Sources of Reactive Oxygen Species in Normoxic and Hypoxic Naked Mole-Rat Brain

Oxygen availability dictates the rate of reactive oxygen species production from various cellular sources; excessive ROS accumulation can be cytotoxic. Mitochondria are usually the primary contributors to basal reactive oxygen species generation in the brain; however, xanthine oxidoreductase and nicotinamide adenine dinucleotide phosphate oxidase can also produce considerable reactive oxygen species during hypoxia/reoxygenation. In the brains of most mammals, cellular death accompanies hypoxia-mediated surges in reactive oxygen species production, but this is avoided in the cortex of hypoxia-tolerant naked mole-rats (Heterocephalus glaber). However, the contributions of various reactive oxygen species generators towards total reactive oxygen species homeostasis in naked mole-rat brain is unknown. We hypothesized that mitochondria remain the primary reactive oxygen species generators in naked mole-rat cortex and predicted that pharmacological inhibition of mitochondrial complex I would induce greater fluctuations in superoxide (O₂^•-) and hydrogen peroxide (H₂O₂) than inhibition of xanthine oxidoreductase or nicotinamide adenine dinucleotide phosphate oxidase. To test this, we used fluorescence microscopy to measure H₂O₂ and O₂^•- production from cortical slices during normoxia and hypoxia while pharmacologically inhibiting mitochondrial complex I, xanthine oxidoreductase, or nicotinamide adenine dinucleotide phosphate oxidase. Unexpectedly, we found xanthine oxidoreductase inhibition induced the greatest increase in O₂^•- during normoxia and hypoxia (~100% and 70%, respectively). Hypoxic inhibition of nicotinamide adenine dinucleotide phosphate oxidase induced the greatest decrease in H₂O₂ by ~35% below baseline. Finally, although inhibition of mitochondrial complex I during hypoxia yielded significant fluctuations in O₂^•- and H₂O₂, these changes were considerably smaller than fluctuations induced by inhibiting xanthine oxidoreductase or nicotinamide adenine dinucleotide phosphate oxidase. Together, and unlike in other rodent brain, our results suggest that xanthine oxidoreductase is the primary contributor to reactive oxygen species production in naked mole-rat cortex.