Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: Effects of age and neurotrophic factors

Axon growth failure in the central nervous system (CNS) of adult animals is thought to be attributable to several factors, including an inadequate intrinsic growth response, the presence of inhibitory molecules, and a lack of adequate neurotrophic support. Here we use a new in vitro assay system to quantitatively assess growth of axons in cortex/spinal cord organotypic co-cultures from neonatal rats. Co-cultures of cortex and spinal cord were prepared from neonatal rats at P3 or P7, and by pairing cortex and spinal cords from different ages. Axon growth from the cortex to the spinal cord was assessed using DiI tract tracing techniques. Axons could be traced from the cortex to the spinal cord in co-cultures in which both tissues were obtained from P3 animals, whereas few axons crossed the cortex/spinal cord boundary in co-cultures from P7 animals. A larger number of axons could be traced across the boundary in co-cultures from P3 animals that were treated with neurotrophins (NGF, BDNF, or NT3), whereas neurotrophins produced minimal growth enhancement in P7 co-cultures. In mixed age co-cultures of P7 cortex with P3 spinal cord, moderate numbers of axons extended between the cortex and spinal cord when cultures were treated with neurotrophins, but few if any crossing axons were detected in co-cultures of P3 cortex with P7 spinal cords. These results indicate that successful growth of axons from the cortex to the spinal cord depends on the developmental age of the tissue terrain (the spinal cord and/or the interface between cortex and spinal cord explants), and to a lesser extent on the developmental state of the cortical neurons, and that axon growth between cortex and spinal cord can be enhanced by exogenous neurotrophins. These co-cultures provide a potentially useful assay for factors that affect axon growth that is intermediate between assays based on dissociated neurons and the intact tissue terrain.