Original Contribution Journal of Cosmetic Dermatology, 14, 178--184
Changes in the sebaceous gland in patients with male pattern hair loss (androgenic alopecia) Katsuhiro Kure, MD,1,2 Tsukasa Isago, MD,1 & Takeshi Hirayama, MD3 1
Department of Plastic and Reconstructive Surgery, Tokyo Women’s Medical University Medical Center East, Tokyo, Japan Plaza Plastic Surgery, Tokyo, Japan 3 Memorial Clinic Hirayama, Tokyo, Japan 2
Summary
Background The sebaceous gland and the follicular bulge region have important role in biology of the hair. They initiate destruction of the hair follicle both in human and animal models in certain conditions. The morphometric feature of the sebaceous gland is not well understood so as the distribution of the bulge stem cells in pathological conditions of male pattern hair loss or androgenic alopecia (AGA). Objectives The authors perform morphometric analysis of the sebaceous gland in AGA patients and also study distribution of the follicular stem cells in the bulge region in these populations. Methods Two hundred and fifty cases of glass slide specimen from Japanese patients with male pattern hair loss were reviewed. Among these, 23 cases of the longitudinal (vertical) sections of the scalp skin with diagnosis of AGA were found and analyzed for the morphometric characteristics. Each sebaceous gland area was measured using NIH IMAGEJ system and statistically analyzed. For the identification of the follicular bulge region, an immunohistochemistry using anticytokeratin 15 (C8/144B clone) was carried out in the cases of AGA. Results The sebaceous gland area of the AGA group was noticeably increased, while the size of each sebaceous gland remains unchanged. It has more lobules in the hair follicular unit in the AGA population. In the immunohistochemistry, the follicular stem cells are present in the bulge regions in cases of AGA. Conclusions The overgrowth (multilobulation) of the sebaceous gland and relative preservation of the follicular stem cells suggest that the changes in the sebaceous gland could be an important factor in the pathology of AGA. Keywords: androgenic alopecia, sebaceous immunohistochemistry, male pattern hair loss
Alopecia (male and female pattern hair loss) is not only a medical condition but often devastating social aspects of the patients involved. More than 30–40% of male population over age 20 years old show androCorrespondence: Dr. Katsuhiro Kure, Department of Plastic and Reconstructive Surgery, Tokyo Women’s Medical University Medical Center East, 2-1-10 Nishioku, Arakawa-ku, Tokyo 116-8567, Japan. E-mail: [email protected] Accepted for publication April 9, 2015
178
gland,
follicular
stem
cell,
genic alopecia (AGA) or male pattern hair loss.1–4 Because of recent introduction of medical treatment using finasteride and minoxidil along with other supportive cares,3,4 fewer patients with AGA undergo scalp biopsy as the first option for the diagnosis. At the plastic surgery clinic of the senior author (Dr. Hirayama), more than 250 Japanese patients of alopecia underwent scalp biopsy for the last 13 years (from 1999 to 2012).
© 2015 Wiley Periodicals, Inc.
Sebaceous gland changes in AGA patients
The sebaceous gland has important roles in biology of the hair. It is thought to initiate destruction of the hair follicle both in human and animal models in certain conditions.5–7 It has been said often in routine clinical setting that patients with AGA show abundant sebaceous glands and the bald skin surface is oily. Such anecdotal comments have been also recently supported by the increased number of fat globules by the use of dermoscopy.8 Previous report also showed that the biopsy specimen from AGA patients had more noticeable sebaceous glands, but detailed morphometric data were lacking.9 The biopsy specimen has been studied in the past mostly using the transverse sections, but it is difficult to analyze volumetric features of the sebaceous gland in such sections unless every transverse section of the biopsy specimen has cut at the same level from the skin surface that contains the largest diameter of the sebaceous gland. On the other hand, sections obtained in vertical fashion along the course and longitudinal axis parallel to the hair shaft demonstrate in general the portion of the sebaceous gland more clearly. To analyze the morphological features of the sebaceous gland, vertical sections of the scalp skin were used and morphometric analysis of the sebaceous gland in the alopecia cases was attempted in this study. Another important role exists in the bulge region of the follicle. The bulge region is located in close proximity to the sebaceous gland. In cicatricial alopecia, both the sebaceous gland and the bulge region are often involved in the pathology process together.10 The bulge region of the hair follicle has been investigated in recent years as it contains the multipotent stem cells that can differentiate into variety of dermal elements10 and also it contributes to wound healing process.11 But, the real distribution of the stem cells in the AGA patients is not well understood. Among immunohistochemical markers that recognize the bulge region, anticytokeratin 15 antibody, especially that of the C8/B144 clone, was chosen in this study as it is a specific marker for the stem cell containing bulge region of the hair follicle.10,12,13 In this retrospective study using pathological slides of the AGA patients, we demonstrate characteristics of the sebaceous gland and also study the distribution of the stem cells.
Methods Hematoxylin and eosin (H&E)-stained slides of the biopsied scalp of 250 Japanese patients were reviewed. These slides were processed from the patients who
© 2015 Wiley Periodicals, Inc.
. K Kure et al.
visited the Memorial Clinic Hirayama, Tokyo, between 1999 and 2012 for loss of scalp hair. The biopsy was achieved under the written consent from each patient in accordance with the Declaration of Helsinki. An explanation was made regarding the purposes of the biopsy, that is, to make a diagnosis and also to use the specimen for the alopecia research. These specimens were formalin-fixed, then processed in paraffin embedding and into glass slides. Using these glass slides, further studies were carried out at the Tokyo Women’s Medical University Medical Center East. Cases with advanced scar changes that might preclude the right diagnosis are excluded from the study. Among these, vertical sections with hair shaft visible in its longitudinal direction are further analyzed for the size of the sebaceous gland. They consist of 23 male cases of AGA, age 21–55 (mean 36), and these were compared with control group (n = 17, male subjects, age 25–65 [mean 42]) consisting of scalp tissues obtained from other conditions such as benign nevus, inclusion cysts, chronic dermatitis, fibroma, uninvolved margins from basal cell carcinoma, and other benign conditions. The cases with clinical impression of scalp lesions but turned out to be histologically unremarkable are also included. Grossly normal appearing regions of these specimens were used. The each sebaceous gland area was measured using NIH IMAGEJ system.14,15 The following parameters were employed. First, to demonstrate the overall population of the sebaceous glands, the sebaceous gland index (SG Index) was created and was defined as the areas of the sebaceous glands in the each specimen per lm length (SG Index = sebaceous gland area (lm2) horizontal length (lm) of the specimen). Second, unit sebaceous gland area (lm2), that is, the size of each sebaceous gland area was calculated. Lastly, the area (lm2) of sebaceous gland in each hair follicular unit was obtained. All the parameters were compared with the control group. For the immunohistochemistry to demonstrate the stem cells in the follicular bulge regions, paraffinembedded section slides (n = 5) of the AGA patients’ scalp were used. After deparaffinization, the slides were treated with the primary antibody (DAKO CD8, clone C8/144B). This antibody is known to recognize cytokeratin 15 due to cross-reaction. The slides were then treated with secondary antibody using EnVisionâ system (DAKO) and further stained with hematoxylin. This antibody (CD8, clone C8/144B) was previously found as the marker for the bulge region of the hair follicles and proved to be a specific marker for cytokeratin 15.13 We used this antibody as it reacts even in
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the formalin-fixed, paraffin-embedded tissue such as in the present retrospective study.
Results Histopathological observations
Finding from H&E-stained sections are summarized as follows. In control group, the vertical section demonstrated thick dermis within which anagen hair follicles are easily seen. They consist of long, thick hair shaft, the inner and outer root sheets, and the bulbs (Fig. 1a). In the AGA group, the hair follicles are much (a)
shorter in their longitudinal direction and the dermis was much thinner than that of normal control group (Fig. 1b). Increase in the number of telogen phase and miniaturization of the hair shaft are noted (Fig. 2). Scattered lymphocytic infiltrates are also noted in some cases. The sebaceous gland showed moderate to marked increase in the two-dimensional area measurement and number in AGA group (Fig. 3). The NIH IMAGEJ system was used to measure each sebaceous gland area (Fig. 4). It shows that the SG Index was significantly higher in AGA group (P < 0.001) indicating the sebaceous glands in AGA group increased in general (Fig. 5). While the unit sebaceous gland area (measurement of the each sebaceous gland) revealed no statistical difference from the control group (Fig. 6), the sebaceous gland area in each hair follicular unit showed higher values in comparison with that of the control groups (P < 0.001) (Fig. 7). These results suggest that the size of each sebaceous gland is unchanged, but they are more lobulated or have more lobules in the follicular unit. Immunohistochemistry
The anticytokeratin 15 (C8/144B clone) antibody is known to immunostain the discrete area of the follicular stem cells in the outer root sheath basal layer below the sebaceous gland duct at the attachment site of the arrector pili muscle of the anagen hair,10,13 but
(b)
Figure 1 (a, b) Vertical section of the scalp from a patient with AGA (b) and control (a). The scalp of the AGA is much thinner than that of control. Bar: 1 mm.
180
Figure 2 The hair follicles of AGA case with telogen phase and miniaturization of the hair shaft. Bar: 1 mm.
© 2015 Wiley Periodicals, Inc.
Sebaceous gland changes in AGA patients
its immunoreactivity is not well understood in clinical setting such as in patients with AGA. Our study revealed that there were still positively stained stem cells in the AGA cases (four of five cases) (Fig. 8). One
. K Kure et al.
case showed overall equivocal staining pattern. When the sebaceous gland and the bulge regions are anatomically not clearly seen, the positively stained cells were either scattered or rarely seen (Fig. 9 – brown particles).
Discussion
Figure 3 The sebaceous glands of AGA patient showing enlarged multiple lobulations. Bar: 100 lm.
Figure 4 NIH
IMAGEJ
As the emergence of effective medications such as finasteride and minoxidil, routine biopsy of the scalp skin in male and female pattern hair loss patients (especially in AGA) became more difficult to obtain these days in Japan. In this retrospective study using pathology glass slide specimen, the size and the area of the sebaceous gland were studied using unconventional vertical section slides of AGA patients. Based on the results from the current study, the sebaceous glands are increased in AGA group with a possibility of more lobulations in comparison with the control group. This was also histologically recognizable. While the control group also had multilobulated sebaceous gland in most cases, the AGA group showed more lobules per each follicular unit. There might be concerns as such that we may be observing falsely enlarged sebaceous glands in the presence of miniaturized hair follicles (dwarfing effect) due to the faulty impression that the sebaceous gland looks bigger and multilobulated when compared to the adjacent vellus-like hair follicle. However, with use of NIH IMAGEJ
system was used to measure the areas of the sebaceous glands.
© 2015 Wiley Periodicals, Inc.
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. K Kure et al.
Figure 5 Sebaceous gland index (SGI) is defined as SGI = sebaceous gland (lm2) area/length of the specimen (lm). SGI was significantly higher in AGA group (P < 0.001).
Figure 6 The unit sebaceous gland area is obtained by the measurement of each sebaceous gland. There was no statistical difference between the AGA and control group suggesting the size of each sebaceous gland does not differ.
system, each sebaceous gland area was objectively measured and the absolute number for the sebaceous gland area was obtained. And the results showed the AGA sebaceous gland area was indeed enlarged.
182
Figure 7 The sebaceous gland area per each hair follicular unit showed a significant difference in AGA and control group (P < 0.001) suggesting that the AGA sebaceous gland is more lobulated or has more lobules.
Figure 8 The immunohistochemistry marker for anticytokeratin 15 demonstrated positively stained stem cells (dark brown) in the bulge regions in the AGA cases. Bar: 100 lm.
The sebaceous gland may be the first to be attacked in some of alopecia patients such as discoid lupus erythematosus (DLE) and lichen planopilaris (LPP) (cicatricial
© 2015 Wiley Periodicals, Inc.
Sebaceous gland changes in AGA patients
Figure 9 When the sebaceous gland and the bulge regions are anatomically not clearly seen, the positively stained cells were scattered or rarely seen (brown particles). Bar: 100 lm.
alopecia).10 There is evidence that the sebaceous gland has specific binding protein and the sebaceous gland of the AGA patients shows higher affinity to dihydrotestosterone (DHT; the causative agent of AGA) than the normal population.16 Overpopulated sebaceous gland and increased affinity to the DHT in AGA patient population might be one of the foreseen factors contributing to the etiology of the disease. For example, the increased affinity of the DHT to the sebaceous gland of AGA patients might lead to the overgrowth of the sebaceous gland, and this could result in early shift to the catagen or telogen phases. It is already known that if the sebaceous gland is involved in pathological process such as in animal model that lacks sebaceous gland or in the human condition of DLE and LPP (cicatricial alopecia), it leads to hair loss. Thus, the oversized sebaceous gland with higher affinity to the DHT might be one of several other androgen-dependent conditions.16 There have been number of immunohistochemical markers used to study or to detect the bulge regions of the hair follicles but many of them such as Ki-67 and c-kit17 fail to show specific staining of the bulge area. While several markers have been reported positive for the vicinity of the bulge stem cells including one for CD 200 and CD34,13,18–20 the clone C8/144b of cytokeratin 15 has consistently demonstrated the bulge
© 2015 Wiley Periodicals, Inc.
. K Kure et al.
stem cell region and it is known to show normal hair differentiation in in vitro study.10,13,19 The antibody for cytokeratin 15 (C8/144B clone) is simply the marker for CD8 which is previously known as lymphocytic marker, but it was “surprisingly discovered during unrelated experiments” to stain the hair follicle bulge keratinocytes as well as a subset of lymphocytes of adult human skin.13 It was found to be very specific marker for the bulge area without staining remaining hair follicle and recognizes cytokeratin 15.13 It works well in formalin-fixed, paraffin-embedded tissue such as in the present retrospective study unlike many other markers for the stem cells. The stem cells in the bulge regions are often present in patients of AGA The current study showed positive immunohistochemical staining with the stem cell marker for the bulge region in AGA patients. While the CK15 expression is diminished in the pathologies involving this area such as in the cutaneous lupus erythematosus,21 its staining remains positive in the tumor cells of the apocrine mixed tumor cases,22 suggesting the differentiation of the follicular stem cell in the pathological process of the latter cases. The current study did not show CK15 immunostaining cells in the sebaceous glands that are involved in the pathological process of AGA. So, the overgrowth or overpopulation of the sebaceous gland in AGA might not be the result of altered follicular stem cell differentiation. Nevertheless, the findings in the sebaceous gland and relative preservation of the follicular stem cells could be important findings. Again, it might be possible that DHT leads to enlargement of the AGA sebaceous gland and miniaturization of the follicle in parallel. More study would be needed to conclude that pathology in the AGA sebaceous gland leads to pathology in the follicle. Lastly, we understand the limitations of the present study. It is important to compare the results of the current study to the observation of the horizontal sectioned specimens. The present study was retrospective. To obtain meaningful results from the horizontal sections, the specimens need to be cut at the depth that contains the major portion of the bulge and the sebaceous glands. It was difficult to obtain such specimens in the current retrospective study, but a prospective study using appropriate horizontal sections will add important data, and we would like to pursue toward that direction in the future.
Acknowledgments We would like to thank Dr. Hiroshi Ito for his contribution to this article. This study was presented in part
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. K Kure et al.
at the 37th annual meeting of Japan Society of Aesthetic Plastic Surgery, Tokyo, Japan, September 2014 and at the 19th meeting of the Japan Society of Clinical Hair Restoration, Okayama, Japan, November 2014.
13
References
14
1 Sinclair R. Male pattern androgenic alopecia. BMJ 1998; 317: 865–9. 2 Mounsey AL, Reed SW. Diagnosis and treating hair loss. Am Fam Physician 2009; 80: 356–62. 3 Springer K, Brown M, Stulberg DI. Common hair loss disorders. Am Fam Physician 2003; 68: 93–102. 4 Thiedke CC. Alopecia in women. Am Fam Physician 2003; 67: 1007–14. 5 Al-Zaid T, Vanderweil S, Zembowicz A et al. Sebaceous gland loss and inflammation in scarring alopecia: a potential role in pathogenesis. J Am Acad Dermatol 2011; 65: 597–603. 6 Stenn KS, Sundberg JP. Hair follicle biology, the sebaceous gland, and scarring alopecias. Arch Dermatol 1999; 135: 973–4. 7 Stenn KS. Insights from the asebia mouse: a molecular sebaceous gland defect leading to cicatricial alopecia. J Cutan Pathol 2001; 28: 445–7. 8 Inui S, Nakajima T, Itami S. Scalp dermoscopy of androgenic alopecia in Asian people. J Dermatol 2009; 36: 82– 5. 9 Sellheyer K, Bergfeld WF. Histopathologic evaluation of alopecia. Am J Dermatopathol 2006; 28: 236–59. 10 Al-Refu K. Stem cell and alopecia: a review of pathogenesis. Br J Dermatol 2012; 167: 479–84. 11 Ito M, Liu Y, Yang Z et al. Stem cells in the hair follicle bulge contribute to the wound repair but not to homeostasis of the epidermis. Nat Med 2005; 11: 1351–4. 12 Pontiggia L, Biedermann T, Meuli M et al. Markers to evaluate the quality and self-renewing potential of the
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17
18
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20
21
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engineered human skin substitutes in vitro and after transplantation. J Invest Dermatol 2009; 129: 480–90. Lyle S, Christofidou-Solomidou M, Liu Y et al. The C8/ 144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. J Cell Sci 1998; 111: 3179–88. Rasband WS. Image J. U.S National Institutes of Health, Bethesda, MD. http://imagej.nih.gov/ij/, 1997-2012. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to Image J: 25 years of image analysis. Nat Methods 2012; 9: 671–5. Sawaya M, Honig LS, Hsia SL. Increased androgen biding capacity in sebaceous glands in scalp of male-pattern baldness. J Invest Dermatol 1988; 92: 91–5. Ashrafuzzaman M, Yamamoto T, Shibata N et al. Potential involvement of the stem cell factor receptor c-kit in alopecia areata and androgenic alopecia: histopathological, immunohistochemical, and semiquantitative investigations. Acta Histochem Cytochem 2010; 43: 9–17. Garza LA, Yang CC, Zhao T et al. Bald scalp in men with androgenic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells. J Clin Invest 2011; 121: 613–22. Roh C, Tao Q, Lyle S. Dermal papilla-induced hair differentiation of adult epithelial stem cells from human skin. Physiol Genomics 2004; 19: 207–17. Ohyama M, Terunuma A, Tock CL et al. Characterization and isolation of stem cell-enriched human hair follicle bulge cells. J Clin Invest 2006; 116: 249–60. Al-Refu K, Edward S, Ingham E et al. Expression of hair follicle stem cells detected by cytokeratin 15 stain: implications for pathogenesis of the scarring process in cutaneous lupus erythematosus. Br J Dermatol 2009; 160: 1188–96. Misago N, Narisawa Y. Cytokeratin 15 expression in apocrine mixed tumors of the skin and other benign neoplasms with apocrine differentiation. J Dermatol 2006; 33: 2–9.
© 2015 Wiley Periodicals, Inc.
Changes in the sebaceous gland in patients with male pattern hair loss (androgenic alopecia) Katsuhiro Kure, MD,1,2 Tsukasa Isago, MD,1 & Takeshi Hirayama, MD3 1
Department of Plastic and Reconstructive Surgery, Tokyo Women’s Medical University Medical Center East, Tokyo, Japan Plaza Plastic Surgery, Tokyo, Japan 3 Memorial Clinic Hirayama, Tokyo, Japan 2
Summary
Background The sebaceous gland and the follicular bulge region have important role in biology of the hair. They initiate destruction of the hair follicle both in human and animal models in certain conditions. The morphometric feature of the sebaceous gland is not well understood so as the distribution of the bulge stem cells in pathological conditions of male pattern hair loss or androgenic alopecia (AGA). Objectives The authors perform morphometric analysis of the sebaceous gland in AGA patients and also study distribution of the follicular stem cells in the bulge region in these populations. Methods Two hundred and fifty cases of glass slide specimen from Japanese patients with male pattern hair loss were reviewed. Among these, 23 cases of the longitudinal (vertical) sections of the scalp skin with diagnosis of AGA were found and analyzed for the morphometric characteristics. Each sebaceous gland area was measured using NIH IMAGEJ system and statistically analyzed. For the identification of the follicular bulge region, an immunohistochemistry using anticytokeratin 15 (C8/144B clone) was carried out in the cases of AGA. Results The sebaceous gland area of the AGA group was noticeably increased, while the size of each sebaceous gland remains unchanged. It has more lobules in the hair follicular unit in the AGA population. In the immunohistochemistry, the follicular stem cells are present in the bulge regions in cases of AGA. Conclusions The overgrowth (multilobulation) of the sebaceous gland and relative preservation of the follicular stem cells suggest that the changes in the sebaceous gland could be an important factor in the pathology of AGA. Keywords: androgenic alopecia, sebaceous immunohistochemistry, male pattern hair loss
Alopecia (male and female pattern hair loss) is not only a medical condition but often devastating social aspects of the patients involved. More than 30–40% of male population over age 20 years old show androCorrespondence: Dr. Katsuhiro Kure, Department of Plastic and Reconstructive Surgery, Tokyo Women’s Medical University Medical Center East, 2-1-10 Nishioku, Arakawa-ku, Tokyo 116-8567, Japan. E-mail: [email protected] Accepted for publication April 9, 2015
178
gland,
follicular
stem
cell,
genic alopecia (AGA) or male pattern hair loss.1–4 Because of recent introduction of medical treatment using finasteride and minoxidil along with other supportive cares,3,4 fewer patients with AGA undergo scalp biopsy as the first option for the diagnosis. At the plastic surgery clinic of the senior author (Dr. Hirayama), more than 250 Japanese patients of alopecia underwent scalp biopsy for the last 13 years (from 1999 to 2012).
© 2015 Wiley Periodicals, Inc.
Sebaceous gland changes in AGA patients
The sebaceous gland has important roles in biology of the hair. It is thought to initiate destruction of the hair follicle both in human and animal models in certain conditions.5–7 It has been said often in routine clinical setting that patients with AGA show abundant sebaceous glands and the bald skin surface is oily. Such anecdotal comments have been also recently supported by the increased number of fat globules by the use of dermoscopy.8 Previous report also showed that the biopsy specimen from AGA patients had more noticeable sebaceous glands, but detailed morphometric data were lacking.9 The biopsy specimen has been studied in the past mostly using the transverse sections, but it is difficult to analyze volumetric features of the sebaceous gland in such sections unless every transverse section of the biopsy specimen has cut at the same level from the skin surface that contains the largest diameter of the sebaceous gland. On the other hand, sections obtained in vertical fashion along the course and longitudinal axis parallel to the hair shaft demonstrate in general the portion of the sebaceous gland more clearly. To analyze the morphological features of the sebaceous gland, vertical sections of the scalp skin were used and morphometric analysis of the sebaceous gland in the alopecia cases was attempted in this study. Another important role exists in the bulge region of the follicle. The bulge region is located in close proximity to the sebaceous gland. In cicatricial alopecia, both the sebaceous gland and the bulge region are often involved in the pathology process together.10 The bulge region of the hair follicle has been investigated in recent years as it contains the multipotent stem cells that can differentiate into variety of dermal elements10 and also it contributes to wound healing process.11 But, the real distribution of the stem cells in the AGA patients is not well understood. Among immunohistochemical markers that recognize the bulge region, anticytokeratin 15 antibody, especially that of the C8/B144 clone, was chosen in this study as it is a specific marker for the stem cell containing bulge region of the hair follicle.10,12,13 In this retrospective study using pathological slides of the AGA patients, we demonstrate characteristics of the sebaceous gland and also study the distribution of the stem cells.
Methods Hematoxylin and eosin (H&E)-stained slides of the biopsied scalp of 250 Japanese patients were reviewed. These slides were processed from the patients who
© 2015 Wiley Periodicals, Inc.
. K Kure et al.
visited the Memorial Clinic Hirayama, Tokyo, between 1999 and 2012 for loss of scalp hair. The biopsy was achieved under the written consent from each patient in accordance with the Declaration of Helsinki. An explanation was made regarding the purposes of the biopsy, that is, to make a diagnosis and also to use the specimen for the alopecia research. These specimens were formalin-fixed, then processed in paraffin embedding and into glass slides. Using these glass slides, further studies were carried out at the Tokyo Women’s Medical University Medical Center East. Cases with advanced scar changes that might preclude the right diagnosis are excluded from the study. Among these, vertical sections with hair shaft visible in its longitudinal direction are further analyzed for the size of the sebaceous gland. They consist of 23 male cases of AGA, age 21–55 (mean 36), and these were compared with control group (n = 17, male subjects, age 25–65 [mean 42]) consisting of scalp tissues obtained from other conditions such as benign nevus, inclusion cysts, chronic dermatitis, fibroma, uninvolved margins from basal cell carcinoma, and other benign conditions. The cases with clinical impression of scalp lesions but turned out to be histologically unremarkable are also included. Grossly normal appearing regions of these specimens were used. The each sebaceous gland area was measured using NIH IMAGEJ system.14,15 The following parameters were employed. First, to demonstrate the overall population of the sebaceous glands, the sebaceous gland index (SG Index) was created and was defined as the areas of the sebaceous glands in the each specimen per lm length (SG Index = sebaceous gland area (lm2) horizontal length (lm) of the specimen). Second, unit sebaceous gland area (lm2), that is, the size of each sebaceous gland area was calculated. Lastly, the area (lm2) of sebaceous gland in each hair follicular unit was obtained. All the parameters were compared with the control group. For the immunohistochemistry to demonstrate the stem cells in the follicular bulge regions, paraffinembedded section slides (n = 5) of the AGA patients’ scalp were used. After deparaffinization, the slides were treated with the primary antibody (DAKO CD8, clone C8/144B). This antibody is known to recognize cytokeratin 15 due to cross-reaction. The slides were then treated with secondary antibody using EnVisionâ system (DAKO) and further stained with hematoxylin. This antibody (CD8, clone C8/144B) was previously found as the marker for the bulge region of the hair follicles and proved to be a specific marker for cytokeratin 15.13 We used this antibody as it reacts even in
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. K Kure et al.
the formalin-fixed, paraffin-embedded tissue such as in the present retrospective study.
Results Histopathological observations
Finding from H&E-stained sections are summarized as follows. In control group, the vertical section demonstrated thick dermis within which anagen hair follicles are easily seen. They consist of long, thick hair shaft, the inner and outer root sheets, and the bulbs (Fig. 1a). In the AGA group, the hair follicles are much (a)
shorter in their longitudinal direction and the dermis was much thinner than that of normal control group (Fig. 1b). Increase in the number of telogen phase and miniaturization of the hair shaft are noted (Fig. 2). Scattered lymphocytic infiltrates are also noted in some cases. The sebaceous gland showed moderate to marked increase in the two-dimensional area measurement and number in AGA group (Fig. 3). The NIH IMAGEJ system was used to measure each sebaceous gland area (Fig. 4). It shows that the SG Index was significantly higher in AGA group (P < 0.001) indicating the sebaceous glands in AGA group increased in general (Fig. 5). While the unit sebaceous gland area (measurement of the each sebaceous gland) revealed no statistical difference from the control group (Fig. 6), the sebaceous gland area in each hair follicular unit showed higher values in comparison with that of the control groups (P < 0.001) (Fig. 7). These results suggest that the size of each sebaceous gland is unchanged, but they are more lobulated or have more lobules in the follicular unit. Immunohistochemistry
The anticytokeratin 15 (C8/144B clone) antibody is known to immunostain the discrete area of the follicular stem cells in the outer root sheath basal layer below the sebaceous gland duct at the attachment site of the arrector pili muscle of the anagen hair,10,13 but
(b)
Figure 1 (a, b) Vertical section of the scalp from a patient with AGA (b) and control (a). The scalp of the AGA is much thinner than that of control. Bar: 1 mm.
180
Figure 2 The hair follicles of AGA case with telogen phase and miniaturization of the hair shaft. Bar: 1 mm.
© 2015 Wiley Periodicals, Inc.
Sebaceous gland changes in AGA patients
its immunoreactivity is not well understood in clinical setting such as in patients with AGA. Our study revealed that there were still positively stained stem cells in the AGA cases (four of five cases) (Fig. 8). One
. K Kure et al.
case showed overall equivocal staining pattern. When the sebaceous gland and the bulge regions are anatomically not clearly seen, the positively stained cells were either scattered or rarely seen (Fig. 9 – brown particles).
Discussion
Figure 3 The sebaceous glands of AGA patient showing enlarged multiple lobulations. Bar: 100 lm.
Figure 4 NIH
IMAGEJ
As the emergence of effective medications such as finasteride and minoxidil, routine biopsy of the scalp skin in male and female pattern hair loss patients (especially in AGA) became more difficult to obtain these days in Japan. In this retrospective study using pathology glass slide specimen, the size and the area of the sebaceous gland were studied using unconventional vertical section slides of AGA patients. Based on the results from the current study, the sebaceous glands are increased in AGA group with a possibility of more lobulations in comparison with the control group. This was also histologically recognizable. While the control group also had multilobulated sebaceous gland in most cases, the AGA group showed more lobules per each follicular unit. There might be concerns as such that we may be observing falsely enlarged sebaceous glands in the presence of miniaturized hair follicles (dwarfing effect) due to the faulty impression that the sebaceous gland looks bigger and multilobulated when compared to the adjacent vellus-like hair follicle. However, with use of NIH IMAGEJ
system was used to measure the areas of the sebaceous glands.
© 2015 Wiley Periodicals, Inc.
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. K Kure et al.
Figure 5 Sebaceous gland index (SGI) is defined as SGI = sebaceous gland (lm2) area/length of the specimen (lm). SGI was significantly higher in AGA group (P < 0.001).
Figure 6 The unit sebaceous gland area is obtained by the measurement of each sebaceous gland. There was no statistical difference between the AGA and control group suggesting the size of each sebaceous gland does not differ.
system, each sebaceous gland area was objectively measured and the absolute number for the sebaceous gland area was obtained. And the results showed the AGA sebaceous gland area was indeed enlarged.
182
Figure 7 The sebaceous gland area per each hair follicular unit showed a significant difference in AGA and control group (P < 0.001) suggesting that the AGA sebaceous gland is more lobulated or has more lobules.
Figure 8 The immunohistochemistry marker for anticytokeratin 15 demonstrated positively stained stem cells (dark brown) in the bulge regions in the AGA cases. Bar: 100 lm.
The sebaceous gland may be the first to be attacked in some of alopecia patients such as discoid lupus erythematosus (DLE) and lichen planopilaris (LPP) (cicatricial
© 2015 Wiley Periodicals, Inc.
Sebaceous gland changes in AGA patients
Figure 9 When the sebaceous gland and the bulge regions are anatomically not clearly seen, the positively stained cells were scattered or rarely seen (brown particles). Bar: 100 lm.
alopecia).10 There is evidence that the sebaceous gland has specific binding protein and the sebaceous gland of the AGA patients shows higher affinity to dihydrotestosterone (DHT; the causative agent of AGA) than the normal population.16 Overpopulated sebaceous gland and increased affinity to the DHT in AGA patient population might be one of the foreseen factors contributing to the etiology of the disease. For example, the increased affinity of the DHT to the sebaceous gland of AGA patients might lead to the overgrowth of the sebaceous gland, and this could result in early shift to the catagen or telogen phases. It is already known that if the sebaceous gland is involved in pathological process such as in animal model that lacks sebaceous gland or in the human condition of DLE and LPP (cicatricial alopecia), it leads to hair loss. Thus, the oversized sebaceous gland with higher affinity to the DHT might be one of several other androgen-dependent conditions.16 There have been number of immunohistochemical markers used to study or to detect the bulge regions of the hair follicles but many of them such as Ki-67 and c-kit17 fail to show specific staining of the bulge area. While several markers have been reported positive for the vicinity of the bulge stem cells including one for CD 200 and CD34,13,18–20 the clone C8/144b of cytokeratin 15 has consistently demonstrated the bulge
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stem cell region and it is known to show normal hair differentiation in in vitro study.10,13,19 The antibody for cytokeratin 15 (C8/144B clone) is simply the marker for CD8 which is previously known as lymphocytic marker, but it was “surprisingly discovered during unrelated experiments” to stain the hair follicle bulge keratinocytes as well as a subset of lymphocytes of adult human skin.13 It was found to be very specific marker for the bulge area without staining remaining hair follicle and recognizes cytokeratin 15.13 It works well in formalin-fixed, paraffin-embedded tissue such as in the present retrospective study unlike many other markers for the stem cells. The stem cells in the bulge regions are often present in patients of AGA The current study showed positive immunohistochemical staining with the stem cell marker for the bulge region in AGA patients. While the CK15 expression is diminished in the pathologies involving this area such as in the cutaneous lupus erythematosus,21 its staining remains positive in the tumor cells of the apocrine mixed tumor cases,22 suggesting the differentiation of the follicular stem cell in the pathological process of the latter cases. The current study did not show CK15 immunostaining cells in the sebaceous glands that are involved in the pathological process of AGA. So, the overgrowth or overpopulation of the sebaceous gland in AGA might not be the result of altered follicular stem cell differentiation. Nevertheless, the findings in the sebaceous gland and relative preservation of the follicular stem cells could be important findings. Again, it might be possible that DHT leads to enlargement of the AGA sebaceous gland and miniaturization of the follicle in parallel. More study would be needed to conclude that pathology in the AGA sebaceous gland leads to pathology in the follicle. Lastly, we understand the limitations of the present study. It is important to compare the results of the current study to the observation of the horizontal sectioned specimens. The present study was retrospective. To obtain meaningful results from the horizontal sections, the specimens need to be cut at the depth that contains the major portion of the bulge and the sebaceous glands. It was difficult to obtain such specimens in the current retrospective study, but a prospective study using appropriate horizontal sections will add important data, and we would like to pursue toward that direction in the future.
Acknowledgments We would like to thank Dr. Hiroshi Ito for his contribution to this article. This study was presented in part
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at the 37th annual meeting of Japan Society of Aesthetic Plastic Surgery, Tokyo, Japan, September 2014 and at the 19th meeting of the Japan Society of Clinical Hair Restoration, Okayama, Japan, November 2014.
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References
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