Abstract
Purpose: :
Stress-induced crystallin expression is commonly viewed as an activation of the cell survival mechanism. We analyzed expression of alpha A and alpha B crystallins in a rat optic nerve transection (ONT) model characterized by specific retinal ganglion cell (RGC) degeneration and determined their role in RGC survival.
Methods: :
ONT was performed on adult male Wistar rats weighing 300-350g. Quantitative and spatial expression were examined with western blot and immunohistochemisty, respectively. Electroporation was used to deliver alpha A and alpha B expression constructs to RGCs. Cell protective effects of alpha A and alpha B overexpression after ONT was determined with RGC density analysis.
Results: :
Alpha A and alpha B mRNA and protein expression in the retina was observed predominantly in the ganglion cell layer, where the majority of crystallin-positive cells were co-localized with RGCs. Crystallin expression was also present in the inner nuclear and to a lesser degree in the outer nuclear layers. Very few crystallin-positive cells were found 14 days after ONT, which leads to more than 90% of RGCs loss. Quantitative analysis showed that the expression level of the alpha A (control vs ONT, n=8, P = 0.016) and alpha B proteins (control vs ONT, n=8, P = 0.005) was reduced by approximately 1.6 fold after ONT compared to the contralateral untreated retinas. The effect of alpha A and alpha B overexpression on RGC survival was evaluated 14 days after axotomy. The numbers of surviving RGCs after alpha A and alpha B transfection were 389±57 and 353.57±60 cells/mm2, respectively, versus 198±29 cells/mm2 following transfection with the vector alone [ONT + EGFP (n=5) vs ONT + alpha A - EGFP (n=8), P = 0.003; ONT + EGFP (n=5) vs ONT + alpha B - EGFP (n=8), P = 0.003] or 206±60 cells/mm2 in non-transfected retinas [ONT (n=9) vs ONT + alpha A - EGFP (n=8), P = 0.001; ONT (n=9) vs ONT + alpha B - EGFP (n=8), P = 0.001].
Conclusions: :
The results indicate a significant neuroprotective effect of alpha-crystallins on RGCs with approximately 95% and 75% increase in the RGC survival rate mediated by alpha A and alpha B overexpression, respectively.
Keywords: ganglion cells • neuroprotection • crystallins