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 -innate免疫系统的相互作用动力学的黏膜。粘膜感染车型数量有限，在斑马鱼进行了描述，都局限于消化道紊乱/感染24,43。这里提出的鳔模型扩展感染的位点，以共享一些相似之处至肺，并提供一个密闭部位的器官感染，对病原体较少可能性被冲走。 存在必须掌握这些技术时必须考虑到几个重要的因素。最重要的一点是用于充气鳔的要求。这就要求鱼被老化至5旦在28℃或4单丝旦在33℃。当饲养这个较高的温度用于加速鱼的发育和更好近似生理范围的哺乳动物上皮温度（小鼠皮肤温度为33.1℃，44）。然而，需要该模型这个特定的温度范围内的限制要牢记和观察到的表型应在哺乳动物系统中更完整的解释进行验证。注射部位（后鳔）也是关键的，因为在气动导管保持在physostomous鱼公开（包括斑马鱼）和高压注射别处挠度在器官可以推病原体过去鳔并进入消化道。接种物定量，也较不准确，如果酵母细胞不是都在鳔的背面，因为在后备箱鱼的厚度使得难以显现鳔的两侧。所述鳔的解剖需要灵巧和成像需要发生后立即以避免工件由于该过程的物理创伤。 该技术的局限性主要涉及鱼的发育阶段。这限制了使用吗啉代技术，研究了几天，和击倒效果，必须仔细测试全部或部分药效区别对待。吗啉这一活动在开发后期先前已报道，我们也成功地使用吗啉在这个模型中，用有效的剪接阻塞当时仍有效的为5 DPF。使用药业的acological药物可能是在这个特定的模型一个很好的选择，因为没有限制为鱼的年龄。过去72小时受精后使用鱼有时需要比幼虫斑马鱼的工作，这取决于机构的动物护理和使用委员会更多的监督。研究的长度也受限，如斑马鱼不以高速率对在静态实验室条件45超过10天存活。另一方面，使用5幼鱼旦发展，代替年轻胚胎或幼虫允许研究，其中所有的器官，如肾脏，肝脏或胰脏的功能完全正常46-49。相比在体外细胞培养或体内鼠模型，用（抗体和化学拮抗剂激动剂）试剂的数目，在斑马鱼被限制;然而，直接添加化学品，以媒体的可能性是有利的。敲除和基因敲除斑马鱼的数量仍然有限，但RISË有针对性的基因组编辑打开的突变体的产生令人兴奋的可能性。相对容易的组合来构造的荧光报告线（细胞类型特异性或信号通路特异性）与少年斑马鱼的透明度仍是该系统的主要和独特的优势之一。 期待斑马鱼的鳔感染模型扩展的问题的范围，可以询问有关在脊椎动物粘膜上皮宿主 - 病原体相互作用，提供感染通过上皮的入侵其他病原体的新途径，并强调该机构的实用程序，用于探测脊椎动物疾病。 感染上皮细胞在黏膜水平斑马鱼模型已经出版了探讨肠道紊乱的药物或病原体24,43的影响。然而，这种粘膜念珠菌病的模型是首次利用swimb梯，因此延伸感染的网站，可以在超出消化道斑马鱼进行调查的范围内。在鳔粘膜表面和哺乳动物肺之间的差异未完全阐明，并从这个模型更高的脊椎动物数据的解释/翻译要慎。 所述鳔感染填补当前间隙在脊椎动物感染模型和打开了一个窗口用于成像先天免疫/上皮细胞的相互作用与病原体。现在的问题，先前禁地一个不同的范围，是探索可行的。我们可以确定不同先天免疫细胞之间的相互作用与彼此以及与上皮细胞，其时空招聘，和C的性质白色念珠菌扩散和相对于免疫攻击和疾病进展中的非免疫抑制的宿主形态切换。的可能性，以图像的完整主机数小时至在GRE天在细节表示该系统的显着和重大的优势。 最后，鳔和哺乳动物肺之间的本体论和基因表达的相似进一步表明，针对本机关与基因敲除或化学药物可能揭示微生物龙互动和免疫50-52保守机制。

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><tbody><tr><td>1.7 ml tubes</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>Deep Petri dishes</td><td>Fisher Scientific</td><td>89107-632</td><td></td></tr><tr><td>Transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>Yeast Extract</td><td>VWR Scientific</td><td>90000-726</td><td></td></tr><tr><td>Peptone</td><td>VWR Scientific</td><td>90000-264</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Agar</td><td>VWR Scientific</td><td>90000-760</td><td></td></tr><tr><td>Fine tweezers (Dumont Dumoxel #5)</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Wooden dowels</td><td>VWR Scientific</td><td>10805-018</td><td></td></tr><tr><td>Low melt agarose</td><td>VWR Scientific</td><td>12001-722</td><td></td></tr><tr><td>Flaming Brown micropipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Borosilicate capillary</td><td>Sutter Instruments</td><td>BF120-69-10</td><td></td></tr><tr><td>MPPI-3 injection system</td><td>Applied Scientific Instrumentation</td><td>MPPI-3</td><td></td></tr><tr><td>Back pressure unit</td><td>Applied Scientific Instrumentation</td><td>BPU</td><td></td></tr><tr><td>Micropipette holder kit</td><td>Applied Scientific Instrumentation</td><td>MPIP</td><td></td></tr><tr><td>Foot switch</td><td>Applied Scientific Instrumentation</td><td>FSW</td><td></td></tr><tr><td>Micromanipulator</td><td>Applied Scientific Instrumentation</td><td>MM33</td><td></td></tr><tr><td>Magnetic base</td><td>Applied Scientific Instrumentation</td><td>Magnetic Base</td><td></td></tr><tr><td>Tricaine methane sulfonate</td><td>Western Chemical Inc.</td><td>MS-222</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZ61 top SZX-ILLB2-100 base</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>IX-81 with FV-1000 laser scanning confocal system</td><td></td></tr><tr><td>20X microscope objective</td><td>Olympus</td><td>UPlanSApo 20x/0.75</td><td></td></tr><tr><td>Roller drum</td><td>New Brunswick Scientific</td><td>TC-7</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Glass culture tubes (16 x 150 mm)</td><td>VWR Scientific</td><td>60825-435</td><td></td></tr><tr><td>NaCl</td><td>VWR Scientific</td><td>BDH4534-500GP</td><td></td></tr><tr><td>KCl</td><td>VWR Scientific</td><td>BDH4532-500GP</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR Scientific</td><td>BDH0246-500GP</td><td></td></tr><tr><td>HEPES (Corning)</td><td>VWR Scientific</td><td>BDH4520-500GP</td><td></td></tr><tr><td>Children clay (Play-Doh)</td><td>Hasbro</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>C69-500</td><td></td></tr><tr><td>Methylene blue</td><td>VWR Scientific</td><td>VW6276-0</td><td></td></tr><tr><td>PTU</td><td>Sigma</td><td>P7629-10G</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Hemocytometer (Hausser scientific)</td><td>VWR Scientific</td><td>15170-172</td><td></td></tr><tr><td>Type A immersion oil</td><td>Blue Marble Products</td><td>51935</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Vortex Genie</td><td>VWR Scientific</td><td>14216-184</td><td></td></tr><tr><td>Agarose (Lonza)</td><td>VWR Scientific</td><td>12001-870</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>Fishing wire</td><td>Stren</td><td></td><td></td></tr><tr><td>96 well imaging plate (Sensoplate)</td><td>Greiner Bio-One</td><td>655892</td><td></td></tr><tr><td>High vacuum grease (Dow Corning)</td><td>VWR Scientific</td><td>59344-055</td><td></td></tr><tr><td>Microslide (25 x 75 mm)</td><td>VWR Scientific</td><td>48300-025</td><td></td></tr><tr><td>Cover slips (18 x 18 mm), No 1.5</td><td>VWR Scientific</td><td>48366-045</td><td></td></tr><tr><td>15 cm Petri dish (Olympus plastics)</td><td>Genesee Scientific</td><td>32-106</td><td></td></tr><tr><td>Glycerol (EMD chemicals)</td><td>VWR Scientific</td><td>EMGX0185-5</td><td></td></tr><tr><td>24-well culture dish (Olympus plastics)</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>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。 所述鳔的准确和可重复的感染是由显微注射靶向器官的后部区域来完成的。注入鳔是更容易学习比幼虫或胚胎斑马鱼（ 如静脉或后脑心室），其他大多数显微注射路线。鱼注射前的准备是西米拉尔到，对于其他注射部位，不同之处在于在鳔需要被充气所以鱼是老年人。大约75％的在33℃下所提出的鱼有一个膨胀鳔由4旦（数据未显示）。需要被斜切的鳔位于下用于注入鱼的表皮以及薄肌肉层和微针。在该鱼被注入的角度（ 图3A）也大大改善了过程的速度和易用性。喷射弹丸在鳔的背面的是重要的，因为它允许所有筛选当酵母细胞的可视化，并且避免酵母细胞对鳔两侧的存在，这将歪斜的选择范围。注入朝向鳔的背面也避免了接种物通过气动导管，然后向下消化道一个可能发生的损失。验证该鱼已被注射相对容易，因为丸取代气泡和背面鳔的随后填充有介质（ 图3C）。另外，也可以注射时使用酚红，这将使得该可视化更容易。因为C的大小白念珠菌的酵母细胞（3 - 4微米），将接种的剂量被限制为低于5×10 7 CFU / ml的浓度，由于微针的堵塞。这使得注射酵母细胞1 250之间和在一个5 NL丸药。在我们的手中，注入4 NL在1-1.5×10 7 CFU /毫升给了最一致的结果。这就产生了一个范围每鱼10-50酵母细胞。缩小接种到15-25酵母细胞（ 图3B）给出了一个紧密的表型和响应。使用这些条件，喷射出的鱼的大约50％具有相应的接种物范围内具有95％以上的存活24小时后喷射（数据未示出）。另外，也可以用较低/较高的接种物保持鱼为了研究上的病原体的负担的效果疾病的发展。 所述鳔是约100 NL总体积（400×200×200微米，长x宽x高，用公式为椭圆体的体积），所以注射更高容量也是可能的（最多3个脉冲的4 NL一直使用而对鱼的生存任何显著影响;数据未显示）。 个别鱼纵向活体成像跟随主机和病原体的行为主机和病原体的行为可以遵循的非侵入性地使用该模型数天。的能力，以图像的感染在高分辨率体内发展能够与已被用来检查粘膜念珠菌病的发病机制。嗜中性粒细胞在抵抗在小鼠和人类35-38中发挥关键作用，以念珠菌病。利用MPO:GFP斑马鱼39（嗜中性粒细胞表达绿色荧光蛋白），和C.白色念珠菌表达dTomATO红色荧光蛋白32，我们已在早期感染（ 图4）观察neutrophil- 念珠菌动力学。 嗜中性粒细胞是第一先天细胞类型被招募到粘膜感染部位和鳔与现场C.感染白色念珠菌引起的中性粒细胞（ 图4A，B）的一个快速和连续的招聘。有趣的是，中性粒细胞基本上不招募到鳔当热灭活酵母细胞注射（ 图4A，B）。 长丝注射酵母细胞和C的后迅速发展白色念珠菌继续发芽为第一个48小时后喷射（HPI）。然而，该点的长丝的数量减少（ 图4D）和酵母的比例增加后（未示出）。的能力，以遵循个体鱼非侵入性地实现这些纵向研究（24孔板组织培养皿）并突出在疾病进展和宿主反应（ 图4C）的个体差异。这样详细的研究在感染部位数天列举真菌和免疫细胞仍不切实际的鼠标。 一种快速鳔切除术来改善影像成像免疫或上皮细胞与C的相互作用白色念珠菌在这里提出的模型比较容易。然而，在某些情况下，气泡的存在并围绕鳔，皮肤和肌肉的组织的厚度，损害详细的空间分辨率。以提高图像质量，我们已经开发出一种方法，以解剖鳔出斑马鱼和快速图像它（在10分钟内），通过共聚焦显微镜（ 图5A）的。成像的转基因鱼线，其中所述荧光普遍表达时，这是特别有用的且有多余的荧光在周围组织，如NF-κB:GFP 40或α-catenin的:黄水晶线41,42（ 图5B，C）。该技术需要灵巧和实践进行迅速，但它是可行的，完全图像的95％的所有尝试的鱼。 <img alt="图1" src="/files/ftp_upload/52182/52182fig1highres.jpg" /> 图1:微针形状鳔注射。微针的形状和测量的（A）的综述。从A的微针尖端的（B）的放大倍数在乙每个主要垂直线为0.2毫米。 <img alt="图2" src="/files/ftp_upload/52182/52182fig2highres.jpg" /> 图2:成像幻灯片 。有2％的LMA滴被放置在4个点的真空润滑脂，1厘米相距在玻片上的中间。在狄氏CTED鳔被放置在LMA的中部和盖玻片被降低上顶，直至它与在LMA与真空润滑脂接触。 <img alt="图3" src="/files/ftp_upload/52182/52182fig3highres.jpg" /> 图3:注射白色念珠菌在鳔仍然局部到注射部位。 （A）的微针注射白色念珠菌进入鳔的背面在4 DPF斑马鱼幼虫的方向和位置的示意图。（B）的荧光显微镜选择为15-25酵母细胞的注射小丸后代表接种3组不同的鱼。平均值和平均值的标准误差来表示，N = 17，17，和20，用于组A，B和C分别。（C）的代表性的C的图像白色念珠菌立即仁济后（氟化钙，dTom，红色荧光表达念珠菌）感染AB斑马鱼幼鱼ction。 （分别为左和右面板，）图像获取用10X和20X的目标，并且在红色通道（12和19片，分别地）与在DIC通道的单个切片最大突起的复合材料。气泡（黄色）和上皮衬里（白色）的轮廓表示在左侧面板上。比例尺代表250和50微米，分别<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52182/52182fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52182/52182fig4highres.jpg" /> 图4:注射鳔允许宿主病原体动力学时空清扫。 （A）MPO的代表形象:GFP鱼线4旦注射活的或热灭活（HK）氟化钙，dTom在鳔和成像48 HPI。图像最大投影上的绿色复合材料第二红色通道（20片），并用DIC通道单个片。刻度条表示100μm的（B，C）。主机响应于C.白色念珠菌感染。中性粒细胞招募感染（SOI）早在6 HPI的网站，并继续在数量上增加了48小时。的可能性的图像，并保持鱼个体分开（24孔板）允许进行详细的免疫动力学来实现可视化。（D）的病原体反应。C.白色念珠菌酵母细胞发芽的第24 HPI但是从24 HPI恢复到酵母细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52182/52182fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52182/52182fig5highres.jpg" /> 图5:鳔夹层允许更精细的细节将被成像。吨的（A）示意图他参与的步骤在4-5 DPF斑马鱼的鳔解剖。蓝色箭头指示的与所述镊子（L代表左和R为右）拉动方向（B，C）。解剖鳔从α-catenin的代表图像:黄水晶鱼线41,42（绿色紧密连接）注入4 DPF白色念珠菌（氟化钙，dTom）和共聚焦显微镜2 HPI成像。板是在红色和绿色通道的最大突起的复合物（25切片为B和3片为C）和用于DIC在面板B.比例尺单个片100微米B和100微米和10微米的三<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52182/52182fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>体积（NL） </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td>五</td></tr><tr><td>直径（mm） </td><td> 0.124 </td><td> 0.156 </td><td> 0.179 </td><td> 0.197 </td><td> 0.212 </td></tr></tbody></table> 表1:丸体积与直径的关系。

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

Universidad San Sebastian

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

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

发育生物学

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

破译和成像发病机制与盘带<em&gt;脓肿分枝杆菌</em&gt;斑马鱼胚胎

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

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.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

为梭状体开发拉瓦尔斑马鱼感染模型

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

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 ...

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 ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

脑室微注射脂多糖到斑马鱼幼虫中以评估神经炎症和神经毒性

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

神经科学

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Source: Thrikawala, S. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2020)
The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

一种可视化真菌病原体感染斑马鱼幼虫的 zWEDGI 技术

1. Preparation of&#160;Aspergillus&#160;spores for injection

	From an&#160;Aspergillus&#160;spore suspension, calculate the volume needed to obtain 1 x ...

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

摘要

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此视频中的章节

播散性念珠菌在斑马鱼幼虫的非侵入性成像

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

破译和成像发病机制与盘带

为梭状体开发拉瓦尔斑马鱼感染模型

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

marinum 分枝杆菌感染成人斑马鱼结核的建模

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

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用

脑室微注射脂多糖到斑马鱼幼虫中以评估神经炎症和神经毒性

一种可视化真菌病原体感染斑马鱼幼虫的 zWEDGI 技术

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

摘要

探索更多视频

此视频中的章节

播散性念珠菌在斑马鱼幼虫的非侵入性成像

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

破译和成像发病机制与盘带

为梭状体开发拉瓦尔斑马鱼感染模型

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

marinum 分枝杆菌感染成人斑马鱼结核的建模

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

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

脑室微注射脂多糖到斑马鱼幼虫中以评估神经炎症和神经毒性

一种可视化真菌病原体感染斑马鱼幼虫的 zWEDGI 技术

斑马鱼幼虫感染阿斯珀吉卢斯孢子分析宿主-病原体相互作用