Name: non-invasive assessment of the efficacy of new therapeutics for intestinal pathologies using serial endoscopic imaging of live mice
Uploaded: 2015-03-10T13:00:00.000+00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice at JoVE.com

We would like to thank CSL Ltd. for supporting the purchase of the endoscopy equipment. The research in the laboratory of ME is supported by the Ludwig Institute for Cancer Research, and the laboratories of TP and ME are supported by the Victorian State Government Operational Infrastructure Support and the National Health and Medical Research Council of Australia. ME is an NHMRC Senior Research Fellow.

医学研究动物伦理委员会的沃尔特伊丽莎研究所厅批准每一个在这些协议中描述的过程。 1.准备实验小鼠<ol><li>构成的至少4性别实验队列匹配6-8周龄C57BL / 6小鼠（M.家鼠 ），它们孕育和容纳在相同的特定的无病原体（SPF）的动物设施/室和设置有蒸压食物和水。使用雌性小鼠将允许共壳体小鼠从不同的线路，限制框号，以及降低笼到笼的变化。 </li><li>确保小鼠是至少6周龄，使用3.0毫米直径的内窥镜护套内窥镜程序。 </li><li>使用的小鼠相同的遗传背景，并且在可能的情况，从存储在同一机架，以减少与肠道菌群的变化相关联的笼中。在动物设施之间菌群变化应当考虑到磨片Ñ ​​确定适当的对照小鼠和基因型每个实验22。 </li><li>马克小鼠耳/趾剪报，纹身，或类似的允许，便于识别。 </li><li>称重在第0天的小鼠，以确定基线实验权重。 </li><li>进行胃镜检查（见第5）小鼠第0天，记录基线结肠表型。 </li></ol> 2，临床前试验中的上皮损伤和急性结肠炎型号<ol><li>制备的治疗剂的溶液，以进行测试。对于通过口服管饲法（PO）递送治疗剂，制备溶液，以100微升的最大体积。通过腹膜内（IP）注射递送治疗剂，制备溶液，以200微升的最大体积。 注:在长时间重复IP或口服给药可能导致改变鼠标的行为。交替腹腔注射部位，以避免长时间的不适。对于口服给药，提供小鼠autoclaved葵花籽作为一个&#39;治疗&#39;，以减少与灌胃过程中的不良联想。 </li><li>在第1天，施用的利息（步骤2.1的方法制备）和适当的载体对照的治疗。在提供的例子中（ 图6A），5微克重组人（rh）下iInterleukin素（IL）-11的蛋白质溶于200微升磷酸盐缓冲盐水（PBS），并施用IP </li><li>确定施用，这将依赖于建立该正被测试的治疗试剂的药物动力学分布的时间和频率。 图1概括了使用每天两次腹腔注射在实验的过程中预防性治疗。 </li><li>监控，包括粪便稠度和血液的存在，并每日称重每只小鼠。在治疗的日子里，称量应与治疗药物的给药重合，以减少应力所引起的反复操作时的小鼠。 </l我&gt; <li>在第3天，制备2.5％（2.5克/ 100毫升）中常规地由动物设施提供给小鼠的饮用水的DSS溶液。大约5毫升/小鼠/的DSS溶液一天所需的实验。如果动物设施的环境温度波动的DSS水的消耗可能会改变。 注:DSS是具有刺激性，并应按照MSDS的指令来进行处理。 </li><li>提供新鲜DSS的小鼠自由采食清水瓶连续5天。在5日的晚上，提供正常的饮用水除泥状食物颗粒和小皮氏培养皿（100克食物颗粒/ 10克蛋白质摇/ 10毫升的水）提供的蛋白质补充。这可以防止脱水，并协助最大限度地减少重症减肥。 </li><li>安乐死小鼠二氧化碳中毒和收集组织进行后续组织学分析的第8天，或早晨，当小鼠经历≥15％减重超过三Consecutive天，以先到为准。 注:调整之间1-4％的DSS剂量（重量/体积）根据动物设施的菌群和批次的DSS。进行，常规批量测试，以建立相应的DSS剂量和规避批次到批次的变化。合适的剂量应根据瘦身（原始重量的不大于15％），并确认结肠炎组织病理学。 </li></ol> 3.临床前试验在慢性结肠炎或结肠炎相关的癌症模型<ol><li>对于慢性结肠炎模型开始从步骤3.7。 </li><li>对于结肠炎相关癌症（CAC）的模式，在第1天，以10毫克/公斤（W / W）氧化偶氮甲烷（AOM，250微升，IP）注射各小鼠。 注:AOM是一种致癌物质，并应按照MSDS的指令来进行处理。 </li><li> AOM的储备溶液制备为10毫克/毫升的等分试样并储存在-20℃。对注射的当天，AOM的原液解冻，并稀释至1毫克/米升的PBS。应避免反复冷冻和AOM的原液解冻。 </li><li> AOM的剂量可以8-12毫克/公斤之间调整。常规批量测试需要建立相应的AOM剂量以规避毒性小鼠由于批次到批次的变化。 </li><li>在第1-7天以下的AOM施用权重将是稳定的，因为这个原因重量监控可以第1-7天期间被省略，以避免处理的动物，这将排泄的代谢物的细胞毒性在其粪便。 </li><li>在第7天全部寝具需要下列细胞毒性程序来改变。一旦被褥变化完成后，该动物不再需要的细胞毒性的处理程序，因为它们将不再被排泄细胞毒性代谢物。 </li><li>在第8天，制备2.5％（2.5克/ 100毫升）的DSS溶液（如在步骤2.5中描述），并提供给小鼠自由进食。 </li><li>每天称量小鼠在整个实验过程和监测疾病的迹象，包括竖起福R，拱起，便血和减少运动。 </li><li>第13天，取出DSS解决方案，并提供小鼠与正常饮水，直到28日8-28天构成慢性结肠炎/ CAC协议的一个"周期"。 </li><li>在13-28天，当小鼠给予正常饮水，提供小鼠捣碎食物颗粒和小皮氏培养皿（100克食物颗粒/ 10克蛋白质摇/ 10毫升的水）提供的蛋白质补充。这可以防止脱水，并协助最大限度地减少重症减肥。 注意:当小鼠接​​受DSS含饮用水，以确保DSS消费一致性蛋白补充剂醪不应该提供的。 </li><li>在20日进行的第二次内镜检查来监察个别鼠标的健康（见第5）。 </li><li>在29天的重复周期DSS（周期2:2.5％（重量/体积）DSS天34-50天期间29-33和正常饮水时提供）。 </li><li>在40日​​进行的THIRD内镜检查来监察个别鼠标的健康，并确定肿瘤负荷（见第5）。 </li><li>如果没有肿瘤是可见的内镜40日，开始在天50-72第三DSS周期（周期3:2.5％（重量/体积）DSS天56-72天期间50-55和正常饮水时提供）。通常，肿瘤是由40天可见，因此，决策支持系统的第三周期可以省略。 </li><li>一旦肿瘤变得可见通过内镜检查，分配给每个鼠标内窥镜肿瘤分数（见第5章 ），并指派个别小鼠同伙有类似的基线肿瘤负荷。 </li><li>第42天（或当肿瘤是可见的），管理治疗化合物或相关的车辆控制（参见2.2节）。剂量的定时将依赖于治疗剂的药代动力学曲线。干预治疗的一个例子的小鼠腹腔注射每周三次，提供建立的肿瘤（ 图2）。 </li><li>执行电子ndoscopic考试每周一次以上的治疗性治疗的持续时间来监测肿瘤负荷。通常，4周治疗性治疗是足够观察客观治疗反应。以下戒烟治疗，小鼠的队列也可以通过内窥镜对肿瘤复发监测。 </li><li>安乐死小鼠二氧化碳中毒和或当小鼠经历≥15％减重连续三天以上，以先到者为准收集组织进行生化和/或组织学分析72日的早晨。 </li></ol> 4.临床前试验的散发性结直肠癌型号<ol><li>在第1天，用10mg / kg的急性中耳炎（参见步骤3.2）注入每只小鼠的IP。 AOM的剂量可以8-12毫克/公斤之间调整。常规批量测试需要建立相应的AOM剂量以规避毒性小鼠由于批次到批次的变化。 </li><li>重复注射，每周的第一天对于以下5个周（ 图3）。小鼠应根据细胞毒性安全程序为整个6周处理。 </li><li>权衡对AOM注射当天每只小鼠以减少痛苦而引起反复操作时的小鼠。 注:AOM是一种致癌物质，并应按照MSDS的指令来进行处理。 </li><li>所有的动物应根据细胞毒性安全程序的6周需要这种协议AOM腹腔注射处理。 </li><li>在8周进行内镜检查，以监测为新兴的肿瘤（见第5）。内镜检查应进行每两个星期，直到肿瘤变得可见。通常，野生型C57BL / 6动物开始的AOM的初始给药后发展的肿瘤约40周。 </li><li>一旦肿瘤变得可见通过内镜检查，分配给每个鼠标内窥镜肿瘤分数（见第5章），并指派个别小鼠类似队列<html>之前治疗的肿瘤开始负担。 </li><li>在40周（或当肿瘤是第一个可见的），管理治疗化合物或相关车辆控制。 </li><li>剂量的定时将依赖于治疗剂的药代动力学特性（参见步骤2.1）。干预治疗的一个例子的小鼠腹腔注射每周3次，用被设置已建立的肿瘤（ 图3）。 </li><li>用于治疗性治疗周期的持续时间来监测肿瘤负荷进行内窥镜检查每周。通常，4周治疗性治疗是足够的客观治疗反应。治疗后停止，小鼠的队列也可以通过内窥镜肿瘤复发监测。 </li><li>安乐死的小鼠用CO 2中毒并在45周，或以下4周治疗收集组织用于结肠和肿瘤的生化和/或组织学分析。 </li></ol>ve_title"&gt; 5。内镜检查<ol><li>该内窥镜设备应当根据标准程序16进行组装。 </li><li>消毒和清洁，用70％的乙醇或抗细菌润滑剂的内窥镜探针。 </li><li>画可以使用笔记本或台式计算机和标准的媒体软件，如iMovie中被记录。一个标准的计算机监视器，而不是医用级监视器，足以显现内镜程序（ 图4）在结肠。 </li><li>麻醉5-6只小鼠的组同时与在100％O 2 3％异氟醚的腔室以0.2-0.4升/分的速率。一旦麻醉的小鼠，它是由脚趾捏确认;在异氟烷水平应改变以0.5-2％以进行维护。 </li><li>从室内除去个别的鼠标，并把它腹侧了它的头固定在鼻锥。充分麻醉应监视和后腿应调整，使它们有stretched淘汰落后的鼠标。 </li><li>放置在老鼠的眼睛兽医药膏，以防止干燥。 </li><li>持在那里它满足下脊椎清楚地揭示了肛门鼠标的尾部。插入硬性内窥镜鞘成水的烧杯中，并调整气流，以允许其中一个小气泡的释放的时间。 </li><li>气流将允许结肠膨胀。如果气流太强，空气将被泵入小鼠的胃。没有额外的润滑是必需的。 </li><li>小心地将硬性内窥镜套入直肠。通常，内窥镜能插入多达3厘米，在这一点，结肠小鼠曲线和不能访问的硬性内窥镜。 </li><li>在内窥镜检查程序的常见困难是获得结肠的内腔堵塞因粪便物。通过之前的程序轻轻按摩小鼠腹部鼓励排便，或精心操纵内窥镜周围的FECA避免这种在手术过程中升的问题。蠕动，也可能发生，在此期间，内窥镜应在适当的位置被保持到结肠肌肉松弛。 </li><li>启动视频录制在内窥镜程序的任何阶段。病的得分可以由有经验的助手在手术期间被记录，或从视频文件稍后的时间点。 </li><li>继内镜检查，返回老鼠笼子，并从麻醉恢复过程中监视他们。动物一般都是清醒和移动在2分钟之后删除所管理的异氟醚鼻锥。 </li><li>确保这些程序是由经验丰富的科学家进行的。每个内窥镜程序将每只小鼠约2分钟。 </li></ol> 6.疾病评分<ol><li>并监控结肠炎，得分视频的小鼠内镜结肠炎的严重程度（MEICS）结肠壁由透明表示的厚度指数16，其（i）中记载的改变，（ⅱ）大便稠度，（ⅲ）血管完整性和存在，（iv）对出现在粒状时尚溃疡和再生区，（v）的出血表示由纤维蛋白沉积（ 图5a）。 </li><li>监测肿瘤的负担，进球视频用于肿瘤的发病率和大小。肿瘤大小通过由肿瘤16（ 图5b）所占据的结肠腔的直径来决定。它通常使用既结肠炎和肿瘤评分参数为单个鼠标。用内窥镜，个体肿瘤和结肠黏膜的健康可随时间被监测，提供一个读出的新的治疗剂的成功在单个动物。 </li><li> （可选）静止图像可以从视频中提取的一代的代表性人物（ 图6）。 </li></ol>

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The authors have nothing to disclose.

The three protocols that are described outline methods of reliable and reproducible induction of colonic disease pathology in mice. When combined with routine endoscopic monitoring and the intervention strategies outlined here, these protocols will provide powerful pre-clinical insight into the efficacy of therapeutics. Our laboratories routinely use all of these protocols to monitor the success of novel therapeutics10,23,24.
There are a number of considerations when choosing a pre-clinical animal model to test new therapeutics. These include relevance of the model to the human disease, and the contribution of the tumor microenvironment to the proposed action of the therapeutic target. Here we provide three protocols for therapeutic intervention in established intestinal disease models. These models are reproducible and the delivery of reagents to induce disease is easy to manage. Importantly, the models are highly relevant to multiple facets and stages of colitis onset, and of tumor initiation and progression. Researchers should take into consideration the genetic background of the mouse strains used when designing experiments, as the susceptibility to disease induced by DSS and/or AOM can vary considerably25. In addition, different microbial communities may have different metabolic capacities in the context of AOM, which is metabolized by bacteria. We caution against using different cohorts of mice that were born into different animal facilities (including commercial vendors) in a single experiment. Similarly, the different microflora in mice used from different facilities may elicit different host responses to DSS-induced epithelial barrier damage11. Moreover, the appropriate analysis of tissue (for example RNA purification) should also be considered, since the ability of DSS to inhibit reverse transcriptase will impact on subsequent molecular analysis26,27.
Mouse endoscopy is a cutting edge technique to repeatedly monitor disease onset and progression in an individual mouse. The ability to record videos and extract still images permits easy monitoring of multiple disease parameters and tumors. In addition to improving animal welfare, endoscopic monitoring also reduces the need for multiple cohorts of experimental mice, which traditionally were culled at different time-points to track disease outcome. The MEICS scoring system is not a substitute for histopathological analysis, but provides an alternative means to monitor animal health and mucosal damage in live mice. Mouse endoscopy is a specialized laboratory technique, and all procedures should be performed by trained personnel to ensure appropriate manipulation and handling of the mice, as well as to provide consistent quality in the images used for disease scoring. In the hands of qualified personal, we have found that endoscopy induces little or no damage to the tumors that would cause intra-tumoral bleeding. For the therapeutic protocols outlined, we consider endoscopy highly advantageous, since it provides a way to determine the initial tumor burden, and allows us to group cohorts of animals with similar tumor burdens prior to the administration of a therapeutic drug. Sequential monitoring of the mice enables researchers to determine the efficacy of novel therapies early on, with the option of terminating unsuccessful experiments in a timely manner.
As our understanding of inflammatory bowel disease and colorectal cancers advance, new targets for therapy will be identified. Appropriate animal models will be integral to ensuring that the most promising new therapies are moved towards clinical trials.

结肠直肠癌（CRC）是恶性全世界1的4 个最常见的原因。尽管我们对这个疾病的家族基础上的理解显著的进步，遗传易感性不仅有助于对CRC的情况下2〜20％。其余都归因于大量的外在因素和环境因素，包括慢性炎症。在人类中，慢性炎症和结肠直肠癌之间的关联是溃疡性结肠炎（UC）的患者，谁具有显影结肠炎相关结肠癌（CAC）的更大的风险明显，取决于炎性疾病3的持续时间，程度和严重性-5。因此，新的治疗方法是在发展中，以控制的免疫反应和相关的生产的生长促进因子的炎症肿瘤微环境6-8。有适当的临床前动物模型中增加的需求来表征的治疗功效这些药物对发育和疾病的进展。 小鼠模型已经明确证实，炎性微环境有助于CRC进展，即使在没有明显的炎症9,10。这些模型包括使用多糖葡聚糖硫酸钠（DSS）的，在小鼠的饮用水提供到上皮损伤和急性和慢性炎性肠疾病（IBD）的11,12建模。虽然通过该DSS诱导粘膜损伤和结肠炎的机理还不完全清楚，一些研究表明，DSS抑制细胞的逆转录酶和核糖核酸酶活动的细胞内，或促进纳米脂质复合物，与结肠膜导致上皮损伤熔合形成13,14。修改标准DSS的模型还提供显著洞察由结肠上皮细胞维持组织稳态和单组机制晚黏膜免疫反应15。 氧化偶氮甲烷（AOM）的腹腔内给药单独，或与DSS的结合，提供了一个模式用于检查的体细胞突变之间的相互作用，在上皮粘膜和炎症和基质微环境16,17。 AOM是致癌物质1,2-二甲基肼（DMH）不直接导致DNA突变的代谢物。相反，AOM水解methylazoxymethanol（MAM）由细胞色素异构体CYP2E1在肝脏，在那里MAM缀合有葡糖醛酸，然后通过胆汁分泌物18输送到肠。据认为，该细菌β葡糖醛酸酶有助于MAM造成DNA烷基化的降解和突变的上皮细胞19的积累。大多数的AOM诱导的结肠肿瘤窝藏错义突变的基因中的编码β连环蛋白，渲染蛋白耐蛋白酶degradati上，这会导致经典Wnt信号通路20的异常活化。当AOM的活性是结合由DSS诱发的粘膜损伤，随后的伤口愈合反应创建一个微环境有利于的诱变上皮的生长和扩增。在此模型中的一个变型中，AOM的单独一段数周重复给药可用于模拟散发性结直肠癌，在没有DSS诱导的结肠炎的10,17。这两种模式的免费提供实验设置分别进行研究的CAC和零星的CRC，这两者都与一个促炎症肿瘤微环境10相关联。 利用小鼠序列内窥镜是由贝克尔和他的同事21首创，并允许结肠炎和肿瘤进展的纵向监测。在这里，我们提供了三个临床前的协议的基础上DSS诱发的粘膜损伤和/或AOM介导的涂铁道部感应可重复诱导特异性结肠病变。第一个协议描述诱发急性胃黏膜损伤响应DSS管理引发许多与IBD相关的病理特点。所述第二协议是基于DSS给药三个连续的周期，以模仿炎症在IBD患者中观察到的耀斑，并且可以进行结合AOM诱导突变。最终的协议是基于AOM诱导零星上皮突变。对于每一个这些协议中，我们在相关标准程序展开以包括我们已经开发并监控的新药物的疗效的预防和治疗性干预的方法。

<table><tbody><tr><td>Dextran sulfate sodium (MW 36,000-50,000)</td><td>MP Biochemicals</td><td>160110</td><td>Requires batch testing.</td></tr><tr><td>Azoxymethane</td><td>Sigma</td><td>A5486-100MG</td><td>Requires batch testing.</td></tr><tr><td>Vanilla protein shake</td><td>N/A</td><td>N/A</td><td>Available from hospital pharmacies.</td></tr><tr><td>Isoflurane</td><td>PPC</td><td>M60303</td><td>This is a restricted reagent, which should be stored under lock and key.</td></tr><tr><td>70% ethanol</td><td>N/A</td><td>N/A</td><td>Standard lab reagent.</td></tr><tr><td>Coloview miniendoscopic system</td><td></td><td></td><td></td></tr><tr><td>Endovision Tricam</td><td>Karl Storz</td><td>20212001-020</td><td></td></tr><tr><td>Xenon 175 light source with anti-fog pump</td><td>Karl Storz</td><td>20134001</td><td></td></tr><tr><td>HOPKINS straight Forward Telescope</td><td>Karl Storz</td><td>64301AA</td><td></td></tr><tr><td>Endoscopic sheath (total diameter 3 mm)</td><td>Kalr Stroz</td><td>61029C</td><td></td></tr><tr><td>Fiber optic light cable</td><td>Kalr Stroz</td><td>69495ND</td><td></td></tr><tr><td>Computer and media player software</td><td>Apple</td><td></td><td>iMovie</td></tr><tr><td>Scale</td><td>Any</td><td></td><td>Any scale suitable for weighing mice.</td></tr></tbody></table>

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.

瘦身用作标准参数以监视与结肠炎，这是经常用于监测小鼠的总体健康相关的疾病。动物一般保持自己的体重，而DSS-含有水的管理，只有开始减肥瘦身的时候都恢复正常饮水。可接受的减肥参数应建立符合该机构的动物伦理委员会。为了防止与长期腹泻相关的脱水，使用例行提供醪（食物颗粒磨碎并与饮用水混合），补充有除了正常饮用水的蛋白质抖动。 作为替代麻醉，氯胺酮/赛拉嗪或类似试剂都可以使用，如果异氟烷设备不可用。由于内窥镜的刚性性质，这些过程只允许的最远端3厘米的小鼠结肠的可视化。新的内窥镜用<html>额外的功能（包括柔性和荧光）可根据实验的需要。然而，由于决策支持系统的有害作用主要限于小鼠的远端结肠，用较温和的病理学在中间结肠观察到的，硬性内窥镜不妨碍监测个体小鼠的粘膜健康。虽然我们描述了一个协议，用于预防治疗急性DSS诱发结肠炎，这个协议可以很容易地修改，以测试干预治疗策略。旨在减轻上皮损伤和结肠炎的药物的功效可以在个体小鼠纵向进行监测，并根据所描述的评分参数（ 图5）定量。这是有利的，在传统的实验设计，其中实验方案中所需要的小鼠在特定时间点的剔除，而不允许该疾病响应于治疗随时间的表征。 <html>吨"&gt;在人体临床研究已经强调了在肿瘤如何不同的个体患者响应处理相当大的变化。在此处所描述的程序，提供给监控总体肿瘤​​负荷，以及个别肿瘤在过程中治疗响应的装置的实验，以考虑所概述的癌症模型中的干预协议不考虑到肿瘤发生疗法的效果，这是很重要的。预防性协议，与之前当肿瘤变得内镜可见提供了治疗，需要获得此信息。这里概述的协议提供上的上进展的治疗作用的信息从个人肿瘤（肿瘤大小测量）。肿瘤消退，也可以通过在可见的肿瘤数量的减少来表示。 G"/&gt; 图1:监视预防性治疗疗法的疗效的急性结肠损伤模型中的实验方案（一）需要8天，从开始到完成。治疗从第1天服用（二）预防性治疗。 DSS是在饮用水（c）从所述的实验方案的第3天提供的。内窥镜进行（D）监测动物的疾病进展。建议的时间点包括第0天（未处理）和第2天（健康监控），5日和8（以确定疾病负担）。在实验终止8.日（e）该早晨在野生型小鼠（F）的疾病的增加随着时间的进展。 <img alt="图2" src="/files/ftp_upload/52383/52383fig2highres.jpg" /> 图2:监控介入治疗的疗效模型Ø˚F结肠炎相关癌症的实验方案（一）需要72天从开始到结束。治疗剂（b）的从46天给予小鼠与已建立的肿瘤干预治疗。 AOM（c）的注入在第1天，和DSS设置在饮用水过度的实验方案的三个周期的过程中，开始在8内窥镜（d）中进行监测，动物的疾病进展日。建议时间点包括第0天（未处理），第20天（健康监控），和40天（至动物组根据肿瘤负荷）。内窥镜进行每周在治疗的过程中，以监测疾病的结果。实验（五）终止72日上午在野生型小鼠（六）肿瘤负荷增加，从40日起实施。 <img alt="图3"SRC ="/文件/ ftp_upload / 52383 / 52383fig3highres.jpg"/&gt; 图3:监测介入治疗的疗效的自发结直肠癌模型中的实验方案（一），需要&gt;50周从开始到完成。治疗剂（b）的施用给小鼠已建立的肿瘤干预治疗。 AOM（c）的注入在第1天，此后，连续6周注射在实验方案的过程中。内镜（d）中进行监测，动物的疾病进展。建议的时间点包括第0天（未处理）和第8周（肿瘤监测）和双周有后（建立肿瘤负荷）。内窥镜进行每周以上治疗过程中监测治疗结果。在实验（e）的终止在周50.在野生型小鼠（F）的肿瘤负荷增加从40周开始。 </<html> P&gt; <img alt="图4" src="/files/ftp_upload/52383/52383fig4highres.jpg" width="650" /> 图4:设备建立的实验设置（a）在所述内窥镜部，与单件设备的指示。的硬性内窥镜（b）中 ，与个别组分表示。比例尺= 2.5公分。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52383/52383fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52383/52383fig5highres.jpg" width="650" /> 图5:得分病参数经胃镜大纲（一）鼠内镜结肠炎严重程度（MEICS）指数。个别肿瘤得分参数的大纲（二）。ES / ftp_upload / 52383 / 52383fig5highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/52383/52383fig6highres.jpg" width="650" /> 图6:代表理疗服务代表减肥，内窥镜图像和分数为:（一）急性DSS诱导粘膜损伤（二）肿瘤，发达国家继AOM / DSS协议（三）肿瘤的发展遵循的顺序。 AOM协议。 N = 3只小鼠每组。 * P &lt;0.05，*** P &lt;0.001（学生t检验）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52383/52383fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice at JoVE.com

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

non-invasive-assessment-efficacy-new-therapeutics-for-intestinal

Universidad San Sebastian

Research

JoVE Journal

Medicine

9.9K 次观看.  The Walter and Eliza Hall Institute for Medical Research. 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.

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

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

使用小鼠化疗引起的粘炎模型评估小肠损伤和适应的重要端点和增殖标记

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

科研

癌症研究

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

医学

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

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

免疫与感染

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 ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

视频成像和时空地图来分析胃肠动力小鼠

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

神经科学

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 ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

毛里小肠内皮淋巴细胞的内质成像

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...