The analyses were performed on bile samples from porcine model donors with mild (heart beating donor [HBD]) and moderate warm ischemia (donation after circulatory death [DCD]) grafts. The obtained livers were subjected to 7 h SCS or NEVLP before transplantation. The SCS group consisted of two subgroups (5 animals in each group): HBD (HBD-SCS) livers and DCD livers with 30 min ischemia time (30′DCD-SCS). The livers from the NEVLP groups (HBD/30′DCD/60′DCD/90′DCD-NEVLP (5 animals in each group)) were stored at 4 °C in histidine-tryptophan-ketoglutarate (HTK) solution during the back-table preparation for ex vivo perfusion and were subsequently subjected to 5 h NEVLP at 37 °C. The livers from the SCS and NEVLP groups were subjected to a preservation time of 7 h, followed by transplantation into the recipient pigs. The recipient pigs were followed for a survival period of 4 days. Bile samples were collected during the peri-transplant period at the time points shown in Figure 7 . The research material was provided by scientists from the Department of Surgery at the Toronto General Hospital (University Health Network, Toronto, ON, Canada).
The HBD pigs received heparin at 500 international units/kg of body weight 5 min prior to cross-clamping and cold flushing. In the case of the DCD grafts, the donor pigs received the same dose of heparin 5 min prior to the induction of cardiac arrest, which was accomplished by the intracardiac infusion of potassium chloride (20 mEq). After the induction of cardiac arrest, the desired warm ischemia time was awaited according to the protocol for the respective DCD model (30 min, 60 min or 90 min). Subsequently, all livers were flushed with a total volume of 3 L cold (4 °C) Custodiol-HTK (Essential Pharmaceuticals, LCC, Ewing, NJ, USA) through the aorta and portal vein. In the SCS groups, the livers were packed in bags filled with Custodiol-HTK and then stored in an icebox (4 °C) for 7 h; in the NEVLP-groups, the livers were cannulated and prepared for perfusion on ice (4 °C). To ensure the preservation time was comparable for all experiments, livers from the NEVLP groups were stored on ice for 2 h before being perfused for 5 h at 37 °C. After 5 h of NEVLP, the livers were flushed with cold (4 °C) Custodiol-HTK and then stored on ice before implantation was performed. Following SCS and NEVLP, the grafts were transplanted into recipient pigs using the method described in [44 (link),45 (link)]. Animals were euthanized under deep anesthesia on postoperative day 4.
The HBD pigs received heparin at 500 international units/kg of body weight 5 min prior to cross-clamping and cold flushing. In the case of the DCD grafts, the donor pigs received the same dose of heparin 5 min prior to the induction of cardiac arrest, which was accomplished by the intracardiac infusion of potassium chloride (20 mEq). After the induction of cardiac arrest, the desired warm ischemia time was awaited according to the protocol for the respective DCD model (30 min, 60 min or 90 min). Subsequently, all livers were flushed with a total volume of 3 L cold (4 °C) Custodiol-HTK (Essential Pharmaceuticals, LCC, Ewing, NJ, USA) through the aorta and portal vein. In the SCS groups, the livers were packed in bags filled with Custodiol-HTK and then stored in an icebox (4 °C) for 7 h; in the NEVLP-groups, the livers were cannulated and prepared for perfusion on ice (4 °C). To ensure the preservation time was comparable for all experiments, livers from the NEVLP groups were stored on ice for 2 h before being perfused for 5 h at 37 °C. After 5 h of NEVLP, the livers were flushed with cold (4 °C) Custodiol-HTK and then stored on ice before implantation was performed. Following SCS and NEVLP, the grafts were transplanted into recipient pigs using the method described in [44 (link),45 (link)]. Animals were euthanized under deep anesthesia on postoperative day 4.
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