Sodium-potassium adenosine triphosphatase inhibition enhances membrane accumulation of DiI in rat hippocampus in vivo.
Transient brain ischemia induces significant alterations in lipid structures of neuronal membranes, which are believed to result from lipid peroxidation and free radical attack. Such a membrane structural change may serve as an important histological marker of cell injury. In the present study, we examined how the dynamics of DiI/membrane incorporation may reflect early membrane metabolism and dynamic changes following sodium-potassium pump inhibition. Ouabain (1mM) was stereotactically co-administered with either 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate DiI (50 microg/ml) or ethidium homodimer (4 microM) into the granule cell layer of the adult rat hippocampus. Tissue was cryosectioned and examined with epifluorescence microscopy at 1, 2, 3, 4, 6, 8 and 72h post-injection. Alternate sections were stained with thionine or haematoxylin and eosin to evaluate morphological changes. Ouabain-induced pump inhibition resulted in a dramatic increase in DiI fluorescence in granule cell layer neurons as early as 4h post-injection. This increase in DiI incorporation coincided both spatially and temporally with the appearance of reactive changes characterizing early neuronal injury. However, the fluorescence increase was not a result of membrane breakdown because ethidium homodimer, a membrane-impermeable nucleic acid probe used for labeling cells with compromised membranes, when applied in a similar fashion, failed to show any fluorescence changes. The results of this study suggest that pump inhibition results in a specific increase in membrane lipophilicity possibly due to altered lipid structure.
Omar
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Loeb Health Research Institute, 1053 Carling Avenue, Ottawa Hospital-Civic Campus, University of Ottawa, Ontario, K1Y 4E9, Ottawa, Canada.
Senatorov
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Hu
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D000818:Animals; D002232:Carbocyanines / Q000008:administration & dosage; D002452:Cell Count; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D004996:Ethidium / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000493:pharmacokinetics; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008845:Microinjections; D008856:Microscopy, Fluorescence; D010042:Ouabain / Q000008:administration & dosage; D051381:Rats; D020318:Rats, Long-Evans; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*
D002232:Carbocyanines; D005456:Fluorescent Dyes; C024286:3,3'-dioctadecylindocarbocyanine; D010042:Ouabain; C018533:ethidium homodimer; D000254:Sodium-Potassium-Exchanging ATPase; D004996:Ethidium