Isolation and culture of primary neurons

Cerebral cortical neurons were dissociated from neonatal Sprague-Dawley rats as previously described with slight modifications³⁷. Briefly, neonatal rats were anaesthetized with 2% isoflurane and the cortex was dissected and digested with 0.125% trypsin for 30 min at 37 °C. Neurons were plated on 0.0125% poly-L-lysine-coated culture dishes in Neurobasal medium (Gibco, USA) with B27 (Gibco, USA) in a humidified atmosphere of 5% CO₂ at 37 °C. After 10 days, neurons were placed into an anaerobic chamber (Coy Laboratories, USA) and the culture medium was replaced with a balanced salt solution (116 mM NaCl, 5.4 mM KCl, 1 mM NaH₂PO₄, 1.8 mM CaCl₂, 26.2 mM NaHCO₃, 5 mM HEPES, pH 7.4) aerated with nitrogen to remove the oxygen. Some cultures were treated with liraglutide peptide (500 nM, PeproTech, USA) and/or LY294002 (10 nM, Sigma, USA) or U0126 (10 nM, Sigma, USA) during the OGD insult. At the end of the insult (2 h), the OGD medium was replaced with the original medium and then incubated for 24 h in a humidified atmosphere of 5% CO₂ at 37 °C for further experiments. The animal experimental protocols were approved by the Animal Care and Use Committee of Jinan University. All experimental protocols involving rats were approved by the ethical committee of Jinan University and performed in accordance with approved guidelines and regulations.