A Simple Method of Transducing Retinal Ganglion Cells Using AAV via Transpupillary Injection in Mice

Abstract

Purpose : Intravitreal injection of adeno-associated virus (AAV) is one of the best approaches to transduce retinal ganglion cells (RGCs) in mice. This is due to its high transduction efficiency and selectivity for RGCs. Many studies use a transscleral approach that has the potential of causing damage to the lens or retina. We tested the hypothesis that a transpupillary approach for intravitreal injections will minimize damage to the eye and efficiently transfect RGCs.

Methods : C57BL/6J mice were deeply anesthetized and the iris dilated with tropicamide 1% drops. The globe of the eye was held with forceps while a small, full-thickness incision was made in the posterior aspect of the cornea just anterior to the limbus. The beveled needle was retracted, and a 35-gauge blunt needle was inserted through the incision. The tip of the needle was moved through the anterior chamber to the distal aspect of the pupil. The needle was passed through the pupil, sweeping around the lens, and into the vitreous. At this point, 2μL of AAV-CMV-GFP (cytomegalovirus promoter driving green fluorescent protein) was delivered into the vitreous chamber. Fourteen days after injection, the animals were deeply anesthetized, and live fluorescent fundus images were taken. The mice were deeply anesthetized and perfused with saline followed by 4% formaldehyde in phosphate buffer (pH 7.3). The retina from the injected eye was dissected out, and flat-mounts were stained for GFP.

Results : After the injection procedure, the lens remained clear and appeared undamaged. Fundus imaging and GFP staining results also revealed >90% of the retinas sustained no visible damage to the lens or retina. Retinas injected via the transpupillary approach exhibited homogeneous transduction throughout the ganglion cell layer with GFP, yielding up to 23000 labeled cells.

Conclusions : This study describes a method of transducing RGCs using AAV via a transpupillary injection approach. The advantageous nature of the transpupillary approach reduces the impact of confounding variables on experimental data compared to the transscleral approach. This method represents a promising and efficient approach for delivering reporter genes to RGCs, creating conditions for high levels of gene expression without damage to the lens or retina. Future studies will define the retinal cell type transduced and the transduction efficiency.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.