Impact of local thermal stimulation on the correlation between oxygen saturation and speed-resolved blood perfusion

The physiologically important relationship between oxygen saturation and blood flow is not entirely understood, particularly with regard to the multiple velocity components of flow and temperature. While our previous studies used classic laser Doppler flowmetry combined with an enhanced perfusion probe to assess local blood flow following thermal stimulation, oxygen saturation signals were not assessed. Thus, the current study used multiscale entropy (MSE) and multiscale fuzzy entropy (MFE) to measure the complexity of oxygen saturation signals following thermal stimulation in healthy subjects. The results indicate that thermal stimulation increases oxygen saturation and affects the measured signal complexity in a temperature-dependent fashion. Furthermore, stimulus temperature not only affects the correlation between speed-resolved blood perfusion and oxygen saturation, but also the correlation between the complexity area indices (CAI) of the two signals. These results reflect the complexity of local regulation and adaptation processes in response to stimuli at different temperatures.

information. Multiscale fuzzy entropy (MFE), as an enhanced MSE method, is also an effective method for evaluating the complexity of time series. Relevant research shows that MFE has a more significant correlation with MSE than other multiscale entropies 19 .
In our previous study, both MSE and MFE were used to assess the complexity of thermal stimulation on blood perfusion in the skin 10,20 . Compared with the calculation procedure of MSE, MFE replaces the Heaviside function with fuzzy membership function, which makes MFE less dependent on data length and more resistance to noise. In the current study, both MSE and MFE analysis were used because of two considerations: First, although MFE and MSE have similar meaning, and MFE as an enhanced MSE method, but after all, they use different functions in the calculation process, and get different types of entropy. Calculating MSE and MFE at the same time can make the conclusion more solid. Second, due to the 3 Hz sampling rate, the data length in current study is relatively short than previous study. MFE and MSE have different sensitivity to data length and we also want to explore whether the MSE and MFE are different under the condition of such data length.
Especially in previous study, both the different velocity components of blood flow and its complexity changes resulting from different temperature stimulation were analyzed. However, the relationship between oxygen saturation and speed-resolved blood perfusion were not analyzed. We hypothesize that thermal stimulation not only alter the relationship between signals of local oxygen saturation and velocity components, but also change the relationship of complexity between two kinds of signals in a temperature-dependent manner.

Results
A total of 60 subjects were recruited in the current study. The results of 59 individuals are reported here (thermal stimulation group, TS, N = 29; blank control group, BC, N = 30). One subject's oxygen saturation data could not be imported into Matlab software and was excluded from statistical analyses. In the TS group, the order of thermal stimulations was randomly generated for each subject. The different conditions of thermal stimulation and oxygen saturation signals are shown in Fig. 1.
Oxygen saturation. The average responses of oxygen saturation to thermal stimulation at the different temperatures over time is shown in Fig. 2. Although the strongest increase generally occurred within 5 min of the start of thermal stimulation, the responses as a whole appear to be temperature dependent. In the case of stimulation at 38 °C, oxygen saturation increased throughout the stimulation period and began to decrease 5 min after the stimulation ended. In contrast, following stimulation at 40 °C, maximum oxygen saturation was reached after 15 min. In response to stimulation at 42 and 44 °C, maximum oxygen saturation was attained even more rapidly (i.e., within 5 min of the start of the stimulation). For the 40, 42, and 44 °C thermal stimulations, the maximal oxygen saturation persisted for at least 30 min, extending to approximately 15 min after the termination of stimulation. For all the temperatures investigated below 44 °C, a decay in the oxygen saturation level occurred in a temperature-dependent manner after the end of each stimulation. No such decay was observed in the case of stimulation at 44 °C over the entire measurement period (Fig. 2a).
Complexity calculation. Considering that MFE has been shown to be more closely correlated with MSE than with other multiscale entropy measures 19 , both MSE and MFE were analyzed in the current study. To determine the local oxygen saturation following thermal stimuli, the complexity of the oxygen saturation signals was analyzed using MSE and MFE. In the resting state, neither the MSE nor the MFE differed significantly between the different stimulation groups (Fig. 3a-f). In the case of MSE, stimulation at 38 or 40 °C significantly reduced the complexity area index (CAI). As the temperature of the stimuli increased, the area index also increased. However, the complexity area index at temperatures greater than 42 or 44 °C was still smaller than that of the blank control group (Fig. 3g-i). The results for MFE ( Fig. 3j-m) were similar to those of MSE. The relationship of CAI between MFE and MSE is shown in Fig. S1.
We combined the aforementioned analysis of the complexity of local oxygen saturation signals with an analysis of the complexity of speed-resolved blood perfusion 10 . The velocity components V1, V2, and V3 of speed-resolved blood perfusion were computed as described in reference 10 . The comparison of these velocity components with the oxygen-saturation time series was quantified in terms of Spearman's correlation coefficient, both before (Fig. 4a) and after (Fig. 4b) the application of thermal stimulation.
While there were no significant differences in the results obtained before the start of thermal stimulation (Fig. 4a), there was a significant difference after thermal stimulation (Fig. 4b). For the V1 speed-resolved component, stimulation at 38 °C increased the correlation coefficient with oxygen saturation, while the decrease in the correlation coefficient in response to stimulation at 40 °C was not significantly different than that of the BC group. Moreover, stimulation at 42 and 44 °C significantly reduced the correlation coefficient (Fig. 4b,c). For the V2 speed-resolved component, stimulation at 38 °C increased the correlation coefficient significantly in comparison to the BC group. For the V2 speed-resolved component at stimulation temperatures of 42 and 44 °C, thermal stimulation resulted in a significant reduction in the correlation coefficient (Fig. 4b,d). As for the V3 speed-resolved component, stimulation at both 38 and 40 °C resulted in significant increases in the correlation coefficients in comparison to those of the BC group, while stimulation at 42 and 44 °C resulted in insignificant decreases in the correlation coefficients in comparison to those of the BC group (Fig. 4b,e).
The correlation analysis was repeated and applied to the CAI values, which were calculated from the oxygen-saturation and blood-perfusion data. There is no apparent correlation between the MSE-based CAI values derived from the oxygen-saturation data or from any of the blood-velocity components prior to thermal stimulation (Fig. 5a). However, there was a weak correlation between V2 speed-resolved blood perfusion and oxygen saturation in response to thermal stimulation at 38 °C, (Fig. 5b). In response to stimulation at 40 °C, both low-speed components and high-speed components (Fig. 5c) were correlated with oxygen saturation. At 42 °C stimulation, the only correlation was between low-speed components and oxygen saturation (Fig. 5d) www.nature.com/scientificreports www.nature.com/scientificreports/ in response to stimulation at 44 °C, there was a negative correlation between oxygen saturation and low-speed components (Fig. 5e), while there was a positive correlation between medium-speed components and oxygen saturation (

Discussion
Our results highlight the dependence of local oxygen saturation on stimulus temperature by considering the correlation between oxygen saturation and speed-resolved blood perfusion in response to thermal stimulation. When stimulated at 38 or 40 °C, the correlation of the two signals increased to various extents, while when stimulated at 42 and 44 °C, the correlation of the two signals decreased significantly. However, from the point of view of complexity, the correlation changes caused by different temperature stimuli are quite different from the similarity of signals in the time domain. According to our results, the correlation between the oxygen saturation and blood flow signals is enhanced at lower temperatures. When the stimulation temperature is increased to a certain extent, www.nature.com/scientificreports www.nature.com/scientificreports/ local oxygen consumption becomes more complex, resulting in a decrease in the correlation between the oxygen saturation signal and blood flow signal.
According to previous studies, there are many frequency bands in the blood flux recorded by LDF [21][22][23][24] , which might reflect different physiological rhythms 25 . Kuliga et al. analyzed the correlation between oxygen saturation and blood flow signals in the frequency domain, and showed that there is a significant correlation between low-frequency components, such as the endothelial band, the neurogenic band, and the myogenic band 9 . From another perspective, the microcirculatory perfusion can also be distinguished according to the different velocity of blood flow, and the EPOS system can distinguish different velocity components. Therefore, in the present study, we merely distinguish the correlation between different velocity components and oxygen saturation, and do not analyze the blood and oxygen saturation signals in the frequency domain. www.nature.com/scientificreports www.nature.com/scientificreports/ Previous results have suggested that the different hemodynamic states can be distinguished by a scale analysis of blood flux 26 . The CAI proposed by Costa et al. 18 was calculated in the current study using the following steps: (1) coarse graining of the oxygen saturation signals to obtain new multiple signals series, each of which acquires the system dynamics at this scale; (2) calculating the fuzzy entropy and SampEn for oxygen saturation; and (3) integrating the values of fuzzy entropy or SampEn within the predefined scale to obtain the CAI 17,27 . Previous studies have shown that the CAI has potential value in the diagnosis and prognosis of specific diseases. Decreased MSE values of Electroencephalogram in patient with neonatal seizures reflect who will be more likely to suffer later epilepsy 28 . MSE complexity index of both glucose dynamics and fetal heart rate decreased in the patient with diabetic 29 and acidemia 30 , respectively.
Similar to the results of conventional LDF 20 and speed-resolved blood flux 10 , stimulation at 38 and 40 °C decreased CAI, while stimulation at 42 and 44 °C increased CAI. These results suggest that in the time domain, the response of the various signals induced by different temperature stimuli is different; however, from the point view of complexity, the change in the complexity of various signals is convergent.
Previous study has suggested that there is a positive correlation between absolute changes in oxygen saturation and relative changes in perfusion induced by thermal stimulation 7 . In the present study, Spearman's correlation coefficient was used to measure the correlation between the time series of speed-resolved blood flux and oxygen saturation, which is a supplement to previous research 7 . However, the results of the present study indicate that the correlation between the two signals is enhanced by stimulation at lower temperatures, while the correlation is significantly reduced by stimulation at higher temperatures. These results suggest that the effect of temperature stimulation on oxygen saturation and blood flow is not a simple linear relationship. With increasing stimulation temperature, the process of local oxygen metabolism tends to be complex.
From the perspective of signal complexity, our results show that there is little correlation between the complexity of oxygen saturation and the complexity of different speed-resolved blood flux signals in the resting state. With increases in stimulation temperature, the changes in correlation between low-velocity perfusion and oxygen saturation became more obvious. With 44 °C stimulation, the correlation even reversed from positive to negative. These data indicate that the relationship between low-velocity components and oxygen saturation in terms of signal complexity is close and complex, which further complements previous studies 7 . www.nature.com/scientificreports www.nature.com/scientificreports/ The current study only investigated temperature stimuli from 38 to 44 °C, and did not observe nociceptive thermal stimulation. Furthermore, the temperature intervals in this study were 2 °C, which is much greater than the resolution of the temperature sensor. Therefore, in future research, we should optimize temperature settings to explore the local response caused by temperature stimulation. The local blood flux response induced by thermal stimulation generally consists of two stages: the first stage is the axonal reflex stage, and the second stage is the plateau stage. Because of the different mechanisms of the two stages, we speculate that the correlation between blood flow and oxygen saturation is different. However, owing to the limited sampling rate, the two stages were not analyzed separately here, which is a limitation of the current study. www.nature.com/scientificreports www.nature.com/scientificreports/ conclusions Our experimental results and analyses demonstrate that thermal stimulation significantly increases local oxygen saturation and affects different measures of complexity of local oxygen saturation in a temperature-dependent manner. The complexity area index is at first significantly reduced in response to 38 and 40 °C but subsequently increases upon stimulation at higher temperatures. The correlation analysis in the time domain shows that the correlation between oxygen saturation and speed-resolved blood flux increases when stimulated at 38 °C but decreases significantly at higher temperatures. When considering the data from the point of view of complexity, we observed little correlation between the oxygen saturation and speed-resolved blood flux in the resting state before stimulation. Upon increasing the stimulation temperature, a correlation gradually appears, making the change between low-velocity components and oxygen saturation more apparent. The correlation between medium-velocity components and oxygen saturation was found at 38 °C and 42 °C stimulation, while there was a significant correlation between high-velocity components and oxygen saturation at 40 °C stimulation.

Materials and Methods
Participants and experimental design. Healthy subjects aged 18 to 60 years were recruited and assigned to one of two categories: "thermal stimulation" (TS, N = 29) and "blank controls" (BC, N = 30). One subject's oxygen saturation data can not be imported into Matlab software and was discarded from the analysis. As reported earlier 10 , there was no significant difference in age, sex and MBI between the two groups. Alcohol, tea, and coffee intake were prohibited for at least 24 h prior to measurements. None of the subjects were taking any medication that would affect cardiovascular or autonomic regulation.
Measurements were performed between 9 a.m. and 5 p.m. in a quiet, temperature-controlled (24-26 °C) laboratory. After a period of cardiovascular stability (30 min), a baseline recording was made for 30 min for all participants to ensure stabilization. Subsequently, the individuals in the TS group received four stimuli at 38, 40, 42, and 44 °C using an EPOS thermal stimulation unit (Perimed AB, Stockholm, Sweden). Each temperature stimulus lasted for 30 minutes, and then the probe automatically turned off the heating function according to the software settings. And then followed by another 30 minutes recording. The whole recording process was completed by software setting without any manual operation (Fig. 1b). To enable the measurement of blood-flow parameters in the resting state, the BC group underwent the same procedure as the TS group except that the probe was not heated.
Oxygen saturation measurements and analysis. During recording sessions, subjects were placed in a supine position and their forearms were fixed with a vacuum pillow (AB Germa, Kristianstad, Sweden). A multimodal assessment of microcirculation via diffuse reflectance spectroscopy (DRS) and laser Doppler flowmetry (LDF) was performed by EPOS (Perimed AB, Stockholm, Sweden) with a 3 Hz sampling rate as previously described 5,6 . The system relies on model-based method were model parameters including oxygen saturation, amount and speed-distribution of blood, scattering properties etc., are iteratively updated until modeled and measured spectra agree. The method has been thoroughly described and verified before 1,31,32 . All measurements were set up within the EPOS management system. The probe, which was fixed using double-sided adhesive tape (PF 105-1, Perimed AB, Stockholm, Sweden), combined both a laser Doppler probe and a thermostatic probe at the stimulated point, which allowed for the controlled and consistent heating of the area of the skin under the surface of the probe. The results recorded were exported directly (in MATLAB format) from the EPOS system into MATLAB 2015b (MathWorks, Natick, MA, USA) for analysis. The mean oxygen saturation was averaged over one minute (30 seconds before and 30 seconds after) for each time point.
Complexity of the oxygen saturation signal. The complexity of the blood flux signal was evaluated by MSE and MFE. The theory behind the effectiveness of MSE in teasing apart the complexity of physiological signals has been previously described by Costa et al. 13,14 , while the analytical methods of the MATLAB toolbox were provided by PhysioNet 33 . MFE was calculated using the MATLAB toolbox provided by Azami, H et al. 34 . In addition, we calculated the complexity area index for each coarse-grained time series, which was defined as the integral of the sample entropy curve 27,35 . The parameters of the analysis were described in a previous study (for MSE: m = 2, r = 0.15; for MFE: m = 2, r = 0.15, fuzzy power = 2) 20 .
Statistical analysis. The data are presented as the mean ± SE. Between-subject factors were analyzed using a mixed repeated-measures ANOVA with SPSS software 23.0 (IBM Corp., Armonk, NY, USA), while a permutation test was used for posttests. Multiple comparisons among groups were performed as an independent design. All permutation test analyses were conducted using MATLAB 2015b (MathWorks, Natick, MA, USA). All reported P values were two-sided, and significance was defined as P < 0.05.

Ethical approval and consent to participate. This study was approved by the Institutional Research
Ethics Boards of Acupuncture & Moxibustion, China Academy of Chinese Medical Sciences. In accordance with the Declaration of Helsinki, each subject provided informed consent and had an adequate understanding of the procedure and purpose of the study.