A Study Of The Role For Spinal CX₃CR₁ And Its Mechanism In The Process Of Morphine Tolerance In Rats With Bone Cancer Pain

BackgroundPain is the common symptom in cancer patients. Approximately 30%-50% of all cancer patients will experience moderate to severe pain, and 75%-95% of patients with advanced-stage or metastatic cancer will experience substantial, life-altering cancer-induced pain. Bone cancer pain, one of the most serious cancer pain, is usually induced by primary bone cancer or secondary bone metastasis from breast, prostate, lung cancer, etc. The severity of the pain is closely correlated with the extent of bone destruction. Understanding biological mechanisms for bone cancer pain largely depends on the use of animal models. In 1999, Schiwei et al described their rat femur cancer pain model with NCTC2472 fibroma sarcomatosum cells, after that researchers have in succession established calcaneum bone cancer pain and humeral bone cancer pain. In 2002, Medhurst et al established tibial bone cancer pain model. In recent decade our national researchers have successfully established rat tibial cancer pain model according to that described by Medhurst. In all these models,the tibial cancer pain model established by breast carcinoma cells is regarded as clinically common cancer pain model,which would contribute to further study of the mechanisms underlying cancer pain.Morphine is clinically common medicine used to treat bone cancer pain. Morphine relieves pain by binding theμopioid receptor (MOR) on the sensory neurons in central and peripheral nerve system (CNS and PNS). Morphine is widely used for chronic and acute pain. However, tolerance to analgesia will develop after repeated usage of morphine, this means the attenuation of analgesic efficacy and the shortened analgesic duration, even hyperalgesia. Therefore, its long-term therapy is limited by the development of tolerance. Previous studies hypothesized several kinds of mechanisms of morphine tolerance, such as uncoupling of MOR with G protein, internalization or downregulation of opioids receptors; upregulation of NMDA receptor number or function; upregulation of intracellular proteins e.g. protein kinase A (PKA), protein kinase C (PKC) or CaMulinⅡ(CaMII). In addition, neurotransmitter such as excitatory amino acid (EAA), substance P (SP), calcium gene-related peptide (CGRP), nitric oxide (NO) and prostaglandins (PGs) are upregulated following the development of morphine tolerance, therefore offset or oppose the morphine analgesic function. Despite of numerous studies in last decades, the exact mechanisms of morphine tolerance are still unclear.Simplex morphine tolerance model were used to study the mechanism of chronic morphine tolerance in the past years. That is, morphine was intrathecally or intraperitoneally continuously administered to establish form morphine tolerance howerver,this kind of animal model has no base of painful damage,which cannot image clinical practice. Recently,someone has established rat morphine tolerance model based on arthritis pain.so we attempt to establish a chronic morphine tolerance modes based on tibial cancer pain model by breast carcinoma cells. CX3CR1 is the only receptor of CX3CL1. CX3CL1 is the only member of the CX3CL subfamily and characterized by two distinct forms:a membrane-bound form that displays adhesion properties and a soluble form that is cleaved from the cell membrane and has chemotactic properties. SNL induces a marked reduction of the membrane-bound CX3CL1 in the DRG, suggesting a possible cleavage and release of this chemokine after nerve injury. in the DRG but predominantly in microglia in the spinal cord. Importantly, CX3CR1 expression in microglia is upregulated under conditions of neuropathic pain induced by SIN, CCI, and SNI.The most dramatic spinal up-regulation of CX3CR1 and the most distinct localization of this receptor in spinal microglia were found after SNL. Behavioral studies show that CX3CL1 induces marked mechanical allodynia and thermal hyperalgesia in naive rats and mice. CX3CL1-inducedpain hypersensitivity is abrogated in CX3CR1 knockout mice. In addition, a neutralizing antibody against CX3CR1 or CX3CL1 attenuates the development of mechanical allodynia in the CCI and SNL neuropathic pain models.Finally, mechanical allodynia after PSNL does not develop in CX3CR1 knockout mice, whereas heat hyperalgesia still develops in these mice, suggesting that this receptor is particularly important for regulating mechanical allodynia. so these researches have demonstrated that CX3CL1 and CX3CR1 play an important role in neuropathological pain. A lot of study results reveal that mophine tolerance has some identical mechanisms with pathological pain, however, it is not clear whether CX3CR1 involves in the mechanisms of morphine tolerance.Based on above theories, in this study we designed four parts to evaluate the role of CX3CR1 and its mechanism in the development of morphine tolerance in rats with bone cancer pain. We attemp to find a new target for treatment to chronic morphine tolerance,and provide new scientific evidences for clinically relieving chronic morphine tolerance and persistantly effectively managing cancer pain.1. We established a morphine tolerance model in rats with bone cancer pain and compared to the simplex morphine tolerance model to evalue the advantages of the former.2. On the basis of the first study, we measured spinal CX3CRl-mRNA level with Realtime-PCR methods to observe the change of CX3CR1 mRNA in spinal dorsal horn of rats with bone cancer pain-morphine tolerance so as to deduce whether CX3CR1 involved in the development of morphine tolerance in rats with bone cancer pain.3. After intrathecal injection of minocycline,we measured spinal CX3CR1 protein and OX-42 with Westernblot and immunohistochemistry methods respectively to observe changes of spinal CX3CR1 protein and OX-42, so as to investigate the relationship between spinal microglia and CX3CR1 in the development of morphine tolerance in rats with bone cancer pain.4. We detected spinal CX3CR1 protein and MOR, TRPV1 with Westernblot and immunohistochemistry methods respectively after intrathecal injection of anti-CX3CR1 neutralizing antibody to observe their changes in spinal dorsal horn, so as to explore the downstream pathway of CX3CR1 involving in the development of morphine tolerance in rats with bone cancer pain.Methods and ResultsOne:Establishment of morphine tolerance model in rats with bone cancer painMethods Thirty-six adult female Sprague-Dawley rats with intrathecal catheterization were randomly devided into three groups(n=12):group S, group M and group BM. The rats in group BM were intrathecally administered morphine 20μg/kg twice daily for seven days after tibial cancer pain models were successfully established by Walker256 carcinoma cells. The rats in group M were intrathecally administered morphine 20μg/kg twice daily for seven days after heat-inactivated Walker256 cells were injected into tibial marrow cavity. The rats in group S received sham operation of tibia. The mechanical withdrawl threshold(MWT), mechanical withdrawl duration (MWD) of the rats were determined.The expression of fos protein was measured in spinal lumber 4-6 tissues with immunohistochemistry methods 3 hours after non-noxious palpation on the last day.Results MWT and MWD of rats post-implantation in group S had no difference compared with baseline (P>0.05). MWT of the rats in group M declined while MWD rose after 5 days intrathecally administering drugs, which had statistic significance compared with that in group S (P<0.05 or P＜0.01). MWT of the rats in group BM was lower while MWD was higher than those in group S and group M on days 6 and 9 post-implantation (P＜0.01).The bone destruction scores of the rats in group BM on day 9 post-implantation were more than one. MWT rose while MWD declined on days 1 and 3 intrathecally administering drugs (P＜0.05). MWT was lower while MWD was higher than those in group S and group M after day 5 intrathecally administering drugs(P<0.05 and P<0.01,respectively). Only a few Fos-Like-Immunoreactive neurons(FLINs) were expressed in spinal dorsal horn in group S. Spinal FLINs in group M were significantly more than those in group S (P＜0.01). FLINs in group BM were more than those in group M (P＜0.05)Two:Changes of CX3CR1 mRNA level in spinal dorsal horn of rats with bone cancer pain-morphine tolerance.Methods Forty female Sprague-Dawley rats were randomly devided into three groups. Group C(n=10) is control. The rats in group S(n=10) received sham operation of tibia. The rats in group BM (n=20)were intrathecally administered morphine 20μg/kg twice daily for seven days after tibial cancer pain models were successfully established by breast neoplasm cells. The mechanical withdrawl threshold(MWT), mechanical withdrawl duration (MWD) of the rats were used to evaluate their behaviour manifestations. Expressions of CX3CR1-mRNA in spinal lumber 4-6 tissues were measured by Realtime PCR methods.Results MWT and MWD of rats in group C and group S had no difference respectively in different time points (P>0.05). MWT of the rats in group BM was lower while MWD was higher than those of rats in group C and group S after 7 days intrathecally administering drugs (P<0.01). Spinal CX3CR1-mRNA in group BM was obviously higher than those in group C and group S (P<0.01) after morphine tolerance occured.Three:Effects of intrathecal minocycline on expressions of CX3CR1 protein in spinal dorsal horn of rats with bone cancer pain-morphine tolerance.Methods 48 adult Sprague-Dawley rats of female were randomized into 4groups(n=12).Group C is control. The rats in group S received sham operation of tibia. The rats in group BM were intrathecally administered morphine 20μg/kg twice daily for seven days after tibial cancer pain models were successfully established by breast neoplasm cells. The rats in group BM+m were intrathecally administered minocycline 0.25 mg/kg for three days following treats to the rats in group BM. The mechanical withdrawl threshold(MWT), mechanical withdrawl duration (MWD) of the rats were used to evaluate their behaviours. The expression of CX3CR1 protein and OX-42 in spinal lumber 4-6 tissues were respectively measured by Westernblot and immunohistochemistry methods.Results MWT of the rats in group BM was lower while MWD was higher than those of the rats in group C and group S after 7 days intrathecally administering drugs (P＜0.01).MWT and MWD of the rats in group BM had statistic significance compared with those in group BM+m on day 9 after intrathecally administering drugs (P<0.05) Expressions of CX3CR1 protein and OX-2 in group BM were obviously higher than those in group C and group S (P＜0.01) after morphine tolerance formed. Expression of CX3CR1 protein and OX-2 of rats in group BM+m significantly decreased after intrathecally administering minocycline (P＜0.05) Four:Effects of intrathecal anti-FKR on morphine tolerence in rats with bone cancer painMethods 48 adult Sprague-Dawley rats of female were randomized into 4groups(n=12): group A, group AM, group BM and group GM. Group A is control. Morphine tolerance models with bone cancer pain were established in the rats of the other three groups by intrathecally administering morphine 20μg/kg twice daily for seven days after tibial cancer pain models were successfully established with breast neoplasm cells. Then, the rats in group BM were intrathecally administered anti-FKR and the rats in group GM were administered IgG like that. The mechanical withdrawl threshold(MWT) of the rats were determined pre-implantation with Walker256 cells, on days 3,6,9 post-implantation, days 3, 7 after intrathecally administering drugs, and day 3 after intrathecally administering anti-FKR(T1～T6). So were the mechanical withdrawl durations (MWD). The expression of CX3CR1 protein and MOR, TRPV1 in spinal lumber 4～6 tissues were respectively measured by Westernblot and immunohistochemistry methods.Results The rats in group AM,GM and BM formed stable morphine tolerance models after 7 days intrathecally administering drugs. MWTs of the rats in these three groups were lower while MWDs were higher than those of the rats in group A (P<0.01).MWT and MWD of the rats in group BM had statistic significance compared with those in group AM and GM on day 3 after intrathecally administering anti-FKR(P<0.05). Compared with those in group A, expressions of CX3CR1 protein and TRPV1 obviously increased while MOR decreased in group AM and group GM after morphine tolerance formed (P<0.01) CX3CR1 protein and TRPV1 of rats in group BM were significantly lower while MOR were obviously higher than those in group AM and group GM after intrathecally administering anti-FKR (P<0.05)Five:Statistical analysis All the analysis were performed by SPSS 11.0 for windows. All of the data are expressed as mean±standard deviation (SD). Differences of comparisons between groups were made by a One-way repeated measures ANOVA.While other results were analysed by ANOVA. The difference for statistical significance was P＜0.05.SummaryMajor Results:1 A morphine tolerance model in rats with bone cancer pain were successfully established and had better clinical significance compared to the simplex morphine tolerance model.2 On the basis of the first study, CX3CR1 mRNA upregulated in spinal dorsal horn of rats with bone cancer pain-morphine tolerance.3 After intrathecal injection of minocycline, the expression of spinal CX3CR1 protein and OX-42 decreased,which revealed the relationship between spinal microglia and CX3CR1 in the development of morphine tolerance in rats with bone cancer pain.4 CX3CR1 protein and TRPV1 downregulated,while MOR upregulated in spinal dorsal horn after intrathecal injection of anti-CX3CR1 neutralizing antibody,which revealed the downstream pathway of CX3CR1 involving in the development of morphine tolerance in rats with bone cancer pain.Major Conclusions:Spinal CX3CR1 may play an important role in the mechanism of bone cancer pain-morphine tolerance of rats, activated microglia can increases expressions of CX3CR1 protein and CX3CR1-mRNA, then downregulate neuronal MOR and upregulate TRPV1,which could be one of the mechanisms of CX3CR1 involving in bone cancer pain-morphine tolerance. Significance:Based on successfully establishing bone cancer pain-morphine tolerance model of rats, this study investigated the role and its mechanism of CX3CR1 in the process of bone cancer pain-morphine tolerance. It is demonstrated that spinal CX3CR1 exerted important functions in bone cancer pain-morphine tolerance. Downregulating neuronal MOR and upregulating TRPV1 could be one of the mechanisms.This study will be helpful to find a new target for treatment to chronic morphine tolerance,and provide theory basis and experiment basis for clinically relieving chronic morphine tolerance and persistantly effectively managing cancer pain.