鞘内注射丙戊茶碱对切口痛大鼠脊髓背角神经胶质细胞活化的影响

Effect of intrathecal propentofylline on activation of microglias in spinal dorsal horn of rats with incisional pain

目的 评价鞘内注射丙戊茶碱对切口痛大鼠脊髓背角神经胶质细胞活化的影响.方法 成年雄性SD大鼠96只,体重250～ 320 g,1.5～2.0月龄,采用随机数字表法,将其分为4组（n=24）：对照组（Ⅰ组）、切口痛组（Ⅱ组）、鞘内注射丙戊茶碱预防性用药组（Ⅲ组）和鞘内注射丙戊茶碱治疗性用药组（Ⅳ组）.Ⅱ组、Ⅲ组和Ⅳ组制备切口痛模型,Ⅲ组和Ⅳ组分别于术前30 min或术后30min时鞘内注射丙戊茶碱10μg,Ⅱ组于术前30 min时注射等容量生理盐水.分别于术前、术后2、6、24、48、96 h时测定机械缩足反应阈（MWT）和热缩足潜伏期（TWL）.于术后24、48、96 h时测定痛阈后处死6只大鼠,取脊髓组织,采用Western blot法测定小胶质细胞活化标记物OX-42和星形胶质细胞活化标记物胶质纤维酸性蛋白的表达水平,分别反映小胶质细胞活化水平和星形胶质细胞活化水平;采用免疫组化法测定IL-1β、TNF-α和P物质（SP）的表达.结果 与Ⅰ组比较,Ⅱ组、Ⅲ组和Ⅳ组术后各时点MWT降低,TWL缩短,脊髓小胶质细胞活化水平和星形胶质细胞活化水平升高,脊髓IL-13、TNF-α和SP表达上调（P＜0.05）;与Ⅱ组比较,Ⅲ组和Ⅳ组术后各时点MWT升高,TWL延长,脊髓小胶质细胞活化水平和星形胶质细胞活化水平降低,脊髓IL-1β、TNF-α和SP表达下调（P＜0.05）;与Ⅳ组比较,Ⅲ组术后2、6、24、48 h时MWT升高,TWL延长,术后24、48 h时脊髓小胶质细胞活化水平和星形胶质细胞活化水平降低,术后24、48 h时脊髓IL-1β、TNF-α和SP表达下调（P＜0.05）.结论 鞘内注射丙戊茶碱预防性用药和治疗性用药均可抑制切口痛大鼠脊髓背角神经胶质细胞活化,且效果无明显差异,因此建议采用鞘内注射丙戊茶碱治疗性用药.

Objective To evaluate the effect of intrathecal propentofylline on activation of microglias in the spinal dorsal horn of rats with incisional pain.Methods Ninety-six adult male Sprague-Dawley rats,weighing 250-320 g,aged 1.5-2.0 months,were randomly divided into 4 groups （n=24 each） using a random number table：control group （group Ⅰ）,incisional pain group （group Ⅱ）,intrathecal propentofylline for prophylaxis group （group Ⅲ）,and intrathecal propentofylline for therapy group （group Ⅳ）.A 1 cm longitudinal incision was made through skin,fascia and muscle of the plantar aspect of the hindpaw in isoflurane-anesthetized rats in Ⅱ,Ⅲ and Ⅳ groups.Propentofylline 10 μg was injected intrathecally at 30 min before and after operation in Ⅲ and Ⅳ groups,respectively,while the equal volume of normal saline was given at 30 min before operation in group Ⅱ.Before operation and at 2,6,24,48 and 96 h after operation,the mechanical paw withdrawal threshold （MWT） and thermal paw withdrawal latency （TWL） were measured.Six rats in each group were sacrificed after measurement of the pain threshold at 24,48 and 96 h after operation,and the L4-6 segment of the spinal cord was removed for determination of the expression of OX-42 （a marker of microglial activation） and glial fibrillary acidic protein （a marker of astrocyte activation）,which was used to reflect the activation of microglias and astrocytes respectively,and the expression of iuterleukin-1beta （IL-1 3）,tumor necrosis factor-alpha （TNF-α） and substance P （SP）（by immunohistochemistry）.Results Compared with group Ⅰ,the MWT was significantly decreased,the TWL was significantly shortened,the activation of microglias and astrocytes was significantly increased,and the expression of IL-1β,TNF-α and SP was significantly up-regulated at each time point after operation in Ⅱ,Ⅲ and Ⅳ groups （P〈0.05）.Compared with group Ⅱ,the MWT was significantly increased,the TWL was significantly prolonged,the activation of microglias and astrocytes was significantly decreased,and the expression of IL-1 3,TNF-α and SP was significantly down-regulated at each time point after operation in Ⅲ and Ⅳ groups （P〈0.05）.Compared with group Ⅳ,the MWT was significantly increased,and the TWL was significantly prolonged at 2,6,24 and 48 h after operation,the activation of microglias and astrocytes was significantly decreased at 24 and 48 h after operation,and the expression of IL-lβ,TNF-α and SP was significantly down-regulated at 24 and 48 h after operation in group Ⅲ （P〈0.05）.Conclusion Both prophylactic and therapeutic intrathecal propentofylline can inhibit activation of microglias in the spinal dorsal horn of rats with incisional pain,the efficacy is comparable,and thus prophylactic intrathecal propentofylline is recommended.