Original Contribution Journal of Cosmetic Dermatology, 12, 254--260
Ultrastructure study of hair damage after ultraviolet irradiation Nehal Mohamed Zuel-Fakkar, MD,1 Ekramy Ahmed El Khateeb, MD,2 Hala Sobhi Cousha, MD,3 & Dina Mohamed Hamed, MSc4 1
Department Department 3 Department 4 Department 2
of of of of
Dermatology, Venereology and Andrology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Dermatology, Venereology and Andrology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Dermatology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Summary
Background Natural ultraviolet exposure induces hair damage, which is difficult to avoid. Most of the research work is focused on the effect of ultraviolet on the epidermis, dermis as well as the immune system, whereas the long-term effect of ultraviolet on hair has not been investigated. Aim: we performed our experiment to find out the changes induced in hair follicle and shaft in those patients exposed to high doses of ultraviolet (A and B) during treatment of other skin conditions. Patients and Methods Light and transmission electron microscopy examination of scalp hair follicles and shafts of 10 patients with vitiligo under psoralen plus ultraviolet A (group 1) and 10 patients with vitiligo under narrow band ultraviolet B (group 2) was carried out and compared with those of 10 healthy volunteers (group 3). Results Physical changes in the appearance of hair were more in groups 1 and 2 than control. Reduced hair follicle thickness and perifollicular infiltrate and hyaline disorganized perifollicular collagen were observed more in group 1 than in group 2 with the absence of these changes in group 3. Transmission electron microscopy showed nonspecific cell injury in hair follicles in group 1 more than the other 2 groups, while the damaging effect on hair was more in the second group than the others. Conclusion Due to the damaging effect of ultraviolet on hair, patients under treatment with this modality should be cautious to protect their hair during treatment. Keywords: narrow band-ultraviolet B, psoralen and UVA, hair
Introduction Human hair is constantly subjected to repeated environmental assaults, commonly termed weathering, which is aggravated by various extrinsic damages among which sunlight plays an important role.1 Prolonged exposure to ultraviolet radiation (UVR) causes
Correspondence: N M Zuel- Fakkar, 3 Kolyet el Banat Buildings, Nasr City, Cairo, Egypt. E-mail: [email protected] Accepted for publication August 25, 2013
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dramatic changes to the physical properties of human hair. In extreme cases, irradiation can cause the whole cuticular layer to disintegrate exposing the cortex.2 Ultraviolet bands vary in their penetrating ability to the skin according to their wave length. In general, they do not reach the deeply situated hair follicle melanocytes, and consequently, the stimulatory effect of UVR on these melanocytes may be through an indirect mechanism rather than a direct stimulation.3 Ultraviolet B (UVB) maintains in the cuticle area and is responsible for hair protein loss, while ultraviolet A
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Ultraviolet and hair damage
(UVA) radiation passes through the cuticle, penetrates to the cortex, and is responsible for color changes of hair. The amino acids of the cuticle are altered to a greater extent than those of the cortex because the outer layers of the fiber receive higher intensities of radiation.4 The damage to hair due to UVR is both physical and chemical. Physical damage involves loss in mechanical properties and texture, while chemical damage mainly leads to degradation of hair tissues. This affects the quality and integrity of hair and in turn its quantity.5 We aimed at this work to study the ultrastructure effect of UVR on hair and hair follicle in those patients who are exposed to high and frequent doses of UVA and narrow band UVB (NB-UVB) during treatment of other skin diseases.
Patients and methods This is a controlled comparative study, carried out between December 2010 and December 2012. Patients
Twenty female patients were randomly selected from Dermatology Outpatient Clinic, Ain Shams University Hospital complaining of vitiligo. Patients were all females, and their age ranged from 20 to 40 (29.85 6.40) years. All patients and controls were same socioeconomic status to avoid the effect of factors like vitamin deficiency on hair. Patients were compared with 10 age- and sex-matched healthy volunteers who were served as a control group. All patients and controls were selected used to wear head cover to exclude the effect of natural sun light. Patients’ hair was examined to be looking normal, healthy, and lustrous (inclusion criteria). They were instructed to expose their hair during the sessions inside the cabinet. Our participants were divided into two groups; group 1 (10 patients) for treatment with systemic psoralen and UVA (PUVA) and group 2 (10 patients) for treatment with NB-UVB, both groups were compared with the controls (group 3). Methods
All patients and volunteers were subjected to complete history taking regarding their general health, skin disease, if there are times that they expose their hair to sun light or if there are medications they receive for their skin lesions or other disease. History taking also included the use of hair dryer, hair dying
© 2013 Wiley Periodicals, Inc.
. N M Zuel- Fakkar et al.
or styling, frequency of shampooing, or the use of any chemical agents (factors that may affect hair) (exclusion criteria). Complete general and local examinations were carried out, and all participants signed a written informed consent, and acceptance of ethics committee was taken before the start of the study. Patients of group 1 were treated for their skin lesions using UVA 320–400 nm, Waldmann lighting 7001 (Schwenningen, Germany), equipped with UVA lamps. The machine has physical irradiance values of 11–13 MW/cm2. Dosing of oral psoralen was given according to body weight as 0.6 mg/kg 2 h before exposure to UVA,6 and UVA doses were started according to Fitzpatrick’s classification as skin type 1 with increments 0.5 J/cm2 every other session, depending on skin response with a frequency of three sessions per week. For the second group, NB-UVB (311 nm) machine used was UV-100L Waldmann lightening (Germany), which was equipped with UVB lamps (TL01 lamps, Eindhoven, The Netherlands) that have physical irradiance values of 7–10 MW/cm2 and biological effective (erythematous) irradiance values of 0.4–0.6 MW/cm2. Dosing and increments were also given as if patient with vitiligo is skin type 1, and the frequency of sessions was three sessions per week according to Akdeniz et al., 2013.7 Punch biopsies of 4 mm were taken from the upper occipital region of all patients after 3 months of treatment as well as from the controls. Biopsies were taken under local anesthesia using lidocaine hydrochloride 2%, and biopsies were bisected into 2 equal halves to be prepared for examination by both light and transmission electron microscopies. Light microscopy: One half of each specimen was fixed in 10% formalin solution, dehydrated with alcohol, and embedded in paraffin. Paraffin blocks were serially cut at 4-micron-thick sections and stained with hematoxylin and eosin for histological examination by light microscope (Trinocular light microscope, Olympusâ CX41, Toronto, Canada). Transmission electron microscopy: The other half of each specimen was fixed as fast as possible in glutaraldehyde, postfixed in 2% buffered osmium tetroxide, and then dehydrated in ascending grades of ethyl alcohol. Materials were embedded in spur and then polymerized at 60 °C for 6–18 h. Blocks were trimmed and sectioned at 1 micron thickness on Reichert Jung Ultra microtome using a glass knife. The spur was then re-trimmed to the selected region, and ultrathin section (60–90 nm thickness) was cut on the same ultramicrotome using diamond knife and picked up on copper
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grids and then double stained using uranyl acetate and lead citrate. After staining, the grids were washed on distilled water and then divided on filter paper. The stained grids were examined and photographed using a Philips 400 T electron microscopy (Rotterdam, Netherlands).
Results Cumulative doses ranged from 190 to 650J/cm2 in PUVA group and from 730 to 1020 mJ/cm2 in NB-UVB group. Clinical results
There was splitting of ends, dryness, and reduced strength together with rough texture and loss of luster. Furthermore, we found that hair became lighter in color, stiffer with decreased elongation. One or more of these findings were reported by 100% of the participants of groups 1 and 2 themselves. In contrary, hair shaft was strong, with intact ends and looked smooth and shiny except in 3 (30%) of the participants in group 3 who showed similar findings as in groups 1 and 2. Histopathological results
Hair follicles were reduced in thickness, with shrunken hair bulbs. Trichilemmal keratin was seen encasing the lower portion of some follicles, with the presence of
(a)
(b)
perifollicular inflammatory infiltrate surrounding the affected follicles. The collagen fibers in the area surrounding the affected follicles were hyalinized and disorganized. The cortical cells showed variable degrees of swelling and vacuolization in their cytoplasm, and some cells showed condensed nuclei surrounded by a bright halo. There were some distorted thick follicles filled with loose laminated keratin and compressed dermal papillae (Fig. 1). Those previous changes were milder in group 2 (treated with NB-UVB) than in group 1 (treated with NB-UVB) and completely absent in group 3 (Fig. 2) except for swelling and vacuolization of the cytoplasm of some cells in one patient. Transmission electron microscope results
Hair follicles In group 1, there was severe nonspecific injury in the cortical keratinocytes in the form of cytoplasmic swelling and vacuolization in 9 of 10 cases (90%) and pouch-like protrusions of the cytoplasm in 6 of 10 cases (60%). Necrotic debris was seen between keratinocytes, and apoptotic bodies were also observed in the keratinocytes with nuclear fragmentation. Some of the cells showed partial apoptosis with persistence of the nucleus, while others showed complete apoptosis with smudged nuclei (Figs. 3 and 4). Focal lysis of the plasma membrane was seen, and melanocytes were few in number. Over all, the nuclear effects were more than the membrane effects in all
(c)
Figure 1 (a) A section in skin with hair follicle after UVA irradiation showing shrunken hair bulb (black arrow) and reduced thickness of hair follicle. The lower portion of the follicle is encased in trichilemmal keratin (tk). The section shows perifollicular disorganized collagen fibers (H&E 200). (b) a section in hair follicle after UVA irradiation, showing swelling and vacuolization of the cytoplasm of cortical cells (2 narrow arrows). Shrunken hair bulb with compressed dermal papilla and the hair follicle is distorted and thick, filled with loose laminated keratin with mild perifollicular infiltrate (i) (H&E 400). (c) A section of hair follicle after UVB irradiation showing cortical cells with mild vacuolization of their cytoplasm (blue arrow) and a very mild inflammatory infiltrate (H&E 400).
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patients, and findings were severe in group 1 than group 2, while group 3 showed cell injury only in 3 cases (30%). Hair shaft Thinning and fusion of the cuticular cells were observed. Other hair shafts showed rupture and detachment, sometimes complete disappearance, of the cuticular layers. Cleavage along the endocuticle was seen in all shafts (100%). Those changes were more severe in group 2 than in group 1, while in group 3,
(a)
(b)
Figure 2 (a, b) shows normal hair follicle with normal pulp area from 2 normal controls.
(a)
(b)
. N M Zuel- Fakkar et al.
the shafts looked undamaged with normal cuticle and intact concentric arrangement of smoothly bounded cuticular cells (Fig. 5).
Discussion Hair appearance is of significant cosmetic relevance, especially in females.8 Although the effect of UVR on the extracutaneously located hair shaft is well recognized, its impact on the hair shaft production is not yet well studied (except in vitro), especially in those patients exposed to high doses of UVR during treatment of their skin lesions; hence, this was our study. In this study, we found shrunken hair bulb and reduced hair follicle thickness in hair follicles exposed to UVR. According to Lee et al.,1 these results are due to the DNA damage and inhibition of cell growth in keratinocytes. Muller-Rover et al., 9 added that hair follicle regression is mainly driven by massive apoptosis of hair bulb keratinocytes. Our study showed that keratin was encasing the lower portion of only few hair follicles, and this can be explained by Sebtic et al.,10 who proved in vitro that absorption of radiations results in amino acids photochemical degradation producing free radicals that in turn have an adverse impact on hair protein, especially keratin.
(c)
Figure 3 (a) An electron micrograph showing cross-section of the cortex of a fully keratinized normal hair showing homogenous condensed keratin (K), pigment granules (P), interlocking cell membranes that appear denser than keratin and nuclear remnant (N) (6000 magnification). (b) An electron micrograph of hair follicle after UVA irradiation showing marked cytoplasmic vacuolization and cytoplasmic protrusions are seen compressing the tonofilaments (t) (4600 magnification). (c) An electron micrograph of hair follicle after UVA irradiation, showing three apoptotic bodies in the lower part of section (arrows), in one of them remnants of nucleus is still seen, while in the other two, it is smudged. Cytoplasmic vacuoles and condensed tonofilaments are seen (4600 magnification).
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. N M Zuel- Fakkar et al. to the induction and activation of death receptors on the cell surface.12 It was reported by Ito et al.,13that human hair follicle displays a functional equivalent of the hypothalamic–pituitary–adrenal axis and synthesize cortisol that moves the hair follicle in the catagen
The apoptotic figures seen in our results can be attributed to the stress induced upon exposure to UVR, with subsequent production of corticotrophin-releasing factors by human melanocytes and keratinocytes.11 It can also be attributed to the UV damage of DNA and
(a)
(b)
Figure 4 (a) An electron micrograph after UVB showing apoptotic keratinocyte with nuclear indentation (thick arrow) and degenerative changes in the form of cytoplasmic vacuoles (6000 magnification). (b) An electron micrograph of a section in the cortex after UVB showing degenerative changes in keratinocytes mainly vacuolization (narrow arrows) of the cytoplasm, and some nuclei are absent. Cytoplasmic protrusions are seen in some of the cells. (2000 magnification).
(a)
(b)
(c)
Figure 5 (a) An electron micrograph for hair shaft from the control group showing concentric arrangement of smoothly bounded cuticular cells (8000 magnification). (b) An electron micrograph for hair shaft after UVA showing cleavage along the endocuticle with cuticular detachment ( magnification). (c) An electron micrograph for hair shaft after UVB showing similar cleavage along the endocuticle with cuticular detachment but with more pronounced changes than those seen after UVA due to the loss of concentric arrangement of the smoothly bounded cuticular cells (8000 magnification).
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phase with appearance of such apoptosis inhibiting hair shaft production due to inhibition of keratinocytes proliferation. The previous changes may explain the decreased elongation of hair and the reduced strength noted from the patients’ history and in agreement with the in vitro results obtained by Lu et al.,14 Reduction in hair pigmentation—both that clinically obtained from patients’ history and observed in the microscopic examination—in the two groups treated with UVR when compared to the control group may be a consequence of hair follicle regression, which is known to be associated with stopped follicular melanogenesis.11,15 It is generally believed that keratinocytes are actively involved in the control of pigmentation and any insult to these cells may affect its ability to accept pigment or perhaps to initiate melanocytes activity.16 Another explanation for that finding is the melanin oxidation through the free radicals production after UV exposure.17 Furthermore, in our patients, reduction in the melanin content of hair may be partially related to the original disease (vitiligo), although we selected patients who have no hair affection, still it could be the pathological changes in unaffected skin. Our study also presented the photodamaging effect of UVR on collagen. The hyalinization with degradation and disorganization of collagen bundles may be the result of the inflammatory cell infiltrate observed in the perifollicular area with subsequent production of inflammatory mediators that control fibroblast function, collagen deposition with a subsequent imbalance between metalloproteinase and synthetase, and improper structure of the newly deposited collagen.18 The swelling, vacuolization of cytoplasm in cortical cells, and the condensed nuclei might be a consequence of the UVR-induced apoptosis and represent a stage toward necrosis and the subsequent observed shrunken bulbs. The changes observed in the cuticular layer were more in patients exposed to NB-UVB than in those treated with PUVA. This can be explained by the fact that although both types of UVR affect this layer of the hair follicle, yet the superficially effecting NB-UVB is more harmful to it than the deeply penetrating UVA that bypasses it to the cortex. Those cuticular changes in the form of thinning, fusion, and sometimes disappearance of the cuticular layer explain the clinically observed loss of luster and split ends of hair in the two groups exposed to UVR.19 Moreover, UVB decreases the water content of the proximal root that may account for changes in the physiochemical properties of hair and consequently to the changes in the external appearance.20
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. N M Zuel- Fakkar et al.
The distorted follicles and compressed dermal papillae found more in PUVA-treated group than NB-UVB group explain the rough texture of hair and the decreased strength, respectively, claimed by the patients of both groups after UV exposure and are the results of UVR-induced damage of the follicular cells.21 It is noteworthy that the disorganized collagen bundles surrounding the hair follicles may have a role in that hair follicle distortion. Additionally, it was found that integral lipid layer in the cuticle acts as hair barrier and exposure to UVA leads to bulging of this layer, while exposure to UVB disrupts this layer. Those findings were found to be more pronounced in African populations due to the ethnic decrease in lipid content and account for the physical changes that observed in our patients 4 The present study provides information that UVR changes the function and physiological characters of hair follicle by exerting a complex direct and indirect effects on hair follicle and shaft, which could be reflected on the appearance and growth of hair. Those changes could be more pronounced in patients receiving UVR in attempts to treat other dermatological diseases or those using UV lamps for tanning, due to the extensive and more frequent direct exposure to UVR. Further studies are still warranted to detect the effect of different UV doses on hair follicle and shaft and the nature of the inflammatory infiltrate with the produced inflammatory mediators. Moreover, we need to study the markers expressed on different cell types upon exposure to UV and the imbalance in collagen production in the perifollicular stroma. Study of the effect of UV on hair before and after treatment is also needed to get more accurate information. Patients under treatment with UVR need to be instructed to cover their heads during treatment to protect their hair; however, we would also like to study whether there is any indirect effect of UV on hair.
References 1 Lee CH, Yu CL, Liao WT et al. Effect and interactions of low doses of arsenic and UVB on keratinocytes apoptosis. Chem Res Toxicol 2004; 17: 1199–1205. 2 Signori V. Review of the current understanding of the effect of ultraviolet and visible radiation on hair structure and options for photoprotection. J Cosmet Sci 2004; 55: 95–113. 3 Kochevar IE, Pathak MA, Parish JA. Photophysics, photochemistry, and photobiology. In: IM Freedberg, AZ Eisen, K Wolf, KF Austen, LA Goldsmith, eds. Fitzpatrick’s Dermatology in General Medicine, 5th edn. chapter 16
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5
6 7
8 9
10 11
12
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(P1). New York: McGraw-Hill, Health Professions Division; 1999: pp. 950–952. Ji JH, Park TS, Lee HJ et al. The ethnic differences of the damage of hair and integral hair lipid after ultraviolet radiation. Ann Dermatol 2013; 25: 54–60. Daud FS, Kaulkarni SB. Comparative evaluation of photo protective effect of Aloe vera Toutn.ex linn on UV damage in different Asian hair types. Indian J of Nat Prod Resour 2011; 2: 179–183. Kar PK, Snehi PS, Jha PK. Treatment of vitiligo with psoralen. Indian J Dermatol Venereol Leprol 1990; 55: 121–122. Akdeniz N, Yavuz IH, Bilgili SG et al. Comparison of Efficacy of Narrow Band UVB-Alone, Combination of Calcipotriol-Narrow Band UVB, and Combination Betamethasone-Calcipotriol- Narrow Band UVB Therapies in Vitiligo. J Dermatolog Treat 2013. [Epub ahead of print] Schlake T. Determination of hair structure and shape. Semin Cell Dev Biol 2007; 18: 267–273. Muller-Rover S, Rossiter H, Paus R et al. Overexpression of Bcl-2 protects from ultraviolet B induced apoptosis but promotes hair follicle regression and chemotherapy induced alopecia. Am J Pathol 2000; 156 (4): 1395–405. Sebtic K, Sjerobabski Masnec I, Cavka V et al. UV damage of the hair. Coll Antropol 2008; 32: 163–165. Solminski A, Baker J, Ermak G et al. Ultraviolet B stimulates production of corticotrophin releasing factor (CRF) by human melanocytes. FEBS Lett 1996; 399: 175–176. Kulms D, Poppelman B, Yarosh D et al. Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB
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15 16
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radiation. Proc Natl Acad Sci USA 1999; 96: 7974–7979. Ito N, Ito T, Kromminga A et al. Human hair follicles display a functional equivalent of the hypothalamic- pituitary- adrenal (HPA) axis and synthesize cortisol. FASEB J 2005; 19: 1332–1334. Lu Z, Fischer TW, Hass S et al. Profiling the response of human hair follicles to ultraviolet radiation. J Invest Dermatol 2009; 129: 1790–1804. Tobin DJ, Paus R. Graying: gerontobiology of the hair follicle pigmentary unit. Exp Gerontol 2001; 36: 29–54. Messenger AG, Bleehen SS. Alopecia areata (1984): light and electron microscopic pathology of the regrowing white hair. Br J Dermatol 110: 155- 162. Fernandez E, Barba C, Alonzo C et al. Photodamage determination of human hair. J Photochem Photobiol, B 2012; 106: 101–106. Averbeck M, Beilharz S, Bauer M et al. In situ profiling and quantification of cytokines released during ultraviolet B-induced inflammation by combining dermal microdialysis and protein microarrays. Exp Dermatol 2006; 15: 447– 454. Jeon SY, Pi LQ, Lee WS. Comparison of hair shaft damage after UVA and UVB irradiations. J Cosmet Sci 2008; 59: 151–156. Kim KS, Shin MK, Park HK. Effects of ultraviolet B radiation on physicochemical properties of human hair shaft. Microsec Res Tech 2012; 75: 949–954. Rabe JH, Mamelak AJ, McElgunn PJ et al. Photoaging: mechanisms and repair. J Am Acad Dermatol 2006; 55: 1–19.
© 2013 Wiley Periodicals, Inc.
Ultrastructure study of hair damage after ultraviolet irradiation Nehal Mohamed Zuel-Fakkar, MD,1 Ekramy Ahmed El Khateeb, MD,2 Hala Sobhi Cousha, MD,3 & Dina Mohamed Hamed, MSc4 1
Department Department 3 Department 4 Department 2
of of of of
Dermatology, Venereology and Andrology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Dermatology, Venereology and Andrology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Dermatology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Summary
Background Natural ultraviolet exposure induces hair damage, which is difficult to avoid. Most of the research work is focused on the effect of ultraviolet on the epidermis, dermis as well as the immune system, whereas the long-term effect of ultraviolet on hair has not been investigated. Aim: we performed our experiment to find out the changes induced in hair follicle and shaft in those patients exposed to high doses of ultraviolet (A and B) during treatment of other skin conditions. Patients and Methods Light and transmission electron microscopy examination of scalp hair follicles and shafts of 10 patients with vitiligo under psoralen plus ultraviolet A (group 1) and 10 patients with vitiligo under narrow band ultraviolet B (group 2) was carried out and compared with those of 10 healthy volunteers (group 3). Results Physical changes in the appearance of hair were more in groups 1 and 2 than control. Reduced hair follicle thickness and perifollicular infiltrate and hyaline disorganized perifollicular collagen were observed more in group 1 than in group 2 with the absence of these changes in group 3. Transmission electron microscopy showed nonspecific cell injury in hair follicles in group 1 more than the other 2 groups, while the damaging effect on hair was more in the second group than the others. Conclusion Due to the damaging effect of ultraviolet on hair, patients under treatment with this modality should be cautious to protect their hair during treatment. Keywords: narrow band-ultraviolet B, psoralen and UVA, hair
Introduction Human hair is constantly subjected to repeated environmental assaults, commonly termed weathering, which is aggravated by various extrinsic damages among which sunlight plays an important role.1 Prolonged exposure to ultraviolet radiation (UVR) causes
Correspondence: N M Zuel- Fakkar, 3 Kolyet el Banat Buildings, Nasr City, Cairo, Egypt. E-mail: [email protected] Accepted for publication August 25, 2013
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dramatic changes to the physical properties of human hair. In extreme cases, irradiation can cause the whole cuticular layer to disintegrate exposing the cortex.2 Ultraviolet bands vary in their penetrating ability to the skin according to their wave length. In general, they do not reach the deeply situated hair follicle melanocytes, and consequently, the stimulatory effect of UVR on these melanocytes may be through an indirect mechanism rather than a direct stimulation.3 Ultraviolet B (UVB) maintains in the cuticle area and is responsible for hair protein loss, while ultraviolet A
© 2013 Wiley Periodicals, Inc.
Ultraviolet and hair damage
(UVA) radiation passes through the cuticle, penetrates to the cortex, and is responsible for color changes of hair. The amino acids of the cuticle are altered to a greater extent than those of the cortex because the outer layers of the fiber receive higher intensities of radiation.4 The damage to hair due to UVR is both physical and chemical. Physical damage involves loss in mechanical properties and texture, while chemical damage mainly leads to degradation of hair tissues. This affects the quality and integrity of hair and in turn its quantity.5 We aimed at this work to study the ultrastructure effect of UVR on hair and hair follicle in those patients who are exposed to high and frequent doses of UVA and narrow band UVB (NB-UVB) during treatment of other skin diseases.
Patients and methods This is a controlled comparative study, carried out between December 2010 and December 2012. Patients
Twenty female patients were randomly selected from Dermatology Outpatient Clinic, Ain Shams University Hospital complaining of vitiligo. Patients were all females, and their age ranged from 20 to 40 (29.85 6.40) years. All patients and controls were same socioeconomic status to avoid the effect of factors like vitamin deficiency on hair. Patients were compared with 10 age- and sex-matched healthy volunteers who were served as a control group. All patients and controls were selected used to wear head cover to exclude the effect of natural sun light. Patients’ hair was examined to be looking normal, healthy, and lustrous (inclusion criteria). They were instructed to expose their hair during the sessions inside the cabinet. Our participants were divided into two groups; group 1 (10 patients) for treatment with systemic psoralen and UVA (PUVA) and group 2 (10 patients) for treatment with NB-UVB, both groups were compared with the controls (group 3). Methods
All patients and volunteers were subjected to complete history taking regarding their general health, skin disease, if there are times that they expose their hair to sun light or if there are medications they receive for their skin lesions or other disease. History taking also included the use of hair dryer, hair dying
© 2013 Wiley Periodicals, Inc.
. N M Zuel- Fakkar et al.
or styling, frequency of shampooing, or the use of any chemical agents (factors that may affect hair) (exclusion criteria). Complete general and local examinations were carried out, and all participants signed a written informed consent, and acceptance of ethics committee was taken before the start of the study. Patients of group 1 were treated for their skin lesions using UVA 320–400 nm, Waldmann lighting 7001 (Schwenningen, Germany), equipped with UVA lamps. The machine has physical irradiance values of 11–13 MW/cm2. Dosing of oral psoralen was given according to body weight as 0.6 mg/kg 2 h before exposure to UVA,6 and UVA doses were started according to Fitzpatrick’s classification as skin type 1 with increments 0.5 J/cm2 every other session, depending on skin response with a frequency of three sessions per week. For the second group, NB-UVB (311 nm) machine used was UV-100L Waldmann lightening (Germany), which was equipped with UVB lamps (TL01 lamps, Eindhoven, The Netherlands) that have physical irradiance values of 7–10 MW/cm2 and biological effective (erythematous) irradiance values of 0.4–0.6 MW/cm2. Dosing and increments were also given as if patient with vitiligo is skin type 1, and the frequency of sessions was three sessions per week according to Akdeniz et al., 2013.7 Punch biopsies of 4 mm were taken from the upper occipital region of all patients after 3 months of treatment as well as from the controls. Biopsies were taken under local anesthesia using lidocaine hydrochloride 2%, and biopsies were bisected into 2 equal halves to be prepared for examination by both light and transmission electron microscopies. Light microscopy: One half of each specimen was fixed in 10% formalin solution, dehydrated with alcohol, and embedded in paraffin. Paraffin blocks were serially cut at 4-micron-thick sections and stained with hematoxylin and eosin for histological examination by light microscope (Trinocular light microscope, Olympusâ CX41, Toronto, Canada). Transmission electron microscopy: The other half of each specimen was fixed as fast as possible in glutaraldehyde, postfixed in 2% buffered osmium tetroxide, and then dehydrated in ascending grades of ethyl alcohol. Materials were embedded in spur and then polymerized at 60 °C for 6–18 h. Blocks were trimmed and sectioned at 1 micron thickness on Reichert Jung Ultra microtome using a glass knife. The spur was then re-trimmed to the selected region, and ultrathin section (60–90 nm thickness) was cut on the same ultramicrotome using diamond knife and picked up on copper
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. N M Zuel- Fakkar et al.
grids and then double stained using uranyl acetate and lead citrate. After staining, the grids were washed on distilled water and then divided on filter paper. The stained grids were examined and photographed using a Philips 400 T electron microscopy (Rotterdam, Netherlands).
Results Cumulative doses ranged from 190 to 650J/cm2 in PUVA group and from 730 to 1020 mJ/cm2 in NB-UVB group. Clinical results
There was splitting of ends, dryness, and reduced strength together with rough texture and loss of luster. Furthermore, we found that hair became lighter in color, stiffer with decreased elongation. One or more of these findings were reported by 100% of the participants of groups 1 and 2 themselves. In contrary, hair shaft was strong, with intact ends and looked smooth and shiny except in 3 (30%) of the participants in group 3 who showed similar findings as in groups 1 and 2. Histopathological results
Hair follicles were reduced in thickness, with shrunken hair bulbs. Trichilemmal keratin was seen encasing the lower portion of some follicles, with the presence of
(a)
(b)
perifollicular inflammatory infiltrate surrounding the affected follicles. The collagen fibers in the area surrounding the affected follicles were hyalinized and disorganized. The cortical cells showed variable degrees of swelling and vacuolization in their cytoplasm, and some cells showed condensed nuclei surrounded by a bright halo. There were some distorted thick follicles filled with loose laminated keratin and compressed dermal papillae (Fig. 1). Those previous changes were milder in group 2 (treated with NB-UVB) than in group 1 (treated with NB-UVB) and completely absent in group 3 (Fig. 2) except for swelling and vacuolization of the cytoplasm of some cells in one patient. Transmission electron microscope results
Hair follicles In group 1, there was severe nonspecific injury in the cortical keratinocytes in the form of cytoplasmic swelling and vacuolization in 9 of 10 cases (90%) and pouch-like protrusions of the cytoplasm in 6 of 10 cases (60%). Necrotic debris was seen between keratinocytes, and apoptotic bodies were also observed in the keratinocytes with nuclear fragmentation. Some of the cells showed partial apoptosis with persistence of the nucleus, while others showed complete apoptosis with smudged nuclei (Figs. 3 and 4). Focal lysis of the plasma membrane was seen, and melanocytes were few in number. Over all, the nuclear effects were more than the membrane effects in all
(c)
Figure 1 (a) A section in skin with hair follicle after UVA irradiation showing shrunken hair bulb (black arrow) and reduced thickness of hair follicle. The lower portion of the follicle is encased in trichilemmal keratin (tk). The section shows perifollicular disorganized collagen fibers (H&E 200). (b) a section in hair follicle after UVA irradiation, showing swelling and vacuolization of the cytoplasm of cortical cells (2 narrow arrows). Shrunken hair bulb with compressed dermal papilla and the hair follicle is distorted and thick, filled with loose laminated keratin with mild perifollicular infiltrate (i) (H&E 400). (c) A section of hair follicle after UVB irradiation showing cortical cells with mild vacuolization of their cytoplasm (blue arrow) and a very mild inflammatory infiltrate (H&E 400).
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patients, and findings were severe in group 1 than group 2, while group 3 showed cell injury only in 3 cases (30%). Hair shaft Thinning and fusion of the cuticular cells were observed. Other hair shafts showed rupture and detachment, sometimes complete disappearance, of the cuticular layers. Cleavage along the endocuticle was seen in all shafts (100%). Those changes were more severe in group 2 than in group 1, while in group 3,
(a)
(b)
Figure 2 (a, b) shows normal hair follicle with normal pulp area from 2 normal controls.
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the shafts looked undamaged with normal cuticle and intact concentric arrangement of smoothly bounded cuticular cells (Fig. 5).
Discussion Hair appearance is of significant cosmetic relevance, especially in females.8 Although the effect of UVR on the extracutaneously located hair shaft is well recognized, its impact on the hair shaft production is not yet well studied (except in vitro), especially in those patients exposed to high doses of UVR during treatment of their skin lesions; hence, this was our study. In this study, we found shrunken hair bulb and reduced hair follicle thickness in hair follicles exposed to UVR. According to Lee et al.,1 these results are due to the DNA damage and inhibition of cell growth in keratinocytes. Muller-Rover et al., 9 added that hair follicle regression is mainly driven by massive apoptosis of hair bulb keratinocytes. Our study showed that keratin was encasing the lower portion of only few hair follicles, and this can be explained by Sebtic et al.,10 who proved in vitro that absorption of radiations results in amino acids photochemical degradation producing free radicals that in turn have an adverse impact on hair protein, especially keratin.
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Figure 3 (a) An electron micrograph showing cross-section of the cortex of a fully keratinized normal hair showing homogenous condensed keratin (K), pigment granules (P), interlocking cell membranes that appear denser than keratin and nuclear remnant (N) (6000 magnification). (b) An electron micrograph of hair follicle after UVA irradiation showing marked cytoplasmic vacuolization and cytoplasmic protrusions are seen compressing the tonofilaments (t) (4600 magnification). (c) An electron micrograph of hair follicle after UVA irradiation, showing three apoptotic bodies in the lower part of section (arrows), in one of them remnants of nucleus is still seen, while in the other two, it is smudged. Cytoplasmic vacuoles and condensed tonofilaments are seen (4600 magnification).
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. N M Zuel- Fakkar et al. to the induction and activation of death receptors on the cell surface.12 It was reported by Ito et al.,13that human hair follicle displays a functional equivalent of the hypothalamic–pituitary–adrenal axis and synthesize cortisol that moves the hair follicle in the catagen
The apoptotic figures seen in our results can be attributed to the stress induced upon exposure to UVR, with subsequent production of corticotrophin-releasing factors by human melanocytes and keratinocytes.11 It can also be attributed to the UV damage of DNA and
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Figure 4 (a) An electron micrograph after UVB showing apoptotic keratinocyte with nuclear indentation (thick arrow) and degenerative changes in the form of cytoplasmic vacuoles (6000 magnification). (b) An electron micrograph of a section in the cortex after UVB showing degenerative changes in keratinocytes mainly vacuolization (narrow arrows) of the cytoplasm, and some nuclei are absent. Cytoplasmic protrusions are seen in some of the cells. (2000 magnification).
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Figure 5 (a) An electron micrograph for hair shaft from the control group showing concentric arrangement of smoothly bounded cuticular cells (8000 magnification). (b) An electron micrograph for hair shaft after UVA showing cleavage along the endocuticle with cuticular detachment ( magnification). (c) An electron micrograph for hair shaft after UVB showing similar cleavage along the endocuticle with cuticular detachment but with more pronounced changes than those seen after UVA due to the loss of concentric arrangement of the smoothly bounded cuticular cells (8000 magnification).
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phase with appearance of such apoptosis inhibiting hair shaft production due to inhibition of keratinocytes proliferation. The previous changes may explain the decreased elongation of hair and the reduced strength noted from the patients’ history and in agreement with the in vitro results obtained by Lu et al.,14 Reduction in hair pigmentation—both that clinically obtained from patients’ history and observed in the microscopic examination—in the two groups treated with UVR when compared to the control group may be a consequence of hair follicle regression, which is known to be associated with stopped follicular melanogenesis.11,15 It is generally believed that keratinocytes are actively involved in the control of pigmentation and any insult to these cells may affect its ability to accept pigment or perhaps to initiate melanocytes activity.16 Another explanation for that finding is the melanin oxidation through the free radicals production after UV exposure.17 Furthermore, in our patients, reduction in the melanin content of hair may be partially related to the original disease (vitiligo), although we selected patients who have no hair affection, still it could be the pathological changes in unaffected skin. Our study also presented the photodamaging effect of UVR on collagen. The hyalinization with degradation and disorganization of collagen bundles may be the result of the inflammatory cell infiltrate observed in the perifollicular area with subsequent production of inflammatory mediators that control fibroblast function, collagen deposition with a subsequent imbalance between metalloproteinase and synthetase, and improper structure of the newly deposited collagen.18 The swelling, vacuolization of cytoplasm in cortical cells, and the condensed nuclei might be a consequence of the UVR-induced apoptosis and represent a stage toward necrosis and the subsequent observed shrunken bulbs. The changes observed in the cuticular layer were more in patients exposed to NB-UVB than in those treated with PUVA. This can be explained by the fact that although both types of UVR affect this layer of the hair follicle, yet the superficially effecting NB-UVB is more harmful to it than the deeply penetrating UVA that bypasses it to the cortex. Those cuticular changes in the form of thinning, fusion, and sometimes disappearance of the cuticular layer explain the clinically observed loss of luster and split ends of hair in the two groups exposed to UVR.19 Moreover, UVB decreases the water content of the proximal root that may account for changes in the physiochemical properties of hair and consequently to the changes in the external appearance.20
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The distorted follicles and compressed dermal papillae found more in PUVA-treated group than NB-UVB group explain the rough texture of hair and the decreased strength, respectively, claimed by the patients of both groups after UV exposure and are the results of UVR-induced damage of the follicular cells.21 It is noteworthy that the disorganized collagen bundles surrounding the hair follicles may have a role in that hair follicle distortion. Additionally, it was found that integral lipid layer in the cuticle acts as hair barrier and exposure to UVA leads to bulging of this layer, while exposure to UVB disrupts this layer. Those findings were found to be more pronounced in African populations due to the ethnic decrease in lipid content and account for the physical changes that observed in our patients 4 The present study provides information that UVR changes the function and physiological characters of hair follicle by exerting a complex direct and indirect effects on hair follicle and shaft, which could be reflected on the appearance and growth of hair. Those changes could be more pronounced in patients receiving UVR in attempts to treat other dermatological diseases or those using UV lamps for tanning, due to the extensive and more frequent direct exposure to UVR. Further studies are still warranted to detect the effect of different UV doses on hair follicle and shaft and the nature of the inflammatory infiltrate with the produced inflammatory mediators. Moreover, we need to study the markers expressed on different cell types upon exposure to UV and the imbalance in collagen production in the perifollicular stroma. Study of the effect of UV on hair before and after treatment is also needed to get more accurate information. Patients under treatment with UVR need to be instructed to cover their heads during treatment to protect their hair; however, we would also like to study whether there is any indirect effect of UV on hair.
References 1 Lee CH, Yu CL, Liao WT et al. Effect and interactions of low doses of arsenic and UVB on keratinocytes apoptosis. Chem Res Toxicol 2004; 17: 1199–1205. 2 Signori V. Review of the current understanding of the effect of ultraviolet and visible radiation on hair structure and options for photoprotection. J Cosmet Sci 2004; 55: 95–113. 3 Kochevar IE, Pathak MA, Parish JA. Photophysics, photochemistry, and photobiology. In: IM Freedberg, AZ Eisen, K Wolf, KF Austen, LA Goldsmith, eds. Fitzpatrick’s Dermatology in General Medicine, 5th edn. chapter 16
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5
6 7
8 9
10 11
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(P1). New York: McGraw-Hill, Health Professions Division; 1999: pp. 950–952. Ji JH, Park TS, Lee HJ et al. The ethnic differences of the damage of hair and integral hair lipid after ultraviolet radiation. Ann Dermatol 2013; 25: 54–60. Daud FS, Kaulkarni SB. Comparative evaluation of photo protective effect of Aloe vera Toutn.ex linn on UV damage in different Asian hair types. Indian J of Nat Prod Resour 2011; 2: 179–183. Kar PK, Snehi PS, Jha PK. Treatment of vitiligo with psoralen. Indian J Dermatol Venereol Leprol 1990; 55: 121–122. Akdeniz N, Yavuz IH, Bilgili SG et al. Comparison of Efficacy of Narrow Band UVB-Alone, Combination of Calcipotriol-Narrow Band UVB, and Combination Betamethasone-Calcipotriol- Narrow Band UVB Therapies in Vitiligo. J Dermatolog Treat 2013. [Epub ahead of print] Schlake T. Determination of hair structure and shape. Semin Cell Dev Biol 2007; 18: 267–273. Muller-Rover S, Rossiter H, Paus R et al. Overexpression of Bcl-2 protects from ultraviolet B induced apoptosis but promotes hair follicle regression and chemotherapy induced alopecia. Am J Pathol 2000; 156 (4): 1395–405. Sebtic K, Sjerobabski Masnec I, Cavka V et al. UV damage of the hair. Coll Antropol 2008; 32: 163–165. Solminski A, Baker J, Ermak G et al. Ultraviolet B stimulates production of corticotrophin releasing factor (CRF) by human melanocytes. FEBS Lett 1996; 399: 175–176. Kulms D, Poppelman B, Yarosh D et al. Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB
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radiation. Proc Natl Acad Sci USA 1999; 96: 7974–7979. Ito N, Ito T, Kromminga A et al. Human hair follicles display a functional equivalent of the hypothalamic- pituitary- adrenal (HPA) axis and synthesize cortisol. FASEB J 2005; 19: 1332–1334. Lu Z, Fischer TW, Hass S et al. Profiling the response of human hair follicles to ultraviolet radiation. J Invest Dermatol 2009; 129: 1790–1804. Tobin DJ, Paus R. Graying: gerontobiology of the hair follicle pigmentary unit. Exp Gerontol 2001; 36: 29–54. Messenger AG, Bleehen SS. Alopecia areata (1984): light and electron microscopic pathology of the regrowing white hair. Br J Dermatol 110: 155- 162. Fernandez E, Barba C, Alonzo C et al. Photodamage determination of human hair. J Photochem Photobiol, B 2012; 106: 101–106. Averbeck M, Beilharz S, Bauer M et al. In situ profiling and quantification of cytokines released during ultraviolet B-induced inflammation by combining dermal microdialysis and protein microarrays. Exp Dermatol 2006; 15: 447– 454. Jeon SY, Pi LQ, Lee WS. Comparison of hair shaft damage after UVA and UVB irradiations. J Cosmet Sci 2008; 59: 151–156. Kim KS, Shin MK, Park HK. Effects of ultraviolet B radiation on physicochemical properties of human hair shaft. Microsec Res Tech 2012; 75: 949–954. Rabe JH, Mamelak AJ, McElgunn PJ et al. Photoaging: mechanisms and repair. J Am Acad Dermatol 2006; 55: 1–19.
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