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

Abstract

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 neonates. 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 300µM whole hemisphere brain slices were obtained. During a 24h rest period, slices were cultured in either non-treated control conditions or with Erastin, a promotor of oxidative stress. Slices were then exposed to 2h of OGD followed by Vitamin E (25-100 IU/kg), Erastin (10µM) or Ferrostatin (1µM), an inhibitor of ferroptosis. Relative cytotoxicity was determined using an LDH assay, cell death was quantified via nuclear propidium iodide (PI) staining, oxidative stress was quantified via cellular GSH (glutathione) 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 brain injury.

Role of Vitamin E in Neonatal Neuroprotection: A Comprehensive Narrative Review

“…Preclinical studies to date have largely used various rodent in vitro and in vivo models of brain injury, where Vit E has been shown to decrease both inflammation and oxidative stress, with resulting neuroprotection [ 22 , 32 , 33 , 34 , 35 , 36 , 37 ]. These effects have been examined most frequently in the hippocampus and cerebellum, with fewer studies looking specifically at other regions such as the cortex and basal ganglia [ 7 , 38 , 39 ] ( Figure 2 ). For example, Vit E deficiency in rodents has been shown to result in axonal degeneration in the hippocampus [ 38 ].…”

“…Studies have also shown that inhibition of GSH recycling with the Xc-inhibitor erastin results in uncontrolled lipid peroxidation, which can be either masked or rescued in part by Vit E [ 25 , 43 ]. In a ferret organotypic brain slice model, Vit E (25 IU/kg) administered after OGD decreased cytotoxicity and inhibited oxidative stress by maintaining higher levels of GSH, and decreased transcription of genes responsible for oxidative stress and inflammation [ 7 ]. Vit E is also thought to dampen microglia activation in both mice and rats via decreased production of proinflammatory nitric oxide (NO), IL-1α, TNF-α, and reduce the expression of inducible nitric oxide (iNOS), though this effect has predominately been described in the hippocampus [ 2 ].…”

“…As the brain continues to mature, neurogenesis and angiogenesis respond to ischemic changes differently [ 59 ], thus dampening those effects may give a plausible mechanism for neuroprotection with Vit E in term infants. Although this has not been assessed clinically, term-equivalent studies such as those performed in ferret organotypic brain slices exposed to OGD suggest that Vit E is neuroprotective after hypoxic-ischemic injury in the term-equivalent brain [ 7 ].…”

“…The exact mechanism(s) by which Vit E may mitigate injury to certain brain regions or cell types has yet to be fully elucidated. Accumulation of Vit E in the cortex, hippocampus, basal ganglia and cerebellum has been described in murine and primate brains, suggesting Vit E may play a specific regional role in neuroprotection [ 27 , 39 , 73 , 74 ] and in the ferret Vit E improves cytotoxicity selectively in the hippocampus and cortex [ 7 ]. Further studies are needed to focus on Vit E effects by cell type to help delineate its potential neuroprotective effects, such as regional differences in treatment response to local microglial responses to injury [ 75 ].…”

“…In very low birth weight (VLBW) infants, clinical studies using high dose intravenous Vit E have also reported adverse effects [ 5 ]. In contrast, more recent positive evidence of Vit E as a neuroprotective agent in a term in vitro ferret model of hypoxic ischemic brain injury suggests promise [ 7 ]. As advances in neonatal care are improving rates of mortality, it is imperative that we continue to probe new therapeutic modalities to combat neurological injury in the neonatal period [ 8 ].…”