A Quantifiable Model of Axonal Regeneration in the Demyelinated Adult Rat Spinal Cord

Strategies to increase the extent of axonal regeneration in the adult CNS must address an array of intrinsic and environmental factors which influence neuritic outgrowth. In order to develop anin vivomodel of axonal regeneration in which potential therapies may be assessed, we have quantified growth cones within demyelinated regions in the dorsal funiculus of the spinal cord, following a discrete axotomy. Demyelinated lesions were produced by the intraspinal injection of galactocerebroside antibodies plus serum complement proteins. Axonal integrity was not compromised by the demyelination protocol. Axonal injury was induced at the caudal extent of the demyelinated region using a micromanipulator-controlled Scouten knife. The severity of axonal injury was varied in different animals at the time of surgery and was quantified 8 days later by counting degenerate axons in transverse 1-μm resin sections. Evidence of axonal regeneration within these animals was assessed by an electron microscopic analysis of growth cone frequency and position relative to the site of axotomy. Growth cones were identified within the region of demyelination only; no growth cones were identified within the dorsal column white matter adjacent to the demyelinated region, or rostral or caudal to the region of demyelination, or in animals with an injury but no demyelination. Quantification of growth cones within regions of demyelination indicated a strong linear relationship (P< 0.001) between the number of growth cones and the number of axons severed. These findings indicate that demyelination facilitates axonal regeneration in the adult rat CNS and illustrate a quantifiable method of assessing axonal regeneration.