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Dose-Dependent Microglial and Astrocytic Responses Associated With Post-ischemic Neuroprotection After Lipopolysaccharide-Induced Sepsis-Like State in Mice.

ABSTRACT: In contrast to lipopolysaccharide (LPS)-induced preconditioning, which has repeatedly been examined in the past, the effects of post-ischemic LPS-induced sepsis, although clinically considerably more important, have not systemically been studied. We exposed mice to transient intraluminal middle cerebral artery occlusion (MCAO) and examined the effects of intraperitoneal LPS (0.1 or 1 mg/kg) which was administered 24 h post-ischemia. Post-ischemic glial reactivity, neuronal survival and neurological outcome were differently modulated by the higher and the lower LPS dose. Although both doses promoted neuronal survival after 72 h, the underlying mechanisms were not similar. Mice receiving 1 mg/kg LPS exhibited transient hypothermia at 1 and 3 hours post sepsis (hps), followed by reduced focal neurological deficits at 24, 48 and 72 hps. The lower dose (0.1 mg/kg) did not induce hypothermia, but reduced microglia/macrophage activation with the appearance of an anti-inflammatory CD206 positive cell phenotype in the brain parenchyma. Together, our results indicate a novel, dose-dependent modulation of microglial cells that is intricately involved in brain protection.

SUBMITTER: Sardari M

PROVIDER: S-EPMC7029732 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Dose-Dependent Microglial and Astrocytic Responses Associated With Post-ischemic Neuroprotection After Lipopolysaccharide-Induced Sepsis-Like State in Mice.

Sardari Maryam M Dzyubenko Egor E Schmermund Ben B Yin Dongpei D Qi Yachao Y Kleinschnitz Christoph C Hermann Dirk M DM

Frontiers in cellular neuroscience 20200212

In contrast to lipopolysaccharide (LPS)-induced preconditioning, which has repeatedly been examined in the past, the effects of post-ischemic LPS-induced sepsis, although clinically considerably more important, have not systemically been studied. We exposed mice to transient intraluminal middle cerebral artery occlusion (MCAO) and examined the effects of intraperitoneal LPS (0.1 or 1 mg/kg) which was administered 24 h post-ischemia. Post-ischemic glial reactivity, neuronal survival and neurologi ...[more]

PMID: 32116567

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Project description:BackgroundAstrocytic glycogen works as an essential energy reserve for surrounding neurons and is reported to accumulate excessively during cerebral ischemia/reperfusion (I/R) injury. Our previous study found that accumulated glycogen mobilization exhibits a neuroprotective effect against I/R damage. In addition, ischemia could transform astrocytes into A1-like (toxic) and A2-like (protective) subtypes. However, the underlying mechanism behind accumulated glycogen mobilization-mediated neuroprotection in cerebral reperfusion injury and its relationship with the astrocytic A1/A2 paradigm is unknown.MethodsAstrocytic glycogen phosphorylase, the rate-limiting enzyme in glycogen mobilization, was specifically overexpressed and knocked down in mice and in cultured astrocytes. The I/R injury was imitated using a middle cerebral artery occlusion/reperfusion model in mice and an oxygen-glucose deprivation/reoxygenation model in cultured cells. Alterations in A1-like and A2-like astrocytes and the expression of phosphorylated nuclear transcription factor-κB (NF-κB) and phosphorylated signal transducer and activator of transcription 3 (STAT3) were determined by RNA sequencing, immunofluorescence and immunoblotting. Metabolites, including glycogen, NADPH, glutathione and reactive oxygen species (ROS), were analyzed by biochemical analysis.ResultsHere, we observed that astrocytic glycogen mobilization inhibited A1-like astrocytes and enhanced A2-like astrocytes after reperfusion in an experimental ischemic stroke model in vivo and in vitro. In addition, glycogen mobilization could enhance the production of NADPH and glutathione by the pentose phosphate pathway (PPP) and reduce ROS levels during reperfusion. NF-κB inhibition and STAT3 activation caused by a decrease in ROS levels were responsible for glycogen mobilization-induced A1-like and A2-like astrocyte transformation after I/R. The astrocytic A1/A2 paradigm is closely correlated with glycogen mobilization-mediated neuroprotection in cerebral reperfusion injury.ConclusionsOur data suggest that ROS-mediated NF-κB inhibition and STAT3 activation are the key pathways for glycogen mobilization-induced neuroprotection and provide a promising metabolic target for brain reperfusion injury in ischemic stroke.

Project description:PurposeMicroglia and Müller cells are prominent participants in retinal responses to injury and disease that shape eventual tissue adaptation or damage. This investigation examined how microglia and Müller cells interact with each other following initial microglial activation.MethodsMouse Müller cells were cultured alone, or co-cultured with activated or unactivated retinal microglia, and their morphological, molecular, and functional responses were evaluated. Müller cell-feedback signaling to microglia was studied using Müller cell-conditioned media. Corroborative in vivo analyses of retinal microglia-Müller cell interactions in the mouse retina were also performed.ResultsOur results demonstrate that Müller cells exposed to activated microglia, relative to those cultured alone or with unactivated microglia, exhibit marked alterations in cell morphology and gene expression that differed from those seen in chronic gliosis. These Müller cells demonstrated in vitro (1) an upregulation of growth factors such as GDNF and LIF, and provide neuroprotection to photoreceptor cells, (2) increased pro-inflammatory factor production, which in turn increased microglial activation in a positive feedback loop, and (3) upregulated chemokine and adhesion protein expression, which allowed Müller cells to attract and adhere to microglia. In vivo activation of microglia by intravitreal injection of lipopolysaccharide (LPS) also induced increased Müller cell-microglia adhesion, indicating that activated microglia may translocate intraretinally in a radial direction using Müller cell processes as an adhesive scaffold.ConclusionOur findings demonstrate that activated microglia are able to influence Müller cells directly, and initiate a program of bidirectional microglia-Müller cell signaling that can mediate adaptive responses within the retina following injury. In the acute aftermath following initial microglia activation, Müller cell responses may serve to augment initial inflammatory responses across retinal lamina and to guide the intraretinal mobilization of migratory microglia using chemotactic cues and adhesive cell contacts. Understanding adaptive microglia-Müller cell interactions in injury responses can help discover therapeutic cellular targets for intervention in retinal disease.

Project description:Orosomucoid (ORM) is an acute-phase protein that belongs to the immunocalin subfamily, a group of small-molecule-binding proteins with immunomodulatory functions. Little is known about the role of ORM proteins in the CNS. The aim of the present study was to investigate the brain expression of ORM and its role in neuroinflammation. Expression of Orm2, but not Orm1 or Orm3, was highly induced in the mouse brain after systemic injection of lipopolysaccharide (LPS). Plasma levels of ORM2 were also significantly higher in patients with cognitive impairment than in normal subjects. RT-PCR, Western blot, and immunofluorescence analyses revealed that astrocytes are the major cellular sources of ORM2 in the inflamed mouse brain. Recombinant ORM2 protein treatment decreased microglial production of proinflammatory mediators and reduced microglia-mediated neurotoxicity in vitro LPS-induced microglial activation, proinflammatory cytokines in hippocampus, and neuroinflammation-associated cognitive deficits also decreased as a result of intracerebroventricular injection of recombinant ORM2 protein in vivo Moreover, lentiviral shRNA-mediated Orm2 knockdown enhanced LPS-induced proinflammatory cytokine gene expression and microglial activation in the hippocampus. Mechanistically, ORM2 inhibited C-C chemokine ligand 4 (CCL4)-induced microglial migration and activation by blocking the interaction of CCL4 with C-C chemokine receptor type 5. Together, the results from our cultured glial cells, mouse neuroinflammation model, and patient studies suggest that ORM2 is a novel mediator of astrocyte-microglial interaction. We also report that ORM2 exerts anti-inflammatory effects by modulating microglial activation and migration during brain inflammation. ORM2 can be exploited therapeutically for the treatment of neuroinflammatory diseases.SIGNIFICANCE STATEMENT Neural cell interactions are important for brain physiology and pathology. Particularly, the interaction between non-neuronal cells plays a central role in regulating brain inflammation, which is closely linked to many brain disorders. Here, we newly identified orosomucoid-2 (ORM2) as an endogenous protein that mediates such non-neuronal glial cell interactions. Based on the critical role of astrocyte-derived ORM2 in modulating microglia-mediated neuroinflammation, ORM2 can be exploited for the diagnosis, prevention, or treatment of devastating brain disorders that have a strong neuroinflammatory component, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis.

Project description:BACKGROUND:Remote ischemic preconditioning (RIPC) of a limb has been reported to protect against ischemic stroke. Our previous results demonstrated that the RIPC-mediated neuroprotection is associated with alterations in circulating immune cell populations. Here, we evaluated the effect of the spleen, the largest reservoir of immune cells, on RIPC-mediated neuroprotection against stroke. METHODS:Noninvasive RIPC was achieved by four repeated cycles of 5-min blood flow constriction in the hindlimbs using a tourniquet. The blood and spleens were collected before and 1 h and 3 days after preconditioning to analyze the effect of RIPC on the spleen and the correlation between splenic and peripheral lymphocytes. Moreover, spleen weight and splenic lymphocytes were compared in stroke rats with or without RIPC. Finally, splenectomy was made 1 day or 2 weeks before RIPC and 90-min middle cerebral artery occlusion (MCAO). The infarct areas and deficits were assessed. Blood was collected 1 h after RIPC and 3 days after MCAO to explore the impact of splenectomy on RIPC-induced neuroprotection and immune changes. The contralateral and ipsilateral hemispheres were collected 3 days after MCAO to detect the infiltration of immune cells after RIPC and splenectomy. RESULTS:Flow cytometry analysis demonstrated that the RIPC promptly increased the percentages of CD3+CD8+ cytotoxic T (Tc) cells in the spleen with a relatively delayed elevation in CD3+CD161+ natural killer T (NKT) and CD3-CD45RA+ B lymphocytes. The percentages of circulating lymphocytes are positively correlated with the percentages of splenic lymphocytes in normal rats. Interestingly, RIPC resulted in negative correlations between the percentages of splenic and circulating T lymphocytes, while the correlation between splenic and circulating B lymphocytes remained positive. For animals subjected to RIPC followed by MCAO, RIPC increased splenic volume with an expansion of splenic lymphocytes 3 days after MCAO. Furthermore, the removal of the spleen 1 day or 2 weeks before RIPC and MCAO reduced the protective effect of RIPC on ischemic brain injury and reversed the effects of RIPC on circulating immune cell composition. RIPC significantly reduced brain infiltration of Tc and NKT cells. Prior splenectomy showed no effect on immune cell infiltration after RIPC and stroke. CONCLUSION:These results reveal an immunomodulatory effect of the spleen, effecting mainly the spleen-derived lymphocytes, during RIPC-afforded neuroprotection against cerebral ischemia.

Project description:Microglia are the primary immunocompetent cells in brain tissue and microglia-mediated inflammation is associated with the pathogenesis of various neuronal disorders. Recently, many studies have shown that mesenchymal stem cells (MSCs) display a remarkable ability to modulate inflammatory and immune responses through the release of a variety of bioactive molecules, thereby protecting the central nervous system. Previously, we reported that MSCs have the ability to modulate inflammatory responses in a traumatic brain injury model and that the potential mechanisms may be partially attributed to upregulated TNF-? stimulated gene/protein 6 (TSG-6) expression. However, whether TSG-6 exerts an anti-inflammatory effect by affecting microglia is not fully understood. In this study, we investigated the anti-inflammatory effects of MSCs and TSG-6 in an in vitro lipopolysaccharide (LPS)-induced BV2 microglial activation model. We found that MSCs and TSG-6 significantly inhibited the expression of pro-inflammatory mediators in activated microglia. However, MSC effects on microglia were attenuated when TSG-6 expression was silenced. In addition, we found that the activation of nuclear factor (NF)-?B and mitogen-activated protein kinase (MAPK) pathways in LPS-stimulated BV2 microglial cells was significantly inhibited by TSG-6. Furthermore, we found that the presence of CD44 in BV2 microglial cells was essential for MSC- and TSG-6-mediated inhibition of pro-inflammatory gene expression and of NF-?B and MAPK activation in BV2 microglial cells. The results of this study suggest that MSCs can modulate microglia activation through TSG-6 and that TSG-6 attenuates the inflammatory cascade in activated microglia. Our study indicates that novel mechanisms are responsible for the immunomodulatory effect of MSCs on microglia and that MSCs, as well as TSG-6, might be promising therapeutic agents for the treatment of neurotraumatic injuries or neuroinflammatory diseases associated with microglial activation.

Dataset Information

Dose-Dependent Microglial and Astrocytic Responses Associated With Post-ischemic Neuroprotection After Lipopolysaccharide-Induced Sepsis-Like State in Mice.

Publications

Dose-Dependent Microglial and Astrocytic Responses Associated With Post-ischemic Neuroprotection After Lipopolysaccharide-Induced Sepsis-Like State in Mice.

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