a 5-10% weight gain reduction compared to CTRL, yet the combination of TBI and CUS does not negatively impact exploration in the open field, or sucrose preference (n = 8–12/group). TBI and CUS rendered cognitive deficits when given alone, as expected, but not when given in combination, suggesting a resilience or compensatory interaction. Ongoing directions include assessing serum levels of immune markers such as IL-4, IL-6, IL-10, IL-12 and TNFα, as well as protein levels in discrete brain regions for serotonergic, dopaminergic and adrenergic markers relevant to neurotransmitter synthesis, release and reuptake. This project will provide novel outcomes pertaining to cognition, anxiety- and depressive-like symptoms following overlapping chronic stress and the recovery phase of TBI.
Supported by NIH grants NS094950, NS099683, UPP/UPMC Academic Foundation and Rehabilitation Institute (COB), HD069620, HD069620-S1, NS060005, NS084967 (AEK).
Keywords: controlled cortical impact, chronic unpredictable stress, western blot, attentional set-shifting, inflammatory markers, open field test
HIPPOCAMPAL NETWORK CHANGES AFTER MILD TRAUMATIC BRAIN INJURY
UCSF, Neurosurgery, San francisco, USA
Traumatic brain injury (TBI) is the primary cause of death and disability in children and young adults. TBI causes substantial cognitive impairments such as learning and memory alteration in humans. However, little is known about the causal mechanisms mediating this pathology. Previous research has demonstrated that TBI preferentially leads to hippocampal damage in both the human condition and rodent models of TBI. However, the precise pathophysiological changes that occur in the hippocampus after TBI are not known. In order to model mild TBI (mTBI) in mice, we employed lateral fluid percussion injury (LFPI) and we investigated electrophysiological alterations in hippocampal activity. Specifically, we recorded hippocampal neural activity using chronic 32-channel silicon probes with recording electrodes distributed across areas CA1, CA3, and DG sub-regions of the hippocampus. We recorded local field potentials across the entire hippocampus while the animals were in the home cage (often resting or sleeping) and while performing hippocampal-dependent memory task interspersed with rest sessions in the home cage. Electrodes were implanted 6–8 days post-injury and we recorded the hippocampal activity 1 week after implantation. We have previously demonstrated that LFPI causes regional hippocampal imbalances in excitatory and inhibitory synaptic transmission. Optimal brain function requires a delicate balance between excitatory and inhibitory neurotransmission (E/I balance). Furthermore, E/I balance is essential for the induction and maintenance of neuronal oscillations, which underlie cognitive function and working memory. In this study, we found a multitude of spectral changes especially in the theta and gamma narrow bands that occur after TBI. Furthermore, we found differential activity in CA1 versus CA3 in a complex interaction depending on behavior performance between TBI animal versus Sham control group. These data demonstrate that simultaneously recording across the hippocampus to identify the network activity and holds significant promise to better understand the link between TBI, hippocampus and memory. Acknowledgements Supported by R37HD059288 and T32HL007713-23.
Keywords: mTBI, hippocampus, theta oscillations, memory
The typical environmental enrichment (EE) paradigm, which consists of continuous exposure after experimental traumatic brain injury (TBI), promotes behavioral and histological benefits. However, rehabilitation is often abbreviated in the clinic and administered in multiple daily sessions. While recent studies have demonstrated that a once daily 6-hr bout of EE confers benefits comparable to continuous EE, breaking the therapy into two shorter sessions may increase novelty and ultimately enhance recovery. Hence, the aim of the study was to test the hypothesis that functional and histological outcomes will be significantly improved by daily preclinical neurorehabilitation consisting of two 3-hr periods of EE vs. a single 6-hr session. Anesthetized adult male rats received a controlled cortical impact of moderate-to-severe injury (2.8 mm tissue deformation at 4 m/s) or sham surgery and were then randomly assigned to groups receiving STD housing, a single 6-hr session of EE, or two 3-hr sessions of EE daily for 3 weeks. Motor function (beam-balance/traversal) and acquisition of spatial learning/memory retention (Morris water maze) were assessed on post-operative days 1–5 and 14–19, respectively. Cortical lesion volume was quantified on day 21. Both EE conditions improved motor function and acquisition of spatial learning, and reduced cortical lesion volume relative to STD housing (p < 0.05), but did not differ from one another in any endpoint (p > 0.05). The findings replicate previous work showing that 6-hr of EE daily is sufficient to confer behavioral and histological benefits after TBI and extend the findings by demonstrating that the benefits are comparable regardless of how the 6-hrs of EE are accrued. The relevance of the finding is that it can be extrapolated to the clinic and may benefit patients who cannot endure a single extended period of neurorehabilitation.
Supported, in part, by NIH grants HD069620, HD069620-S1, NS060005, NS084967 (AEK), NS094950, NS099683 (COB), and the University of Pittsburgh Physicians /UPMC Academic Foundation (COB).
Keywords: Controlled cortical impact, Morris water maze, Environmental enrichment
University of Washington, Seattle, USA
Keywords: Functional Outcomes, Peripheral injuries, Clinical Traisl
Uniformed Services University of the Health Sciences, Department of Anatomy, Physiology & Genetics, Bethesda, MD, USA
There is strong evidence to suggest a link between repeated head trauma and subsequent development of chronic traumatic encephalopathy (CTE), a degenerative neurologic condition resulting in motor, cognitive and neuropsychiatric symptoms. Clinical diagnosis of CTE is only definitive post-mortem, but symptomology and neuropathology can be modeled in pre-clinical settings. In this study, male and cycling female C57BL/6J mice underwent repeated (three) concussive brain injuries (rCBI) delivered via a Leica ImpactOne cortical impact device and were assessed either acutely or chronically (up to one year) on a range of motor, cognitive and neuropsychiatric tests. The day following the final injury, injured mice presented with normal locomotor behavior in the open field (OF) test but were impaired on the rotarod task of motor coordination. Rotarod performance was improved shortly following the injuries, but a state of hyperactivity developed during the month following rCBI as assessed in the OF test. Hyperactivity was especially pronounced in male mice, and persisted for at least one year following the injuries. Injured mice did not show impairments in the y-maze test of working memory shortly after the injuries, but mice tested one year post-rCBI were unable to learn a spatial active place avoidance task. Depressive-like behaviors were not apparent at either an acute or chronic time point. Anxiety-like behaviors assessed in the elevated zero maze were equivalent in injured and sham control mice shortly after injuries, but after one year, injured mice spent greater amounts of time in anxiogenic regions of the apparatus. These data demonstrate that a pathological phenotype with motor, cognitive and neuropsychiatric symptoms can be observed in an animal model of rCBI for at least one year post-injury, providing a pre-clinical setting for the study of the link between multiple brain injuries and neurodegenerative disorders.
Keywords: behavior, mouse, brain injury, sex differences
Traumatic brain injury causes severe and long-lasting cognitive consequences. Many individuals exhibit extended impairments and can even develop de novo psychiatric disorders. A large facet of these deficits and component of many psychiatric disorders is altered decision making. Sadly, these symptoms are grossly under-investigated in rodent models of traumatic brain injury. The current study examined one form of decision-making behavior: allocation of choice to probabilistic outcomes, a phenomenon that is most strongly associated with gambling.
Twenty-four rats were trained on the rodent gambling task (RGT) to a stable baseline (∼25 sessions). The RGT consists of four options, each associated with a reward size (1-4 sugar pellets) and a probability of winning that reward. Generally, optimal choice involves the low-risk, low-reward options which deliver the highest overall rate of reward as contrasted with the high-reward, but high-risk options. After training, one-half of rats were given a bilateral, frontal controlled cortical impact injury (+3.0, +0.0, −2.5 @ 3 m/s), while the other half received sham procedures. After a week of recovery, rats were re-tested on the RGT until a new stable baseline emerged (∼30 sessions). Following the behavioral assessment, rats were transcardially perfused. Brains were sliced and stained with cresyl violet to quantify the lesion.
Rats displayed an initial global disruption in behavioral function, with large increases in omitted trials, and low response rates. Response rates gradually increased over a period of two weeks. In the TBI group, choice was chronically altered, with an increased preference for the high-risk, high-reward options. These data pave the way for using rodent models of TBI to investigate the basis of altered decision making and any treatments that may benefit human patients.
Keywords: decision making, controlled cortical impact, rat, frontal cortex
Naval Medical Research Center/ Henry Jackson Foundation, Neurotrauma, Silver Spring, USA
Converging evidence suggests that changes in the vasculature and perivascular space are contributing factors to traumatic brain injury (TBI) resulting from exposure to blast overpressure (BOP). The injury mechanism is still largely unknown. The aim of this study is to characterize TBI resulting from single 37, 75, or 140 kPa BOP. The functional responses of arterioles in pial microcirculation were assessed in real-time in anesthetized rats using intravital microscopy (IVM). Specifically, cerebrovascular reactivity was studied using hypercapnia (7% CO2), barium chloride (BaCl2) and serotonin (5HT) 24 h after blast. To understand the observed changes in pial arteriolar reactivity with blast intensity, the expression of endothelin-1 (ET-1), endothelial nitric oxide synthase (eNOS), and vasopressin (AVP) was studied in frontal cortex. At 24 h after BOP, BaCl2 had an immediate statistically significant vasoconstrictive effect on pial arterioles, while 5HT and CO2 had a vaso-dilatory effect. The changes in pial arteriolar reactivity varied with blast intensity. The effects of BaCl2 (but not CO2 and 5HT) were different from control. Collectively, IVM data suggest that while pial microvessels remain reactive to vasoactive mediators after blast, their responses tend to be different from those of sham animals. Preliminary RT-PCR data show that blast alters mRNA expression for ET-1, eNOS, and AVP in an intensity-dependent manner 24 h post-BOP. Specifically, the higher the blast intensity, the smaller the observed changes in the ratio of mRNA relative to control. The results suggest the degree of functional changes in cerebrovascular responses may correlate with blast intensity and with molecular changes that affect the function of the endothelium, specifically, the expression of ET-1, eNOS, and AVP. Functional changes from low intensity BOP are influenced by ET-1, eNOS, and AVP but functional changes seen with higher BOP intensities may work through different mechanisms.
Keywords: pial microcirculation, intravital microscopy, vascular function, gene expression
This work was funded by work unit number 603115HP.2380.001.A1304. The study protocol was reviewed and approved by the Walter-Reed-Army Institute of Research/Naval-Medical-Research Center Institutional Animal Care and Use Committee in compliance with all applicable Federal regulations governing the protection of animals in research.
Keywords: Stress, Coagulation, survival, Polytrauma
CHRONIC EFFECTS OF BLAST EXPOSURE: A FUNCTIONAL STUDY IN RATS USING ADVANCED BLAST SIMULATOR
Anecdotal observations of blast victims indicate that significant neuropathological and neurobehavioral defects develop at later stages of life. In this study, we have examined neurobehavioral changes in rats up to 9 months after exposure to single and tightly coupled repeated blasts using an advanced blast simulator. Neurobehavioral changes were monitored at acute, sub-acute and chronic time points till 9 months using elevated plus maze, Y-maze, water maze, open field exploration, rotary pole, rotarod and novel object recognition tests. Single and closely coupled repeated blast exposures resulted in significant functional deficits at acute and chronic time points. In most functional tests, rats exposed to repeated blasts performed more poorly than rats exposed to single blast. Rotating pole and rotarod tests to assess the neuromotor/balance functions revealed significant deficits at acute and sub-acute time points after blast exposures, and all experimental subjects, including sham controls, had difficulties with these tests at chronic time points. Interestingly, several functional deficits were more pronounced in blast exposed rats at 6 months and beyond. The most substantial changes in blast-exposed rats were observed in the center time and margin time legacies in the open field exploration test at 6 and 9 months after blast exposures. Notably, these two outcome measures were minimally altered acutely, fully recovered during sub-acute stages, and were pronounced during the chronic stages after blast exposures. Significant neuromotor impairment occurs at early stages after blast exposure and the severity increases with number of exposures. Water maze test for spatial learning and memory revealed short-term memory impairments at chronic stages, but not at early time points. The pronounced changes in center time and margin time legacies in the open field exploration test after 6 months post-blast implicate development of depressive-like behavior.
Keywords: Chronic effects, Repeated blast, Advanced blast simulator, Overpressure
EPIGENETIC MECHANISMS IN BLAST INDUCED NEUROTRAUMA
Virginia Tech, Biomedical Engineering and Mechanics, Blacksburg, USA
Blast-induced neurotrauma (BINT) is becoming increasingly prevalent in military populations since a majority of combat injuries in current wartime efforts are attributable to explosive mechanisms. Civilian populations are also at risk due to ongoing terrorist events worldwide. BINT often occurs with no external signs of injury, making diagnosis and treatment difficult. Clinical manifestations of BINT include long-term functional and psychological impairments. These symptoms are driven by underlying cellular and molecular sequelae including blood brain barrier disruption, cell death, and sustained glial cell activation. The overall hypothesis of this work is that epigenetic regulatory mechanisms contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms, including DNA methylation and histone acetylation, are important processes that are capable of regulating cellular function in response to various environmental stimuli. An established rodent model of BINT was employed to assess epigenetic changes in the injury pathology. Relevant clinical outcomes including decreased motor function, increased anxiety and decreased memory were observed. Analysis of DNA methylation levels following blast exposure elucidated time-dependent and overpressure-dependent DNA methylation changes in the hippocampus. Increased DNA methylation was observed at four hours following blast which transitioned to decreased DNA methylation at one week before returning to sham levels at one month. Importantly, expression changes in several enzymes that regulate levels of methylated DNA residues were observed and may contribute to the observed pathological DNA methylation levels. We have previously demonstrated decreased histone acetylation of histones H2b, H3, H4 following blast exposure. H3 hypoacetylation was also observed specifically in astrocytes. Further Western Blot analysis showed increased nuclear localization of signaling molecules including nuclear factor kappa B which is a key mediator of inflammation. Taken together, the observed changes to DNA methylation and histone acetylation have a potentially broad impact on cellular function which may be important to the injury progression. Continuing to increase our understanding of BINT will mitigate current clinical obstacles by making the development of treatment strategies more efficient.
Keywords: Epigenetics, DNA methylation, Histone acetylation, Blast Induced Neurotrauma
Blast-induced Traumatic brain injury (bTBI) is a leading cause of morbidity and mortality in soldiers in the combat and in training sites and is becoming an important public health issue. The bTBI simultaneously causes primary (shockwave), secondary (penetrating), and tertiary (blunt-accelerative forces) injuries causing different loading modalities and pathological outcomes compared to pure blunt injury. The brain injury patterns resulting from primary shockwave propagation across different brain structures has not been carefully investigated. We hypothesize that bTBI has a unique histopatholgy and pathophysiology different from blunt TBI. In bTBI all the regions of brain from prefrontal cortex to hippocampus to cerebellum to brain stem are all simultaneously biomechanically loaded as the shock wave propagates unimpeded across the brain; this is different from the blunt TBI. Since oxidative stress has been implicated as a major pathogenic factor in TBI, here we examined the brain spatial resolution and cellular distribution of changes in superoxide producing enzyme NADPH oxidase1 (NOX1) as well as neurodegeneration paradigm during the early phase (4h) of moderate blast TBI (180 Kpa) and compared these data with that of moderate blunt TBI (FPI). Immunofluorescence analysis displayed that NOX1 was upregulated in different brain regions and showed a differential vulnerability with the highest increase found in hippocampus and thalamus followed by frontal cortex and cerebellum. Among different cells, neurons exhibit highest amount of basal NOX1 and its increase was higher in hippocampal and cerebellar regions compared to astrocytes and microglia. Interestingly, blunt injury induced in the cortical region did not display any change in NOX1 expression in cerebellum suggesting that the spread of the injury in blunt TBI is localized. Blast did not display neurodegeneration in any region, while blunt TBI showed focal neurodegeneration in the cortical and hippocampus areas only in ipsilateral side. These studies indicate that the injury pattern and pathological changes in blast TBI are unique compared to blunt injuries.
Keywords: blast TBI, blunt TBI, selective vulnerability
CHARACTERIZATION OF NEUROPATHOLOGY RESULTING FROM PRIMARY BLAST EXPOSURE IN A PORCINE TRAUMATIC BRAIN INJURY MODEL
Virginia Tech, Biomedical Engineering and Mechanics, Blacksburg, USA
There is a critical need for a translatable animal model so that human mild blast traumatic brain injury (bTBI) thresholds can be determined and appropriate standoff times can be implemented. Dissimilarities between a rodent and human brain has led to the need to investigate bTBI in a higher-order animal model. The purpose of this study was to characterize the effects of a single blast exposure on male Yucatan minipigs. Animals (5–7 months; n = 10 blast and 5 sham) were exposed to a single blast overpressure (40–72 psi) using a large-diameter mobile shock tube. Ear, eye, and torso protection was used to prevent other injuries. Brains were collected at 72 hours after injury, sectioned, and embedded in paraffin for immunohistochemical staining for Fluoro-Jade C (FJC), α-smooth muscle actin (α-SMA), macrophages/microglia (co-stain of cluster of differentiation 68 (CD-68)/ionized calcium-binding adapter molecule-1 or Iba-1), and β-amyloid precursor protein (βAPP). Quantitative analysis of the stained sections showed that FJC was significantly increased in the hippocampus (p < 0.01). CD-68, but not Iba-1, was significantly increased in the hippocampus (p < 0.01). α-SMA showed significantly increased surface vessel diameter in the frontal cortex, which may be caused by the activation or deactivation of pathways involved in vascular tone (p = 0.019). βAPP did not reveal any axonal swelling or disconnections. The increase of FJC and microglia/macrophage activation in the hippocampus, as well as lack of elevated βAPP found in this study supports previous research in rodent models of primary blast. In summary, a large animal model of primary blast overpressure demonstrated significant neuropathology. Further studies to characterize other brain regions are continuing and will be used to help determine a mild bTBI threshold that can be translated to a human.
The authors would like to acknowledge Dr. Timothy Bentley at the Office of Naval Research for funding this research.
Keywords: Blast, Neuropathology, Traumatic brain injury, minipig
James J Peters VA Medical Center, R&D, Bronx, USA
Injuries from exposure to explosive blasts rose dramatically during Operations Iraqi Freedom/Enduring Freedom, motivating investigations of blast-related neurotrauma. We have undertaken human studies involving “Breachers”, military personnel who are exposed to repeated blasts as their occupational duty. Breachers report physical, emotional, and cognitive symptoms: headache, sleep issues, anxiety. We aim to identify biomarkers associated with blast exposure. Cell-free DNA, derived from neurological tissue, holds potential as possible indicators of exposure detection. We exploit unique DNA methylation profiles of brain cells to create molecular diagnostic assays, capable of detecting peripheral brain-derived cfDNA. DNA methylation signatures in brain are unique from blood cells. We previously performed whole genome DNA methylation analysis, defining single-base pair maps of neurons (NeuN+) and non-neuronal (NeuN-) cell types. Leveraging from this unique data source, we constructed a targeted next generation sequencing platform to detect brain-derived cfDNA within peripheral blood. Data presented involve a subset of 32 individuals, participating in a 2-week data collection at U.S. Army explosive entry training sites, where blood samples were obtained pre- and post-training for methylation studies. Of these, we profiled brain-derived cell-free DNA within bloods from 6 breachers at days 1 (before training) and at days 7,8 and 9 (during training). On day 7, the cohort of breachers were exposed to higher (12 psi) blast. Generally, we observed higher amounts of brain-derived cfDNA during training (days 7, 8 and 9) than pre-training (day 1). To establish the presence of brain-derived cfDNA within peripheral blood associated with blast exposure represents an important step towards future biomarker development to diagnose and monitor potential blast-related TBI.
Keywords: Epigenetics, Blast, Inflammation
The original New Zealand breacher study (NZBS) reported significant, quantifiable acute neurocognitive deficits without any diagnosable injury; however, there is very limited data on neurocognitive effects immediately following overpressure exposure. This study measured neurocognitive performance immediately following heavy wall breaching during an Urban Mobility Breaching Course. The Defense Automated Neurobehavioral Assessment (DANA) Rapid test battery was administered to 24 volunteers before exposure (baseline), immediately after detonating two heavy wall breaching charges, and at the end of the day (acute). Blast sensors were used to quantify individual blast exposure. There were significant DANA performance changes (> 30% slower response time compared to baseline) in 2/22 volunteers for simple reaction time (SRT); 10/22 for procedural reaction time (PRT); and 5/22 for Go/No-Go (GNG) immediately following blast exposure. Significant acute effects were identified in 3/24 volunteers for SRT; 11/24 for PRT; and 7/24 for GNG. Volunteers with acute effects had significantly higher cumulative overpressure and impulse exposure compared to those who had no performance changes. The relationship between higher overpressure exposure and decrements in acute performance agree with those from the NZBS. The PRT subtask appears to be the most sensitive for quantifying immediate and acute blast exposure effects. SRT may be too effortless while GNG may be too stimulating, or subject recovery from transient blast effects may have occurred amid testing. The current study is one of the first to identify neurocognitive impairments immediately following blast exposure. These effects on higher order mental processes may have a significant impact on operational success and may also be important for determining long term health risk. Additional data from follow-on studies from the same training course will allow for more rigorous statistical analyses. This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, Broad Agency Announcement Award No. W81XWH-16-2-0001. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.
Keywords: occupational blast exposure, neurocognitive performance, neurotrauma, explosive entry training, military
EFFECT OF PHYSICAL PROTECTION AND INCREASED INTRACRANIAL PRESSURE ON THE PROPAGATION OF SHOCK WAVE TO BRAIN IN RATS
Naval Medical Research Center, Neurotrauma, Silver Spring, USA
During blast exposure the shock wave can be transmitted to the brain directly through skull or from the body through blood vessels and cerebrospinal fluid. This study evaluated the contribution of head and/or body in transferring blast to brain by measuring intracranial(ICP), arterial(AP), and venous(VP) pressures. Increasing the cerebral volume(CV) may have a protective effect against blast by limiting the relative motion of intracranial contents. Therefore, the protection conferred by increasing CV by internal jugular vein(IJV) compression was also investigated. Rats were exposed to blast (BOP, ∼47 kPa) with their bodies oriented parallel (front) or perpendicular (side) to the blast. Animals were instrumented with pressure probes in the right lateral ventricle, femoral artery, and femoral vein and were randomly assigned to groups with/without protective shielding. Front exposure groups included no protection (F-NP), full body protection excluding tail (F-FP), head protection (F-HP), and body protection (F-BP). Side exposure groups were exposed with no protection (S-NP), or head protection (S-HP). In IJV compression group, compression was applied by tightening a Velcro tape around the animal's neck. Additionally, BOP-induced(front-3x110 kPa, 30 min apart) effects on aquaporin-4 (AQP-4), 3-nitrotyrosine (3-NT), and endothelin receptor A (ETrA) in three groups of animals (control, blast, and blast-IJV) was determined. A significant decrease in shock wave transmission to brain was observed in F-FP, F-HP, and S-HP groups while F-BP offered no significant protection. AP and VP were significantly reduced in F-BP and F-HP. A significant difference was observed in ICP and AP between F-NP and S-NP as well as F-HP and S-HP groups. IJV compression diminished the blast-induced pressure increase, prevented increase in 3-NT and ETrA in cortex, and attenuated the upregulation of AQP-4 and ETrA immunoreactivity in hippocampus. Body protection has limited preventive effect and for brain protection the head and neck should be completely shielded.
Keywords: Blast propagation, Intracranial volume, Shock wave, Protection against blast
The Defense and Veterans Brain Injury Center (DVBIC) publishes the incidence of service members (SMs) who have sustained at least one traumatic brain injury (TBI) while serving in the military since the year 2000 on its website (
Keywords: Military, concussion, combat, Surveillance, blast
CHARACTERIZATION OF A COMBINED MODEL OF BLAST AND BLUNT INJURY
Soldiers are often exposed to both primary blast waves and blunt impacts due to falls or vehicle crashes. While the cumulative effects of repeated exposure to a blast or blunt traumatic brain injury (TBI) has been under investigation, a combination of blast and blunt impacts has yet to be examined. Our hypothesis is a blast exposure, while not necessarily leading to a diagnosable injury, will predispose the brain to greater injury upon a second blunt injury to the head. Here, rats were exposed to primary blasts (130 KPa - mild or 180 KPa – moderate) followed by a fluid percussion injury (FPI - 29–32 psi) to examine if a blast exposure would exacerbate the injury outcome of FPI in the rat. The animals were first subjected to the blast then the craniectomy was performed while still under anesthesia and 24 hrs later, the rats were subjected to the FPI. Control groups of blast +craniectomy, FPI only and craniectomy alone were also generated. Time-matched to 4 hr after FPI, coronal sections from all experimental groups were stained with fluoro jade C. Acute neuronal degeneration was observed in the cortex, hippocampus and thalamus regions of the animals subjected to the blunt and blast +blunt group while blast alone group showed no neurodegeneration. Neurodegeneration was more pronounced in cortical region in both mild and moderate blast +blunt groups than the blunt impact group alone, while respective controls (blast +craniectomy or craniectomy alone) did not show any neuronal degeneration. Hemorrhage was also observed in the moderate blunt and combined impacts than the blast impacts. Potential changes in apnea and the righting reflex are so far inconclusive. Our data indicate that a prior blast impact increases the induced neuronal degeneration from a subsequent blunt impact. Ongoing studies aim to identify the longer term effects on cell death as well as behavioral and biochemical deficits present in the combined model.
Keywords: Combinational injury, fluid percussion injury
SPATIAL AND TEMPORAL DEFORMATION PATTERNS OF THE BRAIN UNDER BLUNT AND BLAST TRAUMA
It is widely accepted that trauma to the head causes the brain to deform inside the skull, creating large strains that cause damage. It is unknown, however, how the brain deforms in many types of trauma such as sports contact, falls and exposure to blasts. We hypothesize that each trauma to the head will lead to distinct magnitudes and rates of deformation that varies spatially and temporally across the brain. The objective of this study was to recreate spatial and temporal deformations that are likely to occur in blast and blunt injuries. For this study a full scale human head surrogate was created from a PVC skull, ballistic gel brain, and hybrid III anthropomorphic neck. Blunt injuries were created with a 2 kg impactor on a drop tower system at velocities of 3&5 mph. For blast injury overpressures of 180 kPa were generated in a field-validated shock tube. Markers in the brain were motion tracked from high speed video and analyzed to compute skull deflections, principal strains and the associated strain rates across the brain. Contour maps were generated to view the spatial and temporal distribution of strains caused by each injury event. In blunt injury, the span and degree of tissue deformation were localized in particular regions and unique to the biomechanical parameters of the blunt injury event (velocity, momentum, direction). 5 mph impacts induced higher strains than 3 mph while crown injuries caused larger skull deflections as well as higher strains and rates than frontal impacts. Preliminary blast experiments produced more widely distributed contour profiles of strain fields within the brain. Maximum principal tensile, compressive, and shear strains were surprisingly high 52–55% that oscillate and decay over several seconds. The data from these experiments may help to identify vulnerable injury sites with more understanding of the relative effects of specific injury loading conditions. This model can act as a platform to build more complex and biofidelic heads as well as a metric to evaluate helmet designs.
Keywords: TBI, Biomechanics, Blast and Blunt trauma, Strain and rate
ACTIVATION OF NLRP3 INFLAMMASOME AND ITS IMPACT ON CEREBRAL AUTOPHAGY MECHANISMS IN SEVERE BLAST-INDUCED TRAUMATIC BRAIN INJURY
Blast-induced traumatic brain injury (bTBI) is the most frequent cause of morbidity and mortality in soldiers in the combat zone as well as in training facilities, which represents a challenging task for the management of patients returning from combat injuries. The bTBI uniquely impacts on the brain by simultaneously causing primary (shockwave), secondary (penetrating), and tertiary (blunt-accelerative forces) injuries that cumulatively exacerbate the pathological outcome. Of several factors that induce brain pathology in TBI, neuroinflammation is an important pathway triggered by upstream factors including the activation of Toll-like receptor4 (TLR4) that transcriptionally activates the synthesis of pro-IL-1β, a precursor of inflammatory cytokine IL-1β, resulting in the production of functional IL-1β by the activation of NLRP3 inflammasome, an assembly of several proteins including apoptosis-associated speck-like protein (ASC1) and caspase-1 (IL-1β converting enzyme). IL1-β promotes a variety of neuroinflammatory responses by altering cellular homeostatic events such as autophagy, a physiological process responsible for lysosomal-dependent degradation of senescent cytoplasmic proteins, intra-cellular organelles as well as toxic protein aggregates. A hallmark of defective autophagy is the accumulation of autophagosome marker LC3, a Microtubule-associated protein that fuses with autophagosome and promotes its degradation. Here we examined regional variations in the brain (hippocampus, thalamus, visual cortex and cerebellum) levels of TLR4, pro-IL-1β, NLRP3 as well as the levels of autophagy marker LC-3 in acute phase (4 h) of severe-blast TBI (240 Kpa) in rats. Immunoblot analysis showed a regional variability in the protein levels of these markers with hippocampus displaying highest increase in TLR4, Pro-IL1 and NLRP3. Additionally, bTBI caused a pronounced accumulation of LC3 protein in hippocampus, which suggests a defective autophagy, process likely mediated by increased neuroinflammation. These data collectively suggest that severe bTBI induces acute neuroinflammatory response by triggering a cascade of events potentially resulting in perturbations in autophagy that may represent an important neuropathological event in bTBI.
Keywords: Autophagy, Blast-TBI, Inflammasome, Interleukin-1-beta, Toll-like Receptor
EFFECTS OF BLAST TRAUMATIC BRAIN INJURY ON REGIONAL CELLULAR INJURY
Neuronal injury in specific brain regions following impact TBI likely contributes to the nature and degree of brain dysfunction. Previous studies that employed the FluoroJade (FJ) fluorochrome stain for labeled identification of blast-induced TBI (bTBI) neuronal injury or degeneration resulted in significantly increased FJ-positive neurons1 in rat hippocampus,2-3 amygdala4 and pre-frontal cortices5 after 17–19 psi shockwave exposure and in the cerebellum after 62-75 psi exposure.6 We examined neuronal injury through FluoroJade-C (FJC) utilization in three brain regions of rats subjected to mild bTBI using an Advanced Blast Simulator (ABS), a compressed air-driven shock tube.
Adult male Sprague-Dawley rats were surgically prepared for bTBI and randomly assigned to receive (n = 6/group) bTBI (20 psi) or Sham bTBI. 24 or 48 hrs after injury, brains were harvested, sectioned on a Cryostat and stained with FJC. FJC-positive cells were counted in the frontal lobe (F), parietal/temporal lobe (PT) and occipital lobe regions (O) of all bTBI and Sham groups.
FJC-positive cells were significantly greater in both bTBI groups 24 and 48 hrs post-injury compared to Sham (P < 0.05, bTBI vs. Sham), with significantly more FJC-positive cells in all bTBI group brain regions (P < 0.001, bTBI F vs. Sham F; P < 0.05, bTBI PT vs. Sham PT; P < 0.05, bTBI O vs. Sham O). FJC-positive cells were significantly higher in the PT than both the F and O regions amongst the bTBI groups (P < 0.05, bTBI PT vs. bTBI F; P < 0.05, bTBI PT vs. bTBI O) while FJC-positive cells in the F region of both bTBI groups were significantly greater than in their O regions (P < 0.01, 24 hr bTBI F vs. 24 hr bTBI O; P < 0.01, 48 hr bTBI F vs. 48 hr bTBI O).
Our data indicates that mild bTBI results in significantly acute neuronal injury 24 and 48 hrs post-blast with increased numbers of FJC-positive cells throughout the parietal/temporal cortex compared to the frontal or occipital regions.
Studies were conducted as part of a team supported by The Moody Project for Translational Traumatic Brain Injury Research.
Keywords: blast induced neurotrauma, primary blast injury, acute neuronal injury, FluoroJade-C stain, histopathology
RESTORING GM1 GANGLIOSIDE EXPRESSION AMELIORATES AXONAL OUTGROWTH INHIBITION AND COGNITIVE IMPAIRMENTS INDUCED BY BLAST TRAUMATIC
Sourasky Medical Center & Tel Aviv University Sackler Faculty of Medicine, Department of Psychiatry, Tel Aviv, Israel
Keywords: Axonal outgrowth inhibition, Regeneration inhibiton
New Jersey Institute of Technology, Biomedical Engineering, Newark, USA
Proteomics offers unique insight into etiology of brain injury by providing unbiased information about large dataset of proteins involved in variety of cellular processes. Simultaneous label free fingerprinting readily available with an aid of bioinformatics data mining backed by differential expression information allows identification of major biochemical pathways. These attractive traits perfectly suited for cross-sectional characterization were exploited in this work to demonstrate fundamental attributes of blast TBI (bTBI) in hippocampus. Ten weeks old Sprague Dawley rats were exposed to single blast at 130 kPa intensity and sacrificed 24 hours post exposure. Brains were homogenized and thirty micrograms of proteins from each sample were subjected to separation followed by digestion. Peptides were labeled by 8-plex iTRAQ labeling, combined, separated by high pH Reverse-phase Liquid Chromatography (RPLC) and further analyzed by RPLC-MS/MS on Q Exactive™ Hybrid Quadrupole-Orbitrap mass-spec. Protein identification: mass spectra were searched against Uniprot rat database via Mascot 2.4 search engine on Proteome Discovery 1.4 platform. Differential hippocampal proteome analysis in blast exposed rats (n = 4) identified 4991 proteins: 219 upregulated and 29 downregulated (p < 0.05). Further analysis revealed there are two major canonical pathways involved in pathology of blast TBI: oxidative phosphorylation and mitochondrial dysfunction. These pathways include changes in mitochondrial electron transport/respiratory chain proteins: 1) cytochrome c oxidase (6 fragments), 2) NADH: ubiquinone (16 fragments), 3) ATP synthase (H+) (5 fragments), 4) NADH dehydrogenase (2 fragments) and ubiquinol-cytochrome c complex (2 fragments). These proteins were mostly upregulated suggesting rapid activation of repair mechanism merely 24 hours post injury. Overall pathway analysis identified total of 70+ canonical pathways potential for etiological, therapeutic and diagnostic applications.
Keywords: proteomics, hippocampus, oxidative phosphorylation, mitochondrial dysfunction
National Institute on Aging, NIH, Drug Design & Development Section; Translational Gerontology Branch, Baltimore, USA
Blast traumatic brain injury (B-TBI), a prevalent form of injury observed in modern warfare, is caused by generation of a blast shockwave which is induced by the detonation of high explosives. Presently there is no FDA approved medication for the treatment of B-TBI. We investigated the benefits of a FDA approved medicine, Exendin-4 (Exenatide, Ex-4) a long acting glucagon-like peptide 1 receptor agonist, with known neuroprotective/neurotrophic/anti-inflammatory properties (Salcedo et al., Br J Pharmacol 166:1586, 2012) in an open field B-TBI model (Rubovitch et al., Exp Neurol 232:280, 2011). B-TBI induced lasting cognitive impairments in novel object recognition and less severe deficits in Y-maze behaviors. These deficits were associated with B-TBI-dependent diffuse neuronal loss and reductions in the levels of synaptophysin protein staining in cortical and hippocampal tissues. Changes in gene expressions linked to dementia disorders also were evident. Treatment of animals with a clinically translatable dose of Ex-4 delivered by subcutaneous micro-osmotic pumps to maintain steady-state drug levels for 7 days starting either 48 hours prior to or 2 hours immediately following B-TBI prevented the induction of cognitive deficits, injury-induced neuronal loss, changes in synaptophysin staining, and largely reversed gene expression changes. These data indicate that treatment with the clinically approved drug, Ex-4, represents a viable option for the management of secondary events triggered by blast-induced, mild traumatic brain injury that is commonly observed in militarized zones. Current studies are optimizing Ex-4 administration to support rapid clinical translation.
Keywords: blast injury, novel object recognition, Y maze, synaptophysin, Therapeutics
Our goal was to investigate the effects of single vs. repeat blasts on the optic nerve and assess protection by antioxidants. We exposed one eye of an anesthetized C57Bl/6 mouse to one or multiple bursts of overpressure air. Blast and sham mice were given either regular or high vitamin C&E diet ad libitum, beginning one month before blast. At one month post-injury, optic nerves and retinas were collected. Changes in optic nerve histology and the visual evoked potential were quantified. Multiplex ELISAs were performed to assess activation of sterile inflammation. In the C57Bl/6 mouse, axon degeneration and vision loss are first detectable at one month post-trauma in our model, using the single blast paradigm. At one month total axon count and healthy axon count were decreased, and glial area increased, in the repeat blast paradigm as compared to both shams and the single blast paradigm. Markers of sterile inflammation (IL-1a, IL-1b, IL-18) were increased in both blast paradigms at one month post-injury, but not with vitamin C&E treatment. Visual function was also preserved with vitamin C&E treatment. Axon degeneration is increased after repeated blasts as compared to a single blast, even at a lower pressure. Sterile inflammation may contribute to axon degeneration after trauma. Treatment with antioxidants decreases markers of sterile inflammation and mitigates loss of visual function after blast.
Keywords: Repeat Blast, Ocular Trauma, Optic Nerve Injury, Antioxidants
A BIOMECHANICAL, HISTOLOGICAL AND COMPUTATIONAL MODELING STUDY OF BRAIN RESPONSE TO FREE-FIELD BLAST
To date, the underlying mechanisms of primary blast induced traumatic brain injury (pbTBI) are still not fully understood. Much of work on in vivo pbTBI has been conducted with rats and swine using a shock tube. The current communication describes a comprehensive study aimed to characterize the blast physics, biomechanical and pathophysiologic features of the operational relevant animal models subjected to open-field blast. The experiments (animal and human cadavers) also provided data for the validation of the computer models and the development of biomechanical correlates at tissue-cellular level. The open-field blast was generated by 3.6 kg of C4 charge to produce incident overpressures (IOP) of three intensities (148.78 ± 3.70, 278.90 ± 36 09.20 ± 18.89 kPa) in frontal blast. One group of anesthetized swine (male Yucatan, n = 5) was instrumented with sensors for measuring intracranial pressure (ICP) in various regions of the brain and the head motion. The other group of swine (n = 12) was exposed to single blasts of medium or high IOP or no pressure (sham, n = 5) for neuropathological analysis. After a 3-day survival period, beta amyloid precursor protein (β-APP), glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule (Iba1) immunohistochemistry was used to assess axonal injury, astrocyte and microglial proliferation. The measured peak ICP in swine brain increased with increasing IOP levels. Peak resultant head acceleration (< 500g) correlated well with peak IOP. The arrival of the ICP was almost simultaneous with head motion indicating that it was due to the pressure wave and not the blast wind. Significantly high β-APP, astrocyte and microglial counts were found in the blast groups compared to sham. The human head computer model predicted coup-contrecoup phenomena consistent with the ICP data. The peak maximum principal strain in the brainstem and frontal lobe were comparable and higher than responses in other regions. The correlation of the swine model responses with pathological changes may lead to development of tissue-level criteria for primary blast-induced TBI.
Keywords: Open-field blast, Blast induced traumatic brain injury, Swine model, Intracranial pressure measurement, Neuronal injury, Glial change
THE ROLE OF ENDOGENOUS AND EXOGENOUS CORTICOSTERONE LEVELS IN THE (MAL)ADAPTIVE RESPONSE TO LOW-PRESSURE BLAST WAVE
Ben-Gurion University of the Negev, Faculty of Health Sciences, Beer Sheva, Israel
Keywords: HPA-axis, Corticosterone, first aid treatment, Serum
Ben-Gurion University of the Negev, Faculty of Health Sciences, Beer Sheva, Israel
Blast wave-induced minimal traumatic brain injury (mTBI) and post-traumatic stress disorder (PTSD) have become one of the most frequent injuries in the military and civilian health care sectors since the increase in worldwide terrorism and warfare. Those injuries tend to become chronic injuries in the absence of early diagnosis and intervention. Therefore, early identification of the affected population is of great clinical importance for treatment planning, and rehabilitation assessment for patients. In this study, we determined the levels of Cell Free DNA (CFD) in serum at different intervals after low-pressure blast wave exposure to evaluate its potential utility as a sensitive biomarker of blast-induced (psycho)pathology. Non-anesthetized rats were exposed to low pressure blast-wave. Blood was obtained at a number of pre and post exposure time-points. Simultaneously, combining of cognitive and behavioral paradigms were used in order to determine the rats' performance. The results showed that blast exposure caused significant time-dependent increases in serum CFD. The level of CFD increased significantly at 2 h post-exposure and was independent of the psychopathology or to the blast overpressure. For most of the rats, the maximum increase was recorded at 2 h, and CFD levels returned to baseline by 5 h post exposure. In rats displaying PTSD-phenotype or mTBI-phenotype, the maximum level of circulating CFD was observed at 2 h post exposure and was still significantly elevated at 5 h after blast exposures. CFD levels may be used as a marker to assess psychopathology after blast exposure.
Keywords: Cell Free DNA, First aid treatment, Corticosterone, Blood serum
Karyopharm Therapeutics, Newton, USA
Traumatic brain injury (TBI) significantly increases the risk of developing other neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and epilepsy. Simply being part of a high contact activity can increase the likelihood of developing neurodegenerative disorders, e.g., US military veterans are twice as likely as the general population to develop ALS. The connection is strongest with multiple or more severe head injuries and in the years following injury. ALS risk is increased 3-fold within 10 years of a severe head injury and 11-fold within 10 years of multiple head injuries. Epilepsy risk is increased: 10-fold with a skull fracture; 5-fold with a severe TBI; 3-fold with a mild TBI; and 38-fold within the first year of TBI. These correlations are, in part, due to the degradation of the blood brain barrier (BBB), a pathological hallmark of TBI. Karyopharm Therapeutics pioneered the development of selective inhibitors of nuclear export (SINE), which inhibit the nuclear export protein Exportin 1 (XPO1). XPO1 traffics over 200 cargo proteins to the cytoplasm, including critical components of pro-inflammatory pathways. Previous research has demonstrated that the SINE compound KPT-350 significantly reduces post-TBI cortical hyperexcitability (strongly correlated with ALS), as well as epileptiform activity, in a mouse controlled cortical impact (CCI) model of TBI. Here, the effect of KPT-350 on BBB integrity was assessed in a rat CCI model of TBI by administering the compound as a single dose or q.d. for 3 days beginning 2 hours post-injury. Using the Evans Blue extravasation method, it was determined that both groups treated with KPT-350 showed significant improvements in BBB integrity relative to vehicle-treated groups. This set of data suggests that KPT-350 has significant potential as an anti-inflammatory and neuroprotective therapeutic for TBI and associated neurological disorders, possibly by preserving BBB integrity.
Keywords: Amyotrophic Lateral Sclerosis, neuroprotection, anti-inflammatory, neurodegenerative
Virginia Commonwealth University (VCU), Richmond, USA
Keywords: blood-brain barrier, caspase-3-cleaved tau, chronic TBI, cleaved-caspase-3, GFAP
University of Pennsylvania, Neurosurgery & Penn Center for Brain Injury and Repair, Philadelphia, USA
Mild traumatic brain injury (mTBI), or concussion, is a substantial public health problem that can result in persistent and debilitating symptoms in a subset of patients. Moreover, TBI, including repetitive concussion, has been linked to syndromes of progressive neurodegeneration such as chronic traumatic encephalopathy (CTE). However, the biology of mTBI and its link to late neurodegeneration remain poorly understood. A prominent feature of moderate or severe TBI at acute and late survival time-points is widespread disruption in blood brain barrier (BBB) integrity. However, it is unknown whether a single mTBI alone is sufficient to precipitate altered BBB integrity. Using an established swine model of pure rotational mTBI, animals were subjected to single mTBI or sham conditions (n = 3–4 per group) and tissue was prepared for immunohistochemical analyses at 6-72 hours post-injury. Following exposure to mTBI, widespread, abnormal extravasation of blood-borne proteins fibrinogen (FBG) and immunoglobulin-G (IgG) into the brain parenchyma was identified at all time points post-injury, despite an absence of hemorrhage. Typically, this appeared in a stereotyped distribution at structural interfaces, indicating a biomechanical etiology; although, notably, there was incomplete regional overlap between evidence of BBB disruption and axonal pathology. Triple immunofluorescence labeling revealed perivascular cellular uptake of both FBG and IgG in astrocytes (GFAP-positive), but not microglia (Iba1-positive) at these acute post-injury time-points. These data indicate that widespread BBB disruption can be demonstrated as a component of just a single mTBI using this swine model of pure rotational injury. The role of mTBI-induced BBB disruption in the acute presentation and outcomes of concussion, as well as its potential contribution to the late neurodegenerative pathologies of CTE, will be important to examine. Grant Support: DOD: PT110785, NIH: NS056202 and NS038104, The McCabe Fund (Univ. Pennsylvania)
Keywords: TBI model
Yale University, Pharmacology, New Haven, USA
Physical trauma to the nervous system produces abnormal patterns of neuronal firing that can eventually lead to neuronal death through mechanisms such as excitotoxicity. Mutations that alter neuronal excitability can also predispose neurons to death. One example of this is Spinocerebellar Ataxia 13 (SCA13), which is caused by mutations in the Kv3.3 voltage-dependent potassium channel, and leads to either early-or late-onset degeneration of the cerebellum. We have found that depolarization of Kv3.3 channels results activation of TANK-Binding kinase-1 (TBK1), an enzyme that controls autophagy and mitophagy. Activation of this enzyme represent a “non-conducting” function of the channel, as it does not require ion flow through the channel itself, and most likely results from conformational changes in cytoplasmic domains of the channel following depolarization. A late-onset mutation in this channel, G952R Kv3.3, leads to greatly potentiated activation of TBK1. Transfection of cell lines with G952R Kv3.3 leads to activation of caspase 7, dense accumulations of multivesicular bodies and presumed autophagosomes that contain the cell survival protein Hax-1, and a significant increase in the rate of cell death. In cerebellar Purkinje cells in the intact nervous system, plasma membrane Kv3.3 channels are closely associated with underlying mitochondria and ER membranes. As in transfected cells, Purkinje cells in mice expressing the G952R Kv3.3 mutation have elevated levels of TBK1 and enhanced TBK1 activity. Electron immunomicroscopy indicates that this enhanced TBK1 activity is associated with the accumulation of dense multivesicular organelles that contain both TBK1 and Hax-1 and that appear to contain mitochondria destined for degradation. Our results indicate that activation of the Kv3.3 potassium channel directly activates cell survival/death pathways, and suggest that abnormal channel activation, as occurs with channel mutation and may also occur with physical injury, promotes neuronal death.
Keywords: potassium channels, mitochondria, mitophagy, action potential firing
Patients' condition may deteriorate after experiencing brain trauma. Edema and hematoma develop at the site of cortical contusion. An increase in inflammation, cell death, and axonal injury is observed in the affected brain regions. To understand the mechanisms of progression of traumatic brain injury (TBI), we hypothesized that cells in the brain acquire senescence, owing to trauma and contribute to the development of TBI. Cellular senescence refers to irreversible arrest of cell division possibly due to stress and damage from exogenous and endogenous stimuli. Senescent cells (SCs) secrete various factors including inflammatory cytokines and proteases that can contribute to tissue dysfunction. The first marker used for identification of SCs is the senescence-associated β-galactosidase (SA-βgal). In addition, p16, an important regulator of senescence, is useful in detecting SCs. We performed histochemical and immunohistochemical analyses for SA-βgal and p16 in brain sections at 1, 4, 7, and 14 days after trauma in a mouse model of controlled cortical impact. On comparing the data from the control and TBI groups, we found that SA-βgal was preferentially expressed in the cortex ipsilateral to the site of injury at 4, 7, and 14 days after trauma. Immunohistochemical analyses showed that macrophages and microglia expressed SA-βgal. We also observed p16 immunoreactivity in the cortex ipsilateral to the site of injury at 4, 7, and 14 days after trauma. p16 immunoreactivity was observed in astrocytes. These findings suggest that SCs are associated with TBI. Since SCs are deleterious to brain tissue, their presence may contribute to the progression of TBI. SA-βgal and p16 are not exclusively expressed in senescence, but they can also be expressed in other types of stress responses. Further studies are required to understand and interpret these results.
Keywords: Cellular senescence, Senescent cells, Senescence-associated β-galactosidase, p16
SPINAL CORD INJURY-MEDIATED CPLA2 ACTIVATION CONTRIBUTES TO LYSOSOMAL DEFECTS LEADING TO IMPAIRMENT OF AUTOPHAGY
University of Maryland, Baltimore, Department of Anesthesiology & Center for Shock, Trauma and Anesthesiology Research (STAR), Baltimore, USA
The autophagy-lysosomal pathway plays an essential role in cellular homeostasis and a protective function against a variety of diseases. However, under certain circumstances pathologically increased autophagy can contribute to cell death. This may occur particularly when lysosomal function is impaired and autophagic degradation is not able to proceed to completion, leading to pathological accumulation of dysfunctional autophagosomes. We have previously shown that autophagy is inhibited and contributed to injury after SCI. Here we examine mechanism of autophagy and lysosomal defects following SCI. Expression levels and processing of the lysosomal enzyme cathepsin D (CTSD) were decreased at 2 h after SCI. Enzymatic activity of CTSD and another lysosomal enzyme, alkaline phosphatase, were decreased 24 h post-injury, indicating lysosomal damage. Sub-cellular fractionation confirmed lysosomal membrane permeabilization (LMP) and leakage of lysosomal content into the cytosol. cPLA2 is an enzyme that cleaves fatty acyl linkage in the phospholipids of cellular membranes and increased activity of cPLA2 may be involved in membrane damage. cPLA2 was activated in the lysosomal fraction, accompanied by increased accumulation of the autophagosomal marker LC3-II and its substrate p62. To directly assess the extent and mechanism of damage to lysosomal membranes, mass spectrometry (MS)-based lipidomics was applied to compare the lipid composition of lysosomal membranes purified from sham or injured spinal cord at 2 h post-injury. Our data demonstrate increases in several classes of lysosophospholipids- the products of phospholipases (PLAs), as well as accumulation of PLA activator, ceramide. Inhibition of cPLA2 decreased lysosomal damage, restored autophagic flux, and reduced neuronal cell damage. Taken together our data implicate lysosomal defects in the pathophysiology of SCI and further indicate that cPLA2 activation leads to lysosomal damage that causes neuronal autophagosome accumulation associated with neuronal cell death.
Keywords: spinal cord injury, autophagy, lysosomal damage, cPLA2
University of Maryland School of Medicine, Anesthesiology & Shock, Trauma and Anesthesiology Reserch Center, Baltimore, USA
Necroptosis, a form of regulated necrosis mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3, respectively), is induced following spinal cord injury (SCI) and thought to contribute to neuronal and oligodendrocyte cell death. However, mechanisms leading to activation of necroptosis after SCI remain unclear. We have previously shown that autophagy, a catabolic pathway facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner, is inhibited following SCI in the rat. Our current data confirm that inhibition of autophagy is also occurring after T10 contusive SCI in the mouse model, as indicated by accumulation of both the autophagosome marker, LC3-II and autophagy cargo protein, p62/SQSTM1. This was most pronounced in the ventral horn motor neurons and was caused by rapid inhibition of lysosomal function after SCI. Interestingly, RIPK1, RIPK3 and the necroptosis effector protein MLKL also rapidly accumulated after SCI and specifically localized to motor neurons with disrupted autophagy, suggesting that the two events may be related. To determine if lysosomal dysfunction could contribute to induction of necroptosis, we treated PC12 cells and cultured rat cortical neurons with lysosomal inhibitors, Bafilomycin A and Chloroquine. Both treatments led to rapid accumulation of RIPK1 and RIPK3, confirming that they are normally degraded by the lysosomal pathway. In PC12 cells lysosomal inhibition also sensitized cells to necroptosis induced by TNFα and caspase inhibitor Boc-D. Imaging studies confirmed that RIPK1 partially localized to lysosomes in both untreated and lysosomal inhibitor treated cells. Similarly, we observed accumulation of RIPK1 in both cytosol and lysosomes after SCI in vivo. Therefore, lysosomal dysfunction after SCI may contribute to both inhibition of autophagy and sensitize cells to necroptosis by promoting RIPK1 and RIPK3 accumulation.
Support: R01NS094527, R01NS091218
Keywords: necroptosis, autophagy, lysosomal damage, spinal cord injury
University of Maryland School of Medicine, Anesthesiology & Shock, Trauma and Anesthesiology Research Center, Baltimore, USA
Autophagy, a lysosome-dependent intracellular degradation pathway, plays an essential role in neuronal homeostasis and is compromised in neurodegenerative diseases. We recently demonstrated that autophagy is inhibited and contributes to neuronal cell death after traumatic brain injury (TBI) in mice. This was caused by lysosomal dysfunction, evidenced by lower protein levels and activity of lysosomal enzymes in day 1 injured cortex. Furthermore, we observed leakage of soluble lysosomal enzymes into the cytosol, indicating lysosomal membrane permeabilization (LMP). This correlated with the activation of cytosolic phospholipase A2 (cPLA2), en enzyme that cleaves fatty acyl linkage in the phospholipids of cellular membranes. At early time points activation of cPLA2 was observed specifically within neurons and co-localized with markers of lysosomal dysfunction. Consistent with the possibility that cPAL2 activity may damage lysosomal membranes, we detected lysosomal damage and LMP in vitro in H4 cells and rat cortical neurons treated with cPLA2 activator ceramide-1-phosphate (C1P). This was accompanied by inhibition of autophagy flux, and was prevented by knock-down of cPLA2 or pretreatment with cPLA2 inhibitor, AACOCF3. To determine if cPLA2 directly injures lysosomal membranes in vivo after TBI, we performed MS-based lipidomic analysis of purified cortical lysosomes. We detected specific increase in phospholipase products, lysophospholipids, in lysosomal membranes from injured as compared to naive mice. Confirming involvement of cPLA2, treatment with AACOCF3 attenuated lysosomal membrane damage and LMP after TBI. This correlated with decrease in accumulation of autophagy markers LC3-II and p62, indicating improved autophagy flux. At 4 weeks after TBI we observed improved motor and cognitive function in mice treated with AACOCF3 as compared to vehicle controls. Together these data indicate that activation of cPLA2 mediates lysosomal damage after TBI, causing inhibition of autophagy and contributing to neuronal cell death and poor outcomes.
Keywords: lysosomal damage, autophagy, cPLA2, traumatic brain injury, lipidomics
Opioids are among the most effective and commonly prescribed analgesics for the treatment of acute pain after spinal cord injury (SCI). We have shown, however, that administration of morphine in the early phase of SCI undermines locomotor recovery in a rodent contusion model. Further, we found that morphine administration is associated with decreased expression of neuronal and astrocytic markers post SCI. Increased neuronal death may lead to further loss of function. Whether morphine increases cell death or decreases the expression of cell specific markers is unknown. To test this, the current study used Caspase-3, as an apoptotic marker, together with specific neuronal and astrocytic markers, and analyzed the temporal sequence of cell loss with morphine administration. Subjects were given a moderate spinal contusion injury or were sham controls. On the day following surgery, half of the subjects in each injury condition were treated with 10 mg of morphine (i.v.) on days 1–2, 20 mg on days 3–4, and 30 mg on days 5–7. The remaining subjects served as controls, receiving an equivalent volume of 0.9% saline across days. To assess the temporal sequence of cell loss, subjects were euthanized on days 2, 4, or 8 (24 hrs after the final dose of morphine). A 1.5 cm section of injured spinal cord was collected and sectioned for immunohistochemistry. We found that morphine-treated subjects had increased co-localization between Caspase-3 and NeuN at 4 days post injury, relative to controls. By contrast, morphine did not increase co-localization between GFAP and Caspase-3, despite the decreased expression of astrocytes at 7 days. These data suggest that while morphine may induce apoptosis in neurons in vivo, astrocyte loss might be mediated by an alternate mechanism. Given the clinical utility of opioid analgesics, it is imperative that we identify the molecular mechanisms mediating the adverse effects of morphine in the rodent SCI model. We must develop safe and effective therapeutic strategies for the use of opioids in pain management after SCI.
Keywords: Spinal Cord Injury, morphine, apoptosis, opioids, locomotor recovery
EVIDENCE FOR THE IMPORTANCE OF THE RIPK3 SCAFFOLD FUNCTION IN THE PATHOGENESIS OF CELL DEATH, INFLAMMATION, AND FUNCTIONAL OUTCOME
Keywords: TBI, cell death, inflammation, IL-1beta, HMGB1, behavior
New York City Office of the Chief Medical Examiner, New York, USA
Neuropathologic descriptions of abusive head trauma (AHT) are limited to short-term survivors, despite the fact that long-term neurologic sequelae are well-described. In particular, the role of tau protein aggregation in pediatric AHT is unclear. From the New York City Office of the Chief Medical Examiner we report a series of five cases of AHT (1 blunt impact, 4 “shaken”) with long-term post-injury survival. The mean age at injury was 9.8 months (range, 2–18 months) and the mean age at death was 16 years (range, 10–25 years). Mean survival was 15.3 years (range, 9.3–23.5 years). The cause of death was certified as long-term sequelae of head trauma in all cases. Manner of death was homicide in three, and undetermined in two. One case had a history of cocaine intoxication at birth; one had neonatal hyperbilirubinemia, then failure to thrive at time of injury. All had severe intellectual disability post-injury and four (80%) had seizures. All cases showed micrencephaly with relative sparing of the posterior fossa. Mean brain weight was 502 g (range, 310–800 g). Two of five cases (40%) had subdural neomembranes or hygroma at autopsy. All cases showed diffuse parenchymal loss affecting the cerebral cortex and white matter, with relative sparing of the cerebellum and brainstem and secondary degeneration of descending corticospinal tracts. In the one case with blunt impact, the parenchymal loss was ipsilateral to the subdural neomembrane. Tau immunohistochemistry in two cases showed tau-positive threads in both, and neuronal and glial cytoplasmic aggregates in the hypothalamus in one. Beta-amyloid precursor protein immunohistochemistry in two cases showed rare positive axons in the anterior commissure in one. We conclude that long-term neuropathologic sequelae of early-life AHT include evidence of direct trauma (subdural neomembranes), significant hypoxic-ischemic injury, and also focal tau aggregation suggestive of a neurodegenerative component (chronic traumatic encephalopathy).
Keywords: abusive head trauma, shaken infant, tau aggregates, subdural neomembranes
Although the majority of people with mild traumatic brain injury (TBI) recover within 10–14 days, a subset of people exhibit symptoms such as cognitive dysfunction for weeks to months after injury. Factors that contribute to developing prolonged cognitive deficits after mild TBI are still unknown. One known predisposition factor for developing psychiatric disorders is an early-life stress event. Exposure to early-life stress primes hippocampal microglia and potentiates the neuroinflammatory response to a subsequent inflammatory challenge. We hypothesized that stress in early life interacts with a mild TBI experienced in young adulthood to result in persistent learning and memory deficits. To test this hypothesis, Sprague Dawley pups were separated from their dams for 3 hr daily from P2-P14. Non-stressed pups remained undisturbed. At 2 months of age, male rats received sham surgery or mild parasagittal fluid-percussion brain injury (1.4–1.6 atm). Animals were assessed behaviorally for 2–6 weeks post-surgery and hippocampal atrophy was evaluated at 2 months post-surgery. Basal corticosterone levels and response to a restraint stress were assayed. Mild TBI did not result in deficits in contextual fear conditioning or water maze performance. In contrast, early-life stress prior to mild TBI resulted in significant impairments in contextual fear conditioning, water maze retention and working memory ability. Hippocampal atrophy was also significantly increased in early-life stressed, mild TBI animals as compared to non-stressed, mild TBI animals. Basal and restraint-induced corticosterone levels were not significantly different between treatment groups. However, corticosterone levels remained significantly elevated after recovery from restraint in early-life stressed, mild TBI animals as compared to non-stressed, mild TBI animals. These results demonstrate that exposure to early-life stress interacts with mild TBI experienced in young adulthood to result in prolonged cognitive deficits and an increased stress response.
This work was supported by The Miami Project to Cure Paralysis and NIH/NINDS R01 NS069721.
Keywords: stress, early life stress, corticosterone, atrophy, hippocampus
MINOCYCLINE PLUS N-ACETYLCYSTEINE RESTORE SYNAPTIC PLASTICITY AND LIMIT GRAY MATTER INJURY WHEN FIRST DOSED 3 DAYS POST-INJURY
The cognitive losses produced by closed head injury (CHI) include impairments in acquiring Barnes Maze, a behavioral task that requires a single functional hippocampus. CHI-injured mice acquire Barnes maze when MINO plus NAC is first dosed as late as 3 days post-injury. Long-term potentiation (LTP) is a form of hippocampal synaptic plasticity needed to acquire Barnes Maze. CHI-injured mice have impaired LTP in both hippocampi, either ipsilateral or contralateral to the impact site. MINO plus NAC treatment beginning 3 days post-injury more readily restored LTP in the contralateral than the ipsilateral hippocampus. The contralateral hippocampus of MINO plus NAC-treated mice retained more neurons, and had greater expression of the dendritic protein MAP2 and the presynaptic protein synaptophysin than saline-treated injured mice. MINO plus NAC treatment also increased expression of PKMzeta, a PKC isoform that is essential for LTP maintenance and memory storage in the hippocampus. Moreover, preliminary studies suggest that neurons in the contralateral hippocampus stained with the rapid Golgi-Cox method have improved dendritic length and arborization, spine density and spine morphology. Together, these data indicate that MINO plus NAC restores the ability to acquire Barnes maze, in part, by limiting gray matter injury and preserving synaptic plasticity in the contralateral hippocampus. These studies also suggest that injury to regions of the brain distal from a head impact can be targeted with drugs with a clinically relevant window even if injuries proximal to the impact site remain.
Keywords: mouse, long-term potentiation, gene expression, time to first dose, neuronal morphology, hippocampus
CHRONIC HISTOPATHOLOGICAL AND FUNCTIONAL OUTCOME AFTER CONTROLLED CORTICAL IMPACT TRAUMA IN MICE
Institute for Stroke and Dementia Research, University of Munich Medical Center, Munich, Germany
Traumatic brain injury (TBI) is a major cause of death and disability worldwide. In past decades, most research focused on clarifying the mechanisms of acute brain damage. There is, however, increasing evidence that TBI pathology may continue for years after the initial insult causing progressive histopathological changes; this chronic posttraumatic brain damage has been linked to long term functional deficits that can be observed in trauma patients, including progressive neurocognitive decline and psychological abnormalities. In order to identify putative therapeutic strategies for long term sequelae of TBI we need to know more about the mechanisms and time course of chronic posttraumatic brain damage. The aim of the current study was to characterize functional and histopathological outcome in a mouse model of controlled cortical impact (CCI) injury, a widely used experimental model of TBI.
Male C57BL/6N mice (n = 12 per group) underwent CCI or sham operation and were observed for 15 min, 24 hours, 1 week, 1, 3, 6, or 12 months after TBI. Neurological function was assessed over the whole observation period using test paradigms evaluating motor function (Beam Walk test), depression like behavior (Tail Suspension test), as well as spatial learning and memory (Barneś Maze). Furthermore, histopathological outcome was assessed using immunohistochemistry.
CCI induced significant deterioration of motor function compared to sham-operated or naïve animals. At 3, 6, and 9 months after CCI, TBI animals showed depression like behavior and massive deterioration of spatial learning and memory function. Significant hydrocephalus was observed starting 3 months after CCI.
This study provides comprehensive data about extent and time course of chronic brain damage after experimental CCI trauma and may help to further understand the pathomechanisms of chronic TBI as well as serve as a base for further investigations.
Keywords: Traumatic btrain injury, Outcome, Posttraumatic brain damage, Hydrocephalus
UC Davis, Center for Neuroscience, Davis, USA
Repeated sports-related injury can lead to cognitive deficits in the absence of motor dysfunction and gross anatomical damage. We hypothesized that impacting a metal disc (helmet) using an electrically-driven piston would produce spatial learning deficits without skull fracture or hippocampal cell death. We designed a 3D-printed plastic implant to permanently secure the helmet to the skull of adolescent rats, allowing for multiple injuries without multiple survival surgeries. Rats received three TBIs (5 m/s, 5 mm depth) separated by 72 hours, beginning on P35. Motor and spatial performance were tested on the Rotarod and Morris water maze, respectively. There were no differences in biological responses (toe pinch reflex, righting time, weight) to the first two injuries; however, toe pinch reflex time (6.7 ± 1.1 s vs. 11.4 ± 1.8 s; p < 0.05) and righting time (60 ± 9.9 s vs. 152.1 ± 50.7 s; p < 0.05) were significantly longer in injured animals after the final injury, with a trend toward a difference in weight one day post-TBI (p < 0.1). There were no differences in motor performance on Rotarod or swim speed in the water maze between repeat- and sham-injured animals. Spatial learning did not differ across five days of training or during three subsequent working memory trials. However, sham animals spent significantly more time searching the platform location during a probe trial 72 hours later (18.3 ± 1.8% vs. 7.9 ± 2.1%; p < 0.05). Moreover, across three days of working memory trials, sham, but not TBI, animals spent progressively less time searching the original platform location (p < 0.005). These data demonstrate a cumulative biological response to three injuries over a 7-day period. In addition, while both sham and TBI animals demonstrate similar spatial memory performance as determined by latency, there is evidence that injured rats utilize alternate search strategies. Therefore, this new model shows relevance for sports-related repeat mild TBI and potential for assessing mechanisms and therapeutic interventions.
Keywords: Animal Model, Spatial Learning, Repeat TBI, Diffuse Injury
UC Davis, Neurological Surgery, Davis, USA
We hypothesize that disruption of theta oscillations (5–12 Hz) in the septohippocampal circuit results in cognitive dysfunction following traumatic brain injury (TBI). Furthermore, we propose that entraining oscillations with deep brain stimulation (DBS) will improve cognitive outcome post-injury. We previously reported that 7.7 but not 100 Hz square-wave stimulation of the medial septum (MSN) improved spatial learning on the Barnes maze following lateral fluid percussion injury (LFP). However, there are other theta stimulation paradigms including continuous, cycled and burst stimulation that are proposed to have distinct effects at the neuronal network and behavioral level. Given the relevance to the clinical population we now compare continuous 7.7 Hz to cycled theta square-wave (30 seconds ON/ 150 seconds OFF) and theta burst (50 millisecond trains of 200 Hz, five times a second) to evaluate paradigm specific effects. Prior to stimulation, EEG was collected on days 3 and 7 post-surgery while animals explored a novel environment. Barnes maze was evaluated post-injury days 8–11. LFP injury resulted in significantly longer righting time and weight loss, qualitative observations of hemi-neglect during recovery and structural damage over the injury site. Consistent with previous reports, LFP resulted in altered oscillations over the first week post-injury. However, injury was not associated with worse performance on the Barnes maze compared to sham or to any of the injured stimulation groups. The gross pathology was further investigated by comparing the number of parvalbumin interneurons within the CA1 and MSN. Future experiments include more granular analyses of EEG during behavior (e.g. coherence, correlation with speed) and characterizing the expression profile of multiple interneuron types (e.g. Calbindin) in hippocampal regions. These data are critical in understanding the effect of TBI on neural systems as well as addressing the potential for DBS to enhance recovery post-TBI.
Keywords: TBI, Deep brain stimulation, Hippocampus, Theta, Learning and memory
University of Miami Miller School of Medicine, Neurological Surgery, Miami, USA
Alterations in the cyclic AMP (cAMP) signaling pathway is associated with impaired long-term potentiation, memory formation and retrieval after chronic traumatic brain injury (TBI). The cAMP-specific phosphodiesterase 4 (PDE4) is involved in the regulation of cAMP signaling in the brain. The PDE4 subfamily, PDE4D, is of particular interest due to its involvement in learning and memory. Recent advancements in the structural understanding of PDE4 subtypes have led to the development of a novel negative allosteric modulator for PDE4D (D159687). In the present study, we hypothesized that treating animals with D159687 would rescue 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 of recovery, animals were treated with vehicle or D159687 (0.3 mg/kg, 6 ml/kg, intraperitoneally) at 30 min prior to cue and contextual fear conditioning, acquisition in the water maze or during a working memory task. Treatment with D159687 had no significant effect on these behavioral tasksin sham animals. In contrast, D159687 significantly reversed the learning and memory deficits in chronic TBI animals. Assessment of hippocampal slices at 3 months post-TBI revealed that D159687 reversed both the depression in basal synaptic transmission in the Schaffer collateral pathway of area CA1 as well as the expression of hippocampal long-term potentiation. Altogether, these results demonstrate that negative allosteric modulation of PDE4D may be an effective therapeutic strategy to improve chronic cognitive dysfunction following TBI.
Keywords: Traumatic Brain Injury, PDE4D inhibition, Long-term potention, Learning and memory
The University of Texas Medical Branch, Anesthesiology, Galveston, USA
Evidence suggests that formation of soluble tau oligomers may contribute to the early spread of pathology following traumatic brain injury (TBI). We hypothesized that if an immunotherapy targeted at removal of tau oligomers were given an hour post TBI, it would result in decreased tau oligomers and increased brain tissue sparing, thus improving behavior. Adult male Sprague Dawley rats received either a single fluid percussion or sham injury and the return of righting reflex was assessed. An intracerebroventricular injection of either anti-tau oligomer-specific monoclonal antibody (TOMA) or nonspecific Imunoglobulin G control was administered 1 hour post TBI. Animals underwent cognitive behavior testing and then fifteen days later, the animals were perfused for histology. Neuroscience Associates block sectioned the brains and stained them for myelin (Weill) and inflammation markers (Iba-1 and CD-68). Ventricle volumes, Corpus Collossum volume, and cortical thicknesses were assessed using ImageJ, and volumes ipsilateral and contralateral to the lesion were compared. Preliminary evaluation of TOMA TBI animals show cortical thinning, ventricle enlargement, and decreased white matter volume on the injured side, while the IgG control treated animals showed smaller histological changes. Preliminary comparisons suggest that the animals with more severe brain pathology also show learning and memory impairment. Sham animals did not show this pattern of brain pathology and behavioral deficits. These studies were funded by The Moody Project for Translational TBI Research, Darrell K Royal Research Fund for Alzheimer's Disease, Mission Connect (TIRR Foundation) and the UTMB Technology and Commercialization Program.
Keywords: TOMA, Immunotherapy, Histology, MWM
Universtiy of Texas Medical Branch, Anesthesiology, Galveston, USA
Traumatic Brain Injury (TBI) is considered an event that can lead to the development of chronic disease pathology and is thought to induce a predisposition towards dementia. Evidence suggests that formation of soluble tau oligomers may be an early event responsible for the spread of pathology. We designed this study to look at the molecular connections between TBI and Alzheimer's Disease (AD) by using a real-time PCR array following treatment with an immunotherapy designed to reduce the soluble tau oligomers that occur after TBI. Adult male Sprague Dawley rats received either a single fluid percussion or sham injury and the return of righting reflex was assessed. An intracerebroventricular injection of either anti-tau oligomer-specific monoclonal antibody (TOMA) or IgG control was administered 1 hour post TBI. Fifteen days later, brain regions (cortex near the injury site, hippocampus and thalamus) were dissected and homogenized separately. AD pathway-focused PCR arrays for rats, containing 84 genes implicated in AD development, were performed. Normalized gene expression was analyzed by ANOVA for each gene, and differences among treatment groups were assessed by Tukey-adjusted contrasts, followed by Benjamini-Hochberg control of false discovery rate. Results suggest that down-regulation of neurodegenerative genes in the TOMA treated animals was brain region specific, indicating different processes might be occurring and additional time points should be studied. These studies were funded by The Moody Project for Translational TBI Research, Darrell K Royal Research Fund for Alzheimer's Disease, Mission Connect (TIRR Foundation) and the UTMB Technology and Commercialization Program.
Keywords: tau oligomer targeted immunotherapy, fluid percussion TBI, gene expression, Alzheimer's Disease
Neuroinflammation following TBI is a major player in the secondary response post-injury contributing to widespread cell death and tissue loss. Microglial distribution and function plays an important role in the inflammatory response following TBI in the immature brain. In this study we investigated neuroinflammation-induced changes in brain tryptophan-kynurenine pathway and abnormal cognition and sleep pattern. On postnatal day 5–7 (P5-7), New Zealand white rabbits from the same litter were randomized into three groups, naïve (no injury), sham (craniotomy alone) and TBI (controlled cortical impact). Pro- and anti-inflammatory cytokine mRNA expression was measure at 6h, 1, 3, 7 and 21 days post-injury. Tryptophan, kynurenine, serotonin and melatonin protein levels were measured at 7 and 21 days post-injury. Cognitive function and sleep pattern were measured at 1–2 months post-injury. We found that 1) TBI induced a significant upregulation of pro-inflammatory cytokines, including TNF-α and IL-1β that last more than 21 days post-injury, indicating a pro-inflammatory microenvironment. 2) TBI induced a significant upregulation of indoleamine 2,3 dioxygeenase 1 (IDO1), the rate limiting enzyme in the tryptophan-kynurenine pathway, in activated microglial and astrocytes at the surrounding area of brain injury up to 21 days post-injury. 3) TBI induced a significant increase in kynurenine level in the brain, along with a significant reduction in plasma melatonin level, suggesting the shift of tryptophan metabolism towards the kynurenine pathway. 4) Pediatric TBI caused significant deficits in cognitive function at two months post-injury. 5) Pediatric TBI induced abnormal sleep EEG pattern, indicated by decreased REM at two month post-injury. This indicates that targeting kynurenine pathway specifically in activated microglia using dendrimer nanodevices may provide a novel therapeutic opportunity to address sleep disturbance and cognitive dysfunction following TBI.
Keywords: melatonin, tryptophan, pediatric TBI, EEG, secondary injury
Purdue University, Center for Paralysis Research, West Lafayette, USA
Low level DC electric fields (EF) in the range of 5-1000 mV/mm have been shown to guide axon growth and orientation in vitro and in vivo during neural development. Our laboratory has exploited this phenomenon by implanting electrodes that deliver weak DC EFs across the lesion to stimulate axonal regeneration after spinal cord injury (SCI). Initial data in human clinical trials (FDA Phase 1) with this stimulator demonstrated higher sensory but not motor recovery versus historical controls. We postulate such differences may arise from spatial variations in the EFs. However, the magnitudes of EFs permeating through the injured region remain largely unknown. The main objective of this study is to model the EFs emanating from the stimulator electrodes and to optimize electrode placement for maximum therapeutic effect. Simulation studies were performed using Sim4Life finite element analysis package. The computational model consisted of a C3-C7 vertebral section obtained from MRI scans, epidural fat, dura, cerebrospinal fluid (CSF), the spinal cord, and torso musculature. Three active electrodes were placed cranial to the site of injury, attached to the spinous process and each of the transverse processes on the C4 vertebra. Three additional ground electrodes were placed caudal to the site of injury on the C6 vertebra. At a 5V electrode input voltage, a cross section of the spinal cord at the level of the injury (C5) showed electric field values toward the lower end necessary for biological effect but varied with electrode placement. There were also differences between EF maps of injured and intact (uninjured) cord. This was due to the CSF, which acted as a shield in the intact cord, but was removed in the injured cord scenario. The computational findings show the importance of considering post-SCI biological consequences that may alter tissue conductivities. The computed results for electric field strength and distribution may also explain the asymmetry in motor vs sensory recovery from the Phase 1 clinical trials and provide insight into field optimization.
Keywords: implantable device, electromagnetics, spinal cord stimulator, finite element analysis
UCSF, Neurology, San Francisco, USA
Keywords: traumatic brain injury, functional outcome, data-driven
University of Pittsburgh, Physical Medicine and Rehabilitation, Pittsburgh, USA
Mortality rates after severe traumatic brain injury (TBI) are considerable. However, few models accurately predict TBI-associated mortality risk, thereby limiting how to apply reliable baseline risk stratification methods to clinical trials. This knowledge gap may partially explain historical failures with identifying effective TBI neuroprotective agents with previous clinical trials. Classification Regression Trees (CRT) utilize decision-learning to determine optimal variables, and corresponding cut-points that are hierarchically ordered according to their prognostication capacity to discriminate clinical outcomes. We leveraged CRT to determine demographic, clinical (head/non-head injury severity), and acute systemic biomarkers known previously associated with mortality [estradiol (E2), testosterone (T), E2:T ratio, tumor necrosis factor-α (TNFα)], and the CNS marker S100β, to predict 6-month mortality among N = 188 individuals with severe TBI. There was a 26.6% overall 6-mo mortality rate. A preliminary CRT showed the strongest mortality predictor was age ±42; therefore, primary analyses included two age-stratified CRT for those above (N = 73) and below (N = 115) age 42. The model for age <42 captured area under the curve (AUC) of 0.91. S100β was the strongest predictor (cut-point: 10.9pg/mL), with higher levels increasing mortality risk. Among those with high S100β, high E2 (cut-point: 109.5pg/mL) was associated with greater mortality risk. Whereas those with lower S100β (< 10.9 pg/mL), high TNFα (cut-point: 21.2 pg/mL) and less non-head injury severity was associated with mortality. The model for age ≥42 captured an AUC of 0.90. GCS above/below 6 was the strongest predictor. Among those with GCS <6, more men died than women. For those with GCS ≥6, higher TNFα (cut-point: 11.3 pg/mL), followed by less severe non-head injury severity, was associated with mortality. CRT generated strong mortality baseline mortality risk models. However, mortality risk captured by systemic and CNS biomarkers varies considerably by age. Similar methodologies may be considered for early assessment of baseline mortality risk to facilitate pre-randomization cohort stratification with clinical trials. Support: DoD-W81XWH-071-0701; NIDILRR-90DP0041; R49-CCR323155.
Keywords: Statistical Modeling, Hormone Physiology, Mortality Prognostication, Moderate to Severe Traumatic Brain Injury
University of Pittsburgh, Physical Medicine & Rehabilitation, Pittsburgh, USA
Understanding the acute interdependencies of inflammatory mediators following traumatic brain injury (TBI) is essential to providing effective care for TBI patients and minimizing tissue damage. Relative CNS concentrations of cytokines and activated microglia reflect many elements of the complex cascades associated with acute neuroinflammation post-TBI and are often predictive of outcome. However, TBI clinical studies to date have not focused on modeling dynamic temporal patterns of simultaneously evolving inflammatory mediators, which has potential in guiding the design of future immunomodulation studies. We derived a mathematical model of ordinary differential equations (ODE) to represent interactions between pro- and anti-inflammatory cytokines, M1- and M2-type microglia and CNS tissue damage. We incorporated cytokine variables, interleukin (IL)-1β, IL-12, IL-10 and IL-4, known to have an active role in microglial activation and phenotype differentiation. The model was fit to cerebrospinal fluid (CSF) cytokine data, collected the first 5 days post-injury from severe TBI individuals. To address the heterogeneity of CSF inflammatory profiles among individuals in the acute recovery phase of TBI, we performed principal components analysis (PCA) on CSF cytokine samples, which identified inflammatory markers that contributed most to this variance. Patients were clustered by PC score into groups with similar CSF inflammatory profiles day 0-3 post-TBI, and then subdivided by 6-month Glasgow Outcome Score (GOS). These classifications identified three patient subgroups – a fair outcome group (GOS = 4,5) and poor outcome group (GOS = 2,3) with lower inflammatory loads, and a poor outcome group (GOS = 2,3) with a higher inflammatory load. Optimal model fits to data showed different microglial and damage responses by subgroup. Significant differences in parameter distributions were identified statistically, suggesting evidence of mechanistic differences underlying the neuroinflammatory responses of each patient subgroup. Through mathematical characterization and data-driven processes, we have created a system for exploring links between acute neuroinflammatory components and patient outcome.
Keywords: mathematical modeling, CSF inflammatory mediators, microglia, TBI outcome
University of Michigan, Department of Computational Medicine and Bioinformatics, Ann Arbor, USA
Traumatic brain injury (TBI) is a major cause of death and disability among young adults and a serious public health problem in the U.S. as well as developing countries. Outcome evaluation of traumatic brain injuries and predicting the ICU length of stay (LoS) could be beneficial in reducing complications, examining the efficiency of ICU care and hence improving the quality of care for head trauma patients. Here, we focus on TBI patients who have been diagnosed with subdural hematoma (SDH) and develop an automated system to predict the ICU-LOS. We employ demographic, clinical and injury information to develop our predictive model. Demographic data (e.g. age and gender), injury severity score, imaging information, health history, hemorrhage width, hemorrhage location and dementia are among the information that we used to train a Random Forest model. We used the information of 200 patients admitted to the University of Michigan Hospital and classified these patients to two categories; 1) staying at ICU for less than three days and 2) staying at ICU for at least 3 days. For the purpose of validation we used 11-fold cross validation and achieved the area under curve (AUC), specificity and sensitivity of 0.78, 0.47 and 0.88 respectively.
Keywords: subdural hematoma, ICU length of stay, prediction, machine learning
University of Oklahoma Health Sciences Center, Pharmaceutical Sciences, Oklahoma City, USA
Following a TBI, shear force and stress to brain tissue releases a plethora of neurotoxic factors in addition to glutamate that contribute to TBI pathology and a worse prognosis, if left untreated. However, histamine release in cerebral ischemia alleviates neuronal apoptosis and improves neurological function and histamine-3 receptors (H3R) are implicated in neuronal disorders to modulate memory, pain and sleep. The specific objective of this study was to quantify changes in H3R and to correlate these changes with pain and vestibulomotor deficits following a mild TBI (mTBI) in Sprague-Dawley male rats compared to sham rats. Brains were extracted from rats euthanized on day 1 or day 8 post-TBI using the controlled cortical impact model. Levels of H3R and injury proteins were quantified in the ipsilateral (left) and contralateral (right) sensory cortex (SC) using immunoblotting analysis. H3R levels were significantly increased in the ipsilateral SC compared to the contralateral SC of mTBI rats and sham rats (n = 3–4). Rotarod performance, tactile allodynia and thermal hyperalgesia were assessed up to day 8 post-injury/sham. mTBI-induced vestibulomotor deficits were transient and rats showed complete recovery within 7 days of injury, whereas the same animals did not recover from mTBI-induced neurological deficits, tactile allodynia and thermal hyperalgesia during that same post-injury period. Reactive astrogliosis and degranulated mast cells were detected in mTBI rats at day 1 post-injury using immunohistochemistry. In conclusion, changes in H3R levels seem to correlate with the histopathology of reactive astrogliosis and neuroinflammation as well as increased pain sensitivity following a mild TBI.
Funding was provided by: University of Oklahoma College of Pharmacy seed grant (HOA), American Association of Colleges of Pharmacy (AACP) New Investigator award (HOA) and summer INBRE funds by the National Institutes of Health, National Institute of General Medical Sciences (Grant 1U54GM104938), an IDeA-CTR to the University of Oklahoma Health Sciences Center.
Keywords: mild traumatic brain injury, histamine receptor, rotarod, pain, rats
BARROW Neurological Institute at Phoenix Children's Hospital, Phoenix, USA
Affective symptoms, including anxiety and post-traumatic stress disorder, are reported in up to 50% of traumatic brain injury (TBI) survivors. The etiology and persistence of these affective symptoms remains unknown which often makes treatment options ineffective. However, the amygdala processes affective behaviors and demonstrates increased hyperexcitability post-injury. We therefore hypothesized experimental diffuse TBI produces amygdala circuitry dysfunction coinciding with structural and molecular pathology as explanations for late-onset affective symptomatology. Adult, male Sprague-Dawley rats were subjected to midline fluid percussion injury or sham surgery. Anxiety-like behavior was assessed using open-field at 1wk or 1mo post-injury. Immediately following testing, microelectrode arrays placed in the basolateral amygdala (BLA) and central nucleus of the amygdala (CeA) quantified real-time glutamatergic neurotransmission as an index of amygdala circuit function. Circuit structure was evaluated histologically by Golgi, silver, and GFAP stains to assess neuronal complexity, neuropathology, and astrocytosis. We found brain-injured rats spent significantly less time in the center of the open-field compared to shams at 1mo but not 1 wk post-injury. This late-onset, anxiety-like behavior coincided with a significant decrease in CeA glutamate release and significantly slower CeA extracellular glutamate clearance in brain-injured rats compared to shams at 1 mo post-injury. Pyramidal neurons of BLA-CeA circuitry significantly increased dendritic complexity over 1 mo post-injury compared to shams. Increased complexity occurred without neuropathology and transient astrocytosis at 1 wk post-injury, but not 1 mo post-injury, compared to shams. Together, diffuse TBI resulted in late-onset, anxiety-like behavior coinciding with dysfunctional neurotransmission in the amygdala circuit comprised of hypertrophied pyramidal neurons and limited pathology. Subsequent studies of glutamate receptors and transporter levels in the CeA may identify molecular mechanisms. These novel findings demonstrate TBI-induced dysregulation of the amygdala circuit may be a plausible therapeutic target to alleviate affective symptoms for TBI survivors. Funding-ABRC(ADHS14-000003606)-VRP(P1201607)-PCH Director's Fund
Keywords: Diffuse TBI, amygdala, anxiety, glutamate, neuron morphology
Wayne State University, Neurosurgery, Detroit, USA
Traumatic brain injuries (TBIs) increase the risk of developing alcohol use disorders. Using a mouse model of mild TBI (mTBI), we demonstrate that mTBI increases binge and chronic ethanol consumption and chronic ethanol preference compared to sham animals. The mechanism by which TBI escalates drinking behaviors is unknown. However, histone hypoacetylation has been observed following either contusive TBI or ethanol consumption, suggesting a key role for the epigenetic modulators, histone deacetylases (HDACs). Class IIa HDACs are robustly expressed in the brain and regulate synaptic signaling through phosphorylation-induced nuclear-cytoplasmic translocation. Disruption of this balance alters the response to abused substances. Thus, we hypothesized that mTBI will alter Class IIa HDAC cellular localization and that mTBI-induced alcohol use will be significantly reduced following treatment with MC1568, a Class IIa specific inhibitor. Here we show that HDAC4, a specific Class IIa HDAC, is increased in the anterior striatum, a critical region of the reward system, 30 d following mTBI compared to sham-injured mice (n = 4/group). However, the ratio of cytoplasmic HDAC4 (pHDAC4) to total HDAC4 was decreased in mTBI mice compared to sham-injured mice. Chronic intermittent ethanol exposued (CIE) mTBI mice (n = 7) decreased expression of total HDAC4 compared to CIE sham-injured mice (n = 8). Yet, the ratio of pHDAC4:total HDAC4 was similar between CIE groups and mTBI alone, suggesting an increase in nuclear HDAC4 versus cytoplasmic HDAC4 after mTBI and/or ethanol exposure. Next, naïve mice were injected with increasing doses of MC1568 (10–40 mg/kg) or vehicle (n = 7–8/group) 2h prior to and/or just before binge ethanol exposure. Daily ethanol consumption was decreased in MC1568-treated mice compared to vehicle-treated mice. Future studies will evaluate the therapeutic efficacy of MC1568 in mitigating increased ethanol consumption following mTBI. Supported by the John Dingell VAMC, Detroit, MI.
Keywords: Mild TBI, Alcohol, Epigenetics, Histone Deacetylase, MC1568
The Mind Research Network/LBERI, Albuquerque, USA
Although cognitive skills are known to increase as a function of maturation during adolescence, the impact of pediatric mild traumatic brain injury (pmTBI) on cognitive trajectories is still actively debated. Clinical and neuropsychological data were collected from 41 youth (12–18 years old [y.o.]) with sub-acute (6.5 ± 2.4 days post-injury) pmTBI and 37 age- and sex-matched healthy controls (HC). Data have also been collected for 21 pmTBI and 22 HC at early chronic phase (i.e., four months post-injury) in this ongoing study, with additional follow-up planned at one year post-injury. Participants completed computerized (CogState Brief Battery) and traditional neuropsychological tests, including effort and estimated premorbid functioning (single-word reading). Participants and their parents also completed current and retrospective versions of the Post-Concussive Symptom Inventory. Premorbid function was higher in HC compared to pmTBI. Relative to HC, pmTBI exhibited impaired performance on the CogState attention task and on composite measures of working memory and executive function (p's < 0.05), with trends for decreased processing speed (p's < 0.10). Both pmTBI and their parents endorsed increased post-concussive symptoms following injury relative to HC when compared to retrospective recollection (p's < 0.05). Reduced attention and working memory in pmTBI were associated with parental report of symptom burden (p's < 0.05). Follow-up analysis at four months indicated attention deficits persisting into the early chronic phase (p = 0.002), with working memory reduced to a trend (p = 0.080). Patient-reported post-concussive symptoms remained elevated in pmTBI (p = 0.047), albeit at much lower magnitude of difference, whereas parental report fell to trend level (p = 0.053). In summary, current results suggest cognitive dysfunction in the sub-acute injury phase that decreases, but does not fully resolve, at four months post-injury.
Keywords: pediatric mild traumatic brain injury, post-concussive symptoms, neuropsychological battery, longitudinal
University of Virginia, Radiology and Medical Imaging, Charlottesville, USA
Multiple studies have shown mTBI causes functional brain abnormalities detectible by resting state functional MRI (rs-fMRI), with some suggesting imaging abnormalities outlast clinical recovery. These studies generally utilize mass-univariate analysis of case-control data, an approach that limits sensitivity for heterogenous findings across individuals. We hypothesized that delayed imaging recovery after mTBI would be more evident in longitudinally acquired rs-fMRI data analyzed with multivariate methods. This hypothesis was tested using the Maryland MagNeTS prospective cohort available through FITBIR. MagNeTS collected MRI data on TBI patients ranging from GCS 3–15 at four time points (within 10 days of injury, 1-month, 6-month, and 18-month). Our analysis examined the 30 patients with GCS ≥13, no MRI evidence of intracranial hemorrhage, and rs-fMRI data at all time-points. For each rs-fMRI dataset, we calculated six voxelwise connectivity metrics progressing from short-to-long range: amplitude of low frequency fluctuation (ALFF), fractional ALFF (fALFF), regional homogeneity (ReHo), voxel mirrored homotopic connectivity (VMHC), binarized degree centrality (bDC), and weighted degree centrality (wDC). Paired support vector machine (SVM) classification compared individuals across time-points. Comparing injury to 18-month, significant SVM classification accuracy was found for all connectivity metrics (ALFF = 93%, fALFF = 73%, ReHo = 77%, VMHC = 70%, bDC = 73%, wDC = 73%). Significant classification accuracy was also found for all metrics when comparing 1-month to 18-month (ALFF = 93%, fALFF = 73%, ReHo = 83%, VMHC = 73%, bDC = 73%, wDC = 73%). However, comparing injury to 1-month performed at chance, and injury to 6-month showed a non-significant trend toward successful classification for all metrics. The results suggest that rs-fMRI connectivity changes related to mTBI are detectable in individuals when longitudinal individual reference data is available. In most patients, the rs-fMRI connectivity changes persist beyond 1 month, with partial normalization by 6 months, and complete normalization by 18 months. These findings demonstrate how the timeline of imaging recovery from mTBI extends beyond the typically described timeline for clinical recovery.
Keywords: fMRI, machine learning, resting state, connectivity, longitudinal, FITBIR
Boston University, College of Engineering, Boston, USA
The mechanisms underpinning concussion, traumatic brain injury (TBI), and chronic traumatic encephalopathy (CTE), and the relationships between these disorders, are poorly understood. To investigate causal mechanisms, we developed a mouse model of closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of impaired arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioral deficits resembling human concussion. Acute neuropathological neuroexamination of brains from impact-injured mice demonstrated acute and chronic neuropathologies, including phosphorylated tau proteinopathy, that recapitulates early CTE pathologies in humans. Impact-induced CTE brain pathologies are also similar to brain pathology in blast-exposed mice and humans. Moreover, acute neurobehavioral signs of concussion were observed only after impact injury but not after blast exposure under conditions matched for head kinematics. Experimental and computational modeling showed that impact generated ipsilateral point loading on the head and seven-fold greater peak shear stress in the brain compared to blast. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that fast-acting, high-amplitude cortical shear stress triggers experimental concussion, whereas longer duration, lower amplitude shear stress associated with head motion induces structural brain damage and neuropathological sequelae. These results suggest that closed-head impact injuries, independent of concussive signs, can induce TBI as well as early pathologies and functional sequelae associated with CTE. These findings elucidate the origins of concussion and differentiate this condition from TBI and sequelae. Funding: NIH, DoD, WWE, Concussion Legacy Foundation, private foundation.
Keywords: Impact, Chronic Traumatic Encephalopathy, Modeling, Blast, Neurobehavior
The EMMES Corporation, Rockville, USA
Keywords: Common Data Elements, Sports-related, Concussion, CDE
Washington University in St. Louis, Biomedical Engineering, St. Louis, USA
Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disease that has been increasingly linked to patients who have sustained one or more head impacts. The unique neuropathological features of CTE, including phosphorylated tau tangles and tau positive astrocytes in sulcal depths and perivascular regions, that distinguish it from other tauopathies such as Alzheimer's disease and frontotemporal dementia are currently identified using immunohistochemistry, making CTE a post-mortem diagnosis. An animal model that reliably recapitulates the long term tau pathology of CTE would be highly beneficial not only in finding noninvasive diagnostic biomarkers, but also in evaluating the efficacy of potential therapeutics. We therefore have implemented the CHIMERA injury paradigm in a transgenic mouse line expressing all six isoforms of human tau.
Four month old mice were exposed to daily subconcussive (2.1 psi) or concussive (3.0 psi) head impacts for twenty consecutive days, with loss of consciousness measured by the loss of righting reflex (LRR). Neurobehavioral measures of sociability, anxiety, depression, learning and memory were then evaluated acutely, three months, and one year post-injury. Cerebrospinal fluid, plasma and brain tissue were then collected from the mice and analyzed for chronic markers of tau, axonal injury, microgliosis, and astrogliosis. Compared to uninjured shams, mice in the 3.1 psi group showed an initial increase in LRR following the second impact, which decreased and plateaued by the final day of injury. There were no differences related to time or injury when assessing changes in social activity or anxiety related activity at acute or chronic time points. Interestingly, mice exposed to repetitive concussive injuries showed progressive deficits in Morris Water Maze during acquisition and probe trial sessions. These preliminary data may provide improved understanding of the relationship between repetitive head impacts, progressive behavioral deficits, and underlying neuropathology.
Keywords: Non Surgical Repetitive TBI, Chronic phosphorylated tau pathology, Astrocytosis, White matter injury
University of Southern California, Schaeffer Center, Los Angeles, USA
We assessed hospitalization rates and expected medical costs in the 6-12 months following a concussion. We identified patients aged 18 to 65 with an inpatient or emergency department visit with a primary or secondary diagnosis of concussion from 2011–2013 in a 25% sample of the Optum Clinformatics Data Mart (n = 1,872). We matched these to uninjured control patients according to age, gender, and insurance plan type (n = 15,427). We then compared hospitalization rates of concussed patients and controls 6 and 12 months following the index date, defined as the date of discharge for the index trauma event. To control for differences in trauma and control patients, we modeled hospitalizations using multivariate logistic regression. Other covariates included age, sex, race, education, household income, the prevalence of chronic health conditions and total medical spending in the 6 months prior to the index event. In the 6 months following the index event, 6.1% of concussion patients experienced a hospitalization compared to 2.3% of control patients without a traumatic brain injury. After adjusting for patient characteristics using logistic regression, concussed patients had a higher probability of being hospitalized 6 months (OR 2.18, 95% CI [1.73–2.73]) and 12 months (OR 2.53, 95% CI [1.90-3.38]) after the index trauma event compared to uninjured control patients. We conclude that concussions are associated with significantly higher rates of hospitalization in the year following injury, even after accounting for differences at baseline. This implies that patients should be closely monitored after discharge.
Keywords: Concussion, Rehospitalization, Health Care Utilization
BARROW Neurological Institute at Phoenix Children's Hospital, Phoenix, USA
Survivors of diffuse traumatic brain injury (TBI) have impairments in postural control, balance, and motor coordination, which impede rehabilitation efforts and lower quality of life. While primary injury to the cerebellum impairs acute movement, few studies have investigated cerebellar vulnerability to secondary consequences of TBI. Here, we hypothesize that injury-induced inflammation and neuropathology contribute to acute cerebellar functional deficits. Adult male rats were subjected to midline fluid percussion injury (mFPI) or sham surgery and assessed for motor function and coordination (1, 2, 7days post-injury; DPI). Tissue was collected and stained with Iba1 to assess microglial ramification and silver stain for neuropathology. Diffuse TBI resulted in motor and coordination deficits assessed by the rotarod and bilateral tactile adhesive removal task. Brain-injured rats had a significantly decreased latency to fall off the rotarod at 1 and 2DPI and a decreased distance traveled at 1, 2, and 7DPI compared to uninjured shams. The time to make contact and remove the adhesive was significantly increased in injured rats at 1 and 2DPI compared to uninjured shams but was resolved by 7DPI. Semi-quantitative histological assessment for neuropathology revealed ‘stripes’ of activated microglia in the molecular layer of the cerebellar cortex at 1, 2, and 7DPI. Stripes were defined as four or more activated cell bodies clustered perpendicular to the cortical surface. Regions with microglial stripes corresponded to regions of silver-stained neuropathology in the same brain-injured rats at identical time points post-injury. The results support cerebellar vulnerability as a secondary consequence of diffuse TBI. From this pathology, acute motor and coordination deficits track the temporal profile of acute injury-induced neuroinflammation representing a plausible therapeutic target to improve postural, balance, and motor impairments in TBI survivors. Funding: PCH Mission Support
Keywords: diffuse traumatic brain injury, microglia, cerebellum, motor impairment
Keywords: DHA, B-Complex Vitamins, Elemental Amino Acids, MTHFR C677T
ANTI-INFLAMMATORY PROPERTIES OF NANO-PULSED LASER THERAPY (NPLT) AFTER TRAUMATIC BRAIN INJURY
University of Texas Medical Branch, Anesthesiology, Galveston, USA
Keywords: Light, Optoacoustic, Neuroinflammation
CNRM/HJF/NIH, NINDS/SB, Bethesda, USA
Traumatic microbleeds (TMBs) are small foci of hypointensity seen on T2* weighted MRI. TMBs were originally equated to diffuse axonal injury (DAI), in association with focal shearing of axons. Recent pathological evidence indicates that TMBs may be vascular in origin and indicative of broader microvascular injury. This study's purpose was to determine if 7T MRI could improve visualization of the vascular injury underlying TMBs. Subjects were enrolled in an arm of the “THINC” study, with 3T MRI within 48 hours of injury and 7T MRI at 3 months and 1 year post-injury. Subjects met ACRM criteria for mTBI with evidence of injury on baseline MRI. A T2* weighted GRE (TR/TE = 12/700, resolution = 3.5 × 3.5 × 3.0 mm, 4 minutes) and 3D SWI were acquired at 3T. A T2* weighted GRE (TR/TE = 32 & 15/1300, resolution = 1.0 × 1.0 × 1.0mm, 10 minutes) sequence was acquired at 7T. Two independent raters scored both 3T and 7T MRI sequences separately, for conspicuity and location. Over a period of 28 months, thirty-three patients were 7T arm eligible, and twelve patients (median GCS = 15, median age = 52.5) agreed to 7T, three female and nine male. Eight patients with punctate and/or linear microbleeds at baseline 3T were confirmed to have microbleeds on 7T. Clusters with multiple punctate TMBs across axial slices on 3T were visualized on 7T as connecting in branching structures most similar to vascular trees. These branching hypointensities were not visualized on 7T in any of the 4 patients who had no visible microbleeds on 3T. This study provides evidence that TMBs represent traumatic vascular injury rather than isolated axonal damage. Multiple punctate TMBs seen at 3T were visible in connected branching structures when visualized at 7T. These patterns likely represent traumatic injury to the microvasculature, resulting in extravasation of erythrocytes into perivascular spaces along vascular trees.
Keywords: Microbleeds, 7T MRI, Vascular Injury, mTBI
University of Kentucky, Spinal Cord and Brain Injury Research Center, Lexington, USA
Traumatic brain injuries (TBIs) are a major public health concern with over 1.7 million TBIs reported each year in the US. Mild TBIs (mTBIs), accounting for over 80% of TBIs, can cause cognitive impairment. While it is known that mTBI does not cause widespread neuronal death, the mechanisms underlying neurological impairment and increased cellular susceptibility to subsequent head impacts are unknown. To investigate the hypothesis that altered mitochondrial bioenergetics following mTBI underlie cellular vulnerability to repeated insults, we employed a mouse model of closed head injury (CHI) to examine mitochondrial function after mTBI. A single CHI was produced by a pneumatically controlled impact device with a silicone tip at midline to model a bilateral diffuse injury. A novel object recognition (NOR) test was performed at 48 hours post-injury. Mitochondrial function was assayed from ventral (including entorhinal) cortex and hippocampus homogenates collected at 24 and 48 hours post-injury (n = 3-6/group). Oxygen consumption rates (OCRs) were measured from isolated mitochondria using a Seahorse XF24 Flux Analyzer. At 48 hours post-injury, recognition memory was significantly impaired in the CHI group compared to sham (p < 0.05). State III (ADP-mediated) respiration OCRs were significantly decreased in the hippocampal mitochondria of the CHI group compared to sham at 24 hours (p < 0.01) but not at 48 hours post-injury. Conversely, ventral cortex-derived mitochondria exhibited a delayed decrease in State III OCRs at 48 hours post-injury (p < 0.0001). No significant differences were observed in other respiration states. This study establishes that mTBI associated with cognitive impairment results in early mitochondrial dysfunction which may have region-specific temporal characteristics. Future directions will expand the acute time course of mitochondrial bioenergetics.
Keywords: Mild traumatic brain injury, Mitochondrial dysfunction, Closed head injury, Cognitive impairment
Clinical management guidelines for sport-related concussion (SRC) emphasize initial rest followed by a gradual return to activities. How to operationalize “rest,” however, is unclear, because the relationship between activity and SRC recovery is complex and dynamic. The aim of this pilot study was to develop and implement procedures for tracking activities after SRC and to explore associations between activities and recovery. High school and collegiate football players (25 concussed, 15 uninjured contact-controls) enrolled within 48 hours of injury and provided data daily for two weeks through a survey embedded into a smartphone application and a commercial fitness tracker (Fitbit). Metrics collected included magnitude and duration of physical activities, mental activities, sleep, and recovery. Completion rate of the daily data was 85%. As expected, concussed athletes reduced their activity levels (relative to controls) acutely after injury and gradually increased them to normal over the first two weeks post-injury, with somewhat different metrics for the Fitbit and mobile survey showing this pattern. Furthermore, there was significant variability across athletes in activities and recovery rates. For example, about 1/3 of athletes endorsed doing an activity that they believed would not be approved by their athletic trainers or doctors in the first week, and 3/4 avoided an activity that they believed could harm their recoveries. Endorsement rates of activity-related symptom exacerbation were extremely high in the first several days post-injury and diminished over time. However, endorsement of an unapproved activity or activity-related symptom exacerbation was not associated with recovery time. The results support the use of these technologies to remotely track athletes' activities and suggest that activity that follows a graded exertion protocol or athletes' judgment may be safe for clinical recovery after SRC. Future studies will evaluate the subset of athletes (nearly 1/3) who engaged in vigorous activity while symptomatic and will gather more information about the types of avoided and unapproved activities.
Keywords: concussion, sport, activity
CONNECTIVITY DOMAIN ANALYSIS ALLEVIATES CROSS-CENTER DIFFERENCES AND IMPROVES DIAGNOSIS OF MILD TBI
Wayne State University, Department of Biomedical Engineering, Detroit, USA
Identification of biomarkers for mild traumatic brain injury (mTBI) diagnosis and outcome prediction is challenging due to the heterogeneity of mTBI patients. Multi-center studies help to alleviate this, if we can reduce the between-center difference. This is particularly true of functional MRI studies. In this study, we demonstrated the advantage of our recently-developed approach, the connectivity domain analysis (CDA), in reducing susceptibility to cross-center heterogeneity and improving prediction accuracy of classification models using two datasets, from Wayne State University (WSU) and University of Maryland School of Medicine (UMD), with a total of 75 controls and 63 mTBI patients. Independent component analysis (ICA) was applied to the time-domain data and the transformed connectivity domain data to extract the intrinsic brain network features, which were then used to classify patients and controls using five different methods. For all investigated classifiers, CDA significantly improved classification accuracy for single-center, combined, and between-center analyses compared to the conventional time domain analysis (TDA). For instance, for an artificial neural network (multilayer perceptron) classification with 10-fold cross-validation, the prediction accuracies for
Keywords: multi-center analysis, connectivity domain analysis (CDA), classification
Center for Neuroscience and Regenerative Medicine, Bethesda, USA
Injury to meninges and vasculature occurs frequently following head trauma. The meninges conspicuously enhance on post-contrast FLAIR MRI in approximately half of mild traumatic brain injury (mTBI) patients imaged acutely. Enhancement in the falx and convexity typically is limited to the dura or subdural space; however, early, subtle enhancement has been detected in adjacent subarachnoid (SA) space. Here we study delayed contrast dispersion into SA following mTBI. Patients presenting to the emergency department were imaged within 6 hours of head injury on 3T MRI. Following administration of single-dose Gd-DTPA, FLAIR sequence (TR/TE/TI = 9000/120/2500, 0.47x0.47x3.5 mm voxels, 1:21 min) was acquired. Patients with enhancement of dura were reimaged with FLAIR within ∼3 hours without additional contrast. Images were reviewed by two expert raters, blinded to order, for presence/absence of contrast enhancement in SA along falx. Values are n(%) or median(IQR). Over 15 months, 15 subjects with baseline enhancement of falx were studied; 9 (60%) male, median age 58 (40.5-63) years, GCS = 15 in 13 and 14 in 2. Time from injury to baseline MRI 4.4 (4.0–5.4) hours; time from contrast to second scan 2.8 (2.4–3.5) hours. From the blinded read, 2 (13%) patients had detectable enhancement in the SA adjacent to falx on baseline FLAIR and 7 (47%) on SA adjacent to the falx on delayed FLAIR. Extravasation of contrast with propagation/dispersion into SA was found in approximately half the patients studied, for an estimated prevalence of 1 in 4 mTBI patients. This phenomenon seen early after mTBI may be indicative of blood-CSF barrier disruption that permits substances in plasma to reach the pial surface of the brain, and as such, has the potential to be deleterious. Understanding the prevalence, pathological basis, and evolution may provide insight into a target for acute treatment.
Keywords: FLAIR, Acute, Delayed post-contrast imaging, BBB Disruption, Contrast Enhancement
Detecting cognitive impairment in rats after moderate traumatic brain injury can be problematic. Although there is histological evidence of damage produced by moderate fluid-percussion injury (FPI) in specific brain regions (e.g. hippocampus), corresponding cognitive dysfunction may not be detectable. Procedures that increase sensitivity of the Morris watermaze, a widely used test of cognitive function, would be useful to overcome this challenge. In tests of working memory, increasing the delay time (inter-trial interval) between trial 1 (learning) and trial 2 (recall) typically increases the difficulty of the test. Here we tested the hypothesis that increasing the inter-trial interval in the working memory version of the Morris water maze would make this test more sensitive to FPI-induced working memory deficits. We compared two delay times (15-sec and 45-sec) in the working memory test between Trial 1 (finding the platform) and Trial 2 (remembering the platform location). Male Sprague-Dawley rats were anesthetized and intubated before a craniotomy was trephined lateral-right of the sagittal suture. Rats were randomly assigned to receive a moderate (2.0 atm) FPI (n = 13) or SHAM injury (n = 12) before the water maze testing including 15-sec or 45-sec inter-trial delay on post-injury days 11 – 15. Using a mixed model ANOVA, comparisons were made between FPI and SHAM for Trial 1, Trial 2, and Delta (Trial 1 – Trial 2) for each delay time. We also compared SHAM 15-sec to SHAM 45-sec and FPI 15-sec to FPI 45-sec. The results indicated that the longer inter-trial delay time of 45 sec did not produce a greater difference between SHAM and TBI than the standard 15-sec delay. This may be due to the poorer performance of SHAM rats tested with the 45-sec delay. Based on these findings, a longer inter-trial interval in this version of the working memory water maze was not more sensitive to FPI-induced deficits. These studies were funded by The Moody Project for Translational TBI Research
Keywords: Cognitive function, TBI, behavioral measure, water maze
PRE-INJURY PERSONALITY TRAITS PREDICT INCIDENCE OF CONCUSSION IN HIGH SCHOOL AND COLLEGIATE FOOTBALL PLAYERS
An estimated 300,000 sport-related concussions (SRCs) occur annually in the U.S., with the highest incidence in high school and collegiate football. About 25% of football SRCs are due to unsafe playing styles, making behavior—and the motives, attitudes, and personality traits that drive it—a credible SRC risk factor that is largely overlooked. The current study explored associations between self-report personality measures, head impact telemetry system (HITS) data, and SRC incidence in a sample of high school and collegiate football athletes. Data were retrieved from Project Head-to-Head II, a large (N = 917), prospective SRC study conducted over the 2015 and 2016 seasons. SRC incidence was operationalized as “concussed” (those who completed baseline testing and were concussed during the evaluation period; n = 83) or “nonconcussed” (matched controls from the same teams; n = 73). Playing style was operationalized as cumulative risk metric (CRM), a cumulative head impact severity estimate. Results of binomial logistic regression showed that concussion group membership was significantly (p ≤ .05) predicted by negative emotionality, meanness, psychoticism, and alienation to complement related findings linking such traits to potentially hazardous playing styles. Although broad aggression predicted concussion incidence only marginally (p = .065), aggression operationalized as physically expressed anger was strongly predictive (OR = 3.09, p = .004). Nonconcussion group membership was significantly predicted by more years of sports participation and communal positive emotionality, and marginally by social closeness and resilience, suggesting that maturity, playing experience, and having a prosocial temperament may promote on-field behaviors that minimize risk of high-impact contact. There was a great deal of consistency in the personality traits that predicted concussion incidence and the traits that predicted playing style (CRM). These findings accentuate the potential role of personality in concussion risk and can be used by coaches and athletic trainers to develop intervention and prevention strategies that encourage on-field safety and reduce SRC risk.
Keywords: sport-related concussion, high school football, collegiate football, HITS, personality, athletic trainers
OPERATION BRAIN TRAUMA THERAPY: LEVETIRACETAM TREATMENT FOR TRAUMATIC BRAIN INJURY IN THE MICRO PIG
Virginia Commonwealth University, Anatomy and Neurobiology, Richmond, USA
Traumatic brain injury (TBI) is a highly prevalent disease with devastating costs. The unsuccessful clinical translation of many promising therapeutics has triggered the rigorous standardization and refinement of TBI models and the use of higher-order animals before moving to large scale clinical trials. Levetiracetam (LEV) is an antiepileptic agent that was the fifth drug tested by Operation Brain Trauma Therapy (OBTT) in three independent rodent models of moderate to severe TBI. To date LEV is the most promising drug tested by OBTT and was therefore advanced to testing in the micro pig model. Adult male micro pigs weighing 22 ± 5 kg were subjected to a mild (1.7 ± 0.2 atm) central fluid percussion brain injury followed by a 15-min post-injury intravenous infusion of either 170 mg/kg LEV or equal volume of saline (n = 7 pigs for each treatment group). Systemic physiology was assessed throughout the post-injury period. Serial blood samples were obtained pre-craniotomy and post-craniotomy, as well as at 1 min, 30 min, 1 h, 3 h and 6 h post-injury. All blood samples were processed to obtain serum following OBTT standard operations for detailed analysis of the astroglial biomarker glial fibrillary acidic protein (GFAP). Animals were killed 6 h following injury for histological assessment of cellular damage, using hematoxylin and eosin, and diffuse axonal injury, using antibodies against amyloid precursor protein (APP). Group analysis showed no differences in post-injury serum GFAP levels between saline and LEV treated micro pigs. There were also no differences in the numbers of APP+ axonal swellings or cellular damage within the thalamic region of the micro pig brain. However, a significant alteration in the morphological properties of the APP+ axonal swellings, including reduced swelling area and increased swelling roundness was observed, suggesting potentially beneficial effects of LEV in the micro pig that warrant further investigation at later survival time points. Support: USArmy W81XWH-10-1-0623.
Keywords: Traumatic brain injury, Micro pig, Axonal injury, Levetiracetam, Biomarker
Naval Medical Research Center, Neurotrauma Department, Silver Spring, USA
The brain and the enteric system are intricately connected and together form the “gut-brain axis” (GBA). When the brain is exposed to a stressor there can be physiological changes within the gut bacteria (microbiome). Likewise, a stressor to the gut, can contribute to changes in brain function. This bidirectional relationship between the brain and gut can influence anxiety and PTSD-like behavior. Blast-induced mild traumatic brain injury (mTBI) and enteric infections are common health concerns among military members in theatre. Together, they influence the GBA, and may influence behavior, especially the anxiety and PTSD-like behavior that is often reported in service members returning from deployment. The goal of the current study was to assess how a physical trauma to the brain and an enteric infection may impact long-term anxiety-like behavior. To simulate a blast-induced mTBI, rats were anesthetized and exposed to 75 Kpa (∼10 psi) of blast overpressure from a blast tube once a day for three consecutive days. Approximately 24 hours after the final blast exposure, half of the animals were given a gavage of Campylobacter jejuni to induce an enteric infection. About 4 months after the blast exposure and infection, the presence of anxiety behavior was assessed using the elevated zero maze and the light/dark emergence test. Rats exposed to both blast and Campylobacter infection were expected to show the greatest amount of anxiety compared to control animals or animals exposed to only blast or only Campylobacter. Contrary to hypotheses, animals exposed to both blast and Campylobacter demonstrated the least amount of anxiety and showed significantly reduced levels of corticosterone compared to control animals. These results indicate that there may be an interaction between the physical trauma of TBI and enteric infection that contributes to a decrease in anxiety-like behaviors, and/or possibly a dampening of the stress response.
Keywords: campylobacter jejuni, gut brain axis, anxiety behavior, blast TBI
Medical College of Wisconsin, Neurosurgery, Milwaukee, USA
The clinical presentation of sport-related concussion (SRC) is heterogeneous and can include a combination of cognitive, somatic, and mood-related symptoms. Identifying subtypes related to specific symptom domains may reveal unique prognostic trajectories, informing clinical treatment. The present study investigated patterns of functional connectivity associated with mood symptoms following SRC. We hypothesized that a) SRC would be associated with disrupted resting state connectivity in regions associated with emotional processing and that b) concussed athletes with more mood symptoms would have greater disruption of functional connectivity in regions associated with emotional processing. Resting state fMRI and structured interviews for the Hamilton Depression Rating Scale (HAM-D) were collected in collegiate athletes at approximately one day (n = 28), one week (n = 29), and one month post-concussion (n = 26). Non-concussed contact sport athletes served as healthy controls (HC; n = 50). Twenty-three regions-of-interest (ROI) were meta-analytically derived using the Neurosynth neuroimaging database based on the term “emotion.” A connectivity matrix of Fisher-Z transformed correlation coefficients between each ROI was calculated for each subject and time point. Concussed athletes reported significantly higher HAM-D scores at one day and one week relative to one month post-concussion, and relative to controls (p's < 0.001). There was no difference in connectivity between concussed and healthy athletes. However, there was a multivariate effect of HAM-D scores on resting state connectivity in concussed athletes at one day and one week post-concussion (p's < 0.01). Follow-up analyses indicated that this multivariate effect was driven by an inverse relationship between HAM-D scores and functional connectivity of regions of the default mode network (e.g., medial prefrontal cortex) to regions of the salience and dorsal attention networks (e.g., anterior insula and superior parietal lobule). Results suggest that mood symptoms following SRC are associated with attenuated inter-network connections between regions commonly associated with both depression and cognitive impairment.
Keywords: concussion, resting state fMRI, mood symptoms, functional connectivity
The King-Devick (KD) test is a vision-based rapid number-naming task used to detect impairment in concussed individuals, primarily assed on the sideline assessment. We tested the following hypotheses: (1) acute and chronic concussion will affect total KD time and (2) KD time will correlate with symptom recovery. KD was administered as recommended to three groups: High school student athletes (13–18 years, 234 females, 149 males) at preseason baseline evaluations during the 2016–2017 school year, patients diagnosed with an acute concussion ≤28 days (12–18 years, 21 females, 29 males) and patients diagnosed with a chronic concussion >28 days (12–18 years, 26 females, 33 males). [GC1] Each participant completed Graded Symptom Checklist (GSC) providing a total score and four symptom subtypes: somatic, cognitive, sleep and emotional. In hypothesis 1, we examined group differences and found group (baseline, acute and chronic) had a significant effect on KD time (F2,489 = 18.91, p < 0.001). KD time was significantly greater in the acute group than both the baseline and chronic groups (p < 0.01). Though not statistically significant, KD time was greater in the chronic group compared baseline (p = 0.06)[GC2]. In hypothesis 2, we evaluated the robust correlation between GSC total, GSC subtype and KD time in each group. No significant correlations were reported in the baseline or chronic groups (rpb = [GC3] 0.04 and 0.08). KD time. In the acute group, GSC total and KD were significantly correlated (rpb = 0.51). Somatic and cognitive symptom subsets were most strongly correlated to KD (rpb = 0.89 and 0.80). Slower KD times in the acute concussion group show a possible injury-related deficit in KD performance that correlates with concussion symptoms (GSC). Potentially, KD can be an objective measure of concussion and/or concussion related symptoms in the acute clinic setting. GSC and KD did not correlate in the chronic group, suggesting persistent symptoms may be due to other factors. High variability of KD time in all three groups supports the need for an individual baseline. Supported by: UCLA BIRC, UCLA Steve Tisch BrainSPORT Program
Keywords: King-Devick, Vision, Clinical Setting, Recovery
NILVADIPINE AMELIORATES MEMORY IMPAIRMENT IN AGED MICE WITH REPETITIVE MILD TBI
Roskamp Institute, Sarasota, USA
Mild TBI (mTBI) is the most common form of brain trauma worldwide. The effect of mTBI is not well-studied within the elderly population, yet it constitutes a significant part of all mTBI patients. The rate of hospitalization for the mTBI population aged over 65 is two times higher than it is for younger adults, with ground falls and motor vehicle accidents as primary reasons. Age alone is known to slow neurorecovery and exacerbate cognitive decline but also dramatically worsens mTBI outcomes. However, preclinical studies focusing on geriatric TBI and its treatment are scant and are limited to moderate and severe injuries only. We hypothesize that mTBI pathophysiology in the aged brain has distinct characteristics from mTBI in younger adults due to pre-existing age-related deteriorations and might require different therapeutic interventions. Herein, we present a study of 23 months old aged hTau mice after sham injuries or repetitive mTBI (r-mTBI, 5x), modeling forces common in human mTBI with or without nilvadipine treatment. Furthermore, we investigated the effects of three weeks of treatment with Nilvadipine, a spleen tyrosine kinase inhibitor which has been shown to block amyloid production, tau hyperphosphorylation and inflammation in mouse models of Alzheimer's Disease and Tauopathy. Nilvadipine is currently under investigation to treat AD in a Phase III trial in Europe. In our r-mTBI mice, we observed that nilvadipine reversed memory impairments and showed a trend in the reduction of inflammation and tau pathology. To our knowledge, this is the only preclinical study focusing on the treatment of r-mTBI in the aged and these results highlight the therapeutic potential of nilvadipine in this situation. Additional studies are ongoing to investigate the effects of nilvadipine on other outcomes and in other injury paradigms.
Keywords: Repetitive mild TBI, Geriatric TBI, Treatment, Nilvadipine
Repeated mild traumatic brain injuries (rmTBI) are major health concern as they can result in lasting cognitive and neuropsychological complications. These functional alterations are likely due to changes in the neurochemical, cellular, and metabolic environment of the brain elicited during the secondary injury cascade. In moderate to severe TBI, neuroinflammation plays a key role in secondary injury with reactive species (RS), glial activation, and activation of inflammatory signaling pathways, such as nuclear factor kappa B (NF-κB), being major contributing factors. However, the pathophysiological changes that result from rmTBI are less characterized and vary between models. Additionally, many studies focus on sub-acute and chronic time periods post-rmTBI due to the association with neurodegenerative diseases, but the early inflammatory response is also important to understand. Therefore, the purpose of this study was to examine different aspects of neuroinflammation acutely following rmTBI. An impact-acceleration model was utilized to deliver rmTBIs (3) to adult C57BL/6J mice with an inter-injury interval of 24 hours. At 6 or 72 hours following the last mTBI, mice were euthanized and hippocampal tissue was collected for biochemical analysis. Immunoblotting was preformed to assess markers of oxidative/ nitrosative stress (protein carbonyls, 4-HNE, 3-NT), glia response (GFAP, YM1, CD68), and NF-κB activation. Additionally, a panel of 25 chemokines/cytokines was examined with a multiplex assay. At 6 hours post-injury, rmTBI resulted in a significant reduction in the anti-inflammatory M2 microglia marker (YM1) and a significant increase in IL-10. At 72 hours, IL-10 remained elevated and there was also a significant increase in IL-1β after rmTBI. There were no differences observed in oxidative/ nitrosative stress or NF-κB pathway markers. Taken together, our results demonstrate that neuroinflammation has a dynamic role acutely following rmTBI and highlights the need to gain a better temporal resolution of the pathophysiological events occurring following rmTBI. Supported by NIH/NINDS R01NS075162 and F31NS093717, Civitan International Research Center (CIRC) Emerging Scholar Award, and NFL Charities Medical Research Grant.
Keywords: repeated mild TBI, inflammation, oxidative stress, NF-κB
MULTIVARIATE APPROACH TO STUDYING THE EFFECTS OF SUBCONCUSSION ON FUNCTIONAL CONNECTIVITY
Vanderbilt University Medical Center, Institute of Imaging Science, Nashville, USA
Concerns about the effects of subconcussive head impacts in sport and the effects of subconcussion on brain connectivity are not well understood. We hypothesize that college football players experience changes in brain functional connectivity that are not found in athletes competing in lower impact sports (soccer and lacrosse) or healthy controls. Functional connectivity changes are likely to be spatially heterogeneous across participants, requiring analytical methods that go beyond mass-univariate methods commonly used in functional MRI (fMRI). To test these hypotheses, resting-state fMRI data was acquired from 31 college football players, 18 college men's lacrosse players, 14 college men's soccer players, and 30 male controls at preseason and postseason time points. Regional homogeneity and degree centrality were calculated as measures of local and long-range functional connectivity, respectively. We utilized paired support vector machine (SVM) classification to probe subconcussion's effects on local and long-range connectivity. SVM classification had high accuracy for regional homogeneity (87%, p = 0.009) in college football, but not in any other metric or group. This finding suggests that subconcussion results changes in local functional connectivity that may be detectible with multivariate analyses. Across the published findings for regional homogeneity in concussion, our regional trends matched 9 of 13 regions from previously published findings, suggesting that subconcussion in football may produce local functional connectivity changes similar to concussion. Lastly, while SVM classification for degree centrality maps in college football players did not reach statistical significance (p = 0.084), a ranking distance comparison of regional trends for ReHo and DC demonstrated a high degree of correspondence between the measures (p = 0.007). Overall, this research suggests that functional connectivity metrics combined with multivariate analyses may be sensitive to functional changes in the brain in response to subconcussive head impacts.
Keywords: subconcussion, fmri, multivariate
University of Calgary, Clinical Neurosciences, Calgary, Canada
Keywords: Cortisol, Athletes, Neuroendocrine
Mild traumatic brain injury (mTBI) is defined as a trauma-induced alteration in brain function in the absence of structural damage. Recently we described an experimental model of mTBI that resulted in a temporary loss of synapses following a single mTBI. In this study, we examined how 30 mTBI delivered over 6 days (5 mTBI per day) affected outcomes in C57Bl/6 mice. Mice were exposed to either daily isoflurane (3 minutes per day for 6 days) or daily isoflurane followed by 5 rapid succession mTBI for 6 consecutive days (30 mTBI total). Immunohistochemical analyses revealed that 30 mTBI over 6 days did not cause grey matter inflammation, cell death, or inflammation. Golgi staining for dendritic spines indicated that the mouse brain adapted to repeat mTBI, with no excitatory synapse loss detectable compared to sham mice. Despite the normal synapse number, behavioral testing at one-month post-injury showed that repeat mTBI mice have deficits in the acquisition and probe phases of the Morris water maze, indicating impaired learning and memory. Furthermore, repeat mTBI mice displayed enhanced risk-taking behavior on the elevated plus maze. Finally, EEG monitoring revealed chronic disruptions in brain wave activity and in the sleep cycles of repeat mTBI mice. We also examined the effect of mTBI on hippocampal circuitry using extracellular field recording electrophysiology. Input/output curves for repeat mTBI mice were identical to sham mice, indicating that the Schaffer collateral pathway remained undamaged after repeat mTBI – however there were significant impairments in long-term potentiation (LTP). We conclude that high-frequency repeat mTBI causes impaired learning, memory, and overall brain function in the absence of structural damage or a decrease in the number of excitatory synapses. We hypothesize that synaptic adaptation occurs after repeat mTBI causing chronic behavioral changes including impairments in sleep, judgment, and cognition. Future experiments will determine the biochemistry of the synaptic adaptation, and assess strategies to reverse the loss of brain function.
Keywords: Repetitive mTBI, Function, Inflammation, Oscillations
LONGITUDINAL RESOLUTION OF TRAUMATIC MENINGEAL ENHANCEMENT ON POST-CONTRAST FLAIR MRI
John Hopkins Suburban Hospital, Bethesda, USA
Keywords: FLAIR, MRI, Contrast Enhancement, Chronic Blood Brain Barrier Disruption
Naval Medical Research Center, Neurotrauma, Silver Spring, USA
Traumatic brain injury (TBI) has been a frequent injury in recent conflicts, Operation Enduring Freedom/Operation Iraqi Freedom (OEF/OIF). A common mechanism of injury is exposure to overpressure waves from incendiary devices, such as improvised explosive devices (IEDs). Recent literature indicates high rates of posttraumatic stress disorder (PTSD) and post concussive symptoms in military personnel following mTBI. It is currently unclear whether mTBI predisposes an individual to PTSD, or if the two develop independently. If mTBI predisposes an individual to PTSD, the brain structures involved in PTSD should be impacted by mTBI. Two areas involved in the “fear circuitry” are the amygdala and the medial prefrontal cortex (mPFC). When the fear circuitry is disrupted, a potential outcome may be PTSD. Evidence indicates that exposure to blast-overpressure (BOP) damages or causes alteration to other regions of the brain's fear circuitry, but it is currently unknown if the mPFC is also damaged. Our study examines the involvement of the mPFC and the amygdala through behavioral tests and molecular assessments. Two such behavioral assessments are cued-fear conditioning and the elevated zero maze (EZM). Long-Evans rats were exposed to a BOP of 75 kPa once daily over 3 consecutive days. Following the final BOP exposure, animals were assessed for fear and anxiety via cued-fear conditioning and EZM. Rats exposed to BOP exhibited greater freezing during extinction compared to controls in the fear-conditioning paradigm. Additionally, rats exhibited altered Corticosterone and Stathmin levels, indicating the stress pathway and amygdala protein expression may be affected. Taken together these findings indicate blast mTBI may exacerbate the development of anxiety related behaviors such as fear. These changes may be the result of underlying hormonal or protein alterations however; further research is needed to further elucidate the specific mechanisms involved.
Keywords: amygdala, PTSD, TBI, animal model, blast
UTHSCSA, Cell and Integrative Physiology, San Antonio, USA
Traumatic brain injury (TBI) is a risk factor for the development of epilepsy. Here, we are testing the hypothesis that reducing neuronal excitability before the process of epileptogenesis commences, moderates or prevents post-TBI deleterious effects. Using a model of blunt TBI, we pharmacologically enhanced currents from M-type K+ channels, which play a dominant role in limiting neuronal excitability. We tested such effects with the anti-convulsant drug, Retigabine (RTG), which enhances M current. Mice subjected to TBI were administered RTG or vehicle 30 minutes after injury. Twenty-four hours later, occurrence of spontaneous seizures was monitored by EEG and video recording. To assay for seizure susceptibility, animals were challenged with pilocarpine (3X, 75 mg/kg, 5 days after TBI), monitored for 24h by video and EEG recording, and the percentage of animals displaying seizures or abnormal EEG spike activity quantified. An independent cohort of mice were sacrificed 24 h after TBI and brain slices Nissl-stained to quantify TBI-induced changes in cell soma areas. A third dual cohort of animals were sacrificed 6 days after TBI and used for immunoblotting analyses of CD40L levels. After TBI ∼33% of vehicle only-treated mice displayed spontaneous seizures, while no animal treated with RTG had seizures. In addition, RTG treatment significantly reduced abnormal spike activities after pilocarpine injection. RTG also significantly reduced TBI-induced neuronal soma swelling and TBI-induced increases in CD40L levels. Preliminary results indicate that phosphorylation of TAU and total TAU levels were affected by TBI and RTG. Long-term behavioral effects of TBI with or without RTG will be assayed in future experiments. Supported by DoD CDMRP grants W81XWH-15-1-0284 and W81XWH-15-1-0283.
Keywords: Potassium channels, KCNQ, Spikes, CD40L
Keywords: Football, Protective Helmets
NEUROSENSORY DEFICITS VARY AS A FUNCTION OF POINT OF CARE IN PEDIATRIC MILD HEAD TRAUMA
MRN, Albuquerque, USA
Deficits in neurosensory functioning are increasingly recognized as a key component of pediatric mild traumatic brain injury (pmTBI). However, no study has examined how neurosensory dysfunction varies as a function of point of care, or how symptom provocation during neurosensory testing distinguishes patients from controls and predicts recovery. Patients were recruited from either the emergency department at University of New Mexico Hospital (UNMH; N = 35) or a concussion clinic at Children's Hospital of Philadelphia (CHOP; N = 41) along with healthy controls (HC; N = 42). Participants completed a standardized battery examining vestibular, oculomotor and visual functioning. Symptoms were measured pre-exam and immediately following each task. There were no differences in demographics or number of prior concussions (p's > 0.10) between CHOP and UNMH patients, whereas mean days post-injury was higher for CHOP patients (p < 0.001). Both pmTBI cohorts had higher pre-exam symptomatology (p's < 0.001) relative to HC but not different from one another (p's > 0.10). Symptom provocation (i.e., relative to pre-exam ratings) was significantly higher for pmTBI patients relative to HC, with greater provocation observed for CHOP compared to ED patients. Symptom provocation marginally increased total classification accuracy for ED patients relative to HC compared to pre-test symptoms alone (≈5% change), but had a much larger effect on accuracy in CHOP sample (≈17% change). Magnitude of symptom provocation also classified recovered versus non-recovered patients with moderate accuracy (73.7%) as defined by pre-test symptomatology. These results highlight the importance of neurosensory testing and symptom provocation in the clinical management of pmTBI and suggest that point of care represents an important outcome variable.
Keywords: Recovery, Vestibular, Ocular Motor, Neurosensory
Baylor College of Medicine, Physical Med & Rehab, Neurology, Radiology, Houston, USA
Prior imaging studies using DTI in sports-related concussion have demonstrated group differences in several brain regions at both subacute and chronic post-injury intervals, but many imaging studies are limited by small sample sizes and a single comparison group. This study investigates DTI-detectable differences in a multi-site study of collegiate athletes in the following groups: 1) athletes sustaining sports-related concussion at day of injury (TBI; n = 49), 2) non-concussed contact sports athletes (CC; n = 27), and 3) non-concussed, non-contact sports athletes (NCC; n = 20). Groups did not differ in age, sex, race/ethnicity, or handedness. Multiple regression analyses including age and sex indicated that group assignment predicted fractional anisotropy (FA) for the genu, body, and splenium of the corpus callosum, right and left frontal white matter, and right and left cingulum bundles, but not for the uncinate fasciculi. In these models, the TBI and CC groups both generally differed from the NCC group, with the concussed group demonstrating lower FA. Thirty-seven of the 49 participants in the TBI group returned 45 days after return to play for repeat MRI assessment. Repeated measure ANCOVAs disclosed decreased FA in the corpus callosum splenium in concussed athletes between Day 0 and 45 days after return-to-play, but not in other tracts, though trend-level decreases were noted in the right cingulum bundle and the right and left uncinate fasciculi.
DTI-related metrics suggested the possibility of brain tissue alteration that is both related to contact sport exposure and concussion-level events. Future work will focus on the relation of DTI to EEG-based findings as well as other symptom and outcome measures.
This work was supported by the United States Department of the Navy (N6311614MPX083) and Brainscope, Inc.
Keywords: sports-related concussion, diffusion tensor imaging
Emory University, Atlanta, USA
The sports medicine community strongly advocates testing multiple neurological domains to assess sports-related concussion, yet data supporting the combination of different assessment tools is lacking. The purpose of this study was to evaluate the relative abilities of the King-Devick Test (KD), the Balance Error Scoring System (BESS), and the Standardized Assessment of Concussion (SAC) (individually and in combination) to correctly discriminate between concussed and uninjured athletes. Athletes who sustained a concussion were paired with healthy controls and underwent testing at 3, 10, and 30 days post-injury. The ability of the tests to differentiate between concussed and control athletes at each testing day was determined by comparing the area under ROC curves (AUC) generated using logistic regression models. The discriminative abilities of each test varied by testing day. When assessed individually, the BESS showed higher discriminative ability at testing days 3 (AUC: 0.85) and 10 (AUC: 0.72), while SAC had the highest discriminative ability at day 30 (AUC: 0.76). On testing day 3, backward elimination logistic regression procedures resulted in a model that included only the BESS firm score and the total symptom score as predictors of concussion (AUC: 0.85). On day 10, the SAC and the BESS firm score remained in the model (AUC: 0.76), although the BESS firm score alone was not a significant predictor. On day 30, only the SAC remained in the model (AUC: 0.76). The discriminative ability was highest when all three tests were used together on testing day 3 (AUC: 0.88). Multiple domains and tests need to be utilized to improve concussion detection. Additionally, individual tests have different discriminatory abilities depending on when they are deployed after concussion, which should be considered when implementing a recovery/return-to-play program.
Keywords: concussion, Sports concussion, Diagnostics, Assessment tools
Stanford University, Bioengineering, Stanford, USA
Keywords: Neurological Testing, Head Impact Exposure, Oculomotor Function
Texas A&M Health Science Center, Neuroscience and Experimental Therapeutics, Bryan, USA
Previously, we have shown that approximately one-third of spinally injured rats exhibit behavioral, physiological and immunological correlates of depression. This incidence corresponds with the clinical population. Following SCI, 18–26% of patients are diagnosed with depressive disorders, compared to 8–12% in the general population. As increased inflammation strongly correlates with depression in both animal and human studies, we hypothesized that the immune activation inherent to SCI could foster future depression. Thus, we proposed that reducing immune activation with minocycline, a microglial inhibitor, would decrease depression-like behavior following injury. Male Sprague-Dawley rats were given access to minocycline (0, 0.33 or 1 mg/ml) in their drinking water for 14 days following a moderate, mid-thoracic (T12) spinal contusion. An array of depression-like behaviors (social activity, sucrose preference, forced swim, open field activity) were examined prior to injury as well as on days 2, 9, 19, and 30 post-injury. Peripheral cytokine levels were analyzed in serum collected prior to injury and on days 10 and 32 post-injury. Hierarchical cluster analysis divided subjects into two groups based on behavior: depressed and not-depressed. Depressed subjects displayed lower levels of open field activity and social interaction relative to their not-depressed counterparts. Minocycline did not appear to reduce depression; equal numbers of minocycline versus vehicle-treated subjects appeared in both phenotypic groups. Further, minocycline dose did not affect serum cytokine levels, but depressed subjects showed significantly greater expression of pro-inflammatory cytokines (IL-6, TNFa, IL-2, IL-13, IL-5) at 10 days post-injury. Interestingly, depressed subjects also exhibited elevated pro-inflammatory cytokines prior to injury. Subjects who later showed depressive behaviors had higher baseline levels of IL-6, which persisted throughout the duration of the experiment. While others have found that elevated IL-6 alone does not produce depressive behavior, our data suggest that it could predict the development of depression after a physical stressor. Funding: Gillson Logenbaugh Foundation, Mission Connect
Keywords: Spinal Cord Injury, Depression, Proinflammatory cytokines, Interleukin-6, Rat model SCI
Pediatric traumatic brain injury (PedTBI) is a major public health problem due to its highest rate of TBI (∼40%), especially in males aged 0–4 years. PedTBI is also associated with increased long term psychiatric disorders including depression in children that they can carry to their adulthood. However, the molecular mechanisms underlying these signaling, morphological alterations and their association with depression have not yet identified. Here we show that chronic PedTBI increases levels of S100A1, an isoform of S100 Ca++-binding proteins in the brain is associated with a multitude of intracellular functions like learning and memory in mice frontal cortex. This concurrent with a decrease levels of brain derived neurotrophic factor (BDNF), plays important roles in the development and plasticity of neural circuits that is known to be altered in depression. We also found significant decrease in BDNF, but increase in S100A1 mRNA expression in PFC of PedTBI mice. We found that mice genetically deficient in S100A1 showed improved behavioral activities and BDNF signaling deficits caused by chronic PedTBI. These data implicate S100A1 in the development of PedTBI induced depression. Using S100A1−/− mice as a novel resource, functional studies at the cellular and molecular level would reveal more about the involvement of S100A1 in depressive phenotypes. Identifying novel regulatory mechanisms of BDNF, a critical mediator of neuroplasticity, by S100A1 may provide avenues to develop newer therapeutics for depression and psychiatric disorders.
Keywords: S100A1, Pediatric traumatic brain injury (PedTBI), Depression, BDNF
THROMBIN INDUCES DOWN-REGULATION OF GLAST AND GLT-1 AND CONTRIBUTES TO DEPRESSION AFTER TRAUMATIC BRAIN INJURY VIA PAR-1
Keywords: Glutamate transporters, thrombin, Par-1, depression
Kainuu Social and Health Care Joint Authority, Laboratory of Clinical Neurophysiology, Kajaani, Finland
Traumatic brain injury (TBI) is one of the leading causes of disability and disablement in the Western World. The aim of the study is to provide data on incidence, demography and etiology in adults with TBI.
Patients with an ICD-10 diagnosis code indicating TBI or a high probability of TBI were collected from the medical files of Kainuu Central Hospital 2004-12 and Sotkamo Municipality Health Center 2005-12 (67.774 and 8.824 adult inhabitants, respectively). Demographic features, etiology, and clinical findings for the diagnosis of TBI were reviewed.
The first 657 potential TBI patients were reviewed. Twentysix patients were excluded because of TBI before year 2004, 85 patients were children, and 92 patients were living outside the County of Kainuu. Of the remaining 454 patients, 193 were diagnosed non-TBI and 89 uncertain TBI. The remaining 172 had definite TBI, of which 5 TBI incidents were duplicates, making up final study sample. The annual crude incidence per 100.000 adult inhabitants of TBI with medical attendance was 135,47 (365/72/9*100.000/67.774*163) in the Kainuu Central Hospital and 28,73 (365/72/8*100.000/8.824*4) in the Sotkamo Municipality Health Center summing 164 new TBI incidents in the County of Kainuu annually. Falls were the leading cause of TBI (63%) and alcohol consumption was involved in 24% of cases. Evidence of loss of consciousness was found in 40% of patients. Acute CT scan was acquired in 60% of patients; one tenth of patients had acute TBI related findings.
Eighty-three % of newly diagnosed TBI cases are recognized in a hospital and 17% in a basic health care.
Keywords: Traumatic brain injury, incidence, etiology, clinical findings
University of Pennsylvania, Anesthesiology and Critical Care Medicine, Philadelphia, USA
Inhibitory interneurons and their functional integration into the network are thought to play a key role in regulation of cortical excitatory input to the dentate gyrus (DG). After traumatic brain injury, GABAergic inhibition is reduced onto dentate granule cells, and is associated with DG network hyperexcitability. Within the DG, parvalbumin-positive (PV+) interneurons (i.e., fast-spiking basket cells and axo-axonic cells) exert strong perisomatic inhibition onto dentate granule cells. While it is known that PV+ interneuron loss occurs after injury, it is unknown how the intrinsic and synaptic properties of the surviving DG PV+ population is affected. In this study, we examined both the intrinsic membrane properties and excitatory synaptic input onto DG PV+ interneurons one week after lateral fluid percussion injury (LFPI) or sham surgery in transgenic mice expressing the fluorescent marker tdTomato in PV+ interneurons. Preliminary data from whole-cell patch clamp recordings reveal no change in the intrinsic membrane properties—including resting membrane potential, action potential threshold and input resistance—of PV+ interneurons after LFPI. However, voltage clamp recordings demonstrate a reduction in spontaneous and miniature EPSC frequency and amplitude in PV+ neurons from LFPI mice compared to sham controls. These findings suggest that while membrane excitability of PV+ interneurons is left intact after LFPI, excitatory drive onto this inhibitory population may be diminished. Less excitatory activation of PV+ interneurons could provide a mechanism of reduced inhibition onto granule cells, and contribute to shifts in DG network excitability after injury.
Funding provided by: NICHD R37 HD059288
Keywords: lateral fluid percussion injury, dentate gyrus, interneurons, parvalbumin
University of Cincinnati, Neurosurgery and Neurology and Rehabilitation Medicine, Cincinnati, USA
A spreading ‘wave of death’, known as terminal depolarization, is hypothesized to mediate the death of cerebral cortex after survivable ischemic insults and at the end-of-life. To determine whether this phenomenon occurs in the human brain, here we performed electrocorticographic recordings with either subdural electrode strips (n = 3) or intraparenchymal electrode arrays (n = 5) in patients with devastating brain injury from ruptured aneurysms (n = 3) or trauma (n = 5) that resulted in activation of a Do Not Resuscitate-Comfort Care order followed by terminal extubation. Systemic physiology and multimodal brain monitoring were performed. Cortical brain function was measured by spontaneous synaptic activity (0.5–50 Hz) and terminal depolarization was measured by direct-current potentials (<0.02 Hz) from intracranial electrodes. The dying process began with declining cardiac function and brain partial pressure of oxygen. This first resulted in the silencing of cortical synaptic activity (i.e., isoelectricity) when mean arterial pressure fell to ∼20 mm Hg. In every case, isoelectricity developed simultaneously on intracranial as well as scalp EEG electrodes. After a delay of 1 to 5 min, a large-amplitude (2 to 9 mV) negative shift of direct-current cortical potential then developed on intracranial electrodes in all patients, indicating terminal depolarization. In 5 patients, the depolarization was observed to spread across cortex at 3 to 31 mm/min. Terminal depolarizations followed cardiac arrest with median delay of 8.2 min. These results provide fundamental insight into the neurobiology of dying and have further implications for survivable cerebral ischemic insults such as cardiac arrest and stroke.
Keywords: spreading depolarization, spreading depression, cardiac arrest
University of British Columbia, Orthopaedics and Mechanical Engineering, Vancouver, Canada
The timing of surgical decompression following a spinal cord injury (SCI) is controversial, as clinical studies have yet to show strong evidence for the effectiveness of early decompression, despite positive preclinical study results. However, there are no previous preclinical studies which employ the most clinically common SCI mechanism: dislocation. Additionally, most preclinical residual compression studies involve slow force application or a time period after insult when the spinal cord is decompressed before residual compression is applied – both being counter to what happens in real-world injuries. The objective of this study was to determine the acute effect of immediate residual compression following a traumatic dislocation spinal cord injury in a rat using somatosensory evoked potentials. A 1.0 mm dislocation spinal cord injury was performed on twelve rats at C5/C6, then either reduced completely or held in residual compression at 0.8 mm. SSEP signals reached steady state prior to injury, and were recorded for 30 minutes post-injury. When the injury was immediately reduced to 0 mm following dislocation, the SSEP amplitude typically dropped immediately. In four cases the signal was absent for at least 12 minutes, and the average never recovered to above 50% baseline. When the spinal cord was held in compression following injury, in each instance the signal remained steady, or increased. The two groups were significantly different at each time point post injury (p < 0.05). Intuitively, one would expect added compression to the spinal cord would reduce signal conduction, however the results demonstrated the opposite. Two potential hypotheses are: compression activates Piezo2 stretch channels, effectively sensitizing the axons, and also restrict tonic descending inhibitory axons, increasing the probability of ascending axon potentials firing. These results emphasize the need for preclinical studies to replicate the pathophysiology of residual compression immediately following a traumatic spinal cord injury.
Keywords: Dislocation, Residual Compression, Animal Model, Pathophysiology, Decompression, Acute
University of Pennsylvania, Neurosurgery, Philadelphia, USA
Memory disruption is often an indication of neurological deficit due to traumatic brain injury and/or post-traumatic epilepsy. Pigs have been demonstrated as an ideal model of inertial injury given the gyrencephalic aspects of the porcine brain and significant white matter composition that produce diffuse axonal injury. We performed in vivo extracellular recordings in order to investigate changes in hippocampal function post injury. To precisely place silicone in-depth probes into porcine hippocampus, we have implemented MRI-free stereotaxis and single-unit functional mapping. Oscillatory field potentials and single-unit neuronal activity were recorded in the dorsal hippocampus of miniature swine in sham and injured animals. Pathophysiological changes were studied at 7 days following a model of closed-head rotational acceleration that produces diffuse axonal injury. Male Yucatan miniature pigs (6 months) underwent coronal rotational acceleration (220–260 rad/sec) with little or no loss of consciousness and minimal subdural bleeding. Paired pulse facilitation in CA1 was deficient, potentially due to changes in neurotransmitter release probability. Theta burst stimulation in entorhinal cortex used to alter baseline activity induced sub-clinical epileptiform activity in a subset of animals. Pyramidal cells and interneurons in CA1 layer were affected differentially based on waveform and firing rate analysis. These alterations suggest an increased post-synaptic excitability in response to deafferentation, or a shift in the excitation-inhibition balance of the local circuitry. Over time post injury these changes may lead to circuit-level changes in the hippocampus that may elicit sub-clinical epileptiform activity and potentially lower seizure thresholds, as well as disrupting network level memory encoding. This large-animal methodology has since been expanded into awake chronic recordings which are being utilized to evaluate the progression of epileptogenesis post injury, as well as investigating how disruptions in limbic circuitry may contribute to post traumatic stress disorder as a co-morbidity.
Keywords: In-Vivo, Stereotaxis, Silicone Probes, Single-Unit Activity, Oscillatory Field Potentials, Hyperexcitability
Uniformed Services University, Program in Neuroscience, Bethesda, USA
Of the 1.5 million people diagnosed with TBI annually, 15–30% develop neuroendocrine dysregulation, leading to subsequent diagnosis of neuropsychiatric disorders. Among military personnel, mild blast (mb)TBI is the leading diagnosis in active war zones. Currently, little is known on how mbTBI disrupts the limbic-HPA axis differentially among males and females, resulting in a dysregulated neuroendocrine stress response. This study sought to examine the sex-dependent hormonal, neuroanatomical and behavioral responses after mbTBI. Animals were exposed to mbTBI (15.5 psi) utilizing the ORA Advanced Blast Simulator and tested either short- (1 day) or long-term (7–12 days) post-injury. To assess the HPA axis after mbTBI, serum corticosterone (CORT) was measured. mbTBI enhanced the diurnal CORT rise (p < 0.05). In response to restraint, mbTBI diminished short-term CORT in males and long-term CORT in females (p < 0.05). We then examined the central and peripheral components in the limbic-HPA stress response. mbTBI did not alter peripheral adrenal 11β-OHase and MC2R or pituitary POMC and CRFR1 expression. DEX suppressed CORT regardless of injury, pointing to a central dysregulation of the HPA axis. Utilizing a transgenic model, we visualized stress reactive CRF neurons in the stress-relevant limbic-PVN circuitry. cFOS labeling was used to detect restraint-induced CRF neuronal activation after mbTBI. In non-injured animals, proestrus females had greater PVN CRF neuronal activation than males (p < 0.05). mbTBI increased PVN CRF neuronal activation in injured compared to sham females (p < 0.05). Preliminary data examining limbic regions suggest increased PFC CRF neuronal activation after mbTBI. Full analysis on CRF activation in all stress-relevant limbic regions is underway. Exposure to mbTBI increased anxiety-like behaviors in all animals (p < 0.05). Understanding the limbic-HPA disruption can begin to develop a targeted approach to treatment for the long-lasting TBI-induced neuroendocrine dysregulations.
Keywords: sex differences, HPA axis, stress, glucocorticoids
University of Louisville, Department of Neurological Surgery, Louisville, USA
Cardiovascular (CV) dysfunction is arguably the most deleterious secondary outcome of SCI and is one of the leading causes of morbidity in the chronic SCI population. CV decline may be further exacerbated by reduced activity following injury. The objective of the present study was to investigate the cardiac changes induced by limiting an animals' ability to move by reducing their cage size. Adult female Sprague Dawley rats received a T2 25 g-cm SCI, then were housed in either large cages (LC; n = 16) or tiny cages (TC; n = 16). Vascular function from the carotid (CA) and femoral arteries (FA) were assessed at baseline, week 1 post-SCI and every two weeks for 10 weeks using ultrasound. All animals showed a reduced CA diameter at 2 weeks post-injury which recovered significantly by 8 weeks for the LC group only. Interestingly, there were no differences in CA blood flow velocity between the TC and LC animals. In contrast, there were differences in FA peak and mean velocity at weeks 6 and 8 post-SCI between the TC and LC groups. Quantitative PCR analysis showed increased expression of markers of angiogenesis including elastin, ACE (Angiotensin-Converting Enzyme), and CD31 (Platelet Endothelial Cell Adhesion Molecule-1), in LC animals compared to TC. Additionally, there was an increase in markers of adaptive ER stress including Atf4, Chop, Grp78, Gadd3 and Xbp1 in the LC animals compared to TC. These findings show that simply limiting the amount of spontaneous activity after SCI influences vascular structure and function above and below the lesion level.
*Dr. Brabazon and Dr. DeVeau contributed equally to this work.
Keywords: SCI, vascular remodeling, exercise, ultrasound
Keywords: Actigraphy, traumatic brain injury, exercise capacity, heart rate, VO2 max
Spinal cord injury (SCI) results in sudden life changes such as paralysis, loss of bowel and bladder control, spasticity and neuropathic pain (NP). Spinal cord-induced neuropathic pain (SCI-NP) has a massive impact on the patients' quality of life following injury. Of the people who suffer from SCI, 60%–80% experience SCI-NP and 1/3 of those describe the pain as debilitating. Although some of the biological underpinnings of neuropathic pain (NP) have been studied,
Keywords: Nitric Oxide, Neuropathic pain, Spinal Cord Injury
Keywords: severe TBI, brain trauma, genotype, neurological outcomes
Hemorrhage is the primary event in about 15% of strokes, and accompanies most severe traumatic CNS injuries. Blood-brain barrier (BBB) disruption is a key feature of hemorrhagic injuries and may contribute to peri-hematomal cell loss. The authors' previous study demonstrated that treatment with the heme oxygenase-1 (HO-1) inducer hemin improved barrier function and neurological outcome after experimental intracerebral hemorrhage (ICH). Since hemin is already in clinical use to treat acute porphyrias, the present study was designed to test the hypothesis that its protective efficacy on BBB function after ICH was sustained through a clinically-relevant time window. ICH was induced in Swiss-Webster mice by an infusion of autologous blood or bacterial collagenase into the right striatum. Mice were randomly assigned to hemin (4 mg/kg i.p.) or vehicle groups; treatment was initiated at intervals up to 12 hours after the induction of ICH, and was repeated daily for a total of four doses. Blood-brain barrier disruption was assessed by Evans blue assay and perihematomal cell viability by MTT assay. A total of 77 mice were used in this study. Two mice died within 24 hours of ICH; both had been randomized to the vehicle group. Consistent with prior observations, systemic hemin therapy increased striatal HO-1, with expression localized to perivascular cells that were predominantly astrocytes. In the blood injection model, hemin reduced parenchymal extravasation of Evans blue by over 60% when therapy was initiated at up to 6 hours; this was associated with a 40% increase in striatal cell viability at four days. The therapeutic time window was narrower in the collagenase model, with no effect on BBB integrity or perihematomal cell viability when treatment was initiated 6 hours or later after ICH. These results suggest that hemin is protective in mouse ICH models when treatment is initiated within 3–6 hours of hemorrhage. In light of the established safety profile of this drug over decades of clinical use, further investigation as a primary therapy for hemorrhagic CNS injuries seems warranted.
Keywords: intracerebral hemorrhage, stroke, heme oxygenase-1, subarachnoid hemorrhage
PAIN INDUCED HEMORRHAGE AFTER SPINAL CORD INJURY IS BLOCKED BY PENTOBARBITAL ANESTHESIA
We have shown that nociceptive stimulation after a contusion injury increases tissue loss and impairs long-term recovery. We recently showed that the local application of an anesthetic (lidocaine; Turtle et al., 2017, J Neurotrauma, 34, 1200 1208), but not systemic morphine, has a protective effect that counters the adverse effect of nociceptive stimulation. Other work implies that this local effect depends, in part, on communication with rostral (brain-dependent) neural systems. Supporting this, nociceptive stimulation has no effect on tissue sparing in rats that received a rostral spinal transection after a contusion injury (see presentation by Reynolds et al.). This suggests that experimental treatments that generally inhibit brain function may reduce the adverse effects of nociceptive input. The present study explored this possibility by testing the effect of systemic pentobarbital. Rats received a contusion injury at T12 and, 24 hrs later, were given an anesthetic dose of pentobarbital (30 mg/Kg). Subjects then received 6 min of intermittent tailshock or nothing. Three hours after shock, rats were sacrificed and one cm of tissue was collected enveloping the injury site. The extent of hemorrhage was assessed by measuring the absorbance of light at 420 nm. As expected, nociceptive stimulation increased the extent of hemorrhage in vehicle treated (awake) rats. It appears that this effect was attenuated by pentobarbital anesthesia. This is consistent with prior work showing that anesthesia also reduces the adverse effect nociceptive stimulation has on recovery. Further work is exploring the generality of these results.
Supported by the Craig H Neilsen Foundation and the National Institute of Health underaward number R21465891-00001 to JW.
Keywords: Pentobarbital, Contusion, Hemoglobin, anesthesia, shock
ENDOTHELIOPATHY OF TRAUMA AFTER TRAUMATIC BRAIN INJURY
Keywords: hemorrhage, endothelial damage, traumatic brain injury, syndecan-1, endothelial glycocalyx breakdown
TUMOR NECROSIS FACTOR ALPHA (TNF-A) IS ASSOCIATED WITH HEMORRHAGE PROGRESSION AFTER TRAUMATIC BRAIN INJURY
Oregon Health and Science University, Neurology, Portland, USA
Keywords: TNF-alpha, Hemorrhage Progression, Isolated TBI
26 rats were randomized to one of three study groups: (1) sham (n = 5); (2) polytrauma (n = 10); (3) polytrauma with TNS (n = 11). TNS was delivered for 30 seconds every 10 minutes for 2 hours. The survival time, plasma levels of lactate dehydrogenase (LDH), aminotransferase (AST) and creatinine were quantified using commercially available assay kits according to the manufacturer's specifications after 90 minutes polytrauma.
Keywords: traumatic brain injury, hemorrhagic shock, trigeminal nerve stimulation, organ damage
TRIGEMINAL NERVE STIMULATION ATTENUATES SECONDARY BRAIN INJURY AFTER TRAUMATIC BRAIN INJURY AND HEMORRHAGIC SHOCK
In the first study, 3 rats were spinalized at the C6 level and CVR was measured during TNS. In the second study, 40 rats were randomized to one of three groups: (1) sham (n = 4); (2) polytrauma (n = 18); (3) polytrauma with TNS (n = 18). TNS was delivered for 1 minute every 15 minutes for 1 hour. Brain edema (wet-dry-weight method), lesion volumes (cresyl violet staining) and blood-brain barrier permeability (Evans blue dye) were measured at 24 hour after injury.
Keywords: traumatic brain injury, hemorrhagic shock, trigeminal nerve stimulation, secondary brain injury
ALTERATIONS IN PERIPHERAL REDOX SYSTEMS IN A RAT MODEL OF TRAUMATIC BRAIN INJURY ASSOCIATED WITH POLYTRAUMA
Traumatic brain injury (TBI) commonly occurs in polytrauma with concomitant organ dysfunction. However, the pathophysiology of peripheral organs following TBI/polytrauma is poorly understood. Here we examined the status of redox systems, glycogen content, creatinine clearance and mitochondrial health in the peripheral organs following penetrating ballistic-like brain injury (PBBI) combined with hypoxemia and hemorrhagic shock (HH). Rats were assigned into four groups: sham, HH, PBBI, and PBBI combined with HH (PHH). In PHH or HH group, a 30-minute hypoxemia (PaO2<40mmHg) was initiated 5 minutes following TBI followed by a 30-minute hemorrhagic shock (MAP ∼40 mm Hg). Urine output, food and water intake were recorded at baseline and end points. Liver, kidney and heart tissues were collected at 1, 2, 7, 14 and 28 days post-injury (DPI). Reduced/oxidized Glutathione (GSH/GSSG) and glycogen content were quantified in the peripheral organs. Superoxide dismutase (SOD) and cytochrome C oxidase activities were measured using enzyme activity kits. Urine/serum creatinine levels were analyzed. Although polytrauma did not change the mitochondrial cytochrome C oxidase activity, a modest decrease in SOD activity was observed following PBBI on 2DPI. The PHH and sham groups exhibited similar trend over time, except that the kidney SOD activity in the PHH group was lower than the other groups on 14DPI. Hepatic glycogen levels were reduced acutely following polytrauma. Urine/serum creatinine ratio in PHH group was significantly elevated on 7-28DPI. Polytrauma induced a delayed disruption of the GSH/GSSG ratio in the liver and kidney, which resolved within 1–2 weeks post-injury. The increase in ratio was primarily due to the augmented GSH level which might imply a defensive mechanism against oxidative damage in these organs following TBI/polytrauma. Increased liver glycogenolysis in the PHH group might imply the increased energy demand following polytrauma. High urine/serum creatinine ratio in PHH group point to elevated renal clearance. Collectively, these specific stress marker results reflect temporal cytological/tissue level damage to the peripheral organs due to TBI/Polytrauma.
Keywords: Polytrauma, Systemic injury, Oxidative stress
Preclinical models of TBI have rarely shown cognitive or behavioral deficits beyond six weeks post injury, whereas outcome measures in clinical trials of therapeutics for TBI are typically assessed 6–12 months after injury. The absence of detectable behavioral deficits at longer time points in preclinical models may be a contributing factor to the lack of success in translating the relatively short-term efficacy of therapeutics in pre-clinical models to human trials. We have previously developed a murine model of TBI followed by delayed hypoxemia to model the secondary insult of hypoxemia and brain hypoxia occurring in the intensive care setting. Understanding long-term effects of delayed hypoxemia following TBI in our murine model is critical for future testing of candidate therapeutics targeting secondary brain hypoxia. Forty 5 week old male mice (C57BL/6J) were randomized to controlled cortical impact (CCI) (N = 24) or sham surgery (N = 16). One day later, awake animals were randomized to 60 minutes of hypoxemia or normoxemia. Six months after initial injury, animals underwent behavior testing (Morris Water Maze (MWM), social interaction, and tail suspension) before euthanasia for immunohistochemical assessments. At six months post injury, mice experiencing CCI and hypoxemia (CCI + H) had longer swim distances to the hidden platform and spent less time in the target quadrant during probe testing compared with CCI alone or sham animals. During social interaction assessments, CCI + H mice spent less time interacting with novel stimulus mice than CCI alone or sham animals. CCI + H had larger lesion volumes compared with CCI alone (14.0% vs 9.9%, P < 0.003). GFAP immunohistochemistry at six months post injury demonstrated increased astrogliosis in the ipsilateral white matter of CCI + H compared with CCI alone. A clinically relevant model of delayed hypoxia following TBI resulted in long-term behavioral deficits and evidence of white matter injury.
Keywords: hypoxemia, hypoxia, secondary injury, preclinical, long-term outcomes, behavior
PERFLUOROCARBON NVX-108 REDUCED SECONDARY BRAIN INJURY IN A SWINE MODEL OF TRAUMATIC BRAIN INJURY
Naval Medical Research Center, NeuroTrauma Department, Silver Spring, USA
Keywords: TBI, swine, brain tissue oxygenation
Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, Stockholm, Sweden
Prehospital intubation in traumatic brain injury (TBI) focuses on limiting the effects of harmful secondary insults, such as hypoxia and hypotension, but has not shown indisputable evidence to be beneficial for patient outcome. The aim of this study was to explore the caracteristics of patients that undergo pre-hospital intubation and, in turn, what factors that affect long-term functional outcome. All TBI patients ≥15 years admitted to the Department of Neurosurgery in Stockholm, Sweden from 2008 through 2014 were included. Data was extracted from prehospital and hospital charts, including prospectively collected Glasgow Outcome Score (GOS) after 12 months. Univariate and multivariate logistic regression models were employed to examine which parameters were independently correlated to prehospital intubation and outcome. A total of 458 patients (n = 178 unconscious) were included. Multivariate analyses indicated that high energy trauma, prehospital hypotension, pupil unresponsiveness, mode of transportation and distance to the hospital were independently correlated with pre-hospital intubation (model explained 0.393 adjusted pseudo-R2), and among them only pupil responsiveness was independently associated with outcome. Pre-hospital intubation was neither associated with outcome in unconscious TBI patients in univariate regression (p = 0.296), nor did it add independent information in a step-up model of independent parameters versus GOS (p = 0.154). In summary, we showed a distinct discrepancy between factors correlated with pre-hospital intubation and parameters associated with outcome. We believe this indicates that the decisions to intubate or not at the scene are based on judgments that are multi-factorial and difficult to assess in a retrospective analysis, but could be beneficial for the individual patient. Prospective studies with structured protocols are necessary to determine potentially favorable effects of pre-hospital intubation, but will be affected by logistic and ethical considerations.
Keywords: Traumatic brain injury, Advanced airway management, Pre-hospital trauma care, Human, Emergency medical services
Bowel dysfunction, often referred to as neurogenic bowel, is one of the most prevalent and clinically recognized comorbidities associated with spinal cord injury (SCI), yet significant knowledge gaps persist regarding the mechanisms contributing to colonic dysfunction after SCI. Following SCI, there is an immediate loss of vascular sympathetic tone resulting in circulatory hypotension. The gastrointestinal (GI) system is highly dependent upon adequate blood flow for proper functioning. Other models of GI inflammation report even brief periods of visceral hypoxia leads to GI dysmotility, inflammation and neuromuscular remodeling. We hypothesized that decreased blood flow and inflammation accompanies colonic remodeling in adult male Wistar rats following a 300kdyn T3-SCI. Three days or three weeks post-injury, both injured and age-matched controls underwent in vivo experimentation to quantify mesenteric blood flow. Following experimentation, tissue was harvested for histological evaluation. Muscularis propria thickness of the proximal colon was significantly increased in the 3-week SCI rats, while the distal colon demonstrated a significant increase in thickness in both 3-day and 3-week SCI rats. Collagen content within the muscularis propria of both the proximal and distal colon was significantly increased in both groups of rats. Colonic mucosal crypt depth and width was significantly reduced within the proximal colon of both groups but only the 3-week SCI rats displayed significant changes in the distal colon. Mean arterial pressure (MAP) and inferior mesenteric artery (IMA) blood flow were significantly lower in T3-SCI rats. Derangements in collagen networks and shortening of mucosal crypts are hallmarks of animals with colitis, and are associated with bowel motor dysfunctions. Our previous reports of diminished colonic motility, enteric oxidative stress, and our present data suggests that bowel dysfunction following T3-SCI may reflect a rapid, multifactorial loss of extrinsic supraspinal innervation, colonic hypoxia in response to decreased systemic blood pressure, and colonic neuromuscular remodeling that may affect the normal physiology and reflect long-term colonic motor dysfunctions as seen in other disease models.
Keywords: Neurogenic bowel, Gastrointestinal smooth muscle, Fibrosis, Dysmotlity
PROGESTERONE ATTENUATES STROKE-INDUCED BRAIN INJURY VIA INHIBITION OF ENDOPLASMIC RETICULUM STRESS: ROLE OF VITAMIN D DEFICIENCY
Keywords: Stroke, Vitamin D, ER Stress, Behavior
QUANTIFICATION OF IMMUNOHISTOCHEMISTRY ON ADJACENT SECTIONS COMPARING FLUORESCENT AND DAB MARKERS
The location of antibody binding sites in brain tissue sections is commonly detected with fluorescent markers or with colored reaction products such as diaminobenzidine. We sought to answer the question: Do both types of detection schemes reveal the antibody binding sites to the same extent as judged by density of staining?
Adjacent sections of tissue were stained with several antibodies, including GFAP for astrocytes and Iba1 for microglia from rat brains that were unilaterally rendered ischemic by middle cerebral artery occlusion. Sets of adjacent sections were immunohistochemically stained free-floating using standard IHC methods. For one set, the antibody binding site was detected using a secondary antibody conjugated with a fluorescent molecule. The second set was stained using the sequence of a secondary antibody conjugated with a biotin molecule, followed by an avidin-HRP complex and then reacting with diaminobenzidine and H2O2.
Two areas of interest were digitally captured for each detection scheme/stain: An ischemic area in one hemisphere and an unaffected area in the other hemisphere. The same region of interest for each stain was analyzed on adjacent sections to rule out any random differences in brain areas. Images of both the DAB and fluorescent stains were converted to 8-bit grayscale. The 8-bit grayscale images of fluorescent-stained tissue were inverted to mimic the 8-bit grayscale images of the DAB images. All 8-bit grayscale images were converted to a binary image for densitometric analysis. Densitometry (measured as the percent area occupied by signal) was performed to determine any differences between the DAB and fluorescent images. The percent area in both the ischemic and normal areas of interest were analyzed in both DAB and fluorescent images for each stain.
There were dramatic differences observed between the two staining methods. In both the GFAP and Iba1 stains there were obvious variations in staining intensity in both the affected and normal brain regions. This is not surprising as there is considerably greater amplification of the antibody presence using the DAB multiple stage sequence vs. the “single” stage fluorophore-secondary antibody sequence. Nonetheless, the degree of hypertrophied astrocytes and microglia seen with the fluorescent method is meager compared to the images using the DAB sequence.
Keywords: IHC Development Method Comparison, Fluorescent vs DAB, Densitometry, IHC Staining Methods
T-lymphocyte (T cell) invasion of the brain parenchyma is a major consequence of traumatic brain injury (TBI). T cells are potent inflammatory mediator, regulating the function of other immune cells including microglial cells. Restricting T cell invasion into the brain improves the function outcome in several brain injury models. We tested the hypothesis that the magnitude of T cell invasion into the cortex is associated with recovery from early post-TBI functional impairment in rats. TBI was induced in adult male Sprague Dawley rats using the lateral fluid percussion injury (FPI) model. The acute functional impairment was assessed based on the neuroscore test. The recovery index was estimated from 2 d to 7 d post-injury. Animals were killed at different time points and the brains processed for immunohistochemical detection of T cells. The total number of T cells was estimated in different brain areas using unbiased stereology. There was a dramatic surge in the total number of T cells in brain at 1 d post-injury (69 146 ± 8710), peaked at 2 d (106 757 ± 19 342) and then drops progressively to 53 409 ± 20 308 at 4 d and 6983 ± 1 150 at 7d. At all time-points, the number of T cells was higher in the cortex than in the hippocampus and thalamus. High number of T cells in the cortex was associated with poor recovery index. Furthermore, animals with poor acute functional impairment continued to show high inflammatory response at the chronic phase. These data demonstrates that T-cell infiltration was most prominent during the first few days after lateral fluid-percussion injury, and a high infiltration magnitude, particularly in the cortex, was associated with severe functional impairment.
Keywords: Lateral fluid-percussion brain injury, T lymphocytes, Functional impairment
GENDER DIFFERENCES IN THE PERIPHERAL IMMUNE RESPONSE TO SPINAL CORD INJURY
Four times as many men are paralyzed from spinal cord injury (SCI) than women worldwide. Although the difference is often credited to male propensity to take risks, female gender may be neuroprotective as females recover motor function better than males. The mechanism is not known. Recently, Dr. Charlotte Hsu, in her PhD thesis, found Fischer 344 (F344) female rats recovered faster with better locomotion than male F344 rats. She found no gender differences in spinal cord anatomy, lesion volume or lipid peroxidation at 24 hours after injury. We next investigated neuroimmune response, a possible contributor to secondary spinal cord injury not well studied in both genders. We performed a T11 spinal cord contusion in F344 rats. Blood was sequentially collected in the same animal cohort pre-injury, 24 hours, 3 days, 7 days and 14 days after injury. White blood cells (WBCs) were manually counted with Hoechst stain followed by differentiation of blood smears and flow cytometry. We found gender differences in the peripheral immune response to SCI. Blood neutrophils and monocytes increased in males and females at 24 hours but continued to peak only in males 3 days after injury. Neutrophils returned to baseline 7 days after injury. In contrast, blood monocytes returned to baseline at 7 days but increased 14 days after injury in both genders. Blood T cells significantly declined in females 24 hours after injury while there was no change in males. T cells increased at 3 days and 7 days, returning to baseline by 14 days. B cells declined at 24 hours, slightly increased at 3 and 7 days but significantly declined to the lowest counts 14 days after injury. Total circulating WBCs declined in both genders 14 days after injury. Our findings demonstrate significant and gender specific changes in the peripheral blood response to SCI. Significantly lower blood neutrophils and monocytes in females may be neuroprotective acutely after SCI.
Keywords: Gender, Blood, Neutrophil, Monocyte, Lymphocyte
NOX2 DEFICIENCY ALTERS MACROPHAGE PHENOTYPE THROUGH AN IL-10/STAT3 DEPENDENT MECHANISM: IMPLICATIONS FOR TRAUMATIC BRAIN INJURY
University of Maryland, Department of Anesthesiology and Shock, Trauma and Anesthesiology Research Center, Baltimore, USA
NOX2 is an enzyme that generates ROS in microglia and macrophages. ROS production is linked with neuroinflammation and chronic neurodegeneration following traumatic brain injury (TBI). NOX2 inhibition following moderate-to-severe TBI markedly reduces pro-inflammatory activation markers and results in concomitant increases in anti-inflammatory responses. Here, we report the signaling pathways that regulate NOX2-dependent macrophage/microglial phenotype in the TBI brain. Bone marrow-derived macrophages (BMDMs) prepared from wildtype (C57Bl/6) and NOX2 deficient (NOX2−/−) mice were treated with combined lipopolysaccharide/ interleukin-4 (LPS/IL-4; both 10 ng/ml), to investigate signal transduction pathways associated with macrophage activation. Signaling pathways and activation markers were evaluated in ipsilateral cortical tissue obtained from adult male wildtype and NOX2−/− mice that received moderate-level controlled cortical impact (CCI). A neutralizing anti-IL-10 approach was used to determine the effects of IL-10 on NOX2-dependent changes. LPS/IL-4-stimulated BMDMs mimic the pro- and anti-inflammatory responses observed in the injured cortex, we show that NOX2−/− significantly reduces markers of proinflammatory activation. In addition, NOX2−/− results in significantly increased anti-inflammatory marker expression, which seems to be dependent on IL-10-mediated STAT3 signaling. Following moderate-level CCI, IL-10 is significantly increased in microglia/macrophages in the injured cortex of NOX2−/− mice. These changes are associated with increased STAT3 activation and a robust anti-inflammatory response. Neutralization of IL-10 in NOX2−/− BMDMs or CCI mice blocks the anti-inflammatory response, thereby demonstrating a critical role for IL-10 in regulating NOX2-dependent changes. These studies indicate that following TBI NOX2 inhibition promotes a robust anti-inflammatory response that is mediated by the IL-10/STAT3 signaling pathway. Thus, therapeutic interventions that inhibit NOX2 activity may improve TBI outcomes by not only limiting pro-inflammatory, but also enhancing IL-10-mediated anti-inflammatory responses that are neuroprotective.
Keywords: Macrophage, NOX2, Neuroinflammation, Interleukin-10, Traumatic brain injury
Augusta University, Neurosurgery, Augusta, USA
Inflammation is an important component of secondary neurological injury after TBI. Immune cells, particularly macrophages, aid in clearance of cellular debris and tissue repair, but sustained release of pro-inflammatory mediators from infiltrating immune cells may exacerbate neuronal death, increase neurovascular injury, and contribute to long-term loss of white matter. Although the precise mechanisms underlying the dual beneficial and detrimental roles of macrophages after CNS injury remain poorly defined, it has been well established that macrophages polarize along a continuum from a classical pro-inflammatory (M1) state to an alternative anti-inflammatory (M2) state. Thus, modulation of macrophage polarization may identify novel opportunities for therapeutic intervention after TBI. Non-psychoactive cannabinoid receptor 2 (CB2R) is predominantly expressed on immune (lymphocytes, monocytes, macrophages) and endothelial cells in both rodents and humans. In the present study, we hypothesize that activation of CB2R attenuates inflammation post-TBI. Using a moderate controlled cortical impact (CCI) model of murine TBI, we assessed the endogenous upregulation of CB2R after TBI and the effects of CB2R agonist and antagonist post-injury. We observed acute upregulation of CB2R up to 3 days post TBI and moderate elevated expression persisted at 3 weeks. Furthermore, CB2R co-localized with infiltrated myeloid cells, specifically macrophages, after TBI. Finally, administration of selective CB2R agonist GP1a attenuated neuroinflammation, while treatment with antagonist AM630 worsened the injury. Interestingly, attenuation of inflammation by GP1a was associated with increased M2 polarization of macrophages. Our findings suggest that CB2R may play an important role in immune cell regulation; therefore, the development of selective CB2R agonists may provide clinical benefits for brain injury patients, without the psychoactive effects of CB1R activation.
Keywords: traumatic brain injury (TBI), cannabinoid receptor 2 (CB2), inflammation, macrophage
Augusta University, Neurosurgery, Augusta, USA
Traumatic brain injury (TBI) is a major public health issue, producing significant patient mortality and poor long-term outcomes. Increasing evidence suggests an important, yet poorly defined, role for the immune system in the development of secondary neurologic injury over the days and weeks following a TBI. In this study, we tested the hypothesis that peripheral macrophage infiltration initiates long-lasting adaptive immune responses after TBI. Using a murine controlled cortical impact model, we used adoptive transfer, transgenic, and bone marrow chimera approaches to show increased infiltration and proinflammatory (M1) polarization of macrophages for up to 3 wk post-TBI. Monocytes purified from the injured brain stimulated the proliferation of naive T lymphocytes, enhanced the polarization of T effector cells (TH1/TH17), and decreased the production of regulatory T cells in an MLR. Similarly, elevated T effector cell polarization within blood and brain tissue was attenuated by myeloid cell depletion after TBI. Functionally, C3H/HeJ (TLR4 mutant) mice reversed M1 macrophage and TH1/TH17 polarization after TBI compared with C3H/OuJ (wild-type) mice. Moreover, brain monocytes isolated from C3H/HeJ mice were less potent stimulators of T lymphocyte proliferation and TH1/TH17 polarization compared with C3H/OuJ monocytes. Taken together, our data implicate TLR4-dependent, M1 macrophage trafficking/polarization into the CNS as a key mechanistic link between acute TBI and long-term, adaptive immune responses.
Keywords: traumatic brain injury (TBI), inflammation, macrophage polarization, T lymphocyte
Barrow at Phoenix Children's Hospital, Phoenix, USA
Effects of traumatic brain injury (TBI) are not isolated to the central nervous system, but can lead to excessive systemic inflammation, exacerbating neurological symptoms such as sleep disruptions which complicate recovery. In the absence of a pathological inflammatory event, peripheral inflammatory cytokines are regulated by the innate immune response. Subsequent to a pathological inflammatory event, the adaptive immune response then attenuates uncontrolled systemic inflammation, with evidence for action through the cholinergic anti-inflammatory pathway. We show that α9/α10 nicotinic acetylcholine receptors (nAChR) are widely expressed on peripheral immune cells, suggesting that nAChR activation may affect inflammatory cell (e.g., macrophage) function. Specifically, α9/α10-nAChR activation may amplify the migration and production of inflammatory cytokines. Inflammatory cytokines can have dual roles as sleep regulatory substances (IL-1β, IL-6) and we previously showed prolonged sleep may be a physiological indicator of inflammation. Here, we hypothesize that a peripheral injection of lipopolysaccharide-(LPS), a trigger of inflammation, significantly increases peripheral cytokines and sleep in wildtype mice, but not in α9/α10-nAChR double knock-out mice. Mice were acclimated to non-invasive piezoelectric sleep cages and baseline sleep was recorded. Wildtype or transgenic mice were administered intraperitoneal injections of LPS-(3μg) or saline and returned to sleep cages. Following 6 hours of sleep recording, blood was collected for plasma analysis of peripheral cytokines. Results indicated LPS significantly increased sleep in wildtype-LPS mice compared to α9/α10-nAChR-LPS mice and saline-treated mice. As expected, LPS significantly increased IL-1β and IL-6 compared to saline-mice and current analyses comparing cytokine levels in α9/α10-nAChR are underway. Ongoing experiments evaluating sleep and cytokines in α9/α10-nAChR knock-out mice following diffuse TBI may identify a mechanism for the peripheral amplification of central inflammation. The role of α9/α10-nAChR in regulating the immune response is unknown and remains a potential underlying mechanism of action and therapeutic target for uncontrolled inflammation after TBI. Funding: Sleep-Medicine Foundation; BNI@PCH.
Keywords: peripheral, cholinergic, nicotinic
Fluid percussion-induced traumatic brain injury (TBI) is one of the most popular experimental models available. Typically, fluid percussion injury (FPI) is applied at midline or parasagittal to midline in rodents. Midline FPI is broadly defined as a diffuse model of TBI in which both hemispheres are equally injured. Lateral FPI is a mixed model of TBI in which the ipsilateral hemisphere receives a focal injury resulting in diffuse damage to subcortical structures. The present study investigated the acute inflammatory response to midline and lateral FPI, with specific emphasis on leukocyte recruitment and cytokine expression within the brain during the first week post-injury. We hypothesized that the acute inflammatory response to TBI would be enhanced following lateral FPI compared to midline FPI. To test this hypothesis, lateral or midline FPI or sham injury was administered to equal numbers of male and female C57BL6 mice at two months of age. By four hours post-injury, flow cytometric analysis revealed a TBI-induced increase in circulating neutrophils following midline and lateral FPI. Unexpectedly, sham injured mice receiving a midline or lateral craniectomy displayed a higher percentage of patrolling monocytes compared to TBI. Furthermore, both sham and TBI mice in the lateral FPI group displayed a higher percentage of circulating inflammatory monocytes. Brain neutrophils and inflammatory monocytes increased following midline and lateral FPI and occurred in conjunction with an increase in inflammatory cytokines IL1-β, TNF-α, and CCL2 at 4 hours post-injury. By 72 hours post-injury, the percentage of circulating neutrophils and monocytes declined in midline and lateral TBI mice. Similarly, brain leukocytes and inflammatory cytokine expression decreased by 72 hours post-injury, although CCL2 expression remained increased in the ipsilateral hemisphere following lateral FPI. Taken together, these data indicate that the acute inflammatory response to midline and lateral FPI is quite similar with regard to leukocyte recruitment and cytokine expression soon after injury. Further studies are underway to determine differences in behavior and leukocyte recruitment at sub-acute time points.
Keywords: leukocytes, fluid percussion injury
NADPH OXIDASE-2 REGULATES NLRP3 INFLAMMASOME ACTIVATION AFTER TRAUMATIC BRAIN INJURY
Medical College of Georgia - Augusta University, Neuroscience and Regenerative Medicine, Augusta, USA
Traumatic brain injury (TBI) contributes to over 30% of injury related deaths. A complex secondary injury cascade follows the initial mechanical injury and exacerbates the primary injury. Oxidative stress and inflammation play key roles in this TBI pathology. NADPH oxidase-2 (NOX2) and inflammasomes have been reported as major contributors to oxidative stress and inflammation in the injured brain, respectively, and deletion of NOX2 is neuroprotective. In particular, the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome can become activated in response to oxidative stress, but little is known about this mechanism following TBI. In this study we utilize NOX2 knockout mice to study the role of NOX2 in mediating NLRP3 inflammasome expression and activation in a controlled cortical impact model of focal TBI. Expression of NLRP3 inflammasome components (NLRP3, apoptosis speck-like protein containing a CARD (ASC), and caspase-1) was robustly increased in the injured cerebral cortex following TBI. Deletion of NOX2 attenuated the expression and assembly of the NLRP3 inflammasome components, which was coupled to reduced cleavage of the pro-inflammatory factor, interleukin-1β (IL-1β). Further work revealed that NOX2 regulation of NLRP3 inflammasome may be mediated by thioredoxin interacting protein (TXNIP), a sensor of oxidative stress. In conclusion, the results of the current study provide novel evidence that NOX2-dependent inflammasome activation contributes to TBI pathology.
Keywords: NADPH Oxidase 2, NLRP3, Inflammasome, Traumatic Brain Injury, Oxidative Stress, Controlled Cortical Impact
UCSF, Neurological Surgery, San Francisco, USA
The acute inflammatory response following spinal cord injury (SCI) is marked by extensive invasion of myeloid lineage leukocytes that exacerbate the injury site by releasing neurotoxic substances. L-selectin, a C-type lectin adhesion receptor, is broadly expressed on myeloid cells and mediates their recruitment to sites of inflammation. Recent studies have shown that L-selectin is involved in additional functions including the activation of integrins, tethering of myeloid cells along the endothelium, and potentiation of neutrophil effector functions. We have previously demonstrated reduced acute pathology and improved long-term recovery in L-selectin knockout mice (KOs). However, the early pathogenic activities of L-selectin and the specific myeloid subtypes involved have yet to be identified. To address this, we examined L-selectin (CD62L) levels on myeloid lineage cells, as well as accumulation of these cells, in uninjured and acutely injured mice by flow cytometry. We found that L-selectin is dynamically regulated after SCI on circulating inflammatory monocytes (Ly6Chi/Ly6G−), non-classical monocytes (Ly6Clow/Ly6G−) and neutrophils (Ly6Clow/Ly6G+). Reduced neutrophil accumulation was observed in the injured spinal cords of L-selectin KO mice compared to wildtypes (WTs) at 24 hours post-SCI, but not at 72 hrs post-SCI. Furthermore, activation of circulating neutrophils, as shown by CD11b levels, was reduced in KOs compared to WTs at 24 hours post-SCI. Administration of diclofenac, an FDA-approved non-steroidal anti-inflammatory drug (NSAID) which is a potent inducer of L-selectin shedding (mediated by cell surface metalloproteinases), reduced L-selectin on circulating non-classical monocytes and neutrophils. Diclofenac did not affect the number of monocytes or neutrophils that accumulated in the injured spinal cord, but did reduce L-selectin levels on infiltrated non-classical monocytes and neutrophils, suggesting other activities for L-selectin beyond recruitment. These findings implicate L-selectin in early pathogenesis and elucidate a novel mechanism (L-selectin shedding) by which NSAIDs attenuate acute inflammatory damage after SCI. Supported by: Department of Defense SCIRP W81XWH-12-1-0563 and National Institutes of Health NINDS F32NS096883.
Keywords: neutrophil, l-selectin, shedding, diclofenac, NSAID
French Armed Forces Biomedical Research Institute, Bretigny-sur-Orge, France
We tested whether [D-Ala2, D-Leu5]-Enkephalin (DADLE), a synthetic delta-opioid receptor agonist, could mitigate brain inflammation following head trauma in adult rats. DADLE (1 and 5 mg/kg) was intraperitoneally injected to rats 15 minutes, 2 and 5 hours after being exposed to left-side parietal fluid percussion injury. Three hours after the last DADLE injection, rats were euthanized, then, their brain was harvested. Cortical and hippocampal tissues were microdissected from the left (ipsilateral, injured) and right (contralateral) sides of each brain and processed for qPCR quantitation of the expression of several prototypical inflammatory markers: Interleukin-1 beta (IL-1b), IL-6, Tumor necrosis factor alpha (TNFa), Macrophage inflammatory protein 1 alpha (MIP1a) and Monocyte chemoattractant protein 1 (MCP-1). As predicted, the expression of all of the inflammatory markers was increased in the ipsilateral cortex and hippocampus after TBI. To a lower extent, exposure to TBI also increased the expression of IL-1b, TNFa, MIP1a and MCP-1 in the contralateral cortex and of IL-1b, IL-6, TNFa and MCP-1 in the contralateral hippocampus. Treatment with the highest dose of DADLE decreased the expression of IL-1b in the contralateral cortex by 39%, and in the ipsilateral and contralateral hippocampus by 31 and 33%. Low dose DADLE did not influence the expression of IL-1b but rather increased the expression of IL-6 in the contralateral cortex and of TNFa and MIP1a in the contralateral hippocampus. Thus, treatment with a high dose of DADLE can mitigate acute brain inflammatory processes following head trauma. By contrast, treatment with a low dose of DADLE tends to increase post-traumatic brain inflammation. It is possible that the differential effects of DADLE are due to recruitment of different subtypes of opioid receptors. This work was supported by a grant from the Direction Générale de l'Armement (French Ministry of Defense).
Keywords: traumatic brain injury, animal model, delta opioid agonist, inflammation
University of Texas Medical School at Houston, Pediatric Surgery, Houston, USA
Traumatic brain injury (TBI) remains a serious public health problem in the United States. According to the World Health Organization (WHO), TBI is a permanent, chronic disease process, caused by non-reversible pathological alterations, requiring special rehabilitation, and/or a long period of observation, supervision, or care. However, despite intensive investigative endeavors, there are no FDA-approved drugs designed to reduce morbidity and mortality associated with TBI.
Neuroinflammation, which been detected as lasting in humans for up to 17 years, plays a pivotal role in the pathogenesis of TBI. In the acute stage of TBI, inflammation activates and mobilizes immune cells toward the site of injury that interfere with the endogenous capacity of the brain to repair itself, exacerbating neuronal death. In the chronic stage, an inflammatory microenvironment caused by an excessive activation of immune cells further contributes to secondary neuronal death and a subsequent positive feedback inflammatory loop.
Teriflunomide (Aubagio) has been approved by the US Food and Drug Administration (FDA) for use in the treatment of multiple sclerosis since 2012. Teriflunomide has been shown to act as an inhibitor of dihydroorotate-dehydrogenase (DHODH), a key mitochondrial enzyme involved in the de novo synthesis of pyrimidines in rapidly proliferating T lymphocytes and B lymphocytes. Moreover, teriflunomide has been shown to affect immunological responses outside of its ability to inhibit pyrimidine synthesis in rapidly proliferating cells, suggesting Teriflunomide may have additional therapeutic mechanisms.
In here, we studied the treatment effect of teriflunomide in severe/moderate and mild injury models of rats. Our results show that in both the rat models the drug could recover the blood-brain barrier integrity and ameliorate inflammation in brain both short term and long term. Consequently, we saw improvement in memory and special learning abilities after treatment in severe/moderate injury. However, our mild injury model didn't have any behavioral deficits.
This project was funded by Sanofi Genzyme.
Keywords: teriflunomide, neuroinflammation, Traumatic Brain Injury, Aubagio,
Virginia Commonwealth University, Pharmacology & Toxicology, Richmond, USA
The endogenous cannabinoid 2-arachidonyl glycerol (2-AG) serves as a rate-limiting precursor for arachidonic acid (AA), a precursor for the production of pro-inflammatory eicosanoids in brain. In vitro work shows that inhibiting the 2-AG biosynthetic enzyme diacylglyercol lipase-β (DAGL-β), which is highly expressed on CNS resident microglia and macrophages in the periphery, reduces inflammatory responses. Here, we examine whether DAGL-β−/− mice display a protection phenotype from the consequences of experimental traumatic brain injury. Experiment 1: Male DAGL-β−/− and +/+ mice were subjected to a left lateral moderate Fluid Percussion injury (FPI) (1.94 ± 0.1 atm), and then assessed for spatial memory performance in a Morris water maze Fixed Platform task as well as for neurological motor impairments using the Neurological Severity Score (NSS) and the Rotarod assay. Unexpectedly, DAGL-β−/− mice demonstrated a significant survival protective phenotype (100% survival) in response to brain injury compared to DAGL-β+/+ mice (77% survival), despite equal injury severities between groups. However, DAGL-β−/− mice displayed similar magnitudes of FPI-induced cognitive impairments in the Fixed Platform task and neurological motor deficits in the NSS and Rotarod assays as DAGL-β+/+ mice. Experiment 2: We increased the magnitude of FPI to 2.0 ± 0.1 atm and 2.17 ± 0.1 atm in male and female DAGL-β−/− and +/+ mice, and assessed mortality. Male DAGL-β−/− mice demonstrated a significant survival protective phenotype at both 2.0 atm (100% survival) and 2.17 atm (90% survival) compared to DAGL-β+/+ mice (75% survival at 2.0 atm, and 60% survival at 2.0 atm). In contrast, female DAGL-β mice generally survived both injury magnitudes regardless of phenotype, suggesting sex differences in survival from FPI. These findings suggest the provocative possibility that DAGL-β activity contributes to TBI-induced mortality, but not to the evolution of TBI cognitive or motor impairments in mice. Accordingly, DAGL-β inhibition represents a potential strategy to ameliorate post-traumatic fatality.
Keywords: Endogenous Cannabinoid, Mortality, Diacylglycerol lipase-β, 2-arachidonylglycerol (2-AG), Spatial Memory, Sex Differences
Inflammation is a prominent secondary event following traumatic brain injury (TBI). Inflammation is mediated by inflammatory cells and inflammatory cytokines/chemokines. Among pro-inflammatory cytokines, interleukin-1 β (IL-1β) plays a pivotal role in triggering inflammatory cascade. Another cytokine, interleukin-18 (IL-18) is a potent inflammation mediator that initiates/amplifies many inflammatory processes. Recent studies have found that release of IL-1β and IL-18 is mediated by inflammasomes. Inflammasomes are intracellular multiprotein complexes which regulate innate immune response, production of pro-inflammatory cytokines such as IL-1β and IL-18, activation of caspase-1 and induction of cell death (pyroptosis). Among the known inflammasomes, NLRP3 is the most extensively studied and its activation is induced following TBI. As a novel target, drug development targeting formation/activation of inflammasomes could be a prospective therapy for TBI. We have recently developed a small molecule GA3 with specificity on NLRP3 inflammasome. GA3 can selectively inhibit NLRP3 inflammasome formation, activation of caspase-1, and production of IL-1β. In this study, we explored the therapeutic value of GA3 for TBI. Adult male Sprague-Dawley rats were subjected to a moderate cortical impact injury. Following TBI, animals received 4 doses of GA3 treatment 6 hrs in bwtween with the first dose starting at 30 minutes folliwng injury. Sensorimotor functions and cognitive functions were assessed. Animals were sacrificed at 2 or 15 days post-injury. Brain tissues were processed for histological examination to assess degenerative neurons, lesion volume, and inflammatory cell response. We found that post-injury treatment with GA3 significantly decreased the number of injury-induced degenerating neurons in the injured cortex and hippocampus. GA3 treatment also reduced cortical brain tissue damage and inflammatory cell response. Moreover, injured animals treated with GA3 had significant improvement in cognitive functional recovery. Further studies assessing the dose response and therapeutic time window of GA3 treatment in this focal injury model are ongoing. Our data suggest that our novel NLRP3 inhibitor has neuroprotective effects for TBI. Supported by CHRB #236-14-15 (Sun).
Keywords: traumatic brain injury, inflammasomes, neuroinflammation, neuroprotection, cognitive function
TARGETING THE LIVER ACUTE PHASE RESPONSE FOLLOWING TRAUMATIC BRAIN INJURY REDUCES BRAIN INFLAMMATION
Georgetown University, Neuroscience, Washington, USA
Traumatic brain injury (TBI) triggers a loss of tissue followed by a strong and acute inflammatory response. Serum amyloid A1 (SAA1) is an acute phase protein that is mainly produced in the liver in response to inflammatory events such as TBI. SAA1 is released into the bloodstream, establishing a channel of communication between the liver and extra-hepatic organs. To date, no in vivo studies have investigated the role of SAA1 release in the central inflammatory response after TBI. We performed a mild and moderate/severe controlled cortical impact (CCI) injury in male C57BL/6J mice. One week before CCI, we injected antisense oligonucleotides (ASOs) (25 mg/kg/week by i.p.) to suppress mRNA SAA1 production. We wanted to explore the potential application of ASOs by modifying the inflammatory response. Our results revealed that the highest grade of TBI severity was correlated with high levels of SAA1 in plasma at 1 day post-injury, and with a larger lesion volume and inflammation. We also found that SAA1 colocalizes with activated macrophages/microglial cells in the injured cortex and hippocampus; blocking SAA1 production reduced microglia activation and macrophage infiltration into the brain. Finally, we demonstrated that ASOs reduced SAA1 levels by 60–80% in plasma at 1 and 3 days after brain injury. ASO-treated mice displayed better motor skills on the rotarod 1 day post-injury. Collectively, our findings demonstrate that SAA1 levels in plasma after brain damage depend on damage severity, acting as a potential biomarker. Also, we found that SAA1 binds to macrophages/microglial cells in the injured brain, and that ASO treatment reduces the inflammatory response in the damaged brain. Altogether, this suggests that blocking SAA1 production could provide an advantageous environment for repair and functional recovery after brain trauma.
Keywords: peripheral inflammation, liver, microglia, macrophages, acute phase proteins
University of California San Francisco, Weill Institutes for Neurosciences, Brain and Spinal Injury Center, Department of Neurosurgery, San Francisco, USA
The objective of this collaborative study is to provide a proof-of-concept of modern data science tools applied to existing high-density data repositories of preclinical animal studies in traumatic brain injury (TBI) for novel translational discoveries. Multi-modal data from graded TBI injuries with varied treatment conditions were gathered from Moody Project for Translational TBI Research studies in rodents. In a chronic time course study, long term learning and memory deficits persist up to one-year post TBI. To convert these big-data to knowledge and to elucidate connections between behavior and histopathologic, genomic and proteomic changes post TBI, the Moody Project PIs launched a collaboration with data scientists with expertise in preclinical and clinical neurotrauma data. The main purpose of this effort is to create an example repository of completed studies in preclinical TBI, and develop syndromic analytical workflows that 1) capture both the complexity of the study design and 2) render outcome testing through multidimensional machine learning approaches to increase the effect size resolution for hypothesis testing of treatment trials. Prior applications of these methodologies to preclinical TBI and clinical TBI have demonstrated utility of multiscalar ‘omics (syndromics) for TBI and SCI. The current study aims to expand upon these efforts, capturing the full spectrum of bio-behavioral and molecular changes that result from TBI, and help resolve treatment approaches that reproduce results across diverse endpoints, increasing potential for translation into clinical trials in humans. These studies were supported in part by the Moody Project for Translational TBI Research and NIH BD2K RoAD-Trip Fellowship.
Keywords: Syndromics, Statistics, Multimodal, Rats
UTMB, Neuroscience Graduate Program and MD/PhD Program, Galveston, USA
The purpose of this study was to determine the global protein changes in the acute, subacute and chronic stages of traumatic brain injury (TBI). Rats were injured using a fluid percussion injury (FPI) model. Brain tissues were collected at 24 hours, 2 weeks, 3 months, 6 months, 1 year after injury. Ipsilateral rat cortex and hippocampi were isolated and analyzed separately for each time point. Tissues were homogenized and then trypsin digested. Protein fragments were analyzed using an orbitrap mass spectrometer and the relative changes in protein levels were determined by comparison with sham operated and naïve animals of the same age. Numerous protein changes were found at the five different time points and they differed among the two brain regions. There was a dramatic change in the cortex neurotransmitter receptor composition at 3 months that resolved by 6 months. Furthermore, significant changes in the hippocampus were observed at 6 months which resolved at 1 year. Finally, proteins associated with multiple metabolic pathways were found to be altered at the 1 year time point. Changes observed indicate substantial modifications in energy metabolism, calcium homeostasis, neurotransmitter receptor composition and immune response. This study has given us greater insight as to what changes occur in the brain after TBI and is the most extensive study of its kind to date. Most significantly, we find changes at the later time points that could help explain the increased risk for neurological disorders in patients with TBI. We acknowledge the finacial support of the Moody Project for Translational Traumatic Brain Injury Research.
Keywords: Longitudinal Proteomics, Chronic TBI
UCLA, Neurosurgery, Los Angeles, USA
Decreases in energy metabolism following traumatic brain injury (TBI) are attributed to impairment in utilizing glucose as a primary fuel source and supplementation with alternative fuels improves outcomes. Unanswered is when and what fuel to provide? Changes in energy metabolism following TBI happen in two phases; a brief period of hyper-metabolism followed by extensive hypo-metabolism. A fuel may function through different mechanisms depending on timing of delivery. It is hypothesized that outcome measures will differ based on a fuel's timing of delivery. Adult male rats were given sham or controlled cortical impact (CCI) injury, then intravenously infused with either glucose, lactate or beta-hydroxybutyrate for 3 hr (early) immediately after or beginning 6hr (late) after injury. Animals were euthanized 24 hr post-injury; ipsilateral-cortex and blood were collected for analyses. Injury alone significantly impaired mitochondrial respiration and citrate synthase (CS) activity and increased reactive oxygen species (ROS) production. Mitochondrial respiration was significantly improved by both early-lactate and late-BHB. ROS production was decreased by both early- and late-BHB and lactate in part by their scavenging properties and upregulation of antioxidant and uncoupling proteins by BHB. Late-glucose also decreased ROS production and may be due to increased pentose phosphate pathway (PPP) activity. Glucose is a known stimulator of mitochondrial respiration and CS activity was normalized. As expected, BHB decreased CS activity via inhibition by acetyl-CoA production. Both early and late-lactate normalized activity of CS. Untargeted metabolomics found significant group differences in PPP, TCA and glycolytic metabolites. An additional cohort of sham, CCI and CCI + early-lactate animals were euthanized at 1hr post-injury and glycogen was quantified. CCI significantly decreased glycogen, while early-lactate increased glycogen to sham amounts indicating early-lactate may improve lactate shuttle activity between astrocytes and neurons. These data stress there is no one optimal alternative fuel, but rather the fuel type used should be guided by metabolic state. This also suggests combination therapy of fuels may be necessary to ensure best outcome in TBI patients.
Keywords: mitochondria, metabolomics, alternative fuel, timing of delivery, outcome
Children's Hospital of Philadelphia, Philadelphia, USA
Damage to the mitochondrial bioenergetics, including the electron transport chain function (ETC) and substrate oxidation and phosphorylation (OXPHOS), exacerbates traumatic brain injury (TBI) and impedes repair. To identify the time-course of mitochondrial bioenergetics after diffuse TBI, which is critical to defining therapeutic targets and treatment windows. Piglets (N = 15), toddler 4-wk olds 8–9 kg, were randomized to 3 groups: sagittal rapid nonimpact rotations (RNR) with terminal end-points: 6 hours (N = 5) or 7 days (n = 5), and Sham (N = 5). At terminal time-points, cortical and hippocampal tissue were harvested rapidly. Fresh tissue homogenates were assayed utilizing high resolution respirometry (Oroboros) for real-time analysis of ETC function and OXPHOS, via titration of substrates, toxins or uncouplers. Respiration values were normalized by mitochondrial content: citrate synthase (rmol/s*mg/CS) and reported as mean ± SEM. Complex I (CI) respiration was significantly (p < 0.05) decreased in cortex and hippocampus, at 6 hours and 7 days compared to shams. Specifically, cortical CI was 1.98 ± 0.12 at 6 hours, 2.12 ± 0.11 at 7 days, and 4.00 ± 0.23 in sham. Hippocampal CI was 1.55 ± 0.10 at 6 hours, 1.77 ± 0.20 at 7 days, and 3.09 ± 0.20 in sham. Complex II respiration (CII) was significantly decreased compared to sham in the cortex (3.12 ± 0.08 vs. 4.1 ± 0.27) and hippocampus (3.39 ± 0.34 vs. 4.4 ± 0.25) at 6 hours; by 7 days there was no significant difference in CII in either injured region. Maximal OXPHOS CI+CII was significantly decreased at 6 hours and 7 days in both injured brain regions versus sham (cortex: 4.88 ± 0.01 and 5.27 ± 0.10 versus 6.28 ± 0.17; hippocampus: 5.00 ± 0.34 and 5.16 ± 0.06 versus 6.64 ± 0.21). Persistent CI dysfunction is implicated in the pathogenesis of neurodegenerative disease and in murine animal models of focal TBI and ischemia reperfusion injury. Interpreted with our previous reported data at 24 hours, diffuse TBI triggers a tri-phasic response of CI: early decrease at 6 hours, transient recovery by 24 hours, with significant CI dysfunction again at 7 days, contributing to decreased OXPHOS. Future interventions should target CI dysfunction after diffuse TBI.
Keywords: mitochondria, time-course, complex I, porcine, diffuse TBI
University of Cincinnati, Neurosurgery, Cincinnati, USA
Traumatic brain injury (TBI) is a leading cause of accidental death in individuals under the age of 45, affecting 2.8 million people in the United States annually. Acute changes in brain metabolism after TBI are associated with changes in neurologic function in animal models and human subjects. Less is known about chronic changes in brain metabolism after TBI. NMR-metabolomics measures small molecule metabolites that are the substrates and products of biochemical reactions. These metabolites support diverse processes including energy production, protein, nucleotide, and transmitter synthesis, and post-translational modification that are critical to cell function. We hypothesize that metabolic changes in chronic TBI are not uniformly distributed throughout the brain and that different brain regions have unique metabolic signatures after TBI. As brain metabolism is tightly regulated, we further hypothesize that these changes are subtle, reflecting reprioritization of resources rather than changes in tissue concentrations of individual metabolites. We examined metabolic profiles in 5 brain regions and plasma in adult rats 60 days after lateral fluid percussion injury or in naïve animals. We found changes in metabolite concentrations between naïve and TBI animals only in hippocampus. Changes in metabolite levels in hippocampus suggest a bias towards excitatory>inhibitory neurotransmission, with increases in glutamate and glutamine, but not GABA. These data also suggest that in hippocampus molecules involved in processes that span neurons and glia (the glu/gln cycle, the lactate shuttle) may be preferentially altered in chronic TBI. Using a correlation-based analyses, we found both significantly increased and decreased correlations between metabolites in chronic TBI in every brain region looked at. We interpret these findings as alterations in how these molecules are utilized and regulated within cells and between cells types in each brain region.
Keywords: chronic TBI, metabolomics, rat, lateral fluid percussion
Traumatic brain injury (TBI) is the leading cause of permanent life-long disability in children and is characterized by deficits in learning and memory, cognition, attention and sensory-motor integration. Metabolic crisis caused by impaired oxidative glucose metabolism following TBI further contributes to cell death. Although glucose is the primary substrate for brain energy and metabolism, brain is also equipped with the ability to oxidize fatty acids for energy and metabolism. This study aimed to examine alterations in both brain glucose and fatty acid metabolism after TBI. Postnatal day 21–22 male rats were anesthetized with isoflurane and TBI was administered using a controlled cortical impact to the left parietal cortex. Harvested brains were dissected into ipsilateral cortex, ipsilateral hippocampus, contralateral cortex, and hippocampus. Using RT-PCR (Qiagen) we analyzed the expression of genes involved in regulation and enzymatic pathways of glucose and glycogen metabolism, as well as genes involved in fatty acid transport and metabolism at 24 hr, 72 hr and 7days after TBI. We found that both astrocytic (Glut1) and neuronal (Glut3) transporters RNA were peaked at 24hrs in all regions studied, while RNA for pyruvate dehydrogenase E1alpha – a key enzyme, which links glycolysis and Kreb's cycle, was decreased at 24 hrs and 72 hrs post injury. In contrast, Fabp7 (brain specific fatty acid transporter) was increased following TBI and peaked at 72 hrs, while mitochondrial fatty acid metabolism genes (CPT1A, CPT2) were not changed after TBI. TCA cycle genes remained unaffected by TBI. To determine the function, we measured glucose and fatty acid oxidation. Glucose oxidation was significantly decreased in ipsilateral cortex and hippocampus, while fatty acid oxidation was increased after TBI in the ipsilateral hippocampus but not in the cortex. This study provides evidence that brain following TBI is capable to use endogenous substrates (fatty acids) to meet the metabolic demands.
Keywords: glucose
ASSOCIATING THE ALTERED GLUCOSE UPTAKE WITH CHANGES IN GLUCOSE TRANSPORTERS, INFLAMMATION AND PATHOLOGY IN DIFFUSE TBI
National Institutes of Health, Radiology and Imaging Sciences, Bethesda, USA
These results suggest that the injured brain required immediate energy to restore neurological function within 24 hr after injury, where the increased astrocyte-to-neuron lactate shuttle(ANLS) appeared to support the high-energy demands by neurons. This was associated with acute increase expression of Glut1/Glut3. However, the brain then entered a hypometabolism state in week 2–3, decreased glucose uptake was disassociated from the expression of Glut1/Glut3, potentially related to the increased inflammation and apoptosis in this secondary injury phase. The non-invasive GWI affords the sensitivity to identify the glucose level for treatment window to increase neuronal survival in TBI.
Keywords: CEST, 2DG, Astrocyte, DTI, Glucose Metabolism, Neuron
University of Kentucky, Spinal Cord and Brain Injury Research Center, Lexington, USA
Mitochondrial dysfunction has been shown to occur following traumatic brain injury (TBI) and spinal cord injury (SCI). Trauma-induced intracellular Ca2+ influx leads to increased mitochondrial Ca2+ buffering and reduced membrane potential. The link between Ca2+ buffering and shut down of mitochondrial respiration seems to be modulation of mitoNEET, an outer mitochondrial membrane protein. Pioglitazone, which binds mitoNEET, has shown neuroprotective effects following TBI and SCI possibly through its interactions with mitoNEET in a peroxisome proliferator activated receptor (PPARγ) independent manner. Furthermore, NL-1, a pioglitazone derivative lacking PPARγ binding, has been demonstrated to increase cortical tissue sparing following TBI, an effect lost in mitoNEET knock out (KO) mice. These results signify mitoNEET as an important mitochondrial target for the treatment of traumatic CNS injuries. To further elucidate organ-specific mitoNEET-mediated miotcondrial function, we utilized mitoNEET KO mice to assay mitochondrial bioenergetics in multiple organs (brain, heart, liver, muscle, brown and white adipose tissue) and characterize mitoNEET-mediated changes in metabolic profiling and behavioral testing. A significant reduction in cognitive function, measured with a novel object recognition test, was observed in KO mice compared to wild-type (WT) mice. It was observed that mitochondrial respiration in mitoNEET KO mice was altered in a tissue specific manner. Brown adipose tissue showed a significant drop in mitochondrial function whereas heart and brain also showed lowered respiration in mitoNEET KO animals. No changes were observed in glucose metabolism but fat deposition was significantly lowered in KO mice. The current results show unique mitochondrial-mediated phenotyping of mitoNEET KO mice and support the role of mitoNEET as a central modulator of mitochondrial bioenergtics and a novel target for intervention following CNS injury.
Keywords: Mitochondrial dysfunction, Bioenergetics, Traumatic Brain Injury, neuroprotection
Recent data suggest that the small noncoding microRNAs (miR)-155 and −124 control the microglial activation state following CNS injury. The present study examined the expression profiles of these miRs following traumatic brain injury (TBI), and investigated the effects of acute or delayed administration of a selective miR-155 antagomir on neuroinflammation and neurological recovery following injury. Experiment 1: Male C57Bl/6 mice underwent moderate controlled cortical impact (CCI) and cortical tissue was isolated at 6h, 24h, 72h and 7d post-injury for miR expression analysis. Experiment 2: Mice received a single icv injection of miR-155 or control antagomir (0.5nmol) immediately after CCI. Cognitive function (Y maze) was tested at post-injury day (PID) 6, and hippocampal tissue was collected for mRNA analysis of inflammatory genes and miR expression, at PID7. Experiment 3: Mice received a delayed 7 day icv infusion of miR-155 or control antagomir (0.5 nmol/day), beginning at 24 h following CCI. Hippocampal tissue was processed as per experiment 2. Experiment 4: Mice received a delayed 7 day icv infusion of miR-155 or control antagomir as per experiment 3. Motor function was assessed using the beam walk test at PID 1, 3, 7, 14, 21 & 28.CCI increased miR-155 and decreased miR-124 expression at PID7. Acute administration of a miR-155 antagomir, attenuated CCI-induced increases in gene expression of several pro-inflammatory mediators including TNF-α, gp-91 and p22phox, at PID7 and reversed injury-induced impairments in the Y maze. Delayed infusion of the miR-155 antagomir attenuated expression of pro-inflammatory mediators including TNF-α, gp-91 and p22phox and improved motor function at PID 14 and 21. These data demonstrate that TBI causes an altered miR-155/miR-124 expression profile up to 7 days post-injury, and that pharmacological inhibition of miR-155 limits expression of inflammatory markers and improves functional recovery. Thus, inhibiting miR-155 may offer a novel therapeutic approach for targeting neuroinflammatory responses after TBI.
Keywords: TBI, neuroinflammation, micro-RNA
THE IMMUNOPROTEASOME MODULATES MULTIPLE FEATURES OF THE MICROGLIAL INFLAMMATORY RESPONSE
Uniformed Services University of the Health Sciences, Anatomy, Physiology and Genetics, Bethesda, USA
Microglia respond to disruption of brain homeostasis following traumatic brain injury (TBI) by transitioning to a reactive state, characterized by morphological and functional changes to promote immune defense and a robust inflammatory response. Immunoproteasomes, an inducible form of the constitutive proteasome, are elevated in reactive microglia following TBI, stroke and infection, however the exact role of the immunoproteasome in neuropathology remains poorly characterized. Here we report that the proteasome undergoes rapid reorganization in microglia following interferon-gamma exposure, converting the constitutive proteasome to the inducible immunoproteasome. Stranded mRNA sequencing analyses indicate that interferon-gamma-dependent transcriptome changes, which are key features of the immune defense and inflammation activities of microglia, are reversed when the immunoproteasome is inhibited. We show in cultured microglia that the immunoproteasome regulates nitric oxide production, phagocytosis and priming consistent with the transcriptome data. These results imply that targeting immunoproteasome function following brain injury may attenuate select microglial activity to improve the pathophysiology of neurodegenerative conditions or the progress of inflammation-mediated secondary injury following neurotrauma.
Keywords: Immunoproteasome, RNA Sequencing, Proteasome, Priming
University of Arizona College of Medicine - Phoenix, Phoenix, USA
Traumatic brain injury (TBI) is not a transient event from which all people recover; the resulting damage can evolve into neurological disease, with significant cognitive symptoms. Injury stretches and shears axons that disrupt neurons within dedicated neural circuits. Further, microglia are activated in response to neuronal injury, representing histological hallmarks of neuroinflammation and chronic neuropathology following TBI. Microglial activation follows morphological change from an inactive ramified state to an active, deramified or amoeboid state the represents the inflammatory process. Memory circuits throughout the hippocampus, in particular, are vulnerable to pathology and underlie impaired cognitive function 28 days after experimental TBI. Therefore, we evaluated the progression of neuropathology and microglia activation within the hippocampal dentate gyrus (DG), CA1, and CA3 sub-regions following midline fluid percussion injury. At time intervals of 1, 7, and 28 days post-injury, significant increases in neuropathology (Silver stain) and decreases in microglia ramification (Iba-1 immunohistochemistry) were quantified compared to control. The dentate gyrus and CA3 showed significant increases in silver pathology at 1 and 7 days post-injury, which decreased by 28 days. The CA1 region showed a significant overall increase in pathology, but not significant at any one time point. The CA3 and CA1 showed significant increases in microglia number at day 7, which decreased by day 28. The DG showed a significant decrease in microglia number on day 1 followed by a significant increase on days 7 and 28. Microglia process length and end points also significantly decreased following injury in all 3 regions suggesting a conversion from an inactive to an active state following TBI. Thus, TBI harbors cellular pathology along the hippocampal memory circuit, which may contribute to memory deficits at 28 days post-injury. Additionally, injury-induced microglia recruitment and activation suggests sustained inflammation associated with the TBI disease process.
Keywords: Dentate Gyrus, CA1, CA3, microglia, neuroinflammation
Following a traumatic brain injury (TBI), a series of neurochemical and inflammatory events occur to repair and promote healing of neural tissue. Prolonged inflammation however, results in neurodegeneration of healthy tissue and can result in cognitive and behavioral deficits. Juvenile brains are more susceptible to the damaging effects of inflammation after a TBI. Microglia play a crucial role in the inflammatory response demonstrating both pro and anti-inflammatory properties. Chronic activation of pro-inflammatory microglia has been associated with poor outcomes. Nutraceutical treatments have been used to help mediate against the negative events following a TBI. In adult models of TBI, nicotinamide (NAM, vitamin B3) has been shown to decrease behavioral deficits and reduce inflammation. These nutraceutical treatments however, have not been studied in the developing brain following injury. The current study explored the effects of NAM on the microglial response over the course of time following a TBI in juvenile animals. Following a bilateral controlled cortical impact at PND-28, animals were given either 500 mg/kg of NAM or 1 mL/kg of 0.9% saline. NAM was administered at 15 minutes, 24 hours, and 48 hours post-injury. Subjects were euthanized at 3 hours, 72 hours, 1 week, and 1 month after injury. Microglia levels were sampled in the cortex lateral and medial to the injury, the dentate gyrus, CA1, and CA3 of the hippocampus, and the dorsal thalamus. Microglia were detected using anti-Iba1 antibodies. Analysis found NAM treated animals had significantly fewer microglia in all regions of the hippocampus and cortex, and dorsal thalamus when compared to saline treated animals. This NAM-induced reduction was observed following the injury at 3 days. These findings suggest that NAM treatments alone have neuroprotective properties and reduce microglial activation. This data corresponds with previous behavioral data showing NAM treated animals having improved performances in Morris water maze testing compared to saline treated animals. These results, in conjunction with previous behavioral data demonstrate potential clinical use of NAM in juvenile populations following TBI.
Keywords: Juvenile, Nutraceutical, Time Course, Recovery
INHIBITION OF NOX4 REDUCES OXIDATIVE STRESS AND NEURONAL DAMAGE INDUCED BY IRON ACTIVATED MICROGLIA
Iron contributes to the pathophysiology of secondary central nervous system injury, as it can accentuate reactive oxygen species (ROS) production among microglia, leading to increased neuronal damage. However, the mechanism by which iron accentuates ROS production among microglia is unknown. NADPH oxidases (NOX) are a family of enzymes known for ROS production within microglia; NOX4 is one isoform that is constitutively active in microglia and directly produces hydrogen peroxide. We hypothesize that NOX4 inhibition can ameliorate iron induced ROS accentuation among microglia, leading to improved neuronal survivability. We now show, using the microglial BV2 cell line activated with pro-inflammatory stimulants including lipopolysaccharide (LPS) or interferon (IFN) γ, that iron induces a significant increase in ROS release. We also demonstrate that the NOX4 specific inhibitor GKT137831 significantly reduced this ROS production. Co-culture of BV2 with either primary cortical neurons or the neuronal-like PC12 cell line showed that iron in combination with LPS significantly reduced neuronal viability and induced caspase-3 activation. Inhibition of NOX4 in microglia blocked this neuronal toxicity. These findings support the use of GKT137831 as an effective ROS reduction strategy following CNS injury, which may increase neuronal survivability and translate into improved functional outcomes in vivo.
Keywords: NADPH oxidase, cytokine, neurotoxicity, oxidative stress, iron
Bay Pines VA Healthcare System, R&D, Bay Pines, USA
Mild traumatic brain injuries have been experienced by at least 15% of recently deployed military personnel and the worldwide prevalence is approximately 0.5% per year. Mild injuries can have long-term consequences that interfere with the quality of life and our goal is to identify therapeutic targets that will reduce neurodegeneration and improve outcomes. We have previously identified pathways responsive to oxidative attack, e.g., transcription factor Nrf2, as regulators of the consequences of mild TBI. An improved understanding of the changes in dendritic complexity in the brain and reactive astrocytosis could provide a context for the effects of tobacco smoke exposure on traumatic brain injury recuperation and whether increasing antioxidant signaling affected plasticity following injury. To examine the potential influences of second hand smoke during recovery from TBI, we exposed mice to cigarette smoke for three 30 min periods each day for 12 days. The dendritic arbor of neurons was determined by Golgi staining, Sholl, and branch point analysis. Astrocytes were examined by immunohistochemical staining for GFAP. We found that neither the mild injuries nor the smoke exposure produced axonal damage detectable with amino cupric silver staining. However, there was significantly reduced complexity in the dendritic arbors in the after mild TBI plus smoke exposure. In the hippocampus, there was an appearance of reactive astrocytosis after the injury and tobacco smoke insult. Although traumatic brain injuries will not be fully preventable, it is important to understand preventative strategies and lifestyle choices, such as cigarette smoking, that could affect postinjury prognosis.
Keywords: Dendrites, Hippocampus, Smoke, GFAP, Golgi Staining, Oxidative Stress
Traumatic contusions are generally viewed as circumscribed brain lesions, without considering their neocortical, limbic or subcortical connections and related circuits. In the era of parallel/distributive theories of information processing in the brain and the Human Connectome Project it is important to understand these lesions as a network problem according to which a node is eliminated, leading to anterograde or retrograde degeneration of interconnected areas. Post-mortem human brains with ventral frontal (orbitofrontal) contusions or diffuse axonal injury (DAI) were examined using immunohistochemistry for phosphorylated epitopes of heavy and medium-molecular weight neurofilament proteins (SMI 310 antibody) as a marker of retrograde degeneration of neuronal cell bodies in connected neocortical, limbic, and subcortical sites. Some cases with frontal also had temporopolar contusions. Maps of afferent and efferent connections of orbitofrontal cortex were drawn based on existing tract-tracing studies in non-human primates, with input by older reports of retrograde degeneration after leucotomy in humans and data from the human connectome project. We focused on major sources of afferent inputs and identified hippocampus, thalamus and interconnected frontal cortices as target regions for investigation. Here we present initial data from 4 patients with TBI (3 with OFC contusions, 1 with DAI). Large numbers of phosphorylated NF-H/NF-M-immunoreactive neuronal cell bodies were found in the ventral anterior (VA) and ventral lateral (VL) nucleus in all cases. In certain cases, p-NF immunoreactivity was also found in cell bodies of hippocampal neurons. In contrast, frontal neocortex did not show any p-NF (+) cell bodies. In the DAI case that had hemorrhagic lesions in the genu of corpus callosum, frontal sections showed robust p-NF (+) cells throughout cortex. Our findings indicate that ventral frontal contusions lead to retrograde degeneration in thalamus, less so in hippocampus, and none in interconnected frontal cortices. Labeling in thalamus is robust and corresponds nicely to known afferents to primate orbitofrontal cortex from tract-tracing and retrograde degeneration studies. In contrast, DAI leads to extensive retrograde changes in prefrontal cortical regions. These studies demonstrate the power of modern molecular tools to update or “ rewrite” the neuropathology of traumatic contusions and DAI based on impaired neocortical and limbic circuits, with implications for understanding the diverse and severe neuropsychiatric symptoms of such patients.
Keywords: Human TBI, Transsynaptic Degeneration, Thalamus, Orbitofrontal Cortex
DIFFUSION BASIS SPECTRUM IMAGING DETECTS AXONAL LOSS AND DEMYELINATION IN CHRONIC TRAUMATIC BRAIN INJURY
The First Affiliated Hospital, Nanchang University, Radiology, Nanchang, China
Traumatic brain injury (TBI) frequently results in neurodegenerative injuries lasting for months or years post-injury. To investigate white matter changes at the chronic stage of TBI, we examined whole-brain white matter tracts using diffusion basis spectrum imaging (DBSI) and tract-based spatial statistics (TBSS). In this preliminary report, we present DBSI findings from corpus callosum (CC), correlating with Mini-Mental State Examination (MMSE) and Beck Depression Inventory (BDI). Eighteen chronic TBI patients (post-injury >3 months; 9 mild, 2 moderate and 7 severe) and twenty healthy control subjects were included in this study. Diffusion-weighted images were collected with a multi-b value scheme (99 directions, maximum b-value = 2000 s/mm2) at 3T (Trio; Siemens, Erlangen, Germany) with voxel size of 2.5 × 2.5 × 2.5 mm3. DBSI were computed using the in-house software developed using Matlab. DBSI pathological metrics were projected onto the skeleton generated by TBSS for statistical analyses. Student t-test was applied for group comparison. Pearson correlation was tested between DBSI metrics and clinical assessments. Compared with control subjects, significantly decreased (p < 0.05) fiber fraction and increased (p < 0.05) fiber radial diffusivity in TBI were found in CC, indicating the predominant role of axonal loss and demyelination at the chronic stage of TBI. In addition, strong correlation was observed in the splenium between fiber fraction and clinical assessments (MMSE: r = 0.60, p < 0.01; BDI: r = −0.72, p < 0.01). Our results were consistent with previous reports that CC is sensitive to shear strain, especially the splenium. In conclusion, DBSI-derived pathological metrics could be novel imaging biomarkers to monitor the course of white matter abnormalities in TBI.
Keywords: Traumatic Brain Injury, Diffusion MRI, Corpus Callosum, TBSS
Keywords: Chronic, Laser Capture Microdissection, Dentate Gyrus, miRNA, Fluid Percussion Injury
PENETRATING BALLISTIC-LIKE BRAIN INJURY ACTIVATES NEUROGENIC NICHES IN ADULT RATS
Active proliferative response is a common occurrence following traumatic brain injury (TBI) and contributes to both gliogenesis and neurogenesis. While post-injury neurogenesis stimulates neurorepair, gliogenesis may cause neuroinflammation that may be detrimental to reparative processes. To design strategies that promote neurorepair, we need to understand the changes in post-traumatic neurogenesis. Here we examined the effects of penetrating ballistic-like brain injury (PBBI) on immature neuronal population in two important neurogenic niches of the adult brain viz., forebrain sub-ventricular zone (SVZ) and hippocampal sub-granular zone (SGZ). Rats were subjected to PBBI (n = 6/time point) or sham craniotomy (n = 6/time-point). To capture proliferating cells, rats were injected with BrdU (50mg/kg) for 7 days and were euthanized at 10 day post-injury. Doublecortin (DCX) immunolabeling was used to identify immature neurons and BrdU/DCX double-labelling was performed to identify newborn immature neurons. Quantification of immunolabelled cells was done using a fluorescence microscope equipped with multi-channel filter set. To understand forebrain neurogenesis, total immature neurons and BrdU positive newborn immature neurons were counted separately in ipsilateral SVZ, striatum, subcortical white matter (SCWM) and cortex. In the hippocampus, DCX positive and Brdu/DCX co-labelled cells were counted from SGZ. DCX positive neurons and BrdU/DCX co-labelled newborn immature neurons were found in both SVZ and SGZ, regardless of the injury status. However, PBBI appeared to enhance the production of newborn neurons in both SVZ and SGZ areas. Furthermore, PBBI significantly enhanced the migration of newborn neurons from SVZ towards striatum, SCWM and cortex (p < 0.05 compared to sham). In the hippocampal SGZ, PBBI did not alter the total number immature neurons however, increased the number of BrdU/DCX co-labelled newborn neurons (p < 0.05 compared to sham). Overall, our results indicate an activation of neurogenesis in two major neurogenic niches of the adult rat brain following PBBI. Our findings also show that these newborn neurons can migrate to areas of injury, purportedly to modulate neurorepair. Further studies will characterize the anatomical integration of newborn neurons and their role in mediating functional recovery.
Keywords: PBBI, Neurogenesis, Doublecortin, Proliferation
INSULIN-LIKE GROWTH FACTOR-1 OVEREXPRESSION ENHANCES NEUROGENESIS AND ACTIVATES THE MTOR PATHWAY AFTER MODERATE TBI
Nearly 5 million people in the United States are living with TBI related disabilities, in part because of the brain's limited capacity to replace lost and damaged neurons. Immature neurons in the hippocampus are highly vulnerable to trauma, but can be replaced through proliferation and differentiation of neural stem cells in the subgranular zone. The extent of injury-induced neurogenesis, however, may be injury severity dependent. Insulin-like Growth Factor 1 (IGF1) modulates basal and injury-induced hippocampal neurogenesis. Using a transgenic mouse model with IGF1 overexpression restricted to astrocytes (IGF Tg) to raise brain levels of IGF1 by means of injury-induced astrogliosis, we previously showed that IGF1 enhances recovery of the immature neuron population and morphology after severe TBI. Mammalian target of rapamycin (mTOR), a signaling molecule downstream of IGF1, has been identified as a potential target for TBI interventions because of its regulatory role in plasticity and cell survival. We hypothesized that increased IGF1 would stimulate mTOR activity following moderate injury, resulting in improved neurogenesis. To this end three cohorts of IGF Tg and wild-type (WT) mice received moderate controlled cortical impact (CCI, n = 8–11/genotype) and survived 1, 3 or 10d or received sham injury (n = 3/genotype; 72 h survival) At 1 and 3d following moderate 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 (3 sections/animal) suggests that IGF1 stimulates activity of the mTOR pathway following moderate TBI in a region-specific manner. At 10d after moderate injury, IGF1 overexpression enhances recovery of immature neurons.
Funding Sources: Kentucky Spinal Cord and Head Injury Research Trust (KSCHIRT) 14-12A and NIH R01 NS072302-02S1, R01 NS0072302, T32 NS077889, and P30 NS051220.
Keywords: IGF1, Neuronal Survival, Moderate Injury, CCI
MODULATION OF ADULT NEUROGENESIS BY NANO-PULSED LASER THERAPY IN A RAT MODEL OF TRAUMATIC BRAIN INJURY
UTMB, Dept. of Anesthesiology, Galveston, USA
Keywords: miRNA, Gene Expression, Proliferation, Differentiation, Therapy, Hippocampus
Traumatic brain injury (TBI) affects nearly 2.8 million people in the United States per year and often leads to lasting cognitive impairment. A strategy to promote recovery after TBI centers on enhancing or controlling neurogenesis in the dentate gyrus region of the hippocampus. We hypothesize that to directly evaluate the role of neurogenesis in recovery after TBI, experimental methods to selectively ablate neurogenesis are necessary. Thus, the goal of this study was to optimize ablation of dentate gyrus neurogenesis in mice using a transgenic strategy to conditionally express diphtheria toxin receptors (DTR) and then crossing these mice with nestin-CreERT2 mice in which a tamoxifen (TAM)-inducible Cre recombinase is expressed under control of a nestin promoter (Nestin-Cre iDTR). It has been reported previously that TAM administration induces permanent expression of the DTR in neural progenitor cells which targets these cells for subsequent ablation in Nestin-Cre iDTR mice. To optimize this “tag-and-ablate” protocol, we compared two injection methodologies for TAM, comparing daily intraperitoneal (i.p.) injection versus delivery in matrix-driven delivery pellets. The main outcome measure was percentage ablation of nestin positive cells assessed via immunohistochemistry and stereology in brains extracted acutely after completion of diphtheria toxin administration. Mice were genotyped to determine if they were Cre+ or Cre−, then they were randomly assigned to either the TAM+ or TAM– groups. Mice were checked daily for body weight during the duration of the experiment. There were no significant differences in nestin positive cells between Cre+ and Cre– animals at any assessed time points for animals receiving the i.p. injection of TAM. There were also no significant difference in number of nestin positive cells in Cre+ and Cre– animals after administration of TAM via pellet. This work suggests that Nestin-CRE iDTR mice may not be a robust tool for studying the role of neurogenesis in neurotrauma under these conditions. Work supported by NS075162.
Keywords: transgenic mice
University of Alabama at Birmingham, Rehabilitation Science, Birmingham, USA
Spinal cord injury (SCI) often leads to central neuropathic pain. Unfortunately, the pathobiological mechanisms of SCI-induced neuropathic pain are complex and treatment options are few. Thus, translational research utilizing animal models of SCI-induced neuropathic pain is needed. One limitation to the evaluation of pain in animals is the reliance on withdrawal responses. We therefore hypothesize that pain assessments should include both withdrawal and supraspinal responses. The objective of this study was to develop a numerical scale to classify evoked pain in pigs which incorporates both reflexive and supraspinal responses. The scale was adapted from previous work on piglets and other domesticated animals. We applied graded stimuli to able-bodied pigs and simultaneously video-recorded their behavioral responses. Stimuli were selected to model quantitative sensory testing as used in clinical practice and research. Four raters scored the responses according to the scale. Operational definitions of individual behaviors were iteratively revised to refine the scale. Next, graded stimuli were applied to four groups of pigs: able-bodied, laminectomy, mid-thoracic SCI, and formalin-injected to induce a local inflammatory response. Recorded responses were randomized and evaluated by raters who were not informed of the force level of the stimuli. The inter-rater reliability and the scores of responses to the stimuli were evaluated. We found that in general, responses to lower force stimuli were consistently rated on the lower end of the scale and that the higher force stimuli were rated at the higher end. We also found that the inter-rater reliability of the scale was 0.924 for five raters with varying scientific backgrounds and experience scoring animal behavior. These data suggest that the PEPS can effectively classify behavioral responses to graded sensory stimuli with a high degree of inter-rater reliability. Support: Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Rehabilitation Research and Development Service (121RX002200).
Keywords: supraspinal pain, porcine animal model, behavioral assessments
After spinal cord injury (SCI), approximately 80% of individuals develop chronic, severe, unrelenting pain (SCI-pain) that is often resistant to conventional pain medication. Thus, there is critical need to identify new therapeutic targets to reduce or eliminate SCI-pain. NADPH oxidase (NOX2) is an enzyme that induces reactive oxygen species (ROS) and serves as a switch between the pro-inflammatory and neurorestorative microglial/macrophage phenotypes; such changes play an important role in chronic neuropathic pain associated with a variety of conditions including SCI-Pain. Increased NOX2 expression and activity after SCI has been demonstrated and pharmacological inhibition of NOX2 improves motor functional recovery. However, the underlying mechanisms of NOX2 in post-traumatic pain remain unexplored. In the present study we report that genetic deletion of NOX2 in NOX2−/− mice or pharmacological inhibition of NOX2 using NOX2ds-tat administrated intraperitoneally significantly reduced mechanical/thermal hyperesthesia and motor dysfunction after moderate contusion SCI in mice. Western blot (WB) and immunohistochemistry (IHC) showed that SCI elevates NOX2 expression predominantly in microglia/macrophages. Deletion of NOX2 significantly reduced CD11b+/CD45+/F4/80+ macrophage infiltration detected by flow cytometry and 8-OHG+ ROS production by IHC in both lesion area and lumbar enlargement at 24h post-injury. NOX2 deficiency also altered microglial/macrophage M1-/M2-like balance towards the M2-like response. The latter was validated by WB showing robust increased protein expression of Arginase1 and YM1 in NOX2-/- mice. Furthermore, qPCR analysis showed that NOX2 deficiency significantly up-regulates anti-inflammatory cytokine IL10 mRNA levels, associated with reduced microRNA155 expression in injured spinal cord tissue 1d after SCI. These findings were confirmed in CD11b-selective microglia/macrophages isolated from adult spinal cord tissue at 3 days post-injury. Taken together, our data suggest an important role for IL-10/miR155 pathway in regulating NOX2-mediated SCI-pain. Thus, specific targeting of NOX2 enzyme may provide an effective strategy for treating chronic pain in SCI patients.
Keywords: Spinal Cord Injury, NOX2, IL-10, Microglia/Macrophage, MiR155
COMPARISON OF DEFICITS FOLLOWING SPINAL CORD INJURY IN A DORSAL ROOT AVULSION VERSUS CONTUSION/COMPRESSION MODEL IN PIGS
Neuropathic pain (NP) is a chronic and debilitating condition affecting many patients with spinal cord injury (SCI). Since chronic pain is the leading cause of disability in the United States, there is a strong impetus to develop efficacious treatments for NP and animal models are key elements in the therapeutic development process. However, paralysis can make evaluation of reaction to a sensory stimulus more complex. Thus, the goal of the current study is to compare motor and sensory deficits in two models of SCI. One model, the dorsal root avulsion model (DRA), is typically associated with little locomotor deficit. In contrast, the mid-thoracic contusion/compression model induces incomplete hind limb paralysis. Adult, female mixed breed pigs (N = 5/group, 36 ± 2 kg) were randomized to receive either a mid-thoracic contusion/compression SCI at T10 (T10-SCI) or a unilateral DRA at the last thoracic and first lumbar nerve roots. For 8-13 weeks post-injury, animals were evaluated with weekly locomotor and quantitative sensory testing for NP in 8 dermatomes located at or below or the level of injury. In addition, animals that developed NP were administered 3 analgesics, randomized with washout to include: intravenous lidocaine (5 mg/kg/day), oral baclofen (60 mg/day) and oral pregabalin (300 mg/day) for 7 days to determine their analgesic efficacy. All T10-SCI animals showed significant motor impairment compared to DRA animals. Interestingly, NP developed below the level of injury in only T10-SCI animals. This occurred 5 weeks post-SCI and persisted to the end of the study. Lidocaine had the most robust analgesic effect. In summary, T10-SCI animals develop below-lesion NP. This was not observed in the DRA model. Intravenous lidocaine was the most effective analgesic in the treatment of SCI-NP. Further investigation is required to more thoroughly characterize the profile and mechanisms of SCI-NP development in the T10-SCI model. Supported by the UAB TJ Atchison Spinal Cord Injury Research Program.
Keywords: spinal cord injury, pain, model, behavior
A BRIEF PERIOD OF NOCICEPTIVE STIMULATION AFTER SPINAL CORD INJURY CAN EXPAND THE REGION OF SECONDARY INJURY
Spinal cord injuries are often accompanied by injuries to other parts of the body (polytrauma). These injuries can lead to the activation of pain fibers (C-fibers). Research has shown that the activation of C-fibers using electrical stimulation after a spinal cord injury hinders behavioral recovery and increases signs of hemorrhage and inflammation (Grau et al., 2014). From this work we know that stimulation at an intensity of 1.5 mA or greater, and for a duration of 6 minutes or more [for shocks given an average of 2 s apart (0.5 Hz)], induces a form of maladaptive plasticity. In the current study, we examine whether less intense, and briefer, periods of stimulation adversely affect recovery after a contusion injury. The present study examined whether less severe stimulation has an adverse effect and the generality of this finding across age and sex. Twenty-four hours after receiving a moderate spinal cord contusion at T12, Sprague Dawley rats received uncontrollable electrical stimulation at either a lower intensity (0, 0.17, 0.5 or 1.5 mA) or a shorter duration (0, 14.5s, 72, or 360s). Locomotor performance was assessed prior to and 3 hrs after stimulation. The tissue was then collected to examine the extent of hemorrhage. We found that relatively mild (0.5 mA) shocks acutely disrupted locomotor performance and increased the extent of hemorrhage (secondary injury). Likewise, just 72 s of shock impaired performance and increased secondary injury. The adverse effect of nociceptive stimulation on spinal function was greatest in older male rats. On-going experiments are examining whether these effects are regulated by behavioral control and the physiological factors that trigger increased hemorrhage. The work suggests that even brief nociceptive input after injury can augment the extent of injury and undermine long-term recovery.
Keywords: nociception, pain, spinal cord injury, hemorrhage, sex, age
EXAMINING RELATIONSHIP BETWEEN PRE-INJURY PAIN AND FUNCTIONAL STATUS FOLLOWING TRAUMATIC BRAIN INJURY
The University of Washington, Biobehavioral Nursing and Health Informatics, Seattle, USA
Keywords: functional status, quality of life, pain
CNRS, UMR 5287-Institut de Neurosciences Cognitives et Intégratives d'â Aquitaine, Bordeaux, France
Pediatrics mild-TBI is a leading cause of hospital visits and has been shown to lead to long-term disability. The underlying pathomechanisms are often assumed to depend on the type of injury and it is generally considered that increased severity would lead to increased pathology. We hypothesized that different severities could lead to different pathomechanisms, even in the same type of TBI. Therefore, we aimed to identify early severity-associated difference within the corpus callosum (CC). In a new jTBI mouse model,
Mild-TBI (G1) exhibited a distinct acute gliovascular phenotype of damage that was unique in that it was not simply a milder form of the same pathology seen in a more severe impact (G2). Astrocytic AQP4, which had the highest expression in the milder phenotype of TBI, could be a key target for differential treatments based on severity.
Keywords: DTI, Behavior, aquaporin, closed head injury
Massachusetts General Hospital, Neurosurgery, Boston, USA
Hemispheric hypodensity (HH) is a striking injury pattern in young children typically associated with subdural hematoma (SDH) and is characterized by widespread hypodensity on CT scan spanning multiple vascular beds. In the unilateral form, the entire hemisphere or large patches of parenchyma underlying the SDH are damaged with relative sparing of the contralateral hemisphere. The objective of this study is to determine the pattern of tissue damage after abusive head trauma (AHT) focal injuries (cortical impact, mass effect, subdural hematoma, seizures) and global insults (apnea, hypoventilation, hypercapnia). Building upon our previous work in a model of HH, we used an anesthetic regimen that avoided the GABAA receptor, induced seizures with kainic acid applied to the ipsilateral hemisphere, and lengthened survival to 24 hours. Areas of damage were identified by red neurons and/or perineuronal or perivascular vacuolization in serial sections spanning each hemisphere in male, 1 month old piglets receiving the AHT insult/injury (n = 6) or sham surgery (n = 2). In injured piglets, the percentage of tissue damage was greater in the ipsilateral vs. the contralateral hemisphere (41.6 ± 9.9% vs. 11.2 ± 4%; P = 0.017) and greater than either hemisphere of sham piglets (ipsilateral: 1.1 ± 0.19, contralateral 4.8 ± 4.2). A piglet with AHT insult/injury that only had a 5-minute seizure failed to display hemispheric tissue damage. In piglets receiving injuries, the proportion of the damaged hemisphere was positively correlated with SDH area (P = 0.03). Hypoxic-ischemic injury encompassed approximately half of the hemisphere adjacent to the SDH including most of the cortical ribbon with relative sparing of the gyral white matter and deep gray matter. The extent of tissue damage may depend upon seizure duration and subdural blood. We will continue to use this model to investigate the pathophysiological cascades leading to hemispheric demise after AHT injuries, and, ultimately, to determine prevention strategies or interventions to limit damage.
Keywords: Hemispheric hypodensity, abusive head trauma
USC Keck SOM, IGC, INI, Marina del Rey, USA
Post-injury outcome varies considerably in children who have sustained a moderate/severe traumatic brain injury (msTBI), with acute injury factors only accounting for some of this variance. In our study, we found that interhemispheric transfer time (IHTT), a measure of corpus callosum (CC) functional integrity, divided patients into two groups a few months post-injury. We studied 21 children (16M/5F) with msTBI, assessed 2-5 months and 13-19 months post-injury, and 20 well-matched healthy control children. CC function was assessed through IHTT, measured using event-related potentials, and white matter (WM) microstructural organization was modeled using diffusion-weighted magnetic resonance imaging (dMRI). Half of the TBI patients had significantly slower IHTT at the first time-point (TBI-slow-IHTT, N = 11), and half were in the normal range (TBI-normal-IHTT, N = 10). The TBI-normal-IHTT group did not differ significantly from healthy controls in WM organization and showed longitudinal improvements in WM organization. In contrast, the WM organization of the TBI-slow-IHTT group was significantly poorer than healthy controls, and longitudinal analyses showed a progressive decline in WM integrity throughout the brain from 2–5 to 13–19 months post-msTBI. These groups did not differ significantly in demographic or clinical variables. IHTT is a potential biomarker for identifying patients with continuing and progress WM degeneration, although the causative factor is unknown at present. Identifying patients at risk for poorer outcomes will help clinicians know which patients might benefit from targeted treatment.
Keywords: Longitudinal, Multimodal, moderate/severe TBI, dMRI, IHTT
Keywords: arachidonic acid, HET0016, 20-Hete
THE IMPACT OF TREATMENT WITH THE PROBIOTIC, LACTOBACILLUS REUTERI, UPON NEUROPROTECTION AND NEUROINFLAMMATION FOLLOWING JTBI
Southern Illinois University, Psychology, Carbondale, USA
Heightened neuroinflammation and cell loss following juvenile traumatic brain injury (jTBI) frequently results in impaired cognitive function that can last into adulthood. Modification of the microbiome using dietary probiotics has been shown to impact behavior and inflammation. The probiotic bacterial strain Lactobacillus reuteri impacts the brain through modification of the gut-brain axis and has been associated with the attenuation of anxiety deficits. In the current study, L. reuteri was introduced starting immediately following jTBI at post-natal day 28 and continued daily for one month following injury. Subjects were tested in a battery of neurocognitive tasks over the next few weeks following injury. Although L. reuteri treated subjects didn't show improvement in the Morris water maze; they spent significantly more time with a novel object 24 hours following object recognition training suggesting improved object but not spatial long-term memory. Treated subjects spend an increased amount of time freezing compared to uninjured shams, suggesting a potential impact of L. reuteri upon contextual fear memory. Treatment with L. reuteri resulted in a significant reduction in lesion size and reduced number of IBA-1+ microglia, suggestive of increased neuroprotection and attenuated neuroinflammation following jTBI. Fecal samples were collected weekly following injury and throughout treatment to examine changes in the gut microbiome. Using 16s sequence analysis, the trajectories of intestinal bacterial communities from injured and non-injured rats separate by injury status with the community trajectory being more similar between individuals receiving injury than shams. Several Operational Taxonomic Units, revealed differences in abundance in injured versus shams with most bacteria being Firmicutes specifically Lachnospiraceae and Ruminococcaceae. Taken together, these data show that TBI impacts the composition of the gut microbial community, and that introducing a probiotic Lactobacillus strain post-injury can impact recovery following jTBI.
Keywords: Juvenile, Microbiome, Probiotic, Inflammation, Cell Loss
The Pediatric Intensity Level of Therapy (PILOT) scale was published by Shore et al. in 2006 and has been adopted as a means of measuring ICU therapeutic intensity in pediatric TBI. However, it has never been validated in another institution. We performed an IRB-approved retrospective chart review examining patients admitted to the pediatric ICU from July 2015 to March 2016. Critical care patients were included in the study if they were admitted due to trauma and were not deemed futile care by neurosurgery on arrival. Patients were broken down into 3 groups depending on their injuries: (1) severe TBI, (2) mild/moderate TBI, (3) acute trauma but no TBI. Those patients who received hypertonic saline were also scored with a Revised PILOT score that gave additional points for daily peak sodium: 150-159 = 2 additional points; 160–169 = 4 additional points; ≥170 = 6 extra points. 44 patients were divided into 3 groups: severe TBI (16), mild/moderate TBI (18), and extracranial trauma (10). The mean PILOT score in the severe TBI group was 35.5 ± 20.4, the mild/moderate group was 9.61 ± 9.04, and the extra-cranial trauma group was 8.4 ± 7.05. ANOVA showed that the severe TBI group was statistically different from each of the other groups (p < 0.001). Of 16 patients in the severe TBI group, 15 received hypertonic saline. No patient in the extra-cranial trauma group received hypertonic saline. Regression analysis shows that treatment with hypertonic saline increases the 7-day PILOT score by 33 points (95%CI 24-41, p < 0.0001) with an R2 of 0.67. The Revised PILOT score was applied to those 15 patients who received hypertonic saline, and increased the 7-day score by 4.76 points (p < 0.001). This small cohort validates the original findings by Dr. Shore that the PILOT score successfully discriminates severe TBI patients from other trauma patients in the ICU. Additionally, our data show a ceiling effect in the original scoring, which is ameliorated by our revised score which takes into account the daily sodium values.
Keywords: Therapeutic Intensity, Clinical Scores
Spinal cord injury (SCI) can have profound effects on the autonomic and cardiovascular systems, notably with injuries above high-thoracic levels frequently resulting in the development of autonomic dysreflexia (AD) characterized by volatile hypertension in response to exaggerated sympathetic reflexes triggered by afferent stimulation below the injury level. We have reported that in rats with complete T4-transection SCI, the antiepileptic drug gabapentin (GBP) significantly reduced the magnitude of induced AD shortly after administration (50 mg/kg), but that once daily treatment did not significantly reduce the frequency of spontaneous AD events. Since daily GBP treatment is reported to prevent excitatory synapse formation by binding to the a2d1 subunit of voltage-gated calcium channels, we investigated the effects of more prolonged and higher GBP dose administration on the incidence and severity of AD, notably in relation to plasticity within the circuitry underlying AD development. Female Wistar rats implanted with telemetric probes for continuous hemodynamic monitoring underwent T4-transection SCI and immediately received 100 mg/kg (i.p.) of GBP every six hours (400 mg/kg/day) for 4-weeks. Results show that this regimen reduces the frequency of daily spontaneous AD events, as well as the magnitude of weekly-induced AD. Such GBP dosages did not affect daily activity measurements and, unlike reports in the mouse AD model, spleen weights were unaltered weeks after T4 SCI, with or without treatment. Assessments of CGRP+ nociceptive afferent fiber sprouting and synapse formation (VGlut2, PSD-95) in the thoracolumbar spinal cord demonstrated established injury effects, but GBP amelioration of AD did not correlate with reduced measures of plasticity. We are increasing the power of these hemodynamic findings while refining our histological assessments to establish whether prolonged high-dose GBP administration can abrogate AD development, possibly by preventing maladaptive intraspinal plasticity. Funding: SCoBIRC Chair Endowment (AGR), 5T32 NS077889 (KCE).
Keywords: Autonomic Dysreflexia, Cardiovascular, Sprouting, Sympathetic
DEVELOP A NOVEL STRATEGY TO ENHANCE AXON REGENERATION AND NEUROPLASTICITY AFTER CNS INJURY
Temple University School of Medicine, Dept. of Neurology, Philadelphia, USA
Axons in adult mammalian CNS fail to regenerate and there are no effective regenerative strategies to treat patients with CNS injuries. Several genes, including PTEN/mTor, Kruppel-like factors and insulin/insulin-like growth factor 1 receptor, partly regulate axon regeneration failure in adult CNS of mammals. Here we report the crucial role of liver kinase B1 (LKB1) in mediating CNS regeneration after CNS injury. LKB1 is a key serine/threonine kinase required for maintaining cell metabolism, energy homeostasis and cell polarity by activating a number of kinases. As the downstream effector of cAMP/PKA and PI3 kinase pathways, LKB1 is a major determinant for migration and differentiation of various cells, including neurons. We thus evaluate the role of LKB1 in regulating regenerative capacity of neurons in adult mammals using transgenic and adeno-associated virus (AAV) vector approaches. Upregulation of LKB1 significantly enhanced neurite extension of adult neurons cultured on inhibitory substrates CNS myelin and aggrecan. Transgenic overexpression of LKB1 stimulates robust regeneration of corticospinal tract (CST) axons in adult mice with mid-thoracic spinal cord injury. Local injections of AAV2-LKB1 into sensorimotor cortex or systemic application of mutant AAV9-LKB1 promote long distance regeneration of injured CST axons into the caudal spinal cord in adult mice. Systemic injection of mutant AAV9-LKB1 also enhances regrowth of descending serotoninergic and tyrosine hydroxylase fibers. Importantly, LKB1 upregulation by either transgenic or viral approaches improves recovery of locomotor function after CNS injury. Therefore, LKB1 is critical for regulating growth capacity of mature neurons and may become an important target for designing highly effective therapies for CNS injury.
Keywords: Liver kinase B1, spinal cord injury, axon regeneration, Functional recovery, growth capacity
Sackler School of Medicine, Tel-Aviv University, Anatomy and Anthropology, Tel Aviv, Israel
Traumatic brain injury is the most common cause of death and chronic disability among people under-35-years-old and without an effective pharmacological treatment currently existing. Hyperbaric oxygen therapy (HBOT) is defined as the inhalation of pure oxygen in a hyperbaric chamber that is pressurized greater than 1 atmosphere. HBOT offers physiological and mechanical effects by inducing a state of increased pressure and hyperoxia. HBOT has been proposed as an effective treatment for mTBI, yet the exact therapeutic window and mechanism that underlies this effect is not completely understood. HBOT was administrated for 4 consecutive days, post a mouse closed head weight drop mild TBI (mTBI) in 2 different time lines: immediate - initiated 3 hours post injury and a delayed treatment - initiated 7 days post injury. Behavioral cognitive tests and biochemical changes were assessed. The results were similar for both the immediate and the delayed treatments. mTBI mice exhibited impairment in learning abilities, whereas mTBI mice treated with HBO displayed significant improvement compared with the mTBI group, performing similar to the sham groups. mTBI mice had a decline in the myelin basic protein, an increase in neuronal loss (NeuN staining), and an increase in the number of reactive astrocytes (GFAP). The HBO treated mice in both groups did not exhibit these changes and remained similar to the sham group. The delay HBOT has a potential to serve as a neuroprotective treatment for mTBI with a long therapeutic window. Further research is needed for fully understanding the cellular changes.
Keywords: hyperbaric oxygen, mice, neuroprotection, initiated and a delayed treatments
Keywords: brain stimulation, postconcussive symptoms, executive function, transcranial direct current stimulation
IN VIVO REPROGRAMMING OF NG2 GLIA INTO FUNCTIONAL NEURONS IN THE INJURED SPINAL CORD
University of Texas Southwestern Medical Center, Molecular Biology, Dallas, USA
Spinal cord injury (SCI) results in irreversible loss of neurons and disruption of neural circuits, with a consequence of motor and sensory dysfunction. The goal of the present study is to regenerate functional neurons after SCI. A key SCI-induced pathological feature is the formation of glial scars surrounding the injury site. NG2 glia constitute a major component of the glial scars and their presence at the injury sites is inhibitory for axon regrowth. Reprogramming these cells into functional neurons may provide a regeneration-based therapeutic strategy for SCI repair. We established a lentiviral approach to specifically target endogenous NG2 glia by using a human NG2 promoter. SCI was introduced by contusion in the adult mice. Our preliminary data showed that neurogenesis can be induced through injection of SOX2-expressing lentivirus under this promoter in both healthy and injured spinal cord. Immunohistochemistry revealed that induced DCX+ new neurons pass through an ASCL1+ neural progenitor and proliferation phase. Through genetic lineage mappings, we confirmed parenchymal NG2 glia as a cellular source for the newly reprogrammed neurons. We also excluded nestin+ neural stem cells or FOXJ1+ ependymal cells along the central canal as a cellular origin. Importantly, these NG2 glia-derived new neuron can mature into synapse-forming cells in vivo. Neuronal maturation can be further promoted by neurotrophic factors. Our results demonstrate that reactive NG2 glia can be robustly reprogrammed into neurons in situ, opening up the possibility of using endogenous cells for spinal cord regeneration after injury.
Keywords: in vivo reprogramming, neurogenesis, spinal cord injury, NG2 glia
Chronic spinal cord injury (SCI) causes ejaculatory dysfunction in men. Ejaculation is a reflex mediated by a spinal ejaculation generator in the lumbosacral spinal cord. A principle component of this generator is a neuronal population located in spinal levels L3-4 and named for their projections to the thalamus (lumbar spinothalamic cells: LSt cells). In rat, LSt cells co-express the neuropeptides galanin, gastrin releasing peptide (GRP), enkephalin, and cholecystokinin. LSt cells integrate sensory inputs during sexual activity to coordinate autonomic and motor outputs required for ejaculation, via interspinal connections and the release of neuropeptides onto spinal target neurons. We recently showed that SCI caused long term changes in the rat spinal ejaculation generator. Specifically, contusion injury ablated ejaculatory reflexes triggered by sensory stimulation, 6 weeks after injury. Moreover, these effects were independent of supraspinal influences, indicating long term changes within the spinal ejaculation generator itself. Here, we tested the hypothesis that SCI caused long term reduction in the expression of GRP and galanin in LSt cells, thereby disrupting ejaculatory reflexes. Male Sprague Dawley rats received either a contusion injury at spinal levels T6-7 or sham surgery. Six weeks following contusion or sham injury, animals were perfused with paraformaldehyde and spinal cords were immunohistoprocessed for galanin and GRP. Quantitative analysis of numbers of cells single or double labeled for galanin and GRP showed that SCI did not cause a significant reduction in galanin-immunoreactive cells. In contrast, SCI significantly reduced labeling for GRP, and fewer galanin-labeled cells co-localized GRP. These results demonstrate that SCI did not reduce the numbers of LSt cells per se, nor the localization of galanin in LSt cells. However, SCI resulted in a selective loss of GRP in LSt cells. Since our prior studies have shown GRP to be a powerful facilitative neuropeptide for control of ejaculation, such selective loss could contribute to the disruption of ejaculation seen in male SCI rats and human patients.
Keywords: Ejaculation, Gastrin Releasing Peptide, Sexual Reflexes, Spinal Cord Injury, Lumbar Spinal Cord, Urogenital
University of Toronto, Institute of Medical Science, Toronto, Canada
The spleen plays an important role in erythrocyte turnover, adaptive immunity, antibody production and, more recently revealed, the mobilization of monocytes/macrophages (Mφ) following tissue injury. In response to trauma, the spleen initiates production of inflammatory cytokines, by resident Mφ, which in turn influences the recruitment of immune cells to the inflamed tissue and exacerbates damage. Our previous work has shown that intravenous mesenchymal stromal cell (MSC) infusion has potent immunomodulatory effects following traumatic spinal cord injury (SCI), associated with the transplanted cells homing to and persisting within the spleen. Therefore, this work aimed to characterize the relationship between the splenic inflammatory response and SCI pathophysiology, with an emphasis on splenic involvement in the therapeutic use of MSCs post-SCI. A rodent model of SCI (C7-T1, 35gram clip) was used to compare secondary tissue damage and 8-week functional recovery (including grip strength, inclined plane and BBB locomotor rating) between splenectomised rodents versus those with a sham procedure. Subsequently, 2.5 million MSCs derived from the term human umbilical cord matrix (HUCMCs) were infused via tail vein at 1hour post-SCI and immunomodulatory effects assessed in the presence or absence of a spleen. Splenectomy alone had no effect on acute spinal cord lesion volume, hemorrhage or inflammation. There was also no significant difference between the groups in functional recovery and 8-week lesion morphometry. Yet, while the infusion of HUCMCs reduced spinal cord hemorrhage and lesion volume in the presence of a spleen, these effects were lost with splenectomy. The HUCMC-mediated rise in systemic levels of interleukin-10 was also absent in splenectomised rodents, suggesting that the spleen is an important target and site of MSC effects post-SCI. Altogether, our results provide a link between MSC function and splenic inflammation, a finding that can help tailor the cells/transplantation approach to enhance therapeutic efficacy and clinical deployment.
Keywords: Mesenchymal Stromal Cells, Spleen, IL-10, Spinal Cord Injury
Post-traumatic hydrocephalus (PTH) is a secondary neurological insult resulting in the derangement of cerebrospinal fluid (CSF) dynamics ensuing moderate to severe traumatic brain injury (sTBI). Given the high risk of clinical deterioration and documented worse outcomes, the identification of biomarkers indicating the onset of PTH is imperative to allow early clinical detection, and improve neurological outcomes in afflicted patients. This study examined CSF cytokine profile with PTH to elucidate the pathogenesis and aide in the early diagnosis of PTH. We conducted a matched case-control study on 50 patients who sustained a sTBI at a level 1 Trauma facility from 2002-2015. All patients were treated with five days of continuous CSF drainage via an extraventricular drain. CSF research samples was collected on post-trauma days 1, 3 and 5. Patients who incurred CNS infection or died within 6 months were excluded. 25 patients who incurred PTH were matched by age, sex, and initial Glasgow Coma Scale with 25 patients who did not incur PTH. The CSF concentrations of 36 different inflammatory markers were analyzed via a Luminex Array Scanner. There were no PTH differences detected between the groups in CSF RBC, WBC. Across all time points, IL-15 (p = 0.007), IL-5 (p = 0.038) and CX3CL1 (p = 0.031) were significantly lower among PTH patients. CCL4 was significantly higher in the PTH group (p = 0.029). IL-2 levels increased at a significantly slower rate in patients with PTH (p = 0.037). No other statistically significant differences were found in any other of the cytokines assayed. Overall, our data suggests potential differences in the immune responses in patients who develop PTH, which may impede the clearing of debris following sTBI. Lower levels of IL-15 and IL-5 suggest the decreased recruitment and proliferation of natural killer cells, T-cells, B-cells, and eosinophils in PTH patients. High levels of CCL4 may indicate a more macrophage rich environment. A slower increase in IL-2 may be indicative of a global impairment of immune function, particularly T-cell function and B-cell differentiation.
Keywords: Post Traumatic Hydrocephalus, Cerebrospinal Fluid, Biomarkers, severe TBI
CHRONIC UPREGULATION OF CLEAVED-CASPASE-3 AND MYELIN PATHOLOGY IN THE THALAMUS AFTER TRAUMATIC BRAIN INJURY IN RATS
University of South Florida College of Medicine, Neurosurgery, Tampa, USA
Traumatic brain injury (TBI) is associated with long-term disabilities and devastating chronic neurological complications including problems with cognition, motor function, sensory processing as well as behavior and mental health including depression, anxiety, personality changes and social unsuitability. Clinical data suggests that disruption of corticothalamic and thalamocortical networks and acute and chronic anatomical and metabolic changes in the thalamus following TBI might be responsible for chronic neurological deficits and altered brain function. The detailed mechanism of the pathological processes is not completely understood. The goal of this study is to evaluate changes following TBI focusing on cleaved-caspase-3, a specific effector of caspase pathway activation, and myelin and microvascular pathologies using immuno- and histochemisty at different time points from 24 hours to 3 months after controlled cortical impact (CCI) in rats. The results of the study for the first time demonstrate a significant chronic upregulation of cleaved-caspase-3 in selected thalamic regions associated with cortical regions affected by CCI injury at the chronic stages of TBI. The significant increases of cleaved-caspase-3 immunoreactivity in the thalamus were observed starting one month and persisting at least for three month following experimental TBI. Further, the study demonstrated the association of the cleaved-caspase-3 with the demyelination of the neuronal processes and the tissue degeneration in the gray matter of thalamus. However the immunofluorescent experiments did not reveal direct association of cleaved caspase-3 with blood brain barrier damage. In conclusion, this study provides new insights into potential mechanisms involving corticothalamic and thalamocortical networks in the etiopathology of chronic neurological disorders associated with TBI. Moreover, this study for the first time suggests that this upregulation of activated caspase-3 and delayed degeneration of myelinated neurons and nerve fibers in the thalamus might be representative of reciprocal pathological processes affecting neuronal connectivity and overall brain function at the chronic stages of TBI.
Keywords: chronic TBI, cleaved-caspase-3, thalamus, myelin pathology
Spinal cord injury (SCI) is a very severe condition which has far-reaching effects on health and quality of life. While the primary tissue damage after SCI occurs from the trauma itself, secondary damage is caused by a cascade of subsequent events, including hemorrhage and inflammation. It is well established that this secondary reaction contributes significantly to the pathology and thereby to the severity of the functional deficits. Inflammation after SCI is exacerbated and prolonged. The main objective of my research is to investigate and modulate the inflammatory response after SCI in order to minimize tissue damage and to promote an environment that is permissive for healing and repair.
The pro-inflammatory chemokine CCL3 plays an important role in various pathological conditions of the nervous system by initiating and maintaining the pro-inflammatory response. Conditions in which CCL3 has a negative impact include neuropathic pain, autoimmune neuropathy and Multiple Sclerosis and its model Experimental Autoimmune Encephalomyelitis.
This project is aimed at investigating and dissecting the roles of CCL3 and its receptors after SCI in C57BL/6 mice, using a model of moderate contusion injury. Preliminary results suggest that CCL3 mRNA is upregulated early and peaks at 6 hours after SCI. It stays elevated for at least 14 days after injury. The main receptor for CCL3, CCR1, is also upregulated after SCI, as is another receptor (CCR5). CCR4, in contrast, is not changed in response to SCI.
Furthermore, CCL3 knockout mice showed improved functional recovery compared to wild type mice, using the BMS score for locomotor recovery. Ongoing and future experiments will confirm expression patterns and behavioral results and assess the impact of CCL3 neutralization after SCI. Our preliminary data suggest a potential role of CCL3 in SCI.
Funding: Wings for Life.
Keywords: Chemokine, CCL3, microglia, macrophage
DISRUPTION OF NNOS-PSD95 INTERACTION AS A NEUROPROTECTIVE STRATEGY FOR TRAUMATIC BRAIN INJURY
IUSM, Neurological Surgery, Indianapolis, USA
Excessive activation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation have been shown to play a crucial role in the pathogenesis of traumatic brain injury (TBI). However, directly inhibiting NMDARs or nNOS produces adverse side effects because these substances have key physiological functions in the central nervous system. Interaction of nNOS-PSD95 is a key step in NMDAR-mediated excitotoxicity. In this study, we examined whether ZL006, a small-molecule that disrupts the nNOS-PSD95 interaction, would be neuroprotective in in vitro cortical neuronal cultures and an in vivo mouse model of controlled cortical impact (CCI). In vitro, ZL006 treatment (0.1, 1.0, and 10 μM) significantly reduced glutamate-induced neuronal death in a dose-dependent manner as measured by propidium iodide staining and lactate dehydrogenase release assays. In vivo, administration of ZL006 (10 mg/kg) at 30 min post-injury in mice significantly inhibited nNOS-PSD95 interaction in the cortex as compared with the vehicle-treated control. Western blot analysis showed that ZL006 treatment significantly reduced CCI-induced expression of apoptotic markers of active caspase-3 and PARP-1. Additionally, administration of ZL006 at 30 min post-injury followed by daily treatment of ZL006 for 7 days significantly improved neuroscores and sensorimotor performance on a rotarod device after mouse CCI. An adhesive removal test showed significant reduction of somatosensory and motor deficits after CCI and ZL006 treatment. Morris water maze test showed that ZL006 significantly reversed CCI-induced memory deficits. Administration of ZL006 also attenuated cognitive impairment in the direct contact social test and the social activity and novelty test. Histological analysis showed a reduction of brain lesion volume in the ZL006-treated group as compared to the control group. These findings collectively suggest that the glutamate-NMDAR-PSD95-nNOS cascade may mediate secondary TBI and that blocking nNOS-PSD95 interaction may represent an attractive strategy for ameliorating TBI.
Keywords: TBI, nNOS-PSD95 interaction, Neuroprotection, Apoptosis
University of Maryland School of Medicine, Baltimore, USA
Traumatic brain injury (TBI) has complex effects on the gastrointestinal tract that are associated with TBI-related mortality and morbidities, including susceptibility to peripheral infections and septicemia. Although human and animal studies have established a link between TBI and intestinal dysfunction during acute stages after injury, long-term consequences of TBI in the gut are vastly unknown. We examined changes in mucosal properties and enteric glial cells in the gut after experimental TBI in mice, as well as effects of Citrobacter rodentium (Cr)—an enteric murine analog of pathogenic human E. coli—on both gut and brain after chronic TBI. Moderate-level TBI in adult male C57BL/6 mice was induced by controlled cortical impact (CCI; 6 m/sec, 2 mm depth). CCI led to delayed, chronic changes in colon morphology, including increased mucosal depth and smooth muscle thickening. At day 28 post-CCI, increased paracellular mucosal permeability associated with decreased claudin-1 mRNA and protein expression was observed in the absence of inflammation in the colon. Activated enteric glial cells (Sox10+/GFAP+) in the colon were significantly increased 28 days after CCI. When challenged with a Cr infection at 28 days post-CCI, host Th1/Th17 immune response in the colon and clearance of Cr were unaffected by CCI; however, colonic paracellular flux and enteric glial cell reactivity were significantly increased. Importantly, Cr infection in chronically-injured mice worsened TBI neuropathology, as measured stereologically by lesion volume, and increased neuroinflammatory responses (GFAP+ astrocytes, CD68+ microglia/macrophages) in the cortex. These enteric microbial-induced changes in the gut and brain were associated with increased circulating IP-10 levels and decreased serum miR-223 expression. This work demonstrates long-term consequences of TBI on the brain-gut axis, and the impact of gut dysfunction on resolution of chronic brain injury. Future studies aim to elucidate centrally- and intestinally-derived mechanisms of brain-gut modulation during injury and immune responses after chronic TBI.
T32DK067872
Keywords: brain-gut axis, intestinal mucosal barrier, enteric glial cells, Citrobacter rodentium, serum biomarkers
University of Kentucky, Sanders Brown Center on Aging, Lexington, USA
Traumatic brain injury (TBI) is a leading cause of long-term impairments in higher cognitive function. Ongoing destructive secondary injury events occur minutes to days after the initial insult characterized by a cascade of pervasive biochemical and pathophysiological stressors. A rapid and sustained phase of mitochondrial dysfunction after TBI impacts a number of important cellular events. We demonstrated previously that specific hippocampal mitochondria-associated microRNAs (miRNAs) are altered following a controlled cortical impact (CCI) injury in rats. Our new studies reveal that the levels of several mitochondria-associated miRNAs (e.g. miR-142-3p, miR-142-5p, miR-146a, miR-155 and miR-223) are altered early on (3-24 hr), but start to normalize by 72 hr after TBI. Knowledge of the temporal changes in mitochondria associated miRNA levels after TBI provides an opportunity to target specific miRNA at specific time points. We have previously shown that miR-107 is significantly downregulated in hippocampal neurons at 24 hr following TBI. In the present study, we used a 21 amino acid peptide-based nanoparticle approach to test whether delivery of miR-107 mimic alters hippocampal levels of miR-107 and two miR-107 validated targets, granulin and beta secretase 1 (BACE1). At 48 hr following stereotaxic injections of peptide+miR-107 into the dorsal hippocampus, miR-107 level increased by 150% while granulin/BACE1 mRNA levels were downregulated by 20–40% depending upon the peptide+miR-107 ratio/concentration. The outcomes of this study suggest a temporally dynamic interaction of miRNA with mitochondria, and a potential role for mitochondria in dictating miRNAs' cellular function. The study also demonstrates the use of a peptide-based nanoparticle approach to effectively deliver miRNA mimics or inhibitors as a way to target specific miRNA activities following TBI.
Keywords: microRNA, mitochondria, traumatic brain injury, peptide-based nanoparticle, miR-146a, miR-155
The purpose of this study is to investigate an animal model of distress (conditioned suppression) to assess the effects of magnesium (MAG) on recovery of anxiety-like behavior following traumatic brain injury (TBI) in rats. TBI is a national health concern where victims experience cognitive impairments, such as post-traumatic stress disorder (PTSD). PTSD affects a TBI victim's ability to maintain daily activities and integrate back into society. MAG administered immediately after TBI may alleviate anxiety symptoms, helping the patient adjust to life after injury as MAD decreases brain swelling. An established method of conditioned suppression (CS), that generates fear responses in animals, will evaluate this intervention.
This study will compare the acquisition and extinction of CS in 3 groups of 10; sham-control, TBI/placebo, and TBI/MAG. Rats will be trained to lever-press for food to establish constant press-rates. This phase consists of 30-m sessions where lever-pressing is maintained by a variable-interval schedule of 30-sec. After a constant press-rate is reached, rats will receive a severe frontal cortex injury. MAG i.p. injections begin 4-hours after injury, then again at 24-hours, and at 72-hours. Post-injury testing consists of ten 30-m classical conditioning sessions where four 30-second tones will be presented and conclude with delivery of a shock (0.5-mA for 0.5-sec). Pairing tone with shock establishes the tone as a stimulus that elicits fear. Fear is measured by the extent to which lever-pressing is disrupted during the tone. Complete CS is achieved when lever-pressing during the tone decreases across successive conditioning trials, until the rat no-longer presses. The final phase presents the tone without the shock (extinction). Sessions will last 30-minutes with four presentations of a 30-sec tone. Extinction sessions conclude when rats have returned to baseline pressing.
Combination of magnesium is hypothesized to result in quicker extinction of lever-suppression than if one or no-intervention is given. Indicating, the treatment may be helpful as an intervention for decreasing PTSD symptoms after TBI.
Keywords: TBI
Krembil Research Institute, Toronto Western Hospital, Genetics and Development, Toronto, Canada
Reactive astrogliosis is a defining hallmark of secondary pathology following spinal cord injury (SCI), posing a significant challenge to endogenous reparative processes and stem-cell mediated regeneration. While neural progenitor cells (NPCs) are widely recognized for cell replacement and trophic support after injury, concurrent modification of the glial scar may further support tissue repair. Hepatocyte growth factor (HGF) is a potent multifunctional cytokine involved in cell and tissue regeneration that may synergistically compliment NPC-mediated repair. The primary objectives of this study are to assess the effect of HGF on NPC cytokine secretion and the resultant combinatorial effect on astrocyte reactivity after injury. RNA-seq was used to quantify HGF expression after C6 SCI (23g 1 min clip compression) 3-56 days post-injury. Rat spinal cord-derived NPCs were pre-treated with HGF for 4 days and conditioned media (CM) was characterized via Proteome Profiler cytokine array. Primary rat astrocytes were activated using TGFβ1 and cells were then washed and treated with either HGF protein (10-50 ng/mL), NPC-CM, or HGF-treated NPC-CM. Astrocytic GFAP, Vimentin, CSPG, and TGFβ1 production were assessed via Western blot and immunocytochemistry. RNA-seq revealed no significant change in endogenous HGF expression up to 56 days after SCI. HGF-treated NPCs demonstrated a decrease in IL-1α secretion and an increase in TIMP-1 compared to untreated NPCs. Combinatorial HGF+NPC-CM treatment demonstrated a significant reduction in astrocytic GFAP, Vimentin, CSPG, and TGFβ1 production compared to HGF or NPC-CM alone. For the first time, we have shown reduced astrocyte reactivity with HGF+NPC-CM combinatorial treatment. This effect may be mediated via inhibition of the TGFβ signaling pathway. As glial scarring remains a fundamental challenge to endogenous and stem cell mediated-regeneration after injury, combinatory treatment paradigms that strategically modify the scar and promote tissue repair should be further explored to increase therapeutic efficacy.
Keywords: neural stem cells, growth factors, astrogliosis, spinal cord injury
University of Ottawa, Medicine, Ottawa, Canada
Keywords: Neural stem cells, Adult human spinal cord, Proliferation / Differentiation, in vitro
Kangwon national university hospital, Neurosurgery, Chuncheon, Korea South
Keywords: mesenchymal stem cell, Spinal cord injury, white mater sparing, gliosis
University of Texas Health Science Center Houston, Pediatric Surgery, Houston, USA
Chronic neuropathic pain (NP) affects approximately 64-82% of all patients living with a spinal cord injury (SCI). Despite years of research, a thorough understanding of the underlying mechanisms involved in the development and maintenance of SCI-dependent NP remains elusive. Inflammation plays a dynamic role in the development of NP, and is a prime therapeutic target. With potent anti-inflammatory and immune-regulatory characteristics, mesenchymal stem cells (MSC) may provide a safe and effective method for modulating and attenuating NP. The objectives are to determine the efficacy of early MSC delivery to attenuate inflammation and NP development, and improve chronic NP and inflammation with delayed MSC treatment. Adult, male, Sprague-Dawley rats will receive a moderate, spinal contusion at T-10. All subjects and age-matched controls are subjected to neurosensory assessments of mechanical allodynia (von Frey filaments) and thermal hyperalgesia (Hargreave's test) in hind paws. Neurosensory assessments are performed prior to injury and 7, 14, 21 and 28 days post-injury for the acute study and again monthly for the chronic study. MSC (50 × 106 cells/ml) are delivered intravenously and intrathecally at 24 hrs (acute) or 7 months (chronic) post-injury. Spinal cord tissue is harvested at 28 days or 8 months post-injury, sectioned and stained for immunohistochemistry. MSC treatment after CNS injury is appealing due to their immunoregulatory and anti-inflammatory characteristics. The data generated in this study suggests that the administration of MSC after spinal contusion injury partially prevents NP associated with mechanical allodynia in the hind paws. The data produced in our chronic study reveal MSC treatment in the chronic phase of injury effectively improves pain and function scores, and indicate a decrease in neuroinflammation. These results support our hypothesis that MSC are an effective cellular therapy approach to treat SCI-dependent NP.
Funding provided by the UTHSC Bentsen Stroke Center Investigator Program.
Keywords: Animal model, mesenchymal stem cells, chronic SCI, neuroinflammation
Acute spinal cord injury continues to be a devastating problem worldwide with high morbidity and mortality. Long term morbidities include sensory, motor, and autonomic dysfunction. There is currently no consensus on pharmacological intervention to alter the clinical course of acute spinal cord injury and improve neurological outcome. Bone marrow-derived mesenchymal stem cells (MSC) have been shown to modulate the injury sequelae of SCI via paracrine effects, although the mechanisms are not completely clear. One potential modality is through secretion of extracellular vesicles (EV). EV are heterogeneous particles with lipid bilayer, containing growth factors, lipids, microRNAs, mRNAs, and proteins. We are testing the application of MSC-derived extracellular vesicles (MSCEv) to reduce neuroinflammation and improve sensory and motor recovery following SCI. Animals will be randomly assigned to three groups: sham laminectomy, or T10 contusion + vehicle or T10 contusion + MSCEv. 1, 2, 3, 5, 7, 10 and 14 days following injury, locomotor recovery is scored using the Basso, Beattie and Bresnahan (BBB) method. At 14 days post-injury, animals will be tested for mechanical allodynia via the Dixon Up-Down Von Frey method then sacrificed. Spleen will be harvested for neurotransmitter assays, and blood and spinal cord will be harvested for flow cytometry and immunohistochemistry. Preliminary data indicate significantly higher locomotor recovery scores in SCI + MSCEv animals when compared to SCI + vehicle animals on days 5, 7 and 14 post-injury (p < 0.0001, p < 0.001 and p < 0.001, respectively). Animals treated with MSCEv also demonstrate significantly higher force thresholds in the mechanical sensitivity test compared to vehicle treated animals at 14 days post-injury (p < 0.05). Flow cytometry analysis of spinal cord reveals increased activation of M1 and M2 microglia in MSCEv treated animals compared to vehicle. Spleen analysis indicates increased myeloid cells and MDSC with decreased NK cells and leukocytes in MSCEv treated animals. These results support our hypothesis that MSCEv are an effective therapeutic after SCI.
Funding provided by Glassell Family Pediatric Stem Cell Research Fund.
Keywords: Extracellular vesicles, locomotor recovery, neuroinflammation, secondary injury
CO-ENCAPSULATION OF MESENCHYMAL STEM CELLS AND CORD BLOOD IMPROVE LOCOMOTOR RECOVERY AND NEUROINFLAMMATION IN SPINAL CORD INJURY
Over 17,000 new spinal cord injuries (SCI) occur each year in the United States, contributing to approximately 282,000 persons currently living with SCI. Currently, treatment consists of surgical stabilization, physical rehabilitation, and analgesia. There is an unmet need for new treatments to mitigate secondary damage that occurs after SCI. Pro-inflammatory cytokines and leukocyte infiltration play a significant role in the development of secondary injuries and present a specific target for cellular therapy. Bone marrow-derived mesenchymal stem cells (MSC) have been shown to modulate inflammation, reducing secondary injuries in SCI via paracrine effects. Our objective is to evaluate the efficacy of MSC co-encapsulated with umbilical cord blood (CB) effector cells and the potential synergistic effect on local neuroinflammation and behavior. MSC are encapsulated in alginate hydrogel beads that are implanted adjacent to the contused T-10 spinal cord 24hrs after injury. Animals are randomly assigned to one of five groups: Naïve, Sham + encapsulated MSC, Sham + co-encapsulated MSC + CB, SCI + encapsulated MSC, SCI + co-encapsulated MSC + CB, alginate beads alone, encapsulated MSC cells, encapsulated cord blood cells or MSC + cord blood encapsulated. Efficacy is measured by locomotor recovery, mechanical and thermal sensitivity, as well as immunohistochemical staining for leukocyte infiltration, demyelination, inflammatory mediators and blood-spinal cord barrier permeability. Animals are sacrificed 14 days after injury and spinal cords are harvested, sectioned and stained for immunoreactivity. Preliminary data indicates that local application of co-encapsulated MSC and cord blood cells significantly improves locomotor recovery 14 days post-injury when compared to systemic MSC (*, p value <0.05). Interestingly, encapsulated MSC animals showed significantly improved locomotor recovery scores on days 2, 3, 5, 7, 10 and 14 when compared to all other groups. We anticipate histological changes will indicate that encapsulation of MSC and co-encapsulation of MSC+CB cells provides increased protection against secondary injury.
Funding provided by the UTHSC Bentsen Stroke Center Investigator Program and Glassell Family Pediatric Stem Cell Research Fund.
Keywords: Encapsulation, umbilical cord blood cells, mesenchymal stem cells, secondary injury
University of Pittsburgh, Neurosurgery, Pittsburgh, USA
Lasting cognitive impairments have been reported by patients afflicted with a traumatic brain injury (TBI) and replicated in experimental TBI models. Previous work from our lab and others implicates impaired neurotransmission as a contributor to neurobehavioral dysfunction after TBI, but little is known about the mechanisms underlying this pathology. In the synapse, formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is a critical step in vesicle fusion and the release of neurotransmitters into the synaptic cleft. We recently showed severe TBI impairs SNARE complex formation; however, it is unknown if this contributes to synaptic dysfunction across the spectrum of injury severity. We hypothesized that lateral fluid percussion injury (lFPI) decreases SNARE complex formation in multiple brain regions and results in impaired function. To this end, 3 cohorts of male Sprague Dawley rats were subjected to sham or 2atm lFPI and assessed for 1) evoked neurotransmitter release, 2) SNARE protein and complex abundance by immunoblotting, and 3) histopathology and neurobehavioral performance. Preliminary findings from ongoing studies suggest deficits in potassium-evoked striatal dopamine release at 7d post-injury. Synaptosomal lysates were generated to focus on synaptic alterations. At 7d post-injury, lFPI significantly reduced hippocampal SNARE proteins VAMP2 and α-synuclein, and SNAP-25 immunoreactive SNARE complexes, but did not alter synaptophysin abundance. Additional alterations in SNARE protein abundance were observed in other brain regions. NeuN and cresyl violet staining revealed the formation of a characteristic cortical lesion at the white matter and neocortex interface, minor hilar neuron loss, and no overt CA1 or CA3 loss after lFPI. Beam balance and walking tasks revealed transient impairments in vestibulomotor function 1d post-injury. Spatial acquisition and memory, as assessed by the Morris water maze, was significantly reduced following lFPI 3 weeks post-injury, consistent with previously published literature. These data provide novel evidence for TBI-induced SNARE protein changes and synaptic dysfunction after brain injury.
Support: 1-F32NS090748, The Pittsburgh Foundation, NIH-NS40125, NIH-NS060672, VAI01RX001127.
Keywords: neurotransmission, synapse, hippocampus, fluid percussion injury
University of Pittsburgh, Neurosurgery, Pittsburgh, USA
Traumatic brain injury (TBI) has been identified as a risk factor for the development of chronic neurodegenerative diseases, including Parkinson's disease. Pathological protein misfolding is associated with disrupted cellular function and can contribute to subsequent neuronal loss. Current hypotheses suggest that synaptic dysfunction precedes neuronal loss in the pathological progression of neurodegenerative disease. Alpha synuclein (α-synuclein) has been well described for its role in contributing to the pathogenesis of Parkinson's disease. Pathological forms of α-synuclein have been observed at chronic time points following TBI. Emerging evidence highlights a central role for α-synuclein in the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex responsible for neurotransmitter release. We recently reported that TBI results in an acute reduction in monomeric α-synuclein. We hypothesized that TBI results in acute formation of α-synuclein aggregates in synapses. To this end, male Sprague Dawley rats were subjected to sham or controlled cortical impact (CCI) injury (2.7 mm, 4 m/s, 150 msec) and sacrificed at 2, 7, or 14 days post-injury. High molecular weight α-synuclein aggregates, confirmed using two antibodies, were detected in hippocampal whole cell and synaptosomal-enriched lysates at all time points assessed. Immunohistochemical staining revealed aggregated α-synuclein immunoreactivity in pericontusional tissue and the dentate gyrus of the hippocampus at 2 weeks following CCI injury. These findings provide novel evidence for acute α-synuclein aggregates in the injured synapse that may contribute to impaired synaptic and neurobehavioral function after TBI.
Support: 1-F32NS090748, The Pittsburgh Foundation, NIH-NS40125, NIH-NS060672, VAI01RX001127.
Keywords: synapse, traumatic brain injury, alpha synuclein, hippocampus
Traumatic brain injury (TBI) is a highly-prevalent cause of morbidity and mortality worldwide with no effective therapy. The majority of TBI cases are mild or ‘concussive.’ These patients overwhelmingly recover well. Patients with complicated-mild or moderate TBI, however, suffer a dramatically higher burden of long-lasting deficits. These patients are therefore an attractive target for investigation. We developed a non-surgical, tunable, monitored model of mouse TBI based off of the closed-head impact model of engineered rotational acceleration (CHIMERA) platform, called modCHIMERA, that targets this TBI severity group. modCHIMERA is characterized by both impact and inertial loading (linear and rotational), with acceleration exceeding scaled human injury thresholds, and righting times consistent with a moderate injury. modCHIMERA induces diffuse damage characterized by multifocal white matter injury, cell death, neuroinflammation, and multidomain neurobehavioral deficits. Importantly, modCHIMERA does not generate a dominant focal, cavitary lesion as results from most moderate-severe TBI models. This approximates the diffuse injuries commonly observed in patients with complicated-mild and moderate TBI, and facilitates investigation of grey matter cellular and subcellular damage. Such grey matter injury pathways may drive lasting neurological impairment separate from the white matter pathology that has been the primary focus of most studies on post-TBI neuronal damage. Consistent with this, modifications to modCHIMERA to limit axonal injury did not eliminate long-term neurobehavioral deficits. To characterize pathways leading to grey matter injury after TBI, cortical regions were assessed after modCHIMERA for neuronal cell death, dendrite length and arbor complexity, dendritic spine density, and synaptic density. Functional connectivity optical intrinsic signal imaging was used to evaluate local circuits vs. those dependent on projection axons, and demonstrates greater impact on ipsilateral node degree consistent with functionally-significant synaptic or dendritic injury. These studies will lead to a greater understanding of traumatic circuit disruption beyond axonal pathways, and aid in the development of novel diagnostic tools for characterization of grey matter injury after TBI.
Keywords: Grey matter, Synapse, Complicated-mild traumatic brain injury, Moderate traumatic brain injury, Functional connectivity, Optical intrinsic signal imaging
Burke Medical Research Institute, White Plains, USA
Schwann cell (SC) transplants are beneficial in rodent models of spinal cord injury (SCI). In a Phase 1 clinical trial, SCs were demonstrated safe in humans. Previously, we demonstrated that transplanted cells die acutely, raising the concern that acute transplant death may counteract some of the beneficial effects of SC transplants. In the current study, we assessed the effect of transplanted cell death on spared spinal cord tissue and then examined the effects of pharmacologically increasing hypoxia-inducible factor alpha (HIF-α) in SCs as a strategy to promote transplanted cell survival. Following transplantation of 2 × 106 SCs one week post-SCI in the rat, nitrotyrosine (3-NT) was significantly increased within the injured spinal cord, suggesting a detrimental effect of cell transplants on spared spinal cord tissue. We then assessed the ability of HIF-a to enhance transplant survival. HIF-a levels were increased in SCs pharmacologically by manipulating the activity of HIF-proylyl-4-hydoxylases (PHD), enzymes that target HIF-α for proteasomal degradation. Treatment of SCs with 1% O2 (24 h), deferoxamine (DFO: 200 mM, 24h), or adaptaquin (AQ; 10 mM, 16 h) increased the level of both HIF-α and its target genes. In vitro, elevation of HIF-α with DFO and AQ, but not 1% O2, protected SCs against H2O2-mediated cell death. In vivo, HIF pretreatments attenuated the increase in 3-NT observed following SC transplantation, but pretreatment was insufficient to promote transplant survival or functional recovery. Examination of HIF-α and its target gene levels revealed that HIF protein and its targets were only transiently elevated within the spinal cord following pretreatment of the cells. Together, the results demonstrate that transplanted cell death can be deleterious and should be counteracted, and that while elevating HIF-α is protective, the effects of pretreatment are short-lived indicating prolonged treatment is required.
Support: NIH 1R01NS075375.
Keywords: Schwann cells, spinal cord injury, HIF, cell transplantation, cell death, cell survival
Burke Medical Research Institute, White Plains, USA
Cellular transplants provide replacement cells and a substrate for repair following spinal cord injury (SCI). Despite these benefits, the effects of cell transplants on functional recovery following SCI are modest. Previously, we determined that although cell transplants persist and integrate long-term, the majority of cells die acutely post-transplantation. We hypothesize that this acute death could attenuate the beneficial effects of cellular transplants. To address whether the acute death of transplanted cells is detrimental, we are developing approaches to enhance transplant survival. In the current study, we manipulated the expression of the transcription factor hypoxia inducible factor 1 alpha (HIF-1α) in Schwann cells (SCs). Overexpression of VP16-HIF-1α increased nuclear HIF-1α protein levels four-fold and resulted in a one-fold increase in the protein levels of two HIF-1α targets, VEGF and enolase. To test whether elevated levels of HIF-1α in SCs could protect them from transplant-mediated cell death, 2 × 106 SCs (control, VP16, or VP16-HIF-1α) were transplanted seven days post-SCI in the rat. Transplant survival was increased by 34% and 21% in VP16-HIF-1α SCs relative to control and VP16 SCs, respectively. Stereological quantification of transplant survival is time-consuming. To determine if IVIS-based imaging could be used to rapidly screen transplant survival, we quantified light emitted from transplants using IVIS imaging for luciferase in live animals and for GFP fluorescence in ex vivo spinal cords. Luciferase-based IVIS imaging detected differences over time but not between treatment groups. GFP IVIS imaging of ex vivo spinal cords showed a trend for increased transplant survival. This suggests that GFP imaging may be useful for screening pro-survival strategies that lead to large differences in transplant survival. The increase in transplant survival with HIF-1α suggests it may be a useful target for enhancing transplant survival. Additional studies that pharmacologically manipulate HIF levels in SCs are presented separately.
Keywords: Schwann cell, Spinal cord injury, transplantation, transcription factor, cell death
Miller School of Medicine, University of Miami, Miami Project to Cure Paralysis, Miami, USA
Schwann cell (SC) transplantation has been extensively studied for spinal cord injury (SCI) repair. SCs are neuroprotective and promote axon regeneration and myelination in the implant site. However, substantial cell death occurs post-implantation, limiting its therapeutic potential. The use of injectable matrices improves transplanted SC survival, axon extension, and function in a rat contusion model. Yet, clinically relevant products are needed for translational use, as the currently available matrices have variable formulations and derive from animal tumors, as Matrigel. We tested an injectable acellular peripheral nerve (iPN) matrix to improve SC grafts in SCI. Acellular iPNs were processed to be injectable and thermally gelling. SCs in iPN were transplanted into the lesion epicenter of T8 contused rats and compared to SCs implanted in Matrigel. Our results showed a plethora of SC-myelinated axons within dense implants in both conditions. iPN grafts contained twice as many axons as Matrigel. Graft volume in iPN was twice as large compared to Matrigel. SC/iPN rats performed as well as SC/Matrigel rats in BBB testing, and showed a faster recovery in BBB subscores. On the grid walk, SC/iPN rats performed fewer errors at 4 weeks, equalizing at 8 weeks. Immune cell profiling by flow cytometry revealed that SC/Matrigel implants exhibited increased T cell and leukocyte infiltration into the lesion, whereas iPN implants were identical to SCI controls. The fact that this clinically relevant iPN matrix is immunologically tolerated and improves transplanted SC survival and axon growth within the graft offers a translational possibility for improving efficacy of SC treatment after SCI.
Keywords: Schwann Cell
Data Blitz Oral Presentations
University of Pennsylvania, Neurology, Philadelphia, USA
Traumatic Brain Injury (TBI) results in diffuse axonal injury (DAI) and diffuse vascular injury (DVI). Both DAI and DVI result from inertial shearing forces, and the two terms are often used interchangeably, the spatial relationships between DAI and DVI have not been carefully studied. Multimodal magnetic resonance imaging (MRI) can help to distinguish these injury mencahisms: diffusion tensor imaging (DTI) provides information about axonal integrity, while arterial spin labeling (ASL) and functional Blood Oxygen Level Dependent imaging (BOLD) with hypercapnia challenge, reflect cerebral blood flow (CBF) and cerebrovascular reactivity (CVR) respectively. Chronic TBI participants (n = 27) and age- and education-matched healthy controls (n = 15) underwent multimodality MRI. The Freesurfer image analysis suite (MGH, Harvard, MA) was used to segment each MP-RAGE image into regions of interest (ROIs). Mean values of mean diffusivity (MD), fractional anisotropy (FA), CBF, and CVR were extracted for each ROI. Additionally, maps were normalized into a common space (MNI Atlas) and z-score maps were generated based on a pool of healthy controls. Normality of an ROI/voxel was determined based on z-score (abnormal MD: z-score >2.5; abnormal FA, CBF, and CVR: z-score < −2.5). Abnormal ROIs in one modality were not predictive of abnormalities in another modality. Approximately 8–10% of abnormal voxels for CVR and CBF also show an abnormal voxel value for MD, while only 1% of abnormal CVR and CBF voxels show a concomitant abnormal FA value. These data indicate that chronic TBI patients display two distinct endophenotypes: microstructural tissue/axonal injury and vascular injury that are spatially independent.
Keywords: Diffusion Tensor Imaging, Cerebral Vascular Reactivity, Chronic TBI, MRI
Messina University, Messina, Italy
Glibenclamide (GLI) is a sulfonylurea receptor antagonist that has shown promise as a therapy to prevent secondary injury following traumatic brain injury (TBI). The present work, part of the Operation Brain Trauma Therapy (OBTT) multi-center pre-clinical drug screening consortium, investigated the effects of GLI treatment on the levels of circulating glial fibrillary acidic protein (GFAP) and the relationships with histopathological and behavioral outcomes after controlled cortical impact (CCI). OBTT demonstrated that GLI treatment reduced contusion volume in CCI; thus, we sought to determine whether circulating GFAP in the initial 24h could inform theranostically on lesion volume at 21d post-injury. Adult male rats subjected to CCI received a bolus (10μg/kg IP) 15min after-injury, followed by a continuous SQ infusion (0.2μg/h) via osmotic pumps throughout 7d of GLI or vehicle. GFAP levels in blood were measured at 1, 4 and 24h after CCI. Vehicle-treated rats displayed distinct temporal profiles for GFAP vs. GLI-treated rats. GFAP in vehicle-treated animals demonstrated a sustained increase after-injury vs. shams (p < 0.0001) peaking at 4h. Conversely, rats treated with GLI initially had high GFAP levels similar to vehicle. However, by 24h GFAP in GLI-treated rats did not differ from sham and were lower (p < 0.05) than vehicle. Overall, GLI-treated rats had significantly lower GFAP release (area under the curve) throughout the study vs. vehicle. GFAP at 24 h also strongly correlated with contusion volume at 21d (r = 0.88, p < 0.0001), hemispheric tissue loss (r = 0.86, p < 0.0001) and MWM latency (r = 0.68, p = 0.0001). Our findings support a role for GFAP in predicting tissue damage and as a marker of therapeutic response corroborating advantageous effects of GLI in CCI. Circulating GFAP may be useful for high-throughput screening of drugs in pre-clinical investigations. Support: USArmyW81XWH-10-1-0623.
Keywords: Biomarkers, GFAP, TBI, controlled cortical impact, rat, neuroprotection
University of Toronto, Institute of Medical Science, Toronto, Canada
Traumatic spinal cord injury (SCI) is a devastating neurological condition that occurs primarily at the cervical (cSCI, >65%) and thoracic (tSCI) levels. Despite the marked neurovascular distinctions of the two levels and strikingly positive response of cSCI to trial drugs such as cethrin compared to tSCI, the mechanisms driving level-specific heterogeneity between their respective milieu remains elusive. We posit that the increased vascularity and grey-white ratio of the cervical cord—relative to the thoracic—results in greater susceptibility to neurovascular disruption, ultimately manifesting a secondary injury of earlier onset, severity, and chronicity. A rat model of moderate clip compression injury was used to induce SCI at the C6-7 and T6-7 levels, with laminectomy-only animals serving as surgical controls. Following sacrifices at 3, 7, 14, and 56 days, samples were subject to RNA-seq, protein work, imaging, and immunohistochemistry. Results of RNA-sequencing revealed striking differences in the onset and temporal profile of astrocytic and pericytic neurovascular processes with canonic stat3-dependent gliotic markers—lcn2, gfap and serpina3n—being upregulated in the cervical cord across time. Further, 3D ultrasound and immunostaining revealed rapid tissue loss and hemorrhage starting as early as 3 days post-cSCI with increased gfap and cspg4 staining in the cord. Finally, Western blotting confirmed an increase in stat3-dependent gliotic markers accompanied by a loss of key blood-brain-barrier proteins tjp1 and ocln in cSCI across time. Taken together, this data demonstrates—for the first time—the level-specific heterogeneity of SCI, with cSCI having a quicker onset and chronicity compared to tSCI. Further, these results reconcile the potential reasons behind why preliminary tSCI-derived trial paradigms may not be suited–in both strategy and timing—to cSCI, and hopes to engage clinicians and scientists in the design and study of level-specific therapeutics.
Keywords: RNASeq, Neurovascular unit, Secondary injury, Pericyte
University of California, Riverside, of Biomedical Sciences, Division Riverside, USA
Traumatic brain injury (TBI) is a major cause of morbidity and mortality in persons under the age of 45 and is a primary public health concern in both the civilian and military populations. Posttraumatic epilepsy (PTE) is a long-term negative consequence of TBI in which one or more unprovoked seizures occur at least one week after the initial trauma and accounts for up to 20% of all symptomatic epilepsy. Severity of injury and early posttraumatic seizures are risk factors associated with PTE, however, predictors of PTE still remain highly variable. Current studies of PTE have focused on pharmacologically testing seizure threshold using pentylenetetrazole and imaging studies, while limited, have not identified a correlation between trauma and seizure susceptibility. In our study, we utilized optical coherence tomography (OCT) to detect functional and structural changes in the brain in a controlled cortical impact (CCI) injury mouse model of TBI. Additionally, animals underwent long-term video-electroencephalography (vEEG) recording to identify spontaneous seizures after TBI and intrahippocampal electrical stimulation to quantitatively define electrographic seizure threshold (EST) and duration (ESD). OCT imaging detected alterations in optical attenuation, a measure of both scattering and absorption of the biological sample, with and without vascular contribution at different time points after TBI. Additionally, injured animals exhibited altered EST and ESD compared to naïve and sham controls. Altogether, our data suggest that seizure threshold can be quantitatively measured and OCT imaging can potentially offer useful information to identify predictors of PTE in our model.
Keywords: Optical coherence tomography, Seizure threshold, EEG
Boston University School of Medicine, Molecular Aging & Development Laboratory, Boston, USA
Despite growing awareness of links between concussion, traumatic brain injury (TBI), and long-term effects of neurotrauma, the underlying mechanisms in the brain and possible changes in the retina are unknown. In this study, we used a new mouse model of concussion and a state-of-the-art retinal eye scanner to investigate post-traumatic inflammatory responses in the brain and retina. We utilized Ccr2RFP/Cx3cr1GFP mice (Jackson Laboratory) to enable immune cell visualization by class (monocyte, microglia), origin (peripheral, brain, retina), morphology, and location. We used a new closed-head impact injury mouse model (Tagge et al., submitted) that recapitulates key features of human concussion. We used a multimodal adaptive optics small-animal imager (MAOSI) (Physical Sciences, Inc.) for in vivo retinal studies. MAOSI has several channels: adaptive optics optical coherence tomography (AO-OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO) with reflectance and fluorescence channels. Before injury, Ccr2RFP/Cx3cr1GFP mice showed normal cellular distribution and morphological phenotype of microglia in retina and brain. We observed significant increase in microglia in retina and brain post-injury. Moreover, microgliosis in both retina and brain was notable for reactive cellular phenotype transformation with overlapping ramification fields. Closed-head impact injury is associated with similar reactive inflammatory responses and sequelae in both retina and brain. The retina can be used as a “brain proxy” for noninvasive diagnosis, prognosis, and monitoring of neuroinflammation after closed-head injuries.
Keywords: Impact Concussion, Mouse Model, Retinal Imaging, Neuroinflammation
University of Miami, Neurological Surgery, Miami, USA
Acute Lung Injury (ALI) is a common systematic complication of severe Traumatic Brain Injury (TBI). However, the pathophysiologic mechanisms of TBI-induced ALI are not well known. We have previously shown that the inflammasome plays a critical role in TBI-induced secondary pathophysiology and that inflammasome proteins are released in Extracellular Vesicles (EV) after TBI. Here we investigate whether EV-mediated inflammasome signaling contributes to the etiology of TBI-induced ALI. In this study, C57/BL6 mice were subjected to Controlled Cortical Impact Injury (CCI) and brains and lungs were examined for inflammasome activation, pyroptosome formation, and ALI. Also, an adoptive transfer experiment was performed by injecting serum-derived EV from sham and TBI injured mice into healthy mice and lungs were analyzed for inflammasome protein expression and ALI. Our findings indicate that brain and lungs of CCI-injured mice showed a significant increase in the expression of AIM2, IL-1b, caspase-1, IL-18 and HMGB1 acutely after TBI. Moreover, injured lungs also showed evidence of pyroptosis. Lungs of CCI-injured animals demonstrated evidence of ALI as determined by thickening of the alveolar septum, alveolar edema and inflammation, thus resulting in a higher ALI score. Adoptive transfer of serum-derived EV from TBI mice, but not sham mice, into healthy mice induced higher expression of inflammasome proteins in lungs and higher ALI scores. Interruption of this axis by administration of Enoxaparin or an anti-ASC significantly inhibited inflammasome activation and improved ALI scores. In conclusion, our data show that inflammasome activation plays a major role in TBI-induced ALI. These data provide strong evidence for activation of a Neural-Respiratory Inflammasome Axis, and demonstrate that targeting this axis with Enoxaparin or anti-ASC antibody may provide a novel therapeutic approach for neurotrauma-induced ALI.
Funding: Miami Project to Cure Paralysis, NIH grants: R42NS086274, F31 HL132425-01A1.
Keywords: Traumatic Brain Injury, Acute Lung Injury
Minocycline was the 10th drug selected for testing by the OBTT consortium. Minocycline is a broad-spectrum tetracycline antibiotic shown to be neuroprotective in neurodegenerative diseases and experimental models of stroke, spinal cord injury (SCI), and brain trauma. It has been reported to inhibit microglial activation, prevent oligodendrocyte and/or neuronal apoptosis, decrease oxidative damage, and reduce inflammation. A phase II clinical trial for acute SCI found Minocycline safe and tended to improve several motor recovery outcomes. The WRAIR site evaluated the effectiveness of Minocycline in the penetrating ballistic-like brain injury (PBBI) model. PBBI (10%) was performed unilaterally in the right hemisphere of anesthetized rats. A 30mg/kg Minocycline loading bolus was administered intravenously 15mins post-PBBI followed by continuous 72-hour infusion (2 mg/kg/hr). This protocol was shown in PK studies by our group to produce steady state blood levels ranging between 5-10 mcg/mL, mimicking those in the successful phase II clinical trial. Groups consisted of TBI-MIN (n = 14), TBI-Veh (n = 19) or Sham (n = 16). Motor and cognitive performance were assessed using rotarod (days 7 and 10 post-PBBI) and Morris water maze (MWM, days 13-17 post-PBBI). On day 21, brain tissue was processed for histology. Motor testing showed significant injury-induced deficits versus sham (p < .05). Overall rotarod latencies were reduced by 51.8 ± 5.5% (TBI-Veh) and 47.4 ± 8% (TBI-MIN) vs. sham. No significant therapeutic effects were detected on the rotarod task. MWM results revealed significant injury-induced deficits with latencies to locate the submerged platform increased by 56.8 ± 14% (TBI-Veh) and 52.9 ± 14.8% (TBI-MIN) versus sham. Positive trends towards improved memory retention (probe trial) and reduced lesion size were observed, but not significant. Current findings do not support further testing of Minocycline using this dosing regimen. However, additional testing may be warranted using higher doses and/or extended treatment durations in order to overcome peak microglial and inflammatory responses in the PBBI model. Supported by U.S. Army Grant W81XWH-10-1-0623.
Keywords: traumatic brain injury, OBTT
IMPACT OF REPETITIVE MTBI ON FEAR MEMORY, DEPRESSIVE BEHAVIOR, AND MARKERS OF SYNAPTIC PLASTICITY IN A MOUSE MODEL OF CHRONIC PTSD
Roskamp Institute, Sarasota, USA
Keywords: PTSD, mTBI, Fear Memory, Depression, Mouse model, Closed Head Injury
University of Pittsburgh, Safar Center, Pittsburgh, USA
Operation brain trauma therapy (OBTT) is a multi-center pre-clinical drug and biomarker screening consortium supported by the US DoD. OBTT screens therapies across three models in rats (parasagittal fluid percussion injury [FPI], controlled cortical impact [CCI] and penetrating ballistic-like brain injury [PBBI]) reflecting a range of phenotypes. Our goals include 1) to define therapies efficacious across models with the best chance for clinical translation, and 2) to define model-dependent effects to guide trials in targeted pathologies. The results of the first 5 therapies tested (nicotinamide, erythropoietin [EPO], cyclosporine [CsA], simvastatin, and levetiracetam) were published. OBTT has now screened 10 therapies, with ongoing testing of drugs 11-12. Drugs 6-12 include glibenclamide, kollidon-VA64, AER-271, amantadine, minocycline, E-64d and P7C3-A20. Levetiracetam showed benefit in multiple models. The second most successful drug, glibenclamide showed multiple benefits in CCI. Other therapies showed model dependent effects: tissue sparing by nicotinamide in CCI, benefit from CsA in FPI but toxicity in PBBI, benefit from amantadine in PBBI. Serum glial fibrillary acidic protein (GFAP) and Ubiquitin carboxy-terminal hydrolase-L1 were assessed at 4 and 24h. GFAP predicted 21d histology and drug effects. Levetiracetam merits additional testing and is being evaluated in our large animal model. Glibenclamide and Amantadine merit additional testing in contusion and penetrating brain injury, respectively. Our data support theranostic use of serum GFAP.
Support: W81XWH-10-1-0623; WH81XWH-14-2-0018.
Keywords: consortium, rat, reproducibility, rigor, pre-clinical, therapy
There remains a need for more effective therapeutics in the treatment of traumatic brain injury (TBI). In this study, we tested whether an effective TBI intervention could be developed around a post-translational target occurring within an opportune treatment window as guided by temporal proteomics. Of interest were delayed-onset processes best managed after patient stabilization on the intensive care unit, focusing on events initiated a day or more after insult, out to two weeks following controlled cortical impact injury. We identified a subset of protein changes with the temporal profile of interest utilizing a self-organizing map approach. Enriched in this map were post-translational processes tied with ionic dysregulation, among which was a highly dynamic processing of neuron-specific K+-Cl− cotransporter 2 (KCC2), an essential component for maintaining chloride homeostasis that is critical to inhibitory neurotransmission. We identified a potential therapeutic window of opportunity starting on day 1 preceding unique acetylation, phosphorylation and ubiquitination events guiding the functional loss of KCC2. To test this window, we administered the KCC2-targeting compound CLP290 daily (50 mg/kg, p.o.) before, at, and after the identified 1-day point of KCC2 post-translational processing. The therapy was most effective at 1-day, preserving plasmalemmal KCC2 within perilesional somatosensory neocortex needed to maintain chloride homeostasis and effective inhibitory neurotransmission. Furthermore, TBI-impacted sensorimotor integration improved significantly with the 1-day intervention on rotarod and whisker adhesive removal task assessments. Together, our findings demonstrate the therapeutic targeting of post-translational processes revealed with temporal proteomics to effectively preserve KCC2-mediated chloride homeostasis with improved functional recovery. Furthermore, the approach defines an effective administration window that can be extended for the intervention of other post-translational events. Lastly, KCC2-targeted therapy may be extended to other neurological insults known to involve chloride dysregulation such as epilepsy and stroke.
Supported by NINDS NIH 1F31NS100322-01, Department of Anatomy and Neurobiology, and VCU Neuroscience PhD Program.
Keywords: KCC2, Proteomics, Inhibition, CCI, Chloride, Post-translational
University of Pennsylvania, Neurosurgery & Penn Center for Brain Injury and Repair, Philadelphia, USA
Mild traumatic brain injury (mTBI), or concussion, is a substantial public health problem that can result in persistent and debilitating symptoms in a subset of patients. Moreover, TBI, including repetitive concussion, has been linked to syndromes of progressive neurodegeneration such as chronic traumatic encephalopathy (CTE). However, the biology of mTBI and its link to late neurodegeneration remain poorly understood. A prominent feature of moderate or severe TBI at acute and late survival time-points is widespread disruption in blood brain barrier (BBB) integrity. However, it is unknown whether a single mTBI alone is sufficient to precipitate altered BBB integrity. Using an established swine model of pure rotational mTBI, animals were subjected to single mTBI or sham conditions (n = 3-4 per group) and tissue was prepared for immunohistochemical analyses at 6–72 hours post-injury. Following exposure to mTBI, widespread, abnormal extravasation of blood-borne proteins fibrinogen (FBG) and immunoglobulin-G (IgG) into the brain parenchyma was identified at all time points post-injury, despite an absence of hemorrhage. Typically, this appeared in a stereotyped distribution at structural interfaces, indicating a biomechanical etiology; although, notably, there was incomplete regional overlap between evidence of BBB disruption and axonal pathology. Triple immunofluorescence labeling revealed perivascular cellular uptake of both FBG and IgG in astrocytes (GFAP-positive), but not microglia (Iba1-positive) at these acute post-injury time-points. These data indicate that widespread BBB disruption can be demonstrated as a component of just a single mTBI using this swine model of pure rotational injury. The role of mTBI-induced BBB disruption in the acute presentation and outcomes of concussion, as well as its potential contribution to the late neurodegenerative pathologies of CTE, will be important to examine. Grant Support: DOD: PT110785, NIH: NS056202 and NS038104, The McCabe Fund (Univ. Pennsylvania).
Keywords: TBI model
University of Maryland, Baltimore, Department of Anesthesiology & Center for Shock, Trauma and Anesthesiology Research (STAR), Baltimore, USA
The autophagy-lysosomal pathway plays an essential role in cellular homeostasis and a protective function against a variety of diseases. However, under certain circumstances pathologically increased autophagy can contribute to cell death. This may occur particularly when lysosomal function is impaired and autophagic degradation is not able to proceed to completion, leading to pathological accumulation of dysfunctional autophagosomes. We have previously shown that autophagy is inhibited and contributed to injury after SCI. Here we examine mechanism of autophagy and lysosomal defects following SCI. Expression levels and processing of the lysosomal enzyme cathepsin D (CTSD) were decreased at 2 h after SCI. Enzymatic activity of CTSD and another lysosomal enzyme, alkaline phosphatase, were decreased 24 h post-injury, indicating lysosomal damage. Sub-cellular fractionation confirmed lysosomal membrane permeabilization (LMP) and leakage of lysosomal content into the cytosol. cPLA2 is an enzyme that cleaves fatty acyl linkage in the phospholipids of cellular membranes and increased activity of cPLA2 may be involved in membrane damage. cPLA2 was activated in the lysosomal fraction, accompanied by increased accumulation of the autophagosomal marker LC3-II and its substrate p62. To directly assess the extent and mechanism of damage to lysosomal membranes, mass spectrometry (MS)-based lipidomics was applied to compare the lipid composition of lysosomal membranes purified from sham or injured spinal cord at 2 h post-injury. Our data demonstrate increases in several classes of lysosophospholipids- the products of phospholipases (PLAs), as well as accumulation of PLA activator, ceramide. Inhibition of cPLA2 decreased lysosomal damage, restored autophagic flux, and reduced neuronal cell damage. Taken together our data implicate lysosomal defects in the pathophysiology of SCI and further indicate that cPLA2 activation leads to lysosomal damage that causes neuronal autophagosome accumulation associated with neuronal cell death.
Keywords: spinal cord injury, autophagy, lysosomal damage, cPLA2
Boston University, College of Engineering, Boston, USA
The mechanisms underpinning concussion, traumatic brain injury (TBI), and chronic traumatic encephalopathy (CTE), and the relationships between these disorders, are poorly understood. To investigate causal mechanisms, we developed a mouse model of closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of impaired arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioral deficits resembling human concussion. Acute neuropathological neuroexamination of brains from impact-injured mice demonstrated acute and chronic neuropathologies, including phosphorylated tau proteinopathy, that recapitulates early CTE pathologies in humans. Impact-induced CTE brain pathologies are also similar to brain pathology in blast-exposed mice and humans. Moreover, acute neurobehavioral signs of concussion were observed only after impact injury but not after blast exposure under conditions matched for head kinematics. Experimental and computational modeling showed that impact generated ipsilateral point loading on the head and seven-fold greater peak shear stress in the brain compared to blast. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that fast-acting, high-amplitude cortical shear stress triggers experimental concussion, whereas longer duration, lower amplitude shear stress associated with head motion induces structural brain damage and neuropathological sequelae. These results suggest that closed-head impact injuries, independent of concussive signs, can induce TBI as well as early pathologies and functional sequelae associated with CTE. These findings elucidate the origins of concussion and differentiate this condition from TBI and sequelae.
Funding: NIH, DoD, WWE, Concussion Legacy Foundation, private foundation.
Keywords: Impact, Chronic Traumatic Encephalopathy, Modeling, Blast, Neurobehavior
Clinical management guidelines for sport-related concussion (SRC) emphasize initial rest followed by a gradual return to activities. How to operationalize “rest,” however, is unclear, because the relationship between activity and SRC recovery is complex and dynamic. The aim of this pilot study was to develop and implement procedures for tracking activities after SRC and to explore associations between activities and recovery. High school and collegiate football players (25 concussed, 15 uninjured contact-controls) enrolled within 48 hours of injury and provided data daily for two weeks through a survey embedded into a smartphone application and a commercial fitness tracker (Fitbit). Metrics collected included magnitude and duration of physical activities, mental activities, sleep, and recovery. Completion rate of the daily data was 85%. As expected, concussed athletes reduced their activity levels (relative to controls) acutely after injury and gradually increased them to normal over the first two weeks post-injury, with somewhat different metrics for the Fitbit and mobile survey showing this pattern. Furthermore, there was significant variability across athletes in activities and recovery rates. For example, about 1/3 of athletes endorsed doing an activity that they believed would not be approved by their athletic trainers or doctors in the first week, and 3/4 avoided an activity that they believed could harm their recoveries. Endorsement rates of activity-related symptom exacerbation were extremely high in the first several days post-injury and diminished over time. However, endorsement of an unapproved activity or activity-related symptom exacerbation was not associated with recovery time. The results support the use of these technologies to remotely track athletes' activities and suggest that activity that follows a graded exertion protocol or athletes' judgment may be safe for clinical recovery after SRC. Future studies will evaluate the subset of athletes (nearly 1/3) who engaged in vigorous activity while symptomatic and will gather more information about the types of avoided and unapproved activities.
Keywords: concussion, sport, activity
PROLONGED KING-DEVICK TIME IN CONCUSSION PATIENTS
The King-Devick (KD) test is a vision-based rapid number-naming task used to detect impairment in concussed individuals, primarily assed on the sideline assessment. We tested the following hypotheses: (1) acute and chronic concussion will affect total KD time and (2) KD time will correlate with symptom recovery. KD was administered as recommended to three groups: High school student athletes (13–18 years, 234 females, 149 males) at preseason baseline evaluations during the 2016–2017 school year, patients diagnosed with an acute concussion ≤28 days (12–18 years, 21 females, 29 males) and patients diagnosed with a chronic concussion >28 days (12–18 years, 26 females, 33 males). [GC1] Each participant completed Graded Symptom Checklist (GSC) providing a total score and four symptom subtypes: somatic, cognitive, sleep and emotional. In hypothesis 1, we examined group differences and found group (baseline, acute and chronic) had a significant effect on KD time (F2, 489 = 18.91, p < 0.001). KD time was significantly greater in the acute group than both the baseline and chronic groups (p < 0.01). Though not statistically significant, KD time was greater in the chronic group compared baseline (p = 0.06) [GC2]. In hypothesis 2, we evaluated the robust correlation between GSC total, GSC subtype and KD time in each group. No significant correlations were reported in the baseline or chronic groups (rpb = [GC3] 0.04 and 0.08). KD time. In the acute group, GSC total and KD were significantly correlated (rpb = 0.51). Somatic and cognitive symptom subsets were most strongly correlated to KD (rpb = 0.89 and 0.80). Slower KD times in the acute concussion group show a possible injury-related deficit in KD performance that correlates with concussion symptoms (GSC). Potentially, KD can be an objective measure of concussion and/or concussion related symptoms in the acute clinic setting. GSC and KD did not correlate in the chronic group, suggesting persistent symptoms may be due to other factors. High variability of KD time in all three groups supports the need for an individual baseline.
Supported by: UCLA BIRC, UCLA Steve Tisch BrainSPORT Program
Keywords: King-Devick, Vision, Clinical Setting, Recovery
ACTIVATION OF CANNABINOID RECEPTOR 2 ATTENUATES TRAUMATIC BRAIN INJURY-INDUCED INFLAMMATION
Augusta University, Neurosurgery, Augusta, USA
Inflammation is an important component of secondary neurological injury after TBI. Immune cells, particularly macrophages, aid in clearance of cellular debris and tissue repair, but sustained release of pro-inflammatory mediators from infiltrating immune cells may exacerbate neuronal death, increase neurovascular injury, and contribute to long-term loss of white matter. Although the precise mechanisms underlying the dual beneficial and detrimental roles of macrophages after CNS injury remain poorly defined, it has been well established that macrophages polarize along a continuum from a classical pro-inflammatory (M1) state to an alternative anti-inflammatory (M2) state. Thus, modulation of macrophage polarization may identify novel opportunities for therapeutic intervention after TBI. Non-psychoactive cannabinoid receptor 2 (CB2R) is predominantly expressed on immune (lymphocytes, monocytes, macrophages) and endothelial cells in both rodents and humans. In the present study, we hypothesize that activation of CB2R attenuates inflammation post-TBI. Using a moderate controlled cortical impact (CCI) model of murine TBI, we assessed the endogenous upregulation of CB2R after TBI and the effects of CB2R agonist and antagonist post-injury. We observed acute upregulation of CB2R up to 3 days post TBI and moderate elevated expression persisted at 3 weeks. Furthermore, CB2R co-localized with infiltrated myeloid cells, specifically macrophages, after TBI. Finally, administration of selective CB2R agonist GP1a attenuated neuroinflammation, while treatment with antagonist AM630 worsened the injury. Interestingly, attenuation of inflammation by GP1a was associated with increased M2 polarization of macrophages. Our findings suggest that CB2R may play an important role in immune cell regulation; therefore, the development of selective CB2R agonists may provide clinical benefits for brain injury patients, without the psychoactive effects of CB1R activation.
Keywords: traumatic brain injury (TBI), cannabinoid receptor 2 (CB2), inflammation, macrophage
Medical College of Georgia - Augusta University, Neuroscience and Regenerative Medicine, Augusta, USA
Traumatic brain injury (TBI) contributes to over 30% of injury related deaths. A complex secondary injury cascade follows the initial mechanical injury and exacerbates the primary injury. Oxidative stress and inflammation play key roles in this TBI pathology. NADPH oxidase-2 (NOX2) and inflammasomes have been reported as major contributors to oxidative stress and inflammation in the injured brain, respectively, and deletion of NOX2 is neuroprotective. In particular, the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome can become activated in response to oxidative stress, but little is known about this mechanism following TBI. In this study we utilize NOX2 knockout mice to study the role of NOX2 in mediating NLRP3 inflammasome expression and activation in a controlled cortical impact model of focal TBI. Expression of NLRP3 inflammasome components (NLRP3, apoptosis speck-like protein containing a CARD (ASC), and caspase-1) was robustly increased in the injured cerebral cortex following TBI. Deletion of NOX2 attenuated the expression and assembly of the NLRP3 inflammasome components, which was coupled to reduced cleavage of the pro-inflammatory factor, interleukin-1β (IL-1β). Further work revealed that NOX2 regulation of NLRP3 inflammasome may be mediated by thioredoxin interacting protein (TXNIP), a sensor of oxidative stress. In conclusion, the results of the current study provide novel evidence that NOX2-dependent inflammasome activation contributes to TBI pathology.
Keywords: NADPH Oxidase 2, NLRP3, Inflammasome, Traumatic Brain Injury, Oxidative Stress, Controlled Cortical Impact
Virginia Commonwealth University, Pharmacology & Toxicology, Richmond, USA
The endogenous cannabinoid 2-arachidonyl glycerol (2-AG) serves as a rate-limiting precursor for arachidonic acid (AA), a precursor for the production of pro-inflammatory eicosanoids in brain. In vitro work shows that inhibiting the 2-AG biosynthetic enzyme diacylglyercol lipase-β (DAGL-β), which is highly expressed on CNS resident microglia and macrophages in the periphery, reduces inflammatory responses. Here, we examine whether DAGL-β−/− mice display a protection phenotype from the consequences of experimental traumatic brain injury. Experiment 1: Male DAGL-β−/− and +/+ mice were subjected to a left lateral moderate Fluid Percussion injury (FPI) (1.94 ± 0.1 atm), and then assessed for spatial memory performance in a Morris water maze Fixed Platform task as well as for neurological motor impairments using the Neurological Severity Score (NSS) and the Rotarod assay. Unexpectedly, DAGL-β−/− mice demonstrated a significant survival protective phenotype (100% survival) in response to brain injury compared to DAGL-β+/+ mice (77% survival), despite equal injury severities between groups. However, DAGL-β−/− mice displayed similar magnitudes of FPI-induced cognitive impairments in the Fixed Platform task and neurological motor deficits in the NSS and Rotarod assays as DAGL-β+/+ mice. Experiment 2: We increased the magnitude of FPI to 2.0 ± 0.1 atm and 2.17 ± 0.1 atm in male and female DAGL-β−/− and +/+ mice, and assessed mortality. Male DAGL-β−/− mice demonstrated a significant survival protective phenotype at both 2.0 atm (100% survival) and 2.17 atm (90% survival) compared to DAGL-β+/+ mice (75% survival at 2.0 atm, and 60% survival at 2.0 atm). In contrast, female DAGL-β mice generally survived both injury magnitudes regardless of phenotype, suggesting sex differences in survival from FPI. These findings suggest the provocative possibility that DAGL-β activity contributes to TBI-induced mortality, but not to the evolution of TBI cognitive or motor impairments in mice. Accordingly, DAGL-β inhibition represents a potential strategy to ameliorate post-traumatic fatality.
Keywords: Endogenous Cannabinoid, Mortality, Diacylglycerol lipase-β, 2-arachidonylglycerol (2-AG), Spatial Memory, Sex Differences
Temple University School of Medicine, Dept. of Neurology, Philadelphia, USA
Axons in adult mammalian CNS fail to regenerate and there are no effective regenerative strategies to treat patients with CNS injuries. Several genes, including PTEN/mTor, Kruppel-like factors and insulin/insulin-like growth factor 1 receptor, partly regulate axon regeneration failure in adult CNS of mammals. Here we report the crucial role of liver kinase B1 (LKB1) in mediating CNS regeneration after CNS injury. LKB1 is a key serine/threonine kinase required for maintaining cell metabolism, energy homeostasis and cell polarity by activating a number of kinases. As the downstream effector of cAMP/PKA and PI3 kinase pathways, LKB1 is a major determinant for migration and differentiation of various cells, including neurons. We thus evaluate the role of LKB1 in regulating regenerative capacity of neurons in adult mammals using transgenic and adeno-associated virus (AAV) vector approaches. Upregulation of LKB1 significantly enhanced neurite extension of adult neurons cultured on inhibitory substrates CNS myelin and aggrecan. Transgenic overexpression of LKB1 stimulates robust regeneration of corticospinal tract (CST) axons in adult mice with mid-thoracic spinal cord injury. Local injections of AAV2-LKB1 into sensorimotor cortex or systemic application of mutant AAV9-LKB1 promote long distance regeneration of injured CST axons into the caudal spinal cord in adult mice. Systemic injection of mutant AAV9-LKB1 also enhances regrowth of descending serotoninergic and tyrosine hydroxylase fibers. Importantly, LKB1 upregulation by either transgenic or viral approaches improves recovery of locomotor function after CNS injury. Therefore, LKB1 is critical for regulating growth capacity of mature neurons and may become an important target for designing highly effective therapies for CNS injury.
Keywords: Liver kinase B1, spinal cord injury, axon regeneration, Functional recovery, growth capacity
University of Texas Southwestern Medical Center, Molecular Biology, Dallas, USA
Spinal cord injury (SCI) results in irreversible loss of neurons and disruption of neural circuits, with a consequence of motor and sensory dysfunction. The goal of the present study is to regenerate functional neurons after SCI. A key SCI-induced pathological feature is the formation of glial scars surrounding the injury site. NG2 glia constitute a major component of the glial scars and their presence at the injury sites is inhibitory for axon regrowth. Reprogramming these cells into functional neurons may provide a regeneration-based therapeutic strategy for SCI repair. We established a lentiviral approach to specifically target endogenous NG2 glia by using a human NG2 promoter. SCI was introduced by contusion in the adult mice. Our preliminary data showed that neurogenesis can be induced through injection of SOX2-expressing lentivirus under this promoter in both healthy and injured spinal cord. Immunohistochemistry revealed that induced DCX+ new neurons pass through an ASCL1+ neural progenitor and proliferation phase. Through genetic lineage mappings, we confirmed parenchymal NG2 glia as a cellular source for the newly reprogrammed neurons. We also excluded nestin+ neural stem cells or FOXJ1+ ependymal cells along the central canal as a cellular origin. Importantly, these NG2 glia-derived new neuron can mature into synapse-forming cells in vivo. Neuronal maturation can be further promoted by neurotrophic factors. Our results demonstrate that reactive NG2 glia can be robustly reprogrammed into neurons in situ, opening up the possibility of using endogenous cells for spinal cord regeneration after injury.
Keywords: in vivo reprogramming, neurogenesis, spinal cord injury, NG2 glia
Scientific Lectures
University of Utah, Neurosurgery, Salt Lake City, USA
Brain tissue hypoxia is common after traumatic brain injury (TBI). Technology exists to detect hypoxia and guide corrective therapy. Current guidelines for the management of severe TBI recommend maintaining PbtO2 > 15-20 mmHg, however uncertainty persists as to the optimal treatment threshold. PbtO2 measures were prospectively and automatically collected every minute from consecutive patients admitted to the San Francisco General Hospital (SFGH) intensive care unit over a 6-year period. We analyzed mean PbtO2 values in TBI patients and the proportion of PbtO2 values below each of 75 thresholds between 0 mmHg to 75 mmHg over various epochs up to 30 days from time of admission. Patient outcomes were calculated using the Glasgow Outcome Scale. We explored putative treatment thresholds by generating 675 separate receiver operator curves (ROC) and 675 generalized linear models (GLM) to examine each 1 mmHg threshold for various epochs. A total of 1,380,841 PbtO2 values were recorded from 190 patients recovering from TBI. A high proportion of PbtO2 measures were below 20 mmHg irrespective of examined epoch. Time below treatment thresholds was more strongly associated with outcome than mean PbtO2. A treatment window was suggested; a threshold of 19 mmHg most robustly distinguished patients by outcome, especially from days 3–5. Benefit to maintaining values at least as high as 33 mmHg was suggested, however. Our “big data” analysis substantially informs the relationship between PbtO2 values and outcome. We were able to discern a therapeutic window for PbtO2 in TBI patients along with minimum and preferred PbtO2 treatment thresholds. Traditional treatment thresholds which have the strongest association with outcome may not be optimal.
Keywords: Brain oxygenation, PbtO2, Outcome, Threshold, Treatment window
Traumatic brain injury (TBI) which is a leading cause of death and disability is a significant public health problem worldwide. The causes of TBI are road traffic accidents, falls, violence, war accidents and sports-related head injuries including chronic repetitive head trauma seen in boxers and kickboxers. TBI is recognized as one of the most common causes of hypopituitarism. The pooled prevalence rate of any hormone deficiency is reported as 28%. GH is the most commonly lost hormone after TBI. The natural history of hypopituitarism after TBI is not well known. Pituitary function improves over time in a considerable number of patients, but it may also worsen rarely over the 1- and 3-year period after TBI. The mechanisms underlying the recovery and worsening of pituitary function have not been clarified yet. Anti-pituitary antibodies (APAs) are found in 50% of the TBI victims 3 years after TBI. The pituitary dysfunction development ratio is significantly higher in APA-positive patients when compared with APA-negative ones. According to the current data, a substantial number of patients with TBI develop permanent hypopituitarism, GH deficiency in particular, at the 5th year after TBI. There are strong associations between the presence of high titres of APAs and/or AHAs and hypopituitarism at the fifth year after TBI. Recent data demonstrated that sports-related chronic repetitive trauma including boxing and kickboxing may also result in pituitary dysfunction. Both APAs and AHAs are detected in boxers, but only AHA positive boxers may develop pituitary dysfunction. It seems that hypothalamic damage might be more important than pituitary damage in athletes exposed to chronic repetitive head trauma. Pituitary volume in retired boxers with GH deficiency has been found to be smaller than that in GH-sufficient boxers. Pituitary autoimmunity may be responsible for the shrinkage of pituitary volume over the years following TBI. Therefore, autoimmunity may play a role in the pathogenesis of TBI-induced pituitary dysfunction.
Keywords: Traumatic brain injury, hypopituitarism, anti-pituitary antibodies, anti-hypothalamus antibodies
GLUTAMATE RECEPTOR PEPTIDES AND AUTOANTIBODIES IN TBI AND ISCHEMIC BRAIN INJURIES
Brain impairments of traumatic and ischemic origin are connected to heterogenous profile of cerebral blood flow (CBF) deficit and neurovascular unit dysfunctions in cortical (grey matter), subcortical (white matter) and brainstem (cervical area). Ionotropic glutamate receptor (GluR) peptides are proposed as early indicators of vascular vasospasm associated with neurotoxicity and might serve as biomarkers of primary injury. It is hypothesized that neurotoxicity cascade after impact/onset is affecting to neuroplasticity and immune response in certain anatomical locations as well.
Our translational research of GluR biomarkers for concussions and mild TBI are presented in conjunction with neurological and radiological assessments. It was shown that ionotropic AMPAR subtypes are related to white matter denrite-axonal shearing and disconnections causing the cognitive deficit and behavior changes after sport-related injury. Ionotropic kainate receptors are susceptible to carbon dioxide balance that potentially affects venous circulation in brainstem influencing involuntary life-sustaining functions and unconsciousness. At the same time NMDAR subtypes are involved in delayed cerebral ischemia and mental status decline.
Autoantibodies to AMPA, NMDA, and kainite receptor are neuroimmune tracers associated with severity of brain impairments radiologically defined in cortical, subcortical, and brainstem areas. The risk of delayed edema formation and microlesions development is associated with secondary injury and neurological complications.
Brain-derived biomarkers (GluR peptides and autoantibodies) detected in biological fluids by immunoassays may aid in stratification of the mild TBI/TIA/stroke assessments and in selection persons for advanced neuroimaging. Sideline or bedside testing of these biomarkers would assist to clinicians in triaging individuals with microstructural injuries for emergent therapeutic interventions.
Keywords: Translational research, Advanced neuroimaging, Neurological assessment
Pain prevalence is particularly high in acute/subacute stage after a nervous system injury, and therefore a large proportion of patients have to undergo their motor rehabilitation in the context of pain. Surprisingly, very limited attention has been devoted to understanding the potential influence of pain on motor learning capacity and rehabilitation outcomes, despite a large body of evidence in animal models suggesting that central sensitization occurring in nociceptive pathways and plasticity related to motor learning interact with each other. In a series of recent studies, we assessed the effect of experimental nociceptive stimuli on the acquisition and next-day retention of a new locomotor task. Participants performed a locomotor adaptation task (perturbing force field applied to the ankle during swing using a robotized orthosis) on two consecutive days. Motor performance and motor strategies were assessed using kinematic measures and EMG activity. Across the different studies and experimental sessions, participants were either tested pain-free, or with cutaneous pain (topical application of capsaicin cream around the ankle). Pain had no significant effect on baseline gait parameters nor on motor performance (i.e. movement error reduction) on Day 1, but resulted in a different motor strategy (less anticipatory). A pain-free test performed on Day 2 showed that cutaneous pain can greatly interfere with retention, despite apparently normal performance during the acquisition. This finding appears to be explained more by the change in context between the motor acquisition and the retention test (i.e. pain vs. no pain) than to a direct interference of pain with the consolidation processes, as no retention deficit was observed when a similar pain was present on both days. The potential implications of these results for neurorehabilitation will be discussed.
Keywords: motor learning, nociception, locomotion, spinal cord injury
Children's Hospital of Philadelphia, Philadelphia, USA
CMX-2043 (Ischemix) is a plasma stable Lipoic Acid-based small molecule that activates the Akt survival pathway through the PI3-Kinase. Additionally, CMX-2043 is a direct anti-oxidant with the added ability to upregulate endogenous antioxidants. Proof of concept trial for CMX-2043 in a porcine model of focal controlled cortical impact (CCI) traumatic brain injury (TBI) to determine effect on the mitochondrial electron transport chain (ETC), oxidative phosphorylation (OXPHOS), and mitochondrial reactive oxygen species (mtROS) generation. Piglets (N = 16), 4-wk old approximately 8–9 kg, were randomized to 3 groups: Sham (N = 5), CCI+Placebo (N = 6), CCI+CMX-2043 (N = 5). Treated animals received an intravenous loading, bolus of CMX-2043 (13.5 mg/kg) 1 hour post-CCI, and a 4.5 mg/kg bolus at 13 hours. At 24 hours post-TBI, fresh brain tissue was harvested and homogenized from the ipsilateral cortex (IPSI, penumbra zone) and mirrored contralateral region (CONTRA), and from corresponding cortex of shams. Real-time analysis of ETC function and simultaneous, mtROS was measured using a high resolution respirometry system (Oroboros) with an integrated fluorometer. mtROS generated with complex I (CI) and complex II (CII) substrates and the overall mitochondrial control ratio ([OXPHOS CI+CII]/[LEAKCI+CII]), during Oligomycin-induced respiration independent of ATP production were measured and compared across groups (ANOVA). The control ratio significantly decreased in both regions of CCI+placebo animals (IPSI: 6.0 ± 0.26, p < 0.0001; CONTRA: 6.6 ± 0.32, p < 0.0001) compared to sham cortex (19.44 ± 1.37). Treatment significantly improved the IPSI control ratio (CMX-2043 11.61 ± 2.28, p < 0.05), but not the CONTRA (11.05 ± 1.71) compared to CCI+placebo. Treatment did not restore IPSI mitochondrial control ratio to sham levels. mtROS generation (in units of H2O2/(ρmol O2/s*mg) was significantly increased in CCI+placebo on both the IPSI (18.77 ± 1.58, p < 0.0001) and CONTRA (13.7 ± 0.58, p < 0.0001) sides compared to sham (2.96 ± 0.19), with IPSI generation significantly greater than CONTRA. Treatment significantly lowered mtROS production on both sides (IPSI: 8.28 ± 1.47, p < 0.0001; CONTRA: 5.13 ± 1.67, p < 0.0001versus CCI+placebo) to sham levels. CMX-2043 improves mitochondrial bioenergetics and limits mtROS generation following TBI.
Keywords: mitochondria, reactive oxygen species, Preclinical Trial, porcine
MGH-Harvard, Neuroprotection Research Laboratory, Charlestown, USA
Recently, it was suggested that neurons can release and transfer damaged mitochondria to astrocytes for disposal and recycling1. Here, we show that astrocytes can also release functional mitochondria that enter into neurons. Astrocytic release of extracellular mitochondria particles was mediated by a calcium-dependent mechanism involving CD38/cyclic ADP ribose signaling. Transient focal cerebral ischemia in mice induced astrocytic mitochondria entry to adjacent neurons that amplified cell survival signals. Suppression of CD38 signaling with siRNA reduced extracellular mitochondria transfer and worsened neurological outcomes. These findings suggest a new mitochondrial mechanism of neuroglial crosstalk that may contribute to endogenous neuroprotective and neurorecovery mechanisms after stroke.
Keywords: CD38, Extracellular mitochondria, ATP, Mitochondrial transfer
Indiana University School of Medicine, Anesthesia, Indianapolis, USA
Keywords: DAMPs, MODS, polytrauma, extracranial
Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Unfavorable TBI outcomes result from primary mechanical injuries to the brain and ensuing secondary non-mechanical injuries that are not limited to the brain. To investigate the mechanisms underlying TBI pathologies, we developed a model of TBI in Drosophila melanogaster. Closed head TBI is inflicted with a mechanical device that subjects flies to rapid acceleration and deceleration. Similar to humans with TBI, flies with TBI exhibit temporary incapacitation, ataxia, activation of the innate immune response, neurodegeneration, and death. We demonstrate that age and diet activate distinct secondary injuries in a genotype-specific manner, and that concurrent activation of age- and diet-regulated secondary injuries synergistically increase mortality. Our Genome-wide Association study revealed that the probability of death following TBI is associated with single nucleotide polymorphisms in genes involved in tissue barrier function and glucose homeostasis. We found that TBI causes intestinal and blood-brain barrier dysfunction and that intestinal barrier dysfunction is highly correlated with the probability of death. Furthermore, we found that ingestion of glucose after a primary injury increases the probability of death through a secondary injury mechanism that exacerbates intestinal barrier dysfunction. Our results indicate that natural variation in the probability of death following TBI is due in part to genetic differences that affect intestinal barrier dysfunction.
Keywords: Intestine, Drosophila, GWAS, Neurodegeneration, Diet, Blood-brain barrier
Keywords: Parkinson's disease, Lewy bodies, Alzheimer's disease, dementia, Alzheimer's pathology
Cerebral edema represents a major predictor for the patient outcome after several brain injuries. After the first description of water channel proteins, aquaporins (AQPs), in the brain led to intense research in understanding the underlying roles of each of them in the edema process. Here, the recent knowledge on the 3 main AQPs (AQP1, AQP4 and AQP9) will be reviewed with a look on the edema in TBI, SCI and stroke. Despite an evident crucial role of AQP4 in edema process, no specific therapeutic agents have been developed to either inhibit or enhance water flux through these channels.
Keywords: astrocyte, aquaporin, TBI, Edema
NHS GGC, Dept. of Neuropathology, Glasgow, United Kingdom
Few issues in research can have attracted as much attention as the association between traumatic brain injury and late neurodegenerative disease. Hollywood movies, endless news articles, billion-dollar litigation and polarised opinions in sport and research have contributed to the, quite literally, game-changing phenomenon that is currently discussed as chronic traumatic encephalopathy (CTE).
However, despite this attention, current understanding of the neuropathology of CTE is based on observations on remarkably few cases; reflecting the many challenges in research in human TBI neuropathology, not least of which is case accrual from autopsy. Perhaps a consequence of the rarity of access to human tissues and experience in their interpretation, there are inevitable limitations in insight into the complex neuropathology of late survival from TBI.
Through a combination of pre-circulated case material, directed reading and case-based discussions this short interactive workshop is designed to provide attendees with insight into current understanding of the neuropathology of late survival from TBI in humans and the continued challenges to research in this field.
In advance of the workshop attendees will be provided with a link to access scanned tissues sections from exemplar cases, which will provide the context for discussions at NNS.
Hay, J et al (2016). Chronic traumatic encephalopathy: the neuropathological legacy of traumatic brain injury. Ann Rev Pathol, 11, 21–45.
McKee, A, et al (2016). The first NINDS/NIBIB consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. Acta Neuropathol, 131, 75–86.
Keywords: chronic traumatic encephalopathy, neuropathology, human studies, diffuse axonal injury, neuroinflammation, blood-brain barrier
Multiple factors contribute to the challenge of reproducibility in preclinical research including, but not limited to, inconsistent naming conventions of relevant experimental factors. The goal of the TBI Preclinical Working Group effort was to identify experimental factors, variables, and conditions (data elements) that are critical for describing and thus reproducing the most frequently used preclinical outcome measures. Importantly, this effort is not meant to standardize methodology or limit innovation but rather to create a dictionary of common data elements for preclinical outcome measures with the goal of enhancing intra- and inter-laboratory data harmonization. The long-term goal of the TBI Preclinical Working Groups is to address the translational challenge in TBI through rigor and methodological transparency allowing for improved and accelerated therapeutic development that ultimately improve outcomes for TBI patients. The general working group has divided into Sub-Working Groups to create common data elements (CDEs) associated with: (1) General Health and Affective Disturbance tests, (2) Cognition and Motor tests, or (3) Large Animal Models. The resulting CDEs will be available for public comment and will eventually be accessible within the Federal Interagency Traumatic Brain Injury Research (FITBIR) Informatics System platform. These meetings explored the relationship between clinical phenotypes of TBI and animal models, discussing what constitutes useful translational model systems and CDEs for data standardization, particularly in the context of Research and Development decision making.
Keywords: Preclinical, Data, Common Data Elements, Traumatic Brain Injury