在利用老鼠的气管收缩的体外测量
Summary
转基因小鼠已归咎于基因的生理功能极为有用。由于在一般情况下，例如，研究和呼吸道功能的研究，特别是经历了一个对小鼠模型显着的转变。在这里，我们提供的协议<em>在体外</em>气管收缩的研究，以评估在小鼠气道平滑肌功能。
Abstract
转基因和基因剔除小鼠的生理和病理生理学航空公司1,2的调查，已经强大的工具。 在体外离体气管准备tensometry已被证明是有用的气道平滑肌（ASM）的转基因小鼠的收缩反应检测。 在体外气管筹备的这些都是比较简单，提供了强有力的回应，并保留两个功能胆碱能神经末梢和肌肉反应，即使经过长时间的孵化。
气管tensometry还提供了一个功能试验，以研究各种第二信使的影响平滑肌收缩的信号转导通路。在气管的收缩主要是由副交感神经，上的ASM（ 图1），释放乙酰胆碱能神经介导。 ASM乙酰胆碱受体主要是毒蕈M2和M3 G I / O和Gq蛋白偶联受体，分别<suP> 3,4,5。 M3受体引起收缩耦合GQ激活磷脂酶C，增加的IP3生产的IP3介导的钙释放和肌浆网3,6,7。 M2 / G I / O信号被认为是加强宫缩抑制腺苷酸环化酶，减少cAMP水平5,8,9,10。这些途径，构成所谓的气道平滑肌11“药物收缩偶联”。此外，通过胆碱能M2受体信号（和M3的信号调制）涉及的去极化的ASM，这反过来又激活L-型电压依赖性钙通道（ 图1）和钙离子内流（即所谓的“兴奋收缩偶联的途径” ）4,7。控制气道收缩的信号传导通路的更详细的评论，可以发现4,12。出现上述途径，小鼠和其他物种之间的保守。然而，小鼠气管不同于其他物种我N部分信号通路。最突出的是他们缺乏13,14组胺和腺苷的收缩反应，无论是在人类和其他物种5,15知名的ASM调制。
在这里，我们提出了隔离小鼠气管环的协议，并在体外测量其收缩输出。包括设备配置的描述，气管环隔离和收缩测量。例子是唤起收缩间接使用高钾刺激神经 和直接由ASM的肌肉去极化，激活电压依赖性钙离子涌入（1。高K +， 图1）。此外，刺激神经 单独使用电场刺激（EFS时， 图1），或直接刺激肌肉的ASM使用外源性神经递质浴（3， 图1。外生的ACH）方法。这FLExibility和便于编制呈现的离体气管环模型参与气道平滑肌收缩的信号级联的一个强大和功能检测。
Protocol
1。设备收缩测量设备的主要部件示意图如图2A所示）。 组织浴。组织沐浴在温暖的温度保持含氧的生理溶液。对小鼠气管环，我们使用循环变暖的解决方案，1熔块玻璃入口泡沫氧（95％/ 5％的O 2 / CO 2的混合物），进口和出口改变端口10毫升组织洗澡，包含了水套解决方案。水库的PSS解决方案存储不断冒泡的95％/ 5％的O 2</…
Discussion
这里介绍的协议提供了生理的准备，评估气道肌肉功能。我们一般同时操作3-4器官浴准备，但是，打包的系统，可以从供应商，允许同时测量高达8制剂（ADInstruments，世界精密仪器，和哈佛仪器）。我们利用力传感器的数量和组织器官浴相同的结果。然而，我们发现，电场刺激提供一些基于刺激电极大小，电极板之间的距离，和编制内电场的位置之间的细微差别的变异。因此，格外小心，应采取?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是从创新和治疗气道疾病，NINDS的补助金（NS052574），防治中心和哮喘研究桑德勒计划拨款资助。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Analogue-Digital Converter | ADInstruments | PowerLab 4/35 | |
| Carbachol (Carbamoylcholine Chloride) | Sigma-Aldrich | C4832 | 10-2 M in water (aliquots can be stored at -20°C) |
| Charting Software | ADInstrtuments | LabChart | |
| Heating Circulator | Haake | Mixer Mill MM400 | |
| Isometric Force Transducer | Kent Scientific | TRN001 | |
| Stimulator | Grass Technologies | S88 Dual Output Square Pulse Stimulator | |
| Tissue Bath | WPI | 47264 |
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Tags
In Vitro MeasurementsTracheal ConstrictionMiceTransgenic MiceKnockout MiceAirway PhysiologyAirway PathophysiologyIsolated Tracheal PreparationsAirway Smooth MuscleASM Contractile ResponseCholinergic Nerve EndingsMuscle ResponsesSecond Messenger Signaling PathwaysParasympathetic NervesCholinergic NervesAcetylcholine ReceptorsMuscarinic M2 ReceptorsMuscarinic M3 ReceptorsGi/o Coupled ReceptorsGq Coupled ReceptorsPhospholipase CIP3 ProductionIP3-mediated Calcium ReleaseSarcoplasmic ReticulumM2/Gi/o SignalingAdenylate Cyclase InhibitionCAMP LevelsPharmaco-contraction Coupling
Citer Cet Article
Semenov, I., Herlihy, J. T., Brenner, R. In vitro Measurements of Tracheal Constriction Using Mice. J. Vis. Exp. (64), e3703, doi:10.3791/3703 (2012).