【作者】 成浩；

【导师】 杨建平；

【作者基本信息】 苏州大学 ， 外科学， 2007， 博士

【摘要】 第一部分:鞘内注射米诺环素对佐剂性关节炎性痛觉过敏的作用目的研究鞘内(i.t)注射米诺环素对完全氟氏佐剂(CFA)性关节炎性痛觉过敏的作用。方法1.行为学研究蛛网膜下腔置管成功的雄性SD大鼠,在右踝关节外侧皮下注射50μl CFA前30 min分别i.t生理盐水(NS)或12.5、25、50μg不同剂量的米诺环素,i.t每天1次,持续7d,观察后爪热刺激回缩潜伏期(PWTL)和机械缩足反射阈值(MWT)14d。CFA致炎后2d、6d,i.t米诺环素50μg,观察24h PWTL和MWT变化;右踝关节外侧皮下注射50μl NS前30min i.t米诺环素50μg、NS 20μ1,观察24 h的PWTL和MWT变化。2.形态学研究蛛网膜下腔置管成功的雄性SD大鼠分别i.t NS和米诺环素50μg后30min右踝关节外侧皮下注射CFA 50μl,i.t每天1次,持续7d,观察CFA后0、2、6d给药后4h和13d脊髓背角小胶质细胞标志物OX-42、星形胶质细胞标志物GFAP、神经元标志物Fos的表达。结果1.行为学研究结果i.t米诺环素不改变无炎症大鼠的PWTL和MWT;CFA致炎后PWTL缩短、MWT减小,米诺环素抑制致炎后PWTL缩短和MWT减小似呈剂量依赖性;预先及CFA致炎后2d米诺环素i.t可抑制PWTL缩短、MWT减小,第6d则无明显影响。2.形态学研究结果预先i.t米诺环素可以抑制CFA致炎后脊髓背角OX-42、GFAP、Fos的表达;CFA致炎后早期i.t米诺环素可以抑制脊髓背角OX-42、GFAP的表达;CFA致炎后后期i.t米诺环素可以减少脊髓背角OX-42表达,但不能抑制GFAP的表达。结论外周注射CFA可以诱导脊髓小胶质细胞激活;预先和反复鞘内注射米诺环素可以抑制脊髓小胶质细胞激活,减轻炎性痛觉过敏;脊髓小胶质细胞可能参与炎性痛觉过敏的调节。第二部分:鞘内注射米诺环素对CFA诱导的脊髓促炎性细胞因子表达的影响目的观察CFA诱导的外周炎症对脊髓促炎性细胞因子表达的影响和鞘内注射米诺环素的作用。方法蛛网膜下腔置管成功的雄性SD大鼠分别i.t NS和米诺环素30min后右踝关节外侧皮下注射CFA,i.t每天1次,持续7d,观察CFA后4h,2、6d、13d后爪PWTL和MWT的变化及CFA致炎后0,2、6d给药后4h和13d脊髓IL-1β、IL-6、TNF-α表达变化。结果CFA致炎后同侧后肢PTWL缩短和MWT减小,预先和反复i.t米诺环素抑制PTWL缩短和MWT的减小:CFA致炎后0,2、6d给药后4h和13d脊髓IL-1β、IL-6、TNF-α表达明显增加,预先和反复i.t米诺环素减少相应时点脊髓IL-1β、IL-6、TNF-α的表达水平。结论外周注射CFA诱导脊髓促炎性细胞因子分泌增加;预先和反复鞘内注射米诺环素可以减少外周炎症诱导的脊髓促炎性细胞因子分泌;脊髓小胶质细胞可能通过促炎性细胞因子介导炎性痛觉过敏。第三部分:鞘内注射米诺环素对CFA诱导的脊髓Pp38MAPK表达的影响目的观察CFA诱导的外周炎症对脊髓Pp38MAPK蛋白表达变化和米诺环素鞘内注射对其表达的影响。方法蛛网膜下腔置管成功的雄性SD大鼠分别i.t NS和米诺环素30min后右踝关节外侧皮下注射CFA,i.t每天1次,持续7d,观察CFA后4h、2、6、13d的PWTL和MWT的变化及CFA致炎后0,2、6d给药后4h和13d脊髓Pp38MAPK蛋白表达变化。结果CFA致炎后同侧后肢PTWL缩短和MWT减小,预先和反复i.t米诺环素可以抑制PTWL缩短和MWT的减小;CFA致炎后0、2、6d给药后4h和13d脊髓Pp38MAPK蛋白表达增加,预先和反复i.t米诺环素可以抑制相应时点脊髓Pp38MAPK的表达,但仍比基础值增加。结论外周注射CFA可以诱导脊髓Pp38MAPK表达增加;预先和反复鞘内注射米诺环素可以抑制CFA诱导的脊髓Pp38MAPK的表达;脊髓小胶质细胞可能通过p38MAPK调节炎性痛觉过敏。更多还原

【Abstract】 PartⅠthe effect of intrathecal minocycline on CFA-induced arthritis inflammatory pain in rats.Objective To observe the effects of intrathecal（i.t） minocycline on inflammatory hyperalgesia in a rat model of CFA-induced arthritis.Methods 1.Behavioral response:After intrathecal catheter being implanted successfully between L₃ and L₄ vertebrae,adult male Sprague-dawley（SD） rats were pretreated with normal saline or 12.5,25,50μg different dose minocycline intrathecally 30 min prior to subcutaneously injection 50μl complete Freunds adjunvant（CFA） in the right tibio-tarsal joint.Intrathecal normal saline or minocycline was injected once daily for seven consecutive days.Paw withdrawal thermal latency（PWTL） and mechanical withdrawal threshold（MWT） were assessed for 14 successive days.Minocycline 50μg was been given intrathecally on day 2 and day 6 after CFA injection;Intrathecal minocycline 50μg or normal saline was injected 30 min prior to subcutaneously injection 50μl normal saline in the right tibio-tarsal joint.PWTL and MWT were assessed for 24 h.2.Morphologic study:Intrathecal normal saline or minocycline 50μg repectively 30 min prior to subcutaneously injection 50μl CFA in the right tibio-tarsal joint.Intrathecal normal saline or minocycline was injected once daily for seven consecutive days.The changes of spinal dorsal horn microglia,astroglia and neuron response to CFA-induced inflammatory pain were examined by immunohistochemisty methods of OX-42,GFAP and Fos after 4 hours following i.t normal saline or minocycline at 0,2,6 days and on 13 days.Results 1.In the behavioral responses detection:Intrathecal minocycline to noinflamed rats did not alter PWTL and MWT;PWTL was shortened and MWT was decreased after CFA-induced arthritis,pretreated and repeated intrathecal minocycline inhibited the shortening of PWTL and decreasing of MWT to seem in a does-dependent way in inflamed rats.Intrathecal minocycline 50μg inhibited reducing of PWTL and diminishing of MWT on day 2,but failed to alter on day 6 after CFA-induced inflammatory pain.2.Morphologic results:Pretreated and repeated intratheacl minocycline suppressed expressions of OX-42,GFAP and Fos in spinal dorsal horn after CFA-induced imflammatory pain.Intrathecal minocycline inhibited expression of OX-42 and GFAP on day 2,but failed to alter expression of GFAP on day 6 after CFA-induced arthritis.Conclusions Peripheral injection CFA induced spinal microglia activation;Pretreated and repeated intratheacl minocycline inhibited spinal microglia activation that could attenuate inflammatory hyperalgesia;Spinal microglia perhaps involved in inflammatory hyperalgesia.PartⅡThe effect of Intratheacl minocycline on CFA-induced spinal proinflammatory cytokines in the ratObjective To investigate the expression levels of spinal proinflammatory cytokines in CFA-induced arthritis rats and the effect of intratheacl minocycline.Methods After intrathecal catheter being implanted successfully between L₃ and L₄ vertebrae, adult male Sprague-dawley（SD） rats were pretreated with normal saline or minocycline 50μg 30 min prior to subcutaneously injection of 50μl CFA in the right tibio-tarsal joint,intrathecal normal saline or minocycline was injected once daily for seven consecutive days.PWTL and MWT were assessed on 4h,2,6,13 days.The changes of spinal proinflammatory cytokines IL-1β、IL-6、TNF-αlevels were examined by ELISA analysis after 4 hours following intrathecal normal saline at 0,2, 6 days and on 13 days.after CFA injection.Results PWTL was shortened and MWT was decreased after CFA-induced inflammatory pain,pretreated and repeated intratheacl minocycline inhibited shortening of PWTL and decreasing of MWT. Expressions levels of IL-1β,IL-6 and TNF-αin the spine cord increased after 4 hours following intrathecal normal saline at 0,2,6 days and on 13 days after CFA injection, pretreated and repeated intratheacl minocycline markedly reduced increaseing of spinal IL-1β,IL-6 and TNF-αlevels in corresponding time point.Conclusions Peripheral injection CFA could induce the increasing of spinal proinflammatory cytokines levels;Pretreated and repeated intratheacl minocycline could reduce the increasing of CFA-induced spinal proinflammatory cytokines;Spinal microglia perhaps mediate the inflammatory hyperalgesia by proinflammatory cytokines.PartⅢThe effect of Intratheacl minocycline on CFA-induced spinal Pp38MAPK in the ratObjective To investigate the expression of spinal Pp38MAPK in CFA-induced arthritis rats and the effect of intratheacl minocycline.Methods After intrathecal catheter being implanted successfully between L₃ and L₄ vertebrae,adult male Sprague-dawley（SD） rats were pretreated with normal saline or minocycline 50μg 30 min prior to subcutaneously injection 50μl CFA in the right tibio-tarsal joint, intrathecal normal saline or minocycline was injected once daily for seven consecutive days.PWTL and MWT were assessed on 4h,2,6,13 days after CFA injection.The changes of expression of spinal Pp38MAPK（phosphorylated p38 mitogen-activated protein kinase） were examined by Western blot analysis after 4 hours following i.t normal saline or minocycline on 0,2,6 days and on 13 days after CFA injection.Results PWTL was shortened and MWT was decreased after CFA-induced inflammatory pain,pretreated and repeated intratheacl minocycline inhibited shortening of PWTL and decreasing of MWT;Sham rats expressed low level Pp38MAPK;Expression of Pp38MAPK in the spine cord was increased after 4 hours following i.t normal saline or minocycline on 0,2,6 days and on 13 days after CFA injection,pretreated and repeated intrathecal minocycline markedly reduced up-regulation of Pp38MAPK levels of spinal cord in corresponding time point,but is increased compared with baseline level.Conclusions Peripheral injection CFA could induce the expression increasing of spinal Pp38MAPK;Pretreated and repeated intrathecal minocycline could inhibit expression of CFA-induced spinal Pp38MAPK; Spinal microglia perhaps mediated the inflammatory hyperalgesia by p38MAPK pathway.更多还原