Background
There is growing interest in limb contrast-enhanced ultrasound (CEU) perfusion imaging for the evaluation of peripheral artery disease. Because of low resting microvascular blood flow in skeletal muscle, signal enhancement during limb CEU is prohibitively low for real-time imaging. The aim of this study was to test the hypothesis that this obstacle can be overcome by intermediate- rather than low-power CEU when performed with an acoustically resilient microbubble agent.
Methods
Viscoelastic properties of Definity and Sonazoid were assessed by measuring bulk modulus during incremental increases in ambient pressure to 200 mm Hg. Comparison of in vivo microbubble destruction and signal enhancement at a mechanical index (MI) of 0.1 to 0.4 was performed by sequential reduction in pulsing interval from 10 to 0.05 sec during limb CEU at 7 MHz in mice and 1.8 MHz in dogs. Destruction was also assessed by broadband signal generation during passive cavitation detection. Real-time CEU perfusion imaging with destruction-replenishment was then performed at 1.8 MHz in dogs using an MI of 0.1, 0.2, or 0.3.
Results
Sonazoid had a higher bulk modulus than Definity (66 ± 12 vs 29 ± 2 kPa, P = .02) and exhibited less inertial cavitation (destruction) at MIs ≥ 0.2. On in vivo CEU, maximal signal intensity increased incrementally with MI for both agents and was equivalent between agents except at an MI of 0.1 (60% and 85% lower for Sonazoid at 7 and 1.8 MHz, respectively, P < .05). However, on progressive shortening of the pulsing interval, Definity was nearly completely destroyed at MIs ≥ 0.2 at 1.8 and 7 MHz, whereas Sonazoid was destroyed only at 1.8 MHz at MIs ≥ 0.3. As a result, real-time CEU perfusion imaging demonstrated approximately fourfold greater enhancement for Sonazoid at an MI of 0.3 to 0.4.
Conclusions
Robust signal enhancement during real-time CEU perfusion imaging of the limb is possible when using intermediate-power imaging coupled with a durable microbubble contrast agent.
In patients with suspected coronary artery disease, myocardial perfusion imaging is routinely used to quantify ischemia at rest or during stress. For peripheral artery disease (PAD), there is no widely accepted method for evaluating limb perfusion. Recent studies suggest that contrast-enhanced ultrasound (CEU) perfusion imaging provides valuable information on PAD severity beyond that provided by standard-of-care evaluation. However, signal enhancement during limb CEU is relatively low because of low skeletal muscle blood flow (0.05–0.3 mL/min/g) and low functional microvascular blood volume, approximately 50% of that in the myocardium. As a result, conventional perfusion imaging with destruction-replenishment analysis during low-power (mechanical index [MI] < 0.2) real-time imaging is not feasible, even with state-of-the-art multipulse algorithms that maximize microbubble signal relative to tissue. Instead, replenishment kinetics in the limb have been performed with high-power (MI > 0.8) “destructive” intermittent imaging, which increases signal intensity but is more time consuming and technically challenging because of the need to maintain the same imaging plane during leg exercise and at the long pulsing intervals (PIs) needed to reach plateau intensity in skeletal muscle. An alternative approach has been to use transit rate analysis with low-power imaging and intravenous bolus injections. This approach produces high microbubble blood concentrations, yet signal enhancement still remains low (<8 dB).
The ultrasound signal intensity from microbubbles achieved during stable cavitation (nondestructive nonlinear oscillation) increases with the acoustic pressure applied. During real-time imaging, there is a practical limit to increasing the power because of microbubble destruction, or inertial cavitation, which in the diagnostic frequency range of clinical scanners occurs at an MI > 0.2. We hypothesized that real-time imaging with intermediate power (MIs of 0.2 to 0.4) produces high signal intensity during limb CEU perfusion imaging when used in conjunction with acoustically “durable” microbubbles capable of undergoing stable cavitation without destruction at an MI > 0.2. We evaluated two microbubble agents used in humans that vary in their acoustic lability because of composition-related differences in viscoelastic damping. To test our hypothesis, we (1) measured microbubble pressure lability in vitro and in vivo, (2) analyzed microbubble acoustic amplitude-frequency response to differentiate stable from inertial cavitation, and (3) assessed in vivo ultrasound signal generation and performed in vivo CEU limb perfusion imaging with a range of acoustic pressures.
Methods
Animals
The study was approved by the Animal Care and Use Committee at Oregon Health & Science University. For murine imaging, we studied 14 wild-type C57Bl/6 mice aged 8 to 12 weeks. Mice were anesthetized with inhaled isoflurane (1.0%–1.5%), and a catheter was placed in a jugular vein for the administration of microbubbles. For canine imaging, we studied 10 dogs (25–31 kg) that were anesthetized with intravenous pentobarbital sodium and placed on a positive-pressure ventilator. A cannula was placed in the femoral vein for microbubble administration.
Microbubbles
Two microbubble agents were studied that differ in terms of gas and shell composition: (1) Definity (Lantheus Medical Imaging, North Billerica, MA), which is composed of octafluoropropane gas stabilized by pegylated and nonpegylated dipalmitoyl phospholipids, and (b) Sonazoid (GE Healthcare, Little Chalfont, United Kingdom), which is composed of decafluorobutane stabilized primarily by hydrogenated egg phosphatidylserine. The latter agent has been shown to be more resilient and less susceptible to ultrasound-mediated destruction in vitro. Microbubble concentration was measured by electrozone sensing (Multisizer III; Beckman Coulter, Brea, CA).
Microbubble Bulk Modulus
Bulk modulus measured from the relationship between ambient constant pressure and microbubble diameter was used to evaluate compressibility. A syringe containing microbubble suspensions was connected to cellulose tubing with an internal diameter of 200 μm, which was placed in the focal plane of a microscope (Axioskop2-FS; Carl Zeiss, Inc, Thornwood, NY) with a water immersion objective (×63/1.3 N.A.). A side port of the system contained a calibrated micromanometer pressure transducer (SPR-671; Millar Instruments, Inc, Houston, TX). The distal end of the tubing was then clamped, and the plunger was advanced by a syringe pump to increase system pressure ( P ) by approximately 50 mm Hg increments up to 200 mm Hg for Definity and 300 mm Hg for Sonazoid. Microscope images of microbubbles in the cellulose tubing at each pressure were acquired, and microbubble volumes ( V ) were calculated using diameter measurements assuming spherical geometry. Bulk modulus ( K ) for a 200 mm Hg pressure change was calculated assuming isotropic conditions and no significant change in wall thickness as P /(Δ V / V 0 ). Measurements were made only for microbubbles with baseline diameters of 3.0 to 5.0 μm because baseline size independently influences K .
In Vivo Acoustic Lability and Skeletal Muscle Signal Enhancement
To expand the applicability of our results to clinical and preclinical settings, in vivo experiments were performed using two separate ultrasound systems performing at either high or low diagnostic frequency. High-frequency CEU was performed with a linear-array transducer (Sequoia 512; Siemens Medical Systems USA, Inc, Mountain View, CA) at a frequency of 7 MHz and a dynamic range of 55 dB. The proximal hindlimb adductor muscles of mice were imaged in the short axis view. The nonlinear fundamental signal component for microbubbles was detected using multipulse phase inversion and amplitude modulation imaging. For low-MI imaging, the proximal hindlimb adductor muscle group of dogs ( n = 6) was imaged at 1.8 MHz using broadband amplitude modulation imaging with medium line density (iE33; Philips Medical Systems, Andover, MA). Imaging in the mice and dogs was performed at MIs of 0.1, 0.2, 0.3, and 0.4, and for each MI, gain settings were optimized to a level just below that which showed tissue speckle on precontrast imaging. In mice, Definity ( n = 6) or Sonazoid ( n = 8) was infused at 1 × 10 7 min −1 . In dogs ( n = 6), microbubbles were infused at 2 × 10 8 min −1 , with a latent period of 15 min between agents, the order of which was randomized. Images were obtained using PIs that were progressively shortened from 10 sec to 50 msec to assess (1) acoustic lability, defined by the progressive decline in signal enhancement with shorter PI, and (2) maximal signal enhancement, which was measured at a PI of 10 sec, which provides sufficient time for complete ultrasound sector volume replenishment in nonischemic mice and dogs.
CEU Perfusion Imaging
Real-time CEU perfusion imaging of the proximal hindlimb in dogs ( n = 4) was performed using destruction-replenishment analysis and the microbubble infusion rates and imaging settings described above. Perfusion imaging was also performed in mice ( n = 3) with the separate purpose of evaluating Sonazoid signal time-intensity data during real-time imaging (at MIs of 0.2 and 0.4) compared with that during intermittent high-power (MI 1.0) imaging. Once a steady-state concentration of microbubbles was achieved, microbubbles within the ultrasound sector volume were destroyed with a high-power (MI > 0.9) five-frame pulse sequence. Real-time microbubble replenishment was assessed at a PI of 0.5 sec, which was deemed to be the longest interval that still would provide a sufficient number of frames to reconstruct a time-intensity plot. For the high-MI murine protocols, the PI was increased from 0.5 to 15 sec. Background-subtracted intensity was measured using the immediate postdestruction frame as background, and time-intensity data were fit to the function
y = A ( 1 − e − β t ) ,
Microbubble Cavitation Response
Microbubble cavitation during ultrasound exposure (1.8 MHz) at an MI of 0.1 to 0.4 was assessed by passive cavitation detection. A spherically focused broadband (10 KHz to 20 MHz) hydrophone (Y-107; Sonic Concepts, Inc, Bothell, WA) with a focal depth of 20 mm and a focal width of 0.4 mm was confocally positioned with the ultrasound transducer at a 60° relative angle to receive signals from a flow phantom containing microbubbles. Received signals were digitized (100 MHz) and saved in a four-channel oscilloscope (Waverunner; Teledyne LeCroy, Chestnut Ridge, NY) using sequence mode. Data analysis was performed in MATLAB (The MathWorks, Natick, MA) using data averaged over the entire 20-min exposure period.
Ultrasound Pressure Measurement
Acoustic pressure measurement at each MI for both high- and low-frequency transducers was determined with a polyvinylidene fluoride needle hydrophone (HPM075/1; Precision Acoustics Ltd, Dorchester, United Kingdom). During calibration, hydrophones were mounted on a computer-controlled three-axis translation system (Velmex Inc, Bloomfield, NY) with 2.5-μm precision.
Statistical Analysis
Differences between the two microbubble agents for any given condition were assessed using unpaired Student t tests (two sided). Differences in values according to power were examined using one-way analysis of variance, and post hoc differences between groups were assessed with Tukey tests for multiple comparisons. Linear associations were analyzed using regression analysis and Pearson product-moment correlation; nonlinear associations were measured by regression analysis with least squares fit. Differences in the slope or rate constants were evaluated using analysis of covariance. Differences were considered significant at P < .05.
Results
Microbubble Compressibility
The mean diameter of microbubbles used for measurement of bulk modulus was similar for Definity and Sonazoid (4.5 ± 0.2 vs 4.4 ± 0.1 μm, P = .62). With incremental increases in stable ambient pressure, microbubble diameter and microbubble volume progressively decreased for both Definity and Sonazoid ( Figure 1 ). The degree to which size decreased with increasing pressure was greater for Definity than for Sonazoid ( P < .001 on analysis of covariance for both pressure-diameter and pressure-volume relationships). Microbubble bulk modulus measured assuming isotropic conditions and constant wall thickness was greater for Sonazoid than for Definity ( Figure 1 D), indicating lower compressibility from differences in viscoelastic properties.
In Vivo Acoustic Lability and Signal Intensity
Acoustic destruction of microbubbles in vivo was evaluated by the decline in video intensity produced by shortening of the ultrasound PI during a continuous infusion of microbubbles. The peak negative acoustic pressures for the high- and low-frequency transducers for each MI setting are shown in Table 1 . The mean diameter for the two microbubble agents was similar, although Sonazoid possessed a narrower size distribution ( Supplemental Figure 1 ). CEU with variable PIs was performed during high-frequency (7-MHz) contrast-specific imaging of the mouse hindlimb to evaluated signal enhancement and the degree of microbubble destruction. Data are displayed in Figure 2 , which shows the full linear range of PIs, and also in Figure 3 , which uses an abbreviated linear range of PIs ( Figures 3A and 3B ) and a log-compressed scale ( Figures 3C and 3D ) to better depict destruction at shorter PIs. With this agent, microbubble destruction was seen at MIs ≥ 0.2, with nearly complete destruction of the agent at MIs of 0.3 and 0.4 evidenced by nearly complete absence of microbubble signal at the shortest PI. For Sonazoid, signal enhancement was very low at an MI of 0.1, but again, intensity increased with MI. Signal intensity did not change with shortening of the PI, indicating very little destruction at 7 MHz, even at an MI of 0.4. Examples of CEU images from murine experiments are provided in Supplemental Figure 2 .
