Nitrous Oxide Antinociception Research Articles

Nitrous oxide-antinociception is mediated by opioid receptors and nitric oxide in the periaqueductal gray region of the midbrain

Previous studies have shown that nitrous oxide (N2O)-induced antinociception is sensitive to antagonism by blockade of opioid receptors and also by inhibition of nitric oxide (NO) production. The present study was conducted to determine whether these occur within the same brain site. Mice were stereotaxically implanted with microinjection cannulae in the periaqueductal gray (PAG) area of the midbrain. In saline-pretreated mice, exposure to 70% N2O resulted in a concentration-dependent antinociceptive effect in the mouse abdominal constriction test. Pretreatment with an opioid antagonist in the PAG significantly antagonized the antinociceptive effect. Pretreatment with an inhibitor of NO production in the PAG also significantly antagonized the antinociceptive effect. These findings suggest that N2O acts in the PAG via an NO-dependent, opioid receptor-mediated mechanism to induce antinociception.

Dynorphin-mediated antinociceptive effects of l-arginine and SIN-1 (an NO donor) in mice

The antinociceptive response of mice to the amino acid l-arginine ( l-ARG) has been attributed to either an opioid mechanism or a non-opioid but nitric oxide (NO)-dependent mechanism. Earlier it was reported that the mechanism of nitrous oxide-induced antinociception involved opioid components and was also dependent on brain NO. This study was designed to determine whether the antinociceptive effects of l-ARG and the NO donor 3-morpholinosydnoimine (SIN-1) might be mediated by brain mechanisms similar to those that are responsible for nitrous oxide (N 2O) antinociception. l-ARG and SIN-1 were administered to mice intracerebroventricularly (i.c.v.), and antinociception was assessed using the acetic acid abdominal constriction test. Both l-ARG and SIN-1 caused dose-related antinociceptive effects that were blocked by naloxone and norbinaltorphimine. The antinociceptive effects of both SIN-1 and l-ARG were also blocked to a greater extent by i.c.v. administration of a rabbit antiserum against rat dynorphin 1–13 than an antiserum against methionine-enkephalin, suggesting that the SIN-1 and l-ARG effects may be related to stimulated release of dynorphin. The antinociceptive effect of l-ARG was antagonized by an inhibitor of neuoronal NO synthase enzyme, indicating that l-ARG had to be converted to NO for its antinociceptive action. These findings indicate that the mechanisms of antinociceptive action of l-ARG and SIN-1 are both mediated by dynorphin and dependent on NO.

Correlation of inbred mouse sensitivity to nitrous oxide antinociception with brain nitric oxide synthase activity following exposure to nitrous oxide

Inhibition of nitric oxide synthase (NOS) antagonizes nitrous oxide (N 2O)-induced antinociception in mice. This study was conducted to compare brain NOS activity in high responding C57BL/6 mice, low responding DBA/2 mice and S 5 mice selectively bred for low responsiveness to N 2O. Exposure to 70% N 2O suppressed acetic acid-induced abdominal constrictions in C57BL/6 mice but not DBA/2 or S 5 mice. N 2O exposure also elevated NOS activity in brains of C57BL/6 mice but not DBA/2 or S 5 mice. The absence of these effects in DBA/2 or S 5 mice is further support for the hypothesis that nitric oxide (NO) may play a critical role in N 2O-induced antinociception in mice.

General anesthetics inhibit the nitrous-oxide-induced activation of corticotropin releasing factor containing neurons in rats

The activation of intracerebral corticotropin releasing factor (CRF) system is involved in nitrous oxide analgesia. We evaluated the effect of general anesthetics on nitrous-oxide-induced CRF activation and antinociception. Male Sprague–Dawley rats inhaled isoflurane (0%, 0.6%, 1.0% and 1.5%) or were administered with intravenous propofol (0, 0.1 and 0.2 mg/kg/min), with or without 75% nitrous oxide inhalation, for 90 min. The brain was fixed with fixative, and brain sections, including the paraventricular nucleus of the hypothalamus, were double immunostained with c-Fos and CRF antibodies to assess the activation of CRF-containing neurons. In other groups of rats, the effect of propofol on nitrous oxide antinociception was evaluated with tail flick latency tests. Both inhaled isoflurane and intravenous propofol inhibited nitrous-oxide-induced activation of CRF neurons, suggesting that these general anesthetics may inhibit one of the analgesic mechanisms of nitrous oxide. Indeed, propofol inhibited the antinociceptive action of nitrous oxide, as evaluated with tail flick latencies (TFL).

Antagonism of nitrous oxide antinociception in mice by antisense oligodeoxynucleotide directed against neuronal nitric oxide synthase enzyme

Nitric oxide (NO) has been implicated in the antinociceptive effect of nitrous oxide (N 2O) in mice. This study was conducted to determine the sensitivity of N 2O-induced antinociception to antagonism by an antisense oligodeoxynucleotide (AS-ODN) against neuronal nitric oxide synthase (nNOS). The AS-ODN significantly antagonized the antinociceptive effect of N 2O in the abdominal constriction test, but a mismatch ODN was without effect. This result implicates the specific involvement of nNOS in N 2O-induced antinociception.

Detection and mapping of quantitative trait loci that determine responsiveness of mice to nitrous oxide antinociception

Exposure to 70% N 2O evokes a robust antinociceptive effect in C57BL/6 (B6) but not in DBA/2 (D2) inbred mice. This study was conducted to identify quantitative trait loci (QTL) in the mouse genome that might determine responsiveness to N 2O. Offspring from the F 2 generation bred from B6 and D2 progenitors exhibited a broad range of responsiveness to N 2O antinociception as determined by the acetic acid-induced abdominal constriction test. QTL analysis was then used to dissect this continuous trait distribution into component loci, and to map them to broad chromosomal regions. To this end, 24 spleens were collected from each of the following four groups: male and female F 2 mice responding to 70% N 2O in oxygen with 100% response (high-responders); and male and female F 2 mice responding with 0% response (low-responders). Genomic DNA was extracted from the spleens and genotyped with simple sequence length polymorphism MapPairs markers. Findings were combined with findings from the earlier QTL analysis from BXD recombinant inbred mice [Brain Res 725 (1996) 23]. Combined results revealed two significant QTL that influence responsiveness to nitrous oxide on proximal chromosome 2 and distal chromosome 5, and one suggestive QTL on midchromosome 18. The chromosome 2 QTL was evident only in males. A significant interaction was found between a locus on chromosome 6 and another on chromosome 13 with a substantial effect on N 2O antinociception.

Role of nitric-oxide synthase isoforms in nitrous oxide antinociception in mice.

Exposure of mice to the anesthetic gas N2O evokes a prominent antinociceptive effect that is sensitive to antagonism by nonselective nitric-oxide synthase (NOS) inhibitors. The present study was conducted to identify whether a specific NOS isoform is implicated in N2O antinociception in mice. In the abdominal constriction test, exposure of mice to 25, 50, and 70% N2O resulted in a concentration-dependent antinociceptive effect that persisted for up to 6 min following removal of the mice from the N2O atmosphere into room air. This N2O antinociceptive effect was antagonized by pretreatment with S-methyl-l-thiocitrulline (SMTC) and higher doses of l-N5-(1-iminoethyl)-ornithine (l-NIO), which reportedly inhibit the neuronal and endothelial isoforms of NOS, respectively. Nevertheless, the N2O-induced antinociception was unaffected by pretreatment with low doses of either SMTC or l-NIO or by pretreatment with 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), which selectively inhibits inducible NOS. The s.c. pretreatment with SMTC and l-NIO reduced brain NOS activity in a dose-dependent manner, whereas AMT had no such effect. Moreover, in blood pressure experiments, SMTC increased SBP in dose-unrelated fashion, whereas l-NIO showed an appreciably weaker but dose-related increase in SBP. The i.c.v. pretreatment with SMTC also reduced N2O antinociception and brain NOS activity without increasing of SBP. These results suggest that it is the neuronal isoform of NOS that is involved in mediation of the antinociceptive effect of N2O in the mice.

Antagonism of Nitrous Oxide Antinociception in Mice by Intrathecally Administered Antisera to Endogenous Opioid Peptides

Previously it was demonstrated that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by κ-opioid receptors. Since nitrous oxide is thought to cause the neuronal release of endogenous opioid peptide to stimulate opioid receptors, this study was designed to identify the opioid peptides involved, especially in the spinal cord, by determining whether nitrous oxide antinociception can be differentially inhibited by intrathecally (i.t.) administered antisera to different opioid peptides. Male NIH Swiss mice were pretreated i.t. with rabbit antisera to opioid peptides then exposed 24 h later to one of three different concentrations of nitrous oxide in oxygen. Dose-response curves constructed from the data indicated that the antinociceptive effect of nitrous oxide was significantly antagonized by antisera to various dynorphins (DYNs) and methionine-enkephalin (ME), but not by antiserum to β-endorphin (β-EP). The AD₅₀ values for nitrous oxide antinociception were significantly elevated by antisera to DYNs and ME but not β-EP. These findings of this study support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test involves the neuronal release of DYN and ME in the spinal cord.

Antagonism of nitrous oxide antinociception in mice by intrathecally administered antisera to endogenous opioid peptides

Previously it was demonstrated that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by kappa-opioid receptors. Since nitrous oxide is thought to cause the neuronal release of endogenous opioid peptide to stimulate opioid receptors, this study was designed to identify the opioid peptides involved, especially in the spinal cord, by determining whether nitrous oxide antinociception can be differentially inhibited by intrathecally (i. t.) administered antisera to different opioid peptides. Male NIH Swiss mice were pretreated i.t. with rabbit antisera to opioid peptides then exposed 24 h later to one of three different concentrations of nitrous oxide in oxygen. Dose-response curves constructed from the data indicated that the antinociceptive effect of nitrous oxide was significantly antagonized by antisera to various dynorphins (DYNs) and methionine-enkephalin (ME), but not by antiserum to beta-endorphin (beta-EP). The AD(50) values for nitrous oxide antinociception were significantly elevated by antisera to DYNs and ME but not beta-EP. These findings of this study support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test involves the neuronal release of DYN and ME in the spinal cord.

Role of Brain Dynorphin in Nitrous Oxide Antinociception in Mice

Earlier studies indicate that nitrous oxide antinociception is mediated by opioid receptors, and we have hypothesized that nitrous oxide stimulates a neuronal release of an endogenous opioid peptide (EOP) that stimulates opioid receptors. To further test this hypothesis, male NIH Swiss mice were pretreated intracerebroventricularly with rabbit antisera to opioid peptides or with various inhibitors of peptidases involved in the degradation of EOPs. Mice were subsequently exposed to three different concentrations of nitrous oxide in oxygen, and their antinociceptive responsiveness was measured using the acetic acid abdominal constriction test. Nitrous oxide antinociception was significantly attenuated by 24-h pretreatment with antisera to various fragments of dynorphin (DYN) but not by antisera against methionine-enkephalin (ME) or β-endorphin (β-EP). In other experiments, nitrous oxide antinociception was significantly enhanced by 30-min pretreatment with phosphoramidon, an inhibitor of endopeptidase 24.11, which has been implicated in DYN degradation, but not bestatin or captopril, which inhibit aminopeptidase and angiotensin-converting enzyme, respectively. The latter enzymes have been implicated in degradation of certain EOPs albeit not DYN. These findings support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by endogenous DYN acting in the central nervous system.

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

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 not by 1.0 or 5.0 mg/kg L659-066 or by 5.0 or 10 mg/kg naloxone. Xenon antinociception was not affected by any of these drugs. Yohimbine and L659-066 are characterized as alpha2-adrenoceptor antagonists. Although yohimbine penetrates the blood-brain barrier after systemic administration, L659-066 does not penetrate it and act peripherally. Therefore, the results indicate that alpha2-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 alpha2 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 alpha2-adrenoceptors, but not opioid receptors, reverses the antinociception induced by nitrous oxide but not by xenon, which indicates that alpha2-adrenoceptors may play a key role in nitrous oxide antinociception. (Anesth Analg 1997;85:931-6)

Yohimbine and flumazenil: effect on nitrous oxide-induced suppression of dorsal horn neurons in cats.

The purpose of this study was to determine the mechanisms of nitrous oxide (N2O) antinociception at the spinal level with yohimbine (an α2-adrenergic antagonist) and flumazenil (a specific benzodiazepine antagonist) using chemonociceptive stimuli in spinal dorsal horn neurons in the cat. A lumbar laminectomy extending from L4 to L6 was performed to allow insertion of a extracellular recording device via a microelectrode. Additional laminectomy was performed at the T12 level to transect the spinal cord. As a noxious stimulus, bradykinin (BK) was injected via the cannula inserted into the femoral artery. Animals were divided into four treatment groups for subsequent experiments: N2O+flumazenil, N2O+yohimbine, flumazenil (alone), and yohimbine (alone). N2O suppressed BK-induced nociceptive responses in transected feline spinal cords. The BK-induced neuronal firing rates were significantly suppressed: to 69.2%, 61.8%, and 52.2% of the baseline firing rate at 10, 20, and 30 min, respectively, after N2O administration. The 47.8% suppression on BK-induced neuronal responses at 30 min after N2O administration was reversed 5 min after administration of yohimbine (25.2% suppression). Similarly, N2O suppression (42.5%) on chemically induced neuronal responses was reversed by flumazenil (24.9% suppression) at identical postadministration intervals. These data imply that N2O suppresses the nociceptive responses in part probably through its agonistic binding activity to the α2-adrenergic, γ-aminobutyric acid (GABA)-benzodiazepine, or both receptor systems in dorsal born neurons of the feline spinal cord.

Nitrous oxide antinociception in BXD recombinant inbred mouse strains and identification of quantitative trait loci

Among inbred mouse strains, DBA/2 mice are unique because of their poor responsiveness to nitrous oxide (N 2O) antinociception. As a first step towards identifying candidate genes involved in determining antinociceptive responsiveness to N 2O, male mice from the DBA/2 strain, the more responsive C57BL/6 strain, their B6D2F 1 offspring, and 22 BXD recombinant inbred (RI) strains derived from DBA/2 and C57BL/6 mice were exposed to N20 and evaluated using the acetic acid abdominal constriction test. When exposed to 70% N 2O, C57BL/6, DBA/2 and B6D2F 1 mice exhibited antinociceptive responses of 78, 22 and 55%, respectively. The BXD RI strains demonstrated varying degrees of responsiveness to N 2O. Cluster analysis revealed one cluster of 16 strains approximating the C57BL/6 progenitor (61.9–100% antinociceptive response to 70% N 2O) and another of six strains around the DBA/2 progenitor (9.1–40% antinociceptive response to 70% N 2O). The robust strain differences permitted screening the strain means with 1492 marker loci previously mapped in BXD RI strains. Using a QTL analysis specifically tailored to existing mouse RI strains, we found associations at the 0.01 level on seven chromosomes with the most promising marker loci being Il2ra, Hbb, Hmgl rs7 and Gsl5 on chromosomes 2, 7, 16 and 19, respectively ( P < 0.002).

Nitrous oxide antinociception in BXD recombinant inbred mouse strains and identification of quantitative trait loci

Nitrous oxide antinociception in BXD recombinant inbred mouse strains and identification of quantitative trait loci

Effect of opioid peptide antisera on nitrous oxide antinociception in rats

This study was performed to examine the effects of ICV injection of antiserum against β-endorphin (β-EP) or methionine-enkephalin (ME) on nitrous oxide-induced antinociception in rats using the hot plate test. The injection of β-EP antiserum reversed the antinociceptive effect of nitrous oxide in a dose-related manner up to 200 μg/rat. However, antagonism of nitrous oxide by 400 μg β-EP antiserum was comparable to that produced by 200 μg. On the other hand, similar amounts of ME antiserum had little effect against nitrous oxide antinociception. These findings suggest that β-EP may play an important role in the antinociceptive effect of nitrous oxide.

Strain-dependent differences in responsiveness of mice to nitrous oxide (N 2O) antinociception

N 2O antinociception was assessed in eight inbred and two outbred mouse strains. Results indicated the following order of responsiveness among the 10 strains: A/J (most sensitive), C57BL/6ByJ, C57BL/6J, BALB/cByJ, C3H/HeJ, Swiss-Webster, CXBK/ByJ, ICR, CBA/J and DBA/2J (least sensitive). These results demonstrate significant strain-dependent differences in antinociceptive responsiveness to N 2O. The weak antinociceptive response to N 2O in the DBA/2J strain, which is sensitive to morphine and U-50,488H, indicates some underlying neurobiological difference in the DBA/2J mouse that imparts resistance to N 2O. The responsiveness of CXBK/ByJ mice to N 2O indicates that μ-opioid receptors may not play an important role in N 2O antinociception in mice.

Contrasting influences of 5-hydroxytryptamine receptors in nitrous oxide antinociception in mice

5-Hydroxytryptamine (5-HT) mechanisms may play a role in opioid-mediated antinociception. Since opioid mechanisms have been implicated in nitrous oxide antinociception, this study was conducted to determine the possible role of 5-HT receptors in nitrous oxide antinociception. Male Swiss Webster mice were pretreated with one of two 5-HT receptor blockers and then tested in the acetic acid abdominal constriction test for their antinociceptive response to nitrous oxide, the κ-opioid agonist U-50, 488H, or the μ-opioid agonist sufentanil. Results indicate that the 5-HT 3 receptor blocker ICS-205,930 antagonized both nitrous oxide and U-50,488H effects but not that of sufentanil. Mianserin, a 5-HT 1c/5-HT 2 receptor blocker, potentiated effects of both nitrous oxide and U-50,488H but not that of sufentanil. These findings show similarities in nitrous oxide and U-50,488H antinociception and further support our hypothesis that nitrous oxide works through central κ-opioid mechanisms in mice. The results also suggest different roles for 5-HT receptor subtypes in mediating or modulating the antinociceptive effect of nitrous oxide.

Protection by U-50,488H against β-chlornaltrexamine antagonism of nitrous oxide antinociception in mice

Nitrous oxide antinociception in the abdominal constriction test was significantly reduced in mice pretreated intracerebroventricularly (i.c.v.) with beta-chlornaltrexamine (beta-CNA). However, this antagonism was reversed when the beta-CNA was co-administered i.c.v. with the kappa-opioid ligand U-50,488H but not the mu-opioid ligand CTOP. These findings further demonstrate that nitrous oxide antinociception in the mouse abdominal constriction paradigm is mediated by kappa- but not mu-opioid receptors.

Effect of subtype-selective opioid receptor blockers on nitrous oxide antinociception in rats

Nitrous oxide antinociception in rats was evaluated by the warm water tail withdrawal test following central pretreatment with blockers of various opioid receptor subtypes. The analgesic dose, 50% (AD 50) value for nitrous oxide antinociception was significantly elevated by MR-2266 (which is relatively selective for κ-opioid receptors) and increased to a lesser degree by ICI-174,864 (which is selective for δ-opioid receptors). However, pretreatment with β-funaltrexamine (which is selective for μ-opioid receptors), even at extremely high doses, was ineffective in altering the AD 50 for nitrous oxide antinociception. According to these findings, nitrous oxide antinociception, as evaluated in this paradigm, appears to be mediated by κ- and possibly δ- but not μ-opioid receptors.

Enkephalinase inhibition prevented tolerance to nitrous oxide analgesia in rats.

Tolerance to nitrous oxide (N2O) antinociception was studied in rats in accordance with the Randall-Selitto pressure nociception test. Both N2O (70% in 30% O2) and the relatively selective enkephalinase inhibitor phosphoramidon (350 micrograms i.c.v.), which blocks the biotransformation of enkephalins, were administered. They both induced a significant analgesic effect which vanished within 45 min. The rapidly developed tolerance to N2O analgesia does not affect the anaesthetic state since the animals remained motionless for the duration of exposure lasting 3 h. In the animals treated with the enkephalinase inhibitor phosphoramidon, no development of tolerance to N2O-antinociception occurred during the exposure lasting 3 h. The results indicate that tolerance to N2O analgesia can be abolished by activation of the enkephalinergic system, which might suggest a possible insufficiency of this system during tolerance to N2O.