Thirty-six male Wistar rats (250 g) were procured from Bioterium of Metropolitan University of Mexico City and housed in clean plastic cages, separated into 6 groups and treated as follows: group 1, control (NaCl 0.9%); group 2, 3-NPA (20 mg/kg); group 3, B2 (10 mg/kg); group 4, B2 (10 mg/kg) + 3-NPA (20 mg/kg); group 5, B6 (10 mg/kg); group 6, B6 (10 mg/kg) + 3-NPA (20 mg/kg), each group N = 6. The administration of treatments was by i.p. The animals received the drugs every 24 h during 3 days. At the end of the treatment period and 30 min after the last drug administration, the animals were sacrificed with guillotine without anaesthetic procedure. The animal brains were immediately extirpated and put in saline solution (NaCl 0.9%) at 4 °C. Tissues were immediately dissected in regions and used to evaluate reduced glutathione (GSH), H2O2, lipid peroxidation, ATPase, and the levels of DA and 5-hydroxyindolacetic acid (5-HIAA).
The rats or breeds employed in the study were subjected to a selection process based on phenotypic variety; genetic, environmental and compartmental factors. Longitudinal weight curves, weight and physical exploration were the means employed to select a breed for inclusion or exclusion in the study. Also, to select a breed, it is fundamental that inbreeding is non-existence, thus having a hereditary control of traits with continuous variation.
The selected animals were kept in cool and dry place at a temperature of 15–16 °C and with air filter and humidity of between 50 and 60. The place was maintained clean and was continuously sterilized to avoid bacterial and fungal growth.
The breeds were fed with standardized diet based on 3800 kcal/kg, proteins 12%, fat 5%, vitamins and minerals. The selected animals were 3 months old male Wistar of approximately 250 g weight.
Brain extraction
On sacrificing the animals, the brains were excised from the base. Then, the brain tissue was dissected in cortex, striatum and cerebellum/medulla oblongata, weighed and homogenised in 5 volumes of 0.05 M tris–HCl, pH 7.4. An aliquot of the homogenized brain tissue was obtained and again homogenised in 0.1 M perchloric acid (HClO4) (50:50 v/v) using Yamato homogenizer (Yamato lh-21 LSC Lab, Dallas, USA) and stored at − 20 °C until analysed.
Animal management and care was conducted in accordance to the international guidelines for animal care and to the Mexican Guidelines ZOO-062. Besides, the study was approved by the Laboratory Animals Use and Care Committee of National Institute of Pediatrics.
Measurement of Dopamine (DA)
The DA levels were measured in the supernatant of tissue homogenized in HClO4 after centrifugation at 5000g for 10 min in a microcentrifuge (Hettich Zentrifugen, model Mikro 12-42, Tuttlingen, Germany), with a version of the technique reported by Calderon et al. [16]. An aliquot of the HClO4 supernatant, and 1.9 mL of buffer (0.003 M octyl-sulphate, 0.035 M KH2PO4, 0.03 M citric acid, 0.001 M ascorbic acid), were placed in a test tube. The mixture was incubated for 5 min at room temperature in total darkness, and subsequently, the samples were read in a spectrofluorometer (Perkin Elmer LS 55, Buckinghamshire, England) with 282 nm excitation and 315 nm emission lengths. The FL Win Lab version 4.00.02 software was used. Values were inferred in a previously standardized curve and reported as nMoles/g of wet tissue.
Measurement of 5-HIAA
5-HIAA levels were measured in the supernatant of tissue homogenized in HClO4 after centrifugation at 5000g for 10 min in a microcentrifuge (Hettich Zentrifugen), with a modified version of the technique reported by Beck et al. [17]. An aliquot of the HClO4 supernatant, and 1.9 mL of acetate buffer 0.01 M pH 5.5 were placed in a test tube. The mixture was incubated for 5 min at room temperature in total darkness, and subsequently, the samples were read in a spectrofluorometer (Perkin Elmer LS 55) with 296 nm excitation and 333 nm emission lengths. The FL Win Lab version 4.00.02 software was used. Values were inferred in a previously standardized curve and reported as nMoles/g of wet tissue.
Measurement of reduced glutathione (GSH)
GSH levels were measured from the supernatant of the perchloric acid homogenised tissue, obtained after centrifuging at 5000g for 5 min (Hettich Zentrifugen) according to a modified method of Hissin and Hilf [18]. A 1.8 mL phosphate buffer pH 8.0 with EDTA 0.2% plus a 20 μL aliquot of the supernatant and 100 mL of ortho-phthaldehyde (OPT) 1 mg/mL in methanol were put in a test tube and mixed. The mixture was then incubated for 15 min at room temperature in absolute darkness. At the end of the incubation time, the samples were read in a spectrophotometer (Perkin Elmer LS 55), with excitation and emission wavelengths of 350 and 420, respectively. FL Win Lab version 4.00.02 software was used. Values were inferred from a previously standardised curve and expressed as nM/g.
Measurement of lipid peroxidation
The lipid peroxidation across the reactive substances to the thiobarbituric acid (Tbars) determination was carried out using the modified technique of Gutteridge and Halliwell [11], as described below: From the homogenized brain in tris–HCl 0.05 M pH 7.4, 1 mL was taken and to it was added 2 mL of thiobarbituric acid (Tba) containing 1.25 g of Tba, 40 g of trichloroacetic acid (Tca), and 6.25 mL of concentrated chlorhydric acid (HCl) diluted in 250 mL of deionized H2O. The mixture was heated to boiling point for 30 min. (Thermomix 1420) and then cooled in an ice bath for 5 min. after which it was centrifuged at 700g for 15 min. (Sorvall RC-5B Dupont). The absorbance of the floating tissues was read in triplicate at 532 nm in a spectrophotometer (Helios de UNICAM). The concentration of TBARS was expressed in µM of Malondialdehyde/g of wet tissue.
Measurement of total ATPase
The activity of ATPase was assayed according to the method proposed by Calderón et al. [19]. 1 mg (10%) w/v of homogenised brain and heart tissues in tris–HCl 0.05 M pH 7.4 was incubated for 15 min in a solution containing 3 mM MgCl2, 7 mM KCl, and 100 mM NaCl. To this was added 4 mM tris-ATP and incubated for another 30 min at 37 °C in a shaking water bath (Dubnoff Labconco, TX, USA). 100 µL trichloroacetic acid at 10% w/v was used to stop the reaction and the samples were centrifuged at 100g for 5 min at 4 °C. Inorganic phosphate (Pi) was measured in triplicates using one supernatant aliquot as proposed by Fiske and Subarrow [20]. Supernatant absorbance was read at 660 nm in a Helios-α, UNICAM spectrophotometer and expressed as mM Pi/g wet tissue per minute.
Measurement of H2O2
The determination of H2O2 was made using the modified technique of Asru [21]. Each brain region (cortex, striatum, cerebellum/medulla oblongata) was homogenized in 3 mL of tris–HCl 0.05 M pH 7.4 buffers. From the diluted homogenates, 100 µL was taken and mixed with 1 mL of potassium dichromate solution (K2Cr2O7). The mixtures were heat to boiling point for 15 min (Thermomix 1420, CA, USA). The samples were later placed in an ice bath for 5 min and centrifuged at 3000g for 5 min (Hettich Zentrifugen). The absorbance of the floating was read in triplicate at 570 nm in a spectrophotometer (Heλios-α of UNICAM, Bristol, UK). The concentration of H2O2 was expressed in µMoles.
Statistical analysis
One way analysis of variance (ANOVA) or Non parametric Kruskal–Wallis test was used after the data have been subjected to variances homogeneity test. Post hoc Tukey–Kramer or Steel–Dwass contrast was employed. The values of p < 0.05 were considered statistically significant [22]. JMP Statistical Discovery Software version 10.0 from SAS was used.
