Evidence for the induction of analgesic cross-tolerance between opioid and apelin/APJ systems in male rats.

BACKGROUND: Opioids are potent pain relievers for managing severe pain. However, their effectiveness is hindered by tolerance, which causes the need for higher doses and leads to adverse effects. In a previous study, we found that prolonged use of apelin, similar to opioids, results in a tolerance to its analgesic effects. It remains unclear whether there is a cross-tolerance between morphine and apelin, meaning if the analgesic effects of one can reduce the effectiveness of the other. METHODS: The tail-flick test was used to assess the nociceptive threshold. All experiments were carried out on 63 male Wistar rats, which received intrathecal apelin (3µg/rat) or morphine (15µg/rat) for 7 days. To determine cross-tolerance between the analgesic effect of morphine and apelin, the analgesic property of apelin or morphine was assessed in chronic morphine- or apelin-treated groups, respectively. To determine the role of apelin and opioid receptors signaling on the development of analgesic cross-tolerance, F13-A and naloxone, as apelin and opioid receptor antagonists, were injected simultaneously with morphine or apelin. At the end of the tests, the expression levels of apelin and mu-opioid receptors were evaluated by western blotting. RESULTS: The data indicated that chronic apelin or morphine produced tolerance to the antinociceptive effects of each other. F13-A and naloxone could inhibit the induction of such cross-tolerance. The molecular data showed that there was a significant downregulation of apelin receptors in chronic morphine-treated rats and vice versa. CONCLUSION: Chronic administration of apelin or morphine induces analgesic cross-tolerance that may, in part, be mediated through receptor interactions and downregulation. The demonstrated efficacy of F13-A in these experiments highlights its potential as a novel target for improving pain management through the inhibition of the apelin/APJ signaling pathway, meriting further investigation.

Mild traumatic brain injury in Drosophila melanogaster alters reactive oxygen and nitrogen species in a sex-dependent manner.

Traumatic brain injury (TBI) is an outside force causing a modification in brain function and/or structural brain pathology that upregulates brain inducible nitric oxide synthase (iNOS), instigating increased levels of nitric oxide activity which is implicated in secondary pathology leading to behavioral deficits (Hall et al., 2012; Garry et al., 2015; Kozlov et al., 2017). In mammals, TBI-induced NO production activates an immune response and potentiates metabolic crisis through mitochondrial dysfunction coupled with vascular dysregulation; however, the direct influence on pathology is complicated by the activation of numerous secondary cascades and activation of other reactive oxygen species. Drosophila TBI models have demonstrated key features of mammalian TBI, including temporary incapacitation, disorientation, motor deficits, activation of innate immunity (inflammation), and autophagy responses observed immediately after injury (Katzenberger et al., 2013; Barekat et al., 2016; Simon et al., 2017; Anderson et al., 2018; Buhlman et al., 2021b). We hypothesized that acute behavioral phenotypes would be associated with deficits in climbing behavior and increased oxidative stress. Because flies lack mammalian-like cardiovascular and adaptive immune systems, we were able to make our observations in the absence of vascular disruption and adaptive immune system interference in a system where highly targeted interventions can be rapidly evaluated. To demonstrate the induction of injury, ten-day-old transgenic flies received an injury of increasing angles from a modified high impact trauma (HIT) device where angle-dependent increases occurred for acute neurological behavior assessments and twenty-four-hour mortality, and survival was significantly decreased. Injury caused sex-dependent effects on climbing activity and measures of oxidative stress. Specifically, after a single 60-degree HIT, female flies exhibited significant impairments in climbing activity beyond that observed in male flies. We also found that several measures of oxidative stress, including Drosophila NOS (dNOS) expression, protein nitration, and hydrogen peroxide production were significantly decreased in female flies. Interestingly, protein nitration was also decreased in males, but surpassed sham levels with a more severe injury. We also observed decreased autophagy demand in vulnerable dopaminergic neurons in female, but not male flies. In addition, mitophagy initiation was decreased in females. Collectively, our data suggest that TBI in flies induces acute behavioral phenotypes and climbing deficits that are analogous to mammalian TBI. We also observed that various indices of oxidative stress, including dNOS expression, protein tyrosine nitration, and hydrogen peroxide levels, as well as basal levels of autophagy, are altered in response to injury, an effect that is more pronounced in female flies.

Impact of increasing one-carbon metabolites on traumatic brain injury outcome using pre-clinical models.

Traumatic brain injury is a major cause of death and disability worldwide, affecting over 69 million individuals yearly. One-carbon metabolism has been shown to have beneficial effects after brain damage, such as ischemic stroke. However, whether increasing one-carbon metabolite vitamins impacts traumatic brain injury outcomes in patients requires more investigation. The aim of this review is to evaluate how one-carbon metabolites impact outcomes after the onset of traumatic brain injury. PubMed, Web of Science, and Google Scholar databases were searched for studies that examined the impact of B-vitamin supplementation on traumatic brain injury outcomes. The search terms included combinations of the following words: traumatic brain injury, dietary supplementation, one-carbon metabolism, and B-vitamins. The focus of each literature search was basic science data. The year of publication in the literature searches was not limited. Our analysis of the literature has shown that dietary supplementation of B-vitamins has significantly improved the functional and behavioral recovery of animals with traumatic brain injury compared to controls. However, this improvement is dosage-dependent and is contingent upon the onset of supplementation and whether there is a sustained or continuous delivery of vitamin supplementation post-traumatic brain injury. The details of supplementation post-traumatic brain injury need to be further investigated. Overall, we conclude that B-vitamin supplementation improves behavioral outcomes and reduces cognitive impairment post-traumatic brain injury in animal model systems. Further investigation in a clinical setting should be strongly considered in conjunction with current medical treatments for traumatic brain injury-affected individuals.

Effects of isoflurane and urethane anesthetics on glutamate neurotransmission in rat brain using in vivo amperometry.

BACKGROUND: Aspects of glutamate neurotransmission implicated in normal and pathological conditions are predominantly evaluated using in vivo recording paradigms in rats anesthetized with isoflurane or urethane. Urethane and isoflurane anesthesia influence glutamate neurotransmission through different mechanisms; however, real-time outcome measures of potassium chloride (KCl)-evoked glutamate overflow and glutamate clearance kinetics have not been compared within and between regions of the brain. In order to maintain rigor and reproducibility within the literature between the two most common methods of anesthetized in vivo recording of glutamate, we compared glutamate signaling as a function of anesthesia and brain region in the rat strain most used in neuroscience. METHODS: In the following experiments, in vivo amperometric recordings of KCl-evoked glutamate overflow and glutamate clearance kinetics (uptake rate and T80) in the cortex, hippocampus, and thalamus were performed using glutamate-selective microelectrode arrays (MEAs) in young adult male, Sprague-Dawley rats anesthetized with either isoflurane or urethane. RESULTS: Potassium chloride (KCl)-evoked glutamate overflow was similar under urethane and isoflurane anesthesia in all brain regions studied. Analysis of glutamate clearance determined that the uptake rate was significantly faster (53.2%, p < 0.05) within the thalamus under urethane compared to isoflurane, but no differences were measured in the cortex or hippocampus. Under urethane, glutamate clearance parameters were region-dependent, with significantly faster glutamate clearance in the thalamus compared to the cortex but not the hippocampus (p < 0.05). No region-dependent differences were measured for glutamate overflow using isoflurane. CONCLUSIONS: These data support that amperometric recordings of KCl-evoked glutamate under isoflurane and urethane anesthesia result in similar and comparable data. However, certain parameters of glutamate clearance can vary based on choice of anesthesia and brain region. In these circumstances, special considerations are needed when comparing previous literature and planning future experiments.

Carvacrol decreases blood-brain barrier permeability post-diffuse traumatic brain injury in rats.

Previously, we showed that Satureja Khuzestanica Jamzad essential oil (SKEO) and its major component, carvacrol (CAR), 5-isopropyl-2-methylphenol, has anti-inflammatory, anti-apoptotic, and anti-edematous properties after experimental traumatic brain injury (TBI) in rats. CAR, predominantly found in Lamiaceae family (Satureja and Oregano), is lipophilic, allowing diffusion across the blood-brain barrier (BBB). These experiments test the hypothesis that acute treatment with CAR after TBI can attenuate oxidative stress and BBB permeability associated with CAR's anti-edematous traits. Rats were divided into six groups and injured using Marmarou weight drop: Sham, TBI, TBI + Vehicle, TBI + CAR (100 and 200 mg/kg) and CAR200-naive treated rats. Intraperitoneal injection of vehicle or CAR was administered thirty minutes after TBI induction. 24 h post-injury, brain edema, BBB permeability, BBB-related protein levels, and oxidative capacity were measured. Data showed CAR 200 mg/kg treatment decreased brain edema and prevented BBB permeability. CAR200 decreased malondialdehyde (MDA) and reactive oxygen species (ROS) and increased superoxide dismutase (SOD) and total antioxidative capacity (T-AOC), indicating the mechanism of BBB protection is, in part, through antioxidant activity. Also, CAR 200 mg/kg treatment suppressed matrix metalloproteinase-9 (MMP-9) expression and increased ZO-1, occludin, and claudin-5 levels. These data indicate that CAR can promote antioxidant activity and decrease post-injury BBB permeability, further supporting CAR as a potential early therapeutic intervention that is inexpensive and more readily available worldwide. However, more experiments are required to determine CAR's long-term impact on TBI pathophysiology.

Satureja khuzistanica Jamzad essential oil and pure carvacrol attenuate TBI-induced inflammation and apoptosis via NF-κB and caspase-3 regulation in the male rat brain.

Traumatic brain injury (TBI) causes progressive dysfunction that induces biochemical and metabolic changes that lead to cell death. Nevertheless, there is no definitive FDA-approved therapy for TBI treatment. Our previous immunohistochemical results indicated that the cost-effective natural Iranian medicine, Satureja khuzistanica Jamzad essential oil (SKEO), which consists of 94.16% carvacrol (CAR), has beneficial effects such as reducing neuronal death and inflammatory markers, as well as activating astrocytes and improving neurological outcomes. However, the molecular mechanisms of these neuroprotective effects have not yet been elucidated. This study investigated the possible mechanisms involved in the anti-inflammatory and anti-apoptotic properties of SKEO and CAR after TBI induction. Eighty-four male Wistar rats were randomly divided into six groups: Sham, TBI, TBI + Vehicle, TBI + CAR (100 and 200 mg/kg), and TBI + SKEO (200 mg/kg) groups. After establishing the "Marmarou" weight drop model, diffuse TBI was induced in the rat brain. Thirty minutes after TBI induction, SKEO & CAR were intraperitoneally injected. One day after TBI, injured rats exhibited significant brain edema, neurobehavioral dysfunctions, and neuronal apoptosis. Western blot results revealed upregulation of the levels of cleaved caspase-3, NFκB p65, and Bax/Bcl-2 ratio, which was attenuated by CAR and SKEO (200 mg/kg). Furthermore, the ELISA results showed that CAR treatment markedly prevents the overproduction of the brain pro-inflammatory cytokines, including IL-1ß, TNF-α, and IL-6. Moreover, the neuron-specific enolase (NSE) immunohistochemistry results revealed the protective effect of CAR and SKEO on post-TBI neuronal death. The current study revealed that the possible neuroprotective mechanisms of SKEO and CAR might be related to (at least in part) modulating NF-κB regulated inflammation and caspase-3 protein expression. It also suggested that CAR exerts more potent protective effects than SKEO against TBI. Nevertheless, the administration of SKEO and CAR may express a novel therapeutic approach to ameliorate TBI-related secondary phase neuropathological outcomes.

Effects of isoflurane and urethane anesthetics on glutamate neurotransmission in rat brain using in vivo amperometry.

Aspects of glutamate neurotransmission implicated in normal and pathological conditions are often evaluated using in vivo recording paradigms in rats anesthetized with isoflurane or urethane. Urethane and isoflurane anesthesia influence glutamate neurotransmission through different mechanisms; however real-time outcome measures of potassium chloride (KCl)-evoked glutamate overflow and glutamate clearance kinetics have not been compared within and between regions of the brain. In the following experiments, in vivo amperometric recordings of KCl-evoked glutamate overflow and glutamate clearance kinetics (uptake rate and T80) in the cortex, hippocampus and thalamus were performed using glutamate-selective microelectrode arrays (MEAs) in young adult male, Sprague-Dawley rats anesthetized with isoflurane or urethane. Potassium chloride (KCl)-evoked glutamate overflow was similar under urethane and isoflurane anesthesia in all brain regions studied. Analysis of glutamate clearance determined that the uptake rate was significantly faster (53.2%, p<0.05) within the thalamus under urethane compared to isoflurane, but no differences were measured in the cortex or hippocampus. Under urethane, glutamate clearance parameters were region dependent, with significantly faster glutamate clearance in the thalamus compared to the cortex but not the hippocampus (p<0.05). No region dependent differences were measured for glutamate overflow using isoflurane. These data support that amperometric recordings of glutamate under isoflurane and urethane anesthesia result in mostly similar and comparable data. However, certain parameters of glutamate uptake vary based on choice of anesthesia and brain region. Special considerations must be given to these areas when considering comparison to previous literature and when planning future experiments.

Aging with TBI vs. Aging: 6-month temporal profiles for neuropathology and astrocyte activation converge in behaviorally relevant thalamocortical circuitry of male and female rats.

Traumatic brain injury (TBI) manifests late-onset and persisting clinical symptoms with implications for sex differences and increased risk for the development of age-related neurodegenerative diseases. Few studies have evaluated chronic temporal profiles of neuronal and glial pathology that include sex as a biological variable. After experimental diffuse TBI, late-onset and persisting somatosensory hypersensitivity to whisker stimulation develops at one-month post-injury and persists to at least two months post-injury in male rats, providing an in vivo model to evaluate the temporal profile of pathology responsible for morbidity. Whisker somatosensation is dependent on signaling through the thalamocortical relays of the whisker barrel circuit made up of glutamatergic projections between the ventral posteromedial nucleus of the thalamus (VPM) and primary somatosensory barrel cortex (S1BF) with inhibitory (GABA) innervation from the thalamic reticular nucleus (TRN) to the VPM. To evaluate the temporal profiles of pathology, male and female Sprague Dawley rats ( n = 5-6/group) were subjected to sham surgery or midline fluid percussion injury (FPI). At 7-, 56-, and 168-days post-injury (DPI), brains were processed for amino-cupric silver stain and glial fibrillary acidic protein (GFAP) immunoreactivity, where pixel density of staining was quantified to determine the temporal profile of neuropathology and astrocyte activation in the VPM, S1BF, and TRN. FPI induced significant neuropathology in all brain regions at 7 DPI. At 168 DPI, neuropathology remained significantly elevated in the VPM and TRN, but returned to sham levels in the S1BF. GFAP immunoreactivity was increased as a function of FPI and DPI, with an FPI × DPI interaction in all regions and an FPI × Sex interaction in the S1BF. The interactions were driven by increased GFAP immunoreactivity in shams over time in the VPM and TRN. In the S1BF, GFAP immunoreactivity increased at 7 DPI and declined to age-matched sham levels by 168 DPI, while GFAP immunoreactivity in shams significantly increased between 7 and 168 days. The FPI × Sex interaction was driven by an overall greater level of GFAP immunoreactivity in FPI males compared to FPI females. Increased GFAP immunoreactivity was associated with an increased number of GFAP-positive soma, predominantly at 7 DPI. Overall, these findings indicate that FPI, time post-injury, sex, region, and aging with injury differentially contribute to chronic changes in neuronal pathology and astrocyte activation after diffuse brain injury. Thus, our results highlight distinct patterns of pathological alterations associated with the development and persistence of morbidity that supports chronic neuropathology, especially within the thalamus. Further, data indicate a convergence between TBI-induced and age-related pathology where further investigation may reveal a role for divergent astrocytic phenotypes associated with increased risk for neurodegenerative diseases.

Case report: Lingering post-concussive symptoms in a pediatric patient with presumed Ehlers-Danlos syndrome.

Background: Connective tissue disorders such as Ehlers-Danlos Syndrome (EDS) can affect collagen and elastin content and structure, including weakening of tissues and vasculature, thus contributing to multiple systemic manifestations. Prior research has successfully focused on peripheral life-threatening manifestations resulting in increased life expectancy, yet clinical observations have warranted investigation of neurological vulnerability, where little is known. Compromised brain tissues and cerebrovasculature could leave these patients vulnerable to mild traumatic brain injury (TBI), with increased severity and duration of post-concussive symptoms and delayed recovery. Clinical reports in adults indicate that higher severity of symptoms after a mild TBI, such as a concussion, can unmask connective tissues disorders leading toward diagnosis. This clinical case report is an example of a pediatric patient with presumed Ehlers-Danlos syndrome who demonstrates increased vulnerability to mild TBI/concussion. Patient: A pediatric female patient presents with unexplained lingering post-concussive symptoms, including trouble sleeping, nausea, frontal headaches, dizziness, visual changes, fatigue, and left-sided weakness more than 6 months post-mild concussion. Patient history of hypermobility, joint derangement, soft tissue mobility, and bruising suggests a potential diagnosis of Ehlers-Danlos syndrome, which may explain symptom severity and length of recovery. Discussion: This case is the first documented instance of increased vulnerability to TBI in a pediatric patient with presumed Ehlers-Danlos syndrome. It highlights the need for awareness and prevention of injury in this vulnerable patient population, suggests more targeted therapeutic intervention for recovery, and demonstrates the need for preclinical research evaluating the influence of genetic mutations associated with connective tissue disorders on the central nervous system.

Neurotrauma Prevention Review: Improving Helmet Design and Implementation.

Neurotrauma continues to contribute to significant mortality and disability. The need for better protective equipment is apparent. This review focuses on improved helmet design and the necessity for continued research. We start by highlighting current innovations in helmet design for sport and subsequent utilization in the lay community for construction. The current standards by sport and organization are summarized. We then address current standards within the military environment. The pathophysiology is discussed with emphasis on how helmets provide protection. As innovative designs emerge, protection against secondary injury becomes apparent. Much research is needed, but this focused paper is intended to serve as a catalyst for improvement in helmet design and implementation to provide more efficient and reliable neuroprotection across broad arenas.

Involvement of T-type calcium channels in the mechanism of low dose morphine-induced hyperalgesia in adult male rats.

It has been shown that systemic and local administration of ultra-low dose morphine induced a hyperalgesic response via mu-opioid receptors. However, its exact mechanism(s) has not fully been clarified. It is documented that mu-opioid receptors functionally couple to T-type voltage dependent Ca+2 channels. Here, we investigated the role of T-type calcium channels, amiloride and mibefradil, on the induction of low-dose morphine hyperalgesia in male Wistar rats. The data showed that morphine (0.01 µg i.t. and 1 µg/kg i.p.) could elicit hyperalgesia as assessed by the tail-flick test. Administration of amiloride (5 and 10 µg i.t.) and mibefradil (2.5 and 5 µg i.t.) completely blocked low-dose morphine-induced hyperalgesia in spinal dorsal horn. Amiloride at doses of 1 and 5 mg/kg (i.p.) and mibefradil (9 mg/kg ip) 10 min before morphine (1 µg/kg i.p.) inhibited morphine-induced hyperalgesia. Our results indicate a role for T-type calcium channels in low dose morphine-induced hyperalgesia in rats.

Drosophila as a model to explore secondary injury cascades after traumatic brain injury.

Drosophilae are emerging as a valuable model to study traumatic brain injury (TBI)-induced secondary injury cascades that drive persisting neuroinflammation and neurodegenerative pathology that imposes significant risk for long-term neurological deficits. As in mammals, TBI in Drosophila triggers axonal injury, metabolic crisis, oxidative stress, and a robust innate immune response. Subsequent neurodegeneration stresses quality control systems and perpetuates an environment for neuroprotection, regeneration, and delayed cell death via highly conserved cell signaling pathways. Fly injury models continue to be developed and validated for both whole-body and head-specific injury to isolate, evaluate, and modulate these parallel pathways. In conjunction with powerful genetic tools, the ability for longitudinal evaluation, and associated neurological deficits that can be tested with established behavioral tasks, Drosophilae are an attractive model to explore secondary injury cascades and therapeutic intervention after TBI. Here, we review similarities and differences between mammalian and fly pathophysiology and highlight strategies for their use in translational neurotrauma research.

Spatial Distribution of Neuropathology and Neuroinflammation Elucidate the Biomechanics of Fluid Percussion Injury.

Diffuse brain injury is better described as multi-focal, where pathology can be found adjacent to seemingly uninjured neural tissue. In experimental diffuse brain injury, pathology and pathophysiology have been reported far more lateral than predicted by the impact site. We hypothesized that local thickening of the rodent skull at the temporal ridges serves to focus the intracranial mechanical forces experienced during brain injury and generate predictable pathology. We demonstrated local thickening of the skull at the temporal ridges using contour analysis on magnetic resonance imaging. After diffuse brain injury induced by midline fluid percussion injury (mFPI), pathological foci along the anterior-posterior length of cortex under the temporal ridges were evident acutely (1, 2, and 7 days) and chronically (28 days) post-injury by deposition of argyophilic reaction product. Area CA3 of the hippocampus and lateral nuclei of the thalamus showed pathological change, suggesting that mechanical forces to or from the temporal ridges shear subcortical regions. A proposed model of mFPI biomechanics suggests that injury force vectors reflect off the skull base and radiate toward the temporal ridge, thereby injuring ventral thalamus, dorsolateral hippocampus, and sensorimotor cortex. Surgically thinning the temporal ridge before injury reduced injury-induced inflammation in the sensorimotor cortex. These data build evidence for temporal ridges of the rodent skull to contribute to the observed pathology, whether by focusing extracranial forces to enter the cranium or intracranial forces to escape the cranium. Pre-clinical investigations can take advantage of the predicted pathology to explore injury mechanisms and treatment efficacy.

Sex-Dependent Pathology in the HPA Axis at a Sub-acute Period After Experimental Traumatic Brain Injury.

Over 2.8 million traumatic brain injuries (TBIs) are reported in the United States annually, of which, over 75% are mild TBIs with diffuse axonal injury (DAI) as the primary pathology. TBI instigates a stress response that stimulates the hypothalamic-pituitary-adrenal (HPA) axis concurrently with DAI in brain regions responsible for feedback regulation. While the incidence of affective symptoms is high in both men and women, presentation is more prevalent and severe in women. Few studies have longitudinally evaluated the etiology underlying late-onset affective symptoms after mild TBI and even fewer have included females in the experimental design. In the experimental TBI model employed in this study, evidence of chronic HPA dysregulation has been reported at 2 months post-injury in male rats, with peak neuropathology in other regions of the brain at 7 days post-injury (DPI). We predicted that mechanisms leading to dysregulation of the HPA axis in male and female rats would be most evident at 7 DPI, the sub-acute time point. Young adult age-matched male and naturally cycling female Sprague Dawley rats were subjected to midline fluid percussion injury (mFPI) or sham surgery. Corticotropin releasing hormone, gliosis, and glucocorticoid receptor (GR) levels were evaluated in the hypothalamus and hippocampus, along with baseline plasma adrenocorticotropic hormone (ACTH) and adrenal gland weights. Microglial response in the paraventricular nucleus of the hypothalamus indicated mild neuroinflammation in males compared to sex-matched shams, but not females. Evidence of microglia activation in the dentate gyrus of the hippocampus was robust in both sexes compared with uninjured shams and there was evidence of a significant interaction between sex and injury regarding microglial cell count. GFAP intensity and astrocyte numbers increased as a function of injury, indicative of astrocytosis. GR protein levels were elevated 30% in the hippocampus of females in comparison to sex-matched shams. These data indicate sex-differences in sub-acute pathophysiology following DAI that precede late-onset HPA axis dysregulation. Further understanding of the etiology leading up to late-onset HPA axis dysregulation following DAI could identify targets to stabilize feedback, attenuate symptoms, and improve efficacy of rehabilitation and overall recovery.

Traumatic Brain Injury-Induced Sex-Dependent Changes in Late-Onset Sensory Hypersensitivity and Glutamate Neurotransmission.

Women approximate one-third of the annual 2.8 million people in the United States who sustain traumatic brain injury (TBI). Several clinical reports support or refute that menstrual cycle-dependent fluctuations in sex hormones are associated with severity of persisting post-TBI symptoms. Previously, we reported late-onset sensory hypersensitivity to whisker stimulation that corresponded with changes in glutamate neurotransmission at 1-month following diffuse TBI in male rats. Here, we incorporated intact age-matched naturally cycling females into the experimental design while monitoring daily estrous cycle. We hypothesized that sex would not influence late-onset sensory hypersensitivity and associated in vivo amperometric extracellular recordings of glutamate neurotransmission within the behaviorally relevant thalamocortical circuit. At 28 days following midline fluid percussion injury (FPI) or sham surgery, young adult Sprague-Dawley rats were tested for hypersensitivity to whisker stimulation using the whisker nuisance task (WNT). As predicted, both male and female rats showed significantly increased sensory hypersensitivity to whisker stimulation after FPI, with females having an overall decrease in whisker nuisance scores (sex effect), but no injury and sex interaction. In males, FPI increased potassium chloride (KCl)-evoked glutamate overflow in primary somatosensory barrel cortex (S1BF) and ventral posteromedial nucleus of the thalamus (VPM), while in females the FPI effect was discernible only within the VPM. Similar to our previous report, we found the glutamate clearance parameters were not influenced by FPI, while a sex-specific effect was evident with female rats showing a lower uptake rate constant both in S1BF and VPM and longer clearance time (in S1BF) in comparison to male rats. Fluctuations in estrous cycle were evident among brain-injured females with longer diestrus (low circulating hormone) phase of the cycle over 28 days post-TBI. Together, these findings add to growing evidence indicating both similarities and differences between sexes in a chronic response to TBI. A better understanding of the influence of gonadal hormones on behavior, neurotransmission, secondary injury and repair processes after TBI is needed both clinically and translationally, with potential impact on acute treatment, rehabilitation, and symptom management.

Mild and Moderate Traumatic Brain Injury and Repeated Stress Affect Corticosterone in the Rat.

Traumatic brain injury (TBI) survivors suffer from a range of morbidities, including post-traumatic endocrinopathies that can cause physical and mental changes in patients, greatly compromising quality of life. This study tested the hypothesis that mild and moderate diffuse TBI leads to chronic deficiencies in corticosterone (CORT) regulation following repeated exposure to restraint stress over time. Young adult male rats (n = 9-11/group) were subjected to mild or moderate TBI induced by midline fluid percussion injury (mFPI) or control sham surgery. At 6 and 24 h post-injury, both mild and moderate TBI resulted in elevated resting plasma CORT levels compared with uninjured shams. Independent of TBI severity, all rats had lower resting plasma CORT levels at 7, 14, 28, and 54 days post-injury compared with pre-surgery baseline CORT. Circulating levels of CORT were also evaluated under restraint stress and in response to dexamethasone (DEX), a synthetic glucocorticoid. Independent of TBI severity, restraint stress elevated CORT at 30, 60, and 90 min post-stressor initiation at all post-injury time-points. A blunted CORT response to restraint stress was observed with lower CORT levels after restraint at 28 and 54 days compared with 7 days post-injury (DPI), indicative of habituation to the stressor. A high dose of DEX lowered CORT levels at 90 min post-restraint stress initiation compared with low-dose DEX, independent of TBI severity. These results support TBI-induced CORT dysregulation at acute time-points, but additional studies that investigate the onset and progression of endocrinopathies, controlling for habituation to repeated restraint stress, are needed to inform the diagnosis and treatment of such morbidities in TBI survivors.

Experimental Traumatic Brain Injury Induces Chronic Glutamatergic Dysfunction in Amygdala Circuitry Known to Regulate Anxiety-Like Behavior.

Up to 50% of traumatic brain injury (TBI) survivors demonstrate persisting and late-onset anxiety disorders indicative of limbic system dysregulation, yet the pathophysiology underlying the symptoms is unclear. We hypothesize that the development of TBI-induced anxiety-like behavior in an experimental model of TBI is mediated by changes in glutamate neurotransmission within the amygdala. Adult, male Sprague-Dawley rats underwent midline fluid percussion injury or sham surgery. Anxiety-like behavior was assessed at 7 and 28 days post-injury (DPI) followed by assessment of real-time glutamate neurotransmission in the basolateral amygdala (BLA) and central nucleus of the amygdala (CeA) using glutamate-selective microelectrode arrays. The expression of anxiety-like behavior at 28 DPI coincided with decreased evoked glutamate release and slower glutamate clearance in the CeA, not BLA. Numerous factors contribute to the changes in glutamate neurotransmission over time. In two additional animal cohorts, protein levels of glutamatergic transporters (Glt-1 and GLAST) and presynaptic modulators of glutamate release (mGluR2, TrkB, BDNF, and glucocorticoid receptors) were quantified using automated capillary western techniques at 28 DPI. Astrocytosis and microglial activation have been shown to drive maladaptive glutamate signaling and were histologically assessed over 28 DPI. Alterations in glutamate neurotransmission could not be explained by changes in protein levels for glutamate transporters, mGluR2 receptors, astrocytosis, and microglial activation. Presynaptic modulators, BDNF and TrkB, were significantly decreased at 28 DPI in the amygdala. Dysfunction in presynaptic regulation of glutamate neurotransmission may contribute to anxiety-related behavior and serve as a therapeutic target to improve circuit function.

Elucidating opportunities and pitfalls in the treatment of experimental traumatic brain injury to optimize and facilitate clinical translation.

The aim of this review is to discuss the research presented in a symposium entitled "Current progress in characterizing therapeutic strategies and challenges in experimental CNS injury" which was presented at the 2016 International Behavioral Neuroscience Society annual meeting. Herein we discuss diffuse and focal traumatic brain injury (TBI) and ensuing chronic behavioral deficits as well as potential rehabilitative approaches. We also discuss the effects of stress on executive function after TBI as well as the response of the endocrine system and regulatory feedback mechanisms. The role of the endocannabinoids after CNS injury is also discussed. Finally, we conclude with a discussion of antipsychotic and antiepileptic drugs, which are provided to control TBI-induced agitation and seizures, respectively. The review consists predominantly of published data.

Does time heal all wounds? Experimental diffuse traumatic brain injury results in persisting histopathology in the thalamus.

BACKGROUND: Thalamic dysfunction has been implicated in overall chronic neurological dysfunction after traumatic brain injury (TBI), however little is known about the underlying histopathology. In experimental diffuse TBI (dTBI), we hypothesize that persisting histopathological changes in the ventral posteromedial (VPM) nucleus of the thalamus is indicative of progressive circuit reorganization. Since circuit reorganization in the VPM impacts the whisker sensory system, the histopathology could explain the development of hypersensitivity to whisker stimulation by 28days post-injury; similar to light and sound hypersensitivity in human TBI survivors. METHODS: Adult, male Sprague-Dawley rats underwent craniotomy and midline fluid percussion injury (FPI) (moderate severity; 1.8-2.0atm) or sham surgery. At 1d, 7d, and 28days post-FPI (d FPI) separate experiments confirmed the cytoarchitecture (Giemsa stain) and evaluated neuropathology (silver stain), activated astrocytes (GFAP), neuron morphology (Golgi stain) and microglial morphology (Iba-1) in the VPM. RESULTS: Cytoarchitecture was unchanged throughout the time course, similar to previously published data; however, neuropathology and astrocyte activation were significantly increased at 7d and 28d and activated microglia were present at all time points. Neuron morphology was dynamic over the time course with decreased dendritic complexity (fewer branch points; decreased length of processes) at 7d FPI and return to sham values by 28d FPI. CONCLUSIONS: These data indicate that dTBI results in persisting thalamic histopathology out to a chronic time point. While these changes can be indicative of either adaptive (recovery) or maladaptive (neurological dysfunction) circuit reorganization, they also provide a potential mechanism by which maladaptive circuit reorganization could contribute to the development of chronic neurological dysfunction. Understanding the processes that mediate circuit reorganization is critical to the development of future therapies for TBI patients.

Rehabilitation modality and onset differentially influence whisker sensory hypersensitivity after diffuse traumatic brain injury in the rat.

BACKGROUND: As rehabilitation strategies advance as therapeutic interventions, the modality and onset of rehabilitation after traumatic brain injury (TBI) are critical to optimize treatment. Our laboratory has detected and characterized a late-onset, long-lasting sensory hypersensitivity to whisker stimulation in diffuse brain-injured rats; a deficit that is comparable to visual or auditory sensory hypersensitivity in humans with an acquired brain injury. OBJECTIVE: We hypothesize that the modality and onset of rehabilitation therapies will differentially influence sensory hypersensitivity in response to the Whisker Nuisance Task (WNT) as well as WNT-induced corticosterone (CORT) stress response in diffuse brain-injured rats and shams. METHODS: After midline fluid percussion brain injury (FPI) or sham surgery, rats were assigned to one of four rehabilitative interventions: (1) whisker sensory deprivation during week one or (2) week two or (3) whisker stimulation during week one or (4) week two. At 28 days following FPI and sham procedures, sensory hypersensitivity was assessed using the WNT. Plasma CORT was evaluated immediately following the WNT (aggravated levels) and prior to the pre-determined endpoint 24 hours later (non-aggravated levels). RESULTS: Deprivation therapy during week two elicited significantly greater sensory hypersensitivity to the WNT compared to week one (p < 0.05), and aggravated CORT levels in FPI rats were significantly lower than sham levels. Stimulation therapy during week one resulted in low levels of sensory hypersensitivity to the WNT, similar to deprivation therapy and naïve controls, however, non-aggravated CORT levels in FPI rats were significantly higher than sham. CONCLUSION: These data indicate that modality and onset of sensory rehabilitation can differentially influence FPI and sham rats, having a lasting impact on behavioral and stress responses to the WNT, emphasizing the necessity for continued evaluation of modality and onset of rehabilitation after TBI.