Fig. 8

Immunofluorescent co-localization of small fluorescent CSF tracers with meningeal fibroblast markers. A, B, Representative photomicrograph of 3 kDa Red Dextran Tetramethylrhodamine (RD/TMR) (magenta, magenta arrowheads) distribution along the meninges around (A) and within (A, B) the longitudinal fissure 10 min after intraventricular injection into the right lateral ventricle in P7 rodents. RD/TMR distribution is primarily within the meningeal layer(s) inferior to a cellular retinoic acid binding protein 2 (CRABP2) (green, green arrowheads)-positive layer (inferior layer identified as the pia mater), with additional RD/TMR co-localization with the CRABP2 + layer (identified as the arachnoid mater). The putative border between the pia mater and underlying parenchyma is indicated with thick white dashed line; the pia and arachnoid are separated by the medium white dashed line, and the arachnoid and dura (falx) are separated by the thin white dashed line. Very little RD/TMR is seen medial/superior to the CRABP2 + layer (dura mater). A, B scalebars = 50 µm. C, D Representative photomicrographs of RD/TMR distribution in the meninges over the dorsal surface of the brain and CRABP2, retinaldehyde dehydrogenase 2 (RALDH2), and zonula occludens 1 (ZO-1) staining. RD/TMR co-localized with a CRABP2+/RALDH2+ layer (arachnoid mater), in addition to the inferiorly adjacent CRABP2-/RALDH2- layer (pia mater) (C). RD/TMR distribution was inferior to the CRABP2+/ZO-1 + layer (arachnoid barrier cell layer), suggesting it localizes to the arachnoid and pia maters (D). E, Quantification of RD/TMR mean intensity in CRABP2±/RALDH2− meninges (dura), CRABP2+/RALDH2+ meninges (arachnoid), CRABP2−/RALDH2− meninges (pia), and the parenchyma of the cortex. There was significantly less RD/TMR in the dura and cortex parenchyma compared to the pia and arachnoid. Data are mean ± SD, n = 4 per group; One-way ANOVA with post-hoc Tukey. All data are representative of 4 rodents