60 day release pellets

Female Wistar rats that were approximately 8 weeks of age and weighed 120–150 g were anaesthetized with Zoletil (20 mg/kg body weight, Virbac AH, Inc., France) and xylazine (9 mg/kg), and subsequently underwent ovariectomies (OVX) via bilateral dorsal incisions. Two weeks later, slow‐release 17β‐E2 pellets were implanted subcutaneously in the dorsal neck area (0.25 mg 60‐day release pellets, Innovative Research of America, Sarasota, Florida) 24 h after coronary ligation. These pellets ensured physiologic E2 plasma concentrations and prevented fluctuations in E2 level due to the oestrus cycle. Rats that did not undergo OVX and the other rats that did undergo OVX received placebo pellets. Oestrogen status was confirmed according to the weight of the uterus and plasma E2 level. To confirm the importance of the ROS signalling in E2‐induced macrophage skewing, we employed 3‐morpholinosydnonimine (2.5 mg/kg per day once daily, SIN‐1, a peroxynitrite generator; Sigma‐Aldrich, St. Louis, MO, USA). Thus, a total of five experimental groups were studied: sham group and infarction groups (non‐OVX intact, OVX, OVX/E2, and OVX/E2/SIN‐1, Figure 1). The heart was excised at Day 3 or 28 after MI as early and late stages of MI.