Benard Erica L, van der Sar Astrid M, Ellett Felix, Lieschke Graham J, Spaink Herman P, Meijer Annemarie H
Department of Molecular Cell Biology, Institute of Biology, Leiden University, Leiden, the Netherlands.
J Vis Exp. 2012 Mar 15(61):3781. doi: 10.3791/3781.
Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage. The site of micro-injection of bacteria into the embryo determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.
斑马鱼（Danio rerio）胚胎越来越多地被用作研究脊椎动物固有免疫系统在宿主 - 病原体相互作用中功能的模型。固有免疫系统的主要细胞类型，即巨噬细胞和中性粒细胞，在胚胎发育的最初几天形成，早于适应性免疫反应所需的淋巴细胞成熟。易于获得大量胚胎、由于外部发育而易于操作、胚胎和幼虫阶段的光学透明性、广泛的遗传工具、丰富的突变资源以及转基因报告系的收集，所有这些都增加了斑马鱼模型的多功能性。鼠伤寒沙门氏菌（S. typhimurium）和海分枝杆菌可在巨噬细胞内寄生，常用于研究斑马鱼胚胎中的宿主 - 病原体相互作用。比较这两种细菌病原体的感染过程很有趣，因为鼠伤寒沙门氏菌感染在一天内是急性且致命的，而海分枝杆菌感染是慢性的，并且可以在幼虫阶段进行成像。将细菌微注射到胚胎中的部位决定了感染是否会迅速扩散到全身或最初是否仍局限于局部。通过将细菌直接经尾静脉在后部血岛处或通过卵黄囊上连接心脏与躯干脉管系统的宽循环通道——居维叶氏管注入血液循环中，可以建立快速的全身感染。在1日龄时，此时胚胎具有吞噬活性的巨噬细胞但中性粒细胞尚未成熟，将细菌注入血岛是首选。对于在2 - 3日龄时进行注射，此时胚胎也已发育出功能性（产生髓过氧化物酶的）中性粒细胞，居维叶氏管是首选的注射部位。为了研究髓样细胞向局部感染的定向迁移，可以将细菌注入尾肌、耳囊或后脑脑室。此外，脊索是一种髓样细胞通常似乎无法到达的结构，但极易受到局部感染。对于高通量应用，一种有用的替代方法是在受精后的最初几个小时内将细菌注入胚胎的卵黄中。将荧光细菌与具有荧光巨噬细胞或中性粒细胞的转基因斑马鱼系相结合，为宿主 - 病原体相互作用的多色成像创造了理想条件。本文视频将描述用鼠伤寒沙门氏菌或海分枝杆菌对斑马鱼胚胎进行静脉内和局部感染以及随后对与固有免疫系统细胞相互作用进行荧光成像的详细方案。
