Exogenous ketone ester delays CNS oxygen toxicity without impairing cognitive and motor performance in male Sprague-Dawley rats.

Hyperbaric oxygen (HBO2) is breathing >1 atmosphere absolute (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 central nervous system (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 ß-hydroxybutyrate (BHB) levels within 0.5 h and remains elevated for 4 h. Male rats were separated into three groups: control (no gavage), water-gavage, or KE-gavage, and were subjected to behavioral testing while breathing 1 ATA (101.3 kPa) of 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 with 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.

Exogenous ketone salts inhibit superoxide production in the rat caudal solitary complex during exposure to normobaric and hyperbaric hyperoxia.

The use of hyperbaric oxygen (HBO2) in hyperbaric and undersea medicine is limited by the risk of seizures [i.e., central nervous system (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 with control groups (D'Agostino DP, Pilla R, Held HE, Landon CS, Puchowicz M, Brunengraber H, Ari C, Arnold P, Dean JB. Am J Physiol Regu Integr Comp Physiol 304: R829-R836, 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 ([Formula: see text]) 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 h 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-ß-hydroxybutyrate + acetoacetate. All levels of hyperoxia tested stimulated [Formula: see text] production similarly in cSC cells and coexposure 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 [Formula: see text] at the same rate during exposure to control O2 and hyperoxygenation; cells that increased [Formula: see text] production by >25% during hyperoxia in comparison with 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.NEW & NOTEWORTHY Exposure of rat medullary tissue slices to levels of O2 that mimic those that cause seizures in rats stimulates cellular superoxide ([Formula: see text]) production to varying degrees. Cellular [Formula: see text] generation in the caudal solitary complex is variable during exposure to control O2 and hyperoxia and significantly decreases during ketone supplementation. Our findings support the theory that ketone supplementation delays onset of central nervous system oxygen toxicity in mammals, in part, by decreasing [Formula: see text] production in O2-sensitive neurons.

CNS function and dysfunction during exposure to hyperbaric oxygen in operational and clinical settings.

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.

Delaying latency to hyperbaric oxygen-induced CNS oxygen toxicity seizures by combinations of exogenous ketone supplements.

Central nervous system oxygen toxicity (CNS-OT) manifests as tonic-clonic seizures and is a limitation of hyperbaric oxygen therapy (HBOT), as well as of recreational and technical diving associated with elevated partial pressure of oxygen. A previous study showed that ketone ester (1,3-butanediol acetoacetate diester, KE) administration delayed latency to seizures (LS) in 3-month-old Sprague-Dawley (SD) rats. This study explores the effect of exogenous ketone supplements in additional dosages and formulations on CNS-OT seizures in 18 months old SD rats, an age group correlating to human middle age. Ketogenic agents were given orally 60 min prior to exposure to hyperbaric oxygen and included control (water), KE (10 g/kg), KE/2 (KE 5 g/kg + water 5 g/kg), KE + medium-chain triglycerides (KE 5 g/kg + MCT 5 g/kg), and ketone salt (Na+ /K+ ßHB, KS) + MCT (KS 5 g/kg + MCT 5 g/kg). Rats were exposed to 100% oxygen at 5 atmospheres absolute (ATA). Upon seizure presentation (tonic-clonic movements) experiments were immediately terminated and blood was tested for glucose and D-beta-hydroxybutyrate (D-ßHB) levels. While blood D-ßHB levels were significantly elevated post-dive in all treatment groups, LS was significantly delayed only in KE (P = 0.0003), KE/2 (P = 0.023), and KE + MCT (P = 0.028) groups. In these groups, the severity of seizures appeared to be reduced, although these changes were significant only in KE-treated animals (P = 0.015). Acetoacetate (AcAc) levels were also significantly elevated in KE-treated animals. The LS in 18-month-old rats was delayed by 179% in KE, 219% in KE + MCT, and 55% in KE/2 groups, while only by 29% in KS + MCT. In conclusion, KE supplementation given alone and in combination with MCT elevated both ßHB and AcAc, and delayed CNS-OT seizures.

A potential early physiological marker for CNS oxygen toxicity: hyperoxic hyperpnea precedes seizure in unanesthetized rats breathing hyperbaric oxygen.

Hyperbaric oxygen (HBO(2)) stimulates presumptive central CO2-chemoreceptor neurons, increases minute ventilation (V(min)), decreases heart rate (HR) and, if breathed sufficiently long, produces central nervous system oxygen toxicity (CNS-OT; i.e., seizures). The risk of seizures when breathing HBO(2) is variable between individuals and its onset is difficult to predict. We have tested the hypothesis that a predictable pattern of cardiorespiration precedes an impending seizure when breathing HBO2. To test this hypothesis, 27 adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters to assess diaphragmatic/abdominal electromyogram, electrocardiogram, and electroencephalogram. Seven days after surgery, each rat was placed in a sealed, continuously ventilated animal chamber inside a hyperbaric chamber. Both chambers were pressurized in parallel using poikilocapnic 100% O(2) (animal chamber) and air (hyperbaric chamber) to 4, 5, or 6 atmospheres absolute (ATA). Breathing 1 ATA O(2) initially decreased V(min) and HR (Phase 1 of the compound hyperoxic ventilatory response). With continued exposure to normobaric hyperoxia, however, V(min) began increasing toward the end of exposure in one-third of the animals tested. Breathing HBO2 induced an early transient increase in V(min) (Phase 2) and HR during the chamber pressurization, followed by a second significant increase of V(min) ≤8 min prior to seizure (Phase 3). HR, which subsequently decreased during sustained hyperoxia, showed no additional changes prior to seizure. We conclude that hyperoxic hyperpnea (Phase 3 of the compound hyperoxic ventilatory response) is a predictor of an impending seizure while breathing poikilocapnic HBO(2) at rest in unanesthetized rats.

Hyperoxic stimulation of synchronous orthodromic activity and induction of neural plasticity does not require changes in excitatory synaptic transmission.

The first study, described in the companion article, reports that acute exposure of rat hippocampal slices to either hyperbaric oxygen (HBO: 2.84 and 4.54 atmospheres absolute, ATA) or normobaric reoxygenation (NBOreox; i.e., normobaric hyperoxia: 0.6 or 0.0→0.95 ATA) stimulates synchronous orthodromic activity in CA1 neurons, which includes activation of O2-induced potentiation (OxIP) and, in some cases, hyperexcitability (secondary population spikes, sPS). In this second study we tested the hypothesis that HBO and NBOreox increase orthodromic activity of CA1 neurons (oPS, orthodromic population spike) and OxIP via a combination of both increased excitatory synaptic transmission (field excitatory postsynaptic potential, fEPSP) and intrinsic excitability (antidromic population spike, aPS). HBO and NBOreox increased the oPS but rarely increased or potentiated the fEPSP. HBO exposure produced epileptiform antidromic activity, which was abolished during inhibition of fast GABAergic and glutamatergic synaptic transmission. Decreasing O2 from 0.95 ATA (control) to 0.6 ATA (intermediate O2) or 0.0 ATA (hypoxia) reversibly abolished the fEPSP, and reoxygenation rarely induced potentiation of the fEPSP or aPS. Intracellular recordings and antidromic field potential recordings, however, revealed that synaptic transmission and neuronal excitability were preserved, albeit at lower levels, in 0.60 ATA O2. Together, these data indicate that 1) the changes in excitatory postsynaptic activity are not required for stimulation of the oPS during and HBO/NBOreox or for activation of OxIP, suggesting the latter is a form of intrinsic plasticity; 2) HBO disinhibits spontaneous synaptic transmission to induce epileptiform activity; and 3) although synchronous synaptic activation of the CA1 neuronal population requires hyperoxia (i.e., 0.95 ATA O2), synaptic activation of individual CA1 neurons does not.

Hyperbaric hyperoxia and normobaric reoxygenation increase excitability and activate oxygen-induced potentiation in CA1 hippocampal neurons.

Breathing hyperbaric oxygen (HBO) is common practice in hyperbaric and diving medicine. The benefits of breathing HBO, however, are limited by the risk of central nervous system O2 toxicity, which presents as seizures. We tested the hypothesis that excitability increases in CA1 neurons of the rat hippocampal slice (400 microm) over a continuum of hyperoxia that spans normobaric and hyperbaric pressures. Amplitude changes of the orthodromic population spike were used to assess neuronal O2 sensitivity before, during, and following exposure to 0, 0.6, 0.95 (control), 2.84, and 4.54 atmospheres absolute (ATA) O2. Polarographic O2 electrodes were used to measure tissue slice PO2 (PtO2). In 0.95 ATA O2, core PtO2 at 200 microm deep was 115±16 Torr (mean±SE). Increasing O2 to 2.84 and 4.54 ATA increased core PtO2 to 1,222±77 and 2,037±157 Torr, respectively. HBO increased the orthodromic population spike amplitude and usually induced hyperexcitability (i.e., secondary population spikes) and, in addition, a long-lasting potentiation of the orthodromic population spike that we have termed "oxygen-induced potentiation" (OxIP). Exposure to 0.60 ATA O2 and hypoxia (0.00 ATA) decreased core PtO2 to 84±6 and 20±4 Torr, respectively, and abolished the orthodromic response. Reoxygenation from 0.0 or 0.6 ATA O2, however, usually produced a response similar to that of HBO: hyperexcitability and activation of OxIP. We conclude that CA1 neurons exhibit increased excitability and neural plasticity over a broad range of PtO2, which can be activated by a single, hyperoxic stimulus. We postulate that transient acute hyperoxia stimulus, whether caused by breathing HBO or reoxygenation following hypoxia (e.g., disordered breathing), is a powerful stimulant for orthodromic activity and neural plasticity in the CA1 hippocampus.

Effects of hyperbaric gases on membrane nanostructure and function in neurons.

This mini-review summarizes current ideas of how hyperbaric gases (>1-10 atmospheres absolute) affect neuronal mechanisms of excitability through molecular interaction with membrane components. The dynamic nature of the lipid bilayer, its resident proteins, and the underlying cytoskeleton make each respective nanostructure a potential target for modulation by hyperbaric gases. Depending on the composition of the gas mixture, the relative concentrations of O(2) and inert gas, and total barometric pressure, the net effect of a particular gas on the cell membrane will be determined by the gas' 1) lipid solubility, 2) ability to oxidize lipids and proteins (O(2)), and 3) capacity, in the compressed state, to generate localized shear and strain forces between various nanostructures. A change in the properties of any one membrane component is anticipated to change conductance of membrane-spanning ion channels and thus neuronal function.

Hyperbaric oxygen and chemical oxidants stimulate CO2/H+-sensitive neurons in rat brain stem slices.

Hyperoxia, a model of oxidative stress, can disrupt brain stem function, presumably by an increase in O2 free radicals. Breathing hyperbaric oxygen (HBO2) initially causes hyperoxic hyperventilation, whereas extended exposure to HBO2 disrupts cardiorespiratory control. Presently, it is unknown how hyperoxia affects brain stem neurons. We have tested the hypothesis that hyperoxia increases excitability of neurons of the solitary complex neurons, which is an important region for cardiorespiratory control and central CO2/H+ chemoreception. Intracellular recordings were made in rat medullary slices during exposure to 2-3 atm of HBO2, HBO2 plus antioxidant (Trolox C), and chemical oxidants (N-chlorosuccinimide, chloramine-T). HBO2 increased input resistance and stimulated firing rate in 38% of neurons; both effects of HBO2 were blocked by antioxidant and mimicked by chemical oxidants. Hypercapnia stimulated 32 of 60 (53%) neurons. Remarkably, these CO2/H+-chemosensitive neurons were preferentially sensitive to HBO2; 90% of neurons sensitive to HBO2 and/or chemical oxidants were also CO2/H+ chemosensitive. Conversely, only 19% of HBO2-insensitive neurons were CO2/H+ chemosensitive. We conclude that hyperoxia decreases membrane conductance and stimulates firing of putative central CO2/H+-chemoreceptor neurons by an O2 free radical mechanism. These findings may explain why hyperoxia, paradoxically, stimulates ventilation.

Pressure (< or=4 ATA) increases membrane conductance and firing rate in the rat solitary complex.

Neuronal sensitivity to pressure, barosensitivity, is illustrated by high-pressure nervous syndrome, which manifests as increased central nervous system excitability when heliox or trimix is breathed at >15 atmospheres absolute (ATA). We have tested the hypothesis that smaller levels of pressure (