IJCS | Volume 31, Nº2, March / April 2018

116 Martins et al. Flaxseed ReducesTNF-α Int J Cardiovasc Sci. 2018;31(2)114-122 Original Article Histological analysis The thoracic aorta was removed at the level of the aortoiliac bifurcation following a midline thoracotomy and laparotomy, and cross sections with a thickness of 2 to 3 mm were cut from different aortic segments. Each segment was labeled, placed in a microtome (Leica ® RM2145, Leica Microsystems Nussloch GmbH, Germany) for overnight processing, and dehydratedwith a graded ethanol series (70%, 80%, and 90%). The tissue samples were subsequently diaphanized for 12 hours in xylol containing serial concentrations of paraffin. Paraffin blocks were obtained by embedding the samples in hot paraffin using a tissue embedding console system (Leica ® EG1160, Leica Microsystems Nussloch GmbH, Germany), according to the standard procedure. The paraffin blocks were cut into 5 μm sections using themicrotome (Leica ® RM2145). Each sectionwas stained with hematoxylin and eosin (HE) and orcein (elastic tissue staining) according to conventional techniques and mounted on permanent slides. Blocks with four aortic segments were prepared by selecting one proximal segment near the aortic arch (point 1), one segment located in the thoracic aorta (point 2), one proximal segment in the abdominal aorta (point 3), and one distal segment in the abdominal aorta (point 4). These segments were selected according to the histological findings in HE staining. Immunohistochemical analysis The tissue microarray (TMA) technique was used for immunohistochemical analysis. The samples were incubatedwith intercellular adhesionmolecule 1 (ICAM-1) monoclonal antibodies at a dilution of 1:50, andmixedwith tumor necrosis factor alpha (TNF- α ) monoclonal antibody for 1 hour. The antibodies were detected by incubating the slides with the substrate 3,3'-diaminobenzidine- tetrahydrochloride‑dihydrate (DAB; DakoCytomation A/S, Glostrup, Denmark). Counterstaining was performedwithMayer’s hematoxylin, followed by sample dehydrationwith different concentrations of 100%ethanol anddeparaffinizationwith xylol. The slidesweremounted with Canada balsam. The protocol described above was standardized at our department. Positive and negative controls were used for each reaction. The histological sections were digitizedwith a scanner (Axio Scan Z1, Zeiss, Jena, Germany) and the images were analyzed using Image-Pro Plus ® software, version 4.5. The color morphometry method was applied, staining the areas with positive antibody imaging in pink and those with negative imaging in brown. The data were transferred to an Excel spreadsheet and subjected to statistical analysis. Statistical analysis The results are expressed as mean, median, minimum and maximum, and standard deviation values. The assumption of normality of the variables was assessed using the Kolmogorov-Smirnov test. The variables not meeting the normality assumptionwere analyzed using the Kruskal-Wallisnonparametric test. Pvaluesbelow0.05were considered statistically significant. The data were analyzed using the IBM SPSS Statistics software, version 20.0. Results One death occurred in group G3 at experimental week 12, resulting in a total of 29 rabbits in the final study sample. Lipid profile Total cholesterol Serum TC levels increased significantly in groups G2 and G3 compared with G1. This result demonstrates that the diet was effective in increasing cholesterol levels in the rabbits (Table 1). LDL-C and HDL-Cl There were no significant differences in the levels of LDL-C and HDL-C among the groups (Tables 2 and 3). However, serumLDL-C levels in the biochemical analysis were higher in group G2 at the end of the experiment. The increase in LDL-C in group G3 was approximately six times lower than that in G2. HDL-C levels increased in groups G1 (12.93 mg/dL), G2 (82.26 mg/dL), and G3 (12.03 mg/dL), with the highest increase observed in G2. Triglycerides The diet did not increase the TG levels in the study groups. In addition, TG levels in G3 decreased by approximately 70% (Table 4). At the end of the experiment, the results indicated significant differences in TG levels between G1 and G3 (p = 0.034), and between G2 and G3 (p = 0.015).