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Background: Stroke is a sexually dimorphic disease with age-dependent sex differences in incidence, prevalence, and outcome. Delineating the molecular mechanisms that determine sexual dimorphism in stroke is necessary to overcome translational barriers and advance the development of novel stroke treatments. Non-coding RNAs, including microRNAs ( miRs ), are upstream regulators of genes that regulate cell survival. In young adult male animals, miR-181a and miR-200c have been established as central regulators in the cellular response to stroke, however their role in females or aged animal cohorts represents a critical knowledge gap. Therefore, in the present study we investigated the response of miR-181a and miR-200c in the brain after stroke in aged male and female mice. Methods: Aged (20 month old) male and female mice were subjected to 1h middle cerebral artery occlusion (MCAO) followed by 24h reperfusion. RT-qPCR, fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) were used to quantify and localize post-MCAO cellular expression of miR-181a and miR-200c. Results: There was no baseline difference in miR-181a between sexes, however miR-200c expression was ~3-fold higher at baseline in female animals versus males. MCAO induced ~2-fold increase in miR-181a in male animals but post-MCAO expression remained unchanged in females. In contrast, miR-200c expression increased by ~16-fold in males and ~4-fold in females. At baseline, miR-181a and miR-200c primarily localized to cortical neurons (NeuN+). In response to MCAO astrocyte reactivity (GFAP+) coincided with robust expression of miR-200c in both males and females. Conclusions: MiR-181a and miR-200c exhibit sexual dimorphism in response to stroke. Augmented expression of miR-200c appears secondary to astrocyte reactivity. Future experiments will determine if inhibition of miR-181a and miR-200c are protective against ischemic stroke in aged animals.

Background and Purpose— MicroRNA (miR)-200c increases rapidly in the brain after transient cerebral ischemia but its role in poststroke brain injury is unclear. Reelin, a regulator of neuronal migration and synaptogenesis, is a predicted target of miR-200c. We hypothesized that miR-200c contributes to injury from transient cerebral ischemia by targeting reelin. Methods— Brain infarct volume, neurological score and levels of miR-200c, reelin mRNA, and reelin protein were assessed in mice subjected to 1 hour of middle cerebral artery occlusion with or without intracerebroventricular infusion of miR-200c antagomir, mimic, or mismatch control. Direct targeting of reelin by miR-200c was assessed in vitro by dual luciferase assay and immunoblot. Results— Pretreatment with miR-200c antagomir decreased post–middle cerebral artery occlusion brain levels of miR-200c, resulting in a significant reduction in infarct volume and neurological deficit. Changes in brain levels of miR-200c inversely correlated with reelin protein expression. Direct targeting of the Reln 3′ untranslated region by miR-200c was verified with dual luciferase assay. Inhibition of miR-200c resulted in an increase in cell survival subsequent to in vitro oxidative injury. This effect was blocked by knockdown of reelin mRNA, whereas application of reelin protein afforded protection. Conclusions— These findings suggest that the poststroke increase in miR-200c contributes to brain cell death by inhibiting reelin expression, and that reducing poststroke miR-200c is a potential target to mitigate stroke-induced brain injury.