RPE necroptosis in mouse model of sodium iodate–induced RPE degeneration

Abstract

Purpose : The mouse model of sodium iodate (NaIO₃)-induced retina degeneration is widely used to mimic retinal pigment epithelial (RPE) cells degeneration in geographic atrophy (GA). However, RPE cell death in GA remains controversial. The purpose of the study is to analyze RPE cell death in vivo using novel transgenic reporter mouse line in combination with the NaIO₃ model.

Methods : NaIO₃ at 20mg/kg was administered by retro-orbital injection into 4 weeks old C57BL/6J mice. Phenotypic changes of the retina were assessed at 1, 2, and 3 days post injection. For histological evaluation we used H&E staining, methylene blue staining of ultra-thin plastic sections, and electron microscopy. TUNEL staining, propidium iodate (PI) staining, and immunochemistry (ZO-1, active caspase 3, HMGB1) was used to analyze RPE and photoreceptors cell death. To analyze necroptosis in vivo in RPE cells we generated transgenic mouse line that expresses RIPK3-GFP under the control of RPE-specific VMD2 promoter.

Results : Histological analysis revealed that 24 hours after NaIO₃ administration RPE start to loose pigmentation and no effect is observed on photoreceptors inner segments (IS) or outer nuclear layer (ONL). At 48h-72h post injection RPE appear swollen, vacuolized, and breaking off from RPE monolayer, suggesting RPE necrosis. IS and ONL are noticeably disorganized. At molecular level RPE monolayer properties are compromised as visualized by ZO-1 staining, 48h post injection, together with observed PI penetration into the cytoplasm and nucleus of RPE cells. TUNEL positive RPE and photoreceptors are observed as early as 24h posts injection, but active caspase 3 is detected only in the photoreceptors layer. RIPK3 aggregation is a critical step for necroptosis. Using GFP staining we observed RIPK3-GFP aggregation in RPE in response to NaIO₃ at 24h post injection. Additionally, release of HMGB1 (pro-inflammatory protein) associated with necroptosis form RPE was observed by both immunostaining and ELISA.

Conclusions : We provide evidence of RPE necroptosis in NaIO₃-induced retinal degeneration model, and conclude that in this model photoreceptor apoptosis may be secondary to RPE necroptosis.

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