法舒地尔提高猪缺血诱导心脏死亡供者心保存效果的实验研究

Experimental study on fasudil for increasing preserving effect on porcine DCD heart induced by ischemia

目的探讨法舒地尔对缺血诱导的心脏死亡供者(DCD)猪心的保护机制及效果。方法将16只家猪分为实验组和对照组,每组各8只。实验组:正中切口开胸,主动脉根部放血诱导供心停跳,继续热缺血25min,主动脉根部灌注含法舒地尔的冷stanford心肌保护液;切取供心,固定在langendorff离体灌注装置上,氧合温血持续灌注和平衡;含法舒地尔的冷stanford液冷浸浴原位保存2h;之后重新氧合温血持续复灌,心脏复跳,观察1h结束实验;对照组:整个实验过程中不使用法舒地尔,其余同实验组。在不同时间点测定两组心脏血流动力学(LVEDP、LVPDP、±dp/dt)、心肌酶、心肌梗死量、心肌组织含水量,观察心肌超微结构改变。结果所有供心均能正常复跳。供心在缺血前、平衡后,两组心脏血流动力学各项指标、冠状动脉流量、心肌酶差异无统计学意义;供心保存结束后复灌30min、复灌60min,实验组心功能和冠状动脉流量的恢复,优于对照组;心肌酶的漏出量、心肌梗死量、心肌组织含水量,实验组少于对照组;电镜下,实验组心肌纤维排列有序,闰盘、Z带清晰可见,肌原纤维间有杆状线粒体、丰富糖原颗粒,线粒体均匀排列;对照组细胞核椭圆形、不规则形,核膜清晰,肌纤维排列正常,闰盘、Z带欠清晰,线粒体轻度肿胀,部分嵴溶解,糖原颗粒减少。结论缺血诱导的猪DCD心,常温下缺血25 min,体外灌注可以复苏;法舒地尔添加到stanford液中,能减轻缺血诱导的猪DCD心I-R损伤,提高该类供心保存效果。

Objective To evaluate the effects of fasudil on DCD hearts .Methods Sixteen domestic ,mixed‐breed male porcine [mean body weight （28 ± 3）kg] were randomly divide into experiment group and control group （n= 8 in each） .Experiment group ：animals were sedated ,anesthetized and paralyzed .Mechanical ventilation with room air was provided .After thoroctomy ,cardiac arrest was established by bloodletting ,and then keeps at room temperature for additional 25 minutes （warm ischemia） .Hearts were perfused via the aortic root with fasudil （0 .1 mg/kg） enriched cold Stanford solution for coronary artery flushing ,rapidly excised and the aorta was cannulated .Hearts were subjected to isolated Langendorff perfusion （retrograde perfused with warm oxygenated autologous blood） afterwards ,and then underwent 20 minutes of equilibrium ,immersed in fasudil （0 .5 mg/kg） enriched cold Stan‐ford solution for 2 hours in situ cold preservation .Finally ,all the hearts were resuscitated with the warm oxygenated autologous blood perfusion .Control group ：hearts from the animals experienced the same except for fasudil supplement .Left ventricular per‐formances were evaluated .Coronary blood flow ,myocardial infarction volume ,myocardial water content ,and myocardial enzyme were measured .Myocardial electron microscopic examinations were carried out as well .Results All the hearts from both groups were successfully resuscitated ,fasudil significantly decreased water content ,enzyme leakage （P〈 0 .01 each versus control group） , and increased coronary blood flow （P〈0 .01 versus control group） .Left ventricular function were better preserved （P〈 0 .01 each versus control group） .All hearts lacked severe necrosis as determined by tetrazolium staining ,myocardial infarction volume were decresed in experiment group （P〈0 .01 each versus control group） .Intracellular components retained various types of organelle in both group ,but still ultrastructural alterations in control group were more distinctive than in experiment group .Conclusion （1） Donor heart arrested by exsanguinations and plus 25 minutes warm ischemia ,could still be resuscitated with satisfactory result ;（2） The addition of fasudil to Stanford solution （before excise of graft ,during the cold preservation） ,might alleviate DCD heart I‐R in‐jury ,improving the effect of DCD heart preservation ,and hopefully ,might be a novel arsenal in clinical DCD heart transplantation in the future .