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Natalie Pankova, Huiyuan Liang, Xu Zhao, Hai Wang, Shelley R Boyd; Immuno-DNIRA (ImmunoD) is a novel imaging technique that identifies ex vivo labelled macrophages in the eye with confocal Scanning Laser Ophthalmoscopy (cSLO). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4370.
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© ARVO (1962-2015); The Authors (2016-present)
Phagocytic macrophages (Mφs) are found in human disease and in pre-clinical models, but their spatial and temporal dynamics cannot be adequately addressed in serial tissue specimens. We previously developed Delayed Near-Infrared Analysis (DNIRA), a technique by which retinal pigment epithelium (RPE) cells can be viewed in vivo in the days following systemic indocyanine green (ICG) injection using cSLO. The purpose of this study was (1) to determine if Mφs can be safely labeled ex vivo with ICG and detected in the rat eye in vivo by cSLO imaging following their systemic injection, and (2) to determine their spatiotemporal change following retinal damage.
Peritoneal Mφs from Sprague Dawley rats were incubated in culture medium +/- ICG (0.25 mg/ml, 30 min), and viewed by white light and fluorescent scanning at 790 nm. Cells were then permeabilized with Triton-X100 to release the dye, and assayed by 790 nm spectroscopy. RPE and retinal damage was induced in rats by systemic injection of sodium iodate (vs saline control), and 107 ICG-labelled cells were injected per animal via tail vein. Sequential cSLO images were taken in the minutes and days following cell injection.
After ICG incubation, Mφs remain viable, take on a green colour and fluoresce. Cellular permeabilization reverses these findings and releases dye into solution, indicating it was internalized by the cells. Immediately following systemic injection of ICG-labelled Mφs, motile fluorescent spots can be detected in the fundus using the ICG excitation/emission filters of the cSLO. In control (saline) animals, cells are found almost exclusively within the retinal and choroidal vasculature. In toxin-treated animals, cells are seen both within the vasculature and extravasating into areas of outer retinal damage.
These data demonstrate that Mφs can be safely labelled in vitro with ICG, and following their systemic injection can be viewed in vivo both in a healthy and damaged fundus. Labelled cells preferentially localize to areas of outer retinal damage, suggesting they may be recruited during disease. We have termed this novel technique Immuno-DNIRA (ImmunoD). Future studies using ImmunoD will investigate the correlation of Mφ dynamics with disease progression.
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