Subjects
Patients with RLS were included who visited the Parkinson’s Disease and Movement Disorders Clinic in the Department of Neurology between March 2014 and April 2017. Final enrollment consisted of 34 RLS patients and 36 age- and sex- matched normal controls. RLS was diagnosed according to the 2012 updated diagnostic criteria of the International Restless Legs Syndrome Study Group (IRLSSG)14. IRLSSG scale (IRLS) was used to measure the severity of RLS15. The 36 healthy subjects with no active neurologic disorders were included in the control group. A experienced neurologist performed clinical and neurological examinations on all participants. After 2 days of withdrawal of medication, cerebral vasomotor reactivity (VMR) and flow-mediated dilation (FMD) were measured. To exclude a secondary RLS, several laboratory studies of serum ferritin, iron, iron-binding capacity, folic acid, vitamin B12, fasting glucose, BUN, and creatinine levels were investigated. A standard nerve conduction study was also performed to exclude subjects with active neuropathy.
To evaluate the association between responsiveness to dopaminergic treatment and the degree of endothelial dysfunction, we asked to 34 of RLS patients about improvement of their RLS symptoms after administration of dopamine agonist.
The pramipexole or ropinirole were administered for more than two months and then responsiveness was evaluated. Subjects were classified as good response, partial response or no response. Responsiveness was evaluated by patients’ IRLSS after usage of dopamine agonist. Good response was defined as decreased IRLSS more than 50% compared with previous score, and partial response definded as decreased IRLSS between 10% to 50%. Patients with less than 10% decreased or increased IRLSS were sorted to the group of no response.
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The inclusion criteria for patients were as follows: (i) aged 18–70 years and (ii) no prior treatment for RLS. We excluded a patients who might have a secondary RLS, who had comorbidities such as pregnancy, chronic kidney disease, iron deficiency or peripheral neuropathy. Several medical conditions that could mimic RLS symptoms (such as neuropathic pain syndromes, myalgia, nocturnal leg cramps, venous stasis, leg edema, arthritis, positional discomfort, habitual foot tapping) also excluded. Finally, patients with uncontrolled hypertension, diabetes mellitus, and dyslipidemia also excluded.
All protocols were approved by the institutional review board prior to initiation of the study.
FMD protocol
Endothelial function was evaluated at the brachial artery in response to hyperemia16. Participants were instructed to stop smoking, alcohol, and caffeine within 24 hour prior to the examination. All FMD studies were performed during the early morning, around 8:00 am. The probe were fixed by fixing device on the right arm of the subject to ensure that the diameters were measured at the same location.
All procedure was performed by experienced sonographer. We used a 10- to 12-MHz broadband linear-array transducer for brachial artery ultrasound imaging. A Logiq S6 ultrasonographic system (GE Healthcare, Waukesha, WI, USA) was used. To induce the occlusion of brachial artery, a blood pressure cuff was placed on the right forearm and inflating the cuff, at least 50 mmHg above the subject's systolic pressure for 5 min. The pressure was then rapidly released to induce hand and forearm hyperemia and subsequent reactive vasodilation of brachial artery. The procedure was performed twice at 30 minutes intervals, and the brachial artery examined in cross-sectional area. In cross-section imaging, we recorded M-mode tracings from 10 seconds before cuff inflation to 90 seconds after cuff deflation. The minimum
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(systolic) and maximum (diastolic) diameters of the brachial artery were obtained by obtained sonographic images from the M-mode and B-mode studies. Images were measured at 10 seconds before inflation of the pressure cuff (baseline diameter) and every 10 seconds until 90 seconds after deflation. FMD was defined as the maximum percentage change in brachial artery diameter relative to baseline, after reactive hyperemia procedure.
VMR protocol
The VMR values were measured as percentage increase of the mean flow velocity on transcranial Doppler (TCD) in response to hypercapnia induced by the rebreathing method. TCD was performed using PMD 100M (Spencer Technologies).
This system also calculates a power M-mode Doppler (PMD) image with a single-gate spectrogram. VMR was evaluated at the both middle cerebral artery (MCA) and basilar artery (BA) in each patient. Subjects sat in a comfortable and quiet room.
Then, subjects were asked to relax and breathe as usual. The subjects’ cardiac rhythm and HR were continuously monitored. Baseline heart rate (HR) and blood pressure (BP) were checked after 5 minutes of rest. During the examination, BP was recorded at intervals of 30 seconds. The stop value, which means HR that the test would be terminated was defined as a 1.1-fold increase in baseline value. As for the re-breathing, the examiner instructed the subjects to fit the mask on their face and breathe 15 times per minute through a 3-liter, limited volume vinyl re-breather bag.
The re-breathing procedure was terminated if (i) the mean flow velocity no longer increased, (ii) the HR reached the calculated “stop value,” (iii) or the subject no longer be able to withstand the re-breathing. During re-breathing, the velocities and spectra of the MCAs and BA were simultaneously monitored in response to re-breathing hypercapnia. Baseline mean-flow velocity (Vbase) was measured after a 1 minute of rest. The maximal mean velocity (Vmax) was defined as the mean flow
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velocity at which no further velocity increase occurred. The VMR was calculated as (Vmax − Vbase) × 100/Vbase17.
Statistics
The Student's t-test and Fisher’s exact test were used to compare the categorical and continuous variables between groups. The correlation between continuous variables, including FMD, VMR and IRLSS was assessed by Spearman's correlation analysis. All statistical analyses were performed using SPSS, ver. 18.0 and a p-value 0.05 was set to indicate statistical significance.
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