This research protocol was approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Female C57BL/6 mice aged 6 to 8 weeks were used in the following experiments. We induced EDE by subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide (Sigma-Aldrich Corp., St. Louis, MO, USA) four times a day (8 AM, 11 AM, 2 PM, and 5 PM) with exposure to an air draft and 30% ambient humidity, as previously described.
23–25 During these experiments, animal behavior, food, and water intake were not restricted.
The mice were randomly divided into six groups according to the topical treatment administered as follows: (1) untreated control (UT): mice that were not exposed to desiccating stress or treated topically; (2) EDE control: mice that received no eye drops; (3) vehicle control: EDE mice treated with balanced salt solution (BSS; Alcon, Fort Worth, TX, USA); (4) EDE mice treated with 0.05% CsA (Restasis; Allergan, Irvine, CA, USA); (5) EDE mice treated with 0.001% AICAR; and (6) EDE mice treated with 0.01% AICAR. AICAR (Toronto Research Chemicals, Toronto, ON, Canada) was diluted in BSS. Two microliters of the eye drops were applied topically to both eyes of unanesthetized mice three times a day (8 AM, 12 PM, 5 PM), daily until they were killed. Clinical parameters, including tear volume, tear film break-up time (BUT), and corneal fluorescein staining scores, were measured 10 days after treatment. The clinical measurements were made after 3 hours of the last scopolamine injection and eye drops application. After measurement of the clinical parameters, the mice were euthanized, and Western blot, multiplex immunobead assay, histology, flow cytometry, and immunohistochemistry were performed. Each group consisted of five animals, and the experiments were performed on three independent sets of mice.