ShH10, A Novel Müller Glia Cell-specific AAV Vector, Expressing GDNF Promotes Retinal Ganglion Cell Survival Following Neuronal Injury In Thy1-YFP Mice

Abstract

Purpose: : ShH10 is a novel AAV variant that efficiently transfects Müller glia following intravitreal injection. This study aims to determine if an ShH10 vector expressing GDNF secreted from Müller glial cells prevents retinal ganglion cell (RGC) loss following optic nerve crush injury in Thy1-yellow fluorescent protein (Thy1-YFP) mice.

Methods: : Thy-1-YFP mice, which express yellow fluorescent protein as a marker of viable retinal neurons, were imaged using the Micron III fundus camera before and after optic nerve crush injury. At the time of optic nerve crush injury, mice were treated with a 1μl intravitreal injection containing 1x10e10 vector genomes of either ShH10.GDNF or ShH10.GFP in one eye with no treatment in the contralateral eye. Mice were sacrificed at four weeks and retinal flatmounts prepared. Fluorescent spots were counted manually in the same retinal area of each animal. ELISA was performed to quantify GDNF levels in the retina of injected eyes.

Results: : ELISA confirmed that retinal GDNF expression was significantly increased in eyes injected with ShH10.GDNF compared to ShH10.GFP (n=9; p<0.05). In vivo imaging demonstrated less fluorescent retinal neuron loss in ShH10.GDNF treated eyes compared to both ShH10.GFP treated eyes and uninjected control eyes. At four weeks, the number of fluorescent cells in untreated eyes without optic nerve crush injury was 231±144 cells per field (cpf). Eyes that received intravitreal ShH10.GDNF (93.3±28.7 cpf) showed more remaining fluorescent cells than eyes that received intravitreal ShH10.GFP (42.3±10 cpf) and no injection (39.8±11.3 cpf). Thus, the number of of fluorescent cells remaining in optic nerve crushed eyes that received ShH10.GDNF was over 200% greater than optic nerve crushed eyes that received ShH10.GFP or no injection (n=5; p<0.05).

Conclusions: : Our results indicate that increased GDNF expression in Müller glial cells following intravitreal injection of ShH10.GDNF results in protection of retinal neurons in Thy1-YFP mice following optic nerve crush. In Thy1-YFP mice, fluorescent cells predominantly represent retinal ganglion cells; therefore, these findings suggest that intravitreal ShH10.GDNF provides a neuroprotective effect on retinal ganglion cells after optic nerve injury.

Keywords: neuroprotection • optic nerve • imaging/image analysis: non-clinical