该研究是由美国国立卫生研究院HL75503以KP支持。

对动物进行的所有实验均批准的田纳西大学健康科学中心大学的机构动物护理和使用委员会。 1，油管灌注大鼠肺制剂<ol><li>准备一个管道系统用聚乙烯管（＃18），用于血液灌注， 如图1连接的压力传感器（P23XL），并放置在显微镜载物台上的管件。 </li><li>设定水浴温度为37°C。 </li><li>连接肺入口，并与聚乙烯管暂时出口。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> 图1。血液灌注管 。该图显示了用于通过隔离肺制剂血液循环管路的设置。也表明是相关联的组件，通过该管子我s路由。心脏和肺的示意图包括显示管和肺动脉（PA）之间的连接部位，和左心房（LA）套管（蓝色虚线）。 2，制备离体大鼠肺<ol><li>麻醉大鼠（雄性Sprague-Dawley大鼠; 250-300克）用氯胺酮（80〜100毫克/千克），并用甲苯噻嗪（5-10毫克/千克）。后确保手术的麻醉平面，将动物仰卧位置。 </li><li>进行气管切开，插入气管插管（PE-90），并与手术缝合固定。 </li><li>通过心脏穿刺（21克蝴蝶针）注入肝素（100-200 U）到心脏，等待60秒，抽血血（〜12-15毫升血）。 </li><li>准备两个套管（聚乙烯管，直径3毫米，4厘米长，扩口的一端），并填充生理盐水。 </li><li>进行开胸手术。做一个切口（3毫米），在右心室和套管的扩口端滑入朝向肺切口动脉。固定套管肺动脉与手术缝合线。 </li><li>切割（3毫米），左心室心尖和第二套管的扩口端滑入切口。引导导管进入左心房。固定插管至左心室与脐带（2毫米宽）。 </li><li>解剖任何结缔组织，并与所连接的插管取出肺和心脏在一起。将肺和心脏的培养皿。重新定位的肺，使膈面在上面。这三个套管应朝向同一方向。 </li><li>放置在能够在XY方向上进行操作的一个阶段的肺制剂。附带显微镜任何阶段都可以进行修改，以保持管件或内置，如果需要一个特制的阶段。 </li></ol> 3，血液离体大鼠肺灌注<ol><li>混合放血血用等体积的白蛋白（5％）溶液，并添加到储存器。启动蠕动泵和设置流程R吃了14毫升/分钟。使血液填充管上。 </li><li>与分流开，取出肺临时进出水口接头。附加肺动脉和左心房插管至肺入口和出口，分别。确保没有气泡在管。 </li><li>气管插管连接到第三压力传感器（P23XL）。通过气管插管肺膨胀与30％氧气和压力保持5厘米高2 O。 </li><li>关闭分流钳，开始灌注肺。维持肺动脉和〜分别为10 3厘米H 2 O的左心房压力。 </li></ol> 4，准备对肺微血管灌注<ol><li>提高对上面的肺和安全水平的导管延伸管的后端。 </li><li>通过插入一个长40厘米PE10管材成约30厘米长的PE90管长约30厘米准备输液导管。在PE10管的外露端连接到一根针（30克）在上亦有一个注射器（1毫升）。 </li><li>插入导管组合成延伸管的凸起尾部。通过左心房和进入肺引导导管组合，直到遇到阻力。然后，再独自推PE10导管进入肺部，直到它遇到阻力。需要注意的是用力过大，同时插入导管会损害肺。 </li><li>填充1毫升注射器用林格氏溶液和附加到一个注射器泵。设置输液速度以10微升/分钟。 </li><li>相比于肺的其余部分注射部位会变得苍白。 </li><li>润肺用生理盐水，盖上保鲜膜，有孔大到足以离开输液部位发现的肺。 </li><li>棉签上的O形环（定制）的底部部分旋塞润滑脂，并放置在玻璃盖玻片（＃1.5，直径为22mm）上的O形环的底部。轻轻地用在输注部位的盖玻片放置O型圈。固定O形环与标准试管方荣景DER。盖玻片/ O型圈组合不变形的肺泡结构，它的下面，如最近报道15。 </li><li>位置上方的O形环的荧光显微镜的物镜（20X），并集中于肺表面。 </li></ol> 5，成像荧光葡聚糖过境微血管<ol><li>准备显微镜成像FITC荧光。 </li><li>选择要成像的区域，并利用图像采集软件在1/minute的速率采集图像。 </li><li>使用注射泵开始输注FITC-葡聚糖20 KD（0.5毫克/毫升）的。荧光血管会逐渐增加，达到最大值。继续输注60分钟。 </li><li>然后切换到林格氏输液洗掉管腔荧光。输液维持超过10分钟。在洗涤过的继续图像采集在1/min极限。 </li></ol> 6，图像分析<ol><li>打开图像采集文件。 </li><li>地方区域利息在图像帧中的微血管。 </li><li>播放的图像文件的帧接一帧并记录在感兴趣的所有帧的每个区域的荧光强度。 </li><li>绘制荧光强度的变化的利息作为时间的函数的每个区域。 </li><li>量化的最大荧光强度和冲刷10分钟后残留的荧光强度。 </li><li>计算最大值之比，以在每个感兴趣的区域残留的荧光强度，以获得与所关注该区域相关联的微血管的渗透性指数。 </li></ol>

<ol>
	<li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. A., 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+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Bhattacharya, J., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+repair+of+the+alveolar-capillary+barrier+in+acute+lung+injury.">Regulation and repair of the alveolar-capillary barrier in acute lung injury.</a> Annu Rev Physiol. 75, 593-615 (2013).</li><li>Parthasarathi, K., 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=Connexin+43+mediates+spread+of+Ca2+-dependent+proinflammatory+responses+in+lung+capillaries.">Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries.</a> J Clin Invest. 116, 2193-2200 (2006).</li><li>Parthasarathi, K., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Acid+instillation+by+a+wedged-catheter+method+reveals+a+role+for+vascular+gap+junctions+in+spatial+expansion+of+Acid+injury.">Localized Acid instillation by a wedged-catheter method reveals a role for vascular gap junctions in spatial expansion of Acid injury.</a> Anat Rec (Hoboken). 294, 1585-1591 (2011).</li><li>Gorin, A. B., Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+permeability+of+endothelial+and+epithelial+barriers+to+albumin+flux.">Differential permeability of endothelial and epithelial barriers to albumin flux.</a> J Appl Physiol Respir Environ Exerc Physiol. 47, 1315-1324 (1979).</li><li>Boutoille, D., 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=FITC-albumin+as+a+marker+for+assessment+of+endothelial+permeability+in+mice:+comparison+with+125I-albumin.">FITC-albumin as a marker for assessment of endothelial permeability in mice: comparison with 125I-albumin.</a> Exp Lung Res. 35, 263-271 (2009).</li><li>Thorball, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-dextran+tracers+in+microcirculatory+and+permeability+studies+using+combined+fluorescence+stereo+microscopy,+fluorescence+light+microscopy+and+electron+microscopy.">FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy.</a> Histochemistry. 71, 209-233 (1981).</li><li>Stevens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+heterogeneity+of+pulmonary+endothelial+cells.">Functional and molecular heterogeneity of pulmonary endothelial cells.</a> Proc Am Thorac Soc. 8, 453-457 (2011).</li><li>Ofori-Acquah, S. F., 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=Heterogeneity+of+barrier+function+in+the+lung+reflects+diversity+in+endothelial+cell+junctions.">Heterogeneity of barrier function in the lung reflects diversity in endothelial cell junctions.</a> Microvasc Res. 75, 391-402 (2008).</li><li>Kandasamy, K., 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=Real-time+imaging+reveals+endothelium-mediated+leukocyte+retention+in+LPS-treated+lung+microvessels.">Real-time imaging reveals endothelium-mediated leukocyte retention in LPS-treated lung microvessels.</a> Microvasc Res. 83, 323-331 (2012).</li><li>Kandasamy, K., 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=Lipopolysaccharide+induces+endoplasmic+store+Ca2+-dependent+inflammatory+responses+in+lung+microvessels.">Lipopolysaccharide induces endoplasmic store Ca2+-dependent inflammatory responses in lung microvessels.</a> PloS One. 8, (2013).</li><li>Qiao, R. L., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+barrier+properties+of+the+pulmonary+microvascular+bed.">Segmental barrier properties of the pulmonary microvascular bed.</a> J Appl Physiol. 71, 2152-2159 (1991).</li><li>Parthasarathi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+connexin43+mediates+acid-induced+increases+in+pulmonary+microvascular+permeability.">Endothelial connexin43 mediates acid-induced increases in pulmonary microvascular permeability.</a> Am J Physiol Lung Cell Mol Physiol. 303, (2012).</li><li>Wu, Y., Perlman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+methods+for+assessing+alveolar+mechanics.">In situ methods for assessing alveolar mechanics.</a> J Appl Physiol 1985. 112, 519-526 (2012).</li><li>Kuebler, W. M., 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=A+novel+signaling+mechanism+between+gas+and+blood+compartments+of+the+lung.">A novel signaling mechanism between gas and blood compartments of the lung.</a> Journal Clin Invest. 105, 905-913 (2000).</li></ol>

作者什么都没有透露。

孤立的血液灌注肺标本加上实时成像提供了确定在单肺微血管通透性改变一个简单的工具。我们采用该方法来定义响应于LPS输注渗透性的变化。我们的数据清楚地表明，脂多糖注射引起的增加微血管通透性。此外，数据还表明，LPS诱导的通透性的改变是相似的在两个小静脉和毛细血管。因此，这种技术的主要优点是定义单个微血管的泄漏，以及一个全局血管网络的能力。鉴于这种技术也产生全球?...

在肺微血管通透性增加导致肺泡水肿和损害气体交换的发展，是急性肺损伤（ALI）1-3的一大特点。因此，血管通透性的估计是在确定肺损伤，并提出治疗措施疗效的程度很重要。重量分析诸如血液中的游离肺湿重与干重比和微血管过滤系数被广泛使用的方法来估算渗透性4,5。其它方法包括定量肺组织6-8中的放射性或荧光探针的保留。然而，上述方法需要肺组织样品的postexperiment加工朝向阐明的渗透率数据。此外，由于一动物只能用于单一治疗方案，大型动物数​​量，可能需要一个完整的研究。上述方法的一个共同特点是，它们确定平均血管通透性为所有的血管组织样本内。但是，它是公认的肺部微观和宏观血管表型不同9。因此，渗透性反应可能是异构的各种血管段以及9,10之间。因此，定量组织样本中的所有肺血管通透性均可能没有充分反映这种异质性。 在离体血液灌注肺标本，血管对肺表面可以通过一个堂堂正正的显微镜4,11,12可视化。这使得在单个容器的反应特征，因此，解决在应对13任异质性。此外，通过利用微血管的荧光成像，基于荧光测定法可并入。进一步，左心房微导管可被用于运载剂和荧光探针进入血管11,14。微导管限制了传递到小肺部区域，从而前冒充只有区域内的血管的灌注剂和荧光基团。这允许将用于单独的实验相同的肺中的多个小区域，导致所需的研究动物的整体减少。 即时成像能够在血管和微血管单隔离肺准备的荧光血管外动态变化的捕获。因此，对于像场中的每个微血管，输液荧光和冲刷的过程中发生变化的荧光可以被记录，并量化离线14。使用的最大和残余血管荧光值，摄像视场中的每个微血管通透性指数可以被确定。以确定响应于炎性或损害性药物的渗透性的变化，所需的剂可以首先给药，然后通透性指数来确定。此外，该图像字段可以设置为通过注入肺区域内微导管，从而使之在选择所需的血管网的高度灵活性。因此，孤立的血液灌注肺的准备以配合实时成像提供了一个有吸引力的实验模型来量化通透性单肺微血管。

<table><tbody><tr><td>Tygon Tubing</td><td>Fisher Scientific</td><td></td><td>#18</td></tr><tr><td>Pressure Transducer</td><td>Data Sciences International</td><td>P23XL</td><td>Need quantity 3</td></tr><tr><td>Butterfly Needle</td><td>Greiner Bio-One</td><td>450081</td><td>21 G</td></tr><tr><td>Peristaltic pump</td><td>Cole Parmer</td><td>Masterflex L/S</td><td></td></tr><tr><td>PE-90 tubing</td><td>Becton Dickinson</td><td>427421</td><td>30 cm needed</td></tr><tr><td>PE-10 tubing</td><td>Becton Dickinson</td><td>427401</td><td>40 cm needed</td></tr><tr><td>Syringe Pump</td><td>Braintree Scientific</td><td>BS8000</td><td></td></tr><tr><td>O-ring</td><td></td><td></td><td>Custom made with a 20 mm diamter hole and a handle to secure O-ring to holder</td></tr><tr><td>Upright fluorescence microscope</td><td>Olympus America</td><td>BX61WI</td><td></td></tr><tr><td>Image Acquisition Software</td><td>Molecular Devices</td><td>Metamorph</td><td></td></tr><tr><td>FITC Dextran 20KD</td><td>Sigma Aldrich</td><td></td><td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&amp;nbsp;</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma Aldrich</td><td></td><td>Serotype 0111:B4</td></tr></tbody></table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

分离的血液灌注的肺制剂被广泛用于可视化和单微血管定义信令。通过实时成像耦合此制备中，以确定在单个肺微血管通透性的改变成为可能。在这里，我们描述的步骤来隔离大鼠肺组织，并与自体血灌注他们。然后，我们概述的步骤注入通过微导管荧光团或代理人到一个小细胞肺癌区域。使用上述这些程序，我们确定通透性增加大鼠肺微血管针对细菌脂多糖注射。数据显示，脂多糖通过这两个小静脉和毛细血管微血管段增加液体泄漏。因此，该方法使得有可能以比较的血管段中的渗透性的反应，因此，限定在响应中的任何不均匀性。而常用的方法来定义肺通透性要求肺组织样品，进行后处理的使用实时成像消除这一要求作为显而易见的，从目前的方法。因此，隔离肺制剂结合实时成像提供了一些优于传统方法来确定肺微血管的通透性，又是一个简单的方法来制定和实施。

分离的血液灌注肺制剂连接到灌注管道和相关设备示于图2。出于演示的目的，我们使用了一个只SD大鼠，虽然本文所描述的程序，可以用任何鼠种使用。通过左心房微导管输注到达肺的一小区域。该注入区域可通过输注引起的变色（ 图3）来识别。作为定位于实时成像的肺制剂示于图4。 <img alt="图2" fo:content-wid...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

孤立的血液灌注肺的准备，使可视化微血管网络上的肺表面是可行的。在这里，我们描述的方法用实时荧光成像量化单一微​​血管隔离肺的通透性。

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

quantifying-single-microvessel-permeability-isolated-blood-perfused

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.4K 次观看.  The University of Tennessee Health Science Center. 孤立的血液灌注肺的准备，使可视化微血管网络上的肺表面是可行的。在这里，我们描述的方法用实时荧光成像量化单一微​​血管隔离肺的通透性。 

视频: Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

一个<em在体内</em分析测试血管通透性

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

科研

医学

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

在缺血大鼠肺的血管生成

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

使用穆林精密切肺片对肺动脉内下氧肺血管收缩进行视频形态分析

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）:在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

微循环血流速度在大鼠肺转移自动测量

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

表征隔离，通风，和仪表小鼠肺灌注与脉动流

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

生物学

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

分流手术，右心导管，血管形态计量学在大鼠模型中的流量引起的肺动脉高压

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...