Role Of Midbrain Periaqueductal Gray P2X₇ Receptor Activation On Bone Cancer Pain Mechanisms In Rats

Objective:（1）To observe the alterations of thermal pain threshold and mechanical in bone cancer pain（BCP）rats induced by inoculation of SHZ-88 breast cancer cells into the titia,and the expression changes of P2X₇ receptor in ventrolateral periaqueductal gray（vlPAG）;（2）To detect the changes of D-serine levels in vlPAG of BCP rats by microdialysis technology and enzyme linked immunosorbent assay（ELISA）methods;（3）To observe the changes of thermal and mechanical threshold values,and the expression of P2X₇ receptor in vlPAG pre-and post-treatment with tramadol in BCP rats.The relationship between P2X₇ receptor expression in vlPAG and the analgesia of tramadol in BCP rats was investigated,and provided a-theoretical basis for the use of tramadol analgesia clinically.Methods:（1）The BCP Sprague-Dawley（SD）female rat model was established by intratibial cell inoculation of SHZ-88 mammary gland carcinoma cells.The electronic von Frey anaesthesiometer and BME-410C automatic thermal pain stimulator were used to observe the changes threshold and mechanical pain threshold of each group rats on different observe times,respectively.Alterations in the number of P2X₇ receptor-positive cells and P2X₇ protein levels in vlPAG were detected by immunohistochemistry or Western blot assay.（2）The co-expression of P2X₇ receptor with NeuN,GFAP in vlPAG were detected by immuno-double labeling method.（3）The levels of neurotransmitter D-serine in vlPAG of BCP rats were observed by microdialysis and ELISA methods.（4）The BCP rats had administered of different doses of tramadol intraperitoneally,The thermal and mechanical pain threshold values of BCP rats and the expression of P2X₇receptor in vlPAG pre-and post-tramadol treatment were studied.（5）To observe the effect of pre-injection of selective P2X₇ receptor antagonist A- 740003 on the analgesic effect of intraperitoneal injection of tramadol in BCP rats vlPAG.Results:（1）The BCP rats model was successfully established by injecting（SHZ-88）breast cancer cells into the right tibia of SD female rats.After the successful modeling,the thermal pain threshold and mechanical pain threshold values of BCP rats decreased significantly from 5 to 21days.The difference between the normal control group and the sham-operation BCP group was statistically significant（P<0.001）.（2）The number of P2X₇ receptor positive cells in vlPAG of BCP rats upregulated compared with the normal control group and the sham-BCP groups（P<0.05）.The P2X₇receptor protein level in vlPAG in BCP group was increased compared with normal and sham-BCP groups（P<0.05）;（3）After modeling（21d）,the levels of D-serine in BCP rats was increased,but it was reduced after using the P2X₇ receptor antagonist A-438079（P<0.05）;（4）Tramadol（10,20,and 40 mg/kg,i.p.）markedly and dose dependently ameliorated the pain behaviors of BCP rats,upregulated pain thresholds,and further raised the expression level of P2X₇ receptor in vlPAG（P<0.001）.（5）After pre-injection of P2X₇ receptor specific antagonist A-740003 in vlPAG of BCP rats and subsequent intraperitoneal injection of tramadol（40mg/kg）,it was found that the analgesic effect of thermal and mechanical pain threshold were decreased compared with that of tramadol group（P<0.001）.the antinociceptive effect of tramadol was partially but significantly blocked by the vlPAG microinjection of P2X₇ receptor antagonist A-740003.Conclusion:1.Activation of the P2X₇ receptor in vlPAG is involved in the formation and maintenance of bone cancer pain in rats;The activation of P2X₇ receptor promoted the function of endogenous analgesic system and had analgesic effect on bone cancer pain in rats.2.Activation of the P2X₇ receptor in vlPAG promotes D-serine release and participates in the mechanisms of bone cancer pain in rats;3.Activation of the P2X₇ receptor in vlPAG is involved inthe supraspinal analgesic m echanism of tramadol.