High magnification imaging of neuronal somata undergoing axon stretch growth in vitro

Axon Stretch Growth (ASG) is the process by which nerve fibers elongate in conjunction with growth of the enlarging body. During early embryonic development, neuronal growth cones traverse short distances to reach local synaptic targets. Subsequently, neuronal somata are separated from their targets by developmental growth of the enlarging organism. It is believed that mechanical forces, such as those due to skeletal growth, transduce a second mode of neuronal growth unique from growth cone extension. Such mechanotransduction results in the elongation of short processes into the long nerves characteristic of the adult nervous system. The majority of nerve regeneration studies focus heavily on the extension of growth cones, while neglecting affiliated body growth. Currently, regeneration of adult nerves by growth cone extension is limited to 3cm in total overall length [1, 2], despite the initial capacity for 30 Fold this limitation. As biomedical engineers, our objective is to define and characterize the role biomechanical forces play in promoting growth of the nervous system. Here, utilizing custom motion-controlled bioreactors, we developed a method to perform high magnification imaging of neuronal somata undergoing ASG. Embryonic rat cervical dorsal root ganglia neurons were dissociated and plated onto poly-d-lysine coated cover glass and stretch grown at 0.25mm/day for 1 week.