Evidence for the Involvement of Spinal Cord α¹Adrenoceptors in Nitrous Oxide–induced Antinociceptive Effects in Fischer Rats

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WE have sought to characterize the molecular mechanism and neural substrates involved in the antinociceptive action of nitrous oxide (N²O). In brief, N²O induces opioid peptide release in the brain stem, leading to the activation of the descending noradrenergic inhibitory neurons, which results in modulation of the pain–nociceptive processing in the spinal cord. 1Available evidence suggests that at the level of the spinal cord, there appear to be at least two neuronal systems that are involved (fig. 1). In one of the pathways, activation of the α²adrenoceptors produces either direct presynaptic inhibition of neurotransmitter release from primary afferent neurons or postsynaptic inhibition of the second-order neurons. 1In a second hypothetical pathway, we propose that inhibitory γ-aminobutyric acid–mediated (GABAergic) interneurons are activated via α¹adrenoceptors, resulting in either presynaptic inhibition of the nociceptive primary afferent neurons or postsynaptic inhibition of second-order neurons. 2In this study, we sought evidence to link the participation of GABAergic neurons in the antinociceptive effect of N²O to their activation by α¹adrenoceptors.

Adult male Fischer rats (11–12 weeks old) were used throughout the study (B&K Universal, Grimston Aldbrough, Hull, United Kingdom). All animal procedures were carried out in accordance with the United Kingdom (Scientific Procedures) Act of 1986, and the study protocol was approved by the Home Office of the United Kingdom (London, United Kingdom). All efforts were made to minimize animal suffering and reduce the number of animals used.

Animals were injected intraperitoneally with the following drugs 15 min before gas exposure: prazosin, an α¹adrenoceptor antagonist (Cat. No. 0623; Tocris Cookson, Ballwin, MO); yohimbine, an α²adrenoceptor antagonist (Cat. No. Y-3125; Sigma Chemical Co., St Louis, MO); naloxone, an opioid receptor antagonist (Cat. No. N-7758; Sigma Chemical Co.); methysergide, a nonselective 5-HT receptor antagonist (Cat. No. 1064; Tocris Cookson); and tropisetron, a selective 5-HT3 receptor antagonist (Cat. No. T-104; Sigma Chemical Co.). Dosages of each drug examined were derived from the literature. 3–6All drugs were dissolved in saline except for prazosin, which was dissolved in heated distilled water. The injection volume was standardized as 1 ml.

Gas exposure was performed in an acryl plastic exposure chamber (18 in long, 9 in wide, and 8 in high). Either a mixture of 75% N²O and 25% O²or air at a flow rate of 4 l/min was continuously delivered into the exposure chamber via  an inflow port and exhausted via  an outflow port. Gas concentrations, including those for N²O, O², and CO², in the chamber were measured continuously by infrared gas spectrometry (Ohmeda 5250 RGM; Ohmeda, Hatfield, Hertz, United Kingdom). Animals were placed into the chamber through the side door after the desired gas concentrations were achieved and stabilized.

One hour before the experiment (baseline) and 30 min after the initiation of gas exposure (which coincides with the peak antinociceptive effect of N²O), 7thermal nociceptive testing was performed using a plantar test device (Plantar test 7370; Ugo Basile, Comerio, Italy). Radiant heat was applied on the plantar surface of hind paws through the floor of the exposure chamber, and the paw withdrawal latency (PWL), defined as the time between the activation of the heat source and hind-paw withdrawal, was automatically recorded. Heat intensity was adjusted such that the baseline PWL was approximately 4 s. To avoid tissue damage, a predetermined cutoff time of 10 s was imposed. Each PWL data set consisted of a mean of three trials for each animal. From the PWL, the percentage of maximal possible effect (%MPE) was calculated as follows:

In another set of experiments, animals were not subjected to nociceptive testing but were killed by an overdose of sodium pentobarbital (100 mg/kg), intraperitoneal, following 90 min of gas exposure, which is the time to the peak effect of c-Fos induction in the spinal cord after N²O exposure. 2During the terminal anesthetic, the animals were perfused with 0.1 m phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 m phosphate buffer via  a 16-gauge cannula inserted through the left ventricle into the ascending aorta. Following decapitation, the spinal cord was expelled by rapid injection of PBS at the sacral vertebral level and stored in 30% sucrose in 0.1 m phosphate buffer for at least 24 h at 4°C. A 5-mm portion of the spinal cord at the lumbar enlargement was cut by a razor blade and was freeze-mounted in embedding matrix, and 30-μm transverse sections were cut at −15°C; every third section was collected in PBS (approximately 40–50 sections per sample).

Approximately 15–20 undamaged free-floating spinal cord sections were selected and were first incubated at room temperature for 30 min in 0.3% hydrogen peroxide in 70% methanol–PBS and for 1 h in blocking solution consisting of 3% rabbit serum and 0.3% Triton X in PBS (PBT), followed by overnight incubation with goat anti–c-Fos antibody (1:10,000, Cat. No. sc-52-G; Santa Cruz Biotechnology, Santa Cruz, CA) in blocking solution (1% normal rabbit serum in PBS) on a shaker at 4°C. Sections were then rinsed with PBT, incubated for 1 h with biotinylated rabbit antigoat immunoglobulin (1:200; Vector Laboratories, Burlingame, CA) in the same solution, rinsed with PBT, and incubated for 1 h with avidin–biotin–peroxidase complex (Vector Laboratories) in PBT. Visualization of the immunohistochemical reaction was achieved by incubation with DAB with nickel–ammonium sulfate (DAB kit; Vector Laboratories). After the staining procedure was completed, sections were rinsed in PBS followed by distilled water, mounted on slide glasses that were dehydrated in 100% ethanol, and cleared in 100% xylene, and cover slips were applied.

Using a DAB staining with nickel enhancement, c-Fos–positive cells were identified by dense black nuclear staining under a bright field microscope (Olympus Model BX50 Research Photomicroscope; Olympus Optical, Southall, Middlesex, United Kingdom). Five randomly selected, undamaged sections from each rat were photographed using a digital camera (Olympus Digital Camera Model C2020Z; Olympus Optical). The number of c-Fos–positive cells was counted for each area of the spinal cord, i.e. , laminae I–II (superficial area), laminae III–IV (nucleus proprius area), laminae V–VI (neck area), and laminae VII–X (ventral area), according to the method by Presley et al.  8Each group was comprised of at least four animals, and the number of c-Fos–positive cells in each group was calculated as mean ± SD. The investigator was blinded to the treatment cohort.

In some animals, the spinal cord was collected after 90 min of gas exposure (either air or 75% N²O) without drug pretreatment or nociceptive testing. Approximately 6–8 undamaged free-floating spinal cord sections from each specimen were first incubated for 1 h in blocking solution consisting of 3% donkey serum (Chemicon International, Temecula, CA) in PBS. They were then incubated overnight with goat anti–c-Fos antibody (1:1,000, Cat. No. sc-52-G; Santa Cruz Biotechnology) and rabbit anti–α¹adrenoceptor antibody (1:1,000, Cat. No. PC160; Oncogene Research Products, Cambridge, United Kingdom) in 1% donkey serum in PBS on a shaker at 4°C. Sections were rinsed with PBT, incubated for 1 h in darkness with a mixture of Cy3-conjugated donkey antigoat secondary antibody (1:200; Jackson Immuno Research Laboratories, West Grove, PA) and FITC-conjugated donkey antirabbit secondary antibody (1:200; Jackson Immuno Research Laboratories) in 1% donkey serum in PBS, then rinsed with PBS, floated in water, and mounted on slide glasses. After being dried in darkness, cover slips were applied to the slides with one drop of VectaShield (Vector Laboratories) mounting medium for fluorescence. The best-preserved undamaged section was selected from each animal for analysis. In each laminal scheme, all c-Fos–positive cells were examined for colocalization with α¹adrenoceptors under a fluorescent microscope by the investigator who was blinded to the treatment cohort (Leica DMR microscope; Leica, Wetzlar, Germany). Results from four animals for each group were summed, and the prevalence of c-Fos–α¹adrenoceptor colocalization was calculated.

Results from the plantar test, i.e. , %MPE, were compared for each drug treatment within the following groups; air–saline, air–drug, 75% N²O–saline, and 75% N²O–drug. The data were analyzed by one-way analysis of variance, and the Dunn test was used as an a posteriori  test. Results from c-Fos single staining were compared in the same way for the entire spinal cord section and for each laminal scheme. Results from c-Fos and α¹adrenoceptor double staining were compared between the air and 75% N²O groups in each laminal scheme or total of the spinal cord. Data were analyzed using the Fisher exact test. In addition, the number of c-Fos–positive cells among either α¹adrenoceptor–positive or –negative cells was compared between air and 75% N²O groups by one-way analysis of variance. A P  value less than 0.05 was considered to be statistically significant.

The animals exposed to air were awake and active during the experiment, while those animals exposed to N²O were excited for the first 5–10 min of exposure, followed by a relatively calm state. The animals injected with prazosin became deeply sedated after N²O exposure, but other drugs did not have this effect. The results from the plantar test are summarized in table 1. The baseline reaction time was approximately 4.0 s in each group. Exposure to 75% N²O increased the reaction time to 6.3 ± 0.4 s, or 36.8 ± 8.3% of MPE. None of the tested drugs alone showed any effect on reaction time. Prazosin, yohimbine, and naloxone almost completely blocked the N²O-induced antinociceptive effect, i.e. , the reaction time was no different from the baseline value. Methysergide or tropisetron showed no effect on N²O-induced antinociceptive effect.

Results from the c-Fos staining experiments are summarized in table 2. The number of c-Fos–positive cells in the entire area of the spinal cord section in the air–saline group was 69.0 ± 7.9 (mean ± SD). Exposure to 75% N²O increased the number of c-Fos–positive cells approximately twofold, to 142.8 ± 5.2. An increase in c-Fos–positive cells was observed in laminae III–IV, V–VI, and VII–X but not in laminae I–II. None of the tested drugs alone showed an effect on the number of c-Fos–positive cells compared with that of the air–saline group. Prazosin and naloxone significantly reduced the total number of c-Fos–positive cells in the spinal cord when compared with the N²O–saline group. Prazosin nearly completely blocked the c-Fos expression in laminae III–IV (32.2 ± 2.1 vs.  air–saline, 29.0 ± 3.7), but the effect of naloxone in laminae III–IV was only partial (1 mg/kg, 56.2 ± 2.8; 10 mg/kg, 43.5 ± 2.9). Neither drug had an inhibitory effect on c-Fos expression in laminae V–VI and VII–X. Yohimbine, methysergide, and tropisetron had no effect on N²O-induced c-Fos expression in any lamina.

In the control group, 52 (41.6%) of 125 c-Fos–positive cells in four animals examined were colocalized with α¹adrenoceptors. In the N²O group, 119 (50.0%) of 238 cells in four animals examined showed c-Fos colocalization with α¹adrenoceptors. Statistical differences were obtained between the two groups for those in laminae III–IV (table 3). When the results were analyzed separately in α¹adrenoceptor–positive and –negative cells (fig. 2), N²O induced c-Fos expression in α¹adrenoceptor–positive cells in laminae III–IV and V–VI, and also in α¹adrenoceptor–negative cells in laminae V–VI. Representative pictures of double staining for c-Fos and α¹adrenoceptors in laminae III–IV are shown in figure 3(a color version of this figure is available on the Anesthesiology Web site at http://anesthesiology.org).

The primary aim of the current study was to investigate whether α¹adrenoceptors are involved in mediation of N²O-induced antinociceptive effect and activation of GABAergic neurons in the spinal cord. We have shown that systemic administration of the α¹adrenoceptor antagonist prazosin blocks N²O-induced antinociceptive effect as measured by the plantar test (table 1) and inhibits N²O-induced c-Fos expression in the spinal cord (table 2). In addition, double-staining analysis revealed that N²O-induced c-Fos expression in laminae III–IV is strongly colocalized with α¹adrenoceptors (table 3and fig. 2). Apart from the caveats that prazosin was administered systemically, rather than intrathecally, and that a single dose was used, these data support our hypothetical “second” pathway mediating N²O-induced antinociceptive effect in the spinal cord, i.e. , through the activation of inhibitory GABAergic interneurons via α¹adrenoceptors (fig. 1). A previous report in mice in which prazosin blocked the antinociceptive effect of N²O as measured by the tail-flick test in 129/svj strain is consistent with this pathway. 9Further, a recent electrophysiological study demonstrated that norepinephrine applied to the sliced rat spinal cord preparation activates GABAergic inhibitory activity through α¹but not α²adrenoceptors. 10 

Nitrous oxide exposure induced c-Fos expression in the spinal cord in most laminae except for laminae I–II, which is consistent with the findings from our previous study. 2When the results from the double staining were analyzed in each lamina, the cells that expressed c-Fos during N²O exposure showed the highest degree of colocalization with α¹adrenoceptors in laminae III–IV (fig. 2). It is known that descending noradrenergic inhibitory neurons from the brain stem terminate in the spinal cord mainly in laminae I–IV, 11while the distribution of the termini depends on the genetic background of the strain and the origin of the pathway in the brain stem, i.e. , A5, A6 (locus ceruleus), or A7. 12,13The majority of the cells that expressed c-Fos induced by N²O exposure in laminae V–VI (to some degree in laminae VII–X, as well) were not colocalized with α¹adrenoceptors (fig. 2). Although prazosin inhibited N²O-induced c-Fos expression in laminae III–IV, neither prazosin nor other receptor antagonists showed an inhibitory effect on c-Fos expression in laminae V–VI (table 2). In our previous study, we found that nearly all cells that express N²O-induced c-Fos were GABAergic neurons. 2Thus, N²O-induced c-Fos–positive cells in laminae V–VI must be GABAergic neurons, but they are activated through receptors other than α¹adrenoceptors, serotonin receptors, or opioid receptors. Further investigations are necessary to determine the identity of such receptors, although they may not be involved in the antinociceptive effect of N²O.

We also examined the effects of other receptor antagonists on N²O-induced antinociceptive effect, as measured by the plantar test, and c-Fos expression in the spinal cord. Yohimbine, an α²adrenoceptor antagonist, blocked N²O-induced antinociceptive effects, a result in agreement with two previous studies. 3,14Ohara et al.  14reported that intraperitoneal injection of yohimbine (crosses the blood–brain barrier) but not L659–066 (an α²adrenoceptor antagonist that does not cross the blood–brain barrier) almost completely blocked the antinociceptive effects of N²O on the tail-flick test in Sprague-Dawley rats. Guo et al.  3reported that administration of the α²adrenoceptor antagonists (atipamezole, yohimbine, or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) intrathecally but not intracerebroventricularly blocked the antinociceptive effects of N²O on the tail-flick test in Sprague-Dawley rats, indicating that the site of antinociceptive action of α²adrenoceptor antagonists is at the spinal cord level. The result that yohimbine did not block N²O-induced c-Fos expression in the spinal cord is also consistent with hypothesized antinociceptive pathways (fig. 1). Because activation of α¹and α²adrenoceptors generally mediates excitatory and inhibitory neurotransmission, respectively, cells that express N²O-induced c-Fos are activated via α¹adrenoceptors and not by α²adrenoceptors. Thus, α²adrenoceptor antagonists would not be expected to affect N²O-induced c-Fos expression in the spinal cord.

Berkowitz et al.  15were the first to report on the inhibitory effects of opioid receptor antagonists against N²O-induced antinociception in 1976. Since then, many investigators have reported similar inhibitory effects on N²O-induced antinociceptive effect in other experimental paradigms and species, e.g. , in rats, 3,16–21but some have reported that opiate receptor antagonists show no effect on N²O-induced antinociception in humans 22–24or in rats. 25,26Gillman 27considered that these negative reports are due in part to the inappropriate administration of naloxone and lack of consideration of naloxone's rapid decay in the brain after systemic administration. Opioid receptor antagonists appear to act at the supraspinal sites because intrathecal administration of opioid receptor antagonist does not block N²O-induced antinociceptive effects in rats. 3 

In the current study, systemically administered naloxone almost completely blocked N²O-induced antinociceptive effects (table 1), while the inhibitory effect of naloxone against N²O-induced c-Fos expression was only partial (table 2). The reason for this discrepancy is unclear but may be explained by difference in timing of examination after naloxone injection. The plantar test was performed 45 min after administration, whereas the effect on c-Fos was examined 105 min after administration. For c-Fos experiments, we collected the spinal cord after 105 min of naloxone administration because it takes 60–90 min for c-Fos (protein) to be induced after N²O exposure. 2It does not necessarily mean that opioid receptors are needed to be blocked by naloxone during the entire period to attenuate N²O-induced c-Fos expression, but we do not know the exact length of time required.

In addition to noradrenergic and opioidergic neurons, serotonergic neurons also play important roles in the descending inhibitory pain suppression system. 28In this study, we examined two kinds of serotonin receptor antagonists, methysergide (nonselective 5-HT receptor antagonist) and tropisetron (selective 5-HT3 receptor antagonist), and found that neither blocked the antinociceptive effect of N²O or N²O-induced c-Fos expression in the spinal cord. Most descending serotonergic inhibitory neurons originate from serotonergic nuclei in the medulla, e.g. , nucleus raphe magnus and the adjacent reticular formation. In a separate study, we recently demonstrated that N²O exposure does not activate serotonergic nuclei in the medulla in Fischer rats, using a double staining analysis for c-Fos and tryptamine hydroxylase, a serotonin synthesizing enzyme. 29However, one report contradicts this, indicating that the 5-HT3 receptor antagonist, ICS-205930, blocked the antinociceptive effects of N²O as measured by the abdominal constriction test in Swiss-Webster mice, while the 5-HT1C/5-HT2 receptor antagonist, mianserin, potentiated this effect. 30This controversy could be attributed to species or experimental paradigm differences, but further investigation is needed for clarification.

Exposure to 75% N²O alone did not cause a hypnotic effect in rats; rather, initial exposure produced excitation. Interestingly, the combination of prazosin and N²O caused a profound hypnotic effect, which was not observed in other treatment groups. Recent studies have suggested that activation of noradrenergic neurons in the locus ceruleus inhibits inhibitory GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus, which results in activation (disinhibition) of histaminergic neurons in the tuberomammillary nucleus, which releases histamine into the cortex to promote arousal. 31–33This neuronal pathway mediates the hypnotic effect of the α²adrenoceptor agonist dexmedetomidine when microinjected into the locus ceruleus, where it inhibits noradrenergic neurons through α²adrenoceptor activation. 34,35In addition, a recent study in rats has shown that activation of α¹adrenoceptors in the locus ceruleus suppresses the G-protein–coupled inward rectifier potassium (GIRK) conductance induced by α²adrenoceptor or μ-opioid receptor agonists. 36Administration of 75% N²O to the rats results in activation of noradrenergic neurons in the locus ceruleus 29,37and excitation (arousal) rather than hypnosis. In light of the above, we propose that N²O activates noradrenergic neurons that project to the locus ceruleus, which contain both α¹and α²adrenoceptors; in aggregate, the effect of α¹adrenoceptors exceeds that of α²adrenoceptors, resulting in suppression of GIRK and activation of the locus ceruleus. When the effect mediated by α¹adrenoceptors is blocked by prazosin, the action mediated by α²adrenoceptors on GIRK predominates, which results in a hypnotic response.

In summary, we have demonstrated that systemic administration of the α¹adrenoceptor antagonist prazosin blocks N²O-induced antinociceptive effect, as measured by the plantar test, and inhibits N²O-induced c-Fos expression in the spinal cord. Furthermore, double-staining analysis has revealed that N²O-induced c-Fos expression is strongly localized in the cells in laminae III–IV with α¹adrenoceptor immunoreactivity. These findings support our hypothesis that N²O-induced antinociceptive effect is mediated by indirect inhibition of the nociceptive primary afferent neurons and/or postsynaptic inhibition of the second-order neurons by the activation of inhibitory GABAergic interneurons through α¹adrenoceptors. In addition, we confirmed previous reports that the α²adrenoceptor antagonist yohimbine also blocks N²O-induced antinociception, which also agrees with this hypothesized pathway. It appears that two pathways are necessary to induce the antinociceptive effects, and neither is individually sufficient to induce antinociception.