大鼠鞘内注射曲马多的抗伤害性效应及与脊髓α_2受体效应的关系

The relationship between antinociceptive effect of intrathecal tramadol and the a2-adrenoccptor effect of the spinal cord in rats

目的探讨鞘内注射曲马多抗伤害性效应及与脊髓α2受体效应的关系.方法Wistar大鼠蛛网膜下腔埋管后随机分为5组(n=8):生理盐水对照组、曲马多10μg组、育亨宾10μg组、育亨宾10 μg+曲马多10μg组、育亨宾10μg+纳洛酮10μg+曲马多10μg组.于大鼠左后爪掌心皮下注射2.5%福尔马林50μl后鞘内注入上述药物,从福尔马林注射后的30 min开始对大鼠的表现行为进行Dubuisson & Dennis评分.另取一组大鼠断头处死后取腰段脊髓制成细胞膜悬液,以3H-育亨宾为放射性标记配基,盐酸育亨宾为非标记配基,曲马多为竞争剂,行标记配基的饱和结合反应及竞争取代反应,并计算3H-育亨宾与脊髓α2受体结合的平衡解离常数(KD)及曲马多与3H-育亨宾竞争结合脊髓α2受体的抑制常数(KI).结果鞘内注入10μg的曲马多能显著抑制福尔马林注射后大鼠的疼痛反应.10μg育亨宾单独鞘内注入无明显抗伤害性效应(P＞0.05),预处理可使10μg曲马多鞘内注入后大鼠的伤害效应评分最多可增加56%,但仍低于对照组大鼠的伤害性效应评分(P＜0.05);增加纳洛酮10μg预处理可使10μg曲马多鞘内注入后大鼠的伤害效应评分增加200%.受体放射性分析表明3H-育亨宾的KD值为1.79 nmol/L,曲马多的KI值为34.14μmol/L,IC50为68.25μmol/L,提示曲马多与脊髓α2受体的亲合力极低,约为育亨宾的1/19 000.结论在脊髓水平,曲马多具有抗伤害性效应,其抗伤害作用的一部分可能与其间接的脊髓α2受体效应有关.

Objective To investigate the relationship between the antinociception of intrathecal tramadol and Oh -adrenoceptor effect of the spinal cord in rats. Methods Forty adult Wistar rats of both sexes weighing 280-320 g wen- anesthetized with ketamine 40 mg . kg-1 and pentobarbital 20 mg. kg-1 ip and PE-10 polyethylene catheter was placed in subarachnoid space 4-6 days before experiment. Tbe animals were randomly divided into live groups of 8 animals : (1) control group received normal saline; (2) group T tramadol 10 μg; (3) group Y yohimbine 10μg;group Y + T yohimbine 10 μg + tramacol 10 μg and (5) group Y + N + T received yohimbine 10 μg + naloxone 10μg + tramadol 10 μg. The above-mentioned drugs including normal saline were injected intrathecally. 2.5% formalin 50ul was injected subcutaneously into the plantar region of left hindpaw. 15 min later the above-mentioned drugs were injected intrathecally. In group 4 and 5 yohimbine and naloxone were given 5 min before tramadol. The animals were placed in a 30 cm × 30 cm× 30 cm glass box for evaluation of pain behavior, which was graded from 0 ( no pain) to 3 ( severe Pain) according to Dobuisson and Dennis. Another sixteen animals were decapitated and lumbar spinal cord was rapidly removed and made into crude membrane suspension. H-yohimbine was used as radioligand for specific binding, yohimbin HCl for nonspecific binding and tramadol as competitor. Drug displacement studies for specific H-yohimbine binding were carried out under standard incubation conditions with 0.21 nmol.L-1 to 2. 1 nmol.L-1 (10 concentrations) of tramadol. Dissociation constant (Kd) and inhibition constant ( Ki) were obtained by special computerized parameter estimation method program. Results Intrathecal tramadol 10 μg could produce time-dependent antinociception. Yohimbine 10μg alone did not produce antinociception ( P > 0.05), but pretreatment with yohimbine 10 ug significantly reduced the antinociceptive effect of tramadol ( 10ug) at 35 min and 40 min and the nociception score increased by 56% and 41 % respectively ( P <0.01). Addition of naloxone increased the nociception score by 200% and 75% but the score was still lower than that in control group, but the difference was not statistically significant ( P > 0.05). Scatchard analysis of the saturation isotherms showed that H-yohimbine was bound to a single binding site with a Kd value of 1.79 nM. The competition curve of tramadol was sigmoidal with a Ki value of 34.14 uM and an IC50 value of 68.25 uM. Tramadol was 19 000-fold less potent for binding to a2-adrenoceptor of the spinal cord as compared to H-yahimbine. Conclusion Intrathecal tramadol produces time-dependent antinociception. Tramadol has very low affinity with a2-adrenoceptor of the spinal cord. A part of its intrathecal antinociceptive effect was related to indirect a2-adrenoceptor effect of the spinal cord.