Antioxidant suppression of hyperoxia-induced reactive oxygen species in cultured rat hippocampal neurons

Central nervous system oxygen toxicity (CNS-OT) is driven, in part, by excess reactive oxygen species (ROS) generated during hyperoxic exposure. Herein, we developed an in vitro screening platform using embryonic rat hippocampal neurons to evaluate antioxidant candidates under hyperoxic stress (1.34 ATA) and quantify ROS by Dihydrorhodamine (DHR)123 fluorescence imaging and flow cytometry. Neuronal cultures remained viable throughout the assay, with approximately 87% anti-βIII tubulin staining efficiency and 88% neurite outgrowth-associated viability. Among the compounds tested, sulforaphane and thioredoxin produced the greatest reductions in ROS-positive neurons following 15-minute hyperoxic exposure, with reductions of 53.3% and 54.2%, respectively, at 1 µM. Thymoquinone showed its greatest reduction in ROS production at 0.1 µM, with an approximately 48% reduction, whereas its effect was diminished at 1 µM, indicating a non-monotonic response across the concentrations tested. Following a 30-minute hyperoxic exposure, the protective effects of all compounds were reduced. Treatment with drug combinations resulted in a modest improvement in ROS suppression, with reductions ranging from 12.8% to 17.3%.These findings suggest that the proposed in vitro platform can reliably distinguish antioxidant efficacy under controlled hyperoxic conditions and can serve as a practical tool for the preclinical screening and optimization of neuroprotective therapies for CNS oxygen toxicity.