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For each image, the brain was manually masked to exclude extracerebral structures. Individual sections were then converted to 8-bit grayscale and thresholded in the gray channel at the same level using Adobe Photoshop software version CS2 (Adobe Systems Europe Ltd., Maidenhead, UK). Quantification of anti�CVCAM-MPIO binding (defined as focal hypointensities) was performed by observers masked to the identity of the data set. Anti�CVCAM-MPIO binding was quantified in 256 consecutive brain sections for each animal. Segmented images were reconstructed using the 3D Constructor plug-in for Image-Pro Plus (Media Cybernetics Inc., Rockville, MD) to visualize the distribution of MPIO binding throughout the brain, with low-signal Tasisulam areas assigned to the red channel and the anatomical images to the green channel. Voxel volumes were then summed and expressed as raw volumes in microliters, with no surface rendering or smoothing effects. The three-dimensional reconstructions of VCAM-MPIO ISRIB purchase binding were then subtracted from the signal arising from sinuses, which likely resulted from blood pooling. Gd-DTPA contrast enhancement on T1-weighted images was defined as regions of hyperintensity and was quantified in 11 consecutive brain sections for each animal. These volumes were then subtracted from the average volume of Gd enhancement in three naive ABH mice injected with Gd. RNA extraction and real-time quantitative PCR assays were performed from pieces of snap-frozen brain as previously described,24 with standard curves generated from reverse-transcribed Inhibitor Library in vitro single-stranded RNA produced from T7 templates for each probe set.25 Results are expressed as number of copies of target per nanogram of input RNA corrected to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Statistical analysis was performed using SPSS software version 19 (SPSS Inc., Chicago, IL). Paired-samples t-tests or U-tests were used to identify differences between the groups for parametric or nonparametric parameters, respectively. Data are expressed as means �� SEM, and statistical significance was assigned at P