# Time-Domain Near-Infrared Spectroscopy in Subjects with Asymptomatic Cerebral Small Vessel Disease
## 2 Materials and Methods  
The study received approval by the Institutional Ethical Committee and by the Ministry of Health and was conducted in compliance with the Declaration of Helsinki and written informed consent was obtained from each subject. We retrospectively selected healthy subjects from a previous TD-NIRS study [10] meeting the following criteria: (a) age \(\geq\) 55 years old; (b) no clinical history of brain disease; (c) normal neurological examination; (d) absence of stenosis \(>\) 50% of extra- or intra-cranial arteries; (e) available brain magnetic resonance imaging (MRI). The burden of CSVD was assessed according to Fazekas score on brain MRI sequences including axial diffusion-weighted imaging (DWI), T2-weighted, fluid-attenuated inversion recovery (FLAIR), gradient echo, and T1-weighted sequences [8]. The scale divides the white matter hyperintensities in periventricular (Fazekas-pv) and deep white matter (Fazekas-d) accounting for size and confluence of lesions. Periventricular lesions (pvWMHs) scores are: 0 = absent; 1 = \"caps\" or pencil-thin lining; 2 = smooth \"halo\"; 3 =irregular periventricular signal extending into the deep white matter. Deep white matter lesions (dWMHs) scores are: 0 = absent; 1 = punctate foci; 2 = beginning confluence; 3 = large confluent area. Subjects were classified according to Fazekas-pvWMHs and Fazekas-dWMHs score in 4 groups (Fazekas groups): \"noWMHs\" group included subjects with pvWMHs and dWMHs score 0; \"pvWMHs\" included subjects with pvWMHs score \(\geq\) 1 and dWMHs score 0; \"dWMHs\" included subjects with dWMHs score \(\geq\) 1 and pvWMHs score 0; \"d+pvWMHs\" included subjects with pvWMHs and dWMHs score \(\geq\) 1. TD-NIRS measurements were performed in resting conditions, with the subject lying with an angle-of-bed inclination of 30\({}^{\circ}\). TD-NIRS measurements were obtained by placing a pair of illuminating and collecting optical fibers in selected standard positions of frontal, central and parietal brain regions of each hemisphere using an EEG cap (g.EEGcap, g.tec medical engineering GmbH, Schiedlberg, Austria) according to the 10-10 international positioning system (F3-F5, C3-C1, P3-P5 for the left hemisphere and F4-F6, C4-C2, P4-P6 for the right hemisphere). These positions were chosen in the previous TD-NIRS study [10] for two main reasons: (a) to gather standardized information about equidistant areas in the territory supplied by middle cerebral artery [19]; (b) to probe both primary and secondary cortices according to probabilistic anatomical correlation of 10-10 international system to the brain cortex [20]. The recording session consisted of three sets of measurements for each position of interest. We obtained concomitant non-invasive arterial blood pressure, heart rate, SpO\({}_{2}\) and peripheral blood Hb concentrations for each subject. TD-NIRS data were acquired by means of a device designed and developed at Department of Physics of Politecnico di Milano [10]. The light sources are three pulsed diode lasers working at 690 nm, 785 nm and 830 nm, respectively (mod. LDH series, PicoQuant GmbH, Berlin, Germany) and at 80 MHz repetition rate. Laser pulses are injected into the tissue by multimode graded index optical fibers (100/140 um core/cladding diameter) paying attention in maintaining the power density below the maximum permissible exposure of 2 mW/mm\({}^{2}\) thanks to a spacer placed at the tip of the probe. Photons are collected from the tissue in reflectance mode by means of a custom-made three truncate fiber bundle made of graded index plastic fibers (1 mm core diameter, 0.29 NA; mod. OM-GIGA POF, Luceat Srl, Brescia, Italy) and then detected by three hybrid photomultipliers (mod. HPM-100-50, Becker-Hickl, GmbH, Berlin, Germany), each dedicated to a specific wavelength by using band pass filters centered at 690 nm, 785 nm and 830 nm (mod. Hard Coated OD4, 10 nm Bandpass Filters, Edmund Optics GmbH, Mainz, Germany).