【作者】 张重；

【导师】 陈子华；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 目的观察鞘内注射不同浓度的罗哌卡因及其在不同时间点对大鼠脊髓、神经根超微结构的影响,并探讨P38磷酸化及线粒体介导的凋亡途径是否参与罗哌卡因的脊髓神经毒性,为临床上安全使用罗哌卡因提供理论依据。方法1改良Yaksh法鞘内置管成功的雄性SD大鼠30只,体重为250-320g,随机分为生理盐水组(N1组,n=6)、罗哌卡因组(R1组),而R1组大鼠鞘内分别注入0.25%、0.5%、0.75%、1.0%罗哌卡因0.12μl/g,间隔1.5h给药1次,持续给药48h,分别记为“R0.25组、R0.5组、R0.75组、R1.0组”(n=6),N1组则给予等量的生理盐水,给药方式与R1组一样;另取改良Yaksh法暴露寰枕膜后不置管的6只雄性SD大鼠,体重为250-320g,记为假手术组(S1组,n=6),S1组不给药。通过分别检测给药前和最后1次给药1.5h后各组大鼠后爪机械刺激回缩阈值(即机械痛阈)、热刺激回缩阈值(即热痛阈)的最大效应百分比以及最后1次给药后运动功能评分来观察其行为学的变化;在完成行为学检测后处死大鼠,取脊髓腰膨大及相应节段神经根行光镜和电镜检查,并进行病理学评分;TUNEL法检测脊髓组织细胞凋亡;PT-PCR法检测脊髓组织caspase-3,9,8 mRNA的表达;Western Blot法检测脊髓组织磷酸化p38、活化caspase-3蛋白表达及胞质内细胞色素C释放情况。2改良Yaksh法鞘内置管成功的雄性SD大鼠30只,体重为250-320g,随机分为对照组(N2组,n=6)、罗哌卡因组(R2组)。R2组鞘内注射0.75%罗哌卡因0.12μl/g,间隔1.5h给药1次,分别持续给药12h、24h、36h、48h,记为“R12组、R24组、R36组、R48组”(n=6)。通过监测R2组大鼠给药前和最后1次给药1.5h后机械痛阈、热痛阈的最大效应百分比及最后1次给药后运动功能评分来观察其行为学的变化,并在完成行为学检测后处死大鼠;N2组则不给药,监测N2组给药前及R2组大鼠第1次给药时的机械痛阈、热痛阈的最大效应百分比,并在完成行为学检测后处死;各组大鼠处死后取脊髓腰膨大组织及相应节段神经根与第一部分进行相同的检测。3将PC12细胞分为三组:对照组(N3):不予任何特殊处理,常规培养48h;罗哌卡因组(R组):15mmol/L罗哌卡因处理48h;罗哌卡因+p38抑制剂SB203580组(R+S组):15mmol/L罗哌卡因+10μmol/L p38特异性抑制剂SB203580处理48h。检测干预后各组细胞计数以及MTT法测定细胞存活率的变化,Western Blot法检测磷酸化p38、活化caspase-3蛋白表达及胞质内细胞色素C释放的情况。结果1各组大鼠热痛阈和机械痛阈的最大效应百分比无统计学差异(P＞0.05),R1各组大鼠每次注药后30s内出现双后肢的麻痹,R0.25组双后肢恢复最快(P＜0.05),R1.0组双后肢恢复最慢(P＜0.05),R0.5组和R0.75组,介于两者其中;电镜结果显示:R0.5组线粒体结构模糊,部分粗面内质网轻度扩张,部分有髓神经纤维髓鞘板层结构疏松;R0.75组线粒体结构肿胀,粗面内质网轻中度扩张,少数神经元的胞核出现固缩,有髓神经纤维的板层结构疏松;R1.0组线粒体呈空泡样变性;粗面内质网极度扩张,神经元细胞核皱缩、甚至裂解为碎块,产生凋亡小体,髓鞘增厚,甚至崩解消失;R0.75与R1.0组的组织病理学损伤评分、TUNEL染色阳性细胞较N1组高,(P＜0.05);R0.5、R0.75、R1.0组的caspase-3、9的mRNA表达以及磷酸化p38、活化caspase-3的蛋白表达、胞质中细胞色素C的释放较N1组高(P＜0.05);2与N2组比较,R12、R24、R36、R48组大鼠给药前后热痛阈和机械痛阈的最大效应百分比和运动阻滞评分无统计学差异(P＞0.05);电镜结果显示:R12与R24组线粒体轻度水肿,少数内质网轻中度扩张,髓鞘的板层结构疏松,R36组和R48组线粒体、内质网、髓鞘板层结构的肿胀程度更为严重,神经元胞核出现一定程度的固缩,部分染色质出现浓缩状态;R36组和R48组大鼠的组织病理学损伤评分、TUNEL染色阳性细胞数较N2组高(P＜0.05);R24、R36、R48组大鼠脊髓组织中caspase-3、9 mRNA的表达水平较N2组高(P＜0.05);R12、R24、R36、R48组大鼠脊髓组织中磷酸化p38蛋白的表达较N2组高,以R12组磷酸化p38水平最高(P＜0.05);而R24、R36、R48组大鼠脊髓组织中活化caspase-3的蛋白水平、胞质中细胞色素C的释放水平较N2组高,以R48组活化caspase-3蛋白和胞质中细胞色素C水平最高(P＜0.05);3与N3组相比,R组、R+S组的细胞计数和细胞存活率均减少(P＜0.05),而磷酸化P38、活化caspase-3蛋白表达以及胞质中细胞色素C的释放均增高(P＜0.05);与R组相比,R+S组的细胞计数和细胞存活率均增高(P＜0.05),磷酸化p38、活化caspase-3蛋白表达及胞质中细胞色素C的释放均减少(P＜0.05)。结论1鞘内注射0.5%,0.75%,1.0%的罗哌卡因48小时的大鼠脊髓和神经根产生浓度依赖性的超微结构改变,其中0.75%,1.0%罗哌卡因引起的组织病理学改变较对照组重,同时产生的凋亡细胞较对照组多。2鞘内注射0.75%的罗哌卡因24h,36h,48h可使大鼠脊髓和神经根产生时间依赖性的超微结构改变,超过36h可使脊髓组织病理学改变较对照组重,同时产生的凋亡细胞较对照组多。3 P38的磷酸化以及线粒体介导的细胞凋亡参与了罗哌卡因神经毒性的机制,同时P38的磷酸化先于活化caspase-3蛋白和胞质中细胞色素C水平的升高。4 P38特异性抑制剂SB203580的应用可通过降低活化caspase-3蛋白和胞质中细胞色素C的水平来减少罗哌卡因对PC12细胞的毒性作用。更多还原

【Abstract】 ObjectiveTo observe the ultramicrostructure of spinal cords and nerve roots of intrathecal administration of different concentrations of ropivacaine at different time points in rats,and also to investigate whether mitochondrial apoptosis and phosphorylation of p38 were involved in spinal neurotoxcity of ropivacaine,providing theoretical exploration for the application of ropivacaine in intrathecal administration.Methods1 Thirty male Sprague Dawley rats weighing 250-320g were successfully implanted with microspinal catheter following the methods of Yaksh.Rats were randomly divided into two groups:group saline (Group N1,n=6) and group ropivacaine(Group R1).Rats in Group R1 were divided into four subgroups to receive 0.25%,0.5%,0.75%,1.0% ropivacaine 0.12μl/g intrathecal injection with 1.5h interval lasted for 48h.These subgroups were respectively signed with Group R0.25,R0.5, R0.75,R1.0(n=6).Rats in Group N1 were received saline accordingly. Rats in sham group(Group S1,n=6) were suffered the operation of intrathecal catheter as Yaksh’s method but no catheters were implanted. Rats in Group S1 didn’t receive any injection.Moter function assessment was investigated after the last injection,while sensory threshold tested by percent maximum possible effect(%MPE) of the paw withdrawal latency to heat(PWHL) and mechanical stimulate (PWML) were investigated before injection and 1.5h after the last injection in all groups.After finishing all these neurofuctional tests,rats were sacrificed.Their lumber spinal cords and the posterior roots were examined by light and electron microscopy,the spinal cords were also evaluated for apoptosis by an in situ TUNEL assay.Expression of caspase-3,9,8 mRNA were evaluated by PT-PCR,expression of cleaved caspase-3,phosphorylate-p38 protein and release of cytochrome C in cytosolic were evaluated by western blot.2 Thirty male Sprague Dawley rats weighing 250-320g were successfully implanted with microspinal catheter as Yaksh’s methods.Rats were randomly assigned to two groups:group control (Group N2,n=6);group ropivacaine(Group R2).Rats in Group R2 were randomly divided into four subgroups to receive 0.75%ropivacaine 0.12μl/g intrathecal injection with 1.5h interval,lasted for 12h,24h,36h and 48h.These subgroups were respectively signed with GroupR12, R24,R36,R48(n=6).Rats in Group N2 didn’t receive any injection. Moter function assessment was investigated after the last injection, while sensory threshold tested by%MPE of PWHL and PWML in Group R2 were investigated before injection and 1.5h after the last injection.These neurofuctional tests in Group N2 were observed before injection and the time of first injection in Group R2.Rats in all groups were sacrificed after finishing neurofuctional tests,their lumber spinal cords and nerve roots were gained for the same detection as the first experiment.3 Rat pheochromocytoma cells(PC12) were divided into three group,group control(Group N3) without any special treatment,just rountine incubation for 48h,group ropivacaine(Group R) with incubation of 15mmol/L ropivacaine for 48h,group ropivacaine+ SB203580(Group R+S) with incubation of 15mmol/L ropivacaine and SB203580(a p38 special inhibitor) 10μmol/L for 48h.Cell counts and cell survival rate detected by MTT were observed after treatment in each group.Changes of cleaved caspase-3,phosphorylate-p38 protein expression and cytochrome C release in cytosolic were evaluated by western blot.Results1%MPE values of PWHL and PWML in all groups were not significantly different(P＞0.05).The recovery time to ambulation of rats in Group R1.0 was longest,while in Group R0.25 was shortest(P＜0.05).The results of electron microscopy in Group N1,S1 and R0.25 were normal.Mild edema of mitochondria and endoplasmic reticulum,loosen of myelin sheaths showed in Group R0.5,moderate edema of mitochondria and endoplasmic reticulum,neuclear pyknosis were detected in Group R0.75,most severe changes such as serious edema of mitochondria and endoplasmic reticulum,demyelination,degeneration of axons and neuclear pyknosis even apoptotic body in nucleus were presented in Group R1.0.The histologic injury scores and positive cells with TUNEL staining in Group R0.75,1.0 were higher than Group N1(P＜0.05),expression of caspase-3,9 mRNA and cleaved caspase-3, phosphorylate-p38 protein and release of cytosolic cytochrome C in Group R0.5,0.75,1.0 were higher than Group N1(P＜0.05)2%MPE values of PWHL and PWML,the recovery time to ambulation of all rats in Group R2 were not significantly different from those of the Group N2(P＞0.05).The results of electron microscopy in Group R12 and R24 showed mild edema of mitochondria and endoplasmic reticulum,slightly loosen of myelin sheaths,more severe edema of mitochondria and endoplasmic reticulum,neuclear pyknosis presented in Group R36 and R48.The histologic injury scores,positive cells with TUNEL staining in Group R36,48 were higher than Group N2 (P＜0.05).Expression of caspase-3,9 mRNA in Group R24,36,48 were higher than Group N2(P＜0.05),expression of phosphorylate-p38 protein in Group R12,24,36,48 were higher than Group N1(P＜0.05), expression of cleaved caspase-3 protein and release of cytosolic cytochrome C in Group R24,36,48 were higher than Group N2(P＜0.05)3 Cell counts and cell survival rates in Group R and R+S were lower than Group N3(P＜0.05),cleaved caspase-3,phosphorylate-p38 protein expression and cytosolic cytochrome C release in Group R and R+S were higher than Group N3(P＜0.05),Cell counts and cell survival rates in Group R+S were higher than Group R(P＜0.05), expression of cleaved caspase-3,phosphorylate-p38 protein and release of cytosolic cytochrome C in Group R+S were lower than Group R(P＜0.05)Conclusions1 Repeatedly intrathecal administration of 0.5%,0.75%,1.0% ropivacaine for 48h may cause dose-dependent ultramicrostructure changes in spinal cords and nerve root of rats.Among them,0.75%,1.0% ropivacaine could cause the higher histologic injure scores and more apoptosis cells. 2 Repeatedly intrathecal administration 0.75 ropivacaine may cause time-dependent ultramicrostructure changes in spinal cords and nerve roots of rats.Among them,0.75%ropivacaine intrathecal administed beyond 36h could cause the higher histologic injure scores and more apoptosis cells.3.Phosphorylation of p38 protein and mitochondrial apoptosis pathway may be involved in spinal neurotoxicity of ropivacaine, moreover,it is suggested that phosphorylation of p38 protein may happen precede the increase of cleaved-caspase 3 and cytostolic cytochrome C.4.SB203580,a specific p38 inhibitor may decline the spinal neurotoxicity of ropivacaine through the reduction of cleaved-caspase 3 and cytostolic cytochrome C.更多还原