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Mark E. Kleinman, Yoshio Hirano, Valeria Tarallo, Bradley D. Gelfand, Hiroki Kaneko, Benjamin J. Fowler, Sami Dridi, Jayakrishna Ambati; RPE Cell Death Imaging and Inhibition in Mouse Models of Geographic Atrophy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5300. doi: https://doi.org/.
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We previously demonstrated significant death of retinal pigment epithelium (RPE) in mice after intravitreous delivery of double-stranded RNAs (dsRNA). More recently, we discovered endogenous cytotoxic dsRNAs derived from Alu, a mobile genetic element, in human eyes affected with geographic atrophy (GA). Exogenous delivery of identical Alu RNAs in mice similarly leads to RPE atrophy. We hypothesize that a major cell death pathway for this response may require caspase activation. In this study, we evaluated the potential use of a near-infrared fluorescent (NIRF) probe to detect and inhibit caspase 1/3 activation in long synthetic dsRNA and Alu RNA induced mouse models of GA. The overarching goal of this work is to translate a clinical imaging tool to visualize RPE cell death in vivo in dry AMD using standard ophthalmic camera equipment.
Wild-type C57BL/6 mice received subretinal injections of purified Alu-derived dsRNAs, poly I:C or appropriate controls. Additional controls included caspase deficient mice. Caspase 1/3 detection was accomplished utilizing a cell permeant NIRF coupled ZVAD peptide sequence administered intravitreously. Caspase inhibition was performed by co-administering similar antagonist tetramer peptides (naked and tagged) at the time of dsRNA treatment. A time-course of caspase 1/3 activation in the RPE/choroid was quantified using commercially available fluorometric plate assays. Fundus imaging, ZO-1 immunostained RPE/choroid flatmounts and spectral domain OCT were performed serially up to day 7.
Treatment with Alu-derived dsRNAs and poly I:C uniformly induced RPE degeneration in Wild-type mice but not in caspase deficient mice. Caspase activation was significantly increased in the RPE/choroid of treated mice 24 hours after treatment. Bio-imaging for active caspase 1/3 revealed large areas of NIRF signal that was not present in control treated Wild-type or caspase deficient mice. Flatmount analyses revealed significant disruption of ZO-1 immunofluorescence pattern and strong NIRF signal in these lesions. Co-administration of caspase inhibitors rescued treated eyes from geographic RPE cell loss and extinguished NIRF probe signal.
Caspase 1/3 activation is present during the RPE cell death in these mouse models of GA. Non-invasive detection of caspase activation in the RPE in vivo is feasible with a custom engineered NIRF bio-probe that can be visualized using common ophthalmic imaging equipment. The bio-probe may also inhibit caspase activation and thus may serve a dual purpose of detection and arrest of RPE loss secondary to cytotoxic dsRNA accumulation in the human condition of advanced dry AMD.
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