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Measurement of Airway Responsiveness on Vigil and Unrestrained Mouse
在监视和受限小鼠中测定气道反应性   

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Abstract

Airway hyperresponsiveness to methacholine is an important characteristic of asthma. This protocol describes how to measure airway response to methacholine in vigil and unrestraint mouse. The enhanced pause (PenH) is an index of the airway response to increasing doses of methacholine recorded by whole-body barometric plethysmography.

Keywords: Airway responsiveness(气道反应性), Plethysmography(体积描记法), PenH(金边), Respiratory parameters(呼吸参数), Mouse(鼠标)

Materials and Reagents

  1. Sterile saline (0.9% NaCl)
  2. Methacholine (Sigma-Aldrich, catalog number: A2251 )
  3. Drierite (Sigma-Aldrich, catalog number: 238988 )
  4. Nebulization (see Recipes)

Equipment

  1. Whole body plethysmography system (EMKA Technologies), including standard setup (whole-body plethysmograph, differential pressure transducer, ventilation pump, amplifier, interface box, acquisition card and iox2 software with the respiratory flow analyzer module) and standard nebulisation equipment (LS230, SYSTAM, Villeneuve-sur-Lot)

Procedure

  1. Turn the plethysmograph system on, and wait for 15 min to let the airflow and signal be stable.
  2. Control the permeability of each pneumotachograph, and change it for a new pneumotachograph if the permeability is altered.
  3. Control the dryness of the drierite; dry drierite is blue and turns pink in the presence of humidity. Drierite has to be changed regularly.
  4. Control functionality of each ventilation pump in the plethysmograph system; each ventilation pump must be regulated to 0.8 L per minute. Check the balls in each flowmeter: All balls have to be perfectly mobile, and show no sign of oxidation (these 3 controls have to be performed before each experiment).
  5. Turn on the IOX software (delivered with the plethysmograph system).
  6. Proceed to calibration of each functional plethysmograph chamber.
  7. Place the animals carefully into each plethysmograph chamber.
  8. Wait for thirty minutes for the animals to become quiet and calm in the plethysmograph chamber.
  9. Begin the measurement session.
  10. Add saline to the nebulizer, start the first nebulisation (30 sec) and record the enhanced pause (PenH) during 20 min.
  11. Add 0.05 M Methacholine solution, nebulize for 30 sec and record PenH (20 min).
  12. Add 0.1 M Methacholine solution, nebulize for 30 sec and record PenH (20 min).
  13. Add 0.2 M Methacholine solution, nebulize for 30 sec and record PenH (20 min).
  14. Add 0.3 M Methacholine solution, nebulize for 30 sec and record PenH (20 min).
  15. Save data.
  16. Turn off the database (IOX software) and the computer.
  17. Remove each mouse from each chamber.
  18. Clean all devices.
  19. Proceed to data analysis. Use the Datanalyst software (delivered with the plethysmograph system) for data collected by the IOX software. The mean PenH of the data measured during 5 min around the peak PenH is used for each dose of methacholine (as shown by the arrows and dotted lines on the Figure 1).


    Figure 1.

Recipes

  1. Nebulization with the standard nebulization system (LS230, SYSTAM) requires 10 ml of each methacholine solution. Prepare a solution of methacholine at 0.3 M, and dilute it to obtain the 0.2 M, 0.1 M and 0.05 M solutions in sterile saline (0.9% NaCl). Methacholine solution has to be prepared on each experimental day from the methacholine powder.

Notes

  1. If the whole body plethysmograph system is equipped with any other nebulization equipment than LS230 (SYSTAM), we recommend to adapt the methacholine doses.

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., Daubeuf, F., Plantinga M., De, Cauwer, L., Gerlo, S., Waelput, W., Van Calenbergh, S., Tavernier, J., Haegeman, G., Lambrecht, B. N., Frossard, N., De Bosscher, K. (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。 该协议描述如何测量在守夜和无约束小鼠中对乙酰甲胆碱的气道反应。 增强的暂停(PenH)是通过全身气压体积描记法记录的乙酰甲胆碱增加剂量的气道反应的指数。

关键字:气道反应性, 体积描记法, 金边, 呼吸参数, 鼠标

材料和试剂

  1. 无菌盐水(0.9%NaCl)
  2. 甲基乙酰胆碱(Sigma-Aldrich,目录号:A2251)
  3. Drierite(Sigma-Aldrich,目录号:238988)
  4. 雾化(参见配方)

设备

  1. 全身体积描记系统(EMKA Technologies),包括标准设置(全身体积描记器,压差传感器,通气泵,放大器,接口盒,采集卡和具有呼吸流分析器模块的iox2软件)和标准雾化设备(LS230,SYSTAM ,Villeneuve-sur-Lot)

程序

  1. 打开体积描记器系统,等待15分钟,让气流和信号稳定。
  2. 控制每个呼吸速度描记器的渗透性,如果渗透性改变,则更换新的呼吸速度描记器。
  3. 控制干燥剂的干燥; 干燥的甜菜是蓝色的,并在湿度的存在下变成粉红色。 Drierite必须定期更换。
  4. 体积描记器系统中每个通气泵的控制功能; 每个通风泵必须调节到每分钟0.8升。 检查每个流量计中的球:所有球必须完全移动,并且没有氧化迹象(这些3个控制必须在每次实验之前进行)。
  5. 打开IOX软件(随体积描记仪系统提供)。
  6. 继续校准每个功能性体积描记器
  7. 将动物小心地放入每个体积描记室。
  8. 等待三十分钟,动物在体积描记室中变得安静和平静
  9. 开始测量会话。
  10. 向喷雾器中加入盐水,开始第一次雾化(30秒),并在20分钟内记录增强的暂停(PenH)。
  11. 加入0.05M乙酰甲胆碱溶液,喷雾30秒,记录PenH(20分钟)
  12. 加入0.1M乙酰甲胆碱溶液,雾化30秒,记录PenH(20分钟)
  13. 加入0.2M乙酰甲胆碱溶液,喷雾30秒,记录PenH(20分钟)
  14. 加入0.3M乙酰甲胆碱溶液,喷雾30秒,记录PenH(20分钟)
  15. 保存数据。
  16. 关闭数据库(IOX软件)和计算机。
  17. 从每个室中取出每只老鼠。
  18. 清除所有设备。
  19. 继续数据分析。 使用Datanalyst软件(随体积描记仪系统提供)用于IOX软件收集的数据。 在峰值PenH周围5分钟内测量的数据的平均PenH用于每剂乙酰甲胆碱(如图1中的箭头和虚线所示)。


    图1。

食谱

  1. 用标准雾化系统(LS230,SYSTAM)雾化需要10ml的每种乙酰甲胆碱溶液。 制备0.3M的乙酰甲胆碱溶液,并稀释它以获得在无菌盐水(0.9%NaCl)中的0.2M,0.1M和0.05M溶液。 乙酰甲胆碱溶液必须在每个实验日从乙酰甲胆碱粉末制备。

笔记

  1. 如果全身体积描记仪系统配备除LS230(SYSTAM)以外的任何其他雾化设备,我们建议调整乙酰甲胆碱剂量。

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,Daubeuf,F.,Plantinga M.,De,Cauwer,L.,Gerlo,S.,Waelput,W.,Van Calenbergh,S.,Tavernier,J.,Haegeman,G.,Lambrecht, Frossard,N.,De Bosscher,K。(2012)。 解离的糖皮质激素受体调节剂可降低小鼠哮喘模型中的气道高反应性和炎症反应 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 on Vigil and Unrestrained Mouse. Bio-protocol 3(4): e328. DOI: 10.21769/BioProtoc.328.
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