作者感谢乐郑氏博士和博士托宾慷慨地提供了α-catenin的：黄水晶鱼线和比尔·杰克曼允许我们做的拍摄在他的实验室。作者承认的资金来源国立卫生研究院（资助5P20RR016463，8P20GM103423和R15AI094406）和美国农业部（项目＃ME0-H-1-00517-13）。此稿件发布为主要农林试验站出版物号3371。

注：所有斑马鱼护理协议和实验按照根据机构动物护理和使用委员会（IACUC）协议A2012-11-03 NIH的指导方针进行。 1.斑马鱼饲养到4天受精后<ol><li>收集AB斑马鱼，或任何其他的转基因系中，第一3小时受精后内，如图另一视频34。 </li><li> 120孵育鸡蛋含有150ml E3媒体15cm的培养皿（5毫米氯化钠，0.17毫米氯化钾，0.33毫米氯化钙2; 0.33毫米氯化镁2 2毫米的HEPES，pH值6.8）与亚甲蓝（0.3微克/毫升决赛浓度）在33°​​C培养箱12/12光/暗光周期。 </li><li> 6小时与E3 + PTU（1-苯基-2-硫脲，10微克/毫升最终浓度）后更换介质并除去死卵。 </li><li>孵育4天，在33℃下，2天后交换用新鲜的E3 + PTU介质。 </li></ol> 2.jection微针准备<ol><li>拉硼硅毛细管（外径1.2毫米; ID0.69毫米）插入用针拔出器具有以下设置的微针（550热; 0拉; 130速度; 110时间）。 注：设置将有所不同，从灯丝到灯丝，得到类似形状的微针晶（ 见图1），并将需要重新调整每当一个新的灯丝安装。 </li><li>填充的微针与3微升的0.4微米过滤的磷酸盐缓冲盐水（PBS; 137mM的氯化钠; 2.7毫米氯化钾; 10毫的Na 2 HPO 4; pH值7.4; 1.8毫KH 2 PO 4）用20微升微加载移液器提示。 </li><li>设置在连接到一压力喷射系统的显微上的微量夹持微针。设置注射压力到30 PSI和脉冲持续时间为30毫秒。 </li><li>使用培养皿用蒸馏水，降低微针，直到针的1/5是在水中。夹在微针的WiTH略低于水位细镊子。使微针出来的水，并推踏板几次直到大丸剂出现。 </li><li>通过降低上层叠用一滴类型的浸油血球微针测量丸药的直径。调整的脉冲持续时间，以获得所需的体积（ 见表1）。 </li><li>为丸将背压保持稳定的油（降低的压力，如果推货量增加，增加的背压，如果药量减少）。记录脉冲的持续时间对于每个微针。 </li><li>通过使用微加载枪头吸它从微针除去PBS中并排出遗留PBS中通过将其背面上的注射器和翻转的连续脉冲开关。保留每个微针。 </li></ol> 3. 念珠菌准备<ol><li>连胜白色念珠菌转化密码子优化dTomato，GFP，BFP，EOS或远端红色从冷冻的储存（-80℃的库存中的25％的甘油），使用无菌的木销钉到酵母peptine葡萄糖（YPD）琼脂板（10克/升酵母提取物，将20g / L的蛋白胨20克/升的葡萄糖20克/升琼脂;高压灭菌并倾25毫升在培养皿中），并孵育过夜，在30℃。 </li><li>使用无菌木销钉挑选一个菌落，接种到5ml YPD液体（10克/升酵母提取物，将20g / L的蛋白胨20克/升葡萄糖的，高压灭菌并在无菌条件下等分在5毫升在16×150毫米的玻璃培养管）。 </li><li>孵育过夜，在30℃的辊筒在60rpm。 </li><li>收集1毫升C的白色念珠菌培养和移植到1.7毫升无菌离心管中。 </li><li>离心机在5000 XG一对夫妇秒。清空上清液，彻底地加入1毫升无菌PBS，旋涡。重复此两次。 </li><li>在无菌PBS 1000：通过稀释1毫升股票1计数使用血球殖民地。 </li></ol> 4.注射在斑马鱼在鳔<ol><li>用于在33℃的培养箱中30分钟，温热的注入培养皿（2％琼脂糖）。 </li><li>在所需的时间，从含有鱼用25毫升吸管没有吸引任何斑马鱼来注入15厘米培养皿取出的150毫升E3 + PTU 100。 </li><li>麻醉4单丝旦斑马鱼加入2毫升4毫克/毫升的缓冲三卡因甲磺酸库存（TR）至其余的50毫升介质（最终浓度200微克/毫升），并等待15分钟。 </li><li>在解剖显微镜下，用一个充气的鳔鱼的选择。鱼，也可以在那个时候筛选均匀的表型，如在MPO中性粒细胞的分布：使用表面荧光解剖显微镜GFP线。 </li><li>淡化C.白念珠菌库存从其原始浓度（ 步骤3.8），以1.5×10 7菌落形成每毫升（CFU / ml）的单元在PBS。 </li><li>转移30斑马鱼上使用塑料移送管注入培养皿TTE并取出多余的介质。 </li><li>用钓鱼线工具（0.012英寸直径的渔线超粘成硼硅毛细管），使3系的10条鱼，通过举办注射菜纵向和垂直定向的鱼。 </li><li>取出多余的介质。 </li><li>彻底涡管1.5×10 7 CFU /毫升C.白色念珠菌 。 </li><li>填写一个微针3微升的C.使用白色念珠菌新的微加载枪头，并设置脉冲持续时间，以适当的设置（见步骤2.9）。 </li><li>旋转注入培养皿进行的微针和鱼的头部之间20°的角度，针对向着鳔（ 图3A）的背面。 </li><li>推微针插入鳔，确保针的孔是在管腔，并按下踏板一次。 </li><li>重复每个鱼和充斥菜E3的转移到恢复盘（25毫升E3 + PTU）ð排空进入复苏菜。 </li><li>另外30鱼转移到注塑菜。 </li><li>重复注射了新的微针充满了彻底涡旋C.白色念珠菌 。 </li><li>完成注射PBS对照，如果需要的话，使用不同的喷射盘以避免污染，并转移到一个单独的恢复盘（ 步骤4.10）。 </li></ol> 5.筛选鱼的期望接种<ol><li>制备40毫升0.4％低熔点琼脂糖溶液（LMA）加入160毫克低熔点琼脂糖至40的E3毫升，通过微波直至溶解，并冷却到37℃沸腾。添加400微升的TR（最终浓度200微克/ ml）的一次冷却的。 </li><li> 30分钟后恢复，如所述（ 步骤4.3仅使用1毫升TR入25毫升的E3 + PTU，最终浓度200微克/毫升）麻醉的鱼。 </li><li>转让30鱼（用尽可能少的媒体越好）〜2毫升LMA的重量船。 </li><li>抽吸个体鱼用移液管和板用1滴的LMA的鱼到96井板成像，仅​​使用10×6的中央的孔中。 </li><li>重复，直到所有的鱼屏幕加载到成像板。 </li><li>让媒体在室温下固化5分钟。 </li><li>位置就在自己身边的鱼，确保他们在与玻璃底部接触。 </li><li>使用20X客观上倒落射荧光/共聚焦显微镜和适当的过滤器，记录C.数量白色念珠菌酵母细胞中的每条鱼的鳔。 </li><li>选择具有15-25酵母细胞的鱼在鳔（或期望的接种物），安乐死非选择鱼（TR在E3的800微克/毫升），并通过用50 E3 + PTU毫升选定鱼返回深培养皿加入3滴的E3到成像板选择的孔和用塑料移液管吸出鱼。 </li><li>孵育在33℃下进行所需的持续时间。 </li></ol> 6.成像鱼后筛选<ol><li>在所需的时间，除去25的50毫升E3 + PTU介质从含有鱼图像深培养皿。 </li><li>如所述（ 步骤4.3仅使用1毫升TR成25毫升E3 + PTU剩余，最终浓度200微克/毫升）麻醉的鱼。 </li><li>鱼板放入96孔成像板和位置适当（ 步骤5.3 - 5.7）。 </li><li>图像扫描使用适当的激光器和发射滤光片共聚焦显微镜。第一焦点上使用下微分干涉对比（DIC）的4X物镜鱼和取得的使用20X物镜与所述共焦激光扫描模式感兴趣区域的用2微秒/像素的速度和1024×600像素的宽高比的图像。收购Z-堆栈与1微米的步长，应用3帧的卡尔曼滤波模式。 </li><li>返回到深培养皿用50毫升E3 + PTU或进入individua升井（24孔培养皿，3毫升E3 + PTU）和孵化在33°C。 </li></ol> 7.剖析鳔<ol><li>填有10毫升注射器利用高真空润滑脂。 </li><li>准备2％LMA（ 步骤5.3琼脂糖8毫升E3 160毫克低熔点的）。 </li><li>在所需的时间，麻醉鱼（ 步骤5.2）。 </li><li>转移10鱼到1.7毫升离心管中，用1ml的E3，并通过添加200微升4毫克/毫升三卡因（最终浓度800微克/毫升）至离心管中，并温育15分钟，在室温下安乐死。 </li><li>通过将4滴真空润滑脂中的正方形，1厘米相距上的MicroSlide（75×25毫米， 图2）制备成像载玻片。 </li><li>将2滴2％LMA在广场中央。 </li><li>将一条鱼在一个单独的载玻片在解剖显微镜下，并确认心脏已经停止了跳动，去除多余的水分。 </li><l我&gt;使用细镊子解剖鳔通过保持头中与左镊子的固定位置和向下拉动前肠与右镊子（ 图4A）。 </li><li>拿起鳔通过气动导管与左镊子，它从消化道分离，并且将其放置在2％的LMA的中心其固化之前。 </li><li>放置盖玻片（18×18 mm）公摄像载玻片的顶部并推动它直到它触及真空润滑脂以及向LMA（ 图2）。 </li><li>在清扫10分钟的图像，如步骤6.4中所述。重复步骤7.7 - 7.11与其他鱼类。 </li></ol>

The authors have nothing to disclose.

在鳔显微注射疾病模型的研究进展和限制这里提出的模型是在Gratacap 等人描述的粘膜念珠菌浸没模型的扩展（2013）。它增加了一个控制感染时间的优点，一个高度重复性感染的剂量，并因此提高效率。我们在这里表明，允许感染动力学的非常详细的非侵入性颞文档以及鳔的更高分辨率离体成像的新方法。这些程序应有利于C的研究白色念珠菌 -in...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name</td>
			<td style="width:259px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL tubes</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deep Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR Scientific</td>
			<td>90000-726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>VWR Scientific</td>
			<td>90000-264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>VWR Scientific</td>
			<td>90000-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine tweezers (Dumont Dumoxel #5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wooden Dowels</td>
			<td>VWR Scientific</td>
			<td>10805-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melt Agarose</td>
			<td>VWR Scientific</td>
			<td>12001-722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate capillary</td>
			<td>Sutter Instruments</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPPI-3 Injection system</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Back Pressure Unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foot Switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine methane sulfonate</td>
			<td>Western Chemical Inc.</td>
			<td>MS-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scope</td>
			<td>Olympus</td>
			<td colspan="2">SZ61 top SZX-ILLB2-100 base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x microscope objective</td>
			<td>Olympus</td>
			<td>UPlanSApo 20x/0.75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller drum</td>
			<td>New Brunswick Scientific</td>
			<td>TC-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass culture tubes (16 x 150 mm)</td>
			<td>VWR Scientific</td>
			<td>60825-435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>VWR Scientific</td>
			<td>BDH4534-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR Scientific</td>
			<td>BDH4532-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>VWR Scientific</td>
			<td>BDH0246-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (Corning)</td>
			<td>VWR Scientific</td>
			<td>BDH4520-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Children clay (Play-Doh)</td>
			<td>Hasbro</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>VWR Scientific</td>
			<td>VW6276-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer (Hausser scientific)</td>
			<td>VWR Scientific</td>
			<td>15170-172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type A immersion oil</td>
			<td>Blue Marble Products</td>
			<td>51935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie</td>
			<td>VWR Scientific</td>
			<td>14216-184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose (Lonza)</td>
			<td>VWR Scientific</td>
			<td>12001-870</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fishing wire</td>
			<td>Stren</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well imaging plate (Sensoplate)</td>
			<td>Greiner Bio-One</td>
			<td>655892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease (Dow Corning)</td>
			<td>VWR Scientific</td>
			<td>59344-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microslide (25 x 75 mm)</td>
			<td>VWR Scientific</td>
			<td>48300-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips (18 x 18 mm), No 1.5</td>
			<td>VWR Scientific</td>
			<td>48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm Petri dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>32-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol (EMD chemicals)</td>
			<td>VWR Scientific</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well culture dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weight boats (8.9 cm)</td>
			<td>VWR Scientific</td>
			<td>89106-766</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

对粘膜病原体的早期防御由双方的上皮屏障和先天免疫细胞。两者的免疫竞争，他们的互通，是极为重要的保护，防止感染。上皮细胞和先天免疫细胞与病原体的相互作用被最佳研究在体内 ，其中复杂的行为展开在时间和空间。然而，现有的模式不允许与病原体战斗在粘膜水平易时空成像。 这里开发的模型创建粘膜感染通过直接注射的真菌病原体， 白色念珠菌 ，进入斑马鱼幼鱼的鳔的。所得感染使上皮和先天免疫细胞行为高分辨率成像贯穿粘膜病的发展。该方法的多功能性允许主机的询问来探测免疫事件导致的pH的详细序列agocyte招募和检查特定的细胞类型并在保护分子途径中的作用。此外，病原菌的免疫攻击的功能的行为可以同时使用荧光蛋白表达C.成像白色念珠菌 。所述宿主 - 病原体相互作用的增加的空间分辨率，也可以使用所描述的快速鳔夹层技术。 这里所描述的粘膜感染模型很简单，重现性好，使其成为粘膜念珠菌病的研究中一个有价值的工具。该系统还可以是大致平移的其他粘膜病原体如结核分枝杆菌，细菌或病毒的微生物，通常通过上皮表面感染。

在注射后鳔这里介绍的实验方法描述在注射C的一致剂量白念珠菌的酵母细胞在4鳔DPF斑马鱼。以前的工作与浸入式模型表明，到C的鳔免疫反应白色念珠菌是类似于哺乳动物粘膜念珠菌32。在这里，我们展示了修改后的感染的方法是比较简单的，重复性好，快速;几百斑马鱼的可以注射和内几个小时进行筛选。 所述鳔的准确?...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Research

JoVE Journal

Immunology and Infection

10.1K 次观看.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

视频: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

科研

免疫与感染

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

弥散性念珠菌感染模型

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

小鼠肺损伤的诱导,通过博霉素内切注射

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

通过注射药物加载的脂体将药物定位到拉瓦尔斑马鱼巨噬细胞

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...