Hypothermic Liver Machine Perfusion With EKPS-1 Solution vs Aqix RS-I Solution: In Vivo Feasibility Study in a Pig Transplantation Model.

OBJECTIVE: Hypothermic machine perfusion (HMP) is superior to simple cold storage for kidney preservation. We previous observed in a porcine liver transplantation model increased tumor necrosis factor-alpha (TNF-alpha) production eventually leading to poor recipient survival after HMP using standard kidney perfusion solution (KPS-1) compared with simple cold storage. We compared two solutions for HMP preservation of the liver: enriched KPS-1 (EKPS-1) and Aqix RS-I. METHODS: Pig livers were obtained after cold flushing with histidine-tryptophan-ketoglutarate solution. Subsequently, the livers were subjected to dual-vessel perfusion with two preservation solutions: EKPS-1 (n = 6) and Aqix RS-I (n = 3). After HMP preservation and transplantation, graft and recipient survival, hepatocellular damage (aspartate aminotransferase concentration), TNF-alpha production, and endothelial cell damage (hyaluronic acid clearance) were recorded. RESULTS: No primary graft nonfunction was observed. Recipient survival at postoperative day 3 was similar in both groups (33%). Aspartate aminotransferase concentration measured in serum samples after reperfusion was similar in both groups. After reperfusion, TNF-alpha concentration was higher and hyaluronic acid clearance was lower in the EKPS-1 group vs the Aqix RS-I group at 60, 120, and 180 minutes (all P < .05). CONCLUSION: Hypothermic machine perfusion provided adequate longer term graft survival. After reperfusion, TNF-alpha production seems to be reduced, and endothelial cell dysfunction remains pronounced with Aqix RS-1 solution compared with EKPS-1 solution.

Liver transplantation from non-heart-beating donors: current status and future prospects in an experimental model.

The widening gap between supply and demand for liver transplantation has prompted many transplant centers to use donors after cardiac death or non-heart-beating donors. These livers--contrary to "classical" brain-dead donors--are exposed to an unavoidable period of warm ischemia, jeopardizing graft function post-transplantation. In a newly developed preclinical model of liver transplantation, we studied--in a biologically unmodified environment--the exact tolerance of the liver to warm ischemia. Following the evidence that liver transplantation from non-heart-beating donors is feasible and safe, provided that warm and cold ischemia are kept short, a clinical programme of liver transplantation from non-heart-beating donors was successfully initiated in our and other Belgian centers. Recently, we demonstrated that the tolerance of such livers to warm ischemia could be substantially improved when some of the previously identified mechanisms leading to graft non-function were tackled by a multi-factorial pharmacological strategy. Meanwhile, cold storage has proven to be insufficient to optimally preserve organs from non-heart-beating donors. As an alternative, machine perfusion preservation was found to consistently improve outcome in kidney transplantation from non-heart-beating donors. Similarly, machine perfusion preservation could improve the preservation of livers, allowing to predict viability prior to transplantation and to ameliorate tolerance to warm ischemia. At present, the definition and development of optimal machine perfusion settings are under investigation at our institution.

Hemodynamic, biochemical, and morphological characteristics during preservation of normal porcine livers by hypothermic machine perfusion.

In renal transplantation, hypothermic machine perfusion optimizes preservation of marginal grafts, assesses their quality prior to transplantation, improves outcome, and may contribute to an increased number of transplantations. Recently, hypothermic machine perfusion has become increasingly popular given the organ shortage and the "obligatory" utilization of marginal organs. Increasing mortality on the liver transplantation waiting list makes it urgent to develop machine perfusion systems for livers, trying to better preserve marginal livers and perhaps to recover currently discarded livers are for clinical transplantation without an increased risk of graft nonfunction. However, data on machine perfusion of livers and perfusion parameters capable of predicting viability are scarce. The aim of this study was to determine the baseline hemodynamic and metabolic profiles and morphology of livers during hypothermic machine perfusion in a porcine model. We used protocol similar to hypothermic machine perfusion of kidneys. Hemodynamic analysis revealed higher vascular resistance in the hepatic artery versus the portal vein. The arterial resistance gradually decreased during perfusion (similar to kidneys), suggesting progressive relaxation of the arterial vasculature, and perhaps better penetration of the microcirculation by the perfusion solution. During hypothermic machine perfusion, transaminases were gradually (but modestly) released, and livers displayed unequivocal signs of aerobic and anaerobic metabolism. After 24 hours, livers appeared morphologically well preserved. In conclusion, this study showed that hypothermic machine perfusion was feasible. During hypothermic machine perfusion, was easily assessed hemodynamic, biochemical, and morphological parameters.

Hypothermic machine perfusion of the liver: is it more complex than for the kidney?

BACKGROUND: Hypothermic machine perfusion (HMP) is superior to simple cold storage (SCS) for the preservation of kidney grafts. Whether HMP is superior to SCS for liver preservation is not known. Before a HMP system can be used clinically for the liver, its superiority to SCS needs to be demonstrated in an in vivo large animal transplant model. OBJECTIVE: The aim was to compare outcomes after liver transplantation (LT) following preservation by SCS or HMP using technology/perfusion conditions similar to those for kidney HMP. METHODS: Pig livers were perfused via the hepatic artery and portal vein for 4 hours with nonoxygenated 4°C University of Wisconsin machine perfusion solution. In the SCS group, flushed livers were stored in histidine-tryptophan-ketoglutarate solution. After preservation by SCS (n = 6) or HMP (n = 8) and LT, we assessed graft and recipient survivals, pH and lactate, hepatocellular damage [aspartate aminotransferase (AST)], Kupffer cell activation (ß-galactosidase), tumor necrosis factor (TNF) α production, endothelial cell function (hyaluronic acid), and expression of Krüppel-like factor (KLF) 2 and 4, which are mediators of the flow-dependent vasoprotective endothelial phenotype. RESULTS: No primary graft nonfunction was observed; livers recovered equally well from the postanhepatic metabolic acidosis in both groups. Pig survival was 5/6 (83%) in the SCS versus 2/8 (12.5%) in the HMP group (P = .04). Livers from both groups recovered equally well from the postanhepatic metabolic acidosis. AST in liver rinse-out samples obtained before LT were lower in the HMP than in the SCS group (P < .05). After reperfusion, AST and ß-galactosidase were equally increased in both groups (P = .13 and 0.962, respectively); TNF-α and hyaluronic acid levels were higher after HMP versus SCS (P = .001 and 0.043, respectively). KLF-2 and -4 expressions were equally up-regulated after reperfusion in the SCS and HMP groups. CONCLUSIONS: In this in vivo model, liver HMP with subsequent transplantation was feasible. However, we did not demonstrate an advantage of HMP, using perfusion conditions shown to be effective for the kidney, over SCS. Despite similar immediate graft function, TNF-α generation, and endothelial cell dysfunction were more pronounced after HMP.

Presumed and actual concentrations of reduced glutathione in preservation solutions.

Reduced glutathione (GSH), an important radical scavenger, has been added to various organ preservation solutions. Because GSH oxidizes into oxidized glutathione (GSSG) and only GSH has scavenging capacity, only GSH in the solution at the time of clinical use is relevant. The concentrations of GSH (GSH(conc)) and GSSG(conc) were determined in 2 static preservation solutions--University of Wisconsin (UW) and Celsior--and in 1 machine preservation solution--Kidney Preservation Solution 1 (KPS-1). We determined the half-life (T(1/2)) of freshly added GSH. The GSH(conc) in UW and KPS-1 was 0.006 ± 0.0018 mmol/L and 0.13 ± 0.30 mmol/L, respectively. The GSH(conc) in Celsior was 2.7 ± 0.17 mmol/L. The manufacturers of these solutions reported 3 mmol/L GSH. GSSG(conc) in UW, KPS-1, and Celsior was 1.58 ± 0.61 mmol/L, 1.13 ± 0.16 mmol/L, and 0.24 ± 0.01 mmol/L, respectively. T(1/2) of GSH in UW, KPS-1, and Celsior was 18 days, 86 days, and 83 days, respectively. The actual GSH(conc) in UW and KPS-1 at the time of clinical use was substantially lower than reported by the manufacturer, owing to the relatively short T(1/2) of GSH. For Celsior, the GSH(conc) was maintained. Therefore, addition of fresh GSH to UW and KPS-1 before clinical use is recommended.

Attempt to rescue discarded human liver grafts by end ischemic hypothermic oxygenated machine perfusion.

In a porcine liver transplant model, a brief period of oxygenated hypothermic machine perfusion (HMP) at the end of simple cold storage (SCS) has been shown to improve the viability of damaged liver grafts. To test the clinical validity of this strategy, we randomized SCS-discarded human liver grafts to either 4 hours of HMP (n = 13) or an additional 4 hours of SCS (n = 14). All livers were then warm reperfused to mimic ischemia-reperfusion injury ex vivo. The settings for HMP were: portal vein: 3 mm Hg, 300 mL/min and hepatic artery: 20 mm Hg, pO(2): 300 mm Hg. Perfusion used Kidney Machine Perfusion Solution at 4°C to 8°C. During warm reperfusion, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) values were higher (P < .015) among the SCS versus HMP methods at all times. The AST slope was lower in HMP versus SCS (P = .01). The LDH slope tended to be lower for HMP versus SCS (P = .07). Morphological scores were not different between HMP and SCS. At the start of warm reperfusion, MAPK was lower in HMP versus SCS (P = .02). Endothelin-1 (EDN1) and ICAM-1 tended to be lower in HMP versus SCS (P = .1 and .07, respectively). No difference was noted in MAPK, EDN1, and ICAM-1 after 60 or 120 minutes of warm reperfusion. In conclusion, HMP down-regulated MAPK and tended to reduce EDN1 and ICAM-1 mRNA in human liver grafts. During warm reperfusion, HMP versus SCS livers showed reduced AST and LDH release but no morphological difference. Further optimization of liver HMP may require different timing/duration of perfusion and/or an higher perfusion temperature.

Differential gene expression profile of porcine livers subjected to warm ischemia alone.

BACKGROUND: Livers exposed to warm ischemia (WI) are increasingly used for transplantation. The molecular mechanisms activated by WI alone (prior to procurement and transplantation) are not understood. To elucidate the pathways involved, we used microarrays to investigate the gene expression in porcine livers exposed to WI. METHODS: Porcine livers (n = 6 group) were randomly subjected to WI periods of 15, 30 or 45 minutes. mRNA was extracted and gene expression determined by microarray analysis. Using bioinformatics software, we identified differentially expressed genes and related molecular pathways. We used the corresponding human annotation of the porcine microarray for the functional analysis. RESULTS: Between 0 and 15 minutes of WI, 3530 genes were altered with a 2-log-fold change of <-0.58 or >+0.58 and P < .05. Between 0 and 30 minutes of WI, 4141 genes were differentially expressed; and between 0 and 45 minutes of WI, 2814 genes. At each time point, ∼50% of genes were up-regulated, whereas 50% were down-regulated. After pathway mapping, we found that the same pathways were induced for observed clustering of in the three WI periods: cell death, proliferation, inflammation, and metabolism pathways. Among the top genes that were up-regulated after 15 minutes of WI, the majority started to return to but did not reach baseline expression with increasing WI. A similar pattern was observed for the top suppressed genes. CONCLUSIONS: WI causes rapid changes in gene expression that affect several molecular pathways. This phenomenon seems to plateau at 15 to 30 minutes of WI. These new insights in the timing and the nature of molecular pathways induced by WI alone may help to design specific interventions to alter these changes and improve the outcome of livers from cardiac death donors.

Reproducible model for kidney autotransplantation in pigs.

OBJECTIVE: Several porcine models have been employed to study mechanisms of warm ischemia, cold ischemia, and ischemia reperfusion injury, but the technical/surgical aspects of these models and their possible pitfalls have not been fully described in detail. The goal of the present study was to develop and optimize a porcine kidney autotransplantation model. MATERIALS AND METHODS: Eleven female pigs (24-51 kg) underwent a left ureteronephrectomy. The procured kidney was flushed with 500 mL of histidine-tryptophan-ketoglutarate preservation solution and subsequently cold stored in University of Wisconsin preservation solution. An autotransplantation was performed 18 hours later, following contralateral nephrectomy. Serum creatinine and urine production were assessed posttransplantation. Pigs were sacrificed at 10 days posttransplantation. RESULTS: Nine pigs showed functioning grafts, immediately producing urine posttransplantation. The serum creatinine values in these pigs followed a bell-shaped curve with peak values at day (D)2-D3. Two renal artery thromboses were observed; the venous anastomosis was prone to stenosis in 6 cases. These technical complications influenced posttransplant observations. Renal artery thrombosis resulted in anuria and increased serum creatinine levels. A stenotic renal vein was associated with a greater peak of serum creatinine. These complications were prevented by modifying the surgical technique accordingly. CONCLUSIONS: We developed and optimized a reproducible model of porcine kidney autotransplantation. Herein we have demonstrated the importance of a proper surgical anastomotic technique to avoid inflow or outflow obstruction that might jeopardize posttransplant kidney function. This clinically relevant model offers the possibility to study various aspects related to kidney preservation without the interference of an allogeneic immune response.

Discriminate liver warm ischemic injury during hypothermic machine perfusion by proton magnetic resonance spectroscopy: a study in a porcine model.

OBJECTIVE: Proton magnetic resonance spectroscopy ((1)H MRS) is a technique to identify and quantify the composition of biofluids. Hypothermic machine perfusion (HMP) is an organ preservation method that has the potential to evaluate organ viability prior to transplantation by analyzing the composition of the perfusate. The aim of this study was to use (1)H MRS to examine the perfusate during HMP of porcine livers exposed to warm ischemia (WI) and to identify potential biomarkers of liver viability. MATERIALS AND METHODS: Porcine livers underwent 4 hours of HMP using kidney perfusion solution-1 (KPS-1) as perfusate after exposure to in situ WI of 0 (n = 6) or 2 hours (n = 5). Samples of the perfusate were taken at the start/end of HMP. Lactate and aspartate aminotransferase (AST) in the samples were measured biochemically as surrogates of the WI-induced damage. MRS acquisition was conducted on a 9.4 Tesla (400 MHz) high-resolution system. RESULTS: AST increased significantly during 4 hours of HMP within groups (P < .02) and discriminated WI injury significantly from the start to the end of HMP (P < .03). (1)H MRS showed significantly elevated signal intensity of lactate, alanine, and histidine during HMP within both groups (P < .02). Furthermore, alanine and histidine were significantly higher in the WI group than in the control group at the end of HMP (P = .011 and P = .038, respectively). CONCLUSION: AST, alanine, and histidine in HMP perfusate discriminated WI injury on porcine liver grafts and might be potential biomarkers of liver viability.