A Temporal Study of Ultrasound Contrast Agent-Induced Changes in Capillary Density

Ischemia-related heart disease affects more than 80 million people in the United States.1 Current interventions include ablation, angioplasty, revascularization, and bypass surgery. The invasive nature of these techniques excludes patients with myocardial ischemia who are not amenable to surgical intervention. For this reason, alternative methods of revascularization in ischemic cardiac muscle have been explored. Over the past 2 decades, cellular, molecular, and genetic therapy attempts have been made to find a clinically relevant treatment.2–6 Ultimately, it is the invasiveness or lack of site specificity that provides the largest obstacle for therapeutic effectiveness of angiogenic treatments. Current research has suggested that ultrasound (US)-ultrasound contrast agent (UCA) treatment can be therapeutically beneficial, providing a noninvasive way to spatially and temporally target ischemic tissues.7–10 This type of angiogenic therapy can be used as an alternative to high-risk percutaneous intervention or bypass graft surgery. Several studies report a reparative response to US-UCA exposure and state that inertial cavitation, or microbubble collapse, is possibly required for angiogenesis to occur.8,11–14 Numerous small-scale studies have shown promising results; however, when large-scale double-blind studies were conducted, they showed limited effects.15 A major impediment for progress to clinical applicability is, perhaps, the lack of understanding of the biophysical mechanisms that connect US-UCAs to neovascularization. It is important to establish a basic knowledge of capillary changes that occur with US-UCA exposure so that US-UCA-induced angiogenesis can be assessed for therapeutic benefit and so that existing therapies can be improved. Current studies in the literature involving US-UCA-induced angiogenesis use peak rarefactional US pressure (Pr) varying between 0.1 and 1.0 MPa.8,12,16,17 These Pr values have been shown to collapse approximately 20% to 70% of UCAs at 1 MHz.18 Therefore, the question remains as to what causes the angiogenic response. Studies involving US-UCA-induced angiogenesis show increases in the collateral blood supply ranging from 5% to 35% with respect to their controls.8–12 However, the effectiveness of the therapy used in each study cannot be correlated to Pr levels or any other parameter used. Also, these studies do not directly correlate a UCA mechanism to the angiogenic response.8–12 In addition, most studies involving US-UCA therapy use ischemic models without exploring the underlying mechanism. Furthermore, the time points for angiogenic assessment (ie, days postexposure [DPE]) were quite varied, ranging from 3 to 28 DPE.8,16,17,19,20 The goals of this study were to explore the influence of UCA collapse cavitation, the angiogenic progression in a normal animal model, and determine whether and if so when the capillary density (as a measure of angiogenesis) increases subsequent to US-UCA exposure in an effort to connect the acute (0 day) bioeffect to the end goal of angiogenesis. This study used a Pr of 3.8 MPa to collapse about 100% of the UCAs18 in an effort to correlate a mechanism with the angiogenic response. Previous work has shown effectiveness at lower Pr values; however, the underlying premise is that angiogenesis is damage induced and involves a reparative response.9,12,13 This study inspects that underlying premise. Examination of UCA collapse cavitation and subsequent angiogenic effects is integral to understanding how to increase angiogenesis. In this study, rats were randomly assigned to 0, 3, 6, 13, 20, and 27 DPE groups to investigate the progression of capillary density and inflammation in response to a Pr sufficient to cause collapse cavitation of UCAs in a normal animal model. Capillary density was quantified in capillaries per square millimeter via immunohistochemical (IHC) staining (CD31 and CD34).