The authors would like to thank Dr George Balanos for his assistance in the flow-mediated dilatation technique.

注:该协议遵循从达德利集团NHS信托基金会的人类研究伦理委员会的指导方针。执行所有描述的技术在温度控制的实验室（21 - 22℃），具有稳定的灯光和噪音的情况下。问个人经历评估从食物，饮料，吸烟和之前的测试练习12小时，以避免。扣压血管活性药物至少12小时在适当的时候。 1.激光多普勒成像与离子导入<ol><li>接通激光多普勒成像仪（LDI），并允许扫描仪自动稳定大约30分钟。启动LDI的软件，然后点击"测试"（该软件的主屏幕便会显示）。在主屏幕上，选择任务栏位于窗口顶部的"Ionto协议"。 </li><li>手动输入协议（在我们的实验室中使用的协议共涉及13号第罐，具有用于离子电渗药物递送从扫描2中设置电流扫描11以30μA的电压）。设置扫描1为基准，没有电流扫描，并扫描12＆13恢复扫描也没有电流。点击OK确认设置并返回到主屏幕。 </li><li>要求参与者在一个半卧位椅子的前臂休息90度舒适，牢固枕头放松，并把前臂下​​一个黑色的垫子。 注:垫有助于限制由围绕组织背景表面产生假象的测量。重要的是，与会者臂牢固地绑在枕头使得没有运动和相关的伪影。 </li><li>连接有线插头在每个有机玻璃室向离子电渗疗法控制器的另一端。连接含有2.5ml的剂量的1％乙酰胆碱（ACh）的对离子电渗疗法控制器的anodal连接的腔室，并连接含有该第二室2.5ml的剂量的1％的钠，硝普钠（SNP）的阴极连接。在用0.5％盐水溶液的混合室两种药剂。两个腔室连接到参加者的右前臂用双面粘接片的掌侧的方面。 </li><li>覆盖腔室由32毫米盖玻片，以防止流体泄漏。 </li><li>在开始扫描之前，打开位于主屏幕的左上角的"扫描仪设置"窗口。选择"视频和远程"选项卡，选择"自动距离"功能来测量参与者前臂的扫描头的距离。 <ol><li>以下自动测距完成后，选择"图像扫描"标签，并确定将通过点击窗口右下角的"标记"按钮被扫描的区域。如果需要的话，通过手动输入在扫描区域的大小成&#39;扫描区和改变所述感兴趣区域的大小＃8217;部附近的窗口的顶部。确保感兴趣的区域包括的每个的电离子透入腔室的直径和大到足以限制变异性皮肤血流量。 </li></ol></li><li>以下评估完成后，保存数据文件。使用LDI图像分析软件来执行灌注测量打开数据文件。 <ol><li>点击主软件窗口"图像查看&#39;，并打开是要分析的图像文件。 </li><li>使用软件来标记出围绕每个室的外直径的感兴趣区域。调整感兴趣的区域，使之正确地装配上的区域，其中所述腔室在场。然后点击"统计信息"图标，并包含平均灌注单位为每室一栏将显示。注意基线灌注部，以及从前述各12扫描的每个腔室的最高灌注单元。 注意:这种分析方法是具体到我们的实验室;然而，其它方法也可以用来表达由LDI扫描获得的数据。对于一个全面的检讨，请参见从Roustit和Cracowski 12的指导方针。 </li></ol></li><li>要计算百分比变化灌注回应胆碱和SNP，减去高峰灌注基线灌注，除以基线灌注，然后乘以100。 注:在我们的实验室，基线的变化相对灌注显示变异良好的内观察员系数乙酰胆碱（7％）和SNP（6％）。 </li></ol> 2.血流介导的扩张和硝酸甘油介导的扩张<ol><li>切换多普勒超声机及含血管图像分析（VIA）的软件联网的PC上。 注:通过软件捕获实时图像（在每秒25帧），并提供，以及对血管直径信息血管边界的质量由超声波机器被检测。 OT她的软件包，可它可能包含额外的功能和设置。最好是请教操作手册的专用软件。 </li><li>要求参与者在一个半卧位扶手椅放松，把他们一个舒适的枕头手臂自己身边，但水平与心脏。周围放置参与者的手腕血压计。 注:患者应被要求保持他们的手臂，仍可以防止在测量过程中的移动文物。 </li><li>确保从超声波机器的线性阵列换能器成立体钳，并拧紧用蝶形螺母夹紧，使得超声换能器保持在一个固定的位置。 注:钳将确保该超声换能器将保持稳定，一旦血管所在的位置。 </li><li>在超声机，滚动到"菜单"，设置扫描频率为5 MHz和优化的深度（建议的深度设置为3.5厘米）和超声机器上的增益设置。调节增益设置，以确保有对称的亮度为容器的远近壁。 </li><li>采用线阵探头，找到肱动脉通常是发现2-10厘米以上的纵向扫描平面的肘窝。进行任何调整，以澄清在此阶段的图像质量。为了帮助确定动脉，打开彩色多普勒帮助显示搏动动脉血流和持续静脉血流区分开来。查看水平穿过屏幕肱动脉;它应该显示为两个固体的平行线，在其表示容器的内腔的线之间通过一个明确的区域隔开。 </li><li>以允许通过软件自动记录血管直径，使用光标来标记感兴趣的预定区域，以检测和跟踪动脉的前壁和后壁。 注意:所关注的区域的大小可以是增加或使用&#39;x&#39;和&#39;y&#39;的位于主软件屏幕上的按钮降低。 </li><li>点击VIA软件和图像动脉2分钟"开始"。在此之后，按"充气"威盛软件，同时充气血压袖带置于周围的手腕上心脏收缩的压力（通常高于220毫米汞柱）5分钟。 注:手腕袖带的目的是以阻塞血液流向手。 </li><li> 5分钟后，放气血压袖带以诱导反应性充血其中，在一个健康的容器中，将刺激NO介导的血管舒张。 注:峰值扩张可能会发生多达180秒以下袖带放气，所以最好是继续录制血管直径为袖带释放后3分钟。 </li><li>以下一个10分钟的休息时间，使用线性阵列换能器重新定位肱动脉并以相同的方式记录一个2分钟基线直径读数作为步2.7。 </li><li>然后要求参与者把一个500微克舌下甘油，硝酸（GTN）平板电脑在他们的舌头，并继续测量肱动脉直径再5分钟。在此之后，要求参与者以去除GTN平板电脑和监视器的参与者，以确保他们不会遇到任何不良影响的药物。 </li><li>进行数据的离线分析所有。二十五个数据点可用于评估每个第二;折叠此数据到Microsoft Excel中一秒钟的时代。将数据导出到数字信号分析软件，并用3秒移动平均过滤器过滤。 </li><li>建立从120秒的数据的基线直径的袖带充气之前。目视检查基线区域和排除文物。平均剩余基线区域，以产生基准直径。 </li><li>对于血流介导的扩张（FMD）的分析，使用软件自动扫描后cuff-通缩区域峰值扩张和使用光标纪念这个峰值目视检查。如果峰值已经误，使用光标选择一个较小区域内，峰值便可以被识别。记录峰值作为峰直径。 </li><li>为GTN的数据，采用相同的方法来与FMD使用，除了搜索继5分钟用药峰扩张在该地区。 </li><li>要计算FMD％和GTN％，减去高峰直径基准直径除以基准直径，然后乘以100。 注意:在我们的实验室中，变异的帧内观察者系数是口蹄疫11％，而对于GTN 12％。 </li></ol> 3.颈动脉内膜中层厚度<ol><li>要求参与者躺在舒适的床上，并放置一个枕头在头下，提供颈部支撑。 </li><li>连接的心电图（ECG）导致多普勒超声，然后将它们附着在所述p心急的四肢。只有基本的ECG迹线是必需的，所以放置在左侧和右侧臂相应引线，并在左脚踝。 </li><li>通过滚动的&#39;菜单&#39;，并设置扫描频率为10 MHz和优化的深度准备超声波机（推荐深度设置为3 - 第4厘米）和增益设置。调节增益设置，以确保有对称的亮度为容器的远近壁。 </li><li>向参加者稍微倾斜其头部的左侧，并使用该线阵换能器，扫描沿着利用纵向扫描平面，以确定任何斑块的存在其所有部分（共同的，内部和外部颈动脉）的右颈动脉。保存显示斑块的任何证据的图像。为了帮助识别动脉，查找在容器内的分叉点，因为这显示了颈动脉分叉到内部和外部颈动脉。 </li><li>测量Ø˚F颈动脉内膜 - 中膜厚度（CIMT），达到总颈动脉，是免费的斑块，并且为1cm近端颈灯泡的截面中的至少3个图像。达到全部图像上的心电图的R波的峰值，因为这对应于心室舒张并在该点该容器是在最少量的剪切应力。 </li><li>重复步骤在左颈动脉3.4和3.5。请参与者稍微倾斜他们的头，为这种测量的权利。 </li><li>为了协助获得清晰的图像的近和远壁，在评估过程中小心操作超声波探头，以确保该容器是垂直于超声波束。通过巧妙地连同小的调整，以施加到近侧到远侧角探针（踵趾运动）的压力改变换能器的倾斜和旋转实现这一点。 </li><li>图像进行分析，离线使用动脉测量软件（AMS）检测血管奔卡里根据Pignoli的线条。加载图像以进行分析，然后使用光标，在不受斑块容器的部分创建的感兴趣的区域。点击"检测"的软件，记录屏幕的cIMT和管腔直径上显示的值。 注:准确的读数只能从远处的墙壁获得，因此忽略了近壁读数。 </li><li>取三次测量的每个边，然后平均这些得到的平均值的cIMT为左，右颈动脉分开。从两侧进一步平均化的cIMT，得到整体的cIMT。 注:变异的这种技术在我们实验室内观察员系数为9％。 </li><li>执行使用相同的软件通过手动标记出使用光标的牌匾任何牌匾的测量。点击"分类"的AMS根据其易感性破裂自动计算斑块和档次的回声。点击在"牌匾特性"窗口中看到这些信息。 </li></ol>

The authors declare that they have no competing financial interests.

本手稿详细的血管功能和形态可在所述外周脉管进行若干不同的评估的方法。每个评估提供了动脉粥样硬化的不同阶段的信息，并帮助表征不同的血管领土血管轮廓。 我们以前曾报道，微血管内皮功能是独立于大血管内皮功能的类风湿关节炎患者的CVD 39的增加的风险的群体。此外，血管功能和形态的评估也彼此独立，在同一组的患者和患者的心血管疾病40,41。这些发现可通过功能细胞和内皮细胞在不同脉管地域38的结构的异质性，以及可能的时间滞后的功能改变到形态学进展进行说明异常的容器中。一项研究太郎和他的同事42发现，一些与会者与动脉粥样硬化已经下降FMD值，但正常的cIMT值。这些研究结果表明亚临床动脉粥样硬化的使用各种方法，这些方法检查是很重要的，以破译心血管疾病的全球影响。 在健康和疾病的微血管的重要性正在获得越来越多的关注，在医学文献。微血管形成大得多的表面积大于大血管使其显著目标从有害刺激43的损坏。据推测，微血管可能是炎症介质而渗透的，导致损伤形成43的较大血管的内皮细胞的主要来源。在II型糖尿病患者，微血管病变常常先于大血管病变44，以及在其它人群风险增加的心血管疾病，如类风湿arthriTIS，干预措施，降低心血管疾病的风险提高微血管，但不是大血管，血管内皮功能45,46。总的来说，这些研究结果表明，检查微血管功能可以理解这引发动脉粥样硬化的复杂机制帮助。 在目前的工作中，利用LDI与血管活性药物离子导入进行微血管内皮功能的评估。其他几个评估可以用来评估微血管功能，包括甲襞capillaroscopy和静脉闭塞体积描记法。然而，前者的评估提供了关于仅仅微血管形态学信息，而后者则是费时，在由于帧内肱血管活性剂1施用某些协议侵入。与此相反，LDI提供了一种简单，时间效率的方式来测量响应于血管活性剂的皮肤血管微血管灌注其中施用非invas结构延续。皮肤血流量的测定已获得广泛接受的文献，因为它易于访问性和很强的相关性与既定的CVD 12。此外，LDI超过诸如激光多普勒血流仪其他多普勒技术的优点是，它能够同时在一个给定的区域进行扫描的多个点，因此，可占细胞运动伪影和皮肤血液流动的空间差异，这两者可影响灌注容器47,48的。 尽管离子电渗疗法的明显的优势，值得注意的是，当前的诱导血管舒张（CⅣ）从离子电渗疗法可能混淆特别是在阴极的血管活性药物的作用是重要的。车辆的用于药物递送的选择可有助于减少这种影响，用0.5％的氯化钠（如用在当前协议）有效地限制CIV 18。此外，使用大直径腔和低电CUR租金（在当前协议使用的）都有助于减少CIV 18。使用一个控制场地也已建议12。生物和行为因素也可影响该技术的可靠性和可重复性。例如，昼夜变化和吸烟已经显示影响微血管内皮功能49,50。严格的录音条件必须遵守，才能获得准确的结果和设计方案12时制定的准则应遵循。 口蹄疫和硝酸甘油介导的扩张的测量提供了关于在大血管内皮功能障碍的信息，并且被广泛应用于非侵入性血管研究。口蹄疫技术提供了对NO的生物利用度的替代信息，并在不同的临床人群7-9心脏事件的有用的预后标志物。在目前的工作中，协议提出占很多的因素是必要的一氧化氮介导的血管舒张25足够的刺激。例如，所述封堵袖带置于远侧的超声探头和周围的手腕27，局部缺血的持续时间为5分钟，26和足够的时间使以下反应性充血28记录"真"峰直径。不幸的是，该协议不包括剪切应力分布的表征作为自动边缘检测软件没有允许同时记录血管直径和脉搏波传导速度的信号。剪应力的计算是不可或缺的口蹄疫26的精确测量，我们建议，在可能情况下，血管研究团体使用的软件，其允许进行这样的测量。 口蹄疫和硝酸甘油介导的扩张的评估也容易受到环境和生物变化24，如在血管直径的微小变化能引起大的FMD / GTN的反应。例如，典型的口蹄疫值健康参与者的范围从5-10％51，其对应于0.25 -在动脉0.5mm直径变化为具有直径为5mm的动脉。鉴于这样的小改动动脉管径，必须小心注意支付给技术和生物因素，可能会影响测量。的确，口蹄疫可受多种生物学和行为因素如交感神经激活52，剥夺睡眠53，咖啡因消耗54，吸烟55，抗氧化治疗56和57天的时间。因此，重要的是控制这些因素通过从确定的准则24,25利用信息。 做用的cIMT先进的，但亚临床动脉粥样硬化的评估。该技术已应用在多个临床人群，提供非常详细的动脉ST相比，如磁共振成像34更复杂的技术ructure。与其它血管技术的cIMT的测量需要仔细考虑其可以影响测量的技术因素。通常，的cIMT应在无斑块区域中进行，在远壁的颈总动脉。类似口蹄疫，的cIMT的测量使用来执行高分辨率超声等是深得用户依赖。报道的变化（CofV）的范围从2.4系数- 18.3％58，而对于口蹄疫是1 - 84％，59。然而，即使当这两种技术都受到主管的ultrasonographers与良好控制的外部因素进行的，但是仍然高CofV 58,60,61。一个原因可能是，血管边界的分析，进行了使用手工方法60,61。这种分析可以降低可靠性成像的文物，如虚假的边界，从ultraso噪音UND信号，和血管扭曲会影响图像22解释。 连续自动边缘检测软件的最新发展，极大地改善了血管壁的边界21,22的检测。在本研究中，通过软件来测量肱动脉的直径，而AMS被用于检测的cIMT。使用这些软件的大大减少操作员的依赖性，但在AMS的情况下，一定程度的操作员控制的是在的情况下仍然可用，其中的图像质量可能很差62。其中使用自动的边缘检测软件实验室通常倾向于具有低CofV 58,63,64，因此应该是所有血管研究实验室的目标是要将血管边界的自动测量，以确保结果的准确性。这也是很好的做法发表研究成果时，报告重复性研究具体协议的结果。 6-9的证据，还有的已审查穷内皮功能及心血管不良结果之间的关系，诸如心肌梗塞和中风的研究缺乏。进一步的前瞻性研究来解决这些问题。另一个限制是利用人工操作来进行评估，并进行分析。这引入偏见的潜在来源;然而，这可以通过致盲操作的结果或确保读者从操作不同的限制。它同样重要的是，以确保读者遵循标准化协议进行数据分析，以使所有的数据一致地进行分析。 综上所述，本手稿提供必要成功执行微血管和大血管内皮功能的评估以及外周循环的血管形态的方法步骤的详细信息。一起使用时，评估提供动脉粥样硬化的不同阶段的全球信息。进一步的前瞻性研究探讨这些技术的潜在的诊断作用是必要的。

内皮是脉管的最内衬，并参与血管稳态的通过多种血管活性过程的调节的维持。干扰这些过程可能会使该容器动脉粥样硬化和增加心血管疾病（CVD）1的风险。周内皮功能是早期异常血管壁2的良好指标。此外，外围血管内皮功能的措施已被证明，以反映冠状内皮功能3-5，并因此被视为心血管疾病6-9的良好预测因子。这也许是不足为奇鉴于动脉粥样硬化，现在广泛赞赏是一种全身性疾病10。周边血管内皮功能的评估通常量化容器到特定刺激的血管扩张反应，具有指示内皮的拖拉响应的衰减功能障碍11，并且可以在不同的血管床进行测量。在先进的船舶结构变化评估的特点可以用内膜中层厚度的超声检查。 在微循环，激光多普勒血流仪（LDF）和激光多普勒成像（LDI）与血管扩张激动剂离子导入可以提供微血管灌注12的有用信息。这两种技术测量由散射光产生的多普勒频移由移动红细胞。灌注被表示为血通量，而不是血流量（毫升/分钟），与血液通量反射平均红细胞流速和浓度。血液通量的测定直线与实际的血流13相关联。 LDI的评估提供了相当大的优势超过LDF，因为不像LDF，LDI可以扫描过一个广大的地区从而占异质性皮肤的血流量，增加了该技术的重复性12。 通过血管扩张激动剂乙酰胆碱（ACh）和硝普钠（SNP），它使用的是弱电流14分别评估内皮依赖性和内皮依赖性功能，进入皮肤离子电渗疗法中提供的刺激的LDI中增加血液通量。一旦通过皮肤，胆碱结合到内皮细胞毒蕈碱受体释放的血管扩张剂一氧化氮（NO）。使用的SNP的直接激活平滑肌细胞受体，以允许容器的最大舒张及检验平滑肌完整性15。有对乙酰胆碱介导的扩张是否涉及NO可言，因为乙酰胆碱可刺激非NO途径，如环氧化酶介导的通路12一定的不确定性。然而，我们先前报道说，乙酰胆碱和SNP的反应是在16 CVD的风险增加受损的患者人群和运动干预称为改善没有Bioactivity也提高了使用LDI 17乙酰胆碱介导的血流量。用于输送药剂进入皮肤微血管车辆通常包括氯化钠或去离子水18,19。微血管内皮功能可以用不同的方法，以皮肤血管电导进行量化-磁通通过动脉压除以的产物，在研究中使用，其中血压可能在研究持续时间改变（ 即，在运动过程中，或抗高血压治疗）12。另一种常用的定量是计算区域的曲线血液通量或表达从基线的百分比增加通量。需要注意的是，没有建立用于显示数据的指引是非常重要的，但调查人员应该利用的做法，显示良好的重复性。 在大血管，血流介导的扩张（FMD）和甘油，硝酸介导的扩张（GTN）的执行，以驴s内皮依赖性和内皮独立功能分别为20。口蹄疫典型地在肱动脉，其中一个箍用于阻塞动脉血流5分钟;袖带的释放导致通过肱动脉导致容器的剪应力介导的扩张的血流量（反应性充血）的突然增加。执行手动20或使用自动化的边缘检测软件21,22的基线和后袖释放直径由与容器直径的后续评估容器的超声成像定量。使用硝酸甘油的帮助，以确定是否在血管舒张异常是由于损失中的平滑肌细胞的完整性，或NO的受损释放从内皮细胞23。口蹄疫和GTN表示为百分比增加血管直径相对刺激后与基线直径。 FMD的正确评估需要大量的重要的考虑因素，在研究方案24,25。袖口闭塞的持续时间必须仔细定时; 5分钟袖口闭塞足以对NO介导的扩张而较长袖口闭塞导致非NO介导的扩张26。同样地，从周围的超声探头的手腕和远端闭塞袖带放置主要调用NO介导的扩张，而在上臂和近端到探针袖带放置仅部分地刺激编号27。同样重要的是要在最初的60秒以下袖带放气可以通过25低估口蹄疫内测量的峰扩张以下袖带放气在一段延长的时期，随着峰直径的测量- 40％28。事实上，一个周期为180秒的很可能是足够的捕获真峰直径，与发生的第一120秒28内最峰值。 刺激的FMD涉及S中的生产剪切应力，从而激活特定的内皮受体释放NO 29。但是，剪切应力也可以激活其他几个血管活性因子（其中有一些可能引起血管收缩）30，使得它必须使诱发剪切应力刺激反映血管舒张从编号为通路26。同样重要的是考虑到口蹄疫期间的剪切应力的刺激，以剪切速率（速度/直径），其作为剪切应力的适当措施的计算，但不一定反映峰值流量31。近期生理的建议表明，剪切应力剖面应始终被定性时，超声系统允许脉搏波传导速度和积极的B模式成像的双工模式25同时测量。 使用B型超声的颈动脉评估可以提供对颈动脉内膜中层厚度（CIMT）的信息，并且是第一个describ编于1986年Pignoli和他的同事32。的cIMT的评估反映增殖的平滑肌细胞进入血管的内膜，是临床事件的早期动脉粥样硬化33一个有用的预测。颈动脉超声通常可以预测动脉结构比同类技术（如磁共振成像或放射学评估）34更好。此外，相关联的cIMT与一些传统的CVD危险因素，包括衰老，高血压，和血脂异常35。改变颈动脉的壁通常是由在NO的生物利用度的降低而促进了容器36内的炎症引发。颈总动脉，颈内动脉和颈动脉分叉点可以用来确定的cIMT，因为每个站点可以类似预测心血管事件37。 在本手稿中，我们提供了详细的方法对microvascula评估ř内皮功能（LDI与离子导入），大血管内皮功能（FMD和GTN）和血管形态（CIMT）。动脉粥样硬化是一种多级方法，该方法开始于血管内皮功能障碍和局灶性粥样硬化病变中的大动脉结束。对于选择上述评估的理由是，它们反映动脉粥样硬化的不同阶段，有利于占血管38的异质性。此外，我们以前曾表明，患者在心血管疾病的风险增加了人口，微血管内皮功能是独立于大血管内皮功能39，和功能评估，从血管40的结构的评估是独立的。因此，该脉管的全球评估可以帮助破译动脉粥样硬化的不同阶段。

<table><tbody><tr><td>Laser Doppler Imager</td><td>Moor Instruments, Devon, UK</td><td>moorLDI2</td><td></td></tr><tr><td>Iontophoresis Controller</td><td>Moor Instruments, Devon, UK</td><td>MIC2</td><td></td></tr><tr><td>Miochol-E 20 mg</td><td>Novartis UK</td><td>Prescribed by physician</td><td>Acetylcholine for endothelium-dependent function</td></tr><tr><td>Nitroprussiat Fides 50 mg</td><td>Rottapharm Spain</td><td>Prescribed by physician</td><td>Sodium nitroprusside for endothelium-independent function</td></tr><tr><td>Doppler Ultrasound</td><td>Siemens PLC, Camberley UK</td><td>Accuson Antares</td><td></td></tr><tr><td>Glyceryl Trinitrate 500 mcg</td><td>Alpharma, Barnstaple, UK</td><td>Prescribed by physician</td><td></td></tr></tbody></table>

a methodological approach to non-invasive assessments of vascular function and morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

激光多普勒成像与离子导入下面从一个健康的中年女性自由的激光多普勒成像扫描从CVD法中位数血液磁通单元示于图1中 ，有在血液中位数通量为乙酰胆碱和SNP的显着增加。基线血流量是48灌注单位为乙酰胆碱，和67灌注单位SNP。峰值血流量响应乙酰胆碱是455灌注单位，以及SNP 446灌注单位。这产生了在灌注的增加（相对于基线）对乙酰胆碱和SNP 831％和566％，分别。所提供的数值是高度依赖用于检查皮肤血流量随着环境因素的设备。 血流介导的扩张和硝酸甘油介导的舒张 图2显示了基线和峰值直径为口蹄疫和GTN评估，从健康的年轻男性无心血管疾病。该肱动脉基线直径为3.0毫米的口蹄疫和GTN评估。峰值直径在口蹄疫测试为3.3毫米，而对于GTN评估它是3.9毫米，这分别对应于血流增加10和30％，相对于基线。 颈动脉内膜中层厚度 图3示出了健康个体的左颈动脉。使用自动边缘检测软件进行的cIMT值的计算。该的cIMT在远处的墙上是0.83毫米和血管的管腔直径为7.71毫米。结果为在同一个体的右颈动脉被0.87毫米为的cIMT和7.80毫米为管腔直径。当平均双方的阅读，是的cIMT0.85毫米和管腔直径为7.76毫米。 <img alt="图1" src="/files/ftp_upload/52339/52339fig1highres.jpg" /> 图1。陈使用30μA的电流进行GES血液磁通响应于激光多普勒成像使用离子电渗疗法。基线结束后扫描来测量基线血流量，10扫描（扫描1〜10）与乙酰胆碱和SNP的离子电渗疗法。以下离子导入，2复苏扫描进行。乙酰胆碱乙酰胆碱=; SNP =硝普钠。 <img alt="图2" src="/files/ftp_upload/52339/52339fig2highres.jpg" /> 图2.血流介导的和硝酸甘油介导的扩张 。该图显示了基线直径和下列申请的血流介导的和硝酸甘油介导的扩张刺激的明显增加，峰直径。 FMD =血流介导的扩张; GTN =硝酸甘油介导的扩张。 <img alt="图3" src="/files/ftp_upload/52339/52339fig3highres.jpg" /><br/&gt; 图3.超声颈动脉扫描。左侧颈动脉超声扫描显示有感兴趣的区域放置一厘米从颈动脉球（点分叉）。 <a href="https://www.jove.com/files/ftp_upload/52339/52339fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。 。</a>

Watch this Scientific Journal Video about A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology at JoVE.com

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

方法论方法血管功能和形态的非侵入性评估

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

a-methodological-approach-to-non-invasive-assessments-vascular

Research

JoVE Journal

Medicine

16.2K 次观看.  Bangor University. The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

视频: A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

非侵入性的评估微血管和内皮功能

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

科研

医学

Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive methods1.&#160;Subjects are placed in the supine position and allowed to rest quietly for at least 10 min prior to the start of the exam. The proper cuff size is selected and a blood pressure is obtained using an oscillometric device. Once a resting blood pressure has been obtained, pressure waveforms are acquired from the right femoral and right common carotid arteries. The system then automatically calculates the pulse transit time between these two sites (using the carotid artery as a surrogate for the descending aorta). Body surface measurements are used to determine the distance traveled by the pulse wave between the two sampling sites. This distance is then divided by the pulse transit time resulting in the pulse wave velocity. The measurements are performed in triplicate and the average is used for analysis.

Pulse wave velocity (PWV) measurement assesses arterial stiffness by a tonometry-based system that measures the speed with which arterial pressure wave travels along the arterial tree, typically approximating the time that it takes this wave to travel from the descending aorta (using the carotid artery as a surrogate) to the femoral artery. Carotid-femoral PWV increases two- to threefold across the adult lifespan.

脉搏波传导速度测试老化的巴尔的摩纵向研究中

Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

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 ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

应用 Videomicroscopy 评价人脂肪组织微血管功能

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

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 ...

Quantitative MRI of Endothelial Permeability and (Dys)function in Atherosclerosis

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of vascular damage and thought to drive atherosclerosis. A method to identify these changes in vivo would be desirable in the clinic. Magnetic resonance imaging (MRI)-based tools and other technologies have enabled a profound understanding of the role of the endothelium in cardiovascular diseases and risk in vivo. There is, however, a need for reproducible and simple approaches for extracting quantifiable data reflective of endothelial damage from a single imaging study. A non-invasive, easy-to-implement, and quantitative MRI workflow was developed to acquire and analyze images that allow the quantification of two imaging biomarkers of arterial endothelial damage (leakiness/permeability and dysfunction). Here, the protocol describes the application of this method in the brachiocephalic artery of atherosclerotic ApoE-/- mice using a clinical MRI scanner. First, late gadolinium enhancement (LGE) and Modified Look-Locker Inversion Recovery (MOLLI) T1 mapping protocols to quantify endothelial leakage using an albumin-binding probe are described. Second, anatomic, and quantitative blood flow sequences to measure endothelial dysfunction, in response to acetylcholine are described. Importantly, the method outlined here allows the acquisition of high-spatial-resolution 3D images with large volumetric coverage enabling accurate segmentation of vessel wall structures to improve inter- and intra-observer variability and to increase reliability and reproducibility. Additionally, it provides quantitative data without the need for high-temporal resolution for complex kinetic modeling, making it model-independent and even allowing for imaging of highly mobile vessels (coronary arteries). Therefore, the approach simplifies and expedites data analysis. Finally, this method can be implemented on different scanners, can be extended to image different arterial beds, and is clinically applicable for use in humans. This method could be used to diagnose and treat patients with atherosclerosis by adopting a precision-medicine approach.

Quantitative MRI of Endothelial Permeability and Dysfunction in Atherosclerosis

We have developed an accurate, non-invasive, and easy-to-use method to quantify endothelial permeability and dysfunction in the arteries using Magnetic Resonance Imaging (MRI), named qMETRIC. This technique enables assessing vascular damage and cardiovascular risk associated with atherosclerosis in preclinical models and humans.

Cardiovascular diseases are the leading causes of death worldwide. A permeable/leaky and dysfunctional endothelium is considered the earliest marker of ...

生物工程

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

神经科学

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

以血管内炎组织调节为重点, 利用肠系膜孤立动脉评价血管张力

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

生物学

<meta charset="utf8"></head><body>使用动脉硬度和内皮功能进行心血管评估

Pulse-Wave Velocity, Flow-Mediated Dilation, and Carotid Intima-Media Thickness to Assess Cardiovascular Risk in Population with Metabolic Syndrome

Pulse-wave velocity (PWV), flow-mediated dilation (FMD), and carotid intima-media thickness (CIMT) are established methods used in research and clinical settings to assess arterial stiffness, endothelial function, and subclinical atherogenesis. These measurements may reflect vascular disease and atherosclerotic progression, which are major causes of adverse cardiovascular events. These methods are particularly valuable in determining cardiovascular dysfunction among populations with different risk factors, such as diabetes mellitus, hypertension, and other metabolic dysfunction-related conditions. They provide a non-invasive and reliable source of information that complements clinical practice. Early detection, risk assessment, and therapeutic decisions regarding cardiovascular disease can be achieved, ultimately contributing to improved patient outcomes. Traditional tools for evaluating cardiovascular disease do not reveal whether metabolic syndrome affects early subclinical cardiovascular disease in patients with obesity. Recent research has highlighted the importance of including arterial stiffness and endothelial function in a comprehensive cardiovascular assessment. Therefore, the aim of the present study is to describe methods that provide information on early subclinical vascular aging, endothelial dysfunction, and atherogenic disease, enabling vascular-targeted risk stratification among populations with obesity and different metabolic profiles.

<meta charset="utf8"></head><body>作者聚焦：评估代谢综合征患者的心血管特征

Endothelial/vascular aging and atherogenesis are key mechanisms that promote the development of cardiovascular diseases. The present protocol describes methods to evaluate arterial stiffness, endothelial dysfunction, and atherogenesis in patients with related risk factors, which are highly valuable in the cardiovascular research field.

脉搏波速度、血流介导的扩张和颈动脉内膜中层厚度评估代谢综合征人群的心血管风险

Pulse-wave velocity (PWV), flow-mediated dilation (FMD), and carotid intima-media thickness (CIMT) are established methods used in research and clinical ...