Delineation of the evolution of compositional changes in atheroma

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Abstract
In a previous manuscript, we described a method combining immunohistochemistry, confocal scanning laser microscopy, and computer-assisted image analysis for the determination of the composition of atherosclerotic plaques [Taatjes et al. (2000) Histochemistry and Cell Biology 113:161–173). We now present an enhanced technique, and its use for age- and gender-related comparative analysis of lesion composition in ApoE knockout mice. Cryosections from aorta were stained with oil red O to detect lipid, SYTOX Green to detect cellularity, and Picrosirius red to delineate collagen fibers. The stained sections were imaged by brightfield light microscopy, epifluorescence microscopy, and polarized light microscopy. Digital images were collected, processed to isolate the lesions, and subjected to computer-assisted image analysis. The average percentage of the vessel wall occupied by lesion increased 1.5-fold in animals from 10 to 20 weeks. Although the amount of lipid in the lesions increased in animals from 10 to 20 weeks, the percentage composition in the lesion remained constant because of the increase in lesion size. Average cellularity showed a modest decrease over the same interval. However, the percentage composition of plaque attributable to collagen increased 2.5-fold in 20-week-old female animals compared with that in males or females of 10 weeks of age and males of 20 weeks of age.

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