Cell and Molecular Components of Brain Barriers in a Model of Hydrocephalus

Hydrocephalus results from excess cerebrospinal fluid (CSF) in the brain’s ventricles due to imbalances in CSF secretion, flow, or absorption. Treatments include invasive shunting, which commonly fails and requires revision throughout a patient’s life. Cellular and molecular characterization during hydrocephalic development may lead to novel targets for pharmacological treatment. This study hypothesized that 1) Choroid plexus (CP), which production of CSF, will show molecular alterations in the blood-CSF barrier (BCSFB) consistent with altered CSF production; 2) Increased CSF will cause fluid accumulation in the ventricular zone disrupting the blood-brain barrier (BBB); 3) Edema formation and molecular alterations in the BBB will impair normal development in the subventricular zone (SVZ), a niche for neural stem and progenitor cells. In rats, a mutation in the Transmembrane 67 (TMEM67) protein results in hydrocephalus. Ventriculomegaly was apparent in Tmem67 -/- animals before postnatal day (P)10 using MRI and Nissl staining (n=3). Using this model, changes in the BBB, BCSFB, and SVZ were evaluated with immunohistochemistry (n=3/4), real-time quantitative polymerase chain reaction (n=6), and western blot (n=5/6). Claudin-1 (Cl1), claudin-2 (Cl2), zona occludin-1 (ZO1), occludin (Occ), epithelial cadherin (E-cadherin); and the water transporter, aquaporin-1 (AQP1) are all expressed in CP epithelial cells and contribute to BCSFB integrity. Hydrocephalic pups showed increased Cl1, Cl2, ZO1, Occ, and AQP1 immunofluorescence (IF) at P10 and P15. E-cadherin IF only increased in P15 hydrocephalic animals compared to wildtype. AQP1 mRNA was increased in P15 hydrocephalic pups compared to wildtype. Slight changes in Cl1, Cl2, and AQP1 protein expression were observed at P10 and P15 in hydrocephalic animals, although not statistically significant (p>0.05). Post-translational modifications such as phosphorylation, glycosylation, and ubiquitination or changes in membrane localization may explain discrepancies between results. AQP4, the most widely expressed water channel in the brain, can indicate disturbances in the BBB. Increased AQP4 IF appeared in ependymal cells, neuroepithelial cells composing the ventricular wall, and the SVZ of hydrocephalic animals at P10 and P15. AQP4 perivascular localization, determined by co-expression with a vasculature marker, was diffuse in hydrocephalic pups at P15. RTqPCR showed no change in AQP4 mRNA in the periventricular cortex of P15 hydrocephalic animals compared to wildtype. ZO1 IF was also increased in endothelial cells of the BBB. Glial fibrillary acidic protein, a marker of astrocytes, IF and protein amount increased in hydrocephalic animals by P10. Astrocyte processes and branching appeared altered in P15 hydrocephalic animals. These results indicate alterations in the BBB and potential edema. To examine effects on the SVZ, markers of neural stem and progenitor cells, nestin and doublecortin (DCX) respectively, were used with proliferating cell nuclear antigen (PCNA). P15 and P10 hydrocephalic animals displayed intense IF of both DCX and nestin in the SVZ compared to wildtype. PCNA+ cells were decreased in areas with dense DCX and GFAP labeling in P15 and P10 hydrocephalic animals compared to wildtype. This may indicate a problem with neuroblast migration to the rest of the cortex. These results provide characterization of brain barriers in the pathophysiology of hydrocephalus with the aim to produce potential targets for pharmacological treatment in the future. United States Department of Defense Congressionally Directed Medical Research Program Award This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.