2.3. Sampling

Once all of the experiments were completed and after 12 h of overnight fasting, each rat was anesthetized with diethyl ether. A blood sample was then withdrawn from the retro-orbital venous plexus under a complete aseptic condition through the use a one-use plastic syringe. Sera were prepared by centrifugation at 5000 rpm for 15 min and were stored at −80 °C until the biochemical investigations were performed.

The heart was exposed via a thoracic incision for histological and molecular studies. Intra-cardiac perfusion was conducted using normal saline followed by 10% neutral-buffered formalin for the partial fixation of the specimens. The carotids arteries and brains were exposed and were carefully dissected. For the molecular investigations, about 20–40 mg tissue samples from the cerebral cortex and carotid artery were snap-frozen in liquid nitrogen and were stored at −80 °C. For total RNA extraction and for the qPCR analysis of eNOS, caspase-3, Bcl-2, LC-3, BECN-1, CHOP, and TNF-α expression, frozen tissue samples were utilized.