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.