- Email: [email protected]
Local peripheral effects of μ-opioid receptor agonists in neuropathic pain in rats
Neuroscience Letters 360 (2004) 85–89 www.elsevier.com/locate/neulet
Local peripheral effects of m-opioid receptor agonists in neuropathic pain in rats Ilona Obara, Ryszard Przewlocki, Barbara Przewlocka* Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krako´w, Poland Received 14 October 2003; received in revised form 16 January 2004; accepted 16 January 2004
Abstract Our study was designed to demonstrate peripheral antinociception of the m-opioid receptor agonists: morphine (MF), [D -Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO), endomorphin-1 (EM-1) and endomorphin-2 (EM-2) in Bennett’s rat model of neuropathic pain. All the agonists were effective in antagonizing allodynia after their intraplantar (i.pl.) but not subcutaneous (s.c.) administration. Opioid peptides: DAMGO, EM-1 and EM-2 were more effective compared with corresponding doses of morphine (opioid alkaloid) in alleviating chronic pain. Peripheral m-opioid receptors mediated the observed effects, as was evidenced by the i.pl. treatment with naloxone methiodide (active only at the site of injection) and by cyprodime, a selective m-opioid receptor antagonist. These results have shown that opioid peptides are effective also after local treatment, and that their peripheral use may be of therapeutic interest in long-term management of chronic pain. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Nerve ligation injury; Peripheral m-opioid receptor; Opioid; Antinociception; DAMGO; Endomorphins
Pain arising from peripheral nerve injury is one of the more important health problems. Presently available therapeutic approaches reduce but do not eliminate characteristic syndromes of neuropathic pain, such as allodynia and hyperalgesia. Morphine and other classic opiates are not efficient analgesics in relieving neuropathic pain [1,14]; however, it has been suggested that neuropathic pain may be attenuated by higher doses (in comparison with those effective in acute pain) of opiates but this is accompanied by aggravation of unwanted side effects. Therefore, new methods and therapies with more potent analgesics, which would be beneficial to clinical practice, are currently being intensively searched for. Besides the central nervous system all three types of opioid receptors (m, d and k) have been found in dorsal root ganglia on cell bodies of sensory neurons, and on their central and peripheral terminals [16]. It is now well established that activation of these peripheral receptors produces analgesia in models of inflammatory pain [8,15]. This can be achieved by administration of opioid agonists at small, systemically inactive doses directly into injured tissue and by compounds that are unable to cross the blood – * Corresponding author. Tel.: þ 48-12-662-3398; fax: þ48-12-637-4500. E-mail address: [email protected] (B. Przewlocka).
brain barrier [8]. Peripheral opioid receptors are upregulated during inflammation and they can be activated by their endogenous ligands (opioid peptides) that are produced by circulating immune cells which migrate to injured tissues [13,16]. Opioid receptors expressed on peripheral afferent axons may also contribute to the opioid-induced antinociception in neuropathic pain [11,18]. Analgesic effects of opioid receptor agonists have been demonstrated after their systemic intravenous (i.v.) injection [3,6]. Moreover, it appears that the peripheral action of i.v. administered morphine in mononeuropathic rats is not only mediated via peripheral m-opioid receptors [6], but also via peripheral kopioid receptors [3]. In the present study, we investigated the effects of morphine (MF), [D -Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO), endomorphin-1 (EM-1) and endomorphin-2 (EM-2) after their intraplantar (i.pl.) administration directly into injured (by sciatic nerve ligation) hind paw and after subcutaneous (s.c.) administration in neuropathic rats. All experiments were performed according to the NIH Guide for Care and Use of Laboratory Animals and recommendations of the IASP [20], and were approved by the Local Bioethics Committee. Male Wistar rats (200 – 350 g) were housed individually in cages with sawdust bedding
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.01.056
86
I. Obara et al. / Neuroscience Letters 360 (2004) 85–89
under a standard 12:12-h light/dark cycle (lights on at 08:00 h) with food and water available ad libitum. Sciatic nerve injury was performed under pentobarbital anesthesia (60 mg/kg, i.p.) by tying four loose ligations around the sciatic nerve with 1 mm spacing as described by Bennett and Xie (1988). The ligatures were tied until they elicited a brief twitch in the respective hind limb, which prevented us from applying too strong a ligation. In all animals, the paw with a sciatic nerve injured in this way responds to pain stimulus; however, the reaction is different from controls. Testing procedures were carried out on days 12 –16 after the injury. Chemicals and their sources were as follows. Morphine hydrochloride: Polfa, Kutno, Poland; EM-1 and EM-2: Tocris Cookson Ltd., USA; DAMGO and naloxone methiodide (NxM): Sigma Chemical Co., USA. Cyprodime (CYP) was donated by Professor H. Schmidhammer (Innsbruck, Austria). All drugs were dissolved in sterile water and injected i.pl. into the injured hind paw in a volume of 20 ml or s.c. in a volume of 4 ml/kg. Each dose was tested in one experimental group of animals, n ¼ 8 – 10: CYP (274 nmol) and NxM (42.6 nmol) were injected i.pl. simultaneously with the highest effective doses of each used mopioid receptor agonist. After completion of the experiment, the animals were killed with an overdose of pentobarbital. Foot withdrawal threshold in response to mechanical stimuli was measured by the use of von Frey filaments (Stoelting, USA), which are used to apply slight pressure to the midplantar surface of the ipsilateral hind paw with the strength ranging from 0.2 to 26 g. The measurements were carried out 5, 15 and 30 min after i.pl. or s.c. drug injection on days 12– 16 after the sciatic nerve ligation. The results of the experiments were evaluated by analysis of variance (ANOVA) followed by Bonferroni’s post hoc test (P , 0:05 was considered to be statistically significant), and presented as a percentage of maximal possible effect (%MPE ^ S.E.M.) using the equation: %MPE ¼ [(BL 2 TL)/(BL 2 Cutoff)] £ 100%, where BL is baseline latency and TL is respective test latency. Our study showed that i.pl. injections of MF (528, 1056 nmol), DAMGO (4, 9.5 nmol), EM-1 (65, 131, 262 nmol) and EM-2 (70, 140 nmol) produced a dose-dependent, significant antiallodynic effect (Fig. 1A). The most potent effect was observed after DAMGO (9.5 nmol); the effects peaked 5 min after the drug injection (68.8 ^ 9.4% MPE) and later gradually declined. A similar strong antinociceptive effect was observed after EM-1 (131, 262 nmol); the effect peaked 5 min after drug injection (68.3 ^ 2.9% MPE) and was still observed until 30 min (56.5 ^ 8.6% MPE). The time course of antiallodynic effect after EM-2 (70, 140 nmol) administration was quite similar (63.1 ^ 2.4% MPE) to EM-1. In contrast, antinociception induced by MF (1056 nmol) reached the same level as that of DAMGO (9.5 nmol) and EM-1 (131, 262 nmol) at 30–60 min after the injection (in Fig. 1A only the effects observed after 5, 15 and 30 min are presented). We also tested the agonists after i.pl. administration in the cold-water
allodynia test and in the paw withdrawal test (thermal hyperalgesia test). The effects observed in these tests were similar to that observed in the von Frey test (approximately 30–50% MPE, data not shown). The antiallodynic effect demonstrated by the von Frey test carried out 5, 15 and 30 min after i.pl. administration of MF (528, 1056 nmol), DAMGO (4, 9.5 nmol), EM-1 (65, 131, 262 nmol) and EM-2 (70, 140 nmol) was antagonized by treatment with the selective m-opioid antagonist cyprodime (CYP; 274 nmol) as well as with the nonselective, peripherally active antagonist of opioid receptors, naloxone methiodide (NxM; 42.6 nmol) (Fig. 1B). The antiallodynic potencies of all used m-opioid receptor agonists are compared in Fig. 2. DAMGO was much more potent then EMs and MF 5, 15 and 30 min after their administration. The antiallodynic potency of DAMGO and EMs was demonstrable at considerably lower concentration (2 –9.5 nmol and 33 –262 nmol, respectively) in comparison with MF (264 – 1056 nmol). Both EMs antagonized allodynia when given in a similar dose range (33 – 263 nmol and 35 –140 nmol, respectively). To compare the peripheral and local effects we administered s.c. MF (1.5, 0.75 mg/kg), DAMGO (7, 20 mg/kg), EM-1 (150, 300 mg/kg) and EM-2 (150, 300 mg/kg) at a dose comparable to the dose used locally (i.pl.). The antiallodynic effects measured in the von Frey and paw withdrawal tests were not statistically significant versus the respective control group (Fig. 2B, only the effect observed after 15 min is presented). Conventionally, the analgesic action of opioids is attributed to their effects on the central nervous system, hence they have been investigated after their central, mainly spinal administration. In the present study, we demonstrated that also locally, i.pl. administered m-opioid receptor agonists (MF, DAMGO, EM-1 and EM-2) were effective in neuropathic pain, in contrast to noneffective s.c. administration. Naloxone methiodide, a nonselective antagonist of opioid receptors which acts only at the site of injection and cyprodime, a selective m-opioid receptor antagonist, inhibited the antiallodynic effect of locally (i.pl.) administered opioids, and showed that the observed effect was mediated by peripheral m-opioid receptors. These observations are in agreement with recent studies indicating that opioids may also induce antinociceptive effects at peripheral sites in a neuropathic pain model [3,11,18]. Some investigators demonstrated the analgesic effect of morphine and various selective agonists of m- (DAMGO), d- (BUBU, DTLET) and k- (U 69,593, ICI 20448) opioid receptors after their systemic (i.v.) administration which might suggest a limited, but still possible effect on the central opioid receptor (spinal and/or supraspinal) due to an uptake of the drug by the nervous system from the blood [3,4,6]. Therefore, in our study we used local injections to the injured paw in order to exclude the role of central effects, and validate the role of peripheral opioid receptors in neuropathic pain.
I. Obara et al. / Neuroscience Letters 360 (2004) 85–89
87
Fig. 1. Effect of local intraplantar (i.pl.) administration of morphine (MF), DAMGO, endomorphin-1 (EM-1) and endomorphin-2 (EM-2) on mechanical allodynia in rats 12–16 days after the sciatic nerve injury measured in von Frey test. (A) Time course of the drug effects. The mean reaction in the control group subjected to sciatic nerve injury and injected with solvent (open symbols) was 2.1 ^ 0.9 g. (B) The influence of local (i.pl.) treatment with selective m-opioid receptor antagonist, cyprodime (CYP; 274 nmol) and nonselective opioid antagonist, naloxone methiodide (NxM; 42.6 nmol) on the effects of i.pl. administration of selected doses of used m-agonists. The results are shown as a mean percentage of maximal possible effect (%MPE ^ S.E.M.) of 8– 10 animals per group. *P , 0:05; ***P , 0:001 in comparison with the solvent-injected group of animals (broken line) with the injured sciatic nerve.þ þ þ P , 0:001 in comparison with the group of animals with the injured sciatic nerve after i.pl. administration of effective dose of tested agonists (ANOVA, Bonferroni test).
Troung et al. [18] provided evidence that local m-opioid receptors were expressed on peripheral sensory axons and mediated analgesia in the microenvironment of a peripheral nerve injury. Despite the fact that peripheral axotomy and peripheral nerve ligation caused a reduction in the number of m-opioid receptors in the rat dorsal root ganglion ([19] and our unpublished observations), Troung et al. [18] demonstrated an increase in the proportion of neurons expressing m-opioid receptors in sensory neurons ipsilateral to the ligated nerve. Moreover, an enhanced axonal transport of m-opioid receptors in sensory neurons, leading to an increase in their number on peripheral nerve terminals, was reported [5]. Alleviation of allodynia after administration of opioid peptides appears faster (5 min after i.pl. administration) and
the same doses of these drugs are as effective as those reducing acute pain, while the alkaloid acts more slowly (15 –30 min after injection) and only its much higher doses are active in comparison with the doses relieving acute pain. Interestingly, we previously demonstrated that endomorphins were more effective, also after their intrathecal (i.t.) administration, than morphine in neuropathic pain [12,14]. The reason for these differences is still unknown, since all agonists appear to act via the same m-opioid receptor. One possible explanation is the involvement of different subtypes of m-opioid receptor as a target of different agonists [7,9]. Another explanation is a possibility of unequal receptor regulation by peptide agonists and morphine due to differential regulation of trafficking
88
I. Obara et al. / Neuroscience Letters 360 (2004) 85–89
Identification of the differences between these m-opioid receptor agonists could be of great importance to our understanding of the mechanism of opioid action in neuropathic pain. It should also be mentioned that although inflammatory conditions are not obvious 2 weeks after surgery in the chronic constriction injury model [2], some authors suggested involvement of the immune system in the mediation of the nervous system response to damage [15]. This may be linked to the presence of activated macrophages around the injured nerve fibers [17], the role of which is to promote the regeneration process in the peripheral nervous system, to participate in the development of neuropathic pain [17] and to activate opioid receptors located on peripheral sensory neurons [15]. The spinal nerve ligation model of neuropathy is expected to produce a milder inflammatory reaction, particularly in the distal parts of the limb. Pertovaara and Wei [11] reported that spinal nerve injury in the Kim and Chung model (1992) might lead to an enhanced contribution of peripheral mechanisms to morphine-induced antinociception. Moreover, local anesthetic action, such as that of lidocaine, was excluded [6]. In conclusion, in the present study we demonstrated a peripheral antinociceptive effect of the m-opioid receptor agonists morphine, DAMGO and endomorphins, in Bennett’s rat model of neuropathic pain after their i.pl. administration. Our studies, alongside investigations of other authors [11,18] concerning the action of antinociceptive opioid receptor agonists (especially morphine) seem to confirm a lower efficacy of morphine in neuropathic pain in comparison with peptide agonists, also after local administration. Therefore, potent peptide opioid agonists with a peripheral site of action can comprise an important target in the strategy for pharmacological treatment of chronic pain.
Acknowledgements Supported by grant PBZ-MN-001-/P05/2002 (I.O. and B.P.) and EU grant No. QLRT-2001-02919 (R.P.). Fig. 2. Comparison of dose–response curves of m-opioid receptor agonists. (A) After their intraplantar (i.pl.) administration (morphine (MF): 264, 528, 1056 nmol; DAMGO: 2, 4, 9.5 nmol; endomorphin-1 (EM-1): 33, 131, 262 nmol; endomorphin-2 (EM-2): 35, 70, 140 nmol) and (B) after their subcutaneous (s.c.) administration (MF: 0.75, 1.5 mg/kg; DAMGO: 7, 20 mg/kg; EM-1: 150, 300 mg/kg; EM-2: 150, 300 mg/kg) measured by von Frey test in rat model of neuropathic pain 12–16 days after the sciatic nerve injury. The results are shown as a mean percentage of maximal possible effect (%MPE ^ S.E.M.) of 8–10 animals per group.
proteins. Those drugs that do not cause receptor internalization, such as morphine, induce development of tolerance more easily and as a consequence may lose their effectiveness in neuropathic pain [10]. An alternative explanation is that nerve injury followed by development of the neuropathic pain process causes some changes in potency and efficacy of binding parameters of the m-opioid receptors.
References [1] D. Bian, M.H. Ossipov, M. Ibrahim, R.B. Raffa, R.J. Tallarida, T.P. Malan Jr, J. Lai, F. Porreca, Loss of antiallodynic and antinociceptive spinal/supraspinal morphine synergy in nerve-injured rats: restoration by MK-801 or dynorphin antisera, Brain Res. 831 (1999) 55 –63. [2] A.L. Clatworthy, P.A. Illich, G.A. Castro, E.T. Walters, Role of periaxonal inflammation in the development of thermal hyperalgesia and guarding behavior in a rat model of neuropathic pain, Neurosci. Lett. 184 (1995) 5–8. [3] G. Catheline, V. Kayser, G. Guilbaud, Further evidence for a peripheral component in the enhanced antinociceptive effect of systemic morphine in mononeuropathic rats: involvement of kappa-, but not delta-opioid receptors, Eur. J. Pharmacol. 315 (1996) 135 –143. [4] R.D. Egleton, T.J. Abbruscato, S.A. Thomas, T.P. Davis, Transport of
I. Obara et al. / Neuroscience Letters 360 (2004) 85–89
[5]
[6]
[7]
[8] [9] [10]
[11]
[12]
opioid peptides into the central nervous system, J. Pharm. Sci. 11 (1998) 1433–1439. A.P. Jeanjean, S.M. Moussaoui, J.M. Maloteaux, P.M. Laduron, Interleukin-1 beta induces long-term increase of axonally transported opiate receptors and substance P, Neuroscience 68 (1995) 151–157. V. Kayser, S.H. Lee, G. Guilbaud, Evidence for a peripheral component in the enhanced antinociceptive effect of a low dose of systemic morphine in rats with peripheral mononeuropathy, Neuroscience 64 (1995) 537–545. D. Labuz, R. Przewlocki, B. Przewlocka, Cross-tolerance between the different mu-opioid receptor agonists endomorphin-1, endomorphin-2 and morphine at the spinal level in the rat, Neurosci. Lett. 334 (2002) 127–130. H. Machelska, C. Stein, Immune mechanisms in pain control, Anesth. Analg. 95 (2002) 1002–1008. G.W. Pasternak, P.J. Wood, Multiple mu opiate receptors, Life Sci. 38 (1986) 1889–1898. M.B. Patel, C.N. Patel, V. Rajashekara, B.C. Yoburn, Opioid agonists differentially regulate mu-opioid receptors and trafficking proteins in vivo, Mol. Pharmacol. 62 (2002) 1464–1470. A. Pertovaara, H. Wei, Peripheral effects of morphine in neuropathic rats: role of sympathetic postganglionic nerve fibers, Eur. J. Pharmacol. 429 (2001) 139 –145. B. Przewlocka, J. Mika, D. Labuz, G. Toth, R. Przewlocki, Spinal analgesic action of endomorphins in acute, inflammatory and neuropathic pain in rats, Eur. J. Pharmacol. 367 (1999) 189 –196.
89
[13] R. Przewlocki, A.H. Hassan, W. Lason, C. Epplen, A. Herz, C. Stein, Gene expression and localization of opioid peptides in immune cells of inflamed tissue: functional role in antinociception, Neuroscience 48 (1992) 491–500. [14] R. Przewlocki, B. Przewlocka, Opioids in chronic pain, Eur. J. Pharmacol. 429 (2001) 79–91. [15] C. Stein, A.H. Hassan, K. Lehrberger, J. Giefing, A. Yassouridis, Local analgesic effect of endogenous opioid peptides, Lancet 342 (1993) 321–324. [16] C. Stein, M.J. Millan, T.S. Shippenberg, K. Peter, A. Herz, Peripheral opioid receptors mediating antinociception in inflammation. Evidence for involvement of mu, delta and kappa receptors, J. Pharmacol. Exp. Ther. 248 (1989) 1269–1275. [17] G. Stoll, H.P. Hartung, The role of macrophages in degeneration and immune-mediated demyelination of the peripheral nervous system, Adv. Neuroimmunol. 2 (1992) 163 –179. [18] W. Truong, C. Cheng, Q.G. Xu, X.Q. Li, D.W. Zochodne, Mu opioid receptors and analgesia at the site of a peripheral nerve injury, Ann. Neurol. 53 (2003) 366 –375. [19] X. Zhang, G. de Araujo Lucas, R. Elde, Z. Wiesenfeld-Hallin, T. Hokfelt, Effect of morphine on cholecystokinin and mu-opioid receptor-like immunoreactivities in rat spinal dorsal horn neurons after peripheral axotomy and inflammation, Neuroscience 95 (2000) 197–207. [20] M. Zimmermann, Ethical guidelines for investigations of experimental pain in conscious animals, Pain 16 (1983) 109– 110.
Local peripheral effects of m-opioid receptor agonists in neuropathic pain in rats Ilona Obara, Ryszard Przewlocki, Barbara Przewlocka* Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krako´w, Poland Received 14 October 2003; received in revised form 16 January 2004; accepted 16 January 2004
Abstract Our study was designed to demonstrate peripheral antinociception of the m-opioid receptor agonists: morphine (MF), [D -Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO), endomorphin-1 (EM-1) and endomorphin-2 (EM-2) in Bennett’s rat model of neuropathic pain. All the agonists were effective in antagonizing allodynia after their intraplantar (i.pl.) but not subcutaneous (s.c.) administration. Opioid peptides: DAMGO, EM-1 and EM-2 were more effective compared with corresponding doses of morphine (opioid alkaloid) in alleviating chronic pain. Peripheral m-opioid receptors mediated the observed effects, as was evidenced by the i.pl. treatment with naloxone methiodide (active only at the site of injection) and by cyprodime, a selective m-opioid receptor antagonist. These results have shown that opioid peptides are effective also after local treatment, and that their peripheral use may be of therapeutic interest in long-term management of chronic pain. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Nerve ligation injury; Peripheral m-opioid receptor; Opioid; Antinociception; DAMGO; Endomorphins
Pain arising from peripheral nerve injury is one of the more important health problems. Presently available therapeutic approaches reduce but do not eliminate characteristic syndromes of neuropathic pain, such as allodynia and hyperalgesia. Morphine and other classic opiates are not efficient analgesics in relieving neuropathic pain [1,14]; however, it has been suggested that neuropathic pain may be attenuated by higher doses (in comparison with those effective in acute pain) of opiates but this is accompanied by aggravation of unwanted side effects. Therefore, new methods and therapies with more potent analgesics, which would be beneficial to clinical practice, are currently being intensively searched for. Besides the central nervous system all three types of opioid receptors (m, d and k) have been found in dorsal root ganglia on cell bodies of sensory neurons, and on their central and peripheral terminals [16]. It is now well established that activation of these peripheral receptors produces analgesia in models of inflammatory pain [8,15]. This can be achieved by administration of opioid agonists at small, systemically inactive doses directly into injured tissue and by compounds that are unable to cross the blood – * Corresponding author. Tel.: þ 48-12-662-3398; fax: þ48-12-637-4500. E-mail address: [email protected] (B. Przewlocka).
brain barrier [8]. Peripheral opioid receptors are upregulated during inflammation and they can be activated by their endogenous ligands (opioid peptides) that are produced by circulating immune cells which migrate to injured tissues [13,16]. Opioid receptors expressed on peripheral afferent axons may also contribute to the opioid-induced antinociception in neuropathic pain [11,18]. Analgesic effects of opioid receptor agonists have been demonstrated after their systemic intravenous (i.v.) injection [3,6]. Moreover, it appears that the peripheral action of i.v. administered morphine in mononeuropathic rats is not only mediated via peripheral m-opioid receptors [6], but also via peripheral kopioid receptors [3]. In the present study, we investigated the effects of morphine (MF), [D -Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO), endomorphin-1 (EM-1) and endomorphin-2 (EM-2) after their intraplantar (i.pl.) administration directly into injured (by sciatic nerve ligation) hind paw and after subcutaneous (s.c.) administration in neuropathic rats. All experiments were performed according to the NIH Guide for Care and Use of Laboratory Animals and recommendations of the IASP [20], and were approved by the Local Bioethics Committee. Male Wistar rats (200 – 350 g) were housed individually in cages with sawdust bedding
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.01.056
86
I. Obara et al. / Neuroscience Letters 360 (2004) 85–89
under a standard 12:12-h light/dark cycle (lights on at 08:00 h) with food and water available ad libitum. Sciatic nerve injury was performed under pentobarbital anesthesia (60 mg/kg, i.p.) by tying four loose ligations around the sciatic nerve with 1 mm spacing as described by Bennett and Xie (1988). The ligatures were tied until they elicited a brief twitch in the respective hind limb, which prevented us from applying too strong a ligation. In all animals, the paw with a sciatic nerve injured in this way responds to pain stimulus; however, the reaction is different from controls. Testing procedures were carried out on days 12 –16 after the injury. Chemicals and their sources were as follows. Morphine hydrochloride: Polfa, Kutno, Poland; EM-1 and EM-2: Tocris Cookson Ltd., USA; DAMGO and naloxone methiodide (NxM): Sigma Chemical Co., USA. Cyprodime (CYP) was donated by Professor H. Schmidhammer (Innsbruck, Austria). All drugs were dissolved in sterile water and injected i.pl. into the injured hind paw in a volume of 20 ml or s.c. in a volume of 4 ml/kg. Each dose was tested in one experimental group of animals, n ¼ 8 – 10: CYP (274 nmol) and NxM (42.6 nmol) were injected i.pl. simultaneously with the highest effective doses of each used mopioid receptor agonist. After completion of the experiment, the animals were killed with an overdose of pentobarbital. Foot withdrawal threshold in response to mechanical stimuli was measured by the use of von Frey filaments (Stoelting, USA), which are used to apply slight pressure to the midplantar surface of the ipsilateral hind paw with the strength ranging from 0.2 to 26 g. The measurements were carried out 5, 15 and 30 min after i.pl. or s.c. drug injection on days 12– 16 after the sciatic nerve ligation. The results of the experiments were evaluated by analysis of variance (ANOVA) followed by Bonferroni’s post hoc test (P , 0:05 was considered to be statistically significant), and presented as a percentage of maximal possible effect (%MPE ^ S.E.M.) using the equation: %MPE ¼ [(BL 2 TL)/(BL 2 Cutoff)] £ 100%, where BL is baseline latency and TL is respective test latency. Our study showed that i.pl. injections of MF (528, 1056 nmol), DAMGO (4, 9.5 nmol), EM-1 (65, 131, 262 nmol) and EM-2 (70, 140 nmol) produced a dose-dependent, significant antiallodynic effect (Fig. 1A). The most potent effect was observed after DAMGO (9.5 nmol); the effects peaked 5 min after the drug injection (68.8 ^ 9.4% MPE) and later gradually declined. A similar strong antinociceptive effect was observed after EM-1 (131, 262 nmol); the effect peaked 5 min after drug injection (68.3 ^ 2.9% MPE) and was still observed until 30 min (56.5 ^ 8.6% MPE). The time course of antiallodynic effect after EM-2 (70, 140 nmol) administration was quite similar (63.1 ^ 2.4% MPE) to EM-1. In contrast, antinociception induced by MF (1056 nmol) reached the same level as that of DAMGO (9.5 nmol) and EM-1 (131, 262 nmol) at 30–60 min after the injection (in Fig. 1A only the effects observed after 5, 15 and 30 min are presented). We also tested the agonists after i.pl. administration in the cold-water
allodynia test and in the paw withdrawal test (thermal hyperalgesia test). The effects observed in these tests were similar to that observed in the von Frey test (approximately 30–50% MPE, data not shown). The antiallodynic effect demonstrated by the von Frey test carried out 5, 15 and 30 min after i.pl. administration of MF (528, 1056 nmol), DAMGO (4, 9.5 nmol), EM-1 (65, 131, 262 nmol) and EM-2 (70, 140 nmol) was antagonized by treatment with the selective m-opioid antagonist cyprodime (CYP; 274 nmol) as well as with the nonselective, peripherally active antagonist of opioid receptors, naloxone methiodide (NxM; 42.6 nmol) (Fig. 1B). The antiallodynic potencies of all used m-opioid receptor agonists are compared in Fig. 2. DAMGO was much more potent then EMs and MF 5, 15 and 30 min after their administration. The antiallodynic potency of DAMGO and EMs was demonstrable at considerably lower concentration (2 –9.5 nmol and 33 –262 nmol, respectively) in comparison with MF (264 – 1056 nmol). Both EMs antagonized allodynia when given in a similar dose range (33 – 263 nmol and 35 –140 nmol, respectively). To compare the peripheral and local effects we administered s.c. MF (1.5, 0.75 mg/kg), DAMGO (7, 20 mg/kg), EM-1 (150, 300 mg/kg) and EM-2 (150, 300 mg/kg) at a dose comparable to the dose used locally (i.pl.). The antiallodynic effects measured in the von Frey and paw withdrawal tests were not statistically significant versus the respective control group (Fig. 2B, only the effect observed after 15 min is presented). Conventionally, the analgesic action of opioids is attributed to their effects on the central nervous system, hence they have been investigated after their central, mainly spinal administration. In the present study, we demonstrated that also locally, i.pl. administered m-opioid receptor agonists (MF, DAMGO, EM-1 and EM-2) were effective in neuropathic pain, in contrast to noneffective s.c. administration. Naloxone methiodide, a nonselective antagonist of opioid receptors which acts only at the site of injection and cyprodime, a selective m-opioid receptor antagonist, inhibited the antiallodynic effect of locally (i.pl.) administered opioids, and showed that the observed effect was mediated by peripheral m-opioid receptors. These observations are in agreement with recent studies indicating that opioids may also induce antinociceptive effects at peripheral sites in a neuropathic pain model [3,11,18]. Some investigators demonstrated the analgesic effect of morphine and various selective agonists of m- (DAMGO), d- (BUBU, DTLET) and k- (U 69,593, ICI 20448) opioid receptors after their systemic (i.v.) administration which might suggest a limited, but still possible effect on the central opioid receptor (spinal and/or supraspinal) due to an uptake of the drug by the nervous system from the blood [3,4,6]. Therefore, in our study we used local injections to the injured paw in order to exclude the role of central effects, and validate the role of peripheral opioid receptors in neuropathic pain.
I. Obara et al. / Neuroscience Letters 360 (2004) 85–89
87
Fig. 1. Effect of local intraplantar (i.pl.) administration of morphine (MF), DAMGO, endomorphin-1 (EM-1) and endomorphin-2 (EM-2) on mechanical allodynia in rats 12–16 days after the sciatic nerve injury measured in von Frey test. (A) Time course of the drug effects. The mean reaction in the control group subjected to sciatic nerve injury and injected with solvent (open symbols) was 2.1 ^ 0.9 g. (B) The influence of local (i.pl.) treatment with selective m-opioid receptor antagonist, cyprodime (CYP; 274 nmol) and nonselective opioid antagonist, naloxone methiodide (NxM; 42.6 nmol) on the effects of i.pl. administration of selected doses of used m-agonists. The results are shown as a mean percentage of maximal possible effect (%MPE ^ S.E.M.) of 8– 10 animals per group. *P , 0:05; ***P , 0:001 in comparison with the solvent-injected group of animals (broken line) with the injured sciatic nerve.þ þ þ P , 0:001 in comparison with the group of animals with the injured sciatic nerve after i.pl. administration of effective dose of tested agonists (ANOVA, Bonferroni test).
Troung et al. [18] provided evidence that local m-opioid receptors were expressed on peripheral sensory axons and mediated analgesia in the microenvironment of a peripheral nerve injury. Despite the fact that peripheral axotomy and peripheral nerve ligation caused a reduction in the number of m-opioid receptors in the rat dorsal root ganglion ([19] and our unpublished observations), Troung et al. [18] demonstrated an increase in the proportion of neurons expressing m-opioid receptors in sensory neurons ipsilateral to the ligated nerve. Moreover, an enhanced axonal transport of m-opioid receptors in sensory neurons, leading to an increase in their number on peripheral nerve terminals, was reported [5]. Alleviation of allodynia after administration of opioid peptides appears faster (5 min after i.pl. administration) and
the same doses of these drugs are as effective as those reducing acute pain, while the alkaloid acts more slowly (15 –30 min after injection) and only its much higher doses are active in comparison with the doses relieving acute pain. Interestingly, we previously demonstrated that endomorphins were more effective, also after their intrathecal (i.t.) administration, than morphine in neuropathic pain [12,14]. The reason for these differences is still unknown, since all agonists appear to act via the same m-opioid receptor. One possible explanation is the involvement of different subtypes of m-opioid receptor as a target of different agonists [7,9]. Another explanation is a possibility of unequal receptor regulation by peptide agonists and morphine due to differential regulation of trafficking
88
I. Obara et al. / Neuroscience Letters 360 (2004) 85–89
Identification of the differences between these m-opioid receptor agonists could be of great importance to our understanding of the mechanism of opioid action in neuropathic pain. It should also be mentioned that although inflammatory conditions are not obvious 2 weeks after surgery in the chronic constriction injury model [2], some authors suggested involvement of the immune system in the mediation of the nervous system response to damage [15]. This may be linked to the presence of activated macrophages around the injured nerve fibers [17], the role of which is to promote the regeneration process in the peripheral nervous system, to participate in the development of neuropathic pain [17] and to activate opioid receptors located on peripheral sensory neurons [15]. The spinal nerve ligation model of neuropathy is expected to produce a milder inflammatory reaction, particularly in the distal parts of the limb. Pertovaara and Wei [11] reported that spinal nerve injury in the Kim and Chung model (1992) might lead to an enhanced contribution of peripheral mechanisms to morphine-induced antinociception. Moreover, local anesthetic action, such as that of lidocaine, was excluded [6]. In conclusion, in the present study we demonstrated a peripheral antinociceptive effect of the m-opioid receptor agonists morphine, DAMGO and endomorphins, in Bennett’s rat model of neuropathic pain after their i.pl. administration. Our studies, alongside investigations of other authors [11,18] concerning the action of antinociceptive opioid receptor agonists (especially morphine) seem to confirm a lower efficacy of morphine in neuropathic pain in comparison with peptide agonists, also after local administration. Therefore, potent peptide opioid agonists with a peripheral site of action can comprise an important target in the strategy for pharmacological treatment of chronic pain.
Acknowledgements Supported by grant PBZ-MN-001-/P05/2002 (I.O. and B.P.) and EU grant No. QLRT-2001-02919 (R.P.). Fig. 2. Comparison of dose–response curves of m-opioid receptor agonists. (A) After their intraplantar (i.pl.) administration (morphine (MF): 264, 528, 1056 nmol; DAMGO: 2, 4, 9.5 nmol; endomorphin-1 (EM-1): 33, 131, 262 nmol; endomorphin-2 (EM-2): 35, 70, 140 nmol) and (B) after their subcutaneous (s.c.) administration (MF: 0.75, 1.5 mg/kg; DAMGO: 7, 20 mg/kg; EM-1: 150, 300 mg/kg; EM-2: 150, 300 mg/kg) measured by von Frey test in rat model of neuropathic pain 12–16 days after the sciatic nerve injury. The results are shown as a mean percentage of maximal possible effect (%MPE ^ S.E.M.) of 8–10 animals per group.
proteins. Those drugs that do not cause receptor internalization, such as morphine, induce development of tolerance more easily and as a consequence may lose their effectiveness in neuropathic pain [10]. An alternative explanation is that nerve injury followed by development of the neuropathic pain process causes some changes in potency and efficacy of binding parameters of the m-opioid receptors.
References [1] D. Bian, M.H. Ossipov, M. Ibrahim, R.B. Raffa, R.J. Tallarida, T.P. Malan Jr, J. Lai, F. Porreca, Loss of antiallodynic and antinociceptive spinal/supraspinal morphine synergy in nerve-injured rats: restoration by MK-801 or dynorphin antisera, Brain Res. 831 (1999) 55 –63. [2] A.L. Clatworthy, P.A. Illich, G.A. Castro, E.T. Walters, Role of periaxonal inflammation in the development of thermal hyperalgesia and guarding behavior in a rat model of neuropathic pain, Neurosci. Lett. 184 (1995) 5–8. [3] G. Catheline, V. Kayser, G. Guilbaud, Further evidence for a peripheral component in the enhanced antinociceptive effect of systemic morphine in mononeuropathic rats: involvement of kappa-, but not delta-opioid receptors, Eur. J. Pharmacol. 315 (1996) 135 –143. [4] R.D. Egleton, T.J. Abbruscato, S.A. Thomas, T.P. Davis, Transport of
I. Obara et al. / Neuroscience Letters 360 (2004) 85–89
[5]
[6]
[7]
[8] [9] [10]
[11]
[12]
opioid peptides into the central nervous system, J. Pharm. Sci. 11 (1998) 1433–1439. A.P. Jeanjean, S.M. Moussaoui, J.M. Maloteaux, P.M. Laduron, Interleukin-1 beta induces long-term increase of axonally transported opiate receptors and substance P, Neuroscience 68 (1995) 151–157. V. Kayser, S.H. Lee, G. Guilbaud, Evidence for a peripheral component in the enhanced antinociceptive effect of a low dose of systemic morphine in rats with peripheral mononeuropathy, Neuroscience 64 (1995) 537–545. D. Labuz, R. Przewlocki, B. Przewlocka, Cross-tolerance between the different mu-opioid receptor agonists endomorphin-1, endomorphin-2 and morphine at the spinal level in the rat, Neurosci. Lett. 334 (2002) 127–130. H. Machelska, C. Stein, Immune mechanisms in pain control, Anesth. Analg. 95 (2002) 1002–1008. G.W. Pasternak, P.J. Wood, Multiple mu opiate receptors, Life Sci. 38 (1986) 1889–1898. M.B. Patel, C.N. Patel, V. Rajashekara, B.C. Yoburn, Opioid agonists differentially regulate mu-opioid receptors and trafficking proteins in vivo, Mol. Pharmacol. 62 (2002) 1464–1470. A. Pertovaara, H. Wei, Peripheral effects of morphine in neuropathic rats: role of sympathetic postganglionic nerve fibers, Eur. J. Pharmacol. 429 (2001) 139 –145. B. Przewlocka, J. Mika, D. Labuz, G. Toth, R. Przewlocki, Spinal analgesic action of endomorphins in acute, inflammatory and neuropathic pain in rats, Eur. J. Pharmacol. 367 (1999) 189 –196.
89
[13] R. Przewlocki, A.H. Hassan, W. Lason, C. Epplen, A. Herz, C. Stein, Gene expression and localization of opioid peptides in immune cells of inflamed tissue: functional role in antinociception, Neuroscience 48 (1992) 491–500. [14] R. Przewlocki, B. Przewlocka, Opioids in chronic pain, Eur. J. Pharmacol. 429 (2001) 79–91. [15] C. Stein, A.H. Hassan, K. Lehrberger, J. Giefing, A. Yassouridis, Local analgesic effect of endogenous opioid peptides, Lancet 342 (1993) 321–324. [16] C. Stein, M.J. Millan, T.S. Shippenberg, K. Peter, A. Herz, Peripheral opioid receptors mediating antinociception in inflammation. Evidence for involvement of mu, delta and kappa receptors, J. Pharmacol. Exp. Ther. 248 (1989) 1269–1275. [17] G. Stoll, H.P. Hartung, The role of macrophages in degeneration and immune-mediated demyelination of the peripheral nervous system, Adv. Neuroimmunol. 2 (1992) 163 –179. [18] W. Truong, C. Cheng, Q.G. Xu, X.Q. Li, D.W. Zochodne, Mu opioid receptors and analgesia at the site of a peripheral nerve injury, Ann. Neurol. 53 (2003) 366 –375. [19] X. Zhang, G. de Araujo Lucas, R. Elde, Z. Wiesenfeld-Hallin, T. Hokfelt, Effect of morphine on cholecystokinin and mu-opioid receptor-like immunoreactivities in rat spinal dorsal horn neurons after peripheral axotomy and inflammation, Neuroscience 95 (2000) 197–207. [20] M. Zimmermann, Ethical guidelines for investigations of experimental pain in conscious animals, Pain 16 (1983) 109– 110.