The authors are grateful for the support from the Center for Predicative Medicine Animal Models (Carol Vanover, Ashley Biller and Jennifer Kraenzle) and Microbiology (Daniel Cramer and Julie Sotsky) Core Facilities. This work was supported by funding from the NIH (HHSN272201000033I to M.B.L and J.M.W.).

注:所有的此处描述的步骤进行了审查并批准路易斯维尔生物安全委员会（协议＃13-056）和实验动物管理和使用委员会（协议＃13-064）的大学。 1.制备染料<ol><li>稀释的0.1％（重量/体积）考马斯亮蓝的PBS中，并用0.45微米注射器式滤器过滤杀菌。 </li></ol> 2.准备绿脓杆菌文化<ol><li>滴注前15小时，接种3毫升肉汤培养物用单菌落。 </li><li>生长培养物15小时，在37℃下在振荡器上（200rpm）下。 </li><li>离心机1毫升文化在1.5 ml离心管12000×g离心30秒。 </li><li>取出介质悬浮颗粒在PBS 1毫升。 </li><li>稀释的细菌纸浆悬浮液1:10 PBS中的等分试样，并测量经稀释的细菌悬浮液的OD 600，以确定细菌浓度。 </li><li>稀释的细菌储备悬浮在PBS中，以细菌接种物的所需浓度，用50微升为IMIT接种的输送量。 </li></ol> 3. IMIT滴入<ol><li>放置一组小鼠成异氟烷麻醉诱导室和麻醉用2 - 3％异氟烷/氧混合物。 </li><li>在镇静的最初发作，颈背鼠标，按住鼠标直立，并管理10微升通过强饲法针头的2％利多卡因溶液至咽喉后部，并使溶液流失下到会厌。返回鼠标麻醉室。 <ol><li>允许至少5分钟，以允许利多卡因充分效果的局部麻醉剂。 </li></ol></li><li>当小鼠中都取得了镇静所需的水平（呼吸的〜60的BPM率），降低了异氟醚2％保持镇静。 </li><li>预紧染料或细菌接种到250μl的气密精密注射适合用22克长钝针。 <ol><li>先画了150微升的空气，通过注射器的柱塞聚四氟乙烯测量。其次，制定50微升接种通过推进聚四氟乙烯活塞从150微升标记到200微升标记的注射器主体。 注:当样品被喷射到一个插管小鼠中，50微升悬浮液将被首先传送，随后用150μl的气垫将在全肺分发的接种物。 </li></ol></li><li>从感应腔中取出一个鼠标，躺在仰卧插管平台上。通过挂钩的门牙用附着到尼龙搭扣条带的O形环，并固定魔术贴到平台固定鼠标到该平台。抬起鼠标45°倾斜。 </li><li>使用微棉签，缩回舌与滚动运动，用优势手。 </li><li>用非惯用手，使用操作耳镜配合使用的插管之镜既MAintain舌头回缩和可视化声门。 </li><li>用优势手，用导丝通过一个20G的导管螺纹插管鼠标，休息的导管至10毫米深度的小鼠气管（导管适合与10毫米露出导管的硅套筒）。取下耳镜/之镜。 </li><li>确认鼠标已被正确地固定导管与非优势手，而简单地附上含有有色染料1/16"清管鲁尔连接的长度插管。 注:该染料将迅速来回迁移响应于呼吸。 </li><li>不要继续进行后续的步骤，如果确认插管尚未建立，在这一点上。如果插管失败未遂，复位导管和导丝进行一个额外的尝试插管。 注:这是不明智试图鼠标在一个会话中两个以上的插管，而不会造成创伤鼠标。 </li><li>继续固定导管用非惯用手在插入含有液体悬浮/气垫精密注射器/钝针。 </li><li>分配液体/空气直接进入在单个流体运动的肺，并立即从小鼠中取出针/导管。 </li><li>返回鼠标于笼中，允许从麻醉中恢复。 </li></ol> IMT交货4.表征<ol><li> IMIT滴注后，安乐死的小鼠用CO 2窒息在适当的时间在接种后。 </li><li>固定安乐死鼠标上的夹层板浸泡在胸部和腹部，用70％乙醇喷瓶。 </li><li>如果评价显像剂的分布在整个肺，除去从使用无菌技术的动物的肺中，并显示肺部适当成像。 注:肺可通过适当的固定或冷冻保存的其他组织染色技术来制备&lt;/ LI&gt; <li>如果评估感染的肺组织的细菌负荷，从使用无菌技术的动物取出肺。地方肺部进入无菌，预称重1盎司样品袋。称量和记录样品袋+肺的重量。 <ol><li>加入1毫升无菌1X PBS对每个样品袋+组织。重新封装样品袋。 </li><li>均匀轻柔滚动组织25毫升血清吸管在样品袋+组织。 </li><li>产生的肺匀浆物中的无菌PBS中，盘上的琼脂平板（LB，或在适当的细菌种类被研究）系列稀释: <ol><li>进行6倍连续稀释成U形底96孔板中通过多道移液器然后板一式三份样品通过多通道上的琼脂平板。 </li></ol></li><li>培养琼脂平板过夜，在37℃和枚举集落形成单位的第二天。 </li></ol></li></ol>

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The authors have nothing to disclose.

IMIT滴注提供了重要的改进，以现有的呼吸系统疾病模型的可重复直接灌输试剂进入肺部的能力。这是谁执行的IMIT技术快速的办法，地理位置优越，为团队的两名研究人员，其中一个管理的麻醉和笼养的物流，以及其他。每只小鼠3分钟 - 大型研究可以使用IMIT为2的平均时间承诺进行。由于该方法利用异氟醚作为麻醉剂，小鼠从麻醉中快速恢复，减少了通过恢复监测动物的饲养时间。 该IMIT方法的技术上最有挑战性的方面是插管小鼠的初始步骤。个人的学习执行IMIT能够专注于导管放置的该第一步骤并确保插管已通过染料移动的视觉确认已经实现。该方法的好处是，肺规格IFIC灌注是通过使用插管的确认，这增加了两个新的研究者的信任以及专家试图插管困难的动物的保证。优化插管成功的可能性的关键要素是:一）实现了深度镇静，以便有足够的工作时间，二）在口腔中之镜的正确位置，使会厌良好的可视化，三）良好的深度布局之镜，以使舌保持在整个过程缩回，以及iv）使用倾斜平台，以支持研究人员的手，使得程序进行放松和以稳定的方式。 之一的IMIT过程的局限性是关系到IMIT滴注事件频率。由于与未接插管相关的潜在创伤，所以不推荐多于两个插管尝试在单个会话中进行（最多两个未命中）。 IMIT已被使用，它能够极好的潜力提供治疗剂进入鼠肺，但是治疗方案而使用很频繁的递送试剂的进入肺的可能并不适合IMIT。它也可以是:IMIT可每日用于递送试剂到鼠肺而不引起显著创伤，但只有当由高度熟练的研究人员进行的，由于大多数外伤与插管相关联被认为是与一个未接插管事件相关联。这种高频IMIT应与本地兽医和IACUC进行讨论。 IMIT的额外潜在的局限性是正被插管的小鼠的大小。 22克，其中一个20G的导管被认为是一个合适尺寸的小鼠在这个尺寸范围内的气管 - 上述IMIT步骤，使用约17小鼠发展。较大的导管已被成功地用于在老年小鼠;的IMIT米的初步发展ADE使用18G的导管BALB / c小鼠这是"20克。重要的是，如果备用导管的尺寸时，钝针应来源于哪个适合的导管的腔和被修剪到的长度延伸只是1毫米超出导管末端。小鼠大于17 g时的插管可以是可能的，但不推荐使用，由于所需的专业知识，并需要使用更小的导管和之镜比如上所述。 我们已经使用IMIT为若干呼吸道病原体除了P的输送铜绿假单胞菌 ，包括B. 9 杆菌和肺炎克雷伯菌 10。该IMIT模式已经取得了重要进展，以我们的B.研究假单胞菌呼吸系统疾病，已经确定了鼻内接种引起的小鼠，而不是在人类疾病9所观察到的全身性疾病端点的早期，URT-相关的发病率。B.假单胞菌是1级SELEC的生物防御冲击吨剂，正因为如此，呼吸系统疾病模型已被用于气雾剂曝光开发了型号为武器化的病原体条目的潜在生物防御相关的路由。由于目前的气溶胶模式导致两个城市轨道交通和轻轨的感染，同样的潜在发病初期表现型，我们已经确定了B的鼻腔模型假单胞菌呼吸系统疾病，可向气溶胶模型。该IMIT模型的未来适应可以是插管介导的气雾剂递送（IMAD），其中小鼠进行气管插管为目标气溶胶递送只进入肺。机械通气机目前可维持异氟烷麻醉，这可能是一种适于输送气雾化，而不是基于液体，病原体的挑战。 IMIT最初是作为一种方法来优化递送细菌至肺，还具有应用程序交付的其他试剂到小鼠的肺。由于DIS上面讨论过，鼻内递送化合物的入小鼠导致低效率的，高度可变的递送试剂的进入肺的靶器官。鼻腔给药的正电子发射断层扫描（PET）成像试剂的小鼠肺部产生了40％，交付效率11，而我们已经证明，IMIT提供了一个很好的替代其他肺部交货与其&gt; 98％交付疗效和multilobar分配办法。这种改进在靶向递送至肺具有提高的治疗递送的可重复性对于治疗肺部疾病的潜力。 IMIT可以提供类似的好处的研究:一）对环境刺激肺部的影响，二）肺癌表型的研究，III）肺特异性siRNA敲除。

老鼠被用来模拟多种人类疾病的表现，包括呼吸系统疾病万千。替代疾病模型往往无法概括的模型化疾病的各个方面，通常是由于重要的生理或在两个主机模型免疫差异。因此，改善的模型系统的一个目标是开发方法，使代理人更紧密地反映疾病过程或宿主反应如在原始主机系统观察。有在该机制中的小鼠和人类之间的几个关键的生理差异由它们激发空气。包括在这些差异的城市轨道交通与轻轨之间的显著比例的差异的大小。据估计，该小鼠具有相对的URT表面积对人，对归一化的总肺活量1,2-&gt; 100倍。因此，鼻的鼠标鼻甲允许对吸入空气更广泛的筛选，以方便breathi一个更大的速度纳克，这可能对肺炎的研究一显著影响，如果鼻腔感染在疾病进展的一个显著的作用。 几种不同的方法已经被用来灌输细菌到小鼠的肺中，研究人样呼吸系统疾病。最常见的这些方法是鼻内接种，其中液体悬浮液被施加在鼠标的一个或两个鼻孔。虽然比较简单，注意事项，如灌注量使用，可通过鼻内接种3-5冲击灌输的效率成轻铁麻醉和类型。具体地，Miller 等人的已经表明土拉弗朗西斯菌的那鼻内滴注于体积小于50μl的没有导致细菌的滴注到轻轨6。他们还使用相对于注射氯胺酮/甲苯​​噻嗪麻醉吸入异氟醚时观察到更好的轻便滴注。但是，我们的工作经验e为鼠疫鼻内接种表明接种较为一致可以使用氯胺酮/甲苯噻嗪可实现相比，异氟醚（MBL，未发表的数据）。这些差异可以归因于病原体中使用或在实验室程序来变化，但重要的是设计出该技术的潜在的可变性。此外，肺后鼻内滴注表明，初始细菌接种物的比例相对较低到达肺不久收获（在鼠疫菌的情况下，只有10％被回收后1小时滴注7），这表明大量的细菌可以保留在URT（或吞咽到胃肠道中）。在某些疾病模型，对URT粘膜细菌的本显著沉积可能混淆了疾病进展的理解，如果所述生物体是能够定殖鼠的鼻腔中与人类疾病不一致的方式的。例如，使用体内&lt;/ em&gt;的成像中，已经观察到， 类鼻疽伯克霍尔德菌，其不殖于人的URT，当通过鼻内滴注法8递送导致小鼠鼻腔的压倒性的机会性感染。 其他方法来灌输细菌到小鼠的肺部也被应用在传染病研究。然而，相对于滴鼻这些方法往往需要更多的专业技术和/或昂贵的设备没有消除感染引发的多个站点（ 如气溶胶[城市轨道交通和轻轨]的潜力;经口[消化道和轻轨];和手术气管内[轻铁及血流]）。定，可以与感染的辅助站点相关联的潜在的并发症，我们试图开发一种气管内的办法，绕过URT并提供病原体直接进入麻醉小鼠的肺中，也限制无意inoculat离子进入血流或胃肠道。为此，插管介导气管（IMIT）滴注是作为一个非手术的过程，它包括通过一个中间步骤之前，灌输正确的验证导管放置，保证接种的轻轨滴注。此方法是利用染料滴注描述目视证明接种的广泛分布在整个肺和P.假单胞菌滴注以证明该方法对肺的高度有效的输送（&gt; 98％的接种物）。重要的是，虽然最初是为细菌交付，IMIT还提供了一个有效的工具:一）灌输各种分子的其它呼吸系统疾病模型，二）肺特异性治疗交货的研究，以及iii）基础肺功能研究，包括有针对性siRNA递送至肺。

<table><tbody><tr><td>Rodent, Tilting WorkStand</td><td>Hallowell EMC</td><td>000A3467</td><td>Base should be detached when working in a BSC</td></tr><tr><td>Operating Otoscope Head</td><td>Welch Allyn</td><td>21700</td><td></td></tr><tr><td>Otoscope 3.5 V Li Battery</td><td>Welch Allyn</td><td>71900</td><td></td></tr><tr><td>Mouse Intubation Specula short, Autoclaveable</td><td>Hallowell EMC</td><td>200A3589S</td><td></td></tr><tr><td>Incisor Loops</td><td>Hallowell EMC</td><td>210A3490A</td><td></td></tr><tr><td>Cotton fine tip applicator</td><td>Puritan</td><td>871-PC DBL&amp;nbsp;</td><td>Used for tongue retraction</td></tr><tr><td>I.V. Catheter, 20 G</td><td>Exel Int</td><td>26741</td><td>Optional: fit a silicon sleeve with 10 mm exposed catheter surface</td></tr><tr><td>Gas tight syringe, 250 ul</td><td>Hamilton</td><td>81120</td><td>Used for delivery of liquid inoculum by IMIT</td></tr><tr><td>Blunt Needle, 22 G</td><td>Hamilton</td><td>91022</td><td>Trim to length to protrude 1 mm from 20 G catheter</td></tr><tr><td>Guide wire (Fiber optic wire, 0.5 mm)</td><td>TheFiberOpticStore.com</td><td>FOF .50</td><td>Cut to 6&quot; length: used as guide wire for intubation</td></tr><tr><td>Tuberculin syringe, 1 ml</td><td>Becton Dickinson</td><td>309659</td><td>Assemble with fiber optic wire as guide wire</td></tr><tr><td>Brilliant Blue R (Coomassie)</td><td>Sigma</td><td>B0149</td><td></td></tr><tr><td>Tygon tubing, 1/16&quot;</td><td>Saint Gobain</td><td>ALC00002&amp;nbsp;</td><td></td></tr><tr><td>Male Luer 1/16&quot; barb</td><td>Cole Parmer</td><td>45503-22</td><td></td></tr><tr><td>Female Luer 1/16&quot; barb</td><td>Cole Parmer</td><td>45500-00</td><td></td></tr><tr><td>Lidocaine, USP</td><td>Spectrum</td><td>LI102</td><td>pH lidocaine into solution at 2%(w/v) pH7.0</td></tr><tr><td>Sample bag, 1 oz</td><td>Whirl-Pak</td><td>B01067</td><td></td></tr><tr><td>U-bottom 96 well plate, sterile</td><td>Greiner</td><td>650161</td><td></td></tr></tbody></table>

intubation-mediated intratracheal (imit) instillation: a noninvasive, lung-specific delivery system

呼吸系统疾病的研究通常包括使用小鼠模型的作为替代的系统。但是，也有鼠和人呼吸道系统之间显著生理差异，尤其是在它们的上呼吸道（URT）。在一些模型中，在小鼠鼻腔这些差异可以用鼻内滴注技术时，这可能限制了小鼠模型的有用性，研究这些疾病有下呼吸道（LRT）对疾病进展和介绍一个显著影响。由于这些原因，这将是有利的是开发一种技术，以直接灌输细菌进入小鼠肺中，以研究LRT疾病在不存在URT的参与。我们称这种肺部释药技术插管介导气管（IMIT）滴注。这种非侵入性技术，最大限度地减少灌输的潜在进入血液，可发生在更具侵入性的traditional外科气管内感染的方法，并限制附带的消化道递送的可能性。 IMIT是其中小鼠第一插管，用一个中间步骤，以确保正确的导管放置到气管，随后插入一个钝针的进入导管介导的直接输送细菌进入肺一个两步过程。这种方法有利于递送的&gt; 98％的药效成具有优良的试剂的分布在整个肺的肺。因此，IMIT代表了一种新的方法来研究LRT疾病和治疗直接递送至肺，在使用小鼠作为替代物来研究人类呼吸性疾病的能力提高。此外，这种输送系统的准确度和再现性也使得它适合于良好实验室规范标准（GLPS），以及递送的广泛需要较高的效率输送至肺部试剂。

为了显现灌输材料经由IMIT方法的分配，加入50μl的0.1％考马斯亮蓝染料，灌输到麻醉小鼠的肺中。将小鼠立即处死并通过无菌尸检取出肺。 图1示出了将染料递送至肺的所有叶。 以确定细菌经由IMIT方法递送到肺中的量，三组小鼠（n = 3时），滴注用三个不同浓度的P的假单胞菌 （1.21×10 8，1.21x10 7，和1.21×10 6个菌落形成单位每50μl[CFU]）。立即IMIT滴注后，处死小鼠，取出肺部和细菌数量进行计数和比较，接种物（ 图2）。输送的接种物是通过这种方法非常高效，具有&gt; 98％的从肺部回收的接种物的注入动物。此外，IMIT滴注是高度可再现不论接种物（R 2 = 0.9951）的浓度。 <img alt="图1" src="/files/ftp_upload/52261/52261fig1highres.jpg" /> 图1:IMIT滴注整个肺部分布接种物从滴眼用50μl的0.1％考马斯亮蓝显示蓝色染料分布在所有波瓣的小鼠肺组织。 <img alt="图2" src="/files/ftp_upload/52261/52261fig2highres.jpg" /> 图2:细菌IMIT滴注到肺部小鼠滴注P.铜绿假单胞菌和细菌灌输到肺部的数量（登录10 CFU -回收）进行比较，估计接种（登录10 CFU -交付）。每个圆圈代表一个单独的鼠标（的CFU /肺n = 3时为每细菌的剂量）。

Watch this Scientific Journal Video about Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System at JoVE.com

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

试剂插管介导气管（IMIT）滴注是研究呼吸系统疾病，以及用于直接灌输治疗剂进入肺的方法，优良的，非侵入性方法。它是一种快速和高重现性的方法，该方法适合于临床前试验。

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 ...

intubation-mediated-intratracheal-imit-instillation-noninvasive-lung

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

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

24.2K 次观看.  University of Louisville Medical School. 试剂插管介导气管（IMIT）滴注是研究呼吸系统疾病，以及用于直接灌输治疗剂进入肺的方法，优良的，非侵入性方法。它是一种快速和高重现性的方法，该方法适合于临床前试验。 

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

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

Direct Tracheal Instillation of Solutes into Mouse Lung

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 &mu;L instillate volume can be introduced into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

溶质直接气管滴入小鼠肺

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

生物学

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

灌输和固定方法有用的小鼠肺癌研究

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

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 ...

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

直接注射内管理,改善小鼠肺内的选择性代理沉积

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

小鼠干粉配方的内经管理

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

一种新生儿啮齿动物气管内滴注药物治疗肺部疾病的微创方法

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and minimizing systemic drug exposure. The transcutaneous technique is simple and allows for the IT delivery of substances to the lung of prematurely born rabbits shortly after birth. Newborn pups are anesthetized with inhaled Isoflurane before being placed in a supine position with the neck extended. The larynx is identified and stabilized before transcutaneous placement of a 26-gauge (G) catheter into the trachea. Following catheterization of the trachea, a 30 G blunt needle attached to a Hamilton syringe is introduced into the IT catheter and is used for delivering a precise volume into the trachea during spontaneous respiration. After the IT injection is completed, the needle and catheter are withdrawn, and the pup is allowed to recover from anesthesia. Transcutaneous IT injection delivers a large proportion of the injected substance to the lung, with the majority remaining in the lung 3 hours after the intervention. The injections are well tolerated from the day of birth and can be repeated for multiple consecutive days without influencing survival. This technique can be used to investigate the effect of pharmaceutical agents on lung development and in the prevention of neonatal lung injury in preterm rabbits.

Transcutaneous intratracheal injection allows for effective intrapulmonary drug delivery during spontaneous respiration. Single and multiple injections are well tolerated with no effect on survival. The technique is simple to perform and can examine the effect of substances on lung development and the prevention of lung injury in newborn rabbits.

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and ...

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of acute lung injury or acute inflammation. The current most commonly used methods in acute lung injury mouse models are an intraperitoneal injection of LPS and tracheostomy for the tracheal infusion of LPS. However, the former method lacks lung targeting and damages other organs, and the latter method induces operative trauma, infection risk, and a low survival rate. Here, we recommend a noninvasive oropharyngeal endotracheal intubation method for LPS instillation in mice. In this method, LPS is noninvasively introduced into the trachea through the oropharyngeal cavity to be instilled into the lung with the help of an apparatus for endotracheal intubation. This method not only assures lung targeting but also avoids damage and the risk of death in the animals. We expect that this approach will become widely used in the field of acute lung injury.

Here, we propose a protocol for intratracheal lipopolysaccharide (LPS) delivery via noninvasive oropharyngeal endotracheal intubation. This method minimizes the trauma of the surgical procedure for the animal and accurately delivers LPS to the trachea and then to the lungs.

小鼠无创气管内脂多糖滴注

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of ...