CLINICS 2012;67(9):1087-1091 bovine serum albumin (Inlab, Sa˜o Paulo, SP, Brazil) in PBS containing 0.4% Triton X-100 (PBS-Tx; Sigma Chemical Co, St Louis, MO, USA) for 30 min and incubated for 48 h with polyclonal CGRP-antibody (1:2250; courtesy of Dr. Rodrigo, Cajal Institute, Spain) and gentle stirring at 4˚C. The primary antibody was then removed, and the sections were washed in PBS-Tx for 30 min. The sections were then immersed in a secondary antibody (goat anti-rabbit lgG; Sigma Chemical Co., St Louis, MO, USA) diluted 1:50 in PBS-Tx for 2 h at room temperature with gentle stirring. After washing with PBS-Tx three times for 15 min, a soluble complex of horseradish peroxidase and rabbit anti-horseradish perox- idase (Sigma Chemical Co., St Louis, MO, USA) diluted 1:500 was applied for 1 h30 min at room temperature. The reaction was revealed in a medium containing 0.06% 3,39- diaminobenzidine (DAB, Sigma Chemical Co., St Louis, MO, USA) dissolved in PBS for 10 min, and 1 mL of 3% H2O2/mL was then added to the DAB medium for an additional 10 min. Finally, the sections were rinsed in PBS, mounted on glass slides, dehydrated in ethanol, cleared with xylene and covered with Entellan (Merck, Darmstadt, Germany) and coverslips. Control sections were prepared by omitting the primary antibody and replacing it with PBS. Optical Densitometry Semi-quantitative densitometric analysis was used to measure the intensity of the CGRP immunoreaction using a Nikon Optiphot-2 microscope (100x, Tokyo, Japan) coupled to a Micrometrics camera (Accu Scope, Commack, NY, USA) and Image Pro Plus Software 6.0 (Media Cybernetics, Rockville, MD, USA). The digitized images obtained from the dorsal horn of the spinal cords were converted to an 8-bit gray scale (0–255 gray levels). All of the lighting conditions and magnifications were held constant. The picture elements (pixels) employed to measure the optical density were obtained from squares measuring 1160 mm2 (area of interest; AOI) overlaid on the gray scale image. Both the left and right sides of each spinal cord were used. For each rat, 10 measurements were taken from the dorsal horn of the spinal cord. The results for the spinal cords are presented as the total mean value from the three studied regions. The background staining subtraction and correction were performed in accordance with our previously published protocol (17). The optical density (OD) was calculated using the following formula: ODðx,yÞ~{log½ðINTðx,yÞ{BLÞ =ðINC{BLÞ where ‘‘OD(x,y)’’ is the optical density at pixel(x,y); ‘‘INT(x,y)’’ is the intensity at pixel(x,y); ‘‘BL’’, or black, is the intensity generated when no light penetrates the material; and ‘‘INC’’ is the intensity of the incidental light. Statistical analysis The blood glucose and body weight data were analyzed using repeated measures analysis of variance (ANOVA), and the differences between the groups were assessed using the Bonferroni post hoc test. The data obtained from the nociceptive test and the optical densitometry of CGRP-ir were analyzed using a one-way ANOVA and the Bonferroni post hoc test. Statistical significance was set at p,0.05. The data were run on the Statistica 6.0 software package (StatSoft, Exercise and CGRP in the spinal cord do Nascimento PS et al. Figure 1 - Nociceptive sensitivity: the latency to tail deflection on the tail-flick apparatus for the C, D and TD groups. ANOVA followed by Bonferroni’s post hoc test. *corresponds to p,0.001 compared with the C and TD groups. Inc., Tulsa, OK, USA). All of the data are represented as the means¡standard error of mean (SEM). RESULTS Body weight and blood glucose concentrations Before diabetes induction, there were no differences in the body weight between the C (298¡5 g), D (307¡16 g) and TD (297¡9 g) groups (p.0.05). However, 30, 60, and 90 days after diabetes induction, the rats from the D (269¡10 g; 256¡11 g; 254¡11 g, respectively) and TD (281¡11 g; 270¡8 g; 290¡17 g, respectively) groups pre- sented lower body weights than the C group (351¡4 g; 384¡3 g; 406¡3 g, respectively; p,0.001; Table 1). Two days after diabetes induction, the blood glucose concentrations were higher in the D (389¡21 mg/dL) and TD (352¡20 mg/dL) groups than in the C (86¡5 mg/dL) group (p,0.001). The blood glucose concentrations were still higher at 30, 60 and 90 days in the D (525¡25 mg/dL; 534¡7 mg/dL; 527¡26 mg/dL, respectively) and TD (438¡46 mg/dL; 525¡22 mg/dL; 535¡27 mg/dL, respec- tively) groups after diabetes induction compared with the C group (90¡2 mg/dL; 89¡2 mg/dL; 94¡3 mg/dL, respec- tively; p,0.001). There were no differences between the D and TD groups at any time (Table 1). Nociceptive sensitivity The latency to the tail deflection of the C group was expressed as 100%. Group D required 154.7% more time to deflect the tail than the C group (p,0.001) and 120% more time to deflect the tail than the TD group (p,0.001). The TD 1089 Figure 2 - CGRP immunoreactivity (CGRP-ir) in the dorsal horn of the spinal cord from the C, D and TD animals. ANOVA with Bonferroni’s post hoc test. * corresponds to p,0.05 compared with the C and TD groups.