Fig. 5: The pia is not a barrier to CSF tracer transport (size 66 kDa), irrespective of periarteriolar space (PAS) type.

a A CSF tracer (bovine serum albumin conjugated to Texas Red, TxRd-BSA; 66 kDa) was injected into the cisterna magna of live ketamine–xylazine anesthetized mice. After dura had been removed, the pial surface and CSF tracer movement patterns were imaged through a cranial window using two-photon (2P) laser scanning microscopy. b Second harmonic generation (SHG) was used to visualize the collagen fibers in the inner arachnoid and pia. c SHG imaging of an arteriole in the subarachnoid space (SAS), with an orthogonal reconstruction in the XZ plane (top panel), showing the inner arachnoid overlying the vessel surrounded by epipia coursing within the SAS on top the intimal pial layer. d SHG imaging of a penetrating arteriole (PA) showing how the epipia coalesces with the intimal pia as it dives down into cerebral cortex. Bottom right: YZ orthogonal view of the PA entering cortex. e Representative images of each PAS type surrounding penetrating arterioles at −20, −30, −40 µm below the cortical surface: Type A (top panel), Type B (middle panel), Type C (bottom panel). f CSF tracer in the surface perivascular spaces (PVS) is surrounded by the inner arachnoid superiorly, the intimal pia inferiorly, the fusion of the inner arachnoid/intimal pia laterally, and the epipia of the accompanying artery medially. This space coincides with the SAS of the surrounding leptomeningeal arterioles. g CSF tracer enters penetrating PAS and traverses across the merged epipial and intimal pial membranes. h Distribution of PAS by type (n = 42 PVS from 8 mice). i Area (µm²) of tracer coverage in the PVS as a function of depth from the brain surface. One-way ANOVA with Tukey’s post hoc for multiple comparisons, P = 0.4257, ns: not significant. j Probability of observing CSF tracer influx at varying depths of PAS type at 60 min after injection. Log-rank (Mantel–Cox) test, P = 0.7952, ns. k Time to tracer influx after the start of the intracisternal tracer injection in minutes (min). One-way ANOVA, P = 0.6399, ns. Scale bars = b–d, f, g 20 µm. Data are presented as mean ± SEM. Source data are provided as a Source Data file.