Although functional regeneration occurs following peripheral nerve lesions, as yet, it has not been possible to promote clinically significant axonal regeneration in the adult mammalian central nervous system (CNS). Thus, the major emphasis of this research proposal is to analyze various cellular and molecular components of the peripheral nerve environment in order to identify factors that facilitate axonal growth for both PNS and CNS neurons. Since the immature CNS is capable of exhibiting considerable plasticity following injury, cellular and molecular components associated with immature CNS glia (astrocytes) also will be evaluated for their ability to foster axonal growth. Two basic experimental procedures will be used for these studies. First, in vitro septal explants will be grown in partially defined or conditioned media on a variety of substrates composed of glial cell layers (astrocytes and Schwann cells) or extracellular matrix molecules in order to identify culture conditions that facilitate neurite growth. Second, those in vitro environments that promote neurite growth from immature septal explant neurons will be adapted for transplantation in the CNS of adult rodents to further determine whether preparation that enhance neurite growth in culture also enhances regeneration from mature septal cholinergic neurons in vivo. These experiments will specifically evaluate whether: 1) CNS axonal growth is mediated by membrane associated molecules, 2) cell secreted diffusible factors enhance axon growth, and 3) components of the extracellular matrix (ECM) promote regeneration. Once individual cellular components have been identified as promoting axonal growth, then a final series of experiments will evaluate whether a combination of ECM components, diffusible factors and/or membrane associated molecules are necessary to promote optimal CNS regeneration. Immunocytochemical procedures will be used to identify specific cell phenotypes such as astrocytes, Schwann cells and cholinergic neurons and for localizing different molecular components in the culture and transplant substrates. A variety of neuroanatomical techniques will be employed for tracing axonal projections and for identifying the source of regenerating axons within the CNS. Results from the proposed in vitro explant studies and in vivo transplantation experiments will help characterize specific cellular components and extracellular matrix constituents that can stimulate regeneration following injury to the adult mammalian brain and spinal cord.
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