间断性和持续性肝缺血再灌注对线粒体功能的影响

间断性和持续性肝缺血再灌注对线粒体功能的影响孙经建张柏和吴孟超陈汉钱光相作者采用大鼠肝缺血再灌注损伤模型，观察肝线粒体功能，以及肝组织内钙离子、脂质过氧化物（ＬＰＯ）和超氧化物歧化酶（ＳＯＤ）含量的变化，旨在比较间断性与持续性肝血流阻断对上述指标的影...     

热休克蛋白70对肝缺血再灌注损伤的作用及机制探讨

目的 探索热休克蛋白70（Heat shock protein 70,HSP700）对大鼠肝缺血再灌注损伤的作用及机制。方法 实验大鼠分为持续阻断组，间断阻断组和假处理组。用免疫金银法观察再灌注1h后肝组织HSP70蛋白的表达，并检测其肝细胞及线粒体功能，肝组织内钙和脂质过氧化物（Lipid peroxide,LPO)含量，以及超氧化物歧化酶（Superoxide dismutase,SOD)活性。结果 HSP70蛋白在持续性缺血再灌注后无表达，肝细胞功能，线粒体功能和SOD活性均显下降，而肝组织内钙和LPO值均显升高。间断性血流阻断组则有HSP70蛋白的合成表达，该组与假处理组的上述各项指标的差异均无显意义。结论 HSP70蛋白在肝缺血再灌注后的诱导产生有利于保护肝细胞有线粒体的功能，该效应与细胞内钙稳定和SOD活性增高有关。     

热休克蛋白70对肝缺血再灌注损伤的作用及机制探讨

目的 探讨热休克蛋白70（Heat shock protein 70,HSP70）对大鼠肝缺血再灌注损伤的作用及机制。方法 实验大鼠分为持续阻断组、间断阻断组和假处理组。用免疫金银法观察再灌注1h后肝组织HSP70蛋白的表达,并检测其肝细胞及线粒体功能、肝组织内钙和脂质过氧化物（Lipid peroxide,LPO）含量,以及超氧化物歧化酶（Superoxide dismuase,SOD）活性。结果 HSP70蛋白在持续性缺血再灌注后无表达,肝细胞功能、线粒体功能和SOD活性均显著下降,而肝组织内钙和LPO值均显著升高。间断性血流阻断组则有HSP70蛋白的合成表达,该组与假处理组的上述各项指标的差异均无显著意义。结论HSP70蛋白在肝缺血再灌注后的诱导产生有利于保护肝细胞及线粒体的功能,该效应与细胞内钙稳定和SOD活性增高有关。     

热休克蛋白70在肝血流间断阻断再灌注后的表达及作用

目的：探讨热休克蛋白70（HSP70）在肝血流间断和持续阻断再灌注后的表达及作用。方法：将实验大鼠分为持续阻断组，间断阻断组和假处理组，用免疫金银法观察再灌注1h后肝组织中HSP70的表达，并检测肝细胞及线粒体功能。结果：HSP70面肝持续性缺血再灌注后无表达，肝细胞和线粒体功能均显著下降，间断性血流阻断组则有HSP70的合成表达，该组与假处理组的肝细胞和线粒体功能指标的差异均无显著性。结论：HSP70在间断性肝血流阻断再灌注后的早期合成，有利于保护肝细胞和线粒体功能。     

间断性阻断肝血流对线粒体功能的影响及机理探讨

目的 探讨间断性和持续性阻断肝血流对线粒体功能的影响及其作用机理。方法 实验大鼠分为持续阻断组、间断阻断组和假处理组,检测其肝线粒体功能、肝组织内钙和脂质过氧化物（ＬＰＯ）含量,以及超氧化物歧化酶（ＳＯＤ）活性。结果 与间断性阻断肝血流相比,持续性缺血再灌注后大鼠肝线粒体呼吸控制比率（ＲＣＲ）、磷氧比值（Ｐ／Ｏ）、氧化磷酸化效率（ＯＰＲ）和ＳＯＤ活性均显著下降,其值分别为３．０４±０．１７对２．０     

兔肝缺血/再灌注损伤肝组织氧压及线粒体形态观察

目的　观察兔肝缺血 /再灌注损伤不同时相肝组织氧压及线粒体形态变化 ,研究常温下肝脏缺血 /再灌注的损伤及其机制。方法　 4 0只兔随机分为 2组即缺血组和对照组。组织气体分析仪持续测定兔肝组织氧压 (Hepatic tissue oxygeonpressure Ptio2 ) ;电镜观察肝脏病理及线粒体形态改变 ,图像分析仪对线粒体的立体形态计量分析。结果　缺血组兔在肝脏缺血后肝 Ptio2值开始下降 ,再灌注 6 0 min时肝 Ptio2仍未恢复正常 (P<0 .0 5 )。电镜观察发现 :肝缺血 30 min,线粒体肿胀、内质网扩张 ,再灌注 6 0 m in后线粒体肿胀进一步加重 ,部分线粒体破坏 ,结构不清 ,线粒体空泡变性。兔肝缺血再灌注损伤后肝细胞线粒体与对照组相比 ,其比表面减小、平均体积增大 (P<0 .0 5 )。结论　常温下入肝血流阻断可以导致肝脏缺血 /再灌注后肝细胞功能障碍和病理损害。其作用机制与缺血期肝细胞缺氧、再灌注期肝脏微循环障碍和肝细胞线粒体的损伤有关     

肝动脉复流时机对大鼠肝内胆管上皮细胞缺血再灌注后超微结构的影响

目的 观察不同肝动脉复流时机对大鼠肝内胆管上皮细胞缺血再灌注损伤后超微结构的影响。方法 建立大鼠原位部分肝脏缺血再灌注损伤模型，54只大鼠随机分为假手术组（SO）、肝动脉先复流组（IAR）和门静脉先复流组（IPR），IAR组、IPR组分别于复流后0．5、2．0、4．0、12．0h时取材，用透射电镜观察肝内胆管上皮细胞的超微结构，通过计算机图像分析系统对线粒体形态计量分析。结果 （1）随着缺血再灌注后时间的延长，IAR组与IPR组肝内胆管上皮细胞损伤均有加重趋势，表现为线粒体肿胀、嵴模糊或消失、微绒毛减少等超微结构改变。（2）IPR 0．5h组、IPR 2h组胆管上皮细胞与IAR组相应时间点比较，超微结构损害无明显加重，线粒体平均面积、平均周径及数密度差异均无统计学意义（P〉0．05）；但IPR 4h组、IPR 12h组与IAR组相应时间点比较，超微结构损害加重，线粒体平均面积增大，差异有统计学意义（P〈0．05），线粒体平均周径增大，差异有统计学意义（P〈0．05），其中IPR 12h组线粒体数密度减少，差异有统计学意义（P〈0．05）。结论 与门静脉先复流相比较，肝动脉先复流对大鼠肝内胆管上皮细胞缺血再灌注后的超微结构具有保护作用。     

肝硬化鼠肝缺血再灌注胃粘膜病理损伤的研究

目的 探讨肝缺血再灌注对肝硬化鼠胃粘膜的病理损伤过程。方法：实验大鼠制备肝硬化模型。并行肝脏缺血再灌注，取大鼠胃粘膜标本行电镜，光镜检查。并检测血中转氨酶的变化。结果 肝硬化鼠胃粘膜出现充血，水肿，有糜烂及小溃疡形成，电镜见内质网扩张，线粒体肿胀。肝缺血再灌注后病理过程较前加重，表现为溃疡面的扩大，部分出现粘膜上皮脱落等。细胞崩解。内质网扩张明显。核膜不清。结论 肝缺血再灌注加重了门脉高压性胃炎的病理过程。     

缺血后处理对大鼠肝缺血再灌注时肝细胞线粒体损伤的影响

目的 评价缺血后处理对大鼠肝缺血再灌注时线粒体损伤的影响.方法 雄性SD大鼠30只,体重180～230 g,随机分为3组(n=10):假手术组(S组)、肝缺血再灌注组(IR组)和缺血后处理组(Ipo组).IR组和Ipo组采用阻断肝门60 min再灌注6 h的方法 制备肝缺血再灌注模型,Ipo组缺血60 min时再灌注10 s、缺血10 s,反复6次,进行缺血后处理.于再灌注6 h时取静脉血样,测定血清谷丙转氨酶(ALT)及天门冬氨酸氨基转移酶(AST)活性,然后取肝组织,制备病理切片及分离肝细胞,电镜下观察线粒体超微结构,测定线粒体膜电位及线粒体Na+-K+-ATP酶活性.结果 与S组比较,IR组和Ipo组血清ALT和AST活性升高,线粒体Na+-K+-ATP酶活性及线粒体膜电位降低(P<0.01);与IR组比较,Ipo组血清ALT和AST活性降低,线粒体Na+-K+-ATP酶活性及线粒体膜电位升高(P<0.05或0.01).Ipo组线粒体损伤程度轻于IR组.结论 缺血后处理可减轻大鼠肝缺血再灌注时肝细胞线粒体损伤.     

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。     

Role of ischemic preconditioning in hepatic ischemia-reperfusion injury

Background

Investigation into less traumatic method of vascular occlusion during liver resection is the actual problem in hepatic surgery because of high level of complications such as liver failure. In this connection, the goal of our study was to determine the optimal model of vascular clamping. The research showed that vascular occlusion with ischemic preconditioning in the mode 5/10/15 the most delicate technique.

Methods

Forty white giant rabbits were divided randomly into four groups (n=10 in each group). In group I we used continuous Pringle maneuver by 30 min. In group II we used intermittent Pringle maneuver: 15 min of clamping/5 min of unclamping (reperfusion)/15 min of clamping. In group III we used intermittent Pringle maneuver with ischemic precondition: 5 min of ischemia/5 min of reperfusion, 10 min of ischemia/5 min of reperfusion/15 min of ischemia. Group IV (control group) is without hepatic ischemia. All animals were performed a liver biopsy at the end of the surgery. Five rabbits from each group underwent re-laparotomy on day 3 after surgery with biopsy samples being taken for studying reparative processes in liver parenchyma.

Results

Results of morphometric analysis were the best to illustrate different level of liver injury in the groups. Thus, there were 95.5% damaged hepatocytes after vascular occlusion in hepatic preparations in group I, 70.3% damaged hepatocytes in group II, and 42.3% damaged hepatocytes in group III. There were 5.3% damaged hepatocytes in the control group.

Conclusions

Vascular occlusion with ischemic preconditioning in the mode 5/10/15 the most delicate technique that does not involve major structural injuries and functional disorders in the remnant liver. Thus, it is amenable to translation into clinical practice and may improve outcomes in liver resection with inflow vascular occlusion.     

Effect of superoxide dismutase on liver ischemia-reperfusion injury in the rat: a biochemical monitoring

Oxygen free radicals have been implicated in the pathogenesis of postischemic liver injury. High-dose superoxide dismutase (SOD), a radical scavenging enzyme, has been investigated in a rat model of liver ischemia reperfusion by biochemical monitoring. Blood vessels to the median and left lobe were clamped for 1 h and then reperfusion was allowed. The indices used were serial venous blood levels of AST, ALT, calcium, and ATP determination in liver tissue. In SOD-treated animals (7,5000 U i.v.) a significant attenuation of the rise in enzyme levels was observed as well as the absence of the decrease in calcium level in the early phase after reperfusion as compared with control rats, and furthermore ATP restoration was significantly increased.     

异氟醚对家兔肺缺血-再灌注损伤的影响

目的 研究异氟醚对体家兔肺缺血－再灌注（I－R）损伤的影响。方法 32只新西兰家兔根据Eppinger模型加以改进,建立在体兔肺缺血再灌注模型,随机分四组（n＝8）,A组：假手术组,开胸后维持通气120min。B组：缺血再灌注组,阻断左肺门60min,开放再通气60min。C组：异氟醚＋缺血再灌主组,缺血前吸入1MAC异氟醚30min,开放再通气时也吸入1MAC异氟醚。D组：异氟醚组,持续吸入1MAC异氟醚120min,观察各组实验结束时肺湿－干重比（W／D）,肺内二醛含量（MDA）和肺组织学改变以及术中血流动力学的改变。结果 B组及C组W／D,肺MDA含量均较A、D两组显著增高,（P＜0．05）,且病理切片显示,部分肺泡结构破坏,肺泡间隔增宽,肺泡腔水肿并有白细胞渗出,但B组改变明显严重,且B组W／D,肺MDA含量也明显高于C组（P＜0．05）,血液动力学基本维持正常。结论 异氟醚在体家兔肺I－R损伤有一定的保护作用。     

Ischemic preconditioning-induced microvascular protection at a distance

Ischemic preconditioning-induced microcirculatory protection appears to be a systemic rather than a local phenomenon. This protection induced by remote ischemic preconditioning (RIPC) may be attributed to a humoral rather than a neuronal mechanism. An innervated (Inn, Groups 1 and 4) or denervated (Den, Groups 2 and 3) and vascular isolated right cremaster of the rat was prepared. Left femoral vessels were clamped (Groups 1, 2, and 4) for 45 min or unclamped (Group 3) as a control. After 2 hr of reperfusion in the left lower extremity, 4 hr of ischemia in the right cremaster was applied in Groups 1, 2, and 3. However, in Group 4, 4 hr of ischemia in the cremaster muscle was instituted before reperfusion of the left femoral artery was begun. Microcirculatory responses, including terminal arteriole diameter, capillary perfusion, and endothelium function were evaluated. Four groups (six rats per group) were designed: Group 1: RIPC (Inn); Group 2: RIPC (Den); Group 3: sham RIPC (Den); and Group 4: fake RIPC (Inn). Ischemia of 45 min followed by 2 hr of reperfusion in the left lower extremity of the rat induced a significant microvascular protection against subsequent 4-hr ischemia in both innervated and denervated cremasters. This microvascular protection at a distance was lost in sham RIPC and in fake RIPC groups. The results demonstrated that remote protection induced by ischemic preconditioning is a systemic phenomenon and due to a humoral mechanism.     

Hemodynamic interaction between portal vein and hepatic artery flow in small-for-size split liver transplantation

In split-liver transplantation, the entire portal flow is redirected through relatively small-for-size grafts. It has been postulated that excessive portal blood flow leads to graft injury. In order to elucidate the mechanisms of this injury, we studied the hemodynamic interactions between portal vein- and hepatic artery flow in an experimental model in pigs. Six whole pig liver grafts were implanted in Group 1 ( n=6) and six whole liver grafts were split into right and left grafts and transplanted to Groups 2 ( n=6) and 3 ( n=6), respectively. The graft-to-recipient liver volume ratio was 1:1, 2:3 and 1:3 in Groups 1, 2 and 3, respectively. Portal vein- and hepatic artery flows were measured with an ultrasonic flow meter at 60,120 and 180 min after graft reperfusion. Portal vein pressure was also recorded at the same time intervals. Graft function was assessed at 3,6h and 12h, and morphological changes at 12h after reperfusion. Following reperfusion, portal vein flow showed an inverse relationship to graft size, while hepatic artery flow was reduced proportionately to graft size. The difference was significant among the three groups ( P<0.05). Portal vein pressure was significantly higher in group 3, compared to groups 1 and 2 ( P<0.05). Hepatic artery buffer response was significantly higher in Group 3, compared to Groups 1 and 2 in relation to pre-occlusion values ( P<0.05). Split-liver transplantation, when resulting in small-for-size grafts, is associated with portal hypertension, diminished arterial flow, and graft dysfunction. Arterial flow impairment appears to be related to increased portal vein flow.     

胃饲乙醇预处理增强肝脏对缺血再灌注损伤耐受性的实验研究

目的　探讨胃饲乙醇预处理能否延长大鼠肝脏耐受入肝血流阻断的安全时限。方法　Wistar大鼠随机分组 ,动物乙醇预处理后制作缺血再灌注模型 ,于手术后 0h～ 7d内不同时相活杀大鼠 ,留取血及肝脏标本保存待检。结果　入肝血流阻断 12 0min时 ,胃饲乙醇预处理组 14d存活率为10 0 % ,缺血组存活率为 70 % ,差异显著 ;预处理组和缺血组较正常组ALT值明显升高 ,72h后基本恢复 ,缺血末期和再灌注 6h ,预处理组和缺血组两者间差异显著 ;预处理组和缺血组ATP值均低于正常组 ,预处理组再灌注 12h后恢复 ,缺血组再灌注 72h恢复正常 ;病理观察见预处理组较缺血组表现为较轻的病理损害 ;免疫组化结果 :正常肝脏仅见弱阳性表达 ,散在分布于库普弗细胞。预处理组和缺血组表现为肝细胞阳性表达 ,术后随再灌注时相的延长 ,HO 1表达逐渐增强 ,预处理组表达明显强于缺血组 ,相差显著。结论　胃饲乙醇预处理能够增强肝脏对缺血再灌注损伤的耐受性 ,大鼠肝脏耐受入肝血流阻断的安全时限可延长至 12 0min     

Hemoglobin-glutamer 200 reduces reperfusion injury of the cold preserved rat liver by induction of heme oxygenase-1

Microcirculatory failure after cold liver preservation and reperfusion impairs tissue oxygenation and causes additional organ damage. Hemoglobin-glutamer (HbG) 200 is a hemoglobin-based oxygen carrying solution capable to improve organ oxygenation. The aim of this study was to evaluate its potential to decrease reperfusion injury after cold liver preservation. Therefore, Wistar rat livers were stored at 4 degrees C for 24 h and reperfused in the isolated perfused rat liver model with a sanguineous perfusate for 180 min. The perfusate consisted of rat blood and Krebs-Henseleit solution (Group A), supplemented by either HES 6% (Group B), or HbG (Groups C and D). In Group D heme oxygenase (HO) activity was blocked by intraperitoneal tin protoporphyrin-IX application before organ harvest. HbG supplementation increased the perfusate hemoglobin by 3,3 g/dL. After 180 min reperfusion perfusate alanine aminotransferase levels (72 +/- 27 micro/L) were significantly reduced in Group C, compared with Groups A and B (140 +/- 28 micro/L and 203 +/- 62 micro/L, respectively). These results correlated with a significant increase of HO-1 expression and activity during reperfusion. These effects could be abolished by tin protoporphyrin-IX application. HbG has been proven to be effective to reduce cold liver preservation-reperfusion injury. The positive effect on reperfusion injury depends on the induction of HO-1, which increases the bilirubin production, an important antioxidant acting as intracellular radical scavenger.     

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     

Impact of gradual blood flow increase on ischaemia-reperfusion injury in the rat cremaster microcirculation model.

INTRODUCTION: We aimed to evaluate the impact of gradual blood reperfusion on ischaemia-reperfusion injury and to explain the pathophysiology of reperfusion injury in a rat cremaster muscle microcirculation model. MATERIALS AND METHODS: Twenty-four Sprague-Dawley rats weighing 150-200 g were evaluated in three groups. Cremaster muscles were prepared for microcirculatory observations. Group I (n=8, control): no ischemia was induced. Group II (n=8, acute reperfusion): microclamps were applied to the right external iliac vessels for 150 min, then venous and arterial clamps were released at once. Group III (n=8, gradual reperfusion): microclamps were applied to the right external iliac vessels for 150 min, and then the first venous clamp was released; the arterial clamp was opened gradually by a specially designed microclamp holder (Sheey ossicle holding clamp). In all groups, following a wait of 150 min blood flow velocity was measured for 15 min and then the animals were reperfused freely for 1h. Next, red blood cell velocity, vessel diameters, functional capillary perfusion and endothelial oedema index were analysed, and rolling, migrating and adhesing leukocytes and lymphocytes were counted. All observations were videotaped for slow-motion replay. Muscle damage was evaluated histologically. RESULTS: In the acute clamp release group, blood velocities increased up to 600% of their pre-ischaemic values during the post-ischaemia-reperfusion period. The numbers of rolling, adhering and transmigrating leukocytes were significantly higher and histological evaluation revealed more tissue damage in the acute reperfusion group. CONCLUSION: Depending on histological and microcirculatory findings, gradual reperfusion was confirmed to reduce the intensity of reperfusion injury.     

自体原位肝移植大鼠围术期急性肺损伤的研究

目的 建立SD大鼠自体原位肝移植模型,动态观察自体原位肝移植围术期肺损伤特点.方法 选取健康SPF级SD大鼠40只,随机分为假手术对照组(S组,n=8)和模型组(M组),M组进行自体原位肝移植手术,根据肝脏再灌注时间不同再分为4个亚组.S组在麻醉后只进行开腹和血管的分离,不进行肝脏的阻断和灌注.分别在肝脏再灌注后4、8、16、24 h采集动脉血和肺组织标本,分别用于检测动脉氧分压、肺组织病理学.结果 与S组[(120.75 ±6.58) mm Hg(1 mmHg =0.133 kPa)]比较,M2组(97.50±8.22)mm Hg动脉氧分压降低(P＜0.05),其余各组差异无统计学意义(P＞0.05).M组肺组织镜下见大量的炎症细胞浸润,肺泡腔内渗出明显,肺泡腔狭窄,肺间质明显出血充血,肺泡间隔明显增厚,肺实质所占的比例显著增加;这些病理改变在M2组(肝脏再灌注8h)达到高峰,然后逐渐好转.结论 自体原位肝移植后SD大鼠存在肺损伤,在肝脏再灌注后8h达到高峰.