Defining the concentration gradient of nerve growth factor for guided neurite outgrowth.

The developing axon is believed to navigate towards its target tissue in response to a concentration gradient of neurotrophic factors, among other diffusible and surface-bound stimuli. However, the minimum concentration gradient required for guidance over the maximum distance is still unknown, largely because well-defined systems have not been utilized to address this question. In this study, a linear concentration gradient of nerve growth factor was achieved across a 5-mm agarose membrane that separated a nerve growth factor source compartment from a sink compartment. The concentrations in both compartments were maintained constant (and different). Both concentration and concentration gradient were well defined across the membrane, allowing us to study the relative importance of concentration gradient vs concentration for neurite guidance. The orientation of PC12 cell neurites was studied in response to a series of nerve growth factor concentration gradients in vitro. For effective guidance of PC12 cell neurite outgrowth, a minimum concentration gradient of 133ng/ml per mm was required, below which guidance was ineffective. Higher gradients were effective for guidance yet were limited by the concentration of nerve growth factor in the source compartment. At a nerve growth factor concentration of 995ng/ml, the PC12 cells' receptors were saturated, thereby limiting the maximum effective distance for guidance to less than 7.5mm in response to a diffusible nerve growth factor cue. This distance exceeds the 0.5-2mm distance observed by others for effective neurite guidance. Using this model system, we propose that the minimum concentration gradient can be defined for other cells and growth factors. Ultimately, it is anticipated that such concentration gradients could be included in a device to promote regeneration.