Ketone salts inhibit production of superoxide anions during normobaric and hyperbaric hyperoxia in rat solitary complex neurons

The threat of central nervous system‐O 2 toxicity (CNS‐OT; i.e., seizures) is what limits the use of hyperbaric oxygen (HBO 2 ) in diving, submarine and hyperbaric medicine. We previously reported that ketone ester given orally to Sprague‐Dawley (SD) rats 30 min prior to HBO 2 exposure increases the levels of ketone bodies in the blood without inducing pathological metabolic acidosis, and delays the onset of CNS‐OT by nearly 600% (D'Agostino et al., 2013 AJP 304: R829–R836). We hypothesize that therapeutic ketosis induces neuroprotection against CNS‐OT in part by decreasing production of reactive oxygen species during hyperoxia. Tissue slices of the caudal medulla oblongata (300–400mm thick) were harvested from SD rats (P12–P28; n=35 rats). The fluorogenic dye, dihydroethidium (DHE, 2.5 mM) was added to nutrient medium (36–37 °C) containing a cocktail of nitric oxide synthase inhibitors (1 mM N‐w‐nitro‐L‐arginine methyl ester & 100 mM 7‐nitroindazole) and superoxide dismutase inhibitors (10mM diethyldithiocarbamate) to optimize O 2 ‐induced changes in DHE fluorescence (Ciarlone and Dean, 2016 AJP 311: C1014–C1026). DHE fluorescence was detected in neurons of the caudal solitary complex (cSC) using a Nikon TE‐2000 inverted microscope or a custom‐built hyperbaric microscope and Texas Red filter (ex/em 525 nm/590 nm) once every 5 min and expressed in arbitrary units of change in fluorescence intensity unit/minute (DFIU/min). Nutrient media were equilibrated with gases as follows (in ATA): control O 2 : 0.4 O 2 /0.05 CO 2 /bal N 2 (60 min); normobaric hyperoxia, NBO 2 : 0.95 O 2 /0.05 CO 2 (60 min); and HBO 2 : 4.95 O 2 /0.05 CO 2 (60 min). Ketone salts (KS) added to media during hyperoxia (60 min) included 2–2.5 & 5 mM, 50:50 mix of b–hydroxybutyrate + acetoacetate. Data were analyzed by 1‐way ANOVA and appropriate comparisons testing (P<0.05) using Nikon NIS‐Elements and Graphpad Prism. DHE fluorescence increased during 1 hr of NBO 2 (139%) and HBO 2 (240%). Neurons in the cSC had variable sensitivity to hyperoxia exhibiting low to high rates of increased superoxide production. During the 2 nd hr of NBO 2 , DHE fluorescence was unchanged from that in the 1 st hr. Conversely, DHE fluorescence decreased during the 2 nd hr of HBO 2 suggesting cellular dysfunction in 4.95 ATA O 2 . Thus, KS were tested during hr 2 in NBO 2 in the same slices, whereas KS were tested during hr 1 of HBO 2 in a separate set of brain slices. One hour of ketosis significantly decreased DHE fluorescence on average during NBO 2 by 1/3 (2mM) and 1/2 (5mM), and during HBO 2 by 1/3 (5mM); however, not all neurons as defined by their O 2 ‐sensitivity for superoxide production were equally responsive to ketosis. Our findings support the hypothesis that therapeutic ketosis induces neuroprotection during hyperoxia in part by decreasing O 2 ‐induced production of superoxide anions. Support or Funding Information Research supported by ONR Undersea Medicine This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .