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Y. Hu, J. E. Torres-Munoz, J. L. Goldberg; Gene Expression Analysis of Regenerating vs. Non-Regenerating Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):641.
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© ARVO (1962-2015); The Authors (2016-present)
Most retinal ganglion cells (RGCs) die after optic nerve transection. Peripheral nerve grafting can enhance RGC survival; however, only 10% of surviving RGCs grow axons into peripheral nerve graft. To identify specific genes that may be important for RGC regeneration after optic nerve transection and peripheral nerve graft, in the present study we examined the gene expression differences between regenerating and non-regenerating RGCs in vivo.
RGCs from adult female Sprague-Dawley rats were retrogradely labeled from the superior colliculus with 4% Fluorogold. 1 week later, the left optic nerve was completely transected, and a piece of peripheral nerve was grafted to the end of the optic nerve. Rats were allowed to survive for 2 weeks. 3 days before euthanasia, regenerating RGCs were retrogradely labeled with Fluororuby injected into the distal end of the peripheral nerve graft. Fluororuby+/Fluorogold+ regenerating RGCs and Fluororuby-/Fluorogold+ surviving, non-regenerating RGCs were isolated by laser capture microdissection (LCM) or fluorescence-activated cell sorting (FACS). RNA was isolated and RT-PCR and gene array analysis was performed comparing these two samples.
We successfully labeled RGCs with Fluorogold and Fluororuby. We found that approximately 5-10% of surviving RGCs regenerated into the peripheral nerve grafts, as previously published. RGCs were successfully identified in frozen sections by LCM, and RNA isolated from laser-captured RGCs was positive by RT-PCR for housekeeping genes. Gene array analysis is underway.
The present study could help design strategies addressing the >90% of surviving RGCs that normally do not regenerate, and make a significant step forward in our approach to neuroprotection and axon regeneration.
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