Exogenous ketone salts inhibit superoxide production in the rat caudal solitary complex during exposure to normobaric and hyperbaric hyperoxia

The use of hyperbaric oxygen (HBO ) in hyperbaric and undersea medicine is limited by the risk of seizures (i.e., CNS oxygen toxicity, CNS-OT) resulting from increased production of reactive oxygen species (ROS) in the CNS. Importantly, ketone supplementation has been shown to delay onset of CNS-OT in rats by ~600% in comparison to control groups (D'Agostino et al., 2013). We have tested the hypothesis that ketone body supplementation inhibits ROS production during exposure to hyperoxygenation in rat brainstem cells. We measured the rate of cellular superoxide (.O ) production in the caudal Solitary Complex (cSC) in rat brain slices using a fluorogenic dye, dihydroethidium (DHE), during exposure to control O (0.4 ATA) followed by 1-2 hr of normobaric oxygen (NBO ) (0.95 ATA) and HBO (1.95, and 4.95 ATA) hyperoxia, with and without a 50:50 mixture of ketone salts (KS) DL-b-hydroxybutyrate (BHB + acetoacetate (AcAc)). All levels of hyperoxia tested stimulated .O production similarly in cSC cells, and co-exposure to 5 mM KS during hyperoxia significantly blunted the rate of increase in DHE fluorescence intensity during exposure to hyperoxia. Not all cells tested produced .O at the same rate during exposure to control O and hyperoxygenation; cells that increased .O production by >25% during hyperoxia in comparison to baseline were inhibited by KS, whereas cells that did not reach that threshold during hyperoxia were unaffected by KS. These findings support the hypothesis that ketone supplementation decreases the steady state concentrations of superoxide produced during exposure to NBO and HBO hyperoxia.