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Natalie Pankova, Huiyuan Liang, David Sung Hyeon Baek, Xu Zhao, Hai Wang, Shelley R Boyd; Delayed Near-Infrared Analysis (DNIRA) is a Novel Technique That Permits Visualization of Rat Retinal Pigment Epithelium (RPE) Layer In Vivo. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2097.
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To develop a novel technique that permits in vivo detection of RPE loss in the rodent eye. Several forms of blinding eye disease are characterized by atrophy of the RPE that can be detected non-invasively in the clinic as patches of reduced (dark) Fundus Autofluorescence (FAF). However similar patches have not been described in pre-clinical rodent models of disease. We hypothesized that this is due to low autofluorescence in the young rodent eye, and that detection of RPE loss can be enhanced by pre-labelling this monolayer. Toxicity studies have described that RPE cells can internalize the near-infrared (NIR) dye indocyanine green (ICG) in vitro. We proposed that RPE labelling could be achieved in vivo with doses that did not elicit retinal toxicity.
ICG dye at doses of 0.35 and 5.0 mg/kg, or PBS was injected systemically in 46 Sprague Dawley (SD) rats, and the fundus evaluated prior to injection, and up to 1 month thereafter using in vivo imaging and bright flash electroretinography (ERG). Confocal scanning laser ophthalmoscopy (cSLO) was performed using the 830 nm channel to detect Infrared (IR) reflectance, and with the ICG angiography filters (795/810 nm) without angiography to detect ICG fluorescence. In a subset of animals, an RPE toxin was injected to induce RPE loss and observe the effect on the 795/810 nm signal. Rat RPE monolayers were cultured ex vivo to evaluate ICG uptake and release.
Using our newly developed technique that we have termed “Delayed Near Infrared Analysis (DNIRA),” we detected a speckled NIR fluorescent signal up to 1 month following a single tail-vein ICG injection that was in the same focal plane as the outer retina/RPE complex. This signal 1) was apparent only after ICG injection, 2) was dose-dependent, 3) required the presence of the ICG filters, 4) did not change the appearance of the IR reflectance channel, and 5) was lost following damage to the RPE. The ERG remained normal throughout, indicating no retinal toxicity. Rat RPE monolayers were shown to internalize ICG dye that can be released following permeabilization, suggesting the in vivo signal is due to internalization of the dye by RPE cells.
DNIRA following systemic ICG dye injection is a novel and safe non-invasive technique for in vivo labeling of the RPE layer in preclinical rodent models of eye disease.
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