Nonlinear Optical 3-Dimensional Method for Quantifying Atherosclerosis Burden
- 2-photon excitation
- atherosclerosis burden
- infrared pulsed laser
- plaque volume
- second harmonic generation
Changes in atherosclerosis burden provide an experimental measure of the effectiveness of therapeutic strategies. Unfortunately, none of the methods used in mice to assess atherosclerosis burden ex vivo is sufficiently accurate and fast enough to process the number of samples required in preclinical studies. We aimed to design an easy-to-implement and relatively fast method for accurate volumetric quantification of atheroma plaque burden in mice.
The apolipoprotein E–deficient mouse (Apoe−/−) is a widely used preclinical model that reproducibly develops hypercholesterolemia and atherosclerosis.1 Two widely used methods for ex vivo assessment of atherosclerosis burden in mice are histopathologic analysis2 and the en face method.3 The histological method requires serial axial sectioning of arteries and subsequent histopathologic staining. This approach is labor intensive, and its inherent destructiveness undermines the accuracy of lesion-volume determinations. In en face analysis, the aorta is opened longitudinally and stained to reveal lipid-laden plaques; the stained area is quantified from photographs. The speed and ease of en face analysis have made it the most widely used method for measuring atherosclerotic plaques ex vivo, but this method gives only a 2-dimensional (2D) measure of plaque burden.
Here, we describe the nonlinear optical 3D (NLO-3D) method. Aortas from euthanized Apoe−/− mice fed a high-fat diet were dissected, stained with oil red O as described,4 and placed in a holder to permit measurement by either NLO-3D imaging or the en face method (Figure IA in the Data Supplement). We used a single excitation wavelength (800 nm) of a commercially available 2-photon instrument to simultaneously generate 3 confocal signals from these aortas (Figure IB in the Data Supplement): second harmonic generation from collagen and 2-photon excitation fluorescence from elastin and from oil red. A Zeiss 780 upright microscope equipped with a ×10 air objective (Zeiss Plan …