title: AAV-mediated transfer of RhoA shRNA and CNTF promotes retinal ganglion cell survival and axon regeneration.
abstract: The aim of the present study was to determine whether adeno-associated viral vector (AAV) mediated transfer of ciliary neurotrophic factor (CNTF) and RhoA shRNA has additive effects on promoting the survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve crush (ONC). Silencing effects of AAV-RhoA shRNA were confirmed by examining neurite outgrowth in PC12 cells, and by quantifying RhoA expression levels with western blotting. Young adult Fischer rats received an intravitreal injection of (i) saline, (ii) AAV green fluorescent protein (GFP), (iii) AAV-CNTF, (iv) AAV-RhoA shRNA, or (v) a combination of both AAV-CNTF and AAV-RhoA shRNA. Two weeks later, the ON was completely crushed. Three weeks after ONC, RGC survival was estimated by counting βIII-tubulin-positive neurons in retinal whole mounts. Axon regeneration was evaluated by counting GAP-43-positive axons in the crushed ON. It was found that AAV-RhoA shRNA decreased RhoA expression levels and promoted neurite outgrowth in vitro. In the ONC model, AAV-RhoA shRNA by itself had only weak beneficial effects on RGC axon regeneration. However, when combined with AAV-CNTF, AAV-RhoA shRNA significantly improved the therapeutic effect of AAV-CNTF on axon regeneration by nearly two fold, even though there was no significant change in RGC viability. In sum, this combination of vectors increases the regenerative response and can lead to more successful therapeutic outcomes following neurotrauma.
authors.affiliation: Joint Shantou International Eye Center, Shantou University and The Chinese University of Hong Kong, Shantou, PR China. Electronic address: cenlp@hotmail.com.
authors.forename: Ling-Ping
authors.initials: LP
authors.lastname: Cen
authors.affiliation: Joint Shantou International Eye Center, Shantou University and The Chinese University of Hong Kong, Shantou, PR China.
authors.forename: Jia-Jian
authors.initials: JJ
authors.lastname: Liang
authors.affiliation: Joint Shantou International Eye Center, Shantou University and The Chinese University of Hong Kong, Shantou, PR China.
authors.forename: Jian-Huan
authors.initials: JH
authors.lastname: Chen
authors.affiliation: School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA, Australia.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Harvey
authors.affiliation: Department of Ophthalmology and Visual Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, PR China.
authors.forename: Tsz Kin
authors.initials: TK
authors.lastname: Ng
authors.affiliation: Joint Shantou International Eye Center, Shantou University and The Chinese University of Hong Kong, Shantou, PR China.
authors.forename: Mingzhi
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Joint Shantou International Eye Center, Shantou University and The Chinese University of Hong Kong, Shantou, PR China; Department of Ophthalmology and Visual Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, PR China.
authors.forename: Chi Pui
authors.initials: CP
authors.lastname: Pang
authors.affiliation: Joint Shantou International Eye Center, Shantou University and The Chinese University of Hong Kong, Shantou, PR China; Department of Ophthalmology and Visual Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, PR China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Cui
authors.affiliation: Joint Shantou International Eye Center, Shantou University and The Chinese University of Hong Kong, Shantou, PR China; Department of Neurology, Affiliated Hospital of Hubei University for Nationalities, Enshi, PR China. Electronic address: youmingf@aliyun.com.
authors.forename: You-Ming
authors.initials: YM
authors.lastname: Fan
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism* / Q000473:pathology; D002470:Cell Survival / Q000502:physiology; D020934:Ciliary Neurotrophic Factor / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D015316:Genetic Therapy; D005822:Genetic Vectors; D056965:Injections, Intraocular; D008297:Male; D000071446:Neuronal Outgrowth / Q000502:physiology; D020221:Optic Nerve Injuries / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D011916:Rats, Inbred F344; D012165:Retinal Ganglion Cells / Q000378:metabolism* / Q000473:pathology; D020742:rhoA GTP-Binding Protein / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism
chemical_list: D020934:Ciliary Neurotrophic Factor; D034741:RNA, Small Interfering; D020742:rhoA GTP-Binding Protein
keywords: CNTF; RNAi; RhoA; ganglion cell; nerve regeneration; retina