Hypothermic oxygenated perfusion protects from mitochondrial injury before liver transplantation

Abstract

Background Mitochondrial succinate accumulation has been suggested as key event for ischemia reperfusion injury in mice. No specific data are however available on behavior of liver mitochondria during ex situ machine perfusion in clinical transplant models. Methods We investigated mitochondrial metabolism of isolated perfused rat livers before transplantation. Livers were exposed to warm and cold ischemia to simulate donation after circulatory death (DCD) and organ transport. Subsequently, livers were perfused with oxygenated Belzer-MPS for 1h, at hypothermic or normothermic conditions. Various experiments were performed with supplemented succinate and/or mitochondrial inhibitors. The perfusate, liver tissues, and isolated mitochondria were analyzed by mass-spectroscopy and fluorimetry. Additionally, rat DCD livers were transplanted after 1h hypothermic or normothermic oxygenated perfusion. In parallel, perfusate samples were analysed during HOPE-treatment of human DCD livers before transplantation. Findings Succinate exposure during rat liver perfusion triggered a dose-dependent release of mitochondrial Flavin-Mononucleotide (FMN) and NADH in perfusates under normothermic conditions. In contrast, perfusate FMN was 3-8 fold lower under hypothermic conditions, suggesting less mitochondrial injury during cold re-oxygenation compared to normothermic conditions. HOPE-treatment induced a mitochondrial reprogramming with uploading of the nucleotide pool and effective succinate metabolism. This resulted in a clear superiority after liver transplantation compared to normothermic perfusion. Finally, the degree of mitochondrial injury during HOPE of human DCD livers, quantified by perfusate FMN and NADH, was predictive for liver function. Interpretation Mitochondrial injury determines outcome of transplanted rodent and human livers. Hypothermic oxygenated perfusion improves mitochondrial function, and allows viability assessment of liver grafts before implantation. Funding detailed information can be found in Acknowledgments.

HOPE (hypothermic oxygenated perfusion) strategies in the era of dynamic liver graft preservation

“…We read with great interest the latest article by Andrea Schlegel et al. [2] in EBioMedicine . In it, the authors use HOPE to establish beneficial biochemical mechanisms, by restoring mitochondrial function through succinate metabolism and prevention of energy breakdown.…”

“…Another relevant data reported by Andrea Schelegel et al. [2] is that measurement of mitochondrial flavin mononucleotide (FMN) and NAD levels can serve as new predictors of human liver graft function when HOPE is used.…”

New Insights in Molecular Mechanisms and Pathophysiology of Ischemia-Reperfusion Injury 2.0: An Updated Overview

“…Recent efforts have been conducted to avoid the detrimental injury effects that occur during liver resection interventions by inhibiting iNOS with L-NAME [21], as well as those associated with the rewarming injury after cold graft preservation, which are relevant in the context of transplantation [22]. With this in mind, several machine perfusion (MP) strategies [23][24][25] have been introduced to better preserve the graft before the subsequent liver transplantation. In general terms, the main goal is to provide vascular flow and oxygen to the donor graft during preservation; either in hypothermic oxygenated perfusion (HOPE) [23,24] (using a perfusion solution, normally Belzer-MPS) or in normothermic MP perfusion (NMP) [25] (using blood or other fluids with oxygen carrying capacity).…”

“…With this in mind, several machine perfusion (MP) strategies [23][24][25] have been introduced to better preserve the graft before the subsequent liver transplantation. In general terms, the main goal is to provide vascular flow and oxygen to the donor graft during preservation; either in hypothermic oxygenated perfusion (HOPE) [23,24] (using a perfusion solution, normally Belzer-MPS) or in normothermic MP perfusion (NMP) [25] (using blood or other fluids with oxygen carrying capacity). These strategies can be applied either immediately after organ procurement or after static cold storage (SCS).…”

“…These promising MP perspectives in kidney [28][29][30] have been extended to liver by using HOPE to rescue vulnerable livers, such as the ones from donors after cardiac death (DCD) and steatotic ones [24,25]. In this sense, the recent investigations by Schlegel et al [23] have established the protection mechanisms of HOPE based on the metabolism of succinate. However, the establishment of new markers to evaluate the quality of the liver grafts is a pending challenge for the immediate future in IRI investigations [31][32][33].…”