Growth from regenerating goldfish retinal cultures in the absence of serum or hormonal supplements: tissue extract effects

Abstract

The minimal requirements for the regeneration of optic nerve fibers in vitro were established in a serum-free retinal explant preparation. This serum-free preparation was developed as a prerequisite for testing the growth-promoting activity of tissue extracts prepared from the primary target of regenerating fibers. Explants taken from goldfish retinas 14 days after a prior optic nerve crush were capable of long-term survival and regenerated neurite outgrowth without serum or hormonal supplements. Serum-free conditions for explant outgrowth required only a basic Leibovitz (L-15) media containing 0.6% methyl cellulose (MC). Explants were also capable of neurite outgrowth in L-15 media alone when culture dishes were preplated with MC. MC treatment permitted both the regeneration of neurites in serum-free L-15 and a significant increase in the rate and extent of neurite outgrowth when combined with 10% fetal calf serum (FCS). Explants grown in L-15 with both MC and FCS produced a 2.5-fold increase in the length of neurite outgrowth over MC alone and a 1.5-fold increase in the length of neurite outgrowth over FCS alone. MC activity which permitted minimal serum-free regeneration and optimal serum supplemented regeneration was determined to be substrate related. Retinas were dissociated to determine if ganglion cells, like the intact explant, were capable of survival and neurite regeneration in serum-free conditions. These cells survived and extended long neurites when grown in L-15 with FCS or with FCS and MC, but they did not survive in serum-free L-15 with MC. The minimal serum-free conditions for explant survival and neurite regeneration were used as a model system to test the growth-promoting activity of crude tissue extracts prepared from the goldfish brain. Extracts prepared from the primary target region, the optic tectum, stimulated a significant 2.5-fold increase in the length of regenerating neurites. The optic tectal extract (OTex) stimulated outgrowth with significantly high specific activity when compared with extracts of identical protein concentrations prepared from the cerebellum (Cex). At a minimal protein concentration of 150 micrograms/ml, the OTex stimulated a 1.5-fold increase in neurite outgrowth above Cex. These results indicated that a serum-free culture preparation had been established for optic nerve regeneration. This culture system has proven to be an extremely sensitive bioassay model without the masking effect of a serum supplement. Serum-free cultures may be used in further studies to determine the role neurotrophic factors may play in a widely used model of successful central nervous system (CNS) regeneration.