Patients
This was a hospital-based, cross-sectional study. We included all eligible patients who were admitted to the outpatient clinics of our institution between April 2018 and June 2019 for a routine follow-up visit.
The inclusion criteria were
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Being older than 40-year-old
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Having cerebral SVD in magnetic resonance imaging (MRI) based on STRIVE criteria (Wardlaw, Smith, Biessels, et al., 2013)
The exclusion criteria were:
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a debilitating symptomatic ischemic stroke (NIHSS ≥ 5)
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acute or subacute stroke (< 3 months)
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an MRI feature that suggested large artery involvement
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a history of malignancy
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atrial fibrillation (AF), or suspicion of AF
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cognitive impairment due to probable Alzheimer’s disease
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having other causes of SVD (genetic, inflammatory, toxic)
Patients having a history of minor ischemic stroke (NIHSS < 5) or transient ischemic attack (TIA) and having cognitive complaints attributed to cognitive impairment were not excluded, and these patients were grouped as symptomatic group. The diagnosis of dementia or mild cognitive impairment was based on the 2011 AHA/ASA criteria (Gorelick et al., 2011). The remaining patients who had non-specific complaints were classified into the asymptomatic group.
The local ethics committee approved the study protocol (reference number: 08/11/2017-419798), and all participants gave informed consent.
Method: The evaluation included a two-step procedure: laboratory investigation of ED biomarkers and detailed clinic clinical and radiological assessment.
Laboratory evaluation of endothelial dysfunction. The following biomarkers related to endothelial dysfunction, ICAM-1, ADMA, IL-6, and PAI-1, were assayed via enzyme-linked immunosorbent assay (ELISA). CRP was analyzed in the autoanalyzer of the faculty’s central biochemistry laboratory (Immunoturbidimetric assay). Blood samples were taken from the cubital vein between 8–10 a.m. after a night-long fasting. All samples except CRP were evaluated twice. ICAM-1 (Human ICAM-1 Platinum ELISA/ Affymetrix ebioscience Catalog No: BMS 201/ 96 test), ADMA (General Asymmetrical Dimethylarginine ELISA Kit / ABclonal Catalog No: RK00614/ 96 test), IL-6 (Human IL-6 ELISA Kit / İnvitrogen Catolog No: BMS213-2 / 96 test) ve PAI-1 (Human PAI-1 Platinum ELISA Affimetrix eBioscience Catalog No: BMS 2033/ 96 test).
Clinical evaluation
We noted demographic features and vascular risk factors (hypertension, hyperlipidemia, obesity, smoking, diabetes mellitus coronary artery disease) and performed a detailed neurologic examination of each patient. A routine cardiovascular work-up, including electrocardiogram (ECG), 24-hour Holter monitoring, and transthoracic echocardiography, was performed to exclude cardioembolic causes in each case. A standardized mini-mental state test and routine laboratory tests, including complete blood count, glucose, kidney and liver function test, electrolytes, thyroid function test, lipid panel, HbA1c, erythrocyte sedimentation rate, vitamin B12, glomerular filtration rate, anti-HIV, HBsAg, anti-HCV and VDRL were performed in all patients. Lastly, an estimated glomerular filtration rate was calculated for each patient using the modification of diet in renal disease (MDRD) formula.
Neuroimaging
MRI scans were acquired using 1.5T The Siemens Magnetom Avanto (Erlangen, Germany) scanner and The 3T Philips Ingenia MR scanner (Philips, Best, the Netherlands) according to STRIVE criteria.(Wardlaw, Smith, Biessels, et al., 2013) The standard imaging protocol included sagittal TSE T2, coronal TSE T2 (repetition time (TR) 3,000 ms, echo time (TE) 80 ms, field-of-view (FOV) 185 mm × 230 mm, matrix size 400 × 320, slice thickness 5 mm), axial fluid attenuation inversion recovery (FLAIR) (TR 11,000 ms, TE 125 ms, inversion time 2.5 sec, FOV 185 mm × 230 mm, matrix size 370 × 260, slice thickness 5 mm), axial T1-weighted (TE = 17 ms, TR = 600 ms, matrix = 188 × 320, NEX = 1, FOV = 230 mm, flip angle = 90°) and, DWI sequences. Patients were excluded from the study if any sequence was missing. Two experienced neuroradiologists evaluated all MRI data (S.A., O.K.). The WMHs were graded 0–3 for periventricular and deep white matter using the Fazekas scale.(Fazekas et al., 1987) The EPVSs were graded on a 5-point scale both for the centrum semiovale (EPVSs-CS) and the basal ganglia (EPVSs-BG).(Doubal et al., 2010) Lacunes were counted and separated into three groups (none, one, and multiple). Cerebral atrophy was evaluated according to the global cortical atrophy scale.(Pasquier et al., 1996)
Data and statistical analysis
Patients were dichotomized as symptomatic and asymptomatic. Patients with cognitive impairment, according to 2011 AHA/ASA criteria, or minor stroke were considered the symptomatic group. The remaining patients with non-specific complaints, i.e., dizziness, vertigo, or headache, were grouped as the asymptomatic group. First, we compared symptomatic vs. asymptomatic groups. Following, we compared patients with minor stroke, patients with cognitive deficit, and asymptomatic patients.
Continuous variables were indicated as median, minimum-maximum, or mean, and standard deviation as applicable. Categorical variables were shown as n (%). The Shapiro-Wilk test was performed for normality. As all the data were nonhomogeneous, we used the χ 2 test for categorical variables and whichever of the Mann-Whitney U test or the Kruskal-Wallis test was appropriate for continuous variables to compare them between groups. Bonferroni correction was applied if multiple comparison tests were encountered. Spearman correlation analysis was used to evaluate the relationship between radiological markers and biomarkers of ED (scores of WMHs, EPVSs-CS, EPVSs-BG, lacunes, and cerebral atrophy vs. serum levels of ADMA, PAI-1, IL-6, CRP, ICAM-1). Logistic regression analysis adjusted for vascular risk factors, medications, and glomerular filtration rate was performed for significant variables in the abovementioned analysis.
Statistical significance was set at a p-value ≤ 0.05. All calculations were made using IBM SPSS software version 20 (International Business Machines Corp., New Orchard Road, Armonk, NY, USA)