Jacky M K Kwong1, Celia Hoang1, Reshil T Dukes1, Richard W Yee2, Brian D Gray3, Koon Y Pak3, Joseph Caprioli1. 1. Jules Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States. 2. Cizik Eye Clinic, Hermann University Eye Associates, Houston, Texas, United States. 3. Molecular Targeting Technologies, Inc., West Chester, Pennsylvania, United States.
Abstract
PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
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