Rewarming Preservation by Organ Perfusion System for Donation After Cardiac Death Liver Grafts in Pigs N. Matsunoa,b,*, H. Obarab, R. Watanabea, S. Iwataa, S. Konoa, M. Fujiyamaa, T. Hiranoc, H. Kanazawaa, and S. Enosawaa a National Center for Child Health and Development, Tokyo, Japan; bTokyo Metropolitan University, Mechanical Engineering, Tokyo, Japan; and cTokyo University of Pharmacology and Life Science, Clinical Pharmacology, Tokyo, Japan
ABSTRACT Background. Use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. However, this requires the development of novel preservation methods to recover the organ from changes due to warm ischemia time (WIT). Methods. Porcine livers were perfused with a newly developed machine perfusion (MP) system. The livers were perfused with modified University of Wisconsin solution (UW) gluconate. All grafts were procured after acute hemorrhagic shock with the ventilator off. For group 1 (n ¼ 6), grafts were procured after WIT of 60 minutes and preserved by hypothermic MP (HMP) for 3 hours. For group 2 (n ¼ 5), grafts were preserved with 2 hours of simple cold storage (SCS) and HMP for 2 hours. For group 3 (n ¼ 6), grafts were preserved with 2 hours of SCS and rewarming up to 25 C by MP for 2 hours (RMP). The preserved liver grafts were transplanted orthotopically. Results. The alanine aminotransferase level in perfusate in RMP during perfusion preservation was maintained at less than that of HMP. The levels of aspartate aminotransferase and lactate dehydrogenase in the 2 hours after reperfusion were significantly lower in group 3. Histologically, the necrosis of hepatocytes was less severe in group 3. The survival rate in group 3 was 2/4, but 0/4 in the other group. Conclusion. RMP is expected to facilitate the recovery of the DCD liver grafts.
T
HE SUPERIORITY of machine perfusion (MP) preservation to simple cold storage (SCS) was recently reported in clinical kidney preservation [1e3]. The objective of MP is to resuscitate marginal grafts suffering from impaired blood flow by providing nutrients. In addition, the most significant advantage of MP preservation is that it allows the evaluation of the graft viability by monitoring the effluent enzymes in the perfusion solution and determining the flow pressure before transplantation [3,4]. The clinical use of hypothermic MP (HMP) preservation for liver grafts has begun [5]. However, the use of MP for the metabolic organ under hypothermic conditions may not be optimal for a graft obtained from a deceased cardiac donor (DCD) because the metabolic activities are suppressed considerably at low temperatures, whereas the deteriorative effects of cooling remain. Experimentally, Fondevila et al reported that normothermic MP improved further DCD liver
function for an organ that was preserved 90 minutes after cardiac death [6]. However, the reality of clinical organ retrieval might require a period of cold preservation. Therefore, we performed this study to identify the optimum conditions for DCD liver grafts, focusing on preservation temperature. The aim of this study was to evaluate the efficacy of rewarming during MP preservation up to 22 C with
Supported in part by grants from the Scientific Research Fund of the Ministry of Education and by a Research Grant for Immunology, Allergy and Organ Transplant from the Ministry of Health, Labor and Welfare, Japan (Nos. 23390316). *Address correspondence to Naoto Matsuno, MD, PhD, Department of Innovative and Transplant Surgery, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan. E-mail: [email protected]
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.12.035
Transplantation Proceedings, 46, 1095e1098 (2014)
1095
1096
MATSUNO, OBARA, WATANABE ET AL
the use of a temperature-controlled system for porcine liver grafts that were injured by 60 minutes of warm ischemic injury and 5 hours total ischemic time.
conditions and temperatures of the preservation solution. The temperature in the organ chamber is controlled by a heat exchanger and ice-cold water. The flow rate was controlled as 0.22 and 0.06 mL/min per gram for the PV and HA, respectively.
MATERIALS AND METHODS Animals
Preparation and Preservation of the DCD Liver
Domestic female cross-bred Large-Yorkshire, Landrace, and Dulroc pigs (approximately 20 kg, 2 to 3 months old) were purchased from Saitama Experimental Animals Supply Co., Ltd.
Perfusion Preservation Machine We developed an organ preservation system with temperaturecontrolled MP (Figs 1A and B) [7]. The system has separate circulating perfusion circuits for the portal vein (PV) and hepatic artery (HA) allowing nonpulsatile and pulsatile flow, respectively. An oxygenator was installed in the HA circuit. Both circuits were connected via plastic connectors to the hepatic vessels. Water proof thermocouples installed in this system measured the solution and the organ temperatures. A computer records data and controls flow
Fig 1. (A,B) The continuous liver perfusion machine. The system consists of two circuits for the portal vein and hepatic artery. The circuits can control the flow, providing nonpulsatile flow for the portal vein and pulsatile flow for the hepatic artery. The temperature of the perfusate was controlled by a heat exchanger. An oxygenator was installed in the circuit for the hepatic artery.
Seventeen pigs weighing approximately 20 kg each were included as donors and recipients. Under inhalation anesthesia with isoflurane (Forane, Abbott, Japan), cardiac arrest was induced by hemorrhagic shock and removal of ventilation. The conditions of the MP were as follows: Group 1 (n ¼ 6) grafts had a warm ischemia time (WIT) of 60 minutes and preserved MP at 8 C for 4 hours. Group 2 (n ¼ 5) grafts had a WIT of 60 minutes and SCS and MP at 8 C. Group 3 (n ¼ 6) grafts were linearly rewarmed from 4 C to 25 C. All livers were stored for 2 hours at 4 C (groups 2 and 3), then were subsequently treated with MP preservation for 2 hours under hypothermic (group 2) and rewarming (group 3) conditions. Liver grafts in groups 2 and 3 were left for 60 minutes under warm ischemic conditions and perfused with 25 C Euro Collins-based solution containing urokinase, citrate phosphate dextrose, and phentolamine
REWARMING PRESERVATION
1097
mesilate. The solution used for MP was a University of Wisconsin gluconate solution containing low molecular dextran (Dextran 40: 2g) and 200 mL of Aminolevan (Otsuka Pharmaceutical Factory Inc., Japan), which contains branched chain amino acids. The temperature was controlled by the heat exchanger.
Orthotopic Liver Transplantation Orthotopic liver transplantation was performed as described elsewhere [8]. Vascular anastomoses were carried out from the suprahepatic vena cava, PV, infrahepatic vein, and HA. Biliary reconstruction was performed by cholecystojejunostomy. The venous bypass between the left femoral vein and splenic vein to the left external jugular vein was made using a passive bypass. All experiments in the three groups were terminated 5 hours after reperfusion. All had good postoperative courses characterized by sustained blood pressure, spontaneous breathing, and weaning from respiration, and awakening [9].
Viability Assessment Effluent was obtained after 2 hours of MP preservation to analyze the aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels. The recipient laboratory data were also obtained to analyze the AST and LDH levels at 2 hours after reperfusion. Liver biopsy specimens were obtained 1 hour after blood reperfusion. The adenosine triphosphate (ATP) level in liver tissue was measured by the luciferin-luciferase method with the ATP Bioluminescence Assay Kit according to manufacturer’s instructions and normalized to protein content.
Statistical Analysis The SPSS software program was used for the statistical analysis (SPSS version 18.0, SPSS Inc., United States). All results are given as the means standard deviation (SD) and P values
ABSTRACT Background. Use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. However, this requires the development of novel preservation methods to recover the organ from changes due to warm ischemia time (WIT). Methods. Porcine livers were perfused with a newly developed machine perfusion (MP) system. The livers were perfused with modified University of Wisconsin solution (UW) gluconate. All grafts were procured after acute hemorrhagic shock with the ventilator off. For group 1 (n ¼ 6), grafts were procured after WIT of 60 minutes and preserved by hypothermic MP (HMP) for 3 hours. For group 2 (n ¼ 5), grafts were preserved with 2 hours of simple cold storage (SCS) and HMP for 2 hours. For group 3 (n ¼ 6), grafts were preserved with 2 hours of SCS and rewarming up to 25 C by MP for 2 hours (RMP). The preserved liver grafts were transplanted orthotopically. Results. The alanine aminotransferase level in perfusate in RMP during perfusion preservation was maintained at less than that of HMP. The levels of aspartate aminotransferase and lactate dehydrogenase in the 2 hours after reperfusion were significantly lower in group 3. Histologically, the necrosis of hepatocytes was less severe in group 3. The survival rate in group 3 was 2/4, but 0/4 in the other group. Conclusion. RMP is expected to facilitate the recovery of the DCD liver grafts.
T
HE SUPERIORITY of machine perfusion (MP) preservation to simple cold storage (SCS) was recently reported in clinical kidney preservation [1e3]. The objective of MP is to resuscitate marginal grafts suffering from impaired blood flow by providing nutrients. In addition, the most significant advantage of MP preservation is that it allows the evaluation of the graft viability by monitoring the effluent enzymes in the perfusion solution and determining the flow pressure before transplantation [3,4]. The clinical use of hypothermic MP (HMP) preservation for liver grafts has begun [5]. However, the use of MP for the metabolic organ under hypothermic conditions may not be optimal for a graft obtained from a deceased cardiac donor (DCD) because the metabolic activities are suppressed considerably at low temperatures, whereas the deteriorative effects of cooling remain. Experimentally, Fondevila et al reported that normothermic MP improved further DCD liver
function for an organ that was preserved 90 minutes after cardiac death [6]. However, the reality of clinical organ retrieval might require a period of cold preservation. Therefore, we performed this study to identify the optimum conditions for DCD liver grafts, focusing on preservation temperature. The aim of this study was to evaluate the efficacy of rewarming during MP preservation up to 22 C with
Supported in part by grants from the Scientific Research Fund of the Ministry of Education and by a Research Grant for Immunology, Allergy and Organ Transplant from the Ministry of Health, Labor and Welfare, Japan (Nos. 23390316). *Address correspondence to Naoto Matsuno, MD, PhD, Department of Innovative and Transplant Surgery, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan. E-mail: [email protected]
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.12.035
Transplantation Proceedings, 46, 1095e1098 (2014)
1095
1096
MATSUNO, OBARA, WATANABE ET AL
the use of a temperature-controlled system for porcine liver grafts that were injured by 60 minutes of warm ischemic injury and 5 hours total ischemic time.
conditions and temperatures of the preservation solution. The temperature in the organ chamber is controlled by a heat exchanger and ice-cold water. The flow rate was controlled as 0.22 and 0.06 mL/min per gram for the PV and HA, respectively.
MATERIALS AND METHODS Animals
Preparation and Preservation of the DCD Liver
Domestic female cross-bred Large-Yorkshire, Landrace, and Dulroc pigs (approximately 20 kg, 2 to 3 months old) were purchased from Saitama Experimental Animals Supply Co., Ltd.
Perfusion Preservation Machine We developed an organ preservation system with temperaturecontrolled MP (Figs 1A and B) [7]. The system has separate circulating perfusion circuits for the portal vein (PV) and hepatic artery (HA) allowing nonpulsatile and pulsatile flow, respectively. An oxygenator was installed in the HA circuit. Both circuits were connected via plastic connectors to the hepatic vessels. Water proof thermocouples installed in this system measured the solution and the organ temperatures. A computer records data and controls flow
Fig 1. (A,B) The continuous liver perfusion machine. The system consists of two circuits for the portal vein and hepatic artery. The circuits can control the flow, providing nonpulsatile flow for the portal vein and pulsatile flow for the hepatic artery. The temperature of the perfusate was controlled by a heat exchanger. An oxygenator was installed in the circuit for the hepatic artery.
Seventeen pigs weighing approximately 20 kg each were included as donors and recipients. Under inhalation anesthesia with isoflurane (Forane, Abbott, Japan), cardiac arrest was induced by hemorrhagic shock and removal of ventilation. The conditions of the MP were as follows: Group 1 (n ¼ 6) grafts had a warm ischemia time (WIT) of 60 minutes and preserved MP at 8 C for 4 hours. Group 2 (n ¼ 5) grafts had a WIT of 60 minutes and SCS and MP at 8 C. Group 3 (n ¼ 6) grafts were linearly rewarmed from 4 C to 25 C. All livers were stored for 2 hours at 4 C (groups 2 and 3), then were subsequently treated with MP preservation for 2 hours under hypothermic (group 2) and rewarming (group 3) conditions. Liver grafts in groups 2 and 3 were left for 60 minutes under warm ischemic conditions and perfused with 25 C Euro Collins-based solution containing urokinase, citrate phosphate dextrose, and phentolamine
REWARMING PRESERVATION
1097
mesilate. The solution used for MP was a University of Wisconsin gluconate solution containing low molecular dextran (Dextran 40: 2g) and 200 mL of Aminolevan (Otsuka Pharmaceutical Factory Inc., Japan), which contains branched chain amino acids. The temperature was controlled by the heat exchanger.
Orthotopic Liver Transplantation Orthotopic liver transplantation was performed as described elsewhere [8]. Vascular anastomoses were carried out from the suprahepatic vena cava, PV, infrahepatic vein, and HA. Biliary reconstruction was performed by cholecystojejunostomy. The venous bypass between the left femoral vein and splenic vein to the left external jugular vein was made using a passive bypass. All experiments in the three groups were terminated 5 hours after reperfusion. All had good postoperative courses characterized by sustained blood pressure, spontaneous breathing, and weaning from respiration, and awakening [9].
Viability Assessment Effluent was obtained after 2 hours of MP preservation to analyze the aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels. The recipient laboratory data were also obtained to analyze the AST and LDH levels at 2 hours after reperfusion. Liver biopsy specimens were obtained 1 hour after blood reperfusion. The adenosine triphosphate (ATP) level in liver tissue was measured by the luciferin-luciferase method with the ATP Bioluminescence Assay Kit according to manufacturer’s instructions and normalized to protein content.
Statistical Analysis The SPSS software program was used for the statistical analysis (SPSS version 18.0, SPSS Inc., United States). All results are given as the means standard deviation (SD) and P values
