This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (Z01 ES102005).

所有实验均按照动物福利法和人文关怀和实验动物的使用美国公共卫生服务政策审核后，由动物护理和NIEHS的使用委员会进行。 1. K.的制备肺炎文化注意:执行在生物安全2级（BSL2）罩或其他BSL2指定区域的所有步骤，并丢弃每个学院BSL2指引浪费。 <ol><li>对于K的悬浮生长肺炎 ，细菌解冻的甘油和转移1毫升K的接种工作文化肺炎库存到在500毫升或更大烧瓶50毫升胰蛋白酶大豆肉汤（TSB）组成。 </li><li>通过加入900微升培养数期克雷伯使甘油原液肺炎至100μl无菌甘油中，并在-20℃或-80℃冷冻。对于长期储存，建议-80℃。 </li><li>从文化的细菌由隔夜摇瓶TEP 1.1 - 在37℃下在200-225 rpm.In为了促进对数期生长（14 18小时），稀1-5毫升此过夜培养的成含50ml TSB在早晨烧瓶中并放回在37℃下振摇〜2.5小时。 </li><li>转移1毫升K的从最后一步到1.5ml试管和自旋经7,500×g离心2分钟肺炎培养。白色颗粒应该是可见的。不干扰沉淀除去上清液，轻轻地用移液管在1毫升的无菌盐水的悬浮细菌和7,500 xg离心再次旋转2分钟。 <ol><li>执行此清洗步骤2倍，造就了决赛，洗颗粒在1毫升无菌盐水。 </li></ol></li><li>执行此整洁的接种记录明智的连续稀释。例如，添加400微升接种到3.6毫升无菌盐水连续做出10 -1 -10 -9稀释系列。 </li><li>确定冲K的OD 600 肺炎通过将加入1ml的1:10和1:100的稀释液移入一次性试管并测量OD 600，用无菌盐水第一消隐。重复的测量，以确保准确性。的1.0的OD 600大约相当于4-7×10 8 CFU / ml的。注意，外径和CFU之间的关系/ ml的可能不是线性的。 <ol><li>从稀释使用OD 600来计算K的浓度肺炎，运用外径:CFU /毫升以上的转换，以及保理在稀释。 </li></ol></li><li>使用K.肺炎浓度以制备所需接种CFU剂量在&lt;100μl的体积递送到小鼠（见下文）。 </li><li>还板10 -6 -10 -9稀释100微升和100微升无菌盐水到在RT个人胰蛋白酶大豆琼脂（TSA）平板上。在37℃孵育过夜，并以计算出在实验中使用并控制污染确切浓度枚举的细菌菌落。 注:最终的接种实际细菌浓度可能是±500 CFU /毫升的预测被吸收。实验者最好应确认OD 600: 体内小鼠使用前试点研究CFU / ml的关系，凭经验调整的OD 600:根据自己的亲身经历CFU / ml的转换。 </li></ol> K. 2.鼠气管内（IT）吸入从肺炎口咽<ol><li>确保鼠尾标，纹身，或其他经批准的标识符唯一标识。 </li><li>制定给药使用之前，为了加快程序去除异氟醚小鼠P200吸管细菌接种。对于它的愿望最佳效果，使用&lt;100微升的体积，以防止溢出。在这里，使用2000 CFU / 50微升的K.肺炎 。 </li><li>麻醉小鼠用异氟烷气体（以1升/分钟的流速例如 2％异氟烷）的明确室或按institutional准则。小鼠的麻醉一起数被实验者的舒适程度，通常为1-2在一个时间确定。观察呼吸和确认麻醉级别后，深呼吸是可见的，2-3秒可呼吸间进行计数。 注意:如果有关于挥发性（吸入）麻醉药可能的混杂影响表示关切，麻醉可以达到，而不是用氯胺酮/赛拉嗪腹腔注射。用上述方法，深度麻醉只持续几分钟。如果更长时间麻醉诱导，眼膏应该用来防止眼干燥。 </li><li>一旦老鼠被麻醉，其定位在一个半卧位仰卧位，由橡皮筋上颌切牙悬挂在倾斜的有机玻璃板钉（ 图1）之间拉伸。 </li><li>轻轻拉动鼠标舌边有一对钝非脊钳和存款量进入口腔与P200 pipett即十分小心，以避免诱发创伤要么舌或口咽部。 </li><li>在保持缩回舌头，轻轻地用戴手套的手指，直到鼠标吸入堵塞的鼻子。继续以覆盖鼻子直到鼠标已采取两个或两个以上吸入剂，并且没有液体在口腔中可见。覆盖鼻子有助于确保鼠标会吸入细菌进入肺部，因为老鼠是专鼻子呼吸。 </li><li>从接种板上取下鼠标，回到笼子里，把鼠标在它的后面，防止寝具或碎屑堵塞鼻孔，而鼠标从麻醉中复苏。 </li><li>一旦所有的老鼠被给予和从麻醉中苏醒了，小家鼠在一个房间/房间只含老鼠已经与剂量K.肺炎。每天监控小鼠，包括体重，如果超越48小时后感染。 </li><li>如果让老鼠超越48日常使用的混搭和/或补充热量小时。 </li></ol> 3.支气管肺泡灌洗液（BALF）收集和分析<ol><li>每安乐死机构准则老鼠。在这里，使用150毫克/公斤戊巴比妥钠或商业安乐死溶液，然后放血等效剂量注射死刑，因为这避免了可能出现的并发症与CO 2吸入和颈椎脱位合并肺部。 </li><li>在后卫位置上的鼠标和尤其是在胸部和颈部用70％乙醇喷洒消毒皮毛鼠标。 </li><li>使刚刚胸骨下方的纵行剪开，然后拿着用钳子胸骨，尼克隔膜，使肺回落入胸腔。对血管的任一侧切向上穿过胸腔，然后向上通过颈部，露出气管。 </li><li>由于气管被纵肌两边包围，精心裁剪通过中间，推动组织向两侧，随着这避免了切割周边脉管，这可能引入血液进入气管。如果脉管缺口，清洁用纱布周边地区访问气管管腔前。 </li><li>使用外科剪刀或针缺口气管关于从头部向下的方式¼并插入连接于1ml注射器用1ml 1X磷酸盐缓冲盐水（PBS）尾部进气管的预装的插管。如果使用鼠标&lt;8周龄或女性，在灌洗量减少至600-800微升。 </li><li>推体积入肺缓慢，允许所有裂片膨胀，然后用注射器拉动卷退了出来，重复3次。如果PBS正在走出鼻孔插管未按远远不够进入气管或通货膨胀过快发生。备选地，如果肺不充气良好，插管已被推在太多，所以略微撤回。 </li><li>收集在冰上15毫升管汇集洗涤。 </li><li>纺于1200×g离心5分钟空域灌洗液，收集上清到1.5毫升管，并在-80℃下冻结。接着，使用上清液（ 即 ，BALF）用于蛋白质，细胞因子，以及用于各种其它生化分析的测量结果。粒料表示从支气管肺泡空间的细胞。 </li><li>如果基于颜色的红血细胞（红细胞）是在细胞沉淀明显，溶胞用1ml ACK裂解缓冲液中进行1分钟，然后加入5毫升1×PBS中的停止裂解反应和稀释缓冲液中。处理所有标本相同，无论是处理或不ACK裂解液。 </li><li>自旋的细胞以1200 xg离心5分钟，倒出上清液，并且使细胞上于1ml 1×PBS中。 </li><li>涡街流量计和计数细胞的总空域细胞计数血球。 </li><li>基于细胞的密度增加约80-150微升（约80微升的被感染的老鼠和150个为一个天真鼠标）在一个细胞离心离心机滑动室。旋转细胞小号到显微镜玻片后续染色以确定细胞分化的人群。 </li><li>让细胞干通宵幻灯片上。染色细胞用三污渍（ 如 ，HEMA 3 [见表]）通过浸渍将载玻片在固定剂的7倍，在染色1 9倍，7倍污点2（污渍之间没有漂洗），并使其干燥。染色干燥后，盖玻片幻灯片和手动计数显微镜差异。一般地，中性粒细胞和单核细胞/巨噬细胞容易受到它们的尺寸和核形态区分开来。 </li></ol> 4.确定肺癌和周围组织的细菌负荷<ol><li>开始之前:与900微升1×PBS对肺和脾和90微升1×PBS的血液稀释准备试管。标签板用于培养细菌，并在室温集;因此，一旦组织已收集时间将均质化并镀细菌生长之间被最小化。 注:由于异构deposi在可与抽吸发生肺部细菌和灰，建议个体小鼠被用于任一总呼吸道细胞结构或总（双边）肺细菌负荷的分析。 </li><li>安乐死鼠标按步骤3.1和随后收集血液从左心室，放置成肝素管，以避免凝固。除去在含有5ml 1×PBS中，接着除去脾脏和安置的15毫升管所有肺叶和地点于2ml 1×PBS中。试管置于冰上。小心清洁仪器，每个组织/鼠标之间的乙醇。 </li><li>均化在冰上组织，优选与可以每个样本之间改变支配均化。一次性均质提示可高压灭菌实验之间重用。 </li><li>一旦组织被匀浆，进行连续稀释和板。按图纸上的塑料分界线在一个单一的TSA板重复给定组织/稀释板样品。电镀和迪lution建议是如下（在所有情况下，10微升样品被涂布在板），并以24小时后的感染尸检。如果分析48-72小时的样品，稀释液的较大的数目可能需要在稍后的时间点，由于增加的细菌负担电镀。 <ol><li>血液 - 加入10微升的血液进入90微升无菌1X PBS的稀释1:10。板整齐重复1:10稀释样品。一旦血液已被镀，9300 xg离心旋转残留血液10分钟，以对细胞因子和趋化因子的测量中提取血浆。 </li><li>加入100微升肺匀浆入900微升无菌1X PBS的连续100:肺 - 1 - 稀释1:10。板整齐匀浆重复所有稀释。 </li><li>加入100微升匀浆脾为900微升无菌1X PBS的连续100: - 对于脾虚稀释1:10-1。板整齐匀浆一式两份都稀释。 </li></ol></li><li>允许传播样品简单干。 </li&gt; <li>倒置TSA平板及地点在静态37℃培养箱中过夜。 </li><li>通过平均从板的两侧，并从各稀释的细菌菌落确定定植细菌的数量。在5毫升的肺和2毫升脾的初始稀释度因子，以确定总组织的CFU。 </li></ol>

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The authors declare that they have no competing financial interests.

细菌性肺炎小鼠模型，用基因靶向和体内生物和药物干预合作，提供了重要的见解肺宿主防御反应。伟大的进步特别是取得我们的支配中性粒细胞招募到受感染空域10,11趋化因子和粘附分子的理解。 体内肺炎模型，不同于基于细胞或替代办法，还提供了关键洞察内分泌被感染的肺和其他器官，如肝脏（急性期反应）之间发生的通信12和肾上腺（应力糖皮质激素）13。最后，由体内肺炎模型揭示了一个重要和临床高度相关的一点是，成功的病原体清除不等于主机的成功。在许多情况下，一个overexuberant免疫反应可导致宿主死亡由于过度的旁观者肺损伤，甚至在成功的病原体清除8,14的脸。鉴于此，病原体清除和免疫反应的平行测量通常最翔实，并可能最终需要存活研究以显示主机的综合韧性。 这里，我们已经通过抽吸描述了一个简单和非侵入性的方法用于递送细菌对鼠肺。相比雾化，这种方法执行学习人员的呼吸曝光下潜在风险，提供了高浓缩的接种物递送至肺深部，并通过眼和上呼吸道的免疫应答避免潜在的混杂。鼻内接种也携带上呼吸道感染的潜在混杂的关注，包括中枢神经系统4的局部侵袭性感染的可能性，并且还报道了导致在肺高度可变接种5，相对于经由任一口服或经皮途径气管插管，该方法需要很少的培训，是更快速（〜每只小鼠1分钟），而且，至少比后一种方法，具有较低的发病率。其中一些是由其它方法共享- -口咽抽吸方法的潜在缺点包括全身麻醉的要求，片状的概率（ 即 ，非对称和多相）病原体递送，下叶15的主要感染，以及无法直接病原体单方面单肺。由于后抽吸（数据未示出）在肺部片状分布-即以比雾化15其他所有肺部感染的方法中遇到的结果-我们一般不上感染小鼠进行单侧肺测定。两肺要么灌洗在一起，以评估免疫应答，或用于汇集细菌定量尸检一起itation和/或分子分析（ 例如 ，基因表达，髓过氧化物酶测定法，细胞因子ELISA）。该协议的特别关键的步骤是一步1.8 -confirming外径600:CFU / mL的关系之前， 在体内感染，也确认通过每个实验电镀接种量;以及步骤3.6 - 确保具有良好的体积将返回到注射器的气道的非创伤性灌洗。 尽管合并感染口腔菌群（ 即多种微生物肺炎）是一个理论的关注，我们还没有感染K.小鼠肺匀浆中遇到的多态细菌菌落肺炎或S.肺炎 。对于担心出现这种情况的调查，但是，对照组小鼠可如果需要暴露在吸气的车辆（ 即缓冲区）。虽然胃腔（通过残留细菌送气吞咽）的一些污染是可能的机智^ h我们描述的方法，我们从来没有遇到过胃肠临床体征或大体病理变化。此外，这种可能性是共同的气雾和鼻内方法，甚至可以发生以下中的气管内接种方法咳嗽。 一些方法论的关注是普遍的所有车型肺炎。对照组和实验组老鼠应该是年龄相匹配的2-3个星期内彼此理想。虽然我们还没有找到K.病原体清除或气道炎症明显的性别差异肺炎模型，小鼠应该性别匹配，如在先天免疫应答性别之间显著差异已经报道16。仔细考虑对照组小鼠的起源应该放在首位。同窝的控制一般都在购买商业控件不完全回交，在免疫细胞群微生物相关的差异，以及其他epigenet最好集成电路的差异是可能影响免疫应答17,18的所有可能的混淆。仔细注意应给予的特定血清型和感染细菌如致病实质性差别的培养历史可以看出。根据我们的经验K.肺炎 ，建议使用范围感染接种的执行最初的试点研究（ 如 500-2,000 CFU）的文献报道特异性细菌剂量的发病率/死亡率的-即使在相同的细菌血清型和小鼠品系-不得翻译好一个人的自己的实验室，有可能是由于各种技术差异。炎症及宿主防御的结果对照组和实验小鼠之间组间差异可能是接种有关。由于动物养殖和人员的后勤现实中，我们经常与每个实验组小鼠5-6进行实验。虽然这有时会提供了CF足够的统计力量U和细胞计数的结果，我们发现，我们经常需要，以实现充足的电力以重复这种规模的研究一次或多次。 一种用于在给定的实验一些小鼠中，在血液和脾脏没有检测到细菌生长，可能会遇到24小时后感染（ 图5）。我们解释这表明24小时可能接近可检测肺外传播动力学门槛。如果在血液/脾脏低/不可检测的生长，观察，从同一小鼠肺CFU的应始终进行检查，以解决技术错误的可能性（ 即 ，非常低的/不可检测肺部感染可能表明在小鼠的给药错误）。 最后，在小鼠体内细菌性肺炎口咽愿望传递方法是一个强大的技术，它是比较容易从成本，训练和装备的角度来看收购，而现实是即使没有广泛的专业技术实验室肺手续。该方法容易地连接到宿主防御反应的下游微生物和免疫学测定，并且可以另外被用作用于各种非苛性暴露的肺递送方法。

社区获得性肺炎仍然感染在美国死亡的主要原因，死亡率在过去40年，尽管在接种疫苗的改进和抗生素的战略1,2整体变化不大。尽管在公共卫生水平缺乏可察觉进展，近年来引人注目的进展已在我们的肺炎的分子和细胞发病机理的理解制成，具有许多的向前通过使用肺部感染的小鼠模型成为可能这些步骤。小鼠的遗传易处理性，鼠和人的免疫系统，和鼠靶向免疫试剂繁多，已成为商用的相似性都共同促进该领域的快速进步。 在文献中描述的细菌性肺炎的小鼠模型一般在四种情况之一的技术路线依赖于病原体接种:ⅰ）雾化; II）INTRanasal交付; III）口服交货;和iv）手术气管内注射（ 即气管切开术）3。感染的所有路线各有优缺点3。在上气道的特定，相对曝光，电位为口鼻菌群，对于全身麻醉要求的混合，递送至远端肺接种，的递送病原体大叶性肺炎分布，技术专长的要求，和程序的发病率的变异性在这些广泛地变化的方法。 常用的口服感染技术包括通过无论是"瞎子"（非可视）的方式，或直接在可视化喉气管3-5（translaryngeal）插管。这两种方法，而健壮的，需要大量的训练，还可以携带创伤的风险的上气道。在本报告中，我们描述了经口感染的一个技术上的非密集，非侵入性的，廉价且快速的方法，W特此细菌（ 肺炎在所提供的示例克雷伯 ）吸移到麻醉小鼠的口咽通过抽吸（ 即 ，吸入）递送至肺部。我们和其他人已经成功地6-9使用了吸入性肺炎的技术。这种多功能，也容易学习肺递送方法可以扩展至输送许多额外的非苛性剂到肺部，包括细胞因子和其它蛋白质，病原体相关分子（ 如脂多糖），细胞（ 即 ，继转移），和毒素（ 例如 ，博莱霉素）。除了讨论重要的技术方面的考虑，我们还描述了一个综合的，定量的方法来评估随后的宿主反应肺炎，包括细菌清除下游测量（ 即菌落形成单位[CFU]定量肺和外周器官）和白细胞堆积在领空。

<table><tbody><tr><td>Klebsiella pneumoniae, serotype 2</td><td>ATCC</td><td>43816</td><td></td></tr><tr><td>Tryptic soy broth</td><td>Becton Dickenson</td><td>211825</td><td></td></tr><tr><td>Excel Safelet IV Catheters, 18 G x 1 1/4&quot;</td><td>Claflin Medical Equipment</td><td>MEDC-031122</td><td></td></tr><tr><td>Hema 3 Solution 1</td><td>Fisher</td><td>23-122-937</td><td></td></tr><tr><td>Hema 3 Solution 2</td><td>Fisher</td><td>23-122-952</td><td></td></tr><tr><td>Hema 3 Fixative</td><td>Fisher</td><td>23-122-929</td><td></td></tr><tr><td>27&amp;frac12; gauge tuberculin syringes</td><td>Fisher</td><td>14-826-87</td><td></td></tr><tr><td>Lithium heparin plasma collectors</td><td>Fisher</td><td>2675187</td><td></td></tr><tr><td>L-shaped disposable spreaders</td><td>Lab Scientific</td><td>DSC</td><td></td></tr><tr><td>1x PBS, pH 7.4</td><td>prepared in-house</td><td>n/a</td><td>Distilled water (5 L), NaCl (40 g), KCl (1 g), Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (5.75 g), KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (1 g)&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>ACK lysis buffer</td><td>prepared in-house</td><td>n/a</td><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl (4.145 g), KHCO&lt;sub&gt;3&lt;/sub&gt; (0.5 g), EDTA (18.6 mg), bring up to 500 ml with distilled water and pH to 7.4</td></tr></tbody></table>

a non-invasive and technically non-intensive method for induction and phenotyping of experimental bacterial pneumonia in mice

虽然社区获得性肺炎仍然是一个重大的公共卫生问题，细菌性肺炎小鼠模型中我们底层的细胞和分子发病机制的认识，最近促进显著临床进展。 在活体小鼠模型捕捉宿主防御反应的综合生理和弹性的方式不被替代的，简化的离体方法揭示。几种方法已在文献中小鼠细菌肺内接种，包括雾化，鼻内递送，"盲"和可视化的条件下经口气管插管，和经皮气管插管进行了描述。所有的方法都具有相对的优点和局限性。这里，我们详细描述用于细菌的气管内递送，通过鼠标涉及抽吸（ 即 ，吸入）的非侵入性的，在技术上不密集的，廉价且快速的方法的同时，在全身麻醉下吸移到口咽感染接种物。这种方法可用于各种各样的无腐蚀性生物和化学制剂的肺递送，并且相对容易学习，即使对于肺程序最小先前经验的实验室。除了描述的吸入性肺炎的方法，我们也用于测定随后在体内小鼠的肺先天免疫反应，特别是提供一步一步的程序，用于量化细菌清除和感染呼吸道的细胞免疫应答的方法。这种集成和简单的肺炎评估方法允许的遗传和环境操纵在肺先天免疫效果快速和强大的评估。

C57BL / 6小鼠被感染K的2000 CFU 肺炎 43816（2型），通过口咽部吸入肺。在这个剂量，一般老鼠开始出现临床症状12-24小时后感染，包括嗜睡，皱皮，体重减轻5％-10％（ 图2A）的。内48-72小时感染后，许多小鼠表明，通常是由平均20％的重量损失和结果的在具有降低活性驼背姿势之前和响应减少到刺激疾病和发病的症状。更长的持续时间的研究中，可能需要K的低剂量肺炎 。感染K. 500 CFU 肺炎导致较温和的疾病峰值3-5ð感染后，其特点是有恢复和体重增加开始由5-6天（ 图2B）的重量损失10-15％。体重是经常预测的生存，以20％的稀有的重量损失LY与恢复相关。 宿主反应的最终成功可能存活研究，其中小鼠10-14天为濒死的迹象，如最小的响应，以触觉刺激和/或减重监控，安乐死（每制度上认可准则）得到最好的说明&gt; 20％。如果一个目标的侵染接种对照组（ 图2C）在接近50％的致死率（ 即 ，LD 50剂量）存活研究通常最有效的。的LD 50剂量可以检测的两个相对增加，并在实验组存活率降低。差动存活可能是接种相关的，所以几个感染剂量有时可能需要以检测改变的宿主防御的表型。 下呼吸道感染K.肺炎的特征在于洛伊克的健壮涌入ocytes进入气道。免疫细胞计数和分化的BAL细胞沉淀的染色表明6H感染后（ 图3A）内的增加的气道的免疫细胞。 24小时的细胞构成的山峰，中性粒细胞是在气道（ 图3A-B）的主要免疫细胞。 C57BL / 6小鼠的两性表明等效呼吸道白细胞响应于K.肺炎在24小时和48小时后的感染（ 图3C-D）。 细菌性肺炎诱发本地（肺内）和全身促炎介质包括细胞因子和趋化因子。局部细胞因子/趋化因子水平可以在与常规的ELISA中BALF或通过使用多重细胞因子系统进行检测。细胞因子包括IL-6，RANTES，TNFα，G-CSF，和其它的是在两个24小时和48小时后的感染K.可检测肺炎并提供洞察伦克微环境和其免疫细胞的募集（ 图4）。 肺，血液和外周组织（多个）如脾脏的集合允许对组织的细菌负荷的定量，提供洞察微生物的本地肺间隙以及细菌的肺外传播。连续稀释和肺的培养证实的24小时和48小时后的感染（ 图5A）之间的微生物负担扩张。类似的结果在血液和脾（ - C 图5B）指出。值得注意的是，双峰细菌负担可以在血液和脾脏在24小时后感染另外，具有高品位的细菌传播某些小鼠和人，即使在肺中的面部的高档细菌负荷，显示无检测细菌在血液或脾脏。K.在C57BL / 6小鼠的肺炎细菌负载不GEND呃特异性的，如男性和女性表现出在各种组织接种后（ 图5D-F）的等效的负担。 <img alt="图1" src="/files/ftp_upload/54508/54508fig1.jpg" /> 图 1: 程序委员会为吸入性肺炎小鼠的研究小鼠被放置，头，回到板（ 即 ，半卧位），由门牙悬挂于一根橡皮筋拉长的两个销钉安装在一个有机玻璃或类似的主板。 <img alt="图2" src="/files/ftp_upload/54508/54508fig2.jpg" /> 图2: 减肥和评估发病率随着 K 的肺部吸入 肺炎 。C57BL / 6小鼠（N = 10％至20条件）由K的抽吸感染肺炎 43816（2型）。 （A）每日的权重，索引预感染重，从感染2000 CFU小鼠。在这项研究中，没有小鼠存活过去6天（B）感染500 CFU结果在日常减肥经过4天感染后有一个过渡向增重150日起5（C）生存曲线CFU，500 CFU和2000菌落形成单位（CFU）的感染，这表明500 CFU近似于LD50。在板A和B的数据是平均±SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54508/54508fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54508/54508fig3.jpg" /> 图3:感染 K.肺炎 结果在与时间相关的气道白细胞增多。C57BL / 6小鼠给予肺内感染K的2000 CFU 肺炎在不同时间空域流体总白细胞（WBC），嗜中性粒细胞（PMN）和巨噬细胞（Mφ）的即（A）的枚举指向后感染。 （B）在染色，中性粒细胞和巨噬细胞可以通过核形态和大小在细胞离心涂片领域容易识别和计数。 （C - D）的男性和女性感染导致招募炎性细胞的数量相似于24小时和48小时后的感染。数据每6-25条件- / -小鼠中获得，并均数±SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54508/54508fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54508/54508fig4.jpg" /> 图4: 细胞因子和趋化因子诱导的空域响应于细菌感染的C57BL / 6小鼠给予intrapulmon。叉感染K. 2000 CFU 肺炎。BALF的细胞因子和趋化因子是由多重测定在24小时和48小时后的感染定量。数据从5-15只/条件推导，并平均±SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54508/54508fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54508/54508fig5.jpg" /> 图 5: 肺炎 C57BL 期间在肺癌和外周组织的细菌负担的时间依赖性膨胀 / 6小鼠给予肺内感染K的2000 CFU 肺炎。（A）肺实质细菌负担通过TSA 24小时和48小时后的感染电镀肺匀浆连续稀释测量。 （B）的血流和（C）脾细菌负荷进行类似定量。 （ <str翁&gt;ð - F），在各种组织中的细菌负担在肺炎的诱导后雄性和雌性小鼠当量。数据10只小鼠/条件推导，并均数±SEM。在板B，C，E和F的零值分配值0.1，允许对数尺度的图形。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54508/54508fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

几种方法已在文献中的小鼠模型细菌性肺炎进行了描述。在此，我们描述了用于通过移液到口咽细菌接种物的抽吸（ 即 ，吸入）诱导肺炎的非侵入性的，廉价的，快速的方法。对于肺先天免疫应答的评估下游方法也是详细说明。

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 ...

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Immunology and Infection

8.9K 次观看.  National Institutes of Health. 几种方法已在文献中的小鼠模型细菌性肺炎进行了描述。在此，我们描述了用于通过移液到口咽细菌接种物的抽吸（ 即 ，吸入）诱导肺炎的非侵入性的，廉价的，快速的方法。对于肺先天免疫应答的评估下游方法也是详细说明。 

视频: A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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, ...

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, ...

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

通过细胞内分娩诱导小鼠脑膜炎球菌性脑膜炎血清群C

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

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

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

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 ...

医学

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

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 ...

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

摘要

探索更多视频

此视频中的章节

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

肺炎链球菌在鼻内殖民期间炎症反应的特征

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

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

通过细胞内分娩诱导小鼠脑膜炎球菌性脑膜炎血清群C

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

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

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

摘要

探索更多视频

此视频中的章节

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

肺炎链球菌在鼻内殖民期间炎症反应的特征

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

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

通过细胞内分娩诱导小鼠脑膜炎球菌性脑膜炎血清群C

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

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

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