Fujita H, Kuroda Y, Saitoh Y
Department of Surgery, Kobe University School of Medicine.
Kobe J Med Sci. 1995 Jun;41(3):47-61.
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.
我们已经证明，通过双层法在保存过程中对胰腺进行氧合可使ATP持续产生，以维持细胞完整性，并延长胰腺保存活力的时间。本研究的目的是使用线粒体氧化磷酸化解偶联剂2,4-二硝基苯酚，研究ATP与氧合本身对非实质细胞（血管内皮）活力的影响。使用体重为12-18kg的杂种犬，雌雄不限。在全身麻醉下，进行胰腺左叶切除术。节段性胰腺移植物立即自体移植（第1组；对照组）或在48小时保存后自体移植（第2组；使用欧-柯林斯溶液[EC]的简单冷藏法，第3组；使用EC的双层冷藏法，第4组；使用含0.2 mM DNP的EC的双层冷藏法），并在自体移植时切除胰腺的其余部分。通过移植后的移植物存活情况判断移植物活力。移植后2周静脉葡萄糖耐量试验的K值大于1.0被认为移植物存活。使用高效液相色谱法在保存后测定ATP的组织浓度。使用锥虫蓝灌注/固定试验在保存后检查血管内皮的活力。锥虫蓝在伊红复染切片中的核染色表明细胞活力丧失。在再灌注30至240分钟后，还使用H2清除技术测量反映胰腺微循环的胰腺组织灌注。第1、2、3和4组的移植物存活率分别为5/5、0/4、4/4和0/3。与第2组相比，第3组的ATP组织浓度显著更高（7.91±1.21[n = 4]对1.21±0.31[n = 4]μmol/g干重，p < 0.01）。DNP导致第4组的组织ATP显著降低（0.61±0.07[n = 3]对7.91±1.21[n = 4]μmol/g干重，p < 0.01）。第3组非实质细胞核锥虫蓝摄取百分比显著低于第2组（11.29±3.71[n = 3]对26.41±1.66[n = 3]%，p < 0.01），并且DNP（第4组）增加了锥虫蓝摄取（30.10±4.08[n = 3]对11.29±3.71[n = 3]%，p < 0.01）。第3组再灌注2小时后的组织灌注显著高于第2组（68.64±8.62[n = 5]对45.56±12.84[n = 5]ml/min/100g，p < 0.01）。此外，DNP（第4组）导致胰腺组织灌注显著降低（28.84±9.09[n = 5]对68.64±8.62[n = 5]ml/min/100g，p < 0.001）。很明显，双层法（第3组）保护微血管内皮免受冷缺血损伤，而使用DNP抑制ATP产生（第4组）导致内皮损伤、再灌注后微循环紊乱并因此导致移植物活力丧失。我们得出结论，通过双层法在保存过程中维持ATP组织水平可保护胰腺移植物的微血管内皮免受冷缺血损伤。这是双层法成功保存胰腺作用机制之一。
