Abstract
Abstract: :
Purpose: Biochemical and epidemiological evidence suggests that generation of toxic oxidants in the retina/retinal pigment epithelium/choroid complex results in its damage, and may play an important role in the development of age–related macular degeneration (AMD). Our goal is to test this theory in the mouse by using ribozymes to reduce the levels of enzymes such as manganese superoxide dismutase (MnSOD) that mitigate levels of reactive oxygen species (ROS). Previously, we showed that MnSOD ribozyme 432 is able to cause loss of electroretinogram (ERG) response and histological damage in the retinas of DBAJ/1 mice. In our current studies we examine SOD2 RZ432 mediated retinal damage in the DBAJ/1 using electron microscopy. To confirm the changes observed in the DBAJ/1, we also examined the effects of SOD2 RZ432 on the retinas of C57BL/6 mice. Methods: The SOD2 RZ432 was cloned under the control of the CMV–beta actin (CBA) promoter and packaged in AAV1 capsids, which led to expression primarily in the RPE following subretinal injection. Four–week old DBAJ/1 and C57BL/6 mice were injected subretinally in the right eye with 1µL of CBA–SOD2–RZ432 (2x1013p/mL) or CBA–SOD2–inactiveRZ432 (2.5x1013p/mL) in the left eye to serve as a control. Full field scotopic ERG analysis was performed at 2 and 4 months post injection, and at 4.5 months the eyes were processed to examine retinal histology by light and electron microscopy. Results: DBAJ/1 mice showed a progressive loss of a– and b–wave amplitudes in response to the active ribozyme. There was not a significant reduction in amplitudes at 2 months post injection, but an average of 40 percent reduction was observed in a– and b–wave amplitudes at 4 months post injection. C57BL/6 mice showed an average of 40 and 38 percent, respectively, in a– and b–wave amplitudes at both the 2 and 4 months measurements. Light and electron microscopy analysis of retinas injected with the active SOD2 RZ432 showed complete loss or disorganization of photoreceptor outer and inner segments, condensed chromatin, thinning of the outer nuclear layer, massive vacuolization of the RPE, and accumulation of pigment vesicles in the interphotoreceptor space. Conclusions: : Expression of SOD2 RZ432 in the RPE results in significant attenuation in the ERG response and caused substantial histological damage to the retinas of DBAJ/1 and C57BL/6 mice. Since AAV1 leads to transduction of the RPE, these retinal changes were probably the consequences of RPE damage caused by an increased ROS burden.
Keywords: age-related macular degeneration • retinal pigment epithelium • oxidation/oxidative or free radical damage