Seventy-two-hour preservation of porcine liver by continuous hypothermic perfusion with UW solution in comparison with simple cold storage

Porcine livers preserved for 72 hr using continuous hypothermic perfusion (CHP) were studied in order to compare the effects of CHP on energy metabolism with those of simple cold storage (SCS). The livers of the CHP group were perfused in situ for 72 hr at 7 degrees C with University of Wisconsin (UW) solution and FC-43 as an oxygen carrier, while those of the S1 group were stored ex situ for 48 hr at 4 degrees C in UW solution, and those of the S2 group for 72 hr. They were then recirculated with human blood at 37 degrees C for 2 hr for the evaluation of their viability. At the end of preservation, significantly higher levels of total adenine nucleotides (TAN) and energy charge (EC) were observed in the CHP group compared with the S1 and S2 groups. At 2 hr recirculation, the level of TAN was significantly higher in the CHP group than in the S1 and S2 groups. The EC level was also higher in the CHP group than in the S2 group. During recirculation, the ketone body ratio (acetoacetate/beta-hydroxybutyrate) was higher in the CHP group than in the S2 group. The values in the CHP group were above 1.0 after 45 min recirculation. There were no significant differences in the pyruvate/lactate ratio and lactate level between the CHP and S1 groups. However, these values were significantly different from those in the S2 group. The present findings demonstrate that CHP using UW solution and the oxygen carrier was better able to preserve the energy metabolism of the porcine liver for 72 hr than 48-hr SCS in UW solution.     

钙通道阻滞剂对低温保存大鼠肝脏的保护作用

目的：研究钙通道阻滞剂是否减轻低温保存下肝细胞的钙超载并起到保护肝脏的作用。方法：低温保存大鼠肝细胞于不同时限,按保存液的不同成分分组：（1）对照组：DMEM液;（2）实验Ⅰ组：DMEM液＋Verapamil(维拉帕米）;（3）实验Ⅱ组：DMEM液＋Nifedipine(硝苯地平）;（4）实验Ⅲ组：DMEM液＋Diltiazem(硫氮卓酮）。用Fura-2法测定低温保存下的大鼠肝细胞内钙和肝功能,并在光镜和电镜下观察肝脏的结构。结果：细胞内钙及肝功能测定,各实验组与对照组差异显著;组织学观察,保存0－48h各实验组损伤明显低于对照组。结论钙通道阻滞剂对低温保存肝脏有保护作用,且三类药物中帕米作用最强,硝苯地平和硫氮卓酮次之。     

Advantages of normothermic perfusion over cold storage in liver preservation

BACKGROUND: To minimize the ischemia-reperfusion injury that occurs to the liver with the current method of preservation and transplantation, we have used an extracorporeal circuit to preserve the liver with normothermic, oxygenated, sanguineous perfusion. In this study, we directly compared preservation by the standard method of simple cold storage in University of Wisconsin (UW) solution with preservation by perfusion. METHODS: Porcine livers were harvested from large white sows weighing between 30 and 50 kg by the standard procedure for human retrieval. The livers were preserved for 24 hr by either cold storage in UW solution (n=5) or by perfusion with oxygenated autologous blood at body temperature (n=5). The extracorporeal circuit used included a centrifugal pump, heat exchanger, and oxygenator. Both groups were then tested on the circuit for a 24 hr reperfusion phase, analyzing synthetic function, metabolic capacity, hemodynamics, markers of hepatocyte and reperfusion injury, and histology. RESULTS: Livers preserved with normothermic perfusion were significantly superior (P=0.05) to cold-stored livers in terms of bile production, factor V production, glucose metabolism, and galactose clearance. Cold-stored livers showed significantly higher levels of hepatocellular enzymes in the perfusate and were found to have significantly more damage by a blinded histological scoring system. CONCLUSIONS: Normothermic sanguineous oxygenated perfusion is a superior method of preservation compared with simple cold storage in UW solution. In addition, perfusion allows the possibility to assess viability of the graft before transplantation.     

Limitations of heart preservation by cold storage.

Clinical heart preservation is currently limited to only 4-6 hr, while the kidney, liver, and pancreas can tolerate 24-48 hr of cold ischemia. A fundamental difference between these organs is that the heart is contractile, containing large quantities of actin and myosin, and is susceptible to contracture-induced injury caused by energy deprivation. We have quantified and correlated the onset of contracture with levels of ATP and glycogen during cold storage in rabbit hearts flushed with UW solution, with and without 1 mM calcium (Ca), or 3 mM iodoacetate (IAA). A fluid-filled left ventricular balloon was used to generate pressure-volume curves (compliance) at 1, 6, 12, 18, and 24 hr of cold storage. Onset of contracture occurred in UW stored hearts at 18 hr, contracture in hearts exposed to Ca occurred between 6 and 12 hr. Compliance was significantly less in hearts exposed to Ca at 12, 18, and 24 hr (P less than .01) than in hearts without Ca. ATP levels were well maintained for up to 18 hr in the hearts preserved in UW solution (78%), but fell more rapidly in the presence of Ca at 12 hr (P less than .005), 18 hr (P less than .005), and 24 hr (P less than .05). In comparison, the ATP supply of the liver and kidney was exhausted by only 4 hr of cold storage. Onset of myocardial contracture correlated with a decrease in ATP to less than 80% of control, and contracture accelerated ATP decline 3-6-fold. IAA caused nearly complete myocardial contracture and ATP depletion within 2 hr. Isolated heart function was 77% and 73% at 6 and 12 hr of storage, but fell to 54% and 42% at 18 and 24 hr, respectively, coinciding with development of contracture. We conclude that ischemic contracture in this model is a major cause of myocardial damage during cold storage, and is accelerated by the presence of Ca. Other organs can be successfully stored despite exhaustion of ATP reserves. Thus successful cold-storage of the heart is highly ATP-dependent. Since cold storage inevitably leads to ATP depletion, extension of myocardial ischemic tolerance will depend on either reversible inhibition of ATP hydrolysis during storage, reversible uncoupling of contracture development from ATP depletion, or maintaining ATP production by continuous hypothermic perfusion.     

Extended cadaver renal preservation with combined simple cold storage and hypothermic pulsatile perfusion

Although simple cold storage and hypothermic pulsatile perfusion are each accepted methods of cadaver kidney preservation the efficacy of combining these techniques for extended renal preservation is unclear. In 18 patients cadaver allografts were used that had been preserved with combined simple cold storage (range 6 to 20 hours) and hypothermic pulsatile perfusion (range 9 to 38 hours). The total interval of preservation for these kidneys ranged from 25 to 48 hours. Excellent post-transplant graft function was achieved in all cases, with a mean serum creatinine nadir of 1.4 mg./dl. In 14 patients (78 per cent) grafts continued to function at intervals of 3 to 38 months after transplantation. Of the remaining 4 patients 3 lost the grafts to rejection, while 1 died 7 months after transplantation with a well functioning graft. These data suggest that the combination of simple cold storage and hypothermic pulsatile perfusion provides a safe and effective method for extended renal preservation.     

Prolongation of the kidney preservation period by simple cold storage up to 72 hours by human atrial natriuretic peptide.

To prolong the preservation period of donor kidneys, we tried to evaluate the effect of human atrial natriuretic peptide (hANP), which has vasodilator, natriuretic, and GFR-increasing effects. Autotransplantations were successful in 6 of 8 hANP-treated cases, but only one of 7 untreated cases (P less than 0.05) was successful. When removed kidneys were perfused prior to preservation, the gravity perfusion time was shorter in hANP-treated kidneys, resulting in rapid cooling. Renal biopsy 1 hr after transplantation revealed normal histology in the hANP-treated kidneys while a remarkable capillary collapse occurred in controls. A hANP-like immunoreactivity contents of biopsied tissue in the hANP-treated kidneys showed a higher value than in the controls, and cyclic GMP contents were also higher in the hANP-treated kidneys. Microangiography showed complete clarity, down to the peripheral vessels, in the hANP-treated kidneys, but not in the controls. Thus, the present study suggests that hANP is useful for prolonging the preservation time of donor kidneys.     

Dielectric spectrogram for evaluating ischemic microstructural changes of the liver in simple cold preservation

Purpose: Monitoring ischemic liver injury is important in liver transplantation. We previously reported that dielectric properties were correlated with tissue adenosine triphosphate during liver preservation; however, it is unknown what the changes in dielectric properties are based on. Using rats, we studied the relationship between dielectric parameters and microstructural changes in liver tissues during preservation. Methods: We serially measured the percent decrease in conductivity and the percent increase in relative permittivity in rat livers, and determined the areas of cellular and extracellular components using a charge-coupled device microscope and an NIH image. Thereafter, we evaluated the relationship between dielectric parameters and microstructural changes in the liver tissues. Results: A positive linear and exponential correlation was found between the percent decrease in conductivity and the percent decrease in extracellular components (P < 0.01). A positive linear and exponential correlation was also found between the percent increase in relative permittivity and the percent increase in cellular components (P < 0.01). Conclusions: Our results suggest that the serial changes in tissue conductivity and the relative permittivity reflect changes in liver tissue microstructures during cold preservation. Received: July 12, 2001 / Accepted: July 2, 2002 Reprint requests to: T. Yamada     

联合使用库司托啶与异搏定对大鼠肝脏低温保存的作用

目的 研究库司托啶（Ｃｕｓｔｏｄｉｏｌ）与异搏定（Ｖｅｒａｐａｍｉｌ）联合应用对低温保存大鼠肝脏的保护作用。方法 将４０只Ｗｉｓｔａｒ大白鼠制作肝离体灌注模型,库司托啶和异搏定作肝脏灌注并置于４℃保存６、２４和４８ｈ后观察肝窦内皮细胞死亡率,肝功能以及肝细胞形态的的变化。结果 库司托啶和异搏定灌注组明显优于对照组,保存２４ｈ,库司托啶＋异搏定（ＣＶＡ）组,库司托啶组的肝窦内皮细胞死亡率分别为３０．     

Combined effects of fasting and alanine on liver function recovery after cold ischemia

The effect of donor nutritional status on hepatic function recovery after cold ischemia is still debated. We demonstrated previously that a 48-h fast diminished the survival rate of liver-transplanted rats and that the deleterious effect of fasting was prevented by infusion of alanine to the recipient at reperfusion. Whether the duration of fasting influenced the protective effect of alanine and whether this effect was metabolic were not known, and the elucidation of these questions is the aim of this study. The effect on hepatic function recovery of fasting periods of 24 h, 48 h and 72 h prior to cold ischemia were studied in a model of isolated, perfused rat liver. After a cold-ischemic time of 24 h in University of Wisconsin (UW) solution at 4 degrees C, livers were reperfused for 3 h. The combined effect of alanine (8 mM) infusion at liver reperfusion was evaluated for each prior fasting period. The addition of pyruvate (8 mM), a metabolic intermediary of alanine, was only tested in the 72-h fasting group. The evaluation criteria were: liver weight after reperfusion, release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in the perfusate, bile production, vascular resistance and liver histology after reperfusion. The enzyme release at reperfusion was significantly higher when livers were harvested from rats submitted to a 48-h fast (ALT) or a 72-h fast (ALT, AST, LDH), as compared to those from fed rats. Vascular resistance was increased in 72-h fasted livers. An addition of alanine (8 mM) at reperfusion lowered the release of AST, ALT and LDH. This effect was more obvious when the fasting duration was increased. By contrast, the addition of pyruvate at reperfusion did not improve the recovery of livers submitted to a 72-h fasting period before preservation. A long fasting period is deleterious as compared to feeding; however, this effect can be compensated by infusion of alanine at reperfusion. The mechanism involved is not metabolic. In a clinical setting, the infusion of alanine to the recipient at reperfusion may be a convenient way to compensate for donor undernutrition, especially after a long stay in an intensive care unit.     

Protagonism of the arterial circulation in the preservation of the pig's liver by simple perfusion and hypothermic storage

A modified technique to perform the successive perfusions of the liver that are necessary for its preservation by the simple perfusion method and hypothermic storage is presented. This technical variety has been tested on Large White pig's livers and consists in doing the successive perfusions of cooling, preservation and washout, first through the hepatic artery and there after through the portal vein. The macroscopic and biochemical characteristics of the effluents obtained through the infrahepatic inferior vena cava during the perfusion-washout of the livers at the end of the period cold ischemia shows its effectiveness. Likewise, the hepatic perfusion-washout begun via the arterial vein and finished via the portal one avoids post-revascularization hyperpotassemia in the receptor animals. It is hypothesized that the arterial protoganism of perfusions, when the simple perfusion and hypothermic storage method of hepatic preservation is used, could be a prophylaxis against complications of a post-transplant biliary origin.