The effects of laser-mediated hair removal on immunohistochemical staining properties of hair follicles

The effects of laser-mediated hair removal on immunohistochemical staining properties of hair follicles

The effects of laser-mediated hair removal on immunohistochemical staining properties of hair follicles Jeffrey S. Orringer, MD,a Craig Hammerberg, Ph...

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The effects of laser-mediated hair removal on immunohistochemical staining properties of hair follicles Jeffrey S. Orringer, MD,a Craig Hammerberg, PhD,a Lori Lowe, MD,a,b Sewon Kang, MD,a Timothy M. Johnson, MD,a Ted Hamilton, MS,a John J. Voorhees, MD, FRCP,a and Gary J. Fisher, PhDa Ann Arbor, Michigan Background: The mechanisms involved in laser-mediated hair removal remain unclear. One means of reducing hair growth is alteration of follicular stem cells. Objective: We sought to examine the effects of laser hair removal on the immunohistochemical staining properties of human hair follicles, including the putative stem cells of the bulge region. Methods: Treatment of unwanted axillary hair was performed on one side using an 800 nmewavelength diode laser and on the other side using a 1064 nmewavelength neodymium:yttrium-aluminum-garnet laser. Serial skin samples were obtained at baseline and various times after treatment and stained using immunohistochemical techniques. Results: Hair shafts were thermally altered, but the immunostaining properties of much of the follicle, including the bulge region, remained generally unchanged. Limitations: This study only addressed the acute immunohistochemical changes found after a single treatment using specific laser parameters. Conclusions: Laser-mediated hair removal does not appear to work by frank destruction of follicular stem cells. Other mechanisms including functional alteration of these cells may underlie the clinical efficacy of the procedure. ( J Am Acad Dermatol 2006;55:402-7.)

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nwanted hair is an exceedingly common issue for patients of both sexes that potentially carries with it profound effects on one’s psychosocial well-being. Thus, for many years, various methods of hair removal have been attempted, but the long-term efficacy of most such treatments has been limited. During the past decade,

From the Departments of Dermatologya and Pathology,b University of Michigan Medical School. Supported by the University of Michigan Department of Dermatology Cosmetic Dermatology and Laser Research Fund. Conflicts of interest: None identified. Accepted for publication April 16, 2006. Reprint requests: Jeffrey S. Orringer, MD, Department of Dermatology, University of Michigan Medical School, 1500 E Medical Center Dr, 1910 Taubman Center, Ann Arbor, MI 48109-0314. E-mail: [email protected] Published online May 28, 2006. 0190-9622/$32.00 ª 2006 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2006.04.057

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laser-mediated hair removal has become an accepted means of achieving permanent hair reduction.1-3 Although the popularity of laser hair removal continues to increase, a significant knowledge gap regarding the mechanisms involved persists. In fact, exactly how long-standing or permanent removal of hair by laser therapy is accomplished remains unclear, and relatively few studies regarding laser hair removal have reported the histologic changes that result from such therapy.4-8 One potential means by which lasers might provide permanent removal of a hair follicle is by the destruction of the follicular stem cells that normally participate in the regeneration of the epidermis and its adnexal structures. Experimental evidence has suggested that the selective destruction of follicular stem cells alone will prevent the regrowth of hair.9 In fact, one could argue that to ensure truly permanent hair removal, these cells should be eliminated or functionally altered by the treatment. Recent work has suggested that the putative follicular stem cells are located in the bulge region of the follicle, and

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markers of such cells have been identified using immunohistochemical techniques.10-17 We, therefore, sought to examine the effects of laser therapy on the immunohistochemical staining properties of follicular stem cells.

of various follicular compartments. The monoclonal antibodies used were cytokeratin 15 (clone LHK15, Chemicon, Temecula, Calif), cytokeratin 19 (clone Ks19.1, Sigma, St Louis, Mo), and CD34 (clone QBEnd/10, Biogenex, San Ramon, Calif). Staining was performed as previously described.18

METHODS This study was approved by our institutional review board, and written informed consent was obtained from all study participants. Twelve patients aged 22 to 60 years (mean 39.8) with at least moderately dense, dark axillary hair were recruited. Exclusion criteria included a history of laser therapy or electrolysis to the axillae, active infection of the axillae, a history of keloid scar formation, and a history of having taken an oral retinoid within 1 year of study entry. Pregnant women and potential patients not desiring permanent hair reduction at the treated sites were also excluded. This was a prospective, randomized trial in which patients underwent a laser hair removal treatment to the axillae followed by serial biopsies at the treated sites. Patients were randomized to receive laser therapy using an 800 nmewavelength diode laser (Lightsheer, Lumenis Inc, Santa Clara, Calif) to one axilla and treatment using a 1064 nmewavelength neodymium:yttriumaluminum-garnet laser (GentleYAG, Candela Corp, Wayland, Mass) to the contralateral axilla. Before treatment, the axillae were shaved and topical 4% liposomal lidocaine (L.M.X.4 Cream, Ferndale Laboratories Inc, Ferndale, Mich) was applied for approximately 30 minutes. Baseline axillary skin samples (3 mm in diameter) were obtained using locally injected 1% lidocaine with epinephrine for anesthesia. For the diode laser, treatment parameters included the following: the spot size was 12 3 12 mm, the fluence was 40 J/cm2, and the pulse duration was 20 milliseconds. Contact cooling was used throughout the treatment. For the neodymium:yttrium-aluminumgarnet laser, settings included a spot size of 12-mm diameter, fluence of 70 J/cm2, and pulse duration of 3 milliseconds. Because of patient discomfort, the fluence used was decreased to 50 J/cm2 for one patient and to 60 J/cm2 for another. In all cases, the dynamic cooling device was used throughout the procedure. Patients then provided additional 3 mmediameter skin samples taken from the treated sites bilaterally on postprocedure days 1, 3, and 7. In all cases, skin samples were obtained by angling the punch biopsy device in such a way as to follow the apparent track of the hair follicles removed, and tissue was obtained from areas specifically noted to contain rather dense hair at baseline. Vertical sections of skin samples were produced and immunohistochemical staining was performed for known markers

RESULTS All treated participants demonstrated the expected clinical end points during and after treatment that are believed to be consistent with clinical efficacy with each of the devices tested. Diode laser therapy resulted in perifollicular edema and erythema, and singed hair was often seen at the skin surface immediately posttreatment. With the neodymium:yttrium-aluminum-garnet laser, slightly more delayed perifollicular edema was noted, along with some comparatively slightly milder erythema. With this device, hair was often shed during a period of days after the treatment. Although no formal hair counts were performed in this study, with both devices, patients were found to have obvious clinically apparent decreases in hair density of the axillae during the brief follow-up period. Histologically at all posttreatment time points examined, the external root sheath appeared intact except for rare mild focal disruption (Fig 1, A). The hair bulb, including the matrix and dermal papillae, was consistently intact. Some mild but variable thinning of the follicular epithelium was found at the isthmus and infundibulum in some samples. The hair shaft was noted to demonstrate changes consistent with thermal injury including a thinned or shriveled appearance in some instances, whereas many follicles were noted to be entirely devoid of a hair shaft after treatment (Fig 1, B). There was some increase in keratotic debris at the level of the infundibulum often with distorted, curved hairshafts, consistent with trichomalacia entrapped by this debris (Fig 1, C ). Focal alteration of the internal root sheath was seen in some specimens (Fig 1, D). In theses instances, variable, incomplete, and uneven disruption of the internal root sheath was noted, often resulting in a shaggy appearance. Immunohistochemical staining for cytokeratin 15 resulted in variable staining of the infundibulum. Consistent strong expression of cytokeratin 15 was noted at the outer root sheath just below the level of the entrance of the sebaceous gland and involving the putative bulge region with no staining of the hair bulb (Fig 2, A). Staining for CD34 was demonstrated to occur at the outer root sheath above the level of the hair bulb and often involved up to about half of the length of the follicle (Fig 2, B). However, immunostaining for CD34 did not

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Fig 1. A, Mild focal disruption of external root sheath after single laser hair removal treatment. Thinned and shriveled hair shaft demonstrating changes consistent with thermal injury (B), keratotic debris at level of infundibulum (C), and focal disruption of internal root sheath (D) after laser-mediated hair removal. (Original magnification 360.)

colocalize with that of cytokeratin 15 and specifically appeared to spare the bulge region of the follicle. Cytokeratin 19 immunostaining was found at the outer root sheath in two locations within the follicle: one below the entrance of the sebaceous glands in association with cytokeratin 15 staining, and another including the lower portion of the follicle in association with CD34 extending to just above the bulb (Fig 2, C ). Expression of cytokeratin 19 was noted to often be patchy in comparison with that of cytokeratin 15.

DISCUSSION Despite extensive clinical evidence for the effectiveness of laser and light-based treatments in the removal of unwanted hair, relatively little is known about the effects of these devices on the structure and function of hair follicles. We, thus, sought to examine the anatomic results of laser hair removal

using immunohistochemistry to gain insight that might provide clues as to the mechanisms involved. The bulge-activation hypothesis states that multipotent cells located in the bulge region of the follicle contribute to the newly forming hair matrix after induction by the dermal papillae during the late telogen phase of the hair growth cycle.1 The vital role of stem cells in the formation of new anagen hair follicles makes these cells a logical target for destruction during light-based therapy to remove unwanted hair. Recent work has elucidated markers for these multipotent cells residing in the bulge region and for cells within various other regions of the follicle.10-17 We used this information to assess the immunostaining properties of hair follicles after laser therapy. The specific markers used in this study include cytokeratin 15, cytokeratin 19, and CD34. Both cytokeratins 15 and 19 have been proposed to be biomarkers for follicular stem cells. Cytokeratin 15 is

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Fig 2. A, Cytokeratin 15 immunostaining at level of bulge region in human follicle after lasermediated hair removal treatment. B, CD34 immunostaining of suprabulbar outer root sheath after laser therapy. C, Cytokeratin 19 immunostaining at level of bulge region and in suprabulbar region after laser-mediated hair removal. (Original magnification 360; inserts, 3120.)

an intracellular intermediate filament protein preferentially expressed in the cells of the human hair follicle bulge.10 Cytokeratin 19 is a 40-kd protein present in the outer root sheath in hair-bearing adult human skin, including in the bulge region, which is known to be populated with slow-cycling and

[3H]thymidine-labeleretaining cells.14,15 CD34 is a surface protein that has been demonstrated to be overrepresented in the suprabulbar outer root sheath in human follicles. Although CD34 is actually a marker of the bulge region in murine follicles, its expression in the bulge region in human follicles is

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either very low or entirely absent, making it a useful negative marker for the putative stem cells of this area.16 Despite the use of laser parameters that have been reported to clinically achieve permanent hair reduction, the posttreatment follicles examined in this study remained, for the most part, relatively structurally intact. As expected, clinically there was a loss of hair in the treated areas, and histologically hair shafts were found to demonstrate changes consistent with thermal injury including, in many cases, a thinned and shriveled appearance. In addition, in later samples, follicles devoid of hair shafts were often foundeindicative of elimination of the thermally destroyed hair. However, immunohistochemical markers for the multipotent cells of the bulge region stained with a similar pattern and degree of intensity in posttreatment images as compared with baseline. This suggests that if the follicular stem cells are altered in some way by the treatment, the resulting changes must not be detectible by the markers we used. It remains possible that, despite the persistence of the putative follicularly based stem cells in the acute period after laser hair removal, a sublethal thermal injury to these cells might result in functional changes leading to an inability of the follicle to regenerate hair. Alternatively, it is possible that other components of the follicle, such as the dermal papilla or hair matrix, are the true targets of laser-based hair removal. The crucial transient role that epithelial stem cells located in the follicle are believed to play in wound healing actually makes their complete destruction undesirable.9 Had we determined that these cells were entirely destroyed by the laser treatments, there would have been concern regarding the ability of the skin in the treated areas to heal normally after any subsequent injury. Thus, the fact that these cells appear to survive the laser therapy supports the safety of such treatment, and there have been no clinical reports of delayed cutaneous wound healing after laser hair removal to our knowledge. Our study was limited by the technical difficulties inherent in processing hair-containing skin samples. That is, it is exceedingly difficult to vertically section skin samples in such a way as to allow for histologic examination of entirely intact follicles. We strove to increase the chances of obtaining samples that included intact follicles by angling our punch biopsy devices to follow the apparent track of the hair, but despite this, in many instances the follicles had to be examined over serial sections because they were still bisected or trisected during processing. We considered the use of horizontal sections to potentially increase the overall number of follicles captured in

any given image, but this technique would not have enabled us to view the overall follicular architecture and immunohistochemical staining patterns to the extent that vertical sectioning allowed. In addition, we examined skin samples obtained in the fairly acute postprocedure period (ie, during the first week after treatment). It remains possible that more profound changes in the putative follicular stem cells might have been demonstrated at later posttreatment time points, and further research examining skin samples obtained at later time points is warranted. In addition, our study examined the immunohistologic changes that result from a single laser treatment. It is possible that through serial treatments, as are generally provided in the clinical setting, an additive destructive effect on the follicle is created such that the stem cells or other vital structures within the follicle are more profoundly altered. Finally, we used laser parameters that we have found to be clinically efficacious for our patients and that fell within the general ranges suggested by the devices’ manufacturers. However, the use of higher fluences or altered pulse durations, for example, might have resulted in follicular changes not demonstrated in the current study. There are many reports in the literature of the clinical effectiveness of laser-based hair removal, and some studies have suggested long-standing results. Given that the cells apparently most responsible for regeneration of hair appear to remain at least structurally intact suggests that at least one of several possibilities may be true. Laser hair removal may work by impacting an as yet unidentified target within the follicle. Alternatively, follicular stem cells may be functionally altered by these treatments even while apparently remaining structurally intact and maintaining at least some of their vital functions including the ability to participate in cutaneous wound healing. Less likely is the possibility that laser hair removal only generates greatly delayed hair regrowth over time and that much or all of the hair in a treated area will ultimately return. This latter hypothesis is now disputed by a decade of clinical experience with the use of lasers for hair reduction and by several reports of long-term efficacy. It appears clear that many of these light-based systems do achieve long-standing or truly permanent hair removal, but the mechanisms involved still remain elusive. We wish to thank Suzan Rehbine, LPN, for her assistance with patient recruitment and tissue procurement and Heather Y. Kovarik, LPN, for her assistance during the treatment of study patients. We also appreciate the editing assistance of our colleague, Andrzej Dlugosz, MD.

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11. Morris R, Yaping L, Marles L, Yang Z, Trempus C, Li S, et al. Capturing and profiling adult hair follicle stem cells. Nat Biotechnol 2004;22:411-7. 12. Claudinot S, Nicolas M, Oshima H, Rochat A, Barrandon Y. Long-term renewal of hair follicles from clonogenic multipotent stem cells. Proc Natl Acad Sci U S A 2005;102: 14677-82. 13. Liu Y, Lyle S, Yang Z, Cotsarelis G. Keratin 15 promotor targets putative epithelial stem cells in the hair follicle bulge. J Invest Dermatol 2003;121:963-8. 14. Michel M, Torok N, Godbout M, Lussier M, Gaudreau P, Royal A, et al. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci 1996;109: 1017-28. 15. Commo S, Gaillard O, Bernard B. The human hair follicle contains two distinct K19 positive compartments in the outer root sheath: a unifying hypothesis for stem cell reservoir. Differentiation 2000;66:157-64. 16. Ohyama M, Terunuma A, Tock C, Radonovich M, Pise-Masison C, Hopping S, et al. Characterization and isolation of stem cellenriched human hair follicle bulge cells. J Clin Invest 2006;116: 249-60. 17. Cotsarelis G. Gene expression profiling gets to the root of human hair follicle stem cells. J Clin Invest 2006;116:19-22. 18. Fisher G, Choi H, Bata-Csorgo Z. Ultraviolet irradiation increases matrix metalloproteinase-8 protein in human skin in vivo. J Invest Dermatol 2001;117:219-26.