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Haijiang Lin, Miin Roh, Hidetaka Matsumoto, Albert Alhatem, Peggy Bouzika, Yusuke Murakami, Joan Miller, Demetrios Vavvas; Blocking the necroptosis pathway decreases RPE and photoreceptor damage induced by NaIO3. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1957.
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© ARVO (1962-2015); The Authors (2016-present)
Sodium iodate (NaIO3) has been used extensively as a retinotoxin to induce RPE cell damage and degeneration of photoreceptors in vitro and in vivo. RIP-Kinase dependent programmed necrosis is an important redundant cell death pathway that has been shown to be involved in photoreceptor cell death. We wanted to investigate if these pathways are actively involved in RPE and photoreceptor cell death after NaIO3 insult.
ARPE-19 cells were exposed to different concentration of NaIO3 in the presence or absence of various concentration of RIPK inhibitors ( Nec-1) or pan-caspase inhibitor (Z-VAD) individually or in combination. Cell death was determined at different time points by MTT (Sigma Aldrich), LDH (Promega), ATP (Promega) and TUNEL (Millipore) assay. C57BL/6 and RIP3-/- mice were treated with a peritoneal injection of NaIO3 and eyes were enucleated at day 3 or 7. TUNEL staining was used to evaluate photoreceptor cell death. Photoreceptor cell loss was evaluated by measuring the thickness of outer nuclear layer (ONL). Microglias in ONL were quantified in retinal whole mount with Iba-1 antibody. RPE degeneration was also assessed in RPE whole mount with ZO-1 antibody.
NaIO3 resulted in significant cell death of ARPE-19 cells. Treatment with Nec1 resulted in better protection than treatment with zVAD (P<0.01). A synergistic protective effect is observed when co-treating the cells with Nec-1 and Z-VAD. Nec-1 treatment also decreased the ARPE-19 mitochondrial damage caused by NaIO3. In vivo administration of NaIO3 resulted in significant RPE and photoreceptor destruction with significant inflammatory cell infiltration . RIP3 knockout animals displayed significantly less RPE and photoreceptor cell loss as well as significantly less inflammation.
Programmed necrosis is an important cell death pathway mediating NaIO3 RPE and photoreceptor cell toxicity. Therefore, blocking the necroptosis pathway may serve as a novel therapeutic target for various retinal degeneration diseases.
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