OP08 Evidence for early astrocyte activation, cellular stress and compensatory microglial related transforming growth factor-α responses in bile-duct ligated rats
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Introduction Inflammation and ammonia are important mediators in the pathogenesis of hepatic encephalopathy and though the mechanisms are unclear astrocytes are thought to have a central role. Recently Microglia, which also mediate brain inflammation, were implicated in the brain effects of acute liver failure; however their influence in chronic liver disease is unknown. Aim The aim of this longitudinal study was to characterise the early brain responses in bile-duct ligated (BDL) rats in the 4-weeks following ligation. Method Twenty-four male Sprague-Dawley rats were studied after sham-operation or BDL and sacrificed at either 1-day or, 1-, 2- or 4 weeks post-surgery (n=4/group). Consciousness, brain water content, arterial ammonia, plasma biochemistry and proinflammatory (IL-6, TNF-α and γ-IFN) and antiinflammatory (IL-4 and IL-10) cytokines, were analysed. Immunohistochemical markers of activated microglia (ED1, OX6 and Iba-1), astrocytes (GFAP), inflammatory responses (IL-1s and iNOS), cellular stress (HSP-25) and the predominant antiinflammatory and alternative microglial activation marker TGF-s (using RT-PCR), were also analysed. Results Compared to Shams, arterial and brain ammonia (p Conclusion Regional astrocyte activation and cellular stress (indicated by increased HSP-25 expression), are early features of BDL. These events are associated with increased brain proinflammatory cytokine production and iNOS expression. This proinflammatory response is not due to obvious microglia activation, but consequent upon activation of astrocytes, possibly related to hyperammonemia and/or associated cell swelling. However, an observed TGF-s response may reflect compensatory antiinflammatory microglial responses, designed to limit the effect of astrocyte activation; with interventions targeting its brain expression potential novel therapies for hepatic encephalopathy.Keywords:
Proinflammatory cytokine
Neuroglia
Background: Microglia are key mediators of inflammation during perinatal brain injury. As shown experimentally after inflammation-sensitized hypoxic ischemic (HI) brain injury, microglia are activated into a pro-inflammatory status 24 h after HI involving the NLRP3 inflammasome pathway. The chemokine (C-X-C motif) ligand 1 (CXCL1), and its cognate receptor, CXCR2, have been shown to be involved in NLRP3 activation, although their specific role during perinatal brain injury remains unclear. In this study we investigated the involvement of CXCL1/CXCR2 in brain tissue and microglia and brain tissue after inflammation-sensitized HI brain injury of newborn rats. Methods: Seven-day old Wistar rat pups were either injected with vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia (8% O2 for 50 min). Pups were randomized into four different treatment groups: (1) Sham group (n = 21), (2) LPS only group (n = 20), (3) Veh/HI group (n = 39), and (4) LPS/HI group (n = 42). Twenty-four hours post hypoxia transcriptome and gene expression analysis were performed on ex vivo isolated microglia cells in our model. Additionally protein expression was analyzed in different brain regions at the same time point. Results: Transcriptome analyses showed a significant microglial upregulation of the chemokine CXCL1 and its receptor CXCR2 in the LPS/HI group compared with the other groups. Gene expression analysis showed a significant upregulation of CXCL1 and NLRP3 in microglia cells after inflammation-sensitized hypoxic-ischemic brain injury. Additionally, protein expression of CXCL1 was significantly upregulated in cortex of male pups from the LPS/HI group. Conclusion: These results indicate that the CXCL1/CXCR2 pathway may be involved during pro-inflammatory microglia activation following inflammation-sensitized hypoxic-ischemic brain injury in neonatal rats. This may lead to new treatment options altering CXCR2 activation early after HI brain injury.
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Hypoxia
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Abstract In order to evaluate proinflammatory cytokine levels and their producing cell types in the control aged rat brain and after acute excitotoxic damage, both adult and aged male Wistar rats were injected with N‐methyl‐ D ‐aspartate in the striatum. At different survival times between 6 hr and 7 days after lesioning, interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor alpha (TNF‐α) were analyzed by enzyme‐linked immunosorbent assay and by double immunofluorescence of cryostat sections by using cell‐specific markers. Basal cytokine expression was attributed to astrocytes and was increased in the normal aged brain showing region specificity: TNF‐α and IL‐6 displayed age‐dependent higher levels in the aged cortex, and IL‐1β and IL‐6 in the aged striatum. After excitotoxic striatal damage, notable age‐dependent differences in cytokine induction in the aged vs. the adult were seen. The adult injured striatum exhibited a rapid induction of all cytokines analyzed, but the aged injured striatum showed a weak induction of cytokine expression: IL‐1β showed no injury‐induced changes at any time, TNF‐α presented a late induction at 5 days after lesioning, and IL‐6 was only induced at 6 hr after lesioning. At both ages, in the lesion core, all cytokines were early expressed by neurons and astrocytes, and by microglia/macrophages later on. However, in the adjacent lesion border, cytokines were found in reactive astrocytes. This study highlights the particular inflammatory response of the aged brain and suggests an important role of increased basal levels of proinflammatory cytokines in the reduced ability to induce their expression after damage. © 2009 Wiley‐Liss, Inc.
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Background and Purpose— Activation of Notch worsens ischemic brain damage as antisense knockdown or pharmacological inhibition of the Notch pathway reduces the infarct size and improves the functional outcome in a mouse model of stroke. We sought to determine whether Notch activation contributes to postischemic inflammation by directly modulating the microglial innate response. Methods— The microglial response and the attendant inflammatory reaction were evaluated in Notch1 antisense transgenic (Tg) and in nontransgenic (non-Tg) mice subjected to middle cerebral artery occlusion with or without treatment with a γ-secretase inhibitor (GSI). To investigate the impact of Notch on microglial effector functions, primary mouse microglia and murine BV-2 microglial cell line were exposed to oxygen glucose deprivation or lipopolysaccharide in the presence or absence of GSI. Immunofluorescence labeling, Western blotting, and reverse-transcription polymerase chain reaction were performed to measure microglial activation and production of inflammatory cytokines. The nuclear translocation of nuclear factor-κB in microglia was assessed by immunohistochemistry. The neurotoxic potential of microglia was determined in cocultures. Results— Notch1 antisense mice exhibit significantly lower numbers of activated microglia and reduced proinflammatory cytokine expression in the ipsilateral ischemic cortices compared to non-Tg mice. Microglial activation also was attenuated in Notch1 antisense cultures and in non-Tg cultures treated with GSI. GSI significantly reduced nuclear factor-κB activation and expression of proinflammatory mediators and markedly attenuated the neurotoxic activity of microglia in cocultures. Conclusions— These findings establish a role for Notch signaling in modulating the microglia innate response and suggest that inhibition of Notch might represent a complementary therapeutic approach to prevent reactive gliosis in stroke and neuroinflammation-related degenerative disorders.
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Traumatic brain injury (TBI) is a major health problem with high rates of mortality and morbidity worldwide. The response of the brain to TBI is orchestrated by a number of cytokines, including interleukin-6 (IL-6). IL-6 is a major cytokine in the central nervous system and it is produced by different cells, such as neurons, glial cells, and endothelial cells. Since glial cells are one of the most important sources and targets of IL-6, we have examined the role of microglia-derived IL-6 in normal conditions and following a model of TBI, cryolesion of the somatosensorial cortex. To this end, tamoxifen-inducible microglial IL-6-deficient (Il6ΔMic , using Cx3cr1 CreER model) mice and control (Il6lox/lox ) mice were used. In normal conditions, microglial IL-6 deficiency reduced deambulation and exploratory behavior and decreased anxiety in a sex-dependent manner. The transcriptome profile following cryolesion was dramatically altered 1 day post-lesion in Il6ΔMic compared with Il6lox/lox mice. However, the phenotype of Il6ΔMic mice was less compromised in the following days, suggesting that compensatory mechanisms are at play.
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Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2–12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2‐4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba‐1 + ) and astrocytes (GFAP + ), however, were undetected during this 2–12 h timeframe. Increased Iba‐1 immunoreactivity and de‐ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba‐1 or GFAP immunoreactivity after LPS challenge. GLIA 2016;64:300–316
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Hyperammonemia
Aquaporin 4
Neuroglia
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Microglial activation and microglia‐mediated inflammation play an important role in the occurrence, development, and outcome of stroke. Brain injury induces the activation and release of proinflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐ α ), interleukin‐ (IL‐) 1 β , and IL‐6. Many studies have confirmed that acupuncture is effective in treating ischemic stroke. However, its protective mechanism against ischemic brain injury is complex and multifactorial. In this study, we observed the effects of electroacupuncture at Baihui (GV20) and Dazhui (GV14) on microglial activation and inflammation in the cortical ischemic penumbra (IP) of permanent middle cerebral artery occlusion (pMCAO) rats. It was found that electroacupuncture inhibited the degeneration and necrosis of microglia in the cortical IP and ameliorated mitochondrial damage. Immunofluorescence and western blot analysis showed that microglia were in a resting state or weakly activated in the normal brain. After cerebral ischemia, the expression of microglial markers (Iba‐1 and CD11b) increased, and NF‐ κ B p65, IL‐1 β , and TNF‐ α expression gradually increased. The dynamic changes were generally temporally consistent. Electroacupuncture downregulated the expressions of Iba‐1 and CD11b. Additionally, it inhibited the expression of NF‐ κ B p65, IL‐1 β , and TNF‐ α and reduced the conversion of microglia to the M1 phenotype after ischemia. Electroacupuncture regulated the activation of microglia and microglia‐mediated inflammation after cerebral ischemia, confirming the relevant theories regarding the effect of acupuncture treatment on cerebral ischemia and guiding clinical practice.
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Posttraumatic inflammatory processes contribute to pathological and reparative processes observed after traumatic brain injury (TBI). Recent findings have emphasized that these divergent effects result from subsets of proinflammatory (M1) or anti-inflammatory (M2) microglia and macrophages. Therapeutic hypothermia has been tested in preclinical and clinical models of TBI to limit secondary injury mechanisms including proinflammatory processes. This study evaluated the effects of posttraumatic hypothermia (PTH) on phenotype patterns of microglia/macrophages. Sprague-Dawley rats underwent moderate fluid percussion brain injury with normothermia (37℃) or hypothermia (33℃). Cortical and hippocampal regions were analyzed using flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR) at several periods after injury. Compared to normothermia, PTH attenuated infiltrating cortical macrophages positive for CD11b+ and CD45high. At 24 h, the ratio of iNOS+ (M1) to arginase+ (M2) cells after hypothermia showed a decrease compared to normothermia. RT-PCR of M1-associated genes including iNOS and IL-1β was significantly reduced with hypothermia while M2-associated genes including arginase and CD163 were significantly increased compared to normothermic conditions. The injury-induced increased expression of the chemokine Ccl2 was also reduced with PTH. These studies provide a link between temperature-sensitive alterations in macrophage/microglia activation and polarization toward a M2 phenotype that could be permissive for cell survival and repair.
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