Use of explant cultures of peripheral nerves of adult vertebrates to study axonal regeneration in vitro

Explanted preparations of peripheral nerves with attached dorsal root ganglia of adult mammals and amphibia survive for several days in serum-free medium and can be used to study axonal regeneration in vitro. This review outlines the methods which we routinely use and how they may be applied to study different aspects of axonal regeneration. When the peripheral nerves are crushed in vitro, axons regenerate through the crush site into the distal stump within 1 day (mouse) or 3 days (frog). The outgrowth distance of the leading sensory axons can be determined with the use of a simple method based on axonal transport of labelled proteins. A compartmentalised system permits selective application of drugs and other agents to either ganglia or peripheral nerve containing the regenerating axons and has been used to study selected aspects of regeneration including influence of non-neuronal cells, retrograde signalling, axonal release of proteins during regeneration and the role of phospholipase A₂ activity. Explanted preparations may also be cultured in a layer of extracellular matrix material (matrigel), in which spontaneous outgrowth of a large number of naked axons from the cut ends of nerves starts within 1 day and continues for several days. This provides an opportunity to study the direct effects of different agents on axonal elongation. Preparations cultured in collagen gels show sparse spontaneous axonal growth, but this can be increased by addition of certain growth factors. The phenotype of the regenerating axons can be studied using immunohistochemical methods.