Interactions between ultrasound stimulated microbubbles and fibrin clots

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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.