Nine adult male white rabbits weighing 500 gm were observed for 12 weeks after the animals reached at least 1.0 kg adult body weight gain and 12 months age. These animals were scheduled on 12-week diet period on a high trans fatty acid (TFA) rich diet (supplemented with aluminum hydroxide placebo and vegetable ghee 7.2 gm/day) to develop hyperlipidemia and atherosclerosis after which they were randomized to intervention and control groups. Rabbit chow was treated with ferric chloride (5 mg/day) complexed with EDTA in equal proportions to keep ferric iron in the solution to stimulate peroxidation in rabbits. A double dose of vitamin C (10 mg/day) was added to this chow to produce ferrous ion, which is powerful oxidant. After 16 weeks, rabbits were fed oxidized rabbit chow for 4 weeks as described elsewhere 1.

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 mm², 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 mm², 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.

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.

For contrast enhancement plots, only coronary plaques with > 2 mm³ 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.

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.

Magnetic resonance images were manually traced for outer wall and lumen area measurement. The area measurements determined the percentage aortic wall thickness [ × 100%] and the percent stenosis of the lumen area [area of thrombus / (area of lumen) × 100%]. These calculations were performed on the coronary artery MR images showing thrombus and the luminal surface of the aortic 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 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.

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 two-foiled value was < 0.05 10.

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 Figure 3 (arrows on three panels in top row). In Figure 3, T1 weighted (left panel), proton density-weighted (middle panel) and T2-weighted (right panel) showed different signal intensities suggestive of distinct plaque 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.

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 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.

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 proteoglycan shown in Figure 4. Calcification was distinguished as black or no signal (see Figure 4, feature C on top left and feature A at bottom right panels) that showed space in histology sections.

Quantitative comparison of total artery radius and area, thickness of the aortic wall, lumen area from MRM and histological samples was made as shown in table 4 as histogram bars (on left) and corresponding correlation (on right) (figure 5). The MRM values and the histological values showed correlation shown in Figure 5. The linear regression was calculated for total wall areas (r² = 0.498; p value 0.0048); lumen areas (r² = 0.881; p < 0.0001); percent wall thickness (r² = 0.820; p < 0.0001); coronary luminal % stenosis (r² = 0.831; p < 0.0001). The percent luminal stenosis was (mean ± SD) determined by comparing atherosclerotic aorta MRM with their respective control MRM images. The location of thrombus was defined as the most distal point of the thrombus from the artery. Thrombus area showed insignificant correlation (r² = 0.137; p < 0.193) between histology and MRI. Similarly, there was a correlation (r² = 0.771; p < 0.0001) between plaque volumes by MRM and histology. The total area measurement showed correlation by T2-w MRM (r² = 0.646; p 0.008) over MRI (r² = 0.488; p < 0.006) with histology.

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, T2-weighted 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 T1-weighted, T2-weighted or proton density weighted images. So, gray levels for any location on these images may characterize coronary artery better by combining the contrast information of that location on these three images.

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 1718. These developments and such advances may help to determine plaque vulnerability and its type.