To obtain sufficient volume and quality of blood samples, the refined retro-orbital bleeding (ROB) method using the lateral approach was performed as described by Ashish Sharma et al. [33 (link)]. For this purpose, sterile glass Pasteur transfer pipettes (with flat edges) were used. Animals were mildly sedated with diethyl ether (inhalation route), and the eyelid was pulled back to proptose the eye. The flat edge of the pipette was placed at the lateral canthus and was oriented toward the back of the head at an angle of 45° to the sagittal and coronal planes. Then it was twisted gently with pressure against the orbital bone just in front of the zygomatic arch until blood flowed from the capillaries draining the orbital sinus. This way, capillary motion draws blood into the tube. Collected Blood samples were centrifuged at 3000 rpm for 15 min, and serum was isolated and stored at −20 °C until biochemical analysis.
At the end of the experiment, all rats were sacrificed using the decapitation method without chemical anesthetics (collected organs must be fresh and free of chemicals), and the kidney, pancreas, and liver were quickly removed and washed with ice-cold saline solution. Therefore, each organ was finely crushed and homogenized in cold phosphate-buffered (0.1 M; pH 7.4) and centrifuged at 8000× g for 20 min at 4 °C. The supernatant was collected and stored at −20 °C to analyze the oxidative stress parameters [34 (link)].
At the end of the experiment, all rats were sacrificed using the decapitation method without chemical anesthetics (collected organs must be fresh and free of chemicals), and the kidney, pancreas, and liver were quickly removed and washed with ice-cold saline solution. Therefore, each organ was finely crushed and homogenized in cold phosphate-buffered (0.1 M; pH 7.4) and centrifuged at 8000× g for 20 min at 4 °C. The supernatant was collected and stored at −20 °C to analyze the oxidative stress parameters [34 (link)].
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