CYTOLOGICAL STUDIES OF DEVELOPING AND MATURE NEURONS

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The work proposed is a critical portion of one of the central goals of The Miami Project to Cure Paralysis; to thoroughly explore the use of Schwann cell (SC) transplantation in efforts to favorably influence the response of neural tissue to injury and disease. We believe that the remarkable capability of the SC to influence regenerative responses in both peripheral and central neural tissue indicates potential for use in human neural injury, particularly spinal cord injury. We outline a series of complex tissue culture experiments aimed at gaining a fuller understanding of the basic biology of the SC, especially of the human SC. We believe a major effort to study the biology of the human SC is required, for many of its basic characteristics appear to be different from those that have been elucidated from the study of rodent tissue. We first propose to study the mechanisms by which rat SC function is controlled by basal lamina (BL) assembly for we believe this aspect of the regulation of SC function is critical to understanding its ability to attain full functional capacity in central neural tissues. We will use a myelinating tissue culture system to study the linkage between expression of myelin-specific proteins and BL assembly, and will explore the role of the cell adhesion molecule L1 in the signalling which influences early SC development. We seek further to determine whether SC surface integrins are involved in the regulation of SC function and whether the cytoskeleton of the SC is involved in the signalling which allows SC function to go forward as BL is assembled. Having arranged for a reliable source of viable human peripheral nerve, we propose to use tissue culture methods to improve procedures for separating SCs from human adult peripheral nerve, and to study the cellular, soluble and matrix factors which regulate human SC proliferation. Experiments are proposed to determine the capacity of the human SC to produce extracellular matrix (ECM) components and to determine whether ECM deposition regulates SC ensheathment and myelination by the human SC, as it does in the rat. We will study the ability of the human SC to provide trophic support for neural crest- derived sensory neurons. In order not to limit our observations on human SC function to tissue culture studies, we have developed a method of introducing human SCs into guidance channels placed to bridge gaps in the immune-deficient rodent peripheral nerve, and will use this system to study the regulation of human SC proliferation in vivo, as well as the ability of human SCs to promote peripheral axonal regeneration. Finally, we propose to introduce a recombinant retrovirus which contains sequences for both beta-galactosidase and basic fibroblast growth factor and to subsequently compare the ability of the infected cells to support neuronal health and induce neurite growth, as well as to determine how this genetic change alters the ability of the SC in undertaking its basic function of axonal ensheathment, BL assembly, and myelination. These basic studies of SC biology are considered essential to any consideration of use of this cell in autotransplantation in human patients with neural injury. The potential of this cell to influence axonal regeneration as well as to repair demyelination gives this work considerable urgency.