University Of Wisconsin Group Research Articles

Diazoxide, a KATP Channel Opener, Prevents Ischemia–Reperfusion Injury in Rodent Pancreatic Islets

Diazoxide (DZ) is a pharmacological opener of ATP-sensitive K(+) channels that has been used for mimicking ischemic preconditioning and shows protection against ischemic damage. Here we investigated whether diazoxide supplementation to University of Wisconsin (UW) solution has cellular protection during islet isolation and improves in vivo islet transplant outcomes in a rodent ischemia model. C57/B6 mice pancreata were flushed with UW or UW + DZ solution and cold preserved for 6 or 10 h prior to islet isolation. Islet yield, in vitro and in vivo function, mitochondrial morphology, and apoptosis were evaluated. Significantly higher islet yields were observed in the UW + DZ group than in the UW group (237.5 ± 25.6 vs. 108.7 ± 49.3, p < 0.01). The islets from the UW + DZ group displayed a significantly higher glucose-induced insulin secretion (0.97 ng/ml ± 0.15 vs. 0.758 ng/ml ± 0.21, p = 0.009) and insulin content (60.96 ng/islet ± 13.94 vs. 42.09 ng/islet ± 8.15, p = 0.002). The DZ-treated islets had well-preserved mitochondrial morphology with superior responses of mitochondrial potentials, and calcium influx responded to glucose. A higher number of living cells and less late apoptotic cells were observed in the UW + DZ group (p < 0.05). Additionally, the islets from the UW + DZ group had a significantly higher cure rate and improved glucose tolerance. This study is the first to report mitoprotective effects of DZ for pancreas preservation and islet isolation. In the future, it will be necessary to further understand the underlying mechanism for the mitoprotection and to test this promising approach for pancreas preservation and the islet isolation process in nonhuman primates and ultimately humans.

Diazoxide Preconditioning of Non-Human Primate Pancreas During Procurement and Preservation Improves Islet Isolation Outcomes and Function.

Objective: Diazoxide (DZ) has been shown as an effective pharmacological ischemic preconditioning agent. In a previous report, we showed that DZ had a strong protection of rodent islets against ischemia-reperfusion injury. In this follow-up study, we are investigating whether DZ supplementation to University of Wisconsin (UW) solution during pancreas harvesting and preservation has similar cytoprotection in non-human primate. Research Design and Methods: Cynomolgus monkey pancreata were flushed with UW (n = 3) or UW + 150 μM DZ (n = 3) during pancreas harvesting and preserved for 8 hrs prior to islet isolation. Islet yields, in vitro, and in vivo function were evaluated. Results: (i) Significantly higher islet yields were observed in the UW+DZ group than in the UW group (5,7887 vs. 23,574 IEq/pancreas; p = 0.02. and 5,396 vs. 1,646 IEq/gram; p = 0.019). (ii) The DZ-treated islets had higher insulin content (59.22 vs. 40.35 ng/islet; p = 0.006). The islets from the UW + DZ group post culture have significantly higher insulin positive cells per islet (96.15% vs. 82.74%; p = 0.004). (iv)The DZ-treated islets had a significant lower apoptotic cells per islet post culture (1.64% vs. 9.85%, p = 0.0015). (v) The DZ-treated islets had improved in vitro calcium influx in response to glucose challenge and in vivo function reflected in a higher cure rate and shorter days needed to reverse diabetes in a nude mouse transplant model. Conclusion: This study in NHP confirmed our previous observation in rodent model, showing similar DZ cellular protection on islet cells against ischemia-reperfusion injury that provides a strong evidence for future DZ application in human islet and pancreas transplantation.

Sub-normothermic preservation of donor hearts for transplantation using a novel solution, Somah: A comparative pre-clinical study

Hearts preserved ex vivo at extreme hypothermia (4°C) undergo time-dependent irreversible injury. Our studies using a novel solution, Somah, suggest that hearts are viably preserved at 21°C. In this study we evaluate the relative efficacy of Somah for preservation of hearts at 21°C when compared with the clinically used Celsior and University of Wisconsin (UWS) solutions. Porcine hearts arrested by cardioplegia at 21°C using Somah, Celsior or UWS solution were stored in the respective solutions at 21°C (n = 5) for 5 hours and then reperfused ex vivo for functional assessment. We assessed development of edema, cardiac tissue high-energy phosphate (HEP; ATP + creatine phosphate) levels and release of cardiac enzymes. Alterations in left ventricular wall thicknesses and functional parameters were examined by 2-dimensional (2D) echocardiography. Changes in myocardial oxygen consumption (MVO2) and lactate utilization were assessed at reperfusion. Heart weights were unaltered during 5-hour storage in all groups. After storage, HEP levels were 28.33 ± 5.51, 10.20 ± 2.78 and 5.92 ± 1.46 nmol/liter per milligram protein (p < 0.001) in the Somah, Celsior and UWS group hearts, respectively. Upon reanimation, 2D echocardiography showed edema in the Celsior and UWS hearts; prompt attainment of physiologic function was associated with rapid establishment of aerobic metabolism not requiring stimulatory interventions in the Somah hearts, but not in the Celsior/UWS hearts. Percent fractional area change, ejection fraction and stroke volume were significantly higher (p < 0.001) in Somah hearts than in Celsior and UWS group hearts. Increased synthesis of HEP, rapid metabolic switch and optimal function together provide evidence that hearts procured for transplantation are preserved in a superior viable condition at 21°C with Somah, but not with other commonly used clinical preservation solutions.

Amifostine enhances the antioxidant and hepatoprotective effects of UW and HTK preservation solutions.

To investigate whether amifostine contributes to the antioxidant and cytoprotective effects of histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW) preservation solutions. Forty-eight Sprague Dawley male rats were equally divided into six groups: (1) ringer Lactate (RL) group; (2) RL + amifostine (RL + A) group; (3) HTK group; (4) HTK + A group; (5) UW group; and (6) UW + A group. Rats in the RL + A, HTK + A and UW + A groups were administered amifostine intraperitoneally at a dose of 200 mg/kg prior to laparotomy. The RL group was perfused with RL into the portal vein. The RL + A group were perfused with RL into the portal vein after amifostine administration. The HTK group received an HTK perfusion while the HTK + A group received an HTK perfusion after administration of amifostine. The UW group received a perfusion of UW, while the UW + A group received a UW perfusion after amifostine administration. Liver biopsy was performed to investigate histopathological, immunochemical [transferase mediated dUTP nick end labeling (TUNEL), inducible nitric oxide syntetase (iNOS)] and ultrastructural alterations. Biochemical alterations were determined by examining levels of alanine aminotransferase, alkaline phosphatase and nitric oxide in the perfusion fluid. Pathological sinusoidal dilatation and centrilobular hydropic alteration were significantly lower in the groups that received amifostine prior to preservation solution perfusion. Although the best results were obtained in the UW + A group, we did not observe a statistically significant difference between the UW + A and HTK + A groups. iNOS grades were significantly lower in the amifostine groups 12 h after treatment. When the amifostine groups were compared against each other, the iNOS grades obtained from the UW + A and HTK + A groups were similar while the RL + A group had a much poorer score. TUNEL assays demonstrated a lower apoptosis ratio in the amifostine groups than in the non-amifostine groups 12 h after treatment. No statistically significant difference was observed between the UW + A and HTK + A groups for apoptosis. Cellular ultrastructure was best preserved in the UW + A and HTK + A groups. Here, we show that preoperative administration of a single dose of amifostine is sufficient to minimize the preservation damage in hepatic cells.

Muscle is a target for preservation in a rat limb replantation model.

Ischemia exceeding 6 hours makes clinical limb replantation difficult and places the patient at risk of functional deficit or limb loss. We investigated the preservation of muscle function and morphology with solutions in rat hindlimb in vivo and in vitro. Quadriceps femoris muscles from luciferase transgenic rats were preserved for 24 hours at 4°C in extracellular-type trehalose containing Kyoto (ETK), University of Wisconsin (UW), or lactated Ringer's (LR) solution (control). Muscle luminescence was measured with a bioimaging system. Amputated limbs of Lewis rats preserved with ETK, UW, or LR for 6 or 24 hours at 4°C were transplanted orthotopically. At week 8, terminal latency and amplitude were measured in the tibialis anterior muscle. The muscles were also analyzed histologically. Isolated muscles preserved in ETK or UW had significantly higher luminescence than did muscles immersed in LR (P < 0.05). In the 6-hour-preserved limb transplantation model, although the 3 groups had almost the same terminal latency, electrical amplitude was significantly lower in the LR group. Histologically, muscles preserved with LR showed the most atrophic changes. In the 24-hour-preserved model, the survival rate of the LR group was 37.5% in contrast to 80% in the ETK and UW groups. Electrical signals were not detected in the LR group owing to severe muscle atrophy and fibrosis. The ETK and UW groups showed good muscle function electrophysiologically. Preservation solutions can protect muscle function and morphology in ischemia-reperfusion limbs and improve recipient survival rates after transplantation of long-term-preserved limbs.

Comparison of New Preservation Solutions, HN-1 and University of Wisconsin Solution, in Pancreas Preservation for Porcine Islet Isolation.

For pancreatic islet transplantation, maintaining organ viability after pancreas procurement is critical and a major determinant for better graft function and survival. University of Wisconsin (UW) solution is currently the gold standard for abdominal organ preservation and the pancreas in particular. However, in the use of UW preservation solution for islet transplantation, there are disadvantages to be overcome, such as the inhibition of collagenase activity during pancreatic digestion. In this study, we compared UW solution with HN-1 solution in pancreas preservation for islet isolation. Islet yield was significantly greater in the HN-1 group than the UW group both before and after purification. In the in vitro assay, the adenosine triphosphate content in cultured islets was significantly higher in the HN-1 group than in the UW group. Furthermore, in streptozotocin-induced diabetic nude mice, the islet graft function of the HN-1 group was superior to that of the UW group. We concluded that the use of HN-1 solution is a promising approach for optimal pancreas preservation in islet transplantation.

Organ Biology-New Development.

Organ Biology-New Development.

Protective effect of perfluorocarbon emulsions on rat donor lung

Objective To evaluate the protective effect of perfluorocarbon emulsions (FCE) on donor lung of rats during storage.Methods Twenty-four healthy male Sprague-Dawley rats,weighing 350-400 g,were equally and randomly divided into 2 groups using a random number table:University of Wisconsin (UW) solution group (UW group) and FCE group (FCE group).After the model of lung perfusion was established according to the method described by Fischer et al,the lung and heart were removed and perfused with 4 ℃ UW or FCE preservation solutions.The lung was taken out when stored for 6 h for determination of SOD activity (by WST assay),malondialdehyde (MDA) content (by TBA assay),and activity of myeloperoxidase (MPO) and content of interleukin1 β (IL-1β),IL-6,tumor necrosis factor-apha (TNF-α) (using ELISA) in lung tissues and for microscopic examination of pathologic changes.Results MPO activity was significantly lower in UW group than in FCE group (P ＜0.05).There were no significant differences in the SOD activity and content of MDA,IL-1β,IL-6,TNF-α between the two groups (P ＞ 0.05).Conclusion FCE can reduce the neutrophil infiltration in lung tissues,indicating that FCE is more superior to UW solution in reduction of injury to the donor lung of rats. Key words: Fluorocarbons ; Emulsifying agents; Lung transplantation; Cytoprotection

Histidine-Tryptophan-Ketoglutarate and University of Wisconsin Solution Demonstrate Equal Effectiveness in the Preservation of Human Pancreata Intended for Islet Isolation: A Large-Scale, Single-Center Experience

We previously reported a small-scale study on the efficacy of histidine-tryptophan-ketoglutarate (HTK) solution versus University of Wisconsin (UW) solution on pancreas preservation for islet isolation. In this large-scale, retrospective analysis (n = 252), we extend our initial description of the impact of HTK on islet isolation outcomes and include pancreatic digestion efficacy, purification outcomes, and islet size distribution. Multivariable linear regression analysis, adjusted for donor age, sex, BMI, cold ischemia time, and enzyme, demonstrated similar results for the HTK group (n = 95) and the UW group (n = 157), including postpurification islet yields (HTK: 289,702 IEQ vs. UW: 283,036 IEQ; p = 0.76), percentage of digested pancreatic tissue (HTK: 66.9% vs. UW: 64.1%; p = 0.18), and islet loss from postdigestion to postpurification (HTK: 24,972 IEQ vs. UW: 39,551 IEQ; p = 0.38). Changes in islet size between the postdigestion and postpurification stages were comparable within each islet size category for HTK and UW (p = 0.14-0.99). Tissue volume distribution across purification fractions and islet purity in the top fractions were similar between the groups; however, the HTK group had significantly higher islet purity in the middle fractions (p = 0.003-0.008). Islet viability and stimulation indices were also similar between the HTK and the UW groups. In addition, we analyzed a small sample of patients transplanted either with HTK (n = 7) or UW (n = 8) preserved islets and found no significant differences in posttransplant HbA1c, β-score, and frequency of insulin independence. This study demonstrates that HTK and UW solutions offer comparable pancreas preservation for islet transplantation. More in vivo islet outcome data are needed for a complete analysis of the effects of HTK on islet transplantation.

Effect of Prostaglandin E-1 on Wisconsin University and Histidine-Tryptophan-Ketoglutarate Preservation Solutions on Preservation Injury of the Perfused Liver

BackgroundThe aim of this study was to investigate the effects of prostaglandin E-1 (PGE-1) on preservation injury in livers perfused with the University of Wisconsin (UW) or histidine-tryptophan-ketoglutarate (HTK) solutions. Materials and methodsFive groups each including six rats included. Ringer’s lactate RL (group 1), HTK (group 2), HTK + PGE-1 (group 3), UW (group 4), or UW PGE-1 (group 5). Liver tissue and preservation fluid samples were obtained from the perfused lives for pathological and biochemical examinations respectively at 0, 6 and 12 hours. ResultsUpon biochemical examination, aspartate aminotrasnferase and alanine aminotransferase values were highest among the group with RL solution and lowest with PGE-1. Liver structure was found to be damaged immediately after RL solution, whereas it was preserved in the other four groups. Fewer cellular changes were reported at the end of 12 hours in the groups administered PGE-1 compared with the other groups. ConclusionsPGE-1 when applied before preservation protected liver functions, decreased pathologic injury, and delayed changes that occur under cold ischemic conditions.

Role of Hydroxyethyl Starch in Ischemia-Reperfusion Injury in Rat Intestinal Transplantation

ObjectiveThis study was designed to evaluate the role of 0%, 3%, 6% hydroxyethyl starch (HES) and University of Wisconsin (UW) perfusion and preservation solutions on ischemia-reperfusion injury (IRI) of rat intestinal transplantations, solutions, respectively. Materials and methodsRats underwent orthotopic intestinal transplantation (Lewis to Lewis) after using perfusion and preservation saline (group l), 3% HES (group 2), 6% HES (group 3), or UW (group 4) solutions. The change in weight was recorded from preoperative to postoperative day (POD) 30. At 30 minutes after reperfusion, we harvested intestinal juice preoperatively as well as at 30 minutes after reperfusion and on POD 1 and 3 when recipients underwent open surgery for maltose absorption tests and sampling. The Park' scores of IRI were evaluated by light microscopy after hematoxylin and eosin (H&E) staining. ResultsAn increased weight was more evident in group 2 than the other groups, particularly the on POD 1 and POD 3 (P < .05). It was significantly greater than groups 1 and 3 on POD 7 (P < .05). Compared with the other groups, the 30-minute post-reperfusion. Park score and intestinal juice content in group 2 was decreased significantly (P < .01), while in group 3 the Park score was increased, and the maltose absorption level decreased significantly (P < .05). ConclusionThree percent HES solution attenuated IRI in rat intestinal transplantation. High-concentration HES solutions were unfit for intestinal preservation. Thus the adverse effects of UW solution may be attribute at least in part to its high HES, concentration.

HTK preservative solution is associated with increased biliary complications among patients receiving DCD liver transplants: A single center experience

This study compares biliary complication rates associated with use of two different preservative solutions, Histidine-Tryptophan-Ketoglutarate (HTK) and University of Wisconsin (UW), utilized in orthotopic liver transplantation (LT) with donations after cardiac death (DCDs). Between 1997-2010, we retrospectively studied 35 LTs performed utilizing DCD donors, preserved either with HTK (n=17) or UW(n=18). Biliary complications were defined by the presence of anastomotic strictures, non-anastomotic strictures, and/or biliary leak on endoscopic retrograde cholangiopancreatography. HTK and UW cohorts were similar in terms of demographics as well as pre- and post-operative biochemical profile. Donor age was significantly higher among HTK compared to UW recipients (41.5 ± 11.9 vs. 26.2 ± 8.8 years, p<0.001). The incidence of post-LT biliary complications was higher in the HTK group (76% vs. 39% in UW group, p=0.041). Hepatic arterial thrombosis (HAT) was observed among 3 HTK patients (17.7%) and 1 UW patient (5.6%), p=0.33. No patients underwent retransplantation in UW group, five recipients in HTK group underwent retransplantation (29%), p=0.019; 4 due to biliary complications and 1 due to HAT. This single-center study reveals that the use of HTK preservative among DCD donors is associated with an increased risk of biliary complications. Multicenter retrospective studies are suggested to further verify this observation.

Improved Kidney Isograft Survival with a New Histidine-Tryptophan-Ketoglutarate (HTK)-Based Cold Preservation Solution

Introduction: Aim of this study was to evaluate a new Histidine-Tryptophan-Ketoglutarate (HTK)-based cold preservation solution with (N46/25/7.5 Custodiol N; Deferoxamine 25 mmol/l, LK 614 7.5 mmol/l) and without addition of iron-chelators (N46) in comparison with traditional HTK and University of Wisconsin (UW) solution in a rat renal transplant model. Methods: Left kidneys of Lewis rats were perfused with and stored in the respective preservation solution at 0-4°C for 24h (short term series - STS) or 18h (long term series - LTS). Then they were transplanted isotopically into Lewis rats followed by right nephrectomy. A group of animals underwent mobilization of the left kidney and right-sided nephrectomy as sham operation. A second control group consisted of rats receiving HTK-perfused kidneys without cold storage. Observation time was 7 days in the STS and 24 weeks in the LTS. Prior to sacrifice, animals were held for 24h in a metabolic cage for urine collection with free access to food and water. Creatinine and blood urea nitrogen (BUN) were measured in blood and urine. Clearances were calculated. Histological sections were scored for acute tubulus necrosis (ATN; 0=0%, 1=up to 25%; 2=up to 50%; 3=up to 75%; 4=up to 100% of field of view (FOW); mean of 20 FOW's per section at 10x magnification) All investigators were blinded to the groups except to the control and the UW groups (the latter due to the sticky consistency of the UW solution). The pathologist was blinded to all groups. Kaplan-Meier curves, normality tests, ANOVA and Bonferroni's Multiple Comparisons post-hoc test for were performed using Graph pad Prism. Results: In the STS, all rats with an HTK-preserved kidney (n = 11) were dead on day 4. 7 of 10 (70%) animals of the UW group survived up to one week. Rats showed significantly better survival with kidneys preserved with N46 (7 of 8; 89%) and N46/25/7.5 (7 of 7; 100%). All rats in the control groups survived (each n = 5). There was no significant difference between the treated groups in s-Creatinine, BUN, Creatinine or BUN clearance. The ATN score was 1.5±0.2 in the UW group, 1.0±0.3 in the N46 group and 1.0±0.1 in the N46/25/7.5 group but missed statistical significance. In the LTS, both groups, N46 and N46/25/7.5 (each n=12), showed 100% survival after 24 weeks. Significantly fewer rats survived with a UW preserved kidney (n=8 of 11; 73%). All rats in the control groups survived (each n=12). There was no statistical significant difference between the treated groups in s-Creatinine, BUN, Creatinine or BUN clearance. But proteinuria was significantly higher in the surviving UW rats (1292±461 mg/dl) in comparison to N46 (355±55 mg/dl; p< 0.01) and N46/25/7.5 (508±106 mg/dl; p< 0.05). The ATN score was also significantly better in the N46 (0.76±0.06) and N46/25/7.5 group (0.72±0.09) than in the UW group (1.18±0.12; p< 0.01 each). Conclusions: The new HTK-based preservation solution N46/25/7.5 (Custodiol N) and its base solution N46 improve renal isograft survival in comparison to both standard preservation solutions HTK and UW.

Preservation Solutions for Liver Transplantation in Adults: Celsior versus Custodiol: A Systematic Review and Meta-analysis With an Indirect Comparison of Randomized Trials

BackgroundThe University of Wisconsin (UW) solution has been recognized as the gold standard for liver preservation; however, it possesses some limitations, and other solutions exist for organ preservation. The aim of this study was to compare the liver functions of transplanted grafts that had been stored in Celsior and Custodiol solutions. MethodsWe searched the MEDLINE, EMBASE, LILACS, Cochrane Central Register of Controlled Trials, and SCIELO databases. We included randomized and quasi-randomized, controlled trials that compared the efficacy and safety of Celsior and Custodiol with UW solution for liver preservation in adults. The factors that were considered for analysis were their impacts on primary dysfunction (primary nonfunction and initial poor function), ischemic-type biliary lesions, and patient and graft survival rates. Because of the lack of direct evidence, an indirect comparison of Celsior and Custodiol was calculated. ResultsWe identified 3 randomized controlled trials and 1 quasi-randomized, controlled trial to pool in a meta-analysis of Celsior versus UW solutions. The number of episodes of primary dysfunction was lower in the Celsior group (7.4%) than in the UW group (9.8%), but the difference was not significant (relative risk [RR], 0.68; 95% confidence interval [CI], 0.22–1.97). Two randomized controlled trials compared Custodiol and Wisconsin solutions were identified. The number of episodes of primary dysfunction was also lower in the Custodiol group (3.0%) compared with the Wisconsin group (8.4%), but the difference was not significant (RR, 0.36; 95% CI, 0.08–1.70). An indirect comparison using data from the main analysis revealed no difference between the Celsior and Custodiol solutions (RR, 1.88; 95% CI, 0.57–6.16). ConclusionThe Celsior and Custodiol solutions performed similarly to UW solution as preservation solutions in liver transplantation clinical settings.

Comparative Impact on Islet Isolation and Transplant Outcome of the Preservation Solutions Institut Georges Lopez-1, University of Wisconsin, and Celsior

Institut Georges Lopez-1 (IGL-1) is a preservation solution similar to University of Wisconsin (UW) with reversed Na/K contents. In this study, we assessed the impact of IGL-1, UW, and Celsior (CS) solutions on islet isolation and transplant outcome. We retrospectively analyzed 376 islet isolations from pancreases flushed and transported with IGL-1 (n=95), UW (n=204), or CS (n=77). We determined isolation outcome and β-cell function in vitro. Transplanted patients were divided into three groups depending on preservation solution of pancreas, and islet graft function was assessed by decrease in daily insulin needs, C-peptide/glucose ratios, β-scores, and transplant estimated function at 1- and 6-month follow-up. IGL-1, UW, and CS groups were similar according to donor age, body mass index, and pancreas weight. There was no difference in islet yields between the three groups. Success rates, transplant rates, β-cell secretory function, and viability were similar for all three groups. We observed no difference in decreased insulin needs, C-peptide glucose ratios, β-scores, and transplant estimated function at 1- and 6-month follow-up between IGL-1, UW, and CS groups. Our study shows that IGL-1 is equivalent to UW or CS solutions for pancreas perfusion and cold storage before islet isolation and transplantation.

Impact of Normothermic Preservation with Extracellular Type Solution Containing Trehalose on Rat Kidney Grafting from a Cardiac Death Donor

BackgroundThe aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx).MethodsPost-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringer's (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4–6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2).ResultsUsing the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion.ConclusionsNormothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.

Preservation of pancreatic islets in cold UW solution before transplantation

Culture of islets prior to transplantation needs to be revisited for maintaining functional islet capacity. This study was conducted to compare cold UW (University of Wisconsin) preservation with conventional culture based on insulin secretory capacity in vitro and in vivo. Islets isolated from Wistar rats were either cultured for 24 h at 37°C in RPMI1640 medium or DMEM containing various concentrations of glucose or preserved for the same period in UW solution or in DMEM solution at 4°C. The islet yield in UW group, but not in other groups, was maintained as comparable with that of fresh islets. Insulin secretory capacity in response to glucose was maintained only in the islets of UW group, but not in other groups. SCID mice given 300 IEQ islets of UW group showed gradual restoration of normoglycemia as found in the mice given freshly isolated islets. Meanwhile, those mice given cultured islets for 24 h at 37°C in RPMI1640 medium showed rapid decrease of blood glucose levels on day 1 followed by relatively elevated levels on day 2, suggesting unstable insulin secretory capacity of islets. Morphological staining with anti-HMGB1 (high mobility group B1) antibody revealed central damage of islets in all culture groups regardless of glucose concentration and in islets of cold DMEM group, whereas those in the UW group were quite intact. These results suggest that cold preservation in UW solution is simple and beneficial in protecting islets morphologically and functionally before transplantation.

An Effective Method to Release Human Islets From Surrounding Acinar Cells With Agitation in High Osmolality Solution

Introduction Islet purification is mainly performed by the density gradient method. However, purification of the embedded islets that are surrounded by exocrine tissue should be difficult, because their density is similar to exocrine tissue. In this study, we performed chart review to assess the relationship between the ratio of embedded islets and efficacy of purification. Then, we tested several conditions of a new method to free the islets from surrounded exocrine tissues using high osmolality solution with gentle agitation. Materials and Methods First, we performed chart review of our human islet isolation. Second, embedded islet-enriched human islet fractions (embedded islets >50%) were suspended in University of Wisconsin (UW) solution (UW group, 320 mOsm/kg/H 20) or osmolality-adjusted UW solution (400, 500, and 600 mOsm/kg/H 20; 400 group, 500 group, and 600 group, respectively). Each tube was gently shaken at 4°C. The tissue samples were taken before shaking and after 15, 30, and 60 minutes. Islet yield, percentage of embedded islets, and viabilities were assessed. Results The chart review revealed that high ratio of embedded islets deteriorated the efficacy of islet purification. The islet yield in all groups except for the 600 group did not change at 15 minutes, but it decreased in all groups at 60 minutes. The average percentage of embedded islets before shaking was 62.6%. Although percentage of embedded islets were decreasing in all groups, it was < 20% at 15 minutes in the 500 and 600 groups whereas it was >44% in the UW group, which indicated that higher osmolality would have a greater effect. Viability was >95% in all groups at 30 minutes. Conclusions The embedded islets deteriorated the efficacy of islet purification. Gentle agitation of embedded islets in high osmolality (500 mOsm/kg/H 2O, 15 minutes) could release islets from surrounded exocrine tissue.

Liver Preservation with SCOT 15 Solution Decreases Posttransplantation Cholestasis Compared with University of Wisconsin Solution: A Retrospective Study

Background SCOT 15 is a new solution to preserve abdominal organs for transplantation. Its principal characteristic is the use of polyethylene glycol. Herein We report our experience using SCOT 15 compared with the reference University of Wisconsin (UW) solution for hepatic transplantation. Methods We compared 2 groups: SCOT 15 (n = 33; 2009–2010) versus UW (n = 34; 2008–2010), which were paired for cold and warm ischemic times, donor ages, and graft weights. Endpoints were biologic tests in the first 2 months after the operation. A linear mixed model was used to evaluate longitudinal changes and influences of each solution. Results No primary failure was observed. At postoperative day 0, transaminase values were higher in the SCOT 15 than in the UW group: aspartate transaminase: 2,435 ± 399 vs 589 ± 83 IU/L ( P < .01); alanine transaminase: ALT: 1,207 ± 191 vs 484 ± 64 IU/L ( P < .05), then returned to low levels in both groups. From day 0 to 8, coagulation factors reached normal values; there was no difference between the 2 groups. Total bilirubin decreased similarly in the 2 groups. However, from the second postoperative week (W1) to W8, the SCOT 15 group showed a slow decrease in the mean values of gamma-glutamyltranspeptidase (gGT) from 233 ± 125 to 130 ± 161 IU/L, which were significantly lower than those in the UW group, where the gGT remained around 300 IU/L ( P < .01). The End-Stage Liver Disease, Child-Pugh, or United Network for Organ Sharing scores, primary liver diseases, hepatitic C virus status, arterial or biliary complications, and male/female ratio, which was different in the 2 groups, did not statistically influence these results. Conclusions The main effect of cold storage of human liver using SCOT 15 compared with UW solution was to decrease cholestasis following transplantation.

Evaluation of Cysteine Effect on Redox Potential of Porcine Liver Preserved by Simple Hypothermia

Introduction Cysteine (cys), a thiol amino-acid, is involved in de novo glutathione (GSH) synthesis in the extra- and intracellular space. It is also probably involved in the anaerobic glycolysis process. Both these facts may affect the metabolic condition of the liver preserved by simple hypothermia for transplantation. The aim of the study was to verify whether cysteine addition to histidine-tryptophan-ketoglutarate (HTK) organ preservation solution showed a positive effect on liver redox potential after 12-hour preservation in simple hypothermia. Materials and methods After collecting livers of Great White breed pigs that underwent 30 min of warm ischemia, before 30-min perfusion and cooling to 4°C with modified HTK solution containing cysteine prior to 12 h of preservation. Activity of glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) was determined in liver homogenates after perfusion and after the preservation period. The results were compared with pure HTK, Ringer's and reference University of Wisconsin (UW) solutions. Results 30 min of perfusion and 12 h of cold preservation (CIT) in the Ringer's solution markedly increased GPx, SOD, and GR activities in liver homogenates compared with the activity using other fluids. After 12-h CIT the activities of GR, GPx and SOD were significantly higher in cys-modified HTK solution than the control HTK solution. They were comparable to the values recorded for the UW group. Conclusions Addition of cys to the HTK solution positively influenced the total pool of free radical scavengers in a liver undergoing 12-hour ischemia in the simple hypothermia, which was reflected in the elevated redox enzyme activity possibly due to cys participation in GSH synthesis.