In order to understand better how gliotic wound tissue of the CNS can be manipulated in order to allow for improved regeneration following CNS trauma or disease, the goals of the experiments described in this proposal are to understand the mechanisms that trigger astrocytes to become reactive and how to modify astrogliosis in order to overcome the repulsive elements made by reactive cells. We have discovered that insoluble B-amyloid (B-AP) peptides when placed on nitrocellulose into the neonatal rat cortex or when used as a substrate in vitro, trigger the increased synthesis by reactive astroglia of a potent neurite repulsive chondroitin sulfate proteoglycan (CS-PG) containing matrix that is deposited over the surface of the peptide. We will use these assays to test for regional and age specificity of the astroglial response to learn whether all types of astrocytes from various regions of the CNS respond equally to the gliotic trigger. We will study whether various cytokines and trophic factors can modify the production of inhibitory ECM stimulated by B-AP or other potential gliosis triggers. Modifications with chondroitinase or antibodies of the inhibitory factors in the matrix made by reactive astrocytes and culture of neurons on the treated or untreated matrix or on top of the reactive cells themselves will allow us to determine.-whether the reactive cells or the reactive matrix can support robust axonal outgrowth once the suspected inhibitor is nullified. We will purify and characterize in substrate assays the B-AP induced gliotic matrix and compare it to purified inhibitory PG's induced in vivo by trauma. We will generate antibodies specific to the most actively inhibitory PG elements for use in a variety of future experiments. Experiments are planned to nullify the reactive glial matrix inhibitor with the use of enzymes or antibodies in two models of regeneration through the glial scar in vivo. Identification and modification of the molecular components in the scar that give rise to the growth refractory gliotic state will allow us to suggest strategies for enhancing regeneration in situations where gliosis is a barrier to regeneration.
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