The Presence of Calcium Significantly Enhanced Annexin-V-Labelling of Degenerating Axons after Experimental Anterior Ischemic Optic Neuropathy

Abstract

Purpose: Annexin-V binds to exposed phosphyltidylserine on the extracellular membrane in a calcium-dependent fashion and has been shown to label degenerating retinal ganglion cell axons and soma after experimental anterior ischemic optic neuropathy (AION). Because the labeling of degenerating axons with annexin-V is relatively labile, in this study, we determine whether inclusion of calcium in different experimental steps helps to enhance axonal labeling.

Methods: We induced optic nerve head ischemia in adult C57BL/6 mice using laser-assisted photochemical thrombosis. After one week, we performed intravitreal injection of annexin-V-A488 and imaged the retina 2-3 hours later using confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT). Then, we prepared retinal whole mount in calcium-free or 2.5 mM CaCl₂ conditions and imaged the retinae using fluorescence microscopy for up to 7 days after mounting.

Results: On the day of annexin-V-A488 labeling, degenerating axons were easily seen in both Ca²⁺-containing (N = 30) and Ca²⁺-free (N = 10) conditions. Annexin-V-A488 labeling of degenerating axons one week after ischemia correlated well with the presence of optic nerve swelling on day-1 as measured by OCT. No annexin-V-A488 labeling of axons was seen in the control eyes (N = 6), which showed no swelling on OCT. Inclusion of Ca²⁺ to the fixative, wash, and mounting solutions significantly improved the persistence of annexin-V-A488 signal one to seven days after labeling. In contrast to the difficulty of preserving axonal labels, bright annexin-V-A488 signal on degenerating soma was easily seen even one week after labeling. We used these brightly labeled soma to test the lability of annexin-V-A488 labeling over 5, 10, and 20 min of light exposure and found a dramatic, time-dependent decrease in fluorescence signal, consistent with the relative lability of the annexin-V-A488 signal.

Conclusions: Annexin-V-A488 brightly labels degenerating retinal ganglion cell axons one week after experimental anterior ischemic optic neuropathy, and the presence of 2.5 mM Ca²⁺ during retinal tissue preparation helps preserve this relatively labile label. During microscopy, even minutes of bright light exposure can quench annexin-V-A488 signal, and every effort should be made to shield the tissue from bright light during experimentation.