兔肝脏冷灌注后去除门静脉淤血对血清内毒素以及肝组织中核因子-κB活性的影响

目前临床肝移植多采用非转流手术,术中阻断门静脉可导致肠道淤血、肠上皮的缺血缺氧,使肠道内环境改变,引起肠道内菌群失调,大量产牛细胞内毒素被吸收入门静脉[1].尽管肝脏枯否细胞可以清除内毒素,但移植肝的枯否细胞功能减退,移植肝成为内毒素损伤的主要靶器官,甚至引起肝功能衰竭[2]."放血疗法"已为多数临床医牛接受,但肝移植中去除门静脉淤血多为经验性,缺乏实验依据.我们建立兔肝脏原位冷灌注模型,观察恢复灌流时去除门静脉淤血对血清内毒素及肝脏再灌注损伤的影响及其作用机制,为临床提供一种安全有效的方法.     

肝脏缺血再灌注损伤机制和缺血预处理保护作用

缺血再灌注(ischemia reperfusion, IR)损伤是指缺氧器官细胞损伤在恢复氧供之后更加加重的现象。Toledo Pereyra 等在 1975 年首先认识到IR损伤是肝移植过程中一种重要病理损伤状态,可发生移植肝淤血、进行性血栓形成和/或器官坏死,导致移植失败。直到80年代中期,“再灌注损伤(reperfusion injury)”才逐渐在肝移植文献中应用。　　肝脏IR损伤可分为热缺血和冷保存缺血再灌注损伤。热 IR 损伤(warm ischemia reperfu sion injury)与肝脏外科、肝移植、低血容量性休克、毒性肝损害、静脉阻塞性疾病和 Budd Chiari综合征等普遍相关…     

下腔静脉逆行再灌注法在原位肝移植中的应用进展

原位肝移植术中移植肝的再灌注方式对术后肾、肺、胆道等并发症发生率,及移植肝和受体存活率有着重要影响。下腔静脉逆行再灌注法是利用低压力、低流速、低氧饱和度的静脉血液对移植肝进行再灌注。近年来研究表明,下腔静脉逆行再灌注法有利于减轻移植肝的缺血-再灌注损伤、缩短下腔静脉阻断时间,对于肝移植术后移植肝功能的保护、降低再灌注后综合征及相关并发症的发生率具有积极作用。本文将对这一手术方式的研究近况作一综述。     

美心力对肝移植手术患者术中血流动力学及心肌酶的影响

复杂的肝脏手术，如肝切除、肝移植等，术中常需要阻断肝脏血流，从而导致肝脏缺血、缺氧，恢复血流后则会引起肝缺血-再灌注损伤。肝缺血-再灌注损伤不仅会影响肝脏本身，而且对远隔脏器也会产生组织损伤。目前，关于如何减轻肝缺血-再灌注损伤的报道很多，其中大多数是关于缺血-再灌注对移植肝本身影响的报道，关于如何减少肝移植手术中肝缺血-再灌注损伤对心肌影响的问题，国内外尚无相关报道。本研究观察肝移植手术中静脉泵人不同剂量美心力对血流动力学及心肌酶的影响，旨在探索肝移植手术中减轻肝再灌注损伤和心肌损伤的可行途径。     

肝缺血再灌注对肠道损伤及细菌移位的研究进展

肝缺血再灌注损伤除了对肝脏损伤外,还可因肠组织缺血缺氧、再灌注损伤、内毒素血症、免疫屏障受损等引起肠黏膜上皮细胞脱落、坏死,增加小肠通透性,促发肠道细菌和内毒素移位,诱发MODS,并且肝脏损伤和肠道损伤相互形成恶性循环。因此,在肝缺血再灌注过程中,有效的保护肠黏膜屏障,防止细菌和内毒素移位,将减少肝切除、肝移植等手术的并发症,提高其存活率。     

受体低氧预适应后肠黏膜改变对大鼠移植肝的影响

目的:研究低氧预适应对肠道黏膜屏障的作用以及对移植肝再灌注损伤的影响.方法:88只SD大鼠分为原位肝移植组(A组)、受体低氧预适应组(B组)和假手术组(C组)、A、B组均行原位肝移植.术后2 h检测大鼠血清肝功能和内毒素水平、肝组织超氧化物歧化酶(SOD)、丙二醛(MDA)含量,观察肝、肠组织细胞形态学改变以及3d和1周存活率.结果:B组肝功能损害与A组相比明显降低(P<0.01),MDA含量、血清内毒素明显低于A组(P<0.01),SOD活性明显高于A组(P<0.01);B组肝细胞结构改变较小,肠黏膜上皮损伤较轻;术后1周存活率B组较A组高(P<0.01).结论:受体低氧预适应能够保护肠黏膜屏障,减轻移植肝再灌注损伤.     

低氧预适应对肝切除后肝保护作用的分子机制

低氧预适应已被证明对机体抵御应激反应有保护作用,低氧预适应主要通过调节低氧预适应因子1、核因子κB和保护线粒体等方式减弱甚至消除肝切除手术时缺血再灌注损伤对剩余肝脏中肝细胞的损伤,通过远程-异位缺氧预适应和交叉-多能缺氧预适应2个方面对剩余肝发挥保护作用.低氧预适应可以减轻肝癌行肝切除术后残余肝脏的缺血再灌注损伤,但对是否有促进剩余肝脏再生的作用尚无明确证据,有待进一步实验研究.本文主要对低氧预适应对肝癌行肝切除后的影响作一综述.     

肝移植中肠道屏障功能保护的研究进展

肝移植过程中，肝缺血再灌注损伤因素等可导致肠道屏障功能受损。通过谷氨酰胺、益生菌、组胺等制剂的干预，以及低氧预适应等方法可减轻肝移植过程中肠道屏障的损伤作用，进而提高移植肝的功能，提高患者存活率。

     

肝脏缺血再灌注损伤机制和缺血预处理保护作用

缺血再灌注(ischemia reperfusion，IR)损伤是指缺氧器官细胞损伤在恢复氧供之后更加加重的现象。Toledo—Pereyra等在1975年首先认识到IR损伤是肝移植过程中一种重要病理损伤状态，可发生移植肝淤血、进行性血栓形成和／或器官坏死，导致移植失败。直到80年代中期，“再灌注损伤(reperfusion injury)”才逐渐在肝移植文献中应用。     

肠道淤血再灌注在大鼠肝脏缺血再灌注损伤中的作用

目的:探讨肠道淤血再灌注在大鼠肝脏缺血再灌注损伤中的作用。方法:SD大鼠40只被分成4组:单纯肠道淤血再灌注肝脏组(E组)、无肠道淤血肝脏缺血再灌注组(I组)、肝脏缺血再灌注组(H组)、未处理组(S组),每组10只。测定血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、肿瘤坏死因子-α(TNF-α)水平;测定肝组织丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;计算肝、肠系膜淋巴结肠道细菌移位率;镜下观察肝组织、肠黏膜形态变化;Western blot测定并计算肝组织NF-κb活化P65(P-P65)占P65的比例。结果:血清ALT、AST、TNF-α及肝组织MDA含量H组高于I组、E组高于S组(P0.05),肝组织SOD活性则相反。肝组织NF-κb、P-P65/P65值在E组、I组和H组较S组升高(P0.05),其中H组高于I组(P0.05)。结论:肠道淤血再灌注是加重肝脏缺血再灌注损伤的重要因素;肠道淤血再灌注能加重肝缺血再灌注损伤的发生机制可能与肠黏膜屏障受损致肠源性细菌移位,NF-κb被活化致炎症介质TNF-α等释放增加及肝脏抗氧化能力降低,氧化应激性损伤加重有关。     

阻断肝脏神经和抑制库普弗细胞对移植鼠肝保护作用的观察

目的　研究移植肝库普弗细胞和交感神经在再灌注损伤发生中的作用 ,以及阻断供肝交感神经和抑制库普弗细胞对移植肝的保护机制。方法　 13 0对实验大鼠随机分为 4组 ,即正常对照组 (N组 )、氯化钆组 (C组 )、六甲胺组 (H组 )和六甲胺氯化钆组 (HC组 )。同等条件下 ,于肝移植术后 4,8,16,2 4h检测受体的肝功能、肝储备功能 ,并切取肝脏组织行光镜、电镜检查。结果　阻断供肝交感神经和抑制供肝库普弗细胞可明显改善移植肝的肝功能和肝储备功能 ,形态学变化也与之相符。结论　阻断供肝交感神经和抑制供肝库普弗细胞对移植肝有保护作用     

麦角新碱预处理对减轻大鼠移植肝缺血再灌注损伤的作用

目的 探讨使用外源性药物麦角新碱预处理对减轻大鼠移植肝缺血再灌注损伤的作用.方法 在大鼠的门静脉-左肾静脉搭桥、肝后下腔静脉内置管分流法自体原位肝移植模型中,于肝门阻断前10 min经大鼠尾静脉注射麦角新碱;观察移植肝缺血前和再灌注后5 min、30 min、2 h时血清一氧化氮(NO)和血浆内皮素1(ET1)水平以及NO/ET1的比值变化;测定血清丙氨酸转氨酶(ALT)酶学差异和肝组织内三磷酸腺苷(ATP)和丙二醛(MDA)含量变化;再灌注2 h取肝组织检测肝细胞、肝小叶超微结构.结果 应用麦角新碱预处理的大鼠移植肝缺血前门脉血浆中ET1升高(P＜0.01),但再灌注后5 min、30 min时,血浆中ET1水平降低(P＜0.05);而缺血前NO/ET1比值降低(P＜0.01),再灌注后5 min时,NO/ET1比值升高(P＜0.01);再灌注后ALT的升高有逐渐降低趋势;再灌注后2 h肝细胞内超微结构的损害程度减轻.结论 使用麦角新碱预处理能减轻大鼠移植肝缺血再灌注损伤.移植肝缺血再灌注损伤的靶细胞是肝血窦内皮细胞,NO/ET1比值平衡可能是影响移植肝微循环血流量变化的调节因素.     

Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model

BACKGROUND: The use of cell transplantation as an alternative therapy for orthotopic liver transplantation has been widely anticipated due to a chronic donor shortage. We previously reported the method used to enrich hepatic progenitor cells (HPCs) forming cell aggregations. In this study, we transplanted HPCs into the liver injury model mice to determine whether HPC transplantation may improve the liver dysfunction. METHODS: We obtained donor cells from E13.5 fetal livers of green fluorescent protein (GFP) transgenic mice. We transplanted GFP-positive fetal liver cells into the transgenic mice which express diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Subsequently, we induced selective liver injury to recipient mice by DT administration. We then evaluated the engraftment of the transplanted cells and their effect on survivorship. RESULTS: The low dose of DT induced sublethal liver injury and the high dose of DT was lethal to the liver injury model mice. The transplanted GFP-positive cells were engrafted into the recipient livers and expressed albumin, resembling mature hepatocytes. They continued to proliferate, forming clusters. The survival rate at 25 days after transplantation of the cell-transplanted group (8 of 20; 40.0%) was improved significantly (P=0.0047) in comparison to that of the sham-operated group (0 of 20; 0%). CONCLUSIONS: The transplanted cells were engrafted and repopulated the liver of recipient mice, resulting in the improvement of the survival rate of the liver injury model mice. We therefore propose that HPCs are a desirable cell source for cell transplantation.     

Protection of sinusoidal endothelial cells against storage/reperfusion injury by prostaglandin E2 derived from Kupffer cells.

BACKGROUND: In clinical liver transplants, grafts are frequently exposed to endotoxin (lipopolysaccharide, LPS) before harvest and may be predisposed to dysfunction. Because graft failure is linked to sinusoidal endothelial cell injury after storage/reperfusion, we investigated the effect of donor exposure to LPS on graft survival in relation to sinusoidal endothelial cell injury after storage/reperfusion in rats. METHODS: Rats were injected with 0.5 mg/kg LPS. In some rats, 20 mg/kg GdCl3 or 5 mg/kg indomethacin was injected before LPS to ablate Kupffer cells and inhibit prostaglandin (PG) synthesis, respectively. Other rats were injected with 100 microg/kg dimethyl PGE2, a stable PGE2 analog. Rat livers were harvested, stored in cold UW solution and transplanted to non-treated rats for determination of survival and liver injury in recipients. Otherwise, after cold storage, the livers were reperfused briefly with physiological buffer containing trypan blue for determination of sinusoidal endothelial cell injury by counting trypan blue-positive nuclei in histological sections. RESULTS: Donor treatment with LPS increased hepatic PGE2 production before storage and decreased recipient survival, but paradoxically decreased killing of sinusoidal endothelial cells after storage and reperfusion. Pretreatment of donors with GdCl3 or indomethacin prevented the protective preconditioning of sinusoidal endothelial cells by LPS, whereas pretreatment with dimethyl PGE2 protected sinusoidal endothelial cells to the same extent as LPS. Unlike LPS, however, PGE2 attenuated graft injury after liver transplants. CONCLUSION: PGE2 derived from LPS-stimulated Kupffer cells protects sinusoidal endothelial cells against storage/reperfusion injury. Unlike LPS, PGE2 improves graft function after liver transplants. Thus, donor preconditioning with PGE2 may be beneficial in liver transplants.     

Prostanoid release in experimental liver transplantation

Prostanoids are biologically active mediators of inflammation and tissue injury. To investigate the role of prostanoids in orthotopic liver transplantation we used a porcine model and determined prostaglandin E2, 6-keto-prostaglandin F1 alpha, and thromboxane B2 in arterial, portal, and hepatic venous blood during organ harvesting, the recipient operation, and the early postoperative period. There were no significantly increased serum levels during the donor operation or at the end of cold storage. As early as 1 or 5 min after initiation of reperfusion of the transplanted organ, prostanoids in hepatovenous blood increased dramatically (100-500-fold). Changes in arterial and portal blood (10-50-fold) were less pronounced but still statistically significant. In surviving animals these values returned to normal within 24 hr. The hepatic release of metabolites of the arachidonic acid cascade after liver grafting indicates that the synthesis of prostanoids might contribute to morphological and functional alterations of the transplanted graft. In addition, the increased arterial values of circulating prostanoids may potentially participate in severe cardiovascular, hemostatic, and immunological alterations known to occur after liver transplantation.     

VEGF mitigates histone‐induced pyroptosis in the remote liver injury associated with renal allograft ischemia–reperfusion injury in rats

Clinical evidence has indicated a possible link between renal injury and remote liver injury. We investigated whether extracellular histone mediates remote hepatic damage after renal graft ischemia–reperfusion injury, while vascular endothelial growth factor (VEGF) is protective against remote hepatic injury. In vitro, hepatocyte HepG2 cultures were treated with histone. In vivo, the Brown‐Norway renal graft was stored in 4°C preservation solution for 24 hours and then transplanted into a Lewis rat recipient; blood samples and livers from recipients were harvested 24 hours after surgery. Prolonged cold ischemia in renal grafts enhanced liver injury 24 hours after engraftment. Caspase‐1, ASC, NLRP3, and AIM2 expressions in hepatocyte, CD68⁺‐infiltrating macrophages, tissue, and serum interleukin‐1β and ‐18 were greatly elevated, indicating that pyroptosis occurred in the liver and resulted in acute liver functional impairment. Blocking the caspase‐1 pathway decreased the number of necrotic hepatocytes. VEGF treatment suppressed the hepatocyte pyroptosis and liver function was partially restored. Our data suggested that renal allograft ischemia–reperfusion injury is likely associated with acute liver damage due to hepatocyte pyroptosis induced by histone and such injury may be protected by VEGF administration. VEGF, therefore, may serve as a new strategy against other remote organ injuries related to renal transplantation.     

Graft tumor necrosis factor receptor-1 protects after mouse liver transplantation whereas host tumor necrosis factor receptor-1 promotes injury

BACKGROUND: Tumor necrosis factor (TNF)-alpha is a cytokine with pleiotropic effects on the liver. The predominant hepatic receptor for TNFalpha is TNF receptor-1 (TNFR1). TNFR1 mediates liver injury after ischemia/reperfusion but is also mitogenic during hepatic regeneration. This study investigated the role of graft and host TNFR1 in early graft injury after liver transplantation in mice. METHODS: Livers from TNFR1 deficient (TNFR1-/-) and wild type (WT) mice were transplanted into either TNFR1-/- or WT recipients in all four possible combinations after 12 hours of cold storage. After eight hours, alanine transferase (ALT), necrosis, TdT-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining, caspase-3 activation, and myeloperoxidase were determined. RESULTS: When TNFR1-/- livers were transplanted into either WT or TNFR1-/- recipients, ALT was twofold greater than when WT donor livers were used. Necrosis and TUNEL staining also increased twofold and sevenfold, respectively, after transplantation of TNFR1-/- donor livers compared to WT. By contrast, ALT and necrosis decreased when WT or TNFR1-/- livers were transplanted into TNFR1-/- hosts compared to WT, which was associated with decreased neutrophil infiltration. CONCLUSION: In conclusion, graft and recipient TNFR1 has opposing effects. Graft TNFR1 decreases graft injury, whereas recipient TNFR1 mediates an increase of injury associated with enhanced neutrophil infiltration. Cross-transplanting of knockout and wild-type livers provides a new means to investigate graft-host interactions during hepatic injury.     

Transplant surgery injury recruits recipient MHC class II-positive leukocytes into the kidney

BACKGROUND: CD4 T cells, which are stimulated by the "indirect pathway" of antigen-presentation, participate in rejection. These T cells are sensitized by recipient major histocompatibility complex (MHC) class II-positive leukocytes that migrate into the transplant. Therefore, an important early step in rejection is the immigration of these recipient MHC class II-positive leukocytes into the renal transplant. The regulation of this early step is not understood. We now test the hypothesis that such leukocytes immigrate into the renal transplant in response to ischemic injury occurring during the transplant procedure. METHODS: We transplanted Brown Norway (BN) kidneys into F1 Lewis/Brown Norway (L/BN) recipients. The F1 recipients are tolerant to the parental BN antigens, and any infiltration of recipient MHC class II-positive leukocytes results from injury occurring during transplantation surgery. In addition, ischemia/reperfusion injury was also induced by temporarily occluding the native renal arteries for 30 minutes. Transplanted kidneys and native kidneys, which suffered ischemia/reperfusion injury, were studied by immunohistochemistry on days 3, 7, 14, and 28 after surgery. Staining by the new monoclonal antibody (mAb) OX62 and antibodies to MHC class II identified dendritic cells. In addition, the following monoclonal antibodies identified: gamma/delta T cells, V65; B cells, OX33; cells that may be macrophages, dendritic cells, or dendritic cell precursors, ED1 (+) and OX62 (-); and recipient class II MHC, OX3. RESULTS: After transplantation, the serum creatinine increased to 4 mg/dl and then decreased, which was consistent with reversible injury during transplantation and the absence of rejection. We found that the injury of transplantation itself resulted in the infiltration of recipient MHC class II-positive leukocytes into the transplanted kidney. This infiltrate peaked at days 7 to 14 after surgery. The inflammation was peritubular and patchy and involved cortex and outer medulla. Double staining for OX62 and OX3 identified some of the infiltrating leukocytes as dendritic cells. Other recipient leukocytes were MHC class II positive, ED1 positive, and OX62 negative. We also found that MHC class II leukocytes, including dendritic cells, infiltrated native kidneys injured by ischemia/reperfusion injury. CONCLUSION: To our knowledge, this is the first demonstration that injury to the kidney during transplantation recruits recipient MHC class II-positive leukocytes into the kidney. Some of these leukocytes are dendritic cells.     

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.     

低氧预适应对自体原位肝移植大鼠血清内毒素的影响

目的 探讨低氧预适应(HP)对大鼠自体肝移植后血清内毒素的影响.方法 采用改良的自体肝移植模型进行研究.将SD大鼠随机分为正常对照组(NC)、自体肝移植(AT)和低氧预适应+AT(HP)3组,每组24只.HP组术前用8%氮氧混合气体处理90 min.各组分别于术后1,6,12 h处死大鼠取肠标本,抽血检测ALT和AST水平;在光镜及透射电镜下观察各组小肠黏膜损伤程度及线粒体超微结构改变;同时应用ELISA试剂盒测定大鼠血清内毒素水平的变化.结果 HP组血清ALT,AST及内毒素水平均显著低于AT组,但高于NC组,差异均有统计学意义(P＜0.05);光镜下 HP组小肠黏膜损伤程度明显低于AT组;透射电镜下HP组线粒体损伤较AT组明显减轻.结论 HP可减轻肝IRI对肠道黏膜的损伤,减少了肠道内毒素移位而降低肝移植大鼠血清内毒素水平,从而减轻内毒素对移植肝功能的影响,其机制可能与减轻线粒体结构损伤,减轻缺血缺氧后线粒体功能障碍,改善细胞的供能,提高细胞的抗缺氧能力有关.