IJCS | Volume 31, Nº2, March / April 2018

115 Martins et al. Flaxseed ReducesTNF-α Int J Cardiovasc Sci. 2018;31(2)114-122 Original Article antioxidants, and polyphenols, 4 which can significantly decrease the effect of atherosclerotic factors. One such product is flaxseed ( Linum usitatissimum L.). The nutritional composition of flaxseed comprises fats (41%), dietary fibers (28%), proteins (21%), moisture (7.7%), ashes (3.5%), and soluble sugars (1 – 2%), 5,6 and their lipid content includes 51 – 55% of alpha-linolenic acid. 7 Additionally, flaxseed is the richest source of lignan secoisolariciresinol-diglucoside (SDG), which has potent antioxidant and antiatherogenic properties. Based on these considerations, the study of the functional properties of flaxseed becomes essential for the scientific community and the general population. The aim of the present study was to assess the potential effects of flaxseed on atherogenesis in rabbits subjected to a high-cholesterol diet (containing 0.25% cholesterol) of lyophilized eggs. For this purpose, we analyzed the lipid levels and performed immunohistochemical analysis of inflammatorymarkers in the aortic segments of the rabbits. Methods The present study was approved by the Animal Research Ethics Committee of PUC-PR under the protocol number 720/2012 andwas conducted in the Laboratory of Experimental Surgery at Hospital Angelina Caron (HAC), the Experimental Pathology Laboratory at PUC‑PR, and the Central Animal Facility at PUC-PR. Animals Thirty male New Zealand white rabbits ( Oryctolagus cuniculus ) weighing approximately 3 kg and with a mean age of 4 months were selected for the experiment. Calculation of the sample size was based on the study by Prim et al. (2008). 8 The rabbits were kept in the animal facility at PUC-PR in a macroenvironment with light/dark cycles of 12/12 hours, fresh air changes, and temperature controlled between 19°C and 23°C. The animals were identified on an individual basis and maintained in individual cages cleaned daily. Experimental period and group distribution The study was conducted over 12 weeks (84 days). The animals were maintained in individual cages and randomized to three groups according to dietary treatment. The control group (G1, n = 10) received a standard diet for rabbits (Nuvital® Nutrientes S.A., Colombo, PR, Brazil). The high-cholesterol diet group (G2, n = 10) received a standard diet for rabbits (Nuvital®) plus 0.25% cholesterol from lyophilized eggs. The high-cholesterol plus flaxseed diet group (G3, n = 9) received a standard diet for rabbits (Nuvital®), 0.25% cholesterol from lyophilized eggs, and 8 g of ground flaxseed per kg of body weight. Preparation of supplementary feed The high-cholesterol diet offered to groups G2 and G3 throughout the experimental period contained 0.25% cholesterol, which was added to induce atherosclerotic lesions in the aorta. In order to achieve that, 5 kg of standard feed (Nuvital®), weighed and ground for each animal group, was added to 1,800 g of egg powder diluted in 2,000 mL of water. The flaxseed concentration was 8 g per kg of body weight. The seeds were ground in a food processor and stored under refrigeration before added to the feed. The feed given to groups G2 andG3was supplemented with lyophilized eggs. This mixture was processed using an industrial meat grinder (Poli ® , model PCP-22LR-N, feeder #10, Siemsen, Brusque, SC, Brazil). For group G3, the mixture was supplemented with ground flaxseed. After grinding the mixture, the feed was pelleted, and the pellets were heated in an electric oven for 10 min at a temperature of 180°C. The prepared feed was stored under refrigeration until use. Animal preparation and sample collection The animals were anesthetized, and blood was collected according to the method described by Précoma et al. 9 and Alessi et al. 10 Xylazine (Coopazine  ; Schering‑Plough‑Coopers, Cotia, SP, Brazil) at 5 mg/kg mixed with ketamine (Vetanarcol  , König, Mairinque, SP, Brazil) at 30 mg/kg was administered intramuscularly into the rabbits’ thigh area. Biochemical analyses performed on days 1 and 84 included total cholesterol (TC), LDL-C, high-density lipoprotein (HDL-C), and triglycerides (TG), which were assessed using an automated equipment (ADVIA 1200, Siemens Healthcare Diagnostics, Inc., Newark, DE, USA) and commercial kits.