Recombinant tissue plasminogen activator (r-tPA) remains the cornerstone of reperfusion therapy in patients with stroke. However, response to r-tPA is highly variable and not fully explained by clinical factors with many patients not achieving a good outcome despite thrombolysis treatment. In 78 patients with acute ischemic stroke treated with r-tPA, thrombin generation capacity was measured in plasma. Functional outcome was assessed at 90 days using the modified Rankin Scale (mRS). In addition, inflammatory markers were analyzed to identify factors associated with increased thrombin generation in this patient population. Thrombin generation capacity was significantly greater in patients with worse 90-day mRS (mRS > 2) compared to those with good 90-day mRS (mRS ≤ 2; 254 nM vs. 210 nM; p = 0.0007). This association remained significant after adjustment for admission NIHSS, age, sex, stroke etiology, and atrial fibrillation (OR: 1.68, 95
Sphingolipids critically regulate microvascular integrity and function, but the role of glycosphingolipids in endothelial survival and angiogenesis remains poorly defined. Herein, we experimentally deactivated or activated UDP-glucose ceramide glucosyltransferase (UGCG), which converts ceramide to glucosylceramide, by the pharmacological inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), siRNA-mediated knockdown or the pharmacological activator L-PDMP. Sphingolipid and glycosphingolipid profiles were examined by liquid chromatography-tandem-mass spectrometry. Effects on endothelial survival, proliferation, transwell migration, extracellular vesicle (EV) release and tube formation were assessed in human cerebral microvascular endothelial cells (hCMEC/D3). In vitro, pharmacological UGCG deactivation near-completely suppressed hexosylceramide levels and at high dose increased ceramide and sphingosine-1-phosphate (S1P), a known pro-angiogenic sphingolipid, while UGCG knockdown moderately decreased mostly short (C16, C18) hexosylceramides. UGCG activation increased hexosylceramide without significantly altering ceramide and S1P. Pharmacological UGCG deactivation increased endothelial tube formation, a marker of angiogenesis, but at high dose decreased endothelial survival, whereas UGCG knockdown and UGCG activation reduced endothelial tube formation and migration or proliferation, respectively. Pharmacological UGCG deactivation and activation, but not UGCG knockdown increased endothelial release of EVs with anti-angiogenic activity. In mice exposed to transient middle cerebral artery occlusion, pharmacological UGCG deactivation and activation reduced the length and branch density of small-sized (< 4 µm) and intermediate (4–5.4 µm) cerebral microvessels in the reperfused striatum as revealed by 3D light-sheet microscopy, indicative of microvascular endothelial degeneration. Our results suggest that pharmacological UGCG deactivation promotes angiogenesis in vitro probably via S1P elevation. In vivo, UGCG deactivation failed to stabilize microvascular network integrity post-ischemia/reperfusion, presumably due to ceramide-associated cell stress.
Intracerebral hemorrhage (ICH) produces a rapidly evolving and spatially heterogeneous neurovascular microenvironment in which secondary injury is shaped not only by hematoma volume and location, but also by the interaction of blood-derived toxins, blood–brain barrier disruption, edema, oxidative stress, protease activity, and glial responses. Increasing evidence suggests that these processes are better understood as dynamic network events rather than isolated inflammatory pathways. This review applies a network-centered framework to astrocyte–microglia coupling, viewing it as a critical control layer that may either support injury containment and hematoma resolution or drive persistent neurotoxicity and failed repair. Comparisons with ischemic stroke are used to distinguish shared inflammatory modules from hemorrhage-specific drivers, including heme, hemoglobin, iron overload, thrombin, fibrinogen, and clot-associated protease signaling. Integrating findings from single-cell and spatially resolved studies, the review summarizes the temporal and spatial organization of post-hemorrhagic microenvironment remodeling and discusses astrocyte-dependent regulation of barrier function, edema dynamics, immunometabolism, redox buffering, and synaptic homeostasis. It also examines how astrocyte-derived cues influence microglial state transitions through danger sensing, inflammasome signaling, cyclic GMP–AMP synthase–stimulator of interferon (IFN) genes signaling, phagocytic containment, iron-handling programs, complement-mediated synaptic vulnerability, and interaction with infiltrating myeloid cells. Recurring astrocyte–microglia network motifs are further evaluated as therapeutic control points, with emphasis on how lesion stage and spatial compartmentalization shape intervention windows for purinergic, chemokine, cytokine, IFN, complement–coagulation, and lipid/iron signaling pathways. Translational priorities, limitations, and therapeutic opportunities are discussed across hematoma-toxicity reduction, barrier and edema repair, network reprogramming, and regenerative microenvironment shaping. Meaningful improvement in ICH outcome will likely depend on biomarker-guided and stage-specific reprogramming of astrocyte–microglia network dynamics to restore microenvironmental balance, rather than on nonspecific suppression of neuroinflammation.
Despite the remarkable technical success of endovascular thrombectomy (EVT) in achieving high recanalization rates for acute ischemic stroke due to large vessel occlusion (LVO), angiographic success does not consistently translate into functional recovery. Approximately 50
VEGFA plays a pivotal role in angiogenesis and is known to affect clinical phenotypes related to the vasculature in various diseases, such as cancer or stroke. However, it is still unclear how the VEGFA genotype influences the clinical phenotype of moyamoya disease (MMD). This study included 137 Japanese MMD patients (84 adult and 53 pediatric patients) carrying either the heterozygous RNF213 p.R4810K or the RNF213 wild-type genotypes. Patients with homozygous RNF213 p.R4810K genotype or the other rare RNF213 variants were excluded because these genotypes are already known to affect the clinical phenotype. After genotyping VEGFA rs2010963 (NM_001171623.2, c.-634G > C, also known as c.+405G > C), we statistically analyzed the genotype-phenotype correlation. Genotyping and clinical data collection were both blinded. 110 (80.3
Background. Ring finger protein 213 (RNF213) p.R4810K is a genetic susceptibility factor for intracranial arteriopathies. In patients with isolated intracranial arterial steno-occlusive disease (ICAD) without Moyamoya disease (MMD), we developed a phenotype-based triage score to prioritize RNF213 genotyping by enriching the p.R4810K carriage probability. Methods. We retrospectively analyzed 753 patients with isolated ICAD involving the distal internal carotid artery or middle cerebral artery M1 segment who underwent RNF213 genotyping between January 2010 and November 2022, excluding definite MMD or significant extracranial steno-occlusion. A prespecified score incorporated anterior cerebral artery (ACA) laterality (0/1/2), tandem lesion burden ≥ 3 (0/1), and MMD family history (0/1; weighted ×4). Discrimination, prespecified thresholds (≥ 3 and ≥ 4), and bootstrap-corrected calibration were assessed, with external application in an independent cohort. Results. RNF213 p.R4810K was identified in 289 patients (38.4
To investigate the associations of traditional and novel lipid parameters with the hemorrhagic phenotype in adults with primary moyamoya disease (MMD) and to evaluate their value for phenotype discrimination and stratification. This retrospective dual-center cross-sectional study included 1,176 adults with primary MMD treated at Beijing Hospital and Beijing Tiantan Hospital between January 2022 and January 2026, including 857 patients with a non-hemorrhagic phenotype and 319 with a hemorrhagic phenotype. Traditional and derived lipid parameters were analyzed. Missing body mass index values were handled using multiple imputation. Multivariable logistic regression, restricted cubic spline analysis, receiver operating characteristic analysis, incremental discrimination analysis, sensitivity analyses, subgroup analyses, and exploratory mediation analysis were performed. Compared with patients with the non-hemorrhagic phenotype, those with the hemorrhagic phenotype had lower body mass index and higher levels of several cholesterol-related lipid parameters. In fully adjusted models, total cholesterol, low-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol were significantly associated with the hemorrhagic phenotype, with odds ratios for the highest versus lowest quartile of 3.435, 3.197, and 3.150, respectively. These indicators showed modest individual discriminative ability and provided limited incremental improvement when added to the basic clinical model. Sensitivity and subgroup analyses were generally consistent. Exploratory analyses identified BMI-related negative indirect effects. Cholesterol-related lipid parameters, particularly total cholesterol, low-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol, were associated with the hemorrhagic phenotype in adult primary MMD and may serve as supplementary markers for phenotype stratification.
Combined central-peripheral magnetic stimulation shows promise for post-stroke dysphagia (PSD), but its neural mechanisms remain unclear. We evaluated the its efficacy and cortical mechanisms in PSD patients. In this single-blind, parallel-controlled trial, 44 PSD patients were randomized to four groups: active-rTMS + active-rPMS, active-rTMS + sham-rPMS, sham-rTMS + active-rPMS, or sham-rTMS + sham-rPMS. Clinical scales, such as functional oral intake scale (FOIS) and Penetration-Aspiration Scale (PAS), and Videofluoroscopic Swallowing Study (VFSS) were accessed before, after intervention. Cortical activation during rest and swallowing task was measured by functional near-infrared spectroscopy (fNIRS). Significant interaction effects (time × intervention) were found for FOIS (Wald χ2 = 39.816, p < 0.001) and PAS scores (Wald χ2 = 45.433, p < 0.001), with the active-rTMS + active-rPMS group showing the most pronounced therapeutic gains. Concurrently, this group displayed significant improvements of laryngeal vestibular closure duration (LCD, MD = –3.598, 95
Post-stroke cognitive impairment (PSCI) reflects vulnerability to cognitive decline beyond acute brain injury. Lipoprotein(a) (Lp(a)) is a genetically determined vascular risk factor with pro-inflammatory and pro-atherogenic properties, but the pathways linking Lp(a) to cognitive outcomes after stroke remain unclear. We examined whether systemic inflammation mediates or modifies Lp(a)-related cognitive risk after ischemic stroke. We analyzed data from a prespecified substudy of the Third China National Stroke Registry. Baseline serum Lp(a) and high-sensitivity C-reactive protein (hs-CRP) were measured. PSCI was defined as a Montreal Cognitive Assessment score ≤ 22 at 1 year. Multivariable logistic regression, interaction, joint exposure, and causal mediation analyses were performed to evaluate the role of systemic inflammation in the Lp(a)–PSCI association. Among 954 patients with acute ischemic stroke, higher baseline Lp(a) was associated with increased PSCI risk after adjustment for baseline cognitive performance and clinical covariates (highest vs. lowest tertile: OR, 1.479; 95
Background: Stroke is a major complication of atrial fibrillation (AF), and risk prediction using the congestive heart failure, hypertension, age, diabetes, stroke, vascular disease, and sex category score (CHA₂DS₂-VASc) remains limited by residual heterogeneity. We aimed to identify plasma proteins associated with post-AF stroke and evaluate whether a protein score provides incremental predictive information beyond CHA₂DS₂-VASc. Methods: We analyzed 709 AF participants from the UK Biobank Pharma Proteomics Project, with 76 incident strokes. Stroke-related proteins were identified using multivariable Cox regression, least absolute shrinkage and selection operator (LASSO) Cox regression, and machine-learning approaches. A five-protein score was constructed, and its incremental value beyond CHA₂DS₂-VASc was assessed by discrimination, calibration, reclassification, clinical net benefit, and 1000-bootstrap internal validation. Mendelian randomization served as supportive genetic evidence. Results: Five core proteins were identified: epidermal growth factor receptor (EGFR), V-type proton ATPase subunit D (ATP6V1D), neurotrophin 4 (NTF4), amnionless (AMN), and discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2). Adding the five-protein score to CHA₂DS₂-VASc improved discrimination, increasing the concordance index from 0.681 to 0.768. The combined model had 3-, 5-, and 8-year receiver operating characteristic areas of 0.799, 0.775, and 0.806, respectively, and showed favorable five-year prediction error and calibration, with a Brier score of 0.0347, calibration intercept of −0.068, and calibration slope of 0.968. The score improved continuous net reclassification improvement (0.459; P = 0.028). Mendelian randomization provided supportive genetic evidence for AMN, EGFR, and DCBLD2. Conclusions: The five-protein score provided incremental predictive information beyond CHA₂DS₂-VASc for post-AF stroke risk assessment.
Stroke is the second most common cause of death worldwide and predominantly affects individuals over 65 years old. Its prevalence is projected to increase in parallel with the aging global population. Nutrition is a modifiable risk factor for ischemic stroke. Folates, B-vitamins and choline play a central role in one-carbon metabolism (1 C), which is a key metabolic network that integrates nutritional signals with biosynthesis, redox homeostasis, epigenetics, regulation of cell proliferation, and stress resistance. Using preclinical models, our research group has previously shown that deficiencies in 1 C lead to worse stroke outcomes. However, the impact of ischemic stroke on 1 C enzymes in human brain tissue remains unexplored. The objective of this study is to investigate whether ischemic stroke contributes to a change in the levels of 1 C enzymes after ischemic stroke in male and female patients. Cortical brain tissue sections from ischemic stroke patients and controls were stained for enzymes involved in 1 C. All tissue was co-stained with neuronal nuclei (NeuN) and DAPI (4′,6-diamidino-2-phenylindole). The colocalization of all three markers was evaluated by two individuals who were masked to the experimental groups. Ischemic stroke increased neuronal levels of the folate receptor and 1 C enzymes, methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS) and serine hydroxy methyltransferase (SHMT). In male stroke brain tissue was observed to have increased levels of MTHFR, TS, and SHMT. Female brain tissue had increases in the folate receptor and TS. The results suggest that ischemic stroke leads to changes in neuronal levels of FR and 1 C enzymes levels in penumbra. Further clinical investigation is required to determine whether there is increase enzymatic activity and how 1 C is impacted in other cells within the brain, such as glial and endothelial cells.
TMEM16A forms a Ca²⁺-activated Cl⁻ channel in vascular mural cells (smooth muscle cells and pericytes) that generates depolarizing Cl⁻ efflux upon intracellular Ca²⁺ elevation, thereby amplifying agonist-induced vasoconstriction. TMEM16A has been implicated in excessive capillary pericyte constriction following cerebral ischemia, suggesting that its inhibition may improve post-stroke recovery. However, the impact of systemic vascular TMEM16A inhibition on focal reperfusion efficiency and cerebrovascular autoregulation remains unknown. To address this question, mice with inducible mural cell-specific (Myosin Heavy Chain 11 promoter controlled) deletion of TMEM16A were subjected to transient middle cerebral artery occlusion. Reperfusion dynamics and stroke-reperfusion outcome were assessed using laser speckle contrast imaging, cylinder test for motor function, and infarct quantification by 2,3,5-triphenyltetrazolium chloride staining. Systemic cardiovascular parameters were monitored with radiotelemetry. Middle cerebral artery myogenic tone was assessed with pressure myography. Mice lacking TMEM16A in mural cells exhibited impaired reperfusion and worsened stroke outcome compared with wild-type controls, despite unchanged systemic cardiovascular parameters. In wild-type mice, capillary pericytes maintained basal contractile tone in both hemispheres, and this was further enhanced in peri-infarct cortex. In contrast, TMEM16A-deficient capillary pericytes lacked basal tone in both the ipsilateral and contralateral hemispheres. TMEM16A-deficient middle cerebral arteries failed to develop pressure-induced myogenic tone. These findings demonstrate that TMEM16A is required for effective cerebral autoregulation and that its deficiency significantly impairs post-ischemic reperfusion. The results caution against systemic TMEM16A inhibition as a therapeutic strategy for stroke and highlight the need for spatially restricted approaches to modulate cerebral perfusion via the Ca²⁺-activated Cl⁻ channels.
Secondary brain injury (SBI) following intracerebral hemorrhage (ICH) is heavily driven by the mechanical compression of the expanding hematoma, yet how neurons transduce this physical force into pathological intracellular signals remains poorly understood. This study investigates the role of the mechanosensitive ion channel Piezo2 in ICH-induced SBI and its underlying molecular mechanisms. Using a collagenase-induced ICH mouse model and single-cell RNA sequencing analysis, we identified a marked upregulation of Piezo2 in perihematomal neurons. To determine its functional significance, we employed both genetic knockdown (shRNA) and pharmacological modulation with D-GsMTx4 in vivo. Modulation of Piezo2 significantly alleviated acute neurological deficits, reduced brain edema, and improved long-term cognitive performance in ICH mice. Mechanistically, we observed that the neuroprotective effects of Piezo2 inhibition were associated with an attenuation of neuronal endoplasmic reticulum (ER) stress. Specifically, inhibition of Piezo2 preserved ER ultrastructure, which was accompanied by a robust downregulation of the PERK/ATF4/CHOP signaling cascade markers. Collectively, our findings suggest that Piezo2 contributes to neuronal damage following ICH and that its modulation impacts ER stress. Targeting Piezo2 represents a novel experimental concept to mitigate secondary neurodegeneration associated with hematoma-induced mechanical strain, though extensive preclinical optimization is required before considering its clinical viability.
Vascular endothelial injury underlies the development of severe complications following acute ischemic stroke. While mitochondrial microparticles (mtMPs) compromise endothelial integrity, their mechanistic role in endothelial dysfunction and clinical association with malignant cerebral edema (MCE) remain unclear. We analyzed paired intracranial and peripheral arterial blood samples from patients with anterior circulation large vessel occlusion stroke (LVO-AIS) who underwent thrombectomy. Human brain microvascular endothelial cells were stimulated with intracranial versus peripheral mtMPs, followed by RNA sequencing and analysis. The associations between mtMP levels and MCE were evaluated via multivariate logistic regression and receiver operating characteristic (ROC) analyses. Subgroup and sensitivity analyses were conducted to verify the consistency of the findings. Paired samples (n = 16) revealed higher mtMP levels in intracranial blood than in peripheral blood (P = 0.02). RNA sequencing revealed that intracranial mtMPs altered endothelial gene expression (80 upregulated, 52 downregulated), enriching pattern recognition and proinflammatory pathways, alongside enrichment trends in oxidative stress and neutrophil chemotaxis. In the clinical cohort (n = 90, 20
Inflammation contributes to haematoma expansion (HE) and poor prognosis in spontaneous intracerebral haemorrhage (ICH). This study aimed to characterise the temporal profile of soluble Toll-like receptor 4 (sTLR4) during the hyperacute and acute phases of ICH and to evaluate its association with HE. Serum sTLR4 levels were measured at admission, 24 and 72 h from 99 patients with primary hemispheric ICH within 12 h of symptom onset, and at baseline from 39 non-stroke controls using enzyme-linked immunosorbent assay (ELISA). Longitudinal sTLR4 fluctuations were evaluated through linear mixed-effects models. Independent HE prognosticators were identified using multivariable logistic regression, and model performance was assessed via the area under the receiver operating characteristic curve (AUC-ROC). ICH patients exhibited significantly higher baseline sTLR4 levels than controls, which declined over time and were not associated with clinical variables. Notably, patients with HE demonstrated higher admission sTLR4 levels than those without HE (2.50 [95
Chronological age is a strong predictor of poor outcomes after ischemic stroke but may not fully capture underlying biological vulnerability. This study investigated whether age-related brain atrophy and plasma YKL-40, a marker of astroglial inflammation, mediate the association between age and the one-year risk of ischemic stroke recurrence or all-cause mortality. Data were obtained from 4,305 participants enrolled in the Third China National Stroke Registry. Baseline brain atrophy was quantified from structural T1-weighted MRI using an automated deep learning–based pipeline (FastSurfer), yielding hemispheric cortical and white matter volumes that were modeled as indicators of a latent atrophy construct. Structural equation modeling was applied to estimate direct and indirect pathways linking age, brain atrophy, YKL-40, and one-year composite outcomes, adjusting for sex, atrial fibrillation or flutter, hypertension, and diabetes, with indirect effects evaluated using 5,000 bootstrap resamples. The total effect of age on one-year outcomes was not significant (β = −0.011; 95
Ischemic stroke is the most prevalent form of stroke worldwide and remains a major cause of long-term disability. Impaired autophagic flux is a critical pathological mechanism that worsens neuronal injury after ischemia. This study aimed to elucidate the role of phosphatidylinositol-5-phosphate 4-kinase type II alpha (PIP4K2A) and its regulation of autophagy in cerebral ischemia/reperfusion (I/R) injury. Exploratory TMT-based serum proteomics identified PIP4K2Aas an elevated candidate protein in patients with acute ischemic stroke (AIS). Whole-blood RT-qPCR validation showed increased PIP4K2A mRNA in AIS patients and an association between higher PIP4K2A expression and lower NIHSS scores, although the small clinical cohort and peripheral sampling design preclude causal or tissue-origin conclusions. Using a transient middle cerebral artery occlusion (tMCAO) mouse model and a primary neuronal oxygen-glucose deprivation/reperfusion (OGD/R) model, we found that I/R injury markedly upregulated PIP4K2A expression in ischemic brain tissue and primary neurons. AAV-mediated PIP4K2A overexpression in vivo alleviated ischemic brain injury, preserved neuronal survival, reduced infarct volume, and improved long-term cognitive and motor functions, whereas PIP4K2A knockdown exacerbated these outcomes. In vitro, lentiviral-mediated PIP4K2A overexpression improved neuronal viability after OGD/R. Mechanistically, RNA-seq, co-immunoprecipitation, and mCherry-EGFP-LC3 tandem reporter analyses showed that PIP4K2A overexpression was associated with reduced TRIB3 mRNA and protein levels, decreased abundance of the stress-induced TRIB3-p62 complex, improved autophagic flux, and reduced autophagosomal accumulation. Concurrently, PIP4K2A-associated TRIB3 reduction was accompanied by enhanced AKT/mTOR signaling. These findings identify PIP4K2A as an endogenous protective regulator in cerebral ischemia/reperfusion injury and suggest that thePIP4K2A/TRIB3/p62 axis may represent a potential therapeutic target for ischemic stroke.
A significant proportion of ischemic strokes are classified as embolic stroke of undetermined source (ESUS), complicating secondary prevention. Nitric oxide metabolism is regulated by L-arginine (Arg), asymmetric (ADMA) and symmetric dimethylarginine (SDMA), contributing to increased cardiovascular risk. Previous studies suggest that SDMA and the ratio of Arg/SDMA help to detect atrial fibrillation (AF). We hypothesized that SDMA and Arg/SDMA improve diagnosis of cardioembolic stroke (CES), which may enable more accurate secondary prevention. This prospective nested case-control study included ischemic stroke patients (CES or ESUS) at Hannover Medical School between January 2022 and July 2023. Blood samples were collected after 24 h of stroke onset. ADMA, SDMA and Arg concentrations were measured and relevant biomarker ratios were calculated. Logistic regression and receiver operating curve analyses examined discriminatory biomarker performances for CES versus ESUS, alongside established clinical risk scores. 235 patients (107 CES, 128 ESUS) were analyzed. Arg/SDMA ratio and SDMA values showed comparable or better discriminatory power between CES and ESUS than clinical AF scores (AUC SDMA = 0.71 (95
Sedation is frequently required in the acute management of ischemic stroke (AIS), yet its potential to influence secondary brain injury and systemic complications remains insufficiently integrated into clinical practice. This narrative review examines experimental and clinical evidence on the biological effects of commonly used sedatives in neurocritical care, propofol, dexmedetomidine, and ketamine, and explores their potential role as active therapeutic agents in AIS. Experimental studies consistently show that propofol reduces oxidative injury and apoptotic signalling, dexmedetomidine attenuates neuroinflammation and sympathetic overactivation, and ketamine limits excitotoxicity through N-methyl-D-aspartate receptor blockade. Together, these mechanisms provide a plausible basis for mitigating infarct progression and protecting vulnerable neural networks during the acute phase. Clinical data remain limited, predominantly observational, and heterogeneous, but suggest that sedative choice may influence neurological recovery, hemodynamic stability, and extra-cerebral organ function. These potential benefits must be weighed against known risks, including hemodynamic effects of propofol, blood pressure reductions with dexmedetomidine, and historical concerns regarding ketamine and intracranial dynamics. At present, the absence of standardized sedation strategies for AIS and the variability in study design preclude firm conclusions. Recognizing sedation as an intervention with possible neuroprotective and systemic effects, rather than solely a means of comfort or procedural facilitation, may open new avenues for targeted multiorgan protection in early stroke care. Rigorous clinical trials are needed to define how propofol, dexmedetomidine, or ketamine can be optimally integrated into AIS management.
