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     

一氧化氮在缺血预处理保护大鼠移植肝脏再灌注损伤中的作用

目的　探讨一氧化氮 (NO)在缺血预处理 (IP)保护大鼠移植肝脏缺血再灌注损伤中的作用。方法　采用SD大鼠原位肝移植动物模型 ,供肝冷保存时间为 10 0min ,无肝期 2 5min。12 8只大鼠随机分成 4组 (n =32 ) :A组 (对照组 )、B组 (IP组 )、C组 [腺苷 (Ade)组 ]、D组 [NO合成抑制剂N L 精氨酸甲基脂 (NAME)组 ]。其中各组的半数用于观察存活率 ,另一半用于移植肝脏再灌注 2h后取血及肝脏检测。结果　IP组和Ade组的 1周存活率、胆汁分泌量、抗氧化酶活力及血清NO水平均明显高于对照组 (P <0 .0 5 ) ,血清丙氨酸氨基转移酶 (ALT)、肿瘤坏死因子(TNF)及肝组织中的过氧化物含量均明显低于对照组 (P <0 .0 5 )。肝组织损伤以窦状内皮细胞为主 ,并且是以凋亡的方式发生死亡 ,IP组和Ade组窦状内皮细胞损伤明显轻于对照组 (P <0 .0 0 1) ;NAME组的各种观察结果与对照组相近 (P >0 .0 5 )。结论　IP能够通过增加NO的合成来减轻再灌注早期窦状内皮细胞所受到的损伤 ,改善微循环 ,提高移植肝脏的功能。     

常温肝缺血再灌注损伤的实验研究

目的：探讨肝缺血再灌注损伤的作用机制。方法：采用大鼠部分肝缺血再灌注模型，将健康雄性SD大鼠24只随机分为三组：A组（手术对照），B组（肝缺血90min），C组（肝缺血90min再灌注120min）。观察每一动物肝组织病理切片；分别检测血浆谷草转氨酶（AST）、谷丙转氨酶（ALT）、乳酸脱氢酶（LDH）、肿瘤坏死因子（TNF－α）、白介素1β（IL-1β）浓度；测定肝组织中髓过氧化物酶（MPO）含量。结果：肝缺血再灌注后，光镜下大鼠肝组织有明显的肝血窦和中央静脉瘀血，内皮细胞及肝细胞普遍水肿变性；C组肝细胞坏死较B组明显；血浆中肝功能酶学指标显著升高，（B、C组与A组比及C组比B组P均＜0．01）；肝组织中MPO活性升高，以再灌注120min组为著（C组比A组P＜0．01）；与血浆中TNF－α、IL-1β的变化趋势相同（TNF－α：C组比A组P＜0．05，IL－1β：B、C组比A组P均＜0．01）。结论：肝脏微循环障碍是肝缺血再灌注损伤的病理基础；TNF－α、IL-1β介导中性粒细胞参与的肝缺血再灌注损伤过程。     

缺血后处理对大鼠移植肝缺血再灌注损伤的保护作用

目的探讨在体条件下缺血后处理对大鼠移植肝缺血再灌注损伤的保护作用及其可能机制。方法采用SD大鼠原位肝移植模型,供肝冷保存时间100min,无肝期控制于18min以内,60只雄性健康SD大鼠随机分为3组,对照组12只,缺血再灌注损伤组和后处理组各24只。对照组开腹后仅游离肝周韧带;缺血再灌注损伤组受体大鼠供肝切除前仅以肝素化生理盐水经门静脉灌注;后处理组供肝植入后完全再灌注前,给予多次短暂复灌复停作为缺血后处理。缺血再灌注损伤组、后处理组受体一半（6只）于再灌注后2h留取血液及肝组织,另一半（6只）于再灌注后6h留取肝组织。对照组于关腹后相应时间留取血液及肝组织。各组分别检测肝功能,采用酶联免疫吸附法测定血清肿瘤坏死因子Or．和中性粒细胞弹性蛋白酶。根据酶促反应原理,利用分光光度仪测定肝脏谷胱甘肽过氧化物酶、丙二醛、髓过氧化物酶、超氧化物歧化酶。肝组织HE染色后光镜下观察组织学变化。结果缺血再灌注损伤组和后处理组血清肝功能指标、炎性细胞因子水平及肝组织过氧化物含量均高于对照组（P〈0．05）,而后处理组较缺血再灌注损伤组则明显低（P〈0．05）;缺血再灌注损伤组和后处理组肝组织抗氧化酶活力显著低于对照组（P〈0．05）,而后处理组较缺血再灌注损伤组则明显高（P〈0．05）。结论缺血后处理对大鼠移植肝的缺血再灌注损伤有明显的保护作用。提高组织的抗氧化能力和降低炎性细胞因子水平可能是缺血后处理保护作用的机制之一。     

异丙酚对肝移植大鼠肝窦内皮细胞凋亡的影响

目的研究异丙酚对肝移植大鼠肝窦内皮细胞（SEC）凋亡的影响。方法24只健康雄性SD大鼠建立大鼠原位肝移植模型,随机分为3组（n=8）。Ⅰ组（对照组）移植肝再灌注前30min腹腔注射生理盐水15ml/kg；Ⅱ组和Ⅲ组移植肝再灌注前30min分别腹腔注射异丙酚100mg/kg和50mg/ kg。移植肝再灌注6h后取下腔静脉血,测定血浆谷丙转氨酶（ALT）、谷草转氨酶（AST）活性,TUNEL法测定移植肝SEC凋亡,Western blot法测定肝组织Bcl-2及Bax蛋白表达。结果再灌注6h后,与Ⅰ组比较,Ⅱ组、Ⅲ组大鼠血浆ALT、AST活性降低,凋亡SEC减少,Bcl-2蛋白表达上调,Bax蛋白表达下调（P＜0．01）；与Ⅱ组比较,Ⅲ组大鼠血浆ALT、AST活性增高,凋亡SEC增多,Bax蛋白表达上调（P＜0．01）,Bcl-2蛋白表达下调（P＜0．05）。结论异丙酚可剂量依赖性地抑制大鼠移植肝早期再灌注肝窦内皮细胞凋亡,其机制与上调Bcl-2蛋白表达、下调Bax蛋白表达有关。     

内皮素-1与肝脏缺血再灌注损伤的实验研究

目的：探索内皮素－1（ET－1）在肝脏血再灌注损伤中的作用。方法：选择雄性Wistar大鼠80只,分为正常对照组、缺血再灌注组1生理盐水组和ET－1抗体组、观察肝脏缺血60min再灌注3h后血浆ET－1、丙氨酸转氨酶（ALT）、透明质本酸（HA）、以及肝组织中ET－1和丙二醛（MDA）含量的变化,并观察肝组织病理学变化,同时,在缺血再灌注组选择第1、3、6、12和24h时相点观察ET－1的变化规律。结果：肝脏缺血再灌注后,血浆和肝组织中ET－1,血浆HA`ALT肝组织中MDA显著升高,而ET－1抗体组血浆ET－1、HA、ALT与缺血再灌注组相比显著降低（P＜0．01,P＜0．05）,同时,肝组织的瘀血程度和损伤程度显著改善。结论ET－1参与了肝脏缺血再灌注损伤,这种损伤与肝脏微循环障碍有关。     

HHI-Ⅰ对大鼠肝缺血再灌注损伤的影响

目的：研究活血化瘀注射液Ⅰ号（HHI-Ⅰ）对大鼠肝脏缺血再灌注（IR）损伤的影响,并与缺血预处理（IP）比较作用的效果.方法：清洁级健康雄性SD大鼠80只,随机分为4组（Sham组,IR组,IP组,HHI-Ⅰ组）,测血清谷丙转氨酶（ALT）、谷草转氨酶（AST）、乳酸脱氢酶（LDH）的水平;取左肝测组织丙二醛（MDA）、超氧化物歧化酶（SOD）的含量.结果：IR组、IP组、HHI-Ⅰ组的ALT、AST、LDH活性均明显高于Sham组（P＜0.01）,再灌注后SOD值均下降,各组各时间点与Sham组比较均具有统计学差异（P＜0.05）.再灌注后MDA值均升高,缺血再灌注后6 h升至最高点（P＜0.01）,且各时间点预处理组均低于IR组（P＜0.05）,HHI-Ⅰ组MDA（3 h、6 h）明显低于IP组（P＜0.05）.结论：大鼠肝脏缺血再灌注后造成了明显的肝脏损伤,IP与HHI-Ⅰ预处理均可改善IR对肝脏造成的损伤,且后者效果优于前者.     

缺血预处理对大鼠肝脏移植物再灌注早期NF—kB活性的影响及意义

目的 观察缺血预处理对大鼠肝脏移植后再灌注早期核转录因子-kB（NF-kB）活性和TNF—α、细胞间黏附分子-1（ICAM-1）表达的影响，探讨缺血预处理的保护机理。方法 建立大鼠肝脏移植模型，供肝在林格液中保存2h，实验分假手术组、对照组和缺血预处理组（IP组）3组，IP组于供肝切取前夹闭第一肝门10min，然后开放10min。分别于移植再灌注后1、2、4及6h抽血进行肝酶学检查，切取肝脏作NF-kB活性、TNF—α和ICAM-1表达的测定。结果 IP组肝功能得到有效改善。与假手术组比较，对照组及IP组再灌注后移植物的NF-kB活性明显增强，且在1、2h达高峰，4h后减弱，TNF—α和ICAM-1表达也随之增加；同对照组相比，IP组的NF-kB活性降低，并且在1、2h差异具有统计学意义（P〈0．05），TNF—α和ICAM-1表达亦降低。结论 缺血预处理对于供肝的保护作用可能是通过抑制再灌注早期NF-kB的活性，减少了TNF—α和ICAM-1炎症介质的释放，从而减轻了移植物再灌注早期的炎症反应实现的。     

白藜芦醇对肝脏缺血再灌注损伤的保护作用

摘要：探讨白藜芦醇对肝脏缺血再灌注损伤的防护作用。将雄性SD大鼠随机分为空白对照组、缺血再灌注组（I/R组）、I/R加生理盐水处理组和I/R加白藜芦醇处理组。观察肝脏缺血40 min再灌注1，3，6，12h后血清谷丙转氨酶（ALT）、谷草转氨酶（AST）及肝组织丙二醛（MDA）含量的变化以及肝组织病理学改变。结果示肝脏I/R后血清ALT，AST及肝组织MDA含量均显著升高，肝脏缺血再灌注前用白藜芦醇15 mg/kg者，血清ALT，AST及肝组织MDA含量均明显降低，且肝组织病理学损害明显减轻。结果表明白藜芦醇对肝脏I/R损伤具有保护作用     

不同缺血预处理方式对大鼠供肝的保护作用及其机制

目的 探讨不同方式的缺血预处理对大鼠供肝冷缺血—再灌注损伤的防护作用及其机制。方法 192只wistar大鼠做为供、受体行原位肝移植，供肝切取前给予不同方式的缺血预处理(C组为对照组，不行预处理；E1组在供肝冷灌注前行门静脉(PV)和肝动脉(HA)夹闭5min，再灌注10min；E2组PV、HA夹闭5min，再灌注5min，并重复上述过程1次；E3组PV、HA夹闭10min，再灌注15min)，移植完成后，于门静脉复流后0．5、2、6、24h检测血清肝脏酶学、血清肿瘤坏死因子—α(TNF—α)水平及肝组织中细胞凋亡情况。结果 与对照组相比，实验组大鼠在门静脉复流后0．5、2hTNF—α水平明显降低(P＜0．05)，实验组之间相比，E2组大鼠血清TNF—α水平明显低于E1、E3组(P＜0．05)；在24h E2组大鼠血清TNF—α水平明显低于C、E1、E3组(P＜0．05)。在2、6h实验组凋亡指数(AI)明显低于对照组(P＜0．05)，实验组之间相比，E2组AI明显低于EI、E3组(P＜0．05)；24h实验组AI明显低于对照组(P＜0．05)。结论 缺血预处理可能通过减少TNF—α释放，减轻细胞凋亡，从而减轻移植肝的损伤。5min缺血，5min再灌注，并重复1次的缺血预处理方式效果较好。     

Endothelin-1 levels in portal venous blood in relation to hepatic tissue microcirculation disturbance and hepatic cell injury after ischemia/reperfusion

This study was conducted to clarify the role of endothelin-1 in the portal vein after hepatic ischemia/reperfusion and to ascertain whether it is related to hepatic microcirculation disturbance. Using a canine ischemic liver model, the portal and systemic endothelin-1 levels were measured before ischemia, then after 1h and 2h of reperfusion, and comparatively evaluated with the serum levels of GOT and lactic dehydrogenase (LDH). As an indicator of liver tissue microcirculation, tissue blood flow volume (TBF) was also measured in the site subjected to ischemia. The animals were divided into: group 1, which received ischemia for 30 min; group 2, which received ischemia for 60 min; and group 3, which received a sequence repeated four times of 15 min ischemia and 10 min reperfusion. The portal endothelin-1 level became significantly elevated after reperfusion compared to that before ischemia in all groups, being significantly higher in group 2 than in the other groups. The systemic endothelin-1 level also increased after reperfusion; significantly in group 2. The portal endothelin-1 level was generally higher than the systemic level, which again was statistically significant in group 2. After 2h of reperfusion, a significant positive correlation was found between the portal endothelin-1 level and serum LDH, whereas a significant negative correlation was found between the portal endothelin-1 level and TBF. The finding that the portal endothelin-1 level became elevated after hepatic ischemia/reperfusion suggests that it probably plays an essential role in hepatic ischemia/ reperfusion injury by adversely influencing tissue microcirculation.     

150例活体肝移植供肝的后台修整

目的 总结150例活体供肝的后台修整经验.方法 回顾性分析2007年2月至2008年5月间完成的150例活体供肝的修整资料.结果 150例供肝中,包含肝中静脉及尾状叶的左半肝3例,肝左静脉与肝中静脉成形;左外侧叶2例;带肝中静脉右半肝67例,肝右静脉与肝中静脉成形;不带肝中静脉右半肝78例.78例不带肝中静脉右半肝中23例未进行S_5、S_8静脉的重建,供肝血流恢复后S_5、S_8肝组织均有不同程度的淤血.其余55例用不同材料重建S_5、S_8静脉:新鲜尸体髂静51例,受体大隐静脉1例,受体曲张脐静脉1例,受体肝内门静脉1例,受体肝静脉1例.145例右半肝供肝中门静脉为C型的7例,均成形为一个开口.结论 HTK是活体供肝的最佳灌注、保存液,供肝流出道恰当的成形、重建不但可简化供肝植入的操作步骤,还可最大限度的保护有功能的肝组织,是受体术后顺利恢复的关键.     

Attenuation of acute phase shear stress by somatostatin improves small-for-size liver graft survival.

The major concern of living donor liver transplantation is small-for-size graft injury at the early phase after transplantation. Novel therapeutic strategies should be developed. To investigate the protective effect of somatostatin related to hemodynamic stress on small-for-size liver graft injury, we applied a treatment regimen of low-dose somatostatin in a rat orthotopic liver transplantation model using small-for-size grafts (median, 38.7%; range, 35-42%). Somatostatin was given at 5 minutes before total hepatectomy and immediately after reperfusion in the recipient (20 microg/kg). Graft survival, portal hemodynamics, intragraft gene expression and hepatic ultrastructural changes were compared between the rats with or without somatostatin treatment. Seven-day graft survival rates in the somatostatin treatment group were significantly improved compared to the control group (66.7% vs. 16.7%, P = 0.036). In the treatment group, portal pressure and hepatic surface blood flow were significantly decreased within the first 30 minutes after reperfusion, whereas in the control group, transient portal hypertension and excessive hepatic blood flow were observed. Intragraft expression (both messenger RNA and protein) of endothelin-1 was significantly downregulated accompanied with upregulation of heme oxygenase-1 and A20. Better preservation of liver function was found in the treatment group. Hepatic ultrastructure, especially the integrity of sinusoids, was well protected in the treatment group. In conclusion, low-dose somatostatin rescues small-for-size grafts from acute phase injury in liver transplantation by attenuation of acute-phase shear stress that resulted from transient portal hypertension.     

Alterations in intrahepatic hemodynamics of the harvested porcine liver

Hemodynamic properties of a donor liver, during initial reperfusion, are associated with the degree of graft preservation injury and have been proposed to correlate with subsequent markers of liver function. In the present study, hepatic hemodynamics, that is, portal venous pressure, hepatic vascular resistance, and compliance (vascular distensibility), were characterized (1) in situ before porcine livers were manipulated, (2) after these same livers were isolated and perfused within a bypass circuit, and (3) on reperfusion after 2 hours of cold ischemia. Hepatic vascular resistance was determined in each of these three states from the portal vein pressure response to differing hepatic blood flows. In addition, the response of the same livers to norepinephrine and nitroprusside was evaluated in each condition. In the in situ and isolated perfused liver, portal venous pressure increased only modestly despite doubling of hepatic flows. After cold ischemia, the pressure response to higher flows was significantly greater and much less of a reduction in hepatic vascular resistance was noted than in studies prior to cold ischemia. Unlike livers prior to cold ischemia, the pressure response to norepinephrine was attenuated following cold ischemia. The response to nitroprusside, however, remained intact reducing the portal pressure to that of in situ livers. Therefore the portal hypertension that follows cold ischemia appears to be largely provoked by the preservation injury and not by surgical manipulation or the bypass circuit. This increment in portal pressure is responsive to a nitric oxide donor. Presented in part at the Fortieth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Fla., May 16–19, 1999.     

Ischemic preconditioning improves energy state and transplantation survival in obese Zucker rat livers

Livers from obese donors often have fatty infiltrates and are more susceptible to ischemia-reperfusion injury and subsequent graft dysfunction. This often leads to the exclusion of organs from obese donors. We investigated whether ischemic preconditioning (IP, 10 min ischemia, 10 min reperfusion) preserves cellular metabolism in livers from obese Zucker rats during cold ischemia. Liver samples (-IP and +IP) were collected from obese and control lean rats at different time points of cold ischemia (CI) and analyzed by magnetic resonance spectroscopy (1H- and 31P-MRS) to assess whether IP improves hepatic cellular metabolism. IP significantly improved high energy metabolism in IP livers from obese rats when compared with obese controls during the first hours of CI. At 4 h of cold storage, obese IP livers were not different from control lean non-IP livers. The beneficial metabolic effect of IP on livers form obese rats, however, was absent at 8 h of reperfusion. In contrast, in livers from lean rats, IP resulted in improved high-energy metabolism during the entire observation period of 8 h. In a later part of the study, IP of liver grafts from obese rats before 4 h of cold storage improved recipient survival after graft transplantation. IP of liver grafts from obese rats before 4 h of CI increases 24-h survival of recipient animals from 25% to 88%.     

Protective effects of trifluoperazine on the microcirculation of cold-stored livers

Previous studies have shown a protective effect of trifluoperazine (TFP), a calmodulin inhibitor, upon the microcirculation of cold-stored kidneys. The present study points to similar beneficial effects of TFP on the microcirculation of cold-stored livers; 25 canine livers were preserved for 24 hr with Euro-Collins' solution (EC) (n = 8), University of Wisconsin solution (UW) (n = 7), or UW + TFP (n = 10). The stored livers underwent heterotopic transplantation (HLTX); hepatic-artery and portal-vein pressure and flow were monitored; oxygen consumption and extraction were measured before HLTX and at 15-min intervals after reperfusion, for 1 hr. Mean hepatic-artery and portal-vein flow (HAF & PVF) prior to donor hepatectomy were 172 and 530 cc/min, respectively. Poor HAF and PVF occurred in EC-HLTX (mean 35, 175 cc/min, respectively). The damaged EC-flushed livers could not compensate to the decreased hepatic blood flow by increased oxygen extraction (oxygen consumption and extraction, 8.7 vol.% and 48%, respectively). Light and electron microscopy showed severe liver necrosis and periportal hemorrhages. Improved hepatic-artery and portal-vein flows were seen in UW HLTX (105 and 254 cc/min), and oxygen consumption and extraction were 16.4 vol.% and 66%, respectively. Liver biopsy taken just before reperfusion revealed well-preserved liver architecture. Liver biopsy obtained 1 hr after reperfusion revealed marked edema of the portal triad, sinusoid congestion, and hemorrhage. Electron-microscopy biopsies obtained during reperfusion at 15-min intervals revealed severe vasospasm of the terminal hepatic arterioles and progressive damage to the liver microcirculation. The addition of TFP to the UW-flush solution resulted in excellent protection of the liver microcirculation. Marked increase in hepatic-artery and portal-vein blood flow was noted after reperfusion (mean 167 and 421 cc/min, respectively (P 0.02 vs. UW: P 0.001 vs. EC). The recovery of metabolic activity was evident by the high oxygen consumption and extraction (25.8 vol.% and 80%, respectively). And serial liver biopsies obtained after reperfusion have shown excellent protection of liver architecture and the absence of hepatic arteriolar vasospasm. Taken together, these data suggest that the addition of TFP to the UW solution protects the liver microcirculation by rendering the hepatic microcirculation insensitive to vasospastic stimuli during reperfusion, thus permitting better metabolic recovery after transplantation.     

FK 409 ameliorates small-for-size liver graft injury by attenuation of portal hypertension and down-regulation of Egr-1 pathway

OBJECTIVE: To investigate whether low-dose nitric oxide donor FK 409 could attenuate small-for-size graft injury in liver transplantation using small-for-size grafts. SUMMARY BACKGROUND DATA: The major concern of live donor liver transplantation is small-for-size graft injury at the early phase after transplantation. Novel therapeutic strategies should be investigated. METHODS: We employed a rat orthotopic liver transplantation model using small-for-size (40%) graft. FK 409 was given at 30 minutes before graft harvesting (2 mg/kg) to the donor and immediately after reperfusion (1 mg/kg) to the recipient (FK group). Graft survival, intragraft genes expression, portal hemodynamics, and hepatic ultrastructural changes were compared between the 2 groups. RESULTS: Seven-day graft survival rates in the FK group were significantly improved compared with those of rats not receiving FK 409 (control group; 80% versus 28.6%, P = 0.018). In the FK group, portal pressure was significantly decreased within the first 60 minutes after reperfusion whereas in the control group, transient portal hypertension was observed. Intragraft expression (both mRNA and protein) of early growth response-1, endothelin-1, endothelin-1 receptor A, tumor necrosis factor-alpha, macrophage-inflammatory protein-2, and inducible nitric oxide synthase was significantly down-regulated accompanied with up-regulation of heme oxygenase-1, A20, interferon-gamma-inducible protein-10, and interleukin-10 during the first 24 hours after reperfusion. Hepatic ultrastructure, especially the integrity of sinusoids was well protected in the FK group. CONCLUSIONS: Low-dose FK 409 rescues small-for-size grafts in liver transplantation by attenuation of portal hypertension and amelioration of acute phase inflammatory response by down-regulation of Egr-1, together with prior induction of heat shock proteins.