Combined Treatments To Promote Axon Regeneration And The Establishment Of A Novel Model Of Ischemic Optic Neuropathy

Glaucoma, traumatic optic neuropathy, ischemic optic neuropathy and optic neuritis, which mainly affect the retinal ganglion cell (RGC) axons in the optic nerve, are the leading causes of irreversible vision loss worldwide. Since RGC axon fibers can hardly regenerate back to their targets after injury, the way to elicit a sustained and potent axon regeneration is one of the most critical questions in the research field of ophthalmology and neuroscience. In the current study, we combine the treatments that counteract extrinsic inhibitory signals and enhance RGCs axon intrinsic growth capacity, in order to promote substantial axon regeneration. Furthermore, we create a novel, reproducible and reliable rodent model of posterior ischemic optic neuropathy (PION), which provides a handy tool for in vivo axon regeneration research and also helps to understand the pathogenesis and molecular changes in PION.PARTⅠCombined Treatments for Promoting Axon Regeneration after Optic Nerve Injury:Strategies for Overcoming Inhibitory Signals and Activating RGCs’Intrinsic Growth StateObjective:RGC axons fail to regenerate after optic nerve injury, which arises from both extrinsic inhibitory molecules and limited intrinsic regenerative potential. In this study, we combine strategies of both overcoming various inhibitory molecules and promoting axon growth capacity to enhance axon regeneration after optic nerve injury.Methods:The expression of KLF-2,-5,-9,-13,-15,-16and PTEN in adult rat’s RGCs were knocked down by intravitreal injection of AAV8Y733F-shRNA. Injection of AAV2-Cre was employed to knock out PTEN and/or KLF4in RGCs of PTENf1/f1, KLF4f1/f1, and PTENf1/f1&KLF4f1/f1mice. RhoA, a downstream target of many inhibitory environmental signals, was inactivated by intravitreal injection of AAV2-C3. Retrograde labeling of Fluorogold was applied for quantification of RGC survival and CTB-594was used to estimate axon regeneration.Results:Compared to control group, simultaneous application of AAV8Y733F-shRNA-anti KLF-2,-5,-15could significantly increase RGCs survival up to50%. Similar RGC protection effects were observed in both AAV8-shRNA-anti KLF-9,-13,-16and AAV8Y733F-shRNA-anti PTEN applied groups. Moreover, the number of regenerate axons in all these three experiment groups was significantly more than control group, however, most regenerated axons were restrict within1mm away from crush site. Deletion of PTEN in RGCs elicited potent axon regeneration. Meanwhile, deletion of KLF4failed to enhance axon regeneration. Thus the average number of regenerate axons was similar in PTEN deletion animals and PTEN&KLF4double deletion animals. When combined with C3application, the effect of PTEN deletion was further enhanced and the longest regenerate axon reached over4mm away from crush site.Conclusions:Simultaneously knock down multiple KLFs, which suppress RGCs axon growth, increase RGCs survival and induce modest axon regeneration after optic nerve injury. Different from developmental deletion of KLF4, which promotes axon regeneration, deleting KLF4in RGCs of adult mice cannot enhance axon regeneration. Substantial levels of regeneration require treatments that counteract extrinsic inhibitors and promote axon growth capacity.PART ⅡA Novel Rodent Model of Posterior Ischemic Optic NeuropathyObjective:To develop a reliable, reproducible rat model of posterior ischemic optic neuropathy (PION) and study the cellular responses in the optic nerve and retina, and the neuroprotective potential of trophic factors.Methods:PION was induced in adult rats by photochemical-induced ischemia (PCI). Retinal and optic nerve vasculature was examined by FITC-dextran extravasation. Tissue sectioning and immunohistochemistry were employed to investigate the pathological changes. Retinal ganglion cell (RGC) survival at different time points after PION induction, with or without neurotrophic application, was quantified by Fluorogold retrograde labeling. Results:Optic nerve injury was confirmed after PION induction, including local vascular leakage, optic nerve edema and cavernous degeneration. Immunostaining data revealed microglial and macrophage activation and focal loss of astrocytes, with adjacent astrocytic hypertrophy. Up to23%,50%and70%RGC loss was observed at time points of1week,2weeks and3weeks post injury comparing to both non-injury group and sham-treated group (laser only). Within the retina, focal or diffuse RGC loss was specific to strain of rat, consistent with differences in optic nerve axon retinotopy. Experimental treatment by brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) remarkably prevented the RCG loss in PION rats. By3week after injury, more than40%of the RGCs were saved with the application of neurotrophic factors.Conclusions:The rat PION created by PCI is a reproducible and reliable animal model mimicking the key features of human PION. The high resemblance between rat and human PION makes it an ideal model to study the pathophysiology of the disease, most of which remain to be elucidated. Furthermore, it provides an excellent model for testing therapeutic approaches for optic neuropathies and axon regeneration.