Name: in vitro and in vivo model to study bacterial adhesion to the vessel wall under flow conditions
Uploaded: 2015-06-11T15:00:00.000+00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions at JoVE.com

这项工作是由该基金VOOR Wetenschappelijk Onderzoe​​k（FWO）VLAANDEREN G0466.10，11I0113N支持; "埃迪墨克斯研究基金"和"Sporta研究经费"为小儿心脏病，UZ比利时鲁汶（JC）;该中心分子和血管生物学由Programmafinanciering鲁汶（PF / 10/014）的支持下，由鲁汶大学的"Geconcentreerde Onderzoe​​ksacties"（GOA一十三分之二千零九）和勃林格殷格翰研究经费。

动物实验批准了鲁汶的伦理委员会。 体外灌注和体内实验1.准备细菌<ol><li>我们使用S.金黄色葡萄球菌菌株Newman在这个手稿中描述的所有实验。S.金黄色葡萄球菌Newman贮存在脑心浸液（BHI），用10％甘油在-80℃。 </li><li>使用无菌环刮冷冻细菌关闭并接种在5毫升的胰蛋白酶大豆肉汤（TSB）中O / N在37℃（OD 600&gt; 3）。 </li><li>通过离心（2600克，室温，5分钟）洗涤细菌和悬浮细菌沉淀在5ml PBS（磷酸盐缓冲盐水）。 </li><li>准备一个1mg / ml的溶液的5（6） - 羧基荧光素的N-羟基琥珀酰亚胺基酯（羧基荧光素）的乙醇。稀释的1mg / ml的羧基荧光素的溶液，以150微克/毫升在实验室级水（ 例如，的MilliQ水）。避光管用铝箔和储存在-20℃。 </li><li>离心细菌（2600×g离心，室温，5分钟）。重悬细菌沉淀在800微升PBS中，并加入200微升或150微克/毫升羧基荧光素溶液的400微升（终浓度50μg/ ml的为进行体内实验）（30微克/毫升为灌注实验的最终浓度）。保护管从光用铝箔和孵育在室温30分钟在振荡器上。 </li><li>标记后，用在PBS中的6％的牛血清白蛋白（BSA）溶液块。 </li><li>稀释使用光学密度菌（OD）的OD用于体外实验600 0.65（相当于约3×10 8菌落形成单位（CFU）/ ml的金黄色葡萄球菌 ）和一个外径为进行体内实验600 1.8（对应于在PBS中约1×10 9 CFU / ml的金黄色葡萄球菌 ）。从光用铝箔保护管，置于冰上。 </li></ol> 2. 体外灌流实验<ol><li>盖玻片涂层<ol><li>在实验室级水稀von Willebrand因子（vWF）（Haemate磷，库存浓度2400微克/毫升）（用去离子），以50微克/毫升的最终浓度。 </li><li>在等渗葡萄糖溶液（SKF溶液，pH值2.7-2.9，由制造商提供）至160微克/ ml的终浓度稀释胶原。 </li><li>涂层的玻璃盖玻片（24×50毫米）的VWF或胶原滴加200μl的上封口膜涂层并放置盖玻片上的液滴的顶部。液滴将沿盖玻片的表面上扩散。 </li><li>孵育在加湿容器盖玻片为4小时，在室温。小心地提起从封口膜盖玻片用钝针头。安装盖玻片在流动室的底部部分。 </li></ol></li><li>塑料涂布泥釉与内皮细胞<ol><li>外套塑料单（1阱的PCA细胞培养室，Sarstedt的，德国）用1ml在PBS中的1％明胶溶液并孵育30分钟，在37℃。在明胶包被的塑料单种子的人脐静脉内皮细胞（HUVECs）和成长他们70-80％汇合。装入塑料滑移在流动室的底部部分。 </li></ol></li><li>灌注试验 注: 在体外灌注模型的示意图表示在图1。 <ol><li>执行体外细菌粘附研究中微平行板流动室在层流剪切应力2.5达因/厘米2和20达因/厘米2之间，以模拟不同的生理流动条件。 </li><li>流室（室内设计）由金属框架，并提出了有机玻璃的灌注腔（聚（甲基）丙烯酸甲酯（PMMA））的。通过将其连接到一个高精度输注泵（PHD 2000输液，哈佛仪器，USA）中，我们可以生成流岭0.0001微升/分钟和220.82毫升/分钟之间TES。 </li><li>的管路连接到流动室的上部，并在管道注入介质。轻轻放置在底部部件的顶流室的上部和组装流室。要小心，以避免产生气泡。注射1毫升培养基通过腔室以确保该室是不渗漏，并除去多余的涂覆溶液。避免气泡。 </li><li>鼠标放置在一个热控制的加热垫在37℃下在显微镜托盘。由于这是一个终端的程序，也没有必要为严格asceptic程序。使靠近颈静脉切开，轻轻取出右侧颈肌和从周围组织分离出颈静脉。 </li><li>设置输液泵和荧光显微镜。输液泵设置取决于注射器直径和所需的流量（参见2.4节）。从现在起，工作在一个黑暗的房间。 </li><li> VWF涂层: <ol><li>填的注射器用荧光标记的细菌，并将其连接到入口管。避免气泡。启动输液泵10分钟。输注时间取决于所用的剪切速率和涂层，细菌和介质，并应代表粘连的稳定状态。 </li><li> 10分钟后，洗去未结合的细菌用PBS注射器连接到入口管和启动输液泵。 </li><li>至少要花15幅图像或电影在洗涤过程后不同的位置。细菌是小的和潜在的难以集中。在此之前的体外流动的实验中，合适的焦平面可以将一滴荧光标记的细菌对盖玻片和放置盖玻片在流室进行检索。然后，搜索相应的焦平面并保存设置。 注:在体外流动实验，在清洗步骤期间捕获的图像（开始后±5分钟）确保只信号附着的细菌被捕获。 </li></ol></li><li>胶原蛋白涂层: <ol><li>添加60微克/毫升VWF的荧光标记的细菌刚开始灌注前。填的注射器用补充有60微克/毫升的VWF荧光标记的细菌或荧光标记的细菌，并将其连接到入口管。避免气泡。重复步骤2.3.5.2 2.3.5.3至</li></ol></li><li>内皮细胞: <ol><li>激活的内皮细胞通过灌注用的Ca 2+ -ionophore A23187的0.1毫溶液（原液的10mM溶解在二甲基亚砜（DMSO））的DMEM以相同的剪切速率为细菌灌注10分钟通过灌注用0.1毫米。重复步骤2.3.5.2 2.3.5.3来。 </li></ol></li></ol></li><li>计算剪切速率和剪切应力如下。 剪切速率= 6Q / WH 2 其中:Q:流速在毫升/分钟，瓦特:在厘米宽度中，h:高度厘米 剪切应力（τ）=剪鼠前粘度（μ） 其中μ:媒体:0.01达因x秒/厘米2，全血:0.04达因x秒/厘米2 </li><li>图像分析<ol><li>使用带有黑白摄像机倒置荧光显微镜获取实时图像，并使用图像处理软件开发。用1.5秒的曝光时间。采取多个快照（至少15）随机分布在流室的涂层表面，并将其保存在相应的文件格式。 </li><li>与ImageJ的图像进行分析。减去背景来从图像中删除（处理过程 - Substract背景）平滑连续的背景和定义的阈值来设置下限和上限阈值，分割灰度图像转换成所关注的特征。测量限于阈的区域。 </li><li>比较细菌粘附，表示为荧光区域， 例如，使用统计分析软件。比较使用单向ANOVA或叔基团WO尾学生的t检验。报告所有值平均值±标准平均（SEM）的错误。考虑&lt;0.05显著一个P值（* P &lt;0.05; ** P &lt;0.01; *** P &lt;0.001）。 </li></ol></li></ol> 3. 体内肠系膜灌注模型<ol><li>鼠标的制备/手术<ol><li>实验前禁食的小鼠的夜晚，以限制排便。 </li><li>通过皮下注射丁丙诺啡（0.1毫克/公斤体重（BW））20-30之前的手术分钟得到6-8周龄小鼠（C57BL / 6）的手术前镇痛。 </li><li>通过腹膜内注射氯胺酮（125毫克/千克体重）和甲苯噻嗪（12.5毫克/千克体重），麻醉鼠标。通过检查反射踏板。适用兽医药膏，以防止干燥。 </li><li>鼠标放置在一个热控制的加热垫在37℃下在显微镜托盘。由于这是一个终端的程序，也没有必要为严格asceptic程序。使靠近颈静脉切开，轻轻取出右侧颈肌和从周围组织分离出颈静脉。 </li><li>插入一个2法国静脉导管插入右颈静脉输液荧光标记的细菌或其它解决方案。通过中线腹部切口打开腹腔，并用棉签传播系膜并以可视化的肠系膜动脉和小静脉循环。 </li><li>将鼠标在右侧上的透明板和固定套管用胶带。使用热敷，以防止体温过低。为了防止组织脱水，降500微升0.9％氯化钠的肠子。 </li></ol></li><li>细菌粘附的荧光显微镜肠系膜循环<ol><li>在黑暗的房间工作。用棉签来固定船只和可视化他们倒置显微镜下。 </li><li>局部应用5微升10毫米的解决方案邻f显示的Ca 2+ -ionophore A23187溶解在DMSO中。 10秒后，注入100微升标记的细菌（参见步骤1）通过颈静脉导管。花时间推移图像。后的实验结束后，根据机构批准的指南安乐死的小鼠。 </li></ol></li><li>图像分析<ol><li>获得使用倒置荧光显微镜，采用了黑白摄像机和使用任何图像软件开发捕获的实时图像。应用自动曝光时间和特定于所使用的设备的对比度优化。 </li><li>获得使用工具栏中的"收购"工具（多维收购 - 时间）时间推移图像使用1000张/秒的40个循环。将图像保存在一个合适的图像文件格式。 </li><li>使用ImageJ分析软件来测量每幅图像的荧光信号的区域处理的图像。定义阈值来设置下限和上限阈值，分割灰度图像到f中感兴趣的功能设定。标识感兴趣（血管）的区域，并测量限于阈值和感兴趣的区域的面积。比较细菌粘附，使用任何统计或制图软件表示为荧光区域。 </li></ol></li></ol>

<ol>
	<li>Vanassche, T., Peetermans, W. E., Herregods, M. C., Herijgers, P., Verhamme, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-thrombotic+therapy+in+infective+endocarditis.">Anti-thrombotic therapy in infective endocarditis.</a> Expert Rev Cardiovasc Ther. 9 (9), 1203-1219 (2011).</li><li>Heying, R., van de Gevel, J., Que, Y. A., Moreillon, P., Beekhuizen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-binding+proteins+and+clumping+factor+A+in+Staphylococcus+aureus+experimental+endocarditis:+FnBPA+is+sufficient+to+activate+human+endothelial+cells.">Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.</a> Thromb Haemost. 97 (4), 617-626 (2007).</li><li>Pappelbaum, K. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultralarge+von+Willebrand+factor+fibers+mediate+luminal+Staphylococcus+aureus+adhesion+to+an+intact+endothelial+cell+layer+under+shear+stress.">Ultralarge von Willebrand factor fibers mediate luminal Staphylococcus aureus adhesion to an intact endothelial cell layer under shear stress.</a> Circulation. 128 (1), 50-59 (2013).</li><li>Claes, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+Staphylococcus+aureus+to+the+vessel+wall+under+flow+is+mediated+by+von+Willebrand+factor&#8211;binding+protein.">Adhesion of Staphylococcus aureus to the vessel wall under flow is mediated by von Willebrand factor&#8211;binding protein.</a> Blood. 124 (10), 1669-1976 (2014).</li><li>Thiene, G., Basso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+and+pathogenesis+of+infective+endocarditis+in+native+heart+valves.">Pathology and pathogenesis of infective endocarditis in native heart valves.</a> Cardiovasc Pathol. 15 (5), 256-263 (2006).</li><li>Sixma, J. J., Schiphorst, M. E., Verweij, C. L., Pannekoek, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+deletion+of+the+A1+domain+of+von+Willebrand+factor+on+its+binding+to+heparin,+collagen+and+platelets+in+the+presence+of+ristocetin.">Effect of deletion of the A1 domain of von Willebrand factor on its binding to heparin, collagen and platelets in the presence of ristocetin.</a> Eur J Biochem/FEBS. 196 (2), 369-375 (1991).</li><li>Theilmeier, G., Lenaerts, T., Remacle, C., Collen, D., Vermylen, J., Hoylaerts, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+activated+platelets+assist+THP-1+monocytoid/endothelial+cell+interaction+under+shear+stress.">Circulating activated platelets assist THP-1 monocytoid/endothelial cell interaction under shear stress.</a> Blood. 94 (8), 2725-2734 (1999).</li><li>Bjerketorp, J., Jacobsson, K., Frykberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+von+Willebrand+factor-binding+protein+(vWbp)+of+Staphylococcus+aureus+is+a+coagulase.">The von Willebrand factor-binding protein (vWbp) of Staphylococcus aureus is a coagulase.</a> FEMS Microbiol Lett. 234 (2), 309-314 (2004).</li><li>Friedrich, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylocoagulase+is+a+prototype+for+the+mechanism+of+cofactor-induced+zymogen+activation.">Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation.</a> Nature. 425 (6957), 535-539 (2003).</li><li>Vanassche, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+formation+by+staphylothrombin+facilitates+Staphylococcus+aureus-induced+platelet+aggregation.">Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation.</a> Thromb Haemost. 107 (6), 1107-1121 (2012).</li><li>Vanassche, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+staphylothrombin-mediated+fibrin+deposition+in+catheter-related+Staphylococcus+aureus+infections.">The role of staphylothrombin-mediated fibrin deposition in catheter-related Staphylococcus aureus infections.</a> J Infect Dis. 208 (1), 92-100 (2013).</li><li>Buerkle, M. A., Lehrer, S., Sohn, H. Y., Conzen, P., Pohl, U., Kr&#246;tz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+cyclooxygenase-2+enhances+platelet+adhesion+in+hamster+arterioles+in+vivo.">Selective inhibition of cyclooxygenase-2 enhances platelet adhesion in hamster arterioles in vivo.</a> Circulation. 110 (14), 2053-2059 (2004).</li><li>Kim, K. H., Barazia, A., Cho, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+heterotypic+platelet-neutrophil+interactions+on+the+activated+endothelium+during+vascular+inflammation+and+thrombus+formation+in+live+mice.">Real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice.</a> J Vis Exp. 2 (74), (2013).</li></ol>

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剪应力是为早期细菌粘附于血管壁和用于随后产生的血管内或心内膜赘生物和转移性感染4,5的一个关键因素。我们的体外和体内模型中所述互补研究生理剪切应力下的血管内感染的发病机制。这些模型使我们能够确定血管性血友病因子结合蛋白（vWbp）作为主要的S.金黄色葡萄球菌蛋白流下互动与一个受伤的血管壁露出VWF 4。 血管内感染，尤其是感染性心内膜炎，值得关注，不仅是因为败血症所致的器官衰竭而死亡，但由于也本地和遥远（&#39;转移&#39;）的并发症。引起感染性心内膜炎和转移性感染，细菌有附着​​到容器壁上，从而抗蚀剂流动的血液的剪切应力。最对细菌的研究毒力因子已经在静态条件下被执行。然而，这些相互作​​用建立可能无法承受剪切力，并研究流量条件下，可以揭示新的，以前没有认识到的因素在细菌宿主相互作用。 使用微平行流动室，我们和其他人已经表明的VWF对血管粘附的重要性。在剪切应力，VWF逐步展现出来的，从它的休息球状结构，并公开通过其受体GPIb的血小板6交互的A1域。流动腔室已被广泛用来研究血小板功能7。 值得注意的是，还S.下流动葡萄球菌粘附需要的VWF，以及尤其是在剪切露出的A1结构域。我们确定vWbp调解VWF结合。 vWbp是凝固酶，有助于S.金黄色葡萄球菌病理生理通过激活宿主的凝血酶原。 Staphylothrombin，水库ulting细菌凝固酶和凝血酶原复合物，纤维蛋白原转变成不溶性纤维蛋白8,9。我们的研究表明，vWbp不仅激活凝血酶原，但触发细菌血纤维蛋白-血小板聚集，这提高下流动4,10,11的粘附到血管的形成。 体外流室模型允许不同的球员 ​​在细菌粘附研究细胞或基质成分。细菌毒力因子可以通过使用突变体或无害的细菌表达特定表面蛋白进行研究。可替代地，药理抑制剂或阻断抗体可以被添加到在流动室中的介质。的宿主因素如细胞外基质的不同成分的作用可以通过使用盖玻片与不同涂层进行研究。盖玻片也可覆盖有内皮细胞，其中的活化状态可以通过添加特异性刺激进行调制。阿帕室温从血管壁，宿主血细胞和血浆蛋白质的贡献可以通过加入这些因素，流动介质进行研究。因此，增加了复杂性不同的条件可以在层流的标准条件进行研究，以解开，允许细菌附着于血管壁体内的相互作用。 在体外模型中鉴定相互作用随后研究的动物模型，以测试其相关性在一个复杂的有机体。其它体内模型来研究下流动的动态相互作用已被描述，如仓鼠背皮褶腔12和提睾模型13。相比较而言，这里所描述的肠系膜灌注模型提供，因为它易于使用的几个优点，有可​​能以改变承载小鼠的遗传背景，并评价药物干预。 总之，所描述的模型提供研究表面蛋白不仅S的可能性金黄色葡萄球菌 ，但在不同的主机背景许多其它微生物，以更好地理解血管感染的发病机制。

To establish endovascular infections, pathogens require a mechanism to adhere to the endothelium, which lines the vessel wall and the inner surface of the heart, and to persist and establish an infection despite being exposed to the shear stress of rapidly flowing blood. The most frequent pathogen causing life-threatening endovascular infections and infective endocarditis is Staphylococcus aureus (S. aureus)1. 
Various bacterial surface-bound adhesive molecules mediate adhesion to host tissue by interacting with extracellular matrix components. These MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) recognize molecules such as fibronectin, fibrinogen, collagen and von Willebrand factor (VWF). MSCRAMMs are important virulence factors of S. aureus and are implicated in the colonization and invasion of the host2. Most studies on these virulence factors have been performed in static conditions, and thus may not be representative for human infections where initial adhesion of the bacteria occurs in flowing blood. 
In the case of bloodstream infections, bacteria need to overcome the shearing forces of flowing blood in order to attach to the vessel wall. Models that investigate the interaction between bacteria and endothelium or subendothelium under flow conditions are therefore of particular interest. 
A recent study showed that the adhesion of S. aureus to blood vessels under shear stress is mediated by VWF3. VWF, a shear stress-operational protein, is released from endothelial cells upon activation. Circulating VWF binds to collagen fibers of the exposed subendothelial matrix. Our group reported that the von Willebrand factor-binding protein (vWbp) of S. aureus is crucial for shear-mediated adhesion to VWF4. 
In this article, we present an in vitro flow chamber model where bacterial adhesion to different components of the extracellular matrix or to endothelial cells can be evaluated. To validate the findings from in vitro data, we have developed an in vivo model that visualizes and quantifies the direct interaction of bacteria with the vessel wall and the formation of bacteria-platelet thrombi in the mesenteric circulation of mice, using real-time intravital vascular microscopy.

<table><tbody><tr><td>Brain Heart Infusion (BHI)</td><td>BD Plastipak&amp;nbsp;</td><td>237500</td><td></td></tr><tr><td>Tryptic Soy Broth (TSB)</td><td>Oxoid</td><td>CM0129</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Invitrogen</td><td>14190-169</td><td>D-PBS</td></tr><tr><td>5(6)-carboxy-fluorescein N-hydroxysuccinimidyl ester&amp;nbsp;</td><td>Sigma-Aldrich</td><td>21878-25MG-F</td><td>fluorescent labeling&amp;nbsp;</td></tr><tr><td>Bovine Serum Albumin Fraction V (BSA)</td><td>Roch</td><td>10 735 086 001</td><td></td></tr><tr><td>Haemate-P</td><td>CSL Behring</td><td>PL 15036/0010</td><td>VWF</td></tr><tr><td>Horm collagen</td><td>Takeda</td><td>10500</td><td>collagen</td></tr><tr><td>1-well PCA cell culture chambers</td><td>Sarstedt</td><td>94.6140.102</td><td>plastic slips</td></tr><tr><td>Temgesic</td><td>Reckitt Benckiser</td><td>283716</td><td>bruprenorphine</td></tr><tr><td>Anesketin (Ketamin hydrochloride 115 mg/ml (100 mg/ml ketaminum))</td><td>Eurovet</td><td>BE-V136516</td><td>ketamin</td></tr><tr><td>XYL-M 2% (xylazine hydrochloride 23.32 mg/ml (20 mg/ml xylazine))</td><td>VMD Arendonk</td><td>BE-V170581</td><td>xylazine</td></tr><tr><td>2 french intravenous catheter green</td><td>Portex</td><td>200/300/010</td><td></td></tr><tr><td>0,9% Sodium chloride (NaCl)</td><td>Baxter Healthcare</td><td>W7124</td><td></td></tr><tr><td>cotton swabs</td><td>International Medical Product</td><td>300230</td><td></td></tr><tr><td>Ca2+-ionophore solution A23187</td><td>Sigma-Aldrich</td><td>C7522-10 MG</td><td></td></tr><tr><td>26 gauge 1 ml syringe&amp;nbsp;&amp;nbsp;</td><td>BD Plastipak&amp;nbsp;</td><td>300013</td><td></td></tr><tr><td>26 gauge 1 ml syringe&amp;nbsp; with needle</td><td>BD Plastipak&amp;nbsp;</td><td>300015</td><td>intra-peritoneal injection</td></tr><tr><td>Centrifuge 5810-R</td><td>Eppendorf</td><td>5811 000.320</td><td></td></tr><tr><td>Glass cover slips (24x50)</td><td>VWR</td><td>BB02405A11</td><td>Thickness No, 1</td></tr><tr><td>PHD 2000 Infusion</td><td>Harvard Apparatus</td><td>702100</td><td>High-accuracy Harvard infusion pump</td></tr><tr><td>Axio-observer DI</td><td>Carl-Zeiss</td><td>Inverted fluorescence microscope</td><td></td></tr><tr><td>ImageJ</td><td>National Institute of Health</td><td>Analysis software</td><td></td></tr><tr><td>Graphpad Prism 5,0</td><td>Graphpad Software</td><td>Analysis software</td><td></td></tr><tr><td>AxioCam MRm</td><td>Carl-Zeiss&amp;nbsp;</td><td>Black and white camera</td><td></td></tr></tbody></table>

in vitro and in vivo model to study bacterial adhesion to the vessel wall under flow conditions

为了使血管内感染和感染性心内膜炎，细菌必须能够粘附于血管壁，同时被暴露于流动的血液的剪切应力。 要识别有助于微生物血管粘附的细菌和宿主因素，需要的研究生理剪切条件下，这些相互作​​用合适的模型。在这里，我们描述了一种在体外流动室模型，允许调查细菌粘附到细胞外基质的不同成分或与内皮细胞，并开发了一个活体显微镜模型直接可视化的细菌的初始粘合力，以在体内内脏循环。这些方法可用于鉴定所需细菌下流动的粘附的细菌和宿主因素。我们示出了剪切应力的相关性和血管性血友病因子为Staphy的粘合作用黄色葡萄球菌同时使用体外和体内模型。

金黄色葡萄球菌的粘合性的VWF，内皮下基质和内皮细胞是一种剪切应力依赖性现象要强调在间S的相互作用的剪切应力的作用金黄色葡萄球菌和VWF，我们进行灌注过的VWF在不同剪切速率涂覆的盖玻片（ 在体外灌注模型的示意图在图中给出的金黄色葡萄球菌 1。粘附到VWF从250秒的增加而增加的剪切速率-1〜2,000秒-1（ 图2），这表明高的剪切力不抑制，但加强细菌附着到VWF。 为了研究的VWF的细菌粘附于胶原的贡献，内皮下基质的主要成分，我们灌注荧光标记S.金黄色葡萄球菌在胶原中VWF的存在或不存在。在不存在的VWF，S的密合性一个ureus胶原随着剪切速率降低。然而，当VWF是存在于培养基中，S的粘着黄色葡萄球菌的增加而增加的剪切速率（ 图3）。 体外流模型还允许我们检查细菌的粘附下流动于内皮细胞。我们内皮细胞灌注与荧光标记的S.金黄色葡萄球菌在剪切速率为500〜2000秒-1。凡表示，内皮细胞被激活了的Ca 2+ -ionophore，造成VWF释放。内皮细胞的活化和随后的VWF释放，S的增加粘附金黄色葡萄球菌 （ 图4A），从而形成典型的"带状"图案的荧光标记在剪切力（ 图4B）的方向上排列的细菌集群，这表明沿着线性拉伸的VWF分子的细菌的结合。 最初在体内细菌附着在内脏静脉被VWF介由于S.金黄色葡萄球菌是能够坚持的VWF，我们用野生型小鼠（VWF + / +）和VWF缺陷型小鼠（VWF - / - ），调查细菌粘附到活化血管壁体内 。内脏静脉实时电视显微镜允许体内可视化的循环荧光标记S.黄色葡萄球菌 （ 在体内灌注模型的示意图概述示于图5）。 内皮由钙 -ionophore药理活化后，我们观察到个体的细菌和细菌的聚集体的快速局部堆积到WT小鼠的血管壁（补充视频1和2）。细菌几乎没有发生粘着上观察到VWF -deficie的活化的血管壁nt的小鼠（补充视频3）与WT小鼠的附着力相比（ 图6）。不存在的VWF降低S的能力金黄色葡萄球菌坚持以激活血管壁。 <img alt="图1" src="/files/ftp_upload/52862/52862fig1.jpg" /> 图1. 在体外流动模型的示意图。 在体外流动模型是一种多功能模型，它允许不同的剪切依赖性机制的研究，如细 ​​菌粘附于内皮下基质也是血栓形成。微平行流动室被放置在盖玻片（塑料或玻璃）与蛋白质和血管内皮细胞的不同的涂层。不同的细菌（橙色和灰色点）的密合性，可以分析和血浆蛋白，血小板和全血的存在的影响进行评估。荧光标记的血小板（蓝色椭圆形）或fibrinog烯（蓝色串）可以用在具有不同的抑制剂（黑色椭圆）组合来区分细菌和宿主因素。 S的 ​​细菌粘附的代表图像黄色葡萄球菌对VWF胶原涂层的存在（底部）或无（顶部）显示（比例尺为100微米）。 <a href="https://www.jove.com/files/ftp_upload/52862/52862fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52862/52862fig2.jpg" /> 图2.粘附S.黄色葡萄球菌对VWF增加而增加的剪切速率。微平行流动室灌注过涂覆的VWF（50微克/毫升）与荧光标记S.金黄色葡萄球菌Newman在250至2000秒-1（秒-1）在中等剪切速率（正&gt; 5）。所有结果表示为平均值±SEM表示。 * P &lt;0.05，** P &lt;0.01。 <img alt="图3" src="/files/ftp_upload/52862/52862fig3.jpg" /> 图3.粘附S.黄色葡萄球菌对内皮下膜是剪切和VWF依赖性。微平行流动室灌注过涂覆胶原（160微克/毫升）与荧光标记S.金黄色葡萄球菌Newman在250至2000秒-1在中等剪切速率（正&gt; 5）。 VWF（60微克/毫升）存在于培养基中，其中表示。所有结果表示为平均值±SEM表示。 ** P &lt;0.01。 <img alt="图4" src="/files/ftp_upload/52862/52862fig4.jpg" /> 图4.粘附S.黄色葡萄球菌对活化的内皮细胞是剪切依赖性的。微平行流动室灌注过的内皮细胞。 （ 一 ）人脐静脉内皮细胞的钙离子-ionophore一个激活23187（0.1毫摩尔），接着通过一个10分钟的灌注荧光标记S.金黄色葡萄球菌Newman在500至2000秒-1在中等剪切速率（正&gt; 5）。所有结果表示为平均值±SEM表示。 * P &lt;0.05。微平行流室灌注（B）在图像内皮细胞活化与S.金黄色葡萄球菌在1000秒的剪切速率-2 S。金黄色葡萄球菌形成了±200微米长字符串，暗示附着力VWF多（比例尺为100微米）。 <a href="https://www.jove.com/files/ftp_upload/52862/52862fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52862/52862fig5.jpg" /> 图5. 体内肠系膜灌注模型的示意图，一个右颈内静脉置管（黄线）插入氟的管理escently标记的细菌（橙色点），附加的麻醉剂或其它成分，如药物抑制剂和抗体。腹膜腔被打开，肠系膜铺展以可视化的血管（静脉和动脉）在荧光显微镜下。内皮由钙 -ionophore，其诱导的VWF的释放药理活化后，细菌可以通过颈静脉导管注射。实时视频血管显微允许循环荧光标记的细菌在体内的可视化和由此产生的细菌血小板血栓形成。 <a href="https://www.jove.com/files/ftp_upload/52862/52862fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52862/52862fig6.jpg" /> 图6的S的初始粘合金黄色葡萄球菌。 - / -小鼠对激活的内皮是在体内通过的VWF 的体内静脉肠系膜灌注之C57B1 / 6- VWF + / +和C57BL / 6- VWF模型介导的 。粘连的荧光标记S.金黄色葡萄球菌的局部活化血管壁是显著低于VWF - / -小鼠。所有结果表示为平均值±SEM表示。 *** P &lt;0.001，N&gt; 7。 <img alt="视频图1" src="/files/ftp_upload/52862/52862figV1.jpg" /> 视频1:S的实时粘连金黄色葡萄球菌活化血管壁VWF + / +小鼠。 <a href="https://www.jove.com/files/ftp_upload/52862/Video1JorienClaes.mov" target="_blank">请点击此处观看该视频。</a> 视频2:实时聚集形成和栓塞S.金黄色葡萄球菌在VWF + / +小鼠。 <a href="https://www.jove.com/files/ftp_upload/52862/Video2JorienClaes.mov" target="_blank">请点击此处观看该视频。</a> <img alt="视频图3" src="/files/ftp_upload/52862/52862figV3.jpg" /> 视频3:S的实时粘连黄色葡萄球菌对活化血管壁中VWF - - / -小鼠/ -与VWF + / +和VWF 小鼠 体内肠系膜灌注模型。五微升钙 -ionophore（10毫米）的是APPLI编到的可视化血管床的区域。羧基荧光素标记S的悬挂金黄色葡萄球菌是通过颈静脉导管注入。肠系膜循环被倒置显微镜下观察。 <a href="https://www.jove.com/files/ftp_upload/52862/Video3JorienClaes.mov" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions at JoVE.com

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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Immunology and Infection

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

10.6K 次观看.  KU Leuven. To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion. 

视频: In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

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. ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

的剪切粘合地图使用SynVivo合成微血管网络代

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

生物工程

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

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

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

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

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 ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

发育生物学

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

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 ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

研究流动下血管药物靶向的体外3D细胞培养动脉模型

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

恒流中原发性人类内皮细胞的肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

实时研究全血-内皮相互作用和血栓动力学的体外微流体疾病模型

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...