Assessment of microvascular flow in human atherosclerotic carotid plaques using ultrasound localization microscopy

Abstract

Background: Neovascularisation of carotid plaques contributes to their vulnerability. Current imaging methods such as contrast-enhanced ultrasound (CEUS) usually lack the required spatial resolution and quantification capability for precise neovessels identification. We aimed at quantifying plaque vascularisation with ultrasound localization microscopy (ULM) and compared the results to histological analysis.

Methods: We conducted a prospective, monocentric, study involving patients who were undergoing carotid endarterectomy (CEA) for carotid artery stenosis. The day before CEA ultrasound examination coupled with the injection of microbubbles (MB) as a contrast agent (CEUS) to image the MB circulating within and around the carotid plaque was performed. CEUS images analysis classified patients into 2 groups: absence of neovascularisation (group A) or presence of neovascularisation (group B). ULM was performed by localising and tracking individual MB centres to reconstruct the neovessels structure with a resolution of around 60 μm. Plaques were manually segmented on the images to quantify the number of neovessels and various haemodynamic metrics inside the plaques. Histological analysis of the excised carotid plaque specimens classified patients into 2 groups: absence of neovascularisation (group I) or presence of neovascularisation (group II).

Findings: Among the 26 patients included, classification was as follows: group I: n = 8 and group II: n = 18, 18 patients had analysable CEUS images and were classified as follows: group A: n = 10, group B: n = 8. The median (Q1-Q3) number of MB tracked per second inside the plaque was 0.03 (0-0.37) for patients in group I and 0.51 (0-3) for patients in group A versus (vs.) 3.55 (1.26-17.68) for patients in group II and 9.69 (5.83-34.68) for patients in group B (p = 0.00049; p = 0.010 respectively). The length of the MB tracks was 0.02 mm (0-0.16) in group I vs. 0.29 mm (0.22-0.45) in group II (p = 0.0069). The study also showed that flow in the neovessels was greater during systole than during diastole period: 9.38 (1.67-19.17) MB tracked per second vs. 1.35 (0.28-6.56) (p = 0.021).

Interpretation: ULM allows the detection of neovessels within the carotid atherosclerotic plaque. Thus, ULM provides a precise picture of plaque neovascularisation in patients and could be used as a non-invasive imaging technique to assess carotid plaque vulnerability.

Funding: The study was sponsored and funded by Assistance Publique-Hôpitaux de Paris (CRC 1806 APHP INNOVATION 2018). Co-funding by ART (Technological Research Accelerator) biomedical ultrasound program of INSERM, France.

Keywords: Atherosclerosis; Carotid plaque; Contrast-enhanced ultrasound; Neovascularisation; Super-resolution imaging; Ultrasound imaging.

Fig. 1

Summary of the processing pipeline for ULM analysis. (a) Acquisition: ultrasound imaging coupled with injection of MB. Radiofrequency (RF) data are beamformed with a delay-and-sum algorithm. (b) Pre-processing of the data. Cardiac cycle parts are detected based on tissue Doppler: the data is divided between systole and diastole periods on which the ULM processing will be applied separately). Clutter filtering based on Singular Value Decomposition (SVD) and signal renormalisation are applied to extract MB signal. (c) ULM processing on each block of filtered frames: localisation of the centres of the MB & tracking of their positions. (d) Rigid registration of the MB tracks' positions for each cycle part and accumulation of the tracks over the whole acquisition.

Fig. 2

Examples of ULM density and velocity maps of group II (with neovessels) patients with plaque segmentation superimposed.

Fig. 3

Flowchart describing the inclusion of the patients and the groups used for statistical analysis.

Fig. 4

Different imaging modalities in two patients (one from group I (up) & one from group II (down)): (a) microscope transversal view of carotid histological cuts a.1. patient from group I, a.2. patient from group II (red arrows indicate neovessels). & (b) carotid ultrasound images in longitudinal views. b.1. B-Mode image of patient from group I, b.2. Normalised power Doppler of the carotid artery combined with plaque segmentation and ULM map of patient from group I, b.3. Contrast Enhanced Ultrasound image of patient from group I. b.4. B-Mode image of patient from group II, b.5 Contrast Enhanced Ultrasound image of patient from group I, b.6 Normalised power Doppler of the carotid artery combined with plaque segmentation and ULM map of patient from group II (red arrows indicate neovessels).

Fig. 5

Comparison of the performance of ULM and Power Doppler for the classification of patients based on histological assessment of the neovascularisation (n = 26—Mann–Whitney U test): (a) Mean of Power Doppler inside the plaques (p = 0.93), (b) Number of MB tracked per second inside the plaque (p = 0.00049).

Fig. 6

Comparison of three ways of evaluating the presence of neovessels inside the plaque (n = 18—Mann–Whitney U test—p = 0.010): the classification of patients based on CEUS is on the horizontal axis and is indicated by the marker colour, the ULM scores are on the vertical axis, and the classification of patients based on histology is indicated by the marker shape.

Fig. 7

Distribution of velocities of the MB obtained by ULM inside the plaques of group II patients (n = 18).

Fig. 8

Comparison of number of MB tracked per second inside the plaque during systole and diastole for group II patients (n = 18—Sign test—p = 0.021).

Supplementary Figure S1

Examples of 2 ULM maps coming from 2 different acquisitions performed on the same patients. The segmentation of the plaque was performed by a trained MD on the B-mode images. Although the position of the probe has clearly moved between the two acquisitions, we can notice that both the power Doppler and the ULM detections show relatively similar patterns—in both a case where neovessels are detected and a case where neovessels are not detected (confirmed by histology).