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.     

Evaluation of rat liver apoptotic and necrotic cell death after cold storage using UW,HTK, and Celsior

BACKGROUND: The benefit of Celsior in liver graft preservation is controversial. In the isolated perfused rat liver model, we compared the effects of Celsior, University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) preservation solutions on liver cell death. METHODS: Rat livers were stored at 4 degrees C for 0, 8, 16, or 24 hr in either Celsior, UW, or HTK and reperfused for 90 min (37 degrees C). Bile secretion and perfusate levels of liver enzymes and histone-associated DNA fragments were measured. Apoptosis and oncotic necrosis were analyzed in biopsies by DNA gel electrophoresis, hematoxylin and eosin histology, and enzyme histochemistry for lactate dehydrogenase (LDH) and 5'-nucleotidase (5'-NT). RESULTS: Perfusate flow rate through the liver during perfusion did not significantly differ among preservation solutions. Bile secretion was best preserved in UW livers after 16-hr (versus HTK livers) and 24-hr storage (versus HTK and Celsior livers). Enzyme leakage from UW livers was lower compared with HTK livers after 8-hr storage (serum glutamic oxaloacetic transaminase [SGOT], LDH) and with Celsior and HTK livers after 16-hr (SGOT, LDH) and 24-hr storage (SGOT, serum glutamic pyruvic transaminase, LDH, purine nucleoside phosphorylase). In situ LDH and 5'-NT activities were best preserved in UW livers (up to 24 hr), whereas enzyme activities declined remarkably in HTK livers (after 8 hr) and Celsior livers (after 16 hr of cold storage). Although perfusate DNA fragment levels were repeatedly lowest from Celsior livers, apoptotic DNA laddering and the number of fragmented nuclei in hematoxylin and eosin sections was not different among livers after 8, 16, or 24 hr of storage. CONCLUSIONS: Celsior and UW are equally effective in preventing rat liver cell death after 0-16 hr of cold preservation as compared with the less effective HTK solution. After 24-hr cold storage, rat livers were best preserved in UW. Furthermore, there was no significant difference in mode of cell death (apoptosis or oncotic necrosis) after storage in any of the three solutions.     

Verapamil improves rat hepatic preservation with UW solution

Verapamil, a calcium channel blocker, improves myocardial preservation during cold cardioplegia and protects against renal damage during periods of warm and cold ischemia. To determine if verapamil could prevent ischemic damage to livers during and after cold storage, harvested rat livers were flushed with either University of Wisconsin (UW) solution or UW solution with 25 mg/liter verapamil. Twenty rats were used in each group. After 24 hr of storage at 4 degrees C, livers were perfused with oxygenated blood through the portal veins for 2 hr at 37 degrees C and pH 7.4. Liver enzymes, electrolytes, and perfusate flow rate were determined at 30-min intervals. At 90 min of perfusion, the verapamil group of livers had less elevation of AST (110 +/- 17 IU/liter vs 172 +/- 25 IU/liter, P less than 0.05), ALT (115 +/- 21 IU/liter vs 210 +/- 34 IU/liter, P less than 0.05), and LDH (962 +/- 170 IU/liter vs 1452 +/- 253 IU/liter, NS). Verapamil livers produced more bile than controls (6.9 +/- 1.9 microliters/g vs 2.3 +/- 1.7 microliter/g, P less than 0.05) and maintained a higher portal flow rate throughout the perfusion. Both groups showed similar reduction in liver weights after storage (3.9 +/- 0.9% vs 2.8 +/- 0.7%) and required the same amount of bicarbonate for correction of acidosis during perfusion (2.6 +/- 0.2 mM vs 2.8 +/- 0.2 mM). Light microscopic exam after perfusion showed hepatocyte damage in 30% of control livers, but 0% of verapamil livers. We conclude that verapamil-treated rat livers showed less damage and better function upon reperfusion after 24 hr of cold storage. This agent may be clinically useful as an additive to the UW preservation solution for livers.     

Effect of S-nitrosoglutathione (GSNO) added to the University of Wisconsin solution (UW): I) Morphological alteration during cold preservation/reperfusion of rat liver

Cold liver preservation in the University of Wisconsin solution (UW) followed by reperfusion alters hepatic parenchyma and stroma. In this study we demonstrated the benefit of adding S-nitrosoglutathione (GSNO) to the UW solution before cold storage, as an effective Nitric Oxide (NO) donor to prevent hepatic injury. Wistar adult rat livers were stored in UW solution (4 degrees C-48Hs) and then reperfused 60 minutes in the isolated perfused rat liver model (IPRL). Normal untreated livers and perfused livers, but not preserved were used as controls. Parenchymal damages were evaluated with Hematoxylin-Eosin stain and an inmunohistochemistry assay for albumin was used as functional test. To study the stroma, collagen type III and I networks were analyzed using Picro-sirius Red stain and Gordon Sweets' method for reticulin. After 48 Hs of cold preservation in UW solution livers showed few rounded endothelial cells inside sinusoidal lumen and extended areas of cell vacuolation. Albumin distribution was evident only around central veins and middle zones of the hepatic lobule. Collagens III and I networks were disorganized. When preserved with the addition of 100 microM GSNO and then reperfused, the hepatic morphology, in general, was conserved showing little vacuolation, fewer endothelial cells inside sinusoids and good albumin distribution around central veins and middle zones. The stroma had organized networks of collagen III and I. We concluded that the addition of 100 microM GSNO as a NO donor, can improve UW solution properties to preserve rat liver by maintaining the hepatic morphology and avoiding hepatic injury post cold preservation/reperfusion.     

Effects of hypothermic machine perfusion on rat liver function depending on the route of perfusion.

BACKGROUND AND METHODS: The aim of this study was to evaluate the efficacy of hypothermic machine perfusion (HMP) to preserve rat livers according to the route of perfusion, i.e., via portal vein, hepatic veins (retrograde), or hepatic artery. Livers were preserved for 24 or 48 hr by simple cold storage (SCS) or by HMP. Preservation solution was supplemented with (HMP) or without (SCS) hydroxyethyl starch. After preservation, grafts were reperfused for 2 hr with an oxygenated Krebs-Henseleit bicarbonate buffer. RESULTS: After 24 hr of preservation, total glutathione concentrations in HMP livers were similar (1287+/-37, 1418+/-118, and 1471+/-62 nmol/g in hepatic artery, portal vein, and hepatic vein HMP livers, respectively) and higher than in the SCS (833+/-118 nmol/g, P<0.05) group. These higher total glutathione values were due to higher reduced glutathione concentrations. ATP concentrations in the liver tissue were similar in HMP groups (0.75+/-0.4, 0.64+/-0.1, and 0.77+/-0.1 micromol/g in hepatic artery, portal vein, and hepatic vein HMP livers, respectively) and higher than in SCS (0.32+/-0.06 micromol/g, P<0.05). After 2 hr of normothermic reperfusion, bile production in the HMP portal and HMP retrograde groups were similar (391+/-29 ml and 372+/-25 ml) and higher than in the HMP artery or SCS groups (275+/-25 ml and 277+/-32 ml, respectively; P<0.05). Aspartate transaminase, alanine transaminase, lactate dehydrogenase, and purine nucleoside phosphorylase release into the perfusate of HMP portal and HMP retrograde perfused livers was similar and significantly lower compared to the HMP artery and SCS groups. At the end of reperfusion, no statistical differences were found for glutathione concentration and energetic reserves in the livers of each group. After 48 hr of preservation, livers from the HMP portal and HMP retrograde groups did significantly better than livers from the HMP artery or SCS groups. CONCLUSIONS: This study confirms the superiority of HMP over SCS to preserve the liver graft. It shows that retrograde perfusion is similar to PV perfusion and that perfusion by HA is less beneficial.     

Hypothermic machine perfusion of the liver and the critical balance between perfusion pressures and endothelial injury

Hypothermic machine perfusion (HMP) provides better protection against cold ischemic injury than cold storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Rat livers were cold-perfused for 24 hours to study perfusion pressures for both hepatic artery and portal vein. Cold storage served as control and was compared to HMP-preserved livers using a mean arterial perfusion pressure of 25 mm Hg and a portal perfusion pressure of 4 mm Hg (25% of normothermic liver circulation) and to HMP at 50 mm Hg and 8 mm Hg perfusion, respectively (50% of normothermic liver circulation). UW solution was enriched with 14.9 micromol/L propidium iodide (PI) to stain for dead cells and with an additional 13.5 micromol/L acridine orange to stain for viable hepatocytes. A low PI-positive cell count was found using HMP at 25% of normal circulation compared to cold storage. The PI count was high for the HMP group perfused at just 50% of normal circulation compared to HMP at 25% and compared to cold storage. In summary, for liver HMP, perfusion at 25% showed complete perfusion with minimal cellular injury. HMP using perfusion pressures of 25 mm Hg for the hepatic artery and 4 mm Hg for the portal vein is feasible without induction of endothelial injury.     

Improved liver preservation for transplantation due to calcium channel blockade.

Intracellular calcium is an important determinant for cell death in organ hypothermic preservation for transplantation. In this study, we show that prevention of calcium entry improves the result of liver cold storage in UW solution. The isolated perfused rabbit liver was used. After 48 hr of cold storage in UW solution, bile production was reduced by 70% (P less than 0.005). However, by adding the calcium channel blockers verapamil or nifedipine (40 microM) to the UW solution, this reduction was abolished, and the livers produced the same amount of bile as unpreserved livers. Furthermore, addition of the calcium channel activator, BAY K8644 (40 microM), to the UW solution, reduced bile production by 50% (P less than 0.01) already after preservation for 24 hr. We conclude that calcium entry is of importance for liver function after preservation and cold storage, and that including a calcium channel blocker to the preservation solution makes long-term liver preservation safer.     

High-Na + low-K + UW cold storage solution reduces reperfusion injuries of the rat liver graft

The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K ⁺ UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na ⁺ UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na ⁺ UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K ⁺ UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na ⁺ UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na ⁺ UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K ⁺ UW solution. Liver preservation in the high-Na ⁺ UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution. Received: 26 August 1997 Received after revision: 12 November 1997 Accepted: 28 November 1997     

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.     

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.     

Washout of the pig liver with Haemaccel after hypothermic preservation

In hepatic preservation by simple perfusion and hypothermic storage, a portal and hepatic washout before revascularization would avoid receptor hyperkaliema. In this report we study the effectiveness of this washout with Haemaccel at room temperature. Large-White pigs were used and eight livers were perfused "in situ" via the portal wein with Hartmann's solution containing 10,000 IU of heparin at 4 degrees C, and afterwards, via portal and arterial routes with C2 solution at 4 degrees C. After a cold ischemia time of less than 31/2 hours a liver washout via the portal vein and hepatic artery with Haemaccel before portal revascularization was done. The high concentrations of glucose, K+, GOT, GPT and LDH in the effluents obtained during the washout are attributed to Haemaccel hyperosmolarity. A portal and arterial hepatic washout associated with free drainage of the first 50-100 ml of portal venous blood after hepatic portal revascularization through the infrahepatic inferior vena cava (IH-IVC), prevents hyperkaliemia from occurring after a portal and arterial revascularization in the orthotopic liver transplant (OLT) in pigs.     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

目的 比较Celsior(CS)液与UW液对大鼠无心跳供者(NHBD)供肝的保存效果.方法 选取健康雄性SD大鼠作为肝移植的供、受者.通过阻断大鼠主动脉和膈上下腔静脉10 min的方法,制备和获取NHBD供肝,并采用不同的器官保存液灌注和冷保存供肝.随机将受者分为4组.CS8 h组:受者采用经CS液灌注和冷保存8 h的供肝移植;UW8 h组:受者采用经UW液灌注和冷保存8 h的供肝移植;CS16 h组:受者采用经CS液灌注和冷保存16 h的供肝移植;UW16 h组:受者采用经UW液灌注和冷保存16 h的供肝移植.受者门静脉开放前、开放后1、3及6 h,取各组受者的静脉血检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、内皮素1(ET-1)、白细胞介素1(IL-1)及肿瘤坏死因子α(TNF-α)水平;观察和比较各组受者的胆汁生成量、移植肝组织病理学改变及术后7 d内的存活率.结果 NHBD供肝经UW液灌注后呈"花斑"状,肝叶边缘灌注不良,经CS液灌注后肝叶边缘灌注良好.CS8 h组和UW8 h组受者的胆汁生成量分别为(0.21±0.01)ml和(0.10±0.02)ml(P<0.05).门静脉开放后1、3及6 h,CS8 h组受者的血清ALT及AST水平明显低于UW8 h组(P<0.05),门静脉开放后1、3h,CS8 h组受者的血清ET-1、IL-1及TNF-α水平均明显低于UW8 h组(P<0.05);CS8 h组受者移植肝肝窦扩张、门静脉充血及炎症细胞浸润等病理学改变明显轻于UW8 h组,CS8 h组和UW8 h组受者术后7 d的存活率分别为58.3%和25.0%(P<0.05).CS16 h组和UW16 h组受者各时点的胆汁分泌量、血清ALT、AST、ET-1、IL-1及TNF-α水平的比较,差异均无统计学意义(P>0.05),两组受者均在术后3 d内死亡,两组受者移植肝组织病理学改变无明显差异.结论 CS液对大鼠NHBD供肝的保存效果优于UW液,这可能与UW液较CS液粘稠及CS液能够减少枯否细胞的激活有关;NHBD供肝的冷保存时间不宜超过16 h.     

Warm preflush with streptokinase improves microvascular procurement and tissue integrity in liver graft retrieval from non-heart-beating donors

BACKGROUND: Apart from the warm ischemic insult, integrity of liver grafts from non-heart-beating donors (NHBD) is additionally affected by microvascular alterations including erythrocyte aggregation and thrombus formation, which might hamper appropriate equilibration of the preservation solution to the grafts' microvasculature precluding cold preservation. Thus, the objective of our study was to characterize microvascular perfusion quality of University of Wisconsin (UW) solution during initial flushout of livers from NHBD rats, and to analyze the effects of an additional warm preflush with Ringer's lactated solution (RL) and with RL containing streptokinase (SK). METHODS: Cardiocirculatory arrest was induced by phrenotomy. Subsequent to 30 min of warm ischemia, livers were perfused via an aortic catheter by gravity of 100 cm H2O either with 4 degrees C 100 ml UW solution (UW, n=7), or with 25 degrees C 30 ml RL preflush followed by 4 degrees C 100 ml UW solution (RL+UW, n=7), or with 25 degrees C 30 ml SK- (7500 IU) containing RL preflush and 4 degrees C 100 ml UW solution (SK/RL+UW, n=6). Liver microperfusion was quantified using in situ fluorescence epi-illumination microscopy. Liver microcirculation of sham-operated living animals (n=4) served as controls. Enzyme release after a 24-hr cold preservation period was used as an indicator of graft integrity. RESULTS: After 30 min of warm ischemia, microvascular perfusion of UW solution was found markedly altered when compared with that of sham-operated living controls, as indicated by a significant reduction (P<0.05) of acinar and sinusoidal perfusion. Preflush with RL (RL+UW) only slightly attenuated the acinar and sinusoidal perfusion deficits, whereas the addition of SK to RL (SK/RL+UW) resulted in a significant improvement of microvascular graft perfusion (P<0.05). Accordingly, the increased enzyme release observed in solely UW-flushed livers after 24 hr cold preseravtion was only slightly influenced by preflush with RL, but markedly attenuated (P<0.05) by pre-flush with RL containing SK. CONCLUSION: The additive fibrinolytic therapy using SK is effective to improve microvascular procurement of livers after warm ischemia and may thus represent a promising approach to attenuate parenchymal cell injury in liver graft retrieval from NHBD.     

Portal and arterial washout after hypothermic preservation of the pig liver: prevention of hyperkalaemia after revascularization

In an orthotopic liver transplantation (OLT), portal revascularization may produce acidosis and hyperkalaemia due to loss of intracellular acid metabolites and K+ during hypothermic preservation. To verify the effectiveness of portal and arterial washout in preventing hypokalaemia after liver preservation, an OLT was done in 18 large-white pigs. The donor livers were perfused in situ via the portal vein with Hartmann's solution containing 1.000 IU of heparin at 4 degrees C. Afterwards, a cold Collins C2 solution was perfused either in vitro (group A) or in situ (group B). The cold ischemia time in both groups was less than 3 1/2 h. Before doing the portal revascularization of the donor livers, a washout via the portal vein and hepatic artery with saline serum was performed. The concentration of K+, glucose, GOT and LDH in effluents obtained through infrahepatic inferior vena cava were significantly lower in group B than in group A. Simple washout of the livers prior to revascularization prevented hyperkalaemia in both groups.     

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.     

加氧低温灌流下31磷-磁共振频谱与肝酶测定在判断大鼠供肝活力中的意义

目的　探讨判断经受热缺血的肝脏活力的方法。方法　将 18只SD大鼠随机分成 3组 :A组肝脏热缺血时间 (WI)为 0min ;B组WI为 30min ;C组WI为 6 0min。各组肝脏冷保存后 2～2 .5h ,采用31磷 磁共振频谱 (31P MRS)测定各组加氧低温灌流 30min时的离体肝脏频谱 ,同时测定保存液和灌流液中肝酶的变化。结果　β 三磷酸核酸 (β NTP)与亚甲基二磷酸 (MDP)的比值 ,B组与A组比较 ,P >0 .0 5 ;C组与A、B组比较 ,P <0 .0 1。肝酶测定结果 :随着热缺血时间的延长 ,保存液中的肝酶逐渐升高 ,每组之间比较 ,差异均有显著性 (P <0 .0 5 ) ;灌流液中的肝酶 ,A组与B组比较 ,差异无显著性 (P >0 .0 5 ) ,C组与A、B两组比较 ,差异均有极显著性 (P <0 .0 1)。结论　测定加氧低温灌流下大鼠离体肝脏的ATP再生能力能较好判断其活力 ,该方法在临床热缺血供肝活力的判断上具有一定参考价值 ;同时测定保存液和灌流液中的肝酶含量 ,能配合31P MRS判断肝脏的活力。     

Effect of Hepatic Artery Flow on Bile Secretory Function After Cold Ischemia

These studies evaluated the influence of hepatic arterial flow on biliary secretion after cold ischemia. Preparation of livers for transplantation or hepatic support impairs biliary secretion. The earliest indication of cold preservation injury during reperfusion is circulatory function. Arterial flow at this time may be critical for bile secretion. Porcine livers were isolated, maintained at 4 degrees for 2 h and connected in an extracorporeal circuit to an anesthetized normal pig. The extracorporeal livers were perfused either by both the hepatic artery and portal vein (dual) or by the portal vein alone (single). Incremental doses of sodium taurocholate were infused into the portal vein of both the dual and single perfused livers, and the bile secretion was compared. Most endogenous bile acids are lost during hepatic isolation. After supplementation, the biliary secretion of phosphatidyl choline and cholesterol was significantly better in the dual than single vessel-perfused livers; however, no difference was seen in bilirubin output. Single perfused livers were completely unable to increase biliary cholesterol in response to bile acid. The dependence of bile cholesterol secretion on arterial flow indicates the importance of this flow to the detoxification of compounds dependent on phosphatidyl choline transport during early transplantation.     

肝移植术后胆泥及胆结石形成原因分析

目的 探讨肝移植术后胆泥和胆结石形成的原因。方法 对13例肝移植术后发生胆泥和／或胆结石的临床资料进行回顾性分析。结果 13例发生胆泥和／或胆结石的时间为1.5－12个月（平均5.5个月）,供肝热缺血时间平均为5.5min,冷缺血时间11.5h;UW液经门静脉灌注;胆道冲洗时间在热缺血后15-20min;11例供肝动脉与受者的肝动脉吻合,2例与结肠中动脉吻合。结论 肝移植术后发生胆泥及胆结石有多种原因,其中主要是缺血、灌注损伤及肝动脉供血不足。     

Reduction of hepatic ischemia/reperfusion injury by a soluble P-selectin glycoprotein ligand-1.

OBJECTIVE: The authors' goal was to determine the effects of specific binding and blockade of P- and E-selectins by a soluble P-selectin glycoprotein ligand-1 (PSGL-1) in rat models of hepatic in vivo warm ischemia and ex vivo cold ischemia. The authors also sought to determine the effect of selectin blockade on isograft survival in a syngeneic rat orthotopic liver transplant model. SUMMARY BACKGROUND DATA: Ischemia/reperfusion (I/R) injury is a major factor in poor graft function after liver transplantation, which may profoundly influence early graft function and late changes. It is hypothesized that I/R injury leads to the upregulation of P-selectin, which is then rapidly translocated to endothelial cell surfaces within 5 minutes of reperfusion of the liver, initiating steps leading to tethering of polymorphonuclear neutrophil leukocytes to the vascular intima. Local production by leukocytes of interleukin-1, tumor necrosis factor-alpha, or both induces P-selectin expression on the endothelium and continues the cascade of events, which increases cell adherence and infiltration of the organ. METHODS: To examine directly the effects of selectins in a warm hepatic I/R injury model, 100 microg of PSGL-1 or saline was given through the portal vein at the time of total hepatic inflow occlusion. The effects of PSGL-1 in cold ischemia were assessed using an isolated perfused rat liver after 6 hours of 4 degrees C storage in University of Wisconsin (UW) solution, with or without the instillation of PSGL-1 before the storage. To evaluate the effect of selectin blockade on liver transplant survival, syngeneic orthotopic liver transplants were performed between inbred male Sprague-Dawley rats after 24 hours of cold ischemic storage in UW solution. A separate group of animals received two doses of 100 microg of PSGL-1 through the portal vein before storage and before reperfusion of the transplanted liver. Recipient survival was assessed at 7 days, and the Kaplan-Meier product limit estimate method was used for univariate calculations of time-dependent recipient survival events. RESULTS: In an in vivo warm rat liver ischemia model, perfusion with PSGL-1 afforded considerable protection from I/R injury, as demonstrated by decreased transaminase release, reduced histologic hepatocyte damage, and suppressed neutrophil infiltration, versus controls (p < 0.05). When cold stored livers were reperfused, PSGL-1 reduced the degree of hepatocyte transaminase release, reduced neutrophil infiltration, and decreased histologic hepatocyte damage (p < 0.05 vs. UW-only controls). On reperfusion, livers treated with PSGL-1 demonstrated increased portal vein blood flow and bile production (p < 0.05 vs. UW-only controls). In addition, 90% of the rats receiving liver isografts stored in UW solution supplemented with PSGL-1 survived 7 days versus 50% of those whose transplanted syngeneic livers had been stored in UW alone (p < 0.05). CONCLUSIONS: Selectins play an important role in I/R injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreases hepatocyte injury, neutrophil adhesion, and subsequent migration in both warm and cold rat liver ischemia models. In addition, the use of PSGL-1 before ischemic storage and before transplantation prevents hepatic injury, as documented by a significant increase in liver isograft survival. These findings have important clinical ramifications: early inhibition of alloantigen-independent mechanisms during the I/R damage may influence both short- and long-term survival of liver allografts.     

A comparison of UW with Eurocollins preservation solution in liver transplantation

Fifty consecutive liver transplants were performed using livers perfused with and stored in University of Wisconsin preservation solution. These grafts were compared with the preceding 55 consecutive transplants performed using livers perfused and preserved with Eurocollins solution. The purpose of the study was to determine if organs preserved with UW solution functioned better after transplantation than organs preserved with Eurocollins. Extensive retrospective analysis of prospectively accumulated data included enzyme levels through 30 days, cost and length of hospital stay, blood product usage, and ischemia time. Average age of patients in the UW group was 47.1 years compared with 39.6 years in the EC group (P less than 0.05); cold ischemia time was 7.21 hr in the UW group compared with 5.21 in EC (P = 0.0001). Total bilirubin values were significantly lower on days 0-6 and day 14, but not day 30, in the UW group. Aspartate aminotransferase was significantly lower in the UW group on days 0-1, 3-6, and 14, but not on day 3 or day 30. Prothrombin times were significantly lower in the UW group across all times (days 0-6, 14, and 30). Intraoperative and postoperative use of packed red blood cells and fresh frozen plasma was lower in the UW group (P less than or equal to .05). Also, total hospital days, intensive care unit days, and hospital cost to the patient were lower in the UW group (P less than or equal to .05). A second analysis was done comparing only nonemergent transplants from both groups. These results confirmed the initial analysis of a longer cold ischemia time in the UW group (P less than 0.001), and improved enzyme values in the TBR, AST, and PT in the UW group (P less than 0.05). Also, hospital cost in the UW group was again lower (P less than 0.05). In this nonrandomized study, the cold ischemia time was increased but kept close to that of the control group. We conclude that UW solution is an improved donor liver preservation solution on the basis of improved enzyme values, decreased blood usage, shorter hospital stay, and lower hospital charges.