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

Validation of Freeze-Drying to Visualize Percutaneous ³H-Estradiol Transport: The Influence of Skin Hydration on the Efficacy of the Method

“…Because 3 H-estradiol accumulated in the stratum corneum [1], the desired grain density n was reached much sooner over the stratum corneum than over other parts of the section. As a result, shorter autoradiography exposure times were possible, which was described by:…”

“…by previous autoradiography experiments or by layerwise retention measurements in the skin as described by Seta et al [12]. In Neelissen et al [1] it was stated that the distribution factor can also be used to illustrate the effect of donor type on the distribution of 3 H-estradiol in the skin. The equation to calculate the distribution factor can be derived from equations 1 and 2:…”

“…As described in Neelissen et al [1], the exposure time of autoradiographs from cross sections of the skin can be estimated by a mathematical approach: Neelissen/Arth/Schrijvers/Junginger/ Boddé with n the desired grain density in the autoradiographs in grains per square millimeter, d sk the thickness of the skin in millimeters, a s the surface-related activity in disintegrations per minute per square millimeter tissue (defined as the total radioactivity deposited on and taken up in the skin, per unit exposed surface area), · the isotope loss during Spurr infiltration, 1,440 the conversion from minutes to days, d se the section thickness in millimeters, s the sensitivity of the emulsion/ developer combination in grains/disintegration, Û the relative sensitivity factor to compensate for 3 H-isotope absorption in the sections, and t cal the calculated exposure time in days. Because 3 H-estradiol accumulated in the stratum corneum [1], the desired grain density n was reached much sooner over the stratum corneum than over other parts of the section.…”

“…In Neelissen et al [1], freeze-drying followed by light microscopic and EM-ARG was optimized for human dermatomed skin pieces containing 3 H-estradiol after a dry dose permeation experiment. The experiments described here with two additional donor formulations, a patch and a buffer solution, showed that the quality of freeze-drying strongly depended on the level of stratum corneum hydration.…”

“…In Neelissen et al [1] freeze-drying for light and electron microscopic autoradiography of percutaneous estradiol transport was optimized and validated. A 3 H-estradiol solution was applied to dermatomed human skin, resulting in a crystalline 3 H-estradiol layer after the solvent had evaporated.…”