aFGF Promotes Axonal Growth in Rat Spinal Cord Organotypic Slice Co-Cultures

Abstract

This study developed a slice culture model system to study axonal regeneration after spinal cord injury. This model was tested in studies of the roles of acidic fibroblast growth factor (aFGF) and peripheral nerve segments in axonal growth between pieces of spinal cord. Transverse sections of P15–P18 Sprague-Dawley rat spinal cord were collected for organotypic slice cultures. Group I consisted of two slices of spinal cord in contact with each other during the culture period. Group II consisted of two slices that were separated by 3 mm and connected by two segments of intercostal nerves. Group III consisted of single slices for studies of neuron survival. Some cultures from each group included aFGF in the culture medium. Bromodeoxyuridine (BrdU) was included in the medium for some cultures. The results showed three principal findings. First, counts of neurofilament-positive cells demonstrated that treatment with aFGF significantly increased the number of surviving neurons in culture. Second, neurofilament immunostaining and DiI tracing demonstrated axons crossing the junction between the two pieces of spinal cord or growing through the intercostal nerve segments, and these axons were seen only in cultures with aFGF treatment. Third, few cells were double stained for neurofilament and BrdU, and these were found only with aFGF treatment. These results demonstrate that (1) organotypic slice cultures present a useful model to study regeneration from spinal cord injury, (2) aFGF rescues neurons and promotes axonal growth in these cultures, and (3) segments of intercostal nerves promote axon growth between slices of spinal cord.