Stroke is the leading cause of disability-affected life years in China, posing a great social hazard. Diabetes is a chronic disease that seriously affects human health that can aggravate cerebral ischemia - reperfusion injury. Oxidative stress is the main mechanism through which hyperglycemia aggravates cerebral ischemia - reperfusion injury. Our aim is to investigate whether incretins (GLP-1 and GIP) can exert neuroprotective effects by activating the Nrf2/ARE pathway to reduce oxidative stress, and to compare the efficacy of the single GLP-1- receptor liraglutide and the dual GLP-1/GIP receptor agonist DA3-CH. Methods and Results: Male Sprague-Dawley rats were randomly divided into 4 groups and treated with STZ and with either saline, liraglutide or with DA3-CH (10mmol/kg, once-daily ip. for 14 days). Thereafter, rats underwent middle cerebral artery occlusion followed by 24-h reperfusion. The drugs reduced blood glucose levels, decreased the infarct area, and lowered the neurological deficit score. They furthermore mitigated oxidative stress damage, enhanced the ability of brain tissue to scavenge ROS, and increased the expression of proteins in the Nrf2/ARE signaling pathway, including Nrf2, HO-1, and NQO1.Conclusions: The drugs inhibit oxidative stress through activating the Nrf2/ARE pathway. Overall, DA3-CH was superior to liraglutide.
Brain clearance pathways via the interstitial fluid and cerebrospinal fluid are essential for maintaining homeostasis and removing metabolic waste. Disruptions in these processes are linked to neurodegenerative diseases, particularly Alzheimer’s Disease (AD), where an imbalance between amyloid β (Aβ) production and clearance leads to cerebral amyloidosis. Although AD research continues to rely heavily on murine models, including APPswe/PS1dE9 mice, it remains unclear to what extent these models replicate the deficits in brain clearance observed in human AD. We examined AD pathology in transgenic APPswe/PS1dE9 (tg) mice and wildtype (wt) littermates at six, nine and twelve months using immunohistochemistry for Aβ plaques (6E10+) and activated microglia (Iba1+). To assess integrative parameters contributing to brain clearance, we applied two complementary approaches. First, we performed daytime-dependent intrahippocampal injections of a fluorescent tracer in six-month-old tg and wt mice, followed by histological analysis of tracer dispersion 1 h and 3 h after injection. Second, we present a novel, longitudinal and non-invasive approach utilizing washout kinetics of intravenously administered gadolinium-based contrast agents (GBCA) as an integrative proxy to assess brain clearance dynamics. In this novel approach DOTAREM® was administered intravenously and imaging was done with anesthetized tg and wt mice at nine and twelve months of age. Integrity of the blood-brain-barrier was further evaluated via western blot analysis of occludin protein expression. Despite tg mice displayed characteristic AD-like phenotype, with significant, age-dependent Aβ plaque deposition and neuroinflammation no significant differences in the integrative GBCA washout kinetics were detected between wt and tg mice, suggesting that no major deficits in the combined clearance pathways are present. However, contrast-agent uptake showed a general age-dependent increase consistent with aging-related changes known from human studies. Our findings indicate that the APPswe/PS1dE9 mouse model does not exhibit evidence of major integrative brain clearance impairment, despite this being a hallmark of sporadic AD in humans. These results suggest limitations of this model for studying clearance-related mechanisms of late-onset AD, which should be acknowledged when planning future studies using this murine model.
Aging is associated with a decline in blood-brain barrier (BBB) integrity. Interestingly, recent reports suggest that females may be more resistant to BBB disruption than males. Using a robust model of hypoxia-induced BBB disruption, we exposed aged (20 months) male and female mice to chronic mild hypoxia (CMH; 8
The development of tubular organs relies on the presence of a transient luminal extracellular matrix within their internal cavities, which regulates both tubular size and morphogenesis. In the neural tube, the brain cavities are filled with the embryonic cerebrospinal fluid (eCSF), a protein-rich fluid known for its trophic and mechanical roles in neuroepithelial development. However, the molecular organization of the eCSF and the mechanisms coordinating its morphogenetic signaling remain poorly understood. Among the proteins detected in the eCSF, SCO-spondin stands out as a giant glycoprotein containing multiple matricellular domains that mediate interactions with different molecules, as well as domains associated with self-assembly. In this study, we investigated whether the eCSF exhibits a defined structural organization and sought to characterize its molecular components and associated interactions, with special emphasis on SCO-spondin. We examined the structural and molecular composition of eCSF in chick embryos using histochemistry, immunohistochemistry, light-sheet microscopy, and scanning electron microscopy. Co-immunoprecipitation followed by tandem mass spectrometry was used to identify molecular interactors of SCO-spondin. Our findings reveal that the eCSF is not a homogeneous fluid but rather an interlaced, sponge-like fibrillar mesh functioning as a luminal extracellular matrix intimately associated with the neuroepithelium. This network comprises SCO-spondin, fibronectin, proteoglycans, and lipophilic aggregates, forming a porous scaffold that may contribute to the retention of fluid and signaling molecules. Histochemical and immunohistochemical analyses of the isolated matrix indicate that it is acellular and glycosylated, with SCO-spondin and fibronectin among its main components. Proteomic analysis identified 74 SCO-spondin interactors, including morphogen carriers such as retinol-binding protein, transthyretin, riboflavin-binding protein, as well as lipoproteins, extracellular vesicles, and structural proteins. These findings suggest that, at early developmental stages, SCO-spondin is associated with macromolecular complexes involved in molecular transport and extracellular organization within the brain cavities. Our findings redefine eCSF as a structured supramolecular network rather than a simple fluid. This network may contribute to the spatial organization of molecular components within the ventricular cavities and their interaction with the neuroepithelium. We propose that the eCSF represents a structured and dynamic luminal extracellular matrix that may play an important role in brain morphogenesis, with SCO-spondin as a key organizing component.
The blood–brain barrier (BBB) prevents most therapeutics from reaching the brain, and the functionally coupled glymphatic system governs solute clearance. A controlled, non-invasive means of modulating these systems would have diagnostic and therapeutic value. We reviewed whether transcranial magnetic stimulation (TMS) affects BBB permeability and glymphatic clearance. In this scoping review, PubMed and Embase were searched from 1985 to June 2026 for studies applying any form of TMS with outcomes relating to the BBB or glymphatic system, in human, animal, or in-vitro models. Direct permeability measures, glymphatic measures (including DTI-ALPS), and indirect serum markers were eligible. ClinicalTrials.gov was searched for ongoing trials. Forty-five studies were included and grouped into six categories by outcome system and direction of effect. Three main effects emerged. First: single-session, low-frequency rTMS at threshold or supra-threshold intensity produced immediate, focal, dose-dependent BBB opening that reversed within minutes to hours, facilitating delivery of small and large molecules. Second: repeated high-frequency, multi-session protocols were associated with barrier stabilisation and repair in injury models. Third, similar multi-session protocols were associated with enhanced glymphatic and meningeal-lymphatic clearance. Human evidence rested largely on indirect surrogates (ADC, DTI-ALPS, S100B, neuron-specific enolase), with only one study using sensitive contrast-based imaging to demonstrate opening. A further twelve studies using alternative magnetic platforms mapped the field’s conceptual boundary. TMS may induce controlled, transient, reversible BBB opening and longer-term protective and clearance effects, but human data remain limited, warranting dedicated validation and safety characterisation.
Prostate‑specific membrane antigen (PSMA) PET is increasingly used in glioma for tumor delineation and patient selection for PSMA‑targeted radioligand therapy. However, whether high tracer uptake reflects true target‑specific binding or non‑specific leakage across a disrupted blood-brain barrier (BBB) remains unresolved. This ambiguity limits the rational application of PSMA‑based theranostics in neuro‑oncology. We aimed to quantitatively separate these contributions using dynamic 68Ga‑PSMA‑11 PET with pharmacokinetic modeling. This prospective study enrolled 49 consecutive patients with suspected glioma (mean age 50 ± 13 years; 21 male). All underwent static 68Ga‑PSMA‑11 PET/MRI; 16 patients additionally underwent 40‑minute dynamic 68Ga‑PSMA‑11 PET/CT. Pharmacokinetic parameters (K1: blood‑to‑tissue transfer rate; k3: specific binding rate; Ki: net influx rate) were derived using two‑tissue compartment models (irreversible for tumors/parotid, reversible for normal brain). SUVmax was correlated with kinetic parameters and with histopathologic PSMA expression (H‑score, immunoblotting) in spatially matched samples. In BBB‑intact normal brain, K1 and Ki were near zero. In gliomas, both parameters were significantly elevated and correlated strongly with SUVmax (K1: r = 0.67, P = 0.006; Ki: r = 0.70, P = 0.003). In contrast, k3 did not reach statistical significance across tissues (P = 0.074) and showed no significant correlation with SUVmax (r = 0.24, P = 0.37). SUVmax correlated with PSMA expression only within contrast‑enhancing (BBB‑disrupted) intratumoral regions (r = 0.71, P = 0.01), not in non‑enhancing peritumoral tissue or whole‑lesion analysis. These findings indicate that BBB permeability, rather than specific binding rate, is the dominant correlate of PSMA PET signal intensity in glioma. Dynamic 68Ga‑PSMA‑11 PET enables quantitative separation of BBB permeability (K1) from specific target binding (k3) in gliomas. K1 serves as a non‑invasive, quantitative imaging biomarker of regional BBB integrity. Static SUVmax conflates delivery and binding and does not reliably reflect target availability. This “BBB‑gated” paradigm provides a mechanistic framework for interpreting PSMA PET signals in brain tumors and has direct implications for patient selection in PSMA‑targeted radioligand therapy. Chinese Clinical Trial Registry, ChiCTR2300076481. Registered 10 October 2023, https://www.chictr.org.cn.
Choroid plexus dysfunction has been proposed to reduce CSF production and impair brain solute clearance, but the in vivo consequences of sustained clearance failure for biomarker profiles and disease mechanisms remain poorly defined. We examined Skogholt’s disease, a rare, maternally inherited neurodegenerative syndrome as a natural human model to test whether primary choroid plexus failure can reshape CSF dynamics and biomarker profiles independently of overt neurodegeneration. Multimodal assessment of 40 individuals (mean age 57.8 years; 16 affected individuals and 24 controls) was used to evaluate clearance dynamics, diffusion properties, and CSF–plasma biomarker relationships, including model-free analysis of dynamic contrast-enhanced MRI washout of the choroid plexus. We observed convergent evidence for a choroid plexus-centred clearance defect. Affected individuals had markedly reduced choroid plexus volume (Cohen’s |d| = 2.64) and prolonged contrast washout (time-above-half-peak |d| = 4.9) without cerebral parenchymal blood–brain barrier disruption. Diffusion MRI showed widespread reductions in mean diffusivity (|d| ≈ 1.0 in white matter; 0.85–0.89 in cortex), opposite to changes typical of neurodegeneration. CSF tau, amyloid β species, β-trace, neurofilament light and glial fibrillary acidic protein were elevated up to 12-fold, whereas plasma levels were normal or reduced, indicating marked CSF–plasma dissociation. Directional trace-metal shifts suggested impaired ATP-dependent transport. Mitochondrial genome sequencing identified a rare homoplasmic MT-RNR2 (m.1681G > A) variant that segregates with disease and is absent as a homoplasmic variant in public mitochondrial reference datasets. The variant localises to a highly conserved structural element of the mitochondrial 16S ribosomal RNA, supporting its candidacy as a pathogenic variant pending functional validation. These findings provide in vivo evidence for choroid plexus dysfunction in a maternally inherited disorder with convergent evidence supporting a mitochondrial aetiology, associated with reduced CSF production and impaired blood–CSF barrier transport, substantially altering biomarker profiles in the absence of cortical atrophy or blood–brain barrier disruption. Elevated CSF but normal or low plasma neurodegeneration markers most parsimoniously reflect impaired CSF-to-blood clearance, highlighting a clearance-sensitive dimension of biomarker biology. Dynamic contrast-enhanced MRI choroid plexus washout and CSF–plasma coupling emerge as complementary in vivo markers of clearance capacity. This syndrome may represent a prototype of disorders defined by primary CSF clearance failure rather than neuronal degeneration.
Normal pressure hydrocephalus (NPH) is a cause of neurological impairment, with programmable shunt valves serving as the standard treatment. However, magnetic interference can result in altered pressure settings, affecting valve functionality. This systematic review investigates the effects of magnetic resonance imaging (MRI) on programmable shunt valves in NPH patients. Conducted in adherence to PRISMA guidelines, an extensive literature search across PubMed and Embase databases from January 2000 to January 2025, was performed. Studies evaluating the impact of MRI at 1.5T and 3T field strengths on valve settings were included. Valve reprogrammability and outcomes were analysed, with statistical comparisons across MRI strengths and valve types using Mann-Whitney U and ANOVA tests. The pooled analysis included 475 NPH patients across 11 studies. Valve setting alteration was required in 44.2
Recent discoveries focused on the role of intraspinal pressure (ISP) in metabolite clearance after spinal cord injury (SCI) have initiated intense research on CSF inflow and outflow pathways. This study aimed to investigate whether the pulsatile ISP wave serves as the primary driver of posttraumatic syringomyelia (PTS) formation and progression, and to determine if a novel subarachnoid‑subarachnoid (S‑S) bypass procedure can effectively attenuate this abnormal pulsatile ISP wave in affected PTS patients. In this prospective cohort study of patients with PTS, ISP across the injury site was monitored intraoperatively both before and after placement of the S-S bypass tube. Neurological impairment was assessed using standardized scales at baseline, one year postoperatively, and at the final follow-up. A logistic regression model was used to analyze prognostic factors associated with surgical outcomes. All 64 enrolled patients underwent S-S bypass surgery; 54 (84.4
The blood-brain barrier (BBB) functions as a highly selective regulatory system governing molecular transport between systemic circulation and the central nervous system. The maintenance of this strict permeability relies on the continuous, high-energy interactions of the neurovascular unit (NVU) primarily fueled by mitochondria. Sustaining the electrochemical gradients and active transport systems essential for this regulation imposes an immense metabolic burden, rendering the NVU uniquely susceptible to bioenergetic failure. This review critically evaluates the mechanistic link between mitochondrial dysfunction and specific, aberrant BBB phenotypes. We detail evidence surmising how mitochondrial dynamics within multiple NVU cell populations, including brain microvascular endothelial cells, pericytes, astrocytes and microglia, govern barrier stability. We specifically examine how mitochondrial reactive oxygen species (mtROS), aberrant fission/fusion cycles and impaired quality control precipitate tight junction disassembly and inflammatory activation. Furthermore, we explore the emerging paradigm of “mitoceuticals,” a class of therapeutics engineered to correct these bioenergetic deficits. By targeting molecular mechanisms such as mitoNEET stability and mitochondrial dynamics, these agents offer a unique strategic opportunity to preserve NVU function. We conclude by evaluating the potential of these agents to therapeutically reinforce the bioenergetic infrastructure of the NVU as a vital, yet under-explored, avenue for treating ischemic and neurodegenerative disorders.
Blood-brain barrier integrity is essential for central nervous system homeostasis, yet the mechanisms underlying ultra-acute barrier dysfunction after abrupt hemodynamic changes remain incompletely understood. Piezo1 is a mechanosensitive Ca²⁺ channel expressed in endothelial cells and activated by mechanical stimuli such as shear stress, but its contribution to rapid brain endothelial barrier disruption remains unclear. We investigated whether Piezo1 activation induces an ultra-acute disruption of the endothelial barrier and explored the underlying signaling mechanisms. Human brain microvascular endothelial cells (HBEC-5i) were stimulated with the Piezo1 agonist Yoda1 in the presence or absence of the Src inhibitor saracatinib, followed by barrier-function assessment and phosphoproteomic profiling. To assess in vivo relevance, vascular permeability was evaluated within minutes after reperfusion in a mouse cerebral ischemia–reperfusion model treated with the mechanosensitive channel inhibitor GsMTx4. Comprehensive phosphoproteomic profiling after Piezo1 activation revealed enrichment of phosphorylation events associated with cell junctions, adhesion, and cytoskeletal organization. Yoda1 induced rapid phosphorylation of junction-associated proteins, including occludin at Tyr287, and caused acute barrier disruption in HBEC-5i cells. Saracatinib attenuated occludin Tyr287 phosphorylation and preserved barrier integrity, supporting the involvement of Src-family kinase-sensitive signaling in this response. In mice, GsMTx4 significantly attenuated vascular permeability within minutes after reperfusion, consistent with the in vitro observations and supporting a role for mechanosensitive channel-associated signaling in ultra-acute blood–brain barrier leakage immediately after flow restoration. This study provides the first phosphoproteomic characterization of rapid phosphorylation changes associated with Piezo1 activation in human brain endothelial cells. Guided by these phosphoproteomic signatures, our data support the involvement of saracatinib-sensitive signaling, accompanied by occludin Tyr287 phosphorylation, in rapid endothelial barrier disruption. These findings suggest that Piezo1-associated signaling may contribute to ultra-acute BBB dysfunction and provide a rational for further investigation in the context of abrupt hemodynamic stress and cerebral revascularization.
The cerebrospinal fluid (CSF)-contacting nucleus has been well characterized in rats and macaques but not yet precisely defined in mice. This study aims to provide a comprehensive description of the location, adjacent structures, cell morphology, spatial imaging, and genetic architecture of the mouse CSF-contacting nucleus; Previous work has shown that knockout of the rat CSF-contacting nucleus elevates pain sensitivity; accordingly, we further verify certain key molecular substances within the mouse CSF-contacting nucleus may mediate neuropathic pain (NP) via the CSF pathway. Cholera toxin subunit B, Alexa Fluor™ 488/594 Conjugate (CB-AF488/594) or recombinant adeno-associated virus (rAAV) was unilaterally injected into the lateral ventricle (LV) of C57BL/6J mice for specific labeling of the CSF-contacting nucleus. Three-dimensional imaging of the nucleus was obtained using fMOST technology, followed by accurate isolation via laser capture microdissection (LCM) and gene expression profiling using Smart-seq2. Differentially expressed genes (DEGs) between normal mice and NP model mice were subjected to GO and KEGG functional enrichment analysis, and key regulatory genes were identified via bioinformatics and molecular techniques (immunofluorescence, qPCR, Western blot). The mouse CSF-contacting nucleus extends continuously from the ventral periaqueductal gray (vPAG) of the inferior midbrain to the central gray of the fourth ventricle (4 V) floor in the superior pons. fMOST reveals a rivet-like shape. In NP mice, 284 shared-genes were detected between the dorsal root ganglion (DRG) and the CSF-contacting nucleus, 190 of which showed strong interactions. Among seven highly interconnected clusters, chemokine C-C receptor 5 (Ccr5) in cluster 2 had the highest MCODE score. Molecular assays confirmed its significant upregulation in the nucleus versus controls, and intranuclear injection of maraviroc (a CCR5 inhibitor) markedly attenuated NP-induced nociceptive behaviors. Unilateral injection of CB-AF488/594 or rAAV into the LV specifically labels the mouse CSF-contacting nucleus, whose anatomy and cell biology are nearly identical to those of rats and macaques. The mouse CSF-contacting nucleus possesses a unique genetic profile, and CCR5 is a key target for its pain regulation, inhibiting CCR5 may be a potential strategy for pain treatment via CSF pathway.
Tight junctions (TJs) are a major structural component of the blood-brain barrier (BBB), contributing to brain homeostasis by restricting paracellular diffusion. Although BBB maturation begins during embryogenesis, the timing and dynamics of functional barrier maturation perinatally remain unclear. This question is particularly relevant for the striatum, a metabolically demanding brain region that undergoes rapid postnatal maturation and is vulnerable to neonatal injury. We investigated developmental dynamics of BBB tracer permeability in mouse striatum using in situ microperfusion of capillaries (ISMICAP) combined with two-photon microscopy. Small-molecule tracers were applied from late embryonic (E18) to adolescent (P25) stages. Bovine serum albumin (BSA) was applied as a macromolecular tracer at P2 and P25. Small-molecule tracers, namely 7-hydroxycoumarin-3-carboxylic acid (7HCC), sulforhodamine 101 (SR101), and biocytin-tetramethylrhodamine, revealed a pronounced but transient increase in extravascular fluorescence during the neonatal phase (P0-P2), followed by progressive restriction by P12 and P25. During the neonatal permeability window, 7HCC labeled perivascular cells located 2.8 μm from the endothelium and distinct from NT500/525-labeled pericytes, whereas SR101 accumulated within endothelial cytoplasm, indicating tracer-specific vascular and perivascular accumulation patterns. The membrane probe FM1-43 showed a similar temporal pattern, with enhanced diffusion to abluminal membranes and labeling of pericyte-like mural cells. In contrast, BSA showed low extravascular fluorescence at P2 comparable to P25, indicating that increased neonatal small-molecule permeability did not extend to BSA-sized macromolecules. At P25, small-molecule tracer permeability was higher in the striatum than in the cortex, whereas FM1-43 showed the opposite regional pattern. BBB maturation in the striatum is not a linear tightening process but includes a transient neonatal phase of increased TJ-associated permeability to small-molecule tracers, while remaining restrictive to BSA-sized macromolecules. Although tracer-specific vascular and perivascular accumulation patterns were observed, the overall temporal profile supports a discrete perinatal window of decreased barrier restriction. This dynamic permeability window may reflect physiological remodeling of barrier function during the perinatal transition. Defining this window mechanistically may improve understanding of neonatal brain vulnerability and may inform strategies for temporally targeted CNS drug delivery.
Chiari Malformation Type 1 (CM-1) is characterized radiologically by > 5 mm herniation of the cerebellar tonsils below the foramen magnum (FM). Tonsillar descent below the FM does not correlate well with symptom severity nor does it predict outcomes following posterior-fossa-decompression surgery. Using 2D phase-contrast magnetic resonance imaging (MRI) it is possible to measure cerebrospinal fluid (CSF) hydrodynamics non-invasively in CM-1 patients. The volume of fluid moved each heartbeat, or stroke volume, has shown promise as a new metric to characterize CM-1 malformation in adults, but has been relatively underexplored in the pediatric population. We measured CSF stroke volume in a pediatric CM-1 cohort before and after posterior fossa decompression surgery and in age-matched controls. 24 pediatric CM-1 patients (age 9.3 ± 4.5, 7 M, 17 F) underwent posterior fossa decompression surgery to treat CM-1 symptoms and received an MRI pre-operatively and 6 months post-operatively. 8 additional healthy volunteers (age 7.8 ± 5.7, 5 F) were used as control subjects. ECG-gated 2D phase-contrast magnetic resonance (PCMR) scans were acquired in the axial orientation at the foramen magnum (FM). CSF stroke volume was calculated from flow by separately integrating positive and negative flow, then averaging the absolute values. The CSF stroke volumes in the pre-surgical CM-1 subjects (0.26 ml ± 0.21) were lower than control subjects (0.55 ± 0.2, p < 0.01). Post-surgical stroke volumes in CM-1 subjects (0.44 ± 0.3) were not different than controls (p = 0.18). Pre-surgical stroke volumes were lower than post-surgical stroke volumes across all patients (p = 0.03), and in paired patients who had both pre- and post-surgical scans (p = 0.03). There was no difference in peak velocity pre-surgical and control (p = 0.7) or pre- and post-surgical subjects (p = 0.2). Pre-surgical patients with syrinx (10/15) showed no difference in stroke volume to patients without syrinx (p = 0.6). Healthy pediatric control subjects had higher CSF stroke flow volume than CM-1 patients. Surgery increased CSF stroke volume in CM-1 patients to values equal to healthy control subjects, and post-surgical stroke volumes were significantly greater than pre-surgical volumes. CSF stroke volume shows potential for use as a flow metric in assessing pediatric CM-1 patients.
Endothelial cells and astrocytes are critical structural and functional components of the blood-brain barrier. In many neuroinflammatory diseases, endothelial cells are among the first to respond to inflammatory stimuli and release extracellular vesicles (EVs). However, whether inflammatory stimulation alters EV RNA cargo and subsequently regulates astrocyte function remains unclear. In this study, we performed integrated RNA sequencing and proteomic analyses to investigate the effects of TNFα-stimulated endothelial EVs on astrocytes. RNA profiling revealed significant alterations in EV cargo after TNFα stimulation, including 867 upregulated and 577 downregulated mRNAs, 317 upregulated and 15 downregulated lncRNAs, and 88 upregulated and 62 downregulated miRNAs. The results of functional enrichment analysis suggested that altered EV RNAs may primarily promote inflammatory responses, cell migration, and RNA splicing in astrocytes while reducing their regulatory effects on neuronal projection and calcium homeostasis. Further integrative analysis of EV RNAs and astrocytic proteomics revealed key overlapping targets, including upregulated expression of ICAM1, SOD2, TFPI2, and TNFAIP8, whereas NFKBIA expression was consistently decreased. Network analysis revealed NF-κB as the central regulatory node. Reduced levels of EV-derived NFKBIA mRNA were associated with decreased IκBα protein levels in astrocytes, which promoted NF-κB activation and inflammatory cytokine release. Finally, overexpression of IκBα in astrocytes significantly attenuated TNFα EV-induced IL-1β and IL-6 secretion. Collectively, these findings demonstrate that TNFα-stimulated endothelial EVs coordinately regulate astrocyte function through mRNA, lncRNA, and miRNA cargo and that the IκBα/NF-κB axis may be a key mechanism underlying endothelial EV-mediated inflammatory disruption of the blood-brain barrier.
Abstract Background Infantile hydrocephalus is a challenging condition with variable timing of clinical recognition. Early identification of newborns at increased risk could enable closer surveillance and earlier intervention. Neonatal blood samples collected routinely for newborn screening allow us to test whether specific blood biomarkers are associated to hydrocephalus. Methods We used the data from an established nationwide Danish cohort study of 14,866 infants born from March 2009 to March 2011, with blood samples collected 2–8 days after birth and linked to longitudinal health registry data. The data included ten protein biomarkers spanning from inflammatory, cellular stress, growth, and neurotrophic pathways. Associations between neonatal biomarker concentrations and hydrocephalus were investigated using multivariable regression models adjusting for gestational age, age at sampling, and neonatal anthropometrics. Results Among 14,866 infants, 41 were diagnosed with hydrocephalus (approximately 3 per 1,000 births). The primary biomarker analyses included 30 hydrocephalus cases diagnosed no later than the age of 2 years and 14,577 controls. Neonatal heat-shock protein 70 (HSP70) concentrations were significantly higher in infants who developed hydrocephalus. No significant differences were found for the remaining biomarkers, and subgroup analyses related to intraventricular hemorrhage did not show distinct biomarker profiles. Conclusions In this nationwide cohort, elevated HSP70 in neonatal blood samples was associated with development of infantile hydrocephalus. No other of the inflammatory and neurotrophic biomarkers we investigated were significantly associated with hydrocephalus. These findings support further mechanistic investigation of HSP70 as a potential component of multifactorial early-life risk models of hydrocephalus.
Hydrocephalus is common with a mixed phenotype and aetiology. Its treatment is predominantly surgical, with temporary or permanent cerebrospinal fluid (CSF) diversion, which does not address the underlying disease mechanism. The choroid plexus (ChP), a secretory epithelium found in all four ventricles of the brain, produces most CSF within the central nervous system and has been investigated extensively over the past century to understand its function and potential role as a therapeutic target. Past attempts at either medically or surgically controlling its rate of secretion have not significantly altered our approach to the treatment of hydrocephalus, with CSF diversion remaining the main intervention. Rodent models of post-haemorrhagic hydrocephalus (PHH) have advanced our understanding of choroid plexus function in health and disease. Pre-clinical experiments have demonstrated a hypersecretory response in the ChP that may contribute to PHH. Targeting the choroid plexus directly may therefore present a novel therapeutic method. These findings have led to a significant increase in pre-clinical studies exploring this hypersecretory response and how to modulate it. Translating these promising results to clinical practice will rely on the development of large animal hydrocephalus models, which thus far has been limited. This literature review discusses the recent advances in targeting the ChP as a treatment for hydrocephalus, both surgically and non-surgically, and the current barriers to further advancement of this approach.
Using adeno-associated virus to transfer genetic information to the choroid plexus has emerged as a promising route for long-term gene therapy in the brain for a variety of conditions. Overexpression of proteins has proved effective in small animal models but few attempts have been made to translate this technology to clinic, suppress the activity of a protein of interest, or further expand the limited capsid serotypes known to have choroid plexus tropism. We utilise transfer of green fluorescent protein to show choroid plexus epithelium tropism for novel AAV6 derived capsid ShH10Y445F in mouse, rat and porcine tissue explant cultures. In vivo tropism is shown in the mouse following stereotactic intracerebroventricular injection. We examined the distribution of viral transduction across the choroid plexus in all four ventricles following a single unilateral intracerebroventricular injection using both green fluorescent protein as a transgene, but also the CRISPR/Cas9 system to deliver permanent knockdown of apical water channel aquaporin-1. Quantitative immunofluorescence and SURVEYOR assay were used to statistically assess the magnitude and extent of choroid plexus knockdown across the ventricular system. We conclude that serotype ShH10Y445F targets choroid plexus epithelium in mouse, rat and pig; and when carrying the CRISPR/Cas9 system can reduce target protein expression in these cells. Transduced choroid plexus epithelial cells distribute unevenly with a bias toward the lateral ventricle on the injected side and a preferential infection of choroid plexus in the lateral over the third and fourth ventricles. Overcoming irregular distribution represents a challenge for clinical translation of this technology where clinical efficacy may require manipulation of the entire choroid plexus.
The discovery of meningeal lymphatic vessels (mLVs) has reshaped our understanding of brain waste clearance, positioning them as a very important clearance route. However, quantifying the precise hydrodynamics and solute transport mechanisms within the human dura mater in vivo remains highly challenging due to the multiscale nature of cranial fluid dynamics and imaging resolution limits. We integrated patient-specific intrathecal contrast-enhanced MRI (gMRI) data from a single healthy subject with a novel anatomical segmentation of the cranial dura mater. By employing a hierarchy of inverse mathematical and micro-mechanical models, we estimated previously inaccessible physiological transport parameters. To ensure parameter identifiability, we deliberately modeled a scenario where the meningeal lymphatic vessels act as the sole fluid efflux route, thereby establishing an upper limit for their clearance capacity. These baseline estimates subsequently informed a spatially distributed, double-porosity 3D computational model of the human dura to simulate the fluid dynamics of cranial clearance. Our analysis suggests that purely diffusive macroscopic models fail to replicate in vivo tracer distribution. Conversely, a dual-porosity advection-diffusion framework successfully captures the tissue kinetics. The transport within the mLV compartment is characterized by highly convective clearance (with Pe > 1 ), with macroscopic fluid velocities and effective lymphatic diffusion coefficients yielding an upper-bound advective capacity theoretically sufficient to clear the total albumin content in the Cerebrospinal Fluid (CSF). Based on our findings on a single subject (Gonzo) solute clearance within the human cranial dura mater emerges as multiphasic, advection-dominated process. Rather than relying on passive Fickian diffusion, molecules appear to be actively and rapidly cleared through the meningeal lymphatic network via convective mechanisms. While our assumption of exclusively mLV-driven clearance provides a theoretical upper limit for their capacity, this hierarchical modeling approach successfully bridges clinical imaging and microscopic fluid dynamics, offering quantitative insights into the primary mechanical drivers of dural fluid clearance.
Ischemic stroke remains a leading cause of disability and death, yet decades of therapeutic development have yielded remarkably little clinical progress. The limited progress in improving stroke outcomes is closely linked to dysfunction of the blood-brain barrier (BBB), which undergoes profound structural and functional disruption during ischemic injury. Barrier injury under ischemic conditions is driven, in part, by glutamate dysregulation, neuroinflammation, and oxidative stress, which act in concert to destabilize endothelial integrity and exacerbate BBB permeability. Targeting these interconnected mechanisms represents a promising approach to preserve BBB function and limit secondary injury following stroke. Interventions aimed at modulating excitotoxic signaling, inflammatory cascades, and redox imbalance can protect the BBB and the larger neurovascular unit (NVU); however, aging further compromises BBB integrity and resilience, compounding these pathological processes and posing additional challenges for therapeutic development, particularly given the higher incidence of stroke in older individuals. This review highlights current understanding of BBB physiology in health and under ischemic conditions with an emphasis on how excitotoxicity, neuroinflammation, and oxidative stress compromise BBB functional integrity. Additionally, we evaluate therapeutic strategies aimed at preserving or restoring BBB homeostasis. By integrating insights into BBB biology with advances in neurovascular therapeutics, this review provides a forward-thinking assessment on the future of therapeutic development for ischemic stroke.
