Application of Laser Speckle Flowmetry to Vascular Remodeling

描述（由申请人提供）：该提案的主要目标是（1）开发和验证激光散斑成像（LSI），用于重复测量微血管和侧支动脉中的绝对血流速度，以及（2）通过使用这些LSI方法，深入了解剪切应力如何调节微血管重塑和动脉生成。LSI利用相干激光与组织相互作用产生的散斑图案。随着组织中的红细胞移动，图像表现出斑点对比度和变化的降低，这可能与血流有关。LSI以前已被用于测量相对流量变化;然而，基于初步的比较微粒图像测速（PIV）研究，我们认为LSI的绝对血流测量可能是可行的，并适用于微血管重塑和动脉生成的体内模型。我们的总体假设是LSI可用于测量和跟踪单个微血管和侧支动脉中的剪切应力水平，因为这些血管随着时间的推移在结构上重塑。 在目标1中，我们将优化和利用LSI来确定小鼠背侧皮褶窗口室中的单个微血管中的剪切应力水平，因为它们响应于血流动力学扰动而重塑，该扰动将通过微闭塞拱状小动脉或小静脉产生。微血管重塑本质上是维持组织灌注的“长期”自身调节反应;然而，失调的微血管重塑可促进和/或加剧许多病理状况。剪切应力是一个可能的调节功能性微血管重塑，然而，“分子换能器”连接微血管重塑剪切应力基本上是不确定的。我们认为这主要是因为目前还没有低成本的实验方法能够将绝对剪切应力变化与个体血管随时间的微血管重塑联系起来。目标1的结果将填补这一关键的知识空白，并首次使体内实验适应微血管适应。在目标2中，我们将使用优化的LSI方法来确定动脉生成和单核细胞募集如何通过剪切应力大小和/或方向的改变来调节。在外周动脉疾病（PAD）治疗的背景下，治疗性动脉生成临床试验一直不成功。我们认为，这主要是由于我们对内源性反应的持续了解不足。我们实验室有趣的新研究表明，在上游动脉闭塞后暴露于剪切逆转的侧支血管段表现出明显加速的动脉生成。揭示为什么剪切逆转加速动脉生成可以为改进治疗方法提供重要线索。目标2中的研究将产生详细的剪切生长历史，然后可以用于了解内皮细胞如何感知和响应剪切应力大小和方向的精确变化。从本质上讲，这将提供必要的基础机制研究，旨在破译哪些信号通路链接剪切应力加速动脉重塑。

DESCRIPTION (provided by applicant): The major goals of this proposal are (1) to develop and validate laser speckle imaging (LSI) for repeated measurements of absolute blood velocity in microvessels and collateral arteries and (2) to provide, through the use of these LSI methods, insight into how shear stress regulates microvascular remodeling and arteriogenesis. LSI utilizes the speckle pattern generated by the interaction of coherent laser light with tissue. As red blood cells in the tissue move, the image exhibits a decrease in speckle contrast and variation, which can be related to blood flow. LSI has been used previously to measure relative flow changes; however, based on preliminary comparative microparticle image velocimetry (¿PIV) studies, we believe that absolute blood flow measurements with LSI may be feasible and applicable to in vivo models of both microvascular remodeling and arteriogenesis. Our overall hypothesis is that LSI can be used to measure and track shear stress levels in individual microvessels and collateral arteries as these vessels structurally remodel through time. In Aim 1, we will optimize and utilize LSI to determine shear stress levels in individual microvessels in mouse dorsal skinfold window chamber as they remodel in response to a hemodynamic perturbation, which will be generated by micro-occluding an arcade arteriole or venule. Microvascular remodeling is essentially a "long-term" autoregulatory response that maintains tissue perfusion; however, dysregulated microvascular remodeling can facilitate and/or exacerbate many pathological conditions. Shear stress is a likely regulator of functional microvascular remodeling; however, the "molecular transducers" linking microvascular remodeling to shear stress are essentially unidentified. We contend this is primarily because no low-cost experimental approaches capable of linking absolute shear stress changes to microvascular remodeling in individual vessels over time currently exist. The results from Aim 1 will fill this critical knowledge gap and enable, for the first time, in vivo experimental investigtion into microvascular adaptation. In Aim 2, we will use an optimized LSI approach to determine how arteriogenesis and monocyte recruitment are regulated by alterations in shear stress magnitude and/or direction. In the context of the treatment of peripheral arterial disease (PAD), therapeutic arteriogenesis clinical trials have been unsuccessful. We contend this is due largely to our continued poor understanding of the endogenous response. Intriguing new studies from our lab show that collateral segments exposed to shear reversal after upstream arterial occlusion exhibit markedly accelerated arteriogenesis. Uncovering why shear reversal accelerates arteriogenesis could provide important clues for improved therapeutic approaches. The studies in Aim 2 will yield detailed shear-growth histories that can then be used to understand how endothelial cells sense and respond to these precise changes in shear stress magnitude and direction. In essence, this will provide the necessary foundation for mechanistic studies aimed at deciphering which signaling pathways link shear stress to accelerated arteriogenic remodeling.