Microglial depletion worsens lesion in female but not male C57BL/6J mice after P10 hypoxia-ischemia

Background: Rodent models for perinatal hypoxic ischemic (HI) encephalopathy have reported sex differences such as the same injury causing larger lesions in the brains of males compared to females. Microglia, the resident immune cells of the brain that have sex-dependent developmental trajectories and gene expression patterns, likely play a different role in females and males following HI. However, there is conflicting literature on whether depletion of microglia worsens or improves HI-induced lesions and whether this differs by sex. Here we tested the effect of pharmacologic microglia depletion on HI lesion size in male and female mice. Methods: An initial cohort of C57BL/6J mouse pups underwent HI at postnatal day 10 (P10) using a modified Vannucci procedure or a Sham insult followed by brain collection at P13. Another cohort of mice received daily intraperitoneal injections from P7 to P12 of either 25mg/kg PLX3397 (PLX, a CSF1R inhibitor) or vehicle (Veh). These mice also underwent HI or Sham at P10, resulting in four groups (Veh-Sham, Veh-HI, PLX-Sham, PLX-HI). All groups included female and male mice. Behavioral testing was performed both pre-HI (forelimb grasping [P8, P9]) and post-HI (open field traversal [P12], behavior and appearance observations [P13]). P13 brain sections underwent immunohistochemistry for Iba1 or cresyl violet staining for lesion scoring. Results: P13 HI hippocampal sections had more Iba1 signal than Sham, with more variance in Male-HI vs Female-HI mice. PLX led to >95% depletion of Iba1+ cells at P10 or P13, and effective elimination of microglia did not differ by sex. In the hippocampus, Female-PLX-HI mice had worse lesion scores than Female-Veh-HI mice; this was not true in male mice, where there was a trend in the opposite direction. Female-PLX-HI mice also had worse lesion scores than Male-PLX-HI mice. In contrast to this sex-dependent effect of PLX on lesion score, there was no difference among groups in developmental milestones. Conclusion: PLX3397 injection P7-P9 or P7-P12 effectively depletes microglia by P10 or P13, respectively. Microglial depletion via PLX worsens HI-induced injury in female mice but not in male mice. ### Competing Interest Statement The authors have declared no competing interest.