Basic fibroblast and epidermal growth factors stimulate survival in adult porcine photoreceptor cell cultures.
OBJECTIVE
To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas.


METHODS
Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways.


RESULTS
Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands.


CONCLUSIONS
The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.
D000818:Animals; D019393:Arrestin / Q000378:metabolism; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005136:Eye Proteins; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D051597:Hippocalcin; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010766:Phosphorylation; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology* / Q000378:metabolism; D051603:Recoverin; D017299:Rod Opsins / Q000378:metabolism; D013552:Swine; D014443:Tyrosine / Q000378:metabolism
D019393:Arrestin; D002135:Calcium-Binding Proteins; D004338:Drug Combinations; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D017299:Rod Opsins; D016222:Fibroblast Growth Factor 2; D051603:Recoverin; D051597:Hippocalcin; D014443:Tyrosine; D004815:Epidermal Growth Factor; D010751:Phosphopyruvate Hydratase