Survival of severed axons is critically dependent upon an ability to repair the damage (seal off cut ends) within minutes to hours after severance. However, very little is currently known about cellular mechanisms responsible for short-term repair of damaged axons. To obtain such data, we propose to use two invertebrate preparations, the squid giant axon (GA) and the earthworm medial giant axon (MGA) as model systems to: (1) Characterize seal formation and the specific mechanisms involved in effecting repair (e.g., cytoskeletal constriction, axolemmal fusion, and vesicle formation). (2) Determine factors responsible for glial and axonal changes associated with repair and irreversible changes, especially spongiform vacuolation, associated with lack of repair (degeneration). We have chosen to use GA and MGA preparations because they are large, which facilitates application of experimental techniques, and because mechanisms common to the two axons, which have different glial structure and which exist in organisms of different phylogenetic origin, are more likely to be typical of those found in higher organisms and humans. Seal formation will be characterized electrically by complex impedance measurements and ion-selective vibrating probe techniques. Seal formation and modifications of axonal/glial structures will be characterized morphologically by light microscopic (differential- interference-contrast and confocal) and electron microscopic observations. For this purpose, axons will be transected, dialyzed, or injected with solutions containing altered ion concentrations (especially Ca2+) and specific dyes. To localize Ca2+ at the electron microscope level in normal and experimental GAs and MGAs, x-ray microanalysis will be used. From comparisons of the response to stresses of the two model preparations, a better understanding of cellular mechanisms for axonal repair (sealing), and the consequences of not effecting repair (degeneration) will be assessed. This information should help formulate new strategies to improve the repair of injured axons in mammals and to ameliorate the treatment of neurodegenerative diseases displaying the pathology of spongiform vacuolation, such as scrapie, AIDS, Creutzfeldt- Jacob, and Alzheimer's disease.
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