Zeng Cheng, Hu Xiaoyan, Wang Yanfeng, Zeng Xianpeng, Xiong Yan, Li Ling, Ye Qifa
Transplant Center, Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430071, P.R. China.
Institute of Hepatobiliary Diseases, Wuhan University, Wuhan, Hubei 430071, P.R. China.
Exp Ther Med. 2018 Feb;15(2):1410-1416. doi: 10.3892/etm.2017.5587. Epub 2017 Dec 1.
The protective mechanisms for liver preservation associated with hypothermic machine perfusion (HMP) remain unclear. However, the lack of a common and portable HMP system for rat livers limits the study of HMP. The present study aimed to develop a novel, modified HMP system using a LifePort Kidney Transporter for preserving rat livers. A simple 'Y' shunt combined with a pressoreceptor for flow and pressure regulation was adapted to perfuse rat livers via the portal vein continuously using a LifePort Kidney Transporter under its 'prime mode' setting. An electronic scale was installed under the liver container to calculate the portal inflow according to the association with weight, density and volume of the perfusate. A total of 10 rat livers underwent 6 h of HMP using histidine-tryptophan-ketoglutarate solution enriched with acridine orange (AO) and propidium iodide (PI). The perfusion status of HMP was assessed by comparison of AO+PI-positive cell count in core region (CR) and peripheral region (PR) of rat liver under fluorescence microscopy. The dynamics (inflow, pressure and intrahepatic resistance of perfusion) were assessed to identify whether this system met the demands for HMP of rat livers. Biochemical [alanine transaminase (ALT), lactate dehydrogenase (LDH) and endothelin levels] and histological parameters (sinusoidal dilatation, endothelial cell detachment and vacuolization) were measured to determine cellular damage associated with HMP. No significant difference was observed between the CR and PR according to the comparison of the AO+PI-positive cell count, which indicated that complete perfusion was achieved. Intrahepatic resistance significantly decreased during the initial 3 h of HMP (P<0.01), but remained stable during the final 3 h. ALT and LDH levels significantly increased over the 6 h HMP duration: ALT (0 h, 42.67±5.81 U/l; 3 h, 90.67±6.74 U/l; 6 h, 164.33±7.31 U/l; P<0.01) and LDH (0 h, 492.90±90.20 U/l; 3 h, 973.53±97.4; 6 h, 1,843.40±85.78 U/l; P<0.01) However, the levels of endothelin and oxygen consumption were constant throughout HMP. Furthermore, histological analysis indicated sinusoidal dilation was significantly increased in the post-HMP group compared with the pre-HMP group (P<0.01); however, no other significant differences were observed. Combined with the results of ATP test (640.64±29.46 nmol/l) and bile production (4.88±0.69 µl/h/g of liver) at the end of HMP, the present results demonstrated minimal cellular injury associated with HMP while retaining the dependability and portability of the LifePort Kidney Transporter, which suggests the modified HMP system met the demands required and may be suitable for rat liver preservation.
低温机器灌注（HMP）相关的肝脏保存保护机制仍不清楚。然而，缺乏一种用于大鼠肝脏的通用且便携的HMP系统限制了对HMP的研究。本研究旨在开发一种使用LifePort肾脏转运器的新型改良HMP系统来保存大鼠肝脏。一个简单的“Y”形分流器与一个用于流量和压力调节的压力感受器相结合，通过LifePort肾脏转运器在其“灌注模式”设置下经门静脉持续灌注大鼠肝脏。在肝脏容器下方安装一个电子秤，根据灌注液的重量、密度和体积的关系来计算门静脉血流量。总共10个大鼠肝脏使用富含吖啶橙（AO）和碘化丙啶（PI）的组氨酸 - 色氨酸 - 酮戊二酸溶液进行了6小时的HMP。通过荧光显微镜比较大鼠肝脏核心区域（CR）和周边区域（PR）中AO + PI阳性细胞计数来评估HMP的灌注状态。评估动力学参数（灌注的流入量、压力和肝内阻力）以确定该系统是否满足大鼠肝脏HMP的要求。测量生化指标[丙氨酸转氨酶（ALT）、乳酸脱氢酶（LDH）和内皮素水平]和组织学参数（窦状隙扩张、内皮细胞脱离和空泡化）以确定与HMP相关的细胞损伤。根据AO + PI阳性细胞计数的比较，CR和PR之间未观察到显著差异，这表明实现了完全灌注。在HMP的最初3小时内肝内阻力显著降低（P < 0.01），但在最后3小时保持稳定。在6小时的HMP过程中，ALT和LDH水平显著升高：ALT（0小时，42.67±5.81 U/L；3小时，90.67±6.74 U/L；6小时，164.33±7.31 U/L；P < 0.01）和LDH（0小时，492.90±90.20 U/L；3小时，973.53±97.4；6小时，1843.40±85.78 U/L；P < 0.01）。然而，在整个HMP过程中内皮素水平和耗氧量保持恒定。此外，组织学分析表明，与HMP前组相比，HMP后组窦状隙扩张显著增加（P < 0.01）；然而，未观察到其他显著差异。结合HMP结束时的ATP测试结果（640.64±29.46 nmol/L）和胆汁生成量（4.88±0.69 μl/h/g肝脏），目前的结果表明与HMP相关的细胞损伤最小，同时保留了LifePort肾脏转运器的可靠性和便携性，这表明改良的HMP系统满足了所需的要求，可能适用于大鼠肝脏保存。
