The effects of light-induced retinal damage (LIRD) in C57BL6/J mouse is mediated by RPE-65 activity

Abstract

Purpose : Controversy exists in the field regarding the extent of heterogeneity in microglial responses in a damage dependent context in the eye. We investigated the hypothesis that mutations in genes involved in the visual cycle, namely rpe65, can be used to study changes in damage kinetics and immune cell recruitment by modulating the extent of phototoxicity

Methods : Using mice containing the wildtype Rpe-65(Leucine at aa450) and the low activity variant (L450M), we used light-induced retinal damage (LIRD) to study how changes in Rpe-65 affects damage kinetics in the C57BL6/J line. Mice were aged to P60+. We used both L450 and L450M animals that were exposed to LIRD at 50,000 lux for 5 hours during the dark phase of their circadian rhythm (ZT12-ZT17). Animals were then grouped into day 3, 5, 7, and 10 post LIRD groups, at which time retinal thickness and morphology were measured via SD-OCT and cSLO. Animals were sacrificed immediately after terminal in vivo data collection and ocular tissue was collected for RPE flat mounts. RPE flat mounts were stained with markers for microglia (IBA-1), zonula occludins (ZO-1), and cell nuclei (Hoescht). The IBA-1 positive cells that were deposited into the RPE sheet were then counted and examined for morphology.

Results : Preliminary data suggest that post LIRD mice expressing the L450 wildtype version of Rpe65 exhibit early signs of morphological changes in fundus images and SD-OCT images compared to animals expressing the L450M variant. The L450 animals have an increased presence of autofluorescent dots (postulated to be microglia) in cSLO images that are detectable at ~ 3 days within the RPE layer. Additionally, quantification of IBA-1- positive cells in the RPE layer by immunohistochemistry show that there is a significant difference between IBA+ cell deposition between L450 and L450M mice at days 3, 5, and 7 (p-value <0.05). The most significant differences in IBA+ cell counts at the RPE were on day 7 (p-value < 0.001) post LIRD (n=3/group).

Conclusions : The L450M Rpe65 variant has 60% of the wildtype Rpe65 (L450) activity level. Here we show that the L450 are more susceptible to LIRD damage as characterized by SD-OCT, cSLO, and IHC images. This data suggests that changes in RPE function via reduction of Rpe65 can change both the extent of damage and the kinetics of damage resolution.

This is a 2021 ARVO Annual Meeting abstract.