AN ORGANOTYPIC MODEL OF HEXACARBON "DYING-BACK" NEUROPATHY
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This thesis describes the development of an organotypic model of hexacarbon "dying-back" disease and its use in unravelling the etiology and pathogenesis of a toxic neuropathy. "Dying-back" is a common type of neurological degeneration which is associated with exposure to a number of industrial and pharmaceutical compounds, among them the widely used industrial solvents n-hexane and methyl n-butyl ketone (MnBK). Although experimental animal models of hexacarbon disease exists, the development of an in vitro organotypic model using living nerve and muscle tissues, afforded unique advantages in assessing toxicological and pathogenic aspects of this neuropathy.;The aim of this thesis involved developing an organotypic culture model which paralleled the spatial-temporal pattern of nerve fiber damage seen in animals and humans exposed to neurotoxic hexacarbons. Once established, the metabolic competence of this model was examined with respect to hexacarbon conversion. A scheme of relative neurotoxicity among the six interrelated hexacarbon metabolites was established and common neurotoxic metabolites identified. The hypothesis of hexacarbon - inhibition of glycolysis was examined in tissue culture by pyruvate supplementation.;The organotypic complex consisted of fetal mouse spinal cord and ganglion, co-cultured with muscle. These explants were allowed to mature into a functionally and structurally coupled sensory-motor unit. Once established, chronic, acute and threshold levels were established for n-hexane, MnBK and their interrelated metabolites, 2-hexanol, 2,5-hexanediol, 5-hydroxy-2-hexanol, and 2,5-hexanedione and its molecular analogue, 2,4-hexanedione. Using chronic non-cytotoxic levels of each hexacarbon, mature cultures were intoxicated for 4-8 weeks and the sequence of induced pathological changes documented using brightfield, Nomarski differential and electron microscopy. The recovery of toxin-damaged axons were also examined. The pattern and morphological structures associated with this disease in vivo were examined using the organotypic system.;Once confidence was established in the validity of this model, the questions of metabolic competence, relative neurotoxic potency and primary toxicity were addressed. These issues were elusive on an in vivo level because of systemic biotransformational influences. Organotypic cultures were exposed to chronic concentrations of individual metabolices. Exhausted nutrient fluids, removed from each test group, were analyzed for catabolic products using collaborative gas-liquid chromatography (GLC) to examine the cultures ability to metabolize hexacarbons. These data reported that the nerve-muscle explants were able to oxidize and reduce the parent hexacarbons n-hexane and MnBK to their in vivo metabolic counterparts. Thus organotypic metabolism paralleled that seen in vivo. Although each metabolite produced "dying-back" pathology in culture, GLC data reported the recurring presence of 2,5-hexanedione (2.5-HD) in the exhausted nutrient fluids removed from all groups. Homogenizing MnBK treated cultures revealed tissue-bound metabolites which were not detected in the exhausted fluids.;The relative toxicity was established among hexacarbon metabolites by using equimolar concentrations of each compound. A distinct pathological stage was used as an end-point to evaluate their relative neurotoxic potency.;This thesis has established the validity of using organotypic cultures to study toxin-induced neuropathies. Acting in concert with biochemical and in vivo studies, this system provides a promising experimental tool for evaluating the effects of neurotoxic chemicals on the peripheral and central nervous system.