Fig. 2

Structural and functional abnormalities of the Chp and ependyma in MIA offspring. (a) Schematic diagram of the cerebral aqueduct location within the brain. (b) Representative images of brain slices after Evans Blue detection of CSF outflow, with arrows indicating the cerebral aqueduct (Aq) and the fourth ventricle (4 V). (c) Representative H&E staining of brain slices from the anterior to the posterior cerebral aqueduct in offspring. Scale bar, 100 μm. Quantification of the anterior and posterior areas of the cerebral aqueduct (n = 12–13 from 4–5 litters). Two-way ANOVA tests were applied. (d) Schematic diagram of the method for detecting the rate of CSF production in offspring and quantification of CSF production rate (n = 3 from 3 litters). Unpaired T tests were applied. * P < 0.05. (e) Representative IF staining of pNKCC1 in the Chp of offspring and quantification of fluorescence intensity (n = 5–6 from 3 litters). Scale bar, 100 μm. Unpaired T tests were applied. * P < 0.05. (f) Representative IF staining of Iba1⁺ macrophages in the Chps of offspring, and quantification of macrophage infiltration in the total (left), apical (middle), and stromal (right) regions of the Chps (n = 8 from 3–4 litters). Scale bar, 100 μm. Unpaired T tests were applied. * P < 0.05. (g) qPCR analysis of il1β and tnfα mRNA levels in the Chp of offspring (n = 10 from 3–4 litters). Unpaired T tests were applied. ** P < 0.01 and **** P < 0.0001. (h) Schematic diagram of the method for detecting blood-CSF barrier leakage using FITC-dextran and the quantification of fluorescence intensity in the CSF of offspring (n = 6 from 3–4 litters). Unpaired T tests were applied. * P < 0.05. (i) Representative IF staining images and quantification of Occludin and β-Catenin fluorescence intensity in the Chp of offspring (n = 4–5 from 3 litters). Scale bar, 100 μm. Unpaired T tests were applied. ** P < 0.01. (j) Representative images of the movement trajectory of fluorescent microspheres in the LVs of offspring and quantification of the movement speed of fluorescent microspheres, each value represents the average movement speed of fluorescent microspheres in 4 fields of view in the brain sections of each mouse. (n = 5 from 3 litters). Scale bar, 100 μm. Unpaired T tests were applied. * P < 0.05. (k) Representative immunoblot images and quantification of Foxj1 protein level in the samples obtained from ependyma of adult offspring (n = 4 from 3 litters). Unpaired T tests were applied. * P < 0.05. (l) Representative SEM images of ependymal cilia in the LVs of offspring, with red arrows indicating the direction of ciliary tufts. Scale bar, 50 μm (low magnification), and 10 μm (high magnification). (m and o) Representative IF staining images (m) and quantification (o) of ciliary tufts in the LVs of offspring (n = 5–6 from 3–4 litters). Scale bar, 100 μm. Unpaired T tests were applied. ** P < 0.01. (n-r) Representative IF staining images of ECs in the lateral ventricles of offspring (n). Scale bar, 100 μm. Quantification of the density of Foxj1⁺Sox2⁺ ECs (p) and Foxj1⁻Sox2⁺ neural progenitor cells (q) and the ratio of the two (r) (n = 5–6 from 3–4 litters). Unpaired T tests were applied. ** P < 0.01 and * P < 0.05