tometry (Paugh and Joshi 1992; Joshi et al. 1996). The 5-min application protocol (n = 8) demonstrated a slight mean increase (test/control = 1.45) in permeability for the eyes receiving the tear solution. Neither the 3-day nor the 7-day application protocol caused a change in epithelial permeability when comparing the preserved and nonpreserved solutions to baseline data. This study provides information on the design of an exaggerated test application protocol. The acute application 0.01% benzalkonium chloride at the rate of six drops in 5 min did cause a measurable increase in epithelial permeability, but the effect was minimal. Clinical dose rates did not cause a measurable change in permeability.

3.3.5  Eye Bath Technique to Measure Epithelial Permeability

Bathing the corneal surface is the most accurate technique for estimating the corneal epithelial permeability. This method is best used with the rabbit model. The fluorophotometer can be used with either sodium fluorescein or carboxyfluorescein. A discussion of the differences between the two probes is useful. The corneal ­epithelial cell layer is a continuous lipid membrane with leaky intercellular clefts that are closed by zonula occludens or tight junctions. The integrity of the cell layer can be measured by the degree of restraint imposed on the migration of solute ­molecules, which is expressed as epithelial cell layer permeability. The ease with which a solute can penetrate the cell membranes depends on its lipid solubility and the integrity of the cells, i.e., membranes and zonula occludens. Sodium fluorescein is often used in ophthalmology to detect lesions in the corneal epithelium. Sodium fluorescein will slowly diffuse across lipid membranes and into intercellular spaces of normal cornea, but it has difficulty passing through the zone occludens of the epithelial cells (Adler et al. 1971). Once the sodium fluorescein obtains entry into the interior of the cell, it diffused freely to the interior of surrounding cells by ­passing through junction surfaces (Kanno and Loewenstein 1964). Araie (1986) preferred carboxyfluorescein, as compared to fluorescein, as a tracer for in vivo evaluation of cellular barrier function. He observed the permeability value did not change over a wide range of carboxyfluorescein concentrations. Also, the ­carboxyfluorescein did not penetrate the cell membrane; rather it appeared to have a permeability reflecting intercellular junction complex diffusion. The use of ­carboxyfluorescein rather than fluorescein as a permeability marker provides a more sensitive indicator of the integrity of the zonula occludens due to the more lipophobic nature of carboxyfluorescein. (Grimes et al. 1982; Araie 1986). The permeability to a molecule, such as carboxyfluorescein, is a measure of the number of molecules crossing the epithelial barrier relative to the bathing concentration and duration of exposure to the molecule. The initial measure of carboxyfluorescein in the cornea, following a bathing period, is the most appropriate value to calculate the epithelial permeability. Sodium fluorescein is the standard fluorophore used to ­measure corneal epithelial integrity and for this reason sodium fluorescein is selected in our fluorophotometry studies.

The effects on the corneal epithelium from drugs, tear-lubricating solutions, procedures (such as LASIK), etc., are tested for safety and efficacy. Safety testing is best