Mannosamine preserves blood brain barrier integrity and promotes angiogenesis in a stroke model

Mannosamine preserves blood brain barrier integrity and promotes angiogenesis in a stroke model | 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 Mannosamine preserves blood brain barrier integrity and promotes angiogenesis in a stroke model Naresh Kumar R N, Tommaso Mori, Pin Li, Nicole Lummis, Tanja Diemer, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8408292/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cerebral ischemia remains a major global health challenge, contributing significantly to long-term disability and mortality, with limited therapeutic options currently available. This study explores the therapeutic potential of Mannosamine (ManN), an hexosamine previously shown to be a mitogen for endothelial cells (ECs), in the context of cerebral ischemic injury. Using a transient middle cerebral artery occlusion (tMCAO) mouse model, we assessed the effects of ManN administration on ischemic brain damage. Mice receiving ManN exhibited significantly smaller infarct volumes, as measured by magnetic resonance imaging (MRI), and reduced blood–brain barrier (BBB) permeability compared to controls. ManN treatment enhanced pericyte coverage, improved EC survival, and increased vascular density in the ischemic brain regions. Our analyses revealed attenuation of ischemia-induced structural abnormalities, including reduced vacuolation, cellular shrinkage, and nuclear condensation. To elucidate the underlying mechanisms, in vitro experiments with brain endothelial cells (bEND.3) demonstrated that ManN treatment promoted GSK3β phosphorylation and facilitated nuclear accumulation of β-catenin. This activation of the Wnt/β-catenin pathway led to upregulation of key target genes such as LEF1 , TCF7 , AXIN2 , and APCDD1 . Enhanced interaction of β-catenin with LEF1/TCF7 was associated with increased expression of tight junction proteins and transcytosis inhibitors, contributing to BBB stabilization and angiogenic support. Collectively, these findings highlight the ability of ManN to activate endothelial Wnt/β-catenin signaling, thereby preserving BBB integrity and promoting angiogenesis, suggesting its promise as a novel therapeutic strategy for cerebral ischemia. Translational Medicine Cerebral ischemia Glycosylation inhibition Endothelium Vascular permeability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Stroke remains one of the leading causes of death and long-term disability globally and is broadly categorized into ischemic and hemorrhagic types. Approximately 87% of all strokes are ischemic, which occurs when a cerebral artery becomes obstructed, leading to a disruption in blood supply and resulting in significant damage to localized brain regions ( 1 ). Despite a decline in stroke-related mortality over the past decades ( 2 ), more than half of stroke survivors continue to experience long-term disabilities, placing a significant burden on healthcare systems and society ( 3 ). Survivors of an initial ischemic stroke frequently experience complications such as partial paralysis, cognitive impairments, and reduced independence in daily living ( 4 ). Tissue-type plasminogen activator (tPA) remains the only FDA-approved therapy for ischemic stroke that may rapidly dissolve clots and restore blood flow to the brain. However, a narrow therapeutic window of just 4.5 hours often yields unpredictable outcomes ( 5 ). Moreover, there are currently few effective pharmacological or surgical alternatives available for patients who fall outside this critical window to support their functional recovery. Several therapeutic strategies have been proposed to limit post-stroke vascular leakage, including VEGF inhibition to control the acute increase in permeability ( 6 ). In line with this, Paul et al. demonstrated that Src inhibition significantly attenuates VEGF-driven permeability ( 7 ), while Corti et al. reported that targeting syndecan-2 with a neutralizing antibody effectively reduces VEGF-mediated vascular leakage but not angiogenesis ( 8 ). The central nervous system (CNS) vasculature differs from peripheral vessels due to the presence of the blood-brain barrier (BBB), a selective diffusion barrier formed by specialized features of capillary endothelial cells. These include the development of tight junctions, reduced transcytosis, lack of fenestrations, and the expression of various efflux transporters and small molecule carriers ( 9 ). Disruption of cerebral blood flow triggers a series of pathological events, such as oxidative stress and neuroinflammatory responses, that compromise the integrity and permeability of the BBB ( 10 , 11 ). BBB is primarily composed of vascular endothelial cells, basement membrane, pericytes, and astrocytes, components of the neurovascular unit (NVU), which collectively support and preserve its function ( 12 ). Maintaining barrier integrity depends on the interactions between endothelial cells and pericytes, which are closely associated with astrocytic end-feet, collectively forming NVU ( 13 ). Enhanced permeability of the blood-brain barrier (BBB) following cerebral ischemia-reperfusion is linked to poorer clinical outcomes and may lead to lasting disability after a stroke ( 14 ). Thus, there is an unmet need for innovative therapeutic strategies aimed at enhancing functional recovery in individuals suffering from ischemic stroke. D-Mannosamine (ManN) is a naturally occurring hexosamine that was first identified in 1960 as a constituent of bacterial cell walls ( 15 ). It was later recognized as a potential intermediate in the biosynthetic pathway of sialic acids ( 16 ). ManN exhibits a wide range of biological activities, such as influencing enzyme function, altering growth factor–dependent signaling cascades, affecting the stability of various proteins, and modulating cell survival. Additionally, it demonstrated several additional effects, including anti-tumor potential and the ability to promote osteogenic differentiation, and protective effects on articular cartilage ( 17 , 18 ). Recent findings have highlighted ManN as a powerful regulator of endothelial cell function, primarily through its capacity to disrupt both N-linked and O-linked protein glycosylation. In 2000, Zhong et al. reported that treatment with ManN significantly alters the glycosylation patterns in endothelial cells, triggering cellular stress responses, including the unfolded protein response (UPR) and activation of c-Jun N-terminal kinase (JNK) signaling ( 19 ). Interestingly, rather than promoting cell death, these stress-related pathways supported endothelial cell proliferation, enhanced survival, and stimulated angiogenesis ( 19 ). The Wnt/β-catenin signaling pathway is essential for controlling cerebrovascular development and the establishment of the blood-brain barrier (BBB) during embryogenesis ( 20 ). Numerous studies have demonstrated that Wnt/β-catenin signaling activity is markedly reduced in ischemic brain regions in animal models of cerebral ischemia-reperfusion. Although the VEGF signaling pathway has been implicated in angiogenesis in a variety of organs, including the brain ( 21 , 22 ), a characteristic of angiogenesis in the central nervous system (CNS) (but not in other organs) is also its reliance on canonical Wnt signaling, primarily mediated by the ligands Wnt7a and Wnt7b ( 20 ). In the present study, we found that ManN reduces blood-brain barrier (BBB) permeability in the ischemia-reperfusion tMCAO model, enhances vascular density in the ischemic brain region, and specifically activates canonical Wnt/β-catenin signaling in brain endothelial cells. Results Effects of ManN in the transient middle cerebral artery occlusion (tMCAO) model To evaluate whether ManN has therapeutic potential in cerebral ischemia, we employed the transient middle cerebral artery occlusion (tMCAO) model in mice (23). We first optimized the duration of reperfusion following filament insertion into the MCA and found that a 2-hour occlusion period reliably produced cerebral infarction, and we also ensured that the neuroscore did not exceed 2. The timeline of drug administration, surgical procedures, and infarct assessment is illustrated in Figure 1A. Mice pretreated with ManN prior to surgery exhibited significantly smaller infarct volumes compared to PBS-treated controls on days 2, 4, and 6, as determined by T2-weighted MRI (Figure 1B). Quantification of day 4 images was performed using ImageJ (Figure 1C). On day 6, animals were sacrificed, and brain tissues were collected for TTC staining, which confirmed a marked reduction in infarct size in the ManN-treated group (Figure 1D). ManN reduces blood brain barrier (BBB) permeability Ischemic stroke leads to disruption of the blood-brain barrier (BBB), resulting in increased permeability, which can exacerbate neurological damage during cerebral ischemia-reperfusion. This prompted us to investigate the potential effect of ManN on BBB permeability. The experimental timeline, including drug administration, surgical procedure, and evaluation of Evans blue extravasation, is depicted in Figure 2A. Mice treated with ManN exhibited a noticeable reduction in Evans blue leakage into brain tissue compared to PBS-treated controls (Figure 2B). The extravasated dye was quantified through tissue lysis followed by fluorometric analysis, which revealed significantly lower dye levels in the ManN group (Figure 2C). In addition, B20.4.1 (B20), a cross-species neutralizing anti-VEGF monoclonal antibody (24), used as a positive control at the dose of 10 mg/kg, was included to validate the assay (Figure 2D and 2E). Initially, we used PBS as control for ManN, and subsequently, we also tested the N-acetyl derivative of ManN (NAcManN). However, we did not observe any significant difference between PBS and NAcManN in all experimental conditions tested. Thus, they are both valid controls. ManN increases pericyte coverage and endothelial cell survival The integrity of the blood-brain barrier (BBB) is upheld by a coordinated interaction of cellular and molecular components, including endothelial cells, pericytes, astrocytes, and the extracellular matrix, which collectively form the neurovascular unit (25). Given the observed protective effects of ManN in reducing infarct volume and preserving BBB permeability, we sought to investigate its influence on the neurovascular unit architecture in the context of ischemic stroke. At 24 hours post-surgery, ManN-pretreated mice displayed enhanced endothelial cell survival (Figure 3A) and preserved pericyte coverage (Figure 3B). Quantitative analysis using ImageJ confirmed a significant increase in pericyte coverage in the ManN-treated group (Figure 3C). ManN stimulates angiogenesis in tMCAO model To further evaluate angiogenesis, endothelial cells in the ischemic region were stained on day 6 post-stroke. ManN treatment led to markedly higher vascular density compared to PBS-treated controls (Figure 3D), with corresponding quantification of CD31 staining shown in Figure 3E. ManN pretreatment ameliorates histopathological alterations in brains of MCAO mice The MCAO model group exhibited extensive damage to cerebral tissues and cells, characterized by liquefactive changes, prominent spongiform appearance, and swollen, degenerating glial cells. Neurons appeared disorganized with condensed, darkly stained nuclei. However, ManN treatment significantly alleviated these pathological alterations in the ischemic brain regions (Figure 4A). ManN pretreatment preserves the integrity of the vessels in the brain of MCAO mice We investigated the effects of ManN on microvascular structure following transient middle cerebral artery occlusion, using transmission electron microscopy (TEM). In sham-operated mice, cortical capillaries displayed intact endothelial cells, basal lamina, and astrocytic end-feet. In contrast, PBS-treated stroke brains exhibited pronounced swelling of astrocytic end-feet in both the ischemic core and penumbra 24 hours after stroke, along with their detachment from the basal lamina, disruption of the plasma membrane, and endothelial cell swelling. In ManN-treated mice, only minimal edema of astrocytic end-feet was observed at the same time points (Figure 4B&4C). ManN pretreatment activates the JNK pathway To investigate the mechanism underlying the effects of ManN on cerebral ischemia, we initially examined JNK, a member of the MAPK family, which has been previously reported to be selectively activated by ManN in bovine choroidal endothelial cells (BCECs) (19). On day 6 following stroke surgery, mice were euthanized, and brain tissues were collected. The cortex was isolated and subjected to Western blot analysis for JNK and its downstream target, c-Jun (Figure 5A). The analysis revealed a significant increase in the activation of both JNK and c-Jun in the cortical tissue of ManN-treated mice (Figure 5B&5C). In addition, consistent with earlier findings that ManN disrupts protein glycosylation in BCECs (19), we observed a reduction in the molecular weight of VEGFR2 and an upregulation of GRP78 in cortical lysates, indicating glycosylation inhibition (Figure 5A). These molecular alterations were further validated in isolated brain microvessels obtained from the cortex of wild-type mice. The purity of the microvessel preparation, free from neuronal contamination, was confirmed by the absence of β3-tubulin in Western blotting and positive immunostaining for endothelial cells, pericytes, and astrocytes (Supplementary Figure 1). Exposure of these isolated microvessels to ManN led to pronounced activation of JNK (Figure 6A&6B) and c-Jun (Figure 6C&6D). Similar to the cortical tissue, we also detected a decrease in VEGFR2 molecular weight (Figure 6E) and an increase in GRP78 expression (Figure 6F&6G), consistent with inhibition of glycosylation in these vascular structures (19). ManN activates Wnt signaling The Wnt/β-catenin signaling pathway is known to play a pivotal role in brain responses to cerebral ischemia. Its activation has been associated with neuroprotective effects, promotion of neurogenesis and angiogenesis, as well as maintenance of blood-brain barrier (BBB) integrity (26). To determine whether ManN influences this pathway, we treated isolated brain microvessels with ManN and assessed the expression of key Wnt target genes, including LEF1 , TCF7 , AXIN2 , and APCDD1 . ManN treatment led to a significant upregulation of these target genes in the microvessels (Supplementary Figure 2A). To further support these findings, we utilized the mouse brain endothelial cell line bEND.3 (from ATCC) to evaluate the expression of Wnt target genes following ManN exposure. Consistent with the results in microvessels, bEND.3 cells exhibited a dose-dependent increase in Wnt target gene expression upon ManN treatment (Supplementary Figure 2B). GSK3β is a key regulator of the canonical Wnt signaling pathway. In the absence of Wnt signaling, GSK3β promotes β-catenin degradation by phosphorylating it (27). In bEND.3 cells treated with ManN, we observed a marked inhibition of GSK3β activity, as indicated by increased phosphorylation at the serine 9 residue (Figure 7A). Quantification using ImageJ confirms this elevation in GSK3β phosphorylation (Figure 7B). This inhibition facilitates nuclear translocation of β-catenin in ManN-treated cells compared with those treated with PBS, as shown in Figure 7C and its quantification (Figure 7D). Once translocated into the nucleus, β-catenin can induce the transcription of genes essential for maintaining blood-brain barrier (BBB) integrity. ManN treatment markedly increased the mRNA levels of several tight junction–related genes, including Claudin-5 (CLDN5), Tight Junction Protein-1 (TJP1), and Occludin (OCLN) (Figure 8A), which was further supported by corresponding elevations at the protein level (Figure 8B–E). We next evaluated tight junction protein expression in the cortical tissue of mice subjected to tMCAO and subsequently treated with ManN. As shown in Figures 9A and 9B, ManN administration enhanced levels of active (non-phospho) β-catenin and increased the expression of key tight junction proteins compared with PBS-treated controls (Figures 9C–F). Additionally, ManN elevated MFSD2A (Major Facilitator Superfamily Domain Containing 2A), an essential regulator that suppresses endothelial transcytosis and is typically reduced following cerebral ischemia (Figure 8A). Discussion In this study, we demonstrate that ManN treatment alleviates edema following cerebral ischemia. Using the transient middle cerebral artery occlusion (tMCAO) model, we evaluated the therapeutic efficacy of ManN and found that it significantly decreased vascular permeability after ischemia-reperfusion injury, thereby contributing to the preservation of blood-brain barrier (BBB) integrity. ManN also supported pericyte retention and improved endothelial cell survival under acute stress, ultimately promoting angiogenesis within the ischemic brain region. ManN selectively activated the Wnt/β-catenin signaling pathway by inhibiting GSK3β, leading to increased nuclear translocation of β-catenin. In addition, ManN upregulated the expression of tight junction proteins and key transcytosis inhibitors in brain endothelial cells. The blood-brain barrier (BBB) is a highly dynamic interface that regulates the exchange of substances between the bloodstream and brain tissue. Pericytes are essential for both the development and preservation of BBB integrity ( 28 ). Pericytes are key elements of the neurovascular unit and contribute significantly to the structure and function of the BBB. Their coverage along microvessels is critical for maintaining vascular permeability, and they help regulate the BBB by influencing endothelial cell junctions ( 9 ). Reduced pericyte coverage disrupts blood–brain barrier integrity by impairing endothelial junctions ( 29 ). We evaluated pericyte coverage following ManN treatment in the tMCAO model. Our findings show that ManN pretreatment significantly enhances pericyte coverage in ischemic regions, which in turn contributed to a reduction in overall vascular damage. Pericytes also play a vital role in the stabilization of new blood vessels by secreting paracrine signals, including PDGF-BB, VEGF, TGF-β, and Ang-1 (reviewed in ( 30 )) which control endothelial cell migration, proliferation, and differentiation (reviewed in ( 31 )). VEGF administration has been reported to enhance capillary formation and pericyte association, improve cerebral blood flow, and reduce the extent of infarcted brain tissue in the MCAO model ( 32 ). However, enhanced VEGF levels during ischemic conditions have been associated with disrupted endothelial cell junctions, leading to increased vascular permeability and subsequent edema formation (reviewed in ( 33 )). These findings are further supported by studies showing that inhibition of VEGF signaling can mitigate edema formation and limit tissue damage following MCAO in the mouse brain ( 6 , 34 , 35 ). Restoring cerebral blood flow in injured regions is beneficial for stroke recovery. Given their critical involvement in angiogenesis, pericytes are believed to support stroke recovery by modulating the formation of new blood vessels. Collectively, these studies suggest that pericytes play a beneficial role in ischemic stroke by facilitating angiogenic processes. Our findings demonstrate that ManN treatment significantly increased vascular density within the ischemic region, potentially by preserving pericyte coverage, supporting vessel stabilization and new blood vessel formation. Stroke rapidly activates angiogenesis as part of a neurovascular repair program. New vessel growth in injured brain regions supports recovery and is linked to improved outcomes and survival ( 36 ). Following a stroke, the growth of new blood vessels in the brain begins rapidly and can continue for weeks, driven in part by increased endothelial cell activity. Studies indicate that vascular endothelial growth factor (VEGF) plays a key role in this process, enhancing blood vessel formation and aiding functional recovery in experimental stroke models ( 37 , 38 ). However, VEGF plays a dual role after stroke, initially increasing blood-brain barrier permeability but later promoting blood vessel growth and neural repair during recovery ( 22 ). The JNK signaling pathway, known for promoting cell death in the acute phase, also plays important roles in neuronal migration, axonal growth, and angiogenesis during recovery ( 39 ) and reviewed in reference ( 40 ). Given our previous report that ManN selectively activates the JNK pathway in BCECs ( 19 ), we sought to determine whether this signaling axis is similarly engaged under ischemic conditions. Consistent with our earlier findings, ManN treatment led to a robust activation of JNK signaling in the MCAO-induced ischemia/reperfusion model. This upregulation may underlie, at least in part, the pro-angiogenic effects observed, highlighting JNK as a potential mediator of ManN-induced vascular remodeling in the post-ischemic brain. Evidence from studies in the rat brain suggests that endoplasmic reticulum (ER) stress–induced apoptosis plays a significant role in the pathogenesis of neurodegenerative conditions and cerebral ischemia ( 41 – 44 ). Ischemic preconditioning and postconditioning involve brief, repeated interruptions of blood flow at the onset of reperfusion, which mechanically modify the hemodynamic characteristics of the reperfusion process ( 45 ). Postconditioning confers neuroprotection against ischemia/reperfusion injury by suppressing the expression of mitochondrial apoptotic proteins and promoting the activation of intrinsic protective pathways ( 46 ). GRP78 is a major molecular chaperone in the endoplasmic reticulum and increases upon ischemic pre- or postconditioning. It plays a protective role against ER stress-induced apoptosis, as demonstrated in both in vivo ( 42 ) and in vitro studies ( 47 ). This preconditioning effect can also be triggered by pharmacological agents that provide protection against ER stress ( 48 ). Consistent with earlier studies, our results demonstrate that ManN treatment upregulates GRP78 expression. Given GRP78's established role in mitigating ER stress, this induction may contribute to the protective effects of ManN against ER stress triggered by ischemia/reperfusion injury. The Wnt/β-catenin signaling pathway in endothelial cells is crucial for the development and preservation of the blood-brain barrier (BBB) under normal physiological conditions and is also implicated in various neurological disorders, including ischemic stroke, glioblastoma, medulloblastoma, and multiple sclerosis ( 49 – 51 ). Disruption of genes encoding critical components of the Wnt/β-catenin signaling pathway in mice leads to substantial blood-brain barrier (BBB) breakdown, both during embryonic development and in adult stages, underscoring the pathway’s essential role in BBB integrity ( 20 , 52 ). In line with these findings, our study also revealed that ManN treatment upregulated the expression of Wnt-responsive genes in bEND.3 cells and brain microvessels, suggesting activation of this signaling pathway. Glycogen synthase kinase-3β (GSK-3β), as part of the APC/axin complex, plays a pivotal role in regulating β-catenin turnover, a key transcriptional co-regulator in the Wnt/β-catenin pathway ( 53 , 54 ). The GSK-3β/β-catenin signaling axis has been implicated in maintaining blood-brain barrier (BBB) integrity, modulating cerebrovascular pathology, and promoting neuroprotection ( 55 ) ( 56 ). Phosphorylation of GSK-3β at serine 9 renders the enzyme inactive ( 57 ), thereby preventing it from targeting β-catenin for degradation.. In the central nervous system, β-catenin is essential for maintaining blood-brain barrier (BBB) integrity by regulating the expression of tight junction proteins ( 58 ) and limiting caveolae-dependent transcytosis ( 59 ). When stabilized and unphosphorylated, β-catenin translocates to the nucleus, where it partners with LEF1/TCF transcription factors to promote the expression of tight junction components ( 49 ) and molecules that suppress transcytosis ( 59 ). Our results are consistent with earlier studies showing that ManN treatment leads to the inactivation of GSK3β through enhanced phosphorylation. This inhibition facilitates the stabilization and nuclear translocation of β-catenin in endothelial cells. Once in the nucleus, β-catenin promotes the transcription of tight junction proteins and factors known to suppress transcytosis, thereby contributing to the reinforcement of endothelial barrier function. In summary, our study identifies ManN as a promising therapeutic agent for cerebral ischemia, capable of reducing infarct size and preserving BBB integrity. ManN treatment enhanced endothelial cell survival, increased vascular density, and stabilized the BBB by promoting pericyte coverage and upregulating tight junction-associated proteins. Mechanistically, ManN activated the endothelial Wnt/β-catenin signaling pathway, as evidenced by increased GSK3β phosphorylation, nuclear translocation of β-catenin, and the induction of canonical Wnt target genes. These findings highlight a novel role for ManN in modulating vascular responses following ischemic injury and underscore the therapeutic potential of targeting endothelial signaling pathways to preserve neurovascular function in stroke. Methods and Materials Reagents D-Mannosamine hydrochloride (M4670), N-acetyl Mannosamine (A8176), Evans Blue (E2129) were purchased from Sigma. Sulfo-NHS-SS-Biotin (A8001) purchased from ApexBio, USA. Cell culture Mouse brain microvascular endothelial cells (bEND.3, ATCC CRL-2299) were maintained in DMEM high glucose medium supplemented with antibiotics and 10% FBS. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. tMCAO surgery All animal care and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego, and carried out in compliance with the guidelines provided by the Animal Care Program (ACP). Male mice aged seven to eight weeks were anesthetized using a ketamine/xylazine cocktail. A midline incision was made in the neck, and the surrounding soft tissues were gently retracted. The common carotid artery (CCA) was carefully separated from adjacent nerves, and a ligature was placed using 6 − 0 suture thread from Fine Science Tools (FST). The external carotid artery (ECA) was then isolated, and a second ligation was applied. Subsequently, the internal carotid artery (ICA) was exposed, and a loose knot was placed using the same suture. Once a clear view of the ICA was obtained, it was temporarily occluded with a microvascular clamp. A small incision was made in the CCA just proximal to the bifurcation of the ECA and ICA. A silicone-coated monofilament (0.22 mm diameter) was gently inserted into the ICA up to the point of the clamp. While inserting the filament, the clamps on the arteries were released to allow the filament to reach and occlude the origin of the middle cerebral artery (MCA) within the Circle of Willis. The loose suture around the ICA was then tightened to secure the filament in place. The incision was closed using surgical clips, and the mice received a subcutaneous injection of 0.5 mL of normal saline to maintain hydration. They were then placed in a thermoregulated recovery chamber for two hours ( 23 ). Reperfusion was initiated after this period by re-anesthetizing the mice with half of the original anesthetic dose, followed by the careful removal of the monofilament. The incision was then permanently closed with clips. Mice were returned to the thermocage to stabilize body temperature before being transferred back to their standard housing. MRI Imaging On days 2, 4, and 6 following transient middle cerebral artery occlusion (tMCAO), mice were anesthetized using 2.5% isoflurane in oxygen and positioned in a stereotactic frame for magnetic resonance imaging (MRI). Imaging was conducted using a 4.7 T horizontal bore MRI system (MR Solutions 4000MRS) equipped with an MR Solutions platform, integrated anesthesia delivery, and animal monitoring setup. T2-weighted sequences were employed to assess infarct volume. Initially, three orthogonal views were acquired to localize the infarcted region. Subsequently, 12–15 axial T2-weighted slices (0.5 mm thickness) were collected, covering a field of view (FOV) of 15 × 15 mm² with a matrix resolution of 128 × 128, an echo time (TE) of 9 ms, and a repetition time (TR) of 4000 ms, to provide detailed anatomical visualization. Image analysis and infarct volume quantification were performed using ImageJ software ( 60 ). TTC staining On day 6 post-tMCAO, mice were euthanized and brains were carefully harvested. The brains were then coronally sectioned into five uniform slices, each 2 mm thick. These sections were incubated in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 20 minutes to visualize the infarcted regions. Following staining, the slices were photographed and subsequently fixed in 4% paraformaldehyde (PFA). Blood-brain barrier permeability (Evans blue extravasation assay) Blood-brain barrier (BBB) disruption was assessed by measuring Evans blue dye extravasation by fluorescence detection ( 61 ). In brief, 10 minutes after reperfusion, mice received an intravenous injection of Evans blue (4 mg/kg, prepared as a 2% solution in PBS). After 24 hours of reperfusion, mice were perfused transcardially with PBS to remove intravascular dye, and the brains were collected. The ischemic hemispheres were homogenized in 1 mL of 50% trichloroacetic acid, followed by centrifugation at 7000 rpm for 5 minutes. The resulting supernatant was diluted fourfold with absolute ethanol. Fluorescence intensity was then measured using a plate reader (Molecular Devices) at an excitation wavelength of 620 nm and emission at 680 nm. Evans blue levels were quantified and reported as micrograms per ischemic hemisphere. Immunohistochemistry for brain cryosections Following 24 hours of reperfusion, mice were anesthetized using a ketamine/xylazine mixture. Transcardial perfusion was carried out, beginning with 8 mL/min of biotin solution for 6 minutes, followed by perfusion with 4% paraformaldehyde (PFA) for an additional 6 minutes. The brains were then carefully extracted to prevent tissue damage and placed in 30% sucrose solution overnight for cryoprotection, allowing them to fully sink. Once cryoprotected, the brains were coronally embedded in 100% OCT compound and stored at − 80°C. Serial coronal sections of 10 µm thickness were cut using a cryostat at 0.5 mm intervals, spanning from the olfactory bulb to the cerebellum. Brain sections were rinsed in 1X PBS for 5 minutes, permeabilized with ice-cold methanol for 10 minutes, and then washed twice more in PBS (5 minutes each). To block non-specific binding, sections were incubated in 10% normal goat serum containing 0.2% Triton X-100 in PBS for 1 hour at room temperature. Following this, sections were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. The next day, slides were washed three times in PBS (5 minutes each), then incubated with appropriate fluorescent secondary antibodies for 2 hours at room temperature. Fluorescent images were acquired using a Carl Zeiss fluorescence microscope. Transmission electron microscopy (TEM) Mice were restrained and 200 µL of 50 mg/mL of HRP solution injected retro orbitally (10 µL per gram mouse under isoflurane. Mice were returned to the cage for 20 mins. Mice were then anesthetized with ketamine/xylazine. Mice were then transcardially perfused with saline at ~ 8 mL/min for 1 min and fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.15M cacodylate buffer) at ~ 8 mL/min for 3 min. After fixation, mouse brains were dissected out, cerebellum/olfactory bulb were removed, brains were hemisected, and immersion fixed in fixative which contains 5% glutaraldehyde, 4% PFA, 0.1 M sodium cacodylate buffer for 1 h at room temperature. Brains were then transferred to another fixative, which contains 4% PFA, 0.1 M sodium cacodylate buffer, and fixed for 5 h at 4°C. The brain was then transferred to sodium cacodylate buffer and incubated overnight at 4°C. By using a vibratome,100 µm-thick sections of the superficial cortex were obtained and transferred to a 6-well plate containing sodium cacodylate buffer. Sections were then stained with 3,3'-diaminobenzidine for 45 mins. After 45 mins, these sections were transferred to fixative containing 4% PFA, 0.1 M sodium cacodylate buffer. Small cortical tissue blocks were prepared and submitted to the UCSD Electron Microscopy Core for processing. Ultrathin sections were generated using a Leica microtome equipped with a diamond knife, followed by staining with uranyl acetate and lead. Images were acquired on a JEOL 1400Plus transmission electron microscope operating at 80 kV using a 4k × 4k Gatan camera. For each animal, a minimum of 20 distinct vascular cross-sections were imaged. Microvessel isolation Mice were euthanized and their brains were immediately collected and transferred into a Petri dish containing MCDB 131 medium. The meninges were carefully removed by gently rolling the brain on blotting paper. Using a razor blade, the cerebellum was sagittally dissected away, and the cortical regions were separated by eliminating deeper brain structures. Cortical tissue was then homogenized in 1 mL of MCDB 131 medium using a loose-fit 7 mL Dounce homogenizer with ten strokes. An additional 7 mL of MCDB 131 medium was added, followed by two more strokes to complete homogenization. The resulting homogenate was centrifuged at 2000 × g for 5 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL of 15% dextran, then brought to a final volume of 8 mL with the same solution. This suspension was centrifuged at 4700 × g for 15 minutes. After carefully discarding the supernatant, the pellet was resuspended in 1 mL of DPBS and passed through a 40 µm cell strainer, followed by a wash with 10 mL of DPBS. To collect microvessels, the cell strainer was inverted and rinsed with 15 mL of MCDB 131 medium containing 0.5% BSA ( 62 ). Isolated microvessels were treated with ManN for 6 hours in a humidified incubator at 37°C with 5% CO₂. For immunofluorescence studies, microvessels retained on the strainer were fixed with 4% paraformaldehyde and stained with appropriate primary antibodies. Western blotting Isolated microvessels were centrifuged at 10,000 RPM for 5 minutes and subsequently lysed in RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitor cocktail (Cell Signaling Technology). For brain tissue samples, cortices were dissected following the respective treatments and lysed in the same RIPA buffer. Similarly, bEnd.3 cells were lysed with RIPA buffer post-treatment. Equal amounts of total protein from each sample were loaded onto SDS-PAGE gels for electrophoresis and then transferred onto PVDF membranes. The membranes were blocked for 1 hour at room temperature using LI-COR blocking buffer and then incubated overnight at 4°C with the appropriate primary antibodies listed in Table-1 diluted in antibody dilution buffer. The following day, membranes were washed three times with 1X TBST and incubated for 1 hour at room temperature with fluorescent dye-conjugated secondary antibodies. Fluorescent signals were detected using the LI-COR imaging system, and band intensities were quantified using ImageJ software. RNA extraction and qRT-PCR Total RNA was isolated from bEND.3 cells following the respective ManN treatments using the RNeasy Plus Mini Kit (Qiagen). One microgram of RNA was reverse transcribed into complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For quantitative real-time PCR (qRT-PCR), 10 ng of cDNA was used per reaction with TaqMan Fast Advanced Master Mix (Applied Biosystems) on the ViiA™ 7 Real-Time PCR System. Gene expression levels were normalized to the housekeeping gene β-actin, and all samples were analyzed in triplicate. Relative expression was calculated using the 2^−ΔΔCt method. The probe list is given in Table 2. Immunofluorescence bEND.3 cells were seeded onto fibronectin-coated coverslips placed in 24-well plates and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS). Once the cells reached approximately 80% confluence, they were serum-starved overnight in low-glucose DMEM containing 1% FBS. The following day, cells were treated with the indicated concentrations of ManN for 6 hours at 37°C in a CO₂ incubator. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 0.1% Triton X-100. Blocking was performed using 1% bovine serum albumin (BSA). Cells were then incubated overnight at 4°C with β-catenin primary antibody diluted 1:800 in PBST containing 1% BSA. The next day, they were washed and incubated with Alexa Fluor 488-conjugated secondary antibody (Life Technologies) for 2 hours at room temperature, followed by nuclear counterstaining with DAPI. Fluorescent images were acquired using a Keyence microscope, and quantification was performed using ImageJ software. Statistical analysis All experiments were conducted independently in triplicate, except for the reperfusion time optimization for the tMCAO procedure. Bar graphs display the mean values along with standard deviation (SD). Statistical analyses were carried out using GraphPad Prism version 10. Comparisons between two groups were assessed using a two-tailed Student’s t-test. For comparisons involving more than two groups, one-way ANOVA followed by multiple comparisons was applied. In cases involving multiple variables across multiple groups, two-way ANOVA was used. Bonferroni correction was applied in all analyses to adjust for multiple comparisons. A p-value of less than 0.05 (P < 0.05) was considered statistically significant. Declarations Acknowledgements We thank the UCSD Animal Care Facilities for excellent support. We are grateful to Drs. Xinlian Zhang and Karen Messer from the UCSD Moores Cancer Center Biostatistics group for their helpful comments and advice. We thank Genentech, Inc. for the gift of anti-VEGF antibody B.20.4.1. Also, we thank the Moores Cancer Center microscopy facility and the UCSD Electron microscopy core facility for excellent support. References G. B. D. S. Collaborators, Global, regional, and national burden of stroke, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol 18 , 439-458 (2019). R. Waziry et al. , Time Trends in Survival Following First Hemorrhagic or Ischemic Stroke Between 1991 and 2015 in the Rotterdam Study. Stroke 51 , STROKEAHA119027198 (2020). E. S. Donkor, Stroke in the 21(st) Century: A Snapshot of the Burden, Epidemiology, and Quality of Life. Stroke Res Treat 2018 , 3238165 (2018). C. L. Richards, F. Malouin, S. Nadeau, Stroke rehabilitation: clinical picture, assessment, and therapeutic challenge. Prog Brain Res 218 , 253-280 (2015). K. L. Furie, M. V. Jayaraman, 2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke. Stroke 49 , 509-510 (2018). N. van Bruggen et al. , VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain. J. Clin. Invest. 104 , 1613-1620 (1999). R. Paul et al. , Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke. Nature Med. 7 , 222-227 (2001). F. Corti et al. , Syndecan-2 selectively regulates VEGF-induced vascular permeability. Nat Cardiovasc Res 1 , 518-528 (2022). R. Daneman, A. Prat, The blood-brain barrier. Cold Spring Harb Perspect Biol 7 , a020412 (2015). P. T. Do, C. C. Wu, Y. H. Chiang, C. J. Hu, K. Y. Chen, Mesenchymal Stem/Stromal Cell Therapy in Blood-Brain Barrier Preservation Following Ischemia: Molecular Mechanisms and Prospects. Int J Mol Sci 22 (2021). H. Yu et al. , The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice. Proc Natl Acad Sci U S A 119 (2022). X. Peng, Z. Luo, S. He, L. Zhang, Y. Li, Blood-Brain Barrier Disruption by Lipopolysaccharide and Sepsis-Associated Encephalopathy. Front Cell Infect Microbiol 11 , 768108 (2021). C. Cho, P. M. Smallwood, J. Nathans, Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation. Neuron 95 , 1056-1073 e1055 (2017). X. Jiang et al. , Blood-brain barrier dysfunction and recovery after ischemic stroke. Prog Neurobiol 163-164 , 144-171 (2018). O. Luderitz et al. , Identification of D-mannosamine and quinovosamine in Salmonella and related bacteria. J Bacteriol 95 , 490-494 (1968). F. Monaco, J. Robbins, Incorporation of N-acetylmannosamine and N-acetylglucosamine into thyroglobulin in rat thyroid in vitro. J Biol Chem 248 , 2072-2077 (1973). T. Onoda et al. , Antitumor activity of D-mannosamine in vitro: different sensitivities among human leukemia cell lines possessing T-cell properties. Cancer Res 42 , 2867-2871 (1982). Y. J. Chen, C. C. Yao, C. H. Huang, H. H. Chang, T. H. Young, Hexosamine-Induced TGF-beta Signaling and Osteogenic Differentiation of Dental Pulp Stem Cells Are Dependent on N-Acetylglucosaminyltransferase V. Biomed Res Int 2015 , 924397 (2015). C. Zhong et al. , Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways. Nat Commun 11 , 6330 (2020). R. Daneman et al. , Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc Natl Acad Sci U S A 106 , 641-646 (2009). L. Perez-Gutierrez, N. Ferrara, Biology and therapeutic targeting of vascular endothelial growth factor A. Nat Rev Mol Cell Biol 24 , 816-834 (2023). Z. G. Zhang et al. , VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest 106 , 829-838 (2000). O. Engel, S. Kolodziej, U. Dirnagl, V. Prinz, Modeling stroke in mice - middle cerebral artery occlusion with the filament model. J Vis Exp 10.3791/2423 (2011). H. P. Gerber et al. , Mice expressing a humanized form of VEGF-A may provide insights into safety and efficacy of anti-VEGF antibodies. Proc. Natl. Acad. Sci. USA 104 , 3478-3483 (2007). C. Iadecola, The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease. Neuron 96 , 17-42 (2017). Z. Mo et al. , Activation of Wnt/Beta-Catenin Signaling Pathway as a Promising Therapeutic Candidate for Cerebral Ischemia/Reperfusion Injury. Front Pharmacol 13 , 914537 (2022). A. J. Valvezan, P. S. Klein, GSK-3 and Wnt Signaling in Neurogenesis and Bipolar Disorder. Front Mol Neurosci 5 , 1 (2012). R. Daneman, L. Zhou, A. A. Kebede, B. A. Barres, Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468 , 562-566 (2010). B. V. Zlokovic, Cerebrovascular effects of apolipoprotein E: implications for Alzheimer disease. JAMA Neurol 70 , 440-444 (2013). P. Dore-Duffy, J. C. LaManna, Physiologic angiodynamics in the brain. Antioxid Redox Signal 9 , 1363-1371 (2007). Y. Persidsky, S. H. Ramirez, J. Haorah, G. D. Kanmogne, Blood-brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol 1 , 223-236 (2006). A. Zechariah et al. , Vascular endothelial growth factor promotes pericyte coverage of brain capillaries, improves cerebral blood flow during subsequent focal cerebral ischemia, and preserves the metabolic penumbra. Stroke 44 , 1690-1697 (2013). S. M. Weis, D. A. Cheresh, Pathophysiological consequences of VEGF-induced vascular permeability. Nature 437 , 497-504 (2005). I. D. Kim, J. W. Cave, S. Cho, Aflibercept, a VEGF (Vascular Endothelial Growth Factor)-Trap, Reduces Vascular Permeability and Stroke-Induced Brain Swelling in Obese Mice. Stroke 52 , 2637-2648 (2021). R. Kimura, H. Nakase, R. Tamaki, T. Sakaki, Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke 36 , 1259-1263 (2005). J. Krupinski, J. Kaluza, P. Kumar, S. Kumar, J. M. Wang, Role of angiogenesis in patients with cerebral ischemic stroke. Stroke 25 , 1794-1798 (1994). Y. Sun et al. , VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest 111 , 1843-1851 (2003). Y. Wang et al. , VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena. Brain 128 , 52-63 (2005). C. Uchida, E. Gee, E. Ispanovic, T. L. Haas, JNK as a positive regulator of angiogenic potential in endothelial cells. Cell Biol Int 32 , 769-776 (2008). C. Y. Kuan, R. E. Burke, Targeting the JNK signaling pathway for stroke and Parkinson's diseases therapy. Curr Drug Targets CNS Neurol Disord 4 , 63-67 (2005). D. M. Arduino, A. R. Esteves, S. M. Cardoso, C. R. Oliveira, Endoplasmic reticulum and mitochondria interplay mediates apoptotic cell death: relevance to Parkinson's disease. Neurochem Int 55 , 341-348 (2009). T. Hayashi, A. Saito, S. Okuno, M. Ferrand-Drake, P. H. Chan, Induction of GRP78 by ischemic preconditioning reduces endoplasmic reticulum stress and prevents delayed neuronal cell death. J Cereb Blood Flow Metab 23 , 949-961 (2003). V. P. Nakka, A. Gusain, R. Raghubir, Endoplasmic reticulum stress plays critical role in brain damage after cerebral ischemia/reperfusion in rats. Neurotox Res 17 , 189-202 (2010). L. Chen, X. Gao, Neuronal apoptosis induced by endoplasmic reticulum stress. Neurochem Res 27 , 891-898 (2002). H. Zhao, R. M. Sapolsky, G. K. Steinberg, Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. J Cereb Blood Flow Metab 26 , 1114-1121 (2006). B. Xing et al. , Ischemic postconditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury in the rat. Stroke 39 , 2362-2369 (2008). S. Kishi et al. , Nerve growth factor attenuates 2-deoxy-d-glucose-triggered endoplasmic reticulum stress-mediated apoptosis via enhanced expression of GRP78. Neurosci Res 66 , 14-21 (2010). Q. J. Quinones, J. H. Levy, Ischemic Preconditioning and the Role of Antifibrinolytic Drugs: Translation From Bench to Bedside. Anesth Analg 126 , 384-386 (2018). S. Liebner et al. , Wnt/beta-catenin signaling controls development of the blood-brain barrier. J Cell Biol 183 , 409-417 (2008). J. Chang et al. , Gpr124 is essential for blood-brain barrier integrity in central nervous system disease. Nat Med 23 , 450-460 (2017). J. E. Lengfeld et al. , Endothelial Wnt/beta-catenin signaling reduces immune cell infiltration in multiple sclerosis. Proc Natl Acad Sci U S A 114 , E1168-E1177 (2017). J. M. Stenman et al. , Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. Science 322 , 1247-1250 (2008). M. Caspi et al. , Aldolase positively regulates of the canonical Wnt signaling pathway. Mol Cancer 13 , 164 (2014). P. Han, S. Ivanovski, R. Crawford, Y. Xiao, Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration. J Bone Miner Res 30 , 1160-1174 (2015). Y. Zhao et al. , GSK-3beta Inhibition Induced Neuroprotection, Regeneration, and Functional Recovery After Intracerebral Hemorrhagic Stroke. Cell Transplant 26 , 395-407 (2017). W. Wang et al. , GSK-3beta inhibitor TWS119 attenuates rtPA-induced hemorrhagic transformation and activates the Wnt/beta-catenin signaling pathway after acute ischemic stroke in rats. Mol Neurobiol 53 , 7028-7036 (2016). D. M. Chuang, Z. Wang, C. T. Chiu, GSK-3 as a Target for Lithium-Induced Neuroprotection Against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke. Front Mol Neurosci 4 , 15 (2011). K. A. Tran et al. , Endothelial beta-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis. Circulation 133 , 177-186 (2016). Z. Wang et al. , Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier. Sci Adv 6 , eaba7457 (2020). I. A. Mulder et al. , Automated Ischemic Lesion Segmentation in MRI Mouse Brain Data after Transient Middle Cerebral Artery Occlusion. Front Neuroinform 11 , 3 (2017). M. Fujimoto et al. , Tissue inhibitor of metalloproteinases protect blood-brain barrier disruption in focal cerebral ischemia. J Cereb Blood Flow Metab 28 , 1674-1685 (2008). Y. K. Lee, H. Uchida, H. Smith, A. Ito, T. Sanchez, The isolation and molecular characterization of cerebral microvessels. Nat Protoc 14 , 3059-3081 (2019). Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryDocument12192025.docx Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8408292","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":563194119,"identity":"dc2a76fe-348b-4bf7-8327-fbf394b1ed9b","order_by":0,"name":"Naresh Kumar R N","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Naresh","middleName":"Kumar R","lastName":"N","suffix":""},{"id":563197467,"identity":"efafbed9-e5db-4bcb-b5be-8240357a95f4","order_by":1,"name":"Tommaso Mori","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Tommaso","middleName":"","lastName":"Mori","suffix":""},{"id":563197468,"identity":"67dc8c02-6389-45ab-8c87-fdc3d43367fc","order_by":2,"name":"Pin Li","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Pin","middleName":"","lastName":"Li","suffix":""},{"id":563197469,"identity":"5cb760b2-3518-465d-83b3-6106ea4c3658","order_by":3,"name":"Nicole Lummis","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Lummis","suffix":""},{"id":563197470,"identity":"5f5820c6-8ecd-4628-9b6e-8e9336878b30","order_by":4,"name":"Tanja Diemer","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Tanja","middleName":"","lastName":"Diemer","suffix":""},{"id":563197471,"identity":"4354187d-9fd4-4d47-80cc-fe005dbea704","order_by":5,"name":"Reid Laursen","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Reid","middleName":"","lastName":"Laursen","suffix":""},{"id":563197472,"identity":"5f9127e7-6b90-4ffd-8538-591ba98ded67","order_by":6,"name":"Wenjing Zhou","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Zhou","suffix":""},{"id":563197473,"identity":"f476fb0e-b280-4cef-b0bc-e396cd080bf7","order_by":7,"name":"Nilima Biswas","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Nilima","middleName":"","lastName":"Biswas","suffix":""},{"id":563197474,"identity":"3da0a331-cf55-4e00-9d92-bf46621e9d5e","order_by":8,"name":"Hong Xin","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Xin","suffix":""},{"id":563197475,"identity":"53cdd74a-ad82-4074-bc70-fd49a9e9a566","order_by":9,"name":"Richard Daneman","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Daneman","suffix":""},{"id":563197476,"identity":"8de4f734-935e-4e6e-89c8-e04ebf746fd4","order_by":10,"name":"Napoleone Ferrara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYDAC5gMQml8CSDxggPCAbGbcWtgSILTkDCCRkECKFoMbxGoxZ2M+9rmi5p688e3mgx8Sf9xhMDh+/OENhgrrxAYcWizb2JJnnjlWbLjtzrFkiYSEZwwGZ3KMLRjOpOPUYnC/x5ixgS2BcduNHAOglsNAF/KwSTC2Hcat5Rj/Z8aGfwn2m2fkf/4B0cL+TILxHz4tPMyMjW0JiRskctigtjCYSTA24NPCZszY2JeQPONGmplFQtphHkmQXxKOpRvj1sL8mLHhW4Jt/4zkxzc+2ByW4wOF2Icaa1lcWjAAD5hMIFb5KBgFo2AUjAKsAABHhF4m0PL1WwAAAABJRU5ErkJggg==","orcid":"","institution":"University of California, San Diego","correspondingAuthor":true,"prefix":"","firstName":"Napoleone","middleName":"","lastName":"Ferrara","suffix":""}],"badges":[],"createdAt":"2025-12-19 21:43:54","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8408292/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8408292/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99317692,"identity":"59883391-0b36-4e4c-8b49-069b2b22fc32","added_by":"auto","created_at":"2025-12-31 16:30:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1701843,"visible":true,"origin":"","legend":"","description":"","filename":"Kumaretal.Recentversion12192025final.docx","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/9900026e7e44d0f89749b35c.docx"},{"id":99190606,"identity":"1017ae39-65dc-42d7-aaaf-302236f01fce","added_by":"auto","created_at":"2025-12-30 00:52:02","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342,"visible":true,"origin":"","legend":"","description":"","filename":"rs8408292.json","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/a6c240dcd6a52f7ee01d57ba.json"},{"id":99190618,"identity":"7d071b4b-2239-4d41-9197-3f393fa3e0a5","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133911,"visible":true,"origin":"","legend":"","description":"","filename":"rs84082920enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/6e10e8082e6fc3e50a1b8f4e.xml"},{"id":99190614,"identity":"b0b768cf-ab91-4e05-a16f-e3c17ce97012","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152619,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/aa3e228eba5ba48d8ce1cfc4.jpeg"},{"id":99190619,"identity":"6b4e35a6-ba05-490c-8819-fa1de4c70b17","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":447763,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/ffe1580ec38af46831ef0541.jpeg"},{"id":99190609,"identity":"89b5f020-1ae8-49e0-b4d8-66befd229ce5","added_by":"auto","created_at":"2025-12-30 00:52:02","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":251929,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/18f37efe432ce8141f1f5cbe.jpeg"},{"id":99190610,"identity":"7f6dbdca-4680-472a-94db-863282663ea1","added_by":"auto","created_at":"2025-12-30 00:52:02","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":218496,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/0d7fd490ebf74d8cdcd8fe6e.jpeg"},{"id":99190612,"identity":"c3056e4d-5eae-443f-9a0b-0a12116022ae","added_by":"auto","created_at":"2025-12-30 00:52:02","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124558,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/0780c1294505d21ec70a1ce3.jpeg"},{"id":99315837,"identity":"bfacb8f7-8d1b-4f51-a960-539f144b3027","added_by":"auto","created_at":"2025-12-31 16:27:23","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":325104,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/f8579d039ca701f3ce48db3a.jpeg"},{"id":99190617,"identity":"9031f310-d38e-4e10-8b5b-27675f8d3c54","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":198102,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/f4b80a638bb352e637105e2d.jpeg"},{"id":99190624,"identity":"5c9ab922-5cef-4c8d-91dc-830520ca6fb5","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":321129,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/473d5bf6b6cae0c95ff3eb04.jpeg"},{"id":99316281,"identity":"92f678cd-8bbe-47df-8a47-0a50f0f27a37","added_by":"auto","created_at":"2025-12-31 16:28:06","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":341501,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/4d49a4fd822e4a0d43b8727d.jpeg"},{"id":99317091,"identity":"71cde602-d229-4f30-b18e-c3394e04697c","added_by":"auto","created_at":"2025-12-31 16:29:39","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":57129,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/ce876fc36118a62b07592a6b.png"},{"id":99317710,"identity":"a1528153-b6a0-4e56-975d-bff7a0c9e728","added_by":"auto","created_at":"2025-12-31 16:30:37","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108103,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/dd193c16f34098d74be5ece0.png"},{"id":99190622,"identity":"894a8f41-91b2-4b9b-ac23-90a55d06e66a","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107835,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/aa5fee08187004b371e1271d.png"},{"id":99319079,"identity":"671ae12c-38e5-4050-bf78-425bd4758ea5","added_by":"auto","created_at":"2025-12-31 16:36:13","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97376,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/282c41a8fb4acf589e26d23e.png"},{"id":99190635,"identity":"30ca0a05-b71c-4ce8-89a3-1fab1a9eab66","added_by":"auto","created_at":"2025-12-30 00:52:04","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46962,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/9de421127e9d22bb8d0a5db7.png"},{"id":99190626,"identity":"4d089d7a-ebf8-4d7c-be68-d7b614d27526","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91436,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/26470694df577af6facb87c3.png"},{"id":99190636,"identity":"978c8c66-2f78-4556-b2eb-9aa671a8daad","added_by":"auto","created_at":"2025-12-30 00:52:04","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58969,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/2d7f072f9396954e06794f11.png"},{"id":99190630,"identity":"338f5355-707c-4e36-a504-bd5e5c796d26","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66479,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/188d809114cef9fb1d20a382.png"},{"id":99190620,"identity":"b5f5bd26-fea3-4c01-b0d5-e8dff48e6d35","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79918,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/75af77820f8a9425a4fb73d8.png"},{"id":99190633,"identity":"0e4e0dec-3a57-4548-ab1d-ae00660749fa","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131586,"visible":true,"origin":"","legend":"","description":"","filename":"rs84082920structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/55ee0b7ba205288caeb56903.xml"},{"id":99190627,"identity":"2d49af3b-2712-4cd5-b88c-51df178231c6","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145340,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/13a974878cde29be72a39189.html"},{"id":99190605,"identity":"8aae76c6-2bfc-4d56-a082-6546f07ab352","added_by":"auto","created_at":"2025-12-30 00:52:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":152619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMannosamine (ManN) pretreatment attenuates cerebral infarct volume in MCAO mice.\u003c/strong\u003e\u003cbr\u003e\n(A) Schematic diagram outlining the experimental timeline, including administration of treatments and endpoint analysis.\u003cbr\u003e\n(B) T2-weighted MRI scans were used to assess cerebral infarct volume.\u003cbr\u003e\n(C) Quantification of infarct size using ImageJ analysis revealed a significant reduction in infarct volume in the ManN-treated group compared to PBS controls.\u003cbr\u003e\n(D) Representative TTC-stained brain sections on day 6 post-MCAO show visibly smaller infarct areas in ManN-treated mice relative to PBS-treated mice. Data are presented as mean ± SEM (n = 3, Student T-test, two tailed). ****p \u0026lt; 0.001 versus PBS group.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/ba61c4c11167dea6cd4eed16.jpeg"},{"id":99190604,"identity":"e3e2a75a-6211-4a68-94b4-81f6ca753962","added_by":"auto","created_at":"2025-12-30 00:52:02","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":447763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eManN pretreatment mitigates blood-brain barrier (BBB) disruption following MCAO.\u003c/strong\u003e\u003cbr\u003e\n(A) Diagram illustrating the experimental setup and timeline.\u003cbr\u003e\n(B) Representative images showing BBB integrity assessed via Evans Blue dye leakage; ManN pretreatment markedly decreased dye extravasation compared to PBS-treated MCAO mice.\u003cbr\u003e\n(C) Quantification of Evans Blue accumulation in the ischemic hemisphere using a fluorescence microplate reader confirms significant reduction in BBB permeability with ManN treatment.\u003cbr\u003e\n(D, E) Representative images demonstrate BBB protection with anti-VEGF antibody (B20) treatment compared to the IgG control, serving as a positive reference for assay validation. (F, G) Representative image showing Evans blue leakage following N-acetyl ManN treatment and its quantification. Data are presented as mean ± SEM (n = 3, Student T-test, two tailed). ****p \u0026lt; 0.001 versus PBS group.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/750412bbc844630dea6f5ab4.jpeg"},{"id":99317062,"identity":"df55b0ea-ee81-4f7a-b553-052abc4da51a","added_by":"auto","created_at":"2025-12-31 16:29:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":251929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eManN preserves pericyte coverage and endothelial survival in the infarcted region and promotes increased vascular density in the infarcted area.\u003c/strong\u003e\u003cbr\u003e\n(A) Representative images of ischemic brain areas immunostained for CD 31 (Endothelial cells, grey), PDGFRb (pericyte, green). (B) Representative images of ischemic brain areas immunostained for CD13 (pericytes, grey), CD31 (endothelial cells, green), and αSMA (vascular smooth muscle cells, red).\u003cbr\u003e\n(C) Quantification of CD13-positive signal excluding regions co-stained with αSMA was performed using ImageJ. Data are shown as mean ± SEM (n = 3,).\u003c/p\u003e\n\u003cp\u003e(D) Representative immunofluorescence images of the ischemic region stained for CD31 (red), a marker of endothelial cells.\u003cbr\u003e\n(E) Quantitative analysis of CD31 staining was conducted using ImageJ software. Results are expressed as mean ± SEM (n = 3, One-way ANOVA with multiple comparison and Student T-test, two tailed). ***p \u0026lt; 0.001 compared to the PBS-treated group.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/27a3a4c3d938bce384b8cf91.jpeg"},{"id":99315972,"identity":"587124d7-e57b-4782-9e45-bed344dac355","added_by":"auto","created_at":"2025-12-31 16:27:30","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":218496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegeneration of brain tissue in I/R group is ameliorated by ManN pretreatment. Brain microvessels preserved by ManN treatment upon MCAO surgery in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of the brain H\u0026amp;E section upon ManN treatment in tMCAO mice. (B)Representative transmission electron microscopy images of cortical capillaries in the ischemic core or penumbra 24 h after tMCAO. (a) Sham-operated mouse shows a circular vessel lumen (L), a crescent-shaped endothelial nucleus, and a thin, compact endothelial cytoplasm. Perivascular astrocytic end-feet (AP) appear flat and not easily distinguishable. (b)PBS-treated stroke mouse exhibit markedly swollen astrocytic end-feet at 24 h, with detachment of the plasma membrane from the basal lamina. (c) ManN-treated stroke mouse displays preserved vascular ultrastructure with normal endothelial cells (E), basal lamina, and astrocytic end-feet. L, lumen; AP, astrocytic end-feet; E, endothelial cell. (C) Bar diagram represents the quantification of preserved vessels in the ischemic core. Scale bar = 2 μm. Data are shown as mean ± SEM (n = 3, One-way ANOVA with multiple comparisons). *p \u0026lt; 0.05, **p \u0026lt; 0.01 compared to the PBS-treated group.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/86f3c0214030055409b567a8.jpeg"},{"id":99316636,"identity":"ca617dbf-307c-4a7b-b0c1-f215b916372c","added_by":"auto","created_at":"2025-12-31 16:28:45","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":124558,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eManN stimulates the JNK signaling pathway in MCAO brain tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Representative western blot showing expression levels of VEGFR2, BIP, p-JNK/JNK, and p-C-Jun/C-Jun in brain tissue harvested from mice subjected to MCAO/R injury with or without ManN treatment.\u003cbr\u003e\n(B \u0026amp; C) Quantitative analysis of p-JNK/JNK and p-C-Jun/C-Jun normalized to β-actin. Data are presented as mean ± SEM (n = 3, Student T-test, two tailed). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. PBS-treated group.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/c8d1b26f1532dbfff35d8217.jpeg"},{"id":99316561,"identity":"f3e86ca5-11ce-462b-8054-f7eb1e8159ba","added_by":"auto","created_at":"2025-12-31 16:28:35","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":325104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eManN stimulates the JNK signaling pathway and inhibits protein glycosylation and elevates BIP expression in brain microvessels.\u003c/strong\u003e\u003cbr\u003e\n(A) Isolated brain microvessels were exposed to two different concentrations of ManN (40 µM or 400 µM) for 4 hours. A dose-dependent activation of JNK was observed relative to PBS-treated controls.\u003cbr\u003e\n(B) Quantification of phosphorylated JNK relative to total JNK was performed using ImageJ software.\u003cbr\u003e\n(C) C-Jun, a downstream target of JNK, was also markedly upregulated following ManN treatment.\u003cbr\u003e\n(D) The ratio of phosphorylated C-Jun to total C-Jun was quantified using ImageJ.\u003c/p\u003e\n\u003cp\u003e(E) Isolated brain microvessels were treated with ManN at concentrations of 40 µM or 400 µM for 6 hours. A noticeable reduction in the molecular weight of VEGFR2 was observed in ManN-treated samples compared to those treated with PBS, indicating decreased glycosylation.\u003cbr\u003e\n(F) Representative western blot showing increased expression of the ER stress marker BIP following ManN treatment, normalized to β-actin.\u003cbr\u003e\n(G) Quantitative analysis of BIP protein levels reveals a significant upregulation in response to ManN treatment relative to the PBS group. Data are shown as mean ± SEM (n = 3, One-way ANOVA with multiple comparison). **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. PBS-treated group.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/58bf38e3f753936bd4b52035.jpeg"},{"id":99190625,"identity":"d3f073bc-7c8a-4bec-8cbd-f9c844a213e8","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":198102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eManN enhances phosphorylation of GSK3β and promotes β-catenin nuclear translocation in brain endothelial cells (bEND.3).\u003c/strong\u003e\u003cbr\u003e\n(A) bEND.3 cells were exposed to ManN for 15 minutes. After treatment, proteins were extracted and analyzed by western blotting for phosphorylated GSK3β (pGSK3β), total GSK3β, and β-actin. (B) The bar graph illustrates the quantification of pGSK3β levels normalized to total GSK3β.\u003c/p\u003e\n\u003cp\u003e(C) Immunofluorescence analysis showing β-catenin localization following 6 hours of ManN treatment. After treatment, cells were fixed and stained to visualize β-catenin distribution.\u003c/p\u003e\n\u003cp\u003e(D) The bar graph represents the quantification of β-catenin nuclear translocation using ImageJ software. Data are shown as mean ± SEM (n = 3, One-way ANOVA with multiple comparison). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 compared to the PBS-treated group.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/421a44e6a12ed1ffc62c72b5.jpeg"},{"id":99190628,"identity":"3c35545b-39a6-484e-9bd2-9b01ec20c5ae","added_by":"auto","created_at":"2025-12-30 00:52:03","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":321129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eManN enhanced the mRNA expression and protein levels of tight junctions and transcytosis inhibitor in brain microvascular endothelial cells (bEND.3).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) bEND.3 cells were treated with ManN for 4 hours. Following treatment, total RNA was extracted, reverse transcribed into cDNA, and analyzed by qPCR to assess the expression of tight junctions and transcytosis inhibitor. The bar graph shows relative gene expression levels normalized to β-actin. No significant alterations were detected with the N-acetylated ManN form of the compound.\u003c/p\u003e\n\u003cp\u003e(B) bEND.3 cells were exposed to ManN for 24 hours. After treatment, proteins were extracted and analyzed by western blotting for TJP-1, OCLN, CLDN-5, and β-actin. (C) The bar graph illustrates the quantification of TJP-1 levels normalized to β-actin. (D) The bar graph illustrates the quantification of OCLN levels normalized to β-actin. (E) The bar graph illustrates the quantification of CLDN-5 levels normalized to β-actin. Results are presented as mean ± SEM (n = 3, Two-way ANOVA and One-way ANOVA with multiple comparison). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 relative to the PBS control group.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/b4ec1287d86de87d6285b1a2.jpeg"},{"id":99317923,"identity":"75ae8a13-fd7f-4fdf-a51d-5c087c2e2652","added_by":"auto","created_at":"2025-12-31 16:30:57","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":341501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eManN increases non-phospho β-catenin and tight junction proteins in brain cortex upon tMCAO.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Brain cortices were collected from mice after treatment. Proteins were extracted and analyzed by western blotting for non-phospho β-catenin and β-actin. (B) The bar graph illustrates the quantification of β-catenin levels normalized to β-actin. (C) Western blotting for TJP-1, OCLN, CLDN-5, and β-actin. (D) The bar graph illustrates the quantification of TJP-1 levels normalized to β-actin. (E) The bar graph illustrates the quantification of OCLN levels normalized to β-actin. (F) The bar graph illustrates the quantification of CLDN-5 levels normalized to β-actin. Results are presented as mean ± SEM (n = 3, One-way ANOVA with multiple comparison). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 relative to the PBS control group.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/db62c685c148edb6abd670ff.jpeg"},{"id":99323875,"identity":"6d6ca4cc-3ed9-4579-8cb0-ed58789f5c18","added_by":"auto","created_at":"2025-12-31 16:46:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3749745,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/2c5f285a-b730-4bf5-a87a-d0ca28fa99dc.pdf"},{"id":99190603,"identity":"79f4ca71-4102-4884-9e3b-6fecdd05da4f","added_by":"auto","created_at":"2025-12-30 00:52:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":278007,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDocument12192025.docx","url":"https://assets-eu.researchsquare.com/files/rs-8408292/v1/622d18ffad8fab5b625ac136.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eMannosamine preserves blood brain barrier integrity and promotes angiogenesis in a stroke model\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStroke remains one of the leading causes of death and long-term disability globally and is broadly categorized into ischemic and hemorrhagic types. Approximately 87% of all strokes are ischemic, which occurs when a cerebral artery becomes obstructed, leading to a disruption in blood supply and resulting in significant damage to localized brain regions (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite a decline in stroke-related mortality over the past decades (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), more than half of stroke survivors continue to experience long-term disabilities, placing a significant burden on healthcare systems and society (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Survivors of an initial ischemic stroke frequently experience complications such as partial paralysis, cognitive impairments, and reduced independence in daily living (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Tissue-type plasminogen activator (tPA) remains the only FDA-approved therapy for ischemic stroke that may rapidly dissolve clots and restore blood flow to the brain. However, a narrow therapeutic window of just 4.5 hours often yields unpredictable outcomes (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Moreover, there are currently few effective pharmacological or surgical alternatives available for patients who fall outside this critical window to support their functional recovery. Several therapeutic strategies have been proposed to limit post-stroke vascular leakage, including VEGF inhibition to control the acute increase in permeability (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In line with this, Paul et al. demonstrated that Src inhibition significantly attenuates VEGF-driven permeability (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), while Corti et al. reported that targeting syndecan-2 with a neutralizing antibody effectively reduces VEGF-mediated vascular leakage but not angiogenesis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe central nervous system (CNS) vasculature differs from peripheral vessels due to the presence of the blood-brain barrier (BBB), a selective diffusion barrier formed by specialized features of capillary endothelial cells. These include the development of tight junctions, reduced transcytosis, lack of fenestrations, and the expression of various efflux transporters and small molecule carriers (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Disruption of cerebral blood flow triggers a series of pathological events, such as oxidative stress and neuroinflammatory responses, that compromise the integrity and permeability of the BBB (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). BBB is primarily composed of vascular endothelial cells, basement membrane, pericytes, and astrocytes, components of the neurovascular unit (NVU), which collectively support and preserve its function (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Maintaining barrier integrity depends on the interactions between endothelial cells and pericytes, which are closely associated with astrocytic end-feet, collectively forming NVU (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Enhanced permeability of the blood-brain barrier (BBB) following cerebral ischemia-reperfusion is linked to poorer clinical outcomes and may lead to lasting disability after a stroke (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Thus, there is an unmet need for innovative therapeutic strategies aimed at enhancing functional recovery in individuals suffering from ischemic stroke.\u003c/p\u003e \u003cp\u003eD-Mannosamine (ManN) is a naturally occurring hexosamine that was first identified in 1960 as a constituent of bacterial cell walls (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). It was later recognized as a potential intermediate in the biosynthetic pathway of sialic acids (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). ManN exhibits a wide range of biological activities, such as influencing enzyme function, altering growth factor\u0026ndash;dependent signaling cascades, affecting the stability of various proteins, and modulating cell survival. Additionally, it demonstrated several additional effects, including anti-tumor potential and the ability to promote osteogenic differentiation, and protective effects on articular cartilage (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Recent findings have highlighted ManN as a powerful regulator of endothelial cell function, primarily through its capacity to disrupt both N-linked and O-linked protein glycosylation. In 2000, Zhong et al. reported that treatment with ManN significantly alters the glycosylation patterns in endothelial cells, triggering cellular stress responses, including the unfolded protein response (UPR) and activation of c-Jun N-terminal kinase (JNK) signaling (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Interestingly, rather than promoting cell death, these stress-related pathways supported endothelial cell proliferation, enhanced survival, and stimulated angiogenesis (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Wnt/β-catenin signaling pathway is essential for controlling cerebrovascular development and the establishment of the blood-brain barrier (BBB) during embryogenesis (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Numerous studies have demonstrated that Wnt/β-catenin signaling activity is markedly reduced in ischemic brain regions in animal models of cerebral ischemia-reperfusion. Although the VEGF signaling pathway has been implicated in angiogenesis in a variety of organs, including the brain (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), a characteristic of angiogenesis in the central nervous system (CNS) (but not in other organs) is also its reliance on canonical Wnt signaling, primarily mediated by the ligands Wnt7a and Wnt7b (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In the present study, we found that ManN reduces blood-brain barrier (BBB) permeability in the ischemia-reperfusion tMCAO model, enhances vascular density in the ischemic brain region, and specifically activates canonical Wnt/β-catenin signaling in brain endothelial cells.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of ManN in the transient middle cerebral artery occlusion (tMCAO) model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether ManN has therapeutic potential in cerebral ischemia, we employed the transient middle cerebral artery occlusion (tMCAO) model in mice (23). We first optimized the duration of reperfusion following filament insertion into the MCA and found that a 2-hour occlusion period reliably produced cerebral infarction, and we also ensured that the neuroscore did not exceed 2. The timeline of drug administration, surgical procedures, and infarct assessment is illustrated in Figure 1A. Mice pretreated with ManN prior to surgery exhibited significantly smaller infarct volumes compared to PBS-treated controls on days 2, 4, and 6, as determined by T2-weighted MRI (Figure 1B). Quantification of day 4 images was performed using ImageJ (Figure 1C). On day 6, animals were sacrificed, and brain tissues were collected for TTC staining, which confirmed a marked reduction in infarct size in the ManN-treated group (Figure 1D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManN reduces blood brain barrier (BBB) permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIschemic stroke leads to disruption of the blood-brain barrier (BBB), resulting in increased permeability, which can exacerbate neurological damage during cerebral ischemia-reperfusion. This prompted us to investigate the potential effect of ManN on BBB permeability. The experimental timeline, including drug administration, surgical procedure, and evaluation of Evans blue extravasation, is depicted in Figure 2A. Mice treated with ManN exhibited a noticeable reduction in Evans blue leakage into brain tissue compared to PBS-treated controls (Figure 2B). The extravasated dye was quantified through tissue lysis followed by fluorometric analysis, which revealed significantly lower dye levels in the ManN group (Figure 2C). In addition, B20.4.1 (B20), a cross-species neutralizing anti-VEGF monoclonal antibody (24), used as a positive control at the dose of 10 mg/kg, was included to validate the assay (Figure 2D and 2E). Initially, we used PBS as control for ManN, and subsequently, we also tested the N-acetyl derivative of ManN (NAcManN). However, we did not observe any significant difference between PBS and NAcManN in all experimental conditions tested. Thus, they are both valid controls.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManN increases pericyte coverage and endothelial cell survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe integrity of the blood-brain barrier (BBB) is upheld by a coordinated interaction of cellular and molecular components, including endothelial cells, pericytes, astrocytes, and the extracellular matrix, which collectively form the neurovascular unit (25). Given the observed protective effects of ManN in reducing infarct volume and preserving BBB permeability, we sought to investigate its influence on the neurovascular unit architecture in the context of ischemic stroke. At 24 hours post-surgery, ManN-pretreated mice displayed enhanced endothelial cell survival (Figure 3A) and preserved pericyte coverage (Figure 3B). Quantitative analysis using ImageJ confirmed a significant increase in pericyte coverage in the ManN-treated group (Figure 3C). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManN stimulates angiogenesis in tMCAO model\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further evaluate angiogenesis, endothelial cells in the ischemic region were stained on day 6 post-stroke. ManN treatment led to markedly higher vascular density compared to PBS-treated controls (Figure 3D), with corresponding quantification of CD31 staining shown in Figure 3E.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManN pretreatment ameliorates histopathological alterations in brains of MCAO mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MCAO model group exhibited extensive damage to cerebral tissues and cells, characterized by liquefactive changes, prominent spongiform appearance, and swollen, degenerating glial cells. Neurons appeared disorganized with condensed, darkly stained nuclei. However, ManN treatment significantly alleviated these pathological alterations in the ischemic brain regions (Figure 4A).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManN pretreatment preserves the integrity of the vessels in the brain of MCAO mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the effects of ManN on microvascular structure following transient middle cerebral artery occlusion, using transmission electron microscopy (TEM). In sham-operated mice, cortical capillaries displayed intact endothelial cells, basal lamina, and astrocytic end-feet. In contrast, PBS-treated stroke brains exhibited pronounced swelling of astrocytic end-feet in both the ischemic core and penumbra 24 hours after stroke, along with their detachment from the basal lamina, disruption of the plasma membrane, and endothelial cell swelling. In ManN-treated mice, only minimal edema of astrocytic end-feet was observed at the same time points (Figure 4B\u0026amp;4C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManN pretreatment activates the JNK pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the mechanism underlying the effects of ManN on cerebral ischemia, we initially examined JNK, a member of the MAPK family, which has been previously reported to be selectively activated by ManN in bovine choroidal endothelial cells (BCECs) (19). On day 6 following stroke surgery, mice were euthanized, and brain tissues were collected. The cortex was isolated and subjected to Western blot analysis for JNK and its downstream target, c-Jun (Figure 5A). The analysis revealed a significant increase in the activation of both JNK and c-Jun in the cortical tissue of ManN-treated mice (Figure 5B\u0026amp;5C). In addition, consistent with earlier findings that ManN disrupts protein glycosylation in BCECs (19), we observed a reduction in the molecular weight of VEGFR2 and an upregulation of GRP78 in cortical lysates, indicating glycosylation inhibition (Figure 5A). These molecular alterations were further validated in isolated brain microvessels obtained from the cortex of wild-type mice. The purity of the microvessel preparation, free from neuronal contamination, was confirmed by the absence of \u0026beta;3-tubulin in Western blotting and positive immunostaining for endothelial cells, pericytes, and astrocytes (Supplementary Figure 1). Exposure of these isolated microvessels to ManN led to pronounced activation of JNK (Figure 6A\u0026amp;6B) and c-Jun (Figure 6C\u0026amp;6D). Similar to the cortical tissue, we also detected a decrease in VEGFR2 molecular weight (Figure 6E) and an increase in GRP78 expression (Figure 6F\u0026amp;6G), consistent with inhibition of glycosylation in these vascular structures (19).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManN activates Wnt signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Wnt/\u0026beta;-catenin signaling pathway is known to play a pivotal role in brain responses to cerebral ischemia. Its activation has been associated with neuroprotective effects, promotion of neurogenesis and angiogenesis, as well as maintenance of blood-brain barrier (BBB) integrity\u0026nbsp;(26). To determine whether ManN influences this pathway, we treated isolated brain microvessels with ManN and assessed the expression of key Wnt target genes, including \u003cstrong\u003eLEF1\u003c/strong\u003e\u003cstrong\u003e, \u003cstrong\u003eTCF7\u003c/strong\u003e, \u003cstrong\u003eAXIN2\u003c/strong\u003e,\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eAPCDD1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e ManN treatment led to a significant upregulation of these target genes in the microvessels (Supplementary Figure 2A). To further support these findings, we utilized the mouse brain endothelial cell line bEND.3 (from ATCC) to evaluate the expression of Wnt target genes following ManN exposure. Consistent with the results in microvessels, bEND.3 cells exhibited a dose-dependent increase in Wnt target gene expression upon ManN treatment (Supplementary Figure 2B).\u003c/p\u003e\n\u003cp\u003eGSK3\u0026beta; is a key regulator of the canonical Wnt signaling pathway. In the absence of Wnt signaling, GSK3\u0026beta; promotes \u0026beta;-catenin degradation by phosphorylating it (27). In bEND.3 cells treated with ManN, we observed a marked inhibition of GSK3\u0026beta; activity, as indicated by increased phosphorylation at the serine 9 residue (Figure 7A). Quantification using ImageJ confirms this elevation in GSK3\u0026beta; phosphorylation (Figure 7B). This inhibition facilitates nuclear translocation of \u0026beta;-catenin in ManN-treated cells compared with those treated with PBS, as shown in Figure 7C and its quantification (Figure 7D). Once translocated into the nucleus, \u0026beta;-catenin can induce the transcription of genes essential for maintaining blood-brain barrier (BBB) integrity. ManN treatment markedly increased the mRNA levels of several tight junction\u0026ndash;related genes, including Claudin-5 (CLDN5), Tight Junction Protein-1 (TJP1), and Occludin (OCLN) (Figure 8A), which was further supported by corresponding elevations at the protein level (Figure 8B\u0026ndash;E). We next evaluated tight junction protein expression in the cortical tissue of mice subjected to tMCAO and subsequently treated with ManN. As shown in Figures 9A and 9B, ManN administration enhanced levels of active (non-phospho) \u0026beta;-catenin and increased the expression of key tight junction proteins compared with PBS-treated controls (Figures 9C\u0026ndash;F). Additionally, ManN elevated MFSD2A (Major Facilitator Superfamily Domain Containing 2A), an essential regulator that suppresses endothelial transcytosis and is typically reduced following cerebral ischemia (Figure 8A).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrate that ManN treatment alleviates edema following cerebral ischemia. Using the transient middle cerebral artery occlusion (tMCAO) model, we evaluated the therapeutic efficacy of ManN and found that it significantly decreased vascular permeability after ischemia-reperfusion injury, thereby contributing to the preservation of blood-brain barrier (BBB) integrity. ManN also supported pericyte retention and improved endothelial cell survival under acute stress, ultimately promoting angiogenesis within the ischemic brain region. ManN selectively activated the Wnt/\u0026beta;-catenin signaling pathway by inhibiting GSK3\u0026beta;, leading to increased nuclear translocation of \u0026beta;-catenin. In addition, ManN upregulated the expression of tight junction proteins and key transcytosis inhibitors in brain endothelial cells.\u003c/p\u003e\n\u003cp\u003eThe blood-brain barrier (BBB) is a highly dynamic interface that regulates the exchange of substances between the bloodstream and brain tissue. Pericytes are essential for both the development and preservation of BBB integrity (\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e). Pericytes are key elements of the neurovascular unit and contribute significantly to the structure and function of the BBB. Their coverage along microvessels is critical for maintaining vascular permeability, and they help regulate the BBB by influencing endothelial cell junctions (\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e). Reduced pericyte coverage disrupts blood\u0026ndash;brain barrier integrity by impairing endothelial junctions (\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e). We evaluated pericyte coverage following ManN treatment in the tMCAO model. Our findings show that ManN pretreatment significantly enhances pericyte coverage in ischemic regions, which in turn contributed to a reduction in overall vascular damage.\u003c/p\u003e\n\u003cp\u003ePericytes also play a vital role in the stabilization of new blood vessels by secreting paracrine signals, including PDGF-BB, VEGF, TGF-\u0026beta;, and Ang-1 (reviewed in (\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e)) which control endothelial cell migration, proliferation, and differentiation (reviewed in (\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e)). VEGF administration has been reported to enhance capillary formation and pericyte association, improve cerebral blood flow, and reduce the extent of infarcted brain tissue in the MCAO model (\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e). However, enhanced VEGF levels during ischemic conditions have been associated with disrupted endothelial cell junctions, leading to increased vascular permeability and subsequent edema formation (reviewed in (\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e)). These findings are further supported by studies showing that inhibition of VEGF signaling can mitigate edema formation and limit tissue damage following MCAO in the mouse brain (\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e). Restoring cerebral blood flow in injured regions is beneficial for stroke recovery. Given their critical involvement in angiogenesis, pericytes are believed to support stroke recovery by modulating the formation of new blood vessels. Collectively, these studies suggest that pericytes play a beneficial role in ischemic stroke by facilitating angiogenic processes. Our findings demonstrate that ManN treatment significantly increased vascular density within the ischemic region, potentially by preserving pericyte coverage, supporting vessel stabilization and new blood vessel formation.\u003c/p\u003e\n\u003cp\u003eStroke rapidly activates angiogenesis as part of a neurovascular repair program. New vessel growth in injured brain regions supports recovery and is linked to improved outcomes and survival (\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e). Following a stroke, the growth of new blood vessels in the brain begins rapidly and can continue for weeks, driven in part by increased endothelial cell activity. Studies indicate that vascular endothelial growth factor (VEGF) plays a key role in this process, enhancing blood vessel formation and aiding functional recovery in experimental stroke models (\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e). However, VEGF plays a dual role after stroke, initially increasing blood-brain barrier permeability but later promoting blood vessel growth and neural repair during recovery (\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e). The JNK signaling pathway, known for promoting cell death in the acute phase, also plays important roles in neuronal migration, axonal growth, and angiogenesis during recovery (\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e) and reviewed in reference (\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e). Given our previous report that ManN selectively activates the JNK pathway in BCECs (\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e), we sought to determine whether this signaling axis is similarly engaged under ischemic conditions. Consistent with our earlier findings, ManN treatment led to a robust activation of JNK signaling in the MCAO-induced ischemia/reperfusion model. This upregulation may underlie, at least in part, the pro-angiogenic effects observed, highlighting JNK as a potential mediator of ManN-induced vascular remodeling in the post-ischemic brain.\u003c/p\u003e\n\u003cp\u003eEvidence from studies in the rat brain suggests that endoplasmic reticulum (ER) stress\u0026ndash;induced apoptosis plays a significant role in the pathogenesis of neurodegenerative conditions and cerebral ischemia (\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e). Ischemic preconditioning and postconditioning involve brief, repeated interruptions of blood flow at the onset of reperfusion, which mechanically modify the hemodynamic characteristics of the reperfusion process (\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e). Postconditioning confers neuroprotection against ischemia/reperfusion injury by suppressing the expression of mitochondrial apoptotic proteins and promoting the activation of intrinsic protective pathways (\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e). GRP78 is a major molecular chaperone in the endoplasmic reticulum and increases upon ischemic pre- or postconditioning. It plays a protective role against ER stress-induced apoptosis, as demonstrated in both \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e) and \u003cem\u003ein vitro\u003c/em\u003e studies (\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e). This preconditioning effect can also be triggered by pharmacological agents that provide protection against ER stress (\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e). Consistent with earlier studies, our results demonstrate that ManN treatment upregulates GRP78 expression. Given GRP78\u0026apos;s established role in mitigating ER stress, this induction may contribute to the protective effects of ManN against ER stress triggered by ischemia/reperfusion injury.\u003c/p\u003e\n\u003cp\u003eThe Wnt/\u0026beta;-catenin signaling pathway in endothelial cells is crucial for the development and preservation of the blood-brain barrier (BBB) under normal physiological conditions and is also implicated in various neurological disorders, including ischemic stroke, glioblastoma, medulloblastoma, and multiple sclerosis (\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e). Disruption of genes encoding critical components of the Wnt/\u0026beta;-catenin signaling pathway in mice leads to substantial blood-brain barrier (BBB) breakdown, both during embryonic development and in adult stages, underscoring the pathway\u0026rsquo;s essential role in BBB integrity (\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e). In line with these findings, our study also revealed that ManN treatment upregulated the expression of Wnt-responsive genes in bEND.3 cells and brain microvessels, suggesting activation of this signaling pathway.\u003c/p\u003e\n\u003cp\u003eGlycogen synthase kinase-3\u0026beta; (GSK-3\u0026beta;), as part of the APC/axin complex, plays a pivotal role in regulating \u0026beta;-catenin turnover, a key transcriptional co-regulator in the Wnt/\u0026beta;-catenin pathway (\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e). The GSK-3\u0026beta;/\u0026beta;-catenin signaling axis has been implicated in maintaining blood-brain barrier (BBB) integrity, modulating cerebrovascular pathology, and promoting neuroprotection (\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e) (\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e). Phosphorylation of GSK-3\u0026beta; at serine 9 renders the enzyme inactive (\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e), thereby preventing it from targeting \u0026beta;-catenin for degradation.. In the central nervous system, \u0026beta;-catenin is essential for maintaining blood-brain barrier (BBB) integrity by regulating the expression of tight junction proteins (\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e) and limiting caveolae-dependent transcytosis (\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e). When stabilized and unphosphorylated, \u0026beta;-catenin translocates to the nucleus, where it partners with LEF1/TCF transcription factors to promote the expression of tight junction components (\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e) and molecules that suppress transcytosis (\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e). Our results are consistent with earlier studies showing that ManN treatment leads to the inactivation of GSK3\u0026beta; through enhanced phosphorylation. This inhibition facilitates the stabilization and nuclear translocation of \u0026beta;-catenin in endothelial cells. Once in the nucleus, \u0026beta;-catenin promotes the transcription of tight junction proteins and factors known to suppress transcytosis, thereby contributing to the reinforcement of endothelial barrier function.\u003c/p\u003e\n\u003cp\u003eIn summary, our study identifies ManN as a promising therapeutic agent for cerebral ischemia, capable of reducing infarct size and preserving BBB integrity. ManN treatment enhanced endothelial cell survival, increased vascular density, and stabilized the BBB by promoting pericyte coverage and upregulating tight junction-associated proteins. Mechanistically, ManN activated the endothelial Wnt/\u0026beta;-catenin signaling pathway, as evidenced by increased GSK3\u0026beta; phosphorylation, nuclear translocation of \u0026beta;-catenin, and the induction of canonical Wnt target genes. These findings highlight a novel role for ManN in modulating vascular responses following ischemic injury and underscore the therapeutic potential of targeting endothelial signaling pathways to preserve neurovascular function in stroke.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003eReagents\u003c/h2\u003e\n \u003cp\u003eD-Mannosamine hydrochloride (M4670), N-acetyl Mannosamine (A8176), Evans Blue (E2129) were purchased from Sigma. Sulfo-NHS-SS-Biotin (A8001) purchased from ApexBio, USA.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture\u003c/h2\u003e\n \u003cp\u003eMouse brain microvascular endothelial cells (bEND.3, ATCC CRL-2299) were maintained in DMEM high glucose medium supplemented with antibiotics and 10% FBS. Cells were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003etMCAO surgery\u003c/h2\u003e\n \u003cp\u003eAll animal care and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego, and carried out in compliance with the guidelines provided by the Animal Care Program (ACP). Male mice aged seven to eight weeks were anesthetized using a ketamine/xylazine cocktail. A midline incision was made in the neck, and the surrounding soft tissues were gently retracted. The common carotid artery (CCA) was carefully separated from adjacent nerves, and a ligature was placed using 6\u0026thinsp;\u0026minus;\u0026thinsp;0 suture thread from Fine Science Tools (FST). The external carotid artery (ECA) was then isolated, and a second ligation was applied. Subsequently, the internal carotid artery (ICA) was exposed, and a loose knot was placed using the same suture.\u003c/p\u003e\n \u003cp\u003eOnce a clear view of the ICA was obtained, it was temporarily occluded with a microvascular clamp. A small incision was made in the CCA just proximal to the bifurcation of the ECA and ICA. A silicone-coated monofilament (0.22 mm diameter) was gently inserted into the ICA up to the point of the clamp. While inserting the filament, the clamps on the arteries were released to allow the filament to reach and occlude the origin of the middle cerebral artery (MCA) within the Circle of Willis. The loose suture around the ICA was then tightened to secure the filament in place. The incision was closed using surgical clips, and the mice received a subcutaneous injection of 0.5 mL of normal saline to maintain hydration. They were then placed in a thermoregulated recovery chamber for two hours (\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eReperfusion was initiated after this period by re-anesthetizing the mice with half of the original anesthetic dose, followed by the careful removal of the monofilament. The incision was then permanently closed with clips. Mice were returned to the thermocage to stabilize body temperature before being transferred back to their standard housing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eMRI Imaging\u003c/h2\u003e\n \u003cp\u003eOn days 2, 4, and 6 following transient middle cerebral artery occlusion (tMCAO), mice were anesthetized using 2.5% isoflurane in oxygen and positioned in a stereotactic frame for magnetic resonance imaging (MRI). Imaging was conducted using a 4.7 T horizontal bore MRI system (MR Solutions 4000MRS) equipped with an MR Solutions platform, integrated anesthesia delivery, and animal monitoring setup. T2-weighted sequences were employed to assess infarct volume. Initially, three orthogonal views were acquired to localize the infarcted region. Subsequently, 12\u0026ndash;15 axial T2-weighted slices (0.5 mm thickness) were collected, covering a field of view (FOV) of 15 \u0026times; 15 mm\u0026sup2; with a matrix resolution of 128 \u0026times; 128, an echo time (TE) of 9 ms, and a repetition time (TR) of 4000 ms, to provide detailed anatomical visualization. Image analysis and infarct volume quantification were performed using ImageJ software (\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eTTC staining\u003c/h2\u003e\n \u003cp\u003eOn day 6 post-tMCAO, mice were euthanized and brains were carefully harvested. The brains were then coronally sectioned into five uniform slices, each 2 mm thick. These sections were incubated in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 37\u0026deg;C for 20 minutes to visualize the infarcted regions. Following staining, the slices were photographed and subsequently fixed in 4% paraformaldehyde (PFA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eBlood-brain barrier permeability (Evans blue extravasation assay)\u003c/h2\u003e\n \u003cp\u003eBlood-brain barrier (BBB) disruption was assessed by measuring Evans blue dye extravasation by fluorescence detection (\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e). In brief, 10 minutes after reperfusion, mice received an intravenous injection of Evans blue (4 mg/kg, prepared as a 2% solution in PBS). After 24 hours of reperfusion, mice were perfused transcardially with PBS to remove intravascular dye, and the brains were collected. The ischemic hemispheres were homogenized in 1 mL of 50% trichloroacetic acid, followed by centrifugation at 7000 rpm for 5 minutes. The resulting supernatant was diluted fourfold with absolute ethanol. Fluorescence intensity was then measured using a plate reader (Molecular Devices) at an excitation wavelength of 620 nm and emission at 680 nm. Evans blue levels were quantified and reported as micrograms per ischemic hemisphere.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunohistochemistry for brain cryosections\u003c/h2\u003e\n \u003cp\u003eFollowing 24 hours of reperfusion, mice were anesthetized using a ketamine/xylazine mixture. Transcardial perfusion was carried out, beginning with 8 mL/min of biotin solution for 6 minutes, followed by perfusion with 4% paraformaldehyde (PFA) for an additional 6 minutes. The brains were then carefully extracted to prevent tissue damage and placed in 30% sucrose solution overnight for cryoprotection, allowing them to fully sink. Once cryoprotected, the brains were coronally embedded in 100% OCT compound and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Serial coronal sections of 10 \u0026micro;m thickness were cut using a cryostat at 0.5 mm intervals, spanning from the olfactory bulb to the cerebellum.\u003c/p\u003e\n \u003cp\u003eBrain sections were rinsed in 1X PBS for 5 minutes, permeabilized with ice-cold methanol for 10 minutes, and then washed twice more in PBS (5 minutes each). To block non-specific binding, sections were incubated in 10% normal goat serum containing 0.2% Triton X-100 in PBS for 1 hour at room temperature. Following this, sections were incubated overnight at 4\u0026deg;C with primary antibodies diluted in blocking buffer. The next day, slides were washed three times in PBS (5 minutes each), then incubated with appropriate fluorescent secondary antibodies for 2 hours at room temperature. Fluorescent images were acquired using a Carl Zeiss fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e\n \u003cp\u003eMice were restrained and 200 \u0026micro;L of 50 mg/mL of HRP solution injected retro orbitally (10 \u0026micro;L per gram mouse under isoflurane. Mice were returned to the cage for \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e20\u003c/span\u003e mins. Mice were then anesthetized with ketamine/xylazine. Mice were then transcardially perfused with saline at ~\u0026thinsp;8 mL/min for 1 min and fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.15M cacodylate buffer) at ~\u0026thinsp;8 mL/min for 3 min. After fixation, mouse brains were dissected out, cerebellum/olfactory bulb were removed, brains were hemisected, and immersion fixed in fixative which contains 5% glutaraldehyde, 4% PFA, 0.1 M sodium cacodylate buffer for 1 h at room temperature. Brains were then transferred to another fixative, which contains 4% PFA, 0.1 M sodium cacodylate buffer, and fixed for 5 h at 4\u0026deg;C. The brain was then transferred to sodium cacodylate buffer and incubated overnight at 4\u0026deg;C. By using a vibratome,100 \u0026micro;m-thick sections of the superficial cortex were obtained and transferred to a 6-well plate containing sodium cacodylate buffer. Sections were then stained with 3,3\u0026apos;-diaminobenzidine for 45 mins. After 45 mins, these sections were transferred to fixative containing 4% PFA, 0.1 M sodium cacodylate buffer. Small cortical tissue blocks were prepared and submitted to the UCSD Electron Microscopy Core for processing. Ultrathin sections were generated using a Leica microtome equipped with a diamond knife, followed by staining with uranyl acetate and lead. Images were acquired on a JEOL 1400Plus transmission electron microscope operating at 80 kV using a 4k \u0026times; 4k Gatan camera. For each animal, a minimum of 20 distinct vascular cross-sections were imaged.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eMicrovessel isolation\u003c/h2\u003e\n \u003cp\u003eMice were euthanized and their brains were immediately collected and transferred into a Petri dish containing MCDB 131 medium. The meninges were carefully removed by gently rolling the brain on blotting paper. Using a razor blade, the cerebellum was sagittally dissected away, and the cortical regions were separated by eliminating deeper brain structures. Cortical tissue was then homogenized in 1 mL of MCDB 131 medium using a loose-fit 7 mL Dounce homogenizer with ten strokes. An additional 7 mL of MCDB 131 medium was added, followed by two more strokes to complete homogenization.\u003c/p\u003e\n \u003cp\u003eThe resulting homogenate was centrifuged at 2000 \u0026times; g for 5 minutes at 4\u0026deg;C. The supernatant was discarded, and the pellet was resuspended in 1 mL of 15% dextran, then brought to a final volume of 8 mL with the same solution. This suspension was centrifuged at 4700 \u0026times; g for 15 minutes. After carefully discarding the supernatant, the pellet was resuspended in 1 mL of DPBS and passed through a 40 \u0026micro;m cell strainer, followed by a wash with 10 mL of DPBS. To collect microvessels, the cell strainer was inverted and rinsed with 15 mL of MCDB 131 medium containing 0.5% BSA (\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eIsolated microvessels were treated with ManN for 6 hours in a humidified incubator at 37\u0026deg;C with 5% CO₂. For immunofluorescence studies, microvessels retained on the strainer were fixed with 4% paraformaldehyde and stained with appropriate primary antibodies.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blotting\u003c/h2\u003e\n \u003cp\u003eIsolated microvessels were centrifuged at 10,000 RPM for 5 minutes and subsequently lysed in RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitor cocktail (Cell Signaling Technology). For brain tissue samples, cortices were dissected following the respective treatments and lysed in the same RIPA buffer. Similarly, bEnd.3 cells were lysed with RIPA buffer post-treatment.\u003c/p\u003e\n \u003cp\u003eEqual amounts of total protein from each sample were loaded onto SDS-PAGE gels for electrophoresis and then transferred onto PVDF membranes. The membranes were blocked for 1 hour at room temperature using LI-COR blocking buffer and then incubated overnight at 4\u0026deg;C with the appropriate primary antibodies listed in Table-1 diluted in antibody dilution buffer. The following day, membranes were washed three times with 1X TBST and incubated for 1 hour at room temperature with fluorescent dye-conjugated secondary antibodies. Fluorescent signals were detected using the LI-COR imaging system, and band intensities were quantified using ImageJ software.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eRNA extraction and qRT-PCR\u003c/h2\u003e\n \u003cp\u003eTotal RNA was isolated from bEND.3 cells following the respective ManN treatments using the RNeasy Plus Mini Kit (Qiagen). One microgram of RNA was reverse transcribed into complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For quantitative real-time PCR (qRT-PCR), 10 ng of cDNA was used per reaction with TaqMan Fast Advanced Master Mix (Applied Biosystems) on the ViiA\u0026trade; 7 Real-Time PCR System. Gene expression levels were normalized to the housekeeping gene \u0026beta;-actin, and all samples were analyzed in triplicate. Relative expression was calculated using the 2^\u0026minus;\u0026Delta;\u0026Delta;Ct method. The probe list is given in Table\u0026nbsp;2.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescence\u003c/h2\u003e\n \u003cp\u003ebEND.3 cells were seeded onto fibronectin-coated coverslips placed in 24-well plates and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS). Once the cells reached approximately 80% confluence, they were serum-starved overnight in low-glucose DMEM containing 1% FBS. The following day, cells were treated with the indicated concentrations of ManN for 6 hours at 37\u0026deg;C in a CO₂ incubator. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 0.1% Triton X-100. Blocking was performed using 1% bovine serum albumin (BSA).\u003c/p\u003e\n \u003cp\u003eCells were then incubated overnight at 4\u0026deg;C with \u0026beta;-catenin primary antibody diluted 1:800 in PBST containing 1% BSA. The next day, they were washed and incubated with Alexa Fluor 488-conjugated secondary antibody (Life Technologies) for 2 hours at room temperature, followed by nuclear counterstaining with DAPI. Fluorescent images were acquired using a Keyence microscope, and quantification was performed using ImageJ software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll experiments were conducted independently in triplicate, except for the reperfusion time optimization for the tMCAO procedure. Bar graphs display the mean values along with standard deviation (SD). Statistical analyses were carried out using GraphPad Prism version 10. Comparisons between two groups were assessed using a two-tailed Student\u0026rsquo;s t-test. For comparisons involving more than two groups, one-way ANOVA followed by multiple comparisons was applied. In cases involving multiple variables across multiple groups, two-way ANOVA was used. Bonferroni correction was applied in all analyses to adjust for multiple comparisons. A p-value of less than 0.05 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003e We thank the UCSD Animal Care Facilities for excellent support. We are grateful to Drs. Xinlian Zhang and Karen Messer from the UCSD Moores Cancer Center Biostatistics group for their helpful comments and advice. We thank Genentech, Inc. for the gift of anti-VEGF antibody B.20.4.1. Also, we thank the Moores Cancer Center microscopy facility and the UCSD Electron microscopy core facility for excellent support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eG. B. D. S. Collaborators, Global, regional, and national burden of stroke, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. \u003cem\u003eLancet Neurol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 439-458 (2019).\u003c/li\u003e\n\u003cli\u003eR. Waziry\u003cem\u003e et al.\u003c/em\u003e, Time Trends in Survival Following First Hemorrhagic or Ischemic Stroke Between 1991 and 2015 in the Rotterdam Study. \u003cem\u003eStroke\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, STROKEAHA119027198 (2020).\u003c/li\u003e\n\u003cli\u003eE. S. Donkor, Stroke in the 21(st) Century: A Snapshot of the Burden, Epidemiology, and Quality of Life. \u003cem\u003eStroke Res Treat\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, 3238165 (2018).\u003c/li\u003e\n\u003cli\u003eC. L. Richards, F. Malouin, S. Nadeau, Stroke rehabilitation: clinical picture, assessment, and therapeutic challenge. \u003cem\u003eProg Brain Res\u003c/em\u003e \u003cstrong\u003e218\u003c/strong\u003e, 253-280 (2015).\u003c/li\u003e\n\u003cli\u003eK. L. Furie, M. V. Jayaraman, 2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke. \u003cem\u003eStroke\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 509-510 (2018).\u003c/li\u003e\n\u003cli\u003eN. van Bruggen\u003cem\u003e et al.\u003c/em\u003e, VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 1613-1620 (1999).\u003c/li\u003e\n\u003cli\u003eR. Paul\u003cem\u003e et al.\u003c/em\u003e, Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke. \u003cem\u003eNature Med.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 222-227 (2001).\u003c/li\u003e\n\u003cli\u003eF. Corti\u003cem\u003e et al.\u003c/em\u003e, Syndecan-2 selectively regulates VEGF-induced vascular permeability. \u003cem\u003eNat Cardiovasc Res\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 518-528 (2022).\u003c/li\u003e\n\u003cli\u003eR. Daneman, A. Prat, The blood-brain barrier. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, a020412 (2015).\u003c/li\u003e\n\u003cli\u003eP. T. Do, C. C. Wu, Y. H. Chiang, C. J. Hu, K. Y. Chen, Mesenchymal Stem/Stromal Cell Therapy in Blood-Brain Barrier Preservation Following Ischemia: Molecular Mechanisms and Prospects. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e (2021).\u003c/li\u003e\n\u003cli\u003eH. Yu\u003cem\u003e et al.\u003c/em\u003e, The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e (2022).\u003c/li\u003e\n\u003cli\u003eX. Peng, Z. Luo, S. He, L. Zhang, Y. Li, Blood-Brain Barrier Disruption by Lipopolysaccharide and Sepsis-Associated Encephalopathy. \u003cem\u003eFront Cell Infect Microbiol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 768108 (2021).\u003c/li\u003e\n\u003cli\u003eC. Cho, P. M. Smallwood, J. Nathans, Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 1056-1073 e1055 (2017).\u003c/li\u003e\n\u003cli\u003eX. Jiang\u003cem\u003e et al.\u003c/em\u003e, Blood-brain barrier dysfunction and recovery after ischemic stroke. \u003cem\u003eProg Neurobiol\u003c/em\u003e \u003cstrong\u003e163-164\u003c/strong\u003e, 144-171 (2018).\u003c/li\u003e\n\u003cli\u003eO. Luderitz\u003cem\u003e et al.\u003c/em\u003e, Identification of D-mannosamine and quinovosamine in Salmonella and related bacteria. \u003cem\u003eJ Bacteriol\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 490-494 (1968).\u003c/li\u003e\n\u003cli\u003eF. Monaco, J. Robbins, Incorporation of N-acetylmannosamine and N-acetylglucosamine into thyroglobulin in rat thyroid in vitro. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e248\u003c/strong\u003e, 2072-2077 (1973).\u003c/li\u003e\n\u003cli\u003eT. Onoda\u003cem\u003e et al.\u003c/em\u003e, Antitumor activity of D-mannosamine in vitro: different sensitivities among human leukemia cell lines possessing T-cell properties. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 2867-2871 (1982).\u003c/li\u003e\n\u003cli\u003eY. J. Chen, C. C. Yao, C. H. Huang, H. H. Chang, T. H. Young, Hexosamine-Induced TGF-beta Signaling and Osteogenic Differentiation of Dental Pulp Stem Cells Are Dependent on N-Acetylglucosaminyltransferase V. \u003cem\u003eBiomed Res Int\u003c/em\u003e \u003cstrong\u003e2015\u003c/strong\u003e, 924397 (2015).\u003c/li\u003e\n\u003cli\u003eC. Zhong\u003cem\u003e et al.\u003c/em\u003e, Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 6330 (2020).\u003c/li\u003e\n\u003cli\u003eR. Daneman\u003cem\u003e et al.\u003c/em\u003e, Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 641-646 (2009).\u003c/li\u003e\n\u003cli\u003eL. Perez-Gutierrez, N. Ferrara, Biology and therapeutic targeting of vascular endothelial growth factor A. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 816-834 (2023).\u003c/li\u003e\n\u003cli\u003eZ. G. Zhang\u003cem\u003e et al.\u003c/em\u003e, VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 829-838 (2000).\u003c/li\u003e\n\u003cli\u003eO. Engel, S. Kolodziej, U. Dirnagl, V. Prinz, Modeling stroke in mice - middle cerebral artery occlusion with the filament model. \u003cem\u003eJ Vis Exp\u003c/em\u003e 10.3791/2423 (2011).\u003c/li\u003e\n\u003cli\u003eH. P. Gerber\u003cem\u003e et al.\u003c/em\u003e, Mice expressing a humanized form of VEGF-A may provide insights into safety and efficacy of anti-VEGF antibodies. \u003cem\u003eProc. Natl. Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 3478-3483 (2007).\u003c/li\u003e\n\u003cli\u003eC. Iadecola, The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 17-42 (2017).\u003c/li\u003e\n\u003cli\u003eZ. Mo\u003cem\u003e et al.\u003c/em\u003e, Activation of Wnt/Beta-Catenin Signaling Pathway as a Promising Therapeutic Candidate for Cerebral Ischemia/Reperfusion Injury. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 914537 (2022).\u003c/li\u003e\n\u003cli\u003eA. J. Valvezan, P. S. Klein, GSK-3 and Wnt Signaling in Neurogenesis and Bipolar Disorder. \u003cem\u003eFront Mol Neurosci\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1 (2012).\u003c/li\u003e\n\u003cli\u003eR. Daneman, L. Zhou, A. A. Kebede, B. A. Barres, Pericytes are required for blood-brain barrier integrity during embryogenesis. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e468\u003c/strong\u003e, 562-566 (2010).\u003c/li\u003e\n\u003cli\u003eB. V. Zlokovic, Cerebrovascular effects of apolipoprotein E: implications for Alzheimer disease. \u003cem\u003eJAMA Neurol\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 440-444 (2013).\u003c/li\u003e\n\u003cli\u003eP. Dore-Duffy, J. C. LaManna, Physiologic angiodynamics in the brain. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1363-1371 (2007).\u003c/li\u003e\n\u003cli\u003eY. Persidsky, S. H. Ramirez, J. Haorah, G. D. Kanmogne, Blood-brain barrier: structural components and function under physiologic and pathologic conditions. \u003cem\u003eJ Neuroimmune Pharmacol\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 223-236 (2006).\u003c/li\u003e\n\u003cli\u003eA. Zechariah\u003cem\u003e et al.\u003c/em\u003e, Vascular endothelial growth factor promotes pericyte coverage of brain capillaries, improves cerebral blood flow during subsequent focal cerebral ischemia, and preserves the metabolic penumbra. \u003cem\u003eStroke\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 1690-1697 (2013).\u003c/li\u003e\n\u003cli\u003eS. M. Weis, D. A. Cheresh, Pathophysiological consequences of VEGF-induced vascular permeability. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e437\u003c/strong\u003e, 497-504 (2005).\u003c/li\u003e\n\u003cli\u003eI. D. Kim, J. W. Cave, S. Cho, Aflibercept, a VEGF (Vascular Endothelial Growth Factor)-Trap, Reduces Vascular Permeability and Stroke-Induced Brain Swelling in Obese Mice. \u003cem\u003eStroke\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 2637-2648 (2021).\u003c/li\u003e\n\u003cli\u003eR. Kimura, H. Nakase, R. Tamaki, T. Sakaki, Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. \u003cem\u003eStroke\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 1259-1263 (2005).\u003c/li\u003e\n\u003cli\u003eJ. Krupinski, J. Kaluza, P. Kumar, S. Kumar, J. M. Wang, Role of angiogenesis in patients with cerebral ischemic stroke. \u003cem\u003eStroke\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1794-1798 (1994).\u003c/li\u003e\n\u003cli\u003eY. Sun\u003cem\u003e et al.\u003c/em\u003e, VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 1843-1851 (2003).\u003c/li\u003e\n\u003cli\u003eY. Wang\u003cem\u003e et al.\u003c/em\u003e, VEGF overexpression induces post-ischaemic neuroprotection, but facilitates haemodynamic steal phenomena. \u003cem\u003eBrain\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 52-63 (2005).\u003c/li\u003e\n\u003cli\u003eC. Uchida, E. Gee, E. Ispanovic, T. L. Haas, JNK as a positive regulator of angiogenic potential in endothelial cells. \u003cem\u003eCell Biol Int\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 769-776 (2008).\u003c/li\u003e\n\u003cli\u003eC. Y. Kuan, R. E. Burke, Targeting the JNK signaling pathway for stroke and Parkinson\u0026apos;s diseases therapy. \u003cem\u003eCurr Drug Targets CNS Neurol Disord\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 63-67 (2005).\u003c/li\u003e\n\u003cli\u003eD. M. Arduino, A. R. Esteves, S. M. Cardoso, C. R. Oliveira, Endoplasmic reticulum and mitochondria interplay mediates apoptotic cell death: relevance to Parkinson\u0026apos;s disease. \u003cem\u003eNeurochem Int\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 341-348 (2009).\u003c/li\u003e\n\u003cli\u003eT. Hayashi, A. Saito, S. Okuno, M. Ferrand-Drake, P. H. Chan, Induction of GRP78 by ischemic preconditioning reduces endoplasmic reticulum stress and prevents delayed neuronal cell death. \u003cem\u003eJ Cereb Blood Flow Metab\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 949-961 (2003).\u003c/li\u003e\n\u003cli\u003eV. P. Nakka, A. Gusain, R. Raghubir, Endoplasmic reticulum stress plays critical role in brain damage after cerebral ischemia/reperfusion in rats. \u003cem\u003eNeurotox Res\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 189-202 (2010).\u003c/li\u003e\n\u003cli\u003eL. Chen, X. Gao, Neuronal apoptosis induced by endoplasmic reticulum stress. \u003cem\u003eNeurochem Res\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 891-898 (2002).\u003c/li\u003e\n\u003cli\u003eH. Zhao, R. M. Sapolsky, G. K. Steinberg, Interrupting reperfusion as a stroke therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. \u003cem\u003eJ Cereb Blood Flow Metab\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1114-1121 (2006).\u003c/li\u003e\n\u003cli\u003eB. Xing\u003cem\u003e et al.\u003c/em\u003e, Ischemic postconditioning inhibits apoptosis after focal cerebral ischemia/reperfusion injury in the rat. \u003cem\u003eStroke\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 2362-2369 (2008).\u003c/li\u003e\n\u003cli\u003eS. Kishi\u003cem\u003e et al.\u003c/em\u003e, Nerve growth factor attenuates 2-deoxy-d-glucose-triggered endoplasmic reticulum stress-mediated apoptosis via enhanced expression of GRP78. \u003cem\u003eNeurosci Res\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 14-21 (2010).\u003c/li\u003e\n\u003cli\u003eQ. J. Quinones, J. H. Levy, Ischemic Preconditioning and the Role of Antifibrinolytic Drugs: Translation From Bench to Bedside. \u003cem\u003eAnesth Analg\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 384-386 (2018).\u003c/li\u003e\n\u003cli\u003eS. Liebner\u003cem\u003e et al.\u003c/em\u003e, Wnt/beta-catenin signaling controls development of the blood-brain barrier. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e183\u003c/strong\u003e, 409-417 (2008).\u003c/li\u003e\n\u003cli\u003eJ. Chang\u003cem\u003e et al.\u003c/em\u003e, Gpr124 is essential for blood-brain barrier integrity in central nervous system disease. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 450-460 (2017).\u003c/li\u003e\n\u003cli\u003eJ. E. Lengfeld\u003cem\u003e et al.\u003c/em\u003e, Endothelial Wnt/beta-catenin signaling reduces immune cell infiltration in multiple sclerosis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, E1168-E1177 (2017).\u003c/li\u003e\n\u003cli\u003eJ. M. Stenman\u003cem\u003e et al.\u003c/em\u003e, Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e322\u003c/strong\u003e, 1247-1250 (2008).\u003c/li\u003e\n\u003cli\u003eM. Caspi\u003cem\u003e et al.\u003c/em\u003e, Aldolase positively regulates of the canonical Wnt signaling pathway. \u003cem\u003eMol Cancer\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 164 (2014).\u003c/li\u003e\n\u003cli\u003eP. Han, S. Ivanovski, R. Crawford, Y. Xiao, Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration. \u003cem\u003eJ Bone Miner Res\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1160-1174 (2015).\u003c/li\u003e\n\u003cli\u003eY. Zhao\u003cem\u003e et al.\u003c/em\u003e, GSK-3beta Inhibition Induced Neuroprotection, Regeneration, and Functional Recovery After Intracerebral Hemorrhagic Stroke. \u003cem\u003eCell Transplant\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 395-407 (2017).\u003c/li\u003e\n\u003cli\u003eW. Wang\u003cem\u003e et al.\u003c/em\u003e, GSK-3beta inhibitor TWS119 attenuates rtPA-induced hemorrhagic transformation and activates the Wnt/beta-catenin signaling pathway after acute ischemic stroke in rats. \u003cem\u003eMol Neurobiol\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 7028-7036 (2016).\u003c/li\u003e\n\u003cli\u003eD. M. Chuang, Z. Wang, C. T. Chiu, GSK-3 as a Target for Lithium-Induced Neuroprotection Against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke. \u003cem\u003eFront Mol Neurosci\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 15 (2011).\u003c/li\u003e\n\u003cli\u003eK. A. Tran\u003cem\u003e et al.\u003c/em\u003e, Endothelial beta-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis. \u003cem\u003eCirculation\u003c/em\u003e \u003cstrong\u003e133\u003c/strong\u003e, 177-186 (2016).\u003c/li\u003e\n\u003cli\u003eZ. Wang\u003cem\u003e et al.\u003c/em\u003e, Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, eaba7457 (2020).\u003c/li\u003e\n\u003cli\u003eI. A. Mulder\u003cem\u003e et al.\u003c/em\u003e, Automated Ischemic Lesion Segmentation in MRI Mouse Brain Data after Transient Middle Cerebral Artery Occlusion. \u003cem\u003eFront Neuroinform\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 3 (2017).\u003c/li\u003e\n\u003cli\u003eM. Fujimoto\u003cem\u003e et al.\u003c/em\u003e, Tissue inhibitor of metalloproteinases protect blood-brain barrier disruption in focal cerebral ischemia. \u003cem\u003eJ Cereb Blood Flow Metab\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 1674-1685 (2008).\u003c/li\u003e\n\u003cli\u003eY. K. Lee, H. Uchida, H. Smith, A. Ito, T. Sanchez, The isolation and molecular characterization of cerebral microvessels. \u003cem\u003eNat Protoc\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 3059-3081 (2019). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of California, San Diego","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cerebral ischemia, Glycosylation inhibition, Endothelium, Vascular permeability","lastPublishedDoi":"10.21203/rs.3.rs-8408292/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8408292/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCerebral ischemia remains a major global health challenge, contributing significantly to long-term disability and mortality, with limited therapeutic options currently available. This study explores the therapeutic potential of Mannosamine (ManN), an hexosamine previously shown to be a mitogen for endothelial cells (ECs), in the context of cerebral ischemic injury. Using a transient middle cerebral artery occlusion (tMCAO) mouse model, we assessed the effects of ManN administration on ischemic brain damage. Mice receiving ManN exhibited significantly smaller infarct volumes, as measured by magnetic resonance imaging (MRI), and reduced blood\u0026ndash;brain barrier (BBB) permeability compared to controls. ManN treatment enhanced pericyte coverage, improved EC survival, and increased vascular density in the ischemic brain regions. Our analyses revealed attenuation of ischemia-induced structural abnormalities, including reduced vacuolation, cellular shrinkage, and nuclear condensation. To elucidate the underlying mechanisms, in vitro experiments with brain endothelial cells (bEND.3) demonstrated that ManN treatment promoted GSK3β phosphorylation and facilitated nuclear accumulation of β-catenin. This activation of the Wnt/β-catenin pathway led to upregulation of key target genes such as \u003cem\u003eLEF1\u003c/em\u003e, \u003cem\u003eTCF7\u003c/em\u003e, \u003cem\u003eAXIN2\u003c/em\u003e, and \u003cem\u003eAPCDD1\u003c/em\u003e. Enhanced interaction of β-catenin with LEF1/TCF7 was associated with increased expression of tight junction proteins and transcytosis inhibitors, contributing to BBB stabilization and angiogenic support. Collectively, these findings highlight the ability of ManN to activate endothelial Wnt/β-catenin signaling, thereby preserving BBB integrity and promoting angiogenesis, suggesting its promise as a novel therapeutic strategy for cerebral ischemia.\u003c/p\u003e","manuscriptTitle":"Mannosamine preserves blood brain barrier integrity and promotes angiogenesis in a stroke model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 00:51:57","doi":"10.21203/rs.3.rs-8408292/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f34861b6-ae79-4106-85e8-2a16359787c1","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59979067,"name":"Translational Medicine"}],"tags":[],"updatedAt":"2025-12-30T00:51:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 00:51:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8408292","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8408292","identity":"rs-8408292","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}