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

The use of hyperbaric oxygen (HBO2) 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 (.O2‑) production in the caudal Solitary Complex (cSC) in rat brain slices using a fluorogenic dye, dihydroethidium (DHE), during exposure to control O2 (0.4 ATA) followed by 1-2 hr of normobaric oxygen (NBO2) (0.95 ATA) and HBO2 (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 .O2- 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 .O2- at the same rate during exposure to control O2 and hyperoxygenation; cells that increased .O2‑ 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 NBO2 and HBO2 hyperoxia.

Effect of hyperoxic and hyperbaric conditions on the adenosinergic pathway and CD26 expression in rat

The nucleoside adenosine acts on the nervous and cardiovascular systems via the A2A receptor (A2AR). In response to oxygen level in tissues, adenosine plasma concentration is regulated in particular via its synthesis by CD73 and via its degradation by adenosine deaminase (ADA). The cell-surface endopeptidase CD26 controls the concentration of vasoactive and antioxidant peptides and hence regulates the oxygen supply to tissues and oxidative stress response. Although overexpression of adenosine, CD73, ADA, A2AR, and CD26 in response to hypoxia is well documented, the effects of hyperoxic and hyperbaric conditions on these elements deserve further consideration. Rats and a murine Chem-3 cell line that expresses A2AR were exposed to 0.21 bar O2, 0.79 bar N2 (terrestrial conditions; normoxia); 1 bar O2 (hyperoxia); 2 bar O2 (hyperbaric hyperoxia); 0.21 bar O2, 1.79 bar N2 (hyperbaria). Adenosine plasma concentration, CD73, ADA, A2AR expression, and CD26 activity were addressed in vivo, and cAMP production was addressed in cellulo. For in vivo conditions, 1) hyperoxia decreased adenosine plasma level and T cell surface CD26 activity, whereas it increased CD73 expression and ADA level; 2) hyperbaric hyperoxia tended to amplify the trend; and 3) hyperbaria alone lacked significant influence on these parameters. In the brain and in cellulo, 1) hyperoxia decreased A2AR expression; 2) hyperbaric hyperoxia amplified the trend; and 3) hyperbaria alone exhibited the strongest effect. We found a similar pattern regarding both A2AR mRNA synthesis in the brain and cAMP production in Chem-3 cells. Thus a high oxygen level tended to downregulate the adenosinergic pathway and CD26 activity. Hyperbaria alone affected only A2AR expression and cAMP production. We discuss how such mechanisms triggered by hyperoxygenation can limit, through vasoconstriction, the oxygen supply to tissues and the production of reactive oxygen species.