Expressions of nitrotyrosine and TUNEL immunoreactivities in cultured rat spinal cord neurons after exposure to glutamate, nitric oxide, or peroxynitrite

Although excitotoxic and oxidative stress play important roles in spinal ne uron death, the exact mechanism is not fully understood. We examined cell d amage of primary culture of 11-day-old rat spinal cord by addition of gluta mate, nitric oxide (NO) or peroxynitrite (PN) with detection of nitrotyrosi ne (NT) or terminal deoxynucleotidyl transferase-mediated dUTP-biotin in si tu nick end labeling (TUNEL). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immunoreactivity for NT became stronger in the cytoplasm o f large motor neurons in the ventral horn at 6 to 48 hr and positive in the axons of the ventral horn at 24 to 48 hr. TUNEL positive nuclei were found in spinal large motor neurons from 24 hr, and the positive cell number gre atly increased at 48 hr in contrast to the vehicle. Pretreatment of culture s with alpha -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/ka inate receptor antagonist, NO-suppressing agent, and antioxidant protected the immunoreactivity for NT or TUNEL. The present results suggest that both excitotoxic and oxidative stress play an important role in the upregulatio n of NT nitration and the apoptotic pathway in cultured rat spinal neurons. (C) 2001 Wiley-Liss, Inc.