欢迎您, 登录 | 注册

首页 | English

X
加载中

The intratracheal instillation technique is used to deliver a variety of agents to the lungs ranging from pathogens (bacteria, viruses), toxins, to therapeutic agents. To model lung inflammation and injury, LPS can be administrated via intranasal, intratracheal, or aerosol approaches. Each technique has its limitations. The intratracheal technique can involve the non-invasive instillation method (via the oro-tracheal route) or a direct injection into the trachea. Here, we describe an optimized method for direct visual instillation of LPS via the non-invasive oro-tracheal route.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Non-invasive Intratracheal Instillation in Mice
小鼠无创气管滴注法

免疫学 > 动物模型 > 小鼠
作者: Guadalupe Ortiz-Muñoz
Guadalupe Ortiz-MuñozAffiliation: Department of Medicine, University of California, San Francisco, San Francisco, USA
Bio-protocol author page: a2304
 and Mark R. Looney
Mark R. LooneyAffiliation: Department of Medicine, University of California, San Francisco, San Francisco, USA
For correspondence: mark.looney@ucsf.edu
Bio-protocol author page: a2305
Vol 5, Iss 12, 6/20/2015, 4173 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1504

[Abstract] The intratracheal instillation technique is used to deliver a variety of agents to the lungs ranging from pathogens (bacteria, viruses), toxins, to therapeutic agents. To model lung inflammation and injury, LPS can be administrated via intranasal, intratracheal, or aerosol approaches. Each technique has its limitations. The intratracheal technique can involve the non-invasive instillation method (via the oro-tracheal route) or a direct injection into the trachea. Here, we describe an optimized method for direct visual instillation of LPS via the non-invasive oro-tracheal route.
Keywords: Non-invasive(无创), Intratracheal instillation(气管内滴注), Mice(老鼠)

[Abstract]

Materials and Reagents

  1. Lipopolysaccharide (LPS) (Sigma-Aldrich, catalog number: L2880 )
  2. Ketamine (Henry Schein, catalog number: 010177 ) and Xylazine (Henry Schein, catalog number: 033198 ) for mouse anesthesia
  3. Phosphate buffered saline (PBS) (see Recipes)

Equipment

  1. Intubating platform (Figure 1), which can be constructed using 0.5 inch (1.3 cm) boards. The longer board should be roughly 6 inches (15 cm) wide x 14 inches (36 cm) long. The smaller supporting board should be roughly 6 inches (15 cm) wide x 7 inches (18 cm) long. On the back side of the longer board, nail two rectangular pieces of wood that are roughly 2.5 inches (6.4 cm) x 1.5 inches (3.8 cm) in size, rendering a groove that is slightly wider than 0.5 inches (1.3 cm). The smaller board fits into this groove, thus producing the 45 degree angle of the intubating platform. Finally, cut a V-shaped groove on the top of the platform, and string suture (3-0 or larger) between 2 pushpins (or nails). Alternatively, a rodent intubation stand can be purchased (Braintree Scientific, Inc., catalog number RIS 100).


    Figure 1. Representative Model of Intubating Platform

  2. Fiber-optic illuminator (Cole Parmer Fiber-Lite, model: 9745-00 )
  3. Curved blunt-ended forceps
  4. Polyethylene tubing PE-10 (Intramedic, catalog number: 427401 )
  5. Insulin syringe with attached needle (sterile)
  6. Hands-free binocular magnifiers
  7. Warming pad (Gaymar T/Pump Classic, model: TP650 ) @ 38 °C setting

Procedure

This protocol describes the procedure for the direct visual instillation method for intratracheal (i.t.) instillation (Su et al., 2004) of LPS.

  1. Preparation
    All protocols using live animals must be approved by an Institutional Animal Care and Use Committee and must follow officially approved procedures for the care and use of laboratory animals. Furthermore, experimental animals treated with biohazard materials should be handled and disposed using recommended animal biosafety procedures. When appropriate, the person(s) handling the animals must have official certification for performing procedures on animals and for submitting protocols for ethical approval.
    1. Weigh the animal on a balance to calculate the LPS dose required for each mouse.
    2. In an Eppendorf tube, prepare the desired LPS solution (diluted in PBS) in a volume not to exceed 50 μl. The LPS concentration will vary depending on the experimental goals, but is usually between 1 mg/kg (mild lung injury) and 10 mg/kg (severe lung injury). Stock solutions of LPS can be prepared, aliquoted, and frozen at -80 °C to help standardize instillations.
    3. Attach 1 inch (2.5 cm) of PE-10 tubing to the insulin needle and syringe and load it with the LPS solution. Importantly, syringe should be pre-loaded with at least 50 μl of air to insure that all of the liquid volume is expelled from the needle.
    4. Anesthetize animals with Ketamine (50-80 mg/kg) and Xylazine (8-12 mg/kg) injected intraperitoneal using insulin syringes. The anesthetics may be mixed in the same syringe. This anesthetic dose should maintain anesthesia for 20-30 min.

  2. Intratracheal Instillation
    1. Check the level of anesthesia using a paw pinch stimulus a few minutes after delivery of the anesthetics. Once adequate anesthesia is observed, suspend the mouse by its incisors in the supine position on the intubating platform (Figure 2A).
    2. Turn on the fiber-optic illuminator and position it over the trachea. (Figure 2A-B)
    3. Using curved blunt-ended forceps, carefully grasp the tongue and in a upward and leftward motion, position the tongue to gain visualization of the larynx. Hands-free binocular magnifiers can be used for improved visualization of the larynx.
    4. Position the fiber-optic light source to trans-illuminate the tracheal opening
    5. Insert the PE-10 tubing (attached to the insulin needle and syringe) 0.5-1 cm into the trachea and instill the LPS solution (volume not to exceed 50 µl).
    6. To avoid the intratracheal instillate from escaping the trachea, leave the PE-10 tubing in place for 5 sec.
    7. Maintain the mouse in the same position on the intubating platform for at least 30 sec.
    8. Remove the mouse and place it prone on a heating pad for recovery.


      Figure 2. A-B. Mouse positioned on the intubation platform. C. Bronchoalveolar lavage (BAL) accumulation of white blood cells (WBC) 48 h after LPS instillation at dose of 5 mg/kg. WBCs were quantified with a Coulter Counter.

Recipes

  1. Phosphate buffered saline (PBS)
    0.2g/L KH2PO4
    2.16 g/L Na2HPO4
    0.2 g/L KCl
    8.0 g/L NaCl
    Sterile filtered

Acknowledgments

This work was supported by the National Heart, Lung, and Blood Institute grant R01 HL107386 (M.R.L.).

References

  1. Su, X., Looney, M., Robriquet, L., Fang, X. and Matthay, M. A. (2004). Direct visual instillation as a method for efficient delivery of fluid into the distal airspaces of anesthetized mice. Exp Lung Res 30(6): 479-493.

材料和试剂

  1. 脂多糖(LPS)(Sigma-Aldrich,目录号:L2880)
  2. 用于小鼠麻醉的Ketamine(Henry Schein,目录号:010177)和Xylazine(Henry Schein,目录号:033198)
  3. 磷酸盐缓冲盐水(PBS)(见Recipes)

设备

  1. 插管平台(图1),其可以使用0.5英寸(1.3cm)板构造。较长的板应该是大约6英寸(15cm)宽×14英寸(36cm)长。较小的支撑板应该是大约6英寸(15cm)宽×7英寸(18cm)长。在较长板的背面上,钉有大约2.5英寸(6.4cm)×1.5英寸(3.8cm)大小的两个矩形木材,形成比0.5英寸(1.3cm)略宽的凹槽。较小的板配合到该凹槽中,从而产生插管平台的45度角。最后,在平台的顶部切割一个V形槽,并在2个图钉(或指甲)之间缝线(3-0或更大)。或者,可以购买啮齿动物插管架(Braintree Scientific,Inc.,目录号RIS 100)

    图1。 插管平台代表模型

  2. 光纤照明器(Cole Parmer Fiber-Lite,型号:9745-00)
  3. 弯曲的钝端钳
  4. 聚乙烯管PE-10(Intramedic,目录号:427401)
  5. 带有针头的胰岛素注射器(无菌)
  6. 免提双目放大镜
  7. 保暖垫(Gaymar T/Pump Classic,型号:TP650)@ 38°C设置

程序

该方案描述了用于气管内(i.t.)滴注(Su等人,2004)的直接视觉滴注方法的程序。

  1. 准备
    使用活体动物的所有方案必须获得机构动物护理和使用委员会批准,并且必须遵循官方批准的用于实验动物的护理和使用的程序。 此外,用实验动物处理 生物危害材料应使用推荐的动物生物安全程序进行处理和处理。在适当的情况下,处理动物的人必须获得官方认证,以便执行动物程序和提交伦理批准程序。
    1. 称量动物的天平,以计算每只小鼠所需的LPS剂量。
    2. 在Eppendorf管中,制备所需的LPS溶液(稀释于 PBS),体积不超过50μl。 LPS浓度将变化 取决于实验目的,但通常在1mg/kg之间 (轻度肺损伤)和10mg/kg(严重肺损伤)。库存解决方案  可以制备LPS,等分,并在-80℃冷冻以帮助 标准化滴注
    3. 连接1英寸(2.5厘米)的PE-10管  到胰岛素针和注射器并且用LPS溶液加载它。 重要的是,注射器应该预装至少50微升的空气 确保所有液体体积从针排出。
    4. 麻醉动物用氯胺酮(50-80毫克/公斤)和赛拉嗪(8-12 mg/kg)使用胰岛素注射器腹膜内注射。 麻醉剂 可以在同一注射器中混合。 这种麻醉剂量应该保持 麻醉20-30分钟。

  2. 气管内滴漏
    1. 使用爪子夹捏刺激几分钟检查麻醉的水平 麻醉后交付。 一旦观察到适当的麻醉,   通过其门牙悬挂鼠标在仰卧位置 插管平台(图2A)
    2. 打开光纤照明器并将其放置在气管上。 (图2A-B)
    3. 使用弯曲的钝端钳,仔细抓住舌头和在   向上和向左运动,定位舌头以获得可视化 的喉。 免提双筒望远镜放大镜可用于改进 喉的可视化。
    4. 定位光纤光源以透射气管开口
    5. 插入PE-10管(连接到胰岛素针和注射器)   0.5-1厘米进入气管,并滴注LPS溶液(体积不 超过50μl)。
    6. 为了避免气管内滴注物逃离气管,将PE-10管道留在原位5秒钟。
    7. 保持鼠标在插管平台上的相同位置至少30秒。
    8. 取出鼠标,将其放置在加热垫上以便恢复。


      图2。 A-B。 鼠标定位在插管平台上。 C。 支气管肺泡灌洗(BAL)白细胞积聚(WBC)48 h   在LPS滴注后以5mg/kg的剂量。 WBCs用a 库尔特计数器。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    0.2g/L KH 2 PO 4 sub/
    2.16g/L Na 2 HPO 4
    0.2 g/L KCl
    8.0g/L NaCl 无菌过滤

致谢

这项工作得到国家心肺血液研究所拨款R01 HL107386(M.R.L.)的支持。

参考文献

  1. Su,X.,Looney,M.,Robriquet,L.,Fang,X.and Matthay,M.A。(2004)。 直接视觉灌注作为有效地将流体输送到麻醉小鼠远端空间的方法。 Exp Lung Res 30(6):479-493。
English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Ortiz-Muñoz, G. and Looney, M. R. (2015). Non-invasive Intratracheal Instillation in Mice. Bio-protocol 5(12): e1504. DOI: 10.21769/BioProtoc.1504; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
2/27/2016 6:51:36 AM  

Spencer Li
Zhejiang University

i've been performing the intratracheal instillation in mice with your methods. After instillation, the mouse starts coughing which may cause the inconsistent doses of LPS.
how to avoid the coughing during the procedure???

2/29/2016 11:03:23 AM  

Mark Looney (Author)
Department of Medicine, University of California, San Francisco, USA

There will always be some respiratory distress after an intratracheal liquid challenge--that cannot be avoided. I would keep the instillate volume to less than 50 microliters if at all possible. Also, deeper sedation usually helps blunt the coughing response.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook
Guadalupe Ortiz-Muñoz的其他实验方案(1)
Mark R. Looney的其他实验方案(1)