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.     

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.     

Injury to cultured liver endothelial cells during cold preservation: energy-dependent versus energy-deficiency injury

Previously, we demonstrated an energy-dependent injury to cultured liver endothelial cells during cold incubation in University of Wisconsin (UW) solution. Here, the effects of Histidine-Tryptophan-Ketoglutarate (HTK) and Euro-Collins (EC) solutions on these cells were studied. In HTK solution, 83%±4% of the cells had lost viability after 9 h of incubation at 4°C. The addition of cyanide (1 mM) to simulate hypoxic conditions protected the cells to the extent that only 9%±1% of the cells lost viability over the same period; the addition of glucose (10 mM) led to increased cell injury. ATP levels were highest in the incubations with the most rapid loss of viability. In Krebs-Henseleit buffer and EC solution, in contrast, cell injury increased upon addition of cyanide; the addition of glucose to Krebs-Henseleit buffer decreased injury. We conclude that the injury to cultured liver endothelial cells during cold incubation in HTK solution is energy-dependent, as it is in UW solution, whereas cells behave differently in EC solution and Krebs-Henseleit buffer.     

Influence on Energy Kinetics and Histology of Different Preservation Solutions Seen During Cold Ischemia in the Liver

Background and Purpose

Cold flush preservation prolongs tissue viability during ischemia. However, there is little understanding of the effects of various preservation fluids on events during this period. A study of cold ischemia in rat livers was undertaken to compare biochemical and histological changes over time, using three preservation solutions: University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), and Leeds solution (LS) under development at our institution. Leeds solution is a phosphate-based sucrose solution that like UW contains the impermeant lactobionate and the metabolite allopurinol (1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) which acts as a competitive inhibitor of xanthine oxidase, stopping the breakdown of hypoxanthine to xanthine by oxidizing it to alloxanthine, inhibiting both the conversion of hypoxanthine to xanthine and the conversion of xanthine to uric acid.

Materials and Methods

At various time points, samples were analyzed for adenosine triphospate (ATP) and metabolites by high-performance liquid chromatography as well as for histological changes.

Results

In all livers, ATP, ADP, and AMP degraded over 4 hours. In UW and LS groups, degradation beyond hypoxanthine was halted, and it continued in the HTK group. This blockade led to a significant reduction in the accumulation of xanthine and uric acid. Histological analysis showed protected architecture and maintenance of reticulin scaffolds in the UW and LS groups, whereas tissue breakdown was seen from earlier time points in the HTK group. Additionally, throughout ischemia, signs of pathological injury were more pronounced with UW- than with LS-preserved tissue.

Conclusions

These results implied that cold ischemia in the liver is characterized by dynamic biochemical changes coincident with pathological injury which are initiated from the time of organ perfusion and influenced by the choice of the perfusion fluid. Allopurinol in UW and LS appears to be critical. We hypothesized that it may also affect the degree of subsequent reperfusion injury. The data supported the assertion that LS offerred improved preservation over UW, adding to the impetus to shorten ischemic times in clinical transplantation.     

Fibrinolytic preflush upon liver retrieval from non-heart beating donors to enhance postpreservation viability and energetic recovery upon reperfusion.

BACKGROUND: Our objective was to evaluate graft equilibration with high viscosity (University of Wisconsin solution [UW]) or low viscosity (Bretschneider's histidine-tryptophan-ketoglutarate [HTK]) during liver procurement from non-heart beating donors (NHBD) and the potential impact of a preceding fibrinolysis with streptokinase on postpreservation viability. METHODS: After 60 min of cardiac arrest, rat livers were perfused by gravity (60 cm H2O) via the portal vein with either 60 ml of HTK, 20 ml of UW, or 20 ml of Ringer's solution (22 degrees C including 7500U of streptokinase) and, subsequently, 20 ml of UW. After 24 h of storage at 4 degrees C, viability of the livers was assessed upon isolated reperfusion in vitro. RESULTS: Magnetic resonance imaging revealed severe perfusion deficits, which were mildly attenuated with HTK, upon flush-out with UW. After preflush with streptokinase, a mostly homogenous distribution of the preservation solution was observed throughout the liver tissue. The choice of the flush-out solution (UW or HTK) had no influence on parenchymal enzyme leakage, hepatic bile production, or tissue levels of ATP after reperfusion of the livers. Fibrinolytic preflush, however, resulted in a relevant and significant improvement of structural integrity as well as functional and metabolic recovery. CONCLUSIONS: Compromised vascular tissue perfusion upon organ harvest in NHBD triggers graft dysfunction after cold storage and can easily be circumvented by temporary fibrinolysis before graft retrieval.     

Addition of a water-soluble alpha-tocopherol analogue to University of Wisconsin solution improves endothelial viability and decreases lung reperfusion injury.

BACKGROUND: Reperfusion injury following lung preservation has been associated with free radical formation and subsequent endothelial cell damage. Trolox is a water-soluble analogue of the free radical scavenger alpha-tocopherol. We hypothesized that addition of this form of vitamin E to University of Wisconsin (UW) solution would decrease reperfusion injury and improve lung function after cold ischemic preservation. MATERIALS AND METHODS: Bovine aortic endothelial cells were cultured and stored at 4 degrees C for 12, 24, and 48 h in UW or UW + Trolox (UWT). Endothelial cell viability after storage was assessed by dimethylthiazole tetrazolium cytotoxicity assay. An isolated rat perfused lung (IPL) model was used and lungs were flushed with the respective solutions with cold storage times of 6 and 12 h. Following storage, the lungs were reperfused with fresh blood and lung function was assessed by blood gas analysis, alveolar-arterial gradient, and compliance. RESULTS: There was no difference in endothelial cell viability between UW and UWT after 12 or 24 h; however, UWT had higher endothelial cell viability than UW with 48 h of cold ischemic storage. Using the IPL model, the pO2 was higher with UWT than UW after 6 and 12 h of cold ischemia. The alveolar-arterial oxygen difference was significantly lower for UWT versus UW at 6 h. UWT provided increased compliance at 6 and 12 h of ischemia. CONCLUSIONS: The addition of a water-soluble vitamin E analogue to UW solution resulted in increased endothelial cell viability after prolonged storage and improved whole lung preservation in the postreperfusion period as evidenced by higher oxygenation and increased compliance. These results are clinically relevant as the lung is extremely sensitive to reperfusion injury and UW solution is being increasingly used in lung transplantation and remains the predominant solution in abdominal organ transplantation.     

Heart preservation in HTK solution: role of coronary vasculature in recovery of cardiac function

BACKGROUND: Poor myocardial tolerance to prolonged cold ischemia remains a major concern in heart transplantation. In this study, we estimated superiority of Histidine-Tryptophan-Ketoglutarate (HTK) over University of Wisconsin (UW) as a cardiac preservation solution. METHODS: Isolated rat hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function. The hearts were arrested and stored at 4 degrees C in UW and HTK solution for 8 hours, and then reperfused. The aortic flow, coronary flow, cardiac output, rate pressure product, and left ventricular dp/dt in the HTK group recovered significantly more than the UW group. The values of myocardial total adenine nucleotides and the adenosine triphosphate to adenosine diphosphate ratio were higher in the HTK than in the UW group. We also examined coronary vascular responsiveness using left coronary arteries dissected from the rat hearts before flushing, before storage, after storage, and after reperfusion. RESULTS: The maximal relaxation response to acetylcholine was significantly higher in the HTK than in the UW group after reperfusion, although there were no significant differences at each stage before reperfusion. In addition, the endothelium-independent relaxation response to sodium nitroprusside in the HTK group was also well preserved after reperfusion. CONCLUSIONS: These results indicate that HTK is superior to UW solution for cardiac preservation. HTK protects coronary vasculature during preservation, which together with reperfusion might lead to improved functional cardiac recovery following preservation.     

肝移植术中应用S-腺苷-L-蛋氨酸对供肝热缺血损伤的影响

目的探讨术中S-腺苷-L-蛋氨酸（SAMe）加入UW液和血浆冲洗液对热缺血损伤供肝及其恢复的影响。方法建立10min热缺血大鼠肝移植模型，分为A组：UW液灌注＋乳酸钠林格氏液冲洗、B组：UW液灌注＋血浆冲洗、C组：SAMe加入UW液灌注＋血浆冲洗和D组：UW液灌注＋SAMe加入血浆冲洗4组，观察肝组织组织病理学变化和电子显微镜下超微结构变化，并检测血清AST和透明质酸。结果C组和D组术后24h血清AST均低于B组（P〈0．05）。A组术后3h和24h血清HA高于B组（P〈0．05），B组复流后3h及24h血清HA均高于C组和D组（P〈0．05）。组织病理学表现B组复流后3h和24h肝细胞损伤和微循环紊乱较C组和D组明显；超微结构表现，A组复流后3h线粒体肿胀，肝窦内皮细胞肿胀，细胞核不规则，可见内皮细胞凋亡，大部分区域肝窦状隙明显狭窄，内皮层结构模糊，红细胞淤积，受压变形，白细胞附壁，可见内皮层完整性破坏；复流后24h，可见线粒体嵴断裂，核融解。B组内皮细胞损伤较A组轻，C组和D组超微结构表现微循环紊乱和肝细胞损伤表现较B组轻。结论供肝切取术中UW液中加入SAMe灌注保存，血浆冲洗液中加入SAMe可改善热缺血供肝微循环，减轻缺血再灌注损伤，并减轻肝细胞热缺血损伤，有利于10min热缺血供肝功能的恢复。     

Hepatic energy metabolism during hypothermic storage and reperfusion using different protecting solutions.

Effects of 5 cold storage solution on hepatic high energy phosphate metabolism and metabolic function were examined using the isolated perfused rat liver. University of Wisconsin (UW), Euro-Collins (EC), and 2 cardioplegic solutions, Bretschneider's HTK and St. Thomas Hospital solution, were studied for their protective capacity. Krebs-Henseleit bicarbonate buffer (KHB) was used to point out the effect of simple hypothermia. Liver ATP, total adenine nucleotides and energy charge losses were significantly lower during 21 h of storage in UW-preserved livers. Also, only UW-protected livers were able to complete regeneration of ATP and total adenine nucleotides after 1 h of reperfusion, whereas EC, HTK, St. Thomas and KHB stored livers only showed minimal regeneration. Concerning metabolic function, UW protected livers liberated significantly less LDH and sGOT as well in the 21-hour storage solution as into the perfusate under reperfusion conditions. This study demonstrates the capability of UW solution in liver preservation by its ability to maintain and restore high energy phosphates.     

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.     

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.     

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。     

Greater Hemodynamic Instability With Histidine-Tryptophan-Ketoglutarate Solution Than University of Wisconsin Solution During the Reperfusion Period in Living Donor Liver Transplantation

Objective

University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions are the 2 most commonly used liver preservation solutions. The aim of this study was to compare cardiovascular stability, acid-base status, and potassium concentrations between patients who received grafts preserved in either UW or HTK solution in orthotopic liver transplantation (OLT).

Patients and Methods

In this retrospective study, 87 patients who underwent living donor OLT were divided into 2 groups: UW (n = 28) and HTK (n = 59). Group HTK was subdivided into group NF-HTK (n = 31; nonflushed before reperfusion) and group F-HTK (n = 28; flushed before reperfusion). We determined mean arterial pressure (MAP) and heart rate every minute for 5 minutes after reperfusion and the maximum change in these values and incidence of postreperfusion syndrome (PRS). Body temperature, cardiovascular and acid-base parameters, as well as potassium concentrations were compared at 5 minutes before and 5 and 30 minutes after reperfusion.

Results

The maximum decreases in MAP within 5 minutes after reperfusion were significantly greater in both the NF-HTK and the F-HTK groups. The rate of PRS was significantly greater in the NF-HTK compared with the UW group. Flushing with HTK solution decreased the rate of PRS; there was no significant difference between the F-HTK and UW groups. All serial changes in body temperature, cardiovascular and acid-base parameters, as well as potassium concentrations were similar among the 3 groups.

Conclusions

The incidence of PRS was greater using HTK compared with UW solution during the reperfusion period. Therefore, careful hemodynamic management is advised when using HTK solution.     

Histidine–Tryptophan–Ketoglutarate (HTK) Is Associated with Reduced Graft Survival in Deceased Donor Livers, Especially Those Donated After Cardiac Death

Single-center studies have reported that liver allograft survival is not affected by preservation in histidine–tryptophan–ketoglutarate (HTK) versus University of Wisconsin (UW) solution. We analyzed the UNOS database of liver transplants performed from July, 2004, through February, 2008, to determine if preservation with HTK (n = 4755) versus UW (n = 12 673) impacted graft survival. HTK preservation of allografts increased from 16.8% in 2004 to 26.9% in 2008; this was particularly striking among donor after cardiac death (DCD) allografts, rising from 20.7% in 2004 to 40.9% in 2008. After adjusting for donor, recipient and graft factors that affect graft survival, HTK preservation was associated with an increased risk of graft loss (HR 1.14, p = 0.002), especially with DCD allografts (HR 1.44, P = 0.025) and those with cold ischemia time over 8 h (HR 1.16, P = 0.009). Furthermore, HTK preservation was associated with a 1.2-fold higher odds of early (< 30 days) graft loss as compared to UW preservation (OR 1.20, p = 0.012), with a more pronounced effect on allografts with cold ischemia time over 8 h (OR 1.31, p = 0.007), DCD allografts (OR 1.63, p = 0.09) and donors over 70 years (OR 1.67, p = 0.081). These results suggest that the increasing use of HTK for abdominal organ preservation should be reexamined.     

Histidine-Tryptophan-Ketoglutarate and Delayed Graft Function After Prolonged Cold Ischemia

Background

Several articles have compared histidine-tryptophan-ketoglutarate solution (HTK) with other preservation solutions in both liver and kidney transplantation, and the results suggest that HTK is as good or better than the criterion standard University of Wisconsin solution (UW) for short periods of cold ischemia, such as in live donation, but that it is not so efficient for longer periods of cold ischemia, causing a higher incidence of delayed graft function.

Objective

To evaluate energy levels, metabolites, and histologic findings to determine why HTK is inefficient for longer periods of cold ischemia.

Methods

Rat livers were perfused with either HTK or UW, and at various times, tissue samples were obtained for analysis of adenine triphosphate and metabolites using high-performance liquid chromatography or for histologic analysis.

Results

The high energy charge observed with HTK-perfused livers plateaued after 5 minutes, and by 60 minutes began to decrease, following the same trend as other samples. The plateau is due to excess available glucose; however, after 1 hour, it is beginning to be consumed. Low levels of uridine, required for glycogen synthesis, are found in HTK-perfused livers, which suggests that at reperfusion, there is none available, whereas the higher concentrations found in UW-perfused livers may be advantageous after reperfusion. This will be especially detrimental to use of HTK because glycogen is used up rapidly because of the presence of α-ketoglutarate in the solution, enabling continuation of the tricarboxylic acid cycle.

Conclusions

Overall, HTK seems to do well for the first 2 hours, after which any advantage observed initially starts to disappear. A liver perfused in HTK and transplanted after more than 1 hour reacts like an organ from an individual who has been starved, because of the low energy charge and absence of a glycogen store or ability to synthesis glycogen because of lack of uridine. Livers perfused with UW demonstrate higher levels of uridine and do not lose their glycogen content to the same extent as HTK-perfused livers. These findings explain in part why HTK sometimes causes delayed graft function after longer periods of cold ischemia.     

Microcirculatory disturbances and leucocyte adherence in transplanted livers after cold storage in Euro-Collins,UW and HTK solutions

Integrity of the hepatic microcirculation and maintenance of endothelial cell viability are critical components in preventing primary non-function after liver transplantation. Therefore, hepatic microcirculation and leucocyte-endothelial interaction were studied in rat livers stored for 1 h in Euro-Collins (EC), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solutions and subsequently transplanted. One hour after transplantation surgery, the livers were exposed under an intravital fluorescence microscope. After injection of the leucocyte marker acridine orange (1 mol/kg), six pericentral fields were observed for 30 s and experiments were recorded continuously. The percentage of perfused sinusoids was reduced in the livers in the EC group (82.9%) in contrast to the UW (93.2%) and HTK groups (91.0%). Livers in the EC group showed a reduction in the diameters of pericentral sinusoids (7.3±0.2 m; mean±SEM) compared with the UW group (9.5±0.2 m; P<0.05) and HTK group (10.2±0.8 m; P<0.05), indicating substantial cell swelling in livers stored in EC solution. Permanent adherence of leucocytes was most frequently observed in the EC group (33.5±1%), while this phenomenon was less pronounced in the UW group (14.5+1.1%; P<0.05) and HTK group (16.3±0.7%; P<0.05). Conversely, temporary adherence of leucocytes was reduced in the EC group (19.7+1.3%) compared with the UW group (30.5+2.1%) and the HTK group (34.4+0.8%). Microcirculatory failure and cell swelling in the EC group might be due to the lack of osmotic substances or oxygen radical scavengers included in UW (allopurinol, glutathione) and HTK (mannitol) solutions. In conclusion, cold storage of livers in UW and HTK solutions results in better preservation of the microcirculation and prevention of adhesion of leucocytes after transplantation compared with the EC solution.     

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.     

The significance of bile secretion after the transplantation of long-preserved livers in the rat

Although one of the simplest indicators for predicting liver viability is bile secretion, it has never been proven whether it could be a good index for the viability of grafts in liver transplantation after cold ischemia. The present study, conducted on male Wistar rats, was undertaken to determine whether bile secretion reflects the viability of livers which have been preserved long-term. Livers were stored for up to 24 h in Euro-Collins (EC) or University of Wisconsin (UW) solution at 4°C, and transplanted orthotopically. The correlation between 1-week survival, bile flow, and the tissue adenosine triphosphate (ATP) level 4 h after transplantation was then investigated for each subgroup. The survival rates of the animals in the UW subgroups were much higher than those in the EC subgroups. In the rats transplanted with livers preserved for 6h in EC solution (EC-6), in which 100% survival was observed, both bile flow and ATP recovered sufficiently. Conversely, in the EC-12 group, in which only 10% survival was seen, restoration of bile flow, in ml/h per kg body weight, and ATP resynthesis, in pmol/g wet weight, were severely suppressed, with levels of 1.35 ± 1.05 and 0.77 ± 0.34, respectively. Moreover, in the EC-18 group, with 0% survival, neither bile flow nor ATP recovered. In the rats transplanted with livers preserved for 18h in UW solution (UW-18), bile flow and ATP, being 1.03 ± 0.56 and 1.12 ± 0.59, respectively, were much higher than those in the EC-18 group. A good correlation was found between bile flow and ATP (r = 0.84). The results of this study thus led us to conclude that bile secretion is a reliable index for predicting the viability of orthotopically transplanted grafts damaged by cold ischemia.     

Improved microcirculation by low‐viscosity histidine– tryptophan–ketoglutarate graft flush and subsequent cold storage in University of Wisconsin solution: results of an orthotopic rat liver transplantation model

As previously shown in a model of isolated rat liver perfusion, the combined use of an initial graft flush with low‐viscosity histidine–tryptophan–ketoglutarate (HTK) solution followed by cold storage in University of Wisconsin (UW) solution markedly improved the preservation during an extended cold storage period. In this study, we aimed to transfer our results into an in vivo model of orthotopic rat liver transplantation, and to elucidate the potential mechanism of the improved preservation by focusing on the hepatic microcirculation. Livers were harvested from male Wistar rats. Aortic perfusion with a pressure of 100 cm H₂O was performed with either UW (group UW) or HTK (groups UW and HTK_UW), followed by additional back‐table perfusion with UW (group HTK_UW). After 20‐h cold storage at 4 °C, livers were orthotopically transplanted with reconstructing the hepatic artery. As measured by bile flow and liver enzymes, HTK flush followed by UW storage was superior compared to single use of either UW or HTK solution. The hepatic microcirculation was significantly improved, as shown by the increased percentage of reperfused sinusoids and reduced sinusoidal leucostasis. HTK and UW effectively reduce ischaemia‐reperfusion injury after liver transplantation. By combining the comparative advantages of both solutions, a cumulative effect resulting in an improved preservation was shown. Thus, this mechanism improves microcirculatory reperfusion.