The animals used in this study were male Wistar rats (aged three months). They were randomly allocated into two groups: control group (CG, n=10) and resistance trained group (TG, n=10). The rats in both groups were sacrificed at six months of age. Three rats were housed in each cage, provided with standard laboratory chow and water ad libitum throughout the study. Rats were weighed every week. The animal room was maintained at 21±1°C with artificial 12:12 light and dark cycle.

After a period of adaptation (one week) to the device, rats in the TG were trained to climb a 1.1-m vertical ladder (80° incline) with weights tied to their tails.12,13 The rats were trained once a day, five days per week, for 12 weeks. Each training session consisted of six climbs requiring 8–12 dynamic movements per climb.13

Body weight (BW) was measured at the beginning of each week of the experiment, and the new weight to be carried by the animals was adjusted according to their body weight during that week.13 After measurement of the maximum weight lifted (one repetition maximum-1RM), the training load was set at 60% 1RM. The animals did not require any external stimulus to conduct the training. Animals of the CG were trained once a day for five days a week during 12 weeks to undergo the same procedures as the trained rats and standardize the sample, but each training session consisted of just one climb. Blood pressure was measured at the end of the experiment through the tail-cuff plethysmography. The Research Ethics Committee of the Universidade São Judas Tadeu (USJT), São Paulo, SP, Brazil, approved the animals’ handling in compliance with the International Guiding Principles for Biomedical Research involving Animals (Protocol number: 015/2006).

At the end of the experiment (24h after the last RT session), each animal was anesthetized with Pentobarbital sodium (3mg/100g body weight, intra peritoneal) and killed by exsanguination. The rat was weighed, the heart removed and weighed, and the left ventricle (LV) dissected and weighed. The ascending aorta was excised after thoracotomy, and an arterial ring (5mm long) was isolated and cleaned from adherent adipose and connective tissues.

Ten aortas from each group were immersed in the fixative (freshly prepared) 4% (w/v) formaldehyde in 0.1M phosphate buffer, pH 7.2 for 48h.14 The arterial rings were sectioned according to the vertical section method,15 processed according to the routine histological procedures, and embedded in Paraplast plus (Sigma Chemical Co., St Louis, USA).

The volume of smooth muscle cell nuclei (V[smc], μm3) was obtained by measuring the major and minor diameter of the myocyte nuclei in four non-consecutive histological sections per animal. The following formula was used to calculate the nuclear volume: V=a2.b/1.91, where V is the volume; a is the smaller axial diameter; b is the greater axial diameter; 1.91 is constant.16 The final value was the average of the measurements (Fig. 1A and B).

Figure 1.

Histological sections (Verhoeff staining) of the ascending aorta of the CG (A) and the TG (B) rats used for measurement of the major and minor diameter of smooth muscle cell nucleus (arrows). Arrowheads – elastic fibers. (C and D) Verhoeff stained histological sections of the ascending aorta to verify the number of elastic lamellae (arrows) of the CG (C) and TG (D) rats.

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We obtained the number of elastic lamina (N[lamina]) by counting their number at four different points of the aortic wall in three non-consecutive histological sections per animal. The final value was the average of the counts (Fig. 1C and D).

The elastic lamina surface density (Sv[lamina]) was quantified according to the stereological method described by Marques et al.17 We put a 16 cycloid arc test system on the monitor screen, calibrated it (Zeiss micrometer 1mm/100), and arranged the minor axes of the cycloids in parallel with the defined vertical axis. The number of elastic lamina intersections with the cycloid arcs (I[lamina]) was counted, allowing estimation of elastic lamina surface density (Sv[lamina]) according to the formula: (Sv[lamina])=2×I[lamina]/LT (LT is the test line length based on the system calibration).

The collagen content was determined in four fields of four histological non-serial sections of five μm stained with the Picrosirius technique and examined under polarized light. When studied with this method, tissues containing collagen fibers show birefringent thick and thin fibers intensely.18 We entered histological sections into a KS-400 digital analyzing computer program (Zeiss, Germany) to determine the collagen content range in the two groups, and it quantified the area percentage (12,000μm2) of collagen fibers.19 The sections were analyzed using a microscope equipped with appropriate polarization lenses. In each section, we selected four randomized fields and quantified the area percentage of collagen fibers in each field. The aortic wall thickness was measured in four non-consecutive sections stained with hematoxylin and eosin in four fields located at 0°, 90°, 180°, and 270° (Fig. 2A).

Figure 2.

Absolute values for 1RM test. Results are mean±SEM. *p<0.05, compared with the previous test. To compare the means of the groups, we used the t test.

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The maximum and minimum diameters of collagen fibrils were determined in ten electron micrographs of each aorta at a final magnification of 130,000× obtained from regions where the fibrils were transversely sectioned. We used the image analyzer program to measure the diameters of each fibril. By commencing at a corner of each field and radiating outward in an arc, the diameters of the fibrils present in the field were measured.

We used InStat for data analysis and the mean±Standard Error of Mean (SEM, when not specified) for descriptive statistics. The independent Student's t test was used to compare the aortic parameters of the two groups of rats. Two-way ANOVA (group vs. time) was performed to evaluate changes in 1RM. The level of significance was set at p<0.05.

Fig. 2B shows the progressive increase in the weight lifted by the rats of the TG obtained during the repetition maximum test. The CG and TG had similar values for repetition maximum at the beginning of the experiment. After 6 weeks and 12 weeks, the load lifted by the animals in the CG group was similar to that lifted in the first test. At the end of 6 weeks, the TG lifted 1580 (SD=92g), and 2405 (SD=140g) at the end of 12 weeks, which represents 1.6-fold and 3.04-fold the body weight for 6 and 12 weeks, respectively.

After 12 weeks of resistance training, no significant difference was observed between groups for body weight or blood pressure (Table 1A). The ratios of heart and ventricular weights to BW were higher for the TG compared to the CG, indicating that the heart grew more than the BW in the TG. The LVW (left ventricular weight), LV, and wall thickness were significantly higher in the TG rats than in the CG rats. However, LV internal diameter was significantly smaller (10%) in the TG than in the CG rats. These results may indicate the development of concentric hypertrophy in hearts from the TG rats.

The aortic wall thickness was larger in the TG than in the CG rats (p<0.05) (Table 1B and Fig. 3). The volume of smooth muscle cell nucleus was larger in the TG than in the CG rats (p<0.05) (Table 1B). Resistance training also affected aortic stereological parameters. N[lamina] and SV[lamina] were greater in the TG than in the CG rats (p<0.05) (Table 1B).

Figure 3.

(A) Schematic representation of a cross-section of the aorta showing the locations where measurements of the wall thickness were taken. (B and C) Photomicrographs of aortic wall sections from the CG (B) and TG (C) rats stained with hematoxylin and eosin and taken with the same magnification. The wall thickness in (C) seems to be greater than in (B).

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The collagen content was higher in the TG compared to the CG (Fig. 4A and B and Table 1B). In the CG rats (Fig. 4C and D), the collagen fibers were composed mainly of small diameter fibrils. Collagen fibrils of small and large diameter formed the aortic wall in the TG rats (Fig. 4C and D), with predominance of large diameter fibrils. Fig. 4E and F is histograms showing the distribution of collagen fibril diameter in the CG and TG aortas, respectively.

Figure 4.

Picrosirius red-stained aorta cross-sections of the CG (A) and TG (B) rats viewed under polarized light to show the collagen fibers. The number of collagen fibers in A seems to be greater than in B. High-power electron micrographs of transverse sectioned collagen fibers from the aorta wall of the CG (C) and TG (D) groups of rats. The electron micrographs are viewed with the same magnification showing collagen fibrils of large (thick arrows) and short diameter (thin arrows). The number of large fibers seems to be greater in the TG than in the CG. (E and F) Histograms showing the distribution of collagen fibril diameter in CG and TG aortas, respectively.

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