Neuroprotective effects of C3 exoenzyme in excitotoxic retinopathy

The purpose of this study is to evaluate the neuroprotective effects of C3 exoenzyme (C3) on N-methyl-d-aspartate (NMDA)-induced retinopathy in rats. C3 was expressed in Escherichia. coli and purified by affinity chromatography. Immunofluorescence was performed in NIH 3T3 cells treated with C3 to verify the cellular uptake of the protein. NMDA was injected intravitreally into rat eyes with or without C3. At various time points after injection, eyes were enucleated. Hematoxylin/eosin staining was performed on retina cross-sections for morphological analysis. Survival and apoptosis of cells in the ganglion cell layer (GCL) were assessed by cresyl violet staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) on retina flat-mounts. RhoA levels in retina cells were evaluated by Western blot to detect C3 uptake in vivo. The cellular uptake of C3 was verified by immunofluorescence. Damage including a decrease in inner plexiform layer (IPL) thickness and reduction of cell density in the GCL, corresponding to apoptosis of neurons, was induced by intravitreal injection of NMDA. Protection against this damage was observed following co-injection of C3 and NMDA. RhoA ADP-ribosylation induced by C3 was confirmed by Western blot. Our results suggest that C3 exerts neuroprotective effects against excitotoxic damage induced by NMDA.