Preliminary Study On The Role And Mechanism Of Sema3A In Regeneration Of Injuried Optic Nerve

Background:The optic nerve,which belongs to the central nervous system,have difficulty regenerating and recovering once injured.Thus the patients are likely to suffer irreversible visual damage and their life quality is also poor.There is no effective treatment for optic nerve injury at present.Therefore,exploring the key factors to effectively regulate the regeneration process after optic nerve injury has always been a hot and difficult issue in ophthalmology.Sema3 A is a classical axon guidance molecular in vertebrates and plays an important role in the development of nervous system.Researches have demonstrated that Sema3 A can inhibit axon regeneration and promote neuronal apoptosis during central nervous system injury,thus preventing the process of nerve regeneration and restoration.However,the mechanism of Sema3 A in recovery of optic nerve injury still remains unclear.Rho/ROCK pathway,abundantly activated after central nervous system injury as well,can integrate multiple inhibitory signals and produce cascade waterfall signals by acting on various protein substrates to inhibit axon regeneration.There are two forms of ROCK,ROCK1 exist in non-nervous tissues,while ROCK2 are mainly expressed in the brain and spinal cord.Based on literature review and previous work,we proposed a scientific hypothesis that “Sema3A can negatively regulate the regeneration process of optic nerve injury and its mechanism may be related to ROCK2 signaling pathway”,so as to provide a new idea for the restoration of optic nerve injury.Object:The study is aimed to observe the expression of Sema3 A and ROCK2 after optic nerve and retinal ganglion cell(RGC)injury both in vivo and vitro by immunoblotting and qRTPCR,and to investigate the inhibitory effect and possible mechanism of Sema3 A on restoration of optic nerve injury.It will confirm that Sema3 A could inhibit axon regeneration via ROCK2.As a result,it will provide new targets and directions for the therapy of optic nerve injury.Method:1.Optic nerve crush model of C57 mice established,the expression of Sema3 A and ROCK2 protein were detected at 1,3,7 and 14 days after injury.The locations of Sema3 A and ROCK2 in retinae from normal mice and mice with injuried ON for 3 days were observed by immunofluorescence.2.The expression of Sema3 A mRNA and ROCK2 mRNA were investegated at 4,8,16 and 48 h after oxygen-glucose deprivation of primary cultured RGCs.3.Primary RGCs were treated with different concentrations of Sema3 A for different time,and anti-?3-tubulin+/MAP2-was identified as axon by immunofluorescence staining.The length of axons were measured by Image J software.The appropriate concentration of Sema3 A was selected and ROCK2 inhibitor was added in advance with different concentrations to observe whether ROCK2 inhibitor could affect the role of Sema3 A on axon growth..4.Sema3 A,ROCK2 inhibitor,Sema3A+ROCK2 inhibitor and PBS were injected into the vitreous cavity on the 0th and 7th day after ON crush.The expression of GAP43 and Sprr1 a mRNA in the retina were detected on the 5th day after injury.The visual function of each group was detected by flash visual evoked potential.Frozen section of optic nerve was used to display axon regeneration by anti-GAP43 immunofluorescence.5.The primary RGCs were processed with the concentration and time of Sema3 A determined in method 3.Western blotting was used to detect the expression of ROCK2 downstream substrate MLC2 and its phosphorylated molecule pMLC2.Result:1.Sema3 A and ROCK2 expression first increased and then decreased after optic nerve crush,starting to increase at Day 1,peaking at Day 3 and declining at Day 7.Immunofluorescence showed that Sema3 A and ROCK2 were poorly expressed in the normal retina,while the positive cells in the retinal ganglion cell layer increased significantly at Day 3 after injury.2.The expression of Sema3 A and ROCK2 in primary RGCs after oxygen-glucose deprivation experienced an initial increase at 4 h,then a mild decrease was found at 8 h and finally an increase happened again.The expression of Sema3 A and ROCK2 reached its peak at 48 h and 16 h respectively.3.Sema3 A can inhibit the axon growth of RGCs in vitro,and pre-adminstration of ROCK2 inhibitor Y27632 can alleviate the inhibition of Sema3 A on axon growth.4.After optic nerve crush,expression of GAP43,Sprr1 a mRNA and the number of regenerating axons per optic nerve section in Sema3 A group were significantly decreased compared with ROCK2 inhibitor,Sema3A+ROCK2 inhibitor and PBS groups.F-VEP showed that N2-P2 amplitude in Sema3 A group was also the smallest.5.The expression of pMLC2 protein and the ratio of pMLC2/MLC2 increased after adding Sema3 A to cultured RGCs in vitro.Conclusion:1.The expression of Sema3 A and ROCK2 increased both in vitro and in vivo after RGCs injury.2.Sema3 A can inhibit the growth of primary RGCs axons and regeneration of optic nerve after injury in vivo.ROCK2 inhibitor can alleviate the unfavorable effects of Sema3 A.3.Sema3 A can promote the phosphorylation of MLC2,which may help explain the possible mechanism of Sema3 A in inhibiting axon regeneration.This will provide a new way to explore the recovery of optic nerve injury.