A new fluorescent histological marker for ischemic neurons, EA 50: Correlated with Fos and Jun/AP-1 immunoreactivity

Cerebral ischemia/hypoxia induces ischemic neuronal changes characterized by nuclear pyknosis, cytoplasmic shrinkage, and basophilia. The ischemic neurons were shown to exhibit strong and persistent c-fos proto-oncogene. The ischemic neuronal. changes and c-fos expression are thought to be the consequence of release of excessive glutamate by the ischemic neurons. In the present study, we investigated with immunohistochemistry the subcellular distribution of Fos and Jun/AP-1, the protein products of c-fos and c-jun proto-oncogenes, and compared them with histological changes shown by hematoxylin-eosin and by EA 50 stains. The latter is a stain mixture used traditionally in the Papanicolaou procedure and has a specific affinity for ischemic neurons. The active ingredient is eosin Y, a tetrabrominated derivative of fluorescein. With EA 50, the ischemic neurons stain red and emit a yellow fluorescence, while the non-ischemic neurons are green and non-fluorescent. The subcellular site of eosin Y binding corresponds with Fos and Jun/AP-1; all are concentrated in the nuclei and spread into the perikaryon, dendrites, and axons. The eosin Y-binding appears in neurons that have shown advanced ischemic changes. The dye is thus a good histological marker for damaged neurons, but requires freshly fixed tissues and paraffin sections of less than 4 mu m thick. Preincubation of tissue sections in antibodies against Fos and Jun abolishes the eosin Y binding, suggesting that the dye may interact with Fos/Jun/AP-1 protein or other protein products in the ischemic neurons.