In Vitro Ischemic Injury Model for Retinal Ganglion Cells

We established an in vitro mimic of ischemic injury in RGCs according to our previously applied calcium ionophore/ATP depletion injury model (Zhang et al. 2021 (link); Ge et al. 2020 (link); Lee and Emala 2002 (link)). When RGCs were confluent over 90% of the plate, ischemia was simulated for 2 h by changing the medium to Hanks’ balanced salt solution (HBSS) with 10 mM antimycin A (a complex III inhibitor of mitochondrial electron transport; ab141904; Abcam, Cambridge, MA, US) and 2 mM calcium ionophore (A2318; Aladdin, Shanghai, China), which were dissolved in dimethylsulfoxide (DMSO). The complete growth medium was reapplied, and the cells were sustained for 0–4 h. For gene knockdown and overexpression experiments, 24 h before ischemic injury, when the density of the RGCs on the 24-well plate reached 80% to 90%, the cells were transfected lncRNA uc007nnj.1 siRNA/plasmid, miR-155-5p mimic or inhibitor, Tle4 siRNA, and scramble siRNA (Ribobio) using Lipofectamine 2000. After 6–8 h, the transfection solution was removed and replaced with a complete growth medium. Immunofluorescence staining of Tuj1 (1:500, GB11139; Servicebio, Wuhan, China) was used to identify the purity of RGCs.