Ultrasound Mediated Microbubbles Destruction Augmented Sonolysis: An In Vitro and In Vivo Study

Abstract

Objective: This study was aimed at exploring ultrasound mediated microbubbles destruction (UMMD) assisted sonolysis in both the in vitro and in vivo clots.

Methods: Therapeutic ultrasound (TUS) and lipid microbubbles (MBs) were used in whole blood clots and divided into the control, TUS group, and TUS + MB group. Thrombolytic rates and microscopy were performed. Color Doppler flow imaging (CDFI) and angiography were performed to evaluate the recanalization rates and flow scores in femoral arterial thrombus (FAT) in rabbits. FAT were dyed with H&E.

Results: The average thrombolytic ratios of TUS + MB group were significantly higher than those of TUS group and the control group (both P < 0.05). Clots had different pathological changes. Recanalization rates and flow scores in TUS + MB group were significantly higher than the control and TUS group. Flow scores and recanalization ratios were grade 0 in 0% of the control group, grade I in 25% of TUS group, and grade II or higher in 87.5% of TUS + MB group after 30 min sonolysis.

Conclusions: Both the in vitro and in vivo sonolysis can be significantly augmented by the introduction of MBs without thrombolytic agents, which might be induced by the enhanced cavitation via UMMD.

Figure 1

Diagram of experiment setup at in vitro study.

Figure 2

Thrombolytic efficacy and the pathological changes of in vitro clots. (a) Histogram of thrombolytic rate in the three groups in clots. TUS + MB group versus TUS group, P < 0.05; TUS + MB group versus the control group, P < 0.01. (b) Pathological changes of clots stained with HE under light microscopy. In the control group, the surface and the inner fibrin structure of the red thrombus remained normal without micropores or clefts ((B1)-(B2), ×100); in TUS group, a few tiny fractures and vacuoles were observed at the inner structure (black arrowheads) and loosened surface (blue arrowheads) of clots and a small amount of tiny clefts (black asterisks) was found in an almost intact fiber net within the clots ((B3)-(B4), ×100); in TUS + MB group, there were more and larger vacuoles (black asterisks) at the loosened surface (blue arrowheads) area and more irregular clefts (black arrowheads) and tunnel-like disruption within the clots ((B5)-(B6), ×100). (c) Pathological changes of clots under scanning microscopy. Intact structure and fibrin networks (yellow circles) were observed at the surface and inner part of clots in the control group (C1); there were micropores at the surface, and a loose fibrin network with microcavities (blue circles) at the inner part of clots in TUS group (C2) and even larger cavities (blue circles) and disrupted fiber in TUS + MB group (C3). (d) Fluorescent distribution of MBs in clots following TUS irradiation. DiO stained MBs were in bright green particles and evenly distributed under fluorescent microscopy ((D1), ×200). A bright fluorescent band at the surface of clots ((D2), ×100) and a few DiO-positive MBs (white arrowheads) were observed within the clots ((D3), ×100) in MB group; a brighter and broader band at the surface of clots ((D4), ×100) and more DiO MBs in spherical or irregular particles (white arrowheads) at the surface and inner part of clots ((D5), ×100) in TUS + MB group.

Figure 3

Assessment of thrombolytic efficacy and the pathological changes in FAT in rabbits. (a) Assessment of thrombolytic rate with CDFI. In the control group, continuous flow was recorded in the left femoral artery ((A1), white arrows), while a defect in blood flow was found at FAT in the right femoral artery with CDFI ((A2), white arrows); a narrow and strip flow in blue or turbulent in grade I was observed through the edge of clots in TUS group at 30 min ((A3), white arrows); blood flow of grade II or higher ((A4), white arrows) was recorded at 30 min in the TUS + MB group. (b) Assessment of thrombolytic rate with X-ray angiography. No reperfusion in the control ((B1), white arrowheads), grade I flow in TUS group ((B2), white arrowheads) and grade II ((B3), white arrowheads) in TUS + MB group at 30 min. (c) Pathological changes of FAT following sonothrombolytic treatment under light microscopy. An obstructive thrombus consisted of red blood cells and fibrin matrix was observed (C1); small amount of red blood cells destruction was observed associated with partially dissolved FAT ((C2), black asterisk) at 30 min in TUS group; clots were sonolysed in large amount and the recanalized FA was collapsed with residual thrombus in the inner wall of FA ((C3), black asterisks).