Introduction: Intracerebral hemorrhage (ICH) has high mortality and is debilitating for those that do survive. After bleeding into the brain parenchyma, there is massive free iron and heme accumulation as a byproduct of red blood cell lysis. Microglia, as the resident phagocyte of the brain, are tasked with initiating the immune response and clearing the debris. Therefore, it is important to promote microglial survival to induce efficient clearance of the hemorrhage. One way to do this is through inhibition of ferroptosis. Ferroptosis is an iron dependent form of cell death mediated by oxidative injury. While neurons have previously been shown to undergo ferroptosis after ICH, it is unclear if microglia also undergo ferroptosis, and how ferroptotic microglia contribute to ICH recovery. Methods and Results: ICH was induced in 10–12-week-old male mice by stereotaxically injection of collagenase type VII (0.16U in 0.8uL) into the right striatum. We then quantified microglial ferroptosis using flow cytometry and measuring C11-BODIPY (581/591) oxidation of microglia following ICH at various timepoints up to Day 11. We found that microglia undergo ferroptosis which peaks at day 8 and starts to decline by day 11 (Fig 1A). We also utilized in-vitro generated primary mouse microglia created from P1-2 pup brains, and treated with various concentrations of heme for 18 hours. We showed that 10uM of heme significantly induces microglial cell death (Fig 2). To test these findings in vivo, mice after ICH were either treated intraperitonially with either Ferrostatin-1 (5mg/kg once a day) or vehicle control for 6 days following ICH. Mice underwent baseline corner test and then repeated corner test at day 7 following ICH by a blinded observer. We found that mice treated with Ferrostatin-1 exhibited significant motor recovery from ICH compared to vehicle controls in Day 7 corner test (Fig 3A). Also, mice exhibited reduced oxidized microglia, indicating Ferrostatin-1 treatment reduced microglial cell death (Fig 3B). Conclusion: This data suggests that microglial ferroptosis can significantly affect ICH recovery and targeting microglial ferroptosis can be a potential avenue for ICH treatment.
Velazquez et al. (Thu,) studied this question.