Fig. 2: Increased microglial turnover in the cerebellum following 2HIT stress exposure.

a Representative immunostaining of Ki67(+) microglia. Images show Iba-1(+) microglia, DAPI-stained nuclei, and cell proliferation marker Ki67 expression in a male mouse. b, c Bar graphs showing the density and proportion of Ki67(+) microglia. Data from 5- (b) and 9- (c) week-old male Control, RSDS susceptible (RSDS sus), MIA, 2HIT susceptible (2HIT sus), and 2HIT+microglia replacement (rMG) groups are presented across the cerebellar cortex (CbCrx), cerebellar nuclei (CbN), hippocampus (HPC), and ventral tegmental area (VTA) (mean ± SEM). d Time course of the Ki67(+) microglia proportion. The proportion of Ki67(+) microglia in male is plotted at 5 and 9 weeks of age. The proportion of CbCrx Ki67(+) microglia in the 2HIT sus group has increased significantly at 9 weeks under stress conditions than at 5 weeks. e Representative immunostaining of cleaved Caspase 3 (clv-Casp3)-positive microglia. Images show Iba-1(+) microglia, DAPI-stained nuclei, and apoptosis marker clv-Casp3 expression in a male mouse. Bar graphs showing the density and proportion of clv-Casp3(+) microglia. Data from 5- (f) and 9- (g) week-old male Control, RSDS sus, MIA, 2HIT sus, and 2HIT+rMG groups are presented across CbCrx, CbN, HPC, and VTA (mean ± SEM). h Time course of the clv-Casp3(+) microglia proportion. The proportion of clv-Casp3(+) microglia in male is plotted at 5 and 9 weeks of age. The proportion of CbCrx clv-Casp3(+) microglia in the 2HIT sus group has increased significantly at 9 weeks under stress conditions than at 5 weeks. * indicates significance by one-way ANOVA with multiple comparisons (Tukey-Kramer method) (p < 0.05), and † indicates significance by two-sample unpaired Student’s t-test (p < 0.05).