Dry skin conditions are related to the recovery rate of skin temperature after cold stress rather than to blood flow

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1 Report Dry skin conditions are related to the recovery rate of skin temperature after cold stress rather than to blood flow Yasuko Yoshida-Amano 1, PhD, Tomoko Nomura 2, PhD, Yoshinori Sugiyama 1, PhD, Kayoko Iwata 3, MS, Yuko Higaki 4, MD, PhD, and Masanori Tanahashi 1, MS 1 Health Beauty Products, Kao Corporation, Tokyo, 2 Biological Science Laboratory, Kao Corporation, Tochigi, 3 Lifestyle Research Center, Kao Corporation, and 4 Institute of Women s Health, Tokyo Women s Medical University, Tokyo, Japan Correspondence Yasuko Yoshida-Amano, PhD Health Beauty Products, Kao Corporation Bunka, Sumida-ku Tokyo Japan amano.yasuko@kao.co.jp Conflicts of interest: None. Abstract Background Cutaneous blood flow plays an important role in the thermoregulation, oxygen supply, and nutritional support necessary to maintain the skin. However, there is little evidence for a link between blood flow and skin physiology. Therefore, we conducted surveys of healthy volunteers to determine the relationship(s) between dry skin properties and cutaneous vascular function. Methods Water content of the stratum corneum, transepidermal water loss, and visual dryness score were investigated as dry skin parameters. Cutaneous blood flow in the resting state, the recovery rate (RR) of skin temperature on the hand after a cold-stress test, and the responsiveness of facial skin blood flow to local cooling were examined as indices of cutaneous vascular functions. The relationships between dry skin parameters and cutaneous vascular functions were assessed. Results The RR correlated negatively with the visual dryness score of skin on the leg but correlated positively with water content of the stratum corneum on the arm. No significant correlation between the resting state of blood flow and dry skin parameters was observed. In both the face and the body, deterioration in skin dryness from summer to winter was significant in subjects with low RR. The RR correlated well with the responsiveness of facial skin blood flow to local cooling, indicating that the RR affects systemic dry skin conditions. Conclusions These results suggest that the RR but not blood flow at the resting state is associated with dry skin conditions and is involved in skin homeostasis during seasonal environmental changes. 176 Introduction The cutaneous vasculature, which consists of a superficial plexus in the papillary dermis and a deep horizontal plexus located on the dermal side of the dermal subcutaneous interface, 1 plays two important roles in the human body. One role is to adjust the body temperature under thermally stressed conditions, and the other is to supply oxygen and nutritive substances to the skin to maintain its viability. The role of blood flow in controlling body temperature has been well investigated. When the entire body is heated, cutaneous vessels dilate due to the action of sympathetic cholinergic nerves that release several co-transmitters to cause active vasodilation. 2 In contrast, vessels constrict due to sympathetic noradrenergic nerves when exposed to a cold environment. 3 Regarding skin tissue maintenance, it has been clearly shown that increased skin blood flow and/or angiogenesis results in accelerated wound healing. 4,5 However, very little has been reported regarding a potential link between skin conditions at steady state and skin blood flow. Skin blood flow can be measured non-invasively using laser Doppler fluxometry (LDF), laser Doppler perfusion imaging, and photoplethysmography. 6 Additionally, measuring skin temperature is a substitute method to estimate skin blood flow. 6 The cold stress test (CST) is carried out by immersing one or two hands in cold water, then measuring finger skin temperatures from pre-immersion to post-immersion. 7 As vascular function is affected by sympathetic noradrenergic nerves when exposed to cold stimuli, the CST is often used to assess autonomic function. 8 Delayed recovery of skin temperature and/or blood flow after cold water immersion has been reported in patients with Raynaud s phenomenon, 9 11 hand arm vibration syndrome, 7,12 and type II diabetes, 13 and in subjects who have cold hypersensitivity, which refers to a condition in which subjects feel excessively cold in the hands and feet at a low environmental temperature. 14 The majority of individuals with Raynaud s phenomenon and cold hypersensitivity are women, 15,16 which suggests that women tend to have peripheral vascular dysfunction more than men. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

2 Yoshida-Amano et al. Blood vessel functionality is related to dry skin Report 177 Dry skin is a common skin problem both in the cosmetic and dermatological fields, particularly in the winter. Subjective complaints of dry skin are more common for women than for men, suggesting that women perceive dry skin more sensitively than men. 17 Although environmental factors such as low temperature and humidity are causes of dry skin, 18 it remains unclear why the degree of dry skin varies in different individuals. Given that previous reports showed that patients with Raynaud s phenomenon or diabetes often present with dry skin, 19,20 we hypothesized that cutaneous blood flow, particularly the sensitivity to a cold environment, is related to dry skin conditions in the winter even in healthy adults. Few studies have assessed the relationship between blood flow and skin physiology, including dry skin. A decline in the number of capillary vessels was observed in aged skin although the degree varied by body site. 21 Age-related changes in topography occur in skin with decreased numbers of capillary vessels. 22 However, it is still unclear whether cutaneous blood flow and the reactivity to cold stress is related to dry skin conditions in healthy adults. Here, we used the CST to examine the systemic cutaneous blood flow responsiveness to cold stress. There was a significant correlation between the recovery rate (RR) and dry skin parameters, which indicates involvement of the cutaneous blood flow response to skin physiology for the first time. Materials and methods Subjects and study design All studies were conducted according to the principles outlined in the Declaration of Helsinki. All procedures were approved by the Ethics Committee of the Kao Corporation (Tokyo, Japan). Written informed consent was obtained from each subject before participation. For minor subjects, we also obtained written informed consent from their guardians. Three studies were carried out according to the objectives. For analysis of the association between blood flow, RR, and skin physiology, a study was carried out in February on 56 healthy Japanese women (17 67 years old) (study I). As we aimed to evaluate the general relationship between blood flow and skin conditions, the menstrual cycle was not considered in study I. To assess the involvement of the RR with skin temperature after CST in seasonal changes of skin physiology, 37 female subjects (30s, n = 18; 50s, n = 19) were recruited and skin capacitance, transepidermal water loss (TEWL), and visual dryness score were measured in the summer (September) and in the winter (February) (study II). In study II, to minimize factors influencing skin conditions other than the season, subjects in their 30s were studied during the late follicular phase of their menstrual cycle (days 8 12) both in the summer and in the winter. All subjects in their 50s were postmenopausal and were not on hormone replacement therapy and had not previously been on hormone replacement therapy. The relationships between the RR and facial blood flow response to cold stimuli were analyzed in 18 healthy Japanese men aged years old (study III). To evaluate blood flow and RR in relation to dry skin (studies I and II), female subjects were recruited because they complain about dry skin symptoms more than men. 17 Exclusion criteria included systemic or peripheral vascular diseases, hypertension (resting systolic pressure >140 mmhg and/or diastolic pressure >90 mmhg), any medications that could alter any cardiovascular or thermoregulatory variables, and smoking. See Table 1 for a summary of the physical characteristics of the subjects in each study. Experimental conditions In all three studies, skin measurements and visual dryness evaluations were performed under conditions of 20 C and 40% relative humidity, and measurements of skin blood flow at the resting state, CST, and local cooling tests were carried out under conditions of 24 C and 50% relative humidity. Skin measurements were performed for each subject after washing with a commercial facial cleanser and acclimatization to the room conditions for minutes in each study. Skin physiology measurements As an index of skin barrier function, TEWL was measured using a Cutometer â MPA 580 (Courage+Khazaka electronic GmbH, Cologne, Germany) with a Tewameter probe. As an indicator of stratum corneum water content, the skin capacitance at the skin surface was measured using a Cutometer â MPA 580 with a corneometer probe (Courage+Khazaka electronic GmbH). All skin measurements were conducted on one side of the cheek, and the arm and leg of each subject. TEWL and capacitance were measured three and five times, respectively, and the mean values were calculated. Visual assessment of skin dryness The degree of skin dryness was assessed by a single expert (Y.Y-A.) using a five-point visual dryness scale by a modified method as previously described: 23 Table 1 Subject characteristics in studies I, II, and III. Gender Study I Study II Study III Female Female Male Number of subjects Age (years) Height (cm) Weight (kg) BMI (kg/m 2 ) SBP (mmhg) DBP (mmhg) MAP (mmhg) Values are means SD. BMI, body mass index; DBP, diastolic blood pressure; MAP, mean arterial pressure; SBP, systolic blood pressure.

3 178 Report Blood vessel functionality is related to dry skin Yoshida-Amano et al. Score 0: Normal skin no signs of scaling or flaking. Score 1: Slight flaking slight, but definite roughness; may have a powdery or ashy appearance. Score 2: Moderate flaking/scaling moderate roughness; somewhat coarse surface. Score 3: Marked scaling/slight fissuring marked roughness; coarse scaling, cracking evident as uplifted scales. Score 4: Severe scaling/fissuring marked roughness; very coarse scaling; cracking progressing to fissuring. As a preliminary study, we evaluated the visual dryness scale in 76 female subjects with a wide age range (16 67 years old) and confirmed that the inter-evaluator correlation coefficient of visual dryness scores was more than Figure 1 shows representative examples of skin surface images corresponding to each score. Measurement of skin blood flow at the resting state Subjects were seated and acclimatized to the room conditions for 30 minutes before measurements. After the blood flow became stable, measurements were conducted on the face, arm, and leg for 60 seconds using a laser-doppler blood flowmeter (ALF21; Advance, Tokyo, Japan) with a laser-doppler probe (OP type; Advance), and mean values were calculated. Cold stress test As an index of cutaneous vascular and systemic/local autonomic functions, the CST was performed by a modified method as previously described. 8 Briefly, after capture with a Thermo tracer TH9260 (Nippon Avionics, Co., Ltd, Tokyo, Japan), the right hand was immersed to wrist level in cold water at 15 C for 1 minute. Thermal images of the dorsum of both hands were obtained immediately after immersion and 10 minutes later, then the RR of the skin temperature was calculated as follows: RR ¼ðT 10 T 0 Þ=ðT tl T 0 Þ100ð%Þ T bl, baseline skin temperature; T 0, skin temperature immediately after cold stress; T 10, skin temperature 10 minutes after cold stress. Local cooling test protocol As an index of local cutaneous vascular reactivity to cold stress, a local cooling test was performed on the facial skin. Skin blood flow was measured using a laser-doppler blood flowmeter (ALF21; Advance) with a laser-doppler probe (OP type; Advance). Local skin temperature was controlled by a circular probe with a temperature control stimulator (TS-8000; Physio-Tech, Tokyo, Japan). Red blood cell flux was measured with a laser-doppler probe attached at the center of the circular probe. The laser-doppler signal and skin temperature were recorded with a PowerLab (ADInstrument, Otago, New Zealand) using LabChart software (ADInstrument). The mean arterial blood pressure was monitored periodically during the experiment using a finger cuff placed on the middle finger of each subject (FinometerMIDI; Finapres Medical Systems, Amsterdam, the Netherlands). The circular probe was attached to the cheek, and the probe temperature was adjusted and kept at 30 C until the skin blood flow became stable. The baseline LDF flux was then measured for 60 seconds, followed by decreasing the skin temperature at a rate of 1.0 C every 1 second to a temperature of 10 C. After the probe was held constant at 10 C for 600 seconds, the probe temperature was increased at the same rate to 30 C and kept at that temperature for 300 seconds. The cutaneous vascular conductance (CVC) was calculated as the ratio of LDF flux to mean arterial blood pressure and is expressed as a percentage of the baseline CVC value (%CVC Base ). Statistics All associations between parameters except the visual dryness score were analyzed using Pearson s correlation coefficient. The association analysis between the visual dryness score and other parameters was carried out by Spearman s rank correlation coefficient. SPSS version 11 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. Comparisons between high and low RR subjects for seasonal changes of skin parameters are presented as means SD. Values between both groups were compared by Student s t-test except for the visual dryness score. The visual dryness score was Figure 1 Representative images of skin dryness corresponding to visual dryness scores of 0, 1, 2, 3, and 4. Scale bar = 1mm

4 Yoshida-Amano et al. Blood vessel functionality is related to dry skin Report 179 compared by Mann Whitney s U-test. Statistical significance was set at P Results In study I, the associations between skin blood flow in a resting state or the RR evaluated by CST and skin parameters were analyzed in 56 female subjects aged years. Table 2 shows the correlations between age and skin parameters/blood flow in the cheek, arm, and leg. The visual dryness score was negatively correlated with age in the cheek (P < 0.05) and was positively correlated in the leg (P < 0.01). The correlations between TEWL and age varied by body site; there was a negative correlation in the cheek (P < 0.05) and a positive correlation in the arm (P < 0.01). It has been reported that the RR is delayed with aging in healthy adults. 24 In our study, a weak negative correlation was observed between age and the RR although it was not statistically significant (R = 0.159, P = 0.242). There was no significant correlation between blood flow in a resting state and any skin parameter either by direct correlation analysis or by partial correlation analysis, which eliminates the effect of age (data not shown). On the other hand, the RR showed a positive correlation with capacitance in the arm (P < 0.01) and a negative correlation with the visual dryness score in the leg (P < 0.05) (Table 3). Some of those correlations still existed when the effect of age was removed: there was a positive correlation between the RR and capacitance in the arm (P < 0.01). A weak negative correlation was also observed between the RR and visual dryness score in the leg (P < 0.1), although it was not statistically significant (Table 4). Regarding correlations between dry skin condition and physiological parameters of skin, significant positive correlations was found between visual dryness score and TEWL in the face (R = 0.397, P = 0.003). Similar but not significant correlation was found in the arms and legs. Significant negative correlations were found between visual dryness score and capacitance in the arm (R = 0.411, P = 0.002) and in the leg (R = 0.548, P < 0.001). The relationships between the RR and seasonal changes in skin physiology were analyzed in study II. Thirty-seven female subjects were recruited and divided into two groups according to their initial RR measured in the summer, as no significant differences between summer and winter RR were reported when the same individuals were tested for CST. 25 RR profile showed a U-shaped distribution. Using the RR value of 70%, the bottom value of the RR distribution, we could divide the subjects into two groups. An RR of 70% was considered the high RR group and an RR of <70% was considered the low RR group, with the top 40% of all subjects (n = 14) considered the high-value group. There was no significant difference between the high and low RR groups regarding average age ( and ), systolic blood pressure ( and ), or diastolic blood pressure ( and ), respectively. Skin parameters, such as TEWL, conductance, and visual dryness score, were measured in the summer and in the winter. The change in visual dryness score from summer to winter was significantly higher in the low RR group in the cheek (P < 0.05) and the leg (P < 0.05). A similar tendency was observed in the arm but not at a significant level (P < 0.1) (Table 5). To clarify the relationships between the RR and the thermal regulation of facial blood flow, CST and a local cooling test on the face was performed on 18 Japanese males in study III. When facial skin was cooled to 10 C using a circular probe Table 2 Correlations between age (n = 56) and skin parameters/blood flow in study I Cheek Arm Leg R P value R P value R P value Transepidermal water loss * ** Capacitance Visual dryness score * ** Blood flow *P < 0.05; **P < Table 3 Correlations between recovery rate and skin parameters in study I Cheek Arm Leg R P value R P value R P value Transepidermal water loss Capacitance ** Visual dryness score * *P < 0.05; **P < 0.01.

5 180 Report Blood vessel functionality is related to dry skin Yoshida-Amano et al. Cheek Arm Leg R P value R P value R P value Table 4 Partial correlations between recovery rate and skin parameters in study I Transepidermal water loss Capacitance <0.001** Visual dryness score ** P < Table 5 Comparison between subjects with high and low RR for seasonal changes of skin parameters in study II Cheek Arm Leg High RR Low RR P value High RR Low RR P value High RR Low RR P value TEWL BL Δ Capacitance BL Δ Visual dryness score BL Δ * * Values are means SD. BL, baseline value measured in the summer; D, delta value (winter summer); RR, recovery rate; TEWL, transepidermal water loss. *P < 0.05 high RR vs. low RR. (Fig. 2b,d), skin blood flow initially decreased relative to the baseline level (Fig. 2a,c). However, the blood flow behavior varied in the subjects as follows: some subjects showed a gradual blood flow increase above the baseline level (Fig. 2a), whereas others showed a stable blood flow lower than the baseline (Fig. 2c). The sum of %CVC Base during cooling ( s) correlated positively with RR (R = 0.54, P < 0.01) (Fig. 2e). These results suggest that subjects with a higher RR are able to keep the blood flow level from becoming too low during cooling and possibly can better tolerate cold stimuli on their facial skin. Discussion In this study, we demonstrate for the first time that cutaneous blood flow regulation, characterized by the RR of the skin temperature after CST, is involved in dry skin conditions such as TEWL, capacitance, and visual dryness score. Consistent with a previous study, no correlation was found between blood flow in the resting state and any dry skin parameters. 26 As skin is the organ most directly affected by temperature changes in the environment, changes in blood flow after thermo-stimulation are more likely to affect skin physiology than at the resting state. In our study, some skin parameters correlated with the ages of the subjects. By partial correlation analysis (Table 4), which eliminates the influence of age, a significant correlation between RR and capacitance in the arm was observed. The relationship of capacitance, which represents the water content of the stratum corneum, to RR indicates that the RR is involved in skin dryness by controlling the moisture level of the skin surface. The weak correlation between the visual dryness score and the RR observed in the arm and the leg may support our conclusion (Table 4), although it was not statistically significant. The differences in seasonal changes of dry skin parameters observed in the high and the low RR groups represent blood flow responsiveness to cold stress and are crucial to skin conditions in the winter. The seasonal shift from summer to winter leads to an increase of TEWL and decreases the amount of water in the stratum corneum. 27,28 In our visual dryness rating of each subject s body and facial skin, most subjects showed no skin dryness or only a slight dryness in the summer (Table 5). However, the deterioration of skin dryness by the seasonal change to winter is considerably different between the high RR and the low RR groups. The high RR group showed less of a change in skin dryness during the summer and winter, whereas the low RR group showed a noticeable increase of skin dryness in the winter (Table 5). Despite the fact that no difference of the visual dryness score was observed between the high RR and the low RR groups in the summer, the TEWL was slightly higher in the low RR group (P < 0.1). These differences of TEWL in the summer may affect the deterioration of skin dryness in the winter. The skin blood flow functionality measured by CST may be involved in skin homeostasis during the

6 Yoshida-Amano et al. Blood vessel functionality is related to dry skin Report 181 Figure 2 Relationships between RR and facial blood flow under cold stimuli in study III. (a d) Representative tracing of the skin blood flow response to local cooling to 10 C in subjects who showed high (a, b) and low (c, d) values of the sum of the %CVC Base during steady cooling at 10 C ( s). Arrows indicate the start and end points of steady cooling at 10 C. (a, c) Relative skin blood flow response to local cooling. (b, d) Skin temperature change during local cooling. (e) Correlation between the sum of the %CVC Base during steady cooling ( s) and RR. %CVC Base, percentage of the baseline cutaneous vascular conductance value; RR, recovery rate environmental change in seasons and is related to skin dryness in the winter. Separately from study I, we recruited different sets of female subjects in study II. Further studies, to follow the same individuals at the several time points such as menstrual cycle or the seasons, are needed to clarify the role of RR in skin dryness. In relation to diseases, patients with Raynaud s phenomenon or diabetes who are known to show an abnormal RR often present with dry skin. 19,20 In addition to healthy subjects, an impaired RR is possibly involved in the dry skin symptoms of patients with these diseases. Notably, associations of RR and skin conditions were found not only on the arms and legs but also on the face (Table 5). Given that CST can evaluate autonomic function, 8 it is reasonable that RR measured by CST correlates with the whole body skin including the face. To elucidate the relationships between RR and local facial blood flow regulation under cold stimuli, we used a local cooling test of facial skin followed by CST in the same individuals. The results showed a significant correlation between relative total blood flow during facial cooling and RR (Fig. 2e). By local cooling, after the initial blood flow decrease, transient vasodilation occurs followed by continuous vasoconstriction. 29 Our results show that this transient vasodilation varies among subjects, some showing a remarkable increase whereas others show a slight or no increase, which results in the variance of relative total blood flow. A significant positive correlation of RR and relative total blood flow during local cold stimuli on the face indicates that subjects who have a high RR tend to show transient vasodilation compared to subjects with a low RR. It has been reported that the cold-induced vasodilation response, which occurs during continuous cold exposure of the fingers, 30 is reduced with aging. 31,32 Cold-induced vasodilation is thought to be a protective response of the skin to continuous ischemic conditions. 30 Although there is a difference between glabrous and non-glabrous skin, it is possible that the variation of transient vasodilation observed in the face during local cooling is also a protective reaction of the skin from excessive blood flow reduction caused by cold stimuli. Our present data advocate a hypothesis that the RR can be an indicator of facial blood flow response to cold stimuli and that RR measured in the summer might possibly be able to predict seasonal changes in facial skin dryness. Further investigation of relationships between RR and facial blood flow response to thermal stimuli including gender and age difference would help us to understand how systemic vascular function effect on skin dryness. Our present data suggest that the RR can be an indicator of facial blood flow response to cold stimuli and that RR measured in the summer might possibly be able to predict seasonal changes in facial skin dryness. It remains to be elucidated how blood flow regulation under thermal stress is involved in regulating skin characteristics. It is

7 182 Report Blood vessel functionality is related to dry skin Yoshida-Amano et al. possible that oxygen and nutritive substances supplied by the blood flow affect the turnover of skin cells. Blood flow also provides heat and maintains an appropriate skin temperature, which influences enzymatic activities in skin cells. It has been reported that the ceramide composition, which is catalyzed by several enzymes, strongly correlates with dry skin conditions and skin conductance. 23 Other molecules involved in the maturation of keratinocytes can be candidates that are influenced by blood flow. To clarify the mechanisms underlying skin physiology affected by blood flow functionality, further in vivo and/or in vitro analysis is required. Limitations In this research, three different sets of subjects were recruited for each study. To clarify the relationship between RR and dry skin, further long-term trials on the same individuals will be necessary. 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