Rachel K. Rowe

The p38α MAPK Regulates Microglial Responsiveness to Diffuse Traumatic Brain Injury

Neuropathology after traumatic brain injury (TBI) is the result of both the immediate impact injury and secondary injury mechanisms. Unresolved post-traumatic glial activation is a secondary injury mechanism that contributes to a chronic state of neuroinflammation in both animal models of TBI and human head injury patients. We recently demonstrated, using in vitro models, that p38α MAPK signaling in microglia is a key event in promoting cytokine production in response to diverse disease-relevant stressors and subsequent inflammatory neuronal dysfunction. From these findings, we hypothesized that the p38α signaling pathway in microglia could be contributing to the secondary neuropathologic sequelae after a diffuse TBI. Mice where microglia were p38α-deficient (p38α KO) were protected against TBI-induced motor deficits and synaptic protein loss. In wild-type (WT) mice, diffuse TBI produced microglia morphological activation that lasted for at least 7 d; however, p38α KO mice failed to activate this response. Unexpectedly, we found that the peak of the early, acute phase cytokine and chemokine levels was increased in injured p38α KO mice compared with injured WT mice. The increased cytokine levels in the p38α KO mice could not be accounted for by more infiltration of macrophages or neutrophils, or increased astrogliosis. By 7 d after injury, the cytokine and chemokine levels remained elevated in injured WT mice but not in p38α KO mice. Together, these data suggest that p38α balances the inflammatory response by acutely attenuating the early proinflammatory cytokine surge while perpetuating the chronic microglia activation after TBI.

Resolvins AT-D1 and E1 differentially impact functional outcome, post-traumatic sleep, and microglial activation following diffuse brain injury in the mouse.

Traumatic brain injury (TBI) is induced by mechanical forces which initiate a cascade of secondary injury processes, including inflammation. Therapies which resolve the inflammatory response may promote neural repair without exacerbating the primary injury. Specific derivatives of omega-3 fatty acids loosely grouped as specialized pro-resolving lipid mediators (SPMs) and termed resolvins promote the active resolution of inflammation. In the current study, we investigate the effect of two resolvin molecules, RvE1 and AT-RvD1, on post-traumatic sleep and functional outcome following diffuse TBI through modulation of the inflammatory response. Adult, male C57BL/6 mice were injured using a midline fluid percussion injury (mFPI) model (6-10min righting reflex time for brain-injured mice). Experimental groups included mFPI administered RvE1 (100ng daily), AT-RvD1 (100ng daily), or vehicle (sterile saline) and counterbalanced with uninjured sham mice. Resolvins or saline were administered daily for seven consecutive days beginning 3days prior to TBI to evaluate proof-of-principle to improve outcome. Immediately following diffuse TBI, post-traumatic sleep was recorded for 24h post-injury. For days 1-7 post-injury, motor outcome was assessed by rotarod. Cognitive function was measured at 6days post-injury using novel object recognition (NOR). At 7days post-injury, microglial activation was quantified using immunohistochemistry for Iba-1. In the diffuse brain-injured mouse, AT-RvD1 treatment, but not RvE1, mitigated motor and cognitive deficits. RvE1 treatment significantly increased post-traumatic sleep in brain-injured mice compared to all other groups. RvE1 treated mice displayed a higher proportion of ramified microglia and lower proportion of activated rod microglia in the cortex compared to saline or AT-RvD1 treated brain-injured mice. Thus, RvE1 treatment modulated post-traumatic sleep and the inflammatory response to TBI, albeit independently of improvement in motor and cognitive outcome as seen in AT-RvD1-treated mice. This suggests AT-RvD1 may impart functional benefit through mechanisms other than resolution of inflammation alone.

Diffuse brain injury induces acute post-traumatic sleep.

Objective Clinical observations report excessive sleepiness immediately following traumatic brain injury (TBI); however, there is a lack of experimental evidence to support or refute the benefit of sleep following a brain injury. The aim of this study is to investigate acute post-traumatic sleep. Methods Sham, mild or moderate diffuse TBI was induced by midline fluid percussion injury (mFPI) in male C57BL/6J mice at 9:00 or 21:00 to evaluate injury-induced sleep behavior at sleep and wake onset, respectively. Sleep profiles were measured post-injury using a non-invasive, piezoelectric cage system. In separate cohorts of mice, inflammatory cytokines in the neocortex were quantified by immunoassay, and microglial activation was visualized by immunohistochemistry. Results Immediately after diffuse TBI, quantitative measures of sleep were characterized by a significant increase in sleep (>50%) for the first 6 hours post-injury, resulting from increases in sleep bout length, compared to sham. Acute post-traumatic sleep increased significantly independent of injury severity and time of injury (9:00 vs 21:00). The pro-inflammatory cytokine IL-1β increased in brain-injured mice compared to sham over the first 9 hours post-injury. Iba-1 positive microglia were evident in brain-injured cortex at 6 hours post-injury. Conclusion Post-traumatic sleep occurs for up to 6 hours after diffuse brain injury in the mouse regardless of injury severity or time of day. The temporal profile of secondary injury cascades may be driving the significant increase in post-traumatic sleep and contribute to the natural course of recovery through cellular repair.

Recovery of neurological function despite immediate sleep disruption following diffuse brain injury in the mouse: clinical relevance to medically untreated concussion.

STUDY OBJECTIVE We investigated the relationship between immediate disruption of posttraumatic sleep and functional outcome in the diffuse brain-injured mouse. DESIGN Adult male C57BL/6 mice were subjected to moderate midline fluid percussion injury (n = 65; 1.4 atm; 6-10 min righting reflex time) or sham injury (n = 44). Cohorts received either intentional sleep disruption (minimally stressful gentle handling) or no sleep disruption for 6 h following injury. Following disruption, serum corticosterone levels (enzyme-linked immunosorbent assay) and posttraumatic sleep (noninvasive piezoelectric sleep cages) were measured. For 1-7 days postinjury, sensorimotor outcome was assessed by Rotarod and a modified Neurological Severity Score (NSS). Cognitive function was measured using Novel Object Recognition (NOR) and Morris water maze (MWM) in the first week postinjury. SETTING Neurotrauma research laboratory. MEASUREMENTS AND RESULTS Disrupting posttraumatic sleep for 6 h did not affect serum corticosterone levels or functional outcome. In the hour following the first dark onset, sleep-disrupted mice exhibited a significant increase in sleep; however, this increase was not sustained and there was no rebound of lost sleep. Regardless of sleep disruption, mice showed a time-dependent improvement in Rotarod performance, with brain-injured mice having significantly shorter latencies on day 7 compared to sham. Further, brain-injured mice, regardless of sleep disruption, had significantly higher NSS scores postinjury compared with sham. Cognitive behavioral testing showed no group differences among any treatment group measured by MWM and NOR. CONCLUSION Short-duration disruption of posttraumatic sleep did not affect functional outcome, measured by motor and cognitive performance. These data raise uncertainty about posttraumatic sleep as a mechanism of recovery from diffuse brain injury.

Diffuse brain injury does not affect chronic sleep patterns in the mouse

Abstract Primary objective: To test if the current model of diffuse brain injury produces chronic sleep disturbances similar to those reported by TBI patients. Methods and procedures: Adult male C57BL/6 mice were subjected to moderate midline fluid percussion injury (n = 7; 1.4 atm; 6–10 minutes righting reflex time) or sham injury (n = 5). Sleep–wake activity was measured post-injury using a non-invasive, piezoelectric cage system. Chronic sleep patterns were analysed weekly for increases or decreases in percentage sleep (hypersomnia or insomnia) and changes in bout length (fragmentation). Main outcomes and results: During the first week after diffuse TBI, brain-injured mice exhibited increased mean percentage sleep and mean bout length compared to sham-injured mice. Further analysis indicated the increase in mean percentage sleep occurred during the dark cycle. Injury-induced changes in sleep, however, did not extend beyond the first week post-injury and were not present in weeks 2–5 post-injury. Conclusions: Previously, it has been shown that the midline fluid percussion model used in this study immediately increased post-traumatic sleep. The current study extended the timeline of investigation to show that sleep disturbances extended into the first week post-injury, but did not develop into chronic sleep disturbances. However, the clinical prevalence of TBI-related sleep–wake disturbances warrants further experimental investigation.

MW151 Inhibited IL-1β Levels after Traumatic Brain Injury with No Effect on Microglia Physiological Responses

A prevailing neuroinflammation hypothesis is that increased production of proinflammatory cytokines contributes to progressive neuropathology, secondary to the primary damage caused by a traumatic brain injury (TBI). In support of the hypothesis, post-injury interventions that inhibit the proinflammatory cytokine surge can attenuate the progressive pathology. However, other post-injury neuroinflammatory responses are key to endogenous recovery responses. Therefore, it is critical that pharmacological attenuation of detrimental or dysregulated neuroinflammatory processes avoid pan-suppression of inflammation. MW151 is a CNS-penetrant, small molecule experimental therapeutic that restores injury- or disease-induced overproduction of proinflammatory cytokines towards homeostasis without immunosuppression. Post-injury administration of MW151 in a closed head injury model of mild TBI suppressed acute cytokine up-regulation and downstream cognitive impairment. Here, we report results from a diffuse brain injury model in mice using midline fluid percussion. Low dose (0.5–5.0 mg/kg) administration of MW151 suppresses interleukin-1 beta (IL-1β) levels in the cortex while sparing reactive microglia and astrocyte responses. To probe molecular mechanisms, we used live cell imaging of the BV-2 microglia cell line to demonstrate that MW151 does not affect proliferation, migration, or phagocytosis of the cells. Our results provide insight into the roles of glial responses to brain injury and indicate the feasibility of using appropriate dosing for selective therapeutic modulation of injurious IL-1β increases while sparing other glial responses to injury.

Using anesthetics and analgesics in experimental traumatic brain injury.

Valid modeling of traumatic brain injury (TBI) requires accurate replication of both the mechanical forces that cause the primary injury and the conditions that lead to secondary injuries observed in human patients. The use of animals in TBI research is justified by the lack of in vitro or computer models that can sufficiently replicate the complex pathological processes involved. Measures to reduce nociception and distress must be implemented, but the administration of anesthetics and analgesics can influence TBI outcomes, threatening the validity of the research. In this review, the authors present evidence for the interference of anesthetics and analgesics in the natural course of brain injury in animal models of TBI. They suggest that drugs should be selected for or excluded from experimental TBI protocols on the basis of IACUC-approved experimental objectives in order to protect animal welfare and preserve the validity of TBI models.

Aging with a traumatic brain injury: Could behavioral morbidities and endocrine symptoms be influenced by microglial priming?

A myriad of factors influence the developmental and aging process and impact health and life span. Mounting evidence indicates that brain injury, even moderate injury, can lead to lifetime of physical and mental health symptoms. Therefore, the purpose of this mini-review is to discuss how recovery from traumatic brain injury (TBI) depends on age-at-injury and how aging with a TBI affects long-term recovery. TBI initiates pathophysiological processes that dismantle circuits in the brain. In response, reparative and restorative processes reorganize circuits to overcome the injury-induced damage. The extent of circuit dismantling and subsequent reorganization depends as much on the initial injury parameters as other contributing factors, such as genetics and age. Age-at-injury influences the way the brain is able to repair itself, as a result of developmental status, extent of cellular senescence, and injury-induced inflammation. Moreover, endocrine dysfunction can occur with TBI. Depending on the age of the individual at the time of injury, endocrine dysfunction may disrupt growth, puberty, influence social behaviors, and possibly alter the inflammatory response. In turn, activation of microglia, the brains immune cells, after injury may continue to fuel endocrine dysfunction. With age, the immune system develops and microglia become primed to subsequent challenges. Sustained inflammation and microglial activation can continue for weeks to months post-injury. This prolonged inflammation can influence developmental processes, behavioral performance and age-related decline. Overall, brain injury may influence the aging process and expedite glial and neuronal alterations that impact mental health.

Diffuse traumatic brain injury affects chronic corticosterone function in the rat

As many as 20–55% of patients with a history of traumatic brain injury (TBI) experience chronic endocrine dysfunction, leading to impaired quality of life, impaired rehabilitation efforts and lowered life expectancy. Endocrine dysfunction after TBI is thought to result from acceleration–deceleration forces to the brain within the skull, creating enduring hypothalamic and pituitary neuropathology, and subsequent hypothalamic–pituitary endocrine (HPE) dysfunction. These experiments were designed to test the hypothesis that a single diffuse TBI results in chronic dysfunction of corticosterone (CORT), a glucocorticoid released in response to stress and testosterone. We used a rodent model of diffuse TBI induced by midline fluid percussion injury (mFPI). At 2months postinjury compared with uninjured control animals, circulating levels of CORT were evaluated at rest, under restraint stress and in response to dexamethasone, a synthetic glucocorticoid commonly used to test HPE axis regulation. Testosterone was evaluated at rest. Further, we assessed changes in injury-induced neuron morphology (Golgi stain), neuropathology (silver stain) and activated astrocytes (GFAP) in the paraventricular nucleus (PVN) of the hypothalamus. Resting plasma CORT levels were decreased at 2months postinjury and there was a blunted CORT increase in response to restraint induced stress. No changes in testosterone were measured. These changes in CORT were observed concomitantly with altered complexity of neuron processes in the PVN over time, devoid of neuropathology or astrocytosis. Results provide evidence that a single moderate diffuse TBI leads to changes in CORT function, which can contribute to the persistence of symptoms related to endocrine dysfunction. Future experiments aim to evaluate additional HP-related hormones and endocrine circuit pathology following diffuse TBI.

Clinical relevance of midline fluid percussion brain injury: Acute deficits, chronic morbidities and the utility of biomarkers

Abstract Background: After 30 years of characterisation and implementation, fluid percussion injury (FPI) is firmly recognised as one of the best-characterised reproducible and clinically relevant models of TBI, encompassing concussion through diffuse axonal injury (DAI). Depending on the specific injury parameters (e.g. injury site, mechanical force), FPI can model diffuse TBI with or without a focal component and may be designated as mild-to-severe according to the chosen mechanical forces and resulting acute neurological responses. Among FPI models, midline FPI may best represent clinical diffuse TBI, because of the acute behavioural deficits, the transition to late-onset behavioural morbidities and the absence of gross histopathology. Review: The goal here was to review acute and chronic physiological and behavioural deficits and morbidities associated with diffuse TBI induced by midline FPI. In the absence of neurodegenerative sequelae associated with focal injury, there is a need for biomarkers in the diagnostic, prognostic, predictive and therapeutic approaches to evaluate outcomes from TBI. Conclusions: The current literature suggests that midline FPI offers a clinically-relevant, validated model of diffuse TBI to investigators wishing to evaluate novel therapeutic strategies in the treatment of TBI and the utility of biomarkers in the delivery of healthcare to patients with brain injury.