Ahmad Ibrahim. Rasheed* - Zeinab Abdul- Rahman Kamar**

Egyptian Dermatology Online Journal 2 (2): 1, December,  2006

Abstract

Background:

 The effect of lasers in treating unwanted hair varies markedly with any change in pulse characteristics or in the treatment program. Many treatment parameters and regimens have been recommended and many criteria (clinical and histological) have been suggested to judge permanency of the results.

Aim of the work:

To evaluate the hair follicle stem cells and hair matrix cells under the effect of different parameter settings of hair removal lasers.

Material and methods:

 Ten healthy adult females (mean age 33.4 years +/- 8.6) were subjected to treatment of axillary hair by 7 different programs of hair removal laser (alexandrite or Nd:YAG lasers) at different spot sizes (7, 12, 15 & 18 mm) and at different pulse durations (5, 10, 40 and 100 milliseconds). (One program for each one of 8 quadrants of the both axillae with the 8th quadrant left untreated). Clinical evaluation was done 6 weeks and 6 months after 2 sessions. Immunohistochemical skin examination was done for cytokeratin-15 (CK-15) +v (hair follicle stem) cells and LEF-1 +ve (hair matrix) cells 6 weeks after 2 sessions.

Results:

All programs could show statistically significant reduction of hair on both follow up visits as well as significant reduction of LEF-1 score and CK-15 score as evaluated 6 weeks after 2 sessions. Significantly less results could be seen with smaller spot size of 12 mm and with 40 millisecond pulse duration. There was positive correlation between clinical results on both visits. LEF-1 score showed -ve correlation with both clinical (early and late) scores but more with the short term clinical score. CK-15 score showed strong -ve correlation with both clinical scores and positive correlation with LEF-1 score.

Conclusion:

 Hair follicle stem cells are important targets for the efficacy of hair removal lasers. Evaluation of hair follicle stem cells rather than hair matrix cells can represent a reliable predictor of long lasting clinical results.

Introduction

In the last decade, laser and light-based technology for hair removal became one of the fastest growing procedures in modern cosmetic dermatology.[1] Light therapy can be used to destroy hair follicles by one of several mechanisms; thermal (due to local heating), mechanical (due to shock waves or violent cavitations), or photochemical (due to generation of toxic mediators like singlet oxygen or free radicals). Hair removal has been attempted using each of these three means.[1,2,3]

The thermal mechanism employed for hair removal by laser/light-based systems is based on the principle of selective photothermolysis. According to this principle, selective thermal destruction of a target will occur if sufficient energy is delivered at a wavelength well absorbed by the target within a time period less than or equal to the thermal relaxation time (TRT) of the target. Under these conditions, it is possible to selectively target the intended structure (e.g. the hair follicle) while sparing the surrounding tissues.[4]

In the visible to near-infrared region, melanin is the natural chromophore for targeting hair follicles. Lasers or light sources that operate in the red or near-infrared wavelength region all lie in an optical window of the spectrum in which selective absorption by melanin is combined with deep penetration into the dermis. Therefore, deep and selective heating of the hair shaft, the hair follicle epithelium, and the heavily pigmented matrix is possible in the 600- to 1100-nm region. However, melanin in the epidermis presents a competing site for absorption. To obtain spatial confinement of thermal damage, the pulse duration should be shorter than (or equal to) the thermal relaxation time of the hair follicle but longer than that of surface epidermis (about 3 milliseconds). Thermal relaxation time of human terminal hair follicles has never been measured, but is estimated to be approximately 10-100 milliseconds, depending on follicular size. Therefore, most devices for hair removal have pulse durations in the 3-50 millisecond range. Selective cooling of the epidermis has been also applied to minimize epidermal injury.[1,2,3]

As the target is melanin, the follicle must be treated in the anagen cycle. It is during the anagen phase that melanin production occurs and becomes part of the growing follicle.[5] In early anagen (in-particular), the bulb is well melanized and still fairly superficial. This represents the best timing for treatment by laser.[6]

However, the bulge-activation hypothesis maintains that the bulge area of the outer root sheath near the erector pili muscle insertion contains pluripotential cells, which contribute to the new hair matrix when induced by the dermal papillae during the late telogen phase.[7] Thus, to achieve long-term hair removal, it is essential to destroy the structures that are responsible for hair growth: the bulge and bulb.[5]

This has paved the way for the emergence of the so-called propagation theory and the concept of thermal damage time. Since the melanin occupies a much smaller volume compared with the follicle, heat is conducted from the melanin-rich shaft and the melanized portion of the bulb to the surrounding structures according to the laws of thermal diffusion. As it has been demonstrated that the most important targets for permanent hair removal (i.e. the stem cells) are located at the outer root sheath of the follicle at a distance from the hair shaft, and from the base of the follicle, reconsideration as to the appropriate laser parameters particularly pulse width and energy density has been recommended to allow for the propagation of the thermal damage through the entire follicular volume. This takes 3-20 times longer than the thermal relaxation time of the hair follicle. (This duration is referred to as the thermal damage time or TDT). It has been the basis of the new technology of superlong lasers as a method of more permanent hair reduction.[7,8,9]

So, there seems to be some sort of controversy as regards the mechanism of action of laser and the structure(s) to target. This has been reflected on the characteristics of laser invented for the purpose of hair treatment and also on the parameters recommended for better efficacy. In fact, a lot of variables have to be addressed on treating any patient with epilatory lasers. Some are pertaining to the laser itself e.g. pulse characteristics (spot size, wave length, duration and intensity), others to the treatment protocol (e.g. timing, frequency and total number of sessions) in addition to the patient variables (e.g. color of the skin, color and thickness of the hair, other biophysical tissue characteristics, anatomic area, age , sex, and hormonal profile).[1] Putting in consideration the large number of variables to deal with and also the not yet settled nature of mechanism of action, no easy conclusions can be made and the need is always there for an objective way to assess the validity of theoretical postulations and to evaluate the efficacy of any recommended treatment regimen.

The aim of the study was to evaluate the effect of variation in pulse characteristics on the clinical outcome (short and mid-term) and to assess the correlation between clinical results and the ablative effect on hair matrix and. hair follicle stem cells

Material And methods

Ten adult females have been included in the study. Excluded from the study were skin phototype V or VI, cases with recent sun tan, subjects below the age of 18 or above 45 years, photosensitive patients or those on photoactive medications, cases with previous attempts of treatment, patients suffering from major systemic or cutaneous illness, pregnant ladies, patients with clinical &/or laboratory evidence of hormonal troubles or of polycystic ovaries, cases with positive personal or family history of melanoma and those with keloidal tendency. In addition only patients with coarse, dense and black axillary hair occupying a fairly large surface area were accepted in the study.

The material of the study has been subjected to 2 sessions of axillary hair treatment by laser at 6 week interval (after informed consent).

In each case, four quarters have been marked on the hair bearing area of each axilla with a white indicator pencil. Identical marking in the two laser sessions as well as before clinical scoring and on taking biopsies was insured by the use of a premade template applied in fixed relation to the regional anatomic boundaries or to some already existing cutaneous landmarks (e.g. nevus or scar) and aided by digital photography under standard conditions of illumination and angles of exposure.

One quarter in each case was left without treatment (as a control) while each one of the other 7 quadrants was subjected to one of the following 7 parameter settings (or programs):

At 755 nm (wave length):

1) 18 mm (spot size) / 3 milliseconds (pulse duration) /12-14 j/cm2 (fluence)

2) 15 mm (spot size) / 3 milliseconds / 12-15 j/cm²

3) 15 mm (spot size) / 10 milliseconds / 12-14 j/cm²

4) 15 mm (spot size) / 40 milliseconds / 14 j/cm²

5) 12 mm (spot size) / 5 milliseconds / 14-16 j/cm²

At 1064 nm (wave length):

6) 15 mm (spot size) / 5 milliseconds / 25-30 j/cm²

7) 7 mm (spot size) / 100 milliseconds / 80-100 j/cm²

The machines used were Gentle-lase plus (Candela Corp., Wayland, MA, USA), Apogee 9300 and Apogee Elite (Cynosure, Westford, MA, USA).

Cooling was done with dynamic cooling device (DCD) or continuous cold air current (Cryo-5 or Smartcool) (Cynosure, Westford, MA, USA).

Protocol of treatment was as follows: no epilation for 4 weeks, no bleaching for 3 weeks, pretreatment shaving, removal of deodorants and other creams, marking, single pulse per spot, no overlap, post operative application of soothing cream plus usual post operative instructions.

The areas treated were evaluated clinically (for hair density and thickness) where each criterion was given a score of 0 to 6 and the average of each of these two scores was calculated for the 10 quadrants corresponding to each program. The multiplication product of the 2 scores was designated as the overall clinical score. This has been calculated initially as well as 6 weeks and 6 months after the last (2 nd) session.

A small elliptical biopsy was taken from each quadrant 6 weeks after the 2nd session.

The skin specimens were immediately fixed in 10% neutral formalin and embedded in paraffin blocks for subsequent staining. The paraffin blocks were cut into 30 serial 4 um-sections and every third section was subjected to immunohistochemical staining using monoclonal antibodies against LEF-1 (as a marker of hair matrix cells)[10] (ABCAM - Cambridge - UK) or monoclonal antibodies against cytokeratin-15 (as a marker of hair follicle stem cells)[11] (ABCAM - Cambridge - UK). The procedure was done according to the manufacturer instructions. Three sections from each biopsy were also stained with hematoxylin and eosin stain for routine histopathologic examination.

Immunohistochemistry:

i. Four-micron thick tissue sections cut from the representative paraffin-embedded tissue blocks, overlaid on APES (Sigma, St. Louis, USA) coated slides, were deparaffinized (2 changes of xylene X 5 minutes each, 1 change of acetone X 1 min) followed by rehydration in decreasing ethanol concentrations (95% ethanol X 3 mins, 70% ethanol X 3 mins, distilled water X 1 min).

ii. For staining with all the antibodies, the tissue sections were subjected to antigen unmasking by heating the sections immersed in 10 mM citrate buffer pH 6.0 (2.1 gms of anhydrous citric acid crystals dissolved in 1L of distilled water and pH adjusted to 6.0) inside a 600 watt microwave oven in full power for 35 minutes, allowed to cool to room temperature then washed briefly with 0.05 M Tris-Hcl buffer pH 7.4.

ii. Endogenous peroxidase activity was then quenched by immersing the sections in methanolic H₂O₂ (1 part 3% H₂O₂ plus 4 parts absolute methanol) for 30 minutes. After brief rinsing, the sections were placed in 0.05 M Tris-Hcl buffer pH 7.4 for 10 minutes.

iii. Sections were then overlaid with adequate amount of primary antibody diluted optimally using 0.05 M Tris-Hcl buffer pH 7.4 containing 1% bovine serum albumin (Sigma, St .Louis, USA) followed by incubation at 40C overnight.

iv. The slides were then washed with three changes (5 mins each) of 0.05 M Tris-Hcl buffer pH 7.4 followed by incubation for 30 minutes at room temperature after application of biotinylated secondary (link) antibody in phosphate buffered saline containing carrier protein and 15 mM sodium azide (LSAB Plus Kit, DAKO, Denmark).

v. After three washings (5 mins each) in Tris-Hcl buffer, peroxidase conjugated streptavidin was applied to cover the specimens and incubated at room temperature for 30 minutes.

vi. Slides were rinsed with 3 changes of Tris-Hcl buffer for 5 mins each. Sections were then covered with substrate chromogen solution prepared freshly by dissolving 1 mg of 3,3? - diaminobenzidine tetrahydrochloride (Sigma, St. Louis, USA) in 1 ml of 0.05 M Tris-Hcl buffer pH 7.4 containing 1 ?l of hydrogen peroxide. The slides were incubated at room temperature for 5 to 10 minutes under microscopic control till the optimal development of brown colored peroxidase reaction product.

vii. After rinsing in distilled water, the sections were lightly counterstained with Harris' hematoxylin, followed by mounting with cover slips with DPX as mounting medium.

viii. Precaution was taken so that drying of tissue sections strictly did not occur at any time during the entire procedure of immunostaining. All incubations were done inside humid chambers.

ix. Controls: During each batch of staining, positive and negative controls appropriate for the particular antibody were incorporated.

Primary and secondary antibodies used in the study are shown in table (1)
 

*Abcam, Cambridge, UK

Table (1): Primary and secondary antibodies used in the study.
 

Scoring method:

The stained sections were examined under the X40, X100, X200 and X400 magnification lens of light microscopy equipped with SIS image analysis computer system. For each marker, immunoreactivity was determined using a semiquantitative method in which the extent and intensity of staining was assessed on a score of 0 to 6 while the weighted score was the multiplication product of these two scores. The average of weighted score of the 10 sections examined for each biopsy was then calculated. The location and pattern of staining were also recorded. According to the degree of reduction in the average score, a final grade was given to each biopsy according to the following rule: 0-5 % reduction = 1, 5-10 % reduction = 2, 10-15 % reduction = 3, 15-20 % reduction = 4, 20-25 % reduction = 5, 25-30 % reduction = 6, 30-35 % reduction = 7, 35-40 % reduction = 8, 40-45 % reduction = 9, 45-50 % reduction = 10, 50-55 % reduction = 11, 55-60 % reduction = 12, 60-65 % reduction = 13, 65-70 % reduction = 14, 70-75% reduction = 15, 75-80 % reduction =16, 80-85 % reduction = 17, 85-90 % reduction = 18, 90-95 % reduction = 19, 95-100 % reduction = 20. The overall average grade for each set of parameters (or program) in the whole patient group was then calculated and compared with that of untreated control areas.

Statistical analysis:

Comparative analysis between two groups was done through unpaired T-test using graphpad software downloaded from the website: http://www.graphpad.com/quickcalcs/ttest1.cfm[12]

Correlation between different parameters was done through graphpad software downloaded from the website: http://calculators.stat.ucla.edu/correlation.php[13]

Results

The study included 10 healthy females of skin phototype II (3 cases 30 %), type III (5 cases 50 %) and type IV (2 cases 20 %). with the age ranging between 29 and 41 years (mean 33.4 +/- 8.6)

The results of clinical assessment for hair reduction (evaluated 6 weeks and 6 months after 2 sessions) are shown in tables (2)a, b, &c. The different programs showed statistically significant reduction in the early and late clinical scores (p<0.001). The clinical results were more or less comparable for different parameters settings except for the programs number 4 and 5 which gave significantly less hair reduction at the first follow-up visit after 6 weeks, and for program 5 only at the second follow up visit (after 6 months).
 

Table (2)a: Average clinical score after 6 weeks and after 6 months.
 

(N.B. The figures shown refer to p-value for unpaired student T-test comparing the corresponding 2 groups.)

Table(2)b: Comparative analysis of the early clinical score.
(after 6 weeks)