Amelioration of liver graft viability by improvement of sinusoidal microcirculation

【导语】非心脏跳动供体肝脏（NHBD）有望成为解决长期供体短缺问题的又一肝脏移植来源。不受控制的NHBD移植物通常遭受广泛的热缺血损伤，因此移植结果较差。为了明确保存NHBD肝脏的观点，我们比较了目前可用的三种溶液，UW， HTK和ET-Kyoto （ETK）溶液对NHBD肝脏保存后的损伤。[方法]Wistar大鼠在心脏骤停后60分钟或30分钟切除膈肌，取肝。经门静脉注射尿激酶溶液溶解窦内血栓，室温置肝10分钟。用80ml冷UW或HTK或ETK溶液冲洗肝脏，在各自的溶液中4℃保存8小时。以未保存的HBD肝为对照。我们用离体肝灌注模型评估移植物的生存能力。复温20分钟后，用含氧Krebs Henseleit缓冲液灌注更多的肝脏。灌注60分钟。在灌注过程中监测门静脉压和胆汁分泌。测定灌注液中AST水平。TUNEL染色检测细胞凋亡。[结果]注射尿激酶溶液可提高移植物存活率。ETK组门静脉压明显低于UW和HTK组。在再灌注60分钟时，ETK组比UW和HTK组产生更多的胆汁。ETK组与UW组比较差异有统计学意义。然而，ETK组的胆汁产量仅为HBD组的三分之一。ETK组灌注液中AST水平低于UW和HTK组。UW组与ETK组仅在再灌注后5分钟有统计学差异。H-E站立时，UW组肝细胞明显缩小，导致窦腔明显扩大。另一方面，在HTK组，肝脏结构得到了更好的保存。相反，较高的UW溶液渗透压可能会促进NHBD肝脏损伤前肝细胞的损伤。冷藏8小时后TUNEL染色。HTK组虽结构保留，但弥漫性出现大量TUNEL阳性肝细胞。相反，UW组仅见少量TUNEL阳性细胞，肝细胞明显萎缩。[结论]与HBD肝脏相比，较高的UW溶液渗透压可能对NHBD肝脏的保存有害。在NHBD移植物中，冷保存期间细胞凋亡的限制并不一定有助于维持更好的移植物活力。ETK方案似乎比HTK方案有优势。然而，ETK溶液足以维持NHBD移植物的活力。寻找更好的保存方法抢救NHBD肝脏是必要的。少

[Introduction] Non-heart-beating donor (NHBD) livers are anticipated as an additional source of grafts to solve chronic shortage of donors. Uncontrolled NHBD graft usually suffers from an extensive injury of warm ischemia, and the transplantation therefore results in poor outcomes. To make a point for preserving NHBD livers clear, we compared post preservation damage of NHBD livers using three solutions available at present, UW, HTK, and ET-Kyoto (ETK) solutions.[Methods] Wistar rats underwent phrenotomy and livers were retrieved 60 or 30 minutes after cardiac arrest. Urokinase solution was injected via the portal vein in order to dissolve thrombi in sinusoid, then the livers were left 10 minute at room temperature. The livers were flushed by 80ml of cold UW or HTK or ETK solution, and preserved in the respective solution for 8 hours at 4℃. HBD liver without preservation was used as control. We assessed graft viability using isolated liver perfusion model. After 20-minute rewarming, li … More vers were perfused by oxygenated Krebs Henseleit Buffer. Perfusion was carried out for 60 minutes. Portal venous pressure and bile production were monitored during perfusion. AST level in the perfusate was measured. Apoptosis was evaluated by TUNEL staining.[Results] The injection of urokinase solution improved graft viability. Portal venous pressure was lower in ETK group than UW and HTK groups. ETK group produced more bile during 60-minute reperfusion than UW and HTK groups. There was significant difference between ETK group and UW group. However, bile production in ETK group was only one-third of HBD livers. The level of AST in the perfusate was lower in ETK group than UW and HTK groups. There was significant difference only in 5 minute after reperfusion between the groups of UW and ETK. In H-E staning, hepatocyte shrank markedly in the UW group, resulting in remarkable extension of sinusoidal space. On the other hand, in the HTK group, the liver architecture was better preserved. A higher osmolarity of UW solution might, in converse, facilitate the damage in the pre-injured hepatocytes in NHBD livers. TUNEL staining after 8-hour cold storage. In HTK group, in spite of preserved architecture, there appeared many TUNEL positive hepatocytes diffusely. Conversely, only a few TUNEL positive cells were noticed in UW group, despite hepatocytes showed marked shrinkage.[Conclusion] Instead of HBD livers, higher osmolarity of UW solution may be harmful in the preservation of NHBD livers. In NHBD graft, limitation of apoptosis during cold preservation did not necessarily contribute to maintaining a better graft viability. ETK solution seemed to have advantage, compared with HTK solution. However, ETK solution was enough to maintain NHBD graft viability. A better strategy in preservation method for salvaging NHBD livers is necessary. Less