【作者】 谷秋寒；

【导师】 熊利泽； 董海龙；

【作者基本信息】 第四军医大学 ， 麻醉学， 2009， 硕士

【摘要】 自1986年Murry等提出缺血预处理概念以来,人们对预处理进行了大量的研究。诸多措施(如低温、电针等)和药物(如兴奋性氨基酸拮抗剂、自由基清除剂、米诺环素等)均已被证实可诱导缺血耐受。然而,截至目前,大部分预处理相关研究的观察时间均不超过再灌后72小时。有人怀疑,这种耐受所致的保护效应并没有永久性的减少细胞死亡,只不过延缓了细胞死亡的进程。那么,随着再灌注时间的延长,这种保护效应会不会逐渐减弱甚至消失呢?这是一个存在诸多争议,很值得关注的研究热点。我们的前期研究表明高压氧(hyperbaric oxygen, HBO)预处理可模拟“缺血预处理”,减轻随后因阻闭腹主动脉所引起的脊髓缺血再灌注损伤[2],此现象亦存在于大鼠局灶性脑缺血模型[3]。另有研究表明,中枢神经系统缺血再灌注损伤后均可出现细胞凋亡现象,而脊髓缺血再灌注后细胞延迟性的死亡大部分正是凋亡而不是坏死。高压氧预处理可减少缺血区神经元的早期凋亡,阻止早期凋亡向延迟性神经损害转变,甚至可能逆转早期的凋亡,使神经元免于坏死。因此,本研究以“高压氧预处理诱导兔脊髓缺血耐受现象”为切入点,对预处理的神经保护效果进行长期观察,并研究其远期效应是否与抑制了神经元凋亡相关。第一部分高压氧预处理诱导兔脊髓缺血耐受的远期效果观察目的:探讨高压氧预处理诱导的兔脊髓缺血耐受能否长期存在。方法:实验一,16只雄性新西兰大白兔,随机分为2组(n =8):对照组,即常压空气组;HBO组,即高压氧(HBO)预处理组(100% O2,2.5 ATA,1 h/d,5 d),最后一次处理后24 h所有动物均采用肾下腹主动脉阻断法造成脊髓缺血(20 min),观察再灌后1,2,7,14,21 d时的神经功能学评分。实验二,32只雄性新西兰大白兔,分组同实验一(n =16),重复上述处理,并分别于再灌后2, 21 d每组各灌注8只动物取脊髓(L5-7),制作标本,行组织病理学观察。灌注动物前进行神经功能学评分。结果:HBO组神经功能学评分在1, 2, 7,14,21d时均明显优于对照组(P<0.05),但组内比较,HBO组再灌7,14,21d时的神经功能学评分与2d时均无显著差别(P>0.05);HBO组脊髓前角正常神经元计数在2d和21d时均明显优于对照组,但组内比较,HBO组再灌21d时的脊髓前角正常神经元计数与2d时无显著差别(P>0.05);神经功能学评分与其对应脊髓前角正常神经细胞元之间有显著相关性(r2d = 0.903,P<0.01; r21d = 0.922, P<0.01)。结论:高压氧预处理可以诱导兔脊髓缺血耐受,且此效应长期存在。第二部分高压氧预处理诱导兔脊髓缺血耐受的远期效果与神经元凋亡的关系实验一高压氧预处理诱导兔脊髓缺血耐受的远期效果与再灌后早期神经元凋亡的关系目的:探讨高压氧预处理诱导的兔脊髓缺血耐受的远期效果是否通过抑制再灌后早期神经元的凋亡介导。方法:50只雄性新西兰大白兔随机分为2组(n =25):对照组,即常压空气组;HBO组,即高压氧(HBO)预处理组(100% O2,2.5 ATA,1 h/d,5 d),最后一次处理后24 h所有动物均采用肾下腹主动脉阻断法造成脊髓缺血(20 min),于再灌注8 h、24 h、2 d、3 d和5 d时每组各灌注5只动物取脊髓(L5-7),制作标本,TUNEL法检测神经元凋亡情况。灌注动物前进行神经功能学评分。结果:HBO组神经功能学评分在24 h、2 d、3 d和5 d时均明显优于对照组(P<0.05),在8 h时无统计学差异。与对照组相比,HBO组凋亡神经元数在各个时间点均明显少于对照组(P<0.05);对照组与HBO组凋亡神经元均于再灌后8小时即明显出现,凋亡神经元数于再灌后2天达峰值,随后逐渐减少。结论:高压氧预处理诱导兔脊髓缺血耐受的远期效果可能是通过抑制再灌后早期神经元的凋亡产生的。实验二高压氧预处理诱导兔脊髓缺血耐受的远期效果与再灌后晚期神经元凋亡的关系目的:探讨高压氧预处理诱导兔脊髓缺血耐受的远期效果是否通过抑制再灌后晚期神经元的变性和凋亡介导。方法:40只雄性新西兰大白兔随机分为2组(n =20):对照组,即常压空气组;HBO组,即高压氧(HBO)预处理组(100% O2,2.5 ATA,1 h/d,5d),最后一次处理后24 h所有动物均采用肾下腹主动脉阻断法造成脊髓缺血(20min),于再灌注2,7,14,21d每组各灌注5只动物取脊髓(L5-7),制作标本,FJ-B染色检测变性神经元,TUNEL法检测神经元凋亡情况。灌注动物前进行神经功能学评分。结果:HBO组神经功能学评分在2,7,14,21d时均明显优于对照组(P<0.05)。FJ-B染色阳性神经元数仅在2d时明显低于对照组(P<0.05),TUNEL染色阳性神经元数仅在2d和7d时明显低于对照组(P<0.05),其余时间点两组之间比较,结果无统计学差异(P>0.05)。结论:高压氧预处理诱导兔脊髓缺血耐受的远期效果与再灌后晚期神经元的凋亡无明显关系。结论1高压氧预处理对兔脊髓缺血再灌注损伤有明显的保护作用,且此保护作用可能是永久性的。2高压氧预处理对脊髓缺血再灌注损伤的这种永久性的保护作用可能是通过抑制缺血再灌注后早期神经元的凋亡产生的;而与再灌后晚期神经元的变性和凋亡无关。更多还原

【Abstract】 Background Since 1986 the concept of ischemic preconditioning was proposed by Murry, several strategies (such as low temperature, electro-acupuncture, etc.) as well as drugs (such as excitatory amino acid antagonists, free radical scavengers, minocycline, etc.) have been proved to induce ischemic tolerance in the organs. However, most observation time of these studies’did not exceed 72 hours after reperfusion. It was suspected that preconditioning didn’t permanently prevent cell death after ischemic/reperfusion injury, but only postponed the evolution of the progress of cell death. Whether the protective effects will gradually weaken, disappear or not change in a long-term observation is a question disputable but very important that need us to answer. Our previous study showed that hyperbaric oxygen (hyperbaric oxygen, HBO) preconditioning can simulate "ischemic preconditioning" and induce ischemic tolerance in spinal cord ischemic/reperfusion injury. This phenomenon also exists in focal cerebral ischemia model of rats. Another study showed that apoptosis does exist in ischemia/reperfusion injury in the central nervous system, and the delayed cell injury of ischemia/reperfusion in spinal cord is mainly caused by apoptosis rather than necrosis. Hyperbaric oxygen preconditioning can reduce early apoptosis of neurons and may even reverse apoptosis. Therefore, this study take "the ischemic tolerance induced by hyperbaric oxygen preconditioning in rabbit spinal cord" as the breakthrough point, to study the neuroprotective effect of HBO preconditioning for long-term observation and the relationship between the long-term effects and neuron apoptosis.PartⅠLong-standing ischemic tolerance induced by hyperbaric oxygen preconditioning in spinal cord of rabbitsObjective To determine if the ischemic tolerance induced by hyperbaric oxygen preconditioning in spinal cord of rabbits is long-standing. Methods In experiment 1, sixteen male New Zealand rabbits were randomly assigned to 2 groups (8 in each group) : animals in control group received room air and normal ambient pressure (1 h/d, 5 d); animals in HBO group received HBO pretreatment (100% O2, 2.5 atmosphere absolute [ATA], 1 h/d, 5 d). At 24 hours after the last treatment, the spinal cord ischemia was induced for 20 min by infrarenal aortic cross clamping. At 1day, and 2, 7, 14, 21 days after reperfusion, the neurologic function was scored by the Tarlov criteria (in which 4 was normal and 0 was paraplegia). In experiment 2, thirty-two animals were also randomly assigned to HBO group and control group (16 in each group). Animals underwent the same treatment as in experiment 1. Then eight animals in each group were sacrificed at 2 days and 21 days after reperfusion, respectively. And their spinal cords (L5-7) were removed immediately for histopathologic examination. The neurologic functions were scored before they were sacrificed.Results The neurologic function scores in HBO group were significantly greater than in control group at all time points (P<0.05), which had no statistical differences between 7,14 or 21 days and 2days in HBO group (P>0.05). The numbers of normal neurons in the anterior spinal cord in HBO groups were also significantly greater than in control groups at 2 days and 21 days, which had no statistical differences between 2 days and 21 days in HBO groups. There was a strong correlation between the neurologic function scores and the numbers of normal neurons in the anterior horn of the spinal cords (r2d =0.903, P<0.01; r21d =0.922, P<0.01). Conclusion Hyperbaric oxygen preconditioning induced ischemic tolerance in spinal cord of rabbits and the effect is long-standing.PartⅡThe relationship between the long-standing ischemic tolerance induced by hyperbaric oxygen preconditioning and the cell apoptosis after reperfusion in spinal cord of rabbitsExperiment 1 The relationship between the long-standing ischemic tolerance induced by hyperbaric oxygen preconditioning and the early stage apoptosis after reperfusion in spinal cord of rabbitsObjective To determine if hyperbaric oxygen preconditioning induced long-standing ischemic tolerance via reducing the early stage apoptosis after reperfusion in spinal cord of rabbits. Methods Fifty male New Zealand rabbits were randomly assigned to 2 groups (25 in each group) : animals in control group received room air and normal ambient pressure (1 h/d, 5 d); animals in HBO group received HBO pretreatment (100% O2, 2.5 ATA, 1 h/d, 5 d). At 24 hours after the last treatment, the spinal cord ischemia was induced for 20 min by infrarenal aortic cross clamping. Five animals in each group were sacrificed at 8, 24 hours and 2, 3, 5 days after reperfusion, respectively. And their spinal cords (L5-7) were removed immediately for TUNEL staining. The neurologic functions were scored before they were sacrificed. Results The neurologic function scores in HBO groups were significantly less than that in control groups at 24 hours and 2, 3, 5 days after reperfusion(P<0.05), which had no statistical differences at 8 hours (P>0.05). The numbers of TUNEL positive neurons in HBO groups were significantly less than that in control groups at all time points(P<0.05). Conclusion Hyperbaric oxygen preconditioning induced long-standing ischemic tolerance via reducing early stage apoptosis after reperfusion in spinal cord of rabbits.Experiment 2 The relationship between the long-standing ischemic tolerance induced by hyperbaric oxygen preconditioning and the later stage apoptosis after reperfusion in spinal cord of rabbitsObjective To determine if hyperbaric oxygen preconditioning induced long-standing ischemic tolerance via reducing the later stage apoptosis after reperfusion in spinal cord of rabbits. Methods Forty male New Zealand rabbits were randomly assigned to 2 groups (20 in each group) : animals in control group received room air and normal ambient pressure (1 h/d, 5 d); animals in HBO group received HBO pretreatment (100% O2, 2.5 ATA, 1 h/d, 5 d). At 24 hours after the last treatment, the spinal cord ischemia was induced for 20 min by infrarenal aortic cross clamping. Five animals in each group were sacrificed at 2, 7, 14, 21 days after reperfusion, respectively. And their spinal cords (L5-7) were removed immediately for TUNEL staining and FJ-B staining. The neurologic functions were scored before they were sacrificed. Results Neurologic function scores in HBO groups were significantly greater than that in control groups at all time points (P<0.05). The numbers of TUNEL positive neurons in HBO groups were significantly less than in control groups at 2 and 7 days(P<0.05), and FJ-B positive neurons in the anterior spinal cord in HBO groups was only significantly less than that in control group at 2 days(P<0.05), which had no statistical differences at other time points(P>0.05). Conclusion The long-standing ischemic tolerance induced by hyperbaric oxygen preconditioning doesn’t significantly related to later stage apoptosis after reperfusion in spinal cord of rabbits.更多还原