Atherosclerosis is characterized by the accumulation of lipoproteins in arterial walls, leading to plaque formation and reduced blood flow. This article details a method for visualizing lipid-rich atherosclerotic plaques using Sudan IV staining in a mouse model.
Atherosclerosis occurs due to buildup of lipoproteins in the inner lining of the arteries, resulting in plaque accumulation, thickening and narrowing of the arteries, and reduced blood flow.
Plaques consist of a fibrous cap with smooth muscle cells and extracellular matrix, enclosing a core composed of low-density lipoproteins, LDLs - spherical particles with a hydrophobic core of cholesterol esters and triglycerides, surrounded by a phospholipid outer layer - along with inflammatory cells.
To visualize lipid-rich atherosclerotic plaques, obtain a freshly harvested mouse aortic arch. Transfer it onto a dark background-customized platform for improved contrast during wide-area imaging. Add buffer to prevent tissue dehydration.
Remove the perivascular adipose tissue surrounding the aortic arch to prevent false background staining. Incise the aorta and its branches longitudinally through the lumen to expose the innermost surface with atherosclerotic plaque buildup.
Secure the opened aortic arch onto the platform and incubate in ethanol. Ethanol displaces water from the tissue, inactivating proteins, and preserving the tissue architecture.
Treat with Sudan IV stain solubilized in organic solvents. Within the plaque, Sudan IV, a fat-soluble diazo dye having high affinity for triglycerides and cholesterol esters of lipoproteins, moves from the organic solvent phase and binds to the LDLs’ hydrophobic core of lipids, forming stable complexes. Wash away excess stain using ethanol.
Under a stereomicroscope, the stained lipid-rich atherosclerotic plaques appear orange-red, while the non-plaque areas appear pale.
To prepare a pinning bed for en face analysis, fold a segment of paraffin wax film 8 times to make a flat 25 x 25-millimeter surface, and wrap black electric insulation tape around the film to make a dark background for the aorta.
Place a label on the back side of the pinning bed, and use a lead pencil to write down the mouse identification number. Transfer the aortic arch to the pinning bed, and add a drop of PBS to the tissue. Using a stereomicroscope, clean the aorta from the remaining periadventitial adipose tissue, and use Vannas scissors and Dumont forceps to gently peel away all of the surrounding adipose tissue without manipulating or damaging the aorta.
Next, introduce the Vannas scissors into the aortic lumen to expose the intimal surface. Begin cutting the outer curvature of the ascending arch in the distal direction continuing to cut open the branches, including the brachiocephalic artery, and sparing the dorsal part of the descending thoracic region.
To display the intimal surface, cut open the lesser curvature, and fold open the aorta. Using a micro Castroviejo needle holder, secure the open arch to the pinning bed with the blunt end of minutien insect pins without stretching the specimen, gently bending the pins away from the specimen, as the tissue is held in place. Then, store the pinned arch face-down in a Petri dish of PBS at 4 degrees Celsius.
For Sudan IV staining, rinse the specimen for 5 minutes in a Petri dish of 70% ethanol with the arch facing down, before transferring the specimen to a dish of Sudan IV working solution for 7 minutes.
At the end of the incubation, rinse the sample with two 3-minute washes in 80% ethanol to de-stain the normal intimal surface followed by a final rinse in PBS, before returning the tissue to its original Petri dish. Then, acquire micrographs under a stereomicroscope connected to a digital camera at a 10X magnification, obtaining images of the pinned arch submerged in PBS, using small metal weights to hold the pinning bed to the bottom of the Petri dish.
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