Research has suggested that nitrous oxide may be harmful to ischemic neurons; however, the evidence for this is equivocal. The authors used rat hippocampal slices to examine the effects of nitrous oxide on neuronal hypoxic damage.
The evoked population spike (PS) was recorded from hippocampal CA1 pyramidal cells before, during, and after hypoxia. Control groups received nitrogen concentrations equal to nitrous oxide throughout the experiments. Biochemical measurements were made from dissected CA1 regions under experimental conditions that matched the electrophysiology studies.
Recovery of the PS after hypoxia was 18 +/- 7% in slices treated with 50% nitrous oxide before and during 3.5 min of hypoxia; this compares with 41 +/- 9% (P < 0.05) in nitrogen-treated slices. Slices treated with nitrous oxide (95%) only during hypoxia (6 min) also demonstrated significantly less recovery of the PS than did slices treated with nitrogen. There was no significant difference in recovery if nitrous oxide was discontinued after the hypoxic period. Adenosine triphosphate concentrations after 3.5 min of hypoxia in slices treated with nitrous oxide decreased to the same extent as in nitrogen-treated slices (47% vs. 50%). Calcium influx increased during 10 min of hypoxia in untreated slices, but nitrous oxide did not significantly increase calcium influx during hypoxia. The sodium concentrations increased and potassium concentrations decreased during hypoxia; nitrous oxide did not significantly alter these changes.
Nitrous oxide impaired electrophysiologic recovery of hippocampal slices after severe hypoxia. Nitrous oxide did not cause significant changes in the biochemical parameters examined.
Key words: Anesthetics, inhalational: nitrous oxide. Brain, hippocampal slice: ischemia; anoxia; adenosine triphosphate; calcium; sodium; potassium. Ions: sodium; potassium; calcium. Electrophysiology: extracellular recording; evoked population spike.
Some of the debate over the use of nitrous oxide in neuroanesthesia has centered on its neurophysiologic effects and whether it worsens cerebral ischemia. [1-4]In some studies, nitrous oxide appears to enhance anoxic and ischemic damage, whereas in other studies no effect of nitrous oxide on damage was seen. [5-10].
We used a rat hippocampal slice to study the effect of nitrous oxide on hypoxic neuronal damage. We examined electrophysiologic recovery after exposure to hypoxia to determine if nitrous oxide had a detrimental effect. In addition, we measured adenosine triphosphate (ATP), sodium, and potassium concentrations and calcium influx to investigate the possible mechanism of nitrous oxide's effect.
Materials and Methods
The experiments were approved by the Institutional Animal Care and Use Committee of the State University of New York-Health Science Center at Brooklyn. The methods have been reported before. [11,12]Adult (110-120 days old) male Sprague Dawley rats were decapitated and the brains rapidly removed and dissected to obtain the hippocampi. Each hippocampus was sliced transversely to its long axis so that several 500-micro meter-thick slices were obtained. The slices were secured to nylon mesh mounted on a plexiglass grid. To minimize ischemic damage during the preparation, the tissue was kept cold (4 [degree sign] Celsius) in artificial cerebral spinal fluid (aCSF) throughout these steps. The aCSF was composed of 126 mM NaCl, 3 mM KCl, 1.4 mM KH2PO4, 1.3 mM MgSO4, 1.4 mM CaCl2, 26 mM NaHCO3, and 4 mM glucose, pH 7.4. For the electrophysiologic studies, a grid holding several slices was placed in a chamber where the slices were superfused continuously with aCSF at a rate of 60 ml/min. The aCSF was aerated with 95% oxygen and 5% carbon dioxide, and the temperature in the tissue chamber was maintained at 37 [degree sign] Celsius. After incubating the slices for 60 min, a bipolar metal electrode was used to stimulate the Schaffer collateral pathway supramaximally. A single tungsten electrode was placed in the cell body layer of the CA1 region to record a postsynaptic population spike (PS) from the CA1 pyramidal cells (Figure 1). After a stable response was obtained, the slice was stimulated every 10 s with the parameters of stimulation and the position of the electrodes unchanged. The electrophysiologic responses were quantified by measuring the amplitude of the postsynaptic population spike. This response is a negative going field potential and is measured as the average of the difference in millivolts between the first peak and the trough (Figure 1(A to B)) and the difference between the second peak and the trough (Figure 1(C to B)). Signal acquisition and measurements were performed using a 2801A AD converter (Data Translation, Marlboro, MA) with Snapshot Storage Scope software (HEM Data Corp., Southfield, MI). Measurements were taken as the average of five consecutive readings. Untreated slices that are continuously superfused with aCSF aerated with 95% oxygen and 5% carbon dioxide maintain stable responses (+/- 10%) 1-5 h after slices are prepared. .
Hypoxia was generated by superfusing the slice from a reservoir containing aCSF that had been pre-equilibrated with a gas mixture containing no oxygen and 5% carbon dioxide. This reduced oxygenation in the aCSF from 646 +/- 15 to 61 +/- 5, 38 +/- 3, 30 +/- 3, and 30 +/- 2 mmHg after 1, 2, 3, and 6 min of changing to aCSF equilibrated with 95% nitrogen (n = 5 independent runs for all oxygen measurements), respectively. The oxygen concentration within the slice is lower than that in the aCSF and decreases from the slice surface to its interior. This is severe hypoxia. After the hypoxic period, normoxic superfusion (95% oxygen) was resumed and the slices were allowed to recover for 60 min. The ability of the slices to recover from the hypoxic insult was evaluated by dividing the amplitude of the first spike of the population spike complex measured 60 min after hypoxia by its prehypoxic, predrug amplitude and is expressed as a percentage. Recovery was assessed after 60 min of reoxygenation; prolonging the reoxygenation up to 3 h does not affect recovery (unpublished observations).
Nitrous oxide was bubbled into the reservoir containing aCSF from gas tanks containing analyzed gas mixtures supplied by the Liquid Carbonic Corporation (Oak Brook, IL). The concentrations of nitrous oxide, oxygen, and carbon dioxide were measured in a branch of the tubing coming from the gas tank just before it aerates the aCSF throughout the experiments with a Datex 254 airway monitor. Nitrous oxide was tested in two concentrations, 50% and 95%, and its effect was evaluated by comparing it with control groups that received nitrogen instead of nitrous oxide. The oxygen concentration was the same in nitrous oxide and control groups in all experiments at all times. Experimental designs differed according to the time of exposure to nitrous oxide and its concentrations. All mixtures included 5% carbon dioxide as part of the buffering system.
In experimental series 1, slices were exposed to reduced oxygen (45%) and either 50% nitrous oxide or 50% nitrogen for 30 min followed by 95% oxygen for 60 min; in this experiment, the slices were not exposed to hypoxia. In experimental series 2, the slices were treated with 50% nitrous oxide and 45% oxygen (reduced oxygen) for 30 min, followed by 3.5 min of hypoxia with 50% nitrous oxide and 45% nitrogen and 60 min of reoxygenation with 95% oxygen. In experimental series 3, the slices were exposed to 50% nitrous oxide and 45% nitrogen only during 4.5 min of hypoxia, whereas experimental series 4 exposed slices to 6 min of hypoxia with 95% nitrous oxide. In both experimental series 3 and 4, the gas concentration changed directly from 95% oxygen to the hypoxic mixture (hypoxia directly). In the fifth experimental series, the slices were treated with 50% nitrous oxide (reduced oxygen) for 30 min before hypoxia, during 3 min of hypoxia, and for 30 min after hypoxia, followed by 95% oxygen for 60 min. In this experiment, the control group also received 50% nitrous oxide before (reduced oxygen) and during hypoxia but was replaced by nitrogen after the hypoxic period.
For biochemical studies, rat hippocampal slices were obtained as described for the electrophysiology experiments. Plexiglass grids containing slices obtained from one animal were placed in beakers containing aCSF. The aCSF was aerated with 95% oxygen and 5% carbon dioxide. The beakers were placed in a water bath and maintained at 37 [degree sign] Celsius. Hypoxia was generated by aerating the beakers with gas mixtures containing no oxygen; all mixtures had 5% carbon dioxide. The experimental design of the biochemical experiments matched that described for the electrophysiology studies and included several experimental groups. There was always a group with slices that received no drug and remained normoxic.
Adenosine triphosphate was measured in slices incubated as described above and subjected to periods of 3.5 min, 5 min, and 6 min of hypoxia. Before hypoxia, the slices were treated with 50% nitrous oxide or 50% nitrogen and 45% oxygen (reduced oxygen) for 30 min, as in the electrophysiology series 2 experiments. During hypoxia, the slices were treated with either nitrous oxide (50%) and nitrogen (45%) or with nitrogen (95%). Adenosine triphosphate concentrations were also measured in slices that were subjected to hypoxia without prior exposure to a reduced oxygen concentration (hypoxia directly), as in electrophysiology series 3 and 4. After hypoxia, or an equivalent time period, the slices were rapidly frozen in liquid nitrogen and lyophilized. The dry tissue was dissected to obtain the CA1 region extending from the stratum radiatum to the alveus, including the CA1 pyramidal cell layer. The dissected CA1 regions were then weighed. Adenosine triphosphate was extracted by homogenizing the tissue in 3 N ice-cold perchloric acid and measured after neutralization using the firefly luciferin-luciferase assay. .
Calcium influx was measured using radioactive tracers. Radioactive calcium (sup 45 Ca, 0.5 micro Ci/ml) was added to each group for 10 min at the beginning of each treatment period. Control slices were incubated in45Ca for 10 min during normal oxygenation. Because this technique is not sensitive to brief periods of hypoxia, 10 min of hypoxia was used. The slices were treated with nitrous oxide (50%) or nitrogen (50%); (reduced oxygen) for 30 min and then subjected to 10 min of hypoxia with either nitrous oxide (50%) and nitrogen (45%) or nitrogen (95%). At the end of the experiment, the slices were taken from the beakers and immediately placed in agitated ice-cold modified aCSF containing 2 mM LaCl3for 20 min. This was done to wash out the extracellular45Ca while preserving intracellular45Ca. Modified aCSF, which was used to prevent precipitation of the lanthanum, contains 158 mM NaCl, 4 mM KCl, 1.3 mM MgSO4, and 2 mM LaCl3. The slices were then taken from the La wash, blotted, frozen in liquid nitrogen, and lyophilized. The CA1 regions were dissected, weighed, digested in 70% HNO3, diluted with water, and counted in a liquid scintillation counter. .
Sodium and potassium concentrations were measured in the dissected CA1 region after 6 min of hypoxia. The slices were treated as described for the calcium measurements until the end of the experimental period, when they were removed from the beakers and submerged in agitated ice-cold isotonic sucrose for 10 min to wash ions from the extracellular space. The sodium washout time had been evaluated previously by measuring the amount of sodium left in the tissue after different washout periods. Ten minutes in sucrose solution allowed the washout of extracellular sodium with only a minimal effect on intracellular concentrations. The slices were dissected, and the CA1 regions obtained from slices on the same plexiglass grid (5 slices per grid) were pooled and placed in preweighed tubes, dried at 90 [degree sign] Celsius for 18 h, and weighed. After dilute nitric acid (0.1 N) was added, the tissue was shaken for 18 h and the supernatant, after centrifugation, was assayed using a flame photometer.
All values are expressed as mean +/- SEM. Statistical analysis was performed using analysis of variance and two-tailed unpaired Student's t tests. If the standard deviations of the groups tested were significantly different (by the F test), the Welch test was used. The significance level was P < 0.05 unless otherwise indicated. Instat2 from GraphPad Software (San Diego, CA) was used for statistical analysis.
In electrophysiology series 1 studies, the effect of nitrous oxide was examined in tissue not subjected to hypoxia. Slices treated with either 50% nitrous oxide or 50% nitrogen, combined with 45% oxygen, had a rapid and marked decrease in the population spike amplitude; the amplitude of the population spike decreased to about 24% of its amplitude in 95% oxygen in both groups (Figure 2). When the oxygen was subsequently increased to 95%, there was approximately 75% recovery of the population spike amplitude. The amplitude of the population spike in slices treated with nitrous oxide was not significantly different from that in slices treated with nitrogen (Figure 2).
In series 2, the effect of nitrous oxide before and during hypoxia was examined. In the period before anoxia, slices treated with 50% nitrous oxide and 45% oxygen showed a significant reduction of the population spike amplitude to 16 +/- 5% of normoxic levels (95% oxygen); slices treated with 50% nitrogen and 45% oxygen also showed a reduction in population spike amplitude (22 +/- 5%; Figure 3). There was no significant difference between the nitrous oxide- and the nitrogen-treated slices before hypoxia. During hypoxia, the population spike was blocked after 135 +/- 7 s in the nitrous oxide group and after 136 +/- 7 s in the nitrogen group (NS). After return to 95% oxygen, slices subjected to nitrous oxide had significantly less recovery of the population spike: recovery was 18 +/- 7% in nitrous oxide-treated slices and 41 +/- 9% in nitrogen-treated slices (Figure 3(A)). Examples of the individual recordings from one nitrous oxide and one nitrogen control experiment are shown in Figure 3(B).
In experimental series 3, slices were subjected to 50% nitrous oxide only during the hypoxia period. The population spike recovered to 47 +/- 11% of its prehypoxia/pretreatment amplitude, when slices were exposed to 50% nitrous oxide during 4.5 min of hypoxia (Figure 4). Control slices that received nitrogen recovered to 74 +/- 11% (Figure 4); the difference between the nitrous oxide and the nitrogen groups was not significant (P = 0.1). The blockage of the population spike during hypoxia occurred at 192 +/- 7 s in the slices subjected to nitrous oxide, and at 218 +/- 13 s in those subjected to nitrogen (NS). In four experiments from each group, the population spike was not blocked during hypoxia.
When slices were treated with 95% nitrous oxide only during a 6-min period of hypoxia (series 4), the population spike recovered to 17 +/- 7% of its prehypoxic/pretreatment amplitude; this was significantly less than recovery in control slices that were treated with nitrogen (44 +/- 10%; Figure 5). The blockage of the population spike during hypoxia occurred significantly earlier in the nitrous oxide-treated slices (176 +/- 16 s vs. 225 +/- 16 s).
The effect of discontinuing nitrous oxide after hypoxia was examined in experimental series 5. Slices treated with 50% nitrous oxide for 30 min before hypoxia, during 3 min of hypoxia, and for 30 min after hypoxia showed 37 +/- 9% recovery of the population spike 1 h after reoxygenation with 95% oxygen (Figure 6). Replacing nitrous oxide with nitrogen immediately after hypoxia, resulted in 22 +/- 8% recovery; these groups were not significantly different (Figure 6).
Adenosine triphosphate concentrations measured after 30 min of exposure to either 50% nitrous oxide or 50% nitrogen, combined with 45% oxygen, were significantly reduced to about 80% of their concentration in 95% oxygen (reduced oxygen, Table 1). The ATP concentrations measured in nitrous oxide-treated slices were not significantly different from those measured in nitrogen-treated slices. Slices subsequently subjected to hypoxia showed a further reduction in ATP levels (Table 1). Adenosine triphosphate levels measured after 3.5, 5, and 6 min of hypoxia were not significantly different when slices treated with nitrous oxide were compared with slices treated with nitrogen for the same hypoxic times. Slices that underwent a period of 6 min of hypoxia with either nitrous oxide or nitrogen, but were not exposed to these agents before hypoxia (hypoxia directly), had ATP concentrations that were also not significantly different from each other (Table 1).
Calcium uptake in slices treated with nitrous oxide before and during a period of 10 min of hypoxia was significantly increased to 127% its of normoxic uptake (Table 2); nitrogen-treated slices also had a significant increase in calcium uptake (123% of normoxic uptake). These groups were not significantly different from each other. Calcium uptake in untreated slices subjected to 10 min of hypoxia without previous exposure to a reduced oxygen concentration (hypoxia directly) was 118% of normoxic uptake; this was not significantly different from calcium uptake in untreated slices subjected to the same period of hypoxia after 30 min of exposure to 45% oxygen (123%; reduced oxygen, Table 2). Slices treated with 50% nitrogen and 45% oxygen for 30 min and not subjected to hypoxia did not have a significant change in calcium uptake when compared with normoxic slices (101% of normoxic uptake; Table 2).
Intracellular sodium concentrations, measured in slices treated with 50% nitrous oxide and 45% oxygen before and with 50% nitrous oxide and 45% nitrogen during 6 min of hypoxia were significantly increased to 245% of their normoxic concentration (Table 3). Sodium increased significantly to 219% of normoxic concentration after 6 min of hypoxia in slices treated with nitrogen. The difference between nitrous oxide- and nitrogen-treated slices was not significant.
Intracellular potassium concentrations, measured in slices treated with nitrous oxide before and during 6 min of hypoxia, showed a significant reduction to 68% of those in normoxic slices (Table 4). Potassium also decreased significantly after hypoxia in slices treated with nitrogen (78% of normoxic concentrations). Potassium concentrations measured at the end of hypoxia in nitrous oxide-treated and nitrogen-treated slices were not statistically different.
Electrophysiologic recovery after anoxia was examined in hippocampal slices treated with nitrous oxide before and during and only during the hypoxic insult. The inclusion of nitrous oxide in the gas mixture used to aerate the aCSF required a decrease in the oxygen concentration before hypoxia. Because oxygen delivery to the slice occurs by diffusion from the aCSF, decreasing the oxygen concentration reduces the oxygen gradient through the slice and may compromise the viability of cells located in the core of the slice. We chose to study 50% nitrous oxide, because it allowed us to use a concentration of oxygen (45%) that only moderately compromised cell viability. Because the slices were exposed to a reduced concentration of oxygen before hypoxia, we examined short durations of hypoxia; 3.5 min of hypoxia produced about 50% recovery in untreated slices and allowed us to examine beneficial and detrimental effects of nitrous oxide. The duration of treatment with nitrous oxide before hypoxia (30 min) permitted sufficient time for the drug to exert its effects and enabled us to obtain stable recordings before hypoxia. Under these conditions, we showed that nitrous oxide impaired electrophysiologic recovery after hypoxia.
Because exposure to a reduced oxygen concentration before hypoxia influenced the outcome, we examined slices treated with nitrous oxide only during the hypoxic period. Fifty percent nitrous oxide, when present during 4.5 min of hypoxia, had no significant effect on recovery (P = 0.1). Therefore, we examined a higher concentration of nitrous oxide (95%) during 6 min of hypoxia, which resulted in significant impairment (P < 0.05). The impaired electrophysiologic recovery after hypoxia in this experiment had to be due to a direct effect of nitrous oxide during hypoxia.
The decrease in the amplitude of the population spike in the period before hypoxia was the same in slices treated with nitrous oxide and slices treated with nitrogen. Therefore, this decrease was almost certainly caused by the reduced oxygen concentration and not by nitrous oxide. There may well have been electro-physiologic changes induced by nitrous oxide, as seen by others, [16,17]and the changes due to hypoxia may have occluded our ability to observe them.
We evaluated what happened if slices treated with nitrous oxide and 45% oxygen, instead of being subjected to hypoxia, were returned to 95% oxygen. With restoration of the initial oxygen concentration, the population spike recovered to the same extent in both nitrous oxide- and nitrogen-treated slices. This indicates that nitrous oxide does not cause any additional permanent electrophysiologic changes in neurons not subjected to hypoxia.
Discontinuing nitrous oxide after a hypoxic episode did not improve recovery of the population spike. This result reinforces our hypothesis that nitrous oxide impairs recovery through an effect that occurs during the hypoxic period. This finding may be important: It suggests that if hypoxia occurs while neurons are exposed to nitrous oxide, we might expect it to result in neuronal changes that will not be reversed when nitrous oxide is discontinued. These experiments also showed that posthypoxic exposure to nitrous oxide does not increase damage, which corresponds with our finding that nitrous oxide does not alter recovery in nonhypoxic tissue.
We postulated that nitrous oxide might exert its negative effect on electrophysiologic recovery by increasing the decrease in ATP during anoxia. Nitrous oxide increases CMRO2in vivo [18-22]and could increase neuronal metabolism and energy demand, exacerbating the energy imbalance during hypoxia. Previously we showed a correlation between ATP concentrations at the end of anoxia and recovery of the evoked response. [23,24]For the three durations of hypoxia tested (3.5, 5, and 6 min), ATP levels in slices treated with nitrous oxide were not significantly lower than in nitrogen-treated hypoxic slices. Our results show that the impaired electrophysiologic recovery in nitrous oxide-treated slices is not due to alterations in ATP concentrations.
When the oxygen concentration was reduced to 45% before hypoxia, ATP concentrations were reduced to about 80% of normoxic levels. During hypoxia, ATP concentrations decreased further to about 40%. Slices exposed to 45% oxygen had reduced energy stores and increased sensitivity to hypoxia. This may be why ATP concentrations after a brief period of hypoxia (3.5 min) in slices previously exposed to 45% oxygen were similar to the concentrations measured after longer periods of hypoxia (6 min) in slices that underwent hypoxia without prior exposure to 45% oxygen (Table 1).
One possible effect of nitrous oxide is an increase in energy use and thereby a decrease in the concentration of oxygen in the neurons. The concentration of ATP in the slice during hypoxia is a sensitive indicator of the oxygen concentration; when oxygen concentrations decrease. ATP levels decrease concomitantly. In this study there was no significant difference in the ATP concentrations between nitrogen and nitrous oxide, and thus it appears that nitrous oxide does not significantly enhance oxygen use or metabolism.
Increased intracellular concentrations of calcium during hypoxia have been implicated as a cause of irreversible neuronal damage. Because uptake from the extracellular space plays an important role in this increase, we examined nitrous oxide's effect on calcium uptake. Calcium uptake was significantly increased during 10 min of hypoxia. However, there were no differences between nitrous oxide- and nitrogen-treated slices. We conclude that nitrous oxide does not impair electrophysiologic recovery by increasing the uptake of calcium from the extracellular space.
During hypoxia, the increase in intracellular sodium concentrations and the decrease in intracellular potassium concentrations correlate with the amount of damage. [26-28]Drugs that block sodium entry and potassium depletion have been shown to improve recovery after hypoxia. [15,27,28]Therefore, we studied the effect of nitrous oxide on intracellular sodium and potassium concentrations. There were no significant differences in sodium and potassium concentrations between nitrous oxide-treated and nitrogen-treated slices, and thus the reduction in population spike recovery with nitrous oxide cannot be explained by an effect on the concentrations of these ions.
The block of the population spike occurred more rapidly in nitrous oxide-treated slices in the experiment in which the drug was present for 6 min during hypoxia. In the other experiment in which nitrous oxide impaired recovery, hypoxia lasted 3.5 min, which may have been too short to evaluate the block of the population spike. In this experiment, the population spike amplitude in the nitrous oxide-treated slices was significantly lower than in the nitrogen-treated slices after 2 min of hypoxia (Figure 3), suggesting that nitrous oxide could be accelerating the blockage of the signal during hypoxia. This could be due to a more rapid hypoxic depolarization and may help explain the effect of nitrous oxide. However, we did not directly measure hypoxic depolarization, so this inference must remain tenuous.
Nitrous oxide did not exacerbate the changes in ATP, sodium, potassium, or calcium during hypoxia, and thus these biochemical parameters appear not to be the mechanisms by which nitrous oxide enhances damage. Because nitrous oxide has been shown to enhance excitation, [18,22]it may exert its harmful effects through increased activity of excitatory pathways or decreased activity of inhibitory pathways.
Nitrous oxide poses important difficulties to in vivo or clinical studies, because for ethical reasons it cannot be compared with a control group not receiving an anesthetic. An in vitro model may offer advantages, because nitrogen can be used to replace the anesthetic in control groups. To date, all the studies addressing the effect of nitrous oxide in relation to ischemic or hypoxic damage included control groups that received an anesthetic drug. The only study that focused specifically on nitrous oxide included the administration of a large dose of a barbiturate to all the animals; that study probably did not demonstrate a harmful effect of nitrous oxide due to the protection against damage provided by the barbiturate. Our study is the only one to address the effect of nitrous oxide using a control group that was not given an anesthetic.
Nitrous oxide impaired electrophysiologic recovery from hypoxia in the rat hippocampal slice. This effect was observed when 50% nitrous oxide was present before and during hypoxia or when 95% nitrous oxide was present only during hypoxia. Discontinuing nitrous oxide after hypoxia did not improve recovery.