title: The mechanism of action of the two-layer cold storage method in canine pancreas preservation--protection of pancreatic microvascular endothelium.
abstract: We have demonstrated that oxygenation of a pancreas during preservation by a two-layer method leads continued ATP production to maintain cellular integrity and produces an extended period of preserved pancreatic viability. The aim of this study is to examine the effect of ATP vs. oxygenation per se on viability of nonparenchymal cell (vascular endothelium), using 2.4 dinitrophenol, an uncoupler of mitochondrial oxidative phosphorylation. Mongrel dogs of both sexes, weighing 12-18kg were used. Under general anesthesia, a left lobectomy of the pancreas was performed. The segmental pancreas graft was autotransplanted immediately (group 1; control) or after 48-hour preservation (group 2; simple cold storage method with Euro-Collins' solution [EC], group 3; two-layer cold storage method using EC, group 4; two-layer cold storage method using EC with 0.2 mM DNP), and the remainder of the pancreas was excised at the time of autotransplantation. Graft viability was judged from graft survival after transplantation. A K-value of intravenous glucose tolerance test more than 1.0 at 2 weeks after transplantation was considered graft survival. Tissue concentration of ATP was determined after preservation using high-performance liquid chromatography. Viability of vascular endothelium was examined using trypan-blue perfusion/fixation test after preservation. Nuclear staining by trypan blue in eosin-counterstaining sections was indicative of loss of cell viability. Pancreatic tissue perfusions, which reflect pancreatic microcirculation, were also measured using H2 clearance technique after 30 to 240 min of reperfusion. Graft survival rates in groups 1, 2, 3 and 4 were 5/5, 0/4, 4/4 and 0/3 respectively. ATP tissue concentration was significantly higher in group 3 compared with group 2 (7.91 +/- 1.21 [n = 4] vs. 1.21 +/- 0.31 [n = 4] mumol/g dry weight, p < 0.01). DNP caused a significant decrease in tissue ATP in group 4 (0.61 +/- 0.07 [n = 3] vs. 7.91 +/- 1.21 [n = 4] mumol/g dry weight, p < 0.01). The percentage of nuclear trypan blue uptake of nonparenchymal cells in group 3 was significantly lower than group 2 (11.29 +/- 3.71 [n = 3] vs. 26.41 +/- 1.66 [n = 3] %, p < 0.01), and DNP (group 4) increased trypan blue uptake (30.10 +/- 4.08 [n = 3] vs. 11.29 +/- 3.71 [n = 3] %, p < 0.01). Tissue perfusions after 2hr-reperfusion in group 3 were significantly higher than group 2 (68.64 +/- 8.62 [n = 5] vs. 45.56 +/- 12.84 [n = 5] ml/min/100g, p < 0.01). Moreover, DNP (group 4) caused a significant decrease in pancreatic tissue perfusions (28.84 +/- 9.09 [n = 5] vs. 68.64 +/- 8.62 [n = 5] ml/min/100g. p < 0.001). It was clear that the two-layer method (group 3) protected microvascular endothelium against cold ischemic damage and inhibition of ATP production using DNP (group 4) caused endothelial damage, microcirculatory disturbance after reperfusion and consequently loss of graft viability. We conclude that microvascular endothelium of the pancreas graft is protected against cold ischemic injury by maintaining ATP tissue levels during preservation by the two-layer method. This is one of the mechanisms of action of the two-layer method in successful pancreas preservation.
authors.affiliation: Department of Surgery, Kobe University School of Medicine.
authors.forename: H
authors.initials: H
authors.lastname: Fujita
authors.forename: Y
authors.initials: Y
authors.lastname: Kuroda
authors.forename: Y
authors.initials: Y
authors.lastname: Saitoh
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis / Q000096:biosynthesis*; D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D004195:Disease Models, Animal; D004285:Dogs; D004730:Endothelium, Vascular / Q000502:physiology*; D005260:Female; D006085:Graft Survival / Q000502:physiology; D008297:Male; D010179:Pancreas; D016035:Pancreas Transplantation / Q000379:methods
chemical_list: D000255:Adenosine Triphosphate