ABC | Volume 112, Nº2, February 2019

Original Article Maifrino et al Exercise effects on ovariectomized mice Arq Bras Cardiol. 2019; 112(2):180-188 8-OHdG is one of the main markers of DNA oxidative damage induced by reactive oxygen species (ROS). 11,12 MMPs play key roles in the function of various tissues during growth, development and aging of the organism. 13-17 The excessive or unbalanced MMP activity is associated with the pathogenesis of many diseases. 18,19 among them cardiovascular diseases, such as atherosclerosis. 20 The detection of apoptosis in tissues is a marker related to mitochondrial injury, reactive oxygen species production, and oxidative stress. In apoptosis, DNA breakage results in several fragments with free 3’-OH ends. The identification of cells undergoing the process of apoptosis consists in detecting enzymatically the free 3’-OH ends with the addition of nucleotides modified by the TdT enzyme (terminal deoxynucleotidyl transferase). Thus, we aimed to verify the effects of moderate aerobic training on the ascending aorta of, low-density lipoprotein receptor LDL knockout and ovariectomized female mice. Methods Animals and group formation The experiments were performed in 15 female mice C57BL/6 and 15 of low-density lipoprotein receptor knockout female mice (LDL-KO) weighing 20-25g, from the Animal House of the São Judas Tadeu University, São Paulo, Brazil. The mice received the standard laboratory chow and water ad libitum . The animals were placed in cages in a room with controlled temperature (22°C) and a 12-h light-dark cycle. All surgical procedures and protocols were approved by the Experimental Animal Use Committee of Universidade São Judas Tadeu (058/2007). After a simple randomization, the mice were divided into six groups (n = 5): sedentary control (S-C), ovariectomized sedentary control (OS-C), ovariectomized trained control (OT‑C), sedentary LDL KO (S‑LDL KO), ovariectomized sedentary LDL KO (OS‑LDL KO) and ovariectomized trained LDL KO (OT‑LDL KO). The animals were separated physically and randomly between the groups / boxes. The sample size definition was performed according to previous data from other authors, 21-23 which were based on the instructions of CONCEA (Conselho Nacional de Controle de Experimentação Animal) Normative Instruction N°. 27 and determined by the formula n = (2 α /2 δ ) 2/E 24 was used, where n stands for sample size; (2 α ) 2 stands for a critical value that corresponds to the desired degree of confidence, δ stands for the population standard deviation and E stands for the margin of error (difference between the sample mean and the mean of the true population). Ovariectomy At nine months of age, the animals were anesthetized (ketamine 120 mg/kg + xylazine 20 mg/kg), and a small abdominal incision was performed where the ovaries and oviducts were found, sectioned and removed. Then, the skin and muscle wall were sutured. 25,26 The efficacy of the ovariectomy was determined by observation of vaginal secretions during four consecutive days. Training protocol Seven days after the ovariectomy, all animals were adapted on the treadmill for tenminutes during three days before initiating the training. The maximal exercise test was performed in all groups at the beginning and at the ending of the training program, providing the basis for the prescription of physical training, and with the purpose to evaluate the physical capacity of the trained animals. The trained groups were submitted to a moderate physical training protocol on a treadmill, with progressive speed and load (1 hour a day/5 days a week at 50-60% of maximum effort speed) during 4 weeks, as previously described. 27 Histological procedures At the end of the experiment, the animals were weighed and subsequently euthanized by decapitation. Thoracotomy was performed by cutting the ascending aorta at the heart base. The aorta samples were washed (PBS - phosphate buffered saline 0.1M, pH 7.4) and fixed in 10% formaldehyde for 24 hours. Then, they were dehydrated, cleared and embedded in paraffin. The aorta was sectioned transversely (5 μm thick), and the samples were stained in H&E for histomorphometric analysis. 7 In order to analyze the effects of aerobic training on the whole aorta, the tunica intima was not separated from the tunica media. Picrosirius staining was used for classification of collagen fibers I and III. The images were captured at 4 points, at 0º, 90º, 180º and 270º, with x10 magnification to measure the aorta thickness, and x40 for other evaluations, and transferred to an image analysis program (Axion Visio Software, Zeiss ® ). To analyze the volume density of types I and III collagen fibers, the images were captured by light microscope with polarized light, analyzed using a test system of 252 points, and the values were expressed as percentages. Immunohistochemical analysis: 8-OHdG, MMP-2 and MMP-9 Five 4-µm cross-sections, mounted on previously silanized slides, were used to show the expression of 8-OHdG, MMP‑2 and MMP-9. The slides were then deparaffinized, cleared, hydrated and washed in running water. Then, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide, protein blocking was performed with 0.3% skimmilk diluted in PBS and the slides were incubated overnight with anti-8-OHdG (SC66036 Santa Cruz® Biotechnology, CA, USA) primary antibody, titrated 1:100, and MMP2/72KDa and MMP9/KDa (SC-10436 Santa Cruz® Biotechnology, CA, USA; SC-6840 Santa Cruz ® Biotechnology, CA, USA), titrated 1:150 in PBS‑BSA 0.1%. All slides were then placed in a humid chamber at 4°C overnight. The material was washed with PBS buffer and incubated with biotinylated secondary antibody. For revelation, the 3-3´diaminobenzidine chromogenic substrate was used at a ratio of 0.06 g per 100 mL of PBS, and 1 mL of 20-volume H 2 O 2 for five minutes at 37°C and counter-stained withMayer’s hematoxylin for 3minutes. Finally, the slides weremounted with cover slips and entellan® for analysis under light microscopy. Investigation of the apoptotic cell death by TUNEL immunocytochemistry TUNEL staining was performed using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore ® , Germany) according to the manufacturer’s instructions. 181