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

Age‑Dependent and Post‑Intraventricular Hemorrhage Remodeling of the Ependymal Glycocalyx in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Age‑Dependent and Post‑Intraventricular Hemorrhage Remodeling of the Ependymal Glycocalyx in Mice Tomohiro Iida, Kosuke Mori, Hiroyuki Tomita, Ayumi Niwa, Tomohiro Kanayama, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7369159/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Nov, 2025 Read the published version in Fluids and Barriers of the CNS → Version 1 posted 9 You are reading this latest preprint version Abstract 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. ependymal glycocalyx brain–cerebrospinal fluid barrier lectin electron microscopy aging intraventricular hemorrhage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The ependymal cells lining the cerebral ventricles are ciliated glial cells derived from radial glia [ 1 ], and they play a pivotal role in cerebrospinal fluid (CSF) circulation, molecular exchange with the brain parenchyma, and maintenance of the neural stem cell niche [ 2 ]. These cells are covered by the glycocalyx (Gcx), a complex of polysaccharides and proteins [ 3 – 6 ], which functions as the frontline interface mediating cellular interactions with the environment. The ependymal Gcx may contribute to the formation of a negatively charged cell surface, facilitate smooth CSF flow, and act as a physical and immunological barrier against pathogens and metabolic waste [ 3 , 7 ]. However, the structural and compositional alterations of the ependymal Gcx in response to physiological and pathological stressors such as aging or acute brain injury and the subsequent effect on ependymal cell function and overall brain homeostasis remain unclear. Few studies have clearly distinguished between surface Gcx and intracellular glycans. Therefore, in the present study, we conducted a comprehensive histochemical analysis using a panel of lectins with diverse glycan-binding specificities to qualitatively and quantitatively assess changes in the structure, composition, and localization of the lateral ventricular ependymal Gcx in young adult mice, aged mice, and mice with intraventricular hemorrhage (IVH). Through this approach, we aimed to elucidate not only the physiological role of the ependymal Gcx but also its alterations in response to aging and acute brain injury, thereby providing insights that may contribute to the development of diagnostic markers and therapeutic strategies. Methods Mice Wild-type C57BL/6J mice were obtained from the Japan Jackson Laboratory (Japan). Male mice aged 8–10 and 60–62 weeks were used in all experiments. In this study, 8-10-week-old mice were defined as “young adult,” and 60–62-week-old mice were defined as “aged”. All animal procedures were conducted in accordance with the guidelines of the Gifu University International Animal Care and Use Committee (Approval No. A20250001). Mice were housed under standard laboratory conditions with a 12 h light/12 h dark cycle at a controlled temperature of 22 °C, with ad libitum access to food and water. IVH Animal Model The IVH mouse model was established via intracerebroventricular (i.c.v.) injection of 25 μL of autologous whole blood, as previously described [8]. Mice were anesthetized using three types of intraperitoneal administration of mixed anesthesia, as reported previously [9]. A 1-mm burr hole was drilled in the skull at a point 0.5 mm posterior and 1.0 mm lateral to the bregma. A 26-gauge needle was inserted freehand into the right lateral ventricle to a depth of 2.5 mm. A total of 25 μl of autologous whole blood, collected from the orbital venous plexus, was manually injected at the slowest possible rate using a Hamilton micro syringe. After injection, the needle was left in place for an additional 2 min to minimize backflow. The burr hole was sealed with bone wax, and the skin incision was closed with sutures. Tissue preparation of mice Frozen sections: Mice were euthanized, and tissues were collected without perfusion. Samples were embedded in an optimal cutting temperature compound, snap-frozen in liquid nitrogen, and stored at -80 °C. The embedded tissues were coronally sectioned at a thickness of 5 μm near the optic chiasm using a rotary microtome (Leica, Wetzlar, Germany). Paraffin sections: Following anesthesia, the thoracic cavities of the mice were opened, and the inferior vena cava was incised. Perfusion was performed using a drip infusion system with equal volumes of cold 0.1 M phosphate-buffered saline (PBS) and cold 4% paraformaldehyde. Afterward, the tissues were harvested, dissected into smaller pieces, and processed for paraffin embedding and sectioning. Lectin fluorescent staining Three lectin kits (I–III), each comprising a diverse array of lectins with distinct binding specificities for broad screening of glycan structures on cell surfaces, tissues, or purified glycoproteins, were obtained from Vector Laboratories (Burlingame, CA, USA). In addition, SiaFind α2,3-Specific Lectenz, an antibody-based reagent that specifically recognizes α2,3 linked sialic acid residues, was purchased from Lectenz Bio (Athens, GA, USA). Details of the biotinylated lectins included in kits I–III and SiaFind are summarized in Table 1. For double-fluorescence staining using an antibody and biotinylated lectins, tissue sections were fixed in 4% paraformaldehyde in PBS for 15 min. After washing with PBS, sections were blocked with the Histofine Mouse Stain Kit (NICHIREI BIOSCIENCES INC.) for 60 min at room temperature. Subsequently, biotinylated lectins (1:200 dilution) and an ependymal cell marker, the S100β antibody (mouse monoclonal, 1:500 dilution; sc-393919, Santa Curz Biotechnology), were applied and incubated overnight at 4 °C. The following day, sections were washed with PBS, blocked again with the Histofine Mouse Stain Kit, and incubated with Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:250 dilution; ab150165, Abcam) and DyLight 594-conjugated streptavidin (1:250 dilution; Vector Laboratories) for 1 h at room temperature. Finally, after washing with PBS, the nuclei were counterstained with DAPI, and coverslips were mounted using appropriate mounting medium. Table 1. Binding specificities of the lectins that were used in this study. Lectin Common Abbreviation Specificity Concanavalin A ConA αMan > αGlc Dolichos biflorus agglutinin DBA GalNAcα(1,3) Peanut Agglutinin PNA Galβ(1,3) > Galβ(1,4) > Gal Ricinus communis agglutinin Ⅰ RCA Ⅰ GalNAc > αGal Soybean agglutinin SBA αGalNAc > αGal > βGalNAc Ulex Europaeus agglutinin Ⅰ UEA Ⅰ Fucα(1,2) Wheat Germ agglutinin WGA NeuAc >>> GlcNAc Griffonia simplicifolia lectin Ⅰ GSL Ⅰ αGal Len culinaris lectin LCA αMan > αGlc Phaseolus vulgaris Erythroagglutinin PHA E Complex structures Phaseolus vulgaris Leucoagglutinin PHA L Complex structures Pisum sativum agglutinin PSA αMan > αGlc Wheat Germ agglutinin, succinylated succinylated WGA GlcNAc Datura stramonium lectin DSL GlcNAc oligomer Erythrina cristagalli lectin ECL Gal Griffonia simplicifolia lectin Ⅱ GSL Ⅱ GlcNAc Jacalin Jacalin αGal Lycopersicon esculentum lectin LEL GlcNAc oligomer Solanum tuberosum lectin STL GlcNAc Vicia villosa lectin VVL GalNac SiaFind α2,3-Specific Lectenz SiaFind α2,3 linked NeuAc Abbreviation: Man, mannose; Glc, glucose; GalNAc, N-acetylgalactosamine; Gal, galactose; Fuc, fucose; NeuAc, sialic acid; GlcNAc, N-acetylglucosamine Immunofluorescence Staining The paraffin-embedded brain tissues were cut into 4-μm sections. The sections were blocked with bovine serum albumin at 37 °C for 60 min; thereafter, they were incubated with the following primary antibodies overnight at 4 °C: ionized calcium binding adaptor molecule-1 (Iba-1) (1:500, #019-1974, Wako) and Galectin-3 (1:100, #13-5301-85, Bay Biosciences). After washing the sections three times with PBS, they were incubated with secondary antibodies at 37 °C for 60 min. Scanning electron microscopy (SEM) Ependymal Gcx was visualized using SEM, as previously described[10, 11]. Briefly, tissue samples were cut into 5 mm³ pieces and initially fixed for 2 h in a solution of 2% glutaraldehyde, 2% sucrose, 0.1 M sodium cacodylate buffer (pH 7.3), and 2% lanthanum nitrate. Thereafter, the specimens were immersed overnight in a solution containing 2% sucrose, 0.1 M sodium cacodylate buffer (pH 7.3), and 2% lanthanum nitrate, followed by washing in an alkaline solution (0.03 M sodium hydroxide with 2% sucrose). After fixation and washing, the specimens were dehydrated through a graded ethanol series. Subsequently, they were frozen in 100% ethanol and rapidly cooled with liquid nitrogen to create fracture surfaces for SEM observation. Afterward, the frozen tissues were fractured using a carving knife. Finally, the specimens were examined using SEM (S-4800, Hitachi, Tokyo, Japan). Low-vacuum scanning electron microscopy (LVSEM) For LVSEM, we used a modified version of a previously described method [12], the details of which are described in a manuscript in preparation at the time of the present study (Mori, K. et al.). Briefly, mice were deeply anesthetized and perfused with a solution of 10% neutral buffered formalin containing 1% Alcian Blue 8GX and 2% sucrose. Subsequently, the brains were harvested, processed for paraffin embedding, and cut into 2-μm thick serial sections. After deparaffinization, the sections were stained with Periodic Acid-Methenamine silver. The stained specimens were air-dried, mounted onto a sample holder using conductive adhesive tape, and observed without metal coating using a low-vacuum scanning electron microscope (TM3030Plus, Hitachi High-Tech, Tokyo, Japan) at an acceleration voltage of 15 kV. Transmission electron microscopy Tissue fixation was performed as described for the SEM. After fixation, brain samples were postfixed with 2% osmium tetroxide in 0.1 M cacodylate buffer for 2 h at 4 °C. Thereafter, the samples were dehydrated through a graded ethanol series (50–100%), treated with propylene oxide, and embedded in epoxy resin (Quetol812: DDSA: MNA = 7:4:4, with 1.5% DMP-30 catalyst). Polymerization was performed at 40 °C for 8 h, 70 °C for 24 h, and 75 °C for 12 h. Ultrathin sections (90 nm) were cut using an ultramicrotome (Leica EM UC7) and mounted on copper grids. Sections were stained with 2% uranyl acetate for 15 min, followed by lead citrate for 5 min, and dried on filter paper. Images were acquired using a transmission electron microscope (HT7800, Hitachi, Japan). Image Analysis Images used for fluorescence intensity and inter-inflection distance measurements were acquired under consistent imaging conditions to ensure data comparability. Following background subtraction, the mean fluorescence intensities of the Gcx and cytoplasm were measured in 10 randomly selected cells per animal (n = 3; total of 30 cells). The thickness of the ependymal Gcx was quantified as previously described [13]. Briefly, a measurement line was drawn perpendicular to the ependymal cell surface using merged images of lectin and S100β staining. Radial fluorescence intensity profiles for both channels were extracted at the ependymal surface and fitted to a sigmoid curve using the least squares method. The inflection point of the lectin-derived curve was defined as the outer boundary of the Gcx layer, and the inflection point of the S100β curve was defined as the apical surface of the ependymal cell. The distance between these two inflection points was interpreted as the thickness of the ependymal Gcx. Gcx thickness was measured at 16 randomly selected points per animal (n = 3 or 5; total of 48 or 80 points). The extent of periventricular inflammation was evaluated by measuring Iba-1 fluorescence intensity within a 17-µm-wide band region of the periventricular brain parenchyma, as well as Galectin-3 fluorescence intensity in the ependymal cells. In addition, the size of Iba-1-positive Kollmer cells in the choroid plexus was assessed. All image processing and quantification were performed using ImageJ software (image processing software, open source). Data Acquisition and Analysis We performed an integrated analysis of public single-cell RNA-sequencing (scRNA-seq) data comprising 9,406 murine ependymal cells. The dataset was compiled from five independent studies (accession IDs: SCP565, SRP135960, GSE74672, SCP318, and PMID_32669714_FACS) using the Talk2Data platform (v4, BioTuring Inc.). Bioinformatic Analysis and Visualization All bioinformatic analyses, including cell type identification, segregation of young adult and aged adult populations, and differential gene expression analysis, were conducted within the Talk2Data environment. All figures, including the bubble heatmap (Figure 4a), single-cell heatmap (Figure 4b), and Circos plot (Figure 4c), were generated using the visualization tools integrated within the platform. Statistical analysis All data were presented as mean ± standard error of the mean (SEM). Differences in Gcx thickness, periventricular coverage, Iba-1 fluorescence intensity per pixel, Kolmer cell Iba-1-positive area, and Galectin-3 fluorescence intensity between young and aged mice for each lectin were assessed using unpaired t-tests. Changes in Gcx thickness over time following intraventricular hemorrhage were analyzed using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test to correct for multiple comparisons. To validate the use of inflection point distances as a quantitative measure of Gcx thickness, correlations with actual measurements were evaluated using Pearson’s correlation coefficient (r). p -value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 10.4.2 (GraphPad Software, Inc., La Jolla, CA, USA). Results Visualization and Glycan Profiling of the Ependymal Gcx in Normal Mice In SEM observations, the Gcx on the surface of ependymal cells was not detectable under conventional glutaraldehyde fixation. However, when combined with lanthanum staining, a dense Gcx layer was clearly visualized on the apical surface of ependymal cells and surrounding the cilia (Figure 1a). Histologically, Alcian blue staining distinctly labeled the glycan layer on the apical membrane, and low-vacuum SEM enabled three-dimensional visualization of the Gcx structure covering the base of the cilia (Figure 1b, 1c). In lectin staining, ependymal cells were immunolabeled with S100β, and the apical Gcx layer was clearly highlighted (Figure 1d). Furthermore, fluorescence-based screening using 21 lectins with diverse glycan-binding specificities revealed that the ependymal Gcx consists of a wide variety of glycans. Particularly strong signals were observed for ycopersicon esculentum lectin (LEL), Ricinus communis agglutinin I (RCA-I), α2,3-linked sialic acid–specific lectins (SiaFind), Solanum tuberosum lectin (STL), Datura stramonium lectin (DSL), and peanut agglutinin (PNA) (Figure 1f). Representative images ranging from strongly positive to negative lectin signals are shown in Figure 1g. In contrast, cytoplasmic binding was prominent for Vicia Villosa Lectin, Griffonia Simplicifolia Lectin I, Erythrina Cristagalli Lectin, Wheat germ agglutinin (WGA), LEL, and Succinylated Wheat Germ Agglutinin (S-WGA). Notably, lectins such as PNA and α2,3-linked sialic acid–specific lectins, which showed strong affinity to the apical Gcx, were negative in the cytoplasm (Figure 1d), indicating distinct glycan profiles between the apical surface and the intracellular compartments. Structural and Compositional Changes in the Ependymal Gcx Associated with Aging As performed in young mice, the mean fluorescence intensity of the ependymal Gcx was measured in aged mice. Increased intensities were observed with PHA-E and PHA-L staining; however, many lectins, including SiaFind and LEL, showed a decreasing trend. The pattern of lectin positivity and negativity remained unchanged across groups, regardless of lectin type (Figure 2a). Morphologically, the ependymal Gcx in aged mice appeared markedly thinned, and widespread detachment of the layer was observed (Figure 2b). Quantitative analysis of Gcx coverage along the ventricular circumference revealed a significant decrease from 77.25 ± 1.85% in young mice to 33.06 ± 7.14% in aged mice (p < 0.0011) (Figure 2c). These age-related changes were also confirmed by TEM imaging (Figure 2d). In young adult, TEM revealed that not only the apical surface of the ependymal cells (black alllow) but also the microvilli (brack allow head) and cilia (small black allow) were covered with Gcx, whereas in aged, partial loss of Gcx was observed. Furthermore, comparison between TEM and lectin-stained images, based on differences in the thickness of the glycan layer, suggested that the Gcx layer observed in lectin staining corresponds to the layer that includes the microvilli (Figure 2e). Using lectin-stained images, the thickness of the Gcx layer containing microvilli was evaluated by measuring the distance between inflection points in the fluorescence intensity profile (Figure 2f). The validity of this method was confirmed using PNA staining, which were approximately 20 % lower than direct measurements but displayed a strong positive correlation (r = 0.877, p < 0.01), validating this metric for relative thickness quantification and supporting its utility for relative Gcx thickness assessment (Figure 2g). Using this method, significant thinning was observed in aged mice across all three representative lectins: LEL (0.821 ± 0.175 vs. 0.500 ± 0.231 µm), PNA (0.796 ± 0.136 vs. 0.373 ± 0.165 µm), and RCA-I (0.747 ± 0.208 vs. 0.445 ± 0.191 µm) (Figure 2h). Consistent with this baseline pattern, aged mice exhibited ectopic cytoplasmic PNA signals, with PNA-positive cells increasing from 2.97 ± 0.81 % to 20.30 ± 3.93 % in young adult mice (Figure 2i, j), indicating intracellular retention of Galβ1-3GalNAc-terminated glycans. Increased cytoplasmic binding was observed for many other lectins, except for S-WGA, as determined via comparative fluorescence intensity analysis (Figure 2k). Dynamic Changes in the Ependymal Gcx Following IVH Time-course analysis following IVH induction revealed a rapid thinning and loss of the ependymal Gcx layer, as demonstrated using Alcian blue and PNA staining (Figure 3a). The assessment of Gcx detachment based on PNA staining showed maximal damage on day 3, with partial recovery observed on day 7 (Figure 3b). However, quantitative analysis of Gcx thickness using LEL, PNA, and RCA-I demonstrated a sustained reduction in thickness from day 1 through day 7 (Figure 3c). The inflammatory response in the periventricular region, evaluated by Iba-1–positive microglia and Galectin-3 expression in ependymal cells, and inflammation in the choroid plexus, assessed by the size of Iba-1-positive Kolmer cells, peaked on day 1 or day 3 and showed signs of resolution on day 7 (Figure 3d). In contrast, in aged IVH model mice, both Iba-1 and Galectin-3 levels remained elevated even at day 7 (Figure 3e). Age-Related Downregulation of Vesicular Transport and Sialylation Genes in Ependymal Cells Revealed by Integrated Single-Cell Transcriptomic Analysis To investigate age-related transcriptomic changes in murine ependymal cells, we performed an integrated analysis of 9,406 cells derived from five public single-cell RNA-seq studies (SCP565, SRP135960, GSE74672, SCP318, and PMID_32669714_FACS). Our analysis revealed a significant and coordinated downregulation of genes associated with vesicular transport and protein sialylation in aged ependymal cells compared to their younger counterparts. A bubble heatmap demonstrated that both the percentage of expressing cells and the average expression levels of key genes in these pathways—RAB6A, RAB8A, RAB11A, RAB11B, NEU1, NEU2, ST3GAL1, and SLC35A1—were markedly lower in aged adult mice (Figure 4a). This finding was substantiated at the single-cell level, where a heatmap showed consistent and robust expression of these genes across the young cell population, in contrast to the sporadic and weaker expression observed in aged cells (Figure 4b). A Circos plot further visualized the diminished contribution of this entire gene set to the transcriptomic profile of the aged ependymal cell population, confirming a broad suppression of these pathways with age (Figure 4c). Discussion This study provides a comprehensive characterization of the glycan profile of the ependymal Gcx in the murine brain and demonstrates its dynamic alterations in response to aging and acute brain injury (Figure 5). Notably, we adapted a method originally developed for evaluating Gcx thickness via two-photon microscopy [13, 14] and applied it, for the first time, to fluorescence immunohistochemistry. This enabled the objective quantification of glycan distribution beyond conventional qualitative assessments. This approach supports the emerging concept of the "glycocode"—where changes in glycan composition reflect cellular state—and introduces a novel perspective in neuroglycobiology. Previous studies [3, 6, 7], including ours, have shown that the ependymal Gcx is rich in sialylated glycans. Besides, sialic acid is a key component of vascular endothelial Gcx and plays critical roles in intercellular interactions, glycoprotein binding, and the regulation of vascular permeability [15, 16]. It is plausible that in the ependyma, sialic acid contributes similarly to selective permeability. Moreover, its hydrophilic and negatively charged nature may reduce friction with CSF, acting as a lubricant to support smooth CSF flow. Notably, electrohydrodynamic studies suggest that the negative charge of the Gcx may directly facilitate CSF perfusion [17]. Although previous studies [3, 4, 6] have indicated that the ependymal Gcx is PNA-negative unless treated with neuraminidase, our study identified clear PNA-positivity without enzymatic treatment. This discrepancy may arise from differences in tissue preparation; paraffin sectioning often compromises glycan epitopes, whereas our use of cryosections better preserved Gcx structure in a near-native state [18, 19]. Meanwhile, reports of PNA-positive Gcx in vascular endothelium are extremely limited. No studies have demonstrated PNA positivity in normal endothelial Gcx without neuraminidase treatment. Where such staining has been observed, it has been confined to endothelial cells located in the central regions of certain tumors [20]. Our findings suggest that the ependymal Gcx, despite being rich in sialic acid, contains an unusually high abundance of galactose-terminated glycans lacking sialic acid capping. This glycan configuration may represent a structural adaptation that enables molecular exchange with the brain parenchyma while preserving selective barrier function, distinguishing it from vascular endothelium. Age-related thinning and decreased coverage of the ependymal cell Gcx, accompanied by a reduction in terminal galactose residues, α2,3-linked sialic acids, and poly-N-acetyllactosamine structures, suggest impaired molecular exchange with the brain parenchyma, disruption of barrier function, and dysfunction in CSF circulation. Notably, sialic acids function as “self” markers contributing to the maintenance of immune tolerance. Therefore, the loss of this “sialic acid shield” may increase susceptibility to immune factors—including complement activation [21]—and act as a trigger or exacerbating factor for age-associated chronic neuroinflammation. Collectively, these findings suggest that the degradation of the Gcx on the ependymal surface may contribute to the disruption of homeostasis in the cerebral microenvironment. In addition, cytoplasmic PNA binding, which was absent in young mice, was frequently observed in the ependymal cells of aged mice. In most of these PNA-positive cells, thinning or partial loss of the apical Gcx was also detected. Furthermore, several lectins—including those specific for α2,3-linked sialic acids—exhibited significantly increased intracellular binding in aged ependymal cells. Transcriptomic analysis provided further evidence for an age-associated decline in two essential processes within murine ependymal cells: vesicular trafficking and protein sialylation. The coordinated downregulation of multiple Rab GTPases suggests dysfunction of the intracellular transport machinery, a system fundamental to the delivery of glycoproteins and maintenance of the ependymal barrier [22]. Given the critical role of the ependymal layer at the CSF–brain interface, such deficits may impair ciliary function, reduce neurotrophic support, and destabilize epithelial integrity. Simultaneously, reduced expression of key sialylation genes indicates aberrant post-translational protein modification, likely compromising cell-cell adhesion and intercellular signaling [23], which are crucial for supporting the neurogenic niche in the subventricular zone [24]. Taken together, these results suggest that impaired glycoprotein trafficking and glycosylation are hallmarks of ependymal cell aging. Importantly, the observed increase in intracellular lectin binding likely reflects not enhanced glycan biosynthesis but rather ectopic accumulation due to defects in apical delivery and lysosomal degradation of glycoproteins. These alterations are consistent with previous findings [25] and indicate a loss of glycoprotein homeostasis as a defining characteristic of aged ependymal cells. Such disturbances may contribute to age-related pathophysiology, including ventricular enlargement and impaired metabolic waste clearance, as observed in conditions such as normal-pressure hydrocephalus and Alzheimer’s disease. Our IVH model revealed acute-phase disruption of the ependymal Gcx. Decreased binding of LEL, PNA, and RCA-I following IVH suggests Gcx damage and subsequent barrier failure due to hemorrhagic insult, with recovery requiring an extended time. In agreement with prior studies [26], our data show that IVH-induced inflammation was prolonged in aged mice. These results strongly suggest that age-related Gcx fragility not only impedes recovery from acute brain injury but also contributes to persistent inflammation and deterioration of CSF dynamics [27]. The novelty of this study lies in the comprehensive and quantitative profiling of both apical and intracellular glycans of the ependymal Gcx using cryosections. Furthermore, it uniquely demonstrates that these glycan structures undergo dynamic changes in response to aging and brain injury. Maintaining or restoring the integrity of the ependymal Gcx may contribute to the preservation and regeneration of ependymal cell function. In addition, this study provides a foundational framework for the comprehensive understanding of the roles of ependymal glycans in various processes, including cerebrospinal fluid circulation, cell adhesion, and neuroimmune regulation, spanning from molecular mechanisms to systems-level neurobiology. Furthermore, these findings suggest the potential for developing novel anti-aging strategies and therapeutic approaches targeting glycan modifications in neurodegenerative diseases. Limitations This study was limited to lectin-based histochemical analysis in murine models, and caution is required when extrapolating to human physiology. In addition, owing to potential overlap in lectin-binding specificities, detailed structural identification of individual glycans may require complementary high-resolution glycomic approaches such as mass spectrometry. Finally, the metric used in this study—distance between fluorescence intensity inflection points—reflects relative Gcx thickness including the microvilli, but does not represent absolute physical dimensions. Moving forward, several directions may enhance the interpretability and applicability of these findings. The integration of structural and functional analyses—such as tracer-based CSF clearance assays or permeability measurements—could help delineate the physiological roles of Gcx alterations. Causal investigation into how Gcx remodeling influences disease severity, recovery, or therapeutic response—through gain- or loss-of-function strategies, conditional gene manipulation, or targeted enzymatic degradation—may provide mechanistic insight into its role in central nervous system homeostasis. Furthermore, pharmacological modulation using agents such as rapamycin or glycosylation-targeted compounds may inform strategies for preserving or restoring Gcx integrity. Lastly, combining glycomic analysis with single-cell or spatial transcriptomics could reveal cell-type–specific regulation of glycan biosynthesis and its association with neuroimmune or barrier functions. Conclusions This study demonstrated that the ependymal Gcx in the murine brain possesses a glycan profile essential for molecular exchange with the brain parenchyma, CSF circulation, and maintenance of barrier integrity under physiological conditions. In addition, we captured the dynamic alterations in the ependymal glycan profile associated with aging and acute brain injury. Notably, the preservation of ependymal cell function in the aging brain and its restoration following brain injury may represent critical therapeutic targets for delaying the progression of neurodegenerative processes. By elucidating the previously underexplored role of ependymal glycans in maintaining the homeostasis of the brain microenvironment, this study establishes a foundation for a new research direction at the intersection of neuroscience and neuropathology. Abbreviations Gcx, glycocalyx CSF, cerebrospinal fluid IVH, intraventricular hemorrhage scRNA-seq, single-cell RNA sequencing SEM, scanning electron microscopy LVSEM, low-vacuum scanning electron microscopy S.E.M., standard error of the mean RCA-1, Ricinus communis agglutinin I SiaFind, α2,3-linked sialic acid–specific lectins STL, Solanum tuberosum lectin DSL, Datura stramonium lectin (DSL) LEL, Lycopersicon esculentum lectin Declarations Ethics approval and consent to participate This study was approved by the Gifu University International Animal Care and Use Committee (Number:A2025-0001). Consent for publication Not applicable. Availability of data and materials Data is available upon reasonable request. Competing interests The authors declare no conflicts of interest. Funding The research was supported by JSPS KAKENHI grants JP20K0758723 (H.T.), JP23H03326 (H.T.), and JP24K19545 (K.O.), as well as the JST FOREST Program (JPMJFR220W, H.T.). Authors’ contributions Study concept and design: Tomohiro Iida. Data collection and analysis: Tomohiro Iida, Kosuke Mori, Hiroyuki Tomita, and Kohtaro Taguchi. Data interpretation: Tomohiro Iida and Hiroyuki Tomita. Writing of the manuscript: Tomohiro Iida and Hiroyuki Tomita. Review and editing: Tsuyoshi Izumo, and Akira Hara. Supervision: Ayumi Niwa, Tomohiro Kanayama, Kazufumi Ohmura, Shigeyuki Sugie, Hideshi Okada and Akira Hara. All authors approved the final version of the manuscript. Acknowledgments We thank Kyoko Takahashi, Ayako Suga, Masayoshi Shimizu, Reiko Kitazumi, and Kayo Nakamura for their support during the experiments. Figures 2 and 5 schematics were created in BioRender. Iida, T. (2026) https://BioRender.com/tzj5t3p, https://BioRender.com/xfjhwcw. Code availability Not applicable. References Redmond, S.A., et al., Development of Ependymal and Postnatal Neural Stem Cells and Their Origin from a Common Embryonic Progenitor. Cell Rep, 2019. 27 (2): p. 429-441.e3. Deng, S., et al., Roles of Ependymal Cells in the Physiology and Pathology of the Central Nervous System. Aging Dis, 2023. 14 (2): p. 468-483. Grondona, J.M., et al., Ependymal denudation, aqueductal obliteration and hydrocephalus after a single injection of neuraminidase into the lateral ventricle of adult rats. J Neuropathol Exp Neurol, 1996. 55 (9): p. 999-1008. Debbage, P.L., A systematic histochemical investigation in mammals of the dense glycocalyx glycosylations common to all cells bordering the interstitial fluid compartment of the brain. Acta Histochem, 1996. 98 (1): p. 9-28. Rhodes, R.H., Ultrastructure of complex carbohydrates of rodent and monkey ependymal glycocalyx and meninges. Am J Anat, 1987. 179 (4): p. 369-84. Wagner, C., et al., Cellular mechanisms involved in the stenosis and obliteration of the cerebral aqueduct of hyh mutant mice developing congenital hydrocephalus. J Neuropathol Exp Neurol, 2003. 62 (10): p. 1019-40. Jiménez, A.J., et al., Structure and function of the ependymal barrier and diseases associated with ependyma disruption. Tissue Barriers, 2014. 2 : p. e28426. Zhu, W., et al., Mouse models of intracerebral hemorrhage in ventricle, cortex, and hippocampus by injections of autologous blood or collagenase. PLoS One, 2014. 9 (5): p. e97423. Kawai, S., et al., Effect of three types of mixed anesthetic agents alternate to ketamine in mice. Exp Anim, 2011. 60 (5): p. 481-7. Tachi, M., et al., Human Colorectal Cancer Infrastructure Constructed by the Glycocalyx. J Clin Med, 2019. 8 (9). Ando, Y., et al., Brain-Specific Ultrastructure of Capillary Endothelial Glycocalyx and Its Possible Contribution for Blood Brain Barrier. Sci Rep, 2018. 8 (1): p. 17523. Mukai, S., et al., Three-dimensional electron microscopy for endothelial glycocalyx observation using Alcian blue with silver enhancement. Med Mol Morphol, 2021. 54 (2): p. 95-107. Lipowsky, H.H., L. Gao, and A. Lescanic, Shedding of the endothelial glycocalyx in arterioles, capillaries, and venules and its effect on capillary hemodynamics during inflammation. Am J Physiol Heart Circ Physiol, 2011. 301 (6): p. H2235-45. Gao, L. and H.H. Lipowsky, Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes. Microvasc Res, 2010. 80 (3): p. 394-401. Betteridge, K.B., et al., Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function. J Physiol, 2017. 595 (15): p. 5015-5035. D'Addio, M., J. Frey, and V.I. Otto, The manifold roles of sialic acid for the biological functions of endothelial glycoproteins. Glycobiology, 2020. 30 (8): p. 490-499. Tozzi, A., Charged Interfaces in the Brain: How Electrostatic Forces May Guide Cerebrospinal Fluid Dynamics. Eur J Neurosci, 2025. 61 (9): p. e70145. Brandon, D.M., S.K. Nayak, and P.S. Binder, Lectin binding patterns of the human cornea. Comparison of frozen and paraffin sections. Cornea, 1988. 7 (4): p. 257-66. Kuno, M., et al., Evaluating glycocalyx morphology and composition in frozen and formalin-fixed liver tumor sections. Pathol Res Pract, 2024. 263 : p. 155660. Ohmura, K., et al., Visualizing the endothelial glycocalyx in human glioma vasculature. Brain Tumor Pathol, 2025. 42 (2): p. 33-42. Granados-Durán, P., et al., Complement system activation contributes to the ependymal damage induced by microbial neuraminidase. J Neuroinflammation, 2016. 13 (1): p. 115. Stenmark, H., Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol, 2009. 10 (8): p. 513-25. Varki, A., Sialic acids in human health and disease. Trends Mol Med, 2008. 14 (8): p. 351-60. Lim, D.A. and A. Alvarez-Buylla, The Adult Ventricular-Subventricular Zone (V-SVZ) and Olfactory Bulb (OB) Neurogenesis. Cold Spring Harb Perspect Biol, 2016. 8 (5). Muthusamy, N., et al., MARCKS-dependent mucin clearance and lipid metabolism in ependymal cells are required for maintenance of forebrain homeostasis during aging. Aging Cell, 2015. 14 (5): p. 764-73. Wan, Y., et al., Effects of aging on hydrocephalus after intraventricular hemorrhage. Fluids Barriers CNS, 2020. 17 (1): p. 8. Ma, Q., et al., Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun, 2017. 8 (1): p. 1434. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 07 Nov, 2025 Read the published version in Fluids and Barriers of the CNS → Version 1 posted Editorial decision: Revision requested 04 Sep, 2025 Reviews received at journal 04 Sep, 2025 Reviews received at journal 28 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers invited by journal 15 Aug, 2025 Editor assigned by journal 15 Aug, 2025 Submission checks completed at journal 14 Aug, 2025 First submitted to journal 13 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7369159","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501694023,"identity":"a3c6139b-e7fb-4395-badf-477d831d10c8","order_by":0,"name":"Tomohiro Iida","email":"","orcid":"","institution":"Gifu University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tomohiro","middleName":"","lastName":"Iida","suffix":""},{"id":501694024,"identity":"266453a8-75b0-44aa-be4d-4b8ca6bd2a28","order_by":1,"name":"Kosuke Mori","email":"","orcid":"","institution":"Gifu University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kosuke","middleName":"","lastName":"Mori","suffix":""},{"id":501694026,"identity":"bf517284-9d56-4a07-8816-883de8ec1461","order_by":2,"name":"Hiroyuki Tomita","email":"data:image/png;base64,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","orcid":"","institution":"Gifu University Graduate School of 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kohtaro","middleName":"","lastName":"Taguchi","suffix":""},{"id":501694031,"identity":"76ff479c-a6e7-48d6-868e-9701f42138d5","order_by":6,"name":"Kazufumi Ohmura","email":"","orcid":"","institution":"Gifu University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazufumi","middleName":"","lastName":"Ohmura","suffix":""},{"id":501694032,"identity":"46c3bc46-37cc-4c3e-84f0-0e3623b13613","order_by":7,"name":"Shigeyuki Sugie","email":"","orcid":"","institution":"Asahi University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shigeyuki","middleName":"","lastName":"Sugie","suffix":""},{"id":501694033,"identity":"87e490ec-d17c-44b4-806e-676d9e759bbd","order_by":8,"name":"Hideshi Okada","email":"","orcid":"","institution":"Gifu University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hideshi","middleName":"","lastName":"Okada","suffix":""},{"id":501694035,"identity":"8afe017b-fe53-4d37-b6af-55ddabdbab47","order_by":9,"name":"Tsuyoshi Izumo","email":"","orcid":"","institution":"Gifu University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tsuyoshi","middleName":"","lastName":"Izumo","suffix":""},{"id":501694041,"identity":"bd0f79dd-6665-4062-8bdc-07bb1a92eed3","order_by":10,"name":"Akira Hara","email":"","orcid":"","institution":"Gifu University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Hara","suffix":""}],"badges":[],"createdAt":"2025-08-14 02:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7369159/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7369159/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12987-025-00725-x","type":"published","date":"2025-11-07T15:56:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89973868,"identity":"30e51d8c-105f-4175-8096-1a41e4e6681e","added_by":"auto","created_at":"2025-08-27 05:52:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1591221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisualization and glycan profiling of the ependymal Gcx in young adult mice. \u003c/strong\u003ea) Scanning electron microscopy (SEM) images of ventricular ependymal cells. Gcx is not visible under conventional glutaraldehyde fixation (left) but clearly visualized surrounding the cell surface and cilia with lanthanum staining (right).\u003cstrong\u003e \u003c/strong\u003eb) Alcian blue + hematoxylin-eosin (HE) staining. Glycans covering the apical surface of the ependyma are stained blue with Alcian blue. White scale bar: 20 µm. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ec) Low-vacuum SEM image of a serial section corresponding to (b). A three-dimensional view of the Gcx layer covering the base of the cilia and surrounding the ciliary shafts at the apical surface. Enlarged view on the right.\u003cstrong\u003e \u003c/strong\u003ed) Double immunofluorescence staining of lectin (PNA, red) and S100β (green) in frozen sections. A distinct glycan layer is observed at the apical surface of the ependyma, comparable to that seen in electron microscopy. White scale bar: 10 µm.\u003cstrong\u003e \u003c/strong\u003ee) Fluorescence intensity plot along the measurement line (yellow) in (d), from the ventricular lumen (left) toward the brain parenchyma (right); red indicates lectin, green indicates S100β. The plot confirms localization of the Gcx at the ependymal cell surface.\u003cstrong\u003e \u003c/strong\u003ef) Bar graph showing the mean fluorescence intensity of the ependymal Gcx for each of the 21 lectins examined.\u003cbr\u003e\ng) Merged images of lectin (red) and S100β (green) staining. Representative examples are shown: strong positive (RCA-I, STL), moderate positive (WGA, PHA-E), and negative (UEA-I, S-WGA). White scale bar: 10 µm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7369159/v1/6f481e1591884da3811e58e9.png"},{"id":89973873,"identity":"16c5ec97-e51f-4fc1-9146-fefdb6bc09da","added_by":"auto","created_at":"2025-08-27 05:52:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":859976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of ependymal Gcx between young adult and aged mice.\u003c/strong\u003e a) Heatmap representing the mean fluorescence intensity per pixel of the ependymal Gcx in young adults and aged mice. b) Representative images of PNA-based fluorescent staining of the ependymal Gcx in young adults (top) and aged (bottom) mice. White scale bar: 5 µm. c) Gcx coverage along the periventricular circumference of the lateral ventricle in young adults and aged mice (two-sided t-test; mean ± s.e.m.). d) Transmission electron microscopy (TEM) images of ventricular ependymal cells. In the young adult (left two), the Gcx is clearly visible on the ependymal cell apical surface (black arrowheads), microvilli (black arrows) and cilia (small black allow). In the aged mouse (right), loss of the Gcx is evident on both the apical surface (white arrowheads) and microvilli (white arrows). White bar: 500 nm. e) Schematic of the ependymal surface Gcx inferred from lectin staining and TEM images. f) Double fluorescence staining for LEL (red) and S100β (green) (left). Fluorescence intensity plot along the measurement line (yellow) is shown on the right (red: LEL, green: S100β; dots indicate inflection points). White scale bar: 5 µm. g) Correlation between the inflection point distance and manually measured Gcx thickness using PNA staining (Pearson correlation). h) Comparison of Gcx thickness between young adults and aged mice based on inflection point distances using LEL, PNA, and RCA-I (two-sided t-test; mean ± s.e.m.). i) Representative double fluorescence staining for PNA (red) and S100β (green) showing increased cytoplasmic PNA positivity in aged ependymal cells. White scale bar: 5 µm. j) Cytoplasmic PNA-positivity rate per length of the ventricular wall (two-sided t-test; mean ± s.e.m.). k) Heatmap comparing the mean fluorescence intensity in the ependymal cytoplasm for 21 different lectins.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7369159/v1/66fdde2a301e3957bd1bab89.png"},{"id":89976184,"identity":"d6a5669d-7382-4628-966b-d24f859f40a9","added_by":"auto","created_at":"2025-08-27 06:00:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1172402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime-course changes in ependymal Gcx and inflammation following IVH.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Representative images showing the time-course (day 0, 1, 3, 7) of ependymal Gcx (Alcian blue and PNA staining), periventricular inflammation (Iba-1, Galectin-3), and choroid plexus inflammation (Iba-1) in the IVH model. White scale bar: 10 µm. b) Time-course of ependymal Gcx coverage rate during the acute phase of IVH, as assessed using PNA staining (one-way ANOVA with Dunnett’s post hoc test; mean ± s.e.m.). c) Time-dependent changes in ependymal Gcx thickness during the acute phase of IVH, measured using inflection point distances with LEL, PNA, and RCA-I (one-way ANOVA with Dunnett’s post hoc test; mean ± s.e.m.). d) Temporal progression of inflammation during the acute phase of IVH: (left) Iba-1 fluorescence intensity per pixel in the periventricular parenchyma; (center) size of Iba-1-positive Kolmer cells in the choroid plexus; (right) Galectin-3 fluorescence intensity per unit ventricular circumference in ependymal cells (one-way ANOVA with Dunnett’s post hoc test; mean ± s.e.m.). e) Comparison of acute inflammatory responses between young and aged mice at days 0, 3, and 7 after IVH: (left) Iba-1 fluorescence intensity per pixel in the periventricular parenchyma; (center) size of Iba-1–positive Kolmer cells; (right) Galectin-3 fluorescence intensity per unit ventricular circumference in ependymal cells.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7369159/v1/32d2ad75a73d19c9065712e5.png"},{"id":89973875,"identity":"bd08b8b5-35d1-4584-94d9-c08b091caf42","added_by":"auto","created_at":"2025-08-27 05:52:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":169763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAge-associated downregulation of vesicular transport and sialylation pathway genes in murine ependymal cells.\u003c/strong\u003e a) Bubble heatmap showing the percentage of cells expressing each gene (dot size) and the normalized mean expression level (color intensity) in young and aged adult mice. b) Heatmap displaying single-cell expression levels of the indicated genes. Each row represents a cell, grouped by age. c) Circos plot illustrating the relationship between the young and aged adult cell populations and the collective expression of the eight target genes. The width of the ribbon corresponds to the relative expression strength.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7369159/v1/e1a87d7a4ea5ee586a4ba9d5.png"},{"id":89973885,"identity":"5b78914c-e582-45cd-8d81-5055cb7f0863","added_by":"auto","created_at":"2025-08-27 05:52:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":571761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAge- and IVH-associated alterations in the ependymal glycocalyx\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic illustration of ependymal Gcx changes in aging and IVH. In the young state, the ependymal surface is covered by a dense Gcx rich in sialic acid and galactose residues. Aging leads to Gcx thinning and altered glycan composition, while IVH causes acute Gcx loss and epithelial denudation. These changes may compromise CSF–brain barrier integrity and promote neuroinflammation.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7369159/v1/d6790d43e247fd3406b27ce8.png"},{"id":95563920,"identity":"47fa5d1b-50c4-4b88-9760-62207a066366","added_by":"auto","created_at":"2025-11-10 16:03:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5198537,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7369159/v1/905ca67b-be22-40bf-899f-45c01edf026c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Age‑Dependent and Post‑Intraventricular Hemorrhage Remodeling of the Ependymal Glycocalyx in Mice","fulltext":[{"header":"Background","content":"\u003cp\u003eThe ependymal cells lining the cerebral ventricles are ciliated glial cells derived from radial glia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and they play a pivotal role in cerebrospinal fluid (CSF) circulation, molecular exchange with the brain parenchyma, and maintenance of the neural stem cell niche [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These cells are covered by the glycocalyx (Gcx), a complex of polysaccharides and proteins [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which functions as the frontline interface mediating cellular interactions with the environment. The ependymal Gcx may contribute to the formation of a negatively charged cell surface, facilitate smooth CSF flow, and act as a physical and immunological barrier against pathogens and metabolic waste [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, the structural and compositional alterations of the ependymal Gcx in response to physiological and pathological stressors such as aging or acute brain injury and the subsequent effect on ependymal cell function and overall brain homeostasis remain unclear. Few studies have clearly distinguished between surface Gcx and intracellular glycans.\u003c/p\u003e\u003cp\u003eTherefore, in the present study, we conducted a comprehensive histochemical analysis using a panel of lectins with diverse glycan-binding specificities to qualitatively and quantitatively assess changes in the structure, composition, and localization of the lateral ventricular ependymal Gcx in young adult mice, aged mice, and mice with intraventricular hemorrhage (IVH). Through this approach, we aimed to elucidate not only the physiological role of the ependymal Gcx but also its alterations in response to aging and acute brain injury, thereby providing insights that may contribute to the development of diagnostic markers and therapeutic strategies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild-type C57BL/6J mice were obtained from the Japan Jackson Laboratory (Japan). Male mice aged 8\u0026ndash;10 and 60\u0026ndash;62 weeks were used in all experiments. In this study, 8-10-week-old mice were defined as \u0026ldquo;young adult,\u0026rdquo; and 60\u0026ndash;62-week-old mice were defined as \u0026ldquo;aged\u0026rdquo;. All animal procedures were conducted in accordance with the guidelines of the Gifu University International Animal Care and Use Committee (Approval No. A20250001). Mice were housed under standard laboratory conditions with a 12 h light/12 h dark cycle at a controlled temperature of 22 \u0026deg;C, with ad libitum access to food and water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIVH Animal Model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IVH mouse model was established via intracerebroventricular (i.c.v.) injection of 25 \u0026mu;L of autologous whole blood, as previously described [8]. Mice were anesthetized using three types of intraperitoneal administration of mixed anesthesia, as reported previously [9]. A 1-mm burr hole was drilled in the skull at a point 0.5 mm posterior and 1.0 mm lateral to the bregma. A 26-gauge needle was inserted freehand into the right lateral ventricle to a depth of 2.5 mm. A total of 25 \u0026mu;l of autologous whole blood, collected from the orbital venous plexus, was manually injected at the slowest possible rate using a Hamilton micro syringe. After injection, the needle was left in place for an additional 2 min to minimize backflow. The burr hole was sealed with bone wax, and the skin incision was closed with sutures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTissue preparation of mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen sections: Mice were euthanized, and tissues were collected without perfusion. Samples were embedded in an optimal cutting temperature compound, snap-frozen in liquid nitrogen, and stored at -80 \u0026deg;C. The embedded tissues were coronally sectioned at a thickness of 5 \u0026mu;m near the optic chiasm using a rotary microtome (Leica, Wetzlar, Germany).\u003c/p\u003e\n\u003cp\u003eParaffin sections: Following anesthesia, the thoracic cavities of the mice were opened, and the inferior vena cava was incised. Perfusion was performed using a drip infusion system with equal volumes of cold 0.1 M phosphate-buffered saline (PBS) and cold 4% paraformaldehyde. Afterward, the tissues were harvested, dissected into smaller pieces, and processed for paraffin embedding and sectioning.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLectin fluorescent staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree lectin kits (I\u0026ndash;III), each comprising a diverse array of lectins with distinct binding specificities for broad screening of glycan structures on cell surfaces, tissues, or purified glycoproteins, were obtained from Vector Laboratories (Burlingame, CA, USA). In addition, SiaFind \u0026alpha;2,3-Specific Lectenz, an antibody-based reagent that specifically recognizes \u0026alpha;2,3 linked sialic acid residues, was purchased from Lectenz Bio (Athens, GA, USA). Details of the biotinylated lectins included in kits I\u0026ndash;III and SiaFind are summarized in Table 1.\u003c/p\u003e\n\u003cp\u003eFor double-fluorescence staining using an antibody and biotinylated lectins, tissue sections were fixed in 4% paraformaldehyde in PBS for 15 min. After washing with PBS, sections were blocked with the Histofine Mouse Stain Kit (NICHIREI BIOSCIENCES INC.) for 60 min at room temperature. Subsequently, biotinylated lectins (1:200 dilution) and an ependymal cell marker, the S100\u0026beta; antibody (mouse monoclonal, 1:500 dilution; sc-393919, Santa Curz Biotechnology), were applied and incubated overnight at 4 \u0026deg;C. The following day, sections were washed with PBS, blocked again with the Histofine Mouse Stain Kit, and incubated with Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:250 dilution; ab150165, Abcam) and DyLight 594-conjugated streptavidin (1:250 dilution; Vector Laboratories) for 1 h at room temperature. Finally, after washing with PBS, the nuclei were counterstained with DAPI, and coverslips were mounted using appropriate mounting medium.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 566px;\"\u003e\n \u003cp\u003eTable 1. \u0026nbsp; Binding specificities of the lectins that were used in this study.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eLectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eCommon Abbreviation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eSpecificity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eConcanavalin A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eConA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026alpha;Man \u0026gt; \u0026alpha;Glc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eDolichos biflorus agglutinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eDBA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGalNAc\u0026alpha;(1,3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePeanut Agglutinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGal\u0026beta;(1,3) \u0026gt; Gal\u0026beta;(1,4) \u0026gt; Gal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eRicinus communis agglutinin\u0026nbsp;Ⅰ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eRCA\u0026nbsp;Ⅰ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGalNAc \u0026gt; \u0026alpha;Gal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eSoybean agglutinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eSBA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026alpha;GalNAc \u0026gt; \u0026alpha;Gal \u0026gt; \u0026beta;GalNAc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eUlex Europaeus agglutinin\u0026nbsp;Ⅰ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eUEA\u0026nbsp;Ⅰ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eFuc\u0026alpha;(1,2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eWheat Germ agglutinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eWGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eNeuAc \u0026gt;\u0026gt;\u0026gt; GlcNAc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGriffonia simplicifolia lectin\u0026nbsp;Ⅰ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGSL\u0026nbsp;Ⅰ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026alpha;Gal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eLen culinaris lectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eLCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026alpha;Man \u0026gt; \u0026alpha;Glc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePhaseolus vulgaris Erythroagglutinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePHA E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eComplex structures\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePhaseolus vulgaris Leucoagglutinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePHA L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eComplex structures\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePisum sativum agglutinin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003ePSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026alpha;Man \u0026gt; \u0026alpha;Glc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eWheat Germ agglutinin, succinylated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003esuccinylated WGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGlcNAc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eDatura stramonium lectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eDSL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGlcNAc oligomer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eErythrina cristagalli lectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eECL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGriffonia simplicifolia lectin\u0026nbsp;Ⅱ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGSL\u0026nbsp;Ⅱ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGlcNAc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eJacalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eJacalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026alpha;Gal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eLycopersicon esculentum lectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eLEL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGlcNAc oligomer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eSolanum tuberosum lectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eSTL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGlcNAc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eVicia villosa lectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eVVL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eGalNac\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eSiaFind \u0026alpha;2,3-Specific Lectenz\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003eSiaFind\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026alpha;2,3 linked NeuAc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 566px;\"\u003e\n \u003cp\u003eAbbreviation: Man, mannose; Glc, glucose; GalNAc, N-acetylgalactosamine; Gal, galactose; Fuc, fucose; NeuAc, sialic acid; GlcNAc, N-acetylglucosamine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunofluorescence Staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe paraffin-embedded brain tissues were cut into 4-\u0026mu;m sections. The sections were blocked with bovine serum albumin at 37 \u0026deg;C for 60 min; thereafter, they were incubated with the following primary antibodies overnight at 4 \u0026deg;C: ionized calcium binding adaptor molecule-1 (Iba-1) (1:500, #019-1974, Wako) and Galectin-3 (1:100, #13-5301-85, Bay Biosciences). After washing the sections three times with PBS, they were incubated with secondary antibodies at 37 \u0026deg;C for 60 min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eScanning electron microscopy (SEM)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEpendymal Gcx was visualized using SEM, as previously described[10, 11]. Briefly, tissue samples were cut into 5 mm\u0026sup3; pieces and initially fixed for 2 h in a solution of 2% glutaraldehyde, 2% sucrose, 0.1 M sodium cacodylate buffer (pH 7.3), and 2% lanthanum nitrate. Thereafter, the specimens were immersed overnight in a solution containing 2% sucrose, 0.1 M sodium cacodylate buffer (pH 7.3), and 2% lanthanum nitrate, followed by washing in an alkaline solution (0.03 M sodium hydroxide with 2% sucrose). After fixation and washing, the specimens were dehydrated through a graded ethanol series. Subsequently, they were frozen in 100% ethanol and rapidly cooled with liquid nitrogen to create fracture surfaces for SEM observation. Afterward, the frozen tissues were fractured using a carving knife. Finally, the specimens were examined using SEM (S-4800, Hitachi, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLow-vacuum scanning electron microscopy (LVSEM)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor LVSEM, we used a modified version of a previously described method [12], the details of which are described in a manuscript in preparation at the time of the present study (Mori, K. et al.). Briefly, mice were deeply anesthetized and perfused with a solution of 10% neutral buffered formalin containing 1% Alcian Blue 8GX and 2% sucrose. Subsequently, the brains were harvested, processed for paraffin embedding, and cut into 2-\u0026mu;m thick serial sections. After deparaffinization, the sections were stained with Periodic Acid-Methenamine silver. The stained specimens were air-dried, mounted onto a sample holder using conductive adhesive tape, and observed without metal coating using a low-vacuum scanning electron microscope (TM3030Plus, Hitachi High-Tech, Tokyo, Japan) at an acceleration voltage of 15 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTransmission electron microscopy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue fixation was performed as described for the SEM. After fixation, brain samples were postfixed with 2% osmium tetroxide in 0.1 M cacodylate buffer for 2 h at 4 \u0026deg;C. Thereafter, the samples were dehydrated through a graded ethanol series (50\u0026ndash;100%), treated with propylene oxide, and embedded in epoxy resin (Quetol812: DDSA: MNA = 7:4:4, with 1.5% DMP-30 catalyst). Polymerization was performed at 40 \u0026deg;C for 8 h, 70 \u0026deg;C for 24 h, and 75 \u0026deg;C for 12 h. Ultrathin sections (90 nm) were cut using an ultramicrotome (Leica EM UC7) and mounted on copper grids. Sections were stained with 2% uranyl acetate for 15 min, followed by lead citrate for 5 min, and dried on filter paper. Images were acquired using a transmission electron microscope (HT7800, Hitachi, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImage Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages used for fluorescence intensity and inter-inflection distance measurements were acquired under consistent imaging conditions to ensure data comparability. Following background subtraction, the mean fluorescence intensities of the Gcx and cytoplasm were measured in 10 randomly selected cells per animal (n = 3; total of 30 cells). The thickness of the ependymal Gcx was quantified as previously described [13]. Briefly, a measurement line was drawn perpendicular to the ependymal cell surface using merged images of lectin and S100\u0026beta; staining. Radial fluorescence intensity profiles for both channels were extracted at the ependymal surface and fitted to a sigmoid curve using the least squares method. The inflection point of the lectin-derived curve was defined as the outer boundary of the Gcx layer, and the inflection point of the S100\u0026beta; curve was defined as the apical surface of the ependymal cell. The distance between these two inflection points was interpreted as the thickness of the ependymal Gcx. Gcx thickness was measured at 16 randomly selected points per animal (n = 3 or 5; total of 48 or 80 points). The extent of periventricular inflammation was evaluated by measuring Iba-1 fluorescence intensity within a 17-\u0026micro;m-wide band region of the periventricular brain parenchyma, as well as Galectin-3 fluorescence intensity in the ependymal cells. In addition, the size of Iba-1-positive Kollmer cells in the choroid plexus was assessed. All image processing and quantification were performed using ImageJ software (image processing software, open source).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Acquisition and Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed an integrated analysis of public single-cell RNA-sequencing (scRNA-seq) data comprising 9,406 murine ependymal cells. The dataset was compiled from five independent studies (accession IDs: SCP565, SRP135960, GSE74672, SCP318, and PMID_32669714_FACS) using the Talk2Data platform (v4, BioTuring Inc.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBioinformatic Analysis and Visualization\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll bioinformatic analyses, including cell type identification, segregation of young adult and aged adult populations, and differential gene expression analysis, were conducted within the Talk2Data environment. All figures, including the bubble heatmap (Figure 4a), single-cell heatmap (Figure 4b), and Circos plot (Figure 4c), were generated using the visualization tools integrated within the platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were presented as mean \u0026plusmn; standard error of the mean (SEM). Differences in Gcx thickness, periventricular coverage, Iba-1 fluorescence intensity per pixel, Kolmer cell Iba-1-positive area, and Galectin-3 fluorescence intensity between young and aged mice for each lectin were assessed using unpaired t-tests. Changes in Gcx thickness over time following intraventricular hemorrhage were analyzed using one-way analysis of variance (ANOVA), followed by Dunnett\u0026rsquo;s multiple comparison test to correct for multiple comparisons. To validate the use of inflection point distances as a quantitative measure of Gcx thickness, correlations with actual measurements were evaluated using Pearson\u0026rsquo;s correlation coefficient (r). \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 10.4.2 (GraphPad Software, Inc., La Jolla, CA, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVisualization and Glycan Profiling of the Ependymal Gcx in Normal Mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn SEM observations, the Gcx on the surface of ependymal cells was not detectable under conventional glutaraldehyde fixation. However, when combined with lanthanum staining, a dense Gcx layer was clearly visualized on the apical surface of ependymal cells and surrounding the cilia (Figure 1a). Histologically, Alcian blue staining distinctly labeled the glycan layer on the apical membrane, and low-vacuum SEM enabled three-dimensional visualization of the Gcx structure covering the base of the cilia (Figure 1b, 1c). In lectin staining, ependymal cells were immunolabeled with S100\u0026beta;, and the apical Gcx layer was clearly highlighted (Figure 1d).\u003c/p\u003e\n\u003cp\u003eFurthermore, fluorescence-based screening using 21 lectins with diverse glycan-binding specificities revealed that the ependymal Gcx consists of a wide variety of glycans. Particularly strong signals were observed for ycopersicon esculentum lectin (LEL), Ricinus communis agglutinin I (RCA-I), \u0026alpha;2,3-linked sialic acid\u0026ndash;specific lectins (SiaFind), Solanum tuberosum lectin (STL), Datura stramonium lectin (DSL), and peanut agglutinin (PNA) (Figure 1f). Representative images ranging from strongly positive to negative lectin signals are shown in Figure 1g. In contrast, cytoplasmic binding was prominent for Vicia Villosa Lectin, Griffonia Simplicifolia Lectin I, Erythrina Cristagalli Lectin, Wheat germ agglutinin (WGA), LEL, and Succinylated Wheat Germ Agglutinin (S-WGA). Notably, lectins such as PNA and \u0026alpha;2,3-linked sialic acid\u0026ndash;specific lectins, which showed strong affinity to the apical Gcx, were negative in the cytoplasm (Figure 1d), indicating distinct glycan profiles between the apical surface and the intracellular compartments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStructural and Compositional Changes in the Ependymal Gcx Associated with Aging\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs performed in young mice, the mean fluorescence intensity of the ependymal Gcx was measured in aged mice. Increased intensities were observed with PHA-E and PHA-L staining; however, many lectins, including SiaFind and LEL, showed a decreasing trend. The pattern of lectin positivity and negativity remained unchanged across groups, regardless of lectin type (Figure 2a). Morphologically, the ependymal Gcx in aged mice appeared markedly thinned, and widespread detachment of the layer was observed (Figure 2b). Quantitative analysis of Gcx coverage along the ventricular circumference revealed a significant decrease from 77.25 \u0026plusmn; 1.85% in young mice to 33.06 \u0026plusmn; 7.14% in aged mice (p \u0026lt; 0.0011) (Figure 2c). These age-related changes were also confirmed by TEM imaging (Figure 2d). In young adult, TEM revealed that not only the apical surface of the ependymal cells (black alllow) but also the microvilli (brack allow head) and cilia (small black allow) were covered with Gcx, whereas in aged, partial loss of Gcx was observed. Furthermore, comparison between TEM and lectin-stained images, based on differences in the thickness of the glycan layer, suggested that the Gcx layer observed in lectin staining corresponds to the layer that includes the microvilli (Figure 2e). Using lectin-stained images, the thickness of the Gcx layer containing microvilli was evaluated by measuring the distance between inflection points in the fluorescence intensity profile (Figure 2f).\u0026nbsp;The validity of this method was confirmed using PNA staining, which were approximately 20 % lower than direct measurements but displayed a strong positive correlation (r = 0.877, p \u0026lt; 0.01), validating this metric for relative thickness quantification and supporting its utility for relative Gcx thickness assessment (Figure 2g). Using this method, significant thinning was observed in aged mice across all three representative lectins: LEL (0.821 \u0026plusmn; 0.175 vs. 0.500 \u0026plusmn; 0.231 \u0026micro;m), PNA (0.796 \u0026plusmn; 0.136 vs. 0.373 \u0026plusmn; 0.165 \u0026micro;m), and RCA-I (0.747 \u0026plusmn; 0.208 vs. 0.445 \u0026plusmn; 0.191 \u0026micro;m) (Figure 2h).\u003c/p\u003e\n\u003cp\u003eConsistent with this baseline pattern, aged mice exhibited ectopic cytoplasmic PNA signals, with PNA-positive cells increasing from 2.97 \u0026plusmn; 0.81 % to 20.30 \u0026plusmn; 3.93 % in young adult mice (Figure 2i, j), indicating intracellular retention of Gal\u0026beta;1-3GalNAc-terminated glycans. Increased cytoplasmic binding was observed for many other lectins, except for S-WGA, as determined via comparative fluorescence intensity analysis (Figure 2k).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDynamic Changes in the Ependymal Gcx Following IVH\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTime-course analysis following IVH induction revealed a rapid thinning and loss of the ependymal Gcx layer, as demonstrated using Alcian blue and PNA staining (Figure 3a). The assessment of Gcx detachment based on PNA staining showed maximal damage on day 3, with partial recovery observed on day 7 (Figure 3b). However, quantitative analysis of Gcx thickness using LEL, PNA, and RCA-I demonstrated a sustained reduction in thickness from day 1 through day 7 (Figure 3c).\u003c/p\u003e\n\u003cp\u003eThe inflammatory response in the periventricular region, evaluated by Iba-1\u0026ndash;positive microglia and Galectin-3 expression in ependymal cells, and inflammation in the choroid plexus, assessed by the size of Iba-1-positive Kolmer cells, peaked on day 1 or day 3 and showed signs of resolution on day 7 (Figure 3d). In contrast, in aged IVH model mice, both Iba-1 and Galectin-3 levels remained elevated even at day 7 (Figure 3e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAge-Related Downregulation of Vesicular Transport and Sialylation Genes in Ependymal Cells Revealed by Integrated Single-Cell Transcriptomic Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate age-related transcriptomic changes in murine ependymal cells, we performed an integrated analysis of 9,406 cells derived from five public single-cell RNA-seq studies (SCP565, SRP135960, GSE74672, SCP318, and PMID_32669714_FACS). Our analysis revealed a significant and coordinated downregulation of genes associated with vesicular transport and protein sialylation in aged ependymal cells compared to their younger counterparts.\u003c/p\u003e\n\u003cp\u003eA bubble heatmap demonstrated that both the percentage of expressing cells and the average expression levels of key genes in these pathways\u0026mdash;RAB6A, RAB8A, RAB11A, RAB11B, NEU1, NEU2, ST3GAL1, and SLC35A1\u0026mdash;were markedly lower in aged adult mice (Figure 4a). This finding was substantiated at the single-cell level, where a heatmap showed consistent and robust expression of these genes across the young cell population, in contrast to the sporadic and weaker expression observed in aged cells (Figure 4b). A Circos plot further visualized the diminished contribution of this entire gene set to the transcriptomic profile of the aged ependymal cell population, confirming a broad suppression of these pathways with age (Figure 4c).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides a comprehensive characterization of the glycan profile of the ependymal Gcx in the murine brain and demonstrates its dynamic alterations in response to aging and acute brain injury (Figure 5). Notably, we adapted a method originally developed for evaluating Gcx thickness via two-photon microscopy [13, 14] and applied it, for the first time, to fluorescence immunohistochemistry. This enabled the objective quantification of glycan distribution beyond conventional qualitative assessments. This approach supports the emerging concept of the \u0026quot;glycocode\u0026quot;\u0026mdash;where changes in glycan composition reflect cellular state\u0026mdash;and introduces a novel perspective in neuroglycobiology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious studies [3, 6, 7], including ours, have shown that the ependymal Gcx is rich in sialylated glycans. Besides, sialic acid is a key component of vascular endothelial Gcx and plays critical roles in intercellular interactions, glycoprotein binding, and the regulation of vascular permeability [15, 16]. It is plausible that in the ependyma, sialic acid contributes similarly to selective permeability. Moreover, its hydrophilic and negatively charged nature may reduce friction with CSF, acting as a lubricant to support smooth CSF flow. Notably, electrohydrodynamic studies suggest that the negative charge of the Gcx may directly facilitate CSF perfusion [17].\u003c/p\u003e\n\u003cp\u003eAlthough previous studies [3, 4, 6] have indicated that the ependymal Gcx is PNA-negative unless treated with neuraminidase, our study identified clear PNA-positivity without enzymatic treatment. This discrepancy may arise from differences in tissue preparation; paraffin sectioning often compromises glycan epitopes, whereas our use of cryosections better preserved Gcx structure in a near-native state [18, 19]. Meanwhile, reports of PNA-positive Gcx in vascular endothelium are extremely limited. No studies have demonstrated PNA positivity in normal endothelial Gcx without neuraminidase treatment. Where such staining has been observed, it has been confined to endothelial cells located in the central regions of certain tumors [20]. Our findings suggest that the ependymal Gcx, despite being rich in sialic acid, contains an unusually high abundance of galactose-terminated glycans lacking sialic acid capping. This glycan configuration may represent a structural adaptation that enables molecular exchange with the brain parenchyma while preserving selective barrier function, distinguishing it from vascular endothelium.\u003c/p\u003e\n\u003cp\u003eAge-related thinning and decreased coverage of the ependymal cell Gcx, accompanied by a reduction in terminal galactose residues, \u0026alpha;2,3-linked sialic acids, and poly-N-acetyllactosamine structures, suggest impaired molecular exchange with the brain parenchyma, disruption of barrier function, and dysfunction in CSF circulation. Notably, sialic acids function as \u0026ldquo;self\u0026rdquo; markers contributing to the maintenance of immune tolerance. Therefore, the loss of this \u0026ldquo;sialic acid shield\u0026rdquo; may increase susceptibility to immune factors\u0026mdash;including complement activation [21]\u0026mdash;and act as a trigger or exacerbating factor for age-associated chronic neuroinflammation. Collectively, these findings suggest that the degradation of the Gcx on the ependymal surface may contribute to the disruption of homeostasis in the cerebral microenvironment.\u003c/p\u003e\n\u003cp\u003eIn addition, cytoplasmic PNA binding, which was absent in young mice, was frequently observed in the ependymal cells of aged mice. In most of these PNA-positive cells, thinning or partial loss of the apical Gcx was also detected. Furthermore, several lectins\u0026mdash;including those specific for \u0026alpha;2,3-linked sialic acids\u0026mdash;exhibited significantly increased intracellular binding in aged ependymal cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTranscriptomic analysis provided further evidence for an age-associated decline in two essential processes within murine ependymal cells: vesicular trafficking and protein sialylation. The coordinated downregulation of multiple Rab GTPases suggests dysfunction of the intracellular transport machinery, a system fundamental to the delivery of glycoproteins and maintenance of the ependymal barrier [22]. Given the critical role of the ependymal layer at the CSF\u0026ndash;brain interface, such deficits may impair ciliary function, reduce neurotrophic support, and destabilize epithelial integrity. Simultaneously, reduced expression of key sialylation genes indicates aberrant post-translational protein modification, likely compromising cell-cell adhesion and intercellular signaling [23], which are crucial for supporting the neurogenic niche in the subventricular zone [24].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, these results suggest that impaired glycoprotein trafficking and glycosylation are hallmarks of ependymal cell aging. Importantly, the observed increase in intracellular lectin binding likely reflects not enhanced glycan biosynthesis but rather ectopic accumulation due to defects in apical delivery and lysosomal degradation of glycoproteins. These alterations are consistent with previous findings\u0026nbsp;[25] and indicate a loss of glycoprotein homeostasis as a defining characteristic of aged ependymal cells. Such disturbances may contribute to age-related pathophysiology, including ventricular enlargement and impaired metabolic waste clearance, as observed in conditions such as normal-pressure hydrocephalus and Alzheimer\u0026rsquo;s disease.\u003c/p\u003e\n\u003cp\u003eOur IVH model revealed acute-phase disruption of the ependymal Gcx. Decreased binding of LEL, PNA, and RCA-I following IVH suggests Gcx damage and subsequent barrier failure due to hemorrhagic insult, with recovery requiring an extended time. In agreement with prior studies [26], our data show that IVH-induced inflammation was prolonged in aged mice. These results strongly suggest that age-related Gcx fragility not only impedes recovery from acute brain injury but also contributes to persistent inflammation and deterioration of CSF dynamics [27].\u003c/p\u003e\n\u003cp\u003eThe novelty of this study lies in the comprehensive and quantitative profiling of both apical and intracellular glycans of the ependymal Gcx using cryosections. Furthermore, it uniquely demonstrates that these glycan structures undergo dynamic changes in response to aging and brain injury. Maintaining or restoring the integrity of the ependymal Gcx may contribute to the preservation and regeneration of ependymal cell function. In addition, this study provides a foundational framework for the comprehensive understanding of the roles of ependymal glycans in various processes, including cerebrospinal fluid circulation, cell adhesion, and neuroimmune regulation, spanning from molecular mechanisms to systems-level neurobiology. Furthermore, these findings suggest the potential for developing novel anti-aging strategies and therapeutic approaches targeting glycan modifications in neurodegenerative diseases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLimitations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was limited to lectin-based histochemical analysis in murine models, and caution is required when extrapolating to human physiology. In addition, owing to potential overlap in lectin-binding specificities, detailed structural identification of individual glycans may require complementary high-resolution glycomic approaches such as mass spectrometry. Finally, the metric used in this study\u0026mdash;distance between fluorescence intensity inflection points\u0026mdash;reflects relative Gcx thickness including the microvilli, but does not represent absolute physical dimensions. Moving forward, several directions may enhance the interpretability and applicability of these findings. The integration of structural and functional analyses\u0026mdash;such as tracer-based CSF clearance assays or permeability measurements\u0026mdash;could help delineate the physiological roles of Gcx alterations. Causal investigation into how Gcx remodeling influences disease severity, recovery, or therapeutic response\u0026mdash;through gain- or loss-of-function strategies, conditional gene manipulation, or targeted enzymatic degradation\u0026mdash;may provide mechanistic insight into its role in central nervous system homeostasis. Furthermore, pharmacological modulation using agents such as rapamycin or glycosylation-targeted compounds may inform strategies for preserving or restoring Gcx integrity. Lastly, combining glycomic analysis with single-cell or spatial transcriptomics could reveal cell-type\u0026ndash;specific regulation of glycan biosynthesis and its association with neuroimmune or barrier functions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated that the ependymal Gcx in the murine brain possesses a glycan profile essential for molecular exchange with the brain parenchyma, CSF circulation, and maintenance of barrier integrity under physiological conditions. In addition, we captured the dynamic alterations in the ependymal glycan profile associated with aging and acute brain injury. Notably, the preservation of ependymal cell function in the aging brain and its restoration following brain injury may represent critical therapeutic targets for delaying the progression of neurodegenerative processes. By elucidating the previously underexplored role of ependymal glycans in maintaining the homeostasis of the brain microenvironment, this study establishes a foundation for a new research direction at the intersection of neuroscience and neuropathology.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eGcx, glycocalyx\u003c/p\u003e\n\u003cp\u003eCSF, cerebrospinal fluid\u003c/p\u003e\n\u003cp\u003eIVH, intraventricular hemorrhage\u003c/p\u003e\n\u003cp\u003escRNA-seq, single-cell RNA sequencing\u003c/p\u003e\n\u003cp\u003eSEM, scanning electron microscopy\u003c/p\u003e\n\u003cp\u003eLVSEM, low-vacuum scanning electron microscopy\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS.E.M., standard error of the mean\u003c/p\u003e\n\u003cp\u003eRCA-1, Ricinus communis agglutinin I\u003c/p\u003e\n\u003cp\u003eSiaFind, \u0026alpha;2,3-linked sialic acid\u0026ndash;specific lectins\u003c/p\u003e\n\u003cp\u003eSTL, Solanum tuberosum lectin\u003c/p\u003e\n\u003cp\u003eDSL, Datura stramonium lectin (DSL)\u003c/p\u003e\n\u003cp\u003eLEL, Lycopersicon esculentum lectin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Gifu University International Animal Care and Use Committee (Number:A2025-0001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData is available upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by JSPS KAKENHI grants JP20K0758723 (H.T.), JP23H03326 (H.T.), and JP24K19545 (K.O.), as well as the JST FOREST Program (JPMJFR220W, H.T.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStudy concept and design: Tomohiro Iida. Data collection and analysis: Tomohiro Iida, Kosuke Mori, Hiroyuki Tomita, and Kohtaro Taguchi. Data interpretation: Tomohiro Iida and Hiroyuki Tomita. Writing of the manuscript: Tomohiro Iida and Hiroyuki Tomita. Review and editing: Tsuyoshi Izumo, and Akira Hara. Supervision:\u0026nbsp;Ayumi Niwa, Tomohiro Kanayama,\u0026nbsp;Kazufumi Ohmura,\u0026nbsp;Shigeyuki Sugie, Hideshi Okada and Akira Hara.\u0026nbsp;All authors approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgments\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Kyoko Takahashi, Ayako Suga, Masayoshi Shimizu, Reiko Kitazumi, and Kayo Nakamura for their support during the experiments. Figures 2 and 5 schematics were created in BioRender. Iida, T. (2026) https://BioRender.com/tzj5t3p, https://BioRender.com/xfjhwcw.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCode availability\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRedmond, S.A., et al., \u003cem\u003eDevelopment of Ependymal and Postnatal Neural Stem Cells and Their Origin from a Common Embryonic Progenitor.\u003c/em\u003e Cell Rep, 2019. \u003cstrong\u003e27\u003c/strong\u003e(2): p. 429-441.e3.\u003c/li\u003e\n\u003cli\u003eDeng, S., et al., \u003cem\u003eRoles of Ependymal Cells in the Physiology and Pathology of the Central Nervous System.\u003c/em\u003e Aging Dis, 2023. \u003cstrong\u003e14\u003c/strong\u003e(2): p. 468-483.\u003c/li\u003e\n\u003cli\u003eGrondona, J.M., et al., \u003cem\u003eEpendymal denudation, aqueductal obliteration and hydrocephalus after a single injection of neuraminidase into the lateral ventricle of adult rats.\u003c/em\u003e J Neuropathol Exp Neurol, 1996. \u003cstrong\u003e55\u003c/strong\u003e(9): p. 999-1008.\u003c/li\u003e\n\u003cli\u003eDebbage, P.L., \u003cem\u003eA systematic histochemical investigation in mammals of the dense glycocalyx glycosylations common to all cells bordering the interstitial fluid compartment of the brain.\u003c/em\u003e Acta Histochem, 1996. \u003cstrong\u003e98\u003c/strong\u003e(1): p. 9-28.\u003c/li\u003e\n\u003cli\u003eRhodes, R.H., \u003cem\u003eUltrastructure of complex carbohydrates of rodent and monkey ependymal glycocalyx and meninges.\u003c/em\u003e Am J Anat, 1987. \u003cstrong\u003e179\u003c/strong\u003e(4): p. 369-84.\u003c/li\u003e\n\u003cli\u003eWagner, C., et al., \u003cem\u003eCellular mechanisms involved in the stenosis and obliteration of the cerebral aqueduct of hyh mutant mice developing congenital hydrocephalus.\u003c/em\u003e J Neuropathol Exp Neurol, 2003. \u003cstrong\u003e62\u003c/strong\u003e(10): p. 1019-40.\u003c/li\u003e\n\u003cli\u003eJim\u0026eacute;nez, A.J., et al., \u003cem\u003eStructure and function of the ependymal barrier and diseases associated with ependyma disruption.\u003c/em\u003e Tissue Barriers, 2014. \u003cstrong\u003e2\u003c/strong\u003e: p. e28426.\u003c/li\u003e\n\u003cli\u003eZhu, W., et al., \u003cem\u003eMouse models of intracerebral hemorrhage in ventricle, cortex, and hippocampus by injections of autologous blood or collagenase.\u003c/em\u003e PLoS One, 2014. \u003cstrong\u003e9\u003c/strong\u003e(5): p. e97423.\u003c/li\u003e\n\u003cli\u003eKawai, S., et al., \u003cem\u003eEffect of three types of mixed anesthetic agents alternate to ketamine in mice.\u003c/em\u003e Exp Anim, 2011. \u003cstrong\u003e60\u003c/strong\u003e(5): p. 481-7.\u003c/li\u003e\n\u003cli\u003eTachi, M., et al., \u003cem\u003eHuman Colorectal Cancer Infrastructure Constructed by the Glycocalyx.\u003c/em\u003e J Clin Med, 2019. \u003cstrong\u003e8\u003c/strong\u003e(9).\u003c/li\u003e\n\u003cli\u003eAndo, Y., et al., \u003cem\u003eBrain-Specific Ultrastructure of Capillary Endothelial Glycocalyx and Its Possible Contribution for Blood Brain Barrier.\u003c/em\u003e Sci Rep, 2018. \u003cstrong\u003e8\u003c/strong\u003e(1): p. 17523.\u003c/li\u003e\n\u003cli\u003eMukai, S., et al., \u003cem\u003eThree-dimensional electron microscopy for endothelial glycocalyx observation using Alcian blue with silver enhancement.\u003c/em\u003e Med Mol Morphol, 2021. \u003cstrong\u003e54\u003c/strong\u003e(2): p. 95-107.\u003c/li\u003e\n\u003cli\u003eLipowsky, H.H., L. 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Alvarez-Buylla, \u003cem\u003eThe Adult Ventricular-Subventricular Zone (V-SVZ) and Olfactory Bulb (OB) Neurogenesis.\u003c/em\u003e Cold Spring Harb Perspect Biol, 2016. \u003cstrong\u003e8\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eMuthusamy, N., et al., \u003cem\u003eMARCKS-dependent mucin clearance and lipid metabolism in ependymal cells are required for maintenance of forebrain homeostasis during aging.\u003c/em\u003e Aging Cell, 2015. \u003cstrong\u003e14\u003c/strong\u003e(5): p. 764-73.\u003c/li\u003e\n\u003cli\u003eWan, Y., et al., \u003cem\u003eEffects of aging on hydrocephalus after intraventricular hemorrhage.\u003c/em\u003e Fluids Barriers CNS, 2020. \u003cstrong\u003e17\u003c/strong\u003e(1): p. 8.\u003c/li\u003e\n\u003cli\u003eMa, Q., et al., \u003cem\u003eOutflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice.\u003c/em\u003e Nat Commun, 2017. \u003cstrong\u003e8\u003c/strong\u003e(1): p. 1434.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ependymal glycocalyx, brain–cerebrospinal fluid barrier, lectin, electron microscopy, aging, intraventricular hemorrhage","lastPublishedDoi":"10.21203/rs.3.rs-7369159/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7369159/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe 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.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe comprehensively investigated age- and injury-related changes in the ependymal Gcx using young (8\u0026ndash;10-week-old), aged (60\u0026ndash;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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAged 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.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe 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.\u003c/p\u003e","manuscriptTitle":"Age‑Dependent and Post‑Intraventricular Hemorrhage Remodeling of the Ependymal Glycocalyx in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 05:52:27","doi":"10.21203/rs.3.rs-7369159/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-04T19:58:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-04T19:22:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-28T21:17:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14929175565291663340302034784172507509","date":"2025-08-17T19:33:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314725750700259349300840015079178287773","date":"2025-08-15T17:53:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-15T17:49:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-15T11:24:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-15T02:43:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fluids and Barriers of the CNS","date":"2025-08-14T02:40:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"997fe175-ab66-4a01-b1c4-5443efe509c2","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T15:58:57+00:00","versionOfRecord":{"articleIdentity":"rs-7369159","link":"https://doi.org/10.1186/s12987-025-00725-x","journal":{"identity":"fluids-and-barriers-of-the-cns","isVorOnly":false,"title":"Fluids and Barriers of the CNS"},"publishedOn":"2025-11-07 15:56:59","publishedOnDateReadable":"November 7th, 2025"},"versionCreatedAt":"2025-08-27 05:52:27","video":"","vorDoi":"10.1186/s12987-025-00725-x","vorDoiUrl":"https://doi.org/10.1186/s12987-025-00725-x","workflowStages":[]},"version":"v1","identity":"rs-7369159","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7369159","identity":"rs-7369159","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}