Cortical Neuron Isolation and OGD Procedure

All experiments were carried out in accordance with the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. Cortical neurons from B6 Albino mice (B6N-Tyrc-Brd/BrdCrCrl, Charles River Laboratories International, Inc.) were prepared according to previous reports (12 (link), 13 (link)). Briefly, embryonic day 16 fetuses (n = 21) were taken from euthanized, pregnant mice in sterile conditions. Fetal brains were removed and cortical tissues were dissected under a microscope. The meninges were then removed and cortical tissues were chopped into small pieces. Cells were dispersed followed by mechanical trituration using Neuron Dissociation Solutions (Wako Pure Chemical Industries, Ltd., Japan) and filtered through a 70 μm pore-size cell strainer. Cells were then resuspended in neurobasal medium (GIBCO) supplemented with 2% B27 (Invitrogen) and plated onto Poly-L-Lysine Culture Dishes (BioCoat™, Corning Inc. Japan). Cells were cultured in a humidified incubator at 37°C with 5% CO₂, and half of the medium was replaced with fresh solution every 3 days. To reduce contamination by glial cells, 10 μM cytosine arabinofuranoside (Sigma-Aldrich) was added for 24 h on the 4th day of culture. We cultured cortical neurons for 7days, and OGD procedure was performed.