Male Sprague–Dawley rats were injected with GTN (10 mg/kg, i.p.) or vehicle and sacrificed 4 h after the injection. The principles of the Helsinki declaration and IASP’s guidelines for pain research in animal were rigorously applied [25]. Animals were housed in plastic boxes in groups of 2 with water and food available ad libitum and kept on a 12:12 h light–dark cycle. A total of 28 animals were used for the experiments and all procedures were in accordance with the European Convention for Care and Use of Laboratory Animals and were approved by the local animal ethic committee of the University of Pavia (Document n. 2, 2012). GTN [Bioindustria L.I.M. Novi Ligure (AL), Italy] was prepared from a stock solution of 5.0 mg/1.5 mL dissolved in 27 % alcohol and 73 % propylene glycol. For the injections, GTN was further diluted in saline (0.9 % NaCl) to reach the final concentration of propylene glycol (PG) 16 % and alcohol 6 % and administered at a dose of 10 mg/kg. A solution of saline (0.9 % NaCl), PG 16 % and alcohol 6 % was used as vehicle (CT group).