FIGURE 3.

Administration of REL-1017 did not cause neuronal necrosis in brain cingulate cortex. (A) Upper row: histological examination (H&E) of the cingulate cortex at Day 3 and 5. Black asterisk marks indicate the presence of necrotic cells, appearing as shrunken, angular cells with pyknotic nuclei and bright eosinophilic cytoplasm, in the brains of animals treated with MK-801 (positive control). Lower row: representative photographs of Fluoro Jade B immunofluorescence, which is a marker of cellular necrosis. Necrosis was present in the cingulate cortex and piriform lobe of MK-801 treated rats on Day 3 and 5 in 100% of the rats (M/F) (white arrowheads indicate some necrotic neuronal nuclei). Consistent with the histological analysis, Fluoro Jade B immunofluorescence was absent in vehicle, REL-1017, or racemic methadone treated rats. (B) Quantification of the severity of neuronal necrosis showed a statistical increase in cell necrosis in MK-801 treated brains (p < 0.0001 versus all the other groups on Day 3 and 5). Necrotic findings were classified as moderate to marked (ranging from 5 to 40% of necrotic cells).