丙戊茶碱预处理削弱大鼠关节炎痛觉过敏的脊髓机制

目的研究预先鞘内注射丙戊茶碱对大鼠佐剂性关节炎热痛觉过敏的影响及其作用是否与抑制脊髓星形胶质细胞活化相关。方法实验采用大鼠右后爪踝关节外侧皮下注射CFA50μl致炎模型。①SD大鼠48只,随机分为6组(n=8),生理盐水(normal saline,NS)组:鞘内注射(intrathecal injection,it)NS10μl加皮下注射NS50μl;模型组:NSi加皮下注射CFA;单用丙戊茶碱组:10μg/10μl丙戊茶碱i加皮下注射NS;丙戊茶碱治疗组,分别为p2.5、p5、p10组:2.5μg/10μl、5μg/10μl及10μg/10μl丙戊茶碱it加皮下注射CFA。热辐射法测定各组大鼠热缩足反射潜伏期。②SD大鼠30只,随机分为6组(n=5),随机取3组鞘内预先注射生理盐水10μl,30min后注射CFA50μl,并分别于注射CFA5h、3d、7d麻醉;余大鼠预先注射丙戊茶碱(10μg/10μl,it),每天1次,30min后注射CFA50μl制成炎症模型,分别于注射CFA后5h、3d、7d麻醉;灌注,取材,免疫组化分析在炎症的不同阶段,大鼠炎症侧脊髓背角星形胶质细胞标志物GFAP表达。结果①模型组大鼠注射侧d2热缩足反射潜伏期与生理盐水组比较明显缩短(P<0.01),鞘内注射丙戊茶碱(5和10μg/10μl组)5h后大鼠热缩足反射潜伏期明显延长(P<0.01),有效作用时间为d1~d7;2.5μg/10μl组药物有效作用时间为d1~d2;注射对侧肢体大鼠行为学在实验观察期间没有明显变化;②模型组大鼠注射侧脊髓背角星形胶质细胞活化明显,积分光密度值增加(P<0.01)。鞘内注射丙戊茶碱(10μg/10μl组)5h后免疫组织化学染色可看到GFAP染色变浅,星形胶质细胞分支缩短,积分光密度值下降(P<0.01)。结论预先鞘内注射丙戊茶碱可提高大鼠热反射缩足潜伏期,可能通过抑制脊髓部位星形胶质细胞活化发挥抗伤害性作用。

Effect and mechanism of pretreating propentofylline on attenuation of arthritis thermal hyperalgesia

Aim To investigate whether activation of astrocytes is involved in the induction and maintenance of Complete Freund′s Adjuvant(CFA)-induced inflammatory pain, and observe whether intrathecal injection of propentofylline(PPF)reduces the expression of glial fibrillary acidic protein(GFAP)in the spinal cord of the inflammatory rats and have anti-hyperalgesic effect.Methods Seventy-eight male adult Holzman Sprague-Dawley rats(180~220 g)were used in an arthritic pain model induced by injecting CFA 50 μl into right hind paw of ankle mortise. subcutaneously and laterally.Part one:forty-eight rats were randomly divided into 6 groups(n=8 in each). Normal saline (NS) group:normal rats received normal saline (NS) both by intrathecal injection(it) and subcutaneously(sc). Model group: normal rats received 50 μl CFA in right hind paw of ankle mortise subcutaneously. PPF single group: normal rats received PPF it after NS sc PPF therapy groups:rats received CFA injection after PPF it(concentrations were 2.5, 5 and 10 μg/10 μl, re-spectively). Radiant-heat method was adopted to measure TWL(thermal withdrawal latency) of rats. Part two: 30 SD rats were randomly divided into 6 groups(n=5 in each). 3 groups were chosen randomly and pretreated with NS half an hour before CFA injection. Inflammatory rats were killed after being injected CFA 5h in the first, third and seventh day (i.e. model group, CFA5h, CFA3d, CFA7d); another 3 groups were pretreated with PPF (10 μg/10 μl, i.t), once per day,injected CFA half an hour later, and killed in the fifth hour, third and seventh day(i.e. PPF therapy group, PPF5h,PPF3d,PPF7d); Five rats of NS group were used as the blank control.The change of GFAP expression in L4～5 segments of spinal cord was accessed by immunohistochemistry analysis.Results PPF (10 μg/10 μl, i.t) solely had no effect on TWLs of normal rats.PPF (2.5 μg/μl, i.t) had no effect on TWL of inflamed hind paw during the experiment except the first and the second day(P>0.05,n=8). At the dosage of 5 and 10 μg/μl, PPF produced respectively marked anti-hyperalgesia in inflamed hind paw(P<0.05,n=8). Both dosages inhibited the induction of hyperalgesia in both acute and persistent paradigms. The TWL of right hind paw was significantly higher in the PPF groups than that in model group(P<0.01). Astrocytes marker GFAP was deeply stained in spinal cord dorsal horn of model group , PPF it 10 μg significantly attenuated the activation of GFAP in the spinal cord dorsal horn in group PPF(P<0.01).Conclusions The result suggests that PPF has antihyperalgesia properties. The antihyperalgesia effect of PPF is closely related to the suppression of the astrocytes activation in spinal cord during CFA-induced inflammatory pain progress.