Regular article
Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons

https://doi.org/10.1016/S1044-7431(02)00037-4Get rights and content

Abstract

In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.

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.

References (58)

  • S.A. Jo et al.

    Ciliary neurotrophic factor is an axogenesis factor for retinal ganglion cells

    Neuroscience

    (1999)
  • M.R. Mason et al.

    Overexpression of GAP-43 on thalamic projection neurons of transgenic mice does not enable them to regenerate axons through peripheral nerve grafts

    Exp. Neurol.

    (2000)
  • J. Mey et al.

    Intravitreal injection of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo

    Brain Res.

    (1993)
  • A. Meyer-Franke et al.

    Characterization of the signalling interactions that promote the survival and growth of developing retinal ganglion cells in culture

    Neuron

    (1995)
  • A. Meyer-Franke et al.

    Depolarization and cAMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons

    Neuron

    (1998)
  • G. Ming et al.

    cAMP-dependent growth cone guidance by netrin-1

    Neuron

    (1997)
  • G. Mukhopadhyay et al.

    A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration

    Neuron

    (1994)
  • S. Neumann et al.

    Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation

    Neuron

    (2002)
  • J. Qiu et al.

    Spinal axon regeneration induced by elevation of cyclic AMP

    Neuron

    (2002)
  • S. Shen et al.

    Retinal ganglion cells lose trophic responsiveness after axotomy

    Neuron

    (1999)
  • R.L. Snow et al.

    Ganglion cell neurogenesis, migration and early differentiation in the chick retina

    Neuroscience

    (1994)
  • H.J. Song et al.

    Signal transduction underlying growth cone guidance by diffusible factors

    Curr. Opin. Neurobiol.

    (1999)
  • X. Tao et al.

    Calcium influx regulates BDNF transcription by a CREB family transcription factor dependent mechanism

    Neuron

    (1998)
  • H.K. Yip et al.

    Axonal regeneration of retinal ganglion cellseffect of trophic factors

    Prog. Ret. Eye Res.

    (2000)
  • D. Cai et al.

    Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate

    J. Neurosci.

    (2001)
  • A. Cohen et al.

    Neurotrophin-4/5 (NT-4/5) increases adult rat retinal ganglion cell survival and neurite outgrowth

    J. Neurobiol.

    (1994)
  • S. Cohen-Cory

    BDNF modulates, but does not mediate, activity-dependent branching and remodeling of optic axon arbors in vivo

    J. Neurosci.

    (1999)
  • Q. Cui et al.

    At least two mechanisms are involved in the death of rat retinal ganglion cells following target ablation in neonatal rats

    J. Neurosci.

    (1995)
  • Q. Cui et al.

    CNTF promotes the regrowth of retinal ganglion cell axons into murine peripheral nerve grafts

    NeuroReport

    (2000)
  • Cited by (0)

    View full text