Axonal connections within the white matter of the central nervous system play the crucial role of transmitting electrical signals. Common and devastating diseases such as stroke, spinal cord injury and multiple sclerosis almost always cause damage to white matter structures, yet far less is known about the pathophysiology of white matter injury. Despite the lack of synaptic machinery in this tissue, early reports indicate that glutamate-dependent excitotoxic mechanisms play an important role in mediating white matter injury. This application aims to examine in greater detail how endogenous excitotoxins damage myelinated axons. Using the in vitro rat optic nerve and spinal dorsal columns as well studied models of isolated white matter, electrophysiology and immunchistochemistry for injury markers in myelin, axoplasm and glial cytoplasm will be used to examine the effects of exogenously applied excitotoxins such as glutamate, kainite, and ANIPA. Selective inhibitors will be applied to dissect out which subclass of ionotropic glutamate receptor(s) are responsible for injury. Abnormal fluxes of Na and Ca ions will be examined using ion-sensitive dyes and confocal microscopy to see which compartments (myelin, axon cylinder, glia) suffer excess accumulations as a result of glutamate receptor activation. Total (free + bound) elemental analysis of Na and Ca will be performed with electron probe x-ray microanalysis as the ionized fraction may underestimate the total amount of Na or Ca entry and may be a more reliable determinant of subsequent functional injury. The role of endogenous glutamate, released non-synaptically by in vitro anoxia or ischemia, will be studied using a simlar approach, with the goal of determining which sub cellular compartments suffer ionic overload and structural injury that is dependent on activation of glutamate receptors. Immunchistochemistry and high-resolution confocal microscopy, coupled with digital image processing techniques for resolution enhancement and 3-dimensional reconstruction, will be applied to examine the distribution of glutamate receptors in white matter using specific antisera. By elucidating glutamate-dependent injury mechanisms in CNS white matter, it is hoped that an important new avenue wil1 become available for pharmacological protection of this key tissue.
