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?...

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 border="0" cellpadding="0" cellspacing="0">
	<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&#181;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&#160;</td>
			<td>Spectra Physics</td>
			<td></td>
			<td>Ti:sapphire laser 690nm &#8212; 1040nm</td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Olympus</td>
			<td>FV10-MRL/R&#160;</td>
			<td>495 to 540 nm&#160;</td>
		</tr>
		<tr>
			<td>25&#215; 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&#160;</td>
			<td>Warner Instrument&#160;</td>
			<td>SHD-27LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents&#160;</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

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Focal Ca2+ Transient Detection in Smooth Muscle

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+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

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+ ...

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 ...

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.  ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 ...

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 ...

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 ...

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 ...