这项工作得到国家卫生研究院的国家过敏和传染性疾病研究所的支持 [R01 AI117211、R01 AI130060、R21 AI127954 和 R42 AI106375 到 BS] 和美国食品药品监督管理局 [合同HHSF223201710199C 到 BML]。

所有涉及动物的程序必须由研究员的机构动物护理和使用委员会 (IACUC) 批准。
1. 细菌接种的制备
<ol><li>分离细菌菌落。<ol>
<li>在适当的无菌琼脂培养基上 (例如, 胰蛋白酶大豆琼脂) 上 HUMC1 细菌菌株 (例如,鲍曼), 小心产生孤立的菌落。</li>
		<li>在适当的条件下孵化 (例如, 在37摄氏度过夜)。</li>
	</ol>
</li>
	<li>发展隔夜文化。<ol>
<li>从琼脂板中选择一个具有无菌接种循环的具有代表性的孤立菌落, 并用它接种10毫升适当的无菌发酵液培养基 (如胰蛋白酶酱油)。</li>
		<li>允许样品到达固定的阶段在适当的情况下 (例如, 37 °c 与震动在200转每隔夜在一个通气盖, 50 毫升圆的小瓶)。</li>
	</ol>
</li>
	<li>培养亚文化。<ol>
<li>使用吸管将隔夜文化的 100 ul 转移到10毫升新鲜的无菌汤中。</li>
		<li>允许达到指数/对数阶段在适当的条件 (例如, 37 °c 与震动在 200 rpm 3 小时在一个通气帽, 50 毫升圆锥小瓶)。</li>
	</ol>
</li>
	<li>洗净亚文化。<ol>
<li>从孵化环境中移除亚文化。</li>
		<li>离心机在 4000 x g 5 分钟内颗粒细菌。</li>
		<li>吸入并丢弃上清。</li>
		<li>将10毫升无菌磷酸盐缓冲盐水 (PBS) 添加到颗粒和漩涡中, 直到完全悬浮。</li>
		<li>重复步骤 1.4.2-1.4.4 两次, 总共3洗涤。</li>
	</ol>
</li>
	<li>调整细菌悬浮液的浓度。<ol>
<li>使用分光光度法测量 600 nm (OD600) 的光学密度, 测量细菌悬浮液的光学密度。</li>
		<li>将无菌 PBS 添加到细菌悬浮液中, 直到接种的光学密度降至 OD600的 0.5, 使用等式 ci×Vi = cf×Vf, 其中 "c" 是集中, "V" 是容量, "i"是初始的, "f" 是最终的。* Cf应始终为 0.5;Vf是要求解的变量。</li>
		<li>如果细菌悬浮液低于 od600的 0.5, 离心它在 4000 x 克5分钟, 以颗粒细菌和清除适量的上清达到 OD600 0.5。</li>
		<li>在无菌 PBS 中执行串行稀释 (例如, 三1:100 稀释达到 1 x 10-6) 和无菌琼脂板, 以确定每毫升菌落形成单位中细菌浓度的相关系数 (CFUs/毫升)在 OD600的0.5。 注: 此相关系数的值将因每个物种的每种应变而异, 并且必须在接种准备感染前确定。</li>
		<li>在确定相关系数后, 使用它来创建所需浓度的接种 (例如, 2 x 108-1 x 109 CFUs/毫升);如果所需的接种大于 OD600的 0.5, 离心机的细菌悬浮在 4000 x g 5 分钟, 以颗粒的细菌和删除适量的上清达到预期的浓度。</li>
		<li>在体内进行试验性研究, 以确定每株小鼠的每种细菌的毒力, 因为这些值在不同基因背景的同一物种和小鼠的细菌分离之间会有所不同。对于不同的革兰阴性种, LD100在小鼠一般是 1-5 x 108 CFUs/小鼠, 虽然某些菌株是致命的在较低的菌。</li>
	</ol>
</li>
	<li>冻结相同的整除数, 以供将来用作按需、精确菌, 如先前发布的8 (可选)。<ol>
<li>准备 1 L 的细菌接种如上所述, 转移到两个500毫升圆锥小瓶, 和离心机在 4000 x 克5分钟。</li>
		<li>从每个圆锥小瓶中丢弃450毫升的上清液, 并在剩余的上清并用重悬细菌的颗粒。</li>
		<li>将浓缩的细菌接种转移到含有磁性搅拌棒的250毫升烧杯中, 并在 300 rpm 的搅拌板上连续混合。</li>
		<li>仔细转移 600 ul 的浓缩细菌接种 (例如, 1 x 1010 CFUs/毫升), 使用吸管到1.6 毫升低温小瓶, 其中含有完全不育 H300O 和 2 ul 的无菌甘油;混合和存储在-80 摄氏度。 注: 贮存所需的甘油会混淆实验结果, 导致过量死亡, 因此有必要将其冲洗掉。</li>
		<li>当准备使用时, 在室温下将样品解冻10分钟。</li>
		<li>将1毫升转移至50毫升圆锥小瓶, 加入9毫升的无菌 PBS, 并在 4000 x g 的离心机上为5分钟的细菌颗粒。在适当数量的 PBS 中吸入上清和并用重悬预先确定的 CFUs (1 毫升 x 冷冻库存浓度), 以达到所需的传染性接种浓度。</li>
	</ol>
</li>
</ol>2. 麻醉小鼠
<ol><li>氯胺酮/甲苯噻嗪 注: 麻醉与氯胺酮/甲苯噻嗪有一个很长的时间, 这为研究员谁是新的在这项技术完成接种程序之前, 老鼠醒来。<ol>
<li>通过结合8.5 毫升的 pharmaceutifcal 级 PBS, 1 毫升100毫克/毫升氯胺酮库存溶液 (最终浓度10毫克/毫升) 和0.5 毫升的20毫克/毫升甲苯噻嗪库存溶液 (最终浓度10毫克/毫升), 准备10毫升的注射液。</li>
		<li>管理 10 ul/g 的腹腔 (IP) 注射 (例如, 250 ul 的25克鼠标)。</li>
		<li>将眼膏应用于被氯胺酮/甲苯噻嗪麻醉的小鼠眼中, 因为他们的眼睛在麻醉的延长期间容易受损。</li>
		<li>把鼠标放在笼子里, 监视它, 直到它从麻醉中醒来。 注意: 动物不应被留在无人看管, 直到它恢复足够的意识, 以维持胸骨卧床。</li>
	</ol>
</li>
	<li>异 氟 醚 注意: 从诱导室取出后, 小鼠很快从异氟醚引起的麻醉中苏醒, 因此这种麻醉方法只应在精通接种程序后使用。<ol>
<li>把天真的老鼠放在适当大小的感应室里, 氧气流动。</li>
		<li>在密封室关闭后, 麻醉使用精确的蒸发器将异氟醚添加到 4% (v/v), 至少5分钟;通过从感应腔中取出一只老鼠来确认麻醉, 并测量从麻醉中醒来需要多长时间 (~ 六十年代)。 注意: 请注意, 麻醉时间可能因机构指南而异, 有些动物可能需要5分钟以上才能完全麻醉。</li>
		<li>通过将异氟醚浓度调整为 2-3%, 保持麻醉可达10分钟;如果麻醉总持续时间为 > 15 分钟, 可将眼膏应用于麻醉小鼠眼中。</li>
		<li>把鼠标放在笼子里, 直到从麻醉中醒来, 就像在步骤2.1 中一样。</li>
	</ol>
</li>
</ol>3. 接种/感染小鼠
<ol><li>暂停一只麻醉鼠标, 将其顶部门牙挂在一根结实的细弦上 (如牙线), 在高处固定物体的高度大约两倍于操作表面上方 (例如, 20 厘米).、实验室工作台)。</li>
	<li>用无菌的钝端钳轻轻地将老鼠的舌头从嘴里拉出来。将舌头转移到无菌、戴手套的手指上, 以防老鼠的舌头被意外粉碎。把舌头放在嘴外, 允许进入咽, 防止接种的吞咽。</li>
	<li>按住鼠标的舌头, 把 50 ul 接种 (细菌悬浮) 到咽使用微;咽位于口腔的交界处, 咽向口腔后部。 注意: 只有通过吹打在微的第一个停止处传递液体是非常重要的。当接种放置在咽中时, 鼠标将停止呼吸几秒钟;最终, 反身吸入会导致老鼠吸入细菌悬浮液, 这是由液体进入肺部的明显的爆裂性噪声所表明的, 导致肺炎感染。</li>
	<li>如果接种不足以阻挡正常的呼吸, 用镊子捏鼻孔, 以迫使反射吸入通过嘴和随后吸入的接种。</li>
	<li>接种后, 释放舌头, 把鼠标从悬浮绳上移开, 放在笼子里, 直到它从麻醉中醒来, 如步骤2.1。</li>
</ol>4. 监测疾病进展情况
注意: 由于动物的痛苦, 应使用各种指标来表明老鼠奄奄一息;应按照先前批准的 IACUC 议定书, 在这一确定之后执行安乐死;moribundity 的各种标记包括体温、体重减轻、外貌、步态和其他可以从血液中获得的生物标志物 (例如通过国家统计局)9、10、11、12、 13,14,15,16。
<ol><li>根据先前批准的 IACUC 协议 (例如, CO2和颈椎脱位) 弄死小鼠。</li>
	<li>切除肺从安乐死的老鼠通过切开气管, 肺动脉和肺静脉接近肺部。<ol>
<li>显微镜<ol>
<li>将肺部 (或部分) 放置在试样模具中。</li>
			<li>用最佳切削温度 (O.C.T.) 化合物填充试样模, 完全淹没组织。</li>
			<li>将样品冷冻-80 摄氏度。</li>
			<li>将样品送到病理学实验室, 然后在幻灯片上装上显微镜。</li>
		</ol>
</li>
		<li>细菌负担<ol>
<li>在分析天平上称肺组织。</li>
			<li>将肺组织转移到含有 2-5 毫升无菌 PBS 的50毫升圆锥小瓶 (取决于组织的大小)。</li>
			<li>用组织均质体融汇肺组织。</li>
			<li>根据预期肺部的细菌负担, 在无菌 PBS 中进行匀浆的连续稀释, 以达到约 1000 CFUs/毫升。<ol>
<li>例如, 如果预计 1 x 109 CFUs/毫克, 执行三1:100 稀释: 转移 100 ul 的匀浆到9.9 毫升的 PBS 和漩涡;这是第一个1:100 稀释。转移 100 ul 的前1:100 稀释到9.9 毫升的 PBS 和漩涡;这是第二个1:100 稀释。转移 100 ul 第二1:100 稀释到9.9 毫升 PBS 和漩涡;这是第三次和最后1:100 稀释。</li>
				<li>如果肺部的细菌负担不明, 执行一系列的稀释以捕获预期的范围。</li>
			</ol>
</li>
			<li>不育琼脂上的板材稀释。<ol>
<li>用琼脂 (TSA) 盘制备无菌胰蛋白酶大豆汤: 结合30克的, 15 克的琼脂, 1 升的水, 混合与一个磁力搅拌器, 并加热至沸点为100摄氏度为5分钟. 允许冷却, 然后在液体循环蒸压釜15分钟. 允许冷却 ~55°c, 转移10毫升新鲜蒸压 TSA 到无菌的培养皿, 并且允许冷却到室温。让干燥在台式 2-5 d 或发现在生物安全柜之下为10分钟。</li>
				<li>将稀释的 100 ul 转移到含有无菌 tsa 的培养皿中, 并使用吊具或玻璃珠在 TSA 上传播。</li>
				<li>将培养皿隔夜存放在37摄氏度的湿润孵化箱中 (即37 个°c 孵化器, 里面装满水的1升烧杯)</li>
			</ol>
</li>
			<li>根据琼脂电镀确定 CFUs/毫升肺匀浆 (例如, 100 CFUs 在 TSA 板上, 上面描述的第三和最后稀释的 100 ul 是 100 CFUs/100 ul x 100dil#1 x 100dil#2 x 100dil#3 = 1×109CFUs/毫升)。</li>
			<li>用镁肺组织/ml 匀浆法 CFUs/毫升肺匀浆方法计算 CFUs/镁肺组织 (如上文所述样品使用100毫克的肺组织匀质在 PBS 5 毫升, 然后 1 x 109 CFUs/毫升÷ 100 mg/5 毫升 = 5 x 107CFUs/毫克)。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

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作者声明他们没有竞争的财政利益。

可以肯定的是, 老鼠不是微型人类。从老鼠模型得到的结果必须考虑在上下文和随后解释为适用性对人, 根据区别和相似性在两个种类6之间。选择合适的小鼠菌株也很重要, 因为某些感染比其他的更容易发生;同样适用于选择16的病原体菌株。
必须以严格和高度可重复的方式进行感染。菌对老鼠的致命作用是一个价值, 但即使是90% 的价值也无?...

下呼吸道感染是世界上最致命的传染性疾病, 也是1发展中国家最常见的死亡原因。在全球范围内, 这些感染占320万以上死亡人数的1。此外, 医院内的肺炎是最常见和致命的医疗形式的感染, 并由最耐药性的病原体2,3造成。在社区获得和医院肺炎中获得细菌性肺炎的典型途径是口咽内容进入肺泡的愿望。用于研究这些疾病的小鼠模型经常使用鼻腔接种4, 将大部分细菌存放在肺部外, 造成非靶向并发症和症状, 如鼻窦炎和身体创伤, 这些都是不一致的疾病。在人类的进步, 模型设计来效仿。其他模型可能使用吸入室和 micromisting 设备, 更准确地模仿病毒, 结核, 和真菌肺炎, 但不准确地重述正常的采集路线的典型细菌肺炎。
小鼠口咽吸入性肺炎模型可用于模拟细菌性肺炎的自然途径和发病机制。通过用吸管将细菌悬浮剂的50µL 接种到麻醉小鼠的咽中, 随后会产生自反性的吸入, 导致感染性肺炎。利用这个模型, 我们可以研究肺炎引起的病原体的发病机制和新的治疗方法, 以更高的保真度模型来对抗这些疾病, 更类似于人类所观察到的吸入性肺炎感染。此外, 不同于通过口腔5,6感染的相似模型, 这个模型确保完全接种到达肺部而不是肠道, 在那里它可以引起非现场炎症和感染, 如胃炎和肠炎。最后, 不像另一个发布的模型, 需要喉镜和 inoculates 通过气管7, 这个模型不会阻碍呼吸道与饲针, 不需要注射接种分娩。相反, 接种依赖于老鼠的自然吸入反射。

<table><tbody><tr><td>Agar</td><td>BD</td><td>214530</td><td>Combine with TSB to make TSA</td></tr><tr><td>Beads, Borosilicate Glass</td><td>Kimble</td><td>135003</td><td>Sterilize by baking or autoclaving before each use</td></tr><tr><td>Beaker, 250 mL</td><td>Pyrex</td><td>1003</td><td>Used during precise aliquoting of concentrated bacterial inocula</td></tr><tr><td>Centrifuge</td><td>Sorvall</td><td>ST 40R</td><td>Capable of 4,000&amp;times;g at 4&amp;deg;C</td></tr><tr><td>Chamber for Anesthesia</td><td>Kent Scientific Corporation</td><td>VetFlo-0720</td><td>Accommodates up to 5 mice</td></tr><tr><td>Cryomold, Intermediate Size</td><td>Sakura Tissue-Tek</td><td>4566</td><td>Disposable vinyl specimen molds, 15&amp;times;15&amp;times;5 mm</td></tr><tr><td>Dental Floss</td><td>Oral-B</td><td>37000469537</td><td>Tie to stable post approx. 6&quot; above table height</td></tr><tr><td>Forceps</td><td>VWR</td><td>82027-440</td><td>Used to gently pull tongue out of mouse&#039;s mouth</td></tr><tr><td>Homogenizer for Lung Tissue</td><td>Omni International</td><td>TM125-115</td><td>Autoclave before first use; rinse between samples</td></tr><tr><td>Isoflurane for Anesthesia</td><td>Abbott</td><td>10015516</td><td>Alternative drug can be used; modify procedure accordingly</td></tr><tr><td>iSTAT Cartridge</td><td>Abbott</td><td>03P79-25</td><td>Various cartridges are available to suit your needs</td></tr><tr><td>Ketamine, 100 mg/mL</td><td>Western Medical Supply</td><td>4165</td><td>Dilute 1:10 in PBS to 1 mg/mL and combine with Xylazine at 1 mg/mL</td></tr><tr><td>Ointment for Eyes</td><td>Akorn</td><td>Tears Renewed</td><td>Avoid touching eye with tip of dispenser</td></tr><tr><td>Optimal Cutting Temperature (O.C.T.) Compound</td><td>Fisher Scientific</td><td>23-730-571</td><td>Used to freeze lung samples at -80 &amp;deg;C to prepare for pathology sectioning</td></tr><tr><td>Petri Dish</td><td>VWR</td><td>25384-302</td><td>Polystyrene, disposable, sterilized, 100&amp;times;15 mm</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Corning</td><td>21-031-CM</td><td>Dulbecco&#039;s PBS without calcium and magnesium</td></tr><tr><td>Pipette Tips, 200-&amp;mu;L</td><td>VWR</td><td>10017-044</td><td>Autoclave before use</td></tr><tr><td>Pipetter, 200-&amp;mu;L</td><td>Gilson</td><td>Pipetman P200</td><td>Autoclave and calibrate before use</td></tr><tr><td>Spreader, Bacterial Cell</td><td>Bel-Art</td><td>F377360006</td><td>Sterilize by baking or autoclaving before each use</td></tr><tr><td>Stir Bar, Magnetic, 7.9 mm Diameter &amp;times; 38.1 mm Length</td><td>VWR</td><td>58948-150</td><td>Used for stiring concentrated bacterial inocula during aliquoting</td></tr><tr><td>Stir Plate, Magnetic</td><td>Corning</td><td>PC-620D</td><td>Used for stiring concentrated bacterial inocula during aliquoting</td></tr><tr><td>Tryptic Soy Broth (TSB)</td><td>BD</td><td>211822</td><td>Combine with Agar to make TSA</td></tr><tr><td>Vial, Conical, Sterile, 50 mL</td><td>Corning</td><td>431720</td><td>Used for preparing bacterial inocula</td></tr><tr><td>Vial, Conical, Sterile, 500 mL</td><td>Corning</td><td>431123</td><td>Used to concentrate inocula for preparing frozen inocula</td></tr><tr><td>Vial, Cryogenic, 2.0 mL</td><td>Corning</td><td>430659</td><td>Used for cryogenic storage of concentrated bacterial inocula</td></tr><tr><td>Xylazine, 20 mg/mL</td><td>Akorn</td><td>AnaSed Injection</td><td>Dilute 1:20 in PBS to 1 mg/mL and combine with Ketamine at 1 mg/mL</td></tr></tbody></table>

murine oropharyngeal aspiration model of ventilator-associated and hospital-acquired bacterial pneumonia

小鼠感染模型对于了解疾病发病机制和测试旨在对抗致病病原体的新疗法的功效至关重要。感染性肺炎是临床病人最常见的感染之一, 因此需要适当的体内模型。典型的肺炎模型使用鼻腔接种, 将过量的生物体沉积在肺部外, 造成非靶向并发症和症状, 如鼻窦炎、胃炎、肠炎、物理创伤或微粒喷雾模拟气溶胶。传播更典型的病毒, 结核, 或真菌性肺炎。这些模型不能准确地反映典型的社区或医疗保健获得的细菌性肺炎的发病机制。与此相反, 这种小鼠口咽吸入性肺炎模型模仿了医疗获得性肺炎的雾滴路径。接种50µL 的细菌悬浮液进入麻醉小鼠的咽, 引起反射性吸入, 导致肺炎。通过这个模型, 我们可以研究肺炎引起的病原体的发病机制和新的治疗方法来对抗这些疾病。

通过仔细遵循该协议, 可以很容易地获得可重现和健壮的数据。严格遵守自己的定制接种准备协议是至关重要的, 因为实验要相互比较。在感染过程中适当处理小鼠也很重要。一定要把老鼠放进一个没有异氟烷的麻醉室里。老鼠会恐慌, 如果他们被放置在一个房间, 已预先填充异氟醚, 可能会遇到过剩的压力, 这可能会损害实验结果。在固定容器盖后, 缓慢地引入异氟醚, 逐步...

Watch this Scientific Journal Video about Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia at JoVE.com

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

传染性肺炎是人类最常见的感染之一。适当的体内模型对于了解疾病发病机制和检测新疗法的疗效至关重要。与这个小鼠口咽吸入肺炎模型, 你可能审查发病机制和新的治疗反对这些致命的传染。

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...

murine-oropharyngeal-aspiration-model-ventilator-associated-hospital

Research

JoVE Journal

Medicine

11.7K 次观看.  University of Southern California. 传染性肺炎是人类最常见的感染之一。适当的体内模型对于了解疾病发病机制和检测新疗法的疗效至关重要。与这个小鼠口咽吸入肺炎模型, 你可能审查发病机制和新的治疗反对这些致命的传染。

视频: Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

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

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

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

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

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

科研

免疫与感染

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

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 Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

医学

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

评估肺炎消退的实验模型

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

建立用于呼吸道感染研究的 Lung-on-chip 模型

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

作者聚焦：开发微流体 Lung-on-Chip 模型，用于深入研究人类免疫反应和感染机制

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs ...

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

摘要

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此视频中的章节

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

绿脓杆菌引起的肺损伤模型

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

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

评估肺炎消退的实验模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

摘要

探索更多视频

此视频中的章节

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

绿脓杆菌引起的肺损伤模型

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

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

评估肺炎消退的实验模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病