Minor Thomas, Olschewski Peter, Tolba Rene H, Akbar Susanne, Kocálková Mariana, Dombrowski Frank
Surgical Research Division, University Clinic of Surgery, Bonn, Germany.
Clin Transplant. 2002 Jun;16(3):206-11. doi: 10.1034/j.1399-0012.2002.01128.x.
The aim of the present study was to improve the viability of marginal livers from non-heart beating donors upon cold preservation using two different techniques for the provision of tissue aerobiosis. Livers from male Wistar rats (250-300 g bw) were harvested after 60 min of cardiac arrest, flushed via the portal vein with 20 mL of heparinized Ringer's solution and 60 mL of histidine-tryptophan-ketoglutarate (HTK) preservation solution. Control livers were then stored submerged in HTK for 24 h at 4 degrees C while other organs were subjected to aerobic conditions by either insufflation of gaseous oxygen via the venous vascular system of the cold stored organ (VSOP) or pulsatile machine perfusion (MP) with oxygenated HTK at 5 mL/min at 4 degrees C. Superoxide dismutase (SOD) (7500 IU) was added to the last 10 mL of HTK in order to prevent adverse effects of high oxygen tensions at hypothermia. Viability of the livers was assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer at constant flow. VSOP or MP, both significantly improved vascular conductivity upon reperfusion as evaluated by portal venous pressure, reduced hepatic enzyme release and led to a rise in hepatic bile production upon reperfusion. Induction of apoptosis was also looked for in tissue homogenates by Western analysis for cleavage of poly(ADP-ribose)polymerase (PARP). Expression of cleaved PARP fragment could be found in reperfused control livers but also, though to a lesser extend, after VSOP or MP. In conclusion, provision of oxygen during cold preservation significantly contributes to improve organ viability upon reperfusion and must be regarded as a useful adjunct for marginal or pre-damaged livers. HTK has been shown for the first time to be also suitable for long-term MP preservation of the liver, but, as inferred from these data, simple insufflation of gaseous O2 may be considered a feasible alternative.
本研究的目的是通过两种不同的组织需氧供应技术，提高非心脏跳动供体边缘肝脏在冷保存时的活力。雄性Wistar大鼠（体重250 - 300克）在心脏骤停60分钟后摘取肝脏，经门静脉用20毫升肝素化林格氏液和60毫升组氨酸 - 色氨酸 - 酮戊二酸（HTK）保存液冲洗。对照肝脏随后在4℃下浸没于HTK中保存24小时，而其他肝脏通过以下两种方式之一进行需氧处理：通过冷保存器官的静脉血管系统吹入气态氧（VSOP），或在4℃下以5毫升/分钟的速度用含氧的HTK进行搏动性机器灌注（MP）。在最后10毫升HTK中加入超氧化物歧化酶（SOD）（7500国际单位），以防止低温时高氧张力的不良影响。在体外以恒定流量用含氧的克雷布斯 - 亨塞尔特缓冲液进行离体灌注时评估肝脏的活力。VSOP或MP在再灌注时均显著改善了血管传导性（通过门静脉压力评估），减少了肝酶释放，并导致再灌注时肝胆汁分泌增加。还通过蛋白质免疫印迹法分析组织匀浆中聚（ADP - 核糖）聚合酶（PARP）的裂解情况来寻找凋亡诱导现象。在再灌注的对照肝脏中可发现裂解的PARP片段表达，在VSOP或MP处理后也能发现，不过程度较轻。总之，冷保存期间提供氧气显著有助于提高再灌注时器官的活力，对于边缘或预先受损的肝脏而言，应被视为一种有用的辅助手段。首次证明HTK也适用于肝脏的长期MP保存，但从这些数据推断，简单地吹入气态O2可能是一种可行的替代方法。
