Fig. 2

SLYM defines the subarachnoid cisternal system and the pial perivascular spaces. (A) Schematic of the experimental approach. CSF tracers, either Evans Blue (0.9 kDa, 0.5% in aCSF w/v) or BSA-647 (67 kDa, 0.5% in aCSF w/v) were delivered (10 µL, 2 µL/min) through a cisterna magna cannula in K/X-anesthetized Prox1-eGFP mice. After 30 min of tracer circulation, the Prox1-eGFP mice were perfused with phosphate buffered saline (PBS) to remove blood signal interference. In a subset of experiments, wheat germ agglutinin (WGA) conjugated to Alexa Fluor 647 was injected retro-orbitally to outline the vasculature. (B) Macroscopic fluorescent images of the whole brain showing the cisterns and pial perivascular spaces filled with a small size tracer, Evans blue (blue). (C) Representative images of a brain from a Prox1-eGFP (green, middle) mouse injected with a larger CSF tracer, BSA-647 (magenta, left). 2-photon images of middle cerebral artery (right) illustrate the accumulation of the fluorescent tracer below the Prox1-eGFP+ cells. The CSF tracer is taken up by cells bordering the perivascular spaces after death, including the arterial smooth muscle cells and pia [11]. (D) Confocal images of skull and brain from Prox1-eGFP and wildtype mice taken before and after DPP4 staining. Confocal and two-photon XY projections from whole-mount dorsal skull (first row), dorsal brain (second row), ventral brain (third row), and ventral skull (bottom row). Orthogonal projections were obtained as indicated by the dashed line in the first panel, except for the ventral brain after DPP4 staining where the projection was obtained from an area including a DPP4 positive section close to a vessel outline by WGA. A meningeal lymphatic vessel positive for Prox1-eGFP is observed in the dorsal skull of the Prox1-eGFP mouse (green arrow). The analysis showed that SLYM in the Prox1-eGFP reporter mice (green arrowheads) adheres to the brain surface, whereas the ABC layer, immunolabeled for DPP4 (cyan arrowheads), adheres to dura on the skull