Because of toxicity at high concentrations, nitric oxide (NO) contributes to spinal cord injury (SCI) secondary lesions. At low concentrations NO modulates nuclear factor–κB (NF-κB) activation. The authors investigated the activity of neuronal and endothelial NO synthase (nNOS and eNOS) to determine correlations with NF-κB activation and inducible NOS (iNOS) expression soon after SCI.
In 48 adult male Wistar rats clip-based (50 g/mm2/10 seconds) SCI was induced, and spinal cords were removed at different intervals for the following evaluations: 1) assaying specific activity of nNOS and eNOS; 2) electrophoresis mobility shift assay for activated NF-κB; 3) Northern blotting for iNOS; 4) immunohistochemistry for iNOS and NF-κB; and 5) immunofluorescence for iNOS and NF-κB.
At 15 minutes postinjury, eNOS activity decreased significantly (p < 0.001), as did nNOS activity at 1 hour compared with these levels in control animals and rats killed at 15 and 30 minutes after SCI (p < 0.001). Basal NF-κB levels were variable in controls and at 15 and 30 minutes after injury. One hour postinjury, NF-κB activation was diffuse. Inducible NOS messenger RNA localized diffusely, peaking 6 hours after injury and remaining stable until 24 hours postinjury. Immunohistochemical analysis showed diffuse iNOS and NF-κB staining, especially in neurons inside and around the lesion. Immunofluorescence demonstrated that injured neurons were a source of NF-κB and iNOS soon after injury.
Both nNOS and eNOS exhibited different regulation and roles soon after injury: nNOS correlated with NF-κB activation, whereas eNOS may have participated in vascular changes of the injured spinal cord. Neurons seemed to play a pivotal role in modulating and amplifying the inflammatory response in the injured spinal cord.