Fig. 3.

Atherosclerotic lesion size, severity, and composition. APOE∗3-Leiden.CETP mice were exposed to 6-h phase advancement every 3 days (circadian disturbance; CD) or regular 12:12 light–dark cycle (Control), while having either ad libitum food access (AL) or food access during the dark phase only (time-restricted feeding; TRF) for a total duration of 14 weeks after which hearts were collected. Cross sections of the aortic root area were stained with (A) haematoxylin-phloxine-saffron (asterisks are positioned in lumen next to lesions) or with anti-α-actin antibody, Sirius Red (SR), or anti-MAC-3 antibody, and atherosclerotic lesion area was determined and (B) expressed as a function of distance from the appearance of open aortic valves, from which (C) the mean atherosclerotic lesion area was calculated. Lesions were categorized according to lesion severity, expressed as a percentage of total lesions, and shown (D) per lesion severity (mild; type I-III and severe; type IV-V) and (E) lesion type (I–V). Using anti-α-actin antibody, SR, and anti-MAC-3 antibody-stained cross sections, the (F) smooth muscle cell (SMC)-positive lesion area, (G) collagen-positive lesion area, and the (H) macrophage-positive lesion area was calculated (n = 12–16 mice/group). Data are presented as means ± SD. ^$CD + AL vs. CD + TRF; ∗ Control + AL vs. CD + AL. ^$ P < 0.05; ∗∗P < 0.01, according to two-way ANOVA and following Tukey's multiple-comparison test.