Annexin-V Imaging of Degenerating Axons Following Experimental Anterior Ischemic Optic Neuropathy

Abstract

 

Purpose

 

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.

 

Methods

 

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.

 

Results

 

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.

 

Conclusions

 

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.

 

 

In vivo and in vitro images of annexin-V labeled axons.(A) & (B)An example of annexin-V labeled axons in retinal whole mount preparations 7 days post-AION. (C) In vivo image of retina 7 days post-AION, same eye as (B).

 

 

Analysis of fragmentation pattern. (A) Axons indicated by arrows were traced. There is a striking punctate pattern of degeneration. (B) and (C) Fluorescence along the axons traced in (A). (B) corresponds to the left axon, (C) corresponds to the right axon. The plot shows peaks that correspond to the puncta, which could be useful in quantifying this pattern.

 

Keywords: 531 ganglion cells • 426 apoptosis/cell death • 552 imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)