An optimized protocol for the acute isolation of human microglia from autopsy brain samples.

Microglia are increasingly recognized to be crucially involved in the maintenance of tissue homeostasis of the brain and spinal cord. Not surprisingly is therefore the growing scientific interest in the microglia phenotypes associated with various physiological and pathological processes of the central nervous system. Until recently the investigation of these phenotypes was hindered by the lack of an isolation protocol that (without an extended culturing period) would offer a microglia population of high purity and yield. Thus, our objective was to establish a rapid and efficient method for the isolation of human microglia from postmortem brain samples. We tested multiple elements of already existing protocols (e.g., density separation, immunomagnetic bead separation) and combined them to minimize preparation time and maximize yield and purity. The procedure presented in this article enables acute isolation of human microglia from autopsy (and biopsy) samples with a purity and yield that is suitable for downstream applications, such as protein and gene expression analysis and functional assays. Moreover, the present protocol is appropriate for the isolation of microglia from autopsy samples irrespective of the neurological state of the brain or specific brain regions and (with minor modification) could be even used for the isolation of microglia from human glioma tissue.

CXCL10/CXCR3 signaling in glia cells differentially affects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus.

The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell-types involved in CXCL10/CXCR3 signaling under pathological conditions are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes express CXCL10 in response to excitotoxicity. Experiments in OHSCs derived from CXCL10-deficient (CXCL10(-/-) ) and CXCR3-deficient (CXCR3(-/-) ) revealed that in the absence of CXCL10 or CXCR3, neuronal cell death in the CA1 and CA3 regions was diminished after NMDA-treatment when compared to wild type OHSCs. In contrast, neuronal cell death in the DG region was enhanced in both CXCL10(-/-) and CXCR3(-/-) OHSCs in response to a high (50 µM) NMDA-concentration. Moreover, we show that in the absence of microglia the differential changes in neuronal vulnerability between CXCR3(-/-) and wild type OHSCs are fully abrogated and therefore a prominent role for microglia in this process is suggested. Taken together, our results identify a region-specific role for CXCL10/CXCR3 signaling in neuron-glia and glia-glia interactions under pathological conditions.

CCL21-induced calcium transients and proliferation in primary mouse astrocytes: CXCR3-dependent and independent responses.

CCL21 is a homeostatic chemokine that is expressed constitutively in secondary lymph nodes and attracts immune cells via chemokine receptor CCR7. In the brain however, CCL21 is inducibly expressed in damaged neurons both in vitro and in vivo and has been shown to activate microglia in vitro, albeit not through CCR7 but through chemokine receptor CXCR3. Therefore, a role for CCL21 in CXCR3-mediated neuron-microglia signaling has been proposed. It is well established that human and mouse astrocytes, like microglia, express CXCR3. However, effects of CCL21 on astrocytes have not been investigated yet. In this study, we have examined the effects of CCL21 on calcium transients and proliferation in primary mouse astrocytes. We show that similar to CXCR3-ligand CXCL10, CCL21 (10(-9) M and 10(-8) M) induced calcium transients in astrocytes, which were mediated through CXCR3. However, in response to high concentrations of CCL21 (10(-7) M) calcium transients persisted in CXCR3-deficient astrocytes, whereas CXCL10 did not have any effect in these cells. Furthermore, prolonged exposure to CXCL10 or CCL21 promoted proliferation of wild type astrocytes. Although CXCL10-induced proliferation was absent in CXCR3-deficient astrocytes, CCL21-induced proliferation of these cells did not significantly differ from wild type conditions. It is therefore suggested that primary mouse astrocytes express an additional (chemokine-) receptor, which is activated at high CCL21 concentrations.

Enhanced hippocampal neurogenesis in the absence of microglia T cell interaction and microglia activation in the murine running wheel model.

Recently, activated microglia have been shown to be involved in the regulation of several aspects of neurogenesis under certain experimental conditions both in vitro and in vivo. A neurogenesis supportive microglia phenotype has been suggested to arise from the interaction of microglia with homing encephalitogenic T cells. However, a unified hypothesis regarding the exact nature of microglia activity that is supportive of neurogenesis is yet missing from the field. Our aim was to investigate the connection between microglia activity and adult hippocampal neurogenesis under physiological conditions. To address this question we compared the level of microglia activation in the hippocampus of mice, which had access to a running wheel for 10 days and that of sedentary controls. Surprisingly, despite elevated levels of proliferation of neural precursors and survival of newborn neurons in the dentate gyrus microglia remained in a "resting" state morphologically, antigenically, and at the transcriptional level. Moreover, neither T cells nor MHCII expressing microglia were present in the hippocampal brain parenchyma. Though microglia in the dentate gyrus of the runners proliferated at a higher level than in the sedentary controls, this difference was also present in non-neurogenic sites. Therefore, our findings suggest that classical signs of microglia activation and microglia activation arising from interaction with T cells in particular are not a prerequisite for the activity-induced increase in adult hippocampal neurogenesis in C57Bl/6 mice. Thus, our results draw attention on the species and model differences that might exist regarding the regulation of adult hippocampal neurogenesis.

Region-specific expression of immunoregulatory proteins on microglia in the healthy CNS.

In accordance with a high degree of spatial organization in the central nervous system (CNS), most CNS diseases display a regional distribution. Although microglia have been established as key players in various CNS diseases, it is not yet clear whether microglia display region-specific properties. Therefore, this study aimed to evaluate the existence of distinct microglia phenotypes in various regions of the healthy, adult mouse CNS. Using ex vivo flow cytometric analysis surface expression of CD11b, CD40, CD45, CD80, CD86, F4/80, TREM-2b, MHCII, CXCR3, CCR9, and CCR7 were analyzed. Most of these immunoregulatory markers were found on microglia and showed significant region-specific differences in expression levels. These findings considerably corroborate the existence of immunological diversity among microglia in the healthy, unchallenged CNS of adult mice.

Expression of CXCL4 in microglia in vitro and in vivo and its possible signaling through CXCR3.

Signaling through chemokine receptor CXCR3 in the brain has been implicated in various brain diseases, as CXCR3 and its ligands are found under these conditions. Recently, a new chemokine ligand for CXCR3 was reported. In humans, an alternatively spliced variant of CXCR3 expressed on microvascular endothelial cells, named CXCR3b, was shown to bind CXCL4. In the periphery, the cellular expression and functions of CXCL4 are well described but in the brain its expression and function are unknown. Here, we show that brain microglia are a cellular source of CXCL4 in vitro and in vivo under neurodegenerating conditions. Microglial migration induced by CXCL4 is absent in CXCR3-deficient microglia, indicating a role of CXCR3. CXCL4 furthermore attenuates lipopolysaccharide-induced microglial phagocytosis and nitric oxide production in microglia and BV-2 cells. Based on these findings, it is proposed that locally released CXCL4 may control microglia responses.

Optimized isolation enables ex vivo analysis of microglia from various central nervous system regions.

Ex vivo analysis is an accurate and convenient way to study in vivo microglia phenotype and function. However, current microglia isolation protocols for ex vivo analysis show many differences in isolation steps (perfusion, removal of meninges and blood vessels, mechanical dissociation, enzymatic dissociation, density separation, immunomagnetic separation, and fluorescence-activated cell sorting), often without addressing their effects on microglia purity, number, phenotype, and function. Therefore, the aim of this study was to provide an optimized isolation protocol with emphasis on microglia purity and number to enable ex vivo analysis of adult mouse microglia. The application of this protocol for ex vivo phenotype and functional analysis is corroborated by results from flow cytometry, gene expression analysis, chemotaxis, and phagocytosis assays. In addition, this study shows the possibility to analyze microglia isolated from various central nervous system regions such as optic nerve, striatum, hippocampus, spinal cord, cerebellum, and cerebral cortex. Furthermore, this is the first study presenting DRAQ5 as a superior alternative to propidium iodide for the discrimination between living and dead cells. DRAQ5 staining facilitated the identification of microglia upon flow cytometry without the need of additional fluorescent markers. Along with a favorable emission spectrum, DRAQ5 proved a valuable tool for flow cytometry of microglia. The presented optimized microglia isolation protocol for ex vivo analysis offers the opportunity to obtain more insight into both general and region-specific microglia behavior.