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>

<ol>
	<li>Spellberg, B., Talbot, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommended+Design+Features+of+Future+Clinical+Trials+of+Antibacterial+Agents+for+Hospital-Acquired+Bacterial+Pneumonia+and+Ventilator-Associated+Bacterial+Pneumonia.">Recommended Design Features of Future Clinical Trials of Antibacterial Agents for Hospital-Acquired Bacterial Pneumonia and Ventilator-Associated Bacterial Pneumonia.</a> Clinical Infectious Diseases. 51 (S1), S150-S170 (2010).</li><li>Kalil, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+Adults+With+Hospital-acquired+and+Ventilator-associated+Pneumonia:+2016+Clinical+Practice+Guidelines+by+the+Infectious+Diseases+Society+of+America+and+the+American+Thoracic+Society.">Management of Adults With Hospital-acquired and Ventilator-associated Pneumonia: 2016 Clinical Practice Guidelines by the Infectious Diseases Society of America and the American Thoracic Society.</a> Clinical Infectious Diseases. 63 (5), e61-e111 (2016).</li><li>Medina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+pneumococcal+pneumonia.">Murine model of pneumococcal pneumonia.</a> Methods in Molecular Biology. , 405-410 (2010).</li><li>Azoulay-Dupuis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+efficacy+of+a+new+fluoroquinolone,+sparfloxacin,+against+penicillin-susceptible+and+-resistant+and+multiresistant+strains+of+Streptococcus+pneumoniae+in+a+mouse+model+of+pneumonia.">In vivo efficacy of a new fluoroquinolone, sparfloxacin, against penicillin-susceptible and -resistant and multiresistant strains of Streptococcus pneumoniae in a mouse model of pneumonia.</a> Antimicrobial Agents and Chemotherapy. 36 (12), 2698-2703 (1992).</li><li>Mizgerd, J. P., Skerrett, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+pneumonia.">Animal models of human pneumonia.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 294 (3), L387-L398 (2008).</li><li>Rayamajhi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-surgical+intratracheal+instillation+of+mice+with+analysis+of+lungs+and+lung+draining+lymph+nodes+by+flow+cytometry.">Non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry.</a> Journal of Visualized Experiments. (51), (2011).</li><li>Nielsen, T. B., Bruhn, K. W., Pantapalangkoor, P., Junus, J. L., Spellberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+virulent+Acinetobacter+baumannii+to+reduce+variability+of+in+vivo+studies.">Cryopreservation of virulent Acinetobacter baumannii to reduce variability of in vivo studies.</a> BMC Microbiology. 15, 252 (2015).</li><li>Trammell, R. A., Toth, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+predicting+death+as+an+outcome+for+mice+used+in+infectious+disease+research.">Markers for predicting death as an outcome for mice used in infectious disease research.</a> Comparative Medicine. 61 (6), 492-498 (2011).</li><li>Bast, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+murine+model+of+pneumococcal+pneumonia:+use+of+temperature+as+a+measure+of+disease+severity+to+compare+the+efficacies+of+moxifloxacin+and+levofloxacin.">Novel murine model of pneumococcal pneumonia: use of temperature as a measure of disease severity to compare the efficacies of moxifloxacin and levofloxacin.</a> Antimicrobial Agents and Chemotherapy. 48 (9), 3343-3348 (2004).</li><li>Hankenson, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weight+loss+and+reduced+body+temperature+determine+humane+endpoints+in+a+mouse+model+of+ocular+herpesvirus+infection.">Weight loss and reduced body temperature determine humane endpoints in a mouse model of ocular herpesvirus infection.</a> Journal of the American Association for Laboratory Animal Science. 52 (3), 277-285 (2013).</li><li>Adamson, T. W., Diaz-Arevalo, D., Gonzalez, T. M., Liu, X., Kalkum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermic+endpoint+for+an+intranasal+invasive+pulmonary+aspergillosis+mouse+model.">Hypothermic endpoint for an intranasal invasive pulmonary aspergillosis mouse model.</a> Comparative Medicine. 63 (6), 477-481 (2013).</li><li>Nielsen, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+Exacerbates+Infection+via+Hyperinflammation+by+Signaling+through+TLR4+and+RAGE.">Diabetes Exacerbates Infection via Hyperinflammation by Signaling through TLR4 and RAGE.</a> mBio. 8 (4), (2017).</li><li>Nielsen, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+Antibody+Protects+Against+Acinetobacter+baumannii+Infection+by+Enhancing+Bacterial+Clearance+and+Evading+Sepsis.">Monoclonal Antibody Protects Against Acinetobacter baumannii Infection by Enhancing Bacterial Clearance and Evading Sepsis.</a> Journal of Infectious Diseases. 216 (4), 489-501 (2017).</li><li>Cheng, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+serotypes+5+and+8+capsular+polysaccharides+in+protection+against+Staphylococcus+aureus+in+murine+models+of+infection.">Evaluation of serotypes 5 and 8 capsular polysaccharides in protection against Staphylococcus aureus in murine models of infection.</a> Human Vaccine Immunotherapy. 13 (7), 1609-1614 (2017).</li><li>Wong, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+Pathophysiological+Overview+of+Acinetobacter+Infections:+a+Century+of+Challenges.">Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges.</a> Clinical Microbiology Reviews. 30 (1), 409-447 (2017).</li></ol>

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&#215;g at 4&#176;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&#215;15&#215;5 mm</td>
		</tr>
		<tr>
			<td>Dental Floss</td>
			<td>Oral-B</td>
			<td>37000469537</td>
			<td>Tie to stable post approx. 6&#34; 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&#39;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 &#176;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&#215;15 mm</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td>Dulbecco&#39;s PBS without calcium and magnesium</td>
		</tr>
		<tr>
			<td>Pipette Tips, 200-&#956;L</td>
			<td>VWR</td>
			<td>10017-044</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Pipetter, 200-&#956;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 &#215; 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

11.6K 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

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

发光法小鼠肝肿瘤模型

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

科研

医学

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

整骨手法治疗为肺炎是非常好的辅助工具

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

蛛网膜下腔出血的小鼠模型

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

口咽念珠菌病的免疫缺陷小鼠Th17细胞炎症模型

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

的不可逆和可逆单侧输尿管梗阻小鼠模型

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

Use of Electromagnetic Navigational Transthoracic Needle Aspiration (E-TTNA) for Sampling of Lung Nodules

Lung nodule evaluation represents a clinical challenge especially in patients with intermediate risk for malignancy. Multiple technologies are presently available to sample nodules for pathological diagnosis. Those technologies can be divided into bronchoscopic and non-bronchoscopic interventions. Electromagnetic navigational bronchoscopy is being extensively used for the endobronchial approach to peripheral lung nodules but has been hindered by anatomic challenges resulting in a 70% diagnostic yield. Electromagnetic navigational guided transthoracic needle lung biopsy is novel non-bronchoscopic method that uses a percutaneous electromagnetic tip tracked needle to obtain core biopsy specimens. Electromagnetic navigational transthoracic needle aspiration complements bronchoscopic techniques potentially allowing the provider to maximize the diagnostic yield during one single procedure. This article describes a novel integrated diagnostic approach to pulmonary lung nodules. We propose the use of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) for mediastinal staging; radial EBUS, navigational bronchoscopy and E-TTNA during one single procedure to maximize diagnostic yield and minimize the number of invasive procedures needed to obtain a diagnosis. This manuscript describes in detail how the navigation transthoracic procedure is performed. Additional clinical studies are needed to determine the clinical utility of this novel technology.

Use of Electromagnetic Navigational Transthoracic Needle Aspiration E-TTNA for Sampling of Lung Nodules

We describe the novel use of electromagnetic navigational guided transthoracic needle aspiration for the pathologic assessment of human lung nodules.

用于肺内结节样利用电磁导航肺穿刺抽吸术（E-TTNA）的

Lung nodule evaluation represents a clinical challenge especially in patients with intermediate risk for malignancy. Multiple technologies are presently ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.