Optimization and validation of freeze-drying for light and electron microscopic autoradiography of percutaneous steroid transport

Abstract

A freeze-drying/autoradiography procedure to visualize steroid transport across human stratum corneum was optimized and validated. A finite dose of [ 3H]estradiol ( 3H-E2) was applied to human abdominal skin, i.e. isolated stratum corneum and dermatomed epidermal sheets, in an in vitro permeation set-up. The visualization procedure included cryofixation, freeze-drying, osmium tetroxide ( OsO 4) vapor fixation, Spurr resin embedding and autoradiography. The different steps of the sample preparation were carefully examined for morphological quality, extraction and diffusion of 3 H- E2 from the skin. It appeared that dermatomed skin was, for several reasons, more suitable to process than isolated stratum corneum. While cross-sections of dermatomed skin displayed a well-preserved stratum corneum, the viable epidermis suffered from decreased structural preservation due to cryofixation artifacts. The apparent extraction of 3 H- E2 during the infiltration with Spurr resin was due to donor phase residing at the skin surface. In the autoradiographs, there was no indication that 3 H- E2 had diffused out of the skin. Silver grains were mainly localized over the superficial corneocytes. Only a few grains were present over the deeper layers of the stratum corneum, both over the inter- and intracellular domains. An equation was deduced to estimate the exposure time of autoradiographs from skin samples, with the advantage that no time was wasted with empirical range finding tests. In the equation a distribution factor was introduced which can be used to quantify the localization of the silver grains over the different tissue structures. In conclusion, it can be stated that the visualization method is suitable to study the distribution of 3 H- E2 in human stratum corneum following topical application.