特别感谢莎拉Kossak酒店，米切尔Guanzon，宋允杨和李郭嘉，为他们的实验动物的内镜检查过程中他们的合作。

1。设立内窥镜系统<ol><li>设置了内窥镜系统麻醉的小鼠前，按照制造商的说明进行操作。使用视频输尿管镜专为人类获得本协议（ 图1）。插入管的外径是9.9，FR（3.3毫米），其远端是8.5，FR（2.8毫米）和仪器通道的内径为6周五（1.2毫米）。 </li><li>虽然使用内窥镜尚未链接到疾病传播[9]，消毒的内窥镜和透镜与孢子形成微生物工作时，用70％乙醇10或者用适当的技术。准备抑菌消毒润滑剂和非刮拭组织饱和内窥镜末端。 </li><li>附上一个3毫升注射器充满空气（或合适的CO 2气体的混合物）的仪器通道的顶部。 </li><li>打开电源，并设置白平衡正常的白光，以及窄带成像（NBI）模式下，当适用。自2001年以来，NBI提供诊断成像的好处，因为它强调精细的血管结构上粘膜，包括溃疡和肿瘤病变，其中有特征的异常血管11。 NBI过滤模式限制捕获的光的波长为390-445 nm和530-550 nm处，其中突出的结构与血红蛋白。 </li><li>打印考核评分表（ 图2）来记录你的发现。熟悉预期的正常和NBI图像（ 图3A）和定义，以及每项准则的成绩在考试前（见第6.1节）。 </li><li>准备在磷酸盐缓冲盐水（PBS）中的着色剂为基础的解决方案，色素​​，如果需要的话。在色素，使用染料作为造影剂，以突出黏膜变化进行了考试。用亚甲基蓝（0.1-1％）和靛蓝胭脂红（0.1％）解决方案（ 图3B）。注该亚甲蓝是一种氧化还原剂可以制作氧化自由基，间接交联氨基酸残基，和DNA降解12，这可能会干扰样品的分析。 </li></ol> 2。鼠标的全身麻醉<ol><li>选择麻醉剂和给药途径。从有关药物与免疫系统和研究目标的潜在干扰动物资源的设施或机构动物护理和使用委员会（IACUC）请教兽医。有些药物可能会影响细胞因子的表达分布在动物，包括啮齿动物13。使用注射三溴乙醇的终端程序或异氟醚生存内镜，以及适当的控制。 </li><li>用于感应，辖异氟醚4-5％在100％的氧气以0.2-0.5升/分钟的速率;使用0.5-2％，进行维护。这个协议是不适合于所有的动物。请咨询您的兽医机构进行适当的指导和培训。通过有关建议IACUC遵守，以实现动物福利的最高标准。确保鼠标，如果完全麻醉。 </li><li> &#39;带&#39;麻醉鼠标在使用实验室胶带背部或腹部位置的支撑面表。当心你的内窥镜屏幕上的解剖定位。确保其下肢和尾巴。磁带的带也可放置在胸前，以防止运动远离呼吸器，但要避免限制胸廓呼吸运动。 </li></ol> 3。详细内镜检查<ol><li>寻找肛周病变插入内窥镜伸入直肠前，然后经肛门推进范围。 </li><li>大衣与无菌手术润滑剂内窥镜。千万不要涂在CCD（电荷耦合器件）传感器的表面。 </li><li>插入内窥镜，并开始录制。录制的视频是米有用内镜检查，如果需要检查后eticulous评价。尽量在时间，以减少肠/粘膜有刺激性尽可能少的长度进行胃镜检查。 </li><li>通过使用注射器慢慢地分开设置肠壁施加空气。根据鼠标的大小，使用1-2毫升的空气。避免过多的空气吹入，它可能会导致麻醉小鼠的呼吸窘迫和死亡。 </li><li>推进内窥镜前进你开道用空气或范围的轻柔旋转。没有前进的内窥镜，如果你没有看到肠腔。穿孔或直肠/结肠眼泪可以发生在这个阶段，尤其是在年轻小鼠（&lt;8-10周龄）。拍照是必要的。 </li><li>随着范围的进步，评估是否有自发性出血（定义为自然发生粘膜出血与外伤性内镜相关的）。自发出血可作为内窥镜进入直肠和结肠被观察到。 </li><li>如SESS结肠壁的透明度。透明度定义为可视化的壁间血管在结肠和其他周围的内脏（ 图4）的能力。范围温柔的动作（向后转，顺时针，逆时针）将有助于确定血管可以在其他脏器的角度来看待。肠系膜脂肪组织也可见到。注意如果盆腔内直肠似乎比结肠壁在腹腔透明度较低。 </li><li>检查，以确定病灶（水肿区，糜烂，溃疡，肿瘤质量）的类型和数量。这将使用结肠炎得分，并已开发出的小数标识符被记录在计分的形式。 </li><li>一旦其中结肠尾端弯曲成近端结肠的面积达到了，撤内窥镜慢慢寻找接触性出血（的发生是由于后粘膜脆性的存在内窥镜前进或删除）。它没有必要故意刮结肠表面。经过适当的内镜检查健康的大肠黏膜不流血。 </li></ol> 4。活检<ol><li>当需要活检，通过专用仪器内窥镜通道插入活检钳。监控内窥镜屏幕上的镊子的进步，以防止肠穿孔。 </li><li>打开钳活检杯到约60-80℃杯对杯角，并指导他们感兴趣的区域。过度打开钳子或压力可以把握肠组织，这会导致健康透明肠壁活检引起的穿孔的额外层。如果发生穿孔，麻醉期间安乐死的动物按照你IACUC建议。穿孔导致内毒素血症和腹膜炎。 </li><li>关闭活检杯，同时保持钳闭拉出。 </li><li>圣雷莫通过冲洗组织一个解决方案，是温和的组织和钳子已经从钳子试样。后来的RNA和PBS是共同的工作方案，如果需要，使用针撞出样品。避免侵蚀着钳子杯或切削刃。 </li></ol> 5。麻醉恢复期<ol><li>待考试结束，提供一个温馨的环境，鼠标，直到它恢复。使用任一种电光源，或温暖垫定在37°C避免过热;周围血管扩张危及动物的恢复。密切监视鼠标，直到完全康复。 </li></ol> 6。大肠健康与十进制标识符综合内镜评分系统<ol><li>使用评分表（ 图2）及以下的参数定义（ 小鼠大肠炎症和肿瘤的内镜评估使用十进制加权计分法：参数定义和协议 ）实例和可选的&#39;远端近端结肠炎内镜评分系统“（D-PECS），作为无偏差评估和详细记录保存的指引。 </li><li>分数的计分制的形式（ 图2）中所述的四个参数的大肠炎症。精心评估肛周区域，肠透明度，肠出血的类型（如果存在），以及存在和病灶的数量（糜烂，溃疡和/或肿瘤细胞）。 </li><li>在评估肿瘤为研究目的，研究的数量，位置和大小，并使用结肠图和计分表上条形图描述它们。得到的分数和病变的数目（当适用时）的数值总和将确定背景结肠的炎症，最主导型病变，以及病变的数目。 </li></ol>

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</ol>

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有关于修改内窥镜技术在小鼠和故障排除几方面的考虑。需要使用灵活的内镜检查过程中掌握了过程的关键方面包括需要防止腹胀和鼠标的呼吸妥协的风量的调节，以及视图控件的细微的技术方面与协调的角度和转矩控制。转矩控制是很重要的，以减少肠穿孔的危险。如果处理不当，鼠灵活的内窥镜成为一个非常安全的，快速的程序，可以直接可视化和感兴趣的研究领域肠活检取样。 为了解决因粪块（ 图5）查看障碍物，要明白，粪粒甚至禁食或使用泻药后通常观察是很重要的。如果存在，粪便可以很容易地移动近端与温柔的空气插件ufflation。在约11％的情况下，有必要也可以使用1ml的PBS灌肠。在此之前进行灌肠，检查动物，似乎有腹胀或怀孕时，务请审慎行事。对于蠕动它等待几秒钟，直到蠕动波传递，因为空气吹入不会停止蠕动是很重要的。 迄今为止，用于评价小鼠肠道疾病最内镜评估系统是针对要么结肠炎或癌症。鉴于这两个条件往往相伴发生，并可以彼此，标准化的计分制度，同时考虑炎症和肿瘤的形成将大大促进跨学科交流，从而提高信息由癌症研究胃肠研究过程中所获得的可用性进行交互，反之亦然。因为肿瘤没有在大肠癌区域诱导某种形式的炎性改变很少存在我N小鼠​​，我们已经在我们的评分系统中唯一允许肿瘤的肠道健康的范围内研究的十进制标识符部分包括肿瘤。为了提高评分系统的有效性和可靠性，并促进其长期的实验或临床试验中使用，我们还开发了一个标准化的报表一起使用的评分系统（ 图2），其存储系统所需的数据元素计算小鼠肠道炎症评分的基础上，精确的定义每个分类标准。 效力有限多个鼠内镜评分系统的前一个可用性阻碍各研究结果的解释。为了最大限度地减少其公布的方法来使用经历的实验室工作人员的混乱决定，我们建议您使用，可以从二维图像来推断标准进行内镜检查（ 如避免标准THA吨参阅纹理或一致性-粪便;注意，某些小鼠包括无菌可具有软便），并且是不共线（ 即由相同的炎症方面的生理上和时间上驱动）由于主要体征炎症是连续的，由于实足炎症活动，我们推荐以下条件：1）肠透明度，首先由Becker 等4所述，由于壁厚的反映; 2）出血，粘膜和血管完整性的指标。 3）肛周发现，如结肠完整性和对皮肤的刺激或倾向过度炎症的指标，以及4）局灶性病变，进行分级炎症病变（其在小鼠中主要溃疡性）的严重程度。到今天为止，我们还没有看到在3％DSS诱导的小鼠结肠炎的典型模式肿瘤病变。在本文中描述的鼠鼻内镜评分系统提供了第一次，就我们所知定量评估机制的存在和诱导肿瘤性病变中的结肠炎症的上下文。 这是第一次，我们也突出的重要性，包括狭窄的小鼠在内窥镜的一个参数，因为它们似乎是通常存在于肠道炎症， 如 SAMP / YIT / FC的某些动物模型。狭窄代表由于增加纤维化，这是很难内窥镜评估对于新手的粘膜慢性炎症（准）周区域，肠失去弹性。我们已经注意到，在内窥镜定义狭窄影响的小鼠中，存在于片段是近端狭窄相比，远段（其可以内窥镜检查），可能是由于减慢肠道转运更多的炎症。为研究目的，最好是组织学随访动物在实验结束时，以更好地表征炎症侧翼候选人狭窄区域matory反应。 如果没有适当的培训和专家的反馈，任何打分系统可能有变异在任何给定的时间。它强调在老鼠身上进行了内镜培训程序应该由医疗专业人员的培训的兽医进行监测，以确保重要的是：其他科学家或技术人员的我）适当的培训，以及ii）确保动物在不误诊或不慎受伤程序。继续执行这一程序，以确保可能的最佳实践和动物福利前咨询负责你的动物研究机构的专家咨询意见的兽医。明智的做法是定期监测训练有素的内镜医师的性能。用我们这里给出的指引，随机在动物的计算顺序，并评估以盲每一个动物的图像。定期协商的参数定义和协议的标准和k在内镜检查和评分第6 EEP他们的手）。 总之，作为一个团队的医疗和兽医肠胃病学，我们提出了一个标准化的协议和半定量计分系统对结肠炎和肿瘤的小鼠模型的内镜评价。这些研究工具将使研究人员进行例行鼠内镜，并允许研究人员在各级的经验，产生肠道炎症和肿瘤形成的跨实验室和学科之间的重复性和可比性的措施。我们已经开发的系统，以产生有意义的数值分数高的有效性，并规定特定的结果，它可以是自定义的单个实验室内部使用的快速跟踪能力。我们还描述了一种歧视性的打分替代方案，考虑到区域中考虑的易感性差异，疾病结肠内（见第E，̵6，远端近端内窥镜结肠炎的评分系统，简称D-PECS）。数据报表和一些数字可以作为墙上的海报，以帮助缺乏经验的科学家（ 图1，3，5，6）。成功实现小鼠内镜取决于熟悉的皮肤粘膜交界处，在肛门，直肠，结肠，和周围脏器的正常解剖结构，为解剖上的差异占微妙内镜差额17。 虽然目前即使是最小，最灵活，最优秀和内镜技术无法达到近端结肠体内 ，快速移动的胶囊和单纤维内镜领域的技术进步有可能成为可用于小鼠在不久的将来。标准化协议和本文所提出的内镜评分系统代表了可视化和评估小鼠肠道环境活体动物的状态有价值的工具。这些工具将facili论文之间和各学科的成绩大老可比性，也就有了适用于新的和正在出现的鼠内镜技术。

鼠内镜检查已经完成作为一个有用的研究工具超过十年1-3。到目前为止，大多数研究采用小鼠内窥镜使用了硬性内窥镜，虽然有些也使用软式乙状结肠镜。鼠胃镜提供立竿见影的效果和肠道正常，炎症的严重程度和肿瘤进展比较间接的措施，比如形体消瘦，腹泻，和组织学（这是次优的，当病灶呈斑片状）的程度更客观的估计数字，而提供对结肠的整体健康状况的见解。最值得注意的是，内窥镜允许的实验室动物模型随时间的反复评价，相对于传统的组织学检查，需要被实施安乐死，并收获用于分析1冒号的动物。尽管有这些优点，利用鼠内窥镜技术还没有广泛和由缺乏implementati标准化协议的限制在和病理结果的得分。正确实施，鼠胃镜拥有巨大潜力，以进一步促进我们的理解和多种慢性胃肠疾病，包括炎症性肠道疾病，结肠炎，大肠肿瘤动物模型的表征。 小鼠内镜技术的效用有赖于其可重复性和客观性，这就需要标准化的审查程序的存在和一致的，非冗余，可靠的评分系统肠道病变。至少三个描述性的评分系统对结肠炎4-6的内窥镜的评估，以及用于结肠肿瘤的小鼠4,7,8已经出版。然而，这些报道办法和评分系统是不能跨的研究容易比较。在许多情况下，有一种用于病变的分类标准没有明确的定义，和标准给出时，它们有很大的不同。此外，没有报告的评分系统是整合双方结肠炎和肿瘤，二是可以同时发生，对结果的互动效应最常见的结肠病理，到一个单一的测量工具的评估。最后，确实存在对结肠炎的内镜评分系统往往有炎症个类别，有限制的歧视（ 即 ，在短整型尺度窄的得分选择，往往1-4），以正确地表示疾病进展的情况，并能够使用参数统计分析。 在本文中，我们描述了使用柔性内窥镜的评估鼠结肠炎和结肠肿瘤的严重程度和描述的标准化协议，用于采用该技术的肛周区域，直肠和结肠的解剖评估。我们在内窥镜说明有效的故障排除，以减少创伤和图像伪影，和我们描述了一个可重复的评分系统基于验证的已发表的临床参数。该评分系统集成使用的有12个可能的等级炎症，肿瘤测绘和绘图选项，以及一个新的十进制单位制（即小数点标识符）歧视高水平突出与特定的诊断价值发现两者肠道炎症和肿瘤的评价（ 即肿瘤的发展，内镜检查等过程中的并发症）。使用十进制标识符允许对相关的数据集进行进一步的下游分析的快速跟踪。最后，我们在肠炎和结肠炎相关癌（葡聚糖硫酸钠（DSS）诱导的结肠炎， 难辨梭状芽孢杆菌感染，氧化偶氮甲烷/ DSS诱导结肠癌）的多种小鼠模型进行可靠性和评分系统的有效性测试。

<table><tbody><tr><td>Isoflurane, USP</td><td>Webster Veterinary</td><td></td><td></td></tr><tr><td>Surgical lubricant</td><td>Savage laboratories</td><td>surgilube, 0281-0205-45</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo Scientific</td><td>SH30256</td><td></td></tr><tr><td>RNAlater</td><td>Ambion</td><td>AM7021</td><td></td></tr><tr><td>Methylene Blue 1% Aqueous Solution</td><td>Fisher Science Education</td><td>S96393</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Flexible digital ureteroscope</td><td>Olympus America</td><td>URF-V</td><td></td></tr><tr><td>Video system center</td><td>Olympus America</td><td>VISERA Pro OTV-S7 Pro</td><td></td></tr><tr><td>Xenon light source</td><td>Olympus America</td><td>VISERA Pro CLV-S40 Pro</td><td></td></tr><tr><td>Video recorder</td><td>MediCapture, Inc.</td><td>MediCap USB200</td><td></td></tr><tr><td>Flexible biopsy cup forceps</td><td>Olympus America</td><td>FBC-3115</td><td></td></tr><tr><td>Anesthesia machine</td><td>Euthanex Corporation</td><td>EZ-7000 Classic System</td><td></td></tr></tbody></table>

flexible colonoscopy in mice to evaluate the severity of colitis and colorectal tumors using a validated endoscopic scoring system

为研究目的而利用现代内镜，大大方便了我们的胃肠道病变的认识。特别是，实验内窥镜一直需要在一个单独的实验动物反复评估，如那些评价慢性炎症性肠疾病的机制和结直肠癌的进展的研究是非常有用的。然而，对不同的研究中使用的方法是高度可变的。至少有三个内镜评分系统已经出版的小鼠结肠炎和公布结直肠肿瘤的评估，协议未能解决伴随结肠炎症的存在。本研究开发和验证一个可重复的内窥镜计分制度，同时集成了炎症和肿瘤的评价。这种新颖的评分系统由三大部分组成：1）评估大肠炎症的程度和严重性（基于肛周发现，沃尔玛的透明度升，粘膜出血，和局灶性病变），2）肿瘤性病变（格图和条形图），和临床病例通过病理和研究的相关基于十进制单位分配的类别观察病变3）数值排序的定量记录和内镜并发症（十进制标识符）。视频和手稿本文中所呈现的制备，以下IACUC批准的协议，以允许研究者使用经过验证和高度可重复的方法内窥镜得分自己的实验小鼠中，与该系统选项从近端内窥镜结肠炎（D-PECS区分远侧）。

Watch this Scientific Journal Video about Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System at JoVE.com

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

在过去的几年中，新一代内窥镜已成为重要的诊断艾滋病的研究评估小鼠结肠炎和结肠肿瘤。我们在此提出一个详细的协议，用于炎症和大肠肿瘤在小鼠体内的内镜检查，以及使用十进制标识符记录结肠炎和结肠直肠肿瘤的内镜严重性一种新的评分系统。

灵活的结肠镜检查在小鼠使用经过验证的内镜评分系统来评估结肠炎和结肠直肠肿瘤的严重程度

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

flexible-colonoscopy-mice-to-evaluate-severity-colitis-colorectal

Nanyang Technological University

Research

JoVE Journal

Medicine

31.7K 次观看.  Case Western Reserve University School of Medicine, Cleveland. 在过去的几年中，新一代内窥镜已成为重要的诊断艾滋病的研究评估小鼠结肠炎和结肠肿瘤。我们在此提出一个详细的协议，用于炎症和大肠肿瘤在小鼠体内的内镜检查，以及使用十进制标识符记录结肠炎和结肠直肠肿瘤的内镜严重性一种新的评分系统。 

视频: Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

遗传工程小鼠模型的散发性结肠直肠癌

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

科研

癌症研究

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

医学

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

生物化学

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

调查在DSS诱导的鸡传染性法氏囊病模型的肠道炎症

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

生物学

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

活小鼠肠道移植物对宿主病的诱导及微内窥镜评价

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

免疫与感染

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

慢性结肠炎相关结肠直肠癌的肿瘤切除术模型，通过卡卡贴片的精确定位

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Segmental Adeno-Cre Infection: A Technique to Generate Isolated Colorectal Cancer using Genetically Engineered Mouse Models

Source: Betzler, A. M., et al. <a href="https://www.jove.com/v/55952"> A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer.</a>&#160;J. Vis. Exp. (2017).
This video describes the generation of a colorectal mouse model by segmental adeno-cre infection. The infected colon segment leads to tumorigenesis within this segment of the mucosa, resulting in adenomas ultimately progressing to invasive and metastatic carcinoma. This model is an attractive platform for studies of cancer biology and preclinical therapeutic trials.

节段性腺-Cre 感染:一种使用基因工程小鼠模型生成孤立性结直肠癌的技术

Source: Betzler, A. M., et al.  A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer.&#160;J. Vis. Exp. (2017).
This video describes the ...

灵活的结肠镜检查在小鼠使用经过验证的内镜评分系统来评估结肠炎和结肠直肠肿瘤的严重程度

摘要

探索更多视频

遗传工程小鼠模型的散发性结肠直肠癌

建模结肠炎相关的癌症与氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

鼠内镜

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

调查在DSS诱导的鸡传染性法氏囊病模型的肠道炎症

活小鼠肠道移植物对宿主病的诱导及微内窥镜评价

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

慢性结肠炎相关结肠直肠癌的肿瘤切除术模型，通过卡卡贴片的精确定位

节段性腺-Cre 感染:一种使用基因工程小鼠模型生成孤立性结直肠癌的技术

灵活的结肠镜检查在小鼠使用经过验证的内镜评分系统来评估结肠炎和结肠直肠肿瘤的严重程度

摘要

探索更多视频

遗传工程小鼠模型的散发性结肠直肠癌

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

鼠内镜

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

调查在DSS诱导的鸡传染性法氏囊病模型的肠道炎症

活小鼠肠道移植物对宿主病的诱导及微内窥镜评价

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

慢性结肠炎相关结肠直肠癌的肿瘤切除术模型，通过卡卡贴片的精确定位

节段性腺-Cre 感染:一种使用基因工程小鼠模型生成孤立性结直肠癌的技术

建模结肠炎相关的癌症与氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）