Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW,HTK, and Celsior solutions

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4 degrees C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not hanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Improved rat liver preservation by hypothermic continuous machine perfusion using polysol, a new, enriched preservation solution.

For experimental machine perfusion (MP) of the liver, the modified University of Wisconsin solution (UW-G) is most often used. In our search for an enriched MP preservation solution, Polysol was developed. Polysol is enriched with various amino acids, vitamins, and other nutrients for the liver metabolism. The aim of this study was to compare Polysol with UW-G for MP preservation of the liver. Rat livers were preserved during 24 hours with hypothermic MP using UW-G (n = 5) or Polysol (n = 5). Hepatocellular damage (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], alpha-glutathione-S-transferase [alpha-GST]) and bile production were measured during 60 minutes of reperfusion (37 degrees C) with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours of cold storage in UW (n = 5). MP using UW-G or Polysol showed less liver damage when compared with controls. Livers machine perfused with Polysol showed less enzyme release when compared to UW-G. Bile production was higher after MP using either UW-G or Polysol compared with controls. In conclusion, machine perfusion using Polysol results in better quality liver preservation than cold storage with UW and machine perfusion using UW-G.     

Examination of the role of the impermeants lactobionate and raffinose in a modified UW solution

Rat liver transplants were performed in order to assess the importance of the impermeant anion lactobionate and the trisaccharide raffinose on the effectiveness of a simplified variant of UW solution for liver preservation by simple cold storage. Rat livers were stored at 4 degrees C for 18, 24, 30, or 40 hr in a modified UW solution or in one of three variants of UW in which one of these impermeants was replaced by another more permeable agent. Using modified UW solution (solution A), 50% (5/10) of rats receiving livers that had been preserved for 30 hr survived for more than 1 week; with solution B, which differs from A in the replacement of raffinose by glucose, the 1-week survival was 60% (6/10) after 30-hr preservation. Solution C, which is identical to A except for the replacement of lactobionate by gluconate, gave 20% (2/10) survival rate after 30-hr preservation. However, using solution D, which is identical to A except for substitution of chloride for lactobionate, none (0/8) of the rats receiving liver preserved for 30 hr survived. These results suggest that the inclusion of lactobionate as a major anion plays a crucial role in the effectiveness of UW solution, whereas raffinose can be replaced by more permeant glucose without deleterious effect.     

Correlation of donor nutritional status with sinusoidal lining cell viability and liver function in the rat

We have demonstrated that the sinusoidal lining cell injury sustained by rat liver allografts during hypothermic storage is a critical determinant of graft viability. The present study was designed to examine the effect of donor nutritional status on hepatic microcirculation and graft function. Rat livers from four nutritional groups (group I, fasted; group II, fed; group III, intraperitoneal glucose; and group IV, fed plus intraperitoneal glucose) were excised and stored for 24 hr in Marshall's isotonic citrate solution. Then the livers were perfused under anoxic conditions with trypan blue. The percentage of nonviable SLC in each group was 26.7 +/- 8.1, 24.9 +/- 7.9, 17.6 +/- 6.9, and 5.9 +/- 1.9 in groups I, II, III, and IV respectively; i.e., there was a significant improvement in SLC viability with nutritional repletion in group IV. Electron microscopy was performed on livers from groups I and IV following 30-hr preservation in University of Wisconsin solution and after 16-hr preservation in Marshall's isotonic citrate solution. Biopsies were taken at the end of storage and after 1 hr of reperfusion at 37 degrees C. At the end of preservation group IV livers contained glycogen and had much more normal liver ultrastructure than group I livers. After reperfusion there was partial recovery of normal SLC morphology in both groups and depletion of glycogen in group IV. Liver function was studied on the isolated perfused rat liver system at 37 degrees C following 30-hr storage in UW solution. Transaminase release into the perfusate was significantly lower in nutritionally repleted livers than in livers from fasted animals. A significant reduction in perfusate platelet count occurred only in livers from fasted animals. The results show that nutritional repletion can reduce the injury of cold preservation to both hepatocytes and endothelial cells and improve liver function in the postpreservation period.     

Evaluation of a Modified HTK Solution Containing the New Iron Chelator LK 614 in an Isolated Rat Liver Perfusion Model

Background: Cold storage-induced injury to donor organs remains a persistent problem in transplantation medicine. We here evaluated a modified HTK solution including, among others, a new, membrane-permeable iron chelator, LK 614, in the isolated perfused rat liver model. Methods: Rat livers were stored at 4°C for 24 hr in modified HTK solution, in modified HTK solution with LK 614 or in traditional HTK solution, and reperfused for 60 min at 37°C with Krebs-Henseleit buffer (KH buffer). Bile secretion and lactate dehydrogenase (LDH) release into the perfusate were measured, and hepatic microcirculation evaluated optically after the addition of trypan blue to the perfusate. Biopsies were evaluated by a pathologist blinded to the experimental conditions. Results: Compared with HTK-preserved livers, LDH leakage was significantly lower and bile secretion significantly higher in the modified HTK solution with the iron chelator (both p <. 05), while values for the modified solution without the iron chelator were in between (no significant difference to either of the two other solutions). The hepatic microcirculation was also preserved best in livers stored in the modified HTK solution with LK 614. Histomorphological investigation confirmed the improved preservation in the modified HTK solution with LK 614. Conclusions: These results suggest that livers might be better preserved in modified HTK solution containing the iron chelator LK 614 (and also, but less so, in the modified solution without the iron chelator) than in the original HTK solution, and such a modified solution should now be evaluated in an animal model of liver transplantation.     

Seventy-two-hour preservation of the canine liver by machine perfusion

The UW solution effectively preserves the dog liver for up to 48 hr by simple cold storage. This solution contains lactobionate as the primary impermeant. Another solution developed for machine perfusion of the kidney is similar to the UW solution but contains gluconate in place of lactobionate. In this study the UW gluconate solution was used for the continuous hypothermic machine perfusion of dog livers for 72 hr. Dog livers were continuously perfused at 5 degrees C through the portal vein at a pressure of 16-18 mm Hg and transplanted. Seven of 8 dogs survived for 7 or more days following orthotopic transplantation. The livers functioned as well as those preserved for 48 hr by cold storage in the UW solution as indicated by various liver-function tests. Successful machine perfusion was only achieved when the perfusate contained a high concentration of potassium (125 mM) but not with a high concentration of sodium (125 mM). This study demonstrates the feasibility of machine-perfusion preservation of the liver that yields longer preservation of equal quality compared to simple cold storage. For the development of truly long-term preservation (5 or more days) and better quality short-term preservation, machine perfusion may be the method of choice.     

Low viscosity histidine-tryptophan-ketoglutarate graft flush improves subsequent extended cold storage in University of Wisconsin solution in an extracorporeal rat liver perfusion and rat liver transplantation model.

Adequate flushing for liver donation requires large fluid volumes delivered at a high flow. This can be achieved more effectively with crystalloid solutions than with colloid-based solutions. This study examined the combination of initial histidine-tryptophan-ketoglutarate solution (HTK) graft flush and subsequent storage in University of Wisconsin solution (UW) to that of the single use of each solution. Livers from inbred Wistar rats were procured using aortic perfusion with UW or HTK for initial perfusion and reflushed after 30 minutes using either solution. In a third group, after perfusion with HTK, organs were reflushed with UW. A 60-minute in-vitro recirculating perfusion was performed after 24 hours of cold storage in the subsequent solution, as well as allotransplantation after 18 and 24 hours of cold storage. In extracorporeal perfusion, the HTK flush followed by UW storage was superior compared to the single use of either UW or HTK solution, as measured by portal venous pressure, bile flow, liver enzymes released into the effluent perfusate, glycerol leakage, and histological examinations. These data were consistent with the transplantation study. Histological damage and enzyme release after 5-day survival were lowest in the HTK flush and subsequent UW storage groups following 18 hours of cold storage; likewise, the 5-day survival was superior following 24 hours of cold storage. In conclusion, the combined use of HTK solution for initial graft rinse and subsequent storage in UW solution resulted in a cumulative protection. Choosing low-viscosity HTK solution for the initial organ flush may represent a feasible improvement in liver preservation, which also further reduces the required amount of UW solution.     

Comparison of histidine-tryptophan ketoglutarate solution and University of Wisconsin solution in prolonged cold preservation of kidney allografts

Although University of Wisconsin (UW) solution is the standard preservation solution for organ transplantation, Histidine-Tryptophan Ketogluatarate (HTK) solution has been increasingly used. This study compared HTK or UW for cold static storage of kidney allografts. In all, 149 renal transplants were performed with cold ischemic times (CI) greater than 16 hr (UW 87, HTK 62) and a subset analysis was performed with CI over 24 hr (HTK 31, UW 38). Data from receiving renal transplant centers focused on delayed graft function (DGF), patient and allograft survival. In CI greater than 16 hr, graft and patient survival were comparable. HTK cohort had lower DGF. In CI greater than 24 hr, there was no difference in patient survival, a trend towards improved graft survival in HTK, and decreased rate of DGF in HTK. This data suggests that UW and HTK have at least similar efficacy in kidney preservation at longer ischemic times.     

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.     

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.     

Enzymatic and morphological evaluation of the hepatic cytoprotective effect of somatostatin and octreotide during extended experimental liver preservation

Cytoprotective effects on the liver of somatostatin (ST) and octreotide (OT) have been previously described in normothermic ischemia-reperfusion models. The purpose of this study was an enzymatic and morphological assessment of hepatic cytoprotective effects during extended cold storage. Rat livers were washed in situ via the portal vein with University of Wisconsin solution (UW) UW+ST, or UW+OT. After 24 or 48 hours of cold ischemia time (CIT), livers were reperfused for 2 hours via the portal vein with oxygenated KHB at 37 degrees C using a nonrecirculating ex situ isolated perfusion system. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatinine kinase (CK) were assessed in the perfusate during ex situ isolated reperfusion. After a 24-hour cold ischemia time (CIT) ALT, LDH, and CK levels were significantly lower (P < .05) in the UW+ST and the UW+OT livers than the UW livers. After 48-hour CIT, AST, ALT, LDH, and CK levels were significantly lower (P < .05) in the UW+ST and the UW+OT livers than the UW livers. Histopathological examination revealed mild differences after 24-hour CIT but an evidently less ischemically damage organ after 48-hour CIT. With the limitations of an in vitro model, ST and OT showed enzymatic and morphological effects during extended liver preservation.