Department of Cell Biology, The University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2012;7(8):e44018. doi: 10.1371/journal.pone.0044018. Epub 2012 Aug 30.
The role of the endothelium in the pathogenesis of cardiovascular disease is an emerging field of study, necessitating the development of appropriate model systems and methodologies to investigate the multifaceted nature of endothelial dysfunction including disturbed barrier function and impaired vascular reactivity.
We aimed to develop and test an optimized high-speed imaging platform to obtain quantitative real-time measures of blood flow, vessel diameter and endothelial barrier function in order to assess vascular function in live vertebrate models.
We used a combination of cutting-edge optical imaging techniques, including high-speed, camera-based imaging (up to 1000 frames/second), and 3D confocal methods to collect real time metrics of vascular performance and assess the dynamic response to the thromboxane A(2) (TXA(2)) analogue, U-46619 (1 µM), in transgenic zebrafish larvae. Data obtained in 3 and 5 day post-fertilization larvae show that these methods are capable of imaging blood flow in a large (1 mm) segment of the vessel of interest over many cardiac cycles, with sufficient speed and sensitivity such that the trajectories of individual erythrocytes can be resolved in real time. Further, we are able to map changes in the three dimensional sizes of vessels and assess barrier function by visualizing the continuity of the endothelial layer combined with measurements of extravasation of fluorescent microspheres.
We propose that this system-based microscopic approach can be used to combine measures of physiologic function with molecular behavior in zebrafish models of human vascular disease.
内皮细胞在心血管疾病发病机制中的作用是一个新兴的研究领域，因此需要开发适当的模型系统和方法来研究内皮功能障碍的多方面特性，包括受损的屏障功能和血管反应性受损。
我们旨在开发和测试一种经过优化的高速成像平台，以获得血流、血管直径和内皮屏障功能的定量实时测量值，从而评估活体脊椎动物模型中的血管功能。
我们结合了最先进的光学成像技术，包括高速、基于相机的成像（高达 1000 帧/秒）和 3D 共聚焦方法，以收集血管性能的实时指标，并评估血栓素 A2（TXA2）类似物 U-46619（1 μM）对转基因斑马鱼幼虫的动态反应。在 3 天和 5 天孵化后的幼虫中获得的数据表明，这些方法能够在感兴趣的血管的大（1 毫米）段中成像多个心动周期的血流，速度和灵敏度足以实时解析单个红细胞的轨迹。此外，我们能够通过可视化内皮层的连续性并结合测量荧光微球的渗出来映射血管的三维尺寸变化并评估屏障功能。
我们提出，这种基于系统的显微镜方法可用于将人类血管疾病斑马鱼模型中的生理功能与分子行为相结合进行测量。
