HYPOTHERMIC MACHINE PERFUSION OF THE LIVER AND THE CRUCIAL BALANCE BETWEEN PERFUSION PRESSURES AND ENDOTHELIAL INJURY

Abstract

P25 Aims: Hypothermic machine perfusion (HMP) provides better protection against cold-ischemic injury than cold-storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold-storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Methods: Rat livers were preserved using static cold-storage or continuous perfusion with the appropriate UW preservation solution. Cold-storage was compared to HMP preserved livers using an arterial perfusion pressure of 25 mmHg (mean) and a portal perfusion pressure of 4 mmHg (low pressure group), and to HMP at 50 mmHg and 8 mmHg perfusion, respectively (high pressure group). To stain for dead cells, the UW solution was enriched with 14.9 μM propidium iodide (PI), and with an additional 13.5 μM acridine orange, to stain for viable hepatocytes. After 24 h preservation the amount and anatomical localization of PI positive cells were assessed. Acridine orange (AO) used to stain viable hepatocytes, and the RECA-1 (rat endothelial cell antibody-1) and ED-1 (Kupffer cell marker) antibodies were used to identify which cells were PI positive. ATP levels were determined as a viability marker and to confirm that higher energy levels can be obtained with HMP than with cold-storage. Results: All livers in the cold-storage group and both HMP groups were completely perfused as shown with AO staining. Cold-storage preserved livers showed 75.1+/-6.2 dead PI positive cells per microscopic field. PI staining in the low-pressure group showed 64.4+/-7.8 and in the high-pressure group showed 93.4+/-5.9 PI positive cells per microscopic field. PI positive cells were non-parenchymal cells and correlated with the pattern found for RECA-1. The results for ED-1 staining did not correspond with the pattern found for PI positive cells. ATP levels were low after cold-storage (1.2 +/- 0.5) and significantly higher after HMP i.e. 44.5+/-5.9 (low-pressure) and 36.5+/-2.8 pmol/μg-protein (high-pressure), respectively. Conclusions: This study showed better ATP levels for HMP preserved livers in comparison to CS livers. The perfusion pressure showed a low PI-positive cell count in comparison to CS, when the HMP technique was used at a low perfusion pressure. A high HMP perfusion pressure resulted in more PI-positive cells compared to CS, indicating that the perfusion pressure is critical for HMP preservation of the liver. The PI-positive cells were found in the portal triad, sinusoids and central veins and were non-parenchymal in origin. The staining pattern with RECA-1 demonstrated that these non-parenchymal cells can be identified as endothelial cells. HMP preservation is effective for the liver with low-pressure perfusion and it is crucial to fine-tune the perfusion pressure to prevent endothelial injury.