Article
Hypo-osmotic Shock-Induced Subclinical Inflammation of Skin in a Rat Model of Disrupted Skin Barrier Function
Biological Research for Nursing 2015, Vol. 17(2) 135-141 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1099800414532827 brn.sagepub.com
Chihiro Kishi, MHS1, Takeo Minematsu, PhD1, Lijuan Huang, PhD1, Yuko Mugita, MHS1, Aya Kitamura, BHS1, Gojiro Nakagami, PhD1, Takumi Yamane, PhD1, Mikako Yoshida, PhD2, Hiroshi Noguchi, PhD2, Megumi Funakubo, PhD1, Taketoshi Mori, PhD2, and Hiromi Sanada, PhD1
Abstract Aging disrupts skin barrier function and induces xerosis accompanied by pruritus. In many cases, elderly patients complain of pruritus during skin hygiene care, a condition called aquagenic pruritus of the elderly (APE). To date, the pathophysiology and mechanism of action of APE have not been elucidated. We conducted the present study to test the hypothesis that hypoosmotic shock of epidermal cells induces skin inflammation and elongation of C-fibers by nerve growth factor b (NGFb) as a basic mechanism of APE. The dorsal skin of HWY rats, which are a model for disrupted skin barrier function, was treated with distilled water (hypotonic treatment [Hypo] group) or normal saline (isotonic treatment [Iso] group) by applying soaked gauze for 7 days. Untreated rats were used as a control (no-treatment [NT] group). Histochemical and immunohistochemical analyses revealed inflammatory responses in the epidermis and the dermal papillary layer in the Hypo group, while no alterations were observed in the Iso or NT groups. Induction of expression and secretion of NGFb and elongation of C-fibers into the epidermis were found in the Hypo group. In contrast, secretion of NGFb was significantly lower and elongation of C-fibers was not observed in the Iso group. These results suggest that hypo-osmotic shock–induced inflammatory reactions promote hypersensitivity to pruritus in skin with disrupted barrier function. Keywords elderly, hygiene care, hypo-osmotic shock, inflammation, skin barrier function The skin is the largest organ of the human body and forms the boundary between the body and its surroundings. Skin barrier function plays one of the most important roles in protection of the human body against pathogenic bacteria, chemical irritants, and physical stimulants (Nix, 2000). The skin barrier consists of intercellular lipid–lamellar structures and the acidic status of the skin (Berger & Steinhoff, 2011; Landmann, 1988). Many researchers have reported structural and functional alterations in the skin of older people (Berger & Steinhoff, 2011; Luebberding, Krueger, & Kerscher, 2013; Xia et al., 2013). In particular, aging disrupts the barrier function via, for example, reduced cellular activity of keratinocytes for producing intercellular lipids (Finch, 2003; Penzer & Finch, 2001); failure of the intercellular lipid–lamellar structure (Ramos-eSilva, Boza, & Cestari, 2012; Seyfarth, Shliemann, Antonov, & Elsner, 2011); abnormal keratinization (Farage & Miller, 2007; White-Cho & Reddy, 2011); and expansion of the interstitial layer of basal cells, prickle cells, and corneum (Minematsu et al., 2011). These alterations induce xerosis in older people (White-Cho & Reddy, 2011).
Researchers have reported the prevalence of xerosis in elderly nursing home residents to be 37.5% (Smith, Atkinson, Guo, & Yamagata, 2002). Most cases of xerosis in the elderly are accompanied by pruritus called senile pruritus (Finch, 2003). Norman (2003) reported that 1,002 of the 1,556 elderly residents in a nursing home in the United States had senile pruritus. Similarly, 41% of elderly patients suffered from pruritus in a dermatology outpatient ward in Thailand (Thaipisuttikul, 1
Department of Gerontological Nursing/Wound Care Management, Division of Health Sciences and Nursing, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 2 Department of Life Support Technology (Molten), Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Corresponding Author: Hiromi Sanada, PhD, Department of Gerontological Nursing/Wound Care Management, Division of Health Sciences and Nursing, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Email: [email protected]
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1998). Senile pruritus is a serious problem that threatens the well-being of older people owing to the resultant insomnia and restriction of activity (Fagerstro¨m & Hellstro¨m, 2011; Murota et al., 2010). It can also induce scratching behavior that results in secondary conditions such as additional inflammation and excoriation (Penzer & Finch, 2001). Pruritus frequently occurs when patients take a bath, sleep, or change their clothes (Yosipovitch & Hundley, 2004). In particular, nurses commonly report that patients complain of pruritus when they are undergoing skin hygiene care (Dyble & Ashton, 2011). Skin hygiene is an important part of nursing care not only to remove sweat, extra sebum, scum, and dirt but also to invigorate patients. Previous studies have shown elevated expression and secretion of nerve growth factor b (NGFb) in keratinocytes in xerosis (Tominaga, Ozawa, Tengara, Ogawa, & Takamori, 2007). NGFb induces elongation and invasion of nerve C-fibers into the epidermis in xerosis, which results in hypersensitivity for pruritus. Elevation of body temperature, mechanical stimulation with body brushes or towels, and residual shampoo and soap are factors that can induce pruritus during hygiene care (Farage, Miller, Berardesca, & Maibach, 2009). Furthermore, water exposure itself can induce pruritus in elderly patients, a condition that is called aquagenic pruritus of the elderly (APE). The pathophysiology and mechanism of action of APE have not been fully elucidated, although some investigators have reported elevated activity of histamines, neuropeptides, and acetylcholinesterase in aquagenic pruritus due to polycythemia vera (Bircher & Meier-Ruge, 1988; Sa´nchez-Carpintero & Espan˜a-Alonso, 1997; Steinman et al., 1987). Water used in skin hygiene care is a hypotonic solution. Cells are covered with a plasma membrane, which is a semipermeable phospholipid bilayer, and solutions move between inside and outside of the cell in response to the difference in osmotic pressure (Bangham, 1968). When cells are exposed to a hypotonic solution, immediate inflow of the solution occurs. This inflow results in cell swelling, cytoskeletal transformation, and finally cell disruption (D’Alessandro, Russell, Morley, Davis, & Lane, 2002), a series of events known as hypo-osmotic shock. Minematsu et al. (2011) have demonstrated that aging enhances transdermal penetration of fluorescein diluted with normal saline, suggesting that keratinocytes can easily be exposed to hypotonic solutions during hygiene care in aged individuals. In the present study, we hypothesized that contact with hypotonic solutions would induce skin inflammation and elongation of C-fibers into the epidermis due to hypo-osmotic shock in a rat model of disrupted skin barrier function. We used this rat model rather than aged rats because skin aging includes several structural and functional alterations.
Materials and Methods Animals We individually maintained 6-week-old male Hairless Wistar Yagi (HWY) rats (Japan SLC, Shizuoka, Japan) in a
temperature- and humidity-controlled room (23 + 2 C, 45 + 10%) on a 12-hr/12-hr light/dark cycle. We provided standard laboratory diet and filtered water ad libitum. The Animal Ethics Committee of the University of Tokyo approved the experimental protocol, and we treated all animals according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. HWY rats are a mutational line that originated from Wistar rats, which show normal skin histology except for hair follicle dysplasia. In preliminary tests, we compared transepidermal water loss (TEWL), which is a major parameter for skin barrier function (Hodgkinson & Nay, 2005; Nix, 2000), as measured with VapoMeter (Delfin Technologies, Kupio, Finland), between HWY and control (Wistar) rats (six animals per group). The average TEWL in HWY rats (13.14 + 0.95 g/ m2h) was significantly higher than that in the controls (9.21 + 0.93 g/m2h; p ¼ .006). Therefore, we used HWY rats as a model for disrupted skin barrier function.
Experimental Design and Treatments We randomly assigned 18 HWY rats to the hypotonic treatment (Hypo), isotonic treatment (Iso), or no-treatment (NT) group. Rats were gently held in the researcher’s arm during treatment, which was conducted without anesthesia. The dorsal skin was treated with distilled water (Hypo group) or normal saline (Iso group) via application of gauze soaked in these solutions for 1 min. For the NT group, the rats were simply held for 1 min without anesthesia. Treatments were performed once daily (at 16:00 hr) and repeated for 7 days. The effects of treatment were evaluated and compared among the groups by measuring the infiltration of inflammatory cells, expression and secretion of NGFb, and elongation of C-fibers into the epidermis. On Day 8, the treatment regions were macroscopically observed.
Sample Collection On Day 8, we performed skin blotting on the treated area using the method described subsequently. Following euthanasia by overdose application of pentobarbital sodium (Somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan), treated skin tissue was harvested. Skin tissue was divided into two pieces: One was fixed in 4% paraformaldehyde diluted with 0.2 M phosphate buffer for histological analysis and the other was frozen in liquid nitrogen for Western blotting.
Skin Blotting Secretion of NGFb was measured by skin blotting. NGFb is a glycoprotein that consists of 120 amino acids and forms a homodimer. Expression and secretion of NGFb are elevated in the epidermis under inflammatory conditions, whereas they are remarkably low in normal tissue (Wei et al., 2012). Skin blotting was performed as described previously (Minematsu et al., 2014). An 8-mm disk of nitrocellulose membrane was wetted with 2-μl saline and attached to the surface of treated
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skin using plastic tape for 10 min. We stored the collected membranes at 4 C until immunostaining. For immunostaining, membranes were hydrated with phosphate-buffered saline, blocked with blotting solution (Blocking One; Nacalai Tesque, Kyoto, Japan) at room temperature overnight and reacted with rabbit anti-NGFb primary antibody (Abcam, Cambridge, UK; diluted at 1:1000) and horseradish peroxidase (HRP)–conjugated anti-rabbit immunoglobulin secondary antibody (Thermo Fisher Scientific, Waltham, MA; diluted at 1:1000) at room temperature for 30 min each. We visualized the immunoreactivity by incubating with chemiluminescence substrate (Luminata Forte, Merck Millipore, Billerica, MA) and recording with the chemiluminescence imaging system (LumiCube; Liponics, Tokyo, Japan). Intensity of immunoreactivity was measured using the image analysis software ImageJ (National Institutes of Health, Bethesda, MD).
Histological Analysis Fixed skin tissue was divided into two pieces, and paraffin sections of 5-μm thick and frozen sections of 20-μm thick were prepared. For histological observation, we stained the paraffin sections with Mayer’s hematoxylin and eosin (HE; Muto Pure Chemical, Tokyo, Japan). Immunohistochemistry for major histocompatibility complex (MHC)-II and NGFb consisted of the following steps: inactivation of endogenous peroxidase activity by incubating with 0.3% hydrogen peroxide in methanol for 30 min; reaction with primary rabbit anti-MHC-II antibodies (Thermo Fisher Scientific; diluted at 1:200) and rabbit anti-NGFb (diluted at 1:1000) at 4 C for 12 hr; reaction with biotin-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; diluted at 1:1000) at 4 C for 12 hr; formation of the HRP-conjugated avidin–biotin complex (VectaStain ABC Kit, Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions; and visualization with 3,30 diaminobenzidine tetrahydrochloride substrate (Wako Pure Chemical Industries, Osaka, Japan). Finally, we performed counterstaining with Mayer’s hematoxylin. The frozen sections were used for immunofluorescent staining for protein gene product (PGP)9.5 (Jankowski, Rau, Soneji, Anderson, & Koerber, 2010). Sections were blocked at room temperature for 30 min; incubated with rabbit anti-PGP9.5 (GeneTex, Irvine, CA; diluted at 1:100) and DyLight-488conjugated anti-rabbit IgG antibody (Rockland Immunochemicals, Gilbertsville, PA; diluted at 1:1000) at 4 C for 12 hr; and embedded with Fluoromount-G with 40 ,6-diamidino-2phenylindole (Southern Biotech, Birmingham, AL). Fluorescent inverted microscopy (BIOREVO BZ-9000; Keyence, Osaka, Japan) was used for observation of the sections.
Western Blotting We exfoliated the epidermal layer from frozen skin samples and homogenized it in sample buffer that consisted of 2% (w/v) sodium dodecyl sulfate, 50 mM Tris–HCl, 5% (v/v) 2-mercapto
sodium, and 2.5% (w/v) sucrose. After centrifugation (6,000 rpm), 100 μl of supernatant were used. Following supplementation with 0.1% bromophenol blue, the samples were heated at 95 C for 10 min. After separation of 10 μl per lane of sample through 15% polyacrylamide gel electrophoresis (200 V, 40 min), it was transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA) using a semidry blotting method (30 V, 90 min). NGFb was detected using the skin blotting method described previously. b-Actin was immunologically stained with an indirect method using rabbit anti-b-actin (GeneTex; diluted at 1:200).
Statistical Analysis Values are presented as means + SD. Statistical differences among the three groups were examined using the Tukey’s multiple comparison test, with p < .05 considered statistically significant. Statistical analysis was performed with Statistical Package for the Social Sciences v. 20 (IBM, Armonk, NY).
Results Inflammatory Status of Treated Skin We did not observe macroscopic changes in all the groups. HE staining showed normal histology of the epidermis, which consisted of layers of flattened granular cells, prickle cells, and basal cells, in the NT group (Figure 1A). In the Hypo group, we observed marked swelling of the granular and prickle cells, disrupted basal layer, and infiltration of inflammatory cells into the dermal papillary layer (Figure 1B). In contrast, there was no histological alteration in the Iso group (Figure 1C). Immunohistological staining for MHC-II, which is a marker for macrophages, dendritic cells, and Langerhans cells, revealed infiltration of MHC-II-positive cells in the epidermis and dermal papillary layer in the Hypo group, whereas there were few positive cells in the NT and Iso groups (Figure 2). These results indicate the subclinical inflammatory status of the skin in the Hypo group.
Expression and Secretion of NGFb NGFb is a cytokine that is secreted by keratinocytes under inflammatory conditions and induces nerve fiber growth. We identified expression of NGFb in the Hypo and Iso groups by Western blotting, but NT skin was completely negative for NGFb (Figure 3A). Immunohistochemistry showed NGFb-positive secretory granules in clusters of basal cells of the epidermis in the Hypo group. In contrast, there were few NGFb-positive cells in the epidermis of the NT and Iso groups (Figure 3B). Epidermal secretion of NGFb was detected by skin blotting (Figure 3C) and compared among experimental groups following quantification of the intensity of immunoreactivity for NGFb (Figure 3D). The intensity of signals in the Hypo group (16,970.42 + 7,442.28) was significantly higher than that in the NT (3,978.84 + 1,857.57, p ¼ .017) and Iso groups (4,845.26 + 3,641.92, p ¼ .032), whereas there was no significant difference between the NT and Iso groups (p ¼ 1.000).
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Figure 1. Hypo-osmotic treatment–induced histological changes revealed by hematoxylin and eosin staining. We compared the histology of dorsal skin in rats with (A) no treatment (NT) and those treated with (B) distilled water (Hypo) and (C) normal saline (Iso). The Hypo group showed disrupted alignment of the basal cell layer, swelling of granular and prickle cells, and infiltration of inflammatory cells into the dermal papillary layer (arrowheads). In the Iso group, there was no histological change relative to the NT group. Note. e ¼ epidermis, d ¼ dermis, bar ¼ 50 μm.
Figure 2. Hypo-osmotic treatment–induced infiltration of major histocompatibility complex (MHC)-II-positive inflammatory cells. We observed immunoreactivity for MHC-II, which is a marker for macrophages, dendritic cells, and Langerhans cells, in (B) intact untreated dorsal skin (no treatment [NT]), (B) skin treated with hypo-osmotic solution (Hypo), and (C) skin treated with isotonic solution (Iso). There were many MHC-II-positive cells (arrows) in the epidermis and dermal papillary layer in the Hypo group, whereas there were few positive cells in the NT and Iso groups. Note. e ¼ epidermis, d ¼ dermis, bar ¼ 50 μm.
Elongation of C-Fibers into the Epidermis C-fibers reach just below the basement membrane under normal conditions. However, secreted NGFb from epidermal keratinocytes under inflammatory conditions leads to elongation and invasion of C-fibers into the epidermis through the basement membrane. We identified the C-fibers by immunofluorescence for PGP9.5 (Figure 4). PGP9.5-positive nerve fibers were abundant in the dermal papillary layer in all groups, but
we observed invasion of elongated C-fibers only in the Hypo group (Figure 4A–C).
Discussion In this study, we demonstrated that contact with a hypotonic solution induced inflammation and elongation of C-fibers into the epidermis due to hypo-osmotic shock in the skin of HWY
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Figure 3. Acceleration of expression and secretion of nerve growth factor (NGF)b in the epidermis induced by hypo-osmotic shock. (A) Expression of NGFb shown by Western blotting. (B) Distribution of NGFb-positive cells (arrowheads) shown by immunohistochemistry. (C) Secretion of NGFb in the treatment area of the dorsal skin shown by skin blotting. (D) Skin blotting signals (mean + SD) for NGFb. *p < .05. The results show increased expression and secretion of NGFb in the Hypo group. In contrast, secretion of NGFb in the Iso group was significantly lower than that in the Hypo group and similar to that in the NT group, although the induction of a relatively weaker expression of NGFb was detected in the Iso group. Note. NT ¼ no-treatment group; Hypo ¼ group treated with a hypo-osmotic solution; Iso ¼ group treated with an isotonic solution; e ¼ epidermis; d ¼ dermis; sb ¼ stratum basale epidermis; bar ¼ 50 μm (left panels in 3B), 10 μm (right panels in 3B).
rats in which skin barrier function was disrupted. Moreover, contact with isotonic saline induced only slight inflammation and did not result in the elongation of C-fibers in HWY rat skin. Preliminary testing of HWY rats showed failure of dorsal skin barrier function. Upon histological examination, we did not observe changes indicative of aging skin, such as atrophy of collagen and preinflammatory lesions. HWY rats thus showed a decline in skin barrier function, alone, so this model was well suited for our purpose. In the Hypo group, we observed marked swelling of granular and prickle cells by HE staining. We also observed infiltration of inflammatory cells into the epidermis and dermal papillary layer by HE staining and immunohistochemistry for MHC-II, without any macroscopic alteration, suggesting subclinical inflammatory status similar to that of dermatitis. Moreover, we saw increased expression and secretion of NGFb in the Hypo group. In contrast, we did not observe these inflammatory reactions histologically in the Iso group. We did, however, also detect expression of NGFb in the Iso group, which was probably due to friction or maceration by the treatmentaffected skin. Secretion of NGFb in the Iso group was significantly lower than that in the Hypo group, and we did not observe elongation of C-fibers into the epidermis. These results suggest that hypo-osmotic shock is a factor in induction of the inflammatory reaction and elongation of C-fibers in the epidermis. Researchers reported similar histological changes and increased scratching behavior in a mouse model of pruritus in which shaved dorsal skin was treated with acetone/ether followed by water (Miyamoto, Nojima, Shinkado, Nakahashi, & Kuraishi, 2002; Okawa et al., 2012; Tominaga et al., 2007). Future research is needed to reveal hypo-osmotic shock– induced pruritus by the observation of scratching behavior in HWY rats. Nurses provide hygiene care to keep patients clean and refreshed. However, our results suggest that the usual hygiene care using hypotonic solutions might induce pruritus in elderly patients. At present, nurses try to improve xerosis by using bath salts, including natural moisturizing factors such as ceramide, urea, and Vaseline. Our results propose an additional improvement in hygiene care through use of osmolality-regulated bath water. Future research is needed to determine the appropriate osmolality range of bath water. The present study had some limitations. The mutation profile of genes has not been fully elucidated in HWY rats; therefore, further research is required to generalize our findings to normal rats and humans. We used HWY rats in order to reveal hypo-osmotic shock–induced skin inflammation in the presence of disrupted skin barrier function. In future research, we plan to use aged animals to explore the association between hypo-osmotic shock and APE because aging leads to several other structural and functional changes in the skin in addition to decreased barrier function. In conclusion, our findings confirm that hypotonic solutions induce inflammation by hypo-osmotic shock in skin with disrupted barrier function. Further, we found that an isotonic solution does not induce skin inflammation in comparison with a
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Figure 4. Hypo-osmotic treatment–induced elongation of C-fibers into the epidermis. We observed the distribution of C-fibers by immunofluorescence staining for PGP9.5 (A, D, and G) with DAPI counterstaining (B, E, and H) in 20-μm thick frozen sections of the NT (A–C), Hypo (D–F), and Iso (G–I) groups. We found C-fibers only in the epidermis of the Hypo group, although abundantly in the dermal papillary layer in all groups. Note. NT ¼ no-treatment group; Hypo ¼ group treated with a hypo-osmotic solution; Iso ¼ group treated with an isotonic solution; Arrowheads ¼ c-fibers in the dermal papillary layer; Arrows ¼ elongated c-fibers into epidermis; e ¼ epidermis; d ¼ dermis; bar ¼ 200 μm.
hypotonic solution. These findings may lead to improvements in hygiene care in elderly patients. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: A part of this study was supported by the Tokyo Association of Medical Sciences.
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Hypo-osmotic Shock-Induced Subclinical Inflammation of Skin in a Rat Model of Disrupted Skin Barrier Function
Biological Research for Nursing 2015, Vol. 17(2) 135-141 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1099800414532827 brn.sagepub.com
Chihiro Kishi, MHS1, Takeo Minematsu, PhD1, Lijuan Huang, PhD1, Yuko Mugita, MHS1, Aya Kitamura, BHS1, Gojiro Nakagami, PhD1, Takumi Yamane, PhD1, Mikako Yoshida, PhD2, Hiroshi Noguchi, PhD2, Megumi Funakubo, PhD1, Taketoshi Mori, PhD2, and Hiromi Sanada, PhD1
Abstract Aging disrupts skin barrier function and induces xerosis accompanied by pruritus. In many cases, elderly patients complain of pruritus during skin hygiene care, a condition called aquagenic pruritus of the elderly (APE). To date, the pathophysiology and mechanism of action of APE have not been elucidated. We conducted the present study to test the hypothesis that hypoosmotic shock of epidermal cells induces skin inflammation and elongation of C-fibers by nerve growth factor b (NGFb) as a basic mechanism of APE. The dorsal skin of HWY rats, which are a model for disrupted skin barrier function, was treated with distilled water (hypotonic treatment [Hypo] group) or normal saline (isotonic treatment [Iso] group) by applying soaked gauze for 7 days. Untreated rats were used as a control (no-treatment [NT] group). Histochemical and immunohistochemical analyses revealed inflammatory responses in the epidermis and the dermal papillary layer in the Hypo group, while no alterations were observed in the Iso or NT groups. Induction of expression and secretion of NGFb and elongation of C-fibers into the epidermis were found in the Hypo group. In contrast, secretion of NGFb was significantly lower and elongation of C-fibers was not observed in the Iso group. These results suggest that hypo-osmotic shock–induced inflammatory reactions promote hypersensitivity to pruritus in skin with disrupted barrier function. Keywords elderly, hygiene care, hypo-osmotic shock, inflammation, skin barrier function The skin is the largest organ of the human body and forms the boundary between the body and its surroundings. Skin barrier function plays one of the most important roles in protection of the human body against pathogenic bacteria, chemical irritants, and physical stimulants (Nix, 2000). The skin barrier consists of intercellular lipid–lamellar structures and the acidic status of the skin (Berger & Steinhoff, 2011; Landmann, 1988). Many researchers have reported structural and functional alterations in the skin of older people (Berger & Steinhoff, 2011; Luebberding, Krueger, & Kerscher, 2013; Xia et al., 2013). In particular, aging disrupts the barrier function via, for example, reduced cellular activity of keratinocytes for producing intercellular lipids (Finch, 2003; Penzer & Finch, 2001); failure of the intercellular lipid–lamellar structure (Ramos-eSilva, Boza, & Cestari, 2012; Seyfarth, Shliemann, Antonov, & Elsner, 2011); abnormal keratinization (Farage & Miller, 2007; White-Cho & Reddy, 2011); and expansion of the interstitial layer of basal cells, prickle cells, and corneum (Minematsu et al., 2011). These alterations induce xerosis in older people (White-Cho & Reddy, 2011).
Researchers have reported the prevalence of xerosis in elderly nursing home residents to be 37.5% (Smith, Atkinson, Guo, & Yamagata, 2002). Most cases of xerosis in the elderly are accompanied by pruritus called senile pruritus (Finch, 2003). Norman (2003) reported that 1,002 of the 1,556 elderly residents in a nursing home in the United States had senile pruritus. Similarly, 41% of elderly patients suffered from pruritus in a dermatology outpatient ward in Thailand (Thaipisuttikul, 1
Department of Gerontological Nursing/Wound Care Management, Division of Health Sciences and Nursing, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 2 Department of Life Support Technology (Molten), Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Corresponding Author: Hiromi Sanada, PhD, Department of Gerontological Nursing/Wound Care Management, Division of Health Sciences and Nursing, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Email: [email protected]
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1998). Senile pruritus is a serious problem that threatens the well-being of older people owing to the resultant insomnia and restriction of activity (Fagerstro¨m & Hellstro¨m, 2011; Murota et al., 2010). It can also induce scratching behavior that results in secondary conditions such as additional inflammation and excoriation (Penzer & Finch, 2001). Pruritus frequently occurs when patients take a bath, sleep, or change their clothes (Yosipovitch & Hundley, 2004). In particular, nurses commonly report that patients complain of pruritus when they are undergoing skin hygiene care (Dyble & Ashton, 2011). Skin hygiene is an important part of nursing care not only to remove sweat, extra sebum, scum, and dirt but also to invigorate patients. Previous studies have shown elevated expression and secretion of nerve growth factor b (NGFb) in keratinocytes in xerosis (Tominaga, Ozawa, Tengara, Ogawa, & Takamori, 2007). NGFb induces elongation and invasion of nerve C-fibers into the epidermis in xerosis, which results in hypersensitivity for pruritus. Elevation of body temperature, mechanical stimulation with body brushes or towels, and residual shampoo and soap are factors that can induce pruritus during hygiene care (Farage, Miller, Berardesca, & Maibach, 2009). Furthermore, water exposure itself can induce pruritus in elderly patients, a condition that is called aquagenic pruritus of the elderly (APE). The pathophysiology and mechanism of action of APE have not been fully elucidated, although some investigators have reported elevated activity of histamines, neuropeptides, and acetylcholinesterase in aquagenic pruritus due to polycythemia vera (Bircher & Meier-Ruge, 1988; Sa´nchez-Carpintero & Espan˜a-Alonso, 1997; Steinman et al., 1987). Water used in skin hygiene care is a hypotonic solution. Cells are covered with a plasma membrane, which is a semipermeable phospholipid bilayer, and solutions move between inside and outside of the cell in response to the difference in osmotic pressure (Bangham, 1968). When cells are exposed to a hypotonic solution, immediate inflow of the solution occurs. This inflow results in cell swelling, cytoskeletal transformation, and finally cell disruption (D’Alessandro, Russell, Morley, Davis, & Lane, 2002), a series of events known as hypo-osmotic shock. Minematsu et al. (2011) have demonstrated that aging enhances transdermal penetration of fluorescein diluted with normal saline, suggesting that keratinocytes can easily be exposed to hypotonic solutions during hygiene care in aged individuals. In the present study, we hypothesized that contact with hypotonic solutions would induce skin inflammation and elongation of C-fibers into the epidermis due to hypo-osmotic shock in a rat model of disrupted skin barrier function. We used this rat model rather than aged rats because skin aging includes several structural and functional alterations.
Materials and Methods Animals We individually maintained 6-week-old male Hairless Wistar Yagi (HWY) rats (Japan SLC, Shizuoka, Japan) in a
temperature- and humidity-controlled room (23 + 2 C, 45 + 10%) on a 12-hr/12-hr light/dark cycle. We provided standard laboratory diet and filtered water ad libitum. The Animal Ethics Committee of the University of Tokyo approved the experimental protocol, and we treated all animals according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. HWY rats are a mutational line that originated from Wistar rats, which show normal skin histology except for hair follicle dysplasia. In preliminary tests, we compared transepidermal water loss (TEWL), which is a major parameter for skin barrier function (Hodgkinson & Nay, 2005; Nix, 2000), as measured with VapoMeter (Delfin Technologies, Kupio, Finland), between HWY and control (Wistar) rats (six animals per group). The average TEWL in HWY rats (13.14 + 0.95 g/ m2h) was significantly higher than that in the controls (9.21 + 0.93 g/m2h; p ¼ .006). Therefore, we used HWY rats as a model for disrupted skin barrier function.
Experimental Design and Treatments We randomly assigned 18 HWY rats to the hypotonic treatment (Hypo), isotonic treatment (Iso), or no-treatment (NT) group. Rats were gently held in the researcher’s arm during treatment, which was conducted without anesthesia. The dorsal skin was treated with distilled water (Hypo group) or normal saline (Iso group) via application of gauze soaked in these solutions for 1 min. For the NT group, the rats were simply held for 1 min without anesthesia. Treatments were performed once daily (at 16:00 hr) and repeated for 7 days. The effects of treatment were evaluated and compared among the groups by measuring the infiltration of inflammatory cells, expression and secretion of NGFb, and elongation of C-fibers into the epidermis. On Day 8, the treatment regions were macroscopically observed.
Sample Collection On Day 8, we performed skin blotting on the treated area using the method described subsequently. Following euthanasia by overdose application of pentobarbital sodium (Somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan), treated skin tissue was harvested. Skin tissue was divided into two pieces: One was fixed in 4% paraformaldehyde diluted with 0.2 M phosphate buffer for histological analysis and the other was frozen in liquid nitrogen for Western blotting.
Skin Blotting Secretion of NGFb was measured by skin blotting. NGFb is a glycoprotein that consists of 120 amino acids and forms a homodimer. Expression and secretion of NGFb are elevated in the epidermis under inflammatory conditions, whereas they are remarkably low in normal tissue (Wei et al., 2012). Skin blotting was performed as described previously (Minematsu et al., 2014). An 8-mm disk of nitrocellulose membrane was wetted with 2-μl saline and attached to the surface of treated
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skin using plastic tape for 10 min. We stored the collected membranes at 4 C until immunostaining. For immunostaining, membranes were hydrated with phosphate-buffered saline, blocked with blotting solution (Blocking One; Nacalai Tesque, Kyoto, Japan) at room temperature overnight and reacted with rabbit anti-NGFb primary antibody (Abcam, Cambridge, UK; diluted at 1:1000) and horseradish peroxidase (HRP)–conjugated anti-rabbit immunoglobulin secondary antibody (Thermo Fisher Scientific, Waltham, MA; diluted at 1:1000) at room temperature for 30 min each. We visualized the immunoreactivity by incubating with chemiluminescence substrate (Luminata Forte, Merck Millipore, Billerica, MA) and recording with the chemiluminescence imaging system (LumiCube; Liponics, Tokyo, Japan). Intensity of immunoreactivity was measured using the image analysis software ImageJ (National Institutes of Health, Bethesda, MD).
Histological Analysis Fixed skin tissue was divided into two pieces, and paraffin sections of 5-μm thick and frozen sections of 20-μm thick were prepared. For histological observation, we stained the paraffin sections with Mayer’s hematoxylin and eosin (HE; Muto Pure Chemical, Tokyo, Japan). Immunohistochemistry for major histocompatibility complex (MHC)-II and NGFb consisted of the following steps: inactivation of endogenous peroxidase activity by incubating with 0.3% hydrogen peroxide in methanol for 30 min; reaction with primary rabbit anti-MHC-II antibodies (Thermo Fisher Scientific; diluted at 1:200) and rabbit anti-NGFb (diluted at 1:1000) at 4 C for 12 hr; reaction with biotin-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; diluted at 1:1000) at 4 C for 12 hr; formation of the HRP-conjugated avidin–biotin complex (VectaStain ABC Kit, Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions; and visualization with 3,30 diaminobenzidine tetrahydrochloride substrate (Wako Pure Chemical Industries, Osaka, Japan). Finally, we performed counterstaining with Mayer’s hematoxylin. The frozen sections were used for immunofluorescent staining for protein gene product (PGP)9.5 (Jankowski, Rau, Soneji, Anderson, & Koerber, 2010). Sections were blocked at room temperature for 30 min; incubated with rabbit anti-PGP9.5 (GeneTex, Irvine, CA; diluted at 1:100) and DyLight-488conjugated anti-rabbit IgG antibody (Rockland Immunochemicals, Gilbertsville, PA; diluted at 1:1000) at 4 C for 12 hr; and embedded with Fluoromount-G with 40 ,6-diamidino-2phenylindole (Southern Biotech, Birmingham, AL). Fluorescent inverted microscopy (BIOREVO BZ-9000; Keyence, Osaka, Japan) was used for observation of the sections.
Western Blotting We exfoliated the epidermal layer from frozen skin samples and homogenized it in sample buffer that consisted of 2% (w/v) sodium dodecyl sulfate, 50 mM Tris–HCl, 5% (v/v) 2-mercapto
sodium, and 2.5% (w/v) sucrose. After centrifugation (6,000 rpm), 100 μl of supernatant were used. Following supplementation with 0.1% bromophenol blue, the samples were heated at 95 C for 10 min. After separation of 10 μl per lane of sample through 15% polyacrylamide gel electrophoresis (200 V, 40 min), it was transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA) using a semidry blotting method (30 V, 90 min). NGFb was detected using the skin blotting method described previously. b-Actin was immunologically stained with an indirect method using rabbit anti-b-actin (GeneTex; diluted at 1:200).
Statistical Analysis Values are presented as means + SD. Statistical differences among the three groups were examined using the Tukey’s multiple comparison test, with p < .05 considered statistically significant. Statistical analysis was performed with Statistical Package for the Social Sciences v. 20 (IBM, Armonk, NY).
Results Inflammatory Status of Treated Skin We did not observe macroscopic changes in all the groups. HE staining showed normal histology of the epidermis, which consisted of layers of flattened granular cells, prickle cells, and basal cells, in the NT group (Figure 1A). In the Hypo group, we observed marked swelling of the granular and prickle cells, disrupted basal layer, and infiltration of inflammatory cells into the dermal papillary layer (Figure 1B). In contrast, there was no histological alteration in the Iso group (Figure 1C). Immunohistological staining for MHC-II, which is a marker for macrophages, dendritic cells, and Langerhans cells, revealed infiltration of MHC-II-positive cells in the epidermis and dermal papillary layer in the Hypo group, whereas there were few positive cells in the NT and Iso groups (Figure 2). These results indicate the subclinical inflammatory status of the skin in the Hypo group.
Expression and Secretion of NGFb NGFb is a cytokine that is secreted by keratinocytes under inflammatory conditions and induces nerve fiber growth. We identified expression of NGFb in the Hypo and Iso groups by Western blotting, but NT skin was completely negative for NGFb (Figure 3A). Immunohistochemistry showed NGFb-positive secretory granules in clusters of basal cells of the epidermis in the Hypo group. In contrast, there were few NGFb-positive cells in the epidermis of the NT and Iso groups (Figure 3B). Epidermal secretion of NGFb was detected by skin blotting (Figure 3C) and compared among experimental groups following quantification of the intensity of immunoreactivity for NGFb (Figure 3D). The intensity of signals in the Hypo group (16,970.42 + 7,442.28) was significantly higher than that in the NT (3,978.84 + 1,857.57, p ¼ .017) and Iso groups (4,845.26 + 3,641.92, p ¼ .032), whereas there was no significant difference between the NT and Iso groups (p ¼ 1.000).
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Figure 1. Hypo-osmotic treatment–induced histological changes revealed by hematoxylin and eosin staining. We compared the histology of dorsal skin in rats with (A) no treatment (NT) and those treated with (B) distilled water (Hypo) and (C) normal saline (Iso). The Hypo group showed disrupted alignment of the basal cell layer, swelling of granular and prickle cells, and infiltration of inflammatory cells into the dermal papillary layer (arrowheads). In the Iso group, there was no histological change relative to the NT group. Note. e ¼ epidermis, d ¼ dermis, bar ¼ 50 μm.
Figure 2. Hypo-osmotic treatment–induced infiltration of major histocompatibility complex (MHC)-II-positive inflammatory cells. We observed immunoreactivity for MHC-II, which is a marker for macrophages, dendritic cells, and Langerhans cells, in (B) intact untreated dorsal skin (no treatment [NT]), (B) skin treated with hypo-osmotic solution (Hypo), and (C) skin treated with isotonic solution (Iso). There were many MHC-II-positive cells (arrows) in the epidermis and dermal papillary layer in the Hypo group, whereas there were few positive cells in the NT and Iso groups. Note. e ¼ epidermis, d ¼ dermis, bar ¼ 50 μm.
Elongation of C-Fibers into the Epidermis C-fibers reach just below the basement membrane under normal conditions. However, secreted NGFb from epidermal keratinocytes under inflammatory conditions leads to elongation and invasion of C-fibers into the epidermis through the basement membrane. We identified the C-fibers by immunofluorescence for PGP9.5 (Figure 4). PGP9.5-positive nerve fibers were abundant in the dermal papillary layer in all groups, but
we observed invasion of elongated C-fibers only in the Hypo group (Figure 4A–C).
Discussion In this study, we demonstrated that contact with a hypotonic solution induced inflammation and elongation of C-fibers into the epidermis due to hypo-osmotic shock in the skin of HWY
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Figure 3. Acceleration of expression and secretion of nerve growth factor (NGF)b in the epidermis induced by hypo-osmotic shock. (A) Expression of NGFb shown by Western blotting. (B) Distribution of NGFb-positive cells (arrowheads) shown by immunohistochemistry. (C) Secretion of NGFb in the treatment area of the dorsal skin shown by skin blotting. (D) Skin blotting signals (mean + SD) for NGFb. *p < .05. The results show increased expression and secretion of NGFb in the Hypo group. In contrast, secretion of NGFb in the Iso group was significantly lower than that in the Hypo group and similar to that in the NT group, although the induction of a relatively weaker expression of NGFb was detected in the Iso group. Note. NT ¼ no-treatment group; Hypo ¼ group treated with a hypo-osmotic solution; Iso ¼ group treated with an isotonic solution; e ¼ epidermis; d ¼ dermis; sb ¼ stratum basale epidermis; bar ¼ 50 μm (left panels in 3B), 10 μm (right panels in 3B).
rats in which skin barrier function was disrupted. Moreover, contact with isotonic saline induced only slight inflammation and did not result in the elongation of C-fibers in HWY rat skin. Preliminary testing of HWY rats showed failure of dorsal skin barrier function. Upon histological examination, we did not observe changes indicative of aging skin, such as atrophy of collagen and preinflammatory lesions. HWY rats thus showed a decline in skin barrier function, alone, so this model was well suited for our purpose. In the Hypo group, we observed marked swelling of granular and prickle cells by HE staining. We also observed infiltration of inflammatory cells into the epidermis and dermal papillary layer by HE staining and immunohistochemistry for MHC-II, without any macroscopic alteration, suggesting subclinical inflammatory status similar to that of dermatitis. Moreover, we saw increased expression and secretion of NGFb in the Hypo group. In contrast, we did not observe these inflammatory reactions histologically in the Iso group. We did, however, also detect expression of NGFb in the Iso group, which was probably due to friction or maceration by the treatmentaffected skin. Secretion of NGFb in the Iso group was significantly lower than that in the Hypo group, and we did not observe elongation of C-fibers into the epidermis. These results suggest that hypo-osmotic shock is a factor in induction of the inflammatory reaction and elongation of C-fibers in the epidermis. Researchers reported similar histological changes and increased scratching behavior in a mouse model of pruritus in which shaved dorsal skin was treated with acetone/ether followed by water (Miyamoto, Nojima, Shinkado, Nakahashi, & Kuraishi, 2002; Okawa et al., 2012; Tominaga et al., 2007). Future research is needed to reveal hypo-osmotic shock– induced pruritus by the observation of scratching behavior in HWY rats. Nurses provide hygiene care to keep patients clean and refreshed. However, our results suggest that the usual hygiene care using hypotonic solutions might induce pruritus in elderly patients. At present, nurses try to improve xerosis by using bath salts, including natural moisturizing factors such as ceramide, urea, and Vaseline. Our results propose an additional improvement in hygiene care through use of osmolality-regulated bath water. Future research is needed to determine the appropriate osmolality range of bath water. The present study had some limitations. The mutation profile of genes has not been fully elucidated in HWY rats; therefore, further research is required to generalize our findings to normal rats and humans. We used HWY rats in order to reveal hypo-osmotic shock–induced skin inflammation in the presence of disrupted skin barrier function. In future research, we plan to use aged animals to explore the association between hypo-osmotic shock and APE because aging leads to several other structural and functional changes in the skin in addition to decreased barrier function. In conclusion, our findings confirm that hypotonic solutions induce inflammation by hypo-osmotic shock in skin with disrupted barrier function. Further, we found that an isotonic solution does not induce skin inflammation in comparison with a
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Figure 4. Hypo-osmotic treatment–induced elongation of C-fibers into the epidermis. We observed the distribution of C-fibers by immunofluorescence staining for PGP9.5 (A, D, and G) with DAPI counterstaining (B, E, and H) in 20-μm thick frozen sections of the NT (A–C), Hypo (D–F), and Iso (G–I) groups. We found C-fibers only in the epidermis of the Hypo group, although abundantly in the dermal papillary layer in all groups. Note. NT ¼ no-treatment group; Hypo ¼ group treated with a hypo-osmotic solution; Iso ¼ group treated with an isotonic solution; Arrowheads ¼ c-fibers in the dermal papillary layer; Arrows ¼ elongated c-fibers into epidermis; e ¼ epidermis; d ¼ dermis; bar ¼ 200 μm.
hypotonic solution. These findings may lead to improvements in hygiene care in elderly patients. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: A part of this study was supported by the Tokyo Association of Medical Sciences.
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