Temporal and quantitative analysis of atherosclerotic lesions in diet-induced hypercholesterolemic rabbits

Abstract

The diet-induced atherosclerotic rabbit is an ideal model for atherosclerosis study, but temporal changes in atherosclerotic development in hypercholesterolemic rabbits are poorly understood. Japanese white rabbits were fed a high-cholesterol diet to induce sustained hypercholesterolemia, and each group of 10-12 animals was then sacrificed at 6, 12, 16, or 28 weeks. The rabbit aortas were harvested, and the sizes of the gross and intima atherosclerotic lesions were quantified. The cellular component of macrophages (Mφs) and smooth muscle cells (SMCs) in aortic intimal lesions was also quantified by immunohistochemical staining, and the correlation between plasma cholesterol levels and the progress of atherosclerotic lesions was studied. The ultrastructure of the atherosclerotic lesions was observed by transmission electron microscopy (TEM). Widely variable atherosclerotic plaques were found from 6 weeks to 28 weeks, and the lesional progress was closely correlated with cholesterol exposure. Interestingly, a relatively reduced accumulation of Mφ, an increased numbers of SMCs, and a damaged endothelial layer were presented in advanced lesions. Moreover, SMCs were closely correlated with cholesterol exposure and lesional progress for the whole period. Cholesterol exposure directly determines atherosclerotic progress in a rabbit model, and the changes in the cellular component of advanced lesions may affect plaque stability in an atherosclerotic rabbit model.

Figure 1

A schematic of experimental design.

Figure 2

cholesterol exposure and gross atherosclerotic lesions. Cholesterol exposure was evaluated by the area under the curve (AUC) at different experimental time points (a). Representative images of aortic atherosclerosis as stained by Sudan IV (b). Gross lesion areas (left) that were calculated in the aortas of chow- and HCD-fed rabbits at 6, 12, 16 and 28 weeks and the correlations (right) between cholesterol exposure and gross lesion area (c). The data are expressed as the means ± SEM. n = 6–8 for each group. *P < 0.05 and **P < 0.01.

Figure 3

Microscopically atherosclerotic lesions. Representative images of aortic atherosclerosis as stained with H&E and EVG (a). The intima lesional areas (upper) in the sections were measured using the EVG sections and the correlations (beneath) between cholesterol exposure and intima lesions area (b). The data are expressed as the means ± SEM. n = 6–8 for each group. *P < 0.05 and **P < 0.01.

Figure 4

Immunohistochemical investigations of the lesions. Representative images are shown for macrophages (Mφ) and SMC α-actin (a). Mφ- and SMC-positive areas were determined using an image analysis system (b). The Mφs and SMCs were compared with cholesterol exposure (AUC) and intima lesion area, respectively (c). The data are expressed as the means ± SEM. n = 6–8 for each group. *P < 0.05 and **P < 0.001.

Figure 5

Transmission electron microscopic analysis and the evaluation of lesional inflammation. Damaged endothelial layer (left) and remaining endothelial cells (arrows) at 16 weeks and lipids overladen macrophages (right; L = lipid drop, N = nucleus) at 28 weeks.