先天免疫反应中的斑马鱼幼体型号无创成像<em>链球菌iniae</em>感染
Summary
Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.
Abstract
水生病原体， 链球菌iniae，负责超过100亿美元的损失每年用于水产养殖业和能够引起全身性疾病在鱼类和人类。更好地理解S. iniae疾病的发病机制，需要一个合适的模型系统。遗传易处理性和斑马鱼的早期发育阶段的光学透明度允许对转基因品系的产生和非侵入性的成像用荧光标记的免疫细胞。适应性免疫系统不完全功能，直到数周后受精，但斑马鱼幼体有一个保守的脊椎动物先天性免疫系统与两个中性粒细胞和巨噬细胞。因此，幼虫感染模型的生成允许的先天免疫控制S.的具体贡献的研究iniae感染。
注射的部位将决定感染是否全身或局部开始。在这里，我们提出我们的协议耳泡注射的斑马鱼时效2-3天受精后，以及我们的技术感染荧光共焦成像。本地化感染部位可观察初始微生物入侵，招募宿主细胞和传播感染。使用S的斑马鱼幼体模型我们的研究结果iniae感染表明斑马鱼可用于检查主机嗜中性粒细胞和巨噬细胞在局部细菌感染的不同的贡献。此外，我们将描述如何免疫细胞photolabeling可用于感染过程期间跟踪单个宿主细胞的命运。
Introduction
链球菌iniae是一个主要的水生病原体，其能够引起全身性疾病在两个鱼和人1。而S. iniae负责的水产养殖业巨大损失，这也是一个潜在的人畜共患的病原体，能够与类似的其他引起链球菌人类病原体的临床病症引起的疾病免疫功能低下的人类宿主。鉴于其相似性与人类的病原体，它来研究S.是非常重要的在天然宿主的上下文iniae疾病的发病机制。 S的 成年斑马鱼模型iniae感染揭示宿主白细胞向感染的局部部位，以及一个快速时间来承载死亡健壮浸润，时间太短，涉及适应性免疫系统7。为了获得一个深入的研究，以S.先天性免疫应答iniae感染在体内 ，有必要使用一个模型，该模型是更适合为n对创实时成像。
幼虫斑马鱼具有许多优点，使得它越来越具有吸引力脊椎动物模型用于研究宿主 – 病原体相互作用。斑马鱼是相对便宜且易于使用和维护相比哺乳动物模型。适应性免疫并不功能成熟之前4-6周受精后，但幼虫具有高度保守的脊椎动物先天免疫系统与补体，Toll样受体，细胞因子，和嗜中性粒细胞和巨噬细胞具有抗微生物功能，包括吞噬作用和呼吸爆发2-6 8-11。此外，遗传易处理性和发展的胚胎和幼虫阶段的光学透明性允许对稳定的转基因品系的产生与荧光标记的免疫细胞使得能够检查实时体内宿主-病原体相互作用。使用photoconvertible蛋白质，这些转基因品系，如书房的产生dra2允许单个宿主细胞的起源和命运的超过12感染的过程中的跟踪。
当开发一个斑马鱼幼体感染模型，显微注射的选址将确定是否感染最初是局部或全身。全身血液感染入尾静脉或居维叶的导管是最常用的研究微生物病原体在斑马鱼和可用于研究在病原体菌株之间的毒力的主机和微生物细胞，细胞因子应答，和差之间的相互作用是有用的。对于生长较慢的微生物，早期注入的胚胎在16-1,000细胞阶段的卵黄囊可用于产生全身感染13,14，具有最佳的发育阶段对发现是间生长缓慢的微生物的显微注射在16到128细胞阶段15。但是，蛋黄许多微生物囊注射，以后主机的发展阶段往往是致命的吨他主持由于对白细胞浸润16-18的微生物和缺乏营养丰富的环境。
本地化感染通常会导致对感染的部位，可以很容易量化以非侵入性成像的白细胞的定向迁移。这种类型的感染可以允许介导白细胞迁移以及各种白细胞群体的不同迁移和吞噬能力调查机制的解剖。局部感染也是有用的研究中的细菌菌株的毒力之间的差异时，以及研究微生物入侵机制，因为物理主机的障碍必须越过为一个局部感染，成为全身。斑马鱼通常升高的温度下25-31℃的19，但它们也可以被保持在温度高达34-35℃的某些人类病原体侵袭的严格的温度要求的研究毒力20，21。
许多不同的位点已被用来产生一个初始局部细菌感染包括后脑心室22，背侧尾肌18，心包腔23和耳泡（耳）5，16，24。然而，已经发现，注射细菌进入尾肌可造成组织损伤的细菌中，当调查白细胞响应13可能扭曲的结果的和炎症独立。虽然少损伤与注入到相关的后脑，虽然它最初是在缺乏幼胚白细胞，后脑脑室稳步随着时间的推移获得更多的免疫细胞为小胶质细胞定居。后脑心室也是比较困难的位置到图像。耳囊是一个封闭的空腔，没有直接进入脉管25,26。它通常是缺乏列伊kocytes，但白细胞可以响应于炎症刺激如感染被招募到耳泡。它也是细菌微量注射在斑马鱼老化2-3天受精后，因为成像的易用性和喷射的可视化（dpf）的一个优选的位点。因此，我们选择了耳泡作为我们本地化细菌感染的部位。
Protocol
Representative Results
Discussion
此处所用的感染的方法是用于在2-3旦胚胎和幼虫的初始局部感染宿主的免疫应答的研究是有用的。炎症刺激，如感染，在一个封闭的腔体的焦点，如耳泡允许对嗜中性粒细胞和巨噬细胞趋化及吞噬的研究。注入细菌进入耳泡的一个需要注意的是中性粒细胞的能力，以有效地吞噬细菌在填充流体的腔可以依赖于特定的微生物。尽管大肠杆菌和枯草芽孢杆菌 ，不容易通过在耳泡28</sup…
Divulgations
The authors have nothing to disclose.
Acknowledgements
笔者想感谢实验室成员斑马鱼的保养和维护。这项工作是由美国国立卫生研究院，美国国家研究服务奖A155397到EA哈维和NIH R01GM074827安娜Huttenlocher支持。
Materials
| 1.7 ml eppendorfs | MidSci | AVSS1700 | |
| 14 ml falcon tube | BD Falcon | 352059 | |
| 27 G x 1/2 in. needle | BD Biosciences | 305109 | |
| 96 well plate | Corning Incorporated | 3596 | |
| Agar | BD Biosciences | 214030 | |
| CellTracker Red | Molecular Probes, Invitrogen | C34552 | |
| CNA agar | Dot Scientific, Inc | 7126A | |
| Disposable transfer pipets | Fisher Scientific | 13-711-7m | |
| Dissecting Scope | Nikon | SMZ745 | |
| DMSO | Sigma Aldrich | D2650 | |
| Ethanol 200 proof | MDS | 2292 | |
| Fine tweezers | Fine Science Tools | 11251-20 | |
| Gel comb | VWR | 27372-482 | 4.2 mm width, 1.5 mm thick |
| Glass bottom dishes | Custom made by drilling a 16–18 mm hole in the center of a 35-mm tissue culture dish bottom and placing a 22-mm round #1 coverslip in the hole and sealing with a thin layer of Norland Optical Adhesive 68 cured by UV light. | ||
| Glycerol | Fisher Scientific | G33-4 | |
| High melt agarose | Denville Scientific, Inc. | CA3510-6 | |
| Hydrogen peroxide | Fisher Scientific | H325 | |
| Laser Scanning Confocal Microscope | Olympus | with FV-1000 system | |
| Low melt agarose | Fisher | BP165-25 | |
| Magnetic stand | Tritech (Narishige) | GJ-1 | |
| Microinjection system | Parker | Picospritzer III | |
| Microloader pipet tips | Eppendorf | 930001007 | |
| Micromanipulator | Tritech (Narishige) | M-152 | |
| Micropipette puller | Sutter Instrument Company | Flaming/Brown P-97 | |
| Nanodrop spectrophotmeter | Thermo Scientific | ND-1000 | |
| N-Phenylthiourea (PTU) | Sigma aldrich | P7629 | |
| Paraformaldheyde | Electron Microscopy Sciences | 15710 | |
| Petri Dishes | Fisher Scientific | FB0875712 | 100 mm x 15 mm |
| Phenol | Sigma Aldrich | P-4557 | |
| Phenol Red | Ricca Chemoical Company | 572516 | |
| Phosphate Buffered Saline | Fisher Scientific | BP665-1 | |
| Potassium hydroxide | Sigma Aldrich | P-6310 | |
| Pronase | Roche | 165921 | |
| Protease peptone | Fluka Biochemika | 29185 | |
| Small cell culture dish | Corning Incorporated | 430165 | 35 mm x 10 mm |
| Sudan Black | Sigma Aldrich | S2380 | |
| Thin wall glass capillary injection needles | World Precision Instruments, Inc. | TW100-3 | |
| Todd Hewitt | Sigma Aldrich/Fluka Analytical | T1438 | |
| Tricaine (ethyl 3-aminobenzoate) | Argent Chemical Laboratory/Finquel | C-FINQ-UE-100G | |
| Triton X-100 | Fisher Scientific | BP151-500 | |
| Tween 20 | Fisher Scientific | BP337-500 | |
| Yeast extract | Fluka Biochemika | 92144 |
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