Intraperitoneal injection of oxygenated perfluorochemical improves the outcome of intraportal islet transplantation in a rat model.

A 50% to 75% early graft loss upon engraftment has been suggested to in intraportal islet transplantation (IPIT). Hypoxia in the portal vein contributes to graft loss in immediately posttransplantation. Herein we examined the effect on the outcome of IPIT of intraperitoneal oxygenated perfluorochemical (PFC) as an oxygen carrier. Isolated Lewis rat islets were transplanted into the portal vein of a chemically induced diabetic syngeneic rat. First, 1500 IEQ was determined to be the optimal dose in this study. When oxygenated PFC (group 1) was intraperitoneally injected following IPIT of 1500 IEQ, the success rate of transplantation was 5/6, in contrast to 1/6 when PFC with no oxygen was injected (group 2) and 1/6 in IPIT without PFC, respectively. The area under the glucose profile curve on intraperitoneal glucose tolerance tests on posttransplant day 28 in group 1 was significantly smaller than that for group 2. In conclusion, intraperitoneal oxygenated PFC improved the outcome of IPIT.

Augmentation of tissue ATP level during digestion using preoxygenated perfluorocarbon results in high islet yield-new strategy for islet isolation.

BACKGROUND: Recently, preservation using oxygenated perfluorocarbon (the two-layer method) has shown beneficial effects on islet yield and viability. In this paper, we apply this concept on isolation processes to examine the effectiveness of oxygenation. METHODS: Rat pancreata were digested using four different methods: (groups 1A, 1B, 2A, and 2B) with or without oxygenated perfluorocarbon in groups 1 and 2, respectively. Adenosine was added into the collagenase solution in subgroup A whereas it is not added in subgroup B. RESULTS: Tissue oxygen tension in group 1 was about 0 during digestion; whereas it rapidly reached about 300 mm Hg and was maintained in group 2. Tissue ATP level just after laparotomy (control) was 4.2 +/- 0.7 micromol/g dry weight. The ATP levels after digestion were 0.12 +/- 0.03 in group 1A (P < 0.01 vs control); 0.70 +/- 0.10 in group 1B (P < 0.01 vs control); 0.30 +/- 0.18 in group 2A (P < 0.01 vs control); and 2.90 +/- 0.80 in group 2B (P = 0.19 vs control). Islet yields (IEQ/pancreas) were 1600 +/- 400 in group 1B; 1400 +/- 400 in group 1B; 1300 +/- 400 in group 2A; and 2400 +/- 100 in group 2B. The amount in group 2B was significantly greater than that in the other three groups. CONCLUSIONS: Oxygen provision by preoxygenated perfluorocarbon itself showed no beneficial effect on islet yield. However, if oxygen provision was associated with adenosine administration into the pancreas, tissue ATP levels after digestion were well maintained, and a greater number of islets were retrieved.

Successful islet transplantation from a single pancreas harvested from a young, low-BMI, non-heart-beating cadaver.

BACKGROUND: Young donors, donors with low body mass index (BMI), and non-heart-beating (NHB) donors are considered nonideal for islet transplantation. In this report, we successfully used a pancreas from a young, low-BMI, NHB donor for islet transplantation. METHODS: The donor was a 15-year-old adolescent boy whose cause of death was rupture of a primary brain tumor. According to Japanese regulations, his pancreas was procured after cardiac arrest. Warm ischemic time was 3 minutes and cold ischemic time was 300 minutes. The pancreas was digested by the automated method of Ricordi, followed by purification using continuous Euro-Ficoll gradients on a Cobe 2991 device. The recipient was a 35-year-old woman with unstable type 1 diabetes mellitus. Her pretransplant C-peptide level was null. She suffered frequent hypoglycemic unawareness. Her pretransplant M value, which is a good marker for glucose instability, was 125. Islet yield was 252,816 IEQ. There were no signs of contamination. Viability of islets assessed by FDA/PI staining was 83%. Stimulation index was 2.7. RESULTS: The patient received 5160 IEQ/kg of islets via the portal vein under local anesthesia. There were no transplant-related complications. Although she required minimal exogenous insulin, her C-peptide level increased to 0.7 ng/mL at postoperative day (POD) 14. Her M value at POD 15 to 19 decreased dramatically to 23.6, indicating good glycemic control. At 3 months posttransplant, episodes of hypoglycemia disappeared. CONCLUSIONS: Although an additional transplant is mandatory to wean patients from insulin, this case shows the possibility of using marginal donors, such as a young, low-BMI, NHB donor, for pancreas islet transplantation.

The effect of fasting and exogenous adenosine on ATP tissue concentration and viability of canine pancreas grafts during preservation by the two-layer method.

We have demonstrated a direct correlation between a high ATP level in a canine pancreas graft after preservation by the two-layer method and good posttransplant outcome. The purpose of this study was to examine the effect of fasting and exogenous adenosine on the ATP tissue level and viability of the canine pancreas graft during preservation by the two-layer method. Graft viability was judged by graft survival following autotransplantation. Maintenance of normoglycemia for 5 days post-transplant was considered graft survival. The pancreas was harvested from either 72-hr-fated (n = 3) or -fed dogs (n = 4) and preserved by the two-layer (Euro-Collins' solution [EC]/perfluorochemical [PFC]) method for 24 hr. Graft survival rates in fed and fasted groups were 4/4 (100%) and 3/3 (100%), respectively. There was no significant difference in ATP tissue concentrations between the two groups (7.48 +/- 0.55 vs. 7.03 +/- 0.74 mumol/g dry wt, NS). The pancreas was subjected to 60 min warm ischemia and then was preserved by the two-layer method using EC or EC containing 5 mM adenosine for 24 hr. Without adenosine, graft survival rate was 0/3 (0%) and ATP tissue levels were not changed during preservation (1.62 +/- 0.26 vs. 1.56 +/- 0.40 mumol/g dry wt, NS). However, provision of adenosine to the graft during preservation led to the restoration of ATP tissue levels (1.90 +/- 0.54 vs. 8.13 +/- 0.98 mumol/g dry wt, P < 0.01) in 4 of 5 grafts, and these grafts functioned immediately and maintained normoglycemia after transplantation. Graft survival rate was 4/5 (80%). One of 5 grafts, however, did not survive, and the ATP tissue level was not adequately recovered during preservation compared with viable grafts (3.67 vs. 8.13 +/- 0.98 mumol/g dry wt). This study clearly demonstrates that the nutritional state of the donor has no influence on the ATP tissue level and viability of the graft during 24-hr preservation by the two-layer method. On the other hand, provision of adenosine to the graft during preservation stimulates ATP synthesis and improves the viability of the ischemically damaged pancreas.

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.

The mechanism of action of the two-layer (Euro-Collins' solution/perfluorochemical) cold-storage method in canine pancreas preservation--the effect of 2,4 dinitrophenol on graft viability and adenosine triphosphate tissue concentration.

Tissue concentrations of adenosine triphosphate have been previously associated with successful pancreas preservation using the two-layer cold storage method in a canine autotransplantation model. To clarify the role of ATP vs. oxygenation per se, we used 2,4 dinitrophenol, an uncoupler of mitochondrial oxidative phosphorylation. DNP caused no toxicity in pancreas grafts preserved for 24 hr in Euro-Collins' solution or 48 hr in University of Wisconsin solution. Tissue concentration of ATP and viability of pancreas grafts, defined as maintenance of normoglycemia for 5 days following transplantation, were compared among six groups after a preservation interval of 24 or 48 hr. After 24 hr all grafts were viable, whether preserved using simple cold storage in EC (group 1a), two-layer (EC/perfluorochemical [PFC]) method (group 2a), or two-layer (EC+DNP/PFC) method (group 3a); respective graft survival was 4/4 (100%), 5/5 (100%), and 4/5 (80%); one of five dogs in group 3a died of a cause unrelated to the pancreas. ATP levels were higher in group 2a compared with group 1a (7.47 +/- 0.47 vs. 1.41 +/- 0.53 mumol/g dry weight, P less than 0.01) and lower in group 3a compared with group 2a (1.25 +/- 0.37 vs. 7.47 +/- 0.47, P less than 0.01). After 24 hr, we observed no difference in viability despite ATP concentration differences. However after 48 hr preservation, graft viability varied among the groups: 0/4 (0%), 4/4 (100%), and 0/3 (0%) in groups 1b, 2b, and 3b, respectively. ATP tissue concentration was again higher in group 2b after two-layer (EC/PFC) method preservation (7.91 +/- 1.21 vs. 1.21 +/- 0.31 mumol/g dry weight, P less than 0.01) compared with EC preservation (group 1b). DNP again caused a significant decrease in tissue ATP in group 3b (0.61 +/- 0.07 vs. 7.91 +/- 1.21, P less than 0.01). The two-layer (EC/PFC) method clearly protected pancreas viability, and inhibition of ATP production using DNP caused loss of viability in this model. We conclude that oxygenation of the pancreas during preservation by the two-layer method allows continued ATP production within the graft. Metabolic processes vital to cellular integrity can be maintained, which produces an extended period of preserved pancreatic viability.

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.

Protection of canine pancreatic microvascular endothelium against cold ischemic injury during preservation by the two-layer method.

BACKGROUND: Ischemic injury of the microvascular endothelium during cold preservation causes a disturbance of vascular microcirculation after reperfusion and results in graft failure. Recently we have shown that oxygenation of the canine pancreas during preservation by the two-layer method extends the period of preserved pancreatic viability. The aim of this study was to clarify the role of the oxygenation of the pancreas graft by the two-layer method in the viability of the microvascular endothelium during preservation. METHODS: After preservation of the canine pancreas by simple cold storage in Euro-Collins solution (EC) (group 1) or by the two-layer method using EC (group 2) for 48 hr, the viability of vascular endothelial cells was judged from nuclear trypan blue uptake. Pancreatic tissue perfusions were measured with a hydrogen gas clearance technique, and graft survival rates were examined after autotransplantation. In the control group, the grafts were autotransplanted without preservation (group 3). RESULTS: Graft survival rates were 0 of 5 (0%), 5 of 5 (100%), and 5 of 5 (100%) in groups 1, 2, and 3, respectively. The percentage of trypan blue-positive vascular endothelium in group 1 was significantly higher compared with group 3 (control) (26.4+/-1.7 vs. 7.4+/-4.3%, P<0.01). The two-layer method (group 2) decreased trypan blue uptake (11.3+/-3.7 vs. 26.4+/-1.7%, P<0.01). Pancreatic tissue perfusions after 2 hr of reperfusion were in inverse proportion to trypan blue uptake. Namely, pancreatic tissue perfusions in group 1 were significantly lower than group 3 (control) (45.6+/-12.8 vs. 64.5+/-20.6 ml/min/100 g, P<0.01). The two-layer method (group 2) improved pancreatic tissue perfusions (68.8+/-8.6 vs. 45.6+/-12.8 ml/min/100 g, P<0.01). CONCLUSION: We conclude that oxygenation of the pancreas during preservation by the two-layer method protects the microvascular endothelium from cold ischemic injury. Consequently, pancreatic microcirculation can be maintained after reperfusion, thus extending the period of preserved pancreatic viability.

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 O2] 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.

Extending the margin of safety of preservation period for resuscitation of ischemically damaged pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C with thromboxane A2 synthesis inhibitor OKY046.

We have shown that 5-hr preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C allows ATP synthesis and makes it possible to resuscitate a canine pancreas subjected to 90 min of warm ischemia. However, 8 hr of preservation using this method caused a disturbance of vascular microcirculation and did not resuscitate the grafts. The aim of this study was to examine the effect of thromboxane A2 synthesis inhibitor OKY046 on vascular endothelial cells and ATP tissue levels of canine pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C, and vascular microcirculation and pancreas viability after transplantation. Graft viability was judged by graft survival following autotransplantation. ATP tissue levels were measured by high-performance liquid chromatography at the end of preservation. Viability of the vascular endothelial cells was judged using nuclear trypan blue uptake of the graft after preservation. Pancreatic tissue perfusion was measured using an H2 clearance technique after reperfusion. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5). However, 5-hr preservation made it possible to recover the pancreas (5/5); 8-hr preservation was not successful (0/3). ATP tissue levels after 5-hr and 8-hr preservation were 9.40+/-2.09 and 7.37+/-1.06 micromol/g dry weight, respectively, and OKY046 did not affect ATP synthesis during 8-hr preservation (8.44+/-0.92 micromol/g dry weight). The percentage of nuclear trypan blue uptake of endothelial cells in 8-hr-preserved grafts was 37.6+/-11.6% and was significantly higher than the value in 5-hr-preserved grafts (5.0+/-3.0%; P<0.01). However, OKY046 significantly reduced trypan blue uptake in 8-hr-preserved grafts (8.2+/-3.6%; P<0.01). Pancreatic tissue perfusion in 8-hr-preserved grafts after 2 hr of reperfusion was 28.5+/-7.5 ml/min/100 g, and was significantly lower than the value in 5-hr-preserved grafts (57.1+/-4.4 ml/ min/100 g; P<0.01), but OKY046 dramatically improved pancreatic tissue perfusion (97.1+/-14.6 ml/min/100 g; P<0.01). As a consequence, 8-hr-preserved grafts were resuscitated (4/5). We conclude that OKY046 protects the vascular endothelium during preservation by the two-layer method at 20 degrees C and consequently improves vascular microcirculation on reperfusion. Together with ATP synthesis, which is essential for repairing damaged cells, the canine pancreas graft subjected to 90 min of warm ischemia is resuscitated during 8-hr preservation by the two-layer method at 20 degrees C. This method holds promise for pancreas-kidney transplantation from cardiac arrest donors.

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.

Protective effect of preservation of canine pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) method against rewarming ischemic injury during implantation.

Rewarming ischemia during implantation severely compromises posttransplant pancreas graft survival because the graft has already been subjected to warm and cold ischemia before implantation. The purpose of this study was to examine whether preservation of the pancreas graft by the two-layer method ameliorates rewarming ischemic injury of the graft during implantation using a canine model. After flushing with cold University of Wisconsin solution (UW), the pancreas grafts were preserved by the two-layer (UW/perfluorochemical [PFC]) method (group 1) or simple cold storage in UW (group 2) for 24 hr and then autotransplanted. In control, the pancreas grafts were flushed out with cold UW and immediately autotransplanted without preservation (group 3). After completion of vascular anastomosis, vascular clamp was not released until 90, 120, or 150 min of rewarming ischemia, including anastomosis time, had elapsed. After 90 min of rewarming ischemia, graft survival rates were 5/5, 100%, 5/5, 100%, and 5/5, 100%, in groups 1, 2, and 3, respectively. After 120 min, all the grafts in groups 2 and 3 failed (0/5, 0%, and 0/5, 0%, respectively); however, all the grafts in group 1 survived (5/5, 100%). Even after 150 min, 1 of 3 grafts in group 1 survived (1/3, 33%). After 24 hr preservation, tissue ATP levels of the grafts in group 1 were about 2-fold the reference values before harvesting (8.23 +/- 0.72 vs. 4.44 +/- 0.49 mumol/g dry weight, P < 0.05) and significantly higher compared with group 2 (8.23 +/- 0.72 vs. 1.76 +/- 0.52 mumol/g dry weight, P < 0.01). After 120 min of rewarming ischemia, tissue ATP levels in group 1 were 84% of the reference values and significantly higher compared with group 2 (3.75 +/- 0.25 vs. 1.57 +/- 0.48 mumol/g dry weight, P < 0.05). Two hours after reperfusion, ATP levels in group 1 were 42% of reference values but significantly higher compared with group 2 (1.86 +/- 0.36 vs. 1.03 +/- 0.18 mumol/g dry weight, P < 0.05). We conclude that the two-layer (UW/PFC) method ameliorates rewarming ischemic injury of the pancreas graft during implantation by increasing tissue ATP contents during preservation and consequently maintaining tissue ATP levels during implantation.

Effect of pancreatic warm ischemia on islet yield and viability in dogs.

BACKGROUND: Defining tolerable warm ischemia (WI) is mandatory before nonheartbeating cadavers can be used to enlarge the donor pool. No studies to date have precisely evaluated the effect of pancreatic WI on islet yield and viability in a large animal model. METHODS: We used mongrel dogs in our study at the University of Minnesota. Excised pancreases were left in situ for a designated period (0, 30, 45, and 60 min in groups 1 to 4, respectively) of WI. Then, they were digested by the automated collagenase digestion method of Ricordi, purified on Euro-Ficoll discontinuous gradients with the COBE cell processor, and autotransplanted into the liver via a mesenteric vein. We compared the four groups in terms of islet yield, expressed as islet equivalents (IE; diameter standardizing to 150 microm) per pancreas weight (IE/g pancreas), and viability, assessed by functional success (maintenance of normoglycemia for 2 weeks) after transplant. RESULTS: Mean islet yield (+/- SD) and the functional success rate after transplant were as follows: 6200+/-1800 IE/g pancreas and 4 of 4 (100%) in group 1; 6300+/-4400 and 4 of 4 (100%) in group 2; 3800+/-2600 and 2 of 4 (50%) in group 3; and 1400+/-1300 and 0 of 4 (0%) in group 4 (P=0.01 vs. group 1). CONCLUSIONS: With 30 min or less of WI, there are no deleterious effects on islet yield and viability. However, with periods of WI longer than 30 min, the loss in islet yield is severe, resulting in functional failure after autotransplantation. The limit of WI that is tolerable for islets is shorter than for a whole pancreas.

Resuscitation of ischemically damaged pancreas during short-term preservation at 20 degrees C by the two-layer (University of Wisconsin solution/perfluorochemical) method.

We have shown that 24-hr preservation by a two-layer (University of Wisconsin solution [UW]/perfluorochemical [PFC]) cold storage method allows tissue ATP synthesis and makes it possible to resuscitate a canine pancreas subjected to 90 min of warm ischemia. The purpose of this study was to examine whether increasing preservation temperature to 20 degrees C makes it possible to shorten a preservation period for recovery of ischemically damaged pancreas grafts. After 90 min of warm ischemia, canine pancreas grafts were preserved using the two-layer (UW/PFC) method for 1 to 8 hr at 20 degrees C, and then autotransplanted. A K-value of intravenous glucose tolerance test more than 1.0 at 2 weeks after transplantation was considered graft survival. ATP tissue levels were measured by high performance liquid chromatography at the end of preservation. Pancreatic tissue perfusions were measured using an H2 clearance technique after 30 min to 4 hr of reperfusion. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5, control group). However, 3- and 5-hr preservations made it possible to recover the ischemically damaged pancreas (3/5 and 5/5, respectively), although 1- and 8-hr preservations were not successful (0/3 and 0/3, respectively). ATP tissue levels in 1-hr-preserved grafts were 2.55 +/- 0.38 mumol/g dry weight and were significantly lower compared with the levels in 5- and 8-hr-preserved grafts, 9.40 +/- 2.09 (P < 0.01) and 7.37 +/- 1.06 (P < 0.01), respectively. On the other hand, pancreatic tissue perfusions in 8-hr-preserved grafts after 2 hr of reperfusion were 28.50 +/- 7.52 ml/100 g/min and were significantly lower than the values in 1- and 5-hr-preserved grafts, 66.0 +/- 11.22 (P < 0.01) and 57.10 +/- 4.40 (P < 0.01), respectively. It was suggested that 1-hr-preservation was not enough to synthesize ATP, which was essential to repair damaged cells, although vascular microcirculation at reperfusion was maintained and 8-hr preservation incurred microcirculatory disturbances, although ATP for repairing damaged cells was synthesized. We conclude that 3- to 5-hr preservation at 20 degrees C by the two-layer (UW/PFC) method accelerates ATP synthesis, which is essential for repairing damaged cells and protects vascular microcirculation. This makes it possible to resuscitate ischemically damaged pancreases faster. This method holds promise for pancreas-kidney transplantation from cardiac arrest donors.

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

To reduce cold ischemic injury of the heart by supplying sufficient oxygen to the heart during preservation, we have developed a new cavitary, two-layer (University of Wisconsin solution/perfluorochemical) cold storage method. The oxygenation of the heart during preservation by this method allows ATP production within the graft and makes it possible to extend preservation time up to 48 hr in the heterotopic rat heart transplant model.

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.

Posttransplant nonfunction of canine islets in PVG rats deficient in complement component C6.

BACKGROUND: Discordant islet xenografts are immediately nonfunctional in nonimmunosuppressed recipients other than the mouse, a process called primary nonfunction. Although at present it is unknown whether complement is involved, complement might participate in the induction of primary nonfunction through a number of mechanisms. We investigated the potential role of the membrane attack complex of complement in primary nonfunction of transplanted xenoislets. METHODS: Canine islets were transplanted into both nonimmunosuppressed and immunosuppressed normocomplementemic and C6-deficient (C6D) PVG rats. Cyclosporine, rapamycin, deoxyspergualin, and mycophenolate mofetil were used for immunosuppression from day -3 to cessation of islet cell function. Serum glucose was measured at 6 hr after transplant and daily thereafter. Xenograft tissue sections were obtained at various times after transplant and stained for inflammatory cells and insulin. RESULTS: Canine islets grafted in nonimmunosuppressed C6D rats and normocomplementemic rats underwent primary nonfunction in all animals. The incidence of primary nonfunction in animals receiving a four-drug immunosuppressive regimen was 33% in the normocomplementemic rats but only 10% in the C6D rats. The mean functional islet survival time was 1.57+/-0.33 days in the normocomplementemic group and 2.70+/-0.67 days in the C6D group (P=0.38). The islet xenografts showed little difference in degree and composition of cell infiltration between normocomplementemic and C6D rats. CONCLUSION: The membrane attack complex does not appear to play a major role in primary nonfunction of canine islet xenografts in nonimmunosuppressed PVG rats. However, there was a lower incidence of primary nonfunction and a longer posttransplant survival time in immunosuppressed C6D rats, suggesting the membrane attack complex may play a minor role in recipients that are heavily immunosuppressed.

The primary structure of skeletal muscle myosin heavy chain: III. Sequence of the 22 kDa fragment and the alignment of the 23 kDa, 50 kDa, and 22 kDa fragments.

The amino acid sequence of the 197-residue 22 kDa fragment from chicken pectoralis muscle was determined to be as follows: K-K-G-S-S-F-Q-T-V-S-A-L-F-R-E-N-L-N-K-L- M-A-N-L-R-S-T-H-P-H-F-V-R-C-I-I-P-N-E-T-K-T-P-G-A-M-E-H-E-L-V-L-H-Q-L-R- C-N-G-V- L-E-G-I-R-I-C-R-K-G-F-P-S-R-V-L-Y-A-D-F-K-Q-R-Y-R-V-L-N-A-S-A-I-P-E-G-Q- F-M-D-S- K-K-A-S-E-K-L-L-G-S-I-D-V-D-h-T-Q-Y-R-F-G-H-T-K-V-F-F-K-A-G-L-L-G-L-L-E- E-M-R-D- D-K-L-A-E-I-I-T-R-T-Q-A-R-C-R-G-F-L-M-R-V-E-Y-R-R-M-V-E-R-R-E-S-I-F-C-I- Q-Y-N-V-R-S-F-M-N-V-K-H-W-P-W-M-K-L-F-F-K, where h stands for 3-N-methylhistidine. The amino acid sequences of the 22 kDa fragment and its equivalent fragment from chicken ventricle and gizzard muscle myosins were also determined by our group. Predicted secondary structures of these 22 kDa fragment regions and of the reported chicken embryo myosin revealed some possible structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Excellence of the two-layer method (University of Wisconsin solution/perfluorochemical) in pancreas preservation before islet isolation.

BACKGROUND: In islet transplantation pancreatic preservation before islet isolation is an obstacle compromising islet yield and viability. We tested the feasibility of a two-layer method (University of Wisconsin solution [UW]/perfluorochemical) for pancreatic preservation before islet isolation. METHODS: Dog pancreases were processed into pure islets by the method of Ricordi preceded by five different preservations (groups 1-a and 1-b, the two-layer method for 3 and 24 hours; groups 2-a and 2-b, simple cold storage in UW for 3 and 24 hours; group 3, without preservation). Islet yields and functional success after autotransplantation into the liver were compared among the groups. RESULTS: Postpurification islet equivalents (IE)/gm pancreas and functional success rate were 5600 (mean), 83% in group 1-a; 4000, 56% in group 1-b; 4700, 33% in group 2-a; 1300, 0% in group 2-b; and 5000, 89% in group 3 (p < 0.05; 2b versus 1-a, 1-b, and 3), respectively. There was no statistical difference among groups 1-a, 1-b, and 3 in terms of islet yield and function (p > 0.2). CONCLUSIONS: The two-layer method is more effective than conventional simple cold storage in UW for pancreatic preservation before islet isolation. Clinical trials with the two-layer method are warranted.

Biliary-enteric anastomosis by means of a single layer of serosubmucosal sutures without T-tube drainage.

BACKGROUND: To lessen anastomotic stricture after biliary-enteric anastomosis, we developed a new biliary-enteric anastomosis that uses a single layer of interrupted serosubmucosal sutures without T-tube drainage. OBJECTIVE: To evaluate the safety and reliability of this new technique in a canine model of choledochoduodenostomy. METHODS: In 10 beagles, the common bile duct (2 to 3 mm in diameter) was ligated close to the duodenum with 3-0 polyglactin. On the fifth day after operation, the serum bilirubin level was elevated (137 to 205 mumol/L [8 to 12 mg/dL]) and the bile duct was dilated. The anastomosis between serosubmucosal layers of the dilated bile duct (8 to 10 mm in diameter) and duodenum was accomplished with interrupted sutures of 6-0 polyglactin with two needles. Stitches were inserted in the submucosal plane at the cut edge of the duct and duodenum to appose the mucosa accurately and to avoid accidental perforation of the entire thickness of the duct and duodenum. A T tube was not placed. RESULTS: There was no anastomotic leakage and the bilirubin level was normalized (14 to 17 mumol/L [0.8 to 1.0 mg/dL]) 7 days after operation for anastomosis. Histologic examination of specimens removed 6 or 12 months after operation showed good connective-tissue union and good mucosal continuity between the bile duct and the duodenum. There was no mucosal scarring and contracture or stricture formation. CONCLUSION: This new technique is simple and reliable and is recommended as an alternative method for restoring the continuity between the bile duct and intestinal tract after operation for obstructive jaundice caused by benign and malignant stricture of the bile duct.