Rat Retinal Ganglion Cells

The Expression and Function of TRPV4 in Retinal Ganglion Cells at Elevated Pressure

The study investigated TRPV4 expression levels in retinal ganglion cells (RGCs) under different pressures using immunofluorescence and western blotting techniques together with TUNEL assays. Wang's team investigated TRPV4 channels, which play a role in human senses and might be involved in glaucoma-induced retinal damage.

Using immunofluorescence, western blotting, and TUNEL assays, TRPV4 expression in retinal ganglion cells (RGCs) under varying pressures was examined. Western blotting showed reduced TRPV4 levels at 40 mmHg and 60 mmHg for 24 hours compared to normal culture conditions (Fig. 1A-D). TRPV4 levels increased in GRCs at these pressures. Pathway enrichment scores for glaucomatous cells were analyzed using the GSEA algorithm to find correlations between samples and pathways (Fig. 1E-G). TRPV4 expression correlated positively with apoptosis, PI3K-AKT-mTOR, and ROS-upregulated pathways. These pathways are also linked to apoptosis in cancer, rheumatoid arthritis, and acute respiratory conditions.

Fig. 1. TRPV4 expression levels in RGCs in the glaucoma model (Wang L, Zhang W, et al., 2023).

SA-2 Is Neuroprotective to Primary RGCs in the Trophic Factor Deprivation Model

Glaucoma is characterized by degeneration of retinal ganglion cells (RGCs), resulting in optic nerve damage and vision loss. Current treatments largely focus on reducing intraocular pressure (IOP) and do not prevent RGC loss. These neurons, essential for transmitting visual data, are vulnerable to oxidative damage due to high metabolic demands and insufficient antioxidant responses, leading to apoptosis.

Neurotrophic factors are crucial for RGC survival. Pham et al. modeled RGC death by depriving these factors, treating primary RGCs with SA-2 at 0.1 mM, 0.5 mM, and 1 mM for 48 hours. Apoptotic and dead cells were stained with LIVE Green Caspase-3 and -7 Detection Kit. TF deprivation resulted in a 37.83% decrease in RGCs. SA-2 significantly increased RGC preservation across all concentrations (0.1 mM: 108.87% increase, p < 0.01; 0.5 mM: 158.93%, p < 0.001; 1 mM: 74.96%, p < 0.01) compared to vehicle (DPBS) (Fig. 2A-B, teal color). Apoptotic RGCs decreased significantly at 0.1 mM (93.72%), 0.5 mM (99.99%), and 1 mM (92.78%) (Fig. 2A-B, green color). Dead RGCs also reduced significantly at all concentrations (Fig. 2A-B, orange color). In the presence of neurotrophic factors, SA-2 significantly increased RGCs at concentrations of 0.5 mM (232.45% increase, p < 0.0001) and 1 mM (194.74%, p < 0.0001) (Fig. 3A-B, teal). Primary RGCs naturally die even with neurotrophic factors, but SA-2 reduced apoptotic cells at 0.1 mM (52.5% decrease, p < 0.0001), 0.5 mM (30%, p < 0.01), and 1 mM (52.48%, p < 0.001) (Fig. 3A-B, green). SA-2 also decreased dead RGCs at 0.1 mM (95.33% decrease, p < 0.0001) and 0.5 mM (99.99%, p < 0.0001) compared to control (Fig. 3A-B, orange). These findings demonstrate SA-2's potential to protect RGCs from apoptosis within the TF deprivation model.

Fig. 2. Treatment of primary retinal ganglion cells (RGCs) from Sprague Dawley rat pups in the absence of neurotrophic factors (Pham J H, Johnson G A, et al., 2022).

Fig. 3. Treatment of primary RGCs from Sprague Dawley rat pups in the presence of neurotrophic factors (Pham J H, Johnson G A, et al., 2022).