Bone morphogenetic proteins promote neurite outgrowth in retinalganglion cells

摘要

Purpose: The purpose of the present study is to test the ability ofmembers of the transforming growth factor/bone morphogenetic proteinfamily to influence retinal ganglion cell (RGC) survival and neuriteoutgrowth in primary cell culture using a high throughput analysis.Methods: Primary cell cultures were generated using immunoselectionof Thy-1 positive cells from dissociated postnatal rat retina and grownon poly-L-lysine/laminin coated 96 well culture dishes in the presenceor absence of members of the transforming growth factor/bonemorphogenetic protein family. High throughput analysis was performedfollowing fluorescence staining with Hoechst, Calcein AM, and TOTO-3.Outcomes included overall cell survival, survival of cells with neuriteoutgrowth, and a variety of parameters of neurite outgrowth.Results: Immunomagnetic selection led to an enrichment of cellcultures for RGCs (79%±6.8%). While no significant effect on overallsurvival was observed with any of the factors tested, members of thebone morphogenetic protein (BMPs) family (BMP2, BMP13, and GDF8 (growthdifferentiation factor 8)) and BDNF (brain derived neurotrophic factor)increased the number of surviving RGCs with neurite extension in a dosedependent manner. As a group, BMPs increased the number of neurites,length of neurites, and the number of branch points, while BDNFprimarily increased neurite length and branch points.Conclusions: We have developed an efficient system that allows forhigh throughput analysis of cultures enriched for RGCs. Using this assaysystem, we found that BMPs promote the survival of outgrowth neurons andneurite development in RGC culture.