Interactions between ultrasound stimulated microbubbles and fibrin clots

血栓（血凝块）造成的血管闭塞是死亡和发病的主要原因，在许多情况下，如中风，目前的临床方法解决得很差。超声刺激微泡（USMB）正在研究作为一种技术，以“打破”血凝块，并已经历了体外，体内和初步临床试验的测试。尽管有这项工作，USMB实现凝块侵蚀效果的机制在很大程度上仍然是一个猜测的问题，这是改进的“超声溶栓”（STL）技术的发展的障碍。 在最近的工作进行了调查之间的USMB和纤维蛋白凝块的微观尺度的相互作用，以期获得基本的USMB溶栓机制的见解。纤维蛋白凝块是光学透明的并且由纤维蛋白网络组成，纤维蛋白网络是全血凝块（其还包含红细胞和血小板）的主要成分，其负责维持其机械完整性。开发实验以光学检查US暴露期间USMB-凝块相互作用，并使用3D双光子显微镜（2PM）评估超声处理后纤维蛋白凝块结构。我们已经获得了第一个直接证据，即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，这些数据表明有三种不同的情况需要考虑：i）MB在一次辐射力下平移到边界，这可能在二次辐射力的影响下合并; ii）MB在边界处振荡，在其中一部分穿透之前引起边界变形; iii）穿透MB在一次辐射力下平移，吸入液体并导致纤维蛋白网络损坏。目前的STL方法总是以规则的间隔发送等幅US脉冲。然而，由于MB将在这些机制中的每一个中表现不同，因此这些数据表明脉冲方案应该在这些机制中的每一个中不同地暴露MB。 这项工作的总体目标是调查，在微观尺度上，USMBs和凝块之间的相互作用，并使用由此产生的信息来开发改进的STL曝光计划。该提案是锚定在实验工作中，并将检查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（血小板和红细胞的纤维蛋白网络）的相互作用。具体而言，我们将研究传统和新型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，我们将采用我们已建立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，2PM评估液体和MB摄取和凝块损伤。在目标3中，这些单个MB结果将与脉冲方案的研究相结合，以在执行“STL”序列之前以受控方式将MB群体带到凝块边界（目标1和2）。 这项基础研究将为USMB和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，将开发新的暴露方案，这将提高USMB STL的有效性。由于STL是一种有前途的新兴方法，用于影响许多人的临床疾病（例如中风）的溶栓，因此可以合理地将脑卒中的改善描述为高影响。在临床背景下，这些方法可以实现为现有STL系统的改编。

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.