Acoustic radiation force enhances ultrasound contrast agent retention to p-selectin in vivo

Abstract

Recent studies have shown that targeted ultrasound contrast agents known as microbubbles (MB) can achieve specific adhesion to intravascular pathology. This may enable non- invasive diagnosis of disease at the molecular level using contrast- enhanced ultrasound (CEU). However, several investigations have suggested that the ability of injected contrast agents to contact the blood vessel surface, where the molecular markers of disease are located, may to be weak, especially in large or high- flow vessels. This deficiency may limit the usefulness of targeted CEU using these agents. It has been hypothesized that the efficacy of this technique may be improved by increasing the number of circulating MB that contact and adhere to the intended endothelial target site. Low-intensity acoustic radiation has been used to cause the directional migration of targeted MB toward the vessel wall in several in vitro systems. In the current study we present evidence that this technique enhances adhesion of MB targeted to the pro-inflammatory endothelial protein P- selectin in vivo. We assessed the retention of MB bearing the anti- P-selectin antibody Rb40.34 in mouse models of inflammation of the cremaster microcirculation and the femoral vessels. Low- intensity acoustic force was applied for several minutes immediately after MB injection, and MB retention was assessed using intravital microscopy in 10-20 microscopic fields of view. Targeted MB exhibited a 5-fold greater retention following application of acoustic radiation in the cremaster microcirculation, a 2.5-fold increase in the femoral vein, and a 20-fold increase in the femoral artery. These results suggest that low-intensity radiation force is a viable mechanism for enhancing MB retention for imaging inflammation. in several models in vivo (2,3) using the pro-inflammatory endothelial proteins P-selectin or ICAM-1 as targets. However, subsequent in vitro research has revealed that the efficiency of the MB:endothelium interaction is quite low under certain conditions (4), which may limit the diagnostic power of this technique. Previous studies have suggested that rapid rates of blood flow, especially in large vessels, may inhibit MB adhesion. In vitro, the adhesion of MB targeted to P-selectin has been shown to decrease with increasing shear rate (4, 5). This may be due to an inability of free-flowing MB to come into contact with the endothelial surface. Microbubbles reportedly exhibit rheology similar to erythrocytes (6), and thus may travel in the axial center of blood vessels. Hemodynamic forces may bring MB into contact with the endothelium in the small, branching vessels of the microcirculation; however, achieving targeted MB adhesion in large vessels where such forces are absent has may prove difficult. This may limit the ability of targeted CEU to detect several clinically important pathologies, such as atherosclerotic plaque growth in the carotid arteries. Several groups have hypothesized that applied acoustic radiation force may be used as a mechanism to move freely- flowing MB into contact with the endothelium. Applied acoustic radiation induces two forces upon compressible MB: the primary force, which is directed away from the acoustic source and is proportional to the magnitude of the acoustic pressure; and the secondary force, which is primarily attractive, and causes MB aggregation. Pioneering work by Dayton and colleagues (7, 8) revealed that the magnitude of the radiation forces can be many fold greater than that of buoyancy, and subsequent work showed that this technique indeed could enhance MB adhesion in vitro (5, 9). In the current project, we assessed the ability of applied acoustic radiation to enhance MB retention in two models of inflammation. We assessed MB retention in the murine microcirculation using a cremaster model of inflammation similar to the one investigated by Lindner and colleagues (2). To determine whether acoustic radiation force can induce MB adhesion in large, high-flow vessels, we investigated a model of inflammation in the mouse femoral vessels. Our studies suggest that acoustic radiation force can induce a several-fold increase in MB retention in the cremaster microcirculation and femoral vein, although appreciable MB retention in these vessels was also achieved in