Fig. 4

Microglial repopulation restores neuronal activity and drives transcriptomic reprogramming of microglia in the mPFC post irradiation. (A) Schematic of the microglia-depletion experiment. Microglia were depleted by continuous administration of PLX3397-containing diet for 3 weeks, started 1 week before 15 Gy cranial irradiation (R + PLX). As a control, mice receiving 15 Gy irradiation were fed with regular chow diet throughout the whole experiment (R + CON). (B) Representative images (left panel) and quantification (right panel) of IBA1-positive microglial cell density in the mPFC (R + CON: n = 3; R + PLX: n = 5). Scale bar: 100 μm. (C) Representative images (left panel) and quantification (right panel) of c-FOS-positive cell density in the mPFC (R + CON: n = 3; R + PLX: n = 5). Scale bar: 100 μm. (D) Schematic of the microglial repopulation experiment. Microglia were depleted by continuous administration of PLX3397-containing diet for 1 week before receiving 15 Gy cranial irradiation, and returned to normal chow diet immediately after irradiation for two additional weeks. (E) Representative images (left panel) and quantification (right panel) of IBA1-positive microglial cell density in the mPFC (R + CON: n = 6; R + PLX: n = 6). Scale bar: 100 μm. (F) Representative images (left panel) and quantification (right panel) of c-FOS-positive cell density in the mPFC (R + CON: n = 6; R + PLX: n = 6). Scale bar: 100 μm. (G) Representative images of IBA1 (green) and CD68 (red) immunostaining, and three-dimensional (3D) reconstruction (gray) of microglia cells in the mPFC. Scale bar, 20 μm. (H-K) Quantification of CD68 intensity in IBA1 + area (H), IBA1 + cell soma size (I), total process length (J) and total branch points (K) in the mPFC. R + CON: n = 6; R + PLX: n = 6. (L) Sholl analysis of microglial morphology in the mPFC. (M) Schematic of the microglia-repopulation procedures. The mPFC tissues were collected from mice at 2 weeks post irradiation (for male, Sham: n = 4; R + CON: n = 4; R + PLX: n = 3). (N) Volcano plot showing the fold-change and the p-value of gene expression changes comparing R + PLX mice versus R + CON mice. Red: significantly up-regulated expression, blue: significantly down-regulated expression. Only DEGs with p-value < 0.01 were presented with color. (O) Verification of selected DEGs with qPCR analysis. Data were presented as fold-changes over Sham group (Sham: n = 10–11; R + CON: n = 12; R + PLX: n = 10–12; outliers removed: Csf1r-Sham: n = 1; Ephx1-Sham: n = 1; Pros1-Sham: n = 1; Cnp-Sham: n = 1; Csf1r-R + CON: n = 1; Cnp-R + PLX: n = 1). (P) The enrichment analysis of DEGs in specific cell types. (Q) Heatmap showing DEGs enriched in microglia related to microglial survival (upper panel), neuroinflammation (middle panel), and phagocytosis (lower panel). Data are presented as mean density ± s.e.m. and analyzed by Student’s t test (B, C, E, F, H-K), two-way ANOVA (L), or two way ANOVA with Fisher’s LSD (O)