VISUALIZATION OF DIFFUSION PATHWAYS ACROSS THE STRATUM-CORNEUM OF NATIVE AND IN-VITRO-RECONSTRUCTED EPIDERMIS BY CONFOCAL LASER-SCANNING MICROSCOPY

Confocal laser scanning microscopy is a technique that permits the dir ect visualization in unfixed material of diffusion pathways and the ce llular distribution of fluorescent markers after topical applications. This approach, in which the tissue specimen is optically sectioned, a llows the study of changes in distribution pattern of applied compound s depending on the vehicle, time and depth without the interference of chemical alterations induced by most of the current techniques used f or such studies. Using this technique the permeability properties of i n-vitro-reconstructed epidermis were compared with those of the native counterpart. The epidermis was reconstructed by culturing human adult keratinocytes at the air-liquid interface either on fibroblast-popula ted collagen or on de-epidermized dermis. A fluorescent probe - Nile r ed (NR) - was applied in three different vehicles - polyethylene glyco l (PEG) with a molecule mass of 400 (Da), propylene glycol (PG) and di methyl sulphoxide (DMSO) - which perturb the SC barrier function to di fferent extents, When NR was applied in PEG and PG on native epidermis , the amount of NR penetrating into and through the SC was very low bu t was markedly increased when NR was applied in DMSO. Unlike native ep idermis, the reconstructed epidermis allowed rapid NR penetration afte r the application in any of the solvents used, Furthermore, NR applied on reconstructed epidermis, was distributed quite homogeneously betwe en the cellular and the intercellular spaces throughout the SC, sugges ting that not only intercellular lipid structures but also the propert ies of the cornified envelopes differed markedly from those found in n ative epidermis, The differences in transport pathways between reconst ructed and native epidermis may be partially ascribed to the culture c onditions used, since incubation of freshly isolated epidermis under t he same culture conditions as used for the reconstruction of the epide rmis also leads to profound changes in the NR diffusion pathways.