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的氯化钙 ，1.2毫硫酸镁 ，1.2毫KH 2 PO 4，25mM的碳酸氢钠 ，0.026毫摩尔EDTA，和11.1毫D-葡萄糖）。 </li><li>保持在冰上的储备缓冲和通气用95％ 的 O 2和5％的CO 2的缓冲器。 </li><li>开关器官浴系统上，在37℃设定温度，加入5毫升准备使用的缓冲液中向每个腔室并保持曝气用95％ 的 O 2和5％的CO 2的缓冲器。 </li><li>用Krebs-Ringer缓冲液洗室一次30分钟。 </li><li>添加5毫升的Krebs-Ringer缓冲液到每个腔室并覆盖所述室，以避免蒸发。 </li></ol> 2.鼠标主动脉的分离<ol><li>注入将其溶于0.9％NaCl中腹膜内，在150毫克/千克的浓度牺牲小鼠戊巴比妥。 </li><li>仰面躺在鼠标放在手术板上。 </li><li>喷胸部区域的毛皮用70％乙醇消毒和水分的目的。 </li><li>通过在肋切开胸腔，暴露心脏和肺部，并取出肺。 </li><li>在胸腔用纸巾清除血液平缓。 </li><li>用镊子轻轻抓住心脏和切割主动脉和脊椎手术剪的血管周围脂肪组织分离胸主动脉。 </li><li>立即浸入wholÈ组织在冰冷的（4℃）的Krebs-Ringer缓冲液。 </li><li>解剖显微镜下取出心脏和主动脉弓，并保持胸主动脉段在缓冲区中。 </li><li>从解剖与手术剪刀​​和镊子在显微镜下秉承血管周围组织无主动脉。 </li><li>掌握主动脉的端部边缘与镊子，并通过用26 G×1 / 2&quot;注射器轻轻冲洗的Krebs-Ringer缓冲液冲洗掉在血管管腔的血液。 </li><li>切开洗净主动脉环为3毫米长的段。 注意:在这一步要注意不损伤内皮细胞层。 </li></ol> 3. DHE和DAF-2DA染色<ol><li>传送清洗主动脉段用钳子向在37℃下填充有的Krebs-Ringer缓冲液的器官浴室通入95％O 2和5％ 的 CO 2。 注:轻触只用钳子主动脉环的外膜侧，不钳的Blood船只。 </li><li>平衡在器官浴室中的动脉30分钟。 </li><li>添加乙酰胆碱到器官浴中，以最终浓度为1μM，孵育动脉10分钟。 </li><li>制备将1ml DHE / DAF-2DA溶液（每种染料的5微米，从1000×库存稀释）用温热的预的Krebs-Ringer缓冲液中的1.5 ml离心管中。包装用铝箔的离心管中，以避免光线照射。 </li><li>刺激后，从器官浴转移主动脉环到DHE / DAF-2DA溶液在微量离心管在37℃下用95％ 的 O 2和5％CO 2充气和孵化的主动脉环30分钟。 注意:从这个步骤中，在黑暗始终保持主动脉。 </li><li>转移主动脉环至新的离心管中的Krebs-Ringer缓冲液洗涤和重复洗涤1分钟内三次。 </li><li>转移主动脉环至4％多聚甲醛溶液固定30分钟。 </li><li>制备1毫升4-&#39;，6-二脒基-2-苯基吲哚（DAPI）溶液（300纳米，从1000×库存稀释）用磷酸盐缓冲盐水（PBS）在1.5ml微量离心管中缓冲。染液主动脉3分钟。 </li><li>转移主动脉到新的离心管，用PBS缓冲液洗涤1分钟。重复洗涤三次。 注意:注意不要夹住主动脉环或损坏的内皮细胞层。 </li></ol> 4. 恩面对安装<ol><li>加安装介质向滑动的下降。 </li><li>用显微剪刀纵向切开主动脉环镜下，使卷起的主动脉平并挂载恩面对与朝下于载玻片上内皮的安装介质上。不要移动主动脉来回。 </li><li>盖上盖玻片幻灯片和密封用指甲油的幻灯片。 </li><li>在轻保护空气干燥后，用幻灯片成像可怕ctly小时内或将它们存储在-20℃，接下来的几天。 </li></ol> 5.共聚焦显微成像<ol><li>使用共聚焦显微镜检测荧光信号。开动机器，优化成像设置，如放大，扫描速度，分辨率，Z-堆栈。 <ol><li>对于这个协议，请使用200 Hz的扫描速度，分辨率为1,024×1,024像素为0.25微米的Z-步长。从DAF-2DA激发荧光用488nm的氩激光在515nm处检测，而在514毫微米从DHE激发荧光和在605nm发射检测。 </li></ol></li><li>使用10X放大倍率为重点样本，直到DAPI信号是紫外线清晰，然后调整目标，40X的放大倍率。 </li><li>通过调整控制面板上的Z位置定义内皮细胞层的范围内。 DAPI染色细胞核的信号是在内皮细胞层椭圆形或圆形的点。 </li><li>从顶部扫描样品通过内皮信号和记录图像的整个厚度（在管腔边界内皮细胞层）。选择用于扫描每个样品至少3种不同的领域。 </li></ol> 6.图像的分析<ol><li>使用软件来打开通过共聚焦显微镜扫描的数据。选择每个领域中的三个连续的图像进行分析，并评价每个样品的至少3个不同的领域。导出所选图像，并将它们保存为JPEG文件。 </li><li>从量化DAF-2DA，DHE和DAPI与图像处理软件染色图像。从DAPI染色图像，选择"插件"→"分析"→"细胞计数"来算DAPI阳性核的数目。 <ol><li>从DAF-2DA和DHE染色图像，选择"分析"→"措施"来分析信号的强度，并采取"中庸"值的相对信号强度。本然后将结果作为DAF-2DA到比DAPI阳性细胞核或DAPI DHE的比例。对于每个样品，利用每一个图像和场的平均值。 </li></ol></li><li>由平均对照组的划分每个样品的结果，以获得倍数变化。用非配对Student t检验或ANOVA以及Dunnett或邦费罗尼后检验进行统计分析。给数据为平均值±SEM。考虑当p &lt;0.05 14平均值作为显著差异。 </li></ol>

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No conflicts of interest declared.

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，因为在动脉的媒体弹性纤维尤其在冷冻切片主动脉给这可能与特定的NO或O 2的干扰很强的自发荧光信号。 -在内皮细胞中产生的信号。另外，在中层平滑肌细胞中特定的 O 2 .-代由NADPH氧化酶或其它酶也可干涉与在血管部分，它表示具有血管冷冻切片的方法的缺点在血管内皮细胞上的信号。与此相反，在连接面染色法，从一个完整的血管片段的内皮细胞层检测信号具体地，因此给出更精确的分析。低温恒温机冷冻切片准备也是一个昂贵的投资。比较其他生化方法，该方法的主要限制是不太定量的，但它是对样本之间的相对比较的良好选择。此外，这是昂贵的共焦显微镜的要求也可能是这种方法的一个限制。多器官腔系统是方便并且如果这不是可以在实验室中，可以很容易地建立与水浴和它们连接到监管系统的碳储气罐管的系统中，从而使血管中的管子是保持在37℃并通入95％O 2和5％的CO <suB&gt; 2。 有迹象表明，人们必须注意的几个关键步骤。确保血管周围组织清洁易于安装。在血管腔中血液凝块一定会被冲洗走，因为它们可能导致人工信号，并与所述荧光信号干扰。在血管制剂，孵育，洗涤，切割和装配的整个过程中，人们必须非常谨慎不要破坏血管的内皮细胞层。准备在整个过程中不要让血管干。 DHE和DAF-2DA都是荧光探针，所以从染色步骤主动脉应始终避光曝光开始。定影前步骤主动脉的内皮细胞必须是活的，所以主动脉应始终保持在用95％ 的 O 2和5％CO 2充气的的Krebs-Ringer缓冲液。样品的图像应制备后尽快作出。在发出荧光NCE信号将成为在几天弱甚至与保护安装的介质中，这可能会影响结果的准确性。该方法可以不太厚血管壁申请血管。 这种方法能够在完整的血管的内皮细胞层否同时想象力和O 2 .-生产中，如果两种染料加到血管在一起。你也可以用这种方法来评估在体外孤立血管NO和O 2 .-生产药物的药理作用。为了这个目的，该清洗主动脉通常切成两部分，一个是用于控制和另一个是用于药物治疗，药物应该平衡后加入到温育缓冲液之前DAF-2DA和DHE染色步骤。它是特别有用的，如果此方法与另一种方法一起使用， 例如 ，与分离的血液的v的血管舒缩反应分析essels，内皮细胞的生理功能可以得到证实。此方法应也适用于疾病模型的完整的血管和底层机制的任何内皮（DYS）函数的分析。 总之，我们提出了一个简单的协议，以同时检测NO和O 2 .-生产完整的血管用荧光共焦显微镜的内皮细胞层。我们展示了如何这种方法成功地在肥胖小鼠模型的工作。这种方法是在许多血管疾病的条件下的内皮NO的调查和O 2 .-生产一种有用的技术。

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。

<table><tbody><tr><td>Dihydroethidium (DHE)</td><td>Invitrogen</td><td>D 1168</td><td>dissolve with DMSO to 5&amp;nbsp;mmol/L as 1,000x stock, stored at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>Diaminofluorescein-2 Diacetate (DAF-2DA)</td><td>Calbiochem</td><td>251505</td><td>dissolve with DMSO to 5&amp;nbsp;mmol/L as 1,000x stock, stored at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Invitrogen</td><td>D 1306</td><td>dissolve with water to 300&amp;nbsp;&amp;micro;mol/L as 1,000x stock, stored at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Mounting medium</td><td>Vector labor. (reactolab)</td><td>H-1000</td><td></td></tr><tr><td>N&lt;sub&gt;&amp;#969;&lt;/sub&gt;-Nitro-L-arginine methyl ester hydrochloride (L-NAME)</td><td>Sigma-aldrich</td><td>N5751</td><td></td></tr><tr><td>Pentobarbital</td><td>Sigma-aldrich</td><td>P3636</td><td></td></tr><tr><td>Multi-Myograph System&amp;nbsp;</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&amp;nbsp;</td><td>Leica&amp;nbsp;</td><td>DM6000&amp;nbsp;</td><td></td></tr><tr><td>Image processing software</td><td>National Institute of Health (NIH)</td><td>Image J&amp;nbsp;</td><td></td></tr><tr><td>Surgical scissors&amp;nbsp;</td><td>S&amp;amp;T AG</td><td>SDC-11</td><td></td></tr><tr><td>Microsurgical scissors&amp;nbsp;</td><td>F.S.T</td><td>15000-01</td><td></td></tr><tr><td>Forceps</td><td>S&amp;amp;T AG</td><td>JF-5</td><td></td></tr><tr><td>Coverslip round diameter 15&amp;nbsp;mm</td><td>VWR</td><td>631-1579</td><td></td></tr><tr><td>Tips 1 ml</td><td>VWR</td><td>RFL-1200c&amp;nbsp;</td><td></td></tr><tr><td>Tips 200 &amp;#956;l</td><td>VWR</td><td>613.0659</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tubes 1.5 ml</td><td>Eppendorf&amp;nbsp;</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。 在7周的年龄开始，雄性小鼠（C57BL / 6J）期间14周给予到一个正常食物（NC自由进入;能量含量:10.6％的脂肪，27.6％的蛋白质，57％碳水化合物，纤维4.8％ ）或高脂肪饮食（HFD，能量含量:55％脂肪，21％蛋白质，24％碳水化合物）。 14周后HFD只小鼠牺牲和胸主动脉进行解剖，并秉承组织清理。前DAF-2DA和DHE染色工序中，eNOS的抑制剂LNģ-Nitroarginine甲酯（L-NAME）（1毫摩尔）加入到浴室1小时到方框eNOS活性14。代表性共聚焦荧光结果在图1中被示出。在蓝色在上面板表示由DAPI染色内皮细胞的细胞核中。中间面板，绿色是从DAF-2T从非荧光DAF-2 NO，这意味着如果绿信号的强度是较高的转换的荧光信号，有更多的NO在细胞中。同样，红色在下部面板是从DHE被O 2 .-被氧化的荧光信号，所以呈现更红色的样品具有多个O 2 .-在细胞中。 共聚焦荧光显微镜揭示下降d。在小鼠的主动脉内皮细胞层的NO产生（DAF-2DA染色）和增加的L-NAME敏感的 O 2 .-代（DHE染色）馈送HFD相比，小鼠的供给NC（ 图1）14，这表明eNOS的，解偶联肥胖。这些信号进行量化，并在条形图14呈现。 <img alt="图1" src="/files/ftp_upload/53718/53718fig1.jpg" /> 图1. eNOS的，解偶联肥胖。共聚焦显微镜带人脸检测NO和O 2 .-分别DAF-2DA和DHE染色。 N = 5; *** P &lt;0.005 VS NC;†††P &lt;0.005 VS HFD组。比例尺= 0.1毫米。 （数据来自参考14） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53718/53718fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> </p&gt;

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 ...

en-face-detection-nitric-oxide-superoxide-endothelial-layer-intact

Nanyang Technological University

Research

JoVE Journal

Biology

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

9.5K 次观看.  University of Fribourg. 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。 

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

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

为含量测定一氧化氮生物活性分析技术

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

科研

医学

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

肱动脉的临床研究超声评价血管内皮依赖性血流介导的血管舒张功能

The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The ...

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

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

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 ...

生物学

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

<meta charset="utf8"></head><body>使用荧光成像和 EPR 检测血小板中的超氧阴离子

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by&#160;human platelets,&#160;understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually,&#160;identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

<meta charset="utf8"></head><body>作者聚焦：ROS 检测的创新技术及其对血小板研究的影响

The generation of superoxide anion is essential for the stimulation of platelets and, if dysregulated, critical for thrombotic diseases. Here, we present three protocols for the selective detection of superoxide anions and the study of redox-dependent platelet regulation.

检测血小板中超氧阴离子生成的替代方法 

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ...

免疫与感染

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

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

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

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

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

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

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

基于化学发光的检测方法，用于检测自氧化和亚硝化化合物中的一氧化氮及其衍生物

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

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 ...

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

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为含量测定一氧化氮生物活性分析技术

肱动脉的临床研究超声评价血管内皮依赖性血流介导的血管舒张功能

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

板基检测血液中一氧化氮的 vasp 磷酸化

检测血小板中超氧阴离子生成的替代方法

检测血小板中超氧阴离子生成的替代方法

检测血小板中超氧阴离子生成的替代方法

细胞内钙的双光子成像

基于化学发光的检测方法，用于检测自氧化和亚硝化化合物中的一氧化氮及其衍生物

发展与表征