Bone morphogenetic proteins promote neurite outgrowth in retinal ganglion cells.
OBJECTIVE
The purpose of the present study is to test the ability of members of the transforming growth factor/bone morphogenetic protein family to influence retinal ganglion cell (RGC) survival and neurite outgrowth in primary cell culture using a high throughput analysis.


METHODS
Primary cell cultures were generated using immunoselection of Thy-1 positive cells from dissociated postnatal rat retina and grown on poly-L-lysine/laminin coated 96 well culture dishes in the presence or absence of members of the transforming growth factor/bone morphogenetic protein family. High throughput analysis was performed following fluorescence staining with Hoechst, Calcein AM, and TOTO-3. Outcomes included overall cell survival, survival of cells with neurite outgrowth, and a variety of parameters of neurite outgrowth.


RESULTS
Immunomagnetic selection led to an enrichment of cell cultures for RGCs (79%+/-6.8%). While no significant effect on overall survival was observed with any of the factors tested, members of the bone morphogenetic protein (BMPs) family (BMP2, BMP13, and GDF8 (growth differentiation factor 8)) and BDNF (brain derived neurotrophic factor) increased the number of surviving RGCs with neurite extension in a dose dependent manner. As a group, BMPs increased the number of neurites, length of neurites, and the number of branch points, while BDNF primarily increased neurite length and branch points.


CONCLUSIONS
We have developed an efficient system that allows for high throughput analysis of cultures enriched for RGCs. Using this assay system, we found that BMPs promote the survival of outgrowth neurons and neurite development in RGC culture.
Kerrison
John B
JB
Guerrieri Center for Genetic Engineering and Molecular Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. jkerris1@jhmi.edu
Lewis
Robert N
RN
Otteson
Deborah C
DC
Zack
Donald J
DJ
D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D002452:Cell Count; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000502:physiology; D016501:Neurites / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects*
D019485:Bone Morphogenetic Proteins; D019208:Brain-Derived Neurotrophic Factor