Quantifying Intimal Hyperplasia in Mice

Following sacrifice, mice were perfused via a cannula in the left ventricle with phosphate-buffered saline for 5 min, followed by 4% paraformaldehyde for 30 min at 100 cm H₂O. The femoral arteries were embedded in paraffin, cut into 5-μm sections, and prepared for Elastica van Gieson staining. Serial sections of the 1 mm proximal region from the incision site of the wire insertion were evaluated using an Elastica van Gieson stain kit (HT25A-1KT; Sigma-Aldrich, Tokyo, Japan) to visualize the internal elastic lamina, as described previously [14 (link),15 (link)]. Specimens were viewed under a BZ9000 microscope (Keyence, Tokyo, Japan). The intimal and medial areas were measured by computerized morphometry using a BZ-II analyzer (Keyence). Intimal hyperplasia was defined as the formation of a neointimal layer medial to the internal elastic lamina. The medial area represents the area between external and internal elastic laminas. The intima-to-media ratio was calculated as the intimal area divided by the media area, as described previously [[12] (link), [13] (link), [14] (link), [15] (link)].