Markers of instability in high-risk carotid plaques are reduced by statins

Quantification of the content of macrophages and matrix metalloproteinase-9 within atherosclerotic carotid plaques. Panel a, High-resolution image during processing. Single, overlapping images, framed in blue and green, were joined using the Panorama software stitching technique. As a result, we were able to zoom in on the details and quantitatively analyze the image (×50 original magnification). Panel b, Representative cross-section of macrophage immunoreactivity is shown at high resolution (×400 original magnification). Panel c, Representation of Fig 2, b during the process of specific signal detection at ×400 original magnification. A color threshold mask was defined for the total section areas as well as the areas of macrophage infiltration to detect the percentage of red-colored immunostaining areas compared with the total section area (see Evaluation of Immunohistochemical Stainings). We determined the size of the area occupied by CD68-positive cells planimetrically and calculated the total percentage of macrophage-rich areas in relation to the combined surface area measurements of all cross sections per specimen (sum of macrophage-rich areas of all cross sections in μm² × 100/sum of surface area of all cross sections in μm²). The total percentage of MMP-9 positive areas was determined by a corresponding approach.

Validation of immunohistochemical analysis for detection of macrophages and matrix metalloproteinase-9 (MMP-9). Panel a, Representative cross section shows specific immunoreactivity for macrophages. Anti-CD68 staining results in a granular cytoplasmic staining with red dye precipitates (×100 original magnification). Panel b, Positive control shows specific immunoreactivity for macrophages in a tonsil biopsy specimen. Panel c, Representative cross section shows specific immunoreactivity for MMP-9. The color pattern resulting from anti-MMP-9 staining is cytoplasmic (×100 original magnification) Panel d, Positive control shows specific immunoreactivity for MMP-9 in a liver biopsy specimen.

Characteristic morphologic features of indirect plaque instability. Panel a, Representative cross section showing plaque with thin fibrotic cap and a fixed thrombus in the lumen of the vessel (staining with elastin van Gieson, ×15 original magnification). Panel b, Plaque cross-section with a very large lipid core (staining with elastin van Gieson, ×15 original magnification). Panel c, Plaque cross-section shows a plaque rupture with plaque content erupting from the lipid core (staining with elastin van Gieson, ×50 original magnification). Panel d, Plaque cross-section shows an intraplaque hemorrhage (staining with hematoxylin and eosin, ×50 original magnification).

Box and whisker plots show the distribution of percentage of macrophage-rich area (PMA) in relation to the surface area of all cross-sections per specimen and the total percentage of matrix metalloproteinase-9-positive areas (PMMA-9) in relation to the surface area of all cross-sections per specimen in different patient groups. Boxes represent the interquartile range (IQR) and the centerlines represent the median. The whiskers extend to the upper and lower adjacent values (±1.5 × IQR).