Effects of CO2 Fractional Laser on Hair Growth in C57BL/6 Mice and Potential Underlying Mechanisms
Hair loss, or alopecia, significantly impacts quality of life, self-esteem, and social interactions. Recent advancements in laser and light-based therapies have emerged as promising treatments. Among these, the CO2 fractional laser has shown potential for stimulating hair regrowth, particularly in androgenetic alopecia. However, the precise mechanisms underlying its efficacy remain poorly understood. This study systematically investigates the effects of CO2 fractional laser treatment on hair follicle regeneration in C57BL/6 mice and explores the molecular pathways involved in this process.
Experimental Design and Parameter Optimization
The study utilized six-week-old female C57BL/6 mice, housed under controlled conditions. Dorsal hair was shaved, and skin transition from telogen to anagen was monitored via color change (pink to black). A 10,600 nm CO2 fractional laser (Pixel CO2, Alma Lasers Ltd.) was applied to four dorsal skin regions per mouse, testing four energy parameters (6, 12, 18, and 24 mJ/spot) at a fixed density of 361 spots/cm². Skin reactions, hair regrowth, and adverse effects were evaluated over 13 days.
Initial parameter screening revealed dose-dependent outcomes. Lower energies (6 and 12 mJ/spot) induced minimal crust formation but delayed anagen entry (onset on day 11). At 24 mJ/spot, superficial ulceration and scarring occurred, rendering it unsuitable for therapeutic use. The 18 mJ/spot setting produced optimal results: yellow eschar formed without scarring, premature anagen entry (skin darkening by day 7), and visible hair regrowth by day 11. This parameter was selected for subsequent mechanistic analyses.
Histological and Morphological Observations
Longitudinal and transverse skin sections were analyzed via hematoxylin-eosin (HE) staining. Immediate post-treatment effects (day 1) included neutrophil aggregation around microscopic thermal injury zones (MTZs) and hair follicles. By day 3, epidermal exfoliation and re-epithelialization were evident, accompanied by mild dermal inflammation resolving progressively. Hair follicle numbers in deep subcutis increased steadily from day 5 onward, with the most significant rise between days 9–13 (Figure 1C). The proportion of anagen-phase follicles surged from 25.4% ± 4.6% on day 5 to 81.3% ± 7.2% by day 13 (Figure 1D). These findings confirm that CO2 laser treatment accelerates telogen-to-anagen transition, with follicular activation beginning as early as day 5.
Inflammatory Cytokine Dynamics
Real-time PCR quantified mRNA levels of inflammatory cytokines and Wnt signaling components. Interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) exhibited sharp increases immediately post-treatment (day 1), followed by gradual declines. Transforming growth factor-β1 (TGF-β1) expression displayed a triphasic pattern: an initial spike on day 1, a secondary rise on day 5 coinciding with anagen initiation, and sustained elevation from days 7–13. Vascular endothelial growth factor (VEGF) mirrored this trend, with peak expression during early anagen (days 5–7).
Wnt Signaling Activation
Wnt10b, a key regulator of hair follicle cycling, showed progressive upregulation from day 5 onward, aligning with follicular regeneration. Immunohistochemistry confirmed increased Wnt10b protein expression in hair bulb epithelial cells and surrounding dermal structures (Figure 2B). Conversely, Wnt5a remained undetectable, suggesting pathway specificity. VEGF protein localization paralleled mRNA data, with intense staining in dermal vasculature and hair follicles during anagen progression (Figure 2A).
Mechanistic Insights
The study proposes a multifactorial model for CO2 laser-induced hair regrowth. Moderate laser injury creates a controlled inflammatory microenvironment, attracting neutrophils and activating hair follicle stem cells (HFSCs). Acute inflammation resolves by day 5, coinciding with HFSC proliferation and differentiation. Subsequent phases involve VEGF-driven angiogenesis, ensuring nutrient delivery to regenerating follicles, and sustained Wnt10b signaling, which promotes follicular cycling and anagen maintenance.
Notably, excessive energy (24 mJ/spot) disrupts this balance, causing destructive inflammation and scarring. Lower energies (6–12 mJ/spot) fail to generate sufficient stimulus for timely HFSC activation. The 18 mJ/spot setting optimally balances injury and regeneration, inducing transient inflammation without structural damage.
Clinical Implications
Compared to non-ablative fractional lasers (e.g., 1550 nm), the CO2 laser triggers earlier anagen entry (5–7 days vs. 7–9 days). This advantage stems from its ablative nature, which generates deeper MTZs and more robust cytokine release. Combining CO2 laser therapy with topical growth factors could enhance clinical outcomes, as suggested by prior human studies.
Conclusion
This work elucidates the dose-dependent effects of CO2 fractional lasers on murine hair regeneration. At 18 mJ/spot, controlled injury induces transient inflammation, VEGF-mediated angiogenesis, and Wnt10b upregulation, collectively driving HFSC activation and anagen progression. These findings provide a mechanistic foundation for optimizing laser-based alopecia therapies and highlight the importance of energy calibration to avoid scarring.
doi.org/10.1097/CM9.0000000000000220
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