Evaluation of rat liver apoptotic and necrotic cell death after cold storage using UW,HTK, and Celsior

BACKGROUND: The benefit of Celsior in liver graft preservation is controversial. In the isolated perfused rat liver model, we compared the effects of Celsior, University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) preservation solutions on liver cell death. METHODS: Rat livers were stored at 4 degrees C for 0, 8, 16, or 24 hr in either Celsior, UW, or HTK and reperfused for 90 min (37 degrees C). Bile secretion and perfusate levels of liver enzymes and histone-associated DNA fragments were measured. Apoptosis and oncotic necrosis were analyzed in biopsies by DNA gel electrophoresis, hematoxylin and eosin histology, and enzyme histochemistry for lactate dehydrogenase (LDH) and 5'-nucleotidase (5'-NT). RESULTS: Perfusate flow rate through the liver during perfusion did not significantly differ among preservation solutions. Bile secretion was best preserved in UW livers after 16-hr (versus HTK livers) and 24-hr storage (versus HTK and Celsior livers). Enzyme leakage from UW livers was lower compared with HTK livers after 8-hr storage (serum glutamic oxaloacetic transaminase [SGOT], LDH) and with Celsior and HTK livers after 16-hr (SGOT, LDH) and 24-hr storage (SGOT, serum glutamic pyruvic transaminase, LDH, purine nucleoside phosphorylase). In situ LDH and 5'-NT activities were best preserved in UW livers (up to 24 hr), whereas enzyme activities declined remarkably in HTK livers (after 8 hr) and Celsior livers (after 16 hr of cold storage). Although perfusate DNA fragment levels were repeatedly lowest from Celsior livers, apoptotic DNA laddering and the number of fragmented nuclei in hematoxylin and eosin sections was not different among livers after 8, 16, or 24 hr of storage. CONCLUSIONS: Celsior and UW are equally effective in preventing rat liver cell death after 0-16 hr of cold preservation as compared with the less effective HTK solution. After 24-hr cold storage, rat livers were best preserved in UW. Furthermore, there was no significant difference in mode of cell death (apoptosis or oncotic necrosis) after storage in any of the three solutions.     

Celsior solution compared with University of Wisconsin solution (UW) and histidine–tryptophan–ketoglutarate solution (HTK) in the protection of human hepatocytes against ischemia–reperfusion injury

Celsior, a new preservation solution in thoracic organ transplantation was evaluated for efficacy in cold preservation of human hepatocytes and compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). Human hepatocyte cultures were preserved at 4 degrees C in Celsior, UW and HTK for 2, 6, 12, 24 and 48 h with 6 h of reperfusion. Levels of lactate dehydrogenase (LDH; cell necrosis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; mitochondrial function), and adenosine 5'-triphosphate (ATP; loss of intracellular energy) were measured. Cell necrosis, mitochondrial dysfunction, and loss of ATP were significantly ( P<0.001, P<0.001, P<0.002, respectively) lower in Celsior than in HTK. The amount of cell necrosis and mitochondrial dysfunction in Celsior solution (CS) and UW was equal ( P=n.s.) up to 24 h and significantly lower in UW after 48 h ( P<0.001). Additionally, the intracellular level of ATP was significantly higher after ischemia ( P<0.001) and reperfusion from long-term ischemia (24, 48 h) ( P<0.002). We can conclude that Celsior was superior to HTK and equal to UW in the protection of human hepatocytes against cold preservation injury from ischemia and reperfusion. Furthermore, Celsior was effective in long-term preservation of human hepatocytes.     

不同器官保存液对大鼠肝脏缺血再灌注损伤时嘌呤核苷磷酸酶活性和透明质酸吸收率的影响

目的 探讨大鼠肝脏低温保存及常温缺血再灌注过程中在不同的保存液中嘌呤核苷磷酸酶(PNP)活性和透明质酸(HA)吸收率的变化.方法 将大鼠肝脏在三种不同保存液中低温保存16 h和24 h后,用37℃Krebs-Henseleit液连续循环灌注90 min,分别于不同灌注时间检测灌洗液中PNP活性和外源性透明质酸的吸收率的变化.结果 经过16 h的低温保存后,再灌注60 min前,HTK保存的肝脏中PNP明显高于uw和Celsior;60 min后HTK和Celsior保存的肝脏中PNP明显高于UW;经过24 h的低温保存后,再灌注15 min后,HTK保存的肝脏中PNP明显高于Celsior,而Celsior又明显高于UW.低温保存16 h后,再灌注时,3种保存液保存的肝脏对外源性透明质酸的吸收率均为负值,表明肝窦内皮细胞受到一定程度的损伤;保存24 h者,UW液保存肝脏外源性透明质酸的吸收率明显高于Celsior液和HTK液.结论 随着低温保存和再灌注时间的延长,大鼠肝脏中PNP活性逐渐增高,而外源性透明质酸的吸收率下降;二者可作为评价肝脏缺血再灌注损伤的指标.     

UW is superior to Celsior and HTK in the protection of human liver endothelial cells against preservation injury.

Celsior solution (CS), a new preservation solution in thoracic organ transplantation, was evaluated for its efficacy in cold preservation of human liver endothelial cells (HLEC) and was compared to University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). HLEC cultures were preserved at 4 degrees C in CS, UW, and HTK, for 2, 6, 12, 24, and 48 hours, with 6 hours of reperfusion. Levels of lactate dehydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and adenosine 5'-triphosphate (ATP) were measured after each interval of ischemia and the respective phase of reperfusion. Preservation injury of HLEC as measured by LDH release, intracellular ATP level, and MTT reduction were overall significantly (P CS > HTK.     

Influence of preservation solution on the isolation and culture of human hepatocytes from liver grafts

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 +/- 41% vs. 79 +/- 14%, p < 0.05), yield of cells (4.0 +/- 5.2 x 10(6) vs. 8.2 +/- 5.6 x 10(6) cells/g, p < 0.05), and protein content at 24 h of culture (0.6 +/- 0.6 vs. 1.2 +/- 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.     

Three Preservation Solutions for Cold Storage of Heart Allografts: A Systematic Review and Meta‐Analysis

Organ preservation solution has been designed to attenuate the detrimental effects during the ischemic period. The aim of this study was to systematically evaluate the evidence comparing preservation solutions for heart preservation. Studies were searched in PubMed, Embase, the Cochrane Library, the Transplant Library, and the International Clinical Trials Registry Platform. The primary outcomes were patient survival and donor heart dysfunction. The secondary outcomes were in‐hospital mortality and enzyme gene expression. The University of Wisconsin solution (UW) was associated with a significantly improved survival at 30 days and 90 days (hazard ratio = 1.16, 95% confidence interval [CI] = 1.11–1.22, P < 0.00001; risk difference [RD] = 0.03, 95% CI = 0.01–0.05, P = 0.002), compared with Celsior. Hearts preserved with UW exhibited less ischemic necrosis than those preserved with Celsior (RD = ?0.07, 95% CI = ?0.08 to 0.05, P < 0.00001). UW was associated with better survival compared with histidine–tryptophan–ketoglutarate solution (HTK). There was no statistical difference in donor heart dysfunction and in‐hospital mortality outcomes when comparing HTK with Celsior solution. During static cold storage preservation, this study suggests that UW solution has better clinical outcomes for heart transplantation compared with the other two organ preservation solutions. Besides, the protective effect of Celsior solution is similar to HTK solution in donor heart preservation.     

A comparative study of the most widely used solutions for cardiac graft preservation during hypothermia.

BACKGROUND: Reports conflict on the benefits of preservative solutions. We investigated the efficacy of the most widely used cardioplegic solutions by comparing extracellular solutions such as Celsior solution, St. Thomas Hospital solutions 1 and 2 (STH-1, STH-2), the modified University of Wisconsin solution (UW-1), Lyon Preservation solution (LYPS) from our laboratory, and intracellular solutions such as standard University of Wisconsin solution (UW), Bretschneider solution (HTK), Stanford solution (STF), and Euro-Collins solution (EC). METHODS: Male rats (n = 110) were randomized into 11 groups: LYPS, Celsior, STH-1, STH-2, UW-1, UW, HTK, STF, EC, and normal saline solution groups, and a control group. All hearts, except controls, were preserved by cold storage (8 hours at 4 degrees C) in the various solutions. We used an isolated non-working-heart model and biopsy specimens to assess heart preservation (n = 5/group). RESULTS: Hearts stored in the EC and saline solutions had poor left ventricular developed pressure (LVDP) x heart rate (HR) (1,407.5 +/- 154 and 1,390 +/- 439 mm Hg/mn, respectively). In contrast, hearts stored in LYPS and Celsior had a LVDP x HR close to control hearts (31,349 +/- 1,847, 27,620 +/- 1,207, and 36,627 +/- 1,322 mm Hg/mn, respectively), whereas hearts stored in STH-1, STH-2, UW-1, UW, HTK, and STF had intermediate functional response (14,278 +/- 2,176, 12,402 +/- 1,571, 11,428 +/- 1,629, 11,603 +/- 2,521, 7,045 +/- 537, and 7,086 +/- 1,206 mm Hg/mn, respectively). Hearts preserved with STH-2, UW, HTK, STF, EC, and saline solution showed significantly increased release of creatine kinase and lactate dehydrogenase than did control hearts or hearts preserved in Celsior, LYPS, STH-1, and UW-1. The energetic charge (EC = [(0.5 adenosine diphosphate + adenosine triphosphate) / (adenosine triphosphate + adenosine diphosphate + adenosine monophosphate)]) in STH-2, UW, HTK, STF, EC, and saline groups was significantly lower (p < 0.05) than in the other groups. CONCLUSION: Extracellular-type solutions provided better preservation than did intracellular-type solutions. However, UW and UW-1 (intracellular- and extracellular-type solutions) provided equivalent preservation of cardiac function. Preservation quality may be attributed to calcium, often added to extracellular solutions. Among extracellular solutions, Celsior and LYPS solution showed comparable efficacy on left ventricular function and seemed to offer better preservation than the other solutions tested in this study.     

Luminal preservation of rat small intestine with University of Wisconsin or Celsior solution

AIMS: Luminal administration of a preservation solution that prevents mucosal injury may decrease posttransplant complications. However, luminal administration of University of Wisconsin solution (UW) is controversial. In this study, we examined the potential of Celsior as a luminal small bowel preservation solution in comparison to UW or UW enriched with glutamine. METHODS: Small bowels of six normal WagRij rats were excised and divided into six equal segments. Each segment was luminally flushed with 10 mL ice-cold UW, UW with glutamine (20 g/L) or Celsior, and stored for 0, 2.5, and 24 hours at 4 degrees C. LDH, glucose, and lactate concentrations were determined in the preservation solutions. Histologic changes were determined using the Park score. RESULTS: Lactate dehydrogenase (LDH) was increased in all solutions after 2.5- and after 24-hour preservation. However, LDH was lower in Celsior than UW and UW with glutamine. Furthermore, higher glucose and lactate levels were found after 2.5- and 24-hour preservation in UW and UW with glutamine compared to Celsior. Histologically, jejunal segments were more susceptible to preservation than ileal segments, irrespective of the preservation solution used. Mucosal injury was evident after 2.5 hours (Park Scale 0-3) and increased significantly after 24 hours (park scale 3-6). CONCLUSIONS: Based on the lower glucose, lactate, and LDH levels in small intestines stored in Celsior, this study suggests that Celsior is a better luminal preservation solution than UW. Unfortunately, histological evaluations still show severe mucosal injury, indicating that there is a need for better luminal preservation solutions or for concomittant intravascular delivery of a preservation solution.     

Rescue of the Cold Preserved Rat Liver by Hypothermic Oxygenated Machine Perfusion

The aim of the study was to investigate whether hypothermic oxygenated liver perfusion after cold liver preservation resuscitated metabolic parameters and whether this treatment had a benefit for liver viability upon reperfusion.
We preserved rat livers either by cold storage (UW) for 10 h, or by perfusion for 3 h (oxygenated modified UW) after 10 h cold storage. We assessed viability of livers after preservation and after ischemic rewarming + normothermic reperfusion ex vivo . Ten hour cold storage reduced mitochondrial cytochrome oxidase and metabolically depleted the livers. Oxygenated perfusion after cold storage resulted in uploaded cellular energy charge and oxidized mitochondrial cytochrome oxidase. Reperfusion after 10 h cold storage increased formation of superoxid anions, release of cytosolic LDH, lipid peroxidation, caspase activities and led to disruption of sinusoidal endothelial cells. In contrast, reperfusion after 10 h cold storage + 3 h hypothermic oxygenated perfusion resulted in no changes of lipid peroxidation, bile flow, energy charge, total glutathione, LDH release and of caspase activation, as compared to fresh resected livers.
This study demonstrates, that a metabolically depleted liver due to cold storage can be energy recharged by short-termed cold machine perfusion. The machine perfused graft exhibited improved viability and functional integrity.     

Maintenance of cold-preserved porcine hepatocyte function with UW solution and ascorbic acid-2 glucoside

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 microg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 +/- 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 microg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 microg/ml) would be a useful cold preservation means for the development of cell therapies.     

Comparison of solutions for preservation of the rabbit liver as tested by isolated perfusion

The University of Wisconsin (UW) solution consists of a relatively complex mixture of agents. In this study we compared simpler preservation solutions, namely, histidine-tryptophan-ketoglutarate glutarate (HTK) and phosphatebuffered sucrose (PBS) with different compositions of UW solution in the isolated perfused rabbit liver model. Livers were stored cold for 24 and 48 h. After 24 h of preservation, the amount of bile produced in UW-preserved livers was significantly greater (P<0.05) than that in HTK-preserved livers. Also, there was less LDH released into the perfusate in UW-preserved livers. There was more edema and lower K+/Na+ rations in HTK-preserved livers than in UW-preserved livers (all data P<0.05). After 48 h of preservation, the differences between livers preserved in UW or HTK solution were less noticeable than at 24 h and bile production was similar. LDH and AST release were greater in HTK-preserved livers than in UW livers, but these differences were not statistically significant. Preservation in PBS for 48 h was worse than in either UW or HTK solution. Substitution of polyethylene glycol (PEG) for hydroxyethyl starch (HES) in 48-h UW-preserved livers was not effective. We conclude that solutions simpler in composition than UW solution may be effective in kidney transplantation but do not appear suitable for successuful liver preservation.     

Energy metabolism of porcine liver during a 72-hour hypothermic perfusion with UW solution

Porcine livers were preserved for 72 h using continuous hypothermic perfusion with UW solution and fluorocarbon emulsion (FC-43). They were then recirculated with human blood for 2 h at 37 degrees C for the evaluation of their viability. During hypothermic perfusion and recirculation, the main metabolites of the perfusate and adenine nucleotide concentrations were measured in order to examine the energy metabolism of the livers. This study revealed that hypothermically perfused livers, which mainly utilize endogenous fatty acids as an energy substrate during this time were adequately preserved for 72 h. In terms of energy metabolism, it is suggested that when using the method of continuous hypothermic perfusion with UW solution and FC-43, the administration of adequate amounts of certain fatty acids might prolong the preservation period.     

Evaluation of preservation solutions by ESR-spectroscopy: superior effects of University of Wisconsin over Histidine-Tryptophan-Ketoglutarate in reducing renal reactive oxygen species

Despite the causative role of oxidative stress in renal ischemia-reperfusion (I-R) injury effects of preservation solutions on reactive oxygen species (ROS) release have not been sufficiently evaluated. We compared the effects of most common solutions in kidney transplantation, University of Wisconsin (UW) and Histidine-Tryptophan-Ketoglutarate (HTK). ROS formation in isolated perfused rat kidney was detected by electron spin resonance spectroscopy using spin label 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Donor kidneys from Lewis rats were pretreated with saline (controls), in therapeutic groups, kidneys underwent 18 h of cold storage (CS) preserved by HTK or UW solution. Experimental protocol included a stabilization period followed by additional I-R. Kidneys preserved by HTK produced highest ROS values in the control period after CS, whereas levels in UW and control group did not vary significantly. A peak release induced by additional I-R was also significantly highest in HTK kidneys, and UW did not differ from controls. During reperfusion, levels in HTK exceeded control and UW values. Renal vascular resistance, caspase-3-activity, and tissue hydration were enhanced in HTK compared with UW group, whereas ATP concentration was less reduced in UW-preserved tissue. These data show the greater antioxidative potential of UW solution, which also attenuated organ impairment after CS in the early reperfusion period.     

Value of energy substrates in HTK and UW to protect human liver endothelial cells against ischemia and reperfusion injury

Adenosine 5'-triphosphate (ATP) depletion is a major cause of cellular injury during ischemia and reperfusion in organ transplantation. Therefore, histidine-tryptophan-ketoglutarate solution (HTK; alpha-ketoglutarate) and University of Wisconsin solution (UW; adenosine) were supplied with energy substrates to achieve graft viability. Nevertheless, their efficacy for maintaining the ATP level, particularly in human liver endothelial cells, was uncertain. Furthermore, it is of interest whether a high ATP level is beneficial in human liver endothelial cell viability. We used human liver endothelial cells between the 3rd and 6th passages in a cell culture model. Human liver endothelial cells were exposed to hypothermic preservation (4 degrees C) in HTK and UW for 2, 6, 12, 24 and 48 h with subsequent reperfusion of 6 h. ATP and lactate dehydrogenase (LDH) were measured after each interval. In comparison to HTK, UW demonstrates a statistically significantly higher level of ATP after each interval of ischemia (p < 0.001) and reperfusion (p < 0.002). Additionally, UW-preserved human liver endothelial cells exceed the ATP level of the warm control during all intervals of ischemia. The loss of cell viability (LDH) was statistically significantly higher after ischemia (p < 0.01) and reperfusion (p < 0.01) in HTK than in UW except after the interval of 48 h. In conclusion, adenosine was more effective than alpha-ketoglutarate in maintaining a high ATP level in human liver endothelial cells after ischemia and reperfusion. Different pathways of energy substrate utilization were a contributing factor. The beneficial effect of the higher ATP level caused by adenosine to human liver endothelial cell viability was limited to 24 h of ischemia. Beyond this ischemia time we could not prove a favorable impact of adenosine on human liver endothelial cells.     

Preservation solutions for static cold storage of kidney allografts: a systematic review and meta-analysis

Static cold storage is the most prevalent method for renal allograft preservation. Several solutions have been designed to counteract the detrimental effects of cold ischemia and reperfusion. The aim of this study was to appraise the evidence for the currently available preservation solutions. We performed a systematic literature search using MEDLINE, EMBASE, the Cochrane Library, the Transplant Library and trial registries. Inclusion criteria specified any comparative, prospective study for deceased donor renal allografts. Studies were assessed for methodological quality. The primary outcome was delayed graft function (DGF). Fifteen trials with a total of 3584 kidneys were included. Eurocollins was associated with a higher risk of DGF than University of Wisconsin solution (UW) in two randomized controlled trials (RCTs) and histidine-tryptophan-ketoglutarate (HTK) in two RCTs. UW was associated with an equal risk of DGF compared with Celsior in three RCTs and HTK in two RCTs. There was limited data regarding other comparisons and outcomes. The choice of preservation solution has an effect on the incidence of DGF, which might, in turn, affect long-term outcomes. Both UW and HTK have lower rates of DGF than Eurocollins. There is no difference in the incidence of DGF with the use of Celsior, HTK and UW. These findings are supported by registry data.     

The use of myocytes as a model for developing successful heart preservation solutions

The development of a successful method to preserve the heart for relatively long periods (24-48 hr) requires demonstrating successful orthotopic transplantation and long-term survival after preservation. There are, however, multiple variables that may affect the quality of heart preservation, and it is nearly impossible to systematically study all the variables in this complicated model. One model that may be useful to study how preservation parameters affect heart cell preservation is the isolated myocyte preparation. In this study myocytes were isolated from the rabbit heart and the effects of up to 24 hr cold storage on viability measured to determine if this would be a suitable preservation model. Myocytes were stored in various preservation solutions including; EuroCollins (EC), two cardioplegic solutions (Stanford [ST] and Bretschneider solution [HTK]) and the University of Wisconsin solution (UW) with or without the addition of polyethylene glycol. The viability of myocytes was judged by measuring the effects of preservation and rewarming after preservation on cellular morphology (percent rod-shaped cells), ATP concentration, and LDH release. Myocytes preserved in the cardioplegic solutions were least well preserved after 12 and 24 hr storage, as judged by the loss of rod-shaped morphology and lower ATP concentration. Preservation in EC resulted in a decrease in the percent rod-shaped cells after 12 hr and 24 hr storage that was greater than obtained in the UW solutions. The best preservation of myocyte morphology and highest content of ATP was obtained in myocytes stored in the UW solutions, especially those containing PEG. The myocyte model of heart preservation shows a loss of cell integrity that is related to the preservation solution (HTK greater than ST greater than EC greater than UW-PEG) and these results are similar to what has been shown in the past with other models of heart preservation. Thus the myocyte model appears to be a useful method to test how many preservation solutions and preservation variables affect heart cell metabolism. In the future, results from these types of studies may find use in developing improved heart preservation solutions for testing in the orthotopic transplant model.     

Comparison of M-Kyoto solution and histidine-tryptophan-ketoglutarate solution with a trypsin inhibitor for pancreas preservation in islet transplantation

The use of University of Wisconsin (UW) preservation solution in islet transplantation has some disadvantages, including inhibition of collagenase activity for pancreatic digestion. Histidine-tryptophan-ketoglutarate (HTK) solution has demonstrated an efficacy similar to UW solution for organ preservation in clinical pancreas transplantation. Recently, we reported that islet yield from porcine pancreata was significantly gtreater when they were preserved using M-Kyoto solution compared with UW solution. Here, we compared HTK solution with ulinastatin (M-HTK) and M-Kyoto solution for islet yield. In porcine islet isolation, islet yield after purification was significantly greater in the M-Kyoto/perfluorochemical (PFC) group compared with the M-HTK/PFC group. The M-Kyoto/PFC group had a significantly lower ADP/ATP ratio compared with the M-HTK/PFC group, suggesting that different islet yields might be due to the differences as energy sources of the solutions used. In conclusion, M-Kyoto/PFC solution is better for pancreas preservation before islet isolation than M-HTK/PFC solution.     

Hemodynamic and biochemical changes during normothermic and hypothermic sanguinous perfusion of the porcine hepatic graft

Using an ex vivo liver sanguinous perfusion system, hemodynamic and biochemical changes of the porcine livers were studied, which were preserved cold (4 degrees C) for 24 hr in University of Wisconsin solution and reperfused with normothermic (37 degrees C) (n = 8) or hypothermic (32 degrees C) (n = 8) blood for 3 hr. Six more livers were reperfused with normothermic blood (37 degrees C) immediately after procurement as controls. The total hepatic blood flow was adjusted to 1 ml/min/g liver weight, in which hepatic artery and portal vein flows were administered at a 1:2 ratio. In livers stored cold for 24 hr in UW solution and perfused normothermically, a statistically higher hepatic artery resistance was exhibited at 30 and 60 min after reperfusion (P less than 0.05), and there was lower bile output (P less than 0.05) at 90 and 120 min as compared to the controls. In livers stored cold for 24 hr in UW solution and perfused hypothermically, as compared to ones perfused normothermically, statistically higher hepatic-artery and portal-vein resistances (P less than 0.05) were observed throughout the perfusion period and 60 min after reperfusion, respectively. In addition, bile output and oxygen consumption of these livers were statistically lower than those of ones perfused normothermically (P less than 0.05). In contrast, chemistries of the perfusate of livers perfused hypothermically were comparable to ones perfused normothermically. Histologic examination of the liver perfused hypothermically demonstrated hepatic arterial and/or portal venous congestion and mild-to-moderate hemorrhage in the portal triads. This study suggests that livers preserved for a prolonged period of time demonstrate a high hepatic arterial resistance shortly after revascularization, and that recipient hypothermia after revascularization may be a risk factor for the development of hepatic arterial thrombosis following liver transplantation.     

Comparison of HTK- and UW-solution for liver preservation tested in an orthotopic liver transplantation model in the pig

The aim of this experimental study was to compare the preservation potency of University of Wisconsin (UW) and HTK (Bretschneider) solutions in an orthotopic liver transplantation (OLT) model in pigs. Livers were harvested using an in situ perfusion technique, where organs were flushed with the solution being tested, stored on ice — cold storage (CS) — for 2 or 24 h and then transplanted. Parameters monitored were liver enzymes in serum, hepatic water content, high energy phosphates, nuclear magnetic resonance (NMR) relaxation time T2, light microscopy and bile production. CS for 24 h is an extreme in pig liver preservation and is not compatible with animal survival. Biopsies showed drastic morphological changes and grafts did not produce bile in either group. (Bile production 2 h CS: HTK, 5.6 ± 1.8 ml/h; UW, 4.7 ± 2.3 ml/h) Enzyme release after reperfusion (ASGOT, ?LDH) was higher in long-term preservation. Hepatic tissue water content significantly decreased during CS in UW preserved livers. Edema alter reperfusion (?H₂0: HTK 24 h = + 5.6%, UW 24 h= + 4.8%) and regeneration capacity after reperfusion (UW 2 h = 63%, HTK 2 h = 55%, UW 24 h = 30%, HTK 24 h = 30%) were not significantly different. However, we did not observe major differences in preservation potency between the solutions tested. Differences were correlated, rather, with length 9 time of CS, than with the solution used. Therefore, HTK solution seemed to be a low potassium containing alternative to UW solution.     

Cytochrome P-450 content and activity after cold storage of rat hepatocytes in university of wisconsin and sodium-lactobionate-sucrose solutions.

We compared the capacity of University of Wisconsin (UW) and of sodium-lactobionate-sucrose (SLS) hypothermic preservation solutions to maintain the integrity of the hepatic cytochrome P-450-dependent mono-oxygenase system. Isolated rat hepatocytes were stored for 0, 10, 24, and 48 hours in UW or SLS solution and were subsequently cultured shortly at 37 degrees C. Cell viability declined slightly but significantly in a time-dependent manner during cold preservation in either UW or SLS solution, and warm culture exacerbated this effect. Total cytochrome P-450 declined gradually after cold preservation and warm culture to reach values of 70% and 52% of unstored controls in cells preserved for 24 and 48 hours in cold UW solution, respectively. Storage in cold SLS solution yielded a similar decrease to 79% and 59% of unstored controls for the equivalent preservation times. Cytochrome P-450 activity was assessed by the metabolism of theophylline after various cold preservation times in UW or SLS solutions. Production of the major metabolite 1,3-dimethyluric acid was not significantly affected by extended cold preservation periods in either UW or SLS solutions. Similarly, the amount of residual theophylline remained stable in all groups, suggesting that alternative metabolic routes were not modified. These studies show that cold preservation in SLS solution is as effective as that in UW solution in terms of cell viability, cytochrome P-450 content, and activity toward theophylline. In addition, the significant reduction in cytochrome P-450 in conjunction with unaffected theophylline disposition suggests that certain cytochrome P-450 isoforms are specifically damaged by cold preservation and rewarming.