Background
The aim of this study was to evaluate the influence of acoustic power on ultrasound molecular imaging data with targeted microbubbles.
Methods
Imaging was performed with a contrast-specific multipulse method at mechanical indexes (MIs) of 0.18 and 0.97. In vitro imaging was used to measure concentration-intensity relationships and to assess whether damping from microbubble attachment to cultured endothelial cells affected signal enhancement. Power-related differences in signal enhancement were evaluated in vivo by P-selectin-targeted and control microbubble imaging in a murine model of hind-limb ischemia-reperfusion injury.
Results
During in vitro experiments, there was minimal acoustic damping from microbubble-cell attachment at either MI. Signal enhancement in the in vitro and in vivo experiments was 2-fold to 3-fold higher for high-MI imaging compared with low-MI imaging, which was due to greater pixel intensity, the detection of a greater number of retained microbubbles, and increased point-spread function. Yet there was a linear relationship between high-MI and low-MI data indicating that the relative degree of enhancement was similar.
Conclusion
During molecular imaging, high-MI protocols produce more robust targeted signal enhancement than low-MI protocols, although differences in relative enhancement caused by condition or agent are similar.
Molecular imaging with contrast-enhanced ultrasound relies on the detection of microbubbles or nanoparticles that are targeted to disease-specific molecules. In general, targeted agents are administered as boluses and are then imaged using high–mechanical index (MI) ultrasound after a 5-minute to 10-minute pause to allow the clearance of freely circulating microbubbles from the blood pool. Signal from targeted microbubbles is encoded in the first frame obtained upon the resumption of imaging, after which the signal decays from the acoustic disruption of the agent. High-MI imaging has been used in most molecular imaging studies to maximize sensitivity for retained tracer. However, there has been increasing interest in using low-MI imaging to (1) take advantage of frame averaging, (2) choose appropriate frames void of motion artifacts from multipulse contrast techniques, and (3) facilitate 3-dimensional volume scanning. Also, real-time imaging will greatly improve the ease of localizing the tissue of interest, which is important for transitioning ultrasound molecular imaging from anesthetized animals, for which the subject and probes are fixed, to awake humans.
An important consideration when using low-MI imaging is whether signal enhancement will be sufficiently robust to evaluate subtle differences in molecular expression profile. The volumetric vibration of microbubbles in an acoustic field is substantially influenced by viscous and thermal damping from its microenvironment. Microbubble attachment to biosurfaces may disproportionately affect nonlinear signal generation when imaging at a low MI. This notion is supported by the finding that ultrasound frequency and/or amplitude response is altered when microbubbles are attached to particles, cells in suspension, or cell monolayers. The purpose of this study was to determine whether differences in targeted microbubble signal enhancement during high-MI and low-MI imaging in vivo influence molecular imaging data on expression profile.
Methods
Microbubble Preparation
Lipid-shelled decafluorobutane microbubbles were prepared by sonication of a gas-saturated aqueous suspension of distearoylphosphatidylcholine (2 mg/mL) and polyoxyethylene-40-stearate (1 mg/mL). For the preparation of targeted microbubbles, distearoylphosphatidylethanolamine–polyethylene glycol(2000)biotin (0.1 mg/mL) was added to the aqueous suspension. Targeting ligands were conjugated to the microbubble surface using a biotin-streptavidin link, as previously described. Microbubbles were targeted to vascular cell adhesion molecule (VCAM)–1 or P-selectin by the conjugation of rat antimouse immunoglobulin G1 monoclonal antibody from hybridoma (clone MK2.7 for VCAM-1, RB40.34 for P-selectin). Control microbubbles were prepared in a similar fashion with isotype control antibody (R3-34; Pharmingen Inc, San Diego, CA). Microbubble size distribution and concentration were measured by electrozone sensing (Multisizer III; Beckman Coulter, Fullerton, CA).
Contrast Ultrasound
Ultrasound imaging in vitro and in vivo was performed with a linear array transducer (15L8) interfaced with a Sequoia ultrasound system (Siemens Medical Systems, Mountain View, CA). A multipulse algorithm using phase and amplitude modulation (contrast pulse sequencing) was used to detect the nonlinear fundamental component of the microbubble signal. Imaging was performed at 7 MHz with low-MI (0.18) and high-MI (0.97) settings. The dynamic range was set at 55 dB, and gain settings for each MI were adjusted at the beginning of each study to a level just below that which produced visible background speckle. Digital data were transferred to an offline computer for analysis. Intensity on each imaging frame was transformed from a logarithmic to a linear scale using known dynamic range and span of intensity values. To ensure that the relationship between microbubble concentration and signal intensity at 7 MHz was linear with low-MI and high-MI settings, plots of concentration versus intensity were generated by imaging microbubbles suspended in a water bath.
Signal From Microbubble-Cell Complexes In Vitro
Microbubble ligation to cultured endothelial cells was used as an in vitro model to determine the relative effect of attachment on signal intensity during high-MI and low-MI imaging. Murine endothelial cells (SVEC4-10; ATCC, Manassas, VA) that express VCAM-1 were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in culture flasks. For activation, the cells were pretreated with tumor necrosis factor–α (10 ng/mL) for 2 to 4 hours. The cells were then removed from the culture flasks by trypsin digestion, centrifuged, and resuspended in cell culture medium. Microbubbles (1 × 10 6 ) targeted to murine VCAM-1 were added to suspensions of SVEC4-10 in a total volume of 1 mL and incubated at 37°C for 5 minutes with intermittent gentle agitation to allow for microbubble attachment to cells. The percentage of microbubbles that were adhered to cells was varied according to the degree of tumor necrosis factor–α activation and the number of cells added (range of microbubble/cell ratios, 20:1-100:1). The relative proportion of free microbubbles and those ligated to endothelial cells was quantified by light microscopy. The microbubble suspensions were diluted 100-fold and loaded into 3-mL dialysis cassettes (Thermo Scientific, Rockford, IL). The cassettes were suspended 3 cm beneath the ultrasound transducer in a water bath (acoustic focus, 3 cm). Several frames were first acquired at an MI of 0.18, after which the MI was increased to 0.97. Video intensity was measured from the upper third of the cassette. Values were averaged from several frames during low-MI imaging, whereas only the first frame was used from high-MI imaging acquisitions. Pixel intensity histograms were also generated from similar regions of interest using raw images before log-linear conversion.
Molecular Imaging of Inflammation in Acute Ischemia
Differences in molecular imaging signal enhancement during low-MI and high-MI imaging in vivo were evaluated in a murine model of acute ischemia-reperfusion injury of the hind limb. The study was approved by the institutional animal care and use committee. Imaging was performed in 18 wild-type C57Bl/6 mice and in 6 mice with gene-targeted homozygous deletion of P-selectin (P-/-). Anesthesia was induced and maintained with inhaled isoflurane. A jugular vein was cannulated for the administration of microbubbles. Acute hind-limb ischemia was produced in 12 of the wild-type and all 6 of the P-/- mice by the placement of an external occlusion device placed around the proximal hind limb for 10 minutes. Complete cessation of blood flow in the hind limb was confirmed by contrast-enhanced ultrasound perfusion imaging during occlusion.
Molecular imaging was performed 45 minutes after ischemia. The proximal hind-limb adductor muscles were imaged in a transaxial plane leg midway between the inguinal fold and the knee. P-selectin-targeted or control microbubbles (3 × 10 6 for each) were injected intravenously in random order. After each injection, ultrasound imaging was paused for 10 minutes, after which imaging was resumed at an MI of 0.18. Several frames were acquired, after which the MI was increased to 0.97, with acoustic shielding during power adjustment. The signal from retained targeted agent was derived as previously described. The initial frame at an MI of 0.97 was acquired, after which microbubbles in the sector were destroyed by several more frames. Background images representing freely circulating microbubbles were subsequently acquired at both MI settings with a pulsing interval of 10 seconds for high-MI imaging. The molecular signal was derived by digital subtraction of several averaged postdestruction frames at appropriate MI from several averaged predestruction frames for low-MI imaging or the initial frame for high-MI imaging. Regions-of-interest were drawn over the adductor muscle group to measure video intensity. The raw images were used to generate histograms of pixel video intensity and to analyze the number and percentage of pixels that demonstrated video enhancement (>3 standard deviations beyond the mean background signal).
Statistical Methods
Data are expressed as mean values unless otherwise stated. For assessment of microbubble stability when imaged at the low MI, repeated-measures analysis of variance was used. Correlations were made with Pearson’s correlation and least squares fit.
Results
Microbubble Concentration-Intensity Relationships
To validate comparisons between video intensities, the relation between microbubble concentration and video intensity was performed in an in vitro water tank. This relation was relatively linear for both low-MI and high-MI imaging, with some slight flattening of the curves at high concentrations that approached dynamic range saturation ( Figure 1 A). The ratio of signal enhancement at high MI to that at low MI ( Figure 1 B) was approximately 3:1, except at the very lowest microbubble concentrations, at which the signal was extremely low because of the paucity of microbubble events resulting in nearly equal background intensity levels.
Acoustic Response From Microbubbles Ligated to Cells
For the in vitro cell-binding studies, a wide range of attachment efficiency (3%-31% of all microbubbles) was achieved by combining a constant number of VCAM-1-targeted microbubbles with SVEC4-10 cells in suspension in different concentrations and activation states. Clustering of microbubbles to one another on the cell surface was not observed ( Figure 2 A), and there was no preferential attachment for microbubbles according to size. At both low MI and high MI, the percentage of microbubbles attached did not significantly influence background-subtracted video intensity ( Figure 2 B), indicating that microbubble ligation to cells did little to alter signal generation at low or high power under in vitro conditions. Despite similar background (precontrast) levels, video intensity values were 2-fold to 3-fold higher at high MI compared with low MI ( Figure 2 C). High-MI data were only 1.5 to 1.7 higher than low-MI data for similar analysis performed before log-linear conversion of data. Illustrated by the two examples in Figures 3 A and 3B, increasing the MI resulted in a rightward shift in the histogram for pixel intensity, indicating that high signal enhancement was due in part to greater intensity for pixel enhancement.
