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>

<ol>
	<li>Reznik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy,+physiology,+and+function+of+the+upper+respiratory+tract.">Comparative anatomy, physiology, and function of the upper respiratory tract.</a> Environ Health Perspect. 85, 171-176 (1990).</li><li>Hoyt, R. F. J., Hawkins, J. V., St. Clair, M. B., Kennet, M. B., Fox, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2.">Chapter 2.</a> The Mouse in Biomedical Research, Volume 2, Second Edition: Diseases (American College of Laboratory Animal Medicine). 2, 23-90 (2007).</li><li>Visweswaraiah, A., Novotny, L. A., Hjemdahl-Monsen, E. J., Bakaletz, L. O., Thanavala, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+tissue+distribution+of+marker+dye+following+intranasal+delivery+in+mice+and+chinchillas:+a+multifactorial+analysis+of+parameters+affecting+nasal+retention.">Tracking the tissue distribution of marker dye following intranasal delivery in mice and chinchillas: a multifactorial analysis of parameters affecting nasal retention.</a> Vaccine. 20, 3209-3220 (2002).</li><li>Eyles, J. E., Spiers, I. D., Williamson, E. D., Alpar, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+radioactivity+following+intranasal+administration+of+radioactive+microspheres.">Tissue distribution of radioactivity following intranasal administration of radioactive microspheres.</a> J Pharm Pharmacol. 53, 601-607 (2001).</li><li>Southam, D. S., Dolovich, M., O'Byrne, P. M., Inman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+intranasal+instillations+in+mice:+effects+of+volume,+time,+body+position,+and+anesthesia.">Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia.</a> Am J Physiol Lung Cell Mol Physiol. 282, 833-839 (2002).</li><li>Miller, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+murine+intranasal+dosing+efficiency+using+luminescent+Francisella+tularensis:+effect+of+instillation+volume+and+form+of+anesthesia.">Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.</a> PLoS ONE. 7, (2012).</li><li>Lathem, W. W., Crosby, S. D., Miller, V. L., Goldman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+primary+pneumonic+plague:+a+mouse+model+of+infection,+pathology,+and+bacterial+transcriptional+activity.">Progression of primary pneumonic plague: a mouse model of infection, pathology, and bacterial transcriptional activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 17786-17791 (2005).</li><li>Warawa, J. M., Long, D., Rosenke, R., Gardner, D., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+diagnostic+imaging+to+characterize+altered+respiratory+tract+colonization+by+the+Burkholderia+pseudomallei+capsule+mutant.">Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the Burkholderia pseudomallei capsule mutant.</a> Front Microbiol. 2, 133 (2011).</li><li>Gutierrez, M., Pfeffer, T. L., Warawa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+3+Secretion+System+cluster+3+is+a+critical+virulence+determinant+for+lung-specific+melioidosis.">Type 3 Secretion System cluster 3 is a critical virulence determinant for lung-specific melioidosis.</a> Submitted. , (2014).</li><li>Fodah, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+Klebsiella+pneumoniae+comparative+genetic+analyses+with+virulence+profiles+in+a+murine+respiratory+disease+model.">Correlation of Klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model.</a> PLoS ONE. In revision, (2014).</li><li>Soto-Montenegro, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+airway+distribution+of+transnasal+solutions+in+mice+by+PET/CT+imaging.">Assessment of airway distribution of transnasal solutions in mice by PET/CT imaging.</a> Mol Imaging Biol. 11, 263-268 (2009).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1035">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:447px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Rodent, Tilting WorkStand</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">000A3467</td>
			<td>Base should be detached when working in a BSC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Operating Otoscope Head</td>
			<td style="width:160px;">Welch Allyn</td>
			<td style="width:119px;">21700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Otoscope 3.5 V Li Battery</td>
			<td style="width:160px;">Welch Allyn</td>
			<td style="width:119px;">71900</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:311px;">Mouse Intubation Specula short, Autoclaveable</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">200A3589S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Incisor Loops</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">210A3490A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Cotton fine tip applicator</td>
			<td style="width:160px;">Puritan</td>
			<td style="width:119px;">871-PC DBL&#160;</td>
			<td>Used for tongue retraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">I.V. Catheter, 20G</td>
			<td style="width:160px;">Exel Int</td>
			<td style="width:119px;">26741</td>
			<td>Optional: fit a silicon sleeve with 10mm exposed catheter surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Gas tight syringe, 250ul</td>
			<td style="width:160px;">Hamilton</td>
			<td style="width:119px;">81120</td>
			<td>Used for delivery of liquid inoculum by IMIT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Blunt Needle, 22G</td>
			<td style="width:160px;">Hamilton</td>
			<td style="width:119px;">91022</td>
			<td>Trim to length to protrude 1mm from 20G catheter</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Guide wire (Fiber optic wire, 0.5mm)</td>
			<td style="width:160px;">TheFiberOpticStore.com</td>
			<td style="width:119px;">FOF .50</td>
			<td>Cut to 6&#34; length: used as guide wire for intubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Tuberculin syringe, 1ml</td>
			<td style="width:160px;">Becton Dickinson</td>
			<td style="width:119px;">309659</td>
			<td>Assemble with fiber optic wire as guide wire</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Brilliant Blue R (Coomassie)</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:119px;">B0149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Tygon tubing, 1/16&#34;</td>
			<td style="width:160px;">Saint Gobain</td>
			<td style="width:119px;">ALC00002&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Male luer 1/16&#34; barb</td>
			<td style="width:160px;">Cole Parmer</td>
			<td style="width:119px;">45503-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Female luer 1/16&#34; barb</td>
			<td style="width:160px;">Cole Parmer</td>
			<td style="width:119px;">45500-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Lidocaine, USP</td>
			<td style="width:160px;">Spectrum</td>
			<td style="width:119px;">LI102</td>
			<td>pH lidocaine into solution at 2%(w/v) pH7.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Sample bag, 1oz</td>
			<td style="width:160px;">Whirl-Pak</td>
			<td style="width:119px;">B01067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">U-bottom 96 well plate, sterile</td>
			<td style="width:160px;">Greiner</td>
			<td style="width:119px;">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

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

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

科研

医学

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

术中的微妙的子宫内膜异位症的检测:A新型范式为相关联的与的的腹膜的完整性之亏损盆腔疼痛的的的的检测和治疗的

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

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 ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

纤维蛋白胶对小鼠肺移植研究肺特异性血管生成

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

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 ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

评价中的连接有关的大脑兴奋患者癫痫多式联运Imaging-和激励为基础的方法

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal颌下腺灌输和本地化分割照射在涎腺功能减退症大鼠模型

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

聚丙烯酸/纳米水的气管内灌注积液模型

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

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. ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...