Receptor microscopic autoradiography for the study of percutaneous absorption, in vivo skin penetration, and cellular–intercellular deposition

Introduction: Microscopic autoradiography with cellular resolution and preservation of in vivo conditions is potentially the method of choice to gain detailed information about sites of deposition and retention in the epidermis and of penetration to the dermis after topical application of drugs. We tested this using 3 H-Maxacalcitol. Methods: Dorsal skin of adult rats was treated in vivo with ointment containing 1 or 40 μg/kg body weight of the vitamin D analogue 3 H-Maxacalcitol for periods of 0.5, 2, 8, 24, 48, or 168 h. Samples of skin exposed to the ointment and control samples remote from the treatment site were excised and freeze-mounted, and 4-μm frozen sections were exposed to nuclear emulsion. Results: Two penetration routes to the dermis could be distinguished: one via epidermal cell layers and the other via hair follicles. Highest uptake and retention of radiolabeled steroid was observed in stratum corneum and in intercellular spaces of stratum granulosum. By contrast, cell boundaries and intercellular spaces in the stratum spinosum and basale contained low levels of radioactivity. Keratinocytes in these layers showed high concentration in the cytoplasm at early time intervals, when surrounding radioactivity levels were high, but high nuclear and low or no cytoplasmic concentration at late time intervals, when surrounding radioactivity levels were low. Discussion: The autoradiographic method provides detailed information on time- and dose-related distribution of radiolabeled compound at the cellular level that is not obtainable with common radioassays and biochemical procedures. A sustained concentration and retention of radiolabeled steroid in the stratum corneum and intercellular space of the stratum granulosum indicate a selective deposition in components of secreted-membrane-coating granules and suggest a temporary barrier and depot for slow release. The differential cytoplasmic–nuclear distribution in the stratum Malpighi suggests functional correlation to a toxic–hormetic reversal of action on cell proliferation, from high-dose inhibitory effects associated with high extranuclear concentration as utilized in the treatment of psoriasis, to low-dose stimulatory effects associated with high nuclear and low cytoplasmic concentration as applicable in wound healing.