2.2. Duct Ligation and Parotid Gland Weight

Mice were injected intraperitoneally with a mixture of medetomidine hydrochloride (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg). Under anesthesia, the unilateral parotid excretory duct was ligated, according to a previously reported procedure [18]. Salivary glands recover when the ligation is released 7 days after the duct ligation [19, 20]. We previously reported that parotid gland morphology showed weak atrophy of acinar cells in some samples on day 3 of duct ligation, whereas most acinar cells in the parotid gland were atrophied on day 7 of duct ligation [18]. Therefore, the period of duct ligation was set at 7 days for the present study. The mice were weighed on day 7 after ligation, and the parotid glands were excised [18] and weighed immediately. Parotid gland weight was divided by body weight to calculate parotid gland weight per body weight. Mice used as controls (n = 10) underwent sham operations and were maintained for the same period as the experimental group. In brief, a unilateral cheek was incised and sutured.