Animals and initial procedures
The present study was divided into two distinct experiments. The first was composed of 5 female spontaneously hypertensive rats (SHRs) of ~18 weeks old. The second experiment was composed of 12 male SHRs of ~22 weeks old divided into three experimental groups: control group (CG; n = 4), exercised at low intensity (LIG; n = 4), and exercised at high intensity (HIG; n = 4). All animals were obtained from the bioterium of the Federal University of São Paulo, Brazil. Water and food were provided ad libitum and the animals were kept in a 12:12 h dark–light cycle in a room at 23 ± 2°C. The study was approved by the local ethics committee on animal use from the Catholic University of Brasília, Brazil, and procedures were in accordance with the Brazilian College of Animal Experimentation [13]. Before beginning the exercise training, all animals were familiarized with the experimental environment and treadmill platform (Li 870, Letica Scientific Instruments, Barcelona, Spain) and adapted for three weeks as previously described [12].
Experimental design 1
After the adaptation period, five rats (~18 weeks old, 227.4 ± 29.3 g, and 172.4 ± 8.1 mmHg of systolic blood pressure) were submitted to 4 weeks of treadmill running, 5 days per week, 30 min per day at a velocity corresponding to the MLSS (20 m.min−1).
Maximal lactate steady state
The MLSS was previously identified by Almeida, et al. [12] during rectangular tests at three different velocities (15 m.min−1, 20 m.min−1 and 25 m.min−1). In order to verify the effects of the proposed exercise intensity on MLSS at the end of the training period (4 weeks) a novel MLSS was tested in all animals using three other velocities (25 m.min−1, 30 m.min−1 and 35 m.min−1) (Figure 1a). The velocities were set randomly and the tests were carried out at 48 h intervals. The tests lasted for 25 min of continuous exercise (0% graded) or until animal exhaustion. During the tests, capillary blood was collected every 5 min from the distal portion of the animals’ tail for blood lactate concentration analysis. MLSS was considered as the highest intensity of effort where the blood lactate did not vary more than 1 mmol.L−1 from the 10th to the 25th min, as described previously [12].
Blood lactate analysis
10 μL of blood was collected with capillaries from a small incision in the distal tail portion of each animal, rapidly deposited in microtubes (0.6 mL) containing 20 μL of 1% sodium fluoride, and stored at −20°C for further biochemical analysis. The blood lactate concentration was analysed by the electro-enzymatic method from YSI Sports 2700 (Yellow Springs, OH, USA) [14].
Experimental design 2
At the end of the first experiment, a novel group of 12 SHRs (~22 weeks old, 306.8 ± 11.1 g, and 169.8 ± 13.8 mm Hg of systolic blood pressure) was used to verify the effect of two distinct exercise intensities (below and above MLSS) on aerobic fitness and systolic blood pressure status. SHRs were divided into three groups: low intensity group (LIG; n = 4), which trained at a running velocity corresponding to 20% below MLSS (16 m.min−1), high intensity group (HIG; n = 4), which trained at a velocity corresponding to 15% above the MLSS (23 m.min−1), and control group (CG; n = 4), where the animals were not submitted to exercise. The exercise groups underwent 30 min of treadmill exercise (without slope), 5 days per week for 4 weeks.
Incremental test (IT)
Besides the use of MLSS as parameter for exercise prescription, an incremental test (IT) (0% graded test, increments of 3 m.min−1 every 3 min, starting at 5 m.min−1 until animal exhaustion) was also used to determine the maximum velocity (Vmax) in all groups (CG, LIG and HIG). IT was performed previously to the training period (t0) and immediately after four weeks of exercise training (t4) (Figure 1b). In this way, Vmax was used to establish aerobic fitness.
Blood pressure measurements
Systolic blood pressure (SBP) was measured in all animals before the training period started (t0) and at the end of four weeks of exercise training (t4). To carry out the SBP measurements, all animals were lightly soothed with a common combination of 10% ketamine (10 mg.kg−1) and 2% xylazine (10 mg.kg−1), and then SBP was measured by the tail-cuff plethysmography method (LE 5001 Pressure Meter, Letica, Barcelona, Spain). Inconsistencies in diastolic blood pressure were observed throughout the experiment, but these data were not recorded in this study.
Statistical procedures
After verifying data normality (Kolmogorov-Smirnov test), data were presented as mean and standard deviation values in both experiments. Here, the parametric test was able to identify the data normality in a sample size of 4. To observe the effect of exercise training at MLSS intensity on aerobic fitness and the effect of different exercise intensities on SBP, inferential analyses were conducted by One-way ANOVA with Bonferroni post-hoc test. The level of significance was set at P < 0.05.