(a) 12 Weeks
(b) 24 Weeks
(c) 24 Weeks
Figure 2: Diet-induced atherogenesis in 𝐴 𝑛 𝑥 𝐴 2 + / + and 𝐴 𝑛 𝑥 𝐴 2 / mice. 𝐴 𝑛 𝑥 𝐴 2 / mice on the C57Bl/6 background were backcrossed to 𝐴 𝑝 𝑜 𝐸 / mice (Jackson Laboratories) to generate 𝐴 𝑛 𝑥 𝐴 2 / 𝐴 𝑝 𝑜 𝐸 / double knockout mice. At 3 weeks of age, both 𝐴 𝑛 𝑥 𝐴 2 + / + 𝐴 𝑝 𝑜 𝐸 / and 𝐴 𝑛 𝑥 𝐴 2 / 𝐴 𝑝 𝑜 𝐸 / mice male and female mice were placed on a western diet, composed of 1% (wt/wt) adjusted calories from fat and 0.15% (wt/wt) cholesterol (TD88137, Harland Tekland Laboratory) ad libitum, for 12 ( 𝑛 = 1 4 –20 mice/group) or 24 weeks ( 𝑛 = 1 7 –23 mice/group). At 12 or 24 weeks (a and b, resp.), the animals were perfused with PBS for 20 minutes, whereupon the entire aorta from the heart to 5–10 mm below the bifurcation of the iliac arteries was removed and fixed in 4% paraformaldehyde. The aorta was evaluated for lesion development by en face Oil red O staining, and morphometry of digital images of the stained aortas was performed using Adobe Photoshop 7.0 software [24]. For aortic root analyses, hearts removed at 24 weeks (c) were fixed in 3% PFA and paraffin embedded. Serial sections, 10 microns in thickness, obtained from the region of the proximal aorta to the level of the aortic leaflet, were stained with hematoxylin and eosin. Digital microscopic images were analyzed using image analysis software (NIH Image 1.63). Lesion size for each mouse was calculated as the average lesion size in 10–15 sections over a distance of 200 to 300 microns in the aortic root. Representative lesions are shown.