Proteomic analysis identifies plasma correlates of remote ischemic conditioning in the context of experimental traumatic brain injury.

Remote ischemic conditioning (RIC), transient restriction and recirculation of blood flow to a limb after traumatic brain injury (TBI), can modify levels of pathology-associated circulating protein. This study sought to identify TBI-induced molecular alterations in plasma and whether RIC would modulate protein and metabolite levels at 24 h after diffuse TBI. Adult male C57BL/6 mice received diffuse TBI by midline fluid percussion or were sham-injured. Mice were assigned to treatment groups 1 h after recovery of righting reflex: sham, TBI, sham RIC, TBI RIC. Nine plasma metabolites were significantly lower post-TBI (six amino acids, two acylcarnitines, one carnosine). RIC intervention returned metabolites to sham levels. Using proteomics analysis, twenty-four putative protein markers for TBI and RIC were identified. After application of Benjamini-Hochberg correction, actin, alpha 1, skeletal muscle (ACTA1) was found to be significantly increased in TBI compared to both sham groups and TBI RIC. Thus, identified metabolites and proteins provide potential biomarkers for TBI and therapeutic RIC in order to monitor disease progression and therapeutic efficacy.

Acute Post-Traumatic Sleep May Define Vulnerability to a Second Traumatic Brain Injury in Mice.

Chronic neurological impairments can manifest from repetitive traumatic brain injury (rTBI), particularly when subsequent injuries occur before the initial injury completely heals. Herein, we apply post-traumatic sleep as a physiological biomarker of vulnerability, hypothesizing that a second TBI during post-traumatic sleep worsens neurological and histological outcomes compared to one TBI or a second TBI after post-traumatic sleep subsides. Mice received sham or diffuse TBI by midline fluid percussion injury; brain-injured mice received one TBI or rTBIs at 3- or 9-h intervals. Over 40 h post-injury, injured mice slept more than shams. Functional assessments indicated lower latencies on rotarod and increased Neurological Severity Scores for mice with rTBIs within 3 h. Anxiety-like behaviors in the open field task were increased for mice with rTBIs at 3 h. Based on pixel density of silver accumulation, neuropathology was greater at 28 days post-injury (DPI) in rTBI groups than sham and single TBI. Cortical microglia morphology was quantified and mice receiving rTBI were de-ramified at 14 DPI compared to shams and mice receiving a single TBI, suggesting robust microglial response in rTBI groups. Orexin-A-positive cells were sustained in the lateral hypothalamus with no loss detected, indicating that loss of wake-promoting neurons did not contribute to post-traumatic sleep. Thus, duration of post-traumatic sleep is a period of vulnerability that results in exacerbated injury from rTBI. Monitoring individual post-traumatic sleep is a potential clinical tool for personalized TBI management, where regular sleep patterns may inform rehabilitative strategies and return-to-activity guidelines.

Simultaneous Cryosectioning of Multiple Rodent Brains.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Novel TNF receptor-1 inhibitors identified as potential therapeutic candidates for traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) begins with the application of mechanical force to the head or brain, which initiates systemic and cellular processes that are hallmarks of the disease. The pathological cascade of secondary injury processes, including inflammation, can exacerbate brain injury-induced morbidities and thus represents a plausible target for pharmaceutical therapies. We have pioneered research on post-traumatic sleep, identifying that injury-induced sleep lasting for 6 h in brain-injured mice coincides with increased cortical levels of inflammatory cytokines, including tumor necrosis factor (TNF). Here, we apply post-traumatic sleep as a physiological bio-indicator of inflammation. We hypothesized the efficacy of novel TNF receptor (TNF-R) inhibitors could be screened using post-traumatic sleep and that these novel compounds would improve functional recovery following diffuse TBI in the mouse. METHODS: Three inhibitors of TNF-R activation were synthesized based on the structure of previously reported TNF CIAM inhibitor F002, which lodges into a defined TNFR1 cavity at the TNF-binding interface, and screened for in vitro efficacy of TNF pathway inhibition (IκB phosphorylation). Compounds were screened for in vivo efficacy in modulating post-traumatic sleep. Compounds were then tested for efficacy in improving functional recovery and verification of cellular mechanism. RESULTS: Brain-injured mice treated with Compound 7 (C7) or SGT11 slept significantly less than those treated with vehicle, suggesting a therapeutic potential to target neuroinflammation. SGT11 restored cognitive, sensorimotor, and neurological function. C7 and SGT11 significantly decreased cortical inflammatory cytokines 3 h post-TBI. CONCLUSIONS: Using sleep as a bio-indicator of TNF-R-dependent neuroinflammation, we identified C7 and SGT11 as potential therapeutic candidates for TBI.

Midline (central) fluid percussion model of traumatic brain injury in pediatric and adolescent rats.

OBJECTIVE Experimental traumatic brain injury (TBI) models hold significant validity to the human condition, with each model replicating a subset of clinical features and symptoms. TBI is the leading cause of mortality and morbidity in children and teenagers; thus, it is critical to develop preclinical models of these ages to test emerging treatments. Midline fluid percussion injury (FPI) might best represent mild and diffuse clinical brain injury because of the acute behavioral deficits, the late onset of behavioral morbidities, and the absence of gross histopathology. In this study, the authors sought to adapt a midline FPI to postnatal day (PND) 17 and 35 rats. The authors hypothesized that scaling the craniectomy size based on skull dimensions would result in a reproducible injury comparable to the standard midline FPI in adult rats. METHODS PND17 and PND35 rat skulls were measured, and trephines were scaled based on skull size. Custom trephines were made. Rats arrived on PND10 and were randomly assigned to one of 3 cohorts: PND17, PND35, and 2 months old. Rats were subjected to midline FPI, and the acute injury was characterized. The right reflex was recorded, injury-induced apnea was measured, injury-induced seizure was noted, and the brains were immediately examined for hematoma. RESULTS The authors' hypothesis was supported; scaling the trephines based on skull size led to a reproducible injury in the PND17 and PND35 rats that was comparable to the injury in a standard 2-month-old adult rat. The midline FPI suppressed the righting reflex in both the PND17 and PND35 rats. The injury induced apnea in PND17 rats that lasted significantly longer than that in PND35 and 2-month-old rats. The injury also induced seizures in 73% of PND17 rats compared with 9% of PND35 rats and 0% of 2-month-old rats. There was also a significant relationship between the righting reflex time and presence of seizure. Both PND17 and PND35 rats had visible hematomas with an intact dura, indicative of diffuse injury comparable to the injury observed in 2-month-old rats. CONCLUSIONS With these procedures, it becomes possible to generate brain-injured juvenile rats (pediatric [PND17] and adolescent [PND35]) for studies of injury-induced pathophysiology and behavioral deficits, for which rational therapeutic interventions can be implemented.

Aging with Traumatic Brain Injury: Effects of Age at Injury on Behavioral Outcome following Diffuse Brain Injury in Rats.

Development and aging are influenced by external factors with the potential to impact health throughout the life span. Traumatic brain injury (TBI) can initiate and sustain a lifetime of physical and mental health symptoms. Over 1.7 million TBIs occur annually in the USA alone, with epidemiology suggesting a higher incidence for young age groups. Additionally, increasing life spans mean more years to age with TBI. While there is ongoing research of experimental pediatric and adult TBI, few studies to date have incorporated animal models of pediatric, adolescent, and adult TBI to understand the role of age at injury across the life span. Here, we explore repeated behavioral performance between rats exposed to diffuse TBI at five different ages. Our aim was to follow neurological morbidities across the rodent life span with respect to age at injury. A single cohort of male Sprague-Dawley rats (n = 69) was received at postnatal day (PND) 10. Subgroups of this cohort (n = 11-12/group) were subjected to a single moderate midline fluid percussion injury at age PND 17, PND 35, 2 months, 4 months, or 6 months. A control group of naïve rats (n = 12) was assembled from this cohort. The entire cohort was assessed for motor function by beam walk at 1.5, 3, 5, and 7 months of age. Anxiety-like behavior was assessed with the open field test at 8 months of age. Cognitive performance was assessed using the novel object location task at 8, 9, and 10 months of age. Depression-like behavior was assessed using the forced swim test at 10 months of age. Age at injury and time since injury differentially influenced motor, cognitive, and affective behavioral outcomes. Motor and cognitive deficits occurred in rats injured at earlier developmental time points, but not in rats injured in adulthood. In contrast, rats injured during adulthood showed increased anxiety-like behavior compared to uninjured control rats. A single diffuse TBI did not result in chronic depression-like behaviors or changes in body weight among any groups. The interplay of age at injury and aging with an injury are translationally important factors that influence behavioral performance as a quality of life metric. More complete understanding of these factors can direct rehabilitative efforts and personalized medicine for TBI survivors.

Diffuse traumatic brain injury induces prolonged immune dysregulation and potentiates hyperalgesia following a peripheral immune challenge.

BACKGROUND: Nociceptive and neuropathic pain occurs as part of the disease process after traumatic brain injury (TBI) in humans. Central and peripheral inflammation, a major secondary injury process initiated by the traumatic brain injury event, has been implicated in the potentiation of peripheral nociceptive pain. We hypothesized that the inflammatory response to diffuse traumatic brain injury potentiates persistent pain through prolonged immune dysregulation. RESULTS: To test this, adult, male C57BL/6 mice were subjected to midline fluid percussion brain injury or to sham procedure. One cohort of mice was analyzed for inflammation-related cytokine levels in cortical biopsies and serum along an acute time course. In a second cohort, peripheral inflammation was induced seven days after surgery/injury with an intraplantar injection of carrageenan. This was followed by measurement of mechanical hyperalgesia, glial fibrillary acidic protein and Iba1 immunohistochemical analysis of neuroinflammation in the brain, and flow cytometric analysis of T-cell differentiation in mucosal lymph. Traumatic brain injury increased interleukin-6 and chemokine ligand 1 levels in the cortex and serum that peaked within 1-9 h and then resolved. Intraplantar carrageenan produced mechanical hyperalgesia that was potentiated by traumatic brain injury. Further, mucosal T cells from brain-injured mice showed a distinct deficiency in the ability to differentiate into inflammation-suppressing regulatory T cells (Tregs). CONCLUSIONS: We conclude that traumatic brain injury increased the inflammatory pain associated with cutaneous inflammation by contributing to systemic immune dysregulation. Regulatory T cells are immune suppressors and failure of T cells to differentiate into regulatory T cells leads to unregulated cytokine production which may contribute to the potentiation of peripheral pain through the excitation of peripheral sensory neurons. In addition, regulatory T cells are identified as a potential target for therapeutic rebalancing of peripheral immune homeostasis to improve functional outcome and decrease the incidence of peripheral inflammatory pain following traumatic brain injury.

Experimental diffuse brain injury results in regional alteration of gross vascular morphology independent of neuropathology.

PRIMARY OBJECTIVE: A dynamic relationship exists between diffuse traumatic brain injury and changes to the neurovascular unit. The purpose of this study was to evaluate vascular changes during the first week following diffuse TBI. It was hypothesized that pathology is associated with modification of the vasculature. METHODS: Male Sprague-Dawley rats underwent either midline fluid percussion injury or sham-injury. Brain tissue was collected 1, 2 or 7 days post-injury or sham-injury (n = 3/time point). Tissue was collected and stained by de Olmos amino-cupric silver technique to visualize neuropathology or animals were perfused with AltaBlue casting resin before high-resolution vascular imaging. The average volume, surface area, radius, branching and tortuosity of the vessels were evaluated across three regions of interest. RESULTS: In M2, average vessel volume (p < 0.01) and surface area (p < 0.05) were significantly larger at 1 day relative to 2 days, 7 days and sham. In S1BF and VPM, no significant differences in the average vessel volume or surface area at any of the post-injury time points were observed. No significant changes in average radius, branching or tortuosity were observed. CONCLUSIONS: Preliminary findings suggest gross morphological changes within the vascular network likely represent an acute response to mechanical forces of injury, rather than delayed or chronic pathological processes.

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.

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.

Acute over-the-counter pharmacological intervention does not adversely affect behavioral outcome following diffuse traumatic brain injury in the mouse.

Following mild traumatic brain injury (TBI), patients may self-treat symptoms of concussion, including post-traumatic headache, taking over-the-counter (OTC) analgesics. Administering one dose of OTC analgesics immediately following experimental brain injury mimics the at-home treated population of concussed patients and may accelerate the understanding of the relationship between brain injury and OTC pharmacological intervention. In the current study, we investigate the effect of acute administration of OTC analgesics on neurological function and cortical cytokine levels after experimental diffuse TBI in the mouse. Adult, male C57BL/6 mice were injured using a midline fluid percussion (mFPI) injury model of concussion (6-10 min righting reflex time for brain-injured mice). Experimental groups included mFPI paired with either ibuprofen (60 mg/kg, i.p.; n = 16), acetaminophen (40 mg/kg, i.p.; n = 9), or vehicle (15% ethanol (v/v) in 0.9% saline; n = 13) and sham injury paired OTC medicine or vehicle (n = 7-10 per group). At 24 h after injury, functional outcome was assessed using the rotarod task and a modified neurological severity score. Following behavior assessment, cortical cytokine levels were measured by multiplex ELISA at 24 h post-injury. To evaluate efficacy on acute inflammation, cortical cytokine levels were measured also at 6 h post-injury. In the diffuse brain-injured mouse, immediate pharmacological intervention did not attenuate or exacerbate TBI-induced functional deficits. Cortical cytokine levels were affected by injury, time, or their interaction. However, levels were not affected by treatment at 6 or 24 h post-injury. These data indicate that acute administration of OTC analgesics did not exacerbate or attenuate brain-injury deficits which may inform clinical recommendations for the at-home treated mildly concussed patient.

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

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.

Neuropathology in sensory, but not motor, brainstem nuclei of the rat whisker circuit after diffuse brain injury.

Neurological dysfunction after traumatic brain injury (TBI) is associated with pathology in cortical, subcortical, and brainstem nuclei. Our laboratory has reported neuropathology and microglial activation in the somatosensory barrel cortex (S1BF) and ventral posterior medial thalamus (VPM) after diffuse TBI in the rat, which correlated with post-injury whisker sensory sensitivity. The present study extends our previous work by evaluating pathology in whisking-associated sensory and motor brainstem nuclei. Brains from adult, male rats were recovered over 1 month after midline fluid percussion or sham injury. The principal trigeminal nucleus (PrV, sensory nucleus) and facial nucleus (VIIN, motor nucleus) were examined for neuropathology (silver histochemistry) and microglial activation (Iba1). Significant neuropathology in PrV was evident at 2 and 7 days post-injury compared to sham. Iba1-labeled microglia showed swollen somata and thickened processes over 1 month post-injury. In contrast, the VIIN showed non-significant neuropathology and reduced labeling of activated Iba1 microglia over 1 month post-injury. Together with our previous data, neuropathology and neuroinflammation in the whisker somatosensory pathway may contribute to post-injury sensory sensitivity more than the motor pathway. Whether these findings are direct results of the mechanical injury or consequences of progressive degeneration remains to be determined.

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.

Rod microglia: elongation, alignment, and coupling to form trains across the somatosensory cortex after experimental diffuse brain injury.

BACKGROUND: Since their discovery, the morphology of microglia has been interpreted to mirror their function, with ramified microglia constantly surveying the micro-environment and rapidly activating when changes occur. In 1899, Franz Nissl discovered what we now recognize as a distinct microglial activation state, microglial rod cells (Stäbchenzellen), which he observed adjacent to neurons. These rod-shaped microglia are typically found in human autopsy cases of paralysis of the insane, a disease of the pre-penicillin era, and best known today from HIV-1-infected brains. Microglial rod cells have been implicated in cortical 'synaptic stripping' but their exact role has remained unclear. This is due at least in part to a scarcity of experimental models. Now we have noted these rod microglia after experimental diffuse brain injury in brain regions that have an associated sensory sensitivity. Here, we describe the time course, location, and surrounding architecture associated with rod microglia following experimental diffuse traumatic brain injury (TBI). METHODS: Rats were subjected to a moderate midline fluid percussion injury (mFPI), which resulted in transient suppression of their righting reflex (6 to 10 min). Multiple immunohistochemistry protocols targeting microglia with Iba1 and other known microglia markers were undertaken to identify the morphological activation of microglia. Additionally, labeling with Iba1 and cell markers for neurons and astrocytes identified the architecture that surrounds these rod cells. RESULTS: We identified an abundance of Iba1-positive microglia with rod morphology in the primary sensory barrel fields (S1BF). Although present for at least 4 weeks post mFPI, they developed over the first week, peaking at 7 days post-injury. In the absence of contusion, Iba1-positive microglia appear to elongate with their processes extending from the apical and basal ends. These cells then abut one another and lay adjacent to cytoarchitecture of dendrites and axons, with no alignment with astrocytes and oligodendrocytes. Iba1-positive rod microglial cells differentially express other known markers for reactive microglia including OX-6 and CD68. CONCLUSION: Diffuse traumatic brain injury induces a distinct rod microglia morphology, unique phenotype, and novel association between cells; these observations entice further investigation for impact on neurological outcome.