Then Hoechst 33342 was added to stain the nuclei and the coverslips were mounted on the slides. The slides were imaged by an ob server blind to treatments on a Zeiss Axiovert upright fluorescent microscope with identical exposure settings and identical post acquisition processing for each image. Statistical analysis Quantification of selleck catalog band intensities was performed by densitometric analysis using Quantity One 1 D analysis software. The statistical calculations and graphing were performed using GraphPad Prism soft ware, version 5. All data were tested using one way ana lysis of variance with Tukeys post hoc test. A P value of 0. 05 or less was judged significant. Results were expressed as mean SEM.

Results OGD reperfusion induces NO formation and iNOS protein expression The cortical astrocyte culture was subjected Inhibitors,Modulators,Libraries to OGD for 8 h and then exposed to reperfusion for either 16 h or 24 h. The concentration of NO in the culture media and the protein expression of iNOS were then determined. Nitrogen oxide generation was slightly increased after OGD treatment. The increase of NO was exaggerated by the reperfusion treatment. Inhibitors,Modulators,Libraries The concentration of NO reached a maximal level at the latest time studied. A similar result was observed in the iNOS protein assay. Under normal cir cumstances, the iNOS expression was too low to be detected. After OGD treatment, the iNOS expression was up regulated. Inhibitors,Modulators,Libraries The reperfusion treatment led to a dramatic increase of iNOS expression in reactive astro cytes compared with the control cells that were not trea ted.

When iNOS protein expression was quantified, exposure to OGD reperfusion induced a remarkable in crease in the iNOS protein level, especially in astrocytes following OGD 8 h reperfusion 24 h. These cells exhib ited a significant Inhibitors,Modulators,Libraries increase of iNOS expression, about 2. 5 fold as compared with the control. PDI and SOD1 are up regulated after OGD reperfusion treatment, and they were binding to each other We investigated the changes in PDI and SOD1 expres sion levels following OGD reperfusion treatment in cul tured astrocytes. Cell lysates from astrocytes under various treatments were analyzed by immunoblot. Western blot analysis using anti PDI monoclonal antibody revealed an enhancement of PDI expression after OGD at 8 h, and this had reached a maximum by 24 h of reperfu sion. A similar pattern was also observed in SOD1 expression.

Immunoblot analyses confirmed an increased expression of SOD1 in cultured astrocytes when they were exposed to OGD for 8 h. In addition, reperfusion significantly induced appreciable SOD1 ex pression in these cells, yielding a three fold higher abundance of Inhibitors,Modulators,Libraries SOD1 protein in the OGD 8 h reperfu sion 24 h group when compared with the control group. www.selleckchem.com/products/epz-5676.html These results demonstrate that the eleva tion of PDI and SOD1 expression correlate well with the induction of iNOS in activated astrocytes following OGD reperfusion treatment.