Regular articleIntraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons
Introduction
Cyclic AMP (cAMP) plays an important role in diverse neuronal functions including survival (Rydel and Greene, 1988), process expression (Song and Poo, 1999), and modulation of growth cone responses to a range of diffusible and nondiffusible factors (Ming et al., 1997). In tissue culture, cAMP elevation alters neuronal responses to growth factors (Song et al., 1997) and blocks the effects of myelin-associated inhibitory factors (Cai et al., 1999), properties that together may enhance neural repair processes after injury. Recent in vivo studies on neural crest-derived sensory nerves support this suggestion Neumann et al 2002, Qiu et al 2002. Here we use an established, quantifiable model of central nervous system (CNS) injury in adult rats to examine directly the effect of elevated cAMP on axonal regeneration in vivo.
The visual system is an excellent system in which to study neuroprotection and regeneration in the adult CNS. Retinal ganglion cells (RGCs) can be directly targeted by using intravitreal injection techniques, the optic nerve (ON) is a centrally derived tract that is surgically accessible within the orbit, and the extent of RGC axonal regrowth can be monitored by using anatomical, functional, and behavioral methods. In adult rats, the viability of RGCs axotomized after intraorbital ON transection is increased by intravitreal application of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) or neurotrophin 4/5 (NT-4/5) Mansour-Robaey et al 1994, Peinado-Ramon et al 1996, or by using molecules that block death signaling cascades (Bähr, 2000). RGC death is also reduced, and regeneration of axons elicited, by grafting a segment of autologous peripheral nerve (PN) onto a cut ON stump So and Yip 2001, Villegas-Perez et al 1988. In hamsters and mice, intraocular injection of ciliary neurotrophic factor (CNTF) increases the regeneration of RGC axons through such grafts Cui and Harvey 2000, Cui et al 1999.
In the presence of a cocktail of peptide growth factors including BDNF and CNTF, elevation of intracellular cAMP in vitro and in vivo potentiates RGC survival and plasticity Meyer-Franke et al 1995, Shen et al 1999. The aim of the present study, using the PN-to-ON graft approach in adult rats, was to determine whether intraocular elevation of cAMP, alone or in combination with intravitreal injections of either NT-4/5 or CNTF, enhanced RGC viability after axotomy and increased the number of RGCs regenerating axons into PN grafts. To increase cAMP levels in the retina, we used a nondegradable membrane-permeable analogue, 8-(4-chlorophenylthio)-adenosine 3′:5″-cyclic monophosphate (CPT-cAMP), shown previously to be effective in neonatal eyes (Shen et al., 1999). Regenerating RGCs were retrogradely labeled after fluorogold (FG) injections into the distal end of PN grafts. The amount of regeneration was also assessed in animals that received only PN grafts, and in PN-grafted rats that received intraocular injections of either saline, NT-4/5 or CNTF. For each of the seven groups, the number of RGCs with regenerating axons was compared with the number of cells in the ganglion cell layer immunoreactive for βIII-tubulin, a cytoskeletal protein expressed by RGCs Ju et al 2000, Snow and Robson 1994, Watanabe et al 1991.
Section snippets
Viability of axotomized RGCs in PN-grafted animals
We wished to develop a method that would provide reliable postmortem information about the number of RGCs surviving in the retina 3 weeks after ON section and PN transplantation, thus providing information about the proportion of viable RGCs that regenerated axons into grafts in different experimental conditions.
Discussion
In adult rats, intravitreal injections of CNTF significantly increased RGC axonal regeneration into PN grafts, whereas regrowth was decreased after NT-4/5 injections. Elevated cAMP by itself did not affect the number of RGCs regrowing axons into grafts, but when CPT-cAMP was coinjected with CNTF there was a massive increase in regeneration. The negative effect of NT-4/5 on axonal regeneration was partially offset by coinjection with the membrane-permeable cAMP analogue, which brought the level
Experimental methods
All surgical procedures carried out in neonatal and young adult (8-week-old) Sprague-Dawley rats were approved by the Animal Ethics Committee of The University of Western Australia.
Acknowledgements
We thank Genentech for the gift of NT-4/5 and Natalie Symons for technical assistance. This work was supported by research grants to A.R.H. and Q.C. from Australian NHMRC (Nos. 990067 and 000222), Western Australia Neurotrauma Research Program and AA Saw Medical Award, and to H.K.Y. and K.F.S. from The University of Hong Kong and Hong Kong Research Grant Council.
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