The effect of experimental glaucoma on retinal ganglion cell dendritic tree size in the rat

Abstract

Abstract: : Purpose: Electrophysiological and single cell dye injection studies have suggested that changes in the dendritic tree of retinal ganglion cells (RGC) preceed cell death in experimental models of glaucoma, but a limitation of such techniques is the relatively small number of cells that can be studied in each retina. We used a modified adeno–associated virus (AAV) vector to transfect a high proportion of RGC in the rat with green fluorescent protein (GFP). Structural changes in the dendritic tree of large numbers of RGC could then be studied by direct visualization of GFP in retinal wholemounts. Methods:A modified AAV carrying the gene for GFP under the control of a chicken beta–actin promoter and incorporating the woodchuck hepatitis post–transcriptional regulatory element (WPRE) was constructed. One eye of 8 adult Wistar rats received a single intravitreal injection of 2µl viral stock (2x10¹² particles/ml). One week later, experimental glaucoma was induced in the injected eye of 4 rats by 532nm laser treatment to the trabecular meshwork using our previously–reported technique. Intraocular pressure was measured by Tonopen XL tonometry. Two weeks after injection, retinal wholemounts were prepared and examined by masked observers under fluorescence microscopy. The maximum RGC dendritic tree diameter was measured for 50 consecutive cells in each treated and control retina. The density of cells with a dendritic tree diameter greater than twice the cell body diameter was also estimated. Results: The mean density of GFP–positive cells two weeks after AAV injection was 1828±299 cells/mm². Almost all GFP–transfected cells seen in retinal cross sections were localized to the RGC layer. Approximately 1% of GFP–positive cells in both glaucoma and control retinas had a dendritic tree at least twice the diameter of the cell body and an axon projecting towards the optic nerve head, confirming identity as RGC. The number of presumed RGC did not differ significantly between control eyes and experimental glaucoma eyes after 1 week of elevated IOP. The mean dendritic tree diameter was significantly smaller in experimental glaucoma (146+60µm) compared to control eyes (191+90µm) (n=200 cells measured in each group, p<0.00001). Conclusions: Experimental glaucoma in the rat is associated with a decrease in RGC dendritic tree diameter that preceeds significant RGC death.

Keywords: gene transfer/gene therapy • ganglion cells • intraocular pressure