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A total of 7 sections were obtained from each brain. Indirect immunofluorescence against CD31 (PECAM-1, 1:100; Sigma Aldrich, St. Louis, MO) was performed to assess cerebral vascular density. DAPI (4��,6-diamidino-2-phenylindole dihydrochloride; Sigma Aldrich, St. Louis, MO) staining was used for nuclear identification. Stained tissue sections were photographed for the DAPI (blue) and PECAM-1 (red) channels. The number of PECAM-1-positive cells per field was quantified. All images were photographed at the same exposure times and magnification. The obtained values from all animals were averaged, and the results were expressed as the mean number of PECAM-1-positive cells per field?��?SD. WT (2 months old, n?=?2; 10 months old, n?=?2) and mdx (2 months [http://www.selleckchem.com/products/nlg919.html NLG919 price] old, n?=?2; 10 months old, n?=?2) mice were injected intraperitoneally with a bolus of 0.1?mL/10?g Evans blue (10?mg/mL; Sigma Aldrich, St. Louis, MO). After 24?hours, the mice were deeply anesthetized with isoflurane and [http://www.selleckchem.com/products/Romidepsin-FK228.html Romidepsin supplier] the brains were excised and rapidly frozen in OCT media (Electron Microscopy Sciences, Hatfield, PA). Tissues were sectioned at 10?��m slice thickness at the location corresponding to the MR imaging slice. Evans blue extravasation was evaluated by fluorescence in WT versus mdx. The remaining WT (2 months old, n?=?2; 10 months old, n?=?2) and mdx (2 months old, n?=?2, 10 months old, n?=?2) mice were anesthetized with isoflurane and fluorescein isothiocyanate-dextran (FITC-dextran, 10?mg/mL; Sigma Aldrich, St. Louis, MO) was injected intravenously at a dose of 200?mg/kg. After 10?minutes, the brains were excised and rapidly frozen with liquid nitrogen in OCT media (Electron Microscopy Sciences, Hatfield, PA) and mounted to the cryoimaging system. The tissue was sectioned in 25?��m slices and the bright-field and fluorescent images from the cryoimaging system were processed and analyzed with custom-built MATLAB (Mathworks Inc., Natick, MA) and AMIRA (Mercury Computer Systems, San Diego, CA) software ( Roy et al., 2009). The brain contour was traced manually from the bright-field images, and a mask was generated. The mask was applied to fluorescent images, and the 3D cerebral vasculature [http://en.wikipedia.org/wiki/LMTK2 LMTK2] was reconstructed using thresholding and volume rendering methods ( Qutaish et al., 2012). The resolution for the reconstructed 3D images were 10.5?��?10.5?��?25 ��m3. All data are reported as mean?��?SD. A two-tailed, unpaired Student's t-test was performed to compare mdx and WT mice. Statistical significance was established at a level of p?