1. ¹School of Anatomy and Human Biology, The University of Western Australia, Western Australia, Australia
2. ²School of Animal Biology, The University of Western Australia, Western Australia, Australia
3. ³The Western Australian Institute for Medical Research, The University of Western Australia, Western Australia, Australia
4. ⁴Red's Spinal Cord Laboratory, The University of Western Australia, Western Australia, Australia
5. ⁵Netherlands Institute for Neuroscience, Amsterdam, The Netherlands

Correspondence: Dr AR Harvey, School of Anatomy and Human Biology, The University of Western Australia, M309, 35 Stirling Hwy, Crawley, WA 6009, Australia. E-mail: arharvey@anhb.uwa.edu.au

⁶Current address: Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye Hospital, 147 Argyle St., Kowloon, Hong Kong.

Received 9 January 2006; Revised 29 March 2006; Accepted 2 April 2006; Published online 18 May 2006.

Abstract

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 III-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 450358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 2004064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many III-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 (1135367 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.