Monitoring and prevention of ischaemia-reperfusion injury in liver transplantation : experimental and clinical studies

Abstract

Liver function after transplantation is determined not only by the quality of the donor organ or recipient related factors, but also by factors associated with organ preservation. During preservation, the liver graft is exposed to ischaemia and reperfusion with subsequent injury to the transplanted liver. This injury results in primary liver damage with negative consequences in hepatic metabolism. In severe cases, the graft may never start to function. Therefore, methods allowing close monitoring of the liver during and after transplantation are wan-anted. Reduction in injury related to preservation may be achieved by introduction of more effective preservation methods and better preservation solutions. It could also be achieved by donor pretreatment, which leads to increased resistance of the graft prior to the injury. The primary aim of our studies was to evaluate continuous intrahepatic microdialysis as a method for monitoring of liver metabolism during transplantation in humans and in a large animal model (pigs). A second aim was to study the effect of donor pretreatment with the hydrophilic bile acid, ursodeoxycholic acid (UDCA), in experimental models in rats and pigs.

Intrahepatic glucose metabolism based on glucose, lactate and pyruvate, was monitored with microdialysis during the transplantation procedure in a pig model. Additionally, intrahepatic glycerol levels, reflecting cell membrane damage were monitored. In addition, the lactate/pyruvate ratio, which has been shown to be a sensitive marker of ischaemia, was calculated. At the beginning of preservation, after cold perfusion, glucose, lactate and glycerol levels increased, whereas pyruvate levels decreased rapidly. During cold storage, glucose levels increased, whereas lactate was stable and pyruvate levels were below detection limits. A strong correlation between glycerol levels and time of cold storage was observed. When the liver graft was placed in the recipient and was warmed up (by the environment), glucose, lactate and glycerol showed an accelerated increase.

Shortly after reperfusion, these levels peaked and started to normalise thereafter. Pyruvate also increased after reperfusion but peaked later. The lactate/pyruvate ratio increased after cold perfusion and remained stable during cold storage. During implantation it showed an accelerated increase, but decreased rapidly after reperfusion. In livers with longer cold storage, significantly higher levels of glucose and glycerol were observed after reperfusion. These metabolic changes observed in the liver during and after transplantation indicate that rewarming during implantation of the liver graft into the recipient followed by reperfusion exacerbates the injury already incurred during cold storage. High intrahepatic glucose levels appear to be a liverspecific event, which represents glycogen degradation in injured hepatocytes.

The study in rats on oral donor pretreatment with UDCA showed that UDCA was enriched in the bile to 36% of bile acid content in UDCA pretreated rats, which was associated with a higher bile flow after reperfusion. Moreover, the liver transplantation model in rats showed that UDCA protects the liver from ischaemia-reperfusion injury as indicated by lower levels of serum transaminase and reduced induction of hepatocyte apoptosis. In order to obtain more clinically relevant data on protective effect of UDCA on liver grafts, a large animal model of liver transplantation in pigs was tested. Despite an unexpectedly low UDCA enrichment in bile (to 3.6%) in the treated group, significant effects of UDCA on glucose metabolism and reduction of neutrophils infiltration were observed. In fact, after reperfusion, glucose and lactate levels were significantly lower in the UDCA treated animals. Results from studies on pretreatment with UDCA showed that augmented bile saltdependent bile flow is preserved in the liver after cold storage. The pretreatment with UDCA may also lead to unproved liver cell function early after transplantation as indicated by less glycogenolysis and reduced non-oxidative glycolysis.

Based on our experimental studies, intrahepatic microdialysis was evaluated in liver graft monitoring in humans. Ten consecutive patients undergoing liver transplantation were continuously monitored with microdialysis for 72 hours after transplantation. During the first 24 hours, intrahepatic glucose levels were higher than in the systemic circulation. Initial increases in lactate and lactate/pyruvate ratio normalized within the first 24 hours and remained stable. Also initial elevations in glycerol levels decreased within 16 hours and remained stable thereafter. This study is the first to use microdialysis to describe the normal recovery from ischaemia after human liver transplantation.

In conclusion, continuous microdialysis monitoring of the liver graft is a useful tool in research on liver preservation. It is easy to perform and safe in a clinical setting. Its use to detect and monitor pathological changes in the liver graft, such as hepatic artery thrombosis, portal vein thrombosis or early rejection, should be addressed in further studies. The experimental studies on donor pretreatment with UDCA indicate a protective effect on the transplanted liver graft and should, therefore, be tested in controlled clinical trials.