Isolation and Characterization of Microglia from Adult Mouse Brain: Selected Applications for ex Vivo Evaluation of Immunotoxicological Alterations Following in Vivo Xenobiotic Exposure

Abstract

Microglia play a dual role in neuroprotection as well as in neurodegeneration and thus occupy the focal interest in neurodegenerative disease research. In vitro studies either by using cell lines or neonatal mouse primary microglia correlated xenobiotic induced microglial activation and neuronal death. However, these in vitro studies cannot portray the in vivo scenario. Therefore, environmental pollutant induced in vivo alteration in microglial function can be best assessed by ex vivo analysis, which is not in use because of limitations in the isolation procedure. Therefore, in the first part of the study we describe an optimized isolation procedure and characterization of isolated cells. The second part of the study demonstrates the utility of the isolated cells in evaluation of immunotoxicological alterations following arsenic, as a model xenobiotic, exposure. Purity of the isolated microglia was checked by immunostaining of microglial (CD11b and CD68) and nonmicroglial (GFAP) markers. Immunostaining of activation marker Iba1 proves that cells were not activated during the isolation procedure. Microglia yield and viability from the treated group shows no significant alterations compared to that of the control group. Proinflammatory cytokines (IL-6 and TNF-α) were upregulated following arsenic treatment as in the case of the LPS stimulated group without alterations in anti-inflammatory IL-10. Phagocytic potential was affected significantly following arsenic exposure without alteration in viability. Thus, our protocol can be proficiently used for quick isolation of primary microglia from adult mouse brain without altering their activation status, and most importantly, the isolated cells can be of aid to the ex vivo evaluation of immunotoxicological alterations.

Kososan, a Kampo medicine, prevents a social avoidance behavior and attenuates neuroinflammation in socially defeated mice

“…Second, it has been reported that CSDS causes neuroinflammation throughout the brain, including the prefrontal cortex and amygdala [79]. This finding is likely to support our data in the ex vivo microglial response to LPS reflecting the inflammatory response profiles of microglia in the whole brain [11, 46, 47, 80]. However, further studies on whether kososan extract affects stress-induced neuroinflammation in other brain regions are needed.…”

“…Microglia were isolated from adult testing mouse whole brain except the cerebellum as described previously [11, 46, 47] with some modifications. Briefly, following decapitation, the whole brain except the cerebellum was readily extracted and chopped finely with a fine sharp scissor in ice-cold serum-free Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Sigma) containing papain (20 U/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA), DNase I (2 mg/ml, Sigma), and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).…”

Arsenic exposure impels CD4 commitment in thymus and suppress T cell cytokine secretion by increasing regulatory T cells

“…Splenocytes were seeded at a density of 0.1 × 10 6 cells/200 µl/ well in 96 well culture plates and incubated in the absence or presence of concanavalin A (ConA) (2.5 µg/ml; Sigma) in a 5% CO 2 incubator at 37 °C for 72 h. Culture supernatants were collected and pooled from each treatment group, and secreted cytokines were measured in supernatant plated in triplicate using a Milliplex mouse cytokine assay kit as previously described 59 in Bio-Plex MAGPIX multiplex reader (Bio-Rad). Fluorescence associated with different antibody-coated magnetic beads was read to quantitate respective cytokines.…”

Arsenic and the immune system

“…Recent studies have shown that arsenic alters the functions of microglia, which are the resident macrophages found in the central nervous system [12,13]. The metalloid notably increased ex-vivo secretion of IL-6 and TNF- from microglia isolated from mice exposed for 7 days to 0.38 mg/kg arsenite [12]. In vitro, the supernatant of arsenic-exposed-microglial cultures induced murine neuronal cell death [14].…”