On days 1 and 2, morphine administration produced a significant analgesia compared with the vehicle + saline treatment based on mechanical hyperalgesia and thermal nociception (all P < 0.0001; Figs. 1 and 2). The level of morphine analgesia decreased on the consecutive days of chronic morphine treatment compared with day 1 (in von Frey test, day 3: P < 0.001; days 4–8, all P < 0.0001; in hot plate test, days 6–8: all P < 0.0001). Although on day 8 morphine consistently produced significant antimechanical hyperalgesia compared with vehicle + saline treatment (8.93 ± 1.43 vs 4.00 ± 1.70 g, P < 0.0001), this effect of morphine decreased significantly compared with its efficacy on day 1 (57.48 ± 5.00 vs 8.93 ± 1.43 g, P < 0.0001; Fig. 1). Thermal paw withdrawal latency on day 8 of chronic morphine administration did not differ statistically from that of vehicle + saline treatment (10.96 ± 3.18 vs 9.02 ± 2.22 seconds, P = 0.1328; Fig. 2), indicating that the rats receiving vehicle + morphine were tolerant to its analgesic effect of tumor-evoked pain.
The combination of AM1241 and morphine (AM1241 + morphine group) strengthened analgesia of morphine for mechanical stimulus on days 7 and 8 (P < 0.0001) compared with morphine administration alone (Fig. 1). Although mechanical withdrawal thresholds of AM1241-pretreated rats (AM1241 + morphine group) were reduced substantially from day 4 to day 8 compared with day 1 (days 4–8: all P < 0.0001), their analgesic response remained significantly higher than the animals injected with morphine alone on days 7 and 8 (AM1241 + morphine group versus vehicle + morphine group, P < 0.0001). The selective CB2 receptor antagonist AM630 reversed the effects of AM1241 on morphine analgesia and tolerance and displayed no difference between the AM630 + AM1241 + morphine group and vehicle + morphine group (all P > 0.1249). The analgesic response of repeated administration of AM1241 alone (AM1241 + saline group, 0.07 μg, IT) did not differ from the vehicle + saline group. Detailed information of multiple comparisons for mechanical withdrawal threshold (mean difference, 99% confidence interval, and P value) can be found in the Supplemental Digital Content (Supplemental Tables 5 and 6, http://links.lww.com/AA/B343).
The combination of AM1241 and morphine (AM1241 + morphine group) strengthened the analgesia of morphine for thermal nociception on days 6, 7, and 8 (all P < 0.0001) compared with administration morphine alone (Fig. 2). Although the paw withdrawal latency of AM1241-pretreated rats (AM1241 + morphine group) decreased on day 8 compared with day 1 (P < 0.0001), the analgesic response to morphine on day 8 remained significantly greater (21.09 ± 2.61 vs 10.96 ± 3.18 seconds, P < 0.0001) in the AM1241-pretreated rats compared with the morphine alone–injected group, which displayed tolerance to morphine analgesia. The latency values were not statistically different from vehicle + saline-treated rats (10.96 ± 3.18 vs 9.02 ± 2.22 seconds; P = 0.1328). No difference was observed in the analgesic response of the AM630 + AM1241 + morphine group compared with the morphine-injected group in the hot plate test. The analgesic response of repeated administration of AM1241 alone (AM1241 + saline group, 0.07 μg, IT) did not differ from the vehicle + saline group. Detailed information of multiple comparisons for thermal withdrawal latency (mean difference, 99% confidence interval, and P value) can be found in the Supplemental Digital Content (Supplemental Tables 7 and 8, http://links.lww.com/AA/B343).
Western blot analysis revealed a band at approximately 55 kDa corresponding to the MOR in samples obtained from the spinal cord (Fig. 3, A and C) and DRG (Fig. 3, B and D). The vehicle + saline group served as the control group in Figure 3. AM1241-pretreated rats displayed a significant increase in MOR expression in the spinal cord (P < 0.0001) and DRG (P < 0.0001) compared with morphine administration alone. The effect of AM1241 on morphine-induced MOR protein expression was abolished by the selective CB2 receptor antagonist AM630, and there was no difference between the AM630 + AM1241 + morphine group and vehicle + morphine group (P = 0.6541 in spinal cord; P = 0.2427 in DRG). Detailed information of multiple comparisons for MOR protein expression (mean difference, 99% confidence interval, and P value) can be found in the Supplemental Digital Content (Supplemental Table 9, http://links.lww.com/AA/B343).
In the present study, we found that coadministration of a nonanalgetic dose of the CB2 receptor agonist AM1241 with morphine reduced tolerance to the analgesic effects of morphine, increased MOR protein expression in the spinal cord and DRG, and mRNA expression in the spinal cord of tumor-bearing rats. Our findings strongly suggest that a CB2 receptor agonist may play a positive role in attenuating morphine tolerance in cancer pain treatment.
Several studies have shown that coadministration of a low- or nonanalgetic-dose CB circumvented antinociceptive tolerance to morphine in normal rats and animals models of neuropathic pain.6,19,20 However, the effect of a low-dose CB2 receptor agonist on morphine tolerance in cancer pain has not been reported. Our findings indicated that IT administration of a nonanalgetic dose of the CB2 receptor agonist AM1241 increased the analgesic effects of morphine while alleviating morphine tolerance during the treatment of cancer pain. There are several reasons for these results that should be considered. First, the selective CB2 receptor agonist attenuates morphine tolerance via upregulating MOR expression in cancer pain. However, it is possible that the CB2 receptor agonist alleviates morphine tolerance by an interaction between opioid and CB receptors and by reduction of glial and mitogen-activated protein kinase (MAPK) activation.7,24
MOR expression was different in the dorsal horn and DRG in various animal models.25–27 MOR expression was decreased in the ipsilateral DRG based on a neuropathic and bone cancer pain model,25,26 thereby reducing the effects of MOR agonists.28,29 Here, we observed that coadministration of AM1241 with morphine led to an increase in MOR protein expression both in the spinal cord and DRG after chronic morphine exposure in this Walker 256 tumor-bearing animal model. Furthermore, we detected the level of MOR mRNA in the spinal cord and DRG to judge whether upregulation of MOR expression happened in transcription. As we expected, the level of MOR mRNA was increased in the spinal cord of AM1241-pretreated rats, but not in the DRG. In this cancer pain model, the reason for the different effects of AM1241 on mRNA expression between the spinal cord and DRG was not clear. The present results suggest that CB2 receptor agonists could attenuate morphine tolerance via regulating MOR expression in cancer pain.
Although the role of CBs in opioid tolerance has been studied, the mechanism underlying these actions has not been defined. Opioid and CB receptors both belong to the family of Gi/Go-protein–coupled receptors and are often coexpressed in the cells of the nervous system.30 The interaction between opioid and CB receptors may be facilitated by similar intracellular signal pathways.2,24 Previous studies have shown that expression and activation of MOR in the brainstem are attenuated by the CB2 receptor antagonist SR144528 via CB2 receptors.31 The CB2-specific antagonist AM630 inhibited MOR expression, whereas the CB2-specific agonist JWH 015 markedly induced MOR expression in Jurkat T cells.32 These regulatory events were mediated by a signal transducer and activator of transcription (STAT)5-interleukin (IL)-4-STAT6 signaling pathway.32 Expression of IL-4 has also been detected in astrocytes from multiple sclerosis lesions33 and in lipopolysaccharide-activated microglia.34 In addition, IL-4 induces MOR transcription in primary neurons.35 These findings indicate that CBs induce MOR expression via an IL-4-dependent pathway in neurons of the central nervous system.
Opioids and CBs may interact at multiple levels and have been associated with analgesic, psychotrophic, and immunomodulatory effects. Previous studies demonstrated that the activation of glial cells by chronic morphine administration contributes to morphine tolerance.2,18,36 CB2 receptors have been detected in glial cells in distinct regions of the nervous system, such as the spinal cord and DRG.10,11 Glial CB2 receptors are dramatically upregulated in response to damaging stimuli11,37 and opioid agonist treatment.7,16 The CB2 receptor agonist attenuated inflammatory and neuropathic activation of glia in the central nervous system11,38 and coadministration of the selective CB2 receptor agonist AM1241 with morphine reduced morphine-mediated activation of spinal glia.7 MAPK activation2 and glial proinflammatory mediator release39,40 have also been linked to morphine tolerance. Administration of the CB2 receptor agonist reduces MAPK phosphorylation in neuropathic pain24 and morphine-induced inflammatory responses in activated microglial cells.18 Based on these findings, we speculate that there are possible mechanisms by which CB2 receptor agonists attenuate morphine tolerance in cancer pain.
There are some limitations to our study. First, we only used a single dose of a CB2 receptor agonist with morphine to determine the role of a CB2 receptor agonist in morphine tolerance. It would have been much more useful to know the effects over a range of agonist doses. Second, this study used measurement of MOR protein and mRNA expression at a single time point, that is, on day 8, did not determine MOR expression on other tested days. Third, we did not investigate the pathway between the CB2 receptor and MOR. Thus, the cellular and neurophysiologic pathways involved in the development of CB-related morphine tolerance remain uncertain in cancer pain. Further studies are required to address these limitations.
In summary, the present results suggest that CB receptor agonists are involved in opioid-mediated analgesia and attenuate opioid tolerance during management of cancer pain. Upregulation of MOR expression by CB2 receptor agonists may contribute to the synergistic effects of CBs and opioids. The use of CBs for cancer treatment is currently limited to chemotherapy- or radiotherapy-associated nausea.41 Our current data provide a novel pharmacologic approach using CB2 receptor agonists to strengthen morphine analgesia, thereby reducing the doses and side effects of morphine used for severe pain therapies.
We thank professors Yuyan Ma and Hongbo Jin (Cancer Institute of Heilongjiang Province and Department of Physiology, Harbin Medical University) for their assistance with this project. We thank the analyst Shaofei Su (Department of Statistics of Harbin Medical University) for help with statistical analyses.
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