Enhancing 7-dehydrocholesterol suppresses brain ferroptosis and tissue injury after neonatal hypoxia-ischemia

These data present a study seeking to assess the effects of increasing 7-DHC concentrations for neuroprotection in cell culture and an in vivo neonatal (P9) mouse model of unilateral carotid artery ligation followed by hypoxia (30 min at 8%). The tabs correlate to each of the figures in the manuscript: Tab "fig 1B": shows RSL-3 mediated ferroptosis in HT1080 cells pre-treated for 24 h with vehicle (VEH), 50 nM cariprazine (CAR), 500 nM aripiprazole (ARI), 500 nM trazodone (TRZ) or 50 nM lovastatin (LOV). Cell viability was assessed with Alamar blue. CAR, ARI and TRZ are potent DHCR7-inhibiting medications and significantly suppress ferroptotic induced cell death. LOV is a potent HMGCR inhibitor and it does not prevent ferroptosis, indicating that the ferroptosis suppression is not due to cholesterol deprivation, but to an increase in 7-DHC. Tab "fig 2B": shows HT1080 cells pre-treated with 1, 10 and 25 nM CAR for 24h before ferroptosis was induced with 25 nM RSL-3. Cells were incubated with RSL-3 for 24 h and cell viability was assessed with Alamar blue. Cell viability is significantly increased with CAR pre-treatment. Also shows HT1080 cells treated simultaneously with 25 nM RSL-3 and 1, 10 or 25 nM CAR for 24h (co-treatment with CAR and RSL-3). Cell viability was assessed with Alamar Blue. Tab "fig 3B": shows the effects of CAR on neuronal viability after 4 h of oxygen glucose deprivation (OGD). For the pre-treatment experiments, neurons were treated with vehicle, 1 nM, 10 nM or 25 nM CAR for 48 h before OGD. For the post-treatment experiments, neurons were treated with vehicle, 1 nM, 10 nM or 25 nM 30 min after OGD. Cell viability was assessed 24 h after the termination of OGD and were calculated by dividing the numbers of live neurons after OGD by the number of live neurons of the control (not exposed to OGD). 7-DHC levels were assessed by LC-MS/MS at the endpoint of the experiment. Concentrations of 7-DHC are elevated by CAR administration but consumed by OGD. Tab "fig 4": 7-DHC derived oxysterols are increased in the brain tissue of CAR-treated animals after hypoxic-ischemic brain injury (HIBI). Animals were injected with 0.2 mg/kg of CAR as described in Figure 5A. 7-DHC-derived oxysterols DHCEO and ep7DHC were not detected (NC) in the Sham+vehicle or HIBI+vehicle groups but were detected in both CAR groups. Tab "fig 5B-E": For the pre-treatment experiments, postnatal day 7 (P7) mice were injected with vehicle (VEH) or 0.2 mg/kg CAR 48 h before HIBI. For the post-treatment experiments, mice were injected with vehicle or 0.2 mg/kg CAR 30 min after HIBI. MDA levels assessed by TBARS are decreased in animals treated with CAR, indicating that CAR treatment decreases markers of phospholipid oxidation. Tissue viability assessed by 2,3,5-Triphenyltetrazolium Chloride (TTC) reveals increased tissue viability in HIBI animals treated with CAR.