Jordan L. Harrison

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

BackgroundSince 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).MethodsRats 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.ResultsWe 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.ConclusionDiffuse 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.

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.

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

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

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.

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.

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.

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

Abstract 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.

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

Acquired neurological injuries initiate a pathological cascade of secondary injury processes, including inflammation, which continue for days to weeks following injury. Injury-induced neuroinflammation acts as a host defense mechanism contributing to the neutralization of the insult (removing offending factors) and restoring structure and function of the brain (establish homeostasis). The timing of these protective functions of the immune response is vital, since chronic inflammation has been associated with progressive cell loss and neurotoxicity (for review, see Faden and Loane, 2015). The pathophysiology of traumatic brain injury (TBI) includes arachidonic acid derived lipid mediators driving inflammatory conditions that promote the activation of resident microglia and infiltration of neutrophils through the disrupted blood-brain barrier (Figure 1A). A separate sub-class of lipid mediators, termed specialized pro-resolving mediators (SPMs), functions to resolve inflammation (Figure 1B). Endogenous SPMs, notably those derived from omega-3 fatty acids, may represent a valuable target in shifting the balance of neuroinflammatory processes from inflammation-driving to inflammation-resolving conditions in the injured central nervous system (CNS). Enthusiasm for a therapeutic approach involving SPMs comes from the natural routes of administration, such as dietary supplementation of their metabolic precursors, exogenous SPMs, and adjunctive interventions focused on increasing the availability of SPMs after injury. Figure 1 Membrane fatty acid profiles contribute to the balance of inflammation driving and resolving lipid mediators. Biochemically, lipid mediators represent a diverse family of endogenous bioactive molecules enzymatically derived from fatty acid substrates. Prostaglandins, a family of extensively studied lipid mediators, are synthesized from arachidonic acid and are elevated after acquired neurological injury, such as TBI (Yang and Gao, 1999). For years, the predominantly pro-inflammatory nature of prostaglandins contributed to the perspective that lipid mediators singly promote inflammation. Affirming this, administration of other arachidonic-acid derived families, such as leukotrienes, have shown pro-inflammatory effects after TBI, while their inhibition has shown improvement in outcome (Hartig et al., 2013). In contrast, SPMs derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), including families of resolvins and protectins, have demonstrated a role in the resolution of inflammation (for review, Recchiuti and Serhan, 2012). Comprehensive mechanisms of action for SPMs have not been identified, but data indicate g-protein-coupled receptors on leukocytes bind SPMs to reduce infiltration and promote tissue regeneration (Recchiuti and Serhan, 2012). While both prostaglandins and SPMs are lipid mediators, they are distinguished by their roles in promoting versus resolving inflammation, respectively (Hartig et al., 2013). This broader perspective on lipid mediators has established a platform to further investigate the balance between pro-inflammatory and pro-resolving lipids in mediating the course of inflammation in the wake of acquired neurological injury, potentially through dietary supplementation or exogenous administration. Dietary supplementation with metabolic precursors of SPMs has a potential for increasing the availability of SPMs and resolving inflammation after neurological injury. In the brain, fatty acid metabolic precursors of lipid mediators are incorporated into cell membranes (Figure 1). A western diet is especially high in omega-6 fatty acids, such as arachidonic acid, which displace omega-3 fatty acids, such as DHA and EPA, in cell membranes (Bradbury, 2011). In this situation, omega-6 derived lipid mediators, including prostaglandin E2 and leukotriene B4, may prolong inflammation and injury. With omega-3 supplementation, the balance of lipid mediator metabolic precursors can be restored or reversed by providing substrates for SPM production. Metabolic precursors of the inflammation-resolving SPMs, including DHA, have demonstrated potent anti-inflammatory effects in animal models of ischemic stroke (Belayev et al., 2009) and TBI (Wu et al., 2011). In one study, rats were subjected to middle cerebral artery occlusion (MCAO) and subsequently treated intravenously with high (70 mg/kg), medium (16 or 35 mg/kg), or low (3.5 mg/kg) doses of DHA (Belayev et al., 2009). The low and medium doses, but not high dose, of DHA resulted in significant tissue sparing in the peri-infarct region. Treated rats showed significantly better performance in neurological function up to 7 days following MCAO. Following experimental TBI in rats, DHA-enriched diet for 12 days preserved otherwise depleted brain derived neurotrophic factor (BDNF) and improved learning performance (Wu et al., 2011). While the mechanisms of action of DHA in the wake of neurological injury remain widely unknown, its therapeutic potential is compelling. Further support for the efficacy of dietary supplementation is demonstrated by the increased production of SPMs following supplementation after injury. After cellular insult, membrane fatty acids, including DHA, are released and become more readily available for metabolism into SPMs (Martin et al., 2000). To test the endogenous production of SPMs, DHA was administered intravenously after the onset of ischemic stroke in the rat and brain levels of lipid metabolites were measured (Belayev et al., 2011). Mass spectrometric analysis revealed that neuroprotectin D1 (NPD1) was biosynthesized in the peri-infarct region of DHA-treated rats. This study demonstrates that SPM synthesis in the brain can be enhanced through precursor supplementation, which lead to therapeutic efficacy on behavioral performance and histological outcome following neurological injury. While DHA is not a lipid mediator, investigations into its therapeutic effects in neurological injury suggest its effects may be attributable in part to the biosynthesis of SPMs. In addition to dietary supplementation, administration of exogenous SPMs may improve outcome from neurological injury. Using different SPMs and models of neurological injury, two studies demonstrated therapeutic efficacy of exogenous SPMs. Following MCAO, rats were administered DHA, NPD1, or saline. Both DHA and NPD1 treatments independently reduced infarct volume and attenuated behavioral deficits measured by neurological assessment (Eady et al., 2012). Next, our group tested the efficacy of aspirin-triggered resolvin D1 (AT-RvD1) and resolvin E1 (RvE1) on amelioration of functional deficits after diffuse TBI in mice (Harrison et al., 2015). AT-RvD1, but not RvE1, treatment mitigated motor deficits in rotarod performance and cognitive deficits in the novel object recognition task. Together, these experimental data in stroke and TBI indicate a standalone role for SPMs as therapeutics for acquired neurological injury. In light of the outlined therapeutic potential of SPMs through diet and direct administration, practical considerations in the therapeutic use of SPMs are warranted. Effective therapeutic strategies may include continuous prophylactic dietary supplementation of fatty acid precursors, direct administration of exogenous SPMs, or even physical medicine approaches to potentiate SPM production. These strategies are presented in order of patient accessibility, where direct SPM administration may require specialized knowledge, inventory, and administration by a healthcare professional. On the other hand, dietary omega-3 fatty acid supplementation is a tangible home healthcare strategy, but requires time for fatty acids to accumulate into cell membranes, particularly in the brain. Since acquired neurological injuries occur without warning, supplementation after injury may not be effective or practical. Populations at higher risk for stroke and TBI, such as athletes and soldiers, may benefit from continuous prophylactic supplementation with omega-3 fatty acids. Physical medicine approaches to potentiate endogenous production of SPMs have not been investigated, but could include approaches shown to be therapeutic after acquired neurological injury and secondary inflammation, such as remote ischemic conditioning (Joseph et al., 2015). Complementary approaches directed to antagonism or depletion of inflammation-driving prostaglandins or leukotrienes may contribute to shifting the inflammatory balance toward resolution. In conclusion, SPMs provide a cellular target for therapeutic approaches to limit secondary injury processes after acquired neurological injury. The potent anti-inflammatory properties of SPMs in peripheral diseases (see review: Recchiuti and Serhan, 2012) and the therapeutic efficacy of their fatty acid precursors in neurological disease provide a sound basis for further exploration of their neuroprotective efficacy in acquired neurological injury. Of particular value to the clinical problems of stroke and TBI are therapies which are rapid and accessible. The endogenous nature of SPMs makes them promising candidates for readily accessible therapies, which could shift the inflammatory balance toward resolution of cellular pathophysiology and limit the extent of injury. The authors have no conflicts of interest to declare. JLH, RKR and JL contributed to manuscript content and style.

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

Abstract 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.