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Measurement of Airway Responsiveness in the Anesthetized Mouse
测定麻醉后小鼠的气道反应性   

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Abstract

Airway hyperresponsiveness to methacholine is an important characteristic of asthma. Many devices can be used to measure airway responsiveness in the mouse but it is well established that the invasiveness of a measurement technique is correlated with its precision and reproducibility. This protocol describes how to measure airway responses to aerosolized methacholine in anesthetized, tracheotomized, and mechanically ventilated mice using the forced oscillation technique with FlexiVent® (SCIREQ). This is a computer-controlled precision piston pump that can intersperse mechanical ventilation with volume and pressure controlled manoeuvres to obtain accurate, reproducible measurement of respiratory mechanics.

Keywords: Airway responsiveness(气道反应性), Forced oscillation technique(强迫振荡技术), Flexivent®(®FlexiVent), Methacholine(乙酰甲胆碱), Mouse(鼠标)

Materials and Reagents

  1. Sterile saline (0.9% NaCl) (B. Braun)
  2. Acetyl-β-methylcholine chloride, powder (Sigma-Aldrich, catalog number: A-2251 )
  3. Drierite (Sigma-Aldrich, catalog number: 238988 )
  4. Sodium Pentobarbital (5.47%) (CEVA)
  5. Xylazine (2%) (Rompun®, Bayer)
  6. Cotton wire n50 (DMC, catalog number: 334A/50 )

Equipment

  1. FlexiVent® system (SCIREQ Technologies) composed of the following modules: FV-M1 (1.8 ml), FV-PP-M1 , FV-XC , FV-EC , FV-BU and Aeroneb® Lab nebuliser system associated with Aeroneb® Pro (AG-AL-1000)
  2. "1 ml" sterile syringes (Terumo, catalog number: SS+01T1 )
  3. "25-gauge 0.5 mm" sterile needles (Terumo, catalog number: NN-2516R )
  4. 18-gauge metal needle (SCIREQ Technologies, catalog number: 73-0019 )

Procedure

Summary of the protocol to measure airway responsiveness to methacholine:

  • Mice are weighed and anesthetised i.p. with xylasine and pentobarbital.
  • Mice are tracheotomised and a 18-gauge metal needle is inserted into the trachea and connected to the FlexiVent® computer-controlled small animal ventilator.
  • Baseline values are measured.
  • Saline and then methacholine are aerosolized in the ventilator circuit.
  • Airway measurements are performed with the FlexiVent® protocol.
  • Mice are disconnected at the end to ensure that they breathe normally again without the aid of the ventilator.


  1. Anaesthesia
    1. Bring xylasine and pentobarbital solutions to room temperature (18-25 °C), and place methacholine and saline solutions on ice.
    2. Weigh the mouse.
    3. Hold the mouse in your hand by the dorsal skin so that its head is up and its rear legs are down. Maintain its tail with fingers.
    4. Use "1 ml" syringes and "25-gauge" needles to inject solution and administrate 6 ml/kg of the xylasine solution intraperitoneally (i.p.).
    5. Isolate the mouse in another cage. Wait for 10 min.
    6. Hold the mouse in your hand by the dorsal skin so that its head is up and its rear legs are down. Maintain its tail with fingers.
    7. Use another "1 ml" syringe and a "25-gauge" needle to inject 6 ml/kg of pentobarbital i.p.
    8. Replace the mouse in the cage. Wait for 10 min.
  2. Tracheotomy
    1. Check that the mouse is profoundly anesthetised.
    2. Dissect and expose the trachea, place a cotton wire under the trachea, insert the 18-gauge metal needle into the trachea and tie a knot around the trachea with the cotton wire in order to hold the needle in place.
    3. Connect the needle (i.e. connect the mouse) to the computer-controlled small animal ventilator (FlexiVent®).
    4. Follow the "Mouse_AN_v5.2.ext" standard protocol supplied with the FlexiVent®. This protocol consists of 12 cycles of 2 perturbations (snapshot and Prime-8) after 10 sec of nebulization, and allows to measure the resistance, elastance and compliance data.
  3. Measurement processes and methacholine nebulization
    1. Start ventilation when the mouse is connected.
    2. Start the protocol.
    3. Add saline in the nebuliser for the first nebulization step (10 sec) and proceed to the 12 perturbations cycles.
    4. Rinse the nebuliser 3 times with deionized water (eliminate solution with a syringe and replace it with water, repeat 3 times).
    5. Add methacholine in the nebuliser for the second nebulisation step (10 sec) and proceed to the 12 perturbations cycles.
    6. Stop the experiment and stop the ventilation.
    7. Disconnect immediately the 18-gauge metal needle from the apparatus. The mouse should breathe again normally, indicating it was alive during the measures. Then, euthanize the mouse. If the mouse is dead, data should be eliminated.
    8. Rinse the nebulizer 3 times with deionized water.
    9. Begin a new analysis session with the following mouse.
  4. Data analysis
    1. Export data from the Scireq FlexiVent® V5.2 program to an Excel file
    2. For each saline or methacholine nebulization, the peak response is calculated as the mean of the three maximal values (in red on the figure here below), and used for calculation of airway resistance and compliance. Airway resistance is expressed as cm H2O.s.ml-1 and airway compliance as ml.cm H2O-1.


      Figure 1.

Recipes

  1. Prepare a solution of methacholine containing 50 mg/ml methacholine (0.26 M) diluted in sterile saline. Methacholine must be prepared extemporaneously.
  2. Freshly prepare a solution of xylasine containing 0.25% xylasine diluted in sterile saline and a solution of pentobarbital containing 0.9% pentobarbital.

Acknowledgments

This protocol was adapted from a previously published paper: Reber et al. (2012). FD was supported by the “fond de dotation recherche en santé respiratoire”, call for tenders 2010.

References

  1. Reber, L. L., F. Daubeuf, M. Plantinga, L. De Cauwer, S. Gerlo, W. Waelput, S. Van Calenbergh, J. Tavernier, G. Haegeman, B. N. Lambrecht, N. Frossard and K. De Bosscher (2012). A dissociated glucocorticoid receptor modulator reduces airway hyperresponsiveness and inflammation in a mouse model of asthma. J Immunol 188(7): 3478-3487.

简介

气道对乙酰甲胆碱的高反应性是哮喘的一个重要特征。 许多装置可以用于测量小鼠中的气道反应性,但是已经确定的是,测量技术的侵入性与其精确度和再现性相关。 该协议描述了如何使用强制振荡技术用FlexiVent </sup>(SCIREQ)测量在麻醉,气管切开和机械通气的小鼠中气雾化的乙酰甲胆碱的气道反应。 这是一个计算机控制的精密活塞泵,可以穿插机械通气与体积和压力控制操作,以获得准确,可重复的呼吸力学测量。

关键字:气道反应性, 强迫振荡技术, ®FlexiVent, 乙酰甲胆碱, 鼠标

材料和试剂

  1. 无菌盐水(0.9%NaCl)(B.Braun)
  2. 乙酰基-β-甲基胆碱氯化物,粉末(Sigma-Aldrich,目录号:A-2251)
  3. Drierite(Sigma-Aldrich,目录号:238988)
  4. 戊巴比妥钠(5.47%)(CEVA)
  5. 赛拉嗪(2%)(Rompun ,Bayer)
  6. 棉线n50(DMC,目录号:334A/50)

设备

  1. 由以下模块组成的FlexiVent TM系统(SCIREQ Technologies):FV-M1(1.8ml),FV-PP-M1,FV-XC,FV-EC,FV-BU和Aeroneb 与 Pro(AG-AL-1000)相关的实验室雾化器系统
  2. "1ml"无菌注射器(Terumo,目录号:SS + 01T1)
  3. "25号0.5mm"无菌针(Terumo,目录号:NN-2516R)
  4. 18号金属针(SCIREQ Technologies,目录号:73-0019)

程序

测量对乙酰甲胆碱的气道反应性的方案的概述:

  • 小鼠称重并麻醉i.p。 与木糖醇和戊巴比妥。
  • 将小鼠气管切开并将18号金属针插入气管中并连接到计算机控制的小动物通气机。
  • 测量基线值。
  • 盐水和乙酰甲胆碱在呼吸机回路中雾化。
  • 气道测量使用FlexiVent 协议进行。
  • 小鼠在末端断开,以确保他们在没有呼吸机的帮助下再次正常呼吸。


  1. 麻醉
    1. 使木糖醇和戊巴比妥溶液至室温(18-25℃),并将乙酰甲胆碱和盐溶液置于冰上。
    2. 称重鼠标。
    3. 将鼠标握在手中的背部皮肤,使其头部向上,其后腿向下。 用手指保持其尾巴。
    4. 使用"1ml"注射器和"25号"针头注射溶液,并腹膜内(i.p.)给予6ml/kg的二甲苯磺胺溶液。
    5. 在另一个笼子中隔离鼠标。 等待10分钟。
    6. 将鼠标握在手中的背部皮肤,使其头部向上,其后腿向下。 用手指保持尾巴。
    7. 使用另一个"1ml"注射器和"25号"针注射6ml/kg戊巴比妥腹膜内。
    8. 更换鼠标在笼子里。 等待10分钟。
  2. 气管切开术
    1. 检查鼠标是否严重麻醉。
    2. 解剖和暴露气管,在气管下方放置一根棉线,将18号金属针插入气管,用棉线在气管周围打结,以便将针固定到位。
    3. 将针(连接鼠标)连接到计算机控制的小动物呼吸机(FlexiVent ®)。
    4. 遵循FlexiVent ®提供的"Mouse_AN_v5.2.ext"标准协议。 该协议由12个周期的2次扰动(快照和Prime-8)在10秒的雾化后组成,并允许测量电阻,弹性和顺应性数据。
  3. 测量过程和乙酰甲胆碱雾化
    1. 在鼠标连接时开始通气。
    2. 启动协议。
    3. 在雾化器中添加盐水进行第一次喷雾步骤(10秒),然后进行12个扰动周期。
    4. 用去离子水冲洗雾化器3次(用注射器清除溶液,用水替换,重复3次)。
    5. 在雾化器中加入乙酰甲胆碱进行第二次喷雾步骤(10秒),然后进行12个扰动周期。
    6. 停止实验并停止通气。
    7. 立即断开18号金属针与设备的连接。 鼠标应该再次正常呼吸,表明在措施期间它是活的。 然后,安乐死的鼠标。 如果鼠标死了,应该删除数据。
    8. 用去离子水冲洗雾化器3次。
    9. 使用以下鼠标开始新的分析会话。
  4. 数据分析
    1. 将Scireq FlexiVent ® V5.2程序中的数据导出到Excel文件
    2. 对于每个盐水或乙酰甲胆碱雾化,峰值响应计算为三个最大值的平均值(在下文的图上为红色),并用于计算气道阻力和顺应性。 气道阻力表示为cm H 2 Osml -1,气道依从性为ml.cm H 2 O sup>。


      图1。

食谱

  1. 制备乙酰甲胆碱含有50mg/ml乙酰甲胆碱(0.26M)稀释在无菌盐水中的溶液。 乙酰甲胆碱必须临时制备。
  2. 新鲜制备包含稀释于无菌盐水中的0.25%木聚糖的木聚糖溶液和含有0.9%戊巴比妥的戊巴比妥溶液。

致谢

该协议改编自以前发表的论文:Reber等人(2012)。 FD由"fond de dotation recherche ensantérespiratoire"支持,呼吁招标2010年。

参考文献

  1. Reber,LL,F.Daubeuf,M.Plaininga,L.Deauau,S.Gerlo,W.Waelput,S.Van Calenbergh,J.Tavernier,G.Haegeman,BNLambrecht,N.Frossard和K.De Bosscher 2012)。 解离的糖皮质激素受体调节剂降低小鼠哮喘模型中的气道高反应性和炎症。 188(7):3478-3487
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Daubeuf, F., Reber, L. and Frossard, N. (2013). Measurement of Airway Responsiveness in the Anesthetized Mouse. Bio-protocol 3(7): e645. DOI: 10.21769/BioProtoc.645.
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