The rate of diffusion of fluorophores through the corneal epithelium and stroma

The time taken to cross the rabbit corneal epithelium and stroma was estimated for fluorescein (F), carboxyfluorescein (CF), rhodamine B (RB), and sulforhodamine B (SRB). Paired corneas were mounted in vitro; one was intact and the dye solution was kept in continuous contact with its epithelial surface; the epithelium was scraped from the other and the dye was applied as a pulse to the bare stroma. The time course of the dye appearing in a solution rapidly passing over the endothelial surface was determined by fluorometry. This rate of appearance was compared in the two cases and used to estimate the diffusional lag time introduced by the epithelium. For the very hydrophilic CF and SRB, the delay was too short to measure; this is compatible with the passage of these dyes taking place through the paracellular spaces. For the very lipophilic RB, the delay was about 2 min; this was rather too slow for it to be explained as being controlled entirely by diffusion in the cytosol. For the intermediate F, the delay was 5 min; it is suggested that this is a result of it partitioning between the spaces and the cytosol during its passage. The experiments also led to determinations of the permeability of the epithelial and endothelial layers to the dyes. In both cases lipophilicity was a strong determinant of penetration, but not the only one. The permeability of the endothelium to F was unchanged from its in vivo value in these experiments, but that of the epithelium was increased four-fold. The diffusion rate of the dyes across the stroma could also be determined. There was no clear relationship with molecular size or partition coefficient. The rate of diffusion of F across the tissue was about half that in its plane, as determined in previous experiments. This is possibly a result of the anisotropic structure of the tissue.