The Influence Of Slits And EphrinA5on Adult Mouse Optic Nerve Regeneration

In adult mammals, central nervous system (CNS) injuries will cause permanent function deficits due to failure of axon regeneration. There are two major reasons for this phenomenon:the first one is the terminals of injured CNS axons fail to reform growth cones by activating cell intrinsic mechanism after injury; the second one is the inhibitory environment around injured CNS tissues, especially the dramatically up-regulated inhibitory extracellular matrix molecules during the inflammatory stages limit axon regeneration seriously after the CNS trauma. Some research have shown that deletion of phosphatase and tensin homologue (PTEN) or suppressor of cytokine signalling3(SOCS3) gene could activate related PTEN/mTOR and JAK/STAT pathway which would promote protein synthetic progress and axon regeneration. Furthermore, double knock out pten/socs3genes followed increasing the level of CNTF by protein eye injection enabled robust and sustained axon regeneration. Though many optic nerves crossed the lesion site and got a long-distance growth under this condition, most of them couldn’t grow longer when they reached the optic chiasm. This phenomenon triggers us to consider whether there were some inhibitory guidance molecules blocking the axon further re-growth.During mammalian CNS development, neuronal precursor cells have to migrate to their final destinations and axons have to navigate to their correct targets to establish normal connectivity. Neuronal migration and axon path-finding are guided by extracellular cues. In the research of mouse visual system, the Slits guidance molecules were proved that they could repel retinal axons in vivo and involved in axon path-finding, thereby, assisting to define the site in the ventral diencephalon where the optic chiasm forms. In addition, in the study of the possible roles of the Eph receptors in chiasm formation, the isolated hypothalamic inhibited retinal axons in vitro. Add the soluble EphA5-Fc to block the interactions between EphA and its ligands reduced the inhibition effects. It is well known that the major receptor of ephrinA5is EphA5, and it can repel the retinal ganglion cells (RGCs) axon growth. Together with the in vivo distributions, these results suggest the possible roles for EphA5and ephrinA5ligand in retinal axon growth path-finding and/or reorganization during optic chiasm formation.In the first part of this thesis, I used recombinant adeno-associated virus to introduce gene expression in vivo. After injecting virus into PTENf/f or PTENf/f/SOCS3f/f mouse, I compared the different effects to mature RGC axon regeneration among the groups of single knock pten gene, double knock out pten/socs3genes and double knock out pten/socs3gene following scAAV1-CNTF injection. The results showed that there were not obvious differences between PTEN and PTEN/SOCS3knock out mouse. However, additional CNTF expression could increase the axon regrowth ability dramatically.The second part, the potential function of Slit proteins and ephrinA5protein to RGC axon regeneration in vitro system was explored. At the first, the retinas got from E16mouse were co-cultured with beads coated with Slit1,2,3and ephrinA5for3days. The results suggested that Slit1,2and ephrinA5could inhibit the retina axon growth. Then adult PTENf/f/SOCS3f/f mouse were injected scAAV2-Cre and scAAV1-CNTF to do co-culture with beads coated Slitl,2and ephrinA5separately. After4days growing, we could see Sit2and ephrinA5partly disturbed the axons regenerated from adult retinas. These data suggest that the guidance molecules Slit2and ephrinA5potentially affect the mature axon regeneration after optic nerve injury.