鞘内注射CD200Fc对切口痛大鼠脊髓背角小胶质细胞活化的影响

Effect of intrathecal injection of CD200Fc on microglial activation in spinal dorsal horn in model of incisional pain in rats

目的探讨鞘内注射CD200Fc对切口痛大鼠脊髓背角小胶质细胞活化的影响。方法选取健康清洁级雄性SD大鼠54只,体质量280~300 g,6~8周龄。采用随机数字表法分为空白对照组(B组)、切口痛组(IP组)和CD200Fc组(CF组)3组,每组18只。B组仅进行异氟醚麻醉,IP组鞘内注射生理盐水5μl,CF组鞘内注射5μl CD200Fc(4μg/μl),注射结束后IP组和CF组立即在异氟醚麻醉下制备切口痛模型。每组取6只大鼠,分别于术前,术后4 h、1 d、3 d、5 d和7 d时测定机械刺激缩足反射阈值(paw mechanical withdrawal threshold,PMWT)和热刺激缩足反射潜伏期(paw withdrawal thermal latency,PWTL)。每组取模型制备成功大鼠12只,于术后3 d时处死取脊髓背角组织,采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测TNF-α和IL-1β含量,采用Western blot法检测小胶质细胞活化标志物OX42表达水平。结果与B组比较,IP组术后4 h、1 d、3 d和5 d时的PMWT降低[(3.8±1.3)g比(23.2±2.0)g、(5.9±1.7)g比(23.1±1.6)g、(9.9±1.3)g比(21.5±1.4)g和(17.6±3.9)g比(23.9±2.7)g],PWTL缩短[(3.5±1.0)s比(18.2±1.2)s、(6.5±1.1)s比(18.4±1.8)s、(11.6±2.0)s比(18.3±1.8)s和(16.5±1.3)s比(18.3±1.3)s],术后3 d时的脊髓背角TNF-α和IL-1β含量升高[(27.62±3.23)pg/ml比(9.05±1.31)pg/ml,(71.39±7.58)pg/ml比(26.66±3.51)pg/ml],脊髓背角OX42蛋白表达上调,差异均有统计学意义(P<0.05)。CF组术后4 h、1 d和3 d时的PMWT较B组明显降低[(9.1±1.8)g、(12.8±2.8)g和(18.3±2.3)g],PWTL较B组明显缩短[(7.2±1.4)s、(12.2±1.4)s和(15.5±1.4)s];术后3 d时的脊髓背角TNF-α和IL-1β含量明显高于B组[(15.38±1.71)pg/ml,(48.71±6.74)pg/ml],脊髓背角OX42表达上调,差异均有统计学意义(P<0.05)。与IP组比较,CF组术后4 h、1 d、3 d和5 d时的PMWT升高[5 d,(22.0±2.1)g],术后4 h、1 d和3 d时的PWTL延长,术后3 d时的脊髓背角TNF-α和IL-1β含量降低,脊髓背角OX42表达下调,差异均有统计学意义(P<0.05)。结论鞘内注射CD200Fc可以缓解大鼠切口痛,其机制可能与抑制脊髓背角小胶质细胞活化及TNF-α和IL-1β释放减少有关。

Objective To explore the effect of CD200Fc injected intrathecally on the microglial activation in the spinal dorsal horn in model of incisional pain in rats.Methods Fifty-four healthy clean-grade male SD rats,weighing 280-300 g,aged 6-8 weeks,were used in the study.All the rats were divided into three groups using a random number table method:blank control group(group B),incisional pain group(group IP)and intrathecal CD200Fc group(group CF),with 18 rats in each group.Group B only received isoflurane anesthesia.Normal saline 5μl was injected intrathecally in group IP,and CD200Fc 5μl(4μg/μl)was injected intrathecally in group CF.After injection,plantar incision surgery was performed under isoflurane anesthesia in group IP and group CF.Six rats in each group were selected,and the paw mechanical withdrawal threshold(PMWT)and paw withdrawal thermal latency(PWTL)were measured before operation,and at four hours and one day,three days,five days and seven days after operation.Twelve rats with successful model preparation in each group were selected,at three days after operation,the rats were sacrificed,and the lumbar segment(L4-L5)of the spinal cord was removed for determination of TNF-αand IL-1βcontents(by ELISA)and OX42 expression(by Western blot).Results Compared with group B,the PMWT[(3.8±1.3)g vs.(23.2±2.0)g,(5.9±1.7)g vs.(23.1±1.6)g,(9.9±1.3)g vs.(21.5±1.4)g,(17.6±3.9)g vs.(23.9±2.7)g]and PWTL[(3.5±1.0)s vs.(18.2±1.2)s,(6.5±1.1)s vs.(18.4±1.8)s,(11.6±2.0)s vs.(18.3±1.8)s,(16.5±1.3)s vs.(18.3±1.3)s]were significantly decreased at four hours and one day,three days and five days after operation,and the contents of TNF-αand IL-1βin spinal dorsal horn[(27.62±3.23)pg/m vs.(9.05±1.31)pg/ml,(71.39±7.58)pg/ml vs.(26.66±3.51)pg/ml]were increased and the expression of OX42 was up-regulated at three days after operation in group IP,the differences were statistically significant(P<0.05).Compared with group B,and the PMWT[(9.1±1.8)g,(12.8±2.8)g,(18.3±2.3)g]and PWTL[(7.2±1.4)s,(12.2±1.4)s,(15.5±1.4)s]were significantly decreased at four hours and one day and three days after operation,and the contents of TNF-αand IL-1βin spinal dorsal horn[(15.38±1.71)pg/ml,(48.71±6.74)pg/ml]were increased and the expression of OX42 was up-regulated at three days after operation in group CF,the differences were statistically significant(P<0.05).Compared with group IP,the PMWT was significantly increased at four hours and one day,three days and five days[(22.0±2.1)g]after operation,and the PWTL was significantly increased at four hours and one day and three days after operation,and the contents of TNF-αand IL-1βwere decreased and the expression of OX42 was down-regulated at three days after operation in group CF,the differences were statistically significant(P<0.05).Conclusions Intrathecal injection of CD200Fc can relieve incision pain in rats,by inhibiting the microglia activation and the release of TNF-αand IL-1βin spinal cord.