Fig. 4

IL-13 reduces OL/OPC death after exposure to OGD/R and encourages OPC differentiation in a microglia-dependent manner. (A) OL/OPC survival after OGD/R was quantified by live/dead staining. Aa Representative images of live (green) and dead (red) staining of cultured oligodendrocytes or OPCs. Scale bar: 25 μm. Ab Percentages of dead cells were quantified. Data are from 4 independent experiments. B–D NG2 and MBP immunostaining was performed to evaluate OPC differentiation. B Primary OPC cultures were treated with T3 (100 ng/mL) + CNTF (20 ng/mL) or escalating concentrations of IL-13 for 3 days. Ba Cells were triple-stained with MBP (red), NG2 (green), and DAPI (blue). Scale bar: 25 μm. Bb Quantification of MBP⁺ OLs and NG2⁺ OPCs after treated with escalating concentrations of IL-13. Data are from 4 independent experiments. Bc Quantification of OPCs survival after treated with escalating concentrations of IL-13. n = 5/group. C Typical OPC and OL were selected for reconstruction of 3D-rendered images. D Primary OPCs were co-cultured with microglia treated with/without IL-13 (20 ng/ml) for 3 days. Da Cells were then immunostained for MBP (red) and NG2 (green), and nuclei were labeled with DAPI (blue). After skeleton reconstruction, the protrusion length and the number of protrusions of each cell were analyzed. White rectangles illustrates: where skeleton images were taken from. The red reconstruction line in the skeleton images show the longest path of each cell. Scale bar: 25 μm. Db Quantification of MBP⁺ OLs. Data are from 4 independent experiments. Dc–e Quantification of the number, the maximum protrusion length and the longest path of each cell. Each point shown in the results represents a cell. Four independent experimental samples were analyzed in each group, and a total of n = 40 cells were analyzed in each group. All data are presented as the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. One-way ANOVA followed by Bonferroni’s post hoc, or Kruskal–Wallis test followed by Dunn’s post hoc