V. Fontaine et al., SURVIVAL OF PURIFIED RAT PHOTORECEPTORS IN-VITRO IS STIMULATED DIRECTLY BY FIBROBLAST GROWTH FACTOR-2, The Journal of neuroscience, 18(23), 1998, pp. 9662-9672
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.