Fluids and Barriers of the CNS

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

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.

BackgroundChoroid plexus (ChP) produces cerebrospinal fluid (CSF), and regulates brain development and adult subventricular zone (SVZ) neurogenesis, but its role in hippocampal subgranular zone (SGZ) neurogenesis in adulthood and early postnatal stages is not well understood. Current tools to directly manipulate neonatal ChP/CSF volume are very limited, representing an urgent need in the field. MethodsWe first discovered the specific "leaky" expression of DTR gene in the ChP of adult ROSA26-iDTR mice which can be used to specifically ablate ChP in adult brain that generated robust and long-lasting ablation of ChP and reduction of CSF volume. In this study, we the effectiveness of ROSA26-iDTR allele in ablating neonatal ChP. We also developed a novel AAV2/5-CMV-DTR vector with validated ChP tropism in both neonatal and adult mice, which induces substantial CSF loss in both neonates and adult mice. With both the ROSA26-iDTR genetic and AAV2/5-DTR viral-mediated ChP ablation in young adults and at defined postnatal ages, we quantified ventricular CSF volume by MRI and characterized postnatal neurogenesis. Doublecortin-positive (DCX+) neuroblasts, Ki67+ proliferating cells, and TUNEL+ apoptotic cells were quantified in SVZ and SGZ using confocal microscopy and machine learning-assisted cell counting. ResultsWe show that ROSA26-iDTR-mediated ChP ablation is inefficient before postnatal day 10, suggesting that this line may be of limited utility for CSF reduction in the early neonatal period before P10. P3-5 Dtx treatment of a previously used dosage of 20ng/g dosage did not lead to a reduction in CSF volume. Higher dosage of 40ng/gX3 Dtx dosage at p3-5 generated only moderate partial reduction of CSF in third ventricle and total CSF volume, with indication of toxicity associated with high Dtx dosage in general. In contrast, p10-12 injection of 20ng/gX3 Dtx led to robust CSF reduction. To target early neonatal days, AAV2/5 CMV-DTR virus shows high tropism for ChP epithelial cells and leads to near-complete ablation of CSF in neonatal brains. ChP/CSF loss in neonates or young adult mice leads to a substantial reduction of DCX+ cells at the SVZ but a moderate but significant reduction of SGZ DCX+ neuroblasts, without changes in Ki67+ or TUNEL+ cells. ConclusionsThis study reports a novel role of the ChP/CSF in maintaining the neuroblast pool in the neurogenic niches in both early postnatal and adult stages. Moreover, we expand the available tools to target the ChP and CSF production in the neonate, with potential uses in treating conditions such as neonatal hydrocephalus.

During development, the size of the neuroepithelial cell pool plays a key role in establishing brain size, determining the numbers of derived progenitors and subsequent neuronal cell types. While early histogenesis is well modelled in brain organoids, the organ-scale geometry of the telencephalon is not accurately recapitulated. Herein, we present a new approach for generating ventral and dorsal forebrain organoids which develop a large ventricular neuroepithelium, characteristic of the closed telencephalic vesicle. Using a growth medium that supports aerobic glycolysis and is typically used for endothelial cells, we modulate neuroepithelial expansion to induce a more anatomically accurate neuroepithelial layer which, upon maturation, thickens physiologically to generate the typical neurogenic layered architecture. In addition, we present a new method for embedding organoids in miniature collagen spheres which mimics native extracellular matrix, stabilizes the ventricular geometry for dynamic culture conditions, and provides a means for incorporating vascular cells for neurovascular development. Finally, we demonstrate that human organoids grown under these conditions exhibit dramatically enlarged ventricles and delayed maturation compared to mouse. Together, this approach provides a model of the forebrain neuroepithelium with morphogenetic macroscale geometry and tissue architecture, suitable for investigating neurodevelopment and disease.

Extracellular vesicles (EVs) mediate intercellular transfer of lipids, proteins, and nucleic acids between nearly all cell types. We previously showed that astrocyte-derived EVs modulate neuronal mitochondria in vitro, but whether endogenous astrocytic EVs are trafficked to neuronal mitochondria in vivo remained unknown. To address this, we generated an EV reporter mouse, Aldh1l1-Cre; CD9-tGFPfl/fl, in which astrocyte-secreted EVs are labeled with a CD9-turboGFP fusion protein (CD9-tGFP). Astrocyte-specific expression of CD9-tGFP was verified in brain tissue and isolated EVs, comprising 13.2 {+/-} 1.6% of total brain EVs. In primary glial cultures, CD9-tGFP was restricted to astrocytes, localizing to vesicular compartments and cell protrusions (filopodia and cilia), with 89.3 {+/-} 2.2% of astrocyte-derived EVs carrying the label. These EVs were enriched with the sphingolipid ceramide, consistent with its co-distribution with CD9-tGFP in astrocytic cell protrusions. In the cortex, hippocampus, and cerebellum, CD9-tGFP was predominantly detected in astrocytic processes co-labeled with GLAST1 and GFAP, forming contacts with laminin-positive capillaries and parvalbumin-positive neurons. CD9-tGFP-labeled EVs were detected inside capillaries and neurons, and super-resolution STED microscopy revealed partial overlap with neuronal mitochondria. Live-cell spinning disk confocal imaging and AI-assisted proximity analysis confirmed uptake of CD9-tGFP EVs by neuronal cells and trafficking of their cargo to mitochondria in vitro. Biochemical isolation of synaptic and non-synaptic mitochondria confirmed EV-derived cargo on mitochondria in vivo, with 3-fold higher association of CD9-tGFP with synaptic than non-synaptic mitochondria. Together, these findings validate the Aldh1l1-Cre; CD9-tGFPfl/fl reporter mouse as a powerful tool for tracking astrocyte-derived EVs in vivo and provide direct evidence that their cargo is preferentially trafficked to synaptic mitochondria. Graphical AbstractAstrocyte-derived extracellular vesicles target neuronal mitochondria in vivo O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/718987v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@174d92aorg.highwire.dtl.DTLVardef@5d8248org.highwire.dtl.DTLVardef@114483borg.highwire.dtl.DTLVardef@924d55_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

BackgroundExtracellular vesicles (EVs) are nano and macro-sized, lipid-bound particles, involved in cellular communication. Interestingly, cancer-derived EVs show a heterologous and cross-species tumour tropism which makes them a potential tool for efficient delivery of therapeutic small interfering RNA (siRNA) to the tumour cells. MethodsEVs derived from glioblastoma cells (U373P and U373vIII) were loaded with EGFRvIII siRNA to develop a targeted therapeutic strategy against glioblastoma. EV biodistribution was evaluated using fluorescent indocyanine green (ICG) staining followed by ex vivo imaging. Different loading strategies, including passive loading, sonication, saponin-mediated membrane permeabilization, electroporation, and transfection were assessed for their efficiency in loading siRNA into EVs. The efficiency of each method was evaluated by nano flowcytometry, in vitro uptake assay followed by immunoblot (western blot) analysis. Eventually, the most effective formulation was tested for the systemic siRNA administration and selective tumour delivery in vivo, followed by evaluation of tumour size and EGFRvIII expression. ResultsHere, we showed that siRNA transfection into EVs was the most effective loading strategy, as confirmed by nano-flow cytometry, uptake assays, and western blot analysis, achieving over 90% knockdown efficiency in vitro for EVs carrying EGFRvIII siRNA. In vivo, EGFRvIII siRNA-loaded EVs homed to the tumour site and downregulated EGFRvIII expression compared with the PBS-siRNA control group; however, no significant tumour shrinkage was observed. ConclusionEGFRvIII-targeting, glioblastoma cell-derived EVs can be used as siRNA delivery carriers for targeted gene therapy in glioblastoma. However, further optimization of siRNA delivery and treatment duration is required.

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.

Newborns are especially susceptible to bacterial meningitis, primarily caused by Group B Streptococcus (GBS), due to incomplete maturation of immune and barrier systems. While meningitis is well known to break down the blood-brain barrier (BBB), how the meningeal arachnoid barrier, a critical component of the blood-cerebrospinal fluid barrier (B-CSFB), responds to infection is poorly understood. Using a neonatal mouse model of bacterial meningitis, we demonstrate that GBS infection significantly increases arachnoid barrier permeability, coinciding with alterations in Claudin-11 tight junction distribution and elevated meningeal production of proinflammatory cytokines (IL-6, TNF-, CXCL1). CD206+/Lyve1+ border-associated macrophages (BAMs) undergo significant morphological and molecular activation post-infection, but their depletion prior to GBS infection did not attenuate arachnoid barrier leakage or inflammatory cytokine levels during infection. We show that meningeal fibroblasts are a main source of proinflammatory cytokines in response to GBS infection and that exposure to the inflammatory cytokine TNF- alone is sufficient to induce neonatal arachnoid barrier breakdown. These results support neonatal arachnoid barrier is vulnerable to cytokine-induced breakdown in bacterial infection and highlight the role of non-immune meningeal cells like fibroblasts during bacterial infection.

Intracerebral haemorrhage is the most severe subtype of stroke; however, pre-clinical investigation often fails to translate to the clinic. Cerebral organoids offer an adaptable, in vitro model of human brain tissue for pre-clinical investigation of disease. We recently demonstrated that the tissue can be successfully vascularised to mimic the cerebrovasculature. Cerebrovascular weakness was induced with atorvastatin to mimic damage observed in intracerebral haemorrhage and to replicate the diseases pathological features. We used atorvastatin to disrupt functional morphology in human brain microvascular endothelial cells in 2D and 3D model systems. Whole human blood was added to initiate damage to cerebral tissues. Vascularised cerebral organoids exhibited loss of vascular integrity when treated with atorvastatin. Tissue was vulnerable to injury from human whole blood, and an innate immune response was initiated, resulting in increased cell death. Here we show that vascularised cerebral organoids demonstrate a novel model platform for investigating pathology associated with human whole blood insult in intracerebral haemorrhage.

Mathematical descriptions of drug release from polymeric nanocapsules are commonly based on first-order kinetics derived from the Noyes-Whitney equation. However, previous formulations implicitly predict that increasing drug solubility in the oily core accelerates release, which contradicts experimental evidence. In this work, we revisit the modeling framework and derive a physically consistent equation based on diffusion through the polymeric shell coupled with partition equilibrium at the oil-water interface. The resulting model shows that the effective release rate is inversely proportional to solubility. This formulation resolves the apparent paradox, preserves the experimentally observed exponential saturation behavior, and collapses kinetic data into a single intrinsic parameter. We further demonstrate that the only prior model proposed specifically for nanocapsular systems [1] also embeds the solubility paradox, and show that its own published validation data in fact confirm the inverse-solubility scaling derived here, with a deviation of only 9% between two independent formulations. Dimensional consistency, comparison with classical and nanoscale-specific models, and validation using published data support the robustness of the proposed approach.

Intracerebral haemorrhage (ICH) is a severe form of stroke with high morbidity and mortality rates. For survivors, acute haematoma expansion strongly determines neurological outcome. Although blood pressure reduction is widely investigated as a strategy to limit haematoma growth, the haemodynamic mechanisms regulating haemorrhage development remain poorly understood. Zebrafish provide a tractable in vivo model to study cerebrovascular biology and spontaneous ICH, yet the contribution of vascular regulation to haemorrhage onset and expansion has not been explored in this species. Here, we investigated whether pharmacological modulation of vascular dilation influences ICH development in zebrafish larvae. We first characterised vascular changes during the developmental window in which spontaneous ICH occurs and observed increased heart rate and progressive reductions in arterial diameter between 2 and 3 days post-fertilisation, suggesting increased vascular resistance. We then tested whether vasoconstriction promotes haemorrhage using angiotensin II, which induced systemic and cerebrovascular vasoconstriction but did not increase ICH incidence or haematoma size in two independent ICH models. In contrast, pharmacological vasodilation using sodium nitroprusside or isoproterenol significantly reduced haematoma size in a high-incidence model of atorvastatin-induced ICH. Live imaging of cerebral blood flow revealed that vasodilation was associated with the confinement of red blood cells around affected vessels rather than dispersing into the brain ventricles. Together, these findings indicate that vascular dilation modulates haemorrhage progression in zebrafish ICH and establish this model as a platform to investigate haemodynamic mechanisms regulating haematoma expansion.

Caveolae, flask-shaped membrane invaginations highly enriched in endothelial cells, play a central role in buffering membrane tension, yet the principles governing their spatial organization remain elusive. This investigation sought to generate the most comprehensive and systematic analysis of blood vessel caveolar spatial organization. To do so, our group leveraged micropatterning technologies to impose precise biophysical constraints on endothelial cell geometry to probe how caveolae are organized under defined tensional and polarity environments. These experiments were integrated with a high-throughput spatial cell mapping computational pipeline for analyzing thousands of caveolae, providing an extremely high-fidelity analysis. Our results provide a governing framework of how total cellular caveolae are spatially organized during random and directional migration, non-motile polarized, nascent and stable monolayers with differing confinement levels as well as in angiogenic vasculature in vivo. Broadly, our results demonstrated caveolae preferentially organized in the rear of migrating and polarized endothelial cells. In differing monolayer configurations, caveolae default to a peri-junctional spatial organization. Lastly, in mouse retinal blood vessels caveolae are most prominent in the vascular front due to their responsiveness to vascular endothelial growth factor signaling. Overall, these results strongly suggest that caveolae cellular arrangement and number are highly predictive of vascular stability and remodeling states.

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.

The gliovascular unit (GVU), a specialized interface between the brain and the vascular system, assembles and matures after birth and establishes essential homeostatic functions, including blood-brain barrier integrity, metabolic exchanges, fluid drainage, neurovascular coupling, and immune surveillance. Here, we systematically compared the postnatal maturation of the cortical GVU in male vs. female mice. On P15, males exhibited a transiently greater vessel density and a higher level of aquaporin 4 expression in perivascular astrocyte processes. Females exhibited a higher density of perivascular macrophages expressing the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1), along with earlier development of arterial vascular smooth muscle cells and greater cerebral blood flow. Transcriptomic profiling during the P5-P120 period revealed sex-specific developmental trajectories within the GVU, with the most prominent differences on P5. Taken as a whole, our results highlight pronounced sex-dependent differences in GVU assembly, GVU maturation, and the development of molecular programs that might influence brain physiology and vulnerability to neurodevelopmental disorders.

Disturbances of neurovascular coupling (NVC) contribute to metabolic derailment and neurological symptoms associated with epilepsy. While postictal arterial constriction can be alleviated by inhibitors of voltage gated calcium channels (VGCCs), less is known regarding seizure-associated electrical signals in higher-order capillaries and their role in determining pericyte tone during seizures. Here we investigated electrical signaling within the ex vivo neurovascular unit (NVU) derived from rat and human brain tissue. We focused on electrical signal transduction between pericytes and endothelial cells and the potential role of VGCCs in vasomotion. Using dye coupling and paired patch-clamp recordings, we showed that morphologically heterogeneous groups of mid-capillary pericytes build a functional syncytium with endothelial cells. Coupling was asymmetric, allowing for directed propagation of electrical signals. Regardless of their morphology, mid-capillary pericytes responded with depolarization and constriction to metabotropic receptor (GPCR) activation (by thromboxane, norepinephrine and UDP-glucose). However, depolarization via the patch pipette induced neither Ca2+-influx nor constriction, suggesting lack of contribution of VGCCs to vasomotion. On inducing epileptiform activity, A2a adenosine receptors and inwardly rectifying potassium channels hyperpolarized the capillary syncytium, followed by repeated depolarizations due to seizure-associated potassium increase in the parenchyma. Thus, while mid-capillary pericytes are contractile, their tone does not rely on their membrane potential and VGCCs. However, syncytial coupling allows for transmission of seizure-associated hyper- and depolarizing signals to upstream feeding arterioles. Significance statementElectro-metabolic signaling is a mechanism, which couples neuronal metabolic activity to local blood flow, by generation and conduction of hyperpolarizing electrical signals in the vasculature. Repeated seizures are followed by postictal hypoperfusion, suggesting disturbances in this signaling mechanism. Due to the inaccessibility of mid capillary pericytes, little is known about how seizure-associated electrical signals modulate local capillary tone. O_LIRat and human mid-capillary pericytes are contractile and actively regulate capillary diameter upon GPCR activation. C_LIO_LIWhile GPCR-induced vasoconstriction is associated with depolarization of the pericytes, depolarization via the patch pipette induces neither constriction nor intracellular Ca2+ increases. C_LIO_LIDespite differences in their morphology, mesh and thin strand pericytes participate in a common electrical syncytium along with the capillary endothelial cells both in rat and in human tissue. C_LIO_LISignal transmission at electrical synapses between pericyte-pericyte and pericyte-endothelial cell pairs is asymmetric, suggesting a preferred direction of propagation of electrical signals. C_LIO_LIActivation of A2a adenosine receptors and Kir channels mediate capillary hyperpolarization prior to the onset of seizures, which is followed by seizure-associated depolarization due to extracellular potassium accumulation. C_LI

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.