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Gun Ho Lee, M Ali Shariati, Jeffrey Ma, Yaping Joyce Liao; Annexin-V Imaging of Degenerating Axons Following Experimental Anterior Ischemic Optic Neuropathy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2689.
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Ischemic optic neuropathy is the most common acute optic neuropathy in adults older than 50 and leads to retrograde degeneration of retinal ganglion cells. We used annexin-V, an endogenous protein that binds to living cells undergoing apoptosis, to visualize dying retinal axons following ischemia.
We induced optic nerve head ischemia in adult C57BL/6 mice and Thy1-YFP mice using laser-assisted photochemical thrombosis. Thy1-YFP mice were imaged using cSLO and in vitro whole mount dissection within 1 week after ischemic insult. To visualize annexin-labeling of axons, we performed intravitreal injection of annexin-V-A488 one week after induction of AION and imaged the retina 2-3 hours later using confocal scanning laser ophthalmoscopy (cSLO). Then, we prepared retinal whole mount preparations. The retinae were imaged using fluorescence microscopy and quantified in a masked fashion.
At three-to-five days after AION, confocal scanning ophthalmoscopy and histologic analyses showed a decrease in Thy1-YFP expression in the RGC axons. One week after AION, the degenerating RGC axons exhibited dramatic axonal labeling of annexin-V-A488, a marker of apoptosis (N = 14, P = 0.003), with a punctate pattern of distribution with 5.8 ± 0.4 segments/100 µm or inter-segment interval of 20.1 ± 1.4 µm.
Annexin-V-A488 binding is an early marker of retinal ganglion cell axonal degeneration following ischemia. Degenerating retinal ganglion cell axons can be visualized in vivo and has a close correspondence to the whole mount retinal preparations. Study of the damaged axons revealed a fragmented pattern of annexin-V-A488 binding, similar to that of Wallerian degeneration.
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