Rompun

Mice were anesthetized via intraperitoneally injection of a mixture of zoletil (30 mg/kg; Zoletil 50, Virbac, Carros, France) and rompun (10 mg/kg; rompun, Bayer Health-Care, Leverkusen, Germany), and the trachea were exposed and cannulated with silicone tubing attached to a 23-gauge needle on a tuberculin syringe. Bronchoalveolar lavage (BAL) fluid was withdrawn after instillation of 800 μL sterile phosphate-buffered saline (PBS) through the trachea into the lung. The total cell counts in BAL fluid were determined using a hemocytometer. The BAL fluid was cytospun (5 minutes at 750 g) onto microscope slides and stained with Diff-Quick (Sysmax, Kobe, Japan). The percentages of macrophages, lymphocytes, eosinophils, and neutrophils were calculated by counting 500 leukocytes on randomly selected regions of the slides under a light microscope. The supernatants were stored at −70°C.

To perform ovariectomy, a general anesthetic containing a mixture of ketamine hydrochloride (Ketamine HCl; Yuhan Corp., Seoul, Korea) and Rompun (Rompun; Bayer Korea, Seoul, Korea) in the ratio of 1:1 was administered by intraperitoneal injection (0.4 mL); the abdomen was disinfected, the abdominal hair removed, and the depilated area disinfected with 10% betadine solution before the operation.
Using surgical scissors, the abdominal muscles, skin, and peritoneum were cut and the abdominal cavity entered for approximately 2.0 cm, and the uterine tubes connected to the ovaries were ligated to the ovary with the silk thread. The ovaries were completely removed and the peritoneum, abdominal muscles, and skin were sutured. A week-long recovery period was allowed.

Animals of the CCD group were randomly divided into two groups. Eight rats were subjected to optogenetic viral vector AAV-CaMKII-hChR2-EYFP (titer 1 × 10¹³ GC/ml, Korea Institute of Science and Technology, Seoul, South Korea), while the remaining eight rats were subjected to null virus AAV-CaMKII-EYFP (titer 1 × 10¹³ GC/ml, Korea Institute of Science and Technology, Seoul, South Korea) into M1 layer 5 region (anteroposterior (AP): 1 mm, mediolateral (ML): 1.5 mm, dorsoventral (DV): 1.5 mm),^{28 (link)–30 (link, no link found)} by intracranial injection under general anesthesia. Animals of the sham group were also divided into two groups for injection of either the optogenetic virus or null virus. We injected adeno-associated virus carrying the ChR2-EYFP fusion protein under the control of an excitatory neuron-specific calcium/calmodulin-dependent protein kinase II (CaMKII) promoter in our study. Before injecting virus, the animals were anesthesized by an i.p. injection of a mixture of 15 mg/kg Zoletil (Zoletil50®, Virbac Laboratories, Carros, France) and 9 mg/kg Rompun (Rompun®, Bayer, Seoul, South Korea). Then, 2 µl of virus was injected at a rate of 0.3 µl/min using a Hamilton syringe and an automatic microsyringe pump. After injection, the needle was kept in the same place for 5 min to have the virus absorbed and then retracted in a very slow manner.

For the OA model, 33 rats were anesthetized with an intraperitoneal injection of a 1:1 mixture of tiletamine and zolazepam (Zoletil 50; Virbac, Carros, France) with xylazine (Rompun; Bayer, Leverkusen, Germany) at a dose of 30 mg Zoletil and 10 mg Rompun per kilogram of body weight. The anterior surface of the left hind limb was shaved with an electric clipper, and the skin around the incision area was cleansed with Betadine. The skin and fascia on the kneecap region of the left hind limb was vertically incised in the midline for a distance of approximately 4 cm. The patella was retracted laterally to expose the articular cavity. The synovial membrane was excised, and the knee joint was bent to expose the anterior cruciate ligament. The medial collateral ligament was also exposed by retracting the pes anserine muscles aside. Then, the anterior cruciate and medial collateral ligaments were transected, and the medial meniscus was completely removed with surgical scissors. The patella was then relocated back to its original position, and the fascia and skin were closed with 3–0 polydioxanone suture. A single dose of antibiotic cream was applied to prevent postoperative infection.