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Successful use of ocean pout thermal hysteresis protein (antifreeze protein III) in cryopreservation of transplanted mammalian heart at subzero temperature
The Journal of Heart and Lung Transplantation Volume 21, Number 1
Abstracts
Results:There was a significant increase in peak concentrations of CRAB in month 2 and months 2 and 3 combined (p⫽0.009) (Table 1)associated with rejection. Although ␣-tropomyosin was also increased in month 2 during rejection (366⫹/-115 compared to 82⫹/-29) this did not reach statistical significance. MLC1 was not detected in human serum. Conclusion: Myocardial proteins can be detected in the circulation and are elevated during acute rejection; whether this is of clinical use remains to be seen. Month 1, Month 2, Month 3, Months 2 and 3, mean conc. mean conc. mean conc. mean conc. (SE) (SE) (SE) (SE) Rejection 174 (81) Non-rejection 147 (114)
465.9 (131) 42.5 (21)
206.3 (93) 75 (39)
511.4 (125) 87.6 (46)
228 SUCCESSFUL USE OF OCEAN POUT THERMAL HYSTERESIS PROTEIN (ANTIFREEZE PROTEIN III) IN CRYOPRESERVATION OF TRANSPLANTED MAMMALIAN HEART AT SUBZERO TEMPERATURE G. Amir,1 B. Rubinsky,2 A.K. Smolinsky,1 J. Lavee,1 1Heart Transplantation Unit, Department of Cardiac Surgery, Sheba Medical Center, Tel Hashomer, Israel; 2Biomedical Engineering Laboratory, Department of Mechanical Engineering, University of California, Berkeley, Ca Introduction: Freeze tolerant ocean pouts are able to survive subzero temperatures by noncolligatively lowering the freezing temperature of their body fluids using thermal hysteresis proteins (antifreeze proteins, AFP III). We report the successful subzero cryopreservation of mammalian hearts using the ocean pout AFP III. Methods: Heterotopic heart transplantations were performed in Sprague Dawly isoimmunic rats weighing 250-350 mg. Eight hearts were arrested using UW solution and antrogradly perfused with a preservation solution containing AFP III in UW solution. The harvested hearts were preserved for 2-4 hours at subzero temperatures ranging from -0.6°C to -1.8°C. Preservation solution was flushed out using iced (4°C) UW solution with nitroglycerine. Control group of 4 hearts were preserved using UW soultion only at -1.5°C for 2 hours. Viability of the transplanted hearts was visually assessed and scored on a scale of 1-6 (1- poor contraction, 6- excellent contraction). Results: All eight hearts preserved at subzero temperatures using AFP III survived. All four control hearts preserved without AFP III froze and died upon reperfusion. Results are summarized in the table. AFP III concentration
Temp (°C)/Time
3 mg/ml 5 mg/ml 5 mg/ml 5 mg/ml 15 mg/ml 15 mg/ml 15 mg/ml 15 mg/ml
⫺0.6/4 hours ⫺0.6/30 min ⫺0.6/30 min ⫺0.6/3.5 hours ⫺1.5/2 hours ⫺1.5/2 hours ⫺1.8/2 hours ⫺1.8/2 hours
Viability score (1–6) 4 3 4 4 4 4 4 5
Conclusions: Subzero cryopreservation of transplanted hearts using AFP III is possible. Further studies are ongoing to determine whether subzero cryopreservation will enable prolongation of ischemic times while providing good hemodynamic performance.
137
229 VENTILATION WITH NITRIC OXIDE DURING PERFUSION IMPROVES POST-TRANSPLANTATION FUNCTION OF THE NON-HEART-BEATING DONOR LUNG J.D. Aitchison,1 H.E. Orr,2 P.A. Flecknell,2 J.A. Kirby,3 J.H. Dark,1 1Cardiopulmonary Transplantation, Freeman Hospital, Newcastle upon Tyne, United Kingdom; 2Comparative Biology Centre, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom; 3Department of Surgery, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom Aim. We aimed to determine whether inhaled nitric oxide (NO) would ameliorate warm ischaemic lung injury in the Non-HeartBeating-Donor (NHBD), thereby improving function after longer warm ischaemic intervals prior to retrieval, and studied this in our existing pig NHBD lung transplant model.1 Methods. All animal use conformed with “Guiding Principles in the Care and Use of Laboratory Animals”, 1991. Juvenile pigs (groups n⫽6) of weight ⬇50kg were anaesthetised throughout and euthanased without regaining consciousness. Donor lungs were retrieved 1 (N1) or 2 (N2, N2NO) hours post hypoxic death. Left lungs were ventilated with 100% oxygen, with 20ppm NO added for the N2NO group. Perfusion was with deoxygenated, neutrophil-depleted blood and Perfadex; left lungs were then transplanted, total ischaemic times N1 8.1h, N2 9.1h, N2NO 9.3h. Our model allows independent ventilation of the transplanted left lung with 100% oxygen, selective pulmonary arterial and venous sampling and independent pulmonary blood flow measurement over a 12-hour follow-up. Results. Addition of NO to the ventilating oxygen during perfusion significantly improved post-transplant pulmonary venous oxygen tension; time-weighted means N1 51⫾s.d. 14kPa, N2 54⫾16, N2NO 61⫾6: p⫽0.01 (ANOVA). Transplant pulmonary vascular resistance was non-significantly improved; N1 19⫾7 Wood units, N2 29⫾24, N2NO 17⫾6: p⫽0.31. Calculated absolute neutrophil uptake was significantly lowered by NO; N1 0.6⫾1.4 *109per minute, N2 1.2⫾1.0, N2NO 0.4⫾0.9: p⫽0.029 (ANOVA). Conclusions. Adding nitric oxide to ventilation during blood and perfadex perfusion of the NHBD lung significantly improves post-transplant function. NO is associated with a decreased neutrophil uptake post transplantation. This technique offers another source of satisfactory organs for transplantation. 1EurJCTSurg 2001;20(1):187-194. 230 DONOR BRAIN DEATH INTENSIFIES THE RECIPIENT INFLAMMATORY RESPONSE IN CHRONIC RAT CARDIAC ALLOGRAFT REJECTION M.J. Wilhelm,1,2 J. Pratschke,1 F. Beato,1 M. Taal,1 C. Schmid,2 H.H. Scheld,2 W.W. Hancock,3 N.L. Tilney,1 1Surgical Research Laboratory, Harvard Medical School, Boston, MA; 2Department of Cardiothoracic Surgery, University of Muenster, Muenster, Germany; 3Millennium Pharmaceuticals, Inc., Cambridge, MA Introduction: It becomes increasingly evident that brain death may play a role as a donor associated risk factor. The purpose of this study was to investigate the effect of donor brain death on long-term intragraft changes in a model of chronic rat cardiac allograft rejection.
Abstracts
Results:There was a significant increase in peak concentrations of CRAB in month 2 and months 2 and 3 combined (p⫽0.009) (Table 1)associated with rejection. Although ␣-tropomyosin was also increased in month 2 during rejection (366⫹/-115 compared to 82⫹/-29) this did not reach statistical significance. MLC1 was not detected in human serum. Conclusion: Myocardial proteins can be detected in the circulation and are elevated during acute rejection; whether this is of clinical use remains to be seen. Month 1, Month 2, Month 3, Months 2 and 3, mean conc. mean conc. mean conc. mean conc. (SE) (SE) (SE) (SE) Rejection 174 (81) Non-rejection 147 (114)
465.9 (131) 42.5 (21)
206.3 (93) 75 (39)
511.4 (125) 87.6 (46)
228 SUCCESSFUL USE OF OCEAN POUT THERMAL HYSTERESIS PROTEIN (ANTIFREEZE PROTEIN III) IN CRYOPRESERVATION OF TRANSPLANTED MAMMALIAN HEART AT SUBZERO TEMPERATURE G. Amir,1 B. Rubinsky,2 A.K. Smolinsky,1 J. Lavee,1 1Heart Transplantation Unit, Department of Cardiac Surgery, Sheba Medical Center, Tel Hashomer, Israel; 2Biomedical Engineering Laboratory, Department of Mechanical Engineering, University of California, Berkeley, Ca Introduction: Freeze tolerant ocean pouts are able to survive subzero temperatures by noncolligatively lowering the freezing temperature of their body fluids using thermal hysteresis proteins (antifreeze proteins, AFP III). We report the successful subzero cryopreservation of mammalian hearts using the ocean pout AFP III. Methods: Heterotopic heart transplantations were performed in Sprague Dawly isoimmunic rats weighing 250-350 mg. Eight hearts were arrested using UW solution and antrogradly perfused with a preservation solution containing AFP III in UW solution. The harvested hearts were preserved for 2-4 hours at subzero temperatures ranging from -0.6°C to -1.8°C. Preservation solution was flushed out using iced (4°C) UW solution with nitroglycerine. Control group of 4 hearts were preserved using UW soultion only at -1.5°C for 2 hours. Viability of the transplanted hearts was visually assessed and scored on a scale of 1-6 (1- poor contraction, 6- excellent contraction). Results: All eight hearts preserved at subzero temperatures using AFP III survived. All four control hearts preserved without AFP III froze and died upon reperfusion. Results are summarized in the table. AFP III concentration
Temp (°C)/Time
3 mg/ml 5 mg/ml 5 mg/ml 5 mg/ml 15 mg/ml 15 mg/ml 15 mg/ml 15 mg/ml
⫺0.6/4 hours ⫺0.6/30 min ⫺0.6/30 min ⫺0.6/3.5 hours ⫺1.5/2 hours ⫺1.5/2 hours ⫺1.8/2 hours ⫺1.8/2 hours
Viability score (1–6) 4 3 4 4 4 4 4 5
Conclusions: Subzero cryopreservation of transplanted hearts using AFP III is possible. Further studies are ongoing to determine whether subzero cryopreservation will enable prolongation of ischemic times while providing good hemodynamic performance.
137
229 VENTILATION WITH NITRIC OXIDE DURING PERFUSION IMPROVES POST-TRANSPLANTATION FUNCTION OF THE NON-HEART-BEATING DONOR LUNG J.D. Aitchison,1 H.E. Orr,2 P.A. Flecknell,2 J.A. Kirby,3 J.H. Dark,1 1Cardiopulmonary Transplantation, Freeman Hospital, Newcastle upon Tyne, United Kingdom; 2Comparative Biology Centre, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom; 3Department of Surgery, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom Aim. We aimed to determine whether inhaled nitric oxide (NO) would ameliorate warm ischaemic lung injury in the Non-HeartBeating-Donor (NHBD), thereby improving function after longer warm ischaemic intervals prior to retrieval, and studied this in our existing pig NHBD lung transplant model.1 Methods. All animal use conformed with “Guiding Principles in the Care and Use of Laboratory Animals”, 1991. Juvenile pigs (groups n⫽6) of weight ⬇50kg were anaesthetised throughout and euthanased without regaining consciousness. Donor lungs were retrieved 1 (N1) or 2 (N2, N2NO) hours post hypoxic death. Left lungs were ventilated with 100% oxygen, with 20ppm NO added for the N2NO group. Perfusion was with deoxygenated, neutrophil-depleted blood and Perfadex; left lungs were then transplanted, total ischaemic times N1 8.1h, N2 9.1h, N2NO 9.3h. Our model allows independent ventilation of the transplanted left lung with 100% oxygen, selective pulmonary arterial and venous sampling and independent pulmonary blood flow measurement over a 12-hour follow-up. Results. Addition of NO to the ventilating oxygen during perfusion significantly improved post-transplant pulmonary venous oxygen tension; time-weighted means N1 51⫾s.d. 14kPa, N2 54⫾16, N2NO 61⫾6: p⫽0.01 (ANOVA). Transplant pulmonary vascular resistance was non-significantly improved; N1 19⫾7 Wood units, N2 29⫾24, N2NO 17⫾6: p⫽0.31. Calculated absolute neutrophil uptake was significantly lowered by NO; N1 0.6⫾1.4 *109per minute, N2 1.2⫾1.0, N2NO 0.4⫾0.9: p⫽0.029 (ANOVA). Conclusions. Adding nitric oxide to ventilation during blood and perfadex perfusion of the NHBD lung significantly improves post-transplant function. NO is associated with a decreased neutrophil uptake post transplantation. This technique offers another source of satisfactory organs for transplantation. 1EurJCTSurg 2001;20(1):187-194. 230 DONOR BRAIN DEATH INTENSIFIES THE RECIPIENT INFLAMMATORY RESPONSE IN CHRONIC RAT CARDIAC ALLOGRAFT REJECTION M.J. Wilhelm,1,2 J. Pratschke,1 F. Beato,1 M. Taal,1 C. Schmid,2 H.H. Scheld,2 W.W. Hancock,3 N.L. Tilney,1 1Surgical Research Laboratory, Harvard Medical School, Boston, MA; 2Department of Cardiothoracic Surgery, University of Muenster, Muenster, Germany; 3Millennium Pharmaceuticals, Inc., Cambridge, MA Introduction: It becomes increasingly evident that brain death may play a role as a donor associated risk factor. The purpose of this study was to investigate the effect of donor brain death on long-term intragraft changes in a model of chronic rat cardiac allograft rejection.