Inclusion and exclusion criteria
Eligible subjects had to be heathy and between the ages of 18 and 60 years. All participants were requested to avoid alcohol, tea and coffee for at least 24 hours prior to the test. None of the subjects had any diseases or were taking any medication affecting cardiovascular or autonomic regulation.
Participants and groups
A total of 60 healthy subjects were enrolled in the study, and all subjects completed measurements and were included in the statistical analysis. All of the subjects lived in Beijing, and all of them were Chinese. The general characters are presented in Table 1. All experiments took place in a quiet, temperature-controlled (24-26 °C) laboratory. On arrival at the laboratory, subjects were asked to empty their bladders. Following a period of cardiovascular stability (40 min), a baseline recording was then made for at least 15 min. Then, over a 5 min period, the test subjects ingested 500 mL of 0.9% saline at different temperatures, and EGG and skin blood flux were monitored. The experimental design, signal recording and analysis process were shown in Figure S1.
Electrogastrogram measurement protocol and analysis
The subjects maintained a supine position. Eight cutaneous electrodes were placed on the abdomen of the participant and connected to DC amplifiers. The EGG recordings were processed with a NeurOne system (NeurOne, MEGA electronics Ltd, Finland). The data were digitized with a sampling rate of 10000 Hz and then downsampled at 1000 Hz. EGG data were analyzed with the FieldTrip toolbox, and the analysis code for EGG analysis was provided by Rebollo, I.
RMSSD and mean RR interval analysis
The analysis method can be referenced in our previous study[17-19]. The raw data of channel one were exported in EDF format, imported into Kubio HRV software and analyzed . The analysis parameter was default. The root mean square of successive differences (RMSSD) was given by (1).
Protocol for measurement of blood perfusion
Both legs were exposed, and bilateral Zusanli acupoints (ST 36) were marked by senior acupuncture doctors. The acupuncture point ST 36 is located in the tibialis anterior muscle, 4 fingerbreadths below the kneecap and 1 fingerbreadth lateral from the anterior crest of the tibia. Blood perfusion signals were recorded using a PeriFlux System 5000 (Perimed AB, Stockholm, Sweden) with a 64 Hz sample rate and 0.2 s time constant. An optical fiber probe connected with a Periflux 5000 was used to illuminate and collect the scattered light from the skin tissue. The probe was attached to the surface of interest with two-sided adhesive tape.
Mean blood perfusion analysis and wavelet analysis
The recorded file of each subject was opened in PeriSoft (version 2.5.5, Perimed AB, Stockholm, Sweden) for Windows. The detailed data were exported in txt format and then imported into MATLAB software and analyzed. The laterality index of blood flux every 10 min on both sides of ST36 was calculated as (1).
Previous studies indicated that blood flux oscillations at frequencies from 0.0095 to 1.6 Hz might reflect different physiological rhythms, which can be separated into five frequency bands in the frequency domain[23-26]. In the present study, wavelet analysis was performed on the blood flux signal (10 min) using a Morlet wavelet (The Mathworks Inc., Natick, MA, USA). For every frequency interval, the laterality index was also calculated as (2).
Data are presented as the mean±SE. Mixed repeated-measures ANOVA was used to analyze between-subject factors with R software and fdrtool package . All correlation analyses were conducted using MATLAB software (MathWorks, Natick, Massachusetts, USA). All reported P values were two-sided, and the level of significance was defined as P<0.05.