Dual-energy CT imaging of atherosclerotic plaque using novel ultrasmall superparamagnetic iron oxide in hyperlipidemic rabbits

Abstract Background A study using magnetic resonance imaging (MRI) revealed that ultra-small superparamagnetic iron oxide is phagocytosed by macrophages. However, MRI has limitations in obtaining clear images due to its poor spatial and temporal resolutions. Purpose To examine whether the use of dual-energy computed tomography (DECT) facilitated the visualization of carboxymethyl-diethylaminoethyl dextran magnetite ultra-small superparamagnetic iron oxide (CMEADM-U) accumulation in arteriosclerotic lesions using hyperlipidemic rabbits. Material and Methods CMEADM-U at 0.5 mmol Fe/kg was administered to Watanabe hereditary atherosclerotic (WHHL) rabbits (n = 6, 24 sections) and New Zealand white (NZW) rabbits (n = 2, 6 sections). After 72 h, DECT was performed to prepare virtual monochromatic images (35 keV, 70 keV) and an iron-based map. Subsequently, the aorta was collected along with hematoxylin and eosin staining, Berlin blue (BB) staining, and RAM11 immunostaining. Results In the WHHL rabbits, CMEADM-U accumulation was not observed at 70 keV. However, CMEADM-U accumulation consistent with an arteriosclerotic lesion was observed at 35 keV and the iron-based map. On the other hand, in the NZW rabbits, there was no accumulation of CMEADM-U in any images. Further, there were significant differences in the iron-based map value at the site of accumulation among the grades of expression on BB staining and RAM11 immunostaining. In addition, there was a good correlation at 35 kev and iron-based map value (r = 0.42; P < 0.05). Conclusion DECT imaging for CMEADM-U facilitated the assessment of macrophage accumulation in atherosclerotic lesions in an in vivo study using a rabbit model of induced aortic atherosclerosis.


Introduction
Acute coronary syndrome is caused by plaque rupture around the coronary artery wall in approximately two-thirds of patients. As characteristics of plaque that is prone to rupture, a thin, fibrous capsule (<65 μm), positive remodeling, and a presence of abundant oxidized low-density lipoprotein cholesterol and macrophages were reported (1)(2)(3). In particular, the accumulation of macrophages caused by inflammation may play an important role in plaque destabilization, but no molecular imaging method to evaluate coronary plaque inflammation has been established. Ultra-small superparamagnetic iron oxide (USPIO) was developed as a contrast medium for magnetic resonance imaging (MRI). The particle diameter is markedly small, approximately 50 nm, and its blood retention time is long. As USPIO is phagocytosed by macrophages, previous studies using magnetic resonance angiography confirmed the macrophage accumulation of USPIO in arteriosclerotic lesions (4,5). Carboxymethyl-diethylaminoethyl dextran magnetite USPIO (CMEADM-U) was recently developed (6) as a new type, which accumulates in arteriosclerotic plaque macrophages more markedly and is retained for a longer time than conventional USPIO. However, quantitative assessment using MRI is difficult in the analysis of small objects, such as coronary plaque, due to its poor spatial and temporal resolutions. Recently, dual-energy computed tomography (DECT), which is performed with two different energies, has become available. DECT scans can provide various images from two datasets, which is proposed by the utilization of the attenuation coefficient, density, and CT values. Virtual monochromatic imaging (VMI), in which the effective energy can be randomly modified after imaging, can create images from those at lower than 70 keV corresponding to 120 kVp, which is primarily used on single-energy CT (SECT), and enhance the contrast (7). Furthermore, material density images obtained using 2-or 3-material decomposition methods can be visualized based on any substance; substances with similar CT values that cannot be visualized on SECT can be differentiated (8). A study previously reported that DECT images facilitated the visualization and even quantitative assessment of this novel USPIO using a phantom atherosclerotic model (9). However, its usefulness in vivo remains to be clarified.
The aim of the present study was to examine whether DECT facilitates the visualization of arteriosclerotic plaque macrophage accumulation by administering CMEADM-U to a rabbit model for familial hypercholesterolemia.

Study design
The present study was approved by the governmental animal protection committee and the institutional review board for the care of animal subjects. All protocols were approved by the Intervention Technical Center Animal Welfare Committee (IVT-20-66) and experiments were conducted in accordance with the Animal Experiment Welfare Regulations. Our study is reported in accordance with ARRIVE guidelines.

Animal model and experimental protocol
As an atherosclerotic model, four Watanabe hereditary atherosclerotic (WHHL) rabbits (age = 10 months, mean body weight = 3.2 kg) were used. As a control model, two New Zealand white (NZW) rabbits (age = 10 months, mean body weight = 3.9 kg) were used. CMEADM-U at 0.5 mmol Fe/ kg was administered 72 h before CT imaging. The rabbits inhaled isoflurane through a mask and CT was performed under deep anesthesia. Subsequently, after confirming systemic collapse and analgesia under anesthesia control with isoflurane, an axillary incision was made, and all animals were exsanguinated through the axillary artery. They were regarded as being euthanized by confirming an SpO 2 reaching zero on a bedside monitor and dilation of the pupils. Subsequently, the aorta was collected and fixed in paraformaldehyde. Paraffin blocks were prepared, and hematoxylin and eosin (H&E) staining and Berlin blue (BB) staining were conducted using axial sections with a thickness of 3 μm. Furthermore, specimens for immunostaining of macrophages (macrophage clone RAM11: RAM11) were prepared using sections with a thickness of 4 μm. The association with CMEADM-U accumulation was evaluated. Six sections per animal (24 sections in total) from the WHHL rabbits and three (6 sections in total) from the NZW rabbits were prepared. For alignment between CT images and pathological slices, the upper margin of the aortic arch and abdominal aorta-common iliac artery bifurcation were used as a landmark.

CT protocol and CT analysis
All rabbits were scanned using an Aquilion One PRISM Edition machine (Canon Medical Systems Corporation, Otawara, Japan). This system facilitates rapid kVp switchingtype dual-energy scans (135 kVp and 80 kVp). The following imaging conditions were adopted: tube voltage = 135 and 80 kVp; tubal current = 100 and 570 mA; slice thickness = 0.5 mm; tube rotation speed = 0.5 s; rearrangement field of view = 240 mm; and matrix = 512 × 512. For image analysis, a Vitrea workstation (Canon Medical Systems Corporation, Otawara, Japan) was used, and a DE-analyzing application and spectral analysis were adopted . On VMI, image contrasts can be regulated by changing the effective energy. In this study, 35 keV, at which the highest contrast can be obtained, was used. Furthermore, images at 70 keV corresponding to 120 kVp, which is primarily used in clinical practice, were rearranged and compared. In addition, USPIO imaging at different concentrations was conducted, and an iron-based map with the 3-material decomposition method was prepared by measuring the CT value for each concentration at 85 and 65 keV. For image measurement, the region of interest (ROI) for the vascular wall on each image was established. The CT value (Hounsfield unit [HU]) was measured from VMI findings and the iron-based map value was from an iron-based map.

Histopathological examination
In the WHHL sections, the grade of accumulation on BB staining and RAM11 immunostaining was evaluated as follows: grade 0 = no accumulation; grade 1 = ≤25% of the inner circumference of the aorta; grade 2 = 25%-50% of the inner circumference of the aorta; grade 3 = 50%-75% of the inner circumference of the aorta; and grade 4 = the entire inner circumference of the aorta) using a BHS binocular microscope (Olympus Corporation, Japan). The results were compared with those of DECT. In addition, for the quantitative pathological analysis, virtual slides of the specimens (Aperio ImageScope, SVS mode) were prepared and screen shots of 2.0× zoom images were stored in TIFF format. On the TIFF images, the RAM11-positive area (μm 2 ) was measured using ImageJ (National Institute of Health, USA) and compared with the results of DECT. The image size was 1284 × 692 pixels.

Statistical analysis
The data are expressed as the mean ± standard deviation. To compare significant differences between two groups, the Student's t-test was used. For comparison among three or more groups, the analysis of variance (ANOVA) was adopted. For inter-group comparison, the Tukey honestly significant difference test was used. To examine the correlation between the RAM11-positive area obtained on quantitative pathological analysis and DECT, Pearson's correlation coefficient was used. A P value of 0.05 was regarded as significant. Statistical analyses were performed using SPSS version 23 software (IBM Corp., Armonk, NY, USA).

Results
Pathological findings showed that atherosclerosis was not observed on HE staining (Fig. 1a) in the control group, and the immunostaining was negative for either BB (Fig. 1b) or RAM11 (Fig. 1c). On the other hand, in the Table 1. Results of grade classification for positive areas at Berlin blue staining and RAM11 immunostaining.  atherosclerotic group, all slices exhibited atherosclerosis on H&E staining (Fig. 2a). The immunostaining was positive for BB (Fig. 2b) staining and RAM11 (Fig. 2c). In the control group, there was no accumulation on any DECT image of the same site as pathological sections (Fig. 1df). In the atherosclerotic group, accumulation of CMEADM-U was not observed at 70 keV of VMI (Fig. 2d), but contrast enhancement was obtained in the vascular wall at 35 keV (Fig. 2e), where we identified the iron accumulation at an iron-based map (Fig. 2f).

Discussion
To our knowledge, this is the first study in which the accumulation of CMEADM-U was visualized at the site of atherosclerosis consistent with intimal hemosiderosis on BB staining and macrophage RAM11-positive reactions by performing DECT in an in vivo study. Previous studies also reported that USPIO facilitated the visualization of macrophage accumulation in the presence of atherosclerotic lesions using MRI (4,5). However, the clinical application of MRI for the coronary artery was considered difficult due to its poor spatial/temporal resolutions and negative contrast medium. We hypothesized that the accumulation can be visualized using CT, which is clinically applicable for the coronary artery based on spatial/temporal resolutions. Indeed, contrast enhancement that was not expressed at 70 keV, corresponding to an energy zone on conventional SECT, was obtained by performing DECT and adopting VMI at 35 keV. This result supports our previous study in which contrast enhancement effects were noted through the visualization of a low-energy area using DECT in a phantom experiment (9). Furthermore, when comparing CT findings with the pathological results, there was a significant difference in the CT value at the site of accumulation between the grades of RAM11-positive macrophages, but there was no significant difference in the grade of hemosiderosis on BB staining. Therefore, we utilized the 3-material decomposition method for DECT and prepared an iron-based map as the main component of CMEADM-U. In the 3-material decomposition method, the iron-based map can be visualized from different concentrations (2.5 and 50 mg Fe/mL.) of CMEADM-U on DECT, which were based on the CT values at 65 and 85 keV in this study. As a result, there was a significant difference in the grade of hemosiderosis on BB staining and a high correlation with the RAM11-positive area. The quantitative assessment of macrophage accumulation, which reflects inflammation related to arteriosclerotic lesions, was also available.
A long blood retention time is important for USPIO to be sufficiently phagocytosed by intra-plaque macrophages. In this study, imaging was conducted 72 h after administration based on the previous report, which indicated the difference in the signal-to-noise ratio (SNR) 72 h after the administration of USPIO in an animal experiment using rabbits (10). As the blood retention time of CMEADM-U (6) is longer than that of conventional USPIO, the optimal timing of imaging may not be 72 h after administration. However, in this study, serial imaging was not performed. As the halflife of USPIO in blood differs between humans and animals, the optimal timing of imaging for clinical application in humans must be further investigated. Concerning the dose, previous studies examined administration to animals at 1.0 mmol Fe/kg (5,10). This volume is larger than that previously administered to humans (11). However, due to differences in the half-life in blood, 0.2-1.0 mmol Fe/kg must be administered to animals when comparing it with 45 μmol/kg in humans (12). As the particle diameter of CMEADM-U is smaller than that of conventional USPIO, a dose of 0.5 mmol Fe/kg was used considering an improvement in accumulation. In this study, DECT confirmed its accumulation, but the optimal dose may differ between MRI and CT. In the future, the optimal dose for clinical application in humans should be examined. Previous studies reported the administration of USPIO to humans (11,13) and we evaluated its accumulation using DECT, which facilitates coronary-artery assessment; therefore, DECT could be clinically applied for non-invasive molecular imaging of coronary inflammation in near future.
The present study has some limitations. In the rabbits used in this study, CT was performed under deep anesthesia and respiratory depression was not conducted. Therefore, the appearance of motion artifacts related to respiration may have influenced the study results. Furthermore, plain CT was not performed before the administration of CMEADM-U, and it was difficult to differentiate CMEADM-U accumulation from calcification in a highdensity area in arteriosclerotic lesions based on CT findings alone. However, pathologically, there were correlations between the grade of a high-density area and degree of hemosiderosis on BB staining or RAM11-positive macrophages. In addition, pathological findings in a high-density area did not always include calcification; therefore, a highdensity area could reflect accumulation of CMEADM-U.
In conclusion, the macrophage accumulation of CMEADM-U in arteriosclerotic lesions could be evaluated by adopting DECT in vivo in a rabbit model of induced aortic atherosclerosis.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.