Peroxynitrite generated in the rat spinal cord induces neuron death and neurological deficits

Abstract

We previously demonstrated that the concentration of peroxynitrite significantly increases following impact spinal cord injury (SCI). The aim of this study was to test whether the SCI-induced elevation of peroxynitrite induces neuronal death and consequent neurological deficits. Peroxynitrite was generated by administering 5 mM S-morpholinosydnonimine, a donor of peroxynitrite, through a microdialysis fiber into the gray matter of the rat spinal cord for 5 h. This mimics the concentration and duration of peroxynitrite elevation after SCI. Neuron death was assessed by counting the neurons along the fiber track in Cresyl Violet-stained sections removed at different times post-peroxynitrite exposure. Peroxynitrite induced significantly more neuron death than did the artificial cerebrospinal fluid (ACSF) control, with the percentage of neuronal loss being 17+/-2%, 28+/-2%, 39+/-3%, and 43+/-4% at 6, 12, 24 and 48 h post-peroxynitrite exposure (P=0.01-<0.001). The losses of total neurons or motoneurons immuno-stained with anti-neuron-specific enolase or anti-choline acetyltransferase antibodies was significantly higher in the peroxynitrate-exposed group than in ACSF controls at 24 h post-exposure, further confirming peroxynitrate damage to neurons. The susceptibility to oxidative damage in motoneurons was similar to that of other neurons characterized at 24 h post-peroxynitrite exposure. Peroxynitrite-induced neurological deficits were examined by the Basso-Beattie-Bresnahan test (BBB test), the inclined-plane test and footprint analysis. Peroxynitrite significantly (P<0.001) reduced the locomotor rating score (BBB test) and the maximum angle of inclined plane compared to sham and ACSF-exposed animals (repeated measures analysis of variance). The footprint analysis revealed that peroxynitrite significantly increased the distance between the feet and the angle of hindlimb rotation compared to sham (P=0.01 and P<0.001) or ACSF controls (P=0.01 and P=0.005) and significantly shortened the stride length compared to sham (P<0.001) and ACSF control (P=0.005) treatments. Therefore the SCI-produced level of peroxynitrite induced neuron loss and neurological dysfunction, strong evidence that peroxynitrite is a secondary damage agent in SCI.