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Abstract
The analgesic properties of the synthetic cannabinoid WIN55,212-2 were investigated in a model of neuropathic pain. In male Wistar rats, bilateral hind limb withdrawal thresholds to cold, mechanical and noxious thermal stimuli were measured. Following this, unilateral L5 spinal nerve ligation was performed. Seven days later, sensory thresholds were reassessed and the development of allodynia to cold and mechanical stimuli and hyperalgesia to a noxious thermal stimulus confirmed.
The effect of WIN55,212-2 (0.1—5.0mgkg−1, i.p.) on the signs of neuropathy was then determined; there was a dose related reversal of all three signs of painful neuropathy at doses which did not generally alter sensory thresholds in the contralateral unligated limb. This effect was prevented by co-administration of the CB1 receptor antagonist SR141716a, but not by co-administration of the CB2 receptor antagonist SR144528, suggesting this action of WIN55,212-2 is mediated via the CB1 receptor. Administration of SR141716a alone had no affect on the observed allodynia and hyperalgesia, which does not support the concept of an endogenous analgesic tone.
These data indicate that cannabinoids may have therapeutic potential in neuropathic pain, and that this effect is mediated through the CB1 receptor.
Introduction
There have been suggestions that extracts of Cannabis sativa have medicinal properties for thousands of years, but until recently, little sound scientific data has been available to support this hypothesis. However, the structure of Δ9-tetrahydrocannabinol (THC), the major psychoactive component of the 66 known cannabinoids found in marijuana, was elucidated in the early 1960s and since then THC has been shown to be associated with a number of pharmacological effects, including analgesia (Gaoni & Mechoulam, 1964). A further breakthrough in the elucidation of cannabinoid pharmacology came in 1988 with the discovery of a cannabinoid receptor in rat brain (CB1, Devane et al., 1988). This receptor was subsequently cloned in 1990 (Matsuda et al., 1990) and was shown to have a seven transmembrane G-protein coupled structure (Howlett et al., 1986). A second cannabinoid receptor (CB2), with 44% sequence homology to CB1, was identified and cloned in 1993 (Munro et al., 1993) and was found to be predominantly expressed by cells of the immune system. Also in 1992, the endogenous cannabinoid anandamide (AEA) was extracted from the brain and spinal cord (Devane et al., 1992). More recently palmitoylethanolamide (PEA) and 2-arachidonyl glycerol (2-AG) have also been identified as potential endogenous cannabinoids (Mechoulam et al., 1995; Sugiura et al., 1995). The synthesis of the specific high affinity receptor antagonists, SR141716a (SR1) at CB1 (Rinaldi-Carmona et al., 1995; Welch et al., 1998) and SR144528 (SR2) at CB2 (Rinaldi-Carmona et al., 1998; Griffin et al., 1999), and the development of CB1 (Ledent et al., 1999) and CB2 'knockout mice' (Buckley et al., 2000) afforded new techniques for the elucidation of CB receptor mediated effects. Several studies have demonstrated an analgesic effect with synthetic and endogenous cannabinoids in inflammatory pain models (for example Jaggar et al., 1998a, 1998b; Calignano et al., 1998; Richardson et al., 1998.)
Neuropathic pain is defined as 'pain initiated or caused by a primary lesion or dysfunction in the nervous system' (Merskey & Bogduk, 1994) and is an area of largely unmet therapeutic need. Tricyclic antidepressants and certain anticonvulsants are the mainstay of clinical therapy for neuropathic pain (McQuay et al., 1996; Sindrup & Jensen, 1999). However, systematic reviews reveal that only between 30—50% of patients suffering from neuropathic pain achieve clinically significant (>50%) pain relief with any available single therapy (McQuay et al., 1996; Sindrup & Jensen, 1999). Furthermore, side effects of these therapies often limit their usefulness (McQuay et al., 1996). Although controversial, it is generally accepted that opioids are less effective in neuropathic than inflammatory pain. (Rowbotham, 1999). One explanation for this observation is that spinal opioid receptor expression decreases after peripheral nerve injury (Besse et al., 1992). A recent study has demonstrated little decrease of spinal CB receptor binding when compared to μ-opioid receptor binding after neonatal capsaicin treatment (Hohmann & Herkenham, 1998). Furthermore, it has also been demonstrated using immunocytochemistry that, following dorsal rhizotomy, spinal CB1 receptor expression remains unaltered (Farquhar-Smith et al., 2000). This sparing of CB1 receptors following peripheral nerve injury could indicate a potential therapeutic advantage of cannabinoids over opioids in the treatment of neuropathic pain. This hypothesis is further supported by a recent study (Mao et al., 2000), which demonstrated an anti-hyperalgesic effect of THC in an animal model of neuropathic pain and suggested a therapeutic advantage of THC over opioids in painful neuropathy.
Various animal models of neuropathic pain have been developed (Kim et al., 1997). The three most commonly used models share partial injury of the sciatic nerve and a subsequent alteration in hind limb withdrawal thresholds to sensory stimuli as a common feature. In particular, hyperalgesia to a noxious thermal stimulus and allodynia to cold and mechanical stimuli are usually observed. The first model described was the chronic constriction injury (CCI) model in which loose chromic gut ligatures are placed around the sciatic nerve (Bennett & Xie, 1988). A more recent modification was the partial sciatic nerve ligation (PNL) model, in which a tight ligation is placed around 1/3 to 1/2 of the sciatic nerve trunk (Seltzer et al., 1990). A third model is the spinal nerve ligation (SNL) model, in which the L5 and L6 spinal nerves are tightly ligated (Kim & Chung, 1992). A direct comparison of various features of these three models of neuropathic pain has been reported (Kim et al., 1997). In this study, the latency and duration of signs of painful neuropathy were examined, and the authors also examined the effect of sympathectomy on these signs. This study showed that the partial nerve injury evoked signs with roughly the same onset in all three models, but that qualitatively the mechanical and cold allodynia were greatest in SNL. Ongoing pain, as determined by non-weight bearing behaviour, was greatest in CCI. Surgical lumbar sympathectomy caused a reduction in pain behaviour in all three models, with the largest decrease in SNL, suggesting a greater sympathetic nervous system involvement in this model.
There are two reports of the effectiveness of cannabinoids in an animal model of neuropathic pain. One study reported that the synthetic cannabinoid WIN55,212-2 alleviated the allodynia and hyperalgesia associated with the CCI model of neuropathic pain (Herzberg et al., 1997). It was confirmed that this effect was mediated through the CB1 receptor and occurred at a dose that was not associated with obvious side effects. This study also demonstrated an increase in thermal hyperalgesia and mechanical allodynia by administration of SR141716a alone, leading the authors to infer the presence of an endogenous tone of cannabinoid analgesia in neuropathy. However, the evidence for such tone during inflammation is controversial (Beaulieu et al., 2000). A separate study reported that THC, administered intrathecally, also alleviated the hyperalgesia of the CCI model (Mao et al., 2000). This effect was shown to be CB1 receptor mediated.
It has been suggested that the neuropathy in the CCI model is largely dependent upon an inflammatory reaction (Wagner et al., 1998) and therefore the anti-inflammatory effects of cannabinoids may have obfuscated the true effect on neuropathy in this model. Both the PNL and SNL are associated with a lesser inflammatory component. Therefore, in this study we examined whether the anti-allodynic and anti-hyperalgesic effects of cannabinoids were found in a model of neuropathic pain associated with less of an inflammatory component than the CCI model, namely the SNL model. We also investigated the concept of endogenous tone in this model.
Methods
Animal maintenance
All experiments were approved by the Home Office. Animals were housed, six per cage at constant temperature under a 14:10h light/dark cycle, with free access to food and water at all times.
Surgery
A left L5 spinal nerve ligation, a modification of that described by Kim & Chung (1992), was performed on male Wistar rats of between 200—350g in weight (n=126). The rats were anaesthetized (pentobarbitone sodium, 60mgkg−1, i.p.) and the surgery performed using standard aseptic techniques. Using the transverse processes of L6 as a guide, the left paraspinal muscles were exposed and separated from the spinous processes of L4 to S2 by blunt dissection. The L6 transverse process was then removed by hemi-laminectomy and the L5 spinal nerve exposed and identified according to its size and position. This was then ligated tightly with a 3-0 silk suture and sectioned 1—2mm distal to the suture before haemostasis was confirmed and the wound was sutured at both muscle and skin levels. Sham surgery (n=6) was performed by exposing the L5 spinal nerve as described above, but not damaging it.
Sensory testing
Three tests of hind limb withdrawal to thermal, cold and mechanical stimuli were employed in this study. Each test was repeated on both the operated hind paw and the contralateral hind paw with all sensory testing performed by a 'blinded' investigator.
(i)
Cold allodynia was assessed using the acetone drop application technique modified from Carlton et al. (1994). Animals were placed in plexiglass boxes (23×18×14cm) with 0.8cm diameter mesh flooring and allowed to acclimatize for 15min or until exploratory behaviour ceased. Sampling was performed by the application of a single bubble of acetone to the mid plantar surface of each hind paw from the tip of a 1ml syringe. A positive response was recorded if the animal withdrew the paw following application. For each measurement, the paw was sampled five times and a mean calculated. At least 3min elapsed between each test.
(ii)
Thermal hyperalgesia was assessed using an infrared noxious heat stimulus (Plantar test, Ugo Basile, Italy, Hargreaves et al., 1988). Briefly, animals were placed in a clear plexiglass box (23×18×14cm) with a dry glass floor and allowed to acclimatize for 15min or until exploratory behaviour ceased. A focused beam of radiant heat at a constant temperature of 46°C and wavelength of 50nm was applied to the plantar surface of the paw. The hind paw withdrawal latency (s) to this stimulus was tested three times at intervals of not less than 3min and a mean calculated. The device has an automatic cut-off at 21s to avoid the risk of thermal injury to the skin.
(iii)
Mechanical allodynia was assessed using an electronic Von Frey device (Möller et al., 1998; Ahmad & Rice, 1999). Animals were placed into raised plexiglass boxes (23×18×14cm) with 0.8cm diameter mesh flooring and allowed to acclimatize for 15min or until exploratory behaviour ceased. Sampling was conducted by a calibrated nylon electronic force transducer (1.0mm diameter, type 739, Somedic Sales AB, Sweden) which was applied manually to the mid-plantar hind paw at a rate of 0.5—1.0Ns−1 with the withdrawal threshold amplified (Senselab 701, Somedic, Sweden) and displayed on a PC based chart recorder (AcqKnowledge v3.02, Biopac Systems Inc. U.S.A.). The mean withdrawal threshold was taken from a set of five applications, not less than 10s apart.
Pharmacological interventions
Baseline sensory thresholds were measured for each group of animals (n=6) pre-operatively and 7 days post-operatively. Animals displaying thermal hyperalgesia or cold or mechanical allodynia were then administered the relevant drug according to a pre-determined randomization table and testing was re-performed at 20, 40, 60 and 90min post drug administration. Each group of animals was used for only one drug administration protocol to ensure no 'carry over' effects, hence they were used for only a single treatment. WIN55,212-2 (0.1—5.0mgkg−1, i.p.) was administered at t=0, in 40% dimethylsulfoxide (DMSO) in saline solvent, SR141716a (0.5mgkg−1, i.p.) and SR144528 (1mgkg−1, i.p.) were administered at t=−5min and dissolved in 40% DMSO in saline solvent. All animals were humanely culled at the end of the experiment. A summary of the treatment groups is displayed in Table 1.
Statistical analysis
Statistical significance was determined for neuropathy by a paired t-test and for drug effects by one-way ANOVA (Dunnett, compared to post operative values), both taking P<0.05 as statistically significant (SigmaStat v2.0, Jandel Corporation, U.S.A.)
Drugs
WIN55,212-2 was supplied by Tocris Cookson Ltd., U.K., SR141716a was a gift from SRI—NIMH chemical synthesis program and SR144528 was a gift from Sanofi Recherche, France.
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Results
All animals included in the analysis of study exhibited altered sensory thresholds 7 days following SNL (rejection rate=7%). In the sham surgery groups (20—22, Table 1) there was no significant difference from baseline sensory thresholds. In all animals in which solvent only was administered as a control, sensory thresholds were not subsequently altered in either the ipsilateral or contralateral paws.
WIN55,212-2 studies
(i)
Administration of WIN55,212-2 reversed the cold allodynia produced by SNL at a dose of 2.5mgkg−1 over 90min (Figure 1). This effect was observed to be both dose- and time-dependent with a maximum effect at 20—40min. The dose dependency was observed with a non-significant trend towards an effect at 0.5mgkg−1 WIN, but no effect at 0.1mgkg−1.
(ii)
WIN55,212-2 also reversed the thermal hyperalgesia produced by SNL. This effect was again observed to be both dose and time dependent with the results generally concurring with those from the cold allodynia studies (Figure 2). Thermal hyperalgesia was attenuated throughout the entire 90min experiment at a dose of 2.5mgkg−1 WIN55,212-2 and also between 20—40min at a dose of 0.5mgkg−1. No effect was seen at the 0.1mgkg−1 dose.
(iii)
Mechanical allodynia produced by SNL was not as effectively attenuated by WIN55,212-2 as the cold allodynia and thermal hyperalgesia (Figure 3). A significant effect was only observed at the 5.0mgkg−1 dose throughout the entire 90min experiment. However, significant effects were also observed on the sensory thresholds of the contralateral limb at this dose. At 20 and 40min post dosing the 2.5 and 0.5mgkg−1 doses had no effect on mechanical allodynia.
Receptor involvement
The CB1 receptor antagonist SR141716a (0.5mgkg−1) prevented the anti-hyperalgesic or anti-allodynic effects seen at the highest doses of WIN55,212-2 (Figure 4). In the mechanical and thermal stimuli studies, SR141716a prevented the anti-allodynic and anti-hyperalgesic effects of WIN55,212-2 over the 90min investigation period. In the cold stimuli studies, the anti-allodynic effects of WIN55,212-2 were prevented over 40min, but persisted at 60 and 90min at a much reduced level than the 2.5mgkg−1 alone group. Administration of SR141716a alone had no significant effects on any of the sensory thresholds (Figure 5). Co-administration of WIN55,212-2 (2.5mgkg−1) and the CB2 receptor antagonist SR144528 (1mgkg−1) did not prevent the anti-allodynic effect of WIN55,212-2 in cold stimuli over 90min (Figure 6).
Discussion
This study has provided evidence for analgesic actions of the synthetic cannabinoid WIN55,212-2 in the spinal nerve ligation model of painful neuropathy. WIN55,212-2 attenuated cold allodynia and thermal hyperalgesia at a dose of 2.5mgkg−1, i.p and mechanical allodynia at a dose of 5.0mgkg−1, i.p. These effects are CB1 receptor mediated and have been observed at doses of WIN55,212-2 that cause no significant alteration in the sensory thresholds of the uninjured contralateral hind limb. Neither administration of SR141716a alone, administration of solvent nor sham surgery had an effect on the sensory thresholds. These results concur with a previous study which examined the analgesic actions of WIN55,212-2 in the CCI model of neuropathic pain (Herzberg et al., 1997) despite important differences between the models.
The blockade of the anti-hyperalgesic and anti-allodynic effects of WIN55,212-2 by the CB1 receptor antagonist, but not the CB2 receptor antagonist demonstrates that the analgesic effect of WIN55,212-2 is mediated through the CB1 receptor. This is despite the fact that WIN55,212-2, like most other cannabinoids, has significant affinity for the CB2 receptor, as well as CB1. The CB1 receptor is localized throughout the central nervous system, and has been mapped by immunocytochemistry in the brain (Tsou et al., 1998), spinal cord (Farquhar-Smith et al., 2000) and in cultured dorsal root ganglion cells (Ahluwalia et al., 2000). Immunocytochemical study of the spinal cord has shown that the expression of CB1 receptors is localized to areas associated with nociception, for example the superficial dorsal horn and lamina X (Farquhar-Smith et al., 2000). The localization of CB1 receptors in lamina I and lamina IIi/III coincides with the terminals of some of the neurons lost in peripheral nerve injury. It would therefore be expected that CB1 receptor expression would also decrease after peripheral nerve injury. However, Farquhar-Smith et al. (2000) observed no biologically relevant decrease in CB1 receptor after dorsal rhizotomy or rostral hemisection of the spinal cord. The authors postulate that this finding could be explained by the majority of spinal CB1 receptors being localized on spinal interneurons. This concept is supported by electrophysiological studies (Jennings et al., 2000) and changes in spinal cord receptor binding of [3H]-CP55,940 after neonatal capsaicin treatment (Hohmann & Herkenham, 1998), where the authors found only a 16% reduction in CB1 receptor binding after death of small diameter afferents was caused by capsaicin treatment. However, a multi-level dorsal rhizotomy (C3—T2) caused a 46% decrease in [3H]-CP55,940 binding, suggesting a presence of CB1 receptors on central termini of primary afferent neurons (Hohmann et al., 1999), although this finding could be explained by post-synaptic degeneration of neurons after such an extensive surgery.
A possible confounding factor in our experiments is a putative effect of WIN55,212-2 on the sensory thresholds per se. However, we have controlled for such an effect by measuring the sensory thresholds in the contralateral hind paw in all experiments. Only at the highest used dose of WIN55,212-2 was there a significant increase in the sensory threshold of the contralateral paw, suggesting that such an effect is not a confusing factor to the results at doses of WIN55,212-2 of 2.5mgkg−1 or less.
The well-known psychotropic effects of cannabinoids are an obstacle to the development of cannabinoid based analgesics for the treatment of neuropathic pain (Perez-Reyes, 1999). Nevertheless, although not formally measured, we did not observe any obvious abnormal rodent behaviour indicative of psychotropic effects at the doses of WIN55,212-2 studies used in these experiments. However, there are a number of ways in which this issue may be circumvented: for example, structural engineering of cannabinoid molecules may permit certain cannabinoids to retain therapeutic activity whilst being devoid of psychotropic effects. One example of a cannabinoid which retains analgesic activity, in a model of inflammatory arthritis, in the absence of psychotropic effects is cannabidiol (Malfait et al., 2000). Furthermore, in a recent publication, Fox and colleagues have demonstrated that peripherally or spinal intrathecally administered WIN55,212-2, at doses that were not systemically active, attenuated the altered sensory thresholds associated with partial sciatic nerve ligation (Fox et al., 2001). This suggests that either selective delivery of cannabinoids by these routes, or systemic administration of cannabinoids that do not penetrate into the CSF, may be a method of divorcing analgesic from psychotropic effects. Finally, Baker et al. (2001) have demonstrated, using a model of multiple sclerosis, that concentrations of endocannabinoids are selectively elevated in areas of neuronal injury in spinal cord and brain. This finding indicates that it may be possible to selectively manipulate endocannabinoid concentrations in areas of neuronal injury, by inhibiting their degradation.
It has also been demonstrated that [3H]-DAMGO (a μ-opioid receptor agonist) binding was reduced by 60% following neonatal capsaicin treatment (Hohmann & Herkenham, 1998) and by 62% following multi-level dorsal rhizotomy (Hohmann et al., 1999). This consistent loss of opioid receptors following nerve injury is a factor for the relative ineffectiveness of morphine in neuropathic pain. Also, with several studies documenting a stable population of CB1 receptors in the superficial dorsal horn after peripheral nerve injury, a potential therapeutic advantage for cannabinoids over opioids has been found at the level of the spinal cord. This hypothesis is further supported by a study in which it was observed that THC alleviated hyperalgesia in the CCI model of neuropathic pain (Mao et al., 2000). This effect was shown to be on CB1 and not opioid receptor dependent, with no cross tolerance between opioids and cannabinoids, suggesting a different pathway of antinociception for cannabinoids and opioids.
In this study, we were unable to replicate the increase in allodynia or hyperalgesia associated with administration of the CB1 receptor antagonist SR141716a alone to animals with peripheral nerve injury that was observed by Herzberg et al. (1997). In our study, there was no significant alteration in sensory thresholds in SR141716a treated animals when compared to solvent treated controls. The increase of hyperalgesia or allodynia seen after administration of the receptor antagonist has been hypothesized to suggest the presence of an endogenous tone that is active during neuropathy—viz. locally synthesized endocannabinoids act at a low level on the receptors in response to peripheral nerve injury, causing a degree of attenuation of the hyperalgesia or allodynia. Thus, by blocking the action of these endogenous ligands by administration of the specific antagonist, the hyperalgesia or allodynia will be increased. The current data on an endogenous cannabinoid tone in pain models is controversial. Several studies have shown the presence of a SR141716a-associated enhancement of signs of pain or nociception in a variety of models, including the formalin test for inflammation (Calignano et al., 1998; Strangman et al., 1998). This could be interpreted as supporting the hypothesis that an endogenous tone exists in inflammatory pain, but not in neuropathic pain. If this is the case, then the inflammatory component of the CCI model could account for the difference seen between this and the findings of Herzberg et al. (1997). However, other studies of endogenous tone in inflammatory models have found no evidence supporting this concept, either in an inflammatory model (Beaulieu et al., 2000) or when administered to uninjured animals (Rinaldi-Carmona et al., 1995). The use of SR141716a as an experimental tool to reveal endogenous tone could be further confused by the findings of several groups that SR141716a is actually an inverse agonist at the CB1 receptor (Landsman et al., 1997; Pan et al., 1998). Further studies in CB1−/− knockout mice did not support the concept of an endogenous tone (Ledent et al., 1999). It also remains to be determined whether the levels of endogenous cannabinoids found in the spinal cord and dorsal root ganglia are sufficient to activate the receptors. Despite the controversial nature of the endogenous tone, our experiments seem to contradict its existence and agree with the study by Beaulieu et al. (2000) that there is a weak analgesic action at about 60min post-administration, possibly due to SR141716a being able to show weak agonist activity under certain conditions.
This study has demonstrated the existence of CB1 receptor, but not CB2 receptor, mediated analgesia in an animal model of neuropathic pain. In light of the current therapeutic need for neuropathic pain treatments, this study provides evidence for the potential of cannabinoids, or inhibitors of degradation of endocannabinoids, as lead compounds for development of new therapies. The development of cannabinoid receptor specific agonists and antagonists will provide essential new tools for investigating the mechanism of this analgesic action.
Source, Graphs and Figures: The synthetic cannabinoid WIN55,212-2 attenuates hyperalgesia and allodynia in a rat model of neuropathic pain
The analgesic properties of the synthetic cannabinoid WIN55,212-2 were investigated in a model of neuropathic pain. In male Wistar rats, bilateral hind limb withdrawal thresholds to cold, mechanical and noxious thermal stimuli were measured. Following this, unilateral L5 spinal nerve ligation was performed. Seven days later, sensory thresholds were reassessed and the development of allodynia to cold and mechanical stimuli and hyperalgesia to a noxious thermal stimulus confirmed.
The effect of WIN55,212-2 (0.1—5.0mgkg−1, i.p.) on the signs of neuropathy was then determined; there was a dose related reversal of all three signs of painful neuropathy at doses which did not generally alter sensory thresholds in the contralateral unligated limb. This effect was prevented by co-administration of the CB1 receptor antagonist SR141716a, but not by co-administration of the CB2 receptor antagonist SR144528, suggesting this action of WIN55,212-2 is mediated via the CB1 receptor. Administration of SR141716a alone had no affect on the observed allodynia and hyperalgesia, which does not support the concept of an endogenous analgesic tone.
These data indicate that cannabinoids may have therapeutic potential in neuropathic pain, and that this effect is mediated through the CB1 receptor.
Introduction
There have been suggestions that extracts of Cannabis sativa have medicinal properties for thousands of years, but until recently, little sound scientific data has been available to support this hypothesis. However, the structure of Δ9-tetrahydrocannabinol (THC), the major psychoactive component of the 66 known cannabinoids found in marijuana, was elucidated in the early 1960s and since then THC has been shown to be associated with a number of pharmacological effects, including analgesia (Gaoni & Mechoulam, 1964). A further breakthrough in the elucidation of cannabinoid pharmacology came in 1988 with the discovery of a cannabinoid receptor in rat brain (CB1, Devane et al., 1988). This receptor was subsequently cloned in 1990 (Matsuda et al., 1990) and was shown to have a seven transmembrane G-protein coupled structure (Howlett et al., 1986). A second cannabinoid receptor (CB2), with 44% sequence homology to CB1, was identified and cloned in 1993 (Munro et al., 1993) and was found to be predominantly expressed by cells of the immune system. Also in 1992, the endogenous cannabinoid anandamide (AEA) was extracted from the brain and spinal cord (Devane et al., 1992). More recently palmitoylethanolamide (PEA) and 2-arachidonyl glycerol (2-AG) have also been identified as potential endogenous cannabinoids (Mechoulam et al., 1995; Sugiura et al., 1995). The synthesis of the specific high affinity receptor antagonists, SR141716a (SR1) at CB1 (Rinaldi-Carmona et al., 1995; Welch et al., 1998) and SR144528 (SR2) at CB2 (Rinaldi-Carmona et al., 1998; Griffin et al., 1999), and the development of CB1 (Ledent et al., 1999) and CB2 'knockout mice' (Buckley et al., 2000) afforded new techniques for the elucidation of CB receptor mediated effects. Several studies have demonstrated an analgesic effect with synthetic and endogenous cannabinoids in inflammatory pain models (for example Jaggar et al., 1998a, 1998b; Calignano et al., 1998; Richardson et al., 1998.)
Neuropathic pain is defined as 'pain initiated or caused by a primary lesion or dysfunction in the nervous system' (Merskey & Bogduk, 1994) and is an area of largely unmet therapeutic need. Tricyclic antidepressants and certain anticonvulsants are the mainstay of clinical therapy for neuropathic pain (McQuay et al., 1996; Sindrup & Jensen, 1999). However, systematic reviews reveal that only between 30—50% of patients suffering from neuropathic pain achieve clinically significant (>50%) pain relief with any available single therapy (McQuay et al., 1996; Sindrup & Jensen, 1999). Furthermore, side effects of these therapies often limit their usefulness (McQuay et al., 1996). Although controversial, it is generally accepted that opioids are less effective in neuropathic than inflammatory pain. (Rowbotham, 1999). One explanation for this observation is that spinal opioid receptor expression decreases after peripheral nerve injury (Besse et al., 1992). A recent study has demonstrated little decrease of spinal CB receptor binding when compared to μ-opioid receptor binding after neonatal capsaicin treatment (Hohmann & Herkenham, 1998). Furthermore, it has also been demonstrated using immunocytochemistry that, following dorsal rhizotomy, spinal CB1 receptor expression remains unaltered (Farquhar-Smith et al., 2000). This sparing of CB1 receptors following peripheral nerve injury could indicate a potential therapeutic advantage of cannabinoids over opioids in the treatment of neuropathic pain. This hypothesis is further supported by a recent study (Mao et al., 2000), which demonstrated an anti-hyperalgesic effect of THC in an animal model of neuropathic pain and suggested a therapeutic advantage of THC over opioids in painful neuropathy.
Various animal models of neuropathic pain have been developed (Kim et al., 1997). The three most commonly used models share partial injury of the sciatic nerve and a subsequent alteration in hind limb withdrawal thresholds to sensory stimuli as a common feature. In particular, hyperalgesia to a noxious thermal stimulus and allodynia to cold and mechanical stimuli are usually observed. The first model described was the chronic constriction injury (CCI) model in which loose chromic gut ligatures are placed around the sciatic nerve (Bennett & Xie, 1988). A more recent modification was the partial sciatic nerve ligation (PNL) model, in which a tight ligation is placed around 1/3 to 1/2 of the sciatic nerve trunk (Seltzer et al., 1990). A third model is the spinal nerve ligation (SNL) model, in which the L5 and L6 spinal nerves are tightly ligated (Kim & Chung, 1992). A direct comparison of various features of these three models of neuropathic pain has been reported (Kim et al., 1997). In this study, the latency and duration of signs of painful neuropathy were examined, and the authors also examined the effect of sympathectomy on these signs. This study showed that the partial nerve injury evoked signs with roughly the same onset in all three models, but that qualitatively the mechanical and cold allodynia were greatest in SNL. Ongoing pain, as determined by non-weight bearing behaviour, was greatest in CCI. Surgical lumbar sympathectomy caused a reduction in pain behaviour in all three models, with the largest decrease in SNL, suggesting a greater sympathetic nervous system involvement in this model.
There are two reports of the effectiveness of cannabinoids in an animal model of neuropathic pain. One study reported that the synthetic cannabinoid WIN55,212-2 alleviated the allodynia and hyperalgesia associated with the CCI model of neuropathic pain (Herzberg et al., 1997). It was confirmed that this effect was mediated through the CB1 receptor and occurred at a dose that was not associated with obvious side effects. This study also demonstrated an increase in thermal hyperalgesia and mechanical allodynia by administration of SR141716a alone, leading the authors to infer the presence of an endogenous tone of cannabinoid analgesia in neuropathy. However, the evidence for such tone during inflammation is controversial (Beaulieu et al., 2000). A separate study reported that THC, administered intrathecally, also alleviated the hyperalgesia of the CCI model (Mao et al., 2000). This effect was shown to be CB1 receptor mediated.
It has been suggested that the neuropathy in the CCI model is largely dependent upon an inflammatory reaction (Wagner et al., 1998) and therefore the anti-inflammatory effects of cannabinoids may have obfuscated the true effect on neuropathy in this model. Both the PNL and SNL are associated with a lesser inflammatory component. Therefore, in this study we examined whether the anti-allodynic and anti-hyperalgesic effects of cannabinoids were found in a model of neuropathic pain associated with less of an inflammatory component than the CCI model, namely the SNL model. We also investigated the concept of endogenous tone in this model.
Methods
Animal maintenance
All experiments were approved by the Home Office. Animals were housed, six per cage at constant temperature under a 14:10h light/dark cycle, with free access to food and water at all times.
Surgery
A left L5 spinal nerve ligation, a modification of that described by Kim & Chung (1992), was performed on male Wistar rats of between 200—350g in weight (n=126). The rats were anaesthetized (pentobarbitone sodium, 60mgkg−1, i.p.) and the surgery performed using standard aseptic techniques. Using the transverse processes of L6 as a guide, the left paraspinal muscles were exposed and separated from the spinous processes of L4 to S2 by blunt dissection. The L6 transverse process was then removed by hemi-laminectomy and the L5 spinal nerve exposed and identified according to its size and position. This was then ligated tightly with a 3-0 silk suture and sectioned 1—2mm distal to the suture before haemostasis was confirmed and the wound was sutured at both muscle and skin levels. Sham surgery (n=6) was performed by exposing the L5 spinal nerve as described above, but not damaging it.
Sensory testing
Three tests of hind limb withdrawal to thermal, cold and mechanical stimuli were employed in this study. Each test was repeated on both the operated hind paw and the contralateral hind paw with all sensory testing performed by a 'blinded' investigator.
(i)
Cold allodynia was assessed using the acetone drop application technique modified from Carlton et al. (1994). Animals were placed in plexiglass boxes (23×18×14cm) with 0.8cm diameter mesh flooring and allowed to acclimatize for 15min or until exploratory behaviour ceased. Sampling was performed by the application of a single bubble of acetone to the mid plantar surface of each hind paw from the tip of a 1ml syringe. A positive response was recorded if the animal withdrew the paw following application. For each measurement, the paw was sampled five times and a mean calculated. At least 3min elapsed between each test.
(ii)
Thermal hyperalgesia was assessed using an infrared noxious heat stimulus (Plantar test, Ugo Basile, Italy, Hargreaves et al., 1988). Briefly, animals were placed in a clear plexiglass box (23×18×14cm) with a dry glass floor and allowed to acclimatize for 15min or until exploratory behaviour ceased. A focused beam of radiant heat at a constant temperature of 46°C and wavelength of 50nm was applied to the plantar surface of the paw. The hind paw withdrawal latency (s) to this stimulus was tested three times at intervals of not less than 3min and a mean calculated. The device has an automatic cut-off at 21s to avoid the risk of thermal injury to the skin.
(iii)
Mechanical allodynia was assessed using an electronic Von Frey device (Möller et al., 1998; Ahmad & Rice, 1999). Animals were placed into raised plexiglass boxes (23×18×14cm) with 0.8cm diameter mesh flooring and allowed to acclimatize for 15min or until exploratory behaviour ceased. Sampling was conducted by a calibrated nylon electronic force transducer (1.0mm diameter, type 739, Somedic Sales AB, Sweden) which was applied manually to the mid-plantar hind paw at a rate of 0.5—1.0Ns−1 with the withdrawal threshold amplified (Senselab 701, Somedic, Sweden) and displayed on a PC based chart recorder (AcqKnowledge v3.02, Biopac Systems Inc. U.S.A.). The mean withdrawal threshold was taken from a set of five applications, not less than 10s apart.
Pharmacological interventions
Baseline sensory thresholds were measured for each group of animals (n=6) pre-operatively and 7 days post-operatively. Animals displaying thermal hyperalgesia or cold or mechanical allodynia were then administered the relevant drug according to a pre-determined randomization table and testing was re-performed at 20, 40, 60 and 90min post drug administration. Each group of animals was used for only one drug administration protocol to ensure no 'carry over' effects, hence they were used for only a single treatment. WIN55,212-2 (0.1—5.0mgkg−1, i.p.) was administered at t=0, in 40% dimethylsulfoxide (DMSO) in saline solvent, SR141716a (0.5mgkg−1, i.p.) and SR144528 (1mgkg−1, i.p.) were administered at t=−5min and dissolved in 40% DMSO in saline solvent. All animals were humanely culled at the end of the experiment. A summary of the treatment groups is displayed in Table 1.
Statistical analysis
Statistical significance was determined for neuropathy by a paired t-test and for drug effects by one-way ANOVA (Dunnett, compared to post operative values), both taking P<0.05 as statistically significant (SigmaStat v2.0, Jandel Corporation, U.S.A.)
Drugs
WIN55,212-2 was supplied by Tocris Cookson Ltd., U.K., SR141716a was a gift from SRI—NIMH chemical synthesis program and SR144528 was a gift from Sanofi Recherche, France.
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Results
All animals included in the analysis of study exhibited altered sensory thresholds 7 days following SNL (rejection rate=7%). In the sham surgery groups (20—22, Table 1) there was no significant difference from baseline sensory thresholds. In all animals in which solvent only was administered as a control, sensory thresholds were not subsequently altered in either the ipsilateral or contralateral paws.
WIN55,212-2 studies
(i)
Administration of WIN55,212-2 reversed the cold allodynia produced by SNL at a dose of 2.5mgkg−1 over 90min (Figure 1). This effect was observed to be both dose- and time-dependent with a maximum effect at 20—40min. The dose dependency was observed with a non-significant trend towards an effect at 0.5mgkg−1 WIN, but no effect at 0.1mgkg−1.
(ii)
WIN55,212-2 also reversed the thermal hyperalgesia produced by SNL. This effect was again observed to be both dose and time dependent with the results generally concurring with those from the cold allodynia studies (Figure 2). Thermal hyperalgesia was attenuated throughout the entire 90min experiment at a dose of 2.5mgkg−1 WIN55,212-2 and also between 20—40min at a dose of 0.5mgkg−1. No effect was seen at the 0.1mgkg−1 dose.
(iii)
Mechanical allodynia produced by SNL was not as effectively attenuated by WIN55,212-2 as the cold allodynia and thermal hyperalgesia (Figure 3). A significant effect was only observed at the 5.0mgkg−1 dose throughout the entire 90min experiment. However, significant effects were also observed on the sensory thresholds of the contralateral limb at this dose. At 20 and 40min post dosing the 2.5 and 0.5mgkg−1 doses had no effect on mechanical allodynia.
Receptor involvement
The CB1 receptor antagonist SR141716a (0.5mgkg−1) prevented the anti-hyperalgesic or anti-allodynic effects seen at the highest doses of WIN55,212-2 (Figure 4). In the mechanical and thermal stimuli studies, SR141716a prevented the anti-allodynic and anti-hyperalgesic effects of WIN55,212-2 over the 90min investigation period. In the cold stimuli studies, the anti-allodynic effects of WIN55,212-2 were prevented over 40min, but persisted at 60 and 90min at a much reduced level than the 2.5mgkg−1 alone group. Administration of SR141716a alone had no significant effects on any of the sensory thresholds (Figure 5). Co-administration of WIN55,212-2 (2.5mgkg−1) and the CB2 receptor antagonist SR144528 (1mgkg−1) did not prevent the anti-allodynic effect of WIN55,212-2 in cold stimuli over 90min (Figure 6).
Discussion
This study has provided evidence for analgesic actions of the synthetic cannabinoid WIN55,212-2 in the spinal nerve ligation model of painful neuropathy. WIN55,212-2 attenuated cold allodynia and thermal hyperalgesia at a dose of 2.5mgkg−1, i.p and mechanical allodynia at a dose of 5.0mgkg−1, i.p. These effects are CB1 receptor mediated and have been observed at doses of WIN55,212-2 that cause no significant alteration in the sensory thresholds of the uninjured contralateral hind limb. Neither administration of SR141716a alone, administration of solvent nor sham surgery had an effect on the sensory thresholds. These results concur with a previous study which examined the analgesic actions of WIN55,212-2 in the CCI model of neuropathic pain (Herzberg et al., 1997) despite important differences between the models.
The blockade of the anti-hyperalgesic and anti-allodynic effects of WIN55,212-2 by the CB1 receptor antagonist, but not the CB2 receptor antagonist demonstrates that the analgesic effect of WIN55,212-2 is mediated through the CB1 receptor. This is despite the fact that WIN55,212-2, like most other cannabinoids, has significant affinity for the CB2 receptor, as well as CB1. The CB1 receptor is localized throughout the central nervous system, and has been mapped by immunocytochemistry in the brain (Tsou et al., 1998), spinal cord (Farquhar-Smith et al., 2000) and in cultured dorsal root ganglion cells (Ahluwalia et al., 2000). Immunocytochemical study of the spinal cord has shown that the expression of CB1 receptors is localized to areas associated with nociception, for example the superficial dorsal horn and lamina X (Farquhar-Smith et al., 2000). The localization of CB1 receptors in lamina I and lamina IIi/III coincides with the terminals of some of the neurons lost in peripheral nerve injury. It would therefore be expected that CB1 receptor expression would also decrease after peripheral nerve injury. However, Farquhar-Smith et al. (2000) observed no biologically relevant decrease in CB1 receptor after dorsal rhizotomy or rostral hemisection of the spinal cord. The authors postulate that this finding could be explained by the majority of spinal CB1 receptors being localized on spinal interneurons. This concept is supported by electrophysiological studies (Jennings et al., 2000) and changes in spinal cord receptor binding of [3H]-CP55,940 after neonatal capsaicin treatment (Hohmann & Herkenham, 1998), where the authors found only a 16% reduction in CB1 receptor binding after death of small diameter afferents was caused by capsaicin treatment. However, a multi-level dorsal rhizotomy (C3—T2) caused a 46% decrease in [3H]-CP55,940 binding, suggesting a presence of CB1 receptors on central termini of primary afferent neurons (Hohmann et al., 1999), although this finding could be explained by post-synaptic degeneration of neurons after such an extensive surgery.
A possible confounding factor in our experiments is a putative effect of WIN55,212-2 on the sensory thresholds per se. However, we have controlled for such an effect by measuring the sensory thresholds in the contralateral hind paw in all experiments. Only at the highest used dose of WIN55,212-2 was there a significant increase in the sensory threshold of the contralateral paw, suggesting that such an effect is not a confusing factor to the results at doses of WIN55,212-2 of 2.5mgkg−1 or less.
The well-known psychotropic effects of cannabinoids are an obstacle to the development of cannabinoid based analgesics for the treatment of neuropathic pain (Perez-Reyes, 1999). Nevertheless, although not formally measured, we did not observe any obvious abnormal rodent behaviour indicative of psychotropic effects at the doses of WIN55,212-2 studies used in these experiments. However, there are a number of ways in which this issue may be circumvented: for example, structural engineering of cannabinoid molecules may permit certain cannabinoids to retain therapeutic activity whilst being devoid of psychotropic effects. One example of a cannabinoid which retains analgesic activity, in a model of inflammatory arthritis, in the absence of psychotropic effects is cannabidiol (Malfait et al., 2000). Furthermore, in a recent publication, Fox and colleagues have demonstrated that peripherally or spinal intrathecally administered WIN55,212-2, at doses that were not systemically active, attenuated the altered sensory thresholds associated with partial sciatic nerve ligation (Fox et al., 2001). This suggests that either selective delivery of cannabinoids by these routes, or systemic administration of cannabinoids that do not penetrate into the CSF, may be a method of divorcing analgesic from psychotropic effects. Finally, Baker et al. (2001) have demonstrated, using a model of multiple sclerosis, that concentrations of endocannabinoids are selectively elevated in areas of neuronal injury in spinal cord and brain. This finding indicates that it may be possible to selectively manipulate endocannabinoid concentrations in areas of neuronal injury, by inhibiting their degradation.
It has also been demonstrated that [3H]-DAMGO (a μ-opioid receptor agonist) binding was reduced by 60% following neonatal capsaicin treatment (Hohmann & Herkenham, 1998) and by 62% following multi-level dorsal rhizotomy (Hohmann et al., 1999). This consistent loss of opioid receptors following nerve injury is a factor for the relative ineffectiveness of morphine in neuropathic pain. Also, with several studies documenting a stable population of CB1 receptors in the superficial dorsal horn after peripheral nerve injury, a potential therapeutic advantage for cannabinoids over opioids has been found at the level of the spinal cord. This hypothesis is further supported by a study in which it was observed that THC alleviated hyperalgesia in the CCI model of neuropathic pain (Mao et al., 2000). This effect was shown to be on CB1 and not opioid receptor dependent, with no cross tolerance between opioids and cannabinoids, suggesting a different pathway of antinociception for cannabinoids and opioids.
In this study, we were unable to replicate the increase in allodynia or hyperalgesia associated with administration of the CB1 receptor antagonist SR141716a alone to animals with peripheral nerve injury that was observed by Herzberg et al. (1997). In our study, there was no significant alteration in sensory thresholds in SR141716a treated animals when compared to solvent treated controls. The increase of hyperalgesia or allodynia seen after administration of the receptor antagonist has been hypothesized to suggest the presence of an endogenous tone that is active during neuropathy—viz. locally synthesized endocannabinoids act at a low level on the receptors in response to peripheral nerve injury, causing a degree of attenuation of the hyperalgesia or allodynia. Thus, by blocking the action of these endogenous ligands by administration of the specific antagonist, the hyperalgesia or allodynia will be increased. The current data on an endogenous cannabinoid tone in pain models is controversial. Several studies have shown the presence of a SR141716a-associated enhancement of signs of pain or nociception in a variety of models, including the formalin test for inflammation (Calignano et al., 1998; Strangman et al., 1998). This could be interpreted as supporting the hypothesis that an endogenous tone exists in inflammatory pain, but not in neuropathic pain. If this is the case, then the inflammatory component of the CCI model could account for the difference seen between this and the findings of Herzberg et al. (1997). However, other studies of endogenous tone in inflammatory models have found no evidence supporting this concept, either in an inflammatory model (Beaulieu et al., 2000) or when administered to uninjured animals (Rinaldi-Carmona et al., 1995). The use of SR141716a as an experimental tool to reveal endogenous tone could be further confused by the findings of several groups that SR141716a is actually an inverse agonist at the CB1 receptor (Landsman et al., 1997; Pan et al., 1998). Further studies in CB1−/− knockout mice did not support the concept of an endogenous tone (Ledent et al., 1999). It also remains to be determined whether the levels of endogenous cannabinoids found in the spinal cord and dorsal root ganglia are sufficient to activate the receptors. Despite the controversial nature of the endogenous tone, our experiments seem to contradict its existence and agree with the study by Beaulieu et al. (2000) that there is a weak analgesic action at about 60min post-administration, possibly due to SR141716a being able to show weak agonist activity under certain conditions.
This study has demonstrated the existence of CB1 receptor, but not CB2 receptor, mediated analgesia in an animal model of neuropathic pain. In light of the current therapeutic need for neuropathic pain treatments, this study provides evidence for the potential of cannabinoids, or inhibitors of degradation of endocannabinoids, as lead compounds for development of new therapies. The development of cannabinoid receptor specific agonists and antagonists will provide essential new tools for investigating the mechanism of this analgesic action.
Source, Graphs and Figures: The synthetic cannabinoid WIN55,212-2 attenuates hyperalgesia and allodynia in a rat model of neuropathic pain
