The presence of cerebral and/or systemic endothelial dysfunction in patients with leukoaraiosis - a case control pilot study

The study was performed in two age- and gender-matched groups of patients sharing identical VRF. The inclusion age was between 45 and 65 years. The LA group consisted of patients from neurology outpatient clinic with visible LA in brain magnetic resonance imaging (MRI). All patients in LA group with a history of stroke/minor stroke or transient ischaemic attack (TIA) had the index event at least 6 months before the inclusion. The control group consisted of patients who had undergone brain MRI for other reasons and did not have any radiological signs of LA. The study was approved by the National Medical Ethics Committee of the Republic of Slovenia and all subjects gave written informed consent before inclusion. All the procedures followed were in accordance with institutional guidelines.

The VRF were evaluated based on a standardized interview, encompassing patient history, neurological examination, body mass index determination, laboratory tests and electrocardiography (ECG). The laboratory tests consisted of fasting total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG) and glucose. Only subjects with normal fasting serum glucose levels (<6.1 mmol/l) based on regular general practice serum glucose measurements, were included. Patients with diabetes were not included in the study. Additionally, laboratory tests included vitamin B₁₂, folic acid and thyroid stimulating hormone (TSH) in order to exclude other possible secondary causes of cognitive impairment. Subjects with a known source of cardiogenic embolism based on ECG were excluded from the study.

The colour-coded duplex sonography and power Doppler sonography of the carotid and vertebral arteries were performed in all participants. The intima-media thickness (IMT) was measured bilaterally according to the Mannheim intima-media consensus on the far wall of the common carotid artery [19]. Our final IMT value was based on the mean value of three maximal IMT measurements. Eventual plaques were classified into five standard categories according to their echogenicity. All ultrasonographic measurements were done in a blind fashion by the same experienced ultrasonographer unaware of a particular participant’s status.

A brain MRI without contrast medium was performed in all subjects to look for radiological signs of LA (Philips 1.5T Achieva, coil SENSE-NV-16, Eindhoven, the Netherlands). The scans were systematically evaluated for signs of LA in a double-blind fashion employing two experienced neuroradiologists. The following sequences were used: T2 weighted sequence in the sagittal and transversal plane, echo time (TE) = 100 ms, repetition time (TR) = short, slice thickness 5 mm, matrix 512 × 512; T1 weighted sequence in the transversal plane, TR = 450 ms, TE = 15 ms, slice thickness 5 mm, matrix 320 × 320; FLAIR sequence in the transversal plane, TR = 1100 ms, TE = 140 ms, slice thickness 3 mm, matrix 256 × 256; time of flight (TOF) sequence, TE = out of phase, TR = short, matrix 560 × 560; diffusion imaging, b = 0, b = 500, b = 1000 and an apparent diffusion coefficient (ADC) map. Leukoaraiosis was classified into four subgroups according to the Fazekas score: 0–none, 1–mild punctuate hyperintensive lesions, 2–partly confluent hyperintensive lesions and 3–extensive confluent hyperintensive lesions [20].

The study of endothelial function took place in a quiet room under constant conditions between 8.00 a.m. and 11.00 a.m. after a fasting period of at least 10 h. Flow-mediated dilatation of the right brachial artery was studied according to the recommendations of the International Brachial Artery Reactivity Task Force prior to infusion of L-arginine in order to avoid a possible influence of L-arginine on the determination of FMD [21]. A high-resolution B mode ultrasound system (ALOKA Alpha 10, Tokyo, Japan) with a 10 MHz linear array transducer was used. The patients rested in the supine position for 10 min prior to hemodynamic measurements. The right brachial artery was scanned in the longitudinal section 20 to 100 mm above the antecubital fossa to find the clearest images of the anterior and posterior wall layers. The end-diastolic mean arterial diameter was measured at the end of the diastole period, incident with the R-wave on the simultaneously recorded ECG. At least three cardiac cycles were analyzed for each scan and the measurements averaged. The flow velocity was measured at a fixed incident angle of 68° to the vessel with the range gate of 1.3 mm located in the centre of the artery. The baseline blood flow was estimated by multiplying the velocity time integral of the Doppler flow signal (corrected for the incident angle) by the vessel cross-sectional area. A hyperaemic flow increase was induced by inflation of a blood pressure cuff placed around the forearm to a pressure of 300 mmHg for 4.5 min. The hyperaemic flow was recorded for the first 15 to 20 s and diameter measurements took place 60 to 90 s after cuff deflation. The FMD was expressed as the percentage change in the artery diameter after reactive hyperaemia relative to the baseline scan.

After a 15 min pause, the cerebrovascular reactivity to L-arginine (CVR) was determined. In the preparatory phase, each patient rested for 15 min to achieve a sufficiently relaxed state. The experiment consisted of a 15 min baseline period, a 30 min intravenous infusion of 100 ml of 30 % L-arginine solution and a 15 min interval after the cessation of L-arginine application. The patients were instructed to breathe normally and encouraged not to doze off. The mean arterial velocity (v_m) was recorded bilaterally in the trunks of both middle cerebral arteries (MCA) through the temporal acoustic windows at a depth of 50 to 54 mm. Two 2 MHz probes were prevented from moving by a mechanical probe holder (DopplerBox, Sipplingen, Germany). Throughout the procedure, the mean arterial blood pressure (MAP) and heart rate (HR) were measured continuously using non-invasive plethysmography (Colin CBM-7000, Komaki-City, Japan). The plethysmograph was connected directly to the Doppler sonograph. The partial pressure of exhaled CO₂ (CO₂) was measured by an infrared capnograph (Draeger, Capnodig, Lübeck, Germany), which was connected to a breathing mask and the Doppler sonograph. TCD Multi-Dop X4 (DWL, Sipplingen, Germany) software was used to determine v_m during the 10 min rest interval and the 10 min interval following the cessation of L-arginine infusion. The v_m was calculated according to the equation:

The CVR was calculated according to the equation:

The v_ms denotes the mean arterial velocity after L-arginine infusion and the v_mr the mean arterial velocity at rest.

Mean arterial pressure, HR and CO₂ were calculated for the same intervals as v_m using TCD software. All measurements to determine FMD and CVR were carried out by the single experienced investigator who was blind to the patients’ characteristics. The repeatability of TCD measurements of v_m upon which CVR is calculated was 95.8 %.

The following variables were statistically analyzed by statistical software SPSS 20.0.0: FMD, baseline diameter of the right brachial artery (diam_b), posthyperaemic diameter of the right brachial artery (diam_p), v_m, MAP, HR, CO₂ and CVR. The paired t-test was used to compare diam_b and diam_p and additionally v_m, MAP, HR and CO₂ before and after intravenous infusion of L-arginine for each group separately. The unpaired t-test was used to compare diam_b, diam_p, FMD, v_m, MAP, HR, CO₂ and CVR-L-arg between patients with LA and those without LA. One-way ANOVA with Bonferroni correction was used to compare FMD, v_m, MAP, HR, CO₂ and CVR between the subgroups of LA. Fisher’s exact test was used to examine the significance of the association between categorical variables. Linear regression was used to explore the correlation between CVR and FMD in patients with LA. A 95 % confidence interval (CI) was used. For any statistical test used in the study, p ≤ 0.050 was regarded as statistically significant.