Name: murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation
Uploaded: 2014-08-26T14:00:00.000+00:00
Duration: 9 min 42 s
Description: Watch this Scientific Journal Video about Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation at JoVE.com

我们感谢宋佳Dufentester和埃尔克·韦伯的专家技术援助。我们感谢Faekah乔哈尔阿尤校对稿和Stefan布鲁克纳对医疗信息的支持。这项工作是由否则，克朗，费森尤斯-基金会（2012_A94）支持跨学科的资助。 D. Bettenworth是由医学院，WestfälischeWilhelms安大学明斯特的一个研究奖学金支持。 M.布鲁克纳是由德意志研究联合会（DFG SFB1009B8）的"Gerok"旋转位置的支持。 我们感谢海克百隆用于说明鼠标卡通。

所有的动物实验批准根据德国动物保护法的Landesamt献给自然色，UMWELT UND Verbraucherschutz（LANUV）。 1材料与实验装置<ol><li>动物保健<ol><li>使用任何应变体重20〜25克雌性或雄性小鼠，并根据当地的动物保护立法容纳他们。 </li><li>饲料的小鼠与特殊饲料对啮齿动物和应用苜蓿 - 自由饲料至少三天进行荧光检查，以尽量减少腔内自发荧光之前。 </li><li>蒸压提供饮水自由采食 。 </li></ol></li><li>诱导急性DSS诱导的结肠炎<ol><li>将3g决策支持系统于100毫升灭菌水溶液:准备3％（W / V）葡聚糖硫酸酯钠（36,000-50,000大DSS，分子量）。提供这种溶液作为唯一的饮用水的小鼠随意和计算将5ml每小鼠/天DSS的溶液。饲料续ROL小鼠灭菌水，无DSS 自由采食 9。 </li></ol></li><li>诱导大肠癌<ol><li>溶解诱变氧化偶氮甲烷（AOM）（注意！可能导致癌症和遗传损伤！）在无菌等渗盐水，得到1毫克/毫升的最终浓度。应用单剂量10毫克的AOM每公斤体重腹腔内使用1毫升注射器（30克）10。 </li><li>挑战小鼠（不包括对照小鼠）与3％（重量/体积）的DSS从第0天到7天，14天的重复周期，以21天28〜35和第42天至49以诱导炎性从动大肠致癌。只有在这些挑战（ 见图4A进行了详细的时间表）之间喂小鼠高压灭菌的水。蒸压水在整个实验饲料的对照组小鼠。 </li></ol></li><li>荧光内镜的制备（FE） <ol><li>利用荧光素Isothiocyanat（FITC） - 葡聚糖（分子量70000大; FITC标记:葡萄糖= 1:250）的对射探测器结肠腺瘤由相关的不典型增生的血管图案的视觉增强ction。 </li><li>施用60毫克FITC偶联葡聚糖静脉内5分钟前荧光内窥镜检查稀释于100μl的PBS中。 </li></ol></li><li>麻醉<ol><li>提供连续的异氟烷供给麻醉（1.5 LO 2 /分钟; 1.5-2％（体积）的异氟醚[2 -氯- 2 - （二氟甲氧基）-1,1,1 -三氟乙烷]）。使用特殊的兽医麻醉设备，口罩，以严格控制麻醉。 </li></ol></li><li>灌肠剂的制备<ol><li>灌输加入2ml液体灌肠剂（含量:磷酸氢二钠1.5％（重量/体积）和磷酸二氢钠11％（重量/体积））进入结肠如果显著粪便装载怀疑有可能会遮挡图。 </li></ol></li></ol> 2，技术设备<ol><li>使用已制定并批准为使用小动物胃镜兽医内镜工作站。连接WOrkstation到照相机单元，氙光源，空气泵和常规个人电脑监视器，用于白光内窥镜检查。然后连接相机和微型刚性望远镜（1.9毫米外直径，10cm长; 图5）。 </li><li>对活检钳或注射管的应用程序中使用的内窥镜护套与工作通道（ 图5D）。使用无护套工作通道进行诊断结肠镜检查。 </li><li>为荧光内窥镜检查配置光源的设置来激励二手示踪剂（ 例如，490纳米的FITC缀合的葡聚糖）。此外，整合的望远镜和照相机（ 例如，525纳米的FITC偶联的葡聚糖）之间的适当的带通滤波器。 </li><li>通过内窥镜获得的活检的工作通道插入挠性活检钳​​（3夏尔。28厘米）。 </li><li>通过工作通道引入灵活的注射管（0.96 mm）适用外用，粘膜内或腔内àdministration的诊断或治疗剂。 </li><li>使用可加热的体检表与42℃的温度。这可以防止老鼠变成低温在考试期间。 </li></ol>动物麻醉3。 <ol><li>放置鼠标在一个小而防漏盒和管理的异氟醚（100％（体积/体积），5％（体积），3升/分钟）。等到鼠标失去知觉。 </li><li>转移到鼠标的体检表进行内窥镜检查。继续通过面罩吸入异氟醚与剂量100％V / V，1.5％（体积），1.5升/分钟。始终适用眼膏，以防止眼睛干涩，同时在麻醉下。 </li><li>通过检查反射评价麻醉效果。检查"周围反射转向":如果充分麻醉，鼠标放置在它的后面不应该转身。检查"脚趾反射":当麻是足够的，软的捏在动物的脚趾之间不应该​​导致腿部撤离（阶段手术耐受性）。 </li></ol> 4，结肠镜检查<ol><li>打好麻醉小鼠易/它在体检表背面。 </li><li>管理2毫升灌肠通过搞定插管进入结肠，如果显著粪便装载怀疑可能掩盖的看法。待小鼠施用灌肠后大便。将内窥镜非常小心，以免穿孔。 </li><li>与它们中的一个被连接到所述空气泵打开护套的两个阀。密封阀门等用食指免除空气。结肠充气用的空气，慢慢地，仔细地，尤其是在情况下活检或注射。 </li><li>只有尽量提前内窥镜为右结肠弯曲，以避免穿孔（4-5厘米的肛门）。 </li><li>结肠镜检查诊断<ol><li>检查粘膜的炎症或恶性变化而拉回内窥镜。回力慢慢来评估肠的整个圆周。评估腔内PA使用适当的建立评分系统根据需要thologies。 </li><li>到保证期间卷绕区域的重复可视化图像采集相同​​内窥镜的位置，需要注意的鼠肛门粘膜损伤之间的距离。此外，使用活检钳的前端作为间隔物来实现的内窥镜和图像采集过程中的伤口区域之间的相同距离。伤口大小与内窥镜护套，其包括3毫米的尺寸。 注:将内窥镜在同一位置与以前的考试照片文档光盘比较。测量病灶在同一个角度和距离在每次随访内镜检查。 </li></ol></li><li>活检过程<ol><li>取活检与两名调查员的帮助。通过工作通道仔细介绍活检钳，直到镊子的顶端是在监视器的第二研究者上可见。小心翼翼地打开和关闭钳到虚空穿孔。 </li><li>动钳病理部位。 </li></ol></li><li>注射过程<ol><li>在两名调查员的帮助下完成注射程序。预充柔性喷射管（0.96毫米）完全配合剂进行给药。通过工作通道推管，直到套管（30 G）的显示器的第二研究者上可见。制备微细注射器轻轻管理的诊断或治疗剂所需的量。进样量应为50μL最大。 </li><li>将针头插入submocosa在15-30度的角度。面对斜面的黏膜方向。粘膜表明成功注射后的特性提升的迹象。 </li></ol></li><li>荧光内镜（FE） <ol><li>辖60毫克FITC标记的葡聚糖稀释于100微升的PBS静脉注射前，荧光内镜检查。 </li><li>检查注射之间的最佳时间点您的荧光标记的示踪剂和摄像程序，其是依赖于示踪剂药理学。根据所使用的示踪剂激发和发射波长配置的带通滤波器系统的设置。进行静脉注射荧光染料后，立即对非特定血液量的示踪剂的荧光镜检查（ 例如，FITC），以评估在粘膜表面的血管图案。 </li><li>考虑成像示踪剂后，申请好几个小时的情况下，有针对性的示踪剂或"智能探头"，提供给后台比一个更好的目标。 </li><li>执行结果的照片和视频文件。 </li></ol></li></ol> 5，后结肠镜检查<ol><li>在空置笼子分开鼠标，并将其放在纸巾保护鼠标从抽吸垃圾。暖鼠标采用了红灯灯，以防止体温过低。观察鼠标，不要将无人看管，直到它已重新获得足够的自觉维护胸骨斜卧。一旦完全清醒，请将鼠标回到各自的笼子里。 </li><li>在实验中，代替鼠标在一个小而防漏框的端部和管理的CO 2（100％（体积/体积），100％（体积），3升/分钟）。等到鼠标完全失去意识并停止呼吸。调度鼠标由股骨颈骨折。进行腹部剖腹探查术和外植体的结肠。纵向打开结肠和洗进一步的组织学和分子评价。 </li></ol>

<ol>
	<li>Bettenworth, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+18F-FDG+PET/CT+imaging+to+monitor+lesion+activity+in+intestinal+inflammation.">Translational 18F-FDG PET/CT imaging to monitor lesion activity in intestinal inflammation.</a> Journal of nuclear medicine : official publication, Society of Nuclear Medicine. 54, 748-755 (2013).</li><li>Lewis, J. S., Achilefu, S., Garbow, J. R., Laforest, R., Welch, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+imaging.+current+technology+and+perspectives+for+oncological+imaging.">Small animal imaging. current technology and perspectives for oncological imaging.</a> European journal of cancer. 38, 2173-2188 (2002).</li><li>Huang, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonoscopy+in+mice.">Colonoscopy in mice.</a> Surgical endoscopy. 16, 22-24 (2002).</li><li>Becker, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+colitis+and+colon+cancer+development+in+mice+using+high+resolution+chromoendoscopy.">In vivo imaging of colitis and colon cancer development in mice using high resolution chromoendoscopy.</a> Gut. 54, 950-954 (2005).</li><li>Becker, C., Fantini, M. C., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+colonoscopy+in+live+mice.">High resolution colonoscopy in live mice.</a> Nature protocols. 1, 2900-2904 (2006).</li><li>Neurath, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+tumor+development+and+wound+healing+using+endoscopic+techniques+in+mice.">Assessment of tumor development and wound healing using endoscopic techniques in mice.</a> Gastroenterology. 139, 1837-1843 (2010).</li><li>Pickert, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+links+IL-22+signaling+in+intestinal+epithelial+cells+to+mucosal+wound+healing.">STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing.</a> The Journal of experimental medicine. 206, 1465-1472 (2009).</li><li>Shapira, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+murine+laparoscopy+for+continuous+in-vivo+assessment+of+the+liver+in+multiple+disease+models.">Utilization of murine laparoscopy for continuous in-vivo assessment of the liver in multiple disease models.</a> Plos one. 4, e4776 (2009).</li><li>Wirtz, S., Neufert, C., Weigmann, B., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+induced+mouse+models+of+intestinal+inflammation.">Chemically induced mouse models of intestinal inflammation.</a> Nature protocols. 2, 541-546 (2007).</li><li>Neufert, C., Becker, C., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inducible+mouse+model+of+colon+carcinogenesis+for+the+analysis+of+sporadic+and+inflammation-driven+tumor+progression.">An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression.</a> Nature protocols. 2, 1998-2004 (2007).</li><li>Dieleman, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+experimental+colitis+induced+by+dextran+sulphate+sodium+(DSS)+is+characterized+by+Th1+and+Th2+cytokines.">Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.</a> Clinical and experimental immunology. 114, 385-391 (1998).</li><li>Gao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colitis-accelerated+colorectal+cancer+and+metabolic+dysregulation+in+a+mouse+model.">Colitis-accelerated colorectal cancer and metabolic dysregulation in a mouse model.</a> Carcinogenesis. 34, 1861-1869 (2013).</li><li>Foersch, S., Neufert, C., Neurath, M. F., Waldner, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endomicroscopic+Imaging+of+COX-2+Activity+in+Murine+Sporadic+and+Colitis-Associated+Colorectal+Cancer.">Endomicroscopic Imaging of COX-2 Activity in Murine Sporadic and Colitis-Associated Colorectal Cancer.</a> Diagnostic and therapeutic endoscopy. 2013, 250641 (2013).</li><li>Bremer, C., Ntziachristos, V., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical-based+molecular+imaging:+contrast+agents+and+potential+medical+applications.">Optical-based molecular imaging: contrast agents and potential medical applications.</a> European radiology. 13, 231-243 (2003).</li><li>Keller, R., Winde, G., Terpe, H. J., Foerster, E. C., Domschke, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+endoscopy+using+a+fluorescein-labeled+monoclonal+antibody+against+carcinoembryonic+antigen+in+patients+with+colorectal+carcinoma+and+adenoma.">Fluorescence endoscopy using a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen in patients with colorectal carcinoma and adenoma.</a> Endoscopy. 34, 801-807 (2002).</li><li>Jones, M. K., Tomikawa, M., Mohajer, B., Tarnawski, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+mucosal+regeneration:+role+of+growth+factors.">Gastrointestinal mucosal regeneration: role of growth factors.</a> Frontiers in bioscience : a journal and virtual library. 4, 303-309 (1999).</li><li>Mertz, H. R., Walsh, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptic+ulcer+pathophysiology.">Peptic ulcer pathophysiology.</a> The Medical clinics of North America. 75, 799-814 (1991).</li><li>Pantelis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sealing+with+a+fixed+combination+of+collagen+matrix-bound+coagulation+factors+on+the+healing+of+colonic+anastomoses+in+experimental+high-risk+mice+models.">The effect of sealing with a fixed combination of collagen matrix-bound coagulation factors on the healing of colonic anastomoses in experimental high-risk mice models.</a> Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie. 395, 1039-1048 (2010).</li><li>Burk, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+factor+from+a+transformed+cell+line+that+affects+cell+migration.">A factor from a transformed cell line that affects cell migration.</a> Proceedings of the National Academy of Sciences of the United States of America. 70, 369-372 (1973).</li><li>Msaki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+RelA+(p65)+threonine+505+phosphorylation+in+the+regulation+of+cell+growth,+survival,+and+migration.">The role of RelA (p65) threonine 505 phosphorylation in the regulation of cell growth, survival, and migration.</a> Molecular biology of the cell. 22, 3032-3040 (2011).</li><li>Zigmond, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+murine+colonoscopy+for+orthotopic+implantation+of+colorectal+cancer.">Utilization of murine colonoscopy for orthotopic implantation of colorectal cancer.</a> PloS one. 6, e28858 (2011).</li><li>Foersch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+VEGF+in+gastrointestinal+cancer+in+vivo+using+confocal+laser+endomicroscopy.">Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy.</a> Gut. 59, 1046-1055 (2010).</li><li>Mitsunaga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+endoscopic+detection+of+murine+colitis-associated+colon+cancer+by+topically+applied+enzymatically+rapid-activatable+probe.">Fluorescence endoscopic detection of murine colitis-associated colon cancer by topically applied enzymatically rapid-activatable probe.</a> Gut. 62, 1179-1186 (2013).</li></ol>

作者什么都没有透露。

上皮伤口的愈合是一个持续的过程。在胃肠道粘膜内连续生理剥离表层细胞的发生，要求上皮细胞16的频繁再生。因此，伤口愈合不良，对多种疾病产生巨大的影响，包括胃肠道溃疡和17吻合口漏18。可能只能不完全地进行在细胞培养系统中的体外 19,20评价的分子背景，以及潜在的候选药物，刺激上皮的愈合。因此，需要更复杂的实验装置如鼠结肠镜检查以?...

动物模型，极大地丰富了我们的许多肠道病变的认识。实验室小鼠（ 小家鼠 ）已成为生物医学研究的主要动物模型，由于其丰富的遗传和基因组信息，并在转基因和基因敲除株一应俱全。除了增进了解疾病的发病机制，动物模型也重要的是用于测试候选药物以及临床诊断或治疗性干预。然而，尽管有各种​​各样的小鼠模型模仿人类疾病，即在病人护理常规使用的许多诊断和介入选项不可用于小鼠。因此，监测策略，以监测小鼠疾病或治疗干预的效果的过程中往往局限于间接观测或验尸分析。虽然非侵入性程序监测小鼠存在活力样疾病活动指数，曲体重减轻或增加，血液，尿液和粪便antification分析，这些只是间接的指标，并通过个体间变异有偏见。此外， 验尸分析防止纵向观察，在重复的时间点。先进的成像技术来监测小鼠最近才推出1,2疾病活动。虽然这些成像技术允许重复的分析，他们只提供对肠道的描述，往往不准确的观点，并不能够直接可视粘膜或允许诊断或治疗干预措施，如活检收购或候选药物的局部和黏膜内的应用程序。 最近，已经开发出高分辨率内镜系统，在活体小鼠使用3,4。首次这些内窥镜技术允许的腔内结肠疾病的病状，如伤口愈合或肠道炎症提供物镜直接可视ective，实时状态，允许在相同的动物在重复的时间点的纵向研究。除了允许重复活检在个体小鼠，内窥镜系统还可以用于通过允许直接应用一种物质到感兴趣的区域，以治疗影响明显的肿瘤或局部炎症。此外，作为治疗和控制物质可以直接递送到感兴趣的区域，这可以在相同的小鼠进行的，不含个体间变异。这些系统现在已经被用于结肠的炎症，伤口愈合，腹腔镜肝脏活组织检查和原位诱导使用各种评分系统肝肿瘤8和肿瘤发展的评估，如结肠炎的严重性（MEICS）5-7的鼠内窥镜索引。 MEICS由五个参数来评估炎:结肠壁的增厚，血管格局的变化，纤维蛋白存在时，mucos粒度人面，大便一致性。 在这个协议中，我们描述了在肠伤口愈合，炎症和结肠癌的小鼠模型中使用刚性内窥镜。首先，我们证明伤口愈合和结肠炎症的内镜评估以及结肠炎活动的纵向评估和致癌的小鼠结肠的研究。除了 ​​描述性的使用鼠内镜，我们提供了使用内视镜仪器的详细说明，以取得活组织切片检查，并关注不同的组件（ 例如，候选药物或肿瘤细胞）的局部和黏膜内的应用程序。最后，我们证明了利用小鼠荧光内镜，它采用先进的分子成像技术，在结直肠肿瘤的设置。

<table><tbody><tr><td colspan="4">Reagents</td></tr><tr><td>Alfalfa-free diet</td><td>Harlan Laboritories, Madison, USA</td><td>2014</td><td></td></tr><tr><td>Azoxymethane (AOM)</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>A5486</td><td></td></tr><tr><td>Bepanthen eye ointment</td><td>Bayer, Leverkusen, Germany</td><td>80469764</td><td></td></tr><tr><td>Dextran sulphate sodium (DSS)</td><td>TdB Consulatancy, Uppsala, Sweden</td><td>DB001</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>E 4382</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>E 9884</td><td></td></tr><tr><td>Falcon Tube 50 ml</td><td>BD Biosciences, Erembodegem, Belgium</td><td>352070</td><td></td></tr><tr><td>Florene 100 V/V</td><td>Abbott, Wiesbaden, Germany</td><td>B506</td><td></td></tr><tr><td>Haematoxylin</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>HHS32-1L</td><td></td></tr><tr><td>Isopentane (2-Methylbutane)</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>M32631-1L</td><td></td></tr><tr><td>Methylene blue</td><td>Merck, Darmstadt, Germany</td><td>1159430025</td><td></td></tr><tr><td>O.C.T. Tissue Tek compound&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sakura, Zoeterwonde, Netherlands</td><td>4583</td><td></td></tr><tr><td>Omnican F - canula</td><td>Braun, Melsungen, Germany</td><td>9161502</td><td></td></tr><tr><td>Phosphate buffered saline, PBS</td><td>Lonza, Verviers, Belgium</td><td>4629</td><td></td></tr><tr><td>Sodium Chloride 0.9%</td><td>Braun, Melsungen, Germany</td><td>5/12211095/0411</td><td></td></tr><tr><td>Standard diet</td><td>Altromin, Lage, Germany</td><td>1320</td><td></td></tr><tr><td>Tissue-Tek Cryomold</td><td>Sakura, Leiden, Netherlands</td><td>4566</td><td></td></tr><tr><td>Vitro &amp;ndash; Clud&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>R. Langenbrinck, Teningen, Germany</td><td>04-0002&amp;nbsp;</td><td></td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>AIDA Control</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20 096020</td><td></td></tr><tr><td>Bandpass filter</td><td>Semrock, Rochester, USA</td><td>HC 716/40</td><td></td></tr><tr><td>Bandpass filter</td><td>Semrock, Rochester, USA</td><td>HC 809/81</td><td></td></tr><tr><td>Biopsy Forceps, 3 Fr., 28 cm</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>61071ZJ</td><td></td></tr><tr><td>Dell Monitor</td><td>Dell, Frankfurt am Main, Germany</td><td>U2412Mb</td><td></td></tr><tr><td>Examination Sheath, 9 Fr.</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>61029D</td><td></td></tr><tr><td>Examination Sheath, 9 Fr.</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>61029C</td><td></td></tr><tr><td>Fiber Optic Light Cable, 3.5 mm</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>69495NL</td><td></td></tr><tr><td>Fluorescein Blue Filter System</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20100032</td><td></td></tr><tr><td>Fluorescein Barrier Filter</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20100033</td><td></td></tr><tr><td>Foot switch</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20010430</td><td></td></tr><tr><td>HOPKINS Telescope, 1.9 mm, Length 10 cm</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>1830231</td><td></td></tr><tr><td>SCB D-light P&amp;nbsp;</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20 133720</td><td></td></tr><tr><td>SCB tricam SL II</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20 2230 20</td><td></td></tr><tr><td>Tubing set instruments VETPUMP II</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>69811</td><td></td></tr><tr><td>Tricam PDD PAL</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20221037</td><td></td></tr><tr><td>UniVet Porta</td><td>Groppler Medizintechnik, Deggendorf, Germany</td><td>BKGM 0451</td><td></td></tr><tr><td>Vetpump 2</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>69321620</td><td></td></tr></tbody></table>

murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation

小鼠模型被广泛用于研究人类疾病的发病机制和临床前评估诊断程序以及治疗干预。然而，病理性改变的有效评估的通常需要的组织学分析，并且当进行离体，就必须在动物的死亡。因此，在传统的实验环境中，个体内随访检查是几乎不可能。因此，在活体小鼠小鼠内窥镜的发展使得研究者首次两者直接可视化的胃肠道粘膜上，也重复监视变更的程序。众多的应用在体内鼠内镜存在，包括研究肠道炎症或创伤愈合，多次获得粘膜活检组织，并在本地管理使用微型喷射导管的诊断或治疗剂。最近，分子影像扩展了诊断成像月dalities允许使用特定photoprobes鲜明的靶分子的特异性检测。总之，鼠内镜检查已经成为一种新型的尖端技术， 活体成像诊断的实验和在各个领域的临床前研究可能显著影响。

在体内监测肠伤口愈合 （; 图1A等于1毫米）在例行胃镜检查，黏膜伤口是由微型活检钳，直径3法国机械诱发。随后，伤口愈合是通过日常的内镜检查监测和残余创面面积使用图像编辑软件， 如测量量化，ImageJ的（ 图1B）。个体伤口闭合时间的推移由实际创伤面积/初始伤口面积的商表示。例如，在卷绕代后3天，伤口面积的41％±4.1％...

Watch this Scientific Journal Video about Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation at JoVE.com

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

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

小动物成像技术允许串行诊断检查和治疗干预的体内 。近日，应用的范围也显著扩大，目前包括评估结肠肿瘤的发展，促进伤口愈合和监测炎症。该协议说明了鼠内镜这些不同的应用潜力。

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

murine-endoscopy-for-vivo-multimodal-imaging-carcinogenesis

Research

JoVE Journal

Medicine

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

18.6K 次观看.  University Hospital Münster. 小动物成像技术允许串行诊断检查和治疗干预的体内 。近日，应用的范围也显著扩大，目前包括评估结肠肿瘤的发展，促进伤口愈合和监测炎症。该协议说明了鼠内镜这些不同的应用潜力。 

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

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

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 ...

医学

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

生物工程

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 ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

多式联运定量相成像数字全息显微准确评估肠道炎症和上皮伤口愈合

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

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

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

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 ...

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

在小鼠中进行结肠镜引导捏活检并评估后续组织变化

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount ...

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into the cell and tissue scale dynamics that orchestrate intestinal regeneration. Here, we describe an intravital microscopy method that locally induces tissue damage at the single crypt scale and follows the regenerative response of the intestinal epithelium in living mice. Single crypts or larger intestinal fields were ablated by a high-intensity multiphoton infrared laser in a time- and space-controlled manner. Subsequent long-term repetitive intravital imaging enabled the tracking of the damaged areas over time and allowed for the monitoring of crypt dynamics during tissue recovery over a period of multiple weeks. Crypt remodeling events such as crypt fission, fusion, and disappearance were observed in the neighboring tissue upon laser-induced damage. This protocol enables the study of crypt dynamics both in homeostatic and pathophysiological settings, such as aging and tumor initiation.

Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

激光消融和活体显微镜研究肠道重塑

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into ...

鼠内镜

摘要

探索更多视频

此视频中的章节