Fluorescein Staining in an Autoimmune Dry Eye Rat Model to Examine Corneal Damage

Overview

This video demonstrates the staining of the damaged cornea using fluorescein dye to evaluate the extent of autoimmune dry eye disease in a rat model. The green fluorescence spots of the stained collagen fibers indicate corneal epithelial damaged cells.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Assessment of Dry Eye Features

Measure corneal damage with fluorescein staining.

1. Add 2 µL of 0.2% fluorescein to the rat cornea. Passively open and close the rat eyelids 3 times with a gloved finger to spread the fluorescein dye on the surface of the eye.
2. After 1 min, draw 1 mL of saline with a 3 mL syringe (without any needle attachment), position the syringe about 2-3 mm anterior to the cornea, and gently propel saline onto the eye.
3. Acquire images under the cobalt blue filter (i.e., ~400 nm) of an ocular-imaging microscope with the background lights switched off.
   NOTE: Images were subsequently analyzed by a grading system modified from previous publications. Images were then analyzed by subjectively grading the number, area, and intensity of the green spots from 0 to 2, where 0 indicates their absence, 1 indicates punctate staining of less than 50 spots, and 2 indicates punctate staining of more than 50 spots.

Materials

Name	Company	Catalog Number	Comments
Reagent
Fluorescein sodium solution	Bausch & Lomb U.K Limited	NA
Equipment
Stereo microscope with ring light illuminator and camera	Carl Zeiss	NA