Effect of a 30 kD protein from tectal extract of rat on cultured retinal neuron.

For the first time we have shown here that the constituent from tectal extract (Te) which can support and promote the survival and growth of the retinal ganglion cell is a 30 kD protein. (i) Using MTT colorimetric microassay to measure the optical density for the survival and growth of the cultured retinal neuron, it was found that the optical densities for the experimental cultures with either Te, or its 10-30 kD fraction, or its greater than or equal to 30 kD fraction were 2-4 times that of the control culture without Te (P less than 0.01). This indicated that experimental cultures were more active in growth. (ii) The retinal neurons cultured on Te Phast gels showed that large retinal neurons (greater than 18 microns) grew only on the gel region containing the 30 kD protein. The retrograde prelabelled with horseradish peroxidase (HRP) for retinal ganglion cells indicated that these surviving neurons grown on Te Phast gel were HRP positive, i.e. retinal ganglion cells. (iii) The retinal explants cultured with 30 kD gels at a close distance of 1 mm apart revealed that many neurite outgrowths, up to 400 microns, extended from the retinal explants towards 30 kD gels, and that many individual cells and tissue masses migrated out from the explants and grew onto 30 kD gels. They were stained anti-neuron specific enolase and anti-Thy 1.1 positive, indicating they are retinal ganglion cells. Therefore, we concluded that the 30 kD protein is the neuronotrophic factor in the tectal extract specific for retinal ganglion cells.