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...

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 border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Klebsiella pneumoniae, serotype 2</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:119px;">43816</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tryptic soy broth</td>
			<td style="width:169px;">Becton Dickenson</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel Safelet IV Catheters, 18G x 1 1/4&#34;</td>
			<td>Claflin Medical Equipment</td>
			<td style="width:119px;">MEDC-031122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Solution 1</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Solution 2</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-952</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hema 3 Fixative</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">23-122-929</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">27&#189; gauge tuberculin syringes</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">14-826-87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lithium heparin plasma collectors</td>
			<td style="width:169px;">Fisher</td>
			<td style="width:119px;">2675187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">L-shaped disposable spreaders</td>
			<td style="width:169px;">Lab Scientific</td>
			<td style="width:119px;">DSC</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:243px;">1x PBS, pH 7.4</td>
			<td style="width:169px;">prepared in-house</td>
			<td style="width:119px;">n/a</td>
			<td>Distilled water (5 L), NaCl (40 g), KCl (1 g), Na2HPO4 (5.75 g), KH2PO4 (1 g)&#160;&#160;&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:243px;">ACK lysis buffer</td>
			<td style="width:169px;">prepared in-house</td>
			<td style="width:119px;">n/a</td>
			<td>NH4Cl (4.145 g), KHCO3 (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?...

Watch this Scientific Journal Video about A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice at JoVE.com

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 ...

a-non-invasive-technically-non-intensive-method-for-induction

Research

JoVE Journal

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

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

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白细胞的归位和迁移的非侵入性的成像在体内