冷冻保存大鼠肝脏离体再灌注时嘌呤核苷磷酸酶活性和透明质酸吸收率的变化

目的探讨不同保存液保存大鼠肝脏后离体再灌注时嘌呤核苷磷酸酶(PNP)活性和透明质酸吸收率的变化及意义。方法分别采用UW液、HTK液和Celsior液灌洗、冷保存Wistar大鼠肝脏16及24 h,然后用37℃的Kreb-Henseleit液在常温下连续灌注90 min,分别于灌注0、15、30、60和90 min时,从灌注液中取样,测定嘌呤核苷磷酸酶活性和外源性透明质酸吸收率的变化,据此评价肝窦内皮细胞的状况。结果经过16 h的低温保存,在再灌注60 min以前,HTK液组灌注液中PNP的含量明显高于UW液组和Celsior液组(P<0.01);再灌注60 min后,HTK液组和Celsior液组灌注液中PNP的含量明显高于UW液组(P<0.01)。经过24 h的低温保存,在再灌注15 min后,HTK液组灌注液中PNP含量明显高于Celsior液组(P<0.01),而Celsior液组又明显高于UW液组(P<0.01)。透明质酸的吸收率均为负值,说明内源性透明质酸的释放大于外源性透明质酸的吸收,且随着保存时间和再灌注时间的延长,这一趋势更加明显,其中HTK液组最明显,Celsior液组次之。结论随着低温保存和再灌注时间的延长,肝脏中PNP活性逐渐升高,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.     

Comparison of Celsior and UW solution in experimental pancreas preservation

BACKGROUND: The University of Wisconsin solution (UW) is the gold standard for pancreas preservation. Celsior (CEL) was formulated specifically for heart preservation. Recently, experimental and clinical experience has been reported on the application of CEL to abdominal organs. In this animal study, pancreas preservation with CEL was compared with that in UW solution. PATIENTS AND MATERIALS: Heterotopic, allogeneic pancreaticoduodenal transplantation was performed in female G?ttingen Minipigs (n = 12 donors, n = 12 recipients). The grafts were flushed and stored for 6 h at 4 degrees C in UW or CEL. The recipients were randomized into two groups receiving either UW (n = 6)- or CEL (n = 6)-preserved grafts with a follow-up of 5 days. Blood flow (laser Doppler), partial oxygen tension, histological changes, endothelin-1 (plasma, immunohistochemistry), lipase, amylase, trypsinogen activation peptide, and C-reactive protein (CRP) were measured. RESULTS: Partial oxygen tension was lower in the CEL group (P < 0.05). However, blood flow did not differ between UW- and CEL-preserved organs. The histomorphologic analysis of the pancreatic grafts revealed significantly less edema in the UW-preserved organs. Serum levels of amylase, lipase, CRP, and TAP taken from the central venous blood were comparable in the two groups, except for higher amylase values 36 h after reperfusion in the CEL group compared to the UW group (P < 0.05). Likewise, TAP taken from the portal venous effluent of the graft was found to be higher in the CEL group than in UW (P < 0.05). Endothelin-1 serum levels rose significantly during reperfusion without differences between the two groups. ET-1 immunohistochemistry revealed increased local ET-1 during reperfusion in all grafts. However, the ET-1 immunostaining in the CEL group was more pronounced than that in the UW group (P < 0.05). CONCLUSIONS: Our results suggest that CEL solution is not as effective in preventing pancreatic ischemia/reperfusion damage as the standard UW solution in experimental pancreas transplantation. Increased ET-1 immunostaining and reduced p(ti)O(2) in the CEL group indicate increased microcirculatory damage in the CEL group.     

Pulmonary preservation with Bretscheider''s HTK and Celsior solution in minipigs

Background: Pulmonary preservation with high potassium/low oncotic pressure Euro-Collins (EC) solution is associated with endothelial dysfunction and reduced surfactant function. We compared two low potassium solutions, histidine-tryptophane-ketoglutarate (HTK) and Celsior, to EC in lung ischemia-reperfusion injury. Methods: In 19 minipigs, the left lung was perfused in situ with cold preservation solution (EC, n=6; HTK, n=6; Celsior, n=7). Reperfusion was started after 90 min of warm ischemia. The right pulmonary artery and main bronchus were clamped. Bronchoalveolar lavage (BAL) was obtained before ischemia and after 2 h of reperfusion. Surfactant activity was determined from the BAL in a pulsating bubble surfactometer. Results: Animals in the EC group survived 3.7±1.4 h. Six Celsior and five HTK treated animals survived the observation period of 7 h (P<0.001). Compliance of the reperfused lung deteriorated less in both Celsior and HTK groups (P<0.001). In EC and HTK animals, the pO₂/FiO₂ ratio was lower (P=0.002), and pulmonary vascular resistance was higher (P=0.02) than in Celsior animals. Surfactant function was impaired after reperfusion in all groups. Conclusions: Compared to EC, HTK solution showed moderate and Celsior distinct improvement of post-ischemic pulmonary function. However, surfactant function was not well preserved in any group.     

Preliminary results of a clinical randomized study comparing Celsior and HTK solutions in liver preservation for transplantation

INTRODUCTION: A prospective, randomized, multicenter, open clinical trial was performed to compare the main liver function tests, postoperative complications, and early graft and patient survival of recipients transplanted with livers preserved in Celsior (CEL) versus histidine tryptophan ketoglutarate (HTK) solutions. METHODS: We analyzed the data from a single center. Forty livers randomized to CEL (n = 20) or HTK (n = 20) preservation solution were perfused in situ via the aorta and portal vein (CEL, 30 mL/kg via portal vein and 60 mL/kg via aorta; and HTK solution, 30 mL/kg via portal vein and 120 mL/kg via aorta). RESULTS: The groups were comparable with regard to donor, graft, and recipient characteristics. The mean cold ischemia time was 458 minutes (range: 203-667 minutes) in CEL and 450 (range: 310-684 minutes) in HTK. The incidence of initial poor function and primary nonfunction in CEL and HTK were (0 vs 1) and (0 vs 1), respectively. No differences were observed for acute rejection. No vascular or biliary complications were reported in either group. The 3-month graft and patient survival rates were 95% and 95% in CEL and 80% and 90% in HTK. The 12-month graft and patient survival rates were 90% and 90% in CEL and 75% and 85% in HTK. CONCLUSIONS: To our knowledge, this is the first report comparing CEL and HTK preservation solutions in clinical liver preservation. Although a greater 1-year graft and patient survival was observed in the CEL group, a definitive evaluation comparing CEL and HTK solutions in clinical preservation must await completion of the trial.     

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.     

A report of the eurotransplant randomized multicenter study comparing kidney graft preservation with HTK, UW and EC solutions

Abstract Special attention has been focused in this randomized study on the primary function of renal allografts preserved with different solutions. Histidine-Tryptophane-Ketoglutarate (HTK) and University of Wisconsin (UW) solutions provided a significantly lower incidence of delayed graft function compared to Euro-Collins solution. Improved renal function after transplantation was observed in the HTK and UW groups compared to the EC group.     

Pancreas preservation with University of Wisconsin and Celsior solutions

BACKGROUND: Although the use of Celsior has been recently described for heart, lung, liver, and kidney transplantation, no data are available on its use for clinical pancreas preservation. METHODS: We herein describe the results of 112 pancreas transplants preserved with either University of Wisconsin (UW; (n = 56) or Celsior (n = 56) solution at two Italian transplant centers. The groups were comparable with regard to all donor and recipient characteristics. RESULTS: Mean cold and warm ischemia times were 10.1 +/- 2.2 hours and 37.2 +/- 8.2 minutes for UW compared to 10.8 +/- 2.4 hours and 38.3 +/- 6.7 minutes for Celsior (P = NS). Delayed endocrine pancreas function was recorded in two UW-preserved grafts (3.6%). Actuarial 1-year patient survival was 94.6% for UW as compared with 100% for Celsior (P = NS). Equivalent graft survival figures were 91.0% for UW as compared with 96.4% for Celsior (P = NS). CONCLUSIONS: Within the range of cold ischemia times reported in this study, UW and Celsior solutions have similar safety profiles for pancreas transplantation.     

Eurotransplant randomized multicenter kidney graft preservation study comparing HTK with UW and Euro-Collins

The aim was to evaluate the effect of HTK compared to UW and Euro-Collins (EC) on the initial graft function and long term graft survival in two prospective randomized studies. Only kidneys from heart-beating, kidney-only or kidney + heart donors were eligible for entry. Initial non-function (INF) was defined as the absence of life-sustaining renal function, requiring dialysis treatment on two or more occasions, during the first week after transplantation. To evaluate the contribution of the preservation solutions on INF in relation to other factors, a multivariate, 2-step logistic regression model was used. Randomization was performed between July 1990 and September 1992. The UW-HTK study comprised 342 donors and 611 transplants (UW: 168 donors and 297 transplants, HTK: 174 donors and 314 transplants). In the EC-HTK study 317 donors and 569 transplants were included (EC: 155 donors and 277 transplants, HTK: 162 donors and 292 transplants). INF occurred in 33 % of either HTK-(n = 105) or UW-(n = 99) preserved kidneys (P = NS), and in 29 % of the HTK-(n = 85) and in 43 % of the EC-(n = 119) preserved kidneys (P = 0.001). Multivariate analysis showed no significant influence of the preservation solution on the incidence of INF in the UW-HTK study, but factors contributing to INF were donor age, cause of death, retransplantation, and cold ischemic period. The EC-HTK study showed a significantly higher risk of INF, using EC as preservation, in addition to cold ischemic period and donor quality. The 3-year graft survival of HTK-preserved kidneys was 73 %, compared to 68 % for UW-preserved kidneys in the UW-HTK study (P = NS); while the 3-year graft survival of HTK preserved kidneys was 70 % compared to 67 % for EC-preserved kidneys in the EC-HTK study (P = NS). We can conclude that HTK is comparable to UW in its preservative abilities, using kidneys from heart-beating kidney-only donors, whereas EC as renal preservation solution should be avoided. Received: 2 November 1998 Received after revision: 10 August 1999 Accepted: 16 September 1999     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

Objective To compare the effects of Celsior (CS) solution vs University of Wisconsin (UW) solution on preservation of non-heart- beating donor (NHBD) livers in rats. Methods The healthy male SD rats were taken as donors and recipients. The thoracic descending aorta and supradiaphragmatic inferior vena cava were cross-clamped for 10 min to induce ischemia as a NHBD model. After flushed with the cold CS or UW solution, NHBD livers were stored at 4℃ for 8 h or 16 h. Then they were transplanted using the two-cuff technique with arterial reconstruction. Accordingly, the recipients were divided into four groups: CS 8 h, UW 8 h, CS 16 h and UW 16 h groups. The bile discharge, liver enzymes (ALT and AST), ET-1, IL-1 and TNF-alpha levels in serum at 1, 3, and 6 h after reperfusion were measured. Histopathological examination was carried out by HE staining, and the 7-day survival rate was observed. Results As compared with a homogenous appearance in the NHBD livers perfused with CS solution, the NHBD livers perfused with UW solution showed a heterogenous appearance, with more or less mottling. In the CS 8 h group there were more bile discharge at 1 h, lower ALT and AST levels at 1, 3 and 6 h, and lower serum ET-1, IL-1 and TNF-alpha ? levels at 1 and 3 h after reperfusion than the UW 8 h group (P<0.05). Histopathological examination showed a well-preserved hepatic architecture, milder portal congestion and inflammatory infiltration in CS 8 h group as compared with UW 8 h group. The 7-day survival rate in CS 8 h and UW 8 h groups was 58.3% and 25.0% respectively (P<0.05). There were no significant differences in bile discharge, ALT, AST, ET-1, IL-1 and TNF-alpha ? levels at different time points between CS 16 h and UW 16 h groups (P>0.05), and histopathological examination showed no much difference in the tissue damage between two groups; all recipients died within 3 days (P>0.05). Conclusion CS was more effective than UW in preserving NHBD livers after 8 h of cold storage, which may be due to the better perfusion of CS and inhibition of Kupffer cells, but it had better preserve NHBD livers with CS no more than 16 h.     

分离大白鼠肝细胞的低温储存研究

采用Ｃｏｌｌａｇｅｎａｓｅ离体肝门静脉循环灌注的方法制备分离的大白鼠肝细胞，游离的肝细胞储存于４℃低温的Ｌ－１５和ＵＷ液中２４小时和４８小时，通过ＴＢ染色排除试验，细胞形态学观察，乳酸脱氢酶漏出率的测定和肝细胞膜电位的测定来分析比较低温储存后的肝细胞与新分离的肝细胞活力的变化。结果表明：肝细胞在ＵＷ液中低温储存４８小时，其存活率为８０．８％±５．６％，说明ＵＷ储存液对肝细胞有着显著的保护作用。     

The impact of liver preservation in HTK and UW solution on microcirculation after liver transplantation

Severe microcirculatory disturbances due to endothelial cell damage and leukocyte adherence during reperfusion of transplanted livers are considered to contribute to early graft failure. Since the degree of reperfusion injury after liver transplantation depends on the length of preservation time and the solution used for preservation, the aim of our study was to assess three solutions with respect to microvascular perfusion and leukocyte adhesion. Therefore, rat livers were stored up to 24 h in Euro-Collins (EC), University of Wisconsin (UW), or histidin-tryphtophan-ketoglutarate (HTK) solutions prior to orthotopic transplantation. The livers were studied in situ 60 min postoperatively using intravital fluorescence video microscopy. Using simple syringe flushing (10 ml), sinusoidal perfusion decreased below 50% in EC preserved livers after 8 h preservation, in HTK preserved livers after 16 h preservation, and remained higher than 70% in livers preserved in UW up to 24 h. Permanent adhesion of leukocytes was increased more rapidly in organs after 1, 8, 16, and 24 h preservation in HTK (16%, 15%, 34%, and 49.7% ± 4.7%) compared to those preserved in UW (15%, 18%, 17%; and 32.7% ± 3.3%; P < 0.05). Using a 10-fold volumn of the organ weight of HTK solution during the harvesting procedure, with an 8 min equilibration period, sinusoidal perfusion (39.6 ± 4.7%) and leukocyte adhesion (42.7 ± 3.1%) were not improved after 24 h. In contrast, equilibration with a volumn of approximately 40-times the liver weight improved sinusoidal perfusion (70.8% ± 2.7%; P < 0.01) and leukocyte adhesion (24.9% ± 3.1%; P < 0.01) significantly. Thus, using HTK solution, simple flushing prior to long-term cold storage resulted in microcirculatory disturbances when compared to UW solution. Larger volumns of HTK solution with an additional equilibration period of 8 min, however, reduced leukocyte adhesion and improved sinusoidal perfusion to a similar degree as UW solution.     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

Objective To compare the effects of Celsior (CS) solution vs University of Wisconsin (UW) solution on preservation of non-heart- beating donor (NHBD) livers in rats. Methods The healthy male SD rats were taken as donors and recipients. The thoracic descending aorta and supradiaphragmatic inferior vena cava were cross-clamped for 10 min to induce ischemia as a NHBD model. After flushed with the cold CS or UW solution, NHBD livers were stored at 4℃ for 8 h or 16 h. Then they were transplanted using the two-cuff technique with arterial reconstruction. Accordingly, the recipients were divided into four groups: CS 8 h, UW 8 h, CS 16 h and UW 16 h groups. The bile discharge, liver enzymes (ALT and AST), ET-1, IL-1 and TNF-alpha levels in serum at 1, 3, and 6 h after reperfusion were measured. Histopathological examination was carried out by HE staining, and the 7-day survival rate was observed. Results As compared with a homogenous appearance in the NHBD livers perfused with CS solution, the NHBD livers perfused with UW solution showed a heterogenous appearance, with more or less mottling. In the CS 8 h group there were more bile discharge at 1 h, lower ALT and AST levels at 1, 3 and 6 h, and lower serum ET-1, IL-1 and TNF-alpha ? levels at 1 and 3 h after reperfusion than the UW 8 h group (P<0.05). Histopathological examination showed a well-preserved hepatic architecture, milder portal congestion and inflammatory infiltration in CS 8 h group as compared with UW 8 h group. The 7-day survival rate in CS 8 h and UW 8 h groups was 58.3% and 25.0% respectively (P<0.05). There were no significant differences in bile discharge, ALT, AST, ET-1, IL-1 and TNF-alpha ? levels at different time points between CS 16 h and UW 16 h groups (P>0.05), and histopathological examination showed no much difference in the tissue damage between two groups; all recipients died within 3 days (P>0.05). Conclusion CS was more effective than UW in preserving NHBD livers after 8 h of cold storage, which may be due to the better perfusion of CS and inhibition of Kupffer cells, but it had better preserve NHBD livers with CS no more than 16 h.     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

Objective To compare the effects of Celsior (CS) solution vs University of Wisconsin (UW) solution on preservation of non-heart- beating donor (NHBD) livers in rats. Methods The healthy male SD rats were taken as donors and recipients. The thoracic descending aorta and supradiaphragmatic inferior vena cava were cross-clamped for 10 min to induce ischemia as a NHBD model. After flushed with the cold CS or UW solution, NHBD livers were stored at 4℃ for 8 h or 16 h. Then they were transplanted using the two-cuff technique with arterial reconstruction. Accordingly, the recipients were divided into four groups: CS 8 h, UW 8 h, CS 16 h and UW 16 h groups. The bile discharge, liver enzymes (ALT and AST), ET-1, IL-1 and TNF-alpha levels in serum at 1, 3, and 6 h after reperfusion were measured. Histopathological examination was carried out by HE staining, and the 7-day survival rate was observed. Results As compared with a homogenous appearance in the NHBD livers perfused with CS solution, the NHBD livers perfused with UW solution showed a heterogenous appearance, with more or less mottling. In the CS 8 h group there were more bile discharge at 1 h, lower ALT and AST levels at 1, 3 and 6 h, and lower serum ET-1, IL-1 and TNF-alpha ? levels at 1 and 3 h after reperfusion than the UW 8 h group (P<0.05). Histopathological examination showed a well-preserved hepatic architecture, milder portal congestion and inflammatory infiltration in CS 8 h group as compared with UW 8 h group. The 7-day survival rate in CS 8 h and UW 8 h groups was 58.3% and 25.0% respectively (P<0.05). There were no significant differences in bile discharge, ALT, AST, ET-1, IL-1 and TNF-alpha ? levels at different time points between CS 16 h and UW 16 h groups (P>0.05), and histopathological examination showed no much difference in the tissue damage between two groups; all recipients died within 3 days (P>0.05). Conclusion CS was more effective than UW in preserving NHBD livers after 8 h of cold storage, which may be due to the better perfusion of CS and inhibition of Kupffer cells, but it had better preserve NHBD livers with CS no more than 16 h.     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

Objective To compare the effects of Celsior (CS) solution vs University of Wisconsin (UW) solution on preservation of non-heart- beating donor (NHBD) livers in rats. Methods The healthy male SD rats were taken as donors and recipients. The thoracic descending aorta and supradiaphragmatic inferior vena cava were cross-clamped for 10 min to induce ischemia as a NHBD model. After flushed with the cold CS or UW solution, NHBD livers were stored at 4℃ for 8 h or 16 h. Then they were transplanted using the two-cuff technique with arterial reconstruction. Accordingly, the recipients were divided into four groups: CS 8 h, UW 8 h, CS 16 h and UW 16 h groups. The bile discharge, liver enzymes (ALT and AST), ET-1, IL-1 and TNF-alpha levels in serum at 1, 3, and 6 h after reperfusion were measured. Histopathological examination was carried out by HE staining, and the 7-day survival rate was observed. Results As compared with a homogenous appearance in the NHBD livers perfused with CS solution, the NHBD livers perfused with UW solution showed a heterogenous appearance, with more or less mottling. In the CS 8 h group there were more bile discharge at 1 h, lower ALT and AST levels at 1, 3 and 6 h, and lower serum ET-1, IL-1 and TNF-alpha ? levels at 1 and 3 h after reperfusion than the UW 8 h group (P<0.05). Histopathological examination showed a well-preserved hepatic architecture, milder portal congestion and inflammatory infiltration in CS 8 h group as compared with UW 8 h group. The 7-day survival rate in CS 8 h and UW 8 h groups was 58.3% and 25.0% respectively (P<0.05). There were no significant differences in bile discharge, ALT, AST, ET-1, IL-1 and TNF-alpha ? levels at different time points between CS 16 h and UW 16 h groups (P>0.05), and histopathological examination showed no much difference in the tissue damage between two groups; all recipients died within 3 days (P>0.05). Conclusion CS was more effective than UW in preserving NHBD livers after 8 h of cold storage, which may be due to the better perfusion of CS and inhibition of Kupffer cells, but it had better preserve NHBD livers with CS no more than 16 h.