【作者】 王月玲；

【导师】 郭曲练；

【作者基本信息】 中南大学 ， 麻醉学， 2008， 硕士

【摘要】 研究背景布托啡诺是一种合成的阿片受体激动-拮抗剂,其镇痛效应强、作用时间久而副反应（呼吸抑制、成瘾等）发生率较低,已广泛应用于临床,但鞘内给药局限于动物实验阶段。研究表明椎管内NMDA受体拮抗剂和阿片类药物合用有协同镇痛作用,前期动物实验已证实了氯胺酮（非竞争的NMDA受体拮抗剂）和布托啡诺鞘内联合用药可以减少布托啡诺的用药量并起到相似的镇痛效果。PKA、CREB信号转导系统在脊髓水平痛觉传递和调制及疼痛中枢敏感化的形成和维持中起着重要作用,cAMP/PKA信号通路能磷酸化多种底物,是细胞内最重要的信号转导通路之一,CREB可接受多种信号转导通路的调控。在布托啡诺复合氯胺酮镇痛机制中,是否抑制了cAMP/PKA-CREB信号通路值得研究。目的本研究观察鞘内预先注射布托啡诺以及合用鞘内注射非竞争性的N-甲基-D-天门冬氨酸（NMDA）受体拮抗剂氯胺酮对于福尔马林炎性痛大鼠的镇痛作用,采用免疫组化的方法检测大鼠脊髓背角PKA和pCREB的表达来探讨其镇痛机制。方法40只SD雄性大鼠均采用改良Yaksh法进行鞘内置管,置管5天后,被随机分为7组:对照组（C组,n=5）;生理盐水组（NS组,n=5）;低剂量布托啡诺组（LB组,n=6）;高剂量布托啡诺组（HB组,n=6）;低剂量氯胺酮组（LK组,n=6）;高剂量氯胺酮组（HK组,n=6）;低剂量布托啡诺复合低剂量氯胺酮组（LB+LK组,n=6）。在各组大鼠的左后足掌面皮下注射5%福尔马林50μl。致痛前30分钟,C组鞘内不注射任何药物,NS组大鼠鞘内预先注射生理盐水,LB组鞘内预先注射布托啡诺12.5μg,HB组鞘内预先注射布托啡诺25μg,LK组鞘内预先注射氯胺酮50μg,HK组鞘内预先注射氯胺酮100μg,LB+LK组鞘内预先注射布托啡诺12.5μg后立即注射氯胺酮50μg。采用疼痛加权评分法（PIS）评估福尔马林注射后1小时内的疼痛行为,所有大鼠均在注射福尔马林2小时后处死,取大鼠脊髓L₅节段标本,用免疫组化方法测定脊髓背角PKA和pCREB的表达,并进行相关性分析。结果在福尔马林炎性痛的第一时相和第二时相中,C组和NS组的PIS值明显升高,HB组和LB+LK组的PIS值均低于C组（p＜0.05或0.01）,LB组、LK组和HK组的PIS值稍降低,但与C组比较没有统计学差异。在大鼠脊髓背角免疫组化实验中,C组和NS组的PKA和pCREB表达明显增强,HB组和LB+LK组的PKA和pCREB表达均明显弱于C组（p＜0.01）,LB组、LK组和HK组的PKA和pCREB表达稍弱于C组,但与C组比较没有统计学差异。对PKA和pCREB的免疫组化染色评分进行相关性分析,两者呈正相关。结论1.在大鼠福尔马林炎性痛模型中,鞘内预先注射布托啡诺25μg与鞘内预先注射布托啡诺12.5μg复合氯胺酮50μg可产生相似的镇痛效果。2.在大鼠福尔马林炎性痛模型中,PKA与pCREB表达明显升高且呈正相关,表明PKA-CREB通路可能是炎性痛形成和维持的机制之一。3.在大鼠福尔马林炎性痛模型中,鞘内预先注射布托啡诺12.5μg复合氯胺酮50μg,脊髓背角PKA与pCREB表达明显降低且呈正相关,其镇痛作用可能与抑制PKA-CREB信号通路有关。更多还原

【Abstract】 Background Butorphanol is an agitation-antagon opioid receptor, which has stronger analgesia,longer action time and lower side effect （e.g.respiratory depression,addiction）and applied in clinic generally,but intrathecal administration stays at the animal experiment stage nowdays. Recent study found that the antagon of the NMDAR and opioid drug might generate synergistic effect.PKA and CREB play an important role in transmitting and modulating pain and development and maintain of central sensitization of spinal cord.Therefore,it is important meaning to investigate the feasibility of association from intrathecal administration and molecular level mechanisms of analgesia for pain mechanisms and therapy.Objective To investigate the effect of preemptive intrathccal butorphanol tartrate and/or ketamine（non-competitive NMDA receptor antogonist）on PKA and pCREB expression in spinal dorsal horn and evaluate antinociception in the rats of formalin pain.Methods microspinal catheters were inserted intrathecally according to the method of Yaksh.After 5 days,40 male rat were divided into 7 groups randomly:control group（C group,n=5）;saline group（NS group,n=5）;low-dose butorphanol（LB group,n=6）;high-dose of butorphanol（HB group,n=6）;low-dose of ketamine（LK group,n=6）; high-dose of ketamine（HK group,n=6）;low-dose of butorphanol and low-dose of ketamine（LB+LK group,n=6）.At 30 minutes before left palmaris paw subcutaneous formalin injection,the control group received no agent prior to formalin injection,the NS group rats were intrathecally pretreated with saline,the LB group were intrathecally pretreated with low-dose of butorphanol 12.5μg,the HB group were intrathecally pretreated with high-dose of butorphanol 25μg,the LK group were intrathecally pretreated with low-dose of ketamine 50μg,the HK group were intrathecally pretreated with high-dose of ketamine 100μg,the LB+LK group were intrathecally pretreated with butorphanol 12.5μg and ketamine 50μg.Pain intensity scoring（PIS）was utilized to assess pain behavior within 1 hour after formalin injection.2 hours later,PKA and pCREB expression in the spinal dorsal horn of the L₅ segment was assayed using immunohistochemistry and done the analysis of correlation.Result Intrathccal injection high-dose butorphanol 25μg or butorphanol 12.5μg with ketamine 50μg were both capable of inhibition both phases of formalin response,the PIS was decreased（p＜0.05 or 0.01）. However,the expression of both the early and later phases was not significantly affected in other groups.Intrathecal pretreatment of high-dose butorphanol 25μg or ketamine 50μg with butorphanol 12.5μg both lowered the expression of spinal cord PKA and pCREB（p＜0.01）. Conclusion1.In rat of formalin induced inflammatory pain,the effect of the antinociception of the intrathecal pre-treatment of butorphanol 25μg is similar with the effect of the intrathecal administration of the combination of butorphanol 12.5μg and ketamine 50μg.2.The expression of PKA and pCREB in the spinal dorsal horn is higher obviously,as well positive correlation,which indicates that the PKA-CREB pathway may be the formation and maintenance of the formalin induced inflammatory pain.3.In rat of formalin induced inflammatory pain,the expression of PKA and pCREB in the spinal dorsal horn is decreased obviously,as well positive correlation after the intrathecal pre-treatment of butorphanol 12.5μg and ketamine 50μg,the antinociception mechanisms may be connects with the inhibition of the PKA-CREB pathway.更多还原