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Dhanesh Amarnani, Eric Kim, Patricia A D'Amore, Leo A Kim; Chloroquine-induced Retinal Toxicity In Vitro and In Vivo. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1608.
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© ARVO (1962-2015); The Authors (2016-present)
To develop and characterize a murine model of chloroquine-induced retinal toxicity and to determine the mechanisms of chloroquine toxicity
One intravitreal injection of chloroquine (2 mg/kg/2 ul) was administered into adult mice. After one week effects were evaluated by fundus photography, OCT, infrared reflectance, autofluorescence, ERG and by light and electron microscopy. Levels of cell death pathway and anti-oxidant genes in RPE and neurosensory retina were examined by quantitative RT-PCR. ARPE-19 and 661W cells were treated with a dose range of chloroquine (10-500 uM) for 24 hr and cell death was measured by LDH release. The role of lysosomal enzymes, cathepsins and cell death mediators was examined using specific cell permeable cathepsin, caspase, and RIP1 kinase inhibitors.
Fundus and infrared reflectance revealed areas of retinal atrophy and subretinal debris. OCT demonstrated disruption of the RPE and ellipsoid layers. Pinpoint autofluorescence was evident in affected areas. ERG exhibited a decreased scotopic response in both the A and B-waves. Histopathology showed loss of the RPE and disrupted outer segments, OPL and photoreceptors. Phalloidin staining of RPE flat mounts confirmed patches of RPE cell loss and cell enlargement. RPE mRNA levels of BAX, NLRP2, NLRP3, ASC, and cathepsins were significantly upregulated (2-4 fold), suggesting the activation of caspase-dependent cell death pathways. Levels of BAX, RIP3, and GPX4 mRNA were increased primarily within the neurosensory retina by 2-4 fold and 10-fold, respectively, pointing to apoptosis, necroptosis and oxidative stress-mediated photoreceptor cell death. Chloroquine induced cell death in ARPE-19 and 661W cells in a dose-dependent manner; 500 uM chloroquine led death of 30-40% ARPE-19 cells and 40-50% 661W cells at 24 hr. In ARPE-19 cells, inhibition of caspase-3, 8 and 9(mediators of apoptosis) led to a 50, 60 and 30% decrease in cell death, respectively, whereas caspase-1(a mediator of pyroptosis) inhibition resulted in a 30% decrease in cell death. Inhibition of RIP1 kinase did not significantly decrease cell death.
Phenotypic and functional retinal changes induced by chloroquine in the mouse are analogous to those observed in human with chloroquine or hydroxychloroquine retinal toxicity. Chloroquine induces cell death by apoptosis and pyroptosis but does not involve necroptosis, making it distinct from the mechanism of tamoxifen.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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