FIGURE 3.

Deoxyribonucleic acid (DNA) damage accumulates near the region of impact 1 week after rmTBI. DNA damage is evident 1 week post-injuries. Immunohistochemistry for yH2AX in female mice reveals DNA damage in the form of DSBs accumulates after rmTBI in both the cortex at the impact region (A,C, scale bar represents 200 μm) and underlying hippocampal dentate gyrus (B,D, scale bar represents 200 μm), with minimal staining in shams. Staining of dsDNA with S9.6 to assess levels of R-Loop structures revealed a significant main effect of injury (p = 0.05, two-way ANOVA), and post-hoc testing indicates that this increased expression is only significant in the female rmTBI mice compared to their sham counterparts (p = 0.04, Bonferroni). Staining of dsDNA for 8-oxo to assess oxidative base damage shows a similar trend, with a main effect of injury nearing significance (p = 0.08, two-way ANOVA) and post-hoc testing revealing the increased expression to be significant only in female mice (p = 0.04, Bonferroni). Representative images of the dot blot are shown to the right of the quantification plot (E). Using Western Blotting, protein levels of yH2AX were found to be significantly increased in rmTBI mice compared to controls (F) (p = 0.05, main effect of injury, two-way ANOVA), with a significant effect of sex (p = 0.0004, main effect of sex, two-way ANOVA) and no significant interaction. DNA2 trended toward increased expression in female rmTBI mice compared to shams, but did not reach statistical significance (F). 53BP1 was found to have a significant main effect of sex (p < 0.0001, two-way ANOVA) but no significant main effect of injury, however, it was trending toward increased expression in female rmTBI mice compared to shams. Representative blots are shown to the right of the plot (F). Error bars represent standard deviation and there are n = 4 per group. Indication of statistical significance is as follows: *p < 0.05.