The long term objectives of this project are to induce functional regeneration in the adult nervous system. To this end, we are conducting experiments in the well-defined fimbria-fornix (FF) lesion model and in an analogous model using lesions to the perforant path (PP). The FF model has been used to demonstrate the effectiveness of NGF administration to spare lesioned septal neurons and promote their regeneration. Specifically, using the FF model we plan to: 1) Determine if neurotrophic factors other than NGF enhance neuronal survival and regeneration from the lesioned septum and what other neuronal phenotypes are responsive. 2) Determine whether and which specific surface molecules may be located on regenerating axons or substrate cells that are involved in elongation and guidance in regeneration. 3) Determine if providing additional trophic/tropic factors can enhance target innervation and functional recovery.
The specific aims of the PP model experiments are to: 1) Prevent death and degeneration of entorhinal layer II neurons by administration of trophic factors. 2) Induce regeneration of entorhinal fibers across the trophic bridging graft.
These aims will be accomplished by placing collagen-plug bridging grafts containing fibroblasts genetically engineered to produce NGF, NT3, beta-FGF, BDNF, SDGF, or beta-Gal into the lesion cavities. Sections will be assessed by brightfield or multiple-fluorescent immunolabeling using confocal microscopy to determine responsive neuronal phenotypes and extent of regeneration. Quantitation of neuronal number in the septum or entorhinal cortex will be made using unbiased stereology and assessment of fiber density through the graft by computer-assisted optical density measurements. Surface molecules will be investigated using confocal microscopy of identified antibodies against tenascin, laminin, astrotactin, NILE, N-cadherin, and N-CAM. Specificity of innervation will be assessed using electron microscopy. A battery of behavioral tests will be used to determine if and which of the specific neuronal systems regenerated and result in functional recovery. By extending our studies from the FF model to the PP model, we will test the generality of this approach toward adult CNS regeneration.
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