The fluorescence intensities of SMI-71/p65-Thr435 phosphorylation or GFPA/p65-Thr435 phosphorylation were measured using a computer-assisted image analysis program (The University of Texas ImageTool program V. 3.0 and AxioVision Rel. 4.8 software). After regions were outlined, 30 areas/rat (300 μm2/area) were randomly selected within the PC, and double immunofluorescent merge images were captured from the PC (15 sections from each animal). Merge images were digitally separated to red or green image, and converted to grayscale images, respectively (n = 36 per region examined, in non-SE, 12 hr post-SE and 1 day post-SE). The range of intensity values was obtained from the selected images. Based on the mean range of intensity values, each image was normalized by adjusting the black and white range of the image. Manipulation of the images was restricted to threshold and brightness adjustments to the whole image. Intensity measurements are represented as the mean number of a 256 gray scale (NIH Image 1.59 software and AxioVision Rel. 4.8 software). Values for background staining were obtained from the corpus callosum. Optical density values were then corrected by subtracting the average values of background noise obtained from 15 image inputs.