Application of a Simple Quantitative Assessment of Atherosclerotic Lesions in Freshly Isolated Aortas from Rabbits

Rabbits are widely used for the study of atherosclerosis; however, the lack of a unified and quantitative analysis of atheroma limits data interpretation and comparisons between laboratories. In this study, we applied a simple quantitative method, referred to as the oil red O (ORO) dye-eluting method, for analysis of atherosclerotic plaques in freshly isolated aortas. It employs ORO staining of the plaques followed by elution of the dye that is subjected to quantitative measurement. Atherosclerosis was induced in rabbits by feeding a 1% (w/w) high cholesterol diet for 4 or 12 weeks. Thoracic aortas were isolated and sufficiently stained by ORO. These dyes were easily and completely extracted by 100% ethanol and quantified by spectrophotometric measurement at 510 nm. A series of cross-sectional slices at 100-µm intervals were counterstained by elastic van Gieson. It was found that there was a highly positive correlation between the dye concentration and the amount of plaque tissue, determined as volume of plaques (regression coefficient r²: 0.8792, p < 0.001). The color equivalence of the dye content was expressed as µg/mm² of intimal aorta area to allow direct comparisons among aortas. The color equivalences of ORO content in rabbits fed 12 weeks were almost 5.0 times higher than those fed 4 weeks. Thus, this ORO dye-eluting method is useful for quantification of atherosclerotic plaques in aortas in rabbits, as well as other animal models.