NgR RNA Interference Combined With Intravitreal Zymosan-A Injection Enhances Optic Nerve Regeneration

Optic nerve belongs to the central nervous system. After injury, there is not any effective therapy for it, approximately 50 percent patients will lose their vision. Therefore, the research of how to promote retinal ganglion cells (RGCs) survival and axon regeneration after optic nerve injury is of big significance and is the hot and difficult spot in the basic and clinic visual science research.Two major reasons may be responsible for the regrowth failure of RGCs. After optic nerve injury, the neurotrophic factors will decrease for the reason of axoplasm flow difficulty, the myelin-related inhibitory factors will release from the changed microenvironment, which both contribute to the regeneration failure.After optic nerve injury in mature mammals, RGCs'axons normally show a transient local sprouting reaction but no long-range growth. Failure in regeneration has been partly attributed to proteins associated with CNS myelin. Three myelin-associated, structurally distinct proteins, Nogo, named for its inhibitory action on axonal growth, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, have been implicated in this inhibition. Nogo receptor (NgR) which binds to each of the three inhibitors has been proposed to mediate their inhibitory roles, so the NgR may represent a potential convergence point for multiple inhibitory signals. The application of NgR competitive antagonist NEP1–40 could enhance the regeneration ability of injured neuron, the active immunity to adult Sprague-Dawley(SD) rats with myelin protein extract could dramatically make RGCs regrow their axon after optic nerve crush, and the monoclonal antibody to NgR could effectively neutralize the inhibition of myelin protein and promote the neuron to grow in vitro, but all those measures have their disadvantage and could not utmost promote CNS neuron regrowth.RNA interference (RNAi) technology is a relatively new and powerful tool to analyze gene function. RNAi can be induced in cells by the introduction of synthetic double-stranded small interfering RNA (siRNA) that is subsequently caused the target gene degradation by the cellular machinery, and seems to be a far more rapid way to investigate the functions of a gene than generating transgenic animals.The great challenge for RNAi is how to transduce siRNA into target cells or animals body. SiRNA could be carried by plasmid or viral vector system in vivo. Relatively speaking, the latter has more and stable transduction efficiency. However, viral vectors also have their disadvantage, such as safety issue, target question, and so on. Retrovirus has potential carcinogenicity, adenovirus has intensive immunogenicity, and on the target issue, different viral vectors have different cell propensity. For example, recombined adeno-associated virus-2(rAAV-2) has high CNS neuron propensity and can deliver siRNA into rat retinal ganglion cells and exert RNA interference roles.Recent studies have shown several intraocular manipulations, including injuring the lens, injecting the proinflammatory agent Zymosan-A, could induce a aseptic inflammation in eye and make macrophages and other inflammatory cells gathering in vitreous, which could secrete one or several kinds of neurotrophic factors and provide RGCs with a robust growth state and promote their axon regrowth.Up to now, gene therapy through viral vectors carrying siRNA has been in the exploratory stage. After optic nerve injury, there are other inhibitory factors besides the three, glial scar will develop and form the physical barrier. So, it is the endeavour direction of treating optic nerve injury of how to efficiently and utmostly inhibit more inhibitory factors before the formation of glial scar and promote the RGCs regrowth potency. The purposes of this study are: restrainting the expression of NgR gene using RNAi technology so as to reducing or blocking the production of the co-receptor of the three inhibitors; inducing the inflammation and activating inflammatory cells in eye by applying Zymosan-A, increasing regeneration potential of injured optic nerve; offering a new strategy for the treatment of optic nerve injury and providing experimental basis for treating CNS injury.The study is divided into four parts: 1. to screen the siRNA sequence targeted to NgR and verify its inhibition in vitro; 2. to construct the rAAV-2 which could express the specific siRNA targeted to NgR and evaluate its inhibitory effect in vivo; 3.to confirm the effect of intravitreal Zymosan-A injection on promoting RGCs survival and regrowth; 4. to investigate the optic nerve regeneration effect by using NgR RNAi combined with intravitreal Zymosan-A injection in vivo.The main results and conclusions are as follows:1. Our test in vitro showed the siRNA targeted to NgR could efficiently down-regulated NgR expression in hippocampus neuron, but the control siRNA had no influence on the NgR level, which laid the foundation for constructing the recombinant virus vector.2. We had constructed the rAAV-2 for expressing siRNA and made the amplification and depuration of it. Polymerase chain reaction(PCR) confirmed the siRNA sequence had successfully introduced into the vector, sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE) showed the virus had greater than 98 per purity and achieved 1012 genome title, cell transfection exhibited that the enhanced green fluorescent protein (EGFP) expressed normally, all that demonstrated the virus vector had successfully constructed and met the requirement of animal experiment.3. Our in vivo test showed four and eight-week point after rAAVs injection, the EGFP could be detected in retina sections, which meant that rAAVs had successfully transduced into RGCs and the shRNA been being expressed.4. Western bolt test showed the recombinant virus vector could efficiently inhibited the NgR protein in RGCs, the inhibition had been existed 4 weeks after virus injection and maintained till 8 weeks, which confirmed it had been integrated into RGCs genome and might have longer inhibition, but the control viruses had no influence on NgR level.5. Our in vivo test showed optic nerve injury could induce RGCs death, only 2 per RGCs survived 21 days after optic nerve crush, but with 20 per in Zymosan-A treating eyes, which showed the activating inflammatory cells induced by Zymosan-A might have some protective effect to the axon-crushed RGCs.6. Our animal experiment showed both NgR RNAi and intravitreal Zymosan-A injection could partially promote RGCs axon regeneration after optic nerve crush, but combining application could significantly reinforce the repairing efficacy of injury optic nerve in vivo.