Experimental & Clinical Applications of Ex Vivo Liver Perfusion
Creators: Hefler, Joshua J.
Publisher: name: University of Alberta Library
Year: 2023

<p>Normothermic machine perfusion (NMP) is a novel method of organ preservation that has recently risen in prominence. NMP involves the maintenance of an organ outside the body to recreate its physiologic milieu, such as by providing oxygen and nutrients and maintaining temperature. We are only just beginning to understand the variety of ways that NMP<br>
can be implemented to solve clinical problems in liver transplantation, as well as address research questions in transplantation and hepatology, more broadly. This thesis presents a mixture of basic science and clinical studies. <br>
The first studies are dedicated to building an NMP-based large animal model of donation after circulatory death (DCD) to test the effect of cyclosporine A (CsA) on hepatic ischemia/reperfusion injury (IRI). It starts with a systematic review and meta-analysis to identify evidence for CsA in preclinical models of myocardial IRI (in which it has been most studied). It was found that the most common dose was 10mg/kg and the effect was less pronounced in models using pigs and/or female animals and greater when dosed prior to the onset of ischemia. Also, part of the reason why CsA failed to translate to clinical studies of myocardial IRI was the prolonged duration of ischemia compared to preclinical models, which makes transplantation potentially better suited to intervention with CsA. The second study in this series tested the effect of CsA and its nonimmunosuppressive analog, NIM-811, in a murine model of partial warm hepatic IRI. It was found that animals treated with either 10 or 25mg/kg of CsA or 10mg/kg of NIM-811 had decreased<br>
serum alanine aminotransferase (ALT) at six hours post-reperfusion compared to saline treated controls (1793.5 [1603-2157] U/L, 1823 [1668-2932] U/L, 2375 [1963-2919] U/L vs. 3300.5 [2481-4911] U/L; p&lt;0.001, 0.007, and 0.031, for 10mg/kg CsA, 25mg/kg CsA and 10mg/kg NIM- 811, respectively). This corresponded to decreased histological injury scores (p&lt;0.001, 0.003, 0.043, for 10mg/kg CsA, 25mg/kg CsA and 10mg/kg NIM-811, respectively). The final study tests CsA in an NMP-based DCD model. Pigs were pretreated with 20mg/kg of CsA and subject to either 45 minutes of warm ischemia followed by 2 hours of SCS or 60 minutes of warm ischemia followed by 4 hours of SCS. Their livers were reperfused via an experimental NMP setup.<br>
Compared to the livers of animals treated with saline only, livers treated with CsA did not show any difference in markers of injury. Peak values of serum transaminases were no different between groups (p=0.912, 0.455 for ALT and aspartate aminotransferase [AST], respectively). Similarly,<br>
there was no difference in midpoint histological injury score (p=0.271).<br>
The second experimental section focuses on building a model of acute liver failure using an isolated perfused liver. Porcine livers were perfused via NMP and acetaminophen was added<br>
as a hepatotoxic agent. Acetaminophen-treated livers received a median dose of 8.93g (8.21-9.75g) of acetaminophen, achieving a peak acetaminophen level of 3780µmol/L (3189-3913µmol/L). Peak values of ALT (76 vs. 105U/L; p=0.429) and AST (3576 vs. 4712U/L; p=0.429) were not significantly different between treated and untreated livers. However, by the end of perfusion, histology scores were significantly worse in the acetaminophen treated group (p=0.016). Injury<br>
was confounded by the development of significant methemoglobinemia, with a peak methemoglobin level of 19.3%, compared to 2.0% for control livers (p=0.004). As methemoglobinemia is not the mechanism by which acetaminophen is known to cause hepatotoxicity clinically, this was seen as a major  limitation of the model.<br>
The final research section reports clinical outcomes of liver recipients transplanted<br>
following NMP from one of the first centres in North America to implement NMP. From 2015 to 2021, 79 livers were transplanted following NMP compared to 386 after SCS only. NMP livers were preserved for a median time of 847min compared to 288.5min in the SCS cohort (p&lt;0.0001). We observed significantly improved 30-day graft survival (p=0.030), though no differences in long term patient survival, major complications or biliary or vascular complications.<br>
NMP of the liver is an important technique for studying liver injury in the preclinical setting, both as it relates to transplantation and more broadly to other areas of hepatology. The role of NMP in the clinical setting is still being defined and is likely to expand as it becomes increasingly integrated into modern transplant programs.</p>