- Email: [email protected]
Tolerance limits of liver grafts with 30 minutes of warm ischemia to cold preservation in swine
Tolerance Limits of Liver Grafts With 30 Minutes of Warm Ischemia to Cold Preservation in Swine D. Qing and B. Han ABSTRACT Aim. The aim of this study was to investigate the safe time limits of cold preservation in University of Wisconsin (UW) solution of liver grafts subjected to warm ischemia (WI) for 30 minutes from non– heart-beating donors (NHBDs). Methods. The safe time limits were studied in a simple porcine orthotopic liver transplantation (LTx) model. In donors, livers were subjected to 30 minutes of WI and subsequent 6-hour (Group 1, n ⫽ 5), 10-hour (Group 2, n ⫽ 5), and 14-hour (Group 3, n ⫽ 3) cold preservation in UW solution. Results. All 5 animals in Group 1 survived up to 7 days, the survey endpoint. In Group 2, only 2 animals survived to the same survey endpoint. All animals in Group 3 died within 12 hours. The 1-week survival rate of Group 1 was significantly higher than those of the other 2 groups. Group 1 showed a lower level of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) after LTx, less pathological damage, higher concentration of adenosine triphosphate (ATP), and higher microcirculation blood flux in the grafted liver tissue at 1 hour after reperfusion than the other 2 groups. Conclusions. It is concluded that about 6 hours is the safe time limit of cold preservation in UW solution for liver grafts from NHBDs subjected to WI for 30 minutes.
L
IVER transplantation (LTx) has been widely accepted as an effective treatment for end-stage liver disease. But a serious shortage of donor organs has limited its clinical application. To increase the number of livers available for transplantation, graft procurement from non– heart-beating donors (NHBDs) has again become a focus of attention.1,2 It is suggested that warm ischemia (WI), cold ischemia, or ischemia-reperfusion injury are risk factors for postoperative graft dysfunction from NHBDs. Prolonged WI or cold storage time are direct causes of early primary liver graft nonfunction. If the WI time or cold ischemic time exceed the safe limits of tolerance, the LTx will fail. Some reports, including our research, suggest that the safe limit of tolerance to WI of liver grafts from NHBDs is about 30 minutes.3–5 It is not known how long is the safe time limit of cold preservation in University of Wisconsin (UW) solution of liver grafts with WI from NHBDs, a question that we investigated for cold preservation in UW solution of liver grafts from NHBDs after 30 minutes of WI. MATERIAL AND METHODS In accordance with the China legislation on protection of animals and the “Guide for the Care and Use of Laboratory animals” (NIH © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 37, 409 – 412 (2005)
publication no. 8623, revised 1985), 26 Chinese Guangxi BAMA miniature swine weighing 8 to 11 kg were used after 36 hours of fasting. Orthotopic LTx was performed as previously described with minor modifications.6 Five minutes after administration of 12,500 IU intravenous heparin to the donor, the liver was subjected to in situ WI for 20 minutes by clamping all vessels to the liver. At the end of the WI period, the liver was washed out with 4°C Lactated Ringer’s solution and UW solution. After harvesting, the liver was stored for 6, 10, or 14 hours at 4°C in UW solution. During the anhepatic period, VVB was not used in the recipients. At the time of the suprahepatic vena cava anastomosis, the liver allograft was cooled with 500 mL of 4°C Lactated Ringer’s solution. After completion of the suprahepatic: vena cava anastomosis and portal vein anastomoses, the liver allograft was reperfused. Then the From the Hepatobiliary Surgery Centre of Chengdu Military District, Kunming General Hospital of PLA, Kunming, Yunnan Province, and Institute of Hepatobiliary Surgery of PLA, Southwest Hospital, and Third Military Medical University, Chongqing City, People’s Republic of China. Address reprint requests to Deke Qing, Hepatobiliary Surgery Centre of Chengdu Military District, Kunming General Hospital of PLA, Kunming, Yunnan Province, People’s Republic of China, 650032. E-mail: [email protected] 0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2004.11.064 409
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Fig 1. Postoperative survival rate of recipients. Pigs surviving more than 7 days (168 hours) were humanely-killed. Survival in Group 2 was significant lower than Group 1 (P ⬍ .05). Survival in Group 3 was also significantly lower than Group 1 (P ⬍ .01). infrahepatic vena cava and hepatic artery and bile duct were reconstructed by end-to-end techniques. No immunosuppressant was administered during or after the operation. The pigs were divided into 3 groups. Group 1 (n ⫽ 5) liver grafts had 30 minutes WI and a subsequent 6 hours of cold preservation. Group 2 (n ⫽ 5) grafts had 30 minutes WI and a subsequent 10 hours of cold preservation. Group 3 (n ⫽ 3) grafts had 30 minutes WI and a subsequent 14 hours of cold preservation. Postoperative survival was assessed for 7 days. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations in arterial blood were measured using an autoanalyzer. Assays of adenine nucleotides were performed at donor laparotomy, after cold preservation, and at 1 hour at reperfusion in
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Fig 3. Changes in AST concentration in recipient arterial blood (POD1, POD2, POD3, and POD7 ⫽ 1, 2, 3, and 7 postoperative days after LTx, respectively). The serum AST concentration in Group 2 was significantly higher than in Group 1 at 1 hour after reperfusion of the graft (P ⬍ .05) and on the first POD (P ⬍ .05) and on the second POD (P ⬍ .01), AST in Group 3 was significantly higher than in Group 1 at 1 or 6 hours after reperfusion of the graft (P ⬍ .05). the recipient. Graft liver specimens were frozen in situ with stainless steel tongs precooled with liquid nitrogen. The concentration of adenosine triphosphate (ATP) was determined enzymatically.7 Liver microcirculation blood flux was assessed using Laser Doppler Fluxmetry (Perimed AB, Stockholm, Sweden). At the following time points: laparotomy, end of WI and after cold preservation before implantation, and 1 hour after reperfusion. Graft tissue samples underwent histological examination with hematoxylin and eosin staining. Electron microscopic examination also was performed on some tissue samples. Data are expressed as the mean values ⫾ SD. An analysis of variance and paired Student t test were used to determine significance. Differences in survival were determined using the KaplanMeier analysis. P ⬍ .05 was considered significant.
RESULTS Survival
Figure 1 shows the postoperative survival rate of the recipient pigs. All 5 animals in Group 1 survived to 7 days, the survey endpoint. In Group 2, only 1 animal survived to the survey endpoint, the other 4 animals died within 12 hours after LTx because of noncorrectable metabolic acidosis or to coma. All animals in Group 3 died within 12 hours due to primary graft failure. At autopsy, neither intraabdominal bleeding nor anastomotic failure was observed. The 1-week survival rate of Group 1 was significantly higher than those of the other 2 groups. Fig 2. Changes in ALT concentration in recipient arterial blood (postoperative day [POD]1, POD2, POD3, and POD7 ⫽ 1, 2, 3, and 7 postoperative days after LTx, respectively). The serum ALT concentration at 1 hour after reperfusion of the graft in Group 3 was significantly higher than in group 1 (P ⬍ .05), and at 6 hours after reperfusion of the graft, ALT in Group 2 was significantly higher than in Group 1 (P ⬍ .05), ALT in Group 3 was also significantly higher than in Group 1 (P ⬍ .01), and ALT in Group 2 was significantly higher than in Group 1 on the first POD (P ⬍ .05).
Table 1. ATP Concentration of the Liver Grafts (mol/g wet tissue, mean ⴞ SD) Group
At Laparotomy
After Preservation
1 2 3
4.05 ⫾ 0.5 4.3 ⫾ 0.53 4.06 ⫾ 0.57
1.15 ⫾ 0.2 0.82 ⫾ 0.23* 0.77 ⫾ 0.08*
Note: Compare with Group 1: *P ⬍ .05, †P ⬍ .01.
1 hr After Reperfusion
3.35 ⫾ 0.4 2.41 ⫾ 0.72* 1.83 ⫾ 0.22†
TOLERANCE LIMITS OF LIVER GRAFTS
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Table 2. Changes in Microcirculation Blood Flux 1 Hour After Reperfusion in the Graft Livers (LDF) (mv) (mean ⴞ SD) Group
At Paratomy
1 hr After Reperfusion
P
1 2 3
11.17 ⫾ 0.17 11.2 ⫾ 0.19 11.25 ⫾ 0.29
10.32 ⫾ 0.46 9.18 ⫾ 0.9 7.14 ⫾ 0.78
— ⬍.05 ⬍.01
Microcirculation
Microcirculation blood flux at 1 hour after reperfusion in the grafted livers was significantly decreased in Group 2 (P ⬍ .05) and Group 3 (P ⬍ .01) compared with Group 1 (Table 2). Histological Examination
ALT and AST
The serum ALT concentration at 1 hour after reperfusion of the Group 3 grafts was significantly higher than that in Group 1 (P ⬍ .05), after 6 hours of storage. The ALT in Group 2 was significantly higher than that in Group 1 (P ⬍ .05), but less than that in Group 2 (P ⬍ .05). The serum AST concentration in Group 2 was significantly higher than that in Group 1 at 1 hour after reperfusion of the graft (P ⬍ .05) and on the first day postoperatively (P ⬍ .05) and on the second day postoperatively (P ⬍ .01). The AST in Group 3 was significantly higher than in Group 1 at 1 or 6 hours after reperfusion of the graft (P ⬍ .05) (Fig 2 and Fig 3).
One hour after reperfusion, the livers in Group 1 (Fig 4A) showed well-preserved hepatic architecture with middle hepatocyte vacuolization. The liver of the survivor in Group 2 (Fig 4B) showed degeneration and necrosis of hepatocytes with hepatocyte vacuolization and dilatation of the sinusoids. The livers in Group 3 (Fig 4C) showed diffuse hepatocyte degeneration and necrosis and severe damage of the sinusoids, and also severe damage to the hepatic architecture with hepatic cords separating hepatocyte apoptosis and nuclear degeneration (Fig 4D). Two of 5 pigs from Group 1 and Group 2 that survived the study period showed some extent of necrosis of the biliary tract.
ATP
DISCUSSION
After preservation, the ATP concentration decreased significantly in Groups 2 and 3 compared with Group 1 (P ⬍ .05). At 1 hour after reperfusion, the ATP concentration was restored to 80% of the initial value in Group 1. In Group 2, although the survivor showed a similar, successful pattern of ATP recovery, recovery of the nonsurvivors was suppressed. The mean values at 1 hour after reperfusion in that group was significantly lower than that in Group 1 (P ⬍ .05). The recovery rate also was significantly lower in Group 3 (P ⬍ .01) (Table 1).
It has been suggested that 30 hours, even up to 48 hours represents the safe time limit of human liver preservation in UW solution that have not undergone WI based upon canine strokes.8 –10 But the safe time limits of cold preservation in UW solution of liver grafts from NHBDs subjected to WI for 30 minutes has not been fully delineated. By using the incidence of primary liver graft nonfunction, recipient death, necrosis of the graft liver, and other indices, such as liver pathological observations, hepatic function, energy metabolism of the liver, hepatic microcirculation as
Fig 4. (A to C) Light micrograph of the liver graft 1 hour after reperfusion. Hematoxylineosin stain; original magnification ⫻200. (A) Group 1, wellpreserved hepatic architecture with middle hepatocyte vacuolization. (B) Group 2, prominent hepatocyte degeneration and vacuolization and necrosis with dilatation of the sinusoids. (C) Group 3, diffuse hepatocyte degeneration and necrosis and severe damage of the sinusoids. (D) Group 3, electron microscope picture of the liver graft 1 hour after reperfusion, severe damage of the hepatic architecture with hepatic cord separating, hepatocyte apoptosis, and nuclear deformity are evident (original magnification ⫻1500).
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the standards for evaluating the safe time limits, we observed that all animals could survive up to 1 week with little necrosis of liver tissue after 30 minutes WI followed by less than 6 hours of cold preservation. However, when the cold preservation time exceeded 6 hours, the incidence of liver tissue necrosis primary graft nonfunction increased with a significantly decreased 1-week survival rate. The results suggest that liver grafts obtained with 30 minutes WI and less than 6 hours cold preservation in UW solution can be transplanted safely. Prolonging the cold preservation time from 6 to 10 or 14 hours led to increased ALT or AST concentrations, decreased ATP concentration at 1 hour after reperfusion, impaired microcirculation blood flux at 1 hour after reperfusion, and more severe liver pathological injuries. There were significant differences in these indexes between Group 1 and the other 2 groups. Biochemical index changes were consistent with liver pathological results and with animal survival rates. The problem with LTx from NHBDs is the severe and irreversible damage to the intrahepatic biliary tract in grafts of those pigs that survive after transplantation.11 In our experiment, this complication developed in 2 of 5 pigs from Group 1. Group 2 hosts that survived the study period showed prominent necrosis of the biliary tract. More research should be performed on measures to prevent the biliary complications. In recent years, the duration of cold preservation of liver grafts has been gradually prolonged because more organs are transported from one place to another and because the recipient hepatectomy has become more complex. Based on this condition, how do we control the cold preservation time of liver grafts from NHBDs? Our research suggests the one can
QING
avoid primary graft nonfunction LTx, from NHBDs by cold preservation times less than 6 hours with 30 minutes of WI. REFERENCES 1. Reich DJ, Munoz SC, Rothstein KD, et al: Controlled non-heart-beating donor transplantation: a successful single center experience, with topic update. Transplantation 70:1159, 2000 2. Garcia-Valdecasas-Salgado J-C: Non beating heart donors as a possible source for liver transplantation. Acta Chir Belg 100:268, 2000 3. Ong HS, Soo KC, Jeseph VT, et al: The viability of liver graft for transplantation after prolonged warm ischaemia. Ann Acad Med Singapore 28:25, 1999 4. Kimura J: Porcine liver transplantation using warm ischemic grafts. Hokkaido Igaku Zasshi 71:475, 1996 5. Saad S, Minor T, Kotting M, et al: Extension of ischemic tolerance of porcine livers by cold preservation including postconditioning with gaseous oxygen. Transplantation 71:498, 2001 6. Oike F, Uryuhara K, Otsuka M, et al: Simplified technique of orthotopic liver transplantation in pigs. Transplantation 71:328, 2001 7. Jaworek D, Gruber W, Bergmeyer HU: Adenosine-5=diphosphate and adenosine-5 -monophosphate. In Bergmeyer HU (ed): Methods of Enzymatic Analysis. New York: Academic Press; 1974, p 2127 8. Clavien PA, Harvey PRC, Strasberg SM: Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies. Transplantation 53:957, 1992 9. Jamieson NV, Sundberg R, Lindell S, et al: Preservation of the canine liver for 24 – 48 hours using single cold storage with UW solution. Transplantation 46:517, 1988 10. Boudjema K, Lindell SL, Southard JH, et al: The effect of fasting on the quality of liver preservation by simple cold storage. Transplantation 50:943, 1990 11. Lopez-Boado MA, Garcia-Valdecasas JC, Ordi J, et al: Histological changes during and after liver transplantation from NHBD pig. Transplant Proc 29:3471, 1997
L
IVER transplantation (LTx) has been widely accepted as an effective treatment for end-stage liver disease. But a serious shortage of donor organs has limited its clinical application. To increase the number of livers available for transplantation, graft procurement from non– heart-beating donors (NHBDs) has again become a focus of attention.1,2 It is suggested that warm ischemia (WI), cold ischemia, or ischemia-reperfusion injury are risk factors for postoperative graft dysfunction from NHBDs. Prolonged WI or cold storage time are direct causes of early primary liver graft nonfunction. If the WI time or cold ischemic time exceed the safe limits of tolerance, the LTx will fail. Some reports, including our research, suggest that the safe limit of tolerance to WI of liver grafts from NHBDs is about 30 minutes.3–5 It is not known how long is the safe time limit of cold preservation in University of Wisconsin (UW) solution of liver grafts with WI from NHBDs, a question that we investigated for cold preservation in UW solution of liver grafts from NHBDs after 30 minutes of WI. MATERIAL AND METHODS In accordance with the China legislation on protection of animals and the “Guide for the Care and Use of Laboratory animals” (NIH © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 37, 409 – 412 (2005)
publication no. 8623, revised 1985), 26 Chinese Guangxi BAMA miniature swine weighing 8 to 11 kg were used after 36 hours of fasting. Orthotopic LTx was performed as previously described with minor modifications.6 Five minutes after administration of 12,500 IU intravenous heparin to the donor, the liver was subjected to in situ WI for 20 minutes by clamping all vessels to the liver. At the end of the WI period, the liver was washed out with 4°C Lactated Ringer’s solution and UW solution. After harvesting, the liver was stored for 6, 10, or 14 hours at 4°C in UW solution. During the anhepatic period, VVB was not used in the recipients. At the time of the suprahepatic vena cava anastomosis, the liver allograft was cooled with 500 mL of 4°C Lactated Ringer’s solution. After completion of the suprahepatic: vena cava anastomosis and portal vein anastomoses, the liver allograft was reperfused. Then the From the Hepatobiliary Surgery Centre of Chengdu Military District, Kunming General Hospital of PLA, Kunming, Yunnan Province, and Institute of Hepatobiliary Surgery of PLA, Southwest Hospital, and Third Military Medical University, Chongqing City, People’s Republic of China. Address reprint requests to Deke Qing, Hepatobiliary Surgery Centre of Chengdu Military District, Kunming General Hospital of PLA, Kunming, Yunnan Province, People’s Republic of China, 650032. E-mail: [email protected] 0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2004.11.064 409
410
Fig 1. Postoperative survival rate of recipients. Pigs surviving more than 7 days (168 hours) were humanely-killed. Survival in Group 2 was significant lower than Group 1 (P ⬍ .05). Survival in Group 3 was also significantly lower than Group 1 (P ⬍ .01). infrahepatic vena cava and hepatic artery and bile duct were reconstructed by end-to-end techniques. No immunosuppressant was administered during or after the operation. The pigs were divided into 3 groups. Group 1 (n ⫽ 5) liver grafts had 30 minutes WI and a subsequent 6 hours of cold preservation. Group 2 (n ⫽ 5) grafts had 30 minutes WI and a subsequent 10 hours of cold preservation. Group 3 (n ⫽ 3) grafts had 30 minutes WI and a subsequent 14 hours of cold preservation. Postoperative survival was assessed for 7 days. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations in arterial blood were measured using an autoanalyzer. Assays of adenine nucleotides were performed at donor laparotomy, after cold preservation, and at 1 hour at reperfusion in
QING
Fig 3. Changes in AST concentration in recipient arterial blood (POD1, POD2, POD3, and POD7 ⫽ 1, 2, 3, and 7 postoperative days after LTx, respectively). The serum AST concentration in Group 2 was significantly higher than in Group 1 at 1 hour after reperfusion of the graft (P ⬍ .05) and on the first POD (P ⬍ .05) and on the second POD (P ⬍ .01), AST in Group 3 was significantly higher than in Group 1 at 1 or 6 hours after reperfusion of the graft (P ⬍ .05). the recipient. Graft liver specimens were frozen in situ with stainless steel tongs precooled with liquid nitrogen. The concentration of adenosine triphosphate (ATP) was determined enzymatically.7 Liver microcirculation blood flux was assessed using Laser Doppler Fluxmetry (Perimed AB, Stockholm, Sweden). At the following time points: laparotomy, end of WI and after cold preservation before implantation, and 1 hour after reperfusion. Graft tissue samples underwent histological examination with hematoxylin and eosin staining. Electron microscopic examination also was performed on some tissue samples. Data are expressed as the mean values ⫾ SD. An analysis of variance and paired Student t test were used to determine significance. Differences in survival were determined using the KaplanMeier analysis. P ⬍ .05 was considered significant.
RESULTS Survival
Figure 1 shows the postoperative survival rate of the recipient pigs. All 5 animals in Group 1 survived to 7 days, the survey endpoint. In Group 2, only 1 animal survived to the survey endpoint, the other 4 animals died within 12 hours after LTx because of noncorrectable metabolic acidosis or to coma. All animals in Group 3 died within 12 hours due to primary graft failure. At autopsy, neither intraabdominal bleeding nor anastomotic failure was observed. The 1-week survival rate of Group 1 was significantly higher than those of the other 2 groups. Fig 2. Changes in ALT concentration in recipient arterial blood (postoperative day [POD]1, POD2, POD3, and POD7 ⫽ 1, 2, 3, and 7 postoperative days after LTx, respectively). The serum ALT concentration at 1 hour after reperfusion of the graft in Group 3 was significantly higher than in group 1 (P ⬍ .05), and at 6 hours after reperfusion of the graft, ALT in Group 2 was significantly higher than in Group 1 (P ⬍ .05), ALT in Group 3 was also significantly higher than in Group 1 (P ⬍ .01), and ALT in Group 2 was significantly higher than in Group 1 on the first POD (P ⬍ .05).
Table 1. ATP Concentration of the Liver Grafts (mol/g wet tissue, mean ⴞ SD) Group
At Laparotomy
After Preservation
1 2 3
4.05 ⫾ 0.5 4.3 ⫾ 0.53 4.06 ⫾ 0.57
1.15 ⫾ 0.2 0.82 ⫾ 0.23* 0.77 ⫾ 0.08*
Note: Compare with Group 1: *P ⬍ .05, †P ⬍ .01.
1 hr After Reperfusion
3.35 ⫾ 0.4 2.41 ⫾ 0.72* 1.83 ⫾ 0.22†
TOLERANCE LIMITS OF LIVER GRAFTS
411
Table 2. Changes in Microcirculation Blood Flux 1 Hour After Reperfusion in the Graft Livers (LDF) (mv) (mean ⴞ SD) Group
At Paratomy
1 hr After Reperfusion
P
1 2 3
11.17 ⫾ 0.17 11.2 ⫾ 0.19 11.25 ⫾ 0.29
10.32 ⫾ 0.46 9.18 ⫾ 0.9 7.14 ⫾ 0.78
— ⬍.05 ⬍.01
Microcirculation
Microcirculation blood flux at 1 hour after reperfusion in the grafted livers was significantly decreased in Group 2 (P ⬍ .05) and Group 3 (P ⬍ .01) compared with Group 1 (Table 2). Histological Examination
ALT and AST
The serum ALT concentration at 1 hour after reperfusion of the Group 3 grafts was significantly higher than that in Group 1 (P ⬍ .05), after 6 hours of storage. The ALT in Group 2 was significantly higher than that in Group 1 (P ⬍ .05), but less than that in Group 2 (P ⬍ .05). The serum AST concentration in Group 2 was significantly higher than that in Group 1 at 1 hour after reperfusion of the graft (P ⬍ .05) and on the first day postoperatively (P ⬍ .05) and on the second day postoperatively (P ⬍ .01). The AST in Group 3 was significantly higher than in Group 1 at 1 or 6 hours after reperfusion of the graft (P ⬍ .05) (Fig 2 and Fig 3).
One hour after reperfusion, the livers in Group 1 (Fig 4A) showed well-preserved hepatic architecture with middle hepatocyte vacuolization. The liver of the survivor in Group 2 (Fig 4B) showed degeneration and necrosis of hepatocytes with hepatocyte vacuolization and dilatation of the sinusoids. The livers in Group 3 (Fig 4C) showed diffuse hepatocyte degeneration and necrosis and severe damage of the sinusoids, and also severe damage to the hepatic architecture with hepatic cords separating hepatocyte apoptosis and nuclear degeneration (Fig 4D). Two of 5 pigs from Group 1 and Group 2 that survived the study period showed some extent of necrosis of the biliary tract.
ATP
DISCUSSION
After preservation, the ATP concentration decreased significantly in Groups 2 and 3 compared with Group 1 (P ⬍ .05). At 1 hour after reperfusion, the ATP concentration was restored to 80% of the initial value in Group 1. In Group 2, although the survivor showed a similar, successful pattern of ATP recovery, recovery of the nonsurvivors was suppressed. The mean values at 1 hour after reperfusion in that group was significantly lower than that in Group 1 (P ⬍ .05). The recovery rate also was significantly lower in Group 3 (P ⬍ .01) (Table 1).
It has been suggested that 30 hours, even up to 48 hours represents the safe time limit of human liver preservation in UW solution that have not undergone WI based upon canine strokes.8 –10 But the safe time limits of cold preservation in UW solution of liver grafts from NHBDs subjected to WI for 30 minutes has not been fully delineated. By using the incidence of primary liver graft nonfunction, recipient death, necrosis of the graft liver, and other indices, such as liver pathological observations, hepatic function, energy metabolism of the liver, hepatic microcirculation as
Fig 4. (A to C) Light micrograph of the liver graft 1 hour after reperfusion. Hematoxylineosin stain; original magnification ⫻200. (A) Group 1, wellpreserved hepatic architecture with middle hepatocyte vacuolization. (B) Group 2, prominent hepatocyte degeneration and vacuolization and necrosis with dilatation of the sinusoids. (C) Group 3, diffuse hepatocyte degeneration and necrosis and severe damage of the sinusoids. (D) Group 3, electron microscope picture of the liver graft 1 hour after reperfusion, severe damage of the hepatic architecture with hepatic cord separating, hepatocyte apoptosis, and nuclear deformity are evident (original magnification ⫻1500).
412
the standards for evaluating the safe time limits, we observed that all animals could survive up to 1 week with little necrosis of liver tissue after 30 minutes WI followed by less than 6 hours of cold preservation. However, when the cold preservation time exceeded 6 hours, the incidence of liver tissue necrosis primary graft nonfunction increased with a significantly decreased 1-week survival rate. The results suggest that liver grafts obtained with 30 minutes WI and less than 6 hours cold preservation in UW solution can be transplanted safely. Prolonging the cold preservation time from 6 to 10 or 14 hours led to increased ALT or AST concentrations, decreased ATP concentration at 1 hour after reperfusion, impaired microcirculation blood flux at 1 hour after reperfusion, and more severe liver pathological injuries. There were significant differences in these indexes between Group 1 and the other 2 groups. Biochemical index changes were consistent with liver pathological results and with animal survival rates. The problem with LTx from NHBDs is the severe and irreversible damage to the intrahepatic biliary tract in grafts of those pigs that survive after transplantation.11 In our experiment, this complication developed in 2 of 5 pigs from Group 1. Group 2 hosts that survived the study period showed prominent necrosis of the biliary tract. More research should be performed on measures to prevent the biliary complications. In recent years, the duration of cold preservation of liver grafts has been gradually prolonged because more organs are transported from one place to another and because the recipient hepatectomy has become more complex. Based on this condition, how do we control the cold preservation time of liver grafts from NHBDs? Our research suggests the one can
QING
avoid primary graft nonfunction LTx, from NHBDs by cold preservation times less than 6 hours with 30 minutes of WI. REFERENCES 1. Reich DJ, Munoz SC, Rothstein KD, et al: Controlled non-heart-beating donor transplantation: a successful single center experience, with topic update. Transplantation 70:1159, 2000 2. Garcia-Valdecasas-Salgado J-C: Non beating heart donors as a possible source for liver transplantation. Acta Chir Belg 100:268, 2000 3. Ong HS, Soo KC, Jeseph VT, et al: The viability of liver graft for transplantation after prolonged warm ischaemia. Ann Acad Med Singapore 28:25, 1999 4. Kimura J: Porcine liver transplantation using warm ischemic grafts. Hokkaido Igaku Zasshi 71:475, 1996 5. Saad S, Minor T, Kotting M, et al: Extension of ischemic tolerance of porcine livers by cold preservation including postconditioning with gaseous oxygen. Transplantation 71:498, 2001 6. Oike F, Uryuhara K, Otsuka M, et al: Simplified technique of orthotopic liver transplantation in pigs. Transplantation 71:328, 2001 7. Jaworek D, Gruber W, Bergmeyer HU: Adenosine-5=diphosphate and adenosine-5 -monophosphate. In Bergmeyer HU (ed): Methods of Enzymatic Analysis. New York: Academic Press; 1974, p 2127 8. Clavien PA, Harvey PRC, Strasberg SM: Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies. Transplantation 53:957, 1992 9. Jamieson NV, Sundberg R, Lindell S, et al: Preservation of the canine liver for 24 – 48 hours using single cold storage with UW solution. Transplantation 46:517, 1988 10. Boudjema K, Lindell SL, Southard JH, et al: The effect of fasting on the quality of liver preservation by simple cold storage. Transplantation 50:943, 1990 11. Lopez-Boado MA, Garcia-Valdecasas JC, Ordi J, et al: Histological changes during and after liver transplantation from NHBD pig. Transplant Proc 29:3471, 1997