Prolonged exposure of mice to He-O2 at high pressure: effects on seizure and anesthesia liability

Multiday exposures of CD-1 mice to He-O2 atmospheres at pressures from 30 to 100 atm result in marked increases of threshold pressures for type I high-pressure neurological syndrome seizures. The effect develops with a half time (t1/2) of 12 h and is reversible (t1/2 = 7 h). The maximum enhancement of Pc is attained at a conditioning pressure of 80 ATA. Pressure conditioning also results in suppression of the compression rate effect on Pc. Furthermore, reserpine blocks the increase in Pc during prolonged pressure exposure. The entire effect thus appears to be an extension in time of the monoaminergic compression rate effect on Pc. Pressure conditioning does not modify anesthesia tolerance, unlike N2 habituation which affects anesthesia threshold pressure as well as Pc. The results are compared with the effects of habituation to inert-gas narcotics and the implications of the data for an understanding of inert-gas high-pressure antagonism in intact animals are discussed.     

Opposing effects of anesthetics on pressure tolerance and compression rate effect

Inert gas narcotics increase intrinsic pressure tolerance (1,000Pc) in CD-1 mice but interfere with development of the protective responses raising seizure thresholds during slower compression (e.g., 60Pc). This secondary narcotic effect can block up to 40% of the total attainable increase in Pc. The narcosis susceptible moiety of this compression rate effect develops early, whereas a narcosis resistant remnant accounts for increase in Pc occurring after 90 min of compression or pressure exposure. Pressure conditioning by multiday pressure exposure entails increases in both 60Pc and 1,000Pc and in virtual annullment of the compression rate effect. The effect can be completely blocked by narcotic gases in the conditioning atmosphere. In addition to blocking part of the compression rate effect the presence of narcotic gases under these conditions can reverse the effects of previously established pressure conditioning. 60Pc regresses much more slowly under these conditions than 1,000Pc. Either reversal rate is much more rapid in air at 1 ATA than at 80 ATA under 0.9 atm N2O. The implications of these data are discussed with regard to evaluation of the hypothesis of antagonism between inert gas narcotics and high pressures and to elaboration of the monoamine hypothesis to account for the modification of the compression rate effect by narcotic gases.     

Effect of habituation to subanesthetic N2 or N2O levels on pressure and anesthesia tolerance

Exposure of CD-1 mice to subanesthetic partial pressures of N2O (0.5 atm) or N2 (10-20 atm) for periods up to 14 days results in up to 40% decreases in the mean threshold pressure eliciting type I high-pressure neurological syndrome (HPNS) seizures, and in increases up to 38% in the N2 partial pressure producing anesthesia. For all combinations of preexposure time, N2 partial pressure, as well as identity of the conditioning gas the relations between the convulsion threshold pressure (Pc) and the anesthesia N2 pressure (Pa) appear to be uniquely correlated by the equation Pa = 54.5 - 0.2(Pc - 60)1.2. The potency of N2O with respect to these habituation phenomena is between 28 and 33 times higher than that of N2, depending on the aspects compared. Evidence is presented indicating that after 14 days of habituation the animals have attained between 75 and 85% compensation for the anesthetic as well as the anticonvulsant effects of the conditioning gas. The bearing of the results on the problem of the nature of the antagonism between inert gas narcotic agents and high pressure and on the hypothesis that habituation tends toward restoration of isofluidity (or some analogous normalization process) are discussed.     

Effects of helium and other inert gases on Echinosphaerium nucleofilium (protozoa, heliozoa).

The effects of helium, nitrogen, argon and krypton on Echinosphaerium nucleofilum (Heliozoa) have been studied at partial pressures of 10-130 atm. Additional experiments have been carried out with hydrostatic pressure alone. Helium causes shortening of the axopods over the whole range of pressures, and damage to the cell body at pressures of 60-90 atm, both with a maximum at 80 atm. These effects cannot be explained in terms of hydrostatic pressure alone; a 'pressure reversal' effect may be operating, causing the peak at 80 atm. Nitrogen also causes both cell damage and axopod shortening, the severity increasing with increasing pressure. Argon and krypton cause cell damage but no shortening. The order of potency for cell damage is krypton greater than argon greater than nitrogen greater than helium. It is suggested that there may be tuo sites of action, possibly the microtubules (for axopod shortening) and the cell membrane (for cell damage). In appropriate mixtures of helium and argon, both the cell damage usually caused by argon, and the axopod shortening usually caused by helium, are prevented. Possible mechanisms include the effects of hydrostatic pressure on gas solubility coefficients, reversal of the effects of the gases by the increase in total pressure, and competition for sites of action.     

Human skeletal muscle function and metabolism during intense exercise at high O2 and N2 pressures

The maximal contractile force (peak torque) of the quadriceps femoris was studied during 60 repeated unilateral dynamic knee extensions in nine subjects under three different conditions, viz., during air breathing at normal (1 ATA) and raised (6 ATA) ambient pressures and during O2 breathing at 1.3 ATA. In six subjects the electromyographic (EMG) activity of the working muscle was recorded. Muscle biopsies were obtained from the vastus lateralis before, immediately after, and 1 min after exercise. Tissue specimens were subsequently assayed for various muscle metabolites. Peak torque, as an average of the 60 knee extensions, was higher (P less than 0.05) at 1.3 ATA than at 6 or 1 ATA. Peak torque of the exercising muscle declined more rapidly at 1 ATA than at 1.3 ATA, differing in the final 24 contractions by 14%. At 6 ATA peak torque of the initial 12 contractions was 6% lower (P less than 0.05) than at 1 ATA but equaled 1-ATA values in the latter third of the exercise bout. Although the EMG activity at 1 ATA increased relative to that at 6 ATA as exercise proceeded, the rate of force decline was greater at 1 ATA. Despite greater total work produced at 1.3 ATA than at 1 ATA, the metabolic response to exercise was not substantially altered at increased O2 pressure. However, the restitution rate of energy-rich phosphagens and the elimination of lactate during recovery were greater (P less than 0.05) at 1.3 ATA. These results suggest that hyperoxia may enhance the rate of energy release, whereas high N2 pressure and/or high hydrostatic pressure seem to interfere with neuromuscular activity.     

Effects of subanesthetic doses of inert gases on behavioral thermoregulation in mice

Mice exposed to subanesthetic partial pressures of N2O (0.25 to 0.75 atm) or N2 (5.7 or 11.33 atm) and allowed to choose between a warm and a cool environment showed a marked preference for the cooler environment. This behavior was associated with the onset of hypothermia, with deep body temperature falling by up to about 3 degrees C, usually to a new, steady level. Both the length of time spent in the cooler environment and the degree of the hypothermia produced increased with the partial pressure of N2O or N2 used. The effects of N2O on behavioral thermoregulation and body temperature were reversible. There was a correlation between anesthetic potency and the ability of both gases to alter thermoregulation, suggesting that the effect of these agents on thermoregulation was caused by the same molecular interactions as those which underlie anesthesia. Since both gases elicited changes in behavioral thermoregulation promoting rather than opposing the onset of hypothermia, it is concluded that they may have acted to lower the level at which deep body temperature was being regulated.     

Human cold air habituation is independent of thyroxine and thyrotropin.

Thyroxine (T4) is required in species possessing brown adipose tissue (BAT) for the maintenance of cold tolerance and adaptation. In humans, who possess negligible quantities of BAT, the importance of T4 has not been demonstrated. We studied the effects of decreased serum T4 and thyrotropin (TSH) on human cold habituation after repeated cold air exposures. Eight men (T3+) received a single daily dose of triiodothyronine (T3; 30 micrograms/day), and another eight men (T3-) received a placebo. All 16 normal thyroid men underwent a standardized cold air test (SCAT) under basal conditions in January and again in March after eighty 30-min 4.4 degrees C air exposures (10/wk). Measurements of basal metabolic rate (BMR), O2 consumption (VO2), mean arterial pressure (MAP), plasma norepinephrine (NE), serum TSH, free and total T4, and free and total T3 were repeated before and after 8 wk of exposure. TSH, free T4, and total T4 were 50% lower for T3+ than for T3- subjects. Total and free T3 were not different between groups. BMR was unchanged after habituation, whereas the cold-stimulated VO2, MAP, and NE were significantly reduced for all subjects in March. The relationship between VO2 and NE (r2 = 0.44, P less than 0.001) during the initial SCAT was unchanged with habituation. We suggest that human cold habituation is independent of major changes in circulating T4 and TSH.     

Methods for intracellular recording from hippocampal brain slices under high helium pressure

A method for intracellular recording from rat hippocampal brain slices under helium pressure is described. The preparation is mounted on a horizontal mobile platform that is rolled into the pressure chamber and can be viewed at pressure. Remote manipulation of the glass microelectrodes is achieved by a high-resolution electrically driven commercially available system. The slice is superfused continuously from a closed system within the chamber. Temperature is maintained at 37 degrees C and PO2 at 0.5 atm within the pressure chamber. A pressure of 200 ATA can be obtained, although thus far recordings have been made up to only 130 ATA. The experiments demand that a number of sample recordings be made from the same slice at both ambient and high pressure, and tests have proved that, although difficult, this can be achieved. The resting membrane potential, the current-voltage relationship, and the action potential responses to short (8 ms), medium (80 ms), and long (800 ms) depolarizing current pulses have all been measured in CA1 pyramidal neurons.     

Pressure increases oxygen affinity of whole blood and erythrocyte suspensions

Effect of hydrostatic pressure (HP) on whole blood (WB) or erythrocyte suspension hemoglobin (Hb) O2 affinity has been studied using newly developed techniques. O2 partial pressure at which hemoglobin is half-saturated with O2 (P50) measurements were made at 5 HP (1, 26, 51, 76, and 126 ATA) on thin films of human WB or erythrocytes at 37 degrees C. CO2 partial pressure of WB was either 28 or 57 Torr (film pH 7.51 or 7.31). HP increased affinity of erythrocytes and WB. For erythrocytes in tris(hydroxymethyl)aminomethane buffer, the ratio (r) of P50 (1 ATA)/P50 (51 ATA) was 1.089 (P less than 0.01) at pH 7.0. WB P50 decreased with HP at a rate of -3.3 X 10(-2) Torr X atm-1; change in P50 at higher HP vs. 1 ATA was highly significant (P less than 0.01). No effect of HP was seen on the CO2 Bohr coefficient. Inert gas choice, N2 vs. helium (He), had no effect. Measurement of decrease of P50 with HP at 76 ATA in hemolyzed WB gave an r of 1.15, as great or greater than that found in WB, indicates that Donnan equilibrium alteration is not involved. No effect of HP was found in WB on the ratio of P50 of erythrocytes with normal (5 mmol/l erythrocytes) 2,3-diphosphoglycerate (DPG) to P50 of erythrocytes with less than 5% of normal DPG; i.e., no effect of pressure was seen on the independent influence of DPG on P50. WB measurements of Hb O2 uptake under simulated physiological conditions are characterized by a net decrease in partial molal volume on oxygenation of 30-35 ml/mol Hb4.     

Hydrostatic pressure and adrenergic drugs (agonists and antagonists): effects and interactions in fish

Cardiac responses to alpha and beta adrenergic drugs have been studied in the eel firstly at atmospheric pressure (1 ATA), secondly at 101 ATA per se hydrostatic pressure. The exposure of treated eels for 1 hr at 101 ATA cancels the propranolol effect, increases the clonidine effect and induces paradoxal effects (reversal effect) of isoproterenol, phentolamine and phenylephrine. The highly significant interaction (P less than 0.001) between drugs and HP at 101 ATA is discussed and interpreted as effects of hydrostatic pressure inducing a change in receptor function.     

Pentobarbital anesthesia and the response of tumor and normal tissue in the C3Hf/sed mouse to radiation

Studies of the effect of pentobarbital anesthesia on the radiation response have been performed using early generation isotransplants of three spontaneous tumors of the C3H mouse: a mammary carcinoma (MCaIV), a fibrosarcoma (FSaII), and a squamous cell carcinoma (SCCVII). The enhancement ratio of pentobarbital [ER(PB)] for TCD50 as the end point was greater than or equal to 1 for all conditions tested. The ER(PB) for O2 3 ATA conditions and two equal doses was 1.46, 1.72, and 2.21 for MCaIV, FSaII, and SCCVII, respectively. The ER(PB) using MCaIV was the same for O2 and carbogen at 1 or 3 ATA. Also, tumor size of MCaIV did not significantly affect the ER(PB) for O2 3 ATA conditions. Further, with the two-dose protocol the anesthesia and the hyperbaric oxygen needed to be used at the second dose; condition at the first dose was not critical. For fractionated irradiation of MCaIV (10 and 15 equal doses) the ER(PB) was smaller than for two-dose treatment; also the effect was less for intratumor temperature of 35 degrees C than 26-27 degrees C. There was no effect of the anesthesia on the acute response of normal skin of the leg. Lung damage by hyperbaric oxygen was not an important factor in these results. Additionally, ERs were computed for O2 at 3 ATA. This ER(O2 3 ATA) was larger for anesthesized than conscious mice. The ER(O2 3 ATA) for MCaIV was high (greater than 1.5) even for radiation given in 10 or 15 equal doses.     

Induction of Superoxide Dismutase by Molecular Oxygen

Oxygen induces superoxide dismutase in Streptococcus faecalis and in Escherichia coli B. S. faecalis grown under 20 atm of O(2) had 16 times more of this enzyme than did anaerobically grown cells. In the case of E. coli, changing the conditions of growth from anaerobic to 5 atm of O(2) caused a 25-fold increase in the level of superoxide dismutase. Induction of this enzyme was a response to O(2) rather than to pressure, since 20 atm of N(2) was without effect. Induction of superoxide dismutase was a rapid process, and half of the maximal level was reached within 90 min after N(2)-grown cells of S. faecalis were exposed to 20 atm of O(2) at 37 C. S. faecalis did not contain perceptible levels of catalase under any of the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23), and the concentration of catalase in E. coli was not affected by the presence of O(2) during growth. S. faecalis, which had been grown under 100% O(2) and which therefore contained an elevated level of superoxide dismutase, was more resistant of 46 atm of O(2) than were cells which had been grown under N(2). E. coli grown under N(2) contained as much superoxide dismutase as did S. faecalis grown under 1 atm of O(2). The E. coli which had been grown under N(2) was as resistant to the deleterious effects of 50 atm of O(2) as was S. faecalis which had been grown under 1 atm of O(2). These results are consistent with the proposal that the peroxide radical is an important agent of the toxicity of oxygen and that superoxide dismutase may be a component of the systems which have been evolved to deal with this potential toxicity.     

Effects of hydrostatic pressure and inert gases on platelet aggregation in vitro

A novel cuvette was used to subject citrated platelet-rich plasma (PRP) to high hydrostatic pressure with negligible contamination by He (used for compression of the apparatus). Aggregation was induced at pressure by ADP and quantified turbidimetrically. The maximum degree of aggregation (MDA) was reduced from a control level of 82.2 to 53.6% by exposure to 101 ATA. Because decompression bubbles did not form, aggregation was also measured immediately after a compression cycle. After exposure to 101 ATA hydrostatic pressure, platelets responded normally to ADP at 1 ATA. In a matching apparatus, PRP was equilibrated with high partial pressures of inert gases. Normal physiological plasma Po2 and pH were maintained during equilibration. N2O (5 ATA) reduced the MDA from 86.5 (control) to 58.1%. N2 (51 ATA) reduced the MDA from 74.7 (control) to 51.6%, and 101 ATA Pn2 reduced the MDA from 78.0 (control) to 32.3%. He (100 ATA) reduced the MDA from 83.6 to 38.6%. It was concluded that platelet aggregation was relatively sensitive to hydrostatic pressure and less sensitive to inert gases than predicted from their anesthetic potency ratios.     

Nitrogen binding to deoxyhemoglobin at high pressures and its relation to changes in hemoglobin-oxygen affinity

The stoichiometric and thermodynamic properties of nitrogen (N2) binding to human deoxyhemoglobin (Hb) at N2 saturation pressures up to 400 atm were derived from measured N2 solubilities in protein-free buffers (pH 7.1) and in corresponding buffer + Hb (6.5% w/w) solutions at 20.0, 25.0, and 37.0 degrees C. At each temperature, approximately 3 N2 molecules bind per Hb tetramer at N2 pressures of 100 atm, while about 7 N2 molecules bind per tetramer at 400 atm N2 pressure, where available binding sites are still not fully saturated. Calculated N2-Hb binding isotherms are well described by a simple binding model with 12 independent and equivalent binding sites/Hb tetramer. N2 binding at each of the sites is hydrophobic, exhibiting the typical increase of the dissociation enthalpy with temperature. Enthalpies of dissociation are slightly more negative, while corresponding unitary entropies are somewhat less negative than those for the transfer of N2 from olive oil to water. Calculated partial molar volumes of N2 in Hb are positive but less than the corresponding partial molar volumes of N2 in buffer. Results indicate that N2 binding to Hb is accompanied by only small protein conformational changes which entail slight structural destabilization and loss of free volume in the protein that partially accommodates the volume of the N2 ligand. Results are related to previously reported effects of high pressure and high-pressure N2 on HbO2 affinity, illuminating essential features of the molecular mechanisms for these effects.     

Effect of exposure to high pressure on subsequent spatial learning and memory in rats

The effects of high helium pressure on the subsequent acquisition of spatial memory were studied in male rats. Thirty-two rats were exposed to 65 ATA helium-oxygen pressure for 4.2 days, decompressed (total time in chamber 5 days), and then tested in an eight-arm radial maze. Thirty-two control rats were exposed in the chamber to 1 ATA air. Each rat had 20 sessions in the maze (2 sessions/day for 10 days), and the number of correct (visiting an arm not previously visited to obtain the reward pellet) and incorrect choices (visiting a previously visited arm) were recorded. Statistical analysis showed that the rats exposed to 65 ATA performed significantly better than 1-ATA controls during the first 8 of 20 sessions. This effect was most pronounced in sessions 5-8. Results for sessions 9-20 showed that the pressure-treated rats still made more correct choices but to an extent that did not always reach statistical significance. Possible explanations include the pressure-treated rats performing better because of hunger after a lower food consumption at pressure. Alternatively, pressure itself may enhance proposed mechanisms of spatial memory such as long-term potentiation.     

BCNU-induced protection from hyperbaric hyperoxia: role of glutathione metabolism

To explore the role of the glutathione oxidation-reduction cycle in altering the sensitivity of rats to the effects of hyperbaric hyperoxia, we administered N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU) to decrease tissue glutathione reductase activity. We then exposed these animals and their matched vehicle-treated controls to 100% O2 at 4 ATA. Animals that received BCNU and were immediately exposed to hyperbaric O2 showed enhanced toxicity by seizing earlier in the exposure than controls. Animals that received BCNU 18 h before the hyperbaric O2 exposure were paradoxically protected from the effects of the exposure with a prolongation of their time to initial seizure and a marked increase in their survival time during the exposure. Tissue glutathione concentrations were also measured in the various groups and the hyperbaric O2 exposure produced marked decreases in hepatic glutathione levels in all control animals. In animals treated with BCNU 18 h before exposure, hepatic glutathione concentrations also decreased, but the concentrations had significantly increased during the 18-h waiting period, allowing these animals to maintain hepatic levels in the normal range even during their hyperbaric exposures. We conclude that treatment of rats with BCNU 18 h before exposure to hyperbaric hyperoxia results in enhanced protection of the animals during the exposure.     

Effect of High Oxygen Tensions on the Growth of Selected, Aerobic, Gram-negative, Pathogenic Bacteria

The in vitro effects of high O(2) tensions (P(O2)) on aerobic, enteric pathogens were examined at pressures of up to 3 atm absolute. Organisms from the genera Salmonella, Shigella, and Vibrio were usually subjected to 24-hr exposures. Tensions of 0.87, 1.87, and 2.87 atm absolute of O(2) (plus traces of CO(2) and N(2)) became progressively inhibitory for Salmonella and Shigella growth, but were bactericidal only for V. comma strains at tensions greater than 0.87 atm absolute of O(2). Growth inhibition of enteric organisms resulted from increased P(O2), rather than pressure per se, and could be mitigated nutritionally; an appropriate carbohydrate source is at least partially involved. Further studies with vibrios indicated that such mitigation was independent of medium pH. In addition, a synergistic relationship existed between O(2) and sulfisoxazole when tensions from 0.87 to 2.87 atm absolute of O(2) were maintained for 3 to 24 hr. Synergism occurred even under nutritional conditions which negated growth inhibition by O(2) alone. Bactericidal concentrations of sulfisoxazole, in the presence of increased P(O2), were reducible up to 4,000-fold. The combined procedure employed in this investigation, by use of an antimicrobial drug of known action, which also synergizes with O(2), plus nutritional studies, suggests a means for establishing a site of O(2) toxicity. These data support the concept that O(2) inhibition of growth represents a metabolic disturbance and that metabolic pathways involving p-aminobenzoic acid may be O(2)-labile. Such an approach could also guide development of antimicrobial agents as O(2) substitutes for promoting synergism.     

Alteration of Striatal Dopamine Levels Under Various Partial Pressure of Oxygen in Pre-convulsive and Convulsive Phases in Freely-Moving rats

The purpose of this study was to investigate the change in the striatal dopamine (DA) level in freely-moving rat exposed to different partial pressure of oxygen (from 1 to 5 ATA). Some works have suggested that DA release by the substantia nigra pars compacta (SNc) neurons in the striatum could be disturbed by hyperbaric oxygen (HBO) exposure, altering therefore the basal ganglia activity. Such changes could result in a change in glutamatergic and GABAergic control of the dopaminergic neurons into the SNc. Such alterations could provide more information about the oxygen-induced seizures observed at 5 ATA in rat. DA-sensitive electrodes were implanted into the striatum under general anesthesia. After 1 week rest, awaked rats were exposed to oxygen–nitrogen mixture at a partial pressure of oxygen of 1, 2, 3, 4 and 5 ATA. DA level was monitored continuously (every 3 min) by in vivo voltammetry before and during HBO exposure. HBO induced a decrease in DA level in relationship to the increase in partial pressure of oxygen from 1 ATA to 4 ATA (?15 % at 1 ATA, ?30 % at 2 ATA, ?40 % at 3 ATA, ?45 % at 4 ATA), without signs of oxygen toxicity. At 5 ATA, DA level strongly decreases (?75 %) before seizure which occurred after 27 min ± 7 HBO exposure. After the epileptic seizure the decrease in DA level disappeared. These changes and the biphasic effect of HBO were discussed in function of HBO action on neurochemical regulations of the nigro striatal pathway.     

Energetics of streptococcal growth inhibition by hydrostatic pressure.

Growth of Streptococcus faecalis in complex media with various fuel sources appeared to be limited by the rate of supply of adenosine-5' -triphosphate (ATP) at 1 atm and also under 408 atm of hydrostatic pressure. Growth under pressure was energetically inefficient, as indicated by an average cell yield for exponentially growing cultures of only 10.7 g (dry weight) per mol of ATP produced compared with a 1-atm value of 15.6. Use of ATP for pressure-volume work or for turnover of protein, peptidoglycan, or stable ribonucleic acid (RNA) did not appear to be significant causes of growth inefficiency under pressure. In addition, there did not seem to be an increased ATP requirement for ion uptake because cells growing at 408 atm had significantly lower internal K(+) levels than did those growing at 1 atm. Pressure did stimulate the membrane adenosine triphosphatase (ATPase) or S. faecalis at ATP concentrations greater than 0.5 mM. Intracellular ATP levels were found to vary during the culture cycle from about 2.5 mumol/ml of cytoplasmic water for lag-phase or stationary-phase cells to maxima for exponentially growing cells of about 7.5 mumol/ml at 1 atm and 5.5 mumol/ml at 408 atm. N,N'-dicyclohexylcarbodiimide at a 10 muM concentration improved growth efficiency under pressure, as did Mg(2+) or Ca(2+) ions at 50 mM concentration. These agents also enhanced ATP pooling, and it seemed that at least part of the growth inefficiency under pressure was due to increased ATPase activity. In all, it appeared that S. faecalis growing under pressure has somewhat reduced ATP supply but significantly increased demand and that the inhibitory effects of pressure can be interpreted largely in terms of ATP supply and demand.