MECHANISMS OF ACTION OF SOME PENETRATION ENHANCERS IN THE CORNEA - LASER SCANNING CONFOCAL MICROSCOPIC AND ELECTROPHYSIOLOGY STUDIES

The present study investigates the effects of various penetration enchances including EDTA, digitonin, deoxycholic acid, and a cytoskeleton-active agent, cytochalasin B, on the structure and electrophysiological properties of the rabbit cornea. Using confocal fluorescence microscopy, with the aid of a specific membrane probe (3,3'-dioctadecyloxacarbocyanine perchlorate), and a viability polar tracer (propidium iodide), structural changes and cell death as a result of the penetration enhancers can be detected. In addition to its great sensitivity, the technique provides direct observation, through optical sectioning, of the interior structures of living tissues without the usual artifacts associated with tissue preparation which is required in conventional microscopy. In conjunction with the microscopic method, an electrical method based on membrane resistance and capacitance measurements was used to provide quantitative assessment of the promoting effect of the enhancers. Since electrical resistance is a measure of charge flow and ionic membrane permeability, it can be used to indicate changes in dimension of the aqueous transport pathway, i.e., the tight junction and the intercellular space, as well as the degree of penetrability of the charged species. On the other hand, membrane capacitance reflects the ability of the membrane dielectric to store charges, and thus, it can be used to detect structural damage to the membrane surface. Results obtained from this study indicate that all enhancers significantly increase membrane permeability depending on the concentration and exposure time. However, with the exception of the cytoskeleton-active agent, all enhancers also cause severe membrane damage. Mechanistically, this agent acts specifically on the tight junction proteins and consequently increases intercellular permeability. The chelator, EDTA, acts not only on cell junctions by interfering with calcium ions and altering intercellular integrity, but also disrupts the plasma membrane. Digitonin and bile salt interact with membrane lipid structures and disrupt their organization.