Tear secretion and stability of tear film examinations

Tear secretion was evaluated using the phenol red thread test. The mice were fixed in a 50-ml tube with the bottom cut off and phenol red-impregnated cotton threads (Tianjin Jingming New Technological Development Co., Ltd., Tianjin, China) were applied to the lateral canthus for 15 sec. The length of wetting of the thread, which turned red, was measured (mm).

The stability of the tear film was examined using a tear film breakup time (BUT) assay and corneal fluorescein staining. The mice were anesthetized with 50 mg/kg pentobarbital sodium (intraperitoneal) and 1 µl 1% fluorescein sodium (Alcon Laboratories, Inc., Duluth, GA, USA) were applied to the conjunctival sac. The BUT was recorded as the duration between the natural blink response of the mice to the appearance of a dark area, representing tear film breakup, using slit-lamp biomicroscopy and a cobalt blue light (BX900; Haag-Streit AG, Koeniz, Switzerland). The BUT was recorded three times and the average time was used. Then excess fluorescein sodium was rinsed using PBS and the corneal epithelium deficit was evaluated using fluorescein punctate staining the duration of which depended on the natural blink response time of each mouse. The staining was scored according to previously established standards (27).