All rabbits were adopted a week before the experiments in the Laboratory Animal Center of China Medical University (Taiwan, ROC). The animals were kept at a controlled temperature (23 ± 2 °C), in relative humidity (60% ± 10%), with 12 h light–dark cycles (07:00–19:00), and given food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC no. 2021-276) of China Medical University. A total of 30 rabbits were used for the impression cytology study. The rabbits were randomly divided into 3 groups: the normal control group (n = 10), the vehicle group (n = 10), and the anti-miR-328 group (n = 10). Ten rabbits in the normal control were not treated with anything. DED was induced in the other 20 rabbits from days 1 to 21 by instilling 20 µL of 0.15% BAC twice per day (9 am and 5 pm). A quantity of 20 µL of anti-miR-328 (160 µM) or vehicle (PBS) was instilled in both eyes twice per day from day 8 to day 21, whereas BAC was still instilled 10 min after anti-miR-328 or vehicle treatment in these 2 weeks. Conjunctival impression cytology specimens were collected on day 21. After instilling 0.5% Alcaine and wiping away excessive fluid from the eye, a half-circular piece of nitrocellulose filter paper (T ADVANTEC®, Chiyoda-ku, Tokyo, Japan) with a diameter of 5.5 mm was placed on the superior bulbar conjunctiva. The filter paper was held in place for 1 min via slight pressure and was then peeled off from the eye and immediately fixed with 10% neutral buffered formalin. Periodic acid–Schiff (PAS) staining was performed using the PAS Stain Kit (ScyTek, Logan, UT, USA) according to the manufacturer’s protocol. The number of goblet cells was counted under a microscope at a 400× magnification. The density of goblet cells was quantified and expressed as the average number of cells in three random fixed areas (0.36 mm2) of each specimen in a high-powered field.
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