Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB103

Poster Communications

Impedance spectroscopy indicates multiple barriers in stratified epithelia

R. Mannweiler1, S. Bergmann2, J. Brandner2, D. Günzel1

1. Institute of Clinical Physiology, Charité Universitätsmedizin Berlin, Berlin, Berlin, Germany. 2. Klinik und Poliklinik für Dermatologie und Venerologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany.

For the development of efficient and sustainable treatments for skin diseases, it is essential to understand the full mechanism of skin barrier formation. Although the Stratum Corneum (SC) has been identified as an important component of the skin barrier [1], it is still a matter of debate if and to what extend the Tight Junction (TJ) is playing a role in skin barrier function[BJ1] . There is evidence, that the disruption of the TJ protein Claudin (Cldn) 1 affects skin barrier formation [2], and that Cldn1 and Cldn4 are altered in several skin diseases [3,4]. Our goal was to investigate the effects of Cldn1 and 4 disruption on barrier function and the general role of the TJ in skin. As a common method for characterizing the barrier function of epithelia, Ussing chamber based impedance spectroscopy in the kHz domain [5] was performed. Isolated cells from human foreskin were cultivated as 3D reconstructed human epidermis (RHE) in cell culture inserts to mimic skin tissue. The impact of Cldn1 and 4 was studied by comparison of RHE with Cldn1 and 4 knockdown (KD) cells, respectively. 5% Triton X-100 in PBS was applied to the apical side of the cell layers to induce SC disassembly and cell permeabilization. Immunofluorescence staining was carried out to correlate morphological changes with barrier function. Nyquist plots of the impedance spectra show the presence of more than one time constant. Thus, the current is forced to pass further capacitors represented by the underlying cell layers due to a high resistor in the paracellular pathway. This indicates the barrier to be formed by more than the SC. Differences in the epithelial resistance and the distribution of the phase shift over the frequency prove that the absence of Cldn1 and 4 affects barrier formation in different ways (Fig. 1). Treatment with Triton X-100 reveals the presence of multiple time constants, each of which represents a specific compartment within the tissue (Fig. 2). In summary, these findings suggest that the TJ not only influences the formation of the SC but also acts as a barrier in the underlying layers of skin tissue.

Where applicable, experiments conform with Society ethical requirements