Penetrating Ballistic Brain Injury Produces Acute Alterations in Sleep and Circadian-Related Genes in the Rodent Cortex: A Preliminary Study.

Traumatic brain injury (TBI) affects millions of Americans each year, with extremely high prevalence in the Veteran community, and sleep disturbance is one of the most commonly reported symptoms. Reduction in the quality and amount of sleep can negatively impact recovery and result in a wide range of behavioral and physiological symptoms, such as impaired cognition, mood and anxiety disorders, and cardiovascular effects. Thus, to improve long-term patient outcomes and develop novel treatments, it is essential to understand the molecular mechanisms involved in sleep disturbance following TBI. In this effort, we performed transcriptional profiling in an established rodent model of penetrating ballistic brain injury (PBBI) in conjunction with continuous sleep/wake EEG/EMG recording of the first 24 h after injury. Rats subjected to PBBI showed profound differences in sleep architecture. Injured animals spent significantly more time in slow wave sleep and less time in REM sleep compared to sham control animals. To identify PBBI-related transcriptional differences, we then performed transcriptome-wide gene expression profiling at 24 h post-injury, which identified a vast array of immune- related genes differentially expressed in the injured cortex as well as sleep-related genes. Further, transcriptional changes associated with total time spent in various sleep stages were identified. Such molecular changes may underlie the pathology and symptoms that emerge following TBI, including neurodegeneration, sleep disturbance, and mood disorders.

Differential Effects of Caffeine on Motor and Cognitive Outcomes of Penetrating Ballistic-Like Brain Injury.

This study assessed the effect of caffeine on neurobehavioral recovery in the WRAIR penetrating ballistic-like brain injury (PBBI) model. Unilateral frontal PBBI was produced in the right hemisphere of anesthetized rats at moderate (7%-PBBI) or severe (10%-PBBI) injury levels. Animals were randomly assigned to pretreatment groups: acute caffeine (25 mg/kg CAF gavage, 1 h prior to PBBI), or chronic caffeine (0.25 g/L CAF drinking water, 30 days prior to PBBI). Motor function was evaluated on the rotarod at fixed-speed increments of 10, 15, and 20 RPM. Cognitive performance was evaluated on the Morris water maze. Acute caffeine showed no significant treatment effect on motor or cognitive outcome. Acute caffeine exposure prior to 10%-PBBI resulted in a significantly higher thigmotaxic response compared to vehicle-PBBI groups, which may indicate 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. However, chronic caffeine exposure significantly increased the latency to locate the platform in the Morris water maze task at all injury levels. Results indicate that chronic caffeine consumption prior to a penetrating TBI may provide moderate beneficial effects to motor recovery, but may worsen the neurocognitive outcome.

Combination therapy of levetiracetam and gabapentin against nonconvulsive seizures induced by penetrating traumatic brain injury.

BACKGROUND: Posttraumatic seizures are a medical problem affecting patients with traumatic brain injury. Yet effective treatment is lacking owing to the limitations of antiepileptic drugs (AEDs) applicable to these patients. METHODS: In this study, we evaluated the dose-response efficacy of levetiracetam (12.5-100.0 mg/kg) and gabapentin (1.25-25.0 mg/kg) administered either individually or in pairs at fixed-dose ratios as a combination in mitigating posttraumatic nonconvulsive seizures induced by severe penetrating ballistic-like brain injury (PBBI) in rats. Seizures were detected by continuous electroencephalogram (EEG) monitoring for 72 hours postinjury. Animals were treated twice per day for 3 days by intravenous injections. RESULTS: Both levetiracetam (25-100 mg/kg) and gabapentin (6.25-25 mg/kg) significantly reduced PBBI-induced seizure frequency by 44% to 73% and 61% to 69%, and seizure duration by 45% to 64% and 70% to 78%, respectively. However, the two drugs manifested different dose-response profiles. Levetiracetam attenuated seizure activity in a dose-dependent fashion, whereas the beneficial effects of gabapentin plateaued across the three highest doses tested. Combined administration of levetiracetam and gabapentin mirrored the more classic dose-response profile of levetiracetam monotherapy. However, no additional benefit was derived from the addition of gabapentin. Furthermore, isobolographic analysis of the combination dose-response profile of levetiracetam and gabapentin failed to reach the expected level of additivity, suggesting an unlikelihood of favorable interactions between these two drugs against spontaneously occurring posttraumatic seizure activities at the particular set of dose ratios tested. CONCLUSION: This study was the first attempt to apply isobolographic approach to studying AED combination therapy in the context of spontaneously occurring posttraumatic seizures. Despite the failure to achieve additivity from levetiracetam and gabapentin combination, it is important to recognize the objectivity of the isobolographic approach in the evaluation of AED combination therapy against seizures directly associated with brain injuries.

Functional and Molecular Correlates after Single and Repeated Rat Closed-Head Concussion: Indices of Vulnerability after Brain Injury.

Closed-head concussive injury is one of the most common causes of traumatic brain injury (TBI). Isolated concussions frequently produce acute neurological impairments, and individuals typically recover spontaneously within a short time frame. In contrast, brain injuries resulting from multiple concussions can result in cumulative damage and elevated risk of developing chronic brain pathologies. Increased attention has focused on identification of diagnostic markers that can prognostically serve as indices of brain health after injury, revealing the temporal profile of vulnerability to a second insult. Such markers may demarcate adequate recovery periods before concussed patients can return to required activities. We developed a noninvasive closed-head impact model that captures the hallmark symptoms of concussion in the absence of gross tissue damage. Animals were subjected to single or repeated concussive impact and examined using a battery of neurological, vestibular, sensorimotor, and molecular metrics. A single concussion induced transient, but marked, acute neurological impairment, gait alterations, neuronal death, and increased glial fibrillary acidic protein (GFAP) expression in brain tissue. As expected, repeated concussions exacerbated sensorimotor dysfunction, prolonged gait abnormalities, induced neuroinflammation, and upregulated GFAP and tau. These animals also exhibited chronic functional neurological impairments with sustained astrogliosis and white matter thinning. Acute changes in molecular signatures correlated with behavioral impairments, whereas increased times to regaining consciousness and balance impairments were associated with higher GFAP and neuroinflammation. Overall, behavioral consequences of either single or repeated concussive impact injuries appeared to resolve more quickly than the underlying molecular, metabolic, and neuropathological abnormalities. This observation, which is supported by similar studies in other mTBI models, underscores the critical need to develop more objective prognostic measures for guiding return-to-play decisions.

Subacute Changes in Cleavage Processing of Amyloid Precursor Protein and Tau following Penetrating Traumatic Brain Injury.

Traumatic brain injury (TBI) is an established risk factor for the development of Alzheimer's disease (AD). Here the effects of severe penetrating TBI on APP and tau cleavage processing were investigated in a rodent model of penetrating ballistic-like brain injury (PBBI). PBBI was induced by stereotactically inserting a perforated steel probe through the right frontal cortex of the anesthetized rat and rapidly inflating/deflating the probe's elastic tubing into an elliptical shaped balloon to 10% of total rat brain volume causing temporary cavitation injury. Separate animals underwent probe injury (PrI) alone without balloon inflation. Shams underwent craniectomy. Brain tissue was collected acutely (4h, 24h, 3d) and subacutely (7d) post-injury and analyzed by immunoblot for full length APP (APP-FL) and APP beta c-terminal fragments (ßCTFs), full length tau (tau-FL) and tau truncation fragments and at 7d for cytotoxic Beta amyloid (Aß) peptides Aß40 and Aß42 analysis. APP-FL was significantly decreased at 3d and 7d following PBBI whereas APP ßCTFs were significantly elevated by 4h post-injury and remained elevated through 7d post-injury. Effects on ßCTFs were mirrored with PrI, albeit to a lesser extent. Aß40 and Aß42 were significantly elevated at 7d following PBBI and PrI. Tau-FL decreased substantially 3d and 7d post-PBBI and PrI. Importantly, a 22 kDa tau fragment (tau22), similar to that found in AD, was significantly elevated by 4h and remained elevated through 7d post-injury. Thus both APP and tau cleavage was dramatically altered in the acute and subacute periods post-injury. As cleavage of these proteins has also been implicated in AD, TBI pathology shown here may set the stage for the later development of AD or other tauopathies.

The Revised Neurobehavioral Severity Scale (NSS-R) for Rodents.

Motor and sensory deficits are common following traumatic brain injury (TBI). Although rodent models provide valuable insight into the biological and functional outcomes of TBI, the success of translational research is critically dependent upon proper selection of sensitive, reliable, and reproducible assessments. Published literature includes various observational scales designed to evaluate post-injury functionality; however, the heterogeneity in TBI location, severity, and symptomology can complicate behavioral assessments. The importance of choosing behavioral outcomes that can be reliably and objectively quantified in an efficient manner is becoming increasingly important. The Revised Neurobehavioral Severity Scale (NSS-R) is a continuous series of specific, sensitive, and standardized observational tests that evaluate balance, motor coordination, and sensorimotor reflexes in rodents. The tasks follow a specific order designed to minimize interference: balance, landing, tail raise, dragging, righting reflex, ear reflex, eye reflex, sound reflex, tail pinch, and hindpaw pinch. The NSS-R has proven to be a reliable method differentiating brain-injured rodents from non-brain-injured rodents across many brain injury models.

Neuroprotection and anti-seizure effects of levetiracetam in a rat model of penetrating ballistic-like brain injury.

PURPOSE: We assessed the therapeutic efficacy of FDA-approved anti-epileptic drug Levetiracetam (LEV) to reduce post-traumatic nonconvulsive seizure (NCS) activity and promote neurobehavioral recovery following 10% frontal penetrating ballistic-like brain injury (PBBI) in male Sprague-Dawley rats. METHODS: Experiment 1 anti-seizure study: 50 mg/kg LEV (25 mg/kg maintenance doses) was given twice daily for 3 days (LEV3D) following PBBI; outcome measures included seizures incidence, frequency, duration, and onset. Experiment 2 neuroprotection studies: 50 mg/kg LEV was given twice daily for either 3 (LEV3D) or 10 days (LEV10D) post-injury; outcome measures include motor (rotarod) and cognitive (water maze) functions. RESULTS: LEV3D treatment attenuated seizure activity with significant reductions in NCS incidence (54%), frequency, duration, and delayed latency to seizure onset compared to vehicle treatment. LEV3D treatment failed to improve cognitive or motor performance; however extending the dosing regimen through 10 days post-injury afforded significant neuroprotective benefit. Animals treated with the extended LEV10D dosing regimen showed a twofold improvement in rotarod task latency to fall as well as significantly improved spatial learning performance (24%) in the MWM task. CONCLUSIONS: These findings support the dual anti- seizure and neuroprotective role of LEV, but more importantly identify the importance of an extended dosing protocol which was specific to the therapeutic targets studied.

Synthesis of Findings, Current Investigations, and Future Directions: Operation Brain Trauma Therapy.

Operation Brain Trauma Therapy (OBTT) is a fully operational, rigorous, and productive multicenter, pre-clinical drug and circulating biomarker screening consortium for the field of traumatic brain injury (TBI). In this article, we synthesize the findings from the first five therapies tested by OBTT and discuss both the current work that is ongoing and potential future directions. Based on the results generated from the first five therapies tested within the exacting approach used by OBTT, four (nicotinamide, erythropoietin, cyclosporine A, and simvastatin) performed below or well below what was expected based on the published literature. OBTT has identified, however, the early post-TBI administration of levetiracetam as a promising agent and has advanced it to a gyrencephalic large animal model--fluid percussion injury in micropigs. The sixth and seventh therapies have just completed testing (glibenclamide and Kollidon VA 64), and an eighth drug (AER 271) is in testing. Incorporation of circulating brain injury biomarker assessments into these pre-clinical studies suggests considerable potential for diagnostic and theranostic utility of glial fibrillary acidic protein in pre-clinical studies. Given the failures in clinical translation of therapies in TBI, rigorous multicenter, pre-clinical approaches to therapeutic screening such as OBTT may be important for the ultimate translation of therapies to the human condition.

Simvastatin Treatment in Traumatic Brain Injury: Operation Brain Trauma Therapy.

Simvastatin, the fourth drug selected for testing by Operation Brain Trauma Therapy (OBTT), is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor used clinically to reduce serum cholesterol. In addition, simvastatin has demonstrated potent antineuroinflammatory and brain edema reducing effects and has shown promise in promoting functional recovery in pre-clinical models of traumatic brain injury (TBI). The purpose of this study was to assess the potential neuroprotective effects of oral administration of simvastatin on neurobehavioral, biomarker, and histopathological outcome measures compared across three pre-clinical TBI animal models. Adult male Sprague-Dawley rats were exposed to either moderate fluid percussion injury (FPI), controlled cortical impact injury (CCI), or penetrating ballistic-like brain injury (PBBI). Simvastatin (1 or 5 mg/kg) was delivered via oral gavage at 3 h post-injury and continued once daily out to 14 days post-injury. Results indicated an intermediate beneficial effect of simvastatin on motor performance on the gridwalk (FPI), balance beam (CCI), and rotarod tasks (PBBI). No significant therapeutic benefit was detected, however, on cognitive outcome across the OBTT TBI models. In fact, Morris water maze (MWM) performance was actually worsened by treatment in the FPI model and scored full negative points for low dose in the MWM latency and swim distance to locate the hidden platform. A detrimental effect on cortical tissue loss was also seen in the FPI model, and there were no benefits on histology across the other models. Simvastatin also produced negative effects on circulating glial fibrillary acidic protein biomarker outcomes that were evident in the FPI and PBBI models. Overall, the current findings do not support the beneficial effects of simvastatin administration over 2 weeks post-TBI using the oral route of administration and, as such, it will not be further pursued by OBTT.

Intravenous Administration of Simvastatin Improves Cognitive Outcome following Severe Traumatic Brain Injury in Rats.

Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor commonly used to reduce serum cholesterol. The beneficial effects of oral simvastatin have been reported in pre-clinical models of traumatic brain injury (TBI). The current study was designed to evaluate the potential beneficial effects of simvastatin in a model of severe penetrating TBI using an intravenous (IV) route of administration. Rats were subjected to unilateral frontal penetrating ballistic-like brain injury (PBBI), and simvastatin was delivered intravenously at 30 min and 6 h post-injury and continued once daily for either 4 or 10 days post-PBBI. Motor function was assessed on the rotarod and cognitive performance was evaluated using the Morris water maze (MWM) task. Serum levels of inflammatory cytokines and the astrocytic biomarker, glial fibrillary acidic protein (GFAP), were quantified at 1 h, 4 h, and 24 h post-injury. Histopathological damage was assessed at the terminal end-point. Rotarod testing revealed significant motor deficits in all injury groups but no significant simvastatin-induced therapeutic benefits. All PBBI-injured animals showed cognitive impairment on the MWM test; however, 10-day simvastatin treatment mitigated these effects. Animals showed significantly improved latency to platform and retention scores, whereas the 4-day treatment regimen failed to produce any significant improvements. Biomarker and cytokine analysis showed that IV simvastatin significantly reduced GFAP, interleukin (IL)-1α, and IL-17 serum levels by 4.0-, 2.6-, and 7.0-fold, respectively, at 4 h post-injury. Collectively, our results demonstrate that IV simvastatin provides significant protection against injury-induced cognitive dysfunction and reduces TBI-specific biomarker levels. Further research is warranted to identify the optimal dose and therapeutic window for IV delivery of simvastatin in models of severe TBI.

Sialylation regulates brain structure and function.

Every cell expresses a molecularly diverse surface glycan coat (glycocalyx) comprising its interface with its cellular environment. In vertebrates, the terminal sugars of the glycocalyx are often sialic acids, 9-carbon backbone anionic sugars implicated in intermolecular and intercellular interactions. The vertebrate brain is particularly enriched in sialic acid-containing glycolipids termed gangliosides. Human congenital disorders of ganglioside biosynthesis result in paraplegia, epilepsy, and intellectual disability. To better understand sialoglycan functions in the nervous system, we studied brain anatomy, histology, biochemistry, and behavior in mice with engineered mutations in St3gal2 and St3gal3, sialyltransferase genes responsible for terminal sialylation of gangliosides and some glycoproteins. St3gal2/3 double-null mice displayed dysmyelination marked by a 40% reduction in major myelin proteins, 30% fewer myelinated axons, a 33% decrease in myelin thickness, and molecular disruptions at nodes of Ranvier. In part, these changes may be due to dysregulation of ganglioside-mediated oligodendroglial precursor cell proliferation. Neuronal markers were also reduced up to 40%, and hippocampal neurons had smaller dendritic arbors. Young adult St3gal2/3 double-null mice displayed impaired motor coordination, disturbed gait, and profound cognitive disability. Comparisons among sialyltransferase mutant mice provide insights into the functional roles of brain gangliosides and sialoglycoproteins consistent with related human congenital disorders.

The WRAIR projectile concussive impact model of mild traumatic brain injury: re-design, testing and preclinical validation.

The WRAIR projectile concussive impact (PCI) model was developed for preclinical study of concussion. It represents a truly non-invasive closed-head injury caused by a blunt impact. The original design, however, has several drawbacks that limit the manipulation of injury parameters. The present study describes engineering advancements made to the PCI injury model including helmet material testing, projectile impact energy/head kinematics and impact location. Material testing indicated that among the tested materials, 'fiber-glass/carbon' had the lowest elastic modulus and yield stress for providing an relative high percentage of load transfer from the projectile impact, resulting in significant hippocampal astrocyte activation. Impact energy testing of small projectiles, ranging in shape and size, showed the steel sphere produced the highest impact energy and the most consistent impact characteristics. Additional tests confirmed the steel sphere produced linear and rotational motions on the rat's head while remaining within a range that meets the criteria for mTBI. Finally, impact location testing results showed that PCI targeted at the temporoparietal surface of the rat head produced the most prominent gait abnormalities. Using the parameters defined above, pilot studies were conducted to provide initial validation of the PCI model demonstrating quantifiable and significant increases in righting reflex recovery time, axonal damage and astrocyte activation following single and multiple concussions.

Ethosuximide and phenytoin dose-dependently attenuate acute nonconvulsive seizures after traumatic brain injury in rats.

Acute seizures frequently occur following severe traumatic brain injury (TBI) and have been associated with poor patient prognosis. Silent or nonconvulsive seizures (NCS) manifest in the absence of motor convulsion, can only be detected via continuous electroencephalographic (EEG) recordings, and are often unidentified and untreated. Identification of effective anti-epileptic drugs (AED) against post-traumatic NCS remains crucial to improve neurological outcome. Here, we assessed the anti-seizure profile of ethosuximide (ETX, 12.5-187.5 mg/kg) and phenytoin (PHT, 5-30 mg/kg) in a spontaneously occurring NCS model associated with penetrating ballistic-like brain injury (PBBI). Rats were divided between two drug cohorts, PHT or ETX, and randomly assigned to one of four doses or vehicle within each cohort. Following PBBI, NCS were detected by continuous EEG monitoring for 72 h post-injury. Drug efficacy was evaluated on NCS parameters of incidence, frequency, episode duration, total duration, and onset latency. Both PHT and ETX attenuated NCS in a dose-dependent manner. In vehicle-treated animals, 69-73% experienced NCS (averaging 9-10 episodes/rat) with average onset of NCS occurring at 30 h post-injury. Compared with control treatment, the two highest PHT and ETX doses significantly reduced NCS incidence to 13-40%, reduced NCS frequency (1.8-6.2 episodes/rat), and delayed seizure onset: <20% of treated animals exhibited NCS within the first 48 h. NCS durations were also dose-dependently mitigated. For the first time, we demonstrate that ETX and PHT are effective against spontaneously occurring NCS following PBBI, and suggest that these AEDs may be effective at treating post-traumatic NCS.

Longitudinal assessment of gait abnormalities following penetrating ballistic-like brain injury in rats.

Traumatic brain injury (TBI) results in enduring motor and cognitive dysfunction. Although gait disturbances have been documented among TBI patients, few studies have profiled gait abnormalities in animal models of TBI. We sought to obtain a comprehensive longitudinal analysis of gait function following severe penetrating ballistic-like brain injury (PBBI) in rats. Rats were subjected to either unilateral frontal PBBI, probe insertion alone, or sham surgery. Sensorimotor performance was assessed using the CatWalk automated gait analysis system. Baseline measurements were taken 3 days prior to injury and detailed analysis of gait was performed at 1, 3, 7, 14, and 28 days post-injury. Both PBBI and probe-inserted rats displayed altered static and dynamic gait parameters that were primarily evident during the early (<7 days) post-injury phase and were resolved by 1 month post-injury. PBBI produced more severe deficits compared to probe-alone which were reflected in the number, magnitude, and resolution time of abnormal gait parameters. While altered parameters were detected in all four paws, they were more apparent on the contralateral side. Gait parameters including paw pressure, print area, swing speed, and stride length were significantly decreased whereas stance, swing, and step cycle duration were increased compared to sham. Overall, altered gait patterns detected using the CatWalk system in the PBBI model were injury-severity dependent, resolved at later time points, and appeared similar to those reported in severe TBI patients. These results indicate that the CatWalk may be most useful for neuroprotection studies that focus on the acute/subacute recovery period after TBI.

Sialidase, chondroitinase ABC, and combination therapy after spinal cord contusion injury.

Axon regeneration in the central nervous system is severely hampered, limiting functional recovery. This is in part because of endogenous axon regeneration inhibitors that accumulate at the injury site. Therapeutic targeting of these inhibitors and their receptors may facilitate axon outgrowth and enhance recovery. A rat model of spinal cord contusion injury was used to test the effects of two bacterial enzyme therapies that target independent axon regeneration inhibitors, sialidase (Vibrio cholerae) and chondroitinase ABC (ChABC, Proteus vulgaris). The two enzymes, individually and in combination, were infused for 2 weeks via implanted osmotic pumps to the site of a moderate thoracic spinal cord contusion injury. Sialidase was completely stable, whereas ChABC retained>30% of its activity in vivo over the 2 week infusion period. Immunohistochemistry revealed that infused sialidase acted robustly throughout the spinal cord gray and white matter, whereas ChABC activity was more intense superficially. Sialidase treatment alone resulted in improved behavioral and anatomical outcomes. Rats treated exclusively with sialidase showed significantly increased hindlimb motor function, evidenced by higher Basso Beattie and Bresnahan (BBB) and BBB subscores, and fewer stepping errors on a horizontal ladder. Sialidase-treated rats also had increased serotonergic axons caudal to the injury. ChABC treatment, in contrast, did not enhance functional recovery or alter axon numbers after moderate spinal cord contusion injury, and dampened the response of sialidase in the dual enzyme treatment group. We conclude that sialidase infusion enhanced recovery from spinal cord contusion injury, and that combining sialidase with ChABC failed to improve outcomes.

Similarities and differences of acute nonconvulsive seizures and other epileptic activities following penetrating and ischemic brain injuries in rats.

The similarities and differences between acute nonconvulsive seizures (NCS) and other epileptic events, for example, periodic epileptiform discharges (PED) and intermittent rhythmic delta activities (IRDA), were characterized in rat models of penetrating and ischemic brain injuries. The NCS were spontaneously induced by either unilateral frontal penetrating ballistic-like brain injury (PBBI) or permanent middle cerebral artery occlusion (pMCAO), and were detected by continuous electroencephalogram (EEG) monitoring begun immediately after the injury and continued for 72 h or 24 h, respectively. Analysis of NCS profiles (incidence, frequency, duration, and time distribution) revealed a high NCS incidence in both injury models. The EEG waveform expressions of NCS and PED exhibited intrinsic variations that resembled human electrographic manifestations of post-traumatic and post-ischemic ictal and inter-ictal events, but these waveform variations were not distinguishable between the two types of brain injury. However, the NCS after pMCAO occurred more acutely and intensely (latency=0.6 h, frequency=25 episodes/rat) compared with the PBBI-induced NCS (latency=24 h, frequency=10 episodes/rat), such that the most salient features differentiating post-traumatic and post-ischemic NCS were the intensity and time distribution of the NCS profiles. After pMCAO, nearly 50% of the seizures occurred within the first 2 h of injury, whereas after PBBI, NCS occurred sporadically (0-5%/h) throughout the 72 h recording period. The PED were episodically associated with NCS. By contrast, the IRDA appeared to be independent of other epileptic events. This study provided comprehensive comparisons of post-traumatic and post-ischemic epileptic profiles. The identification of the similarities and differences across a broad spectrum of epileptic events may lead to differential strategies for post-traumatic and post-stroke seizure interventions.

Neuroprotective effects of sulforaphane after contusive spinal cord injury.

Traumatic spinal cord injury (SCI) leads to oxidative stress, calcium mobilization, glutamate toxicity, the release of proinflammatory factors, and depletion of reduced glutathione (GSH) at the site of injury. Induction of the Keap1/Nrf2/ARE pathway can alleviate neurotoxicity by protecting against GSH depletion, oxidation, intracellular calcium overload, mitochondrial dysfunction, and excitotoxicity. Sulforaphane (SF), an isothiocyanate derived from broccoli, is a potent naturally-occurring inducer of the Keap1/Nrf2/ARE pathway, leading to upregulation of genes encoding cytoprotective proteins such as NAD(P)H: quinone oxidoreductase 1, and GSH-regulatory enzymes. Additionally, SF can attenuate inflammation by inhibiting the nuclear factor-κB (NF-κB) pathway, and the enzymatic activity of the proinflammatory cytokine macrophage inhibitory factor (MIF). Our study examined systemic administration of SF in a rat model of contusion SCI, in an effort to utilize its indirect antioxidant and anti-inflammatory properties to decrease secondary injury. Two doses of SF (10 or 50 mg/kg) were administered at 10 min and 72 h after contusion SCI. SF (50 mg/kg) treatment resulted in both acute and long-term beneficial effects, including upregulation of the phase 2 antioxidant response at the injury site, decreased mRNA levels of inflammatory cytokines (i.e., MMP-9) in the injured spinal cord, inactivation of urinary MIF tautomerase activity, enhanced hindlimb locomotor function, and an increased number of serotonergic axons caudal to the lesion site. These findings demonstrate that SF provides neuroprotective effects in the spinal cord after injury, and could be a candidate for therapy of SCI.

A novel animal model of closed-head concussive-induced mild traumatic brain injury: development, implementation, and characterization.

Closed-head concussive injury is one of the most common causes of traumatic brain injury (TBI). While single concussions result in short-term neurologic dysfunction, multiple concussions can result in cumulative damage and increased risk for neurodegenerative disease. Despite the prevalence of concussion, knowledge about what occurs in the brain following this injury is limited, in part due to the limited number of appropriate animal research models. To study clinically relevant concussion we recently developed a simple, non-invasive rodent model of closed-head projectile concussive impact (PCI) TBI. For this purpose, anesthetized rats were placed on a platform positioned above a torque-sealed microcentrifuge tube packed with fixed amounts of dry ice. Upon heating, rapid sublimation of the dry ice produced a build-up of compressed CO(2) that triggered an eruptive force causing the cap to launch as an intact projectile, resulting in a targeted PCI head injury. A stainless steel helmet was implemented to protect the head from bruising, yet allowing the brain to sustain a mild PCI event. Depending on the injury location and the application of the helmet, PCI-induced injuries ranged from severe (i.e., head injury with subdural hematomas, intracranial hemorrhage, and brain tissue damage), to mild (no head injury, intracranial hemorrhage, or gross morphological pathology). Although no gross pathology was evident in mild PCI-induced injury, the following protein changes and behavioral abnormalities were detected between 1 and 24 h after PCI injury: (1) upregulation of glial fibrillary acidic protein (GFAP) in hippocampal regions; (2) upregulation of ubiquitin carboxyl-terminal hydrolase L1 (UCHL-1) in cortical tissue; and (3) significant sensorimotor abnormalities. Overall, these results indicated that this PCI model was capable of replicating salient pathologies of a clinical concussion, and could generate reproducible and quantifiable outcome measures.

Sialidase enhances recovery from spinal cord contusion injury.

Axons fail to regenerate in the injured spinal cord, limiting motor and autonomic recovery and contributing to long-term morbidity. Endogenous inhibitors, including those on residual myelin, contribute to regeneration failure. One inhibitor, myelin-associated glycoprotein (MAG), binds to sialoglycans and other receptors on axons. MAG inhibition of axon outgrowth in some neurons is reversed by treatment with sialidase, an enzyme that hydrolyzes sialic acids and eliminates MAG-sialoglycan binding. We delivered recombinant sialidase intrathecally to rats following a spinal cord contusive injury. Sialidase (or saline solution) was infused to the injury site continuously for 2 wk and then motor behavior, autonomic physiology, and anatomic outcomes were determined 3 wk later. Sialidase treatment significantly enhanced hindlimb motor function, improved bulbospinally mediated autonomic reflexes, and increased axon sprouting. These findings validate sialoglycans as therapeutic targets and sialidase as a candidate therapy for spinal cord injury.

Sialidase enhances spinal axon outgrowth in vivo.

The adult CNS is an inhibitory environment for axon outgrowth, severely limiting recovery from traumatic injury. This limitation is due, in part, to endogenous axon regeneration inhibitors (ARIs) that accumulate at CNS injury sites. ARIs include myelin-associated glycoprotein, Nogo, oligodendrocyte-myelin glycoprotein, and chondroitin sulfate proteoglycans (CSPGs). Some ARIs bind to specific receptors on the axon growth cone to halt outgrowth. Reversing or blocking the actions of ARIs may promote recovery after CNS injury. We report that treatment with sialidase, an enzyme that cleaves one class of axonal receptors for myelin-associated glycoprotein, enhances spinal axon outgrowth into implanted peripheral nerve grafts in a rat model of brachial plexus avulsion, a traumatic injury in which nerve roots are torn from the spinal cord. Repair using peripheral nerve grafts is a promising restorative surgical treatment in humans, although functional improvement remains limited. To model brachial plexus avulsion in the rat, C8 nerve roots were cut flush to the spinal cord and a peroneal nerve graft was inserted into the lateral spinal cord at the lesion site. Infusion of Clostridium perfringens sialidase to the injury site markedly increased the number of spinal axons that grew into the graft (2.6-fold). Chondroitinase ABC, an enzyme that cleaves a different ARI (CSPGs), also enhanced axon outgrowth in this model. In contrast, phosphatidylinositol-specific phospholipase C, which cleaves oligodendrocyte-myelin glycoprotein and Nogo receptors, was without benefit. Molecular therapies targeting sialoglycoconjugates and CSPGs may aid functional recovery after brachial plexus avulsion or other nervous system injuries and diseases.