The cannabinoid receptor agonist WIN 55,212-2 mesylate blocks the development of hyperalgesia produced by capsaicin in rats

Pain 81 (1999) 25–33

The cannabinoid receptor agonist WIN 55,212-2 mesylate blocks the development of hyperalgesia produced by capsaicin in rats Jun Li a, Randy S. Daughters a, Christopher Bullis b, Rimon Bengiamin a, Mark W. Stucky c, John Brennan b, Donald A. Simone a , d ,* a

Department of Psychiatry, University of Minnesota, 420 Delaware St. S.E., Box 392, Minneapolis, MN 55455, USA b Department of Biology, Hamline University, St. Paul, MN 55104, USA c Department of Anesthesiology, University of Minnesota, Minneapolis, MN 55455, USA d Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA Received 30 March 1998; received in revised form 23 October 1998; accepted 30 November 1998

Abstract Although it is well known that cannabinoids produce antinociception in acute pain models, there is less information on the ability of cannabinoids to alleviate hyperalgesia. In the present study, we determined whether cannabinoids attenuated the development of hyperalgesia produced by intraplantar injection of capsaicin in rats. In normal, untreated animals, intraplantar injection of 10 mg capsaicin produces nocifensive behavior (elevation of the injected paw) suggestive of pain, an increase in the frequency of withdrawal from punctate mechanical stimuli applied to the paw (mechanical hyperalgesia) and a decrease in the latency of withdrawal from noxious heat (heat hyperalgesia). Separate groups of animals were pretreated intravenously with vehicle, the cannabinoid receptor agonist WIN 55,212-2 at doses of 1,10 100 or 200 mg/kg, or the enantiomer WIN 55,212-3 (100 mg/kg) 5 min before intraplantar injection of capsaicin into one paw. The duration of nocifensive behavior was measured during the first 5 min after capsaicin injection. Withdrawal responses to mechanical and heat stimuli applied to the plantar surface of both hindpaws were measured before and at 5 and 30 min after capsaicin. Pretreatment with WIN 55,212-2 produced a dose-dependent decrease in nocifensive behavior and in hyperalgesia to mechanical and heat stimuli produced by capsaicin, as compared with vehicle pretreatment. Doses of 100 and 200 mg/kg WIN 55,212-2 completely blocked the development of hyperalgesia to mechanical and heat stimuli without altering withdrawal responses on the contralateral control paw. Furthermore, these doses of WIN 55,212-2 had no effect on basal withdrawal responses to heat in animals that did not receive capsaicin. The inactive enantiomer WIN 55,212-3 did not alter the development of capsaicin-evoked pain or hyperalgesia. These data suggest that low doses of cannabinoids, which do not produce analgesia or impair motor function, attenuate chemogenic pain and possess antihyperalgesic properties.  1999 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Pain; Cannabinboids; CB1 receptor; Thermal hyperalgesia; Mechanical hyperalgesia

1. Introduction Although cannabinoids have been used therapeutically as an analgesic for over 100 years (Reynolds, 1890), it is only recently that we have begun to understand how cannabinoids affect neural function. The discoveries of a cannabinoid receptor in the mammalian brain (Devane et al., 1988; Herkenham et al., 1991) and an endogenous cannabinoid ligand, anandamide (Devane et al., 1992), suggest that cannabinoids within the central nervous system may have a * Corresponding author. Tel.: +1-612-625-6464; fax: +1-612-624-8935.

neurotransmitter or neuromodulator function. Although the functional significance of endogenous cannabinoids is not fully understood, the possibility that cannabinoids are involved in modulation of nociception is supported by several lines of evidence. First, cannabinoid receptors are widely distributed throughout the central nervous system, including areas known to be involved in pain modulation, such as the periaquaductal grey and spinal cord (Herkenham et al., 1990; Jansen et al., 1992). Second, the antinociceptive properties of cannabinoids have been well documented in animal studies using a variety of nociceptive measures (Buxbaum, 1972; Sofia et al., 1973; Kaymakc¸alan et al.,

0304-3959/99/$20.00  1999 International Association for the Study of Pain. Published by Elsevier Science B.V. PII: S03 04-3959(98)002 63-2

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1974; Bloom and Dewey, 1978; Sanders et al., 1979; Moss and Johnson, 1980; Yaksh, 1981). Third, intravenous administration of cannabinoids decreased evoked responses of wide dynamic range neurons in the spinal cord (Hohmann et al., 1995) and thalamus (Martin et al., 1996). Hyperalgesia, defined as a decrease in pain threshold and/ or increased pain to a normally painful stimulus, often occurs following tissue injury and inflammation. Hyperalgesia can be located not only at the site of injury (primary hyperalgesia) but also within surrounding, uninjured tissue (secondary hyperalgesia) as well (Lewis, 1936; Hardy et al., 1950). Primary hyperalgesia, particularly hyperalgesia to heat, occurs at least in part by sensitization of nociceptors (Fitzgerald and Lynn, 1977; Campbell et al., 1979; Meyer and Campbell, 1981; LaMotte et al., 1982, 1983). It is believed that changes in the central nervous system underlie secondary hyperalgesia. It has been shown, for example, that tissue injury and inflammation produce sensitization of spinal dorsal horn neurons as evidenced by enlarged receptive fields (McMahon and Wall, 1983; Cook et al., 1987; Hylden et al., 1989) and enhanced synaptic efficacy and excitability (Woolf and King, 1990). Capsaicin, the pungent ingredient in hot peppers, has been used to produce hyperalgesia in humans. When applied topically or injected into the skin, capsaicin produces burning pain, hyperalgesia to heat within the immediate vicinity of capsaicin application, and mechanical hyperalgesia within a large surrounding area (Szolcsa´yni, 1977; Carpenter and Lynn, 1981; Simone et al., 1989; Culp et al., 1989; LaMotte et al., 1991). Burning pain and hyperalgesia to heat following capsaicin are believed to occur, at least in part, by excitation and sensitization of C-fiber nociceptors (Kenins, 1982; Konietzny and Hensel, 1983; Szolcsa´yni et al., 1988; Baumann et al., 1991; LaMotte et al., 1992). Psychophysical studies of the neural mechanisms underlying secondary (mechanical) hyperalgesia produced by intradermal injection of capsaicin suggested that secondary hyperalgesia is mediated by changes in the central nervous system (LaMotte et al., 1991; Torebjo¨rk et al., 1992). In correlative electrophysiological studies in monkeys, it was found that excitation and sensitization of spinothalamic tract (STT) neurons to mechanical stimuli following intradermal injection of capsaicin correlated with pain and hyperalgesia in humans (Simone et al., 1991). We recently developed a model of capsaicin-produced hyperalgesia in rats in order to perform correlative behavioral, pharmacological and physiological studies (Gilchrist et al., 1996). Intraplantar injection of capsaicin evoked nocifensive behavior characterized by lifting and shaking the injected paw for several minutes after injection, and decreased paw withdrawal latency to heat (heat hyperalgesia). In addition, the threshold (mN) for withdrawal from punctate stimuli applied to the injected paw decreased after capsaicin and the frequency of paw withdrawal evoked by suprathreshold stimuli increased, suggestive of mechanical hyperalgesia.

In the present study we investigated whether a cannabinoid receptor agonist, WIN 55,212-2, inhibited nocifensive behavior and hyperalgesia produced by intraplantar injection of capsaicin in rats. A preliminary report has been published in abstract form (Daughters et al., 1997).

2. Methods 2.1. Subjects Eighty eight adult, male, Sprague–Dawley rats (Harlan Industries, Indianapolis, IN), weighing 250–350 g were used. Rats were paired in plastic housing cages with soft bedding and had access to food and water ad libitum. Animals were maintained on a 12:12 light–dark cycle. Each animal was used in one experiment only. All protocols were approved by the Animal Care Committee of the University of Minnesota. 2.2. Intraplantar injection of capsaicin Capsaicin (8-methyl-N-vanillyl 6-nonamide) at a concentration of 1 mg/ml (0.1%) was prepared for injection as described previously (Gilchrist et al., 1996). Capsaicin was dissolved in vehicle containing 7% polyxyethylene(20)sorbitan monooleate (Tween 80) in normal saline. The capsaicin solution was passed through a Millipore filter (0.22 mm) and stored in a sterile glass injection vial. Intraplantar injections of 10 mg were given in a volume of 10 ml using a 0.5 ml insulin syringe with a 28 ga needle. Before any injection, a 5 mm diameter circle was marked on the center of the plantar surface of one randomly selected hindpaw with a felt-tip pen. This marked area served as the injection site. In a pilot study it was determined that an intraplantar injection of the vehicle produced a bleb approximately 4 mm in diameter. During an injection the needle penetrated the skin just distal to the marked area on the selected hindpaw and the injection was made into its center. Care was taken to deliver each injection superficially in the skin. A successful intraplantar injection was noted by the appearance of bleb within the marked injection site on the hindpaw. 2.3. Withdrawal responses to mechanical stimuli Rats were placed on an elevated plastic mesh (1 cm2 perforations) under a clear plastic cage and were adapted to the testing environment for at least 15 min. A von Frey monofilament with a calibrated bending force of 94.9 mN was used to deliver a punctate stimulus to the plantar surface of the hindpaw and was applied from below the mesh floor. The stimulus was applied for a duration of approximately 1 s with an interstimulus interval of approximately 5 s. Withdrawal responses evoked by the monofilament were obtained from ten consecutive trials, were tallied and then

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divided by the total number of trials to produce a withdrawal response frequency. Care was taken to stimulate random locations on the plantar surface. The toes and heel were avoided. Only robust and immediate withdrawal responses from the stimulus were considered. Mechanical hyperalgesia was defined as a significant increase in the frequency of withdrawal evoked by the mechanical stimulus.

(Research Biochemicals) and its enantiomer WIN 55,2123 were dissolved in a vehicle containing 40% dimethyl sulfoxide (DMSO) in normal saline. WIN 55,212-2 (1, 10, 100 and 200 mg/kg), WIN 5,212-3 (100 mg/kg) and the vehicle were administered intravenously via the lateral tail vein using a 0.5 ml tuberculin syringe. All injections were given in a volume of 1 ml/kg body weight.

2.4. Withdrawal responses to heat stimuli

2.7. Experimental design

Radiant heat was applied to the plantar surface of each hindpaw, and withdrawal response latencies were determined according to a method similar to that described previously (Hargreaves et al., 1988). Rats were placed under a clear plastic cage (23 × 13 × 13 cm) on a clear 3-mm thick glass plate which was elevated to allow maneuvering of a radiant heat source underneath. Each rat was adapted to the testing environment for at least 15 min prior to any stimulation. Heat stimuli were produced by a 50 W light bulb placed in a custom-built case which allowed focusing of the light source. The heat source was positioned such that the focused beam of radiant heat (8 mm diameter) was applied to the middle of the plantar surface of the hindpaw. Withdrawal latencies to the nearest 0.1 s were measured automatically by use of a photocell which terminated each trial and stopped the timer upon withdrawal of the hindpaw. Each hindpaw received four stimuli, alternating between paws. The interstimulus interval for each paw was at least 1 min and a 20 s cutoff was imposed on the stimulus duration to prevent tissue damage. Withdrawal latency for each paw was defined as the mean of the last three trials to eliminate the large variability found in the initial latency measurement. Withdrawal latencies were measured daily for each hindpaw during a training period of at least 4 consecutive days, or until latencies were stable. The heat source was maintained at a constant intensity which produced stable withdrawal latencies of approximately 12 s during the training period. Hyperalgesia to heat was defined as a decrease in withdrawal latency.

In all studies of nociception, baseline measures of withdrawal responses evoked by heat and/or mechanical stimuli were determined for each hindpaw and obtained for at least 4 consecutive days until withdrawal responses were stable. Animals were divided into six groups and were matched according to their withdrawal latencies to heat, so that mean withdrawal latencies did not differ between the groups. On the test day, each group of animals was pretreated with either the vehicle (n = 8), WIN 55,212-2 at doses of 1, 10, 100 or 200 mg/kg (n = 8 per group), or 100 mg/kg WIN 55,212-3 (n = 6) at 5 min before the capsaicin injection. The duration of nocifensive behavior, defined as an abnormal posturing of the capsaicin-injected hindpaw, was determined during the first 5 min after the capsaicin injection. Withdrawal responses to mechanical and heat stimuli were obtained 30 min before, and at 5 and 30 min after capsaicin. Since cannabinoids are known to produce antinociception, we determined the effect of WIN 55,212-2 on basal withdrawal responses to mechanical and heat stimuli in the absence of capsaicin. Four additional groups of animals (n = 6 per group) were given an intravenous injection of 10, 100 or 200 mg/kg WIN 55,212-2, or an equal volume of the vehicle. Withdrawal responses were obtained from both hindpaws 30 min before and at 10 and 30 min after the intravenous injection. These are the same times at which behavioral measures were made in animals that received capsaicin. Eighteen additional animals were randomly divided into three groups of six rats each and used to determine the effect of WIN 55,212-2 on motor coordination. Each group received an intravenous injection of vehicle or WIN 55,212-2 at doses of 100 or 200 mg/kg. The amount of time (up to 5 min) the animals were able to remain on the treadmill was measured 15 min before and at 10 min after injection, the same time at which withdrawal responses were measured in animals that received capsaicin. During the test session, animals were allowed only one attempt on the treadmill.

2.5. Assessment of motor function The rota-rod test (Dunham and Miya, 1957; Kinnard and Carr, 1957) was used to determine the effect of WIN 55,212-2 on motor function. Animals were trained to run continuously on a rota-rod treadmill (Ugo Basile, Stoelting, Chicago, IL) for a period of 5 min. The speed of the treadmill was 16 rpm. Each animal underwent two training sessions each day until the criterion of 5 min was achieved for at least 2 consecutive days. Each training session lasted 15 min, and animals usually achieved the criterion of remaining on the treadmill for 5 min within 2 weeks. 2.6. Cannabinoid administration The cannabinoid receptor agonist WIN 55,212-2

2.8. Data analyses The effect of pretreatment with WIN 55212-2 and WIN 55,212-3 on the duration of nocifensive behavior produced by capsaicin was determined using a one-way ANOVA. Three-way ANOVAs with repeated measures were used to

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determine the effect of WIN 55,212-2 on withdrawal latency to heat and on frequency of withdrawal from mechanical stimuli. Independent factors were group (drug), paw (capsaicin injected versus contralateral noninjected) and time (before and at 5 and 30 min following capsaicin). Comparisons between the capsaicin-injected paw and the contralateral, untreated paw were made between groups. Mean values of various treatments at different times were compared using the Tukey HSD test. The antinociceptive effect of WIN 55,212-2 on basal withdrawal responses to heat were analyzed using a two-way ANOVA with repeated measures. For each animal, the withdrawal latencies from each hindpaw were averaged. Since the mechanical stimuli rarely evoked a withdrawal response under normal conditions, only the withdrawal latencies to heat were analyzed statistically. The effect of WIN 55,212-2 on the amount of time animals ran on the treadmill was analyzed by one-way ANOVA. All data are expressed as the mean ± SEM and a P-value ,0.05 was considered significant.

3. Results 3.1. Nocifensive behavior following capsaicin Injection of capsaicin produced immediate guarding behavior characterized by lifting the injected paw or not applying pressure on it, as described previously (Gilchrist et al., 1996). In animals pretreated with the vehicle, nocifensive behavior after injection of capsaicin persisted for an average duration of 187.3 ± 43.1 s. WIN 55,212-2 produced a dose-dependent decrease in the duration of nocifensive behavior which is shown in Fig. 1. A one-way ANOVA

Fig. 2. Mean (±SEM) frequency of withdrawal evoked by mechanical stimuli before (baseline) and at 5 and 30 min after intraplantar injection of capsaicin. Animals were pretreated with vehicle (veh), WIN 55,212-2 (1–200 mg/kg), or the inactive enantiomer WIN 55,212-2 (200 mg/kg) 5 min before capsaicin. Data shown are for the injected paw only. Pretreatment with the 100 and 200 mg/kg doses of WIN 55,212-2 blocked the increase in withdrawal frequency produced by capsaicin. *Indicates a significant difference from the vehicle-treated group. †Indicates a significant difference from baseline.

revealed a significant difference in the duration of nocifensive behavior between the groups (F5,37 = 9.41, P , 0.001). Post-hoc comparisons indicated that pretreatment with doses of 10, 100 or 200 mg/kg produced a significant decrease in capsaicin-evoked nocifensive behavior compared to vehicle pretreatment (P , 0.05). The magnitude of the attenuation of nocifensive behavior produced by these doses did not differ. Pretreatment with WIN 55,212-3 did not alter nocifensive behavior relative to the vehicle-treated group. Ongoing nocifensive behavior always disappeared by the time testing for mechanical and heat hyperalgesia began (5 min after injection). 3.2. Withdrawal responses to mechanical stimuli

Fig. 1. Mean ( ± SEM) duration of nocifensive behavior produced by intraplantar injection of capsaicin. Nocifensive behavior was measured for 5 min following capsaicin. Animals were pretreated with vehicle, various doses of WIN 55,212-2, or the less active enantiomer WIN 55,212-3, 5 min before the capsaicin injection. WIN 55,212-2 produced a dose-dependent decrease in nocifensive behavior. *Indicates a significant difference from the vehicle-treated group.

All animals pretreated with vehicle exhibited an increase in the frequency of withdrawal following intraplantar injection of capsaicin. Capsaicin increased the mean frequency of withdrawal of the injected paw from 3.8 ± 0.2% before injection to 48 ± 10% at 5 min and 26 ± 10% at 30 min after injection (Fig. 2). This is comparable to what was observed in untreated animals (Gilchrist et al., 1996), and indicates that intravenous injection of vehicle did not alter the development or magnitude of mechanical hyperalgesia produced by capsaicin. In contrast, pretreatment with WIN 55,212-2 produced a dose-dependent decrease in mechanical hyperalgesia produced by capsaicin. Analysis of variance revealed significant differences in withdrawal response frequency between the groups (F5,40 = 3.62, P , 0.01). Before capsaicin, the mean frequency of withdrawal for both hindpaws did not differ between the groups. Although the 1 and 10 mg/kg doses of WIN 55,212-2 did not significantly attenuate the mean frequency of withdrawal at 5 min after capsaicin injection (29 ± 13 and

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36 ± 15%, respectively), four animals in each of these groups did not exhibit an increase in frequency of withdrawal. As shown in Fig. 2, higher doses of WIN 55,212-2 completely blocked the development of mechanical hyperalgesia produced by capsaicin. In animals pretreated with the 100 or 200 mg/kg doses, the mean frequency of withdrawal for the injected paw at 5 min following capsaicin was 11.2 ± 7 and 2.5 ± 4.6%, respectively, and differed significantly from the vehicle-treated group (P , 0.05). Also, animals pretreated with these doses exhibited lower withdrawal response frequencies at 30 min after capsaicin compared with the vehicle-treated group (3.8 ± 2.0%, P , 0.05 and 3.8 ± 5.2%, P , 0.05, respectively). Pretreatment with the enantiomer, WIN 55,212-3, did not alter the development of mechanical hyperalgesia. Also, the frequency of withdrawal for the contralateral non-injected paw was not changed in any of the groups after capsaicin. 3.3. Withdrawal responses to heat stimuli In normal animals, intraplantar injection of 10 mg capsaicin produces approximately a 50% decrease in withdrawal latency to heat at 5 min after capsaicin and which recovers by 30 min (Gilchrist et al., 1996). A similar effect was observed in the present study in animals pretreated with the vehicle (Fig. 3). Mean withdrawal latencies decreased from 11.5 ± 1.2 s before capsaicin to 5.2 ± 1.8 s at 5 min after capsaicin. As for mechanical hyperalgesia, pretreatment with the vehicle did not alter the development or magnitude of hyperalgesia to heat. Analysis of variance indicated significant differences in withdrawal latency between groups (F5,40 = 3.26, P , 0.05), and as shown in Fig. 3, WIN 55,212-2 attenuated capsaicin-evoked heat hyperalgesia in a dose-dependent fashion. There were no differences in withdrawal latency between the groups before any injection. Although animals that were pretreated with the 10 mg dose exhibited a significant decrease in withdrawal latency at 5 min following capsaicin (as compared with the contralateral paw), the magnitude of hyperalgesia was less than that observed in vehicle-pretreated animals (P , 0.04). In animals pretreated with vehicle, withdrawal latency for the injected paw decreased an average of 6.3 ± 0.99 s. However, in animals given 10 mg/kg WIN 55,212-2, withdrawal latency decreased only 2.5 ± 1.40 s. Mean withdrawal latency for the injected paw of animals pretreated with 100 mg/kg (P , 0.01) and 200 mg/kg (P , 0.01) also differed significantly from the vehicle-treated group at 5 min after capsaicin and produced a significantly greater attenuation of heat hyperalgesia than the 10 mg/kg dose (P , 0.05). In fact, these higher doses of WIN 55,212-2 prevented the development of hyperalgesia to heat. Mean withdrawal latency for the injected paw of animals that were pretreated with the 100 and 200 mg/kg doses were 12.7 ± 1.4 and 11.2 ± 0.50 s, respectively, before capsaicin. After capsaicin, mean latencies were 12.4 ± 1.4 and 14.3 ± 1.0 s for doses of 100 and 200 mg/kg, respectively.

Fig. 3. Mean (±SEM) withdrawal latency to heat before and at 5 and 30 min after intraplantar injection of capsaicin. For each panel, the solid symbols represent the injected paw and the open symbols represent the contralateral paw. Animals were pretreated with either vehicle, WIN 55,212-2 (1–200 mg/kg), or the inactive enantiomer WIN 55,212-3 (100 mg/kg). Arrows indicate time of pretreatment; capsaicin was given at time ‘0’. Note that the y-axis scale differs for the 200 mg/kg dose of WIN 55,212-2. *Indicates a significant difference from the contralateral paw.

Following these doses of WIN 55,212-2, no significant differences were observed between the capsaicin-injected and control paws. Although pretreatment with the enantiomer, WIN 55,212-3, did not alter the development of hyperalgesia to heat at 5 min after capsaicin injection, it prolonged the duration of hyperalgesia. The effect of intravenous administration of the vehicle, and 10, 100 and 200 mg/kg WIN 55,212-2 on basal withdrawal latencies to heat was investigated in naive animals (without capsaicin). Neither the vehicle nor WIN 55,212-2 altered withdrawal latency at 10 or 30 min after injection. In animals pretreated with the 100 and 200 mg/kg doses heat withdrawal latencies increased an average of 2.6 and 3.6 s at 10 min after injection, but this was not statistically significant. 3.4. Motor coordination following WIN 55,212-2 Administration of the vehicle or WIN 55,212-2 at doses of 100 and 200 mg/kg did not alter significantly the amount of time animals were able to run on a treadmill (Fig. 4). Before and at 10 min following injection of vehicle and 100 mg/kg WIN 55,212-2 all animals were able to run continuously on the treadmill for 5 min. After injection of 200 mg/ kg WIN 55,212-2, only two of six animals were unable to remain on the treadmill for the full 5 min. These animals

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al., 1997a) or oligonucleotides directed at the CB1 receptor (Richardson et al., 1998) produces hyperalgesia, suggesting that endogenous cannabinoids tonically modulate nociceptive transmission in the spinal cord. 4.1. Attenuation of hyperalgesia to mechanical and heat stimuli following WIN 55,212-2

Fig. 4. Mean (±SEM) duration of running time on a treadmill at 10 min after intravenous injection of vehicle or WIN 55,212-2 doses of 100 and 200 mg/kg. Before injection, all animals were trained to run on the treadmill continuously for 5 min.

remained on the treadmill for 59 and 82 s, respectively. The remaining four animals in this group were each capable of running on the treadmill for the entire 5 min period.

4. Discussion Cannabinoids, including the endogenous cannabinoid anandamide, have been shown to produce antinociception to a variety of stimuli including heat (Kaymakc¸alan and Deneau, 1971; Buxbaum, 1972; Chesher et al., 1973; Sofia et al., 1973; Bloom et al., 1977; Yaksh, 1981; Lichtman and Martin, 1991; Martin et al., 1991, 1996; Fride and Mechoulam, 1993) and mechanical stimuli (Sofia et al., 1973; Martin et al., 1996), and to electrical stimulation of teeth (Kaymakc¸alan et al., 1974). In addition, D9-tetrahydrocannabinol inhibits the first (phasic) and second (tonic) phases of nocifensive behavior produced by subcutaneous injection of formalin into the hindpaw (Moss and Johnson, 1980). The finding that cannabinoids attenuate chemogenic pain is supported by the present study in which intravenous administration of the cannabinoid receptor agonist WIN 55,212-2 dose-dependently inhibited nocifensive behavior produced by intraplantar injection of capsaicin. It is believed that antinociception produced by WIN 55,212-2 and other cannabinoids is receptor-specific and thought to be mediated primarily by actions at the CB1 receptor. The CB1 receptor antagonist, SR141716A (Rinaldi-Carmona et al., 1994), has been shown to block cannabinoid, but not morphine-induced antinociception, following systemic or intracerebroventricular administration using the tail-flick test (Lichtman and Martin, 1997). In addition, intracerebroventricular injection of antisense oligodeoxynucleotide directed at the CB1 receptor inhibits antinociceptive responses normally produced by cannabinoids (Edsall et al., 1996). Moreover, intrathecal administration of the CB1 antagonist SR141716A (Richardson et

We have shown previously that intraplantar injection of capsaicin produces a dose-dependent increase in the frequency of withdrawal from mechanical stimuli decreases withdrawal latency to heat (Gilchrist et al., 1996) suggestive of hyperalgesia. In the present study, it was found that the cannabinoid WIN 55,212-2 blocked the development of mechanical and heat hyperalgesia. Since cannabinoids are known to depress motor function (Compton et al., 1993), it is possible that the apparent ‘antihyperalgesia’ produced by the 100 or 200 mg/kg doses of WIN 55,212-2 may actually have been due to motor dysfunction. However, we found that 100 mg/kg WIN 55,212-2 did not impair motor coordination at 10 min following injection; the time at which behavioral measures of hyperalgesia were made. It is, therefore, unlikely that attenuation of capsaicin-evoked hyperalgesia by doses of 10 and 100 mg/kg WIN 55,212-2 were attributed to motor dysfunction. Since some of the animals exhibited impaired motor coordination after the 200 mg/kg dose, the blockade of hyperalgesia by this dose may have been attributed to motor deficits in some of the animals. In a previous study, 0.25 mg/kg WIN 55,212-2 produced analgesia in response to noxious pressure and heat stimuli, but also affected motor function (Martin et al., 1996). However, the decreased motor coordination following this high dose of WIN 55,212-2 was short lasting, and the duration of analgesia was significantly longer than the duration of motor impairment. This finding lends additional support to the suggestion that attenuation of capsaicin-evoked hyperalgesia produced by the doses of WIN 55,212-2 in the present study was not due to motor dysfunction. Cannabinoids have been shown to attenuate hyperalgesia in a model of neuropathic pain produced by loose ligation of the sciatic nerve in rats (Herzberg et al., 1997). In that study, intraperitoneal administration of 2.14 mg/kg WIN 55,212-2 completely blocked hyperalgesia to heat and mechanical stimuli ipsilateral to the injury without significantly altering withdrawal responses on the contralateral paw. The antihyperalgesic effect of WIN 55,212-2 was attenuated by the CB1 antagonist SR141716A suggesting a receptor-specific effect. These studies support the notion that cannabinoids can block hyperalgesia at doses which do not produce analgesia or affect motor function. Similarly, the doses of WIN 55,212-2 used in the present study did not significantly alter withdrawal responses in the paw contralateral to the capsaicin injection. Furthermore, WIN 55,212-2 did not alter basal heat withdrawal latency. This demonstrates that the doses used produced antihyperalgesia but not analgesia.

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4.2. Cannabinoid antinociception: sites of action The present study employed the intravenous route of administration, and it is therefore unknown whether the antihyperalgesic properties of WIN 55,212-2 were due to spinal or supraspinal mechanisms, or both. However, regardless of the site(s) of action for cannabinoids, it is likely that pain and hyperalgesia produced by capsaicin were mediated, at least in part, by inhibiting nociceptive transmission in the spinal cord. Our preliminary data suggest that intravenous administration of 100 mg/kg WIN 55,212-2, the dose that attenuated nocifensive behavior and blocked the behavioral expression of hyperalgesia in the present study, decreases responses of nociceptive dorsal horn neurons evoked by intraplantar injection of capsaicin and blocks subsequent sensitization to heat and mechanical stimuli (Simone et al., 1997). This could occur through supraspinal mechanisms which activate descending pain modulatory systems. This possibility is supported by the finding that microinjection of WIN 55,212-2 into the dorsal lateral periaquaductal gray, dorsal raphe, or rostral ventromedial medulla, areas known to be involved in descending modulation (Yaksh and Rudy, 1978; Basbaum and Fields, 1984), elevate tail-flick latencies (Martin et al., 1995, 1998). Alternatively, direct activity of WIN 55,212-2 at the spinal cord level could also mediate its effect of blocking sensitization of dorsal horn neurons since a spinal component of cannabinoid antinociception has been demonstrated (Lichtman and Martin, 1991). Finally, cannabinoids may also act through peripheral mechanisms to produce antinociception. Cannabinoid receptors have been identified on dorsal root ganglion neurons (Hohmann et al., 1995), and intraplantar injection of anandamide, the endogenous ligand for cannabinoid receptors, has been reported to inhibit hyperalgesia to heat produced by inflammation of the hindpaw and to block capsaicin-evoked plasma extravasation and peripheral release of calcitonin gene-related peptide (Richardson et al., 1997b). Recent evidence suggests that cannabinoids produce antinociception by acting at peripheral CB1 and CB2like receptors (Calignano et al., 1998). In that study, anandamide or the synthetic cannabinoid receptor agonists HU210 and WIN 55,212-2 co-injected with formalin into the hindpaw attenuated formalin-induced nocifensive behavior. The analgesic effect of these compounds was blocked by systemic administration of the CB1 antagonist SR141716A but not by the CB2 antagonist SR144528, further suggesting that the analgesic effects of WIN 55,212-2 are mediated through CB1 receptors. Interestingly, it is possible that WIN 55,212-2 attenuates activity of capsaicin-sensitive primary afferent neurons and thereby, prevents sensitization of dorsal horn neurons.

5. Conclusions The present results indicate that cannabinoids possess

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antihyperalgesic properties at subanalgesic doses, and at doses that do not appear to affect motor function. Although the mechanisms underlying the antihyperalgesic effects of cannabinoids are unknown, our preliminary studies suggest that cannabinoids prevent the hyperexcitability of dorsal horn neurons that contributes to hyperalgesia. Since hyperalgesia is associated with many chronic painful syndromes, low doses of cannabinoids may represent a novel therapeutic approach for alleviating hyperalgesia. Our data suggest that low doses of cannabinoids might be effective without the unwanted side effects typically associated with these compounds. Additional behavioral, pharmacological and electrophysiological studies are needed to determine mechanisms and anatomical sites that underlie cannabinoid-induced antinociception.

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