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Perforating artery flow velocity and pulsatility in patients with carotid occlusive disease. A 7 tesla MRI study

Highlights • Patients with COD show lower perforating artery flow velocity in watershed areas.• Reduced perforating artery flow seems to be related to subcortical ischemic burden.• Perforating artery function may be relevant for clinical outcome in COD patients.


Introduction
Patients with uni-or bilateral carotid occlusive disease are known to express post-occlusive hemodynamic abnormalities in the cerebral vasculature. These include a lower perfusion pressure in the absence of sufficient collaterals [1,2] as well as abnormalities in cerebral blood flow (CBF) and cerebrovascular reactivity [3][4][5][6][7]. Furthermore, patients may develop low-flow infarcts, mainly in border zone areas of the brain, the deep white matter (WM) of the semi-oval center (CSO), and the gray matter of the basal ganglia (BG) [1,3,[7][8][9]. Moreover, patients with carotid occlusive disease can also express lesions typically attributed to small vessel disease (SVD), such as lacunar infarcts and white matter hyperintensities (WMH) [9]. It has been suggested that the burden of these lesions is higher at the ipsilateral side of the occlusion or stenosis, although results from previous studies are conflicting [10,11]. This raises the question if hemodynamics of small vessels, in particular in perforating arteries, are also affected in patients with carotid occlusive disease.
With the advance of 7 tesla (T) MRI we are now able to measure blood flow velocity and pulsatility in the small perforating arteries of the brain [12]. In this study, we assessed if perforating artery flow measures are altered in patients with carotid occlusive disease. We compared flow velocity and pulsatility in the cerebral perforating arteries in the CSO and BG between patients with carotid occlusive disease and matched controls, as well as interhemispherically in patients with unilateral carotid occlusive disease. In addition, we investigated if perforating artery flow measures are related to lesions typically attributed to SVD.

Study population
Participants of the Heart Brain Connection study were included and all participants provided written informed consent prior to enrolment in the study [13]. We included participants with asymptomatic or symptomatic > 80% stenosis of the internal carotid artery (ICA) who were not eligible for a carotid endarterectomy, as well as patients with an occlusion of the ICA. Patients with an ICA occlusion should not have had a brain infarct or transient ischemic attack in the three months prior to inclusion. Subjects in the control group had no history of carotid occlusive disease. All participants were aged > 50 years and independent in daily life and had a 7T MRI available, acquired according to protocol (for all in-and exclusion criteria we refer to Table 1). Patients and controls were matched for age and sex through frequency matching. All participants underwent clinical evaluation, a 3T brain MRI [13] and within three months a 7 tesla MRI. We obtained informed consent in 58 participants, four were excluded due to claustrophobia, seven dropped out due to errors with the scanner at the study visit and inability to reschedule the participant. In 21 patients with carotid occlusive disease and 19 controls the 7T MRI scans were successfully obtained (for more details we refer to Fig. 1).
The Heart-Brain Connection study was approved by the Medical Ethics Committee of the Leiden UMC (LUMC) and local boards of the participating UMCs (LUMC, Maastricht UMC, Utrecht UMC and VU UMV) [14]. The study was conducted in accordance with the declaration of Helsinki and the Medical Research Involving Human Subjects Act (WMO).

Clinical characteristics
For the patients with carotid occlusive disease the side of the stenosis or occlusion was recorded and whether the stenosis or occlusion had been symptomatic, which was defined as a transient ischemic attack (TIA) or ischemic stroke. This was based on medical records and questionnaires. Possible stenosis or occlusions in other cervical arteries (i.e. left and right internal carotid arteries and left and right vertebral arteries) were recorded from medical records as well.
Vascular risk factors were recorded for patients and reference participants. Hypertension was determined by measurement on two study visits. Each study visit systolic and diastolic blood pressure were measured on each arm twice, at least one minute apart. Mean systolic and diastolic blood pressure were averaged out of four measurements on two study visits. The criteria for hypertension were: the use of blood pressure medication, or a mean systolic blood pressure above 140 mmHg or a mean diastolic blood pressure higher than 90 mmHg. The criteria for hyperlipidemia were: the use of cholesterol lowering medication, or a diagnosis of hyperlipidemia by a physician based on earlier blood cholesterol and LDL levels (even if a patient did not use medication, but for example would adhere to a diet) or a present LDL above 3,5 mmol/L. The present LDL cholesterol level (mmol/L) was determined from a blood sample taken from the participant the first study visit. The criteria for diabetes were: the use of antidiabetic medication, or a diagnosis of diabetes by a physician (even if a patient did not use medication, but for example would adhere to a diet) based on glucose levels or on previous or present HbA1c levels above 53 mmol/mol. The present HbA1c level (mmol/mol) was determined from a blood sample taken from the participant at the first study visit. Both current and previous smoking was noted, as well as the number of years of smoking, years stopped and amount of smoked cigarettes/cigars per day.

Image acquisition
2D phase contrast (PC) acquisitions aimed at small perforating arteries in the semioval center (CSO) and the basal ganglia (BG) were performed on 7T MRI (Philips) as previously described [12,15]. Slice planning and scanning parameters of the 2D PC scans are shown in Fig. 2 and Table 2. Of note, slice planning of the phase contrast acquisitions was individually adapted, based on a T1 image. In addition, 2D-Qflow measurements at the first branch of both left and right middle cerebral arteries (MCA, M1) were acquired. A peripheral pulse oximeter was used for retrospective gating and cushions were placed beside the subject's head to minimize head movement during scanning.

First segment of the middle cerebral artery (M1)
Velocity (cm/s) and pulsatility measurements at the first segment of both left and right middle cerebral arteries were acquired on 7T MRI (0.5 × 0.5 mm resolution, Venc=120 cm/s) using a 2D-Qflow sequence. Contouring of the M1 was done semi-automatically using MeVisLab (v3.1.1, VS2017, MeVis Medical Solutions AG, DE) and M1 flow velocity was defined as the average over the contoured lumen. Pulsatility was derived from the velocity (V) using the following formula where V max , V min and V mean are the maximum, minimum and mean flow velocity, respectively. Flow velocity and pulsatility measures of left and right M1 were averaged. During data analysis we learned that the echo time of the M1 2D-Qflow was too long, resulting in flow voids. Due to this problem data of three participants with flow voids covering a large part of the contoured lumen were not used in the analyses. Showing the selection criteria relevant to our study. For more elaborate in-and exclusion criteria on the overall Heart Brain Connection study we refer to the design article. [14].

Cerebral perforating arteries
2D PC scans were first visually checked for sufficient quality; scans with poor image quality due to severe subject movement were excluded from analysis.
Perforating artery flow measures were assessed in the two regions of interest using a 2D WM mask at the CSO and a manual delineation of the BG, both excluding infarcts [15]. WM masks and lacunar infarcts in these regions were delineated from 3T MRI T1-weighted and FLAIR images using the Quantib Brain Segmentation Tool and the FMRIB Software Library (FSL version 6.0.1, Oxford, UK)(Sup. Table 1) [16]. The lacunar infarct masks were dilated with a 3 × 3 mm kernel. The dilated lacunar infarcts were removed from the WM mask, and the resulting mask was further eroded by including only the central white matter, which was defined as white matter further than 16 mm away from the outer edge of the brain. This additional erosion was performed because the poor gray/white matter contrast in these regions on the PC image makes it difficult to recognize a mismatch between the WM mask and the underlying anatomy as described before [17]. Small perforating arteries in the CSO and BG were detected as previously published [15], also automatically excluding CSO perforating arteries located in ghosting artifact regions [17] and perforating arteries in the BG oriented non-perpendicularly to the scanning plane. In addition, apparent perforating arteries located within a 1.2 mm radius from each other were excluded, as these are mostly multiple detections located on larger and/or non-perpendicular vessels (Fig. 2). Of note, the 2D WM masks where not limited to the perfusion territory of the carotid artery. Our technique does not permit separating perforating arteries fed by the carotids from those fed by the vertebrobasilar system. Yet, based on the vascular anatomy, the majority of the included vessels should originate from the carotids, although this can vary between subjects, also due to collateral flow in the patients.
The perforating artery density, mean blood flow velocity and pulsatility were assessed separately for the BG and CSO. The cerebral perforating artery density is defined as the number of perforating  arteries per cm 2 of the mask. Averaging over all perforating arteries results in the mean blood flow velocity. To calculate the pulsatility the perforating arteries' velocity curves are first normalized by division with V mean and averaged before the pulsatility is calculated using formula [1] (due to the normalization procedure V mean equals 1.0).

SVD manifestations
Automated delineation of WMH was based a previous method using a FLAIR scan (sup. Table 1 and sup. Fig. 1). [18] Visual assessment of lacunar infarcts, microbleeds, enlarged perivascular spaces, and WMH grade was performed by a neuroradiologist. SVD burden score ranged 0-4, and was based on i) presence of one or more lacunar infarcts (1 point), ii) microbleeds present (1 point), iii) moderately to severely enlarged perivascular spaces in basal ganglia (1 point), iv) Fazekas 3 (1 point), according to an established scale. [19,20] To limit the number of correlational analyses perforating artery flow measures were primarily related to WMH volume and the SVD burden score.

Statistical analysis
Demographics and vascular risk factors were compared between patients and controls with unadjusted independent t-test or Chi square test for proportions. Perforating artery flow measures were compared between the groups with ANOVA, in secondary analyses also adjusted for age, sex and hypertension. In patients with unilateral carotid occlusive disease, flow measures were compared between hemispheres with paired samples t-tests. In the patients, the relation between perforating artery flow measures and SVD manifestations was assessed with Spearman correlation. All data analyses were executed in IBM SPSS statistics (v. 22, SPSS Inc., Chicago, IL) and a p-value ≤0.05 was considered statistically significant. With the achieved sample size of 21 patients and 19 controls the study was powered to detect differences between the groups with an effect size ≥0.9 SD units for continuous variables (alpha 0.05, power 80%).

Results
Of 27 patients with carotid occlusive disease and 20 controls, 21 patients and 19 controls (mean age) had sufficient 2D PC scan quality at the CSO or BG level, and these 40 subjects constituted the study population (characteristics in Table 3). The reason for low quality scans were artifacts resulting from subject movement during scanning. Among the 40 study subjects, patients and controls were well matched for age (mean age 64±7 years, 65±10 years, respectively), but among the patients the proportion of males was higher (15 (71%) versus 9 (47%)). The carotid occlusion was unilateral in 18 patients and bilateral in three.
Sixteen patients and four controls were diagnosed with hypertension ( Table 2). Four patients were diabetic. Fifteen patients were symptomatic, of which eleven with ischemic stroke, and fifteen with TIA.

Perforating artery flow measures of patients with carotid occlusive disease vs. controls
In the CSO, N density and pulsatility of the perforating arteries were similar in patients with carotid occlusive disease as compared to controls, but flow velocity tended to be lower in patients (mean difference [95%CI]: 0.08 cm/s [0.00 -0.16], p = 0.053). In the BG, N density , flow velocity and pulsatility were similar between patients and controls (Table 4). Adjustment for age, sex, smoking status and hypertension did not influence these results.

Perforating artery flow measures between hemispheres of patients with carotid occlusive disease
In the 18 patients with unilateral carotid occlusive disease there were no differences in Ndensity or the perforating artery flow measures between hemispheres in the CSO (Table 5). In the BG, perforating artery flow velocity and Ndensity also did not differ between hemispheres. However, BG perforating artery pulsatility tended to be lower in the ipsilateral than the contralateral hemisphere (0.11 [-0.00 -0.22], p = 0.08).
Within patients, no significant relations between perforating artery flow measures and SVD manifestations were found, although a lower CSO flow velocity (r=− 0.56; p = 0.06) and BG flow velocity (r=− 0.43; p = 0.11) tended to be related to larger WMH volume (Table 6; and for controls sup. Table 2).

Discussion
In this exploratory study we found that several measures of cerebral perforating artery flow were unaffected in patients with carotid occlusive disease, although there were indications of reduced perforating artery flow velocity at the level of the CSO. Lower CSO artery flow velocity also tended to be associated with larger WMH volume in these patients and the same was found for artery flow velocity at the BG level.
Observed values for CSO perforating artery flow velocity and pulsatility of our controls correspond with previous findings in elderly healthy participants [12,21]. This is also true for the pulsatility values of perforating arteries in the BG [12,21]. BG flow velocities are slightly lower, likely due to the exclusion of non-perpendicular perforating arteries at this level in the current study. These are often larger vessels with higher velocities. A relatively large number of subjects was excluded due to poor scan quality resulting from subject movement during scanning (patients: 33% in CSO and 21% in BG; controls: 25% in CSO and 18% in BG). A previous study about perforating artery flow reported lower exclusion numbers due to subject motion (patients: 18% in CSO and 0% in BG; controls: 0% in CSO and 8% in BG) [15]. The difference may result from the slightly higher mean age of our cohort (64 vs. 59 years for patients; 65 vs. 61 years for controls), and this has been noted before in studies of perforating artery flow in older subjects [12]. This is a known phenomenon for older people which tend to move more during MRI scanning than younger people [22]. The burden of SVD manifestations in our patients with carotid occlusive disease was somewhat lower than previously described [11,23,24]. This may be caused by the lower age of our included patients and the fact that they are still independent in daily life, suggesting a patient cohort with relatively normal and stable hemodynamic function.
Carotid occlusive disease is characterized by hemodynamic impairments, such as decreased CBF and cerebrovascular reactivity, and lower perfusion pressure in the absence of sufficient collaterals [1][2][3][4][5][6][7]. These hemodynamic impairments can vary between carotid occlusive disease patients, depending on the degree of collateral flow and on the patient's hemodynamic stability, and is also reflected in ischemic symptoms. PET, MRI and transcranial Doppler measurements have shown that hemodynamic impairments are typically most pronounced in border zone areas, including the CSO and BG [3][4][5][6]. In patients with carotid occlusive disease, these regions also frequently present with low-flow infarcts (i.e. rosary-like infarctions) and WMH [8,9]. Previous research has shown that hemodynamic alterations as well as cerebral lesions are more common in the CSO than in other border zone regions [3][4][5][6]25]. These previous findings agree with lower flow velocity in the CSO perforating arteries in patients with carotid occlusive disease and its relation with WMH volume as observed in the present study. Hemispherical differences concerning hemodynamic impairments and lesion occurrence and severity in patients with carotid occlusive disease are not unambiguously shown in literature [9]. This agrees with the current finding that  Abbreviations: N density = number of detected cerebral perforating arteries/cm 2 ; CSO = semioval center; BG = basal ganglia; CI = confidence interval.

Table 6
Relations between cerebral perforating artery flow and visible manifestation of small vessel disease in patients with carotid occlusive disease. apart from lower M1 pulsatility, no differences between hemispheres were found in hemodynamic parameters of flow. Because we did not obtain an MR angiography of the circle of Willis, we cannot evaluate how patterns of collateral flow in the proximal arteries affect perforating artery flow measures. Our results suggest that hemodynamic changes occur in the distal small perforating arteries of CSO border zone areas, which likely reflect the vulnerability of these regions in patients with carotid occlusive disease, despite the fact that our patients otherwise appeared to be relatively hemodynamically stable, being asymptomatic for the past period and also having normal flow in proximal arteries. Nevertheless, the hemodynamic changes in the small perforating arteries may be clinically relevant also in this setting as reflected in the relation with WMH volume. In light of these observations the question arises whether lower perforating artery flow and subcortical ischemic lesions in patients with carotid occlusive disease are a direct consequence of hemodynamic changes at the level of the carotid artery, or rather reflect cooccurring SVD. The latter can be due to shared risk factors between atherosclerosis and SVD [9,26]. Our research cannot distinguish between these two possibilities, but does indicate that hemodynamic changes in small perforating arteries may contribute to overall brain damage in patients with carotid occlusive disease.
Some limitations of our study need to be considered. First, the perforating arteries assessed in this study have sub-voxel sizes, indicating the presence of partial volume effects which generally lead to underestimation of the velocity and an overestimation of the pulsatility [12]. However, it appears unlikely that partial volume effect drive our results, because a drop in perfusion pressure and CBF has been shown to result in vasodilation in an attempt to maintain CBF [27]. Therefore, in patients with carotid occlusive disease, partial volume effects are expected to be less, leading to less velocity underestimation. Nevertheless, despite less velocity underestimation, results indicate lower flow velocities in patients with carotid occlusive disease. Furthermore, the statistical power of this study was limited. Therefore, we could only detect relations with medium or large effect. Limited power was due to i) the challenging acquisition of good quality 7T MRI data of the cerebral perforating arteries due a motion sensitive 2D CP sequence, reflected in the relatively small sample size included in the analysis; ii) even after optimization of the signal to noise ratio to the extent of what is currently feasible, the perforating artery flow measurements have a relatively large amount of noise, introducing measurement uncertainty; and iii) the inclusion of a reasonably hemodynamically stable subject cohort, reflected by the absence of symptoms three months prior to inclusion. In hemodynamically less stable subjects larger effects are expected to be found.

Conclusion
Our observations in a group of patients with carotid occlusive disease without recent ischemic symptoms, indicate that although several measures of cerebral perforating artery flow were unaffected, there were indications of reduced perforating artery flow velocity, particularly in the watershed areas, and reduced perforating artery flow velocity tended to be related to a higher burden of subcortical ischemic injury. This suggests that the small perforating arteries contribute to damage and possibly also to clinical outcome in patients with carotid occlusive disease. This offers value to future research into the small cerebral perforating arteries in larger cohorts and preferably longitudinal studies.

Disclosures
The authors have no conflicts of interest to declare.

Sources of funding
This work is part of the Heart-Brain Connection crossroads (HBCx) consortium of the Dutch CardioVascular Alliance (DCVA The funding sources had no involvement in the study design, study conduct, interpretation of data, preparation of the manuscript, or decision to publish.