AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.
We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of betaIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450+/-358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200+/-4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many betaIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135+/-367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (+/-2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (+/-1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.
Leaver
S G
SG
School of Anatomy and Human Biology, The University of Western Australia, Western Australia, Australia.
Cui
Q
Q
Plant
G W
GW
Arulpragasam
A
A
Hisheh
S
S
Verhaagen
J
J
Harvey
A R
AR
D000818:Animals; D019771:Axotomy; D002470:Cell Survival; D020934:Ciliary Neurotrophic Factor / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007150:Immunohistochemistry; D007267:Injections; D009416:Nerve Regeneration; D020221:Optic Nerve Injuries / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D051381:Rats; D017208:Rats, Wistar; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000473:pathology / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods*; D014822:Vitreous Body
D020934:Ciliary Neurotrophic Factor; D049452:Green Fluorescent Proteins