Head and spinal cord injury are major health care issues, causing immense hardships to the victims and their families. The initial trauma is worsened by secondary destructive processes, including lipid peroxidation. Better understanding the mechanisms of secondary damage accompanying central nervous system trauma should pave the way to developing improved pharmacological methods of disrupting the cascade of processes contributing to secondary damage. It has been hypothesized that free radicals attack polyunsaturated fatty acids in cell membranes, triggering free radical chain reactions that destroy membrane phospholipids. Although a variety of free radical species may be involved in peroxidative damage hydroxyl radical is the most destructive of these. Two major pathways for generating hydroxyl radical in vivo have been proposed. One is the iron-catalyzed Haber-Weiss reaction. Another is NO-medicated OH generation. This project is to unequivocally determine whether free radicals in these two pathways contribute to secondary injury through peroxidative degradation of membrane phospholipids and to provide information of the roles of these free radicals in spinal cord injury.
The specific aims are: 1. To obtain time courses of superoxide, hydroxyl radical, nitric oxide and peroxynitrite release following in vivo impact injury to the rat spinal cord. 2. To generate the free radicals involved in the two pathways in vivo in the rat spinal cord at the concentration and duration that mimic release upon trauma and characterize the damage they cause neurochemically and histologically. A model well established in the P.I.'s laboratory will be used.
for Aim 1, a microdialysis fiber or a push-pull cannula will be inserted laterally into the spinal cord, and released species will be analyzed by measuring products of reactions of free radicals in microdialysates or perfusates. The measurements will be made specific by administering free radical scavengers or inhibitors through the dialysis fiber or cannula. The results will provide sound in vivo tests of whether these free radicals are indeed released following trauma.
For Aim 2, microdialysis and a push-pull cannula will be used to apply free radical precursors and pertinent enzymes into the spinal cord to generate free radicals, and to sample released substances. As a marker of peroxidation, malonyldialdehyde (MDA) will be measured from microdialysates during application of the free radicals to correlate free radical production and peroxidative damage in in vivo experiments. The other radicals measured from microdialysates during one radical generation will also provide information on the pathways of hydroxyl radical generation. Histological examination will reveal whether these free radicals damage neurons.
