Name: evaluating vascular hyperpermeability-inducing agents in the skin with the miles assay
Uploaded: 2018-06-19T13:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay at JoVE.com

我们感谢劳拉 Denti、瓦伦蒂娜 Senatore 和伦敦大学科学学院生物资源股的工作人员帮助小鼠饲养和卡米尔 Charoy 的技术支持。这项工作得到了医学研究理事会赠款 (MR/N011511/1) 的支持, Ruhrberg 和英国心脏基金会博士奖学金对 j.t. 傲慢 (FS/13/59/30649)。

所有的动物工作都是在英国内政部和机构动物福利和道德审查机构 (AWERB) 指导下进行的。
1. 鼠标准备
注: 对至少8周龄的成年小鼠进行实验, 年龄可达6月。为了演示在不同动物之间的每一个步骤的技术重现性和一致的时间, 每一个实验性的会议使用至少2和最多6只老鼠。为了评估突变对转基因小鼠的渗透诱导物质的影响, 理想情况下, 每项实验使用2变种小鼠和 2 littermate 控制。
<ol><li>24小时前刺激血管 hyperpermeability, 麻醉小鼠使用适当的吸入麻醉, 如异氟醚。使用3% 异氟醚进行麻醉诱导, 直到矫正反射丢失, 鼠标对外部刺激反应迟钝。使用1.5% 异氟醚进行麻醉维护 (确保鼠标有恒定的呼吸速率)。在麻醉下, 小心地用电动剃须刀剃掉每只老鼠的两侧, 避免皮肤受伤。 注: 麻醉是用来避免对动物造成压力, 并尽量减少剃须期间的运动, 这可能导致皮肤损伤。</li>
	<li>把剃光的老鼠送回家里的笼子里。对于雄性小鼠, 在麻醉恢复后将每个雄性置于一个单独的笼子里, 以防止打斗, 从而损害皮肤。对于雌性老鼠, 把他们送回他们的家庭笼子。 注: 如果在笼子里的雄性小鼠身上观察到打斗行为, 在手术前, 在实验前3天将它们分离成单独的笼子, 让潜在的皮肤损伤愈合。</li>
	<li>监视老鼠直到他们恢复知觉 (通常在几秒钟之内) 并且移动在笼子 (通常在1分钟之内)。</li>
</ol>2. 静脉注射埃文斯蓝染料
<ol><li>在层流柜中, 制备马来酸 pyrilamine 胺抑制剂 (4 µg/µL 0.9% 生理盐水) 和埃文斯蓝染料 (1% 瓦特/v 0.9% 生理盐水) 的分离无菌溶液。通过22µm 过滤器通过解决方案进行消毒。</li>
	<li>使用无菌的1毫升注射器与30克针腹腔注射每只老鼠与10µL pyrilamine 马来酸溶液/克的体重 (图 1A)。要执行注射, 首先, 把鼠标的背上, 然后倾斜, 使头部朝向地面, 腹部向上方向。在腹部的下象限内注射, 远离中线以避免撞击膀胱。 注意: 小鼠在8周至6月之间的体重通常介于15至30克之间. 马来酸 Pyrilamine 将抑制内源组胺的释放, 否则会促进血管渗漏, 独立于所测试的试剂。</li>
	<li>将鼠标放置在37摄氏度的热室中, 10 分钟促进血管舒张。或者, 使用一盏合适的热灯聚焦在尾部, 以促进血管舒张, 如果使用的热灯是允许的地方道德准则。</li>
	<li>一次将一只鼠标移动到鼠标抑制剂, 用70% 乙醇擦拭尾部以清洁注射区, 进一步促进血管舒张。</li>
	<li>使用无菌1毫升注射器与30G 针, 以管理100µL 埃文斯蓝色染料静脉注射通过尾静脉 (图 1B)。立即通过握住手指和拇指之间的尾巴来对注射部位施加压力, 以防止出血。 注意: 可以通过将灯光定向到注射区来改善尾静脉的可视化。关于对鼠标尾静脉进行静脉注射的进一步细节, 可在已发布的18号议定书中找到。</li>
	<li>允许染料循环30分钟。</li>
</ol>3. 刺激血管 Hyperpermeability
<ol><li>在层流柜中使用无菌溶液, 将感兴趣的渗透诱导剂稀释成浓度, 使最终剂量能在20µL 的体积内输送。 注: 在图 1所示的示例实验中-图 2中, VEGFA 用于刺激 PBS 中 2.5 ng/µL 浓度的血管 hyperpermeability, 总剂量为 50 ng, pbs 用作车辆控制。</li>
	<li>将渗透诱导剂的兴趣和车辆控制成2分不育300µL 注射器与31克针。加载足够的解决方案, 每三个注入20µL 的解决方案 (即准备60µL 的代理和车辆每只老鼠, 加上额外的体积, 以解释针的死容量)。</li>
	<li>麻醉第一只用异氟醚测试的老鼠 (3% 用于麻醉诱导, 直到矫正反射丢失, 鼠标对外部刺激反应迟钝, 1.5% 的麻醉维护, 确保老鼠有恒定的呼吸速率)。</li>
	<li>Intradermally 注入20µL 的渗透促进剂到鼠标的侧面 (图 1C)。确保针的角度与皮肤的一角。检查皮肤内凸起气泡的形成, 表明注射成功。重复2个额外的部位, 至少1厘米的皮注射液。</li>
	<li>转动鼠标暴露第二侧面和重复三个注射与控制车解决方案。在纸上记录每个注射部位的位置, 以方便他们识别随后收集的皮肤样品。 注意: 在这个阶段小心处理鼠标皮肤;例如, 在执行皮下注射时避免捏皮肤。</li>
	<li>将注入的鼠标返回到其主笼子并监视鼠标, 直到它恢复知觉 (通常在几秒钟内) 并在笼子周围移动。</li>
	<li>当第一个鼠标恢复时, 立即重复步骤3.3 和3.5 与第二个鼠标, 等等 (最多6小鼠每会话最大)。记录每个鼠标被注入的时间, 以调整进行后续步骤的时间。</li>
</ol>4. 在真皮内积累的埃文斯蓝的量化
<ol><li>在接受了皮下注射后, 用20分钟的宫颈脱位来剔除每只老鼠, 观察与注射小鼠相同的顺序。 注: 血管渗漏的持续时间可能根据所使用的渗透诱导剂而异, 因此, 皮下注射与剔除之间的时间可能需要优化。</li>
	<li>将每只被宰杀的老鼠放在背上, 把脚钉在一个裹着干净纸巾的软木板上 (图 1D)。</li>
	<li>使用钝剪刀, 使垂直切口大约 3-4 cm 从下腹部到死的老鼠的胸口。用镊子和手术刀, 从两侧的皮肤中梳理出真皮的内侧和埃文斯蓝堆积的部位 (图 1E)。针下松散的皮肤和清除周围地区的脂肪与手术刀。如果需要, 采取一个代表性的照片, 以证明埃文斯蓝色泄漏到皮肤的程度。</li>
	<li>使用镊子和手术刀, 消费的皮肤区域包括泄漏埃文斯蓝色染料为每个地点注入渗透诱导剂或车辆。 注: 注意在皮肤上同样大小的区域, 以确保在随后的步骤 4.7, 甲酰胺体积的类似部分将被每个皮肤样品吸附, 使提取的染料被稀释在类似的剩余的甲酰胺体积.步骤3.5 中记录的注入图将有助于确定要切除的区域。</li>
	<li>将每个样品放入1.5 毫升管中, 相应地贴上标签, 确保每个样品都放在管子的底部。将样品储存在-20 摄氏度, 直到进一步使用。如果立即处理, 请按照下列步骤操作。</li>
	<li>将打开的管子放在烤箱中或加热块的井中, 在55摄氏度的情况下, 在一夜之间干燥皮肤样品。</li>
	<li>要提取埃文斯蓝染料, 添加250µL 的去离子甲酰胺的样品在通风罩, 关闭管。确保所有的皮肤样品都覆盖的甲酰胺和孵化一夜之间的烤箱或加热块在55摄氏度。</li>
	<li>离心机样品在台式离心机以最大速度 (> 1万 x g) 为40分钟。</li>
	<li>在通风罩中, 将含有染料的上清液的100µL 从每个试样转移到一个透明的、平坦的底部96井板 (图 2A) 的一个独立井中。</li>
	<li>测量在620毫微米的峰值埃文斯蓝色吸光度与参考读数 740 nm 在分光光度计。 注: 620 nm 是埃文斯蓝吸光度的峰值, 而 740 nm 不被埃文斯蓝色吸收, 并充当参考波长。</li>
	<li>平均每三个相同的药剂或车辆的吸光度读数为每个鼠标, 以说明技术的变异性 (图 2B)。</li>
	<li>计算渗透诱导剂与车辆控制读数的折差 (图 2C)。</li>
</ol>

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自从171952年的第一个描述以来, 英哩法为研究者提供了一种相对快速、简单、可靠的方法来研究血管 hyperpermeability 的分子机制。例如, 英里分析被用来测试不同药剂的能力和/或效力, 以诱导 hyperpermeability19,20,21或测试 hyperpermeability 阻滞剂的功效8 ,22,23。作为另一个例子, 诱导血管通透性的药物已经在转基因小鼠及其 littermate 控制中进行了测试, 以确定配体诱导的特异受体和信号传感蛋白的要求 hyperpermeability答复10、11、12、13、14、15、20、23。从那以后, 已经使用了好几种改良版本的迈尔斯化验, 例如关于使用的示踪剂。因此, 埃文斯蓝已经取代了一些研究与荧光标记 dextrans 不同大小或微球11,13。
迈尔斯对其他检测血管 hyperpermeability 机制的研究的主要优点是它比较容易执行, 不需要昂贵的设备。此外, 作为一种体内技术, 这种检测模型在完整的血管背景下的血管渗漏, 而不是通过体外检测, 如通井流量测定或反式内皮电阻测量渗漏 (志愿者) 化验, 仅集中于内皮细胞单层。在体内测定血管 hyperpermeability 的方法可能不同于这里描述的英里分析, 利用不同的传递路线, hyperpermeability 诱导剂或分析不同部位的血管渗漏,例子通过全身交付代理通过尾巴静脉然后血管渗漏检查在肺或气管11,13,15。迈尔斯试验的一个局限性是, 某些制剂的皮下注射在原则上也可能影响血压和流量, 除了内皮屏障的破坏, 因此, 也间接影响血管通透性。然而, 这项检测最近被证明是测量 VEGF 诱发的血管通透性-独立于对全身血压的影响15。迈尔斯测定的另一个局限性是, 不同组织的血管床对渗透性促进剂的反应可能不同, 因此, 在皮肤中用英里数测定得出的结果可能不具代表性, 例如, 在肺或脑。
动物的年龄和体重可能会影响英里测定中所观察到的渗漏。为了尽量减少这些变量的影响, 研究人员应使用窝或类似大小和衰老的小鼠作为对照。当评估特定基因的小鼠突变体时, 应通过异型与异型育种策略和 wildtype 窝作为对照来生成小鼠。此外, 最好使用快速可逆的气体麻醉剂, 如异氟醚, 以避免血管收缩, 这已经描述了一些麻醉药物通过注射注射24,25。将埃文斯蓝染料注入小鼠侧尾静脉是本协议的关键步骤, 对实验和数据的质量有很大的影响。因此, 研究人员必须有能力执行尾静脉注射, 并可能需要事先的经验或实践之前开始的迈尔斯化验实验。另外, 研究人员也可以使用其他的静脉通路来提供埃文斯蓝色, 例如, 如果他们有经验的话, 可采用复古轨道注射。皮下注射渗透性促进溶液可以引起对皮肤的独立剂损伤。因此, 皮下注射不应超过20µl 的体积, 应始终进行在存在的组胺抑制剂和规范化的车辆控制注射。
许多研究人员已经用英里数法对 vegf 的成分进行了评估, 比如信号通路, 因为 vegf A 诱导的血管通透性会导致癌症患者16的腹水和视力损害在几个新生血管中的水肿眼部疾病7。因此, 我们和其他人已经使用了英里度分析来比较 vegf--一种诱导的小鼠血管渗漏, 基因修饰, 缺乏 vegf 的特定成分-信号级联12,13,14,15,20,23. 我们最近使用这种方法的研究表明, 主要的病理 VEGF--一种异构体, VEGF165, 通过 VEGFR2 和 NRP1 的复合物发出信号, NRP1 细胞质领域促进 SRC 家族激酶介导的内源性激酶的活化。唤起一个 hyperpermeability 的反应14。VEGFA 还促进血管的生长, 因此, 可以使用治疗, 以恢复血液流向缺血性组织, 如果其 hyperpermeability 活动可以特别抑制。研究表明, 引导 VEGF 的分子机制-血管内皮细胞对血管 hyperpermeability 的反应可能, 因此, 确定可选择性地操纵的途径, 以抑制病理 VEGF-诱发水肿疾病, 如癌症或缺血性眼病。迈尔斯的化验无疑将继续是一个有用的方法, 以支持这些和许多其他类型的功能研究, 以阐明分子调节因子和血管 hyperpermeability 的效应。

心血管系统的主要功能是使所有器官的循环和组织之间的气体、养分和废料的转移。血管有特定器官水平的基底通透性, 允许这种交流1。例如, 肾脏的血管是高度渗透性的, 而血脑屏障形成一个紧密的, 高度坚不可摧的界面2,3,4。形成血管内衬的内皮细胞在循环和底层组织之间提供了物理屏障, 并以特定器官的方式调节血管通透性。然而, 某些刺激导致内皮屏障的局部击穿, 以增加从循环的液体渗出到间质以上的基础水平1。例如, 在组织外伤、炎症、肿瘤、脓毒症、脑血管疾病或大脑和心脏等部位, 当组织缺血发生于中风或心肌梗塞时, 这些 hyperpermeability 被观察到, 分别5,6,7,8. 当长期升高时, hyperpermeability 会导致水肿, 进而导致组织损伤, 如眼部疾病中视力丧失9。因此, 对血管 hyperpermeability 反应进行建模, 对于了解增强内皮通透性的机制以及测试旨在抑制这种作用的药物的功效是可取的。
迈尔斯检测是一种公认的、常用的和相对简单的技术, 用于测量体内血管渗漏作为血管 hyperpermeability 的替代品。尽管它没有考虑到可能增加血管渗漏的复合因子, 如血压或血流等, 但通常认为英里法是一种可靠的方法来评估物质的渗透性调节活性, 并识别促进其活动的信号介质。因此, 迈尔斯的化验已经成为许多研究的一部分, 已经确定了血管 hyperpermeability 的调解人及其作用机制10,11,12,13, 14、15, 如血管内皮生长因子 VEGF-A 最初被确定为血管通透性因子 VPF16。
最初开发了几英里和英里的研究豚鼠血管通透性17, 他们的化验后来适应了使用小鼠, 这是目前的模式选择的有机体, 以阐明分子机制的血管通透性条例由于其精湛的适用性, 基因操纵。简言之, 埃文斯蓝染料是静脉注射到成年小鼠, 并允许循环30分钟 (图 1)。渗透诱导剂与车辆控制, 然后 intradermally 注射在多个站点上的反对侧翼的鼠标, 以诱发血管泄漏 (图 1)。因此, 白蛋白结合的埃文斯蓝染料 extravasates 和积累在真皮 (图 1)。在人道地剔除老鼠之后, 埃文斯蓝从真皮中提取出来, 并以测试物质的比率计算出的血管泄漏水平 (图 2)。

<table><tbody><tr><td>Pyrilamine maleate salt</td><td>Sigma-Aldrich</td><td>P5514-5G</td><td>Resuspend in PBS</td></tr><tr><td>Deionised Formamide</td><td>Sigma-Aldrich</td><td>S4117</td><td>Use in fume cupboard</td></tr><tr><td>Microlance Needles 30g x 0.5&quot;</td><td>BD&amp;nbsp;</td><td>305106</td><td></td></tr><tr><td>Sterile Plastipak 1ml Luer</td><td>BD&amp;nbsp;</td><td>309659</td><td></td></tr><tr><td>Sterile MicroFine syriinges 0.3 ml - 8 mm - 30G&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>324826</td><td></td></tr><tr><td>Evans blue</td><td>Sigma Aldrich</td><td>E2129</td><td></td></tr><tr><td>Mouse restrainer</td><td></td><td></td><td></td></tr><tr><td>Heat chamber</td><td></td><td></td><td></td></tr><tr><td>Hair clippers</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Merial</td><td>ap/drugs/220/96</td><td></td></tr><tr><td>Scalpal&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Blunt scissor</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>Heatblock</td><td></td><td></td><td></td></tr><tr><td>Cork board</td><td></td><td></td><td></td></tr><tr><td>Benchtop refrigerated centrifuge</td><td></td><td></td><td></td></tr><tr><td>Recombinant Vascular Endothelial Growth Factor 165</td><td>Reliatech</td><td>M30-004</td><td></td></tr></tbody></table>

evaluating vascular hyperpermeability-inducing agents in the skin with the miles assay

血管内皮在脊椎动物体内的主要功能是在血液和身体的每个组织之间起屏障作用, 从而使内皮细胞渗透到血球、血浆大分子和水中, 可以根据生理需要。在某些疾病中, 细胞因子和生长因子被释放, 靶向内皮屏障瞬时增加血管通透性;然而, 他们长期存在可能导致慢性血管 hyperpermeability, 从而组织损伤水肿。迈尔斯检测是一种体内技术, 允许研究人员通过代理测量血管渗漏来研究血管 hyperpermeability。在这里, 我们提供了一个详细的协议, 如何执行这个过程在鼠标, 这是最广泛使用的模型有机体, 研究哺乳动物的生理和病理学。该程序包括静脉注射埃文斯蓝染料标签的循环白蛋白后, 多次皮下注射渗透诱导剂和车辆控制解决方案, 以反对侧翼的鼠标。因此, 埃文斯蓝染料逐渐泄漏到真皮中, 它积累和可提取的量化作为渗透诱导剂相对于车辆的泄漏。英里分析可以在野生类型或转基因小鼠模型中进行, 并可与药物管理结合, 研究调节血管通透性的分子机制, 识别能够诱发或阻断的药物/靶点。hyperpermeability。

我们使用英里分析法评估血管内皮生长因子 A 在 wildtype C57/Bl6 小鼠中诱导的相对于车辆控制 (PBS) 的动脉泄漏的能力。在这里, 我们展示了一个典型的实验, 使用 wildtype 小鼠刺激与皮下注射20µL 溶液中含有 50 ng VEGF-a 在 pbs 或车辆 pbs 仅。在注射 VEGF 的皮肤样品中, 埃文斯蓝染料的渗漏明显增加--与 PBS 相比, 在原位可见 (图 1E) 和萃取后甲酰胺中的染料 (图 2A)。定量的多项实验表明, 50 的 VEGF-A 显着诱导更多的埃文斯蓝泄漏比 PBS 单独 (图 2B), 平均3倍增加的血管渗漏比车辆 (图 2C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57524/57524fig1.jpg"> 图 1: 在迈尔斯化验中诱导血管渗漏.在鼠标中执行英里检测时, 顺序步骤的示意图表示 (顶部面板) 和相应的图像 (底部面板)。(A) 马来酸 Pyrilamine 注射腹腔, 以防止内源组胺释放10至30分钟 (B) 静脉注射100µl 1% 埃文斯蓝到鼠尾静脉。(C) 30 分钟后, 血管渗漏是由制剂的皮下注射 (VEGF A; 绿色圆圈) 与车辆控制 (PBS; 白色圆圈) 引起的.(D、E)为了访问埃文斯蓝积累的网站, 老鼠被宰杀20分钟后, 皮下注射和皮肤两侧解剖和固定下来。腹腔内;静脉注射: 静脉;证件: 皮;刻度条, 1 厘米.<a href="/files/ftp_upload/57524/57524fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57524/57524fig2.jpg"> 图 2: 在迈尔斯化验中量化血管渗漏.(A) 埃文斯蓝染料是从图 1中每种皮样中提取的皮肤样品中萃取的, 并装入一个96井板中, 用于吸光度法阅读。(B、C)通过绘制多项实验的吸光度读数的图形表示 (B) 渗透诱导剂 (VEGF A; 深绿色圆圈) 和车辆 (PBS; 灰色) 的归一化吸收值 (光密度、OD);圆圈), 或 (C) 代理与车辆 (误差条: SEM) 的 OD 变化。三种 PBS 和 VEGF 的吸光度读数 (a) 中的样品分别为白色或绿色圆圈, 以 (B、C) 显示。<a href="/files/ftp_upload/57524/57524fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay at JoVE.com

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

在这里, 我们提出了一个协议, 以测量的血管泄漏诱导的皮下渗透促进剂进入小鼠皮肤。该技术可用于确定分子促进或抑制血管渗漏的能力, 或研究调节血管通透性的分子机制。

用迈尔斯测定法评价皮肤血管 Hyperpermeability 诱导剂的含量

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

evaluating-vascular-hyperpermeability-inducing-agents-skin-with-miles

Research

JoVE Journal

Biology

14.7K 次观看.  University College London. 在这里, 我们提出了一个协议, 以测量的血管泄漏诱导的皮下渗透促进剂进入小鼠皮肤。该技术可用于确定分子促进或抑制血管渗漏的能力, 或研究调节血管通透性的分子机制。

视频: Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

科研

癌症研究

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

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

生物工程

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

小鼠量度本地过敏反应

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

免疫与感染

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

隔离和使用的牛肠系膜动脉和静脉部分，作为生物测定在小肠测试的Vasoactivity

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

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 ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

精确实施最小红斑剂量（MED）测试，以评估人体炎症反应中的个体变异

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

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 ...

生物学

研究人类微血管功能障碍的新机制

Intradermal Microdialysis: An Approach to Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally invasive technique used to investigate mechanisms of vascular smooth muscle and endothelial function in the cutaneous circulation. This technique allows for the pharmacological dissection of the pathophysiology of microvascular endothelial dysfunction as indexed by decreased nitric oxide-mediated vasodilation, an indicator of cardiovascular disease development risk. In this technique, a microdialysis probe is placed in the dermal layer of the skin, and a local heating unit with a laser Doppler flowmetry probe is placed over the probe to measure the red blood cell flux. The local skin temperature is clamped or stimulated with direct heat application, and pharmacological agents are perfused through the probe to stimulate or inhibit intracellular signaling pathways in order to induce vasodilation or vasoconstriction or to interrogate mechanisms of interest (co-factors, antioxidants, etc.). The cutaneous vascular conductance is quantified, and mechanisms of endothelial dysfunction in disease states can be delineated.

作者聚焦:一种揭示心血管疾病风险人群机制的药物解剖方法 

Intradermal microdialysis is a minimally invasive technique used to investigate microvascular function in health and disease. Both dose-response and local heating protocols can be utilized for this technique to explore mechanisms of vasodilation and vasoconstriction in the cutaneous circulation.

皮内微透析:研究人类微血管功能障碍新机制的方法

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally ...

用迈尔斯测定法评价皮肤血管 Hyperpermeability 诱导剂的含量

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利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

一个

一个完整的皮肤缺损模型，以评估生物材料的血管化

小鼠量度本地过敏反应

隔离和使用的牛肠系膜动脉和静脉部分，作为生物测定在小肠测试的Vasoactivity

方法论方法血管功能和形态的非侵入性评估

精确实施最小红斑剂量（MED）测试，以评估人体炎症反应中的个体变异

微灌技术在调查微血管渗透性的大鼠肠系膜条例

皮内微透析:研究人类微血管功能障碍新机制的方法

皮内微透析:研究人类微血管功能障碍新机制的方法