The role of ischemic preconditioning in rat liver graft

OBJECTIVE: The objective of this study was to investigate the protective effects of different modes of ischemic preconditioning (IPC) on an ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: A total of 192 Wistar rats were randomly allocated into 4 groups, each including 48 rats: control group (C), experimental group 1 (E(1)), experimental group 2 (E(2)), and experimental group 3 (E(3)). IPC was not performed in group C. Among the animals in the experimental groups, IPC was performed by blocking blood flow by the portal vein and the hepatic artery followed by reperfusion by removal of the clamp before donor liver resection: Group E(1), 5-minute ischemia and 10-minute reperfusion; Group E(2), 5-minute ischemia and 5-minute reperfusion and immediately the same procedure; and Group E(3), 10-minute ischemia and 15-minute reperfusion. Liver transplantations were performed 4 hours after IPC. At 0.5 hour, 2 hours, 6 hours, and 24 hours after portal vein reperfusion recipient blood and graft samples were obtained to determine the levels of ALT, AST, TNF-alpha, and apoptosis index (AI). RESULTS: At 0.5 hour and 2 hours after portal vein reperfusion, serum tumor necrosis factor (TNF)-alpha in the experimental groups (E(1), E(2), and E(3)) was significantly lower than in the control group (P < .05). The values in group E(2) were significantly lower than those in groups E(1) and E(3) (P < .05). At 24 hours serum TNF-alpha in group E(2) was significantly lower than groups C, E(1), and E(3) (P < .05). At 2 hours and 6 hours, AI values in experimental groups (E(1), E(2), and E(3)) were significantly lower than in group C (P < .05). AI in group E(2) was significantly lower than that in groups E(1) and E(3) (P < .05). At 24 hours, AI values among experimental groups (E(1), E(2), and E(3)) were significantly lower than that in the control group (P < .05). CONCLUSION: IPC may attenuate liver graft injury by decreasing apoptosis of hepatocytes and production of TNF-alpha. The method of IPC with 5-minute ischemia and 5-minute reperfusion followed immediately by another cycle of the same procedure was a better way to protect a liver graft from I/R injury.     

缺血后处理对大鼠肝缺血再灌注损伤早期的保护作用

目的 探讨缺血后处理（IPC）对大鼠肝脏缺血再灌注损伤早期的保护作用机理。方法 建立大鼠肝脏缺血再灌注模型，54只健康雄性SD大鼠随机分为假手术组（SO组）、对照组（IR组）和缺血后处理组（IPC组）。后处理组于完全再灌注前，给予多次短暂复灌复停作为缺血后处理。分别于再灌注后1h、3h及6h抽血进行血清ALT、AST活性及TNF-α表达测定，免疫组化测定肝脏NF-kB活性及ICAM-1的表达。结果 与SO组比较，IR组及IPC组再灌注后大鼠血清ALT、AST活性明显增高，肝脏NF-kB活性明显增强，TNF-α和ICAM-1表达也随之增加；同IR组相比，IPC组的血清ALT、AST活性明显降低，NF-kB活性及TNF-α和ICAM-1表达亦明显降低，差异均具有统计学意义（P〈0．01）。结论 缺血后处理能够减轻肝脏缺血再灌注损伤。其保护作用可能与通过抑制再灌注早期NF-kB的活性，降低了TNF-α和ICAM-1等炎性细胞因子水平有关。     

缺血或药物预处理对大鼠供肝缺血再灌注损伤的抑制作用

目的　探讨缺血预处理 (IPC)或阿霉素预处理 (DPC ,模拟IPC)对大鼠供肝延迟性保护作用的发生机制。方法　将供鼠分为 3组。IPC组 :供鼠采用肝脏预先缺血 10min后再开放 ;DPC组 :供鼠经静脉注射阿霉素 (1mg/kg体重 ) ;对照组 :供鼠用等量生理盐水注射。观察各组预处理后血红素氧化酶 1(HO 1)和热休克蛋白 70 (HSP70 )含量 ;建立上述各组大鼠原位肝移植模型 ,并设假手术对照组 ,观察肝移植后各组对供肝缺血再灌注损伤的影响。结果　IPC组HO 1、HSP70含量分别于预处理 12h和 2 4h达到高峰 ;IPC和DPC组预处理 2 4h ,诱导的HSP70、HO 1含量差异无显著性 (P >0 .0 5 )。对照组肝移植后 6h ,肝组织中ICAM 1mRNA表达和内皮细胞ICAM 1分子表达明显增强 ,髓过氧化物酶 (MPO)活性增高 ,血清中天冬氨酸转氨酶 (AST)、丙氨酸转氨酶 (ALT)、乳酸脱氢酶 (LDH)及肝组织湿重 /干重 (W/D)水平明显升高 ,和假手术组相比 ,差异有显著性 (P <0 .0 1)。IPC或DPC组肝移植后减弱了ICAM 1mRNA和蛋白表达及MPO活性 ,AST、ALT、LDH及W/D的水平亦明显降低 ,与对照组比较 ,差异有显著性 (P <0 .0 5 )。结论　IPC的延迟保护作用是通过降低中性粒细胞的粘附浸润来实现的 ,这与IPC诱导生成HSP70和HO 1有关。DPC可以模拟IPC的延迟性保护     

缺血预处理对大鼠小体积供肝的保护作用及机制

目的探讨缺血预处理（IPC）对大鼠小体积供肝的保护作用及其机制。方法120只SD大鼠随机分为3组（每组20对）：无热缺血组（NWI）、缺血再灌注组（WI）和缺血预处理组（IPC）。用双袖套法建立大鼠小体积肝移植模型。各组10只受体大鼠于术前1d、术后1、2、3、5d取血，用自动生化分析仪检测AST和ALT。NWI组于供肝灌注前及植入后0．5、1、2、3h，WI组于热缺血前及植入后0．5、1、2、3h，IPC组于IPC前、IPC后及植入后0．5、1、2、3h取肝组织，用硝酸还原法检测其NO浓度。结果IPC可降低大鼠小体积肝移植术后血清AST和ALT浓度，提高再灌注早期肝脏组织NO的浓度，降低再灌注晚期肝脏组织NO的浓度（P〈0．05）。结论NO在大鼠肝脏的缺血再灌注损伤中可能具有双重作用。IPC对大鼠小体积供肝的缺血再灌注损伤有保护作用。其机制可能是通过促进供肝再灌注后早期NO合成，改善肝脏微循环，同时抑制供肝再灌注后晚期NO合成，减轻过量NO的损伤作用，从而保护移植肝脏功能。     

缺血预处理对大鼠移植肝脏缺血再灌注损伤的保护作用

目的　探讨缺血预处理 (ischemicpreconditioning ,IP)对大鼠移植肝脏缺血再灌注损伤的保护作用。 方法　采用SD大鼠原位肝移植动物模型 ,供肝冷保存时间 10 0min ,无肝期 2 5min。 64只SD大鼠随机均分成两组 :对照组 ,获取供肝前仅以肝素生理盐水经门静脉灌注 ;IP组 ,获取供肝前阻断肝门血供 10min ,再灌注 10min ,然后再以肝素生理盐水经门静脉灌注。每组受体的一半 (n =8)用于观察存活率 ,另一半 (n =8)用于移植肝脏再灌注 2h后取血及肝脏检测。结果　IP组的 1w存活率、胆汁分泌量、抗氧化酶活力、血清NO水平均明显高于对照组 (P<0 .0 5 ) ,血清ALT、AST、LDH、TNF及肝组织中的过氧化产物含量均明显低于对照组 (P<0 .0 5 ) ,组织的病理改变也轻于对照组。结论　IP能够提高血清NO水平 ,降低血清TNF含量 ,对大鼠移植肝脏的缺血再灌注损伤具有保护作用     

大鼠边缘性体积供肝肝移植模型的实验研究

目的通过建立不同体积供肝的大鼠肝移植模型,探索边缘性体积供肝大小的适宜范围,为研究小肝综合征的发病机理及防治措施提供一种易于复制的小动物模型。方法将192只大鼠随机分为全肝移植组、半肝移植组、小体积供肝移植组及超小体积供肝移植组,每组48只,分别建立全肝、半肝、小体积供肝和超小体积供肝的大鼠肝移植模型。移植术后,4组各抽取24只大鼠用于观察存活率;抽取12只大鼠于移植术前,移植术后5、15、30、45及60min测定门静脉压力;另12只大鼠于术后24h测定谷丙转氨酶（ALT）水平。结果全肝移植组的7d累积生存率为100％（24／24）,半肝移植组为87．5％（21／24）,小体积供肝移植组为37．5％（9／24）,超小体积供肝移植组均于术后48h内死亡。全肝移植组术中开放门静脉后,60min内其门静脉压力稳定;半肝移植组大鼠在开放门静脉后,其门脉压力虽有小幅升高,但仍保持相对稳定;而小体积供肝移植组和超小体积供肝移植组开放门静脉后,其门静脉压力均显著升高,15min时均达到高峰,之后该2组的门静脉压力开始回落,至45～60min时逐渐稳定。供肝的体积大小与开放门静脉后5（r=-0．942）、15（r=-0．947）、30（r=-0．900）、45（r=-0．825）和60min（r=-0．705）时的门静脉压力均呈负相关关系（P〈0．001）。肝移植术后24h,全肝移植组和半肝移植组ALT水平均低于小体积供肝移植组及超小体积供肝移植组（P〈0．05）,且小体积供肝移植组ALT水平低于超小体积供肝移植组（P〈0．05）。供肝体积的大小与大鼠移植术后的ALT水平呈负相关关系（r=-0．685,P〈0．001）。结论大鼠部分肝移植模型中,供肝与受体标准肝脏体积比（Gv／sLV）的安全界限为50％,GV／SLV为30％～35％的供肝可视为边缘性体积供肝,小于30％的供肝可视为超小体积供肝。     

间充质干细胞移植对大鼠移植肝脏缺血再灌注损伤的影响

目的 研究骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)移植促进大鼠减体积肝移植术后肝脏缺血再灌注损伤修复及再生的作用.方法 本研究①取同龄健康Wistar大鼠骨髓进行BMSCs体外培养,用第3代细胞制备悬液.②建立同龄健康Wistar大鼠50%减体积肝移植模型,分为实验组(BMSCs经门静脉植入)和对照组(生理盐水经门静脉注射)观察大鼠术后生存状态,术后不同时间留取血清和肝组织标本,采用免疫组化、流式细胞等方法进行检测.结果 成功体外分离培养BMSCs和建立50%减体积肝移植模型;大鼠肝移植术后2 h,自由活动和饮水.实验组丙二醛(MDA)、超氧化物歧化酶(SOD)、丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)不同时间点与相应的对照组相比差异有统计学意义(均P＜0.05).实验组细胞周期:术后第2、3天时肝组织的G0/G1期细胞比例显著低于对照组(分别F=22.061,44.572,均P＜0.01).增殖细胞核抗原(PCNA)阳性细胞数增高,术后第2、3和7天时比较,差异有统计学意义(分别F=30.716,28.281,8.975,均P＜0.05).结论 移植肝局部植入BMSCs后,可以促进大鼠肝细胞增殖和加快减体积肝移植术后缺血再灌注损伤的修复.     

缺血预处理对大鼠减体积肝移植术后Akt生存信号通路的影响及其意义

目的 探讨缺血预处理(IPC)对大鼠减体积肝移植术后Akt生存信号通路的影响及意义.方法 将36只成年雄性SD大鼠随机分为2组:50%减体积肝移植组(Control组)和IPC组,Western blot检测肝组织总Akt和p-Akt及其下游的p-Bad和p-GSK3β蛋白水平,同时结合血清学和组织病理学分析Akt生存信号通路变化的意义.结果 与Control组比较,IPC组术后6、24 h丙氨酸转氨酶(ALT)水平显著下降(6 h:1064.49±126.53比802.90±82.39;24 h:1401.13±172.73比943.80±116.25,P<0.01);Control组术后24 h,肝细胞明显空泡样变性伴局部坏死,小叶结构破坏,门脉周围水肿、充血,炎症细胞浸润明显,而IPC组损伤减轻;Western blot结果显示:与Control组比较,IPC组术后2、6、24 h肝组织中p-Akt、p-Bad、p-GSK3β蛋白水平上调.结论 IPC明显减轻减体积肝移植术后再灌注损伤,其机制可能与激活Akt生存信号通道密切相关.     

不同时限缺血预处理对硬化肝脏保护作用的实验研究

目的: 探讨缺血预处理对硬化肝脏缺血再灌注的作用,并寻找一种有效缺血预处理的时间窗和理想方案. 方法: 将64只雄性、肝硬化SD大鼠随机分为八组,每组八只:假手术组(SO组);缺血再灌注组(I/R组);缺血预处理1、2、3、4、5和6组(IPC1、IPC2、IPC3、IPC4、IPC5和IPC6).以肝组织ATP、ADP、AMP及EC(用高效液相色谱法测定),血清ALT、AST、LDH(用全自动生化仪测定)和肝脏胆汁分泌量来评价肝功能. 结果: 再灌注末,IPC3组、IPC4组、IPC5组ATP含量均明显高于I/R组(P分别为0.01、0.07和0.000);同样,测定EC时发现,IPC3组、IPC4组和IPC5组均明显高于I/R组(P=0.000).与I/R组比较,IPC4组和IPC5组的血清ALT差异有显著性(P分别为0.013和0.000);其血清LDH差异亦有显著性(P=0.023,P=0.000),而血清AST却只有IPC5组显著低于I/R组(P=0.001).IPC3组、IPC4组和IPC5组肝脏的胆汁分泌量明显高于I/R组(P=0.028,P=0.023,P=0.008). 结论: 5~10min予一次或两次缺血预处理,能启动IPC对肝硬化大鼠肝脏I/R损伤的保护作用;10 min的缺血预处理,对肝硬化大鼠肝脏I/R损伤的保护作用最强.     

Activation of intracellular neutrophil elastase in the transplantation of ischemic liver.

Ischemia-reperfusion injury is an important cause of primary nonfunction of transplanted organs, and neutrophil elastase has been implicated in the pathophysiology of ischemia-reperfusion injury. We assessed the kinetics of intracellular neutrophil elastase (INE) activity in canine liver transplantation. Mongrel dogs underwent orthotopic whole-liver transplantation. The animals in group I (n = 6) received fresh liver grafts, and all of the dogs survived longer than 24 h. The animals in group II (n = 5) received liver grafts injured by 30 min of warm ischemia. Only 1 animal survived longer than 24 h after reperfusion. A significant increase in the serum ALT and LDH levels was observed in group II after reperfusion of the graft. Isolated peripheral neutrophils were homogenized, and the neutrophil elastase activity in the supernatant was determined by using a spectrophotometric assay. The INE activity was expressed as the neutrophil elastase value per 1 x 10(10) peripheral neutrophils. In group I, the INE activity 10 min and 2 h after reperfusion was 7.6 +/- 2.6 and 6.1 +/- 2.4 U, respectively. In group II, this activity was 25.9 +/- 7.4 and 44.3 +/- 23.7 U, respectively. There was a significant correlation between serum LDH levels and INE activity 10 min after reperfusion (gamma = 0.70, p < 0.02). In conclusion, the INE activity increased more sharply after the reperfusion of ischemically injured liver grafts. The INE activity correlates with serum LDH levels immediately after reperfusion, suggesting that the increase in the INE activity depends on the severity of ischemic damage.     

缺血预适应对肢体缺血再灌注大鼠肝脏的保护效应

目的 观察缺血预适应(IPC)对大鼠肢体缺血再灌注后肝脏损伤的影响,以进一步探讨IPC对肢体缺血再灌注后肝脏功能的保护作用。方法 实验用雄性Wistar大鼠18只,随机分为对照(Control)组,缺血再灌注(IR)组和缺血预处理(IPC IR)组．每组6只。分别测定血浆谷草转氧酶(ALT)、谷丙转氨酶(AST)、乳酸脱氢酶(LDH)．血浆和肝组织超氧化物歧化酶(SOD)、黄嘌呤氧化酶(XOD)、丙二醛(MDA)的含量变化及肝组织的湿/干重比值(W／D)、髓过氧化物酶含量(MPO)及DNA双链百分率(Ratio of DNA Chain％)。结果 发现IPC减轻了肢体IR后引起的ALT、AST、LDH、XOD、MDA、MPO、W／D含量的升高．并且增加了SOD以及肝组织中DNA双链百分率。结论 IPC对肢体IR继发的肝脏功能损伤具有保护作用。     

缺血预处理对肝脏缺血再灌注损伤中MPO、TNF-α、ET、NO的影响

目的：探讨缺血预处理（IPC）对肝脏缺血再灌注（I/R）损伤中的中性粒细胞和某些细胞因子的影响。方法：采用大鼠部分肝脏原位缺血再灌注损伤模型,30只Wistar大鼠随机分成：①正常对照组;②缺血再灌注对照组;③预处理组。②、③组均在60min再灌注完成后取血及肝组织标本,检测血清AST、ALT、LDH、NO、ET-1、TNF-α、及肝组织中髓过氧化酶（MPO）活性和肝细胞病理改变。结果：预处理组与再灌注对照组比较,肝功明显改善,NO含量升高,ET-1、TNF-α浓度和MPO活性明显降低,两组比较差异具有显著性。结论：缺血预处理对肝脏缺血再灌注损伤有明显保护作用,可能与提高内源性NO水平、减轻中性粒细胞在肝脏中的渗出和聚集、抑制ET-1、TNF-α的合成有关。     

Delivery of antioxidative enzyme genes protects against ischemia/reperfusion-induced liver injury in mice.

Hepatic ischemia/reperfusion (I/R) injury is characterized by the generation of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The aim of this study is to investigate whether antioxidative gene delivery by our polylipid nanoparticles (PLNP) is an effective approach for prevention of the injury. Polyplexes of extracellular superoxide dismutase (EC-SOD) and/or catalase genes were injected via the portal vein 1 day prior to a warm I/R procedure in mice. The effects of the gene delivery were determined 6 hours after starting reperfusion. PLNP-mediated antioxidative gene delivery led to a marked increase in human EC-SOD and catalase gene expression in the liver. Liver superoxide dismutase (SOD) and catalase activity both increased approximately 10-fold. Increased liver superoxide anion levels caused by the I/R procedure were reduced to normal levels by EC-SOD gene delivery. The overexpression of these 2 antioxidative genes significantly suppressed the I/R-induced elevation of serum alanine aminotransferase (ALT) levels, decreased liver malondialdehyde content, restored glutathione reserve, and improved liver histology. In conclusion, EC-SOD or catalase gene delivery by PLNP resulted in high levels of the transgene activity in the liver, and markedly attenuated hepatic I/R injury. The protection is directly associated with elevated antioxidative enzyme activity as the result of the gene delivery. This novel approach may become a potential therapy to improve graft function and survival after liver transplantation.     

Synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin reduces hepatic ischemia-reperfusion injury in rats

BACKGROUND: In hepatic surgery and liver transplantation, ischemia-reperfusion (I/R) is an unavoidable process, and protection against hepatic I/R injury is a major unresolved problem. In this study, we investigated whether 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol bound to saturated C18 fatty acids (beta-SQAG9), which was derived from sea urchin intestines, could reduce this injury. This agent was recently reported to have immunosuppressive effects in allogeneic rat skin grafts. MATERIALS & METHODS: Male Lewis rats were divided into two experimental groups. Group 1 rats were injected with SQAG9 (50 mg/kg) into the penile vein 15 minutes before the induction of ischemia and into the portal vein just reperfusion. The same amounts of normal saline were injected into rats in the control group (group 2). Each experimental groups included six rats. Seventy percent hepatic ischemia (20 minutes) was induced by occluding the blood vessels and bile duct with a vascular clamp. For examination of hepatic function, serum levels of aspartate aminotransferase, (AST) alanine transaminase (ALT), and lactic dehydrogenase (LDH) were measured. In addition, histological examination was also assessed. RESULTS: Three hours after reperfusion, the mean plasma concentration of AST, ALT, LDH in group 1 was suppressed compared with group 2. Six hours after reperfusion, the hepatic damage in group 1 was mild in comparison with that in group 2. CONCLUSIONS: Our data demonstrated that SQAG-9 reduced the warm hepatic I/R injury.     

Bcl-2基因修饰的肝细胞移植对肝脏缺血再灌注损伤的影响

目的观察人Bcl-2(hBcl-2)基因修饰的肝细胞移植对肝脏缺血再灌注损伤的影响。方法将包含hBcl-2基因阅读框架(0·7kb)的核苷酸亚克隆至Psectag2A载体,脂质体转染balb/c小鼠胎肝细胞(BNL.CL2),经门静脉移植入balb/c小鼠(2×106细胞)。移植72h后左肝后叶常温下缺血45min后再灌注8h,比较测定凋亡细胞,血清ALT、AST、LDH,以及组织学的改变。结果转染hBcl-2基因的balb/c小鼠在肝脏缺血再灌注后ALT(P<0·05或0·01)、AST(P<0·05)、LDH(P<0·05)、凋亡细胞(P<0·05)均较对照组显著降低,组织学改变减轻。进行缺血再灌注的各组细胞损伤均以细胞凋亡为主(P<0·05或0·01)。结论hBcl-2基因修饰的肝细胞移植对肝脏缺血再灌注损伤具有保护作用,主要通过抑制缺血再灌注后的细胞凋亡。     

Cellular damage and early metabolic function of transplanted livers stored in Eurocollins or University of Wisconsin solution.

In a clinical setting, the effect of Eurocollins (EC) and University of Wisconsin solution (UW) on liver grafts were studied in the early reperfusion phase of liver transplantation. Blood samples were drawn before and after declamping of the portal vein in a group of 11 transplants with EC-perfused livers, and a group of 12 transplants with UW-perfused livers. Parenchymal damage was assessed by the LDH, AST, and ALT, and purine degradation by measuring the uric acid levels. Metabolic function was determined by the serum bile acids and the plasma amino acids, i.e. (valine + leucine + isoleucine)/(phenylalanine + tyrosine) ratio. Donor and pretransplant recipient parameters were almost identical. The cold ischemia time of both groups differed significantly. The results show the following: a significant difference between both the LDH and the uric acid levels in the two groups was revealed, with a smaller increase of the LDH levels and no increase of the uric acid levels in the UW group. Metabolic activity, as measured from the bile acids and the amino acid profile in the peripheral blood, was identical in both groups. We conclude that both EC-stored and UW-stored liver grafts show immediate metabolic function after reperfusion. The amount of metabolic function was equal in both groups, notwithstanding longer cold ischemia time in the UW group. In addition, more parenchymal damage occurred in the EC group.     

N-acetylcysteine attenuates the increase in alpha-glutathione S-transferase and circulating ICAM-1 and VCAM-1 after reperfusion in humans undergoing liver transplantation.

BACKGROUND: Oxidative stress and leukocyte-endothelial interactions contribute significantly to the reperfusion injury of the transplanted liver. Therefore, we investigated the effect of N-acetylcysteine (NAC) on reperfusion injury and circulating adhesion molecules during human liver transplantation. METHODS: In a prospective study, 10 orthotopic liver transplantation patients were treated with high-dose NAC and 10 patients were treated with 5% glucose (placebo group) immediately before and during reperfusion of the donor liver. Parameters of hepatocellular injury, cellular oxygenation, plasma cytokines, and circulating adhesion molecules were determined at various time points during the liver transplantation. RESULTS: NAC had no significant effect on the arterial lactate/pyruvate or hydroxybutyrate/acetoacetate ratio during the liver transplantation. At baseline, liver transplantation patients exhibited elevated levels of cytokines and circulating adhesion molecules compared with healthy volunteers (n=7). While no significant effect of NAC on circulating L- and P-selectin was observed, it significantly inhibited the increase in circulating ICAM-1 and VCAM-1 24 hr after reperfusion. There were no significant differences in maximal postoperative values of serum aspartate transaminase (peak AST) or alanine transaminase (peak ALT) between both groups. However, NAC significantly reduced the rise in alpha-glutathione S-transferase after reperfusion of the donor liver. CONCLUSIONS: NAC attenuated the increase in alpha-glutathione S-transferase and circulating ICAM-1 and VCAM-1 after reperfusion of the donor liver, indicating possible cytoprotective effects of NAC.     

阿霉素诱导热休克蛋白72对大鼠肝脏冷缺血-再灌注损伤的保护作用

目的观察阿霉素（DXR）预处理诱导大鼠肝脏热休克反应在肝脏长时间冷缺血一再灌注损伤中对肝细胞的保护作用。方法供体大鼠术前按1mg／kg由外周静脉注射DXR（DXR组）,对照组注射生理盐水。48h后行肝脏原位冷灌注,获取肝脏后将其在4℃UW液中保存48h,然后行原位肝移植,再灌注1、3h。逆转录-聚合酶链反应法测定肝组织肿瘤坏死因子-α（TNF-α）mRNA、中性粒细胞化学趋化性细胞因子（CINC）mRNA、巨噬细胞炎症蛋白-2（MIP-2）mRNA的表达,蛋白质印迹法测定肝组织热休克蛋白72（HSP72）、核转录因子-κB（NF-κB）的表达,测定血清谷丙转氨酶、TNF-α、CINC、MIP-2水平。同时观察7d生存率。结果DXR组TNF-α mRNA、CINCmRNA、MIP-2mRNA的表达均低于对照组。DXR组HSP72表达显著,对照组基本无表达;DXR组NF-κB无表达,对照组显著表达。DXR组血清TNF-α、CINC、MIP-2显著低于对照组（P〈0．05）。DXR组7d生存率为50％,对照组为0（P〈0．05）。结论DXR预处理大鼠供肝可使肝脏长时间冷缺血-再灌注损伤显著减轻;HSP72的诱导可抑制NF-κB激活导致的炎症反应,对肝实质细胞提供保护作用。     

Effect of cold-ischemia time on C-X-C chemokine expression and neutrophil accumulation in the graft liver after orthotopic liver transplantation in rats

BACKGROUND: The precise mechanisms leading to polymorphonuclear neutrophil (PMN) recruitment and activation in the extended cold-preserved liver after transplantation are not yet fully understood. METHODS: We histologically evaluated the number of accumulated PMNs in graft livers, with varying time periods of cold ischemia (1, 6, and 24 hr in University of Wisconsin solution at 4 degrees C), after liver transplantation in rats. Intragraft expression of macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) mRNA, as well as immunohistochemical expression of MIP-2 and CINC in the graft liver, were investigated after reperfusion. The levels of MIP-2 and CINC in the hepatic vein, and tumor necrosis factor (TNF)-alpha, which stimulates these chemokine production, were also monitored. RESULTS: The number of accumulated PMNs in sinusoids significantly increased in the 24-hr cold-ischemia group within 3 hr after reperfusion, compared with the 1-hr and 6-hr groups. Serum MIP-2 levels in the 24-hr group significantly increased at 3, 6, and 12 hr after reperfusion, compared with the other groups. Intragraft MIP-2 mRNA was also up-regulated to a greater extent in the 24-hr group. Similarly, serum CINC levels in the 24-hr group significantly increased at 3 hr, compared with the 1-hr group. CINC mRNA also increased as cold-ischemia time was prolonged. Immunohistochemical staining revealed that hepatocytes were the main source of both MIP-2 and CINC protein. In addition, TNF-alpha in the hepatic vein was detected only in the 24-hr group after reperfusion. CONCLUSION: Extended cold preservation of the graft liver might up-regulate MIP-2 and CINC expression of hepatocytes, most probably through elevated TNF-alpha, and might contribute to PMN recruitment and activation after reperfusion.     

Reperfusion injury associated with portal venous and hepatic arterial perfusion in liver transplantation

BACKGROUND: Although reperfusion injury in organ transplantation is presently well established, its exact role in liver transplantation still has to be defined. The aim of this part of the study was to document the reperfusion injury associated with porcine liver transplantation and to evaluate the different components of the reperfusion injury associated with arterial and portal reperfusion. METHODS: Large white X Landrace pigs were randomized into two groups: group 1, initial portal reperfusion, and group 2, initial arterial reperfusion. Several indicators of reperfusion injury, endothelial cell function, and hepatocellular damage were assessed. Early histopathological findings in biopsy specimens can predict poor graft outcome, therefore histological findings of the liver biopsies after liver transplantation were also studied. RESULTS: Malondialdehyde concentrations were lower and vitamin A concentrations were higher in the animals subjected to initial portal reperfusion. Serum amino aspartate transferase and serum hyaluronic acid concentrations were higher in the animals subjected to initial portal reperfusion. Histological results showed that hepatocyte vacuolization, neutrophil infiltration, single hepatocyte necrosis, and group cell necrosis of the hepatocytes were all significantly reduced in group 2 compared to group 1. CONCLUSION: Results of this study indicate that the major part of reperfusion injury is constituted during the portal venous reperfusion and that this injury can be, at least partially, attenuated by initial arterial reperfusion.