Name: bile duct ligation in mice: induction of inflammatory liver injury and fibrosis by obstructive cholestasis
Uploaded: 2015-02-10T15:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis at JoVE.com

笔者要感谢德国研究基金会（SFB / TRR57，Q3和Q2）的资金支持。作者感谢Mareike舒尔茨，帕斯卡Paschenda和Klaudia Warzecha为他们准备的照片帮助。

注:所有实验均经国家官方动物护理和使用委员会（LANUV，雷克林豪森，德国）。老鼠是根据联邦实验动物科学协会（FELASA）的指引，安置特定病原体的条件下。所有实验均符合有关动物保护和"指南实验动物的护理和使用"（卫生出版第 8版全国学院，2011年），德国联邦法律进行。 1.术前准备注:执行干净的，但在非无菌条件下的所有程序。这是用来进行手术的所有仪器等可重复使用的设备，如手术钳，剪刀和科利柏拉钩必须根据是在严格遵守与动物进行手术的机构准则的协议，使用前消毒。;对于必需的试剂，材料和设备的详细清单，请参考具体的材料/设备的列表。 <ol><li>在完成实验，整体保持在加温板的动物在温度为37℃，永久地连接到一个麻醉系统，并且盖的操作区域与不透液性，自粘窗帘。妥善安排所有的仪器及那些在实验过程中，手术前使用的解决方案（ 图1）。 </li><li>麻醉小鼠吸入在100％的氧气4体积％异氟烷以4升/分钟的麻醉诱导的流速。麻醉的深度足以以下重要标准达到时:定期自主呼吸，疼痛刺激脚趾间的设置后无反射，并且疼痛没有反应。 </li><li>刮胡子鼠标的腹部的皮毛用电动剃须刀的皮草，保护眼睛免受干燥了的眼睛和正使用OSE软膏。 </li><li>放置在鼠标上的37℃下加热的热板，插入鼠标吻在Fluovac麻醉系统的Fluovac掩模，并固定动物的腿部用丝绸带的条纹。 </li><li>维持小鼠通过吸入在100％的氧气1.5-3体积％异氟烷的麻醉以1升/分钟的流速，并通过腹腔注射丁丙诺啡溶液（0.1毫克/公斤体重溶解在0.9％NaCl溶液）导入围手术期的镇痛。 </li><li>消毒剃腹部皮肤与湿润与标准的防腐剂，准备用酒精溶液手术前的治疗皮肤的纱布拭。注意:在我们的协议，我们使用一种多元醇皮肤antisepticum。这是完全按照有关动物保护和联邦的实验动物科学协会的指导原则德国联邦法律。这种防腐剂含有70％（V / V）2-丙醇，丁1,3.diol和喹啉的痕迹黄色和perfum即</li></ol> 2.外科手术<ol><li>通过切割表皮加上筋膜同时用11.5厘米外科剪刀打开用的约2厘米的长度的中线剖腹腹部。 </li><li>通过使用剪刀作为吊具解剖上腹膜顶部的结缔组织。 </li><li>切开腹膜沿白线，打开腹腔。 </li><li>通过在胸骨插入夹持缝合，提高缝线的丝，并在Fluovac掩模的顶部将其固定放大空腔。 </li><li>通过插入一个科利柏牵开器在腹膜腔（ 图2）传播的操作区域。 </li><li>解除用湿润（0.9％NaCl溶液）棉签肝脏，使得它的腹部侧粘到隔膜和脐是清晰可见的。 </li><li>露出胆管由肠道（ 图3）的尾部运动。 </li><li>仔细分离从胆管侧翼门静脉和肝动脉用微型细齿钳（图4A）。 </li><li>周围放置胆管5-0缝合并用两个手术结固定。当捆扎结连续增加的牵引力，以确保有效梗阻无需切断胆管（ 图4B）。 </li><li>添加第二颅结扎以相同的方式，但不解剖中（ 图4C）之间的胆管。否则，存在着相当大的风险，胆漏，如果一个结是不安全的，而动物没有经验，但胆汁淤积形成严重的腹膜炎。 </li><li>切开缝合（ 图4D）的两端，降低胸骨，然后取出牵引器。 </li><li>冲洗腹腔用0.9％NaCl溶液和替换腹部器官的生理位置。 </li><li>关闭两个腹部层（腹膜和表皮加上仪表板）具有独立运行的缝线用6-0 Mersilk。 </li><li>切电子商务缝线的NDS和消毒用纱布蘸拭用消毒液的操作区域。 注:当第一次执行的手术，在16X-20X的放大倍数进行手术显微镜下的所有程序。这允许更好的识别胆管和清楚地从门静脉和肝动脉（ 图5和6）的区别所在。一些实验室建议在这两个之间结扎胆管的解剖。离开胆管完好因为在结中的一个潜在的泄漏会导致严重的急性腹膜炎，腹水，和全身内毒素血症时胆管解剖。 </li></ol> 3，术后治疗和随访<ol><li>让鼠标在热身通过红外线灯笼子里恢复，直到鼠标完全清醒和活跃。 </li><li>随后，将鼠标移动到一个正常的笼子，并提供自由采食获得水和食物。 </li><li>一压脚提升的手术，监测动物以规则的时间间隔，并进行后续的术后治疗与合适的镇痛（ 例如 ，丁丙诺啡溶液）后的内部动物护理的本地建议和使用委员会。 注:3天进行镇痛治疗。任何异常行为可能表明罕见的并发症，如腹膜炎，败血症或内脏出血，应处理为人类的端点终止实验。 </li><li>动物保持有自由获取食物和水随意直至实验结束。没有必要对采血因为正在进行的纤维形成用黄疸表示。 </li><li>当动物被处死，收集血液用于临床化学参数（AST，ALT，胆红素等 ）和肝脏中检索用于组织化学和生物化学分析的测定。 </li></ol>

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The authors have nothing to disclose.

胆汁淤积性肝损伤是主要的致病因素为肝纤维化和肝硬化的慢性肝病的发展中的一个。基于这样的事实，这些疾病产生无法估计的医疗保健费用，这是可以理解的，许多研究人员试图了解正在进行肝纤维化的发病机制。因此，实验模型已生成的模拟，导致肝脏炎症，纤维化和肝硬化1的复杂机制的各个方面。 外科BDL]是用于诱导小鼠和大鼠4,23,24阻塞性胆汁淤积损伤最普遍的实验模型中的一个。在大多数的协议，将动物麻醉并一个中间段剖腹被执行。接着，胆管，从腹腔发现并使用外科麻线结扎两次。其结果是，在小鼠和大鼠接受本手术ðevelop强烈的纤维化反应，在首先从汇管区25场起源。年期间几个不同的外科技术和修改已被描述。特别程序甚至允许重新连接或再吻合术BDL 23后。其他技术是基于局部BDL导致显著少坏死形成并因此肝细胞增殖24。部分BDL在随后切除胆囊（胆囊切除术）相结合，防止胆囊炎的形成也代表了一个极好的实验模型急性胆汁淤积。有人提议是一个模型，更接近人类的情况24。事实上，建立这种模式的过程中，它已经表明，它重复导致只有很少的病理组织损伤胆汁淤积，不进行慢性胆汁淤积25。因此，有人提出，这种模式是理想的学习纬度逆转胆汁淤积24电子商务的影响。甚至更复杂的方法是基于显微并允许快速且可重复的方式，以仅在肝中26的选定部分造成胆汁性肝损伤。 虽然从最初的BDL协议这些复杂的改型已被证明在调查的特定研究问题非常有益的，许多实验室全世界基本上瞄准采用BDL模型作为胆汁淤积性纤维化重复性，可靠性高的模型。然而，许多并发症可以发生，可以显着地改变了由该模型得到的，如果技术错误都不能避免的结果的可重复性和可靠性。例如，出血有关的伴随胆管血管损伤的并发症（ 见图3 - 4）可以期间或手术后迅速发生。随后心脏抑制或r过量麻醉espiratory失败也是程序可以避免的并发症。严重的感染，从腹膜炎败血症，在实验的整个期间，可能会发生，如果缝线不正确地进行和胆汁泄漏到腹腔。意外伤害的肠道手术过程中也可能会导致腹膜炎。因此，很明显，这是符合严格操作准则标准化协议强烈要求。这一规定也于近日欧盟，其在2013年1实施新的动物福利规则的国家内的要求。与此相关的规定各自的要求是不是新的，并已经提出在1959年，当罗素和伯奇认为道德框架进行的，主要是基于更换，细化和还原（3R）原则上27动物科学实验。 继outlin当ED的协议有可能产生的技术错误只有少数并发症。可能发生的，而低频三个具体问题。 如同所有的外科手术，麻醉剂的过量是危险的动物特别是在与低​​温组合的潜在来源。如果在手术过程中的心血管并发症发生，麻醉剂的供给，应立即停止，并且操作者应尽量提供尽可能氧气鼠标。这可以通过被填充有空气和泵入受损动物的嘴的小塑料注射器的手段来完成。可替代地，使用小Peleusball为受影响动物的通风是振兴过程中经常有帮助的。 在伤口愈合的问题在BDL手术后，老鼠会咬自己的痛处接缝或其他动物。如果出现这种情况，各小鼠应分别关不住的。动物与开放性伤口应麻醉，伤口周围的区域轻轻消毒用标准的防腐剂，而应伤口被缝了起来。在接下来的3天里，这些动物的伤口应定期检查（一天两到三倍）。 腹部或腹水形成的次膨胀是指示为细菌感染。这些可能发生由于在手术过程中未消毒的工作。所有类型的感染应无例外处理的人性化的终点，应该被牺牲的动物受到影响。 我们提供了一个易于遵循协议，允许BDL的小鼠的表现是实现简单并且唤起只有低动物死亡率结合具有高再现性。所有的手术方案可以通过熟练的科学家迅速抓获。在完成实验，将动物保持在一个加温板，在3​​7℃和永久地连接到麻醉系统，减少疼痛和痛苦。对于手术，腹部被打开了中线剖腹手术和胆管双重结扎不解剖它。这里讨论的代表性结果证明，表型改变对于肝脏形态（炎症，纤维化，肝硬化）是高度可再现的，并允许研究纤维化的不同方面（ 例如 ，起始，炎症，进展，终末期疾病）在定义的时间点。 我们希望，我们的协议的总结将有助于缩短学习曲线是必要的成功建立在其他实验室这一纤维化模型，并保证在不同地点可靠和可重复的结果。因此我们认为，所提出的协议支持的3R原则，即在1959年应该由拉塞尔和Burch和代表的是在许多国家无线当前实施的新的动物福利规则的基础薄的欧洲框架。

肝纤维化被定义为过量产生和细胞外基质（ECM）的积累源自基质产生肝星状细胞的相互作用的复杂网络和各种肝脏驻留和浸润细胞的血细胞1,2。虽然肝纤维化可通过多种不同的刺激而引起底层纤维化的分子机制一般都非常相似。下面的肝损害，分子和细胞的变化高度精心策划的程序启动。在这个程序中炎症信号，单核/巨噬细胞和肝星状细胞之间的密切互动发生，在最终结果中的星状细胞的活化和转分化为肌成纤维细胞，细胞外基质沉积和连续的解剖和功能改变的肝组织的完整性3。肝星状细胞的活化是特别由炎症信号和相互作用与肝从动巨噬细胞的住宅（ 即枯否细胞）。病原体相关分子模式是由专门的模式识别受体识别，如Toll样受体，当通过触发大量的炎性细胞因子和趋化因子驱动的炎性过程3中的表达和分泌的不同途径的复杂网络激活信号。当疾病产生因素被消除的炎症反应，所形成的肝损伤只是暂时的。与此相反，如果损伤仍然存在，肝脏和在导致正常肝实质由瘢痕组织形成渐进取代前景中的表达和ECM观众的堆积内的慢性炎症的发展。 由于在人类肝纤维化是一个世界性的临床问题，已在过去几十年建立的急性和慢性肝衰竭几种实验啮齿动物模型。在万亩海相系统，例如，共同模式是多种不同的肝毒素的施用，胆总管结扎，诱导免疫介导的肝损伤，并且有针对性地引入基因缺陷或转基因的，反之亦然的过度影响临界信令涉及肝纤维化4的发病机制的途径。 在啮齿类动物的胆总管结扎已经进行了作为实验过程中研究了很多年5-8。现在已经提出了超过三十年前9第一高度重复性的协议延长胆管结扎（BDL）在啮齿动物。在该协议中双方插管/梗阻和结扎诱导高产肝硬化大鼠与具有可比性的那些人胆汁性肝硬化9观察形态变化。各自的协议是简单，在外科手术过程是相当快地适用和动物的存活率是高的超过95％。在经典大鼠插管/梗阻协议，2厘米的短切口，略低于剑突。此后，套管被插入到胆管的近端部和固定在其与丝缝线的位置。在下一步骤中，套管的远侧部分被遮挡用3节，通过中线切口的下端在右下腹9把埋皮下。底，腹部被关闭，动物被允许恢复。在结扎协议，将大鼠经受胆总管双重结扎具有或不具有所述连字9之间的胆管解剖。 该实验模型被广泛接受，并广泛应用于全球数百个实验室诱导肝胆汁淤积和纤维化。它诱导的肝内胆管上皮细胞的增殖，肌纤维母细胞differentiatioÑ ​​门户成纤维细胞的增殖周围胆管上皮细胞，导致细胞外基质10,11的高度重现，大规模表达和沉积。因此，这种模式在大鼠和小鼠中的应用正在被科学家，旨在了解肝脏炎症和纤维化的发病机理中流行。 在我们的实验室，我们已经广泛使用该协议在过去的大鼠在几个实验研究，旨在探讨肝纤维化的特殊的分子和细胞方面，并测试新型抗肝纤维化概念和药物12-15。 最近，我们采用了这种方法，以鼠系统，发现胆管结扎手术也是一个有吸引力的平均成立时间依赖性纤维化低的变化和死亡率在小鼠16-18。由于动物的更小的尺寸，然而，对于麻醉的一些重要的修改，外科一位软件ention和后处理的观察是必要的，以获得可靠的和可再现的结果在该模型中。完全自适应概括在以下方案和所附的视频文档。

<table><tbody><tr><td>Isoflurane</td><td>Forene Abbott</td><td>B 506</td><td></td></tr><tr><td>Shaver Favorita II</td><td>Aeskulap</td><td>GT104</td><td></td></tr><tr><td>Cutter head</td><td>Aeskulap</td><td>GT730</td><td></td></tr><tr><td>Bepanthen eye and nose ointment</td><td>Bayer Vital GmbH</td><td>6029009.00.00</td><td></td></tr><tr><td>Warming plate and controller</td><td>Labotect</td><td>HP 062</td><td></td></tr><tr><td>Fluovac anesthesia system</td><td>Harvard Apparatus</td><td>34-1030</td><td></td></tr><tr><td>ISOFLO (Isoflurane Vapor) vaporiser</td><td>Eickemeyer</td><td>4802885</td><td></td></tr><tr><td>Scotch Tape</td><td>commercially available</td><td></td><td></td></tr><tr><td>Tissue paper</td><td>commercially available</td><td></td><td></td></tr><tr><td>Durapore silk tape</td><td>3M</td><td>1538-1</td><td></td></tr><tr><td>Cotton Gauze swabs</td><td>Fuhrmann GmbH</td><td>32014</td><td></td></tr><tr><td>Poly-Alcohol Haut&amp;hellip;farblos Antisepticum</td><td>Antiseptica GmbH</td><td>72PAH200</td><td></td></tr><tr><td>Raucodrape OR adhesive drapes</td><td>Lohmann &amp;amp; Rauscher GmbH</td><td>33013</td><td></td></tr><tr><td>Scissor</td><td>Fine Science Tools Inc.</td><td>14074-11</td><td></td></tr><tr><td>Graefe forceps straight</td><td>Fine Science Tools Inc.</td><td>11050-10</td><td></td></tr><tr><td>6-0 Mersilk suture</td><td>Ethicon</td><td>K889H</td><td>Silk, non-absorbable/Abdominal closure</td></tr><tr><td>Needle holder Mathieu</td><td>Fine Science Tools Inc.</td><td>12010-14</td><td></td></tr><tr><td>Colibri retractor</td><td>Fine Science Tools Inc.</td><td>17000-03</td><td></td></tr><tr><td>Cotton swabs</td><td>Noba Verbandmittel</td><td>974202</td><td></td></tr><tr><td>Cotton swabs</td><td>Heinz Herenz Medizinalbedarf GmbH</td><td>1032238</td><td></td></tr><tr><td>25mL beaker</td><td>Schott Duran</td><td>50-1150</td><td></td></tr><tr><td>Isotonic (0.9%) NaCl solution</td><td>DeltaSelect GmbH</td><td>PZN 00765145</td><td></td></tr><tr><td>Micro-serrations forceps Moria MC31</td><td>Fine Science Tools Inc.</td><td>11370-31</td><td>Bile duct separation</td></tr><tr><td>5-0 Mersilene suture</td><td>Ethicon</td><td>EH6731H</td><td>Polyester, non-absorbable/Bile duct ligation</td></tr><tr><td>5mL syringe</td><td>BD Discardit II</td><td>300296</td><td></td></tr><tr><td>1mL syringe</td><td>BD Plastipak</td><td>300013</td><td></td></tr><tr><td>Sterican needle 26 G x 1</td><td>B. Braun</td><td>4657683</td><td></td></tr><tr><td>Buprenorphine</td><td>Essex Pharma</td><td>997.00.00</td><td>Analgeticum, 0.1 mg/kg</td></tr><tr><td>Infrared lamp</td><td>Petra Electric</td><td>IR 11</td><td></td></tr></tbody></table>

bile duct ligation in mice: induction of inflammatory liver injury and fibrosis by obstructive cholestasis

在大多数脊椎动物中，肝脏产生胆汁所必需的乳化吸收的脂肪和使脂质在小肠中的消化以及排泄胆红素和其他代谢产物。在肝脏中，实验梗阻肝外胆道系统的启动病态事件的复杂级联，导致胆汁淤积和炎症导致强烈的纤维化反应从门静脉周围字段始发。因此，胆总管结扎手术已成为最常用的模型来诱导啮齿类动物阻塞性胆汁淤积损伤，并研究背后诱导不适当胆汁流量这些病理生理机制的分子和细胞事件。近年来，不同的外科技术已经描述，要么允许重新连接或再吻合胆管结扎（BDL）之后， 例如 ，局部的BDL，或其它显微方法具体的研究问题。然而，最常用的模型是胆总管诱导后21至28天强烈的纤维化反应的完全阻塞。死亡率会很高因感染并发症或技术错误。在这里，我们提供了详细的手术过程为BDL模型小鼠诱导按照到3R规则动物福利由罗素和伯奇在1959年假设一个高度重复性的纤维化反应。

在一个典型的实验BDL在40雄性C57BL / 6野生型小鼠加权约18-20克执行。这个实验是为了研究肝纤维化，在起始阶段（3和7天），进展（10，14，和20天）期间和长期（30和60天）16中。在这种模式下，persinusoidal纤维化已经在手术后10天的发展，而门静脉周围纤维化是永久增大至实验结束后20天被充分开发。在所提到的实验中，所有接收到的一个简单的假手术的动物存活，而只有两个40只小鼠（5％），该接收到的BDL的开发了一般状况较差，并因此在实验计划结束点之前过早处死。假手术动物的活性是无一例外已经正常1天剖腹后，在最初三天而大部分经受BDL动物表现出降低的活性。 Ĵaundiced皮肤有BDL 16的设定后，已经已经很明显在所有BDL动物一两天。 两个谷丙转氨酶（ALT）和天冬氨酸转氨酶（AST），代表肝损伤的行之有效的血清标志物的值迅速增加和BDL（见表1）后，在第​​7天和第20天达到高峰。此后，ALT和AST持续下降，直到第30天，并保持稳定，直到手术后60天。与胆汁淤积损伤线，总胆红素血清浓度升高逐渐达到7天后16高原。对ALT和AST血清的活动类似的时间当然也报道了老鼠进行BDL。在最近的研究中它被证明血清AST和ALT水平升高达5或10倍的正常的第一周BDL手术后两周19后下降。 通常情况下，假手术的动物的肝脏仍然期待斯穆特H在实验结束时，而接收BDL动物的肝脏中显示的架构改变其主要特征在于水肿和纤维化结节的形成对应的肝和胆囊的填充有大量积水的表面上胆（ 图7）。特性形态学改变肝脏了由BDL手术诱导也可证明在标准的组织学分析（ 图8）。 在同一组实验中，肝纤维化的发展是基于肝脏组织学通过使用评分系统，其中门静脉周围纤维化是从0-4和窦周纤维化上演0-2盲病理学家评估半定量评估，得到最大值，该值相当于6.肝硬化正如预期的，平均纤维化评分的组假手术的动物中为0.00±0.00。与此相反，该组的动物吨帽子收到BDL，比分稳步上升，直到60天至4.83±0.17的数值。的3对门脉周围纤维化的最大值在20天达到在分析的所有动物中，窦周纤维化是在第10天的实验的缺席，是先显后两周。此后，它逐渐增大至实验结束，以1.8±0.17（ 表1）的值。 另外，在已执行的其他几个独立的动物实验中，观察到纤维化的形成是肝内胶原表达和沉积的高度重现放映时间依赖性增加正在进行纤维化（ 图9）的结果。同样地，正在进行纤维化的过程是显着的升高的表达α平滑肌肌动蛋白（α-SMA），表示成纤维细胞的标记， 即活化的肝星状细胞和成肌纤维细胞的门户，和肝脏中羟脯氨酸的氨基酸在胶原丰富地发现矩阵18（ 图9）。此外，波形蛋白是表示增加肌纤维母细胞或成纤维细胞的数量表达的BDL手术20的设定后增加。炎症在受伤的肝脏通过相伴2脂质运载蛋白（LCN2）是急性和慢性肝损伤强烈诱导和演进过程中的急性肝损伤21,22保肝作用的表达增加，进一步体现。 可以通过特异性染色的抗体，其特异于CD45（ 图10）检测到的炎性细胞浸润所接收BDL动物的肝脏。这种细胞表面标记物也被称为PTPRC（蛋白质酪氨酸磷酸酶，受体型）是在所有的分化的造血细胞，除了红细胞和血浆细胞中特异性表达。 <table fo:keep-together.within页="总是"边界="0"的cellpadding ="0"CELLSPACING ="0"&gt; <tbody><tr><td rowspan="2"> 胆管结扎后时间（天） </td><td> 总胆红素 </td><td rowspan="2"> AST（U / L） </td><td rowspan="2"> ALT（U / L） </td><td rowspan="2"> 门户纤维化 </td><td rowspan="2"> 窦周纤维化 </td><td rowspan="2"> 总比分 </td></tr><tr><td> （毫克/ dL）的 </td></tr><tr><td> 0（N = 3） </td><td> 0.17±0.06 </td><td> 192.67±30.50 </td><td> 50.33±6.03 </td><td> 0.0±0.0 </td><td> 0.0±0.0 </td><td> 0.0±0.0 </td></tr><tr><td> 3（N = 5） </td><td> 6.85±2.21 </td><td> 1159.25±319.27 </td><td> 566.50±335.25 </td><td> 0.0±0.0 </td><td> 0.0±0.0 </td><td> 0.0±0.0 </td></tr><tr><td> 7（N = 5） </td> &lt;TD&gt; 14.38±2.14± </td><td> 976.60±477.16 </td><td> 448.20±259.47 </td><td> 0.60±0.25 </td><td> 0.0±0.0 </td><td> 0.60±0.25 </td></tr><tr><td> 10（N = 5） </td><td> 15.92±2.60 </td><td> 1916.60±868.25 </td><td> 560.40±80.88 </td><td> 1.40±0.25 </td><td> 0.25±0.25 </td><td> 1.67±0.25 </td></tr><tr><td> 14（N = 5） </td><td> 17.90±3.84 </td><td> 1088.60±276.32 </td><td> 505.00±96.15± </td><td> 2.4±0.25 </td><td> 1.0±0.0 </td><td> 3.40±0.24 </td></tr><tr><td> 20（N = 4） </td><td> 18.00±2.12 </td><td> 1072.67±364.27 </td><td> 404.00±195.48 </td><td> 3.0±0.0 </td><td> 1.0±0.0 </td><td> 4.0±0.0 </td></tr><tr><td> 30（N = 5） </td><td> 16.04±4.79 </td><td> 446.40±169.75 </td><td> 260.20±126.97 </td><td> 2.8±; 0.2 </td><td> 1.4±0.25 </td><td> 4.20±0.20 </td></tr><tr><td> 60（6） </td><td> 16.02±1.19 </td><td> 484.67±117.79 </td><td> 257.17±50.97 </td><td> 3.0±0.0 </td><td> 1.8±0.17 </td><td> 4.83±0.17 </td></tr></tbody></table>使用缩写是:ALT，谷丙转氨酶; AST，天冬氨酸转氨酶。 表1:纤维化得分在一个代表性实验该表的数据是从在其中肝纤维化是在C57BL / 6小鼠16诱导胆管结扎研究再现。在这项研究中，死亡率的BDL手术后为5％（2 40只动物处死过早因为差的动物条件开发）。 <img alt="图1" src="/files/ftp_upload/52438/52438fig1highres.jpg" /> 图1:实验装置用于执行胆管结扎的动物保持在加热板上，在37℃的温度和操作区域覆盖整体与不透液性，自粘窗帘。在完成手术后，动物被永久地连接到一个麻醉系统。所有仪器及解决方案（镇痛药，麻醉剂，消毒液，0.9％氯化钠）排列清晰。 <img alt="图2" src="/files/ftp_upload/52438/52438fig2highres.jpg" /> 图2:手术区的制备腹腔开放前，腹部皮肤，应剃光用电动剃须刀的皮毛和消毒与杀菌纱布拭。该手术区域，再覆盖不透液性，自粘窗帘。腹部被打开了中线剖腹手术（〜2厘米长）。该空腔是通过将在胸骨的保持缝线和操作区域散布的放大插入科利柏牵引器允许在手术过程中不受阻碍地实验。 <img alt="图3" src="/files/ftp_upload/52438/52438fig3highres.jpg" /> 图3:曝光胆管。 （A）为进行胆管结扎，肝脏的腹侧被抬起，以便它可以粘到隔膜和肝门变得清晰可见。（B）中为了更好地暴露胆管，食道被尾侧移动了与一个加湿棉签。胆管是用箭头标记。 <img alt="图4" src="/files/ftp_upload/52438/52438fig4highres.jpg" /> 图4:结扎胆管。 （A）在第一步骤中，胆管小心地从使用微锯齿镊子侧翼门静脉和肝动脉分开。（B）接着，缝合线周围放置胆管和固定用外科结。（℃）之后，将第二缝合线放置在靠近第一缝合线和周围的胆管癌打结。（D）中的缝合线被缩短，腔冲洗用0.9％NaCl溶液和所有器官来代替他们的生理位置。 <img alt="图5" src="/files/ftp_upload/52438/52438fig5highres.jpg" /> 图5:胆管与打结的精确表示要记录的放置缝合线和两个结防止胆汁流的设定，在图4中所概述的相同的程序下双目是记录（A）的约束第一缝合线。在第一缝线的（B）中打结。胆管与第二缝合的（C）的约束。（D）的所述第二缝线打结。（E） -双结扎胆汁ðUCT后缩短多余缝线。（E&#39;），此面板描绘了（E）的草图。肝脏的右侧（RL），左（LL）和中值（毫升）裂片以及胆管，胃，十二指肠和所述位置字母。在草图胆管由两个双缝线结扎。 <img alt="图6" fo:content-width="4in" src="/files/ftp_upload/52438/52438fig6highres.jpg" width="400" /> 图6:胆管，门静脉和肝动脉小鼠解剖对于胆总管更好解剖位置，门静脉和肝动脉被描述为一个方案。标示也右侧（RL）的位置，左（LL）中位数（毫升）和肝脏的尾状（CL）瓣。 <img alt="图7" src="/files/ftp_upload/52438/52438fig7highres.jpg" /> 图7:代表appearaNCE肝脏术后2周假手术组和BDL的。的C57BL / 6小鼠进行假手术或BDL手术。两周后的内脏腔被打开了。而假手术动物的肝脏显示纤维化的迹象，动物接受胆管结扎的肝脏对应肝脏的表面上有一个不规则的表面结构，形成水肿和纤维化结节。 <img alt="图8" src="/files/ftp_upload/52438/52438fig8highres.jpg" /> 图8:HE染色染色肝切片从C57BL / 6野生型动物，是假手术（A）或接收BDL三周（B）制备的。将切片用苏木精和曙红下列标准程序。请注意，BDL肝脏内的典型的改变，包括炎症的迹象（浸润细胞），实质（肝CYTE）坏死，胆管增生。在每幅图板比例尺条为50微米。 <a href="https://www.jove.com/files/ftp_upload/52438/52438fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/52438/52438fig9highres.jpg" /> 图9:组织学和生物化学读数为肝纤维化。 （A）的肝脏切片从该接收的假手术或BDL 2周的动物制备并用天狼星红（ 上面板，在红色的胶原纤维 ），或为α平滑肌肌动蛋白（α-SMA）的表达（ 下图进行分析，α-SMA棕色阳性细胞 ）免疫组化法。在各图面板的比例尺条为100μm左右。（B）中 ，从肝脏的蛋白质提取物进行Western印迹和expre分析裂变I型胶原，α-SMA，波形蛋白和被公认的肝纤维化的标志物。脂笼蛋白-2（LCN2）的表达指示与正在进行的纤维形成相关的炎症反应。在这种分析中，平等的蛋白质负荷证明了探测印迹特定的β肌动蛋白的抗体。 <a href="https://www.jove.com/files/ftp_upload/52438/52438fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图10" src="/files/ftp_upload/52438/52438fig10highres.jpg" /> 图10:浸润炎症细胞的免疫学染色串行肝脏切片从该接收到的胆管结扎3周的动物的肝脏制备。将切片用苏木精和曙红（A）或与一种抗体，其特异于CD45（B）中 。请注意，高数周围胆管的CD45阳性细胞。 （未示出），这些大规模浸润，表明炎症不在从假手术动物的肝脏切片可见。在每幅图板比例尺条为500微米。 <a href="https://www.jove.com/files/ftp_upload/52438/52438fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis at JoVE.com

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

胆管结扎小鼠:感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

bile-duct-ligation-mice-induction-inflammatory-liver-injury-fibrosis

Research

JoVE Journal

Medicine

52.0K 次观看.  RWTH Aachen University. Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

视频: Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

Creation of Reversible Cholestatic Rat Model

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and affect the nutritional status and surgical outcomes. Study of the pathophysiological changes occurring in the liver and other organs is of importance. Various studies have been done in cholestatic rat models.
We used a reversible cholestatic rat model in our recent study looking at the role of methylprednisolone in the ischemia reperfusion injury. Various techniques for creation of a reversible cholestatic model have been described. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.
This model can be used in various studies, such as looking at the changes in nutritional, physiological, pathological, histological and immunological changes in the gastrointestinal tract. This model can also be used to see the effects of cholestasis and various therapeutic interventions on major hepatic surgeries.

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.

可逆的胆汁淤积大鼠模型的建立

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and ...

科研

医学

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

确定小鼠肝脏中的胆汁密度

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

发育生物学

The Influence of Liver Resection on Intrahepatic Tumor Growth

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not detectable by routine clinical imaging, may be stimulated by hepatic regeneration factors after liver resection. It is not entirely clear, however, which factors are crucial for tumor recurrence.
The presented mouse model may be useful to explore the mechanisms that play a role in the development of recurrent malignant lesions after liver resection. The model combines the easy-to-perform and reproducible techniques of defined amounts of liver tissue removal and tumor induction (by injection) in mice. The animals were treated with either a single laparotomy, a 30% liver resection, or a 70% liver resection. All animals subsequently received a tumor cell injection into the remaining liver tissue. After two weeks of observation, the livers and tumors were evaluated for size and weight and examined by immunohistochemistry.
After a 70% liver resection, the tumor volume and weight were significantly increased compared to a laparotomy alone (p &#60;0.05). In addition, immunohistochemistry (Ki67) showed an increased tumor proliferation rate in the resection group (p &#60;0.05).
These findings demonstrate the influence of hepatic regeneration mechanisms on intrahepatic tumor growth. Combined with methods like histological workup or RNA analysis, the described mouse model could serve as foundation for a close examination of different factors involved in tumor growth and metastatic disease recurrence within the liver. A considerable number of variables like the length of postoperative observation, the cell line used for injection or the timing of injection and liver resection offer multiple angles when exploring a specific question in the context of post-hepatectomy metastases. The limitations of this procedure are the authorization to perform the procedure on animals, access to an appropriate animal testing facility and acquisition of certain equipment.

A high incidence of tumor recurrence after resection of liver metastases remains an unsolved problem. The illustrated mouse model may be useful to investigate the reasons for such recurrences. It combines a liver resection model with intrahepatic tumor cell injection for the first time.

肝切除肝内肿瘤生长的影响

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

银纳米粒子法改善小鼠胆道闭锁综合征

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

免疫与感染

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

回肠致胆汁积滞在小鼠肠道中

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

慢性肝纤维化小鼠模型用于研究胆道闭锁

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

通过胆管 TNBS 输液治疗慢性胰腺炎的鼠标模型

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

生物学

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

小鼠部分胆管结扎术: 局部梗阻性胆汁淤积症的对照模型

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...