Author + information
- Received April 8, 2013
- Revision received July 1, 2013
- Accepted July 8, 2013
- Published online November 5, 2013.
- Ahmed M. Mahmoud, PhD∗,†,
- Debaditya Dutta, PhD∗,
- Linda Lavery, BS∗,
- Douglas N. Stephens, MS‡,
- Flordeliza S. Villanueva, MD∗ and
- Kang Kim, PhD∗,§,‖∗ ()
- ∗Center for Ultrasound Molecular Imaging and Therapeutics, Department of Medicine, University of Pittsburgh School of Medicine, Heart and Vascular Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
- †Department of Biomedical Engineering and Systems, Cairo University, Giza, Egypt
- ‡Department of Biomedical Engineering, University of California, Davis, California
- §Department of Bioengineering, University of Pittsburgh School of Engineering, Pittsburgh, Pennsylvania
- ‖McGowan Institute for Regenerative Medicine, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
- ↵∗Reprint requests and correspondence:
Dr. Kang Kim, 950 Scaife Hall, Center for Ultrasound Molecular Imaging and Therapeutics, Department of Medicine, University of Pittsburgh School of Medicine, HVI, UPMC, 3550 Terrace Street, Pittsburgh, Pennsylvania 15213.
Objectives This study sought to examine the feasibility of in vivo detection of lipids in atherosclerotic plaque (AP) by ultrasound (US) thermal (or temporal) strain imaging (TSI).
Background Intraplaque lipid content is thought to contribute to plaque stability. Lipid exhibits a distinctive physical characteristic of temperature-dependent US speed compared with water-bearing tissues. As tissue temperature changes, US radiofrequency (RF) echoes shift in time of flight, which produces an apparent strain (thermal or temporal strain [TS]).
Methods US heating-imaging pulse sequences and transducers were designed and integrated into commercial US scanners for US-TSI of arterial segments. US-RF data were collected while gradually increasing tissue temperature. Phase-sensitive speckle tracking was applied to reconstruct TS maps coregistered to B-scans. Segments from injured atherosclerotic and uninjured nonatherosclerotic common femoral arteries (CFA) in cholesterol-fed New Zealand rabbits, and segments from control normal diet–fed rabbits (N =14) were scanned in vivo at different time points up to 12 weeks.
Results Lipid-rich atherosclerotic lesions exhibited distinct positive TS (+0.19 ± 0.08%) compared with that in nonatherosclerotic (–0.10 ± 0.13%) and control (–0.09 ± 0.09%) segments (p < 0.001). US-TSI enabled serial monitoring of lipids during atherosclerosis development. The coregistered set of morphological and compositional information of US-TSI showed good agreement with histology.
Conclusions US-TSI successfully detected and longitudinally monitored lipid progression in atherosclerotic CFA. US-TSI of relatively superficial arteries may be a modality that could be integrated into a commercial US system for noninvasive lipid detection in AP.
Atherosclerosis is characterized by vessel wall inflammation and thickening, where lipids, cells, and scar tissue deposit (1,2). These compositional changes in the vascular wall lead to the formation of atherosclerotic plaque (AP) (3). APs can rupture and cause major cardiovascular events such as acute coronary syndromes or ischemic stroke (4). Post-mortem correlational studies linking morphological and compositional features of plaque to acute cardiovascular events have motivated efforts to develop imaging methods for interrogating plaques in vivo; some of these imaging approaches are in clinical use, undergoing clinical trials, or still under pre-clinical development (5). To the extent that lipids in a plaque may be prognostically important (6,7), we sought to develop a method for detecting lipids in AP.
Ultrasound (US) thermal (or temporal) strain imaging (TSI) can identify lipid-bearing tissue (LBT) surrounded by water-bearing tissue (WBT) (8,9). US-TSI is based on the observation that the speed of sound is temperature-dependent in LBT versus WBT in opposite directions (10). As sound speed increases with a temperature rise in WBT, radiofrequency (RF) US echoes arrive back sooner to the US transducer (10). Conversely, sound speed decreases with increasing temperature in LBT, such that echoes from LBT return later. Because in US imaging, time of flight translates into distance, these phenomena make LBT appear further away during imaging, which can be estimated as positive thermal (or temporal) strain (TS), whereas WBT appears closer, which can be estimated as negative TS (9). These apparent thermal or temporal strains have no relation to the mechanical strains generated by tissue deformation in elastography.
Based on these considerations, we hypothesized that lipids in AP can be detected by US-TSI. Using a rabbit atherosclerotic model, we present the first in vivo study demonstrating the feasibility of detecting lipids in AP noninvasively using US-TSI.
Fourteen male New Zealand white rabbits (3.5–4 kg) were studied (4 controls, 10 accelerated atherosclerosis) (11) under the approval of the Institutional Animal Care and Use Committee of the University of Pittsburgh. The atherosclerosis rabbit group was fed an atherogenic diet (peanut oil 6%, cholesterol 1%) for 5 weeks. One week after commencing the diet, a balloon catheter (2-F Fogarty, Edwards Lifesciences, Irvine, California) was introduced into the common femoral artery (CFA) to induce injury. Injured right CFAs served as “atherosclerotic” vessels (n = 10), while uninjured contralateral CFAs in the same atherosclerosis group were used as “nonatherosclerotic” vessels (n = 10). In 4 rabbits without balloon injury that were fed a normal diet, the right CFA served as a negative control for atherosclerosis (“normal diet control”).
Imaging and heating were performed using a single US transducer at 6 MHz (axial resolution ≈ 200 μm) attached to a clinical US scanner (SonixTOUCH, Ultrasonix Medical Corp., Richmond, British Columbia, Canada). Rabbit electrocardiographic signal served as a trigger to synchronize US-TSI frame acquisition to end-systole (or end-diastole) to eliminate the mechanical strain from cardiac pulsation. An US-TSI sequence (12) was adapted as follows: The sequence began with imaging for 5 ms upon receiving the trigger, followed by heating for 192 ms, and then a short pause until the next trigger.
In a subset of 8 animals, high-resolution US-TSI was investigated using an electrocardiographic synchronized high-frequency US machine (Vevo2100, VisualSonics Inc., Toronto, Ontario, Canada). A custom US heating array transducer was attached to the imaging transducer (axial resolution ≈75 μm) to efficiently increase tissue temperature (13). An US center frequency of 21 MHz was used for imaging, while 3.55 MHz was used to operate the heating array.
US-RF data were processed offline using Matlab 7.12.0 (The MathWorks, Natick, Massachusetts). A temperature increase of 1.1 ± 0.1°C in 5 s was measured in vivo near the CFA using a temperature sensor. In this study, a number of US frames (heating time) corresponding to approximately 1.5°C temperature rise was used.
US-RF frames were acquired before and after a temperature rise. Due to temperature-induced sound speed change, US-RF echoes arrive sooner or later based on tissue composition, which appears as negative or positive time shift, respectively. These shifts were tracked using a phase-sensitive speckle tracking technique (14). TS was then estimated as the derivative of time shifts along the US propagation direction (9). TS maps for arterial segments, color coded such that red and blue indicated the positive and negative strain, respectively, were superimposed on B-mode images.
Post-mortem, CFAs were perfusion-fixed, excised, embedded in optimal cutting temperature compound, and frozen at –80°C. Vessel cross-sections were stained with general hematoxylin and eosin for nuclei staining, and oil red O for lipid staining. Histology sections were identified relative to the distance from CFA bifurcation to enable comparison with US at anatomically concordant sites.
To quantify lipid progression relative to an atherosclerotic segment, a region of interest, from lumen boundary to adventitial layer, was manually segmented. Then, the area of red stained lipids (oil red O) was divided by the total segmented area to estimate percentage lipid. Similar procedures were followed to quantify percentage lipid in approximately matched segments within US-TSI for comparison. Histological quantitative measurements were performed using Image J 1.46r (National Institutes of Health, Bethesda, Maryland).
Data analyses were performed using the Statistics Toolbox of Matlab 7.12.0. All values are expressed as the mean ± SD. TS assessments in the atherosclerotic, nonatherosclerotic, and control vessel groups were compared at terminal days using Student's t test. Linear regression analysis was performed to compare US and histology measurements. A p value <0.05 was considered significant (2-tailed).
Serial US-TSI, coregistered to Duplex US, was performed at week 0 (day of injury), and 4, 6, 8, 10, or 12 weeks post-injury. US-TSI was performed for multiple views of the CFA from the CFA bifurcation and up to ∼12 mm proximally. Regions of interest in the CFA were within the heating beam and at depths ranging from 9 to 13 mm from the skin surface. Rabbits were euthanized at week 4 (n = 1), 6 (n = 1), 8 (n = 2), 10 (n = 1), and 12 (n = 5) post-injury.
US-TSI of atherosclerotic and nonatherosclerotic arteries
Figure 1 compares US-TSI of atherosclerotic and contralateral nonatherosclerotic CFAs in a cholesterol-fed rabbit 12 weeks post-injury. The dashed lines in the B-scans approximately mark the heated area. US-TSI (Fig. 1B) for the atherosclerotic vessel (Fig. 1A) shows positive TS at sites, where histology (Fig. 1C) indicated lipids. In the nonatherosclerotic vessel (Fig. 1D), US-TSI (Fig. 1E) did not show positive TS near the lumen. Histology (Fig. 1F) showed normal vessel.
Monitoring lipid progression using US-TSI
The B-mode of the atherosclerotic vessel (Fig. 2A) shows a noticeable luminal stenosis (arrows) and corresponding reduced US Doppler signal (Fig. 2B) at 10 weeks post-injury. Histology (Fig. 2C) confirmed the presence of lipid-laden AP within the segment. Serial US-TSI was performed for this CFA at 6, 8, and 10 weeks post-injury. Positive TSs were observed in the segment at week 6 (Fig. 2D), and progressed in intensity and spatial extent in weeks 8 and 10 (Figs. 2E and 2F).
Quantitative assessment of lipid-rich AP using US-TSI
TS in lipid-rich atherosclerotic segments (n = 10) was significantly higher (p < 0.001) than that measured in both uninjured nonatherosclerotic (n = 10) and control segments (n = 4) (Fig. 3A). Atherosclerotic segments exhibited positive strains of +0.19 ± 0.08%, whereas nonatherosclerotic and control segments exhibited mostly negative strains of –0.10 ± 0.13% and –0.09 ± 0.09%, respectively. No significant difference in TS was observed between nonatherosclerotic and control segments (p = 0.748).
A close correlation was found between histological and US-TSI measurements of percentage lipid (n = 10) (Fig. 3B).
Using a rabbit atherosclerotic model, this study is the first in vivo demonstration that differences in TS between atherosclerotic and normal segments can be spatially mapped and those areas with positive TS colocalize with histologically proven lipid-laden AP. These preliminary data have major implications for the in vivo detection of lipid-rich plaques.
Atherosclerotic segments exhibited distinct positive TS as would be expected in LBT, whereas control and nonatherosclerotic segments possessed negative TS typical of WBT. Similar findings were observed in vitro using excised tissue (9). We measured a relatively large SD of TS in atherosclerotic segments, probably for several reasons. First, because we combined measurements at different terminal days (4 to 12 weeks), variations in lipid content increased the range of positive TS. Second, atherosclerotic segments contain WBT such as smooth muscle cells, connective tissue, and fibrosis, which have negative temperature dependences of sound speed. This would result in heterogeneous TS maps, as the varied tissue components cannot be completely excluded during image segmentation. TS in control and nonatherosclerotic segments also had large standard deviations, as some areas exhibited positive TS, which could be due to normal adventitial fat (15). Three common factors that may vary TS in all groups are the variation in the heating beam, US attenuation in muscle/vascular tissue of ∼1 dB/(MHz·cm) (16), and potential residual mechanical deformation due to physiological motion. Notwithstanding these inherent limitations, the average TS clearly differentiated lipids from WBT in AP with high significance (p < 0.001).
Terminal study points were varied to compare US-TSI and histology at different stages of atherosclerosis progression. Although some atherosclerotic vessels did not show a significant stenosis by duplex US, US-TSI was able to detect non–flow-limiting AP fatty lesions. Furthermore, US-TSI serially tracked lipid accumulation during AP progression, exhibiting a continuous change in the area and/or value of positive TS in AP over time (Figs. 2D to 2F). The continuous remodeling in the shape of AP at different stages (17) may suggest corresponding changes in lipid distribution. Although a slight misalignment in imaging planes between each week is possible, the overall observations were consistent with the interpretation of AP progression.
Relatively few animals were studied in this proof-of-concept study, precluding complete statistical analysis and quantitative measurements of the sensitivity and specificity of US-TSI. However, our quantitative analysis, albeit limited, showed good correlation between US and histology (Fig. 3B). US and histology measurements did not match in their absolute magnitudes, which could be due to the limited resolution of US compared to high-magnification histology images, and/or the potential post-mortem changes in vessels during histology processing (18). Although this rabbit model exhibited some features of AP similar to human disease, the pathophysiology and extreme nature of the lesions may not be fully representative of those causing human carotid plaque or acute coronary syndromes (11). Nonetheless, these lipid-rich arterial lesions (19) enabled us to demonstrate the feasibility of noninvasive lipid detection in AP.
US-TSI relies on inducing a gentle temperature rise in tissue, so temperature control is important for safe in vivo use. At this small temperature (<10°C), a linear relationship between sound speed and temperature can be assumed (20). Accordingly, the temperature rise during US-TSI can be estimated using the linear relationship between heating time and TS (12). According to the American Institute of Ultrasound in Medicine (21), “there have been no significant, adverse biological effects observed due to temperature increases less than or equal to 2°C above normal, for exposure durations up to 50 hours.” For future clinical implementation, we chose an US-TSI temperature increase of ∼1.5°C in <10 s, which generated sufficient signal/noise without conferring significant adverse biological effects. The peak negative pressure of US heating pulse was estimated experimentally. The clinical system exhibited a pressure amplitude of –1.27 MPa, while it was –1.40 MPa using the custom heating array. We did not observe adverse biological effects due to US-TSI. Also, to the best of our knowledge, there have been no previous reports to suggest adverse effects of the temperature rises used in this study on plaque stability. Our limited observations thus far and the acoustic parameters used would predict the safety of our approach, although important next steps at this juncture would be to perform serial studies on the heating effect on plaque biology and other tissues along with rigorous measurements for temperature and time-average power. However, a comprehensive systematic investigation of bioeffects was outside the scope of the current proof-of-concept study.
US-TSI was able to detect and longitudinally monitor lipids in AP. Lipids in atherosclerotic arterial segments exhibited distinct positive TS, whereas control and nonatherosclerotic segments, comprising mostly WBT, showed predominantly negative TS. Lipid-rich lesions identified by US-TSI colocalized and were quantitatively concordant with histology. These preliminary findings demonstrate the potential utility of US-TSI for noninvasive lipid detection in AP in vivo. This novel strategy is worthy of additional study to determine its sensitivity and specificity in detecting lipids in AP and the potential clinical utility of these findings.
The authors would like to thank Dr. Andrew Carson for technical discussion in histology.
This study was supported by the National Institute of Health (NIH) grant R01 HL098230-01A1; small animal imaging system (Vevo2100) was supported by the NIH grant 1S10RR027383-01. Dr. Villanueva has made material transfer agreements with Lantheus Imaging and GE Global Research. All authors have reported that they have no conflicting relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- atherosclerotic plaque
- common femoral artery
- lipid-bearing tissue
- thermal (or temporal) strain
- thermal (or temporal) strain imaging
- water-bearing tissue
- Received April 8, 2013.
- Revision received July 1, 2013.
- Accepted July 8, 2013.
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