Biliary tract injury caused by different relative warm ischemia time in liver transplantation in rats

BACKGROUND:There is a controversy over the degree of liver and biliary injury caused by the period of secondary warm ischemia.A liver autotransplantation model was adopted because it excludes the effects of infection and immunological rejection on bile duct injury.This study was undertaken to assess biliary tract injury caused by relative warm ischemia(secondary warm ischemia time in the biliary tract)and reperfusion. METHODS:One hundred and two rats were randomly divided into 5 groups:groupⅠ(control);group...     

苦参碱对大鼠原位肝移植冷缺血再灌注损伤的保护作用

目的:探讨苦参碱对大鼠原位肝移植中供肝冷缺血再灌注损伤的保护作用及其机制方法:应用延长保存的大鼠原位肝移植模型, 大鼠224只随机分为对照组、低剂量(40 mg／ kg)、高剂量苦参碱治疗组(80 mg／kg)和假手术组,将供肝在4℃林格液中保存5 h后植入受体,分别观察移植术后1 wk生存率,并且检测移植术后1,2,4,24 h血ALL TNF-α,内毒素 (ET),透明质酸(HA),一氧化氮(NO)及肝组织丙二醛(MDA),超氧化物歧化酶(SOD),细胞间黏附分子-1(ICAM-1)的含量,并观察移植肝脏病理形态学的改变．结果:与对照组比较,苦参碱低剂量、高剂量治疗组术后1 wk生存率显著增加(75％,75％ vs 0,P<0．01),肝功能改善,血清HA(277．62 ±29．06,406．84±95．04 μg／L vs 1109．42± 110．28 μg／L,P<0．01)和肝组织ICAM-1表达均显著减少,血清NO含量增加(53．1±5．1,54．2 ±4．9 μmol/L vs 30．2±2.3 μmol／L,P<0．01), TNF-α(1．69±0．22,1．29±0．33 U／L vs 5．96 ±0．59 U／L,P<0．01)、ET(0．343±0．111, 0_302±0．059 kEU／L vs 0．643±0．110 kEU／L． P<0．01)以及肝组织MDA(0．87±0．41,0．69± 0．22 μmol／g vs 2．35±0．54 μmol／g,P<0．01) 水平均明显降低,肝组织SOD(19．89±1．84, 21．04±1．86 kU／g vs 13．39±0．85 kU／g,P<0．01) 水平均明显升高,肝细胞和肝窦内皮细胞形态也发生改善．结论:苦参碱可以通过减轻再灌注后内毒素血症,抑制库氏细胞激活及释放TNF-α, ICAM-1等炎症性细胞因子,清除氧自由基,促进NO合成等途径,减轻肝细胞及肝窦内皮细胞的损伤．     

Effects of warm ischemia time on biliary injury in rat liver transplantation

AIM: To investigate the effect of different secondary warm ischemia time (SWIT) on bile duct injury in liver-transplanted rats.METHODS: Forty-eight male inbred Sprague-Dawley rats were randomly assigned into four groups: a sham-operation group and three groups with secondary biliary warm ischemia time of 0 min, 10 min and 20 min. A rat model of autologous liver transplantation under ether anesthesia was established, and six rats were killed in each group and blood samples and the median lobe of the liver were collected for assay at 6 h and 24 h after hepatic arterial reperfusion.RESULTS: With prolongation of biliary warm ischemia time, the level of vascular endothelial growth factor-A was significantly decreased, and the value at 24 h was higher than that at 6 h after hepatic arterial reperfusion, but with no significant difference. The extended biliary SWIT led to a significant increase in bile duct epithelial cell apoptosis, and a decrease in the number of blood vessels, the bile duct surrounding the blood vessels and bile duct epithelial cell proliferation in the early postoperative portal area. Pathologic examinations showed that inflammation of the rat portal area was aggravated, and biliary epithelial cell injury was significantly worsened.CONCLUSION: A prolonged biliary warm ischemia time results in aggravated injury of the bile duct and the surrounding vascular plexus in rat autologous orthotopic liver transplantation.     

Synergistic effect of cold and warm ischemia time on postoperative graft outcome in human liver transplantation

BACKGROUND/AIMS: Prolonged cold ischemia time (CIT) during graft preservation and warm ischemia time (WIT) defined as a rewarming time have been reported to cause postoperative graft dysfunction after orthotopic liver transplantation (OLT). However, a synergistic effect of both CIT and WIT on patients of graft survival has not been confirmed. The aim of this study was to determine whether simultaneously prolonged CIT and WIT was associated with early graft outcome after clinical OLT. METHODOLOGY: Between May 1997 and July 1998, 186 consecutive OLT cases were divided into 4 groups as follows: group A, CIT < or =12 hrs and WIT < or =45 min; group B, CIT >12 hrs and WIT < or =45 min; group C, CIT < or =12 hrs and WIT >45 min; and group D, CIT > 12 hrs and WIT >45 min. Liver graft survival within 90 days of OLT and early postoperative graft function were analyzed. RESULTS: The graft loss rates were 5.4% in group A, 9.8% in group B, 11.1% in group C, and 42.9% in group D. The mean highest aspartate aminotransferase (AST) values after OLT in group D (3352.3+/-569.4 U/L) was significantly higher than those in groups A (1411.7+/-169.2 U/L) and B (1931.3+/-362.6 U/L). CONCLUSIONS: The simultaneously prolonged cold and warm ischemia time significantly caused hepatic allograft injury and failure, suggesting some synergistic effects of CIT and WIT on postoperative graft function.     

Calcium mobilization in liver transplantation graft with warm ischemia

The influence of warm ischemia on calcium mobilization in liver transplantation was investigated. Twenty-four porcine orthotopic liver transplantations were performed by a temporary portal arterialization technique. Swine were divided into three groups according to warm ischemia time; I (0 min,n=9), II (30 min,n=8), and III (60min,n=7). Ionized calcium was measured in arterial and hepatic venous blood, in initial perfusate, and in initial perfused blood. In group I, all the pigs survived, while in group III all succumbed. In group II, four survived and four died. Ionized calcium level in influx showed no differences, but the level in the initial perfusate in group I was significantly higher than that in group III. The level in the initial perfused blood in group I was significantly higher than levels in groups II and III. Retrospective analysis in group II showed that ionized calcium value in the initial perfused blood in the survivors was significantly higher than that in the non-survivors. A substantial amount of ionized calcium accumulated after revascularization in the graft loaded with warm ischemia, and, in group II, significantly more ionized calcium accumulated in the non-survivors.     

Mechanism of cold ischemia-reperfusion-induced graft injury after orthotopic liver transplantation in rats

BACKGROUND/AIMS: The purpose of this study was to clarify the mechanism of cold ischemia-reperfusion-induced graft injury after liver transplantation, especially with regard to the relationship between hepatocyte, sinusoidal endothelial cell injury, and hepatic hemodynamic alteration. METHODOLOGY: We evaluated changes in hepatocyte and sinusoidal endothelial cell function, and hepatic hemodynamics after reperfusion in an isogeneic rat liver-transplantation model. The livers of male LEW rats were stored in 4 degrees C lactated Ringer's solution for 1 hr, 3 hr (viable graft), and 6 hr (nonviable graft) before implantation. After reperfusion, hepatocyte function was assessed by serum alanine aminotransferase level and bile output; sinusoidal endothelial cell function was evaluated by serum hyaluronic acid level. Furthermore, we measured hepatic venous oxygen saturation, and portal venous blood flow using a transit time blood flow meter. RESULTS: At 2 hr after reperfusion, the hepatocyte function was similar in all groups. However, the sinusoidal endothelial cell function deteriorated severely in the nonviable graft group, and significantly decreased hepatic venous oxygen saturation levels were observed, suggesting poor hepatic circulation. At 4 hr after reperfusion, the hepatocyte injury was markedly increased in the nonviable graft group. Although systemic blood pressure remained stable, significantly decreased portal venous blood flow in the nonviable graft group was found compared with the viable graft groups. Histopathological studies showed that massive ischemic necrosis was seen in zone III (central) of hepatic lobule 8 hr after reperfusion in the nonviable graft group. CONCLUSIONS: These data suggest that the sinusoidal endothelial cell injury was predominant in the early phase of reperfusion, and might cause microcirculatory disturbances, resulting in decreased portal venous blood flow. This phenomenon may subsequently cause ischemic damage to the hepatocyte, with eventual graft failure.     

Energy metabolism and survival of liver grafts from non-heart-beating donor rats with warm ischemia injury

Introduction The quality of liver graft is a key factor for liver transplantation. Organs from non- heart-beating donors (NHBDs) seem to bean effective option to alleviate the problem of liver donor shortage.[1-3] However, the main obstacle to the use of livers from NHBDs is that warm ischemia injury to the liver is related to cardiac arrest.[4-6] Moreover, in liver transplantation, the allograft sustains inevitable cold ischemia in addition to re- warming injury during liver reperfusion. …     

供肝冷热缺血移植后损伤与炎症细胞的关系

目的 探讨冷、热缺血移植术后引起供肝损伤的炎性细胞是否相同。方法 雄性SD大鼠随机分成2组,每组各24只。供肝分别在乳酸林格氏中保存120或240min后行原位肝移植术;另雄性SD大鼠随机分成3组,每组各24只供肝分别经心跳停搏90、120和150min后行原位肝移植术。大鼠分别于术后1、3、6和24h处死采样。结果 随着冷、热缺血时间的延长,移植后血清ALT等值不断升高,如冷缺血120min和240min移植术后1h ALT值(U/L)分别为454.8±45.2和1063.0±166.1,3h分别为712.0±65.9和1639.0±241.2,6h分别为1702.0±169.2和4193.0±672.7,24h分别为1067.0±141.2和1316.7±205.2,热缺血90、120和150min移植术后1hALT值(U/L)分别为5035.2±786.7、6075.3±1613.1和 7449.5±1052.0,3h分别为5564.5±696、4、7900.7±863.0和8854.8±2089.3,6h分别为7363.8±616.7、10459.3±1573.7和10294.0±530.0,24h90min组为1942.5±188.5;且术后移植肝的病理损害也逐渐加重。冷缺血移植术后3、6h肝细胞出现坏死,且有大量中性粒细胞浸润;热缺血移植术后3、6h肝细胞也有大量坏死,但却出现淋巴细胞趋润。电镜表现与镜下基本一致,旦电镜证实热缺血移植后浸润的淋巴细胞为T淋巴细胞。结论 冷、热缺血移植术后供肝的损伤似乎是由两种不同的炎性     

Mechanisms of reperfusion injury after warm ischemia of the liver

The review highlights recent advances in our understanding of basic mechanisms of reperfusion injury after warm hepatic ischemia. Kupffer cells play a central role as the initial cytotoxic cell type and as a source of many proinflammatory mediators. Subsequently, neutrophils are activated and recruited into the liver. Factors and conditions are outlined that determine whether neutrophils undergo apoptosis without causing damage or migrate out of the sinusoids and attack parenchymal cells. In addition to the inevitable inflammatory response during reperfusion, microcirculatory perfusion failure, due to an imbalance between the actions of vasodilators and vasoconstrictors, also has a serious impact on reperfusion injury. A better understanding of the basic pathophysiology will reveal potential targets for therapeutic interventions and will show us how to avoid risk factors that may aggravate reperfusion injury.     

Prolonging warm ischemia reduces the cold preservation limits of liver grafts in swine

Introduction Liver transplantation (LT) is widely accepted as an effective treatment for end-stage liver disease, but a serious shortage of donor organs limits itsclinical application. To increase the number of livers available for transplantation, graft procurement from non-heart-beating donors (NHBDs) has again become a focus of attention.[1-3] It is suggested that warm ischemia (WI), cold ischemia (CI) or ischemia/reperfusion injury of the graft are risk factors for postoperative graft …     

热缺血供肝耐受冷保存时限的实验研究

心脏停搏供体是肝移植供肝来源的重要途径之一。有研究表明，热缺血30min以内的供肝可考虑应用，但此类供肝本身存在热缺血损伤，在后续的威斯康星大学溶液（UW液）中能冷保存多久能为临床所用尚未可知。本实验探讨了存在热缺血10min的供肝在UW液中冷保存的安全时限。     

Desferrioxamine in warm reperfusion media decreases liver injury aggravated by cold storage

AIM:To evaluate whether desferrioxamine decreases ischemia and perfusion injury aggravated by cold storage(CS)in a rat liver perfusion model. METHODS:Isolated rat livers were kept in CS in University of Wisconsin Solution for 20 h at 4℃,then exposed to 25 min of warm ischemia(WI)at 37℃ followed by 2 h of warm perfusion(WP)at 37℃with oxygenated(95%oxygen and 5%carbon dioxide) Krebs-Henseleit buffer.Desferrioxamine(DFO),an iron chelator,was added at different stages of storage,ischemia and perfusion:in CS only,in WI only,in WP only, in WI and perfusion,or in all stages.Effluent samples were collected after CS and after WI.Perfusate samples and bile were collected every 30 min(0,0.5,1,1.5 and 2 h)during liver perfusion.Cellular injury was assessed by the determination of lactate dehydrogenase(LDH) and aspartate aminotransferase(AST)in the effluent and perfusate samples.Total iron was analysed in the perfusate samples.After WP,the liver was collected for the determination of liver swelling(wet to dry ratio) and liver morphological examination(hematoxylin and eosin staining). RESULTS:Increased CS time caused increased liver dysfunction during WP.After 2 h of WP,liver injury was indicated by increased release of AST(0.5 h CS:9.4± 2.2 U/g liver vs 20 h CS:45.9±10.8 U/g liver,P<0.05) and LDH(0.5 h CS:59±14 U/g liver vs 20 h CS:297 ±71 U/g liver,P<0.05).There was an associated increase in iron release into the perfusate(0.5 h CS:0.11 ±0.03μmoL/g liver vs 20 h CS:0.58±0.10μmoL/g liver,P<0.05)and reduction in bile flow(0.5 h CS: 194±12μL/g vs 20 h CS:71±8μL/g liver,P<0.05). When DFO was added during WI and WP following 20 h of CS,release of iron into the perfusate was de- creased(DFO absent 0.58±0.10μmoL/g liver vs DFO present 0.31±0.06μmoL/g liver,P<0.05),and liver function substantially improved with decreased release of AST(DFO absent 45.9±10.8 U/g liver vs DFO present 8.1±0.9 U/g liver,P<0.05)and LDH(DFO absent 297±71 U/g liver vs DFO present 56±7 U/g liver,P<0.05),and inc     

Oxidative stress in hepatocytes and stimulatory state of Kupffer cells after reperfusion differ between warm and cold ischemia in rats

Rat liver was kept at 4°C or 37°C in MEM, and reperfused through a closed circulation from the hepatic vein to the portal vein at 37°C with the same solution. Although purine nucleoside phosphorylase and ALT activities were increased in the perfusate, depending on the duration of ischemia at both 4°C and 37°C, the ratio of the latter to the former was significantly higher after 37°C-ischemia than after 4°C-ischemia. The stimulation stage of Kupffer cells evaluated in situ by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was elevated after 4°C-ischemia longer than 1 h, but not after 37°C-ischemia. In contrast, the degree of oxidative stress in hepatocytes assessed by formazan deposition after liver perfusion with nitro blue tetrazolium alone was greater after 37°C-ischemia than after 4°C-ischemia. These results suggest that oxidative stress in hepatocytes and the stimulatory state of Kupffer cells after ischemia-reperfusion may differ between 4°C-ischemia and 37°C-ischemia, probably leading to different development of liver damage.     

Dynamical changing patterns of histological structure and ultrastructure of liver graft undergoing warm ischemia injury from non-heart-beating donor in rats

AIM: To investigate the histological and ultra-structural characteristics of liver graft during different of warm ischemia time (WIT) in rats and to predict the maximum limitation of liver graft to warm ischemia. METHODS: The rats were randomized into 7 groups undergoing warm ischemia injury for 0, 10, 15, 20, 30,45 and 60 min, respectively. All specimens having undergone warm ischemia injury were investigated dynamically by light and electron microscopy, and histochemistry staining. After orthotopic liver transplantation (OLT), the recovery of morphology of liver grafts after 6, 24 and 48 h was observed. RESULTS: The donor liver from non-heart-beating donors (NHBD) underwent ischemia injury both in the warm ischemia period and in the reperfusion period. Morphological changes were positively related to warm ischemia injury in a time-dependent manner during the reperfusion period. The results demonstrated that different degrees of histocyte degeneration were observed when WIT was within 30 min, and became more severe with the prolongation of WIT, no obvious hepatocyte necrosis was noted in any specimen. In the group undergolng warm ischemia injury for 45 min, small focal necrosis occurred in the central area of hepatic lobule first. In the group undergoing warm ischemia injury for 60 min, patchy or diffused necrosis was observed and the area was gradually extended, while hepatic sinusoid endothe lial cells were obviously swollen. Hepatic sinusoid was obstructed and microcirculation was in disorder. CONCLUSION: The rat liver graft undergoing warm ischemia injury is in the reversible stage when the WIT is within 30 min. The 45 min WIT may be a critical point of rat liver graft to endure warm ischemia injury. When the WIT is over 60 min, the damage is irreversible.     

Dynamical changing patterns of histological structure and ultrastructure of liver graft undergoing warm ischemia injury from non-heart-beating donor in rats

AIM: To investigate the histological and ultra-structural characteristics of liver graft during different of warm ischemia time (WIT) in rats and to predict the maximum limitation of liver graft to warm ischemia. METHODS: The rats were randomized into 7 groups undergoing warm ischemia injury for 0, 10, 15, 20, 30, 45 and 60 min, respectively. All specimens having undergone warm ischemia injury were investigated dynamically by light and electron microscopy, and histochemistry staining. After orthotopic liver transplantation (OLT), the recovery of morphology of liver grafts after 6, 24 and 48 h was observed. RESULTS: The donor liver from non-heart-beating donors (NHBD) underwent ischemia injury both in the warm ischemia period and in the reperfusion period. Morphological changes were positively related to warm ischemia injury in a time-dependent manner during the reperfusion period. The results demonstrated that different degrees of histocyte degeneration were observed when WIT was within 30 min, and became more severe with the prolongation of WIT, no obvious hepatocyte necrosis was noted in any specimen. In the group undergoing warm ischemia injury for 45 min, small focal necrosis occurred in the central area of hepatic lobule first. In the group undergoing warm ischemia injury for 60 min, patchy or diffused necrosis was observed and the area was gradually extended, while hepatic sinusoid endothe-lial cells were obviously swollen. Hepatic sinusoid was obstructed and microcirculation was in disorder. CONCLUSION: The rat liver graft undergoing warm ischemia injury is in the reversible stage when the WIT is within 30 min. The 45 min WIT may be a critical point of rat liver graft to endure warm ischemia injury. When the WIT is over 60 min, the damage is irreversible.     

Establishment of a rat liver transplantation model with prolonged biliary warm ischemia time

AIM:To investigate the impact of different time points of secondary warm ischemia on bile duct in a rat autologous liver transplantation model with external bile drainage.METHODS:One hundred and thirty-six male inbred SD rats were randomly assigned to one of four groups(Ⅰ-Ⅳ) according to the secondary warm ischemia time of 0,10,20 and 40 min.A rat model of autologous liver transplantation with continuous external biliary drainage under ether anesthesia was established.Ten rats in each group were used to evaluate the one-week survival rate.At 6 h,24 h,3 d and 7 d after reperfusion of the hepatic artery,6 rats were killed in each group to collect the blood sample via the infrahepatic vena cava and the median lobe of liver for assay.Warm ischemia time of liver,cold perfusion time,anhepaticphase,operative duration for biliary external drainage and survival rates in the four groups were analyzed for the establishment of models.RESULTS:No significant difference was shown in warm ischemia time,anhepatic phase and operative duration for biliary external drainage among the four groups.Five of the 40 rats in this study evaluated for the one-week survival rate died,including three deaths of severe pulmonary infection in group Ⅳ.A significant decrease of one-week survival rate in group Ⅳ was noted compared with the other three groups.With the prolongation of the biliary warm ischemia time,the indexes of the liver function assessment were significantly elevated,and biliary epithelial cell apoptosis index also increased.Pathological examinations showed significantly aggravated inflammation in the portal area and bile duct epithelial cell injury with the prolonged secondary warm ischemia time.Microthrombi were found in the micrangium around the biliary tract in some sections from groups Ⅲ and Ⅳ.CONCLUSION:The relationship between secondary warm ischemia time and the bile duct injury degree is time-dependent,and 20 min of secondary warm ischemia time is feasible for the study of bile duct injury.