【作者】 苏斌虓；

【导师】 熊利泽； 董海龙；

【作者基本信息】 第四军医大学 ， 麻醉学， 2009， 博士

【摘要】 背景缺血再灌注损伤是引起胸主动脉及脊柱手术后发生截瘫的最主要原因。随着大量新技术的应用,此类手术术后并发症已经大大降低,但是近期的一项统计结果显示,其发病率仍然超过了10%。因此如何预防胸主动脉及脊柱手术后的缺血再灌注损伤,仍然是个值得关注的课题。远程缺血预处理是指,在耐受缺血能力较强的器官诱导短暂缺血,使得重要脏器产生缺血耐受的预处理方式,多选择肢体作为诱导器官。由于其安全,容易被患者和医生所接受,因此其临床应用前景巨大。近来研究表明,内源性大麻素在中枢神经系统损伤方面扮演着重要角色,并且参与了缺血预处理的发生机制。泛素系统功能异常是导致神经元延迟死亡的重要原因。脊髓缺血损伤后泛素早期的持续表达增加,被证实与脊髓运动神经元缺血易感性相关。本研究旨在探讨远程缺血预处理对脊髓缺血损伤的保护作用及其机制,为远程缺血预处理直接应用于临床提供理论基础。实验一肢体远程缺血预处理在诱导大鼠脊髓缺血耐受中的作用目的探讨肢体远程缺血预处理在诱导大鼠脊髓缺血耐受中的作用,为进一步机理研究奠定基础。方法采用Fogarty导管阻断胸主动脉血流合并系统低血压(放血)的方法,制作大鼠脊髓缺血模型。暴露右侧股动脉,在股动脉分叉以上处阻断/开放,进行肢体远程缺血预处理。雄性SD大鼠40只(300~350g),随机分为5组(n=8):假手术组,大鼠接受和其它组动物一样的手术操作包括置管及麻醉,但不诱导脊髓缺血及远程缺血预处理;单纯缺血对照组,1~2%异氟醚麻醉60min,暴露右侧股动脉,诱导脊髓缺血12min;远程缺血预处理2min组,暴露右侧股动脉,阻断/开放(预处理)3个循环,每次为2min阻断/2min开放,远程缺血预处理30min后,诱导脊髓缺血12min;远程缺血预处理3min组,暴露右侧股动脉,在股动脉分叉以上处阻断/开放(预处理)3个循环,每次为3min阻断/3min开放,远程缺血预处理30min后,诱导脊髓缺血12min。远程缺血预处理5min组,暴露右侧股动脉,在股动脉分叉以上处阻断/开放(预处理)3个循环,每次为5min阻断/5min开放,远程缺血预处理30min后,诱导脊髓缺血12min。再灌注24h、48h,由一不了解分组情况的观察者,评估并记录运动缺陷评分。评分方法:a)后肢行走:0分正常;1分行走时脚趾在肢体下伸展;2分趾关节行走;3分靠后肢挪动身体,但是无下肢行走;4分无行走并且后肢无运动。b)放置/踱步反射:0分正常;1分反应弱;2分无踱步反射。每只动物的运动缺陷评分(MDI)为得分a+b。总分6分表示完全瘫痪,0分为功能正常。评分完毕后,截取腰段脊髓组织(L4～L6),行HE染色。光镜下观察HE染色结果并对脊髓前角正常神经元计数。结果1.生理学参数:脊髓缺血模型制作过程中缺血组间血流动力学指标(PMAP、DMAP、HR),直肠温度,pH、PaO2、PCO2及血糖变化在各组间均无明显差异。脊髓缺血期间,各组远端平均动脉压力(DMAP)均维持在DMAP<5mmHg,证实导管阻断合并系统低血压的方法制作大鼠的脊髓缺血模型阻断确实,缺血效果可靠。缺血组近端动脉压(PMAP),在脊髓缺血1min内均由90mmHg降至45mmHg,表明各缺血组在系统低血压的产生时间及水平上保持一致。4个缺血组大鼠,在脊髓缺血期间,都表现为心率下降,低于缺血前,有统计学差异,但是组间没有差异。再灌注后5分钟之内,HR、血压,都可以恢复到缺血前水平,且缺血组间没有差别。缺血组再灌注后血糖高于缺血前(p<0.05),pH低于缺血前(p<0.05),整个手术中未出现低氧及二氧化碳潴留。手术过程中,直肠温度维持在37.2°C～37.5°C之间。各组动物体重,放血量没有差异。假手术组动物各项生理学参数在整个手术操作过程中保持稳定。2.运动缺陷评分(MDI):再灌注24h、48h,分别对所有动物进行运动缺陷评分。假手术组没有神经功能改变。预处理3min3个循环组,MDI低于对照组及其它两组(P < 0.01),表明运动功能好于其它3组。单纯缺血对照组、预处理2min组、预处理5min组MDI没有统计学差异。3.前角正常神经元计数:在假手术组,未见损伤神经元。预处理3min3个循环组,前角正常神经元数量多于其它三个缺血组,包括对照组、预处理2min组及预处理5min组(P<0.01)。对照组、预处理2min组及预处理5min组之间,前角正常神经元计数无统计学差异。结论短暂下肢远程缺血预处理可以诱导大鼠脊髓的缺血耐受。实验二大麻素受体在远程缺血预处理诱导大鼠脊髓缺血耐受中的作用机理研究目的探讨大麻素受体拮抗剂对远程缺血预处理诱导脊髓缺血耐受的影响,初步证明内源性大麻素在远程缺血预处理诱导大鼠脊髓缺血耐受的作用机制。方法雄性SD大鼠64只,随机分为8组(n=8)。预处理方案为: 3个循环,每次为3min阻断/3min开放(预处理方案根据实验一的结果制定)。分组:①单纯缺血对照组;②远程缺血预处理组;③远程缺血预处理+AM251组,每次预处理15min前静脉注射大麻素受体(1CB1)拮抗剂AM251(1mg/kg体重);④远程缺血预处理+AM630组,每次预处理15min前静脉注射大麻素受体2(CB2)拮抗剂AM630(1mg/kg体重);⑤远程缺血预处理+溶剂组;⑥AM251组,每次麻醉前15min静脉注射AM251(1mg/kg体重);⑦AM630组:每次麻醉前15min静脉注射AM630(1mg/kg体重);⑧溶剂组:每次麻醉前15min静脉注射大麻素受体拮抗剂溶剂(1ml/kg体重)。所有组动物均接受脊髓缺血损伤,其方法如前所述。再灌注24h、48h,由一不了解分组情况的观察者,评估并记录运动缺陷评分。评分完毕后,截取腰段脊髓组织(L4～L6),行HE染色。光镜下观察HE染色结果并对脊髓前角正常神经元计数。结果1.生理学参数:各组未见差异。2.运动缺陷评分(MDI)远程缺血预处理组MDI低于单纯缺血对照组,远程缺血预处理+AM251组,(P<0.05)。远程缺血预处理组与RIPC+AM630组,及远程缺血预处理+Vehicle组,MDI无差别。单纯缺血对照组,远程缺血预处理+AM251组,AM251组,AM630组,Vehicle组之间没有统计学差异。3.前角正常神经元计数:远程缺血预处理组前角正常神经元数量多于单纯缺血对照组和预处理+AM251组(P<0.05)。远程缺血预处理组与远程缺血预处理+AM630组,及远程缺血预处理+Vehicle组前角正常神经元计数无差别。单纯缺血对照组,远程缺血预处理+AM251组,AM251组,AM630组,溶剂组之间前角正常神经元计数没有统计学差异。结论CB1受体拮抗剂逆转了远程缺血预处理诱导的脊髓缺血耐受,表明CB1受体介导了远程缺血预处理诱导脊髓缺血耐受的机制。实验三远程缺血预处理诱导脊髓缺血耐受过程中内源性大麻素含量的变化目的探讨远程缺血预处理诱导脊髓缺血耐受过程中内源性大麻素含量的变化。方法雄性SD大鼠24只,随机分为2组(n=12):①缺血对照组。②预处理组。在缺血前即刻,以及缺血再灌注后1h两个时间点,截取腰段脊髓组织(L4～L6),每个时间点取动物数量为6只。提取内源性大麻素并利用液相质谱仪(HPLC/MS)检测内源性大麻素AEA和2-AG的含量。结果1.生理学参数:生理学参数各组未见差异。2.腰段脊髓AEA和2-AG的含量变化:缺血前,远程缺血预处理组脊髓节段内AEA含量要高于缺血对照组(p<0.05)。缺血后两组间AEA含量并未见统计学差异。单纯缺血对照组,缺血后AEA含量高于缺血前(p<0.05)。两组2-AG的浓度则在缺血前和缺血后均未见明显变化。结论远程缺血预处理可以诱导脊髓节段内内源性大麻素AEA在脊髓缺血前释放增加,起到诱导脊髓缺血耐受的作用。实验四远程缺血预处理对大鼠脊髓缺血后泛素化的调节及内源性大麻素的作用目的探讨远程缺血预处理对大鼠脊髓缺血后泛素化的调节及内源性大麻素在此过程中的作用。方法雄性SD大鼠24只,随机分为3组(n=8)①单纯缺血对照组;②远程缺血预处理组;③远程缺血预处理+AM251组。缺血再灌注后3h,及24h,组织灌注,并截取腰段脊髓组织(L4～L6),每个时间点动物数量为4只,制作冰冻切片。行免疫组织化学染色观察脊髓前角泛素的表达及TUNEL染色。免疫组化切片利用image pro plus 6.0软件计算平均光密度值,分析结果。结果1.泛素在脊髓前角的表达:免疫组织化学染色结果显示,泛素在脊髓前角的神经元内有表达,染色阳性信号为淡兰色颗粒,每张切片随机选择10个点计算其平均光密度值,远程缺血预处理组3小时的泛素表达明显低于对照组(P<0.001),和远程缺血预处理+AM251组(P<0.01);再灌注24h后,远程缺血预处理组泛素表达明显低于对照组(P<0.01),和远程缺血预处理+AM251组(P<0.01)。2.细胞凋亡实验:在缺血再灌注后3h(早期),各组均未见凋亡细胞。结论远程缺血预处理可以抑制脊髓缺血后早期的泛素表达,其原因与远程缺血预处理诱导内源性大麻素释放,减轻脊髓损伤有关。脊髓缺血后泛素的表达明显早于细胞凋亡发生的时间。小结1.短暂下肢远程缺血预处理可以诱导大鼠脊髓的缺血耐受。2.内源性大麻素CB1受体介导了远程缺血预处理诱导的脊髓缺血耐受。3.远程缺血预处理可以诱导脊髓节段内内源性大麻素AEA在脊髓缺血前释放增加,通过CB1受体,调节脊髓局部血流,起到了脊髓保护作用。4.远程缺血预处理可以抑制脊髓缺血后早期的泛素表达,其原因与远程缺血预处理诱导内源性大麻素释放,减轻脊髓损伤有关。脊髓缺血后泛素的表达明显早于细胞凋亡发生的时间,结果提示泛素可能是脊髓损伤的一个早期标志。更多还原

【Abstract】 Background: Ischemia-reperfusion injury was the most important cause of paraplegia after thoracic aorta and spine surgery. The incidence of paraplegia was over10% as reported in thoracoabdominal aneurysm repairs surgery. Although a number of studies have shown that the risk of postoperative paraplegia was decreased with the application of new techniques, preventing spinal cord ischemic injury is still a big concern in the thoracic aorta and spine surgery. Recent studies also found that the IPC induced ischemic tolerance was occurred not only within the same piece of tissue, but also between different regions as well as different organs. This phenomenon was called remote ischemic preconditioning (RIPC). RIPC has greater potential for clinical application than classic ischemic preconditioning, since it can be performed in a non-vital organ, avoiding the high risk of preconditioning in the vital organ, such as the brain or the spinal cord. Numerous studies have suggested that the endocannabinoids were implicated in the protective effects of IPC through cannabinoid (CB) receptor-dependent mechanisms. Vulnerability of motor neurons in the spinal cord against ischemia is considered to play an important role in the development of delayed paraplegia after surgery of the thoracic aorta. Recently, the ubiquitin system has been reported to participate in neuronal cell death. This study was designed to explore the protective effects and mechanism of RIPC, providing evidence of clinical application for RIPC.Experiment 1 Effect of repeated limb ischemic preconditioning on spinal cord ischemia-reperfusion injuryObjective To invest the effect of repeated limb ischemic preconditioning on spinal cord ischemia-reperfusion injury in rat. Methods The rat spinal cord ischemia model was used. A total of 40 male Sprague-Dawley rats were randomly divided into five groups (n=8 in each group):①. Sham group: Rats were operated in the same way as in other groups but without limb ischemia preconditioning and spinal cord ischemia.②Control group: After rats were anesthetized with 1-2% isoflurane for 30min, the spinal cord ischemia was induced.③RIPC2min-3c group: Three cycles of 2 minutes right femoral artery occlusion and reperfusion were induced prior to spinal cord ischemia-reperfusion.④RIPC3min-3c group: Three cycles of 3 minutes right femoral artery occlusion and reperfusion were induced prior to spinal cord ischemia-reperfusion.⑤RIPC5min-3c group: Three cycles of 5 minutes right femoral artery occlusion and reperfusion were induced prior to spinal cord ischemia-reperfusion. The animals were neurologically assessed at 24 and 48h after reperfusion by an observer who was unaware of the grouping. To evaluate the neuronal damage, motor function deficits in the hind limbs were evaluated according to the criteria as reported. Motor deficit scores were graded according to the following scale: a) walking/use of hind limbs: 0 scored as normal, 1 for toes flat under the body when walking, 2 for knuckle walking, 3 for movement of the hind limbs but unable to walk; and 4 for no movement, dragging hind limbs; b) placing/stepping reflex: 0 scored for normal, 1 for weak, and 2 for not stepping. Each motor deficit score was obtained by adding the scores for scales a and b. A histopathologic evaluation was performed in the spinal cord for 48h after reperfusion. Three representative sections taken from L-4 to L-6 segments were stained with Haematoxylin and Eosin (HE) for histopathologic evaluation. Neuronal injury was evaluated at a magnification of×200 by an observer who was unaware of the grouping. The remaining normal neurons in the ischemic ventral spinal cord were counted. Results 1. Physiologic variables. Except for sham group, the rectal temperature, arterial pH, PaCO2, PaO2, and blood glucose concentrations were similar in other four groups. In groups that spinal cord ischemia was induced, blood glucose was elevated and pH was dropped after reperfusion, but there is no difference among groups. Five minutes after the reperfusion, the value of the distal blood pressure was recovered to nearly preischemic level. 2. Neurologic outcome.All animals had survived until the final neurologic behavior assessment at 48 h after reperfusion. There was no neurologic change in sham group, and all of other groups of rats showed motor deficit in varying degrees. The neurologic outcome in the RIPC3min-3c group was better than that of control, RIPC2min-3c, and RIPC5min-3c groups, respectively (P < 0.01). Three rats in RIPC3min-3c group showed complete normal motor function (motor deficit index,MDI = 0). The hind-limb MDI of five groups at 48h after reperfusion were shown in. 3. Histopathologic evaluation.The number of normal neurons in the RIPC3min-3c group was significantly more than that in the RIPC2min-3c, RIPC5min-3c, and control group (P<0.01). No difference was found in the number of normal neurons at the anterior spinal cord among the control, RIPC2min-3c and RIPC5min-3c groups. Conclusion Brief episode of femoral artery occlusion and reperfusion can induce spinal cord ischemia tolerance in a rat model, and RIPC for 3min and 3 cycles is the best choice to prevent against spinal cord ischemia for 12min.Experiment 2 Effect of cannabinoid receptor antagonist on the ischemic tolerance induced by RIPCObjective To invest the effect of cannabinoid receptor antagonist on the ischemic tolerance induced by RIPC. Methods 64 male Sprague-Dawley rats were randomly divided into 8 groups averagely (n=8 in each group):①Control group: Right femoral artery was isolated without occlusion, followed by spinal ischemia/reperfusion protocol.②Remote preconditioning(RIPC) group: Three cycles of 3 minutes right femoral artery occlusion and reperfusion were induced prior to spinal cord ischemia/reperfusion.③AM251+RIPC: CB1 receptor antagonist AM251 (1 mg/kg) was intravenously administered 15 min before right femoral artery occlusion.④AM630+RIPC: Cannabinoid CB2 receptor antagonist AM630 (1 mg/kg) was intravenously administered 15 min before right femoral artery occlusion.⑤Vehicle+RIPC: Vehicle was intravenously administered 15 min before right femoral artery occlusion (3 min) and reperfusion (3 min, three cycles) prior to spinal cord ischemia/reperfusion protocol.⑥AM251: AM251 (1 mg/kg) was intravenously administered 15 min before sham operation of RIPC.⑦AM630: AM630 (1mg/kg) was intravenously administered 15 min before sham operation of RIPC.⑧Vehicle: Vehicle was intravenously administered 15 min before sham operation of RIPC. Thirty minutes after preconditioning or 45 minutes after chemical pretreatment, the rats were subjected to spinal cord ischemia for 12 mins. The time of total anesthesia was identical in 8 groups. The animals were neurologically assessed at 24 and 48h after reperfusion. A histopathologic evaluation was performed in the spinal cord for 48h after reperfusion. Three representative sections taken from L-4 to L-6 segments were stained with Haematoxylin and Eosin (HE) for histopathologic evaluation. Neuronal injury was evaluated at a magnification of×200 and the remaining normal neurons in the ischemic ventral spinal cord were counted. Results 1. Physiologic variables. The hemodynamics, rectal temperature, arterial pH, PaCO2, PaO2, and blood glucose concentrations changes were similar with those in Experiment 1. 2. Neurologic outcome.All animals had survived until the final neurologic behavior assessment at 48 h after reperfusion. The neurologic outcome in the RIPC group was better than that of the Control (p<0.05) and AM251+RIPC group (p<0.01). The MDI in RIPC, AM630+RIPC, and Vehicle+ RIPC3 groups did not show significant difference. There was no significant difference among Control, AM251+RIPC, AM251, AM630, and Vehicle groups.3. Histopathologic evaluation.The number of normal motor neurons in the RIPC group was significantly more than that in the Control (p<0.05), AM251+RIPC groups (p<0.05). There was no significance among RIPC, AM630+RIPC, and Vehicle +RIPC groups. The ventral neurons in AM251, AM630, and Vehicle groups were damaged seriously, and the number of normal neurons among these groups did not show significant difference. Conclusion Cannabinoid 1 receptor has mediated the protective mechanism of RIPC. Experiment 3 The contents of AEA and 2-AG in lumbar spinal cord samples pre- and post- spinal cord ischemia.Objective To in vest the contents of two kind of endocannabinoids ,AEA and 2-AG, in spinal cord samples pre- and post- spinal cord ischemia. Methods 24 rats were divided into 2 groups averagely: Control group and RIPC group (n=12 in each group). Animals were sacrificed at prior (30 minutes after remote preconditioning or sham operation) or 1h after spinal cord ischemia. The spinal cord was removed within 1 minute (-20°C). A sample of 1 cm from L-4 to L-6 segments of spinal cord was frozen in dry ice and then stored in -80°C refrigerator. For endocannabinoid measurement, spinal cord from the rats of control or remote preconditioning groups (n=6 in each time point) were sampled. The endocannabinoid was detected by HPLC-MS. Results The preischemic AEA level in RIPC group was higher than that in Control group (p<0.05). The results indicated that RIPC (3 cycles of 3 min limb ischemia/reperfusion) stimulated the formation of AEA in the lumbar spinal cord. The postischemic AEA level did not show significant difference between two groups. There was no significant difference on 2-AG content in lumber spinal cord at corresponding time between two groups. Conclusion RIPC induces AEA increasing before ischemia in spinal cord which play a key role in the mechanism of spinal cord ischemia tolerance induced by RIPC.Experiment 4 The expression of ubiquitin in lumbar spinal cord after RIPC and the role of endocannabinoids.Objective To invest expression of ubiquitin, and the relationship among the ubiquitin molecular, cell apoptosis and endocnnabinoids after ischemia tolerance in the spinal cord induced by RIPC. Methods 24 rats were divided into 3 groups averagely: Control group and RIPC group (n=8 in each group). Animals were sacrificed at 3h and 24h after spinal cord ischemia. The section of L-4 to L-6 spinal cord segments was removed after fixation with routine method. The freezing section of 14μm were kept in -20°C refrigerator for immunohistochemical studies and apoptosis detection. Results Mean optical density value of ubiquitin in RIPC group is lower than the value in other two groups after reperfusion for 3h and 24h (p<0.05).The cell apoptosis in all three groups is not found after reperfusion for 3h in the sections. Conclusion RIPC suppressed the expression in spinal cord after ischemia, which is intercepted by AM251. Ubiquitin maybe a earlier marker of neuron stress when the cell apoptosis has not been detected.Conclusions1. Brief episode of femoral artery occlusion and reperfusion can induce spinal cord ischemia tolerance in a rat model.2. Cannabinoid 1 receptor has mediated the protective mechanism.3. RIPC induces AEA increasing before ischemia in spinal cord which play a key role in the mechanism.4. RIPC suppressed the expression in spinal cord after ischemia, which is intercepted by AM251. Ubiquitin maybe a earlier marker of neuron stress when the cell apoptosis has not been detected.更多还原