Morphological Changes In Microglia Research Articles

The protective role of proton-sensing TDAG8 in the brain injury in a mouse ischemia reperfusion model

Extracellular acidification in the brain has been observed in ischemia; however, the physiological and pathophysiological implications of the pH reduction remain largely unknown. Here, we analyzed the roles of proton-sensing G protein-coupled receptors, including T-cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) in a mouse ischemia reperfusion model. Cerebral infarction and dysfunctional behavior with transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion were exacerbated by the deficiency of TDAG8, whereas no significant effect was observed with the deficiency of OGR1 or GPR4. We confirmed that the pH of the predicted infarction region was 6.5. TDAG8 mRNA was observed in Iba1-positive microglia in the mouse brain. The tMCAO increased the mRNA expression of tumor necrosis factor-α in the ipsilateral cerebral hemisphere and evoked morphological changes in microglia in an evolving cerebral injury. These tMCAO-induced actions were significantly enhanced by the TDAG8 deficiency. Administration of minocycline, which is known to inhibit microglial activation, improved the cerebral infarction and dysfunctional behavior induced by tMCAO in the TDAG8-deficient mouse. Thus, acidic pH/TDAG8 protects against cerebral infarction caused by tMCAO, at least due to the mechanism involving the inhibition of microglial functions.

Gas6 Inhibits Toll-Like Receptor-Mediated Inflammatory Pathways in Mouse Microglia via Axl and Mer.

Background: Microglia are well known key regulators of neuroinflammation which feature in multiple neurodegenerative disorders. These cells survey the CNS and, under inflammatory conditions, become “activated” through stimulation of toll-like receptors (TLRs), resulting in changes in morphology and production and release of cytokines. In the present study, we examined the roles of the related TAM receptors, Mer and Axl, and of their ligand, Gas6, in the regulation of microglial pro-inflammatory TNF-α production and microglial morphology.Methods: Primary cultures of murine microglia of wild-type (WT), Mer−/− and Axl−/− backgrounds were stimulated by the TLR4 agonist, lipopolysaccharide (LPS) with or without pre-treatment with Gas6. Gene expression of TNF-α, Mer, and Axl was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) was used to measure TNF-α release from microglia. Immunofluorescence staining of β-actin and the microglial marker Iba1 was performed to reveal microglial morphological changes, with cellular characteristics (area, perimeter, Feret’s diameter, minimum Feret, roundness, and aspect ratio) being quantified using ImageJ software.Results: Under basal conditions, TNF-α gene expression was significantly lower in Axl−/− microglia compared to WT cells. However, all microglial cultures robustly responded to LPS stimulation with the upregulation of TNF-α expression to similar degrees. Furthermore, Mer receptor expression was less responsive to LPS stimulation when in Axl knockout cells. The presence of Gas6 consistently inhibited the LPS-induced upregulation of TNF-α in WT, Mer−/− and Axl−/− microglia. Moreover, Gas6 also inhibited LPS-induced changes in the microglial area, perimeter length, and cell roundness in wild-type cells.Conclusion: Gas6 can negatively regulate the microglial pro-inflammatory response to LPS as well as via stimulation of other TLRs, acting through either of the TAM receptors, Axl and Mer. This finding indicates an interaction between TLR and TAM receptor signaling pathways and reveals an anti-inflammatory role for the TAM ligand, Gas6, which could have therapeutic potential.

Erythropoietin modulates striatal antioxidant signalling to reduce neurodegeneration in a toxicant model of Parkinson's disease

The current study sought to characterize the pro-survival effects of erythropoietin (EPO) in a toxicant model of Parkinson's disease (PD). EPO treatment induced time-dependent elevations of antioxidant glutathione peroxidase (GPx) and anti-apoptotic factors (pAkt and pBad/Bad) within the striatum and substantia nigra pars compacta (SNc). Intriguingly, our results indicated a region- and lesion size- dependence of pro-survival effects of EPO. Indeed, intra-striatal (but not intra-nigral) infusion of EPO was effective at preventing dopaminergic terminal degeneration and sSNc neuronal loss induced by two different doses of 6-OHDA. These neuroprotective consequences were paralleled by a diminution of microglial morphological changes, along with enhanced motor functioning seen through a reduction in apomorphine-induced rotational behaviour. Finally, in the context of the 6-OHDA lesion, EPO again induced anti-apoptotic (Bcl-2) and antioxidant (GPx) factors within the striatum. Taken together, these results raise the possibility of EPO's potential use as an adjuvant therapy in the treatment of PD, or at least, suggest possible brain-region specific targets for the protective effects of EPO.

A maternal Western diet during gestation and lactation modifies offspring’s microglial cell density and morphology in the hippocampus and prefrontal cortex in Yucatan minipigs

Changes in microglial development and morphology can be induced by inflammatory conditions and associated with eating or mood disorders, such as hyperphagia or depression. In a previous paper in the minipig model, we showed that maternal Western diet during gestation and lactation decreased hippocampus neurogenesis and food-rewarded cognitive abilities in the progeny. Whether these alterations are concomitant with a central inflammatory process in brain structures involved in learning and memory (hippocampus, HPC), cognitive (prefrontal cortex, PFC), or hedonic (orbitofrontal cortex, OFC) control of food intake is still unknown. In the present study, Yucatan minipigs (Sus scrofa) sows were exposed to two different diets during gestation and lactation (standard, SD N = 7 vs. Western diet, WD N = 9). Iba1 is a calcium-binding protein specifically expressed in microglia in the brain, which plays an important role in the regulation of the microglia function. Iba1 expression was examined by immunohistochemical analyses in the PFC, OFC and HPC of piglets. The density of microglial cells, as well as their morphology, were assessed in order to have an indirect insight of microglial cell activation state possibly in relationship with neuroinflammation. The density of Iba1-positive cells was higher in the PFC but not in the HPC of WD compared to SD piglets (p < 0.001). In the HPC, anterior and dorsolateral PFC, WD piglets had more unipolar cells, contrary to SD that had more multipolar cells (P < 0.0001). Opposite effects were observed in the OFC, with SD presenting more unipolar (P < 0.001) microglial cells compared to WD. We showed here that maternal diet during pregnancy and lactation had significant effects on morphological changes of microglial cells in the offspring, and that these effects differed between the HPC and PFC, suggesting different response mechanisms to the early nutritional environment.

Atypical Cadherin FAT3 Is a Novel Mediator for Morphological Changes of Microglia.

Microglia are resident macrophages that are critical for brain development and homeostasis. Microglial morphology is dynamically changed during postnatal stages, leading to regulating synaptogenesis and synapse pruning. Moreover, it has been well known that the shape of microglia is also altered in response to the detritus of the apoptotic cells and pathogens such as bacteria and viruses. Although the morphologic changes are crucial for acquiring microglial functions, the exact mechanism which controls their morphology is not fully understood. Here, we report that the FAT atypical cadherin family protein, FAT3, regulates the morphology of microglial cell line, BV2. We found that the shape of BV2 becomes elongated in a high-nutrient medium. Using microarray analysis, we identified that FAT3 expression is induced by culturing with a high-nutrient medium. In addition, we found that purinergic analog, hypoxanthine, promotes FAT3 expression in BV2 and mouse primary microglia. FAT3 expression induced by hypoxanthine extends the time of sustaining the elongated forms in BV2. These data suggest that the hypoxanthine-FAT3 axis is a novel pathway associated with microglial morphology. Our data provide a possibility that FAT3 may control microglial transitions involved in their morphologic changes during the postnatal stages in vivo.

Chronic intermittent ethanol and lipopolysaccharide exposure differentially alter Iba1-derived microglia morphology in the prelimbic cortex and nucleus accumbens core of male Long-Evans rats.

Accumulating evidence has linked pathological changes associated with chronic alcohol exposure to neuroimmune signaling mediated by microglia. Prior characterization of the microglial structure-function relationship demonstrates that alterations in activity states occur concomitantly with reorganization of cellular architecture. Accordingly, gaining a better understanding of microglial morphological changes associated with ethanol exposure will provide valuable insight into how neuroimmune signaling may contribute to ethanol-induced reshaping of neuronal function. Here we have used Iba1-staining combined with high-resolution confocal imaging and 3D reconstruction to examine microglial structure in the prelimbic (PL) cortex and nucleus accumbens (NAc) in male Long-Evans rats. Rats were either sacrificed at peak withdrawal following 15days of exposure to chronic intermittent ethanol (CIE) or 24hr after two consecutive injections of the immune activator lipopolysaccharide (LPS), each separated by 24hr. LPS exposure resulted in dramatic structural reorganization of microglia in the PL cortex, including increased soma volume, overall cellular volume, and branching complexity. In comparison, CIE exposure was associated with a subtle increase in somatic volume and differential effects on microglia processes, which were largely absent in the NAc. These data reveal that microglial activation following a neuroimmune challenge with LPS or exposure to chronic alcohol exhibits distinct morphometric profiles and brain region-dependent specificity.

Stimulator of IFN genes mediates neuroinflammatory injury by suppressing AMPK signal in experimental subarachnoid hemorrhage

BackgroundNeuroinflammation is closely associated with the poor prognosis in subarachnoid hemorrhage (SAH) patients. This study was aimed to determine the role of stimulator of IFN genes (STING), an essential regulator to innate immunity, in the context of SAH.MethodsA total of 344 male C57BL/6 J mice were subjected to endovascular perforation to develop a model of SAH. Selective STING antagonist C-176 and STING agonist CMA were administered at 30 min or 1 h post-modeling separately. To investigate the underlying mechanism, the AMPK inhibitor compound C was administered intracerebroventricularly at 30 min before surgery. Post-SAH assessments included SAH grade, neurological test, brain water content, western blotting, RT-PCR, and immunofluorescence. Oxygenated hemoglobin was introduced into BV2 cells to establish a SAH model in vitro.ResultsSTING was mainly distributed in microglia, and microglial STING expression was significantly increased after SAH. Administration of C-176 substantially attenuated SAH-induced brain edema and neuronal injury. More importantly, C-176 significantly alleviated both short-term and persistent neurological dysfunction after SAH. Meanwhile, STING agonist CMA remarkably exacerbated neuronal injury and deteriorated neurological impairments. Mechanically, STING activation aggravated neuroinflammation via promoting microglial activation and polarizing into M1 phenotype, evidenced by microglial morphological changes, as well as the increased level of microglial M1 markers including IL-1β, iNOS, IL-6, TNF-α, MCP-1, and NLRP3 inflammasome, while C-176 conferred a robust anti-inflammatory effect. However, all the mentioned beneficial effects of C-176 including alleviated neuroinflammation, attenuated neuronal injury and the improved neurological function were reversed by AMPK inhibitor compound C. Meanwhile, the critical role of AMPK signal in C-176 mediated anti-inflammatory effect was also confirmed in vitro.ConclusionMicroglial STING yielded neuroinflammation after SAH, while pharmacologic inhibition of STING could attenuate SAH-induced inflammatory injury at least partly by activating AMPK signal. These data supported the notion that STING might be a potential therapeutic target for SAH.

Transgenic Overexpression of GPNMB Protects Against MPTP-Induced Neurodegeneration.

Parkinson's disease (PD) is a progressive neurodegenerative disease highlighted by a marked loss of dopaminergic cell loss and motor disturbances. Currently, there are no drugs that slow the progression of the disease. A myriad of factors have been implicated in the pathogenesis and progression of PD including neuroinflammation. Although anti-inflammatory agents are being evaluated as potential disease-modifying therapies for PD, none has proven effective to date, suggesting that new and novel targets are needed. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a transmembrane glycoprotein that has recently been shown to reduce inflammation in astrocytes and to be increased in post-mortem PD brain samples. Here we show that transgenic overexpression of GPNMB protects against dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropridine mouse model of Parkinson's disease. Furthermore, GPNMB overexpression reduces gliosis and prevented microglial morphological changes following MPTP treatment compared with wild-type MPTP-treated mice. Additionally, recombinant GPNMB attenuates LPS-induced inflammation in primary mouse microglia. These results suggest a neuroprotective and anti-inflammatory role for GPNMB and warrant further investigation for GPNMB as a novel therapy for PD.

MP07-19 ACYLOXYACYL HYDROLASE REGULATES MICROGLIAL PHENOTYPE IN A MOUSE MODEL OF INTERSTITIAL CYSTITIS

INTRODUCTION AND OBJECTIVE: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a devastating condition of chronic pelvic pain. We previously linked a locus encoding acyloxyacyl hydrolase (Aoah) with pain severity in a murine model of IC. AOAH-deficient mice develop pelvic allodynia and exhibit symptoms co-morbid with IC/BPS, such as altered voiding and a depressive phenotype. Microglia are the resident immune cells of the central nervous system (CNS), and studies have linked altered microglial morphology and activation to neuropathic pain and depression. It is also well established that microglial activation can be modulated by the gut microbiota, and AOAH dysbiosis has been reported in IC/BPS patients. Therefore, we examined the microglial phenotype in AOAH-deficient mice and the potential role of microglia in modulating pelvic pain. METHODS: To analyze changes in microglial morphology, brains from wild type (WT) C57BL/6 and AOAH-deficient mice were sectioned and stained for the microglial marker P2RY12. Following immunohistochemistry, z-stacks were acquired and skeletal analyses were performed using Image J. Activation of microglia was measured through Western blotting and a proteome profiler array of mouse cytokines using brain lysates. To address the functional role of microglia in pelvic pain, microglia were eliminated from the CNS of AOAH-deficient and WT mice by oral administration of chow containing PLX5622. Mice were exposed to mechanical stimulation of the pelvic area with von Frey filaments prior to and following microglial elimination. RESULTS: Our preliminary data revealed altered microglial morphology in AOAH-deficient mice compared to WT mice. AOAH-deficient mice exhibited hyper-ramified microglia, suggesting a surveillance phenotype, in the cortex and Barrington’s nucleus, an essential region for micturition. We also observed hyper-ramified microglia in the paraventricular nucleus, a regulator of stress and depression. Microglia were less ramified in the hippocampus, consistent with an activated phenotype. Due to the morphological changes, we expect to observe differences in cytokine release between WT and AOAH-deficient mice, reflecting these differential activation states. In addition, we expect that microglia elimination will alleviate pelvic allodynia and depression in AOAH-deficient mice. CONCLUSIONS: Overall, we observed that microglial phenotype is dependent on AOAH, suggesting microglia-specific receptors or cytokines may be considered as future therapeutic targets for treating IC/BPS. Source of Funding: NIDDK awards U01 DK082342 and R01 DK066112-06S1

Impact of Reduced ASIC2a on Cortical Thickness, Microglia Density, and Brain Water Content in E18.5 Fetuses

Acid‐sensing Ion Channels (ASICs) are ligand‐gated cation channels that respond to acidic stimuli. ASIC2 is widely expressed in neurons of the central and peripheral nervous system as well as on microglia. Although ASIC2 plays a role in cell migration, their role in brain development is not fully known. We tested the hypothesis that reduction or absence of ASIC2a contributes to changes in microglia density, cortical thickness, and brain water content during development. Brains were collected from ASIC2a wild type (WT, n=6), heterozygous (HET, n=4), and homozygous (HOMO, n=3) fetuses at E18.5. We used hematoxylin &amp; eosin (H&amp;E) staining for cortical thickness measurements, immunofluorescence staining using ionized calcium‐binding adapter molecule (Iba‐1) to measure microglia density, and wet weight to dry weight ratio for brain water content. Reduction of ASIC2a had no impact on cortical thickness (WT=499±21, HET=509±7, and HOMO=478±21 μm; p=0.60) or microglia density in the subventricular zone near the lateral ventricle (WT=22±2, HET=18±2, and HOMO=24±3; p=0.34) at E18.5. Interestingly, microglia morphology in HET fetuses were different compared to WT and HOMO mice with thinner and longer processes. Brain water content increased in HOMO mice (88.2±0.7%) compared to WT (86.5±0.3%, p&lt;0.01) and HET (86.8±0.2%, p=0.02) fetuses. Our results indicate that while reduced ASIC2a does not contribute to changes in microglia density or cortical thickness in E18.5 mouse fetuses, reduced ASIC2a induces morphological changes in microglia and ASIC2 knockout increases brain water content at E18.5. Increased brain water content could be an indicator of hydrocephalus. In ongoing studies, we are determining whether reduced ASIC2a mediates structural and functional impairments in the neurovasculature of the maternal and developing brain.Support or Funding InformationNIH Grants: R00HL129192, R00HL129192‐S1

Microglia Activation and IL‐1β Expression in Select Ventilatory Control Regions Following Exposure to Chronic Sustained Hypoxia

Ventilatory acclimatization to hypoxia (VAH) is defined as the time‐dependent increase in ventilation which occurs with chronic sustained hypoxia (CSH) of several hours to months. Previous research has shown that astrocytes and microglia undergo a morphology shift, indicating a possible change in activity, upon exposure to hypoxic conditions and may contribute to VAH. Understanding when and how the different cell types in respiratory control regions are activated is pertinent to understanding ventilatory control during hypoxic conditions. The first part of this study aimed to optimize the Sholl analysis method to detect morphological changes in microglia. Two different microglia antibodies, Iba‐1 and CD11b[Ox42], were compared using Sholl analysis to determine which antibody best represents the branching pattern of the individual microglia. Analysis indicated that neither antibody was statistically different from the other in terms of microglia branching (p&gt;0.05), so CD11b[Ox42] was chosen to simplify the immunohistochemistry protocol. The second part of this study assessed the morphology shift of microglia in the nucleus tractus solitarius (NTS) and Pre‐Bötzinger Complex (PBC) following CSH exposure. Based on previous research, we hypothesized that microglia in the NTS and PBC would be activated following CSH exposure, as assessed via a morphology shift to a more amoeboid state indicated by less microglia branch crossings of the Sholl brackets. To address this hypothesis, rats were exposed to either normoxic, 60‐minutes of CSH, or 12‐hours of CSH. Microglia morphology was assessed in perfused brainstem tissue via immunohistochemistry, confocal imaging, and image analysis. In the NTS, microglia branching analysis revealed a trend towards a more amoeboid morphology at the 60‐minute CSH time point, but only one of the Sholl analysis brackets, 21–30μM, was statistically significant from normoxic conditions (p&lt;0.05). In the PBC, microglia branching analysis also revealed a trend towards a more amoeboid morphology at the 60‐minute CSH time point with statistically significant (p&lt;0.05) branch patterns at the 11–20μM, 21–30μM, and 31–40 μM Sholl brackets. A morphology shift of microglia to a more amoeboid state could indicate their localized response to neurotransmitters or cytokines. In a follow‐up study we investigated the expression of IL‐1β in the NTS region using immunofluorescent labeling. Rat brainstem tissue from normoxic conditions, 15‐minute CSH, and 60‐minute CSH was labeled with antibodies against IL‐1β, GFAP, and Cd11b[Ox42]. IL‐1β positive cells were counted in the NTS region. This preliminary set of cell counts (n=2) suggests that there is an increase in IL‐1β positive cells with CHS exposure (normoxic 17 ± 2 IL‐1β positive cells, 15‐minute CSH 31.5 ± 0.5 IL‐1β positive cells, 60‐minute CSH 29.5 ± 9.5 IL‐1β positive cells). Taken together these data provide a great starting point in assessing the activation profiles of glial cells and cytokine activity in select respiratory control regions in the brainstem.Support or Funding InformationCentenary College Student‐Faculty Summer Research Award 2018 and 2019 (JRF and JAS); NIH R01HL081823 (FLP)

Microglia depletion and alcohol: Transcriptome and behavioral profiles.

Alcohol abuse induces changes in microglia morphology and immune function, but whether microglia initiate or simply amplify the harmful effects of alcohol exposure is still a matter of debate. Here, we determine microglia function in acute and voluntary drinking behaviors using a colony-stimulating factor 1 receptor inhibitor (PLX5622). We show that microglia depletion does not alter the sedative or hypnotic effects of acute intoxication. Microglia depletion also does not change the escalation or maintenance of chronic voluntary alcohol consumption. Transcriptomic analysis revealed that although many immune genes have been implicated in alcohol abuse, downregulation of microglia genes does not necessitate changes in alcohol intake. Instead, microglia depletion and chronic alcohol result in compensatory upregulation of alcohol-responsive, reactive astrocyte genes, indicating astrocytes may play a role in regulation of these alcohol behaviors. Taken together, our behavioral and transcriptional data indicate that microglia are not the primary effector cell responsible for regulation of acute and voluntary alcohol behaviors. Because microglia depletion did not regulate acute or voluntary alcohol behaviors, we hypothesized that these doses were insufficient to activate microglia and recruit them to an effector phenotype. Therefore, we used a model of repeated immune activation using polyinosinic:polycytidylic acid (poly(I:C)) to activate microglia. Microglia depletion blocked poly(I:C)-induced escalations in alcohol intake, indicating microglia regulate drinking behaviors with sufficient immune activation. By testing the functional role of microglia in alcohol behaviors, we provide insight into when microglia are causal and when they are consequential for the transition from alcohol use to dependence.

Effects of age on retinal macrophage responses to acute elevation of intraocular pressure

There is accumulating evidence that aging shifts the central nervous system milieu towards a proinflammatory state, with increased reactivity of microglia in the aging eye and brain having been implicated in the development of age-related neurodegenerative conditions. Indeed, alterations to microglial morphology and function have been recognized as a part of normal aging. Here, we sought to assess the effects of age on the retinal microglial and macrophage response to acute intraocular pressure (IOP) elevation. Further, we performed experiments whereby bone marrow from young or middle-aged mice was used to reconstitute the bone marrow of whole-body irradiated 12 month old mice. Bone marrow chimeric mice then underwent cannulation and IOP elevation 8 weeks after whole-body irradiation and bone marrow transplantation in order to determine whether the age of bone marrow alters the macrophage response to retinal injury. Our data show retinal macrophage reactivity and microglial morphological changes were enhanced in older mice when compared to younger mice in response to injury. When IOP elevation was performed after whole-body irradiation and bone marrow rescue, we noted subretinal macrophage accumulation and glial reactivity was reduced compared to non-irradiated mice that had also undergone IOP elevation. This effect was evident in both groups of chimeric mice that had received either young or middle-aged bone marrow, suggesting irradiation itself may alter the macrophage and glial response to injury rather than the age of bone marrow.

Temporal analysis of histopathology and cytokine expression in the rat cerebral cortex after insulin‐induced hypoglycemia

Hypoglycemic coma causes neuronal death in the cerebral neocortex; however, its unclear pathogenesis prevents the establishment of preventive measures. Inflammation plays a pivotal role in neuronal damage in the hypoglycemic state; however, the dynamics of glial cell activation or cytokine expression remain unknown. Here, we aimed to elucidate the spatiotemporal morphological changes of microglia and time-course cytokine expression profiles in the rat cerebral cortex after hypoglycemic coma. We performed histopathological and immunohistochemical (Iba1, neuronal nuclei, glial fibrillary acidic protein) analyses in the cingulate cortex and four areas of the neocortex: hindlimb area (HL), parietal cortex area 1 (Par1), parietal cortex area 2 (Par2), and perirhinal cortex (PRh). We measured tumor necrosis factor alpha (TNFα) and interleukin-6 messenger RNA (mRNA) expression by real-time reverse transcriptase-polymerase chain reaction. Necrotic neurons appeared in the neocortex as early as 3 h after hypoglycemic coma, while they were absent in the cingulate cortex. Neuronal nuclei-immunopositive neurons in the HL, Par2, and PRh were significantly less abundant than in the control at day 1. In Iba1 immunostaining, large rod-shaped cells were detected at 3-6 h after hypoglycemia, and commonly observed in the HL, Par2, and PRh. After 6 h, rod-shaped cells were rarely observed; instead, there was a prominent infiltration of hypertrophic and ameboid-shaped cells until day 7. The mRNA expression of TNFα was significantly higher than the control at 3-6 h after hypoglycemia in the neocortex, while it was significantly higher only at 3 h in the cingulate cortex. Our results indicate that early and transient appearance of rod-shaped microglia and persisting high TNFα expression levels characterize inflammatory responses to hypoglycemic neuronal damage in the cerebral neocortex, which might contribute to neuronal necrosis in response to transient hypoglycemic coma.

Abstract TP337: Microglial Activation and Neuroinflammation Are Mediated by Over-Activation of Cofilin After Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a major health concern associated with high mortality and morbidity. 50% of the stroke victims die within the first month and the rest of the survivors have to deal with long-term disabilities. The understanding of the molecular mechanism of the secondary injury-induced neuroinflammation or microglial activation is not studied well. Our previous study demonstrated a major causative role of the cytoskeletal protein, cofilin in ICH-induced brain injury. We demonstrated that knockdown of cofilin in a mouse model of collagenase induced-ICH improved neurobehavioral deficits, decreased hemorrhagic volume and microglia activation. In the present study, we aimed to study the cofilin signaling up to 14 days following ICH. We subjected different cohorts of mice to intrastriatal collagenase injection-induced ICH and mice were sacrificed at different time-points of 1, 3, 7, and 14 days. Using Western blotting (WB) and quantitative real-time PCR, we observed an upregulation of cofilin protein and mRNA levels in the ipsilateral striatum on day 3 and then a decreasing trend was observed from day 7 to day 14. Using immunohistochemistry analysis, activated microglia were observed to be increased after ICH from day 1, especially around the hematoma and lasted until day 7 and there was a gradual decrease observed at day 14. Activated microglia were associated with morphological changes from ramified into an amoeboid shape particularly around the hematoma from day 1 up to day 14. Human ICH autopsy brain sections also indicated that intracellular cofilin is localized within microglia and is associated with microglia morphological changes and activation of surrounding microglia. In conclusion, we believe that cofilin overactivation plays a causative role in the activation of microglia and subsequently leads to widespread neuroinflammation following ICH. Developing cofilin inhibitors might provide novel alternatives for ICH treatment.

Microglial activation and responses to vasculature that result from an acute LPS exposure

Bacterial cell wall endotoxins, i.e. lipopolysaccharides (LPS), are some of the original compounds shown to evoke the classic signs of systemic inflammation/innate immune response and neuroinflammation. The term neuroinflammation often is used to infer the elaboration of proinflammatory mediators by microglia elicited by neuronal targeted activity. However, it also is possible that the microglia are responding to vasculature through several signaling mechanisms. Microglial activation relative to the vasculature in the hippocampus and parietal cortex was determined after an acute exposure of a single subcutaneous injection of 2 mg/kg LPS. Antibodies to allograft inflammatory factor (Aif1, a.k.a. Iba1) were used to track and quantify morphological changes in microglia. Immunostaining of platelet/endothelial cell adhesion molecule 1 (Pecam1, a.k.a. Cd31) was used to visualize vasculature in the forebrain and glial acidic fibrillary protein (GFAP) to visualize astrocytes. Neuroinflammation and other aspects of neurotoxicity were evaluated histologically at 3 h, 6 h, 12 h, 24 h, 3 d and 14 d following LPS exposure. LPS did not cause neurodegeneration as determined by Fluoro Jade C labeling. Also, there were no signs of mouse IgG leakage from brain vasculature due to LPS. Some changes in microglia size occurred at 6 h, but by 12 h microglial activation had begun with the combined soma and proximal processes size increasing significantly (1.5-fold). At 24 h, almost all the microglia soma and proximal processes in the hippocampus, parietal cortex, and thalamus were closely associated with the vasculature and had increased almost 2.0-fold in size. In many areas where microglia were juxtaposed to vasculature, astrocytic endfeet appeared to be displaced. The microglial activation had subsided slightly by 3 d with microglial size 1.6-fold that of control. We hypothesize that acute LPS activation can result in vascular mediated microglial responses through several mechanisms: 1) binding to Cd14 and Tlr4 receptors on microglia processes residing on vasculature; 2) damaging vasculature and causing the release of cytokines; and 3) possibly astrocytic endfeet damage resulting in cytokine release. These acute responses may serve as an adaptive mechanism to exposure to circulating LPS where the microglia surround the vasculature. This could further prevent the pathogen(s) circulating in blood from entering the brain. However, diverting microglial interactions away from synaptic remodeling and other types of microglial interactions with neurons may have adverse effects on neuronal function.

Microglia alter the threshold of spreading depolarization and related potassium uptake in the mouse brain

Selective elimination of microglia from the brain was shown to dysregulate neuronal Ca2+ signaling and to reduce the incidence of spreading depolarization (SD) during cerebral ischemia. However, the mechanisms through which microglia interfere with SD remained unexplored. Here, we identify microglia as essential modulators of the induction and evolution of SD in the physiologically intact brain in vivo. Confocal- and super-resolution microscopy revealed that a series of SDs induced rapid morphological changes in microglia, facilitated microglial process recruitment to neurons and increased the density of P2Y12 receptors (P2Y12R) on recruited microglial processes. In line with this, depolarization and hyperpolarization during SD were microglia- and P2Y12R-dependent. An absence of microglia was associated with altered potassium uptake after SD and increased the number of c-fos-positive neurons, independently of P2Y12R. Thus, the presence of microglia is likely to be essential to maintain the electrical elicitation threshold and to support the full evolution of SD, conceivably by interfering with the extracellular potassium homeostasis of the brain through sustaining [K+]e re-uptake mechanisms.

Transient neonatal sleep fragmentation results in long-term neuroinflammation and cognitive impairment in a rabbit model

Sleep fragmentation is an increase in sleep-wake transitions without an overall decrease in total sleep time. Sleep fragmentation is well documented during acute and chronic hospitalization and can result in delirium and memory problems in children. Sleep fragmentation is also often noted in neurodevelopmental disorders. However, it is unclear how sleep fragmentation independent of disease affects brain development and function. We hypothesized that acute sleep fragmentation during the neonatal period in otherwise healthy animals would result in neuroinflammation and would be associated with abnormalities in cognitive development. The orbital shaker method was used to fragment sleep for 72 h in postnatal day 3 New Zealand white rabbit kits (fragmentation group). To control for maternal separation, the sham group was separated from the dam and maintained in the same conditions without undergoing sleep fragmentation. A naïve control group remained with the dam. Kits underwent behavioral testing with novel object recognition and spontaneous alternation T-maze tests at 2–3 weeks post-fragmentation and were sacrificed 3–50 days after fragmentation. Sleep fragmentation resulted in acute and chronic changes in microglial morphology in the hippocampus and cortex, and regional differences in mRNA expression of pro- and anti-inflammatory cytokines at 3, 7 and 50 days post-fragmentation. Impaired novel object recognition and a longer latency in T-maze task completion were noted in the fragmented kits. This was in spite of normalization of sleep architecture noted at 2 months of age in these kits. The results indicate that transient neonatal sleep fragmentation results in short-term and long-term immune alterations in the brain, along with diminished performance in cognitive tasks long-term.

Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue

Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.SIGNIFICANCE STATEMENT Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.

Inhibition of Microglial Activation in the Amygdala Reverses Stress-Induced Abdominal Pain in the Male Rat.

Background & AimsPsychological stress is a trigger for the development of irritable bowel syndrome and associated symptoms including abdominal pain. Although irritable bowel syndrome patients show increased activation in the limbic brain, including the amygdala, the underlying molecular and cellular mechanisms regulating visceral nociception in the central nervous system are incompletely understood. In a rodent model of chronic stress, we explored the role of microglia in the central nucleus of the amygdala (CeA) in controlling visceral sensitivity. Microglia are activated by environmental challenges such as stress, and are able to modify neuronal activity via synaptic remodeling and inflammatory cytokine release. Inflammatory gene expression and microglial activity are regulated negatively by nuclear glucocorticoid receptors (GR), which are suppressed by the stress-activated pain mediator p38 mitogen-activated protein kinases (MAPK).MethodsFisher-344 male rats were exposed to water avoidance stress (WAS) for 1 hour per day for 7 days. Microglia morphology and the expression of phospho-p38 MAPK and GR were analyzed via immunofluorescence. Microglia-mediated synaptic remodeling was investigated by quantifying the number of postsynaptic density protein 95–positive puncta. Cytokine expression levels in the CeA were assessed via quantitative polymerase chain reaction and a Luminex assay (Bio-Rad, Hercules, CA). Stereotaxic infusion into the CeA of minocycline to inhibit, or fractalkine to activate, microglia was followed by colonic sensitivity measurement via a visceromotor behavioral response to isobaric graded pressures of tonic colorectal distension.ResultsWAS induced microglial deramification in the CeA. Moreover, WAS induced a 3-fold increase in the expression of phospho-p38 and decreased the ratio of nuclear GR in the microglia. The number of microglia-engulfed postsynaptic density protein 95–positive puncta in the CeA was increased 3-fold by WAS, while cytokine levels were unchanged. WAS-induced changes in microglial morphology, microglia-mediated synaptic engulfment in the CeA, and visceral hypersensitivity were reversed by minocycline whereas in stress-naïve rats, fractalkine induced microglial deramification and visceral hypersensitivity.ConclusionsOur data show that chronic stress induces visceral hypersensitivity in male rats and is associated with microglial p38 MAPK activation, GR dysfunction, and neuronal remodeling in the CeA.