威康信托基金会提供财政支持（贵宾奖JSY和074128研究奖学金KLB）。医学研究理事会助学金，以RJA Leica_SP2_Stacker，用于进口“重建”成系列的TIFF的算法，是基于Leica_tiff_sequence，读他的许可徕卡SP2的TIFF格式，由托尼柯林斯开发和使用在ImageJ插件。 （ <a href="http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html">http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html</a> ）StackReg和TurboReg，用正确的组织运动的算法，是基于钍venaz和他的同事（1998）9 。 Excel_writer.jar写由Kurt德沃斯（http://rsb.info.nih.gov/ij/plugins/excel-writer.html）。 JNCT0.08JoVE.ijm，粒子探测算法，是基于书面吉隆坡脑，以前5详细的算法。

第1部分：制备的Ca 2 +指示剂 （俄勒冈州绿488 BAPTA - 1上午） <ol><li>俄勒冈州绿488 BAPTA - 1上午有少量粉（50微克）500μL小瓶。添加40μL聚醚F - 127解决方案的粉末，轻轻摇动30秒，然后加入460μLPSS的，并轻轻摇晃30秒最终的解决方案，10微米俄勒冈绿488 BAPTA - 1上午，提供了4 × 125μL分装。避免暴露于光线和储存在-20℃ </li></ol>第2部分： 钙离子加载<ol><li>取出的Ca 2 +（-20℃）冷冻指标（俄勒冈州绿488 BAPTA - 1上午），并允许在室温下解冻。 </li><li>取1毫升PSS在试管中，在水浴地方。与95％的O 2 / 5在泡沫出现每秒约1率2％的二氧化碳气泡。起泡需要将试管与塞或封口膜，例如担保。 </li><li>从动物解剖组织，在Sylgard林立的培养皿中，小心地取出结缔组织。无论平滑肌器官类型，PSS的应更换一次10分钟和组织应清洗（例如，通过更换PSS三次）以下清扫。输精管：考虑器官切割长度的方法来生产的组织表。膀胱：膀胱颈（后）纵向打开圆顶的顶部（前）。准备4条，6 - 8毫米长和1 - 2毫米宽，沿背水面的逼尿肌，切割中的主导肌束方向。 </li><li>解剖中的“预热和冒泡”的PSS的组织。 </li><li>加入125μL10μmol俄勒冈绿488 BAPTA - AM试管，并给予一点点的动摇，用手混合。 </li><li>继续冒泡离开，用铝箔纸覆盖。时间是需要足够的组织采取的Ca 2 +指示剂组织和平滑肌层厚度的大小而异。例如：70分钟“（小鼠逼尿肌，大鼠anococcygeus）为120分钟（大鼠逼尿肌，小鼠输精管）。 </li></ol>第3部分：准备显微镜“的Ca 2 +装”组织仔细组织穿针，以尽量减少组织的自发运动（一个相对完整的组织的平滑肌细胞成像的财产）和一个合适的地点，以方便成像的位置（如平片的组织可能是必要的调查在两个层面，而不是三个，）。 <ol><li>冒泡PSS的组织冲洗至少5分钟。 </li><li>安装在Sylgard列队准备菜，拉伸组织（略），形成一个平板。避免使用长的“管脚”可能划伤的目标;使用直径为25-50微米的银线，而不是短期的长度为“管脚”，并插入一个角度。 </li><li>显微镜舞台上的位置准备的菜，要小心，以确保PSS的不逃避的菜Sylgard林立的区域（因为这可能会导致大面积覆盖在PSS灌流开始时）。 </li><li>为了泵切换到superfuse PSS的准备。每小时100毫升的速度往往是就业。 </li><li>暖风机上的开关。 </li><li>广场上的准备菜（贴上磁铁底座上的每个钢带）的流入和流出管和温度探头。流出管尖的高度，将确定的PSS的深度。要小心，以确保流出实际上是消除PSS的（因为它有时并不最初）。 </li><li>设置的暖风机上的温度达到所需的温度，如33-35 C. </li><li>允许组织以平衡至少10分钟。 </li></ol>第4部分：图像选址曝光激发照明光漂白的指标，这样可以避免使用超过几秒钟的时间。 <ol><li>随着低功耗目标（10X或20X）使用蓝色光epifluorescence的，令人振奋，以查看平滑肌，并确定一个合适的区域监测。尽量挑一个网站的基础上：运动（这应该是最小的）和平滑肌细胞的数量在该领域。明亮的“结构”，可能会导致大量的“误报”图像检测算法，这样就可以避免（如）穿插上皮细胞或成纤维细胞中有大量的网站。 </li><li>切换到一个更高的功率目标（40X）。 </li><li>旋转舞台或光轴，使平滑肌细胞（S）位于水平（即快速扫描轴平行）。 </li></ol>第5部分：共聚焦显微镜共聚焦显微镜的细节是“成立”是具体到每个显微镜类型，但关键因素是：速度（最低为5 Hz大部分的Ca 2 +瞬变的要求）;分辨率和大小的领域（这两项交易，与速度） 。以下协议的一些具体徕卡SP2直立共聚焦显微镜。一个典型的协议是：100架系列，在5 Hz（256 × 256像素;约100 x 100微米。）收购或13.5赫兹（256 x 64像素，约100 × 25微米。）。扫描头设置双向移动（最大限度地采集速度），并获得在每行的1000赫兹。 <ol><li>关闭针孔一个“通风磁盘”。 </li><li>设置的AOTF的25至35％（488纳米）的激光功率，这个值是亮度和漂白之间的权衡。 （后者是在长时间录音的一个特别的问题。） </li><li>采集每个站点的六个系列，及四 - &#39;待遇&#39;，每六个网站。它的共同目标显示器相同的六个地点前的药物（即“控制”）和药后，虽然显着的漂白，可能会导致实验者偏离这个。 </li><li>执行，“时间控制”，它是必不可少与 Ca 2 +成像的。 </li></ol>第6部分：分析的准备工作<ol><li>下载并安装图像j从：http://rsb.info.nih.gov/ij/download.html。下载特定于平台的版本，然后从http://rsb.info.nih.gov/ij/upgrade/ij.jar下载最新的imageJ.jar文件，替换旧的文件更新到最新版本。苹果Mac用户：如果它的运行速度很慢，确保您使用最新版本的Java虚拟机（JVM）可从苹果（http://www.apple.com/macosx/features/java/）。 </li><li>下载Excel_writer.jar：http://rsb.info.nih.gov/ij/plugins/excel-writer.html和插件安装到[目录]&gt; jar目录。 </li><li>复制的Plugins目录中的下列文件：Leica_SP2_Stacker.class; JNCT0.08Release.ijm。这些脚本安装使用插件[菜单]&gt;宏&gt;安装... ImageJ功能。 </li><li>我们使用可视作为一个单一的&#39;电影&#39;在软件生成的每一个系列的Leica SP2激光共聚焦显微镜，该软件（莱卡，LCS），但保存为单独的帧。要改造成系列的帧：（一）确保所有的“框架”，以重建在一个文件夹（如“DataFolder&gt; TIFF格式”）。 （ii）选择插件[菜单]&gt;宏&gt; Leica_SP2_Stacker。选择根目录（例如，“DataFolder”）。从下拉列表（例如&#39;TIFF格式/&#39;）选择所需的文件夹。选择输出目录或生成一个新的（如“DataFolder&gt;书库”）。然后，该脚本将重建一系列单个帧。 </li></ol>第7部分：运动更正虽然运动的影像网站可能具有良好的钉最小化和均匀拉伸的组织，一些平滑肌机关表现出自发性收缩，不能小心寄托单独取消。在这里，我们描述了使用免费提供的算法，该算法在一系列的帧对齐或匹配。 <ol><li>下载“StackReg”（http://bigwww.epfl.ch/thevenaz/stackreg/）和“TurboReg”（http://bigwww.epfl.ch/thevenaz/turboreg/）。 </li><li>安装到每个插件使用的插件目录&gt;宏&gt;安装... </li><li>加载要处理的堆栈（文件&gt;打开... ...），然后转移到一个框架，清楚地显示感兴趣的领域。其他帧将对准这个参照系。 </li><li>插件&gt;书库&gt; StackReg。尝试每个不同的“转变”（即翻译，刚体，缩放旋转，仿射），以确定哪些是最有效的的。 （进一步的细节：http://bigwww.epfl.ch/thevenaz/stackreg/） </li></ol>第8部分：粒子探测算法成像对于每一个“地盘”，这些因素会有所不同，影响的Ca 2 +瞬变检测。通过测量每个站点的某些属性（“粒子参数” - 详列如下），在检测过程中提供便利。因此，为每个站点，减去一个计算“门槛”，“阈值的比例”和“细胞边缘门槛”。 <ol><li>插件[菜单]&gt;宏&gt;安装，并选择“JNCT0.08Release.ijm”。 </li><li>插件[菜单]&gt;宏&gt;装入堆栈（快捷键：L）。选择一个系列，并计算出粒子参数： <ol style="list-style:none;"><li> A.阈值减去（背景） </li></ol></li><li>您认为是什么“背景”，选择一个长方形的投资回报率。 </li><li>测量（快捷键：M）。记下的意思。 <ol style="list-style:none;"><li> B.为比例计算的门槛</li></ol></li><li>选择一个矩形细胞（S）的光学平面面积的投资回报率大约。 </li><li>测量（快捷键：M）。注意上下的均值和标准差。 </li><li>重复再两次或一次，可以得出按“转向”三盒。 </li><li>计算并记下为比例阈值=（1 + SD /平均值）× 32，平均每三个值复制/网站。 <ol style="list-style:none"><li> C.小区边缘的门槛</li></ol></li><li>图像&gt;书库&gt; Z项目...选择投影类型：“平均强度” </li><li>图像&gt;调整&gt;阈值。 </li><li>选择黑色和白色。 </li><li>定义“下的”（上滑块）限制，并记下相应的值（滑块权）。只有在黑色区域发生的事件将被计算在内。 </li><li>点击“重置”。 </li><li>分析栈（快捷键：2） </li><li>在此阶段只选择一个系列，即“第一个文件”和“最后文件”应该是相同的。 </li><li>输入值的“门槛减去”和“细胞边缘阈值”，“最低比例”。保留其他设置为默认值。 </li><li>该算法会产生，其中加工系列是显示在左侧和右侧的原的双窗格窗口。经过仔细评估的事件，用肉眼可见，有没有被发现的误报和场合。为了得到更准确的检测钙瞬变可能需要稍微调整的“粒子参数” 。虽然过程可能会显得有点“碰运气”在第一次尝试，很快就会以什么参数是关键的感觉。 </li><li>对于每一个系列，该算法产生一个Excel文件，检测到的事件的细节特征（如幅度，面积和位置）。 “误报” - 可能会被删除，由专人从这个Excel文件，审查的图像算法的输出文件（如步骤8.17） - 确定。 </li></ol>

<ol>
	<li>Haugland, R. P., Gregory, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indicators+for+Ca2+,+Mg2+,+Zn2++and+other+metal+ions.">Indicators for Ca2+, Mg2+, Zn2+ and other metal ions.</a> Handbook of fluorescent probes and research products. , 771-826 (2002).</li><li>Boland, B., Himpens, B., Gillis, J. M., Casteels, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+force+and+Ca2++in+anococcygeal+and+vas+deferens+smooth+muscle+cells+of+the+mouse.">Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse.</a> Pflugers Arch. 421, 43-51 (1992).</li><li>Hashitani, H., Fukuta, H., Takano, H., Klemm, M. F., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+propagation+of+spontaneous+excitation+in+smooth+muscle+of+the+guinea-pig+urinary+bladder.">Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder.</a> J Physiol. 150, 273-286 (2001).</li><li>Heppner, T. J., Bonev, A. D., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elementary+purinergic+Ca2++transients+evoked+by+nerve+stimulation+in+rat+urinary+bladder+smooth+muscle.">Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle.</a> J. Physiol. 564, 201-212 (2005).</li><li>Brain, K. L., Jackson, V. M., Trout, S. J., Cunnane, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+ATP+release+from+nerve+terminals+elicits+focal+smooth+muscle+Ca2++transients+in+mouse+vas+deferens.">Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2+ transients in mouse vas deferens.</a> J. Physiol. 541, 849-862 (2002).</li><li>Fleichman, M., Schneider, T., Fetscher, C., Michel, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+underlying+carbachol-induced+contraction+of+rat+urinary+bladder+II.+Protein+kinases.">Signal transduction underlying carbachol-induced contraction of rat urinary bladder II. Protein kinases.</a> J. Pharmacol. Exp. Ther. 308, 54-58 (2004).</li><li>Young, J. S., Meng, E., Cunnane, T. C., Brain, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+purinergic+neurotransmission+in+the+mouse+urinary+bladder.">Spontaneous purinergic neurotransmission in the mouse urinary bladder.</a> J. Physiol. 586, 5743-5755 (2008).</li><li>Nelson, M. T., Cheng, H., Rubart, M., Santana, L. F., Bonev, A. D., Knot, H. J., Lederer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxation+of+arterial+smooth+muscle+by+calcium+sparks.">Relaxation of arterial smooth muscle by calcium sparks.</a> Science. 270, 633-637 (1995).</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans. Image Process. 7, 27-41 (1998).</li></ol>

作出了努力，以尽量减少使用动物的数量和他们的苦难，所有实验均按照英国动物（科学规程）行动1986年，欧洲共同体理事会指令86/09/EEC。

荧光灯的Ca 2 +指示剂的选择俄勒冈州的绿488 BAPTA凌晨1点被选择的Ca 2 +指示剂 ，因为它（我）是光明的，相比其他指标（如氟- 4和钙绿 ）1，（ii）是敏感的生理变化， 在Ca 2 +浓度一个类似规模的1细胞内的Ca 2 +浓度的[Ca 2 +] I（VAS如输精管2）K D;（三）加载许多平滑肌细胞，使平滑肌细胞的耦合进行监测。不过，俄勒冈州绿488 BAPTA - 1上午是一个非ratioable的Ca 2 +指示剂 ，因此不能轻易地被用来确定绝对的[Ca 2 +] i的。此外，它也可能缓冲的Ca 2 +如果存在高浓度，并成为高振幅变化的[Ca 2 +] i的饱和。 自发性收缩的抑制作用自发收缩平滑肌机关可能减少，如药理，通过阻断运动的Ca 2 +通过L型Ca 2 +通道（例如通过：硝苯地平，地尔硫4）。请注意，这些药物将大大降低全细胞的Ca 2 +闪烁/瞬变的幅度。 输精管收缩输精管主要是嘌呤和去甲肾上腺素能神经递质结合的结果。然而，焦距的Ca 2 +在这个组织中的瞬变，去甲肾上腺素受体 ​​阻断剂（如α1受体，哌唑嗪 5），这样的运动可减少不影响焦距的Ca 2 +瞬变不敏感。 另外，可能被阻止收缩，“下游”，通过抑制肌球蛋白轻链激酶如渥曼青霉素6。 结论在亚微米分辨率的Ca 2 +荧光监测，是一个功能强大的技术来研究神经递质的释放5,7后交界的影响和释放的Ca 2 +从内部商店（如“ 火花 ”8 ）。分析的Ca 2 +瞬变的使用方法这里列出的成本效益比市售的图像分析软件包，可以通过自定义编写的Java插件ImageJ度身订造的分析。

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Oregon Green 488 BAPTA-1 AM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>O6807</td>
<td>10 x 50 &mu;g</td>
</tr>
<tr>
<td>Pluronic F-127 solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P3000MP</td>
<td>20% solution in DMSO</td>
</tr>
<tr>
<td>Leica SP2 upright confocal microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Air table (&#x2018;vibration isolated workstation&#x2019;)</td>
<td>&nbsp;</td>
<td>Newport</td>
<td>M-VW-3046-OPT-012140</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 elastomer</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
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		</div>

focal ca2+ transient detection in smooth muscle

的Ca 2 +平滑肌的成像提供进入细胞的机制，可能无法导致膜电位的变化，如从内部存储的Ca 2 +释放，的洞察力，并允许同时监控多个单元格，例如，评估，耦合合胞体组织。亚细胞的Ca 2 +瞬变共同平滑肌，但因为他们随机发生的看法往往是广泛的领域，造成的问题，器官，难以准确衡量，它容易产生合同。为了克服这个问题，我们已经开发了一系列的成像协议和分析程序的收购，然后分析，以自动方式，频率，此类事件的位置和幅度。虽然这种方法可应用在其他情况下，我们自己的工作，涉及当地嘌呤钙的检测，定位与亚微米分辨率的递质释放的瞬变。 从cotransmitter植物神经，它结合P2X1受体对逼尿肌和输精管的平滑肌ATP释放。 钙离子进入平滑肌，造成嘌呤neuroeffector的Ca 2 +瞬变（NCTS）。协调中心的Ca 2 +瞬变允许的方式，对电的诸多优势中的神经递质释放的光学监测。除了大大提高了空间分辨率，光学记录允许从许多同时分辨网站的记录递质释放的额外优势。此外，光学平面的重点是更容易保持正确或在长时间录音系列比细胞内的尖锐的微电极的重新定位。 总之，概述的Ca 2 +荧光成像的方法，详细介绍了组织准备，数据的采集和分析。我们概括了几个剧本， 如钙瞬变分析使用。

Watch this Scientific Journal Video about Focal Ca2+ Transient Detection in Smooth Muscle at JoVE.com

Focal Ca2+ Transient Detection in Smooth Muscle

高分辨率CA的详细信息的方法<sup&gt; 2 +</sup&gt;成像在孤立的器官，包括平滑肌组织，图像采集和数据分析的准备。

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

focal-ca2-transient-detection-in-smooth-muscle

Research

JoVE Journal

Biology

Focal Ca2+ Transient Detection in Smooth Muscle

11.2K 次观看.  University of Oxford. 高分辨率CA的详细信息的方法 2 +成像在孤立的器官，包括平滑肌组织，图像采集和数据分析的准备。

视频: Focal Ca2+ Transient Detection in Smooth Muscle

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

人脐动脉平滑肌细胞的分离（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

科研

生物学

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

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 ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

肺动脉平滑肌细胞从新生小鼠隔离

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

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.

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.  ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

小鼠主动脉夹层损伤:高效<em&gt;体内</em&gt;平滑肌细胞增殖和内皮功能模型

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...