Animals
Male and female adult C57BL/6 mice (20–30 g) were maintained in a
humidity level (55–65%) and temperature-controlled room on a 12-hours
light/dark cycle with food (pelleted form) and water ad libitum .
Animals were housed 8 per cage, with nesting material. All experiments
were carried out in the light phase (between 07:00 a.m. and 7:00 p.m)
and the animals were acclimatized to the laboratory room for at least
1 hour before the experiments. The protocols employed in our study were
approved by the Institutional Committee for Animal Care and Use of the
Federal University of Santa Maria (UFSM; #9818180820).
The experimental protocols followed the guidelines of Animal Research
Reporting In Vivo Experiments (ARRIVE) [39]. Also, experiments were
performed using the current ethical guidelines for the investigation of
experimental pain in conscious animals, and the minimum necessary number
of animals and the intensity of noxious stimuli were used to demonstrate
the consistent effects of the treatments. Group size of n=8 animals for
behavioral experiments was determined using G*Power (v3.1).
All measurements of animal behavior were always performed by the same
experimenter and blinded to drug administration or the group to be
tested. ST (stressed) and NST (non-stressed) groups (both female and
male) were divided and studied on the same day, and experiments were
replicated on different days to generate results from the required
number of mice (See on Fig 1. schematic representation of experimental
design).