Chronic Restraint Stress Inhibits Hair Growth via

Substance P Mediated by Reactive Oxygen Species in

Mice
Nan Liu1., Lin-Hui Wang2., Ling-Ling Guo3., Guo-Qing Wang2, Xi-Ping Zhou2, Yan Jiang2, Jing Shang4,
Koji Murao5, Jing-Wei Chen6, Wen-Qing Fu1*, Guo-Xing Zhang2*
1 Department of Clinical Psychology, Medical College of Soochow University, Suzhou, P.R. China, 2 Department of Physiology, Medical College of Soochow University,
Suzhou, P.R. China, 3 Department of Pathology, Medical College of Soochow University, Suzhou, P.R. China, 4 New Drug Screening Center, China Pharmaceutical
University, Nanjing, P.R. China, 5 Department of Clinical Laboratory, Faculty of Medicine, Kagawa University, Kita-gun, Kagawa, Japan, 6 Department of Internal Medicine,
Suzhou Chinese Traditional Medicine Hospital, Suzhou, P.R. China

Abstract
Backgrounds: Solid evidence has demonstrated that psychoemotional stress induced alteration of hair cycle through
neuropeptide substance P (SP) mediated immune response, the role of reactive oxygen species (ROS) in brain-skin-axis
regulation system remains unknown.

Objectives: The present study aims to investigate possible mechanisms of ROS in regulation of SP-mast cell signal pathway
in chronic restraint stress (CRS, a model of chronic psychoemotional stress) which induced abnormal of hair cycle.

Methods and Results: Our results have demonstrated that CRS actually altered hair cycle by inhibiting hair follicle growth in
vivo, prolonging the telogen stage and delaying subsequent anagen and catagen stage. Up-regulation of SP protein
expression in cutaneous peripheral nerve fibers and activation of mast cell were observed accompanied with increase of
lipid peroxidation levels and reduction of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px)
in CRS mice skin. In addition, SP receptor antagonist (RP67580) reduced mast cell activations and lipid peroxidation levels as
well as increased GSH-Px activity and normalized hair cycle. Furthermore, antioxidant Tempol (a free radical scavenger) also
restored hair cycle, reduced SP protein expression and mast cell activation.

Conclusions: Our study provides the first solid evidence for how ROS play a role in regulation of psychoemotional stress
induced SP-Mast cell pathway which may provide a convincing rationale for antioxidant application in clinical treatment
with psychological stress induced hair loss.

Citation: Liu N, Wang L-H, Guo L-L, Wang G-Q, Zhou X-P, et al. (2013) Chronic Restraint Stress Inhibits Hair Growth via Substance P Mediated by Reactive Oxygen
Species in Mice. PLoS ONE 8(4): e61574. doi:10.1371/journal.pone.0061574
Editor: Johanna M. Brandner, University Hospital Hamburg-Eppendorf, Germany
Received September 30, 2012; Accepted March 8, 2013; Published April 26, 2013
Copyright: ß 2013 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (30900464, 81270316), the Research Program of Soochow University
(Q413400111). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: zhangguoxing@suda.edu.cn (GXZ); fuwenqing@suda.edu.cn (WQF)
. These authors contributed equally to this work.

Introduction Substance P (SP) is a stress-related neuropeptide, synthesized in

dorsal root ganglion small neuronal cell body, and released from
Hair is an important skin appendage whichhas wide range of cutaneous peripheral nerve terminals [13–14]. As a member of
functions including thermoregulation, physical protection, sensory neuropeptides family, there are three principal neurokinin
activity, and social interactions [1]. Up to date, basic and clinical receptors have been described: NK-1, NK-2, and NK-3, which
observations have demonstrated that psychoemotional stress was a bind with high affinity to SP. Remarkably, the NK1 receptor is
potential cause of hair loss [2–5]. Further study revealed that hair widely distributed to the central and peripheral nervous system
loss was highly related with hair follicle pathophysiological changes especially in mast cell in mammals [4,15–18]. Previous studies
[6]. Hair follicle has its own specific cycle which is defined as rapid have demonstrated SP induced neurogenic inflammation and mast
growth (anagen), regression (catagen), and resting periods (telogen) cell degranulation which were involved in skin disorders, such as
according to histological morphology [7–10]. Numerous clues atopic dermatitis and psoriasis [4–5,19–21]. Therefore, mast cell
have revealed that psychoemotional stress played an important
has been regarded as a ‘‘switchboards’’ of neurogenic inflamma-
role in abnormal hair cycling [4–5,11–12]; however, the
tion induced by stress [14]. Arck and her colleagues proposed that
mechanisms of psychoemotional stress induced alterations of hair
SP was the key mediator of the ‘‘brain-hair follicle axis’’ through
cycling still remains many unknown elements.

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 Role of Oxidative Stress in Hair Cycle

mast cell activation [4,22]. Their further study clearly confirmed Anagen Induction and CRS Application
that the center position of substance P in regulating human hair Method of depilation to induced anagen of hair cycle was as
follicle [23]. These observations indicated that the interaction described previously [4]. Briefly, wax/rosin mixture (1:1 on
between SP and mast cell via NK-1 receptor might be one of the weight) was applied to the dorsal skin (from neck to tail) of mice,
most important pathways in stress which induced abnormal of hair then waiting for it dried and peeling-off the wax/rosin mixture
cycle. removed all hair shafts and immediately induced a highly
In recent decades, oxidative stress and excessive reactive oxygen synchronized hair growth, as evidenced by the homogeneously
species (ROS) generation, have been generally accepted, played a pink skin color in the back, which indicated all hair follicles in
critical role in various diseases, including skin diseases [24–25]. telogen [8–10]. CRS application began on day 1 after depilation.
Some allergic and inflammatory skin diseases, for example, atopic Following the previously reported method, mice were placed in a
dermatitis, urticaria and psoriasis were demonstrated to be closely 50 mL conical centrifuge tube with multiple punctures to allow
related to oxidative stress [26–27]. Several researches also revealed ventilation, then plugged the tube to prevent shuttle of mice [40].
that the mast cell function could be modulated by the balance of Tubes were held slantly (head elevation) in the cage. Application of
oxidative/antioxidative system which could induce sensitization of CRS was carried out for 6 hours daily (10:00 a.m.–16:00 p.m.)
mast cell by increase of cytokine mRNA expression [28–29]. In while food and water were prohibited. Period of CRS was lasted
addition, previous study also confirmed that oxidative stress for 9 and 18 days and the control mice were kept in their original
involved in skin and hair aging [30–31]. These studies indicated cage, but food and water were not provided during the CRS
that oxidative stress may play a vital role in mast cell activation period.
and hair follicle pathological change [32]. Recently, it was
reported that ROS were second messengers of neurokinin Reagents
signaling in peripheral sensory neurons [33]. Although acute and RP67580 (Santa Cruz biotechnology,CA, USA),a selective
chronic psychoemotional stress both induced oxidative stress in antagonist of the tachykinin NK1 receptor [41–42] was dissolved
many organs such as brain, liver, kidney, heart and stomach of rats in ethanol and then diluted with PBS to make a final concentration
[34–39], and SP-mast cell pathway was demonstrated to play a key of 1 mg/ml. RP67580 was injected intraperitoneally at a dose of
role in psychoemontional stress induced abnormal of hair cycle. 200 mg/mouse every other day [4]. 4-Hydroxy-2, 2, 6, 6-
However, the role of oxidative stress in chronic psychoemotional tetramethyl piperidinoxyl (Tempol, Sigma, MO, USA) is a stable
stress induced abnormal of hair cycle, especially its role in SP-mast membrane-permeable superoxide dismutase (SOD) mimetic that
cell pathway in skin has not been investigated. exhibits potent antioxidant activity against superoxide as well as
For this reason, the present study aims to explore the possible hydroxyl radicals [43]. Our previous studies have demonstrated
role of oxidative stress in SP-mast cell pathway in chronic restraint that Tempol decreased vascular superoxide anion production in
stress mice (CRS, a chronic psychoemoational stress model). We conscious chronic and acute angiotensin II induced hypertensive
find that CRS could: (1) inhibit weight gain and increase plasma rats [44–45]. In the present study, Tempol was injected
cortisol content; (2) inhibit the hair growth observed by visual and intraperitoneally to against oxidative stress in skin at a dose of
microscopic method; (3) induce increase of oxidative stress marker, 200 mg/kg/day because that the maximum tolerated dose of
such as lipid peroxidation levels determined thiobarbituric acid Tempol administered intraperitoneally was found to be 275 mg/
reactive substances (TBARS), and alter the activities of antioxidant kg [46]. The mice were randomly divided into the following four
enzymes, such as superoxide dismutase (SOD) and glutathione groups: (1) Control group; (2) CRS group: no drug injection and
peroxidase (GSH-Px) in skin; (4) increase the number of SP application of CRS; (3) Tempol group: application of CRS
immunoreactive nerve fibers and activate mast cell by observing concomitant with 200 mg/kg/day of Tempol injection intraper-
immunofluorescence and toluidine blue stain in skin. (5) CRS- itoneally for 9 days or 18 days; (4) RP67580 group: application of
induced pathological changes in skin could be normalized by SP CRS concomitant with 200 mg/mouse of Tempol injection
receptor specific antagonist (RP67580). (6) CRS-induced SP-mast intraperitoneally for 9 days or 18 days (every other day).
cell pathway could be suppressed by antioxidant (Tempol), which
also normalizes hair growth. Tissue and Plasma Preparation
In order to explore the change of whole hair cycle, we chose two
Materials and Methods time points which indicted different hair cycle stage. The first step,
Animals on day 9 after depilation when hair cycle of control mice was in
the late anagen [9–10], blood samples were collected by
Six to eight weeks old male C57BL/6 mice (Provided by center
enucleated eyeball under anesthesia and added heparin for
of experimental animal, Soochow University) were used because
anticoagulation, then centrifuged at 3000 rpm for 15 minutes to
the dorsal hair follicles in such strain mice at this age were in
obtain plasma, after that stored at280uC immediately. Skin
telogen stage naturally [9–10]. All mice were acclimated for a
specimens from back skin were harvested about 265 cm, the
week under the following conditions: room temperature was
region of neck, which was an essential section for the quantitative
2261uC; humidity was 5065% and with a 12-hour light: dark
histomorphology of the hair cycle [4]. It was fixed in 4%
cycle (lights on at 6:00 a.m. and off at 18:00 p.m.). During this
paraformaldehyde then paraffin-wax embedded. The remaining
period, food and water were provided ad libitum. The present study
skin specimens were cryopreserved at280uC immediately. The
was conformed to the Guide for the Care and Use of Laboratory Animals
second step, on day 19 after depilation when hair cycle of control
published by the US National Institutes of Health (NIH
mice just spontaneously entered the catagen transformation [9–
Publication No. 85–23, revised 1996). All procedures were
10], mice were sacrificed; plasma and skin tissues were sampled
approved by the Animal Care and Use Committee of the
according to above mentioned method.
University of Szeged.

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 Role of Oxidative Stress in Hair Cycle

Measurement of Body Weight and Plasma Corticosterone method for measurement of TBARS levels has been described
Concentration previously [41–42]. Briefly, 100 mL sample solution was mixed
All mice were recorded body weight on day 0, 4, 9, 14, 19 after with 15% trichloroacetic acid and 0.375% thiobarbituric acid.
depilation. Plasma corticosterone concentration was determined Butylated hydroxytoluene (0.01%) was added to the assay mixture
by mouse corticosterone RIA kit following manufacturer’s to prevent autoxidation of the sample and the mixture was heated
instruction (Beyotime, China). at 100uC for 15 minutes. After cooling, the mixture was
centrifuged at 14,000 rpm for 5 minutes and the absorbance of
Protein Concentration Measurement the organic phase was measured at 535 nm. The amount of
The skin tissues were homogenized in a solution containing thiobarbituric acid reactive-substances (TBARS) was determined
0.15 mol/L KCl and 0.02 mol/L Tris-HCl (pH 7.4) then used by the malondialdehyde standard curve and expressed as mmol/L
BCA Protein Assay Kit (Beyotime, China) to determined protein in 100 mg protein.
concentration. Finally, the skin tissue homogenates were adjusted
at a concentration of 1 mg/ml for further analysis. Measurement of SOD & GSH-Px Activities
SOD activity was detected according to previous report [48]. In
Measurement of Lipid Peroxidation Levels in Skin this method, xanthine–xanthine oxidase complex produced
Thiobarbituric acid reactive substances (TBARS) is a well- superoxide radicals which reacted with water-soluble tetrazolium
recognized parameter for lipid peroxidation levels which is also as (WST-1). GSH-Px activity using H2O2 as a substrate was assayed
a biomarker of tissue damage caused by oxidative stress [47]. The by an adaptation of the method reported by Lawrence and Burk

Figure 1. Macroscopic observations of the hair cycle. The hair shafts of murine skin were shaven by animal clippers beforehand. A: The
significant area of color in the mouse dorsal skin was surrounded by a red line. B: The corresponding skin color gray-scale ratio on day 9 was shown
on the left and day 19 on the right. Data were presented as mean 6 SEM, n = 9 in each group, * P,0.05 compared with control group, { P,0.05
compare with CRS group.
doi:10.1371/journal.pone.0061574.g001

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 Role of Oxidative Stress in Hair Cycle

[49–51]. Using 96-well plate, SOD activity was measured at Assessment of Hair Cycle
450 nm and GSH-Px activity was measured at 412 nm by All mice were taken pictures with a digital camera (Panasonic,
Microplate reader (Thermo Scientific, USA), and both of the Japan) every day after depilation. The grayscale (0–255) of specific
data were presented as U/100 mg protein. area in photograph (the region of neck) were analyzed by Image J
software and presented as ratios (grayscale/255). The HE stain
Immunofluorescence was used to quantify the stage of the hair follicles using a published
The specimens were cut into 14 mm sections. Standard classification technique based on the morphology of the dermal
immunofluorescence method for a double labeled immunofluo- papilla and sebaceous glands [9]. To identify accurately, a
rescence study was applied to identify the nerve and the minimum of 100 back skin hair follicles per mouse were scored
neuropeptide as previous method [52]. Briefly, polyclonal anti- which assigned to the telogen, anagen and catagen stages in
MAP2 antibody (Cell Signaling Technology, MA, USA) was used ascending numerical order (anagen I–VI: 1–4; catagen I–VIII: 5–
to stain the cutaneous nerves and polyclonal antibody for SP 8; telogen: 9) according to their characteristic, stage-specific
(Santa Cruz biotechnology, CA, USA) were used to stain the histological appearance as described in previous reports [10–
neuropeptides. The tissues were stained with anti-MAP2 antibody 11,53]. The number of hair follicles in each specific hair cycle
in a dilution of 1:500 and with anti-SP antibody in a dilution of stage was then multiplied by its corresponding score. The resulting
1:500. All sections were examined with Confocal Laser Scanning sum was divided by the total number of hair follicles per visual
Microscope (Leica, Germany). Only structures with strong, field to obtain the average hair cycle score which was defined in
continuous linear staining were counted as nerve fibers. relevant hair cycle stage.

Figure 2. Effects of stress on the hair cycle stage. A: A representative area of each group on day 9 and day 19 after depilation with the majority
of hair follicles. Original magnification was6100. B: The results of hair follicles score for day 9 were shown on the left and the data for day 19 on the
right. The Y axis depicted histometric score assessed anagen induction. For each mouse a minimum of 10 individual visual fields were assigned to
define hair cycle stages. Data were presented as mean 6 SEM, n = 9 in each group, * P,0.05 compared with control group, { P,0.05 compared with
CRS group.
doi:10.1371/journal.pone.0061574.g002

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 Role of Oxidative Stress in Hair Cycle

Toluidine Blue Staining after depilation were examined in mice exposed to CRS compared
Mast cells in the skin section were stained with toluidine blue. In to control mice. On day 9 after depilation (late anagen), the skin
brief, the deparaffinized sections were soaked in a solution color of neck region in control mice was darker than the CRS mice
containing 0.1 M citric acid, 0.2 M dibasic sodium phosphate (Figure 1A) and the corresponding skin grayscale ratio was
and 0.5% toluidine blue for 20 minutes at room temperature. significantly lower than CRS mice (Figure 1B). Morphological
Mast cells were classified as degranulated when eight or more study showed most of hair follicles in control mice entered anagen
granules were found outside the cell membrane at 2006magni- V/VI and the majority of hair follicles in CRS mice was in anagen
fication as described previously [10,16]. Number of positively II/III (Figure 2A). On day 19 after depilation (catagen), skin color
stained cells per section of the entire dermis was counted and the of neck region in control mice was lighter than the CRS group
ratio of degranulated mast cells to all mast cells was calculated. (Figure 1A) and the corresponding skin grayscale ratio was
significantly higher than CRS mice (Figure 1B); meanwhile, most
Statistical Analysis of hair follicles in control mice entered catagen V/VI, CRS mice
All data were presented as the means 6 S.E.M. Statistical was still in anagen VI (Figure 2A). On day 9 and day 19, scores of
significance between more than two groups was tested using two hair cycle in control mice were both significantly higher than in
way ANOVA followed by the Newman-Keel test or an unpaired CRS mice (Figure 2B). These results suggested CRS inhibited hair
two tail Student’s t-test. P,0.05 were considered statistically growth by prolonging the telogen stage and delaying subsequent
significant. anagen and catagen stage.

Results CRS Inhibits Mice Body Weight Gain and Increases

Plasma Corticosterone Concentration
CRS Induces Alteration of Hair Cycle To confirm whetherCRS successfully induced psychoemtional
To investigate whether CRS affects the hair growth to influence stress, we monitored mice body weight gain and measured plasma
hair cycling in vivo, the onset and termination of anagen stage corticosterone concentration. Our results showed that CRS

Figure 3. Effects of CRS on mice body weight gain and plasma corticosterone concentrations. A: On day 4, 9, 14, and 19 after depilation,
body weight gain was significantly inhibited in CRS group compared with control group. B: The plasma corticosterone concentrations of mice for day
9 were shown on the left and the data for day 19 on the right. Data are presented as mean 6 SEM, n = 9 in each group, * P,0.05 compared with
control group.
doi:10.1371/journal.pone.0061574.g003

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 Role of Oxidative Stress in Hair Cycle

Figure 4. CRS induces skin tissue oxidative stress. A: TBARS levels in mice skin tissue on day 9 shown on the left and on day 19 shown on the
right. B: SOD activities in skin on day 9 shown on the left and on day 19 shown on the right. C: GSH-Px activities in skin on day 9 shown on the left
and on day 19 shown on the right. Data were presented as mean 6 SEM, n = 9 in each group, * P,0.05 compared with control group, { P,0.05
compared with CRS group.
doi:10.1371/journal.pone.0061574.g004

inhibited mice body weight gain significantly on day 4, 9, 14 and CRS Induces activation of SP-Mast Cell Pathway
19 compared with control group (Figure 3A). Plasma concentra- To confirm the CRS induces increase of SP expression in
tions of corticosterone on day 9 and day 19 after depilation both peripheral nerve fibers, SP and nerve fibers marker, MAP2 were
significantly increased in CRS group compared with control group co-immunostained in the dermis. On day 9 and day 19 after
(Figure 3B). These results confirmed that CRS indeed induced depilation, the number of SP+ nerve fibers was increased
psychoemotional stress. significantly in CRS mice than in control mice (Figure 5, 6).
Furthermore, to confirm CRS induces mast cell activation, we
CRS Increases TBARS Levels and Decreases SOD&GSH-Px performed mast cell specific staining, toluidine blue staining.
Activities in Skin Results showed that the ratio of degranulated mast cells also
The effects of CRS on oxidative stress in skin tissue were increased significantly in CRS mice compared with control mice
examined by measurements of skin tissue lipid peroxidation (Figure 7). These results suggested that CRS induced increase of
marker, TBARS levels. On day 9 and day 19 after depilation, peripheral nerve SP expression and inflammatory response which
TBARS levels in CRS group were significant increased compared is concordant with previous reports [4–5].
with control group (Figure 4A). Activities of SOD and GSH-Px in
CRS group were markedly decreased than control group in skin NK1 Receptor Antagonist Normalizes Most of CRS-
(Figure 4B, C). These results indicated that CRS induced increase Induced Alterations
of oxidative stress might be through reduction of antioxidant To investigate the possible mechanisms involved in CRS-
enzyme activity. induced alterations in mice skin, NK1 receptor antagonist
RP67580 was intraperitoneal injected in CRS mice. Although
RP67580 did not influence CRS-induced decrease of body weight

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 Role of Oxidative Stress in Hair Cycle

Figure 5. CRS increases amount of SP+ nerve fibers in dermis on day 9. A representative area of dermis in each group on day 9 after
depilation was shown. The nerve fiber was stained continuous green linear and SP+ nerve fiber was stained red. Original magnification was6500.
n = 6 in each group,
doi:10.1371/journal.pone.0061574.g005

gain and increase of plasma concentrations of corticosterone number (Figure 5, 6) and ratio in degranulated mast cells (Figure 7)
(Figure 3A, 3B), but RP67580 significantly ameliorated CRS- compared with CRS group on day 9 and day 19. These results
induced alterations in skin gray scale and hair follicle score both on clearly showed that ROS regulated CRS-induced alterations not
day 9 and day 19 (Figure 1 and Figure 2). In addition, only in the down-stream of SP-mast cell pathway, but also in the
administration of RP67580 significantly reduced CRS induced up-stream of SP-mast cell pathway (Figure 8).
increase of SP+ nerve fibers number (Figure 5, 6), and inhibited
increases of ratio in degranulated mast cells (Figure 7) in dermis Discussion
both on day 9 and day 19.
Furthermore, RP67580 also normalized CRS-induced increase One of the main findings of the previous studies was that
of TBARB levels and reduction of SOD and GSH-Px activities psychoemotional stress could inhibit hair growth in vivo through
(Figure 4A, B, C). These results suggested that CSR-induced SP-Mast cell pathway [4–5]. Using the mouse model of CRS, we
alterations were mediated by SP-NK1-mast cell-ROS pathway. firstly demonstrated the complicated role of ROS in regulation of
this pathway by two aspects. One is through SP induces mast cell
to generate ROS to exert pathological effects while the other is
Antioxidant Normalizes Most of CRS-Induced Alterations
through other possible ligands, such as glucoticoid, cortisol to
To investigate the possible role of ROS in CRS-induced SP-
stimulate ROS generation, thereafter regulates SP synthesis.
mast cell pathway activation, antioxidant Tempol was intraper-
Skin oxidative stress inhibited hair growth and delayed onset of
itoneal injected in CRS mice. Our results showed that Tempol
the anagen in mice hair cycle that induced by CRS, including
also did not affects CRS-induced decrease of body weight gain and
change of hair follicles morphology and skin color which were
increase of plasma concentration of corticosterone (Figure 3A, 3B),
reversed by administration of antioxidant Tempol, confirming the
but significantly ameliorated CRS-induced alterations in skin gray
undoubted role of oxidative stress in psychoemotional stress
scale and hair follicle score both on day 9 and day 19 (Figure 1 and
induced abnormal of hair growth (Figure 1 & Figrue 2). In order to
Figure 2). As a strong antioxidant, Tempol reasonable reduces
investigate the whole changes of hair cycling, we chose two time
CRS-induced increase of TBARB levels. Our results also showed points for observation. The first one is on day 9 after depilation
Tempol could increase CRS-induced reduction of GSH-Px while hair follicles of control mice enter into late anagen; The
activity but did not affect CRS-induced reduction of SOD activity second one is on day 19 after depilation while hair follicles of
(Figure 4A, B, C). Last but not the least, we found that Tempol control mice just enter catagen [9–10]. Compared withpreviously
significantly reduced CRS induced increases of SP+ nerve fibers studies, we find that CRS delays both anagen and catagen in first

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 Role of Oxidative Stress in Hair Cycle

Figure 6. CRS increases amount of SP+ nerve fibers in dermis on day 19. A representative area of dermis in each group on day 19 after
depilation was shown. The nerve fiber was stained continuous green linear and SP+ nerve fiber was stained red. Original magnification was6500.
n = 6 in each group.
doi:10.1371/journal.pone.0061574.g006

hair cycle after depilation which has been considered refractory to control mice. This indicated that CRS enhanced lipid peroxida-
systemic stress [11]. Furthermore, we do not observe the tion and production of ROS and inhibited the antioxidant system
premature catagen in CRS model compared with sonic stress for in skin. Moreover, the activity of SOD decreased significantly in
24 hours [4]. This discrepancy may be due to the different models mice treated with Tempol compared to control mice. This
used in the experiments. Our model started the stress exposure on phenomenon may be due to that Tempol is a SOD mimetic and
day 1 after depilation (thus at onset of anagen development) and there may be a competitive inhibition between exogenous Tempol
ended it on day 19 when catagen could be expected after anagen and endogenous SOD.
onset on day 1. This covers the entire time during which depilation In the present study, we chose C57BL/6 mice for animal model
induced hair cycle should take place. Previous studies started because this strain is an ideal model for hair follicle research.
expouse of stress in mice when that was in anagen VI [4] or in the Depilation induced anagen development and consecutive stages
telogen phase [11] and did than not enter anagen. This (catagen and telogen) were no significant difference with the
phenomenon indicated that: (1) hair cycle is likely to be more natural hair cycling in C57BL/6 mice [56]. Moreover, the
sensitive to restraint stress than foot shock and CRS may be an melanin of C57BL/6 mice only synthesized in the anagen lead to
appropriate model for hair cycling research. (2) Chronic and acute the skin color varies periodically. Therefore, we can roughly
stimulation may have different effects on hair cycling. (3) CRS determine the hair cycling by mouse skin color [57].
could not shorten but prolong the telogen stage and delay Unlike the other models which have been described for studies
subsequent anagen and catagen stage. of hair cycling constantly such as sonic stress for 24 hours [4–5] or
Recently, the role of oxidative stress in skin disorders has been foot shock for 2–4 weeks [11–12], we now used an animal model
characterized. Many pathogenesis of skin disease might be of chronic restraint stress (CRS) to research whether oxidative
attributed to ROS and oxidative stress, especially in hair aging stress is implicated in progression of hair loss. CRS is an emotional
and skin carcinogenesis [25,54–55]. We showed here for the first stress which contains more psychoemotional component and can
time that chronic psychoemotional stress causes oxidative stress in well simulated social psychological stressors by anxiety and fear in
mice dorsal skin by measurement of TBARS levels, SOD and animals and can also increase plasma corticosterone concentration
GSH-Px activity. The results showed that on day 9 and day 19 and inhibit body weight gain [33,38]. Meanwhile, we applied
after depilation, skin tissues of the stressed mice had high TBARS antioxidant to inhibit the effects of oxidative stress to observe if
levels and low activity of SOD and GSH-Px compared with

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 Role of Oxidative Stress in Hair Cycle

Figure 7. Effects of CRS on skin mast cell activation in dermis. A: A representative area of dermis in each group on day 9 and day 19 after
depilation was shown. Original magnificaiton was6400. Dark blue stained cells are mast cells. B: The degranulated mast cells in each group on day 9
were shown on the left and day 19 on the right. Data were presented as mean 6 SEM, n = 6 in each group, * P,0.05 compared with control group, {
P,0.05 compared with CRS group.
doi:10.1371/journal.pone.0061574.g007

antioxidants have any therapeutic effects in psychoemotional stress thereafter mediate SP expression, according to well established
induced pathological changes, focused on changes of hair cycle. stress-response theory, we reasonably speculate CRS increase
Similar with previous studies [4–5], we also observed that CRS stress-induced hormones through classic hypothalamic-pituitary-
increased amount of SP+ nerve fibers, degranulation ratio of mast adrenal (HPA) axis and these hormones could stimulate ROS
cells. Furthermore, we also observed CRS increased TBARS generation [58], thereafter to affect SP expression in peripheral
levels, decreased antioxidant enzyme activities. CRS induced nerves.
alterations can be reversed by NK1 receptor antagonist. These In addition to physical immobilization, psychoemotional stress
findings suggest that SP could change local tissue redox condition plays a significant part in CRS [59]. After chronic restraint,
by NK1 receptor which is widely expressed in mast cell, thereafter, whether on day 9 or day 19 after depilation compared with control
it is reasonable to speculate that CRS induces SP-NK1-ROS mice, the stressed mice body weight gain was obviously inhibited
pathway which may be similar with previous reported mechanisms and the concentration of corticosterone in the plasma was
of SP in nociceptive neurons [38]. In addition, we also observed significantly increased, which suggest that mice are under stressful
that antioxidant could affect SP expression, suggesting ROS conditions [11]. CRS increased plasma corticosterone concentra-
regulated CRS induced increase of SP expression. Although in the tion have been examined in the greatest detail and have been seem
present study we did not demonstrate how CRS increases ROS as an indicator to measure systemic stress response [34,59–60].

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 Role of Oxidative Stress in Hair Cycle

that CRS model is successfully established in our research.

Although administration of Tempol or RP 67580 could not reduce
plasma corticosterone concentration, suggesting that antioxidant
and SP receptor antagonist have few effects on systemic stress
responses, at least we explore its possible therapeutic effects in
CRS-induced hair loss. Limitation of the present study is CRS-
induced loss of body weight gain may also affect hair growth.
Recent clinical trials have confirmed that the activities of GSH-
Px and SOD were increased in scalp of patients with alopecia
areata [61]. Relatively, the activity of GSH-Px in plasma of
patients with alopecia was significantly decreased [62]. These
clinical evidences indicated that antioxidant enzyme system played
a critical role in the occurrence and development of alopecia. Our
study now reports the first experimental evidence that oxidative
stress involved in the abnormal of hair cycle as well as its role in
regulation of SP-Mast cell pathway. The present data provide a
convincing rationale for antioxidant application in clinical
treatment with psychoemotional stress induced hair growth
disorders.

Figure 8. Schematic diagram of oxidative stress affects hair Acknowledgments

follicles cycling induced by psychological stress. Oxidative stress
We thank Yong-ping Gu for his excellent technical assistance.
delay the anagen of hair cycle and activate SP-Mast cell pathway, and
SP also affects oxidative stress in skin.
doi:10.1371/journal.pone.0061574.g008 Author Contributions
Conceived and designed the experiments: WQF GXZ. Performed the
This indicated that psychoemotional factor, at least, plays a role in experiments: NL LHW LLG. Analyzed the data: GQW XPZ. Contributed
a series of changes of stressed mice, and the above evidence suggest reagents/materials/analysis tools: YJ JS KM JWC. Wrote the paper: GXZ.

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