Yasuki Tanioka

Clinical application of the two-layer (University of Wisconsin solution/perfluorochemical plus O2) method of pancreas preservation before transplantation.

Background. The two-layer method [University of Wisconson solution (UW)/perfluorochemical plus O 2] for pancreas preservation has been demonstrated to be superior to simple UW storage alone in the canine model.For the first time, we applied the two-layer method to clinical whole-pancreas transplantation. Methods. Pancreases were placed in the two-layer method in 10 cases and UW alone in 44 cases before transplant. The mean cold ischemic time was 16.5 hr in the two-layer group versus 18.1 hr in the UW group (P =NS). We compared the condition of graft at the time of reperfusion, and then 3 months posttransplant graft function and complications. Results. At the time of reperfusion, no grafts in the two-layer group were edematous, compared with 10(23.3%) of 43 in the UW group (P =0.18). Seven (70%) of 10 grafts in the two-layer group obtained the best overall quality score, compared with 24(57.1%) of 42 in the UW group (P =0.72). Nine (90%) of 10 recipients in the two-layer group became insulin-independent during hospitalization, compared with 31(70.5%) of 44 in the UW group (P =0.26). Time to insulin independence was no different between the two groups. No pancreas grafts preserved by the two-layer method suffered acute rejection. Conclusions. The two-layer preservation method is feasible in human clinical transplantation. It was at least equivalent and may be superior to UW alone in both morphologic and functional assessment of the transplanted pancreas.

Small bowel preservation using a cavitary two-layer (University of Wisconsin solution/perfluorochemical) cold storage method.

Preservation of the small bowel by a cavitary two-layer (University of Wisconsin solution [UW]/perfluorochemical) cold storage method was evaluated in the heterotopic rat segmental small bowel transplant model. Simple cold storage UW was effective only for 24-hr preservation. However, the cavitary two-layer method using UW made it possible to prolong preservation time up to 48 hr. Furthermore, without oxygen bubbling, the cavitary two-layer method was not effective for 48-hr preservation. Histologic studies of grafts preserved by the cavitary two-layer method for 48 hr at 7 days after transplantation showed normal architecture of the intestinal mucosa. This study demonstrates that the oxygenation of the small bowel during preservation by the cavitary two-layer method using UW makes it possible to extend preservation time up to 48 hr in the heterotopic rat segmental small bowel transplant model.

Pancreas preservation by the 2-layer cold storage method before islet isolation protects isolated islets against apoptosis through the mitochondrial pathway

BACKGROUND Apoptosis in isolated islets has been implicated in primary nonfunction or early graft failure after islet transplantation. Recently, pancreas preservation by the 2-layer method (TLM) before islet isolation has been proved to improve the islet yield, quality, and transplant results not only in experimental models, but also in clinical settings. We examined the influence of TLM on apoptosis of isolated islets. METHOD Rat islets freshly isolated and after pancreas preservation by TLM or conventional cold storage in University of Wisconsin solution (UW) were examined and compared. Islet apoptosis was assessed by TUNEL and annexin V assays. The apoptosis pathways involved were investigated by measurement of caspase 3, 8, and 9 activities and by immunoblotting for total and phosphorylated c-Jun NH2-terminal kinase (JNK) and p38. RESULTS Islet apoptosis in the UW group was significantly increased compared with the fresh and TLM groups. Both caspase 3 and 9 activities in the UW group were higher than in the fresh and TLM groups with an approximate increase of 2- to 3-fold. On the other hand, there was no significant difference in caspase 8 activity among these 3 groups. JNKs were strongly activated both in the TLM and UW groups; although they were not activated in the fresh group, p38 was activated to almost the same levels in these 3 groups. CONCLUSIONS Pancreas preservation by TLM before islet isolation protects isolated islets against apoptosis mainly through the mitochondrial pathway. Pancreas storage before islet isolation even with TLM triggers activation of JNKs in isolated islets.

The effect of ouabain (inhibitor of an ATP-dependent Na+/K+ pump) on the pancreas graft during preservation by the two-layer method

To clarify how synthesized ATP is utilized to maintain cellular integrity during preservation by the two-layer method, we examined the effect of ouabain, inhibitor of ATP dependent Na+/K+ pump, on ATP tissue levels, graft weight, and vascular endothelial cells during 48 hr preservation by the two-layer method and pancreatic tissue perfusion and graft survival after transplantation in a canine model. Ouabain treatment did not affect ATP production but prevented its utilization by the sodium pump, and actually significantly increased ATP levels and decreased the weight loss of the graft. In addition, ouabain caused a significant increase of nuclear trypan-blue staining in vascular endothelial cells and a significant decrease of pancreatic tissue perfusion at reperfusion. Consequently, the grafts did not survive. We conclude that the two-layer method allows sufficient synthesis of ATP to drive the sodium pump and maintains membrane integrity of parenchymal cells and vascular endothelial cells, thus extending the period of preserved pancreatic viability.

Successful extended preservation of ischemically damaged pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) cold storage method.

We have demonstrated that a two-layer (University of Wisconsin solution [UW]/perfluorochemical [PFC]) cold storage method restores the function of ischemically damaged pancreas during 24-hr preservation in canine autotransplantation model. The purpose of this study was to examine the possibility of a long-term preservation of the ischemically damaged pancreas by the two-layer (UW/PFC) method. After 60 or 90 min of warm ischemic time, pancreas grafts were preserved by the two-layer (UW/PFC) method or a simple cold storage in UW alone for up to 96 hr. A K value of i.v. glucose tolerance test more than 1.0 2 weeks after autotransplantation was considered successful preservation. After 60 min warm ischemia, limitation of preservation time by the simple cold storage in UW was 24 hr (5/5 100% and 0/5 0%; 24- and 48-hr preservation, respectively). However, the two-layer method made it possible to extend the preservation time up to 48 hr (5/5 100%, 5/5 100%, 2/5 40%, and 0/5 0%; 24-, 48-, 72-, and 96-hr preservation, respectively). After 90 min warm ischemia, the simple cold storage in UW was not effective even for 24-hr preservation (0/5 0%). However, 48-hr preservation was successful by the two-layer (UW/PFC) method (5/5 100%, 5/5 100%, and 0/5 0%; 24-, 48-, and 72-hr-preservation, respectively). After preservation by the two-layer (UW/PFC) method, ATP tissue concentrations of viable grafts were significantly higher compared with nonviable grafts (9.11 +/- 3.05 (n = 22) versus 5.22 +/- 1.02 (n = 13) mumol/g dry wt, P < 0.001). Based on analysis of individual ATP for each graft, if an ATP concentration of 6.0 mumol/g dry weight was determined as a critical value for doing the transplant, sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 84.6%, 91.7%, and 94.3%, respectively. This study clearly demonstrated that 48-hr preservation of the canine pancreas subjected to either 60 or 90 min warm ischemia was successfully achieved by the two-layer (UW/PFC) cold storage method, and ATP tissue concentration at the end of preservation by this method would predict the post-transplant outcome of the ischemically damaged pancreas just prior to transplantation.

Role of adenosine in preservation by the two-layer method of ischemically damaged canine pancreas

The purpose of this study was to clarify the role of adenosine in preservation of ischemically damaged pancreas by the two-layer (Euro-Collins solution [EC]/perfluorochemical [PFC]) method using a canine model. Twenty-four-hour preservation of the pancreas graft subjected to 60-min warm ischemia was successful by the two-layer (EC with adenosine/PFC) method (4/5, 80%), but neither simple cold storage in EC (0/5, 0%), nor EC with adenosine (1/5, 20%), nor the two-layer (EC/PFC) method (0/3, 0%) was successful. Tissue ATP concentrations at the end of preservation by the two-layer (EC with adenosine/PFC) method were significantly higher compared with the two-layer (EC/PFC) method (7.23 +/- 2.17 vs. 1.56 +/- 0.40 mumol/g dry weight, P < 0.01). Studies with [2-3H]adenosine demonstrated that only part of adenosine was converted to inosine, hypoxanthine, and adenine, whereas the remainder was incorporated into adenine nucleotides in the pancreas graft. In addition, hypoxanthine, inosine, and adenine did not substitute for adenosine. We conclude that provision of adenosine to ischemically damaged pancreas during preservation by the two-layer (EC/PFC) method allows ATP synthesis within the graft via direct phosphorylation of adenosine. Metabolic processes vital to repair damaged cells and maintain cellular integrity can be maintained, which makes it possible to preserve ischemically damaged pancreas.

Application of the two-layer method on pancreas digestion results in improved islet yield and maintained viability of isolated islets

Background. Oxygenation of the pancreas during preservation by the two-layer method (TLM) has shown beneficial effects in islet transplantation. Here, we apply this concept (oxygenation) to the isolation process. Methods. Rat pancreases were digested using four different methods. Pancreases were digested with preoxygenated perfluorocarbon (PFC) in group 2 and without it in group 1. Additionally, adenosine was included in the collagenase solution in subgroups B but not in subgroups A. Islet yields and viability were compared between groups. Results. Tissue oxygen tension in group 1 was essentially zero during digestion, but rapidly reached around 300 mm Hg and was maintained in group 2. The tissue adenosine triphosphate (ATP) level in rat pancreas just after laparotomy (control) was 4.2±0.7 &mgr;mol/g dry weight; after digestion, it was 0.12±0.03 &mgr;mol/g, 0.70±0.10 &mgr;mol/g, 0.30±0.18 &mgr;mol/g, and 2.90±0.80 &mgr;mol/g in groups 1A, 1B, 2A, and 2B, respectively. No significant differences were observed between group 2B and control (P=0.19). Islet yields (IEQ/pancreas) were 1600±400, 1400±400, 1300±400, and 2400±100 in groups 1A, 1B, 2A, and 2B, respectively. The islet yield of group 2B was significantly higher than other groups (P<0.05). The cure rate after transplanting 200 islets into athymic nude mice did not differ (80% in all groups). The stimulation indices in the four groups were also the same. Conclusions. Tissue ATP levels after digestion were well maintained using TLM with adenosine digestion method. Consequently, greater numbers of islets could be retrieved. The new method was at least equivalent to islet function isolated by conventional method. Clinical study is therefore warranted.

Protection against experimental small intestinal ischaemia–reperfusion injury with oxygenated perfluorochemical

Intestinal ischaemia–reperfusion (IR) injury frequently occurs in abdominal surgery. Perfluorochemical (PFC) can be used to oxygenate intestinal organs directly and allows adenosine 5′‐triphosphate (ATP) production within the submerged organs during ischaemia. This study was designed to evaluate the protective effect of PFC in IR injury, focusing on cytokine production in rat small intestine.

No Mortality among 100 Consecutive Pancreaticoduodenectomies in a Middle-volume Center

Mortality rate following pancreaticoduodenectomy has markedly decreased in high-volume centers. We achieved zero mortality among 100 pancreaticoduodenectomies in a middle-volume center. The purpose of this study was to review our experience and analyze factors contributing to the zero mortality. Patient backgrounds, intraoperative variables, postoperative complications, and surgical, radiologic, and other medical interventions for the complications were retrospectively analyzed for 100 consecutive pancreaticoduodenectomies for malignant or benign disease. The mean age of the patients was 63 years. Altogether, 59 patients had preoperative co-morbidity, and 35 had a past history of abdominal surgery. The median operating time and blood loss were 525 minutes and 1215 ml, respectively. Postoperative complications occurred in 42 patients. The most frequent complication was pancreatic stump leak (n = 12), but no life-threatening pancreatic anastomotic leak occurred. This may result from the duct invagination anastomosis applied to 67 pancreases with a small duct. Serious complications were seen in six patients; two patients required surgical intervention, but four were successfully treated with the help of interventional radiologists or internists. Radiologic intervention was applied to 13 patients: drainage of an intraabdominal abscess/collection and vascular intervention. In addition to advances in surgical techniques to reduce local complications, particularly pancreatic anastomotic leak, intimate collaboration with experienced interventional radiologists and internists allows zero mortality even in middle-volume centers.

Resection of the Colon Simultaneously with Pancreaticoduodenectomy for Tumors of the Pancreas and Periampullary Region: Short-term and Long-term Results

Simultaneous resection of the colon with pancreaticoduodenectomy (PD) is occasionally inevitable to accomplish curative resection in instances when a periampullary tumor involves the mesentery of the colon. However, there is little information regarding short- and long-term outcomes of this aggressive surgery. Among 95 consecutive patients who underwent PD for periampullary malignant tumors, 12 had simultaneous resection of the right colon (group 1) and 83 underwent PD alone (group 2). Intraoperative variables, postoperative morbidity and mortality, and the length of the hospital stay were comparatively analyzed. Survival was also compared between the groups in a subset of 36 pancreatic adenocarcinoma patients. Group 1 included more patients with pancreatic cancer, and portal vein resection was more frequently performed, which seemed to be associated with a significantly longer operating time (640 vs. 510 minutes) and increased total blood loss (1965 vs. 1220 ml). However, morbidity and mortality rates did not differ between the groups (50,0% and 0%, respectively, in group 1; 44.6% and 1.2%, respectively, in group 2). The median hospital stays were 67 and 48 days in groups 1 and 2, respectively. In a subset of 36 pancreatic adenocarcinoma patients, the median progression-free survivals were 6 months in both groups 1 and 2; the median overall survivals were 14 months in group 1 and 12 months in group 2. There was no statistically significant difference in survival between the groups. Simultaneous right hemicolectomy with curative intent at the time of PD could thus be performed safely and may offer a survival benefit even for individuals who have advanced pancreatic cancers with involvement of the transverse mesocolon.