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Hydrocortisone stimulates neurite outgrowth from mouse retinal explants by modulating macroglial activity.

ABSTRACT: PURPOSE:There is mounting evidence that retinal ganglion cells (RGCs) require a complex milieu of trophic factors to enhance cell survival and axon regeneration after optic nerve injury. The authors' goal was to examine the contribution of components of a combination of hormones, growth factors, steroids, and small molecules to creating a regenerative environment and to determine if any of these components modulated macroglial behavior to aid in regeneration. METHODS:Postnatal day 7 mouse retinal explants embedded in collagen were used as an in vitro model of neurite regeneration. Explants were treated with the culture supplements fetal bovine serum, N2, and G5 and a mixture of G5 and N2 components, designated enhanced N2 (EN2). Explants were evaluated for neurite outgrowth over 7 days in culture. The effects of each treatment were also evaluated on cultured RGCs purified by Thy1 immunopanning. Immunohistochemistry and qPCR analysis were used to evaluate differences in gene expression in the explants due to different treatments. RESULTS:EN2 stimulated significant neurite outgrowth from explants but not from purified RGCs. Elimination of hydrocortisone (HC) from EN2 reduced the mean neurites per explant by 37%. EN2-treated explants demonstrated increased expression of Gfap, Glul, Glt1, Cntf, Pedf, and VegfA compared with explants treated with EN2 without HC. Subsequent experiments showed that increased expression of Cntf and Glul was critical to the trophic effect of HC. CONCLUSIONS:These data suggest that the HC in EN2 indirectly contributed to neurite outgrowth by activating macroglia to produce neurotrophic and neuroprotective molecules.

SUBMITTER: Toops KA

PROVIDER: S-EPMC3704200 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Hydrocortisone stimulates neurite outgrowth from mouse retinal explants by modulating macroglial activity.

Toops Kimberly A KA Berlinicke Cynthia C Zack Donald J DJ Nickells Robert W RW

Investigative ophthalmology & visual science 20120424 4

<h4>Purpose</h4>There is mounting evidence that retinal ganglion cells (RGCs) require a complex milieu of trophic factors to enhance cell survival and axon regeneration after optic nerve injury. The authors' goal was to examine the contribution of components of a combination of hormones, growth factors, steroids, and small molecules to creating a regenerative environment and to determine if any of these components modulated macroglial behavior to aid in regeneration.<h4>Methods</h4>Postnatal day ...[more]

PMID: 22395888

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Project description:We have undertaken a genome-wide study of transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root ganglia (DRG) during a time course of neurite outgrowth in vitro. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection. Comparison across both models revealed many genes with similar gene expression patterns during neurite outgrowth. These patterns were minimally affected by exposure to the potent inhibitory cue Semaphorin3A, indicating that this extrinsic cue does not exert major effects at the level of nuclear transcription. We also compared our data to several published studies of DRG and SCG gene expression in animal models of regeneration, and found the expression of a large number of genes in common between neurite outgrowth in vitro and regeneration in vivo. Experiment Overall Design: We wished to determine the transcriptional profiles of neurons undergoing neurite outgrowth in vitro. We were particularly interested in finding genes whose expression is generally associated with the process of neurite outgrowth, rather than with cell type-specific effects. Thus, in order to avoid focusing on transcripts unique to one tissue type versus another, we used a comparative strategy to look for effects that were common to two tissue types and therefore more likely to be involved in the general process of neurite outgrowth. While these explants contain multiple cell types, we felt this was preferable to the more disruptive conditions required to dissociate neurons or obtain a pure neuron population. To this end, we monitored gene expression in cultured explants from SCG and DRG using DNA microarrays. We initiated our studies by culturing embryonic day 13 (E13) mouse SCG in vitro and harvesting tissue for RNA isolation at time points from 2 to 65 hours. Time points were selected to detect both fast, short-term responses (2, 5 and 12 hours), as well as sustained, long-term changes (24, 40, and 65 hours). Samples were hybridized to Affymetrix MG-U74v2 A and B microarrays, with RNA from acutely dissected explants serving as a baseline reference. We followed these experiments with a parallel analysis of a more heterogeneous tissue type, the DRG, which is more frequently used than SCG for in vivo studies of neurite regeneration. Cervical and upper thoracic DRG from E12 embryos were cultured with NGF (the same trophic support as in SCG cultures), harvested at time points from 2 to 40 hours, and hybridized to Affymetrix MOE 430A microarrays.

Dataset Information

Hydrocortisone stimulates neurite outgrowth from mouse retinal explants by modulating macroglial activity.

Publications

Hydrocortisone stimulates neurite outgrowth from mouse retinal explants by modulating macroglial activity.

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