Flat, whole-mount nerve preparations: a useful tool for studying the process of regenerating axon outgrowth

A method, which is based on the use of flat, whole-mount nerve preparations, has been developed for studying the process of regenerating axon outgrowth, employing the rat sciatic nerve as a model. At various intervals after a nerve crush, animals are perfused with aldehyde fixatives, the nerve dissected out, and its epineurium removed. Next the nerve is flattened between two glass slides, removed and reacted (floating), then whole-mounted on a micro slide and cover-slipped. Regenerating axons have been labeled by means of the horseradish peroxidase tracing technique, a histochemical technique for acetylcholinesterase, or an indirect immunocytochemical technique utilizing antibodies against tubulin. With all these techniques, individual outgrowing axons and their bundles can be clearly visualized. Regenerating axons labeled by horseradish peroxidase are readily traced along their entire undulating courses from the distal margin of the crush zone to axonal tips, which mark the leading edge of several waves of outgrowing axons. It appears that such flat, whole-amount nerve preparations can be useful for obtaining: (1) accurate estimates of the rate of regenerating axon elongation, (2) values characterizing the duration of the initial delay of axonal outgrowth, and (3) information concerning the nature of axonal subpopulations that elongate at different rates.