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Jacky M. K. Kwong, Celia Hoang, Reshil T. Dukes, Richard W. Yee, Brian D. Gray, Koon Y. Pak, Joseph Caprioli; Bis(Zinc-Dipicolylamine), Zn-DPA, a New Marker for Apoptosis. Invest. Ophthalmol. Vis. Sci. 2014;55(8):4913-4921. doi: 10.1167/iovs.13-13346.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the labeling of apoptotic cells with a molecular probe of bis(zinc(II)-dipicolylamine) (Zn-DPA) conjugated with a fluorescent reporter in a rat model of retinal ganglion cell (RGC) degeneration induced by N-methyl-D-aspartate (NMDA).
Adult Wistar rats were given unilateral intravitreal injections of 3 μL 40 mM neutralized NMDA and euthanized at 1, 2, 4, 24, and 48 hours. One hour before euthanasia, 3 μL Zn-DPA conjugated with fluorescein (Zn-DPA 480) was intravitreally injected. Prelabeling of RGC with retrograde fluorogold (FG), TUNEL, and immunohistochemistry with III β-tubulin and vimentin were performed.
Fluorescence labeling of Zn-DPA 480 was observed in the retinas from 1 hour up to 24 hours after NMDA injection, whereas the labeling was reduced at 48 hours postinjection. At both 4 and 24 hours postinjection, most Zn-DPA 480–positive cells in the RGC layer were labeled by FG and III β-tubulin. The number of TUNEL-positive cells increased from 4 to 24 hours. At 24 hours, 95.7% of Zn-DPA 480–positive cells were TUNEL positive, whereas 95.1% of TUNEL-positive cells were Zn-DPA 480 positive. The numbers of Zn-DPA 480–positive cells at 1 and 2 hours after NMDA injection were significantly higher than TUNEL.
Our findings demonstrate that intravitreal injection of fluorescent Zn-DPA 480 labels retinal neurons undergoing apoptosis and that recognition of exposed phosphatidylserine appears earlier than detection of DNA fragmentation, indicating the potential of Zn-DPA as an imaging probe for tracking degenerating retinal neurons.
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