Purpose: To evaluate the effect of autophagy inducers on damage caused by vital dye in adult human RPE (ARPE) cells and in a rat model.
Methods: ARPE-19 cells were exposed to ICG or BBG (0.05 mg/ml) with rapamycin (200 nM) or metformin (2 mM) for 30 min and treated with or without 20 μM chloroquine (CQ) to identify the protein levels of LC3 and SQSTM1 by immunoblotting. In vivo study was performed by injecting 10 μl 0.05% ICG and 0.25% BBG into the subretinal space of the rat eyes, and/or co-treated them with metformin and rapamycin. The retinas were used to determine autophagy with the LC3-II level and apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay.
Results: In this study, both ICG and BBG inhibited autophagy flux in adult human retinal pigment epithelium cells (ARPE-19), whereas only ICG consistently reduced autophagy in the retina of rats. Moreover, rapamycin and metformin induced autophagic flux in ARPE-19 cells and increased the LC3-II level in retinal tissues exposed to vital dyes. Both ICG and BBG increased apoptosis in the retina of rats. However, both rapamycin and metformin induced autophagy and reduced the apoptosis caused by vital dyes.
Conclusion: Taken together, these results suggest that rapamycin and metformin may diminish vital dye-induced retinal damage in vivo through activation of autophagy.
Keywords: Autophagic flux; In vivo; Retinal pigment epithelium; Vital dyes.