2.6. In Vivo Rat Anterior Cruciate Ligament (ACL) Transection-Induced Osteoarthritis (OA) Model

Ten to 11-week-old male Sprague Dawley (SD) rats with an average weight range from 230 g to 270 g (bought from Harbin Medical University, Harbin, China) were used to establish an experimental model by anterior cruciate ligament transection (ACLT). Rats were raised at the experimental animal center in Northeast Agricultural University on a standard 12 h dark/light cycle. All animal experiments were carried out in accordance with the guidelines of the China Ethical Committee for Animal Experiments.

A total of 72 rats were anesthetized (about 3.5% isoflurane, obtained from Shenzhen Ruiwode Life Technology Co., Ltd., Shenzhen, China) and the joint capsule incision and ACLTs were performed under an operating microscope. Rats were randomly divided into 6 groups: (1) ACLT group (no treatment), (2) Sham group (capsule incision only, PBS treatment), (3) Low-dose L-theanine (50 mg/kg), (4) Moderate-dose L-theanine (100 mg/kg), (5) High-dose L-theanine (200 mg/kg), (6) Celecoxib group (2.86 mg/kg [28]). The drug delivery route and dosage are presented in Figure 1. L-theanine was bought from Shanghai yuanye Bio-Technology Co., Ltd. (BR, 99%).

The intervention time point in vivo; osteoarthritis (OA) was induced in 10- to 11-week-old male Sprague Dawley (SD) rats by anterior cruciate ligament transection (ACLT) of the right knee. Sham surgery was performed on the right knee by incision of joint capsule, and sham operation was performed from a separated group of rats. L-theanine (50, 100 or 200 mg/kg, once a day gavage, until the rats were sacrificed), was administrated one day after ACLT surgery. Phosphate-buffered saline (PBS, 1 mL) was used for treatment after sham surgery as a control. Celecoxib (2.86 mg/kg) was used for treatment 4 weeks after ACLT surgery as a positive control.