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Antinociceptive role of galanin in periaqueductal grey of rats with experimentally induced mononeuropathy
Neuroscience Vol. 96, No. 4, pp. 767–771, 2000 767 Copyright q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
Antinociception of galanin in PAG in neuropathic rats
Pergamon PII: S0306-4522(00)00005-1 www.elsevier.com/locate/neuroscience
ANTINOCICEPTIVE ROLE OF GALANIN IN PERIAQUEDUCTAL GREY OF RATS WITH EXPERIMENTALLY INDUCED MONONEUROPATHY D. WANG,* T. LUNDEBERG† and L.-C. YU*‡ *Department of Physiology, College of Life Sciences, and National Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871, People’s Republic of China †Department of Physiology and Pharmacology, and Department of Medical Rehabilitation, Karolinska Institutet, 171 77 Stockholm, Sweden
Abstract—The present study was performed in rats with experimentally induced mononeuropathy after left common sciatic nerve ligation. The hindpaw withdrawal latencies to thermal and mechanical stimulation increased significantly after intra-periaqueductal grey injection of 2 or 3 nmol, but not 1 nmol of galanin in rats with mononeuropathy. Intraperitoneal administration of 4.5 mg/kg morphine induced significant increases in hindpaw withdrawal latencies to both noxious stimulation, which were attenuated by following intra-periaqueductal grey injection of 2 nmol of the galanin antagonist galantide. Furthermore, the antinociceptive effect induced by intra-periaqueductal grey injection of 26.6 nmol of morphine was attenuated significantly by following intraperiaqueductal gray administration of 2 nmol of galantide. The results demonstrated that in periaqueductal grey galanin plays an antinociceptive role in rats with mononeuropathy and galanin is involved in the mechanisms of opioid-induced antinociception. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: galanin, opioid peptides, periaqueductal grey, mononeuropathy, antinociception, hindpaw withdrawal latency.
Bennett and Xie set up a mononeuropathic pain model by placing four loose ligatures around one sciatic nerve in rats. 3 In the chronic constriction injury model, rats developed a marked and long-lasting but reversible hyperalgesia to thermal and/or mechanical stimulation. Damage to both myelinated and unmyelinated peripheral axons has been described, though the damage was greater to the myelinated fibers. 3 Neurochemical changes were also seen in the primary afferent fibers, spinal cord and dorsal root ganglia (DRG) at 14–28 days following chronic constriction injury. 15 At that time the mononeuropathic rats displayed marked decreases in withdrawal latencies to stimulation interpreted as signs of hyperalgesia. 15 Some neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) were down-regulated, while others such as vasoactive intestinal peptide and galanin were up-regulated. 6,12 Among these neuropeptides, galanin is of particular interest because galanin may be an endogenous analgesic compound as elegantly demonstrated by WiesenfeldHallin and collaborators. 27 Administration of a galanin antagonist induced a higher rate of autotomy after axotomy. 21 However, Munglani et al. reported that 100–120 days after the constriction, when the hindpaw withdrawal latencies reached the same level as they were before the constriction, galanin-immunoreactivity decreased ipsilaterally in laminae 1 and 2 of the dorsal horn of the chronic constriction injury rats. 15 Yu et al. reported that the hindpaw withdrawal latency to thermal and mechanical stimulation increased significantly after intrathecal injection of galanin in mononeuropathic rats.25 The results indicate that galanin may participate in the modulation of chronic pain following peripheral nerve injury. 12
Opioid binding sites in the superficial dorsal horn of the spinal cord in rats also changed after nerve chronic constriction injury. A significant up-regulation in m binding sites was found at the spinal cord level following nerve ligation. 4 The up-regulation appeared to be bilateral although, on the ipsilateral side, this effect might be masked by fiber degeneration. 4 Recent studies indicated that there may be a close interaction between galanin and opioids in the mechanisms of endogenous antinociception in intact rats. 17,20,22,25 Recent studies in our laboratory demonstrated that intrathecal injection 1 or 3 nmol of galantide, an antagonist of galanin, 2 attenuated the antinociceptive effects induced by intrathecal injection of morphine in rats with mononeuropathy. 28 Furthermore, our study demonstrated that intra-periaqueductal grey (PAG) administration of galanin induced marked antinociceptive effects in intact rats. 22 The present study was performed to investigate the role of intra-PAG injection of galanin on nociception and the presumable interaction between galanin and opioids in PAG in mononeuropathic rats. EXPERIMENTAL PROCEDURES
Animal preparation Freely moving male Sprague–Dawley rats weighing 250–300 g (Experimental Animal Center of Beijing Medical University, Beijing, China) were used in the present experiments, which were conducted according to the guidelines of the animal ethical committee of Karolinska Institutet and every effort was made to minimize both the animal suffering and the number of animals used. Surgical procedures
‡To whom correspondence should be addressed. Tel.: 1 86-10-62751867; fax: 1 86-10-62751526. E-mail address: [email protected] (L.-C. Yu). Abbreviations: CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; HWL, hindpaw withdrawal latency; PAG, periaqueductal grey.
Rats were anaesthetized with intraperitoneal sodium pentobarbital (45 mg/kg). To produce a mononeuropathic model, the rat’s left sciatic nerve was exposed for 8–10 mm at the level of the midthigh. Four loose ligatures (4.0 chromic gut) were made around the dissected nerve with a 1.0–1.5-mm interval between each of them. The ligation was 767
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carefully manipulated so that the nerve was barely constricted. 3 The skin incision was closed with 4-0 silk sutures. Intra-periaqueductal grey injection The animals were anaesthetized by intraperitoneal sodium pentobarbital (45 mg/kg) and were mounted on a stereotaxic instrument. A stainless steel guide cannula of 0.8 mm o.d. was directed to PAG (AP 5.5, L 0.5, H 6.0 mm from the surface of the skull) according to Paxinos and Watson 16 and was fixed to the skull by dental acrylic. On the experimental day a stainless steel needle with 0.4 mm diameter was directly inserted into the guide cannula, with 1 mm beyond the tip of the latter. One microliter of solution was thereafter infused into PAG over 1 min. Nociceptive tests All rats were accustomed to the testing conditions for five days before the experiment was conducted. The latencies to hindpaw withdrawal during thermal and mechanical stimulation were measured. The thermal response was assessed by the hot-plate test. The entire ventral surface of the rat’s hindpaw was placed manually on the hot-plate which was maintained at a temperature of 528C (51.8–52.48C). 25,26 The time to hindpaw withdrawal was measured in seconds to be referred to as the hindpaw withdrawal latency (HWL). The Randall Selitto Test (Ugo Basile, Type 7200, Italy) was used to assess the HWL to mechanical stimulation. A wedge-shaped pusher at a loading rate of 30 g/s was applied to the dorsal surface of the manually handled hindpaw and the latency required to initiate the struggle response was assessed and expressed in seconds. 25,26 The average values obtained before intra-PAG injection were regarded as the basal HWL. The HWLs recorded during subsequent experiments were expressed as percentage changes of the basal level for each rat (% change of the HWL). Each rat was tested with both types of stimulation. Histological test At the end of the experiment rats were killed with intraperitoneal high dose of sodium pentobarbital (80 mg/kg) and the rat’s heads were fixed in 10% formalin for one week with the injecting tube in situ. The location of the tip of the injecting tube was verified in serial 50-mm crystal sections. Only the results from nociceptive tests with the tip of the injecting tube placed within PAG area were used for statistical analysis.
Chemicals Morphine hydrochloride (morphine hydrochloride, Shenyang First Pharmaceutical Factory, Shenyang, China) was diluted to 1 mg/ml with sterilized saline and was administrated intraperitoneally at a dose of 4.5 mg/kg. Solutions for intra-PAG administration were prepared with sterilized saline, each with a volume of 1 ml: (i) 1, 2 or 3 nmol of galanin (galanin, Bachem, Feinchemikalien AG, Switzerland), respectively; (ii) 2 nmol of galantide [Galanin (1–13)Substance P (5–11) amide, Bachem, Feinchemikalien AG, Switzerland]; (iii) 26.6 nmol of morphine. Control groups were given 1 ml of 0.9% saline injected into PAG. Statistical analysis Data from nociceptive tests were presented as mean ^ S.E.M. The difference between groups was determined by two-way analysis of variance (ANOVA) for repeated measures. *P , 0.05, **P , 0.01 and ***P , 0.001 were considered as significant differences. RESULTS
Effects of intra-periaqueductal grey administration of galanin on hindpaw withdrawal latencies to noxious stimulation in rats with mononeuropathy Rats received intra-PAG injection of 1 nmol (n 9), 2 nmol (n 8) or 3 nmol (n 9) of galanin, or 1 ml of 0.9% saline (n 14) as the control group. As shown in Fig. 1, the HWLs to thermal and mechanical stimulation increased significantly after intra-PAG injection of 2 nmol (Thermal test: Fleft/left 45.43, P , 0.001; Fright/right 13.73, P , 0.001. Mechanical test: Fleft/left 18.39, P , 0.001; Fright/right 7.49, P , 0.01) or 3 nmol of galanin (Thermal test: Fleft/left 65.23, P , 0.001; Fright/right 71.70, P , 0.001. Mechanical test: Fleft/left 44.11, P , 0.001; Fright/right 44.00, P , 0.001) compared with the control group. In the group receiving intra-PAG injection of 1 nmol of galanin there were no significant changes in HWLs in comparison with the control group (Thermal test: Fleft/left 2.18, P 0.14; Fright/right 3.28, P 0.07. Mechanical test: Fleft/left 2.26, P 0.14; Fright/right 0.15,
Fig. 1. Effects of intra-periaqueductal grey injection of 1, 2 or 3 nmol of galanin on left (A, C) and right (B, D) hindpaw withdrawal latencies to thermal (A, B) and mechanical (C, D) stimulation in rats with mononeuropathy. Control group received intra-periaqueductal grey injection of 1 ml of 0.9% saline. One microliter of 0.9% saline (W, n 14); 1 nmol of galanin (A, n 9); 2 nmol of galanin (X, n 8); 3 nmol of galanin (B, n 9). The data are described as % change of HWL. Mean ^ S.E.M., two-way ANOVA, *P , 0.05 and ***P , 0.001 compared with the control group.
Antinociception of galanin in PAG in neuropathic rats
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Fig. 2. Effects of intra-periaqueductal grey injection of galantide on intraperitoneal morphine-induced increases in HWL in rats with sciatic nerve ligation. Left HWL: A and C; right HWL: B and D. HWLs to thermal (A, B) and mechanical (C, D) stimulation. Time 0 min: intraperitoneal injection of 4.5 mg/kg of morphine; time 10 min: intra-periaqueductal grey injection of 2 nmol of galantide or 1 ml of 0.9% saline as a control. Morphine 1 1 ml of 0.9% saline (A, n 8); Morphine 1 2 nmol of galantide (B, n 8); Saline 1 2 nmol of galantide (W, n 6). The data are expressed as % change of HWL. Mean ^ S.E.M., two-way ANOVA, ***P , 0.001 compared with the control group.
P 0.70). The effects of intra-PAG administration of 3 nmol of galanin reached the peak between 10–20 min after the injection and then began to recover from 30 min. Effects of intra-periaqueductal grey injection of galantide on intraperitoneal morphine-induced increases in hindpaw withdrawal latency Rats with mononeuropathy received intraperitoneal injection of 4.5 mg/kg morphine, followed 10 min later, by intra-PAG injection of 2 nmol of galantide (n 8) or 1 ml of 0.9% saline (n 8) as the control group. The results are shown in Fig. 2. In the control group, the HWLs to both thermal and mechanical stimulation increased and lasted for more than 60 min. In the group receiving intraperitoneal morphine followed by intra-PAG injection of 2 nmol of galantide, the increased HWLs were attenuated significantly in both tests (Thermal test: Fleft/left 70.61, P , 0.001; Fright/right 132.40, P , 0.001. Mechanical test: Fleft/left 37.02, P , 0.001; Fright/right 49.05, P , 0.001) compared with the control group. In another group, rats receiving intraperitoneal administration of 1 ml of 0.9% saline, followed 10 min later, by intra-PAG administration of 2 nmol of galantide (n 6), there were no marked changes in the HWL to thermal and mechanical stimulation, as shown in Fig. 2. Effects of intra-periaqueductal grey injection of galantide on the increased hindpaw withdrawal latency induced by intra-periaqueductal grey injection of morphine Rats with mononeuropathy received intra-PAG injection of 26.6 nmol of morphine, followed 10 min later, by 2 nmol of galantide (n 6) or 1 ml of 0.9% saline (n 6) as a control. The results are shown in Fig. 3. After intra-PAG injection of morphine, the HWL to both noxious stimulation increased. In
the group receiving intra-PAG injection of morphine, followed by 2 nmol galantide, the increased HWLs were attenuated significantly (Thermal test: Fleft/left 37.18, P , 0.001; Fright/right 25.64, P , 0.001. Mechanical test: Fleft/left 19.08, P , 0.001; Fright/right 28.21, P , 0.001) as compared with the control group. In another group, rats receiving intra-PAG administration of 1 ml of 0.9% saline, followed 10 min later, by intra-PAG administration of 2 nmol of galantide (n 6), there were no marked changes in HWLs to both noxious thermal and mechanical stimulation. DISCUSSION
The results of the present study show that intra-PAG administration of 2 or 3 nmol of galanin induced a dosedependent antinociceptive effect in rats with mononeuropathy, while 1 nmol of galanin did not. Intraperitoneal injection of 4.5 mg/kg of morphine induced significant increases in HWLs to thermal and mechanical stimulation, and the effects were attenuated by intra-PAG injection of 2 nmol of the galanin receptor antagonist galantide. Furthermore, intra-PAG administration of 2 nmol of galantide attenuated the antinociceptive effects induced by intra-PAG injection of 26.6 nmol of morphine. The results indicate that galanin may have an antinociceptive role in the PAG in mononeuropathic rats, and that galanin interacted with opioid peptides in this brain area. Galanin is a neuropeptide with mainly inhibitory effects in central nervous system. 1,9,10 There was a marked increase of galanin-immunoreactivity in primary sensory neuron and DRG after loose sciatic nerve ligation. 6,12 Interestingly, the change was paralleled by changes in nociceptive behaviors. 15 It has, therefore, been suggested that galanin may be involved in the endogenous analgesic system, especially after peripheral nerve injury. 6,24,25 Nerve injury, such as loose sciatic nerve ligation and axotomy, led to hyperactivity in
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Fig. 3. Intra-periaqueductal grey injection of galantide attenuated the increased HWL induced by intra-periaqueductal grey morphine in rats with sciatic nerve ligation. Left HWL: A and C; right HWL: B and D. HWLs to thermal (A, B) and mechanical (C, D) stimulation. Time 0 min: intra-periaqueductal grey injection of 26.6 nmol of morphine; time 10 min: intra-periaqueductal grey injection of 2 nmol of galantide or 1 ml of 0.9% saline as a control. Morphine 1 1 ml of 0.9% saline (A, n 6); Morphine 1 2 nmol of galantide (B, n 6); Saline 1 2 nmol of galantide (W, n 6). The data are expressed as % change of HWL. Mean ^ S.E.M., two-way ANOVA, ***P , 0.001 compared with the control group.
the somatosensory system and spontaneous firing of some primary afferent fibers. 7 It is possible that the release of galanin is due to the abnormal impulses of these afferents. 6 Another reason could be diffusion and/or rapid enzymatic breakdown of accumulated peptide. It is also possible that there may be a slowing down of peripheral axonal transport. 11 Also, the accumulation of galanin at the injury site might play a role in the initiation or modulation of ectopic neural activity. 11 Furthermore, Carlton et al. found that many galanin terminals no longer co-localized with CGRP after peripheral nerve lesion, possibly indicating there was an increased antinociceptive activity after nerve lesions. 5 In conclusion, the up-regulation of galanin might respond to the increased nociceptive input induced by partial nerve injury. Another possible and important role of galanin after nerve injury may be to re-establish the neural target contact. The cytokine leukemia inhibitory factor produced by the degenerated nerve possibly up-regulates the galanin in the DRG large neurons in chronic constriction injury rats. 12 This is in line with the finding that galanin has trophic effects on axotomized neurons which are temporarily deprived of their target-derived trophic factors. 27 This may partly explain why some axotomized afferents cease synthesizing their “normal” neuropeptides (e.g. substance P and CGRP) and start synthesizing others such as galanin. Also, Ma and Bisby reported that partial sciatic nerve injury induced greater galanin up-regulation in medium- and large-size DRG neurons than complete sciatic nerve injury. 12 Moreover, according to the report of Munglani et al., the level of galanin-immunoreactivity was down-regulated 100–120 days after sciatic nerve lesion, by which time resolution of the hyperalgesia and peripheral nerve injury has occurred. 15 All the above results indicate that the change in galanin expression observed in peripheral neurons after nerve injury
could be characterized as reflecting a shift in the “focus” of the neuron from synaptic transmission to regeneration. 29 Previous studies showed that galanin co-exists with several other neurotransmitters in the CNS in animals, including opioids. 10 Both galanin receptors 9,14,19 and opioid receptors, including m-, d- and k-receptors 13 are found in rat PAG, an essential structure for opioid analgesia. 8 Galanin has been proposed to interact with opioids in modulation of the transmission of nociceptive information at the spinal cord level 18,21,23 and in PAG 22 in intact rats. In the present study, the increased HWL induced by intra-PAG or intraperitoneal administration of morphine was significantly attenuated by intra-PAG administration of the galanin receptor antagonist galantide, indicating a possible interaction in antinociception between galanin and opioids in PAG in rats with mononeuropathy. Interestingly, m-opioid receptor binding sites were also upregulated in the spinal cord in chronic constriction injury rats, possibly due to the activation of descending control systems in response to increased nociceptive inputs following sciatic nerve lesion. 4 After binding to its receptor, galanin may facilitate the inhibitory effects of opioid peptides, and/or enhance the affinity of opioid peptides to their own receptors. When galantide, the antagonist of galanin receptors, 1 was applied, it prevented galanin from binding to its receptors, and thus indirectly attenuating the opioid analgesia, as we observed in the present study. Also, in support of our findings is the study by Reimann et al. who investigated whether endogenous galanin, by the use of the galanin antagonist galantide, interacts with exogenously administered morphine, in the rat spinal cord. 18 They reported that in the rat tail-flick test, intrathecal injection of 3 mg of morphine had a significant antinociceptive effect which was almost completely antagonized by co-injection of 2 mg of galantide. Also, this would
Antinociception of galanin in PAG in neuropathic rats
suggest that galantide may have a pro-nociceptive effect, a suggestion supported by the finding that galantide induced a higher rate of autotomy after axotomy. 21 CONCLUSION
Intra-PAG injection of galanin induced a significant antinociceptive effect in rats with sciatic nerve ligation. The increased HWLs induced by intraperitoneal administration of morphine were significantly attenuated by following intra-PAG injection of galantide, the antagonist of galanin receptors, indicating that PAG is one of the key sites of the interaction between galanin and opioids in rats with
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mononeuropathy. Furthermore, intra-PAG injection of galantide also reversed the antinociceptive effects induced by intra-PAG administration of morphine. The data suggest an antinociceptive role of galanin in the PAG of mononeuropathic rats, and a possible interaction between galanin and opioid peptides in this brain area.
Acknowledgements—This study was supported by funds from the National Natural Science Foundation of China (NSFC), the Natural Science Research Foundation of Peking University, the Karolinska Institutet Foundation and the Foundation of Acupuncture and Alternative Methods.
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(1996) Stereological analysis of galanin and CGRP synapses in the dorsal horn of neuropathic primates. Brain Res. 711, 16–25. 6. Colvin L. A., Mark M. A. and Duggan A. W. (1997) The effect of a peripheral mononeuropathy on immunoreactive (ir)-galanin release in the spinal cord of the rat. Brain Res. 766, 259–261. 7. Devor M. (1994) The pathophysiology of damaged peripheral nerves. In Textbook of Pain (eds Wall P. D. and Melzack R.), 3rd edn, pp. 79–100. Churchill Livingstone, Edinburgh. 8. Fields H. L. and Basbaum A. I. (1994) Central nervous system mechanisms of pain modulation. In Textbook of Pain (eds Wall P. D. and Melzack R.), 3rd edn, pp. 243–257. Churchill Livingstone, Edinburgh. 9. Kask K., Berthold M. and Bartfai T. (1997) Galanin receptors: involvement in feeding, pain, depression and Alzheimer’s disease. Life Sci. 60, 1523–1533. 10. Kask K., Langel U. and Bartfai T. (1995) Galanin—a neuropeptide with inhibitory actions. Cell. molec. Neurobiol. 15, 653–673. 11. Long A., Bongenhielm U., Boissonade F. M., Fried K. and Robinson P. P. (1998) Neuropeptide immunoreactivity in ligature-induced neuromas of the inferior alveolar nerve in the ferret. Brain Res. 791, 263–270. 12. Ma W. and Bisby M. A. (1997) Differential expression of galanin immunoreactivities in the primary sensory neurons following partial and complete sciatic nerve injuries. Neuroscience 79, 1183–1195. 13. Mansour A., Fox C. A., Akil H. and Watson S. J. (1995) Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications. Trends Neurosci. 18, 22–29. 14. Merchenthaler I., Lopez F. J. and Negro-Vilar A. (1993) Anatomy and physiology of central galanin-containing pathway. Prog. Neurobiol. 40, 711–769. 15. Munglani R., Harrison S. M., Smith G. D., Bountra C., Birch P. J., Elliot P. J. and Hunt S. P. (1996) Neuropeptide changes persist in spinal cord despite resolving hyperalgesia in a rat model of mononeuropathy. Brain Res. 743, 102–108. 16. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic, Sydney. 17. Przewlocka B., Machelska H., Rekowski P., Kupryszewski G. and Prezewlocki R. (1995) Intracerebroventricular galanin and N-terminal galanin fragment enhance the morphine induced analgesia in the rat. J. neural Transm. 102, 229–235. 18. Reimann W., Englberger W., Friderichs E., Selve N. and Wilffert B. (1994) Spinal antinociception by morphine in rats is antagonized by galanin receptor antagonists. Naunyn-Schmiedeberg’s Arch. Pharmac. 350, 380–386. 19. Ryan M. C. and Gundlach A. L. (1996) Localization of preprogalanin messenger RNA in rat brain: identification of transcripts in a subpopulation of cerebellar purkinje cells. Neuroscience 70, 709–728. 20. Selve N., Englberger W., Friderichs E., Hennies H.-H., Reimann W. and Wilffert B. (1996) Galanin receptor antagonists attenuate spinal anti-nociceptive effects of DAMGO, tramadol and non-opioid drugs in rats. 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(1996) Retrograde and paracrine influences on neuropeptide expression in sympathetic neuron after axonal injury. In Cytokines and the CNS (eds Ransohoff R. M. and Benveniste E. N.) Vol. 1, pp. 169–186. CRC, Florida. (Accepted 6 January 2000)
Antinociception of galanin in PAG in neuropathic rats
Pergamon PII: S0306-4522(00)00005-1 www.elsevier.com/locate/neuroscience
ANTINOCICEPTIVE ROLE OF GALANIN IN PERIAQUEDUCTAL GREY OF RATS WITH EXPERIMENTALLY INDUCED MONONEUROPATHY D. WANG,* T. LUNDEBERG† and L.-C. YU*‡ *Department of Physiology, College of Life Sciences, and National Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871, People’s Republic of China †Department of Physiology and Pharmacology, and Department of Medical Rehabilitation, Karolinska Institutet, 171 77 Stockholm, Sweden
Abstract—The present study was performed in rats with experimentally induced mononeuropathy after left common sciatic nerve ligation. The hindpaw withdrawal latencies to thermal and mechanical stimulation increased significantly after intra-periaqueductal grey injection of 2 or 3 nmol, but not 1 nmol of galanin in rats with mononeuropathy. Intraperitoneal administration of 4.5 mg/kg morphine induced significant increases in hindpaw withdrawal latencies to both noxious stimulation, which were attenuated by following intra-periaqueductal grey injection of 2 nmol of the galanin antagonist galantide. Furthermore, the antinociceptive effect induced by intra-periaqueductal grey injection of 26.6 nmol of morphine was attenuated significantly by following intraperiaqueductal gray administration of 2 nmol of galantide. The results demonstrated that in periaqueductal grey galanin plays an antinociceptive role in rats with mononeuropathy and galanin is involved in the mechanisms of opioid-induced antinociception. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: galanin, opioid peptides, periaqueductal grey, mononeuropathy, antinociception, hindpaw withdrawal latency.
Bennett and Xie set up a mononeuropathic pain model by placing four loose ligatures around one sciatic nerve in rats. 3 In the chronic constriction injury model, rats developed a marked and long-lasting but reversible hyperalgesia to thermal and/or mechanical stimulation. Damage to both myelinated and unmyelinated peripheral axons has been described, though the damage was greater to the myelinated fibers. 3 Neurochemical changes were also seen in the primary afferent fibers, spinal cord and dorsal root ganglia (DRG) at 14–28 days following chronic constriction injury. 15 At that time the mononeuropathic rats displayed marked decreases in withdrawal latencies to stimulation interpreted as signs of hyperalgesia. 15 Some neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) were down-regulated, while others such as vasoactive intestinal peptide and galanin were up-regulated. 6,12 Among these neuropeptides, galanin is of particular interest because galanin may be an endogenous analgesic compound as elegantly demonstrated by WiesenfeldHallin and collaborators. 27 Administration of a galanin antagonist induced a higher rate of autotomy after axotomy. 21 However, Munglani et al. reported that 100–120 days after the constriction, when the hindpaw withdrawal latencies reached the same level as they were before the constriction, galanin-immunoreactivity decreased ipsilaterally in laminae 1 and 2 of the dorsal horn of the chronic constriction injury rats. 15 Yu et al. reported that the hindpaw withdrawal latency to thermal and mechanical stimulation increased significantly after intrathecal injection of galanin in mononeuropathic rats.25 The results indicate that galanin may participate in the modulation of chronic pain following peripheral nerve injury. 12
Opioid binding sites in the superficial dorsal horn of the spinal cord in rats also changed after nerve chronic constriction injury. A significant up-regulation in m binding sites was found at the spinal cord level following nerve ligation. 4 The up-regulation appeared to be bilateral although, on the ipsilateral side, this effect might be masked by fiber degeneration. 4 Recent studies indicated that there may be a close interaction between galanin and opioids in the mechanisms of endogenous antinociception in intact rats. 17,20,22,25 Recent studies in our laboratory demonstrated that intrathecal injection 1 or 3 nmol of galantide, an antagonist of galanin, 2 attenuated the antinociceptive effects induced by intrathecal injection of morphine in rats with mononeuropathy. 28 Furthermore, our study demonstrated that intra-periaqueductal grey (PAG) administration of galanin induced marked antinociceptive effects in intact rats. 22 The present study was performed to investigate the role of intra-PAG injection of galanin on nociception and the presumable interaction between galanin and opioids in PAG in mononeuropathic rats. EXPERIMENTAL PROCEDURES
Animal preparation Freely moving male Sprague–Dawley rats weighing 250–300 g (Experimental Animal Center of Beijing Medical University, Beijing, China) were used in the present experiments, which were conducted according to the guidelines of the animal ethical committee of Karolinska Institutet and every effort was made to minimize both the animal suffering and the number of animals used. Surgical procedures
‡To whom correspondence should be addressed. Tel.: 1 86-10-62751867; fax: 1 86-10-62751526. E-mail address: [email protected] (L.-C. Yu). Abbreviations: CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; HWL, hindpaw withdrawal latency; PAG, periaqueductal grey.
Rats were anaesthetized with intraperitoneal sodium pentobarbital (45 mg/kg). To produce a mononeuropathic model, the rat’s left sciatic nerve was exposed for 8–10 mm at the level of the midthigh. Four loose ligatures (4.0 chromic gut) were made around the dissected nerve with a 1.0–1.5-mm interval between each of them. The ligation was 767
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carefully manipulated so that the nerve was barely constricted. 3 The skin incision was closed with 4-0 silk sutures. Intra-periaqueductal grey injection The animals were anaesthetized by intraperitoneal sodium pentobarbital (45 mg/kg) and were mounted on a stereotaxic instrument. A stainless steel guide cannula of 0.8 mm o.d. was directed to PAG (AP 5.5, L 0.5, H 6.0 mm from the surface of the skull) according to Paxinos and Watson 16 and was fixed to the skull by dental acrylic. On the experimental day a stainless steel needle with 0.4 mm diameter was directly inserted into the guide cannula, with 1 mm beyond the tip of the latter. One microliter of solution was thereafter infused into PAG over 1 min. Nociceptive tests All rats were accustomed to the testing conditions for five days before the experiment was conducted. The latencies to hindpaw withdrawal during thermal and mechanical stimulation were measured. The thermal response was assessed by the hot-plate test. The entire ventral surface of the rat’s hindpaw was placed manually on the hot-plate which was maintained at a temperature of 528C (51.8–52.48C). 25,26 The time to hindpaw withdrawal was measured in seconds to be referred to as the hindpaw withdrawal latency (HWL). The Randall Selitto Test (Ugo Basile, Type 7200, Italy) was used to assess the HWL to mechanical stimulation. A wedge-shaped pusher at a loading rate of 30 g/s was applied to the dorsal surface of the manually handled hindpaw and the latency required to initiate the struggle response was assessed and expressed in seconds. 25,26 The average values obtained before intra-PAG injection were regarded as the basal HWL. The HWLs recorded during subsequent experiments were expressed as percentage changes of the basal level for each rat (% change of the HWL). Each rat was tested with both types of stimulation. Histological test At the end of the experiment rats were killed with intraperitoneal high dose of sodium pentobarbital (80 mg/kg) and the rat’s heads were fixed in 10% formalin for one week with the injecting tube in situ. The location of the tip of the injecting tube was verified in serial 50-mm crystal sections. Only the results from nociceptive tests with the tip of the injecting tube placed within PAG area were used for statistical analysis.
Chemicals Morphine hydrochloride (morphine hydrochloride, Shenyang First Pharmaceutical Factory, Shenyang, China) was diluted to 1 mg/ml with sterilized saline and was administrated intraperitoneally at a dose of 4.5 mg/kg. Solutions for intra-PAG administration were prepared with sterilized saline, each with a volume of 1 ml: (i) 1, 2 or 3 nmol of galanin (galanin, Bachem, Feinchemikalien AG, Switzerland), respectively; (ii) 2 nmol of galantide [Galanin (1–13)Substance P (5–11) amide, Bachem, Feinchemikalien AG, Switzerland]; (iii) 26.6 nmol of morphine. Control groups were given 1 ml of 0.9% saline injected into PAG. Statistical analysis Data from nociceptive tests were presented as mean ^ S.E.M. The difference between groups was determined by two-way analysis of variance (ANOVA) for repeated measures. *P , 0.05, **P , 0.01 and ***P , 0.001 were considered as significant differences. RESULTS
Effects of intra-periaqueductal grey administration of galanin on hindpaw withdrawal latencies to noxious stimulation in rats with mononeuropathy Rats received intra-PAG injection of 1 nmol (n 9), 2 nmol (n 8) or 3 nmol (n 9) of galanin, or 1 ml of 0.9% saline (n 14) as the control group. As shown in Fig. 1, the HWLs to thermal and mechanical stimulation increased significantly after intra-PAG injection of 2 nmol (Thermal test: Fleft/left 45.43, P , 0.001; Fright/right 13.73, P , 0.001. Mechanical test: Fleft/left 18.39, P , 0.001; Fright/right 7.49, P , 0.01) or 3 nmol of galanin (Thermal test: Fleft/left 65.23, P , 0.001; Fright/right 71.70, P , 0.001. Mechanical test: Fleft/left 44.11, P , 0.001; Fright/right 44.00, P , 0.001) compared with the control group. In the group receiving intra-PAG injection of 1 nmol of galanin there were no significant changes in HWLs in comparison with the control group (Thermal test: Fleft/left 2.18, P 0.14; Fright/right 3.28, P 0.07. Mechanical test: Fleft/left 2.26, P 0.14; Fright/right 0.15,
Fig. 1. Effects of intra-periaqueductal grey injection of 1, 2 or 3 nmol of galanin on left (A, C) and right (B, D) hindpaw withdrawal latencies to thermal (A, B) and mechanical (C, D) stimulation in rats with mononeuropathy. Control group received intra-periaqueductal grey injection of 1 ml of 0.9% saline. One microliter of 0.9% saline (W, n 14); 1 nmol of galanin (A, n 9); 2 nmol of galanin (X, n 8); 3 nmol of galanin (B, n 9). The data are described as % change of HWL. Mean ^ S.E.M., two-way ANOVA, *P , 0.05 and ***P , 0.001 compared with the control group.
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Fig. 2. Effects of intra-periaqueductal grey injection of galantide on intraperitoneal morphine-induced increases in HWL in rats with sciatic nerve ligation. Left HWL: A and C; right HWL: B and D. HWLs to thermal (A, B) and mechanical (C, D) stimulation. Time 0 min: intraperitoneal injection of 4.5 mg/kg of morphine; time 10 min: intra-periaqueductal grey injection of 2 nmol of galantide or 1 ml of 0.9% saline as a control. Morphine 1 1 ml of 0.9% saline (A, n 8); Morphine 1 2 nmol of galantide (B, n 8); Saline 1 2 nmol of galantide (W, n 6). The data are expressed as % change of HWL. Mean ^ S.E.M., two-way ANOVA, ***P , 0.001 compared with the control group.
P 0.70). The effects of intra-PAG administration of 3 nmol of galanin reached the peak between 10–20 min after the injection and then began to recover from 30 min. Effects of intra-periaqueductal grey injection of galantide on intraperitoneal morphine-induced increases in hindpaw withdrawal latency Rats with mononeuropathy received intraperitoneal injection of 4.5 mg/kg morphine, followed 10 min later, by intra-PAG injection of 2 nmol of galantide (n 8) or 1 ml of 0.9% saline (n 8) as the control group. The results are shown in Fig. 2. In the control group, the HWLs to both thermal and mechanical stimulation increased and lasted for more than 60 min. In the group receiving intraperitoneal morphine followed by intra-PAG injection of 2 nmol of galantide, the increased HWLs were attenuated significantly in both tests (Thermal test: Fleft/left 70.61, P , 0.001; Fright/right 132.40, P , 0.001. Mechanical test: Fleft/left 37.02, P , 0.001; Fright/right 49.05, P , 0.001) compared with the control group. In another group, rats receiving intraperitoneal administration of 1 ml of 0.9% saline, followed 10 min later, by intra-PAG administration of 2 nmol of galantide (n 6), there were no marked changes in the HWL to thermal and mechanical stimulation, as shown in Fig. 2. Effects of intra-periaqueductal grey injection of galantide on the increased hindpaw withdrawal latency induced by intra-periaqueductal grey injection of morphine Rats with mononeuropathy received intra-PAG injection of 26.6 nmol of morphine, followed 10 min later, by 2 nmol of galantide (n 6) or 1 ml of 0.9% saline (n 6) as a control. The results are shown in Fig. 3. After intra-PAG injection of morphine, the HWL to both noxious stimulation increased. In
the group receiving intra-PAG injection of morphine, followed by 2 nmol galantide, the increased HWLs were attenuated significantly (Thermal test: Fleft/left 37.18, P , 0.001; Fright/right 25.64, P , 0.001. Mechanical test: Fleft/left 19.08, P , 0.001; Fright/right 28.21, P , 0.001) as compared with the control group. In another group, rats receiving intra-PAG administration of 1 ml of 0.9% saline, followed 10 min later, by intra-PAG administration of 2 nmol of galantide (n 6), there were no marked changes in HWLs to both noxious thermal and mechanical stimulation. DISCUSSION
The results of the present study show that intra-PAG administration of 2 or 3 nmol of galanin induced a dosedependent antinociceptive effect in rats with mononeuropathy, while 1 nmol of galanin did not. Intraperitoneal injection of 4.5 mg/kg of morphine induced significant increases in HWLs to thermal and mechanical stimulation, and the effects were attenuated by intra-PAG injection of 2 nmol of the galanin receptor antagonist galantide. Furthermore, intra-PAG administration of 2 nmol of galantide attenuated the antinociceptive effects induced by intra-PAG injection of 26.6 nmol of morphine. The results indicate that galanin may have an antinociceptive role in the PAG in mononeuropathic rats, and that galanin interacted with opioid peptides in this brain area. Galanin is a neuropeptide with mainly inhibitory effects in central nervous system. 1,9,10 There was a marked increase of galanin-immunoreactivity in primary sensory neuron and DRG after loose sciatic nerve ligation. 6,12 Interestingly, the change was paralleled by changes in nociceptive behaviors. 15 It has, therefore, been suggested that galanin may be involved in the endogenous analgesic system, especially after peripheral nerve injury. 6,24,25 Nerve injury, such as loose sciatic nerve ligation and axotomy, led to hyperactivity in
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Fig. 3. Intra-periaqueductal grey injection of galantide attenuated the increased HWL induced by intra-periaqueductal grey morphine in rats with sciatic nerve ligation. Left HWL: A and C; right HWL: B and D. HWLs to thermal (A, B) and mechanical (C, D) stimulation. Time 0 min: intra-periaqueductal grey injection of 26.6 nmol of morphine; time 10 min: intra-periaqueductal grey injection of 2 nmol of galantide or 1 ml of 0.9% saline as a control. Morphine 1 1 ml of 0.9% saline (A, n 6); Morphine 1 2 nmol of galantide (B, n 6); Saline 1 2 nmol of galantide (W, n 6). The data are expressed as % change of HWL. Mean ^ S.E.M., two-way ANOVA, ***P , 0.001 compared with the control group.
the somatosensory system and spontaneous firing of some primary afferent fibers. 7 It is possible that the release of galanin is due to the abnormal impulses of these afferents. 6 Another reason could be diffusion and/or rapid enzymatic breakdown of accumulated peptide. It is also possible that there may be a slowing down of peripheral axonal transport. 11 Also, the accumulation of galanin at the injury site might play a role in the initiation or modulation of ectopic neural activity. 11 Furthermore, Carlton et al. found that many galanin terminals no longer co-localized with CGRP after peripheral nerve lesion, possibly indicating there was an increased antinociceptive activity after nerve lesions. 5 In conclusion, the up-regulation of galanin might respond to the increased nociceptive input induced by partial nerve injury. Another possible and important role of galanin after nerve injury may be to re-establish the neural target contact. The cytokine leukemia inhibitory factor produced by the degenerated nerve possibly up-regulates the galanin in the DRG large neurons in chronic constriction injury rats. 12 This is in line with the finding that galanin has trophic effects on axotomized neurons which are temporarily deprived of their target-derived trophic factors. 27 This may partly explain why some axotomized afferents cease synthesizing their “normal” neuropeptides (e.g. substance P and CGRP) and start synthesizing others such as galanin. Also, Ma and Bisby reported that partial sciatic nerve injury induced greater galanin up-regulation in medium- and large-size DRG neurons than complete sciatic nerve injury. 12 Moreover, according to the report of Munglani et al., the level of galanin-immunoreactivity was down-regulated 100–120 days after sciatic nerve lesion, by which time resolution of the hyperalgesia and peripheral nerve injury has occurred. 15 All the above results indicate that the change in galanin expression observed in peripheral neurons after nerve injury
could be characterized as reflecting a shift in the “focus” of the neuron from synaptic transmission to regeneration. 29 Previous studies showed that galanin co-exists with several other neurotransmitters in the CNS in animals, including opioids. 10 Both galanin receptors 9,14,19 and opioid receptors, including m-, d- and k-receptors 13 are found in rat PAG, an essential structure for opioid analgesia. 8 Galanin has been proposed to interact with opioids in modulation of the transmission of nociceptive information at the spinal cord level 18,21,23 and in PAG 22 in intact rats. In the present study, the increased HWL induced by intra-PAG or intraperitoneal administration of morphine was significantly attenuated by intra-PAG administration of the galanin receptor antagonist galantide, indicating a possible interaction in antinociception between galanin and opioids in PAG in rats with mononeuropathy. Interestingly, m-opioid receptor binding sites were also upregulated in the spinal cord in chronic constriction injury rats, possibly due to the activation of descending control systems in response to increased nociceptive inputs following sciatic nerve lesion. 4 After binding to its receptor, galanin may facilitate the inhibitory effects of opioid peptides, and/or enhance the affinity of opioid peptides to their own receptors. When galantide, the antagonist of galanin receptors, 1 was applied, it prevented galanin from binding to its receptors, and thus indirectly attenuating the opioid analgesia, as we observed in the present study. Also, in support of our findings is the study by Reimann et al. who investigated whether endogenous galanin, by the use of the galanin antagonist galantide, interacts with exogenously administered morphine, in the rat spinal cord. 18 They reported that in the rat tail-flick test, intrathecal injection of 3 mg of morphine had a significant antinociceptive effect which was almost completely antagonized by co-injection of 2 mg of galantide. Also, this would
Antinociception of galanin in PAG in neuropathic rats
suggest that galantide may have a pro-nociceptive effect, a suggestion supported by the finding that galantide induced a higher rate of autotomy after axotomy. 21 CONCLUSION
Intra-PAG injection of galanin induced a significant antinociceptive effect in rats with sciatic nerve ligation. The increased HWLs induced by intraperitoneal administration of morphine were significantly attenuated by following intra-PAG injection of galantide, the antagonist of galanin receptors, indicating that PAG is one of the key sites of the interaction between galanin and opioids in rats with
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mononeuropathy. Furthermore, intra-PAG injection of galantide also reversed the antinociceptive effects induced by intra-PAG administration of morphine. The data suggest an antinociceptive role of galanin in the PAG of mononeuropathic rats, and a possible interaction between galanin and opioid peptides in this brain area.
Acknowledgements—This study was supported by funds from the National Natural Science Foundation of China (NSFC), the Natural Science Research Foundation of Peking University, the Karolinska Institutet Foundation and the Foundation of Acupuncture and Alternative Methods.
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