Name: measurement of smooth muscle function in the isolated tissue bath-applications to pharmacology research
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The Watts Laboratory through the years, and Dr. Marlene Cohen and Kathy Schenck for teaching us this assay over two decades ago. NIGMS R25GM074119 supported development of this teaching module for a Short Course in Integrative and Systems Pharmacology held on the campus of MSU from 2005 to 2013.

注:本文中所描述的所有程序都是根据密歇根州立大学的机构动物护理和使用委员会（IACUC）制定的准则进行。 1.系统准备和设置<ol><li>使5升的生理盐溶液（PSS），这是需要的，它使用高达50毫升组织浴的组织浴中的收缩实验的量; 见表2 PSS配方。乘以组织浴的数目乘以浴体积，然后通过所需的组织洗涤次数乘以计算总所需体积。 <ol><li>使用表2为使PSS的指南。盐溶解在约4升的水。注:HPLC - I类水推荐</li><li> （如果使用的二水合物盐147克/ L）的溶液中，所以最终的解决方案是1.6毫钙加入8毫升的1M的CaCl 2溶液。 </li><li>量子sufficit PSS解决5 L. </li></OL&gt; </li><li>通过打开循环恒温水浴预热组织浴系统至37℃。关键的步骤:在系统的每一个组件是水夹套，确保它们串联连接到彼此。流的方向是至关重要 - 确保在最高有倒钩连接的水流入各组分在最低的有倒钩的连接和缩小。 </li><li>开启数据采集系统。电源上的力传感器至少15分钟前实验平衡温度。 注:大多数力传感器采用应变计是温度变化敏感，表现出热漂移最初通电后。 </li><li>启动数据采集软件，并确保与数据采集系统连接。请按照制造指令使数据记录。 </li><li>确保力传感器组织放置在组织浴和数据记录开始之前，前被校准; FOllow制造商的说明进行校准。 </li><li>组织浴系统连接到一个95％ 的 O 2/5％CO 2的医用级气筒和检查气体泄漏，然后加压系统。 </li><li>填充组织浴储层PSS和允许溶液的时间来达到最佳温度。首相系统并删除系统和管道内的气泡。 </li><li>检查组织浴充气器，以确保一致溶液曝气，其中增氧PSS缓冲，并提供布朗运动来分发，这将在组织浴中的试验过程中被引入药物。确保通风/气泡不会引起组织运动，这将扰乱数据记录;降低气体流量是必要的。 注:该组织浴和数据采集系统现在已经准备好开始实验。 </li></ol> 2.组织准备<ol><li>理想地，解剖来自动物组织之前立即使用，并直接将我n要PSS。 注意:有些组织可以在一夜之间在4℃保存在PSS，但适当的控制必须做到以验证长期存放组织功能;见第6。 </li><li>使用胸主动脉作为这项工作的组织。 </li><li>麻醉按照机构准则大鼠和向下放置鼠背侧。通过脚趾捏并为此疼痛刺激反射反应的损失确认麻醉。 注:此协议使用70毫克/公斤的戊巴比妥通过腹腔注射交付。对啮齿动物的麻醉制度的指导方针可能会有所不同。 </li><li>通过创建一个切口沿隔膜在肋条笼形件的底部剪刀创建气胸。然后，继续从胸腔的底部切口的胸骨和平分。 </li><li>解剖远或放置一旁那些器官是在脊柱的顶部和定位在主动脉，其直接位于沿脊柱。 注意:这个STEP提供了明确的工作空间解剖出主动脉。 </li><li>断绝一切联系的主动脉;从食道，肺等，并切断主动脉垂直于脊柱在膜片的水平。 </li><li>轻轻握住钳主动脉，用剪刀从脊柱解剖主动脉。开始在邻近的脊椎和工作朝向心脏的下隔膜面积的夹层。请务必采取额外的预防措施不拉或拽主动脉组织，这可能会损坏组织。 </li><li>立即将含有PSS的准备解剖盘的主动脉。 注:主动脉成环的解剖是在解剖盘，有一个黑色的硅橡胶基础最好的做法。这允许使用的电线可用于导管插入主动脉和固定到盘的，从而提供稳定性，同时在解剖盘中。黑色背景对比提供了有助于解剖。应注意在使用cannulating线为endothel胶质细胞层可以用与容器的内腔的导线的多余的摩擦被除去。 </li><li>取的线和一个小的90°角，另一端成角度的端部推入硅橡胶基础锚定导丝。放置在解剖盘整个主动脉。 </li><li>持导丝与镊子的末端，然后将金属丝轻轻拧到主动脉的内腔。 </li><li>使用镊子和主动脉滑到线。当导丝的自由端是可见的，曲线的丝轻轻并将自由端插入皿的硅橡胶基础，以稳定的组织。 </li><li>使用小vannas剪刀和镊子，取出血管周围脂肪组织和所有其他外部组织，血块等直到胸主动脉出现白色和略有纤维性。 </li><li>从电线拆下清洗主动脉，用剪刀剪下的戒指是大约3-5毫米宽的主动脉。 </li><li>放置一个主动脉环上的一对钛中的一个ssue钩握住钩子和使用镊子轻轻滑动环到挂钩。与第二钩状重复此过程。请注意，以确保挂钩不纠结;参见图3。 </li><li>检查每个钩具有连接不同的丝线。检查一个钩丝具有允许用于连接到一个玻璃或不锈钢棒的小打结循环，而另一种是10-4厘米长片缝合，就可以用手绑在力传感器;参见图3。 注意:一旦主动脉固定在钩，所述组织是准备被安装在组织浴;参见图3。 </li><li>重复步骤2.14-2.15，安装第二主动脉环在第二组织浴室。 </li></ol>巴斯3.组织安置<ol><li>注意，在这个系统中，组织浴本身是固定的。此时，填充温热，充气的PSS的组织浴中，并允许该溶液来。温度即</li><li>用丝线缝合，打结钩在组织上，以制备在不锈钢棒上的塞销中的一个。将为此进入组织浴室。杆连接到环立场，并将在染色菜充满了PSS，以保持组织沉浸在缓冲区中的另一端;参见图3。 <ol><li>放置杆和组织在组织浴室，并确保组织被完全沉浸在PSS和杆是安全的;参见图3。 </li><li>配合其他缝合力传感器，并确保离开松弛的组织和力传感器之间的缝合。 注:此松弛会通过调整微米被删除;参见图3。 </li></ol></li><li>重复这些步骤，第二主动脉瓣环和组织洗浴室。 </li></ol> 4.设置被动张力注意:每个组织具有长度（螺），在该平滑肌细胞响应最优LLY。初步实验，以确定最佳的拉伸张力而实现待检查该长度必须为每个单独的组织类型进行。大鼠胸主动脉有4个g的最优被动伸展紧张。 <ol><li>使用千分尺/齿条和小齿轮，以增加张力至2g，并等待所述组织，以达到平台期。一旦达到平稳状态，增加紧张又2克，等待组织高原。 注:在主动脉，后2克的初始张力树立和平台达到，组织应再放宽到〜1.6克2克的第二应用将使组织到〜3.6克，从该组织将再次放松和张力将减小。因此，具有加入4- g总张力之后，该软件应说明大致3.2克张力。 </li><li>重复这些步骤，第二主动脉瓣环和组织洗浴室。 </li></ol> 5.平衡和药品制作<ol><li>平衡组织的施加被动张力至组织后的60分钟。 </li><li>在平衡期，洗组织每次15-20分钟。沥干组织浴缸和一个温暖水库替换PSS的PSS。 注意:在此期间，该组织将放松，这是由被动张力的损失表示。 </li><li>在此期间，准备药物用于实验，这需要至少1000倍，在浴中的实际浓度越浓，所以只有小体积药物库存的需要以达到所需的浓度。 </li></ol> 6.初次挑战<ol><li>在平衡期结束时，洗组织最后一次，保存数据，并重新零的所有输入。 </li><li>挑激动剂（化合物，这将导致活性的收缩），以使组织响应。 注:在大鼠胸主动脉，动脉平滑肌将积极签约，α-肾上腺素能受体激动剂由于该神经支配组织由交感神经系统。因为肾上腺素受体激动剂引起松弛肾上腺素能激动剂不会在肠组织非常有用。在这种情况下，也可以使用一个受体激动剂如乙酰胆碱（胆碱受体）。一种替代既福林和乙酰胆碱是使用非受体激动剂如高钾（K），为第一个挑战。在平滑肌，高钾操作来间接开放的钙通道，因而被视为平滑肌完整性的综合指数。 <ol><li>加入10 -5 M去氧向组织浴执行第一询问或唤醒挑战。为了达到此浓度的50毫升组织浴中，加50微升的10 -2 M溶液去氧肾上腺素。 注:50微升到50ml的PSS是一个1 / 1,000稀释，使最终的浓度比库存1000倍以下，或10 -5 M.内的组织浴中的知识量和一贯维护的体积的hamber是确定最终药物浓度是至关重要的。 </li></ol></li><li>添加10 -5 M去氧向组织浴，并允许收缩到峰值，然后高原，当数据的斜率变为零。确保记录每个实验活动，以帮助后的实验分析。 </li><li>高原后，排空和重新填充组织浴室新PSS彻底清洗激动剂出来。请务必记录这些事件也是如此。 注:经验法则是，激动剂在浴的浓度降低10倍每次洗涤。虽然，超过3-4次洗涤可能是必要的，以使所述组织回落到其平衡基线音（张力）。 </li><li>让组织其余基线的基调，然后再继续〜10分钟。 </li></ol> 7.实验注:哌唑嗪，α-肾上腺素受体拮抗剂，将被引入到组织浴室转移的浓度RESPONSE曲线去氧肾上腺素（PE）;这是可证明的拮抗作用。 <ol><li>到1浴中，添加车辆（H 2 O），其中哌唑溶解。到另一个，添加哌唑嗪的适当的浓度。在这种情况下，加入25微升车辆以一浴和25微升哌唑嗪的10 -5 M浓度的其他实现5×10 -9 M或在浴中的5nM的浓度。 注意:虽然除了车辆的在本实施例看来是不必要的，它使用具有潜在的是血管活性（ 例如，二甲基亚砜，乙醇，乙酸乙酯）其他车辆时必须这样做。添加相应的车辆即使没有证据存在它是血管活性。 </li><li>离开车辆/哌唑嗪在浴室和不洗组织1小时。 <ol><li>而组织的平衡，准备多个浓度激动剂（PE）的。包括一个宽范围的浓度，从浓度引发无反应（ 例如，&lt;/ em&gt;的10 -9 M 的 PE）到超过最大应答（浓度如 10 -4 M的PE）。自浓度-响应曲线是对数性的，从10 -1 1M贮存液使通过连续稀释六个单独的储备溶液的PE（10 -6 -10 -1 M）。确保所需要的各浓度的体积比三重所需的所有浴（ 即 &gt; 300微升）的总体积稍大。 </li></ol></li><li>着手加入激动剂，如表1所述。 注意:这个实验会产生累积浓度响应曲线PE;每次迭代考虑到物质的已加入到浴中的量。增加发生，直到他们不再增加收缩，或整个曲线趋于平稳。 <ol><li>添加每个药物浓度分别直到组织达到阈值。 </li><li>如果没有发生变化，添加在接下来的浓度系列; 见表2。 注意:这些添加的时机取决于所用的激动剂。例如，去甲肾上腺素和肾上腺素收缩的快速发展，并应在几分钟之内的高原。相比之下，内皮素-1收缩发展缓慢，不得高原近45分钟。其他的实验可以在组织进行施工的曲线，如本后，只要（1）该物质冲刷;和（2）它可以证明所述组织返回到正常收缩行为和不受先前刺激。 </li></ol></li></ol> 8.数据分析<ol><li>确保数据保存立即之前和之后的干预（ 例如，唤醒或全曲线）。 </li><li>找到并设置组织基线对每个组织浴室/输入通道，这将在数据分析提供帮助。 </li><li>一旦所述基线设置，发现对于每个浓度的最大反应，并记录瓒GE从基线。依次移动通过每个浓度并记录基线偏移。 </li><li>格拉夫将所得的数据。在任何图形程序做到这一点。 注意:图表垫棱镜设计用于药理学家，这是理想的这里提出的实验的类型。 </li></ol>

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M., O'Connor, C., McLoughlin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+changes+in+pH+on+wall+tension+in+isolated+rat+pulmonary+artery:+role+of+the+RhoA/Rho-kinase+pathway.">Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 287, L673-L684 (2004).</li><li>Tykocki, N. R., Thompson, J. M., Jackson, W. F., Watts, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ryanodine+receptors+are+uncoupled+from+contraction+in+rat+vena+cava.">Ryanodine receptors are uncoupled from contraction in rat vena cava.</a> Cell Calcium. 53, 112-119 (2013).</li></ol>

Authors have nothing to disclose.

的等长收缩力作为一种研究工具测量超过150年的历史，但它仍然是收缩组织2原型技术受体表征。该技术的功率是在其简单性和通用性:通过记录通过增加拮抗剂的存在或不存在激动剂的浓度引起的反应，信息无数可导出关于每种药物的药理学特性和受体向其中它结合3-5。这种类型的实验也争取有关的竞争与所使用的拮抗剂的非竞争性的信息，以及受体的异质性和非特异性药物作用6-8。因此，简单的排列组合，以这种孤立的组织浴实验，可以生成介导激动剂诱发肌肉收缩的受体的一个比较完整的药理学特性。 在激动方面，第在给定组织的激动剂电子相对效力（E MAX）和效力（EC 50）可被计算并与其它激动剂在相同组织1的响应。在我们的实验中，将大鼠胸主动脉孵育用载体或α1肾上腺素能受体拮抗剂哌唑嗪（5 nm）的一小时中添加α1肾上腺素能激动剂苯向组织浴并产生收缩之前。 图2给出修改的结果图1的，绿色的响应已被标以最大收缩由聚乙烯取得了明显的最大值，该半最大收缩标记为这样，然后与PE浓度相关联的原因造成的响应。确定这些值是识别实现了半最大（50％）的反应或EC 50值的激动剂的有效浓度。 不幸的是，使用激动剂依赖性参数独自一人来确定受体结合特性可并发9。理想的是，使用受体拮抗剂附加实验允许计算的两个重要参数是关键限定药物与受体之间的相互作用:所述拮抗剂离解常数（pK值B）的-log 10和摩尔拮抗剂的-log 10必要浓度以引发在浓度-反应曲线2倍向右移位（PA 2）10。无论K B＆和PA 2增益的一个事实，即他们是兴奋剂无关的值，保持不变，甚至不同组织11之间有用。从图2中的数据，PK B能够使用以下等式来计算，并根据欧共体别处计算50值: EC 50在没有哌唑嗪= 2×10 -8 M的 EC 50在预哌唑嗪SENCE = 7×10 -6 M 在下面的等式求解在K B: 登录（DR-1）=日志[B] -日志K B＆ 其中: [B] =拮抗剂浓度，或5×10 -9 M.登录（5×10 -9 M）= -8.3 DR =的EC 50值拮抗剂/ EC 50的情况下存在剂量率。如果有一个向右移位，则此值将大于1。因此: DR = 7×10 -6 M / 2×10 -8 M或700×10 -8 M / 2×10 -8 M =二分之七百= 350 代替[B]和DR: 登录（350-1）= -8 -登录K B＆ 2.54 = -8 -登录KB 的pK B = 10.54 该值哌唑嗪与获得的值一致时，哌唑嗪与α1肾上腺素RECEPT交互或12,13，这表明肾上腺素受体 ​​介导的收缩，以苯为α1肾上腺素能受体。 几个步骤是这些实验的成功是至关重要的。解剖后的组织必须保持在PSS，以防止组织的生存力的丧失。任何肌肉等长制剂，产生最大的力量对被动紧张14,15的最佳长度张力关系。在这个协议中所描述的实验中，最佳的被动张力是得到0.5克被动张力到组织的简要添加增量预先确定的。组织应该开始无音，然后平衡30分钟后，将组织用KCl（80毫米）的最大浓度，其产生的活动音调的挑战。然后，将组织洗涤并允许返回到基线音。 15分钟后，在基线，另一个0.5克被动张力置于与该过程重复，直到一个高原活性张力用另外的被动张力实现。在初步实验中，将4g总被动张力的测定，以达到最大活性的张力产生在主动脉环，因而张力这个总量前平衡放置在环。在程序上，这是最好的零之前的被动张力的应用，但可以在之前的实验中的任何时间进行。过度伸展的组织，在实验或夹层的任一点，将带负组织的生存力和实验的结果产生影响。放置组织在洗澡时，因为此步骤具有过度伸展的最高似然，这是最必要的。充分洗涤，在数量和持续时间，需要为可重现的效果。第二个挑战太快经过最初的挑战，日前组织已经回到基线紧张，会造成异常反应。如果就读可逆拮抗剂/抑制剂，组织不应该事先洗涤以激动剂另外，作为INHIBI器的浓度将降低。 有显着的优点和缺点，以分离的组织浴测定。 缺点:组织可能在钩上手术切除环或安置体验不同程度的损伤。由于内皮细胞是主动脉环的内衬，必须小心在放置在环上的挂钩，以便不破坏该细胞层。组织也可能有时间明显不同的长度，其中它们是可行的，在组织浴，而这必须被确定;不是所有的组织是相同的。沿着这些线路，组织可在全天的反应改变这样的时间控制成为了必要的控制每一个实验。这方面的一个很好的例子就是豚鼠气管，在一个典型的8小时的实验提高了收缩最大100％。药物是难溶于水的可沉淀出来的PSS。最后，一​​个累计第二药物是一种激动剂，可能会导致不同的结果，如果受体脱敏的激动剂发生的非累积加法;血管紧张素II是一种这样的药物，其示出快速快速耐受。 优点:其中的组织浴实验的主要优点是，它是实时;人们可以看到实验，因为它展现，并且可以迅速地作出结论，并在实验过程中规划后续步骤，以及故障排除。实验需要每天做。多个组织通常可以从一个动物制备，使得动物可以作为其自身的对照，而增加了强度以一个实验。人们也可以隔离所述组织从其他因素，以便测试该组织对药物的相对较纯的响应， 在体外实验中，如组织浴系统还允许使用相比， 在体内实验中少量的药物。 本文所描述的基本实验设计可以广泛修改，以允许更多的参数记录或引入其他外界刺激。例如，另外的电极允许支配神经16,17的电场刺激。通过加入热或pH探针，温度和pH值对收缩反应的影响也可以测量18,19。同样地，氧可以部分或全部用N 2以评估缺氧诱导的作用取代。此外，可以使用等距收缩的测量在此视频中使用的相同的基本原理来开发系统，允许等长张力的发展和变化的伴测量细胞内钙20。信号转导也容易了研究，由于系统存在的响应时能迅速冷冻组织样本，使得一个信号转导途径体系的活性可以生物化学进行验证。 EQU的变化ipment可以用来做，这是巨大的。这整个系统，建造或自动左右手，可以从多个不同的公司购买。在这个协议中使用的组织浴和组织持有人手工吹制（组织浴）和手工建造（持有人）通过一个内部的密歇根州立大学机械车间。

药理学学科采用了分离组织浴系统超过150年。该系统的多功能性使得科学家在世界各地以表征受体和受体信号转导，这方面的知识形成已处理的数以百万计的疾病或病症，例如高血压，心脏衰竭，糖尿病，胃肠道疾病，膀胱的个体的疗法的基础功能障碍，哮喘和吞咽障碍，仅举几例。为了这一天，分离的组织浴保持药物开发和基础研究的一个重要方面，因为它允许所述组织以用作组织。在此朱庇特的课，正式的协议共享演示视觉和虚拟实验，从一个孤立的组织浴实验，测量等长收缩，这使得受体的表征利用数据。 该技术的主要优点在于，所述组织是过着第二功能为一体的组织，用生理结果（收缩或松弛）是相关于身体。它是一种合成的步骤（药物 - 受体相互作用，信号传导，第二信使的产生，改变平滑肌兴奋性，并改变组织功能）。而其它技术允许每个步骤的研究（ 例如，放射性配体结合药物的亲和力，第二信使测定）中，分离的组织浴技术允许集成的所有这些步骤1。另一个优点是，保持组织功能允许的重要的药理学变量是在一个组织中的VS的蜂窝环境更有意义的计算;它更接近如何检查药品将在工体作为一个整体。

<table><tbody><tr><td>LabChart Software</td><td>ADInstruments</td><td></td><td>7.2</td></tr><tr><td>PowerLab (4 channel)&amp;nbsp;</td><td>ADInstruments</td><td>ML760</td><td></td></tr><tr><td>QuadBridge (4 channel)</td><td>ADInstruments</td><td>ML112</td><td>ML112</td></tr><tr><td>Grass Adapter Cable</td><td>ADInstruments</td><td>MLAC11</td><td>MLAC11</td></tr><tr><td>Grass Force-displacement Transducer</td><td>Grass Instrument Co</td><td>FT03</td><td>FT03</td></tr><tr><td>Grass Transducer Cable</td><td>Grass Instrument Co</td><td>TAC-7 REV-1</td><td></td></tr><tr><td>BNC to BNC Cable</td><td>ADInstruments</td><td>MLAC01</td><td></td></tr><tr><td>IsoTemp 2100</td><td>Fisher Scientific</td><td></td><td>IC-2100</td></tr><tr><td>Tissue Bath</td><td>Multiple Sources&amp;nbsp;</td><td></td><td></td></tr><tr><td>Physiological Salt Solution</td><td></td><td></td><td>PSS</td></tr><tr><td>Braided Silk Suture</td><td>Harvard Apparatus</td><td>51-7615</td><td>SP104</td></tr><tr><td>Ring Stand</td><td>Humboldt MFG Co</td><td>H-2122 7</td><td></td></tr><tr><td>Dissecting Dishes</td><td>Handmade with Silicone</td><td></td><td></td></tr><tr><td>Tygon Tubing</td><td>VWR Scientific</td><td>63010-100</td><td>R-3603</td></tr><tr><td>Hose Clamps</td><td>Cole-Parmer Instrument Co</td><td>06832-08</td><td>SNP-8</td></tr><tr><td>50 ml Muscle Bath</td><td>Eberhartglass Blowing</td><td>Custom</td><td></td></tr><tr><td>250 ml Warming Chambers</td><td>Eberhartglass Blowing</td><td>Custom</td><td></td></tr><tr><td>Gas Dispersion Tube</td><td>Ace Glass</td><td>7202-06</td><td>7202-02</td></tr><tr><td>Micrometer</td><td></td><td></td><td></td></tr><tr><td>Custom Stands</td><td></td><td></td><td></td></tr><tr><td>Three-prong Clamps</td><td>VWR International</td><td></td><td>Talon</td></tr><tr><td>S-connector</td><td>VWR International</td><td></td><td>Talon</td></tr><tr><td>Tissue Hooks</td><td>Hand Made in House</td><td>Custom</td><td></td></tr><tr><td>Tissue Dissection</td><td></td><td></td><td></td></tr><tr><td>Leica Stereomicroscope MZ6</td><td>Leica</td><td></td><td>10447254</td></tr><tr><td>Stereomaster Microscope Fiber-optic Light Source</td><td>Fisher Scientific</td><td>12562-36</td><td>12562-36</td></tr><tr><td>Culture Petri Dish</td><td>Pyrex</td><td></td><td>7740 Glass</td></tr><tr><td>Sylgard Silicone Elastomer</td><td>Dow Corning</td><td></td><td>Sylgard 170 Kit</td></tr><tr><td>Vannas Scissors</td><td>George Tiemann &amp;amp; Co</td><td>160-150</td><td>160-150</td></tr><tr><td>Splinter &amp;amp; Fixation Forceps</td><td>George Tiemann &amp;amp; Co</td><td>160-55</td><td>160-55</td></tr></tbody></table>

measurement of smooth muscle function in the isolated tissue bath-applications to pharmacology research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

在激动而言，在给定组织的激动剂的相对效力（E MAX）和效力（EC 50）可被计算并与其它激动剂在相同组织1的响应。在我们的实验中，将大鼠胸主动脉孵育用载体或α1肾上腺素能受体拮抗剂哌唑嗪（5 nm）的一小时中添加α1肾上腺素能激动剂苯向组织浴，并产生收缩（ 图1）之前。 图2呈现改性图1的结果，绿色响应有显着的最大收缩由聚乙烯取得了明显的最大值，该半最大收缩标记为这样，然后与PE浓度相关联的原因造成的响应。确定这些值是识别实现了半最大（50％）的反应或EC激动剂的有效浓度<sUB&gt; 50值。这是donegraphically在使用可用于曲线EC 50值的拟合和计算逻辑函数为S形曲线的这个example.Computer软件。 在产生和解释这些结果，使用激动剂的低和高浓度来创建浓度 - 响应曲线是重要的。如果没有足够的积分以示稳定的基线和最高的高原，EC 50和E MAX只能估计。这是在本实施例以图形方式进行。使用逻辑函数为S形曲线的计算机软件，可用于曲线EC 50值的拟合和计算。 <img alt="图1" src="/files/ftp_upload/52324/52324fig1highres.jpg" /> 图1:组织浴示意图卡通代表组织的环放置在双壁，水夹套组织巴日。缓冲流入和流出由一个3路玻璃阀集成到室的底部调节。 的 O 2 / CO 2通过该密封用垫片，以防止PSS或气体的泄漏的曝气装置中鼓泡。再循环输入低于以保持适当的再循环和防止形成气穴会导致温度失调的输出。 <img alt="图2" src="/files/ftp_upload/52324/52324fig2highres.jpg" /> 图2:大鼠胸主动脉内皮+上图描绘了四个独立的实验（不同的动物并通过在由不同的颜色代表不同的一组窗口不同研究者进行），以测试哌唑嗪的转移在PE诱导的收缩的能力。哌唑嗪（5纳米）清楚地错开的PE诱导的收缩曲线向右以并行方式。 图3:大鼠胸主动脉内皮+ EC 50值的PE图形估计在不存在（载体）和拮抗剂的存在下（哌唑嗪）。绿线（C组）只被标记。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> 盐 </td><td> MW（克/摩尔） </td><td> 5升 </td><td> FINAL毫米 </td></tr><tr><td></td><td></td><td></td><td> （克） </td></tr><tr><td>氯化钠</td><td> 58.45 </td><td> 37.99 </td><td> 130 </td></tr><tr><td>氯化钾</td><td> 74.56 </td><td> 1.75 </td><td> 4.7 </td></tr><tr><td> KH2PO4 </td><td> 136.1 </td><td> 0.8 </td><td> 1.18 </td></tr><tr><td>硫酸镁•7H 2 O </td><td> 246.5 </td><td> 1.45 </td><td> 1.17 </td></tr><tr><td>碳酸氢钠</td><td> 84.21 </td><td> 6.25 </td><td> 14.9 </td></tr><tr><td>葡萄糖</td><td> 180.16 </td><td>五</td><td> 5.5 </td></tr><tr><td> EDTA </td><td> 380 </td><td> 0.05 </td><td> 0.03 </td></tr></tbody></table> 表1:正常生理盐溶液（PSS）的配方在本实验中所用的碳酸氢盐缓冲的PSS的含量。包括的是所有的盐的分子量，并加入到5升的dh 2 O，以获得所需浓度的量。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 在浴浓度 </td><td> 除了 ​​激动剂洗澡做 </td></tr><tr><td> 1×10 -9 M的 </td><td> 50毫升1×10 -6 M的 </td></tr><tr><td> 3×10 -9 M的 </td><td>百毫升1×10-6 M </td></tr><tr><td> 1×10 -8 M的 </td><td> 35毫升1×10 -5 M </td></tr><tr><td> 3×10 -8 M的 </td><td>百毫升1×10 -5 </td></tr><tr><td> 1×10 -7 M的 </td><td> 35毫升1×10 -4 M </td></tr><tr><td> 3×10 -7 M的 </td><td>百毫升1×10 -4 M </td></tr><tr><td> 1×10 -6 M的 </td><td> 35毫升1×10 -3 M </td></tr><tr><td> 3×10 -6 M的 </td><td>百毫升1×10 -3 M </td></tr><tr><td> 1×10 -5 M </td><td> 35毫升1×10 -2 M </td></tr><tr><td> 3×10 -5 M </td><td>百毫升1×10 -2 M </td></tr><tr><td> 1×10 -4 M </td><td> 35毫升1×10 -1 M </td></tr></tbody></table> 表2:组织浴添加剂激动剂浓度要加入激动剂的量，以每一个浴中以亲perly构造累积浓度 - 响应曲线。所需的浴浓度是通过加入增加激动剂的浓度的连续量来实现的。每次加入考虑激动剂已经存在于浴中的浓度。这样做是为了最大限度地减少在组织浴中，会导致使用激动剂的单一浓度的体积增加的增加量。

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Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

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138.1K 次观看.  Michigan State University. This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition. 

视频: Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

肠系膜动脉收缩和舒张的研究，使用自动线Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

科研

医学

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

膀胱平滑肌收缩带作为一种方法来评估下尿路药理学

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

隔离和使用的牛肠系膜动脉和静脉部分，作为生物测定在小肠测试的Vasoactivity

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

人子宫平滑肌的收缩力测量对药物开发的帮助

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

生物学

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

以血管内炎组织调节为重点, 利用肠系膜孤立动脉评价血管张力

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

<meta charset="utf8"></head><body>研究胃肠道动力学和药物对运动影响的方案

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug digestion and absorption and plays a crucial role in treating these diseases. The intestinal tube movement experiment, a common and essential in vitro method, is utilized to study gastrointestinal dynamics. This includes the preparation of the isolated intestinal tube, as well as the suspension of the prepared intestinal tube in the bath and its connection to a signal detector. This is followed by the recording and analysis of a series of parameters, such as tension, which can be used to assess intestinal motor function, as well as considerations for keeping the intestinal tube active in vitro. The standardized program from sampling to data collection greatly improves the repeatability of the experimental data and ensures the authenticity of the recording of intestinal tension after physiological, pathological, and drug intervention. Here we present the key problems in experimental operation and a valuable reference experimental protocol for studying drugs that regulate gastrointestinal motility.

<meta charset="utf8"></head><body>作者聚焦:药物对大鼠肠管影响的可靠且可重复的体外评估

We present a method for isolating rat intestinal tubes and assessing the impact of drugs on their tension, frequency, and amplitude in vitro. This method offers a valuable approach for researchers investigating intestinal tubes.