carbocyanines and ethidium-homodimer

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.

Topics: Animals; Carbocyanines; Cell Count; Cell Death; Cell Membrane; Cell Size; Ethidium; Fluorescent Dyes; Hippocampus; Male; Microinjections; Microscopy, Fluorescence; Ouabain; Rats; Rats, Long-Evans; Sodium-Potassium-Exchanging ATPase