Optic Nerve Crush Induces a Reactive Oxygen Species-Sensitive Reporter Gene in Retinal Ganglion Cells

Abstract

Purpose:: Retinal ganglion cells (RGCs) die as a result of axonal injury in a variety of optic neuropathies. We previously showed that there is a superoxide burst in RGCs following axotomy in vitro (Lieven CJ et al; IOVS 47:1477, 2006), suggesting that superoxide induction is an intracellular signaling event preceding RGC apoptosis. To determine whether these in vitro findings could be confirmed in vivo, we used a transgenic reporter mouse line.

Methods:: Mice transgenic for the human placental alkaline phosphatase (hPAP) reporter gene coupled in cis to the antioxidant response element (ARE) underwent unilateral optic nerve crush or intravitreal injection of H₂O₂ (100 µM). The contralateral eye was untreated. At various times following treatment, eyes were rapidly enucleated, fixed, retinas flat mounted, and processed immunohistochemically using rabbit anti-hPAP and AlexaFluor 488 goat anti-rabbit antibody.

Results:: hPAP expression was observed at 5 days after crush, but not 1 or 3 days. Staining was limited to the ganglion cell layer in crushed eyes, with none present in control eyes. This staining pattern differed from H₂O₂ treated eyes, which showed diffuse staining of cells in all retinal layers.

Conclusions:: These data provide the first in vivo evidence for reactive oxygen species induction in RGCs following axotomy.

Keywords: ganglion cells • oxidation/oxidative or free radical damage • signal transduction