In vivo multiphoton imaging for non-invasive time course assessment of retinoids effects on human skin.

BACKGROUND: In vivo multiphoton imaging and automatic 3D image processing tools provide quantitative information on human skin constituents. These multiphoton-based tools allowed evidencing retinoids epidermal effects in the occlusive patch test protocol developed for antiaging products screening. This study aimed at investigating their relevance for non-invasive, time course assessment of retinoids cutaneous effects under real-life conditions for one year. MATERIALS AND METHODS: Thirty women, 55-65 y, applied either retinol (RO 0.3%) or retinoic acid (RA 0.025%) on one forearm dorsal side versus a control product on the other forearm once a day for 1 year. In vivo multiphoton imaging was performed every three months, and biopsies were taken after 1 year. Epidermal thickness and dermal-epidermal junction undulation were estimated in 3D with multiphoton and in 2D with histology, whereas global melanin density and its z-epidermal distribution were estimated using 3D multiphoton image processing tools. RESULTS: Main results after one year were as follows: a) epidermal thickening with RO (+30%); b) slight increase in dermal-epidermal junction undulation with RO; c) slight decrease in 3D melanin density with RA; d) limitation of the melanin ascent observed with seasonality and time within supra-basal layers with both retinoids, using multiphoton 3D-melanin z-epidermal profile. CONCLUSIONS: With a novel 3D descriptor of melanin z-epidermal distribution, in vivo multiphoton imaging allows demonstrating that daily usage of retinoids counteracts aging by acting not only on epidermal morphology, but also on melanin that is shown to accumulate in the supra-basal layers with time.

In vivo multiphoton multiparametric 3D quantification of human skin aging on forearm and face.

Quantifying skin aging changes and characterizing its 3D structure and function in a non-invasive way is still a challenging area of research, constantly evolving with the development of imaging methods and image analysis tools. In vivo multiphoton imaging offers means to assess skin constituents in 3D, however prior skin aging studies mostly focused on 2D analyses of dermal fibers through their signals' intensities or densities. In this work, we designed and implemented multiphoton multiparametric 3D quantification tools for in vivo human skin pigmentation and aging characterization. We first demonstrated that despite the limited field of view of the technic, investigation of 2 regions of interest (ROIs) per zone per volunteer is a good compromise in assessing 3D skin constituents in both epidermis and superficial dermis. We then characterized skin aging on different UV exposed areas-ventral and dorsal forearms, face. The three major facts of aging that are epidermal atrophy, the dermal-epidermal junction (DEJ) flattening and dermal elastosis can be non-invasively quantified and compared. Epidermal morphological changes occur late and were only objectified between extreme age groups. Melanin accumulation in suprabasal layers with age and chronic exposure on ventral and dorsal forearms is less known and appears earlier. Superficial dermal aging changes are mainly elastin density increase, with no obvious change in collagen density, reflected by SHGto2PEF ratio and SAAID index decrease and ImbrN index increase on all skin areas. Analysis of the z-dermal distribution of these parameters highlighted the 2nd 20 µm thickness normalized dermal sub-layer, that follows the DEJ shape, as exhibiting the highest aging differences. Moreover, the 3D ImbrN index allows refining the share of photoaging in global aging on face and the 3D SAAID index on forearm, which elastin or fibrillar collagens densities alone do not allow. Photoaging of the temple area evolves as a function of chronic exposure with a more pronounced increase in elastin density, also structurally modified from thin and straight elastic fibers in young volunteers to dense and compact pattern in older ones. More generally, multiphoton multiparametric 3D skin quantification offers rich spatial information of interest in assessing normal human skin condition and its pathological, external environment or product induced changes.