The Role of IGL-2 Preservation Solution on Rat Livers during SCS and HOPE

Abstract

The scarcity of livers for transplantation is rising, and new strategies to extend the donor pool are being explored. One solution is to use marginal grafts from extended criteria donors, presenting, for example, liver steatosis. As current preservation solutions (UW, HTK, and IGL-1) were mainly designed for static cold storage (SCS) only, IGL-2, a modified version of IGL-1, was developed to be suitable for SCS and dynamic preservation, such as hypothermic oxygenated perfusion (HOPE). In this study, we investigated the combined effect of IGL-2, SCS, and HOPE and compared it to the most used preservation solution (UW and Belzer MPS). Four experimental groups with six rats each were designed using Zucker rats. All groups underwent 24 h of SCS (in IGL-2 or UW) + 2 h of normothermic machine perfusion (NMP) at 37 °C to mimic transplantation. HOPE (IGL-2 or Belzer MPS) was performed before NMP on half of the rats. The IGL-2 group demonstrated lower transaminases and a significantly low level of glycocalyx proteins, CASP3, and HMGB1 in the perfusates. These data suggest the protective role of IGL-2 for fatty livers in preserving the endothelial glycocalyx, apoptosis, and inflammation.

Keywords: fatty liver; glycocalyx; ischemia-reperfusion; machine perfusion.

Scheme 1

Experimental groups and study design description. Preservation solutions are highlighted in grey and black: IGL-2: Institut Georges Lopez-2; Reperfusion: albumin-based reperfusion solution; UW: University of Wisconsin; MPS: machine perfusion solution (Belzer MPS). Preservation protocols are highlighted in blue, dark blue, and red: SCS: static cold storage; NMP: normothermic machine perfusion; HOPE: hypothermic oxygenated machine perfusion.

Figure 1

Liver histology after 24 h SCS + 2 h NMP. (A) IGL2.SCS (B) BELGEN.SCS (C) The damage grade score (DGS) was assessed by an expert pathologist and tended to be higher in the IGL2.SCS Group (p = 0.68) (Kruskal–Wallis test).

Figure 2

Hepatocellular injury markers quantification and anaerobic state evaluation. (A) Aspartate aminotransferase (AST) was significantly lower in the IGL2 group than in the BELGEN group after 2 h of reperfusion (p < 0.01). (B) Alanine aminotransferase (ALT) expression tended to be decreased in the IGL2 group compared to the BELGEN group after 2 h of reperfusion (p = 0.95). (C) No differences were observed in the lactate levels between the two groups. n = 6/group. * = p < 0.05.

Figure 3

Glycocalyx integrity during reperfusion at 37 °C following 24 h of SCS. Proteins expression of SDC1 and HPSG by ELISA were measured from perfusates. (A) SDC1 levels were significantly lower after 2 h of reperfusion in the IGL2 group (p = 0.04). (B) HPSG levels were significantly lower after 2 h of reperfusion in the IGL2 group (p = 0.01). n = 6/group. * = p <0.05.

Figure 4

Expression of apoptosis-related marker active caspase 3 and inflammation marker HMGB1 in the perfusate during reperfusion following 24 h of SCS and 2 h of HOPE. (A) We observe no significant difference in the caspase 3 levels at T0 and T120. (B) HMGB1 (p < 0.01) is significantly less expressed in the IGL2 group after 2 h of reperfusion. n = 6/group. ** = p < 0.01.

Figure 5

Liver histology after 24 h SCS + 2H HOPE + 2H NMP. (A) IGL2.HOPE (B) PERFGEN.HOPE (C) The damage grade score (DGS) was assessed by an expert pathologist and was significantly lower for the IGL2 group (p = 0.02). (Kruskal–Wallis test). * = p < 0.05.

Figure 6

Hepatocellular injury markers quantification and anaerobic state evaluation. (A) Aspartate aminotransferase (AST) levels tended to be lower in the IGL2 group than in the PERFGEN group after 2 h of reperfusion (p = 0.53). (B) Alanine aminotransferase (ALT) expression tended to be decreased in the IGL2 group compared to the PERFGEN group after 2 h of reperfusion (p = 0.28). (C) No differences were observed in the lactate levels between the two groups. n = 6/group.

Figure 7

Glycocalyx integrity during reperfusion at 37 °C following 24 h of SCS and 2 h of HOPE. Proteins expression of SDC1 and HPSG by ELISA were measured from perfusates. (A) SDC1 levels were significantly lower after 2 h of reperfusion in the IGL2 group (p < 0.01). (B) HPSG levels were significantly lower after 2 h of reperfusion in the IGL2 group (p < 0.01). n = 6/group. ** = p < 0.01.

Figure 8

Expression of apoptosis-related marker active caspase 3 and inflammation marker HMGB1 in the perfusate during reperfusion following 24 h of SCS and 2 h of HOPE. Caspase 3 (p = 0.02) and HMGB1 (p = 0.03) were significantly less expressed in the IGL2 group after 2 h of reperfusion. n = 6/group. * = p < 0.05.

Figure 9

The percentage of weight change between the PRE-SCS and POST-SCS phases shows that the livers from the IGL2 groups seemed to have lost weight after NMP throughout preservation. In contrast, the livers from the BELGEN and PERFGEN groups tended to follow the opposite trend. n = 6/group.

Figure 10

Experimental ex vivo machine perfusion system. Ex vivo machine perfusion system is adapted from liver/kidney systems (A) Bubble trap; (B) Membrane oxygenating; (D) Liver perfusion chamber (Radnoti LLC, Covina, CA, USA) assembled by the author (NA) (C) Peristaltic pump (MasterFlex^®, Thermo Fisher Scientific, Waltham, MA, USA); (E) Flow sensor (TS410 Tubing Module, Transonic Systems Inc., Ithaca, NY, USA), a water bath circulator pump (9102A12E, 6-liter High-Stability Digital Controller Refrigerated/Heated Circulating Bath, Polyscience, Niles, IL, USA), and an oxygen bottle (95% O₂/5% CO₂, Air Liquide, Paris, France).