IJCS | Volume 31, Nº6, November / December 2018

612 Moura et al. Diabetic rats and aerobic training Int J Cardiovasc Sci. 2018;31(6)610-618 Original Article Exercise training Swimming In the first week of the experiment, the animals from the DMS group were placed in a pool with 700 L of water, divided by glass tanks of different sizes. The water temperature was maintained at 33 ± 1º C. The training protocol consists of 1 session of 60 min/day, 5 days/week and intensity determined by the MLSS, for 8 weeks. At midpoint of the protocol, that is, the fourth week of training, there was a new lactate test to readjust the intensity of training, as animals suffer physiological adaptations and the intensity established at the beginning of the protocol would satisfy a lower intensity of animal Lan. Treadmill After the week of adaptation and determination of MLSS, the animals of the DMT group started the ET protocol over 8 weeks, 5 days/week, 60 min/day at an intensity corresponding to MLSS. At midpoint of the protocol, that is, the fourth week of training, a new lactate test was performed in order to readjust the intensity of training. Glucose levels and body weight Glucose measurement was performed at the tip of the tail in the following phases of the trial period: after 12 h of fasting; every 7 days during the experimental period. For the fasting blood glucose test, reagent strips were used and measured by a glucometer. Bodyweight was evaluated under the same conditions and at the same time as glycemic control on a scale. Euthanasia of animals At the end of the experiment, rats were not handled for 24 h and after that they were anesthetized with ketamine (0.2 mL/100 g) and xylazine (0.1 mL/100 g), sacrificed by decapitation, and their tissues harvested. Cardiac chambers were dissected, and the left ventricle was weighed, as well as the right soleus and right extensor digitorum longus (EDL) muscles. Skeletal muscle cross-sectional area Soleus and EDL muscles were cut into 5-µm-thick sections using a cryostat and stained with hematoxylin and eosin for examination under light microscopy. Whole muscle cross-sectional area was evaluated at 200× magnification and further analyzed on a digitizing unit connected to a computer using the Axiovision program. All analyses were conducted by a single observer (EM), blinded to the rat’s group. Skeletal muscle glycogen content The soleus and EDL muscles were digested in 30% KOH at 100°C and glycogen was precipitated by the addition of 100% ethanol. After precipitation, the sample was centrifuged at 3500 rpm for 30 min. The supernatant was then decanted off and the precipitated glycogen was obtained quantitatively by two successive extractions with trichloroacetic acid 5%. Glycogen was estimated using a colorimetric assay with an anthrone reagent (0.2% solution in 95% sulfuric acid). The protocol was adapted for skeletal muscle tissue from Balmain et al., 17 and previously used by Voltarelli et al. 18 The values are expressed in microgram per gram of fresh weight. Cardiac structural analysis The left ventricles were then embedded in paraffin for histological processing. Sections (5 μm) were stained with hematoxylin and eosin for examination under light microscopy. Only nucleated myocytes from the transversally-cut muscle fiber areas were included in the cross-sectional diameter of cardiomyocyte analysis. 19 Quantification of left ventricular fibrosis was achieved using picrosirius red staining. Analyses were performed in a computer-assisted morphometric system. Statistical analysis The data are expressed as mean ± standard deviation. The normality of the data was verified through the Kolmogorov-Smirnov test. The effect of exercise training protocols was tested by one or two-way analysis of variance (ANOVA), as appropriate. When a statistically significant differencewas achieved, post hoc comparisons between groups were performed using Bonferroni test. Statistical analyses were performed using Dell Statistica (version 12). The level of significance was set at p < 0.05. Results As expected, the diabetic groups (DMC, DMS andDMT) displayed statistically significant (p< 0.05) higher glycemia at the beginning of the protocol (Table 1) and the glycemia remained high at the end of the protocol. Moreover, DMT