Li Junkai, Ünal Can M, Namikawa Kazuhiko, Steinert Michael, Köster Reinhard W
Division of Cellular and Molecular Neurobiology, Zoological Institute, Technische Universität Braunschweig.
Department of Molecular Biotechnology, Turkish-German University.
J Vis Exp. 2020 Feb 14(156). doi: 10.3791/60793.
Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.
艰难梭菌感染（CDI）被认为是美国最常见的医疗保健相关胃肠道感染之一。针对艰难梭菌的固有免疫反应已有描述，但中性粒细胞和巨噬细胞在CDI中的确切作用尚不太清楚。在当前研究中，斑马鱼幼体被用于建立艰难梭菌感染模型，以在体内成像这些固有免疫细胞的行为和协作。为了监测艰难梭菌，已建立了使用荧光染料的标记方案。通过显微注射标记的艰难梭菌实现局部感染，其在斑马鱼肠道中活跃生长并模拟CDI中的肠道上皮损伤。然而，这种直接感染方案具有侵入性并会造成微观伤口，这可能影响实验结果。因此，此处描述了一种更具非侵入性的微量灌胃方案。该方法包括通过经开口口插管将艰难梭菌细胞直接递送至斑马鱼幼体的肠道。这种感染方法紧密模拟了艰难梭菌的自然感染途径。
