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Tonic endogenous opioid inhibition of visceral noxious information in rabbits
GASTROENTEROLOGY 1996;111:78–84
Tonic Endogenous Opioid Inhibition of Visceral Noxious Information in Rabbits FINN MOLKE BORGBJERG,* CHARLOTTE FRIGAST,‡ and JØRN BO MADSEN§ *Multidisciplinary Pain Center, Herlev Hospital, and Pain Clinic, Bispebjerg Hospital, University of Copenhagen, Copenhagen; ‡ Department of Anatomy and Physiology, Royal Danish Veterinarian and Agricultural University, Frederiksberg; and §Department of Anesthesiology, University of Copenhagen, Glostrup Hospital, Glostrup, Denmark
Background & Aims: A tonic intrinsic spinal inhibitory system on spinal motor reflexes in rabbits has been shown earlier. The aim of this study was to examine the effects of different opioid antagonists against visceral noxious stimulation in awake rabbits. Methods: The opioid receptor antagonists examined were naloxone (nonselective), MR2266 (k), and naltrindole (d). The effects on the visceromotor response thresholds induced by colorectal distention in rabbits were determined after intrathecal and intramuscular administration of the antagonists. Results: Intrathecal naloxone resulted in a dose-dependent decrease of visceromotor response thresholds. The selective antagonists MR2266 and naltrindole had no significant effects. In the presence of MR2266, intrathecal naloxone reduced thresholds to the same degree as when given alone. Analysis of the data from all rabbits showed a statistically significant reduction in visceromotor response thresholds after intrathecal naloxone compared with intramuscular administration. Conclusions: In rabbits, tonic active intrinsic spinal and supraspinal endogenous opioids modulate visceral noxious information. This inhibition is exerted at the m opioid receptor.
D
escending inhibitory control of monosynaptic spinal reflexes was discovered almost a century ago (for review, see Duggan and Morton1). The discovery of the endogenous opioids and their localization within the central nervous system has stimulated research on inhibitory control. Using the opioid antagonist naloxone, several investigators have shown a facilitatory effect on different spinal reflexes in both cats and rabbits,2 – 5 concluding that endogenous opioids produce a tonic inhibition of motor reflexes within the spinal cord. Electrical stimulation and opioid injections into the periaquaductal gray and nucleus raphe magnus produce antinociception6 – 10 mediated at the spinal level, because intrathecal (IT) injections of different monoaminergic, serotonergic, and opioid antagonists eliminate these effects.7,11 – 14 In humans, naloxone antagonizes pain relief induced by electrical stimulation of the central gray matter.15 Direct applications of opioids in the vicinity of the spinal cord / 5E0F$$0017
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produce profound analgesia in both animals and humans (for review, see Yaksh et al.16). Although substantial information exists on the segmental and suprasegmental control of somatic sensory transmission through the spinal cord, far less is known about spinal and supraspinal inhibition of visceral sensory messages.17 – 21 m, k, and d opioid receptors are localized in the spinal cord of the rabbit,22 and endogenous opioids such as leucine-enkephalin and methionine-enkephalin are present in visceral afferent terminations in the dorsal horn of the spinal cord.23 Exogenously administered opioids from all three major opioid receptor classes have been shown to modulate visceral noxious information (for review, see Ness and Gebhart24; for update, see Borgbjerg et al.25). These anatomic and pharmacological observations support the existence of an endogenous opioid system in the visceral noxious pathways. The purpose of the present study was to examine whether endogenous opioids exert a tonic inhibition of visceral noxious information induced by colorectal distention in rabbits and to determine the contribution of the different opioid receptors, m, k, and d receptors, involved. A previous investigation has shown increasing visceromotor response thresholds (VRTs) after colorectal distention in rabbits with increasing age.26 This study includes experiments in older rabbits to elucidate the nature of this observation.
Materials and Methods Thirty-six female New Zealand albino rabbits (weight, 2.5–4.5 kg) were used. The Danish Committee for Animal Research under the Department of Justice accepted the protocol, and the experiments were performed according to the ethical guidelines for experiments in conscious animals. The weight increased during the study because of the time span allowing the rabbits to be old enough to show an increase in their VRT. Twelve rabbits were used for each drug, i.e., Abbreviations used in this paper: IT, intrathecal; VRT, visceromotor response threshold. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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naloxone, MR 2266, and naltrindole. Isotonic saline, 0.5 mL, and sterile water acidified to a pH of 2.2 were tested to obtain control values representing the injection procedure and the effect of a solution with a low pH (performed because of the pH value of the MR2266 solution). Eight rabbits from the naloxone batch and 8 rabbits from the MR2266 batch were allowed to get old enough to develop an increase in VRTs. The rabbits were kept in spacious cages, 2 rabbits in each cage, with free access to food and water. All experiments were performed between 8 AM and 4 PM. The animals had a 2-week period of acclimatization to minimize the stress associated with the experiments. The procedure involved handling to and from the cages, placement in the box (without a top), gentle fixation by the hands, and introduction of the balloon catheter into the anus, without subsequent balloon inflation. This procedure was performed once daily during the period. At the termination of the experiments, the animals were killed by intravenous injection of pentobarbital.
Catheter Implantation During general anesthesia induced by a combination of intramuscular (IM) ketamine (25 mg) and xylazine (10 mg), supplemented with subcutaneous lidocaine infiltration, IT catheters (Portex; OD, 0.9 mm; Hythe, Kent, England) were inserted surgically in the area between L7 and S1. The free end of the catheter was mounted with a rubber membrane and passed subcutaneously to the area of the neck. After closure of the skin incision, the rabbits had a totally subcutaneously implanted IT injection system.27 The presence of motor abnormalities after catheter implantation resulted in exclusion from the study. Injection of 0.5 mL of 1% lidocaine before and at the end of the study verified catheter position. Injection of this amount of local anesthetic in IT catheters invariably resulted in paralysis of the hind legs, whereas previous studies using epidural catheters have shown it to produce motor weakness instead of paralysis.27 All catheters were placed correctly. The animals recovered 1 week after surgery.
Visceral Noxious Stimulation To assess responses to visceral noxious stimulation, a standardized colorectal distention was performed. This test has been described previously.26 Briefly, it consists of water inflation (15 mL/min) of a balloon attached to a double-lumen catheter allowing water infusion and simultaneous pressure registration within the balloon. The nondistended volume of the balloon was 0 mL, the wall of the balloon was made of a thin polyvinyl chloride film, and the balloon contained 50 mL at full distention. The balloon was lubricated with a siliconecontaining gel and inserted intra-anally into the descending colon. The proximal end of the balloon was positioned approximately 8 cm from the anus in awake animals without any signs of discomfort. The water infusion resulted in a steady increase in balloon pressure until a sudden contraction of the abdominal and hind leg musculature. The pressure inside the balloon producing this motor response defined the VRT. The procedure was repeated four times, and the mean of the last
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three values was used as the test parameter. Cutoff distention pressure was 100 mm Hg to prevent tissue damage. Two investigators participated in the tests, which were performed blinded to the observer of the VRT. The normal baseline value of the VRT is 55 { 4 mm Hg (mean { SEM); as a result, 99% of the normal reactions appear in the range between 35 and 75 mm Hg (mean { 2SD). Rabbits with a baseline value below 75 mm Hg are considered normally reacting rabbits.
Somatic Noxious Stimulation Transcutaneous electrical stimulation was applied to examine the existence of segmental effects. The current was applied through two metal electrodes, 2 cm apart. It was generated by a constant current stimulator (Elpha 500; Biometer, Odense, Denmark), and the characteristics were 100 Hz and a pulse width of 0.2 milliseconds. The animals were tested in the area of the neck, cervical region, and lumbar region. The noxious electrical stimulation threshold was the intensity (milliamperes) at which rabbits tried to escape the stimulus. The test value was the average of three ascending determinations. Again, two investigators took part in the procedure to obtain the results in a blinded fashion.
Drug Protocol Drugs for IT injections were mixed so that all doses were administered in a 0.5-mL solution followed by a 0.2-mL isotonic saline solution to flush the catheter. Control values were obtained by injection of 0.5 mL isotonic saline. IM injections of the drugs were given in a volume of 1 mL. The compounds used were 0.002–0.2 mg naloxone (5.5–550 nmol; nonselective opioid receptor antagonist), 0.25 mg MR2266 (770 nmol; selective k receptor antagonist28), and 0.5 mg naltrindole (1100 nmol; selective d receptor antagonist29). Naloxone solutions were made from commercially available solutions without preservatives. MR2266 and naltrindole solutions were made from the powder form of the compounds on the day of the experiments. Naltrindole was dissolved in a 20% (wt/vol) concentration of 2-hydroxypropyl-b-cyclodextrin. MR2266 was dissolved in a combination of sterile water and 10 mmol/mL HCl (pH 2.2). MR2266 was a gift from Boehringer Ingelheim (Ingelheim am Rhine, Germany). Naloxone and naltrindole were purchased from Research Biochemicals Inc. (Natick, MA). Before drug administration, control values of the VRT and the electrical stimulation threshold were obtained; postinjection thresholds were measured 15 minutes later. The doses for MR2266 and naltrindole were chosen, because they were the highest doses obtainable in a volume of 0.5 mL. The following experiments were performed for each compound. Initially, the response from the maximum dose was determined after IT injection. If a significant response was obtained, a dose response was performed, effect after IM administration of the largest dose, and segmental distribution of the IT effect were assessed. At least 3 days were allowed to pass between experiments, and the same rabbits were used.
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Statistical Evaluation Data are expressed as mean { SEM values unless otherwise stated. Despite the use of nonparametric statistics, mean values were chosen to present the data because of the relatively small number of animals in each group. Statistical computations were performed using the actual threshold values from each animal. To obtain the dose-response curve, the dose was plotted against the percent change as follows: % Change Å Postdrug Threshold 0 Predrug Threshold 1 100. Predrug Threshold Statistical evaluations of paired data were performed using Friedman’s analysis of variance (ANOVA) when more than two groups were present and using Pratt’s test for two groups. Unpaired data were evaluated by Kruskall–Wallis test for more than two groups and the Mann–Whitney U test for two groups. To eliminate the risk of mass significance, a significant P value of the ANOVA was requested before comparing different doses or times with placebo or control values, respectively. Linear regression was performed between dose response and log dose values. An F test was performed to test the variance of the regression. A P value of õ0.05 was chosen as the level of significance.
Results Influence of Age on VRTs Eight rabbits from the naloxone batch and 8 rabbits from the MR2266 batch were allowed to get old enough to increase their VRT values. The initial mean threshold for these rabbits was 52.8 { 1.7 mm Hg; with increasing age, the value changed to 93.2 { 2.0 mm Hg. This difference was highly significant (P õ 3 1 1009). Effects on Motor Function In 1 rabbit with a high VRT, IT MR2266 (0.25 mg) produced a staggering walk for approximately 5 minutes. This effect was interpreted as a sign of minor motor dysfunction caused by injection of MR2266. At the time of testing, the rabbit showed a normal ability to walk and jump. At the end of the study, 1 rabbit received an IT injection of 0.5 mg of MR2266, resulting in a paralysis of the lower part of the body for 20 minutes, after which the animal regained normal motor function. Neither naltrindole nor naloxone produced any observable effects on motor function. No other adverse reactions were observed throughout the study, and the rabbits thrived and gained weight. / 5E0F$$0017
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Figure 1. Naloxone-induced decrease in VRTs in rabbits. % Change represents the difference in VRTs after colorectal distention expressed in percentage of the control values. The log-dose response is linear (P õ 0.05; F test). Values are mean { SEM; n Å 8 for each point representing naloxone values. 䊏, Naloxone; 䊊, 0.5 mL IT isotonic saline (n Å 6). *P õ 0.05 compared with saline.
Effects of Isotonic Saline and Acidified Sterile Water Injection of 0.5 mL isotonic saline had no effect on the VRTs: 49.1 { 6 mm Hg before vs. 49.2 { 6.7 mm Hg after administration (P Å 1.0; n Å 6). Sterile water acidified to a pH of 2.2 also had no effect: 51.6 { 3.5 mm Hg before vs. 48.8 { 6.5 mm Hg after administration (P Å 0.8; n Å 6). Effects of Naloxone IT naloxone resulted in a linear log-dose response (P Å 0.01; F test) in rabbits with normal VRTs (Figure 1). Compared with placebo, a significant difference was observed after IT injection of 0.2 mg naloxone (49.3 { 2.5 vs. 37.8 { 3.8 mm Hg; P Å 0.04). IM administration of 0.2 mg naloxone also reduced normal VRTs significantly (60.0 { 2.7 vs. 52.7 { 3.8 mm Hg; P Å 0.02; Figure 2). IT naloxone, 0.2 mg, did not affect electrical stimulation thresholds at either lumbar or cervical level. In contrast, IM naloxone reduced electrical stimulation thresholds at the lumbar (16.2 { 0.7 vs. 13.8 { 0.7 mA; P õ 0.05) and cervical region (8.8 { 0.7 vs. 6.7 { 0.8 mA; P õ 0.01). Although statistical comparison between the VRT values obtained after IT and IM administration did not reach significance in rabbits with normal VRTs, the reduction after IM naloxone was less than after IT naloxone; 12.5% { 3.6% reduction (baseline value, 60.0 { 2.7 mm Hg) in VRT after IM administration vs. 23.8% { 6.2% reduction (baseline value, 49.3 { 2.5 mm Hg) WBS-Gastro
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after IT administration. This tendency was also observed in rabbits with high control VRTs: 20.1% { 5.0% (baseline value, 98 { 1.6 mm Hg) vs. 31.2% { 5.3% (baseline value, 92.8 { 1.8 mm Hg) for IM and IT, respectively (Figure 2). Statistical evaluation of the pooled data from rabbits with normal and high VRTs resulted in a significant difference between IT and IM administration (26.9% { 3.9% [baseline value, 67.9 / 6.2 mm Hg] vs. 16.3% { 3.1% [baseline value, 79.0 / 5.1 mm Hg] reduction for IT and IM, respectively; P õ 0.05) (Figure 2). IT (31.2% { 5.3%) and IM (20.1% { 5.0%) naloxone produced a significant reduction in VRTs in older rabbits with high control VRTs (Figure 2). Statistical comparison in the percentage reduction after IT and IM naloxone in rabbits with normal and high thresholds did not show any significant difference (P Å 0.18 and P Å 0.21 after IT and IM administration, respectively). Effects of MR2266 and Naltrindole Figure 3 shows the results after IT and IM injection of MR2266 and naltrindole. No significant effects were observed by either IT administration (MR2266, 49.5 { 3.6 vs. 54.3 { 7.1 mm Hg; naltrindole, 51.2 { 4.1 vs. 54.7 { 2.7 mm Hg) or IM administration (MR2266, 56.2 { 3.5 vs. 53.8 { 3.7 mm Hg; naltrindole, 62.8 { 4.1 vs. 60.0 { 2.7 mm Hg). IT MR2266 was also unable to produce significant changes in VRTs in rabbits with high values (92.2 { 4.1 vs. 81.8 { 7.3 mm Hg), and additional administration of IT naloxone
Figure 3. The effects of IT and IM MR2266 (k antagonist) and naltrindole (d antagonist) on VRTs. No significant changes were observed. 䊐, MR2266 (0.25 mg); , naltrindole (0.5 mg). Results are mean { SEM; n Å 8 for MR2266 and n Å 6 for naltrindole. % Change represents the difference in VRTs after colorectal distention expressed in percentage of the control values.
after MR2266 produced a significant reduction in VRTs in rabbits with both normal and elevated thresholds (Figure 4). Comparisons of the changes obtained after IT and IM MR2266 (15.4 { 18.6 vs. 03.3% { 3.9%) and naltrindole (11.2 { 12.6 vs. 03.7 { 5.2) did not show any significant differences (Figure 3). Naltrindole was not examined in rabbits with high thresholds.
Discussion
Figure 2. Changes in VRTs after IT and IM naloxone (0.2 mg). Values are expressed in percentage of the control values. Each bar represents values from 8 rabbits (mean { SEM). Values were obtained from rabbits with normal (õ75 mm Hg; 䊐) and high (ú75 mm Hg; ) baseline thresholds to colorectal distention. Pooled data (⽧) represent data from both normal- and high-threshold rabbits. *P õ 0.05 compared with the corresponding control values. -∗-P õ 0.05.
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In the present study, IT and IM naloxone produced a significant decrease in VRTs. The selective d antagonist naltrindole and the selective k antagonist MR2266 had no effect in the dose ranges studied. On a molar basis, the doses of naltrindole and MR2266 exceeded the highest dose of naloxone. Naltrindole and MR2266 have higher affinity for their respective receptors than naloxone and were given in larger doses in the present study; therefore, the effects observed after naloxone were not caused by d or k antagonism. Because of motor impairment, MR2266 was given in the highest dose possible, and the amount of naltrindole was limited by the high lipid solubility of the compound. Furthermore, the present doses of naltrindole and MR2266 antagonized the profound effects on VRTs caused by IT administration of their corresponding agonists D-pen25 D-pen-enkephalin and U50488H. Although naloxone antagonize all three opioid receptors, it shows the highest affinity for the m receptor and is the drug of reference in the existing literature concerning endogenous opioid modulation of motor reflexes.2 – 5 The more selective anWBS-Gastro
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Figure 4. Changes in VRTs after IT MR2266 (0.25 mg) in normalthreshold (䊐) and high-threshold ( ) rabbits (see Figure 2). No significant changes were observed. Administration of IT naloxone in the presence of MR2266 significantly reduced the thresholds compared with the control values obtained before administration of MR2266. Each bar represents values from 8 rabbits (mean { SEM). *P õ 0.05, # P õ 0.01.
tagonists b-funaltrexamine is an irreversible antagonist at the m receptor,30 which would have caused problems in the setup of the present study because the rabbits were examined more than once. The selective m receptor antagonist CTOP is a peptide and has no effect after parenteral administration.31 The involvement of m but not d and k receptors in the tonic inhibition of spinal motor reflexes has been shown earlier2; the effect at the k receptor was considered to be indirect. The present study is the first to show a lack of effect after IT administration of a selective k antagonist. Tattersall et al.17 showed the presence of both descending and intrinsic spinal inhibition of visceral noxious stimuli in cats, but they did not examine the neurotransmitters involved. Tonic opioid inhibition of visceral noxious information has not been reported previously. In a study on colorectal distention, Ness and Gebhart32 administered naloxone to antagonize the effect after morphine. They found that naloxone (3 mg/kg intravenously) antagonized the effect of morphine on the VRT, but the threshold after naloxone did not differ from control value, i.e., there were no signs of endogenous opioid antagonism implicating the existence of a tonic active system. The dose of naloxone used was larger than the highest dose used in the present study (0.07 mg/kg), which produced a significant reduction in VRTs after IM administration. These results in rats obtained by Ness and Gebhart32 were confirmed in other studies33,34 by the same laboratory; naloxone eliminated the inhibitory effects of different opioids but did not change the baseline firing rate of / 5E0F$$0017
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nociceptive neurons or the VRTs. These studies were not designed to detect the effect of naloxone, and the numbers of animals receiving naloxone were too low to perform statistical evaluations of the results. Thus the results do not exclude the possibility of a tonic opioid system involved in the modulation of noxious visceral information in the spinal cord of the rat. Furthermore, the results were obtained in the presence of morphine and cannot be interpreted as an effect after naloxone alone. The same argument can be used against the work by Kawamata et al.19 They administered IT naloxone to antagonize the effects of parenterally administered morphine. Naloxone reduced the effect of morphine on VRTs but did not reduce the thresholds below the control values. The existence of descending inhibition of visceral noxious information has been shown previously.17 – 21 Studies by Kawamata et al.19 and Iwasaki et al.20 indicate that part of this inhibition is caused by a descending endogenous opioid system, which can be activated by parenteral administration of opioids. IT naloxone inhibited the effects of subcutaneous morphine on VRTs,19 and because the effects following parenteral administration of drugs are exerted mainly at the supraspinal level, as shown by Vigouret et al.,35 these results imply that parenterally administered opioids activate a descending inhibitory opioid system, which terminates at the spinal level. Vigouret et al.35 showed that the effect of parenteral morphine is not only mediated by a descending opioid system but also through descending serotonergic, a-adrenergic, and cholinergic systems. Iwasaki et al.20 showed that 1 mg/kg of subcutaneous naloxone antagonized the pregnancy-induced increase in VRTs. The data from the present study on IM naloxone support these observations and confirm the existence of a tonic endogenous opioid system in the modulation of noxious visceral information. Occurrence of increasing VRTs to colorectal distention with increasing age has been reported earlier anecdotally.26 In the present study, this observation was verified and the result was highly significant. Age-related changes in the nociceptive system have been reported previously in rats.36 The biological significance of this observation is unclear, and the present results imply that this observation is not caused by an increased activity in the endogenous opioid system in older rabbits, because no significant differences were found between the effects of naloxone in young and old rabbits. Although no significant difference could be detected between IT and IM naloxone in the two separate groups of rabbits (with normal or high thresholds), the tendency towards a more pronounced effect after IT administration in both groups and the significant difference between IT and IM administration in the pooled data point to the WBS-Gastro
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existence of both supraspinal and intrinsic spinal opioid inhibitory systems. It is beyond the scope of the present study to determine and compare the potency of the two systems. The effects of naloxone on electrical stimulation thresholds did not supply further information concerning the segmental effect but indicate that somatic electrical noxious stimuli are only modulated by a supraspinal opioid system, i.e., a significant reduction in electrical stimulation thresholds after IM naloxone and no effect after IT administration were observed. Theoretically, the effect observed after naloxone administration could be a direct action at the motor neurons resulting in increasing spinal reflexes. Duggan and Zhao37 used intracellular recordings from motor neurons and injections of naloxone extracellularly and found that naloxone had no direct effect on the motor neurons. They concluded that the effects of naloxone on muscle reflexes are caused by antagonism of spinal interneurons. The differences observed between rats and rabbits cannot be explained from the present study, but the use of different species may produce different results. It has been shown that the presence of opioid receptors varies between species.38 – 42 Rabbits have a high density of k receptors in the spinal cord,40 whereas adult rats have few receptors.41,42 Age-related changes36 and genetic differences43 have also been reported. The density of k receptors in the spinal cord of rats diminishes with increasing age42; in mice, the sensitivity to both m and k agonists varies from 0% to 90%43 within different genetic strains. Older rats do not develop allodynia after sciatic nerve ligation,44 and older rabbits respond with higher thresholds to CD (Jensen et al.26 and present study). These differences may be a result of changes in the transmission at the spinal cord caused by excitatory amino acids45 or, as indicated in the present study, differences in the activity of the intrinsic opioid system. In conclusion, the present study shows tonic active intrinsic spinal and descending opioid inhibitory systems modulating visceral noxious stimuli in rabbits, probably caused by interactions at the m opioid receptor.
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oxone near motorneurones fails to produce the effects of systemic naloxone in anaesthetized cats. Neurosci Lett 1984;45: 305–310. Rothman RB, Bykov V, Xue BG, Xu H, De Costa BR, Jacobson AE, Rice KC, Kleinman JE, Brady L. Interaction of opioid peptides and other drugs with multiple kappa receptors in rat and human brain. Evidence for species differences. Peptides 1992;13:977– 987. Gillan MGC, Kosterlitz HW. Spectrum of the mu-, delta- and kappa-binding sites in homogenates of rat brain. Br J Pharmacol 1982;77:461–469. Meunier JC. Mu and kappa opiate binding sites in the rabbit CNS. Life Sci 1982;31:1327–1330. Lahti RA, Mickelson MM, McCall JM, Vonvoigtlander PF. [3H]U69593, a highly selective ligand for the opioid kappa receptor. Eur J Pharmacol 1985;109:281–284. Wood MS, Rodriguez FD, Traynor JR. Characterization of kappa opioid binding sites in rat and guinea-pig spinal cord. Neuropharmacology 1989;28:1041–1046. Pasternak GW. Progress in opiate pharmacology. In: Chapman CR, Foley KM, eds. Current and emerging issues in cancer pain: research and practice. New York: Raven, 1993:113–127. Tanck EN, Kroin JS, McCarthy RJ, Penn RD, Ivankovich AD. Effects of age and size on development of allodynia in a chronic pain model produced by sciatic nerve ligation in rats. Pain 1992;51: 313–316. Gibbs LM, Kendig JJ. Substance P and NMDA receptor–mediated slow potentials in neonatal rat spinal cord: age-related changes. Brain Res 1992;595:236–241.
Received October 23, 1995. Accepted March 25, 1996. Address requests for reprints to: Finn Molke Borgbjerg, M.D., Pain Clinic, Department of Anesthesiology, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark. Fax: (45) 3531-39-58. Supported by grants from the Danish Medical Associations Foundation (086.51) and Christine Jensa La Cours Foundation. The authors thank William Cook, Europe, for preparing the balloon catheters and Jens Randrup for technical assistance.
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Tonic Endogenous Opioid Inhibition of Visceral Noxious Information in Rabbits FINN MOLKE BORGBJERG,* CHARLOTTE FRIGAST,‡ and JØRN BO MADSEN§ *Multidisciplinary Pain Center, Herlev Hospital, and Pain Clinic, Bispebjerg Hospital, University of Copenhagen, Copenhagen; ‡ Department of Anatomy and Physiology, Royal Danish Veterinarian and Agricultural University, Frederiksberg; and §Department of Anesthesiology, University of Copenhagen, Glostrup Hospital, Glostrup, Denmark
Background & Aims: A tonic intrinsic spinal inhibitory system on spinal motor reflexes in rabbits has been shown earlier. The aim of this study was to examine the effects of different opioid antagonists against visceral noxious stimulation in awake rabbits. Methods: The opioid receptor antagonists examined were naloxone (nonselective), MR2266 (k), and naltrindole (d). The effects on the visceromotor response thresholds induced by colorectal distention in rabbits were determined after intrathecal and intramuscular administration of the antagonists. Results: Intrathecal naloxone resulted in a dose-dependent decrease of visceromotor response thresholds. The selective antagonists MR2266 and naltrindole had no significant effects. In the presence of MR2266, intrathecal naloxone reduced thresholds to the same degree as when given alone. Analysis of the data from all rabbits showed a statistically significant reduction in visceromotor response thresholds after intrathecal naloxone compared with intramuscular administration. Conclusions: In rabbits, tonic active intrinsic spinal and supraspinal endogenous opioids modulate visceral noxious information. This inhibition is exerted at the m opioid receptor.
D
escending inhibitory control of monosynaptic spinal reflexes was discovered almost a century ago (for review, see Duggan and Morton1). The discovery of the endogenous opioids and their localization within the central nervous system has stimulated research on inhibitory control. Using the opioid antagonist naloxone, several investigators have shown a facilitatory effect on different spinal reflexes in both cats and rabbits,2 – 5 concluding that endogenous opioids produce a tonic inhibition of motor reflexes within the spinal cord. Electrical stimulation and opioid injections into the periaquaductal gray and nucleus raphe magnus produce antinociception6 – 10 mediated at the spinal level, because intrathecal (IT) injections of different monoaminergic, serotonergic, and opioid antagonists eliminate these effects.7,11 – 14 In humans, naloxone antagonizes pain relief induced by electrical stimulation of the central gray matter.15 Direct applications of opioids in the vicinity of the spinal cord / 5E0F$$0017
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produce profound analgesia in both animals and humans (for review, see Yaksh et al.16). Although substantial information exists on the segmental and suprasegmental control of somatic sensory transmission through the spinal cord, far less is known about spinal and supraspinal inhibition of visceral sensory messages.17 – 21 m, k, and d opioid receptors are localized in the spinal cord of the rabbit,22 and endogenous opioids such as leucine-enkephalin and methionine-enkephalin are present in visceral afferent terminations in the dorsal horn of the spinal cord.23 Exogenously administered opioids from all three major opioid receptor classes have been shown to modulate visceral noxious information (for review, see Ness and Gebhart24; for update, see Borgbjerg et al.25). These anatomic and pharmacological observations support the existence of an endogenous opioid system in the visceral noxious pathways. The purpose of the present study was to examine whether endogenous opioids exert a tonic inhibition of visceral noxious information induced by colorectal distention in rabbits and to determine the contribution of the different opioid receptors, m, k, and d receptors, involved. A previous investigation has shown increasing visceromotor response thresholds (VRTs) after colorectal distention in rabbits with increasing age.26 This study includes experiments in older rabbits to elucidate the nature of this observation.
Materials and Methods Thirty-six female New Zealand albino rabbits (weight, 2.5–4.5 kg) were used. The Danish Committee for Animal Research under the Department of Justice accepted the protocol, and the experiments were performed according to the ethical guidelines for experiments in conscious animals. The weight increased during the study because of the time span allowing the rabbits to be old enough to show an increase in their VRT. Twelve rabbits were used for each drug, i.e., Abbreviations used in this paper: IT, intrathecal; VRT, visceromotor response threshold. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00
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naloxone, MR 2266, and naltrindole. Isotonic saline, 0.5 mL, and sterile water acidified to a pH of 2.2 were tested to obtain control values representing the injection procedure and the effect of a solution with a low pH (performed because of the pH value of the MR2266 solution). Eight rabbits from the naloxone batch and 8 rabbits from the MR2266 batch were allowed to get old enough to develop an increase in VRTs. The rabbits were kept in spacious cages, 2 rabbits in each cage, with free access to food and water. All experiments were performed between 8 AM and 4 PM. The animals had a 2-week period of acclimatization to minimize the stress associated with the experiments. The procedure involved handling to and from the cages, placement in the box (without a top), gentle fixation by the hands, and introduction of the balloon catheter into the anus, without subsequent balloon inflation. This procedure was performed once daily during the period. At the termination of the experiments, the animals were killed by intravenous injection of pentobarbital.
Catheter Implantation During general anesthesia induced by a combination of intramuscular (IM) ketamine (25 mg) and xylazine (10 mg), supplemented with subcutaneous lidocaine infiltration, IT catheters (Portex; OD, 0.9 mm; Hythe, Kent, England) were inserted surgically in the area between L7 and S1. The free end of the catheter was mounted with a rubber membrane and passed subcutaneously to the area of the neck. After closure of the skin incision, the rabbits had a totally subcutaneously implanted IT injection system.27 The presence of motor abnormalities after catheter implantation resulted in exclusion from the study. Injection of 0.5 mL of 1% lidocaine before and at the end of the study verified catheter position. Injection of this amount of local anesthetic in IT catheters invariably resulted in paralysis of the hind legs, whereas previous studies using epidural catheters have shown it to produce motor weakness instead of paralysis.27 All catheters were placed correctly. The animals recovered 1 week after surgery.
Visceral Noxious Stimulation To assess responses to visceral noxious stimulation, a standardized colorectal distention was performed. This test has been described previously.26 Briefly, it consists of water inflation (15 mL/min) of a balloon attached to a double-lumen catheter allowing water infusion and simultaneous pressure registration within the balloon. The nondistended volume of the balloon was 0 mL, the wall of the balloon was made of a thin polyvinyl chloride film, and the balloon contained 50 mL at full distention. The balloon was lubricated with a siliconecontaining gel and inserted intra-anally into the descending colon. The proximal end of the balloon was positioned approximately 8 cm from the anus in awake animals without any signs of discomfort. The water infusion resulted in a steady increase in balloon pressure until a sudden contraction of the abdominal and hind leg musculature. The pressure inside the balloon producing this motor response defined the VRT. The procedure was repeated four times, and the mean of the last
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three values was used as the test parameter. Cutoff distention pressure was 100 mm Hg to prevent tissue damage. Two investigators participated in the tests, which were performed blinded to the observer of the VRT. The normal baseline value of the VRT is 55 { 4 mm Hg (mean { SEM); as a result, 99% of the normal reactions appear in the range between 35 and 75 mm Hg (mean { 2SD). Rabbits with a baseline value below 75 mm Hg are considered normally reacting rabbits.
Somatic Noxious Stimulation Transcutaneous electrical stimulation was applied to examine the existence of segmental effects. The current was applied through two metal electrodes, 2 cm apart. It was generated by a constant current stimulator (Elpha 500; Biometer, Odense, Denmark), and the characteristics were 100 Hz and a pulse width of 0.2 milliseconds. The animals were tested in the area of the neck, cervical region, and lumbar region. The noxious electrical stimulation threshold was the intensity (milliamperes) at which rabbits tried to escape the stimulus. The test value was the average of three ascending determinations. Again, two investigators took part in the procedure to obtain the results in a blinded fashion.
Drug Protocol Drugs for IT injections were mixed so that all doses were administered in a 0.5-mL solution followed by a 0.2-mL isotonic saline solution to flush the catheter. Control values were obtained by injection of 0.5 mL isotonic saline. IM injections of the drugs were given in a volume of 1 mL. The compounds used were 0.002–0.2 mg naloxone (5.5–550 nmol; nonselective opioid receptor antagonist), 0.25 mg MR2266 (770 nmol; selective k receptor antagonist28), and 0.5 mg naltrindole (1100 nmol; selective d receptor antagonist29). Naloxone solutions were made from commercially available solutions without preservatives. MR2266 and naltrindole solutions were made from the powder form of the compounds on the day of the experiments. Naltrindole was dissolved in a 20% (wt/vol) concentration of 2-hydroxypropyl-b-cyclodextrin. MR2266 was dissolved in a combination of sterile water and 10 mmol/mL HCl (pH 2.2). MR2266 was a gift from Boehringer Ingelheim (Ingelheim am Rhine, Germany). Naloxone and naltrindole were purchased from Research Biochemicals Inc. (Natick, MA). Before drug administration, control values of the VRT and the electrical stimulation threshold were obtained; postinjection thresholds were measured 15 minutes later. The doses for MR2266 and naltrindole were chosen, because they were the highest doses obtainable in a volume of 0.5 mL. The following experiments were performed for each compound. Initially, the response from the maximum dose was determined after IT injection. If a significant response was obtained, a dose response was performed, effect after IM administration of the largest dose, and segmental distribution of the IT effect were assessed. At least 3 days were allowed to pass between experiments, and the same rabbits were used.
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Statistical Evaluation Data are expressed as mean { SEM values unless otherwise stated. Despite the use of nonparametric statistics, mean values were chosen to present the data because of the relatively small number of animals in each group. Statistical computations were performed using the actual threshold values from each animal. To obtain the dose-response curve, the dose was plotted against the percent change as follows: % Change Å Postdrug Threshold 0 Predrug Threshold 1 100. Predrug Threshold Statistical evaluations of paired data were performed using Friedman’s analysis of variance (ANOVA) when more than two groups were present and using Pratt’s test for two groups. Unpaired data were evaluated by Kruskall–Wallis test for more than two groups and the Mann–Whitney U test for two groups. To eliminate the risk of mass significance, a significant P value of the ANOVA was requested before comparing different doses or times with placebo or control values, respectively. Linear regression was performed between dose response and log dose values. An F test was performed to test the variance of the regression. A P value of õ0.05 was chosen as the level of significance.
Results Influence of Age on VRTs Eight rabbits from the naloxone batch and 8 rabbits from the MR2266 batch were allowed to get old enough to increase their VRT values. The initial mean threshold for these rabbits was 52.8 { 1.7 mm Hg; with increasing age, the value changed to 93.2 { 2.0 mm Hg. This difference was highly significant (P õ 3 1 1009). Effects on Motor Function In 1 rabbit with a high VRT, IT MR2266 (0.25 mg) produced a staggering walk for approximately 5 minutes. This effect was interpreted as a sign of minor motor dysfunction caused by injection of MR2266. At the time of testing, the rabbit showed a normal ability to walk and jump. At the end of the study, 1 rabbit received an IT injection of 0.5 mg of MR2266, resulting in a paralysis of the lower part of the body for 20 minutes, after which the animal regained normal motor function. Neither naltrindole nor naloxone produced any observable effects on motor function. No other adverse reactions were observed throughout the study, and the rabbits thrived and gained weight. / 5E0F$$0017
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Figure 1. Naloxone-induced decrease in VRTs in rabbits. % Change represents the difference in VRTs after colorectal distention expressed in percentage of the control values. The log-dose response is linear (P õ 0.05; F test). Values are mean { SEM; n Å 8 for each point representing naloxone values. 䊏, Naloxone; 䊊, 0.5 mL IT isotonic saline (n Å 6). *P õ 0.05 compared with saline.
Effects of Isotonic Saline and Acidified Sterile Water Injection of 0.5 mL isotonic saline had no effect on the VRTs: 49.1 { 6 mm Hg before vs. 49.2 { 6.7 mm Hg after administration (P Å 1.0; n Å 6). Sterile water acidified to a pH of 2.2 also had no effect: 51.6 { 3.5 mm Hg before vs. 48.8 { 6.5 mm Hg after administration (P Å 0.8; n Å 6). Effects of Naloxone IT naloxone resulted in a linear log-dose response (P Å 0.01; F test) in rabbits with normal VRTs (Figure 1). Compared with placebo, a significant difference was observed after IT injection of 0.2 mg naloxone (49.3 { 2.5 vs. 37.8 { 3.8 mm Hg; P Å 0.04). IM administration of 0.2 mg naloxone also reduced normal VRTs significantly (60.0 { 2.7 vs. 52.7 { 3.8 mm Hg; P Å 0.02; Figure 2). IT naloxone, 0.2 mg, did not affect electrical stimulation thresholds at either lumbar or cervical level. In contrast, IM naloxone reduced electrical stimulation thresholds at the lumbar (16.2 { 0.7 vs. 13.8 { 0.7 mA; P õ 0.05) and cervical region (8.8 { 0.7 vs. 6.7 { 0.8 mA; P õ 0.01). Although statistical comparison between the VRT values obtained after IT and IM administration did not reach significance in rabbits with normal VRTs, the reduction after IM naloxone was less than after IT naloxone; 12.5% { 3.6% reduction (baseline value, 60.0 { 2.7 mm Hg) in VRT after IM administration vs. 23.8% { 6.2% reduction (baseline value, 49.3 { 2.5 mm Hg) WBS-Gastro
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after IT administration. This tendency was also observed in rabbits with high control VRTs: 20.1% { 5.0% (baseline value, 98 { 1.6 mm Hg) vs. 31.2% { 5.3% (baseline value, 92.8 { 1.8 mm Hg) for IM and IT, respectively (Figure 2). Statistical evaluation of the pooled data from rabbits with normal and high VRTs resulted in a significant difference between IT and IM administration (26.9% { 3.9% [baseline value, 67.9 / 6.2 mm Hg] vs. 16.3% { 3.1% [baseline value, 79.0 / 5.1 mm Hg] reduction for IT and IM, respectively; P õ 0.05) (Figure 2). IT (31.2% { 5.3%) and IM (20.1% { 5.0%) naloxone produced a significant reduction in VRTs in older rabbits with high control VRTs (Figure 2). Statistical comparison in the percentage reduction after IT and IM naloxone in rabbits with normal and high thresholds did not show any significant difference (P Å 0.18 and P Å 0.21 after IT and IM administration, respectively). Effects of MR2266 and Naltrindole Figure 3 shows the results after IT and IM injection of MR2266 and naltrindole. No significant effects were observed by either IT administration (MR2266, 49.5 { 3.6 vs. 54.3 { 7.1 mm Hg; naltrindole, 51.2 { 4.1 vs. 54.7 { 2.7 mm Hg) or IM administration (MR2266, 56.2 { 3.5 vs. 53.8 { 3.7 mm Hg; naltrindole, 62.8 { 4.1 vs. 60.0 { 2.7 mm Hg). IT MR2266 was also unable to produce significant changes in VRTs in rabbits with high values (92.2 { 4.1 vs. 81.8 { 7.3 mm Hg), and additional administration of IT naloxone
Figure 3. The effects of IT and IM MR2266 (k antagonist) and naltrindole (d antagonist) on VRTs. No significant changes were observed. 䊐, MR2266 (0.25 mg); , naltrindole (0.5 mg). Results are mean { SEM; n Å 8 for MR2266 and n Å 6 for naltrindole. % Change represents the difference in VRTs after colorectal distention expressed in percentage of the control values.
after MR2266 produced a significant reduction in VRTs in rabbits with both normal and elevated thresholds (Figure 4). Comparisons of the changes obtained after IT and IM MR2266 (15.4 { 18.6 vs. 03.3% { 3.9%) and naltrindole (11.2 { 12.6 vs. 03.7 { 5.2) did not show any significant differences (Figure 3). Naltrindole was not examined in rabbits with high thresholds.
Discussion
Figure 2. Changes in VRTs after IT and IM naloxone (0.2 mg). Values are expressed in percentage of the control values. Each bar represents values from 8 rabbits (mean { SEM). Values were obtained from rabbits with normal (õ75 mm Hg; 䊐) and high (ú75 mm Hg; ) baseline thresholds to colorectal distention. Pooled data (⽧) represent data from both normal- and high-threshold rabbits. *P õ 0.05 compared with the corresponding control values. -∗-P õ 0.05.
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In the present study, IT and IM naloxone produced a significant decrease in VRTs. The selective d antagonist naltrindole and the selective k antagonist MR2266 had no effect in the dose ranges studied. On a molar basis, the doses of naltrindole and MR2266 exceeded the highest dose of naloxone. Naltrindole and MR2266 have higher affinity for their respective receptors than naloxone and were given in larger doses in the present study; therefore, the effects observed after naloxone were not caused by d or k antagonism. Because of motor impairment, MR2266 was given in the highest dose possible, and the amount of naltrindole was limited by the high lipid solubility of the compound. Furthermore, the present doses of naltrindole and MR2266 antagonized the profound effects on VRTs caused by IT administration of their corresponding agonists D-pen25 D-pen-enkephalin and U50488H. Although naloxone antagonize all three opioid receptors, it shows the highest affinity for the m receptor and is the drug of reference in the existing literature concerning endogenous opioid modulation of motor reflexes.2 – 5 The more selective anWBS-Gastro
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Figure 4. Changes in VRTs after IT MR2266 (0.25 mg) in normalthreshold (䊐) and high-threshold ( ) rabbits (see Figure 2). No significant changes were observed. Administration of IT naloxone in the presence of MR2266 significantly reduced the thresholds compared with the control values obtained before administration of MR2266. Each bar represents values from 8 rabbits (mean { SEM). *P õ 0.05, # P õ 0.01.
tagonists b-funaltrexamine is an irreversible antagonist at the m receptor,30 which would have caused problems in the setup of the present study because the rabbits were examined more than once. The selective m receptor antagonist CTOP is a peptide and has no effect after parenteral administration.31 The involvement of m but not d and k receptors in the tonic inhibition of spinal motor reflexes has been shown earlier2; the effect at the k receptor was considered to be indirect. The present study is the first to show a lack of effect after IT administration of a selective k antagonist. Tattersall et al.17 showed the presence of both descending and intrinsic spinal inhibition of visceral noxious stimuli in cats, but they did not examine the neurotransmitters involved. Tonic opioid inhibition of visceral noxious information has not been reported previously. In a study on colorectal distention, Ness and Gebhart32 administered naloxone to antagonize the effect after morphine. They found that naloxone (3 mg/kg intravenously) antagonized the effect of morphine on the VRT, but the threshold after naloxone did not differ from control value, i.e., there were no signs of endogenous opioid antagonism implicating the existence of a tonic active system. The dose of naloxone used was larger than the highest dose used in the present study (0.07 mg/kg), which produced a significant reduction in VRTs after IM administration. These results in rats obtained by Ness and Gebhart32 were confirmed in other studies33,34 by the same laboratory; naloxone eliminated the inhibitory effects of different opioids but did not change the baseline firing rate of / 5E0F$$0017
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nociceptive neurons or the VRTs. These studies were not designed to detect the effect of naloxone, and the numbers of animals receiving naloxone were too low to perform statistical evaluations of the results. Thus the results do not exclude the possibility of a tonic opioid system involved in the modulation of noxious visceral information in the spinal cord of the rat. Furthermore, the results were obtained in the presence of morphine and cannot be interpreted as an effect after naloxone alone. The same argument can be used against the work by Kawamata et al.19 They administered IT naloxone to antagonize the effects of parenterally administered morphine. Naloxone reduced the effect of morphine on VRTs but did not reduce the thresholds below the control values. The existence of descending inhibition of visceral noxious information has been shown previously.17 – 21 Studies by Kawamata et al.19 and Iwasaki et al.20 indicate that part of this inhibition is caused by a descending endogenous opioid system, which can be activated by parenteral administration of opioids. IT naloxone inhibited the effects of subcutaneous morphine on VRTs,19 and because the effects following parenteral administration of drugs are exerted mainly at the supraspinal level, as shown by Vigouret et al.,35 these results imply that parenterally administered opioids activate a descending inhibitory opioid system, which terminates at the spinal level. Vigouret et al.35 showed that the effect of parenteral morphine is not only mediated by a descending opioid system but also through descending serotonergic, a-adrenergic, and cholinergic systems. Iwasaki et al.20 showed that 1 mg/kg of subcutaneous naloxone antagonized the pregnancy-induced increase in VRTs. The data from the present study on IM naloxone support these observations and confirm the existence of a tonic endogenous opioid system in the modulation of noxious visceral information. Occurrence of increasing VRTs to colorectal distention with increasing age has been reported earlier anecdotally.26 In the present study, this observation was verified and the result was highly significant. Age-related changes in the nociceptive system have been reported previously in rats.36 The biological significance of this observation is unclear, and the present results imply that this observation is not caused by an increased activity in the endogenous opioid system in older rabbits, because no significant differences were found between the effects of naloxone in young and old rabbits. Although no significant difference could be detected between IT and IM naloxone in the two separate groups of rabbits (with normal or high thresholds), the tendency towards a more pronounced effect after IT administration in both groups and the significant difference between IT and IM administration in the pooled data point to the WBS-Gastro
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existence of both supraspinal and intrinsic spinal opioid inhibitory systems. It is beyond the scope of the present study to determine and compare the potency of the two systems. The effects of naloxone on electrical stimulation thresholds did not supply further information concerning the segmental effect but indicate that somatic electrical noxious stimuli are only modulated by a supraspinal opioid system, i.e., a significant reduction in electrical stimulation thresholds after IM naloxone and no effect after IT administration were observed. Theoretically, the effect observed after naloxone administration could be a direct action at the motor neurons resulting in increasing spinal reflexes. Duggan and Zhao37 used intracellular recordings from motor neurons and injections of naloxone extracellularly and found that naloxone had no direct effect on the motor neurons. They concluded that the effects of naloxone on muscle reflexes are caused by antagonism of spinal interneurons. The differences observed between rats and rabbits cannot be explained from the present study, but the use of different species may produce different results. It has been shown that the presence of opioid receptors varies between species.38 – 42 Rabbits have a high density of k receptors in the spinal cord,40 whereas adult rats have few receptors.41,42 Age-related changes36 and genetic differences43 have also been reported. The density of k receptors in the spinal cord of rats diminishes with increasing age42; in mice, the sensitivity to both m and k agonists varies from 0% to 90%43 within different genetic strains. Older rats do not develop allodynia after sciatic nerve ligation,44 and older rabbits respond with higher thresholds to CD (Jensen et al.26 and present study). These differences may be a result of changes in the transmission at the spinal cord caused by excitatory amino acids45 or, as indicated in the present study, differences in the activity of the intrinsic opioid system. In conclusion, the present study shows tonic active intrinsic spinal and descending opioid inhibitory systems modulating visceral noxious stimuli in rabbits, probably caused by interactions at the m opioid receptor.
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Received October 23, 1995. Accepted March 25, 1996. Address requests for reprints to: Finn Molke Borgbjerg, M.D., Pain Clinic, Department of Anesthesiology, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark. Fax: (45) 3531-39-58. Supported by grants from the Danish Medical Associations Foundation (086.51) and Christine Jensa La Cours Foundation. The authors thank William Cook, Europe, for preparing the balloon catheters and Jens Randrup for technical assistance.
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