Primary neuron cultures and oxygen-glucose deprivation modeling

Primary cortical neurons were obtained from C57BL/6 newborn mice, 24 hours postnatal, as described in previous studies (Jiang et al., 2015).

The OGD model is the most common in vitro cell model used to study ischemic diseases. We established an OGD model to simulate TBI-induced secondary brain injury (Salvador et al., 2015) because hypoxic changes caused by cell ischemia are often accompanied by secondary brain injury. After the OGD model was established, the cell viability of neurons decreased significantly, indicating that model generation was successful. All neurons were randomly allocated to the following four groups, without blinding: the control neuron group experienced neither OGD nor Neat1 downregulation or overexpression (n = 3 replicates). The OGD neuron group experienced OGD and was transduced with the Ad-GFP virus (n = 3). The Neat1 overexpression neuron group experienced OGD and was transduced with the Ad-Neat1 overexpression virus (n = 3). The Neat1 downregulation neuron group experienced OGD and was transduced with the Ad-Neat1 downregulation virus (n = 3).

The maintenance medium was neurobasal-A (#10888022, Gibco Life Technologies, New York, USA) with 1× B27 supplement and 0.5 mM L-glutamine. The cell density on six-well plates was approximately 2.5 × 10⁶/mL. Neat1 overexpression and downregulation adenovirus were used to infect primary neurons 24 hours after plating, using multiplicities of infection of 5 and 240, respectively. Polybrene (#H9268, Sigma-Aldrich, Shanghai, China) was also added at 2 µg/mL. Neurons were photographed using a laser-scanning confocal microscope. Four groups were established as described.

The primary neurons were subjected to the OGD model as previously described (Pei et al., 2019). In brief, the neurons were washed twice with phosphate-buffered saline (PBS) and incubated in glucose-free DMEM (#2044490, Gibco Life Technologies, New York, USA) in a hypoxic chamber (1% O₂, 5% CO₂, and 94% N₂) at 37°C for 3 hours. Afterward, the neurons were incubated in normal media under normoxic conditions (5% CO₂ and 95% O₂) at 37°C for 24 hours. All cells were counted using ImageJ software (NIH, Bethesda, MD, USA).