A convenient in vitro assay for the inhibition of neurite outgrowth by adult mammalian CNS myelin using immortalized neuronal cells

Abstract

The adult mammalian CNS contains molecules which inhibit neurite outgrowth and which may be responsible for the lack of successful axonal regeneration after injuries in the brain and spinal cord. We describe an in vitro assay to measure the ability of primary and established lines of neuronal cells to produce neurites in the presence of CNS inhibitory molecules. The assay is suitable for identification of agents and treatments to overcome neurite growth inhibition. Assays are carried out in 96-well plates with CNS myelin substrates using NG108-15 cells, an immortalized cell line that can be induced to produce extensive neuritic growth. The inhibition of neurite outgrowth by CNS myelin observed in this assay is: (1) observed for NG108-15 cells and also PC12 cells and primary superior cervical ganglion neurons, (2) contact dependent, (3) half-maximal at 5 μg/cm 2 of myelin, and (4) trypsin-labile. This assay is quantitative, rapid, highly reproducible, convenient and can be used to test compounds which have the potential to overcome the growth inhibitory molecules present in CNS myelin.