BUT measurement and corneal damage scoring in mice

Masatoshi Miyamoto, Takayuki Sassa, and Akio Kihara

This protocol was utilized for the measurement of tear film break-up time (BUT) and the corneal damage scoring in Tg-Cyp4f39^+/+ and Tg-Cyp4f39^−/− mice at 8–17 months.

Preparation of fluorescein solution and adjustment of slit-lamp microscope

Five mg of Uranine (fluorescein sodium salt, F0096; Tokyo Chemical Industry, Tokyo, Japan) was dissolved in 1 mL of phosphate-buffered saline to make 0.5% (w/v) of fluorescein solution. The solution was made on the day of the experiment and stored in the dark at room temperature until use.
Measurement of tear film break-up time was performed using a slit lamp microscope equipped with an LED light source and cobalt blue and yellow filters (Slit lamp RO8000; Visionix, Pont-de-I'Arche, France). The magnification of the slit-lamp microscope was adjusted so that the eye of the mouse occupied 70–80% of the visual field.

BUT measurement

Measurements were performed by two experimenters (A and B). Both were blind to the genotype of each mouse.
An unanesthetized mouse was held and laid on its left side on the stage of the slit-lamp microscope by experimenter A.
While holding the mouse on the stage, experimenter A focused on the right-eye surface of the animal under the slit-lamp microscope and set the cobalt blue filter for observation.
Experimenter B applied 3 μL of the fluorescein solution onto the lower-half area of the surface of the right eye using a micropipette (Pipetman P10; Gilson, Middleton, WI, USA). The fluorescein solution was carefully loaded so as not to damage the eye surface by the tip of the pipette.
Soon after the application of fluorescein solution, the mouse was manually forced to blink three times by experimenter A while observing under the slit-lamp microscope, to ensure that the fluorescein solution covered the entire surface of the eyeballs.
Once the uniform distribution of fluorescein was confirmed, experimenter A held the mouse eye open and kept observation.
BUT represents the elapsed time (in seconds) from the moment when the uniform distribution of fluorescein was confirmed until it was destroyed.

Corneal damage scoring

Corneal damage scoring was performed on each mouse following BUT measurement by a single experimenter, who was blind to the genotype.
Mouse was anesthetized by intraperitoneal injection of 0.05 mg/g (body weight) pentobarbital sodium salt (P0776; Tokyo Chemical Industry) dissolved in saline.
The eye of the anesthetized mouse was washed with saline to remove unbound fluorescein, and the excess saline after washing was removed with paper wipes.
The eye surface of the mouse was observed for the fluorescein staining of the damaged corneal surface under the slit-lamp microscope with cobalt blue and yellow filters.
Scoring of corneal damage was performed as follows. The eye surface was divided into five sections (Fig. 1), and the corneal damage in each section was evaluated as grade 0 to 3. The grade in each section was determined based on the fraction of the area over which punctate staining was observed: 0, < 1/3 area; 1, 1/3 to 2/3 area; 2, 2/3 to 3/3 area; 3, entire area or presence of filamentary keratitis. The corneal damage score was calculated as the sum of the section scores.

Fig. 1 Partitioning of the eye surface into 5 sections for corneal damage scoring