Fig. 3

Radiation induces transcriptomic shifts and alterations in microglial density as well as properties in the mPFC. (A) Schematic of experimental procedures. The mPFC tissues were collected from mice at 2 weeks post irradiation (RAD, n = 3 for male, n = 4 for female), or non-irradiated control mice (Sham, n = 3 for male, n = 3 for female). (B) Heatmap showing relative expression levels of the differentially expressed genes (DEGs) (filtered by p-adjust < 0.1). (C) Volcano plot showing the fold-change and the p-adjust value of gene expression changes comparing RAD versus Sham groups. Red: significantly up-regulated expression, blue: significantly down-regulated expression. (D) Verification of selected DEGs by qPCR analysis in Sham and RAD mice. Data were presented as fold-change over Sham group (Sham: n = 5 or 7; RAD: n = 8–9; removed outliers: Nrarp-Sham: n = 2; Nrarp-RAD: n = 2). (E) Heatmap showing genes related to cell death and survival in the mPFC. Scale bar on the left: standardized rlog-transformed values across samples. (F) Gene set enrichment analysis (GSEA) of the transcriptomic changes in the mPFC. The length of the bars indicates the normalized enrichment scores (NES), and the color indicates false discovery rate (FDR). The top enriched gene sets are shown on the right side while the top depleted gene sets are on the left. (G) Top panel: Schematic of the experimental procedures. Mice were sacrificed at different time points after cranial irradiation. Bottom panel: Representative images of IBA1-positive microglia in the mPFC. Scale bar, 100 μm. (H) Quantification of IBA1-positive microglia in the mPFC (48 h: n = 5, 5; 1 wk: n = 7, 6; 2 wk: n = 8, 9; 4 wk: n = 7, 7; 3 m: n = 5, 5). (I) Representative images of IBA1 (green) and CD68 (red) immunostaining, and three-dimensional (3D) reconstruction (gray) of microglia cells in the mPFC of the Sham and RAD groups. Scale bar, 20 μm. (J-M) Quantification of CD68 intensity in IBA1 + area (J), IBA1 + cell soma size (K), total process length (L) and total branch points (M). Sham: n = 5; RAD: n = 7. (N) Sholl analysis of microglial morphology in Sham or RAD groups. Data are presented as mean ± s.e.m. and analyzed by multiple t tests (D, H), Student’s t-test (J-M) or two-way ANOVA (N)