A Comparative Study of the Antinociceptive Action of Xenon and Nitrous Oxide in Rats

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1 A Comparative Study of the Antinociceptive Action of Xenon and Nitrous Oxide in Rats Akitoshi Ohara, MD, Takashi Mashimo, MD, Ping Zhang, MD, Yoshimi Inagaki, MD, Satoshi Shibuta, MD, and Ikuto Yoshiya, MD Department of Anesthesiology, Osaka University Medical School, Suita, Osaka, Japan We attempted to clarify the mechanism of antinociceptive action induced by xenon and nitrous oxide. Eighty percent of nitrous oxide or 80% xenon was applied to rats inside enclosed clear plastic glass cylinders with their tails protruding for assessment of the tail-flick response to radiant heat. With repeated testing, there was a rapid reduction to nitrous oxide antinociception within 90 min, which was interpreted as development of tolerance, but not to xenon antinociception. Nitrous oxide antinociception was blocked by the intraperitoneal administration of 0.1 or 1.0 mg/ kg yohimbine, but notby1.0or5.0mg/kgl orby5.0or10mg/kg naloxone. Xenon antinociception was not affected by any of these drugs. Yohimbine and L are characterized as a,-adrenoceptor antagonists. Although yohimbine penetrates the blood-brain barrier after systemic administration, L does not penetrate it and act peripherally. Therefore, the results indicate that a,-adrenoceptors, but not opioid receptors, may play a key role in antinociception induced by nitrous oxide in the central nervous system. Furthermore, the mechanism of xenon antinociception differs from that of nitrous oxide because it does not involve either CQ or opioid receptors. Implications: The precise mechanism of antinociceptive action of nitrous oxide and xenon remains unknown. It is still controversial whether an opioid system plays a role in antinociception induced by nitrous oxide. The results of the study showed that antagonism of central oc,-adrenoceptors, but not opioid receptors, reverses the antinociception induced by nitrous oxide but not by xenon, which indicates that q- adrenoceptors may play a key role in nitrous oxide antinociception. (Anesth Analg 1997;85:931-6) T here has been concern that nitrous oxide might be toxic because of its inhibition of cobalamine (1). Perhaps nitrous oxide causes teratogenic effects (2,3) and spinal cord symptoms (4). Xenon has qualities similar to nitrous oxide. Since the efficacy of xenon as an anesthetic for humans was established and reported by Cullen and Gross (5) in 1951, many authors have examined the anesthetic properties of xenon (6-10). These investigators indicate that xenon possesses advantages over nitrous oxide; it probably does not undergo biotransformation, it is nontoxic, it offers rapid induction and recovery from anesthesia, and it is free of cardiovascular side effects. Were it not for its high cost, xenon would be a valuable alternative to nitrous oxide. However, costs may be minimized by using an anesthesia system with minimal fresh gas flow. Luttropp et al. (11) reported that xenon expenditure during two hours of xenon anesthesia via a Accepted for publication June 18, Address correspondence and reprint requests to Takashi Mashimo, MD, Department of Anesthesiology, Osaka University Medical School, Yamadaoka 2-2, Suita, Osaka 565, Japan. Address to mashimo@anes.med.osaka-u.ac.jp. fully automated minimal flow system was 7.6 L in pigs weighing kg. Both nitrous oxide and xenon have a remarkable antinociceptive effect in humans and animals (12-16). We have demonstrated that the equipotent dose of 0.3 minimum alveolar anesthetic concentration of xenon and nitrous oxide significantly increased the pain threshold as measured by using a radiant heat algometer in human volunteers (16). The antinociceptive effects of both xenon and nitrous oxide were greater than those of halothane, enflurane, isoflurane, and sevoflurane, which exerted no significant antinociceptive effect in subanesthetic concentrations (15). The precise mechanism of the potent antinociceptive action of nitrous oxide and xenon remains unknown. It is still controversial whether an opioid system plays a role in the antinociception induced by nitrous oxide. Numerous reports have demonstrated that nitrous oxide produces a dose-related antinociception and that large doses of opioid antagonists reduce its analgesic action (17-22). It is speculated that nitrous oxide could release endorphins or activate endorphin systems in the central nervous system (CNS). In contrast, other reports (23-25) have shown by the International Anesthesia Research Society /97/$5.00 An&h Analg 1997;85:

2 932 OHARA ET AL. ANTINOCICEPTIVE ACTION OF XENON AND NITROUS OXIDE ANESTH ANALG 1997;85:931-6 that the relevant dose of opioid antagonists could not reverse the antinociceptive action of nitrous oxide. We also demonstrated that the clinical dose of naloxone did not reverse the antinociceptive action induced by either nitrous oxide or xenon at subanesthetic concentrations in humans (16). These results may indicate another antinociceptive mechanism irrelevant to the opioid system. Therefore, we conducted the present study to clarify the possible involvement of a nonopioid system in the antinociception induced by xenon or nitrous oxide in rats. Methods The study protocol was approved by our institution s animal care committee. Male Sprague-Dawley rats ( g; Nihon SLC, Hamamatsu, Japan) were obtained 1 wk before each experiment. They were housed under a 12-h light/ dark cycle (lights on at 8:00 AM) and fed rat chow and water ad libifum until they were transported to the laboratory 1 h before the experiments. All experiments were performed between 10:00 AM and 5:00 PM under normal room light and temperature (24-25 C). Nociception was evaluated by the radiant heat tailflick (TF) test. A rat was placed in a clear plastic glass tube having a 300-mL volume, rubber stoppers in both ends, a rostra1 inlet, and a caudal outlet. Xenon/ oxygen, nitrous oxide/ oxygen, or air was delivered through the rostra1 inlet at the rate of 1 L / min. The tail of each rat was allowed to protrude through the cauda1 hole. The TF apparatus (Tail Flick Unit; Ugo Basile, Italy) focuses a beam of radiant heat, generated by a loo-watt projector lamp, on the underside of the tail 5 cm from the tip. A cutoff latency of 12 s was used to avoid tissue damage to the tail. The nociceptive testing was assessed by curtailment or prolongation of the time required to induce TF after applying radiant heat to the skin of the tail. TF tests were performed five times, with 30-s intervals between tests, and the mean of three values was taken as the TF latency. The algometer was adjusted to the thermal energy output, which produces the control TF latency of approximately 4 s. The mean 5 SE of control TF latencies was s. The TF data are expressed as a percentage of the maximum possible effect (%MPE), which was calculated by the formula (trial TF latency- control TF latency) / (cutoff TF latency-control TF latency) x 100. The Antinociceptive Nitrous Oxide Action of Xenon and Twenty-six rats were allocated to three groups as follows: air inhalation group (n = 8), 80% xenon inhalation group (n = 9), or 80% nitrous oxide inhalation group (n = 9). Animals placed in the tubes started to inhale air, and the baseline measurements were made after 30 min inhalation. Thereafter, rats in the xenon group were exposed to 80% xenon/20% oxygen, and in the nitrous oxide group, animals were exposed to 80% nitrous oxide/20% oxygen at a flow rate of 1 L/min for 2 h, and the measurements of trial TF latency were repeated every 15 min. Nitrous oxide or xenon was then discontinued, and 1 L / min of air was started again. Measurements were repeated every 15 min for 1 h after the discontinuation of nitrous oxide or xenon. The rats in the control air group were placed in 1 L/min of air flow for 3 h after the baseline measurement, and measurements were repeated every 15 min. Anesthetic gases and air were given in a randomized and blind fashion. Reversal of Xenon and Nitrous Oxide Antinociception with Various Antagonists One hundred sixteen rats were used in this study. Forty-five rats that inhaled xenon were allocated to five groups according to the study drug as follows: yohimbine 0.1 mg/kg (n = 9) and 1 mg/kg (n = 9), naloxone 5.0 mg/kg (n = 9) and 10 mg/kg (n = 9), or saline (control) (n = 9). Another 71 rats that inhaled nitrous oxide were allocated to eight groups: yohimbine 0.01 mg/kg (n = S), 0.1 mg/kg (n = 9), and 1 mg/kg (n = 9); L mg/kg (n = 9) and 5.0 mg/kg (n = 9); naloxone 5.0 mg/kg (n = 9) and 10 mg/ kg (n = 9); or saline (control) (n = 9). All drugs were dissolved in 0.9% saline shortly before administration, and the injected volume was always 1 ml. After the drug solution was administered intraperitoneally (II ), the animal was placed in the tube. Administration of air was started at a flow rate of 1 L /min, and the baseline measurements were made after 5 min of air inhalation. Administration of 80% xenon/ 20% oxygen or 80% nitrous oxide / 20% oxygen was then started, and the second measurements were made after 30 min of inhalation. Drugs were randomly administered. The concentration of nitrous oxide was monitored using a mass spectrometer (Perkin-Elmer Cetus, Norwalk, CT), and xenon was monitored using a Thermomat (Fuji Electric, Tokyo, Japan), which is a thermal conductivity gas monitor with a precision of within 1%. Xenon gas (>99.995%) was a gift from Kyodo Oxygen Company (Wakayama, Japan). Yohimbine and naloxone were obtained from Sigma Chemical Company (St. Louis, MO). L was a gift from Merck & Co., Inc. (Whitehouse Station, NJ). Values were expressed as the means 5 SE. Data among groups were analyzed by using one-way analysis of variance and assessed by using Scheffe s test. Data within each group were analyzed by using a

3 ANESTH 1997;85,931-6 ANALG OHARA ET AL. 933 ANTINOCICEPTIVE ACTION OF XENON AND NITROUS OXIDE two-way analysis of variance with repeated measurements and assessed by using the paired t-test. A P value of less than 0.05 was considered statistically significant. Results Slight or moderate excitation was observed for 5-15 min after the start of inhalation of nitrous oxide, followed by a sedative and immobile state. After the inhalation of xenon, however, no excitation was observed in animals. During the period of measurements, rats were immobile in the sedative state in both of the anesthetic gas groups. During inhalation of 80% xenon and 80% nitrous oxide, blood gas analysis data did not show either hypoxemia or hypercapnia. The mean values of ph, Po2, and Pco, were 7.433, 87.8 mm Hg, and 44.3 mm Hg, respectively, for xenon (n = 3) and 7.381,89.2 mm Hg, and 42.1 mm Hg, respectively, for nitrous oxide (72 = 3). %MPE during xenon inhalation did not change through the experiment, and the average %MPE was 90.3? 5.3 (Fig. 1). After discontinuation of xenon, the %MPE decreased close to the baseline shortly. The %MPE increased significantly during 80% nitrous oxide inhalation and showed the maximum increase of min after the start of nitrous oxide. It gradually decreased to at the end of 80% nitrous oxide inhalation. The values of %MPE at 90, 105, and 120 min after the start of nitrous oxide inhalation were significantly less than the maximal %MPE, which was reached at 30 min. The %MPE transiently but not significantly increased by min after discontinuation of 80% nitrous oxide and then decreased to the baseline. The %MPE did not change during air inhalation. The TF time after II administration of yohimbine, L , or naloxone was not different from that with control saline before xenon or nitrous oxide inhalation. The %MPE after II administration of yohimbine or naloxone did not differ from that with saline control during xenon inhalation (Fig. 2). On the other hand, the %MPE during 80% nitrous oxide inhalation significantly decreased with yohimbine in a doserelated manner. It decreased to 11? 4.6 and (P < ) with yohimbine at 0.1 and 1.0 mg/kg, respectively. However, the %MPE did not change with L or naloxone. Discussion The present study shows that the antinociceptive effects of nitrous oxide peaked 30 min after the start of inhalation, but tolerance to nitrous oxide developed B Time (min) N20 (80%) or Air- Air loo? go{ q-& + t N20 t Air / I / I / Time (min) Figure 1. Time course of the effect of xenon (A) or nitrous oxide (B) inhalation on the latency of the thermal nociceptive tail-flick reflex in rats. + = maximum possible effect (%MPE) during inhalation of 80% xenon/20% oxygen (n = 9), and %MPE of 80% nitrous oxide/ 20% oxygen (n = 9). n = %MPE of air (control) (n = 8). All values are mean i SE. P c 0.05, significantly different from the value 30 min after the start of xenon or nitrous oxide inhalation. +P < , $P < 0.001, significantly different from air control group. within 90 minutes in rats (Fig. 1). Tolerance to antinociceptive effects of nitrous oxide has been established in animals (X3,26,27) and humans (28,29). Rupreht et al. (27) demonstrated the development of tolerance and its prevention by pretreatment with enkephalinase inhibitor, suggesting that tolerance may be due to an acute depletion of endorphins. Ngai and Finck (30) suggested that a decrease in opioid receptor density resulting from exposure to nitrous oxide might explain the development of tolerance. However, a decrease in opioid receptor density was produced by prolonged exposure of nitrous oxide for 18 hours, not by short-term exposure for 30 minutes. Because the time required for the development of tolerance to the antinociceptive effect of nitrous oxide ranged from 15 minutes to 2.5 hours, a decrease in opioid receptor density cannot explain the tolerance. Other mechanisms might be involved in the tolerance to nitrous oxide antinociception.

4 934 OHARA ET AL. ANTINOCICEFTWE ACTION OF XENON AND NITROUS OXIDE ANESTH ANALG 1997;85:931-6 OV I Control Yohimbine Naloxone Control Yohimbine L659,066 Naloxone Figure 2. Effects of intraperitoneal administration of saline (control), yohimbine, L , or naloxone on maximum possible effect (%MPE) during inhalation of 80% xenon/20% oxygen (A) and 80% nitrous oxide/20% oxygen (B) in rats. All values are mean + SE. +P < , significantly different from the maximum possible effect of control. Shingu et al. (31) reported that acute tolerance did not develop to nitrous oxide antinociception in rats using a TF latency test, a similar experimental variable. In contrast, others have demonstrated the development of acute tolerance to nitrous oxide actions on electroencephalograph activity and the wake-sleep cycle in cats (32), and the development of tolerance to an anticonvulsant action of nitrous oxide in cats (33). They speculate that tolerance to all actions of nitrous oxide may not always occur because different actions involve different neuronal pathways and/or different neurotransmitters. Nitrous oxide produces good antinociceptive action, but the antinociceptive properties of xenon first observed by Lachmann et al. (14) have not been fully investigated. Quantitative antinociceptive assay using tooth pulp evoked potentials showed a dosedependent antinociception during xenon inhalation in pigs. Xenon had a more potent antinociceptive effect compared with the same concentrations of nitrous oxide in concentrations between 50% and 70%. We also reported that the subanesthetic concentration of xenon produced antinociception comparable to nitrous oxide in human volunteers (16). The present study demonstrated that xenon exerted potent antinociceptive action in rats, and no animal showed a tendency to develop tolerance to xenon during the inhalation. These results indicate that the antinociceptive action of xenon may result from mechanisms different from those of nitrous oxide. The results of the reversal study show that yohimbine antagonized the antinociceptive action induced by nitrous oxide, but that neither L nor naloxone did. In contrast, none of these antagonists affected the antinociceptive action induced by xenon (Fig. 2). Yohimbine and L are characterized as potent a,-adrenoceptor antagonists. The doses of yohimbine and L used in this study were consistent with those that antagonized the analgesic effect elicited by clonidine (34). Although yohimbine penetrates the blood-brain barrier after systemic administration, L does not; it instead acts peripherally (35). Therefore, the results clearly indicate that the cr,-adrenoceptor, but not the opioid system, plays a key role in the antinociception induced by nitrous oxide in the CNS. Guo et al. (36) recently reported that antinociceptive action of nitrous oxide is mediated by the spinal a,-adrenoceptor in addition to the supraspinal opioid receptor in the rat. The present results also suggest that either the endorphin system or the ct!!2- adrenoceptor system may not be involved in antinociception induced by xenon. a,-adrenoceptor activation seems to produce a potent antinociceptive response involving both supraspinal and spinal sites. Fleetwood-Walker et al. (37) and Jones and Gebhart (38) showed that a,-adrenoceptors play an important role in the antinociceptive actions of descending noradrenaline tracts. An electrophysiological study also demonstrated that medetomidine, a highly selective cy,-adrenoceptor agonist, suppresses the nociceptive reflex response through both spinal and supraspinal mechanisms (39). (Y* Binding sites are dense in the substantia gelatinosa of the spinal cord dorsal horn (40). Nicholas et al. (41) indicated that $Aand ozc- receptors located in dorsal ganglia may participate in spinal antinociception. The anatomical sites involved in the supraspinal antinociceptive action of a,-adrenoceptor agonists have not been determined, and they may involve more than one receptor subtype (42). The present results showed that naloxone, administered II at 5 mg/kg or 10 mg/kg, did not antagonize nitrous oxide antinociception in rats. The doses of naloxone used in this study far exceed those required to antagonize the action of opiates at their receptors in

5 ANESTH ANALG OHARA ET AL ;85:9314 ANTINOCICEPTIVE ACTION OF XENON AND NITROUS OXIDE rats (43), which suggests that the antinociceptive action of nitrous oxide may not be mediated through opioid receptors. It is, however, still controversial whether an opioid system plays a role in antinociception induced by nitrous oxide. Our previous study (16) showed that the clinical dose (0.01 mg/kg) of naloxone did not reverse the antinociceptive action of nitrous oxide in humans. Other human and animal studies also demonstrated that a relevant dose of opioid antagonists could not reverse nitrous oxide-induced antinociception (23-25). Large doses of naloxone were used in the studies, which support the reversal effect of opioid antagonists (17-22). Larger doses (5-30 mg/ kg) of naloxone antagonized nitrous oxide antinociception in rats, but smaller doses of the antagonist were not effective (17). Larger doses of naloxone might be required to block the effects of endogenous opioids produced by nitrous oxide compared with exogenous opioids, which is likely explained by opioid receptor multiplicity. Moody et al. (22) also demonstrated the stereoselectivity of naloxone antagonism of nitrous oxide antinociception as evidence of opioid receptor involvement. The existence of a close interaction between central a-adrenergic systems and central opioid systems has become apparent in recent years. Clonidine suppresses neuron firing elicited by morphine withdrawal in the locus coeruleus (44). This indicates that some neurons in the CNS are under the control of both a-adrenergic and opioid receptors (45). Post et al. (46) demonstrated the existence of cross-tolerance to the antinociceptive effects of morphine and c+adrenoceptor agonists (i.e., norepinephrine and clonidine) in rats and suggested that there is a substantial opioid-adrenoceptor interaction in the antinociceptive processes. Flacke et al. (47) reported that clonidine prevents the adrenergic response to opioid reversal with naloxone in dogs. These various findings lead us to believe that naloxone may influence the a,-adrenoceptor system through an opioidadrenoceptor interaction. Interaction between the central opioid and adrenergic pathways may explain the partial reversal of nitrous oxide antinociception by naloxone reported by several investigators (17-22). The present data show that acute tolerance to the antinociceptive effect rapidly develops with nitrous oxide but not with xenon, and that antagonism of central a,-adrenoceptor reverses the antinociception during inhalation of nitrous oxide but not of xenon. This indicates that the a,-adrenoceptor, but not the opioid receptor, may play a key role in the antinociception induced by nitrous oxide in the CNS, and that the mechanism of xenon antinociception may be different from that of nitrous oxide. 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