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Dhanesh Amarnani, Leo A Kim, Patricia A D'Amore, Lindsay Wong; Chloroquine toxicity of the retinal pigment epithelium and retina in vitro and a mouse model of retinal toxicity. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6040.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the cell death mechanisms of chloroquine-induced toxicity of human cultured epithelial cells(RPE). To develop and characterize a murine model of chloroquine-induced retinal toxicity
ARPE-19 cells were cultured until confluence, treated with 500uM chloroquine for 24 hours and cell death was measured by lactate dehydrogenase(LDH) release. Cytoskeletal structure and lysosomal permeability were assessed using phalloidin and lysosomal-associated membrane protein(LAMP-1) staining, respectively. The roles of lysosomal enzymes cathepsin B and L were examined by blocking their activity using specific cell permeable inhibitors. The contribution of different cell death mediators was evaluated by specific inhibition of caspase-1, 3, 8, 9 and receptor-interacting protein(RIP) kinases. To develop an animal model of retina chloroquine toxicity, subretinal chloroquine (0.2 mg/kg) injections were performed. Phenotypic changes were evaluated using optical coherence tomography(OCT) and functional assessment was performed by electroretinography(ERG). Histological changes to the RPE were assessed.
500uM chloroquine induced 30-40% of cell death at 24 hours. Lysosomal permeabilization and cytoskeletal structure disruption were observed at 3 and 6 hours. Blockage of cathepsin B/L activity resulted in a significant decrease in cell death, indicating that lysosomal destabilization and resulting cathepsin release are upstream of these cell death pathways. Partial rescue of cell death was observed with caspases and RIP kinase inhibitors. Preliminary results from OCT demonstrate loss of the RPE layer and thinning of the outer retina within two weeks of injection. ERG revealed a decreased scotopic response in both the A and B-waves of the ERG waveform. H&E staining of retinal cross sections in chloroquine-treated mice revealed loss of RPE and disruption of outer segments. ZO-1 staining of RPE flat mounts demonstrated RPE cells death, enlarged cells and loss of cell junctions.
Chloroquine-induced cell death occurs through multiple cell death mechanisms. Inhibition of upstream activators such as cathepisn B/L may be a feasible approach to block multiple cell death pathways. Some of the phenotypic and functional changes observed in our mouse model are similar to those observed in human subjects with chloroquine toxicity, and may be model of RPE and retinal degeneration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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