2.1. Animals and pain model.
A total of 124 Sprague-Dawley rats (Envigo, Barcelona, Spain) of 7-8 weeks old at the beginning of the behavioural experiments, 100 females (170-200 g) and 24 males (260-300 g) were used. Animals were housed individually in standard plastic cages (42 x 27 x 18 cm3) provided with shredded aspen bedding (Teklad, Barcelona, Spain) and cotton enrichment (iso-BLOXTM; Teklad) and were maintained in 12/12 hours light/dark cycles (light off at 07:00 am, light on at 07:00 pm), at 23 ± 1 º C and 60% humidity. All behavioural tests were conducted during the dark cycle and at least 2 hours after the lights turn off. Food and tap water were available ad libitumthroughout the experimental period. The protocols used were approved by the Animal Care Committee from the University of Valencia and authorised by the regional government, and the studies were performed in strict accordance with Spanish laws (RD 53/2013) and European Directives (EC 2010/63). Animals were sacrificed by isoflurane overdose for further biochemistry analysis.
Rats received a subcutaneous injection of 0.1 mL of the complete Freund adjuvant (CFA; diluted at 1:2 in sterile saline to create an emulsion) in the hindpaw (Hipolito et al., 2015). This animal model of pain based on the CFA administration has been broadly used for replicating aspects of arthritis (Fischer et al., 2017).