Aims: To fully characterise the RGC-5 cell line and determine whether it would be a suitable substitute for primary RGC cell culture. To optimise a CNTF Nogo-P4 inhibitory assay and establish whether (a) CNTF alone is capable of stimulating RGC neurite outgrowth and survival, or (b) an increase in intracellular cAMP is required for CNTF to be effective. To determine whether recombinant AAV2 viral constructs were capable of producing detectable levels of CNTF in HEK-293 transfected conditioned media. To optimise AAV2-eGFP delivery and establish whether AAV2-CNTF-hrGFP and AAV2-CNTF-shRhoA-hrGFP could promote RGC survival and regeneration after optic nerve crush surgery. Methods: The RGC-5 cell line was characterised using semi-quantitative PCR, sequencing and immunocytochemistry. RGC-5 cells were screened for a selection of neuronal, glial, progenitor, oligodendroglial lineages and cone photoreceptor cell markers to identify the cells origin. Retinal cultures were treated with recombinant CNTF and/or Forskolin to promote RGC neurite outgrowth and survival - this was quantified after 3 d. Retinal wholemounts were prepared to assess GFP transduction and survival after intravitreal delivery of AAV2-eGFP. Axonal regeneration and RGC survival were assessed through histological examination of optic nerves and retinal sections. Results: The RGC-5 cell line predominantly expressed oligodendroglial lineage markers and only weakly expressed \(\beta\)III-Tubulin mRNA. RGC-5 cells did not express mRNA for many of the phenotypic markers of RGC. CNTF was effective at stimulating RGC neurite outgrowth without the need for cAMP elevation - furthermore recombinant CNTF could disinhibit Nogo-P4 treated RGC in vitro. GFP transduction was low when injected alone, however, when administered with Pronase-E there was a significant increase in GFP expression. AAV2-CNTF-hrGFP and AAV2-CNTF-shRhoA-hrGFP did not promote RGC survival or regeneration 23 d post optic nerve crush. Conclusions: RGC-5 cells are not an appropriate substitute for primary retinal cell culture in vitro as they express many of the same markers as oligodendrocyte progenitors. CNTF is capable of stimulating RGC neurite outgrowth without an additional elevation of cAMP. AAV2-mediated GFP expression could be enhanced through the partial digestion of the inner limiting membrane - this seems to be the major obstacle in achieving optimal AAV2 transduction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:558918 |
| Date | January 2012 |
| Creators | O'Neill, Jenna Teri |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/3664/ |
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