Effects of MK801 on nitrotyrosine formation, iNOS and nNOS expression. iNOS expression was evaluated by immunoistochemical analysis in the spinal cord section 24 h after SCI. Spinal cord section from sham-operated mice did not stain for iNOS (a), whereas spinal cord section obtained from SCI-operated mice exhibited positive staining for iNOS (b) mainly localized in various inflammatory cells in the gray matter. MK801 treatment (2 mg/kg 30 min and 6 h after SCI induction) reduced the degree for positive staining for iNOS in the spinal cord tissue (c). Moreover the tissue sections obtained from SCI mice group demonstrate a positive staining also for nitrotyrosine mainly localized in inflammatory, in nuclei of Schwann cells in the white and gray matter (e). MK801 treatment (2 mg/kg 30 min and 6 h after SCI induction) reduced the degree of positive staining for nitrotyrosine (f). On the contrary spinal cord obtained from Sham-operated mice did not staining for nitrotyrosine (d). Densitometry analysis of immunocytochemistry photographs (n = 5 photos from each sample collected from all mice in each experimental group) for iNOS and nitrotyrosine (g) from spinal cord tissues was assessed. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as % of total tissue area. Moreover spinal cord sections were processed at 24 h after SCI to determine also the nNOS levels by western blot analysis; nNOS expression was significantly increased in the spinal cord from SCI mice (h and h1). On the contrary, MK801 treatment (2 mg/kg 30 min and 6 h after SCI induction) significantly restores the expression of nNOS (h and h1). The relative expression of the protein band was standardized for densitometry analysis to α-tubulin levels and reported in panel h1, and expressed as mean ± s.e.m. from n = 5/6 spinal cord for each group. This figure is representative of at least 3 experiments performed on different experimental days. *p < 0.01 vs Sham; °p < 0.01 vs SCI.
Esposito et al. BMC Neuroscience 2011 12:31 doi:10.1186/1471-2202-12-31