Supplementary MaterialsAdditional document 1: Figure S1. However, the effects of dasatinib on microglial and/or astrocytic neuroinflammatory responses and its mechanism of action have not been studied in detail. Methods BV2 microglial cells, primary astrocytes, or primary microglial cells were treated with dasatinib (100 or 250?nM) or vehicle (1% DMSO) for 30?min or 2?h followed by lipopolysaccharide (LPS; 200?ng/ml or 1?g/ml) or PBS for 5.5?h. RT-PCR, real-time PCR; immunocytochemistry; subcellular fractionation; and immunohistochemistry were subsequently conducted to determine the effects of dasatinib on LPS-induced neuroinflammation. In addition, wild-type mice were injected with dasatinib (20?mg/kg, intraperitoneally (i.p.) daily for 4?days or 20?mg/kg, orally administered (p.o.) daily for 4?days or 2?weeks) or vehicle (4% DMSO + 30% polyethylene glycol (PEG) + 5% Tween 80), followed by injection with LPS (10?mg/kg, i.p.) or PBS. Then, immunohistochemistry was performed, and plasma IL-6, IL-1, and TNF- levels were analyzed by ELISA. Results Dasatinib regulates LPS-induced proinflammatory cytokine and anti-inflammatory cytokine levels in BV2 microglial cells, primary microglial cells, and primary astrocytes. In BV2 microglial cells, dasatinib regulates LPS-induced proinflammatory cytokine levels by regulating TLR4/AKT and/or TLR4/ERK signaling. In addition, intraperitoneal injection and oral administration of dasatinib suppress LPS-induced microglial/astrocyte activation, proinflammatory cytokine levels (including brain and plasma levels), and neutrophil rolling in the brains of wild-type mice. Conclusions Our results suggest that dasatinib modulates LPS-induced microglial and astrocytic activation, proinflammatory cytokine levels, and neutrophil rolling in the brain. Electronic supplementary material The online version of this article (10.1186/s12974-019-1561-x) contains supplementary material, which is available to authorized users. 10?mg/kg, i.p.) or PBS. In addition, wild-type mice were orally administered dasatinib (20?mg/kg, p.o.) or vehicle (4% DMSO + 30% PEG + 5% Tween 80) daily for 4?days or daily for Amyloid b-Peptide (1-40) (human) 2?weeks and injected with LPS (Sigma, 10?mg/kg, i.p.) or PBS. Three hours after LPS or PBS injection, the mice were perfused and fixed with 4% paraformaldehyde (PFA) solution, and mouse brain tissues were flash-frozen and sliced using a cryostat (35?m thickness). Each brain section was rinsed with PBS three times and permeabilized with PBS containing 0.2% Triton X-100 and 1% BSA for 1?h at room temperature. The brain sections were then washed twice with 1% BSA and incubated with primary anti-Iba-1, anti-GFAP, anti-COX-2, anti-IL-6, anti-Ly-6B (neutrophil marker), or anti-ICAM-1 (endothelial cell marker) antibodies at 4?C overnight. The next day, the brain sections were washed three times with PBS and incubated with Alexa 555-conjugated anti-rabbit IgG (1:200, Life Technologies), anti-goat IgG (1:200, Life Technologies), or anti-rat IgG (1:200, Abcam) for 1?h 30?min at room temperature. The brain sections had been rinsed 3 hucep-6 x with PBS after Amyloid b-Peptide (1-40) (human) that, mounted on the glass slip, and protected with DAPI-containing mounting remedy (Vector Laboratories). Pictures had been acquired with a fluorescence microscope at ?5 or ?10 (DMi8, Leica Microsystems, Wetzlar, Germany). For this scholarly study, we utilized 8C9 man wild-type mice per group, and 2C3 pieces of each mind from ??1.70 to ??2.06?mm in accordance with the bregma in stereotaxic coordinates were utilized to quantify the fluorescence strength of anti-Iba-1, anti-GFAP,anti-COX-2, and anti-IL-6 in the cortex and hippocampus (O111:B4 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell viability assays MTT assayTo determine the consequences of dasatinib on cytotoxicity in BV2 microglial cells and mouse major astrocytes, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BV2 microglial cells and mouse major astrocytes had been separately seeded in 96-well plates (4??104 cells/well) and treated with various concentrations of dasatinib (100, 250, 500, 750, 1000?nM) for 24?h. The cells were then treated with 0.5?mg/ml MTT and incubated in a 5% CO2 incubator at 37?C for 3?h, and the absorbance was measured at 570?nm. In addition, to test the cytotoxic effects of dasatinib on BV2 or mouse primary astrocytes in the presence of LPS, BV2 microglial cells and mouse primary astrocytes were separately seeded in 96-well plates and treated with dasatinib (250?nM) or vehicle for 30?min followed by LPS (200?ng/ml) or PBS for 23.5?h. The cells were then treated with Amyloid b-Peptide (1-40) (human) 0.5?mg/ml MTT and incubated in a 5% CO2 incubator at 37?C for 3?h, and the absorbance was measured at 570?nm. BrdU assayTo investigate the effects of dasatinib on the proliferation of BV2 microglial cells via a non-metabolic assay, a BrdU assay kit (Cell Signaling, Danvers, MA, USA) was used. BV2 microglial cells were seeded in 96-well plates at a density of 4??104 cells/well and treated with various concentrations of dasatinib (100, 250, 500,.