Singlet Oxygen Production in the Caudodorsal Medulla of Rat Brain Slices Increases During Hyperoxia at Normobaric Pressure and Following Decompression from Iso‐oxic Hyperbaric Nitrogen

Abstract

Previous studies in brain slices harvested from the medulla oblongata of Sprague‐Dawley (SD) rats indicate that neurons in the caudal solitary complex (cSC) are stimulated by normobaric hyperoxia (NBO2) and hyperbaric O2 (HBO2). We've also reported that during NBO2, cSC cells produce a variety of reactive O2 and N2 species, including superoxide, nitric oxide, peroxynitrite, hydrogen peroxide, hydroxyl radicals, carbonate radicals, & nitrogen dioxide. These findings support the premise that cSC neurons employ redox signaling mechanisms that are activated by increasing pO2. Presumably, this explains, in part, the paradoxical stimulation of breathing by prolonged exposures to NBO2 and HBO2. We have tested the hypothesis that singlet oxygen (1O2) production increases in cSC cells exposed to a) NBO2 in the presence of nominal N2 and b) iso‐oxic hyperbaric nitrogen (HBN2). In the case of HBN2, we wanted to determine if inert gas molecules react with O2 molecules at pressure to form collision complexes that yield 1O2. The fluorogenic dye, Singlet Oxygen Sensor Green (SOSG, 2.5μM), was used to measure SOSG intensity of cSC neurons (P16–40 M/F SD rats, 400μm). Fluorescent images were captured at 1–2.5 min intervals and correlated with 1O2 production using one of two Nikon TE2000 inverted microscopes; one microscope was automated for use inside a hyperbaric chamber. The HCO3−‐buffered nutrient medium was equilibrated at room pressure with either (in atmospheres absolute, ATA) 0.4 O2 + 0.05 CO2 + 0.55 N2 (control) or 0.95 O2 + 0.05 CO2 + “0.0” N2 (NBO2).* For iso‐oxic HBN2, the air‐filled hyperbaric chamber (0.8 ATA N2) was compressed (0.25 ATA/min) with 100% N2 to 80–85 psig (5.4–5.8 ATA N2), thereby increasing atmospheric pN2 to 6.2–6.6 ATA (0.95 O2 + CO2 in the nutrient medium; pN2 increases in the medium and slice from HBN2 diffusing in from the overlying atmosphere). Nutrient media were delivered to the brain slice at 1.5 ml/min using an HPLC pump (30–31°C). Results show that increasing pO2 from 0.4 to 0.95 ATA at room pressure increased the rate of 1O2 fluorescence. 1–4 mM ascorbate, but not <1mM, decreased the rate of 1O2 fluorescence in 0.4 ATA O2 and NBO2 in a dose‐dependent manner. Exposure to iso‐oxic (0.95 ATA O2) HBN2 (10–30 min) did not increase 1O2; however, decompression (0.5ATA/min) back to sea level induced a post‐dive increase in 1O2 fluorescence that persisted for 10–30 min. The post‐dive increase in 1O2 fluorescence was inhibited by 4mM ascorbate. These findings suggest that 1O2 production is activated in cSC cells by hyperoxia, compression to at least 6.2 ATA N2, decompression, or the combination of compression/decompression in hyperoxic HBN2. We postulate that 1O2 generated in cSC neurons may contribute, in part, to CNS O2 toxicity, inert gas narcosis, and decompression sickness.Support or Funding InformationONR N000141512572 & N000141612537