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K. A. Toops, A. Messing, R. W. Nickells; The Effect of Hydrocortisone on Neurite Outgrowth and Gfap Expression in Retinal Explants. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6106.
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© ARVO (1962-2015); The Authors (2016-present)
We examined the contribution of components of a well-defined minimally complex combination of hormones, growth factors, steroids, and small molecules to a regenerative environment for retinal ganglion cells after axotomy. We investigated if any components indirectly aided regeneration by reducing reactive gliosis in the macroglia surrounding the RGCs.
Neurite outgrowth from PN7 and PN14 mouse retinal explants in a collagen matrix was monitored for one week. The effect of different components in the media on reactive gliosis was examined using explants from dual transgenic mice expressing firefly luciferase under the control of the human glial fibrillary acidic protein promoter (hGFAP-fLuc) and Renilla luciferase under the control of the human glyceraldehyde 3 phosphate dehydrogenase promoter (hGAPDH-RLuc). Explants were assayed for changes in fLuc activity normalized to RLuc activity.
Using culture supplements G5 and N2 (Invitrogen) as starting points, we determined that a combination of components from both, termed enhanced N2 (EN2), stimulated substantial neurite outgrowth from PN7 explants, but had a less dramatic effect on PN14 explants. Removing hydrocortisone (HC) reduced neuritogenesis by 37% (P<0.02) in PN7 explants, but produced no change in PN14 explants. A glucocorticoid receptor inhibitor reduced neuritogenesis by 50% (P<0.02), but a mineralcorticoid receptor inhibitor had no effect. This indicated that HC exerted its effect through the glucocorticoid receptor, which is highly enriched in the retinal macroglia. HC was capable of modulating GFAP expression but this was dependent on the age of the explants. In PN14 explants HC decreased hGFAP-FLuc expression by 2 fold while in PN7 explants HC increased hGFAP-FLuc expression 2.5 fold.
We identified a base combination of molecules that increases the neuritogenic capacity of retinal explants. HC appears to exert its effect through the macroglia as indicated by its ability to modulate GFAP expression. GFAP transcript levels will be assessed with quantitative PCR to confirm these data. Mice expressing varying levels of GFAP, from GFAP knock-outs to GFAP over-expressers, will be used to test whether the amount of GFAP in the macroglia directly affects the regenerative capacity of RGCs.
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