Abstract
Purpose: :
Oxidative stress has been implicated in retinal ganglion cell (RGC) death induced by optic nerve transection (ONT) and during glaucomatous neuropathy. We investigated the changes in regulation of thioredoxins 1 and 2 (Trx1 and Trx2) expression after ONT and evaluated their roles in RGC protection.
Methods: :
ONT was performed on adult male Wistar rats. Retrograde labeling to identify RGCs was performed by placing a Gelfoam soaked with 6% Fluorogold (FG) to the proximal cut surface of the optic nerve after ONT. RGC isolation was performed with magnetic beads coated with Thy-1 monoclonal antibody. The levels of Trx1 and Trx2 in RGCs were examined with Western blot analysis. Immunohistochemisty was used to localize expression of Trx1 and Trx2 in the rat retina. Trx1 and Trx2 expression constructs were delivered to RGCs by intravitreal injection of the plasmid DNA followed by electroporation (ELP). The effect of Trx1 and Trx2 overexpression on RGC survival was determined one and two weeks after ONT.
Results: :
Immunohistochemical analysis showed Trx1 expression primarily in the inner nuclear layer (INL), ganglion cell layer (RGCL), and retinal pigment epithelium, whereas Trx2 immunoreactivity was present in all retinal layers. In the RGCL, both Trx1- and Trx2-positive cells were co-localized with FG-labeled RGCs. Analysis of the endogenous expression of Trx1 and Trx2 in RGCs at 1, 3, and 7 days after axotomy showed that up-regulation of mitochondrial Trx2 and cytosolic Trx1 was observed 3 and 7 days after ONT, respectively. The efficiency of RGC transfection with Trx1 or Trx2 expressing plasmids was 34.73 ± 6.38 % and 26.83 ± 8.33 %, respectively. Both Trx1 and Trx2 overexpression increased the survival rate of RGCs approximately 35% and 135% one and two weeks after ONT.
Conclusions: :
Our findings suggest the involvement of oxidative stress in RGC degeneration induced by ONT and provide evidence for a neuroprotective role of Trx1 and Trx2 in RGC survival.
Keywords: neuroprotection • retina • ganglion cells