Noninvasive Detection of Macrophage-Rich Atherosclerotic Plaque in Hyperlipidemic Rabbits Using "Positive Contrast" Magnetic Resonance Imaging
Grigorios Korosoglou, MD*, ,*,
Robert G. Weiss, MD, ,
Dorota A. Kedziorek, MD,
Piotr Walczak, MD,
Wesley D. Gilson, PhD,
Michael Schär, PhD, ,
David E. Sosnovik, MD||,
Dara L. Kraitchman, VMD, PhD,
Raymond C. Boston, PhD¶,
Jeff W.M. Bulte, PhD,
Ralph Weissleder, MD, PhD|| and
Matthias Stuber, PhD
* Department of Cardiology, University of Heidelberg, Heidelberg, Germany
Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland
Department of Medicine, Cardiology Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland
Philips Medical Systems, Cleveland, Ohio
|| Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
¶ School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania.
Manuscript received October 31, 2007;
revised manuscript received February 12, 2008,
accepted March 19, 2008.
* Reprint request and correspondence: Dr. Grigorios Korosoglou, University of Heidelberg, Department of Cardiology, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany. (Email: grigorios_korosoglou{at}med.uni-heidelberg.de).
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Abstract
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Objectives: This study was designed to identify macrophage-rich atherosclerotic plaque noninvasively by imaging the tissue uptake of long-circulating superparamagnetic nanoparticles with a positive contrast off-resonance imaging sequence (inversion recovery with ON-resonant water suppression [IRON]).
Background: The sudden rupture of macrophage-rich atherosclerotic plaques can trigger the formation of an occlusive thrombus in coronary vessels, resulting in acute myocardial infarction. Therefore, a noninvasive technique that can identify macrophage-rich plaques and thereby assist with risk stratification of patients with atherosclerosis would be of great potential clinical utility.
Methods: Experiments were conducted on a clinical 3-T magnetic resonance imaging (MRI) scanner in 7 heritable hyperlipidemic and 4 control rabbits. Monocrystalline iron-oxide nanoparticles (MION)-47 were administrated intravenously (2 doses of 250 µmol Fe/kg), and animals underwent serial IRON-MRI before injection of the nanoparticles and serially after 1, 3, and 6 days.
Results: After administration of MION-47, a striking signal enhancement was found in areas of plaque only in hyperlipidemic rabbits. The magnitude of enhancement on magnetic resonance images had a high correlation with the number of macrophages determined by histology (p < 0.001) and allowed for the detection of macrophage-rich plaque with high accuracy (area under the curve: 0.92, SE: 0.04, 95% confidence interval: 0.84 to 0.96, p < 0.001). No significant signal enhancement was measured in remote areas without plaque by histology and in control rabbits without atherosclerosis.
Conclusions: Using IRON-MRI in conjunction with superparamagnetic nanoparticles is a promising approach for the noninvasive evaluation of macrophage-rich, vulnerable plaques.
Key Words: atherosclerosis  vulnerable plaque superparamagnetic nanoparticles molecular imaging inversion recovery with ON-resonant water suppression (IRON) positive contrast magnetic resonance imaging
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Abbreviations and Acronyms
| | CNR = contrast-to-noise ratio | | FA = flip angle | | IRON = inversion recovery with ON-resonant water suppression | | MION = monocrystalline iron-oxide nanoparticle | | MRA = magnetic resonance angiography | | MRI = magnetic resonance imaging | | NER = normalized enhancement ratio | | ROI = regions of interest | | SNR = signal-to-noise ratio | | TE = echo time | | TR = repetition time |
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Atherosclerosis is the underlying cause of most cardiovascular diseases. Despite major advances in the treatment of coronary artery disease, it remains the leading cause of death in Western societies and the predominant underlying cause of sudden cardiac death (1,2). The role of inflammation in all stages of atherosclerosis is now widely appreciated (3).
Currently, coronary angiography is the clinical gold standard for diagnosing the presence and extent of coronary artery disease. However, this technique is invasive and provides limited information on the presence of inflammation within the vessel wall and future cardiac events (4). Therefore, a noninvasive technique that can identify macrophage-rich plaques and thereby assist with risk stratification of patients with coronary artery disease would be of great potential clinical utility. Several recent molecular imaging strategies have been proposed to detect inflamed atherosclerotic plaque noninvasively, using targeted gadolinium agents (5,6) and superparamagnetic nanoparticles (7–11). The appeal of superparamagnetic nanoparticles lies in the fact that these agents have completed phase III clinical trials and thus currently have the greatest potential for clinical translation (12). In previous studies, superparamagnetic nanoparticles resulted in local signal loss in areas of plaque. Concerns have been raised, however, that signal loss may also arise from other sources, such as motion, absence of tissue, "fibrous cap," or calcification (8,13,14).
Recently, new magnetic resonance imaging (MRI) methods were reported that create positive signals in areas of superparamagnetic materials (15–18). The purpose of our study was to test whether macrophage-rich plaque can be visualized with positive signals using inversion recovery with ON-resonant water suppression (IRON)-MRI (18) and whether the degree of signal enhancement is related to the number of macrophages in atherosclerotic plaque. Experiments were conducted in Watanabe heritable hyperlipidemic and in New Zealand White rabbits as the control group on a clinical 3-T scanner.
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Methods
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Animals.
This study was approved by the Institutional Animal Care and Use Committee of Johns Hopkins University School of Medicine. Experiments were conducted in 7 mature male heritable hyperlipidemic Watanabe rabbits (14.9 ± 1.3 months old, 3.0 ± 0.3 kg body weight, Brown Family Enterprises, Odenville, Alabama). At this age, Watanabe rabbits exhibit active plaque formation within their aortic wall (7). Further progression of atherosclerotic lesions was induced by a high-cholesterol diet (1% cholesterol, Dyets Inc., Bethlehem, Pennsylvania) for 6 weeks (19). After 6 weeks of a high-cholesterol diet, blood samples were taken from the ear vein to measure total serum cholesterol levels. Four male New Zealand White rabbits (6.6 ± 0.5 months old, 2.9 ± 0.4 kg body weight, Myrtle's Rabbitry, Inc., Thompson Station, Tennessee) served as negative control rabbits for atherosclerosis.
Superparamagnetic nanoparticles.
Monocrystalline iron-oxide nanoparticle (MION)-47 (Center for Molecular Imaging Research, Harvard Medical School, Charlestown, Massachusetts) is a stable colloid, which shortens the longitudinal (T1)- and transverse (T2)-relaxation times of tissue (20,21). MION-47 consists of a central monocrystalline magnetitelike single crystal core coated by multiple 10-kD dextran molecules. The mean size of the nanoparticles is 27.5 ± 6.8 nm, and the R1- and R2-relaxivities are 25.5 mM–1s–1 and 53.7 mM–1s–1, respectively, in an aqueous solution at 37°C and 0.47 T. The plasma half-life of MION-47 is 11.4 ± 0.6 h in mice (21). MION-47 is a laboratory preparation developed by Josephson and Weissleder, which has similar size, relaxivity, and biological properties as ferumoxtran-10, which is a Food and Drug Administration–compliant clinical preparation (12).
Experimental design.
The experiments included MRI before and after the administration of MION-47. Animals were imaged before and after the injection of 250 µmol Fe/kg on day 0. Imaging was repeated on days 1, 3, and 6. Based on quantitative T1-measurements of blood performed in preliminary experiments (T1 of arterial blood of 37 ± 6 ms within 2 h after injection, 125 ± 32 ms on day 1, 1,629 ± 75 ms on day 3, and 1,652 ± 58 ms at baseline), we observed that the T1 value of blood on day 3 approached the T1 value of blood at baseline, which indicated complete clearance of the agent from blood pool at this point. To maximize the time of exposure of the macrophages to the iron particles, a reinjection of 250 µmol Fe/kg was performed on day 3 and animals were imaged again on day 6 and were subsequently sacrificed with an overdose of thiopental (Fig. 1). All rabbits that were followed on this protocol survived until day 6 and showed no clinical signs of respiratory or cardiac failure during the study.
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Figure 1 Experimental Protocol
Experimental protocol illustrating the serial magnetic resonance imaging (MRI) and the contrast agent injections performed in Watanabe and control rabbits. MION-47 = monocrystalline iron-oxide nanoparticle.
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MRI.
Animals were imaged under general anesthesia in a Philips 3-T Achieva system (Philips Medical Systems, Best, the Netherlands) and using a 4-element human carotid receiver coil (Pathway MRI Inc., Seattle, Washington). A standardized protocol was followed, aiming at the visualization of the aortic wall with high-spatial resolution.
T1-Weighted Conventional Magnetic Resonance Angiography (MRA)
Coronal 3-dimensional gradient-echo images were obtained using the following sequence parameters: repetition time/echo time (TR/TE) = 25/2.7 ms, 20° flip angle (FA), 200 x 100 mm2 field of view, and a 0.5 x 0.5 x 1 mm3 voxel size. Fifty sagittal (thoracic aorta) and 50 coronal (abdominal aorta) slices were acquired.
Black-Blood Turbo Field-Echo Imaging
For black-blood turbo field-echo imaging, a dual-inversion pre-pulse with an inversion delay of 370 ms was used for blood-signal nulling. Typical imaging parameters were: TR/TE = 21/10 ms, 30° FA, 100 x 100 mm2 field of view, and a 0.35 x 0.35 x 2 mm3 voxel size.
Black-Blood Turbo Spin-Echo Imaging
For black-blood turbo spin-echo imaging, blood-signal nulling was obtained (as mentioned in the previous paragraph) and typical imaging parameters were: TR/TE = 857/8.2 ms, 90° FA, 100 x 100 mm2 field of view, and a 0.35 x 0.35 x 2 mm3 voxel size. Seven coronal and 20 axial slices were acquired to include the abdominal aorta and 7 sagittal and 20 axial slices were acquired for the thoracic aorta.
IRON
The concept of IRON imaging comprises the use of a spectrally selective saturation pre-pulse ON-resonance with the bandwidth of BW
IRON to suppress the signal originating from ON-resonant protons (18). This saturation pulse does not affect OFF-resonant protons in close proximity to the superparamagnetic nanoparticles. Therefore, signal enhancement adjacent to these particles can be generated, while the ON-resonant background appears signal attenuated. For IRON imaging, an ON-resonant IRON pre-pulse with a BWIRON of 100 Hz and a FA of 100° was used. This IRON pre-pulse was followed by a frequency selective pre-pulse for fat saturation and was combined with turbo field-echo and turbo spin-echo imaging sequences.
Image analysis.
Aortic Wall Thickness
The thickness of the aortic wall was measured on pre-contrast black-blood turbo field-echo imaging images using the Deriche edge detection, as previously shown (22).
Enhancement of the Aortic Lumen on Conventional and on Iron-MRA
For quantification of signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR), regions of interest (ROI) were placed manually in the aortic lumen to measure the mean blood signal (S
Blood). Signal intensity of adjacent muscle (S
Muscle) was measured by choosing a ROI of similar size in a muscle adjacent to the aorta. The ROI were also placed in the air outside of the rabbit to measure the standard deviation of the background signal ( Background), and CNR was calculated as follows:
CNRBlood = (S
Blood – S
Muscle)/Background
Enhancement of the Aortic Wall on Iron Images
To quantify signal enhancement in the aortic wall, signal intensity measurements were performed as follows. The ROI were manually positioned in the vessel wall and the vessel lumen in 5-mm intervals extending from the aortic root to the iliac bifurcation to measure the vessel wall signal S
Wall and mean blood signal S
Blood, respectively. To allow for exact matching between the 4 imaging sessions for each rabbit, anatomical landmarks (the position of the aortic arch, renal arteries, and iliac bifurcation) were used to guide ROI positioning. For each matched slice, the normalized enhancement ratio (NER) of the aortic wall (5) was calculated by dividing the post-contrast SNR of the vessel wall by baseline SNR as follows:
NER = (SNRWall-Post-contrast/SNRBlood-Post-contrast)/(SNRWall-Baseline/SNRBlood-Baseline)
With this approach, the baseline measurements of each animal serve as their own internal reference. Vessel wall enhancement will be indicated by a ratio of NER >1. To investigate the contribution of partial volume effects on positive signal detected by IRON in areas of plaque, the mean NER on day 6 was compared between areas with plaque (confirmed by histology) in Watanabe rabbits and in areas without plaque in control rabbits, which were matched for wall thickness (range 0.4 to 0.6 mm). To achieve matched wall thicknesses between Watanabe and control rabbits, different parts of the aortic wall were compared (abdominal wall with plaque in Watanabe rabbits vs. thoracic aortic wall without plaque in control rabbits).
Post-mortem analysis.
Euthanasia was performed after the final imaging session on day 6. Subsequently, in vivo perfusion fixation was performed and the entire aorta from the aortic root to below the iliac bifurcation was harvested. To account for in vitro tissue shrinkage of the aorta, which may confound the matching of histologic slides and magnetic resonance (MR) images, the aorta was aligned to a hard copy of a 3-dimensional multiplanar reformatted MRA and was then cut at 5-mm intervals. Coregistration was performed carefully, considering anatomical landmarks. The specimens were then frozen, and serially prepared cryosections (10-µm thick) were obtained. Sections of the aorta were stained with: 1) oil red O and counterstained with hematoxylin for specific lipid staining; 2) fast nuclear red for determination of morphology and for measurement of wall thickness; 3) acid phosphatase (Sigma-Aldrich Corp., St. Louis, Missouri) to detect phagocytic cells and counterstained with Prussian blue to detect superparamagnetic nanoparticles (7,13); and 4) RAM-11 (Dako Corp., Carpinteria, California), a marker of the rabbit macrophage cytoplasm. These sections were counterstained with 4',6-diamidino-2-phenylindole-dihydrochloride reagent and were processed for immunofluorescence.
For quantitative analysis, the area of acid phosphatase-positive red cells was determined by manual contouring and was related to the total vessel wall area on the same histology slide. By this approach, a measure for macrophage "density" was assessed on histology slides, which was then related to the magnitude of signal enhancement in corresponding slices on IRON images. A cutoff value of macrophage density >5% was selected to differentiate between macrophage-rich plaque and areas with plaque but with a low density of macrophages. Quantitative analysis of histological specimen was performed using Image J software (National Institutes of Health, Bethesda, Maryland). For the determination of intramacrophage iron-oxide uptake, a quantitative ferrozine-based spectrophotometric assay was performed (23).
Statistical analysis.
Statistical analysis was performed using Stata 9.2 (Stata Corp., College Station, Texas). Data are presented as mean ± 1 SD. Differences in aortic wall thickness and differences in iron-oxide uptake between Watanabe and control rabbits were compared using repeated measures regression analysis and unpaired, heteroskedastic t tests. To correct for the lack of independence of our data, a clustered regression approach was used to compare differences in NER between different time points and between different groups of rabbits, taking into account the repeated measures performed in rabbits. Furthermore, receiver-operating characteristics were used and a cutoff value was selected for NER to provide an optimal trade-off between sensitivity and specificity for the detection of macrophage-rich plaque in matched slices on histology. Concordance analysis using the Lin concordance correlation coefficient (24) was used for the comparison of vessel wall diameter between MRI and histology. Because this approach does not accommodate clustering considerations, we refined a bootstrapping approach for this aspect of our analysis. Differences were considered statistically significant at p < 0.05.
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Results
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Cholesterol levels and characterization of the atherosclerotic lesions.
For the Watanabe rabbits, the serum cholesterol was 2,119 ± 243 mg/dl after 6 weeks of a high-cholesterol diet. The animals exhibited lipid-rich (Fig. 2A) and macrophage-rich (Fig. 2B) atherosclerotic plaque formation. The plethora of macrophages in the atherosclerotic plaques was confirmed by RAM-11 immunostaining (Figs. 2C and 2D). As expected, no plaque and no macrophages were found in control rabbits (Figs. 2E to 2H).
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Figure 2 Histology of Hyperlipidemic Versus Control Rabbits
Watanabe rabbits exhibited lipid-rich plaque formation (A) with a high density of macrophages (acid phosphatase staining in panel B). The high density of macrophages in the atherosclerotic plaque could be confirmed by RAM-11 immunostaining (macrophages stained green by RAM-11 and cell nuclei stained blue by 4',6-diamidino-2-phenylindole-dihydrochloride [DAPI], panels C and D). No plaque formation was observed in control rabbits (E to H).
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Aortic wall thickness.
Figure 3
illustrates representative baseline images of the thoracic and the abdominal aortic wall of Watanabe (Figs. 3A to 3C) and of control (Figs. 3D to 3F) rabbits. Watanabe rabbits exhibited increased thickness both in the thoracic (0.76 ± 0.24 mm vs. 0.42 ± 0.13 mm, p < 0.001) and in the abdominal aortic wall (0.30 ± 0.16 mm vs. 0.21 ± 0.05 mm, p < 0.01) (Fig. 3G) compared with control rabbits. Wall thickness, measured on MR images, correlated closely with that measured by histology (asymptotic concordance of 0.73, p < 0.0001) (Fig. 3H).
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Figure 3 Increased Wall Thickness in Hyperlipidemic Rabbits
Representative black-blood turbo field-echo imaging (A, B, and D) and black-blood turbo spin-echo imaging baseline images (C, E, and F) of the aortic wall in Watanabe (A to C) and control rabbits (D to F). Watanabe rabbits exhibited increased wall thickness in the thoracic (*p < 0.001 vs. control rabbits) and in the abdominal aorta (p < 0.001 vs. control rabbits) (G). Wall thickness measured with magnetic resonance imaging (MRI) correlated closely with histology on matched slices (H) (asymptotic concordance of 0.73, p < 0.0001).
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Detection of superparamagnetic nanoparticles in the aortic lumen.
Immediately after MION-47 injection (day 0), intravascular signal decreased on conventional T1-weighted MRA, reflecting T2*-shortening of the blood pool. Signal intensity returned on day 1 and approached baseline on days 3 and 6 (Figs. 4A to 4D and 4I). Using IRON, superparamagnetic nanoparticles contributed to strong intravascular OFF-resonance enhancement in the lumen of the aorta in both control and Watanabe rabbits. The intraluminal signal remained high on day 1 and approached baseline on days 3 and 6, allowing for better definition of the aortic wall (Figs. 4E to 4I).
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Figure 4 Intravascular OFF-Resonance Enhancement After MION-47 Administration
Conventional T1-weighted magnetic resonance angiography (MRA) in a Watanabe rabbit at baseline (A), on day 0 (B), on day 1 (C), and on day 6 (D) after the injection of superparamagnetic nanoparticles are shown. Injection of the contrast agent resulted in decreased intravascular signal, due to T2*-shortening of the blood. Using inversion recovery with ON-resonant water suppression (IRON), blood, fat, and muscle were homogenously suppressed at baseline (E). Monocrystalline iron-oxide nanoparticle (MION) 47 injection contributed to strong intravascular OFF-resonance enhancement on day 0 (F) and day 1 (G), which approached baseline at later times (H and I), allowing for better judgment of the aortic wall.
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Deposition of superparamagnetic nanoparticles in the aortic wall.
Using conventional images, on day 6, areas of hypointensity (negative contrast) could be detected in the wall of the thoracic (Fig. 5B) and abdominal aorta (Fig. 5D), which were not present at baseline (Figs. 5A and 5C). However, these signal voids were subtle and could not exclusively be attributed to contrast agent deposition, due to respiratory motion artifacts or absence of tissue in the same areas. Using IRON, a striking contrast enhancement was observed on day 6 in the aortic wall of Watanabe rabbits (Figs. 5F and 5H) compared with baseline (Figs. 5E and 5G). This positive signal on MR images corresponded to the deposition of superparamagnetic nanoparticles in macrophage-rich atherosclerotic plaques on histology (Figs. 5I to 5L). Enhancement was not observed in the aortic wall of control rabbits after MION-47 injection (Figs. 5M and 5O), which as expected, showed no evidence of atherosclerosis (Figs. 5N and 5P).
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Figure 5 Striking Positive Signal Enhancement Corresponding to Macrophage-Rich Plaque on IRON Images
On conventional magnetic resonance (MR) images, subtle areas of signal hypointensity (negative contrast) were detected in the aorta (solid arrows and overlaid regions of interest in panels B and D) of hyperlipidemic rabbits after MION-47 injection compared with baseline images (A and C). On IRON images, tissue was homogeneously suppressed at baseline (E and G), while positive signal was seen after MION-47 injection (solid arrows in panels F and H). The positive signal corresponds to iron deposition in matched slices on histology (fast nuclear red staining in panels I and K and combined acid phosphatase and Prussian blue staining in panels J and L). The dotted red lines on the MR images correspond to the cross section of the vessel. In both Watanabe and control rabbits, positive signals were present in paraspinal ribs (dotted arrows in panels F, M, and O) and in lymph nodes (arrowheads in panel O). As expected, normal rabbits showed no plaque on histology (N and P). Abbreviations as in Figure 4.
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Quantitative analysis of IRON images.
Quantification of the aortic wall enhancement showed that NER significantly increased in areas of plaque (confirmed by histology on matched slices) in Watanabe rabbits on day 3 (1.48 ± 0.25, p < 0.001 vs. baseline) and even more by day 6 (1.72 ± 0.46, p < 0.05 vs. day 3). The NER of the aortic wall did not significantly change over time in remote areas without plaque in both Watanabe and control rabbits (Fig. 6A). A comparison of areas with plaque in Watanabe rabbits to areas without plaque in control rabbits (n = 20), which were matched for wall thickness (0.51 ± 0.07 mm in Watanabe vs. 0.51 ± 0.06 mm in control rabbits), showed that the mean NER on day 6 was significantly higher in areas with plaque (1.64 ± 0.43 vs. 1.15 ± 0.19, p < 0.001).
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Figure 6 Correlation of Positive Signal on IRON Images With Macrophage Density in Plaque
(A) Normalized enhancement ratio (NER) increased in areas of plaque in Watanabe rabbits on day 3 (*p < 0.001 vs. baseline) and further increased on day 6 (p < 0.05 vs. day 3) only in the hyperlipidemic group. (B) The NER of the aortic wall on day 6 correlated with macrophage density in atherosclerotic plaques on matched slices (p < 0.001 by clustered regression analysis, taking into account the repeated measures performed in the same rabbit). (C) A cutoff value of NER = 1.27 provided sensitivity of 91% and specificity of 89% (area under the curve [AUC]: 0.97, standard error [SE]: 0.02, 95% confidence interval: 0.87 to 0.99, p < 0.001) for the detection of macrophage-rich atherosclerotic plaque. The dashed curves represent the 95% confidence bounds of the receiver-operating characteristics curve. IRON = inversion recovery with ON-resonant water suppression.
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Furthermore, NER of the aortic wall on day 6 significantly correlated with the macrophage density within atherosclerotic plaques, and for each unit increase of NER, the odds of observing a macrophage increased by 10.8% (95% confidence interval: 5% to 17%, p < 0.001) (Fig. 6B). Selecting a cutoff value of NER = 1.27 provided high sensitivity (91%) and acceptable specificity (89%) (area under the curve: 0.97, SE: 0.02, 95% confidence interval: 0.87 to 0.99, p < 0.001) for the detection of macrophage-rich atherosclerotic plaque (macrophage density >5%), (Fig. 6C).
Ferrozine assays.
Ferrozine-based spectrophotometric assays showed that the deposition of superparamagnetic nanoparticles was significantly higher in the aortic wall of Watanabe rabbits compared with control rabbits (0.64 ± 0.67 mg Fe/g vs. 0.12 ± 0.08 mg Fe/g tissue, p < 0.001) but was of the same order or higher in organs of the reticuloendothelial system including lymph nodes, liver, and spleen (0.83 ± 0.48, 1.14 ± 0.73, and 3.34 ± 1.45 mg Fe/g tissue, respectively).
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Discussion
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The central findings of this study are: 1) macrophage-laden atherosclerotic plaques can be noninvasively detected using IRON-MRI combined with the systemic administration of MION-47; and 2) the magnitude of the contrast enhancement by IRON-MRI in areas of plaque correlates with the number of macrophages. Because superparamagnetic particles have already completed phase III clinical trials (12), the translation of these findings to the clinical realm appears promising.
Previous approaches for imaging of atherosclerotic plaque.
After the study of Ruehm et al. (7), which reported that superparamagnetic nanoparticles are phagocytosed by macrophages, several studies have employed superparamagnetic contrast agents with the goal of visualizing atherosclerotic plaque in animals (8,10,11) and humans (9,13). In all of these studies, superparamagnetic nanoparticles accumulated in regions of atherosclerotic plaque, shortening the local T2- and T2*-relaxation rates, which caused signal loss on MR images. A fundamental drawback of negative contrast techniques, however, is that the agent cannot be distinguished from other sources of signal loss in the image, such as the absence of tissue, motion artifacts, hemorrhage, signal cancellations at water-fat interfaces or calcifications (8,13,14). Therefore, most of the previous approaches reported visual findings without providing quantification of SNR or CNR and determined the presence of inflammation within plaque categorically. Consistent with these concerns, in our study, the signal voids in areas of the aortic wall were subtle and could not be exclusively localized due to competing sources of signal voids.
Detection of macrophages in the aortic wall using IRON-MRI.
To visualize early atherosclerotic changes with high-spatial resolution, a small receiver coil was used in our study at 3 T. Hyperlipidemic rabbits exhibited increased wall thickness compared with control rabbits and showed pronounced intimal thickening, associated with lipid- and macrophage-rich plaque formation. Using IRON, a striking positive signal could be readily detected in areas of macrophage-rich plaque on day 3, which further increased on day 6 after the second injection of 250 µmol Fe/kg MION-47. The increase in positive signal correlated with the macrophage density in areas of plaque and allowed for detection of macrophages with high sensitivity and specificity. Areas with plaque in Watanabe rabbits exhibited a significantly higher positive signal on IRON images after contrast compared with that of wall thickness–matched areas without plaque in control rabbits. Thus, in contrast to conventional approaches, IRON-MRI can accurately detect superparamagnetic nanoparticles in the vessel wall, independent of partial volumes effects.
Iron-oxide deposition was not present exclusively in the aortic wall of the Watanabe rabbits, but also in other organs of the reticuloendothelial system with a high density of macrophages, such as para-aortic lymph nodes and paraspinal ribs. Indeed accumulation can be used to assess the morphological and functional state of these tissues and the number and size of para-aortic lymph nodes may correlate with active inflammation in atherosclerosis. Furthermore, recent studies have introduced alternative techniques for positive contrast MRI of superparamagnetic nanoparticles (15–17). Although the data on imaging of atherosclerotic plaque with these techniques is still limited, the comparison of IRON to other positive contrast techniques merits further investigation.
Study limitations.
The number of rabbits examined was small and the total dose of MION-47 injected to the animals was 500 µmol/kg, which exceeds the currently approved clinical dose. Although human plaques are expected to be more advanced than the early atherosclerotic changes observed in Watanabe rabbits (9,13), the optimal human dose will clearly have to be established in further studies. Furthermore, a repeated injection scheme was performed to reduce the per-session administrated dose for toxicity reasons and to simultaneously prolong the total duration, during which macrophages are exposed to superparamagnetic nanoparticles. This repeated administration and scanning protocol did not allow the evaluation of the clearance of the superparamagnetic nanoparticles, which would necessitate serial MR imaging over longer periods (up to 30 days). However, the aim of our study was to establish a proof-of-principle for the ability of IRON to quantify inflamed atherosclerotic plaque. The current findings warrant further studies in which IRON-MRI is used to study the clearance pattern of the contrast agents. Furthermore, as an important next step, the relation between local T2* measurements and NER needs to be established. As part of the present protocol, additional T2* measurements would have significantly increased overall scanning time and the duration of anesthesia for the rabbits. For these reasons, quantitative T2* measurements were not obtained, which is a limitation. The positive contrast on IRON images originated from superparamagnetic nanoparticles, which have been engulfed by macrophages in inflamed plaques of the hyperlipidemic animals. However, other sources of iron-oxide particles contained, for instance, in areas of intraplaque hemorrhage in rupture-prone lesions (25) may also give rise to positive signal on baseline IRON images. Furthermore, even though the degree of background suppression can be chosen by the parameter settings of the IRON pre-pulse, suboptimal magnetic field shimming or differences in the T1 of the background tissue may additionally lead to positive signal on IRON images as discussed in Stuber et al. (18). This should be considered for the correct interpretation of pre- and post-contrast IRON images. Atherosclerotic plaques were imaged in the aortic wall and not in moving coronary arteries. Furthermore, imaging was performed using a dedicated carotid coil, and although rabbit aortas are similar in size to human coronaries (26), the distance between the coil and human coronaries versus rabbit aortas is substantially larger than the distance to the rabbit aorta studied here, which may result in a reduced spatial resolution secondary to an SNR loss in human studies. From a technical standpoint, however, IRON can easily be combined with whole-heart imaging (27) that incorporates sophisticated motion-compensation strategies. Because the present study was conducted on a human MR system, a translation to human studies seems feasible.
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Conclusions
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In this study, IRON-MRI is combined with superparamagnetic nanoparticles to generate positive contrast for atherosclerotic plaque macrophage imaging. Using this methodology, areas of macrophage-rich plaques are highlighted, and the magnitude of enhancement is significantly related to the number of macrophages as confirmed by histology. Thus, the proposed method may be valuable for the noninvasive evaluation of macrophage-rich, vulnerable plaques in humans and may be useful to monitor therapeutic interventions in atherosclerosis.
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Footnotes
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This study was partially supported by National Institutes of Health grants (1 K08 EB004922-01, R01 HL084186, R01 HL61912, R24 CA92782) and by the Donald W. Reynolds Foundation.
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