河豚毒素心脏停搏液用于离体大鼠心肌保护的实验研究
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摘要
目的:
研究河豚毒素(tetrodotoxin,TTX)心脏停搏液对离体大鼠缺血/再灌注心肌在器官水平和细胞水平的保护作用,探索新的心肌保护方法。
方法:
实验Ⅰ. 取 Wistar 大鼠心脏建立 Langendorff 灌注模型和 Neely左心室工作灌注模型,分成 2 组:即 TTX 停搏组和 STH-2 停搏组。TTX组用 TTX 心脏停搏液停搏,STH-2 组用 St.Thomas No.2 cardioplegia(STH-2)停搏。停搏后将鼠心置于相应配伍的停搏液中,7℃保存 5小时后恢复灌注。观察停搏前和再灌注后各组心脏血液动力学、心肌酶学、能量物质腺苷酸含量和心肌超微结构等方面的改变。
实验Ⅱ. 用酶解法分离Wistar大鼠心脏获得具有搏动性的心室肌细胞悬液,随机分成基础组、STH-2 组和 TTX 组。基础组仅以 K-H 液处理,STH-2 组和 TTX 组分别应用 STH-2 和 TTX 心脏停搏液处理,建立模拟缺血/再灌注损伤的停搏/复搏心肌细胞模型,应用激光扫描共聚焦显微镜与荧光探针相结合的技术,测定各组心肌细胞不同时期的[Na+]i和[Ca2+]i。用倒置显微镜观察细胞搏动情况和形态学变化。
结果:
1.离体大鼠心脏 Langendorff 灌注模型停搏前两组之间的指标无
Objective:
To investigate the protective effects of cardioplegia with sodium channel inhibitor tetrodotoxin(TTX) on ischemia/reperfusion models of isolated rat hearts and isolated cardiomyocytes so as to explore a new myocardial protective method.
Methods:
Test 1. Langendorff perfusion models and Neely working left ventricular perfusion models of isolated rat hearts were sequentially created in Group TTX and Group STH-2. The hearts in each group were arrested by corresponding cardioplegia in Group TTX(study group, TTX ) and Group STH-2 (control group, only STH-2). The arrested hearts were preserved in the corresponding cardioplegia solution respectively at 7℃ for 5 hours, and then reperfused. Pre-ischemic and post-ischemic indexes were studied including hemodynamic parameters, myocardial enzymology, ATP content and ultrastructural changes.
Test 2. Beating ventricular cardiomyocytes were isolated from adult rat hearts by enzymatic dissociation, and randomly created in Group Base, Group STH-2 and Group TTX. Group Base was only treated with K-H buffer solution. Group STH-2 and Group TTX were treated with STH-2 and TTX cardioplegia respectively, and the arrest/re-beating cell models
imitating ischemia-reperfusion injury were established. All three groups were imaged by laser scanning confocal microscope with fluorescent probe for measuring [Na+]i and [Ca2+]i of cardiomyocytes in different stages. The beating status and morphology of cardiomyocytes were observed under the inverted microscope.
Results:
There were no differences in two groups of Langendorff rat hrart models before myocardial ischemia. But the recovery of hemodynamic parameters during reperfusion in Group TTX were better than those in Group STH-2(P<0.01); the releases of CPK and LDH in Group TTX were less than those in Group STH-2(P < 0.05); the activities of Na~+-K~+-ATPase, Ca~(2+)-ATPase, Ca~(2+)-Mg~(2+)-ATPase , and contents of ATP in Group TTX kept relative higher level after reperfusion. Compared with Group STH-2, myocardial ultrastructure in Group TTX was better protected . The elevation of [Na~+]i and [Ca~(2+)]i of cardiomyocyte models during arrest in Group TTX was significantly lower than that in Group STH-2. [Na~+]i and [Ca~(2+)]i in both Group STH-2 and Group TTX after re-beating were higher than those in Group Base (P<0.01), but [Na~+]i and [Ca~(2+)]i in Group TTX were significantly lower than those in Group STH-2(P<0.01). There was no difference in beating rate between Group STH-2 and Group TTX(P>0.05). Active cardiomyocytes with normal morphology in Group TTX after re-beating were more counted than those in Group STH-2 (P<0.01).
Conclusion:
Compared with conventional hyperkalemic depolarizing cardioplegia, polarizing cardioplegia with sodium channel inhibitor TTX could alleviate intracellular Na+ -overload and Ca2+-overload in isolated cardiomyocytes of rat; reduce the consumption of energy; decrease the releases of CPK and
LDH after reperfusion; avoid ultrastructural damage and prevent ischemia-reperfusion injury by arresting heart under polarizing condition. Therefore, polarizing cardioplegia with sodium channel inhibitor TTX might be a new and better cardioplegia in myocardial protection and preservation .
研究河豚毒素(tetrodotoxin,TTX)心脏停搏液对离体大鼠缺血/再灌注心肌在器官水平和细胞水平的保护作用,探索新的心肌保护方法。
方法:
实验Ⅰ. 取 Wistar 大鼠心脏建立 Langendorff 灌注模型和 Neely左心室工作灌注模型,分成 2 组:即 TTX 停搏组和 STH-2 停搏组。TTX组用 TTX 心脏停搏液停搏,STH-2 组用 St.Thomas No.2 cardioplegia(STH-2)停搏。停搏后将鼠心置于相应配伍的停搏液中,7℃保存 5小时后恢复灌注。观察停搏前和再灌注后各组心脏血液动力学、心肌酶学、能量物质腺苷酸含量和心肌超微结构等方面的改变。
实验Ⅱ. 用酶解法分离Wistar大鼠心脏获得具有搏动性的心室肌细胞悬液,随机分成基础组、STH-2 组和 TTX 组。基础组仅以 K-H 液处理,STH-2 组和 TTX 组分别应用 STH-2 和 TTX 心脏停搏液处理,建立模拟缺血/再灌注损伤的停搏/复搏心肌细胞模型,应用激光扫描共聚焦显微镜与荧光探针相结合的技术,测定各组心肌细胞不同时期的[Na+]i和[Ca2+]i。用倒置显微镜观察细胞搏动情况和形态学变化。
结果:
1.离体大鼠心脏 Langendorff 灌注模型停搏前两组之间的指标无
Objective:
To investigate the protective effects of cardioplegia with sodium channel inhibitor tetrodotoxin(TTX) on ischemia/reperfusion models of isolated rat hearts and isolated cardiomyocytes so as to explore a new myocardial protective method.
Methods:
Test 1. Langendorff perfusion models and Neely working left ventricular perfusion models of isolated rat hearts were sequentially created in Group TTX and Group STH-2. The hearts in each group were arrested by corresponding cardioplegia in Group TTX(study group, TTX ) and Group STH-2 (control group, only STH-2). The arrested hearts were preserved in the corresponding cardioplegia solution respectively at 7℃ for 5 hours, and then reperfused. Pre-ischemic and post-ischemic indexes were studied including hemodynamic parameters, myocardial enzymology, ATP content and ultrastructural changes.
Test 2. Beating ventricular cardiomyocytes were isolated from adult rat hearts by enzymatic dissociation, and randomly created in Group Base, Group STH-2 and Group TTX. Group Base was only treated with K-H buffer solution. Group STH-2 and Group TTX were treated with STH-2 and TTX cardioplegia respectively, and the arrest/re-beating cell models
imitating ischemia-reperfusion injury were established. All three groups were imaged by laser scanning confocal microscope with fluorescent probe for measuring [Na+]i and [Ca2+]i of cardiomyocytes in different stages. The beating status and morphology of cardiomyocytes were observed under the inverted microscope.
Results:
There were no differences in two groups of Langendorff rat hrart models before myocardial ischemia. But the recovery of hemodynamic parameters during reperfusion in Group TTX were better than those in Group STH-2(P<0.01); the releases of CPK and LDH in Group TTX were less than those in Group STH-2(P < 0.05); the activities of Na~+-K~+-ATPase, Ca~(2+)-ATPase, Ca~(2+)-Mg~(2+)-ATPase , and contents of ATP in Group TTX kept relative higher level after reperfusion. Compared with Group STH-2, myocardial ultrastructure in Group TTX was better protected . The elevation of [Na~+]i and [Ca~(2+)]i of cardiomyocyte models during arrest in Group TTX was significantly lower than that in Group STH-2. [Na~+]i and [Ca~(2+)]i in both Group STH-2 and Group TTX after re-beating were higher than those in Group Base (P<0.01), but [Na~+]i and [Ca~(2+)]i in Group TTX were significantly lower than those in Group STH-2(P<0.01). There was no difference in beating rate between Group STH-2 and Group TTX(P>0.05). Active cardiomyocytes with normal morphology in Group TTX after re-beating were more counted than those in Group STH-2 (P<0.01).
Conclusion:
Compared with conventional hyperkalemic depolarizing cardioplegia, polarizing cardioplegia with sodium channel inhibitor TTX could alleviate intracellular Na+ -overload and Ca2+-overload in isolated cardiomyocytes of rat; reduce the consumption of energy; decrease the releases of CPK and
LDH after reperfusion; avoid ultrastructural damage and prevent ischemia-reperfusion injury by arresting heart under polarizing condition. Therefore, polarizing cardioplegia with sodium channel inhibitor TTX might be a new and better cardioplegia in myocardial protection and preservation .
引文
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with cation channel blockers[J]. Eur J Pharmacol,1999 May 7,372(1):
37～48
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[2] 龙村.体外循环中的心肌保护:有关心肌保护的几个热点问题[M].中国医学科学院协和医科大学,1997,1～4
[3] Chambers DJ,Hearse DJ. Developments in cardioprotection:“polarized” arrest as an alternative to “depolarized” arrest [J] . Ann Thorac Surg,1999,68(5):1960～1966
[4] Racay P,Matejovicova M,Drgova A.The effect of ischemia and ischemia～reperfusion on ion transport systems[J]. Bratisl Lek Listy, 1998, 99(7):386～394
[5] Jayawant M , Stephenson ER , Damiano RJ. Advantages of continuous hyperpolarized arrest with pinacidil over St.Thomas’ hospital solution during prolonged ischemia[J].J Thorac Cardiovasc Surg,1998,116(1):131～138
[6] Lawton JS,Hsia PW,Allen CT,et al. Myocardial protection in the acutely injury heart : hyperpolarizing versus depolarizing hypothermic cardioplegia[J]. J Thorac Cardiovasc Surg,1997,113(3):567～575
[7] Damiano RJ.The electrophysiology of ischemia and cardioplegia :implications for myocardial protection[J].J Card Surg ,1995,10(4 suppl):445～453
[8] Maskal SL, Cohen NM, Hsia PW, et al. Hyperpolarized cardiac arrest with a potassium～channel opener, aprikalim[J]. J Thorac Cardiovasc Surg, 1995, 110(4): 1083～1095
[9] Snabaitis AK,Shattock MJ,Chambers DJ.A comparison of polarized and depolarized arrest in the isolated rat heart for long-time preservation[J]. Circulation,1997,96:3148～3156
[10] Tanonaka K,Kajiwara H,Kameda H,et al. Relationship between myocardial cation content and injury in reperfused rat hearts treated
with cation channel blockers[J]. Eur J Pharmacol,1999 May 7,372(1):
37～48
[11] LoPachin RM,Gaughan CL,Lehning EJ,et al.Effects of ion channel blockade on the distribution of Na,K,Ca and other elements in oxygen-glucose deprived CA1 hippocampal neurons[J].Neuroscience,2001;103(4):971～83
[12] Garvin PJ,Niehoff ML,Robinson SM.Effects of tetrodotoxin and OKY-046 in renal ischemia reperfusion[J]. J Surg Res,1999 Aug,85(2):273～8
[13] Snabaitis AK , Chambers D. Long-term myocardial preservation :beneficial and additive effects of polarized arrest (Na+channel blockade) , Na+/H+exchange inhibition , and Na+/K+/2Cl ～ cotransport inhibition combined with calcium desensitization[J]. Transplantation,1999 Nov 27,68:1444～1453
[14] Attwell D,Cohen I, Eisner D,et al.The steady state TTX-sensitive (“window”) sodium current in cardiac Purkinje fibres[J]. Pflügers Arch,1979,379 (2):137～142
[15] Chambers DJ.Polarization and myocardial protection[J] . Curr Opin Cardiol,1999,14(6):495～500
[16] 徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社.1991,854～862
[17] Neely JR,Liebermeister H,Batterby EJ,et al.Effect of pressure development on oxygen consumption by isolated rat heart[J].Am J Physiol,1967,212(6):804～814
[18] 肖颖彬,杨素娟,韩本立,等.附加 N～乙酰半胱氨酸心停搏液对低温缺血～再灌注大鼠心脏的保护作用[J]. 第三军医大学学报,1999,21(1):45～48
[19] Juengling E,Kammermeier H.Rapid assay of adenine nucleotides or creatine compounds in extracts tissue by paired-ion reverse-phase high performance liquid chromatography[J].Anal Biochem,1980,102:358
[20] 朱珠,陈烯伟.一种快速、简便测定腺苷酸含量的高效液相色谱法[J].第三军医大学学报,1995,17(4):351～353
[21] Balderman S,Bhayana J,Binette P,et al. Perioperative preservation of myocardial ultrastructual and high-energy phosphates in man[J]. J Thorac Cardiovasc Surg,1981,82:860～874
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