Quantitative Analysis of Meningeal Lymphatics

The images of whole-mount staining meninges were acquired under the 10× lens of a Leica DM4 B with a resolution of 1024×1024 resolution and a z-step of 4 µm. Image J software was used to perform quantitative evaluations of the micrographs. Images of the same region of the superior sagittal sinus (SSS), the confluence of sinuses (COS), and the transverse sinus (TS) were acquired in a confocal microscope. The means of 30 individual lymphatic vessel diameter measurements. The percentage of meningeal lymphatics labeled by AF488 LYVE-1 antibody (i.c.m.) was defined by dividing the area of AF488 LYVE-1 antibody (i.c.m.) labeled by the area of meningeal lymphatics. Fluorescent stereomicrographs of labeled skullcap were obtained with Leica. To quantify the number of total OVA-FITC or Aβ₄₂-FITC in the brain, the images of brain sections were obtained using the confocal microscope. The area of OVA-FITC or Aβ₄₂-FITC was determined by dividing the area labeled OVA or Aβ₄₂ per section by the area of the brain section. The area of OVA-FITC or Aβ₄₂-FITC in the dCLNs was also calculated with the same method. The area of LYVE-1 near the cribriform plate was determined by dividing the area of labeled LYVE-1 per section by the area of the cribriform plate section. All fluorescence micrographs were managed with equally constant exposure time and brightness/contrast and analyzed using the Image J software.