M. Wallace1, J. MacLean1, M. Thornbury3, A. Venkatachalam1, Q. Hu2, N. Ridgway4,5, I. Alwayn1,2,3 1Dalhousie University,Surgery,Halifax, Nova Scotia, Canada 2Dalhousie University,Pathology,Halifax, Nova Scotia, Canada 3Dalhousie University,Microbiology & Immunology,Halifax, Nova Scotia, Canada 4Dalhousie University,Biochemistry & Molecular Biology,Halifax, Nova Scotia, Canada 5Dalhousie University,Pediatrics,Halifax, Nova Scotia, Canada
Introduction:
The number of patients on the waiting list for a liver transplant is increasing, but unfortunately the availability of donor livers does not meet the demand. With rising obesity rates in North America, many livers are discarded because of steatosis, the accumulation of fat within the liver. These extended criteria donor organs are more susceptible to ischemia-reperfusion injury (IRI) and are associated with reduced viability and increased complication rates after transplantation. Normothermic ex vivo liver perfusion (NEVLP) has emerged as a means of reducing the IRI associated with cold static storage of livers. NEVLP may also play a role in actively defatting steatotic livers and thus protect them from IRI. In this pilot study, we hypothesize that four hours of oxygenated NEVLP will preserve the hepatic architecture of steatotic rat livers, and that supplementing the perfusate with defatting compounds will result in a reduction of hepatocellular lipid content.
Methods:
Livers were harvested from obese Zucker rats or lean Wistar rats and perfused ex vivo at 37°C for four hours. NEVLP consisted of membrane oxygenated machine perfusion with a flow rate of 10 mL/min and an initial portal vein pressure of 10 mm Hg. Livers were perfused with Minimum Essential Medium Alpha Modification medium. The perfusate was supplemented with bovine serum albumin, fetal bovine serum, human insulin, lactic acid, and heparin at pH 7.4 with or without the addition of four compounds known to stimulate lipid metabolism by consuming or secreting intracellular triglycerides. Liver tissue biopsies were collected after perfusion for hematoxylin and eosin (H&E) and Oil Red O staining, and triglyceride (TAG) measurement. Perfusate samples were collected throughout NEVLP for quantification of TAG and aspartate aminotransferase (AST), a marker of hepatocyte injury.
Results:
H&E results suggest that the viability of steatotic livers can be maintained using NEVLP under physiological conditions. Steatotic livers are more susceptible to hepatocyte injury during ex vivo perfusion than non-fatty livers. The defatting agents may provide some protection from this injury as indicated by lower AST levels. Perfusion of steatotic livers with the defatting agents results in a reduction of intracellular lipid droplets as shown by Oil Red O staining. However, there is no associated increase in perfusate TAG levels, suggesting that defatting may be occurring by mechanisms other than lipid secretion such as hydrolysis for ketogenesis or beta-oxidation.
Conclusion:
We have shown that four hours of oxygenated NEVLP successfully preserves liver architecture. Supplementing the perfusate with compounds that promote metabolism through export or oxidation may result in defatting of steatotic livers. Defatting steatotic livers could improve transplantation success and increase the donor pool.