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Jacky Man Kwong Kwong, Celia Hoang, Reshil Torrevillas, Joseph Caprioli, Richard Yee, Brian Gray, Jeffrey Mattis, Koon Pak; A New Marker for Detecting Retinal Ganglion Cell Apoptosis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4810. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To detect retinal ganglion cell (RGC) apoptosis using molecular probes comprising, bis(zinc(II)-dipicolylamine) (Zn-DPA) conjugated with fluorescent dyes, in N-methyl-D-aspartate (NMDA)-induced excitotoxicity in rats.
Adult Wistar rats, 3 months old, were given unilateral intravitreal injection of 3uL of 40mM neutralized NMDA in 0.1M phosphate buffered saline. Groups of animals were euthanized at 2, 4, 6, 9, 12, 24 and 48 hours after injection and their eyes were enucleated (N=4 per group). One hour prior to euthanasia, 3uL of 1mM Zn-DPA conjugated with either fluorescein (Zn-DPA480) or rhodamine (Zn-DPA550) was intravitreally injected. Zn-DPA binds to the anionic membrane of cells undergoing apoptosis and necrosis. For quantitative analysis, TdT-mediated biotin-dUTP nick end labeling (TUNEL) and immunohistochemistry of beta-tubulin (RGC marker) and vimentin (Muller cell marker) were performed on retinal sections. Prelabeling of RGC with retrograde dyes including fluorogold and DTMR was performed and the retinal flatmount was examined using fluorescence microscopy.
Remarkable fluorescence labeling of Zn-DPA480 and Zn-DPA550 was observed in the cells in the RGC layer from 2 hours up to 24 hours after NMDA injection. There was absence of Zn-DPA labeling at 48 hours post-injection and with all controls. At 4 hours post-injection, 53.1±10.1% of Zn-DPA 480 positive were beta-tubulin positive while 16.4±9.0% of Zn-DPA480 positive cells were vimentin positive. Prelabeling of fluorogold and DTMR confirmed that the majority of Zn-DPA480 and Zn-DPA550 positive cells found in retinal flatmount were RGCs. The TUNEL positive cells appeared at 4 hours and the number peaked at 18 hours post-injection but disappeared at 48 hours. At 4 hours post-injection, 90.1±4.6% of Zn-DPA480 positive cells were TUNEL positive while over 96.4±3.5% of TUNEL positive cells were Zn-DPA480 positive.
Our findings demonstrate that intravitreal injection of fluorescent Zn-DPAs labels RGCs undergoing apoptosis suggesting the potential as an imaging probe for tracking degenerating retinal neurons in vivo.
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