Abstract
Purpose:
Sodium iodate (NaIO3)-induced cell death can be used to study degeneration of retinal pigment epithelial (RPE) and photoreceptor (PR) cells in vitro and in vivo. Thereby, in vivo studies have shown that RPE cells undergo necrosis and induce apoptosis in PR cells. Additionally, necroptosis could also be involved besides these two classical cell death pathways. Herein, we investigated which cell death pathway is actively involved in RPE and PR cell death after NaIO3 treatment in vitro.
Methods:
Murine primary RPE and retinal cone-derived 661W cells were exposed to different concentrations of NaIO3. The degree of cell death was determined at different time points colorimetrically (XTT assay) or spectrometrically (ApoTox-Glo assay). In a first step, RPE and 661W cells were treated with 1.5, 3, 6, 12, 24, or 48 mM NaIO3 and cellular metabolic activity was detected 24 hours post-treatment by using the XTT assay. Additionally, viability as well as necrosis and apoptosis were assessed in both cell types at 6, 14 and 24 h post-treatment by the ApoTox-Glo assay. Quantitative RT-PCR was performed to determine relative gene expression of the necroptosis-associated kinases RIP1/RIP3 after NaIO3 treatment in the presence or absence of pan-caspase inhibitor Z-VAD and/or RIP kinase inhibitor Nec-1.
Results:
In vitro administration of NaIO3 resulted in a significant concentration-dependent decrease in cell survival in both cell lines. Furthermore, RPE cells showed a higher susceptibility to NaIO3 than 661W cells. A significant time-dependent effect was also observed in both cell lines. While necrosis was prominent in RPE cells with a significant peak at 14 h post-treatment, apoptotic cell death in 661W cells reached its maximum at 6 h after NaIO3 treatment. Co-treatment with Z-VAD and Nec-1 displayed a significant downregulation of RIP1/RIP3 kinases in both cell types.
Conclusions:
In vitro NaIO3-treated RPE cells undergo necrosis, whereas photoreceptor-derived 661W cells die from apoptosis. Necroptosis is not involved in NaIO3-induced cell death. These results confirm our in vivo studies and may serve as an alternative to investigate neuroprotective approaches in the NaIO3 induced mouse model.
Keywords: 701 retinal pigment epithelium •
648 photoreceptors