Methods 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). On the basis of the distribution of the nuclei that are known to be activated by GTN and involved in migraine pain, the following discrete brain areas were dissected out 4 h after GTN or vehicle administration and used for analysis: medulla-pons, containing nucleus trigeminalis caudalis (NTC), nucleus tractus solitarius and area postrema; mesencephalon, containing ventrolateral column of the periaqueductal grey and parabrachial nucleus, and hypothalamus, containing the paraventricular and supraoptic nuclei of the hypothalamus. Western blotting Rats (N = 6 per experimental group) were perfused transcardially with 250 ml cold saline, 4 h after GTN or vehicle administration. Brains were immediately removed and chopped into parts; brain areas of interest were dissected out and used for the preparation of total extracts. The samples were homogenized on ice with a homogenizer in at least 5 volumes of modified RIPA buffer (Tris 50 mM, pH 7.4, NaCl 150 mM, EDTA 1 mM, SDS 0,2 %) supplemented with cocktail inhibitors protease. Then, they were incubated on ice for 20 min. The tissue lysate was centrifuged at 10,000 × g for 45 min at 4 °C and supernatants stored at −80 °C. Protein assay was performed by bicinchoninic acid (BCA) method. A 20 μg of protein were submitted to SDS-poliacrylamide gels 10 % and transferred onto a PVDF membrane (Amersham Biosciences). After blocking with 5 % dry milk, the blots were probed overnight at 4 C° with rabbit polyclonal anti-nNOS serum (1:1000; Cayman Chemical) or anti-eNOS serum (1:1000; Santa Cruz Bioctenology) and then probed for 1 h with an anti-rabbit horseradish peroxidase coupled secondary antibody (1:10000; Amersham Biosciences). An enhanced chemiluminescence system (ECL Advance; Amersham Biosciences) was used for visualization. Membranes were also probed with a rabbit polyclonal anti-β actin antibody (1:1000; Santa Cruz Biotechnology) as a housekeeping protein. For semiquantitative analysis, a Bio-Rad GS800 densitometer was used. NOS expression was evaluated in each sample by dividing the optical density of the NOS band by the intensity of the optical density of the band corresponding to the housekeeping protein. The specificity of the antibodies was confirmed by immunoprecipitation with a specific blocking peptide. Enzyme-linked immunosorbant assays (ELISA) Rats (N = 8 per experimental group) were injected with GTN (10 mg/kg i.p.) or vehicle and then killed with a lethal dose of anaesthetic 4 h after treatment. Their brains were immediately chopped into parts; brain areas of interest were dissected out and frozen at −80 °C until further processing. Blood was drawn from the vena cava and centrifuged at 3000 g for 10 min at 4 °C. ADMA levels (ng/mg proteins or nmol/ml) were quantified by ELISA kit (Antibodies Online) according to the manufacturer’s instructions. Real-time polymerase chain reaction Rats (N = 6 per experimental group) were injected with GTN (10 mg/kg i.p.) or vehicle and then killed with a lethal dose of anaesthetic 4 h after treatment. Their brains were immediately chopped into parts and frozen at −80 °C until further processing. DDAH-1 mRNA expression was analyzed by a real-time polymerase chain reaction (RT-PCR) and total RNA was isolated from the cerebral samples with Trizol reagent in accordance with the method of Chomczynski and Mackey [26]. RNA was quantified by measuring the absorbance at 260/280 nm. cDNA was generated using the iScript cDNA Synthesis kit (Bio-Rad) following the supplier's instructions. Gene expression was analyzed using the Fast Eva Green supermix (Bio-Rad). As regards housekeeping, gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used. The expression of the housekeeping gene remained constant in all the experimental groups considered. The amplification was performed through two-step cycling (95–60 °C) for 45 cycles in a light Cycler 480 Instrument RT-PCR Detection System (Roche) following the supplier's instructions. All samples were assayed in triplicate. Gene expression was calculated using the ΔCt method. Statistical evaluation Data are expressed as mean ± SD. Comparisons between groups (GTN and CT) were performed using the Mann Whitney test. The minimum level of statistical significance was set at p < 0.05.