| Objective:In order to investigate the mechanisms of bone cancer pain, a mouse bone cancer pain model is established to study the role of NR2B in the initiation and maintenance of bone cancer pain by observing the expression and distribution of NR2B in the anterior cingulate cortex and L4-6 spinal cord of the animal.1. The establishment and evaluation of the new model of mouse cancer pain:thirty adult male C57BL/6 mice were randomly assigned to four groups:the test group (n=15):10μl PBS containing 2×106 Lewis lung cancer cells were inoculated into the marrow cavity of distal femur of the animal; the heat-inactivated group (n=5):the same number of Lewis lung cancer cells that had been heat-inactivated were inoculated; PBS group (n=5):the same volume of PBS solution was inoculated; normal control group (n=5):no treatment. We observed the spontaneous lifting time and mechanical allodynia of mice on alternate days from the 7th day after inoculation. The structural damage of the operated femur induced by tumor was monitored by radiological analysis by comparing with the other femur bone of the animal and observed respectively on the 7th,15th,23rd day. In the test group, five femurs from operated animals that were randomly selected were stained with hematoxylin and eosin (HE) after radiography; bone destruction was observed on 7th,15th,23rd day, respectively. Other groups were observed only on the 23rd day.2. The expression and distribution of NR2B in the anterior cingulate cortex and spinal cord in the test group:eighty-one adult male C57BL/6 mice were randomly assigned to three groups:cancer pain group (N=27); sham-operated group (N=27); normal control group (N=27).10μl of D-Hanks solution containing 2×106 Lewis lung cancer cells were inoculated in the cancer pain group; the same volume of solution without cancer cells was used in the sham-operated group; the normal control group:no treatment. Spontaneous lifting time and mechanical allodynia of mouse hind paw were measured on the 6th,10th,14th,18th,22nd day after inoculation. Three mice were taken to observe the expression of NR2B mRNA by using the qRT-PCR detection at the same time. According to the results of the PCR, three mice from each group were decapitated to extract the L4-6 spinal cord and anterior cingulate cortex to do fluorescence quantitative RT-PCR and immunohistochemical staining to observe the expression and distribution of NR2B, the remaining three mice were used to detect NR2B protein by Western Blot on the 14th and 18th day after inoculation.3. The effects of NR2B gene silencing in bone cancer pain mouse: 72 adult male C57BL/6 mice were randomly divided into three groups: cancer pain group (N=21):Lewis lung cancer cells 2×106/10ul were inoculated as stated above; interference group (N=21):PEI/NR2B siRNA complex was intrathecally injected 12 days later after cancer cell inoculation; control group of the interference (N=21):the PEI/Negative control siRNA complex was used in intrathecal injection. Spontaneous lifting time and mechanical allodynia were measured pre-operation and on the 6th,10th,12th,13th,14th,15th,16th,18th and 22nd day after inoculation. L4-6 spinal cord segments were isolated for fluorescence quantitative RT-PCR and Western Blot to detect the expression levels of NR2B in each group.4. The effects of NR2B were mediated by MAPK signaling pathway in the mouse bone cancer pain:forty-eight adult male C57BL/6 mice were randomly divided into four groups:normal group, cancer pain group, interference group and control of interference group. Spontaneous lifting time and mechanical allodynia in each group were measured pre-inoculation and on the 10th,12th,14th,16th and 18th day after inoculation. L4-6 spinal cord of the animals from each group was used to detect the expression levels of NR2B, SP, c-fos and GFAP gene by real-time quantitative PCR, and the expression levels of NR2B, p-ERK, p-p38 and GFAP protein were detected by Western Blot on the 14th and 18th day.1. Evaluation of bone cancer pain model:compared with the controls, the test group displayed a gradual development of spontaneous pain showing longer flinch time from the 11th day on; there was obvious mechanical allodynia from the 13th day on after inoculation as demonstrated by a 50% reduction of von Frey threshold. On the 23rd day after operation, X-ray showed that medullary cavity of lower end of femur on ipsilateral side had disappeared and some cortical bone had been lost. Pathological examination showed that tumor cells penetrated the medullary canal and invaded peripheral tissues. Therefore, the model was validated by data from the above three aspects.2. The expression and distribution of NR2B in the anterior cingulate cortex and spinal cord of mouse with bone cancer pain:Real-time quantitative florescence PCR detection in the anterior cingulate cortex:there was no significant difference between the cancer pain group and the sham operated group on the 6th and 10th day after inoculation, but the NR2B expression in both groups was higher than that in the control group; on the 14th and 18th day, the expression of NR2B in the cancer pain group increased significantly as compared to the groups of normal control and the sham operated (p<0.05), while there was no significant difference between the sham operated group and the normal control group. On the 22nd day, the expression of NR2B in the cancer pain group was reduced, but there was no statistical significance as compared to the other two groups. Real-time quantitative florescence PCR detection of NR2B expression in L4-6 spinal cord:on the 6th day after inoculation, there was no significant difference between the cancer pain group and the sham operated group, but the expression of NR2B in these two groups was increased significantly as compared to the normal control group. On the 10th,14th,18th and 22nd day, the expression of NR2B in the cancer pain group was increased significantly as compared to the groups of normal control and the sham operated (p<0.05), while there was no significant difference between the sham operated and the normal control group.(2) The results of immunohistochemical examination in the anterior cingulate cortex and L4-6 spinal cord:the expression of NR2B in the cancer pain group was mainly located in the superficial dorsal horn, central aqueduct, anterior horn of the spinal cord and the anterior cingulate cortex, the expression level of NR2B was highest in the anterior horn, followed bysuperficial dorsal horn and lowest levels were observed in the central aqueduct. The optical density (OD) values of NR2B-positive cells in L4-6 spinal cord and area of contralateral anterior cingulate cortex in the cancer pain group were significantly different as compared to the sham-operated and the normal controls on the 14th and 18th day after inoculation (P<0.05), whereas there was no significant difference between the later two groups (P>0.05).(3) The results of the Western Blot in L4-6 spinal cord:the expression of NR2B in the spinal cord of cancer pain group was significantly increased as compared to the groups of the normal control and the sham operated on day 14 and 18 after inoculation (p<0.05), while there was no significant difference between the later two groups (P>0.05).3. The effects of NR2B gene silencing in cancer bone pain mouse:(1) Real-time quantitative PCR detection:the expression of NR2B in L4-6 spinal cord in the interference group was reduced significantly as compared to the cancer pain group and the control group of the interference group on the 14th and 18th day after inoculation (P<0.05). The most obvious change in the expression of NR2B in the interference group was observed on the 14th day after inoculation, showing 74% lower expression in the test group as compared to that in the cancer pain group and the control group of the interference. There was no significant difference between the later two groups (P>0.05).(2) The results of Western Blot:the expression of NR2B in the L4-6 spinal cord in the interference group was reduced significantly as compared to that in the cancer pain group on the 14th and 18th day after inoculation (P<0.05). The most obvious change in the expression of NR2B was observed on the 18th day after inoculation, demonstrating 67% lower expression in the interference group than that in the cancer pain group. There was no significant difference between the later two groups (P>0.05).(3) Spontaneous lifting time was decreased and mechanical allodynia was increased obviously in the interference group as compared to the cancer pain group and the control group of the interference.4. The effects of NR2B were mediated by MAPK signaling pathway in bone cancer pain in mouse: (1) Real-time quantitative PCR detection:the expression levels of NR2B, SP, c-fos and GFAP genes in the L4-6 spinal cord of cancer pain group was significantly increased as compared to the normal control group on the 14th and 18th day after inoculation (P<0.05); the expression of these genes in the L4-6 spinal cord in the interference group was significantly lower than that of the cancer pain group; while there was no significant difference between the cancer pain group and the control of interference group(p>0.05).(2) Western Blot detection results:the expression of NR2B, p-ERK, p-p38 and GFAP protein in the spinal cord of cancer pain group was significantly increased as compared to that in the normal group on the 14th and 18th day after inoculation (P<0.05); the expression of these proteins in the L4-6 spinal cord of the interference group was significantly lower than that in the cancer pain group (P<0.05); while there was no significant difference between the cancer pain group and the control group of interference group(p>0.05).1. Inoculation of Lewis lung cancer cells into the marrow cavity of distal femur of C57BL/6 mice can effectively establish a mouse bone cancer pain model. There was much similarity between human cancer pain and this model.2. The observation that NR2B was expressed in large quantity in both the L4-6 spinal cord and anterior cingulate cortex suggests that the generation and maintenance of bone cancer pain not only can be activated in the spinal cord, but also is related to the anterior cingulate cortex.3. In this study we found that the expression of NR2B was strongly positive in the anterior horn cells of spinal cord of the bone cancer pain model, which provided some new insights about the mechanism of cancer pain and might be useful in later research.4. The expression of NR2B was reduced in the L4-6 spinal cord after intrathecal injection of PEI/NR2B-siRNA complex in the mouse with cancer pain, which was consistent with the behavior changes of the animals, and provided further evidence showing that NR2B might play an important role in the initiation of bone cancer pain.5. The MAPK signaling pathway was activated in the bone cancer pain model, suggesting that MAPK signal transduction pathway (ERK pathway and p38 pathway) is involved in the generation and maintenance of bone cancer pain.6. Bone cancer pain is mediated by NR2B receptor system, and may be related to the over-expression of GFAP, yet the mechanisms involved need to be further explored. |
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