Name: assessing leukocyte-endothelial interactions under flow conditions in an ex vivo autoperfused microflow chamber assay
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R01EY022084 / S1（KMC），T32EY007145（HS）和P30EY014104:研报本出版物中由美国国立卫生研究院国家眼科研究所在奖项数量得到了支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。额外的支持是由马萨诸塞州狮子会眼研究基金（KMC）和特别学者奖从研究到防止失明（以KMC）提供。

对动物的所有实验按照研究协会视觉与眼科声明在眼科和视觉研究使用动物进行了处理，并指导方针和条例规定由马萨诸塞州眼耳医院动物护理委员会。 在实验的前一天: 1.准备管的流室<ol><li>为了制备颈边，1厘米PE10聚乙烯管连接至6cm硅管随后5厘米聚乙烯管。对于颈动脉侧，同样准备的颈静脉侧增加了一个T型管的6厘米硅管内约1.5厘米的距离的1厘米聚乙烯管（ 图1A，B）。为了插入T型管，用细镊子如果有必要扩大硅管。 </li><li> 10厘米硅胶管的一端连接到压力传感器再插入颈动脉的S T端IDE T形管插入的另一端。确保连接部位是否紧固和无泄漏。应用一个矩形小片封口膜过那里的管道一起配合，如果需要的话，为了防止泄漏（ 图1C）。 </li></ol> 2.涂料商会<ol><li>制备内皮表面涂层蛋白（ 例如，P选择，细胞间粘附分子-1（ICAM-1），血管细胞粘附蛋白-1（VCAM-1））中的0.1％BSA中。作为指导原则，1微克蛋白在200μlBSA足以涂覆4-5腔室。用1ml注射器连接到一个地下25针，注入每个室（0.04×0.4×50毫米）与内皮表面涂层的蛋白质。存储在一个15毫升管的涂覆腔室于4℃过夜。 </li></ol>在实验的日子 3.准备商会<ol><li>除去从4℃涂覆的腔室和所述颈和颈动脉导管连接到每个侧。密封connecti附件小心地伸展小一块长方形封口膜在哪里管符合室。使用注射器，填充腔室和管路用0.1％BSA（检查渗漏）。孵育45分钟。 </li><li>制备35毫米培养皿中通过切割凹口上的培养皿的每一侧，以保持腔室。稳定的槽口室;然后用一小滴硅氧烷贴上室向培养皿在切口部位。确保室是看在显微镜下的水平。允许硅胶干燥至少15分钟。 </li><li>填充60ml的注射器的锁定用肝素，并连接到压力传感器。压力换能器连接到颈动脉管按以下顺序:换能器，硅管，和T型管。 <ol><li>使用硅管连接到所述换能器和T型管，以确保在连接之间1cm的PE50。通过压力钳（ 图1D）的颈动脉硅管。运行100USP肝素救援人员到场啊管道和腔室，直到没有气泡。 </li></ol></li></ol> 4.设置软件<ol><li>要准备的软件，打开电脑，显微镜，和开放实验室设备。双击徕卡应用套件（LAS）（或同等学历），并准备一个文件夹来保存视频:浏览→采集→电影→定义剪辑序列（剪辑= 15，记录时间= 30秒，间隔= 15秒）然后点击"+"号确认。 </li><li>要记录流量压力打开LabChart（或同等学历）文件，并协调文件名 ​​记录文件夹在步骤4.1，上面。 </li></ol> 5.手术过程<ol><li>麻醉8-10周龄的鼠标使用氯胺酮（60毫克/公斤）和甲苯噻嗪（6毫克/千克）的组合注入腹腔内（IP）。确认深度镇静是在5-10分钟由踏板撤回反射损耗实现。如果需要的话，异氟烷经面罩递送可以根据需要进行说明。 </li><li>而在麻醉下，放置一个变暖垫笼下并设置到37℃以维持体温。一旦适当的麻醉确认后，将鼠标在腹位置，大衣眼睛杆菌肽软膏，防止眼部干涩。 </li><li>此后，用刀片剃除毛发在切口位点并彻底清洗站点与酒精。 </li><li>一旦完全麻醉的安全用胶带保持鼠标的腹部位置的动物。露出用剪刀颈部区域，并删除甲状腺以暴露气管和血管（ 图2A）。 </li><li>清洁颈动脉，直至其完全暴露，注意不要损伤迷走神经（ 图2B）。折片缝合，并通过颈动脉，然后在弯曲所以现在有2条缝合线的颈动脉下方切下的弯曲。每次循环缝合成结（不要拧紧节）。 </li><li>重复同样的步骤，以暴露左颈静脉（ 图2C），制备缝线的相同方法（ 图2D）。 </li><li>管连接到鼠标脉管。 <ol><li>通过注射将1ml肝素的IP Heparinize鼠标。 </li><li>拧紧颈动脉上缝合。放置在容器夹具低，因为它可以在颈动脉（ 图3A）。使用镊子保持动脉由上缝合，使一个小切口，约1/8的动脉的圆周的，使用microscissors（ 图3B）。 </li><li>通过第二结于上部缝合并进入动脉制成切口通过颈动脉导管。打结下部缝合，以使动脉绕油管密封（几节提出，要确保紧密的密封; 图3C）。 </li><li>重复同样的过程用于插入颈静脉管（容器钳不需要用于颈一侧; 图3D）。 </li><li>通过释放血管钳测试流程。如果没有泄漏将鼠标移动到显微镜和连接管颈动脉和颈静脉腔管（见示范手术附视频）。 </li></ol></li></ol> 6.录音滚动速度<ol><li>打开颈动脉夹钳以填充腔室，使得用鼠标循环系统的电路。允许血液循环3-5分钟，以使流动条件稳定。在3-5分钟的等待期间，调整显微镜阶段，使得该腔室是电平从一端到另一与显微镜物镜和它的边缘与屏幕上的视频记录窗平行。 </li><li>填充含有玻璃室用水然后降低10X水浸物镜放入盘中，使得流过腔室中的细胞是可见的改性培养皿。的光强度可能必须被调整为适当的观看的细胞。通常较低的强度更好。 </li><li>调整管的夹具以使所述换能器读出一个稳定的30毫米汞柱，关联到一个介质生理流动压力，或根据需要。 </li><li>按"开始"开始记录视频，并与第一时间间隔接近颈端和移动对朝向颈端的流动，直到完成了压力两者。 注意:几个涂覆条件使用不同的粘附分子可以通过使用多个腔室进行研究，在相同的鼠标。只有一个室被记录的时间。 <ol><li>当录制完成后，关闭阀门在输入网站，停止血液流动。取出室，并使用在一方面细镊子和钳子开槽在另一方面适应新的室（涂有不同的粘附分子）的电路。简历中的步骤概述录制新室6.1-6.4。 </li></ol></li><li>用CO安乐死所有小鼠2窒息之后是脊髓错位在实验结束。这个程序是终端，因此，将小鼠用脊髓错位在实验结束时安乐死。 </li></ol> 7.解释结果<ol><li>分析记录的数据使用"M TrackJ"插件ImageJ的软件23。 </li><li>转换文件通过选择文件→导入→AVI→转换为灰度到灰度。 </li><li>通过标准化→选择→图像→财产帧进入帧间隔（I）→全球。通过将总的视频的持续时间（一般为30秒），通过帧的数量在一个视频（在的Image J视频窗口的上角示出）计算的帧间隔。 </li><li>测量每个白细胞通过选择插件流动→M TrackJ→添加→曲目选择白细胞→每帧15跟踪它 - 20秒→合并。 </li><li>在视图中的所有白细胞重复步骤7.4。 </li><li>确定白细胞的平均滚动速度被选择的措施→两列显示→复制所有M TrakJ的:跟踪到电子表格中。标有"意思是V"的列是像素/秒的测量白细胞滚动。转换成微米/秒由2/3分。 </li><li>分析压力数据的亮点，出口到电子表格中，然后执行以下计算:ΔP（毫米汞柱）和T（达因/厘米2）每个实验组之间的比较。一旦计算，将数据复制到棱镜软件进行绘图。 </li></ol>

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The authors (L.M., H.S., and K.M.C.) have no competing financial interests to disclose.

白细胞募集的方法，是在炎症反应的关键步骤;它涉及到从向靶组织，在那里它们都能够发挥它们的效应子功能的循环系统的白细胞的迁移。白细胞募集是积分在多种炎性病症，如动脉粥样硬化斑块，心肌梗塞，缺血/再灌注，和移植手术1，以及多种与中枢神经系统有关的神经发炎条件10-12,20,24,25。考虑到本病条件白细胞募集跨度的自动注入微腔提供了一个不可或缺的工具，允许研究者研究白细胞迁移动态能力的多样性。 在过去的几十年里，各种体外测定中已开发研究的白细胞粘附26的动态。不幸的是，所有这些测定的要求的提取从血液中的白细胞，引入机械活化。为了更接近体内环境，适应了以收 ​​集全血样本，控制血流动力学流动条件16,17。在这里，我们通过将一涂覆室向小鼠循环系统延伸在该领域中以往的进展。我们能够调节血液的流动的生理范围和研究白细胞滚动的动态。该涂层室让我们来研究具体的粘附分子白细胞相互作用的能力。由于该系统被用作由一个活老鼠操作的单元中，它更紧密地模仿自然的环境中，让我们来研究在多种免疫小鼠模型白细胞 - 内皮相互作用。此外，该系统使我们能够提供的多种基因小鼠模型的优势。而该系统不完全复制的体内环境，它提供了研究specifi一个平台白细胞的生理条件下用C元素，还没有以前是不可能的壮举。虽然这使我们更接近了一步一个更加符合生理环境有限制的制度。它不完全复制的脉管系统的复杂的三维矩阵​​，所以我们只能够评价被限制在腔室涂层白细胞相互作用的某些要素。此外，大，应注意，以确保封闭系统到鼠标循环由以下中的所有协议中提到的步骤。气泡的引入将大大影响该实验的准确度和可重复性。 尽管我们描述了使用的自动注入微流室的评估白细胞轧制动力学过程必须由调查员进行个性化，研究各种疾病的潜力。例如，癌细胞转移通过表达许多白细胞共享共同整合。学习在克其轧制动力学在一个设置更密切模仿体内环境可能在我们的知识帮助，以及可能的预测的某些癌细胞的侵袭性质，。一种可能的方法可以是一个标记技术结合的癌细胞，如GFP 27，伴随着流室测定法来跟踪表达GFP的肿瘤细胞的轧制动态。定涂覆室与各种物质和连接多个腔室，以相同的鼠标这将是有趣地看到这个过程是如何修改后用于与转基因小鼠和疾病模型组合在其他实验室使用的灵活性。我们在这里描述的技术中仅仅触及一个更广泛的应用潜力，只限于由研究者的创造力。

炎症是机体对损伤的普遍反应，在这两个固有免疫和适应性免疫系统功能的关键一步。响应于损伤和/或炎症刺激，内皮细胞特异性上调粘附分子;这导致通过微血管内皮细胞白细胞外渗，主要是毛细血管后静脉。这个过程开始于自由流动的白细胞在内皮血流的束缚。稳定的轧制和白细胞，这反过来又导致轮回和细胞毒性剂的分泌的牢固粘附，按照此圈养1,2。选择素是已知介导的早期步骤的级联3-5;整合是负责对后面的步骤牢固粘附和轮回1,6-8的。 越来越多的证据表明，人体在缺血再灌注损伤的动物模型白细胞和内皮细胞黏附分子有重要作用，哮喘，银屑病，多发性硬化症，和年龄相关的黄斑变性9-12。在这些条件下，炎症反应是误导攻击自己的身体，导致健康组织的破坏。现有的抗炎剂（如非类固醇抗炎药，皮质类固醇，或其他化学治疗剂）携带的严重副作用长期使用13的风险。因此，它是非常感兴趣的有适当的工具，以确定疾病特异性分子的能力，其可最终定位到具有期望的消炎作用，而剩余的非毒性14。 现有的体外方法如静态白细胞粘附测定法如早在1976年15使用。该平行流动室首次在体外用在1987，研究流动条件下白细胞-内皮相互作用。在这些实验中，刺激呼玛N从静脉血多形核白细胞（PMN）灌注过的原代人脐静脉内皮细胞（HUVECs）的单层。为了控制血液动力学流动条件下，哈佛器械注射泵是采用16。可替代地，为了避免人为白细胞隔离，全血组合使用涂布有固定化的粘附分子17在玻璃室中。 为了避免刺激白细胞和机制上研究近似生理条件下其与粘附分子的相互作用，在体外自动注入，体外循环，动静脉电路开发16。在该电路中，血液中的活老鼠的右颈总动脉和左侧颈外静脉之间流动麻醉下，使玻璃微流腔室涂覆有单个或共同固定化的粘附分子内天然的PBL的相互作用。一个主要的优势，这种SYSTE米是采用基因工程小鼠，其中炎性途径是直接或间接地操纵的能力。此外，还有以查明白细胞粘附分子炎症，游离外部活化的分离的贡献，流动性条件下的能力。一个流量调节器，在流动室的输入点中的应用提供了广泛的剪切力的变化的实验以模拟或者动脉或静脉系统18-22。在这里，我们非常详细地描述关于体外自动注入微流室法的编制和性能的协议。

<table><tbody><tr><td>Micro glass chamber 0.4 x 0.04 x 50 mm</td><td>VitroCom</td><td>2540-050</td><td></td></tr><tr><td>Polyethylene tubing PE 10</td><td>Fisher Scientific</td><td>427400</td><td></td></tr><tr><td>Polyethylene tubing PE 60</td><td>Fisher Scientific</td><td>427416</td><td></td></tr><tr><td>Silicone tubing 002</td><td>Fisher Scientific</td><td>11-189-15A</td><td></td></tr><tr><td>Y tube</td><td>Value Plastics</td><td>Y210-6</td><td></td></tr><tr><td>T tube</td><td>value plastics</td><td>T410-6</td><td></td></tr><tr><td>Silicone gel</td><td>Hardware store - Home Depo</td><td></td><td></td></tr><tr><td>35 mm Petri dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>Parafilm</td><td>Pechiney Plastic Packaging</td><td>PM996</td><td></td></tr><tr><td>Fine forceps</td><td>FST</td><td>11253-25</td><td></td></tr><tr><td>Fine scissors</td><td>FST</td><td>15000-08</td><td></td></tr><tr><td>Tube holder</td><td>FST</td><td>00608-11</td><td></td></tr><tr><td>Clamp applicator</td><td>FST</td><td>18057-14</td><td></td></tr><tr><td>Vascular clamp</td><td>FST</td><td>18055-04</td><td></td></tr><tr><td>6-0 silk sutures</td><td>George Tiemann &amp;amp; Co</td><td>160-1215-6/0</td><td></td></tr><tr><td>25 x 1 G needles</td><td>BD</td><td>305125</td><td></td></tr><tr><td>30 x 1/2 G needles</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Heparin 100 USP units/ml</td><td>Hospital pharmacy</td><td></td><td></td></tr></tbody></table>

assessing leukocyte-endothelial interactions under flow conditions in an ex vivo autoperfused microflow chamber assay

白细胞内皮细胞相互作用的急性和慢性炎症的早期和关键事件，并可以，失调的时候，调解组织损伤导致永久性的病理损害。现有的常规测定法允许白细胞粘附分子的分析只白细胞从血液中提取后。这需要血液经过几个步骤之前外周血白细胞（PBL中）可以准备用于分析，这反过来又可以刺激影响的研究结果的PBL。该微自动注入流动室检测，然而，让科学家用活老鼠的全身流动，同时有操纵涂层室的自由研究早期白细胞功能失调。通过疾病模型，白细胞粘附分子的功能性表达，可以评估和量化在涂覆有固定化的内皮粘连分子体外微玻璃室中。在这个模型中，血液流右颈总动脉和麻醉下活老鼠的左外部颈静脉，允许在腔室天然的PBL的相互作用之间。实时实验分析与活体显微镜的帮助下，以及哈佛仪器加压装置实现的。在该玻璃室中的输入点的流调节器的应用允许可比生理流动条件之间的实验。白细胞滚动速度的主要成果，并采用健康的开放式访问软件ImageJ的全国学院进行测量。综上所述，微自动注入流动室法提供了一个最佳的生理环境研究白细胞内皮细胞的相互作用，并允许研究人员研究炎症时，得出准确的结论。

<html>在每个记录只有相互作用的白细胞是显而易见的上腔室。结果，从一个代表性实验可以看出，在图4中的白色箭头指向由感兴趣的粘附分子被捕获的，并沿之间血流内的其它自由流动的那些腔室被轧制个别的白细胞。室内外的普通神器可以看出，在所有的图像大黑球。暗球帮助读者理解有关的白细胞移动到固定点。白细胞前进一段22秒可以看出，在相对于所述球体。在同一图中的曲线示出了其中数据的Tx1降低轧制速度（移位到左边）和Tx2增加与对照相比的速度（移位到右侧）的一个最后的曲线图。 0"/&gt; 图1:流动室的制备管材。所需的管道和室组件（微玻璃室0.4×0.04×50毫米，聚乙烯管材PE10，聚乙烯管PE60（A）工具，硅管002，Y管，T管35毫米的培养皿，细镊子，剪刀精，管夹持器，血管钳，7-0丝线缝合。（B）中制备的颈动脉和颈静脉导管，用于通过涂覆室重新引导小鼠的血流。（C）的连接颈动脉管至压力换能器。 （D）通过钳位来调节血流的速度传递来自传感器的油管。 <a href="https://www.jove.com/files/ftp_upload/52130/52130fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/52130/52130fig2.jpg" width="700" /> 图2:暴露颈动脉和颈静脉（A）中小心切开颈部，露出气管（B）的清洁的右颈动脉，使得一块缝线可以在容器的下方传递（点缀黄色。线勾勒的颈动脉）。（C）的清洁左侧颈静脉以便一块缝线可以在容器下被传递（虚线黄线勾勒的颈静脉）。（D）的安排松散的上部和下部区域并列缝线颈动脉和颈静脉（箭头所指的船只上下地区）。 <a href="https://www.jove.com/files/ftp_upload/52130/52130fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/52130/52130fig3.jpg" width="700" /> 图3:连接管到t他颈动脉和颈静脉。 （A）拧紧颈动脉上打结，并将低于下结夹。（B）使用microscissors，做一个小切口，在颈动脉，约1/8圆周（点缀黄色圆圈表示切口）。（ C）的插入管插入颈动脉和具有至少两个缝合线（绿色虚线示出了管道和黄色虚线颈动脉）固定。（D）中同样插入管道中的颈静脉（绿色虚线示出了管道和黄色虚线颈静脉）。 <a href="https://www.jove.com/files/ftp_upload/52130/52130fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/52130/52130fig4.jpg" width="700" /> 图4:白细胞通过流动湛滚动的代表时程误码率（比例尺= 100微米）（白色箭头指出轧制白细胞）。图描绘了出口到一个绘图程序之后代表性结果（Tx1和Tx2的是实验性治疗组和点击率是对照组）。 TX1表示其中白细胞减慢导致白细胞轧制速度和图形的左移位相比，未治疗组（CTR）的降低的处理，而Tx2的引出右移位反映出增加白细胞滚动速度。 <a href="https://www.jove.com/files/ftp_upload/52130/52130fig4highres.jpg" target="_blank">请这里查看这个数字的放大版本。</a>

Watch this Scientific Journal Video about Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay at JoVE.com

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

在这里，我们提出了一个协议，允许调查人员通过连接涂上内皮源性黏附分子鼠标循环系统室，以评估白细胞招聘动态体外 。此方法提供了显著优势，因为它允许相对生物学条件下白细胞的评估。

评估白细胞内皮的相互作用下水流条件的<em&gt;离体</em&gt;自动注入微流室法

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

assessing-leukocyte-endothelial-interactions-under-flow-conditions-an

Research

JoVE Journal

Bioengineering

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

9.6K 次观看.  Harvard Medical School, Massachusetts Eye & Ear Infirmary. 在这里，我们提出了一个协议，允许调查人员通过连接涂上内皮源性黏附分子鼠标循环系统室，以评估白细胞招聘动态体外 。此方法提供了显著优势，因为它允许相对生物学条件下白细胞的评估。 

视频: Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

科研

生物工程

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

成像白细胞粘附到血管内皮细胞在高腔内压力

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

免疫与感染

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

人中性粒细胞流动室粘附实验

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

微流控法评价人诱导的多能干细胞内皮细胞 (hipsc-ec) 对白细胞粘附的评价

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

发育生物学

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Intravital Microscopy to Analyze Blood Cell-Endothelial Interactions in an Inflammation Mouse Model

Source: Herr, N. et al., <a href="https://app.jove.com/v/53077">Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the real-time visualization of blood cell-endothelial interactions in the mesenterial veins of a mouse model injected intraperitoneally with lipopolysaccharide.

活体显微镜分析炎症小鼠模型中的血细胞-内皮相互作用

Source: Herr, N. et al., Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

An In Vitro Flow Investigation of the Effect of Stromal Cells on Leukocyte Recruitment

Source: Munir, H. et al., <a href="https://app.jove.com/v/52480">Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow.</a> J. Vis. Exp.&#160;(2015)
The video demonstrates a method for investigating the impact of stromal cells on leukocyte recruitment. In a parallel plate flow chamber model, pro-inflammatory cytokines induce the expression of adhesion receptors by endothelial cells, facilitating neutrophil recruitment and migration. The cross-talk between stromal cells and endothelial cells diminishes neutrophil recruitment to the endothelial cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation and Culture of Primary ...

评估白细胞内皮的相互作用下水流条件的

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