3 morpholinosydnonimine

Mouse NSC-34 hybrid cell line, a widely used motor neuron-neuroblastoma fusion line was chosen, as it expressed many of the morphological and physiological properties of motor neurons [26 (link)]. Cellswere grown in Dulbecco’s modified Eagle’s (DMEM)/F-12 medium supplemented with 10% fetal calf serum and 1% of a mixture of penicillin/streptomycin at 37°C in an atmosphere of 5% CO2 in air. Human neuroblastoma cells, SH-SY5Y cells were obtained from the ATCC (Rockville, USA), and were grown on tissue culture plates (Greiner, Hessle, UK) in DMEM, supplemented with 10% fetal calf serum, 1% L-glutamine and 1% SPN antibiotics (Biological Industries, Bet Haemek, Israel). Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂. Cells were exposed to peroxynitrite ion generator 3-morpholinosydnonimine (SIN-1, Sigma–Aldrich) or to glutamate (Sigma—Aldrich).

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.