Is Estrogen Effective for Full-Thickness Cutaneous Wound Healing in Young Male Mice?

The aim of this study is to show the effects of estrogen upon its topical application on the wound healing process in young male mice. Fifty-six male mice aged 7 weeks old were divided into 4 groups: sham operation, castration, estrogen treatment after sham operation, and estrogen treatment after castration. Wound healing was observed daily until day 14 after wounding. Specimens were harvested on days 3, 7, 10, and 14, and stained to evaluate reepithelialization, inflammation, contraction, and collagen accumulation. Wound healing periods of all groups were almost the same, although the concentration of serum estrogen in the estrogen-applied mice was very high, and that in the nonapplied groups was low. The numbers of macrophages in the castrated, estrogen-treated after sham operation, and estrogen-treated after castration groups were significantly decreased compared with that in the sham group in the inflammatory phase; however, the ratio of wound area in these groups did not decrease, and other histological data did not reveal any effects of estrogen. These results indicate that estrogen may show limited effectiveness for full-thickness cutaneous wound healing in young male mice, and decreased inflammation may not always be associated with decreased wound area.

The skin, the largest organ in the body, undergoes numerous age-related structural and functional changes, including a delayed wound healing response. Delayed or failed healing of wounds poses a major problem, leading to increased morbidity and mortality, as well as increased costs for health care systems.¹   In recent years, the important role of hormones in age-related delayed healing has become apparent, and skin has emerged as a clear target of hormonal action. Cutaneous wounds heal more slowly in elderly males than in elderly females, suggesting a role of the sex hormones in the healing process.^2-4 In the context of wound healing, hormonal factors act to mask underlying differences in repair. The influence on repair of intrinsic gender differences in such processes as tissue morphogenesis and perinatal hormonal imprinting may be exposed only when these hormonal factors are removed.⁵ Estrogens, especially 17β-estradiol, have long been known to accelerate healing in females.⁶ These compounds decrease wound size⁷; affect macrophages by reducing the production of a range of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and macrophage migration inhibitory factor (MIF)^1,5; stimulate keratinocyte proliferation; and accelerate reepithelialization and fibroblasts, increasing dermal collagen production.¹ On the other hand, wounds in ovariectomized (OVX) mice, by virtue of negligible systemic estrogen, take substantially longer to heal and contain significantly increased numbers of inflammatory cells compared with intact mice.^1,8   In contrast, androgens inhibit wound healing in males. Testosterone, the major androgen in circulation, which is mostly produced by Leydig cells in the testes, appears to modulate healing by directly altering wound cell populations and cytokine profiles, thereby enhancing the inflammatory response and reducing matrix deposition.^1,9 In addition, recent studies demonstrated that androgen receptors (AR) suppressed wound healing by enhancing the inflammatory response through a localized increase in TNF-α expression via multiple mechanisms, including increasing the inflammatory monocyte population, enhancing monocyte chemotaxis, and enhancing TNF-α expression in macrophages.^2,10 Castrated mice with reduced systemic testosterone display accelerated healing through a dampened inflammatory response, increased matrix deposition, down regulation of TNF-α,^3,4,11 and reduced wound area.^5,9   Many studies have suggested that estrogen promotes wound healing in females. However, there is little understanding of the roles of estrogen in wound healing in males. Ashcroft et al⁷ reported that estrogen treatment decreased wound size and neutrophil numbers, and increased collagen levels in elderly human males. On the other hand, Gilliver et al⁶ reported that sustained exposure to estrogen markedly delays wound reepithelialization, while estrogen minimally influences wound inflammation and reduces collagen accumulation by increasing gelatinase activities in the wounds of estrogen-treated male mice. The role of estrogens in males remains to be established, and further studies on this issue are necessary. Therefore, the aim of this study was to explore the effects of topically applied estrogen on the wound healing process in young male mice.

Animals. Fifty-six BALB/cCrSlc male mice aged 7 weeks (Sankyo Labo Service Corporation, Inc, Toyama, Japan) and weighing 16.9-21.8 g were used. They were caged individually in an air-conditioned room at 25.0o C ± 2.0o C, with a 12 hour light/dark cycle. Water and laboratory chow were given ad libitum. The experimental protocol and animal care were in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University, Japan (AP-101739).   Wounding. Nine-week-old male mice that had undergone castration or sham operation 2 weeks previously were used for the experiments on wound healing. The mice were divided into 4 groups: sham-operated (sham), castrated (CSX), sham-operated treated with 17β-estradiol (sham + E), and CSX treated with 17β-estradiol (CSX + E). The mice were anesthetized by an intraperitoneal injection of pentobarbital sodium (0.05 mg/g weight) and the dorsum was shaved. Two circular full-thickness skin wounds (4 mm in diameter), including the panniculus carnosus muscle on both sides of the dorsum of the mouse, were made with a sterile disposable biopsy punch (Kai Industries Co, Gifu, Japan). Wounds were covered with a hydrocolloid dressing (Tegaderm, 3M Health Care, Tokyo, Japan) to maintain a moist environment, and then mice were wrapped twice with adhesive bandages (mesh pore tape, Nichiban, Tokyo, Japan). The bandages were changed every day and wound healing was observed daily until day 14 after wounding.   Estrogen treatment and measurement of hormone levels. Using a 1 mL syringe tube without needle, 0.01mL of 17β-estradiol gel (l’estrogel 0.06%, Bayer, Tokyo, Japan) was dribbled on the sterilization gauze 28 mm x 10 mm in size. The gauze was then topically applied on the left abdominal side (not on the wound site) every day until day 14. The gauze was wrapped with adhesive bandage so that it did not slip out of position. Serum 17β-estradiol and testosterone levels on day 14 were determined by radio immunoassay (Mitsubishi Chemical Medience Corporation, Tokyo, Japan) on each mouse. The limit of detection of serum estradiol was 10 pg/ml. Values of less than 10 pg/ml were regarded as 10 pg/ml.   Macroscopic observation. The day wounds were created was designated as day 0, and the process of wound healing was observed from day 0 to 14 days after wounding. Wounded edges were traced on polypropylene sheets and photographs were taken every day. The traces on the sheets were captured with a scanner onto a personal computer (Adobe Photoshop Elements 7.0, Adobe System Inc, Tokyo, Japan), and the areas of wounds were calculated using image analysis software (Scion Image Beta 4.02, Scion Corporation, Frederick, MD).   Tissue processing. On days 3, 7, and 10 after wounding, 3 mice from each group were euthanized by a massive pentobarbital sodium intraperitoneal injection. On day 14 after wounding, 5 mice from each group were euthanized using the same method. The wounds and the surrounding intact skin were harvested, stapled onto transparent plastic sheets to prevent over-contraction of specimens, and fixed in 4% paraformaldehyde in 0.2 mol/L phosphate buffer (pH 7.4) for 15 hours. Specimens were dehydrated in an alcohol series, cleaned in xylene, and embedded in paraffin to prepare 5 µm serial sections. These sections were stained with hematoxylin-eosin (HE) or subjected to Azan staining, and immunohistologically stained with anti-neutrophil antibody (Abcam Japan, Tokyo, Japan) for detecting neutrophils, anti-mouse Mac-3 antibody (BD Pharmingen, Tokyo, Japan) for detecting macrophages, or anti-α-smooth muscle actin (α-SMA) antibody, prediluted (Abcam KK, Tokyo, Japan) for detecting myofibroblasts. The procedure for unmasking antigens was antigen-dependent.   Immunohistochemical staining. After deparaffinization and rehydration, antigen unmasking was accomplished by heating slides in a water bath containing sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 20 minutes near 100° C. Slides for Mac-3 antibody and α-SMA were washed with phosphate-buffered saline (PBS), and slides for anti-neutrophil antibody were washed with 0.3% TritonX-100 in PBS. Then, slides were incubated with anti-neutrophil antibody or Mac-3 antibody at a concentration of 1:100 in PBS, or undiluted anti-α-SMA, at 4° C overnight. Slides were again washed with PBS or 0.3% TritonX-100 in PBS. For detection of primary antibodies, slides for Mac-3 antibody and anti-neutrophil antibody were incubated with polyclonal rabbit anti-rat immunoglobulins/HRP (Dako North America, Carpinteria, CA) at a concentration of 1:300 in 0.3% mouse serum (normal) (Dako North America, Carpinteria, CA) in PBS for 30 minutes at 4° C, and slides for α-SMA antibody were exposed to HRP labelled polymer anti-rabbit (ready to use) (Envision+ System-HRP Labelled Polymer Anti-Rabbit, Dako North America, Carpinteria, CA) for 30 minutes at room temperature. Slides were again washed with PBS or 0.3% TritonX-100 in PBS and then incubated in brown chromogen (Dako Liquid DAB+ Substrate Chromogen System, Dako North America, Carpinteria, CA) for 5 minutes or until staining was detected at room temperature. Light hematoxylin counterstaining for 1 minute was applied for visualization of cell nuclei. Finally, slides were rinsed in distilled water, dehydrated, cleared, and mounted for analysis. Negative control slides were obtained by omitting each primary antibody.   Microscopic observations. In accordance with Haryanto et al,¹² for evaluation over the course of the wound healing process, photographic images were prepared, and quantifications of wound areas and the extent of reepithelialization, as well as the cell numbers of each cell type per unit area (neutrophils and macrophages, central area of granulation tissue; myofibroblasts, average value from both edges of the wound and either top or bottom of the wound at ×400 magnification), were undertaken using a microscope digital camera system (DP72, Olympus, Tokyo, Japan). The wound area was defined as being the area bounded by the panniculus carnosus muscle at the base of the wound, the scab and migrating epithelium at the top of the wound, and the margins of normal skin on either side.   The authors measured the ratio of reepithelialization (%) = length of new epithelium/length of wound between wound edges, the number of neutrophils/mm² granulation tissue, macrophages/mm² granulation tissue, myofibroblasts/mm² granulation tissue, and the ratio of collagen fibers in granulation tissue = number of pixels of collagen fibers/number of pixels of granulation tissue area using Adobe Photoshop Element 7.0.

Data are expressed as mean ± SD and analyzed with ANOVA and multiple comparison Tukey-Kramer using JMP 8.0.1 (SAS, Cary, NC). The differences were considered significant at P < 0.05.

Serum estradiol and testosterone levels on day 14. Estrogen-treated groups revealed significantly higher estradiol levels than the untreated groups: sham + E and CSX (P < 0.0001), CSX + E and CSX (P < 0.0001), sham + E and sham (P < 0.0001), CSX + E and Sham (P = 0.0001). These levels reached about 3 times those of the untreated groups (Table 1). Testosterone somewhat decreased in sham + E on day 14 and this value was almost the same as in the castrated groups. There were trends between sham and CSX (P = 0.06), and sham and CSX + E (P = 0.071). These results suggest that estrogen treatment influences systemic androgen levels, while endogenous androgen does not affect exogenous estrogen levels.   Effect of estrogen on wound areas. In all groups, the wound healing processes seemed to be almost the same. New epithelium was observed at the wound edge on day 3 after wounding and, for some wounds, the whole wound surface was covered with new epithelium on day 10. On day 14, all wounds had completed reepithelialization.   On days 0-14, the ratios of wound areas to the initial wound area on day 0 were calculated. The wound area of mice in the CSX + E group significantly increased on day 1 after wounding (Figure 1). There were significant differences between sham and CSX + E (P < 0.0001), CSX and CSX + E (P = 0.001), and sham + E and CSX + E (P = 0.003). Wound areas in all groups increased after wounding. In the sham group, the wound area peaked on day 4 at 1.04 ± 0.35 compared with the initial wound care on day 0, and decreased gradually to 0.84 ± 0.40 on day 7, and 0.12 ± 0.06 on day 14. In the CSX group, the wound area peaked on day 2 after wounding at 1.12 ± 0.22 compared with the initial wound area, and decreased to 0.66 ± 0.14 on day 7 and 0.17 ± 0.06 on day 14. In the sham + E group, wound area peaked on day 2 at 1.25 ± 0.25 compared with that on day 0 after wounding, and decreased to 0.82 ± 0.22 on day 7 and 0.18 ± 0.08 on day 14. In the CSX + E group, the wound area peaked on day 1 after wounding at 1.29 ± 0.27 compared with the initial wound area, and decreased rapidly to 0.74 ± 0.38 on day 7 and 0.12 ± 0.06 on day 14. There were no significant differences between all groups on days 2-14.   Effect of estrogen on reepithelialization. There were no significant differences between all groups in terms of the ratio of reepithelialization. On day 10 after wounding, the ratios of reepithelialization were 91.3% ± 17.4% in sham, 87.0% ± 18.9% in CSX, 100% ± 0.0% in sham + E, and 98.7% ± 3.0% in CSX + E (Figure 2).   Effect of estrogen on inflammation. On day 3 after wounding, the number of neutrophils in CSX + E seemed to be lower than those of sham, CSX, and sham + E (Figures 3A-E). There was a trend between sham and CSX + E (P = 0.0926) on day 3. On day 10, the numbers of neutrophils/mm² in sham, CSX, sham + E, and CSX + E were 692.45 ± 523.33, 663.92 ± 453.84, 215 ± 226.65, and 85.59 ± 134.13, respectively. There was a significant difference between sham and CSX + E (P = 0.0463), and a trend between CSX and CSX + E (P = 0.0607) on day 10 (Figures 3F-I).   On day 3 after wounding, the numbers of macrophages/mm² in sham, CSX, sham + E, and CSX + E were 1250.03 ± 235.71, 762.47 ± 364.43, 868.80 ± 230.26, and 894.73 ± 152.70, respectively (Figure 3J). The numbers of macrophages in CSX, sham + E, and CSX + E seemed to be lower than that of sham on day 3 (Figures 3K-N). There was a significant difference between only sham and CSX (P = 0.0216), and trends between sham and sham + E (P = 0.0707), and sham and CSX + E (P = 0.0996), on day 3. On day 7, there were significant differences between sham and CSX (P = 0.0248), sham and sham + E (P = 0.0072), and sham and CSX + E (P = 0.0293). There were no significant differences between all groups on day 10 and day 14 (Figure 3O-R).   Effect of estrogen on contraction. On day 3 after wounding, a few myofibroblasts were observed along the wound edge in all groups (Figure 4A). On day 7, there were significant differences between sham and CSX (P = 0.0175), and CSX and CSX+E (P = 0.0029). In this regard, CSX peaked on day 7, and the other groups did so on day 10 (Figures 4B-E). All groups decreased on day 14. There were no significant differences on days 3, 10, and 14.   Effect of estrogen on collagen accumulation. On day 3 after wounding, collagen fibers were already synthesized in all wounds (Figure 4F). There were significant differences between CSX and CSX+E (P = 0.0285), and sham + E and CSX + E (P = 0.0228) on day 7 (Figures 4G-J), and between sham and CSX (P = 0.0232), and CSX and CSX + E (P = 0.0175) on day 14 (Figures 4K-N). The ratio of collagen fibers in CSX + E rapidly increased (Figure 4J); however, it eventually decreased to half the peak value (Figure 4N). In CSX, it increased gradually and peaked on day 14 (Figure 4L).

It is well known that estrogen has a beneficial effect on cutaneous wound healing in females.^1,5-7,13 However, its effect in males has remained unclear¹³; one report stated a beneficial effect of estrogen,⁷ while another reported a limited effect.⁶   Although some amount of the 17β-estradiol may be soaked up into the gauze, the serum estradiol levels in the sham + E and CSX + E groups are almost the same as that in the proestrus female mice, 20-30 pg/ml.¹⁴ Thus, this indicates that the applied estradiol is absorbed enough to act on the wounds. Gilliver et al⁶ reported that estrogen treatment from 2 weeks before wounding in 8-week-old male mice increased day 3 wound areas in intact male mice. Ashcroft et al⁷ reported that estrogen treatment for 24 hours from wounding decreased wound size at day 7 in both elderly males and females. This study’s results revealed an increase in wound areas on day 1 in only CSX + E; subsequently, there were no significant differences between all groups (Figure 1). Considering these results, estrogen may be less effective at reducing wound areas, and it may increase wound area in castrated young male mice with low androgen levels. Moreover, Gilliver et al⁶ reported that estrogen treatment greatly retarded wound reepithelialization in castrated estrogen-treated mice compared with the level in castrated mice on day 3, while it did not affect the extent of reepithelialization at that time in intact mice. In the current study’s results, there was no significant difference between all groups until day 14 (Figure 2). This indicates that estrogen treatment does not retard or promote reepithelialization in young male mice.   The authors evaluated the inflammation by counting the numbers of neutrophils and macrophages. According to Gilliver et al⁶, the numbers of neutrophils were comparable in estrogen-treated and untreated castrated mice on day 3. The authors result also supported this, and there were no significant differences between all groups on day 3 (Figure 3A). Estrogen impairs neutrophil chemotaxis, reducing the rate of migration to the wound site while increasing phagocytic function,¹ while testosterone increases the production of proinflammatory mediator, TNF-α, by macrophages via AR and wound TNF-α production is down-regulated by castration.¹⁵ These facts indicate that acute estrogen treatment decreases the numbers of neutrophils in castrated mice during the inflammatory phase; however, the current result did not show a reduction in neutrophil numbers. Therefore, estrogen treatment in young male mice may be less effective for decreasing neutrophil numbers. Similarly, estrogens inhibit the inflammatory functions of macrophages.¹⁶ Gilliver et al⁶ showed that macrophage population sizes were unaffected by estrogen, and Ashcroft et al^4,7 stated that estrogen affects macrophages by reducing the production of a range of proinflammatory cytokines including IL-6, TNF-α, and macrophage migration inhibitory factor (MIF). In review, chronic exposure of peritoneal macrophages to estradiol in vivo was shown to increase their subsequent secretion of IL-1 and IL-6.¹⁶ These results showed the reduction of macrophage population size in estrogen-treated mice and castrated estrogen-treated mice (Figures 3J-N). In addition, CSX decreased the number of macrophages on days 3 and 7. This study supported previous studies,^2-4 and these findings show that macrophages were regulated by both androgen and estrogen, in particular, estrogen treatment in young males. However, the ratio of wound area did not decrease in the inflammatory phase, which suggests that decreased inflammation may not always be an index of decreasing wound area.   In vitro preliminary studies suggest that estrogen stimulates fibroblast matrix production.⁷ However, Liman et al¹⁷ reported that estradiol benzoate inhibits fibroblast proliferation in wound healing upon tracheal surgery by intramuscular administration for 4 weeks. In the author’s results, the estrogen-treated group did not reveal an increase in myofibroblasts differentiated from fibroblasts; therefore, the authors think that estrogen treatment in young male mice has less effect on increasing myofibroblast numbers. On the other hand, castrated mice with reduced systemic testosterone display accelerated healing through a dampened inflammatory response and increased matrix deposition.⁴ The number of myofibroblasts in CSX peaked on day 7, and CSX significantly increased its number (Figure 4A). These data may indicate that the proliferation of myofibroblasts is promoted by castration rather than estrogen treatment.   The authors calculated the ratio of blue collagen fibers on Azan staining to evaluate tissue remodeling. Estrogens, especially 17β-estradiol, have long been known to increase dermal collagen production.¹ Results of the current study revealed increased collagen deposition in CSX + E on day 7 (Figure 4F) and agreed with this previous study. However, this rate had decreased by about half on day 14 (Figures 4G-N) as opposed to the reduction of wound area. Gilliver et al⁶ reported that the collagen of the lower dermis was highly disorganized in estrogen-treated castrated mice compared with that of castrated mice, which suggests that reduced wound collagen accumulation in estrogen-treated mice resulted from increased wound MMP-2 activity. Moreover, Liman et al¹⁷ reported that estradiol benzoate inhibits massive collagen deposition in wound healing upon tracheal surgery by intramuscular administration. Therefore, it is suggested that estrogen reduces wound collagen accumulation in castrated estrogen-treated mice, but has less effect in estrogen-treated mice. This issue should be examined by further studies.

This study showed the effects of estrogen upon its topical application during the wound healing process in male mice. Estrogen reduced macrophage numbers in the inflammatory phase; however, its effects did not lead to a reduction in wound area. These results suggest that estrogen treatment in young male mice has less effect on the promotion of wound healing in terms of reducing wound area, promoting reepithelialization, increasing contraction, and accumulating collagen fibers.

The authors wish to thank Tamae Urai, MSN, Kanazawa University Hospital, Kanazawa, Japan, and Haryanto, MSN, The Muhammadiyah Institute of Nursing, Pontianak, Indonesia, for their useful advice regarding the experimental techniques. Part of this work was supported by a Grant-in-Aid for Scientific Research, Japan (no. 22592363).

The authors are from Kanazawa University, Kanazawa, Japan.

Address correspondence to: Professor Toshio Nakatani, MD, PhD Department of Clinical Nursing Graduate Course of Nursing Science Division of Health Sciences Graduate School of Medical Science Kanazawa University 5-11-80 Kodatsuno Kanazawa 9200942 Japan nakatosi@staff.kanazawa-u.ac.jp

Disclosure: The authors disclose no financial or other conflicts of interest.