Time course of neurotrophic factor upregulation and retinal protection against light-induced damage after optic nerve section.
OBJECTIVE
To assess neurotrophic factor upregulation in the retina after damage to the optic nerve and relate that regulation to changes in photoreceptor stability and function.


METHODS
Retinas of adult pigmented (Long-Evans) rats were examined at successive times (1-60 days) after unilateral optic nerve section. The distribution and expression of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (FGF-2) and their receptor elements FGFR1 and CNTFRalpha were studied with immunohistochemistry and Western blot analysis. FGF-2 and CNTF mRNA levels were also assessed, with semiquantitative reverse transcription-PCR. Levels and localization of the intracellular signaling molecule ERK and its activated, phosphorylated form pERK, were examined by immunohistochemistry. To assess the correlation between neurotrophic factor levels and their protective effect against light damage, albino (Sprague-Dawley) rats were exposed to bright continuous light (1000 lux) for 24 or 48 hours at successive times after nerve section. The TUNEL technique was used to visualize neuronal cell death in the retina.


RESULTS
CNTF upregulation was detected 1 week after optic nerve section, peaked at 2 weeks, and fell to control levels at 4 weeks. CNTF appeared first in the inner retina in the ganglion cells, then in the Muller cells in which it became prominent at the outer limiting membrane (OLM) and in the outer segment (OS) region of photoreceptors. FGF-2 upregulation became prominent, particularly in photoreceptors, 21 to 28 days after surgery, continued to 2 months, and slowly declined thereafter. Double labeling with antibodies to ligand and the receptor showed colocalization of CNTF to its receptor at the OS region, whereas FGF-2-to-FGFR1 binding was found in the outer nuclear (ONL) and outer plexiform (OPL) layers. Optic nerve section provided a significant protective effect against light-induced damage in the first 2 weeks. There was no protection when animals were exposed to damaging light 1 month after nerve section.


CONCLUSIONS
The upregulation of CNTF 7 to 14 days after nerve section correlates with a reduction in the a-wave described previously. Colocalization of CNTF and CNTFRalpha on the outer segments suggests that CNTF acts at the photoreceptor membrane. The slower upregulation of FGF-2 correlates with a reduction of the b-wave. FGF-2/FGFR1 colocalization in the OPL suggests that this factor acts at the synaptic terminals of photoreceptors, modulating the release of neurotransmitters. The time course of pERK upregulation suggests that the successive upregulation of CNTF and FGF-2 activates the ERK pathway. Based on the time course of protection against bright continuous light, it seems that CNTF plays a major role in this effect, and FGF-2 has a less important role in the protection against light-induced damage.
Valter
Krisztina
K
CNS Stability and Degeneration Group, Research School of Biological Sciences, The Australian National University, Canberra City, Canberra, ACT 2601, Australia. valter@rsbs.anu.edu.au
Bisti
Silvia
S
Gargini
Claudia
C
Di Loreto
Silvia
S
Maccarone
Rita
R
Cervetto
Luigi
L
Stone
Jonathan
J
D000818:Animals; D015153:Blotting, Western; D020934:Ciliary Neurotrophic Factor / Q000235:genetics / Q000378:metabolism*; D003714:Denervation; D004596:Electroretinography; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D020287:In Situ Nick-End Labeling; D008027:Light; D009900:Optic Nerve / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D011833:Radiation Injuries, Experimental / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D051381:Rats; D020318:Rats, Long-Evans; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D020801:Receptor, Ciliary Neurotrophic Factor / Q000378:metabolism; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D012160:Retina / Q000378:metabolism / Q000528:radiation effects*; D012162:Retinal Degeneration / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation
D020934:Ciliary Neurotrophic Factor; D012333:RNA, Messenger; D020801:Receptor, Ciliary Neurotrophic Factor; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; C496350:Fgfr1 protein, rat; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1