这项研究得到了防御威胁减少代办处 (DTRA) 根据合同 HDTRA1-14-C-0116 和卓越的中心在传染基因组学 (DOD #W911NF-11-1-0136)。

这项工作是严格按照国家卫生研究院的实验室动物护理和使用指南中的建议进行的。涉及啮齿动物的动物协议是由德克萨斯大学在圣安东尼奥机构动物保育和使用委员会 (IACUC) 根据协议 MU009 (RA) 批准的. 
 1. 准备导管、气管指示器和 F. tularensis 接种 <ol> <li> 准备导管 (20 g x 2 英寸) <ol> <li> 将 20 g x 2 针切割, 并将针头的尖端磨成钝而平滑的完成旋转工具. 
		注: 钝针的长度应允许金属针尖在完全固定在钝针上时, 将大约3毫米的导管套筒解除。钝针使导管结构刚性, 使导管在气管内放置, 并没有钝针尖端凸出从导管套筒防止损伤的气管. </li>
		<li> 用70% 乙醇清洗钝针和导管, 在紫外线照射下进行15分钟消毒. </li>
	</ol> </li> <li> 准备气管指示器 <ol> <li> 修剪一个1000和 #181; L 吸管尖端, 让尖端坐在导管口, 形成一个密封. </li>
		<li> 从200和 #181 中移除筛选器; l 气溶胶屏障吸管尖和在先前修剪过的1000和 #181 中的位置; l 吸管尖. </li>
		<li> 确保过滤器可以在1000和 #181 内自由移动; 当刀尖尖 tip-down 时, L 吸管尖. </li>
	</ol> </li> <li> f. tularensis 接种 <ol> <li> tularensis 在适当的琼脂板上过夜, 在37和 #176; 刮约100和 #181; 从琼脂板上的细菌草坪, 无菌接种环路和用于接种500毫升锥形烧瓶, 其中含有250毫升适当的液体生长培养基 (Fn: 胰酱油), 辅以 0.1% l-半胱氨酸盐酸盐一水合物, 并在37和 #176 夜间孵育摇晃; C. 
		注意: 视频中使用的 F. novicida 在胰大豆琼脂 (TSA) 板上生长, 并辅以 0.1% l-半胱氨酸盐酸盐一水合物. </li>
		<li> 在室温下将夜间生长的液体培养在 4221 x g 处离心10分钟, 除去上清液而不扰乱细菌颗粒. 
		注意: 请遵循机构和 #39 的生物安全建议, 放弃上清. </li>
		<li> 温和地 re-suspend 25 毫升适当液体培养基 (Fn: 补充 0.1% l-半胱氨酸盐酸盐一水合物) 的细菌颗粒, 使用25毫升吸管. </li>
		<li> 使用1毫升吸管, 分悬浮细菌培养成500和 #181; 等分在瓶子里, 在干冰/乙醇浴中冷冻, 然后在-80 和 #176 储存; C. </li>
		<li> 要确定冷冻培养瓶的效价, 请取出存储在-80 和 #176 上的两个冰冻小瓶; C、在冰上解冻并在磷酸盐缓冲盐水 (PBS) 中执行串行稀释, 然后在适当的培养基上电镀 (Fn: TSA 含有 0.1% l-半胱氨酸盐酸盐)。在37和 #176 中孵育 24-48 h; C 和菌落形成单元 (CFU) 被列举来计算冷冻培养瓶内细菌细胞的数量 (平均两个小瓶). </li>
		<li> 在冰上解冻冰冻小瓶, 然后用 PBS 将培养基稀释到最后浓度为 10 7 CFU/100 和 #181, 准备细菌接种;接种应在100和 #181 的浓度, 将产生期望的 CFU; L (在接种的最后浓度的甘油不能超过 3%, 以防止窒息的大鼠)。冷冻培养瓶储存在-80 和 #176; C 可在准备后六月内使用. </li>
	</ol> </li> </ol> 2。大鼠麻醉 <ol> <li> 将麻醉室连接到麻醉机的正常操作的异氟醚蒸发器. </li>
	<li> 将麻醉箱上的气体清理管连接到气体清除系统. </li>
	<li> 打开麻醉机上的氧气流量为4升/分. </li>
	<li> 将异氟醚蒸发器设置为 5%. </li>
	<li> 将老鼠放在麻醉室里. 
	注意: 我们通常使用 8-10 周大的老鼠 (130-180 克);年轻的老鼠很难通过这种技术接种, 因为小的口腔腔的大小. </li>
	<li> 在进行麻醉诱导时, 保持5% 异氟醚的大鼠, 2 升/分氧作用。这需要大约 3-10 min. </li>
	<li> 根据呼吸和心跳的质量和速率确定麻醉深度, 以及在脚趾夹紧试验中对反射刺激的反应。理想的麻醉深度是由 1-1.5 s 计数之间的每次呼吸. </li>
</ol> 3。气管内接种 <ol> <li> 将大鼠从麻醉室中取出, 并将鼠背放在啮齿动物插管支架上。将大鼠的前牙附着在支架上以保持老鼠的位置. 
	注意: 如果在手术过程中, 老鼠开始苏醒, 导管可以被移除, 老鼠回到麻醉室, 到达更深的麻醉面. </li>
	<li> 用惯用手将舌头移到同一侧, 用宽点修整拇指钳. </li>
	<li> 使用支持的手和喉镜对大鼠和 #39 的下颌, 并可视化动物的气管和食道。当老鼠呼吸时, 气管就会打开和关闭. </li>
	<li> 插入导管, 其中包含20口径钝针, 在步骤1.1 中准备, 放入气管。可能有轻微的阻力, 并插入气管可能和 #34; 颠簸和 #34; 由于导管摩擦气管软骨。由于湿气或浅呼吸, 气管可以覆盖的会厌, 防止可视化的气管。轻轻地触摸会厌软骨的边缘会导致软骨瓣打开并揭开气管. </li>
	<li> 从导管中取出钝针, 同时确保导管仍留在气管内. </li>
	<li> 允许几秒钟, 使老鼠能够在导管插入气管内呼吸. </li>
	<li> 牢牢地将气管指示器固定在导管的开口上。气溶胶屏障的运动将指示导管正确插入气管. </li>
	<li> 确保大鼠放置在插管支架上, 使动物的胸部朝向垂直于插管架的平面. </li>
	<li> 卸下气管指示器, 并提供100和 #181; 包含 tularensis 的接种 (10 7 CFU), 使用200和 #181; l 吸管尖, 将尖端固定在导管口上. </li>
	<li> 将1毫升滑尖结核注射器附在空气中, 并提供300和 #181; L 空气以确保接种到达鼠的肺部. </li>
	<li> 从气管取出导管, 从手术台上取出大鼠. </li>
	<li> 允许老鼠苏醒并回到笼子里。确保呼吸恢复正常. </li>
	<li> 确定 F. tularensis 中的 CFU 接种如步骤1.3.5 所述, 以确认 CFU 在气管内的传递. </li>
	<li> 重复步骤 3.1-3.9 接种 (na 和 #239; ve) 动物与100和 #181; L PBS 代替接种. </li>
</ol>

<ol>
	<li>Ellis, J., Oyston, P. C. F., Green, M., Titball, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tularemia.">Tularemia.</a> Clin Microbiol Rev. 15 (4), 631-646 (2002).</li><li>Lyons, C. R., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Francisella+tularensis+infection.">Animal models of Francisella tularensis infection.</a> Ann N Y Acad Sci. 1105, (2007).</li><li>Hutt, J. A., Lovchik, J. A., Dekonenko, A., Hahn, A. C., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Natural+History+of+Pneumonic+Tularemia+in+Female+Fischer+344+Rats+after+Inhalational+Exposure+to+Aerosolized+Francisella+tularensis+subspecies+tularensis+Strain+Schu+S4.">The Natural History of Pneumonic Tularemia in Female Fischer 344 Rats after Inhalational Exposure to Aerosolized Francisella tularensis subspecies tularensis Strain Schu S4.</a> Am J Pathol. 187 (2), 252-267 (2017).</li><li>Ray, H. 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作者没有透露

费希尔344大鼠正在成为兔疫苗开发的重要模型3。通过肺部通道暴露于f. tularensis对于演示对F. tularensis的武器化形式的有效性至关重要, 因为这些方法是作为气溶胶提供的。大鼠的气管内接种有助于大鼠肺部暴露于F. tularensis , 而不需要大型、昂贵和复杂的气溶胶发电设备。使用F. tularensis的选择代理形式的所有实验还需要 BSL3 的包容, 这通常发生在严格限制的空间中。因此, 此技术可将需要安装在该工作环境中的额外设备的数量降至最低。
因为 Fn 是用于录像的F. tularensis应变, 所以所有的技术都是在 BSL2 控制下进行的。这项技术的适应 BSL3 环境包括所有的程序在生物安全柜内执行的人员佩戴生物安全装备 (全罩, 动力空气净化呼吸器 (比峰), 保护罩所有与遮光罩, 双手套, 赃物),这些适应性降低了机动性、灵活性和能见度。气管指示器是一个重要的组成部分, 它允许确认导管正确放置在气管内, 考虑到在 BSL3 条件下工作时通常很难做出这种判断。
有解剖正确的老鼠 "模拟器", 有一个气管和食道, 这些都是有用的完善技术之前, 与活的动物。然而, 使用模拟器与使用活鼠不一样。一种方法是确定这种技术是否正确地在活的动物中进行, 是利用台盼蓝染料作为接种的麻醉大鼠, 并在手术后立即安乐动物。解剖肺组织和胃将揭示, 如果染料被交付到肺部, 而不是食道。通过这种技术接种了 Fn/Ft 疫苗的老鼠也可以在接种后不久进行安乐死, 并在肺组织上镀以确定肺内的实际沉积物。
正确的气管内接种将是重要的评估兔疫苗的有效性, 在 Fischer 344 大鼠, 但它也可能是有益的其他疫苗和/或治疗应用的大鼠, 包括生物。因此, 这种技术可以适应各种肺应用的大鼠模型。虽然气溶胶产生装置的交付可能更类似于 biothreat 的情况下, F. tularensis的气管内接种是兔疫苗开发的一个相对简单、cost-effective 的替代方案。

F. tularensis (Ft) 导致人类疾病, 兔。当细菌通过肺通路获得, 这导致肺炎兔, 有高发病率和死亡率1。由于与雾化表单相关的危险, F. tularensis被认为是 biothreat 剂, 目前在美国没有批准用于人类使用的疫苗。目前正在加紧努力, 研制抗肺兔的疫苗和治疗措施, 以保护人类不受非法使用这种细菌 biothreat。
由于小鼠对F. tularensis感染的极度敏感, 以及试剂的流行, 兔的研究大多集中在鼠标模型上。然而, 由于在这一模型2中展示疫苗功效的难度, 老鼠已经证明是疫苗开发的一个困难模型。最近, Fischer 344 鼠已被开发成一个模型的兔疫苗开发3。Fischer 344 大鼠的敏感性对各种各样的F. tularensis亚种模仿人的敏感性4, 并且鼠可以被保护免受F. tularensis肺挑战接种与活疫苗应变已知保护人5,6,7。由于费舍尔344大鼠模型的一些特点, f. tularensis感染的人, 它可能是一个非常有用的模型, 开发的疫苗, 以防止肺部的F. tularensis暴露。
一种有效的疫苗需要保护人类免受肺部接触到F. tularensis。最有可能的肺部暴露从武器化的F. tularensis将雾化细菌吸入肺部8。然而, 气溶胶生成的F. tularensis既危险又繁琐, 需要专门的设备和控制。在大鼠肺部暴露的另一条途径, 可能更适合于多实验室缺乏专门设备是通过气管内接种6。该技术利用喉镜正确放置在麻醉大鼠气管内的导管。在气管内的位置, 而不是食道, 通过一个简单的装置来证实肺部的气流。F. tularensis随后通过导管将注射器传递到肺部, 然后在导管中引入空气, 以确保细菌的肺部传递。相比之下, Jemski5以前报告说, 通过鼻腔通道将F. tularensis接种到 Fischer 344 大鼠体内, 直到3天 post-inoculation 才从肺部培养出来, 表明在大鼠鼻腔接种不会导致细菌直接输送到肺部。
选择f. tularensis的代理窗体 (f. tularensis亚种tularensis, f. tularensis 亚种holarctica) 要求安全级别 3 (BSL3) 控制程序, 这将防止录像。但是, F. novicida (Fn) 由于其在健康人中的无毒而免受选择代理状态的限制, 并且可在生物安全级别 2 (BSL2) 条件910下安全地使用。此外, Fn 还可作为减毒活疫苗的基础, 在通过气管接种的情况下, 可预防F. tularensis肺暴露, 其方法是:11,12,13。这里介绍的技术允许研究通过肺路线发生的感染, 利用大鼠作为人类的模型。这种技术可以在不需要专门的气溶胶产生设备的情况下进行。Fn 被用于这里拍摄的技术。

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intratracheal inoculation of fischer 344 rats with francisella tularensis

肺部感染的细菌土 tularensis可能导致严重的和潜在的致命疾病, 兔, 在人类。由于目前没有批准的人类兔疫苗, 研究的重点是利用适当的动物模型开发疫苗。费希尔344大鼠已经成为一个模型, 反映了人类对F. tularensis感染的敏感性, 因此是兔疫苗开发的一个有吸引力的模型。344只大鼠的气管内接种与F. tularensis模拟人肺暴露。成功交付到大鼠气管是至关重要的肺分娩。用带光照的喉镜对麻醉大鼠的气管进行适当插管;正确放置在气管内是由一个简单的装置来检测呼吸。在插管后, F. tularensis培养通过注射器在测量剂量内传递。这项技术规范了大鼠气管内的肺部分娩tularensis , 以评估疫苗的功效。

对大鼠气管内接种的体液反应可以通过酶联免疫吸附试验 (ELISA) 对紫外线灭活细菌进行测定, 如前所述, 11。在14和天 28 (图 1) 中, 对344大鼠的总免疫球蛋白 G (IgG) 反应进行了 post-intratracheal 接种, 并对 Fn (107 CFU 接种) 的减毒菌株进行了评估。模拟接种的大鼠接受 PBS intratracheally。相对于天真的模拟接种动物, 血清抗体滴对 Fn post-inoculation 的增加表明气管内接种的效果。低血清反应性可能表明气管插管不正确。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56123/56123fig1.jpg"> 
图 1: 在 F344 大鼠中, 对活减毒的 Fn 气管接种的总 IgG 反应.F344 大鼠血清 (n = 5) 接种 intratracheally 与活减毒 fn 应变 (107 CFU) 分析了总 IgG 水平反应与整个细胞 fn 在天14和天 28 post-inoculation。模拟接种 (天真) 大鼠 (n = 5) 接种 intratracheally 与 PBS。红色区域表示天真的血清的反应性 (F344 鼠模拟接种与 PBS)。误差线代表 SEM.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56123/56123fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis at JoVE.com

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

本议定书描述的是344大鼠气管内接种的土 tularensis.这个过程模仿人的肺暴露对这个潜在的 biothreat 代理并且可以用于测试疫苗和治疗疗效对肺兔。

费希尔344大鼠气管内接种土 tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the ...

intratracheal-inoculation-fischer-344-rats-with-francisella

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

7.1K 次观看.  University of Texas San Antonio. 本议定书描述的是344大鼠气管内接种的土 tularensis.这个过程模仿人的肺暴露对这个潜在的 biothreat 代理并且可以用于测试疫苗和治疗疗效对肺兔。

视频: Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

科研

免疫与感染

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

无创性气管内插管，以研究小鼠肺的病理生理

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

医学

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

舌下免疫治疗作为一种替代起到保护作用对急性呼吸道感染

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

插管介导气管内（IMIT）灌输:一种无创，龙专用输送系统

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

隔离方法弗朗西斯tularensis外膜

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

生物学

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

评估肺炎消退的实验模型

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

费希尔344大鼠气管内接种土 tularensis

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