Research subjects
Patients with ≥50% ipsilateral internal carotid artery stenosis confirmed by carotid ultrasound who had a recent (< 30 days) noncardiac ischemic cerebrovascular event (ischemic stroke, mRS ≤3 or TIA) were prospectively and continuously included in this study. All patients underwent bilateral carotid multimodality ultrasound at baseline, and the largest plaque causing carotid stenosis was included in the final analysis.
Inclusion criteria: 1. age ≥ 50 years; 2. anterior circulation ischemic ischemic stroke or TIA occurring within 30 days; 3. ipsilateral internal carotid artery stenosis ≥50%; and 4. mRS ≤ 3. Exclusion criteria: 1. history of neck radiotherapy; 2. diagnosis of cardiogenic stroke, small vessel strokes, stroke of unknown origin according to TOAST criteria; 3. carotid stenosis not due to atherosclerosis; 4. impaired consciousness to cooperate in completing ultrasound examination; 5. poor quality of ultrasound images; 6. history of carotid endarterectomy or stent placement; or 7. patient lost to follow-up. The study protocol was approved by the ethics committee, and all patients provided written informed consent before the ultrasound examination was performed.
Ultrasonography
Images were acquired by one sonographer with more than 5 years of experience in vascular ultrasound and analyzed by two physicians with more than 5 years of experience in vascular ultrasound using a double-blind method. Ultrasound equipment (Canon Aplio 900, Canon Medical Systems Corporation, Japan) and high-frequency line array probes with frequencies of 5–14 MHz were used. The following ultrasound parameters were obtained at baseline using a multimodal ultrasound protocol: degree of carotid stenosis, length of stenotic segment, maximum plaque thickness, plaque size (area), plaque echogenicity, plaque surface morphology, plaque calcification, number of intraplaque neovascularizations, and plaque stiffness. The patient was supine, and his bilateral carotid arteries (including the common carotid artery, carotid bifurcation and internal carotid artery) were scanned in longitudinal, transverse and oblique views.
Conventional ultrasound imaging
A carotid plaque was defined as a carotid artery with a local intima-media thickness greater than 1.5 mm or focal wall thickness greater than at least 50% of the surrounding vessel wall. Plaque echogenicity was classified according to grayscale ultrasound images: type 1, homogeneous hypoechoic plaque; type 2, hypoechoic predominant plaque; type 3, hypoechoic predominant plaque; and type 4, homogeneous hypoechoic plaque [18, 19]. Plaque ulceration was defined as the presence of an echogenic defect (at least 2 × 2 mm) on the plaque surface with colored flow signal filling visible on CDFI. The degree of internal carotid artery stenosis was classified as 50–69% and 70–99% with reference to peak systolic flow velocity and end-diastolic flow velocity, respectively [20].
SMI
After conventional ultrasonography, the SMI mode was activated to display the target plaque in grayscale mode and monochrome SMI mode in dual format in real time. The SMI specific region of interest box adjustment included the plaque. The SMI scan parameters were set as follows: mechanical index 1.5, frame rate 50–60 fps, dynamic range 55–60 dB, and speed range 1.0–2.0 cm/s. The SMI video images of the plaque of interest were stored in transverse and longitudinal sections for 1 minute each. The intraplaque dynamic enhancement signal was defined as intraplaque microvascular flow (IMVF), and static enhancement was considered an artifact. The IMVF class was classified according to the following visual scale: 1. no IMVF or IMVF confined to the extravascular membrane; 2. dynamic IMVF reaching the shoulder of the plaque; 3. IMVF reaching the plaque core; and 4. extensive neovascularization within the plaque. IPN was classified as limited (IMVF level 1 or 2) and extensive (IMVF level 3 or 4) according to the IMVF level.
SWE imaging
SWE software was enabled after SMI examination, and the elasticity map and quality control map of the plaque of interest were displayed in dual-amplitude real-time. Young’s modulus (YM, kPa) was calculated according to the shear wave propagation equation: YM = ρc2, where YM is the tissue elasticity, ρ is the tissue density, and c is the shear wave velocity. SWE parameter settings were as follows: resolution = 3; smoothing = 3; FR control = 3; and focus = 75%. The SWE region of interest sampling frame was adjusted to include the entire plaque. During the SWE examination, the probe was placed lightly on the surface of the skin to reduce the effect of pressure on the measurement results, and the elastography imaging was stabilized for approximately 3 seconds for the elasticity measurement. Plaque elasticity measurements were performed by manually outlining the entire plaque to measure the overall elasticity of the plaque. Each plaque was measured three times and averaged. The elastography system also generates a quality control map, and measurements are more reliable and accurate when the propagation lines are parallel to each other; otherwise, if the propagation lines are distorted or missing, a new measurement is needed.
Clinical variables
The following variables were collected by a neurologist using a blinded method: (1) age and sex; (2) past medical history, including hypertension, diabetes mellitus, dyslipidemia, history of ischemic cerebrovascular events and coronary artery disease; (3) history of smoking and alcohol consumption; (4) history of drug use; (5) height, weight and body mass index; (6) modified Rankin score (mRS) after stroke; and (7) etiology of ischemic cerebrovascular events.
Ending events and follow-up
After completion of baseline ultrasound, all patients were followed up at a standardized 90-day post ultrasound visit to assess their neurological function (mRS) status after the occurrence of an ischemic cerebrovascular event. Neurological function score assessment was determined by microphone or telephone interview and confirmed by reviewing hospital records. Neurological function outcomes included a 90-day mRS of 2–6/3–6. Clinical events were determined, and ultrasound image results were interpreted by a blinded method.
Statistical analysis
All statistical analyses were performed using SPSS 26.0 statistical analysis software (IBM Corporation, New York, USA). Continuous variables are expressed as the mean ± standard deviation, and categorical variables are expressed as frequencies or percentages. Patients were divided into four groups according to plaque hardness for baseline comparisons. The nonparametric Wilcoxon or Kruskal–Wallis test was used for continuous variables, and the χ2 test was used for categorical variables. Continuous variables were included in the regression model in the form of quartile spacing. Cox proportional risk regression was used to analyze the association between carotid plaque risk biomarkers and adverse functional outcomes. Variables with a univariate analysis of P < 0.1 and clinically accepted predictive parameters were included as confounding variables in the multifactorial model. Uncorrected and corrected hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated. The predictive efficacy of the model was evaluated by applying the subject operating characteristic (ROC) curve and the area under the curve (AUC). P < 0.05 was defined as a statistically significant difference.