常温肝缺血再灌注肝血窦内皮细胞损伤

目的 探讨肝脏常温 因再灌注稆肝血窦内皮细胞的结构变化及其在再灌注损伤中的作用。方法 将４４只大鼠在常温下分别阻断人肝血流２０、４０、６０和９０ｍｉｎ,然后再开放血流２ｈ,制备成缺血再灌注模型,对肝血窦内皮细胞进行扫描和透射电镜观察,正常对照组大鼠５只。结果 肝血流阻断２０、４０ｍｉｎ时内皮细胞受损;血流开放后２ｈ内皮变化可恢复。肝血流肝断６０、９０ｍｉｎ后内皮细胞出现损伤,使部分内皮缺损,上直接     

Reperfusion injury to endothelial cells after cold storage of rat livers: protection by mildly acidic pH and lack of protection by antioxidants

Lethal reperfusion injury to sinusoidal endothelial cells occurs after cold ischemic storage of livers and may be responsible for liver graft failure from storage injury. Here, we evaluated potential mechanisms underlying this reperfusion injury. In rat livers stored in Euro-Collins solution for 24 h and reperfused with Krebs-Henseleit bicarbonate buffer, nonparenchymal cell killing showed periportal predominance as assessed by nuclear staining with trypan blue. In livers reperfused in the retrograde direction, the lobular distribution of cell killing was reversed, indicating that cell killing was more rapid in oxygenrich upstream regions. However, antioxidants, including allopurinol, desferrioxamine, catalase, superoxide dismutase, superoxide dismutase plus catalase, and U74006F, did not reduce cell killing. Similarly, reperfusion with anoxic buffer did not prevent lethal injury. Antioxidants and anoxic reperfusion also did not improve cell viability in livers stored in UW solution. Nevertheless, superoxide generation, as identified by formazan formation from nitroblue tetrazolium, was increased in Kupffer cells after lives storage and reperfusion as compared to unstored livers. Acidification of the reperfusion buffer from pH 7.4 to pH 7.15 reduced overall nonparenchymal cell killing from about 40% to 10%. Moreover, a pH gradient developed across the liver lobule during reperfusion with the effluent 0.2–0.4 pH units more acidic than the influent. This intralobular pH gradient appears to account for the relative sparing of cells in more acidic downstream regions of the lobule. Lower temperatures of reperfusion also reduced lethal injury. In conclusion, Kupffer cells generated superoxide after perfusion of stored rat livers, but formation of oxygen free radicals did not appear to contribute to lethal reperfusion injury to endothelial cells. Rather, mildly acidotic pH was protective against lethal injury. Thus, hydrogen ion concentration may be a critical determinant of reperfusion injury to sinusoidal endothelial cells.     

Liver sinusoidal endothelial cell function in rejected and spontaneously accepted rat liver allografts.

Studies have suggested that liver sinusoidal endothelial cells (LSEC) may play an important role in tolerance induction. In this study, we evaluated the functional difference of LSEC in rejection and spontaneous acceptance of liver allografts by using rat liver transplant model. LSEC function was determined by circulating hyaluronic acid (HA) levels and fluorescein isothiocyanate-labeled formaldehyde-treated serum albumin (FITC-FSA) uptake. Additional parameters include the number of circulating lymphocytes and LSEC apoptosis. In spontaneously accepted group, we found (i) significantly lower serum HA levels (P = 0.002), (ii) a more rapid uptake of FITC-FSA, and (iii) a reduced number of circulating CD8a+ cells when compared with the rejection group. Strikingly, HA levels in spontaneously accepted group are even lower than syngeneic control group. Further investigation revealed that interleukin-1beta, a cytokine that promotes LSEC function, was higher in DA than in Lewis rats. In summary, our study demonstrates that LSEC function is better preserved in spontaneously accepted rat liver allografts than in those which are rejected. These findings warrant further studies to verify if LSEC actively contributes to liver transplant outcome or just a target of different immunologic responses.     

Injury to cultured liver endothelial cells during cold preservation: energy-dependent versus energy-deficiency injury

Previously, we demonstrated an energy-dependent injury to cultured liver endothelial cells during cold incubation in University of Wisconsin (UW) solution. Here, the effects of Histidine-Tryptophan-Ketoglutarate (HTK) and Euro-Collins (EC) solutions on these cells were studied. In HTK solution, 83%±4% of the cells had lost viability after 9 h of incubation at 4°C. The addition of cyanide (1 mM) to simulate hypoxic conditions protected the cells to the extent that only 9%±1% of the cells lost viability over the same period; the addition of glucose (10 mM) led to increased cell injury. ATP levels were highest in the incubations with the most rapid loss of viability. In Krebs-Henseleit buffer and EC solution, in contrast, cell injury increased upon addition of cyanide; the addition of glucose to Krebs-Henseleit buffer decreased injury. We conclude that the injury to cultured liver endothelial cells during cold incubation in HTK solution is energy-dependent, as it is in UW solution, whereas cells behave differently in EC solution and Krebs-Henseleit buffer.     

Warm flush at 37 °C following cold storage attenuates reperfusion injury in preserved rat livers

Abstract Pretransplant rinse solutions have been shown to reduce reperfusion injury in cold-stored liver grafts, especially at the nonparenchymal level in sinusoidal endothelial cells (SEC). In this study, different rinse temperatures were tested in a rat liver preservation model. Livers were washed out in situ via the portal vein with cold (4°C) University of Wisconsin (UW) solution, and after hepatectomy (t0), were stored for 8,16, or 24 h of cold ischemia time (CIT). After storage, livers were flushed with UW solution at either 4 °C, 20 °C, or 37 °C and reperfused for 90 min (37 °C). Control livers were reperfused at t0 without preflush. Levels of hyaluronic acid (HA), purine nucleoside phosphorylase (PNP), AST, and LDH were measured in the reperfusion medium. Bile production was monitored during reperfusion. At the end of reperfusion, liver biopsies were taken for enzyme histochemistry (5′-nucleotidase and LDH). After 8-h CIT and a flush at 4°C, a release of endogenous HA (-7 %) was observed, whereas uptake of exogenous HA occurred after the 20°C flush (2 %, P = NS) and after the 37°C flush (24 %, p < 0.001). HA release occurred at all three preflush temperatures after the 16-h CIT but was significantly lower when flushed at 37 °C (-10 %) that at 4 °C and 20 °C (-64 % and -17 %, respectively, p =0.05). After the 24-h CIT, the release of endogenous HA increased in the 4 °C and 20 °C preflush groups, but not in the 37 °C group. Levels of PNP and AST increased until the 24-h CIT in all groups but were significantly lower after preflush at 37 °C. Release of LDH did not increase with increasing periods of cold storage in any of the flush series. Compared to control livers, mean bile production during reperfusion was significantly decreased following preflush at 4°C or 37 °C after all periods of CIT. No differences in mean bile production could be demonstrated in the three preflush groups after any period of CIT. LDH activity in liver tissue was best preserved after the 8 and 16-h CIT in combination with the 37 °C preflush, indicating less hepatocellular damage. In conclusion, in cold stored rat livers flushed at 37 °C before reperfusion, SEC and hepatocellular damage is attenuated.     

金纳多对大鼠供肝冷保存再灌注中肝窦内皮细胞损伤的防护作用

目的　探讨金纳多对大鼠供肝冷保存再灌注中肝窦内皮细胞 (SEC)损伤的防护作用。方法　 119只Wistar大鼠 ,随机取出 7只为正常对照组 (N组 ) ,另外 112只分别作为供、受体 ,随机分为 4个实验对照组 (C1、C2、C3、C4)和 4个实验组 (E1、E2、E3、E4)。实验对照组供肝保存于4℃Euro collins液 ,实验组供肝保存于含有 0 .2 g/L金纳多的 4℃Euro collins液。C1与E1组、C2与E2组、C3与E3组、C4与E4组供肝冷保存时间分别为 3、6、8、12h ,之后行异体原位肝移植。于受体门脉复流后 15、3 0、60min检测肝脏酶学、透明质酸 (HA)、血浆内皮素 (ET )水平 ,观察移植肝SEC超微结构变化。结果　与实验对照组相比 ,各实验组大鼠门脉复流后 60minALT、AST及门脉复流后 15、3 0、60minHA、ET水平均明显降低 (P <0 .0 5 ) ;随着冷保存时间的延长 ,实验对照组与实验组的上述指标均渐升高 ,但各实验组均低于对应的实验对照组 (P <0 .0 5 )。与实验对照组相比 ,各实验组大鼠门脉复流后 60minSEC的损伤较轻。结论　SEC的损伤随着冷保存时间的延长而加重 ;金纳多能够有效地减轻肝脏冷保存再灌注中SEC的损伤 ,改善供肝术后功能。     

Serum hyaluronic acid for the assessment of graft viability in porcine liver transplantation

For the assessment of graft viability, serum hyaluronic acid (HA) levels during porcine orthotopic liver transplantation were measured in two groups: group 1 (viable:n = 5) in which allografts were transplanted following a minimal cold (4°C) preservation, and group 2 (nonviable:n = 4) in which allografts were transplanted after cold static storage (4°C) for 24 h in University of Wisconsin solution. The changes in the HA levels reached a significant difference between the two groups at 30 min after reperfusion (P < 0.02). In group 1, all animals survived for over 4 days, while all animals in group 2 died within 24 h. The serum HA also demonstrated a significant correlation with prothrombin time, ß-glucuronidase, and aspartate aminotransferase at 120 min after reperfusion. These results suggest that the measurement of serum HA is a potentially effective index for evaluating hepatic allograft viability.     

供肝灌洗液中透明质酸含量与肝脏冷保存损伤及术后成活率的关系

目的 :观察冷保存后供肝灌洗液中透明质酸 (Hyaluronic Acid,HA)水平与肝功能及成活率的关系。方法 :行 2 3例猪原位肝移植术 ,供肝植入前 ,用 4℃乳酸林格氏液经门静脉内灌洗肝脏 ,测量灌洗液中的HA含量。同时 ,分别于术后 2小时、2 4小时及 72小时取血测肝功能 (AL T及 L DH ) ,取其中最高值 (即峰值 )。结果 :以 HA值为 40 0μg/ L作为界限 ,把实验猪分为两组 ,结果发现血清 AL T及 L DH峰值在两组间无显著差异 ,而 2 4小时及 2成活率也没有显著差异。结论 :供肝灌液洗中 HA含量与术后早期肝功能和成活率无关。即测量灌洗液中的 HA含量不能预测供肝功能和术后成活情况     

Venous air embolism,preservation/reperfusion injury,and the presence of intravascular air collections in human donor livers: a retrospective clinical study

In human liver transplantation, air embolism is seldom encountered after graft reperfusion. Nevertheless, despite adequate flushing and clamping routines, air emboli have been reported in transesophageal echocardiography (TEE) studies performed during the reperfusion phase. We retrospectively investigated whether air in the donor liver — as observed with pretransplant magnetic resonace imaging (MRI) — resulted in clinical air embolism or contributed to preservation/reperfusion injury. Clinical air embolism was assessed by intraoperative hemodynamics and end-tidal CO₂ monitoring. Preservation/reperfusion injury was assessed in postoperative biochemical measurements. The outcomes were compared between patients receiving livers containing significant intrahepatic air and patients receiving livers without intrahepatic air. Forty-three livers were studied, seven of which had major intrahepatic air and ten of which had no evidence of air collections. Twenty-six livers showed minor amounts of air and were excluded from further study. One patient who received a liver that did not contain intrahepatic air had clinical evidence of air embolism. Clinical air embolism did not appear to be associated with the presence of significant intrahepatic air based upon pretransplant MRI. Intrahepatic air did not seem to affect the amount of preservation/reperfusion injury. Our data indicate that air bubbles in the portal and arterial branches are absorbed during reperfusion and that the majority of intrahepatic air is effectively removed by the specific flushing routines.     

体外持续肝脏灌注常温保存和HTK液低温保存无心跳供肝效果的比较

目的 比较应用组氨酸-色氨酸-酮戊二酸(HTK)液低温保存和体外持续肝脏灌注(ECLP)系统常温保存无心跳供肝的效果.方法 按保存方法不同将供肝随机分为A组和B组:供肝切取后,A组用HTK液在低温下保存10 h;B组用ECLP系统在常温下用稀释的自体血液持续灌注10 h.两组供肝再经过60 min冷缺血期后,连接ECLP系统用稀释的自体血液再灌注4 h.观察再灌注后1、2、3、4 h四个时间点的胆汁分泌量,门静脉和肝动脉的压力,肝脏耗氧率的变化,灌注液中丙氨酸转氨酶(ALT)、乳酸脱氢酶(LDH)、葡萄糖水平以及灌注后供肝的常规病理和超微病理变化.结果 B组再灌注后1、2、3、4 h时间点的胆汁分泌量,门静脉和肝动脉的压力,灌注液中ALT、LDH和葡萄糖水平,以及2、3、4 h时间点的耗氧率与A组比较,差异均有统计学意义(P＜0.05或P＜0.01);B组供肝的病理损害程度较A组轻.结论 供肝切取后10 h内,利用ECLP系统持续灌注常温保存比用HTK液单纯低温保存在维持无心跳供肝的功能和生理活性方面效果更好.     

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     

Microcirculatory disturbances and leucocyte adherence in transplanted livers after cold storage in Euro-Collins,UW and HTK solutions

Integrity of the hepatic microcirculation and maintenance of endothelial cell viability are critical components in preventing primary non-function after liver transplantation. Therefore, hepatic microcirculation and leucocyte-endothelial interaction were studied in rat livers stored for 1 h in Euro-Collins (EC), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) solutions and subsequently transplanted. One hour after transplantation surgery, the livers were exposed under an intravital fluorescence microscope. After injection of the leucocyte marker acridine orange (1 mol/kg), six pericentral fields were observed for 30 s and experiments were recorded continuously. The percentage of perfused sinusoids was reduced in the livers in the EC group (82.9%) in contrast to the UW (93.2%) and HTK groups (91.0%). Livers in the EC group showed a reduction in the diameters of pericentral sinusoids (7.3±0.2 m; mean±SEM) compared with the UW group (9.5±0.2 m; P<0.05) and HTK group (10.2±0.8 m; P<0.05), indicating substantial cell swelling in livers stored in EC solution. Permanent adherence of leucocytes was most frequently observed in the EC group (33.5±1%), while this phenomenon was less pronounced in the UW group (14.5+1.1%; P<0.05) and HTK group (16.3±0.7%; P<0.05). Conversely, temporary adherence of leucocytes was reduced in the EC group (19.7+1.3%) compared with the UW group (30.5+2.1%) and the HTK group (34.4+0.8%). Microcirculatory failure and cell swelling in the EC group might be due to the lack of osmotic substances or oxygen radical scavengers included in UW (allopurinol, glutathione) and HTK (mannitol) solutions. In conclusion, cold storage of livers in UW and HTK solutions results in better preservation of the microcirculation and prevention of adhesion of leucocytes after transplantation compared with the EC solution.     

冷保存供肝肝移植大鼠肝窦内皮细胞损伤机制的研究

目的 探寻肝移植大鼠肝窦内皮细胞(SEC)损伤的详细过程、方式及机制,为冷保存再灌注损伤的保护研究开辟新的途径.方法 雄性SD大鼠随机分为假手术组(n=6)、UW 1 h肝移植组(n:48)、Uw 12 h肝移植组(n=48).大鼠原位肝移植采用双袖套法,分别于术后不同时相点采取血液及组织标本,检测血清丙氨酸氨基转移酶(ALT)及透明质酸(HA)水平;HE染色观察肝脏病理学变化;TUNEL法检测凋亡,免疫组化法检测Bcl-2及Cleaved Caspase-3的表达状况.结果 UW 1 h、UW 12 h组肝移植后血清ALT、HA均较假手术组明显升高(P<0.05),UW 12 h组又明显高于Uw 1 h组(P<0.05).UW 12 h组ALT水平于术后6 h达高峰,而HA水平却在术后1 h、24 h呈双峰表现.Uw 12 h组首先出现SEC的凋亡继而出现肝细胞的坏死,且UW 12 h组细胞凋亡指数(apoptosis index,AI)明显高于UW 1 h组(P<0.01).两组大鼠SEC的AI均于术后6 h达高峰,与血中ALT的高峰时相点一致.肝移植术后Bcl-2表达明显减弱(P相似文献   

Lipid peroxidation after cold storage and normothermic reperfusion: the effect of trimetazidine

Abstract Ischemia reperfusion injury is still a leading cause of early graft dysfunction after transplantation. Trimetazidine (TMZ) has been postulated to be protective against renal damage from oxygen free radicals. The aim of this study was to assess the effect TMZ during cold storage (CS) and normothermic reperfusion in an isolated perfused pig kidney model. Three groups were studied: control group, immediately perfused (GO), 48 h CS in Euro-Collins solution (G1), and 48 h CS in Euro-Collins solution plus TMZ (G2). Glomerular filtration rate (GFR) and fractional sodium reabsorption (FRNa⁺) were calculated during reperfusion from urine and perfusate samples. Lipid peroxidation was determined by the renal tissue level of Schiff bases (SB) and malondialdehyde (MDA) after reperfusion. A histological evaluation was performed after reperfusion. Renal function was significantly improved and lipid peroxidation reduced after preservation in Euro-Collins solution plus TMZ. Functional data were closely related to histological damage. In conclusion, TMZ is a useful protective agent against renal damage induced by CS.     

不同器官保存液对大鼠肝脏透明质酸吸收率的影响

目的　探讨不同器官保存液对大鼠肝脏透明质酸吸收率的影响 ,以评价它们对肝窦内皮细胞的保护作用。方法　大鼠肝脏原位灌洗后 ,分别在UW液、Celsior液或Histidine Tryptopan Ketoglutarate液 (HTK液 )中低温保存 16和 2 4h ,然后用含透明质酸的Kreb Henseleit液在 37℃下连续循环灌注 90min ,分别于灌注 0、15、30、6 0和 90min时检测肝脏对外源性透明质酸的吸收率。结果　低温保存 16h ,再灌注 0、15、30、6 0和 90min时 ,3种保存液保存的肝脏对外源性透明质酸的吸收率均为负值 ,表明肝窦内皮细胞受到一定程度的损伤 ,但UW液和Celsior液对肝窦内皮细胞的保护作用较HTK液为优 (P <0 .0 1) ;保存 2 4h者 ,UW液对肝窦内皮细胞的保护作用优于Celsior液和HTK液。结论　UW液对肝窦内皮细胞具有较强的保护作用     

冷保存再灌注损伤对肝移植大鼠肝脏再生功能的影响及其调节机制

目的 探讨冷保存再灌注损伤对肝移植大鼠肝脏再生功能的影响及其调节机制.方法 雄性SD大鼠分为假手术组(6只)、UW 1 h肝移植组(48只)、UW 12 h肝移植组(48只).HE染色及透射电镜观察肝组织形态学变化;大鼠内皮细胞抗原和5-溴-2脱氧尿嘧啶核苷(BrdU)双染法检测肝实质细胞及肝窦内皮细胞(SECs)的增殖状况;免疫组织化学法检测VEGF及其受体flt-1、flk-1的表达;RT-PCR法检测flt-1 mRNA的表达.计量资料采用单因素方差分析或t检验.结果 UW 12 h组肝实质细胞和SECs的BrdU标记指数均显著高于UW 1 h组(F=61.45,41.4,P<0.05).UW 1 h组和UW 12 h组大鼠肝实质细胞BrdU标记指数于48 h达高峰,而SECs的BrdU标记指数分别于术后72、96 h达高峰.UW 1 h组和UW12 h组大鼠肝移植术后VEGF表达较假手术组明显增强.UW 1 h组flt-1及flk-1表达较假手术组明显增强,阳性表达主要位于SECs,其表达高峰与SECs增殖高峰一致.UW 12 h组flt-1 mRNA表达较假手术组明显减弱(F=141.67,P<0.05).结论 flt-1表达下调是导致冷保存肝移植大鼠SECs再生高峰延迟,从而减缓移植肝脏功能恢复的重要原因.     

Physiological role of sinusoidal endothelial cells and Kupffer cells and their implication in the pathogenesis of liver injury

We reviewed the morphological characteristics and physiological functions of hepatic sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), both of which are major components of the hepatic sinusoid, and we showed the implication of these hepatic sinusoidal lining cells in the pathophysiology of the liver, based on our experimental studies. The most outstanding feature of SECs is that they are provided with numerous fenestrae, thereby allowing direct communication between the sinusoidal lumen and the space of Disse. Physiologically, SECs play a role in filtration function, endocytic function, and putative participation in the regulation of sinusoidal blood flow. As for KCs, they account for major portion of fixed macrophages in the entire body, and exhibit vigorous activity for phagocytosis, and produce many kinds of soluble mediators such as cytokines, prostanoids, oxygen radicals, and proteases. To determine whether these cells are implicated in pathophysiological processes in the liver we directed our attention to liver injury associated with sepsis and cold-preservation injury of liver tissue. In a septic rat model, we found that when KCs that included hepatic macrophages were activated, they released excess tissue-toxic mediators, probably leading to SEC damage. In the cold-preserved liver,we demonstrated that KCs were functionally activated and that the morphology of SECs was destroyed. When the liver was reperfused with plasma and a leucocyte suspension, hypercoagulability and increased leucocyte adherence occurred. In both experimental models, we demonstrated that KC blockade ameliorated the liver injury, and this was associated with the morphological improvement of SECs. Thus, we showed the pathogenetic implication of KCs and SECs, due possibly to microcirculatory disturbance in the hepatic sinusoid, and further emphasized the involvement of activated KCs in SEC impairment. Received for publication on Nov. 19, 1998; accepted on March 1, 1999     

UTI和丹参在鼠肝脏缺血再灌注中对肝窦内皮细胞损伤保护效应的实验研究

目的：探讨UTI和丹参在肝脏缺血再灌注中对肝窦内皮细胞损伤的保护作用。方法：将大鼠肝脏缺血再灌注模型30只大鼠分为5组，A组为空白对照组（n=3），B组为缺血再灌注组（n=7），C组为UTI组（n=6），D组为丹参组（n=6），E组为UTI＋丹参组（n=8）。除A组外，在缺血30min再灌注120min后分别取血检测AST、ALT、LDH、HA及ET，并取肝组织行光镜和电镜观察。结果：C组、D组较B组各指标明显下降（P〈0．01），E组各指标较C组或D组均有下降，（P〈0．05）。光镜和电镜观察形态学损伤程度，B组最重，C组、D组次之，E组最轻。结论：UTI、丹参在缺血再灌注中能有效减轻对肝窦内皮细胞的损伤，二药合用有协同作用。