No description available.

No description available.

[Truncated abstract] Formation and consolidation of the retinotectal projection during optic nerve regeneration has been associated with two major interlinked processes. Initially, retinal ganglion cell (RGC) axons are guided by molecular guidance cues, such as the Eph receptor tyrosine kinases and their ligands, the ephrins, to their approximately correct location and form a coarse topographic map in the optic tecum. Such axon guidance occurs in the absence of neural activity and is considered to be activity-independent. The second process involves glutamatergic excitation, whereby correctly located connections are strengthened by correlated neural activity, whilst removing inappropriately located ones thereby sharpening the topography.The second process is considered to be activitydependent. Here, a number of experiments were undertaken to further examine the interrelationships of activity-dependent and independent processes with respect to functional outcomes. Two models of optic nerve regeneration were studied. In goldfish, following optic nerve crush, regeneration is successful. … In goldfish, guidance along the medio-lateral tectal axis may occur through preordering of axons prior to entering the tectum via the appropriate medial and lateral brachium, with EphA/ephrin-A then guiding axons over the rostral-caudal axis establishing gross topography. The increase in involvement of NMDA-mediated transmission during the period of activity-dependent refinement consolidated the role of this receptor in synapse plasticity. However what triggers NMDA-mediated activity to increase is still largely unresolved, although as the factors governing receptor trafficking during development and synaptic plasticity become better understood, these can be applied to the period of plasticity associated with regenerating axons. And finally, as GABAergic inhibition appears to suppress activity-dependent refinement, means of overcoming this inhibition through 4 such methods as visual training or pharmacological intervention may have significance for mammalian regeneration.

Teleost retinal ganglion cells can regenerate severed axons following injury, something their mammalian counterparts cannot do. In the teleost, successful regeneration has been attributed in part to microglial cell activities including the phagocytosis of myelin. Although the regulation of microglial phagocytosis has been studied in mammals, in the teleost it is largely unexamined. The present study was designed to identify mediators of microglial phagocytosis released by injured goldfish optic nerve during the course of regeneration. We found that microglial phagocytosis was significantly enhanced in the presence of a 7 day regenerating nerve or medium conditioned by the nerve (CM). When either nerve or CM was incubated with microglia along with an antibody against tumour necrosis factor alpha (TNFalpha), this effect was neutralized. The L929 cell cytotoxicity assay further demonstrated TNFalpha activity in the CM. However, Western blot analysis did not confirm this result. Therefore, further work is necessary to clearly establish the presence of TNFalpha.

No description available.

[Truncated abstract] Developmentally, the rodent retinocollicular projection is often thought of as a homogenous projection of retinal ganglion cell (RGC) axons, however the extensive period of RGC neurogenesis and sequential arrival of their axons into central targets such as the superior colliulus (SC) suggests otherwise. RGC axons are already present in the developing SC at embryonic (E) day 16.5-17. RGCs born on E15 have innervated the SC by birth, whereas axons derived from RGCs that are born last (E19) do not grow into the SC until postnatal (P) days 4-6 (Dallimore et al., 2002). These observations may go someway to explaining why, after SC lesions in rats at P2, there is greater growth distal to the lesion site compared to lesions made at P6 (Tan and Harvey, 1997b). It may be that the post lesion growth is simply de novo growth of axons from late-born RGCs rather than regeneration of pre-existing, injured axons. Early and late cohorts of growing RGC axons presumably encounter different developmental terrains as they grow from retina to central targets, possibly resulting in differences in developmental milestones and growth potentials. There may also be differences in guidance cues, further suggesting that gene expression in early vs late born RGCs may differ. To examine differences between early (E15) and late (E19) born RGCs during development, the time-course and extent of programmed RGC death in normal rat pups, and RGC death following the removal of target-derived trophic factors, was assessed. ... On the other hand, LCM captured GCL analysed for gene expression at P0 and P7 revealed decreases in AKT, Math5, Notch1, c-jun, DCC, Arginase-1 mRNA levels and a considerable decrease in GAP-43 expression. It is not surprising to see differences in gene expression between whole eye and the more specific GCL samples, as the cells in all layers of the retina have very different functions and different developmental profiles. It is important to note decreases in mRNA expression in the GCL for a number of the genes analysed at P0 and P7, reflecting cessation of RGC death and completion of axonal growth into central visual targets. I also examined at the protein level expression of DCC, Arginase1, c-Jun and Bcl-2 at birth (P0) in BrdU labeled RGCs born on E15 or E19. When comparing the percentage of double labelled cells compared to the total number of cells expressing each protein, Bcl-2, c-Jun and Arg1 were expressed more in E15 RGCs (22.90%, 72.71%, and 16.44% respectively in E15 RGCs, compared with 0.52%, 13.17% and 3.59% in E19 RGCs). In contrast, DCC was expressed more at birth in E19 RGCs (18.05% in E19 RGCs compared with 9.23% in E15 RGCs). This shows there is clearly a difference in the expression of proteins in the two cohorts of RGCs, which is consistent with PCR data and with their growth state as their axons encounter the changes in the newborn brain. The overall findings of this research suggest that seemingly homogenous populations of neurons are quite different in their developmental profile and in their response to injury. This work may provide new ways of determining better strategies for CNS repair and the most effective way of targeting cells for regeneration and survival.

[Truncated abstract] There is limited intrinsic potential for repair in the adult human central nervous system (CNS). Dysfunction resulting from CNS injury is persistent and requires prolonged medical treatment and rehabilitation. The retina and optic nerve are CNSderived, and adult retinal ganglion cells (RGCs) and their axons are often used as a model in which to study the mechanisms associated with injury, neuroprotection and regeneration. In this study I investigated the effects of a variety of strategies on promoting RGC survival and axonal regeneration after optic nerve injury, including the use of reconstructed chimeric peripheral nerve (PN) grafts, gene therapy, and intraocular application of pharmacological agents and other factors . . . C3 transferase is an enzyme derived from Clostridium botulinum that inactivates Rho GTPase. Because SC myelin contains MAG and PN also contains CSPGs, I tested the effects of intraocular injection of a modified form of C3 (C3-11), provided by Dr Lisa McKerracher (CONFIDENTIAL data, under IP agreement with Bioaxone Therapeutic, Montreal) on RGC axonal regeneration into PN autografts. My results showed that there was significantly more RGC survival and axonal regeneration in PN autografts after repeated intraocular injection of C3. I also tested whether intraocular injections of CPT-cAMP and/or CNTF can act in concert with the C3 to further increase RGC survival and/or regeneration. Results showed that the effect of C3 and CPT-cAMP plus CNTF were synergistic and partially additive. The use of combination therapies therefore offers the best hope for robust and substantial regeneration. The overall results from my PhD project will help determine how best to reconstruct nerve pathways and use pharmacological interventions in the clinical treatment of CNS injury, hopefully leading to improved functional outcomes after neurotrauma.