Scopolamine hydrobromide

To induce the DED model, adult female mice were exposed to an Intelligently Controlled Environmental System in which the relative humidity, airflow, and temperature were maintained at 15.0% to 20.0%, 2.2 ± 0.2 m/s, and 22 ± 2°C, respectively.⁴⁴ (link) Then, mice were subjected to subcutaneous injection of scopolamine hydrobromide (0.5 mg/0.2 mL; Sigma-Aldrich) 3 times a day for 5 days to decrease tear production. For the untreated group, mice were age and gender matched and fed in a normal environment with normal humidity (relative humidity, 60%–80%; no airflow; temperature, 21°C –23°C). Mice were sacrificed and the conjunctiva were collected and assayed for several experiments on the sixth day of this murine DED model.

This study was approved by the Committee on Animal Research at Yonsei Medical Center. All animal studies were performed in accordance with the Yonsei Medical Center Animal Research Guidelines, which adhere to the standards articulated in the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Dry eye mouse models were developed as described previously [19 (link)]. Eight-week-old C57BL/6 female mice, purchased from Orient Bio Inc. (Sungnam, Korea), were used for this experiment. Experimental dry eye was induced by the subcutaneous injection of 5 mg/mL scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) three times a day in a standard desiccating environment created by placing the mice in a chamber with a continuous air flow (15 L/min) in a room at 25 °C with an ambient humidity of 35%. Fourteen days after the initiation of the experimental dry eye, the mice were treated with or without 5 μL eye drops that contained Cact-3 (48 μM) in 1% polysorbate 80 in phosphate 4 times a day for 10 days. Ten days after the treatment, the measurement of tear volume using the phenol red thread test (Zone-Quick), corneal staining score, and relative mRNA level were performed.

All studies performed were approved by The Institutional Animal Care and Use Committees at Baylor College of Medicine and comply with the ARVO Statement for the Use of Animals in Vision Research. Female C57BL/6 mice aged 6–8 weeks old were purchased from Jackson Laboratories (Bar Harbor, ME). DS was induced by subcutaneous injection of scopolamine hydrobromide (0.5 mg/0.2 ml; Sigma-Aldrich, St. Louis), QID (08:00, 12:00, 14:00, and 17:00 h), for 3, 5 or 10 consecutive days in 6–8 week old female C57BL/6 mice and euthanized at the end of each variable (DS3, DS5 and DS10), as previously published (de Paiva et al., 2009 (link); de Paiva et al., 2006a (link); de Paiva et al., 2006b (link)). Mice were placed in a cage with a perforated plastic screen on one side to allow airflow from a fan placed six inches in front of it for 16 h/day. Room humidity was maintained at 30–35%. Control mice were maintained in a non-stressed (NS) environment containing 50–75% relative humidity without exposure to forced air. Since dry eye is more prevalent in women and male mice do not respond well to desiccation, only female mice were used (Gao et al., 2015 (link); Schaumberg et al., 2003 (link), 2009 (link)).