2.2.1. Ischemia/Reperfusion on Langendorff Isolated and Perfused Heart

The animals were treated with intraperitoneal injection (i.p.) of naringenin (100 mg/Kg), vehicle (dimethyl sulfoxide, DMSO, 1 mL/Kg) or grapefruit IntegroPectin (45 mg/kg; 135 mg/kg; 450 mg/kg), 2 h before heart removal.

The animals were subjected to an i.p. injection of heparin (2500 I.U.) (Sigma-Aldrich, St. Louis, MO, USA), and to a subsequent one of pentothal Sodium (100 mg/kg) (MSD Italia) to induce a deep anesthesia. Each animal was sacrificed, and the explanted heart was immersed in a Krebs solution (NaHCO₃ 25.0 mM, Glucose 11.7 mM, NaCl 117.9 mM, KCl 4.8 mM, MgSO₄ 2.5 mM, CaCl₂ 2.2 mM, KH₂PO₄ 1.2 mM; clioxicarb: 95% O₂ and 5% CO₂; pH 7.4; 4 °C). Then, the heart was set up on a Langendorff apparatus and perfused with Krebs saline solution at 37 °C and constant pressure of 70–80 mmHg, through a peristaltic pump (Peri-Star, 2Biological Instruments, Besozzo, Italy). A latex balloon filled with bidistilled water at a pressure of 5–10 mmHg was placed inside the left ventricle through the mitral valve to monitor the functional parameters of the heart. The ischemia/reperfusion protocol consisted of 30 min stabilization time, 30 min of global ischemia and 120 min of reperfusion. At the end of reperfusion time, the heart was removed, dried, weighed, then the left ventricle was isolated. This was sliced into cross sections and immersed in a 1% w/v solution of 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich) dissolved in PBS (pH 7.4) (Sigma-Aldrich) for 20 min at 37 °C, in the dark. Finally, the slices were fixed in a 10% v/v aqueous formaldehyde solution. As a result, it gave the vital areas a red color.

The latex balloon was connected to a pressure transducer (Bentley Trantec, mod. 800, Ugo Basile, Comerio, Italy) and a data acquisition system (Biopac Systems Inc., Goleta, CA, USA). Recorded parameters were left ventricular pressure (LVDP), heart rate (HR), and contraction/relaxation time (dp/dt). The rate-pressure product (RPP) was calculated as the product of the first two parameters. The effects of the different doses of grapefruit IntegroPectin on cardiac functional parameters (RPP, dp/dt) were expressed in % of the basal values measured at the end of the stabilization period.

The cardiac areas involved in ischemia/reperfusion damage, necrotic or apoptotic, remained white or pale pink. Ischemic area was calculated as % of left ventricle total area (Ai/Avs %) using the software GIMP (release 2.10.32, Spencer Kimball and Peter Mattis, Berkeley, CA, USA).

All data were obtained from 6 different animals and graphed using the GraphPad Prism 8.0 program and expressed as mean ± standard error of mean (SEM). Statistical analyzes were performed with the Student’s t-test, with p < 0.05 considered as an indicator of significant difference.