Intermittent Flying Prior to a Deep Dive in Pure Oxygen Induces Neuroprotection Against Seizures Raffaele Pilla, Csilla Ari, Tina Fiorelli, Heather E. Held, Carol S. Landon and Jay B. Dean 1Department of Molecular Pharmacology & Physiology, Hyperbaric Biomedical Research Laboratory, Morsani College of Medicine, University of South Florida, Tampa, FL Introduction Methods Results Conclusions Oxygen toxicity is thought to occur from a buildup of reactive oxygen and nitrogen species (RO/NS) in the brain. Antioxidant defenses have been shown to increase in response to mild oxidative stresses. Effects of Preconditioning on Latency to Seizure Effects of Preconditioning on Latency to Seizure Seizure Detection Hyperbaric and hyperoxic preconditioning did not delay onset of CNSOT. Normoxic hyperbaric preconditioning significantly decreased latency to seizure. Hypobaric preconditioning conferred neuroprotection, apparently independently of the associated hypoxia. 24h after the last preconditioning, rats were exposed to 5 ATA O2 until the onset of CNS-OT. Figure 1. A) Normal EEG recording; B) EEG recording showing seizure activity; C) rat exhibiting normal body posture; D) rat exhibiting typical seizure posturing (weight borne on hind limbs, forelimbs exhibiting tonic-clonic movement). A B C D Hypothesis Hyperbaric hyperoxia would increase latency to seizure by reducing cellular oxidative stress (i.e. superoxide levels), presumably by up-regulating endogenous antioxidants. Effects of Preconditioning on Cellular Superoxide Production Hyperbaric treatment produced a significant increase in mean RFU, which may indicate a higher number of dead cells. Hypobaric treatment reduced mean RFU, likely indicating lower overall oxidative stress and a smaller number of dead cells. When fluorescence intensity was normalized for cell size, high fluorescence (high superoixde production) was still observed following hyperbaric heliox treatment, but was lower following hyperbaric nitrox treatment and hypobaric treatment. Given the pattern of results in this study, we interpret the data to mean that hyperbaric preconditioning worsens oxidative stress in neurons, leading to increased likelihood of seizure and increased neuronal cell death. Hypobaric exposure apparently protects against these effects. Cellular Studies Cortical and hippocampal neurons dissected from E18 (embryonic) rats were cultured for 14 days on on poly-D-lysine coated multi-well dishes (24 wells). HEPES buffer was added to cell media just prior to 1 hr normoxic gas mix exposure. Gases used were: Air at 1 atmosphere absolute (ATA, control) 10% O2, bal N2 at 2 ATA (hyperbaric normoxia) 10% O2, bal He2 at 2 (hyperbaric normoxia) 40% O2 at 0.5 ATA (hypobaric normoxia) Cells were then incubated for 30 min with dihydroethidium (selective dye for superoxide anion detection, 2.5 µM), then processed through a spectrophotometer for optical density measurements. Cells were promptly fixed in 4% paraformaldehyde in PBS for 1 hour, then rinsed and conserved in sterile PBS at 4o C. The next day, cell fluorescence in 3 fields/well was analyzed using a Nikon© fluorescence microscope and Element© software. Fluorescence intensity was analyzed offline, using the Regions of Interest (ROI) method: fluorescent cells were selected by the operator, and the fluorescence intensity per unit of cellular area was determined. Methods Telemetry Unit Implantation Adult Sprague-Dawley rats were surgically implanted with radio-telemetry units to record diaphragmatic electromyogram, electrocardiogram, electroencephalogram, body temperature, and physical activity. Figure 2. Latency to Seizure as a Function of Preconditioning Treatment. Contrary to our hypothesis, hyperbaric and hyperoxic preconditioning decreased latency to seizure. Hypoxia, on the other hand, appeared to confer a protective effect. Preconditioning Effects of Preconditioning on Cellular Superoxide Production After healing (1 wk), rats were preconditioned with 1 hr treatment/day, every other day (total 3x over 5 days): Air at 1 atmosphere absolute (ATA, control) Air at 2 ATA (hyperbaric normoxia) 100% O2 at 1 ATA (normobaric hyperoxia) 100% O2 at 2 ATA (hyperbaric hyperoxia) 10% O2, bal N2 at 2 ATA (hyperbaric normoxia) 10% O2, bal He2 at 2 (hyperbaric normoxia) Following initial observations, pretreatments were expanded to include hypoxic and hypobaric exposures: 10% O2 at 1 ATA (normobaric hypoxia) 40% O2 at 0.5 ATA (hypobaric normoxia) Air at 0.5 ATA (hypobaric hypoxia) A B Support and Disclosures Fig 3. A) Mean of Relative Fluorescent Units (RFU) calculated from multiple wells. Superoxide levels increased following hyperbaric exposures but decreased following hypobaric treatments. B) The ratio between fluorescence intensity and the selected area in the ROI (superoxide anion production) increased significantly following hyperbaric heliox preconditioning (10% O2, bal He at 2 ATA), but decreased following hyperbaric nitrox (10% O2, bal N2 at 2 ATA) and hypobaric normoxia. Sponsored by Divers Alert Network, Office of Naval Research (ONR #N000140710890, N000141210801). The authors do not have any conflicts of interest to disclose.