Name: the bovine lung in biomedical research: visually guided bronchoscopy, intrabronchial inoculation and in vivo sampling techniques
Uploaded: 2014-07-03T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques at JoVE.com

The authors are very thankful to the Federal Ministry of Education and Research (BMBF) of Germany for the funding of their work. Also, the authors thank Ines Lemser, Sylke Stahlberg, Ingolf R&#252;cknagel and all the other colleagues working in the team of the animal house (FLI, Germany) for their technical assistance with the bronchoscopies. They are very thankful to Maria-Christina Haase (FLI, Germany) for her help in providing literature. Furthermore, the authors wish to express their gratitude to Dr. Angela Berndt (FLI, Germany) and Nicolette Bestul (University of Wisconsin-River Falls) for critical reading of the manuscript.

操守准则 这项研究进行了严格按照欧洲和国家法律的动物护理和使用。该协议是在国图林根，德国（：04-004/11许可证编号）批准委员会关于动物实验的伦理与动物保护。根据监管机构的授权代理动物保护被执行的所有实验。在全身麻醉下被严格执行支气管镜检查。在研究过程中，尽一切努力，以减少不适或痛苦。 总论 所描述的技术已经开发了用于约6-8周龄的小牛，重约60-80千克。在其他大型动物或牛犊在年龄和体重的不同的使用，该技术必须适于配合的尺寸和重量，并考虑到该特定动物物种的肺解剖结构。所有使用的设备必须是无菌的。 鹦鹉热衣原体是一种人畜共患的细菌，会导致呼吸和一般人类疾病。的非禽类C。鹦鹉热衣原体菌株DC15本议定书中所使用必须在生物安全2级处理。与病原体和感染动物的所有工作都必须进行穿着个人防护装备，如呼吸器，飞溅防水连身衣，橡胶靴子和手套。戴合适的呼吸器是非常重要的，因为感染的C.自然路线鹦鹉热是aerogen。包装和样品的标识必须是消毒证明，因为所有的事情都要用消毒有效，根据制造商的前离开畜舍单元的指令被视为对衣原体。 1，准备动物的支气管镜检查<ol><li>确定小腿对麻醉剂的剂量的重量。 </li><li>放置静脉注射（IV）访问我n中左侧颈静脉。 </li><li>首先，慢慢地注入甲苯噻嗪（0.2 mg / kg体重），在约30秒，然后，镇静发生后，注入氯胺酮（2.0 mg / kg体重）。 </li><li>提起动物搬上了餐桌，并将其放置在右侧卧。一旦动物有足够的放置在桌子上，检查静脉通路仍然存在，并在必要时重新调整。在麻醉过程中，定期检查眼睑反射来判断麻醉深度。 </li><li>一旦动物呼吸平稳，有专人拉舌头出来，伸展颈部。将一个金属管窥器在动物的嘴，用轻微的旋转运动。向前推窥器视域下的控制，使用手电筒，直到喉部可见。 </li><li>根据需要注射的7毫克甲苯噻嗪和70mg氯胺酮丸剂维持麻醉整个内窥镜手术。 </li></ol> 2，接种（接种地点：图1） </p&gt; <ol><li>制备3注射器接种物，含有1，2，和5ml的接种物。 </li><li>插入聚四氟乙烯管插入内窥镜的工作通道。管不应该从内窥镜的前端突出。 </li><li>通过金属窥器插入内窥镜。窥器轻微的调整可能是必要的，使喉的传球。的支气管气管肌 ，它关闭，以右侧分支，有助于对齐图像在监视器上。 </li><li>用5毫升的接种物附着在注射器的聚四氟乙烯管的末端。浏览管进入那里的接种应交存的分支机构和应用所需的金额（右肺：Lobus肌 ：0.5毫升，Lobus accessorius：0.5毫升，Lobus尾侧 ：0.5至1.2ml;左肺：Lobus cranialis，PARS cranialis 0.5毫升， 帕尔斯尾侧 ：0.5毫升，Lobus尾侧 ：1.5毫升）中。附上注射器1＆＃160;接种到管毫升，然后导航到支气管气管平滑肌和存款接种（Lobus cranialis，PARS尾侧 ：1.0毫升）。是有帮助的始终接近本地化中的顺序相同。 </li><li>取出内窥镜和窥器。 </li><li>喷雾器将1ml接种到每个鼻孔用的致动器。 </li><li>把动物回到稳定，并将其放置在俯卧位用于唤醒。不要将无人看管的动物或其他动物的公司，直到它恢复足够的意识，以保持胸骨斜卧。恢复稳定的应该是空调，因为动物的体温调节能力是在全身麻醉下下降。 注意：第一个临床症状应该发生大约24-36小时接种后，根据所用的病原体。 </li></ol> 3，抽样程序（采样地点：图2） <ol><li>按照步骤1.1-1.6的介绍准备的动物。 </li><li>支气管肺泡灌洗<ol><li>放置5注射器各含有20毫升无菌等渗盐水，在水浴并允许它们加热至约38℃。 </li><li>插入灌洗导管插入内窥镜的工作通道，然后将内窥镜插入金属窥器和向前导航到左肺主支气管，直到“楔子位置”到达那里的内窥镜不能推进一步前进。 </li><li>此起彼伏，重视与温暖的NaCl溶液的灌洗导管的注射器，注入液体，直接吸它。支气管肺泡灌洗液必须存储在硅化玻璃瓶和恢复后立即置于冰浴中，以防止肺泡巨噬细胞附着到玻璃表面上。注滴注生理盐水的量和回收的流体的量。 </li><li>从工作通道取出灌洗导管。 </li></ol></li><li>支气管刷<ol><li>导航内窥镜所需的采样位置，在描述的协议，这是Bifurcatio气管 。 </li><li>将其插入内窥镜的工作通道前盖刷试管，直到刷的尖端出现在监视器上。 </li><li>推刷塑料管着约5厘米，通过推动手柄从塑料管揭开它，那么它导航到要刷的位置。 </li><li>擦掉轻轻推拉来回刷，而导航内窥镜，以保证电刷和支气管壁之间的接触上皮电话。当停止出血发生摩擦。拉出的工作通道中的前盖的刷子与管。 </li><li>准备多达五个涂片在显微镜玻片上轻轻滚动刷过的幻灯片。在-20°C固定在冷甲醇涂片10分钟，空气干燥和储存</li><li>刷子可以在漂洗各种介质，这取决于所采样的细胞的目的。如果服用多种刷牙用相同的刷子，​​一定要只冲洗介质中不刺激粘膜。 </li></ol></li><li>支气管肺活检<ol><li>导航内窥镜所需的采样位置，在描述的协议，这是Lobus cranialis的帕尔斯尾侧 。之前插入活检钳进入工作通道打开和关闭几次，以确保其工作正常顺利进行。 </li><li>推活检钳进入支气管的尾支气管平滑肌 ，直到有轻微的阻力发生。回力2-3厘米，打开钳，推动约2厘米，关闭钳，拉回来，并从工作通道取出镊子。这需要一些练习。 </li><li>小心地从活检钳取出组织，用针或小镊子。根据进一步利用组织的，它存储在液体NITROGEN或合适的固定介质。这应该去除，以防止自溶过程之后发生的权利。 </li></ol></li><li>后处理程序<ol><li>把动物回到稳定，并将其放置在俯卧位用于唤醒。不要将无人看管的动物或其他动物的公司，直到它恢复足够的意识，以保持胸骨斜卧。恢复稳定的应该是空调，因为动物的体温调节能力是在全身麻醉下下降。 </li><li>密切监测动物气胸的迹象在未来24小时。提供饲料和新鲜的水，当动物已经恢复全意识。 </li></ol></li></ol>

作者什么都没有透露。

接种的支气管镜方法的开发和各种支气管镜抽样方法进行了调整，可应用于大型动物的实验条件下。所描述的技术是简单易学，即使是考官，在内窥镜的经验。支气管镜检查的过程是微创，与接种的方法，以及所描述的抽样方法（BAL，支气管肺活检，支气管刷）没有相关的不良反应，都见过的任何动物。在人类支气管肺活检相关的并发症是出血和气胸48，所有这些被认为在经历了此过程中的牛犊。在支气管肺活检是比较耗时，而且需要比皮的方法更多的设备，但它是创伤小，不承担伤口感染的风险。 inoculat的视觉控制，内镜方法离子允许定义病原体的量沉积在肺部的特定网站。因此，它会导致在所有接种动物32-34非常一致的临床和病理表现。然而，它并不像自然感染的所有功能于小牛。在呼吸C的模型鹦鹉热衣原体感染，接种所描述的技术导致了与病原体的位置34的位点相关联的肺损伤，而在天然获得性感染的小牛通常开发心尖瓣的肺炎。这一事实解释了实验结果的相关性中的牛自然获取肺部感染的上下文时要考虑进去。 内视镜BAL允许抽检中定义的肺区。对于实验目的，这是一个优势相比盲的条件下使用的鼻导管。由于牛肺的解剖，盲目插尿管会推教育署的右侧膈叶在大多数情况下，53,54和考官对被灌洗肺的区域没有影响。在侧卧麻醉小腿的内镜下灌洗的另一个优点是超过80％灌输流体的高回收率。与其他研究的比较显示，在站立，镇静牛犊，133.3±1.6毫升46恢复和127.13±3.53毫升45 240毫升液体进入尾叶灌注后报道。在镇静小牛胸骨斜卧的液体滴入51％可以从尾叶43颅叶和62％被回收。这意味着，大约一半的灌输流体可在小腿垂直位置恢复。根据需要作进一步的样品制备支气管肺泡灌洗液的量，这可能不会留下足够的材料来进行所有需要的实验。 BAL牛已被许多研究小组和许多在各种条件下不同的参数已被检查。多数作者进行的基部裂片43,45,46的灌洗，但用于灌洗流体的量的研究组之间不同。这导致不一致的稀释回收的细胞，蛋白质和其他物质，使其难以从不同的出版物进行比较的结果。因此，在牛的使用建议灌洗五个级为20ml（ 即 100毫升总）身体温暖，等渗盐水，其中滴入后立即恢复。当使用灌洗导管具有大直径（ 即 “2毫米），每个级分的体积需要被稍微增加，这取决于流体的将保留在导管的量。 牛肺的高度细分的解剖结构导致了有条不紊的限制;从肺的一部分获得的结果可能并不适用于肺的其余部分。由于没有经支气管活检和灌洗探讨全肺区的视线控制，考官不知道采样的区域是否是健康或患病。因此，这是很重要的样品，其中所述病原体为了有病原体的回收率较高，并具有取样病变肺区域的更高的可能性之前接种的位置。另一个限制是在较差的临床状况的动物增加了麻醉的风险。所描述的方法应只用于在轻度模型到中度疾病保持负担为动物尽可能低。在反刍动物的全身麻醉应始终保持尽可能短，因为在瘤胃中的气体的发展增加了这些物种的麻醉风险。动物必须被放置在俯卧位后立即支气管镜检查，以允许开发气体外排，必须密切监测，直到它们完全从麻醉中恢复。此外，所描述的技术并不适合初学者竹叶提取的用于进行采样的次数少于24小时的时间间隔。 所描述的协议可以适于其它传染性物质。内窥镜接种各种病原体进行了说明，例如C。鹦鹉热 32-34， 溶血巴斯德氏菌 38-40,42， 嗜血SOMNI 55，和牛病毒性腹泻病毒44。此外，病原体存于肺的网站可以适应所需的型号。当选择的采样点，一些重要的事实，必须考虑到：（ⅰ）采样点应根据接种的位置和在期望病理结果进行选择。 （二）剖检时要执行必须注意要留下足够的未取样的肺部区域为前活体取样。 （三）采样站点位置必须选择，使他们能够与设备到达。尤其是对支气管肺活检，有因活检钳的长度的限制。 （四）笔他为了取样是很重要的，支气管刷和支气管肺活检可能导致轻微出血，这会污染支气管肺泡灌洗液。因此，支气管肺泡灌洗液必须先获得。当使用在其它物种中的协议中，物种特异性肺解剖必须加以考虑。

大动物模型的生物医学研究中的价值在现代生物医学跨学科研究，动物模型仍然不可或缺阐明复杂的相互作用 - 有关健康或疾病状态 - 哺乳动物的生物体内。尽管17诺贝尔奖被授予科学家认为，研究牛，马，羊，家禽或作为生物医学研究1模型，动物实验时下绝大多数都从事与啮齿动物，而研究的不足1％正与家畜或牲畜。 小动物提供了许多实用的优点（ 即成本低，遗传可塑性，高通量，众多遗传的可用性和免疫工具和工具包），和转基因小鼠模型是公认的执行机制研究发现特定的分子途径。在复杂的系统中，T生物医学研究他的生物相关性和小鼠模型的临床应用正在成为越来越多的质疑。他们可能会产生误导，并承担生物复杂性2-9简单化的风险。 由于跨物种的特殊性，没有一个单一的动物物种将彻底反映了人类的处境，以及使用多个模型似乎是有益的一个跨学科的生物医学研究的方法。在转化医学的背景下，大型动物提供了机会，作为对比车型提供具有双重用途的人类和动物健康1高生物相关的结果。值得注意的是，在人类基因组是更像由牛基因组比实验室啮齿动物的基因组中。它还最近已经证实，相对于其他类群，小鼠的基因组是更重排10-12。 在一个复杂的研究设计，利用牲畜提供的uniq个体内，长期受各种体内样品从1 -和-的-同individuum反复收集，而不会牺牲动物研究中的UE的机会。因此，功能性，炎症性和形态的变化可以在超过13时在一定期间内就同一主题进行监控。 牛龙作为一个合适的呼吸模式由于大量的肺解剖，呼吸生理和肺免疫显著差异，小鼠不会重现人类肺疾病的许多重要的病理生理方面。这必须在使用它们作为呼吸系统疾病2,9,14-16的动物模型中进行考虑。虽然解剖和结构的特殊性，存在于每一哺乳动物的肺，功能特性（ 即肺容积，气流和呼吸力学）是成年人和小腿由于类似的体重之间的更好的可比性（50-100公斤）。 牛肺的特定物种的特征总结如下：左肺由两个波瓣（lobus cranialis，它被分成两个部分，并lobus尾侧 ），右侧肺由四个凸角（lobus cranialis，lobus中肌，lobus尾侧 ，并lobus accessorius）。不同于大多数其他哺乳动物的肺解剖，右颅瓣直接分支从气管的右横向侧的支气管。相对于subgross解剖，牛肺呈现出高度的分叶和间质组织17,18的高比例，导致相对低的特定肺顺应性和较高的肺组织的电阻19。因此，相比于其他物种20,21所需要的呼吸活性是相当高的。分叶的高度导致段的独立性强。因此，INFlammatory过程是受结缔组织隔膜，以及患病和健康领域往往在于同一肺叶内。由于缺乏抵押品气道，牛肺特别适合于反射镜阻塞性肺机能障碍13。就在牛肺血管，肺小动脉表现出非常显着的平滑肌层。因此，小腿也可作为肺动脉高压或血管行之有效的动物模型重构22-24。 对于呼吸道感染，家畜存在自然发生的疾病，随着疾病相媲美的人有许多相似之处。典型的例子是牛结核病25，呼吸道合胞病毒（RSV）感染的牛犊26-28，或自然获得衣原体感染29。因此，大动物模型并酷似天然宿主的情况。因此，他们最USEFUL为研究宿主-病原体相互作用和相应的疾病的人类30,31的复杂的病理生理学。 由于呼吸道鹦鹉热衣原体感染的生物相关模型，小腿被选中，因为牛代表自然宿主对这种病菌32-35。从这个模型中获得，对于动物和人类之间的疾病或可能的传播途径的发病信息，将有助于同为牛和人的影响扩大我们的知识。该模型还可以帮助核实普遍接受和替代治疗方案消除肺C的鹦鹉热衣原体感染在兽医和人类医学的兴趣，这一点，再次。 技术从牛呼吸系统申请并获得标本本文介绍并说明了技术和诊断方法applicable来的牛肺和用在我们的模型中，评价对哺乳动物的肺和治疗性干预的功效的病原体两者的影响。 支气管镜检查已在人类医学自1960年以来进行的，被认为是一个安全的程序36。在小腿，实验支气管镜在1968年被描述为在第一时间37。病原体的支气管内的应用就是由Potgieter 等。作为一种可靠的方法来产生下呼吸道病犊牛38，现在是牛的研究34,39,40的普遍做法。支气管内接种一定义下视镜控制病原体的金额允许在肺部感染因子的选择性安置。这导致了一致的临床和病理结果所有动物34，并允许预期因接触病原体被改变的肺区域的针对性抽样。 <p级=“jove_content”&gt; 支气管肺泡灌洗液 （BALF）用于肺部炎症的存在和严重程度以及所述指示器。支气管肺泡灌洗（BAL）是一种标准的过程在人类医学用于多种呼吸道疾病41的诊断。在活牛，BAL是由威尔基和马卡姆在上世纪42七十年代末推出。它被认为是安全的和可重复的方法来研究牛的下呼吸道。由于缺乏对健康的小牛与灵活的纤维支气管镜支气管肺泡灌洗液上的参数足够的数据在健康动物，在1988年的Pringle 等进行灌洗。作者还指出了需要的实验条件下获得类似的结果43下BAL协议的标准化。 BAL仍然用作小牛44-46 体内的抽样方法。 支气管刷通常用于人类医学的一个诊断工具来样肿瘤性病变或微生物分析36。为研究目的，通过细胞学刷牙收获上皮细胞的原代细胞培养可以得到47。在牛中，使用支气管刷的微生物分析已经描述了表征肺43的微生物环境。 支气管肺活检提供了肺组织标本，是弥漫性肺部疾病在人类中一个有价值的诊断工具。医源性气胸手术相关出血与此技术相关的并发症。其发生率报道为小于1％的人类患者48。支气管肺活检不是常见的方法，在牛的使用中，由于所需的设备的成本高，需要获得活检组织的时间。相反，经皮肺活检是野外条件下49-51更方便。

<table><tbody><tr><td>Veterinary Video Endoscope</td><td>Karl Storz GmbH &amp;amp; Co. KG, Tuttlingen, Germany</td><td>PV-SG 22&amp;ndash;140</td><td>diameter: 9 mm, working channel: 2.2 mm, working length 140 cm</td></tr><tr><td>Lavage catheter</td><td>Karl Storz GmbH &amp;amp; Co. KG, Tuttlingen, Germany</td><td></td><td>diameter: 2 mm; length: 180 cm, Luer-lock-adapter</td></tr><tr><td>Actuator</td><td>WEPA Apothekenbedarf GmbH &amp;amp; Co KG, Hillscheid, Germany</td><td>32660</td><td>length: 60 mm</td></tr><tr><td>Biopsy forceps</td><td>Karl Storz GmbH &amp;amp; Co. KG, Tuttlingen, Germany</td><td>REF 60180LT</td><td>1.8 mm, serrated, oval</td></tr><tr><td>Omnifix 20 ml, Luer-Lock</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>4617207V</td><td></td></tr><tr><td>Cytology brush</td><td>mtp GmbH, Neuhausen ob Eck, Germany</td><td>110240-10</td><td>working length 180 cm, brush length: 15 mm, diameter 1.8 mm</td></tr><tr><td>iv acess</td><td>Henry Schein Vet GmbH, Hamburg, Germany</td><td>370-211</td><td>diameter: 1.2 mm; length: 43 mm</td></tr><tr><td>Rompun 2% (xylazin)</td><td>Bayer Vital GmbH, Leverkusen, Germany</td><td></td><td>0.2 mg/kg bodyweight</td></tr><tr><td>Ketamin 10% (ketamine)</td><td>bela-pharm GmbH &amp;amp; Co. KG, Vechta, Germany</td><td></td><td>2.0 mg/kg bodyweight</td></tr><tr><td>Isotonic saline solution</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>3200950</td><td></td></tr><tr><td>SUB 6 waterbath</td><td>CLF analytische Laborger&amp;auml;te GmbH, Emersacker, Germany</td><td>n/a</td><td></td></tr><tr><td>Metal tube speculum</td><td>n/a</td><td>n/a</td><td>diameter: 3.5 cm; length: 35 cm</td></tr><tr><td>Flashlight</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Siliconized glass bottles</td><td>n/a</td><td>n/a</td><td>siliconize with Sigmacote (Sigma-Aldrich Co. LLC)</td></tr><tr><td>Omnifix Luer 3 ml</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>4616025V</td><td></td></tr><tr><td>Omnifix Luer 5 ml</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>4616057V</td><td></td></tr><tr><td>Sealing plugs</td><td>Henry Schein Vet GmbH, Hamburg, Germany</td><td>900-3057</td><td></td></tr><tr><td>Inoculum</td><td></td><td>n/a</td><td>dilute pathogen in 8 ml buffer</td></tr></tbody></table>

the bovine lung in biomedical research: visually guided bronchoscopy, intrabronchial inoculation and in vivo sampling techniques

有在呼吸内科的研究不断寻找替代动物模型。根据不同的研究目的，大动物肺疾病模型往往类似于人类的肺的情况要好得多比老鼠做。与大型动物的工作也提供了机会，反复品尝同一动物在一段时间，这使得长期研究，而不会牺牲动物一定的过程。 目的是要建立体内抽样方法，在一个呼吸鹦鹉热衣原体感染的牛模型的使用。采样应该在不同时间点的学习过程中执行在每个动物，样品应该是合适的，研究的实验条件下的宿主反应，以及病原体。 支气管镜检查是在人类医学和兽医学有价值的诊断工具。它是一种安全，微创手术。这北极勒描述牛犊的支气管内接种以及抽样方法，下呼吸道。内视镜，支气管内接种导致非常一致的临床和病理表现在所有接种动物的，因此，非常适合在肺部感染疾病模型的使用。所描述的抽样方法是支气管肺泡灌洗，支气管刷和支气管肺活检。所有这些都是有价值的诊断工具，在人类医学和可适应的实验目的，以6〜8周龄的小牛。所得到的样品是同时适合病原体检测的肺部炎症在宿主的严重程度和特性。

病程对动物的健康的病原体的效果可以通过临床检查来评估。在我们的呼吸道感染模型，动物进行了检查，每天两次，并用评分系统临床观察记录。附加信息被抓获的从事其他活体取样的方法， 例如 ，采集血液和拭子或肺功能测量。病理组织学检查进行了在不同时间点后接种来描述感染32-34的进度。 支气管肺泡灌洗液回收率在灌输流体的回收率为83.05±4.58％（平均值±标准差）。 病原体检测病原体可复垦支气管刷检进行。另外，各种样品的PCR筛选能够检测的病原体， 例如钛SSUE活检，细胞学刷取样，支气管肺泡灌洗液细胞52或咽拭。病原体的可视化是可能的通过进行免疫组化分析的肺活检和支气管肺泡灌洗液细胞的沉淀制剂的冷冻切片（ 图3）。在以前的实验中，血样和棉签（结膜，粪便，鼻）的PCR方法进行表征和传播的病原体32的脱落。 肺组织局部炎症标志物 BALF中，肺部炎症的各种参数进行研究。总细胞计数和嗜中性粒细胞的比例通常增加时肺部炎症的存在。对于细胞分化，BALF细胞的沉淀制剂可以根据吉姆萨染色，并使用油镜（ 图4）区分。对支气管肺泡灌洗液中细胞和液体的比例通过离心（300×g离心，20分钟）分离。对支气管肺泡灌洗液上清液中包含在肺的炎性过程会改变和实验条件下，可以研究各种标记。例子包括总蛋白和类花生酸类物质29,34。 的电位进一步用所描述的样品的示意图示于图5。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51557/51557fig1highres.jpg" width="500" /> 牛肺与接种位点（黄色）的图1。方案。中的数字表示在该接种物被施用到不同的支气管的顺序。记：权; L：左。右肺：1 Lobus肌 ：0.5毫升，2 Lobus accessorius：0.5毫升，3 Lobus尾侧 ：0.5 ml和4 1.0毫升;左肺：Lobus cranialis， <strong&gt; 5 帕尔斯cranialis：0.5毫升，6 帕尔斯尾侧 ：0.5毫升，7 Lobus尾侧 ：1.5毫升，8 Lobus cranialis，帕尔斯尾侧为1.0 mL。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51557/51557fig2highres.jpg" width="500" /> 。请注意，所有的样品都来自那里的病原体被沉积区域获得支气管肺泡灌洗（蓝色），支气管刷（绿），和肺活检（橙色）：图2中的牛肺与接种位点（黄色）和采样点的计划之前。记：对，L：左。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51557/51557fig3highres.jpg" width="500" /> 图3。一 ）龙从接种叶绿素a小牛活检<html> amydia鹦鹉热接种后4天（DPI），B），支气管肺泡灌洗液中从小腿细胞沉淀接种C。鹦鹉热 9 dpi的。免疫组化标记为衣原体。衣原体包涵体（箭头）是存在于肺（a）和肺泡巨噬细胞（B）。苏木素染液。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51557/51557fig4highres.jpg" width="500" /> 图4。支气管肺泡灌洗液中从接种C.小腿细胞沉淀物鹦鹉热 9 dpi的。肺泡巨噬细胞（＃）是BALF中的主要细胞类型。当炎症过程中存在嗜中性粒细胞的数量（*）增大。修改巴本海姆染色。 es.jpg“宽度=”600“/&gt; 图5。可能的方法的样品制备。 <a href="https://www.jove.com/files/ftp_upload/51557/51557fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

本文介绍了在牛肺支气管镜技术的实验条件下， 即支气管镜引导下接种，支气管肺泡灌洗，支气管刷和支气管肺活检。

牛龙在生物医学研究：视觉引导支气管镜，支气管内接种和<em&gt;在体内</em&gt;抽样技术

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

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Research

JoVE Journal

Medicine

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

23.1K 次观看.  Friedrich-Loeffler-Institut. 本文介绍了在牛肺支气管镜技术的实验条件下， 即支气管镜引导下接种，支气管肺泡灌洗，支气管刷和支气管肺活检。 

视频: The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

科研

免疫与感染

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

支气管肺泡灌洗（BAL）的研究，获得适当的采样率

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

医学

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 ...

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 ...

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 ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

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 ...

Transbronchial Lung Cryobiopsy for Diagnosing Interstitial Lung Diseases and Peripheral Pulmonary Lesions - A Stepwise Approach

Transbronchial lung cryobiopsy (TBLC) is an invasive procedure increasingly implemented during the last decade as an alternative to video-assisted thoracic surgery lung biopsy (SLB) for diagnosing interstitial lung diseases (ILDs). The indication for TBLC has primarily been to sub-classify a specific ILD subtype when this cannot be achieved on the basis of a preceding multidisciplinary team discussion. Although SLB is considered the gold standard for establishing a histological diagnosis, TBLC has been gradually suggested as the first-choice histological diagnostic modality in patients with unclassified ILDs due to a comparable diagnostic yield with SLB, but superior to SLB in terms of complications, including mortality. During recent years, radial endobronchial ultrasound (R-EBUS) and electromagnetic navigation bronchoscopy (ENB)-guided TBLC for peripheral pulmonary lesions have also been described as safe procedures, which may improve the diagnostic yield compared to forceps biopsies. Still, the diagnostic properties of TBLC rely on the quality of the procedure&#39;s performance. This article aims to describe the stepwise approach to conducting TBLC with a flexible bronchoscope for the different indications mentioned, which might be helpful for novice bronchoscopists performing TBLC.

<meta charset="utf8"></head><body>经支气管肺冷冻活检诊断间质性肺疾病和外周肺病变 - 一种阶梯式方法

Transbronchial lung cryobiopsy (TBLC) for diagnosing interstitial lung disease and peripheral pulmonary lesions is a high-yield diagnostic and safe procedure. We describe a stepwise approach to conduct TBLC for the different indications mentioned with a flexible bronchoscope, which might be helpful for novice bronchoscopists performing TBLC.

经支气管肺冷冻活检诊断间质性肺疾病和外周肺病变 - 一种阶梯式方法

Transbronchial lung cryobiopsy (TBLC) is an invasive procedure increasingly implemented during the last decade as an alternative to video-assisted ...

Intrabronchial Delivery: A Technique to Administer an Experimental Agent Selectively Into a Mouse Lung

Source: Liao, S. et. al. <a href="https://www.jove.com/v/59450/direct-intrabronchial-administration-to-improve-selective-agent">Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung</a>. J. Vis. Exp. (2019).
This video describes an intrabronchial approach to administer an experimental agent selectively into distal right and left lung. In the featured protocol, we will administer bleomycin intrabronchially into a mouse to obtain a better model for pulmonary fibrosis.

支气管内输送:一种选择性地将实验药物施入小鼠肺的技术

Source: Liao, S. et. al. Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung. J. Vis. Exp. (2019).
This ...

牛龙在生物医学研究：视觉引导支气管镜，支气管内接种和

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此视频中的章节

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

支气管肺泡灌洗（BAL）的研究，获得适当的采样率

无创性气管内插管，以研究小鼠肺的病理生理

研究微生物群落

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

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

直接注射内管理,改善小鼠肺内的选择性代理沉积

经支气管肺冷冻活检诊断间质性肺疾病和外周肺病变 - 一种阶梯式方法

经支气管肺冷冻活检诊断间质性肺疾病和外周肺病变 - 一种阶梯式方法

支气管内输送:一种选择性地将实验药物施入小鼠肺的技术