[Truncated abstract] During development of the visual system, topographic connections between the retina and the superior colliculus are established using guidance molecules. The EphA family of tyrosine kinase receptors and their ephrin-A ligands are important for establishing topography between the temporo-nasal axis of the retina and the rostro-caudal axis of the superior colliculus. After injury to the visual system via unilateral optic nerve transection, adult mammalian retinal ganglion cells fail to regenerate axons spontaneously to their main visual centre, which in rodents, is the superior colliculus. The EphA5 gradient is down-regulated from a temporalhigh to nasallow gradient to a uniform low level in the few surviving retinal ganglion cells, but ephrin-A2 is upregulated back to a significant rostrallow to caudalhigh gradient in the superior colliculus, similar to that seen during development. In this thesis, a number of experiments have been undertaken to investigate further how EphA5 and ephrin-A2 are regulated after injury and how they may play a role once regeneration has been encouraged through surgical intervention. In the first study, targeted unilateral retinal laser lesions were used to ablate either dorso-nasal or ventro-temporal quadrants of the retina. ... Surviving and regenerating retinal ganglion cells in the retina, and axons in the optic nerve, were analysed. The data suggest that metallothionein-I/II increases axonal regeneration through the optic nerve injury site but, at the dose administered, had no neuroprotective effects on retinal ganglion cells. This thesis provides further insight into the response of guidance molecules to injury, and the potential of metallothionein-I/II as a neuroregenerative factor in the adult mammalian visual system. The regulation of both EphA5 and ephrin-A2 through transsynaptic connections may be a response common to other guidance molecules. Such connectivity now needs to be studied further to understand how it may impact on various treatments designed to increase re-connectivity after other brain injuries, including stroke. The ectopic expression of ephrin-A2 at the insertion site of a peripheral nerve graft in the superior colliculus, implicate this guidance molecule in the glial scar for the first time. Therefore, to overcome inhibition by the glial scar, axons must also overcome ephrin-A2 mediated inhibition, potentially by the addition of EphA5 fusion proteins. Metallothionein-I/II?s effect of increasing axonal regeneration through the optic nerve injury site suggests that it could be used to increase the number of regenerating axons reaching their target. Such strategies to increase the absolute number of regenerated axons should enable these axons to better use the EphA5 and ephrin-A2 topographic gradients to optimize regenerative success.

[Truncated abstract] Microenvironmental changes associated with apoptotic neural degeneration may instruct a proportion of newly transplanted donor cells to differentiate towards the fate of the deteriorating host cellular phenotype. In the work described in this thesis, this hypothesis was tested by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult rats and mice, and then examining whether intravitreally grafted cells from a range of sources of donor neural tissue became incorporated into these selectively depleted retinae. Donor tissues were: a postnatal murine cerebellar-derived immortalised neural precursor cell line (C17.2); an adult rat hippocampal-derived clonal stem-like line (HCN/GFP); mouse embryonic day 14 (E14) primary dissociated retinal cells (Gt[ROSA]26); and adult mouse ciliary pigmented margin-derived primary neurospheres (Gt[ROSA]26). In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), and in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Some adult hosts received ON transection coupled with an autologous peripheral nerve (PN) graft. Donor cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0, 5, 7 or 14 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified, quantified and their differentiation fate within host retinae was assessed. Transplanted male C17.2 cells were identified in host retinae using a Y-chromosome marker and in situ hybridisation, or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal) using Xgal histochemistry or a beta-gal antibody. No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were, however, stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin (TUJ1), a protein highly iii expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased C17.2 cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.