Name: en face detection of nitric oxide and superoxide in endothelial layer of intact arteries
Uploaded: 2016-02-25T15:00:00
Description: Watch this Scientific Journal Video about En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries at JoVE.com

This work was supported by the Swiss National Science Foundation (310030_141070/1), Swiss Heart Foundation, and National Center of Competence in Research (NCCR-Kidney.CH) Switzerland. Yu Yi is supported by the Chinese Scholarship Council.

动物的工作被批准瑞士弗里堡的兽医办公室的伦理委员会。该协议是继动物护理和实验的指导方针在我们的机构。 1.建立用于隔离动脉孵化的制备<ol><li>构造的器官浴系统，其可以被加热到37℃，并与来自碳气体罐95％O 2和5％CO 2通气。 </li><li>根据需要与下列组合物的浓度制备尽可能多的Krebs-Ringer碳酸氢盐缓冲液：（118毫摩尔NaCl，4.7毫摩尔的KCl，2.5mM的氯...

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NO或O 2的检测.-用荧光染料在培养的内皮细胞的许多研究经常被使用，也组织冰冻切片23。在这里，我们扩展了这种方法，以完整的活的血管， 即 ， 连接面部检测NO的和O 2 .-与DAF-2DA和DHE，分别，这是有效的，比较简单，和直观的内皮细胞层的水平。与在血管冷冻切片的方法相比，这种方法显示出较低的背景并且更定量的，因为在动脉的媒体?...

血管内皮细胞通过释放血管活性因子1保持血管功能和结构的完整性。在这些因素中，内皮衍生的一氧化氮（NO）通过内皮NO合酶（eNOS）的由L-精氨酸产生的是在心血管生理学2的最重要的和最特征的因素。否引起平滑肌松弛，抑制细胞增殖，抑制血小板聚集和炎性细胞的粘附和浸润到内皮下间隙，因此防止血管疾病发展3。在许多生理和病理条件，包括衰老，高血压，糖尿病，高脂血症等 ，血管内皮功能障碍特征在于减少NO的生物利用度，增加的 O 2 .-生产存在，促进动脉粥样硬化2的发病机理。从近几年的研究表明，eNOS的解偶联是一种为内皮功能障碍，其中，所述的eNOS酶生成O 2 .-代替否重要机制，各种上述条件1,4下。因此，血管内皮功能的分析，特别是，内皮NO生产和O 2 .-的产生是关键对心血管疾病和并发症的实验研究。 有迹象表明，已经开发了分析和测量生物样品中的NO产生无数的方式方法。由于NO的极其不稳定性质而容易被氧化为NO 2 -和NO 3 -中的3至6秒的半衰期，这是困难的否直接测量。因此测定的NO 2 - / NO 3 -中的流体样品中的被用作从细胞或组织5释放NO的索引。虽然过程是比较容易的，该方法是，然而，EA/ NO 3 - -溶液中所含的由稳定的 NO 2的高背景sily影响。因为没有刺激可溶性鸟苷酸环化酶产生环磷酸鸟苷（cGMP）的6，细胞的cGMP水平也被确定为估算释放NO 7。再次，这是一种间接的估计和可能不是特定的，因为某些内皮源性因子如C型利钠肽（CNP）也可以通过微粒的鸟苷酸环化酶8的激活提高cGMP水平。否是从L-精氨酸与代L-瓜氨酸的生产中作为副产物9，L-瓜氨酸生产测量因此也用作间接方法来估算的NO产生。这种方法的主要缺点是，它是放射性的，它并不能测量生物活性NO水平，因为释放被O 2 NO能够迅速灭活.-;此外，L-瓜氨酸可以再循环到L精氨酸10。如化学发光检测11，电子自旋共振12，或电化学卟啉NO传感器13其他化学方法被一些研究者使用。这些方法通常是不容易在操作中，检测程序，并需要特殊的设备。也提到，许多研究应用器官浴实验孤立血管有或没有内皮评估内皮功能和间接测量内皮衍生NO介导的血管松弛。然而，这种方法，虽然它主要是接近生理状况，但是严格的说，不能测量NO的功能，它比较评估一般反映eNOS的功能的净效果，生产的内皮介导的血管舒缩反应其它内皮源性舒张因子和内皮衍生收缩因子，生产O 2 .-的，和平滑肌细胞还响应到这些因素。 eNOS的功能或NO生产的具体分析，通常需要3。 许多研究小组包括我们具有在所使用的荧光染料的方法来检测细胞内产生NO 14-19近年来。在此方法中使用的细胞可渗透荧光指示剂diaminofluorescein -2-二乙酸酯（DAF-2DA）来测量在活体外或离体的细胞和组织的分类与NO，NOS功能。其原理是，在活细胞，DAF-2DA由胞内酯酶脱乙酰非荧光-4,5- diaminofluorescein（DAF-2），然后将其转换成由与NO反应以荧光DAF-2三唑（DAF-2T） 。从DAF-2T的荧光可在荧光显微镜或荧光共聚焦显微镜14下进行观察。因此，细胞内荧光强度反映在细胞中的细胞内NO的产生或一个在完整血液vesse内皮湖与特定的荧光染料，如二氢乙（DHE）相结合，可以同时评估细胞内的NO和O 2 .-代细胞或血管14。同样，DHE也是被O 2氧化的细胞渗透性化合物.-在细胞内，和氧化产物，然后用核酸插层以发射明亮的红色荧光显微镜或荧光共聚焦显微镜定量检测的。 DHE是用于检测的 O 2 .-从生物样品中的一个非常具体的染料，因为它检测基本上超氧自由基，通过细胞保持良好，甚至可能容忍温和定影20。一的该荧光染料的方法的一个优点是，它检测到并直接为生血管的完整内皮层上可视NO和/或O 2的.- 烯面 。 在本文中，我们描述这种荧光染色法检测NO和O 2 .-这是我们改编为EN人脸检测NO和O 2 .-在高脂饮食（HFD）喂养引起的肥胖小鼠模型的完整的主动脉。我们表明，该方法能够成功，可靠地测量NO和O 2 .-水平及肥胖新鲜分离的完好鼠标主动脉内皮细胞层的eNOS评估（DYS）函数。

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	<tbody>
		<tr>
			<td>Dihydroethidium (DHE)</td>
			<td>Invitrogen</td>
			<td>D 1168</td>
			<td>dissolve with DMSO to 5 mmol/L as 1000X stock, stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>Diaminofluorescein-2 Diacetate (DAF-2 DA)</td>
			<td>Calbiochem</td>
			<td>251505</td>
			<td>dissolve with DMSO to 5 mmol/L as 1000X stock,stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Invitrogen</td>
			<td>D 1306</td>
			<td>dissolve with water to 300 &#181;mol/L as 1000X stock,stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Vector labor. (reactolab)</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name (N o-nitro-l-argininemethylester.HCl)</td>
			<td>Sigma-aldrich</td>
			<td>A5751</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma-aldrich</td>
			<td>P3636</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Myograph System&#160;</td>
			<td>Danish Myo Technology A/S</td>
			<td>Model 610M</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>DM6000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td>National Institute of Health(NIH)</td>
			<td>Image J&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors&#160;</td>
			<td>S&#38;T AG</td>
			<td>SDC-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissors&#160;</td>
			<td>F.S.T</td>
			<td>15000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>S&#38;T AG</td>
			<td>JF-5</td>
			<td></td>
		</tr>
		<tr>
			<td>26G&#215;1/2&#698; syringe</td>
			<td>Becton Dickinson</td>
			<td>305501</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip round diam15mm</td>
			<td>vwr</td>
			<td>631-1579</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 1 mL</td>
			<td>vwr</td>
			<td>RFL-1200c&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 200 uL</td>
			<td>vwr&#160;</td>
			<td>613.0659</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 1.5 mL</td>
			<td>Eppendorf&#160;</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine chloride</td>
			<td>Sigma-aldrich</td>
			<td>A-6625</td>
			<td></td>
		</tr>
	</tbody>
</table>

en face detection of nitric oxide and superoxide in endothelial layer of intact arteries

内皮源性一氧化氮（NO）从内皮NO合酶（eNOS）的生成是在心血管生理学的最重要的血管保护分子中的一个。功能失调性的eNOS如eNOS的解偶联导致在超氧阴离子NO生物利用度和增加而减少（O 2 .-）生产，并反过来促进心血管疾病。因此，NO和O 2 .-在内皮细胞水平的适当的测量是关键对心血管疾病和并发症的研究。因为NO和O 2 .-的极不稳定的性质，它是难以测量NO和O 2 .-直接在血管中。许多方法已被开发来测量NO和O 2 .-生产。它是，然而，无论是不敏感的，或非特异性，或技术要求高，并需要特殊的设备。在这里，我们描述了一个适应为连接面同时检测和细胞内NO和O 2的可视.-使用细胞渗透性diaminofluorescein -2-二乙酸酯（DAF-2DA）和二氢乙（DHE），分别在感应的肥胖小鼠模型中的完整主动脉的荧光染料的方法的高脂饮食喂养。相比于对照贫鼠标我们能证明降低细胞内NO和增强的 O 2 .-水平肥胖小鼠的新鲜分离完整主动脉。我们表明，该方法是对NO的直接检测和可视化和O 2的简单的技术.-在完整的血管，可广泛适用于（生理）病理条件下的内皮（DYS）函数的调查。

肥胖是缺血性冠状动脉心脏疾病的重要危险因素，并与相关联的减少内皮NO的生物利用度，动脉粥样硬化性血管疾病21的一个标志。的eNOS-解偶联已被证明是血管内皮功能障碍的重要机制众多生理和病理条件，包括老化22，动脉粥样硬化和肥胖症14下。因此，在这里我们比较瘦和肥胖小鼠显示NO代表的结果，O 2 .-在主动脉水平。 <p c...

Watch this Scientific Journal Video about En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries at JoVE.com

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

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

在这篇文章中我们将介绍使用荧光染料diaminofluorescein-2双乙酸钠（DAF-2DA）和二氢乙啶（DHE）对恩面对同时检测和细胞内一氧化氮的可视化（NO）和超氧阴离子（O 2的调整相对容易的方法.- ）分别在新鲜分离的肥胖小鼠模型中的完整主动脉。

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

Research

JoVE Journal

Biology

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

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

科研

生物学

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

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 ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Analysis of Oxidative Stress in Zebrafish Embryos

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

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

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

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

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

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 ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

用化学发光法测定硝酸盐和亚硝酸盐，代谢产物中的一氧化氮途径，在生物材料

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

En Face Preparation of Mouse Blood Vessels

En Face Preparation of Mouse Blood Vessels

面对小鼠血液制备

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

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

Application of Genetically Encoded Fluorescent Nitric Oxide NOÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬Ãƒâ€šÃ‚Â¢ Probes, the geNOps, for Real-time Imaging of NOÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬Ãƒâ€šÃ‚Â¢ Signals in Single Cells

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 ...