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。

1。设备收缩测量设备的主要部件示意图如图2A所示）。 <ol><li> 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 / CO 2的混合物在37℃水浴（未显示）。对于解决方案交流，PSS的解决方案是从水库抽水组织浴进口约100毫升每分钟（底部端口），允许较快的解决方案交流。解决方案的插座是通过溢流口（顶部的端口），允许在组织蝙蝠体积不变（〜10毫升）Ĥ在解决方案交流。我们使用了的哈克加热循环泵通过组织浴夹克的温水（保持37℃）。从供应商的数量可以得到组织浴场和各种尺寸和款式，以满足研究者的实验需要。 </li><li> 一个力传感器测量等距紧张，气管内管螺纹超过L形两端的两个不锈钢棒（ 图2A）。应小心使用不锈钢的类型是兼容生物材料。顶端杆连接通过等距力传感器剪辑。底部杆保持在一个固定位置的气管，并安装一个被动张力和/或肌肉长度调整微米。气管收缩创建力传感器的张力，这是一个前置放大器转换为电压信号。底部杆也可以被配置包括两个矩形铂板（4 mm处）T<html>帽子侧面的气管（ 图2B）。铂金板被连接到一个基层S88的刺激，使整个气管电场交付。涂开放的电线和焊料，Sylgard（Sylgard 184硅橡胶，道康宁公司，美联，心肌梗死），沐浴液，防止浸金属。 </li><li>上MacLab 8的A / D系统的A / D转换器，计算机和收购的软件，从前置放大器信号记录。这是当前ADInstrument PowerLab系统硬件的旧版本。我们使用的程序图（的ADInstruments），使整个实验过程中张力的连续记录。气管肌肉产生的张力是相当缓慢的，因此，我们发现，每秒100点的收购是足够的。每次实验前用已知重量（5克），张力测量校准。类似的系统是从其他厂商（例如，BIOPAC，毛重仪器）。 </li></ol>标题“&gt; 2。气管分离<ol><li>组织分离之前的力传感器校准与已知的重量，并组织浴充满正常的PSS（见表一）。进气口进行调整，以获得的O 2 / CO 2的光流。 </li><li>从2个月或年纪较大的老鼠的气管是最优的。可用于年轻的动物，但是从这些获得的气管，需要更多的技能，由于其体积小，安装力传感器导线。解剖前，小鼠深感异氟醚镇静。适当的镇静水平达到时，用钳子捏脚趾是无法引起反应。立即处死小鼠颈椎脱位的一个重要说明：我们已经看到，Avertin（Tribromoethanol），小鼠常用的一种镇静，具有强大的气道平滑肌的松弛作用，因此不应使用气管收缩研究。 </li><li>从胸部到喉咙的皮肤和毛皮被删除。 ŕIBS是削减从胸骨的基础上，横向（双方）的心脏上方。胸骨和肋骨，然后向前拉的喉咙，揭示了心脏/肺，胸腺，气管和食管（腹）（附 - 背到气管）。 </li><li>由切割低于支气管分叉以上咽，气管切除。气管被放入冰冷的含氧（95/5）PSS的解决方案（表一中给出的组成）。 </li><li>气管周围组织解剖干净。在清理过程中，气管，可在咽部或低于分叉举行。然而，应谨慎采取不直接适用于气管本身的镊子。罚款剪刀可用于切断周围组织，但始终平行于气管切，以避免损坏。促进这个过程的一部分，如果气管准备下面的分岔和咽部以上固定到Sylgard涂层菜（Sylgard 184硅胶弹性体，道康宁公司，美联，心肌梗死）。 </li><li>消除周围组织后，气管被切断以下咽和支气管分叉以上，并轻轻地装上力传感器导线。 </li><li>气管螺纹超过两个L形金属插脚（ 图2A）。一个插脚连接到一个等距紧张的连续记录的力 - 位移传感器。另一个插脚连接到一微米。然后抬起，使气管沉浸在PSS组织浴。气管的安装应尽快完成，以尽量减少时间，气管外的PSS举行。与实践，气管安装，可以在一分钟之内完成，但我们一般避免时间超过3分钟，以避免损失的可行性。 </li><li>慢慢调整到微米〜10分钟（约1克力）获得的被动张力。确定最佳的休息紧张经验，我们发现，被动张力 - 1〜50 MN结果，在同等的，最大的高钾刺激的反应。这是利用16,17,18在此范围内的被动张力的其他研究一致。在5-10分钟，气管被动紧张倾向于有所下降（应力松弛的现象）和千分尺是用来调整在平衡〜10分钟的被动张力。气管被允许平衡实验的挑战前至少1小时。 </li></ol> 3。含钾高的刺激下面的平衡，气管被两次挑战与高钾PSS解决方案（67毫米氯化钾，表一）。收缩一般〜需要5-10分钟，以达到稳定状态的时间，组织浴是正常的PSS冲洗时间，完全放松气管。钾收缩重复第二次，第三次，直到重复性收缩（如有必要）获得。 <p=“jove_title”的4课。通过电刺激诱发的收缩气管两侧由两个矩形铂板（电极），使电场刺激（EFS）的准备。 EFS的收缩反应的频率和电压的功能。它也受到物理参数，如电极的面积和它们之间的距离。刺激的功率特性也影响等，在更高的电压和电流输出的刺激可以达到其最大的反应。任何EFS系统的特点，应确定通过检查肌肉收缩反应，在不同的刺激持续时间，频率，电压和脉冲持续时间。对于我们的实验装置，我们已经发现，分开〜4毫米的电极，刺激五（0.5毫秒脉冲）44和30 Hz振幅最优实现重复性附近最大收缩反应。 5。收缩EV由胆碱能刺激oked 此外，由一个单一剂量药物的兴趣或外加的化合物气管的反应进行评估，由多种药物的累积剂量的时尚增加。对于气管，我们实验室经常使用卡巴激活乙酰胆碱受体的卡巴，因为不同的乙酰胆碱，乙酰胆碱酯酶不降解。一个合理的剂量响应范围是从10-8到10-5中号卡巴。 [卡巴]收缩与山型方程的响应曲线拟合的日志允许欧共体50（半最大有效浓度）这是一个衡量气管收缩的敏感性胆碱能激动剂19的估计。值得注意的是，定剂量的卡巴将略大于作为一个单一的剂量累积剂量反应曲线的一部分的反应。 6。代表结果</p&gt; 如图3A所示的收缩反应含钾高的一个例子。收缩达到最大，约10分钟内，但以后可能会出现小幅下降。在早期高钾冲刷，肌肉可能会出现短暂的增加，是由于在温度下降，体积小，加热的PSS解决方案解决方案中的线路瞬时perfuses准备在收缩。这可以通过连接油管加热PSS水库和组织浴中有一个最低的死体积最小，也相对快速交换解决方案（一般在100毫升/分钟泵解决方案）。每个准备将收缩反应有一些差异，由于肌肉质量或剥离过程中所造成的危害后果的差异。 图3B显示了两个不同的肌肉质量高的钾和卡巴的挑战气管。虽然胆碱诱发的收缩不同，y是正常化后，以高钾溶液的反应（ 图3C）类似。 图4显示了一个1卡巴（胆碱）使用单剂量（A）和累积增加（二）诱发收缩的例子。卡巴的解决方案是直接添加到洗澡和冒泡的气体有助于在快速搅拌。这是值得注意此外，单剂量（即1微米， 图4A），比在累积剂量反应曲线（1μM， 图4B）当量浓度稍大的响应。 图4C显示了收缩力的阴谋作为卡巴浓度使用数据从图4B功能。卡巴的影响在10 10-5 M浓度饱和。虽然胆碱能激动剂启动通过钙释放机制的收缩，收缩的主要组成部分，也是介导的去极化和激活电压依赖性钙通道20。 图5A显示EFS的诱发宫缩的一个例子。气管受到刺激，使用时间0.5毫秒，40伏脉冲收缩，直到达到高原（见插图A1）。在刺激频率的增加，导致增加的收缩反应（频率响应曲线绘制图5B）。已被证明电场刺激唤起主要通过激活突触前神经收缩。肉毒毒素的作用，阻断神经递质的释放，阻止大多数的EFS诱发的收缩气管21证明了这一点。此外，河豚毒素，阻止Na +通道，也能抑制神经活动和消除气管回应的EFS代理。 <img alt="图1" src="/files/ftp_upload/3703/3703fig1.jpg" /> T图图1。他的主要信号通路在离体气管准备。胆碱能支配气管平滑肌细胞的轴突终端显示。主要的信号转导通路是货币供应M3和M2-毒蕈乙酰胆碱受体激活（mAchR的）导致的IP3受体（M3）和cAMP的减少量（M2）钙释放。 M2受体（和一些M3受体的贡献）也引起胆碱诱发的去极化，激活L-型电压依赖性钙通道和钙离子内流。共同收缩剂和其效应是：1。高钾（平滑肌细胞和胆碱能神经轴突的去极化），2。电场刺激（EFS，胆碱能神经轴突的去极化）和3。外源性应用程序（如乙酰胆碱或卡巴胆碱剂直接激活毒蕈碱受体）。 <img alt="图2" src="/files/ftp_upload/3703/3703fig2.jpg" /> 用来测量气管收缩的设备示意图如图2。。力传感器，千分尺和组织浴室安装支持通过螺丝钳棒。气管环螺纹在顶部和底部的棒。在图中，组织浴定位下面的准备（即到力传感器安装在气管）。在收缩的研究，组织浴垂直移动洗澡的准备。 B.对于电场刺激的底部杆进行了修改，包括两个铂金板气管控股线横向安装。铂金板是由电线连接到一个刺激。 <img alt="图3" src="/files/ftp_upload/3703/3703fig3.jpg" /> 图3。含钾高的例子（67毫米）气管收缩反应。 （A）显示，含钾高的重复性重复反应。 （二）两种不同的气管收缩反应卡巴的例子。 （三）在B的气管反应是类似的，当正常化d到高的钾反应。 <img alt="图4" src="/files/ftp_upload/3703/3703fig4.jpg" /> 图4。卡巴引起的收缩的例子。 （一）使用单一剂量的卡巴胆碱诱导的收缩后冲洗。 （二）例气管（三）在A从B的峰值收缩的累积剂量反应曲线绘制作为卡巴浓度的功能。 <img alt="图5" src="/files/ftp_upload/3703/3703fig5.jpg" /> 图5。由电场刺激诱 ​​发的收缩的例子。 （一）使用40伏，0.5毫秒脉冲，以及各种刺激频率的气管收缩电场刺激表示。插图是时间膨胀收缩在30赫兹。 （二）从A山顶收缩是plotte的作为刺激频率的函数D。 正常的PSS <table border="1"><tbody><tr><td> 盐 </td><td> 浓。（MM） </td><td> 量（克/ 2升） </td></tr><tr><td>氯化钠</td><td> 119 </td><td> 13.91 </td></tr><tr><td>氯化钾</td><td> 4.7 </td><td> 0.7 </td></tr><tr><td> KH 2 PO 4 </td><td> 1.18 </td><td> 0.32 </td></tr><tr><td>硫酸镁4 x 7H 2 </td><td> 1.17 </td><td> 0.58 </td></tr><tr><td> 碳酸氢钠 </td><td> 18 </td><td> 3.02 </td></tr><tr><td> EDTA的</td><td> 0.026 </td><td> 0.5米0.1毫升</td></tr><tr><td>葡萄糖</td><td> 11 </td><td> 3.96 </td></tr><tr><td>蔗糖</td><td> 12.5 </td><td> 8.56 </td></tr><tr><td> 氯化钙 </td><td> 2 </td><td> 400毫升10毫米</td></tr></tbody></table>高K + PSS的（NACl和氯化钾调整） <table border="1"><tbody><tr><td> 盐 </td><td> 浓。 （MM） </td><td> 量（克/ 2升） </td></tr><tr><td>氯化钠</td><td> 56.7 </td><td> 6.628 </td></tr><tr><td>氯化钾</td><td> 67 </td><td> 9.991 </td></tr></tbody></table> 表1配方的PSS解决方案注 ：解每周新鲜，用超纯水水质，并储存在冰箱不超过5天，以避免污染增长。

<ol>
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J. 21, 812-822 (2007).</li><li>Scheerens, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+topical+exposure+to+toluene+diisocyanate+in+mice+leads+to+antibody+production+and+in+vivo+airway+hyperresponsiveness+three+hours+after+intranasal+challenge.">Long-term topical exposure to toluene diisocyanate in mice leads to antibody production and in vivo airway hyperresponsiveness three hours after intranasal challenge.</a> Am. J. Respir. Crit. Care. Med. 159, 1074-1080 (1999).</li><li>Kenakin, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A pharmacology primer : theory, applications, and methods. , (2009).</li><li>Semenov, I., Wang, B., Herlihy, J. T., Brenner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BK+Channel+{beta}1+Subunits+Regulate+Airway+Contraction+Secondary+to+M2+Muscarinic+Acetylcholine+Receptor+Mediated+Depolarization.">BK Channel {beta}1 Subunits Regulate Airway Contraction Secondary to M2 Muscarinic Acetylcholine Receptor Mediated Depolarization.</a> J. Physiol. , 1803-1817 (2011).</li><li>Moffatt, J. D., Cocks, T. M., Page, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+epithelium+and+acetylcholine+in+mediating+the+contraction+to+5-hydroxytryptamine+in+the+mouse+isolated+trachea.">Role of the epithelium and acetylcholine in mediating the contraction to 5-hydroxytryptamine in the mouse isolated trachea.</a> Br. J. Pharmacol. 141, 1159-1166 (2004).</li><li>Bachar, O., Adner, M., Uddman, R., Cardell, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+enhances+cholinergic+innervation+and+contractile+response+to+electric+field+stimulation+in+a+murine+in+vitro+model+of+chronic+asthma.">Nerve growth factor enhances cholinergic innervation and contractile response to electric field stimulation in a murine in vitro model of chronic asthma.</a> Clin. Exp. Allergy. 34, 1137-1145 (2004).</li><li>Weigand, L. A., Myers, A. C., Meeker, S., Undem, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-cholinergic+nerve+interaction+in+mouse+airways.">Mast cell-cholinergic nerve interaction in mouse airways.</a> J. Physiol. 587, 3355-3362 (2009).</li><li>Kao, J., Fortner, C. N., Liu, L. H., Shull, G. E., Paul, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+the+SERCA3+gene+alters+epithelium-dependent+relaxation+in+mouse+tracheal+smooth+muscle.">Ablation of the SERCA3 gene alters epithelium-dependent relaxation in mouse tracheal smooth muscle.</a> Am. J. Physiol. 277, 264-270 (1999).</li><li>Krane, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquaporin+5-deficient+mouse+lungs+are+hyperresponsive+to+cholinergic+stimulation.">Aquaporin 5-deficient mouse lungs are hyperresponsive to cholinergic stimulation.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 14114-14119 (2001).</li><li>Semenov, I., Wang, B., Herlihy, J. T., Brenner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BK+channel+beta1-subunit+regulation+of+calcium+handling+and+constriction+in+tracheal+smooth+muscle.">BK channel beta1-subunit regulation of calcium handling and constriction in tracheal smooth muscle.</a> Am. J. Physiol. Lung. Cell Mol. Physiol. 291, L802-L810 (2006).</li><li>Fortner, C. N., Breyer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EP2+receptors+mediate+airway+relaxation+to+substance+P+ATP,+and+PGE2.">EP2 receptors mediate airway relaxation to substance P ATP, and PGE2.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 281, 469-474 (2001).</li><li>Hay, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+modulation+of+endothelin+ligand-induced+contraction+in+isolated+tracheae+from+endothelin+B+(ET(B))+receptor+knockout+mice.">Differential modulation of endothelin ligand-induced contraction in isolated tracheae from endothelin B (ET(B)) receptor knockout mice.</a> Br. J. Pharmacol. 132, 1905-1915 (2001).</li><li>Stengel, P. W., Yamada, M., Wess, J., Cohen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M(3)-receptor+knockout+mice:+muscarinic+receptor+function+in+atria,+stomach+fundus,+urinary+bladder,+and+trachea.">M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea.</a> Am. J. Physiol. Regul. Integr. Comp Physiol. 282, R1443-R1449 (2002).</li><li>Trevisani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+in+vitro+expression+of+B1+receptor+in+the+mouse+trachea+and+urinary+bladder.">Evidence for in vitro expression of B1 receptor in the mouse trachea and urinary bladder.</a> Br. J. Pharmacol. 126, 1293-1300 (1038).</li><li>Mehats, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDE4D+plays+a+critical+role+in+the+control+of+airway+smooth+muscle+contraction.">PDE4D plays a critical role in the control of airway smooth muscle contraction.</a> FASEB J. 17, 1831-1841 (2003).</li><li>Kumar, R. K., Herbert, C., Foster, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"classical"+ovalbumin+challenge+model+of+asthma+in+mice.">The "classical" ovalbumin challenge model of asthma in mice.</a> Curr. Drug Targets. 9, 485-494 (2008).</li></ol>

没有利益冲突的声明。

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?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>模拟 - 数字转换器</td><td> ADInstruments </td><td> PowerLab系统4/35 </td><td></td></tr><tr><td>卡巴（Carbamoylcholine氯化物） </td><td> Sigma-Aldrich公司</td><td> C4832 </td><td> 10 -2米的水（等分可以储存在-20°C） </td></tr><tr><td>制图软件</td><td> ADInstrtuments </td><td> LabChart </td><td></td></tr><tr><td>加热循环器</td><td>哈克</td><td>调音台轧机MM400 </td><td></td></tr><tr><td>轴测力传感器</td><td>肯特科学</td><td> TRN001 </td><td></td></tr><tr><td>刺激</td><td>草技术</td><td> S88的双输出SQU脉冲刺激</td><td></td></tr><tr><td>组织浴</td><td> WPI的</td><td> 47264 </td><td></td></tr></tbody></table>

in vitro measurements of tracheal constriction using mice

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&gt; 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。

Watch this Scientific Journal Video about In vitro Measurements of Tracheal Constriction Using Mice at JoVE.com

In vitro Measurements of Tracheal Constriction Using Mice

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&gt;在体外</em&gt;气管收缩的研究，以评估在小鼠气道平滑肌功能。

在利用老鼠的气管收缩的体外测量

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

in-vitro-measurements-of-tracheal-constriction-using-mice

Research

JoVE Journal

Medicine

In vitro Measurements of Tracheal Constriction Using Mice

20.6K 次观看.  UT Health Science Center, San Antonio. 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，我们提供的协议在体外气管收缩的研究，以评估在小鼠气道平滑肌功能。

视频: In vitro Measurements of Tracheal Constriction Using Mice

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

慢性缩窄大鼠坐骨神经疼痛过敏测试

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

科研

医学

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

大鼠建立压力超负荷心肌肥厚模型在升主动脉缩窄

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

心脏重构通过大鼠腹主动脉缩窄的模型

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

微创横向主动脉缩窄小鼠

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

小鼠横主动脉狭窄诱导左心室肥厚的技术研究

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

诱导小鼠横向主动脉狭窄的闭合胸模型

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Sleeve Gastrectomy in Mice using Surgical Clips

The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.

The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.