Osmotic stress in an in vitro model of the outer blood-retinal barrier

Because of the difficulty involved in in vivo experimental manipulations of the outer blood-retinal barrier, we developed an in vitro model. Retinal pigment epithelium (RPE) from bovine eyes was grown on semipermeable membranes. A continuous monolayer of RPE developed a measurable transepithelial electrical resistance (TER). TER was used as a parameter of barrier function. The tightness of the cellular monolayer was also tested with fluorescein. The TER increased in confluent cultures by 60-70 ohms-cm2, and there was no fluorescein leakage. After exposure of the cultures to 0.2% trypsin or 1% ethylenediaminetetraacetic acid (ETDA), we detected a reversible breakdown of the epithelial barrier function. Under transmission electron microscopy, we detected no disassembly of the junctional components after breakdown of the barrier function with trypsin or EDTA. A hyperosmotic NaCl solution in the basal medium as well as a hypoosmotic NaCl solution in the apical medium induced a breakdown in the TER, whereas a hyperosmotic NaCl solution in the apical medium induced only a slight decrease in the TER. Under transmission electron microscopy, we detected an intracellular blistering and large intercellular widening. A hypo-osmolar NaCl solution in the basal medium induced a rise in the TER. With regard to these experiments, it seems, at least in vitro, that the function of occluding junctions in bovine RPE as permeability barriers depends on extracellular calcium. Furthermore, our results were such as if the outer blood-retinal barrier were designed to prevent subretinal inflow of free water.