MRI of coronary artery atherosclerosis in rabbits: Histopathology-MRI correlation and atheroma characterization

Background and objectives We report in vivo magnetic resonance imaging (MRI) characteristics and histopathology correlation of the thrombus formation in atherosclerosis the rabbit animal model. Design and methods Atherosclerosis was induced in white male rabbits with vegetable ghee followed oxidized diet. Baseline MRI of atherosclerosis-recruited rabbits was done and later animals were used for atheroma histopathology characterization. Contiguous cross-sectional T2-weighted fast spin echo MRI images were compared by coronary histopathology. In all animals, coronary aortic wall thickening and atheroma size was measured using MRI. Results MRI images and digitized histological sections confirmed intraluminal thrombus in 6 (67%) of the 9 animals. MRI data showed correlation with the histopathology for aortic wall thickness (R2 = 0.82, P < 0.0001), lumen area (R2 = 0.88, P < 0.0001) and plaque size (R2 = 0.77, P < 0.0001). Optimized TE and TR parameters and multicontrast enhancement generated better MRI visibility of vulnerable plaque components. The MRI data evaluated % stenosis, plaque burden. Frequency of plaques, plaque height in aorta and coronary artery atheroma was also assessed by histology. In vivo, MRI determined the presence and size of the thrombus in this animal model of atherosclerosis and histopathology defined the plaque disruption. Conclusion The combination of in vivo MRI and comparison with histopathology images of rabbit coronary thrombus may be a research tool for understanding of the pathogenesis of acute coronary plaques.


Introduction
Cardiovascular risk assessment was reported by the identification of flow-limiting coronary stenoses, wall thickness, and plaque burden and its components in rabbit experimental model [1] and risk dependence on lipid intake in diet [2]. The mechanism of acute coronary syn-dromes is understood due to the disruption of an atherosclerotic coronary plaque with an overlying thrombus [3a, 3b, 4]. Still coronary artery atheroma development and plaque constituents are not well documented. In vivo/ex vivo MR microimaging (MRM) is emerging as a noninvasive modality in assessment of cardiovascular risk but has

In vivo MRI study protocol
Rabbits were anesthetized by using intramuscular ketamine (30 mg/kg), xylazine (5 mg/kg). The animals were placed prone in the MRI scanner table under respiratory gating. All scans were performed on a 1.5-T LX Horizon GE Signa MR scanner (GE Medical Systems) by use of an UltraImage cardiac phased-array coil for better signal noise ratio. MRI of normal rabbits was performed followed by 8-week high cholesterol diet and these animals underwent MRI examination. The coronary aortic localizer consisted of a fast multislice multistack (transverse) segmented Gradient Echo (GE) localizer scan of the thorax. Scan parameters were: TR = 10 ms, TE = 2.5 ms, flip angle 35°, slice thickness 1 mm, field of view 128 × 128 mm 2 , and matrix 128 × 64. Transverse images of the coronary aorta (1 mm) were obtained along the major axis of the right coronary aorta (RCA), left anterior descending (LAD) and left circumflex (LCx) arteries. For transverse coronary wall imaging, fat-suppressed T2-weighted fast spin echo scans were acquired to assess cross-sectional views of aortic vessel. Scan parameters were: 24 contiguous slices, TE = 80 ms, TR 5 R-R intervals, slice thickness 3 mm, field of view 128 × 128 mm 2 , and matrix 256 × 256, echo train length 10 and minimum spacing ~9 ms [6]. The total imaging time for the T2-weighted scan was 15-18 minutes at the heart rate 140 bpm.

Multislice Gradient Echo (GE) sequence
The basic gradient-echo sequence has two distinct sections: slice selection and image encoding. Applying a magnetic field gradient perpendicular to the desired slice orientation performed slice selection. The applied gradient varied linearly along the gradient axis. This gradient induced a radiofrequency (RF) pulse, which generated resonance spread over a thin band of tissue to give off a magnetic resonance detectible signal. Images were acquired as 2D slices. For coronary artery imaging, the basic GE sequence was applied many times in rapid succession so that Gradient-echo last for 8 msec. During each application the amplitude of the phase encoding gradient was stepped to a new phase encoding value. Slices were acquired in contiguous manner while slices were selected in rapid succession. Motion was minimum between adjacent slices that provided reasonable continuity of coronary sections between slices.

Contrast enhancement and parametric MR microimaging at 9.4 T
For contrast enhancement plots, only coronary plaques with > 2 mm 3 volume were obtained fresh in saline and later cold packed for immediate MR imaging (n = 8). For scan parameters optimization, 9.4 T multi-slice spin echo images were obtained on 'Varian 400 Utility' at 7 discrete echo times (TE = 20-80 ms) with repetition time (TR = 4 sec) and 8 transients per acquisition. TE and TR values were plotted in 3D. These images sets were analyzed by Optimas 6.5 software to produce a parametric T2 image for selected slices. With sample position maintained, an additional image data set was collected at TE = 30 ms and TR arrayed at 8 discrete delays (0.3-8 sec) for saturation recovery. This image set was also analyzed to yield a parametric T1 image for selected slices [5].
With a multicontrast protocol using T1-weighted, proton density-weighted and T2-weighted images, area of coronary tissue features were measured [5].

Tissue preparation for histopathology
The coronary arteries from the rabbits were immediately excised after MR imaging and cut into 16 serial 2.5-mm segments matching corresponding MR images. Using landmark coronary branch performed coregistration. For histological examination, aorta were decalcified in 1% formalin, 4-micron sections were cut, mounted and processed for staining with hematoxylin-eosin and trichrome staining of neutral lipid, calcium, fibrous and cellular components. The morphology of aortic lesions and atheroma pathological changes were assessed using a Polaroid photo scanner at 335 dpi. Each single section was scanned for the numbers of atherosclerosis plaques in different coronary arteries (n = 16). Exact diameter of coronary artery on slide was measured by view-scale to create absolute scale for computing outer radius-wall thickness ratio and vessel area from MR images of the coronary arteries and the matched histopathology sections [6].
The height of the largest plaque in coronary artery of each rabbit was measured. The total extent of atherosclerotic coronary involvement was assessed by the coronary atherosclerosis index [7]. The frequency of plaques, plaque height in aorta and coronary artery were obtained by atheroma scoring for each animal and the product was divided by the body weight of the animal [8]. Disrupted plaque sections stained with Hematoxylin and Eosine. Disrupted plaques had overlying thrombus. Non-disrupted plaques did not have overlying thrombus [9].    Table represents     wall [8]. The corresponding histological sections of the aorta stained with trichrome-mason were put on a projecting microscope (Microprojector model 1202, Carl Zeiss, Inc) and were projected onto a digitizing tablet. The luminal, medial, and outer wall areas were manually traced and quantified by using NIH Image 1.63 Analysis Software program (Scion Image). The percent aortic wall thickness and percent stenosis were determined histologically by the manner as described above for MRI. By using the distance from the right coronary artery as the point of reference, comparative analyses were performed with the MRI scans at the same level [9].

MR microimaging and histology image postprocessing
MR microimages were adjusted for high contrast using 'Sun VNMR' software. These images were displayed on a Sun Sparc 5 workstation. Similarly, histology slides were photographed with Olympus D-320 L Digital Vision camera and the colored digital images were projected with a microimage Video system and Ikegami 370 M control unit onto a SONY Trinitron super fine pitch screen for area computation using Optimas 6.5 software. Data for histology and MR images were compared and statistically analyzed using PRISM 3 software [10].

Statistical analysis
For optimization, MRI data were reported as % signal intensities. MRI and histology data were reported as mean ± 1 SD. Comparisons were done by percent difference and correlations were made by paired t test, least-squares linear regression. Differences were significant if the twofoiled value was < 0.05 [10].

MR imaging and coronary artery specimens
In vivo MR images and corresponding ex vivo MR images with serial histology sections are shown in Figure 1. Gradient echo images showed right coronary artery as bright (panels on top row). Spin echo images enlarged view showed main three coronary arteries as shown with arrows in Figure 1(panels on middle rows). Coronary artery endarterectomy specimens were removed from these three coronary arteries viz. RCA (top), LAD (middle) and LCx (bottom) as shown in Figure 2.
Selection of MRI image scan parameters generated three T1-weighted, T2-weighted and proton density-weighted images that further enhanced the contrast as shown in components. Each plaque component showed specific signal intensity at different T1/T2 and proton density weighting. Delineation of wall thickness, lumen area on ex vivo MRI images and corresponding in vivo images distinguished atheroma. The Wall thickness measurement in a normal coronary artery distinguished the thrombosis seen as thick wall and atheroma seen as narrow lumen in its cross-sectional magnetic resonance images. The thrombosis and control coronary aorta are shown in Figure 3 (panels at middle and bottom rows). Thick coronary aorta with thrombosis had circular lumen. Similarly, control coronary aorta cross-sections showed circular lumen from another rabbit as shown in Figure 3.

Extent and quality of atheroma formation
The mean score of sudanophilia was greater in atherosclerosis group rabbits. The aortic and coronary artery plaque heights, as revealed by micrometer, were significantly greater in the atherosclerosis group compared with control group with no plaque (see Table 1). Coronary Three endarterectomy coronary artery specimens are shown on left panel atherosclerosis index, aortic atherosclerosis score, and coronary atherosclerosis score were significantly lower in control group animals. A variable degree of medial thickening was observed in both rabbit groups, as revealed by paraffin sections of aortic and coronary arteries. Fatty streaks, atherosclerotic plaques and fibrous plaques were observed in atherosclerosis group animals. Rabbits showed myocardial muscle degeneration and mural thrombus in coronary arteries. On histology, all rabbits had evidence of atherosclerosis, as indicated by medial thickening.
The quality of atheroma revealed that control group rabbits showed significantly more ulceration, hemorrhage, and thrombosis, indicating complicated lesions in the coronary arteries. Assessment in general by appearance revealed the plaques as atheromatous, fragile, with cracks or fissures and appeared lipid rich.

Comparison and analysis of the histomorphometric and ex-vivo MRI features of coronary aortic images
A contiguous series of representative ex vivo MR microimaging (MRM) slices 1 mm thick with in-plane resolution of 0.1 mm is shown (Figure 4, right panels in 2 rows). These slices enabled the identification of heterogeneous atheroma components appearing as dark, gray and bright areas. In these images, extensive occlusion of coronary artery showed as darker calcification and crystalline cholesterol ( Figure 4, left panels). Prominent necrotic core (~0.25 mm diameter), media thickening (0.05 -0.15 mm), circular lumen diameter (1-2 mm) with massive atheroma accumulations was a main feature. MRM visualized ( Figure 4, panels in 2 rows) specific features of atheroma size, % wall thickness, % stenosis and plaque volume. Figure 4 shows atherosclerosis-rabbit coronary artery magnetic resonance images of the aortic cross sections, with each showing a crescent shaped mass/thrombus. The corresponding histological sections confirmed the thrombus within the lumen of the aorta at these levels.
A thrombus was also seen in MRI image and was confirmed by histological analyses of the corresponding sections.
Another example of registered MRM image slice and histologic image showed a intact lumen, stenosed necrotic core occluded with thrombi ( Figure 4, right panel at bottom). Histopathology features represented several distinct plaque type III and IV components possibly fibrin intermixed with cholesterol and necrotic core, fibrous cap intact towards lumen feature and collagen or The figure represents four coronary plaques ex vivo T2-w MRI images acquired with a 9.4 T MRI scanner for the segmentation of different plaque components at different levels

Contrast enhancement and resolution
Two approaches were used to improve resolution viz. 3D plots for lipid rich atheroma / fibrous tissue at 9.4 T and parametric imaging. Figure 6 and Table 5 show 3D plots revealing that a high contrast of 0.25 can be achieved using a very short TE (~4 ms) and short TR (~500 ms) and lower contrast of ~0.12 may be obtained at more accessible parameters TR = 2-3 sec; TE = 50-100 ms. In second 'discriminant analysis' approach, T1-, T2-and proton density-weighted image intensities categorized tissue components with color display (right panel of Figure 6).
With a multicontrast protocol using T1-weighted, T2weighted images and proton density-weighted images showed coronary tissue features as shown in Figure 6. Image intensities of different locations on these images indicated that some areas were better MRI-visible on T1weighted, T2-weighted or proton density weighted images. So, gray levels for any location on these images may characterize coronary artery better by combining the Comparison as histogram bars (panels on left) and regression analysis (panels on right) showing correlations of total area, lumen area, wall thickness, atheroma area, artery luminal stenosis, plaque volume by histology method with respective MRI Figure 5 Comparison as histogram bars (panels on left) and regression analysis (panels on right) showing correlations of total area, lumen area, wall thickness, atheroma area, artery luminal stenosis, plaque volume by histology method with respective MRI. Plaque feature areas for distinct histological features are shown comparable with MRI image measurement.
contrast information of that location on these three images.
Sensitivity and specificity Table 6 shows the results of the relative signal intensity (CV %), sensitivity and specificity of the MRI criteria for evaluation of T2 weighted parametric images (panels at bottom of Figure 7). Thrombus component showed low-est sensitivity. However, thrombi next to calcification were poorly visible on MRI because of dark areas of calcified tissue. Other regions of collagen, lipid, and fibrous features were better MRI visible and distinct by histology examination.
Dependence of image contrast for lipid rich atheroma and fibrous tissue is shown as 3D plot (left panel) Figure 6 Dependence of image contrast for lipid rich atheroma and fibrous tissue is shown as 3D plot (left panel). Note the image contrast between atheroma and fibrous tissue as a function of unique combination of scan optimized parameters-TE and TR, at 9.4 T. Multicontrast method is represented to demonstrate different feature space intensity 3D distributions of plaque components using combined T1-w, T2-w and proton density weighting. Table 5: Optimization of MRI spin echo parameters TE and TR for T2 parametric contrast enhancement between atheroma and fibrous tissue at 9.4 T (see Figure 6). Different combinations of TE and TR generate T1-, proton density-and T2-weighting and their relative appearance on gray scale.  Table 7 summarizes the relative ex vivo MRI visibility of different excised tissues using T1-, T2-and proton density weighted (multicontrast) approach with their relaxation times.

Discussion
An atherosclerotic rabbit model of coronary plaque rupture was used. In vivo MRI and ex vivo MRM identify and quantify atheroma contents and plaque volume in rabbits after treatment of vegetable ghee. MRI data and histology data showed correlation between measurements of aortic wall thickness, aortic luminal area, % stenosis and thrombus size. Present study adds new information of coronary thrombus MR characteristics and relaxation times, optimization of MRI scan parameters and comparison of MRIhistopathological quantitation for coronary artery morphometric features. The changes seen in control and atherosclerosis MRI support the development of thrombus in experimental rabbits, occurs after treatment of vegetable ghee. Coronary thrombosis was observed during lipid peroxidation. Since the control group animals received no antioxidants as a result showed more atherosclerosis.
Wall shear stress, turbulence, atheroma formation affect the MR visibility and make routine coronary magnetic resonance angiography inconclusive for composition of the wall, thrombus from underlying atheroma and plaque disruption [11]. As a result, there has been considerable interest in the use of MRI and MRM for the noninvasive assessment of coronary atherosclerosis by using gradient echo (bright blood pool) and spin-echo (dark blood pool) imaging. MRM can evaluate the composition of vulnerable plaque to disruption in order to assess the cardiovascular risk [12][13][14]. The present study evaluates these observations. Due to paucity of sufficient information on in vivo MR image coronary plaque characterization, ex vivo MR image data validation was needed for it. The present study demonstrates the power of in vivo MR imaging accuracy in determination of normal and thrombosed arteries. It further highlights the coronary wall and vulnerable plaque characterization of its components using 1 mm MR slice registration with 4-micron histology section. Thrombi, lumen and wall thickness were  Table 6. On histology sections, matrix appears bluish, calcification show gaps, atheroma as red and continuous fibrous layer.
measurable by using in vivo MRI in an animal model more accurately than previous reports [12][13][14]. The MR visibility was based upon the assumption that plaque components viz. lipid, collagen, calcium, cholesterol, hemorrhage, fibrin etc. show different T1, proton density and T2 signal intensities depending upon their chemical structure and relaxation values as shown in Table 7.
Multi-contrast approach identified dense fibrocellular, lipid rich and calcified regions of atherosclerotic plaque. Plaque atheroma appeared as less dense as corroborating with earlier report [16]. Our data using spin-echo short TE, T2 weighted MRM confirm the sensitivity of MRI in the evaluation of stenosis and aortic wall thickness reported earlier [9]. Our MRI results did not demonstrate thrombi and measurable degree of stenosis on control coronary artery images. It confirms the previous report [1] on development of a thrombus after treatment of vegetable ghee leading to vulnerable plaques or stenotic plaques. However, the role of excessive trans-fatty acids in thrombus development due to vegetable ghee rich diets is poorly known [2].
At 9.4 T, in-plane resolution of 512 × 256 micrometers distinguished the plaque components in contiguous MRM slices e.g. lipid-rich protons, fibrous tissue appearing as brighter, calcium deposits appearing as darker, and measured the size of coronary wall thickness, lumen, and plaque area. However, histological specimen shrinkage caused overestimation of wall area, lumen area and stenosis if compared with true measurements.
In coronary magnetic resonance imaging, contrast enhancement was reported by using T1 and T2 different preparation phases and fat-suppressed gradient echo images. Proton density weighted and T2 weighted MRM images at different levels of brightness provided high SNR and distinct plaque atheroma components [8]. Most investigators reported spin-echo sequences for atheroscle-rosis plaque showing darker lipid rich core and lighter fibrocellular tissue by T2 weighted sequences. However, T2-weighted hypointensity and T1-weighted hyperintensity of hemorrhage and fibrous tissue made distinction of hemorrhage, fibrous tissue and atheroma [15,16]. In present report, atheroma and fibrous content in coronary plaque images generated optimized contrast at different TE and TR values. Present study corroborates with earlier observations of T2 weighted contrast using broad range of TR and TE values [5].
Parametric imaging of segmentation and quantification of plaque components based on their TE and TR values could be accomplished with high degree of sensitivity and specificity. These TE/TR optimized values appear to provide information of noninvasive in vivo application of MRI criteria. T2-weighted signal intensities micro-dissected the necrotic core at different sets of TE values. However, T2-w images showed plaque thrombus and fibrous components as darker. T1 weighted images provided less information than T2 weighted images or proton density weighted images. So, it needs appropriate TE and TR optimized choice for T2-weighted in vivo MR microimaging application.

Limitations of the study
The comparative analysis of MRI with the histological sections showed measurable differences. The fresh frozen tissue histology technique suffers from shrinkage in thrombus size. This results in tissue shrinkage and shape deformation, which may lead to underestimation of the true lumen size. Furthermore, the ex vivo rabbit aorta is also subject to shrinkage lengthwise after dissection. The accuracy of thrombus location by histology is further compromised by the estimation of the distance of the thrombus from the ends of the 1-mm slices. It needs correction factor to minimize the impact of shrinkage. Our previous study suggested that the lipid content in vegetable ghee lipids as important factor in determining plaque vulnerability as lipid disorder in rabbit experimental model [2]. However, differentiation of lipid from fibrous or small thrombotic components of the coronary artery with the use of MRI is difficult because of the large pixel size compared with smaller coronary plaque thickness in this animal model. In the vicinity of coronary stenosis region, the magnetic resonance signal void is minimum due to turbulence and results with loss in stenosis details. Availability of intravascular MRI coils may enhance the chance of better plaque thrombus characterization. Unfortunately, such technical advanced modifications are both invasive and expansive, therefore, less promising. Other possible improvements of MRI techniques such as diffusion weighting, high gradient strength, and a combination of T2-weighted imaging with 1-H spectroscopy have been reported [17,18]. These developments and such advances may help to determine plaque vulnerability and its type.

Conclusion
In a rabbit model of atherosclerosis and plaque rupture, we demonstrate the multicontrast approach of in vivo and ex vivo MRI to determine the plaque burden, size of a thrombus and its accuracy by histology in the rabbit aorta after vegetable ghee treatment.