The Expression And Distribution Of NR2B In The Anterior Cingulate Cortex And L4-6 Spinal Cord Of The Bone Cancer Pain Mice

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.Method: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.ResultsThe expression and distribution of NR2B in the anterior cingulate cortex and spinal cord of mouse with bone cancer pain:(1) 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 superficial dorsal horn, followed by anterior 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 Blotting 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).Conclusion:1. 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.2. 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.