Bi-directional neuro-immune dysfunction after chronic experimental brain injury.

BACKGROUND: It is well established that traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function and that systemic immune changes contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. METHODS: To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham (i.e., 90 days post-surgery) congenic donor mice into otherwise healthy, age-matched, irradiated CD45.2 C57BL/6 (WT) hosts. Immune changes were evaluated by flow cytometry, multiplex ELISA, and NanoString technology. Moderate-to-severe TBI was induced by controlled cortical impact injury and neurological function was measured using a battery of behavioral tests. RESULTS: TBI induced chronic alterations in the transcriptome of BM lineage-c-Kit+Sca1+ (LSK+) cells in C57BL/6 mice, including modified epigenetic and senescence pathways. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI at 8 weeks and 8 months post-reconstitution showed that longer reconstitution periods (i.e., time post-injury) were associated with increased microgliosis and leukocyte infiltration. Pre-treatment with a senolytic agent, ABT-263, significantly improved behavioral performance of aged C57BL/6 mice at baseline, although it did not attenuate neuroinflammation in the acutely injured brain. CONCLUSIONS: TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in hematopoiesis, innate immunity, and neurological function, as well as altered sensitivity to subsequent brain injury.

Fundamental Neurochemistry Review: Microglial immunometabolism in traumatic brain injury.

Traumatic brain injury (TBI) is a devastating neurological disorder caused by a physical impact to the brain that promotes diffuse damage and chronic neurodegeneration. Key mechanisms believed to support secondary brain injury include mitochondrial dysfunction and chronic neuroinflammation. Microglia and brain-infiltrating macrophages are responsible for neuroinflammatory cytokine and reactive oxygen species (ROS) production after TBI. Their production is associated with loss of homeostatic microglial functions such as immunosurveillance, phagocytosis, and immune resolution. Beyond providing energy support, mitochondrial metabolic pathways reprogram the pro- and anti-inflammatory machinery in immune cells, providing a critical immunometabolic axis capable of regulating immunologic response to noxious stimuli. In the brain, the capacity to adapt to different environmental stimuli derives, in part, from microglia's ability to recognize and respond to changes in extracellular and intracellular metabolite levels. This capacity is met by an equally plastic metabolism, capable of altering immune function. Microglial pro-inflammatory activation is associated with decreased mitochondrial respiration, whereas anti-inflammatory microglial polarization is supported by increased oxidative metabolism. These metabolic adaptations contribute to neuroimmune responses, placing mitochondria as a central regulator of post-traumatic neuroinflammation. Although it is established that profound neurometabolic changes occur following TBI, key questions related to metabolic shifts in microglia remain unresolved. These include (a) the nature of microglial mitochondrial dysfunction after TBI, (b) the hierarchical positions of different metabolic pathways such as glycolysis, pentose phosphate pathway, glutaminolysis, and lipid oxidation during secondary injury and recovery, and (c) how immunometabolism alters microglial phenotypes, culminating in chronic non-resolving neuroinflammation. In this basic neurochemistry review article, we describe the contributions of immunometabolism to TBI, detail primary evidence of mitochondrial dysfunction and metabolic impairments in microglia and macrophages, discuss how major metabolic pathways contribute to post-traumatic neuroinflammation, and set out future directions toward advancing immunometabolic phenotyping in TBI.

Understanding microglial responses in large animal models of traumatic brain injury: an underutilized resource for preclinical and translational research.

Traumatic brain injury (TBI) often results in prolonged or permanent brain dysfunction with over 2.8 million affected annually in the U.S., including over 56,000 deaths, with over 5 million total survivors exhibiting chronic deficits. Mild TBI (also known as concussion) accounts for over 75% of all TBIs every year. Mild TBI is a heterogeneous disorder, and long-term outcomes are dependent on the type and severity of the initial physical event and compounded by secondary pathophysiological consequences, such as reactive astrocytosis, edema, hypoxia, excitotoxicity, and neuroinflammation. Neuroinflammation has gained increasing attention for its role in secondary injury as inflammatory pathways can have both detrimental and beneficial roles. For example, microglia-resident immune cells of the central nervous system (CNS)-influence cell death pathways and may contribute to progressive neurodegeneration but also aid in debris clearance and neuroplasticity. In this review, we will discuss the acute and chronic role of microglia after mild TBI, including critical protective responses, deleterious effects, and how these processes vary over time. These descriptions are contextualized based on interspecies variation, sex differences, and prospects for therapy. We also highlight recent work from our lab that was the first to describe microglial responses out to chronic timepoints after diffuse mild TBI in a clinically relevant large animal model. The scaled head rotational acceleration of our large animal model, paired with the gyrencephalic architecture and appropriate white:gray matter ratio, allows us to produce pathology with the same anatomical patterns and distribution of human TBI, and serves as an exemplary model to examine complex neuroimmune response post-TBI. An improved understanding of microglial influences in TBI could aid in the development of targeted therapeutics to accentuate positive effects while attenuating detrimental post-injury responses over time.

Microglial Depletion with CSF1R Inhibitor During Chronic Phase of Experimental Traumatic Brain Injury Reduces Neurodegeneration and Neurological Deficits.

Chronic neuroinflammation with sustained microglial activation occurs following severe traumatic brain injury (TBI) and is believed to contribute to subsequent neurodegeneration and neurological deficits. Microglia, the primary innate immune cells in brain, are dependent on colony stimulating factor 1 receptor (CSF1R) signaling for their survival. In this preclinical study, we examined the effects of delayed depletion of chronically activated microglia on functional recovery and neurodegeneration up to 3 months postinjury. A CSF1R inhibitor, Plexxikon (PLX) 5622, was administered to adult male C57BL/6J mice at 1 month after controlled cortical impact to remove chronically activated microglia, and the inhibitor was withdrawn 1-week later to allow for microglial repopulation. Following TBI, the repopulated microglia displayed a ramified morphology similar to that of Sham uninjured mice, whereas microglia in vehicle-treated TBI mice showed the typical chronic posttraumatic hypertrophic morphology. PLX5622 treatment limited TBI-associated neuropathological changes at 3 months postinjury; these included a smaller cortical lesion, reduced hippocampal neuron cell death, and decreased NOX2- and NLRP3 inflammasome-associated neuroinflammation. Furthermore, delayed depletion of chronically activated microglia after TBI led to widespread changes in the cortical transcriptome and altered gene pathways involved in neuroinflammation, oxidative stress, and neuroplasticity. Using a variety of complementary neurobehavioral tests, PLX5622-treated TBI mice also had improved long-term motor and cognitive function recovery through 3 months postinjury. Together, these studies demonstrate that chronic phase removal of neurotoxic microglia after TBI using CSF1R inhibitors markedly reduce chronic neuroinflammation and associated neurodegeneration, as well as related motor and cognitive deficits.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is a debilitating neurological disorder that can seriously impact the patient's quality of life. Microglial-mediated neuroinflammation is induced after severe TBI and contributes to neurological deficits and on-going neurodegenerative processes. Here, we investigated the effect of breaking the neurotoxic neuroinflammatory loop at 1-month after controlled cortical impact in mice by pharmacological removal of chronically activated microglia using a colony stimulating factor 1 receptor (CSF1R) inhibitor, Plexxikon 5622. Overall, we show that short-term elimination of microglia during the chronic phase of TBI followed by repopulation results in long-term improvements in neurological function, suppression of neuroinflammatory and oxidative stress pathways, and a reduction in persistent neurodegenerative processes. These studies are clinically relevant and support new concepts that the therapeutic window for TBI may be far longer than traditionally believed if chronic and evolving microglial-mediated neuroinflammation can be inhibited or regulated in a precise manner.

Interferon-ß Plays a Detrimental Role in Experimental Traumatic Brain Injury by Enhancing Neuroinflammation That Drives Chronic Neurodegeneration.

DNA damage and type I interferons (IFNs) contribute to inflammatory responses after traumatic brain injury (TBI). TBI-induced activation of microglia and peripherally-derived inflammatory macrophages may lead to tissue damage and neurological deficits. Here, we investigated the role of IFN-ß in secondary injury after TBI using a controlled cortical impact model in adult male IFN-ß-deficient (IFN-ß-/-) mice and assessed post-traumatic neuroinflammatory responses, neuropathology, and long-term functional recovery. TBI increased expression of DNA sensors cyclic GMP-AMP synthase and stimulator of interferon genes in wild-type (WT) mice. IFN-ß and other IFN-related and neuroinflammatory genes were also upregulated early and persistently after TBI. TBI increased expression of proinflammatory mediators in the cortex and hippocampus of WT mice, whereas levels were mitigated in IFN-ß-/- mice. Moreover, long-term microglia activation, motor, and cognitive function impairments were decreased in IFN-ß-/- TBI mice compared with their injured WT counterparts; improved neurological recovery was associated with reduced lesion volume and hippocampal neurodegeneration in IFN-ß-/- mice. Continuous central administration of a neutralizing antibody to the IFN-α/ß receptor (IFNAR) for 3 d, beginning 30 min post-injury, reversed early cognitive impairments in TBI mice and led to transient improvements in motor function. However, anti-IFNAR treatment did not improve long-term functional recovery or decrease TBI neuropathology at 28 d post-injury. In summary, TBI induces a robust neuroinflammatory response that is associated with increased expression of IFN-ß and other IFN-related genes. Inhibition of IFN-ß reduces post-traumatic neuroinflammation and neurodegeneration, resulting in improved neurological recovery. Thus, IFN-ß may be a potential therapeutic target for TBI.SIGNIFICANCE STATEMENT TBI frequently causes long-term neurological and psychiatric changes in head injury patients. TBI-induced secondary injury processes including persistent neuroinflammation evolve over time and can contribute to chronic neurological impairments. The present study demonstrates that TBI is followed by robust activation of type I IFN pathways, which have been implicated in microglial-associated neuroinflammation and chronic neurodegeneration. We examined the effects of genetic or pharmacological inhibition of IFN-ß, a key component of type I IFN mechanisms to address its role in TBI pathophysiology. Inhibition of IFN-ß signaling resulted in reduced neuroinflammation, attenuated neurobehavioral deficits, and limited tissue loss long after TBI. These preclinical findings suggest that IFN-ß may be a potential therapeutic target for TBI.

Enhanced Akt/GSK-3ß/CREB signaling mediates the anti-inflammatory actions of mGluR5 positive allosteric modulators in microglia and following traumatic brain injury in male mice.

We have previously shown that treatment with a mGluR5 positive allosteric modulator (PAM) is neuroprotective after experimental traumatic brain injury (TBI), limiting post-traumatic neuroinflammation by reducing pro-inflammatory microglial activation and promoting anti-inflammatory and neuroprotective responses. However, the specific molecular mechanisms governing this anti-inflammatory shift in microglia remain unknown. Here we show that the mGluR5 PAM, VU0360172 (VuPAM), regulates microglial inflammatory responses through activation of Akt, resulting in the inhibition of GSK-3ß. GSK-3ß regulates the phosphorylation of CREB, thereby controlling the expression of inflammation-related genes and microglial plasticity. The anti-inflammatory action of VuPAM in microglia is reversed by inhibiting Akt/GSK-3ß/CREB signaling. Using a well-characterized TBI model and CX3CR1gfp/+ mice to visualize microglia in vivo, we demonstrate that VuPAM enhances Akt/GSK-3ß/CREB signaling in the injured cortex, as well as anti-inflammatory microglial markers. Furthermore, in situ analysis revealed that GFP + microglia in the cortex of VuPAM-treated TBI mice co-express pCREB and the anti-inflammatory microglial phenotype marker YM1. Taken together, our data show that VuPAM decreases pro-inflammatory microglial activation by modulating Akt/GSK-3ß/CREB signaling. These findings serve to clarify the potential neuroprotective mechanisms of mGluR5 PAM treatment after TBI, and suggest novel therapeutic targets for post-traumatic neuroinflammation. Cover Image for this issue: https://doi.org/10.1111/jnc.15048.

Acute colitis during chronic experimental traumatic brain injury in mice induces dysautonomia and persistent extraintestinal, systemic, and CNS inflammation with exacerbated neurological deficits.

BACKGROUND: Disruptions of brain-gut axis have been implicated in the progression of a variety of gastrointestinal (GI) disorders and central nervous system (CNS) diseases and injuries, including traumatic brain injury (TBI). TBI is a chronic disease process characterized by persistent secondary injury processes which can be exacerbated by subsequent challenges. Enteric pathogen infection during chronic TBI worsened cortical lesion volume; however, the pathophysiological mechanisms underlying the damaging effects of enteric challenge during chronic TBI remain unknown. This preclinical study examined the effect of intestinal inflammation during chronic TBI on associated neurobehavioral and neuropathological outcomes, systemic inflammation, and dysautonomia. METHODS: Dextran sodium sulfate (DSS) was administered to adult male C57BL/6NCrl mice 28 days following craniotomy (Sham) or TBI for 7 days to induce intestinal inflammation, followed by a return to normal drinking water for an additional 7 to 28 days for recovery; uninjured animals (Naïve) served as an additional control group. Behavioral testing was carried out prior to, during, and following DSS administration to assess changes in motor and cognitive function, social behavior, and mood. Electrocardiography was performed to examine autonomic balance. Brains were collected for histological and molecular analyses of injury lesion, neurodegeneration, and neuroinflammation. Blood, colons, spleens, mesenteric lymph nodes (mLNs), and thymus were collected for morphometric analyses and/or immune characterization by flow cytometry. RESULTS: Intestinal inflammation 28 days after craniotomy or TBI persistently induced, or exacerbated, respectively, deficits in fine motor coordination, cognition, social behavior, and anxiety-like behavior. Behavioral changes were associated with an induction, or exacerbation, of hippocampal neuronal cell loss and microglial activation in Sham and TBI mice administered DSS, respectively. Acute DSS administration resulted in a sustained systemic immune response with increases in myeloid cells in blood and spleen, as well as myeloid cells and lymphocytes in mesenteric lymph nodes. Dysautonomia was also induced in Sham and TBI mice administered DSS, with increased sympathetic tone beginning during DSS administration and persisting through the first recovery week. CONCLUSION: Intestinal inflammation during chronic experimental TBI causes a sustained systemic immune response and altered autonomic balance that are associated with microglial activation, increased neurodegeneration, and persistent neurological deficits.

Early or Late Bacterial Lung Infection Increases Mortality After Traumatic Brain Injury in Male Mice and Chronically Impairs Monocyte Innate Immune Function.

OBJECTIVES: Respiratory infections in the postacute phase of traumatic brain injury impede optimal recovery and contribute substantially to overall morbidity and mortality. This study investigated bidirectional innate immune responses between the injured brain and lung, using a controlled cortical impact model followed by secondary Streptococcus pneumoniae infection in mice. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Adult male C57BL/6J mice. INTERVENTIONS: C57BL/6J mice were subjected to sham surgery or moderate-level controlled cortical impact and infected intranasally with S. pneumoniae (1,500 colony-forming units) or vehicle (phosphate-buffered saline) at 3 or 60 days post-injury. MAIN RESULTS: At 3 days post-injury, S. pneumoniae-infected traumatic brain injury mice (TBI + Sp) had a 25% mortality rate, in contrast to no mortality in S. pneumoniae-infected sham (Sham + Sp) animals. TBI + Sp mice infected 60 days post-injury had a 60% mortality compared with 5% mortality in Sham + Sp mice. In both studies, TBI + Sp mice had poorer motor function recovery compared with TBI + PBS mice. There was increased expression of pro-inflammatory markers in cortex of TBI + Sp compared with TBI + PBS mice after both early and late infection, indicating enhanced post-traumatic neuroinflammation. In addition, monocytes from lungs of TBI + Sp mice were immunosuppressed acutely after traumatic brain injury and could not produce interleukin-1ß, tumor necrosis factor-α, or reactive oxygen species. In contrast, after delayed infection monocytes from TBI + Sp mice had higher levels of interleukin-1ß, tumor necrosis factor-α, and reactive oxygen species when compared with Sham + Sp mice. Increased bacterial burden and pathology was also found in lungs of TBI + Sp mice. CONCLUSIONS: Traumatic brain injury causes monocyte functional impairments that may affect the host's susceptibility to respiratory infections. Chronically injured mice had greater mortality following S. pneumoniae infection, which suggests that respiratory infections even late after traumatic brain injury may pose a more serious threat than is currently appreciated.

Neutral Sphingomyelinase Inhibition Alleviates LPS-Induced Microglia Activation and Neuroinflammation after Experimental Traumatic Brain Injury.

Neuroinflammation is one of the key secondary injury mechanisms triggered by traumatic brain injury (TBI). Microglial activation, a hallmark of brain neuroinflammation, plays a critical role in regulating immune responses after TBI and contributes to progressive neurodegeneration and neurologic deficits following brain trauma. Here we evaluated the role of neutral sphingomyelinase (nSMase) in microglial activation by examining the effects of the nSMase inhibitors altenusin and GW4869 in vitro (using BV2 microglia cells and primary microglia), as well as in a controlled cortical injury (CCI) model in adult male C57BL/6 mice. Pretreatment of altenusin or GW4869 prior to lipopolysaccharide (LPS) stimulation for 4 or 24 hours, significantly downregulated gene expression of the pro-inflammatory mediators TNF-α, IL-1ß, IL-6, iNOS, and CCL2 in microglia and reduced the release of nitric oxide and TNF-α These nSMase inhibitors also attenuated the release of microparticles and phosphorylation of p38 MAPK and ERK1/2. In addition, altenusin pretreatment also reduced the gene expression of multiple inflammatory markers associated with microglial activation after experimental TBI, including TNF-α, IL-1ß, IL-6, iNOS, CCL2, CD68, NOX2, and p22phox Overall, our data demonstrate that nSMase inhibitors attenuate multiple inflammatory pathways associated with microglial activation in vitro and after experimental TBI. Thus, nSMase inhibitors may represent promising therapeutics agents targeting neuroinflammation.

Sexual dimorphism in the inflammatory response to traumatic brain injury.

The activation of resident microglial cells, alongside the infiltration of peripheral macrophages, are key neuroinflammatory responses to traumatic brain injury (TBI) that are directly associated with neuronal death. Sexual disparities in response to TBI have been previously reported; however it is unclear whether a sex difference exists in neuroinflammatory progression after TBI. We exposed male and female mice to moderate-to-severe controlled cortical impact injury and studied glial cell activation in the acute and chronic stages of TBI using immunofluorescence and in situ hybridization analysis. We found that the sex response was completely divergent up to 7 days postinjury. TBI caused a rapid and pronounced cortical microglia/macrophage activation in male mice with a prominent activated phenotype that produced both pro- (IL-1ß and TNFα) and anti-inflammatory (Arg1 and TGFß) cytokines with a single-phase, sustained peak from 1 to 7 days. In contrast, TBI caused a less robust microglia/macrophage phenotype in females with biphasic pro-inflammatory response peaks at 4 h and 7 days, and a delayed anti-inflammatory mRNA peak at 30 days. We further report that female mice were protected against acute cell loss after TBI, with male mice demonstrating enhanced astrogliosis, neuronal death, and increased lesion volume through 7 days post-TBI. Collectively, these findings indicate that TBI leads to a more aggressive neuroinflammatory profile in male compared with female mice during the acute and subacute phases postinjury. Understanding how sex affects the course of neuroinflammation following brain injury is a vital step toward developing personalized and effective treatments for TBI.

NOX2 deficiency alters macrophage phenotype through an IL-10/STAT3 dependent mechanism: implications for traumatic brain injury.

BACKGROUND: NADPH oxidase (NOX2) is an enzyme system that generates reactive oxygen species (ROS) in microglia and macrophages. Excessive ROS production is linked with neuroinflammation and chronic neurodegeneration following traumatic brain injury (TBI). Redox signaling regulates macrophage/microglial phenotypic responses (pro-inflammatory versus anti-inflammatory), and NOX2 inhibition following moderate-to-severe TBI markedly reduces pro-inflammatory activation of macrophages/microglia resulting in concomitant increases in anti-inflammatory responses. Here, we report the signaling pathways that regulate NOX2-dependent macrophage/microglial phenotype switching in the TBI brain. METHODS: Bone marrow-derived macrophages (BMDMs) prepared from wildtype (C57Bl/6) and NOX2 deficient (NOX2-/-) mice were treated with lipopolysaccharide (LPS; 10 ng/ml), interleukin-4 (IL-4; 10 ng/ml), or combined LPS/IL-4 to investigate signal transduction pathways associated with macrophage activation using western immunoblotting and qPCR analyses. Signaling pathways and activation markers were evaluated in ipsilateral cortical tissue obtained from adult male wildtype and NOX2-/- mice that received moderate-level controlled cortical impact (CCI). A neutralizing anti-IL-10 approach was used to determine the effects of IL-10 on NOX2-dependent transitions from pro- to anti-inflammatory activation states. RESULTS: Using an LPS/IL-4-stimulated BMDM model that mimics the mixed pro- and anti-inflammatory responses observed in the injured cortex, we show that NOX2-/- significantly reduces STAT1 signaling and markers of pro-inflammatory activation. In addition, NOX2-/- BMDMs significantly increase anti-inflammatory marker expression; IL-10-mediated STAT3 signaling, but not STAT6 signaling, appears to be critical in regulating this anti-inflammatory response. Following moderate-level CCI, IL-10 is significantly increased in microglia/macrophages in the injured cortex of NOX2-/- mice. These changes are associated with increased STAT3 activation, but not STAT6 activation, and a robust anti-inflammatory response. Neutralization of IL-10 in NOX2-/- BMDMs or CCI mice blocks STAT3 activation and the anti-inflammatory response, thereby demonstrating a critical role for IL-10 in regulating NOX2-dependent transitions between pro- and anti-inflammatory activation states. CONCLUSIONS: These studies indicate that following TBI NOX2 inhibition promotes a robust anti-inflammatory response in macrophages/microglia that is mediated by the IL-10/STAT3 signaling pathway. Thus, therapeutic interventions that inhibit macrophage/microglial NOX2 activity may improve TBI outcomes by not only limiting pro-inflammatory neurotoxic responses, but also enhancing IL-10-mediated anti-inflammatory responses that are neuroprotective.

Microglial-derived microparticles mediate neuroinflammation after traumatic brain injury.

BACKGROUND: Local and systemic inflammatory responses are initiated early after traumatic brain injury (TBI), and may play a key role in the secondary injury processes resulting in neuronal loss and neurological deficits. However, the mechanisms responsible for the rapid expansion of neuroinflammation and its long-term progression have yet to be elucidated. Here, we investigate the role of microparticles (MP), a member of the extracellular vesicle family, in the exchange of pro-inflammatory molecules between brain immune cells, as well as their transfer to the systemic circulation, as key pathways of inflammation propagation following brain trauma. METHODS: Adult male C57BL/6 mice were subjected to controlled cortical impact TBI for 24 h, and enriched MP were isolated in the blood, while neuroinflammation was assessed in the TBI cortex. MP were characterized by flow cytometry, and MP content was assayed using gene and protein markers for pro-inflammatory mediators. Enriched MP co-cultured with BV2 or primary microglial cells were used for immune propagation assays. Enriched MP from BV2 microglia or CD11b-positive microglia from the TBI brain were stereotactically injected into the cortex of uninjured mice to evaluate MP-related seeding of neuroinflammation in vivo. RESULTS: As the neuroinflammatory response is developing in the brain after TBI, microglial-derived MP are released into the circulation. Circulating enriched MP from the TBI animals can activate microglia in vitro. Lipopolysaccharide stimulation increases MP release from microglia in vitro and enhances their content of pro-inflammatory mediators, interleukin-1ß and microRNA-155. Enriched MP from activated microglia in vitro or CD11b-isolated microglia/macrophage from the TBI brain ex vivo are sufficient to initiate neuroinflammation following their injection into the cortex of naïve (uninjured) animals. CONCLUSIONS: These data provide further insights into the mechanisms underlying the development and dissemination of neuroinflammation after TBI. MP loaded with pro-inflammatory molecules initially released by microglia following trauma can activate additional microglia that may contribute to progressive neuroinflammatory response in the injured brain, as well as stimulate systemic immune responses. Due to their ability to independently initiate inflammatory responses, MP derived from activated microglia may provide a potential therapeutic target for other neurological disorders in which neuroinflammation may be a contributing factor.

Bidirectional brain-gut interactions and chronic pathological changes after traumatic brain injury in mice.

OBJECTIVES: Traumatic brain injury (TBI) has complex effects on the gastrointestinal tract that are associated with TBI-related morbidity and mortality. We examined changes in mucosal barrier properties and enteric glial cell response in the gut after experimental TBI in mice, as well as effects of the enteric pathogen Citrobacter rodentium (Cr) on both gut and brain after injury. METHODS: Moderate-level TBI was induced in C57BL/6mice by controlled cortical impact (CCI). Mucosal barrier function was assessed by transepithelial resistance, fluorescent-labelled dextran flux, and quantification of tight junction proteins. Enteric glial cell number and activation were measured by Sox10 expression and GFAP reactivity, respectively. Separate groups of mice were challenged with Cr infection during the chronic phase of TBI, and host immune response, barrier integrity, enteric glial cell reactivity, and progression of brain injury and inflammation were assessed. RESULTS: Chronic CCI induced changes in colon morphology, including increased mucosal depth and smooth muscle thickening. At day 28 post-CCI, increased paracellular permeability and decreased claudin-1 mRNA and protein expression were observed in the absence of inflammation in the colon. Colonic glial cell GFAP and Sox10 expression were significantly increased 28days after brain injury. Clearance of Cr and upregulation of Th1/Th17 cytokines in the colon were unaffected by CCI; however, colonic paracellular flux and enteric glial cell GFAP expression were significantly increased. Importantly, Cr infection in chronically-injured mice worsened the brain lesion injury and increased astrocyte- and microglial-mediated inflammation. CONCLUSION: These experimental studies demonstrate chronic and bidirectional brain-gut interactions after TBI, which may negatively impact late outcomes after brain injury.

CD38 Knockout Mice Show Significant Protection Against Ischemic Brain Damage Despite High Level Poly-ADP-Ribosylation.

Several enzymes in cellular bioenergetics metabolism require NAD+ as an essential cofactor for their activity. NAD+ depletion following ischemic insult can result in cell death and has been associated with over-activation of poly-ADP-ribose polymerase PARP1 as well as an increase in NAD+ consuming enzyme CD38. CD38 is an NAD+ glycohydrolase that plays an important role in inflammatory responses. To determine the contribution of CD38 activity to the mechanisms of post-ischemic brain damage we subjected CD38 knockout (CD38KO) mice and wild-type (WT) mice to transient forebrain ischemia. The CD38KO mice showed a significant amelioration in both histological and neurologic outcome following ischemic insult. Decrease of hippocampal NAD+ levels detected during reperfusion in WT mice was only transient in CD38KO animals, suggesting that CD38 contributes to post-ischemic NAD+ catabolism. Surprisingly, pre-ischemic poly-ADP-ribose (PAR) levels were dramatically higher in CD38KO animals compared to WT animals and exhibited reduction post-ischemia in contrast to the increased levels in WT animals. The high PAR levels in CD38 mice were due to reduced expression levels of poly-ADP-ribose glycohydrolase (PARG). Thus, the absence of CD38 activity can not only directly affect inflammatory response, but also result in unpredicted alterations in the expression levels of enzymes participating in NAD+ metabolism. Although the CD38KO mice showed significant protection against ischemic brain injury, the changes in enzyme activity related to NAD+ metabolism makes the determination of the role of CD38 in mechanisms of ischemic brain damage more complex.

NOX2 drives M1-like microglial/macrophage activation and neurodegeneration following experimental traumatic brain injury.

Following traumatic brain injury (TBI), activation of microglia and peripherally derived inflammatory macrophages occurs in association with tissue damage. This neuroinflammatory response may have beneficial or detrimental effects on neuronal survival, depending on the functional polarization of these cells along a continuum from M1-like to M2-like activation states. The mechanisms that regulate M1-like and M2-like activation after TBI are not well understood, but appear in part to reflect the redox state of the lesion microenvironment. NADPH oxidase (NOX2) is a critical enzyme system that generates reactive oxygen species in microglia/macrophages. After TBI, NOX2 is strongly up-regulated in M1-like, but not in M2-like polarized cells. Therefore, we hypothesized that NOX2 drives M1-like neuroinflammation and contributes to neurodegeneration and loss of neurological function after TBI. In the present studies we inhibited NOX2 activity using NOX2-knockout mice or the selective peptide inhibitor gp91ds-tat. We show that NOX2 is highly up-regulated in infiltrating macrophages after injury, and that NOX2 deficiency reduces markers of M1-like activation, limits tissue loss and neurodegeneration, and improves motor recovery after moderate-level control cortical injury (CCI). NOX2 deficiency also promotes M2-like activation after CCI, through increased IL-4Rα signaling in infiltrating macrophages, suggesting that NOX2 acts as a critical switch between M1- and M2-like activation states after TBI. Administration of gp91ds-tat to wild-type CCI mice starting at 24h post-injury reduces deficits in cognitive function and increased M2-like activation in the hippocampus. Collectively, our data indicate that increased NOX2 activity after TBI drives M1-like activation that contributes to inflammatory-mediated neurodegeneration, and that inhibiting this pathway provides neuroprotection, in part by altering M1-/M2-like balance towards the M2-like neuroinflammatory response.

Downregulation of miR-23a and miR-27a following experimental traumatic brain injury induces neuronal cell death through activation of proapoptotic Bcl-2 proteins.

MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level. To identify miRs that may regulate neuronal cell death after experimental traumatic brain injury (TBI), we profiled miR expression changes during the first several days after controlled cortical impact (CCI) in mice. miR-23a and miR-27a were rapidly downregulated in the injured cortex in the first hour after TBI. These changes coincided with increased expression of the proapoptotic Bcl-2 family members Noxa, Puma, and Bax. In an etoposide-induced in vitro model of apoptosis in primary cortical neurons, miR-23a and miR-27a were markedly downregulated as early as 1 h after exposure, before the upregulation of proapoptotic Bcl-2 family molecules. Administration of miR-23a and miR-27a mimics attenuated etoposide-induced changes in Noxa, Puma, and Bax, reduced downstream markers of caspase-dependent (cytochrome c release and caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways, and limited neuronal cell death. In contrast, miRs hairpin inhibitors enhanced etoposide-induced neuronal apoptosis and caspase activation. Importantly, administration of miR-23a and miR-27a mimics significantly reduced activation of Puma, Noxa, and Bax as well as attenuated markers of caspase-dependent and -independent apoptosis after TBI. Furthermore, miR-23a and miR-27a mimics significantly attenuated cortical lesion volume and neuronal cell loss in the hippocampus after TBI. These findings indicate that post-traumatic decreases in miR-23a and miR-27a contribute to neuronal cell death after TBI by upregulating proapoptotic Bcl-2 family members, thus providing a novel therapeutic target.

Experimental Models of Hospital-Acquired Infections After Traumatic Brain Injury: Challenges and Opportunities.

Patients hospitalized after a moderate or severe traumatic brain injury (TBI) are at increased risk of nosocomial infections, including bacterial pneumonia and other upper respiratory tract infections. Infections represent a secondary immune challenge for vulnerable TBI patients that can lead to increased morbidity and poorer long-term prognosis. This review first describes the clinical significance of infections after TBI, delving into the known mechanisms by which a TBI can alter systemic immunological responses towards an immunosuppressive state, leading to promotion of increased vulnerability to infections. Pulmonary dysfunction resulting from respiratory tract infections is considered in the context of neurotrauma, including the bidirectional relationship between the brain and lungs. Turning to pre-clinical modeling, current laboratory approaches to study experimental TBI and lung infections are reviewed, to highlight findings from the limited key studies to date that have incorporated both insults. Then, practical decisions for the experimental design of animal studies of post-injury infections are discussed. Variables associated with the host animal, the infectious agent (e.g., species, strain, dose, and administration route), as well as the timing of the infection relative to the injury model are important considerations for model development. Together, the purpose of this review is to highlight the significant clinical need for increased pre-clinical research into the two-hit insult of a hospital-acquired infection after TBI to encourage further scientific enquiry in the field.