Hyperbaric oxygen (HBO2) is breathed during hyperbaric oxygen therapy and during certain undersea pursuits in diving and submarine operations. What limits exposure to HBO2 in these situations is the acute onset of central nervous system oxygen toxicity (CNS-OT) following a latent period of safe oxygen breathing. CNS-OT presents as various non-convulsive signs and symptoms, many of which appear to be of brainstem origin involving cranial nerve nuclei and autonomic and cardiorespiratory centers, which ultimately spread to higher cortical centers and terminate as generalized tonic-clonic seizures. The initial safe latent period makes the use of HBO2 practical in hyperbaric and undersea medicine; however, the latent period is highly variable between individuals and within the same individual on different days, making it difficult to predict onset of toxic indications. Consequently, currently accepted guidelines for safe HBO2 exposure are highly conservative. This review examines the disorder of CNS-OT and summarizes current ideas on its underlying pathophysiology, including specific areas of the CNS and fundamental neural and redox signaling mechanisms that are thought to be involved in seizure genesis and propagation. In addition, conditions that accelerate the onset of seizures are discussed, as are current mitigation strategies under investigation for neuroprotection against redox stress while breathing HBO2 that extend the latent period, thus enabling safer and longer exposures for diving and medical therapies.

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.

Hyperbaric oxygen (HBO2) is breathing greater than 1 ATA (101.3 kPa) O2 and is used in HBO2 therapy and undersea medicine. What limits the use of HBO2 is the risk of developing CNS oxygen toxicity (CNS-OT). A promising therapy for delaying CNS-OT is ketone metabolic therapy either through diet or exogenous ketone ester (KE) supplement. Previous studies indicate that KE induces ketosis and delays the onset of CNS-OT; however, the effects of exogeneous KE on cognition and performance are understudied. Accordingly, we tested the hypothesis that oral gavage with 7.5 g/kg induces ketosis and increases the latency time to seizure (LSz) without impairing cognition and performance. A single oral dose of 7.5 g/kg KE increases systemic b-hydroxybutyrate (BHB) levels within 0.5 hr and remains elevated for 4 hr. Male rats were separated into 3 groups: control (no gavage), water-gavage, or KE-gavage; and were subjected to behavioral testing while breathing 1 ATA (101.3 kPa) air. Testing included the following: DigiGait (DG), Light/Dark (LD), open field (OF), and novel object recognition (NOR). There were no adverse effects of KE on gait or motor performance (DG), cognition (NOR), and anxiety (LD, OF). In fact, KE had an anxiolytic effect (OF, LD). The LSz during exposure to 5 ATA (506.6 kPa) O2 (£90 min) increased 307% in KE-treated rats compared to control rats. In addition, KE prevented seizures in some animals. We conclude that 7.5 g/kg is an optimal dose of KE in the male Sprague-Dawley rat model of CNS-OT.

Prolonged breathing of hyperbaric oxygen (HBO2) results in central nervous system O2 toxicity (CNS‐OT; i.e., seizures, Sz's). The threat of CNS‐OT is what limits the use of HBO2 in hyperbaric, diving and submarine medicine. Past studies have identified CNS‐OT as the onset of visibly detectable tonic‐clonic Sz's preceded by increased cortical EEG activity. It has been our experience, however, that onset of 1st Sz in Sprague‐Dawley (SD) rats is variable between animals and, in some cases, difficult to confirm in real time. This is problematic because the latency time to seizures (LSz) is how the risk of CNS‐OT is evaluated in hyperbaric research. Consequently, we have begun using longer exposures to HBO2 to capture recurring changes in EEG activity and behaviors that can be analyzed offline to redefine the criteria for 1st Sz. We also wanted to know if subsequent Sz's increased in intensity. Accordingly, cortical EEGs were recorded in 27 adult male SD rats that were implanted with a DSI 4‐ET radio transmitter. At least 1 week following surgery, each rat was placed in a hyperbaric chamber and “dived” to 5 ATA O2. 71 Sz's were identified in 27 rats based on quantified changes in cortical EEG activity (NeuroScore 2.1) and our modified Racine scale of six Sz‐related behaviors. A cortical seizure (CSz) was defined as a series of EEG polyspikes that crossed a threshold of 200μV, lasted ≥15 seconds, and contained an increase in theta wave activity coupled with at least one of the Sz‐related behaviors (65% of the Sz were classified as CSz). Two other Sz types were also recorded: neurological seizures (NSz) that included only the EEG profile described above, but without any detectable behavior changes (14%); and behavioral seizure (BSz) that included one or more of the behavioral components without any detectable changes in cortical EEG activity (21%). LSz was calculated from the moment the chamber pressure reached 5 ATA O2 until the onset of CSz, NSz or BSz. The LSz to 1st seizure was 7.2 ± 5.3 min (n= 27), while the 2nd and 3rd Sz's occurred at 26.2 ± 29.88 min (n=25) and 40 ± 40.86 min (n=13), respectively. Remarkably, in between bouts of Sz's, animals exhibited normal behaviors (grooming, exploring, chewing, etc.) and normal EEG activity in 5 ATA O2. There was also a shift in the type of Sz between the 1st and 2nd Sz such that the fraction of BSz + NSz significantly increased from 22% to 44%. Moreover, intensity of the Sz increased progressively with each successive Sz based on our modified Racine score. We conclude that the majority of HBO2 Sz's in SD rats can be readily identified using a combination of defined EEG and behavioral criteria. However, not all Sz's during HBO2 fit both criteria. The occurrence of BSz suggests that neurological Sz's originate outside the cerebral cortex. The occurrence of NSz indicate that some Sz's will be missed in studies that do not measure EEG activity. The fact that 1st Sz is often underwhelming compared to subsequent Sz's suggests the LSz may be overestimated in some animals. To better understand the SD rat model of CNS‐OT, we recommend that future studies need to 1) implant telemetric leads into multiple cortical and subcortical areas to localize the site of Sz‐genesis; and 2) determine the degree of performance impairment during 1st, 2nd & 3rd Sz's.Support or Funding InformationONR N000141310405 & N000141612537

Hyperbaric oxygen (HBO2) is breathed in HBO2 therapy and in undersea medicine; however, what limits the use of HBO2 in these situations is the risk of CNS oxygen toxicity (CNS‐OT; i.e. generalized seizures, Sz). Thus, we are interested in developing strategies that extend the duration of the safe latency period for breathing HBO2 by delaying or abolishing Sz. Increased production of reactive O2 & N2 species is involved in the neuropathology of CNS‐OT. Accordingly, we tested the following two hypotheses in male Sprague‐Dawley rats: a single predive intra peritoneal treatment given 30min before exposure to 5 ATA O2 of either 1) N(g)‐Nitro‐L‐arginine methyl ester (L‐NAME), which inhibits nitric oxide synthase (NOS) and thus nitric oxide (·NO) production, or Mitoquinone (MitoQ), which inhibits mitochondrial superoxide (·O2‐), delays Sz genesis in freely behaving, adult rats. Each rat was dived twice, separated by ≥1 week, receiving either control saline, L‐NAME (30mg/kg ip), or MitoQ (see below) in randomized order. Each rat was exposed to 1 ATA air (10‐15min), 1ATA O2 (10‐15min), compressed (1 ATG/min) to 5 ATA O2. The latency time to first Sz (LSz1) was measured from time zero (3 ATA O2) until onset of Sz at 5 ATA O2 or 60min (MitoQ) or 75min (L‐NAME), whichever came first. For L‐NAME dives, LSz1 in control dives = 10.8 ± 2.5min (avg ± SD; n=14) whereas L‐NAME delayed Sz significantly, increasing LSz1= 31.5 ± 24.3min, including 3 rats that did not seize (O) by 75min of HBO2 (Fig. 1; P= 0.0064). An additional 3 rats were surgically implanted with radio telemetric modules (4‐ET, DSI Inc.; motor cortex (ECoG) and dorsal medulla oblongata (EBulboG). Increased ECoG and EBulboG activities were delayed and blunted in rats treated with L‐NAME vs.control dives. For MitoQ dives, animal toxicity was evaluated first using up‐and‐down toxicity testing (1 ATA air). The maximum safe dose (ip) was 22g/kg MitoQ. During exposure to HBO2, MitoQ, likewise, delayed Sz or prevented Sz (n=5). Unexpectedly, however, all rats became lethargic during the dive and 3/5 rats died within 24 hr following decompression. A second up‐and‐down toxicity test was conducted using 11‐22mg/kg plus5 ATA O2. Most rats dosed with 14‐22mg/kg MitoQ and exposed to HBO2 did not Sz before 1 hr or delayed Sz; however, 11/16 died within 24 hr following decompression. By contrast, rats treated with 11mg/kg MitoQ plus HBO2 survived; however, the LSz1 was no different compared to control dives (n=2). We conclude that L‐NAME delays Sz genesis during HBO2, presumably by decreasing ·NO production and blocking ·NO‐induced cerebral hyperemia prior to Sz during HBO2. Doses of MitoQ (14‐22mg/kg) that are non‐toxic in 1 ATA air, and delay Sz during exposure to HBO2, however, become lethal when combined with HBO2. We postulate that MitoQ + HBO2 toxicity is due to unbridled ·NO production in the face of diminished production of mitochondrial ·O2‐, which normally reacts with ·NO, and thus accelerates onset of pulmonary O2 toxicity. Cocktails of L‐NAME, ketone ester, and MitoQ a...