大鼠肝移植重建肝动脉对胆管超微结构和并发症的影响

目的 观察大鼠原位肝移植重建肝动脉对肝内胆管上皮细胞缺血再灌注损伤后超微结构及术后胆道并发症的影响.方法 228只SD大鼠分为假手术组(8只)、肝移植重建肝动脉组(55对)和未重建肝动脉组(55对).重建肝动脉组和未重建肝动脉组分别于肝脏复流后0.5、3、6、12、24、36、48 h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;观察术后胆道并发症.结果 两组肝内胆管上皮细胞损伤均有加重,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,至24 h达高峰,以后逐渐恢复.术后两组线粒体平均面积和周径随时间的延长逐渐增大,线粒体数密度随时问延长而减少.在24 h,两组缺血再灌注损伤最显著,之后均开始缓解.在24、36、48 h,两组线粒体平均面积、平均周径比较,差异均有统计学意义(t=-3.566,-7.780,-4.730,-4.610,-2.599,-5.370,P＜0.05);在36、48 h,两组线粒体平均数密度比较,差异有统计学意义(t=-4.619,4.000,P＜0.05).重建肝动脉组的胆道并发症发生率低于未重建肝动脉组(x2=4.286,P＜0.05).结论 大鼠肝移植重建肝动脉对肝内胆管上皮细胞缺血再灌注损伤后的超微结构具有保护作用,有利于术后恢复和减少胆道并发症的发生.     

肝移植术中应用S-腺苷-L-蛋氨酸对供肝热缺血损伤的影响

目的探讨术中S-腺苷-L-蛋氨酸（SAMe）加入UW液和血浆冲洗液对热缺血损伤供肝及其恢复的影响。方法建立10min热缺血大鼠肝移植模型，分为A组：UW液灌注＋乳酸钠林格氏液冲洗、B组：UW液灌注＋血浆冲洗、C组：SAMe加入UW液灌注＋血浆冲洗和D组：UW液灌注＋SAMe加入血浆冲洗4组，观察肝组织组织病理学变化和电子显微镜下超微结构变化，并检测血清AST和透明质酸。结果C组和D组术后24h血清AST均低于B组（P〈0．05）。A组术后3h和24h血清HA高于B组（P〈0．05），B组复流后3h及24h血清HA均高于C组和D组（P〈0．05）。组织病理学表现B组复流后3h和24h肝细胞损伤和微循环紊乱较C组和D组明显；超微结构表现，A组复流后3h线粒体肿胀，肝窦内皮细胞肿胀，细胞核不规则，可见内皮细胞凋亡，大部分区域肝窦状隙明显狭窄，内皮层结构模糊，红细胞淤积，受压变形，白细胞附壁，可见内皮层完整性破坏；复流后24h，可见线粒体嵴断裂，核融解。B组内皮细胞损伤较A组轻，C组和D组超微结构表现微循环紊乱和肝细胞损伤表现较B组轻。结论供肝切取术中UW液中加入SAMe灌注保存，血浆冲洗液中加入SAMe可改善热缺血供肝微循环，减轻缺血再灌注损伤，并减轻肝细胞热缺血损伤，有利于10min热缺血供肝功能的恢复。     

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。     

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。     

大鼠供肝冷缺血再灌注损伤中TNF的作用及己酮可可碱预处理的效果

目的 探讨肿瘤坏死因子（TNF）与大鼠供肝冷缺血再灌注损伤中的关系及观察己酮可可碱（PTX）预处理的防护作用。方法 Wistar大鼠32只，随机分为受体组与供体组。两两随机配对后再随机分为对照组（C组）和实验组（即预处理组，E组）。E组供体大鼠预处理，开腹前1h腹腔注射PTX50mg/kg，对照组供体不进行预处理。供肝均保存6h后行原位肝移植。结果 （1）门静脉复流后30min及3h时血清中TNF水平E组显著低于C组（P＜0．05）；（2）30min及3h谷胱甘肽－S转换酶（GST－S）水平E组显著低于C组（P＜0．05）。（3）30min及3h血清中ALT，AST水平E组显著低于C组（P＜0．050。结论 TNF居大鼠供肝冷缺血再灌注损伤中起重要作用，己酮可可碱预处理可以减轻冷缺血再灌注损伤。     

影响肝缺血再灌注损伤主导因素的肌理

我们动态观察ＳＤ大鼠肝缺血４５．６０分钟复流０．１０，６０，１２０分钟，人体肝门阻断１５分钟复流５－１０分钟，４０分钟不同时限肝细胞内游离钙浓度，肝组织脂质过氧化自由基电子自旋共振信号最大幅值及脏脏超微结构病理变化，结果发现，在再灌注早期（大鼠肝血４５分钟复流１０分钟，人体肝缺血１５分钟复流５－１０分钟已可观察到（「Ｃａ＾２＋）」ｉ）增高现象，而ＲＯＯ．Ｙｍａｘ变化不显著，肝细胞超微结构仅表现为线     

缺血预处理对肝大部切除术中残肝缺血再灌注损伤的保护作用

目的 观察缺血预处理对大鼠肝大部切除术中残肝缺血再灌注损伤的保护作用。方法 健康的雌性SD大鼠随机分为3组：即单纯肝叶切除组（PH组）、缺血再灌注损伤状态下肝叶切除组（IR组）及缺血预处理组（IP组）。分别取术前及术后0．5、6、12、24、48h等时间点，应用全自动生化分析仪检测血清ALT、AST含量，通过免疫组织化学法检测残肝组织中Ki67和Cyclin D1表达变化，采用放免法检测血清中透明质酸（HA）含量。结果 IP组术后24h内各检测点的AST和ALT值明显高于PH组和IR组（P〈0．05）。术后早期IP组大鼠的血清HA表达量明显高于PH组和IR组（P〈0．05）。PH组大鼠肝细胞Ki67和Cyclin D1表达在术后24h达到峰值，并且明显高于IR组和IP组大鼠（P〈0．05）。其中IP组大鼠术后Ki67和Cyclin D1表达量降低地最显著。结论 在合并肝组织大部缺失时，缺血预处理对残留肝组织的缺血再灌注损伤的保护效应消失，它损害了大鼠残肝再生功能。     

不同胆道灌洗方法对大鼠肝内胆管冷保存再灌注损伤的影响

目的观察不同胆道灌洗方法对大鼠移植肝肝内胆管冷保存再灌注损伤的影响。方法应用大鼠原位肝移植模型，将88只SD大鼠随机分为假手术组、胆道非灌洗组、UW液胆道灌洗组、生理盐水（NS）胆道灌洗＋UW液肝内胆道灌注保存组、HTK液胆道灌洗＋UW液肝内胆道灌注保存组、HTK液胆道灌洗＋HTK液肝内胆道灌注保存组。移植肝置于4℃林格液中保存2h后行原位肝移植。移植肝再灌注后24h，检测血清总胆红素（TB）、直接胆红素（DB）、碱性磷酸酶（AKP）、γ-谷酰转肽酶（GGT）及胆汁中GGT、葡萄糖（Glu）含量。在光镜及电镜下观察肝内胆管上皮细胞的形态学变化。结果与非灌洗组比较，胆道灌洗组术后各项指标明显改善（P〈0．01）；HTK液及NS灌洗组较UW液灌洗组术后指标改善明显（P〈0．05）。病理检测发现非灌洗组胆道损伤明显，各灌洗组胆道损伤程度明显改善，HTK液灌洗＋UW或HTK液灌注组对胆管上皮细胞的损伤较轻。结论移植肝冷保存前进行胆道灌洗可以明显减轻胆管上皮细胞的损伤，4℃HTK液灌洗＋4℃UW或HTK液灌注保存效果比较理想。     

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。     

银杏叶提取液对缺血再灌注骨骼肌线粒体的保护作用

目的：观察银杏叶提取液对肢体缺血再灌注损伤的影响，方法：制作兔肢体缺血再灌注损伤动物模型，实验分对照组，再灌注组和治疗组，检测骨骼肌三磷酸腺苷（ATP）水平并观察线粒体超微结构的变化，测定线粒体丙二醛（MDA），还原型谷胱甘肽（GSH）和线粒体Ca含量。结果：再灌注组和对照组比较，以上各项指标差异显著（P＜0．01，P＜0．05），银杏叶提取液促进骨骼肌能量代谢，维持线粒体结构完整性，降低线粒体MDA含量，提高线粒体GSH水平并抑制线粒体Ca超载，治疗组各项测定指标较再灌注组比较明显改善（P＜0．05，P＜0．01），结论：银杏叶提取液对缺血再灌注骨骼肌线粒体有保护作用。     

控制性再灌注防止肺再灌注损伤的研究

目的　探讨控制性再灌注在预防肺缺血再灌注 (I/R)损伤中的作用及其机制。方法将猪分为 2组 ,10只猪切取左肺作供体 ,4℃改良的E C液灌洗和保存 ,4h后进行左肺移植。对照组常规操作 ,实验组采用控制性再灌注 :灌注液 (去白细胞血 :改良Buckberg液 =4∶1) ;灌注压 2 0mmHg ;灌注时间 10min。 0 .5、1和 2h后测血氧分压、肺血管阻力、肺顺应性、一氧化氮 (NO)含量、丙二醛 (MDA)含量、肺干 /湿重比。结果　实验组的左肺氧合功能 ,肺顺应性明显好于对照组 ,肺循环阻力、MDA值及肺含水量均低于对照组 (P <0 .0 1) ;实验组肺中NO含量较对照组明显升高 (P <0 .0 1)。结论　控制性再灌注能明显降低肺I/R损伤 ,起到了较好的移植肺保护效果。     

Controlled postcardioplegia reperfusion: mechanism for attenuation of reperfusion injury

OBJECTIVE: Controlled reperfusion and secondary cardioplegia are used to minimize reperfusion injury. The mechanisms for their benefit are incompletely defined and may include attenuation of myocyte sodium uptake. METHODS: Pigs had 1 hour of cardioplegic arrest followed by reperfusion with blood (control) or warm cardioplegic solution followed by blood (test). Reperfusion injury in the control and test groups was quantified by measuring changes of intramyocyte ion content with atomic absorption spectrometry and by analyzing electrophysiologic recovery from recordings of reperfusion arrhythmias. RESULTS: Control animals had an increase in intramyocyte sodium content at 5 minutes after initiating reperfusion (+20.2 micromol/g dry weight, P <.04), whereas the test group had an insignificant decrease (-14.0 micromol/g dry weight, P =.33). The first rhythm after initiating reperfusion was more often ventricular fibrillation in the control group (100% vs 50%, P <.02), and the control group required more defibrillations to establish a nonfibrillating rhythm (4.5 +/- 1.2 vs 1.1 +/- 0.3, P <.03). CONCLUSIONS: Controlled reperfusion eliminated the increase in intramyocyte sodium that was observed in the control group at 5 minutes after cardioplegic arrest. This improvement in myocyte ion homeostasis during postcardioplegia reperfusion was associated with fewer reperfusion arrhythmias. These data support the hypothesis that attenuation of myocyte sodium gain during postischemic reperfusion is a mechanism by which controlled reperfusion and secondary cardioplegia are beneficial.     

Rate of reperfusion blood flow modulates reperfusion injury in skeletal muscle

The mechanisms of ischemia-reperfusion (I-R) injury in skeletal muscle remain controversial. We investigated the effect of the rate of reperfusion blood flow on I-R injury in an isolated in vivo canine gracilis muscle model in six anesthetized dogs. In all animals, both gracilis muscles were subjected to 6 hr of ischemia followed by 1 hr of reperfusion. During reperfusion, one gracilis artery was partially occluded to limit the rate of reperfusion blood flow to its preischemic rate (limited reperfusion, LR), while the contralateral artery was allowed to perfuse freely at a normal rate (normal reperfusion, NR). Muscle injury was quantified by histochemical staining (triphenyltetrazolium chloride, TTC) with computerized planimetry of the infarct size, and by spectrophotometric determination of technetium-99m pyrophosphate uptake. Endothelial permeability was quantified by measurement of gracilis muscle weight gain and 125I-albumin radioactivity after intravenous injection. Results are presented as the means +/- SEM, and differences are considered to be statistically significant if P less than 0.05 by Student's t test for paired data. LR resulted in significantly less blood flow (9.7 +/- 1.7 cc/min/100 g) when compared to NR (55.7 +/- 11.6 cc/min/100 g). I-R injury was significantly reduced by LR as evidenced by a decrease in TTC infarct size from 41 +/- 7% to 11 +/- 5%, and a decrease in technetium-99m pyrophosphate uptake from 512 +/- 20 to 163 +/- 44 X 10(3) counts/min/g. LR also significantly decreased the postreperfusion edema formation as evidenced by a reduction in the muscle weight gain from 27 +/- 6 to 9 +/- 1 g, and a reduction in the 125I-albumin radioactivity from 45 +/- 14 to 32 +/- 8 counts/min/g. These data suggest that the hyperemic rate of reperfusion blood flow is a significant factor in the pathophysiology of postreperfusion edema and that clinical control of reperfusion injury in skeletal muscle may be achieved by limiting the rate of reperfusion blood flow.     

Controlled perfusion decreases reperfusion injury after high-flow reperfusion.

INTRODUCTION: Some investigators have suggested that high pulmonary artery flow rates increase the risk of severe reperfusion injury after lung transplantation. We hypothesized that controlling the initial flow rate and pulmonary artery pressure would decrease the severity of lung dysfunction in the setting of high-flow reperfusion. METHODS: Using our isolated, ventilated, blood-perfused rabbit lung model, all groups underwent lung harvest, 4-hour storage (4 degrees C), and blood reperfusion. We measured pulmonary artery pressure, peak inspiratory pressure, arterial oxygenation, and wet-to-dry weight ratio. Group 1 (control, n = 8) underwent reperfusion at 60 ml/min for 30 minutes. Group 2 (high flow, n = 8) underwent reperfusion at 120 ml/min for 30 minutes. Group 3 (controlled flow, n = 8) underwent initial reperfusion at 60 ml/min for 5 minutes, followed by reperfusion at 120 ml/min for 25 minutes. RESULTS: Group 1 had significantly improved pulmonary artery pressure, peak inspiratory pressure, arterial oxygenation, and wet-to-dry weight ratio measurements compared with groups 2 and 3 after 30 minutes of reperfusion. However, Group 3 had improved pulmonary artery pressure, peak inspiratory pressure, arterial oxygenation, and wet-to-dry weight ratio measurements compared with Group 2. CONCLUSIONS: High-flow reperfusion results in severe reperfusion injury after lung transplantation. Controlled reperfusion using a low initial flow rate decreases the severity of reperfusion injury associated with high-flow rates.     

Early reperfusion phenomena

Cycles of ischemia-reperfusion are ubiquitous in clinical cardiology. Depending on the duration and intensity of the ischemic episode as well as its repetition mode, several pathophysiologic syndromes have been identified, such as myocardial stunning, hibernation, and preconditioning. It remains a difficult exercise to distinguish ischemic from reperfusion damage. Production of oxygen free radicals and alteration in calcium homeostasis are major players during early reperfusion, responsible for the pathologic and functional alterations. At the molecular level, upregulation and downregulation of a number of genes have been observed in stunned myocardium, pointing toward some inborn survival adaptive mechanism. The no-reflow phenomenon, a most paradoxic event after reperfusion, usually occurs after more prolonged episodes of ischemia. The underlying mechanism involves additional lesions to the microvasculature interacting with myocytes lesions. Further insight into molecular and genomic adaptation to ischemia and reperfusion will undoubtedly help to improve our ability to fight reperfusion injury.     

Leukocyte-depleted reperfusion of transplanted human hearts prevents ultrastructural evidence of reperfusion injury.

The present study examines whether leukocyte depletion can prevent postreperfusion ultrastructural injury in transplanted human hearts. Thirty-two patients undergoing orthotopic cardiac transplantation were randomized to receive either enriched, warm, whole blood (Group I; n = 16) or enriched, warm, leukocyte-depleted blood (Group II; n = 16) reperfusion. Donor hearts were arrested with 1 liter of 4 degrees C crystalloid cardioplegia and topically cooled. RV endomyocardial biopsies taken at end-ischemia and following reperfusion were assessed in a blinded fashion and graded according to injury (1 = minimal to 4 = severe). The mean ischemic time (Group I = 142 min, Group II = 153 min) was similar in the two groups. End-ischemic biopsies showed mild-moderate interstitial edema and mild capillary endothelial swelling in both groups with similar injury scores (Group 1 = 1.3 +/- 0.09 (means +/- SEM), Group 2 = 1.25 +/- 0.08). Postreperfusion biopsies in Group I showed nuclear chromatin clumping, moderate mitochondrial swelling, marked capillary endothelial swelling, and marked interstitial edema with a grade of 2.6 +/- 0.14 (P less than 0.001, paired t test). In contrast, postreperfusion biopsies in Group II showed minimal changes with a grade of 1.33 +/- 0.09, P less than 0.0001 in comparison to Group I Leukocyte-depleted reperfusion of human transplanted hearts prevents ultrastructural injury. This may allow safe extension of the ischemic period and result in improved graft function.     

Mechanisms of myocardial reperfusion injury

Reperfusion of the ischemic myocardium results in irreversible tissue injury and cell necrosis, leading to decreased cardiac performance. While early reperfusion of the heart is essential in preventing further tissue damage due to ischemia, reintroduction of blood flow can expedite the death of vulnerable, but still viable, myocardial tissue, by initiating a series of events involving both intracellular and extracellular mechanisms. In the last decade, extensive efforts have focused on the role of cytotoxic reactive oxygen species, complement activation, neutrophil adhesion, and the interactions between complement and neutrophils during myocardial reperfusion injury. Without reperfusion, myocardial cell death evolves slowly over the course of hours. In contrast, reperfusion after an ischemic insult of sufficient duration initiates an inflammatory response, beginning with complement activation, followed by the recruitment and accumulation of neutrophils into the reperfused myocardium. Modulation of the inflammatory response, therefore, constitutes a potential pharmacological target to protect the heart from reperfusion injury. Recognition of the initiating factor(s) involved in myocardial reperfusion injury should aid in development of pharmacological interventions to selectively or collectively attenuate the sequence of events that mediate extension of tissue injury beyond that caused by the ischemic insult.