Name: intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation
Uploaded: 2016-03-02T15:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.

所有实验均按照贝勒研究院的机构动物护理和使用委员会（IACUC）的指导方针进行。 1.培养不可分型流感嗜血杆菌 （NTHi的），并准备接种<ol><li>在巧克力琼脂平板板的NTHi和在37℃和5％CO 2在加湿的 CO 2培养箱中保持板倒置过夜。翌日，培养在脑心脏浸液肉汤的细菌。然后，加入1毫升肉汤进入无菌1.5毫升管。 </li><li>离心以4,000 xg离心在室温下5分钟。然后，弃去上清液并在1ml无菌1×PBS中重新悬浮沉淀。重复此步骤一次。 </li><li>在分光光度计转移100微升的再悬浮溶液注入900微升1×PBS中的与测量稀释的培养的吸光度在600nm处的波长。 </li><li>确定的生存能力和集落形成单位秒（CFU的）接种的通过培养在巧克力琼脂平板上1点10系列稀释，并在37℃下温育过夜，5％ 的 CO 2。 </li><li>基于从前面的步骤确定的生存能力和的CFUs，调整接种物，以20×10 6 CFU / ml的终浓度。 </li></ol> 2.气管内（IT）滴注<ol><li>用一个150毫克/毫升氯胺酮+ 20毫克/毫升甲苯噻嗪液取0.1毫升/ 10克体重小鼠注入小鼠腹腔内（IP）。该过程应在通风橱中进行与光源保持动物保暖。 </li><li>捏住鼠标的叉指空间，以验证该鼠标充分麻醉。 </li><li>将在它的后面鼠标。剃颈部腹侧区域，并与氯己定和70％的乙醇消毒。 </li><li>解除用鼠齿钳的颈部的上部腹侧部分的皮肤上。做一个小切口（0.5〜1.0厘米）胸腺上面用手术刀然后小心地解剖肌肉以暴露气管。 <ol><li>用1ml注射器用27克针，缓缓注入0.05毫升空气，接着从第1或盐水作为对照获得0.05毫升的NTHi，接着另一0.05毫升空气。保持动物的垂直约1分钟。 </li></ol></li><li>关闭与手术胶水切口。按住鼠标横向躺着和温暖约10分钟。 </li></ol> 3.收集肺泡灌洗液<ol><li>牺牲颈椎脱位24小时小鼠感染后。 </li><li>打开解剖剪鼠标的腹腔和切腹主动脉流血。揭露肺的腹一部分。 </li><li>暴露气管并用20号的导管插管它。灌输0.8毫升BALF缓冲液（1×PBS中，0.1％葡萄糖，10 U / ml肝素），并收集轻轻吸出灌洗。重复此步骤三次。 </li><li>算在100μlBALF悬浮液用血细胞的细胞的总数在放大10倍与台盼蓝染色米。 </li><li>准备修改姬姆萨染色幻灯片。等份加入100μlBALF进入细胞离心涂片和离心机在500×g离心5分钟的适当的孔中。干燥10分钟的幻灯片，并根据制造商的协议使用改性Giemsa染色细胞染色。 </li><li>使用剩余的细胞用于随后的分析，例如荧光激活细胞分选（FACS），共焦显微镜，基因表达和蛋白质印迹11-13。 </li></ol> 4.组织病理学准备<ol><li>对于组织病理学分析，感染与NTHi的小鼠如在步骤2.4中描述。感染后的24小时，通过颈脱位牺牲动物。切断与解剖剪刀以暴露肺和如前所述气管打开胸腔。然后，将20号导管插入气管。 </li><li>填充在4％福尔马林制备2％温暖琼脂糖溶液的肺部。一旦肺完全膨胀，地方冰对肺部的顶部，以巩固琼脂糖溶液。 </li><li>然后，小心地取出胸插头，把它在1X PBS 4％福尔马林50毫升的解决方案，直到它被用于苏木精伊红（H＆E）的部分进行处理。 </li></ol> 5. FACS BALF中染色的细胞<ol><li>把从BALF流体1×10 5个细胞在96孔V形底微量滴定板。 </li><li>在4℃下洗涤细胞用冷的1×PBS中的300微升和自旋在4000 xg离心3分钟。 </li><li>染色与细胞染色液细胞在100μl1×PBS中（1:1000稀释度），并保持在室温下15分钟。 </li><li>在4℃下洗涤细胞用300μl冷1×PBS中的在4000 xg离心3分钟。 </li><li>保持细胞在100μl含有1 FACS洗涤缓冲液[洗涤缓冲液（1×PBS，2％FBS，1mM EDTA中，0.1％叠氮化钠]的:100稀释的Fc块在4℃下15分钟。 </li><li>离心细胞以4000×g离心3分钟，并弃去SUPernatant。 </li><li>染色的细胞用100μl表面染色鸡尾酒（1:100稀释）在FACS洗涤缓冲液在4℃，30分钟。 </li><li>在4℃以4000 xg离心用300μlFACS洗涤缓冲液中洗涤细胞三次3分钟。 </li><li>固定的细胞在100μl的4％多聚甲醛在室温下15分钟。 </li><li>在4℃下用300μl1×PBS中洗涤细胞两次，在400×g离心3分钟，并在300微升的FACS洗涤缓冲液中重新悬浮。 </li><li>请使用流式细胞仪珠单色染色控制。 <ol><li>添加珠粒一滴到96孔V形底微量滴定板并用200μl1×PBS洗涤。 </li><li>加入1μl染色抗体与100μl的1×PBS中并珠和在室温下孵育5分钟。 </li><li>用300μl1X PBS洗两次珠。 </li></ol></li><li>通过流式细胞仪分析14单染色控制和细胞。 </li></ol> 6.均质肺组织来隔离RNA <ol><li>感染后，收集一小块肺（20-40毫克），并保持它的RNA在4℃稳定试剂过夜。然后，在-80℃储存，直到下一个步骤。 </li><li>用冷1X PBS肺组织摆脱RNA稳定试剂。 </li><li>放置组织到1.5毫升离心管中含有裂解缓冲液（1毫升的裂解缓冲液+10μl的β巯基乙醇）。 </li><li>使用尖剪刀切碎的组织切成小块并采用无绳马达粒料杵20-40秒均质。 </li><li>保持在冰上裂解5分钟，并与步骤7.1进行。 </li></ol> 7.从肺RNA分离<ol><li>离心溶胞产物18000 xg离心3分钟。通过移液除去上清液，并仔细转移到一个新的1.5毫升管。 </li><li> 1体积的70％乙醇加入到裂解物。吹打立即混匀，等待2-4分钟。 </li><li>转移到700微升裂解物以置于旋转柱在2 ml收集管。 </li><li>离心机以9,600×g离心15秒，弃去流过。 </li><li>加入350微升严格的洗涤液，离心9,600 XG为15秒。丢弃的流量通过。 </li><li> 80微升DNA酶添加我直接向旋转柱膜并保持在室温下15分钟。 </li><li>添加350微升严格洗涤缓冲液到旋转柱和离心机以9,600×g离心15秒。丢弃的流量通过。 </li><li>以9,600 XG加入500μlRNA洗涤液，离心15秒。丢弃的流量通过。 </li><li>以9,600 XG加入500μlRNA洗涤液和旋转2分钟。丢弃的流量通过。 </li><li>放置在新的2ml收集管中，离心旋转柱以9,600 xg离心1分钟。丢弃的流量通过。 </li><li>直接以9,600 xg离心添加30-50微升不含RNA酶的水，以旋转柱，离心1分钟。在-80°C储存RNA。 </li><li>执行RNA测序分析14。 </li></ol>

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The authors have nothing to disclose.

在此，我们描述了一个独特和微创手术用细菌肺病原体接种的小鼠的肺。我们证明，这个过程可用于研究是否可使用那些在炎症信号传导途径的基因缺陷小鼠不同基因的功能。这个过程也可以用来研究病毒和真菌的肺部感染的炎症反应。此过程在其他方法，例如鼻内或气溶胶吸入的优点是:（1）在此过程中，致病性接种物被直接滴注至下呼吸道; （2）来控制接种物大小的能力;和（3）减少细菌暴露...

炎症是针对传染剂的防御的基本免疫机制。它促进了消灭病原体和受损组织的修复。它还有助于恢复到正常的健康状态1。然而，失调炎症常常导致慢性炎症性疾病2。气道炎症是对于不同的肺部疾病，如慢性阻塞性肺疾病（COPD），哮喘和肺纤维化3的初始触发。 的不可分型（未包囊） 流感嗜血杆菌 （NTHi的）与慢性上下肺部炎性疾病4,5相关联。它是儿童和成人的下呼吸道分离慢性阻塞性肺疾病4,6,7的主导品种。的NTHi感染后的炎症反应的特征在于促炎性细胞因子（如TNF和IL-1β）的上调，并且它介导bý丝裂原活化蛋白激酶（MAPK）和核因子-κB（NF-κB）通过toll样受体（TLR）8。 小鼠模型是用于分析的，因为不同的基因缺陷线的可用性肺部炎性疾病的潜在病理非常有用的工具。几种方法已被用于接种活的/减毒的细菌和细菌产物，包括鼻内滴注和雾化吸入9,10。在这里，我们表现出内气管滴入。虽然不经常使用的，这种方法是更有效的和高重现性的，因为到下呼吸道的直接递送接种物的。

<table><tbody><tr><td>Chocolate agar plate</td><td>Fisher Scientific</td><td>CAS50-99-7</td><td></td></tr><tr><td>Dextrose Anhydrous</td><td>Themo Scientific</td><td>R01300</td><td></td></tr><tr><td>Heparin</td><td>Hospira,Inc</td><td>RL-3010</td><td></td></tr><tr><td>Deft quick solution</td><td>Sigma</td><td>GS500-500ML</td><td></td></tr><tr><td>Syringe needle 20/26G&amp;nbsp;</td><td>BD</td><td>(REF305115/175)</td><td></td></tr><tr><td>Iml syringe</td><td>BD</td><td>REF 309602</td><td></td></tr><tr><td>Catheter 20GA</td><td>BD</td><td>REF 381433</td><td></td></tr><tr><td>Dissecting Scissors, straight, 10 cm long</td><td>kentscientific</td><td>INS600393</td><td></td></tr><tr><td>Iris Forceps, serrated, 10cm long</td><td>kentscientific</td><td>INS650915</td><td></td></tr><tr><td>Tweezer #5 Stainless steel, 11cm long</td><td>kentscientific</td><td>INS600095</td><td></td></tr><tr><td>10% Formalin</td><td>Fisher Scientific</td><td>CAS 67-56-1</td><td></td></tr><tr><td>Agarose</td><td>peqlab</td><td>35-1020</td><td></td></tr><tr><td>5ml polystyrene round-bottom tubes</td><td>BD</td><td>REF 352058</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tubes</td><td>Light Labs</td><td>A-7001-R</td><td></td></tr><tr><td>Reasy Mini&amp;nbsp; kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Pellet pestile motor (Tissue homoginizer)</td><td>Sigma</td><td>Z359971-1EA</td><td></td></tr><tr><td>96 well microtiter plates V bottom</td><td>Thermo</td><td>2605</td><td></td></tr><tr><td>1X PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111-42</td><td></td></tr><tr><td>CD45.2-APC</td><td>eBioscience</td><td>17-0454-81</td><td>Working dilution 1:100</td></tr><tr><td>Ly-6G-eFlor 450</td><td>eBioscience</td><td>48-5931-82</td><td>Working dilution 1:100</td></tr><tr><td>BSA</td><td>HyClone</td><td>SH30574.03</td><td></td></tr><tr><td>RBC Lysis Buffer (10X)</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>Live/Dead fixable aqua dead cell stain kit</td><td>Invitrogen</td><td>L-34957</td><td></td></tr><tr><td>EDTA (0.5M)</td><td>lifetechnologies</td><td>15575-020</td><td></td></tr><tr><td>CD16/CD32 FcBlock</td><td>BD</td><td>553142</td><td></td></tr><tr><td>Facs tubes polystyrene round bottom tube</td><td>BD</td><td>352052</td><td></td></tr><tr><td>Formaldehyde</td><td>Polyscience</td><td>4018</td><td></td></tr></tbody></table>

intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation

在这里，我们有效地描述的详细步骤和直接递送流感嗜血杆菌 到小鼠的下呼吸道。我们演示程序编制H.接种流感 ，H的内气管滴入流感进入肺，支气管肺泡灌洗液（BALF），BALF中的免疫细胞的分析，和RNA隔离差异基因表达分析的汇编。此过程可用于研究的任何细菌，病毒或真菌的肺部炎症反应。直接气管滴入被过度鼻内或气溶胶吸入程序大多优选的，因为它更有效地以较少的歧义开细菌接种到下呼吸道。

气管内滴注导致BALF中（ 图1A，左面板）比安装用盐水一个白血球数显着增加。白细胞的分类计数分析清楚地显示增加的中性粒细胞浸润（ 图1，右图）。在BALF中的细胞的FACS分析进一步证实嗜中性粒细胞数量的增加（ 图1B）。肺组织的H＆E染色的切片的组织学分析显示增加的气道炎症（ 图1C）。这些数据共同表明，气管内?...

Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

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 ...

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Research

JoVE Journal

Immunology and Infection

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

10.1K 次观看.  Baylor Research Institute. 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. 

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

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

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

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 ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

的气管内管理

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