Enhanced affinity for lipophilic dyes in neurons damaged by ischemia or hypoxia.

BACKGROUND

Techniques for detecting neuronal death not only facilitate tracking disease progression but also serve as essential tools for validating experimental models and evaluating neuroprotective interventions. However, there is a scarcity of fluorescence methods that effectively reveal neuronal death and support multiplex fluorescent detection.

NEW METHOD: We present a method that utilizes several lipophilic dyes, including rhodamine R6 and DiOC6(3), designed for fluorescence microscopic detection of dying and dead neurons in both in vivo and in vitro ischemic models.

RESULTS

All the dyes tested exhibited either no staining or only faint staining in healthy neurons and non-neuronal cells but produced intense staining in ischemia-damaged neurons. Adjacent sections stained with DiOC6(3) and Fluoro-Jade B exhibited remarkable consistency in the spatiotemporal patterns of neuronal degeneration. Dual staining with rhodamine R6/Fluoro-Jade B, rhodamine R6/DiOC6(3), DiOC6(3)/Nile Red, and DiOC6(3)/Filipin III confirmed that different dyes shared identical cellular localization. DiOC6(3)/immunofluorescence co-staining showed that lipophilic dyes exclusively labeled damaged or dead neurons, with no detectable signal in reactive astrocytes or microglia. Moreover, lipophilic dyes effectively detected neuronal death induced by oxygen-glucose deprivation in vitro.

COMPARISON WITH EXISTING

METHODS

Rhodamine R6 and DiOC6(3) are known to primarily target the lipid bilayer of biomembranes. Consequently, the new detection technique is highly sensitive to organic solvents. These dyes provide staining with distinct fluorescence characteristics and can be easily combined with other fluorescent dyes or used in immunocytochemistry for multiplex fluorescence labeling.

CONCLUSIONS

This method offers several unique advantages and may have broad applicability in neuropathological research.