Primary Cortical Neuronal Culture and Oxygen-Glucose Deprivation/Reoxygenation (OGD/R) Modeling

In vitro experiment, 12 pregnant SD rats on gestational day 17-19 were provided, and a total of 50 rat fetuses were used. Primary cortical neurons were prepared from the SD rat embryo brains. Briefly, the pregnant SD rats were anesthetized and the embryos were detached from the uterus. After the meninges were carefully discarded from the embryo brain, the cortical tissues were harvested and removed into a culture dish filled with DMEM-F12 medium (Boster, China) containing 10% fetal bovine serum. Then the cortical tissues were cut into 0.5-1 mm³ small pieces, and digested with trypsin (3-4 ml) (Solarbio, China) and DNA ligase (1-2 ml) (MACKLIN, China) in CO₂ incubator at 37°C for 20 minutes. Slightly shake the tissues 4 times every 5 minutes to make it fully contact with the enzyme. Digestion was terminated by adding DMEM-F12 medium containing 10% fetal bovine serum for 10 minutes. Tissues were gently stirred with a pipette to yield a uniform suspension. Cells were then seeded on a tissue culture dish that had been pre-coated using Poly-L-Lysine (PLL) (Sigma, USA). 100 × 10⁴ cells were planted in one well of the 6-well plates, 8 × 10⁴ cells in one well of the 96-well plates, 400 × 10⁴ cells in one well of the 6 cm plate. Following a 4 h period, the medium was exchanged for Neurobasal medium (Gibco, USA) containing 2% B27 (Gibco, USA) and 1% glutamine (Gibco, USA). All reagents used for these experiments were phenol red-free.

To simulate cerebral I/R injury in vitro, the OGD/R model was established. Briefly, cells were pretreated for 24 h with appropriate biochanin A concentrations. OGD was then induced by culturing primary cortical neurons in glucose-free EBSS and incubated in a hypoxic chamber (Billups-Rothenberg, CA, USA) in a 5% CO₂ and 95% N₂ atmosphere at 37°C for 2 h or other appropriate time. For control cells, glucose-containing EBSS was instead used. After this OGD period, cells were returned to complete Neurobasal medium and incubated at 37°C for 24 h in a standard humidified incubator to mimic reperfusion. Control cells underwent a similar procedure, but did not undergo OGD/R.