ABC | Volume 112, Nº2, February 2019

Original Article Maifrino et al Exercise effects on ovariectomized mice Arq Bras Cardiol. 2019; 112(2):180-188 Figure 1 – Mouse aorta cross-sections showing the arrangement of elastic fibers. The control groups showed a similar pattern of arrangement and thickness of the elastic fibers. The LDL KO groups showed more spaced fibers and thicker vessel walls compared to the controls. Photomicrographs, H&E. Calibration Bar = 100 µm. 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). Quantification of apoptotic cells For the quantification of immunostained cells for apoptosis, 30 images of the intima-media layer were captured (10 images/ animal; n = 3 animals/group; with x10 magnification to measure the aorta thickness, and x40 for other evaluations, and transferred to an image analysis program (Axion Visio Software, Zeiss ® ) for each experimental group. For each image, the total number of immunostained cells was obtained as a relative frequency (%) in relation to the total number of cells. The light microscope coupled to a digital camera (Zeiss, Germany) was used to obtain the images, and the photomicrographs were scanned with the AxioVision software (Zeiss®, Germany). Statistical analysis The results were presented as mean and standard deviation. Analysis of variance (ANOVA) and Tukey’s post-hoc tests were properly applied in data analysis. The significance level for all tests was p < 0.05. The data were evaluated using the software Stata 7.0. All continuous variables were normally distributed (Shapiro-Wilk test). To evaluate the normality of the data, the Shapiro-Wilk calculation was used, which found that the data were allocated within the Gaussian curve. Considering a level of significance of 5%, the test assumed the normality hypothesis for the variable with normal distribution. Results Histopathological and histomorphometric analysis The histopathological analysis showed that the animals from the control groups (S-C, OS-C and OT-C) did not exhibit changes in the elastic fiber arrangement and thickness pattern. However, the dyslipidemic groups (S-LDL KO, OS-LDL KO and OT-LDL KO), showed greater spacing between elastic fibers (Figure 1). Thickness of the tunica media - intima (µm) We observed a significant increase in the thickness of the tunica media and intima in dyslipidemic animals, when compared to the control group animals. The ovariectomy and the exercise in the LDL KO groups were a determining factor for the increase of this variable. In both control and LDL KO groups the training exercise did not reverse this process (Figures 1 and 2). Volume density of types I and III collagen fibers in the intima-media and adventitia tunica Similar behavior of the type III collagen fiber was observed between the intima-media and adventitia tunica. We observed a significant decrease in the volume density of the type III collagen fiber in the LDL KO groups, when compared to the S-C (Figure 3). The type I collagen fibers of the tunica adventitia and media-intima showed increase in volume density, influenced by training in the Control groups. The dyslipidemia induces an increase in type I collagen fibers in the LDL KO groups when compared to the Control groups and did not undergo any change by ovariectomy, or by training (Figure 3). Immunohistochemical analysis Figure 4 shows the tissue staining caused by the oxidative stress marker 8-OHdG. Note that the immunoexpression of the marker occurred in all groups. The staining was moderate for LDL KO and Control groups, both the Sedentary and the Ovariectomized Sedentary groups. However, for the Ovariectomized and trained groups, the observed staining was intense. In all control groups (S-C, OS-C and OT-C) the immunoexpression of MMP-2 occurred both in the tunica intima (arrow) and in the tunica adventitia (arrowhead) of 182