Effect Of Nogo-A ASODN And Ciliary Neurotrophic Factor On Repairing Optic Nerve Injury

Optic nerve injury is a common ocular trauma, up to now, however, there is not any effective therapy for it. Regenerative repair of optic nerve injury has been paid attention to by a lot of ophthalmologists. As a classical experimental modal for repair of central nervous system injury, the result of this study will play an important theoretic role in repair of central nervous system injury. Now, it has proved that supplying exterior neurotrophic factors and neutralizing or reducing nerve growth inhibitors can promote the regeneration of retinal ganglion cells(RGCs) in adult mammalian. A lot of neurotrophic factors (NTFs) including NGF, BDNF, FGF, and CNTF can promote RGCs survival both in vitrol and in vivo. Majority of NTFs can promote RGCs survival, but fail to promote long-distance axonal regeneration and functional restoration of the optic nerve. A monoclonal antibody, IN-1 can neutralize the inhibition of nerve growth, and promote axons regeneration in vitro, but the effect is greatly limited in vivo because of its transient activity. In 1999, Cui et al reported that ciliary neurotrophic factor(CNTF) can significantly promote axonal regeneration of RGCs. The amount of regenerated axons enhanced by CNTF was as much as 4-25 times in comparison with the control group. The proximally axotomized RGCs appeared to be more sensitive to CNTF than the distally axotomized RGCs for regeneration. Cui believed that the regeneration was inhibited by the neurite growth inhibitor derived from oligodendrocytes in the distal axotomy model because there was still a 7-mm-long segment of optic nerve (ON) attached to the eye and this segment of the ON contained a large number of oligodendrocytes. It has been confirmed that neurite growth inhibitor derived from oligodendrocytes is a membrane protein, which has a strong inhibitory activity for axonal regeneration of the optic nerve. In 2000, three research groups declared at the same time that they indentified a new gene called Nogo gene in rats and humans. This gene encodes the inhibitory protein. This important result brings a new hope for the study on repair of optic nerve lesion. Based on the result mentioned above, it is suggested that the optic nerve regeneration andfunctional restoration can be achieved and blindness caused by optic nerve injury will be reduced by depressing expression of Nogo gene and supplying exterior neurotrophic factors.The purposes of the study are: depressing the expression of Nogo gene using Antisense oligonucleotides; reducing or blocking the production of neurite outgrouth inhibitor (e.g. Nogo-A); supplying exterior CNTF and increasing potential of regeneration and repairing effect on injured optic nerve; offering a new strategy for the treatment of optic nerve injury and providing experimental basis for development of antisense oligonucleotides drug to treat optic nerve injury in clinic.The study was divided into five parts: 1. to investigate the difference in the expression and the distribution of Nogo-A mRNA in central nervous system and peripheral nervous system by In situ hybridization; 2. to investigate expressive changes of Nogo gene in optic nerves after injury; 3. to filtrate antisense oligonucleotides and its effctive concentration by culture of oligodendrocytes in the optic nerve; 4.to confirm the effect of CNTF on promoting RGCs survival and clarify the mechanism in vitro; 5. to investigate the effect of CNTF and ASODN on repairing optic nerve injury in vivo.The main results and conclusions are as follows:1. We establish a new technique of oligodendrocyte culture with optic nerve tissue block and chemically definitive medium. It is convenient and effective for oligodendrocyte culture. By using this method, we can get a greater number of and more purified oligodendrocytes than those by traditional method. It is a simple and effective method and lays a foundation for filtrating antisense Nogo-A oligonucleotides in the study. 2. Using in situ hybridization method it indicated that the expression of Nogo-A mR...