【作者】 王丽萍；

【导师】 李文献； 孙学军； 邓小明；

【作者基本信息】 第二军医大学 ， 麻醉学， 2008， 硕士

【摘要】 胸腹主动脉瘤传统手术中,为移除病变段血管而需较长时间阻断主动脉,远短缺血及重建后的血液复灌易造成脊髓不同程度的缺血及再灌注损伤,引起神经功能障碍,表现为急性或迟发性截瘫。脊髓缺血-再灌注损伤（ischemia and reperfusion injury,IRI）是大血管术后严重而难以预料的并发症,其发生率为11%～40%。探寻及时而有效预防脊髓缺血-再灌注损伤的方法是目前研究的热点之一。1986年,Murry首次运用数次短暂阻断/开通犬心脏冠状动脉的方法来抵御之后更长时间的缺血-再灌注打击,这一被称之为“缺血预处理”（ischemia preconditioning,IPC）的手段此后逐渐成为预防各脏器缺血-再灌注损伤的有效方法。传统概念认为,IPC之后有两个“治疗时间窗”,即快速预处理（rapid precondition,RPC）和延迟预处理（delayed preconditioning,DPC）。RPC发生在预处理刺激进行的数分钟内,其效应在刺激结束后仅持续1～2 h;DPC开始于预处理刺激结束后12～24 h,其效应可持续3～4 d。经典的IPC机制中线粒体的作用已为很多研究者所关注。预处理早期产生的腺苷、阿片肽等物质,通过激活PKC,后转录修饰线粒体ATP酶敏感性钾通道(mitochondria ATP sensitive potassium channel,mitoK_ATP),使其短暂开放,从而促进钾内流使线粒体的膜电位降低,减少钙离子进入线粒体,防止线粒体内钙超载;预处理晚期由PKC等介导作用于转录因子,促进合成Bcl-2抗凋亡蛋白而抑制线粒体渗透性转换孔（mitochondria permeability transition pore,MPTP）发生渗透性转换。RPC抵御缺血性损伤远期效应的不稳定性以及RPC与DPC之间较长的“无效应期”成为IPC预防器官缺血-再灌注损伤最主要的“瓶颈”。高压氧（hyerbaric oxygen,HBO）,即大于一个绝对大气压（atmospheres absolute,ATA）的100%纯氧,临床上已用于治疗一氧化碳中毒、减压病、动脉气体栓塞及缺血缺氧等相关性疾病。实验证明,HBO也是预防脑、心脏、脊髓等脏器缺血-再灌注损伤的有效预处理方式。目前关于HBO预处理（HBO preconditioning,HBO-PC）的实验研究大都着眼于其延迟性保护效应,即采用2.5 ATA的HBO,每次暴露1 h,每天1次,连续3 d或5 d,以预防24 h后缺血-再灌注损伤。这一保护效应的产生可能有赖于HBO-PC过程中反应性氧自由基（reactive oxygen species,ROS）的生成,从而促进抗氧化物酶、低氧诱导因子-1（hypoxia-inducible factor-1,HIF-1）、Bcl-2等耐缺血缺氧蛋白的表达。但这一预处理方案对于主动脉瘤夹层撕裂、急性破裂等急诊手术没有可操作性。2005年,韩国学者Choi在运用短间隔数次HBO-PC（即20 min/次,间隔20 min,连续4次）来减轻心肌缺血-再灌注损伤,结果发现,在传统概念中预处理的“无效应期”出现了心脏保护作用,并有抗凋亡蛋白Bcl-2的mRNA转录与表达。最近美国Stanford大学脑外科研究小组报道了应用结扎大鼠单侧后肢股动脉远隔预处理（remote preconditioning,RP）方法预防局部脑缺血损伤,他们采用15 min阻断/15 min开放的间隔、连续3次进行预处理刺激,显示对预处理后1～24 h发生的局灶性脑缺血皆有保护作用;但是,如果将处理间隔改为5 min阻断/5 min开放,保护作用则消失。以上研究带给我们的启示是,短期内应用HBO进行多次预处理刺激对脊髓缺血-再灌注损伤是否亦具有保护作用?如果有保护作用,那么以何种预处理刺激间隔才可产生“连续型治疗时间窗”?其可能的机制是什么?这些问题目前尚未见到系统的研究报告。本课题通过在短期内数次HBO暴露,之后在数个时间点建立稳定的大鼠脊髓缺血-再灌注损伤模型,进行IRI打击,分析刺激次数、间隔与治疗时间窗的关系。并着眼于预处理机制的效应器——线粒体,以及基因转录的调节子核因子-κB（NF-κB）,探讨其可能的分子机制。为此,我们设计了以下实验:1、Fogarty球囊导管阻断大鼠胸降主动脉合并降低阻断近端动脉压（proximalarterial pressure.PAP）至40 mmHg建立大鼠脊髓缺血-再灌注损伤模型,选定适合评价预处理效应的脊髓缺血损伤程度。2、根据不同的暴露方案,设立4个HBO预处理组及Control组。HBO-Ⅰ组:进行1次20 min的暴露;HBO-Ⅱ组:20 min/次,间隔10 min,连续4次;HBO-Ⅲ组:20min/次,间隔30 min,连续4次;HBO-Ⅳ组:20 min/次,间隔50 min,连续4次;Control组不进行预处理。3、各组分别于HBO-PC后0.5、2、4、8、12、24、48 h建立大鼠脊髓缺血-再灌注损伤模型,于缺血后12、24 h对大鼠后肢神经功能评分,Nissl染色法观察脊髓前角正常神经元数目。4、各组分别于HBO-PC后0.5、2、4、8、12、24、48 h制备大鼠脊髓线粒体。用可见光分光光度法进行线粒体mitoK_ATP开放度测定,在高Ca²⁺环境下测定给予mitoK_ATP抑制剂5-hydroxydecanoate（5-HD）前后线粒体在520nm吸光度的改变。电镜下观察线粒体显微结构,并应用Western-blot测定线粒体Bcl-2蛋白表达情况。5、运用电泳迁移率变动分析法（electrophoretic mobility shift assay,EMSA）观察单次HBO-PC后大鼠脊髓神经元NF-κB DNA结合活性的时相差异。并用NF-κB抑制物吡咯烷二硫基甲酸盐（pyrrolidine dithiocarbamate,PDTC）和ROS清除剂N-乙酰半胱氨酸（N-acetylcysteine,NAC）干扰Bcl-2的表达。主要实验结果及结论如下:1、Fogarty球囊导管阻断大鼠胸降主动脉合并控制性低血压,可造成稳定的脊髓缺血;阻断9 min构建的大鼠脊髓缺血-再灌注损伤模型,再灌注期神经行为学评分平稳,神经元多数以凋亡的形式死亡,对IPC等神经保护措施应较敏感。2、神经行为学和脊髓组织病理学结果显示,相对于单次HBO-PC,短间隔反复预处理可加强早期预处理效应（HBO-PC后0.5～2 h）;预处理间隔延长至30 min可诱导持续性缺血耐受;预处理间隔如继续延长至50 min,治疗时间窗则恢复呈“二相型”。3、与单次HBO-PC相比,短间隔多次预处理可使预处理早期mitoK_ATP开放幅度增加;mitoK_ATP开放时间不会随预处理间隔的增大而延长。提示短间隔HBO-PC诱导的持续性缺血耐受不由mitoK₍ATP0介导。4、间隔10 min或50 min的HBO-PC可使线粒体Bcl-2的蛋白含量在预处理早期（0.5、2、4 h）与晚期（24、48 h）明显增加。间隔30 min则可使Bcl-2的表达于处理后0.5、2、4、8、12 h及24、48 h皆显著提高,这可能与短间隔HBO-PC诱导持续性缺血耐受相关。5、EMSA显示单次HBO-PC后脊髓组织NF-κB的DNA结合活性于20～40 min和24 h呈现两次转录峰值。PDTC、NAC可完全抑制HBO-Ⅲ（HBO-PC间隔为30 min）组0.5、2 h,4、8、12 h和24 h三个时相的神经保护作用,亦可阻滞Bcl-2在三个时相的表达增加。以上结果表明,短间隔HBO-PC可诱导脊髓持续性缺血耐受,其机制可能是:HBO-PC生成的ROS诱导NF-κB DNA结合活性于预处理早期、晚期两时相增高,并且反复预处理刺激后的时相延长作用导致Bcl-2在预处理后早、中、晚三个时相表达皆增高。更多还原

【Abstract】 During descending thoracic aortc aneurysm repair,the aorta was cross-clamped and unclamped subsequently which may result in spinal cord transient ischemia followed by reperfusion.Ischemia and reperfusion injury（IRI） of spinal cord after thoracoabdominal aortic aneurysm repair remains a devastating complication with an incidence of 11%～40%in postoperative paraplegia and paraparesis.First described in 1986,Murry and colleagues exposed open-chest dogs to a set of non-harmful ischemic insults consisted of four independent cyclic episodes of a 5-min coronary occulusion followed by 5-min reperfusion.This so called "ischemic preconditioning"（IPC） showed some beneficial effects to the pretreated heart against subsequent prolonged ischemia.Following the novel observation of Murry et al.,IPC has become an effective means of reducing harmful ischemic events.Two types of IPC,rapid and delayed,have been reported.Rapid ischemic tolerance occurs within a few minutes of the preconditioning ischemia and lasts for 1-2 hrs.Delayed ischemic tolerance starts at 12-24 hrs and will last for more than 3-4 days.The different molecular mechanisms in the mitochondria,which are involved in transient open of phosphorylated ATP sensitive potassium channel(mitoK_ATP) in the rapid tolerance and transcription of Bcl-2 family genes to inhibit the mitochondrial permeability transition,contribute to each type of tolerance.Both the instability of rapid tolerance and the followed non-protective effect window till the adventure of delayed ischemic tolerance greatly limit the application of IPC as preventive measures in against ischemia and reperfusion insults.Hyperbaric oxygenation（HBO） treatment is a mode of therapy for this serious complication with a established protocol of oxygen pressure not exceeding 3.0 atmospheres absolute（ATA） and a duration limited to a maximum of 2 hrs.HBO has been safely used for the patients with carbon monoxide poisoning,decompression sickness,and arterial gas embolism.It has also been used as an adjunctive therapy for reviving a patient with impaired oxygen delivery.Up to now,HBO has been reported as an effective method of preconditioning in preventing ischemia and reperfusion insults in the brain,heart and spinal cord.In most published experiments,the HBO preconditioning（HBO-PC） protocol comprises 3 or 5 consecutive cycles of 1-h HBO exposure（2.5 atmospheres absolute [ATA]） at a 1-day interval against ischemic attack 24 hrs later.The mechanism of late ischemic tolerance induced by HBO may be related to ROS generated in the preconditioning which upregulated endogenous antioxidant enzymes,hypoxia-inducible factor-1（HIF-1） and Bcl-2 to resist ischemia.Apparently this protocol is impractical in emergency scenarios for patients with thoracoabdominal aortic aneurysm.In 2005,Choi et al pretreated with HBO at 3 ATA for 20 mins,four cycles with 20-min intermission,and isolated hearts were subjected to 45-min ischemia followed by 90-min reperfusion.This protocol induced mRNA transcription of anti-apoptotic protein even in the traditionally non-protective window between the two stages of rapid and delayed tolerance.In the study of remote preconditioning performed in the ipsilateral hind limb against focal stroke,the results showed that rapid preconditioning with three cycles of 15-min occlusion followed with the same period of reperfuison of the left hind femoral artery could reduce infarct size of stroke that occurred 1-24 h after preconditioning.But at three cycles of 5 min,infarct was not reduced.However,whether cyclic short episodes of HBO-PC protects against spinal cord ischemia is not established.Moreover,few have addressed the sustained therapeutic time windows for HBO-PC.In the present study,the rats were preconditioned with repeated HBO of short interval and underwent spinal cord ischemia induced by occlusion of the descending thoracic aorta at different time point after HBO-PC.The purpose of this study was to analyze the relationship between different preconditiong paradigms and therapy time window,and examine the related mitochondrial mechanisms with its upstream transcription regulation.The main experimental procedures are as follows:1.Spinal cord ischemia was induced by occlusion of the descending thoracic aorta with 2F Fogarty catheter in combination with maintaining systemic hypotension（40 mmHg） during the procedure,and determined a suitable time of ischemia to establish basis for next experiments.2.The rats were divided into four HBO-PC and Control groups as follows:the rats in HBO-Ⅰgroup were only pretreated with one cycle HBO of 20 min;the rats of HBO-Ⅱgroup were preconditioned with four cycles HBO of 20 min with interval of 10 min and the rats in the HBO-Ⅲand HBO-Ⅳgroups underwent four episodes of 20 min with intermission of 30 min or 50 min respectively.Control group only underwent Sham preconditioning.3.At 0.5,2,4,8,12,24 and 48 h after HBO-PC,spinal cord ischemia was induced. Neurological scores were evaluated 12 and 24 h after reperfusion.After that,the spinal cords（L3-5） were harvested and sections were stained by Nissl and normal neruons in the anterior spinal cord were counted by means of light microscopy.4.Mitochondria in lumbar spinal cord were extracted at 0.5,2,4,8,12,24 and 48 h.Measurments of mitoK_ATP open extent was conducted by the decrease in light scattering closely paralles the percentage of the mitochondrial population undergoing permeability transition under the circumstance of Ca²⁺-induced mitochondrial swelling.Mitochondrial microstructure was observed by electron microscopy.Bcl-2 protein levels in the mitochondria were analyzed by Western blot using samples taken from spinal cord tissues in different time windows after HBO-PC.5.The changes of DNA-binding activity of nuclear factor-κB（NF-κB） after single HBO-PC were assessed by electrophoretic mobility shift assay（EMSA）.The effects of NF-κB inhibitor pyrrolidine dithiocarbamate（PDTC） and antioxidant N-acetylcysteine （NAC） on HBO-PC and Bcl-2 expression were also evaluated.The main results are as follows: 1.Evaluation for rat model of ischemia and reperfusion injury.The rats underwent 9 min of spinal cord ischemia were stable in neurological scores during reperfusion and were susceptible to preconditionng.The rat model of spinal cord induced by occlusion of the descending thracic aorta is stable for study on mechanism of preconditioning.2.As displayed in neurological scores and pathological results,cyclic short episodes of HBO-PC could improve the rapid tolerance,and the sustained protective effect was induced if suitable interval of HBO-PC was used.But biphasical therapy window with an early and delay effect was presented again after HBO-PC of prolonged intermission.3.The extent of mitoK_ATP was increased after cyclic short episodes of HBO-PC compared with one cycle,and not be affected by the prolonged period of HBO-PC.4.Bcl-2 was expressed both in the early preconditioning and late preconditioning with 20-min or 50-min intermission of HBO-PC in HBO-ⅡandⅣgroups.Moreover,Bcl-2 also increased in the intermediate preconditioning between the conventional two time windows with 30-min interval in HBO-Ⅲgroup.5.DNA-binding activity of nuclear factor-κB（NF-κB） peaked at 20-40 min and 24 h after single HBO-PC.PDTC and NAC could both abrogated the neuroprotective effect and Bcl-2 expression in HBO-Ⅲgroup.In conclusion,cyclic short episodes of HBO-PC can induce sustained tolerance against spinal cord ischemia.This mechanism may be involved in anti-apoptotic protein expression of Bcl-2 in the early and late periods of preconditioning as well as in the intermediate time windows which may be related to peak of NF-κB DNA-binding activity induced by HBO-derived ROS.更多还原