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

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.

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.

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.

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.

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