The efficiency of glymphatic clearance was evaluated using in vivo two-photon imaging [7 (link)]. Briefly, mice (n = 6 per group, 3 male and 3 female) were anesthetized and a thin cranial window was created at the parietal. Fluorescein isothiocyanate (FITC)-dextran (70 kDa; Sigma-Aldrich, USA) was dissolved in artificial cerebrospinal fluid at a concentration of 1%; 10 μl of FITC was injected into the cisterna magna using a microsyringe connected with a syringe pump controller. 0.2 ml of 1% rhodamine B (Sigma-Aldrich, USA) in saline was injected intravenously to show the brain vascular before imaging. Two-photon imaging on the right parietal cortex (2 mm caudal from bregma, and 1.7 mm lateral from the midline) was performed using a two-photon laser scanning microscope (Leica, Germany) equipped with a water immersion objective (25×). To monitor the clearance of FITC-dextran injected into the brain parenchyma, three-dimensional (3D) xyz stacks (512 × 512 pixels, 2-μm resolution) were taken up to 300 μm below the cortical surface at 5, 15, 30, 45, and 60 min after the injection of the FITC-dextran, the overall fluorescence intensities were analyzed. Besides, images 100 μm below the cortical surface were obtained and the fluorescence intensities in the paravascular space were analyzed to examine the efficiency of glymphatic clearance.
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