Induction of OA and experimental timeline
The animals were briefly anaesthetized with a sevoflurane-oxygen mixture. Once withdrawal reflexes were no longer present, the knee joint was shaved, swabbed with 75% ethanol, and flexed at a 90⁰ angle. OA was induced with a single injection of MIA (2 mg in 25 µL of 0.9% saline) through the infrapatellar ligament of the right knee using a 26-gauge needle. The knee was then manually extended and flexed for 30 seconds to distribute the substance into the joint cavity. Control rats received the intra-articular injection of saline solution in the same volume („saline control“). The health status of the animals was then checked regularly by the experimenter and the animal care staff. The day of MIA/saline injection is referred as day 0 (Figure 1).
The animals were habituated to the behavioural test devices 3 days before the basal measurement (before MIA injection) and 30 minutes before each subsequent testing. Behavioural assessment of pain hypersensitivity was performed before and on days 4, 7, 10, 14, 21 and 28 post-injection using the weight-bearing and von Frey tests. The motor performance of the animals was assessed on the 8th, 13th, 20th and 27th day after injection of MIA using the rotarod apparatus (Figure 1). All assessments were performed between 8:00 and 16:00 to avoid diurnal variations in behavioural tests. The measurements were performed by one experimenter, who was blinded to the treatment conditions. The rats were randomly assigned to experimental conditions. Vortioxetine (2 and 10 mg/kg/day) and duloxetine (15 and 25 mg/kg/day) were administered orally, once daily for 28 days after the injection of MIA. Drug doses were selected on the basis of our preliminary results and data from the literature [18, 19, 20].
After the behavioural tests were completed, the rats were sacrificed and the knee joint tissue, the ipsilateral L3-L5 DRGs and the L3-L5 segment of the spinal cord were harvested for the corresponding quantitative real-time PCR (qRT-PCR) and redox status analysis.