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K.-S. Cho, D.-F. Chen; Promoting Optic Nerve Regeneration by Suppressing the Function of Nogo Receptor. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3243. doi: https://doi.org/.
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Optic nerve of adult mammal does not regenerate. Recently, we showed that lack of growth intrinsic factor such as Bcl-2, and the presence of injury-induced glial scars are the key factors blocking optic nerve regeneration in mice. Triple mutant (TM) mice over-express Bcl-2 (Bcl-2tg) and, simultaneously, deficit in glial fibrillary acidic protein (GFAP) and vimentin (Bcl-2tg/GFAP-/-Vim-/-) that results in impairment in glial scar formation supports optic nerve regeneration up to P14. It suggests the appearance of an additional barrier to block optic nerve regeneration after P14. The timing parallels to myelin maturation and peak expression of myelin-derived axon growth inhibitors. We hypothesize that blocking Nogo receptor (NgR) activity, a common receptor for myelin-derived growth inhibitors, may allow optic nerve regeneration in adult TM mice.
Adeno-associate virus (AAV) carrying a dominant negative form of NgR (AAV-NgRDN) or GFP (AAV-GFP) was injected into the vitreous of adult Bcl-2tg, GFAP-/-/Vim-/- double mutant (DM) and Bcl-2tg/GFAP-/-/Vim-/- (TM) mice. Six weeks after AAV injection, mice were anesthetized, and the optic nerve of right eye was crushed. An anterograde tracer, Cholera toxin B subunit (CTB), was injected into vitreous one week after nerve injury. Mice were sacrificed 2 week after nerve injury. The retinas and optic nerves were dissected and fixed in 4% paraformaldehyde, cryoprotected in 30 % sucrose, and embedded in OCT. Frozen retinal and optic nerve sections (10 um) were processed for immunohistochemistry using goat anti-CTB, mouse anti-neurofilament and rabbit anti-GAP43 antibodies followed by reactions with corresponding fluorescence-conjugated secondary antibody.
The mice receiving control AAV-GFP showed negative regeneration after optic nerve injury. AAV-NgRDN induced optic nerve sprouting posterior to the crush site in Bcl-2tg and DM mice. In TM mice, injection of AAV-NgRDN induced robust optic nerve regeneration.
Robust optic nerve regeneration can be induced in adult mice using a combinational approach that maintains the intrinsic axon growth capacity by overexpressing Bcl-2 and simultaneously blocks the growth inhibitory signals presented by scar and myelin-derived molecules.
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