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 eff ects of FGF-2 on photoreceptor survival, we developed a purified photo receptor culture system. The outer nuclear layers of postnatal day 5-1 5 rat retinas were isolated by vibratome sectioning, and the photorece ptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defin ed medium. Immunocytochemical labeling showed that >99.5% of cells wer e photoreceptors, and glial contamination represented similar to 0.2%. Photoreceptors from postnatal day 5-9 retinas survived for at least 2 4 hr in vitro, whereas cells from postnatal day 10-15 retinas died rap idly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner a fter the addition of FGF-2. In control cultures, 36% of originally see ded 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 no t caused by proliferation of photoreceptor precursors. Denaturing or b locking FGF-2 prevented enhancement of survival. Conversely, only 25.5 % of photoreceptors survived in the presence of epidermal growth facto r (EGF). FGF- and EGF-receptor mRNA and proteins were detected in puri fied photoreceptors in vitro, and addition of FGF-2 or EGF led to tyro sine phosphorylation of photoreceptor proteins. These data support a d irect mechanism of action for FGF-2 stimulation of photoreceptor survi val.