Neurotrophic stimulation of fetal rat retinal explant neurite outgrowth and cell survival: age-dependent relationships

Abstract

Serum-free tissue culture conditions have been defined where stimulation of neurite outgrowth from fetal rat retinal explants occurred only in the presence of an active fraction (BE) prepared from a pig brain extract purification procedure. Under these conditions, 18-20-day fetal retinal explants survived and continued to extend long radial neurites for at least 3 weeks in the presence of BE. However, if fibronectin was not equilibrated onto the basic collagen/poly-L-lysine substrate the neurite outgrowth was restricted to a short halo about the circumference of the explant. In addition, a dose-response relationship was demonstrated in the presence of increasing concentrations of BE with respect to the neurite growth index. The half-maximal response for BE was estimated to be between 5 and 10 micrograms/ml. In addition a number of important age-dependent relationships were observed with respect to BE stimulation of retinal neurite outgrowth and cell survival. An inverse relationship was demonstrated between increased developmental age and responsiveness to BE. After 1 week in culture, there was a 3-fold reduction in retinal neurite length measured from the 2-day neonatal explant when compared to that of the 18-day fetus. There was also a significant inverse relationship demonstrated between the length of time before BE was added to the culture medium and the ability of 20-day fetal explants to extend neurites onto the culture substrate. If BE was added as late as 2 weeks after initial explant culture, the various neurite outgrowth indices were significantly lower than in those situations where BE was added at the time of culture or 1 week later. These results imply that BE not only is required for stimulating neurite outgrowth from fetal rat retinal explants, but may be important in survival and maturation of developing retinal neurons. This hypothesis was confirmed when morphometric analysis was performed on 16- and 20-day explants cultured for a week in the presence or absence of BE. The number of necrotic cells in the developing retinal ganglion plexiform-cell layer of 20-day fetal explants was significantly lower when treated with BE. Conversely, the density of identifiable differentiating retinal ganglion-like cells was significantly greater in response to BE treatment in both 16- and 20-day retinal explants.