The authors acknowledge the funding support from the Ministry of Education, Singapore, grant number MOE2010-IF-1-025.

所有的动物实验是由应用科学学院，淡马锡理工学院的动物护理和使用委员会批准。 1. DMN的制备<ol><li>吸取200μl的DMN（1微克/毫升原液），并添加19.8毫升PBS中以制备用于注射10毫克/毫升DMN溶液。 注:DMN是致癌物质。使用通风橱准备DMN和执行动物注射。 </li></ol> 2. DMN腹腔注射<ol><li>使用雄性Wistar大鼠，平均体重为150 - 100克在实验开始前7天 - 适应大鼠4。 </li><li>在22±1℃的室温下保持大鼠，12小时光照和12小时黑暗的周期。提供大鼠食物和水随意 。 </li><li>每天测量食物和水的摄入量。测量人体体重每周。 </li><li>计算DMN的量为基于剂量为10毫克/公斤体重各大鼠。用1ml注射器用25号针头吨Ø绘制DMN溶液放入注射器中计算的体积。 </li><li>为抑制腹腔注射21大鼠。 </li><li>与大鼠适当抑制，引入针到腹部的右下象限，抽回注射器（没有应该吸气）的柱塞腹部空间内检查针的正确位置，并缓慢地注入DMN溶液。 </li><li>当DMN的剂量已被施用，慢慢抽出针弃置于尖锐件箱。 </li><li>执行DMN的腹膜内注射，连续3天，每周4周。每天在同一时间施用的注射剂。重新计算基于所述体重在注射每一周的开始的剂量。 </li><li>从尾静脉每周采集血液测定生化指标:谷丙转氨酶（ALT）和天冬氨酸氨基转移酶（AST）22。 </li><li>测定血清ALT和AST使用商用兽医测试化学分析仪。 </li></ol> 3.肉眼检查和肝脏的收获<ol><li>安乐死大鼠戊巴比妥腹膜内注射，剂量为100毫克/公斤体重。通过检查缺乏心跳确保死亡。 </li><li>制备为后验和如帕金森等人 23将死老鼠背斜卧与腹部朝上描述到解剖板评估动物的状况。 </li><li>消毒和滋润用70％乙醇的皮肤。 </li><li>使用剪刀，切开皮肤从肛门到下巴的ventrum的全长，和反映皮肤。用剪刀，切割从肛门腹壁剑突软骨，暴露腹腔脏器。 </li><li>检查腹部器官原位检查任何异常。注意在颜色，大小和任何变化，器官的位置的流体的腹腔内的存在。检查器官和一致性注意任何气味的存在。 </li><li>用剪刀和镊子，解剖整个肝脏免费的韧带和附件。 <ol><li>开始在其被连接至该膜片的门区和工作以释放附件的所有肝叶。小心切割所有韧带和血管。 </li></ol></li><li>转移肝脏到培养皿中，并称重肝脏。 </li><li>使用手术刀，切开2 - 5 mm产品肝叶厚的部分。为了保持一致性，总是从同一个肝叶样本。取一个楔直径约5毫米的厚度，从右侧侧叶24从lobe.Immediately处的边缘约1cm到10％缓冲的福尔马林固定。确保组织福尔马林体积为1:10或更多。 </li><li>如果需要其他研究收获，并立即在这一点上冻结部分（用液氮）肝叶。作为（3.8），从相同的肝叶一致性样品。 </li></ol><ol><li>固定在10％缓冲福尔马林的肝样品最少24小时。 </li><li>固定后，修剪肝，放入磁带并将其加载到自动组织处理器的篮子里。 </li><li>放置带盒在其中含有10％缓冲的福尔马林中，第一站篮筐。确保纸盒完全淹没。他们可以留在这个房间长达12小时，直到周期开始。 </li><li>编程组织处理器旋转盒的每个1小时，通过含有以下的溶液连续工位:乙醇以50％，70％，90％，100％（2站），二甲苯（2站）和液体石蜡的浓度（ 2站）。 </li><li>转移肝脏样品放入嵌入站的蜡浴。保证嵌入机器的至少一个小时前接通。 </li><li>使用预热钳（65°C），来自W删除的每个磁带斧头浴缸和放置到包埋机的温暖板（65°C）。分配液体石蜡的足量到不锈钢母模具（预加热至65℃），直到半满。从盒转移肝脏的部分到模具中。确保在试样被置于与切割表面（待检查显微镜）平放在模具的底部。 </li><li>用液体石蜡完全填充模具，安装在顶部的空盒和模具放置到包埋机的4°C板来冷却至少30分钟。 </li><li>检查该蜡已经冷却并固化，从模具中取出的石蜡块。块应该很容易蹦出来，而不是固守或裂纹。如果发生这种情况，熔块，并重复此步骤。一旦它固化的石蜡块可切片。块可在室温下保存了很多年。 </li><li>放置蜡块放入切片和部分在10微米厚，直到蜡层被充分除去嵌入的肝组织可见。 </li><li>变化对切片机的设置在5微米厚的削减。使用镊子，通过在边缘保持它捡起一个井切断部，并仔细漂浮在水浴部分在40℃的温度。 </li><li>轻轻地点下的浮动部分干净的载玻片上，然后将其在玻璃载片表面。放置滑动到滑动，在37℃过夜温暖。 </li></ol> 5. Masson染色<ol><li>施加污渍之前，治疗的幻灯片以除去蜡和再水合的组织如下: <ol><li>沉浸在二甲苯中的部分5分钟。重复2倍。 </li><li>浸泡在100％的乙醇的章节3分钟。重复1X。 </li><li>浸入部分的每个中的乙醇以下解决方案1分钟:90％，80％和70％。 </li></ol></li><li>冲洗自来水下滑动删除在幻灯片上的任何现有的乙醇。 </li><li>沉浸在铁苏木载玻片10分钟进行染色细胞核。 </li><li>冲洗载玻片用水从幻灯片除去过量的污渍。 </li><li>沉浸在比布列希猩红-酸性品红幻灯片2分钟进行染色细胞质中。 </li><li>用水冲洗的幻灯片。 </li><li>放置在磷钨/磷钼酸溶液的载玻片10分钟，以促进苯胺蓝的摄取。经过2轮的使用改变磷钨/磷钼酸溶液。 </li><li>放置在苯胺蓝溶液的载玻片10分钟，进行染色胶原纤维。 </li><li>冲洗过量的染色与水，并放置在1％乙酸溶液的幻灯片1分钟，以允许分化发生呈现幻灯片的颜色更细腻和透明。 </li><li>用水冲洗的幻灯片，并继续脱水组织切片。脱水步骤如下: </li><li>沉浸载玻片依次对每个时间10秒，在以下conce乙醇ntrations:70％，80％，95％和100％。 </li><li>浸入的幻灯片在100％乙醇30秒。重复1X。 </li><li>冲洗通过二甲苯的3个站的幻灯片，每次5分钟，以除去乙醇。 </li><li>挂载盖玻片上有安装介质（ 如 DPX封固）试样并晾干。 注意:DPX是干得很快，蜜饯染色和保护组织部分的合成树脂。 </li></ol> 6.纤维化评分对肝脏Masson的腺毛染色切片<ol><li>有兽医病理学家根据纤维化得分25评估纤维化的严重性。如果纤维化得分是由两个或三个独立地病理学家以盲方式进行这将是最佳的。在这种情况下，获得讨论和小组审核后的最终得分进行梳理得分的任何差异。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>得分了</td><td>描述</td></tr><tr><td> 0 </td><td>无纤维化</td></tr><tr><td> 1 </td><td>一些门户区域扩张的纤维，带或不带短纤维隔</td></tr><tr><td> 2 </td><td>大多数门户区纤维状扩大，有或无短纤维隔</td></tr><tr><td> 3 </td><td>大多数门户区纤维状扩大，偶尔门户门户网站（PP）桥接</td></tr><tr><td> 4 </td><td>门脉区纤维状扩张与标桥接（门户网站，门户网站（PP）以及门户中心（PC） </td></tr><tr><td>五</td><td>标志着桥接（PP和/或PC）偶尔结节（不完全肝硬化） </td></tr><tr><td> 6 </td><td>肝硬化，可能或肯定</td></tr></tbody></table> 表1:纤维化评分用于读取马森的腺毛彩绘肝脏切片 25。

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作者宣称，他们没有竞争的经济利益。

我们已经描述了使肝纤维化的动物模型，并评估肝纤维化的严重性的方法。它提供DMN正确的剂量，坚持每周腹腔注射的日程安排是非常重要的。随着实验的进行，它是衡量老鼠和DMN注射每星期开始重新计算剂量的关键。请记住，DMN是有毒的，需要在通风柜进行处理。我们在通风柜进行腹腔注射为好。随着需要反复注射，注射的正确的克制和技术是重要的，可以避免污染物引入和内脏损害。我们很少有从腹腔注射的死亡率。 大鼠应2倍整个实验期间每天进行监测。在上周DMN注射，动物应该更频繁和密切观察垂死的迹象。这是外周OD当肝脏损害最为严重和受影响的老鼠会食欲不振，失去体重和状态。 25 - 大鼠的40％可以变成在此期间严重患病和死亡，如果没有兽医干预。因此，密切监测允许研究者评估动物的条件，并计划实验合适的终端，以便器官可以从大鼠安乐死，而不是失去了从腐烂意外死亡进行新收获的。 我们已经发现ALT和AST水平的每周测量是肝损伤的进展的有用的指标。我们还观察到，AST比ALT不太具体，并在除了肝从受损红细胞和肌肉组织将此归因于它的释放。 确保血液足够体积被收集以得到血清用于分析所需的体积。我们通常收集200 - 300微升每鼠血。 过程中与C肝组织作病理检查的ollection，我们建议将样品从同一肺叶得到的。这是检查在肝脏中的变化是均匀后进行​​。 DMN导致在肝脏广义变化。在研究的早期阶段，我们从左侧外侧和内侧裂片以及右内侧叶采样。我们没有观察到这些叶之间的微观变化的差异。 肝切片应固定在10％缓冲的福尔马林中至少24小时，之后不久进行处理。长时间浸泡超过一个星期可导致组织变脆，难以部分石蜡包埋后。切割适合5微米的薄片，需要练习和耐心。被评定为适合用肉眼（步骤4.11）部分可以用显微镜进行检查，以检查他们没有像部分内的褶皱或撕裂文物。染色过程是最好的完成根据:Ø在没有休息时间，每一步幻灯片浸泡的时间段。在染色过程中，确保部分是在任何时候都保持湿润，并尽快从一个染色溶液转入下一越好。 对于纤维化程度的Masson的腺毛染色的切片的评估需要兽医病理学家的输入端。他/她是谁应该是从一开始就研究的一部分，他/她将能够建议，并在实验的各个阶段参与器官的正确收获了至关重要的团队成员。因此，目前的黄金标准，肝纤维化评分是适当的染色组织切片由病理学家的评估。虽然这将是最佳的，以有一个以上的病理学家的输入，这可能不是在较小的组织是可能的。在这种情况下，客观性可以通过使用成像技术26来改进。我们已发现，从成像技术的结果媲美的病理评分（未发表数据），并建议将其作为评价的辅助方法。 总之，肝纤维化的DMN诱导模型具有比其他动物模型中的许多优点。它是一个比较容易的模型进行，因为它不要求手术技巧。 DMN是对环境更安全和毒性对人类和动物比的CCl 4以下，需要更短的时间以诱导疾病比TAA。相比由四氯化碳 ，TAA产生肝病，和胆管结扎，DMN产生人肝纤维化的接近表示。由于这些原因，我们预计，该模型将继续被广泛用于研究肝纤维化的机制，以及筛选潜在抗纤维化剂。

DMN是一种强效的肝毒素特异性。报道了马吉1其代谢，组织分布，并造成伤害到大鼠的肝脏能力，和由Pritchard的和巴特勒2中记载的细胞凋亡的肝细胞损伤和细胞死亡的机制。该化合物的间歇给药报道以诱导在狗和大鼠3,4-肝纤维化。 该机制与肝纤维化的形态学变化，采用这种模式被广泛研究。在使用大鼠早期研究中，DMN 3周的治疗产生小叶中央出血性坏死后肝硬化结节无脂肪变性5。它表明，在早期纤维化，所形成的胶原是更多的交叉与III型挂钩比I型6更为突出。在与其他原因常见，DMN诱发纤维性变化用的增加Kupffer细胞相关联;驻留在我的肝巨噬细胞n个血窦。这些细胞演变成肌成纤维细胞，并产生细胞外基质这是纤维化7的首要问题过量。 在细胞信号方面，Nakamura等人证明了TGF-β在肝纤维化8的进展中起关键作用。他们用表达截短的II型TGF-β受体的腺病毒，以专门抑制TGF-β信号传导。在这些大鼠肝纤维化与对照组比较时，被壮观DMN治疗期间停止。其他研究已经证实，TGF-β抑制导致肝纤维化的发展9,10缓解。该模型也被用于在一个全局基因表达谱研究，以确定其中可以作为药物靶点抗纤维化疗法11其他纤维化标记物和蛋白质。 其他化学试剂用于诱导肝纤维化包括硫代乙酰胺（TAA）和c阿尔邦四氯化碳（CCL 4）。 TAA首次使用大鼠，后来在小鼠12。这种模式的优点包括:易于管理化学品的饮用水，以特色小结节性肝硬化生化变化模型的重复性。缺点包括:3个月肝纤维化的长时间周期来开发和缺乏对肝纤维化的诱导分子机制的理解。至于四氯化碳 ，它的使用有所下降，原因如下:它不模仿人类肝脏疾病，具有对臭氧层有害的影响，引起疼痛和痛苦动物，是非常对人体有毒，并要求其处理额外的预防措施和处置13,14。 长时间胆道梗阻（通过外科手术），作为肝硬化实验模型最早由Kountouras 等 15日报道。这种方法是无毒的人类和动物。 HH但是，开发所需的肝纤维化的时间变化。由马克斯等人 16 30份报告进行审查，发现花了由七天到四个星期后手术治疗肝纤维化的发展。所描述的病理变化模仿那些人慢性胆道纤维化和模型会更适合有意在这一领域的研究人员。 总之，DMN的大鼠在4周内恒定剂量的间歇给药产生肝纤维化模仿人类疾病。给药的大鼠显示肝损伤和肝纤维化实质的4,17逐步发展。疾病进展和严重性可以通过血样或处死动物在特定时间点进行监测和效果是高度可再现18。因此该模型已被广泛用于研究肝纤维化和肝硬化的机制，以及筛选潜在抗纤维化剂10,19,20。

<table><tbody><tr><td>Dimethylnitrosamine</td><td>Wako</td><td>147-03781</td><td></td></tr><tr><td>Formalin</td><td>Sinopharm chemicals</td><td>F63257009</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>64-17-5</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>1330-20-7</td><td></td></tr><tr><td>Masson trichome stains</td><td></td><td></td><td></td></tr><tr><td>Aniline Blue</td><td>Electron Microscopy Sciences</td><td>#42755</td><td></td></tr><tr><td>Acid Fuschin</td><td>Electron Microscopy Sciences</td><td>RT42685</td><td></td></tr><tr><td>Scarlet Red</td><td>Electron Microscopy Sciences</td><td>#26905</td><td></td></tr><tr><td>Phosphotungstic Acid Hydrate</td><td>ALFA AESAR</td><td>ALFA40116.4</td><td></td></tr><tr><td>Phosphomolybdic Acid Hydrate</td><td>Sigma SG</td><td>221856-25G</td><td></td></tr><tr><td>Weigert&#039;s Iron Hematoxilyn</td><td>Merck</td><td>1.15973.0002</td><td></td></tr><tr><td>DPX Mounting Medium</td><td>Merck</td><td>HX066873</td><td></td></tr><tr><td>Tissue processor</td><td>Leica</td><td>Leica TP 1020</td><td></td></tr><tr><td>Embedding machine</td><td>Sakura</td><td>&amp;nbsp;Sakura Tissue Tek TEC5 Embedding System</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>Leica RM 2235</td><td></td></tr><tr><td>Vet Test Analyzer</td><td>Idexx</td><td>Vet Test 8008</td><td></td></tr></tbody></table>

the dimethylnitrosamine induced liver fibrosis model in the rat

四至六周龄，雄性Wistar大鼠用于产生肝纤维化动物模型。该方法需要四个星期10毫克/公斤的二甲基亚硝胺（DMN）施用，每周连续三天腹膜内给予。作为DMN是一个已知hepatoxin和致癌物质腹膜内注射是在通风柜进行。该模型具有若干优点。首先，肝变化可依次或在感兴趣的特定阶段的研究。其次，肝脏疾病的阶段可通过血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）的酶测定进行监测。第三，在不同阶段的肝损伤的严重程度可以通过在指定的时间点处死动物来确认，随后Masson的腺毛染色的肝组织的组织学检查。 4周DMN给药后，典型的纤维化评分是对伊沙克比例5至6。该模型可以一致地被再现，并已广泛用于评估潜在的抗纤维化剂的功效。

DMN组大鼠减肥，成为竖起被毛缺乏活力。有一个在DMN治疗的大鼠的平均体重显著的损失;第一可检测2周后DMN的治疗，并且这种差异仍然通过DMN处理（ 图1a）周后3和4。由于老鼠收到DMN在连续数周，对肝脏的损害使之变得更小。肝指数;这是在最终体重肝重的百分比是该DMN治疗的大鼠（ 图1b）显著低。 <img alt="图1" src="/files/ftp_upload/54208/54208fig1.jpg" /> 图1:一个）DMN的体重治疗的大鼠。数据被表示为平均值±标准差（n = 6 - 8）。 * P &lt;0.05与正常对照组二）肝脏指数比较。在最后的体重DMN肝重量百分比4周DMN TRE后处理的大鼠atment。数据被表示为平均值±标准差（n = 6 - 8）。 * P &lt;0.05与正常对照组比较， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54208/54208fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 在牺牲4周DMN的治疗后，肝脏相比，那些从老化匹配的对照动物更小和更硬（ 图2a）（ 图2b）。血纤维蛋白可以存在于肝表面和相邻的肝叶粘接。大约20％的大鼠有腹水。 <img alt="图2" src="/files/ftp_upload/54208/54208fig2.jpg" /> 图2:从大鼠一个）肝4周DMN治疗二）肝脏从老年匹配的对照鼠后。在（一）肝脏缩小，坚定和苍白黄色色彩时肝脏相比，从年龄匹配的对照。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54208/54208fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 损害肝脏导致肝细胞细胞膜的通透性增加。提高血清ALT和AST是肝细胞损伤的指标。 DMN的治疗组血清ALT（ 图3a）和AST（ 图3b）是周2和DMN注射后4比对照组显著高。血清ALT和AST水平一般DMN每个治疗一周后增加。 <img alt="图3" src="/files/ftp_upload/54208/54208fig3.jpg" /> 图3: 一个）血清丙氨酸转氨酶（ALT）和b）血清谷草转氨酶（AST）在0周，第2和4的最后DMN注射后DMN处理的大鼠的水平。数据是再版esented作为平均值±标准差（n = 6 - 8）。 * P &lt;0.05与正常对照组比较， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54208/54208fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 从DMN处理的大鼠肝脏组织学检查表明，与对照组相比，有逐步增加和纤维间隔的扩大，与肝细胞的损失，随着时间的推移。 Masson的腺毛染色通常用于突出肝组织中的胶原蛋白沉积:这些被染成蓝色。 <img alt="图4" src="/files/ftp_upload/54208/54208fig4.jpg" /> 图4:用Masson的腺毛染色的肝切片的显微照片:a）从正常对照大鼠肝部;接收1周dimethylnitrosamin的b）由大鼠肝脏部分之后E（DMN）;接收2周DMN后，从大鼠三）肝切面。有偶尔接收门户3周DMN后，门静脉从大鼠的桥接。 四）肝切面大部分门脉区纤维性扩张。注门脉区有明显的门户纤维膨胀收到4周DMN后从大鼠门以及门户中心桥接。 五）肝部分。有肝硬化结节的形成。控制肝脏是选自4周DMN注射后连同大鼠处死。有从0纤维化评分的逐步增加的图案中的（a）; 2（b）中; 3（d）中，以5/6（e）中（C）4。所有显微照片是在40X放大倍数下通过比例尺当量的1个单位长度取为100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54208/54208fig4large.jpg" target="_blank">请Ç</a>舔此处查看该图的放大版本。

Watch this Scientific Journal Video about The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat at JoVE.com

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

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 ...

the-dimethylnitrosamine-induced-liver-fibrosis-model-in-the-rat

Nanyang Technological University

Research

JoVE Journal

Medicine

12.0K 次观看.  Temasek Polytechnic. 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.

视频: The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

科研

发育生物学

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

医学

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

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 as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

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 ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

免疫与感染

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

生物工程

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

对TNBS介导肠纤维化发展的力学洞察与拉帕霉素抑制作用的评价

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

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 ...

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

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

在二氧化硅暴露小鼠模型中使用尼古丁促进肺上皮-间充质转化

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...

该二甲基亚硝胺诱发肝纤维化模型大鼠

摘要

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此视频中的章节

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

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

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

对TNBS介导肠纤维化发展的力学洞察与拉帕霉素抑制作用的评价

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

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

在二氧化硅暴露小鼠模型中使用尼古丁促进肺上皮-间充质转化

该二甲基亚硝胺诱发肝纤维化模型大鼠

摘要

探索更多视频

此视频中的章节

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

二硝基苯磺酸/ TNBS结肠炎模型：提供洞察炎症性肠病和膳食脂肪的影响

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

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

非酒精性脂肪性肝炎合并2型糖尿病的一种先进的小鼠模型

对TNBS介导肠纤维化发展的力学洞察与拉帕霉素抑制作用的评价

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

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

在二氧化硅暴露小鼠模型中使用尼古丁促进肺上皮-间充质转化

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生