Identification of neovascularization by contrast–enhanced ultrasound to detect unstable carotid stenosis

Fig 1. Contrast–enhanced ultrasound (CEUS) for assessing neo-vascularization of carotid plaque. (A) Color Doppler ultrasound of carotid bifurcation. The dotted area represents the stenotic carotid plaque. CCA: Common carotid artery. ICA: Internal carotid artery. ECA: External carotid artery. (B) CEUS of the same area as shown in A. Note yellow-orange color of the contrast agent filling the lumen of the carotid artery. Furthermore, CEUS contrast effects are visible within the carotid plaque (yellow square), indicating plaque neovascularization. (C) Immunohistological evaluation of the plaque area shown in B with anti-CD31 antibody staining. The arrows mark CD31-positive neovessels.

Fig 2. Grades of intraplaque neovascularization as assessed by contrast–enhanced ultrasound (CEUS). The arrows indicate specific plaque characteristics. (A) Grade II: very low diffuse enhancement, no focal intraplaque enhancement. (B) Grade III: clear focal plaque shoulder enhancement in proximal part of carotid plaque. (C) Grade IV: clear focal plaque shoulder enhancement (blue arrow) with strong diffuse intraplaque enhancement in an additional region (red arrow).

Fig 3. Immunohistochemical evaluation of carotid plaque. Immunohistochemical stainings of macrophages (CD68), T-cells (CD3), and neovascularization (CD31). Arrows highlight distinctive features of the immunohistological staining patterns. (A) Macrophages often appear as confluent infiltrates. (B) T cells typically present as single cells. (C) CD31-positive vessels within atherosclerotic plaque.

Fig 4. Neovascularization on contrast-enhanced ultrasound (CEUS) and immunohistochemistry. (A) Contrast-enhanced ultrasound (CEUS) of carotid stenosis. The dotted yellow lines mark the proximal beginning of the carotid plaque. The red arrow marks an area of focal neovascularization. (B) The area corresponding to the white square in A was further analyzed with anti-cd31 immunohistochemistry. Note pronounced neovascularization (red arrows) in the surgical specimen.

Fig 5. Plaque neovascularization and intra-plaque hemorrhages. (A) Hematoxylin/eosin staining of carotid plaque reveals intraplaque vessels densely filled with erythrocytes (red arrows) alongside acute intraplaque hemorrhages (blue arrows). (B) Higher magnification demonstrates extravascular erythrocytes indicative of intraplaque hemorrhages (black arrow). The intraplaque hemorrhages shown here must be relatively fresh because individual erythrocytes are still clearly demarcated. (C) Axial imaging of carotid artery by contrast-enhanced ultrasound (CEUS). The red arrow points to the area that corresponds to the histological image (A). CEUS is not able to distinguish between contrast agent located within neovessels and contrast agent moving freely within the plaque, such as may be the case in neovessel rupture followed by intraplaque hemorrhage.

Fig 6. Massive infiltration of inflammatory cells in area of intraplaque neovascularization. (A) CD31 antibody staining reveals intraplaque neovascularization. Black arrows mark individual neovessels. The dotted lines delineate an area within the plaque characterized by a high density of blue-stained nuclei suggesting strong infiltration of inflammatory cells in close proximity to the area of neovascularization. (B) Higher magnification of boxed area shown in A. Yellow arrows mark individual inflammatory cells. Cell shape and nuclear geometry suggest the presence of inflammatory cells (predominantly macrophages and T cells).