Abstract
The presence of cholesterol crystals is a hallmark of atherosclerosis, but until recently, such crystals have been considered to be passive components of necrotic plaque cores. Recent studies have demonstrated that phagocytosis of cholesterol crystals by macrophages may actively precipitate plaque progression via an inflammatory pathway, emphasizing the need for methods to study the interaction between macrophages and crystalline cholesterol. In this study, we demonstrate the feasibility of detecting cholesterol in macrophages in situ using Micro-Optical Coherence Tomography (µOCT), an imaging modality we have recently developed with 1-µm resolution. Macrophages containing cholesterol crystals frequently demonstrated highly scattering constituents in their cytoplasm on µOCT imaging, and µOCT was able to evaluate cholesterol crystals in cultured macrophage cells. Our results suggest that µOCT may be useful for the detection and characterization of inflammatory activity associated with cholesterol crystals in the coronary artery.
Highlights
Cholesterol crystals are generally considered hallmarks of atherosclerosis, though their roles have long been thought to be passive elements of necrotic cores [1,2], imparting mechanical stability and stiffness to atherosclerotic lesions [3]
We have developed new Optical coherence tomography (OCT) technology termed Micro-OCT, which exhibits ten-fold improvement in resolution along every spatial direction compared to conventional OCT. Micro-Optical Coherence Tomography (mOCT) has shown an improved capability to visualize subcellular features of the human coronary artery compared with conventional OCT, including the visualization of individual macrophages and detailed morphology of extracellular cholesterol crystals [16]
On mOCT, the cholesterol crystal-containing macrophages demonstrated highly scattering inclusions within the cytoplasm matching the location of birefringent crystals visualized under polarization microscopy
Summary
Cholesterol crystals are generally considered hallmarks of atherosclerosis, though their roles have long been thought to be passive elements of necrotic cores [1,2], imparting mechanical stability and stiffness to atherosclerotic lesions [3]. After cholesterol crystals are phagocytosed by macrophages, lysosomal destabilization and leakage of cathepsin B into the cytoplasm follow, where the enzyme indirectly activates the NLRP3 inflammasome. These findings have heightened interest in the interaction between macrophages and cholesterol crystals and have created a demand for an imaging modality capable of visualizing macrophage phagocytosis of cholesterol crystals as a tool for evaluating inflammatory activity in atherosclerosis. Cholesterol monohydrate crystals are birefringent and change the polarization of transmitted light. For this reason, polarization microscopy is considered a gold standard optical microscopy technique for visualization of cholesterol crystals in cells in vitro. Techniques for assessment of crystalline cholesterol in situ are lacking. [6,7]
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