Pain has a negative impact on the quality of life in cancer patients. Almost half of all patients with cancer experience moderate to severe pain, which is even more severe in patients with metastasis or those in advanced stages. Recent clinical trials have demonstrated that cannabinoids may have significant positive effects in refractory chronic and cancer pain.1 The cannabinoids are thought to exert most of their effects by binding to G protein–coupled cannabinoid receptors, which include 2 cloned metabotropic receptors: cannabinoid (CB)1 and CB2. Numerous studies indicated that these receptors are distributed widely across many key loci in pain-modulating pathways, including the peripheral and central terminals of primary afferents, second-order spinal dorsal horn neurons.2 Although many studies have demonstrated that activation of each of the cannabinoid receptors at several of these diverse loci can reduce nociceptive transmission, the contributions of CB1 and CB2 receptors, respectively, and of cannabinoids acting at peripheral or central sites to the global analgesic effects of systemic cannabinoids, remain ambiguous. CB2 receptors are considered peripheral and central receptors, and they have been found in distinct areas of the central nervous system such as the spinal cord, dorsal root ganglia, and microglia.3 Several studies have shown that systemic, local, peripheral, or intrathecal administration of CB2 receptor agonists produces antinociception without overt behavioral effects in neuropathic, inflammatory, postoperative, and acute pain models.4–8 Previous studies found that tumor-evoked pain was dose dependently attenuated by administration of the cannabinoid receptor agonist WIN-55212-2, CP55940 in a murine model of bone cancer pain.9,10 In particular, findings from a propensity score analysis of data obtained from advanced cancer patients suggested that nabilone administration improved treatment of pain and was associated with less overall use of other drugs such as opioids and nonsteroidal antiinflammatory drugs.11 Based on the animal and clinical trials mentioned above, we hypothesized that a cannabinoid receptor agonist might be a novel therapy for cancer pain. Taking into consideration the side effects of a CB1 receptor agonist, such as hypothermia and catalepsy (which limits their clinical application), we chose a CB2 receptor agonist to investigate its effect in cancer pain.
Previous studies have shown that cannabinoid receptor activation protects neurons from N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in vitro.12 Richardson et al.13 found that hypoactivity of the spinal cannabinoid system results in NMDA-dependent hyperalgesia. Hampson et al.14 also found that the endogenous cannabinoid anandamide can modulate the activity of NMDA. NMDA receptors (NMDARs) are a major class of ionotropic glutamate receptors that consist of heteromeric complexes composed primarily of NR1, NR2A–D, and NR3A–B subunits.15 Considerable evidence suggests that the NR2B-contaning NMDAR has a critical role in spinal nociceptive processing. In addition, the roles of NR2B receptor activation in the development of persistent neuropathic pain and cancer pain have been demonstrated.16,17 Our previous studies also demonstrated that bone cancer results in significant up-regulation of spinal NR2B expression.18 Guo et al.19 have found that signal transduction upstream to NR2B tyrosine phosphorylation involves G protein– coupled receptors. In this study, we examined the effects of a CB2 receptor agonist on NR2B mRNA expression. These experiments were performed in the spinal cord, which expresses CB2 receptors and NMDAR. We tested the hypothesis that activation of CB2 receptors inhibits NMDA expression in the spinal cord to relieve cancer pain.
All experiments complied with the guidelines set forth by the International Association for the Study of Pain for the use of experimental animals and were approved by the Institutional Animal Care and Use Committee at the medical college of Nanjing University. For all the experiments, the observer was blinded to the treatment administered. Adult (5–6 weeks old) male C3H/Hej mice (Vital River Experimental Animal Corporation, Beijing, China) weighing 20 to 25 g were used in all experiments. Mice were housed 4 per box, had free access to mouse chow and water, and were maintained on a 12-hour light/dark schedule.
Cell Culture and Implantation
NCTC 2472 fibrosarcoma cells (American Type Culture Collection) were maintained as described previously.20 Cells were kept under 5% CO2 at 37°C and passaged twice a week using nonenzymatic cell dissociation solution. Briefly, fibrosarcoma cells were grown to confluency in 75 cm2 flasks in NCTC 135 medium (pH 7.4) containing 10% horse serum and prepared for implantation by creating a cell suspension with trypsin. Fibrosarcoma cells were counted using a hemocytometer, pelleted, and resuspended in α-MEM (minimal essential medium, α modification) for implantation. Fibrosarcoma cells were implanted into the hindpaw as described by Schwei et al.21 Mice were briefly anesthetized with pentobarbital (50 mg/kg), and 20 μL α-MEM containing no or 2 × 105 NCTC2472 cells was injected into the femoral bone of the right hindpaw using a 25-μL syringe. No mice showed signs of motor dysfunction after implantation of fibrosarcoma cells. Osteolytic NCTC2472 cells were implanted into the intramedullary space of right femurs of mice to induce ongoing bone cancer–related pain behaviors in tumor groups, whereas sham group mice were injected only with α-MEM. Afterward, the injection hole was sealed with dental amalgam, followed by copious irrigation with normal saline. The wound was then closed. Control group mice received no operation.
The CB2 receptor agonist JWH015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone] (CB2 Ki = 13.8 nM, 28-fold selectivity versus CB1) (catalog #J4252; Sigma, St. Louis, MO) and the CB2 receptor antagonist AM630 [6-iodo-2-methyl-1-(2-(4-morpholinyl) ethyl)-1H-indol-3-yl) (4-methoxyphenyl)methanone] (Sigma) were dissolved in 20% dimethyl sulfoxide (DMSO) (in normal saline) and intrathecally administered in a volume of 5 μL. Drugs were administered to conscious animals and were injected into the subarachnoid space through the intervertebral foramen between L5 and L6 according to the method described by Hylden and Wilcox.22 The dose of JWH015 was 0.5 μg, 1 μg, and 2 μg, respectively, and AM630 was 2 μg. The antagonist AM630 was injected 30 minutes before the agonist.
Pain Behaviors Over Time
Seventy-two mice were randomly placed into 7 groups: sham mice (n = 6), control mice (n = 6), and tumor-bearing mice, which included 5 groups, V, J1, J2, J3, and J4 (n = 12 each group). All mice were tested for pain-related behaviors during a 2-week period: day 0 before operation and days 3, 5, 7, 10, and 14 after operation. At day 14, tumor-bearing mice received treatment with JWH015 0.5 μg (J1), JWH015 1 μg (J2), JWH015 2 μg (J3), AM630 2 μg plus JWH015 2 μg (J4), and DMSO (V) after pain-related behavioral tests, respectively. Pain-related behaviors were then measured at 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours after administration. The data measured before administration were regarded as baseline data. Before each test, mice were allowed to acclimatize for at least 30 minutes.
Tactile Allodynia Test
Tactile allodynia was assessed using von Frey filaments (Stoelting, Wood Dale, IL) applied to the right hindpaw. Mice were placed on a wire mesh platform, covered with a glass container (10 × 10 × 15 cm). Each von Frey filament was applied to the plantar surface of the hindpaw at random locations avoiding the heel, toes, and pads. For each trial, the filament was applied for 6 to 8 seconds with an interstimuli interval of approximately 15 seconds. To determine paw withdrawal mechanical thresholds (PWMT), testing was initiated with the moderate von Frey filament. If the mouse began to walk while being probed, the same filament was reapplied for another 6 seconds. If the mouse did not respond to the touch of the filament, testing proceeded with the next higher filament until hindlimb movement occurred or cutoff (highest filament used was 2.0 g) was reached. The bending force of von Frey filaments ranged from 0.16 g to 2.0 g (0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g) using the up-down method. Only vigorous paw withdrawals were counted. Subsequent stimuli were presented based on the response to the previous stimulus. Five responses, starting with the negative response immediately before the first paw withdrawal, were recorded. Each mouse was tested 5 times per stimulus strength. The lowest von Frey filaments that had ≥3 positive responses were regarded as PWMT. The resulting pattern was tabulated. Tactile allodynia was defined as a decrease in paw withdrawal threshold.
Thermal Hyperalgesia Test
To determine paw withdrawal thermal thresholds, a method to measure cutaneous hyperalgesia to thermal stimulation in unrestrained animals is described by Hargreaves et al.23 Mice were placed in individual transparent plexiglas compartments (10 × 10 × 5 cm) on a 3-mm-thick glass floor. The testing paradigm used an automated detection of the behavioral end point; repeated testing does not contribute to the development of the observed hyperalgesia. A radiant thermal stimulator (BME410A; Institute of Biological Medicine, Academy of Medical Science, China) was focused onto the plantar surface of the hindpaw through the glass floor. The nociceptive end points in the radiant heat test were the characteristic lifting or licking of the hindpaw, and the time to the end points was recorded as the paw withdrawal thermal latency (PWTL). To prevent tissue damage, the cutoff time was 25 seconds. Each mouse was measured 5 times and the interstimuli interval was approximately 10 minutes. Mean PWTL was calculated after discarding the highest and lowest values.
Reverse Transcription–Polymerase Chain Reaction
At 12 hours and 72 hours after intrathecal administration, NR2B mRNA levels were measured in tissues from the spinal cords of tumor mice receiving JWH015, AM630, and vehicle using reverse transcription–polymerase chain reaction (RT-PCR) analyses.
Six mice in each group were killed to collect specimens for RT-PCR analyses just after behavioral tests at the corresponding time point under pentobarbital sodium anesthesia, and the L3-5 lumbar spinal cord segments were immediately frozen in liquid nitrogen and stored at −80°C. The specimens for RT-PCR were collected from the same mice just after behavioral tests, to more precisely explain the correlation between the changes in mRNA level and the changes in pain behaviors. Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA), and a 5-μg portion of it was used for cDNA synthesis with M-MLV reverse transcriptase (Promega, Madison, WI). The cDNA was used as a template for PCR amplification with Taq DNA polymerase (Takara, Dalian, China) and NR2B primers (upstream primer, 5′-GCATTCCTACGACACCTTCG-3′, and downstream primer, 5′-GACCACCA-CTGGCTTATTGG-3′) and β-actin primers (upstream primer, 5′-GAGACCTTCAACACCCCAGC-3′, and downstream primer, 5′-ATGTCACGCACGATTTCCC-3′). PCR amplification was performed for 5 minutes at 94°C, 35 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C (for NR2B) or 1 minute at 94°C, 1 minute at 58°C, and 1 minute at 72°C (for β-actin), then 7 minutes at 72°C. Five microliters of amplified cDNA was electrophoresed on 2% agarose gel and stained with ethidium bromide. The intensity of each PCR band was analyzed using a gel imaging analytical system (GDS-8000; UVP, Upland, CA).
All data are expressed as mean ± SD. Animals were randomly assigned to different treatment groups. In experiment 1, to determine whether the implantation of tumor cells induced bone cancer pain, 6 of the tumor-bearing mice were randomly chosen. To compare the data before operation with days 3, 5, 7, 10, and 14 after operation and to assess the differences among groups C, S, and T, repeated-measures analysis of variance (ANOVA) with least significant difference (LSD) post hoc test was used. In experiments 2 and 3, each dependent measure was compared between treatment groups (dose of JWH015 or vehicle) and across test times (predrug, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours) using repeated-measures ANOVA with the LSD post hoc test. One-way ANOVA with the LSD post hoc test was used to determine differences in the expression of NR2B mRNA among treatment groups. A P value <0.05 was considered statistically significant.
Experiment 1: Tumor-Evoked Pain Behavior
Implantation of fibrosarcoma cells into the femoral bone of the right hindpaw of mice produced tactile allodynia and thermal hyperalgesia as indicated by a decrease in paw withdrawal threshold (Fig. 1). Before implantation of fibrosarcoma cells (at day 0), the threshold force for evoking a paw withdrawal did not differ among groups C, S, and T. Paw withdrawal thresholds began to decrease from baseline values in groups T and S 3 days after implantation (P < 0.001) and recovered to the level after operation at day 5 (Fig. 1). PWMT and PWTL remained lower than baseline values and lower than those values of group S in all postimplantation tests after day 7 (Fig. 1A) and day 10 (P < 0.001) (Fig. 1B), respectively. Hence, implantation of fibrosarcoma cells into the femoral bone produced a decrease in PWMT and PWTL. Thus, these 2 criteria for hyperalgesia changed in the same way during the development of tumor-evoked pain behaviors. In addition, no significant differences in PWTL and PWMT were observed in group C at all test points and in group S between days 5, 7, 10, 14, and day 0.
Experiment 2: JWH015 Attenuated Tumor-Evoked Pain Behavior
Intrathecal administration of JWH015 attenuated tumor-evoked pain behavior. At day 14 after the implantation of fibrosarcoma cells, there were no differences in paw withdrawal thresholds among groups (predrug) (Fig. 2). The CB2 receptor agonist JWH015 reversed tumor-induced hypersensitivity in a dose-related manner compared with group V. The lowest dose reversed tumor-induced hypersensitivity at 6, 12, and 24 hours after treatment compared with the predrug withdrawal threshold, and the same difference was found when compared with the intrathecal vehicle group. The highest dose significantly reversed tumor-induced PWMT compared with predrug withdrawal thresholds from 6 hours to 48 hours, especially at 12 hours and 24 hours (P < 0.001). The highest dose of JWH015 also reversed PWTL at all the tested times (P < 0.001), as did group J3 when compared with the vehicle group at all the tested time points after treatment. However, the antihyperalgesia effect of intrathecal JWH015 was prevented by previous intrathecal injection of the CB2 receptor antagonist AM630. Intrathecal administration of JWH015 also attenuated tumor-evoked pain in a time-dependent manner. The largest increase in PWMT and PWTL occurred at 12 hours (P < 0.001) and gradually returned to predrug values at 72 hours.
Experiment 3: mRNA Levels of NR2B in Mouse Spinal Cords After Intrathecal Treatment with JWH015
Results from the behavioral tests suggested that pain relief occurred in mice administered intrathecal JWH015. In our previous study by RT-PCR analysis, we found that NR2B mRNA levels of L3-5 lumbar spinal cord segments in tumor-bearing mice increased significantly at day 14 after operation. To quantify the expression of NR2B in the dorsal horn during the maintenance of antihyperalgesia induced by JWH015, RT-PCR analyses were performed. The expression of NR2B in mice receiving JWH015 (groups J1, J2, and J3) was significantly down-regulated compared with mice receiving DMSO (group V) (P < 0.01). Interestingly, NR2B mRNA in group J3 was significantly decreased in the spinal cord (P < 0.001). Conversely, pretreatment with AM630 (J4) increased the level of NR2B in the spinal dorsal horn compared with mice receiving JWH015 (J3) (P < 0.01) (Fig. 3A). At 72 hours, NR2B mRNA in group J3 was still down-regulated compared with group V control (P < 0.001) (Fig. 3B). No significant change was found in group V between 12 hours and 72 hours (P > 0.05).
Chronic pain generally develops as a consequence of changes in nociceptive processing at the level of primary nociceptive afferents and central neurons. In recent years, cannabinoids have emerged as attractive alternatives or supplements to therapy for chronic pain states.24 Romero-Sandoval and Eisenach25 found that central CB2 receptor activation by intrathecal JWH015 relieved paw incision–induced hypersensitivity and that the efficacy of intrathecal injection of the CB2 receptor agonist was not affected by antagonist injection in the hypersensitive paw. They also found that the maximal effective intrathecal dose of the agonist was inactive when administered systemically, which suggested that such central sites are important. Although numerous studies have shown that cannabinoid agonists are antinociceptive in various kinds of pain models and attenuate hyperalgesia, less is known about the central actions of CB2 agonists in cancer pain states.
Animal models of cancer pain have been developed to examine the mechanisms that underlie tumor-evoked pain and hyperalgesia. Using models in which osteolytic fibrosarcoma cells are implanted into the humerus, femur, tibia, or calcaneus bone, investigators have begun to elucidate the pathophysiological processes by which cancer produces pain.26 These models have also been used to evaluate novel approaches for treating cancer pain.27
In this study, we used an animal model of bone cancer metastases by injecting sarcoma cells that result in behavioral signs of spontaneous and evoked pain. Similar to what was reported by Schwei et al.,21 we found that animals developed severe bone loss during 14 days of observation after inoculation with the sarcoma cells.
Using a model in which fibrosarcoma cells are implanted into and around the calcaneus bone,26 Cain et al.28 found that approximately 35% of C nociceptors exhibited spontaneous activity (0.2–3.4 Hz) and a decrease in heat threshold. Although C nociceptors exhibited ongoing activity and sensitization to heat stimuli, several studies suggested that central sensitization also contributed to cancer pain.29 For example, neurochemical changes in the dorsal horn such as massive astrocyte hypertrophy, c-Fos expression, and internalization of the substance P receptor that occur during tumor growth are consistent with central sensitization.21,30,31 In the rat model of cancer pain, direct evidence for central sensitization has been shown in electrophysiological studies.32 At day 7, 23% of neurons were characterized as wide dynamic range (WDR). At day 9, 30% of neurons were characterized as WDR and these showed a trend toward an increased, but not statistically significant, response to noxious electrical, thermal, and mechanical stimuli. By day 11, 44% of neurons were characterized as WDR, and these showed significantly increased responses to all stimuli modalities, akin to days 15 to 17. Thus, in this model, the alterations in neuronal responses are a viable substrate for pharmacological studies on suprathreshold stimuli. In addition, the clear temporal link between behavioral hyperalgesia and altered neuronal responses may provide an opportunity to investigate changes in dorsal horn gene expression in hyperalgesia.
As shown in a previous study, various degrees of bone destruction were observed in animals that had received injection of 2472 sarcoma cells into the femur. The observed extensive bone destruction was similar to that noted in clinical settings and mainly occurred in the tibia, femur, fibula, jaw, forelimbs, pelvis, and sometimes in scapula and spine as detected by whole-body microcomputed tomographic imaging. Histological examination found that the tumors occupied the entire bone marrow canal and their growth resulted in severe destruction of both cortical and trabecular bone.33
As reported in our previous study,18 we found that the thresholds of tactile allodynia and thermal hyperalgesia were both decreased in tumor-bearing and sham mice at day 3, and recovered to the baseline levels before operation at day 5. These data suggested that the change in pain behaviors at day 3 was attributable to arthrotomy, and the injury quickly healed. The tactile allodynia and thermal hyperalgesia thresholds of tumor-bearing mice decreased with time. At day 14, we observed significant bone cancer pain–related behaviors. In our previous study, we found that NR2B mRNA was significantly increased in tumor-bearing mice on day 14, compared with mice implanted with α-MEM. The significant bone cancer pain–related behaviors that were accompanied by the increased levels of NR2B suggest that NR2B in the spinal cord was involved in the development of bone cancer pain. JWH015 was used to explore the role of NR2B in the development of further bone cancer pain in the later experiment. Therefore, we investigated the effect of JWH015 on bone cancer pain and detected the levels of NR2B in the spinal cord.
Spinal administration of nonselective cannabinoid receptor agonists reduces hypersensitivity in several pain models.34 In this study, we showed a dose-dependent antihypersensitivity effect after intrathecal JWH015, a CB2 receptor–preferring agonist. However, the antihyperalgesia effect of JWH015 was blocked by the CB2 receptor antagonist AM630. The dose-response relationship for the antihyperalgesia effect of JWH015 at 12 hours after administration showed that JWH015 at 0.5, 1, and 2 μg produced an increase in PWMT and PWTL compared with that produced by vehicle. At the highest dose, tumor-induced pain behavior was significantly reversed compared with predrug administration, and when compared with the vehicle group at all the time points tested. The antihyperalgesia effect of JWH015 achieved the maximum at 12 hours; however, the pain behavior still was not totally reversed.
Convincing evidence demonstrated that the development of spinal hyperexcitability and persistent pain involved activation of NMDARs.18 Functional NMDAR consisted of NR1 subunits and one or more subunits of NR2. Among the signal transduction pathways for NMDAR activation involving protein phosphorylation, tyrosine phosphorylation of the NR2 subunits had a key role.35 Furthermore, the signal transduction upstream to NR2B tyrosine phosphorylation involved G protein–coupled receptors.19 The present study further demonstrated that the expression of NR2B mRNA was significantly up-regulated in tumor-bearing mice and that intrathecal administration of JWH015 attenuated the expression. We found additional evidence that signal transduction upstream to NR2B involves G protein–coupled receptors, which include cannabinoid receptors that can be bound with cannabinoid agonists such as JWH015.
A study by Zhuo36 showed that central signaling pathways contribute to chronic pain. Glutamate is the major fast excitatory transmitter between input fibers and pyramidal cells. Peripheral injury such as tissue inflammation or nerve injury triggers a burst of abnormal activity and subsequently activates postsynaptic NMDARs on cingulate pyramidal cells located in layer II–III. Activation of the NMDAR triggers calcium influx. Most NMDARs are the combination of NR1-NR2A, NR1-NR2B with possible minor component of currents made of NR1-NR2A-NR2B. The increase of postsynaptic Ca2+ leads to activation of Ca2+-calmodulin–dependent pathways. Among them, Ca2+ and Ca2+-calmodulin activated adenylyl cyclase, and this activation leads to the generation of the key second-messenger cyclic adenosine monophosphate (cAMP). Subsequently, cAMP activated protein kinase A. Protein kinase A then translocated to the nucleus and phosphorylated CREB. The NR2B gene is linked to a CREB binding domain that might couple the increase of intracellular calcium with the increase of NR2B expression. As a result, postsynaptic synthesis of NR2B was increased, along with the endogenous motor protein KIF17, and these new NR2B subunits were added to postsynaptic NMDARs. Such positive feedback control might further enhance neuronal excitability and contribute to chronic pain.
The CB2 receptor is coupled to Gi/o proteins and thereby negatively couples to adenylyl cyclase and the cAMP pathway in various types of cells.37 Activation of CB2 receptors by cannabinoids leads to inhibition of adenylyl cyclase, which can affect the generation of cAMP, which leads to the inhibition of CREB phosphorylation, and thereby down-regulating the expression of NR2B.
JWH015 was administered intrathecally, because intrathecal injection was most frequently performed at the time of surgery. In our research, we found that intrathecal administration of the CB2 receptor agonist JWH015 significantly reduced tumor-induced NR2B activation, which was consistent with its behavioral effects. Whether JWH015 should be administered at earlier times, before such activation, is uncertain. It is conceivable that JWH015 could act on spinal cord neurons or afferents because CB2 receptors are expressed in neurons.38 This suggests that NR2B inhibition by JWH015 would be enough to induce a reduction of spinal neural activity and hypersensitivity. Patients with cancer pain might require treatments that provide analgesia for weeks or longer. Thus, whether repeated administration of JWH015 for more days would continue to attenuate tumor-evoked pain needs further research. Whether repeated administration of cannabinoids, including JWH015, would produce tolerance to their antihyperalgesia effects in animal models of cancer pain needs to be studied. We showed that central CB2 receptor activation relieved bone cancer–induced hyperalgesia. The antihyperalgesia of spinal CB2 receptor stimulation was consistent with significant inhibition of NR2B mRNA expression, suggesting a primary action on NR2B. The use of a CB2 receptor agonist could be a novel option for treatment of cancer pain.
1. Farquhar-Smith WP. Do cannabinoids have a role in cancer pain management? Curr Opin Support Palliat Care 2009;3:7–13
2. Pacher P, Barkai S, Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev 2006;58:389–462
3. Pertwee RG. Cannabinoid receptors and pain. Prog Neurobiol 2001;63:569–611
4. Malan TP Jr, Ibrahim MM, Deng H, Liu Q, Mata HP, Vanderah T, Porreca F, Makriyannis A. CB2 receptor cannabinoid receptor-mediated peripheral antinociception. Pain 2001;93:239–45
5. Valenzano KJ, Tafesse L, Lee G, Harrison JE, Boulet JM, Gottshall SL, Mark L, Pearson MS, Miller W, Shan S, Rabadi L, Rotshteyn Y, Chaffer SM, Turchin PI, Elsemore DA, Toth M, Koetzner L, Whiteside GT. Pharmacological and pharmacokinetic characterization of the cannabinoid receptor 2 agonist, GW405833, utilizing rodent models of acute and chronic pain, anxiety, ataxia and catalepsy. Neuropharmacology 2005;48:658–72
6. Malan TP Jr, Ibrahim MM, Lai J, Vanderah TW, Makriyannis A, Porreca F. CB2 receptor cannabinoid receptor agonists: pain relief without psychoactive effects? Curr Opin Pharmacol 2003;3:62–7
7. Quartilho A, Mata HP, Ibrahim MM, Vanderah TW, Porreca F, Makriyannis A, Malan TP Jr. Inhibition of inflammatory hyperalgesia by activation of peripheral CB2 receptor cannabinoid receptors. Anesthesiology 2003;99:955–60
8. Romero-Sandoval A, Nutile-McMenemy N, DeLeo JA. Spinal microglial and perivascular cell cannabinoid receptor type 2 activation reduces behavioral hypersensitivity without tolerance after peripheral nerve injury. Anesthesiology 2008;108:722–34
9. Potenzieri C, Harding-Rose C, Simone DA. The cannabinoid receptor agonist, WIN 55, 212-2, attenuates tumor-evoked hyperalgesia through peripheral mechanisms. Brain Res 2008;1215:69–75
10. Hamamoto DT, Giridharagopalan S, Simone DA. Acute and chronic administration of the cannabinoid receptor agonist CP 55,940 attenuates tumor-evoked hyperalgesia. Eur J Pharmacol 2007;558:73–87
11. Pisanti S, Malfitano AM, Grimaldi C, Santoro A, Gazzerro P, Laezza C, Bifulco M. Use of cannabinoid receptor agonists in cancer therapy as palliative and curative agents. Best Pract Res Clin Endocrinol Metab 2009;23:117–31
12. Van Sickle MD, Duncan M, Kingsley PJ, Mouihate A, Urbani P, Mackie K, Stella N, Makriyannis A, Piomelli D, Davison JS, Marnett LJ, Di Marzo V, Pittman QJ, Patel KD, Sharkey KA. Identification and functional characterization of brainstem cannabinoid CB2 receptors. Science 2005;310:329–32
13. Richardson JD, Aanonsen L, Hargreaves KM. Hypoactivity of the spinal cannabinoid system results in NMDA-dependent hyperalgesia. J Neurosci 1998;18:451–7
14. Hampson AJ, Bornheim LM, Scanziani M, Yost CS, Gray AT, Hansen BM, Leonoudakis DJ, Bickler PE. Dual effect of anandamide on NMDA receptor-mediated response and neurotransmission. J Neurochem 1998;70:671–6
15. Cull-Candy S, Brickley S, Farrant M. NMDA receptor subunits: diversity, development and disease. Curr Opin Neurobiol 2001;11:327–35
16. Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science 2000;288:1765–9
17. Ma ZL, Zhang W, Gu XP, Yang WS, Zeng YM. Effects of intrathecal injection of prednisolone acetate on expression of NR2B subunit and nNOS in spinal cord of rats after chronic compression of dorsal root ganglia. Ann Clin Lab Sci 2007;37:349–55
18. Gu XP, Zhang J, Ma ZL, Wang JH, Zhou XF, Jin YQ, Xia XP, Gao Q, Mei FM. The role of N-methyl-D-aspartate receptor subunit NR2B in spinal cord in cancer pain. Eur J Pain 2010;14:496–502
19. Guo W, Zou S, Guan Y, Ikeda T, Tal M, Dubner R, Ren K. Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord during the development and maintenance of inflammatory hyperalgesia. J Neurosci 2002;22:6208–17
20. Compton DR, Rice KC, De Costa BR, Razdan RK, Melvin LS, Johnson MR, Martin BR. Cannabinoid structure-activity relationships: correlation of receptor binding and in vivo activities. J Pharmacol Exp Ther 1993;265:218–26
21. Schwei MJ, Honore P, Rogers SD, Salak-Johnson JL, Finke MP, Ramnaraine ML, Clohisy DR, Mantyh PW. Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci 1999;19:10886–97
22. Hylden JL, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980;67:313–6
23. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988;32:77–88
24. Ashton JC, Milligan ED. Cannabinoids for the treatment of neuropathic pain: clinical evidence. Curr Opin Investig Drugs 2008;9:65–75
25. Romero-Sandoval A, Eisenach JC. Spinal cannabinoid receptor type 2 activation reduces hypersensitivity and spinal cord glial activation after paw incision. Anesthesiology 2007;106:787–94
26. Wacnik PW, Eikmeier LJ, Ruggles TR, Ramnaraine ML, Walcheck BK, Beitz AJ, Wilcox GL. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci 2001;21:9355–66
27. Sevcik MA, Ghilardi JR, Peters CM, Lindsay TH, Halvorson KG, Jonas BM, Kubota K, Kuskowski MA, Boustany L, Shelton DL, Mantyh PW. Anti-NGF therapy profoundly reduces bone cancer pain and the accompanying increase in markers of peripheral and central sensitization. Pain 2005;115:128–41
28. Cain DM, Wacnik PW, Turner M, Wendelschafer-Crabb G, Kennedy WR, Wilcox GL, Simone DA. Functional interactions between tumor and peripheral nerve: changes in excitability and morphology of primary afferent fibers in a murine model of cancer pain. J Neurosci 2001;21:9367–76
29. Coderre TJ, Katz J, Vaccarino AL, Melzack R. Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 1993;52:259–85
30. Medhurst SJ, Walker K, Bowes M, Kidd BL, Glatt M, Muller M, Hattenberger M, Vaxelaire J, O'Reilly T, Wotherspoon G, Winter J, Green J, Urban L. A rat model of bone cancer pain. Pain 2002;96:129–40
31. Zhang RX, Liu B, Wang L, Ren K, Qiao JT, Berman BM, Lao L. Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain 2005;118:125–36
32. Donovan-Rodriguez T, Dickinson AH, Urch CE. Superficial dorsal horn neuronal responses and the emergence of behavioural hyperalgesia in a rat model of cancer-induced bone pain. Neurosci Lett 2004;360:29–32
33. Strube A, Stepina E, Mumberg D, Scholz A, Hauff P, Kakonen SM. Characterization of a new renal cell carcinoma bone metastasis mouse model. Clin Exp Metastasis 2010;27:319–30
34. Scott DA, Wright CE, Angus JA. Evidence that CB-1 and CB-2 cannabinoid receptors mediate antinociception in neuropathic pain in the rat. Pain 2004;109:124–31
35. Xiong ZG, Pelkey KA, Lu WY, Lu YM, Roder JC, MacDonald JF, Salter MW. Src potentiation of NMDA receptors in hippocampal and spinal neurons is not mediated by reducing zinc inhibition. J. Neurosci 1999;19:1–6
36. Zhuo M. Plasticity of NMDA receptor NR2B subunit in memory and chronic pain. Mol Brain 2009;2:4
37. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, Felder CC, Herkenham M, Mackie K, Martin BR, Mechoulam R, Pertwee RG. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev 2002;54:161–202
38. Wotherspoon G, Fox A, McIntyre P, Colley S, Bevan S, Winter J. Peripheral nerve injury induces cannabinoid receptor 2 protein expression in rat sensory neurons. Neuroscience 2005;135:235–45