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Z. Fan, K. Cho, M. Sofroniew, D. Chen; Optic Nerve Regeneration in Adult Mice Overexpressing Bcl–2 and Selectively Eliminating Reactive Astrocytes via Genetic Approaches . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1580.
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© ARVO (1962-2015); The Authors (2016-present)
Optic nerve losses its ability to regenerate in adult mammals. Our previous data showed that loss of Bcl–2 expression in retinal ganglian cells (RGCs) and appearance of reactive astrocytes are two elements of optic nerve regenerative failure. Overexpressing Bcl–2 in neurons and deleting glial fibrillary acidic protein (GFAP) and vimentin in astrocytes successfully sustained the regenerative ability of RGC axons up to 2 weeks in young mice (but didn't in adult mice). Here, we asked: (1) Is the failure of optic nerve regeneration in the adult mutant mice due to incomplete suppression of astrocyte activation after deleting GFAP/Vimentin? Or (2) Does mature CNS present other inhibitors to prevent optic nerve regeneration? To answer this, we introduced another transgenic mouse line that enables selective elimination of reactive astrocytes.
Double transgenic (Bcl–2tg /GFAP–TKtg) mice were produced by crossing Bcl–2 transgenic (Bcl–2tg) male with female mice carrying thymidine kinase (TK) transgene specifically in astrocytes (GFAP–TKtg). Optic nerve crush procedure was performed in wildtype, Bcl–2tg, GFAP–TKtg and Bcl–2tg /GFAP–TKtg mice aged 8–12 weeks old. Anterograde tracer CTB or adeno–associated virus carrying enhanced green fluorescence protein (AAV–EGFP) was injected into the vitreous cavity to label axons. Ganciclovir (GCV) was administered through osmotic mini–pump at 100mg/kg/day for 7 days to selectively ablate replicating reactive astrocytes in mice carrying TK transgene. Ten days after surgery, optic nerve and brain sections were prepared. GAP–43 and GFAP immunofluorescence labeling were performed to assess axon regeneration and astrocytes elimination, respectively.
In mice carrying TK transgene, reactive astrocytes were eliminated around the crush site following GCV administration. However, only in Bcl–2tg /GFAP–TKtg mice, moderate axon regeneration was observed. The farthest regenerating axons reached 3–4 mm distal to the crush site, with an estimated growth rate of 300–400µm/day. Only degenerating axons were observed in all the other mice.
These findings further demonstrate the essential role for Bcl–2 and reactive astrocytes in the regulation of optic nerve regeneration. The data suggest that reactive astrocytes are not the only factor contributing to the inhibitory environment in adult CNS. Other inhibitors, such as myelin–related inhibitors, may also participate in preventing optic nerve regeneration.
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