The caspase inhibitor IDN-6556 prevents caspase activation and apoptosis in sinusoidal endothelial cells during liver preservation injury

Cold ischemia (CI)-warm reperfusion (WR) liver injury remains a problem in liver transplantation. CI-WR initially causes sinusoidal endothelial cell (SEC) apoptosis through a caspase-dependent mechanism. We previously showed that the caspase inhibitor IDN-1965 prevents CI-WR-induced SEC apoptosis. However, this agent required to be administered to the donor, preservation solution, and recipient for efficacy. Here, we show that a second-generation caspase inhibitor, IDN-6556, effectively prevents CI-WR-induced SEC injury when added only to University of Wisconsin (UW) cold storage media. Rat livers were stored in UW solution for 24 hours at 4 degrees C and reperfused for 1 hour at 37 degrees C. Apoptosis was quantitated using terminal deoxynucleotide transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and caspase 3 activation determined by biochemical measurement and immunohistochemical analysis. Pan-caspase inhibitors (IDN-8066, IDN-7503, IDN-7436, IDN-1965, and IDN-6556) were applied at preischemic, cold preservation, or reperfusion periods. TUNEL-positive SEC and caspase 3-like activity in the liver was increased by CI-WR. Three caspase inhibitors (IDN-8066, IDN-1965, and IDN-6556) effectively attenuated SEC apoptosis and caspase 3 activation. The most potent inhibitor, IDN-6556, reduced SEC apoptosis and caspase 3 activity by 55% and 94%, respectively. Prevention of SEC apoptosis by IDN-6556 was not reduced when this agent was administered only during the cold preservation period. When added to the preservation solution, the caspase inhibitor IDN-6556 appears to be a feasible therapeutic agent against ischemia-reperfusion injury in liver transplantation.     

Clinical Trial of the Pan-Caspase Inhibitor, IDN-6556, in Human Liver Preservation Injury

Cold ischemia/warm reperfusion (CI/WR) injury remains a problem in liver transplantation. The aim of the current study was to assess the utility of the pan-caspase inhibitor IDN-6556 on CI/WR injury during human liver transplantation. This report is a post hoc analysis of a Phase II, multi-center, randomized, placebo-controlled, double-blinded, parallel group study. Subjects were assigned to four treatment groups: Group 1 (Organ storage/flush: Placebo-Recipient: Placebo); Group 2 (Organ storage/flush: 15 microg/mL-Recipient: Placebo); Group 3 (Organ storage/flush: 5 microg/mL-Recipient: 0.5 mg/kg); and Group 4 (Organ storage/flush: 15 microg/mL-Recipient: 0.5 mg/kg). Liver cell apoptosis was assessed by serum concentrations of the apoptosis-associated CK18Asp396 ('M30') neo-epitope, TUNEL assay and caspase 3/7 immunohistochemistry. Liver injury was assessed by serum AST/ALT determinations. Serum markers of liver cell apoptosis were reduced in all groups receiving drug as compared to placebo. However, TUNEL, caspase 3/7 positive cells and serum AST/ALT levels were only consistently reduced in Group 2 (drug exposed to organ only). This reduction in serum transaminases was significant and observed across the study. In conclusion, IDN-6556 when administered in cold storage and flush solutions during liver transplantation offers local therapeutic protection against CI/WR-mediated apoptosis and injury. However, larger studies are required to confirm these observations.     

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.     

Hepatic control of perfusate homeostasis during normothermic extrocorporeal preservation

BACKGROUND: We investigated the ability of the isolated porcine liver to maintain acid-base homeostasis in the perfusate and the impact of ischemia-reperfusion injury without or with extracorporeal perfusion. METHODS: Harvested livers were either stored for 24 hours in cold University of Wisconsin solution or preserved by continuous, normothermic, oxygenated sanguineous perfusion with supplemental nutrition, prostacyclin, and bile salts. After a further 24-hour period of reperfusion of both groups on an extracorporeal circuit, the perfusate was assessed for both biochemical indices of synthetic and metabolic liver function as well as hepatocellular injury and blood gas analysis. RESULTS: Livers injured by cold ischemia during preservation displayed inferior synthetic and metabolic functions. Perfused livers, which displayed minimal ischemic injury, produced more bicarbonate than the cold-stored organs, suggesting autoregulation of pH homeostasis in perfused livers in contrast to progressively worsening acidosis in cold-stored organs. CONCLUSIONS: Given proper physiologic substrate the porcine liver has the ability to maintain acid-base homeostasis, provided there is not a significant ischemia-reperfusion injury.     

Ischemic preconditioning and liver tolerance to warm or cold ischemia: experimental studies in large animals

BACKGROUND: In the rodent, ischemic preconditioning (IPC) has been shown to improve the tolerance of the liver to ischemia-reperfusion under normothermic or hypothermic conditions. The aim of the present study was to test this hypothesis in a dog model, which may be more relevant to the human. METHODS: Beagle dogs were used in two distinct animal models of hepatic warm ischemia and orthotopic liver transplantation (hypothermic ischemia). IPC consisted of 10 minutes of ischemia followed by 10 minutes of reperfusion. In the first model, livers were exposed to 55 minutes prolonged warm ischemia and reperfused for 3 days (n = 6). In the second model, livers were retrieved and preserved for 48 hours at 4 degrees C in University of Wisconsin solution, transplanted, and reperfused without immunosuppression for 7 days (n = 5). In each model, nonpreconditioned animals served as controls (n = 5 in each group). Also, isolated dog hepatocytes were subjected to warm and cold storage ischemia-reperfusion to model the animal transplant studies using IPC. RESULTS: In the first model (warm ischemia), IPC significantly decreased serum aminotransferase activity at 6 and 24 hours post-reperfusion. After 1 hour of reperfusion, preconditioned livers contained more adenosine triphosphate and produced more bile and less myeloperoxidase activity (neutrophils) relative to controls. In the second model (hypothermic preservation), IPC was not protective. Finally, IPC significantly attenuated hepatocyte cell death after cold storage and warm reperfusion in vitro. CONCLUSIONS: IPC is effective in large animals for protecting the liver against warm ischemia-reperfusion injury but not injury associated with cold ischemia and reperfusion (preservation injury). However, the IPC effect observed in isolated hepatocytes suggests that preconditioning for preservation is theoretically possible.     

Preservation of porcine non-heart-beating donor livers by sequential cold storage and warm perfusion

BACKGROUND: Normothermic perfusion has been shown to resuscitate and maintain viability of non-heart-beating donor (NHBD) livers that have undergone significant warm ischemic injury. However, the logistics of clinical organ retrieval are complex, and a period of cold storage before warm preservation would simplify the process. We have investigated the effects of short duration of cold preservation before normothermic preservation on the function of porcine NHBD livers. METHODS: Porcine livers were subjected to 60 minutes of warm ischemia and then assigned to the following groups: group W (n=5), normothermic preservation for 24 hours; and group C (n=4), cold preservation in University of Wisconsin solution for 4 hours followed by normothermic preservation for 20 hours (total preservation time 24 hours). Outcome parameters that were measured included bile production, serum transaminases and hyaluronic acid levels (cellular damage), and base deficit and glucose use (metabolic function). RESULTS: Group W livers had superior bile production, metabolic activity (base deficit and greater glucose use), and less evidence of hepatocellular damage (alanine aminotransferase, aspartate aminotransferase), and sinusoidal endothelial cell dysfunction (hyaluronic acid). Group C livers showed greater necrosis and destruction of architecture on histology. CONCLUSION: Normothermic perfusion failed to resuscitate porcine livers after 60 minutes of warm ischemia and 4 hours of cold preservation. Even a short period of cold ischemia is significantly deleterious to the function of ischemically damaged (NHBD) livers.     

Effects of warm Carolina rinse on microvascular reperfusion injury in rat liver transplantation

Abstract Recently, it has been demonstrated that the use of both cold Carolina rinse (CR, 4°C) as well as warm Ringer's lactate (RL, 37°C) attenuates microvascular perfusion failure and leukocyte (WBC) accumulation in liver grafts. The aim of this study was to analyse in vivo whether warming of CR can also lead to a reduction in microvascular reperfusion injury in rat liver transplantation. Syngeneic orthotopic liver transplantation, including arterial reconstruction, was performed in male Lewis rats (180–300 g). Livers were stored in University of Wisconsin (UW) solution for 24 h and rinsed with 15 ml CR which was either cold 4 °C ( n = 7) or warm 37 °C ( n = 8) prior to reperfusion. Hepatic microcirculation and WBC accumulation were assessed by intra-vital fluorescence microscopy, and graft function was determined by analysis of bile flow during the 90-min reperfusion period. Warm CR yielded significantly ( P < 0.01) improved sinusoidal perfusion when compared with cold CR; however, the extent of WBC adherence in both sinusoids and postsinusoidal venules did not vary between the groups. In addition, bile flow was slightly increased after warm CR. We conclude that after 24 h of cold storage in UW solution, warming of CR may offer additional benefit in the prevention of microcirculatory reperfusion injury without affecting WBC accumulation.     

Pharmacologic preconditioning effects: Prostaglandin E1 induces heat-shock proteins immediately after ischemia/reperfusion of the mouse liver

Prostaglandin E1 (PGE1) has several potential therapeutic effects, including cytoprotection, vasodilation, and inhibition of platelet aggregation. This study investigates the protective action of PGE1 against hepatic ischemia/reperfusion injury in vivo using a complementary DNA microarray. PGE1 or saline was continuously administered intravenously to mice in which the left lobe of the liver was made ischemic for 30 minutes and then reperfused. Livers were harvested 0, 10, and 30 minutes postreperfusion. Messenger RNA was extracted, and the samples were labeled with two different fluorescent dyes and hybridized to the RIKEN set of 18,816 full-length enriched mouse complementary DNA microarrays. Serum alanine aminotransferase and aspartate aminotransferase levels at 180 minutes postreperfusion were significantly lower in the PGE1-treated group than in the saline-treated group. The cDNA microarray analysis revealed that the genes encoding heat-shock protein (HSP) 70, glucose-regulated protein 78, HSP86, and glutathione S-transferase were upregulated at the end of the ischemic period (0 minutes postreperfusion) in the PGE1 group. Our results suggested that PGE1 induces HSPs immediately after ischemia reperfusion. HSPs might therefore play an important role in the protective effects of PGE1 against ischemia/reperfusion injury of the liver.     

Hydrogen Gas Ameliorates Hepatic Reperfusion Injury After Prolonged Cold Preservation in Isolated Perfused Rat Liver

Hydrogen gas reduces ischemia and reperfusion injury (IRI) in the liver and other organs. However, the precise mechanism remains elusive. We investigated whether hydrogen gas ameliorated hepatic I/R injury after cold preservation. Rat liver was subjected to 48‐h cold storage in University of Wisconsin solution. The graft was reperfused with oxygenated buffer with or without hydrogen at 37° for 90 min on an isolated perfusion apparatus, comprising the H₂(+) and H₂(?) groups, respectively. In the control group (CT), grafts were reperfused immediately without preservation. Graft function, injury, and circulatory status were assessed throughout the perfusion. Tissue samples at the end of perfusion were collected to determine histopathology, oxidative stress, and apoptosis. In the H₂(?) group, IRI was indicated by a higher aspartate aminotransferase (AST), alanine aminotransferase (ALT) leakage, portal resistance, 8‐hydroxy‐2‐deoxyguanosine‐positive cell rate, apoptotic index, and endothelial endothelin‐1 expression, together with reduced bile production, oxygen consumption, and GSH/GSSG ratio (vs. CT). In the H₂(+) group, these harmful changes were significantly suppressed [vs. H₂(?)]. Hydrogen gas reduced hepatic reperfusion injury after prolonged cold preservation via the maintenance of portal flow, by protecting mitochondrial function during the early phase of reperfusion, and via the suppression of oxidative stress and inflammatory cascades thereafter.     

Preservation of pig liver allografts after warm ischemia: normothermic perfusion versus cold storage

Warm ischemia is known to induce substantial damage to the liver parenchyma. With respect to clinical liver transplantation, the tolerance of the liver to warm ischemia and the preservation of these organs have not been studied in detail. In isolated reperfused pig livers we proceeded according to the following concept: Livers were subjected to 1 or 3 h of warm ischemia. Subsequently, these organs were preserved by either normothermic perfusion or cold storage (histidine-tryptophan-α-ketoglutarate, HTK) for 3 h each. After storage, liver function was assessed in a reperfusion circuit for another 3 h. Parameters under evaluation were bile flow, perfusion flow, oxygen consumption, enzyme release into the perfusate (creatine kinase, glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase, and glutamic pyruvic transaminase), and histomorphology. Damage to the liver was lowest after warm ischemia of 1 h. The results after cold storage were superior to those after normothermic perfusion (GOT: 3.2±0.3 and 2.6±0.2 U/g liver; cumulative bile production: 14.7±2.1 and 9.4±1 ml, respectively;P<0.05). In contrast, we found substantial damage at the end of reperfusion in livers undergoing 3 h of warm ischemia under both preservation techniques with severe hepatocellular pyknoses and essentially altered nonparenchymal cells. The results suggest that pig livers undergoing 1 h of warm ischemia and cold storage for 3 h with HTK solution may lead to functioning after transplantation.     

Protection of the rat liver against rewarming ischemic injury by University of Wisconsin solution

BACKGROUND/AIM: University of Wisconsin (UW) solution has been proven able to prevent liver injury during cold ischemia. During rewarming ischemia, however, the efficacy of this solution in preserving hepatocyte function is unclear. The aim of the present study was to investigate to what extent UW solution protects rat liver during rewarming ischemia. METHODS: Livers were washed out with cool physiologic saline or with UW solution and subjected to rewarming ischemia for periods of 20 min or 45 min followed by reperfusion using a blood-free perfusion model. RESULTS: In comparison with controls, ischemia for 20 min in saline-treated livers led to mild depression of hepatocyte function, while UW solution afforded complete protection of the liver. In UW-treated livers, compared with saline-treated livers exposed to ischemia for 45 min, portal flow was slightly but significantly higher, bile production was increased by 62%, and lactate dehydrogenase leakage into the perfusate was reduced by 61%. In an attempt to explain mechanisms of liver protection by UW solution, we found that UW solution inhibited conversion of hypoxanthine into uric acid, but this effect was not associated with decreased degradation of adenine nucleotides in the liver during ischemia. Following 30 min reperfusion, UW solution increased tissue levels of adenosine triphosphate (not significantly) and adenosine diphosphate (significantly). Further, UW solution markedly reduced tumor necrosis factor-alpha release by the liver both after ischemia and after reperfusion. CONCLUSIONS: These results create the hypothesis that UW solution may protect liver tissue during ischemia in liver surgery as well as during the implantation stage of liver transplantation.     

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.     

Apoptosis of sinusoidal endothelial cells occurs during liver preservation injury by a caspase-dependent mechanism.

BACKGROUND: Cold ischemia/warm reperfusion (CI/WR) liver injury remains a problem in liver transplants. Sinusoidal endothelial cells (SEC) are a target of CI/WR injury, during which they undergo apoptosis. Because caspase proteases have been implicated in apoptosis, our aim was to determine whether liver CI/WR injury induces a caspase-dependent apoptosis of SEC. METHODS: Rat livers were stored in the University of Wisconsin (UW) solution for 24 hr at 4 degrees C and reperfused for 1 hr at 37 degrees C in vitro. Apoptosis was quantitated using the TUNEL assay, and caspase 3 activation determined by immunohistochemical analysis. Rat liver orthotopic liver transplants (OLT) were also performed using livers stored for 30 hr. RESULTS: Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive hepatocytes were rare and did not increase during CI/WR injury. In contrast, TUNEL positive SEC increased 6-fold after reperfusion of livers stored under cold ischemic conditions, compared with controls or livers stored but not reperfused. Immunohistochemical analysis demonstrated active caspase 3 only in endothelial cells after CI/WR injury. When IDN-1965, a caspase inhibitor, was given i.v. to the donor animal and added to UW solution and the reperfusion media, TUNEL positive endothelial cells were reduced 63+/-11% (P<0.05). Similarly, the duration of survival after OLT was significantly increased in the presence of the inhibitor. CONCLUSION: During liver CI/WR injury: 1) selective apoptosis of endothelial cells occurs; 2) caspase 3 is activated only in endothelial cells; and 3) a caspase inhibitor reduces endothelial cell apoptosis and prolongs animal survival after OLT. The pharmacologic use of caspase inhibitors could prove useful in clinical transplantation.     

Effects of taurine on liver preservation in UW solution with consecutive ischemic rewarming in the isolated perfused rat liver

Taurine (2-aminoethane sulfonic acid) is a physiologic amino acid involved in cellular osmoregulation in various species including man. This study was intended to compare the respective effects of cold storage and consecutive ischemic rewarming of the liver on postischemic hepatic flow and hepatocellular outcome upon reperfusion with or without the addition of taurine to the preservation medium. Livers from male Wistar rats were rinsed free of blood via the portal vein and stored ischemically at 4 °C in UW solution. Livers from group 1 were then rinsed again with 10 ml Ringer's solution and reperfused with Krebs-Henseleit buffer at a constant pressure of 10 mmHg for 45 min at 37 °C in a nonrecirculating manner. Livers from groups 2 and 3 were subjected to 30 min of warm ischemia subsequent to cold storage and prior to reperfusion with 10 mM taurine added to the UW solution in group 3. While there were only very few signs of hepatic injury in group 1, the additional period of warm ischemia (group 2) led to a significant reduction in early perfusate flow and enhanced enzyme leakage from the livers during postischemic rinse and reperfusion. Livers in group 3 exhibited an amelioration in hepatic circulation and significantly reduced enzyme release as compared to group 2. The results clearly demonstrate a remarkable impact of postischemic rewarming on graft viability. Furthermore, the addition of taurine to the preservation medium was shown to improve hepatic circulation and enhance viability of the liver upon reperfusion.     

大黄素对大鼠肝脏缺血再灌注损伤的预防作用

目的:探讨大黄素对肝脏缺血-再灌注损伤的保护作用及其机制.方法:将45只成年雄性SD大鼠随机分成三组:假手术对照组(A组)15只,肝缺血30 min、再灌注90min组(B组)15只,术前5 d给予大黄素灌胃60mg/(kg·d),5次 肝缺血30 min、再灌注90min(C组)15只;观察血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、透明质酸酶(HA)、肝组织丙二醛(MDA)含量的变化及肝脏组织病理学改变情况,同时测定各组大鼠胆汁流量情况.结果:C组与B组相比,MDA含量显著降低(P＜0.05),HA浓度明显降低(P＜0.01),胆汁流量显著增加(P＜0.01),同时C组的肝细胞功能明显改善,且肝脏超微结构改变也较轻.结论:大黄素对肝脏缺血-再灌注损伤具有保护作用,该作用与减轻肝脏缺血再灌注后脂质过氧化程度和肝窦内皮细胞损伤有关.     

Effects of a p38 mitogen-activated protein kinase inhibitor as an additive to university of wisconsin solution on reperfusion injury in liver transplantation

BACKGROUND: Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in the development of ischemia/reperfusion injury in nonhepatic organs, such as the heart. However, the role of p38 MAPK activation in the liver is unclear. We examined the effects of FR167653, a novel p38 MAPK inhibitor, as an additive to University of Wisconsin (UW) solution in rat liver transplantation. METHODS: Rat orthotopic liver transplantation was performed after 30 hr of cold storage using UW solution with or without FR167653. Ten-day survival rates, serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels, liver tissue blood flow, histological findings, and activities of p38 MAPK and p46/p54 c-Jun N-terminal kinase (JNK) in liver grafts were evaluated. RESULTS: The addition of FR167653 significantly increased animal survival rates. FR167653 significantly suppressed serum ALT and LDH levels and improved liver tissue blood flow after transplantation. FR167653 also ameliorated histological damage to the liver graft. Neither p38 MAPK nor p46/p54 JNKs was activated during cold storage, whereas both were markedly activated within 30 min of reperfusion and remained activated until 60 min after reperfusion. FR167653 inhibited the activation of p38 MAPK both 30 and 60 min after reperfusion, but it did not affect the activation of p46/p54 JNKs. CONCLUSIONS: The addition of FR167653 to UW solution improved liver graft viability and animal survival rates associated with the inhibition of p38 MAPK activation. These results suggest that inhibiting the activation of p38 MAPK may attenuate ischemia/reperfusion injury in liver transplantation.     

Preimplant Normothermic Liver Perfusion of a Suboptimal Liver Donated After Circulatory Death

Livers retrieved after circulatory death are associated with an increased incidence of primary nonfunction, early allograft dysfunction, and biliary strictures. The authors report a case of preimplant normothermic perfusion of a suboptimal liver from a 57‐year‐old donor after circulatory death who had been hospitalized for 9 days; predonation alanine transaminase level was 63 IU/L, and the period from withdrawal of life‐supporting treatment to circulatory arrest was 150 minutes. After 5 hours of static cold storage, the liver was subject to normothermic machine perfusion with a plasma‐free red cell–based perfusate. Perfusate lactate level fell from 7.2 to 0.3 mmol/L within 74 minutes of ex situ perfusion, at which point perfusate alanine transaminase level was 1152 IU/L and urea concentration was 9.4 mmol/L. After 132 minutes, normothermic perfusion was stopped and implantation begun. After transplantation, the patient made an uneventful recovery and was discharged on day 8; liver biochemistry was normal by day 19 and has remained normal thereafter. Donor common bile duct excised at implantation showed preservation of peribiliary glands, and cholangiography 6 months posttransplantation showed no evidence of cholangiopathy. Preimplant ex situ normothermic perfusion of the liver appears to be a promising way to evaluate a marginal liver before transplantation and may modify the response to ischemia.     

Adding Pulsatile Vascular Stimulation to Venous Systemic Oxygen Persufflation of Liver Grafts

The effect of adding pulsatility to gaseous oxygen persufflation during liver preservation was studied in an isolated rat liver model. Livers from male Wistar rats were retrieved 30 min after cardiac arrest of the donor and subjected to 18 h of cold storage. Some grafts were subjected to nonpulsatile or pulsatile gaseous oxygen persufflation. Graft viability was assessed thereafter upon warm reperfusion in vitro (n = 5 per group). Pulsatile persufflation significantly improved parenchymal integrity (enzyme release, bile flow) upon reperfusion, with respect to nonpulsatile persufflation or cold storage (CS) (e.g., max. release of alanine aminotransferase: 44 ± 10 vs. 178 ± 29 vs. 345 ± 100 U/L; pulsatile vs. nonpulsatile persufflation vs. CS).The effect was associated with the prevention of the ischemic decline in gene and protein expression of the vasoprotective Krüppel‐like factor 2, increased perfusate levels of the endogenous vasodilator nitric oxide, and reduced portal vascular resistance upon reperfusion, while nonpulsatile persufflation was less effective (e.g., vascular resistance: 1235 ± 108 vs. 1607 ± 155 vs. 2215 ± 208 Pa s/mL; pulsatile vs. nonpulsatile persufflation vs. CS). In conclusion, pulsatile mechanostimulation of the hepatovasculature seems a genuine protective mechanism, affecting early graft recovery upon reperfusion.     

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.