【作者】 陈琼；

【导师】 熊利泽； 王强；

【作者基本信息】 第四军医大学 ， 麻醉学， 2008， 硕士

【摘要】 主动脉瘤手术或脊髓手术等因术中阻断主动脉,均可能引起不同程度的脊髓缺血,导致神经功能障碍,严重者表现为急性或迟发性截瘫。虽然临床已采用脑脊液引流、低温、缺血和药物预处理等各种方法加以防治,但胸腹主动脉瘤手术患者的脊髓损伤发生率仍高达3%¹8%。因此,能否寻找有效措施减轻再灌注损伤已成为人们研究的热点。缺血后处理（ischemic postconditioning）概念的提出,为器官保护提供了新选择和新思路,因为后处理是在恢复全面血液灌注之前施予,方法简单、操作方便,无疑具有更直接的临床应用价值。本实验室以前的研究已经证明缺血后处理可以减轻兔脊髓再灌注损伤,其可能的机制与诱导热休克蛋白表达和抑制再灌注时氧自由基（OFR）的过量生成有关。麻醉药物如吸入麻醉药后处理可否模拟缺血后处理效应减轻脊髓再灌注损伤尚未见报道。七氟烷（Sevoflurane）是一种新型吸入麻醉药,因其麻醉诱导迅速、维持平稳、苏醒快速完全而广泛应用于临床麻醉,而且研究已证实七氟烷对心肌缺血再灌注损伤有一定的保护作用。因此,本研究采用兔脊髓缺血再灌注模型,研究七氟烷后处理能否减轻兔脊髓再灌注损伤,并探讨测定七氟烷后处理与氧自由基、机体抗氧化酶活性以及线粒体ATP敏感性钾通道(K_ATP)的关系,明确七氟烷后处理保护脊髓损伤的部分机制。实验一七氟烷后处理减轻兔脊髓缺血再灌注损伤目的探讨七氟烷后处理是否能减轻兔脊髓缺血再灌注损伤。方法清洁级雄性新西兰大白兔48只,随机分为6组（n=8）:假手术组（Sham组）,仅行单纯手术操作但不阻闭腹主动脉;对照组（Control组）,行单纯缺血再灌注,即阻闭腹主动脉20 min后进行再灌注;纯氧后处理组（O₂组）,在再灌注前5 min给予100% O₂,持续13 min。七氟烷后处理组（Sevo 0.5组/Sevo 1.0组/Sevo 1.5组）,分别在再灌注前5 min给予0.5、1.0及1.5MAC七氟烷处理,持续10 min,然后100% O₂洗脱3 min。再灌注48 h对所有动物的后肢运动功能进行评分并取脊髓(L_5～7)行HE染色对脊髓前角正常神经元进行计数。结果再灌注48 h,七氟烷后处理组动物后肢运动功能评分显著于高于对照组（P<0.05）而Sevo 0.5、Sevo 1.0和Sevo 1.5组间无显著差异。七氟烷后处理组脊髓前角正常神经元数量明显多于对照组（P<0.05）,而且Sevo 1.0组脊髓前角正常神经元数量明显多于Sevo 0.5及Sevo 1.5组（P<0.05）。结论七氟烷后处理可减轻兔脊髓缺血再灌注损伤,1.0MAC七氟烷后处理的保护效应较强。实验二氧自由基清除剂MPG对七氟烷后处理脊髓保护作用的影响目的探讨氧自由基在七氟烷后处理减轻兔脊髓缺血再灌注损伤中的作用。方法雄性新西兰大白兔36只,随机分为6组（n=6）。纯氧组（O₂）及七氟烷（Sevo）组,分别接受纯氧和1.0MAC七氟烷后处理并于再灌注前15 min开始静脉泵注20 min去离子水0.5 ml·kg^-1·min^-1;MPG+七氟烷（MPG+Sevo）及MPG+纯氧组（MPG+O₂）,分别接受1.0MAC七氟烷和纯氧后处理并于再灌注前15 min开始静脉泵注20 min 2%氧自由基清除剂2-巯基丙酰甘氨酸（MPG）0.5 ml·kg^-1·min^-1;七氟烷+MPG（Sevo+MPG）及纯氧+MPG组（O₂+MPG）,分别接受1.0MAC七氟烷和纯氧后处理并于再灌注后15 min静脉泵注20 min 2% MPG 0.5 ml·kg^-1·min^-1。各后处理组后处理方法同第一部分实验后处理条件。再灌注48 h对所有动物的后肢运动功能进行评分并取脊髓（L5～7）行HE染色观察对脊髓前角正常神经元进行计数。结果再灌注48 h,Sevo及Sevo+MPG组后肢运动功能评分显著高于O₂组及MPG+Sevo组（P<0.05）,并且脊髓前角正常神经元数量显著多于O₂组及MPG+Sevo组（P<0.05）,而Sevo和Sevo+MPG组后肢运动功能评分和脊髓前角正常神经元计数差异无显著意义。结论氧自由基清除剂MPG可消除七氟烷后处理的脊髓保护作用,表明氧自由基参与七氟烷后处理减轻兔脊髓缺血再灌注损伤。实验三七氟烷后处理对兔脊髓再灌注后抗氧化酶活性及MDA含量的影响目的探讨七氟烷后处理是否通过增加再灌注期间内源性抗氧化物酶的活性减轻兔脊髓缺血再灌注损伤。方法雄性新西兰大白兔80只,随机分为4组（n=20）:假手术组（Sham组）:操作同对照组,但不阻闭腹主动脉;对照组（Control组）:阻闭腹主动脉20 min后再灌注;纯氧（O₂组）及七氟烷后处理组（Sevo组）:阻闭腹主动脉20 min,采用纯氧以及1.0MAC七氟烷后处理,后处理方法同第一部分实验后处理条件。在再灌注的1 h、6 h、24 h和48 h分别取脊髓（L5⁷）,采用分光光度法测定脊髓超氧化物歧化酶（SOD）、过氧化氢酶（CAT）及谷胱甘肽过氧化物酶（GSH-px）活性及丙二醛（MDA）含量测定（各时间点n=5）。再灌注6 h、24 h和48 h在取材前分别行后肢运动功能评分。结果（1）再灌注6 h、24 h和48 h时Sevo组后肢运动功能评分显著高于Control组（P<0.05）。（2）Sevo组脊髓组织中SOD活性在再灌注早期1 h、6 h及24 h显著高于Control组（P<0.05）; CAT活性在再灌注1 h、6 h显著高于Control组（P<0.05）。Sevo组各时间点脊髓组织GSH-px活性与Control组相比差异无显著意义。（3）Sevo组脊髓组织MDA含量在再灌注6 h、24 h和48 h明显低于Control组（P<0.05）。结论七氟烷后处理可通过上调再灌注后脊髓SOD及CAT活性,减少脊髓脂质过氧化,从而减轻脊髓缺血再灌注损伤。实验四氧自由基清除剂MPG对七氟烷后处理后脊髓抗氧化酶活性及MDA含量的影响目的探讨氧自由基清除剂MPG对七氟烷后处理后脊髓抗氧化酶活性及MDA的影响。方法雄性新西兰大白兔24只,随机分为4组（n=6）:对照组（Control组）:阻闭腹主动脉20 min后再灌注并于再灌注前15 min开始静脉泵注去离子水0.5 ml·kg^-1·min^-1持续20 min;七氟烷后处理组（Sevo组）:阻闭腹主动脉20 min,采用1.0MAC七氟烷后处理,并于再灌注前15 min开始静脉泵注去离子水0.5 ml·kg^-1·min^-1持续20 min;MPG+七氟烷组（MPG+Sevo组）:阻闭腹主动脉20 min,采用1.0MAC七氟烷后处理,并于再灌注前15 min开始静脉泵注2% MPG 0.5 ml·kg^-1·min^-1持续20 min;MPG组（MPG组）:阻闭腹主动脉20 min,再灌注前15 min开始静脉泵注20 min 2% MPG 0.5 ml·kg^-1·min^-1持续20 min。各后处理组方法同第一部分实验后处理条件。再灌注6 h行后肢运动功能评分,并取脊髓（L5～7）用分光光度法测定超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性及丙二醛（MDA）含量测定,并在取材前分别行后肢运动功能评分。结果再灌注6 h时Sevo组后肢运动功能评分和脊髓SOD、CAT活性均显著高于Control组、MPG组以及MPG+Sevo组（P<0.05）;脊髓MDA含量低于Control组、MPG组以及MPG+Sevo组（P<0.05）;而Control组、MPG组以及MPG+Sevo组之间无显著差异。结论氧自由基清除剂MPG通过抑制七氟烷后处理引起的抗氧化酶活性增高而取消了七氟烷后处理的脊髓保护作用。实验五线粒体ATP敏感性钾通道抑制剂5-HD对七氟烷后处理脊髓保护作用的影响目的探讨线粒体ATP敏感性钾通道抑制剂5-HD对七氟烷后处理保护兔脊髓缺血再灌注损伤的影响。方法雄性新西兰大白兔32只,随机分为4组（n=8）:纯氧（O₂）组及七氟烷（Sevo）组:阻闭腹主动脉20 min后再灌注,分别接受纯氧和1.0 MAC七氟烷后处理并于再灌注前10 min静脉注射生理盐水2 ml/kg;5-HD+七氟烷（5-HD+Sevo）组及5-HD+纯氧（5-HD+O₂）组:分别接受1.0MAC七氟烷和纯氧后处理并于再灌注前10min静脉注射10%选择性线粒体ATP敏感性钾通道抑制剂5-羟基癸酸甘油酯（5-HD）2 ml/kg。再灌注48 h对所有动物的后肢运动功能进行评分,并取脊髓（L5～7）行HE染色观察脊髓前角正常神经元计数。结果再灌注48 h,Sevo组后肢运动功能评分显著高于O₂组、5-HD+Sevo组和5-HD+O₂组（P<0.05）,并且脊髓前角正常神经元数量显著多于O₂组、5-HD+Sevo组和5-HD+O₂组（P<0.05）,而O₂组、5-HD+Sevo组和5-HD+O₂组后肢运动功能评分和脊髓前角正常神经元计数差异无显著意义。结论线粒体ATP敏感性钾通道抑制剂5-HD可消除七氟烷后处理的脊髓保护作用,表明七氟烷后处理通过激活线粒体ATP敏感性钾通道的开放从而减轻兔脊髓缺血再灌注损伤。小结1. 0.5、1.0、1.5MAC七氟烷后处理均可减轻兔脊髓缺血再灌注损伤,其中1.0MAC七氟烷后处理的保护效应较强。2.七氟烷后处理可以诱导氧自由基的适度产生从而上调内源性抗氧化酶的活性,抑制再灌注期间脂质过氧化,产生脊髓保护效应。3.七氟烷后处理通过激活线粒体ATP敏感性钾通道开放从而减轻兔脊髓缺血再灌注损伤。更多还原

【Abstract】 Temporary aortic occlusion in surgical repair of thoracic and thoracoabdominal aneurisms can cause different extent of spinal cord ischemia which leads to the nerve functional impairment, and then come to acute or delayed paraplegia. Thereby it calamitously affects the surgical outcomes, results in a great of the burden of psycho-economy and remains a persistent clinical problem. To date, numerous clinical and laboratory studies, including our studies, in attempt to decrease the risk of this devastating complication have been reported. Despite various refinements in surgical techniques and developments in adjunctive measures, including hypothermia, cerebrospinal fluid drainage, ischemic and chemicals preconditioning, have been used for reducing the impact of the ischemia and preserving spinal cord function, spinal cord ischemia is present in approximately 3% to 18% of patients, depending on the type of aneurysm and other combined diseases. Therefore, other novel measures to protect against spinal cord ischemic injury require further investigation. Postconditioning, defined as the process of limiting ischemia reperfusion injury-induced damage by application of repetitive short ischemic windows during early reperfusion, provides a new option and good ideal for neurprotection. Clinically, postconditioning is well suited for vaso-occlusive emergencies and elective surgical settings involving clamping of arteries and subsequent release. Our previous studies demonstrated that ischemic postconditioning could alleviate spinal cord ischemia/reperfusion （I/R） injury in rabbits through induction of heat shock proteins （HSP） expression and inhibition of overdose production of oxygen free radicals （OFR） during reperfusion. Recent evidences have opened exciting research avenues into pharmacotherapeutic agents mimicking postconditioning-induced protection.Sevoflurane, a novel inhalation anesthetic, is wildly applied in clinic anesthesia because of its rapid induction, stable maintainence and complete recovery. Moreover, studies have demonstrated that sevoflurane protected myocardial ischemia/reperfusion （I/R） injury. However, it is unknown whether postconditioning with sevoflurane could alleviate spinal cord I/R injury. Thus, in the present study, using the model of the spinal cord ischemia/reperfusion （I/R） injury in rabbits induced by infrarenal aorta occlusion, we investigated that the protective effect of sevoflurane postconditioning on spinal cord ischemia/reperfusion injury in rabbits and its mechanisms including the role of oxygen free radicals, antioxidase, mitochondrial ATP-sensitive potassium channel.Experiment 1. Sevoflurane postconditioning induces neuroprotection against spinal cord ischemia/reperfusion injury in rabbits Objective To investigate whether postconditoning with sevoflurane could alleviate spinal cord I/R injury in rabbits. Methods Forty-eight male New Zealand white rabbits were randomly assigned to six groups （n=8 each）. Animals in the sham group only underwent sham-operation. Animals in the control group underwent spinal cord ischemia for 20 min without postconditioning. Animals in O₂ postconditioning group （Group O₂） inhaled 100% O₂ at 5 min before reperfusion and last for 13 min. Animals in sevoflurane postconditioning groups （Group Sevo 0.5, Sevo 1.0 and Sevo 1.5） inhaled 0.5, 1.0, 1.5 minimum alveolar concentration （MAC） sevoflurane in 100% O₂ at 5 min before reperfusion for 10 min, and then inhaled 100% O₂ for 3 min to wash out the remaining sevoflurane. Forty-eight hours after reperfusion, hind-limb motor function and histopathology of the spinal cord were assessed in a blinded fashion. Results （1） The neurologic and histopathologic outcomes in the sevoflurane postconditioning groups were better than those in the control group （P<0.05）. （2） The histopathologic outcomes in Sevo 1.0 group were better than that in Sevo 0.5 and Sevo 1.5 groups （P<0.05）. Conclusion Postconditoning with sevoflurane could alleviate spinal cord I/R injury in rabbits. Postconditioning with 1.0 MAC sevoflurane offers better neuroprotective effect.Experiment 2. Effect of 2-mercaptopropionylglycine on the neuroprotection of sevoflurane postconditioning against spinal cord ischemia/reperfusion injuryObjective To investigate whether the benefical effect induced by sevoflurane postconditioning against spinal cord I/R is depend on oxygen free radicals in rabbits. Methods Thirty-six male New Zealand White rabbits were randomly assigned to six groups （n=6 each）. Animals in O₂ and Sevo groups received deionized water (0.5 ml·kg^-1·min^-1 intravenously) 10 min before postconditioning with 100% O₂ and 1.0 MAC sevoflurane for 20 min, respectively. In the MPG+Sevo and MPG+O₂ groups, 2% 2-mercaptopropionylglycine （MPG, a potent oxygen free radical scavenger, dissolved in deionized water） was administered (0.5 ml·kg^-1·min^-1 intravenously) at the same time point. In the Sevo+MPG（Sevo+MPG）and O₂+MPG（O₂+MPG）groups, 2% MPG was administered (0.5 ml·kg^-1·min^-1 intravenously) 10 min after postconditioning with 100% O₂ and 1.0 MAC sevoflurane for 20 min, respectively. Spinal cord ischemia was induced by an infrarenal aorta clamping for 20 min. Forty-eight hours after reperfusion, hind-limb motor function and histopathology of the spinal cord were assessed in a blinded fashion. Results （1） The neurologic and histopathologic outcomes in the Sevo and Sevo+MPG groups were better than those in the others groups （P<0.05）. （2） There were no significant diffierences in hind-limb motor function scores and the number of normal neurons at the anterior spinal cord of animals in the Sevo group in comparison with the Sevo+MPG group. Conclusion 2-mercaptopropionylglycine, a scavanger of reactive oxygen species, completely abolished the neuroprotection induced by sevoflurane postconditioning, which indicated that sevoflurane postconditioning against spinal cord I/R injury via release of oxygen free radicals in rabbits.Experiment 3. Effect of sevoflurane postconditioning on antioxidant enzymes activies and malondialdehyde content of spinal cord tissue during reperfusion in rabbitsObjective To investigate the effect of sevoflurane postconditioning on antioxidant enzymes activities and malondialdehyde content of spinal cord tissue after spinal cord I/R injury in rabbits. Methods Eighty male New Zealand rabbits were randomly divided into 4 groups （n=20 each）. Sham group: the procedure was the same as the I/R group without aortic occlusion; I/R group: rabbits underwent occlusion of the infrarenal abdominal aorta for 20 min, followed by reperfusion; O₂ group （100% O₂ postconditioning） and Sevo group （sevoflurane postconditioning）: rabbits were subjected to 100% O₂ and 1.0 MAC sevoflurane postconditioning, respectively, other procedures were the same as in the I/R group. After the neurological outcomes were assessed, the spinal cord tissue （L5⁷） was sampled at 1 h, 6 h, 24 h and 48 h after reperfusion （n=5 at each time point）, and frozen at -70℃for the determination of superoxide dismutase （SOD）, catalase （CAT） activities and malondialdehyde （MDA） content by spectrophotometric methods. The neurological outcomes were assessed before sampled at 6h, 24h and 48h after reperfusion, respectively. Results （1） The motor function scores of the Sevo group were significantly better than those of I/R group at 6h, 24h and 48h after reperfusion （P<0.05）. （2） SOD activities of spinal cord tissue were significantly higher compared with those in I/R group at 1 h ,6 h and 24 h after reperfusion （P<0.05）. CAT activities of spinal cord tissue in Sevo group were significantly higher compared with those in I/R group at 1 h and 6 h after reperfusion （P<0.05）. No significant difference was observed between Sevo and I/R groups at corresponding time points on the activities of glutathione peroxidase （GSH-px）. （3） Compared with the I/R group, Sevo group produced a significant reduction of the MDA content at 6 h , 24 h and 48 h after reperfusion （P<0.05）. Conclusion The protective effects of sevoflurane postconditioning against spinal cord ischemic/reperfusion injury is mediated, at least partially, by up-regulating the activities of SOD and CAT of spinal cord tissue during early reperfusion phase.Experiment 4. Effect of 2-mercaptopropionylglycine on antioxidant enzymes activies and malondialdehyde content of spinal cord after sevoflurane postconditioningObjective To investigate the effect of 2-mercaptopropionylglycine （MPG） on the antioxidant enzymes activies and malondialdehyde content of spinal cord tissue after sevoflurane postconditioning. Methods Twenty-four male New Zealand rabbits were randomly divided into 4 groups （n=6 each）. All animals underwent spinal cord ischemia for 20 min. Animals in control group received deionized water (0.5 ml·kg^-1·min^-1 intravenously) 15 min before reperfusion for 20 min. Sevo groups received deionized water (0.5 ml·kg^-1·min^-1 intravenously) 10 min before postconditioning with 1.0 MAC sevoflurane for 20 min. In the MPG+Sevo group, 2% MPG was administered (0.5 ml·kg^-1·min^-1 intravenously) 10 min before postconditioning with 1.0 MAC sevoflurane for 20min. In the MPG group, 2% MPG group was administered (0.5 ml·kg^-1·min^-1 intravenously) 15 min before reperfusion. After the neurological outcomes were assessed at 6 h after reperfusion, the spinal cord tissue was sampled at 6 h after reperfusion, and frozen at -70℃for the determination of antioxidant enzyme activities and malondialdehyde （MDA） content by spectrophotometric methods. The neurological outcomes were assessed before sampled at 6h after reperfusion. Results （1） The motor function scores of the Sevo group were significantly better than those of the control group, MPG+Sevo group and MPG group at 6 h after reperfusion （P<0.05）. （2） Activities of superoxide dismutase （SOD） and catalase （CAT） of spinal cord tissue in Sevo group were significantly higher compared with those in the control group, MPG+Sevo group and MPG group 6 h after reperfusion.（P<0.05）. （3） Compared with the others groups, Sevo group produced a significant reduction of the MDA level at 6h after reperfusion（P<0.05）. Conclusion MPG administration before each session of sevoflurane postconditioning completely abolished the increase of CAT and SOD activities and the reduction of the MDA level induced by sevoflurane postconditioning.Experiment 5. Effect of 5-hydroxydecanoate on neuroprotection of sevoflurane postconditioning against spinal cord ischemia/reperfusion injuryObjective To investigate whether the neuroprotective effectS induced by sevoflurane postconditioning against spinal cord I/R is depend on mitochondrial ATP-sensitive potassium （KATP） channels in rabbits. Methods Thirty-two male New Zealand rabbits were randomly divided into 4 groups （n=8 each）. Animals in O₂ and Sevo groups received saline （2 ml/kg intravenously） 10 min before postconditioning with 100% O₂ and 1.0 MAC sevoflurane, respectively. In the 5-HD+Sevo and 5-HD+O₂ groups, 10% 5-hydroxydecanoate （5-HD, a specific mitochondrial KATP channel blocker, dissolved in saline） was administered （2 ml/kg intravenously） at the same time point. Forty-eight hours after reperfusion, hind-limb motor function and histopathology of the spinal cord were assessed in a blinded fashion. Results The neurologic and histopathologic outcomes in the Sevo group were better than those in the others groups （P<0.05）. There were no significant diffierences in the neurologic and histopathologic outcomes among the O₂ group, 5-HD+Sevo group and 5-HD+O₂ group. Conclusion 5-hydroxydecanoate, a specific inhibitor of mitochondrial ATP-sensitive potassium channels, abolishes sevoflurane postconditioning-induced protective effects against spinal cord ischemia, suggesting postconditoning with sevoflurane alleviates spinal cord ischemia/reperfusion injury via the activation of mitochondrial ATP-sensitive potassium channels in rabbits.Results1. Postconditoning with 0.5、1.0、1.5 MAC sevoflurane could alleviate spinal cord ischemia/reperfusion injury in rabbits. The neuroprotective effects of postconditioning with 1.0 MAC sevoflurane is better.2. The protective effects of sevoflurane postconditioning against spinal cord ischemia injury via release of oxygen free radicals then up-regulating the activities of SOD and CAT in spinal cord during early reperfusion phase in rabbits.3. Postconditoning with sevoflurane could alleviate spinal cord ischemia reperfusion injury via the activation of mitochondrial ATP-sensitive potassium channels in rabbits.更多还原