Name: two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta
Uploaded: 2015-06-10T13:00:00.000+00:00
Duration: 8 min 8 s
Description: 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

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...

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?...

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

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

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

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

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 ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

体外细胞生理学研究中视网膜微动脉的分离

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while 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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