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Machine perfusion preservation of the non–heart-beating donor rat livers using polysol, a new preservation solution
Machine Perfusion Preservation of the Non–Heart-Beating Donor Rat Livers Using Polysol, A New Preservation Solution M. Bessems, B.M. Doorschodt, A.K. van Vliet, and T.M. van Gulik ABSTRACT Aims. The increasing shortage of donor organs has led to a focus on extended criteria donors, including the non– heart-beating donor (NHBD). An optimal preservation method is required to facilitate successful transplantation of these ischemically damaged organs. The recent literature has shown clear advantages of hypothermic machine perfusion (MP) over cold storage (CS). For MP, modified University of Wisconsin perfusion solution (UW-G) is often used, which, however, is known to cause microcirculatory obstruction, is difficult to obtain, and is expensive. Therefore, Polysol was developed as a MP preservation solution that contains specific nutrients for the liver, such as amino acids, energy substrates, and vitamins. The aim of this study was to compare Polysol with UW-G in a NHBD rat liver model. Methods. After 24 hours hypothermic MP of NHBD rat livers using UW-G or Polysol, liver damage and function parameters were assessed during 60 minutes of reperfusion with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours CS in UW. Results. Liver enzyme release was significantly higher among the CS-UW group compared to MP using UW-G or Polysol. Flow during reperfusion was significantly higher when using Polysol compared to UW-G. Bile production and ammonia clearance were highest when using Polysol compared to UW-G. There was less cellular edema after preservation with Polysol compared to UW-G. Conclusions. MP of NHBD rat livers for 24 hours using UW-G or Polysol resulted in less hepatocellular damage than CS in UW. MP of NHBD livers for 24 hours using Polysol is superior to MP using UW-G.
T
HE CURRENT status of organ transplantation, with patients succumbing while awaiting transplantation, has led to a focus on suboptimal donors, for example the non– heart-beating donor (NHBD).1,2 In these donors the circulation has ceased before harvesting of the organ has commenced, resulting in warm ischemic damage. To achieve successful transplantation, these organs need optimal preservation conditions. The recent literature has shown the advantages of oxygenated hypothermic machine perfusion (MP) over cold storage (CS) for these suboptimal organs, which can even be resuscitated.3–5 A modified University of Wisconsin solution (UW-G) is widely used as a MP preservation solution for livers. However, this solution causes microvasculatory obstructions is hard to obtain, and is expensive.6 Therefore we developed Polysol, a preservation solution based on a colloid with more free radical scavengers, potent buffers, 0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.01.039 326
and specific nutrients for the liver. These nutrients, such as amino acids and vitamins, support metabolism under hypothermic conditions, thereby facilitating resuscitation of ischemically damaged organs. In previous studies this MP preservation solution was shown to result in equal or better preservation of rat livers obtained under optimal conditions.7 The aim of the present study was to compare the MP solution Polysol with UW-G in a NHBD rat liver model.
From the Surgical Laboratory, Academic Medical Center Amsterdam, Amsterdam, The Netherlands. Address reprint requests to M. Bessems, MD, Surgical Laboratory, Department of Surgery, Academic Medical Center, IWO1-173, P.O.B. 22700, 1100 DE Amsterdam, The Netherlands. E-mail: [email protected] © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 37, 326 –328 (2005)
MACHINE PERFUSION PRESERVATION
METHODS Male Wistar rats (weighing 250 to 300 g; Harlan, The Netherlands) were used as liver donors. After a midline laparotomy, warm ischemia (WIT) was induced by phrenotomy. After canulation of the bile duct and preparation of the portal and caval veins, the liver was washed with Ringer’s Lactate (29 mmol/L lactate; Baxter, Utrecht, The Netherlands) for 30 minutes of WIT. The harvested liver was weighed and thereafter preserved hypothermically for 24 hours by either CS (n ⫽ 6), MP-UW-G (n ⫽ 6), or MP-Polysol (n ⫽ 6). For MP and reperfusion a recirculating setup was used consisting of a reservoir, roller pump, glass oxygenator, bubble trap, and heat exchanger. This is a pressure-driven (15 mm Hg) perfusion system. Using an isolated, perfused rat liver model, we assessed hepatocellular damage and liver functions. To determine liver parenchymal damage, aspartate aminotransferase (AST) levels were measured during 60 minutes of normothermic reperfusion (37°C) with oxygenated Krebs-Henseleit buffer (KHB). Liver function was assessed by bile collection and measurement of ammonia clearance rate. For this purpose 5 mmol/L ammonium chloride was added to the KHB prior to reperfusion. Dry/wet liver weight ratios were determined for evaluation of cellular edema. Control livers were reperfused after 24 hours of CS in UW (n ⫽ 6). The statistical analysis was performed using the Kruskall-Wallis test, followed, if appropiate, by the Mann-Whitney U test. For ammonia clearance rates, analysis of repeated measurements was used, with a post hoc test according to Bonferroni. Statistical significance was indicated when P ⬍ .05. Values are expressed as means ⫾ SEM.
RESULTS
Liver damage was significantly higher among the CS group compared to MP-UW-G or MP-Polysol cohorts (Fig 1). Although the results seem to favor Polysol compared to UW-G, these were not significant (AST, t ⫽ 60 minutes: 30 ⫾ 3 versus 43 ⫾ 10 IU/L). Flow at reperfusion was significantly higher at all time points when using Polysol compared to UW-G (t ⫽ 60: 33 ⫾ 1 versus 12 ⫾ 3 mL/min; P ⬍ .01) and at t ⫽ 45 and 60 compared to CS (t ⫽ 60: 33 ⫾ 1 versus 25 ⫾ 3 mL/min) However, after CS perfusate flow (at a constant pressure of 15 mm Hg) was significantly
Fig 1. AST levels measured during normothermic reperfusion with KHB after 24 hours of hypothermic preservation by CS-UW, MP-UW-G, or MP-Polysol. For reference: total AST amount in rat liver is ⫾ 3500 IU. Values are expressed as mean ⫾ SEM, P ⬍ .05 is considered significant. *Significant compared to CS-UW.
327
higher at-all time points compared to MP-UW-G. Bile production was highest using Polysol (390 ⫾ 23 L/h) compared to UW-G (153 ⫾ 55 L/h) and CS (34 ⫾ 19 L/h) (Fig 2). Ammonia clearance rate was highest after MP-Polysol compared to UW-G (Fig 3). Ammonia clearance rates appeared higher after CS compared to MPUW-G; however, this did not reach significance. There was less cellular edema (wet/dry ratio in %) after preservation with Polysol compared to UW-G (72.57 ⫾ 0.01 versus 75.17 ⫾ 0.01), but it was only significant for Polysol compared to CS (72.57 ⫾ 0.01 versus 77.00 ⫾ 0.01). DISCUSSION
The use of organs from marginal donors, like the NHBD, has become an important issue in expanding the donor pool. For these organs the preservation method is of even greater importance than for optimal donor organs. Although the benefits of MP over CS have been shown in clinical NHBD kidney transplantation, only animal studies have been performed regarding MP of liver grafts.8 –11 In this study, three preservation methods for NHBD rat livers were compared: the “gold standard” CS using UW; MP using UW-G; and MP using the newly developed solution Polysol. Regarding both liver damage and liver function, the results were significantly better after 24 hours MP using Polysol, compared to CS in UW or MP using UW-G. Possible explanations may be not only the general advantages of MP over CS, but also the composition of Polysol, which contains the colloid polyethyleneglycol instead of hydroxyethylstarch (HES), resulting in a lower viscosity, the high sodium/low potassium content, the superior buffering capacity, and the enriched nutrients (Table 1). Although liver metabolism at 4°C is decreased to 7% to 40% of the physiologic level, the liver does need some support during the preservation period. Further, it was shown that perfusate flow and ammonia clearance were better after CS compared to MP using UW-G. This might
Fig 2. Bile production measured during normothermic reperfusion with KHB after 24 hours of hypothermic preservation by CS-UW, MP-UW-G, or MP-Polysol. Values are expressed as mean ⫾ SEM, P ⬍ .05 is considered significant. *Significant compared to MP-Polysol.
328
BESSEMS, DOORSCHODT, VAN VILET ET AL Table 1. Important Components of the Preservation Solutions Components
Colloid Na/K ratio Buffer
Antioxidants
Fig 3. Ammonia clearance rates during normothermic reperfusion with KHB after 24 hours of hypothermic preservation by CS-UW, MP-UW-G, or MP-Polysol. Prior to reperfusion 5 mmol/L NH4CI was added to KHB. Values are expressed as means ⫾ SEM, P ⬍ .05 is considered significant. *Significant compared to MP-Polysol.
be explained by the use of HES in UW-G in a continuous perfusion setup, causing shear stress and microcirculatory disturbances.6 In general, MP using Polysol provided us with better reperfusion results compared to both CS in UW and MP using UW-G. Validation of these results will be carried out in a pig liver preservation and transplantation model. In conclusion, 24-hour machine perfusion preservation of NHBD rat livers using the newly developed preservation solution Polysol results in less hepatocellular damage and better liver function compared to cold storage in UW or machine perfusion using UW-G. ACKNOWLEDGMENTS We thank Dr A.J. Meijer from the Department of Medical Biochemistry at the Academic Medical Center for his contribution to this study.
REFERENCES 1. D’alessandro AM, Hoffmann RM, Knechtle SJ, et al: Liver transplantation from controlled non-heart-beating donors. Surgery 128:579, 2000 2. Kootstra G, Daemen JH, Oomen AP: Categories of nonheart-beating donors. Transplant Proc 27:2893, 1995 3. Southard JH, Lindell S, Ametani M, et al: Kupffer cell activation in liver preservation: cold storage vs machine perfusion. Transplant Proc 32:27, 2000 4. Wight J, Chilcott J, Holmes M, et al: The clinical and cost-effectiveness of pulsatile machine perfusion versus cold stor-
Energy substrates Impermeants
Amino acids Vitamins pH indicator
UW
UW-G
Polysol
HES
HES
PEG
KH2PO4
KH2PO4 HEPES
Allopurinol Glutathion
Allopurinol Glutathion
KH2PO4 HEPES Histidine Allopurinol Glutathion Alpha-tocopherol Ascorbic acid Glucose
Glucose Lactobionate Na-Gluconate K-Gluconate Mg-Gluconate Raffinose Raffinose
Na-Gluconate K-Gluconate Trehalose Raffinose
— — —
Miscellaneous Miscellaneous Phenol-red
— — —
The most important differences in composition of the three preservation solutions are mentioned in this table. All three solutions have an osmolality of 320 –340 mOsm and a pH of 7.4. The total amount of components in these solutions is 13 (UW), 14 (UW-G) and 61 (Polysol).
age of kidneys for transplantation retrieved from heart-beating and non-heart-beating donors. Health Technol Assess 7:1, 2003 5. Xu H, Lee CY, Clemens MG, et al: Prolonged hypothermic machine perfusion preserves hepatocellular function but potentiates endothelial cell dysfunction in rat livers. Transplantation 77:1676, 2004 6. Morariu AM, Vd PA, Oeveren V, et al: Hyperaggregating effect of hydroxyethyl starch components and University of Wisconsin solution on human red blood cells: a risk of impaired graft perfusion in organ procurement? Transplantation 76:37, 2003 7. Bessems M, Doorschodt BM, Hooijschuur O, et al: Liver preservation by hypothermic, continuous machine perfusion using a new colloid based preservation solution. Gut 52(supplVI):160A, 2003 8. Guarrera JV, Polyak M, O’Mar AB, et al: Pulsatile machine perfusion with Vasosol solution improves early graft function after cadaveric renal transplantation. Transplantation 77:1264, 2004 9. van der Vliet JA, Kievit JK, Hene RJ, et al: Preservation of non-heart-beating donor kidneys: a clinical prospective randomised case-control study of machine perfusion versus cold storage. Transplant Proc 33:847, 2001 10. Manzarbeitia CY, Ortiz JA, Jeon H, et al: Long-term outcome of controlled, non-heart-beating donor liver transplantation. Transplantation 78:211, 2004 11. Lee CY, Jain S, Duncan HM, et al: Survival transplantation of preserved non-heart-beating donor rat livers preservation by hypothermic machine perfusion. Transplantation 76:1432, 2003
T
HE CURRENT status of organ transplantation, with patients succumbing while awaiting transplantation, has led to a focus on suboptimal donors, for example the non– heart-beating donor (NHBD).1,2 In these donors the circulation has ceased before harvesting of the organ has commenced, resulting in warm ischemic damage. To achieve successful transplantation, these organs need optimal preservation conditions. The recent literature has shown the advantages of oxygenated hypothermic machine perfusion (MP) over cold storage (CS) for these suboptimal organs, which can even be resuscitated.3–5 A modified University of Wisconsin solution (UW-G) is widely used as a MP preservation solution for livers. However, this solution causes microvasculatory obstructions is hard to obtain, and is expensive.6 Therefore we developed Polysol, a preservation solution based on a colloid with more free radical scavengers, potent buffers, 0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.01.039 326
and specific nutrients for the liver. These nutrients, such as amino acids and vitamins, support metabolism under hypothermic conditions, thereby facilitating resuscitation of ischemically damaged organs. In previous studies this MP preservation solution was shown to result in equal or better preservation of rat livers obtained under optimal conditions.7 The aim of the present study was to compare the MP solution Polysol with UW-G in a NHBD rat liver model.
From the Surgical Laboratory, Academic Medical Center Amsterdam, Amsterdam, The Netherlands. Address reprint requests to M. Bessems, MD, Surgical Laboratory, Department of Surgery, Academic Medical Center, IWO1-173, P.O.B. 22700, 1100 DE Amsterdam, The Netherlands. E-mail: [email protected] © 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 37, 326 –328 (2005)
MACHINE PERFUSION PRESERVATION
METHODS Male Wistar rats (weighing 250 to 300 g; Harlan, The Netherlands) were used as liver donors. After a midline laparotomy, warm ischemia (WIT) was induced by phrenotomy. After canulation of the bile duct and preparation of the portal and caval veins, the liver was washed with Ringer’s Lactate (29 mmol/L lactate; Baxter, Utrecht, The Netherlands) for 30 minutes of WIT. The harvested liver was weighed and thereafter preserved hypothermically for 24 hours by either CS (n ⫽ 6), MP-UW-G (n ⫽ 6), or MP-Polysol (n ⫽ 6). For MP and reperfusion a recirculating setup was used consisting of a reservoir, roller pump, glass oxygenator, bubble trap, and heat exchanger. This is a pressure-driven (15 mm Hg) perfusion system. Using an isolated, perfused rat liver model, we assessed hepatocellular damage and liver functions. To determine liver parenchymal damage, aspartate aminotransferase (AST) levels were measured during 60 minutes of normothermic reperfusion (37°C) with oxygenated Krebs-Henseleit buffer (KHB). Liver function was assessed by bile collection and measurement of ammonia clearance rate. For this purpose 5 mmol/L ammonium chloride was added to the KHB prior to reperfusion. Dry/wet liver weight ratios were determined for evaluation of cellular edema. Control livers were reperfused after 24 hours of CS in UW (n ⫽ 6). The statistical analysis was performed using the Kruskall-Wallis test, followed, if appropiate, by the Mann-Whitney U test. For ammonia clearance rates, analysis of repeated measurements was used, with a post hoc test according to Bonferroni. Statistical significance was indicated when P ⬍ .05. Values are expressed as means ⫾ SEM.
RESULTS
Liver damage was significantly higher among the CS group compared to MP-UW-G or MP-Polysol cohorts (Fig 1). Although the results seem to favor Polysol compared to UW-G, these were not significant (AST, t ⫽ 60 minutes: 30 ⫾ 3 versus 43 ⫾ 10 IU/L). Flow at reperfusion was significantly higher at all time points when using Polysol compared to UW-G (t ⫽ 60: 33 ⫾ 1 versus 12 ⫾ 3 mL/min; P ⬍ .01) and at t ⫽ 45 and 60 compared to CS (t ⫽ 60: 33 ⫾ 1 versus 25 ⫾ 3 mL/min) However, after CS perfusate flow (at a constant pressure of 15 mm Hg) was significantly
Fig 1. AST levels measured during normothermic reperfusion with KHB after 24 hours of hypothermic preservation by CS-UW, MP-UW-G, or MP-Polysol. For reference: total AST amount in rat liver is ⫾ 3500 IU. Values are expressed as mean ⫾ SEM, P ⬍ .05 is considered significant. *Significant compared to CS-UW.
327
higher at-all time points compared to MP-UW-G. Bile production was highest using Polysol (390 ⫾ 23 L/h) compared to UW-G (153 ⫾ 55 L/h) and CS (34 ⫾ 19 L/h) (Fig 2). Ammonia clearance rate was highest after MP-Polysol compared to UW-G (Fig 3). Ammonia clearance rates appeared higher after CS compared to MPUW-G; however, this did not reach significance. There was less cellular edema (wet/dry ratio in %) after preservation with Polysol compared to UW-G (72.57 ⫾ 0.01 versus 75.17 ⫾ 0.01), but it was only significant for Polysol compared to CS (72.57 ⫾ 0.01 versus 77.00 ⫾ 0.01). DISCUSSION
The use of organs from marginal donors, like the NHBD, has become an important issue in expanding the donor pool. For these organs the preservation method is of even greater importance than for optimal donor organs. Although the benefits of MP over CS have been shown in clinical NHBD kidney transplantation, only animal studies have been performed regarding MP of liver grafts.8 –11 In this study, three preservation methods for NHBD rat livers were compared: the “gold standard” CS using UW; MP using UW-G; and MP using the newly developed solution Polysol. Regarding both liver damage and liver function, the results were significantly better after 24 hours MP using Polysol, compared to CS in UW or MP using UW-G. Possible explanations may be not only the general advantages of MP over CS, but also the composition of Polysol, which contains the colloid polyethyleneglycol instead of hydroxyethylstarch (HES), resulting in a lower viscosity, the high sodium/low potassium content, the superior buffering capacity, and the enriched nutrients (Table 1). Although liver metabolism at 4°C is decreased to 7% to 40% of the physiologic level, the liver does need some support during the preservation period. Further, it was shown that perfusate flow and ammonia clearance were better after CS compared to MP using UW-G. This might
Fig 2. Bile production measured during normothermic reperfusion with KHB after 24 hours of hypothermic preservation by CS-UW, MP-UW-G, or MP-Polysol. Values are expressed as mean ⫾ SEM, P ⬍ .05 is considered significant. *Significant compared to MP-Polysol.
328
BESSEMS, DOORSCHODT, VAN VILET ET AL Table 1. Important Components of the Preservation Solutions Components
Colloid Na/K ratio Buffer
Antioxidants
Fig 3. Ammonia clearance rates during normothermic reperfusion with KHB after 24 hours of hypothermic preservation by CS-UW, MP-UW-G, or MP-Polysol. Prior to reperfusion 5 mmol/L NH4CI was added to KHB. Values are expressed as means ⫾ SEM, P ⬍ .05 is considered significant. *Significant compared to MP-Polysol.
be explained by the use of HES in UW-G in a continuous perfusion setup, causing shear stress and microcirculatory disturbances.6 In general, MP using Polysol provided us with better reperfusion results compared to both CS in UW and MP using UW-G. Validation of these results will be carried out in a pig liver preservation and transplantation model. In conclusion, 24-hour machine perfusion preservation of NHBD rat livers using the newly developed preservation solution Polysol results in less hepatocellular damage and better liver function compared to cold storage in UW or machine perfusion using UW-G. ACKNOWLEDGMENTS We thank Dr A.J. Meijer from the Department of Medical Biochemistry at the Academic Medical Center for his contribution to this study.
REFERENCES 1. D’alessandro AM, Hoffmann RM, Knechtle SJ, et al: Liver transplantation from controlled non-heart-beating donors. Surgery 128:579, 2000 2. Kootstra G, Daemen JH, Oomen AP: Categories of nonheart-beating donors. Transplant Proc 27:2893, 1995 3. Southard JH, Lindell S, Ametani M, et al: Kupffer cell activation in liver preservation: cold storage vs machine perfusion. Transplant Proc 32:27, 2000 4. Wight J, Chilcott J, Holmes M, et al: The clinical and cost-effectiveness of pulsatile machine perfusion versus cold stor-
Energy substrates Impermeants
Amino acids Vitamins pH indicator
UW
UW-G
Polysol
HES
HES
PEG
KH2PO4
KH2PO4 HEPES
Allopurinol Glutathion
Allopurinol Glutathion
KH2PO4 HEPES Histidine Allopurinol Glutathion Alpha-tocopherol Ascorbic acid Glucose
Glucose Lactobionate Na-Gluconate K-Gluconate Mg-Gluconate Raffinose Raffinose
Na-Gluconate K-Gluconate Trehalose Raffinose
— — —
Miscellaneous Miscellaneous Phenol-red
— — —
The most important differences in composition of the three preservation solutions are mentioned in this table. All three solutions have an osmolality of 320 –340 mOsm and a pH of 7.4. The total amount of components in these solutions is 13 (UW), 14 (UW-G) and 61 (Polysol).
age of kidneys for transplantation retrieved from heart-beating and non-heart-beating donors. Health Technol Assess 7:1, 2003 5. Xu H, Lee CY, Clemens MG, et al: Prolonged hypothermic machine perfusion preserves hepatocellular function but potentiates endothelial cell dysfunction in rat livers. Transplantation 77:1676, 2004 6. Morariu AM, Vd PA, Oeveren V, et al: Hyperaggregating effect of hydroxyethyl starch components and University of Wisconsin solution on human red blood cells: a risk of impaired graft perfusion in organ procurement? Transplantation 76:37, 2003 7. Bessems M, Doorschodt BM, Hooijschuur O, et al: Liver preservation by hypothermic, continuous machine perfusion using a new colloid based preservation solution. Gut 52(supplVI):160A, 2003 8. Guarrera JV, Polyak M, O’Mar AB, et al: Pulsatile machine perfusion with Vasosol solution improves early graft function after cadaveric renal transplantation. Transplantation 77:1264, 2004 9. van der Vliet JA, Kievit JK, Hene RJ, et al: Preservation of non-heart-beating donor kidneys: a clinical prospective randomised case-control study of machine perfusion versus cold storage. Transplant Proc 33:847, 2001 10. Manzarbeitia CY, Ortiz JA, Jeon H, et al: Long-term outcome of controlled, non-heart-beating donor liver transplantation. Transplantation 78:211, 2004 11. Lee CY, Jain S, Duncan HM, et al: Survival transplantation of preserved non-heart-beating donor rat livers preservation by hypothermic machine perfusion. Transplantation 76:1432, 2003