Abstract
Purposewas to create an in vitro model of human retinal detachment (RD) to study the mechanisms of photoreceptor death.MethodsHuman retinas were obtained through eye globe donations for research purposes and cultivated as explants. Cell death was investigated in retinas with (control) and without retinal pigment epithelium (RPE) cells to mimic RD. Tissues were studied at different time points and immunohistological analyses for TUNEL, Cleaved caspase3, AIF, CDK4 and the epigenetic mark H3K27me3 were performed. Human and monkey eye globes with retinal detachment served as controls.ResultsThe number of TUNEL-positive cells, compared between 1 and 7 days, increased with time in both retinas with RPE (from 1.2 ± 0.46 to 8 ± 0.89, n = 4) and without RPE (from 2.6 ± 0.73 to 16.3 ± 1.27, p < 0.014). In the group without RPE, cell death peaked at day 3 (p = 0.014) and was high until day 7. Almost no Cleaved-Caspase3 signal was observed, whereas a transient augmentation at day 3 of AIF-positive cells was observed to be about 10-fold in comparison to the control group (n = 2). Few CDK4-positive cells were found in both groups, but significantly more in the RD group at day 7 (1.8 ± 0.24 vs. 4.7 ± 0.58, p = 0.014). The H3K27me3 mark increased by 7-fold after 5 days in the RD group (p = 0.014) and slightly decreased at day 7 and was also observed to be markedly increased in human and monkey detached retina samples.ConclusionAIF expression coincides with the first peak of cell death, whereas the H3K27me3 mark increases during the cell death plateau, suggesting that photoreceptor death is induced by different successive pathways after RD. This in vitro model should permit the identification of neuroprotective drugs with clinical relevance.
Highlights
Retinal detachment (RD) is one of the most common causes of photoreceptor cell death worldwide (Subramanian and Topping, 2004)
apoptosis inducing factor (AIF) release and Caspase-9 activation were observed in RD human retinas (Hisatomi et al, 2008), but the use of anti-apoptotic factors such as Z-VAD in rodent models of RD did not significantly reduced photoreceptor death (Murakami et al, 2013), suggesting that other pathways are activated during the cell death process
Seizing the opportunity to obtain sections from 4 eye globes containing a uveal melanoma resulting in retinal detachment, we investigated the distribution of the H3K27me3 mark in these retinas
Summary
Retinal detachment (RD) is one of the most common causes of photoreceptor cell death worldwide (Subramanian and Topping, 2004). Various pathological changes occur in detached retinas (Anderson et al, 1981; Lewis et al, 1994; Jablonski et al, 2000), studies on experimental models have shown that photoreceptor cell death is immediately induced, as early as 12 h after the RD event, and peaks after 2 or 3 days (Cook et al, 1995; Hisatomi et al, 2001; Arroyo et al, 2005). AIF release and Caspase-9 activation were observed in RD human retinas (Hisatomi et al, 2008), but the use of anti-apoptotic factors such as Z-VAD in rodent models of RD did not significantly reduced photoreceptor death (Murakami et al, 2013), suggesting that other pathways are activated during the cell death process
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