Ultramorphological analysis of plaque advancement and cholesterol crystal formation in Ldlr knockout mouse atherosclerosis

Backgound and aims: The low-density lipoprotein receptor-deficient (Ldlr(-/-)) mouse has been utilized by cardiovascular researchers for more than two decades to study atherosclerosis. However, there has not yet been a systematic effort to document the ultrastructural changes that accompany the progression of atherosclerotic plaque in this model. Methods: Employing several different staining and microscopic techniques, including immunohistochemistry, as well as electron and polarized microscopy, we analyzed atherosclerotic lesion development in Ldlr(-/-) mice fed an atherogenic diet over time. Results: Lipid-like deposits occurred in the subendothelial space after only one week of atherogenic diet. At two weeks, cholesterol crystals (CC) formed and increased thereafter. Lipid, CC, vascular smooth muscles cells, and collagen progressively increased over time, while after 4 weeks, relative macrophage content decreased. Accelerated accumulation of plate-and needle-shaped CC accompanied plaque core necrosis. Lastly, CC were surrounded by cholesterol microdomains, which co-localized with CC through all stages of atherosclerosis, indicating that the cholesterol microdomains may be a source of CC. Conclusions: Here, we have documented, for the first time in a comprehensive way, atherosclerotic plaque morphology and composition from early to advanced stages in the Ldlr(-/-) mouse, one of the most commonly used animal models utilized in atherosclerosis research.