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Kimberly A. Toops, Albee Messing, Robert W. Nickells; GFAP Expression Supports Neurite Outgrowth In Retinal Explants. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1858.
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Increased expression of glial fibrillary acidic protein (GFAP) by macroglial cells has been correlated with inhibition of axonal regeneration of CNS neurons in co-culture experiments. Previously, we showed that mouse retinal explants grown in media containing hydrocortisone (HC) had more neurite outgrowth and higher GFAP promoter activation and mRNA levels than explants grown in media without HC. We examined the direct impact of GFAP expression on the ability of PN7 explants to generate neurites by titrating GFAP expression with transgenic mice expressing no GFAP to 20 times normal GFAP.
Neurite outgrowth from PN7 mouse retinal explants in collagen matrix was monitored for one week. Explants were grown in media supplemented with either 10% FBS, 10% N2 (Invitrogen), 10% EN2 (N2 with bFGF, EGF, biotin, and HC), or 10% EN2-HC (EN2 without HC). Mice were from four transgenic strains that expressed increasing levels of GFAP: no or reduced GFAP (tm1MES-/- or +/-) < moderately increased GFAP (170.1) < highly increased GFAP (170.2) < very highly increased human GFAP (Tg73.7). GFAP expression from samples of retina or from pooled explant samples was measured using sandwich ELISAs. GFAP expression was normalized to total protein concentration for retinal samples or to average explant size for explant samples.
Consistent with our previous data, regardless of genotype, explants grown in EN2 had more GFAP and neurite outgrowth than explants grown in medias without HC (p<0.05). Regardless of media condition explants expressing the highest level of GFAP (170.2 and 73.7) exhibited the most neurite outgrowth (p<0.005). When neurite outgrowth was evaluated strictly as a function of GFAP level per explant, we observed that the worst growth conditions (65±10.4 neurites/explant) were obtained in the absence and lowest levels of GFAP (less than 0.01 pg/mm²), while the best growth conditions (123±12.8 neurites/explant) were obtained at the highest levels of GFAP expression (more than 0.5 pg/mm², p<0.002).
Increasing GFAP expression supports neurite outgrowth from retinal explants. This contradicts findings obtained by in vitro co-culture experiments.
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