鞘内间断注射不同浓度罗哌卡因在长时间对大鼠脊髓神经毒性影响及机制研究
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摘要
目的：1.观察鞘内间断注射0.5%和1%罗哌卡因后28天对大鼠脊髓、神经根超微结构的影响及机制研究,探讨ERK信号通路、Akt信号通路是否参与了罗哌卡因的脊髓神经毒性；观察鞘内间断注射1%罗哌卡因+神经营养素-3(neurotrophin-3)后28天对大鼠脊髓、神经根超微结构的影响及机制,探讨neurotrophin-3(NT-3)是否能减轻和预防罗哌卡因的神经毒性作用。2.体外观察NT-3对于罗哌卡因细胞毒性的影响。为临床上连续腰麻更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144只,体重280-320g,月龄为1.5月。随机分为生理盐水组(NS组,n=36),0.5%罗哌卡因组(M组,n=36),1%罗哌卡因组(R组,n=36),1%罗哌卡因+NT-3组(T组,n=36)。NS组大鼠鞘内注入0.9%生理盐水、M组大鼠鞘内注入0.5%罗哌卡因、而R组和T组大鼠鞘内注入1%罗哌卡因各0.12ml/kg,间隔1.5h给药一次,持续给药12h。其中T组同时再鞘内注入0.1mg/kg的NT-3。各组均观察1d、3d、5d.7d、28d。NS组记为"NS1组、NS3组、NS5组、NS7组、NS14组、NS28组”(n=6)。M组记为“M1组、M3组、M5组、M7组、M14组、M28组”(n=6)。R组记为"R1组、R3组、R5组、R7组、R14组、R28组”(n=6)。T组记为"T1组、T3组、T5组、T7组、T14组、T28组”(n=6)。分别检测给药前和各时间点处死前各组大鼠后肢机械刺激缩足反应阈值即机械痛阈和热潜伏缩爪阈值即热痛阈以及最后一次给药后运动功能评分来观察其行为学的变化；完成行为学检测后的各组大鼠,取脊髓腰膨大及相应节断行光镜和透射电镜观察,并进行病理学评分；Tunel法检测脊髓凋亡；免疫组化检测各组大鼠腰膨大脊髓ERK1(P44MAPK)、Akt及caspase-3蛋白的表达；Western-blot检测各组磷酸化ERK和磷酸化Akt蛋白表达及相对定量比情况。
2.细胞实验：体外观察NT-3对于罗哌卡因细胞毒性的影响将PC12细胞分为三组：对照组(NS组)：不予任何处理,常规培养48小时；罗哌卡因组(R组)：15mM罗哌卡因干预48h；罗哌卡因+NT-3组(T组)：15mM罗哌卡因干预48h并同时给予NT-3100ng/ml处理48小时。检测干预后每组细胞计数；MTT方法测定细胞成活率的变化；台盼兰染色测定每组细胞活性；Western blot法检测各组细胞磷酸化ERK和磷酸化Akt蛋白表达及相对定量比情况。
结果：1.NS组和M组各对应时间组相比,大鼠机械性触诱发痛阈和热刺激诱发痛阈无显著性差异。各组大鼠机械性触诱发痛阈与NS组和M组各对应时间组相比：R1、R3、R5组和T1、T3组大鼠机械触诱发痛闽升高(P<0.05),其中以R1组升高最明显；与R组相对应时间组相比：T5组大鼠机械痛阈显著降低(P<0.05)。与NS组和M组各对应时间组相比：R1、R3、R5组和T1、T3组大鼠热刺激诱发痛阈升高(P<0.05),其中以R1组升高最明显。与R组相对应时间组相比：T5组大鼠热刺激诱发痛阈显著降低(P<0.05)。
2.电镜和光镜结果。电镜：R组给药后R1、R3、R5、R7、R14R28组电镜超微结构有不同程度的改变。R1组水肿明显,神经细胞呈固缩状,核碎裂,大多数空泡状,有凋亡小体出现,R3组神经细胞线粒体清晰,核皱缩,神经细胞局灶水肿,R5组部分神经细胞核皱缩,内质网轻度扩张,神经细胞有部分肿胀,R7组局灶水肿明显,部分线粒体结构模糊,内质网有轻度扩张,部分神经细胞有皱缩,R14组神经细胞肿胀、水肿,线粒体丢失,呈空泡状,内质网有轻度扩张,R28组郎飞氏结正常。而T组中的对应时间组：T1组胞核花边状,皱缩,有凋亡小体出现的趋势,线粒体结构完整,T3组神经细胞核圆,线粒体结构清晰,轻度水肿,T5组线粒体清晰,内质网有轻度扩张,核完整,轻度水肿,T7组神经细胞核轻度皱缩,部分线粒体空泡状,水肿部明显,T14组趋向正常,细胞核圆,线粒体嵴清晰,内质网结构正常,T28组形态正常。光镜：R1、R3、R5、R7组神经元数量较少,体积明显缩小,核明显固缩、深染。个别神经元坏死,部分胶质细胞有增生。R14及R28组神经元数量较多,部分有轴突,部分体积缩小,核固缩、深染。T1、T3、T5组神经元数量减少,体积缩小,核固缩、深染,T5组神经元数量比R5组多,有点状坏死。T7组部分神经元细胞形态正常,大部分可见长轴突,部分神经元体积缩小,核固缩、深染,胶质细胞增生。T14组部分神经元轴突消失,胞浆少,T28组神经元细胞数量比R28多,形态正常。R组和T组的各对应时间组的组织病理学评分在1d、3d、5d、7d,14d分别较NS组和M组各对应时间高,以R组最高(P<0.05)；与R组的各对应时间组比较, T组各对应时间点的T5、T7、T14组的组织病理学评分显著降低(P<0.05)。NS组和M组的电镜结果基本正常。
3.TUNEL染色阳性细胞计数的结果：与NS组的同时间对应组比较,M组染色阳性细胞无显著性增多。与NS组和M组的同时间对应组比较T1、T3、T5组TUNEL染色阳性细胞增多(P<0.05)；R组的R1、R3、R5、R7组染色阳性细胞增多(P<0.05)。与R组同时间对应组比较T组的R1、R5、R7组染色阳性细胞显著减少(P<0.05)。
4.免疫组化检测的Akt、ERK1、caspase-3Akt:R1、R3、R5组以及T1、T3组与NS和M组的对应时间组比较,脊髓灰质后角Akt阳性表达显著升高(P<0.05)；T3、T5、T8组与R组对应时间组比较,Akt阳性表达显著降低(P<0.05)。ERK1:R1、R3、R5R7、R14组以及T1、T3、T5、T7组与NS和M组的对应时间组比较,脊髓灰质神经元细胞的ERK1阳性表达显著升高(P<0.05); T1T3、T5、T14组与R组对应时间组比较,ERK1阳性表达显著降低(P<0.05)。caspase-3:R1、R3、R5、R7、R14组以及T1、T3、T5、 T7组与NS和M组的相对应时间组比较,脊髓灰质后角caspase-3阳性表达显著升高(P<0.05); T1、T3、T5、T14组与R组相对应时间组比较,caspase-3阳性表达显著降低(P<0.05)。与NS组的同时间对应组比较,M组免疫组化检测的Akt、ERK1、caspase-3无显著性差异。
5.Western blot法测定脊髓腰膨大组织磷酸化ERK以及磷酸化Akt结果与NS组各对应时间组相比,M组脊髓腰膨大组织磷酸化ERK蛋白表达水平以及磷酸化Akt蛋白表达水平无显著性差异。与NS组各对应时间组相比：R1、R3、R5、R7、R14、R28组和T1、T3、T5、T7、T14组磷酸化Akt蛋白表达水平降低(P<0.05),其中以R1、R3组水平降低最明显；与R组相对应时间的各组相比：T组与之相对应时间的T5、T7、T28组磷酸化Akt蛋白表达水平显著升高(P<0.05)。与NS组各对应时间组相比：R1、R3、R5、R7组和T1、T3、T5、T7组磷酸化ERK蛋白表达水平降低(P<0.05),其中以R1、R3组降低最明显；与R组相对应时间的各组相比：T1、T3组磷酸化ERK蛋白表达水平显著升高(P<0.05)。
6.体外细胞实验说明：与NS组比较,R组和T组的细胞计数和细胞存活率均减少、细胞活性降低(P<0.05),而蛋白表达均降低(P<0.05),与R组相比较,T组的细胞计数和细胞存活率、细胞活性均增高(P<0.05),蛋白表达均升高(P<0.05)。
结论：
1.间断鞘内注射0.5%罗哌卡因12h的各组大鼠脊髓神经根超微结构、组织病理学在1-28天无改变。
2.间断鞘内注射1%罗哌卡因12h的各组大鼠脊髓神经根超微结构、组织病理学在1-7天发生改变较重,产生的凋亡细胞较多。随着时间的延长,病理改变逐渐恢复,在28天时大部分恢复正常。
3.间断鞘内注射1%罗哌卡因+NT-312h的各组大鼠脊髓神经根超微结构、组织病理学在1-7天发生改变轻微,产生的凋亡细胞少。随着时间的延长,病理改变逐渐恢复,在28天时完全恢复正常。
4.Akt及ERK信号通路介导的细胞凋亡参与了罗哌卡因的神经毒性机制。
5.NT-3的应用可能对罗哌卡因神经毒性有保护作用。图201副,表3个,参考文献135篇
Objective:1. To observe ultrastructural changes and mechanisms of spinal cords and nerve roots of intrathecal administration of0.5%or1%concentrations of ropivacaine after twenty-eight days in rats, and also to investigate whether ERK and Akt signaling pathways and mitochondrial opotosis were involved in spinal neurotoxicity of ropivacaine. To observe the ultrastructural changes of spinal cords and nerve roots of intrathecal administration of1%concentrations of ropivacaine and NT.-3after days in rats, and investigate the therapeutic potential of NT-3in local anesthetic toxicity.2. To examine the effects of NT-3on ropivacaine cell toxicity in vitro, providing theoretical and therapeutic exploration for application of ropivacaine in intrathecal administration.
Methods:1. One hundred and forty-four/144male Sprague Dawley rats weighing280-320g were successfully implant with microspinal cather following the methods of Yaksh. Rats were randomly divided into four groups:group saline (Group NS, n=36、group0.5%ropivacaine (Group M, n=36)、group1%ropivacaine (Group R, n=36) and group ropivacaine+NT-3(Group T, n=36). Rats in group M were received0.5%ropivacaine or rats in group R and T were received1%ropivacaine, or rats in group NS were received only saline0.12ml/kg intrathecal injection with1.5h interval lasted for12h. And at the meantime Group T gives NT-30. lmg/kg (30μg) again. Each group was observed on1d、3d、5d、7d、14d、28d. The group NS was respectively signed with NS1、 NS3、NS5、NS7、NS14、NS28(n=6). The group M was respectively signed with M1、M3、M5、M7、M14、M28(n=6). The group R was respectively signed with R1、R3、R5、R7、R14、R28(n=6). The groupT was respectively signed with T1、T3、T5、T7、T14、T28(n=6). Moter function assessment was investigated after the last injection, while sensory threshold tested by effect of the paw withdrawal latency to heat (PWHL) and mechanical stimulate (PWML) were investigated both before injection and1d、3d、5d、7d、24d、28d after the last injection in all groups. After finishing all these neurofunctional tests, rats were sacrified. Their lumber spinal cords and posterior roots were examined by light and electron microscopy. The spinal cords were also evaluated for apoptosis by an in situ TUNEL assay. Expression cleaved ERK、Akt proteind by western blot. Spinal caspase-3、ERK1、Akt expression were determined by immunohistochemistry.2. Cell experiments were done in order to examine the effects of NT-3on ropivacaine cell toxicity in vitro. Rat pheochromocytoma cells (PC12) were divided into three group, group control (Group NS) without any special treatment, just routine incubation for48h, group ropivacaine (Group R) with incubation of15mmol/L ropivacaine for48h, group ropivacaine+NT-3(Group T) with incubation of15mmol/L ropivacaine and NT-3100ng/ml for48h. Cell counts and cell survival rate detected by MTT were observed after treatment in each group. Measurement of cell viability trypan was observed after treatment in each group. Changes of Akt、ERK protein expression were evaluated by western blot.
Results:1The changes of PWML and PWHL in each groups: PWML increased in group R1、R3、R5and T1、T3compared with that of group NS and M in each time-course (P<0.05). Group R1showed the most significant increase.Group T5showed the significant decrease compared with R groups in each time-course (P<0.05). PWHL increased in group R1、R3、R5and T1、T3compared with that of group NS and M in each time-course (P<0.05). Group R1showed the most significant increase. Group T5showed the significant decrease compared with R groups in each time course (P<0.05).
2.The results of Electron and optical microscope technique:Electron microscope result:different dgrees of changes appeared in group R1、R3、 R5、R7、R14、R28after admistration. Significant edema, mostly turned to pyknosis、karyorrhexis and vacuole, apoptotic body appeared in group R1. Clear mitochondrion, Nuclear shrinkage, focal edema in the nerve cells appeared in group R3. Parts of nerve cells in group R5showed nuclear shrinkage、slight expansion of the endoplasmic reticulum、partly swelling. Group R7showed focal edema, fuzzy structure of several mitochondrion、slight expansion of the endoplasmic reticulum and several cells shrinkage. Group R14showed nerve cells swelling, edema, Loss and vacuole of mitochondrial, slight expansion of the endoplasmic reticulum. Group R28showed nomal ranvier's node. While correspondingly, the group T1showed lace shaped and wizened nucleus, emerging apoptotic bodies, normal chondriosomes. Group T3showed round neurons nucleus clear structure of apoptotic bodies but slightly edema. Group T5showed clear structure of apoptotic bodies, slightly expandation of endoplasmic reticulum, normal nucleus but slightly edema. Group T7showed slightly wizened neurons nucleus. Partial apoptotic bodies were vesicular but no edema. Group T14were almost normal, round nucleus, clear mitochondrialcristae and normal endoplasmic reticulum. Group T28were normal. Optical microscope technique:nerve cells in Group R1、R3、R5、R7were rarely, nucleus showed significant karyopyknosis and darkstainednucleus. Some nerve cell appeared necrosis and partial glial cells appeared hyperplasia. There were more nerve cells, some of which had axone and some of which appeared atrophia and karyopyknosis and darkstainednucleus. The cells in Group T1、T3and T5were decreased in mount and reduced in size. They appeared karyopyknosis and darkstainednucleus. The number of nerve cell in Group T5was more than that in Group R5, and the cells showed spotty necrosis. Partial nerve cells in Group T7were normal, but most cells had long axone and some appeared atrophia, karyopyknosis and darkstainednucleus, glial cells hyperplasia. Some nerve cell in Group T4showed no axone and the little hyalomitome. Compared with R28, nerve cells are more than that in T28, and the cell shape were normal. Compared with Group NS and M in each corresponding time-points, the histopathological of group Rand group T were higher in the1d、3d、5d、7d and14d, the group R was the highest (p<0.05). Compared with Group R in each corresponding time-points, the histopathological assessment scores of Group T5、T7and T14were significantly decreased (p<0.05).
3.The results of Postivie-staining cells counts by TUNEL staining: Positive-staining cells in group T1、T3、T5as well as group R1、R3、 R5、R7were more than that in group NS and M (P<0.05) in the same time-point; However, positive cells in group R1、R5、R7were more than that in corresponding T groups.(P<0.05)
4.The detectived Akt、ERK1、caspase-3by Immunohistochemistry (IHC) Akt:compared group R1、R3、R5as well as group T1、T3with the group NS or M in the same time-point, the positive expression of Akt in Posterior horn of the spinal cord gray matter increased significantly (P<0.05).The positive expression of Akt protein decreased significantly in group T3、T5、T8compared with group R (P<0.05).
ERK1:compared group R1、R3、R5、R7、R14as well as group T1、 T3、T5、T7with the group NS or M in the same time-point, the positive expression of ERK1in the spinal cord gray neurons matter increased significantly (P<0.05). The positive expression of ERK1protein decreased significantly in groupT1、T3、T5、T14compared with group R (P<0.05).
Caspase-3:compared group R1、R3、R5、R7、R14as well as group T1、T3、T5、T7with the group NS in the same time-point, the positive expression of caspase-3in Posterior horn of the spinal cord gray matter increased significantly (P<0.05). The positive expression of caspase-3protein decreased significantly in group T1、T3、T5、T14compared with group R (P<0.05).
5. The examined results of Phosphorylation of Akt and ERK in spinal cord by western blot:
Compared to group NS or M in each time-course, group R1、R3、 R5、R7、R14. R28and group T1、T3、T5、T7、T14showed a decreased expression of Akt protein (P<0.05), and the group R1、R3were the most significantly. Compared to group R in each time-course, there was a significant increase of Akt protein expression in group T5、T7and T28(p<0.05).
Compared to group NS or M in each time-course, group R1、R3、 R5、R7and group T1、T3、T5、T7showed a decreased expression of Akt protein (P<0.05), and the group R1、R3were the most significantly. Compared to group R in each time-course, there was a significant increase of ERK protein expression in group T1、T3(p<0.05).
6. Cell manipulation in vitro:there are decreases of Cell counts and cell survival rate and cell viability in group R and group T compared with group NS (P<0.05). However, the protein expression decreased also in all groups (P<0.05). Compared to group R, the cell counts and cell survival rate and cell viability increased in group T (p<0.05), protein expression increased also in all groups (P<0.05).
Conclusion:1. Repeatedly intrathecal administration of0.5%ropivacaine for12h does not cause ultramicrostructure and histology changes in spinal cords and nerve root of rats from lth day to28th day.2. Repeatedly intrathecal administration of1%ropivacaine for12h may cause the heavier ultramicrostructure and histology changes and also could cause more apoptosis cells in spinal cords and nerve root of rats from lth day to7th day. And along with the time extension, most pathological changes are back to normal on28th day.3. Repeatedly intrathecal administration of1%ropivacaine for12h may cause the lighter ultramicrostructure and histology changes and also could cause less apoptosis cells in spinal cords and nerve root of rats from lth day to7th day. And along with the time extension, all pathological changes are back to normal on14th day.4. Phosphorylation of Akt and ERK apoptosis pathway may be involved in spinal neurotoxicity of ropivacaine.5. NT-3may decline the spinal neurotoxicity of ropivacaine through the inhibition of Akt and ERK apoptosis pathway. There are figures201and tables3and references135.
方法：1.改良Yaksh法鞘内置管成功的雄性SD大鼠144只,体重280-320g,月龄为1.5月。随机分为生理盐水组(NS组,n=36),0.5%罗哌卡因组(M组,n=36),1%罗哌卡因组(R组,n=36),1%罗哌卡因+NT-3组(T组,n=36)。NS组大鼠鞘内注入0.9%生理盐水、M组大鼠鞘内注入0.5%罗哌卡因、而R组和T组大鼠鞘内注入1%罗哌卡因各0.12ml/kg,间隔1.5h给药一次,持续给药12h。其中T组同时再鞘内注入0.1mg/kg的NT-3。各组均观察1d、3d、5d.7d、28d。NS组记为"NS1组、NS3组、NS5组、NS7组、NS14组、NS28组”(n=6)。M组记为“M1组、M3组、M5组、M7组、M14组、M28组”(n=6)。R组记为"R1组、R3组、R5组、R7组、R14组、R28组”(n=6)。T组记为"T1组、T3组、T5组、T7组、T14组、T28组”(n=6)。分别检测给药前和各时间点处死前各组大鼠后肢机械刺激缩足反应阈值即机械痛阈和热潜伏缩爪阈值即热痛阈以及最后一次给药后运动功能评分来观察其行为学的变化；完成行为学检测后的各组大鼠,取脊髓腰膨大及相应节断行光镜和透射电镜观察,并进行病理学评分；Tunel法检测脊髓凋亡；免疫组化检测各组大鼠腰膨大脊髓ERK1(P44MAPK)、Akt及caspase-3蛋白的表达；Western-blot检测各组磷酸化ERK和磷酸化Akt蛋白表达及相对定量比情况。
2.细胞实验：体外观察NT-3对于罗哌卡因细胞毒性的影响将PC12细胞分为三组：对照组(NS组)：不予任何处理,常规培养48小时；罗哌卡因组(R组)：15mM罗哌卡因干预48h；罗哌卡因+NT-3组(T组)：15mM罗哌卡因干预48h并同时给予NT-3100ng/ml处理48小时。检测干预后每组细胞计数；MTT方法测定细胞成活率的变化；台盼兰染色测定每组细胞活性；Western blot法检测各组细胞磷酸化ERK和磷酸化Akt蛋白表达及相对定量比情况。
结果：1.NS组和M组各对应时间组相比,大鼠机械性触诱发痛阈和热刺激诱发痛阈无显著性差异。各组大鼠机械性触诱发痛阈与NS组和M组各对应时间组相比：R1、R3、R5组和T1、T3组大鼠机械触诱发痛闽升高(P<0.05),其中以R1组升高最明显；与R组相对应时间组相比：T5组大鼠机械痛阈显著降低(P<0.05)。与NS组和M组各对应时间组相比：R1、R3、R5组和T1、T3组大鼠热刺激诱发痛阈升高(P<0.05),其中以R1组升高最明显。与R组相对应时间组相比：T5组大鼠热刺激诱发痛阈显著降低(P<0.05)。
2.电镜和光镜结果。电镜：R组给药后R1、R3、R5、R7、R14R28组电镜超微结构有不同程度的改变。R1组水肿明显,神经细胞呈固缩状,核碎裂,大多数空泡状,有凋亡小体出现,R3组神经细胞线粒体清晰,核皱缩,神经细胞局灶水肿,R5组部分神经细胞核皱缩,内质网轻度扩张,神经细胞有部分肿胀,R7组局灶水肿明显,部分线粒体结构模糊,内质网有轻度扩张,部分神经细胞有皱缩,R14组神经细胞肿胀、水肿,线粒体丢失,呈空泡状,内质网有轻度扩张,R28组郎飞氏结正常。而T组中的对应时间组：T1组胞核花边状,皱缩,有凋亡小体出现的趋势,线粒体结构完整,T3组神经细胞核圆,线粒体结构清晰,轻度水肿,T5组线粒体清晰,内质网有轻度扩张,核完整,轻度水肿,T7组神经细胞核轻度皱缩,部分线粒体空泡状,水肿部明显,T14组趋向正常,细胞核圆,线粒体嵴清晰,内质网结构正常,T28组形态正常。光镜：R1、R3、R5、R7组神经元数量较少,体积明显缩小,核明显固缩、深染。个别神经元坏死,部分胶质细胞有增生。R14及R28组神经元数量较多,部分有轴突,部分体积缩小,核固缩、深染。T1、T3、T5组神经元数量减少,体积缩小,核固缩、深染,T5组神经元数量比R5组多,有点状坏死。T7组部分神经元细胞形态正常,大部分可见长轴突,部分神经元体积缩小,核固缩、深染,胶质细胞增生。T14组部分神经元轴突消失,胞浆少,T28组神经元细胞数量比R28多,形态正常。R组和T组的各对应时间组的组织病理学评分在1d、3d、5d、7d,14d分别较NS组和M组各对应时间高,以R组最高(P<0.05)；与R组的各对应时间组比较, T组各对应时间点的T5、T7、T14组的组织病理学评分显著降低(P<0.05)。NS组和M组的电镜结果基本正常。
3.TUNEL染色阳性细胞计数的结果：与NS组的同时间对应组比较,M组染色阳性细胞无显著性增多。与NS组和M组的同时间对应组比较T1、T3、T5组TUNEL染色阳性细胞增多(P<0.05)；R组的R1、R3、R5、R7组染色阳性细胞增多(P<0.05)。与R组同时间对应组比较T组的R1、R5、R7组染色阳性细胞显著减少(P<0.05)。
4.免疫组化检测的Akt、ERK1、caspase-3Akt:R1、R3、R5组以及T1、T3组与NS和M组的对应时间组比较,脊髓灰质后角Akt阳性表达显著升高(P<0.05)；T3、T5、T8组与R组对应时间组比较,Akt阳性表达显著降低(P<0.05)。ERK1:R1、R3、R5R7、R14组以及T1、T3、T5、T7组与NS和M组的对应时间组比较,脊髓灰质神经元细胞的ERK1阳性表达显著升高(P<0.05); T1T3、T5、T14组与R组对应时间组比较,ERK1阳性表达显著降低(P<0.05)。caspase-3:R1、R3、R5、R7、R14组以及T1、T3、T5、 T7组与NS和M组的相对应时间组比较,脊髓灰质后角caspase-3阳性表达显著升高(P<0.05); T1、T3、T5、T14组与R组相对应时间组比较,caspase-3阳性表达显著降低(P<0.05)。与NS组的同时间对应组比较,M组免疫组化检测的Akt、ERK1、caspase-3无显著性差异。
5.Western blot法测定脊髓腰膨大组织磷酸化ERK以及磷酸化Akt结果与NS组各对应时间组相比,M组脊髓腰膨大组织磷酸化ERK蛋白表达水平以及磷酸化Akt蛋白表达水平无显著性差异。与NS组各对应时间组相比：R1、R3、R5、R7、R14、R28组和T1、T3、T5、T7、T14组磷酸化Akt蛋白表达水平降低(P<0.05),其中以R1、R3组水平降低最明显；与R组相对应时间的各组相比：T组与之相对应时间的T5、T7、T28组磷酸化Akt蛋白表达水平显著升高(P<0.05)。与NS组各对应时间组相比：R1、R3、R5、R7组和T1、T3、T5、T7组磷酸化ERK蛋白表达水平降低(P<0.05),其中以R1、R3组降低最明显；与R组相对应时间的各组相比：T1、T3组磷酸化ERK蛋白表达水平显著升高(P<0.05)。
6.体外细胞实验说明：与NS组比较,R组和T组的细胞计数和细胞存活率均减少、细胞活性降低(P<0.05),而蛋白表达均降低(P<0.05),与R组相比较,T组的细胞计数和细胞存活率、细胞活性均增高(P<0.05),蛋白表达均升高(P<0.05)。
结论：
1.间断鞘内注射0.5%罗哌卡因12h的各组大鼠脊髓神经根超微结构、组织病理学在1-28天无改变。
2.间断鞘内注射1%罗哌卡因12h的各组大鼠脊髓神经根超微结构、组织病理学在1-7天发生改变较重,产生的凋亡细胞较多。随着时间的延长,病理改变逐渐恢复,在28天时大部分恢复正常。
3.间断鞘内注射1%罗哌卡因+NT-312h的各组大鼠脊髓神经根超微结构、组织病理学在1-7天发生改变轻微,产生的凋亡细胞少。随着时间的延长,病理改变逐渐恢复,在28天时完全恢复正常。
4.Akt及ERK信号通路介导的细胞凋亡参与了罗哌卡因的神经毒性机制。
5.NT-3的应用可能对罗哌卡因神经毒性有保护作用。图201副,表3个,参考文献135篇
Objective:1. To observe ultrastructural changes and mechanisms of spinal cords and nerve roots of intrathecal administration of0.5%or1%concentrations of ropivacaine after twenty-eight days in rats, and also to investigate whether ERK and Akt signaling pathways and mitochondrial opotosis were involved in spinal neurotoxicity of ropivacaine. To observe the ultrastructural changes of spinal cords and nerve roots of intrathecal administration of1%concentrations of ropivacaine and NT.-3after days in rats, and investigate the therapeutic potential of NT-3in local anesthetic toxicity.2. To examine the effects of NT-3on ropivacaine cell toxicity in vitro, providing theoretical and therapeutic exploration for application of ropivacaine in intrathecal administration.
Methods:1. One hundred and forty-four/144male Sprague Dawley rats weighing280-320g were successfully implant with microspinal cather following the methods of Yaksh. Rats were randomly divided into four groups:group saline (Group NS, n=36、group0.5%ropivacaine (Group M, n=36)、group1%ropivacaine (Group R, n=36) and group ropivacaine+NT-3(Group T, n=36). Rats in group M were received0.5%ropivacaine or rats in group R and T were received1%ropivacaine, or rats in group NS were received only saline0.12ml/kg intrathecal injection with1.5h interval lasted for12h. And at the meantime Group T gives NT-30. lmg/kg (30μg) again. Each group was observed on1d、3d、5d、7d、14d、28d. The group NS was respectively signed with NS1、 NS3、NS5、NS7、NS14、NS28(n=6). The group M was respectively signed with M1、M3、M5、M7、M14、M28(n=6). The group R was respectively signed with R1、R3、R5、R7、R14、R28(n=6). The groupT was respectively signed with T1、T3、T5、T7、T14、T28(n=6). Moter function assessment was investigated after the last injection, while sensory threshold tested by effect of the paw withdrawal latency to heat (PWHL) and mechanical stimulate (PWML) were investigated both before injection and1d、3d、5d、7d、24d、28d after the last injection in all groups. After finishing all these neurofunctional tests, rats were sacrified. Their lumber spinal cords and posterior roots were examined by light and electron microscopy. The spinal cords were also evaluated for apoptosis by an in situ TUNEL assay. Expression cleaved ERK、Akt proteind by western blot. Spinal caspase-3、ERK1、Akt expression were determined by immunohistochemistry.2. Cell experiments were done in order to examine the effects of NT-3on ropivacaine cell toxicity in vitro. Rat pheochromocytoma cells (PC12) were divided into three group, group control (Group NS) without any special treatment, just routine incubation for48h, group ropivacaine (Group R) with incubation of15mmol/L ropivacaine for48h, group ropivacaine+NT-3(Group T) with incubation of15mmol/L ropivacaine and NT-3100ng/ml for48h. Cell counts and cell survival rate detected by MTT were observed after treatment in each group. Measurement of cell viability trypan was observed after treatment in each group. Changes of Akt、ERK protein expression were evaluated by western blot.
Results:1The changes of PWML and PWHL in each groups: PWML increased in group R1、R3、R5and T1、T3compared with that of group NS and M in each time-course (P<0.05). Group R1showed the most significant increase.Group T5showed the significant decrease compared with R groups in each time-course (P<0.05). PWHL increased in group R1、R3、R5and T1、T3compared with that of group NS and M in each time-course (P<0.05). Group R1showed the most significant increase. Group T5showed the significant decrease compared with R groups in each time course (P<0.05).
2.The results of Electron and optical microscope technique:Electron microscope result:different dgrees of changes appeared in group R1、R3、 R5、R7、R14、R28after admistration. Significant edema, mostly turned to pyknosis、karyorrhexis and vacuole, apoptotic body appeared in group R1. Clear mitochondrion, Nuclear shrinkage, focal edema in the nerve cells appeared in group R3. Parts of nerve cells in group R5showed nuclear shrinkage、slight expansion of the endoplasmic reticulum、partly swelling. Group R7showed focal edema, fuzzy structure of several mitochondrion、slight expansion of the endoplasmic reticulum and several cells shrinkage. Group R14showed nerve cells swelling, edema, Loss and vacuole of mitochondrial, slight expansion of the endoplasmic reticulum. Group R28showed nomal ranvier's node. While correspondingly, the group T1showed lace shaped and wizened nucleus, emerging apoptotic bodies, normal chondriosomes. Group T3showed round neurons nucleus clear structure of apoptotic bodies but slightly edema. Group T5showed clear structure of apoptotic bodies, slightly expandation of endoplasmic reticulum, normal nucleus but slightly edema. Group T7showed slightly wizened neurons nucleus. Partial apoptotic bodies were vesicular but no edema. Group T14were almost normal, round nucleus, clear mitochondrialcristae and normal endoplasmic reticulum. Group T28were normal. Optical microscope technique:nerve cells in Group R1、R3、R5、R7were rarely, nucleus showed significant karyopyknosis and darkstainednucleus. Some nerve cell appeared necrosis and partial glial cells appeared hyperplasia. There were more nerve cells, some of which had axone and some of which appeared atrophia and karyopyknosis and darkstainednucleus. The cells in Group T1、T3and T5were decreased in mount and reduced in size. They appeared karyopyknosis and darkstainednucleus. The number of nerve cell in Group T5was more than that in Group R5, and the cells showed spotty necrosis. Partial nerve cells in Group T7were normal, but most cells had long axone and some appeared atrophia, karyopyknosis and darkstainednucleus, glial cells hyperplasia. Some nerve cell in Group T4showed no axone and the little hyalomitome. Compared with R28, nerve cells are more than that in T28, and the cell shape were normal. Compared with Group NS and M in each corresponding time-points, the histopathological of group Rand group T were higher in the1d、3d、5d、7d and14d, the group R was the highest (p<0.05). Compared with Group R in each corresponding time-points, the histopathological assessment scores of Group T5、T7and T14were significantly decreased (p<0.05).
3.The results of Postivie-staining cells counts by TUNEL staining: Positive-staining cells in group T1、T3、T5as well as group R1、R3、 R5、R7were more than that in group NS and M (P<0.05) in the same time-point; However, positive cells in group R1、R5、R7were more than that in corresponding T groups.(P<0.05)
4.The detectived Akt、ERK1、caspase-3by Immunohistochemistry (IHC) Akt:compared group R1、R3、R5as well as group T1、T3with the group NS or M in the same time-point, the positive expression of Akt in Posterior horn of the spinal cord gray matter increased significantly (P<0.05).The positive expression of Akt protein decreased significantly in group T3、T5、T8compared with group R (P<0.05).
ERK1:compared group R1、R3、R5、R7、R14as well as group T1、 T3、T5、T7with the group NS or M in the same time-point, the positive expression of ERK1in the spinal cord gray neurons matter increased significantly (P<0.05). The positive expression of ERK1protein decreased significantly in groupT1、T3、T5、T14compared with group R (P<0.05).
Caspase-3:compared group R1、R3、R5、R7、R14as well as group T1、T3、T5、T7with the group NS in the same time-point, the positive expression of caspase-3in Posterior horn of the spinal cord gray matter increased significantly (P<0.05). The positive expression of caspase-3protein decreased significantly in group T1、T3、T5、T14compared with group R (P<0.05).
5. The examined results of Phosphorylation of Akt and ERK in spinal cord by western blot:
Compared to group NS or M in each time-course, group R1、R3、 R5、R7、R14. R28and group T1、T3、T5、T7、T14showed a decreased expression of Akt protein (P<0.05), and the group R1、R3were the most significantly. Compared to group R in each time-course, there was a significant increase of Akt protein expression in group T5、T7and T28(p<0.05).
Compared to group NS or M in each time-course, group R1、R3、 R5、R7and group T1、T3、T5、T7showed a decreased expression of Akt protein (P<0.05), and the group R1、R3were the most significantly. Compared to group R in each time-course, there was a significant increase of ERK protein expression in group T1、T3(p<0.05).
6. Cell manipulation in vitro:there are decreases of Cell counts and cell survival rate and cell viability in group R and group T compared with group NS (P<0.05). However, the protein expression decreased also in all groups (P<0.05). Compared to group R, the cell counts and cell survival rate and cell viability increased in group T (p<0.05), protein expression increased also in all groups (P<0.05).
Conclusion:1. Repeatedly intrathecal administration of0.5%ropivacaine for12h does not cause ultramicrostructure and histology changes in spinal cords and nerve root of rats from lth day to28th day.2. Repeatedly intrathecal administration of1%ropivacaine for12h may cause the heavier ultramicrostructure and histology changes and also could cause more apoptosis cells in spinal cords and nerve root of rats from lth day to7th day. And along with the time extension, most pathological changes are back to normal on28th day.3. Repeatedly intrathecal administration of1%ropivacaine for12h may cause the lighter ultramicrostructure and histology changes and also could cause less apoptosis cells in spinal cords and nerve root of rats from lth day to7th day. And along with the time extension, all pathological changes are back to normal on14th day.4. Phosphorylation of Akt and ERK apoptosis pathway may be involved in spinal neurotoxicity of ropivacaine.5. NT-3may decline the spinal neurotoxicity of ropivacaine through the inhibition of Akt and ERK apoptosis pathway. There are figures201and tables3and references135.
引文
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