ABC | Volume 110, Nº6, June 2018

Original Article Oishi et al Endothelial dysfunction and blood pressure in rats Arq Bras Cardiol. 2018; 110(6):558-567 Methods Animals and dietary treatments The experimental protocol was in accordance with the guidelines of the Brazilian College for Animal Experimentation (COBEA) and was approved by the Ethical Committee of the Federal University of São Carlos (026/2013). Seventy male (8-week-old) Wistar rats (250–300 g) were assigned to two experimental groups with food and water ad libitum for 24 weeks: Control (CT, n=35) was fed a standard diet or HFD (n = 35) fed with high-fat diet, that consisted of a standard rat diet plus peanuts, milk chocolate, and biscuits at a proportion of 3:2:2:1 as previously described. 11 Standard diet and high-fat diet contained, respectively, 20/20% of protein, 4.5/20% of fat and 55/40% of carbohydrate. 11 The caloric values of the diets were approximately 4.07 kcal/g for the standard diet and 5.12 Kcal/g for HFD. At time 0 and after every 6 weeks, 7 rats from CT and 7 from HFD group were randomly euthanized, and blood was collected for experimental analysis. Blood pressure measurements in Conscious Rats Indirect systolic blood pressure (SBP) was measured two days before euthanasia every 6 weeks using tail-cuff plethysmography (Power Lab 8/35, AD Instruments, Pty Ltda, CO), as described by Rodrigues et al. 12 Mean SBP was calculated from an average of four successive measurements in each animal. Vascular reactivity studies The animals were anaesthetized with isoflurane and euthanized by decapitation. Thoracic aortas were isolated and cleaned of adherent connective tissues, and placed in a Krebs solution, as described previously. 13 Aortas were carefully mounted as ring preparations ( ≅  4 mm in length) and placed in bath chambers containing Krebs solution at 37°C continuously bubbled with 95% O 2 and 5% CO 2 , pH 7.4 in an isometric myograph (model 610 DMT-USA) and recorded by a PowerLab8/SP data acquisition system (AD Instruments Pty Ltd., Colorado) . The aortic rings were submitted to a tension of 1.5 g, which was readjusted every 15 min during a 60 min equilibration period before adding the given drug. Experiments were done in aortic rings with intact endothelium and also in denude endothelium aortic rings. Endothelial integrity was assessed by the degree of relaxation induced by 1 μmol/l acetylcholine (ACH) in the presence of contractile tone induced by phenylephrine (0.1 μm/l). The ring was considered with intact endothelium if the relaxation with acetylcholine was higher than 80%. In endothelium-denuded aortas, the relaxation to ACH was less than 5%. After the endothelial integrity test, aortic rings were pre-contracted with phenylephrine (100 nM). When the plateau was reached, concentration–effect curves to acetylcholine (0.1nM to 0.1mM) in intact endothelium aortic rings or concentration–effect curves for NO donor sodium nitroprusside (SNP) in denude endothelium aortic rings were constructed. Concentration curves were fitted with a sigmoidal dose-response equation which disclosed the maximal effect (MaxE) and the negative logarithm of the agonist that produces half-maximal response (pD2) using GraphPad Prism (GraphPad Software In, USA). Body fat composition Visceral adipose tissue (VAT) was dissected (mesenteric, epididymal and retroperitoneal white adipose tissues) and weighed to evaluate central adiposity. Aorta lipid peroxidation (Ferrous oxidation-Xylenol Orange – FOX) Thoracic aortas were isolated and cleaned of adherent connective tissues. The methodology was described by Jiang et al. 14 The ferrous oxidation−xylenol orange (FOX), measures lipid peroxides (cumene hydroperoxide – CHP), one of themain products of lipid peroxidation. For the standard assay, the following reagents were added sequentially: 0.25mMFeSO 4 , 25 mM H2SO 4 , 0.1 mM xylenol orange, and water to a total of 0.9 ml. A sample of tissue extract (20‑100 µL) was added, and the final volume was adjusted to 1 ml with water. Blanks were prepared by replacing tissue extract with water. Samples were incubated at room temperature until the reaction was complete (40 min), and absorbance at 560 nm was measured. Serum nitrite and nitrate (NOx) Serum nitric oxide levels were obtained by measuring the serum concentrations of its stable end-products nitrite (NO 2 - ) and nitrate (NO 3 - ), collectively known as NOx, as described previously. 15 The NO/ozone chemiluminescence method was performed using the NO Analyzer 280i (Sievers, Boulder, CO, USA). Determination of adiponectin and inflammatory cytokines Quantification of adiponectin and inflammatory cytokines tumor necrosis factor- α (TNF- α ), interleukin 6 (IL-6) and C-reactive protein (CRP) in serum was performed using the enzyme-linked immunosorbent assay (ELISA) kit. IL-6 and TNF- α were evaluated using commercial OptEIA kits (BD Biosciences, Pharmingen, USA). Adiponectin and CRP were analyzed using Duo Set kits (R&D Systems, USA). All kits were used according to the manufacturers' instructions, and the results were expressed in pg/mL for all cytokines evaluated. Morphological and histological evaluation Aorta segments were quickly cleaned from the surrounding tissues and blood, cut into rings fixed in formalin 37% and embedded in paraffin blocks. Later, 4-μm thick sections were cut with a microtome (Leitz 1512, IMEB, USA), placed onto glass microscope slides and stained with hematoxylin and eosin using the standard methods. Images of transverse sections of the arterial segments were captured using a camera connected to an optical microscope (Leica DM 2000). External diameter (ED) was obtained by measuring the surfaces of the adventitia and internal diameter (ID) from the endothelium surface. The media thickness was obtained by dividing the difference ED - ID by 2 ( δ = ED - ID/2). The media/lumen ratio was calculated from the area data. The images were analyzed using the ImageJ analysis software, as described previously. 16 559