Immunotherapies have substantially advanced cancer treatment; however, their efficacy in gliomas remains limited. This observation cannot be fully explained by tumour-intrinsic factors and may rather be linked to the distinct relationship between the central nervous system (CNS) and the immune system, commonly described as CNS immune privilege. CNS immune privilege is maintained by specialized brain barriers that divide the CNS into compartments with distinct accessibility to immune mediators and immune cells. Although maintaining homeostasis of the CNS parenchyma, these brain barriers direct CNS immune surveillance to the subarachnoid and the perivascular spaces at the CNS borders. Consequently, tumours arising in the CNS parenchyma are shielded from effective immune detection, limiting the efficacy of immunotherapies such as immune checkpoint inhibitors, cancer vaccines and adoptive T cell therapies such as chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) transgenic T cells by restricting their access. Importantly, emerging evidence also indicates that gliomas actively remodel brain barrier functions to reinforce immune evasion. Failure to adequately consider brain barrier function in the context of immunotherapy strategies and clinical trial design therefore represents a major gap in the field. Understanding the orchestrated function of the brain barriers as neuroimmunological interfaces is essential for enhancing immune surveillance and improving immunotherapy responses in patients with brain tumours.

Methylglyoxal (MGO), a highly reactive by-product of glycolysis, has been associated with cognitive decline and Alzheimer’s disease, though the mechanistic role of MGO remains unclear. Moreover, conflicting findings exist regarding MGO’s toxicity on the blood-brain barrier (BBB). This study investigated whether MGO can cross the BBB under physiologically relevant conditions and whether MGO affects BBB permeability. Mice were intravenously injected with highly purified home-made MGO or PBS, and MGO concentration was measured at five timepoints in the cerebral cortex up to 4 hours after injection. MGO toxicity was screened on a human brain endothelial cell line (hCMEC/D3) using a live/dead assay prior to the study of selected MGO concentrations and on hiPSC-derived brain microvascular endothelial cells (EECM-BMECs). EECM-BMECs were cultured on Transwell® inserts, and barrier function was assessed by sodium fluorescein permeability and transendothelial passage of 13 C3-MGO quantified by UPLC-MS/MS. MGO levels in the mouse cortex did not increase post-injection. MGO was not toxic to hCMEC/D3 cells, and it had no impact on barrier properties of EECM-BMECs. After 1-hour exposure, ∼13% of total 13 C3-MGO was recovered in its free form, and only ∼1% of supplemented MGO was recovered from the abluminal side. MGO does not cross the BBB in vivo and does not affect barrier properties of a human in vitro model of the BBB. In vitro MGO passage across the BBB is minimal. These findings suggest that circulating MGO is unlikely to directly affect neuronal function via BBB disruption or enter the brain in its free from.

Current intravital imaging techniques for the mouse central nervous system (CNS) do not simultaneously provide micrometer-scale spatial resolution, whole-brain coverage, and sub-minute temporal resolution, limiting organ-wide interrogation of CNS fluid dynamics in vivo. Here, we introduce intravital synchrotron radiation-based hard X-ray micro computed tomography (SRµCT), a modality that enables dynamic whole-brain imaging at micrometer-scale spatial resolution in living mice. We performed intravital SRµCT of mouse CNS fluid spaces at three synchrotron radiation facilities, imaging both anesthetized free-breathing and mechanically ventilated animals, with and without retrospective cardiac gating. This approach achieves complete brain coverage with temporal resolution of up to 23 s and voxel sizes down to 6.3 µm, at an effective spatial resolution better than 20 µm, enabling time-resolved visualization of cerebrospinal fluid (CSF) contrast distribution and quantitative analysis of tissue motion across the entire brain. By combining micrometer-scale resolution, whole-organ field of view, and dynamic intravital imaging, SRµCT closes a long-standing methodological gap between optical microscopy and magnetic resonance imaging. Intravital SRµCT provides access to spatiotemporal information that cannot be obtained with existing techniques and establishes a framework for testing and integrating mechanistic models of CSF dynamics and solute transport at the scale of the whole brain.

Glioblastoma (GBM) is the most aggressive primary brain tumor characterized by significant cellular and molecular complexity. Central to this complexity are glioma stem cells (GSCs) and the perivascular niche, a specialized microenvironment that integrates signals from the systemic circulation with cues arising from tumor development. Acting as a regulatory hub, the perivascular niche influences GSC proliferation, migration, and resistance to therapy through multiple interactions among vascular, immune, and tumor cell populations. In this review, we focus on GSCs and examine the cellular composition and molecular mechanisms operating within the perivascular niche, highlighting key modes of crosstalk that sustain tumor progression. We furthermore critically evaluate experimental strategies used to study this compartment, including 2D and 3D cell cultures, organoids, and in vivo models, discussing their respective strengths and limitations for testing intercellular interactions and therapeutic approaches. A comprehensive understanding of tumor heterogeneity and the interactions between different tumor cell populations, combined with well-characterized experimental models, is essential for deriving mechanistic insights and informing the development of more effective targeted therapies for GBM, ultimately advancing our knowledge of how tumor–microenvironment interactions drive GBM progression.

Immune surveillance of the central nervous system (CNS) is regulated by the brain barriers. Antigen presentation at the blood-brain barrier (BBB) has been proposed to promote antigen-specific T-cell entry into the CNS, largely based on in vitro studies. Recent in vivo and transcriptomic studies call for a reassessment of this concept. In healthy mouse and human CNS endothelium, major histocompatibility complex (MHC) class I expression is low, and MHC class II is minimal to absent. During neuroinflammation, brain microvascular endothelial cells (BMECs) can acquire antigen-presenting features, predominantly in the context of strong or prolonged inflammation. We propose that BMEC antigen presentation amplifies vascular pathology rather than initiating CNS T-cell entry during immune surveillance or disease.

The endothelial blood–brain barrier was discovered in the 19th century, and, since then, additional epithelial, glial and meningeal brain barriers have been described. In this issue of Nature Neuroscience, Verhaege and colleagues describe the discovery of an entirely overlooked brain barrier localized at the base of the choroid plexus.

The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the central nervous system (CNS). In neuroinflammatory diseases such as multiple sclerosis, this tight control is, however, disturbed, leading to immune cell infiltration into the CNS. The development of in vitro models of the BBB combined with microfluidic devices has advanced our understanding of the cellular and molecular mechanisms mediating the multistep T-cell extravasation across the BBB. A major bottleneck of these in vitro studies is the absence of a robust and automated pipeline suitable for analyzing and quantifying the sequential interaction steps of different immune cell subsets with the BBB under physiological flow in vitro. Here, we present the under-flow migration tracker ( UFM Track) framework for studying immune cell interactions with endothelial monolayers under physiological flow. We then showcase a pipeline built based on it to study the entire multistep extravasation cascade of immune cells across brain microvascular endothelial cells under physiological flow in vitro. UFM Track achieves 90% track reconstruction efficiency and allows for scaling due to the reduction of the analysis cost and by eliminating experimenter bias. This allowed for an in-depth analysis of all behavioral regimes involved in the multistep immune cell extravasation cascade. The study summarizes how UFM Track can be employed to delineate the interactions of CD4 + and CD8 + T cells with the BBB under physiological flow. We also demonstrate its applicability to the other BBB models, showcasing broader applicability of the developed framework to a range of immune cell-endothelial monolayer interaction studies. The UFM Track framework along with the generated datasets is publicly available in the corresponding repositories.

The complex etiology of neurological disorders is a major challenge to the identification of therapeutic candidates. Tackling brain vascular dysfunction is gaining attention from the scientific community, neurologists and pharmaceutical companies, as a novel disease-modifying strategy. Here, we provide evidence that at least 41

Pericytes play a key role in the brain where they support brain microvascular endothelial cells (BMECs) in forming the tightly regulated blood-brain barrier (BBB). The loss of pericytes, and corresponding weakening of the BBB, has been reported in response to episodes of systemic inflammation and in neurodegenerative disease. We recently demonstrated that iPSC-derived pericyte-like and BMEC-like cells form a nascent, 3D basement membrane when cultured across an ultrathin (100 nm thick) and highly nanoporous membrane (McCloskey, Ahmed et al., AHCM 2024). We also concluded that the pericyte-like cells did not contribute soluble factors to enhance permeability. Given the structural role of pericytes in vivo, here we sought to engineer defects in the basement membrane to see if pericytes could repair them. In BMEC-like monocultures, we found that micropore (3 μm and 5 μm) patterns in nanomembranes appeared as corresponding discontinuities in basement membrane laminin and destabilized barrier function. Both the laminin defects and the baseline barrier function were restored with the addition of pericytes on the basal side of the membrane. We further found that: 1) BMECs transmigrate through large micropores in monocultures but not in co-culture with pericytes, and 2) pericytes stabilized barrier function. Our results align with the role of pericytes as structural support cells for the microvasculature and encourage the use of our tissue barrier platform (the μSiM) to model acute and chronic neurological disorders involving pericyte dysfunction and/or disruption of basement membrane integrity.

Current approaches to in vivo imaging of the mouse central nervous system (CNS) do not offer a combination of micrometer resolution and a whole-brain field of view. To address this limitation, we introduce an approach based on synchrotron radiation-based hard X-ray micro computed tomography (SRμCT). We performed intravital SRμCT acquisitions of mouse CNS fluid spaces at three synchrotron radiation facilities. Imaging was conducted on both anesthetized free-breathing and ventilated animals, with and without retrospective cardiac gating. We achieved whole-brain imaging at 6.3 μm uniform voxel size, observed the distribution of cerebrospinal fluid (CSF) contrast agent over time and quantified choroid plexus movement. SRμCT bridges the gap between multiphoton microscopy and magnetic resonance imaging, offering dynamic imaging with micrometer-scale resolution and whole-organ field of view. Intravital SRμCT will play a crucial role in validating and integrating hypotheses on CSF dynamics and solute transport by providing unique data that cannot be acquired otherwise.

Multiple Sclerosis (MS), an autoimmune disorder, is characterized by severe neuroinflammation, leading to demyelination and neuronal damage in the CNS, resulting in significant clinical impairment. MS progression involves complex pathological processes like immune cell invasion and cytokine-mediated recruitment to the CNS. Experimental autoimmune encephalomyelitis (EAE), widely used as a model for MS, despite its translational limitations, has been crucial for identifying effective treatments. Recent studies have shown that sodium channel (NaV) blockers and monoamine oxidase- (MAO) B inhibitors can alleviate symptoms of EAE and optic neuritis (ON), but their mode of action remains partially unclear. To evaluate the effects and understand the action mechanism of NaV blockers and MAO-B inhibitors (rasagiline, safinamide, flecainide and phenytoin) in neurological conditions, various techniques were used, including optical coherence tomography (OCT), optomotor response measurement (OMR), flow cytometry, histological evaluations, Evans blue assay, blood-brain barrier (BBB) permeability assay, western blot, proliferations assay, and gene expression analyses. The study found that the primary therapeutic effect comes from inhibiting the NaV 1.5 sodium channel, not MAO-B inhibition. Flecainide, a NaV 1.5 channel blocker, significantly reduced EAE disability scores, mitigated neurodegeneration, preserved visual function, and restricted immune cell migration into the CNS. Importantly, blocking the NaV 1.5 channel had an effect on the BBB, limiting lymphocyte entry into the CNS. This research highlights sodium channel blockers' potential in treating EAE. The findings demonstrate induced neuroprotection and reduced disease progression, suggesting a novel therapeutic approach. Crucially, it reveals for the first time that NaV 1.5 channel blockade leads to neuroprotection primarily by affecting the BBB, a key factor in controlling immune cell migration, thus addressing a critical aspect of neuroinflammation.

BACKGROUND: Gadolinium (Gd) deposition in the brain was observed in patients with history of gadolinium-based contrast agent (GBCA) administration. However, the exact mechanism behind this deposition remains unclear, especially given that an intact blood-brain barrier (BBB) is considered impermeable to GBCA. In this study, we propose that immune cells might play a role in facilitating GBCA entry into the brain despite an intact BBB. METHODS: Gadoterate meglumine, gadoteridol, gadobutrol and gadodiamide were investigated as GBCAs. Immune cells from human donor buffy coats were isolated, incubated with the GBCA and used in the experiments. Gd associated with the immune cells were measured using single-cell inductively coupled mass spectrometry (SC-ICP-MS). Flow cytometry analysis was performed to characterise the adhesion molecule expression profile on the immune cells and binding assay was employed to check the binding of Gd treated immune cells with endothelial ligands in static conditions. An in vitro model of the human BBB that prevents free diffusion of GBCA across was further used to observe immune cell behaviour at the BBB under physiological flow, in vitro. RESULTS: Our findings confirm that various immune cells, including CD4+ T cells, CD8+ T cells, monocytes, NK cells and B cells are capable of taking up the different GBCAs. Furthermore, we demonstrate that GBCA loading does not impair immune cell interaction with the endothelial ligands required for successful extravasation across the BBB under static conditions. Most importantly, we show that T cells and monocytes, loaded with the different contrast agents, extravasated across an in vitro BBB model under physiological flow conditions in a comparable manner to non GBCA loaded cells. CONCLUSIONS: Taken together, our in vitro observations show that immune cells can transport GBCA across the BBB and could lead to permanent deposition of Gd in the brain.

Lymphatic capillaries continuously take up interstitial fluid and adapt to resulting changes in vessel calibre 1–3 . The mechanisms by which the permeable monolayer of loosely connected lymphatic endothelial cells (LECs) 4 maintains mechanical stability remain elusive. Here we identify dynamic cytoskeletal regulation of LEC shape, induced by isotropic stretch, as crucial for the integrity and function of dermal lymphatic capillaries. We found that the oak leaf-shaped LECs showed a spectrum of VE-cadherin-based junctional configurations at the lobular intercellular interface and a unique cytoskeletal organization, with microtubules at concave regions and F-actin at convex lobes. Multispectral and longitudinal intravital imaging of capillary LEC shape and actin revealed dynamic remodelling of cellular overlaps in vivo during homeostasis and in response to interstitial fluid volume increase. Akin to puzzle cells of the plant epidermis 5,6 , LEC shape was controlled by Rho GTPase CDC42-regulated cytoskeletal dynamics, enhancing monolayer stability. Moreover, cyclic isotropic stretch increased cellular overlaps and junction curvature in primary LECs. Our findings indicate that capillary LEC shape results from continuous remodelling of cellular overlaps that maintain vessel integrity while preserving permeable cell–cell contacts compatible with vessel expansion and fluid uptake. We propose a bellows-like fluid propulsion mechanism, in which fluid-induced lumen expansion and shrinkage of LEC overlaps are countered by actin-based lamellipodia-like overlap extension to aid vessel constriction.

The glia limitans ensheathes the entire central nervous system (CNS) parenchyma towards the outer surfaces and the perivascular spaces and is formed by a subset of astrocytes strategically localized at these outer parenchymal borders. Barrier properties of the glia limitans during health and neuroinflammation are incompletely understood. By developing an aquaporin-4 (Aqp4)-mRuby3 knock-in reporter mouse that allows for in vivo imaging of the superficial and perivascular glia limitans, we here show that the glia limitans forms a barrier for soluble mediators, beads and immune cells. Combining the Aqp4-mRuby3 reporter strain with additional reporter alleles for vascular, leptomeningeal or myeloid cells ensures precise localization of immune cells to CNS border zones versus the CNS parenchyma allowing to assign functional roles in CNS immune surveillance versus neuropathology. Availability of the Aqp4-mRuby3 reporter mouse will further advance our understanding of the active role of the glia limitans in CNS immune privilege.

The endothelial blood-brain barrier (BBB) tightly controls T cell entry into the central nervous system (CNS). T cell extravasation across the BBB involves a multi-step cascade with a predominant role of α4β1-integrins. In contrast to CD4 T cells, α4β1-integrin mediated CD8 T cell interaction with the BBB was proposed to involve the tight junction protein junctional adhesion molecule (JAM)-B. Here, we made use of ODC-OVA mice expressing ovalbumin as neo-self-antigen in oligodendrocytes that is solely visible to CD8 T cells, allowing to investigate CD8 T cell-mediated autoimmune neuroinflammation. We generated JAM-B-deficient ODC-OVA mice (ODC-OVA; JAM-BKO mice) and compared CD8 T cell mediated autoimmune neuroinflammation to their ODC-OVA; JAM-BWT littermates. ODC-OVA; JAM-BKO mice developed ameliorated clinical disease, which was associated with a marked reduction in CD8 T cell infiltration into the CNS parenchyma. Surprisingly, lack of JAM-B did not affect CD8 T cell arrest or extravasation in spinal cord microvessels but rather resulted in CD8 T cell accumulation in the subarachnoid space and perivascular spaces in ODC-OVA; JAM-BKO mice. Detection of Jam-2 RNA expression in cells other than BBB endothelial cells contributing to CNS barriers including astrocytes forming the glia limitans, Bergmann glial cells, meningeal fibroblasts and choroid plexus epithelial cells suggests that JAM-B may regulate CD8 T cell entry into the CNS at barriers other than the BBB, particularly at the glia limitans. Thus, targeting JAM-B could provide a therapeutic strategy for treating neuroinflammation without disrupting T cell-mediated immune surveillance in CNS border compartments.

Multiple sclerosis (MS) is an inflammatory disease of the CNS influenced by a combination of genetic predisposition and environmental factors. Vitamin D (VitD) deficiency is considered a major risk factor for MS. While VitD is associated with immunomodulatory roles, the exact mechanisms by which VitD protects from disease are still largely unknown. CD4 T cells play a key role in MS pathogenesis with autoimmune effector T cells (Teff) infiltrating the CNS across the blood-brain barrier (BBB) and regulatory T cells (Treg) displaying impaired functions. Here we show that treatment of human CD4 T cells with the active form of VitD (1,25-Dihydroxyvitamin D3; 1,25(OH)2D3) decreased cell-surface expression of α4β1- and αLβ2-integrins on Teff but not Treg and reduced Teff adhesion to their endothelial ligands VCAM-1 and ICAM-1. By employing live cell imaging, we observed that VitD treatment reduced arrest of Teff but not Treg to the BBB as well as ICAM-1 and VCAM-1 under physiological flow in vitro and differentially affected post-arrest behaviour of Teff versus Treg on the BBB under physiological flow. Furthermore, VitD treatment favoured the migration of Treg over Teff across the BBB under static and flow conditions in vitro. In vivo live cell imaging showed that VitD reduced T cell arrest on the inflamed BBB during autoimmune neuroinflammation. Finally, VitD also reduced expression of integrins mediating CNS homing on pathogenic CD4 T cells isolated from the CSF of persons with MS (PwMS). As VitD treatment did not alter barrier properties or adhesion molecule profile of our BBB model we propose a beneficial effect of VitD supplementation in PwMS by reducing CNS trafficking of pro-inflammatory T cells while leaving CNS entry of Treg unaffected. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, 310030E_189312, CRSII3_154483 Swiss MS Society European Research Council, ERC-2013-ADG 340733 Agence Nationale de la Recherche, ANR-19-CE14-0043 Foundation France SEP Multiple Sclerosis Society, 859/07, 41 Eugène Devic EDMUS Foundation Fondation d'Aide pour la Recherche sur la Sclerose en Plaques Hubert Curien/germaine de Stael Bangerter Rhyner Foundation Edinburgh Neuroscience funds Uehara Memorial Foundation, https://ror.org/00gc20a07 European Committee for Treatment and Research in Multiple Sclerosis

The blood-brain barrier (BBB) maintains cerebral homeostasis and protects the central nervous system (CNS) during systemic inflammation. Advanced in vitro models integrating circulation, a functional BBB, and reactive glial cells are essential for studying the link between peripheral inflammation and neuroinflammation. Fluid shear stress, a key hemodynamic parameter, strengthens microvascular barriers. This study examines endothelial shear conditioning on barrier function in a fluidic µSiM-BBB (Microphysiological System featuring a Silicon Membrane -BBB). hiPSC-derived brain microvascular endothelial cell monocultures are conditioned with 0.5 Pa shear stress for 48 h. Shear conditioning lowers baseline permeability, increases glycocalyx production, and reduces responses to inflammatory challenges, including barrier breakdown, ICAM-1 upregulation, and neutrophil transmigration. Shear conditioning produces a resilient barrier function against a low-dose inflammatory challenge (10 pg mL-1 TNF-α/IL1-β/INF-γ) but a high-dose challenge (50 pg mL-1) disrupts the barrier. Adding astrocytes as neuroinflammatory "sensors" reveals that a high-dose inflammatory challenge activates astrocytes but only in combination with fibrinogen-a plasma protein known to trigger astrogliosis in multiple neurological conditions. This study highlights the utility of fluidic-enabled µSiM-BBB for investigating acute peripheral inflammation and brain injury relationships, serving as a foundation for more advanced models, including more cells of the neurovascular unit and brain parenchyma.

The meninges, consisting of the dura, arachnoid and pia mater that surround the brain and spinal cord, have been recognized from the earliest anatomical studies. First identified in 1787, lymphatic vessels in the dura are now receiving greater attention as their contribution to cerebrospinal fluid (CSF) clearance in diverse neurological conditions is being investigated. New methods have increased the understanding of dural lymphatics, but much is still being learned about their heterogeneity, intracranial and extracranial connections, and factors that govern their functions and maintenance. Current research is striving to understand the regulation of CSF drainage and influence of brain antigen and immune cell transit through dural lymphatics on aging impairments and the severity of neurodegenerative and neuroimmune diseases, traumatic brain injury, stroke and other neurological disorders. Achieving these goals should lead to safe and effective methods for manipulating CSF clearance through dural lymphatics for therapeutic benefit. McDonald et al. review studies of lymphatic vessels in the dural layer of the meninges and discuss the role of lymphatics in the function and maintenance of the central nervous system, aging, neuroimmunity and the progression of neurological disorders such as Alzheimer’s disease and Parkinson’s disease.

Introduction There is renewed interest in how central nervous system (CNS) fluids and barriers contribute to neurological disorders, including cerebral amyloid angiopathy (CAA). Evidence suggests that impaired clearance of amyloid-β (Aβ) plays a role in CAA formation, highlighting the need to better understand brain fluid clearance mechanisms. Nonetheless, the mechanisms for fluid circulation and solute exchange in the brain remain debated, and many studies overlook the brain’s compartmentalizing barriers. To address this gap, we developed new transgenic mouse models to visualize key CNS barriers. Methods We have developed two dual-reporter mouse strains: a Vascular reporter (Claudin5-GFP for cerebral vessels, Prox1-tdTom for lymphatics) and a Border reporter (Aqp4-mRuby3 for astrocytic glia limitans, VE-cadherin-GFP for leptomeninges and blood vessels). These two strains were crossed with the ArcAβ transgenic line, yielding two triple- transgenic models of cerebral amyloidosis. We analyzed Aβ deposition at different disease stages and examined solute distribution after parenchymal tracer infusion using ex vivo imaging of decalcified skulls and fixed brains. Results Aβ deposition patterns and tracer studies revealed multiple drainage pathways that are dependent on the region of the brain and the size of the solute. These include transport along white matter tracts and across the glia limitans—findings that diverge from the popular “glymphatic” model of perivascular bulk flow. Aβ was found to accumulate at the leptomeninges in a pattern that was not always associated with pial blood vessels. The potential links between Aβ accumulation and changes in barrier integrity are under further investigation. Conclusions Our novel reporter models provide powerful tools to study CNS barriers and solute clearance in CAA. These findings offer new insights into alternative drainage mechanisms besides glymphatic flow. The involvement of each pathway upon the onset of Aβ deposition remains to be elucidated and we speculate that certain types of deposition result from the rerouting of clearance due to the failure of other pathways. Future validation with in vivo imaging techniques, such as two-photon microscopy and synchrotron X-ray imaging, will further elucidate the spatial and functional dynamics of fluid movement in the diseased brain.