Sample collection and esophageal/gastric tissue preparation
After 33 weeks, animals were allowed to fast and then were killed by cervical dislocation. Blood samples were collected by puncturing the orbital venous plexus using heparinized capillary glass tubes. Blood was used for measurement of total free radicals. Hemolysates were used for measurement of the levels of the following parameters: malondialdehyde-MDA, Glutathione (GSH), and endogenous antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). In addition, plasma was used to determine total antioxidant capacity (TAC) level.
Regarding the gastric redox biomarkers and histopathology examination, the stomach was excised and divided into 2 parts symmetrically along the greater and lesser curves. Part 1 was used to evaluate the redox biomarkers and was washed and homogenized in ice-cold phosphate buffer (0.1 mol/l, pH 7.4) using a Potter-Elvehjem homogenizer to give a 10% w/v homogenate. Part 2 was used for histopathology examination and was fixed along with esophageal tissues in 10% formaldehyde.