ABC | Volume 114, Nº1, January 2019

Original Article Brianezi et al. Effects of physical training on the myocardium Arq Bras Cardiol. 2020; 114(1):100-105 parameters: volume density of types I and III collagen fibers, metalloproteinases type 2 and type 9 expression, in addition to COX2 and 8-OhdG expression. Methods Animals Thirty femalemice aged 10months were used: 15 genetically modified female mice, with knockout of the low-density lipoprotein receptor (LDLr Knockout), and 15 female wildtype mice (C57BL/6J) obtained from the University of São Paulo vivarium. The animals were kept in a USJT vivarium at a controlled temperature (22-24°C) and lighting (12 hours of light cycle and 12 hours of dark) receiving commercial feed (NUVILAB CR1, Nuvital Nutrients LTDA, Curitiba, PR) and water "ad libitum". The animals were divided into 6 groups (n = 5): sedentary non‑ovariectomized control (CS), sedentary ovariectomized control (COS); trained ovariectomized control (COT); non-sedentary ovariectomized LDL knockout (LDL-S), sedentary ovariectomized LDL knockout (LDL-OS) and trained ovariectomized LDL knockout (LDL-OT). The division of the animals in the groups was performed by convenicence. The experimental protocol was approved by the Research Ethics Committee of Universidade São Judas Tadeu (CEP‑Protocol: 058/2007) and the research was conducted according to the Principles of Laboratory Animal Care formulated by the National Institutes of Health. Ovariectomy At nine months of age animals underwent the ovariectomy procedure. The animals were anesthetized with a ketamine and xylazine solution (120:20 mg/Kg, im) and placed in supine position and a small incision in the lower third of the abdominal region, parallel to the line of the body, was made. The ovaries, the uterine horns, and the blood vessels were located, sectioned, and removed. After that, the musculature and the skin were sutured. Confirmation of the efficacy of the ovariectomy was determined through colpocytology of the vaginal secretion performed over four consecutive days. On the last day of analysis, euthanasia was performed on these animals. 16 Training Protocol Maximal training Test A maximal training test was performed on all the groups at the beginning and at the end of the exercise training program. The test consists of placing the animal to run on an ergometric treadmill at 0.3 km/h for 3 minutes, and this workload was increased by 0.3 km/h every 3 minutes until the animal reached exhaustion. The time of the test and the speed of the last workload were noted and served to determine the mean value of aerobic capacity of each group. Exercise training Exercise training began 7 days after the ovariectomy surgery; the trained groups were subjected to a physical training protocol on an ergometric treadmill at low-moderate intensity (≈50% to 70%maximal running speed) for 1 hour a day, 5 days a week, for 4 weeks, with a gradual increase in speed from 0.3 to 1.2 km/h. The animals were adapted to the treadmill for 10 minutes on three days prior to beginning the training. Euthanasia and tissue preparation At the end of the training the animals were sacrificed by decapitation. A thoracotomy was performed in which the heart and atria were removed and the right and left ventricles were sectioned. Left ventricle samples were fixed in 10% buffered formalin for 24 hours. Afterwards, the tissue was transferred to a 70% ethyl alcohol solution, dehydrated in increasing ethanol series, diaphanized in xylol and embedded in paraffin. Non-serial, 5 μm thick cuts were performed in which each section received a total of 6 cuts. The sections were stained with the Picrosirius technique, for the analysis of collagen fibers I and III in the left ventricle and visualized by polarized light microscopy. The volume density of collagen fibers I and III (Vv[fc]) expresses the fraction of the volume occupied by the collagen fibers in relation to the total volume of the analyzed image. For this analysis, the test systemwas used with a total of 475 points, which corresponds to 100% of the image, using the program Image J. (version 1.47 - National Intitutes of Health-(Collins). Immunohistochemistry For the immunohistochemical techniques, 3 to 4micrometer thick cuts were made and mounted on slides previously silanized using 4% silane. After the slides were dewaxed, using a heating oven overnight set at 57ºC, after which they were immersed in xylol baths for 30 minutes. Afterwards, they were hydrated in ethyl alcohol with decreasing concentrations of 100%, 80% and 70%, each with a duration of 5 minutes, then washed in running water. In the next step, the antigenic recovery was performed in a water bath at 90°C, the slides were placed in citrate buffer for 30 minutes, then washed in running water. Endogenous peroxidase blockade was performed in H 2 O 2 for 15 minutes, followed by washing in distilled running water and in PBS pH = 7.4. For each slide, a specific antibody was used, table 1, and the antibody was placed overnight in a humid chamber at 4°C. The material was washed with PBS buffer and incubated with a biotinylated secondary antibody for 30 minutes, washed again with PBS and incubated with a streptoavidin-peroxidase antibody for another 30 minutes. A chromogenic substrate, DAB (33-Diaminobenzidine) solution in the ratio of 1:1 were used for five minutes at room temperature to wash with PBS solution and to reveal the reaction. When the presence of a dark brown precipitate was observed the slides were placed in running water, after which they were counterstained with "Hematoxylin Harrys" for 2 minutes. Afterwards, they were submitted to 3 xylol baths to diafieze and to 2 alcohol baths. The slides were mounted with coverslips and Entellan®. In all the techniques, observing the presence of a dark brown precipitate, the sample was visualized under an optical microscope. A quantitative analysis of the images was performed using the program ImageLab 2000, where the brown markers were selected and the program quantified immunoexpression intensity, figure 1. 101