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B. R. Schwechter, L. A. Levin; Optic Nerve Crush Induces a Reactive Oxygen Species-Sensitive Reporter Gene in Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):637.
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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.
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.
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.
These data provide the first in vivo evidence for reactive oxygen species induction in RGCs following axotomy.
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