Click on a number from above to reveal the corresponding step instructions. The links below are permanent and bookmarkable.
Isolation and Mounting of Corneas
Upon receipt in the lab, the corneas are immediately prepared. The bovine corneas are excised and mounted in specially designed chambers. The chambers are then filled with media in a way that allows the corneas to retain their natural shape.
The mounted corneas are incubated to allow them to equilibrate for 1 hour. This is followed by a visual inspection to examine for imperfections (such as cuts, scratches, small opaque areas). Any corneas exhibiting such imperfections are removed from any further testing and discarded.
After the corneas have been examined, an initial baseline opacity measurement is performed for all the corneas. The initial opacity reading is representative of a normal, untreated cornea. Using a calibrated opacitometer, each cornea is compared against an air-filled chamber. After the initial opacity reading, each cornea is labeled with the appropriate test article or control treatment group and prepared for the dosing and rinsing procedure.
Dosing and Rinsing
The medium is removed from the anterior chamber of the holder. The test article or control is dosed on the epithelial surface of the cornea for its designated exposure period. Test articles may be applied neat or in dilution. For particularly viscous materials or slurries, the formulation may be applied directly to the cornea by removing the anterior chamber window.
After each designated exposure time, the corneas are thoroughly rinsed and the chambers are refilled. A post exposure opacity is taken (for liquid protocols) and the corneas are returned to the incubator for a specified post-exposure “expression” incubation. A second opacity measurement may be taken after the post-exposure “expression” incubation.
After the post-exposure opacity measurement, both chambers of the holder are aspirated, the posterior chamber is refilled with fresh media, and fluorescein is added to the anterior chamber. The chambers are then incubated vertically for 90 minutes. After 90 minutes, all of the medium in the posterior chamber is removed and then placed into a prelabeled glass tube.
The medium from each numbered tube is transferred to its corresponding well on a 96-well plate. The optical density at 490 nm is determined using a spectrophotometer.
Preparation for Histology (optional)
Corneas may be saved for histology for analysis of the degree and depth of corneal damage. Each cornea is carefully placed into a plastic cassette, fixed in formalin and processed for hematoxylin and eosin staining.
Histological analysis can be performed at IIVS or through a subcontracted board certified pathologist.
If histology is elected, an additional histology report is prepared complete with photographic representations of the corneas and a full histological evaluation for each test article and control treatment group. Please refer to Histology section for more information.
Calculate In Vitro Score
The opacity and permeability measurements are then evaluated in the calculation of the In Vitro Score. For example, a very mild ocular irritant may result in Mean Opacity Value of 1.4 and a Mean OD490 value of 0.158, resulting in an In Vitro Score of 3.77.
The following classification system (established by Sina et al.) provides a good initial guide to interpretation of these in vitro data. Please note that these specific ranges may not be applicable to all classes of materials.
In Vitro Score: * from 0 to 25 = mild irritant * from 25.1 to 55 = moderate irritant * from 55.1 and above = severe irritant
- Sina, J.F., Galer, D.M., Sussman, R.G., Gautheron, P.D., Sargent, E.V., Leong, B., Shah, P.V., Curren, R.D., and Miller, K. (1995) A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates. Fundamental and Applied Toxicology 26: 20-31