Interactions between ultrasound stimulated microbubbles and fibrin clots

血栓（血凝块）引起的血管闭塞是死亡和发病的主要原因，在许多情况下，例如中风，目前的临床方法很难解决这一问题。超声波刺激微泡 (USMB) 作为一种“分解”血栓的技术正在接受研究，并已在体外、体内和初步临床试验中进行了测试。尽管开展了这项工作，USMB 实现血栓侵蚀作用的机制在很大程度上仍然是猜测，这阻碍了改进的“声溶血栓”（STL）技术的发展。 最近的工作致力于研究 USMB 和纤维蛋白凝块之间的微观相互作用，以期获得 USMB 溶栓的基本机制见解。纤维蛋白凝块是光学透明的，由纤维蛋白网络组成，纤维蛋白网络是全血凝块（还含有红细胞和血小板）的主要成分，负责维持其机械完整性。开发了实验来光学检查 USMB 与凝块在 US 暴露期间的相互作用，并使用 3D 双光子显微镜 (2PM) 评估超声处理后的纤维蛋白凝块结构。我们获得了第一个直接证据，证明 USMB 可以渗透到凝块中，破坏纤维蛋白网络结构，并且渗透性 MB 可以从凝块外部运输液体。重要的是，这些数据表明，需要考虑三种不同的情况：i）在初级辐射力下转移到边界的MB，可能在次级辐射力的影响下合并； ii) 微气泡在边界处振荡，在它们的一部分穿透之前引起边界变形； iii) 穿透性微球在初级辐射力的作用下发生平移，吸引液体并造成纤维蛋白网络损伤。目前的 STL 方法总是定期发送等幅 US 脉冲。然而，由于 MB 在每种情况下的表现不同，这些数据表明脉冲方案应在每种情况下以不同的方式暴露 MB。 这项工作的总体目标是在微观尺度上研究 USMB 和血栓之间的相互作用，并利用所得信息来开发改进的 STL 暴露方案。该提案以实验工作为基础，将检查 USMB 与光学透明血浆凝块（含有纤维蛋白网络和血小板）和全血凝块（含有血小板和红细胞的纤维蛋白网络）的相互作用。具体来说，我们将研究传统和新颖的脉冲方案对位于血浆凝块（目标 1）和全血凝块（目标 2）边界附近的单个 MB（作为大小的函数）的影响。在这项工作中，我们将采用我们既定的方法来实现这一目标：光学镊子操纵单个 MB，高速成像记录 MB 动态，以及 2PM 评估液体和 MB 吸收以及凝块损伤。在目标 3 中，这些单独的 MB 结果将与脉冲方案的调查相结合，以便在执行“STL”序列之前以受控方式将 MB 群体带到血块边界（目标 1 和 2）。 这项基础研究将为 USMB 与含有血细胞的凝块之间的相互作用提供重要的新见解。人们强烈期望基于这些微观物理见解开发新的曝光方案，这将提高 USMB STL 的有效性。由于 STL 是一种有前途的新兴溶栓方法，适用于影响许多人的临床病症（例如中风），因此 SLT 的改善可以合理地描述为具有高影响力。在临床环境中，这些方法可以作为现有 STL 系统的改编来实施。

The occlusion of blood vessels by thrombus (blood clots) is a major cause of mortality and morbidity which is in many circumstances, such as stroke, poorly addressed with current clinical approaches. Ultrasound stimulated microbubbles (USMBs) are under investigation as a technique to ‘break-up’ blood clots, and have undergone testing in vitro, in vivo and in initial clinical trials. Despite this work, the mechanisms by which USMBs achieve clot erosion effects have remained largely a matter of speculation, which is an impediment to the development of improved ‘sonothrombolysis’ (STL) techniques. In recent work undertook to investigate micro-scale interactions between USMBs and fibrin clots with a view to gaining basic mechanistic insights into USMB thrombolysis. Fibrin clots are optically transparent and comprised of a fibrin network, which is the primary constituent of whole blood clots (which also contain red blood cells and platelets) that is responsible for maintaining their mechanical integrity. Experiments were developed to optically examine USMB-clot interactions during US exposures and to assess post-sonication fibrin clot structure with 3D two-photon microscopy (2PM). We have obtained the first direct evidence that USMBs can penetrate into clots, disrupt fibrin network structure, and that penetrating MBs can transport with them fluid from outside the clot. Importantly these data indicate that there are three distinct regimes to consider: i) MBs translating to the boundary under primary radiation forces, which may coalesce under the influence of secondary radiation forces; ii) MBs oscillating at the boundary, inducing boundary deformations before a subset of them penetrate; iii) penetrating MBs translate under primary radiation forces drawing fluid with them and causing fibrin network damage. Current STL approaches invariably send equal amplitude US pulses at regular intervals. However, as MBs will behave differently in each of these regimes, these data suggest that pulsing schemes should expose MBs differently in each of these regimes. The general objective of this work is to investigate, at a micro-scale, the interaction between USMBs and clots and to use the resulting information to develop improved STL exposure schemes. The proposal is anchored in experimental work and will examine USMB interactions with both optically transparent plasma clots (containing fibrin networks and platelets) and whole blood clots (fibrin networks with platelets and red blood cells). Specifically, we will investigate both conventional and novel pulsing scheme effects on individual MBs (as a function of size) situated near the boundaries of plasma clots (Aim 1) and then whole blood clots (Aim 2). In this work we will employ our established approaches to achieve this: optical tweezers to manipulate individual MBs, high speed imaging to record MB dynamics, and 2PM to assess fluid and MB uptake and clot damage. In Aim 3 these individual MB results will be coupled with an investigation of pulsing schemes to bring populations of MBs to the clot boundary in a controlled manner prior to executing the ‘STL’ sequences (Aims 1 and 2). This basic research will provide significant new insights into the interaction between USMBs and clots containing blood cells. There is a strong expectation that new exposure schemes will be developed, based on these micro-scale physical insights, that will improve the effectiveness of USMB STL. As STL is a promising emerging approach for thrombolysis in clinical conditions that affect many people (e.g. stroke), improvements in SLT can therefore be reasonably characterized as being high impact. In a clinical context, these methods can be implemented as adaptations of existing STL systems.