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Vincent Pernet, Sandrine Joly, Noémie Jordi, Martin E Schwab; Three-dimensional analysis of transparent optic nerves reveals enhanced regeneration and reduced branching of axons in EphA4 KO mice after traumatic lesion.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2967.
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© ARVO (1962-2015); The Authors (2016-present)
In the present study, we investigated the influence of the guidance molecules EphA4 and ephrinA3 on retinal ganglion cell (RGC) survival and the regeneration of injured optic nerve axons in adult mice.
Axonal regeneration was examined in 3D in the injured mouse optic nerves of wild-type (WT), EphA4 knock-out (KO) and ephrinA3 KO C57BL/6 mice. Intraorbital traumatic lesion of the optic nerve was performed with a 9.0 suture. Thirteen days later, optic nerve axons were anterogradely traced by injecting cholera toxin β subunit coupled to alexa 594 (CTb-594) in the vitreous space. The day after, animals were intracardially perfused with 4% of paraformaldehyde, optic nerves and retinal flat-mounts were processed for axonal growth and RGC survival analysis respectively. To allow 3D analysis of labeled axons, optic nerves were dehydrated in ethanol and cleared in a solution of benzylbenzoate/benzyl alcohol. CTb-594-stained axons were imaged by confocal microscopy and reconstructed in 3D with the Imaris software (Bitplane). The number of regenerating axons, the directionality and branching of individual axons were examined. The density of surviving RGCs was calculated on retinal flat-mounts after immunostaining for β3tubulin.
The number of regenerating axons was markedly increased in EphA4 KO optic nerve compared with WT controls, at 100, 200, 300, 400, 500 μm from the lesion site. In contrast, ephrinA3 KO mice did not show more growing fibers than WT littermates. The frequency of axonal U-turns was not different between the 3 experimental groups. However, the number of branched axons was reduced in EphA4 KO relative to WT optic nerves. The measurement of growth-associated protein mRNA (Sprr1a, Gap-43 and ATF3) by qRT-PCR did not show stronger growth program activation in retinal lysates from injured EphA4 KO mice. In EphA4 optic nerves, glial fibrillary acidic protein (GFAP)-positive astrocytes retracted from the injury site at 5 days post-lesion, suggesting that decreased gliosis in the optic nerve may enhance RGC axon sprouting after trauma. The survival of RGCs was not significantly affected in animals deprived of EphA4 or ephrinA3.
Our results suggest that EphA4 negatively regulates axonal regeneration in a mouse model of optic nerve trauma, presumably by reducing the glial scar formation.
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