Protocol for the purification and culture of primary retinal ganglion cells and development of common pathological models

To establish a standardized protocol for purifying and culturing primary RGC from postnatal KM mice and to optimize the establishment of three in vitro injury models that mimic hyperglycemia, oxidative stress, and H/R. Retinas from 15 postnatal KM mice (≤ 24 h old) were dissociated and purified via Thy1.2 monoclonal antibody-based immunopanning. RGC identity was confirmed by Brn3a (an RGC-specific marker) immunofluorescence, Tuj1 (a neuronal marker) immunostaining, flow cytometry, and trypan blue exclusion. Pathological models were constructed as follows: ①. Hyperglycemia: RGC were treated with 40-80 mM glucose for 24/48 h. ②. Oxidative stress: RGC were exposed to 80-320 μM H₂O₂ for 24 h. ③. H/R injury: Hypoxia (1% O₂, 4 h) followed by reoxygenation (21% O₂, 12 h), with/without AS-IV (50-200 μM) pretreatment. Purified RGC exhibited characteristic morphology and robust viability (93.33% ± 2.1%). Brn3a immunostaining confirmed the identity of the RGC (95.07% purity via flow cytometry). ① Hyperglycemia model: IC₅₀ values were 67.143 mM (24 h) and 58.406 mM (48 h) (P < 0.05 vs. control).② In the oxidative stress model, the IC₅₀ H₂O₂ concentration was 255.262 μM (24 h, P < 0.05), accompanied by dose-dependent increases in ROS levels and HO-1 mRNA upregulation (P < 0.05). ③. H/R model: AS-IV (100 μM) maximally preserved RGC viability (80% survival, P < 0.05 vs. the injury group), downregulating HIF-1α expression postreoxygenation. This study provides a reproducible protocol for high-purity RGC isolation and validates three pathophysiological models that recapitulate key drivers of optic neuropathies. These models offer a robust platform for mechanistic studies and neuroprotective drug screening.