Fluids and Barriers of the CNS

Liver sinusoidal endothelial cells (LSEC) rapidly dedifferentiate in 2D-monoculture, losing their high endocytic activity and characteristic morphology, limiting their use in mechanistic studies. We established and validated culture conditions that preserve LSEC endocytic capacity for at least 10 days, enabling efficient in vitro siRNA-mediated gene silencing. Mouse LSEC were cultured in 5% oxygen, growth media partially exchanged daily and assessed for cell viability, endocytic capacity, morphology and ultrastructure. Despite typical culture-induced defenestration, the cells showed high viability and efficient endocytosis via scavenger-receptors. This allowed for siRNA-mediated mannose receptor knockdown exemplified by 96% and 76% reduction in Mrc1 mRNA and protein expression at 72h (validated by qPCR and Western blot), with functional assays confirming decreased mannose-receptor-mediated endocytosis. Extended maintenance of LSEC viability and functions, previously restricted to complex co-culture systems, provide a practical platform for investigating LSEC-specific molecular mechanisms and hepatic sinusoid physiology.

Understanding intracranial pressure (ICP) dynamics is essential for interpreting clinical infusion tests used in the diagnosis of cerebrospinal fluid circulation disorders. However, the complex coupling between vascular pulsations, cerebrospinal fluid flow, and intracranial compliance makes quantitative interpretation of these tests challenging. Here, I present a patient specific simulation framework based on an extended electrical analog model that reproduces intracranial pressure dynamics observed during clinical infusion tests. The model integrates physiological inputs including arterial blood pressure, heart rate, respiratory rhythm, and resistance to cerebrospinal fluid outflow derived from clinical data. Built upon the classical Ursino framework, the model incorporates several modifications enabling realistic representation of physiological pulsations and infusion test conditions. The resulting system functions as a hybrid electrical-numerical simulation model representing a simplified digital electrical twin of intracranial hydrodynamics. The model was validated using data from 21 clinical infusion tests performed in patients with suspected normal pressure hydrocephalus. Simulated intracranial pressure recordings were compared with clinical measurements using regression and residual analysis. The simulations demonstrated strong agreement with measured data, with a mean correlation coefficient of r = 0.95 (95% CI 0.94 - 0.96), mean residual values within -1.71 to +1.68 mmHg, and a mean root mean square error (RMSE) of 2.07 mmHg. These results demonstrate that the proposed model accurately reproduces the dynamic behavior of intracranial pressure observed during clinical infusion tests. The framework provides a physiologically grounded computational tool for studying patient specific intracranial dynamics and may support improved interpretation of infusion test results in clinical practice.

A growing number of studies indicate the possible involvement of astrocytes in triggering or modulating neurovascular coupling (NVC), i.e. the local dilation of blood vessels in the brain in response to neuronal activity. Astrocytes possess specialized subcellular compartments, named endfeet, that surround arterioles and capillaries, ideally positioned to mediate NVC. Various vasodilators have been shown to contribute to NVC, such as epoxyeicosatrienoic acid (EET), nitric oxide (NO), or prostaglandin E2 (PGE2), but the precise mechanisms underlying NVC and their variability remain to be fully elucidated. In particular, the involvement of astrocytes in this process is controversial. Recent translatome and proteomics data reveal that astrocytes and in particular endfeet are enriched in the proteins of the PGE2 pathway. However, how the latter could contribute to NVC remains to be characterized. Here, we develop a computational model of astrocyte-mediated NVC that recapitulates these findings and describes Ca2+ and PGE2 signaling in astrocytes, NO release by neurons, and arteriole diameter dynamics using ordinary differential equations. The model successfully reproduces the dynamics of arteriole diameter change during hyperemia from in vivo neocortical recordings in awake mice. Our simulations suggest that the astrocyte PGE2 pathway could be responsible for the late response of NVC at the arteriolar level. We further observe that PIP2-derived diacylglycerol plays a major role in driving arteriole diameter dynamics in our model, while phosphatidic acid-derived diacylglycerol, which is calcium-dependent, mainly acts as an amplifier of this response. Finally, a spatial implementation of the model using a simplified astrocyte geometry suggests that NVC is more efficient when synaptic stimulation occurs at the endfoot level rather than at other astrocytic compartments. Overall, this computational study suggests a partial role for astrocyte-mediated PGE2 release in NVC and points to astrocyte perivascular processes as sub-compartments that are ideally positioned and equipped to mediate NVC. Author summaryIn the brain, the local blood flow is regulated to meet neuronal energy demand by modulating the dilation of neighboring blood vessels. The mechanisms driving this process, known as neurovascular coupling (NVC), remain debated and are likely to differ depending on the physiological context. Recent evidence points to astrocytes, a cell type possessing specialized protrusions called "endfeet", that envelop the entire brain vascular tree. Contacts between synapses and endfeet have recently been reported, positioning the latter as ideal mediators of NVC. Here, we developed a computational model that simulates the signaling between neurons, astrocytes, and blood vessels. Our model successfully reproduces experimental recordings of blood vessels dilation in the brains of awake mice. Our simulations suggest that a specific signaling pathway in astrocytes, involving a molecule called prostaglandin E2, is a key driver of the late phase of NVC, occurring a few seconds after neuronal activity. Furthermore, our model indicates that the location of the stimulated synapses matters: signals sent to the astrocyte endfeet are particularly effective at controlling blood flow. This work helps clarify the active role of astrocytes in brain blood flow regulation, a process critical for healthy brain function.

Cerebral small vessel disease (cSVD) is a major contributor to stroke and cognitive decline, ultimately leading to vascular dementia (VaD). Genetic factors play a key role in the disease susceptibility and progression, and variants in COL4A1 cause one of the most common genetic cSVD. COL4A1 encodes the 1 subunit of type IV collagen, the principle extracellular matrix (ECM) protein in the basement membrane of vasculature. In the central nervous system (CNS), the neurovascular unit (NVU) has the unique astrocyte-derived parenchymal basement membrane (pBM), in addition to the vascular basement membrane (vBM), which together contributing to the regulation of the blood-brain barrier (BBB) function. However, the role of pBM in cSVD remains under investigated and poorly understood. The lack of relevant human models has limited our ability to dissect specific cell-cell and cell-matrix interactions, hindering the identification of effective therapeutic targets. In this study, we hypothesised that astrocyte-mediated ECM remodelling contributes to BBB dysfunction in COL4A1-associated cSVD. To investigate this, human induced pluripotent stem cells (hiPSCs) derived from a patient carrying the COL4A1G755R variant and its isogenic control line were differentiated into astrocytes and brain microvascular endothelial cells (BMECs). Comparing to isogenic controls, the COL4A1G755R astrocytes significantly reduced the expression of ECM-related genes and abnormally increased glutamate uptake. ECM preparations from COL4A1G755R astrocytes significantly damaged the tight junction (TJ) structure formed by control iPSC-derived BMECs and failed to rescue the compromised TJ integrity in COL4A1G755R BMECs. The secretome from COL4A1G755R astrocytes exaggerated the ECM abnormality in COL4A1G755R BMECs. Most importantly, reduced expression of HTRA1, a crucial serine protease known to regulate both ECM turnover and homeostasis, and increased TGF-{beta} signalling was observed in COL4A1G755R astrocytes. Functional rescue by recombinant human HTRA1 protein restored the disrupted TJ continuity in COL4A1G755R BMECs and normalized TGF-{beta} signalling and glutamate uptake in astrocytes. Together, these findings defined a previously unrecognised astrocyte-driven pBM mechanism in COL4A1-associated cSVD and highlight HTRA1 in ECM remodelling as a therapeutic target.

Extracellular vesicles (EVs) are promising nanocarriers for therapeutic delivery; however, the factors governing EV uptake by recipient cells remain incompletely understood. In this study, we investigated whether EV internalization is primarily influenced by donor-cell origin or recipient-cell phenotype. Fluorescently labeled EVs derived from HEK293T, or SKBR-3 cells were incubated with a range of human epithelial, immune, and murine cancer cell lines at different doses and time points. HEK293T-derived EVs showed highly variable uptake across recipient cells, with hepatocellular carcinoma cell lines Huh7 and HepG2 exhibiting the highest internalization, while parental HEK293T cells showed the lowest. THP-1 immune cells also demonstrated strong uptake, whereas Jurkat cells showed moderate uptake. In murine melanoma models, Yummer cells internalized more EVs than B16F10 cells. Importantly, similar uptake trends were observed using SKBR-3-derived EVs, where Huh7 and HepG2 again displayed the highest uptake despite originating from a different donor cell source. EV internalization increased with dose and incubation time until saturation at higher concentrations. Together, these results demonstrate that EV uptake is predominantly determined by recipient-cell characteristics rather than EV source. These findings provide important mechanistic insight for the development of EV-based therapeutics and suggest that optimizing recipient-cell targeting is essential for efficient vesicle-mediated delivery. Graphical abstractEV uptake is determined by cell membrane properties rather than by the source of the EVs. The image was created by Biorender. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/726167v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f5c1cborg.highwire.dtl.DTLVardef@860962org.highwire.dtl.DTLVardef@1d20239org.highwire.dtl.DTLVardef@9003af_HPS_FORMAT_FIGEXP M_FIG C_FIG

The poor prognosis of brain tumors, including IDH-wild-type glioblastoma (GB), as well as brain and leptomeningeal metastases, is partly related to the blood-brain barrier (BBB), which limits the delivery of hydrophilic anticancer drugs to the tumor site and surrounding brain parenchyma. Early studies using vital dyes demonstrated that intracranial injection could bypass the BBB in cats. We confirmed that, in guinea pigs, the vital dye Bleu Patente V diffused efficiently into the brain after a bolus intracranial injection, whereas the brain remained unstained after intravenous administration. Similarly, brain concentrations of the hydrophilic anticancer drug gemcitabine were significantly higher following intracranial injection than after intravenous administration. Consistent with these findings, Bleu Patente penetrated deeply into the cerebral cortex of sheep after a 24-hour intraventricular infusion. At the end of a 24-hour intraventricular infusion of 20 mg gemcitabine in sheep, mean gemcitabine concentrations reached 1,415 {micro}g/L in cerebrospinal fluid and 850 {micro}g/kg in brain tissue. These concentrations exceeded the IC90 values of gemcitabine for A172, U87-MG, and U118-MG human glioblastoma cell lines, as determined in vitro after 24 hours of incubation. We hypothesize that Bleu Patente dye and gemcitabine circumvent the blood-brain barrier (BBB) by utilizing the glymphatic system. Tolerance of a single 24-hour intraventricular infusion of gemcitabine at doses of 5, 10, and 20 mg was good. Taken together, these encouraging preclinical results support the resumption of Phase I clinical trials evaluating intraventricular infusion of gemcitabine in patients with refractory primary or secondary brain tumors.

ObjectiveHereditary hemorrhagic telangiectasia (HHT) is a vascular genetic disorder caused by endothelial cell dysfunction and characterized by telangiectasias and arteriovenous malformations (AVMs). HHT results primarily from loss-of-function mutations affecting components of the BMP9-ALK1-ENG-SMAD signaling cascade, a pathway essential for endothelial quiescence and vascular homeostasis, and currently lacks a cure. Here, we investigated whether nitazoxanide, an orally bioavailable drug with extensive clinical use, can modulate endothelial signaling relevant to HHT. Approach and ResultsNitazoxanide treatment activated SMAD1/5/8 signaling and increased expression of the downstream target ID1 in endothelial cells, while concurrently inhibiting mTOR signaling, indicating a dual modulatory effect on pathways implicated in HHT pathogenesis. In vivo, nitazoxanide activated SMAD signaling in BMP9/10-immunoblocked mice and significantly reduced AVM formation and hypervascularization. Importantly, nitazoxanide restored SMAD1/5/8 activation and ID1 expression in patient-derived blood outgrowth endothelial cells harboring loss-of-function mutations in ALK1 or SMAD4, which exhibit impaired BMP signaling. ConclusionThese findings identify nitazoxanide as a pharmacological modulator capable of activating BMP-SMAD signaling while restraining mTOR activity, thereby overcoming key signaling defects in HHT endothelial cells. Collectively, our results highlight nitazoxanide as a promising therapeutic candidate to target endothelial dysfunction in HHT.

Oligodendrocyte-enriched cortical organoids (OCOs) are a powerful platform for modeling oligodendrogenesis in a human cellular context. However, neuronal activity is impaired in conventional culture media, limiting assessment of neuronal function in conjunction with oligodendrocyte biology. To address this, we used a modified BrainPhys medium termed neuronal activity medium (NAM) and defined the optimal developmental window for NAM exposure to generate OCOs with robust neuronal activity (NAM-OCOs). Stage-specific exposure to NAM, prior to oligodendrocyte expansion, leads to enhanced structural maturation, as evidenced by increased organoid size, heightened synaptogenesis, and upregulation of transcripts associated with neuronal complexity. Further, NAM-OCOs display increased cellular heterogeneity, including greater representation of GABAergic interneurons while preserving oligodendrocyte development and maturation. Altogether, our studies demonstrate that stage-specific exposure to an activity-permissive environment enhances neuronal activity, establishing an OCO model which integrates neuronal activity with oligodendrocyte development and maturation. HighlightsO_LIIncreased neuronal activity in oligodendrocyte-enriched cortical organoids (OCOs) C_LIO_LIStage-specific Neuronal Activity Medium (NAM) optimizes activity C_LIO_LINAM-OCOs display increased cellular heterogeneity and neuronal maturation C_LIO_LIOligodendrogenesis is preserved in NAM-OCOs C_LI eTOC blurbIn this article, Chung et al enhance neuronal activity in oligodendrocyte-enriched cortical organoids (OCOs) through stage-specific exposure to Neuronal Activity Medium (NAM). OCOs exposed to NAM display elevated cellular heterogeneity, structural maturation, and synaptogenesis, while preserving oligodendrocyte development and maturation. These results establish an increasingly comprehensive OCO model for studying neuronal function and oligodendrogenesis.

BackgroundImpairment of brain waste removal contributes to Alzheimers disease etiology and progression. Although hypertension is a risk factor for dementia, little is known about how it affects measures of clearance in human brain. MethodsCross-sectional (n=159) and longitudinal (n=94) analysis of the relationship between blood pressure (BP) and brain clearance. The estimate of brain clearance was measured using positron emission tomography (PET) as the rate of radiotracer (MK-6240) efflux from the lateral ventricles in the 10-30-minute window after tracer injection. We also examined cerebral blood flow, PET-derived tau deposition in the medial temporal lobe, cognition and plasma biomarkers of neurodegeneration. At baseline we compared participants with (n=88) and without (n=71) hypertension. For longitudinal analyses we defined two groups based on systolic BP trajectories from baseline to follow-up: as long-term controlled (n=76) or uncontrolled BP (n=18). ResultsAt baseline, subjects with hypertension had lower ventricular clearance than normotensive controls (Cohens d=0.53, p=0.001). Over the course of the observation period (median 1.85 years) subjects in the uncontrolled BP group experienced a steeper reduction in clearance rates ({beta}=-5.88) than subjects in the controlled BP group ({beta}=-0.81, interaction p=0.039). ConclusionsOur study suggests that hypertension impairs brain clearance of fluids.

Purpose. The choroid plexus (ChP) is the primary source of cerebrospinal fluid and an emerging marker of cerebral health, with enlargement and hypoperfusion reported in aging and neurodegeneration. However, frequent ChP calcifications can confound volumetric and perfusion measures. Although computed tomography (CT) is the gold standard for detecting calcification, it is rarely available in research MRI. Quantitative susceptibility mapping (QSM) offers an alternative sensitive to diamagnetic mineralization but lacks validated susceptibility thresholds. Method. Participants underwent CT and MRI within four weeks, including 3D T1-weighted and a multi-echo gradient echo QSM MRI. ChP calcifications were identified on CT using standard diagnostic criteria. Using the Bayes decision boundary framework, we identified optimal susceptibility thresholds for detecting diamagnetic signals consistent with calcification and compared these thresholds with multiple density levels measured on gold standard CT images. Results. Across all participants (n=20; age=62.2+-12.0 yrs), the optimal susceptibility threshold separating background ChP signal from calcifications was -0.10 ppm at 60 HU (low-density) and -0.15 ppm at 100 HU (high-density). Susceptibility values within calcified tissue exhibited a linear relationship with CT-derived tissue density. A significant positive association was observed between ChP volume and calcification volume among participants with detectable calcification (beta=2.26, p=0.047). Conclusion. This work should provide a practical framework for quantifying ChP calcifications routinely from MRI. The observed relationship between ChP volume and calcification volume highlights the importance of accounting for calcified tissue, particularly when calcification burden is substantial, when investigating ChP abnormalities in aging and neurodegenerative disease.

Antibody-based therapeutics have revolutionized disease treatment, and recent advances in messenger RNA (mRNA) technologies have opened new opportunities for their intracellular production. In particular, in vitro-transcribed mRNA encapsulated in lipid nanoparticles (LNPs) enables targeted delivery to specific cells, where it can enable the synthesis of therapeutic antibodies with prolonged half-lives in a cost-effective manner. Despite rapidly growing experimental data, a modeling framework that integrates mRNA delivery, intracellular expression kinetics, and whole-body antibody disposition remains unavailable. To address this gap, we extended a Physiologically Based Pharmacokinetic model with a novel multiscale layer describing mRNA trafficking, cellular uptake, translation, and degradation. The integrated model was calibrated and validated using five datasets of mRNA-based cancer therapeutics, demonstrating strong predictive performance for the biodistribution of mRNA-encoded antibodies. The newly introduced mRNA layer, while minimally parameterized, effectively represents complex intracellular and systemic processes, enabling quantitative investigation of antibody biodistribution, optimization of dose scheduling, and providing an initial framework for future exploration of how LNP-mRNA formulation influences delivery and pharmacokinetics.

Organotypic slice cultures (OSCs) are widely used to study cellular properties in a functional and developmental tissue context. With the recent advent of transgenic mouse lines and viral tools we postulated that OSCs may enable the study of multicellular glial and neuroglial interactions in development, as well homeostatic and pathological conditions. Here, we made mouse cortical OSCs and used markers for oligodendroglial, microglial states and neuronal types between 1 to 28 days in vitro (DIV). The OSC was characterized by in-vivo like cortical layering, including layer 5 pyramidal neurons and produced highly robust synchronized period bursts resembling Up- and Down states. Glial cells showed a strong cortical layer- and time-dependent development pattern: in the first week (DIV 1-7), slicing-related debris clearance and developmentally restricted sparse oligodendroglial myelination created an environment with highly phagocytic, non-homeostatic microglia (assessed with CD68 and purinergic receptor P2Y12, respectively). Between DIV 14 and 21, however, slices showed stereotypical cortical myelin patterns and the emergence of a homeostatic microglia phenotype while exhibiting continued phagocytosis. Furthermore, live two-photon imaging and morphometric analyses revealed highly ramified microglia and myelinated axons with compact myelination, exceeding lamellae count compared to age-matched in vivo axons. Lastly, from DIV 28 and onwards, myelin integrity became impaired and associated with phagocytic microglia. Together, the results indicate that between DIV14 and 21 cortical OSCs are well suited for live imaging of homeostatic and activity-dependent neuron-glia interactions, bridging the gap between in vivo investigations and primary cell cultures.

Cryopreservation offers an option for long-term storage and global distribution of complex in vitro models, yet protocols for multicellular microphysiolgocial systems (MPS) such as brain organoids/spheroids remain limited. Here, we systematically compared three commercially available cryopreservation (mFreSR, CryoStorCS10, and 3dGRO) and two freezing time points, and established a robust workflow for freezing and recovering brain organoids. After defrosting, we assessed morphology and metabolic activity. We also evaluated electrophysiology, calcium transients, and neurite outgrowth. In addition, we measured astrocyte migration, apoptosis, mitochondrial integrity, microglia survival, and neural marker expression. We found that organoids require a 4-week recovery period to regain structural and functional stability. Although organoids frozen at week 6 showed higher metabolic activity after recovery, organoids cryopreserved at week 2 had clearly better functional outcomes. They exhibited stronger spontaneous network firing and maintained calcium transients. Finally, incorporated microglia-like cells survived the freezing and displayed comparable morphology to unfrozen controls. Across the endpoints measured here, 3dGRO showed the most favorable overall performance; formal ranking across media awaits harmonized normalization, single-organoid electrophysiology, and prespecified QC thresholds. Together, these results define a practical and reproducible cryopreservation strategy that preserves key physiological features of brain organoids and supports the establishment of ready-to-use organoid banks. The ability to reliably store and distribute complex brain-like tissues represents an essential step toward global standardization, scalable experimentation, and wider adoption of human-relevant microphysiological systems. Together, these results demonstrate recovery of key physiological features in the subset of organoids that remain viable after thaw and support the feasibility of brain organoid banking.

Colony stimulating factor 1 receptor (CSF1R) is a tyrosine kinase receptor that is expressed exclusively in microglia within the CNS. Its endogenous ligands, colony stimulating factor-1 (CSF1) and interleukin-34 (IL-34), are released from neurons, positioning CSF1R as a key mediator receptor of neuron-glia communication. CSF1R is considered not only a potential drug target, but also a biomarker of neuroinflammation. From that perspective, selective radioligands for neuroimaging are of great interest for imaging neuroinflammation and determining drug occupancy. In this study, we have validated the binding characteristics of a CSF1R inhibitor, 4-((5-MethOxy-6-((5-methoxypyridin-2-yl)methoxy)pyridin-3-yl)methyl)-2-(1-methyl-1H-pyrazol-4-yl)pyrimidine (5-MOP) as a novel CSF1R radioligand, by performing in vitro saturation binding experiments in human and murine tissues. 5-MOP was found to be selective for CSF1R among a broad range of kinases. Autoradiography revealed that [3H]5-MOP binds with high affinity (KD = 9.8 nM) to a single saturable binding site in human meningioma tissues, and this binding was displaced with known CSF1R inhibitors, including CPPC, sCSF1inh and GW-2580. In contrast, CPPC, which has been extensively used as a CSF1R radioligand showed substantial cross-reactivity to other brain kinases, including Trk A/B/C, and [3H]CPPC could only be displaced with CPPC itself, not by other ligands, including 5-MOP. These results identify [3H]5-MOP as the most selective radioligand currently available, enabling accurate detection of drug occupancy and activated microglia. Significance of the studyThis study identifies and validates a novel selective radioligand that binds CSF1R with high selectivity and low nanomolar affinity. Because CSF1R is selectively expressed in activated microglia, this radioligand could be useful for detecting neuroinflammatory activity.

The metabolic demands of the human brain are met by a complex vascular architecture, yet our characterization of this network remains incomplete. While we have mapped the macroscopic vessels on the brains surface and the microscopic capillaries within small tissue samples, the mesoscopic scale consisting of the penetrating vessels that plunge through cortex remains an anatomical terra incognita. Mapping the interface between the macroscopic and microscopic scales is essential to understanding the critical vascular supply that sustains brain health. Here, we leveraged the BigBrain dataset and developed custom detection and tracing algorithms to reveal a whole-cortex record of the mesoscopic vascular network. We find that vascular density is not uniform across the cortex, but is a heterogeneous landscape that shows clear relationships to traditional areal boundaries. While based on a single human specimen, our results constitute a reference for human mesoscopic angioarchitecture and demonstrates the power of repurposing high-resolution histological atlases. Ultimately, this work lays the groundwork for validating recently developed in vivo MRI techniques for imaging the human cerebrovascular system at mesoscale.

BACKGROUNDHereditary hemorrhagic telangiectasia (HHT) is a genetic disorder caused by pathogenic variants in the endothelial TGF{beta}/BMP pathway, crucial for the vascular arterial-venous differentiation. Vascular defects result in fragile and malformed vessels. The precise mechanisms driving vascular network failure remain incompletely understood, complicating the design of targeted therapies. METHODSNasal telangiectasias from HHT patients carrying variants in ACVRL1 or ENG were used to perform scRNA-seq (2 ACVRL1- and 1 ENG-patient) and spatial transcriptomics (1 ACVRL1 and 1 ENG) to uncover endothelial cells (EC) populations. Vascular characteristics within biopsies were evaluated using transmission electron microscopy (TEM) (1 ACVRL1 and 1 ENG) and histological analyses (23 ACVRL1 and 7 ENG), with particular attention to regions exhibiting varying degrees of damage. RESULTSComparing our HHT tissues with healthy donor from the literature, we identified cellular heterogeneity within EC populations, revealing two distinct venous clusters: a stable, quiescent population (Mature Vein) and an activated, pro-inflammatory population (HHT Vein). The coexistence of these two clusters suggests cellular diversity within the biopsy, further validated by TEM and histology, revealing a juxtaposition of well-organized collagen and cellular architecture with severely disrupted, fibrotic regions. Moreover, cellular crosstalk analyses allowed us to identify critical ligands in ECs that interact with fibroblasts and mural cells. In particular, we found Midkine (MDK) lost in HHT Vein ECs with further validation in vitro, suggesting its potential role in cellular stability. Furthermore, spatial transcriptomics allowed to further uncover pathologic phenotypes in cells neighboring HHT Vein ECs. CONCLUSIONSHHT biopsies exhibit localized inflamed and fibrotic vascular areas with the presence of different transcriptional sub-populations of EC. Within the same tissue, stable and activated ECs can be distinguished. The pathologic-like EC cluster, present exclusively in the HHT samples, may contribute to vascular leakage through the loss of important ligands involved in cellular communication.

The mechanisms that drive myelin damage as seen in demyelinating disorders such as multiple sclerosis remain incompletely understood. Much of our current knowledge is derived from animal models, but interspecies differences limit their relevance in the context of human pathology and could explain why various promising preclinical therapies failed during clinical translation. Human post-mortem organotypic brain slice cultures provide a unique platform to study human myelin biology, as they preserve genetic, cytoarchitectural, pathological and species-specific context. Here, we evaluated myelin integrity in a human post-mortem brain organotypic slice culture model and experimentally induce focal myelin damage. Human post-mortem organotypic slices cultures retain key features throughout the culturing period, but exhibit gradual cellular and myelin loss over time. Myelin fibres within the white matter remain detectable and present preserved structural and chemical integrity up to 13 days in vitro, indicated by the conserved paranodal and nodal organization and stable myelin spectroscopic signature. Delivery of lysophosphatidylcholine using cryogel scaffolds enables focal drug administration throughout the full depth of the slice with minimal diffusion into surrounding tissue and induces localized demyelination after lysophosphatidylcholine application. Similar focal application of the selective Nav1.6 stimulator {beta}-mammal scorpion toxin Cn2 induces subtle myelin destabilization. Overall, our results demonstrate the suitability of a human post-mortem brain organotypic slice culture model as an adequate platform for studying myelin damage in a human disease context.

Perivascular spaces (PVS) are central to cerebrospinal fluid (CSF)-interstitial fluid (ISF) exchange in the brain, yet their brain-wide organization and in vivo accessibility remain poorly resolved due to limited spatial resolution and contrast specificity of existing imaging approaches. Prior gadolinium (Gd)-enhanced MRI studies demonstrate global tracer distribution but yield spatially diffuse signals that do not resolve PVS at the level of individual vessels. Here we introduce an ultra-high-resolution dual-contrast MRI framework that enables vessel-resolved mapping of PVS across the whole brain in vivo. This approach combines ultra-high-field imaging, an implantable radiofrequency coil for enhanced local sensitivity, and intraventricular Gd delivery to achieve sufficient contrast and spatial specificity for detecting vessel-associated perivascular signal. Using this framework, we show that PVS are hierarchically organized along vascular trees, extending from major surface arteries into deep cortical and subcortical regions. Signal patterns along arterial branches and junctions indicate that PVS follows vascular topology. Quantitative analysis reveals that only a subset of penetrating vessels ([~]6%) exhibits detectable PVS signal, indicating heterogeneous organization across vascular networks independent of vessel caliber. Widespread detection of PVS, including in the hippocampus, further demonstrates that ventricularly delivered tracers access a distributed, vessel-associated perivascular network in vivo. These results establish an anatomical framework for mapping perivascular transport pathways across the brain, bridging global tracer imaging with vessel-resolved organization, and enabling investigation of how vascular architecture and fluid dynamics shape CSF-ISF exchange.

IntroductionPerivascular spaces (PVS) support brain homeostasis through metabolite delivery and waste clearance, yet the genetic determinants of PVS morphology during childhood remain unknown. Here, we leveraged cross-sectional Adolescent Brain Cognitive Development Study data (N = 6,600; ages 9-10), including genomics and 3T structural MRI. MethodsLinear mixed-effects models examined associations between 45 single nucleotide polymorphisms (SNPs) previously linked to adult PVS structure or function and PVS count and volume fraction (VF) across six macroregions and 28 Desikan-Killiany subregions. ResultsFifteen SNPs demonstrated significant associations with PVS macroregion morphology, predominantly VF; 21 SNPs demonstrated associations with subregion morphology. Variants near SLC13A3 showed the strongest, most widespread associations with PVS VF. Additional replicated variants implicated Wnt signaling, cell adhesion, apoptosis, and glymphatic function. ConclusionThese findings suggest genetic associations with PVS morphology are detectable in childhood, while highlighting developmental and regional specificity. Longitudinal studies are now needed to determine whether childhood PVS genetic architecture predicts trajectories of glymphatic maturation and associated cognitive outcomes. Key PointsO_LIPerivascular space morphology, mapped across 28 white matter subregions, displays substantial regional and interindividual variability during childhood. C_LIO_LISix genetic associations with perivascular morphology were only detectable at subregion resolution, demonstrating that lobar averaging can obscure spatially restricted signals. C_LIO_LINearly 50% of adult-identified perivascular space risk variants replicated in 9-10 year-olds, with anatomically patterned associations reflecting patterns of neurovascular complexity and neurodevelopmental timing. C_LI

BackgroundCurrent microphysiological models do not support long-term investigations into the chronic effects of vascular risk factors and the development of vascular diseases. Prolonged culture frequently leads to cellular senescence and loss of functional integrity, resulting in variability and inconsistency in modeling chronic vascular responses. Here we aimed to develop and sustain a long-term multicellular human vascular avatar, addressing the critical need for long-term disease modeling and drug testing. MethodsTo identify the optimal media for longevity, cell identity and function were assessed by morphology, qPCR, beta-gal staining, ELISA, bulk RNA-seq and single cell RNA-seq analysis. After optimizing the culture media, iPSCs-derived ECs and VSMCs from unaffected and Hutchinson-Gilford Progeria Syndrome (HGPS) donors were grown in Gravitational Lumen Patterning (GLP) Vessel- Chips for 1-6 months to generate a long-lived vascular avatar for the study of vascular aging. ResultsGuided by quantitative morphological analyses and bulk RNAseq profiling, we generated a novel optimized culture media VSL (VEGF, SB431542 as a TGF-{beta} inhibitor, low fetal bovine serum) that enhances the long-term health of vascular endothelial cells (ECs). Furthermore, we modified the VSL formulation (mVSL) by modulating 8Br-cAMP, FGF, PDGF, and a cell viability enhancer HMH1015 levels to enhance EC-VSMC (vascular smooth muscle cell) crosstalk and support long-term cellular viability. Subsequently, we maintained and characterized a human vascular avatar with a three-dimensional extracellular matrix environment and 3D vascular architecture for over 180 days. Finally, we demonstrated that this long-lived human vascular avatar enabled modeling vascular aging using iPSC-derived vascular cells from patients with Hutchinson-Gilford Progeria Syndrome (HGPS). ConclusionsWe have successfully engineered and maintained a human vascular avatar for over 180 days. The vascular avatar provides a robust platform for modeling disease-associated vascular aging and for evaluating therapeutic strategies targeting chronic vascular disorders.