Background: Dry skin induces antihistamine-resistant itch, as well as epidermal hyperinnervation, which is partly responsible for peripheral itch sensitization. In acute dry skin, topical application of emollients prevents the penetration of nerve fibers into the epidermis. However, the effects of emollients on itch and epidermal hyperinnervation in individuals with chronic dry skin are poorly understood. Objective: This study examined the effects of Tenshino-softgel TM (TSG) on itch-related behavior, epidermal hyperinnervation and skin barrier function in a chronic dry skin model mouse. Methods: Chronic dry skin was induced by application of acetone/ether (1:1) mixture and water (AEW) to the rostral parts of the back of hairless mice twice daily for six consecutive days. As treatment, TSG or, as control, Vaseline (V) was applied to the same areas twice daily. Skin barrier function was evaluated by measuring transepidermal water loss (TEWL) before each treatment. Scratching behavior was recorded and analyzed using a SCLABA<sup>®</sup> -real system, and skin samples were collected for immunohistochemical assays. Results: TEWL tended to be lower and scratching bouts fewer in AEW + TSG- than in AEW-treated mice. The numbers of protein gene product 9.5-immunoreactive fibers and substance P-immunoreactive fibers were each significantly lower in the epidermis of AEW + TSG- than of AEW-treated mice, but the expression of nerve growth factor in epidermis was similar in the three groups. Semaphorin 3A expression was significantly higher in the epidermis of AEW + TSG- than of AEW- and AEW + V-treated mice. Conclusion: Topical application of TSG may attenuate itch induced by chronic dry skin through a mechanism involving the inhibition of epidermal hyperinnervation.
Dry skin, as observed in patients with senile xerosis and atopic dermatitis, is a very common dermatologic problem frequently presenting with pruritus, defined as an unpleasant sensation and a desire to scratch frequently [
Tenshino-softgelTM (TSG) is a gel-like moisturizing lotion made by Ina Food Industry Co., Ltd. TSG contains water, glycerin, urea, methyl paraben, propyl paraben and agar. Agar is widely used as a food and gelling agent in Asian countries. Solutions of glycerol and/or urea in water are not sufficiently viscous, but the addition of agar was found to enhance the viscosity of TSG. Although agar may have moisturizing and/or anti-inflammatory actions [
Male HR-1 hairless mice (Hoshino Laboratory Animal Inc., Ibaragi, Japan), aged 10 weeks, were maintained under clean conditions, with a 12 h light: 12 h dark cycle at 22˚C - 24˚C and food and tap water provided ad libitum. Care and handling of these mice conformed to the NIH guidelines for animal research. All animal procedures were approved by the Institutional Animal Care and Use Committee at Juntendo University Graduate School of Medicine.
TSG was obtained from Ina Food Industry Co., Ltd. (Nagano, Japan), hydrophilic petrolatum (Vaseline, ointment base) from Maruishi Seiyaku Inc. (Osaka, Japan), optimal cutting temperature (O.C.T.) compound from Sakura Finetechnical Co., Ltd. (Tokyo, Japan), normal donkey serum (NDS) from Merck Millipore (Darmstadt, Germany), bovine serum albumin (BSA) from Sigma-Aldrich (St. Louis, MO, USA), Vectashield mounting medium with DAPI from Vector Laboratories Ltd. (Peterborough, UK), and sevoflurane from Abbott Japan (Osaka, Japan).
Primary antibodies used in this study included rabbit anti-protein gene product 9.5 (PGP 9.5, 1:400 dilution; Enzo Life Sciences Inc., Farmingdale, NY, USA), rat anti-substance P (SP, 1:100 dilution; Merck Millipore), rabbit anti-NGF (1:500 dilution; Merck Millipore), and rabbit anti-Sema3A (1:200 dilution; Abcam Inc., Cambridge, MA, UK). Secondary antibodies conjugated with Alexa Fluor dye (1:300 dilution) were purchased from Molecular Probes (Eugene, OR, USA).
Acetone/ether (1:1) mixture (AE), followed by water (AEW) was applied cutaneously as described [
Before each of the AEW treatment, TEWL of the treated area (the rostral parts of the back of mice) was measured under sevoflurane anesthesia using a Tewameter®TM210 (Courage & Khazawa, Cologne, Germany), as described [
Following the second treatment on the sixth day, the behavior of the mice was recorded using a SCLABA®- Real system (Noveltec Inc., Kobe, Japan), as described [
Skin from the dorsal neck of each mouse was taken under sevoflurane anesthesia on the seventh day. Half of each skin sample was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 4 hours. After washing with phosphate-buffered saline (PBS, pH7.4), small pieces of the skin were immersed in PBS containing 20% sucrose overnight at 4˚C. The skin specimens were embedded in O.C.T. compound, frozen on dry ice, and cut into cryosections (20 μm thick for PGP9.5 and SP staining or 8 μm thick for NGF) using a CM1850 cryostat (Leica Microsystems, Wetzlar, Germany). The sections were mounted onto silane-coated glass slides. After blocking in PBS containing 5% NDS, 2% BSA and 0.2% TritonX-100 (blocking solution), the sections were incubated with primary antibodies overnight at 4˚C. The next day, the sections were washed with PBS containing 0.05% Tween 20(PBS-T) and incubated with secondary antibodies for one hour at room temperature. After washing with PBS-T, the sections were mounted in Vectashield mounting medium with DAPI.
For immunofluorescence staining of Sema3A, the other half of each skin sample was embedded in O.C.T. compound without fixation, and cut into cryosections (8 μm thick) using a CM1850 cryostat, mounted onto silane-coated glass slides. The sections were fixed in ice-cold acetone for 10 minutes at −20˚C, rehydrated in PBS-T, blocked in blocking solution, and then incubated with anti-Sema3A antibody overnight at 4˚C. The next day, the sections were washed with PBS-T, incubated with secondary antibody for one hour at room temperature. After washing with PBS-T, the sections were mounted in Vectashield mounting medium with DAPI.
Six random fields per mouse were viewed with a confocal laser-scanning microscope (DMIRE2; Leica Microsystems), with optical sections 0.9 μm thick scanned through the z-plane of the stained specimens (thickness 20 μm). Three-dimensional images were reconstructed using Leica Confocal Software (Leica Microsystems). The numbers of nerve fibers penetrating into the epidermis and intraepidermal nerve fibers were hand-counted separately. The average number of six observed fields per mouse was calculated and used for statistical analysis.
Six random fields per mouse were observed with a confocal laser-scanning microscope, with exposure and acquisition settings such that no signal saturation occurred. The sum of the fluorescence intensity of the epidermis and the area of the epidermis in each observed field was measured using Leica Confocal Software. Fluorescence intensity per unit area was calculated and used for statistical analysis.
Data were analyzed using Prism 5 (Graph Pad software Inc., La Jolla, CA, USA). Statistical analyses were performed by analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test, with P < 0.05 regarded as statistically significant.
Repeated AEW treatment induced the symptoms of dry skin, such as scaling and deep wrinkles (
TEWL tended to be lower in AEW + TSG-treated mice, becoming significantly lower in AEW + TSG-treated than in AEW-treated mice on the fourth day (
The density of PGP9.5-immunoreactive (PGP9.5+) fibers in epidermis was examined immunohistochemically in each group using confocal microscopy (
of SP-immunoreactive (SP+) fibers in the epidermis (
Immunohistochemical examination for the effects of TSG on epidermal NGF and Sema3A expression showed that the expression level of NGF in the epidermis was similar in mice treated with AEW, AEW + V, and AEW + TSG (
This study showed that topical application of TSG significantly reduced the densities of PGP9.5+- and SP+-epi- dermal nerve fibers in a mouse model of chronic dry skin (
In this study, emollients were applied immediately after skin barrier disruption by AEW treatment. Application of emollients such as heparinoid cream to mice with acute dry skin resulted in greater improvements in epidermal nerve fiber density and epidermal NGF levels, but had no effect on epidermal Sema3A levels [
This study showed that repeated AEW treatment elicited spontaneous scratching (
the mechanisms are unclear, scratching behavior in mast cell-deficient mice may be caused, at least in part, by increases in epidermal nerve fibers or pruritogens secreted by other dermal cells and/or keratinocytes [
In conclusion, these findings suggest that topical application of TSG restrains the progression of barrier disruption and improves the imbalance of axon guidance molecule by increasing the expression of Sema3A, possibly resulting in a decrease in epidermal nerve fiber density. TSG may therefore be useful as antipruritic therapy in patients with dry skin-based skin diseases.
We thank Ina Food Industry Co., Ltd. for providing TSG and valuable scientific support. This work was partly supported by a grant Strategic Research Foundation Grant-aided Project for Private Universities from MEXT (Grant number S1311011).
The authors state no conflict of interest.
AtsushiNoguchi,MitsutoshiTominaga,Kyi ChanKo,HironoriMatsuda,YasushiSuga,HideokiOgawa,KenjiTakamori, (2015) Topical Application of Tenshino-SoftgelTM Reduces Epidermal Nerve Fiber Density in a Chronic Dry Skin Model Mouse. Journal of Cosmetics, Dermatological Sciences and Applications,05,254-261. doi: 10.4236/jcdsa.2015.54031