我们感谢威廉Cashdollar博士和西北互助基金会影像中心在威斯康星血液研究所的帮助，成像研究。我们也感谢达里娅Ilatovskaya博士这个手稿和有益的讨论，批判性阅读。这项研究是由卫生赠款HL108880国家研究院​​（以AS）和DP2-OD008396（以AMG）的支持。

下面描述的实验程序批准的机构动物护理和使用委员会（IACUC）在威斯康星医学院并且是按照卫生指南实验动物的护理和使用的国家机构。 1.隔离的大鼠主动脉<ol><li>麻醉大鼠用异氟烷（5％诱导，1.5至2.5％维护）或另一IACUC批准的方法。 </li><li>将鼠在仰卧位置。以暴露腹腔器官，使透过皮肤和皮下腹部组织的小腹侧正中切口。使用纱布垫轻轻偏转肠内以暴露主动脉和腔静脉， 如图1。 </li><li>简言之，钝解剖从结缔组织，并从腔静脉主动脉。使用缝合，打结主动脉尽可能靠近肾脏。解剖约1 - 主动脉为2cm，并放置在一个15毫升的锥形涂是含有正常生理盐溶液（PSS;以mM:140氯化钠，1.2的MgCl 2，2的CaCl 2，4.5氯化钾，6葡萄糖，10 HEPES）在冰上。 </li><li>一旦主动脉分离，按照批准的IA​​CUC协议安乐死的动物。在完成所有的非存活的程序安乐死深麻醉动物通过开胸诱导气胸，以确保动物的人道消亡。3 </li><li>使用解剖显微镜，细尖镊子和microscissors解剖脂肪和结缔组织关闭主动脉。一旦主动脉是干净的，纵向切割主动脉，并将其放在PSS在冰上购买。 </li></ol> 2.染料加载和孵化<ol><li>吸管7微升1mM的的Fluo-4AM染料，它是在基于DMSO的溶液供给到已用铝箔覆盖个别500μl的试管中。存储在冷冻在-20℃以上的时间来保存染料性质。注意:这些分装等等决方案应当为至少六个月是稳​​定的。 </li><li>在使用前，使染料溶液温热至RT开口之前。在即将使用前准备工作的解决方案。不存储上述稀释试剂供以后使用。 </li><li>加入现成的1毫荧光 - 上午04时染料溶解在DMSO溶液，以不含钙450 UL PSS的7微升。加入40微升非基于DMSO 10％聚醚酸溶液的样混合物，以帮助分散乙酰氧基甲基（AM）酯和改善负载钙染料。 </li><li>将解剖主动脉到含有上样缓冲液（如在2.3中描述）1小时的500微升试管中。盖上箔和地点在旋转振动筛在室温下所有管。 </li><li>要监视在血管NO生产，使用DAF-FM双乙酸钠染料。孵育在含有450微升的PSS，40微升聚醚酸和5微升10毫DAF-FM二乙酸酯的溶液主动脉。类似于钙孵育协议（在2.3中描述），将主动脉在含有覆盖在箔和地点在一个旋转摇床上30分钟，在4℃，接着通过下一个在室温30分钟的NO-染料溶液的500微升管。 </li><li>当NO产生被测量，使用内皮NO合成酶阻断剂，L-NAME，阻止NO产生作为对照， 如图4B所示 。该过程中，添加到混合物（如在2.5中描述）为100nM L-NAME对孵化的后30分钟（在室温下）。 </li></ol> 3.激光扫描双光子显微镜协议<ol><li>在1小时孵育后，洗主动脉2 - 3次清洁PSS去除细胞外染料。 </li><li>主动脉转移到含有或者是正常的PSS或无Ca 2+缓冲液（取决于实验方案）的硅氧烷-涂布的菜。在与硅氧烷接触外膜向下到硅酮涂层针主动脉（ 即，暴露于盘开口内皮管腔），使用μ形销。或者，使用切片锚网格或胶水来固定组织。 注:为了减轻压力和可能的机械文物，传输和钙修复主动脉2+的解决方案，并等待5 - 10分钟开始实验前。 </li><li>连接蠕动泵或注射器的硅氧烷涂覆的板的药物自动或手动的应用程序或用于改变细胞外溶液。 </li><li>将鼻一块直立双光子显微镜下的菜，以及安装和放置一个25×（NA 1.05和工作距离2mm）的水浸泡物镜标本上面。注意:如果该特定透镜不可用，使用专门为双光子成像。制造目标，因为它可以增加信号分辨率大大具有规则目标相比较。 </li><li>激活使用软件的双光子激光（激光开始泵和打开）。调谐激光器激发到820纳米。 </li><li>使用萤光，找到主动脉和重点目标的内皮表面用粗调。 </li><li>切换显微镜成两个光子激光扫描模式，确保了激励激光器模式锁定，非退扫描探测器啮合并适合于预期的发射光谱的发射过滤器安装。注:在这里，FV10-MRL / R（495至540纳米）的使用。 </li><li>开始实时扫描用约5％的激光功率和精细聚焦的目的，以可视化的内皮细胞。 注:当正常的PSS培养内皮细胞会表现出强烈的荧光信号。这将明显较弱时血管孵育在无钙缓冲液中。内皮细胞将存在于打开主动脉的顶部和平滑肌细胞中可以看出只是内皮细胞（参见图2）所示。因为血管既不光滑也不均匀，将有其中两个平滑肌细胞和内皮细胞存在的地方。&lt;/ LI&gt; <li>一旦各信元在焦点，程序显微镜软件采集顺序图像在快速扫描模式（光栅扫描，512×512像素的窗口的1.1秒的帧集合频率）。在与荧光 - 凌晨4点荧光平行，收集为了检测变化血管收缩和更好的可视化实验条件透射光图像。 </li><li>收集基线信号的图像后，改变的Ca 2+离子浓度在外部溶液或慢慢使用蠕动泵同时成像应用药理刺激剂。观察装载在细胞内的荧光信号的增加。 注意:内皮细胞的变化的流动反应，因此，建议应用的Ca 2+或NO信号传导的药理活性剂的前使用低层的应用程序和车辆测试。要重新计算细胞内钙浓度的纳米值装有荧光 - 4（解离常数荧光 - 4艘我s 345纳米），荧光强度可能被记录在基线和另外霉素和氯化锰2后4 </li></ol> 4.图像处理和计算<ol><li>下载并安装斐济图像处理包。 </li><li>打开斐济的图像文件。一旦导入文件，出现提示时拆分通道（透射光和荧光信号）。仅使用荧光信号进行数据分析。 </li><li>在斐济程序，请单击分析选项卡，向下滚动到工具选项。在工具选项中，找到ROI说经理选项卡并单击就可以了;会出现一个新的窗口。 </li><li>在此之后，点击设置测量分析选项卡下。选择感兴趣的特定测量。对于钙瞬变测量，用平均灰度值的选项。 </li><li>与圆形轨迹的工具，开始跟踪感兴趣区域（ROI）的区域，然后单击"添加"的投资回报率经理屏幕上每一个řOI。一旦所有感兴趣的细胞已被跟踪，在投资回报率管理器窗​​口，在"更多"选项卡下选择多措施。请记住，无论是直接保存测量或复制和粘贴这些测量到电子表格中。 </li><li>打开*从斐济.txt文件或Excel电子表格和数字转移到新的工作表起源。注:另外，使用类似适马剧情或棱镜进行进一步分析任何程序。 </li><li>在起源的程序，使用绘图→线+符号菜单遵守所有瞬态响应的概述。取消不响应的细胞。 </li><li>重新计算跟踪号码的时间尺度（X轴），使用列→设置列值，乘以跟踪数列的图像采集频率（在我们的例子1.1s）。 </li><li>使用行上的所有选定的记录分析→统计，计算平均值（Y轴）和标准错误跟踪。最终绘制的图形单元的电流组绘制→线+符号。 </li><li>该MEAñ钙瞬变计算被描述为其次。 <ol><li>对于起源计划内总钙释放，点击图，然后点击菜单→分析→微积分整合。面积曲线下总积分出现在结果日志。 </li><li>对于瞬态动力学，点击图，然后使用菜单:分析→非线性曲线拟合→选择数据集（选择X轴范围从最大瞬态响应的结束）。开始配件→选择功能→指数衰减1→完成。 注:指数拟合出现在图形窗口和结果日志。获得的数据（值）表示的钙释放的总金额，并且通道的动力学介导的瞬态响应。 </li></ol></li></ol>

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实验概述。为了更好地理解钙的贡献和NO血管生理学，一种新颖的方法测定制定的[Ca 2+] i和编号为内平滑肌和离体完整主动脉内皮细胞。一起，这种协议包括以下关键步骤:1）机械分离和制备（未酶促消化）容器。重要的是要保持组织健康和完好尽可能获得最佳生理记录是重要的。 2）培育在钙 - 标记染料或NO标记染料与聚醚酸是至关重要的，但是，对于某些其它容器的类型，不同的聚醚酸和孵育时间可能需要进行测试。 3）正确的固定主动脉与硅氧烷涂层盘与电网和销将成像与双光子显微镜期间提供稳定的图。因为该容器将收缩和扩张，以确保该容器是prope是很重要RLY固定在盘子，以避免可能出现的运动伪影。 4）定量的结果。请注意，瞬时幅度可能不总是以比较细胞的生理特性的最佳参数。短暂的积分可能会表现出更好的区别，由于总的[Ca 2+] i或 NO释放。 当实验都正确执行，有可能监视的[Ca 2+] i的（或NO水平），为显示在图3和4和视频1和2。 离体制剂的组合响应于刺激的变化和使用双光子成像能够提供更好的信号分辨率和血管组织细胞内过程的精确测量。在这里，这些技术的组合应用了与经典的非比例的Ca 2+和NO指标（荧光-凌晨4点和DAF-FM双乙酸钠，分别），以测量钙瞬变和在一个孤立的整个主动脉的单个细胞NO的产生。我们相信，这一技术将有助于我们转发知识的机制调节的[Ca 2+] i和在不同的血管细胞类型NO水平。 修改和故障排除 。测量的Ca 2+信号在单个平滑肌细胞敏感离子载体的基本原理最初发表于1985年7大部分的最近的方法进行成像的Ca 2+水平的个体的血管细胞所采用的激光扫描共聚焦显微镜和分离的或培养的准备。对于像血管的动态组织中，这是难以测量钙或使用显微镜为基础的技术NO水平在体内 。2这里的主要限制是，几乎所有的血管上的循环的动脉侧具有内部弹性薄片那之间在于在endot氦和平滑肌细胞的最内层。8此外，大多数的平滑肌和外膜层之间的细胞外物质的是胶原蛋白。8弹性蛋白和胶原蛋白是自体荧光的具有高强度和大范围的激发波长的主要来源。强劲的背景荧光期间共焦成像再加上增加的光散射产生低信号的分辨率，因此，减少钙信号或NO生产检测。装载荧光探针成血管细胞以足够的浓度，以使有意义的测量的限制因素可以通过使用一个体外制剂，双光子显微镜，并应用新的类离子载体，如阿桑特红色，发出荧光在长的可以解决波长的光，其中，存在从自发荧光的结缔组织和纤维的干扰较少。然而，这种方法的主要限制之一是，它是昼夜温差ficult到成像期间将两个或更多的染料。的激发光谱的大部分为双光子成像的可用染料有宽，多形态分布特征，这使得它难以同时测量两个不同的离子载体。然而，低噪声的混合探测器可用于缩小检测的发射光谱假设离子载体发射不同波长的光。例如，该所述的过程中，不可能同时测量NO的产生和Ca 2+浓度的变化，因为发射光谱重叠。 血管内皮细胞，与血流直接接触的暴露于流体剪切应力和调节血管稳态9的电流的方法还可以用于监测流动引起的钙瞬变和在内皮细胞中离体产生NO。此外，该方法可成功地应用到学习的Ca 2+ concentra在从肾脏提取小阻力动脉的平滑肌细胞和灰的变化。在这种情况下，解剖小阻力动脉应的Ca孵育2+溶液，以避免平滑肌细胞损伤由于钙超载。此变型也可应用到主动脉，以减少细胞死亡。 观点 。基于荧光的成像已被广泛用来研究在体外细胞生理学;然而，分离的细胞模型不总是概括当细胞处于其天然环境中发生的生理过程。这已经细胞系的适用性限于转化研究。10此处所述，利用体外制剂给了我们机会，监测新鲜分离的组织细胞行为，所有地方的生理过程和互动与其他类型的细胞仍然存在。随着越来越多的遗传ENGIN的可用eering技术和其概括人类疾病模型的发展， 离体的制剂将是对于转化研究的有用工具。对于这里所描述的研究中，达尔盐敏感（SS / JrHsdMcwi）高血压大鼠品系中。该SS老鼠是盐敏感高血压的概括渐进人高血压的许多方面自然发生的近交遗传模型。被广泛使用这种模式，并提供关键洞察盐敏感性和血管功能障碍11-13背后有了这个大鼠模型的机制，研究人员使用的ZFN能够测试的关键机制，复杂疾病的贡献（如高血压） ，TALENS和CRISPR / CAS基于基因编辑技术。6,14-17使用这里描述连同这些基因编辑资源的实验程序，现在有可能开始测试钙和其它关键因素的盐的贡献敏感的高血压和在许多不同的动物模型中的血管功能障碍。

钙是血管细胞如内皮细胞和平滑肌细胞内的一个基本的第二信使。它是主要的刺激血管收缩和起主要作用于血管扩张，包括通过NO的生成的内皮细胞内的效果。由于成像技术的局限性，它已经几乎不可能完整的容器内观察钙处理。双光子成像系统和建立新的钙或NO标记染料的发展，使得能够对图像以足够的深度和分辨率开始了解钙动力学和脉管系统中NO的产生。 双光子显微镜最近在组织，器官，因为其卓越的能力，深入穿透组织与低背景荧光和高灵敏度的信号，甚至是整个动物研究中得到应用。1,2双光子激发的补偿发光的窄谱离子焦点和使用非退扫描探测器的原因双光子显微镜优于传统共聚焦显微镜。在必要的组织深度由于自发荧光和的离焦的光的散射进共焦针孔共焦显微镜不能产生高质量的图像。因此，我们开发了使用双光子显微镜来测量的[Ca 2+] i的信令和方法在完整的，独立的血管细胞具有高分辨率和低信噪比的NO产生。

<table><tbody><tr><td>DAF-FM</td><td>Life Technologies</td><td>D-23842</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Life Technologies</td><td>F14217</td><td>500 &amp;micro;l in DMSO</td></tr><tr><td>Pluronic F-68 solution</td><td>Sigma-Aldrich</td><td>P5556</td><td></td></tr><tr><td>L-NAME</td><td>Tocris</td><td>0665</td><td></td></tr><tr><td>Olympus upright microscope</td><td>Olympus</td><td>Fluoview FV1000</td><td></td></tr><tr><td>MaiTai HP DeepSee-OL&amp;nbsp;</td><td>Spectra Physics</td><td></td><td>Ti:sapphire laser 690nm &amp;mdash; 1040nm</td></tr><tr><td>Filters</td><td>Olympus</td><td>FV10-MRL/R&amp;nbsp;</td><td>495 to 540 nm&amp;nbsp;</td></tr><tr><td>25&amp;times; water-immersion objective lens</td><td>Olympus</td><td>XLPL25XWMP</td><td>N.A. 1.05 and working distance 2 mm</td></tr><tr><td>Slice Anchor grid&amp;nbsp;</td><td>Warner Instrument&amp;nbsp;</td><td>SHD-27LH</td><td></td></tr><tr><td>Other basic reagents&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta

钙是许多生理过程中血管组织的一个非常重要的调节器。最内皮细胞和平滑肌功能高度依赖于变化的细胞内钙（[Ca2 +浓度i）和一氧化氮（NO）。为了理解如何的[Ca 2+] i的，编号和下游分子通过响应于血管收缩剂和血管扩张剂的血管进行处理，我们开发了一种适用钙标记的新技术（或NO标签）染料与双光子显微镜衡量钙处理（或NO生产）在离体血管。这里所描述的是一个详细的一步一步的过程，演示如何从大鼠，标签钙或NO内的内皮细胞或平滑肌细胞中分离出的主动脉和图像钙瞬变（或NO生产），使用双光子显微镜下列生理或药物刺激。使用该方法的好处是多方面的:1）能够同时进行LY测量两种内皮细胞和平滑肌细胞响应于不同的刺激钙瞬变; 2）它允许一个图像的内皮细胞和平滑肌细胞在其天然设置; 3）该方法是于细胞内钙或NO的变化，并产生高分辨率的图像进行精确的测量很敏感;和4）中描述的方法可以适用于其它分子，如活性氧的测量。总之，应用程序的两个光子激光发射显微镜监测钙瞬变和在一个孤立的血管的内皮细胞和平滑肌细胞的NO产生提供了高品质的量化的数据，并促进了调节血管功能的机制的理解。

为了准确地评估钙血管生理学（血管舒张和血管收缩）的贡献，协议被设计为装载钙染料到两个内皮细胞和平滑肌细胞中分离的完整主动脉。一般实验设置如图1所示，示出了用于分离和制备成像之前该船只的基本策略。简要地说，从大鼠主动脉的分离后，它应清洗的脂肪和结缔组织和狭缝纵向。打开主动脉然后应放置在如在协议中所述和在室温下孵育1小时，用轻摇动含有事先准备好的样混合物的箔覆盖的500μl的管。继温育后，主动脉应洗净，牵制到硅酮覆盖板与最接近的硅氧烷和管腔向上的外膜，并转移到一个直立双光子显微镜成像。 ONCE中的主动脉一直放在显微镜和船只的重点是，内皮细胞应该很容易通过其强大的荧光发射（ 图2A）来定位。内皮细胞也将完成聚焦，因为在容器中，主要由血管内皮细胞的管腔，应当朝上当容器被固定下来的第一个对象。除了 ​​成像内皮细胞，会出现在那里的平滑肌细胞是可见的（ 图2B）的位置。因此，两个内皮细胞和平滑肌细胞可以在同一摄像视野（ 图2C）中被识别。具有的能力，以图像既血管细胞类型相同的摄像视野内可导致更好地理解如何将这些细胞共同的，但独立地响应不同激动剂和刺激。 为了证明该方法的效用，主动脉小区的测量响应性增加了一个cetylcholine胆碱（ACh），一种强效血管扩张剂特异性导致内皮内的变化，进行测定。 如图3A所示 ，内皮细胞可以很容易地看到，而在正常的PSS孵育。在加入ACh（ 图3B）后，将内皮细胞荧光增加表明细胞内钙含量也越来越多。从感兴趣区，或内皮细胞发射的荧光的定量，示于图3C。在加入ACh后，将细胞内钙含量迅速增加，然后缓慢返回到基线。响应于乙酰胆碱的血管内皮细胞内的增加钙是与先前的报告一致。5,6- 类似于钙研究，这个协议是适合于内皮NO生成的检测。对于这些实验，主动脉内皮细胞加载DAF-FM二乙酸酯染料上所示图4A。这种方法最近被用来证明NO产生增加一个的Ca 2+依赖性的方式响应于胆碱刺激。6重要的是，有L-NAME，内皮NO合成酶抑制剂治疗后，这种反应被阻断（ 图4B）。 如何使用此技术可以应用到测量细胞内钙另一个代表性例子示于视频1，其中含有钙的溶液加入到该培养在无钙缓冲液的容器中。当主动脉最初培养在无钙缓冲液（在视频的开始），所述内皮细胞为弱荧光指示在细胞钙的低的水平。然而，除了正常的PSS后，将细胞立即通过增加的[Ca 2+] i的浓度和发光强度高的荧光响应。该实验表明，该细胞是活的和响应于外部刺激。该实验还证实了此方法的高品质数据其产量的生存能力。 <img alt="图1" src="/files/ftp_upload/52734/52734fig1.jpg" /> 图1.主动脉分离实验装置。1）麻醉大鼠后，使腹部的切口，然后从结缔组织和腔静脉分离主动脉。 2）提取后的主动脉，清洗脂肪和结缔组织的容器中，并通过切割纵向打开容器。 3）孵育在将Fluo-4AM染料容器1小时，在室温将其与轻微摇动。 4）孵育后，洗主动脉，其引脚到一个硅涂层板，管腔朝上，并将其传输到直立双光子显微镜的阶段。 <a href="https://www.jove.com/files/ftp_upload/52734/52734fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </一&gt; <img alt="图2" src="/files/ftp_upload/52734/52734fig2.jpg" /> 图2.成像平滑肌和内皮细胞的影响。在足够的负载钙染料的，也可以将图像的平滑肌细胞和内皮细胞。 （A） 的顶部，是在分离的大鼠胸主动脉的Fluo 4荧光内皮细胞的一个例子。底，是用透射光视图（类似地对于B和C）的荧光图像合并。 （B）中 ，是在一个分离的大鼠主动脉的Fluo 4荧光的平滑肌细胞的一个例子。 （C）由于主动脉的非均匀的厚度，它也有可能在那里平滑肌和内皮细胞存在的图像区域。比例尺显示， <a href="https://www.jove.com/files/ftp_upload/52734/52734fig2large.jpg" target="_blank">请点击这里查看大图</a>这个数字。 <img alt="图3" src="/files/ftp_upload/52734/52734fig3.jpg" /> 图3.测量在血管内皮细胞的钙瞬变响应乙酰胆碱。（A）中的主动脉最初制备并培养在正常的PSS缓冲器为基线记录 ​​。 （B）在加入乙酰胆碱（ACh），通过增强表示的钙水平的细胞内增加的内皮细胞（投资回报）发射的荧光信号。显示比例尺。 （ 三）使用了内皮细胞的响应乙酰胆碱的钙瞬变计算Origin软件。 <a href="https://www.jove.com/files/ftp_upload/52734/52734fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52734/52734fig4.jpg" /><b转/&gt; 图4.成像一氧化氮的内皮细胞:（A）装载主动脉与DAF-FM二乙酸酯染料，并把它放在一个双光子显微镜的阶段后，可以将图像的内皮细胞内NO水平使用发出的荧光。 （B）中温育后的内皮NO合成酶抑制剂L-NAME，整体荧光内的感兴趣区减少，说明NO:1的细胞内水平降低。比例尺显示。 <a href="https://www.jove.com/files/ftp_upload/52734/52734fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="视频1" src="/files/ftp_upload/52734/52734figV1.jpg" /> <a href="https://www.jove.com/files/ftp_upload/52734/Video1.avi" target="_blank">内皮细胞响应视频1例，以变化的钙Concentratio</a>ñ。在制备和温育在将Fluo-4AM染料，主动脉用无钙缓冲，并放置在双光子显微镜阶段。当容器被放置在无钙缓冲液中，大部分的内皮细胞是弱荧光。在加入正常的PSS，内皮细胞作出反应，增加细胞内钙离子浓度，并散发出强烈的荧光信号。 <img alt="视频2" src="/files/ftp_upload/52734/52734figV2.jpg" /> <a href="https://www.jove.com/files/ftp_upload/52734/52734_Palygin_Video2.avi" target="_blank">视频中的isoltated大鼠主动脉血管内皮细胞。NO产生2.实施例</a>以下制备和温育在DAF-FM二乙酸酯染料，主动脉放置在双光子显微镜阶段。继在洗澡液加入乙酰胆碱，内皮细胞作出反应，增加细胞内ñØ生产和散发出强烈的荧光信号。请点击此处观看该视频。

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

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 ...

two-photon-imaging-intracellular-ca2-handling-nitric-oxide-production

Nanyang Technological University

Research

JoVE Journal

Biology

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

11.2K 次观看.  Medical College of Wisconsin. 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. 

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

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

科研

医学

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

从鼠标阻力动脉分离微血管内皮管

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

生物学

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+ ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

活体转基因小鼠脑围产层钙和血液学成像

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

神经科学

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

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 ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, myocardial infarction, and angina pectoris, which seriously threaten the life and health of people all over the world. However, how to record the dynamic biomechanical characteristics of isolated blood vessels has long puzzled people. Meanwhile, precise positioning and isolation of coronary arteries to measure in vitro dynamic vascular tension changes have become a trend in CAD drug development. The present protocol describes the macroscopic identification and microscopic separation of rat coronary arteries. The contraction and dilation function of the coronary artery ring along the vessel diameter was monitored using the established multi myograph system. The standardized and programmed protocols of coronary ring tension measurement, from sampling to data acquisition, tremendously improve the repeatability of the experimental data, which ensures the authenticity of vascular tension records after physiological, pathological, and drug intervention.

The present protocol describes the wire myograph technique for measuring vascular reactivity of the rat coronary artery.

标准化大鼠冠状动脉环制备及实时记录血管直径动态张力变化

As a key event of cardiovascular system diseases, coronary artery disease (CAD) has been widely regarded as the main culprit of atherosclerosis, ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function.

细胞内钙的双光子成像

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