Histopathological changes in the hair follicle after irradiation of long-pulse Alexandrite laser equipped with a cooling device

ARTICLE

Procedures for treating hemangiomas and pigmentation disorders of the skin with lasers which give good stable results have been established [1, 2]. In the fields of dermatology and plastic surgery attention is now focusing on epilation with lasers [3-27]. Laser-assisted hair removal has recently received considerable attention due to its noninvasive nature and speed, which is faster than conventional electrolysis.

In this study, the histopathological changes in hair follicles after epilation laser irradiation are discussed and the application of irradiation energy at the time of treatment is described. Currently, long-pulsed ruby lasers [3, 4, 7, 10-13, 16, 17, 23-27], long pulsed alexandrite lasers [14, 16, 18, 20, 26], Q-switched Nd:YAG lasers [6, 11, 16, 19], diode lasers [16, 26], and intense laser-like light source flash lamps [5, 11, 16, 21] are employed as so-called epilation lasers, and many studies on their efficacy have been reported. However, there are few studies of the histopathological changes resulting from these epilation treatments. There are a large number of clinical reports of the efficacy of epilation lasers, but scientific data regarding their exact mechanism of action are still lacking [11, 22, 26, 27].

Materials and methods

Normal hairy skin from the scalps of two volunteers, who needed excision of sebaceous nevi in spindle shape under local anesthesia, was taken right after irradiation with an alexandrite laser with a pulse duration of 3 msec and various levels of fluency. Histopathological examination of the skin was conducted. Skin specimens from the scalp of a patient who had an auricular deformity were also taken during surgery two months after treatment with the same laser and a histopathological examination of the skin was conducted. A GentleLASE Plus™ (Candela Corporation, Wayland, MA, USA) which is a flashlamp excited long-pulse alexandrite laser equipped with a computer controlled cooling device [16] was used. This device has an oscillating wavelength of 755 nm, a pulse duration of 3 msec, an irradiation interval of one pulse/sec and a fluence range adjustable from 6 J/cm² to 100 J/cm². Being able to select spot sizes from 8 mm up to 18 mm is a great advantage of this device. Specific features of this laser system are a lens coupled optical fiber beam delivery and an intelligent cooling device called Dynamic cooling device, which cools the skin with gas immediately before laser irradiation. The coolant gas is tetrafluoroethane (C₂H₂F₄ [freon 134A]) which has a boiling point of ­ 26.2° C and is an environmentally friendly, nontoxic nonflammable freon substitute which is approved by the FDA [16]. A feature of this device is that the spray duration, that is, the duration of the spurt of the coolant gas by the dynamic cooling device, and the delay time, that is, the relative timing before laser treatment, are accurately controlled by a computer and adjustable to 30-100 msec and 20-150 msec, respectively. The laser is programmed to spurt the coolant gas at first through the dynamic cooling device and, after a delay time, to irradiate the skin while the skin, especially the epidermis, is sufficiently cooled and protected from irradiation damage. This function is considered to cause minimum epidermal injury and destroy the follicles selectively at sufficient fluence and also to reduce the pain from the irradiation [26].

In this study, the irradiation fluences were set at 16, 18, 20, 25 and 30 J/cm² with a spot diameter of 15 mm and 20 J/cm² with a spot diameter of 18 mm. The skin was irradiated with the laser with or without the dynamic cooling device. Histopathological examinations were performed to compare the degrees of degeneration in the epidermis and follicles which had been treated with and without the cooling device. The spray time of the dynamic cooling device was set at 50 msec for irradiation at 16 and 18 J/cm², at 60 msec for irradiation at 20 and 25 J/cm² with a spot size of 15 mm and at 70 msec for irradiation at 20 J/cm² with a spot size of 18 mm. The delay duration was set at 20 msec for all irradiations. Immediately after the irradiation, the irradiated skin was resected and the site was sutured. The collected tissue was immediately placed in neutral buffered formalin solution, embedded in paraffin, and hematoxylin & eosin (HE) staining and Fontana-Masson (FM) staining were performed.

Results

The follicles of the skin were irradiated with the long-pulse alexandrite laser at fluences of 16, 18, 20, 25 and 30 J/cm² with a spot diameter of 15 mm and 20 J/cm² with a spot diameter of 18 mm according to the method described above, after which the histopathological changes in the follicles were investigated in detail. When the fluence was gradually increased, degeneration of hair shafts became conspicuous, and deeper. At larger magnification, in the follicular region of the skin irradiated at fluences of 20, 25 and 30 J/cm² with a spot diameter of 15 mm and 20 J/cm² with a spot diameter of 18 mm, the hair shafts were almost completely destroyed in the upper layer of the dermis and the external root-sheaths in the periphery were also destroyed (Fig. 1a). On the other hand, hardly any change in the collagen fibers of the dermis was noted in the periphery of the follicles. Similarly, when the condition of the deep layer of the dermis of the same tissue was examined, the hair shafts and external root-sheaths in the periphery were destroyed but there was no change in the collagen fibers of the dermis, indicating the deep penetration and selectivity of this laser equipment. When the condition of the hair papilla of the tissue irradiated at fluence of 25 J/cm² with a spot size of 15 mm was investigated thoroughly, in comparison with the normal hair papilla, swollen papilla and selective destruction of the site (Fig. 1b) were observed, while the surrounding collagen in the upper and mid dermis was not affected. The condition of the hair papilla of the tissue irradiated at a fluence of 30 J/cm² with a spot size of 15 mm was investigated, almost complete destruction of the site was observed. Distention of the site, injury of melanin containing cells and gaps between the hair shafts and external root-sheaths in the periphery were observed (Fig. 1c). The histopathological condition after irradiation at 20 J/cm² with a spot diameter of 18 mm was almost the same as that after irradiation at 25 J/cm² with a spot diameter of 15 mm, showing selective destruction of the hair bulb (Fig. 1d). On the other hand, no change was detected in the collagen fibers of the dermis, again demonstrating the deep penetration and selectivity of this laser equipment. When the hair papilla was examined at high magnification after F. M. staining, the destruction of melanin-containing cells was noted more clearly. When the membranous bulge of the follicle, where stem cells are considered to play an important role in the hair cycle, was observed at high magnification, the destruction of melanin-containing cells was also observed, in addition to that of the hair shafts (Fig. 1e). Even though the follicles in the deep region of the dermis were selectively destroyed, there was no change in the epidermis if the cooling device was turned on. On the other hand, when the dynamic cooling device was turned off and the tissue was irradiated at a fluence of 25 J/cm² with a spot size of 15 mm, injury of the basal layer of the epidermis was extensive, demonstrating the efficacy of the dynamic cooling device attached to the laser (Fig 2a, b).

Subsequently, the specimens from the patient with an auricular deformity who had auricular surgery 2 months after laser treatment at 20 J/cm² with an irradiation field diameter of 15 mm were investigated to compare the histopathology of the treated and untreated sites. While normal follicles were observed in the site deeper than the dermis in the untreated specimen, reduced follicles and dwarfing of the remaining follicles were clearly observed in the treated specimen (Fig. 3a, b). In the latter, only the follicles assumed to be in the telogen phase at the time of irradiation were observed in the lower layer of the dermis. The patient received another treatment with the long-pulse alexandrite laser 3 months after the initial treatment. As a result, the site which was previously hairy, became almost hairless 8 months after the initiation of the treatment.

Discussion

Although electrolysis has long been employed for the successful and permanent removal of unwanted hair, laser-assisted hair removal has recently received considerable attention since it is noninvasive and faster than electrolysis [11, 17, 28, 29]. The most comprehensive long-term studies with a ruby laser were published by Grossman et al. [3] and Lask et al. [4]. Grossman [3] used a 0.27 to 3 msec pulse duration at 20-50 J/cm² energy fluence and a repetition rate of 2 sec. The results were generally considered to be very successful although long-term expectations must be made clear to the patients. A machine which utilizes a laser light wavelength with larger absorbance differentiates between melanin and hemoglobin [1, 18, 19] and a wavelength which reaches deep into the skin is appropriate for epilation [18, 19]. In this regard, alexandrite and ruby lasers are both considered appropriate [3, 4, 17, 18]. However, it is important to define the optimal pulse duration and irradiation treatment protocol for epilation [6, 15]. The follicle is one of the most important of the skin appendages. Many cell layers are concentrically aligned in the hair in the anagen stage, and the hair grows after the cells divide, by elongating upward in the hair matrix around the hair-bulb in the deepest region [28, 29]. At this time melanin, which is the target of laser treatments [10, 16], exists in large quantities in the hair shafts themselves and in the hair bulb. The theory about the irradiation energy and time required for follicular destruction which is based on the "selective photothermolysis" theory of Grossman et al. [3] states that "the optimal pulse duration for selective photothermolysis is less than or approximately equal to the thermal relaxation time of the target structure". They estimated the thermal relaxation time to be about 40 to 100 msec. for follicles 200 to 300 mum in diameter [3]. It is likely, therefore, that pulse durations of approximately 10 to 50 msec could damage hair follicles with less epidermal injury. However the theory is still controversial, and Finkel et al. [8] proposed that a pulse duration should not exceed the thermal relaxation time of a typical follicle radius (40-100 mum), which is 0.5-10 msec. Because there are so many types of laser systems, whose pulse durations vary from nsec to 20 msec [16], a longer clinical observation period may be necessary to find out whether it is applicable for actual treatment when determining which machine is the most effective when a refined cooling device is used [15].

As mentioned above, the hair cycle is present in the follicles [28-30]. The hair matrix cells are present in the hair bulb region and are actively dividing [19]. The follicular stem cells are present in the bulge portion and are highly differentiated [31]. They are considered to play an important role in the regeneration of hair and in wound healing. Recently the destruction of the bulge portion without damaging the epidermis is considered to be necessary for epilation laser treatment [13, 18]. For this purpose, as shown by these experiments, a technique may be required which simultaneously destroys the peripheral cells and the cells showing some differentiation into melanocytes by transmitting energy from the hair shafts in the site. When such a technique is applied, irradiation at relatively high fluences of 25 J/cm² with a spot diameter of 15 mm, and 20 J/cm² with a spot diameter of 18 mm are required as demonstrated in this study. The fluence of 30 J/cm² with a spot diameter of 15 mm may be too strong because it resulted in complete destruction of the papilla. In addition, concomitant use of some cooling device is essential to prevent injury of the epidermis, pigmentation, depigmented spot formation and ulcer formation with treatment at a high fluence, especially when the patient has skin type III or higher (a high melanin content in the epidermis) according to Fitzpatrick's classification [15, 16, 18, 22, 26]. In this regard, Ash et al. [18] pointed out that "theoretically the alexandrite may be somewhat safer than the ruby for darker skin types because the 755 nm wavelength is less well absorbed by melanin than the 694 nm wavelength". However, the details have not been not fully clarified, and further investigation is required for the development and improvement of efficient epilation. According to our clinical experiences, when GentleLASE Plus™ equipped with a dynamic cooling device is used, which reduces pain, even infants tolerate the irradiation. Though persistent erythema and swelling was noted in the site where follicles were present, it faded within several hours after application of an ointment containing adrenocortical hormone and antibiotic, and almost complete recovery was observed the next day. From our experience, adverse events including ulceration, pigmentation, depigmented spot formation, etc. were not observed for 12 months in any of our patients treated under the above conditions.

The following should be considered for developing ideal epilation laser equipment:

1) The laser should be capable of destroying hair and follicles selectively without injuring the epidermis.

2) The laser irradiation should cause minimum pain and should not require local anesthesia.

3) The laser irradiation should cause no adverse reactions such as pigmentation and depigmentation after the treatment.

4) As to the treatment itself, it is necessary to repeat irradiation several times with an interval of generally 2 to 4 months while considering the hair cycle.

5) It is necessary to aim for "physiological depilation" in view of the physiological function of hair mentioned above, that is, preserve inconspicuous vellus hair and remove only hard hair rather than total depilation.

6) Efficient and safe equipment requiring the least treatment time is desirable.

7) Considering the depth of the photo-transmission of laser light into deep dermis where the hair follicles exist, a large spot size is also desirable.

Although many kinds of equipment with various pulse durations are now commercially available, the presence or absence of a cooling device is also an important point to consider in order to selectively destroy follicles with the least injury to the epidermis and minimum pain at the time of treatment [16, 22]. In this regard, the histopathological results of this study indicate that the efficacy and safety of the GentleLASE Plus^TM, which is a 3 msec pulse duration alexandrite laser equipped with a dynamic cooling device, were very high. It is known that the higher the wavelength, the higher the photo-transmission to the skin. From this standpoint, the wavelength of 755 nm oscillated by the alexandrite laser is considered to be optimal for treatments targeting follicles because it penetrates deeply and provides big differences in the absorption of melanin and hemoglobin. In other words, the machine itself may injure the epidermis but when it is equipped with a dynamic cooling device, it can selectively destroy deep follicles while preventing injury to the epidermis. It is considered that at a given energy level, the diffusion in the skin is more extensive and the laser light reaches deeper, if the area of irradiation is larger [4, 11, 15, 16, 18, 19]. In this regard, the larger irradiation fields of 15 mm (177 mm²) or 18 mm (254 mm²) in diameter with this equipment is advantageous compared to other equipment with smaller irradiation fields. The indications for using this epilation laser machine include depilation after treatment of nevus spilus such as Becker's nevus, hirsutism as an adverse reaction to steroid hormone treatment or hypothyroidism, depilation before and after plastic surgery of auricular deformation, other indications such as congenital anomalies [11, 28, 29], and even depilation after transsexual surgery in addition to so-called cosmetic depilation which is currently a frequent treatment.

The following summarizes the current status and problems of epilation laser treatment. Long wave length and long pulse lasers and optical treatment devices have been utilized for depilation. To determine which device is the most effective, it is necessary to compare the actual results of clinical applications in addition to the histopathological results rather than depend just on theoretical discussions. In addition, it is urgent that the depilation mechanism be elucidated accurately to find the ideal equipment and establish the conditions of treatment [27, 31].

We are convinced that the histopathological results of this study strongly indicate that the long-pulsed alexandrite laser equipped with a cooling device controlled by a computer is an efficacious tool for clinical depilation and the most suitable conditions for treatment are 25 Jcm² with a spot diameter of 15 mm or 20 Jcm² with a spot diameter of 18 mm.

Article accepted on 27/4/00

CONCLUSION

Acknowledgements

The authors are grateful to Ms. Naoko Suzuki and Ms. Yukiko Horikoshi for their technical assistance.

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