Fig. 4

Novel CH25H⁺ microglia cluster identified with neuroprotective effect after stroke. A t-SNE representation and top up-regulated genes of MG6. B Violin plots showing specific marker of the MG6. C, D Dual immunofluorescence confirms the co-expression of CH25H with IBA1 in the ischemic penumbra and quantification of the percentage of CH25H⁺ IBA1⁺ cells at different timepoints after tMCAO (n = 5–6,). Scale bars, 40 μm. (n = 5/6 per group, one-way ANOVA with Bonferroni multiple comparisons test). The data are shown as means ± SD. *p < 0.05, ***p < 0.001, n.s. no significance. E GSEA using DEGs of MG6 reveals positive enrichment of neuron death, wound healing and collagen-containing extracellular matrix. F Flow cytometric analysis of CD206⁺M2-microglia and CD86⁺M1-microglia among CD11b^high CD45^int microglia from WT and Ch25h^−/−mice at 3 days following tMCAO. G Quantification of flow cytometry of CD206⁺CD86⁻ cells in the brain at 3d after tMCAO. (H–I) Quantification of flow cytometry H and immunofluorescence of I internalized fluorescent latex beads. BV2 cells were subjected to hypoxia and glucose deprivation for 3 h, followed by treatment with 25-HC (1 mM) or vehicle for 6 h. And then, BV2 cells were incubated with the beads at a ratio of 1:10 for 2 h, followed by treatment washed and fixed for analysis. Scale bar: 50 μm. (t-SNE t-distributed stochastic neighbor embedding, MG microglia, CH25H Cholesterol 25-Hydroxylase, IBA-1 ionized calcium binding adapter molecule 1, ANOVA analysis of variance, QuSAGE quantitative set analysis for gene expression, GSEA gene set enrichment analysis, DEG differentially expressed genes, 25-HC 25-hydroxycholesterol.)