| BackgroundCancer-induced bone pain (CIBP) is the most common symptom caused by tumor metastasis to bone. The most three types of cancer prone to occur bone metastasis are breast cancer, lung cancer and prostate cancer. The characteristics of CIBP are continuously ongoing pain, breakthrough pain, and movement-related pain. Currently, the primary treatment on cancer pain is utilization of opioids and other adjuvants, such as NSAIDs and cannabinoids. However,45%of patients bearing cancers have not acquired adequate analgesia, who are suffering moderate to severe pain. Two reasons may demonstrate this phenomenon. One is that chronic use of opioids could induce antinociceptive tolerance, respiratory depression, nausea and vomitting. The other is that the mechanism of cancer pain is significantly different with that in neuropathic pain or inflammatory pain, or rather, it is a combination of neuropathic pain and inflammatory pain. The process of cancer pain is a complex result of neuropathic, inflammatory and necrotic pathologic changes in CNS and peripheral tissues.The mechanism of cancer-induced bone pain involves peripheral factors and central sensitization. Considering firstly the tumor itself, the presentation of CIBP may be modulated by tumor type, sites and osteolysis. Tumor cells and the surrounding immune cells can release pro-hyperalgesic mediators, such as prostaglandins, endothelins, bradykinin, TNF-a, and a range of growth factors. The hypermetabolism of tumor tissue induces changes in the microenvironment that may affects CIBP, such as low oxygen levels, acidic pH, and high extracellular calcium concentrations. All these above may sensitize peripheral nociceptors to subsequent stimuli, or directly activate specific receptors on the primary afferent neurons, then produce pain signals. Within the central nervous system, decrease of mu opioid receptors (MOR) in specific subpopulations of primary afferent neurons in the dorsal root ganglia or spinal cord has been found. Pro-hyperalgesic molecules released by activated astrocytes or microglia, sensitization of Wide Dynamic Range (WDR) neurons in the spinal cord, hyperexcitability in DRG neurons, excitatory synaptogenesis, and up-regulation of cytokines in spinal cord in bone cancer rats contribute to the central sensitization and the development of cancer pain.In the past five years, the role of chemokines (CX3CL1, CCL2and CCL5) in the development of CIBP was studied. The up-regulation of chemokines in spinal cord is essential to the development of cancer pain. For example, blocking the function of CX3CR1and CCR2by neutralizing antibodies could prevent the development of CIBP. The increase of CCL5in the tissues surrounding tumor and spinal cord also contributed to CIBP. In our previous study, we screened genes differentially expression in spinal cord of CIBP rat. As the microarray analysis showed, both chemokine CXCL10and its receptor CXCR3were increased in CIBP rats. We supposed that activation of CXCL10/CXCR3pathway may be essential to the development of cancer pain. Based on this background, we investigated the role of chemokine CXCL10in the development and maintenance of cancer-induced bone pain in rat models. Whether up-regulation of CXCL10influences morphine analgesia in animals was also studied. Methods and Results1. The role of CXCL10/CXCR3in the development of CIBP in rats MethodsExperiment1:To measure the change of CXCL10and CXCR3levels in spinal cord of CIBP rats, on days7,14, and21post CIBP modeling, rats were euthanatized and the spinal cord were removed. The expression of CXCL10and CXCR3in the spinal cord were measured by real-time PCR. On days14post modeling, the expressions of CXCL10and CXCR3in L3-L5spinal cord were detected by immunofluorescence. Experiment2:To investigate whether CXCL10was necessary for inducing pain, recombinant rat CXCL10protein (10ng, i.t.) was injected into naive rat. PWTs were measured at5,15,30,45,60,90, and120min after injection. Experiment3:To detect whether CXCL10or CXCR3was required for the development of CIBP, we inhibited the CXCL10/CXCR3function via chronic injecting rabbit anti-CXCL10neutralizing antibody or CXCR3antogonist-AMG487from days1to14. PWTs were measured at30min after drug injection on days1,3,5,7,10, and14.ResultsThe expressions of CXCL10and CXCR3in the spinal cord of CIBP models were increased compared with those in sham rats. Remarkably, the up-regulation of CXCL10and CXCR3was most significant on day14. The optical density mean of CXCL10and CXCR3-positive cells were increased in both contralateral and ipsilateral dorsal horn of the spinal cord of CIBP rats, as showed by immunohistochemistry. Recombinant CXCL10proein decreased PWTs in normal rats immediately from30min after injection. The mechanical allodynia induced by rrCXCL10lasted up to120min. This suggested CXCL10could directly induce algesia. PWTs failed to decrease until day14in anti-CXCL10neutralizing antibody-treated or AMG487-treated rats. However, vehicle of normal rabbit IgG treated rats and CIBP rats showed decreased PWTs after day3. These demonstrated that activation of CXCL10/CXCR3pathway participaited in the development of CIBP.2. The role of microglia in CXCL10-mediated cancer-induced bone pain in ratMethods Experiment1:Spinal cord of CIBP rats were removed on day14post-modeling. To detect which type of cells express CXCR3in spinal cord, the sections from spinal cord were incubated with a mixture of the following primary antibodies:CXCR3antibody and antibody to nerve cells (anti-NeuN, anti-GFAP, or anti-CD11b). Experiment2:To investigate the change of microglial activation in the AMG487-treated CIBP models, CIBP rats received chronic AMG487(20μg, i.t, once daily) after bone cancer modeling. The activation of microglia in the spinal cord was detected on day14by immunohistochemistry. Experiment3: To investigate whether microglial activation was involved in rrCXCL10-induced mechanical allodynia, minocycline, an inhibitor of microglia, was intrathecally injected into CIBP rats once per day under stages of development, early-(days1-7) or later-stage (days11-14). RrCXCL10was administrated1h after the last minocycline injection. PWTs were measured30min after rrCXCL10injection.ResultsCXCR3was co-localized with NeuN and CD11b, but not GFAP. These results suggested that CXCR3was expressed in neuron and microglia in spinal cord. Microglia was significantly activated in the spinal cord of vehicle-treated CIBP rats, displaying a characteristic ramified structure with increased cytoplasmic and dendrite volume. However, after chronic AMG487treatment, the microglial activation was attenuated. These suggested that CXCR3signaling mediated microglial activation. Minocycline treatment ameliorated pain in both stages. Minocycline pre-treatment fully prevented rrCXCL10-induced mechanical allodynia on day14, but only slightly attenuated rrCXCL10-induced mechanical allodynia on day7in CIBP rats and sham rats. These mean rrCXCL10-induced allodynia at the later-stage of CIBP was regulated by microglial activation. 3. The role of CXCL10in morphine analgesiaMethodsExperiment1:To detect the effect of CXCL10on morphine analgesia in CIBP models, on day14after modeling, CIBP rats received intrathecally (i.t.) injection of100ng neutralizing rabbit anti-CXCL10antibody, followed by i.t. injection with10μg morphine. Thirty minutes after the last drug injection, the PWTs of rats in each group were measured, respectively. Experiment2:To detect if overexpression of CXCL10could influence the antinociceptive effect of morphine, mice were i.t. administrated with30ng rmCXCL10, followed by injection of morphine (10mg/kg, s.c). The PWTs were measured at15,30,60,90, and120min after morphine injection.ResultsBoth anti-CXCL10and morphine treatment attenuated the decrease of PWTs in CIBP rats (P<0.01). While blocking the function of CXCL10by pre-treatment with anti-CXCL10, the analgesic effect of morphine in CIBP rats was enhanced compared with that in rats treated by morphine only. In morphine-treated mice, the PWTs were increased from15min and reached a peak of14.17±2.04g at60min after drug administration. However, in rmCXCL10-pretreated mice, the increase of PWTs induced by morphine reached a lower level of7.00±2.76g than that in morphine-treated mice at60min (P<0.01). These results suggested overexpression of CXCL10in spinal cord partly inhibited morphine-induced analgesic effect.4. Statistical analysis All data were presented as mean±SEM. Statistical significance (P<0.05) was determined using a two-way ANOVA (treatment group×min) to detect the changes to PWTs after drug injection over time. Individual comparisons were made with unpaired t-test. The change of CXCL10and CXCR3expression were tested using one-way ANOVA.Conclusion1. Increase of CXCL10/CXCR3in spinal cord contributed to the development and maintenance of CIBP in rats.2. Microglial activation participated in CXCL10-induced algesia in the later-stage of CIBP in rats.3. Blocking CXCL10function by neutralizing anti-CXCL10antibody enhanced morphine analgesia in CIBP rats. Overexpression of CXCL10in spinal cord by injection of recombinant CXCL10protein attenuated morphine analgesia in normal mice.SignificanceThis study firstly demonstrates that up-regulation of CXCL10in spinal cord plays an important role in the development and maintenance of cancer-induced bone pain in rats via microglial activation. It also suggested that increase of CXCL10in spinal cord plays a negative role in morphine analgesia in relief of cancer pain. This study provides a novel insight on the mechanism of cancer pain and provides a new target for cancer pain management. |
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