Photoreceptor damage and immune responses after optogenetic RPE ablation in a mouse model of geographic atrophy

Abstract

Purpose : Geographic atrophy (GA) in advanced age-related macular degeneration (AMD) is characterized by photoreceptor degeneration resulting from chronic oxidative damage to retinal pigment epithelium (RPE), but effective treatments are limited due in part to the absence of adequate animal models for preclinical testing. Here, we characterize the photoreceptor damage and immune response profile in a novel mouse model of GA where we optogenetically ablate RPE cells after AAV-mediated transduction of RPE mitochondria with reactive oxygen species (ROS)-generating proteins.

Methods : We subretinally injected AAV8 vectors expressing ROS-generating mCherry under an RPE-specific Bestrophin (VMD2) promoter with a mitochondrial targeting sequence into wild-type C57BL/6J mice eyes. We focally accelerated oxidative stress and triggered RPE ablation using a custom-built scanning-laser ophthalmoscopy (SLO) system with 561 nm light at 700 μW power over 20 minutes. We measured anatomic changes in vivo using SLO and optical coherence tomography (OCT), then collected ocular tissues immediately, 24 hours, and 1 week after exposure for TUNEL staining to assess apoptosis, and immunohistochemistry for Iba1 and C3 to assess microglial and complement activation, respectively.

Results : Photoactivation of mCherry in RPE mitochondria triggered subsequent focal RPE loss with secondary photoreceptor degeneration resembling GA on SLO and OCT imaging. TUNEL+ cells within the outer nuclear layer, consistent with photoreceptor apoptosis, could be detected maximally within the first 24 hours. Iba1+ microglia appeared to migrate towards the area of RPE and photoreceptor damage starting at 24 hours, and adopted more amoeboid morphology suggesting microglial activation that persisted to 1 week. C3 immunofluorescence was detected within the sub-RPE Bruch’s membrane area, and did not appear to change after RPE photoablation.

Conclusions : Optogenetic RPE ablation in mouse eyes triggered focal oxidative stress, photoreceptor degeneration, and microglial activation. This inducible animal model of GA may enable preclinical testing of novel therapies that target photoreceptor neuroprotection or immune regulation.

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