Ubated in 0.25 M L-arginine for 60 minutes at 37 to produce urea fromUbated

Ubated in 0.25 M L-arginine for 60 minutes at 37 to produce urea from
Ubated in 0.25 M L-arginine for 60 minutes at 37 to produce urea from arginine and the reactions were stopped by adding Stop solution (H2SO4/H3PO4/ H2O, 1:3:7). Then, 1 (final concentration) 1-phenyl-1, 2-propanedione-2-oxime (ISPF) in ethanol was added to the solution, which was heated at 100 for 45 min. The reaction between urea and ISPF produced a pink color, and absorption was measured at 540 nm.Statistical analysisData are expressed as mean ?SE for in vivo experiments. Data are expressed as mean ?SD for in vitro experiments Statistical comparisons were performed using the Student’s t tests and two-way analysis of variance (ANOVA) as appropriate. P values less than 0.05 were considered statistically significant.Levels of NO and TNFa production are markers of classically activated microglia [20]. NO production was measured using the Griess method (Dojindo, Kumamoto, Japan) as total NO (NO2- and NO3-). TNFa production was measured by enzyme-linked immunosorbent assay using the Duoset ELISA Development System (R D Systems, Minneapolis, MN).ResultsGp91phox is upregulated in the peri-contusional region after traumatic brain injuryFig 1 shows the results of immunoblotting experiments to describe the expression of gp91phox after TBI. Protein levels of gp91phox were not increased after TBI in the contralateral hemisphere of Wt mice. In the ipsilateralDohi et al. Journal of Neuroinflammation 2010, 7:41 http://www.jneuroinflammation.com/content/7/1/Page 4 ofFigure 1 Characterization of gp91phox after traumatic brain injury (TBI) using western immunoblots. (A) Immunoblotting signals before and after TBI in wild-type (Wt) mice (left) and gp91phox-/- mice (right). (B) In Wt mice, gp91phox levels were significantly increased on day 1 and day 2 after TBI (*p < 0.05 relative to sham). Gp91phox levels on the ipsilateral side were greater than on the contralateral side on day 1 and day 2 after TBI (*p < 0.05). Each value is the mean ?SE (n = 3). Note that the intensity of each sample was quantified and corrected relative to the labeling control, actin. Increasing levels of gp91phox were not seen in gp91phox-/- mice.hemisphere of Wt mice, protein levels of gp91phox were greater at 1 and 2 days after TBI relative to the contralateral hemisphere (Fig 1A, B). The data indicate that gp91phox increased in the ipsilateral hemisphere following TBI stress. The results of single immunohistochemical detection of gp91phox after TBI are presented Fig 2A. Two days after TBI, gp91phox immunoreactivity was dramatically increased in the peri-contusion region.Gp91phox is mainly expressed by cytotoxic-type classically activated microglia in the peri-contusional regions after TBIGp91phox inhibition reduces the severity of TBI in vivoTTC staining was used to evaluate the role of gp91phox on the severity Sodium lasalocid site pubmed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 of TBI in gp91 phox-/- and Wt mice (Fig 3A, B). Images of the TTC-stained anterior surface of coronal sections demonstrated that the injured brain area in gp91phox-/- mice was significantly smaller (P < 0.01) than that of the Wt mice (Fig. 3B). We verified the prevention of the cortical injury with TUNEL staining, which was used to identify apoptotic-like cell death at 48 hours after TBI (Fig 3C, D). The number of TUNELpositive cells of gp91phox-/- mice was significantly less (p < 0.05) compared with Wt mice (Fig 3D).Gp91phox gene deletion reduces superoxide radical (O2-) production after TBITo identify the cell types expressing gp91 phox in the peri-contusional area,.