Animals
Experimental procedures were carried out following protocols approved by the Ethical Committee for Use of Animals of the Federal University of Lavras (Approval # 025/16), which complies with Brazilian and international guidelines for animal use and welfare. Twenty-five male Wistar rats (60 days old, weighing 230–270 g) were used in the present study. The animals were obtained from the animal breeding facility of São Paulo State University (UNESP) (Botucatu, SP, Brazil) and kept in the Animal Care Unit of the Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo. The animals were kept under a 12-h/12-h light/dark cycle (lights on between 7:00 AM and 7:00 PM) and had free access to water and standard rat food. We chose all group size of all experiments shown in the present study based on previously reported data which are comparable with our experiments (Barretto-de-Souza, Benini, Reis-Silva, & Crestani, 2021; Gomes-de-Souza, Costa-Ferreira, Oliveira, Benini, & Crestani, 2020).
The use of wistar rats for these experiments is justified by the fact that this species of animal has some characteristics that allow us to experiment with it, among them: Viability, similarities with human beings (Leong, Ng, & Jaarin, 2015). Moreover, the choice of using male wistar rats for these experiments was to avoid the possible interference of hormonal variation throughout the estrous cycle of female and autonomic responses during restraint stress (Mahmoodzadeh, Fliegner, & Dworatzek, 2013; Miller et al., 2017; Miller et al., 2011).