Name: complete thymectomy in adult rats with non-invasive endotracheal intubation
Uploaded: 2014-12-29T13:00:00.000+00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation at JoVE.com

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。打开气管插管技术，其中气管被刺破，有6％的死亡率2。使用非侵入性的气管插管已报道由Na和同事;然而，存活率不报告5。如这里所描述插管与套管制备与经皮气管照明允许清晰声带一贯成功插管的可视化。没有先进的设备是需要的提出的方法，它不同于其他报道的非侵入性技术2,5- 8。我们的钝的血管导管针的端部和产生在端向上弯曲的方法可以更容易地避免无意食管插管并最大限度地减少气管创伤。这些是使用改良的血管导管9改善插管也的前述方法。 我们发现保持胸骨正中切口到最低下降与主要纵隔血管接触和出血的风险相关联，同时允许足够的曝光完全切除胸腺。从理论上讲，小切口也应减轻后手术疼痛和恢复时间。利用纤维蛋白胶被首次报道作为一个开放的气管插管的修改程序，以防止气胸及气道损伤2。我们已经发现，少量的胶在同时施加正压的缝合预气管肌肉应用适当地密封所述胸膜腔和有助于防止手术后呼吸并发症。 有协议的几个方面是重要的是强调这样的程序可以被成功地进行。它以固定麻醉导管具有足够胶带，因为它有可能在操作过程中断开气流作为外科医生的手被定位靠近鼠的头部和可视化只集中在显微镜下手术部位是很重要的。施加到前插管套管的最小可能的润滑防止套管滑动到食道。在多层封闭，这也有助于通过小规格针用注射器，以应用氰基丙烯酸酯的组织粘合剂，其<html>可以精确的应用程序，并防止意外过的应用程序的胶水。 此过程不允许再血管化除去的胸腺在受体鼠，它已在别处10描述。我们还没有尝试此过程中的小鼠，其经常被用于免疫研究。在成年小鼠的胸腺切除过程的最近的描述涉及曝光后的胸腺，它具有较高的并发症发生率在成年大鼠或解剖方法是类似于我们的程序11,12的任一真空抽吸。未在层进行封闭，和麻醉是通过腹膜内注射戊巴比妥，这并不强制插管实现的。在任何啮齿类动物的胸腺切除过程的主要限制是用于在手术显微镜，它可以是非常昂贵的要求，并且所需要的专业技术，在放大下操作，并迅速执行该过程。 内容"&gt;完整胸腺切除术的成年大鼠有利于涉及T细胞发育，T细胞反应性和耐受性移植和肿瘤研究涉及T细胞耗竭的无胸腺大鼠13-15免疫学研究，研究。

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><tbody><tr><td>2 inch 14 G angiocatheter</td><td></td><td></td><td></td></tr><tr><td>Operating microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Warming pad</td><td></td><td></td><td></td></tr><tr><td>Heart rate and blood oxygenation monitor for rodents with foot sensors</td><td>Harvard Apparatus</td><td>ST1 72-8010, ST1 72-8098 (Rat foot sensor)</td><td></td></tr><tr><td>Intubation apparatus (plastic with metal bar at the top)</td><td></td><td></td><td>See &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>Small animal anesthesia system with induction box, isoflurane tank and O&lt;sub&gt;2&lt;/sub&gt; tank</td><td>Harvard Apparatus</td><td>ST1 72-6420</td><td></td></tr><tr><td>Small animal ventilator with tubing</td><td>CWE</td><td>12-02000 (ventilator) and 12-04000 (external valve assembly for mice/rats)</td><td></td></tr><tr><td>High-intensity fiber optic Illuminator</td><td>Dolan Jenner</td><td>EEG 2823M</td><td></td></tr><tr><td>Student standard pattern forceps</td><td>Fine Science Tools</td><td>91100-16</td><td></td></tr><tr><td>Fine straight scissors</td><td>Fine Science Tools</td><td>14060-09</td><td></td></tr><tr><td>Blunt-tipped Shea scissors</td><td>Fine Science Tools</td><td>14105-12</td><td></td></tr><tr><td>Small Alm retractor (for sternum)</td><td>Fine Science Tools</td><td>17008-07</td><td></td></tr><tr><td>Blunt Graefe forceps</td><td>Fine Science Tools</td><td>11050-10</td><td></td></tr><tr><td>Fine Dumont forceps</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>5-0 Maxon sutures</td><td>Ethicon</td><td></td><td></td></tr><tr><td>4-0 Silk sutures (with cutting needle)</td><td>Ethicon</td><td></td><td></td></tr><tr><td>6-0 Nylon suture</td><td>Ethicon</td><td></td><td></td></tr><tr><td>Cyanoacrylate glue (Endermil)</td><td></td><td></td><td></td></tr><tr><td>Lubrication gel</td><td>Akorn Animal Health</td><td>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。大鼠恢复与完全没有进一步的呼吸异常。一个老鼠对不明原因POD死亡7例。 后胸腺切除纵隔是检查是否保留胸腺及胸腺去除组织呈Visually检查缺失的部分，后来组织学检查。在尸检，胸部组织进行了检查，苏木精 - 伊红（H＆E）染色。免疫组织化学（IHC），细胞角蛋白然后进行区分胸淋巴结（LN），尤其侧重于与H＆E水平较高的核染色的部分作为是正常的胸腺组织残留胸腺组织。使用兔泛抗细胞角蛋白作为主AB，随后生物素化的山羊抗兔IgG和优秀的Vectastain ABC试剂盒滑轨进行角蛋白染色进行反染色，于显微镜评价之前苏木。胸腺组织可以从胸淋巴结由H＆E（ 图3A和3B）更致密核染色及细胞角蛋白染色，这不同于没有角蛋白染色见于淋巴结（ 图3C和3D）的特性花边图案来区分。 得到的外周血样品从安乐死的大鼠为幼稚T细胞的持久性消耗进行分析。简言之，将红血细胞用ACK裂解缓冲液和外周血白细胞（PBL中）用PBS洗涤两次，2％FBS的之前的抗体染色30分钟，在4℃。的PBLs然后用4％（重量）在分析前/ v的多聚甲醛在中性pH缓冲盐水通过多色流式细胞术。总T细胞进行鉴定染色的PBMCs（外周血单核细胞）用抗CD45 +和CD3 +和幼稚T细胞通过用抗CD45RC，抗CD62L，要么抗CD4或抗CD8染色进行鉴定。每个T细胞亚群的百分比乘以绝对淋巴细胞计数（由杜克大学兽医临床诊断实验室获得），以确定细胞计数。代表性的幼稚的CD4 +和CD8 + T细胞和总T细胞计数示于图4中 。胸腺切除大鼠维持总体T细胞计数相比，控制研究ATS但展示了幼稚T细胞群的损失。 <img alt="图1" src="/files/ftp_upload/52152/52152fig1highres.jpg" /> 图插管插管1.设计。套管是由钝一地下14×2英寸的血管导管的探针的端部形成。远端3毫米短剑是稍微弯曲，引导导管腹，方便气管插管。金属管芯（A）侧视图。血管导管口针以上的（B）侧视图。管心针钝尖（C）放大图。 <img alt="图2" src="/files/ftp_upload/52152/52152fig2highres.jpg" /> 图2.倾斜插管平台用于定位大鼠插管的大鼠从插管平台通过其上门牙金属棒暂停。光源程序Ë将被定位腹侧老鼠的脖子上跨照亮咽。钳用于保存舌头和揭露喉的光圈。（A）顶视图和插管平台（B）侧视图。声门的可视化（C）图悬架大鼠的喉和曝光后。图中的喉部是从Motifolio购买的幻灯片。 <img alt="图3" src="/files/ftp_upload/52152/52152fig3highres.jpg" width="650" /> 图3. H＆E和纵隔组织细胞角蛋白染色可从淋巴结区分胸腺，HE染色（H＆E）正常胸腺（A）和胸LN（B）的染色。胸腺组织（C）和胸椎LN的角蛋白染色恢复后胸腺切除术<strong&gt;（D），放大倍率为200倍。酒吧是100微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52152/52152fig3highres.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/52152/52152fig4highres.jpg" /> 图4.磨耗外周血幼稚T细胞后胸腺切除的总 T细胞，幼稚的CD4 + T细胞和幼稚CD8 + T细胞从PBL中预胸腺切除术（预）和大鼠后4周胸腺切除术，其中定量流式细胞术（邮）。数据示于每组一只大鼠和有代表性的所得总的结果。

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

16.5K 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

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

气管插管小鼠<em&gt;通过</em&gt;直接喉镜使用耳镜

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

科研

医学

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

微循环血流速度在大鼠肺转移自动测量

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

肝硬化大鼠门脉高压综合征的侵袭性血流动力学特征

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

左肺切除术联合单氯联氨酸或苏根为大鼠肺动脉高压的模型

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

口腔插管和通气肺缺血再灌注手术小鼠模型

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

免疫与感染

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

小鼠重复的奥内切管插管

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

探索肺移植中的闭塞性气道疾病

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially reported by our team, this model has gained use in the study of OAD due to its high technical reproducibility and suitability for investigating immunological behaviors and therapeutic interventions.
In the IPTT model, a rodent tracheal graft is directly inserted into the recipient's lung through the pleura. This model is distinct from the heterotopic tracheal transplantation (HTT) model, wherein grafts are transplanted into subcutaneous or omental sites, and from the orthotopic tracheal transplantation (OTT) model in which the donor trachea replaces the recipient's trachea. 
Successful implementation of the IPTT model requires advanced anesthetic and surgical skills. Anesthetic skills include endotracheal intubation of the recipient, setting appropriate ventilatory parameters, and appropriately timed extubation after recovery from anesthesia. Surgical skills are essential for precise graft placement within the lung and for ensuring effective sealing of the visceral pleura to prevent air leakage and bleeding. In general, the learning process takes approximately 2 months.
In contrast to the HTT and OTT models, in the IPTT model, the allograft airway develops airway obliteration in the relevant lung microenvironment. This allows investigators to study lung-specific immunological and angiogenic processes involved in airway obliteration after lung transplantation. Furthermore, this model is also unique in that it exhibits tertiary lymphoid organs (TLOs), which are also seen in human lung allografts. TLOs are comprised of T and B cell populations and characterized by the presence of high endothelial venules that direct immune cell recruitment; therefore, they are likely to play a crucial role in graft acceptance and rejection. We conclude that the IPTT model is a useful tool for studying intrapulmonary immune and profibrotic pathways involved in the development of airway obliteration in the lung transplant allograft.

作者聚焦：调查肺移植后闭塞性毛细支气管炎的关键因素

The murine intrapulmonary tracheal transplantation (IPTT) model is valuable for studying obliterative airway disease (OAD) after lung transplantation. It offers insights into lung-specific immunological and angiogenic behavior in airway obliteration after allotransplantation with high reproducibility. Here, we describe the IPTT procedure and its expected results.

小鼠肺内气管移植：肺移植后闭塞性气道疾病研究模型

Murine intrapulmonary tracheal transplantation (IPTT) is used as a model of obliterative airway disease (OAD) following lung transplantation. Initially ...

完整的胸腺切除术的成年大鼠非侵入气管插管

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气管插管小鼠

微循环血流速度在大鼠肺转移自动测量

在小鼠气管插管快速的改进方法

肝硬化大鼠门脉高压综合征的侵袭性血流动力学特征

左肺切除术联合单氯联氨酸或苏根为大鼠肺动脉高压的模型

口腔插管和通气肺缺血再灌注手术小鼠模型

小鼠重复的奥内切管插管

小鼠肺内气管移植：肺移植后闭塞性气道疾病研究模型

小鼠肺内气管移植：肺移植后闭塞性气道疾病研究模型

小鼠肺内气管移植：肺移植后闭塞性气道疾病研究模型