Optic Nerve Regeneration and Target Recognition in Postnatal Mice

Abstract

Abstract: : Purpose: The optic nerve regenerates poorly after injury in postnatal mammals. Recent data from our group suggest that loss of Bcl–2 expression by RGCs and activation of glial scarring are two important elements of optic nerve regenerative failure in postnatal mice. Concurrent induction of Bcl–2 and suppression of glial scarring using a genetic mouse model reversed the failure of optic nerve regeneration and led to robust regrowth of severed optic nerve fibers up to 2 weeks of age. In this study, we asked whether regenerating axons reach the appropriate targets and establish functional connectivity in the brain. Methods: Optic nerve crush procedure was carried out in Bcl–2 transgenic mice that are also deficient for glial fibrillary acid protein (GFAP) and vimentin (Bcl–2tg/GFAP–/–Vim–/–). Age–matched GFAP–/–Vim–/– mice were used as controls. Immediately following the optic nerve crush, an anterograde tracer cholera toxin B subunit conjugated with fluorescein or rhodomin (CTB) was injected into the vitreous to reveal axon trajectory after injury. Mice were sacrificed at 4 to 8 days after crush and perfused with saline followed by 4% paraformaldehyde. The optic nerve and brain were removed, post–fixed for 24 hr, cryoprotected, and sectioned. Optic nerve sections were then reacted with primary antibodies against GAP–43 or neurofilament. Functional connectivity of regenerating axons was determined by physiological recording in brain slices and by examination of pupillary reflex in mice. Results: Both the CTB labeling and immunohistochemistry revealed robust axonal regeneration and brain innervation in Bcl–2tg/GFAP–/–Vim–/– mice, but not in GFAP–/–Vim–/– mice. The majority of regenerating axons grew along the optic tract ipsilateral to the injury and reached the lateral geniculate nucleus (LGN) and superior colliculus in the same side after entering the brain. Aberrant projections of regenerating axons were also observed in other parts of the brain, including external capsule, mammilary peduncle and so forth. Only immature synaptic activities were recorded from the brain slices containing the LGN. Conclusions: These findings further demonstrate the essential role of Bcl–2 and glial activity in the regulation of optic nerve regeneration. However, the data also suggest that inducing synapse formation and functional recovery after nerve regeneration may remain to be a challenge.

Keywords: regeneration • ganglion cells • transgenics/knock-outs