Department of Pathology, University of Michigan, Ann Arbor.
Department of Pathology, University of Michigan, Ann Arbor;
J Vis Exp. 2021 Feb 11(168). doi: 10.3791/62093.
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.
肠黏膜由单层上皮细胞组成，形成一个动态屏障，允许营养物质和水经细胞旁途径运输，同时防止腔内容物中的细菌和外源性物质通过。这种屏障的破坏会导致对腔内容物的通透性增加，并招募免疫细胞，这两者都是肠道病理状态的标志，包括炎症性肠病（IBD）。由于缺乏允许定量分析的实验体内方法，因此，调节上皮屏障功能和多形核中性粒细胞（PMN）经上皮迁移（TEpM）的机制还不完全清楚。在这里，我们描述了一个强大的小鼠实验模型，该模型采用回肠或近端结肠的体外肠段。体外肠袢（iLoop）完全血管化，与通常用于研究跨上皮细胞单层通透性和PMN迁移的基于腔室的体外方法相比具有生理优势。我们详细介绍了该模型的两种应用：（1）通过检测荧光标记的葡聚糖在腔内注射后血清中的含量来定量测量肠通透性，（2）通过腔内引入趋化因子定量评估PMN穿过肠上皮迁移到肠腔中的情况。我们证明了该模型的可行性，并提供了使用 iLoop 的结果，结果表明与对照组相比，缺乏上皮紧密连接相关蛋白 JAM-A 的小鼠的 iLoop 模型可行。已经表明 JAM-A 可调节上皮屏障功能以及PMN 在炎症反应中的TEpM。我们使用 iLoop 的结果证实了先前的研究结果，并强调了 JAM-A 在调节体内稳态和疾病期间肠道通透性和PMN TEpM 中的重要性。iLoop 模型为肠稳态和炎症的体内重复性研究提供了一种高度标准化的方法，将极大地增强对肠道屏障功能和黏膜炎症的理解，如 IBD 等疾病。
