A novel detection method for neuronal death indicates abnormalities in intracellular membranous components in neuronal cells that underwent delayed death

• Published in: Progress in neurobiology Vol. 226; p. 102461
• Main Authors: Wu, Shuang, Zhang, Yan-Feng, Gui, Yue, Jiang, Tian, Zhou, Cheng-Mei, Li, Jing-Yi, Suo, Jia-Le, Li, Yong-Nan, Jin, Rui-Lin, Li, Shu-Lei, Cui, Jia-Yue, Tan, Bai-Hong, Li, Yan-Chao
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-07-2023
• Subjects: Animals; Brain; Detection method; DiOC6; Fluorescent Dyes; Hippocampus; Kainic Acid; Mice; Neuronal death; Neurons; Rhodamine; Rhodamines; Rhodamine; rhodamine R6; FJ; PFA; OGD; DiOC6; Detection method; SE; TUNEL; DiOC2; KA; Neuronal death; Kainic acid; HE; Hippocampus; DiOC
• ISSN: 0301-0082; 1873-5118
• DOI: 10.1016/j.pneurobio.2023.102461

Acute neuronal degeneration is always preceded under the light and electron microscopes by a stage called microvacuolation, which is characterized by a finely vacuolar alteration in the cytoplasm of the neurons destined to death. In this study, we reported a method for detecting neuronal death using two membrane-bound dyes, rhodamine R6 and DiOC6(3), which may be associated with the so-called microvacuolation. This new method produced a spatiotemporally similar staining pattern to Fluoro-Jade B in kainic acid-damaged brains in mice. Further experiments showed that increased staining of rhodamine R6 and DiOC6(3) was observed only in degenerated neurons, but not in glia, erythrocytes, or meninges. Different from Fluoro-Jade-related dyes, rhodamine R6 and DiOC6(3) staining is highly sensitive to solvent extraction and detergent exposure. Staining with Nile red for phospholipids and filipin III for non-esterified cholesterol supports that the increased staining of rhodamine R6 and DiOC6(3) might be associated with increased levels of phospholipids and free cholesterol in the perinuclear cytoplasm of damaged neurons. In addition to kainic acid-injected neuronal death, rhodamine R6 and DiOC6(3) were similarly useful for detecting neuronal death in ischemic models either in vivo or in vitro. As far as we know, the staining with rhodamine R6 or DiOC6(3) is one of a few histochemical methods for detecting neuronal death whose target molecules have been well defined and therefore may be useful for explaining experimental results as well as exploring the mechanisms of neuronal death.
•KA-injured neurons show increased staining of rhodamine R6 and DiOC6(3).•Increased staining is present only in damaged neurons but not healthy neurons or glia.•The staining is spatiotemporally consistent with that obtained by Fluoro-jade B.•The staining is sensitive to solvent extraction and detergent exposure.•The staining is associated with increased membranous components in damaged neurons.