Name: evaluation of bioenergetic function in cerebral vascular endothelial cells
Uploaded: 2016-11-19T15:00:00.000+00:00
Duration: 6 min 15 s
Description: Watch this Scientific Journal Video about Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells at JoVE.com

This work was supported by the following grants: AHA 16SDG31170008 to X.R. and NIH P20 GM109098, P01 AG027956, and U54 GM104942 to J.W.S.

注:该协议的方案示于图1，其中包括的步骤的时间表。 1.文化和CVE细胞通道<ol><li>培养CVE细胞（bEnd.3细胞）在完全生长培养基（高葡萄糖的Dulbecco改良的Eagle氏培养基，DMEM）中补充有10％胎牛血清（FBS）和1％青霉素/链霉素的T75厘米2组织培养烧瓶中。在一个5％CO 2培养箱生长于37℃。 </li><li>卸下并丢弃细胞培养基。冲洗用0.25％胰蛋白酶0.03％的EDTA溶液的细胞层，然后添加1 - 2毫升胰蛋白酶-EDTA溶液到烧瓶中，并保持该烧瓶在37℃，3 - 5分钟。观察的倒置显微镜下细胞直至细胞层被分离。加入10 mL的完全生长培养基中，并轻轻吹打吸出细胞。 </li><li>滗流体和细胞进入有15毫升离心管中并旋转，在700×g离心5分钟，除去细胞碎片。 </l我&gt; <li>丢弃从离心管的上清液。加入10 mL的完全生长培养基中，并重新悬浮细胞沉淀。旋转细胞在700×g离心5分钟。 </li><li>丢弃从离心管的上清液。重悬在完全生长培养基将细胞沉淀在2.0×10 5个细胞/ mL的浓度（通过使用血细胞计数器计数测定;可以通过台盼蓝染色排除死细胞）。 注:新鲜解冻细胞对于至少3个通道用于实验亚培养。 </li></ol>在细胞培养板2.种子和细胞治疗<ol><li>上的96孔细胞外磁通细胞培养板的种子细胞:80微升/孔。放置在37℃的细胞培养板在5％CO 2的培养箱中。注:每孔16×10 3个细胞将达到60 - 24小时内80％汇合。 </li><li>如果在细胞被暴露于化学，添加处理一旦将细胞附着在板的底部（8 - 24小时后看到鼎）。 注:对于转染实验中，不完全生长培养基中添加抗生素。 </li></ol> 3.线粒体功能的评价使用生物分析仪<ol><li>前测定的开始，通过加入外磁通校准溶液的150微升到板，并在37℃下无CO 2的孵育O / N水合的一个细胞外磁通传感器盒与校准。 </li><li>使用洗板站，细胞培养基改变成pH为7.0的细胞外磁通测定培养基。孵育在37℃的细胞无CO 2的30 - 60分钟。 </li><li>准备8μM寡，4.5微米FCCP，10μM鱼藤酮，并在10 DMEMμM抗霉素A的工作方案。装载25μL储备液成盒注射端口:端口A，寡;端口B，FCCP;端口C，鱼藤酮和抗霉素A与细胞外通量分析网上平台。如表1中所述实施分析方案。 </li><li>加载水合盒插入生物分析仪，并通过单击"开始"按钮进行校准。校准完成后，取出硒鼓底板，然后通过单击"卸载磁带"的提示加载单元板。继续试验直至所有测量的结束。 </li><li>打开数据文件并获得生物分析仪的速度值。将数据导出到电子表格文件。 </li></ol> 4.数据分析注:数据分析的计算如下制定。作为OCR 图2中呈现的值是从生物分析仪获得的。 <ol><li>为了计算基础呼吸 ，减去非线粒体呼吸（测量10 - 12）从基础值（测量3）。 注:基础呼吸=基础 - 非线粒体呼吸=率③ - 费率⑩⑫〜。 </li><li>为了计算ATP生产图片上，减去的ATP联呼吸（测量4 - 6）从基值（测定3）。 注:ATP生产=基础 - ATP挂钩呼吸=率③ - ④率⑥〜。 </li><li>要计算最大呼吸 ，减去非线粒体呼吸（测量10 - 12）最大呼吸（测量7 - 9）。 注:最大呼吸=最大呼吸 - 非线粒体呼吸=率⑦〜⑨ - 费率⑩⑫〜。 </li><li>要计算余力 ，减去最大呼吸基础值（测量3）（测量7 - 9）。 注:备用容量=最大呼吸 - 基底=率⑦〜⑨ - 率③。 </li><li>计算质子泄漏 ，减去非线粒体呼吸（测定10 - 12）从ATP联呼吸（测定4）。 注:质子漏= ATP挂钩呼吸 - 非线粒体升呼吸率=④ - 费率⑩⑫〜。 </li></ol>

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The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Open Access fees for this article were provided by Agilent Technologies.

该协议代表了脑血管内皮细胞的生物能量表型的评估的方法。它用作用于内皮细胞线粒体评价基本分析，它是最佳的设计，调查刺激可能影响在CVE细胞线粒体信号传导途径的机制的实验。它也提供了用于测试可能的治疗剂为BBB-破坏相关疾病的方法。 该议定书中的关键步骤胞磁通bioanalyzers必须测量的OCR实时能力。在该测定中，确定适当的细胞密度是至关重要的。如果细胞密度低于每孔8×10 3个细胞，基底OCR太低待分析（ 图2和图3）;如果细胞密度高于每孔32×10 3个细胞，该细胞不响应寡或FCCP treatmeNTS（ 图2和图3）。在我们的实验模型，该细胞系的最佳细胞密度为每孔，这表明复制的结果和敏感性刺激7和治疗10,14 16×10 3细胞。如果使用24孔生物分析仪，细胞密度的新滴定将需要用于测定。 据记载，的CVE具有大的体积线粒体与其它内皮细胞或组织类型15,16相比，表明更大的能源生产和更少的细胞，用于测量生物能量代谢的需要。我们已经测试了几种类型的脑内皮细胞，如初级和永生化的鼠脑的内皮细胞7和永生化人脑内皮细胞（未公布的数据）。要求这些细胞相似的细胞密度40内达到可接受的OCR（基础呼吸OCR - 160皮摩尔/分钟96孔板和50 - 400皮摩尔/ 24孔板分钟）时，进行的生物能学测定法。 Kaczara 等报道测量生物能量代谢中的人脐静脉内皮细胞（HUVEC），它使用了更高的细胞密度17。 我们的团队没有评估来自其他组织或器官血管细胞。从理论上讲，该生物分析仪可能被在任何细胞类型使用，但它需要优化细胞密度，细胞培养基，和培养条件（某些特定的细胞可能需要涂层板生长良好）测定。它要求该实验完成，以确保OCR率在可接受的范围之内。如果OCR在从其它器官的内皮细胞比从大脑的CVE下，增加细胞密度可以帮助到可接受的OCR的范围内得到。另外，如果电池不使用氧化磷酸化作为其主要能源，生物分析仪不会是一个最优升检测。 生物分析仪允许电子传递链的连续中断，基于在施加试剂的顺序。首先，寡被应用，其抑制线粒体复合物V（ATP合酶）。第二，FCCP，电子解偶联剂被施加，这导致质子梯度的干扰。最后，鱼藤酮和抗霉素A，其抑制线粒体复合物I和III分别被施加到导致电子流的总抑制。药物暴露的顺序是必要的，因为该药物阻断的特定电子传输链反应，和顺序的变化可以被测量以反映线粒体功能。 修改和故障排除以评估线粒体响应在CVE细胞对刺激，则建议的刺激被（见时间轴施加到细胞后的细胞粘附到细胞培养板底部在图1中示出;它通常需要至少6小时）。由处理获得可复制的结果，以保持细胞密度一致是非常重要的。如果预处理之前将细胞接种设计的，对于每个前处理细胞密度应仔细测量，但不包括用于播种的死细胞。如果转染是包含在实验设计，请参照制造商提供的转染方案。在图4给出的数据证明中，miR-34a的转染到使用脂质体转染试剂盒，这要求无抗生素的细胞培养基和丝裂应力测试用miR-34a的表达，以评估在代谢功能的变化CVE细胞。质粒的剂量也可以被优化，如先前发表13。可以对协议进行另一个变化是改变试剂的浓度。寡，FCCP，和/或鱼藤酮和抗霉素A的滴定曲线可以完井特德。 此外，如果各种药物该测定被用于高通量分析，建议包含一个平行试验，以测量细胞生存力和细胞增殖，这是在我们以前的出版物7,13-说明。 OCR的值由细胞数目（ 图2和3）显著的影响，并在细胞增殖和活力测定法中获益的数据的正常化。然而，这些试验的完成是在每个单独的实验室的判断。 该技术的局限性这个协议的主要限制是，我们仅仅在研究中使用的体外细胞培养物模型。目前，没有可用的体外模型或动物模型可针对内皮细胞的线粒体功能。预期新的模型生物能量函数的体内评估待开发和<eM&gt;体外在未来的研究。 另一个限制是继生物能措施的障碍评估的扩展。因为，首先，将生物能量测量完成后，细胞存活率减弱的电子传递链的完整的中断不能进行的实验;第二，需要特定的细胞培养板和插入检测生物能量值，不适合障碍评估插入。因此，没有能生物能学分析后可以完成很多功能分析。然而，可以预期的特殊装置，可开发来执行前的生物能测定功能测定。 关于到现有/替代方法的技术意义先前的技术来评价线粒体功能必要线粒体的分离从细胞18。该网元使用生物分析仪W¯¯技术允许完整细胞线粒体活性，它保留超过评估分离线粒体细胞环境的测量。 掌握这一技术后，未来的应用方向或该协议被设计和用于bEnd.3细胞系开发的，但它也与初级脑血管内皮（pCVE）细胞7或其他内皮兼容，我们已经在我们以前使用pCVE电池7出版物表明了这一点。当使用其他类型的内皮细胞的，可能需要在培养板的涂布和使用的生长因子。然而，滴定试验被推荐用于其它细胞类型为好。这个协议提供了在CVE细胞的生物能学的评价将要通过通常的方法，它可以进一步施加到机构的研究或以这种方式治疗反应。

人们普遍认识到，通过脑血管的内皮（CVE）细胞所形成的血 - 脑屏障（BBB）具有极为重要的血管生物学非常不同和独特的功能。这些内皮细胞使用线粒体生成最三磷酸腺苷的细胞供应（ATP）的化学能量源。除了用于CVE细胞中提供的ATP，线粒体调节在血管内皮细胞，如细胞信号1-4的各种细胞过程，细胞凋亡5，和细胞周期控制和细胞生长6。在CVE细胞线粒体生物能量功能的失调可能影响线粒体生物合成，从而导致受损血管内皮活性，并加剧脑血管疾病和神经变性疾病， 如中风7,8和阿尔茨海默氏病（AD）9。我们已经表明，暴露于lipopolysac侮辱CVE细胞charide（LPS）损害的CVE细胞线粒体功能和恶化中风成果7。叔丁基氢醌（TBHQ）通过在CVE电池10的氧化磷酸化的干扰增加中风的死亡率。因此，生物能量功能的CVE细胞不仅导频用于中枢神经系统的一系列机制相关的研究（CNS）的疾病的定量模型，而且还提供了在治疗中血管生物学靶向线粒体功能的药物筛选的一个突破。 分离线粒体已被用来测量生物能的功能。我们7和其他11个通过使用外通量生物分析报告完整CVE细胞生物能量的细胞的评估。该分析仪提供了内源性细胞生物能量评估的优势。这个敏感和一致的测定测量CVE细胞的线粒体代谢和可用于评估由STI生物能障碍木里，调查线粒体信号通路的影响在CVE细胞线粒体功能的机制，和屏幕药物或治疗。耗氧率（OCR）是通过使用药理分析方法相结合，使用四个线粒体干扰生物分析仪记录:寡，羰基氰化物-4（三氟甲氧基）苯腙（FCCP），鱼藤酮，抗霉素和A.在该协议中，我们详细的优化方法，使该测量和CVE细胞线粒体机能参数，包括基底线粒体呼吸，ATP产生，质子泄漏，最大呼吸，和备用呼吸容量的计算，在一个单一的检测。

<table><tbody><tr><td>bEnd.3 cell line</td><td>ATCC</td><td>CRL-2299</td><td>25 - 30 passages</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S12450</td><td>10% final concentration</td></tr><tr><td>Penicillin/Steptomycin</td><td>Hyclone</td><td>SV30010</td><td>1&amp;times;100 stocking</td></tr><tr><td>0.25% trypsin, 0.03% EDTA solution</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Corning</td><td>25-000-CI</td><td>1.0 &amp;micro;M final concentration</td></tr><tr><td>Glucose</td><td>Sigma</td><td>CAS 50-99-7</td><td>25 mM final concentration</td></tr><tr><td>Oligomycin</td><td>Sigma</td><td>O4876</td><td>1.0 &amp;micro;M final concentration</td></tr><tr><td>Carbonilcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)</td><td>Sigma</td><td>C2920</td><td>0.5 &amp;micro;M final concentration</td></tr><tr><td>Rotenone</td><td>Sigma</td><td>R8875</td><td>1.0 &amp;micro;M final concentration</td></tr><tr><td>Antimycin A</td><td>Sigma</td><td>CAS 1397-94-0</td><td>1.0 &amp;micro;M final concentration</td></tr><tr><td>Plate wash station</td><td>Seahorse Bioscience</td><td></td><td></td></tr><tr><td>Extracellular flux bioanayzer&amp;nbsp;&amp;nbsp;</td><td>Seahorse Bioscience</td><td>XFe96</td><td></td></tr><tr><td>Extracellular flux cell culture plate</td><td>Seahorse Bioscience</td><td>102416-100</td><td></td></tr><tr><td>Extracellular flux sensor cartridge</td><td>Seahorse Bioscience</td><td>102416-100</td><td></td></tr><tr><td>Extracellular flux calibrant solution</td><td>Seahorse Bioscience</td><td>100840-000</td><td></td></tr><tr><td>Extracellular flux assay medium</td><td>Seahorse Bioscience</td><td>102365-100</td><td>PH buffered prior to assay</td></tr></tbody></table>

evaluation of bioenergetic function in cerebral vascular endothelial cells

血 - 脑屏障（BBB）的完整性是关键的，以防止脑损伤。脑血管内皮（CVE）细胞是包含血脑屏障的细胞类型中的一种;这些细胞具有非常高的能量需求，这需要最佳线粒体功能。在疾病或损伤的情况下，在这些细胞中的线粒体功能可以被改变，从而导致疾病或血脑屏障的开口。在这个手稿中，我们将介绍使用的整体，完整的细胞和生物分析仪测量CVE细胞线粒体功能的方法。一个三刀应力测定是用来挑战已经被扰动，物理或化学的细胞，并评估其生物能量的功能。此外，这种方法还提供了筛选具有对线粒体功能的直接影响新的治疗剂的有用方法。我们优化所需的细胞密度，以产生氧消耗速率，其允许多种线粒体段的计算平方米，包括ATP的产生，最大的呼吸和备用容量。我们还通过证明引入微小RNA中，miR-34a的，导致线粒体活性显和检测到的降低表明该测定的灵敏度。而在本文中示出的数据是针对bEnd.3细胞系的优化，我们还进行了优化初级CVE细胞协议，进一步表明在临床前和临床模型的效用。

评估响应于氧化应激CVE细胞的生物能量函数，我们选择了鼠脑微血管内皮细胞系bEnd.3，其显示相同的屏障特性如原发性脑微血管内皮细胞12。鉴于动力学和响应的相对强度是在各种细胞类型中改变，第一系列的实验中，设计通过识别在测定代谢分析使用bEnd.3细胞的最佳数量，以获得可测的OCR的水平，显示在图2.继评估，数据被量化，并在图3中。基础呼吸，最大呼吸呈现，和备用呼吸容量显示出与细胞密度的比例响应。然而，当选择了每孔64×10 3个细胞的ATP产量下降，这表明在汇合细胞培养物是不适合于该实验。最优细胞密度发生8之间-每孔的细胞的32×10 3个的基础上，响应于线粒体干扰。 对于随后的实验，每孔16×10 3个细胞的接种密度被用来允许在OCR变化的最佳检测。用每孔16×10 3个细胞，我们观察到在OCR所期望的响应，并表明微RNA的miR-34a的减少CVE细胞线粒体功能（ 图4）。我们以前曾报道了miR-34a的在这些细胞中13减少氧化磷酸化。重复实验还表明，最大呼吸和备用容量由在的CVE的miR-34a的表达在24小时后转染显著下降，即使基础呼吸，ATP产生，和质子漏无显著变化（ 图4） 。这些数据证明生物分析仪的灵敏度来检测的CVE中线粒体功能的变化。 <img alt="图1" src="/files/ftp_upload/54847/54847fig1.jpg" /> 图1.战略规划实验。时间线为单元，板和墨盒准备指示。 <a href="http://ecsource.jove.com/files/ftp_upload/54847/54847fig1large.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/54847/54847fig2.jpg" /> 在CVE细胞不同的细胞密度测量图2. CVE生物能细胞功能的各种细胞密度的代表性原始数据。耗氧率。数据代表平均值±标准差（n = 5）; OCR:耗氧率; FCCP:羰基氰化物-4-（三氟甲氧基）苯腙;<html>腐/防答:鱼藤酮和抗霉素A.①，②，③和基础注明呼吸; ④，⑤，⑥和ATP注明链接呼吸; ⑦，⑧，⑨和最高指示呼吸; ⑩，⑪，⑫和指示非线粒体呼吸。 <a href="http://ecsource.jove.com/files/ftp_upload/54847/54847fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54847/54847fig3.jpg" /> 图3.在用各种细胞密度CVE细胞生物能功能的代表结果。基底呼吸，ATP产生，最大呼吸，和备用容量是从由生物能产生的原始数据计算出的图使用公式2功能测定在第4所示。数据代表平均值±标准差（n = 5）; OCR:耗氧率。<html> F ="http://ecsource.jove.com/files/ftp_upload/54847/54847fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54847/54847fig4.jpg" /> 图4:受miR-34a的生物能降低功能代表性的成果 （A）以下24小时后转染的miR-34a的表达线粒体功能的原始数据。 （B）的基础呼吸，ATP产生，最大呼吸，和备用容量是从由生物能在图4A功能测定法产生的原始数据计算;该参数是由在第4节的miR-34a的表达所指示的公式来计算减少了在24小时后转染CVE细胞线粒体功能。数据代表平均值±标准差（n = 5）; OCR:耗氧率。 ****，P &lt;0.0001。文件/ ftp_upload / 54847 / 54847fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/54847/54847table1.jpg" /> 表1仪器运行协议。

Watch this Scientific Journal Video about Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells at JoVE.com

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

内皮细胞线粒体是维持血脑屏障完整性的关键。我们引入一个协议来测量脑血管内皮细胞生物能量的功能。

在脑血管内皮细胞生物能量功能的评价

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

evaluation-bioenergetic-function-cerebral-vascular-endothelial

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.2K 次观看.  West Virginia University. 内皮细胞线粒体是维持血脑屏障完整性的关键。我们引入一个协议来测量脑血管内皮细胞生物能量的功能。 

视频: Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

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

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

神经科学

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

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

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

肱动脉的临床研究超声评价血管内皮依赖性血流介导的血管舒张功能

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

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.

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 ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

一个简单的生物测定血管内皮生长因子的评价

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

生物学

跨内皮抗性测量以评估细胞屏障完整性

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

作者聚焦:TEER 检测在评估细胞屏障完整性中的应用 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

在脑血管内皮细胞生物能量功能的评价

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非侵入性的评估微血管和内皮功能

代的永生化小鼠脑微血管内皮细胞株作为

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

方法论方法血管功能和形态的非侵入性评估

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

一个简单的生物测定血管内皮生长因子的评价

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量