王修含診所皮膚科王修含醫師-電波拉皮

Identification of methods to enhance anagen entry can be helpful for alopecia. Recently, nonablative laser has been proposed as a potential treatment for alopecia. However, how the laser parameters affect stem cell activity, hair cycles and the associated side effects have not been well characterized. Here we examine the effects of irradiation parameters of 1,550-nm fractional laser on hair cycles.

The dorsal skin of eight-week-old female C57BL/6 mice with hair follicles in synchronized telogen was shaved and irradiated with a 1,550-nm fractional erbium-glass laser (Fraxel RE:STORE (SR1500) Laser System, Solta Medical, U.S.A.) with varied beam energies (5–35 mJ) and beam densities (500–3500 microthermal zones/cm²). The cutaneous changes were evaluated both grossly and histologically. Hair follicle stem cell activity was detected by BrdU incorporation and changes in gene expression were quantified by real-time PCR.

Direct thermal injury to hair follicles could be observed early after irradiation, especially at higher beam energy. Anagen induction in the irradiated skin showed an all-or-non change. Anagen induction and ulcer formation were affected by the combination of beam energy and density. The lowest beam energy of 5 mJ failed to promote anagen entry at all beam densities tested. As beam energy increased from 10 mJ to 35 mJ, we found a decreasing trend of beam density that could induce anagen entry within 7–9 days with activation of hair follicle stem cells. Beam density above the pro-regeneration density could lead to ulcers and scarring followed by anagen entry in adjacent skin. Analysis of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, revealed that transient moderate inflammation was associated with anagen induction and intense prolonged inflammation preceded ulcer formation.

Fig. 1. Laser irradiation. A. The skin on sides of the trunk was firmly stretched and irradiated with a 1,550nm fractional erbium-glass laser. B. The red rectangle indicated the irradiated area. C. Schematic diagram of the post-natal synchronized hair cycles on the back of C57BL/6 mice. The second telogen started at week 7 and persisted for about 5 weeks. Laser irradiation was performed on week 8. M: morphogenesis; C: catagen; T: telogen; A: anagen.

Fig. 2. Laser beam energy and MTZ. Irradiated skin was sampled 1 day after irradiation. A. Histology. MTZ could be identified by denatured collagen that showed a homogenized bluish change. H&E staining. Bar¼100 mm. B. There was an increasing trend of the depth of MTZ as beam energy was elevated. *P<0.05 compared with 5mJ group (N¼5). C. There was an increasing trend of the width of MTZ as beam energy was elevated. *P<0.05 compared with 5mJ group (N¼5).

Fig. 3. The effect of laser irradiation parameters on anagen induction and ulcer formation. A. Laser beam energy was increased from 5 to 35mJ and laser beam density was increased from 500 to 3500MTZ/cm2. Yellow color indicated the therapeutic window in which premature anagen was induced without ulcer formation. B. Quantification of the dynamics of anagen entry at various irradiation parameters. P<0.05 compared with day 0 (N=5) in each group. 80103mm.

Fig. 4. Changes of skin and hair cycles. Laser beam energy was 15 mJ. At beam density of 552 MTZ/ cm2, no premature anagen entry was induced. At beam density of 1048MTZ/cm2, irradiated skin started to turn grayish on day 9 to day 11(blue arrow head), indicating initiation of anagen. No skin erosion or ulcer was observed. At beam density of 1600MTZ/cm2 and 2010MTZ/cm2, an ulcer developed in the center of irradiated skin on day 5 and day 7 respectively (red arrow head). Premature anagen entry surrounding the scar tissue was observed (yellow arrow head).

Fig. 5. Histological changes during hair cycle progression. Beam energy was 15mJ and beam density was 1048MTZ/cm2. A. Comparison of gross skin change and histology. H&E staining. Bar?100mm. B. Quantification of anagen entry area. * P<0.05 compared with day 0 (N=5). C. Quantification of hair follicle length. * P<0.05 compared with day 0 (N=5).

Fig. 6(A). Dynamic changes of cell proliferation in hair follicles. Beam energy was 15mJ and beam density was 1048MTZ/cm2. A. Double staining for BrdU and p-cadherin.

Fig. 6(B). Dynamic changes of cell proliferation in hair follicles. Beam energy was 15mJ and beam density was 1048MTZ/cm2. A. Double staining for BrdU and p-cadherin. B. Double staining for BrdU and K15. Secondary hair germ was positive for p-cadherin and HFSC was identified by K15. From day 1 to day 5, BrdU was positive above bulge (yellow arrow heads). On day 7, BrdU was positive in secondary hair germ to lower bulge (white arrow heads). On day 9 and day 11, BrdU was positive in bulge (red arrow heads). Many BrdU positive cells could be found in the hair matrix on day 9 to day 13 (green arrow heads). DP: dermal papilla; SG: secondary hair germ; BG: bulge. Bar=50mm.

Fig. 7. Expression of IL-6 (A), TNF-a (B), and IL-1b (C) after laser irradiation of varied beam densities. Laser beam energy was 15 mJ. At beam density of 552 MTZ/cm2, very slight increase of IL- 6, TNF-a, and IL-1b was detected after irradiation. At beam density of 1048MTZ/cm2, a moderate increase of IL-6, TNF-a, and IL-1b was observed from day 1 to day 3. At beam density of 1600MTZ/cm2, prolonged and higher levels of IL-6, TNF-a, and IL-1b were observed.

To avoid side effects of hair follicle injury and scarring, appropriate combination of beam energy and density is required. Parameters outside the therapeutic window can result in either no anagen promotion or ulcer formation.



Lasers Surg. Med. 47:331–341, 2015. © 2015 Wiley Periodicals, Inc.