Psychopharmacology (1988) 96:36-39 Psychopharmacology Springer-Verlag 1988

"Paradoxical" analgesia induced by and

Janet D. Greeley*, A.D. L~, Constantine X. Poulos, and Howard Cappell Addiction Research Foundation, 33 Russell Street, Toronto, Ontario, Canada, M5S 2St, and Departments of Psychology and Pharmacology, University of Toronto, Toronto, Ontario, Canada, M5S 1At

Abstract. Analgesic effects of pellet implantation of the upregulation in receptors that occurs when opiate opiate antagonists naloxone and naltrexone and of chronic antagonists are administered chronically (Bardo et al. 1984; administration of naloxone by subcutaneous injection were Tempel et al. 1985; Yoburn et al. 1986). examined. Rats were implanted with a slow-release pellet In our own laboratory, we have found that chronic ad- containing 10 mg naloxone or 10 mg naltrexone and tested ministration of naloxone induces analgesia as measured on for paw-lick latency on a hotplate apparatus. Controls were a hotplate (Greeley et al. 1985). Repeated subcutaneous in- implanted with placebo pellets or given saline injections jection was the only pharmacologic method used in that as appropriate. There were five test trials at intervals up study. We decided to pursue this issue by validating our to 72 h after implantation of naloxone and up to 120 h original findings with a number of methodological varia- after the implantation of naltrexone. In a separate experi- tions; below we report three experiments in which analgesia ment, 5 mg/kg naloxone was injected; there were single was progressively recruited in rats repeatedly exposed to trials on 5 consecutive days. All drug-treated animals dis- painful stimulation following injection or implantation with played clear and substantial analgesia by their second test the opiate antagonists naloxone and naltrexone. trial. This "paradoxical" analgesia was gradually reversed in the pellet-implant groups as tissue levels of the antago- General methods nists declined, but increased progressively with each trial involving injections. It was hypothesized that blockade of Animals. The animals used throughout these studies were endogenous by antagonists resulted in a form of male Sprague-Dawley rats weighing in the range of "super-pain" on the hotplate, which in turn activated a 220-280 g, obtained from Charles River (Montreal, Que- normally redundant "backup" analgesic system. The re- bec). They were housed individually and allowed free access sults with naloxone injections show that unlike opiate-me- to standard laboratory chow and tap water. The tempera- diated analgesia, this hypothetical system is resistant to tol- ture of the colony room was maintained at 22___ 1 ~ C. erance. Key words: Analgesia - Pain - Naloxone - Naltrexone - Apparatus and procedure. Analgesia was assessed through- out using a hotplate apparatus consisting of a plastic stor- Opiates age tub with an aluminum plate on the surface (Grant In- strument Water Bath Model #ABO2). The temperature of the surface was monitored continually with a thermistor Since its initial development, naloxone has been considered (Yellow Springs Instruments Model # 43 TC) and probes to be an antagonist of the actions of opiates with little (Yellow Springs Instruments banjo surface probes Model agonist action of its own. To the extent that analgesia is #408) taped securely to the surface of the aluminum plate. mediated by the activation of endogenous opiates (Akil Rats were confined to the surface of the hotplate with a et al. 1976), naloxone should be expected only to antagonize clear plexiglass box (27 x 16.5 x 9 cm high) and lid. The in- analgesia, and indeed, when administered acutely in the dex of analgesia was latency to lick either the front or back absence of drugs or other analgesia-inducing manipula- paw following placement on the surface of the hotplate tions, naloxone tends to increase responsiveness to pain which was maintained at 49.5 ~ C (Jacob and Ramadabran (Kirchgessner et al. 1982; Mayer 1983; Grevert and Gold- 1978; Cordere and Rollman 1983; Greeley 1987). Each rat stein 1985). However, chronic administration of naloxone was tested from three to five times in baseline determina- or naltrexone has had the theoretically interesting conse- tions that were used to assign animals to experimental con- quence of enhancing the analgesic effect of a later injection ditions. The maximum latency permitted in any test was of (Bardo et al. 1984; Tempel et al. 1985; Yoburn 30 s; if a pawlick response did not occur within this time, et al. 1986). This enhancement has been attributed to the the rat was removed form the apparatus and assigned a score of 30 s. * Present address: National Drug and Alcohol Research Centre, University of New South Wales, Kensington, New South Wales, Naloxone and naltrexone pellets. Slow-release pellets of nal- Australia oxone and naltrexone were prepared following the method Offprint requests to." H.D. Cappell of Misra and Pontani (1978) and Bardo et al. (1984). Each 37

50-rag pellet contained 10 mg naloxone or naltrexone as 30 Naloxone Pellet Naltrexone Pellet .Naloxone injection base. Placebo pellets contained 45 mg cholesterol and 5 mg glyceryl tristerate. Pellets were implanted under halothane anesthesia in the dorsal area behind the right front limb of the animal. •2 0 co v ..J Experiments 1A and 1B: pellet implantation _J n Io! Naloxone and naltrexone are both opiate antagonists that are efficacious when implanted subcutaneously (Bardo et al. 1985; Tempel et al. 1985). The principal pharmacological difference between the two agents is that naltrexone is lon- O' 4 2'4 218 4=8 7'2 214 48 72 96 120 0 24 48 7~ 9'6 ger-acting. Experiments IA and 1B permitted a test of the Hours efficacy of these agents in inducing analgesia with repeated Fig. 1. Mean paw-lick latencies (PLL)+_standard error of rats painful stimulation using the hotplate technique. treated with various forms of opiate antagonist. Solid circles repre- sent latencies of antagonist-treated rats; open circles represent pla- Experiment 1A : naloxone pellet cebo controls. In the case of pellet implantation (left and center panels), the horizontal axis represents the intervals between hotplate Procedures. The subjects were 56 male Sprague-Dawley rats tests following a single implant. In the case of injections (right weighing approximately 280 g at the beginning of the exper- panel), the horizontal axis represents the intervals at which separate iment. They were assigned to groups matched on the mean injections were administered followed by hotplate testing latencies determined on the basis of three baseline screening trials. Twenty-eight rats (group nalox/p) were implanted groupsx trials interaction (F=3.00, df=4,216, P<0.05). subcutaneously with a pellet containing naloxone; an equal The mean paw-lick latency of group naltrex/p exceeded that number were implanted with a placebo pellet (group plac/ of group plac/p on trial 2 (P<0.01) and on trial 3 (P< p). Rats were retested on the hotplate 4, 24, 28, 48 and 0.05). By the fourth and fifth trials, the mean latencies of 72 h following implantation. All tests were conducted dur- the two groups were not statistically different. ing the light-phase of the subjects' natural diurnal cycle. Once more, the result was a progressive increase in anal- gesia in the group treated with the opiate antagonist. More- Results and discussion. The results are illustrated in the left over, to the extent that analgesia was evident on two consec- panel of Fig. 1. An ANOVA yielded a significant group x utive trials as opposed to only a single trial, there was a trial interaction (F= 2.65, df= 4,224, P < 0.05). correspondence between the duration of action of naltrex- Tests of main effects on each trial showed that there one in comparison to naloxone and the relative duration was no group difference in latency on the first hotplate of the efficacy of the two antagonists in producing analge- exposure after implantation. On trial 2, the latency of of sia. In both cases, the effect did not emerge until the second group nalox/p was significantly longer (P < 0.05) than that test on the hotplate. The length of the interval between of group plac/p. The difference was no longer significant the implantation and testing was not critical to the delayed by trial 3, and by trial 4 the latencies of the two groups emergence of the effect since the same pattern was evident were again quite similar. with hotplate test intervals of both 4 h (naloxone) and 24 h In this experimental paradigm, naloxone produced a (naltrexone). The delayed emergence of the effect suggests progressive increase in analgesic response compared to sa- that the display of analgesia was dependent upon a recruit- line controls. However, the effect was modest in size and ment process that was initiated with the first exposure to shortlived. One hypothesis is that this was due to the rela- painful stimulation with the antagonist present. Although tively short duration of the pharmacologic effectiveness of the effect was longer-lived in experiment 1B than in experi- the implant of naloxone. If so, the longer-acting antagonist ment 1 A, the analgesic response was not very persistent naltrexone should produce a similar but stronger effect. with either naloxone or naltrexone. The most obvious ex- This hypothesis was tested in experiment 1B. planation for this is that once recruited, the time course of the analgesic action followed the time course of the tissue Experiment lB." naltrexone pellet levels of the antagonists. Therefore, repeated testing after Procedures. The subjects were 56 male Sprague-Dawley rats acute injections of an antagonist should show the same weighing approximately 280 g at the beginning of the exper- evidence of a recruitment process, but with an effect that iment. The procedures were generally the same as those persists for as long as the antagonist is present in sufficient followed in experiment 1 A with several changes: five base- quantity on each test trial. This hypothesis was tested in line screening tests on the hotplate were administered; the experiment 2. first test after implantation was delayed until 24 h following surgery; subsequent tests were conducted 48, 72, 96, and Experiment 2: naloxone injections 120 h following the implant; there was no 30-s upper limit Procedure. Forty-eight rats, each weighing approximately imposed on response latency. Twenty-eight experimental 300 g when experiment 2 began, were assigned in equal animals (naltrex/p) were implanted with a pellet containing numbers to two groups matched in initial sensitivity as de- naltrexone, and an equal number of controls (plac/p) were termined in baseline tests on the hotplate. Rats in group implanted with a pellet composed of base alone. nalox/sc were administered 5 mg/kg naloxone subcutane- ously 15 rain prior to testing on the hotplate, and controls Results and discussion. The results are shown in the center (plac/sc) were given physiological saline. There were single panel of Fig. 1. As in experiment 1, there was a significant trials on each of 5 consecutive days. 38

Results and discussion. The results of this comparison are sia in saline-treated controls. In fact, such controls typically illustrated in the fight panel of Fig. 1. The increasing paw- became more sensitive with repeated hotplate testing (cf. lick latency over trials among rats in group nalox/sc com- also Kayan etal. 1973; Siegel 1976; Frederickson 1978; pared to plac/sc was reflected in a significant groups x trials Cordere and Rollman 1983; Greeley 1987; Rochford and interaction (F= 4.52, df= 4,184, P < 0.01). Analyses on indi- Stewart 1987). The implication of this line of thinking is vidual trials revealed that as in the previous experiments, that naloxone converted an otherwise inefficacious sensory there was no difference on the first test trial. By the second stimulus into one that was sufficiently intense to provoke trial, the latency of group nalox/sc was significantly greater the recruitment of analgesia. Rochford and Stewart (1987) than that for controls (P<0.001). The difference between reported a series of experiments in which analgesic effects the two groups was maintained over the final four trials. of naloxone were examined. Since the parameters and pro- The results of experiment 2 clearly confirmed that the cedures employed by Rochford and Stewart were so differ- analgesia produced by an opiate antagonist would be main- ent from the ones used here, comparison of the results is tained if there was an adequate drug level on each test difficult. In one series of studies, they found that naloxone- trial. Moreover, in experiment 2 there was no sign of habit- induced analgesia was only relative; although naloxone pre- uation or tolerance over the four trials following the emer- vented the increase in pawlick latencies that was observed gence of the effect. in saline controls, there was no progressive increase in anal- gesia as was observed in the present work. Therefore, they proposed that naloxone exerted its effect by interfering with General discussion the habituation of stress-induced analgesia. At a physiologi- In three separate experiments with different pharmacologic cal level, they suggested that naloxone blockade of opiate- procedures, repeated painful stimulation resulted in analge- mediated analgesia may stimulate the recruitment of non- sia in rats treated with an opiate antagonist. This confirms opiate analgesia through a process of collateral inhibition. and extends our earlier findings (Greeley et al. 1985). How- Reciprocal inhibition between distinct analgesic systems has ever, even with antagonist-induced analgesia firmly estab- been proposed by others (Kirchgessner et al. 1982) and it lished as an empirical phenomenon, the question of mecha- is consistent with our data. However, the progressive abso- nism arises. Any postulated mechanism must be capable lute increase in analgesia over trials, especially as seen in of addressing several features of the phenomenon. It must experiment 2 with repeated acute administrations of nalox- take into account that some form of recruitment may be one, is difficult to explain as an attenuated form of habitua- required, since in none of the three experiments did the tion. analgesic effect appear until after the application of some Our findings suggest that there is redundancy in an or- painful stimulation with the antagonist present. It must also ganism's ability to respond to painful stimulation; what take into account the magnitude of the effect, which was remains a puzzle is how the action of antagonists brings considerable; by the fifth test in experiment 2, the latency this normally redundant system into play when painful of rats injected with naloxone was three times that of con- stimulation in saline-treated controls appears not to engage trols. Finally, it must be able to integrate the observation such a system at all. Although there was no evidence for that there was no indication of tolerance or habituation, this in response latencies on the first hotplate exposure with at least within the five trials of experiment 2. This is in an antagonist present, perhaps painful stimulation in the contrast to the analgesia produced by morphine, to which presence of an antagonist results in pain so intense (" super- some tolerance is evident even on a single exposure (Kayan pain") that a nonopiate "backup" system is activated. It et al. 1973). is especially interesting that if our findings reflect the opera- One type of mechanism that warrants consideration is tion of such a "backup" system, it has the special feature the upregulation of opiate receptors. This provides an ade- of being relatively resistant to tolerance within the limits quate explanation for the enhancement of analgesia by mor- studied in experiment 2. Opiate-mediated analgesia, in con- phine following treatment with an antagonist (Bardo et al. trast, is subject to rapid tolerance. 1984; Tempel et al. 1985; Yoburn et al. 1986). However, Although we cannot answer the numerous questions it remains to be established how such a mechanism could that these data provoke, it is clear that we have affirmed account for the analgesia produced by an antagonist on the occurrence of an intriguing phenomenon in three inde- its own. Opiate receptors could only be involved if they pendent validations. Whatever the mechanism, the phenom- were induced at a rate and extent that exceeded the capacity enon of naloxone-induced analgesia bespeaks a remarkable of antagonists to block their effects. Perhaps there could plasticity of the system for the regulation of pain. be an upregulation of specific opiate receptors that are rela- tively refractory to the effects of naloxone (i.e., kappa or Acknowledgements. This research was supported in part by a grant delta receptors, Lord et al. 1977; Martin 1978). If so, the (A2612) from the Natural Science and Engineering Research Coun- process of upregulation must be such that it can account cil of Canada to C.X.P and H.C. We gratefully acknowledge the for an increasing efficacy of the same dose of antagonist the support of E.]. duPont de Nemours & Company, which gener- with chronic treatment as in experiment 2. Without these ously supplied the naloxone used in these experiments. properties, there could not have been the conditions neces- sary for endogenous opiates to mediate the analgesia that was observed. References Alternatively, the effect could reflect a nonopiate-me- Akil H, Mayer D J, Liebeskind JC (1976) Antagonism of stimula- diated analgesia (Bodnar et al. 1978; Spiaggia et al. 1979; tion-produced analgesia by naloxone, a antagonist. Kirchgessner et al. 1982). 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