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X. Liu, T. Ishimaru, D. Sretavan; Slit and EphB Axon Guidance Molecules in Optic Nerve Injury . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5221.
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Purpose: After optic nerve injury, retinal ganglion cell (RGC) axons initially extend growth cones in an aborted attempt to re-grow past the injury site. The molecular triggers of this early response and the subsequent failure of RGC axons to regenerate successfully are not well understood. Developing RGC axons respond to axon guidance molecules such as Slit and EphB to find their way in the embryonic visual system. Here, we examined whether axon guidance molecules are re-expressed in the injured adult optic nerve and are capable of influencing the growth of adult RGC axons. Methods: The optic nerves of adult mice (6-24 mos.) were crushed and the optic nerve injury site examined after 2-14 days. In situ hybridization was performed to determine expression of genes encoding the guidance molecules Slit1, 2, 3 and EphB1, B2, B3 as well as the Slit receptors, Robo1, 2. Expression of EphrinB1, B2, B3, which serve as receptors for EphB proteins, was examined by immunostaining. Retinal explants obtained from optic nerve crush animals were cultured and the responses of RGC axons to point sources of axon guidance molecules delivered by micropipettes were studied using time lapse microscopy. Results: Slit1 and EphB3 mRNA were detected in cells at the injury site beginning at day 4, and reached maximum expression at day 12. After optic nerve injury, RGCs expressed mRNA encoding the Slit receptors Robo1, 2 and anti-EphrinB3 immunoreactivity. In timelapse studies, EphB3 protein released from micropipettes triggered the turning of RGC axons (n=19) towards the pipette with a mean turning angle of 27.8+/-5 degrees while heat-inactivated EphB3 resulted in a mean turning angle of -1.1+/-4 degrees (n=14, p <0.001). Conclusions: mRNA encoding the axon guidance molecules Slit1 and EphB3 are expressed in the injured optic nerve. Adult RGC axons express the corresponding receptor proteins and respond to EphB3 by growing to a point source of this guidance molecule. Thus axon guidance molecules involved in embryonic development of the visual system are re-expressed in the adult optic nerve after injury and may influence RGC axon regenerative behavior after trauma.
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