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
Abstract Astrocyte swelling and the subsequent increase in intracranial pressure and brain herniation are major clinical consequences in patients with acute hepatic encephalopathy. We recently reported that conditioned media from brain endothelial cells ( EC s) exposed to ammonia, a mixture of cytokines ( CK s) or lipopolysaccharide ( LPS ), when added to astrocytes caused cell swelling. In this study, we investigated the possibility that ammonia and inflammatory agents activate the toll‐like receptor 4 ( TLR 4) in EC s, resulting in the release of factors that ultimately cause astrocyte swelling. We found a significant increase in TLR 4 protein expression when EC s were exposed to ammonia, CK s or LPS alone, while exposure of EC s to a combination of these agents potentiate such effects. In addition, astrocytes exposed to conditioned media from TLR 4‐silenced EC s that were treated with ammonia, CK s or LPS , resulted in a significant reduction in astrocyte swelling. TLR 4 protein up‐regulation was also detected in rat brain EC s after treatment with the liver toxin thioacetamide, and that thioacetamide‐treated TLR 4 knock‐out mice exhibited a reduction in brain edema. These studies strongly suggest that EC s significantly contribute to the astrocyte swelling/brain edema in acute hepatic encephalopathy, likely as a consequence of increased TLR 4 protein expression by blood‐borne noxious agents. image Ammonia and inflammatory agents, such as lipopolysaccharide (LPS) or cytokines (CKs), activate the toll‐like receptor 4 (TLR4) in endothelial cells (ECs) ultimately resulting in astrocyte swelling. TLR4 protein upregulation was detected in rat brain ECs in acute hepatic encephalopathy (AHE), whereas TLR4 knock‐out mice exhibited a reduction in brain edema after AHE. These studies suggest that ECs significantly contribute to the astrocyte swelling/brain edema in AHE.
Thioacetamide
Hepatic Encephalopathy
Cerebral edema
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Background and Purpose: Astrocytes play a pivotal role in post-ischemic brain inflammation, but the relevant astrocyte-derived mediators of ischemic brain injury remain to be defined. This study aims to investigate the impact of astrocyte-derived factors such as IL-15 on ischemic brain injury. Methods and Results: We show that IL-15 is a prominent factor relased by astrocytes after brain ischemia in a mouse model of transient focal brain ischemia. To further eluciate the biological functions of astrcoyte-derived IL-15 in ischemic stroke, we generated a glial fibrillary acidic protein (GFAP) promoter-controlled IL-15-expressing transgenic mouse line (GFAP-IL-15 tg ). We demonstrate that astrocyte-specific overproduction of IL-15 leads to larger brain infarcts, worse neurodeficits and enhanced lymphocyte infiltration. In GFAP-IL-15 tg mice, we found increased accumulation and activation of CD8 + T and natural killer (NK) cells after cerebral ischemia. Importantly, depletion of either CD8 + T or NK cells in GFAP-IL-15 tg mice prior to cerebral ischemia attenuates the accelerated brain infarction and neurodeficits. Of note, CD8 + T and NK cells are within the proximity of astrocytes in the post-ischemic brain and either knockdown of IL-15 receptor α or blockade of cell-to-cell contact diminishes the activation and effector function of CD8 + T and NK cells caused by astrocytic overproduction of IL-15, suggesting that astrocytic IL-15 is delivered in trans to target cells. Additionally, CD8 + T and NK cells are seen closely adjacent to IL-15-producing astrocytes in acute ischemic lesions of postmortem human brain tissues, implying that astrocytic IL-15-mediated brain injury may be relevant to human stroke. Conclusion: These findings indicate that astrocytic IL-15 contributes to post-ischemic brain damage.
Brain ischemia
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Proinflammatory cytokine
Piriform cortex
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Hypertension is the major risk factor for stroke. Recent work unveiled that hypertension is associated with chronic neuroinflammation; microglia are the major players in neuroinflammation, and the activated microglia elevate sympathetic nerve activity and blood pressure. This study is to understand how brain homeostasis is kept from hypertensive disturbance and microglial activation at the onset of hypertension.Hypertension was induced by subcutaneous delivery of angiotensin II, and blood pressure was monitored in conscious animals. Microglial activity was analyzed by flow cytometry and immunohistochemistry. Antibody, pharmacological chemical, and recombinant cytokine were administered to the brain through intracerebroventricular infusion. Microglial depletion was performed by intracerebroventricular delivering diphtheria toxin to CD11b-diphtheria toxin receptor mice. Gene expression profile in sympathetic controlling nucleus was analyzed by customized qRT-PCR array.Transforming growth factor-β (TGF-β) is constitutively expressed in the brains of normotensive mice. Removal of TGF-β or blocking its signaling before hypertension induction accelerated hypertension progression, whereas supplementation of TGF-β1 substantially suppressed neuroinflammation, kidney norepinephrine level, and blood pressure. By means of microglial depletion and adoptive transfer, we showed that the effects of TGF-β on hypertension are mediated through microglia. In contrast to the activated microglia in established hypertension, the resting microglia are immunosuppressive and important in maintaining neural homeostasis at the onset of hypertension. Further, we profiled the signature molecules of neuroinflammation and neuroplasticity associated with hypertension and TGF-β by qRT-PCR array.Our results identify that TGF-β-modulated microglia are critical to keeping brain homeostasis responding to hypertensive disturbance.
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Microgliosis and astrogliosis are standard pathological features of neurodegenerative disease. Microglia are primed by chronic neurodegeneration such that toll-like receptor agonists, such as LPS, drive exaggerated cytokine responses on this background. However, sterile inflammatory insults are more common than direct CNS infection in the degenerating brain and these insults drive robust IL-1β and TNF-α responses. It is unclear whether these pro-inflammatory cytokines can directly induce exaggerated responses in the degenerating brain. We hypothesized that glial cells in the hippocampus of animals with chronic neurodegenerative disease (ME7 prion disease) would display exaggerated responses to central cytokine challenges. TNF-α or IL-1β were administered intrahippocampally to ME7-inoculated mice and normal brain homogenate-injected (NBH) controls. Both IL-1β and TNF-α produced much more robust IL-1β synthesis in ME7 than in NBH animals and this occurred exclusively in microglia. However, there was strong nuclear localization of the NFκB subunit p65 in the astrocyte population, associated with marked astrocytic synthesis of the chemokines CXCL1 and CCL2 in response to both cytokine challenges in ME7 animals. Conversely, very limited expression of these chemokines was apparent in NBH animals similarly challenged. Thus, astrocytes are primed in the degenerating brain to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. Furthermore, this results in markedly increased neutrophil, T-cell, and monocyte infiltration in the diseased brain. These data have significant implications for acute sterile inflammatory insults such as stroke and traumatic brain injury occurring on a background of aging or neurodegeneration.
Astrogliosis
CXCL1
Gliosis
Proinflammatory cytokine
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Neuroinflammation mediated by activated microglia plays a pivotal role in the pathogenesis of neurological disorders, including hypoxic injury of the developing brain. Thymosin β4 (Tβ4), the major G-actin-sequestering molecule, has an anti-inflammatory effect and has been used to treat various neurological diseases. However, the effect of Tβ4 on hypoxia-induced microglia activation in the developing brain remains unclear. We investigate here the effect of Tβ4 on microglia activation of neonatal rats after hypoxia exposure. Tβ4 treatment was carried out on 1-day-old rats and BV-2 cells. Tβ4 expression in microglia was determined by quantitative real time-PCR, western blotting, and immunofluorescence staining. Secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and nitric oxide (NO) was assessed by enzyme-linked immunosorbent assay and colorimetric assay. mRNA expression of TNF-α and IL-1β, and microRNA 146a expression was determined by quantitative real time-PCR. We showed that Tβ4 treatment significantly inhibited secretion of inflammatory mediators in the cerebellum of neonatal rats following hypoxia injury. Increased expression of endogenous Tβ4 in microglia was observed both in hypoxic rats and in BV-2 cells. Tβ4 treatment significantly inhibited the expression and secretion of hypoxia-induced TNF-α, IL-1β, and NO. Remarkably, microRNA 146a expression was found to have increased in Tβ4-treated BV-2 cells. We demonstrated the anti-inflammatory effect of Tβ4 in neonatal rats following hypoxic brain injury. More importantly, our data reveal, for the first time, that Tβ4 inhibits microglia activation in vitro. Therefore, this study contributes to understanding the role and mechanism of Tβ4 function in central nervous system diseases.
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Thymosin
Brain damage
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Microglia and astrocyte related pro-inflammatory responses are thought to underpin cerebral sequelae of acute liver failure. Conversely, despite background pro-inflammatory responses in cirrhosis, overt brain swelling and coma associated with acute-on-chronic liver failure, is infrequent unless precipitated (e.g. sepsis). Moreover in other chronic neurodegenerative disorders and sepsis, the brain is protected from recurrent microbial insults by compensatory microglial-associated immune responses. To characterise longitudinal cerebral immune responses in a bile duct-ligated (BDL) rat model of cirrhosis.Rats underwent BDL or sham operation before sacrifice at either 1-day, 1, 2 and 4 weeks post-surgery. We analysed consciousness, brain water, biochemistry and immunohistochemistry to assess activation of microglia (ED-1, OX6 and Iba-1), astrocytes (Glial fibrillary acidic protein - GFAP), cellular stress (Heat shock protein - Hsp 25) and pro-inflammatory mediator expression (inducible nitric oxide synthase (iNOS), interleukin-1beta (IL-1β) and tumour growth factor-beta (TGF-β)).BDL significantly increased ammonia and bilirubin (P < 0.01 respectively). The classical microglial markers OX6, ED1 and Iba-1 and pro-inflammatory IL-1β and iNOS were not significantly increased. However, the alternative microglial marker and regulatory cytokine TGF-β was elevated from day 1 to 4 weeks post-BDL. GFAP expression was significantly increased in corpus callosum in all groups. In BDL rats, Hsp 25 was also increased in the corpus callosum, peaking at 2 weeks.BDL triggers early alternative, but not classical, microglial activation. There was a correlation between astrocyte activation and cellular stress. These findings indicate early cerebral immune responses, which may be associated with immune tolerance to further challenge.
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Macrophages are viewed as amplifiers of ischemic brain injury, but the origin of injury-producing macrophages is poorly defined. The role of resident brain macrophages—microglial cells—in stroke remains controversial. To determine whether microglial cells exert injurious effects after neonatal focal stroke, we selectively depleted these cells with intracerebral injection of liposome-encapsulated clodronate before transient middle cerebral artery occlusion in postnatal day 7 rats. Phagocytosis of apoptotic neurons by activated microglia was poor in animals with unmanipulated microglia, and depletion of these cells did not increase the number of apoptotic neurons. Lack of microglia increased the brain levels of several cytokines and chemokines already elevated by ischemia–reperfusion, and also increased the severity and volume of injury, suggesting that microglial cells contribute to endogenous protection during the subacute injury phase. Then, to determine whether accumulation of reactive oxygen species in microglia adversely affects phagocytosis of dying neurons and contributes to injury, we delivered reduced glutathione (GSH) into microglia, again using liposomes. Remarkably, pharmacologically increased intracellular GSH concentrations in microglia induced superoxide accumulation in lipid rafts in these cells, further increased the brain levels of macrophage chemoattractants, and exacerbated injury. Together, these data show that microglia are part of the endogenous defense mechanisms and that, while antioxidants can protect the injured neonatal brain, high levels of reducing equivalents in activated microglia, GSH, trigger superoxide production, favor the reorganization of lipids, amplify local inflammation and exacerbate injury.
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Within hours after stroke, potentially cytotoxic pro-inflammatory mediators are elevated within the brain; thus, one potential therapeutic strategy is to reduce them and skew the brain toward an anti-inflammatory state. Because interleukin-4 (IL-4) treatment induces an anti-inflammatory, "alternative-activation" state in microglia and macrophages in vitro, we tested the hypothesis that early supplementation of the brain with IL-4 can shift it toward an anti-inflammatory state and reduce damage after transient focal ischemia. Adult male rat striata were injected with endothelin-1, with or without co-injection of IL-4. Inflammation, glial responses and damage to neurons and white matter were quantified from 1 to 7 days later. At 1 day, IL-4 treatment increased striatal expression of several anti-inflammatory markers (ARG1, CCL22, CD163, PPARγ), increased phagocytic (Iba1-positive, CD68-positive) microglia/macrophages, and increased VEGF-A-positive infiltrating neutrophils in the infarcts. At 7 days, there was evidence of sustained, propagating responses. IL-4 increased CD206, CD200R1, IL-4Rα, STAT6, PPARγ, CD11b, and TLR2 expression and increased microglia/macrophages in the infarct and astrogliosis outside the infarct. Neurodegeneration and myelin damage were not reduced, however. The sustained immune and glial responses when resolution and repair processes have begun warrant further studies of IL-4 treatment regimens and long-term outcomes.
Transient (computer programming)
Interleukin 1β
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Interleukin‐1 (IL‐1) is induced immediately after brain imjury and elevated levels of IL‐1 have been strongly implicated in the neurodegeneration that accompanies stroke, Alzheimer's disease and Multiple Sclerosis. Antagonizing IL‐1 reduces cell death; however, the basis for this protection has not been elucidated. Here we analyzed the response to penetrating brain injury in mice lacking the type 1 interleukin receptor (IL‐1R1) to determine which cellular and molecular mediators of tissue damage require IL‐1 signaling. At the cellular level fewer amoeboid microglia/macrophages appeared adjacent to the injured brain tissue in IL‐1R1 null mice, and those microglia present at early postinjury intervals retained their resting morphology. Astrogliosis also was mildly abrogated. At the molecular level, cyclooxygenase 2 and IL‐6 expression were depressed and delayed. Interestingly, basal levels of cyclooxygenase 2, IL‐1 and IL‐6 were significantly lower in the IL‐1R1 null mice. Additionally, stimulation of VCAM‐1 mRNA was depressed in the IL‐1R1 null mice, and correspondingly, there was reduced migration of peripheral macrophages into the IL‐1R1 null brain after injury. This observation correlated with a reduced number of cyclooxygenase 2+ amoeboid phagocytes adjacent to the injury. By contrast, the production of nerve growth factor was only mildly affected. Since antagonizing IL‐1 protects neural cells in experimental models of stroke and multiple sclerosis, our data suggest that cell preservation is achieved by abrogating microglial/macrophage activation and the subsequent self‐propagating cycle of inflammation. Acknowledgements: Supported by NMSS Award #RG 3837.
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Gliosis
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