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C. Perez-Marín, P. Sobrado, M. Salinas, I. Canovas, M. Vidal-Sanz, M. Agudo; Retinal Localization and Protein Regulation of Death Related Genes After Optic Nerve Transection and Crush. Invest. Ophthalmol. Vis. Sci. 2007;48(13):638.
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© ARVO (1962-2015); The Authors (2016-present)
To understand the molecular mechanisms underlying retinal ganglion cell (RGC) death following optic nerve (ON) injury (transection and crush). By means of DNA arrays, injury specific regulated genes along time postlesion have been identified recently (Agudo y cols., 2006, IOVS. 47: E-Abstract 4896, 2005, IOVS 46: E-Abstract 3086.and unpublished data.). Here, we investigate if some of the genes associated with death and inflammatory response are also regulated at the protein level.
We selected 3 time-points (12h, 48h and 7d post-lesion) after intraorbital optic nerve Otransection (IONT) or optic nerve crush (IONC) and a control situation (n=4, per treatment and time point). Retinas were either fresh dissected for protein extraction and western blotting, or perfused, frozen and cut on the cryostat on 15µm thick radial sections. RGCs were labelled with Fluorogold (FG) to univocally identify them in the immunofluorescence experiments. Sections and westerns were incubated with primary antibodies against TNFR1, caspase11, STAT1 and cathepsin C and revealed with secondary antibodies fluorescently and HRP labelled, respectively.
Western blotting assays revealed upregulation of all the proteins analysed (TNFR1, caspase 11, STAT1 and cathepsin C) and this is consistent with RNA up-regulation observed in the array experiments. Immunohistofluorescence to radial sections of control and injuried retinas showed that all these proteins but STAT1 were expresed by RGCs as seen by their co-localization with FG.
The RNA regulation of all the genes analyzed in this study is followed by the same protein regulation. Furthermore, the studied proteins are implied in apoptosis death triggered by either inflammatory response or severe insult. This up-regulation, together with our immunohistofluorescence data suggests a functional role for these genes in axotomy induced RGC death.
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