This work was supported by AI101263 from the National Institutes of Health (T.V.B), and by a grant from The Hartwell Foundation (M.L.M). M.L.M. is a member of the Duke Comprehensive Cancer Center.

注：所有涉及使用大鼠实验过程中按照批准的杜克大学的动物护理和使用委员会的协议进行。 1.准备气管插管套管<ol><li>切断2英寸的地下14 angiocathether针剪钳将针结束。 </li><li>捏针的内腔与尖嘴钳闭。 </li><li>向下切夹持部与切割钳打开腔部的边缘，再切上的剩余端的两侧的边缘，在30 - 45°角到针刃。 </li><li>将底部3 - 4毫米套管的成直线滑动关节钳的开口面积和拉向上直到略微向上弯曲已经形成在末端。 注意：这将有助于引导导管进入气管（ 图1）。 </li><li>用细砂纸平滑下来在套管的端部的边缘。 </li><li&gt;返回钝仪器进入血管导管管。确保该管道比所述钝针稍短。 </li></ol> 2.预外科手术<ol><li>通过放置在手术显微镜设定为10X放大倍率在操作区设置站点的程序。 </li><li>将变暖垫的操作区域，并盖上干净的吸收垫。 </li><li>成立了心脏率和血液充氧显示器附近的手术视野。 </li><li>称量大鼠体重为基础的药物的给药（ 例如，术后镇痛，抗生素，或淋巴细胞消耗性抗体）。 注：典型的成年大鼠体重350 - 450克</li><li>稳重与使用感应室汽化3％异氟烷-O 2（3L /分钟）大鼠排放到包含活性炭浪费麻醉气体清除系统。允许5分钟的麻醉诱导提供必要的气管插管全麻深。 </li><li>辖卡洛芬或Meloxicane（4.4毫克/千克）经皮下对大鼠在进行插管和手术之前。 </li><li>使用电推剪剃光之前插管麻醉大鼠的颈部和胸部。 </li></ol> 3.插管<ol><li>通过摩擦在插管套管的端部有少量的润滑（ 如，KY凝胶）制备插管设置。打开呼吸机上通过连接管以开始异氟烷气体的流动。 </li><li>大鼠转移到插管装置和由其上门牙（ 图2A）暂停所述金属条上的老鼠。 注：某些插管设备可以在线购买。这里展示的模型是用压克力板材四周使用丁烷火炬2×4英寸的腰片切片成型自制，和增强连接用氯仿。杆（或金属丝）装在由遮蔽胶带钻孔置于上一次一丙烯酸的IDE，防止分裂或开裂。 </li><li>从颈部（ 图2B）的腹面2厘米-通过定位一个灵活的高强度光源1反式照亮大鼠的颈部。 </li><li>使用一对学生标准图案镊子将舌头轻轻向上和向下方的齿的侧面。握拇指和一只手同时将靠在舌的下端钳子的一侧的平坦内表面食指之间的舌头。按腹侧以暴露喉部（ 图2C）的会厌和光圈。 </li><li>可视化的声带，并通过开放声门引导插管插管的上翘，最终减弱前方进气管，直到血管导管的枢纽接触门牙。 </li><li>去除金属探针，并附加麻醉管的血管导管开放，开始通风与异氟醚。 注：典型的氧气FLO瓦特率与呼吸机是3L /分钟，用3％异氟烷。 <ol><li> 14毫米汞柱 - 以60呼吸/分钟的体积控制通气模式的速率，可实现的〜12的压力下将呼吸机。使用3厘米H 2 O.的积极呼气末正压（PEEP） </li></ol></li><li>观察双边胸壁扩张与呼吸同步，以确保所述气管插管的正确放置。 </li><li>应用兽医推荐眼膏到老鼠的眼睛，以防止干燥，而在麻醉下。 </li><li>适用于老鼠的眼睛兽医软膏，防止干燥，而在麻醉下。 </li><li>固定气管导管的大鼠的头与布带的带材。确保气管插管和麻醉流入管道之间的连接牢固。 </li><li>附加的血氧和心脏监测仪对老鼠的脚，并开始监控。通过确认，确认正确的麻醉，没有反应到电子捏。 </li></ol> 4.开胸和胸腺切除术<ol><li>遵循标准的无菌技术的程序的全部内容。 <ol><li>清洁工作区和手术台和消毒用70％的乙醇溶液中。 </li><li>在手术过程中使用无菌手术手套，并在高压灭菌过程中使用的所有工具和材料。 </li><li>适用providone碘对整个胸部区域，并允许它干燥。然后清洗用纱布上70％的乙醇在皮肤的表面上。覆盖大鼠保鲜膜切割一个洞，露出无菌操作领域。 </li></ol></li><li>确定在上胸部胸骨上切迹。使通过原料2的皮肤2厘米中线切口 - 3毫米以上的胸骨上切迹和使用钝头牛油树剪刀沿胸骨中线向远侧延伸。 </li><li>执行从胸骨上切迹用钝头剪刀乳木果再次1.5厘米正中。 ķEEP正下方胸骨并前进缓慢剪刀的底部边缘。 </li><li>插入一个小阿尔姆牵正下方的胸骨分离和开放揭示预气管带状肌（胸骨舌骨和sternothyroid肌肉）。单独使用钝格雷夫钳预气管带状肌。注意：在这一点上，在气管中可以看出，与插管应气管内部可视化。 </li><li>放置小阿尔姆牵开器的尖头的分离带状肌和胸骨下方。打开牵开，以暴露胸腺的优越方面。 </li><li>用细杜蒙钳，释放胸腺组织的横向边缘和暴露下胸腺瓣。 </li><li>轻轻优拉胸腺到打开切口部位，注意避免与上腔静脉，锁骨下和颈动脉血管接触，并尽量减少胸腺和肺之间的微妙胸膜衬里的破坏。 </li><li>为t他胸腺血管解剖，并透露，使用微型剪刀将两者隔开。用棉签如果需要按住血管止血压力。 </li><li>提供较低的胸腺叶入切口，然后迅速裂解后的附件。取出完整的胸腺和仔细检查胸腺删除任何缺少的部分。 注意：小型胸廓淋巴结可可视包围胸腺，并且通常难以从胸腺组织区分。这些将是具有相似外观胸腺组织离散圆节点，但是将不会在与胸腺连续性。 </li><li>取出阿尔姆牵开和关闭胸骨舌骨和sternothyoid肌肉有两个中断5-0 Maxon公司缝合。 </li><li>适用于2滴手术级氰基丙烯酸酯组织粘合剂跨越缝线密封正压通气纵隔。 注意：这将减少气胸和血肿的发生率。 </li><li>关闭STernum有两个中断对切割针4-0丝线缝合。插入穿过所述肋条之间的间隙针，同时注意引导刚下胸骨针，以避免潜在的肌肉层。 </li><li>与正在运行的4-0尼龙缝合关闭皮肤层。终止异氟烷在这一点上，以缩短术后麻醉恢复期间。 </li><li>清洁切口部位及周围皮肤用生理盐水浸湿的纱布。 </li><li>应用数滴布比卡因（0.25％），局部麻醉，随后1 - 2滴氰基丙烯酸酯胶在切口以密封切口。 </li><li>继续到通风直到大鼠显示独立呼吸努力的迹象，并开始移动至其末端。拔管大鼠，并允许它在一个笼子放置在一个加热垫下密切观察恢复。 </li><li>施用丁丙诺啡（0.01 - 0.05毫克/千克）经皮下对大鼠立即手术后，重复每8-12小时48小时婆ST-操作（通常是5总剂量）。在24和48小时手术后，管理卡洛芬或Meloxicane（4.4毫克/公斤）皮下注射（3总剂量，包括术前剂量）。 </li><li>继续监测，直到老鼠四处移动的笼子里。在呼吸窘迫的任何迹象，执行重新插管和再探索的胸部。 <ol><li>不要让动物无人看管，直到它恢复足够的意识，保持胸骨斜卧。 </li><li>不返回已经历手术给其他动物的公司，直到完全恢复的动物。 </li></ol></li><li>去除皮肤层缝合后1周，可操作。 </li></ol>

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The authors have nothing to disclose.

本协议为完整的胸腺切除术提供了一个三层封切口手术胶水应用程序，旨在最大限度地减少并发症，微创的方法。完整切除胸腺所表明的幼稚T细胞的损失和由角蛋白纵隔淋巴组织的免疫组织化学染色。 成年大鼠胸腺切除术的过程变得复杂化的死亡率为1.5不等 - 20％至围手术并发症，其中大部分是归因于胸腺的与胸膜，心脏和肺下叶的紧密结合，由于纵隔大血管2-4。?...

自20世纪60年代初，胸腺已被确认为中央免疫耐受的发展具有至关重要的作用。啮齿动物胸腺切除术已被证明是在确定同种异体移植和肿瘤转移的设定胸腺的淋巴细胞分化，自身耐受和免疫耐受中的作用的重要步骤。切除大鼠胸腺允许涉及T细胞耗竭或定义T细胞群的过继转移没有再出现本土幼稚T细胞的研究。 新生啮齿类Thymectomies可以使用抽吸技术提供可靠的结果1来实现。在成年大鼠中，这种技术具有一个近似20％的死亡率，并经常导致不完整的胸腺切除2。不断获得成年大鼠胸腺切除彻底，纵隔通过正中开曝光是必需的。但是，此6％，2-4 -过程并发症，包括气管损伤，出血，气胸导致的总死亡率为1.5相关。 在过去的二十年中的改善在胸腺切除术的技术已经减少手术并发症和具有改善的存活率。气管插管允许正压通风，减少气胸率5。插管的方法前面所述范围开放暴露气管使用直接声带可视微创的方法。与插管过程相关的并发症包括气管损伤，声带破裂，无意食管插管，和出血从心脏穿刺或上腔静脉的裂伤所得。此外，下部胸腺裂片到胸膜衬里的接近会导致气胸。 在这里，我们描述的技术通过微创2厘米皮肤切口下气管插管使用钝端血管导管和皮气管照明的简单方法胸腺切除术。在胸腺切除术涉及1.5厘米胸骨切开术和三层闭合与手术胶水的应用程序，以密封纵隔和尽量减少出血和呼吸系统并发症的发生率。此方法可靠地导致完全胸腺切除术所证明的CD4 +和CD8 +幼稚T细胞的下列胸腺切除和缺乏胸腺组织对IHC染色的消失。手术时间和围手术期死亡率保持在最低水平。

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Name of Reagent/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:152px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">2&#34; 14-gauge angiocatheter</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Operating microscope</td>
			<td style="width:117px;">Zeiss</td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Warming pad</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Heart rate and blood oxygenation monitor for rodents with foot sensors</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-8010, ST1 72-8098 (Rat foot sensor)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Intubation apparatus (plastic with metal bar at the top)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;">See Figure 2&#160;</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:283px;">Small animal anesthesia system with induction box, isoflurane tank and O2 tank</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-6420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:283px;">Small animal ventilator with tubing</td>
			<td style="width:117px;">CWE</td>
			<td style="width:152px;">12-02000 (ventilator)and 12-04000 (external valve assembly for mice/rats)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High-intensity Fiber-Optic Illuminator</td>
			<td style="width:117px;">Dolan Jenner</td>
			<td style="width:152px;">EEG 2823M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Student standard pattern forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">91100-16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine straight scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14060-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt-tipped Shea scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14105-12</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Small Alm retractor (for sternum)</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">17008-07</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt Graefe forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11050-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine Dumont forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5-0 Maxon sutures</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">4-0 Silk sutures (with cutting needle)</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">6-0 Nylon suture</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cyanoacrylate glue (Endermil)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Lubrication gel</td>
			<td style="width:117px;">Akorn Animal Health</td>
			<td style="width:152px;">NDC 17478-162-35</td>
			<td></td>
		</tr>
	</tbody>
</table>

complete thymectomy in adult rats with non-invasive endotracheal intubation

胸腺切除术在新生儿啮齿动物是既定的和可靠的程序，免疫学研究。然而，在成年大鼠，出血和气胸从胸膜破坏并发症可导致显著死亡率。这个协议是鼠胸腺切除术，其利用一台迷你胸骨切开术和气管插管的一个简单方法。插管是通过一非侵入性和容易再现方法，并允许正压通气，以防止气胸和可控气道，允许有足够的时间进行认真胸腺解剖，以尽量减少胸膜中断。甲1.5厘米胸骨正中切口减小纵隔血管和胸膜接触，同时仍然提供胸腺充分可视化。继纵隔曝光，胸腺是在放大镜下钝性分离去除。胸膜腔，然后由预气管肌肉随后的手术胶水的应用的缝合线闭合密封。该胸部然后通过缝合封闭胸骨，其次是皮肤缝合闭合封闭。所有thymectomies均完整就证明了免疫组织化学（IHC）染色纵隔组织，并且不存在幼稚T细胞通过流式细胞术，并且过程进行了96％的存活率。这种方法适用于当完成胸腺切除以最小的并发症是期望在无胸腺成年大鼠进一步免疫学研究。

对成年Lewis大鼠（N = 26）中进行此过程。平均手术时间为15±3分钟。没有术中死亡。平均插管时间 - 从老鼠的气管插管设备，建立通风的位置 - 为45±5秒。 24只已经通过手术后一天（POD）的POD 4 14.一位大鼠呼吸道开发难度完全恢复的操作，没有证据呼吸困难或出血，并接受再次手术，探讨纵隔。老鼠被发现有一个折叠的肺叶。以下再膨胀肺，纵隔下正压通气重新密封带手术胶水和胸部被重新关闭。?...

Watch this Scientific Journal Video about Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation at JoVE.com

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

完整的胸腺切除术的成年大鼠非侵入气管插管

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

complete-thymectomy-adult-rats-with-non-invasive-endotracheal

Research

JoVE Journal

Immunology and Infection

16.4K 次观看.  Duke University Medical Center. Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described. 

视频: Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

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

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 ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological 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 ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

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

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

从结膜分离和表型细胞的非侵入性方式

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...