Comparison of University of Wisconsin (UW) and Eurocollins (EC) preservation solutions in a rat liver transplant model

The Eurocollins (EC) and University of Wisconsin (UW) preservation solutions were compared in a rat liver transplant model. After hepatectomy, 48 rat livers were flushed with either EC or UW preservation solution and were randomly assigned to 1, 12, 24, and 30 h of preservation at 4°C, resulting in eight groups each containing six livers. Following preservation, orthotopic liver transplantation with reconstruction of the hepatic artery was performed. The efficacy of the preservation solution was assessed at 48 h post-transplantation by survival histological features and aspartate transaminase assay (AST) values. None of the rats survived 30 h of liver preservation with EC whereas five out of six rats did with UW preservation. After 24 h of liver preservation, three of the six rats in the EC group survived, compared to all six rats in the UW group. Histological evidence of severe ischemia was found in both groups in all but one survivor (UW, 24 h). After 12 h of EC preservation, one rat died within 48 h and severe ischemic changes were found in the remaining five rats. Among the rats with 12 h of UW preservation, only two out of six showed ischemic changes, and all six rats survived beyond 48 h. Without preservation (1 h), ischemic damage was found in two out of six rats in each group and all rats survived. The median AST values were higher in the EC groups than in the UW groups; the difference became significant after 12-h preservation (EC 900 IU/l versus UW 465 IU/l) and 24-h preservation (EC 5220 IU/l versus UW 631 IU/l). However, the median AST value in the five surviving rats whose livers had been preserved for 30 h in UW climbed to 1880 (950–2240) IU/l.. We conclude that UW solution provides better long-term preservation than EC solution. However, even with UW solution, the observed mortality, the severity of ischemic changes, and the pronounced increase in the median AST value cast doubt upon the safety of liver preservation beyond 24 h.     

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.     

供肝热缺血后对冷保存的耐受时限初步探讨

目的探讨供肝热缺血后耐受冷保存的安全时限。方法利用本组所建立的小型猪肝移植模型,设定供肝热缺血时间为20min,根据在UW液中的冷保存时间不同分为3组,分别冷保存12、16、20h,于肝移植术中及术后检测肝功能、肝脏病理、肝组织ATP含量、移植肝脏再灌注后微循环血流量及动物术后1周存活率。结果UW液冷保存12h组肝移植后小型猪1周内全部存活,而冷保存16、20h组存活率分别为20%、0%;随着冷保存时间的从12h延长到20h,ALT、AST逐渐上升,肝脏ATP含量、肝脏微循环血流量逐渐下降,形态学结果显示肝组织细胞变性、坏死及超微结构损害的程度逐渐加重。冷保存12h组与后两组上述指标存在显著性差异,生化及肝脏微循环指标的改变与病理结果及动物生存率相符合。结论在本实验条件下,热缺血时间为20min的供肝耐受冷保存的安全时限约为12h。     

The use of the University of Wisconsin (UW) and Euro-Collins (EC) solutions either alone or in a combined method

From June 1988 to October 1990, a total of 100 orthotopic liver transplantations (OLTs) in 91 patients were performed at the Hospital Clínic of Barcelona. Euro-Collins (EC) solution was used as the flush and storage solution in 29 livers, and the University of Wisconsin (UW) solution was used in 24. A combined method, consisting of flushing and harvesting the liver with UW solution through the portal vein and with EC solution through the aorta, was used in the remaining 47 livers. Livers harvested using such a combined method showed substantially better postoperative function in terms of AST, ALT, and prothrombin activity than those harvested in EC solution alone. Although AST and ALT values were lower in patoents whose livers were harvested using the combined method than with UW alone, differences were not significant. On the other hand, prothrombin activity was consistently better in the UW group. Bilirubin levels, platelet count, and bile output showed no difference among the three groups. We conclude that the combined use of UW and EC solutions for flushing and harvesting is not hazardous to human liver preservation and, in fact, may considerably reduce the amount of UW solution needed and, consequently, the costs.Preliminary results from this study were presented at the First International Congress of the Society for Organ Sharing in Rome in June 1991 and will also appear in Transplantation Proceedings.     

Preservation of dog liver, kidney, and pancreas using the Belzer-UW solution with a high-sodium and low-potassium content

The UW solution developed for cold storage of the liver, pancreas, and kidney was used in a modified form in this study and tested in the orthotopic transplantation of dog livers, kidneys, and pancreases preserved for 48 hr. The modification was the alteration of the concentrations of potassium and sodium. The original UW solution contained 120 mM K+ and 30 mM Na+. In this study the Na+ was 140 mM and the K+ only 9 mM, all other agents were identical to the original UW solution. Six of 11 dogs survived with livers preserved for 48 hr. The five deaths were due to technical complications and unrelated to preservation failure. Postoperative AST and partial thromboplastin time (PTT) values were lower (statistically significant on days 1, 3, and 4) in livers preserved in the high Na+ UW solution than as previously shown in the high-k+ UW solution. Other measures of liver function (bilirubin and fibrinogen) were similar between the high-Na+ and high-K+ groups. Six dogs survived with kidneys preserved for 48 hr in the high-Na+ UW solution. The results were comparable to those obtained with the high K+ solution. Four of six dogs survived for up to 28 days with pancreases preserved for 48 hr. The two deaths were due to technical complications unrelated to preservation failure. Three of the four dogs had normal blood glucose values for one month, and intravenous glucose tolerances test on day 7 and 28 were identical to those obtained in pancreases preserved with the high-K+ UW solution. The high-Na+ version of the UW solution appears equally or slightly more effective for 48-hr organ preservation than the original high-K+ UW solution. The use of a high-Na+ UW solution reduces the problems of hyperkalemic cardiac arrest in in situ flushing of the donor for multiple organ harvesting and in transplantation of the liver. Thus, with this solution livers do not need to be flushed with a low K+-containing solution prior to transplantation.     

Assessment of hepatic viability during cold ischemic preservation

A reliable and easy method for assessing the viability of a cold ischemia-preserved donor liver prior to transplanation into the recepient is needed. Based on an earlier study, we hypothesized that liver free fatty acid (FFA) leakage into the preservation fluid may be a useful, atraumatic indicator of irreversible ischemic injury. The aim of the present study was to determine the time course and magnitude of liver FFA release into the preservation solution and its correlation with the duration of cold ischemic preservation compatible with survival after transplantation. Rat livers (n=48) were flushed and preserved with University of Wisconsin (UW) solution at 4°C for 0, 12, 24, and 48 h. Thereafter, half of the livers were analyzed for preservation fluid FFA (gas-liquid chromatography) and protein. The other half were perfused with Krebs-Henseleit (KH) solution at 37°C for 1 h. Bile secretion and liver enzyme release (SGOT, SGPT, and LDH) were measured in addition to perfusate FFA and protein. Total FFA in the preservation fluid was 24 g/g wet tissue after 12 h; it increased sharply 2.6-fold after 24 h and 3.7-fold after 48 h of preservation. Bile production was normal after 12 h of preservation but fell by 20% and 54% after 24 h and 48 h, respectively. LDH release rose from a value of 20 U/l at 0 time to 120 U/l and 260 U/l after 24 h and 48 h of preservation. These results suggest that liver viability declines sharply between 12 and 24 h of cold ischemic preservation, which corresponds with a sharp decrease in the 1-week survival from 100% to 33% after 12 h and 24 h, respectively, of cold ischemic preservation. We conclude that measuring FFA and LDH in the preservation solution of donor livers may be a useful means of assessing the quality of the cold-preserved liver before insertion into the recipient. We also speculate that a threshold FFA level in the UW preservation fluid indicating irreversible damage may be in the order of 35 g total FFA/g liver. Studies on the clinical applicability of our findings are currently under way.     

High-Na + low-K + UW cold storage solution reduces reperfusion injuries of the rat liver graft

The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K ⁺ UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na ⁺ UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na ⁺ UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K ⁺ UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na ⁺ UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na ⁺ UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K ⁺ UW solution. Liver preservation in the high-Na ⁺ UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution. Received: 26 August 1997 Received after revision: 12 November 1997 Accepted: 28 November 1997     

SX-1液对肝脏保存效果的实验研究

目的 应用改良后的Kamada二袖套法大鼠原位肝移植模型,检验SX-1液、HC-A液和UW液对肝脏保存的效果.方法 在无菌条件下配制肝脏保存液.建立大鼠原位肝移植模型.使用SX-1液、UW液和HC-A液保存大鼠肝脏2、8、24 h后行大鼠原位肝移植,于移植后6 h比较各项肝脏功能.结果 对于ALT、AST,SX-1液组(2、8、24 h)与UW液组同步升高,分析无统计学意义(P＞0.05),与HC-A液组相比,差异有统计学意义(P＜0.05).对于LDH,SX-1液组(2 h、8 h、24 h)与HC-A液组同步升高,差异无统计学意义(P＞0.05),与UW液组相比,差异有统计学意义(P＜0.05).对于分泌胆汁的肝脏个数,各组别与分泌胆汁的肝脏个数无差别(P＞0.05).本组内各时间点分泌胆汁个数有差别(P＜0.05).随肝脏保存时间增长,分泌胆汁的肝脏个数减少.结论 经大鼠原位肝移植模型证实,SX-1液在肝脏酶学方面与UW液作用相当,超过HC-A液水平.肝移植后6 h肝脏分泌胆汁的个数方面,SX-1液与HC-A液、UW液间无明显差别.     

HX—3液和UW液保存大鼠肝脏效果的比较

采用大鼠肝脏非循环离体灌注模型比较自制的ＨＸ－３液和ＵＷ液对大鼠肝脏的保存效果。实验结果显示，经ＨＸ－３液原位灌洗并保存４８小时的肝脏肝组织含水量正常，而同等条件下换用ＵＷ液，肝组织的含水量虽无明显变化，但都低于正常值；随着保存时间的延长，两组肝窦内皮细胞死亡率逐渐上升，但在２４小时以内两组肝窥内皮细胞死亡率的差异不显著．     

Verapamil improves rat hepatic preservation with UW solution

Verapamil, a calcium channel blocker, improves myocardial preservation during cold cardioplegia and protects against renal damage during periods of warm and cold ischemia. To determine if verapamil could prevent ischemic damage to livers during and after cold storage, harvested rat livers were flushed with either University of Wisconsin (UW) solution or UW solution with 25 mg/liter verapamil. Twenty rats were used in each group. After 24 hr of storage at 4 degrees C, livers were perfused with oxygenated blood through the portal veins for 2 hr at 37 degrees C and pH 7.4. Liver enzymes, electrolytes, and perfusate flow rate were determined at 30-min intervals. At 90 min of perfusion, the verapamil group of livers had less elevation of AST (110 +/- 17 IU/liter vs 172 +/- 25 IU/liter, P less than 0.05), ALT (115 +/- 21 IU/liter vs 210 +/- 34 IU/liter, P less than 0.05), and LDH (962 +/- 170 IU/liter vs 1452 +/- 253 IU/liter, NS). Verapamil livers produced more bile than controls (6.9 +/- 1.9 microliters/g vs 2.3 +/- 1.7 microliter/g, P less than 0.05) and maintained a higher portal flow rate throughout the perfusion. Both groups showed similar reduction in liver weights after storage (3.9 +/- 0.9% vs 2.8 +/- 0.7%) and required the same amount of bicarbonate for correction of acidosis during perfusion (2.6 +/- 0.2 mM vs 2.8 +/- 0.2 mM). Light microscopic exam after perfusion showed hepatocyte damage in 30% of control livers, but 0% of verapamil livers. We conclude that verapamil-treated rat livers showed less damage and better function upon reperfusion after 24 hr of cold storage. This agent may be clinically useful as an additive to the UW preservation solution for livers.     

Electrical impedance of the liver during experimental long-term liver preservation

Measurements of electrical impedance were performed to assess ischemic damage in the rabbit liver during long-term preservation with University of Wisconsin (UW) or histidine-tryptophan-ketoglutarate (HTK) solution. The impedance was measured at a frequency of 200 Hz after in situ perfusion and after cold storage for 24 and 48 hours in UW or HTK solution (six livers per group). Z(200 Hz) was significantly higher (P < .01) after 48 compared with 24 hours of cold storage with both protection solutions without significant differences between the livers preserved with both solutions. Electrical impedance was observed to be a sensitive indicator of liver damage during long-term protection, showing similar preservation quality for both preservation solutions.     

自制CMU-1液保存大鼠肝脏的实验研究

目的　探讨CMU 1液保存大鼠肝脏的效果。方法　根据灌注液和保存液的种类将Wistar大鼠分为两组 :UW组和CMU 1组 ,每组分 6h、12h、2 4h 3个保存时限 ,每亚组 6只大鼠。采用离体循环灌注模型 ,研究CMU 1保存液对保存肝脏能量代谢、生化功能、胆汁分泌及形态学方面的影响。结果　随着保存时间延长 ,肝组织TAN含量及AEC逐渐降低 ,CMU 1组较UW组下降略缓慢 ,保存 2 4h后高于UW组 (P <0 0 5 )。再灌注 12 0min后CMU 1组的肝脏分泌胆汁量较UW组多 (P <0 0 5 )。相同时限相比 ,灌出液中ALT、LDH值两组之间无显著差异 (P >0 0 5 )。肝脏组织学变化两组间无明显差异。保存 6h后 ,保存液pH值无明显变化 ;保存 12h后pH值下降 ,两组无明显差异 ;保存2 4h后 ,UW组pH值下降较CMU 1组明显。结论　CMU 1保存液保存大鼠肝脏效果与UW液相似 ,在改善保存肝脏能量代谢、预防细胞内酸中毒、胆汁分泌方面略优于UW液。     

Postoperative recovery of mitochondrial function of the human liver graft procured and preserved with University of Wisconsin (UW) solution

Abstract. Changes in arterial blood ketone body ratio (KBR) were investigated in 47 human liver transplantations. Of the 20 grafts preserved with University of Wisconsin (UW) solution, 10 had a cold preservation period of less than 10 h (UWS group) and 10 of more than 10 h (UWL group). In 27 other cases, grafts were preserved with EuroCollins (EC) solution for less than 10h (EC group). In the EC group, KBR increased over 0. 7 within 6h after reperfusion of the graft in 17 cases (63%) and within 24 h in 7 cases (26%). In the 3 other cases, KBR failed to recover, and these patients underwent retransplantation. In the UW group, KBR recovered within 6 h in 13 cases (65%) and within 24 h in 7 cases (35%). There were no significant differences between the UWS and UWL groups. It is shown that the mitochondrial function of liver grafts preserved with UW solution can be well maintained even after extended preservation periods of more than 10 h.     

Postoperative recovery of mitochondrial function of the human liver graft procured and preserved with University of Wisconsin (UW) solution

Changes in arterial blood ketone body ratio (KBR) were investigated in 47 human liver transplantations. Of the 20 grafts preserved with University of Wisconsin (UW) solution, 10 had a cold preservation period of less than 10 h (UWS group) and 10 of more than 10 h (UWL group). In 27 other cases, grafts were preserved with EuroCollins (EC) solution for less than 10 h (EC group). In the EC group, KBR increased over 0.7 within 6 h after reperfusion of the graft in 17 cases (63%) and within 24 h in 7 cases (26%). In the 3 other cases, KBR failed to recover, and these patients underwent retransplantation. In the UW group, KBR recovered within 6 h in 13 cases (65%) and within 24 h in 7 cases (35%). There were no significant differences between the UWS and UWL groups. It is shown that the mitochondrial function of liver grafts preserved with UW solution can be well maintained even after extended preservation periods of more than 10 h.     

Curcumin has potent liver preservation properties in an isolated perfusion model

BACKGROUND: Curcumin has profound antioxidant and anti-inflammatory properties. This research assessed the effect of curcumin on liver preservation. METHODS: Sprague-Dawley rat livers were flushed with different preservation solutions [Euro-Collins solution (EC), phosphate buffer saline (PBS), University of Wisconsin solution (UW)] with or without curcumin (25-200 microM) and stored at 4 degrees C for 24-48 hours. Livers were then perfused for 120 minutes via the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer solution at a pressure of 18 cm H2O in a perfusion apparatus. The livers in the normal (NL) group were flushed with EC, PBS, or UW, then immediately perfused (zero preservation time). RESULTS: We found that curcumin at 100 microM concentration had the optimal preservation characteristics. Portal flow rates and bile production were significantly higher and liver enzymes (alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase) were significantly lower in the EC+C livers and PBS+C livers than in the EC or PBS with optimum concentration of 100 microM of curcumin. Comparing UW+C vs. UW livers, at 24 hours there was no difference in these parameters; however, at 36 hours and 48 hours, portal flow rates and bile production were significantly higher in UW+C livers. CONCLUSIONS: We found that curcumin has inherent organ preservation quality as it enhanced liver preservation in PBS. In addition, curcumin enhanced the preservation quality of EC and UW solutions, thereby extending the preservation time while maintaining the organ quality.     

Glycogen storage of the liver: a determining factor of initial function of the hepatic graft]

In this study we have investigated the effects of hepatocytes glycogen storage on the quality of livers for transplantation. Rats were fed or fasted for 24 h and hepatocytes isolated and cold stored in UW solution for 24 and 48 hours. Viability of the cells was analyzed by LDH release after 2 hours incubation in L15 with O2. Also, rabbits were fed, fasted (48 h) or glucose fed (48 h) and livers cold stored for 6, 24 and 48 h in UW solution. Functions of the livers were analyzed by isolated perfusion for 2 hours. Hepatocytes from fasted rats released significantly more LDH than hepatocytes from fed rats after 24 and 48 h cold storage. In rabbit livers, fasting depleted glycogen by 85% but had no effect on ATP or glutathione concentration. Livers from fasted rabbits produced similar amount of bile, released similar concentrations of lactate dehydrogenase and aspartate transaminase into the perfusate, maintained similar concentrations of glutathione after 24 hours preservation when compared to fed animals. After 48 h preservation livers from fasted animals were less viable than livers from fed animals and the decrease of liver functions in livers from fasted animals preserved for 48 hours was prevented by feeding glucose. This study shows that liver glycogen storage in hepatocyte is an important metabolite for successful liver preservation. Glycogen may be a source for ATP and antioxydant synthesis during the early period of reperfusion.     

A comparison of Collins and UW solutions for cold ischemic preservation of the rat liver

A new solution which can extend successful preservation times for hepatic allografts was recently developed at the University of Wisconsin (UW). To examine the mechanism of improved viability using this solution, we developed a model of orthotopic hepatic transplantation in the rat. As a baseline study, we compared parameters of viability of allografts preserved in Collins solution to those preserved in UW, including survival, bile output, peak AST, and allograft weight change during storage. Seventy-four rats were transplanted following storage in Collins solution and 70 rats were transplanted after storage in UW. Cold-storage time varied between 2 and 24 hr. The survival with preservation in UW was significantly better than that with Collins when storage time was greater than 2 hr. The preservation time for a viable organ using UW was greater than double that using Collins. The peak AST using UW was lower than that with Collins for cold ischemic times (CIT) up to 10 hr, with significance demonstrated at 5-6 and 7-8 hr when compared with Collins. Prolonged CIT resulted in an increase in liver weight with Collins-preserved livers and a decrease in weight with UW-preserved livers. Using a model of orthotopic liver transplantation in the rat, we demonstrated a doubling of preservation time when UW solution was substituted for Collins. Similar improvements in recipient survival and biochemical parameters of injury have been demonstrated in the canine model and in human clinical trials.     

SWH液对大鼠肝脏保存有效时间的探讨

目的通过与UW液进行比较,探讨SWH液对SD大鼠肝脏保存的有效时间.方法应用大鼠原位肝移植模型,通过高效液相色谱法（HPLC）,检测肝组织ATP、ADP、AMP,计算总腺苷酸含量（TAN）及Atkinson＇s能量转换（EC）,判断肝脏能量代谢状态;通过检测谷丙转氨酶（ALT）、谷草转氨酶（AST）、乳酸脱氢酶（LDH）含量,判定肝脏功能;观察移植术后7d动物存活率,判定SWH液对大鼠肝脏有效保存的时间.结果保存18 h组的ATP、TAN、EC恢复水平明显低于保存8 h组,相差显著,保存12 h组与保存8 h组比较相差不显著;保存18 h组的ALT、AST、LDH明显高于保存8h组,差异显著,保存12 h组与保存8 h组比较相差不显著;SWH液组移植术后7 d动物存活率有高于UW液组的趋势.结论无论使用SWH液还是UW液,SD大鼠肝脏有效保存的时间都不超过12 h.     

Protective effect of fructose-1,6-bisphosphate in the cold storage solution for liver preservation in rat hepatic transplantation

Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect on liver injury following ischemic/reperfusion periods. FBP maintains ATP levels and thereby cellular energy metabolism, which is important to the liver during cold preservation. In the present study, we evaluated the effects of FBP on the composition of storage solutions for cold liver preservation. Adult male Wistar rats were randomly divided into three experimental groups. Hepatic perfusion and preservation were performed with UW, UW plus 10 mmol/L FBP (UWM), and FBP 10 mmol/L (FBPS) alone solutions. Biochemical measurements of AST, ALT, and TBARS were performed on samples of the cold storage solution at 0, 12, 18, and 24 hours preservation. FBPS and UW solutions showed similar preservation grades during 18 hours. Addition of 10 mmol/L of FBP to UW solution induced liver injury and a poor preservation grade. FBP appears to protect the liver from injury caused by free radicals when the preservation time is less than 18 hours. Therefore, FBP may exert a protective effect for the preservation of livers during cold storage, and could represent an important component of new cold storage solutions.     

SWH液对大鼠肝脏保存中肝细胞凋亡的影响

目的　通过与UW液进行比较 ,观察SWH保存液对大鼠肝细胞凋亡及其对肝移植受者存活率的影响。方法　应用大鼠原位肝移植模型 ,通过HE染色、电镜和TUNEL法检测肝细胞凋亡以及观察移植术后 7d动物存活率。结果　随着保存时间的延长 ,两组的凋亡指数 (AI)均无明显升高 ,保存 18h后 ,两组的AI亦无明显改变 ,仅在行肝移植恢复血流循环后AI才明显上升 ,移植前后存在显著性差异 ,P <0 .0 1,SWH液组略低于UW液组 ;SWH液组移植术后 7d动物存活率有高于UW液组的趋势。结论　有效地防止肝细胞凋亡可以提高肝脏的保存效果 ,在防止肝细胞凋亡方面 ,SWH液优于UW液。