In situ precipitation: A novel cytochemical technique for visualization of permeability pathways in mammalian stratum corneum

Abstract

Although the stratum corneum has been traditionally treated as a homogeneous film across which substances penetrate without regard to cellular substructure, recent evidence indicates that the stratum corneum comprises a heterogeneous layer of corneocytes embedded in expanded, neutral, lipid-rich intercellular domains. One might predict, therefore, that lipophilic substances should preferentially traverse the stratum corneum between cells rather than through them. In this study, transport pathways were visualized by first perfusing freshly obtained neonatal mouse and human stratum corneum sheets with n-butanol for 5 min to 2 hr, followed by in situ precipitation of n-butanol with osmium vapor (GF Bahr, Exp Cell Res. 7:457, 1954). Control samples were treated similarly with osmium alone or with rapidly penetrating agents (e.g., n-hexanol) that are nonreactive with osmium. Ultrathin sections of tissues perfused with n-butanol, but not with n-hexanol, demonstrated expanded stratum corneum interstices filled with electron-dense precipitate, identified as osmium by energy-dispersive x-ray analysis (EDAX). About eight times more osmium was demonstrated in intercellular domains of n-butanol-treated samples than in comparable control locales by EDAX. Furthermore, when randomly selected negatives were analyzed by scanning densitometry there was approximately three times more osmium precipitate in the interstices than in the cytoplasm in n-butanol perfused tissues. These results suggest that in situ precipitation represents a useful approach to the study of percutaneous transport and may be generally applicable as a technique for studying lipid versus aqueous channels in other tissues. Finally, the results suggest that the stratum corneum intercellular spaces may serve as a preferential transport pathway for certain lipid-soluble substances.