simultaneous-cryosectioning-of-multiple-rodent-brains

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 ...

University of Arizona College of Medicine

Barrow Neurological Institute at Phoenix Children's Hospital

Phoenix Veteran Affairs Healthcare System

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.

The p38α MAPK regulates microglial responsiveness to diffuse 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.

Using anesthetics and analgesics in experimental traumatic brain injury.

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.

Diffuse brain injury induces acute post-traumatic sleep.

We investigated the relationship between immediate disruption of posttraumatic sleep and functional outcome in the diffuse brain-injured mouse.

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

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.

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

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.

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

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.

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

Lipid mediators of inflammation in neurological injury: shifting the balance toward resolution.

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.

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

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.

Midline (Central) Fluid Percussion Model of Traumatic Brain Injury.

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.

Partial cage division significantly reduces aggressive behavior in male laboratory mice.

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.

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

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.

Article	Year
The p38α MAPK regulates microglial responsiveness to diffuse traumatic brain injury. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 23554495	2013
Using anesthetics and analgesics in experimental traumatic brain injury. Lab animal\| PubMed ID: 23877609	2013
Diffuse brain injury induces acute post-traumatic sleep. PloS one\| PubMed ID: 24416145	2014
Recovery of neurological function despite immediate sleep disruption following diffuse brain injury in the mouse: clinical relevance to medically untreated concussion. Sleep\| PubMed ID: 24899763	2014
Resolvins AT-D1 and E1 differentially impact functional outcome, post-traumatic sleep, and microglial activation following diffuse brain injury in the mouse. Brain, behavior, and immunity\| PubMed ID: 25585137	2015
Experimental diffuse brain injury results in regional alteration of gross vascular morphology independent of neuropathology. Brain injury\| PubMed ID: 26646974	2016
Aging with a traumatic brain injury: Could behavioral morbidities and endocrine symptoms be influenced by microglial priming? Brain, behavior, and immunity\| PubMed ID: 26975888	2017
Lipid mediators of inflammation in neurological injury: shifting the balance toward resolution. Neural regeneration research\| PubMed ID: 26981087	2016
Diffuse traumatic brain injury induces prolonged immune dysregulation and potentiates hyperalgesia following a peripheral immune challenge. Molecular pain\| PubMed ID: 27178244	2016
Midline (Central) Fluid Percussion Model of Traumatic Brain Injury. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27604721	2016
Partial cage division significantly reduces aggressive behavior in male laboratory mice. Laboratory animals\| PubMed ID: 29409371	2018
Midline (central) fluid percussion model of traumatic brain injury in pediatric and adolescent rats. Journal of neurosurgery. Pediatrics\| PubMed ID: 29676680	2018
Novel TNF receptor-1 inhibitors identified as potential therapeutic candidates for traumatic brain injury. Journal of neuroinflammation\| PubMed ID: 29789012	2018

Rachel K. Rowe

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