缺血预处理与缺氧后处理对离体心脏保存的心肌保护作用
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摘要
目的：观察缺血预处理与复跳后心脏进行缺氧处理对离体保存心脏的心肌保护作用。
方法: 将27只大鼠的离体鼠心脏用langendorff装置灌注稳定20分钟后，随机分为3组，每组9只，给予不同的处理。对照组连续灌注K-H液15分钟后，灌注St.Thoma’s2号液，使心脏停跳并将心脏保存在4℃St.Thoma’s2号液中，6小时以后使心脏复跳并再连续灌注K-H液60分钟。缺血预处理组给予5分钟的缺血灌注和10分钟的再灌注完成缺血预处理,以后的处理与对照组相同。缺氧后处理组与对照组前面的处理相同,仅在复跳后给予反复多次的缺氧后处理,再连续灌注K-H液35分钟，一共60分钟。各组分别测量各项心功能指标的恢复率、冠脉流出量、冠脉回流液中碱性磷酸激酶同工酶(CK-MB)的浓度、心肌含水量、严重心律失常的发生率以及左室心内膜下心肌的电镜下超微结构观察。
结果: 缺血预处理可以显著提高保存后鼠心的LVESP、+dp/dtmax、-dp/dtmax（P<0.01），缺氧后处理也提高了鼠心的LVESP、+dp/dtmax、-dp/dtmax（P<0.05），而前者在+dp/dtmax、-dp/dtmax恢复方面优于后者 （P<0.05）。缺血预处理组的LVEDP低于对照组（P<0.05），缺血预处理组与缺氧后处理组的CK-MB漏出量均低于对照组（P<0.05），而两组之间对比无统计学意义。两组的严重心律失常发生率均低于对照组。缺氧后处理组的心肌含水量与对照组比有明显减轻（P<0.05）。电镜超微结构显示缺血预处理与缺氧后处理两组的心肌结构要好于对照组。
结论: 缺血预处理对离体心脏的保存有明显的保护作用；缺氧后处理对离体心脏的保存也有一定的保护作用，此结果为离体心脏的保存提供了新的思路。
Objective: This study was designed to assess the protective effects of ischemic preconditioning and hypoxic postconditioning on long-term myocardial preservation in isolated rat hearts by using the Langendorff Model.
Method: 27 isolated rat hearts were perfused with oxygen-bicarbonated Krebs-Henseleit buffer in the Langendorff mode for 20 minutes. These hearts were then randomly divided into three groups : Group A(n=9, sham controls, SC), the hearts were perfused continuously for another 15 min; Group B(n=9, ischemic preconditioning, IP) the hearts were delt with 5 min of global ischemia and 10 min of buffer-perfusion, both group A and group B were then arrested and preserved for 6 h with St.Thoma’s solution II at 4℃, followed by 60 min of reperfusion; Group C(n=9, hypoxic postconditioning, HP), the isolated hearts were perfused and preserved in the same way as group A, but during the reperfusion period the hearts were treated with hypoxic postconditioning, they were induced with repeated ischemia and reperfusion four times and followed by 35 min of reperfusion. Observed indexes include: 1)recovery of cardiac function; 2) coronary flow; 3)myocardial QCK—MB leakage; 4)myocardial water content; 5) incidence of serious arrhythmia; 6）myocardial ultrastructure observation.
Results: The results demonstrated that ischemic preconditioning significantly improved myocardial contractility and relaxation as compared with sham control group during reperfusion period(P<0.01)，so was the hypoxic postconditioning group (P<0.05). But the hearts that underwent IP showed better recovery of cardial function than those underwent HP(P<0.05). IP group showed a lower left ventricular end-diastolic pressure level(P<0.05). Both IP and HP group showed less CK-MB leakage(P<0.05), but there was no statistical difference between IP group and HP group. The serious arrhythmia incidence of
both groups was less than SC group. Myocardial water content in HP group was less than SC group. Myocardial electron microscope observation showed that myocardial ultrastructure of IP group and HP group was better than that of SC group.
Conclusion: These data suggested that ischemic preconditioning can significantly enhance cardio-protective effects on long-term myocardial preservation. Hypoxic postconditioning can also improve the ventricular function in some degree. Ischemic preconditioning and hypoxic postconditioning might be effective methods for clinical myocardial protection in myocardial preservation for heart transplantation.
方法: 将27只大鼠的离体鼠心脏用langendorff装置灌注稳定20分钟后，随机分为3组，每组9只，给予不同的处理。对照组连续灌注K-H液15分钟后，灌注St.Thoma’s2号液，使心脏停跳并将心脏保存在4℃St.Thoma’s2号液中，6小时以后使心脏复跳并再连续灌注K-H液60分钟。缺血预处理组给予5分钟的缺血灌注和10分钟的再灌注完成缺血预处理,以后的处理与对照组相同。缺氧后处理组与对照组前面的处理相同,仅在复跳后给予反复多次的缺氧后处理,再连续灌注K-H液35分钟，一共60分钟。各组分别测量各项心功能指标的恢复率、冠脉流出量、冠脉回流液中碱性磷酸激酶同工酶(CK-MB)的浓度、心肌含水量、严重心律失常的发生率以及左室心内膜下心肌的电镜下超微结构观察。
结果: 缺血预处理可以显著提高保存后鼠心的LVESP、+dp/dtmax、-dp/dtmax（P<0.01），缺氧后处理也提高了鼠心的LVESP、+dp/dtmax、-dp/dtmax（P<0.05），而前者在+dp/dtmax、-dp/dtmax恢复方面优于后者 （P<0.05）。缺血预处理组的LVEDP低于对照组（P<0.05），缺血预处理组与缺氧后处理组的CK-MB漏出量均低于对照组（P<0.05），而两组之间对比无统计学意义。两组的严重心律失常发生率均低于对照组。缺氧后处理组的心肌含水量与对照组比有明显减轻（P<0.05）。电镜超微结构显示缺血预处理与缺氧后处理两组的心肌结构要好于对照组。
结论: 缺血预处理对离体心脏的保存有明显的保护作用；缺氧后处理对离体心脏的保存也有一定的保护作用，此结果为离体心脏的保存提供了新的思路。
Objective: This study was designed to assess the protective effects of ischemic preconditioning and hypoxic postconditioning on long-term myocardial preservation in isolated rat hearts by using the Langendorff Model.
Method: 27 isolated rat hearts were perfused with oxygen-bicarbonated Krebs-Henseleit buffer in the Langendorff mode for 20 minutes. These hearts were then randomly divided into three groups : Group A(n=9, sham controls, SC), the hearts were perfused continuously for another 15 min; Group B(n=9, ischemic preconditioning, IP) the hearts were delt with 5 min of global ischemia and 10 min of buffer-perfusion, both group A and group B were then arrested and preserved for 6 h with St.Thoma’s solution II at 4℃, followed by 60 min of reperfusion; Group C(n=9, hypoxic postconditioning, HP), the isolated hearts were perfused and preserved in the same way as group A, but during the reperfusion period the hearts were treated with hypoxic postconditioning, they were induced with repeated ischemia and reperfusion four times and followed by 35 min of reperfusion. Observed indexes include: 1)recovery of cardiac function; 2) coronary flow; 3)myocardial QCK—MB leakage; 4)myocardial water content; 5) incidence of serious arrhythmia; 6）myocardial ultrastructure observation.
Results: The results demonstrated that ischemic preconditioning significantly improved myocardial contractility and relaxation as compared with sham control group during reperfusion period(P<0.01)，so was the hypoxic postconditioning group (P<0.05). But the hearts that underwent IP showed better recovery of cardial function than those underwent HP(P<0.05). IP group showed a lower left ventricular end-diastolic pressure level(P<0.05). Both IP and HP group showed less CK-MB leakage(P<0.05), but there was no statistical difference between IP group and HP group. The serious arrhythmia incidence of
both groups was less than SC group. Myocardial water content in HP group was less than SC group. Myocardial electron microscope observation showed that myocardial ultrastructure of IP group and HP group was better than that of SC group.
Conclusion: These data suggested that ischemic preconditioning can significantly enhance cardio-protective effects on long-term myocardial preservation. Hypoxic postconditioning can also improve the ventricular function in some degree. Ischemic preconditioning and hypoxic postconditioning might be effective methods for clinical myocardial protection in myocardial preservation for heart transplantation.
引文
Cooper DKC. The transplantation and replacement of thoracic organs [M]. Lancaster. Kluwer Academic Publishers, 1990,67(2):245~247.
Murry CE，Jennings RB，Reimer KA. Preconditioning with ischemia：a delay of lethal cell injury in ischemic myocardium [J]. Circulation, 1986,74(5):1124～1136.
高 峰，Yan WL, Geng YJ et al. “缺氧后处理”对大鼠缺氧－复氧心室肌细胞的保护作用［J］. 心功能杂志，1999；11(4)：241-243.
Ravinerova T, Tribulova N, Slezak J, et al. Brief intermediate and prolonged ischemia in isolated crystalloid perfused rat heart: relationship between susceptibility to arrhythmias and degree of ultrastructural injury[J]. Mol Cell Cardiol, 1995,27:1937-1951.
Grunnet N, Asmundsson P, Madsen M, et al. Organ donation, allocation and transplantation in the Nordic countries is cardiatranslant 1999 [J]. Transplant Proc, 2001,34(4):2505-2513.
Michel P, Vial R, Rodriguez C, et al. A comparative study of the most widely used solutions for cardiac graft preservation during hypothermia [J]. J Heart Lung Transplant, 2002,21(9):1030~1039
Leesar MA, Stoddard M, Ahmed. Preconditioning of human myocardium with adenosine during coronary angioplasty. Circulation. 1997,95(11):2500-2504.
Crestanello JA, Doliba NM, Babsky AM, Doliba NM, Niibori K, Osbakken MD, Whitman GJ. Mitochondrial function during ischemic preconditioning. Surgery. 2002 Feb;131(2):172-8.
Illes RW, Swoyer KD. Prospective, randomized clinical study of ischemic preconditioning as an adjunct to intermittent cold blood cardio plegia [J]. Ann Thorac Surg, 1998,65(3):748~753.
Kaneda T, Zhang ZW, Ku K, et al. Ischemic preconditioning and nicorandil pretreatment improve donor heart preservation [J]. Jpn Circ, 2001,65(7):678~82.
Taylor SG, Southerton JS, Weston AH, Baker JRJ. Endothelium-dependent effects of acetylcholine in rat aorta;a comparison with sodium nitroprusside and cromakalim.Br J Pharmacol 1988;94;853-63
Kosieradzki M. Mechanisms of ischemic preconditioning and its application in transplantation [J]. Ann Transplant, 2002,7(3):12~20.
张建军,胡大一,刘秀兰,等. 缺血预适应抗心律失常机制的实验研究 [J]. 中国心脏起搏与心电生理杂志, 1999,13(3):178～181
臧益民，朱妙章. 冠状循环生理与临床的关系［M］. 见臧益民，朱妙章. 临床心血管生理学及其进展，1993:136-147.
Cave AC, Collis CS, Downey JM et al. Improved functional recovery by ischemic preconditioning is not mediated by adenosine in the globally ischemic isolated rat heart［J］. Cardiovasc Res,1993;27(3):663-670.
Murry CE，Jennings RB，Reimer KA. Preconditioning with ischemia：a delay of lethal cell injury in ischemic myocardium [J]. Circulation, 1986,74(5):1124～1136.
高 峰，Yan WL, Geng YJ et al. “缺氧后处理”对大鼠缺氧－复氧心室肌细胞的保护作用［J］. 心功能杂志，1999；11(4)：241-243.
Ravinerova T, Tribulova N, Slezak J, et al. Brief intermediate and prolonged ischemia in isolated crystalloid perfused rat heart: relationship between susceptibility to arrhythmias and degree of ultrastructural injury[J]. Mol Cell Cardiol, 1995,27:1937-1951.
Grunnet N, Asmundsson P, Madsen M, et al. Organ donation, allocation and transplantation in the Nordic countries is cardiatranslant 1999 [J]. Transplant Proc, 2001,34(4):2505-2513.
Michel P, Vial R, Rodriguez C, et al. A comparative study of the most widely used solutions for cardiac graft preservation during hypothermia [J]. J Heart Lung Transplant, 2002,21(9):1030~1039
Leesar MA, Stoddard M, Ahmed. Preconditioning of human myocardium with adenosine during coronary angioplasty. Circulation. 1997,95(11):2500-2504.
Crestanello JA, Doliba NM, Babsky AM, Doliba NM, Niibori K, Osbakken MD, Whitman GJ. Mitochondrial function during ischemic preconditioning. Surgery. 2002 Feb;131(2):172-8.
Illes RW, Swoyer KD. Prospective, randomized clinical study of ischemic preconditioning as an adjunct to intermittent cold blood cardio plegia [J]. Ann Thorac Surg, 1998,65(3):748~753.
Kaneda T, Zhang ZW, Ku K, et al. Ischemic preconditioning and nicorandil pretreatment improve donor heart preservation [J]. Jpn Circ, 2001,65(7):678~82.
Taylor SG, Southerton JS, Weston AH, Baker JRJ. Endothelium-dependent effects of acetylcholine in rat aorta;a comparison with sodium nitroprusside and cromakalim.Br J Pharmacol 1988;94;853-63
Kosieradzki M. Mechanisms of ischemic preconditioning and its application in transplantation [J]. Ann Transplant, 2002,7(3):12~20.
张建军,胡大一,刘秀兰,等. 缺血预适应抗心律失常机制的实验研究 [J]. 中国心脏起搏与心电生理杂志, 1999,13(3):178～181
臧益民，朱妙章. 冠状循环生理与临床的关系［M］. 见臧益民，朱妙章. 临床心血管生理学及其进展，1993:136-147.
Cave AC, Collis CS, Downey JM et al. Improved functional recovery by ischemic preconditioning is not mediated by adenosine in the globally ischemic isolated rat heart［J］. Cardiovasc Res,1993;27(3):663-670.