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Regulatory Peptides 86 (2000) 89­94 www.elsevier.com / locate / regpep

An interaction of opioids and galanin in dorsal horn of the spinal cord in mononeuropathic rats

a a, b Yan-Ping Zhang , Long-Chuan Yu *, Thomas Lundeberg

a

Department of Physiology, College of Life Science, and National Laboratory of Biomembranes and Membrane Biotechnology, Peking University, Beijing 100871, People' s Republic of China b Department of Physiology and Pharmacology, and Department of Surgery and Rehabilitation, Karolinska Institutet, 171 77 Stockholm, Sweden Received 29 May 1999; received in revised form 22 August 1999; accepted 1 September 1999

Abstract The present study was performed in rats with experimentally induced mononeuropathy after common sciatic nerve ligation. The hind-paw withdrawal latencies to thermal and mechanical stimulation were increased significantly after intrathecal injection of 3 nmol of galanin. The increased hind-paw response latencies induced by galanin were attenuated by following intrathecal injection of 22 nmol, but not 11 or 2.75 nmol of the opioid receptor antagonist naloxone. Further, the increased hind-paw response latencies induced by galanin were prevented by following intrathecal injection of 10 nmol of mu-opioid receptor antagonist, b-funaltrexamine (b-FNA), but not by 10 nmol of delta-opioid receptor antagonist, natrindole or 10 nmol of kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI). Intrathecal 10 nmol of b-FNA alone had no significant effects on the hind-paw withdrawal responses. These results demonstrate the existence of a specific interaction between galanin and opioids in the transmission of presumed nociceptive information in the spinal cord of mononeuropathic rats. This interaction involves the activation of mu-opioid receptor. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Intrathecal; Naloxone; b-Funaltrexamine; Natrindole; Nor-Binaltorphimine

1. Introduction Painful syndromes following injury of the peripheral nerve are usually characterized by spontaneous burning pain combined with hyperalgesia and allodynia to thermal and / or mechanical stimulation [1]. To investigate the mechanisms of neurogenic pain Bennett and Xie set up an experimental model of mononeuropathy, induced by four loose ligatures placed around the common sciatic nerve, in rats [2]. It has been suggested that this model is more similar to those seen in patients than complete sciatic nerve section [2,3]. Studies have demonstrated that the neuropeptide galanin

*Corresponding author. Tel.: 1 86-10-6275-1867; fax: 1 86-10-62751850. E-mail address: [email protected] (L.-C. Yu)

may be involved in endogenous anti-nociception in the spinal cord [4,5]. The mRNA of galanin is up-regulated in dorsal root ganglion (DRG) cells and in the superfacial laminae of the dorsal horn of the spinal cord following peripheral nerve injury [6­8]. These regions of the spinal cord are known to receive the terminations of primary afferent neurons that respond to noxious stimuli. Also spinal opioids are known to be associated with endogenous anti-nociception. In a recent study we have demonstrated that intrathecal administration of galanin (1 or 3 nmol) resulted in an anti-nociceptive effect in mononeuropathic rats [9]. Also the antinociceptive effects of morphine were antagonized by the galanin antagonist, galantide (M-15) [10]. These observations suggest that there is a close interaction between galanin and opioids in the spinal cord in mononeuropathic rats. The aim of the present study was to further the

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investigation on the interaction of opioids and galanin in the presumed spinal anti-nociception in mononeuropathic rats by using non-selective opioid receptor antagonist naloxone, and the selective mu-opioid receptor antagonist b-funaltrexamine (b-FNA), the delta-opioid receptor antagonist natrindole, or the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI).

2.3. Nociceptive tests

Intrathecal administrations were performed during 5­12 days after left sciatic nerve ligation as during these days the abnormal presumed ``pain'' behaviour is at a relatively stable level [2,3]. Also, 5­12 days after left sciatic nerve ligation a dramatic increase in galanin mRNA is detected [11­14]. The hind-paw withdrawal latency (HWL) in seconds to thermal stimulation and the hind-paw withdrawal threshold (HWT) in grams to mechanical stimulation were measured. The thermal response was assessed using the hot-plate test. To ensure that the entire ventral surface of the hind-paw was in contact with the hot-plate the knee joint was kept extended manually during the test. The rats left or right hind-paw was separately placed manually on the hot-plate which was maintained at a temperature of 528C (51.8­52.48C) [9,15­17]. The response to mechanical stimulus was determined using the Randall Selitto Test (Ugo Basile, Type 7200, Italy). Mechanical stimulation was applied to the dorsal surface of the hind-paw by a wedge-shaped pusher at a loading rate of 30 g / s and the threshold required to initiate the struggle response was measured. The values obtained before intrathecal injection were regarded as the basal HWL or HWT. The HWL and HWT recorded during subsequent experiments were expressed as percentage change of the mean basal level for each rat (% change of HWL or HWT). Each rat was tested with both nociceptive tests.

2. Materials and methods

2.1. Animal preparation

Adult male Sprague­Dawley rats (Experimental Animal Centre of Beijing Medical University, Beijing, China) weighing 250­350 g were used. The animals were individually housed in cages with free access to water and food ad libition, and maintained in a room temperature of 24628C with a normal day / night cycle. All rats were accustomed to the testing conditions for five days before starting the experiment in order to decrease the stress induced by handling and measurements. All experiments were conducted according to the guidelines of the animal ethical committee of Karolinska Institutet and every effort was made to minimize animal suffering.

2.2. Surgical procedures and intrathecal catheter implantation

Rats were anesthetized with intra-peritoneal sodium pentobarbital (45 mg / kg). In order to create a mononeuropathy model, nerve ligation was performed according to the method of Bennett and Xie [2]. Briefly, rat's left sciatic nerve was exposed for 8­10 mm at the level of the mid thigh. Four loose ligatures (4.0 chronic gut) were made around the dissected nerve with a 1.0­1.5 mm interval between each of them. The ligation was carefully manipulated so that the nerve was barely constricted. The skin incision was closed with 4-0 silk sutures. An intrathecal catheter was implanted in each rat immediately following nerve ligation. A sterilized salineflushed polyethylene (PE-10) tube was inserted into the subarachnoid space through a slit in the atlanto-occipital membrane. The caudal end of the catheter was gently threaded into the lumbar enlargement. The catheter was fixed and the skin wound was closed with 4-0 silk sutures. All rats received an intra-muscular injection of potassium penicillin (30,000 IU / rat) after operation in order to prevent infection. Approximately 10­20% of the operated rats exhibited post-surgical motor deficient (e.g. limb paralysis), and these rats were excluded from the experiment.

2.4. Chemicals

Solutions for intrathecal administration were prepared with sterilized saline, each with a volume of 10 ml: (1) 3 nmol of galanin (Galanin, Bachem, Feinchemikalien AG, Switzerland); (2) 2.75 (0.5 mg), 11 (4 mg) or 22 (8 mg) nmol of naloxone (naloxone hydrochloride, Sigma Chemical Company, St. Louis, MO) respectively; (3) 10 nmol of b-funaltrexamine (b-FNA hydrochloride; Research Biochemicals, MA), natrindole (naltrindole hydrochloride; Research Biochemicals), or nor-binaltorphimine (nor-BNI hydrochloride; Research Biochemicals). Control groups were given 10 ml of 0.9% saline.

2.5. Statistical analysis

Data from nociceptive tests were presented as mean6SEM. The difference between groups was determined by two-way analysis of variance (ANOVA) for repeated measures or Student's t-test where applicable, and *P , 0.05, **P , 0.01 and ***P , 0.001 were considered as significant differences. The notation of Fleft / left , Fleft / right or Fright / right expresses the F value in ANOVA, and left or right means left HWL (or HWT) or right HWL (or HWT).

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3. Results

3.1. Effects of intrathecal administration of naloxone on the galanin-induced increase in HWL and HWT in mononeuropathic rats

Thirty-two rats received intrathecal injection of 3 nmol of galanin followed, 10 min later, by either 2.75 (n 5 8), 11 (n 5 8) or 22 nmol (n 5 8) of naloxone, or 10 ml of 0.9% saline as a control (n 5 8). As shown in Fig. 1, after injection of 3 nmol of galanin, both the HWL and HWT to thermal and mechanical stimulation increased in the four groups. In the control group, after administration of 10 ml of 0.9% saline, the galanin-induced increases in HWL and HWT lasted for more than 40 min. After intrathecal injection of 22 nmol of naloxone, the increased HWL and HWT was attenuated significantly (in the thermal test: Fleft / left 5 7.44, P , 0.01; Fright / right 5 11.20, P , 0.01; and in the mechanical test: Fleft / left 5 12.72, P , 0.001) compared to the control group, except right hind-paw in the mechanical test (Fright / right 5 2.19, P 5 0.15). In the groups of receiving

intrathecally 2.75 (Thermal test: Fleft / left 5 0.26, P 5 0.62; F right / right 5 1.02, P 5 0.32; mechanical test: Fleft / left 5 0.37, P 5 0.54; Fright / right 5 0.04, P 5 0.84) and 11 nmol of naloxone (Thermal test: Fleft / left 5 0.24, P 5 0.62; Fright / right 5 0.01, P 5 0.92; mechanical test: Fleft / left 5 0.25, P 5 0.62; F right / right 5 0.03, P 5 0.86), there were no significant changes in HWL and HWT in comparison with the control group.

3.2. Effects of intrathecal administration of b -FNA, natrindole and nor-BNI on galanin-induced increase in HWL and HWT

In order to investigate which type of opioid receptors may be involved in the modulation of galanin-induced increases in HWL and HWT, three types of selective opioid antagonists were used. Twenty-eight rats received intrathecal injections of 3 nmol of galanin followed, 10 min later, by either 10 nmol of b-FNA (mu-opioid antagonist; n 5 7), 10 nmol of natrindole (delta-opioid antagonist; n 5 7), 10 nmol of nor-BNI (kappa-opioid

Fig. 1. Effects of intrathecal injection of naloxone on the galanin-induced increase in HWL and HWT. Time 5 0 min: 3 nmol of galanin was injected intrathcally; Time 5 10 min: intrathecal administration of 22 nmol (n 5 8), 11 nmol (n 5 8) or 2.75 nmol (n 5 8) of naloxone or 10 ml of 0.9% saline as a control (n 5 6). HWL: hind-paw withdrawal latency to thermal test, HWT: hind-paw withdrawal threshold to mechanical test, vertical bars indicate SEM, two-way ANOVA, **P , 0.01, ***P , 0.001 compared with control group.

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antagonist; n 5 7), or 10 ml of 0.9% saline (n 5 7) as control. The results are shown in Fig. 2. After intrathecal injection of galanin the HWL to thermal and HWT to mechanical stimulation increased significantly in the all four groups. In the control group, after administration of 10 ml of saline, the increase in HWL and HWT to both stimuli lasted for about 40 min. 20 min after intrathecal injection of 10 nmol of b-FNA, the increased HWL and HWT were prevented significantly in the thermal test (Fleft / left 5 22.53, P , 0.001; Fright / right 5 16.79, P , 0.001) and in the mechanical test (Fleft / left 5 11.33, P , 0.01; Fright / right 5 10.63, P , 0.01) compared to the control group. However, in the groups of receiving 10 nmol of natrindole (Thermal test: Fleft / left 5 1.67, P 5 0.21; Fright / right 5 0.45, P 5 0.51; mechanical test: Fleft / left 5 0.09, P 5 0.76; Fright / right 5 2.26, P 5 0.14), and of receiving 10 nmol of nor-BNI (Thermal test: Fleft / left 5 1.41, P 5 0.25; Fright / right 5 0.26, P 5 0.61; mechanical test: Fleft / left 5 0.13, P 5 0.72; Fright / right 5 2.44, P 5 0.13), there were no significant changes in HWL and HWT in comparison with control group. Rats with left sciatic nerve ligation received intrathecal administration of 10 nmol of b-FNA (n 5 5). The results

are shown in Fig. 3. Comparing with pre-injection, the injection of b-FNA alone showed no significant influence on HWL and HWT.

4. Discussion Recent work in our laboratory has shown that intrathecal administration of galanin induce a significant increase in hind-paw withdrawal latency in intact and ligated rats [9,10]. Furthermore, the selective galanin receptor antagonist galantide attenuated the anti-nociceptive effect of morphine. These results indicate that galanin and opioids, in some extent, are involved in the transmission of presumed nociceptive information in the spinal cord of rats. The results of present study confirmed the existence of specific interaction between galanin and opioids in the spinal cord of neuropathic rats, show that this interaction is related to the activation of mu-opioid receptors. Chronic constriction injury produced by loosely tying four ligatures around the sciatic nerve is commonly used as an animal model of neuropathic pain [2]. Large myelinated fibres degenerate after constriction injury, but thinly myeli-

Fig. 2. Effects of intrathecal administration of b-FNA, natrindole and nor-BNI on the galanin-induced increase in HWL and HWT. Time 5 0 min: 3 nmol of galanin was injected intrathecally; Time 5 10 min: intrathecal administration of 10 nmol of b-FNA (n 5 7), 10 nmol of natrindole (n 5 7), and 10 nmol of nor-BNI (n 5 7). HWL: hind-paw withdrawal latency to thermal test, HWT: hind-paw withdrawal threshold to mechanical test, vertical bars indicate SEM, two-way ANOVA, **P , 0.01, ***P , 0.001 compared with control group.

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Fig. 3. Effects of intrathecal injection of 10 nmol of b-FNA on HWL to thermal (A) and HWT to mechanical (B) stimulation in mononeuropathic rats. Number of rats in each group: n 5 5, data measured at pre-injection, 5 min and 10 min after the injection. Mean. SEM, vertical bars indicate SEM. Student's t-test (two tails). Left: ligated side, right: contralateral side. HWL: hind-paw withdrawal latency; HWT: hind-paw withdrawal threshold.

following a complete sciatic nerve section. The inhibitory action of galanin on transmission of nociceptive information is supported by several studies [4,5,9,10]. It is therefore tempting to suggest that the up-regulation of galanin induced by peripheral nerve lesion is a compensatory response. Data from the study of Przewlocka et al. [22] have shown that the anti-nociceptive effects derived from opioid agonist morphine can be enhanced by co-administration or following injection of galanin or N-terminal galanin fragment. Also, Selve and Reimann [23,24] have reported that the galanin receptor antagonist galantide and M35 attenuate spinal anti-nociceptive effects of opioid agonist DAMGO, or morphine. All of these observations and together with our recent and present results suggest that galanin and opioids in the dorsal root ganglion and / or spinal cord may serve as some intrinsic anti-nociceptive factors that attenuate the transmission of afferent nociceptive information. The inhibitory effect of opioids on the spinal nociceptive transmission has been well documented, and both pre-and post-synaptic mechanisms have been suggested to play a role [1,15]. It is likely that the effect of opioids is mediated at the dorsal horn level, as an increase in mu-opioid receptors in the superficial dorsal horn has seen in mononeuropathic rats [25,26]. Also, using immunocytochemistry, Goff and collaborators [12] reported that muopioid receptor immunodensities in the superficial dorsal horn was found to be increased from 4 to 28 days after sciatic nerve constriction. The increases of mu-opioid receptors observed after chronic constriction injury might explain why an increased efficacy of opiates has been reported in this model [27]. Also, the attenuated effects of anti-nociception of galanin by mu-opioid receptor antagonist b-FNA in present experiment may relate to the increase of mu-opioid receptors induced by peripheral lesion.

nated and unmyelinated fibres are less severely damaged [18]. In rats with this injury, the decreases of HWL and HWT in the affected hind limb are also observed but to a lesser degree on the intact side, an effect also seen in our previous studies [3,16,18]. Peripheral nerve injury induces significant synthesis and up-regulation of galanin and galanin mRNA in the dorsal root ganglion cells and dorsal horn neurons of the spinal cord [6­8,11,19], which contrasts with the down-regulation of substance P [20]. Ma and Bisby [21] have reported that after sciatic nerve section galanin immuno-reactive neurons were increased in the ipsilateral dorsal root ganglia, and that galanin immuno-reactive axonal fibres were increased in the superficial laminae of the dorsal horn. Furthermore, they found that partial sciatic nerve injuries induced greater galanin up-regulation in mediumand large-sized dorsal root ganglion neurons than that seen

Acknowledgements This study was supported by funds from the Karolinska Institutet Foundation, Foundation of Acupuncture and Alternative Methods, the National Natural Science Foundation of China (NSFC) and the Natural Science Pre-Research Foundation of Peking University.

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