A new model of ischemia/reperfusion injury using precision-cut liver slices

Abstract

Background: Primary cells isolated from the liver are widely used to investigate hepatic pathophysiology. However, these cells do not represent cell-to-cell interactions, three dimensional architecture, and zonal structure in the liver. Furthermore, characterization of cellular and molecular events during ischemia/reperfusion (I/R) is challenging in liver biopsies. Thus, a new model of I/R injury resembling the native liver is needed. Methods: We generated precision-cut liver slices (PCLS) from mouse and human livers using a Krumdieck automatic tissue slicer (Alabama R & D, Munford, AL). Thickness of individual PLCL was 200 microns. To simulate anoxia, nutrient depletion, and tissue acidosis during ischemia, PCLS were incubated in Krebs-Ringer-N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) solution at pH 6.2 in the anaerobic chamber (Coy Laboratory, Grass Lake, MI) for 30 min. To simulate normoxia, nutrient repletion, and restoration to normal pH during reperfusion, ischemic PCLS were aerobically incubated in Dulbecco's modified eagle medium (DMEM) at pH 7.4 for up to 4 h. Changes in tissue viability were assessed with the release of lactate dehydrogenase (LDH) into the extracellular medium. Some PCLS were labeled with green fluorescent rhodamine-123 (Rd-123, mitochondrial membrane potential indicator) and red fluorescent propidium iodide (PI, necrosis marker). Confocal images of PCLS were collected after I/R. Results: LDH release assay revealed that while PCLS from chow diet (CD)-fed lean mice tolerated I/R injury well, those from high fat diet (HFD)-fed steatotic mice were susceptible to I/R injury. Confocal microscopy showed substantial mitochondrial depolarization (loss of Rd-123) and necrosis (nuclear labeling of PI) in the HFD group, which was, however, minimally observed in the CD group. Of importance, mitochondrial dysfunction and cell death after reperfusion occurred predominantly in hepatocytes. Heightened I/R injury in steatotic livers was also confirmed in primary hepatocytes isolated from either CD- or HFD-fed mice. To compare the similarities and differences between mouse and human livers, PCLS were generated from discarded human livers deemed unsuitable for transplant and subjected to 30 min of in vitro ischemia. The loss of mitochondrial membrane potential and hepatocyte death progressively increased after I/R in discarded human livers. At 1 h after reperfusion, diffusive fluorescence of Rd-123 and nuclear labeling of PI became evident, indicative of mitochondrial dysfunction and tissue injury after I/R. SUMMARY: LDH assay and confocal analysis of PCLS demonstrated heightened susceptibility of steatotic livers to I/R injury. In contrast to PCLS from lean livers, those from steatotic livers rapidly developed mitochondrial depolarization and onset of necrosis, cardinal features of I/R injury, even after short ischemia. The early phase of reperfusion injury in steatotic livers occurred predominantly in parenchymal cells. CONCLUSION: In vitro I/R with PCLS is a model suitable for hepatic I/R injury. This work (J-S K) was funded by the National Institutes of Health (DK079878), Mid-America Transplant Foundation (07201903), Foundation for Barnes-Jewish Hospital (4776, 5153), and Washington University Department of Surgery Pilot Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.