ABC | Volume 112, Nº5, May 2019

Original Article Gomes et al Thermoregulation in hypertensive rats Arq Bras Cardiol. 2019; 112(5):534-542 n = 8), trained Wistar (T-WIS, n = 8), control SHR (C-SHR, n = 8) and trained SHR (T-SHR, n =8). Sample size was determined based on sample size calculation. 11 The animals were housed in group cages in a temperature‑controlled room under a 12-h light-dark cycle, and had free access to water and food. Systolic blood pressure (SBP), diastolic blood pressure (DPB) and mean blood pressure (MBP) were measured using tail plethysmography (LE5001; Panlab, Spain). Resting heart rate (RHR) was measured through the sensor placed on the tail, connected to a computer system (PowerLab 4/30; LabChart/ADInstruments, USA) before the first and 48 hours after the last session of physical training. All exercise protocols were approved by the Ethics Committee of Universidade Federal de Viçosa (Protocol # 76/2014) and conducted according to the Helsinki declaration. Physical training protocol Prior to the beginning of exercise training, rats were adapted to a motorized treadmill (Insight Instruments, Brazil), five minutes/day at 5 m/min for five days. In addition, all animals underwent an incremental exercise test (starting at 5 m/min, increasing by 3 m/min every 3 minutes until fatigue) at the beginning of the study, at week 4 and at week 8 of training to determine total exercise time (TET) and maximum running speed (MRS). The exercise programwas performed five days a week, 60 minutes/day, at 50-60% MRS for 12 weeks, in a temperature-controlled room (approximately 22 o C). Both intensity and duration of exercise were gradually increased as proposed by Lavorato et al. 12 Animals of the control group were handled in the same manner as the hypertensive group and underwent the same treadmill exercise program two days a week, 5 minutes/day at 5 m/minute. 12 Experimental protocol following the physical training Familiarization with the experimental protocol The animals were familiarized with the treadmill (Panlab, Harvard Apparatus, Spain) – five minutes per day, 5 degrees of inclination for three consecutive days, at 11 m/min, 13 m/min and 15 m/min). A thermocouple was taped to the tail of the rat and the electrical stimulation delivered at 0.4 – 0.6 mA. 7 This protected the animals from having their legs wrapped around the thermocouple wire and reduced their exposure to electrical stimulation during the running test. 13 Temperature sensor implantation Immediately prior to the surgery, the animals received a prophylactic dose of antibiotic (enrofloxacin 10 mg. kg-1, intramuscular) and analgesics (tramadol, 4 mg .kg -1 , subcutaneously). Anesthesia was induced with 1.5% isoflurane (BioChimico, Brazil) and 100% oxygen (White‑Martins, Brazil) at constant flow of 1L/min. Following preparation of the incision site, a temperature sensor (G2 E-Mitter, Mini‑Mitter, USA) was implanted in the abdominal cavity. 14 After this procedure, the animals were housed in individual boxes and received two additional doses of tramadol in regular intervals of 8 hours. Acute physical exercise protocol After 48 hours of recovery from the surgery, each animal underwent to two exercise sessions at constant speed (60% of MRS),5 o slopeandelectricalstimulation(0.4-0.6mA)untilfatigue. Treadmill speed was 16.0 ± 0.4 m/min; 23.0 ± 0.7 m/min; 16.2 ± 0.5 m/min; 19.6 ± 0.8 m/min for C-WIS, T-WIS, C-SHR and T-SHR, respectively. Fatigue was defined as the point when the animals were unable to keep pace with the treadmill. The animals received electrical stimulation up to ten seconds. 15 The experimental conditions were randomized and balanced. All exercise sessions were carried out from 7 to 12 o’clock, with 48-hour interval between the sessions. During each session, Tcore, skin temperature (T skin ) and VO 2 were recorded every minute. Measurements of the Tcorewere made by telemetry (ER-4000 energizer/receptor, Mini-Mitter Respironics, USA). T skin was measured using a thermometer (THR-140, Instrutherm Instruments, Brazil) connected to a thermocouple (S-09K, Instrutherm Instruments, Brazil) using an impermeable adhesive tape at approximately 20 mm from the lateral base of the tail. 16 VO 2 (ml.Kg -0.75 .min -1 ) was measured by an open-circuit indirect calorimetry system (Panlab, Harvard Apparatus, Spain). The temperature was maintained at 25 o C throughout the exercise session. Calculations Work (W) = body mass (Kg)·force of gravity (9.8 m/s 2 ) ·TET (min)·treadmill speed (m.min -1 )·cos θ (treadmill slope). 17 ME = (W/energy cost)·100. 7 The threshold for cutaneous heat loss was defined as the mean Tcore registered at the time when T skin significantly increased from the lowest measure registered during exercise. 8 Heat loss sensitivity was calculated from the regression slope of Tcore and T skin during the first four minutes after the threshold was achieved. 8 Heat accumulation (HA) = (ΔTcore)·body mass (g)·h, where ΔTcore corresponded to variation of Tcore (T final ‑T initial ), and h corresponds to specific heat of body tissues (0.826 cal.g -1 .ºC -1 ). 18 HA was normalized by 100 g of body mass. The HA/W ratio (cal.j -1 ) was considered an index of thermal efficiency. Statistical analysis Data normality was tested using the Shapiro-Wilk test. Normally distributed variables were expressed as mean ± SD. Tcore, T skin and VO 2 were compared using the two-way ANOVA followed by post-hoc analysis with t-test (LSD, Least Significant Difference) or the Scott Knott test, as appropriate. TET, E, ME, SBP, diastolic blood pressure (DBP), MBP and RHR were analyzed by two-way ANOVA followed by Tukey’s post-hoc test. Paired t-test was used to assess the effects of low-intensity exercise on body mass, SBP, DBP, MBP and RHR. The level of significance was set at 5%. All statistical analyses were performed using the Sisvar software, version 5.6 (Brazil). Results The effects of physical training on body mass, SBP, DBP, MBP, RHR and TET are described in Table 1. Body mass 535