BackgroundMicroglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC). The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors.ResultsTo address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection. The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis identified genes that are specific to AMC and RMC.ConclusionsThe novel and specific molecules identified from the trancriptome explains the quiescent state functioning of microglia in its two distinct morphological states.

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BackgroundIn response to cerebral ischemia, activated microglia release excessive inflammatory mediators which contribute to neuronal damage. Therefore, inhibition of microglial over-activation could be a therapeutic strategy to alleviate various microglia-mediated neuroinflammation. This study was aimed to elucidate the anti-inflammatory effects of Scutellarin and Edaravone given either singly, or in combination in activated microglia in rats subjected to middle cerebral artery occlusion (MCAO), and in lipopolysaccharide (LPS)-induced BV-2 microglia. Expression of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and inducible nitric oxide synthase (iNOS) was assessed by immunofluorescence staining and Western blot. Reactive oxygen species (ROS) and nitric oxide (NO) levels were determined by flow cytometry and fluorescence microscopy, respectively.ResultsIn vivo, both Edaravone and Scutellarin markedly reduced the infarct cerebral tissue area with the latter drug being more effective with the dosage used; furthermore, when used in combination the reduction was more substantial. Remarkably, a greater diminution in distribution of activated microglia was observed with the combined drug treatment which also attenuated the immunoexpression of TNF-α, IL-1β and iNOS to a greater extent as compared to the drugs given separately. In vitro, both drugs suppressed upregulated expression of inflammatory cytokines, iNOS, NO and ROS in LPS-induced BV-2 cells. Furthermore, Edaravone and Scutellarin in combination cumulatively diminished the expression levels of the inflammatory mediators being most pronounced for TNF-α as evidenced by Western blot.ConclusionThe results suggest that Edaravone and Scutellarin effectively suppressed the inflammatory responses in activated microglia, with Scutellarin being more efficacious within the dosage range used. Moreover, when both drugs were used in combination, the infarct tissue area was reduced more extensively; also, microglia-mediated inflammatory mediators notably TNF-α expression was decreased cumulatively.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-014-0125-3) contains supplementary material, which is available to authorized users.

BackgroundNox-2 (also known as gp91phox), a subunit component of NADPH oxidases, generates reactive oxygen species (ROS). Nox-dependent ROS generation and nitric oxide (NO) release by microglia have been implicated in a variety of diseases in the central nervous system. Dexamethasone (Dex) has been shown to suppress the ROS production, NO release and inflammatory reaction of activated microglial cells. However, the underlying mechanisms remain unclear.ResultsThe present study showed that the increased ROS production and NO release in activated BV-2 microglial cells by LPS were associated with increased expression of Nox-2 and iNOS. Dex suppressed the upregulation of Nox-2 and iNOS, as well as the subsequent ROS production and NO synthesis in activated BV-2 cells. This inhibition caused by Dex appeared to be mediated by upregulation of MAPK phosphatase-1 (MKP-1), which antagonizes the activity of mitogen-activated protein kinases (MAPKs). Dex induced-suppression of Nox-2 and -upregulation of MKP-1 was also evident in the activated microglia from corpus callosum of postnatal rat brains. The overexpression of MKP-1 or inhibition of MAPKs (by specific inhibitors of JNK and p38 MAPKs), were found to downregulate the expression of Nox-2 and iNOS and thereby inhibit the synthesis of ROS and NO in activated BV-2 cells. Moreover, Dex was unable to suppress the LPS-induced synthesis of ROS and NO in BV-2 cells transfected with MKP-1 siRNA. On the other hand, knockdown of Nox-2 in BV-2 cells suppressed the LPS-induced ROS production and NO release.ConclusionIn conclusion, it is suggested that downregulation of Nox-2 and overexpression of MKP-1 that regulate ROS and NO may form the potential therapeutic strategy for the treatment of neuroinflammation in neurodegenerative diseases.

BackgroundActivated microglial cells release an excess of inflammatory mediators after an ischemic stroke. We reported previously that scutellarin effectively suppressed the inflammatory response induced by activated microglia in rats subjected to middle cerebral artery occlusion (MCAO); however, the mechanism via which scutellarin exerts its effects on microglial activation has not been explored. This study aimed to elucidate if scutellarin can regulate the Notch pathway that is linked to microglia activation in MCAO rat, and in lipopolysaccharide (LPS)-induced BV-2 microglia. Along with this, we also investigated some characteristic behavioral responses of activated microglia.MethodsExpression of various members of the Notch pathway, including Notch-1, intracellular Notch receptor domain (NICD), recombining binding protein suppressor of hairless (RBP-JK) and transcription factor hairy and enhancer of split-1 (Hes-1) in activated microglia was assessed by immunofluorescence staining and western blot after experimental MCAO and in vitro LPS activation. The effect of scutellarin on migration of microglia was determined by the transwell chamber assay as well as expression of monocyte chemoattractant protein-1 (MCP-1). The morphological change of microglia induced by scutellarin was detected by F-actin staining and electron microscopy.ResultsScutellarin markedly attenuated the expression of NF-κB, Notch-1, NICD, RBP-JK and Hes-1 both in vivo and in activated microglia. It decreased the expression of MCP-1 and microglial migration, but increased the ability of microglia adhesion. Scutellarin also altered the phenotype of microglia by causing rearrangement or reorganization of its cytoskeleton.ConclusionsThe results suggest that scutellarin regulates the activation of microglia via the Notch pathway and concurrently induces morphological and functional changes in activated microglia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0226-z) contains supplementary material, which is available to authorized users.