Adeno-associated viral vector labeling of retinal ganglion cells for in vivo imaging

Abstract

Purpose : The ability to detect and measure early retinal ganglion cell (RGC) loss is important for the diagnosis and monitoring of diseases such as glaucoma. We tested the hypothesis that adeno-associated viral (AAV) vectors would facilitate green fluorescent protein (GFP) labeling of RGCs in mice for in vivo non-invasive and real-time imaging as a means of RGC quantification.

Methods : 1.5 μl of vector with a ubiquitous (AAV2-CAG-GFP) or RGC selective (AAV2-DCX-GFP) promoter was injected into the vitreous of the left eye of C57BL/6 mice (n = 6). The retina was imaged non-invasively by fluorescence imaging (488 nm excitation) with a modified Spectralis (Heidelberg Engineering) device weekly post-injection. All procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. Immunohistochemistry was performed in retinal flat-mount and section preparations for RGC (RNA binding protein with multiple splicing, RBPMS) and amacrine (choline acetyltransferase, ChAT) cell labelling, respectively.

Results : In vivo imaging detected fluorescently labeled cells 1-week post-injection of either vector. Labeling was visible in vivo continuously for at least 5 weeks. Expression profiles demonstrated increased transduction occurred up until approximately 4 weeks post-injection. The in vivo mean (standard error, SE) central retina cell density for the AAV2-CAG-GFP vector was 97 (25) cells/mm² at week-1 and 638 (90) cells/mm² at week-4. For the AAV2-DCX-GFP vector, cell density was 29 (20) cells/mm² at week-1 and 288 (54) cells/mm² at week-4. Immunohistochemistry demonstrated that 5-weeks post-injection, the mean (SE) GFP+ cells that were RBPMS+ was significantly higher (p < 0.05) for the AAV2-DCX-GFP vector, 89 (2)%, compared to the AAV2-CAG-GFP vector, 76 (2)%.

Conclusions : AAV mediated GFP expression labeled RGCs with strong and long-lasting fluorescence when administered by intravitreal injection. Furthermore, AAV specificity to RGCs can be improved with tissue specific promoters such as DCX. This labeling and longitudinal imaging technique can be used to confirm the presence of healthy RGCs and detect changes in the number of RGCs over time.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

In vivo images of GFP labeled retinal neurons in mouse post-intravitreal injection of a ubiquitous (AAV2-CAG-GFP) or RGC selective (AAV2-DCX-GFP) vector.