我们要感谢丹尼斯·马蒂亞斯他说明工作。这项工作是由美国国立卫生研究院T32AI 106704-01A1和俄亥俄州立大学T·弗莱希基金器官移植，灌注，工程和再生支持。

根据机构动物护理和国家研究理事会指南的人文关怀和实验动物的使用（IACUC）的指导方针进行所有的程序，并经历了由美国俄亥俄州立大学IACUC委员会的批准。 1.初始设置<ol><li>建立手术显微镜和手术室（ 图3， 图4）。打开所有设备包括用于维持麻醉和监测生命体征。打开电单元和气候变暖垫。手术台附近输液泵的位置。 <ol><li>制定10毫升液体异氟烷在麻醉注射器吸入（分子量184.5），并将其放置在麻醉单元。 </li><li>建立手术台附近的液氮的200mL容器中，并在那里另一血样将被处理的近离心机。 </li><li>波什银行足球比赛的手术器械，4-0和7-0的编织丝缝合线，无菌棉签，4×4非织造海绵，5个毫升注射器，和手术台附近27个规格胰岛素注射器。 </li></ol></li><li>制备异氟醚腔室和确保足够的异氟烷在麻醉诱导递送系统灌输。 </li></ol> 2.麻醉诱导<ol><li>处理提上了以下的个人防护装备（PPE）大鼠前:口罩，手术手套和一次性礼服。 </li><li>称量大鼠和记录重量。 注:SD大鼠应使用。 </li><li>将大鼠在麻醉室，并开启异氟醚和氧气。使用异氟醚室诱导麻醉。 </li><li>使用电动理发器，以允许清洁剂曝光（ 图5）夹在动物的腹部毛发。 </li><li>地方的additio异氟醚室动物背最终一分钟。执行脚趾捏来验证麻醉深度。 </li></ol> 3.程序<ol><li>与鼻锥动物的鼻子和固定与气候变暖垫限制或磁带四肢老鼠的位置。 </li><li>使用麻醉递送系统，鼻锥和异氟醚麻醉在3.6％动物200和与动物体重大于250 g的250克4％称量继续麻醉。通过执行脚趾捏和皮肤捏确认麻醉深度。 </li><li>使从耻骨中线腹部切口通过使用锋利的剪刀（ 图6）皮肤剑突。 </li><li>请从耻骨到剑突并进入腹部，注意不要损伤膀胱或肠道沿白线腹膜切口。由于肝脏也坚持腹膜向前靠近剑突，确保其发布在这个AR切开腹壁前EA。 </li><li>使通过皮肤和横向切口在肝脏右叶下缘水平腹膜。 </li><li>转动麻醉下降到1.6％动物200和和2对动物称重大于250 g的250克％称量。 </li><li>缩回使用弯曲蚊式钳剑突。 </li><li>地方肋牵开器从所述中线拉动肋尽可能远离可能的（ 图7）。切镰状，膈胃韧带。翻转肝起来使用的润湿无菌棉签。 </li><li>必要时切断附加韧带来访问的PORTA。用生理盐水弄湿的纱布（ 图7）执行内脏转动。 </li><li>取出的疏松结缔组织使用尖锐的或钝的解剖覆盖在门户脐。取出的疏松结缔组织覆盖在门静脉的长度。 </li><li>使用镊子通过疏松的吨至推发行后，以左门静脉，动脉和胆管制作窗口，然后将4-0波茨缝合，但不收紧下来（ 图8）。 </li><li>清除掉疏松结缔组织覆后支随附右肾在近似水平的门静脉。这静脉将用于插管。 </li><li>绘制0.5mL血液出下腔静脉（IVC）用胰岛素注射器（ 图9）的。放置0.5毫升的血液在小瓶，离心机在135×g离心12分钟。尝试抽出血清。 <ol><li>如果一个不同的线不能红血细胞和血清之间可以理解，试图离心另外2 - 在135×g下3分钟。抽出血清并将其放置在小瓶中丙氨酸氨基转移酶（ALT）。卡扣通过直接将其置于液氮中冷冻该标本。 </li></ol></li><li>切7-0缝合静脉近的地方的两片将用于套管灰。将围绕这一思路的第一个7-0环路内侧远越好。扎该循环，并使用它使用弯曲蚊式钳（ 图10）缩回。广场上，将用于插管的交叉口附近与门静脉和放置一个领带静脉第二循环7-0，但不肚带下来。 </li><li>准备用5毫升的注射器用3mL试剂的输液泵。总理油管。 </li><li>采用显微外科钳钳夹远端门静脉。 注:当静脉切开插管为这将减少出血。 </li><li>切割在静脉0.5mm的孔在7-0停留缝合和其与使用小的显微剪刀门静脉交点之间。使用27-0导管将导管插入左侧门静脉系统（ 图11， 图12）。插入导管过去左，右门静脉的分叉。 </li><li>通过infus检查套管的位置荷兰国际集团生理盐水1毫升并观察肝脏灼的左侧和中位数裂片。手动确认导管过去是门静脉右支的起飞，但不超过门静脉喂养中叶的腾飞。 </li><li>不在话下下来波茨缝合，并开始缺血时间。拧紧周围静脉7-0缝线和27-0导管举行的地方，并从远端门静脉取出钳。 </li><li>开始使用输液泵聚乙二醇 - 超氧化物歧化酶（PEG-SOD，0.00067克/毫升）的输注。开始输液尽可能接近到的缺血时间开始。 </li></ol> 4.监测<ol><li>继续监控整个输液动物的生命体征。交付2毫升0.9％生理盐水或2毫升PEG-SOD的（0.00067克/毫升）溶解在0.9％生理盐水中历时15分钟。 </li></ol> 5.再灌注<ol><li>允许一个小时，从我一开始传递schemic时间。这是热缺血时间1小时。 </li><li>取出波茨缝合。取下导管27-0。倒不在话下周围静脉缝合7-0。注意时间。这标志着再灌注的时间。 </li></ol> 6.续取样<ol><li>绘制0.5mL血液出IVC的在120分钟再灌注后。绘制血慢慢地避免溶解红细胞。慢慢地滴血液进入小瓶。保证从下腔静脉出血是每个抽血后控制。 <ol><li>如果继续有出血轻轻向下按压，用无菌棉签或小的1cm乘由丝网1厘米部分切断。 </li></ol></li><li>离心机中以135×g离心12分钟。如果没有实现充分的分离，尝试另外的2 - 在135×g下3分钟。 </li><li>将一半的血清在小瓶为ALT以后处理。捕捉冻结这些标本。 </li></ol> 7.安乐死<ol><li>虽然老鼠仍然是麻醉下切开下腔静脉和superior韦纳·卡瓦（SVC）和监控，直到血液流动，呼吸和心脏搏动停止。 </li><li>切开隔膜和通过在一个圆圈切开隔膜和切开残留在肝脏连接到腹膜腔附加结缔组织执行简短肝切除。取下腹腔肝。 </li><li>就拿从肝脏的左和中位数叶四个样本和四个样品从肝脏右叶。样品应尽可能大，它们的大小将仅受可用肝组织量的限制。放置这些小，标记的小瓶中，并且在捕捉液氮冷冻。使用这些供以后处理组织三磷酸腺苷（ADP），丙二醛（MDA）和谷胱甘肽（GSH）。 </li></ol> 8.后的实验分析<ol><li>使用诊断试剂盒确定的谷胱甘肽（GSH），丙二醛（MDA）和丙氨酸转氨酶（ALT）肝组织中的活动和血清样品根据制造商的说明。 </li><li>均质化的肝组织用裂解缓冲液，并使用Bradford测定法量化。分析由使用抗切割capase-3和肌动蛋白十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和免疫印迹组织裂解物。量化与公开可用的软件进行蛋白质印迹。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylene+glycol-superoxide+dismutase+inhibits+lipid+peroxidation+in+hepatic+ischemia/reperfusion+injury.">Polyethylene glycol-superoxide dismutase inhibits lipid peroxidation in hepatic ischemia/reperfusion injury.</a> Crit Care. 3, 127-130 (1999).</li></ol>

所有作者报告说，他们没有披露。

这一系列的实验表明，PEG-SOD进入左，导致显著跌幅ALT的发布中位数叶，细胞膜的脂质过氧化（MDA）和谷胱甘肽的维护（GSH）的注射时，与对照组相比（生理盐水）。肝组织转氨酶包括丙氨酸转氨酶（ALT）建立肝细胞损伤的标记。当左叶被注射PEG-SOD中ALT的降低表明PEG-SOD的保护作用。增加组织MDA指示增加的脂质过氧化，并且被认为氧化应激和组织损伤的标志物。活性氧物质的过量产生导致制造MDA 26的增加。在组织MDA当PEG-SOD注入的显著减少动物的左侧和中间叶表明PEG-SOD的保护作用。这是与当前的理解是，PEG-SOD免受损伤细胞一致引起的部分还原的活性氧27。另外，在活性氧的存在下，谷胱甘肽二硫化物被还原成还原型谷胱甘肽（GSH）28。与PEG-SOD注入进一步的肝左和中位数叶中GSH维修加固PEG-SOD的保护作用。另外据证实存在增加的裂解的caspase-3，细胞凋亡的产物，在暴露于缺血再灌注损伤的组织。在当PEG-SOD治疗左叶中裂解的caspase-3的下降表明，PEG-SOD导致凋亡减少。 超氧化物歧化酶（SOD）是在活性氧物质的解毒的关键酶。所述酶催化两个超氧阴离子转化成过氧化氢和水。过氧化氢酶则过氧化氢转化为水和氧气，从而完成过程25。的半衰期天然SOD限制了它在实验模型中使用的共轭直到聚乙二醇 - 超氧化物歧化酶（PEG-SOD）的发展。 SOD与聚乙二醇缀合的6分钟增加其半衰期14个小时。 Nguyen 等。展示了其减轻脂质过氧化肝缺血的大鼠模型中的能力，使用全身递送29。 有多种此处详细介绍和一些先前已经在文献中描述的技术的潜在修改。根据不同的模型中使用的分子已被使用吸入11，硬膜外输注12，腹膜内注射17，18，21，22，静脉内给药10，14，15传递<suP&gt;，19，23，24或注射到外围肠系膜上静脉8。 还有在这个协议的几个关键步骤。最重要的是门静脉插管。必须注意的是切静脉孔不会太大。该组织是非常有弹性和孔将扩大自身。我们建议通过切割孔，其为0.5mm与显微剪刀开始。插管可以通过孔使用仪器，其允许更大的灵活性比如果试图手工执行的过程的这个部分被馈送。此外，虽然最初进料套管，应当直接瞄准朝向左侧和右侧门静脉的分叉，以避免穿过静脉的后壁戳一个孔。当所述套管尖端到达分岔，它可以然后被馈入特定左静脉盟友。一旦插管被送入左门静脉，它提供两个左和中间凸角，它的位置可以被手动感觉就能静脉内确认。其位置也可以通过注入冷盐水的少量，看到对肝脏的供给段热烫效果证实。 在大鼠肝门部钳模型提供了证明肝脏缺血再灌注损伤可重复和稳定的平台。可变肺门夹具模型已被用于研究人员研究抗氧化剂和其他小分子6，7，8，9，10，11，12，13，14的保护作用。变化点包括血管是钳位<html>版，其中段是由局部缺血，胆管是否被包括在内和再灌注6，7，8，9，10，11，12，13，14，15，16，17，18的期间的长度， 19，20，21。 此外，当此模型被用于研究分子的给药途径的给药的影响也异构8，10，11，"&gt; 12，14，15，17，18，19，21，22，23，24。有几个优点对所描述的方法。首先，门户提供给局部缺血段的直接插管允许的直接节段性肝输送所研究的药理物质。这使得肝脏作为内部对照的其它叶的利用率。其次，节段性肝插管允许正在研究分布在分子的一个减小的体积，这种方法从而减少了全身性副作用的风险该物质直接喷射到感兴趣的肝段，肝段的直接插管允许物质交付预缺血内，缺血或缺血后。这允许在缺血再灌注损伤周期的任何分子的作用研究。随着缺血时间长度的增加，增加的伤害额外的机会来研究肝再生是可利用的水平。 也有这种做法的一些限制。首先是启动成本。购买手术显微镜的可能是不已经拥有一个实验室一个显著启动成本。这种技术可以是没有显微镜困难或不可能。二是学习曲线时间。虽然这个过程是比较简单，它确实需要一些做法，很可能是一个新手，需要的程序显著数成为一个专家。 总之，这种模式允许可重复的，简单，经济高效的平台来研究肝脏缺血再灌注损伤。尽管在这里描述的协议聚乙烯GLycol超氧化物歧化酶，自由基清除剂25，灌注，此模型可用于注入各种不同的药物物质，以评估其对I / R损伤肝脏的影响。

主要肝手术血流阻断，肝移植，必要一段时间的热缺血，并在一段再灌注导致缺血/再灌注（I / R）损伤1。 I / R损伤的肝脏的后果已被广泛1，2，3中详述。的我后果/ R损伤在文献中详述的包括:生成活性氧，炎症级联的引发，包括嗜中性粒细胞的活化，Kupffer细胞和内皮细胞，血红素加氧酶系统的活化和活化toll样受体，一个内皮素和一氧化氮，核因子κB的活化，和促进促炎细胞因子和粘附分子的合成1，2，3之间的不平衡。这些促炎事件可能升EAD凋亡，坏死，器官功能障碍和最终器官功能衰竭3。 I / R损伤注定肝移植器官可导致早期移植物损失和边际器官损伤3更容易促成当前的供体短缺。目前在2015年5进行了在美国4肝移植等待名单上的15226个潜在受益者，只有5,950肝脏移植手术。由于在器官可用性这种极端限制，研究探索需要以优化移植物的功能和器官利用肝I / R损伤。 用于研究肝I / R损伤动物模型包括大鼠肺门钳模型和大鼠肝移植模型。有很多种目前正在使用大鼠肺门钳模型。最常见的是在其中门静脉，肝动脉和胆管杜克拉供给左横向和中值瓣采用显微夹子6，7，8，9，10，11，12 30至60分钟6，7，10，13，14，然后从60分钟的期间再灌注的夹紧至24小时7，9，10，13，14是允许的。大鼠肝脏的左外侧和中位数裂片包括肝实质9的约70％。旨在研究缺血预处理一些协议包括门血管的间歇性夹紧或后肢之前缺血通过夹紧门血管9,13诱导较长的时间。也有在文献中描述了几种变型。第一种是夹持门静脉和肝动脉供给左横向和中值裂片，但不包括胆管15。第二变形例是通过分割16，17，18，19，20之前，夹持门静脉，肝动脉和胆管诱导全肝缺血。第三修改包括肺门血管30至60分钟8肝右叶夹紧。一个额外的修饰包括夹紧在一个后肢维管束以诱导损伤的肝脏13，21 </suP&gt;。肺门夹紧过程的各种方法在图1A-D中示出。 大鼠肝肺门夹具模型已被用于研究不同的分子和化合物对肝I / R造成的影响。根据不同的模型中使用这些分子使用吸入11，硬膜外输注12，腹膜内注射17，18，21，22，静脉内给药10，14，15，19，23，24或注射已交付到外围肠系膜上静脉8 。 在本报告includ详细大鼠肝门部钳模型ES就可通过门静脉的侧分支门户提供给局部缺血段（ 图2），其允许所研究的药理物质的直接节段性肝输送的直接插管。我们的方法是，以诱导局部缺血的左横向和中值裂片60分钟，在此期间所研究的物质的注入，在这种情况下，聚乙二醇化超氧化物歧化酶，自由基清除剂25，被直接注入缺血段。血液样品在诱导缺血前和在120分钟再灌注后服用。在这一点上，大鼠处死，样品从左侧和中间叶取出。此外，将样品从肝右叶取以用作内部对照。 有许多优点，以这种方式。首先，当所研究的药理物质可以直接注射到缺血段的体积ÒF分布是相比于注射的分布体积进入全身循环或腹膜腔相当低。此外，这种方法减少了，但并没有消除，全身副作用的可能性。

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</table>

method of direct segmental intra-hepatic delivery using a rat liver hilar clamp model

主要肝手术血流阻断，肝移植，必要一段时间的热缺血，并在一段再灌注导致缺血/再灌注（I / R）与无数的负面后果的伤害。在目的地为肝移植边缘器官潜在I / R损伤有助于电流供体短缺继发于降低的器官利用率。一个显著存在需要探讨，以介导其在移植对移植肾功能的影响肝I / R损伤。大鼠肝门部钳模型用于研究不同分子对肝I / R损伤的影响。根据不同的模型，这些分子已使用吸入，硬膜外输注，腹膜内注射，静脉内给药或注射到外围肠系膜上静脉递送。一只老鼠肝门部钳模式已被开发用于研究在改善I / R损伤药物分子的影响。该describED模型大鼠肝门部夹具包括通过门静脉的侧分支门户供给到缺血性肝段的直接插管，从而允许直接节段性肝递送。我们的方法是，以诱导局部缺血的左横向和中值裂片60分钟，在此期间所研究的物质被注入。在这种情况下，聚乙二醇化超氧化物歧化酶（PEG-SOD），自由基清除剂，被直接注入缺血段。这一系列的实验表明，PEG-SOD的输液对肝脏I / R损伤具有保护作用。这种方法的优点包括直接注射该分子与在全身性副作用分布和减少的体积随之减少缺血段。

该实验用2组的n = 3只大鼠各自执行。三个大鼠肝脏注射用2mL生理盐水（NS）与输液泵历时15分钟。三个大鼠肝脏用2mL生理盐水（NS）与聚乙二醇化超氧化物歧化酶（PEG-SOD，0.00067克/毫升）与输液泵在15分钟内混合注入。如在上述方案中所述，取血样前肺门钳，并在120分钟再灌注后。另外，再灌注4肝组织样品的120分钟完成了从左侧和中间叶和四个肝脏样品取出后，从大鼠肝脏的右叶服用。 血清丙氨酸转氨酶（ALT），测定预肺门夹具和在控制120分钟再灌注后（NS）和实验（PEG-SOD）的动物。有控制的ALT水平之间的差异显著（NS）动物预肺门钳，并在120分钟再灌注后。有控制的ALT水平（NS）和实验动物（PEG-SOD）在120分钟（ 图13A）之间的显著差异。组织丙二醛（MDA）是为控制（NS）和实验（PEG-SOD）的动物在肝脏中的右侧和左侧肺叶测量。组织MDA在右叶（非肺门钳）与对照注射（NS）和实验注射（PEG-SOD）表明无显著差异。左叶（后肺门钳和再灌注）组织MDA与对照注射（NS）大于右叶（非肺门钳）P &lt;0.001显著不同。左叶（后肺门钳和再灌注）具有与对照注射（NS）与试验喷射（PEG-SOD）P &lt;0.005（ 图13B）显著不同层次组织MDA的。组织谷胱甘肽（GSH）进行了测定，在右叶（非肺门钳）与对照注射（NS）的组织谷胱甘肽第二实验注射（PEG-SOD）表明无显著差异。左叶（后肺门钳和再灌注）组织GSH与对照注射（NS）大于右叶（非肺门钳）显著不同，与对照注射（NS）P &lt;0.05。左叶（后肺门钳和再灌注）具有显著不同水平与对照注射（NS）与试验喷射（PEG-SOD）P &lt;0.005（ 图13C）的组织谷胱甘肽。进行Western印迹比较控制动物的左，右叶，显示了提高裂解的caspase-3在肺门钳和再灌注（ 图13D）后，左叶。进行第二次免疫印迹比较控制，并与PEG-SOD（ 图13E）处理的动物的左侧瓣。这表明与PEG-SOD治疗动物的肝组织减少裂解的caspase-3。密度还进行demonstra婷认为肝组织裂解的caspase-3的水平在左侧相对于对照动物的（ 图13F）的右叶被显著增加。在比较实验动物的左叶肝组织，用PEG-SOD输注，和对照动物的左叶肝组织，用生理盐水灌注，密度演示显著与PEG-SOD处理的动物相比，动物降低裂解的caspase-3与对照（ 图13G）处理。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/54729/54729fig1.jpg" /> 图1: 解剖插图。大鼠肝脏的解剖A.例证。 B.解剖图的大鼠肝脏。该门蒂到肝脏的左侧和中间叶被夹住。左和中位数叶是缺血性。 C.解剖图示中的大鼠肝的。该门蒂的左叶夹紧。左叶缺血。 D.解剖图的大鼠肝脏。该门蒂以右叶夹住并肝右叶是缺血性。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/54729/54729fig2.jpg" /> 图2: 解剖插图。门静脉的大鼠肝脏的解剖图示通过侧分支静脉插管。门户椎弓根到肝脏的左，中位数裂片是通过缝合线包围和微血管钳已被用于收紧围绕维管束。左和中位数叶是缺血性。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/54729/54729fig3.jpg" /> 图3: 仪器设置。该图表明日<html> Ë仪器设置。 <img alt="图4" class="xfigimg" src="/files/ftp_upload/54729/54729fig4.jpg" /> 图4: 手术室了。该图展示了手术室设置。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54729/54729fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/54729/54729fig5.jpg" /> 图5: 腹毛的剪裁。该图演示了腹毛的剪裁。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54729/54729fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 负载/ 54729 / 54729fig6.jpg" /&gt; 图6: 固定化和皮肤切口。该图显示大鼠和皮肤切口的固定。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54729/54729fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" class="xfigimg" src="/files/ftp_upload/54729/54729fig7.jpg" /> 图7: 肋骨牵安置和去脏。该图演示了肋骨牵放置和取出内脏。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54729/54729fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" class="xfigimg" src="/files/ftp_upload/54729/54729fig8.jpg" /> 图8: 将<html>缝合换货。这个数字表明了缝合的位置。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54729/54729fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" class="xfigimg" src="/files/ftp_upload/54729/54729fig9.jpg" /> 图9: 从下韦纳·卡瓦抽血。该图显示从下腔静脉抽血。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54729/54729fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图10" class="xfigimg" src="/files/ftp_upload/54729/54729fig10.jpg" /> 图10: 静脉分支结扎和收回。该图证明静脉分支结扎和收回。<html> ecsource.jove.com/files/ftp_upload/54729/54729fig10large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。 <img alt="图11" class="xfigimg" src="/files/ftp_upload/54729/54729fig11.jpg" /> 图11: 插管的过程。该图证明插管的过程。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54729/54729fig11large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图12" class="xfigimg" src="/files/ftp_upload/54729/54729fig12.jpg" /> 图12: 插管。这个数字说明了插管。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54729/54729fig12large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 内容" FO:保持-together.within页= "1"&gt; <img alt="图13" class="xfigimg" src="/files/ftp_upload/54729/54729fig13.jpg" /> 图13: 代表性的成果:PEG化的超氧化物歧化酶的直接节段性肝内交付使用大鼠肺门钳模型。 NS =生理盐水。 PEG-SOD =聚乙二醇化超氧化物歧化酶，ALT =谷丙转氨酶，MDA =丙二醛。 A.血清丙氨酸转氨酶（ALT，MU / mL）的比较前肺门钳和120分钟再灌注后之间。有控制（NS）预肺门夹具和控制（NS）在120分钟再灌注后（P &lt;0.001）之间的差异显著。还有，在120分钟再灌注后（P &lt;0.05）对照组（NS）和实验组（PEG-SOD）之间的显著差异。被使用的学生t检验。误差条表示标准偏差。在右叶（非肺门钳）B.组织丙二醛与对照注射（NS）的ð实验注射（PEG-SOD）表明无显著差异。左叶（后肺门钳和再灌注）组织丙二醛与对照注射（NS）大于右叶（非肺门钳）P &lt;0.001显著不同。左叶（后肺门钳和再灌注）具有显著不同水平与对照注射（NS）与试验喷射（PEG-SOD）P &lt;0.005组织丙二醛。被使用的学生t检验。误差条表示标准偏差。 C.在右叶（非肺门钳）组织谷胱甘肽与对照注射（NS）和实验注射（PEG-SOD）表明无显著差异。左叶（后肺门钳和再灌注）组织谷胱甘肽与对照注射（NS）大于右叶（非肺门钳）显著不同，与对照注射（NS）P &lt;0.05。左叶（后肺门钳和再灌注）具有与对照注射液显著不同级别的组织谷胱甘肽的N（NS）与试验喷射（PEG-SOD）P &lt;0.005。被使用的学生t检验。误差条表示标准偏差。控制动物的左叶（后肺门钳和再灌注）与右叶（非肺门钳）（生理盐水）的肝组织D.印迹表明增加裂解的caspase-3。 E. Western印迹示范用PEG-SOD在比较处理，以用对照（生理盐水）处理的动物的动物的肝组织中降低裂解的caspase-3。 F.肝组织裂解的caspase-3的水平在后肺门钳和再灌注动物（P &lt;0.05）被显著增加。被使用的学生t检验。误差条表示标准偏差。 G.在比较实验动物的左叶肝组织（与PEG-SOD输注）和对照动物的左叶肝组织（用生理盐水灌注），有显著在降低裂解的caspase-3用PEG-SOD在比较处理，以用对照（生理盐水）处理的动物imals。被使用的学生t检验。误差条表示标准偏差。

Watch this Scientific Journal Video about Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model at JoVE.com

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

独特的大鼠肝门部钳模型，为研究药物分子在改善缺血再灌注损伤的影响发展。该模型包括通过门静脉的一个分支门供应到缺血性肝段直接插管，从而允许直接肝递送。

直接节段性肝内交付使用大鼠肝门部夹具模型方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

method-direct-segmental-intra-hepatic-delivery-using-rat-liver-hilar

Nanyang Technological University

Research

JoVE Journal

Medicine

14.0K 次观看.  The Ohio State University Wexner Medical Center. 独特的大鼠肝门部钳模型，为研究药物分子在改善缺血再灌注损伤的影响发展。该模型包括通过门静脉的一个分支门供应到缺血性肝段直接插管，从而允许直接肝递送。 

视频: Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

在自觉大鼠高胰岛素 - 正常血糖钳

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

科研

医学

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

部分原位肝移植大鼠模型的肝动脉重建的外科手术

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型:教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

该二甲基亚硝胺诱发肝纤维化模型大鼠

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

生物血管内送货到大鼠肾脏

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

应用新型磁吻合技术 Suprahepatic 腔静脉重建大鼠原位肝移植模型的研究

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

通过结合70%部分切除术和对乙酰氨基酚，生成急性肝衰竭大鼠模型

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

大鼠胡萝卜动脉压力控制段气球损伤与腹腔ventit 治疗应用

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

矫形肝移植大鼠模型中动脉重联后并发症减少

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...