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Global Translational Medicine Quantification of atherosclerosis

ambiguity in the visualization of lesions. On the basis of hematoxylin and eosin (H&E) staining, Verhoeff van
our long-term experience, we recommend that lesions Geison staining, and Masson trichrome staining. Many
are measured manually while observing the pinned aorta works as described in published articles used Oil red O
simultaneously under a dissecting microscope. For en staining (Figure 2A) to visualize lesions since lipid-laden
face analysis, it is preferable that lesion size be presented macrophages are the major cells in lesions. However,
as percent lesion area that is normalized by the intimal this staining does not incorporate extracellular matrix,
surface area of the aortic regions measured. This is critical unesterified cholesterol, smooth muscle cells, and some
because each aorta has a different surface area; also, it is other components. In contrast, H&E staining (Figure 2B)
hard to be uniformly consistent in dissecting and opening can both visualize the plaques readily and distinguish
aortas. the lesion boundaries from the internal elastin lamina.
It is also an easy and quick staining process. Therefore,
3. Quantification of atherosclerosis using we recommend using H&E staining for quantification of
cross-sections of the aortic root lesion areas.

Quantification of atherosclerosis in cross-sections of aortic To keep consistency for lesion quantification in the
roots in mice was initially described by Paigen et al. in the same location for each aortic root, many articles used
1980s . This method has become a common approach the presence of the three leaflets of the aortic valve as a
[10]
for quantifying atherosclerosis in mice since then. The landmark. Based on our own experience, this landmark
recent American Heart Association (AHA) statement is not easily distinguished because of many variations
recommends cutting serial sections from the aortic valves caused by the anatomical differences and the nature of
to the ascending aorta (~8 – 10 μm/section for fresh-frozen tissue sectioning. Anatomically, the three leaflets are not
tissues embedded in optimal cutting temperature [O.C.T.] on the same level in the heart; technically, it is difficult to
compound) . Since region-specific differences of lesion set the three aortic leaflets on the same level for cutting.
[1]
size in aortic roots have been noted frequently, the AHA Furthermore, it is hard to appropriately define where the
statement suggested to measure lesion area throughout initial appearance of the three leaflets is. Rather than using
the aortic root . Despite lack of standard criteria, we the initial appearance of the three leaflets, we recommend
[1]
recommend at least measuring lesion areas on five serial using the disappearance of the three leaflets as the
sections for each aortic root. landmark (“0” in Figure 2C). Although this landmark does

To quantify atherosclerotic lesions in multiple serial not completely overcome the issues mentioned above, it is
sections, it is critical to set up and section aortic roots a more optimal and consistent landmark than the initial
appropriately. This technique requires the researcher to appearance of three leaflets. We label the cross-section with
discern the anatomy of the aortic root that starts from disappearance of the three leaflets as the transition point
the aortic valves at the left ventricular outlet that locates “0,” which represents the ending of the aortic sinus and the
approximately the top 1/4 of the heart. The heart tissue (the beginning of the ascending aorta (“0” in Figure 2C). We
top 1/4) containing the aortic root should be placed in a routinely measure at least 3–4 serial sections before “0”,
base mold that is positioned perpendicularly to the bottom and at least 2–4 sections after “0”. Therefore, lesion areas
surface of the mold and completely covered with O.C.T. on 6–9 sections (including the “0”) through the aortic root
Sectioning the tissue starts from the ventricular side. The are measured (Figure 2C). Recently, we have also noted
ventricular tissue is sectioned and discarded until the aortic that atherosclerotic lesions may be predominant toward
sinus is reached. This should be identified by frequently a specific location of the aortic valve leaflets. Therefore,
checking sections under a microscope until the first two we recommend that the presentation of the aortic root
aortic valves appear. We suggest collecting sections once sections always follows the same orientation using right
the first two aortic valves appear. Frozen aortic samples and left coronary arteries as the orientation landmarks
should be collected serially at 10 μm per section on ~10 (Figure 2C).
slides until the aortic wall disappears or is not intact Plaque areas are quantified using image analysis software.
anymore. Appropriate setting and successful sectioning As noted for en face analysis, automated quantification of
should allow the collection of at least nine serial sections lesion size is not recommended because most staining
per slide for ten slides. We do not recommend sectioning methods, particularly Oil red O staining, cannot provide
< 8 μm per section for fresh frozen aortic samples due to uniform staining that covers the entire area of the lesions
the fragile nature of the tissue. with comparable intensity. The most accurate method is
For cross-sections, lesions can be easily visualized with to manually trace each lesion on cross-sections, although
several types of staining including Oil red O staining, many recent software packages have advanced functions

Volume 1 Issue 1 (2022) 3 https://doi.org/10.36922/gtm.v1i1.76