Augmentation of Tissue Perfusion in Patients With Peripheral Artery Disease Using Microbubble Cavitation

Abstract

Objectives: The authors investigated ideal acoustic conditions on a clinical scanner custom-programmed for ultrasound (US) cavitation-mediated flow augmentation in preclinical models. We then applied these conditions in a first-in-human study to test the hypothesis that contrast US can increase limb perfusion in normal subjects and patients with peripheral artery disease (PAD).

Background: US-induced cavitation of microbubble contrast agents augments tissue perfusion by convective shear and secondary purinergic signaling that mediates release of endogenous vasodilators.

Methods: In mice, unilateral exposure of the proximal hindlimb to therapeutic US (1.3 MHz, mechanical index 1.3) was performed for 10 min after intravenous injection of lipid microbubbles. US varied according to line density (17, 37, 65 lines) and pulse duration. Microvascular perfusion was evaluated by US perfusion imaging, and in vivo adenosine triphosphate (ATP) release was assessed using in vivo optical imaging. Optimal parameters were then used in healthy volunteers and patients with PAD where calf US alone or in combination with intravenous microbubble contrast infusion was performed for 10 min.

Results: In mice, flow was augmented in the US-exposed limb for all acoustic conditions. Only at the lowest line density was there a stepwise increase in perfusion for longer (40-cycle) versus shorter (5-cycle) pulse duration. For higher line densities, blood flow consistently increased by 3-fold to 4-fold in the US-exposed limb irrespective of pulse duration. High line density and long pulse duration resulted in the greatest release of ATP in the cavitation zone. Application of these optimized conditions in humans together with intravenous contrast increased calf muscle blood flow by >2-fold in both healthy subjects and patients with PAD, whereas US alone had no effect.

Conclusions: US of microbubbles when using optimized acoustic environments can increase perfusion in limb skeletal muscle, raising the possibility of a therapy for patients with PAD. (Augmentation of Limb Perfusion With Contrast Ultrasound; NCT03195556).

Keywords: cavitation; microcirculation; muscle perfusion; peripheral artery disease; ultrasound.

Copyright © 2020 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Figures

FIGURE 1. Ultrasound Schemes for Producing Cavitation

(A) Schematic showing that at low line density, microbubble cavitation within each electronically focused line occurs at the intended high acoustic pressure, yet between-line gaps can result in regions where high-pressure cavitation does not occur. At high line density, gaps are eliminated, but microbubble inertial cavitation from pressures above the CT can occur from neighboring lines at a lower than intended pressure. (B) Relative pressure measurements from calibrated hydrophone and those generated by the simulation program illustrating close correlation with regard to pressure distribution in the lateral dimension for a 3-line frame. (C) Simulated line profiles in the axial-lateral dimensions for the 17-, 33-, and 65-line settings; elevational pressure dimensions. Side-lobes or “grating lobes” are detected near the focus. Relative pressure scale is shown at left. CT = cavitation threshold.

FIGURE 2. Spatial Assessment of Microbubble Cavitation

(A) Example of images obtained using contrast-enhanced ultrasound imaging showing the spatial pattern of inertial cavitation (destruction) of stationary microbubbles in a gel phantom with the therapeutic probe oriented at orthogonal plane. Images are shown before exposure and every 5 frames. Therapeutic beam elevational plane is represented by the vertical dimension of microbubble clearing with thin flanking elevational side-lobes. The dashed and dotted lines illustrate ROI to span the elevational plane with and without side-lobes. Graphs illustrate mean (±SEM) normalized contrast intensity in the elevational plane ROI without (B) and with side-lobes (C); and the contrast-free area in the elevational plane with (D) and without (E) side-lobes. ROI = regions of interest.

FIGURE 3. Flow Augmentation in the Murine Hindlimb According to Line Density and Pulse Duration

(A) Example of time-intensity data (graph) and corresponding background-subtracted color-coded contrast-enhanced ultrasound images at various time intervals (seconds) after the destructive pulse-sequence illustrating flow augmentation in the hindlimb exposed to US (33 line-density; 40-cycle pulse) compared with the contralateral control leg. The regions of interest are shown by dashed lines in the first frame. The bar graphs illustrate mean (±SEM) microvascular flux rate (β-value) for (B) 17-line, (C) 33-line, and (D) 65-line density settings using different pulse durations. *p < 0.05 versus corresponding control limb; †p < 0.05 versus 5-cycle. US = ultrasound.

FIGURE 4. Optical Imaging of Adenosine Triphosphate After Limb Contrast US

Mean (±SEM) photon flux in the US-exposed and contralateral limb measured 5 min (A) and 20 min (B) after contrast-enhanced ultrasound. *p < 0.01 versus contralateral. Inset images show examples from mice treated with 17-line, 40-cycle conditions at each time interval. Abbreviation as in Figure 3.

FIGURE 5. Augmentation in Limb Skeletal Muscle Perfusion in Humans

Examples of (A) contrast-enhanced ultrasound images of the calf after a destructive pulse sequence, and (B) corresponding time-intensity curves from a healthy subject before and after therapeutic cavitation with contrast-enhanced ultrasound. An increase in flow (A×β) was attributable to an increase in flux rate (β-value) and microvascular blood volume (plateau intensity). Graphs display MBF in the US-exposed and contralateral calf before, immediately after, and 1 h after exposure to (C) US alone in healthy controls; (D) CEU in healthy controls; and (E) contrast-enhanced ultrasound in subjects with peripheral artery disease. *p < 0.05 versus baseline. BG = background; MB = microbubbles; MBF = microvascular blood flow. Other abbreviation as in Figure 3.

CENTRAL ILLUSTRATION. Ultrasound Exposure of the Calf Skeletal Muscle During Infusion of Ultrasound Contrast Agents Results in Microbubble Inertial Cavitation

The shear-mediated release of ATP results in production of various vasodilator compounds including NO, prostanoids, and adenosine, all of which produce regional vasodilation. ATP = adenosine triphosphate; NO = nitric oxide.