Seventy-two-hour preservation of the canine pancreas by the two-layer (Euro-Collins' solution/perfluorochemical) cold storage method

A 72-hr preservation of canine pancreas by a 2-layer (Euro-Collins'/Perfluorochemical) cold storage method was tested in the canine model of segmental pancreas autotransplantation. The functional recovery of the grafts by this method (group 1) was determined by daily fasting blood glucose concentration and intravenous glucose tolerance test at 2 weeks after autotransplantation and compared with simple cold storage with Euro-Collins' solution (group 2) and control (no preservation) (group 3). Maintenance of normoglycemia for at least 5 days after transplantation was considered a successful preservation. The functional success rates after 72-hr preservation were 100%, 0%, and 100% for groups 1, 2, and 3, respectively. The mean K values of group 1 after 72-hr preservation was 1.78 +/- 0.42 compared with 2.05 +/- 0.32 for group 3 at 2 weeks after transplantation. Biopsies of grafts of group 2 after 72-hr preservation showed remarkable autolytic changes in exocrine and endocrine tissues. In contrast, biopsies of grafts of group 1 after 72-hr preservation showed almost normal architecture in both tissues. In addition, biopsies of 72-hr preserved grafts of group 1 at 4 weeks after after autotransplantation showed almost normal pancreas architecture with minimal fibrotic changes in the exocrine tissue. This study demonstrated the possibility of 72-hr preservation of the pancreas for transplantation.     

The mechanism of action of the two-layer (Euro-Collins' solution/perfluorochemical) cold storage method in canine pancreas preservation

To clarify the mechanism of action of a two-layer [Euro-Collins' solution (EC)/perfluorochemical (PFC) ] cold storage method in the preservation of the pancreas, pancreatic viability and tissue concentrations of adenosine triphosphate (ATP) were examined in the canine model of pancreatic autotransplantation after preservation for 24 and 48 h by simple cold storage in EC (group 1), the two-layer, EC/PFC, method (group 2) and the two-layer, EC + 2, 4 dinitrophenol (DNP)/PFC, method (group 3). DNP is an uncoupler of oxidative phosphorylation. Maintenance of normoglycemia for at least 5 days after transplantation was considered a successful preservation. After preservation for 24 h, the functional success rates of groups 1, 2 and 3 were 100% (4/4), 100% (5/5) and 80% (4/5) respectively. One of five dogs in group 3 died of a cause unrelated to the pancreas. ATP tissue concentrations in group 2 were significantly higher than in group 1 (7.47 ± 0.47 gmol/g dry weight vs 1.41 ± 0.53 gmol/g dry weight, P < 0.01) and ATP tissue concentrations in group 3 were significantly lower than in group 2 (1.25 ± 0.37 gmol/g dry weight vs 7.47 ± 0.47 gmol/g dry weight, P < 0.01). It was apparent that ATP was not an essential factor for successful 24-hour preservation of the canine pancreas in EC because all the pancreatic grafts except one of five grafts in group 3 remained viable after preservation for 24 h, regardless of ATP tissue concentrations. On the other hand, after preservation for 48 h, the functional success rates for groups 1, 2 and 3 were 0% (0/4), 100% (4/4) and 0% (0/3) respectively. ATP tissue concentrations in group 2 were significantly higher than in group 1 (7.91 ± 1.21 gmol/g dry weight vs 1.21 ± 0.314tmol/g dry weight, P < 0.01) and ATP tissue concentrations in group 3 were significantly lower than in group 2 (0.61 ± 0.07 gmol/g dry weight vs 7.91 ± 1.21 µmol/g dry weight, P < 0.01). It was clear that preservation of the pancreas for 48 h was unsuccessful by simple cold storage in EC (group 1) and the two-layer method (group 2) made preservation for 48 h possible by increasing ATP tissue concentrations. However, DNP (group 3) inhibited the synthesis of ATP and the effectiveness of the two-layer method for 48-hour preservation of the pancreas. It was clear that maintenance of high ATP tissue concentrations during preservation was essential for the successful preservation of the canine pancreas in EC by the two-layer method for more than 48 h. We concluded that an adequate supply of oxygen to the pancreas during preservation by the two-layer method led to sufficient production of ATP to maintain cellular integrity and permitted the improvement of pancreatic preservation.     

Characterization of islet-infiltrating immunocytes after pancreas preservation by two-layer (UW/perfluorochemical) cold storage method

Clinical islet transplantation offers the prospect of good blood glycemic control without major surgical risks. Nevertheless, long-term function of the transplanted islets is seldom appreciated because rejection is followed by the graft failure. Although it has been implicated that islets have high immunogenicity, characterization of the islet-infiltrating immunocytes, such as leukocytes and macrophages, has not been extensively studied. Rat islets were isolated by the collagenase digestion method and separated by handpicking under the microscope. The islets were further dispersed into individual cells for flow cytometric analysis. Monoclonal antibodies directed toward T cells, B cells, and macrophages as well as ICAM-1, and MHC class I and II were used to enumerate cells. Pancreatic islets contained 6.3 +/- 2.9% immunocytes; T cells (39.6 +/- 4.2%), B cells (44.7 +/- 5.8%), and macrophages (1.7 +/- 0.6%). MHC class I was expressed on 85.6 +/- 2.8%, MHC class II on 36.8 +/- 2.9%, and ICAM-1 on 39.9 +/- 7.0%. The results of islets from preserved pancreas also showed the same tendency. As these islet-infiltrating immunocytes within the grafts may contribute to the rejection, one potential strategy to prevent early graft loss might start to eliminate or inactivate the islet-infiltrating immunocytes.     

A comparison of cold storage solutions for pancreas preservation prior to islet isolation

BACKGROUND: Several solutions are used to preserve the pancreas prior to islet isolation. This study sought to assess whether the type of solution had an impact on the isolation outcome. METHODS: We reviewed data from 125 islet isolation procedures performed from January 2002 to January 2005. Pancreata were preserved in University of Wisconsin (UW) (n = 101), Celsior (CS) (n = 19), or IGL-1 (n = 5) solutions. Islet isolation results and transplantation rates were compared between groups. RESULTS: UW, CS, and IGL-1 groups were similar according to donor's age, weight, and body mass index. Weight of undigested pancreas was 20 +/- 13.1, 21.4 +/- 15.7, and 17.4 +/- 8.7 g for UW, CS, and IGL-1, respectively (P > .2). Final total number of IEQ was 267,000 +/- 132,000, 277,000 +/- 155,000, and 311,000 +/- 163,000, respectively (P > .4). Success rate (defined as >250,000 IEQ) was 55.5%, 52.9%, and 60% for UW, Celsior, and IGL-1 (P > .9); the transplantation rate was 42.2% for UW, 36.8% for Celsior, and 80% for IGL-1 preservation (P > .2). CONCLUSIONS: In this preliminary study, UW, Celsior, and IGL-1 solutions demonstrated similar islet isolation results. The new IGL-1 solution appears promising.     

自制多器官保存液对肾皮质能量代谢的影响

目的寻找理想的器官保存液，以提高器官移植的长期疗效。方法应用高效液相系统（HPLC），检测自制多器官保存液（SMO液）和对照组（HC-A液和UW液）对犬肾低温保存中皮质腺苷酸含量改变的影响。取皮质标本匀浆、沉淀蛋白、离心，HPLC检测上清中腺苷酸含量，根据标准曲线方程和加样回收率计算ATP、ADP、AMP含量、比值和能荷（EC）。结果随保存时间延长，各组皮质腺苷酸含量下降；保存12、36、72h后SMO组皮质ATP含量（3．7±0．5、3．1±0．3、2．3±0．5nmol／mg蛋白）高于HC—A组（2．8±0．2、2．0±0．2、1．5±0．2nmol／mg蛋白），P〈0．05，SMO组EC（0．38±0．04、0．37±0．05、0．35±0．04）高于HCfA组（0．32±0．03、0．30±0．04、0．28±0．03），P〈0．05，在多数保存时点SMO组ADP、ATP／AMP高于HC—A组（P〈0．05），但2组间AMP、ATP／ADP比值差异无统计学意义（P〉0．05）；犬肾保存期间SMO组腺苷酸含量及EC与UW组比较差异无统计学意义（P〉0．05）。结论低温保存期间SMO液能够维持皮质内较高的ATP和EC水平，减轻肾脏低温缺血损伤。     

SMO保存液对犬肾低温保存期间线粒体功能的保护作用

目 的研究SMO保存液对犬肾低温保存期间线粒体功能的保护作用。方法 建立犬离体肾脏单纯低温保存模型，分别采用0～4℃的SMO保存液、HTK保存液和UW保存液对犬肾进行灌注和保存，并根据所用保存液的不同分为SMO组、HTK组和UW组。分别于低温保存2、24、48和72h后取各组的肾组织皮质标本，进行病理学观察并应用氧电极（Clark）法测定肾组织细胞线粒体的呼吸控制率（RCR）和磷／氧比（P／O）。结果 犬肾在保存48h和72h后，SMO组肾组织病理学改变较HTK组轻；保存24h内肾组织RCR与HTK组无明显差异，但其余时间点均高于HTK组（P〈0．05），保存72h时差异最为明显（P〈0．01）。SMO组犬肾保存2h后，肾组织的P／O比值与HTK组无明显差异（P〉0．05），但在保存24h后则明显高于HTK组（P〈0．05）。SMO组与UW组各项观察指标比较，差异无统计学意义（P〉0.05）。结论 SMO保存液在减轻细胞形态学改变和保持线粒体整体功能方面显著优于HTK保存液，与UW保存液效果相当。     

Extended cadaver renal preservation with combined simple cold storage and hypothermic pulsatile perfusion

Although simple cold storage and hypothermic pulsatile perfusion are each accepted methods of cadaver kidney preservation the efficacy of combining these techniques for extended renal preservation is unclear. In 18 patients cadaver allografts were used that had been preserved with combined simple cold storage (range 6 to 20 hours) and hypothermic pulsatile perfusion (range 9 to 38 hours). The total interval of preservation for these kidneys ranged from 25 to 48 hours. Excellent post-transplant graft function was achieved in all cases, with a mean serum creatinine nadir of 1.4 mg./dl. In 14 patients (78 per cent) grafts continued to function at intervals of 3 to 38 months after transplantation. Of the remaining 4 patients 3 lost the grafts to rejection, while 1 died 7 months after transplantation with a well functioning graft. These data suggest that the combination of simple cold storage and hypothermic pulsatile perfusion provides a safe and effective method for extended renal preservation.     

Improved lung preservation with cold air storage

Conventional topical slush cooling limits lung transport to 4 to 6 hours. For this canine study of an alternate air cooling system, 37 canine lungs were removed: 24 were placed in plastic bags, and inserted in a Transplanthermm container at core air temperatures (n = 6 lungs each) of (A) 4 degrees C, (B) 8 degrees C, (C) 12 degrees C, and (D) 20 degrees C; 6 were stored conventionally in ice slush (E); and 7 were transplanted immediately (F). After 8 hours, the stored lungs were transplanted and the contralateral pulmonary artery was ligated. Survival, arterial oxygen tension, and extravascular lung water were monitored at 15 minutes and every hour for 4 hours. Four-hour survival was 100% in groups A, B, and F; 83% in group C, 50% in group D, and 17% in group E. The mean arterial oxygen tension at 1 hour was lower in group E (6.4 +/- 2.4 kPa) than in group A (39.8 +/- 13.2 kPa) (p = 0.0002) or in group F (42.0 +/- 16.2 kPa) (p = 0.0035). Extravascular lung water in group E was higher at 15 minutes (15.44 +/- 5.63 mL/kg) than in group A (3.76 +/- 0.63 mL/kg) (p = 0.0001) and group F (4.69 +/- 1.65 mL/kg) (p = 0.003). Cold air storage appears to provide better lung preservation than hypothermic immersion in ice slush.     

Preservation of the canine liver for 24-48 hours using simple cold storage with UW solution

The results of a series of 29 orthotopic liver transplants in the dog are described. The livers were preserved in a new cold storage fluid, UW solution, and were successfully transplanted after periods of storage of 24, 30, 36, and 48 hr. All six animals transplanted after 24 hr survived beyond 5 days after transplantation and had excellent graft function. Four of six survived for at least 5 days after 30 hr of cold storage, and five of five after 36 hr. Five of six consecutive dogs that received transplants that had been cold-stored for 48 hr survived for 5 or more days. This solution represents a substantial advance over all existing cold storage solutions for liver preservation.     

The importance of a colloid in canine pancreas preservation.

The role of hydroxyethyl starch (HES), the colloid component of the UW solution, was tested in canine pancreas preservation. Segmental pancreatic autografts were preserved for 48 hr cold storage with UW solution with HES (group 1) or UW solution without HES (group 2). After preservation, the pancreas was transplanted, and survival, serum glucose, serum amylase, intravenous glucose tolerance tests, tissue water content, and histology were compared between groups. In group 1 (with HES), 9/10 dogs were long-term survivors with one dog dying due to causes unrelated to preservation failure. In group 2 (without HES), 3/6 dogs died due to graft loss within one week posttransplant (P = 0.01). No graft failure occurred in group 1 (0/9) versus graft loss in 4/6 dogs in group 2 (P = 0.04). All animals in group 1 (with HES) showed normal serum glucose and amylase concentrations postoperatively, normal tissue water values after preservation, k values comparable to those observed after segmental autotransplantation without preservation, and relatively good histology. In group 2 (without HES), in 4/6 dogs graft failure occurred that led to the death (3 dogs) of the animals or to a diabetic state (1 dog). After 48-hr cold storage without HES, a significant increase in tissue water content, glucose and amylase levels was seen. After transplantation, hyperglycemia, hyperamylasemia, and clinical diabetes were observed in 4/6 dogs. Autopsy and histological evaluation showed evidence of thrombosis and ischemic insult. Two of 6 dogs in group 2 remained normoglycemic during follow-up with borderline k values. The results suggested that for consistently successful 48-hr preservation of the pancreas, HES is an important component of the UW solution. Although a colloid may not be essential for short-term preservation of kidney and liver, it appears to be an important factor in successful pancreas preservation.