Study population
From April 2018 to February 2019, all patients from our in- and outpatient clinic undergoing carotid ultrasound were consecutively and prospectively screened for eligibility. Inclusion criteria were: ≥50 years of age, arterial hypertension and at least one additional risk factor (smoking, diabetes mellitus, hyperlipidemia, peripheral arterial disease (PAD), coronary artery disease (CAD), history of ischemic stroke or transient ischemic attack (TIA)) and evidence of at least one 1.5 mm thick plaque of the ICA or the distal common carotid artery (CCA) in ultrasound.
Exclusion criteria were: contraindications to 3 T CMR such as ferromagnetic implants, claustrophobia, poor clinical condition (modified ranking scale (mRS) > 3), atrial fibrillation or other relevant cardiac arrhythmias interfering with the electrocardiographic (ECG)-trigger), ICA-stenosis > 50% or ICA-occlusion according to North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria [18], expectation of life < 2 years, pregnancy, distance to place of residence > 100 km and refusal of study participation.
Six hundred and thirty-one patients fulfilled the inclusion criteria of which 494 were excluded (Fig. 1) leaving 121 patients for analysis. Patients’ demographics and cardiovascular disease risk factors were obtained from patients’ electronic charts and personal interviews before CMR study.
Written informed consent was obtained from all participants. The local ethics committee approved the study and all ultrasound and CMR procedures were in accordance with institutional guidelines.
Ultrasound examination
All patients underwent 2D Duplex sonography of extracranial and intracranial arteries (IU22, Philips Healthcare, Bothwell, Washington, USA). A linear array probe (L 9–3 MHz, Philips Healthcare) was used for extracranial and a curved array probe (S 5–1 MHz, Philips Healthcare) for intracranial examination. Ultrasound was performed by two medical technical assistants with experience in neurovascular ultrasound > 20 years or by a physician with several years of ultrasound experience. Patients were examined in a supine position and maximum wall thickness and plaque thickness were measured manually by electronic calipers at the far wall of the left and right CCA and ICA. ICA stenosis was graded according to NASCET criteria based on 2D Duplex sonography [18].
CMR-protocol
Multi-contrast CMR included 3D time of flight (TOF), T1-, T2-, and proton density (PD)-weighted imaging. For T1-, T2- and PD-weighted imaging a variable-flip-angle 3D turbo spin echo (TSE) sequence (Sampling Perfection with Application optimized Contrasts using different flip angle Evolution–SPACE) with fat saturation and dark-blood preparation was used. Imaging was executed on a 3 T CMR scanner (Prisma, Siemens Healthineers, Erlangen, Germany) with an 8-channel surface coil (NORAS MRI products GmbH, Hoechberg, Germany).
An isotropic spatial resolution of 0.6mm3 was used for all sequences except for 3D-TOF (0.5 × 0.5 × 0.6 mm). Other sequence parameters were: 3D-TOF: repetition time (TR)/echo time (TE) = 21/3.3 ms, field of view (FOV) = 180 × 135 mm2, matrix 384 × 290, flip angle = 20°, pixel bandwidth (BW) = 250 Hz/Px, total acquisition time (TA) = 4:32 min. T1-weighted SPACE: TR/TE = 900/26 ms, BW = 405 Hz/Px, echo train length (ETL) = 36, TA = 7:32 min. T2-weighted SPACE: TR/TE = 2000/159 ms, BW = 405 Hz/Px, ETL = 61, TA = 7:22 min. PD-weighted SPACE: TR/TE = 1900/26 ms, BW = 405 Hz/Px, ETL = 61, TA = 8:04 min.
4D flow data (spatial/temporal resolution = 0.8 mm3/52.8 ms) were acquired with prospective ECG-triggering using a k-t-accelerated time-resolved 3D phase contrast sequence [19, 20]. Other sequence parameters were: TR/TE = 52.8/3.9 ms, flip angle = 12°, peak GRAPPA acceleration factor = 5, FOV = 140x140mm2, slice thickness = 0.8 mm, BW = 460 Hz/Px, VENC (in-plane) = 0.6 m/s, VENC (through plane) = 1.0 m/s).
Blood pressure levels of the upper right arm were recorded before and after CMR examination after resting in a supine position for at least 5 min. Heart rate was documented every 4 min during 4D flow CMR.
Data analysis
For image processing data sets were imported into a custom-made extension (CaroTo) of the MEVISFlow research software (Fraunhofer MEVIS, Bremen, Germany [21]). Preprocessing of the 4D flow data included noise filtering, correction for Eddy currents and velocity aliasing. Then, a carotid artery mask (3D-phase-contrast CMR (PC-CMR)) was calculated from the 4D flow CMR data. In the next step, a centerline was created in each carotid bifurcation and landmarks (flow diverter and ICA) were marked. From these landmarks predefined points on the centerline were automatically generated, indicating the level of each analysis plane, which was initialized automatically in the next step. If necessary, analysis planes were manually oriented perpendicular to the carotid lumen. For quantitative image analysis we used a plane- and segment-based model as described in a previous study [8]. This was performed in 8 cross-section planes (Fig. 2a). The first plane was positioned on the CCA centerline 1 cm below the flow diverter. The second plane was placed within the ICA bulb, planes 3 to 6 along the ICA with the starting point at the flow diverter and oriented perpendicularly to the centerline with a spacing of each 3 mm. Plane 7 was the most distal ICA plane outside the plaque and positioned manually by the user. Plane 8 was placed automatically in the proximal external carotid artery (ECA). As described previously [8] each analysis plane was divided into 12 wall segments. Segment 1 was located at the posterior bulb (Fig. 2b). The remaining segments were numbered clockwise for the left and counterclockwise for the right carotid arteries. Plaques and stenoses are typically located in the distal CCA and proximal ICA at the posterior wall segments [7]. These were represented by segments 1–3; 11 and 12 in planes 2–6 (Fig. 2b), defined as atherosclerosis-prone region and used to evaluate the distribution of WSS parameters and of wall thickness. The required processing time of one 3D CMR dataset, including the analysis of bifurcation geometry, hemodynamics and wall thickness was 45–60 min.
Geometry of the carotid bifurcation
Geometry of all 242 bifurcations was analyzed automatically based on the 3D-TOF CMR data after manually defining CCA, ICA, and ECA as start/endpoints for centerline computation and based on the location of the flow diverter (FD) as illustrated in Fig. 3. Geometric parameters were described and successfully used previously [8, 10]. They included a) ICA/CCA-diameter ratio, which was calculated using maximum ICA diameter in plane 6 and CCA diameter in plane 1, respectively; b) bifurcation angle, which was measured by two tangential lines of the first 1 cm of the outer wall starting at the FD; c) CCA-ICA tortuosity, which was assessed by calculating the ratio of the direct line and the centerline connection of the CCA in plane 1 and plane 6 in the ICA.
Hemodynamic parameters
For each carotid bifurcation, the vessel-lumen boundary was manually outlined in the magnitude images of the 4D flow CMR data and propagated for each timeframe. Absolute WSS (in N/m2 = 1 Pa) was time-averaged over the cardiac cycle and derived for each vessel segment and for each plane. Systolic WSS was derived by averaging the time frames 2–5 in systole, which presumably contained peak systole. OSI (in %) was calculated as the degree of WSS inversion over the entire cardiac cycle as described previously [8].
Wall thickness
Post-processing of vessel wall thickness was based on 3D-T1-weighed CMR using the same custom-made software (CaroTo, Fraunhofer MEVIS, Bremen, Germany) comprising centerline computation after manual initialization and marking flow diverter and ICA. Quantitative assessment of vessel lumen and wall thickness was carried out in the same eight analysis planes and the 12 segments, which were positioned in the same fashion as described for WSS calculation. Wall thickness was measured after manual delineation of the inner and outer contours of the vessel wall (illustration of workflow in Fig. 4).
Statistical analysis
Data are presented as mean and standard deviation or median (interquartile range) for continuous variables and as absolute frequencies and percentages for categorical variables. Departures from normality were detected with the Shapiro-Wilk and Kolmogorow-Smirnow statistics. Depending on data distribution two-tailed t tests or non-parametric tests were applied as appropriate for continuous variables.
In order to describe the distribution of wall thickness and hemodynamic parameters (absolute/systolic WSS and OSI) mean plots by side and stenosis are presented of the means in anterior and posterior segments. For each of these parameters a corresponding linear mixed model was performed with the respective parameter as dependent variable and a patient’s identity (ID) as random effect. Independent variables were anterior segment (yes/no), right side (yes/no), stenosis (yes/no, side specific), and planes 2–6 (see Fig. 2). In the following, the model corresponding to the primary outcome parameter “wall thickness” is called base model.
In order to investigate the impact of geometric (ICA/CCA-diameter ratio, bifurcation angle, tortuosity) and hemodynamic parameters (absolute/systolic WSS, OSI) on wall thickness, the base model was expanded with the respective parameters as independent variables. The base model was expanded to the final model by adding geometric and haemodynamic parameters while adjusting for risk factors (male gender, age, body mass index (BMI), diabetes mellitus, PAD, smoking, history of stroke or TIA, CAD, hypercholesteremia, hemoglobin A1c (HbA1c) and low density lipoprotein (LDL)-cholesterol). Multiple measures in each patient were taken into account with the mixed effect in the model.