ABC | Volume 114, Nº1, January 2019

Original Article Alegre et al. Açaí and myocardial ischemia-reperfusion in rats Arq Bras Cardiol. 2020; 114(1):78-86 cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, which are responsible for the purple color of the fruit. 13 Ferulic acid, p-hydroxybenzoic, gallic, protocatechuic, ellagic, vanillic, p-coumaric acids, and ellagic acid glycoside are the most abundant phenolic compounds. 13,14 In experimental models, açaí supplementation reduced pulmonary 9 and cerebral oxidative stress, 15 reduced ROS formation in polymorphonuclear cells, 11,16 decreased DNA damage, and presented anti-carcinogenic activity in bladder cancer. 10 Oral açaí administration was able to attenuate hypertrophy and left ventricle dysfunction in rats subjected to myocardial infarction, 17 but no studies on the effect of açaí in the global ischemia model or its effect on reperfusion injury were found. The aim of our study was to evaluate the infarct area, left ventricle function, oxidative stress and the activity of enzymes involved in myocardial energy metabolism in the global ischemia-reperfusion model in rats after açaí supplementation. Method Study design The experimental protocol of this study was approved by the Ethical Committee on the Use of Animals of the Botucatu Medical School (CEUA 1111/2014), and it is in accordance with the norms established by the National Council of Control of Animal Experimentation. Twenty two-months-oldmaleWistar rats weighing 250-300 g were assigned to two groups: control (C; n = 10) and açaí (A; n = 10). Sample size was determined by convenience based on previous studies that used the same experimental model. Animals were kept in an environment with controlled temperature (23°C) and a 12-hour light-dark cycle in individual boxes to control feed intake. Group C received a standard chow, and the group A a standard one supplemented with 5% açaí 18 for six weeks. After the supplementation period, all animals were anesthetized with sodium thiopental (80 mg/kg, IP ) to induce the global ischemia-reperfusion protocol, after which the heart was dissected. A sectional cut of the left ventricle was made to determine the infarct area, and the rest was stored in a freezer at -80°C for further analysis. One rat from the control group was lost due to technical problems during the ischemia‑reperfusion protocol and the study was concluded with 9 rats in the control group and 10 rats in the açaí one. Preparation of the chow supplemented with açaí Commercialized açaí pulp (Icefruit®) was defrosted and incorporated into crushed Nuvilab chow (Nuvital®). After homogenization, the chow was pelleted again, dried at 32°C and stored in a freezer at -20°C until the moment of use. The dose used in the studywas 5%, as proposed by Fragoso et al. 18 Induction of global ischemia, reperfusion and evaluation of cardiac function The rats were anesthetizedwith thiopental sodium (80mg/kg, IP), heparinized (2,000 IU, IP) and subjected to positive pressure ventilation with 100% oxygen. Then, median sternotomy was performed, and the ascending aorta was cannulated to start retrograde perfusion with a modified Krebs-Henseleit solution (NaCl 115mmol/L, KCl 5.4mmol/L, CaCl 2 1.25mmol/L; MgSO 4 1.2 mmol/L, NaH 2 PO 4 1.15 mmol/L, NaHCO 3 25 mmol/L, 11 mmol/L glucose, and 8 mmol/L mannitol). The hearts were transferred to a Langendorff apparatus (Model 830 Hugo Sachs Eletronik, Germany) with perfusion pressure at 75 mmHg. The nutrient solution was constantly oxygenated with a gas mixture of 95% O 2 and 5% CO 2 , and the temperature was maintained at 37°C. A pacemaker was used to maintain controlled heart rate (250 bpm). Left atrium was removed, and a latex balloon was inserted into the left ventricular cavity. The balloon was coupled to a pressure transducer and to a syringe, which allowed variation in the volume of the balloon. After 10 minutes of stabilization, the hearts were subjected to a 30-minute period of global ischemia followed by 30 minutes of reperfusion. 3 Global ischemia was induced by completely stopping the flow of Krebs-Henseleit solution to the heart. After ischemia and reperfusion periods, an evaluation of left ventricular function was performed. The volume inside the balloon was progressively increased to obtain left ventricular diastolic pressure variation of 0 to 25 mmHg. In addition, for each increase in volume to the balloon, the diastolic and systolic pressure, the maximum left ventricular pressure development rate (+dP/dt) and the maximum left ventricular pressure decrease rate (-dP/dt) were recorded. Diastolic pressure-volume curves were constructed. Analysis of infarcted myocardial area A cross-sectional cut of the left ventricle (LV) was made – 5 mm from the apex, with a thickness of 2 mm – and incubated in phosphate buffer with 7.4 pH and 1% triphenyltetrazolium chloride (Sigma Aldrich) for 30 minutes at 37°C. After that, the sections were incubated in a 10% formaldehyde solution overnight. The ventricle sections were positioned between two glass slides and scanned to obtain the images. Infarct area was measured through the ImageJ program by planimetry and expressed as the percentage of infarcted over total areas. In live cells the dye is reduced by dehydrogenases and appears with a dark red coloration. Dead cells lacking the enzymes are not stained and remain pale in color. 19 Analysis of antioxidant enzymes and lipid hydroperoxide Samples of approximately 100 mg of LV tissue were homogenized in a sodium phosphate buffer (0.01 M) with a pH of 7.4 and centrifuged for 30 minutes at -4°C; the total proteins in the samples were quantified by Bradford method. The activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase in the cardiac tissue were determined by spectrophotometry according to previously described methods. 20,21 Lipid hydroperoxide concentration in the cardiac tissue was measured by the oxidation of ammoniacal ferrous sulfate and determined by spectrophotometry. 22 79