DNA methylation: A mechanism for sustained alteration of KIR4.1 expression following central nervous system insult.

Kir4.1, a glial-specific inwardly rectifying potassium channel, is implicated in astrocytic maintenance of K+ homeostasis. Underscoring the role of Kir4.1 in central nervous system (CNS) functioning, genetic mutations in KCNJ10, the gene which encodes Kir4.1, causes seizures, ataxia and developmental disability in humans. Kir4.1 protein and mRNA loss are consistently observed in CNS injury and neurological diseases linked to hyperexcitability and neuronal dysfunction, leading to the notion that Kir4.1 represents an attractive therapeutic target. Despite this, little is understood regarding the mechanisms that underpin this downregulation. Previous work by our lab revealed that DNA hypomethylation of the Kcnj10 gene functions to regulate mRNA levels during astrocyte maturation whereas hypermethylation in vitro led to decreased promoter activity. In the present study, we utilized two vastly different injury models with known acute and chronic loss of Kir4.1 protein and mRNA to evaluate the methylation status of Kcnj10 as a candidate molecular mechanism for reduced transcription and subsequent protein loss. Examining whole hippocampal tissue and isolated astrocytes, in a lithium-pilocarpine model of epilepsy, we consistently identified hypermethylation of CpG island two, which resides in the large intronic region spanning the Kcnj10 gene. Strikingly similar results were observed using the second injury paradigm, a fifth cervical (C5) vertebral hemi-contusion model of spinal cord injury. Our previous work indicates the same gene region is significantly hypomethylated when transcription increases during astrocyte maturation. Our results suggest that DNA methylation can bidirectionally modulate Kcnj10 transcription and may represent a targetable molecular mechanism for the restoring astroglial Kir4.1 expression following CNS insult.

Preclinical assessment of a noncooled MR thermometry-based neurosurgical laser therapy system.

OBJECTIVE: MRI-guided laser interstitial thermal therapy (MRgLITT) has recently gained interest as an ablative stereotactic procedure for intractable epilepsy, movement disorders, and brain tumors. Conventionally, a LITT system consists of a laser generator and cooled laser applicator, which is a fiber optic core surrounded by a sheath through which cooled fluid is pumped. However, this footprint can make the system bulky and nonmobile, limit the maximum depth of targeting, and increase the chances of breakdown. Herein, the authors conduct a preclinical assessment of a noncooled MRgLITT system in a porcine model. METHODS: Three-tesla MRI was used to guide the in vivo placement of noncooled laser applicators in the porcine brain. The study consisted of a survival arm and terminal arm. The laser was activated at a power of 4-7 W for ≤ 180 seconds. Temperature changes were monitored using the MR thermometry software ThermoGuide in the survival arm (n = 5) or both ThermoGuide software and adjacently inserted thermal probes in the terminal arm (n = 3). Thermal damage was determined by the software using the temperature-time relationship of cumulative equivalent minutes at 43°C (CEM43). Temperatures calculated by the software were compared with those recorded by the temperature probes. The dimensions of thermal damage thresholds (TDTs; 2-9, 10-59, 60-239, ≥ 240 CEM43 isolines) given by MR thermometry were compared with the dimensions of irreversible damage on histopathological analysis. RESULTS: There was a strong correlation between temperature recordings by ThermoGuide and those by thermal probes at both 4 mm (r = 0.96) and 8 mm (r = 0.80), with a mean absolute error of 0.76°C ± 2.13°C and 0.17°C ± 1.65°C at 4 and 8 mm, respectively. The area of 2-9 CEM43 was larger than the area of irreversible damage seen on histopathological analysis. The dimensions of the 10 and 60 CEM43 correlated well with dimensions of the lesion on histopathological analysis. A well-defined border (≤ 1 mm) was observed between the area of irreversible damage and healthy brain tissue. CONCLUSIONS: This preclinical assessment showed that the noncooled LITT system was able to precisely reach the target and create well-defined lesions within a margin of safety, without any adverse effects. MR thermometry software provided an accurate near-real-time temperature of the brain tissue, and dimensions of the lesion as visualized by the software correlated well with histopathological findings. Further studies to test the system's efficacy and safety in human subjects are in progress.

A Touchscreen Device for Behavioral Testing in Pigs.

Pigs are becoming more common research models due to their utility in studying neurological conditions such as traumatic brain injury, Alzheimer's disease, and Huntington's Disease. However, behavioral tasks often require a large apparatus and are not automated, which may disinterest researchers in using important functional measures. To address this, we developed a touchscreen that pigs could be trained on for behavioral testing. A rack-mounted touchscreen monitor was placed in an enclosed, weighted audio rack. A pellet dispenser was operated by a radio frequency transceiver to deliver fruit-flavored sugar pellets from across the testing room. Programs were custom written in Python and executed on a microcomputer. A behavioral shaping program was designed to train pigs to interact with the screen and setup responses for future tasks. Pigs rapidly learned to interact with the screen. To demonstrate efficacy in more complex behavior, two pigs were trained on a delay discounting tasks and two pigs on a color discrimination task. The device held up to repeated testing of large pigs and could be adjusted to the height of minipigs. The device can be easily recreated and constructed at a relatively low cost. Research topics ranging from brain injury to pharmacology to vision could benefit from behavioral tasks designed to specifically interrogate relevant function. More work will be needed to develop tests which are of specific relevance to these disciplines.

Spinal cord injury causes a wide-spread, persistent loss of Kir4.1 and glutamate transporter 1: benefit of 17 beta-oestradiol treatment.

During neuronal activity astrocytes function to remove extracellular increases in potassium, which are largely mediated by the inwardly-rectifying potassium channel Kir4.1, and to take up excess glutamate via glutamate transporter 1, a glial-specific glutamate transporter. Here we demonstrate that expression of both of these proteins is reduced by nearly 80% following a crush spinal cord injury in adult male rats, 7 days post-injury. This loss extended to spinal segments several millimetres rostral and caudal to the lesion epicentre, and persisted at 4 weeks post-injury. Importantly, we demonstrate that loss of these two proteins is not a direct result of astrocyte loss, as immunohistochemistry at 7 days and western blots at 4 weeks demonstrate a marked up-regulation in glial fibrillary acidic protein expression. Kir4.1 and glutamate transporter 1 expression were partially rescued by post-spinal cord injury administration of physiological levels of 17beta-oestradiol (0.08 mg/kg/day) in vivo. Utilizing an in vitro culture system we demonstrate that 17beta-oestradiol treatment (50 nM) is sufficient to increase glutamate transporter 1 protein expression in spinal cord astrocytes. This increase in glutamate transporter 1 protein expression was reversed and Kir4.1 expression reduced in the presence of an oestrogen receptor antagonist, Fulvestrant 182,780 suggesting a direct translational regulation of Kir4.1 and glutamate transporter 1 via genomic oestrogen receptors. Using whole-cell patch-clamp recordings in cultured spinal cord astrocytes, we show that changes in protein expression following oestrogen application led to functional changes in Kir4.1 mediated currents. These findings suggest that the neuroprotective benefits previously seen with 17beta-oestradiol after spinal cord injury may be in part due to increased Kir4.1 and glutamate transporter 1 expression in astrocytes leading to improved potassium and glutamate homeostasis.

An Audit and Comparison of pH, Measured Concentration, and Particulate Matter in Mannitol and Hypertonic Saline Solutions.

Background: The preferred hyperosmolar therapy remains controversial. Differences in physical properties such as pH and osmolality may be important considerations in hyperosmolar agent selection. We aimed to characterize important physical properties of commercially available hyperosmolar solutions. Methods: We measured pH and concentration in 37 commonly-used hyperosmolar solutions, including 20 and 25% mannitol and 3, 5, 14.6, and 23.4% hypertonic saline. pH was determined digitally and with litmus paper. Concentration was determined by freezing point and vapor pressure osmometry. Salinity/specific gravity was measured with portable refractometry. Particulate matter was analyzed with filtration and light microscopy and with dynamic light scattering nephelometry. Results: pH of all solutions was below physiological range (measured range 4.13-6.80); there was no correlation between pH and solution concentration (R 2 = 0.005, p = 0.60). Mannitol (mean 5.65, sd 0.94) was less acidic than hypertonic saline (5.16, 0.60). 14/59 (24%) pH measurements and 85/111 concentration measurements were outside manufacturer standards. All 36/36 mannitol concentration measurements were outside standards vs. 48/72 (67%) hypertonic saline (p < 0.0001). All solutions examined on light microscopy contained crystalline and/or non-crystalline particulate matter up to several hundred microns in diameter. From nephelometry, particulate matter was detected in 20/22 (91%) solutions. Conclusion: We present a novel characterization of mannitol and hypertonic saline. Further research should be undertaken, including research examining development of acidosis following hyperosmolar therapy, the relevance of our findings for dose-response, and the clinical relevance of particulate matter in solution.

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

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

17ß-estradiol protects Schwann cells against H2O2-induced cytotoxicity and increases transplanted Schwann cell survival in a cervical hemicontusion spinal cord injury model.

Schwann cell (SC) transplantation is a promising repair strategy after spinal cord injury (SCI); however, a large number of SCs do not survive following transplantation. Previous studies have shown that 17ß-estradiol (E2) protects several cell types against cytotoxicity. Thus, this study evaluated the protective potential of E2 on SCs in vitro and investigated the effect of E2 on transplanted SC survival in a rat model of SCI. Primary SC cultures were found to robustly express estrogen receptors (ER) and incubation with E2 protected SCs against hydrogen peroxide-induced cell death. This protection was not inhibited by the ER antagonist ICI 182,780, suggesting that genomic signaling is not necessary for protection. In a subsequent experiment, cervical hemicontusion SCI was induced in male rats followed by sustained administration of E2 or placebo. Eight days after SCI, SCs were transplanted into the injury epicenter. E2 treatment significantly increased the number of surviving labeled transplanted SCs evaluated 7 days after transplantation. These data demonstrate that E2 protects SCs against oxidative stress and improves transplanted SC survival, which suggests that E2 administration may be an intervention of choice for enhancing survival of transplanted SCs after SCI.

Acute administration of perampanel, an AMPA receptor antagonist, reduces cognitive impairments after traumatic brain injury in rats.

Traumatic brain injury (TBI) is a major cause of death and physical as well as cognitive disability for which an effective treatment option remains to be identified. Evidence in preclinical models has indicated that antagonists of the α-amino-3-hydroxy-5-methyl-4-isozazole propionate (AMPA) receptor exert neuroprotective effects after mechanical injury in vitro and in vivo. In particular, 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile hydrate (perampanel), a selective AMPA receptor antagonist with good bioavailability, was recently shown to therapeutically protect against the sequelae of TBI in the rodent controlled cortical impact model. However, this model induces a largely focal injury and is less representative of diffuse injury components that occur in TBI resulting from acceleration/deceleration forces. Here, we investigated the neuroprotective effects of perampanel in the rodent lateral fluid percussion injury model (LFPI), which produces both focal and diffuse injury. Pre- or post-injury administration of perampanel in male adult rats attenuated the injury-induced increase in the pro-apoptotic bax/bcl-xL ratio in the hippocampus; reduced impairments in learning and memory, assessed by the Morris water maze test; and reduced impairments in reward-seeking behavior, assessed by a female encounter test. Although additional studies are needed to determine the sex-related differences in the neuroprotective effects, these results provide support for the therapeutic potential of perampanel in TBI.

Estradiol targets synaptic proteins to induce glutamatergic synapse formation in cultured hippocampal neurons: critical role of estrogen receptor-alpha.

Estradiol mediates structural changes at synapses of the hippocampus, an area in the brain important for learning and memory. This study was designed to test the hypothesis that estradiol mediates subcellular changes of synaptic proteins to induce new synapses via an estrogen receptor (ER)-mediated process. To elucidate the mechanisms involved in glutamatergic synapse formation, we investigated effects of estradiol on synaptic proteins in cultured hippocampal neurons using immunocytochemistry and confocal microscopy. Synaptic protein distribution and size were identified with antibodies to the presynaptic vesicular glutamate transporter protein (vGlut1) and postsynaptic NMDA receptor (NR1 subunit). We observed an increase in synapse density, as detected by NR1 and vGlut1 colocalization, along dendrites of neurons cultured in steroid-stripped media and exposed to estradiol (10 nM) for 48 h. Additionally, the NR1 subunit was enriched at synaptic clusters. Immunocytochemistry and confocal imaging revealed punctate staining of extranuclear ERs along dendrites of hippocampal neurons expressing NR1. Estradiol increased the density of both ER-alpha and ER-beta protein clusters along dendrites. To test whether ERs play an important functional role in the estradiol-induced synaptogenesis, we used the ER antagonist [7alpha,17beta-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780)] and the ER-alpha- and ER-beta-specific agonists [1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively]. ICI 182,780 blocked the increase in synapse density. Treatment with PPT, but not DPN, induced significant increases in synapse density that mimicked treatment with estradiol. Together, our results demonstrate that estradiol stimulates glutamatergic synapse formation in the developing hippocampus through an ER-alpha-dependent mechanism. These findings carry profound implications regarding the potential of estrogen to influence learning, memory, and possibly hormone-modulated neurodegeneration.

Astroglia: important mediators of traumatic brain injury.

Traumatic brain injury (TBI) research to date has focused almost exclusively on the pathophysiology of injured neurons with very little attention paid to non-neuronal cells. However in the past decade, exciting discoveries have challenged this century-old view of passive glial cells and have led to a reinterpretation of the role of glial cells in central nervous system (CNS) biology and pathology. In this chapter we review several lines of evidence, indicating that glial cells, particularly astrocytes, are active partners to neurons in the brain, and summarize recent findings that detail the significance of astrocyte pathology in traumatic brain injury.

Evaluation of Touchscreen Chambers To Assess Cognition in Adult Mice: Effect of Training and Mild Traumatic Brain Injury.

Cognitive impairments are often experienced after a mild traumatic brain injury (mTBI). In the clinical arena, neuropsychological assessments are used frequently to detect cognitive deficits. Animal models of mTBI, however, rely on an assortment of behavioral tasks to assess cognitive outcome. Computer-based touchscreen systems have been developed for rodents and are hypothesized to offer a translational approach to evaluate cognitive function because of the similarities of tasks performed in rodents to those implemented in humans. While these touchscreen systems have been used in pre-clinical models of neurodegenerative diseases and psychiatric disorders, their use in assessing cognitive impairment after mTBI has not been investigated. We hypothesized that mTBI would result in impaired cognitive performance on touchscreen tasks, particularly those with hippocampal-based learning components, including the paired associate learning (PAL) task and the location discrimination (LD) task. Adult male, C57BL/6 mice received a single impact-acceleration mTBI. We found that training mice before injury to perform to criteria is arduous and that performance is sensitive to many environmental variables. Despite extensive optimization and training, mice failed to perform better than chance in the PAL paradigm. Alternatively, mice demonstrated some capacity to learn in the LD paradigm, but only with the easier stages of the task. The mTBI did not affect performance in the LD paradigm, however. Thus, we concluded that under the conditions presented here, the PAL and LD touchscreen tasks are not robust outcome measures for the evaluation of cognitive performance in C57BL/6 mice after a single impact-acceleration mTBI.

Post-injury administration of a combination of memantine and 17ß-estradiol is protective in a rat model of traumatic brain injury.

Traumatic brain injury (TBI) often leads to substantial adverse cognitive and health outcomes, including permanent disability and death. Preventing these outcomes requires attenuation of the secondary biochemical damage that follows the initial biomechanical insult, but a clinically proven pharmacotherapeutic capable of such has not been identified. In fact, the heterogeneous nature of TBI and the complexity of secondary injury cascades suggest a polytherapeutic approach that targets multiple pathways might be necessary. We and others have reported that 17ß-estradiol (E2) is neuroprotective in models of central nervous system injury. Although E2 is neuroprotective and favorably modulates several key components of secondary injury, it does not effectively block the destructive excitotoxic cascade. Thus, administering E2 in combination with a second drug that targets excitotoxicity, such as the FDA-approved uncompetitive NMDA receptor antagonist memantine hydrochloride, may provide additional benefits. Here, we assessed the neuroprotective potential of an acutely administered intravenous bolus dose of a combination of memantine and E2 after induction of experimental TBI in the clinically relevant lateral fluid percussion model. Our results indicate that the combination of these drugs conferred neuroprotection by increasing neuronal survival and decreasing neuronal degeneration in the hippocampus and cortex ipsilateral to injury. Furthermore, administration of this combination improved vestibulomotor deficits and modestly reduced anxiety. We conclude that further investigation of the neuroprotective potential of memantine administered with E2 is warranted.

RNA Binding Protein Human Antigen R Is Translocated in Astrocytes following Spinal Cord Injury and Promotes the Inflammatory Response.

Inflammation plays a prominent role in the events following traumatic injury to the central nervous system (CNS). The initial inflammatory response is driven by mediators such as tumor necrosis factor α and interleukin 1ß, which are produced by activated astrocytes and microglia at the site of injury. These factors are regulated post-transcriptionally by RNA binding proteins (RBP) that interact with adenylate and uridylate-rich elements (ARE) in the 3'-untranslated region of the messenger RNA (mRNA). Human antigen R (HuR) is one of these RBPs and generally functions as a positive regulator of ARE-containing mRNAs. Here, we hypothesized that HuR plays an important role in the induction of cytokine and chemokines in astrocytes following traumatic injury. Using a mouse model of spinal cord injury, we found HuR to be extensively translocated to the cytoplasm in astrocytes at the level of injury, consistent with its activation. In an in vitro stretch injury model of CNS trauma, we observed a similar cytoplasmic shift of HuR in astrocytes and an attenuation of cytokine induction with HuR knockdown. RNA kinetics and luciferase assays suggested that the effect was more related to transcription than RNA destabilization. A small molecule inhibitor of HuR suppressed cytokine induction of injured astrocytes and reduced chemoattraction for neutrophils and microglia. In summary, HuR is activated in astrocytes in the early stages of CNS trauma and positively regulates the molecular response of key inflammatory mediators in astrocytes. Our findings suggest that HuR may be a therapeutic target in acute CNS trauma for blunting secondary tissue injury triggered by the inflammatory response.

Astrocytic expression of the RNA regulator HuR accentuates spinal cord injury in the acute phase.

We recently showed that the RNA regulator, HuR, is translocated to the cytoplasm in astrocytes in the acute phase of spinal cord injury (SCI), consistent with its activation. HuR positively modulates expression of many pro-inflammatory factors, including IL-1ß, TNF-α, and MMP-12, which are present at high levels in the early phase of SCI and exacerbate tissue damage. Knockdown of HuR in astrocytes blunts expression of these factors in an in vitro stretch injury model of CNS trauma. In this report, we further investigate the impact of HuR in early SCI using a mouse model in which human HuR is transgenically expressed in astrocytes. At 24h following a mid-thoracic contusion injury, transgenic HuR translocated to the cytoplasm of astrocytes, similar to endogenous HuR, and consistent with its activation. Compared to littermate controls, the transgenic mice showed a global increase in astrocyte activation at the level of injury and a concomitant increase in vascular permeability. There was a significant decrease in neuronal survival at this time interval, but no differences in white matter sparing. Long term behavioral assessments showed no difference in motor recovery. In summary, transgenic expression of HuR in astrocytes accentuated neuronal injury and other secondary features of SCI including increased vascular permeability and astrocyte activation. These findings underscore HuR as a potential therapeutic target in early SCI.

Application of the Rat Grimace Scale as a Marker of Supraspinal Pain Sensation after Cervical Spinal Cord Injury.

Experimental models of neuropathic pain (NP) typically rely on withdrawal responses to assess the presence of pain. Reflexive withdrawal responses to a stimulus are used to evaluate evoked pain and, as such, do not include the assessment of spontaneous NP nor evaluation of the affective and emotional consequences of pain in animal models. Additionally, withdrawal responses can be mediated by spinal cord reflexes and may not accurately indicate supraspinal pain sensation. This is especially true in models of traumatic spinal cord injury (SCI), wherein spastic syndrome, a motor disorder characterized by exaggeration of the stretch reflex that is secondary to hyperexcitability of the spinal reflex, can cause paroxysmal withdrawals not associated with NP sensation. Consequently, the aim of this study was to utilize an assessment of supraspinal pain sensation, the Rat Grimace Scale (RGS), to measure both spontaneous and evoked NP after a contusion SCI at cervical level 5 in adult male rats. Spontaneous and evoked pain were assessed using the RGS to score facial action units before and after the application of a stimulus, respectively. Rodents exhibited significantly higher RGS scores at week 5 post-injury as compared to baseline and laminectomy controls before the application of the stimulus, suggesting the presence of spontaneous NP. Additionally, there was a significant increase in RGS scores after the application of the acetone. These data suggest that the RGS can be used to assess spontaneous NP and determine the presence of evoked supraspinal pain sensation after experimental cervical SCI.

Traumatic Brain Injury Induces Alterations in Cortical Glutamate Uptake without a Reduction in Glutamate Transporter-1 Protein Expression.

We hypothesize that the primary mechanism for removal of glutamate from the extracellular space is altered after traumatic brain injury (TBI). To evaluate this hypothesis, we initiated TBI in adult male rats using a 2.0 atm lateral fluid percussion injury (LFPI) model. In the ipsilateral cortex and hippocampus, we found no differences in expression of the primary glutamate transporter in the brain (GLT-1) 24 h after TBI. In contrast, we found a decrease in glutamate uptake in the cortex, but not the hippocampus, 24 h after injury. Because glutamate uptake is potently regulated by protein kinases, we assessed global serine-threonine protein kinase activity using a kinome array platform. Twenty-five kinome array peptide substrates were differentially phoshorylated between LFPI and controls in the cortex, whereas 19 peptide substrates were differentially phosphorylated in the hippocampus (fold change ≥ ± 1.15). We identified several kinases as likely to be involved in acute TBI, including protein kinase B (Akt) and protein kinase C (PKC), which are well-characterized modulators of GLT-1. Exploratory studies using an inhibitor of Akt suggest selective activation of kinases in LFPI versus controls. Ingenuity pathway analyses of implicated kinases from our network model found apoptosis and cell death pathways as top functions in acute LFPI. Taken together, our data suggest diminished activity of glutamate transporters in the prefrontal cortex, with no changes in protein expression of the primary glutamate transporter GLT-1, and global alterations in signaling networks that include serine-threonine kinases that are known modulators of glutamate transport activity.

Glutamate Neurotransmission in Rodent Models of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading cause of death and disability in people younger than 45 and is a significant public health concern. In addition to primary mechanical damage to cells and tissue, TBI involves additional molecular mechanisms of injury, termed secondary injury, that continue to evolve over hours, days, weeks, and beyond. The trajectory of recovery after TBI is highly unpredictable and in many cases results in chronic cognitive and behavioral changes. Acutely after TBI, there is an unregulated release of glutamate that cannot be buffered or cleared effectively, resulting in damaging levels of glutamate in the extracellular space. This initial loss of glutamate homeostasis may initiate additional changes in glutamate regulation. The excitatory amino acid transporters (EAATs) are expressed on both neurons and glia and are the principal mechanism for maintaining extracellular glutamate levels. Diffusion of glutamate outside the synapse due to impaired uptake may lead to increased extrasynaptic glutamate signaling, secondary injury through activation of cell death pathways, and loss of fidelity and specificity of synaptic transmission. Coordination of glutamate release and uptake is critical to regulating synaptic strength, long-term potentiation and depression, and cognitive processes. In this review, we will discuss dysregulation of extracellular glutamate and glutamate uptake in the acute stage of TBI and how failure to resolve acute disruptions in glutamate homeostatic mechanisms may play a causal role in chronic cognitive symptoms after TBI.

17beta-estradiol is protective in spinal cord injury in post- and pre-menopausal rats.

The neuroprotective effects of 17 beta -estradiol have been shown in models of central nervous system injury, including ischemia, brain injury, and more recently, spinal cord injury (SCI). Recent epidemiological trends suggest that SCIs in elderly women are increasing; however, the effects of menopause on estrogen-mediated neuroprotection are poorly understood. The objective of this study was to evaluate the effects of 17beta-estradiol and reproductive aging on motor function, neuronal death, and white matter sparing after SCI of post- and pre-menopausal rats. Two-month-old or 1- year-old female rats were ovariectomized and implanted with a silastic capsule containing 180 microg/mL of 17beta-estradiol or vehicle. Complete crush SCI at T8-9 was performed 1 week later. Additional animals of each age group were left ovary-intact but were spinal cord injured. The Basso, Beattie, Bresnahan (BBB) locomotor test was performed. Spinal cords were collected on post-SCI days 1, 7, and 21, and processed for histological markers. Administration of 17beta-estradiol to ovariectomized rats improved recovery of hind-limb locomotion, increased white matter sparing, and decreased apoptosis in both the post- and pre-menopausal rats. Also, ovary-intact 1-year-old rats did worse than ovary-intact 2-month-old rats, suggesting that endogenous estrogen confers neuroprotection in young rats, which is lost in older animals. Taken together, these data suggest that estrogen is neuroprotective in SCI and that the loss of endogenous estrogen-mediated neuroprotective seen in older rats can be attenuated with exogenous administration of 17beta-estradiol.

Greater neurobehavioral deficits occur in adult mice after repeated, as compared to single, mild traumatic brain injury (mTBI).

Mild traumatic brain injury (mTBI) accounts for the majority of all brain injuries and affected individuals typically experience some extent of cognitive and/or neuropsychiatric deficits. Given that repeated mTBIs often result in worsened prognosis, the cumulative effect of repeated mTBIs is an area of clinical concern and on-going pre-clinical research. Animal models are critical in elucidating the underlying mechanisms of single and repeated mTBI-associated deficits, but the neurobehavioral sequelae produced by these models have not been well characterized. Thus, we sought to evaluate the behavioral changes incurred after single and repeated mTBIs in mice utilizing a modified impact-acceleration model. Mice in the mTBI group received 1 impact while the repeated mTBI group received 3 impacts with an inter-injury interval of 24h. Classic behavior evaluations included the Morris water maze (MWM) to assess learning and memory, elevated plus maze (EPM) for anxiety, and forced swim test (FST) for depression/helplessness. Additionally, species-typical behaviors were evaluated with the marble-burying and nestlet shredding tests to determine motivation and apathy. Non-invasive vibration platforms were used to examine sleep patterns post-mTBI. We found that the repeated mTBI mice demonstrated deficits in MWM testing and poorer performance on species-typical behaviors. While neither single nor repeated mTBI affected behavior in the EPM or FST, sleep disturbances were observed after both single and repeated mTBI. Here, we conclude that behavioral alterations shown after repeated mTBI resemble several of the deficits or disturbances reported by patients, thus demonstrating the relevance of this murine model to study repeated mTBIs.

Large animal and primate models of spinal cord injury for the testing of novel therapies.

Large animal and primate models of spinal cord injury (SCI) are being increasingly utilized for the testing of novel therapies. While these represent intermediary animal species between rodents and humans and offer the opportunity to pose unique research questions prior to clinical trials, the role that such large animal and primate models should play in the translational pipeline is unclear. In this initiative we engaged members of the SCI research community in a questionnaire and round-table focus group discussion around the use of such models. Forty-one SCI researchers from academia, industry, and granting agencies were asked to complete a questionnaire about their opinion regarding the use of large animal and primate models in the context of testing novel therapeutics. The questions centered around how large animal and primate models of SCI would be best utilized in the spectrum of preclinical testing, and how much testing in rodent models was warranted before employing these models. Further questions were posed at a focus group meeting attended by the respondents. The group generally felt that large animal and primate models of SCI serve a potentially useful role in the translational pipeline for novel therapies, and that the rational use of these models would depend on the type of therapy and specific research question being addressed. While testing within these models should not be mandatory, the detection of beneficial effects using these models lends additional support for translating a therapy to humans. These models provides an opportunity to evaluate and refine surgical procedures prior to use in humans, and safety and bio-distribution in a spinal cord more similar in size and anatomy to that of humans. Our results reveal that while many feel that these models are valuable in the testing of novel therapies, important questions remain unanswered about how they should be used and how data derived from them should be interpreted.