254 Automated immuno-histo-enzymatic investigation of metabolic enzyme activity in cryosections of skin and epidermal equivalents

Abstract

Metabolic activity of cells within the skin depends on cell type, nutrient and oxygen availability, position, age, and other parameters. We developmed an automated microscopy method to relate the enzymatic activity of single cells to immunohistochemical marker expression and its position within the epidermis or a 3D epidermal equivalent model. We adapted a StrataQuest software algorithm to predict the epidermis and its strata based on nuclear density mapping and distance-based modeling on automated scans of skin sections. The sections were IF-stained for differentiation markers. The activity assay for the enzyme glucose-6-phosphate dehydrogenase (G6PD) was performed with enzyme specific substrates. The automated prediction of the basal layer of the epidermis wrongly allocated less than five percent of keratin 10 positive cells in skin sections and epidermal equivalents. Next, we analyzed the enzymatic activity of G6PD within the predicted strata and cell shapes predicted from the nuclear distances in epidermal equivalents. The activity was significantly elevated from the basal to the suprabasal layers. We exposed the epidermal equivalents to Metformin, the pro-glycolytic anti diabetes drug, seven- or two days before cryosampling. We could detect a significant increase in measured G6PD activity in all strata of samples that had received metformin two but not seven days before sampling, most dominantly in the basal layer. In conclusion, we present an automated assay for epidermal image analysis that reliably identifies the basal and suprabasal strata of the human epidermis and of epidermal models, and could prove responsiveness of measurable enzyme activity to a candidate longevity drug.