Search

Following a traumatic brain injury (TBI), sleep-wake disturbances are one of the most prevalent and debilitating symptoms. A better understanding of the impact that sleep disturbances have on chronic TBI symptomatology is likely to maximize long-term recovery. While the current treatments of sleep-wake disturbances following TBI are disorder-specific, identifying the underlying pathology may lead to improved pharmacological and rehabilitative treatments. A TBI initiates secondary cellular and molecular cascades that include inflammation and the production of cytokines, as well endocrine dysfunction and concomitant disturbances in hormone secretions. Hormones and cytokines are mediators of the inflammatory response that also regulate endocrine function, thus, communication between the immune and endocrine systems is bidirectional. Interestingly, both the immune and endocrine systems play a critical role in sleep regulation. This narrative review summarizes sleep-wake disturbances reported after TBI and synthesizes the current human and animal literature centered on the hypothesis that immune-endocrine interactions after TBI may induce both acute and chronic disturbances of sleep and wakefulness. Furthermore, we discuss how the immune and endocrine systems may be plausible therapeutic targets to treat TBI-induced sleep disturbances.

Intimate partner violence (IPV) increases risk of traumatic brain injury (TBI). Physical assaults increase in frequency and intensity during pregnancy. The consequences of TBI during pregnancy (gravida TBI; gTBI) on offspring development is unknown, for which stress and inflammation during pregnancy worsen fetal developmental outcomes. We hypothesized that gTBI would lead to increased anxiety- and depression-related behavior, altered inflammatory responses and gut pathology, and distorted brain circuitry in mixed-sex offspring compared to mice born to control mothers. Pregnant dams received either diffuse TBI or sham injury (control) 12 days post-coitum. We found that male gTBI offspring were principal drivers of the gTBI effects on health, physiology, and behavior. For example, male, but not female, gTBI offspring weighed significantly less at weaning compared to male control offspring. At post-natal day (PND) 28, gTBI offspring had significantly weaker intralaminar connectivity onto layer 5 pre-frontal pyramidal neurons compared to control offspring. Neurological performance on anxiety-like behaviors was decreased, with only marginal differences in depressive-like behaviors, for gTBI offspring compared to control offspring. At PND42 and PND58, circulating neutrophil and monocyte populations were significantly smaller in gTBI male offspring than control male offspring. In response to a subsequent inflammatory challenge at PND75, gTBI offspring had significantly smaller circulating neutrophil populations than control offspring. Anxiety-like behaviors persisted during the immune challenge in gTBI offspring. However, spleen immune response and gut histology showed no significant differences between groups. The results compel further studies to determine the full extent of gTBI on fetal and maternal outcomes.

Traumatic brain injury (TBI) can induce acute lung injury (ALI). The exact pathomechanism of TBI-induced ALI is poorly understood, limiting treatment options. Remote ischemic conditioning (RIC) can mitigate detrimental outcomes following transplants, cardiac arrests, and neurological injuries. In this study, we hypothesized that RIC would reduce TBI-induced ALI by regulating the sphingosine-1-phosphate (S1P)-dependent pathway, a central regulator of endothelial barrier integrity, lymphocyte, and myokine trafficking. Male mice were subjected to either diffuse TBI by midline fluid percussion or control sham injury and randomly assigned among four groups: sham, TBI, sham RIC, or TBI RIC; RIC was performed 1 h prior to TBI. Mice were euthanized at 1-h postinjury or 7 days post-injury (DPI) and lung tissue, bronchoalveolar lavage (BAL) fluid, and blood were collected. Lung tissue was analyzed for histopathology, irisin myokine levels, and S1P receptor levels. BAL fluid and blood were analyzed for cellularity and myokine/S1P levels, respectively. One-hour postinjury, TBI damaged lung alveoli and increased neutrophil infiltration; RIC preserved alveoli. BAL from TBI mice had more neutrophils and higher neutrophil/monocyte ratios compared with sham, where TBI RIC mice showed no injury-induced change. Further, S1P receptor 3 and irisin-associated protein levels were significantly increased in the lungs of TBI mice compared with sham, which was prevented by RIC. However, there was no RIC-associated change in plasma irisin or S1P. At 7 DPI, ALI in TBI mice was largely resolved, with evidence for residual lung pathology. Thus, RIC may be a viable intervention for TBI-induced ALI to preserve lung function and facilitate clinical management.

Microglia undergo dynamic structural and transcriptional changes during the immune response to traumatic brain injury (TBI). For example, TBI causes microglia to form rod-shaped trains in the cerebral cortex, but their contribution to inflammation and pathophysiology is unclear. The purpose of this study was to determine the origin and alignment of rod microglia and to determine the role of microglia in propagating persistent cortical inflammation. Here, diffuse TBI in mice was modeled by midline fluid percussion injury (FPI). Bone marrow chimerism and BrdU pulse-chase experiments revealed that rod microglia derived from resident microglia with limited proliferation. Novel data also show that TBI-induced rod microglia were proximal to axotomized neurons, spatially overlapped with dense astrogliosis, and aligned with apical pyramidal dendrites. Furthermore, rod microglia formed adjacent to hypertrophied microglia, which clustered among layer V pyramidal neurons. To better understand the contribution of microglia to cortical inflammation and injury, microglia were eliminated prior to TBI by CSF1R antagonism (PLX5622). Microglial elimination did not affect cortical neuron axotomy induced by TBI, but attenuated rod microglial formation and astrogliosis. Analysis of 262 immune genes revealed that TBI caused profound cortical inflammation acutely (8 hr) that progressed in nature and complexity by 7 dpi. For instance, gene expression related to complement, phagocytosis, toll-like receptor signaling, and interferon response were increased 7 dpi. Critically, these acute and chronic inflammatory responses were prevented by microglial elimination. Taken together, TBI-induced neuronal injury causes microglia to structurally associate with neurons, augment astrogliosis, and propagate diverse and persistent inflammatory/immune signaling pathways.

Clinically, traumatic brain injury (TBI) results in complex heterogeneous pathology that cannot be recapitulated in single pre-clinical animal model. Therefore, we focused on evaluating utility of nanoparticle (NP)-based therapeutics following three diffuse-TBI models: mildclosed-head injury (mCHI), repetitive-mCHI and midline-fluid percussion injury (FPI). We hypothesized that NP accumulation after diffuse TBI correlates directly with blood–brainbarrier permeability. Mice received PEGylated-NP cocktail (20-500 nm) (intravenously) after single- or repetitive-(1 impact/day, 5 consecutive days) CHI (immediately) and midline-FPI (1 h, 3 h and 6 h). NPs circulated for 1 h before perfusion/brain extraction. NP accumulation was analyzed using fluorescent microscopy in brain regions vulnerable to neuropathology. Minimal/no NP accumulation after mCHI/RmCHI was observed. In contrast, midlineFPI resulted in significant peak accumulation of up to 500 nm NP at 3 h post-injury compared to sham, 1 h, and 6 h groups in the cortex. Therefore, our study provides the groundwork for feasibility of NP-delivery based on NPinjection time and NPsize after mCHI/RmCHI and midline-FPI.

Elderly populations (≥65 years old) have the highest risk of developing Alzheimer's disease (AD) and/or obtaining a traumatic brain injury (TBI). Using translational mouse models, we investigated sleep disturbances and inflammation associated with normal aging, TBI and aging, and AD. We hypothesized that aging results in marked changes in sleep compared with adult mice, and that TBI and aging would result in sleep and inflammation levels similar to AD mice. We used female 16-month-old wild-type (WT Aged) and 3xTg-AD mice, as well as a 2-month-old reference group (WT Adult), to evaluate sleep changes. WT Aged mice received diffuse TBI by midline fluid percussion, and blood was collected from both WT Aged (pre- and post-TBI) and 3xTg-AD mice to evaluate inflammation. Cognitive behavior was tested, and tissue was collected for histology. Bayesian generalized additive and mixed-effects models were used for analyses. Both normal aging and AD led to increases in sleep compared with adult mice. WT Aged mice with TBI slept substantially more, with fragmented shorter bouts, than they did pre-TBI and compared with AD mice. However, differences between WT Aged and 3xTg-AD mice in immune cell populations and plasma cytokine levels were incongruous, cognitive deficits were similar, and cumulative sleep was not predictive of inflammation or behavior for either group. Our results suggest that in similarly aged individuals, TBI immediately induces more profound sleep alterations than in AD, although both diseases likely include cognitive impairments. Unique pathological sleep pathways may exist in elderly individuals who incur TBI compared with similarly aged individuals who have AD, which may warrant disease-specific treatments in clinical settings.

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.

A high-resolution, three-dimensional, optical imaging technique for the murine brain was developed to identify the effects of different therapeutic windows for preclinical brain research. This technique tracks the same cells over several weeks. We conducted a pilot study of a promising drug to treat diffuse axonal injury (DAI) caused by traumatic brain injury, using two different therapeutic windows, as a means to demonstrate the utility of this novel longitudinal imaging technique. DAI causes immediate, sporadic axon damage followed by progressive secondary axon damage. We administered minocycline for three days commencing one hour after injury in one treatment group and beginning 72 hours after injury in another group to demonstrate the method’s ability to show how and when the therapeutic drug exerts protective and/or healing effects. Fewer varicosities developed in acutely treated mice while more varicosities resolved in mice with delayed treatment. For both treatments, the drug arrested development of new axonal damage by 30 days. In addition to evaluation of therapeutics for traumatic brain injury, this hybrid microlens imaging method should be useful to study other types of brain injury and neurodegeneration and cellular responses to treatment.

Identifying differential responses between sexes following traumatic brain injury (TBI) can elucidate the mechanisms behind disease pathology. Peripheral and central inflammation in the pathophysiology of TBI can increase sleep in male rodents, but this remains untested in females. We hypothesized that diffuse TBI would increase inflammation and sleep in males more so than in females. Diffuse TBI was induced in C57BL/6J mice and serial blood samples were collected (baseline, 1, 5, 7 days post-injury [DPI]) to quantify peripheral immune cell populations and sleep regulatory cytokines. Brains and spleens were harvested at 7DPI to quantify central and peripheral immune cells, respectively. Mixed effects regression models were used for data analysis. Female TBI mice had 77–124% higher IL-6 levels than male TBI mice at 1 and 5DPI, whereas IL-1β and TNF-α levels were similar between sexes at all timepoints. Despite baseline sex differences in blood-measured Ly6C(high) monocytes (females had 40% more than males), TBI reduced monocytes by 67% in TBI mice at 1DPI. Male TBI mice had 31–33% more blood-measured and 31% more spleen-measured Ly6G(+) neutrophils than female TBI mice at 1 and 5DPI, and 7DPI, respectively. Compared with sham, TBI increased sleep in both sexes during the first light and dark cycles. Male TBI mice slept 11–17% more than female TBI mice, depending on the cycle. Thus, sex and TBI interactions may alter the peripheral inflammation profile and sleep patterns, which might explain discrepancies in disease progression based on sex.

OBJECTIVEExperimental 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.METHODSPND17 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.RESULTSThe 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.CONCLUSIONSWith 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.

Sleep-wake disturbances are both a risk factor and reported morbidity for intracerebral hemorrhage (ICH). ICH begins with a ruptured blood vessel and blood leakage into the parenchyma. In response to initial damage, pathophysiological processes ensue that both exacerbate and repair damage. Inflammation is a hallmark process of ICH, which includes microglia activation and increased cytokine signaling. Due to the dual role of cytokines as inflammatory signaling proteins and sleep regulatory substances (SRSs), we hypothesized that ICH would activate microglia, increase SRSs, and alter sleep-wake patterns following an experimental model of ICH in the mouse. Male mice were randomized to receive an injection of collagenase (ICH; n = 8) or saline (sham; n = 11) in the striatum of the right hemisphere. Sleep-wake activity was recorded for 6 full days after ICH via noninvasive sleep cages. Blood and tissue were collected at 7 days after ICH to quantify pro-inflammatory cytokines/SRSs (IL-1β, TNF-α, IL-6) and microglia deramification by skeleton analysis. There was an overall injury effect on sleep in mice subjected to ICH at the transition from dark (wake) to light (sleep) at 2, 3, 4, 5, and 6 days after ICH compared with shams. Further analysis confirmed that ICH mice had significantly earlier wake offsets at the dark/light transition and more robust circadian patterns of wake behavior than saline control mice. Spatiotemporal skeleton analysis indicated an increase in microglial cell number with a decrease in endpoints per cell (decreased ramification) for the ipsilateral ICH perihematomal region compared with saline control. There were no changes to plasma cytokine levels at 7 days after ICH when comparing each condition. This is the first known study to show changes in sleep-wake patterns after experimental ICH. Elucidation of mechanisms that link sleep, inflammation, and ICH offers new pharmacological opportunities and rehabilitative strategies to improve recovery in stroke patients.

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.

Up to 80% of people meeting DSM-IV definitions for schizophrenia will exhibit difficulties with sleep, along with a breakdown in circadian entrainment and rhythmicity. The changes to the sleep and circadian systems in this population are thought to be interdependent, as evidenced by the frequent use of the combined term “sleep and circadian rhythm disruption” or “SCRD” to describe their occurrence. To understand links between sleep and circadian problems in the schizophrenia population, we analyzed the duration and rhythmicity of sleep behavior in mice lacking function of the immediate early gene early growth response 3 ( Egr3). EGR3 has been associated with schizophrenia risk in humans, and Egr3-deficient (-/-) mice display various features of schizophrenia that are responsive to antipsychotic treatment. While Egr3-/- mice slept less than their wildtype (WT) littermates, they showed no evidence of circadian disorganization; in fact, circadian rhythms of activity were more robust in these mice compared with WT, as measured by time series analysis and the relative amplitude index of Van Someren’s suite of non-parametric circadian rhythm analyses. Differences in circadian robustness were maintained when the animals were transferred to several weeks of housing under constant darkness or constant light. Together, our results suggest that Egr3-/- mice retain control over the circadian timekeeping of sleep and wake, while showing impaired sleep. The findings are compatible with those from a surprising array of mouse models of schizophrenia and raise the possibility that SCRD may be 2 separate disorders in the schizophrenia population requiring different treatment strategies.

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. 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. 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.

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.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.In M2, average vessel volume (p0.01) and surface area (p0.05) 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.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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12974-018-1200-y) contains supplementary material, which is available to authorized users.

Aggression in mice often results in injury leading to unplanned euthanasia or the initiation of protocols to isolate animals, thereby increasing research costs and straining resources. Here, we tested if adding a partial cage divider into existing mouse cages affected aggressive-like behavior in group-housed male mice (18 mice; 3 per cage). Mice were randomly assigned to one of two groups upon arrival to the vivarium: (1) standard cage; (2) cage with a partial cage divider. Behavioral observation over 12 hours were conducted at day one, two, and seven after receipt at the facility in order to assess aggression during the course of establishing dominance hierarchies. Observers blinded to study design and hypothesis scored each video for the number and type of aggressive behaviors, which were summed for each hour and analyzed. Results indicated a statistically significant decrease in aggressive behaviors of mice in cages with dividers compared to mice in standard cages. We conclude that cage dividers, which resemble burrows and provide access to common food/water, may promote rigorous research by reducing the number of animals used in a study and refining housing, thus, improving animal welfare.

Determining regions of altered brain physiology after diffuse brain injury is challenging. Microglia, brain immune cells with ramified and dynamically moving processes, constantly surveil the parenchyma for dysfunction which, when present, results in a changed morphology. Our purpose was to define the spatiotemporal changes in microglia morphology over 28 days following rat midline fluid percussion injury (mFPI) as a first step in exploiting microglia morphology to reflect altered brain physiology. Microglia morphology was quantified from histological sections using Image J skeleton and fractal analysis procedures at three time points and in three regions post-mFPI: impact site, primary somatosensory cortex barrel field (S1BF), and a remote region. Microglia ramification (process length/cell and endpoints/cell) decreased in the impact and S1BF but not the remote region (p

The use of animal modeling in traumatic brain injury (TBI) research is justified by the lack of sufficiently comprehensive in vitro and computer modeling that incorporates all components of the neurovascular unit. Valid animal modeling of TBI requires accurate replication of both the mechanical forces and secondary injury conditions observed in human patients. Regulatory requirements for animal modeling emphasize the administration of appropriate anesthetics and analgesics unless withholding these drugs is scientifically justified. The objective of this review is to present scientific justification for standardizing the use of anesthetics and analgesics, within a study, when modeling TBI in order to preserve study validity. Evidence for the interference of anesthetics and analgesics in the natural course of brain injury calls for consistent consideration of pain management regimens when conducting TBI research. Anesthetics administered at the time of or shortly after induction of brain injury can alter cognitive, motor, and histological outcomes following TBI. A consistent anesthesia protocol based on experimental objectives within each individual study is imperative when conducting TBI studies to control for the confounding effects of anesthesia on outcome parameters. Experimental studies that replicate the clinical condition are essential to gain further understanding and evaluate possible treatments for TBI. However, with animal models of TBI it is essential that investigators assure a uniform drug delivery protocol that minimizes confounding variables, while minimizing pain and suffering.

Research models of traumatic brain injury (TBI) hold significant validity towards the human condition, with each model replicating a subset of clinical features and symptoms. After 30 years of characterization and implementation, fluid percussion injury (FPI) is firmly recognized as a clinically relevant model of TBI, encompassing concussion through severe injury. The midline variation of FPI may best represent mild and diffuse clinical brain injury, because of the acute behavioral deficits, the late onset of subtle behavioral morbidities, and the absence of gross histopathology. This chapter outlines the procedures for midline (diffuse) FPI in adult male rats and mice. With these procedures, it becomes possible to generate brain-injured laboratory animals for studies of injury-induced pathophysiology and behavioral deficits, for which rational therapeutic interventions can be implemented.

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.

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.

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.

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 brain's 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.

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.

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.

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.

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.

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.

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, usingin vitromodels, 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.