Fluids and Barriers of the CNS

Background: Cerebrospinal fluid (CSF) circulation is important for maintaining homeostasis of the central nervous system. Previous studies have largely focused on the ventricular system, the craniocervical junction, or local spinal segments, leaving the overall and spatially heterogeneous characteristics of CSF flow across the craniospinal axis insufficiently characterized. The spinal subarachnoid space (SAS) is often treated as a homogeneous annular compartment surrounding the spinal cord, an approach that may obscure directional differences among its internal regions. Methods: This single-center, exploratory, prospective imaging study enrolled 15 healthy volunteers. All participants underwent 3.0-T electrocardiography-gated two-dimensional cine phase-contrast magnetic resonance imaging (Cine PC-MRI) and high-resolution T2-weighted imaging. CSF was evaluated at the level of the cerebral aqueduct outlet/fourth-ventricle inlet, C1-C2, C5-C6, T5-T6, L1-L2, and the lumbar cistern. Region-of-interest (ROI)-based quantitative analysis using Q-Flow software recorded mean velocity, absolute peak velocity, and directional peak velocity. Results: Multiplanar Cine PC-MRI showed that CSF phase signals within the spinal SAS were not uniformly distributed but formed two principal flow regions, ventral and dorsal. Mean velocity and absolute peak velocity were similar between the ventral and dorsal regions, whereas directional peak velocity differed (1.50 +/- 2.98 cm/s vs. -0.38 +/- 3.23 cm/s, P = 0.036). High-resolution T2-weighted imaging showed denticulate ligaments, nerve roots, and associated fibrous connective tissue in the lateral transition zones between the two regions. Conclusions: In healthy adults, CSF flow in the spinal SAS was not synchronous motion within a single homogeneous compartment; rather, it showed longitudinal oscillatory flow in ventral and dorsal regions coupled to the cardiac cycle. These findings provide preliminary in vivo evidence for studies of CSF hydrodynamics across the craniospinal axis and an imaging basis for investigating CSF circulation disturbances in conditions such as hydrocephalus, Chiari malformation, syringomyelia, and arachnoid adhesions.

Hydrocephalus arises from pathological disturbances in cerebrospinal fluid (CSF) homeostasis, yet current treatment relies almost exclusively on neurosurgical diversion procedures that frequently require surgical revision. No specific and efficient pharmacological alternative is available as a complement to the invasive neurosurgery due to our lack of understanding of the molecular regulators of CSF secretion. By in vivo determination of CSF dynamics in rats and in vitro quantification of choroid plexus transporter activity, we demonstrate that the STE20-proline-alanine-rich kinase (SPAK) is a critical modulator of CSF secretion via its regulation of the Na+/K+/2Cl- cotransporter 1 (NKCC1) and the Na/K-ATPase. Systemic administration of a SPAK inhibitor after a mimicked hemorrhagic event attenuated posthemorrhagic hydrocephalus formation 24h post-hemorrhage. Activation of the choroid plexus transient receptor potential vanilloid 4 (TRPV4) ion channel induced hydrocephalus through CSF hypersecretion. This TRPV4-mediated hypersecretion occurred via activation of NKCC1, not the Na+/K+-ATPase, and required SPAK activity as a molecular link. Together, these findings identify SPAK as a central integrator of TRPV4-dependent signaling and choroid plexus transporter activity, positioning the TRPV4-SPAK axis as a potential pharmacological target for modulating CSF dynamics in hydrocephalus and other pressure-related pathologies.

Purpose In idiopathic normal pressure hydrocephalus (iNPH), longitudinal monitoring of ventricular size is important for diagnosis and treatment follow-up. This study aimed to validate a fully automated AI model for CT ventricular volumetry with subcompartments and to compare AI-derived volume changes with routine radiology assessments. Methods This retrospective, single-center study included 88 patients with iNPH and 456 non-contrast-enhanced head CT examinations. The model was trained on 38 manually labeled CT scans with 12 ventricular subcompartments. Outcomes included segmentation accuracy, correspondence between AI-derived longitudinal ventricular volume changes and radiology report categories (decreased, unchanged, increased), radiologist detection thresholds for ventricular change, and paired pre- and postoperative volume changes in 22 patients with ventriculoperitoneal shunt. Results Mean segmentation accuracy was high (Dice, 0.83). 91% of 100 segmentations were rated as excellent by an expert neuroradiologist. AI-derived ventricular volume changes corresponded well to radiology report categories (median total ventricular volume changes of -17% in cases reported as decreased, 0% in unchanged cases, and +22% in increased cases; all p < 0.001). Radiologists reported ventricular volume change in 50% of cases at an AI-measured relative volume change of +/-6%, and in 90% of cases at +21% for enlargement and -18% for decrease. After shunt placement, ventricular volume decreased by -8% (median), with the largest relative reductions observed in the right temporal and occipital horns. Conclusions Automated AI-based ventricular segmentation on CT enables accurate and reproducible assessment of ventricular volume changes in iNPH and complements routine radiological evaluation for longitudinal and postoperative monitoring.

BACKGROUND: Cerebral ischemia following subarachnoid hemorrhage (SAH) has traditionally been considered transient because functional alterations of the cerebral microcirculation are thought to be self-limiting. However, we identified a previously unrecognized vasculopathy, perivascular fibrosis of the cerebral microcirculation (PFCM), characterized by excessive type I collagen deposition after SAH. This study investigated the mechanisms underlying PFCM and its subsequent effects on cerebral hemodynamics. METHODS: In vivo SAH was modeled in mice by autologous blood injection, whereas oxygenated hemoglobin (OxyHb) exposure was used to mimic SAH in vitro. Pericyte-deficient mice (Pdgfr{beta}+/-) and pericyte-specific vestigial-like family member 3 (VGLL3) conditional knockout mice (Vgll3{Delta}PC) were generated. Pericyte contractility was measured by nanoindentation and traction force microscopy. Molecular mechanisms were examined using Western blotting, immunofluorescence, CUT&Tag, RNA-seq, transmission electron microscopy, and molecular docking. PFCM, impaired dilation of the cerebral microcirculation, and cerebral autoregulation were assessed by two-photon imaging, transcranial Doppler with continuous blood pressure monitoring, super-resolution ultrasound imaging, and photoacoustic imaging. RESULTS: After SAH, mice developed long-term cerebral autoregulation dysfunction marked by impaired dilation of the cerebral microcirculation, with the abnormality being most evident within the relatively lower blood pressure range. The marked reduction in PFCM in Pdgfr{beta}+/- mice indicated that pericytes were the principal cellular contributors. Mechanistically, OxyHb-induced cytoskeletal remodeling in vitro increased pericyte contractility and promoted nuclear translocation of SAH-upregulated VGLL3. This was followed by increased genomic occupancy, Col1a1 transcriptional activation, and type I collagen deposition. Pericyte-specific VGLL3 knockout abolished PFCM and, consequently, significantly alleviated long-term cerebral autoregulation dysfunction. CONCLUSIONS: Our findings identify PFCM mediated by pericytic VGLL3 as a novel vasculopathy leading to long-term cerebral autoregulation dysfunction after SAH.

Chiari I malformation (CM-I) is conventionally defined by cerebellar tonsil position, yet tonsil position is an indirect surrogate for the anatomic obstruction that impairs cerebrospinal fluid (CSF) flow across the craniocervical junction (CCJ). We hypothesized that CCJ crowding, quantified as subarachnoid space narrowing at the foramen magnum and C1, would explain CSF flow impairment more directly than tonsil position. Using non-invasive low b-value diffusion-weighted MRI (low-b dMRI), we quantified effective CSF motility, indexed by mean pseudo diffusivity (M{psi}), across the upper cervical spine, CCJ, and posterior fossa. Voxel-wise CCJ CSF pseudo-diffusion spatial statistics were integrated with CSF Waterways atlas-based regional analyses. We applied this approach in 81 pediatric and adult participants with CM I to determine how CCJ structural features shape regional CSF dynamics and clinical outcomes. Voxel wise analyses revealed that crowding at the foramen magnum was the dominant structural determinant of reduced intracranial CSF effective motility across the CCJ, basilar cisterns, and fourth ventricular outflow pathways (family-wise error corrected p < 0.05). While lower tonsil position and C1 level crowding were also associated with reduced CSF effective motility across the CCJ and fourth ventricular outflow pathways, but their associations within the basilar cisterns were spatially restricted to regions adjacent to the Liliequist membrane. Atlas-based region-of-interest analyses confirmed that greater foramen magnum crowding was associated with lower M{psi} across multiple basilar cisterns, but with higher M{psi} in the ventral spinal CSF compartment. Mediation analyses indicated that CCJ crowding at the foramen magnum and C1 accounted for the majority of the relationship between tonsil position and reduced CSF motility in the basilar cisterns. Multivariate M{psi} profiles across the CSF regions identified data-driven foramen magnum crowding thresholds of 69.5% and 77.5%, stratifying patients into mild, moderate, and severe physiological crowding groups. Exploratory analyses linked lower pre-operative CSF M{psi} to greater pain-related functional impairment, reduced cognitive function, and a higher likelihood of subsequent decompression surgery. Together, these findings demonstrate that CCJ crowding, particularly at the foramen magnum, exerts a quantifiable, region specific impact on CSF effective motility in CM I, and that low b dMRI provides a sensitive, complementary marker of CSF flow impairment. This integrative CCJ structural and CSF flow imaging framework establishes a mechanistic link between CCJ anatomy, CSF dynamics, and symptom burden, offering a scalable tool for phenotyping CM I and informing clinical decision making.

Objective: Shunt-dependent hydrocephalus is a common and costly complication of aneurysmal subarachnoid hemorrhage (aSAH), affecting up to 28% of survivors. Existing prediction tools, including the Chronic Hydrocephalus Ensuing from SAH Score (CHESS), have limited discriminative accuracy. We developed the CHECKMATE score, a clinically practical tool to improve prediction of ventriculoperitoneal shunt dependency after aSAH. Methods: In this multicenter retrospective cohort of 486 patients with aSAH from Mayo Clinic (January 1, 2006-December 31, 2021), we used multivariable logistic regression and machine learning to identify independent predictors of ventriculoperitoneal shunt placement. The CHECKMATE score was derived from 5 weighted variables: symptomatic hydrocephalus (10 points), intraventricular hemorrhage (5 points), SAH volume greater than 10 mL (3 points), neutrophil-to-lymphocyte ratio greater than 12 (2 points), and 10-year incremental age thresholds starting at older than 60 years (1 point each). Results: Of 486 patients (mean age, 56.3 years; 64.6% female), 137 (28.2%) required ventriculoperitoneal shunt placement. The CHECKMATE score achieved an area under the curve of 0.808 (compared to 0.737 for CHESS), with a sensitivity of 0.85, specificity of 0.67, and negative predictive value of 0.92 at the optimal cutoff of 14 points. Conclusions: The CHECKMATE score outperforms CHESS for predicting ventriculoperitoneal shunt dependency after aSAH and is easily used at the bedside. Its high negative predictive value helps identify low-risk patients who may benefit from earlier external ventricular drain weaning and shorter hospital stays.

Cerebrospinal fluid (CSF) provides a specialized extracellular environment for the central nervous system, which is predominantly produced by the choroid plexus, a highly vascularized epithelial structure whose ion transport processes are fundamental to CSF secretion, composition, and homeostasis. The mechanisms of Na+ entry into choroid plexus epithelial cells (CPECs) from the interstitial side remain disputed. The slc4a10 gene product encoding the Na+-dependent Cl-/HCO3- exchanger, Ncbe, was suggested as a key transport mechanism based on its impact on the cell's Na+-dependent regulation of intracellular pH and its basolateral membrane expression. The current study was undertaken to directly assess the contribution of Ncbe to the Na+ uptake into CPECs. Intracellular Na+ was recorded by fluorometry using the Na+ probe Sodium Binding Fluorescent Indicator in clusters of CPECs with access to both the luminal and basolateral membranes. Removal of extracellular Na+ reduced the apparent ex vivo intracellular [Na+] to ~5 mM from a baseline of ~43 mM in the absence of CO2/HCO3- and ~54 mM in the presence of CO2/HCO3-. Flame photometry estimated the intracellular [Na+] ex vivo to ~28 mM. The CO2/HCO3--dependent rate of [Na+] recovery amounted to ~53% of the total recovery rate upon re-addition of Na+. Experiments with access to only the luminal membrane show a [Na+] recovery of a similar rate as observed in the absence of CO2/HCO3- in the clusters. The CO2/HCO3--independent [Na+] recovery was inhibited to ~50% by the NKCC1 inhibitor bumetanide and to ~30% by the TRPv4 inhibitor RN1734. NHE contributed to a minor extent to the CO2/HCO3--independent transport. The HCO3- transport inhibitor DIDS, however, inhibited the total [Na+] recovery rate to ~50%, indicating a role for Ncbe rather than NBCn1 in the cellular [Na+] recovery. Indeed, docking of DIDS into Ncbe and NBCn1 indicated that both proteins can accommodate the binding of DIDS. However, the orientation of the DIDS poses in Ncbe suggests a binding mode more similar to that found in the Anion Exchangers (SLC4A1-3), which seems to accommodate the covalent-type docking more than NBCn1. The Ncbe inhibition by DIDS was supported by the rate of [Na+] recovery that was significantly higher in CPECs from Ncbe-wt than Ncbe-ko mice in the presence of CO2/HCO3-. As both NKCC1 and TRPv4 are localized to the luminal membrane, the findings collectively suggest that Ncbe is the most prominent mechanism for Na+ entry into CPECs expressed at the basolateral side. We suggest Ncbe as the rate-limiting mechanism in the vectorial Na+ transport driving CSF secretion.

Introduction: Anesthesia is known to modulate glymphatic clearance and cerebrospinal fluid (CSF) transport in rodents, but how these effects translate to a larger, gyrencephalic brain is unknown. With its anatomical similarity to the human brain, the pig offers a valuable translational model for examining anesthesia-dependent CSF-to-brain transport. Methods: We used dynamic in vivo SPECT/CT imaging for six hours following cisterna magna injection of [99mTc]-DTPA to quantify CSF-to-brain tracer transport in pigs under two anesthesia regimens: ketamine/dexmedetomidine (K/D, n=5) which previously has been shown in rodents to enhance glymphatic influx relative to GABAergic anesthesia, and propofol (PRO, n=5). Brain and CSF spaces were delineated using a data-driven non-negative matrix factorization approach, and tracer kinetics were quantified using a one-tissue compartment model. Results: Brain influx could be stably estimated from 2 hours post-injection. Hierarchical sub-division of the brain parenchyma identified two kinetically distinct components with different anatomical distributions: a surface component, located ventrally and within the interhemispheric fissure, showed faster kinetics than the anatomically deeper and lateral-dorsal component. Consistent with rodent findings, K/D-anesthetized pigs showed 62% (p=0.002) greater brain tracer accumulation than PRO-anesthetized pigs. However, while the brain influx rates did not differ substantially (p=0.047), a 52% higher cumulative CSF tracer concentration (p=0.047) could account for most of the difference by providing greater tracer availability for brain entry. Conclusions: In the larger gyrencephalic pig brain, we found higher brain tracer accumulation under K/D anesthesia compared to PRO anesthesia. A significant portion of this difference is readily explained by higher CSF retention, likely driven by a slower CSF turnover. This underscores the necessity of dynamic CSF tracer concentration measurements when assessing CSF-brain influx, a factor we suggest that future glymphatic studies should take into account.

In the brain, the water channel Aquaporin 4 (AQP4) is largely restricted to astrocytes, enriched at perivascular astrocytic processes, and involved in fluid balance and neurological disease. The perivascular pool is functionally unique as it is depolarized in diseases, targeted by neuromyelitis optica autoantibodies, and implicated in the clearance of brain metabolites such as amyloid beta. The perivascular AQP4 was recently shown to be AQP4X, an extended isoform arising from readthrough, where approximately 20% of translating ribosomes continue through the Aqp4 stop codon. Because Aqp4 terminates with a UGA, here we tested whether Aqp4 readthrough can be enhanced by 2,6-diaminopurine, a purine analog known to promote UGA decoding. Using dual luciferase and immunoblotting for AQP4X in cultured cells, we show that the drug promotes Aqp4 readthrough and increases AQP4X levels. For in vivo validation, we use the wild-type mouse as well as a genetically engineered mouse with a stop-to-sense mutation allowing Aqp4 readthrough at 100%. 2,6-diaminopurine elevates perivascular AQP4X levels by approximately 10% after 20 hours of single intracranial injection. It fails to enhance AQP4X levels in animals with stop-to-sense mutation, suggesting its action involves Aqp4 readthrough. Aqp4 readthrough can be a pharmacological target for modulating perivascular AQP4X in neurological disease models.

Human iPSC-derived brain organoids are revolutionizing tools to study layers biology, synergize disease modeling, and accelerate therapeutic discoveries that overcome obstacles in monolayer cell culture or animal models. The neurovascular unit including vasculature and microglia is critical for brain development, maintenance of synaptic plasticity and neural activity, and the high metabolic demands of long-term culture. We present a methodology to incorporate these important components during organoid generation and discuss potential approach, aiming consistent production of vascularized organoids for longitudinal study. We also demonstrate that this vascularized organoid is a versatile platform to model brain cancer and traumatic brain injury.

BackgroundFlow diversion stent treatment of cerebral aneurysms has demonstrated high rates of aneurysm occlusion and long-term durability. However, complete endothelization of the flow diverting stent is required for parent vessel healing which closes the aneurysm and metal stent from the circulation. Therefore, stent coatings which enhance endothelial migration and attachment may increase the rate of aneurysm occlusion and reduce complications associated with flow diversion treatment. Here we investigate the use of gelatin as a stent surface coating to enhance the rate of stent endothelialization and coverage and increase aneurysm healing. MethodsNitinol- Neuroform stents (Stryker, Kalamazoo, MI) and cobalt-chromium-Pipeline Flex flow diverting stents (Medtronic, Minneapolis, MN) were used for this study. The stents were coated with gelatin and endothelial cell attachment, proliferation and stent coverage were determined in vitro and compared to uncoated stent controls. A rabbit elastase-aneurysm model was used to determine the effects of endothelial cell seeded-gelatin coated flow diverting stents on aneurysm obliteration and parent vessel healing. ResultsIn vitro, gelatin coating of nitinol stents did not significantly alter endothelial cell attachment, proliferation, or stent coverage. However, gelatin coating of cobalt-chromium stents significantly increased endothelial cell attachment, proliferation and migration. In fact, gelatin coating significantly (p< 0.001) increased the rate of complete stent endothelization by 33% compared to unmodified controls. In vivo, treating aneurysm with endothelial cell seeded-gelatin coated stents resulted in aneurysm occlusion in 8 of 8 (100%) rabbit aneurysms at 90 days compared to only 4 of 7 (57%) in unmodified controls (p< 0.001). Histologically, there were trends in increased neoarterial wall thickness across the aneurysm neck and neointimal formation in the parent artery. Angiographic assessment demonstrated strong parent and side branch patency. ConclusionsGelatin coating enhances EC attachment and stent coverage which is dependent upon the type of stent. Endothelial cell seeded-gelatin coated-flow diverting stents allowed 100% aneurysm obliteration and neoarterial formation without affecting side branch patency or parent artery perfusion. Gelatin coating therefore represents a valuable strategy to enhance stent cellularization and aneurysm occlusion rates.

Maternal thermal strain is associated with adverse pregnancy outcomes, yet fetal temperatures cannot currently be directly measured, limiting quantification of fetal thermal strain. Here, we develop two steady-state models for estimating internal temperatures in a near-term fetus. First, we improve the only previously published human fetal thermoregulation model, deriving a closed-form solution within its simplified uniform-cylinder representation. Second, we introduce a multilayer, anatomically segmented model that resolves tissue-specific temperatures. Both couple the fetal body to central blood pool and amniotic fluid compartments and incorporate a new placenta-umbilical cord heat-exchanger representation. Predictions agree with available intrauterine scalp measurements, with fetal core and head-center temperatures approximately 0.5{degrees}C and 0.8{degrees}C above maternal core, respectively. Physiologically plausible changes in umbilical cord heat-exchanger effectiveness or blood flow increased fetal temperatures by approximately 0.3{degrees}C. These models enable estimation of otherwise inaccessible temperatures, while the multilayer formulation lays a foundation for transient, coupled maternal-fetal thermoregulation modeling.

Despite major advances in the study of cerebellar neurogenesis, cerebellar angiogenesis during embryogenesis remains poorly described. Recent advances in tissue clearing, light-sheet microscopy, and artificial intelligence have increasingly enabled detailed 3D modelling of cerebellar vasculature at early developmental stages. Here, vascular networks in mouse embryos from E11 to birth (P0) were labelled with podocalyxin, SMA, and PECAM-1 antibodies together with the nuclear marker TO-PRO-3 iodide, cleared, imaged by light-sheet microscopy, and finally modelled and quantitatively analyzed using Imaris and VesselVio. Our mapping reveals that the three main paired cerebellar arteries--the superior (SCA), anterior inferior (AICA), and posterior inferior (PICA) cerebellar arteries--emerge sequentially between E11 and E13 and display significant topographical variability comparable to that observed in humans. Morphometric analysis demonstrates distinct developmental dynamics, with SCA growth proportional to cerebellar expansion, whereas the AICA and PICA exhibit accelerated extension during later embryonic stages. Interestingly, the PICA does not reach the cerebellum before birth, highlighting the question of its contribution to embryonic cerebellar vascularization. The intrinsic vascular network evolves from a rudimentary bilayer at E11 into a highly branched architecture organized around radial penetrating vessels, giving rise to collaterals that progressively colonized the cerebellar parenchyma during foliation and lobulation. These vascular changes temporally coincided with the successive stages of cerebellar neurogenesis, supporting an interplay between vascular and neuronal development. Together, our findings provide the first spatio-temporal three-dimensional atlas of cerebellar vascularization during mouse embryogenesis, establishing a reference framework for investigating cerebellar angiogenesis in developmental and pathological conditions. HighlightsO_LIThis work is the first 3D modelling of the cerebellar vasculature in mouse embryo. C_LIO_LISCA, AICA, PICA develop through distinct spatial and temporal growth programs. C_LIO_LIPICA does not contribute to cerebellar vascularization before birth. C_LIO_LIThe intra-cerebellar vascularization evolves at E11 from a simple vessel bilayer. C_LIO_LIBetween E13 and P0, radial vessels form collaterals colonizing cerebellum. C_LIO_LIThe vascular changes temporally coincided with cerebellar neurogenesis. C_LI

Background: The hydrodynamic model of hydrocephalus proposes that ventriculomegaly is driven by exaggerated intraventricular pulsations rather than impaired CSF circulation alone. Under this model, endoscopic third ventriculostomy with choroid plexus cauterization (ETV/CPC) treats hydrocephalus by creating a pulsation absorber and by reducing a primary source of intraventricular pulsation. However, direct intraoperative human evidence supporting this two-step mechanism is lacking. This study aimed to test the hypothesis that ETV followed by CPC would produce measurable, stepwise decreases in mean intraventricular pressure (ICP) and pulsation amplitude in infants with hydrocephalus. Methods: This single-institution proof-of-concept study included infants with symptomatic hydrocephalus undergoing ETV/CPC as the first definitive treatment. A fiber-optic ICP sensor was attached to the operative ventriculoscope and passively recorded mean and pulsatile ICP (pulsation amplitude) throughout the procedure. Longitudinal brain parenchymal volume (BPV) and cerebrospinal fluid volume (CSFV) were obtained through segmentation of clinically acquired T2-weighted MRI and converted to age- and sex-matched z-scores. All patients were followed for a minimum of 6 months postoperatively. Results: Five infants (median corrected age at ETV/CPC 8 months) were included. No surgical complications occurred, and no ETV/CPC failures were observed during follow-up. Overall, mean ICP decreased by 56-97% after the combined procedure in four patients. In three patients (Patients 1, 3, and 5), both mean ICP and pulsation amplitude decreased stepwise following ETV and then CPC, consistent with the hypothesized therapeutic mechanism. Patient 4 demonstrated a large reduction in mean ICP after ETV with minimal additional effect from CPC and no significant change in pulsation amplitude. Patient 2 demonstrated neither a reduction in mean ICP nor a meaningful change in pulsation amplitude after either procedure; this patient also had a delayed and atypical clinical response. Intracranial segmentation demonstrated BPV z-score stabilization within normal range and CSFV plateau in all patients after surgery. Conclusions: This proof-of-concept study provides the first direct intraoperative human evidence supporting the hydrodynamic mechanism of ETV/CPC in a subset of infant with hydrocephalus. Our findings suggest that determination of intraoperative ICP parameters is feasible, safe and might ultimately prove helpful in improving patient selection for ETV/CPC, warranting further investigation in larger cohorts.

The Notch signaling pathway plays a central role in development and cell fate determination. Its function depends on tightly regulated intracellular trafficking of the Notch receptor and the Notch intracellular domain (NICD) after cleavage by {gamma}-secretase. Notch signaling is essential for principal cell differentiation within the renal collecting duct and for proximal-distal patterning during kidney development. Notch activity has also been shown to influence the trafficking of several membrane proteins, including nephrin in kidney cells and monocarboxylate transporter 1 in brain endothelial cells. Aquaporin-2 (AQP2) is the key vasopressin-regulated water channel in the collecting duct, and proper AQP2 trafficking and recycling are required for physiologically appropriate urine concentration. To determine whether and, if so, how Notch signaling modulates AQP2 trafficking, we performed studies using LLCPK1 renal epithelial cells stably expressing AQP2 (LLCPK1-AQP2). Exposing cells to 35 M DAPT (which inhibits y-secretase, preventing cleavage and activation of Notch receptor signaling) for 30 min significantly increased AQP2 membrane accumulation in LLCPK1-AQP2 cells as revealed by immunofluorescence staining. Using a rhodamine-transferrin internalization assay, we found that DAPT reduced clathrin-mediated endocytosis by 60%. This blockade increases AQP2 membrane accumulation by preventing the reinternalization of AQP2 that is delivered to the plasma membrane by exocytosis during its constitutive recycling pathway. Using an F-actin polymerization assay, we then found that Notch inhibition decreases F-actin polymerization by de-activating the small GTPase RhoA, using GSTRBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Because actin polymerization is required for AQP2 endocytosis, RhoA inhibition by DAPT would result in the decreased internalization of AQP2 that we observed by immunofluorescence. While the mechanism by which DAPT inhibits RhoA activity remains to be determined, our study shows that AQP2 trafficking is regulated by the Notch signaling pathway in vitro and suggests that modulation of Notch signaling may represent a novel strategy to address water balance disorders that involve defects in the AQP2 trafficking process.

Ependymal (E1) cells, with their tufts of [~]50 motile cilia, line the walls of the brain ventricles and help propel the cerebrospinal fluid (CSF). The CSF is rich in signaling molecules, but the cellular targets that detect these signals and their function remain unknown. Here, we describe a distinct population of ependymal cells (E2) in the forebrain of mice and humans, the majority having only 1 or 2 cilia. These cilia were motile, but unlike E1 cells cilia, their pattern of motility and high expression of Arl13b and Inpp5e suggest a sensory function. E2 cells were characterized by an enormous, donut-like basal body that contained an increased number and size of subdistal appendages. In mice, E2 cells were mostly born in the embryo, but completed their differentiation in juveniles and young adults; they were found at higher densities in regions of high CSF flow and neurogenesis. E2 cilia contained the G protein-coupled receptor Smoothened, which accumulated in their cilia upon exposure to Sonic Hedgehog (Shh). Together, these findings identify E2 cells as a novel CSF-sensing ependymal cell type and provide a cellular target for the CSF signaling.

Objectives To validate two computational fluid dynamics (CFD) models derived from computed tomography angiography (CTA) for estimating trans-stenotic pressure gradients, using invasive intra-arterial pressure measurements as the reference standard in patients with renal artery stenosis (RAS). Background We assessed whether non-invasive assessment of the pressure gradient using CFD could be a reliable alternative to intra-arterial measurements for identifying hemodynamically significant RAS. Methods We performed intra-arterial measurements at rest and during dopamine-induced hyperemia to assess the trans-stenotic pressure gradient in 28 patients with RAS. A pre-intervention CTA scan was used to simulate the pressure gradient with a CFD model using a strategy based on Murray's law (CFD-Mu) and cortical volume (CFD-C). The agreement between the simulated and measured pressure gradients was assessed using intraclass correlation coefficients (ICC), Bland-Altman analysis and diagnostic agreement on the presence of a hemodynamically significant stenosis. Results In 20 patients, successful measurements and simulations were obtained. The ICC between measured pressure gradient and the CFD pressure gradient was 0.78 and 0.94 during baseline and 0.86 and 0.72 during hyperemia, for CFD-Mu and CFD-C, respectively. The sensitivity of CFD-Mu and CFD-C was 70% for both models at rest and 100% compared to the hyperemic measurements, whereas the specificity was 90% and 70% at rest and 79% and 72% during hyperemia, respectively. Conclusions The results support the use of individualized CFD simulations for hemodynamic assessment of RAS using CTA as input. The CFD models demonstrated high accuracy for the identification of a hemodynamically significant stenosis.

BackgroundOwing to their potent immunomodulatory properties, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (EVs) have emerged as promising neuroprotective treatments for ischemic stroke. Preclinical studies using MSC-EVs have mainly been performed in young, otherwise healthy rodents. Stroke patients frequently carry vascular risk factors and comorbidities. We herein investigated whether MSC-EVs retain neuroprotective activity in hyperlipidemic mice on cholesterol-rich Western diet. MethodsMale C57BL/6J mice were exposed to regular normal diet or Western diet for 6 weeks. At the age of 9-10 weeks, mice were exposed to transient intraluminal middle cerebral artery occlusion (MCAO). Vehicle or MSC-EVs (2x106 or 6x106 cell equivalents) were intravenously administered immediately after reperfusion, and vehicle or rosuvastatin (5 mg/kg/day) were intraperitoneally applied starting immediately after or seven days before MCAO. Neurological deficits, ischemic injury, and immune responses were evaluated up to 72 hours post-ischemia. To investigate the hyperlipidemia-associated immune dysregulation, mice received DNase-I before or immediately after MCAO. In defined subgroups, monocytes/ macrophages or neutrophils were additionally depleted by clodronate liposomes or anti-Ly6G antibodies, respectively. ResultsIn contrast to normolipidemic control mice, MSC-EVs failed to induce post-ischemic neuroprotection in hyperlipidemic mice. Neither MSC-EV dose escalation nor rosuvastatin co-treatment restored the therapeutic efficacy of MSC-EVs. Hyperlipidemia induced systemic innate immune dysregulation characterized by reduced monocyte/ macrophage activation, increased neutrophil activation, and elevated circulating cell-free DNA. DNase-I treatment before, but not after MCAO reversed these immune abnormalities and restored neuroprotection by MSC-EVs, decreasing neurological deficits, infarct volume and brain edema. Depletion of either monocytes/ macrophages or neutrophils abolished the neuroprotective effects of MSC-EVs in DNase-I-pretreated hyperlipidemic mice. ConclusionsImmune dysregulation abolishes MSC-EV-induced neuroprotection after ischemic stroke in hyperlipidemic mice. DNase-I priming restores MSC-EV responsiveness through mechanisms critically involving monocyte/ macrophage and neutrophil rebalancing. Our data highlight the host immune status as determinant of EV therapeutic efficacy.

Computational models coupling one-dimensional vascular networks with three-dimensional tissue domains are widely used for predicting blood flow distribution in tumor perfusion, drug delivery, and therapeutic planning. Two prominent coupling paradigms have emerged: the Lateral Average Model (LAM) which implements distributed transmural exchange via a vessel wall conductivity parameter{gamma} (m Pa-1 s-1), and the Sphere of Influence (SOI) model, which employs localized terminal coupling via a source sphere radius{varepsilon} (m). Despite their broad application, systematic quantitative comparisons of their parametric behavior and predictive equivalence remain lacking. We compare LAM and SOI in 3D-1D simulations on a benchmark vascular network and a porcine liver study with a hepatic arterial network reconstructed from CT arteriography. Across a benchmark vascular network under three sink configurations, the LAM net flow rate rose smoothly with{gamma} and saturated at a plateau, while the SOI net flow rate increased with{varepsilon} without saturating; as a result, global-flow equivalence between the two formulations exists only for particular boundary geometries, and not at all within the tested parameter range for one of the three configurations examined. Despite this partial agreement in total flow, the two models diverged substantially in regional perfusion: in a porcine hepatic arterial network reconstructed from CT arteriography, SOI predicted stable perfusion fractions to two regions of interest across its full tested parameter range, whereas LAM predictions for the same regions varied several-fold with vessel wall permeability and, at low permeability, could invert which region received more flow. These results indicate that the choice of coupling model has limited consequence for predicted total organ flow but substantial consequence for predicted local drug delivery, and we provide guidance for selecting between the two formulations depending on the clinical or research question being asked. Author SummaryWhen doctors plan treatments for liver cancer, they often rely on computer simulations to predict how blood flows through the liver and how well a drug will reach the tumor. These simulations depend on mathematical models that describe how blood moves from vessels into surrounding tissue. Two commonly used approaches exist for building these models, but researchers have generally chosen between them based on habit or convenience rather than on a principled understanding of how their predictions differ. In this work, we directly compared these two approaches, one that spreads blood exchange continuously along the vessel wall, and one that delivers blood from the vessel tips into a surrounding spherical zone, using both a simple test network and a realistic pig liver reconstructed from medical imaging. We found that the two approaches can agree on the total amount of blood reaching the liver, but disagree substantially on where that blood goes within the tissue. This distinction matters enormously for treatment planning: a model that predicts the right total blood flow but delivers it to the wrong region of the liver could lead to an inaccurate forecast of drug concentration at the tumor site. Our results provide practical guidance for researchers on which approach to use depending on what information is available and what question is being asked.

Background and aims Model systems for bile duct disorders are needed for testing therapeutic interventions. Current models have poor human relevance or limited potential for recreating the complex bile duct microenvironment at scale. We aimed to generate a humanized microphysiological system to model and treat cholangiopathies. Methods An in vitro bile duct was created using 3D printed microfluidic chips containing a collagen-embedded canal seeded with patient-derived primary human cholangiocytes. Barrier permeability and compound transport across the epithelium was measured, and disruption of the barrier was performed with lipopolysaccharide treatment. The duct was challenged with the known hepatotoxicant Chlorpromazine. Biliatresone was used to model biliary-atresia and treated using N-acetyl-L-cysteine. Results Cholangiocytes in the bile duct chip established a tight, polarized epithelial barrier. Verapamil and Linerixibat inhibited transport of rhodamine 123 and cholyl-lys-fluorescein respectively with 66 % (p = 0.0004) and 57 % (p = 0.03) reduction. 10 g/mL lipopolysaccharide led to a loss of epithelial barrier integrity, measured by an increase of over 1000 % in leakage of both 3 kDa (p = 0.0002) and 10 kDa dextran (p = 0.0001) along with upregulation of cytokines. Chlorpromazine displayed dose-dependent toxicity with EC50 values of 84, 140 and 96 M for three patient lines. Biliatresone induced a dose-dependent abnormal phenotype with loss of viability. The induced phenotype could be treated with N-acetyl-L-cysteine, improving viability from 23 % to 59 % (p < 0.0001) with treatment of 2 g/mL Biliatresone. Conclusions Our novel platform allows complex studies of bile duct biology, testing of off-target effects from drugs and treatment of a disease phenotype.