Lamprey axons regenerate accurately after spinal transection. In lamprey and other animals that show regeneration of CNS axons, glial cells are connected through a network of desmosomes and contain keratin intermediate filaments instead of glial fibrillary acidic protein (GFAP). The absence of this type of glial cell in mammals may be an important reason for failure of regeneration in human spinal cord injury. Based on preliminary data, it is postulated that desmosomes and their keratin filament network prevent glial cells from migrating in response to injury. This preserves glial pathways that guide regenerating axons in the proximal and distal stumps. The anchored glial cells revert to a less mature phenotype and send longitudinally oriented processes into the injury that form a scaffold along which axons may regenerate in their correct orientation. In order to test this hypothesis, desmosomes will be disrupted by applying ECTA and antidesmosomal antibodies to a spinal transection and the effect on glial migration and axonal regeneration will be compared with that in control animals and similarly lesioned rats. Glial migration will be determined by fluorescence microscopy after microinjecting Di-I near a transection. The effect of perturbing the glial microenvironment on the probability of regeneration will be measured by injecting HRP caudal to the transection and counting retrogradely labeled reticulospinal neurons. The effect on directional specificity of regeneration will be assessed in Muller and Mauthner axons by intra- axonal injection of HRP. The orientation of glial processes and small axons in the scar will be evaluated by immunohistochemistry and EM. Behavioral measures will determine the functional significance of altered regeneration patterns. Preliminary data suggest that the ability of glial cells to extend long processes into a lesion is accomplished by a reversion to a less mature form, similar to that found in the brain. This is reflected in both their morphology and keratin composition. Immunohistochemical, biochemical and EM studies will be performed on brains and spinal cords of animals at different developmental stages to determine whether brain glia are less mature than spinal cord glia in the same animal and whether injured spinal cord glia revert to this less mature phenotype. Finally, the elements responsible for attracting longitudinally oriented glial processes into the lesion will be identified by replacing short excised lengths of spinal cord with grafts containing various cell types. Glial fiber elongation and axonal orientation within the lesion will then be assessed. Once the tissue elements responsible for inducing glial fiber elongation are found, future experiments will determine the molecular mechanisms involved. These experiments will help to explain why regeneration of central axons is more successful in certain lower vertebrates than in mammals and may lead to more effective ways to induce functional regeneration in human spinal cord or brain injury.
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