Ehpds ol

Cranial windows were prepared as we previously described.^{31,32 (link)} Briefly, mice were anesthetized with 1% to 1.5% isoflurane in 30% oxygen and 70% nitrous oxide and kept on a heating plate (37°C ± 0.5°C). After fixation in a custom-made head holder, a window 2 mm in diameter was made in the parietal bone (centered 2.5 mm lateral and 1 mm posterior to the bregma) using a high-speed micro drill (Stoelting). A sterile 5 × 5 mm cover glass (World Precision Instruments) was placed above the window and fixed with dental cement. For in vivo imaging, an Olympus FluoView FVMPE-RS upright multiphoton laser-scanning system mounted on an Olympus XL Plan N 25×/1.05 WMP ∞/0-0.23/FN/18 dipping objective was used. Two-photon excitation was performed with Mai Tai eHPDS-OL (Santa Clara, CA) and Spectra Physics InSight DS-OL lasers (Santa Clara, CA). Emitted fluorescence was detected through a 495- to 540-nm band pass filter. To analyze cortical cerebrovascular permeability, FITC-dextran (molecular weight, 40 KDa; Sigma-Aldrich; 0.1 mL of 10 mg/mL) was injected IV, and time lapse imaging of FITC-dextran was acquired every 3 minutes for 30 minutes. The fluorescence of randomly chosen 20 × 20 μm² regions of interest within the vessel and corresponding areas within the perivascular brain parenchyma was recorded as described.^{16 (link)}