Name: microperfusion technique to investigate regulation of microvessel permeability in rat mesentery
Uploaded: 2015-09-12T14:00:00.000+00:00
Duration: 12 min 48 s
Description: Watch this Scientific Journal Video about Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery at JoVE.com

这项工作是由卫生部资助HL44485和HL28607国家机构的支持。

伦理声明:所有的程序进行审查和批准的机构动物护理和使用委员会。 微量移液器，限位装置和拦截1.初步加工<ol><li>使用电子牵拉调整，使得，当拉动时，在管的拉伸部分的长度约为1厘米和两半偏于对称拉在半几个清洁硼硅玻璃毛细管。确保该锥度是与图1的尺寸相一致。使用两个一半为微量，限位和阻滞剂。 <ol><li>使用斜角的气动砂轮30具有0.5微米的沐浴用水砂纸的微量。设置微量保持器的角，使其从水平约30度。 </li><li>彻底清洗，并通过使用抽吸穿过尖端和向上的轴去拉清洁丙酮干燥微量。检查微量（图1A）用一个小直立显微镜配有鳄鱼夹微量持有者和目镜标线4×和40×的目标。 </li><li>选择微量与前端开口约50μm长，有一个斜面，其长度/宽度比为3.1至3.5，并用一个尖锐的锥形点，这有助于穿透在肠系膜坚韧胶原纤维。 </li><li>存储15-20微量略微不同尺寸的在无尘框。 </li></ol></li><li>用在步骤1.1准备拉毛细管）限制部分。持拉玻璃毛细管简要地靠近一个微火以形成钝端（图1B）。存储若干在无尘框。 </li><li>用在步骤1.1准备拉毛细管）microoccluders。持拉毛细管下一个微火轻轻弯曲（使用管状适配器，例如，23G）的细尖（约4从前端毫米），使接近120度的角度，以在轴（ <s仲&gt;图1C）。制作和存储多个。 </li></ol> 2.动物准备和手术<ol><li>麻醉雄性（350-450克）或雌性Sprague-Dawley大鼠（200-300克）用戊巴比妥钠（80-100毫克/公斤，Sigma-Aldrich公司，P3761）通过皮下注射保护肠系膜不使用腹腔注射。 </li><li>在整个手术过程中和实验方案，维持麻醉根据需要补充注射（30毫克/千克）。确定由脚趾捏或角膜反射麻醉深度。的实验程序完成后，安乐死通过戊巴比妥过量或心内注射的饱和氯化钾麻醉下大鼠。 </li></ol> 3.准备解决方案和红细胞用作流标记<ol><li>制备哺乳动物林格氏溶液灌流和控制灌注溶液（通常为哺乳动物含10mg林格氏液/毫升脂肪酸游离牛血清白蛋白，BSA）中。 <ol><li>通过0.2微米注射器过滤器过滤灌流以去除可能会阻止微量移液器尖端细小颗粒;过滤成小干净的容器中。 </li></ol></li><li>通过绘制约0.2毫升的血液从尾静脉麻醉大鼠的成20G针上含有0.05 ml肝素（1000单位/毫升）的小注射器准备红细胞和转移到含约14毫升的哺乳动物林格氏溶液溶液15ml离心管。 <ol><li>离心机轻轻地收拾红细胞（约最大200×g的7分钟）。绘制上清，重悬浮红细胞在林格氏溶液中。 </li><li>离心并除去上清液后三次留在管供以后使用的底部的封装红细胞。注意，此过程减少了在最终灌流液血小板正常水平31的低于0.1％。 </li></ol></li></ol> 4.安排在动物组织盘子<ol><li>剃麻醉的老鼠的腹部从下到肚脐剑突。确保剃区域周围的右侧延伸（对于大鼠置于右侧），使得头发不会形成燃烧芯吸取灌流出组织的良好。用湿纱布或纸巾取出切下的毛发。 <ol><li>握住钝锯齿钳的皮肤，使用一个坚固的剪刀中线切口（2-3厘米长），通过皮肤。用细（IRIS）的剪刀做一个类似的切口，通过白线，解除了腹壁锯齿钳，以免损坏肠道。 </li><li>在其一侧的动物托盘上的动物，用棉签涂抹（木棍）浸泡在林格氏液和钝锯齿钳轻轻拔出从腹腔内脏。安排在组织中的肠道也使肠系膜被披在支柱观看通过显微镜注意避免接触肠系膜（POTEntially损害微血管）与任一钳或棉花。 注:动物托盘（图2A）可以从有机玻璃构成的，需要的组织以及（约3毫米深），光学透明的支柱（例如，抛光的石英约2厘米直径和5mm高），和一暖垫调整，以保持动物的体温。柱的厚度不能超过物镜的工作距离。 </li></ol></li><li>定位显微镜舞台上的动物托盘使得肠系膜是通过目镜可见。 </li><li>放置一个重力给料滴线，不断superfuse保持在35-37℃，哺乳动物的林格氏液肠系膜。用纱布垫，以保持肠道中的位置，有利于保持水分和灯芯多余的林格氏液从表面。调节流量，吸出水保持一致的灌流厚度。 </li><li>确定目标小静脉的微血管，这是收敛流，一个或两个分支远端真毛细管的下游无支链段。发现无支链的容器中（理想600〜1000微米长）具有轻快血流和游离的白细胞粘附在血管壁上。 </li><li>定位测试微血管在显微镜场的中心，并移动阻挡进入靠近选择的插管站点位置。 </li></ol> 5.填写微管和安装在支架<ol><li>就在cannulating之前，暂停红细胞的灌流液。加7微升封装红细胞的0.5 ml的灌注液在一个干净的硼硅酸盐试管。注:1-3％血球计数都可以使用，但一致的血细胞比容将​​导致从红细胞释放的任何物质一致灌洗液浓度。 </li><li>适合1毫升注射器，用23G油管接头和PE 50管（5-15厘米长）。绘制灌流/红细胞混合物倒入注射器。倒置注射器混合和驱逐所有气泡管和适配器。为了提高效率，适合管道的几个注射器在实验开始前，让他们在无尘盒或塑料袋。 </li><li>通过推进管导入微量的较大端，直到它抵靠锥形区填充微量。注射器柱塞申请一个温柔的快速推进，填补了枪头。注:涓涓溪流或滴应该是可见的尖端证据完全填充。 </li><li>拔出油管，同时轻轻推柱塞，填补了微量轴的较宽的部分。通过仔细轻弹微量轴卸下在宽轴小气泡。注意:类似的程序已经示出32。 </li><li>将微量成与侧端口，其允许连续流体连接至一个水压力计的吸移管支架。确保该保持器，其附连到用于控制微量的非常细的前进液压驱动，是在一个微小的角度（15-25度）对于水平，使得微量锥形的边缘不会撞到肠道或托盘。 </li><li>安装在一个可移动的微操作，它具有的x，y和z的驱动器，以使附近的对准到测试容器（图2）的液压驱动。 </li><li>调整使用注射器或充满水的橡胶管来改变的流体的高度，在压力计的水柱至约40 水柱肠系膜上述静水压力施加到流体中的微。 </li><li>填充吸管后尽快导管插入微血管;红细胞沉降到移液管的底部，以使大约40分钟后很少红细胞会流入容器中。 </li></ol> 6. Microcannulation和微血管压力测量<ol><li>在显微镜下定位填补微量，轻轻按下阻挡到附近施加足够的力夹住组织的微血管组织。绘制阻挡器背部，略微拉伸与微血管线的组织，以便在肠系膜胶原纤维的应力与容器对准。 <ol><li>对齐容器中的微管，降低其进入视野通过目镜观察。将吸头所选择的插管部位的上游和降低它到组织上，使得它部分地阻挡所述容器内的流动，但不会堵塞它。 </li></ol></li><li>通过驱动移液管尖端慢慢进入使用液压驱动的血管腔导管插入血管。注意不要推动通过该容器的下侧。 注意:作为微量进入内腔，灌注液迅速洗血液在血管出管腔和灌注液自由地流入到动物的循环远端测试微血管，取代某些正常血液灌注在下游容器。 </li><li>降低通过调节压力计直到血液灌注压力（所指示通过动物的红细胞）被抽回到所述灌流段;这决定了平衡的压力，其中所述红细胞轻轻摆动在血管腔和测量静水微血管压力（P c）应微血管段的远端。保持容器灌注在这个平衡压力高于一切的压力。 </li></ol> 7. Microocclusion和信息的收集<ol><li>任选的步骤:在使用微封堵以阻止容器中，将其压入在肠系膜的未使用区域中的组织远离任何容器，使尖端聚集组织的微细垫，在反复微保护实验容器之前闭塞。 </li><li>放置阻滞剂显微镜视野的下边缘附近的灌注容器的上方。 </li><li>记录有视频和音频以下。 <ol><li>口头记录块站点的位置和观察在目镜标线片的下屏幕边缘。 </li><li>与顶端阻塞置于正好微血管以上的，可使用显微的微细Ž控制轻轻降低阻滞剂直到在容器的流动闭塞。注意音频压力表压力（P C）。典型的应用闭塞3-10秒，然后松开，恢复自由的灌注。移动块站点向上朝向枪头容器中基于时间:5-10微米的步骤（&gt;初始块后5分钟，在一个位点），闭塞的数目（3-5），为防止血管壁损伤，在块站点。 </li></ol></li></ol>数据和L 的p测量8.分析（渗水） <ol><li>在视频播放期间，通过使用一个阶段微米和在图像上的已知位置的图像的确定，从一个标记红细胞的闭塞部位的初始距离（ℓ0）。在3-10秒记录图像的过程中确定它的位置在若干帧标记单元的初始速度（分升/ DT）。阻止矿容器半径（r）从阻塞（ 图3）期间拍摄的图像。 </li><li>计算ĴV / S为每个闭塞作为（分升/ DT）×（1 /ℓ0）×（R / 2）。计算部分 L P以恒定压力（j V / S）由驱动压力除以（微血管压力-有效胶体渗透压;见讨论）。 </li></ol>

The authors have nothing to disclose.

部分 L P的计算的详细情况。虽然经血管的流体运动发生同时容器自由地灌注，例如交换是太小期间自由灌注被测定，因为它通常是在容器的灌注率小于0.01％。然而，当灌注瞬时通过堵塞微血管，经血管流动停止（即，过滤）从标记红细胞在管腔作为流体的标记物红细胞和闭塞的位点之间的列中的运动测量缩短所示图3。根据实验方案的非支链容器600〜1000微米的?...

微灌在脉管嗣继承建立经由微量吸管在通常小于40微米的直径的血管受控流动的已知组合物的人工灌注液。灌注容器保持在其正​​常组织环境内，并灌注动物的血液到插管的时间。当结合的范围内的视频成象或荧光技术，原位微灌用于支持跨微血管的壁水和溶质的流动的条件下，其中为这些流动的驱动力是已知的并且在血管壁的渗透性属性可以是测量直接评估。此外，通过控制周围组织中的微血管（灌注液和灌流）的流体的组合物，微血管通透性和交换的调节可以通过使内皮细胞形成的微血管壁被暴露于各种电子商务调查xperimental条件（激动剂，修改灌注的条件下，荧光指标来衡量细胞内的成分和信令）的时间精确测量周期（秒小时）。此外，关键的细胞的分子结构调节屏障的超微结构或细胞化学评价所用的相同的微血管，其中渗透性直接测量进行调查。该方法因此形成的细胞和分子机制的研究之间的桥梁来修改内皮屏障功能中培养的内皮细胞单层和调查在完整微血管。请参阅下面的评论作进一步评估1-6。 微灌的一个限制是，它只能用于在微血管床是薄的，透明的，并具有足够的结构完整性，以使插管用玻璃微量吸管。虽然早期的研究中使用的青蛙微血管肠系膜和薄皮肤心绞痛米uscle 7,8，迄今为止最常用的制备在哺乳动物模型是大鼠肠系膜9-15。大多数研究都集中在研究过1-4小时的时期血管通透性的急性变化，但最近的调查已经扩展到测量个体船舶24-72小时的初始灌注12,16后。最近开发的CRISPR技术，这将让更多的转基因大鼠模型可用于研究血管通透性第 17条应启用该通信所描述的方法，在这些重要的新模型大鼠肠系膜小静脉的微血管被应用。 该方法要求一个倒置显微镜配备有定制的显微镜阶段足以容纳动物制备既和用于位置微型工具靠近灌注容器中并对准一个灌注微量与容器的至少三个微操作内腔。例如定制平台的xy显微镜阶段（约90×60厘米）能够从一个1厘米厚的钢板用防锈涂料来制造。该级被连接到一个工程索引表或两个燕尾滑动在水平面安装成直角并支撑在特氟隆支柱或球转移运动。一个典型的钻机（ 见图 2）有许多共同之处用于一系列的活体微循环实验，如测量单血管的血液血管平滑肌的流量和红细胞压积，局部氧输送血液灌注微血管，调节显微镜和微定位设备肌张力，并注入到整个循环荧光示踪剂的局部微血管积累。18-26 该技术的基本方面是体积流量（j v）的横跨微血管壁的规定表面面积（S）的测量。去完成这通过本文所述的改性兰迪斯技术简单的倒置显微镜是足够的。一个小的摄像机安装在图像端口和视频信号上，具有添加的时间的基础上，被显示在视频监视器上，要么在计算机上的数字形式或者作为在视频记录器的数字或模拟信号记录。一旦微血管插管微血管可见的摄像机的一部分可以通过移动台和操纵器作为一个单元而不中断插管被改变。 经血管流动的测量，也可以使用与适当的过滤器一复杂的荧光显微镜更详细的调查，如用于溶质渗透率，细胞质钙或其它细胞机制，以及共焦成像6,12,13的荧光比率监测测量钻机组合， 27。所有微灌方法的主要优点是使重复测量的能力，同船根据驱动力，如静水和肿胀压力，或引起的变化血管反应，炎症性疾病的控制变化。最常见的设计是测量水力传导率（L p）的对经由填充有控制灌注液和红细胞悬液微量第一灌注的容器相同的容器中的配对比较，以建立基线渗透性状态，然后用第二吸移管与测试试剂加入到灌注液。多插管是可能的再灌注控制吸管经过反复的恶性循环。 本协议演示了一个小静脉血管的插管和微灌大鼠肠系膜记录水通量穿过微血管壁并测量血管壁的部分 L P，公用路径的渗透性为整个完好水和溶质的一个有用的指标内皮屏障。该过程被称为改性兰迪斯techniqUE因为原来兰迪斯原理用红细胞的相对运动作为灌注被阻止被保留28后经血管的液体交换的量度的，但实验条件的范围 （例如，横跨微血管壁的静水和白蛋白肿胀压力差异）微灌后可用远比uncannulated血液灌注微血管8,29更大。

<table><tbody><tr><td>MICROSCOPE, TABLE AND STAGE</td><td></td><td></td><td></td></tr><tr><td>inverted microscope (metallurgical type) with trinocular head for video: example</td><td>Olympus</td><td>CK-40</td><td>try to place eyepieces higher relative to stage--you have to look through eyepieces while reaching around to top of stage over intervening micromanipulators</td></tr><tr><td>inverted microscope (metallurgical type) with trinocular head for video: example</td><td>Leica</td><td>DMIL</td><td>try to place eyepieces higher relative to stage--you have to look through eyepieces while reaching around to top of stage over intervening micromanipulators</td></tr><tr><td>narrow diameter, long working distance objective: example</td><td>Nikon</td><td>Nikon E Plan 10&amp;times;/0.25 LWD</td><td></td></tr><tr><td>stage platform--1/2 inch or 1 cm sheet steel</td><td>welding shop</td><td></td><td>this should be heavy to reduce vibration</td></tr><tr><td>Unislide x-y table: dove tail slides</td><td>Velmex</td><td>AXY4006W1</td><td></td></tr><tr><td>VIDEO</td><td></td><td></td><td></td></tr><tr><td>CCD video camera: example</td><td>Pulnix</td><td>TM-7CN (no longer available)</td><td>no color needed</td></tr><tr><td>video capture system with audio--generic</td><td></td><td></td><td></td></tr><tr><td>video playback system (completely still frame, single frame motion)</td><td></td><td></td><td></td></tr><tr><td>small microphone</td><td></td><td></td><td></td></tr><tr><td>MICROMANIPULATORS, HOLDERS</td><td></td><td></td><td></td></tr><tr><td>micromanipulator, XYZ (3)</td><td>Prior/Stoelting (no longer available)</td><td></td><td>look for fine Z, and larger range of travel in coarse drives for ease of positioning</td></tr><tr><td>hydraulic probe drive, one way</td><td>FHC</td><td>50-12-1C</td><td>need to buy either manual drive or electronic drive</td></tr><tr><td>manual drum drive&amp;nbsp;</td><td>FHC</td><td>50-12-9-02</td><td></td></tr><tr><td>or hydraulic drive, 3 way</td><td>Siskiyou Corporation</td><td>MX610 (1-way) or MX630 (3-way)</td><td>great for short arms, water filled and must be sent back for refill ~every 2 years</td></tr><tr><td>connectors/rods/holders</td><td>Siskiyou Corporation</td><td>MXC-2.5, MXB etc.</td><td></td></tr><tr><td>pin vise</td><td>Starrett</td><td>162C</td><td>to hold restrainer</td></tr><tr><td>pipette holder</td><td>World Prescision Instruments</td><td>MPH3</td><td></td></tr><tr><td>water manometer ~120 cm</td><td></td><td></td><td></td></tr><tr><td>MICROSCOPE TRAY</td><td></td><td></td><td></td></tr><tr><td>clear Plexiglas for microscope tray for animal</td><td></td><td></td><td></td></tr><tr><td>3/4 inch polished quartz disc ~1/4 inch tall</td><td>Quartz Scientific Inc.</td><td>custom</td><td>&amp;nbsp;(or polished plexiglass, glass); make sure the height is less than working distance of objective</td></tr><tr><td>Plexiglas glue (Weld-on 4: CAUTION CARCINOGEN)</td><td></td><td></td><td></td></tr><tr><td>medical adhesive for tissue well</td><td>NuSil</td><td>MED-1037</td><td></td></tr><tr><td>All-purpose silicone rubber heat mat, 5&quot; L x 2&quot; W</td><td>Cole Parmer</td><td>EW-03125-20</td><td>heater for microscope tray--needs cord and controller--240V version available</td></tr><tr><td>Power Cord Adapter for Kapton Heaters and Kits, 6 ft, 120 VAC</td><td>Cole Parmer</td><td>EW-03122-75</td><td></td></tr><tr><td>STACO 3PN1010B Variable-Voltage Controller, 10 A; 120 V In, 0-140 V Out</td><td>Cole Parmer</td><td>EW-01575-00</td><td></td></tr><tr><td>PIPET MANUFACTURE</td><td></td><td></td><td></td></tr><tr><td>vertical pipette puller</td><td>Sutter Instrument Company</td><td>P-30 with nichrome filament</td><td></td></tr><tr><td>1.5 mm OD thin wall capillary tubing</td><td>Sutter Instrument Company</td><td>B150-110-10</td><td></td></tr><tr><td>pipette grinder air stone and dissection microscope--see reference in text</td><td></td><td></td><td>or purchase a package from Sutter Instruments or World Precision Instruments</td></tr><tr><td>RX Honing Machine, System II</td><td>RX Honing Machine Corporation</td><td>MAC-10700 Rx System II Machine</td><td>alternative for air stone, use with a dissecting microscope mounted at an angle</td></tr><tr><td>&amp;nbsp;&amp;nbsp; with ceramic sharpening disc</td><td>RX Honing Machine Corporation</td><td></td><td>use &quot;as is&quot; or attach lapping film</td></tr><tr><td>lapping film sheets, 0.3 or 0.5 um</td><td>3M</td><td>part no. 051144 80827</td><td>268X Imperial lapping film sheets with adhesive back--can be purchased from Amazon</td></tr></tbody></table>

microperfusion technique to investigate regulation of microvessel permeability in rat mesentery

实验测量的单独灌注微血管渗透性属性提供的调节中培养的内皮细胞单层的整个微血管床功能交换性能血管通透性分子和细胞机制调查之间的桥梁。 A到导管插入并灌注大鼠肠系膜小静脉的微血管，测量微血管壁的渗透系数方法。所需要的主设备包括一个活体显微镜用支持显微操作以定位三个不同的微型工具的大改性阶段:（1）一个斜面玻璃微量吸管到导管插入并灌注微血管; （2）的玻璃微封堵以瞬时方框灌注和使经血管水流运动的测量在测量流体静压力，以及（3）一个钝玻璃棒以稳定肠系膜组织在插管的部位。修改后的兰迪斯微闭塞techniqUE使用红细胞悬浮在人工灌注液的液体经血管运动的标志，同时也使这些流动的实验条件变化以及水压和胶体渗透压差穿过微血管的重复测量得到精心控制。第一使用控制灌注液，然后经过重新插管相同微血管与测试灌流液的水力传导率的测量，使成对的这些精心控制的条件下的微血管应答的比较。尝试的方法延伸到微血管小鼠预期修改血管通透性遗传修饰的肠系膜是因为不存在的长的直链和无支链的微血管在小鼠肠系膜严重的限制，但在大鼠相似遗传修饰的最近情况使用所述CRISPR / Cas9技术开料调查的，其中本文描述的方法可以应用的新领域。 </P&gt;

图4示出了结果从测量变化期L p的时间过程在大鼠小静脉微血管具有四个灌流液依次插管33以恒定的压力计算部分 L P的大小被用作变化微血管壁渗透性的量度，第一在用含有1％牛血清白蛋白那么当容器暴露使用含有10nM的浅滩的第二微量的抗炎剂缓激肽（BK）灌注液中的控制状态。缓激肽引起的对控制值返回在随后的10-15分钟一过性升高的部分 L P。然后...

Watch this Scientific Journal Video about Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery at JoVE.com

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

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 ...

microperfusion-technique-to-investigate-regulation-microvessel

Research

JoVE Journal

Biology

9.7K 次观看.  University of California Davis. 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. 

视频: Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

一个<em&gt;离体</em&gt;方法培养大鼠肠系膜微血管网络的时间推移成像

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

科研

发育生物学

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 ...

医学

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

评估肌反应及血管活性阻力肠系膜动脉使用压力肌动描记

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

免疫与感染

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

生物学

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

大鼠肠系膜外化：调查涉及在血管生成细胞动力学模型

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

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 ...

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

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

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 ...

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 ...

<meta charset="utf8"></head><body>内皮 PAR1 在 I/R 损伤期间白细胞粘附和 NET 形成中的作用

Visualization of Neutrophil Extracellular Traps in Mesenteric Venules After Mesenteric Ischemia-Reperfusion Injury via Intravital Microscopy

Intestinal ischemia-reperfusion (I/R) injury is an acute condition characterized by tissue damage resulting from restricted blood flow to the mesenteric vessels, leading to both local and systemic pathologies with a poor prognosis. Both ischemia and reperfusion trigger a series of cellular and molecular responses, with inflammatory cells serving as key regulators of the pathology. These interactions with the ischemic endothelium are mediated by multiple adhesion receptors. Several animal models have been established to mimic this pathology and investigate the involved molecular pathways. In this study, a microsurgical model of I/R injury is combined with intravital microscopy to visualize leukocyte rolling, adhesion, and neutrophil extracellular trap (NET) formation. This model is applied to transgenic mice deficient in endothelial PAR1 (F2r) to assess the impact of PAR1 on leukocyte rolling and NET formation 1 h after ischemia and immediately following reperfusion. In vivo, Acridine Orange leukocyte staining was employed, and NETs were visualized using a nucleic acid stain. Interestingly, reduced leukocyte adhesion and NET formation were observed in mice lacking the endothelial PAR1 receptor. This model enables the in vivo analysis of key regulators involved in I/R injury.

<meta charset="utf8"></head><body>通过活体显微镜观察肠系膜缺血再灌注损伤后肠系膜小静脉中中性粒细胞细胞外陷阱的可视化

This protocol presents a method for inducing mesenteric ischemia-reperfusion injury and visualizing neutrophil extracellular traps (NETs) in mesenteric venules using intravital microscopy. Blood flow in the superior mesenteric artery was restricted for 1 h, followed by reperfusion, allowing direct quantification of leukocyte adhesion and NETosis.

通过活体显微镜观察肠系膜缺血再灌注损伤后肠系膜小静脉中中性粒细胞细胞外陷阱的可视化

Intestinal ischemia-reperfusion (I/R) injury is an acute condition characterized by tissue damage resulting from restricted blood flow to the mesenteric ...