Hyperbaric fluorescence microscopy: imaging reactive O 2 and N 2 species in rat brain slices during exposure to normobaric and hyperbaric hyperoxia

Hyperbaric oxygen (HBO) is breathed in hyperbaric and undersea medicine. Protracted exposure to HBO, however, is toxic to the mammalian brain and results in central nervous system‐O 2 toxicity (CNS‐OT; i.e., seizures). Thus, the use of HBO in military diving operations is limited by maximum time at depth; e.g., 10 min at 50 fsw on 100% O 2 . Our lab is interested in the mechanisms and mitigation of CNS‐OT for safer, longer dives. The present study reports our methods for measuring the rate of reactive species production in real time using fluorescence microscopy as a function of PO 2 at normobaric and hyperbaric pressures. Our recent improvements in hyperbaric fluorescence microscopy include the following: 1) quantifying the effect of pressure on focal length of the microscope—during compression the focal length increases and during decompression the focal length shortens (“baro‐blurring”); 2) visual monitoring of the brain slice bath inside a sealed, dark pressure chamber without exciting fluorescent dyes (DHE ex/em: 525/590nm; DAF‐FM ex/em: 495/515nm) by continuous IR illumination (770nm) of the microscope objective and slice bath while imaging with a CCD camera (heat filter removed); 3) an internal pump for recirculating medium containing fluorescent dye; 4) addition of a Lumencor SPECTRA Light Engine; and 5) upgrades in remote controls for x‐y translation of the slice chamber. Experiments using hyperbaric fluorescence microscopy show that while normobaric hyperoxia (NBO; 0.4 – 0.95 ATA O 2 ) and hyperbaric oxygen (HBO; 0.4 – 1.95 ATA O 2 ) increase the rate of superoxide production (2.5uM DHE), not all cells produce superoxide at the same rate. Most DHE‐loaded cSC neurons exhibited varying fluorescence intensities initially in 0.4 ATA O 2 that we labeled “Dim cells” (fluorescence intensity units (FIU) < average FIU at t=0) and “Bright cells” (FIU ≥ average FIU at t=0). O 2 ‐ sensitive cells significantly increased superoxide production (by at least 25% ΔFIU/min) in both Dim and Bright cells after exposure to 0.95/1.95 ATA O 2 . Certain Bright and Dim cells, however, were O 2 ‐insensitive cells (< 25% ΔFIU/min). After measuring the effects of NBO and HBO on superoxide production, we tested the effects of a 1:1 mixture of ketone salts (KS) on ΔFIU/min during increased background oxygenation. The rationale for this is that ketone metabolic therapy delays onset of CNS‐OT seizures. 5mM KS significantly decreased the production superoxide in O 2 ‐sensitive Bright cells at 0.95/1.95 ATA O 2 . Our findings suggest that cells in the cSC are heterogeneous in their capacity for superoxide production and neuroprotection by ketone bodies during exposure to hyperoxia. Studies are underway to determine the effects of NBO & HBO ±KS on nitric oxide production in cSC cells using 1 uM DAF‐FM. Support or Funding Information Research funded by ONR Undersea Medicine Program