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.

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.

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.

The blood-brain barrier (BBB) is crucial for neural homeostasis, tightly regulating molecular exchange between the circulation and brain. However, this selective protection also greatly limits drug delivery to the central nervous system, posing a major challenge for treating neurological disorders. Pharmacological strategies that transiently and safely increase BBB permeability could therefore transform brain drug delivery, yet systematic discovery of such modulators remains hampered by the limitations of current in vitro and in vivo approaches. Here we present FishNAP, a non-invasive, high-throughput zebrafish platform for real-time assessment of BBB permeability in vivo. FishNAP captures developmental changes in barrier function and detects dysfunction in genetic mutants. Using this platform, we screened 2,320 FDA-approved small molecules for compounds capable of opening an intact BBB and identified 11 that reproducibly increased permeability. Seven of these molecules allowed entry of a 1 kDa tracer into brain tissue, and five also permitted passage of a larger 10 kDa Dextran. Barrier integrity recovered within 24 hours for all seven compounds, indicating reversible modulation. Finally, testing three representative molecules (Calcitriol, Lovastatin, and Sunitinib) in adult mice revealed increased BBB permeability and reduced Claudin-5 expression, demonstrating conserved mechanisms of BBB-regulation across vertebrates. FishNAP thus enables systematic discovery of BBB modulators with direct translational potential for brain drug delivery.

Intestinal organoids are three-dimensional in vitro structures derived from stem cells and serve as a valuable model for studying intestinal biology and pathophysiology. This study optimized the isolation, expansion, and differentiation of canine intestinal organoids from duodenum and colon. Organoids were generated from canine intestinal crypts and cultured in Matrigel with a growth factor cocktail. The impact of prostaglandin E2 (PGE2) concentration on organoid growth was evaluated, and a two-phase differentiation protocol--comprising patterning and differentiation media--was implemented, including interleukin (IL)-22 in the duodenal differentiation phase. Organoids cultured with 100 nM PGE2 exhibited increased crypt budding and organoid-forming efficiency, indicative of enhanced stem cell proliferation. Differentiated organoids expressed key intestinal markers (VIL1, SI, CHGA, MUC2), and forskolin-induced swelling demonstrated functional Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) activity. Although the sample size (n=2) limits generalizability, this optimized protocol provides a relevant in vitro model for studying canine intestinal function. The model can be used in future research for disease modelling and translational applications, supporting downstream studies in gastrointestinal disease, drug permeability, and comparative One Health research.

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder marked by numerous renal cysts that impair kidney function, with about half of affected individuals progressing to kidney failure by midlife. Patients exhibit reduced circulating apelin, a ligand of the apelin receptor, known to regulate cardiovascular function including hypertension. We tested whether diminished apelin signaling contributes to cystogenesis and if exogenous apelin receptor activation can improve disease outcomes. MethodsPlasma samples from age- and sex-matched healthy controls and ADPKD participants were analyzed for circulating apelin peptides. To assess direct cystic effects, primary ADPKD renal epithelial cells were grown as 3D collagen-embedded cysts and treated with apelin agonists. Male and female Pkd1RC/RC; Pkd2+/- (PKD) mice were treated for 27 days with apelin agonists, vehicle, or the standard of care drug, Mozavaptan. Kidney and heart weight ratios, BUN, renal cAMP, and kidney transcriptional profiles were evaluated. ResultsCirculating apelin peptides were significantly reduced in ADPKD patients despite normal kidney function (eGFR, BUN, and creatinine). In vitro, both apelin and the small molecule apelin receptor agonist Azelaprag inhibited cyst growth. Apelin and Mozavaptan reduced kidney weight, cystic index, blood urea nitrogen and renal cAMP in PKD mice, whereas Azelaprag did not. Apelin downregulated expression of genes associated with cyst progression, including Lcn2 (Ngal), Postn, and Havcr1 (Kim-1). Mozavaptan, but not apelin, induced diuresis and reduced urinary concentration. ConclusionApelin receptor activation by exogenous apelin inhibited cAMP synthesis and cyst growth and improved kidney function in an orthologous mouse model of ADPKD. We propose that the apelin receptor may be a potential therapeutic target in ADPKD.

Angiogenesis, the formation of new vessels from pre-existing ones, is essential for embryonic development and tightly regulated by VEGFA signaling. However, the contribution of additional modulators remains poorly defined. The co-receptor NRP1 is crucial for hindbrain vascularization, yet how its activity is spatiotemporally controlled is unclear. We identify the glycosylphosphatidylinositol (GPI)-anchored protease MT4-MMP as a key regulator of developmental angiogenesis. Endothelial cell-specific, inducible deletion of MT4-MMP (Mt4-mmpi{Delta}EC mice) causes an exacerbated vascular plexus in the E11.5 embryonic hindbrains. In vitro, loss of MT4-MMP in endothelial cells disrupts cell polarization and migration and enhances VEGFA-induced ERK signaling. Consistently, pERK levels are increased in hindbrain vessels from Mt4-mmpi{Delta}EC embryos, whereas they are reduced in mice with constitutive and global MT4-MMP deficiency. By combining co-expression analysis in cultured cells and embryonic hindbrains with proteomics, in silico protein modeling, and in vitro digestion assays, we identify NRP1 as a previously unrecognized MT4-MMP substrate in this context. Accordingly, inhibition of VEGFA binding to NRP1 partially rescues the aberrant angiogenic phenotype in the embryonic hindbrain of Mt4-mmpi{Delta}EC mice. Our findings reveal that MT4-MMP shapes developing brain vasculature by modulating NRP1-dependent VEGFA/ERK signaling. This newly identified MT4-MMP/NRP1 axis may have potential relevance in CNS vascular abnormalities in development and disease, as well as other pathophysiological contexts.

Scaling up microvessel culture systems is essential for producing vascularized clinically relevant tissues, yet current platforms offer little guidance on how to preserve flow conditions during scale-up. Here, we present a computational-experimental framework using computational fluid dynamics (CFD) to guide the design and scaling of microvessel bioreactors. Interstitial flow distributions were pre-dicted in two perfusion-based platforms-a permeable insert and a rhomboidal microfluidic chamber-across multiple scaling factors and hydrostatic pressures. CFD identified IF ranges conducive to vascu-logenesis and quantified how geometry and pressure modulate flow uniformity. Scaled-up bioreactors generated microvessel networks with consistent morphology and connectivity over a 30-fold increase in culture volume, confirming that maintaining equivalent IF ensures reproducible outcomes. The permeable insert platform maintained uniform IF across scales, while the rhomboidal chamber produced spatially varying IF resulting in heterogeneous but physiologically relevant networks. These findings establish CFD as a predictive tool for rationally scaling perfusion bioreactors, enabling microvessel production at clinically relevant scales with controllable morphology. Significance StatementScaling up microvessel bioreactors is critical for engineering large pre-vascularized tissues. However, larger scales may disrupt flow conditions that drive vessel formation. This study demonstrates that computational fluid dynamics (CFD) can predict interstitial flow and guide the rational scale-up while preserving the vasculogenic microenvironment. Experiments across 30+-fold size increase confirmed that matching inter-stitial flow results in morphologically identical microvessel networks. By linking simulation-based design with experimental validation, this work establishes CFD as design tool for scalable perfusion bioreactors for production of microvessel networks at clinically relevant scales.

Extracellular vesicles are key intercellular messengers that modulate the function of target cells by carrying effectors, either at their surface or in their lumen. In the latter case, their action depends on the ability to deliver their content into the cytosol of target cells. How efficiently EVs deliver their content upon interaction with their target cell is thus a central question for understanding the functional impact of this mode of action. To address this question, signal-driven bimolecular interactions between two partners located respectively in the EV lumen and the target cell cytosol have become a widely used strategy to detect the cytosolic delivery EV content. However, the detection of cytosolic delivery with these assays was often tributary to the artificial enhancement of the fusion between EV and cell membranes, through for instance VSV-G fusogenic protein expression. Here we provide a robust and quantitative LUCiferase-based complementation assay (HiBiT/LgBiT), to quantify the Internalization and cytosolic Delivery of EV content: LUCID-EV. By optimizing the signal-to-noise ratio of the assay, the method for loading HiBiT fragment into EVs (fusion to a lipid-binding domain rather than to tetraspanins), and the intracellular position of LgBiT (associated to membranes), we could quantify cytosolic delivery from various non-VSV-G-expressing EVs into target immune dendritic cells. Importantly, this delivery did not involve the acidic late endosomes environment required for VSV-G-dependent EV cytosolic delivery. The limited efficacy of the process highlights the need for highly sensitive assays like the one described here. Further development of the LUCID-EV assay could help identifying EV/target cells pairs with enhanced cytosolic delivery properties and characterize the cellular route for delivery.

BackgroundPrimary astrocyte cultures derived from neonatal rodent cortices provide a controlled system for investigating astrocyte-specific mechanisms. However, mixed glial preparations frequently contain contaminating microglia and oligodendrocyte progenitor cells, and most existing protocols require pooling tissue from multiple mouse pups to obtain sufficient astrocyte yields. This approach is impractical as it obscures sex and genotype, limits investigations of sex dependent astrocyte phenotypes, and precludes studies in certain transgenic models. To address this gap, our protocol achieves a high astrocyte yield from a single neonatal mouse brain, enabling sex- and genotype-specific cultures without the need for pooling. Mechanical removal of oligodendrocyte progenitors combined with pharmacological depletion of microglia using a Colony Stimulating Factor 1 Receptor (CSF1R) inhibitor produces highly enriched astrocytes suitable for functional assays, including those focused on sex-specific biology. MethodsCortical tissue was isolated from a single mouse pup is mechanically dissociated in astrocyte media. Cell suspensions are transferred to poly-D-lysine-coated flasks in astrocyte media. After 10-15 days in culture, OPCs are mechanically removed by horizontal shaking and microglia are selectively depleted by incubating cultures with CSF1R inhibitor PLX5622 for 24, 48, 72 and 96 hours. After PLX treatment, media is replaced and enriched astrocytes were maintained or passaged for experimentation. The sex of the pups is determined by PCR performed on DNA extracted from tail biopsies. ResultsImmunocytochemical analysis for astrocyte and microglia markers (GFAP and Iba1, respectively) showed that 24 hours of PLX5622 treatment did not fully eliminate microglia from mixed glial cultures. Extending treatment to 48 hours effectively depleted microglia while minimizing cytotoxicity and astrocyte loss and produced a pure, high-yield, sex-specific primary astrocyte culture. PCR reliably enabled the sex identification of pups used in culture using DNA extracted from tail biopsies. DiscussionThis protocol provides an efficient and reproducible method for generating high-purity, sex-specific primary astrocyte cultures from a single mouse brain. It improves consistency and purity while eliminating the need to pool tissue, preserving sex and genotype and enabling studies in transgenic mouse lines of both sexes.

IntroductionNeuroinflammation is increasingly implicated in age-related cognitive decline, neurodegeneration and neuropsychiatric disorders. During systemic inflammation, microglia are rapidly activated, simultaneously changing their shape and releasing cytokines that perturb neuronal function. This change in glial morphology alters their intracellular diffusion properties and provides a potentially measurable signature of their activation state. Diffusion-weighted magnetic resonance spectroscopy (dMRS) shows promise in detecting these changes. Here, we combined IFN-{beta} challenge with dMRS to assess changes in metabolite diffusion in healthy young and older adults. We hypothesised that IFN-{beta} would increase diffusion of choline-containing compounds (tCho) but not N-acetylaspartate + N-acetylaspartylglutamate (tNAA), age would be associated with an increase in tCho diffusion and concentration, lower tNAA concentration and increased effects of IFN-{beta}. MethodsWe recruited 15 young (mean 25.2 {+/-} 5.1 years, 6 male) and 15 older (mean 62.6 {+/-} 4.1 years, 5 male) healthy volunteers, each tested twice, once after IFN-{beta} and once after placebo. Physiological and behavioural responses were recorded hourly, and blood samples taken at baseline, 4 and 6.5 hours post-injection. dMRS occurred at [~]4.5 hours at 3T, using a semi-LASER sequence with four diffusion weightings (b = 0 and 3 x 3500 s/mm{superscript 2}), in 4.5 cm3 VOIs in the left thalamus and corona radiata. Apparent Diffusion Coefficients (ADCs) of tCho, tNAA and creatine+phosphocreatine (tCr) were calculated from averaged spectra using custom MATLAB software. ResultsIFN-{beta} administration produced a significant increase in thalamic tCho diffusivity compared with placebo (t(28) = -2.15, p = 0.040), with no change in tNAA or tCr ADC, or tCho concentrations. IFN-{beta}-related increases in tCho ADC positively correlated with increases in circulating IL-6 (R{superscript 2} = 0.14, p = 0.040). Age-related effects were also evident during the placebo condition, with older participants showing lower thalamic tNAA diffusivity (t(27) = 2.86, p = 0.008), lower tNAA/tCr in both grey and white matter (grey: t(27) = 2.49, p = 0.023; white: t(27) = 2.94, p = 0.007), and higher white-matter tCho/tCr (t(27) = -2.23, p = 0.034). ConclusiondMRS detected IFN-{beta}-induced increases in thalamic tCho diffusivity corresponding with peripheral inflammation, supporting its sensitivity to acute inflammation-induced changes in glial morphology. Age-related differences in tNAA diffusion and concentrations further highlight metabolite-specific ageing effects. HighlightsO_LIdMRS detects increased thalamic total choline diffusivity following IFN-{beta}-induced inflammation. C_LIO_LIIFN-{beta}-related changes in total choline diffusivity are associated with peripheral IL-6 responses. C_LIO_LIAgeing is linked to reduced NAA diffusion and higher white-matter tCho/tCr C_LIO_LIdMRS is sensitive to inflammation- and age-related neurochemical changes in vivo. C_LI

Understanding how specific VWF variants disrupt endothelial processing and function is central to elucidating von Willebrand disease (VWD) pathophysiology. However, current in vitro systems lack either the endothelial specificity or the genetic flexibility required for systematic variant characterization. Here, we present a genetically defined VWF-knockout cord-blood-derived endothelial colony-forming cell (VWF-KO cbECFC) model that enables controlled reintroduction of VWF variants in a physiologically relevant endothelial context. Using a patient with type 3 VWD carrying the homozygous pathogenic variant p.M771V and a second homozygous variant of uncertain significance p.R2663P as a reference, we demonstrate that expression of p.M771V in VWF-KO cbECFCs reproduces the patients intracellular processing defect and loss of high-molecular-weight multimers, whereas p.R2663P behaves as a benign allele. These findings establish the models ability to accurately distinguish pathogenic from non-pathogenic variants. Comparative analyses with HEK293 cells show that VWF-KO cbECFCs provide superior subcellular resolution, reliably forming authentic Weibel-Palade bodies (WPBs) and faithfully revealing ER retention phenotypes that remain ambiguous in non-endothelial systems. The proliferative capacity of cbECFCs further enables scalable and reproducible experimentation, overcoming major limitations associated with patient-derived ECFCs. Looking ahead, the VWF-KO cbECFC platform offers broad potential for VWF and VWD research. Its endothelial identity and genetic flexibility make it suitable for investigating VWF biosynthesis and trafficking, secretion dynamics, WPB biology, angiogenic processes, and shear-dependent VWF function. This system therefore provides a versatile foundation for mechanistic studies, systematic variant assessment, and future translational applications.

PurposeMitochondrial dysfunction contributes to major blinding diseases, including age-related macular degeneration and glaucoma. Although mitochondrial transplantation has shown therapeutic potential in multiple organ systems, translation to the eye remains limited, partly due to uncertainty regarding optimal delivery. We summarize the biologic rationale and preclinical evidence supporting ocular mitochondrial transplantation and present feasibility data evaluating clinically relevant delivery routes. MethodsWe conducted a focused narrative review of ocular mitochondrial transplantation. For feasibility experiments, mitochondria with an endogenous fluorescent dye were isolated from liver donor mice. Postnatal day 7 pups received subretinal injections, and adult CD1 mice received intravitreal injections, including optic nerve head directed delivery. Eyes were analyzed using fluorescence microscopy and immunohistochemistry. Mitochondrial uptake was assessed in cultured retinal pigmental epithelial (RPE) cells using co-incubation assays. Suprachoroidal delivery feasibility was evaluated in cadaveric human near-real surgical specimens using a novel dedicated suprachoroidal injector. ResultsThe literature on ocular mitochondrial transplantation remains limited and consists primarily of small preclinical studies using intravitreal delivery and imaging-based detection. In our experiments, intravitreal delivery produced donor signals predominantly within inner retinal layers, with enrichment along retinal nerve fiber bundles when directed toward the optic nerve head. Cultured RPE cells demonstrated dose-dependent uptake of exogenous mitochondria. Subretinal delivery localized donors signal to the RPE and adjacent outer retina. Suprachoroidal injections demonstrated procedural feasibility with reliable access to the suprachoroidal space and visible injectate distribution. ConclusionsOcular mitochondrial transplantation is in an early stage of investigation. Our feasibility data indicate that established posterior-segment delivery routes expose distinct retinal compartments and that route selection strongly influences anatomic distribution. Further studies are needed to verify intracellular uptake, define dosing and durability, and evaluate safety in disease-relevant models.

The liver is widely considered to be one of the most conserved organs amongst vertebrates, with it being involved in blood detoxification, bile production and the metabolism of xenobiotic compounds. Liver organoids have previously been derived from several species and used as models of drug metabolism, toxicity, and fundamental tissue biology. To date, however, these models have not been developed from ruminant species, specifically cattle and sheep. Here we present the first report of the development and comprehensive characterisation of bovine and ovine liver organoids derived from primary liver tissue. When initially established, organoids from both species were comprised of KRT19- and KRT18-positive cholangiocytes. The capacity for organoids to differentiate into hepatocyte-enriched cultures was evaluated and it was noted that there was an increase in hepatocyte markers in bovine cultures. A comparative analysis of the liver tissue and organoids of both species revealed species-specific differences in gene expression, which were conserved within organoid cultures. Most notably, bovine liver tissue and organoids had enriched expression of genes associated with fatty acid uptake and storage whereas ovine samples had higher expression of genes associated with fatty acid conversion, highlighting fundamental differences between these two ruminant species. Differences in expression of cytochrome P450 family genes were identified alongside those associated with an inflammatory response specifically in bovine samples, whereas ovine samples had higher expression of genes associated with a protective immune response. Despite this, transcriptomic analysis of organoids from both species, cultured in both growth and differentiation media, revealed preserved expression of genes associated with key liver functions, including gluconeogenesis and xenobiotic metabolism. Transcripts associated with the flavin-containing monooxygenases (FMO) family were expressed in both organoid growth media and organoid development media (OGM and ODM respectively), and both species could metabolise triclabendazole into its primary metabolite triclabendazole sulfoxide, therefore validating the potential of the organoids to be applied as in vitro models of metabolism and/or toxicity. Overall, this study provides novel insights into differences in liver composition and function between ruminant species, as well as providing novel experimental models of the liver for both cattle and sheep.

BackgroundPleuroparenchymal fibroelastosis (PPFE) is a rare, fibrotic lung disease with poor prognosis, usually affecting adults which most commonly occurs idiopathically. Biallelic pathogenic variants in DGUOK cause mitochondrial DNA (mtDNA) depletion syndrome, predominantly affecting infants with severe hepatic and neurological symptoms. Detailed description of pulmonary manifestations with late-onset presentation have not been reported. MethodsWe describe nine patients with PPFE and DGUOK-associated mitochondriopathy. Clinical, radiological, histopathological, and genetic data were systematically collected from all patients. Functional studies, single nucleus RNA sequencing (snRNAseq), immunofluorescence staining, transmission electron microscopy and respiratory chain enzyme activity assays were conducted on patient-derived fibroblasts, muscle or lung tissues. mtDNA content quantification was performed on whole genome sequencing (WGS) data. ResultsAll patients (ages 5-36) presented with progressive dyspnea, weight loss and some with spontaneous pneumothoraces. Chest computed tomography and lung biopsies showed features of PPFE. Biallelic pathogenic DGUOK variants were identified in all patients, seven of them carry an unreported intronic variant leading to mtDNA depletion. snRNAseq of lung tissue from four pediatric patients identified Aberrant Basaloid cells and intermediate cells as their precursor localized at the fibrotic edge. Mitochondrial alterations were identified by electron microscopy. ConclusionPPFE in children and young adults is associated with DGUOK-related mitochondriopathy. For the first time, we demonstrate Aberrant Basaloid cells in pediatric fibrotic lung tissue. Since pulmonary involvement may be underrecognized or misinterpreted and the clinical presentation may not always be typical of a mitochondriopathy, we recommend genetic testing in all patients with PPFE of unknown origin.

Preeclampsia (PE), or gestational hypertension, affects around 5% of pregnancies and leads to approximately 70,000 maternal and 500,000 fetal deaths per year worldwide, with increased cardiovascular and metabolic disease in survivors. PE is associated with elevated circulating levels of the alternative splice isoform of VEGF receptor 1 (sFlt1), defects in placental vasculature, kidney damage and, in severe disease, fetal growth restriction. Current mouse models induce PE via direct expression of sFlt1 or elevation of blood pressure, which bypass the natural risk factors for human disease, such as age, obesity, hypertension and diabetes. These risk factors have in common reduced expression of Kruppel-like factors 2 and 4 (KLF2/4), the endothelial transcription factors that protect against cardiovascular disease. We now report that inducible deletion of KLF4 in maternal endothelium (KLF4iECKO) results in gestational hypertension, elevated sFlt1, defective placental vasculature, kidney damage and fetal growth restriction. KLF4iECKO may thus serve as a mouse PE model suitable for mechanistic analysis and screening of treatments that address upstream risk factors.

The bone marrow haematopoietic niche is composed of a diverse array of cell types and extracellular matrix components that together support healthy haematopoiesis. However, live imaging of the bone marrow microenvironment is hampered by tissue accessibility limitations. Using intravital imaging through a titanium imaging window, we investigated the dynamics of human haematopoietic cells and mesenchymal stromal cells within an ectopically implanted humanised scaffold in an immunodeficient murine host. These cell populations expand and differentiate over time, accompanied by progressive remodelling of the scaffold. We observe migration of murine endothelial cells into the scaffold, leading to the formation of a vascular network during the initial development of the humanised niche. Subsequently, the dense collagen matrix that makes up the implanted niche is altered and larger gaps form in regions populated by mesenchymal stroma cells. Collectively, our findings demonstrate dynamic remodelling of the extracellular milieu that supports haematopoietic cell development and establish a platform for longitudinal, in vivo investigation of these processes. Altogether, we describe a novel model that aligns with the 3R guiding principles and enables real-time assessment of bone marrow cell dynamics in vivo. Summary statementRatcliffe and Mian et al. image in vivo dynamics of a bone marrow haematopoietic niche model.

Normal pressure hydrocephalus (NPH) is a potentially reversible neurological disorder characterized by urinary incontinence, gait impairment, and cognitive decline. However, postoperative improvement after shunt placement is variable, and reliable preoperative predictors are lacking, leaving patients exposed to uncertain surgical benefit and procedural risk. We therefore asked whether preoperative cerebrospinal fluid (CSF) metabolic profiles capture biological states associated with recovery potential. We analyzed ventricular CSF from patients undergoing shunt placement and identified metabolic patterns that differed between patients who improved postoperatively and those who did not. These signatures were detectable prior to intervention and were consistent across analytical approaches and patient cohorts. Multivariate models based on metabolite features were associated with postoperative improvement, with strongest performance observed for cognitive outcomes. Pathway-level analyses indicated coordinated alterations in processes related to redox balance, immune-metabolic signaling, and energy substrate utilization. These findings indicate that preoperative CSF metabolite profiles reflect biological states associated with recovery potential in NPH. The results further suggest that metabolic and immune-metabolic processes contribute to variability in surgical responsiveness and support the development of predictive biomarkers for patient stratification.

The hippocampal CA1 subregion supports learning, memory formation, and spatial navigation. Although its three-layered architecture has been described in ex-vivo investigations, the in-vivo microstructural profile of CA1 and its relation to individual variations in memory performance remain poorly characterized. In this study, we used ultra-high field structural MRI at 7 Tesla to investigate the depth-dependent myelination patterns (measured by quantitative T1) of CA1 in younger adults, their relation to the local arterial architecture, and their association with individual differences in cognitive functions, specifically memory performance. Results show that left and right CA1 present depth-dependent patterns of myelination, with the outer and inner compartments showing higher myelination than the middle compartment. No significant relationship between layer-specific myelination of CA1 and distance to the nearest artery was observed. Right CA1 was found to be more myelinated than left CA1. Pairwise correlations and regression models showed that higher left CA1 myelination is linked to higher accuracy in object localization. Together, our data demonstrates the feasibility of describing the three layered myelin architecture of CA1 in vivo, and provides information on how alterations in the architecture of CA1 may relate to alterations in cognitive performance in younger adults.