Yasuhiro Iwanaga

Ultrastructural analyses of pancreatic grafts preserved by the two-layer cold-storage method and by simple cold storage in University of Wisconsin solution

Abstract The two‐layer cold storage method (TLM) using University of Wisconsin (UW) solution supplies sufficient oxygen to pancreatic grafts during preservation and extends pancreas preservation time to up to 96h in the canine model. Simple cold storage in UW (UWM) on the other hand, preserves canine pancreas grafts for up to 72h by preventing cell swelling, mainly because of its high osmotic pressure. The aim of this study is to analyze morphologically dog pancreatic grafts preserved by these two methods with their different mechanisms. Immediately after preservation of canine pancreata by TLM for 72h and 96h (group 1 and group 3, respectively), and by UWM for 72h and 96h (group 2 and group 4, respectively), tissue ATP levels were determined using high‐performance liquid chromatography (HPLC), and detailed morphological analyses of intragraft components were performed using light‐ and electron microscopy. The mean areas of one mitochondrion and rough endoplasmic reticulum (PER) vacuolization were calculated by computer‐graphic analyses using NIH image 1.62f soft. The tissue ATP levels were significantly higher in groups 1 and 3 than groups 2 and 4 (P<0.05). Light microscopy demonstrated no marked difference among the 4 groups. By electron microscopy however, mitochondrial swelling and RER vacuolization were observed in acinar cells to various extents in the 4 groups. They were significantly more evident in group 2 than group 1 (P<0.05), and in group 4 than group 3 (P<0.05). In conclusion, TLM demonstrated excellent protection of intracellular organelles, mitochondria, and RER, up to 72–96h. Well‐maintained graft ATP levels in TLM groups may result in maintaining the integrity of intracellular organelle membranes as well as cellular membranes.

An Experimental Study on the Pharmacokinetics of 5-Fluorouracil Regional Chemotherapy for Pancreatic Cancer

Background: Recently a few centers reported promising results of regional intra-arterial chemotherapy for pancreatic cancer. However, the detailed pharmacokinetics and the side effects of anticancer agents remain unclear.Methods: Catheters were introduced into the gastroduodenal artery and the splenic artery of dogs. In group I, arterial infusion of 5-fluorouracil (5-FU) was performed over 10 minutes. In group II, 5-FU was infused systemically. In group III, an intra-arterial infusion was repeated weekly three times. Blood samples and liver and pancreas tissue samples were obtained to determine 5-FU levels. In a subset of each group, the pancreas, duodenum, and liver were excised for histological analyses.Results: Immediately after the infusion of 5-FU, the portal level in group I was higher than that in group II. However, the mean systemic level in group I was lower than in group II. The mean tissue concentration in the pancreas in group I was significantly higher than that of group II. Histological examination revealed no microscopic alterations after treatment in all groups, including group III.Conclusions: This fundamental study suggested that intra-arterial chemotherapy of 5-FU for pancreatic cancer allows higher regional drug delivery without adverse effects on normal regions of the pancreas, the duodenum, and the liver.

Superiority of Mild Hypothermic (20??C) Preservation for Pancreatic Microvasculature Using the Two-Layer Storage Method

Hypothermia causes vascular endothelial damage that leads to graft microcirculation disorder and eventually thrombosis after reperfusion. The two-layer cold storage method (TL) was previously demonstrated to supply oxygen to the pancreas graft and maintain high adenosine triphosphate tissue concentration. In this study, we evaluated whether mild hypothermic (20°C) preservation using the TL method could reduce endothelial damage while maintaining parenchymal viability. Graft survival by 20°C preservation was investigated using a dog segmental pancreas autotransplantation model (simple storage in University of Wisconsin solution (UW) for 5 and 8 hours or TL for 5, 8, 12, and 24 hrs, respectively). Subsequently, the grafts were preserved in four different conditions (4 and 20°C UW, 4 and 20°C TL) for 8 hours to evaluate microvascular endothelial damage. Trypan blue uptake of vascular endothelium and pancreatic tissue perfusion were evaluated. No graft preserved by 20°C UW for 5 and 8 hours survived (0/7 and 0/4). In contrast, the graft survival rates by 20°C TL for 5, 8, 12, and 24 hours were 100% (5/5), 80% (4/5), 20% (1/5), and 0% (0/4), respectively. In trypan blue uptake analysis, there were significant differences between 4 and 20°C in both UW and TL (4°C UW, 37% [n = 5) vs. 20°C UW, 13% [n = 4] [p < 0.01]; 4°C TL, 29% [n = 5] vs. 20°C TL, 10% [n = 5] [p < 0.01]). The perfusion values in 20°C TL were significantly higher than those in other groups at least for up to 120 minutes after reperfusion (p < 0.01). In short-term pancreas preservation, mild hypothermic TL reduced vascular endothelial cell damage and ameliorated graft microcirculation while maintaining parenchymal viability. Mild hypothermic TL may lessen vascular complications in clinical pancreas transplantation when used for several-hour preservation.