Activation Of Primary Microglia Research Articles

Minocycline alleviates depression-like symptoms by rescuing decrease in neurogenesis in dorsal hippocampus via blocking microglia activation/phagocytosis

Clinical studies examining the potential of anti-inflammatory agents, specifically of minocycline, as a treatment for depression has shown promising results. However, mechanistic insights into the neuroprotective and anti-inflammatory actions of minocycline need to be provided. We evaluated the effect of minocycline on chronic mild stress (CMS) induced depressive-like behavior, and behavioral assays revealed minocycline ameliorate depressive behaviors. Multiple studies suggest a role of microglia in depression, revealing that microglia activation correlates with a decrease in neurogenesis and increased depressive-like behavior. The effect of minocycline on microglia activation in different areas of the dorsal or ventral hippocampus in stressed mice was examined by immunohistochemistry. We observed the increase in the number of activated microglia expressing CD68 after exposure to three weeks of chronic stress, whereas no changes in total microglia number were observed. These changes were observed throughout the DG, CA1 and CA2 regions in dorsal hippocampus but restricted to the DG of the ventral hippocampus. In vitro experiments including western blotting and phagocytosis assay were used to investigate the effect of minocycline on microglia activation. Activation of primary microglia by LPS in vitro causes and ERK1/2 activation, enhancement of iNOS expression and phagocytic activity, and alterations in cellular morphology that are reversed by minocycline exposure, suggesting that minocycline directly acts on microglia to reduce phagocytic potential. Our results suggest the most probable mechanism by which minocycline reverses the pathogenic phagocytic potential of neurotoxic M1 microglia, and reduces the negative phenotypes associated with reduced neurogenesis caused by exposure to chronic stress.

Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

Cascade Signals of Papaverine Inhibiting LPS-Induced Retinal Microglial Activation

Studies have shown that papaverine can inhibit lipopolysaccharide (LPS)-induced microglial activation. The retinal primary microglia of newborn SD rats were isolated and purified, and a LPS-induced microglia activation model was established. The protein phosphorylation level of the signaling pathway was detected by western blotting. The transcription and expression of TNF-α, IL-1β, and IL-10 were respectively detected by RT-PCR and ELISA to observe the abnormal activation of primary microglia. The cAMP inhibitor Rp-isomer, PKA inhibitor H89, and MEK inhibitor U0126 were separately added to further investigate the role of MEK/Erk in PAP inhibition of primary microglial activation and the relationship between cAMP/PKA and MEK/Erk. It was found that the level of MEK phosphorylation was upregulated after LPS stimulation, which was blocked by 10 μg/ml of papaverine.10μM U0126 significantly inhibited TNF-α and IL-1β and increased IL-10 transcription and expression in retinal microglia (P < 0.01). Both Rp-isomer and H89 upregulated the phosphorylation levels of MEK and Erk. Papaverine may inhibit inflammatory factors and promote the expression of anti-inflammatory factors through the cAMP/PKA and MEK/Erk pathway, thereby inhibiting LPS-induced activation of primary retinal microglia, and the MEK/Erk pathway may be partially regulated by cAMP/PKA, which can provide theoretical basis and experimental basis for its protection of the central nervous system.

Loss of Par1b/MARK2 primes microglia during brain development and enhances their sensitivity to injury

BackgroundMicroglia, the resident immune cells of the brain, exhibit various morphologies that correlate with their functions under physiological and pathological conditions. In conditions such as aging and stress, microglia priming occurs, which leads to altered morphology and lower threshold for activation upon further insult. However, the molecular mechanisms that lead to microglia priming are unclear.MethodsTo understand the role of Par1b/MARK2 in microglia, we first expressed shRNA targeting luciferase or Par1b/MARK2 in primary microglial cells and imaged the cells using fluorescent microscopy to analyze for morphological changes. A phagocytosis assay was then used to assess functional changes. We then moved in vivo and used a Par1b/MARK2 knockout mouse model to assess for changes in microglia density, morphology, and phagocytosis using immunohistochemistry, confocal imaging, and 3D image reconstruction. Next, we used two-photon in vivo imaging in live Par1b/MARK2 deficient mice to examine microglia dynamics. In addition, a controlled-cortical impact injury was performed on wild-type and Par1b/MARK2-deficient mice and microglial response was determined by confocal imaging. Finally, to help rule out non-cell autonomous effects, we analyzed apoptosis by confocal imaging, cytokine levels by multiplex ELISA, and blood-brain barrier permeability using Evans Blue assay.ResultsHere, we show that loss of the cell polarity protein Par1b/MARK2 facilitates the activation of primary microglia in culture. We next found that microglia in Par1b/MARK2 deficient mice show increased density and a hypertrophic morphology. These morphological changes are accompanied with alterations in microglia functional responses including increased phagocytosis of neuronal particles early in development and decreased surveillance of the brain parenchyma, all reminiscent of a primed phenotype. Consistent with this, we found that microglia in Par1b/MARK2 deficient mice have a significantly lower threshold for activation upon injury.ConclusionsTogether, our studies show that loss of Par1b/MARK2 switches microglia from a surveillant to a primed state during development, resulting in an increased neuroinflammatory response to insults.

Inhaled hydrogen sulfide prevents neuropathic pain after peripheral nerve injury in mice

Increasing evidence suggests that the pathogenesis of neuropathic pain is mediated through activation of microglia in the spinal cord. Hydrogen sulfide attenuates microglial activation and central nervous system inflammation; however, the role of hydrogen sulfide in neuropathic pain is unclear. In this study, we examined the effects of hydrogen sulfide breathing on neuropathic pain in mice. C57BL/6J mice were subjected to chronic constriction injury (CCI) of the sciatic nerve. After CCI, mice breathed air alone or air mixed with hydrogen sulfide at 40 ppm for 8 h on 7 consecutive days. The expression levels of inflammatory cytokines including interleukin 6 (IL-6) were measured in the spinal cord. Effects of hydrogen sulfide on IL-6-induced activation of microglia were examined in primary rat microglia. Mice that breathed air alone exhibited the neuropathic pain behavior including mechanical allodynia and thermal hyperalgesia and increased mRNA levels of IL-6 and chemokine CC motif ligand 2 (CCL2) after CCI. Inhaled hydrogen sulfide prevented the neuropathic pain behavior and attenuated the upregulation of inflammatory cytokines. Sodium sulfide inhibited IL-6-induced activation of primary microglia. These results suggest that inhaled hydrogen sulfide prevents the development of neuropathic pain in mice possibly via inhibition of the activation of microglia in the spinal cord.

Quetiapine Attenuates Glial Activation and Proinflammatory Cytokines in APP/PS1 Transgenic Mice via Inhibition of Nuclear Factor- B Pathway

Background:In Alzheimer’s disease, growing evidence has shown that uncontrolled glial activation and neuroinflammation may contribute independently to neurodegeneration. Antiinflammatory strategies might provide benefits for this devastating disease. The aims of the present study are to address the issue of whether glial activation and proinflammatory cytokine increases could be modulated by quetiapine in vivo and in vitro and to explore the underlying mechanism.Methods:Four-month–old amyloid precursor protein (APP) and presenilin 1 (PS1) transgenic and nontransgenic mice were treated with quetiapine (5mg/kg/d) in drinking water for 8 months. Animal behaviors, total Aβ levels, and glial activation were evaluated by behavioral tests, enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot accordingly. Inflammatory cytokines and the nuclear factor kappa B pathway were analyzed in vivo and in vitro.Results:Quetiapine improves behavioral performance, marginally affects total Aβ40 and Aβ42 levels, attenuates glial activation, and reduces proinflammatory cytokines in APP/PS1 mice. Quetiapine suppresses Aβ1-42-induced activation of primary microglia by decresing proinflammatory cytokines. Quetiapine inhibits the activation of nuclear factor kappa B p65 pathway in both transgenic mice and primary microglia stimulated by Aβ1–42.Conclusions:The antiinflammatory effects of quetiapine in Alzheimer’s disease may be involved in the nuclear factor kappa B pathway. Quetiapine may be an efficacious and promising treatment for Alzheimer’s disease targeting on neuroinflammation.

Effects of human marrow stromal cells on activation of microglial cells and production of inflammatory factors induced by lipopolysaccharide

There has been an increasing appreciation of the role that microglial cells play in neural damage. Marrow stromal cells (MSCs) can dramatically lessen neural damage in animal models, but the mechanisms involved have not been defined. This study aimed to investigate the effects of human MSCs (hMSCs) on the activation of primary microglia and the attendant production of pro-inflammatory factors stimulated by bacterial endotoxin lipopolysaccharide (LPS). Our study showed that hMSCs in co-cultures and in transwell cultures inhibited the activation of microglial cells, reduced the production of tumor necrosis factor-α (TNF-α) and nitric oxide (NO), downregulated the expression of inducible nitric oxide synthase (iNOS) and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK), whereas hMSCs conditioned medium did not have any effect on microglial inflammation. To further investigate the mechanisms by which hMSCs exert anti-inflammatory effects, we examined the production of neurotrophic factors by hMSCs with enzyme linked immunosorbent assay (ELISA). Our results showed that the production of insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and hepatocyte growth factor (HGF) was significantly increased by hMSCs when cultured in the conditioned medium from activated microglia. We conclude that hMSCs can inhibit microglial activation and the production of attendant inflammatory factors. In addition, hMSCs can interact with microglial cells through diffusible soluble factors, whereas cell contact is not a prerequisite for anti-inflammatory effects. Finally, hMSCs within inflammatory environment can significantly increase the production of neurotrophic factors, which may involve with the anti-inflammatory mechanisms.