The long-term objectives of this project are to increase our knowledge of CNS trauma on the single cell level and to determine conditions which improve survivability of neurons and enhance recovery of injured cells. Modern warfare and the increased mobility of our technological society has greatly increased the risks of CNS injuries. However, limitations of existing techniques have forced investigators to focus primarily on damage at the organ and tissue level. An adequate understanding of the mechanisms underlying cell reactions to physical injury has not been attained. The introduction of methods of laser microbeam cell surgery developed in this laboratory constitutes a major step in the study of trauma on the single cell level. Three techniques can simulate transection, perforation, pressure wave and/or distortion injuries: (a) cytoplasmic vaporization and production of internal pressure waves; (b) substrate vaporization and production of external pressure waves; (c) photobiologically induced Ca++ release from mitochondria and subsequent local collapse of the cytoskeleton. The use of the near UV (337.1 nm) and short pulse lengths (12ns) minimizes heat diffusion and causes physical injuries that can be applied with a positioning accuracy of (plus and minus) 0.5 Mum at any locus along the cell. The size of the lesions range from a minimum of 0.7 Mum to more than 5 Mum depending on objectives and energy densities used. The lesions are applied through the same objectives used for microscopic observation making optical monitoring and electrophysiological recording of cellular reactions during and after the injury possible. The different types of lesions will be investigated with electron and light microscopy to determine characteristic sequences of deterioration. Membrane potential and injury currents will be monitored to determine the dynamics of injury development, consolidation and recovery and to define degrees of injury from which recovery is not possible in normal culture medium. We will also study the contributions made by the cellular locus of the lesion and determine the role played by exoplasmic transport in damage spread. We will test physical, chemical and pharmacological factors that promote cell survival and speed up recovery with emphasis on restoration of cytoskeletal stability and membrane integrity. Finally we will assess the regenerative potential of neurons after trauma.
