Exercise and CGRP in the spinal cord do Nascimento PS et al. Table 1 - Body weight and blood glucose in the studied groups. Group Weight (g) C D TD Before induction 298¡5 307¡16 297¡9 48 h after induction Glycemia (mg/dL) 86¡5 389¡21* 352¡20* 30 days 60 days 90 days Weight (g) 351¡4 269¡10* 281¡11* Glycemia (mg/dL) 90¡2 525¡25* 438¡46* Weight (g) 384¡3 256¡11* 270¡8* Glycemia (mg/dL) 89¡2 534¡7* 525¡22* Weight (g) 406¡3 254¡11* 290¡17* Glycemia (mg/dL) 94¡3 527¡26* 535¡27* CLINICS 2012;67(9):1087-1091 laboratory conditions with food and water available ad libitum and a 12:12 h light/dark cycle. All efforts were made to minimize the number of animals studied and their suffering. The animals were cared for in accordance with Arouca Brazilian law (11794/2008) and the recommendations of the Brazilian Society for Neurosciences, the Review Committee of the School of Veterinary Surgery, University of Buenos Aires, and the International Brain Research Organization. Moreover, the study methods were in com- pliance with the National Institute of Health’s Guidelines for the Care and Use of Laboratory Animals (publication no. 85- 23, revised 1985), and the Ethical Committee of the UFRGS approved the study under protocol number 2008-062. Experimental design The rats were divided into three groups: non-diabetic rats (C), diabetic rats (D), and diabetic rats subjected to treadmill training (TD) for eight weeks. For the nociceptive analysis using the tail-flick test, ten animals were used per group. For the immunohistochemistry studies, six animals were randomly selected per group. Diabetes induction After an overnight fasting period (6 h), the rats from the D and TD groups received a single intravenous injection of streptozotocin (STZ; 50 mg/kg of body weight; Sigma Chemicals Co., St Louis, MO, USA) diluted in 10 mM of citrate buffer (pH 4.5), whereas the C group received only the citrate buffer (9,17). Blood collected from the rats’ tails was used to measure the blood glucose concentrations using test strips (Performa, Roche, USA). Diabetes was defined as fasting glucose .300 mg/dL in the tail vein blood 48 h after the STZ injection (12). The body weights were measured before diabetes induction and every 30 days thereafter, and the blood glucose concentrations were measured 48 h after the induction of diabetes and every 30 days thereafter. Maximal exercise test and treadmill training During the 4th week after diabetes induction, all of the animals underwent adaptation to a treadmill that was originally designed for human use (Runner, Sa˜o Paulo, SP, Brazil) and was modified for use by rats by walking for 10 min at 5 m/min for four days. On the 5th day, the rats were submitted to a maximal exercise test (MET) consisting of a graded exercise on the treadmill with speed increments of 5 m/min every 3 min, starting at 5 m/min and continuing up to the maximal intensity attained by each rat. The exercise was stopped when each animal remained for more than 50% of the time without run or walk. (13,14). The values obtained in the MET were used to plan the treadmill training program, which began during the 5th week after diabetes induction. To correct the exercise intensity, a second MET was performed during the 5th training week. 1088 Exercise was performed on a treadmill twice per day with an interval of 4 h between each session, five days per week. The training intensity was increased gradually according to the MET results. During the first week, the running sessions lasted 10 min, with increases in duration each week, reaching 60 min in the 7th week, which was maintained until the 8th week. Each training session consisted of a warm-up period, a main period and a cool-off period. During the warm-up period, the rats ran 15% of the session time at 30% of the maximum velocity determined by the MET; during the main period, the rats ran 70% of the session time at 60% of the maximum velocity; and during the cool-off period, the rats ran 15% of the session time at 30% of the maximum MET values (10). Nociceptive sensitivity analysis Nociceptive sensitivity was assessed using the tail-flick apparatus (Insight, Ribeira˜o Preto, SP, Brazil). The rats were wrapped in a towel and placed on the apparatus. A light source positioned below the tail was focused on a point 2– 3 cm rostral to the tip of the tail (15). The deflection of the tail activated a photocell and automatically terminated the test. The animals were exposed to the tail-flick apparatus every week to familiarize them with the procedure, as the novelty of the apparatus can itself induce antinociception (16). The tail-flick latency represented the period of time from the beginning of the test to the tail deflection. The final data are presented as a percentage of the control group, which was assigned a value of 100%, and the values are expressed as the means¡standard error of the mean (SEM). Immunohistochemical procedure One day after the nociception analysis, rats were anesthe- tized with sodium thiopental (i.p.; 50 mg/kg; Cristalia, Brazil). Heparin (1000 IU; Cristalia, Itapira, SP, Brazil) was injected into the left cardiac ventricle, and the animals were then transcardially perfused through the left ventricle using a peristaltic pump (Control Company, Friendswood, TX, USA, 20 mL/min) with 400 mL of 0.9% saline solution followed by 400 mL of a fixative solution of 4% paraformaldehyde (Synth, Diadema, SP, Brazil) in 0.1 M phosphate buffer, pH 7.4 (PB). The lumbar spinal cords were removed, post-fixed in the same solution at room temperature for 4 h and cryoprotected by immersion in 15% and 30% sucrose (Synth, Diadema, SP, Brazil) solutions in PB at 4˚C until they sank. After these procedures, the spinal cords were quickly frozen in isopentane (Merck, Darmstadt, Germany) cooled in liquid nitrogen and kept in a freezer (-70˚C) for further analyses. Transverse sections (35 mm) from the lumbar spinal cords were obtained using a cryostat (CM1850, Leica, Wetzlar, Germany) at -20˚C and collected in PB saline (PBS), pH 7.4. The free-floating sections were pre-treated with 3% hydro- gen peroxide for 30 min, carefully washed, treated with 2%