Zoletile 50

For serum biochemistry, 1 mL of whole blood was collected from vena cava at sacrifice under Zoletile mixture (Zoletile 50; Virbac Lab., Paris, France) anesthesia and separated the serum. All serum samples were frozen at −150°C until they were assayed. Serum IL-6 levels werosteocalcin levels were detected by enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA) as pg/mL according to previously established method [30 (link)], and serum IFN-γ levels were also calculated using mouse IFN-γ ELISA kit (BD Biosciences/Pharmingen, San Diego, CA, USA) according to manufacturer's recommended protocols as pg/mL levels.

After acclimatization for 7 days, rats were anesthetized with an intraperitoneal injection of 25 mg/kg of Zoletile (Zoletile 50™; Virbac Laboratories, Carros, France) and maintained with 1%–1.5% isoflurane (Hana Pharmaceutical Co., Hwasung, Korea) in a mixture of 70% N₂O and 28.5% O₂. Surgery was performed according to established methods [16 (link)]. The second group of rats underwent a sham operation in which a similar incision in the linea alba was made but bilateral ovariectomy was not performed.

Rats were anesthetized with an intraperitoneal injection (25 mg/kg) of Zoletile mixture (Zoletile 50, Virbac Lab., Carros Cedex, France). The surgery was carried out according to the method of Kamekura et al. [17 (link)]. Briefly, the OA treatment group underwent open surgery involving ACL transection and partial medial meniscectomy via an incision in the medial aspect of the joint capsule, anterior to the medial collateral ligament. The operation was performed using a 15° 5-mm blade micro-surgical knife, micro-iris scissors, and micro-corneal suturing forceps and #11 and #15 blades. Following surgery, saline was used for washing tissue debris; the incision was closed in two layers. The joint capsule was sutured independently from peripheral tissues using dissolvable 5–0 Vicryl sutures, and the skin was closed by interrupted sutures using silk. This treatment was used to induce OA pathogenesis and referred to as operated-induced sides. Conversely, the right (non-operated intact side) knee joint was used as the contralateral treatment. The second group of rats underwent a sham operation in which a similar incision in the joint capsule was made, but ACL transection and partial medial meniscectomy were not performed. Only the left knees of sham animals were used as controls for disease progression.