Vitamin E Decreases Cytotoxicity and Mitigates Inflammatory and Oxidative Stress Responses in a Ferret Organotypic Brain Slice Model of Neonatal Hypoxia-Ischemia

The gyrencephalic ferret brain is an excellent model in which to study hypoxia-ischemia (HI), a significant contributor to neurological injury in neonates. Vitamin E, an essential fat-soluble antioxidant, reduces oxidative stress and inflammation in both animal models and human infants. The aim of this study was to assess the effects of vitamin E after oxygen-glucose deprivation (OGD) in an organotypic ferret brain slice model of neonatal HI. We hypothesized that vitamin E would decrease cytotoxicity, inflammation, and oxidative stress in OGD-exposed brain slices. Term-equivalent ferrets were sacrificed at postnatal (P) day 21-23 and 300 µM whole-hemisphere brain slices were obtained. During a 24-h rest period, slices were cultured in either nontreated control conditions or with erastin, a promotor of oxidative stress. Slices were then exposed to 2 h of OGD followed by vitamin E (25-100 IU/kg), erastin (10 µM), or ferrostatin (1 µM), an inhibitor of ferroptosis. Relative cytotoxicity was determined using a lactate dehydrogenase assay, cell death was quantified via nuclear propidium iodide staining, oxidative stress was quantified via cellular glutathione (GSH) levels, and target genes responsive to oxidative stress and inflammation were evaluated by qRT-PCR. OGD increased cytotoxicity, which was significantly reduced by treatment with vitamin E. Vitamin E also preserved GSH after OGD and decreased amplification of certain markers of oxidative stress (CHAC1, SLC7A11) and inflammation (TNF-alpha, IL-8). Vitamin E remained protective after pretreatment with erastin and was more protective than ferrostatin, presumably due to its added anti-inflammatory properties. Results from the ferret whole-hemisphere OGD model support the premise that vitamin E neuroprotection is mediated by restoring GSH and acutely decreasing inflammation and oxidative stress after neonatal HI.