INTRODUCTION

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CENTRAL NERVOUS SYSTEM (CNS) oxygen toxicity can appear in humans exposed to oxygen pressures above 180 kPa as convulsions (similar to epileptic seizures, grand mal) and loss of consciousness without any premonitory symptoms. It is known that the risk of CNS oxygen toxicity is greater when CO₂ is present in the inspired gas (2, 7, 8, 13, 19, 21, 23) or in tissue (25), mainly due to its effect on cerebral vasodilatation and increased brain tissue PO₂ (15) and acidity-enhanced reactive oxygen species (ROS) (6, 11, 22). In underwater diving where the risk of CNS oxygen toxicity is also encountered due to elevated PO₂, other extraneous circumstances leading to the elevation of PCO₂ may increase this risk even more (20, 24).

We have recently shown (2) that the latency to CNS oxygen toxicity decreased linearly as a function of the inspired PCO₂ and that it may even be affected by a PCO₂ as low as 1 kPa. These studies were conducted using PO₂ that cause CNS oxygen toxicity without the presence of CO₂ in the inspired gas. PO₂ during closed-circuit diving and hyperbaric oxygen therapy is usually kept at levels that do not cause CNS oxygen toxicity. All of the studies on the effect of CO₂ on CNS oxygen toxicity were conducted at a PO₂ that can cause CNS oxygen toxicity without the added effect of CO₂ (7, 9, 10, 23). Almost nothing is known about the effect of CO₂ on CNS oxygen toxicity at a PO₂ below the level that will on its own produce this toxic effect. We hypothesized that CO₂, which causes vasodilatation in the brain and therefore increases the tissue oxygen pressure, together with acidity-enhanced production of ROS (6, 11, 22), would cause CNS oxygen toxicity at a PO₂ below the level that causes CNS oxygen toxicity on its own. The level of inspired CO₂ at which the breach occurs (the threshold for CNS oxygen toxicity) is the subject of the present study.

The first electrical discharge (FED), which precedes the clinical convulsions, in the electroencephalogram (EEG) is a well-defined phenomenon and may be used to validate the effect of PCO₂ on CNS oxygen toxicity. We used a previously described rat model (1, 3, 4) to investigate the effect of CO₂ in the inspired gas on the threshold for CNS oxygen toxicity as a function of PO₂.

	METHODS

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Animals

Experimental System and Procedures

Experimental cage. The experimental cage is a metal, double-walled cage (25 × 20 × 12 cm; volume = 6.0 liters). One wall for observation of the animal and the top cover, which can be opened, are made of Plexiglas. Thermoregulated water can be pumped through the double wall to control the ambient temperature. The incoming gas flows through a metal container attached to the cage wall for temperature equilibration before entering the cage itself. The metal walls of the cage are covered on the outside with thermal insulating material. A cable with a male miniconnector for EEG recording passes through the top cover. Lin and Jamieson (18) suggested that humidity in the inspired oxygen might affect latency to CNS oxygen toxicity; therefore, the humidity in the cage was monitored throughout the experiment. A humidity and temperature measuring device (EE20FT, EE Electronics, Linz, Austria) was inserted into the top cover of the cage.

Experimental system. The miniconnectors were mated, and the rat was placed in the experimental cage, which was placed in a 150-liter hyperbaric chamber (Roberto Galeazzi, La Spezia, Italy). The flow of gas through the cage was controlled by two needle valves: one controlling the flow of air or oxygen and the other controlling the flow of CO₂. The flow was checked by observation of flowmeters situated inside the hyperbaric chamber. The outgoing gas exited via a bypass tube into the atmosphere of the hyperbaric chamber. A small portion of the outgoing gas was directed out of the pressure chamber (this was controlled by another needle valve), passed through a flowmeter, and sampled by a CO₂ analyzer (CD-3A, Ametek, Pittsburgh, PA). Water hoses were connected to ports in the pressure chamber and to ports in the experimental cage for recirculation of the thermoregulated water (C/H temperature controller bath and circulator 2067, Forma Scientific, Marietta, OH). The temperature in the cage was maintained at thermoneutral range to avoid any effect of temperature on the CNS oxygen toxicity (1). EEG was recorded on a chart recorder (Gould, Cleveland, OH).

Experimental procedure. When the pressure in the chamber was being raised (at 100 kPa/min), the gas flowing through the cage was air. When the desired pressure was reached, a period of 20 min was allowed for acclimation to the experimental conditions, during which time air flowed through the cage at ~8 l/min and CO₂ was added to produce the desired concentration of CO₂ in the cage. The flow of CO₂ was adjusted by means of a fine needle valve to yield the desired concentration, which was kept constant throughout the exposure. After the end of the acclimation period, the flow of air was immediately replaced by pure oxygen at a high flow rate of ~30 l/min for 1 min for fast replacement of the cage's atmosphere, while the flow of CO₂ remained constant. The flow rate was then reduced to 8 l/min, thus restoring the CO₂ concentration within 1 min (a flow of 8 l/min through a 6-liter cage). The EEG signal was amplified and recorded continuously on a chart recorder. The fraction of CO₂ in the inspired gas, ambient temperature, and humidity were read and recorded. The rat was observed through a window in the pressure chamber. When FED, which precedes the clinical convulsions, was seen on the recorder, decompression commenced (at 100 kPa/min). The cage was removed from the pressure chamber, and the rat was freed from the experimental system. The time beginning 12 s from the start of high flow oxygen (30 l/min) until the appearance of FED was noted as the latency to CNS oxygen toxicity.

Experimental protocol. Each rat was subjected to a different exposure (the combination of one PO₂ and one PCO₂) every 2-3 days. In previous studies (3, 4), we showed that the preceding exposure has no effect on the time to FED in the following one if there is a 2-day interval in between. If no FED was recorded within 1 h of commencing the exposure, that exposure was recorded as being without CNS oxygen toxicity. Exposure to hyperbaric oxygen for longer than 1 h may cause other forms of oxygen toxicity like pulmonary oxygen toxicity, which, according to our experience, is expressed by heavy breathing beyond 1 h of hyperbaric oxygen, and we assumed that most of the CNS oxygen toxicity events would have a latent period of <1 h.

Calculations. calculation of the percentage of rats exhibiting the fed as a function of pco₂. All measured data (rat number, PCO₂, latency to the FED in min, or 1 for no-FED) for each of the five PO₂, excluding those obtained at a PO₂ of 507 kPa, were entered into a computer program. For each PO₂, the span of PCO₂ over which FED occurred was determined from the lowest and highest PCO₂ at which a rat exhibited FED. Because PCO₂ values were not grouped under a few selected values but ranged over a continuum, we divided the continuum into a few selected intervals to assess whether the interval selection might influence the final results. Within this range of PCO₂, intervals 5, 6, 7, 8, and 9 were selected one after the other. For each chosen number of intervals, a table was constructed. Each row, representing one rat, had either 1 for no-FED or the number of minutes to FED at the appropriate PCO₂ interval (columns). Because the number of exposures per rat was usually less than the number of intervals, there were empty cells at PCO₂ intervals for which there was no exposure. When two exposures of the same rat happened to have their PCO₂ in a single interval, the last one recorded was entered into the appropriate cell. Thus less exposures were used by the program as the number of PCO₂ intervals decreased because each interval spans a greater range of PCO₂. In a second step, the minutes were converted to +1, and extrapolation and interpolation filled the empty cells. We assumed that the FED would occur at all PCO₂ above the threshold, whereas it would not appear at any PCO₂ below the threshold. Empty cells having +1 at a PCO₂ lower then their own and no data above their own were extrapolated to +1, and empty cells having only 1 at a PCO₂ higher than their own and no data below their own were extrapolated to 1. Empty cells having +1 on both of the neighboring sides were interpolated to +1, and the same was done with 1. The ratio of CNS oxygen toxicity (number of +1 signs) to the total (number of +1 and 1 signs) in each column (PCO₂ interval) and mean PCO₂ were calculated. This allowed us to present the percentage of rats having the FED in the selected PCO₂ intervals.

	RESULTS

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The results for each rat were tabulated in ascending order of PCO₂. Therefore, if a threshold is included in the measured PCO₂ range, no-FED may be expected below the threshold, whereas a latency time will be expected above this threshold. For example, when we take rat 150 in PO₂ of 304 kPa, there was no-FED at PCO₂ of 2.2, 3.8, 5.3, 6.6, and 7.1, whereas latencies of 21 and 38 min are seen at PCO₂ of 7.5 and 9.7 kPa, respectively. Thus the threshold for rat 150 at a PO₂ of 304 kPa is at a PCO₂ between 7.1 and 7.5 kPa. There was a total of 381 hyperbaric exposures (51, 65, 70, 100, 63, and 32 exposures at 253, 304, 355, 405, 456, and 507 kPa PO₂, respectively) in 91 animals. The mean number of exposures per rat was 4.7 ± 3.5 (range = 1-13). The combined mean humidity at the end of the experiment was 46.8 ± 2.6%, and the ambient temperature was 27.5 ± 0.4°C.

The mean latencies to FED were 31.7 ± 10.8, 26.0 ± 7.2, 26.3 ± 13.7, 20.9 ± 9.2, 22.8 ± 11.5, and 26.1 ± 10.9 min at 253, 304, 355, 405, 456, and 507 kPa PO₂, respectively. The difference in the latencies was significant (P < 0.009, ANOVA). The distribution of measured latencies for the four lower PO₂ is shown in Fig. 1 at intervals of 10-min bin. The distribution of the number of measured latencies is largely affected by PCO₂ levels of 0-9.7 kPa as detailed in Experimental protocol. However, at the four PO₂, the number of latencies is reduced from the 25-min bin to the 35-min bin and to the 45-min bin and so forth. Even at the lowest PO₂, most of the CNS oxygen toxicity events occurred within the first 40 min.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Frequency of the latencies to the first electrical discharge (FED) preceding convulsions for 4 PO2. The latencies to the FED from PO2 405 (A), 355 (B), 304 (C), and 253 (D) kPa were grouped into 10-min intervals. The number of latencies in each time interval is presented as a function of the time interval. Only the data from the 4 lower PO2 are shown to confirm that a 1-h exposure limit does not exclude the longest latencies.

We did not use the PCO₂ interval selection for 507 kPa O₂ because virtually all of the animals (91%) convulsed without any CO₂, and, at any level of added CO₂, all of the rats had FED. The cumulative percentage of rats exhibiting FED for all six pressures, as a function of inspired PCO₂, is shown in Fig. 2 for both intervals 5 and 9 PCO₂. The lines connecting the symbols deviate more from a smooth response with the selection of nine intervals because each interval contains less data. But the details of the sigmoid response are better demonstrated by the nine-interval selection. The selection of the number of intervals has little effect on the overall response when the risks at different PO₂ are compared (Fig. 2). As the PO₂ was lowered from 507 to 456 and 405 kPa, the percentage of rats exhibiting FED without addition of CO₂ was reduced from 91% to 62 and 8, respectively, until none convulsed at 355 kPa. At a PO₂ lower than 355 kPa, the risk curve was shifted toward increasing levels of CO₂. The overall cumulative response seems to correspond to a sigmoidal curve.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Percentage of rats that exhibited FED as a function of PCO2 at 6 oxygen pressures when either 5 (A) or 9 (B) intervals of PCO2 were selected. The total number of rats that had FED at the highest PCO2 interval was taken as 100%. Then the percentage of rats with FED in the other PCO2 intervals was calculated. For example, at a PO2 of 304 kPa (A), the rat that did not exhibit FED in PCO2 at the 3- to 4-kPa interval and had FED in the 4- to 5-, 5- to 6-, 6- to 7-, and 7- to 8-kPa intervals. Therefore, of the total of 25 rats (100%) in 304 kPa, the 4% related to this rat is added from the second interval onward.

	DISCUSSION

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The hypothesis that elevated PCO₂ will cause CNS oxygen toxicity at oxygen pressures that do not on their own induce toxicity has been proven correct. Our assumption that there is a PCO₂ threshold for each individual rat, above which the rat will suffer CNS oxygen toxicity and below which it will not, has been validated. Of 381 measurements, there were only four cases in which a rat had FED at a PCO₂ below another PCO₂ at which it did not have FED. However, the difference in PCO₂ for these opposite responses was small (0.7 ± 0.1 kPa), whereas for greater differences in PCO₂ the response of these rats was also in keeping with our assumption.

The sudden switch from no effect to the development of CNS oxygen toxicity may be ascribed to the effect of CO₂ on brain vasodilatation and elevated oxygen pressure together with other effects that may be ascribed to increased acidity. The extensive literature on ROS postulates that acidity enhances ROS production. Acidity affects the release of Fe²⁺ that enhance hydroxyl radical production by the Fenton reaction (6, 11). The hydrogen ion is a reactant in hydrogen peroxide production. The dismutation process is most rapid at an acidic pH, and acidity enhances nitrogen oxide and hydroxyl radical production (22).

In our previous studies on CNS oxygen toxicity in rats, the exposure duration was limited to 1 h to avoid other complications of oxygen toxicity (1, 3, 4). Those studies were conducted at PO₂ above 400 kPa. Because the latency to FED increased as PO₂ decreased (4), it is possible that a condition that did not result in CNS oxygen toxicity within 1 h will produce FED if the exposure lasts beyond the 1-h limit. If, however, latencies to FED longer than 1 h are expected, when we search for the effect of CO₂ near the threshold, latencies close to 1 h may be expected there too. Figure 1 shows that, at all PO₂ in the low range, most of the latencies are below 40 min. The percentages of latencies at the 40- to 50-min bin were only 8, 11, 3, and 12% at a PO₂ of 405, 355, 304, and 253 kPa, respectively; at the 50- to 60-min bin, these percentages were only 6 and 8% at a PO₂ of 355 and 253 kPa, respectively. We therefore conclude that the 1-h limit did not affect the results by excluding longer latencies.

Although the difference in the latencies to FED at the different PO₂ was significant, the values are close to each other because of the additive effect of CO₂ in the low range of PO₂. Thus the difference in latency between 507 and 253 kPa O₂ was only 5.6 min compared with a 19.3-min difference between 709 and 507 kPa O₂ (4). We have previously shown that CO₂ at various PO₂ can reduce the latency to FED to the same minimal level (2), which may explain the similarity here between the various PO₂.

The cumulative probability of CNS oxygen toxicity is most likely not related to random differences but to the different inherent sensitivity of individual rats. We have previously shown that the variability of sensitivity to CNS oxygen toxicity in the rat is greater between than within animals (4, 5). Our data suggest that for each individual rat there is a PCO₂ threshold that remains constant at least over the span of days during which it was determined. To test for individual sensitivity, we selected the rats that had been measured at at least two oxygen pressures. We then scored all PCO₂ thresholds at each oxygen pressure as its PCO₂ interval. PCO₂ threshold scores were analyzed with repeated-measures ANOVA. There was no significant difference between the first and second oxygen pressures, but there was a significant difference between rats (P < 0.0009).

No probability equation was solved for FED at 507 kPa O₂ because virtually all animals (91%) convulsed without any CO₂, and, at any level of added CO₂, all the rats exhibited FED. Thus F₀ was the only parameter derived at 507 kPa O₂. Nonlinear regression was applied to the percentage of rats exhibiting FED as a function of PCO₂. The number of PCO₂ intervals had no significant effect on the solved parameters F₀, N, and P₅₀. The solved FED probability equations for the six PO₂ are shown in Fig. 3. Superimposing Fig. 3 on either panel of Fig. 2 presents a good agreement between data points and the lines. The curve for 507 kPa O₂ (dotted) was calculated from extrapolated parameters. As PO₂ decreased from 507 kPa, the intersection of the sigmoid curve with the ordinate moved down along the percentage of rats that convulsed. With a further reduction in PO₂, the whole sigmoid response shifted along the PCO₂ axis to higher levels. At all PO₂ except 304 kPa, most of the response is completed within a range of 3 kPa CO₂. We do not have an explanation for the broader range at 304 kPa. The PCO₂ above which F starts to rise can be defined by a low value of F and not by a fixed threshold (D in the method). If the value F = 1% is chosen, then the thresholds for PO₂ of 355, 304, and 253 kPa are at PCO₂ of 0.3, 2.4, and 7.1 kPa, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Percentage of rats with central nervous system (CNS) oxygen toxicity as a function of PCO2 (abscissa) and PO2 (values are written on each curve). The lines represent the solution of the FED probability equation. The dotted curve for 507 kPa was calculated with extrapolated parameters because only the intersection with the ordinate is known.

For a quantitative description of the risk of CNS oxygen toxicity as a function of both PCO₂ and PO₂ (in the low hyperoxic range), the parameters of the FED probability equation were plotted against the oxygen pressure (Fig. 4). P₅₀ decreased linearly as the oxygen pressure increased from 250 to 350 kPa and remained stable with further elevation of the PCO₂. F₀ was zero when the oxygen pressure increased from 250 to 350 kPa and increased linearly as the oxygen pressure rose above 350 kPa. N of the term (P₅₀/PCO₂) (on a logarithmic scale) decreased linearly as the oxygen pressure increased from 250 to 350 kPa and remained constant with further elevation of PO₂. These relations can be summarized as follows


Because the physiology causing CNS oxygen toxicity is similar in humans and rats, with the appropriate parameters (as yet unknown), this same model may be used to help define the risk of CNS oxygen toxicity in humans.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   The parameters of the FED probability equation (shown at top) as a function of PO2. Equations that describe the dependent part are also shown. N, power; F0, rats that exhibited FED without added CO2; P50, PCO2 for the half response.

When various agents that affect CNS oxygen toxicity are compared at high and low oxygen pressures, their effect is more prominent at the low pressure. Thus increasing metabolic rate reduced latency to FED more at 450 kPa than at 500 kPa (1), the effect of inspired CO₂ on shortening the latency is greater at 450 kPa than at 500 kPa (2), and the effect of cinnarizine on prolongation of the latency is more prominent at 500 kPa compared with at 600 kPa (5). To compare the effect of oxygen pressure on the sensitivity of CNS oxygen toxicity to CO₂ at the CO₂-affected range, we calculated the slope (change in population with FED/change in PCO₂) of the sigmoid curve at P₅₀ from the derivative of the FED probability equation, and we also measured the slope using the values adjacent to the P₅₀. The slope is shown in Fig. 5 as a function of PO₂. With the exception of the values at 304 kPa, the slope increased as oxygen pressure decreased. That is, at low hyperoxic PO₂ in the PCO₂-affected range, a smaller increase in PCO₂ will place more rats at risk of oxygen toxicity.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   The slope of CNS oxygen toxicity risk (curves in Fig. 3) at PCO2 = P50, as calculated from the FED probability equation (shown at top) and from the measured data adjacent to P50.

Divers using closed-circuit breathing apparatuses, in which the expired CO₂ is absorbed within the system, can be exposed to CO₂ in the inspired gas due to failure of the CO₂ absorption unit. Professional divers have an inherent or acquired tendency to hypoventilate during strenuous underwater work and therefore have elevated alveolar and tissue PCO₂ (16). It is also possible that divers with low sensitivity to CO₂ are at an increased risk of oxygen toxicity (17) as, for example, in nitrox diving (14). When divers encounter increased resistance to their breathing, the level of PCO₂ in their tissues will increase (24).

Closed-circuit or semiclosed-circuit diving apparatuses are designed for operation at oxygen pressures below the level that can cause CNS oxygen toxicity. Because all the studies of the effects of CO₂ on CNS oxygen toxicity have been conducted at oxygen pressures that cause CNS oxygen toxicity without CO₂ in the inspired gas (7, 9, 10, 23), the present study completes the picture for low hyperbaric oxygen. This study demonstrates that at a PO₂ of 355 kPa, which does not cause CNS oxygen toxicity in the resting rat, just a small concentration of CO₂ can turn a normal scenario into a risk situation (Fig. 3). Such a possibility should be considered in divers who are CO₂ retainers (14) or in conditions where high resistance may be expected with elevation of CO₂ (12, 20 , 24).