Abstract
Purpose: :
To characterize the progression of retinal ganglion cell (RGC) axon regeneration in vitro and in vivo.
Methods: :
To determine factors enhancing optic nerve (ON) regeneration, RGC5 cells were cultured on tissue culture dishes in DMEM+10%FBS with different neurotrophic/growth factors, individually, and in combination (ciliary neurotrophic factor [CNTF], neurotrophin3 [NT3], brain derived neurotrophic factor [BDNF], glial-cell-line derived neurotrophic factor [GDNF], nerve growth factor [NGF], insulin-like growth factor [IGF], Sema7a, staurosporine [positive control]) and increasing concentrations of a PTEN (phosphatase and tensin homolog) inhibitor ([BPV(Phen)] 0.2nM to 200nM). Cells were imaged at 15 hours, and at 3, 5 and 7 days to check for survival and neurite outgrowth (>25μm in length).The optic nerve of Thy1-YFP mice was transected either partially or totally with preservation of meninges. Mice were sacrificed at weeks 1, 2, and 3 and optic nerves and retinas were imaged with the Leica DM-IRE2 confocal fluorescent microscope (single and z-stack images). Images were analyzed using Image J software.
Results: :
At 15hrs, all growth factors promoted similar neurite outgrowth. At 3 days, staurosporine and NGF increased the number of cells; BDNF and Sema7a plates showed the most number of arborizations; and Sema7a prolonged RGC survival. 2nM BPV(Phen) was the most effective neurite outgrowth promoter, and combined with IGF/Sema7a/CNTF led to the longest and most numerous outgrowth.Both partial and total optic nerve transections resulted in a significant decrease in fluorescence at all time points, both in the optic nerve distal to the transection site, and also in the retina. No regain in fluorescence distally to the transection site was detected at any time point after transection.
Conclusions: :
Our preliminary experiments show that PTEN inhibitors promote in vitro RGC regeneration. Thy1-YFP mice provide a model to characterize optic nerve regeneration after injury.
Keywords: optic nerve • regeneration • ganglion cells