Neuroinflammatory and functional outcomes after TBI are sex-dependent: Lessons from estrous-phase stratified female mice

Traumatic brain injury (TBI) initiates complex neuroinflammatory cascades that significantly influence recovery outcomes. Although sex differences in neuroinflammation have been reported previously, findings remain inconclusive as the role of the female estrous cycle in post-injury responses is not well understood. In this study, we aimed to characterize sex-based differences in neuroinflammatory, vascular, and behavioral outcomes following TBI, with particular emphasis on the different phases of the estrous cycle in female mice. Male and female mice were subjected to controlled cortical impact (CCI) and subsequently assessed for behavioral deficits, cerebral blood flow (CBF), immune cell infiltration, and inflammatory genes expression. Although TBI induced robust neuroinflammation and reduction in CBF in both sexes, female mice showed significantly increased myeloid, microglial and T-cell presence, as well as elevated expression of key inflammatory transcripts (Btk, Inpp5d, and Tmem173), while downregulation in Grm2 expressions. Stratified cohort of female mice as per phases in estrous cycle (proestrus, estrus, metestrus, and diestrus) before injury showed alterations in Inpp5d, Csf3r, Csf1r, CD84, Tmem173, Homer1, Grm2 and Supt7l. Notably, female injured mice showed differential improvements in select parameters, although estrous cycle phase at the time of impact had limited but phase-dependent impact on CBF. Female mice did not show significant changes in the behavioral tests as compared to male or different estrous phenotypes. However, female mice showed higher frequency to visit center in an open arena. These findings highlight sex-specific neuroinflammatory and transcriptional responses to TBI, with moderate modulation by estrous cycle. Our study further suggests that one estrous phase could be more vulnerable to neuroinflammation or neurovascular injury than others on cellular and molecular level. However, this interphase difference might be masked in the studies including randomly cycled female population. Thus, our results underscore the importance of incorporating sex, estrous and hormonal status into sex-dependent studies on TBI and other brain diseases research to inform the development of targeted therapeutic strategies.