Collection of Samples

Each animal's body weight was measured 48 hours before the last dose of DOX. We used ketamine at a dose of 100 mg/kg and xylazine at 10 mg/kg to anesthetize the animals. First, animals were immobilized, a thoracoabdominal incision was made, and blood was drawn immediately from the heart's left ventricle through a heart puncture. Then the serum was collected by centrifugation at 5,000 rpm for 10 minutes. After that, blood samples were placed in tubes with the clot activator gel. The serum obtained was used to evaluate cTnI, IL-6, and TNF-α using the ELISA technique.

The hearts were taken out, cleaned, and weighed before being sliced into apical and basal sections. Clots were removed by rinsing the basal side of the heart with ice-cold saline and then stored in a deep freeze (-80 C). Each part was weighed after melting and homogenized using an ultrasonic liquid processor with high intensity in 1:10 (w/v) 0.1-M phosphate-buffered saline (pH-7.4) containing 1% triton x-100 and protease-inhibitors cocktails [18]. The homogenate was centrifuged at 5,000 rpm at 4℃ for 10 minutes. The supernatants were utilized to evaluate malondialdehyde (MDA) level, caspase-3, and Bcl-2 using available ELISA kits and total antioxidant capacity (TAC) using colorimetric assay kits as directed by the manufacturer guidelines (Elabscience, U.S.A.).

For histopathological exams, the apical portion was saved and fixed in 10% neutral formalin, then embedded in paraffin block and sliced into parts with a thickness of 5 µM using a microtome. Light microscopy was used to investigate tissue sections stained with hematoxylin and eosin (H&E) [19].