ABC | Volume 110, Nº3, March 2018

Original Article Lemos et al Exercise improves splenic arterioles In SHR Arq Bras Cardiol. 2018; 110(3):263-269 not well established. Thus, the aim of this study was to assess the effects of exercise training on sympathetic activity and arteriolar damages in spleens of spontaneously hypertensive rats (SHR). Methods Animal model and exercise training protocol Forty male SHR and Wistar Kyoto rats (WKY) aged 45‑50 weeks were randomly assigned into four experimental groups of 10 rats each: SHR T and WKY T (that were submitted to exercise training protocol by swimming) or SHR S and WKY S (that were kept sedentary for a similar period of time). The sample size (n) was determined based on studies that evaluated the effects of exercise training on hypertension. These studies served as the basis for the present study that investigates the cardiovascular effects of the accumulated exercise. 11,12 All animals were kept in grouped cages (n = 3) at room temperature around 23°C, humidity of 40-70% and photoperiod of 12-hour light/dark cycle. Efforts were made to avoid any unnecessary distress to the rats, in accordance to the Brazilian Council for Animal Experimentation. All animal protocols were approved by the local Experimental Animal Use Committee (#271/2013), and were performed according to the regulations set forth by the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The swimming exercise protocol was performed in a glass tank and ambient water temperature was kept at 30° ± 1°C. The trained animals received a 20-min adaptation period on the first day, with increases of 10 min each day until reaching 1 hour on the fifth day. 13 After this period, the rats trained 5 days/week with a gradual progression toward a 2-hour session during nine weeks. This protocol is defined as an aerobic endurance and low-intensity training, as the animals swam without additional work load, this method corresponds the intensity below the anaerobic threshold in rats. 14 Sedentary animals were placed in the swimming apparatus for 10 min twice a week to mimic the water stress associated with the experimental protocol. Surgical procedures and hemodynamic parameters recording Twenty-four hours after the last exercise training session, all animals were anesthetized with sodium pentobarbital (40 mg/kg ip) and cannulas of polyethylene (PE-10) were implanted into the femoral artery for cardiovascular recording and into the femoral vein for drug infusion. Then, the polyethylene catheters were exteriorized at the posterior neck region of the animal. Rats received food and water ad libitum and were studied 1 day after catheter placement. Prophylactic treatment with antibiotics and anti-inflammatory drugs were performed to prevent postsurgical infections and inflammation, respectively. 15 After 48 hours of recovery from the anesthesia and surgery, the arterial cannula was connected to an AP transducer and a signal amplifier (Model 8805A, Hewlett-Packard, USA) was converted by the analog‑digital signal plate (sampling frequency - 1000 Hz) by a computerized system data acquisition (Aqdados, Tec Lynx. Eletron. SA, Sao Paulo, Brazil) and stored on computer. The animals were maintained in a peaceful environment for a period of 15 minutes and adaptive later pulsatile AP was continuously recorded at baseline for 30 minutes. During the experimental procedure, systolic AP (SAP), diastolic AP (DAP), mean AP (MAP) and heart rate (HR) were derived from pulsatile AP. Cardiac autonomic tonus To evaluate the exercise training influence on the tonic autonomic control of the heart, we also performed the sympathetic and vagal autonomic blockade after propranolol (5 mg/kg, i.v.) and atropine (4mg/kg, i.v.) injections, respectively, to calculate the sympathetic and vagal effects, as well as the intrinsic HR (iHR) and tonic sympathovagal index. 14 The autonomic blockers were administered in a random sequence with a 15-min interval between them. After double blockade, the cardiovascular recordings lasted for 15 min. Briefly, the sympathetic effect was considered as the difference between the HR after sympathetic blockade and resting HR. Vagal effect was calculated as the difference between HR after vagal blockade and resting HR. The tonic sympathovagal index was obtained as the ratio between resting HR and iHR, considering that the iHR was the HR obtained after double autonomic blockade. 16 Analysis of splenic arteriolar morphometry All animals were anesthetized with sodium pentobarbital and euthanatized with a lethal dose of potassium chloride. Their spleens were excised postmortem and immersed in saline (0.9%) to remove excess blood. Shortly after, the organs were placed on foil, previously treated and weighed in a semi‑analytical Gehaka BG2000 ® . Subsequently, the material was cut and placed inside a sterilized glass with 10% formaldehyde. Thereupon, the material was dehydrated using ethanol at concentrations of 80%, 90% and 95%. Diaphanization was performed with xylol. The material was placed in containers containing liquid paraffin at 60°C. Then, the material was placed in blocks. Histological 2-µm cuts were performed using a microtome and then the material were mounted in glass slides and stained with Masson's Trichrome Blue. The area of the inner and outer layers of each arteriole was quantified by using common light microscope for capturing the images and the imageJ program to check the area of each layer. At the end of the procedures for quantification of the area of each layer, the thickness of each arteriole was obtained. Statistical analysis Shapiro-Wilks and Levene's tests were used to evaluate the normality and homogeneity of the sample. Results were expressed as mean ± SD (for normally distributed variables) or median with upper and lower quartiles (for non-normally distributed variables). For parametric data, we used two‑way ANOVA (etiology vs. intervention), with the Tukey as a post hoc test. The nonparametric data were analyzed by the Mann‑Whitney test. Pearson coefficient was used to test the correlation between sympathetic effect with area of outer wall thickness and total area thickness. Probability values of P < 0.05 were considered statistically significant. Analyses were performed using SigmaStat® v. 2.03 (SPSS, Chicago, IL, USA). 264