Age‑Dependent and Post‑Intraventricular Hemorrhage Remodeling of the Ependymal Glycocalyx in Mice

Background The ependymal glycocalyx (Gcx) is a glycan-rich apical structure that lines the ventricular brain surface. It plays a central role in maintaining cerebrospinal fluid (CSF) dynamics and brain homeostasis by forming a selective molecular barrier, thereby preserving surface negative charge, and supporting ciliary function and CSF flow. Despite its importance, the structural integrity and glycan composition of the ependymal Gcx remain poorly understood, particularly in the context of physiological aging and acute neurological injury, such as intraventricular hemorrhage (IVH). We aimed to elucidate the physiological role of the ependymal Gcx and its alterations in response to aging and acute brain injury. Methods We comprehensively investigated age- and injury-related changes in the ependymal Gcx using young (8–10-week-old), aged (60–62-week-old), and IVH model mice. The Gcx structure was visualized using lanthanum-enhanced electron microscopy, and glycan profiles were assessed through double immunofluorescence staining with S100β and a panel of 21 fluorescent lectins. Gcx thickness was quantitatively analyzed using a novel image analysis approach based on fluorescence intensity profiles. Single-cell RNA sequencing (scRNA-seq) was performed on lateral ventricular tissue to identify transcriptional changes in aged ependymal cells related to glycosylation and vesicular trafficking. Results Aged mice exhibited marked thinning and detachment of the Gcx and a significant reduction in terminal sialic acid residues compared to young controls. Transcriptomic profiling revealed coordinated downregulation of genes essential for sialylation, glycoprotein processing, and autophagic clearance. Increased Peanut Agglutinin binding suggested cytoplasmic accumulation of immature O-glycans. Following IVH, Gcx disruption peaked at day 3 and correlated with periventricular inflammation. Aged IVH mice, characterized by marked thinning and detachment of the ependymal Gcx, exhibited sustained inflammatory responses. Conclusions The ependymal Gcx is a dynamic and injury-sensitive structure whose integrity is compromised by aging and IVH. Its disruption impairs CSF regulation and promotes neuroinflammation, potentially contributing to the development of hydrocephalus and neurodegeneration. Therapeutic modulation of glycosylation pathways may provide a promising strategy to preserve Gcx function and protect the internal brain environment.