International Journal of Radiation Biology, February 2014; 90: 127–132 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.868618
Exposure to gamma-rays at the telogen phase of the hair cycle inhibits hair follicle regeneration at the anagen phase in mice Kimihiko Sugaya1,2 & Tomohisa Hirobe1 1Fukushima Project Headquarters, and 2Research Center for Radiation Protection, National Institute of Radiological Sciences,
Chiba, Japan
mice and humans. The bulge region provides the insertion point for the arrector pili muscle and marks the lowermost permanent portion of the hair follicle during the hair cycle (Nishimura et al. 2010, Pincelli and Marconi 2010, Tanimura et al. 2011). Regeneration of hair follicle structure begins with keratinocyte and melanocyte progenies derived from keratinocyte and melanocyte stem cells present in the hair bulge. In mice, the 1st and 2nd hair cycles are synchronized and proceed in waves all over the body (Dry 1926). Ionizing radiations, such as X-rays and γ-rays, affect mammalian keratinocytes, melanoblasts, melanocytes and fibroblasts during all stages of development. In embryonic mice exposed to ionizing radiations, patches of pigment-less white hair (white spots) can be found in the mid-ventrum of offspring (Russell and Major 1957, Fahrig 1975, Hirobe 1994). However, it is not fully understood whether keratinocyte and melanocyte stem cells in the bulge of postnatal skin are affected by this exposure. While there are few reports showing the changes of hair with regard to the hair cycle after γirradiation, a reduction in the diameter of irradiated hair follicles has been demonstrated in humans (Sieber et al. 1992) and mice (Geng and Potten 1990). These circumstances prompted us to investigate in detail how ionizing radiations affect the regeneration of hair follicles by evaluating changes in the number and morphology of hairs and by observing changes in the frequency of abnormal hair follicles due to defects in keratinocytes and melanocytes using light microscopy.
Abstract Purpose: The effects of ionizing radiations on somatic stem cells largely remain to be studied. Hair follicles are self-renewing structures that reconstitute themselves throughout the hair cycle, which is comprised of the following phases: Anagen (growth), catagen (regression) and telogen (resting), suggesting the presence of their own stem cells. Materials and methods: The whole bodies of C57BL/10JHir mice in the 1st telogen phase were irradiated with γ-rays. Mice were examined for effects on hair follicles, including their number, morphology and pigmentation in the 2nd anagen phase. Results: Decreased hair follicle density and induction of curved hair follicles were observed in the dermal skin of irradiated mice. In addition to these keratinocyte-derived anomalies, melanocyte-derived anomalies including white hair and hypopigmented hair bulbs were found. The decrease in hair follicle density and the increase in the frequency of hypopigmented hair bulbs were dependent on the dose of γ-rays. Conclusions: These results suggest that γ-rays damage stem cells and progenitors for keratinocytes and melanocytes, thereby affecting the structure and character of regenerated hair follicles. The density of hair follicles and pigment production in hair bulbs are established as criteria for the effects of γ-rays on the hair cycle. Keywords: Bulge, γ-rays, hair, keratinocyte, melanocyte, stem cell
Introduction
Materials and methods
Hair follicles are self-renewing structures that reconstitute themselves, which suggests the presence of their own stem cells. The hair cycle consists of three phases: Anagen (growth), catagen (regression) and telogen (resting) (Dry 1926, Chase 1954, Figure 1A). In vivo and in vitro studies have revealed that keratinocyte (Cotsarelis et al. 1990, Kobayashi et al. 1993) and melanocyte (Nishimura et al. 2002, 2005) stem cells are present in the bulge of the hair follicles in both
Mice C57BL/10JHir (nonagouti black, B10) mice were given water and a commercial diet (OA-2; Clea Japan, Tokyo, Japan) ad libitum and maintained at 24 ⫾ 1°C with 40–60% relative humidity and 12 h of fluorescent light/day. This study was approved by the ethics committee of the National Institute of Radiological Sciences (Japan), in accordance with the guidelines of the National Institute of Health.
Correspondence: Dr Kimihiko Sugaya, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 2638555, Japan. Tel: ⫹ 81 43 206 3143. E-mail: [email protected] (Received 26 June 2013; revised 25 October 2013; accepted 28 October 2013)
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Radiation
(A)
Phase: 1st anagen Day: 0–17
B10 mice from 22–24 days after birth (P22–P24) were placed in a box with walls made of acrylic resin (thickness 10 mm), and given a single dose of whole-body irradiation with acute 60Co γ-rays at a dosage rate of approximately 0.3 Gy/min. The effects were investigated in the same mice at P35–P37. Irradiation of all mice was performed at the same time in the morning. The control mice were concurrent with each radiation group. In total, 115 mice were used in four independent experiments, in which four dose groups (0, 0.5, 1 and 2.5 Gy) were referred. catagen 18–20
(B)
2nd anagen 30–45
telogen 21–29
Skin was removed from the mid-dorsal region of the trunk at P35–P37 and fixed with 10% neutral formalin for 16–18 h at room temperature, then washed with distilled water and transferred through a graded series of ethanols into xylol. The specimens were mounted in Canada balsam on microscope slides under cover slips with the epidermis upwards (Hirobe and Zhou 1990). Whole-mount skin preparations were examined under a light microscope to detect anomalies in hairs.
(C)
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Whole-mount skin preparations
P < 0.05 P < 0.001
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Histological analysis Dorsal skin samples were fixed with 10% neutral formalin, washed with distilled water, transferred through a graded series of ethanols into xylol and embedded in paraffin. Serial 8-μm sections were deparaffinized and stained with hematoxylin and eosin (Hirobe and Takeuchi 1977). The number of hair follicles/0.1 mm2 skin and the frequency of abnormal hair follicles (curved), hairs (white) and hair bulbs (hypopigmented) were counted every three sections using light microscopy. The number of melanosomes per hair bulb melanocyte was counted.
Statistical analysis (E)
(F)
0 Gy
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Figure 1. Histological sections of the dorsal skin of control and irradiated mice at the 2nd anagen phase. (A) Diagram showing the hair cycle in mice. The anagen (growth) phase of the 1st hair cycle extends from midgestation-postnatal day 17 (P17). The 1st catagen (regression) phase continues from P18–P20. The 1st telogen (resting) phase continues from P21–P29. The 2nd anagen phase continues from P30–P45. Abbreviations: H, hair shaft; E, epidermis; D, dermis; SG, sebaceous gland; HB, hair bulge; HF, hair follicle; HM, hair matrix; DP, dermal papilla; Arrow, γirradiation; the region above the dotted line indicates the permanent portion of hair follicles. (B) A section of control skin contains 10 hair follicles of the 2nd hair cycle. White arrowhead indicates a typical hair of the 1st hair cycle. (C) Gamma-irradiation (2.5 Gy) decreased the density of hair follicles in the skin. Only 4 hair follicles of the 2nd hair cycle are observed in this section. (D) Quantitative analysis of the effect of γ-rays on hair follicle density in the 2nd anagen phase. Error bars indicate the standard error of the mean for n ⫽ 28, 29, 28 and 30 at dose of 0, 0.5, 1 and 2.5 Gy, respectively (taken together from 4 independent experiments). P values for comparisons of control mice (0 Gy) were calculated by two-tailed Student’s t-test for comparison of groups of
The statistical significance of differences in (a) the number of hair follicles/0.1 mm2 skin and the frequency of abnormal hairs, hair follicles and hair bulbs, or (b) the number of melanosomes per hair bulb melanocytes were determined by two-tailed Student’s t-test for comparison of groups of unequal or equal size, respectively.
Results Effects of γ-rays on hair follicle structure Since newly formed hair follicles are derived solely from keratinocyte and melanocyte stem cells, we hypothesized that damage caused in keratinocyte and melanocyte stem cells in the 1st telogen could be detected as a phenotype of descendant hair follicle structures in the 2nd anagen (Figure 1A). Whole-mount preparations of dorsal skin in mice exposed unequal size. The decrease in the number of hair follicles/0.1 mm2 skin appears to be dose-dependent. (E) No abnormalities are observed in hair follicle structures of the control mice. (F) However, γ-irradiation (0.5 Gy) resulted in curved hair follicles (arrows) in the dorsal skin of mice. One or two constrictions in hair follicles are observed. Scale bars, 100 μm. This Figure is reproduced in color in the online version of the International Journal of Radiation Biology.
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Effects of γ-rays on pigmentation of regenerated hair follicles We found that irradiation with γ-rays (2.5 Gy) at the 1st telogen induced a white hair patch in the trunk skin of B10 mice at the 2nd anagen. Whole-mount preparations also revealed complete white hair shafts in mice exposed to 2.5 Gy γ-rays (Figure 2B), whereas no white hairs were found in control mice (Figure 2A). We then decided to analyze histological sections of the dorsal skin with regard to anomalies derived from melanocyte stem cell, such as the frequency of white hairs and hypopigmented hair bulbs (Supplementary Table I available online at http://informahealthcare. com/abs/doi/10.3109/09553002.2014.868618). Histological sections confirmed the presence of white hair shafts in the growing hair follicles of mice exposed to 0.5 Gy γ-rays (Figure 2D), whereas no white hairs were found in control mice (Figure 2C). However, the frequency of white hairs was very low and the differences between control and irradiated mice were not significant (Supplementary Table I available online at http://informahealthcare.com/abs/doi/10.3109/ 09553002.2014.868618). We also studied the degree of pigmentation of hair bulb melanocytes. Histological sections of control mice contained fully pigmented melanosomes in the hair bulb (Figure 2E). However, mice exposed to 2.5 Gy γ-rays possessed hair
(A)
0 Gy
(B)
2.5 Gy
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0.5 Gy
(E)
0 Gy
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0 Gy
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(I)
40
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30 Frequency (%)
to 2.5 Gy γ-rays showed relatively sparse hair in comparison to control mice. We decided to analyze histological sections of the dorsal skin with regard to anomalies derived from keratinocyte stem cells, such as hair follicle density and the frequency of curved hair follicles (Supplementary Table I available online at http://informahealthcare.com/abs/doi/ 10.3109/09553002.2014.868618). Histological sections of mouse dorsal skin showed mature hair follicles at the 2nd anagen with some regressed hair follicles at the 1st anagen (Figure 1B). In mice exposed to 2.5 Gy γ-rays, there was a decrease in the number of hair follicles per 0.1 mm2 skin section in the 2nd hair cycle (Figure 1C). Gamma-rays at doses of 0.5 and 1 Gy also caused a significant decrease in the number of hair follicles per 0.1 mm2 skin (Supplementary Table I available online at http://informahealthcare. com/abs/doi/10.3109/09553002.2014.868618). Hair follicle density decreased as the dose increased (Figure 1D). Interestingly, the decreasing rate appeared to reach a plateau at a dose of 1 Gy. An additional malformation was observed in the induction of curved hair follicles (Figure 1F). One or two constrictions in the hair follicles were seen in the curved follicles, even in mice exposed to 0.5 Gy γ-rays, whereas no abnormalities were seen in control mice (Figure 1E). The frequency of curved hair follicles in irradiated mice was significantly different to that in the control group (p ⬍ 0.01, p ⬍ 0.01, p ⬍ 0.05 at 0.5, 1 and 2.5 Gy, respectively) (Supplementary Table I available online at http://informahealthcare.com/abs/doi/ 10.3109/09553002.2014.868618). However, these differences did not appear to be dose-dependent. These results suggest that γ-rays affected hair follicle number and structure in the 2nd anagen phase when stem cells and committed progenitors for keratinocytes were irradiated in the 1st telogen.
2.5 Gy
2.5 Gy
P < 0.05 P < 0.01 P < 0.001
** 20
10
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0 0
0.5
1
1.5 2 γ-rays (Gy)
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Figure 2. Effect of γ-rays on the development of progenies of melanocyte stem cells. (A, B) Whole-mount preparations of dorsal skin show the presence of complete white hair shafts (arrow) in mice exposed to 2.5 Gy γ-rays, but not in control mice. Scale bar, 50 μm. (C) A histological section of the dorsal skin of control mice at the 2nd anagen phase. (D) White hair shaft (arrow) of the 2nd hair cycle was observed in mice exposed to 0.5 Gy γ-rays. (E, F) Mice exposed to 2.5 Gy γ-rays possess hair bulb melanocytes with extremely reduced pigmentation (F, arrow) in contrast to the heavily pigmented hair bulbs of control mice. Scale bar, 50 μm. (G, H) Higher magnification views of hair bulbs of control and irradiated mice. Mice exposed to 2.5 Gy γ-rays possess hair bulb melanocytes with fewer and smaller melanosomes compared with control melanocytes, though melanosomes were mature. Arrows indicate hair bulb melanocytes. Scale bar, 20 μm. (I) Quantitative analysis of the effect of γ-rays on the frequency of hypopigmented hair bulbs. Error bars indicate the standard error of the mean for n ⫽ 28, 29, 28 and 30 at dose of 0, 0.5, 1 and 2.5 Gy, respectively (taken together from 4 independent experiments). P values for comparisons of control mice (0 Gy) were calculated by two-tailed Student’s t-test for comparison of groups of unequal size. The increase in the frequency of hypopigmented hair bulbs is also dose-dependent. This Figure is reproduced in color in the online version of the International Journal of Radiation Biology.
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bulb melanocytes with extremely reduced pigmentation (Figure 2F). The number of hair bulb melanocytes did not appear to be reduced. Melanosomes in irradiated hair bulbs appeared mature but they were much fewer in number and smaller in size in the exposed mice than they were in the control mice (Figure 2G, 2H; Supplementary Table II, available online at http://informahealthcare.com/abs/doi/10.3109/ 09553002.2014.868618). In contrast to the frequency of white hairs, the frequency of hypopigmented hair bulbs increased in a dose-dependent manner and reached more than 18% (Figure 2I). There was a significant increase in the frequency of hypopigmented hair bulbs, even in skin exposed to 0.5 Gy γ-rays (Supplementary Table I available online at http://informahealthcare.com/abs/doi/10.3109/09553002. 2014.868618). These results suggest that γ-irradiation affects stem cells and committed progenitors for melanocytes at the 1st telogen and, as a result, inhibits the formation of melanosomes at the 2nd anagen.
Discussion In the present study, γ-irradiation led to a decrease in hair follicle density and an increase in the frequency of hypopigmented hair bulbs; the effects were dose-dependent. These findings suggest that when keratinocyte and melanocyte stem cells and/or their committed progenies are exposed to γ-rays at the 1st telogen, follicle regeneration is inhibited at the 2nd anagen. Ionizing radiations are known to induce damage in DNA and cause DNA double-strand breaks (Ward 1988, Sotiropoulou et al. 2010). It is possible that DNA damage in both the stem and progenitor cells induces hair follicle decline and abnormal hair follicle structures. However, this hypothesis remains to be investigated using markers for DNA damage and stem cells (Pincelli and Marconi 2010). We found that irradiation with γ-rays resulted in a significant decrease in the density of hair follicles at the 2nd anagen. Interestingly, the decreasing rate appears to reach a plateau at the dose of 1 Gy, suggesting the existence of cells showing radiation resistance. In fact, several types of stem cells and corresponding committed progenitor cells for keratinocytes are thought to exist in the bulge region (Mascré et al. 2012). It may be possible that the subpopulations of stem and/or corresponding progenitor cells in the bulge region differ in radiation sensitivity. This might account for our finding of reduced hair follicle density. Further study is needed to clarify the characteristic features of cells showing radiation resistance. Our results showed that irradiation with γ-rays induced a significant decrease in the density of hair follicles, even at a dose of 0.5 Gy, suggesting that a relatively low dose of γ-rays may affect the regeneration of hair follicles. This dose is below that previously reported to inhibit hair follicle regeneration in young and adult mice (Inomata et al. 2009, Sotiropoulou et al. 2010, Aoki et al. 2013). Therefore, it is reasonable to surmise that our experimental system is useful for determining the minimum dose of ionizing radiations required to inhibit postnatal regeneration of hair follicles in mice. The present results showed that γ-rays caused curved hair follicles, suggesting the possibility that γ-rays induce
abnormal keratinocyte differentiation. Hair morphogenesis is known to depend on transforming growth factor (TGF)-α/ epidermal growth factor receptor (EGFR) signaling (Mann et al. 1993, Luetteke et al. 1994). Defective TGF-α/EGFR signaling leads to abnormal hair morphogenesis (curved or misaligned hair follicles), which is manifested by the wavy hair phenotype of spontaneous loss-of-function murine mutations in TGF-α (known as waved-1 or wa1) and EGFR (waved-2 or wa2) (Mann et al. 1993, Luetteke et al. 1994). Moreover, keratinocyte transient receptor potential cation channel, subfamily V, member 3 (TRPV3), a member of the transient receptor potential (TRP) family of Ca2⫹-permeant channels, forms a signaling complex with TGF-α/EGFR and affects hair morphogenesis (Cheng et al. 2010). TRPV3 knockout mice developed a similar curved or misaligned hair follicle structure, suggesting that the TRP channel plays an important role in the formation of new hair follicles (Cheng et al. 2010). Hair morphogenesis is also regulated by the Wnt/β-catenin signaling pathway. Wnt3a signaling was activated in mouse hair follicle melanocytes during the anagen phase of C57BL/6J (B6) mice (Guo et al. 2012). In addition to melanocytes, adjacent keratinocytes expressed Wnt3a during the anagen phase. A large number of growth factors are related to hair morphogenesis, acidic and basic fibroblast growth factors (aFGF, bFGF), insulin-like growth factor (IGF), hepatocyte growth factor and platelet-derived growth factor (see review of Peus and Pittelkow 1996). FGF-13 (Kawano et al. 2004) and IGF-binding protein 5 (Sriwiriyanont et al. 2011), genes of trichorhinophalanged syndrome, which is the regulator of the Wnt signaling pathway (Fantauzzo and Christiano 2012) and prostaglandin D2 (Garza et al. 2012), are also reported to be related to hair morphogenesis in mice and humans. Although many important signaling pathways are suggested to be involved in the regulation of hair follicle formation and growth, the precise nature, timing and intersection of these inductive and regulatory signals remains largely unknown (Festa et al. 2011, Sennett and Rendl 2012). Hypopigmentation in the hair bulb melanocytes was found in numerous hair follicles, even among the 0.5-Gy irradiation group, and gradually increased with dosage. The number of melanocytes did not appear to be reduced, but melanosomes in the melanocytes of irradiated hair bulbs were decreased and their size was greatly reduced in comparison to control mice. These results suggests that γ-ray exposure inhibited melanosome development rather than maturation in hair bulbs in the subsequent anagen phase after melanocyte stem cells were irradiated with γ-rays at the 1st telogen. A previous study showed that graying of the dorsal hairs was induced in the subsequent anagen phase of 6–7-week-old B6 mice (2nd telogen) irradiated with 5 Gy X-rays at one day after plucking (Inomata et al. 2009). The same report showed that melanocyte stem cells were depleted in numerous hair bulges of the 2nd anagen. X-rays gradually decreased the number of melanocyte stem cell-containing bulges in the subsequent hair cycles with a gradual increase in ectopically pigmented mature melanocytes. Their results suggest that melanocyte stem cells lose their stem cell immaturity and commit to differentiate in the niche after X-ray exposure, resulting in stem cell depletion and subsequent hair graying. Their data show
Gamma-rays and hair follicle stem cells 131 that at least 5 Gy X-rays were required for the stable induction of hair graying. In the present study, however, white hairs and hypopigmented hair bulb melanocytes occurred even at 0.5 Gy γ-rays. Although the discrepancies between their results and the present findings cannot be explained well at present, they might be due to the differences in the types of ionizing radiations (X-rays vs. γ-rays), strains (B6 vs. B10), ages (7–8-week-old vs. 22–24-day-old), hair cycle phase (2nd telogen vs. 1st telogen) and plucking (hair cycle after plucking vs. natural hair cycle without plucking). Hair-plucking could induce several kinds of growth factors and cytokines that affect the proliferation and differentiation of keratinocytes, melanocytes and fibroblasts. In order to exclude the effects of these factors on the behavior of melanocyte stem cells, analysis using the natural hair cycle might be preferable. In addition to keratinocyte stem cells and/or melanocyte stem cells in the bulge, fibroblasts in the dermal papilla and adipocytes near the dermal papilla may be targets of ionizing radiations, as both fibroblasts and adipocytes have been shown to play an important role in hair follicle cycling (Festa et al. 2011, Sennett and Rendl 2012, Chi et al. 2013). Further studies are needed to analyze the effect of ionizing radiations on fibroblasts and adipocytes with regard to the hair cycle. In conclusion, the decrease in the density of hair follicles in the skin at the 2nd anagen and the increase in the frequency of hypopigmented hair bulbs are dependent on the dose of γ-rays. Furthermore, these irradiations induced abnormal hair follicle curvature and white hairs, suggesting that γ-irradiation in the 1st telogen strongly affects regeneration of hair follicles, which are produced by the progenies of keratinocyte and melanocyte stem cells, in the 2nd anagen.
Acknowledgements We thank the members of the Laboratory Animal Science Section of NIRS and Ms. Y. Ishihara, H. Nakamura and S. Inoue for their technical assistance. We also thank Drs H. Mizuno and K. Eguchi-Kasai for their advice on γ-irradiation.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Exposure to gamma-rays at the telogen phase of the hair cycle inhibits hair follicle regeneration at the anagen phase in mice Kimihiko Sugaya1,2 & Tomohisa Hirobe1 1Fukushima Project Headquarters, and 2Research Center for Radiation Protection, National Institute of Radiological Sciences,
Chiba, Japan
mice and humans. The bulge region provides the insertion point for the arrector pili muscle and marks the lowermost permanent portion of the hair follicle during the hair cycle (Nishimura et al. 2010, Pincelli and Marconi 2010, Tanimura et al. 2011). Regeneration of hair follicle structure begins with keratinocyte and melanocyte progenies derived from keratinocyte and melanocyte stem cells present in the hair bulge. In mice, the 1st and 2nd hair cycles are synchronized and proceed in waves all over the body (Dry 1926). Ionizing radiations, such as X-rays and γ-rays, affect mammalian keratinocytes, melanoblasts, melanocytes and fibroblasts during all stages of development. In embryonic mice exposed to ionizing radiations, patches of pigment-less white hair (white spots) can be found in the mid-ventrum of offspring (Russell and Major 1957, Fahrig 1975, Hirobe 1994). However, it is not fully understood whether keratinocyte and melanocyte stem cells in the bulge of postnatal skin are affected by this exposure. While there are few reports showing the changes of hair with regard to the hair cycle after γirradiation, a reduction in the diameter of irradiated hair follicles has been demonstrated in humans (Sieber et al. 1992) and mice (Geng and Potten 1990). These circumstances prompted us to investigate in detail how ionizing radiations affect the regeneration of hair follicles by evaluating changes in the number and morphology of hairs and by observing changes in the frequency of abnormal hair follicles due to defects in keratinocytes and melanocytes using light microscopy.
Abstract Purpose: The effects of ionizing radiations on somatic stem cells largely remain to be studied. Hair follicles are self-renewing structures that reconstitute themselves throughout the hair cycle, which is comprised of the following phases: Anagen (growth), catagen (regression) and telogen (resting), suggesting the presence of their own stem cells. Materials and methods: The whole bodies of C57BL/10JHir mice in the 1st telogen phase were irradiated with γ-rays. Mice were examined for effects on hair follicles, including their number, morphology and pigmentation in the 2nd anagen phase. Results: Decreased hair follicle density and induction of curved hair follicles were observed in the dermal skin of irradiated mice. In addition to these keratinocyte-derived anomalies, melanocyte-derived anomalies including white hair and hypopigmented hair bulbs were found. The decrease in hair follicle density and the increase in the frequency of hypopigmented hair bulbs were dependent on the dose of γ-rays. Conclusions: These results suggest that γ-rays damage stem cells and progenitors for keratinocytes and melanocytes, thereby affecting the structure and character of regenerated hair follicles. The density of hair follicles and pigment production in hair bulbs are established as criteria for the effects of γ-rays on the hair cycle. Keywords: Bulge, γ-rays, hair, keratinocyte, melanocyte, stem cell
Introduction
Materials and methods
Hair follicles are self-renewing structures that reconstitute themselves, which suggests the presence of their own stem cells. The hair cycle consists of three phases: Anagen (growth), catagen (regression) and telogen (resting) (Dry 1926, Chase 1954, Figure 1A). In vivo and in vitro studies have revealed that keratinocyte (Cotsarelis et al. 1990, Kobayashi et al. 1993) and melanocyte (Nishimura et al. 2002, 2005) stem cells are present in the bulge of the hair follicles in both
Mice C57BL/10JHir (nonagouti black, B10) mice were given water and a commercial diet (OA-2; Clea Japan, Tokyo, Japan) ad libitum and maintained at 24 ⫾ 1°C with 40–60% relative humidity and 12 h of fluorescent light/day. This study was approved by the ethics committee of the National Institute of Radiological Sciences (Japan), in accordance with the guidelines of the National Institute of Health.
Correspondence: Dr Kimihiko Sugaya, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 2638555, Japan. Tel: ⫹ 81 43 206 3143. E-mail: [email protected] (Received 26 June 2013; revised 25 October 2013; accepted 28 October 2013)
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Radiation
(A)
Phase: 1st anagen Day: 0–17
B10 mice from 22–24 days after birth (P22–P24) were placed in a box with walls made of acrylic resin (thickness 10 mm), and given a single dose of whole-body irradiation with acute 60Co γ-rays at a dosage rate of approximately 0.3 Gy/min. The effects were investigated in the same mice at P35–P37. Irradiation of all mice was performed at the same time in the morning. The control mice were concurrent with each radiation group. In total, 115 mice were used in four independent experiments, in which four dose groups (0, 0.5, 1 and 2.5 Gy) were referred. catagen 18–20
(B)
2nd anagen 30–45
telogen 21–29
Skin was removed from the mid-dorsal region of the trunk at P35–P37 and fixed with 10% neutral formalin for 16–18 h at room temperature, then washed with distilled water and transferred through a graded series of ethanols into xylol. The specimens were mounted in Canada balsam on microscope slides under cover slips with the epidermis upwards (Hirobe and Zhou 1990). Whole-mount skin preparations were examined under a light microscope to detect anomalies in hairs.
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Whole-mount skin preparations
P < 0.05 P < 0.001
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Histological analysis Dorsal skin samples were fixed with 10% neutral formalin, washed with distilled water, transferred through a graded series of ethanols into xylol and embedded in paraffin. Serial 8-μm sections were deparaffinized and stained with hematoxylin and eosin (Hirobe and Takeuchi 1977). The number of hair follicles/0.1 mm2 skin and the frequency of abnormal hair follicles (curved), hairs (white) and hair bulbs (hypopigmented) were counted every three sections using light microscopy. The number of melanosomes per hair bulb melanocyte was counted.
Statistical analysis (E)
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0 Gy
0.5 Gy
Figure 1. Histological sections of the dorsal skin of control and irradiated mice at the 2nd anagen phase. (A) Diagram showing the hair cycle in mice. The anagen (growth) phase of the 1st hair cycle extends from midgestation-postnatal day 17 (P17). The 1st catagen (regression) phase continues from P18–P20. The 1st telogen (resting) phase continues from P21–P29. The 2nd anagen phase continues from P30–P45. Abbreviations: H, hair shaft; E, epidermis; D, dermis; SG, sebaceous gland; HB, hair bulge; HF, hair follicle; HM, hair matrix; DP, dermal papilla; Arrow, γirradiation; the region above the dotted line indicates the permanent portion of hair follicles. (B) A section of control skin contains 10 hair follicles of the 2nd hair cycle. White arrowhead indicates a typical hair of the 1st hair cycle. (C) Gamma-irradiation (2.5 Gy) decreased the density of hair follicles in the skin. Only 4 hair follicles of the 2nd hair cycle are observed in this section. (D) Quantitative analysis of the effect of γ-rays on hair follicle density in the 2nd anagen phase. Error bars indicate the standard error of the mean for n ⫽ 28, 29, 28 and 30 at dose of 0, 0.5, 1 and 2.5 Gy, respectively (taken together from 4 independent experiments). P values for comparisons of control mice (0 Gy) were calculated by two-tailed Student’s t-test for comparison of groups of
The statistical significance of differences in (a) the number of hair follicles/0.1 mm2 skin and the frequency of abnormal hairs, hair follicles and hair bulbs, or (b) the number of melanosomes per hair bulb melanocytes were determined by two-tailed Student’s t-test for comparison of groups of unequal or equal size, respectively.
Results Effects of γ-rays on hair follicle structure Since newly formed hair follicles are derived solely from keratinocyte and melanocyte stem cells, we hypothesized that damage caused in keratinocyte and melanocyte stem cells in the 1st telogen could be detected as a phenotype of descendant hair follicle structures in the 2nd anagen (Figure 1A). Whole-mount preparations of dorsal skin in mice exposed unequal size. The decrease in the number of hair follicles/0.1 mm2 skin appears to be dose-dependent. (E) No abnormalities are observed in hair follicle structures of the control mice. (F) However, γ-irradiation (0.5 Gy) resulted in curved hair follicles (arrows) in the dorsal skin of mice. One or two constrictions in hair follicles are observed. Scale bars, 100 μm. This Figure is reproduced in color in the online version of the International Journal of Radiation Biology.
Gamma-rays and hair follicle stem cells 129
Effects of γ-rays on pigmentation of regenerated hair follicles We found that irradiation with γ-rays (2.5 Gy) at the 1st telogen induced a white hair patch in the trunk skin of B10 mice at the 2nd anagen. Whole-mount preparations also revealed complete white hair shafts in mice exposed to 2.5 Gy γ-rays (Figure 2B), whereas no white hairs were found in control mice (Figure 2A). We then decided to analyze histological sections of the dorsal skin with regard to anomalies derived from melanocyte stem cell, such as the frequency of white hairs and hypopigmented hair bulbs (Supplementary Table I available online at http://informahealthcare. com/abs/doi/10.3109/09553002.2014.868618). Histological sections confirmed the presence of white hair shafts in the growing hair follicles of mice exposed to 0.5 Gy γ-rays (Figure 2D), whereas no white hairs were found in control mice (Figure 2C). However, the frequency of white hairs was very low and the differences between control and irradiated mice were not significant (Supplementary Table I available online at http://informahealthcare.com/abs/doi/10.3109/ 09553002.2014.868618). We also studied the degree of pigmentation of hair bulb melanocytes. Histological sections of control mice contained fully pigmented melanosomes in the hair bulb (Figure 2E). However, mice exposed to 2.5 Gy γ-rays possessed hair
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40
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30 Frequency (%)
to 2.5 Gy γ-rays showed relatively sparse hair in comparison to control mice. We decided to analyze histological sections of the dorsal skin with regard to anomalies derived from keratinocyte stem cells, such as hair follicle density and the frequency of curved hair follicles (Supplementary Table I available online at http://informahealthcare.com/abs/doi/ 10.3109/09553002.2014.868618). Histological sections of mouse dorsal skin showed mature hair follicles at the 2nd anagen with some regressed hair follicles at the 1st anagen (Figure 1B). In mice exposed to 2.5 Gy γ-rays, there was a decrease in the number of hair follicles per 0.1 mm2 skin section in the 2nd hair cycle (Figure 1C). Gamma-rays at doses of 0.5 and 1 Gy also caused a significant decrease in the number of hair follicles per 0.1 mm2 skin (Supplementary Table I available online at http://informahealthcare. com/abs/doi/10.3109/09553002.2014.868618). Hair follicle density decreased as the dose increased (Figure 1D). Interestingly, the decreasing rate appeared to reach a plateau at a dose of 1 Gy. An additional malformation was observed in the induction of curved hair follicles (Figure 1F). One or two constrictions in the hair follicles were seen in the curved follicles, even in mice exposed to 0.5 Gy γ-rays, whereas no abnormalities were seen in control mice (Figure 1E). The frequency of curved hair follicles in irradiated mice was significantly different to that in the control group (p ⬍ 0.01, p ⬍ 0.01, p ⬍ 0.05 at 0.5, 1 and 2.5 Gy, respectively) (Supplementary Table I available online at http://informahealthcare.com/abs/doi/ 10.3109/09553002.2014.868618). However, these differences did not appear to be dose-dependent. These results suggest that γ-rays affected hair follicle number and structure in the 2nd anagen phase when stem cells and committed progenitors for keratinocytes were irradiated in the 1st telogen.
2.5 Gy
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1.5 2 γ-rays (Gy)
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Figure 2. Effect of γ-rays on the development of progenies of melanocyte stem cells. (A, B) Whole-mount preparations of dorsal skin show the presence of complete white hair shafts (arrow) in mice exposed to 2.5 Gy γ-rays, but not in control mice. Scale bar, 50 μm. (C) A histological section of the dorsal skin of control mice at the 2nd anagen phase. (D) White hair shaft (arrow) of the 2nd hair cycle was observed in mice exposed to 0.5 Gy γ-rays. (E, F) Mice exposed to 2.5 Gy γ-rays possess hair bulb melanocytes with extremely reduced pigmentation (F, arrow) in contrast to the heavily pigmented hair bulbs of control mice. Scale bar, 50 μm. (G, H) Higher magnification views of hair bulbs of control and irradiated mice. Mice exposed to 2.5 Gy γ-rays possess hair bulb melanocytes with fewer and smaller melanosomes compared with control melanocytes, though melanosomes were mature. Arrows indicate hair bulb melanocytes. Scale bar, 20 μm. (I) Quantitative analysis of the effect of γ-rays on the frequency of hypopigmented hair bulbs. Error bars indicate the standard error of the mean for n ⫽ 28, 29, 28 and 30 at dose of 0, 0.5, 1 and 2.5 Gy, respectively (taken together from 4 independent experiments). P values for comparisons of control mice (0 Gy) were calculated by two-tailed Student’s t-test for comparison of groups of unequal size. The increase in the frequency of hypopigmented hair bulbs is also dose-dependent. This Figure is reproduced in color in the online version of the International Journal of Radiation Biology.
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bulb melanocytes with extremely reduced pigmentation (Figure 2F). The number of hair bulb melanocytes did not appear to be reduced. Melanosomes in irradiated hair bulbs appeared mature but they were much fewer in number and smaller in size in the exposed mice than they were in the control mice (Figure 2G, 2H; Supplementary Table II, available online at http://informahealthcare.com/abs/doi/10.3109/ 09553002.2014.868618). In contrast to the frequency of white hairs, the frequency of hypopigmented hair bulbs increased in a dose-dependent manner and reached more than 18% (Figure 2I). There was a significant increase in the frequency of hypopigmented hair bulbs, even in skin exposed to 0.5 Gy γ-rays (Supplementary Table I available online at http://informahealthcare.com/abs/doi/10.3109/09553002. 2014.868618). These results suggest that γ-irradiation affects stem cells and committed progenitors for melanocytes at the 1st telogen and, as a result, inhibits the formation of melanosomes at the 2nd anagen.
Discussion In the present study, γ-irradiation led to a decrease in hair follicle density and an increase in the frequency of hypopigmented hair bulbs; the effects were dose-dependent. These findings suggest that when keratinocyte and melanocyte stem cells and/or their committed progenies are exposed to γ-rays at the 1st telogen, follicle regeneration is inhibited at the 2nd anagen. Ionizing radiations are known to induce damage in DNA and cause DNA double-strand breaks (Ward 1988, Sotiropoulou et al. 2010). It is possible that DNA damage in both the stem and progenitor cells induces hair follicle decline and abnormal hair follicle structures. However, this hypothesis remains to be investigated using markers for DNA damage and stem cells (Pincelli and Marconi 2010). We found that irradiation with γ-rays resulted in a significant decrease in the density of hair follicles at the 2nd anagen. Interestingly, the decreasing rate appears to reach a plateau at the dose of 1 Gy, suggesting the existence of cells showing radiation resistance. In fact, several types of stem cells and corresponding committed progenitor cells for keratinocytes are thought to exist in the bulge region (Mascré et al. 2012). It may be possible that the subpopulations of stem and/or corresponding progenitor cells in the bulge region differ in radiation sensitivity. This might account for our finding of reduced hair follicle density. Further study is needed to clarify the characteristic features of cells showing radiation resistance. Our results showed that irradiation with γ-rays induced a significant decrease in the density of hair follicles, even at a dose of 0.5 Gy, suggesting that a relatively low dose of γ-rays may affect the regeneration of hair follicles. This dose is below that previously reported to inhibit hair follicle regeneration in young and adult mice (Inomata et al. 2009, Sotiropoulou et al. 2010, Aoki et al. 2013). Therefore, it is reasonable to surmise that our experimental system is useful for determining the minimum dose of ionizing radiations required to inhibit postnatal regeneration of hair follicles in mice. The present results showed that γ-rays caused curved hair follicles, suggesting the possibility that γ-rays induce
abnormal keratinocyte differentiation. Hair morphogenesis is known to depend on transforming growth factor (TGF)-α/ epidermal growth factor receptor (EGFR) signaling (Mann et al. 1993, Luetteke et al. 1994). Defective TGF-α/EGFR signaling leads to abnormal hair morphogenesis (curved or misaligned hair follicles), which is manifested by the wavy hair phenotype of spontaneous loss-of-function murine mutations in TGF-α (known as waved-1 or wa1) and EGFR (waved-2 or wa2) (Mann et al. 1993, Luetteke et al. 1994). Moreover, keratinocyte transient receptor potential cation channel, subfamily V, member 3 (TRPV3), a member of the transient receptor potential (TRP) family of Ca2⫹-permeant channels, forms a signaling complex with TGF-α/EGFR and affects hair morphogenesis (Cheng et al. 2010). TRPV3 knockout mice developed a similar curved or misaligned hair follicle structure, suggesting that the TRP channel plays an important role in the formation of new hair follicles (Cheng et al. 2010). Hair morphogenesis is also regulated by the Wnt/β-catenin signaling pathway. Wnt3a signaling was activated in mouse hair follicle melanocytes during the anagen phase of C57BL/6J (B6) mice (Guo et al. 2012). In addition to melanocytes, adjacent keratinocytes expressed Wnt3a during the anagen phase. A large number of growth factors are related to hair morphogenesis, acidic and basic fibroblast growth factors (aFGF, bFGF), insulin-like growth factor (IGF), hepatocyte growth factor and platelet-derived growth factor (see review of Peus and Pittelkow 1996). FGF-13 (Kawano et al. 2004) and IGF-binding protein 5 (Sriwiriyanont et al. 2011), genes of trichorhinophalanged syndrome, which is the regulator of the Wnt signaling pathway (Fantauzzo and Christiano 2012) and prostaglandin D2 (Garza et al. 2012), are also reported to be related to hair morphogenesis in mice and humans. Although many important signaling pathways are suggested to be involved in the regulation of hair follicle formation and growth, the precise nature, timing and intersection of these inductive and regulatory signals remains largely unknown (Festa et al. 2011, Sennett and Rendl 2012). Hypopigmentation in the hair bulb melanocytes was found in numerous hair follicles, even among the 0.5-Gy irradiation group, and gradually increased with dosage. The number of melanocytes did not appear to be reduced, but melanosomes in the melanocytes of irradiated hair bulbs were decreased and their size was greatly reduced in comparison to control mice. These results suggests that γ-ray exposure inhibited melanosome development rather than maturation in hair bulbs in the subsequent anagen phase after melanocyte stem cells were irradiated with γ-rays at the 1st telogen. A previous study showed that graying of the dorsal hairs was induced in the subsequent anagen phase of 6–7-week-old B6 mice (2nd telogen) irradiated with 5 Gy X-rays at one day after plucking (Inomata et al. 2009). The same report showed that melanocyte stem cells were depleted in numerous hair bulges of the 2nd anagen. X-rays gradually decreased the number of melanocyte stem cell-containing bulges in the subsequent hair cycles with a gradual increase in ectopically pigmented mature melanocytes. Their results suggest that melanocyte stem cells lose their stem cell immaturity and commit to differentiate in the niche after X-ray exposure, resulting in stem cell depletion and subsequent hair graying. Their data show
Gamma-rays and hair follicle stem cells 131 that at least 5 Gy X-rays were required for the stable induction of hair graying. In the present study, however, white hairs and hypopigmented hair bulb melanocytes occurred even at 0.5 Gy γ-rays. Although the discrepancies between their results and the present findings cannot be explained well at present, they might be due to the differences in the types of ionizing radiations (X-rays vs. γ-rays), strains (B6 vs. B10), ages (7–8-week-old vs. 22–24-day-old), hair cycle phase (2nd telogen vs. 1st telogen) and plucking (hair cycle after plucking vs. natural hair cycle without plucking). Hair-plucking could induce several kinds of growth factors and cytokines that affect the proliferation and differentiation of keratinocytes, melanocytes and fibroblasts. In order to exclude the effects of these factors on the behavior of melanocyte stem cells, analysis using the natural hair cycle might be preferable. In addition to keratinocyte stem cells and/or melanocyte stem cells in the bulge, fibroblasts in the dermal papilla and adipocytes near the dermal papilla may be targets of ionizing radiations, as both fibroblasts and adipocytes have been shown to play an important role in hair follicle cycling (Festa et al. 2011, Sennett and Rendl 2012, Chi et al. 2013). Further studies are needed to analyze the effect of ionizing radiations on fibroblasts and adipocytes with regard to the hair cycle. In conclusion, the decrease in the density of hair follicles in the skin at the 2nd anagen and the increase in the frequency of hypopigmented hair bulbs are dependent on the dose of γ-rays. Furthermore, these irradiations induced abnormal hair follicle curvature and white hairs, suggesting that γ-irradiation in the 1st telogen strongly affects regeneration of hair follicles, which are produced by the progenies of keratinocyte and melanocyte stem cells, in the 2nd anagen.
Acknowledgements We thank the members of the Laboratory Animal Science Section of NIRS and Ms. Y. Ishihara, H. Nakamura and S. Inoue for their technical assistance. We also thank Drs H. Mizuno and K. Eguchi-Kasai for their advice on γ-irradiation.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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