After injury, the immature central nervous system (CNS) has a greater propensity to promote wound repair and regeneration than the mature CNS. This difference is most likely due to the reduced growth-supportive environment of the adult CNS. For example, our previous studies show that CNS wound healing decreased after injury to older rodents. This decrease is paralleled by a decrease in the expression of NCAM, N-cadherin, and laminin, in which only laminin is reexpressed after injury to adults. Injury to the adult CNS but not the immature CNS also results in the increased expression of the growth-inhibitory molecules tenascin and chondroitin sulfate proteoglycan (CS-PG). Our preliminary results show that activated macrophages release cytokines and growth factors that stimulate astrocytcs to increase their expression of these molecules and that immunosuppressive drug treatments reduce the presence of these molecules after brain injury. This proposal is designed to test the hypothesis that wound healing and axonal regeneration within the CNS can be improved by controlling the regulatory mechanisms that induce the expression of growth-inhibitory molecules and by reintroducing specific adhesion molecules back into the wound cavity. To determine the factors that regulate the expression of laminin, tenascin, and CS-PG, astrocytes both in vitro (Specific Aim I) and in vivo (Specific Aim II) will be treated with cytokines and growth factors. Several immunosuppressive drugs will be tested to examine their effect in reducing the expression of the growth-inhibitory molecules (Specific Aim II) and promoting wound closure and axonal regeneration (Specific Aim III). Finally, immunosuppressive drug treatment will be coupled with the transplantation of astrocytes genetically engineered to express high amounts of NCAM, N-cadherin, or both to examine their combined effect on promoting wound closure and axonal regeneration (Specific Aim IV). Relative changes in the expression of these molecules will be quantified using Northern, immunoblot, and ELISA analyses. Immunohistochemistry will also be used to identify the location and number of astrocytes expressing these molecules and to examine the extent of glial scar formation and axonal regeneration. The importance of this work is threefold: (1) it will identify the injury- induced factors that regulate astrocyte production of growth-inhibitory molecules; (2) it will determine if reductions in the production of growth-inhibitory molecules improve wound healing and axonal regeneration; and (3) it will examine the interactions between the growth-supportive and growth-inhibitory molecules and how they effect CNS wound healing and axonal regeneration. The experiments outlined in this proposal will define the role of adhesion and extracellular matrix molecules in mediating astrocyte responsiveness in wound repair, thus forming the basis for future experiments related to the long term goal of reestablishing structural and functional continuity across lesions in the CNS.
