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Relationship between hypothalamic-pituitary-adrenal activity and blockade of tolerance to morphine analgesia by pain: a strain comparison
Pain, 63 (1995)385-389
385
© 1995Elsevier Science B.V. All rights reserved 0304-3959/95/$09.50
PAIN 2859
Relationship between hypothalamic-pituitary-adrenal activity and blockade of tolerance to morphine analgesia by pain: a strain comparison A n t h o n y L. Vaccarino * and Leland C. Couret Jr. Department of Psychology, University of New Orleans, New Orleans, LA 70148 (USA)
(Received9 November1994,revisionreceived20 February 1995,accepted2 March 1995)
Summary We previously reported that morphine fails to produce analgesic tolerance when administered in the presence of formalin-induced pain. The hypothalamic-pituitary-adrenal (HPA) axis is known to respond to stressful stimuli, including pain. To examine whether the blockade of tolerance by pain is related to HPA activity, we assessed the development of tolerance to morphine analgesia in an inbred strain of rats that lack typical stress-induced HPA responses (Lewis strain). Lewis rats lack typical stress-induced activation of corticotropin-releasing hormone, adrenocorticotropin hormone and glucocorticoids. Female Lewis rats were injected with morphine (20 mg/kg, i.p.) or saline for 4 consecutive days in the presence or absence of pain induced by injection of formalin into the hind-paw. The analgesic effect of morphine (5, 10 or 20 mg/kg, i.p.)was then measured in the tail-flick test 24 h after tolerance induction. Inbred female Fischer rats, which show significant stress-induced HPA activity, were used for comparison. Analgesic tolerance was produced in both strains when morphine was delivered in the absence of pain. However, the presence of pain during morphine administration prevented the development of analgesic tolerance in Fischer, but not in Lewis, rats. The differential effects of pain on the development of tolerance to morphine analgesia are suggested to be related to genetically determined differences in stress-induced HPA activity. Key words: Morphine; Analgesic tolerance; Formalin-induced pain; Tail flick; Hypothalamic-pituitary-adrenal axis; Strain comparison; Fischer; Lewis; (Rat)
Introduction
Clinical studies have indicated that when opiates are used to control pain, tolerance and dependence are not a major concern (Melzack 1991; Portenoy 1994; Twycross 1994). In support of this, we recently reported that analgesic tolerance (Vaccarino et al. 1993) and dependence (Vaccarino and Couret, Jr. 1993) in rats do not develop when morphine is administered repeatedly in the presence of formalin-induced pain. A similar finding was also reported by Colpaert et al. (1980) in which tolerance to the analgesic effects of the
* Corresponding author: A.L. Vaccarino, Department of Psychology,
University of New Orleans, New Orleans, LA 70148, USA. Tel.: (504) 286-6771;FAX: (504) 286-6049. SSDI 0304-3959(95)00069-0
opiate, fentanyl, was blocked in rats exposed to a noxious mechanical stimulus during tolerance induction. The hypothalamic-pituitary-adrenal (HPA) axis responds to stressful stimuli (Friedman et al. 1967; Sternebreg et al. 1992; Aloisi et al. 1994), including formalin injection (Linton et al. 1985; Aloisi and Carli 1992). Adrenocorticotropin hormone (ACTH) has been found to prevent the development of tolerance to morphine analgesia (Hendrie 1988), and stress blocks the development of morphine tolerance in intact mice, but not in adrenalectomized mice (Takahashi et al. 1989). In addition, both adrenalectomy (Gebhart and Mitchell 1972; Wei 1973) and hypophysectomy (Holaday et al. 1979) have been shown to potentiate the magnitude of opiate tolerance, with the effects of hypophysectomy being reversed by replacement of ACTH (Holaday et al. 1979). This raises the possibility that blockade of tolerance development by formalin-induced pain is re-
386
lated to an activation of the HPA axis following formalin injection. Observations of different strains of rats suggest a genetic basis for variability of neuroendocrine and behavioural responses to stress (Keim and Sigg 1976; Pare and Glavin 1986; Sternberg et al. 1992). Recently, the Lewis strain of rat has been identified to lack typical stress-induced HPA activity. As compared to Fischer rats, Lewis rats lack or show low increases of hypothalamic corticotropin hormone (CRH), plasma ACTH and corticosterone following a variety of stressors, including restraint, open field, swim or ether (Sternberg et al. 1992). In the present study, we examined the relationship between stress-induced HPA activity and the blockade of tolerance to morphine analgesia by pain. In particular, we wished to find out whether Lewis rats, which show deficits in stress-induced HPA activity, do not show the antagonistic effect of pain on tolerance development. The existence of such strain differences may be useful for studying the 'natural' relationship between neuroendocrine ac-
tivity, pain, stress, and the development of morphine tolerance.
Methods
Subjects Inbred female Lewis (n = 120) and Fischer (n = 120) rats (Harlan Laboratories, USA) weighing 150-200 g served as subjects. Rats were housed individually and allowed free access to food and water, and were maintained on a 12-h light cycle (light onset at 07:00 h).
Tolerance induction Morphine sulphate (Malincrodt, USA) was dissolved in physiological saline and administered intraperitoneally (i.p.) in a volume of 0.1 ml/100 g of body weight. To examine the effects of morphine administration in the presence of pain, rats received 20 mg/kg morphine 10 min after a subcutaneous (s.c.) injection of 50 /zl of 2.5% formalin (Dubuisson and Dennis 1977) into 1 hind paw ('morphine/pain'). To examine the effects of morphine administration in the absence of pain, rats received 20 mg/kg morphine 10 min after an s.c. injection of 50 ~,1 of saline into 1 hind paw ('morphine/no pain'). An additional 2 control groups received saline 10 min after
FISCHER pain
no pain
70" 60" 50" 40" 30" 20 ~ 10" 0
5
1'0
(o
2'0
2o
LEWIS 50
pam
no pare
40
20
10
0
1"o
2'0
5
10
20
morphine (mg/kg) Fig. 1. Percentage MPE (±SEM) in the tail-flick test following 5, 10 or 20 mg/kg morphine in Fischer (top) and Lewis (bottom) rats. Rats received morphine ( - e - ) or saline ( - © - ) for 4 consecutive days in the absence of pain (no pain) or in the presence of pain (pain). See text for significance levels.
387 injection of formalin ('saline/pain') or saline ('saline/no pain') into 1 hind paw. This procedure was repeated for 4 consecutive days and on each of these days the formalin/saline injection was given into a different paw site (day 1: right/dorsal; day 2: left/ventral; day 3: right/ventral; day 4: left/dorsal).
50,
40'
Morphine analgesia Twenty-four hours after tolerance induction (day 5) the rats were taken from the vivarium to the testing room. Following a 1-h habituation period, the rats were tested for analgesia in the tail-flick test. With the animal loosely wrapped in a cloth, the distal portion of the tail was immersed in water maintained at 52°C. The latency to flick its tail was recorded and a 10-see cut-off period was imposed to avoid tissue damage. Three baseline tail-flick latencies were taken at 10-min intervals, with the 2nd and 3rd latencies serving as baseline measures. The rats were then injected with 1 of 3 test doses of morphine (5, 10 or 20 mg/kg) and tested for analgesia at 15, 30, 45, 60 and 90 rain after morphine. Thus, there was a total of 12 groups for each strain (2 × 2 x 3 factorial design; p a i n / n o pain × morphine/ saline x 5 / 1 0 / 2 0 mg/kg morphine) with 10 rats per group.
30'
20'
10'
o
1'0
20
morphine (mg/kg)
Fig. 2. Percentage MPE ( + SEM) in the tail-flick test following 5, 10 or 20 mg/kg morphine in Fischer ( - • - ) and Lewis ( - [] - ) rats that were not exposed to morphine during tolerance induction (combined saline/pain + saline/no pain groups). See text for significance levels.
Results
The means of the 5 post-morphine tail-flick latencies were expressed as percentage of maximum possible effect: % M P E = ( test latency - baseline latency) ~ ( c u t - o f f value - baseline latency ) x
100
Thus, 0% indicates no change from baseline and 100% indicates maximum possible change. The data were analyzed separately for each strain using a 2 X 2 x 3 analysis of variance (ANOVA) with the factors of Pain Exposure ( p a i n / n o pain), Morphine Exposure (morphine/saline) and Test Dose ( 5 / 1 0 / 2 0 m g / k g morphine). In the Fischer strain, ANOVA revealed a significant 3-way interaction for Pain Exposure x Morphine Exposure X Test Dose (F(2, 119) = 3.51, P < 0.05, Fig. 1). Post-hoc comparisons (Tukey's HSD) indicated that significant tolerance to the 5 and 10 m g / k g morphine test doses was produced in rats that received morphine in the absence of pain (morphine/no pain vs. saline/ no pain, P < 0.05), but not in rats that received morphine in the presence of pain (morphine/pain vs. saline/pain, n.s). In the Lewis strain, ANOVA revealed a significant 2-way interaction for Morphine Exposure x Test Dose (F(2, 119) = 3.99, P < 0.05, Fig. 1). As revealed by the non-significant 2-way interactions for Pain Exposure x Morphine Exposure (F(1, 119)=0.36, n.s.) and for Pain Exposure x Test Dose (F(2, 119) --- 0.21, n.s.), the presence of pain during morphine exposure had no effect on tolerance development. Analysis of simple
effects on morphine exposure revealed that significant tolerance was produced in both the morphine/no pain and morphine/pain groups at all doses tested (5 m g / kg: F(1, 38)= 4.33, P < 0.05; 10 mg/kg: F(1, 38)= 5.22, P < 0.05); 20 mg/kg: F(1, 38) = 17.40, P < 0.01). To determine whether Lewis and Fischer rats also differed in sensitivity to morphine analgesia, %MPE for rats that were not exposed to morphine during tolerance induction (combined saline/pain + saline/ no pain groups) was analyzed using a 2 X 3 ANOVA with the factors of Strain (Fischer/Lewis) and Test Dose ( 5 / 1 0 / 2 0 m g / k g morphine). It was found that although morphine produced dose dependent analgesia in both strains (F(2, 114)= 17.29, P < 0.01), the Fisher strain was more sensitive to morphine analgesia than the Lewis strain at all doses tested (F(1, 114)= 20.67, P < 0.01, Fig. 2). No significant Strain x Test Dose interaction was found (F(2, 119) = 0.06, n.s.).
Discussion
In the present study it was found that morphine produced significant analgesic tolerance in both Lewis and Fischer rats when administered in the absence of pain. Strain differences in analgesic tolerance were observed, however, when rats were exposed to pain during tolerance induction. The finding that morphine failed to produce analgesic tolerance in Fischer rats when administered in the presence of pain is consistent with our previous studies using outbred Long-Evans Hooded rats (Vaccarino et al. 1993). On the other hand, pain did not block the development of analgesic tolerance in the Lewis strain. Lewis rats have been shown to lack typical stress-induced HPA activity
388 (Sternberg et al. 1992). For example, following a 3-h restraint stress, Lewis rats do not show any significant increases in hypothalamic CRH content, plasma ACTH or plasma corticosterone. In contrast, Fischer rats show a 1.3-fold increase in CRH, a 1.7-fold increase in plasma ACTH and a 17-fold increase in plasma corticosterone following the same stressor (Sternberg et al. 1992). Both stress, via adrenal glucocorticoids (Takahashi et al. 1989), and ACTH (Hendrie 1988) have been shown to prevent the development of tolerance to morphine analgesia, whereas adrenalectomy (Gebhart and Mitchell 1972; Wei 1973) or hypophysectomy (Holaday et al. 1979) enhance analgesic tolerance. It is possible, therefore, that genetically determined differences in stress-induced HPA activity may contribute to the differential effects of pain on the development of morphine tolerance that was observed in this study. This is further supported by a recent observation in our laboratory that suppression of formalin-induced HPA activity with dexamethasone pretreatment attenuates the blockade of tolerance by pain in outbred LongEvans rats (unpublished observations). Glucocorticoids have been shown to modulate the activity of several neurotransmitter systems, including serotonin (De Kloet et al. 1986), GABA (Majewska 1987) and excitatory amino acids (Tischler et al. 1988). It is possible that the antagonistic effect of pain on morphine tolerance may be mediated by the actions of glucocorticoids on one of these systems. Recent studies have suggested that opiate tolerance may be mediated by non-opiate mechanisms involving the N-methyl-Daspartate (NMDA) receptor (Marek et al. 1991a,b; Trujuillo and Akil 1991; Ben Eliyahu et al. 1992; Tiseo and Inturrisi 1993). Kynurenic acid is an endogenous metabolite of tryptophan that acts as a post-synaptic NMDA antagonist (Ganog et al. 1983). Administration of kynurenic acid during morphine exposure prevents the development of analgesic tolerance, but does not block morphine analgesia (Marek et al. 1991a). Although no direct evidence exists regarding elevated levels of kynurenic acid during pain or stress, glucocorticoids can indirectly increase levels of kynurenic acid by stimulating the induction of tryptophan pyrrolase, which is responsible for the metabolism of tryptophan (Bowman and Rand 1980). In addition, a recent study by Mao et al. (1994) has demonstrated that hyperalgesia develops in association with morphine tolerance, which is also prevented by NMDA antagonists. An alternative explanation, therefore, is that exposure to pain or stress during morphine administation prevents the development such morphine-induced hyperalgesia, It was also found that non-tolerant Fischer rats were more sensitive to morphine-induced analgesia as compared to non-tolerant Lewis rats (see Fig. 2). It is possible that these differences in sensitivity to morphine analgesia are also related to the strain differ-
ences in stress-induced neuroendocrine activity. Stress is known to potentiate morphine analgesia (Appelbaum and Holtzman 1984), and the integrity of HPA axis appears to be critical for mediating the analgesic effects of stress (Bodnar et al. 1979; Lewis et al. 1980; MacLennan et al. 1982). In the present study, rats were tested for analgesia in the tail-flick test on day 5 in the absence of stress produced by formalin injection. However. the handling of the rats and the injection procedures prior to analgesic testing may have produced sufficient stress effects to modify morphine analgesia. It would be reasonable to assume, therefore, that Lewis rats, which show deficits in stress-induced HPA activity, would not show a stress-induced potentiation of morphine analgesia. Animal studies have shown that significant analgesic tolerance develops following exposure to morphine in the absence of pain (Abbott et al. 1982; Louie and Way 1991; Connell et al. 1994). The present and previous studies have suggested that pain (Colpaert et al. 1980; Vaccarino et al. 1992), or the stress associated with pain (Hendrie 1988; Takahashi et al. 1989), may also activate a parallel system involving the HPA axis that prevents the development of analgesic tolerance. Indeed, clinical studies have indicated that when morphine is taken for pain relief, analgesic tolerance is not common (Melzack 1991; Portenoy 1994; Twycross 1994). Patients in pain experience both the physical and psychological stress directly associated with ongoing pain, as well as the stress that the chronic pain may represent an underlying disease, change in lifestyle, and in some cases the reality of an ensuing death. The stress produced by such physical, psychological and cognitive variables may also contribute to the development of analgesic tolerance. The existence of a stress-activated system that prevents the development of analgesic tolerance may be adaptive during natural processes of pain inhibition, such as stress-induced analgesia. Beta-endorphin and ACTH are released concomitantly from some pituitary cells during stress (Guillemin et al. 1977). Pituitary beta-endorphin is believed to mediate some forms of stress-induced analgesia (Lewis et al. 1980; Akil et al. 1984). Therefore, two parallel systems may be activated during stress: an opiate-mediated analgesia, as well as a non-opiate system that prevents the occurrence of "rapid' tolerance to that analgesia. The existence of such a dual system would be adaptive during prolonged stressful situations.
Acknowledgements This research was supported by a University of New Orleans Research Council Grant and an LSU Neuroscience Incentive Grant to A.L.V.L.C.C., Jr., was
389
supported by a Rene and Glorain Curry Scholarship for Cancer Research.
References Abbott, F.V., Melzack, R. and Leber, B.F., Morphine analgesia and tolerance in the tail-flick and formalin tests: dose response relationships, Pharmacol. Biochem. Behav., 17 (1982) 1213-1219. Akil, H., Watson, S.J., Young, E., Lewis, M.E., Khachaturian, H. and Walker, M.J., Endogenous opioids: biology and function, Ann. Rev. Neurosci., 7 (1984) 223-255. Aloisi, A.M. and Carli, G., Formalin-induced pain does not affect ACTH and corticosterone levels in the rat, Soc. Neurosci. Abst., 18 (1992) 500. Aloisi, A.M., Steenbergen, H.L., Van de Poll, N.E. and Farabollini, F., Sex-dependent effects of restraint on nociception and pituitary-adrenal hormones in the rat, Physiol. Behav., 55 (1994) 789-793. Applebaum, B.D. and Holtzman, S.G., Characterization of stress-induced potentiation of opioid effects in the rat, J. Pharmacol. Exp. Ther., 231 (1984) 555-565. Ben-Eliyahu, S., Marek, P., Vaccarino, A.L., Mogil, J.S., Sternberg, W.F. and Liebeskind, J.C., The NMDA receptor antagonist MK801 prevents long-lasting non-associative morphine tolerance in the rat, Brain Res., 575 (1992) 304-308. Bodnar, R.J., Glusman, M., Brutus, M., Spiagga, A. and Kelly, D.D., Analgesia induced by cold-water stress: attenuation following hypophysectomy, Phsiol. Behav., 23 (1979) 53-62. Bowman, W.C. and Rand, M.J., Texbook of Pharmacology, Blackwell, Oxford, 1980, pp. 240. Colpaert, F.C., Niemegeers, C.R.E. and Jansen, P.A.J. and Maroli, A.N., The effects of prior fentanyl administration and pain on fentanyl administration: tolerance to and enhancement of narcotic analgesia, J. Pharmacol. Exp. Ther., 213 (1980) 418-426. Connell, B.J., Barnes, J.C., Blatt, T. and Tasker, R.A.R., Rapid development of tolerance to morphine in the formalin test, NeuroReport, 5 (1994) 817-820. De Kloet, E.R., Sybesma, H. and Reul, J.M.H.M., Selective control by corticosterone of serotonin 1 receptor capacity in raphe-hippocampal system, Neuroendocrinology, 42 (1986) 513-521. Dubuisson, D. and Dennis, S.G., The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats, Pain, 4 (1977) 161-174. Friedman, S.B., Ader, R., Grota, L.J. and Larson, T., Plasma corticosterone response to parameters of electric shock stimulation in the rat, Psychosom. Med., 29 (1967) 323-328. Ganog, A.H., Lanthorn, T.H. and Cotman, C.W., Kynurenic acid inhibits synaptic and acidic amino-acid induced responses in the rat hippocampus and spinal cord, Brain Res., 273 (1983) 170-174. Guillemin, R., Vargo, T., Rossier, J., Minick, S., Ling, N., Rivier, C., Vale, W. and Bloom, F., Beta-endorphin and adrenocorticotrophin are released concomitantly by the pituitary gland, Science, 197 (1977) 1367-1369. Hendrie, C.A., ACTH: a single pretreatment enhances the analgesic efficiency of and prevents the development of tolerance to morphine, Physiol. Behav., 42 (1988) 41-45, Keim, K.L. and Sigg, E.B., Physiological and biochemical concomitents of restraint stress in rats, Pharmacol, Biochem. Behav., 4 (1976) 284-297. Lewis, J.W., Cannon, J.T. and Liebeskind, J.C., Opioid and non-
opioid mechanisms of stress analgesia, Nature, 208 (1980) 623625. Linton, E.A., Tilders, F.J.H., Hodgkinson, S., Berkenbosch, F., Vermes, I. and Lowry, P.J., Stress-induced secretion of adrenocorticotrophin in rats is inhibited by administration of antisera to ovine corticotrophin-releasing factor and vasopressin, Endocrinology, 116 (1985) 966-970. Louie, A.K. and Way, E.L., Overview of opiate tolerance and physical dependecne. In: O.F. Almeida and T.S. Shippenberg (Eds.), Neurobiology of Opioids. Springer, New York, 1991. MacLennon, A.J., Drugan, R.C., Hyson, R.L., Maier, S.F., Madden, J. and Barchas, J,D., Corticosterone: a critical factor in an opioid form of stress-induced analgesia, Science, 215 (1982) 1530-1532. Majewska, M.D., Antagonist-type interaction of glucocorticoids with GABA recepter-coupled chloride channel, Brain Res., 418 (1987) 377-382. Mao, J., Price, D.D. and Mayer, D.J., Thermal hyperalgesia in association with the development of morphine tolerance in rats: role of excitatory amino acid receptors and protein kinase C, J. Neurosci,, 14 (1994) 2301-2312. Marek, P., Ben Eliyahu, S., Gold, M. and Liebeskind, J.C., Excititory amino acid antagonists (kynurenic acid and MK-801) attenuate the development of morphine tolerance in the rat, Brain Res., 547 (1991a) 77-8l. Marek, P., Ben-Eliyahu, S., Vaccarino, A.L and Liebsekind, J.C., Delayed application of MK-801 attenuates development of morphine tolerance in rats, Brain Res., 558 (1991b) 163-165. Melzack, R., The tragedy of needles pain, Sci Am., 262 (1991) 27-33. Pare, W.P. and Glavin, G.B., Restraint stress in biomedical research: a review, Neurosci. Biobehav. Rev., 10 (1986) 339-370. Portenoy, R.K., Opioid therapy for chronic non-malignant pain: current status. In: H.L. Fields and J.C. Liebeskind (Eds.), Progress in Pain Research and Management, 1ASP Press, Seattle, WA, 1994, 247-287. Sternbreg, E.M., Glowa, J.R., Smith, M.A,, Calogero, A.E., Listwak, S.J., Aksentijevich, S., Chrousos, G.P., Wilder, R.L. and Gold, P.W., Corticotrophin releasing hormone related behavioral and neuroendocrine responses to stress in Lewis and Fischer rats, Brain Res., 570 (1992) 54-60. Takahashi, M., Sugimachi, K. and Kaneto, H., Role of adrenal glucocorticoids in the blockade of analgesic tolerance to morphine by footshock stress exposure in mice, Jpn J. Pharmacol., 51 (1989) 329-336. Tischler, M.E., Hendricksen, E.J. and Cook, P.H., Role of glucocorticoids in increase muscle glutamine production in starvation, Muscle Nerve, 11 (1988) 752-756. Tiseo, P.J. and Inturrisi, C.E., Attenuation and reversal of morphine tolerance by the competitive N-methyl-D-aspartate receptor antagonist, LY274614, J. Pharmacol. Exp. Ther., 264 (1993) 10901096. Trujillo, K.A. and Akil, H., Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801, Science, 251 (1991) 85-87. Twycross, R.G., Opioids. In: P.D. Wall and R. Melzack (Eds.), The Textbook of Pain, Churchill Livingston, London, 1994, pp. 943962. Vaccarino, A.L. and Couret Jr., L.C., Formalin-induced pain antagonizes the development of opioid dependence in the rat, Neurosci. Lett., 161 (1993) 195-198. Vaccarino, A.L., Marek, P., Kest. B., Ben-Eliyahu, S., Couret, Jr., L.C., Kao, B. and Liebeskind, J.C., Morphine fails to produce tolerance when administered in the presence of formalin pain in rats, Brain Res., 627 (1993) 287-290.
385
© 1995Elsevier Science B.V. All rights reserved 0304-3959/95/$09.50
PAIN 2859
Relationship between hypothalamic-pituitary-adrenal activity and blockade of tolerance to morphine analgesia by pain: a strain comparison A n t h o n y L. Vaccarino * and Leland C. Couret Jr. Department of Psychology, University of New Orleans, New Orleans, LA 70148 (USA)
(Received9 November1994,revisionreceived20 February 1995,accepted2 March 1995)
Summary We previously reported that morphine fails to produce analgesic tolerance when administered in the presence of formalin-induced pain. The hypothalamic-pituitary-adrenal (HPA) axis is known to respond to stressful stimuli, including pain. To examine whether the blockade of tolerance by pain is related to HPA activity, we assessed the development of tolerance to morphine analgesia in an inbred strain of rats that lack typical stress-induced HPA responses (Lewis strain). Lewis rats lack typical stress-induced activation of corticotropin-releasing hormone, adrenocorticotropin hormone and glucocorticoids. Female Lewis rats were injected with morphine (20 mg/kg, i.p.) or saline for 4 consecutive days in the presence or absence of pain induced by injection of formalin into the hind-paw. The analgesic effect of morphine (5, 10 or 20 mg/kg, i.p.)was then measured in the tail-flick test 24 h after tolerance induction. Inbred female Fischer rats, which show significant stress-induced HPA activity, were used for comparison. Analgesic tolerance was produced in both strains when morphine was delivered in the absence of pain. However, the presence of pain during morphine administration prevented the development of analgesic tolerance in Fischer, but not in Lewis, rats. The differential effects of pain on the development of tolerance to morphine analgesia are suggested to be related to genetically determined differences in stress-induced HPA activity. Key words: Morphine; Analgesic tolerance; Formalin-induced pain; Tail flick; Hypothalamic-pituitary-adrenal axis; Strain comparison; Fischer; Lewis; (Rat)
Introduction
Clinical studies have indicated that when opiates are used to control pain, tolerance and dependence are not a major concern (Melzack 1991; Portenoy 1994; Twycross 1994). In support of this, we recently reported that analgesic tolerance (Vaccarino et al. 1993) and dependence (Vaccarino and Couret, Jr. 1993) in rats do not develop when morphine is administered repeatedly in the presence of formalin-induced pain. A similar finding was also reported by Colpaert et al. (1980) in which tolerance to the analgesic effects of the
* Corresponding author: A.L. Vaccarino, Department of Psychology,
University of New Orleans, New Orleans, LA 70148, USA. Tel.: (504) 286-6771;FAX: (504) 286-6049. SSDI 0304-3959(95)00069-0
opiate, fentanyl, was blocked in rats exposed to a noxious mechanical stimulus during tolerance induction. The hypothalamic-pituitary-adrenal (HPA) axis responds to stressful stimuli (Friedman et al. 1967; Sternebreg et al. 1992; Aloisi et al. 1994), including formalin injection (Linton et al. 1985; Aloisi and Carli 1992). Adrenocorticotropin hormone (ACTH) has been found to prevent the development of tolerance to morphine analgesia (Hendrie 1988), and stress blocks the development of morphine tolerance in intact mice, but not in adrenalectomized mice (Takahashi et al. 1989). In addition, both adrenalectomy (Gebhart and Mitchell 1972; Wei 1973) and hypophysectomy (Holaday et al. 1979) have been shown to potentiate the magnitude of opiate tolerance, with the effects of hypophysectomy being reversed by replacement of ACTH (Holaday et al. 1979). This raises the possibility that blockade of tolerance development by formalin-induced pain is re-
386
lated to an activation of the HPA axis following formalin injection. Observations of different strains of rats suggest a genetic basis for variability of neuroendocrine and behavioural responses to stress (Keim and Sigg 1976; Pare and Glavin 1986; Sternberg et al. 1992). Recently, the Lewis strain of rat has been identified to lack typical stress-induced HPA activity. As compared to Fischer rats, Lewis rats lack or show low increases of hypothalamic corticotropin hormone (CRH), plasma ACTH and corticosterone following a variety of stressors, including restraint, open field, swim or ether (Sternberg et al. 1992). In the present study, we examined the relationship between stress-induced HPA activity and the blockade of tolerance to morphine analgesia by pain. In particular, we wished to find out whether Lewis rats, which show deficits in stress-induced HPA activity, do not show the antagonistic effect of pain on tolerance development. The existence of such strain differences may be useful for studying the 'natural' relationship between neuroendocrine ac-
tivity, pain, stress, and the development of morphine tolerance.
Methods
Subjects Inbred female Lewis (n = 120) and Fischer (n = 120) rats (Harlan Laboratories, USA) weighing 150-200 g served as subjects. Rats were housed individually and allowed free access to food and water, and were maintained on a 12-h light cycle (light onset at 07:00 h).
Tolerance induction Morphine sulphate (Malincrodt, USA) was dissolved in physiological saline and administered intraperitoneally (i.p.) in a volume of 0.1 ml/100 g of body weight. To examine the effects of morphine administration in the presence of pain, rats received 20 mg/kg morphine 10 min after a subcutaneous (s.c.) injection of 50 /zl of 2.5% formalin (Dubuisson and Dennis 1977) into 1 hind paw ('morphine/pain'). To examine the effects of morphine administration in the absence of pain, rats received 20 mg/kg morphine 10 min after an s.c. injection of 50 ~,1 of saline into 1 hind paw ('morphine/no pain'). An additional 2 control groups received saline 10 min after
FISCHER pain
no pain
70" 60" 50" 40" 30" 20 ~ 10" 0
5
1'0
(o
2'0
2o
LEWIS 50
pam
no pare
40
20
10
0
1"o
2'0
5
10
20
morphine (mg/kg) Fig. 1. Percentage MPE (±SEM) in the tail-flick test following 5, 10 or 20 mg/kg morphine in Fischer (top) and Lewis (bottom) rats. Rats received morphine ( - e - ) or saline ( - © - ) for 4 consecutive days in the absence of pain (no pain) or in the presence of pain (pain). See text for significance levels.
387 injection of formalin ('saline/pain') or saline ('saline/no pain') into 1 hind paw. This procedure was repeated for 4 consecutive days and on each of these days the formalin/saline injection was given into a different paw site (day 1: right/dorsal; day 2: left/ventral; day 3: right/ventral; day 4: left/dorsal).
50,
40'
Morphine analgesia Twenty-four hours after tolerance induction (day 5) the rats were taken from the vivarium to the testing room. Following a 1-h habituation period, the rats were tested for analgesia in the tail-flick test. With the animal loosely wrapped in a cloth, the distal portion of the tail was immersed in water maintained at 52°C. The latency to flick its tail was recorded and a 10-see cut-off period was imposed to avoid tissue damage. Three baseline tail-flick latencies were taken at 10-min intervals, with the 2nd and 3rd latencies serving as baseline measures. The rats were then injected with 1 of 3 test doses of morphine (5, 10 or 20 mg/kg) and tested for analgesia at 15, 30, 45, 60 and 90 rain after morphine. Thus, there was a total of 12 groups for each strain (2 × 2 x 3 factorial design; p a i n / n o pain × morphine/ saline x 5 / 1 0 / 2 0 mg/kg morphine) with 10 rats per group.
30'
20'
10'
o
1'0
20
morphine (mg/kg)
Fig. 2. Percentage MPE ( + SEM) in the tail-flick test following 5, 10 or 20 mg/kg morphine in Fischer ( - • - ) and Lewis ( - [] - ) rats that were not exposed to morphine during tolerance induction (combined saline/pain + saline/no pain groups). See text for significance levels.
Results
The means of the 5 post-morphine tail-flick latencies were expressed as percentage of maximum possible effect: % M P E = ( test latency - baseline latency) ~ ( c u t - o f f value - baseline latency ) x
100
Thus, 0% indicates no change from baseline and 100% indicates maximum possible change. The data were analyzed separately for each strain using a 2 X 2 x 3 analysis of variance (ANOVA) with the factors of Pain Exposure ( p a i n / n o pain), Morphine Exposure (morphine/saline) and Test Dose ( 5 / 1 0 / 2 0 m g / k g morphine). In the Fischer strain, ANOVA revealed a significant 3-way interaction for Pain Exposure x Morphine Exposure X Test Dose (F(2, 119) = 3.51, P < 0.05, Fig. 1). Post-hoc comparisons (Tukey's HSD) indicated that significant tolerance to the 5 and 10 m g / k g morphine test doses was produced in rats that received morphine in the absence of pain (morphine/no pain vs. saline/ no pain, P < 0.05), but not in rats that received morphine in the presence of pain (morphine/pain vs. saline/pain, n.s). In the Lewis strain, ANOVA revealed a significant 2-way interaction for Morphine Exposure x Test Dose (F(2, 119) = 3.99, P < 0.05, Fig. 1). As revealed by the non-significant 2-way interactions for Pain Exposure x Morphine Exposure (F(1, 119)=0.36, n.s.) and for Pain Exposure x Test Dose (F(2, 119) --- 0.21, n.s.), the presence of pain during morphine exposure had no effect on tolerance development. Analysis of simple
effects on morphine exposure revealed that significant tolerance was produced in both the morphine/no pain and morphine/pain groups at all doses tested (5 m g / kg: F(1, 38)= 4.33, P < 0.05; 10 mg/kg: F(1, 38)= 5.22, P < 0.05); 20 mg/kg: F(1, 38) = 17.40, P < 0.01). To determine whether Lewis and Fischer rats also differed in sensitivity to morphine analgesia, %MPE for rats that were not exposed to morphine during tolerance induction (combined saline/pain + saline/ no pain groups) was analyzed using a 2 X 3 ANOVA with the factors of Strain (Fischer/Lewis) and Test Dose ( 5 / 1 0 / 2 0 m g / k g morphine). It was found that although morphine produced dose dependent analgesia in both strains (F(2, 114)= 17.29, P < 0.01), the Fisher strain was more sensitive to morphine analgesia than the Lewis strain at all doses tested (F(1, 114)= 20.67, P < 0.01, Fig. 2). No significant Strain x Test Dose interaction was found (F(2, 119) = 0.06, n.s.).
Discussion
In the present study it was found that morphine produced significant analgesic tolerance in both Lewis and Fischer rats when administered in the absence of pain. Strain differences in analgesic tolerance were observed, however, when rats were exposed to pain during tolerance induction. The finding that morphine failed to produce analgesic tolerance in Fischer rats when administered in the presence of pain is consistent with our previous studies using outbred Long-Evans Hooded rats (Vaccarino et al. 1993). On the other hand, pain did not block the development of analgesic tolerance in the Lewis strain. Lewis rats have been shown to lack typical stress-induced HPA activity
388 (Sternberg et al. 1992). For example, following a 3-h restraint stress, Lewis rats do not show any significant increases in hypothalamic CRH content, plasma ACTH or plasma corticosterone. In contrast, Fischer rats show a 1.3-fold increase in CRH, a 1.7-fold increase in plasma ACTH and a 17-fold increase in plasma corticosterone following the same stressor (Sternberg et al. 1992). Both stress, via adrenal glucocorticoids (Takahashi et al. 1989), and ACTH (Hendrie 1988) have been shown to prevent the development of tolerance to morphine analgesia, whereas adrenalectomy (Gebhart and Mitchell 1972; Wei 1973) or hypophysectomy (Holaday et al. 1979) enhance analgesic tolerance. It is possible, therefore, that genetically determined differences in stress-induced HPA activity may contribute to the differential effects of pain on the development of morphine tolerance that was observed in this study. This is further supported by a recent observation in our laboratory that suppression of formalin-induced HPA activity with dexamethasone pretreatment attenuates the blockade of tolerance by pain in outbred LongEvans rats (unpublished observations). Glucocorticoids have been shown to modulate the activity of several neurotransmitter systems, including serotonin (De Kloet et al. 1986), GABA (Majewska 1987) and excitatory amino acids (Tischler et al. 1988). It is possible that the antagonistic effect of pain on morphine tolerance may be mediated by the actions of glucocorticoids on one of these systems. Recent studies have suggested that opiate tolerance may be mediated by non-opiate mechanisms involving the N-methyl-Daspartate (NMDA) receptor (Marek et al. 1991a,b; Trujuillo and Akil 1991; Ben Eliyahu et al. 1992; Tiseo and Inturrisi 1993). Kynurenic acid is an endogenous metabolite of tryptophan that acts as a post-synaptic NMDA antagonist (Ganog et al. 1983). Administration of kynurenic acid during morphine exposure prevents the development of analgesic tolerance, but does not block morphine analgesia (Marek et al. 1991a). Although no direct evidence exists regarding elevated levels of kynurenic acid during pain or stress, glucocorticoids can indirectly increase levels of kynurenic acid by stimulating the induction of tryptophan pyrrolase, which is responsible for the metabolism of tryptophan (Bowman and Rand 1980). In addition, a recent study by Mao et al. (1994) has demonstrated that hyperalgesia develops in association with morphine tolerance, which is also prevented by NMDA antagonists. An alternative explanation, therefore, is that exposure to pain or stress during morphine administation prevents the development such morphine-induced hyperalgesia, It was also found that non-tolerant Fischer rats were more sensitive to morphine-induced analgesia as compared to non-tolerant Lewis rats (see Fig. 2). It is possible that these differences in sensitivity to morphine analgesia are also related to the strain differ-
ences in stress-induced neuroendocrine activity. Stress is known to potentiate morphine analgesia (Appelbaum and Holtzman 1984), and the integrity of HPA axis appears to be critical for mediating the analgesic effects of stress (Bodnar et al. 1979; Lewis et al. 1980; MacLennan et al. 1982). In the present study, rats were tested for analgesia in the tail-flick test on day 5 in the absence of stress produced by formalin injection. However. the handling of the rats and the injection procedures prior to analgesic testing may have produced sufficient stress effects to modify morphine analgesia. It would be reasonable to assume, therefore, that Lewis rats, which show deficits in stress-induced HPA activity, would not show a stress-induced potentiation of morphine analgesia. Animal studies have shown that significant analgesic tolerance develops following exposure to morphine in the absence of pain (Abbott et al. 1982; Louie and Way 1991; Connell et al. 1994). The present and previous studies have suggested that pain (Colpaert et al. 1980; Vaccarino et al. 1992), or the stress associated with pain (Hendrie 1988; Takahashi et al. 1989), may also activate a parallel system involving the HPA axis that prevents the development of analgesic tolerance. Indeed, clinical studies have indicated that when morphine is taken for pain relief, analgesic tolerance is not common (Melzack 1991; Portenoy 1994; Twycross 1994). Patients in pain experience both the physical and psychological stress directly associated with ongoing pain, as well as the stress that the chronic pain may represent an underlying disease, change in lifestyle, and in some cases the reality of an ensuing death. The stress produced by such physical, psychological and cognitive variables may also contribute to the development of analgesic tolerance. The existence of a stress-activated system that prevents the development of analgesic tolerance may be adaptive during natural processes of pain inhibition, such as stress-induced analgesia. Beta-endorphin and ACTH are released concomitantly from some pituitary cells during stress (Guillemin et al. 1977). Pituitary beta-endorphin is believed to mediate some forms of stress-induced analgesia (Lewis et al. 1980; Akil et al. 1984). Therefore, two parallel systems may be activated during stress: an opiate-mediated analgesia, as well as a non-opiate system that prevents the occurrence of "rapid' tolerance to that analgesia. The existence of such a dual system would be adaptive during prolonged stressful situations.
Acknowledgements This research was supported by a University of New Orleans Research Council Grant and an LSU Neuroscience Incentive Grant to A.L.V.L.C.C., Jr., was
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supported by a Rene and Glorain Curry Scholarship for Cancer Research.
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