ABC | Volume 113, Nº5, November 2019

Original Article Sousa et al. Antioxidant effect of rat environmental enrichment Arq Bras Cardiol. 2019; 113(5):905-912 are mitigated. Environmental enrichment of rodents reliably increases performance in learning and memory tasks, and improves overall health and well-being, compared with standardly housed conspecifics. 14,17,18 Such enrichment typically involves introducing stimuli such as toys, tubes, balls, igloos, running wheels, or any items that provoke exploration, social interaction, sensorial and motor activity. Non-motor environmental enrichment (e.g. without wheel running) also appears to exert an antioxidant effect in the hippocampus and cerebral cortex of normotensive rats. 15,16 Thus, environmental enrichment may reduce oxidative stress in the ventrolateral medulla and contribute to hypertension control. Yet despite noted effects on cognitive activity, no consistent data are available regarding exactly how enrichment influences oxidative stress in the ventrolateral medulla of hypertensive animals. The aim of this study is to evaluate the effect of environmental enrichment on oxidative stress in the ventrolateral medulla, heart, and kidneys of renovascular hypertensive rats. Methods Animal experiments All animal experiments in the study were approved by the Ethics Committee on Animal Use of the Universidade Federal de Ouro Preto by protocol number 2016/24. Subjects were forty adult Fischer male rats (6 weeks old; 140–170 g). Renovascular hypertension was induced as previously described. 19 Briefly, animals were anaesthetized with 50 mg/ kg of ketamine and 10 mg/kg of xylazine (i.p.). Next, a silver clip (2K1C; inner diameter, 0.2 mm) was placed around the left renal artery through a midline incision. Normotensive groups (Sham) were subjected to similar procedures but without clips. Four groups were formed: standardly housed hypertensive (2K1C-St) (n = 12), enriched hypertensive (2K1C-EE) (n = 8), standardly housed normotensive (Sham‑St) (n = 7), and enriched normotensive (Sham-EE) (n = 13). The sample size was defined by convenience. Environmental enrichment and standard condition protocol Immediately post-surgery, rats were housed in enriched (EE) or standard (St) cages (Figure 1). The former contained six different objects (plastic coloured balls, igloo, wooden chew sticks, curved tubes, straight tubes); cages were organised with four rats in each cage. Eight different objects combinations were used, with one weekly change to encourage exploration. The control environment consisted of standard cages (St) identical to those used for housing subjects during non‑experimental periods. All groups were kept in the same room, on different racks, to control for lighting (12-12 h light‑dark cycle), temperature (24 ± 1 o C), humidity, and ambient noise. Rat chow and tap water were provided ad libitum. The enrichment procedure lasted for four weeks. At the end of that time, cardiovascular measurements were performed. Cardiovascular measurements Within 2 days of experiment end, rats were anaesthetised with urethane (1.2 g/kg body weight, ip; Sigma-Aldrich, USA). Polyethylene catheters (PE-10 connected to PE-50, Clay Adams, Parsippany, NJ, USA) filled with heparinized saline (400 IU/mL) were inserted into the right femoral artery for measuring pulsatile arterial pressure. Blood pressure was recorded at 1000 Hz sampling rate and 20 mV range digitizing window. Heart rate and mean arterial pressure (MAP) were derived on-line from the pulsatile arterial pressure signal with LabChart for Windows software (ADInstruments Pty Ltd, Australia). Experimental protocols were carried out in anaesthetized rats. After cardiovascular measurements, rats were decapitated and the left ventricle was perfused with saline solution to remove blood from tissues. Brain, hearts, kidneys and the ventrolateral medulla was dissected, and all samples were frozen in liquid nitrogen before storage at -80 o C. Figure 1 – Representation of the environments that rats were exposed (A) standard cage (B) enriched cage. 906