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 border="0" cellpadding="0" cellspacing="0" width="1174">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Veterinary Video Endoscope</td>
			<td style="width:419px;">Karl Storz GmbH &#38; Co. KG, Tuttlingen, Germany</td>
			<td style="width:121px;">PV-SG 22&#8211;140</td>
			<td style="width:443px;">diameter: 9 mm, working channel: 2,2 mm, working length 140 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lavage catheter&#160;</td>
			<td>Karl Storz GmbH &#38; Co. KG, Tuttlingen, Germany</td>
			<td></td>
			<td>diameter: 2 mm; length: 180 cm, Luer-lock-adapter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acuator</td>
			<td>WEPA Apothekenbedarf GmbH &#38; Co KG, Hillscheid, Germany</td>
			<td>32660</td>
			<td>length: 60 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">biopsy forceps</td>
			<td>Karl Storz GmbH &#38; Co. KG, Tuttlingen, Germany</td>
			<td>REF 60180LT</td>
			<td>1.8 mm, serrated, oval</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnifix 20 ml, Luer-Lock</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>4617207V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cytology brush</td>
			<td>mtp GmbH, Neuhausen ob Eck, Germany</td>
			<td>110240-10&#160;</td>
			<td>working length 180 cm, brush length: 15 mm, diameter 1.8 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iv acess&#160;</td>
			<td>Henry Schein Vet GmbH, Hamburg, Germany</td>
			<td>370-211</td>
			<td>diameter 1.2 mm, length 43 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rompun 2% (xylazin)</td>
			<td>Bayer Vital GmbH, Leverkusen, Germany</td>
			<td></td>
			<td>0.2 mg/kg bodyweight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamin 10% (ketamine)</td>
			<td>bela-pharm GmbH &#38; Co. KG, Vechta, Germany</td>
			<td></td>
			<td>2.0 mg/kg bodyweight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isotonic saline solution</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>3200950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUB 6 waterbath</td>
			<td>CLF analytische Laborger&#228;te GmbH, Emersacker, Germany</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">metal tube speculum&#160;</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>diameter: 3.5 cm, length: 35 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">flashlight</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">siliconized glass bottles</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>siliconize with Sigmacote (Sigma-Aldrich Co. LLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnifix Luer 3 ml</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>4616025V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnifix Luer 5 ml</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>4616057V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sealing plugs</td>
			<td>Henry Schein Vet GmbH, Hamburg, Germany</td>
			<td>900-3057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inoculum</td>
			<td></td>
			<td>n/a</td>
			<td>dilute pathogen in 8mL 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 ...

the-bovine-lung-biomedical-research-visually-guided-bronchoscopy

Research

JoVE Journal

Medicine

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

23.0K 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

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力:IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型:教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

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 ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

猪生物医学模型生物库组织样品的取样策略与处理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

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 ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

角膜上皮机械和化学损伤的离体和体内动物模型

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine. Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely. Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.

Experimental methods to harvest adult stem cells from canine intestinal and hepatic tissues to establish 3D organoid cultures are described. Furthermore, the laboratory techniques to ensure consistent growth and provide standard operating procedures to harvest, biobank, and revive canine intestinal and hepatic organoid cultures are discussed.