Visualization of diffusion pathways across the stratum corneum of native and in-vitro-reconstructed epidermis by confocal laser scanning microscopy

Publisher Website
Google Scholar
Abstract
Confocal laser scanning microscopy is a technique that permits the direct visualization in unfixed material of diffusion pathways and the cellular distribution of fluorescent markers after topical applications. This approach, in which the tissue specimen is optically sectioned, allows the study of changes in distribution pattern of applied compounds depending on the vehicle, time and depth without the interference of chemical alterations induced by most of the current techniques used for such studies. Using this technique the permeability properties of in-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-populated collagen or on de-epidermized dermis. A fluorescent probe — Nile red (NR) — was applied in three different vehicles — polyethylene glycol (PEG) with a molecule mass of 400 (Da), propylene glycol (PG) and dimethyl sulphoxide (DMSO) — which perturb the SC barrier function to different 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, but was markedly increased when NR was applied in DMSO. Unlike native epidermis, the reconstructed epidermis allowed rapid NR penetration after the application in any of the solvents used. Furthermore, NR applied on reconstructed epidermis, was distributed quite homogeneously between the cellular and the intercellular spaces throughout the SC, suggesting that not only intercellular lipid structures but also the properties of the cornified envelopes differed markedly from those found in native epidermis. The differences in transport pathways between reconstructed and native epidermis may be partially ascribed to the culture conditions used, since incubation of freshly isolated epidermis under the same culture conditions as used for the reconstruction of the epidermis also leads to profound changes in the NR diffusion pathways.