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Departments of *Physiology, Faculty of Medicine, and Physiotherapy, Faculty of Health Sciences, University of Szeged, Hungary; and Department of Anesthesia and Intensive Care, Danube Hospital, Vienna, Austria
Address correspondence to Gyongyi Horvath, Department of Physiology, Faculty of Medicine, University of Szeged, P.O. Box 427, H-6701, Szeged, Hungary. Address e-mail to horvath{at}phys.szote.u-szeged.hu.
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Abstract |
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2-adrenergic agonists, but the characteristics of the delayed type of acute tolerance have not been analyzed in acute and inflammatory thermal pain tests. We investigated the long-term changes in the antinociceptive potency of morphine (10 mg/kg) injected intraperitoneally and the 2-adrenoceptor agonist dexmedetomidine (150 µg/kg intraperitoneally) on acute heat pain (tail-flick test) sensitivity and on carrageenan-induced inflammatory thermal hyperalgesia (paw withdrawal test) after a second injection 7 days later. The first treatment did not influence the baseline values on Day 8 in either test. In the tail-flick test, the antinociceptive potency of morphine, but not that of dexmedetomidine, was significantly decreased after repeated administration, suggesting a delayed type of acute tolerance to morphine. In contrast, the antihyperalgesic effect of morphine in the paw withdrawal test did not change after repeated injection, whereas the potency of dexmedetomidine was increased on Day 8. There were significant differences between the inflamed and noninflamed sides on Day 1 but not on Day 8, revealing an increased potency of the drugs on the inflamed side. There was no sign of cross-tolerance between the two drugs in either pain test. These data indicate long-term changes in the antinociceptive potency of morphine or dexmedetomidine after single treatment in different heat pain tests. |
Introduction |
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Only a few data are available regarding the duration of acute tolerance, and the results are controversial (2,4,6–10). More than 30 years ago, Shuster et al. (6) observed that the running response to morphine increased when this drug was administered once a week. This effect was not associated with analgesic tolerance, as measured by the tail-flick assay, in Week 5. In contrast, it was reported some years later that a single dose of morphine produced two different types of tolerance—acute and delayed—to hypothermic and antinociceptive effects (2,7,8). Acute tolerance was apparent by 4.5 h and lasted for at least 20 h after morphine administration, whereas the long-term tolerance developed within 24 h, reached the maximal level on Day 3, and persisted for 11 days. The long-lasting effects of systemic heroin and fentanyl were also revealed in rats during the paw pressure vocalization test, i.e., delayed hyperalgesic effects were observed, which might be linked to the delayed type of tolerance described by Yamazaki and Kaneto and other investigators (8–10).
Tolerance to other potent analgesics, such as 2-adrenoceptor agonists (clonidine or dexmedetomidine), can also develop after repeated administration, and cross-tolerance between 2-adrenoceptor agonists and morphine has also been observed (11). However, it has additionally been demonstrated that clonidine is able to inhibit some precipitated withdrawal symptoms in morphine-dependent rats. This effect of clonidine, similarly to its analgesic property, is naloxone insensitive (12). Only one study has shown that a single dose of clonidine resulted in long-lasting tolerance, which was nearly equipotent to and of somewhat shorter duration than that of morphine (8). That study further demonstrated that clonidine-tolerant animals were also tolerant to morphine and vice versa and that this was not suppressed by naloxone.
These earlier studies investigated the delayed type of acute tolerance in acute mechanical pain tests. Because the mechanisms that play roles in the heat versus mechanical pain pathways differ, we investigated the long-lasting effects of morphine and dexmedetomidine on pain sensitivity and the changes in their potency after a second injection 7 days later in acute heat pain (tail-flick test). We applied dexmedetomidine instead of clonidine because it has much higher selectivity for the 2-adrenoceptors (13,14). Furthermore, no data are available regarding the effects of these drugs during thermal hyperalgesia conditions after repeated administration. Accordingly, the second goal of this study was to determine whether the administration of these drugs could induce a delayed type of acute tolerance in rats with carrageenan-induced hindpaw inflammation.
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Methods |
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Acute nociceptive sensitivity was assessed by using the tail-flick test. The reaction time in the tail-flick test was determined by immersing the lower 5-cm portion of the tail in hot water (51°C) until a tail-withdrawal response was observed (cutoff time, 20 s). Baseline latencies were obtained immediately before and then 10, 30, 60, and 90 min after drug injections. The same paradigm was repeated 7 days later.
The inflammatory pain sensitivity was determined by the paw withdrawal (PWD) test after carrageenan administration (a detailed description of this method has been published by Hargreaves et al. (15). Briefly, the rats were placed on a glass surface in a plastic chamber and were allowed to acclimatize to their environment for 15–20 min before testing, and heat stimulus was then directed onto the plantar surface of each hindpaw. The cutoff time was set at 20 s to avoid tissue damage. After obtaining the baseline hindpaw withdrawal latencies (precarrageenan baseline values at –180 min), unilateral inflammation was induced by the intraplantar injection of 1.5 mg of 
-carrageenan in 0.1 mL of physiological saline into the right hindpaw. The PWD latencies were determined again 3 h after carrageenan injection (postcarrageenan baseline values at 0 min) and then 10, 30, 60, and 90 min after drug injection. The same paradigm was performed 7 days later, and carrageenan was injected again into the right hindpaw. Thus, all of the animals were injected on 2 occasions 7 days apart.
The schedule of the experiments and the names of the groups are indicated in Table 1. Morphine hydrochloride (10 mg/kg; Alkaloida, Tiszavasvari, Hungary) and dexmedetomidine (150 µg/kg; a generous gift from Orion-Farmos, Finland) were dissolved in sterile, physiological saline and were injected intraperitoneally (4 mL/kg). Our preliminary experiments indicated the rationale for the use of these doses of morphine and dexmedetomidine: both drugs were effective in both tests.
Data are presented as mean ± sem. Data sets were examined by two-way analyses of variance (ANOVA) (time and treatment factors) and correlation analysis by using STATISTICA software (Statsoft Inc, Tulsa, UK). Detailed analyses of area under the curve (AUC) values were performed by calculating the area during 10–90 min. We did not include the predrug value because we were interested in only the drug effects, and the baseline value did not differ significantly among groups. These data sets were also examined by two-way (time and treatment) ANOVA followed by subsequent one-way ANOVA (at each time of the experiment) and the Fisher least significance difference post hoc test when significant treatment effects were observed. A P value of <0.05 was considered significant.
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Results |
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In respect to the tail-flick test, both morphine and dexmedetomidine caused significant antinociception throughout the testing period (Fig. 1A). The two-way ANOVA of these time-course curves showed significant differences in tail-flick latencies by treatments, by time, and for the interactions (P < 0.001). The post hoc analysis showed that there were no significant differences between the morphine-dexmedetomidine (MO-DEX) and morphine-morphine (MO-MO) groups and similarly between the dexmedetomidine-morphine (DEX-MO) and dexmedetomidine-dexmedetomidine (DEX-DEX) groups at the first treatment, but the effects of the two drugs were different at several time points, i.e., the morphine was more effective than dexmedetomidine at 30 and 60 min.
The detailed statistical analysis of the AUC bars revealed that the antinociceptive potency of morphine was significantly higher than that of dexmedetomidine on Day 1 (Fig. 2A). In the DEX-DEX group, the effect of the drug was unchanged on Day 8 as compared both with Day 1 and the SAL-DEX group, whereas in the MO-MO group, a significant decrease in antinociception was observed on Day 8. In the DEX-MO group, the DEX pretreatment did not influence the effect of morphine, whereas in the MO-DEX group, there was a slight, nonsignificant increase (P = 0.11) in the effectivity of DEX as compared with the DEX-DEX group. Thus, the significant difference between the effects of morphine and dexmedetomidine was not observed on Day 8 between the MO-MO group versus the DEX-DEX or MO-DEX groups.
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Regarding the noninflamed side at the PWD test, neither the precarrageenan nor the postcarrageenan baseline values differed significantly among the groups; furthermore, neither the Day 1 treatment nor the inflammation on the opposite side significantly influenced the baseline values on Day 8 (Fig. 1B). The carrageenan injection resulted in a decrease in PWD latency on the noninflamed side in more than half of the rats, but these changes were similar in all groups and did not correlate with the treatment. The time response curves (Fig. 1B) revealed that both morphine and dexmedetomidine caused significant antinociception throughout the whole test period on both occasions. The AUC analysis (Fig. 2B) showed that the effects of the two drugs did not differ significantly on Day 1, but dexmedetomidine seemed to be slightly more effective in this test. However, on Day 8 there were significant differences between the MO-MO group and the MO-DEX and DEX-DEX groups, showing that dexmedetomidine had a significantly higher potency than morphine.
Neither the precarrageenan nor the postcarrageenan baseline values differed among the groups at the inflamed side; furthermore, these values were also similar on Day 1 and Day 8. The pain threshold for the inflamed paw decreased significantly after carrageenan administration (Fig. 1C). The time response curves (Fig. 1C) revealed that both morphine and dexmedetomidine caused significant antinociception throughout the whole test period on both occasions (at Days 1 and 8).
The detailed AUC analysis on the noninflamed side in the PWD test (Fig. 2B) showed that the effects of the two drugs did not differ significantly on Day 1, but dexmedetomidine seemed to be slightly more effective. However, on Day 8 there were significant differences between the MO-MO group (not the DEX-MO group) and the MO-DEX and DEX-DEX groups, i.e., dexmedetomidine had a significantly higher potency than morphine in this case, suggesting that morphine pretreatment exerted some influence on the effect of the second morphine treatment.
Neither the precarrageenan nor the postcarrageenan baseline values differed among the groups; these values were also similar on Day 1 and Day 8. The pain threshold for the inflamed paw decreased significantly after carrageenan administration (Fig. 1C). The carrageenan injection resulted in a decrease in PWD latency on the noninflamed side in more than half of the rats, but these changes were similar in all groups and did not correlate with the treatment. The saline treatment neither caused any changes in the pain threshold nor influenced the effects of these drugs in either test during the period of observation (SAL-SAL, SAL-MO, and SAL-DEX groups; Fig. 1). This means that the effects of morphine and dexmedetomidine were similar on Day 8 and Day 1 if saline was administered on the first occasion. Both morphine and dexmedetomidine caused significant antinociception throughout the whole testing period, as compared with the baseline saline-treated groups on occasions (Days 1 and 8) and in both tests (Fig. 1). The two-way analysis of these time-course curves showed significant differences in the tail-flick and PWD tests on Days 1 and 8 by treatment, by time, and for the interactions (P < 0.001).
The AUC analysis on the inflamed side showed that, similar as that of the noninflamed side, there were no significant differences in effectivity among the groups during the Day 1 treatment (Fig. 2C). However, the Day 8 treatment led to different results. The effectivity of morphine (MO-MO and DEX-MO groups) was unchanged, independently of the first treatment, whereas dexmedetomidine displayed an enhanced effect on Day 8 (DEX-DEX and MO-DEX groups). Furthermore, on Day 8, the effect of dexmedetomidine was significantly stronger than that of morphine.
The comparison of the inflamed and noninflamed sides revealed significant differences on Day 1 but not on Day 8, which was due to the higher potency of the drugs on the inflamed side 1 week later (Fig. 2D). The quotient of the inflamed versus noninflamed AUC values (AUCinflamed/AUCnoninflamed) was approximately 0.5 for the saline-treated groups, whereas for the other groups it ranged between 0.64 and 0.98. The differences between the SAL-SAL group versus the DEX-DEX and MO-DEX groups were significant at the second treatment, demonstrating that, in these cases, dexmedetomidine was more effective on the inflamed side than on the noninflamed side.
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Discussion |
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Our results for the delayed type of acute tolerance to morphine in the tail-flick test are in agreement with those of Yamazaki and Kaneto’s (8) mechanical test. In contrast, we could not observe delayed tolerance after dexmedetomidine administration, and there were no signs of cross-tolerance between these drugs, which is the opposite of their results. The difference might be explained by the different strains used (mice versus rats), the different drugs applied (clonidine versus dexmedetomidine), and the different pain tests (mechanical versus thermal). Dexmedetomidine has a higher specificity for 2-adrenoceptors and a faster metabolism (13,14), which could influence the development of a delayed type of tolerance. Because the half-life of dexmedetomidine is shorter than that of clonidine, the activation of the 2-adrenoceptor was shorter, and therefore, the sensitivity of the receptor could be normalized within a week.
Some data suggest that other µ-opioid receptor agonists, i.e., heroin and fentanyl, induce a delayed enhancement of mechanical pain sensitivity (hyperalgesia) for several days in rats (9,10). We did not find a decrease in baseline pain sensitivity in either test, which might have been because the drugs applied have different pharmacokinetic properties or the differences in the applied doses or tests. Célérier et al. (10) administered large doses that caused maximal antinociception immediately, whereas the dose in our study was not sufficient to provide the maximal analgesic effect.
We applied both acute heat and inflammatory thermal pain models and found different results in these two tests. Some earlier data likewise suggest differences in the potencies of drugs between acute and inflammatory pain tests. Thus, similarly to our results, morphine was found to be more potent than clonidine in the tail-flick test (16,17), whereas the PWD assay is not as sensitive a method as the tail-flick assay for testing the analgesic activity of opioids (18). Our results have revealed that the antinociceptive potencies of 2-adrenoceptor and opioid receptor agonists are qualitatively opposite in the two tests. Thus, the tail-flick test was more sensitive to opioid receptor agonists and less sensitive to 2-adrenoceptor agonists in comparison with the inflammatory test. These differences might be caused by a difference in bioassay sensitivity because both a learning effect and stress may diminish the sensitivity of the method. Moreover, inflammation significantly changes the levels of different endogenous ligands (19–21).
A rat model of repeated acute inflammation induced by two hindpaw injections of carrageenan 7 days apart was developed by Guilbaud et al. (22), this model at least partly mimicking some recurrent inflammatory pain states encountered in clinical situations. It was demonstrated that the first inflammation enhanced the mechanical pain relating to a second inflammation induced 7 days later at the same or a distant site (21,22). However, we did not find increased thermal hyperalgesia after the second inflammation. This might have been due to the difference in the tests (mechanical versus thermal) or in the dose of carrageenan applied (2 mg versus 1.5 mg).
The enhanced antinociceptive action of opioid agonists during inflammation is well established (18,23). Perrot et al. (21) investigated the potency of IV morphine (1 mg/kg) after a first and second carrageenan injection, applying a mechanical pain test, and found that morphine had a significantly higher potency after the second carrageenan injection. We performed a similar experimental paradigm in our pain test, but morphine (10 mg/kg) did not display an increased potency as compared with the first carrageenan injection (Figs. 1C and 2C). We again suggest that the differences in the pain tests or the applied doses might be the cause of these significant discrepancies. Some data have demonstrated that 2-agonists exhibit enhanced antinociceptive potency during inflammation, which are in agreement with our results (18,24). A likely interpretation of these data is that both the endogenous adrenergic and opioidergic systems might be modified in arthritic animals. Thus, a combined biochemical and behavioral study in polyarthritic rats suggested that in this state, the levels of certain endogenous opioids increase in the spinal cord (25). In addition, the spinal noradrenergic pathways possibly play a role in opioid antinociception during inflammation/hyperalgesia (18).
Chronic pain may alter morphine tolerance development, but chronic morphine induces tolerance in arthritic rats (23). However, the development of acute tolerance to opioids in long-lasting tonic pain may be different. Constant pain could, in principle, counteract the mobilization of antianalgesia systems participating in the development of acute tolerance. Kissin et al. (26) have shown that the inflammatory nociceptive input does not prevent the development of acute tolerance to opioid-induced analgesia in a mechanical pain test. It has been concluded that acute tolerance to the analgesic effect of opioids is profound and develops very rapidly, even in the presence of a constant nociceptive input. Because there was no decrease in the potency of morphine on Day 8 in the inflammatory model, we suggest that the inflammatory state might activate the endogenous antinociceptive systems and may change receptors’ sensitivities, which may mask the delayed type of tolerance to morphine and even increase the potency of dexmedetomidine (20,27).
The implications of these results are manifold. First, we have found that, in contrast with the mechanical pain test, in the heat pain tests, the delayed type of tolerance displayed different features. Second, our results lend further support to the well known fact that repeated treatments in animal studies may influence the results, even if there is a relatively large interval between the two experiments. Finally, because the potency of the dexmedetomidine injection repeated 7 days later was increased in the inflammatory pain model, it may be expected that 2-adrenoceptor agonists may still be effective analgesics when acute tolerance to morphine has developed. Thus, intermittent treatment with these two types of drugs could cause more effective antinociception.
The authors are grateful to D. Durham for editorial assistance with the manuscript.
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Footnotes |
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Supported, in part, by Hungarian Scientific Grant (OTKA) T-34741 and Health Scientific Grant (ETT) 042/2001.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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2-adrenoceptor agonist. Eur J Pharmacol 1988;150:9–14.[ISI][Medline]
2-agonist tolerance is blocked by NMDA receptor and nitric oxide synthase inhibitors. Anesthesiology 2001;95:184–91.[ISI][Medline]
-opioid antagonist. Neurosci Lett 1995;184:173–6.[ISI][Medline]
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