Survival of purified rat photoreceptors in vitro is stimulated directly by fibroblast growth factor-2.
Basic fibroblast growth factor (FGF-2) influences the differentiation and survival of retinal photoreceptors in vivo and in vitro, but it is not known whether it acts directly on photoreceptor FGF receptors or indirectly through activation of surrounding cells. To clarify the effects of FGF-2 on photoreceptor survival, we developed a purified photoreceptor culture system. The outer nuclear layers of postnatal day 5-15 rat retinas were isolated by vibratome sectioning, and the photoreceptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defined medium. Immunocytochemical labeling showed that >99.5% of cells were photoreceptors, and glial contamination represented approximately 0. 2%. Photoreceptors from postnatal day 5-9 retinas survived for at least 24 hr in vitro, whereas cells from postnatal day 10-15 retinas died rapidly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner after the addition of FGF-2. In control cultures, 36% of originally seeded photoreceptors were alive after 5 d in vitro, and in the presence of 20 ng/ml FGF-2 this number was doubled to 62%. This increase was not caused by proliferation of photoreceptor precursors. Denaturing or blocking FGF-2 prevented enhancement of survival. Conversely, only 25.5% of photoreceptors survived in the presence of epidermal growth factor (EGF). FGF- and EGF-receptor mRNA and proteins were detected in purified photoreceptors in vitro, and addition of FGF-2 or EGF led to tyrosine phosphorylation of photoreceptor proteins. These data support a direct mechanism of action for FGF-2 stimulation of photoreceptor survival.
Fontaine
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Laboratoire de Physiopathologie Rétinienne, Université Louis Pasteur, 67091 Strasbourg Cedex, France.
Kinkl
N
N
Sahel
J
J
Dreyfus
H
H
Hicks
D
D
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D019393:Arrestin / Q000235:genetics; D001798:Blood Proteins / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D009410:Nerve Degeneration / Q000503:physiopathology; D009457:Neuroglia / Q000737:chemistry / Q000166:cytology; D020419:Photoreceptor Cells, Vertebrate / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics; D013498:Suramin / Q000494:pharmacology; D020032:Tyrphostins / Q000494:pharmacology
D000970:Antineoplastic Agents; D019393:Arrestin; D001798:Blood Proteins; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D020032:Tyrphostins; D016222:Fibroblast Growth Factor 2; D013498:Suramin; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; C060641:tyrphostin A23