title: Sodium-potassium adenosine triphosphatase inhibition enhances membrane accumulation of DiI in rat hippocampus in vivo.
abstract: 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.
authors.affiliation: Loeb Health Research Institute, 1053 Carling Avenue, Ottawa Hospital-Civic Campus, University of Ottawa, Ontario, K1Y 4E9, Ottawa, Canada.
authors.forename: A I
authors.initials: AI
authors.lastname: Omar
authors.forename: V V
authors.initials: VV
authors.lastname: Senatorov
authors.forename: B
authors.initials: B
authors.lastname: Hu
mesh_terms: 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*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; C024286:3,3'-dioctadecylindocarbocyanine; D010042:Ouabain; C018533:ethidium homodimer; D000254:Sodium-Potassium-Exchanging ATPase; D004996:Ethidium