ラットの断頭脳および superoxide 処理脳における Na^+-K^+ATPase 活性の電顕酵素組織化学的研究

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Na^+-K^+-adenosine triphosphatase (Na^+-K^+ATPase) was studied ultracytochemically in decapitated and superoxide-treated rat brains in order to elucidate the relationship between enzyme activity and damage of membranous structures. All experiments were performed on male adult Wistar rats. The decapitated group consisted of 12 animal brains removed 3, 6, and 12 hours after decapitation. The normal control consisted of 4 animal brains removed immediately after decapitation. The superoxide-treated group consisted of 9 animal prefixed brains immersed in a hypoxan-thine-xanthine oxidase system for 20, 60, and 120 minutes. In both groups, 1 mm sagittal sections of cerebellar vermis were fixed for 50 minutes in a mixed solution of 0.25% glutaraldehyde and 1% paraformaldehyde (0.1 M cacodylate buffer, pH 7.3) at 0℃. Non-frozen sections, approximately 15 μm thick for light microscopy and 40 μm thick for electron microscopy, were prepared by a microslicer. Histochemical procedure was carried out by incubating specimens in the substrate medium using lead citrate as capturing reagent for 40 minutes at 37℃. Following the incubation, specimens were embedded in Spurr's resin. Ultrathin sections were stained in 2% uranyl acetate for 3 minutes at 20℃. Three different kinds of medium (substrate free, substitution of Na^+ for K^+, and ouabain addition) were utilized for control in each experiment. In the normal control Na^+-K^+ATPase reaction was microscopically detected mainly at the cerebellar glomerulus. Electron microscopy revealed the reaction product localized on postsynaptic membranes, plasmalemmas of both axon and dendrite, and filamentous structures of axonal cytoplasm in glomerulus. However, the reaction was negative in mossy fibers and soma of granular cells. Na^+-K^+ATPase activity was almost unchanged 3 hours after decapitation. It decreased considerably in 6 hours, and disappeared in 12 hours. In contrast, Na^+-K^+ATPase activity changed dynamically after superoxide treatment. 60-minute peroxidation caused considetable increase in the activity as compared with both normal control and 20 minute-treated brains, although apparently irreversible histological changes had already occurred. Such increase in the enzyme activity seemed to be rather larger than the degree of membranous damages. Na^+-K^+ATPase had completely diminished by 120-minute peroxidation. These findings show that damage of the plasmalemma precedes the disappearance of Na+-K-F-ATPdse activity in ischemic brains. The remaining Na^+-K^+ATPase activity does not always suggest the reversibility of ischemic lesions.
日本脳神経外科学会の論文
1986-11-15

ラットの断頭脳および superoxide 処理脳における Na^+-K^+ATPase 活性の電顕酵素組織化学的研究

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