Fig. 4: Influx of CSF tracer is delayed, but not restricted across “covered” Type A and Type B PAS.

Distribution of an immunofluorescent tracer (Texas Red conjugated to bovine serum albumin, TxRd; mw 66 kDa) was evaluated in mice sacrificed at 15 versus 30 min following slow intracisternal infusion (a), and revealed heterogeneous spatiotemporal deposition patterns as shown in right-hand images that represent the boxed areas. Enlargement of the small box at lower right panel in a illustrates tracer passage into a Type B PAS (b). Enlarged inset depicts pooling of tracer in PAS, creating a scalloped appearance around smooth muscle cells (b, inset). Cross-sectional images from axial sections (labeled 1–3) of another Type B PAS is shown in the middle panel and the anatomic relationships are summarized in axial schematics on right-hand side. Triple labeling of a Type B penetrating arteriole (shown in longitudinal section) depicts TxRd tracer signal around the ERTR7-labeled pial cell elements (c). Volumetric lightsheet image of a tissue-cleared (CLARITY) specimen further demonstrates variable penetration of the tracer (d). Examples of tracer-positive PAS are shown (e) and distributions are quantified in different brain regions at 15 min (f, h) and 30 min (g, i) post-infusion; n = 57 vessels from 6 mice. j At 30 min post-infusion, tracer was mostly found around large diameter arteries; n = 141 vessels from 6 mice. Red/Texas Red, TxRd; green/FITC, ERTR7; white/CY5, SMA; blue, DAPI or lectin; scale bars = a 500 μm; b–e 10 μm. Source data are provided as a Source Data file.