O. Simonetti et al., VISUALIZATION OF DIFFUSION PATHWAYS ACROSS THE STRATUM-CORNEUM OF NATIVE AND IN-VITRO-RECONSTRUCTED EPIDERMIS BY CONFOCAL LASER-SCANNING MICROSCOPY, Archives of dermatological research, 287(5), 1995, pp. 465-473
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.