Abstract
Purpose: :
Generation of oxidative stress in the retina and retinal pigment epithelium (RPE) complex has been suggested as a main factor leading to the development of age-related macular degeneration (AMD). Our goal is to test this theory in the mouse by reducing RPE specific MnSOD2. Previously, we showed that a MnSOD specific ribozyme Rz432 diminished the electroretinogram (ERG) response and caused histological damage in the retinas of mice. Here we utilized an alternative approach to determine if knockdown of SOD2 cause an increased level of reactive oxygen species in the retina/RPE complex leading to the pathogenesis similar to AMD.
Methods: :
To confirm that subretinally injected VMD2-CRE (AAV1) virus into the floxed-SOD2 mice mediates RPE-specific recombination, VMD2-CRE (AAV1) virus was injected subretinally into the reporter mouse strain which contains a floxed PGK-neo cassette and an enhanced yellow fluorescent protein (EYFP) gene inserted into the Gt(ROSA)26Sor locus. The expression of YFP was detected by immunostaining. To reduce MnSOD2 in the RPE, we utilized Cre-loxP recombination system. Cre was packaged into AAV1 under the control of the VMD2 promoter which leads to expression in the RPE. Mice bearing a floxed allele of SOD2 were injected subretinally in the right eye with AAV1-VMD2-cre. Left eyes were injected with an AAV1-GFP as controls. Electroretinogram (ERG) analysis was performed at 4 or 8 and 14 weeks post injection, and eyes were processed at 8 weeks post injection to examine oxidative damage markers. To see the oxidative stress in these mice, immunostaining was done using anti-HNE, a marker of lipid peroxidation. Eyes of mice injected with AAV-Rz432 were examined by histology.
Results: :
In the reporter mouse line, YFP expression was detected only in the RPE layer after injection with AAV-VMD2-CRE. In mice bearing the floxed SOD2 allele, a 30 percent reduction was observed in both a-wave and b-wave amplitudes of those eyes injected with the VMD2-CRE virus as compared to control. The level of 4-HNE was increased in the right eye as compared with left eye. In continuation of examining the effect of MnSOD2 ribozyme we analyzed retinas of mice 7 and 12 months post injection with AAV-Rz432. By 7 months, retinas exhibited deposits in RPE layer, shortened outer and inner segments of photoreceptors, and the thinning of outer nuclear layer indicating loss of photoreceptor cells. By 12 month of post injection with AAV-Rz432, we detected more pronounced changes to the RPE such as vacuole formation and atrophy.
Conclusions: :
As previously shown, reduction of MnSOD2 by using ribozyme results in the damage of outer retina of mice. RPE-specific down-regulation of SOD2 by using cre-mediated recombination leads to increased oxidative stress in the RPE and reduced ERG response.
Keywords: age-related macular degeneration • retinal pigment epithelium • genetics