Retinal Ganglion Cell and Axonal Loss After Intraretinal Axotomy by Diode-Pumped Laser Combined With Confocal Scanning Laser Ophthalmoscopy

Abstract

Purpose: : Axotomy of retinal ganglion cells (RGC) causes cell death by apoptosis and induces Wallerian (orthograde) and retrograde axonal degeneration. Confocal scanning laser ophthalmoscopy (CSLO) has been used to assess RGC soma loss after optic nerve transection. We combined a dual laser CSLO with a diode-pumped solid-state (DPSS) laser to assess the time course of RGC axonal and soma loss after intraretinal axotomy in the rat in vivo.

Methods: : A 532 nm DPSS laser was mounted on an aluminium optical breadboard positioned in front of the CSLO telescope lens. . Using a 30/70 beam splitter, the 532 nm DPSS laser spot was imaged on the retina while RGC axon bundles were visualized with the 488 nm CSLO laser. At various times after application of DPSS laser burns, the following were quantified: (1) Axonal loss, measured by red-free imaging or CMFDA labeling (Kanamori et al, IOVS, in press); (2) the size of the laser burn at the nerve fiber and RPE layers; (3) counts of RGC somas retrograde labeled with DiR at 300 µm and 900 µm away from the edge of the laser lesion.

Results: : DPSS laser burns resulted in reproducible axon loss distal and proximal to the site of injury. Axonal loss and the size of the laser lesion correlated with the duration of the burn. Axonal loss was complete at 3 weeks after intraretinal axotomy and could be confirmed by immunostaining retinal whole mounts for neurofilaments. Imaging of CMFDA labeled axons demonstrated Wallerian degeneration distal to the laser burn.

Conclusions: : Intraretinal axotomy by DPSS laser combined with multilaser CSLO imaging can be used to study the kinetics of RGC soma loss and axon loss degeneration.

Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • apoptosis/cell death • imaging/image analysis: non-clinical