Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice

Abstract

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis-the ApoE-deficient mouse-by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet-fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.

Fig. 1.

Schematic of aortic tissue preparation for en face imaging. Tissue samples were cut once along the length of the artery and laid flat between two glass coverslips. The lumenal side was placed face-down on the stage of the inverted microscope so that the imaging orientation was consistently directed from the intimal layer out to the adventitial surface. Total tissue surface area was approximately 150 mm².

Fig. 2.

Representative composite images of the entire descending aorta in ApoE-deficient mice fed a standard low-fat diet. Images from three separate channels—TPEF (A), SHG (B), and CARS (C)—were acquired simultaneously, with all three channels visible in D. Each image consists of several individual image tiles, cropped and stitched together (approximately 8 × 10 tiles per composite image). Scale bar, lower left of each image = 500 µm. CARS, coherent anti-Stokes Raman scattering; SHG, second harmonic generation; TPEF, two-photon excitation fluorescence.

Fig. 3.

Representative composite images of the entire descending aorta in ApoE-deficient mice fed a Western diet. Images from three separate channels—TPEF (A), SHG (B), and CARS (C)—were acquired simultaneously, with all three channels visible in D. Each image consists of several individual image tiles, cropped and stitched together (approximately 8 × 10 tiles per composite image). Scale bar, lower left of each image = 500 µm. CARS, coherent anti-Stokes Raman scattering; SHG, second harmonic generation; TPEF, two-photon excitation fluorescence.

Fig. 4.

CARS composite images of the entire descending aorta in ApoE-deficient mice fed either a standard low-fat diet or a Western diet, with specific regions of lipid accumulation highlighted. Enlarged images (B and D) to the right of composite images (A and C) represent uncropped, single-image tiles at full magnification (20×) of the regions highlighted in A and C. Distinct lipid deposits and collagen fibers are evident. Corresponding scale bars are located at the lower right of each image. Apo, apolipoprotein.

Fig. 5.

XZ cross-sectional images of lipid-rich region in the standard diet mouse of Figure 4. CARS/SHG (A) and TPEF/SHG (B) images were taken separately to facilitate visualization of unique structures corresponding to signal origin (i.e., lipid deposits from red CARS, collagen structure from blue SHG, and elastin fibers from green TPEF). Scale bar, lower right = 200 µm. CARS, coherent anti-Stokes Raman scattering; SHG, second harmonic generation; TPEF, two-photon excitation fluorescence.

Fig. 6.

CARS images of type II and III plaque lesions within standard diet–fed and Western diet–fed atherosclerotic ApoE-deficient mice. Lesions are mostly characterized by intracellular lipid accumulation within macrophage cells, which is indicative of Type II lesions. However, in the Western diet–fed mice, some small pools of extracellular lipids are also evident (highlighted with boxes), indicating Type III lesions. Scale bar, lower right = 100 µm. Apo, apolipoprotein; CARS, coherent anti-Stokes Raman scattering.

Fig. 7.

Representative CARS images of macrophage-like cellular structures within plaque lesions, at 40× magnification and two different levels of electronic optical zoom. Clusters of macrophage cells, defined by dark nuclei, are indicated by arrows. Corresponding scale bars are located at the lower right of each image. CARS, coherent anti-Stokes Raman scattering.

Fig. 8.

Images of macrophage cells in atheromatous plaque lesions obtained from simultaneously acquired CARS and TPEF signals. On the left, CARS (red) images of lipids from standard (upper) and western (lower) diet animals. In the middle, TPEF (green) images of CD68-stained, immunofluorescent (Alexafluor 488) macrophage cells from standard (upper) and western (lower) diet animals. On the right, coregistered CARS and TPEF shows colocation of lipid and CD68, respectively, confirming CARS identification of macrophage cells in plaques. Scale bar, lower left = 200 µm. CARS, coherent anti-Stokes Raman scattering; TPEF, two-photon excitation fluorescence.

Fig. 9.

Lipid accumulation in the arterial wall of standard diet–fed and Western diet–fed ApoE-deficient mice. Total lipid content is defined as the ratio of lipid area to total tissue area. Normal C57BL/6 mice (non-ApoE^−/−) fed a standard diet and a Western diet were included as negative controls. Apo, apolipoprotein.

Fig. 10.

General intensity profile of atheromatous plaque lesions in the arterial wall of standard diet–fed and Western diet–fed ApoE-deficient mice. Lesion profile is defined by the overall CARS signal intensity from lipids within the plaque lesion. Apo, apolipoprotein; CARS, coherent anti-Stokes Raman scattering.

Fig. 11.

Collagen distribution within plaque lesions and in the surrounding structural matrix of standard diet–fed and Western diet–fed ApoE-deficient mice. Colocalization of collagen and lipids within the plaque lesion (i.e., plaque-associated collagen area) is defined as the ratio of total collagen content within lipid-rich plaque regions to the total area of those lipid regions. Nonplaque-associated collagen is defined as the ratio of nonplaque-associated collagen area to total tissue area. Apo, apolipoprotein.