In vivo Confocal Scanning Laser Imaging of Retinal Ganglion Cell Superoxide After Optic Nerve Transection

Abstract

Purpose: : We previously used in vitro and ex vivo imaging to demonstrate generation of superoxide within axotomized retinal ganglion cells (RGCs). We now describe the use of longitudinal in vivo imaging to study superoxide within retinal ganglion cells after optic nerve transection.

Methods: : The right optic nerves of Long-Evans rats were transected within the meninges by a transconjunctival approach, sparing the retinal circulation. Both eyes were intravitreously injected with hydroethidine (HEt), which reacts with superoxide to form a fluorophore with a distinct emission profile, to a final concentration of 100 µM. Retinal ganglion cells (RGCs) were identified with retrograde labeling by overlaying the superior colliculi with DiR (20 mg/ml). Confocal scanning laser imaging was performed with a Heidelberg Retina Angiograph 2 (HRA2) using the fluorescein channel for HEt and the indocyanine green channel for DiR. Results were confirmed by immunohistofluorescence.

Results: : HEt-positive cells could be imaged in vivo within the ganglion cell layer 1 day after optic nerve transection. The number of HEt-positive cells increased to a maximal level 4 days after transection. HEt-positive cells were not present in eyes untransected eyes. HEt-positivity co-localized with RGCs retrograde labeled with DiR. Intravitreal pegylated superoxide dismutase significantly reduced the number of HEt-positive cells (40.6 ± 18.5 vs. 4.8 ± 6.3; p < 0.001).

Conclusions: : An increase in RGC superoxide can be identified by confocal scanning laser ophthalmoscopy at an early stage after axonal injury.

Keywords: ganglion cells • apoptosis/cell death • imaging/image analysis: non-clinical