Novel Models for Assessing and Pathophysiology of Hepatic Ischemia-Reperfusion Injury Mechanisms

Abstract

Hepatic ischemia-reperfusion injury (IRI) is a major cause of postoperative hepatic dysfunction and liver failure involving cellular damage to previously ischemic tissues to which blood flow is restored. The reestablishment of blood flow is essential for salvaging ischemic tissues. The reperfusion itself, however, can paradoxically lead to further cellular damage, which involves a multi-factorial process resulting in extensive tissue damage, which can threaten the function and viability of the liver and other organ systems. The following review outlines multiple models for in-lab analysis of the various hepatic IRI mechanisms, including murine, porcine, cell lines, and machine perfusion models.

Keywords: cell line; ischemia–reperfusion injury; machine perfusion; murine; porcine; reactive oxygen species.

Figure 1

The cellular and molecular basis of hepatocellular ischemia–reperfusion injury: #1: Ischemia → anoxia → inhibits Electron Transport Chain-mediated cellular respiration and ATP generation → enzymes inhibited + pumps stop working + acidosis. #2: ATP depletion, iron overload-induced mitochondrial fragmentation, and membrane rupture releasing calcium + free iron + primary ROS shower. #3: Kupffer Cells activation → (a) generation of primary ROS, (b) activation of TNF (#4) and Fas (#5) receptor-mediated pathway triggering the Caspase cascade (#6) leading up to terminal apoptotic effector Caspase 3, (c) recruitment of neutrophils causing membrane and organelle damage and potentiation of Caspase 3 (#7). #8: Primary ROS-induced Caspase 1/11-mediated Gasdermin cleavage → Gasdermin binds to membrane lipids, creating transmembrane pores. #9: ATP depletion-induced calcium pump failure → loss of transmembrane calcium gradient → open voltage-gated calcium channels → massive calcium influx (#10) → organelle disruption and membrane rupture. This adds to calcium efflux from damaged mitochondria and other organelles. Iron is already transported intracellularly via Transferrin Receptor (#11) from ferric to ferrous state and in equilibrium with cytosolic and mitochondrial Heme, Ferritin, and iron–Sulfur Clusters. #12: Kupffer Cells, neutrophils, and mitochondria-generated primary ROS initiate and potentiate the generation of secondary ROS. #13: Fenton Reaction and Haber–Weiss Reactions converting ferrous back to ferric ions and generating the most reactive ROS: the hydroxyl radical—the terminal effector ROS for lipid peroxidation via enzymes LOX [Lipoxygenases] and POR [CytP450-OxidoReductases] and nonenzymatically by ferrous ions. #14: Enmeshed deposits of calcium + iron in peroxidized membrane lipids → cellular and organelle membrane rupture. #15: Caspase 3-induced apoptotic pathways: pyknosis + Karyorrhexis + karyolysis potentiated by primary and secondary ROS hydroxyl radicals (#13), calcium influx (#10), and ATP depletion. Apoptosis also releases substantial amounts of Damage-Associated Membrane Patterns [DAMPs]. DAMPs are also released by other ischemic processes. #15: DAMPs: the final common pathway for cell and organelle membrane rupture. #16: System xc⁻ receptor-mediated transport of Cystine is critical for production of the tripeptide Glutathione [GSH]. The lipid repair enzyme Glutathione Peroxidase [GPX4] catalytically utilizes GSH to inhibit lipid peroxidation. Abbreviations: TNF-r, Tumor Necrosis Factor receptor; Fas-r, Fas receptor; ETC, Electron Transport Chain; NaKA, Sodium–Potassium ATPase; ADP, Adenosine Diphosphate; ATP, Adenosine Triphosphate; M(i) Intact Mitochondria; M(f), Fragmented Mitochondria; N, Neutrophils; Gd, Gasdermin; ATPCal, ATP-dependent Calcium Export Pump; VoltC, Voltage-dependent calcium channels; Nu-k, Karyorrhexis of Nucleus; TF-r, Transferrin Receptor; 1-ROS, primary reactive oxygen species; 2-ROS, secondary reactive oxygen species; KC, Kupffer Cells; Sys-xc⁻, Cystine Transporter; GSH, Glutathione; GPX₄, Glutathione Peroxidase; DAMPs, Damage-Associated Membrane Patterns; L, Normal lipids; L(p), peroxidized lipids; Fe³⁺, ferric ion; Fe²⁺, ferrous ion; Ca²⁺, calcium ion; OH, hydroxyl free radical; O₂, oxygen Molecule.

Figure 2

Schematic of machine perfusion model.