Interactions between ultrasound stimulated microbubbles and fibrin clots

血栓(血栓)阻塞血管是死亡率和发病率的主要原因，在许多情况下，如中风，目前的临床方法处理得很差。超声刺激的微泡(USMB)是一种“分解”血栓的技术，目前正在进行研究，并已进行了体外、体内和初步临床试验。尽管有这项工作，但USMB实现血栓侵蚀效应的机制在很大程度上仍是一个猜测的问题，这阻碍了改进的“超声溶栓”(STL)技术的发展。 在最近的工作中，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。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(也包含红细胞和血小板)的主要组成部分，负责维持其机械完整性。开展了实验，以光学检查USMB-凝块在超声暴露期间的相互作用，并使用3D双光子显微镜(2 PM)评估超声后纤维蛋白凝块的结构。我们已经获得了第一个直接证据，证明USMB可以穿透到血栓中，破坏纤维蛋白网络结构，并且穿透的MBS可以从血栓外输送液体。重要的是，这些数据表明有三种不同的机制需要考虑：i)在一次辐射力作用下移动到边界的MBS，它可能在二次辐射力的影响下结合；ii)MBS在边界处振荡，在它们的子集穿透之前导致边界变形；iii)穿透性MBS在一次辐射力作用下转换，与它们一起吸引流体并造成纤维蛋白网络破坏。当前的STL方法总是以规则的间隔发送相等幅度的US脉冲。然而，由于MBS在每一种制度下的表现不同，这些数据表明，脉冲方案在每一种制度下应该以不同的方式暴露MBS。 这项工作的总体目标是在微观上调查USMB和血块之间的相互作用，并利用由此产生的信息来制定改进的STL暴露方案。该提案以实验工作为基础，将研究USMB与光学透明的血浆凝块(包含纤维蛋白网络和血小板)和全血凝块(与血小板和红细胞的纤维蛋白网络)的相互作用。具体地说，我们将研究传统和新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，我们将使用我们已有的方法来实现这一点：光钳操作单个MB，高速成像记录MB动态，以及2 PM评估液体和MB摄取和凝块损害。在目标3中，这些单独的MB结果将与脉冲方案相结合，以在执行‘STL’序列之前以受控的方式将MB群体带到血栓边界(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，基于这些微观物理洞察，将开发新的暴露方案，从而提高USMB STL的有效性。由于STL是一种在影响许多人(如中风)的临床条件下进行溶栓治疗的有前途的新兴方法，因此SLT的改善可以被合理地描述为高影响。在临床背景下，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.