Abstract
Purpose :
Retinal degeneration is a major cause of blindness in ocular diseases such as glaucoma and ischemic retinopathies. Our previous observations suggested that Nogo-A gene ablation and antibody-based function blocking increased retinal recovery and improved vision after retinal injury. The goal of this current work was to determine the role of endogenous Nogo-A on the maintenance and function of the adult mouse retinae in physiological conditions.
Methods :
Nogo-A was silenced by intravitreal injection of adeno-associated virus serotype 2.2 containing a short hairpin RNA sequence (AAV2.2 shRNA-Nogo-A) in adult C57BL/6J mice. A GFP reporter gene was comprised in this AAV construct. An empty AAV2.2 vector was used as a control vector. Retinal cell infection with AAV2.2-shRNA-Nogo-A was controlled by fluorescent fundoscopy. Changes in the expression of Nogo-A protein were assessed by Western blotting in whole retinal lysates. Visual acuity was tested using the optokinetic reflex (OKR) in freely moving mice. Retinal activity was monitored by electroretinogram (ERG) recording. Retinal ganglion cells (RGCs) and Müller glia were examined by immunofluorescence on retinal histological sections using RNA-binding protein with multiple splicing (RBPMS) and Sex-determining region Y-box 2 (Sox-2) as specific cell markers, respectively.
Results :
One month after AAV2.2-shRNA-Nogo-A injection, GFP fluorescence revealed cell transfection in the RGC and Nogo-Athe inner nuclear layers. Nogo-A protein expression was decreased by ~75% in AAV2.2-shRNA-Nogo-A (n=3 mice/group) relative to control (n=3). The visual acuity of AAV2.2-shRNA-Nogo-A injected mice (n=10) was reduced by 43.7% as compared to AAV2.2-empty (n=7) control, at two months of infection. Consistently, ERG b-wave and a-wave amplitudes were respectively decreased by 35% and by 24.4% relative to control mice. At 60 days of infection, the number of RGCs was reduced by 29.72% in AAV2.2-shRNA-Nogo-A (n=4) in comparison with contralateral control eyes (n=4). Qualitatively, the survival of Müller cells did not seem affected by Nogo-A gene silencing.
Conclusions :
Acute silencing of Nogo-A in the retina exerts detrimental effects on the mouse vision and neuronal maintenance, suggesting an important physiological function of Nogo-A at this level.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.