Journal of Cerebral Blood Flow & Metabolism

Moderate hypoxia is increasingly recognized as a physiological driver of neuroprotection and neuroregeneration. In this first randomised, double-blind, controlled, four-arm trial, we demonstrate the cognitive and neuroplastic effects of cognitive training under moderate inspiratory hypoxia in humans. Healthy volunteers underwent three weeks of either cognitive or sham training under normobaric hypoxia (12% O2) or normoxia (20% O2) for 3.5 hours daily, six days per week. Participants were assessed at baseline, treatment completion, and one-month follow-up. The primary outcome was change in a broad cognitive composite score. Additional cognitive, blood-based, and neuroimaging outcomes were assessed, including measurement of the presynaptic protein SV2A with [11C]UCB-J positron emission tomography (PET) and neural activity through functional magnetic resonance imaging (fMRI). In total, 126 participants were randomised to hypoxia-cognitive training (H-CT: n=36), hypoxia-sham training (H-ST: n=30), normoxia- cognitive training (N-CT: n=30), or normoxia-sham training (N-ST: n=30). Intention-to-treat analyses showed no effect of H-CT relative to N-ST in the primary outcome at treatment completion (primary endpoint; treatment effect=0.11, 95% CI=[-0.06;0.28], p=0.19), but improvements emerged at follow-up (treatment effect=0.17, 95% CI=[0.01;0.34], p=0.04). N-CT induced transient improvement in the primary outcome at treatment completion (treatment effect=0.20, 95% CI=[0.02;0.38], p=0.03), which rendered non-significant at follow-up. Finally, H-ST showed no significant cognitive change relative to N-ST. Moderate hypoxia was safe and well-tolerated. Cognitive benefits were accompanied by decreased hippocampal presynaptic density measured with [11C]UCB-J PET. In conclusion, three weeks of H-CT can enhance cognition with associated effects on neuroplasticity, although with a delayed onset of effects on cognition.

Cerebral blood flow (CBF) is a quantitative metric for mapping perfusion. While the prototypical MRI approach arterial spin labelling (ASL) is well-validated in humans, the reproducibility of rodent ASL mapping remains poor, limiting translational impact. To address this gap, we used both newly acquired and analysis of previously published data to illustrate biological and physical sources of variation in CBF measured with ASL. Via a meta-analysis, we quantified the variation in CBF reported from the cortex of healthy rodents. A total of 23 mouse studies (343 data points) and 5 rat studies (41 data points) met the inclusion criteria. We demonstrate that reported CBF values exhibit a broad variability (50-400 ml/100g/min) driven primarily by experimental confounds rather than physiological differences. Our meta-analysis explores which factors cause variance in perfusion rates measured. Our experimental data highlight biological factors, particularly the choice of anaesthesia (e.g., isoflurane vs. medetomidine) and strain variations, that alter baseline CBF. Our work, reflecting both state-of-the-art and conventional practice in preclinical imaging, highlights the need to account for multiple sources of variability. Establishing community guidelines for rigorous ASL calibration and physiological monitoring will support improved study design and accelerate translational alignment between rodent and human perfusion measurements.

Accurate assessment of cerebral hemodynamics impairment traditionally relies on arterial metrics, yet often overlooks venous drainage and arteriovenous dynamics, thereby limiting the evaluation of ischemia-induced microvascular dysfunction. To address this limitation, we implemented a signal-averaging framework, combined with co-fluctuation analysis, to extract predominantly arterial and venous hemodynamic signals and construct a dynamic arteriovenous co-fluctuation index that quantifies frame-by-frame coordination between arterial inflow and venous outflow activity. This time-resolved index enables spatial characterization of large-scale cortical arteriovenous coordination beyond conventional static correlation-based analyses. Comparative analyses between healthy controls and acute ischemic stroke mice demonstrated that the arteriovenous co-fluctuation index sensitively detects disruption of vascular coordination, revealing a slower state transition that occurs alongside distinct temporal abnormalities and regional heterogeneity between ischemic core and penumbral regions. These findings underscore the utility of arteriovenous coordination as a sensitive indicator of microcirculatory dysfunction, offering a practical analytical tool for assessing stroke-induced microvascular impairment.

IntroductionChemogenetic tools such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) provide a powerful means to causally manipulate defined neuronal populations in vivo. While chemogenetic fMRI studies have consistently demonstrated robust hemodynamic responses following circuit perturbation, considerably less is known about the accompanying metabolic consequences. Functional magnetic resonance spectroscopy (fMRS) offers the potential to probe these neurochemical processes, yet the relationship between hemodynamic and metabolic responses remains poorly understood. Here, we combined chemogenetics, pharmacological fMRI (ph-fMRI), and proton magnetic resonance spectroscopy (1H-MRS/fMRS) at 7 T to investigate the temporal evolution of metabolic and hemodynamic responses in the rat motor cortex. MethodsFemale Fischer rats received viral injections in the motor cortex to express either a pan-neuronal hM3D(Gq) DREADD construct (hSyn-hM3Dq) or an interneuron-targeted construct (hDlx-hM3Dq). Ph-fMRI, fMRS, and 1H-MRS measurements were performed before, during, and following systemic administration of clozapine-N-oxide (CNO, 1 mg/kg). Functional MRS was acquired during the acute response phase (0-60 min post-injection), while conventional 1H-MRS measurements were obtained at a delayed time point (70 min post-injection). ResultsChemogenetic modulation produced robust and opposing hemodynamic responses. Pan-neuronal activation elicited focal positive BOLD responses (+3.5 {+/-} 1.5%), whereas interneuron-targeted activation generated significant negative BOLD responses (-3.3 {+/-} 0.8%). In contrast, acute fMRS measurements revealed no significant changes in Glx or GABA concentrations during the first hour following CNO administration, despite the presence of strong hemodynamic effects. However, delayed metabolic alterations were detected 70 min after CNO administration. Animals expressing the pan-neuronal construct exhibited significant increases in GABA (+14.4%) and total choline compounds (+57.8%), whereas interneuron-targeted animals displayed reductions in several metabolites, including Glx (-15.6%), total NAA (-16.9%), glucose (-25.9%), and total creatine (-25.4%). ConclusionChemogenetic perturbation of cortical circuits produced robust hemodynamic responses but more subtle and temporally complex metabolic effects. The absence of detectable acute changes in Glx and GABA despite strong BOLD responses, together with the emergence of delayed neurochemical alterations, highlights the challenges of interpreting metabolic signals in relation to circuit activity.

Background: Mu-opioid receptors (MORs) are expressed throughout the body including in the brain and gastrointestinal (GI) tract. Total-body PET imaging of the brain and GI tract offers a promising approach for cross-sectional in vivo evaluation of the MOR brain-GI axis. However, intestinal motility and bladder filling introduce motion throughout the GI tract over the scan window. Here we establish analysis methodology to account for motion for dynamic imaging of the brain-GI axis, to further characterize peripheral MORs throughout the body and provide a framework for semi-automatic total-body PET modeling. Methods: 4 subjects underwent 90-min dynamic [11C]-carfentanil (cfn) total-body PET acquisitions at baseline, after intravenous naloxone (central antagonist) administration, and after orally administered loperamide (peripheral agonist and P-glycoprotein substrate). Thalamic MOR availability was measured using the Logan reference tissue model. Using CT-based segmentation, the GI tract was subdivided into anatomical segments, in addition to other peripheral organs (e.g., liver, psoas muscle). Frame-by-frame semi-automatic motion correction was performed with three distinct reference frames (11-14 min post-injection, p.i., 35-40 min p.i., and 85-90 min p.i.). The performance of these three were compared to manual correction. Compartment modeling and Logan graphical analysis were performed to estimate relevant kinetic parameters (K1, VT, VTLogan). Results: Across the 4 subjects and regions, kinetic parameter estimates were highly correlated (r>0.7) for K1, VT and VT Logan when comparing semi-automatic (reference frame at 35-40 min p.i.) and manual correction. With semi-automatic motion correction, graphical-based estimation of VTLogan in the gastrointestinal tract was significantly decreased with loperamide relative to baseline (p<0.05). As expected, naloxone decreased brain thalamic MOR availability but loperamide did not. Conclusions: With semi-automatic motion correction and [11C]-cfn total-body PET, pharmacologic perturbations of MOR brain-GI axis can be quantitatively characterized, reducing the burden of image analysis for these studies.

Background The ischemic penumbra, a metabolically compromised yet potentially salvageable region surrounding the ischemic core, is a prime target for acute stroke intervention. Yet an objective molecular definition of the penumbra, particularly during the earliest stages of ischemia, remains lacking. Methods and Results We applied principal component analysis (PCA) followed by k-means clustering to high-resolution mass spectrometry imaging data covering multiple metabolic pathways to identify a metabolically defined penumbra in a mouse model of hyperacute stroke (45 min middle cerebral artery occlusion, MCAO). Targeted spatial metabolomic profiling by matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) reveals a distinct penumbral metabolic profile, marked by relative preservation of high-energy phosphates, comparable lactate accumulation, and reduced succinate accumulation relative to the core. Spatial transcriptomics revealed selective induction of immediate-early genes, including Npas4, Fos and Junb, within the penumbra. Consistently, imaging mass cytometry shows enrichment of phospho-histone H3 (pHH3) within the penumbra, suggesting a chromatin-associated response potentially linked to immediate-early gene activation. Conclusion Together, these findings provide a multimodal molecular atlas of the hyperacute metabolically defined penumbra and reveal molecular features that facilitates its identification and inform future therapeutic strategies.

Trimethylamine n-oxide (TMAO) is a plasma metabolite linked to adverse cardiometabolic health with complex regulation involving diet, sex, and host genetics. We explored the role of these factors in the genetic regulation of TMAO by performing a primary-level meta-analysis in 1,482 female and male Diversity Outbred (DO) mice from five distinct studies conducted in various regions of the United States. We identified a quantitative trait locus (QTL) associated with TMAO concentration at [~]86 megabase pairs on mouse chromosome 12 with a highly significant LOD score of 67.67. Alleles at the chromosome 12 QTL inherited from the Cast/EiJ (CAST) and PWK/PhJ (PWK) mouse strains primarily drove the association with reduced TMAO concentrations. The chromosome 12 QTL remained significant in sex-stratified analyses and the mode of inheritance appeared additive; furthermore, the QTL was regulated by sex-by-genotype and sex-by-diet interactions. Using a CAST/EiJ X C57BL/6J F2 cross, positional candidates were prioritized by eQTL analysis. Further analysis in a study utilizing the eight DO founding strains identified that Acyp1 was differentially expressed in hepatic tissue from CAST mice, prompting investigation into its genetic regulation. Acyp1 demonstrated relevant cis- and trans-regulation and was significantly correlated with TMAO and hepatic Fmo3. However, no significant relationships between Acyp1 and TMAO were identified in mice inactivated for Acyp1 or with AAV overexpression of Acyp1 in the liver. Genes within the chromosome 12 QTL have synteny with humans and may translate to the genetic regulation of human plasma TMAO concentrations and atherosclerosis. Author SummaryWe explored the roles of diet, sex, and genetics on the regulation of fasting plasma trimethylamine n-oxide (TMAO) concentration by performing a meta-analysis in 1,482 female and male Diversity Outbred (DO) mice from five unique studies. We identified a QTL associated with TMAO concentration on chromosome 12 at [~]86 mega base pair (Mb) with a highly significant LOD score of 67.67. The locus is modified by both sex and diet.

Ischemic stroke remains a leading cause of death and disability, and current reperfusion therapies do not address the secondary neuroinflammatory response following blood-brain barrier (BBB) disruption. Purinergic signaling critically regulates this process: extracellular ATP promotes inflammation, whereas its enzymatic conversion into adenosine exerts tissue-protective effects. Notably, the ectonucleotidase CD73 (NT5E), which catalyzes AMP-to-adenosine conversion, is highly expressed by human but not murine brain endothelial cells (BECs). Here, we investigated the role of endothelial CD73 in ischemic stroke using an AAV vector engineered for selective transduction of murine BECs to induce BBB-specific CD73 expression. Endothelial CD73 enhanced extracellular ATP degradation toward adenosine generation and established a purine metabolism profile resembling that of human BECs. In the transient middle cerebral artery occlusion (tMCAO) mouse model, BBB-targeted CD73 expression reduced infarct volume by 40% and prevented early mortality within 48 h after reperfusion. Transcriptomic and flow cytometric analyses revealed altered leukocyte responses, including increased recruitment of monocytes/macrophages whose gene expression signatures were consistent with inflammation-resolving programs. These findings identify endothelial CD73 as an important regulator of post-ischemic neuroinflammation and highlight species-specific differences in BBB purine metabolism with implications for translational stroke research.

Background: Net Water Uptake (NWU) is a non-contrast CT (NCCT) biomarker of early cerebral edema in ischemic stroke, calculated from attenuation differences between ischemic and contralateral non-ischemic brain regions. Manual NWU quantification is labor-intensive and prone to inter-operator variability, limiting clinical uptake and research scalability. We developed and internally validated a fully automated NWU evaluation pipeline. Methods: We analyzed 24-hour follow-up NCCT scans from the AcT (Alteplase compared to Tenecteplase) trial. Infarcts were automatically obtained by segmentation framework based on a synchronous image-label diffusion probability model. The images and extracted infarcts were registered to the standard MNI152 space, allowing us to mirror the infarct onto the contralateral hemisphere symmetrically, regardless of size or tilt angle. Subsequently, the mirrored region was inversely transformed to return to its original space. Voxels outside the range of 20-80 Hounsfield Units (HU) were excluded to remove non-parenchymal tissue. Automated NWU was computed as the percentage difference in mean HU between infarct and mirrored contralateral regions. The agreement with manually determined NWU was evaluated using Pearson correlation, mean absolute error (MAE), and Bland-Altman analysis. Results: Of 1,327 patients in the trial, 298 (22.5%) met predefined imaging-quality criteria for the manual validation analysis, including well-aligned raw NCCT scans in the axial plane and clear parenchymal infarct segmentations. Automated 24-hour NWU showed excellent agreement with manual measurements (r = 0.99). Mean absolute error was 0.18% (95% CI: 0.01-0.46). Bland-Altman analysis demonstrated minimal bias (0.09%) and satisfactory limits of agreement (-4.05% to +4.24%). Ninety-nine percent of cases fell within {+/-}5% of the manually determined value. Conclusions: Our automated mirrored segmentation pipeline enables accurate and reproducible NWU quantification from routine 24-hour NCCT scans, matching expert manual measurements with minimal bias.

Purpose: {beta}-hydroxybutyrate (BHB), a ketone body and alternative cerebral energy substrate, can be measured in vivo using J-difference edited proton magnetic resonance spectroscopy (1H-MRS). Oral ketone supplementation with substrates such as the ketone monoester (R)-3-hydroxybutyl-(R)-3-hydroxybutyrate (KME) and 1,3-butanediol (BD) have gained attention as a mechanism to elevate circulating BHB and induce ketosis without dietary restrictions. Elevated brain ketone availability is of growing therapeutic interest as a strategy to support neuronal energetics in conditions such as epilepsy, neurodegenerative disease, and alcohol use disorder (AUD). However, both pathways introduce BD into the bloodstream, which crosses the blood-brain barrier. Critically, BD exhibits a spectral signature that closely resembles the prominent BHB peak in JDE-MR spectroscopic imaging (MRSI), identified in a pilot AUD study. Methods: Two separate JDE-MRSI acquisitions tailored for BHB and BD editing were implemented, exploiting frequency separation between the BHB (4.14ppm) and BD (3.95ppm) coupling partners of the observed 1.2ppm resonance to independently quantify each metabolite. Results: Brain BD concentrations (0.25-0.58mM) were comparable to or exceeded corresponding BHB concentrations (0.20-0.27mM) in all volunteers after consumption of a single dose of the KME, indicating that BD constitutes a major fraction of the signal conventionally attributed to BHB. Combined BHB+BD concentrations (~0.45-0.85mM) were consistent with brain BHB values reported in prior studies employing similar doses of the KME, indicating that those measurements likely reflect a combined BHB+BD signal. Conclusions: Separate quantification of the two metabolites is important for interpreting brain ketone studies and for understanding the full pharmacology of KME supplementation.

Perinatal ischemic stroke is an early developmental brain injury caused by obstruction of cerebral blood vessels and is a leading cause of cerebral palsy and cognitive disability in survivors. However, progress in understanding its impact on the brain and other organ systems, as well as in developing effective therapies, remains limited, in part due to the scarcity of relevant preclinical models. Here, we induced ischemic stroke via middle cerebral artery occlusion in perinatal mice and investigated its effects within and beyond the brain across development into adulthood. We found that perinatal stroke disrupted fine motor development and impaired memory. In addition, it induced structural alterations in skeletal muscle and significant changes in gut microbiota composition. Notably, gut-targeted intervention using fecal microbiota transplantation improved fine motor function. Our findings demonstrate, for the first time, the multisystem developmental impact of perinatal stroke, extending beyond the brain, and identify gut microbiota modulation as a promising and potentially safe therapeutic strategy to improve motor outcomes after stroke.

Somatic activating mutations in KRAS are found in the endothelium of the majority of sporadic brain arteriovenous malformations (bAVMs), yet the consequences of oncogenic KRAS signaling in endothelial cells during active vessel morphogenesis remain incompletely characterized. We expressed KRASG12V in primary human umbilical vein endothelial cells using a doxycycline-inducible lentiviral system and examined morphogenic behavior, proliferation, migration, and transcriptional output in a three-dimensional planar co-culture angiogenesis assay. KRASG12V-expressing cells failed to organize into vessel-like networks, instead forming compact sheet-like structures that persisted through day 12. A transient proliferative phase at days 3-5 resolved to control levels by day 12, consistent with preserved sensitivity to contact inhibition rather than unrestricted growth. Enhanced migration at day 5 was accompanied by upregulation of a focal adhesion and matrix remodeling program centered on ITGB3, PLAU, PLAUR, and PIK3CG. Translating ribosome-affinity purification sequencing (TRAP-seq) of the EC-specific translatome across four independent donor pools revealed progressive acquisition of an AVM-associated transcriptional identity by day 12, including upregulation of ACVRL1, ENG, JAG1, NOTCH1, ANGPT2, and TEK, with concordance to human bAVM nidus endothelium at both the gene and pathway level. Pharmacological inhibition with Alpelisib (PI3K), Trametinib (MEK), and Pazopanib (VEGFR2) demonstrated that PI3K is the principal organizer of the morphogenic phenotype. These findings characterize a KRASG12V-driven program in endothelial cells that recapitulates core transcriptional features of bAVM endothelium in a primary cell model.

Atrial fibrillation (AF) is associated with an increased risk of neurological morbidity, yet its impact on cerebral perfusion and neuro-cardiorespiratory regulation remains incompletely understood. We used arterial spin labeling, blood oxygenation level-dependent functional MRI (BOLD-fMRI), and a breath-hold challenge to characterize alterations in 14 AF patients compared with 14 age-matched healthy controls. We also examined the changes after catheter ablation with pulmonary vein isolation (PVI) in a subset of patients. Compared with controls, AF patients exhibited widespread reductions in basal cerebral perfusion, including in brainstem regions involved in cardiorespiratory regulation, and a higher prevalence of periodic breathing during wakeful rest. During breath-hold challenge, the coupling between heart rate and BOLD signal changes ({Delta}BOLD) was smaller in AF, whereas {Delta}BOLD coupling with breath-by-breath O2-CO2 exchange ratio was greater at rest within pontine respiratory centers, indicating altered cardiac and respiratory contributions to cerebral hemodynamic regulation. Follow-up MRI scans 1-6 months after PVI demonstrated that restoration of sinus rhythm was associated with stronger heart rate-{Delta}BOLD coupling during breath-hold challenge, whereas basal cerebral perfusion showed no significant change. This dissociation suggests distinct temporal responses of neuro-cardiorespiratory coupling and cerebral perfusion after sinus rhythm restoration, while the timing of cerebral perfusion recovery remains unresolved.

BackgroundQuantitative dynamic contrast-enhanced myocardial perfusion cardiovascular magnetic resonance (CMR) enables estimation of myocardial blood flow (MBF) and myocardial perfusion reserve (MPR). These measurements require an arterial input function (AIF), which is typically derived from the left ventricular blood pool. However, the contrast agent bolus undergoes dispersion during transport through the coronary vasculature before reaching the myocardial microcirculation. This may introduce systematic and spatially heterogeneous errors in MBF and MPR estimates. PurposeThis work provides an extended segmental analysis of bolus-dispersion-induced errors in quantitative myocardial perfusion MRI using previously established computational fluid dynamics (CFD) simulations in realistic porcine coronary artery models. The focus of the present analysis is the assignment of coronary outlets to myocardial segments and the resulting segmental variability of MBF and MPR errors. MethodsRealistic three-dimensional models of the left and right coronary arteries were extracted from an ex-vivo porcine imaging cryomicrotome dataset. The models extended down to the pre-arteriolar level and included 364 outlets for the left coronary artery and 104 outlets for the right coronary artery, with an average outlet diameter of 383 {+/-} 85 {micro}m. Blood flow was simulated under rest and stress conditions using OpenFOAM. Contrast agent transport was then modeled by solving the advection-diffusion equation using a gamma-variate bolus as input. Outlet concentration-time curves were analyzed using an indicator-dilution model to estimate MBF and MPR errors. Outlets were assigned to standardized myocardial segments, and segmental averages were evaluated with respect to coronary supply territory and travel distance from the model inlet. ResultsThe simulations demonstrated marked segmental heterogeneity of volume blood flow and bolus-dispersion-induced MBF and MPR errors. Errors increased with travel distance from the coronary artery inlet and were more pronounced in regions supplied by the right coronary artery, consistent with lower flow velocities and stronger bolus dispersion. The resulting systematic errors led to underestimation of MBF and overestimation of MPR, with segmental deviations reaching up to approximately 60%. ConclusionBolus dispersion in the coronary vasculature may lead to substantial segmental and location-dependent errors in quantitative myocardial perfusion MRI. This extended analysis indicates that dispersion-related bias is not spatially uniform, but depends on coronary supply territory, travel distance, and flow conditions. These effects should be considered when interpreting regional MBF and MPR estimates, particularly as automated quantitative myocardial perfusion CMR becomes more widely used.

Background: Cerebral vasospasm is a frequent complication after aneurysmal subarachnoid hemorrhage (aSAH) and is associated with delayed cerebral ischemia (DCI) and unfavorable outcome. While CTA-based vasospasm grading is frequently used, its relationship with actual cerebral perfusion remains incompletely understood. This study investigates the association between vasospasm severity and distribution and territorial perfusion deficits. Methods: In this retrospective single-center study, 513 CT examinations (CTA and CT perfusion) from 194 patients with aSAH were analyzed. Vasospasm was graded per vessel segment using the CTA Vasospasm Score, and perfusion deficits were assigned to corresponding vascular territories (left/right anterior circulation, posterior circulation). Vasospasm distribution was further classified by severity and multifocality. Associations between vasospasm score and perfusion deficits were assessed using a generalized linear mixed model with binomial distribution, adjusting for Hunt & Hess grade, modified Fisher score, and days since hemorrhage. Results: Vasospasm was detected in 79.3% of examinations, and a perfusion deficit in at least one territory was present in 62.6%. The proportion of perfusion deficits increased progressively with both vasospasm severity and multifocality, ranging from 21.7-25.0% in the absence of vasospasm to 81.2-82.2% in severe multifocal vasospasm. The CTA Vasospasm Score was significantly associated with perfusion deficits in all territories (OR 1.36-1.50), with stronger associations in the anterior than posterior circulation. Conclusion: Vasospasm severity and distribution are strongly associated with perfusion deficits, supporting a continuum model of ischemic risk. However, the substantial proportion of perfusion deficits occurring independent of vasospasm suggests additional microcirculatory mechanisms not captured by CTA. CT perfusion should be considered complementary to CTA, particularly in clinically deteriorating or non-assessable patients.

Acute ischemic stroke (AIS) induces profound systemic immune alterations that contribute to infection susceptibility. Here, we identify lipocalin-2 (LCN-2) as a rapidly induced and conserved regulator of stroke-associated immunosuppression. Using 3-MACE-Seq, cytokine profiling, and immunofluorescence in C57BL/6J mice subjected to transient middle cerebral artery occlusion (tMCAO), we show that LCN-2 is strongly upregulated in splenic red pulp macrophages (RPMs) within 24 hours and again 7 days post-tMCAO. LCN-2-expressing RPMs form immunological synapses with CD3+ T cells, thereby impacting T cell trafficking. Recombinant LCN-2 directly reprogrammed T cells and monocytes toward hyporesponsive, tolerogenic phenotypes by suppressing inflammatory cytokines, impairing chemotaxis, enhancing phagocytosis, and uncoupling oxidative burst. Human spleens likewise displayed LCN-2-expressing CD68+ RPMs, and LCN-2 preconditioning of monocytes reproduced reduced HLA-DR, CD80, CD206, and ROS with increased uptake of E. coli bioparticles. These findings identify LCN-2 signaling as a central orchestrator of stroke-induced peripheral immunoreprogramming and a potential therapeutic target to mitigate post-stroke immunodepression. SummaryAcute ischemic stroke induces LCN-2 in splenic red pulp macrophages, which reprogram T cells and monocytes toward tolerogenic, hyporesponsive states. Mouse and human data identify LCN-2 as a driver of peripheral immunodepression and a potential target to reduce infection risk. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/733904v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@149bd46org.highwire.dtl.DTLVardef@2974aaorg.highwire.dtl.DTLVardef@1aa7a52org.highwire.dtl.DTLVardef@144dbe5_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Quantifying blood-brain barrier (BBB) integrity from fluorescence microscopy remains limited by subjective scoring and categorical classification methods that lack reproducibility. For objective and consistent BBB phenotyping, we present two semi-automated image-analysis pipelines that replace manual scoring with quantitative, continuous-variable measurements. Our in vitro pipeline, implemented in Python, quantifies the connectivity of tight junction structures by measuring discrete ZO-1 fragment objects within manually traced junction regions. It outputs continuous metrics including average fragment area, total junctional area, and a junctional fragmentation ratio that captures degree of ZO-1 continuity versus discontinuity. In human brain microvascular endothelial cells subjected to glycocalyx component knockdown, the pipeline detected significantly reduced fragment area (37% decrease for both CD44 and syndecan-1 (SDC1) knockdown, p = 0.0148 and 0.0084) and junctional fragmentation ratio (p = 0.0061 and 0.0137). Our in vivo pipeline integrates ilastik-based pixel classification with FIJI macro automation to quantify vascular marker colocalization and to separate vessel signal from microglial contamination within a single fluorescence channel, eliminating the need for dedicated counterstains. Applied across four mouse cohorts [young, aged, Alzheimer's, traumatic brain injury (TBI)] and three brain regions [prefrontal cortex (PFC), hippocampus, midbrain], the pipeline revealed concurrent ZO-1 loss and ICAM-1 elevation in the PFC and hippocampus of aged and Alzheimer's mice, with Alzheimer's-specific doubling of eNOS occurring in the PFC (p = 0.0013). TBI mice showed persistent ZO-1 loss with transient ICAM-1 and eNOS changes. Both deterministic pipelines are available on GitHub and designed for adoption beyond the specific markers and systems analyzed here.

Purpose: To develop an accelerated motion-compensated diffusion-weighted pseudo-continuous arterial spin labeling (MCDW-pCASL) method using a spatial subspace low-rank reconstruction method for efficient quantification of blood-brain barrier (BBB) water exchange (kw) and permeability (PSw). Methods: An accelerated multidelay MCDW-pCASL sequence was developed to simultaneously encode intravascular and extravascular diffusion-weighted ASL signals across multiple post-labeling delays (PLDs). A spatial subspace low-rank reconstruction framework was optimized to enable joint estimation of cerebral blood flow (CBF) and BBB water exchange rate and permeability. Fourteen young healthy adults underwent test-retest scans (separated by ~1 week) at 3T with both the accelerated MCDW-pCASL and a conventional diffusion-prepared (DP) pCASL sequence. Whole-brain, gray-matter, and white-matter CBF and kw values were quantified to assess test-retest repeatability and cross-method agreement. An additional cohort of 30 older adults underwent single-session MCDW and DP scans to evaluate age-related perfusion and BBB kw/PSw differences. Intraclass correlation coefficients (ICCs) were used to assess reliability and agreement. Results: Accelerated MCDW-pCASL demonstrated excellent agreement with DP-pCASL for CBF (ICC = 0.89) and fair agreement for kw (ICC = 0.56). Test-retest repeatability of MCDW-pCASL was good for CBF, BBB kw and PSw (ICC {approx} 0.6). Across both sequences, younger subjects exhibited significantly higher CBF and kw compared with older adults. Conclusion: Incorporating a spatial low-rank subspace reconstruction enables accelerated MCDW-pCASL acquisition with reliable simultaneous quantification of CBF, BBB kw and PSw. Clinical applications of this method for assessing perfusion and BBB function are warranted.

Background: Coronary artery calcium (CAC) is an established measure of coronary atherosclerosis from computed tomography (CT). While deep learning (DL) can quantify CAC from non-dedicated CT, the accuracy is limited by image quality. Purpose: We derived and validated a novel method for DL CAC segmentation on ultra-low dose CT attenuation correction (CTAC) scans that is trained with synthetic low-dose, ungated images. Materials and Methods: Models were trained using one center and externally tested in two other centers. Synthetic, ungated CT scans were generated so that expert segmentations from dedicated CAC scans could be used as ground truth for perfectly registered synthetic images through knowledge adaptation (KAD-CAC). We evaluated agreement between CAC scoring methods vs expert readers on a per-patient and per-vessel basis, as well as associations with the primary outcome of death or myocardial infarction (MI). Results: The DL models were externally tested on 5969 patients with a median age of 64 (IQR 56 - 73), of whom 50.2% were male. The KAD-CAC model had higher Cohens kappa K (0.86, 95% CI 0.85 - 0.87) compared to previous convolutional LSTM model (K 0.78, 95% CI 0.76 - 0.80, p<0.01), or models trained with only gated images (K 0.81, 95% CI 0.80 - 0.82, p<0.01). Net reclassification improvement for CAC stratified risk of death or MI, was greatest for the KAD-CAC model over baseline including age, sex, hypertension, diabetes, dyslipidemia, family history, smoking, stress total perfusion deficit, and left ventricular ejection fraction. Conclusion: We use paired synthetic ungated scans to transfer expert gated CAC annotations into the ungated domain, resulting in substantially better vessel-level CAC scoring and improved risk stratification.

Background: Nuclear medicine and radiopharmaceutical development require coordinated radiochemistry, dosimetry, molecular imaging, radiation-safety and clinical decision processes. Current workflows remain fragmented, difficult to audit and poorly standardised for evaluating domain-specific AI support. Methods: We developed RadGuide AI, a nuclear medicine agent built around a traceable data-model-tool loop. Patent, literature and clinical-trial records were converted into 15,596 initial QA items; relevance screening, completeness checks, semantic deduplication and cross-validation retained 5,474 core QA items. MedGemma-27B-Instruct served as the foundation model and was adapted with LoRA. The system incorporated 55 MCP-wrapped tools covering radiopharmaceutical R&D, clinical decision support, imaging analysis and radiation-safety/dosimetry. Evaluation used a locked N=200 benchmark with predefined denominators, leakage control, expert scoring, statistical procedures, factuality audits and tool-execution metrics. Results: RadGuide-LLM achieved 88.5% answer accuracy (177/200; 95% CI, 83.3-92.2%) and a Macro-Average score of 21.5/25 (bootstrap 95% CI, 20.9-22.0), exceeding GPT-4o, DeepSeek-V3.2 and the base MedGemma model in this technical evaluation. Supplementary audits reported guideline compliance, terminology recall, knowledge coverage, tool-routing success and preclinical/phantom dosimetry agreement with explicit denominators and confidence intervals. Interpretation: RadGuide AI converts nuclear medicine queries into auditable retrieval, tool selection, calculation, verification and reporting workflows. The findings support technical feasibility, not definitive patient-level clinical validation; prospective multicentre studies and external benchmark release remain required before clinical deployment.