Muscle mass as a modifier of stress response in acute ischemic stroke patients.

Stroke triggers a systemic inflammatory response over the ensuing days after the cerebral insult. The age and comorbidities of the stroke population make them a vulnerable population for low muscle mass and sarcopenia, the latter being another clinical condition that is closely associated with inflammation, as shown by increased levels of pro-inflammatory biomarkers, including neutrophil-to-lymphocyte ratio (NLR). In this study, we evaluated the relationship between post-stroke NLR changes and muscle mass in a prospective cohort of acute ischemic stroke patients (n = 102) enrolled in the Muscle Assessment in Stroke Study Turkey (MASS-TR). Admission lumbar computed tomography images were used to determine the cross-sectional muscle area of skeletal muscles at L3 vertebra level and calculate the skeletal muscle index (SMI). The median (IQR) SMI was 44.7 (39.1-52.5) cm2/m2, and the NLR at admission and follow-up were 4.2 (3.0-10.5) and 9.4 (5.7-16.2), respectively. While there was no relationship between SMI and admission NLR, a significant inverse correlation was observed between SMI and follow-up NLR (r = - 0.26; P = 0.007). Lower SMI remained significantly associated (P = 0.036) with higher follow-up NLR levels in multivariate analysis. Our findings highlight the importance of muscle mass as a novel factor related to the level of post-stroke stress response.

Ischemic stroke outcome after promoting CD4+CD25+ Treg cell migration through CCR4 overexpression in a tMCAO animal model.

The importance of neuroinflammation during the ischemic stroke has been extensively studied. The role of CD4+CD25+ regulatory T (Treg) cells during the recovery phase have shown infarct size reduction and functional improvement, possibly through the mitigation of inflammatory immune responses. We aimed to investigate the molecular factors involved in microglia-Treg cell communication that result in Treg trafficking. First, we observed the migration patterns of CD8+ (cytotoxic) T cells and Treg cells and then searched for chemokines released by activated microglia in an oxygen-glucose deprivation (OGD) model. The transwell migration assay showed increased migration into OGD media for both cell types, in agreement with the increase in chemokines involved in immune cell trafficking from the mouse chemokine profiling array. MSCV retrovirus was transduced to overexpress CCR4 in Treg cells. CCR4-overexpressed Treg cells were injected into the mouse transient middle cerebral artery occlusion (tMCAO) model to evaluate the therapeutic potential via the tetrazolium chloride (TTC) assay and behavioral tests. A general improvement in the prognosis of animals after tMCAO was observed. Our results suggest the increased mobility of CCR4-overexpressed Treg cells in response to microglia-derived chemokines in vitro and the therapeutic potential of Treg cells with increased mobility in cellular therapy.

Monocyte-Derived Macrophages Aggravate Cardiac Dysfunction After Ischemic Stroke in Mice.

BACKGROUND: Cardiac damage induced by ischemic stroke, such as arrhythmia, cardiac dysfunction, and even cardiac arrest, is referred to as cerebral-cardiac syndrome (CCS). Cardiac macrophages are reported to be closely associated with stroke-induced cardiac damage. However, the role of macrophage subsets in CCS is still unclear due to their heterogeneity. Sympathetic nerves play a significant role in regulating macrophages in cardiovascular disease. However, the role of macrophage subsets and sympathetic nerves in CCS is still unclear. METHODS AND RESULTS: In this study, a middle cerebral artery occlusion mouse model was used to simulate ischemic stroke. ECG and echocardiography were used to assess cardiac function. We used Cx3cr1GFPCcr2RFP mice and NLRP3-deficient mice in combination with Smart-seq2 RNA sequencing to confirm the role of macrophage subsets in CCS. We demonstrated that ischemic stroke-induced cardiac damage is characterized by severe cardiac dysfunction and robust infiltration of monocyte-derived macrophages into the heart. Subsequently, we identified that cardiac monocyte-derived macrophages displayed a proinflammatory profile. We also observed that cardiac dysfunction was rescued in ischemic stroke mice by blocking macrophage infiltration using a CCR2 antagonist and NLRP3-deficient mice. In addition, a cardiac sympathetic nerve retrograde tracer and a sympathectomy method were used to explore the relationship between sympathetic nerves and cardiac macrophages. We found that cardiac sympathetic nerves are significantly activated after ischemic stroke, which contributes to the infiltration of monocyte-derived macrophages and subsequent cardiac dysfunction. CONCLUSIONS: Our findings suggest a potential pathogenesis of CCS involving the cardiac sympathetic nerve-monocyte-derived macrophage axis.

Human-Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Showed Neuronal Differentiation, Neurite Extension, and Formation of Synaptic Structures in Rodent Ischemic Stroke Brains.

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation.

Isobavachalcone alleviates ischemic stroke by suppressing HDAC1 expression and improving M2 polarization.

Ischemic stroke is a serious cerebrovascular condition. Isobavachalcone (ISO) has been documented to exhibit an anti-inflammatory effect across a variety of diseases; however, its protective impact on ischemic stroke remains unexplored. In this study, we evaluated the influence of ISO in both transient middle cerebral artery occlusion/reperfusion (tMCAO/R) rat models and oxygen-glucose deprivation/reperfusion (OGD/R) cell models. We observed that pretreatment with 50 mg/kg ISO diminished the volume of brain infarction, reduced brain edema, and ameliorated neurological deficits in rats. A reduction in Nissl bodies was noted in the tMCAO/R group, which was reversed following treatment with 50 mg/kg ISO. TUNEL/NeuN double staining revealed a decrease in TUNEL-positive cells in tMCAO/R rats treated with ISO. Furthermore, ISO treatment suppressed the expression of cleaved caspase-3 and BAX, while elevating the expression of BCL-2 in tMCAO/R rats. The levels of CD86 and iNOS were elevated in tMCAO/R rats; conversely, ISO treatment enhanced the expression of CD206 and Arg-1. Additionally, the expression of TNF-α, IL-6, and IL-1ß was elevated in tMCAO/R rats, whereas ISO treatment counteracted this effect. ISO treatment also increased the expression of TGF-ß and IL-10 in the ischemic penumbra of tMCAO/R rats. It was found that ISO treatment hindered microglial M1 polarization and favored M2 polarization. Histone Deacetylase 1 (HDAC1) is the downstream target protein of ISO, with ISO treatment resulting in decreased HDAC1 expression in both tMCAO/R rats and OGD/R-induced cells. Overexpression of HDAC1 was shown to promote microglial M1 polarization and inhibit M2 polarization in OGD/R+ISO cells. Overall, ISO treatment mitigated brain damage following ischemic stroke by promoting M2 polarization and attenuated ischemic injury by repressing HDAC1 expression.

Knockdown of lncRNA AU020206 could inhibit microglia apoptosis in ischemic stroke.

The diagnostic biomarkers associated with ischemic stroke (IS) that may have clinical utility remain elucidated. Thus, the potential functional lncRNAs in IS were explored. The Gene Expression Omnibus database provided the transcriptome profile of IS for download. WGCNA analysis and integrated bioinformatics were used to find genes that were differentially expressed (DEGs). The Starbase database created the lncRNA-based ceRNA network. In order to investigate the molecular mechanism and involved pathways of DEGs in IS, functional enrichment analysis was carried out. Using qRT-PCR, lncRNAs identified as putative IS biomarkers were confirmed to be expressed in a permanent middle cerebral artery occlusion (MCAO) model. Using the annexin V/PI apoptosis test, the amount of apoptosis in oxygen-glucose deprivation (OGD) cells was measured. A total of 1600 common differentially expressed - protein-coding RNA (DE-pcRNAs) and 26 DE-lncRNAs were identified. The results of enrichment analysis indicate that the cytokine may be regulated by common DE-pcRNAs and are vital in the progress of IS. A lncRNAs-mediated ceRNA network including lncRNAs AU020206, Brip1os, F630028O10Rik and 9530082P21Rik was constructed. The expression of these lncRNAs was significantly increased in MCAO model. Knockdown of lncRNA AU020206 inhibited microglia apoptosis in OGD cell model. We constructed a lncRNAs-mediated ceRNA network and found that lncRNA AU020206 inhibited microglia apoptosis in OGD cell model. These findings provided further evidence for the diagnosis and a novel avenue for targeted therapy of IS.

Decreased connexin 40 expression of the sinoatrial node mediates ischemic stroke-induced arrhythmia in mice.

BACKGROUND: Arrhythmia is the most common cardiac complication after ischemic stroke. Connexin 40 is the staple component of gap junctions, which influences the propagation of cardiac electrical signals in the sinoatrial node. However, the role of connexin 40 in post-stroke arrhythmia remains unclear. METHODS: In this study, a permanent middle cerebral artery occlusion model was used to simulate the occurrence of an ischemic stroke. Subsequently, an electrocardiogram was utilized to record and assess variations in electrocardiogram measures. In addition, optical tissue clearing and whole-mount immunofluorescence staining were used to confirm the anatomical localization of the sinoatrial node, and the sinoatrial node tissue was collected for RNA sequencing to screen for potential pathological mechanisms. Lastly, the rAAV9-Gja5 virus was injected with ultrasound guidance into the heart to increase Cx40 expression in the sinoatrial node. RESULTS: We demonstrated that the mice suffering from a permanent middle cerebral artery occlusion displayed significant arrhythmia, including atrial fibrillation, premature ventricular contractions, atrioventricular block, and abnormal electrocardiogram parameters. Of note, we observed a decrease in connexin 40 expression within the sinoatrial node after the ischemic stroke via RNA sequencing and western blot. Furthermore, rAAV9-Gja5 treatment ameliorated the occurrence of arrhythmia following stroke. CONCLUSIONS: In conclusion, decreased connexin 40 expression in the sinoatrial node contributed to the ischemic stroke-induced cardiac arrhythmia. Therefore, enhancing connexin 40 expression holds promise as a potential therapeutic approach for ischemic stroke-induced arrhythmia.

Sulfated Polysaccharide-Based Nanocarrier Drives Microenvironment-Mediated Cerebral Neurovascular Remodeling for Ischemic Stroke Treatment.

Stroke is a leading cause of global mortality and severe disability. However, current strategies used for treating ischemic stroke lack specific targeting capabilities, exhibit poor immune escape ability, and have limited drug release control. Herein, we developed an ROS-responsive nanocarrier for targeted delivery of the neuroprotective agent rapamycin (RAPA) to mitigate ischemic brain damage. The nanocarrier consisted of a sulfated chitosan (SCS) polymer core modified with a ROS-responsive boronic ester enveloped by a red blood cell membrane shell incorporating a stroke homing peptide. When encountering high levels of intracellular ROS in ischemic brain tissues, the release of SCS combined with RAPA from nanoparticle disintegration facilitates effective microglia polarization and, in turn, maintains blood-brain barrier integrity, reduces cerebral infarction, and promotes cerebral neurovascular remodeling in a mouse stroke model involving transient middle cerebral artery occlusion (tMCAO). This work offers a promising strategy to treat ischemic stroke therapy.

4'-O-methylbavachalcone alleviates ischemic stroke injury by inhibiting parthanatos and promoting SIRT3.

Cerebral ischemia-reperfusion injury (CIRI) can induce massive death of ischemic penumbra neurons via oxygen burst, exacerbating brain damage. Parthanatos is a form of caspase-independent cell death involving excessive activation of PARP-1, closely associated with intense oxidative stress following CIRI. 4'-O-methylbavachalcone (MeBavaC), an isoprenylated chalcone component in Fructus Psoraleae, has potential neuroprotective effects. This study primarily investigates whether MeBavaC can act on SIRT3 to alleviate parthanatos of ischemic penumbra neurons induced by CIRI. MeBavaC was oral gavaged to the middle cerebral artery occlusion-reperfusion (MCAO/R) rats after occlusion. The effects of MeBavaC on cerebral injury were detected by the neurological deficit score and cerebral infarct volume. In vitro, PC-12 cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R), and assessed cell viability and cell injury. Also, the levels of ROS, mitochondrial membrane potential (MMP), and intracellular Ca2+ levels were detected to reflect mitochondrial function. We conducted western blotting analyses of proteins involved in parthanatos and related signaling pathways. Finally, the exact mechanism between the neuroprotection of MeBavaC and parthanatos was explored. Our results indicate that MeBavaC reduces the cerebral infarct volume and neurological deficit scores in MCAO/R rats, and inhibits the decreased viability of PC-12 cells induced by OGD/R. MeBavaC also downregulates the expression of parthanatos-related death proteins PARP-1, PAR, and AIF. However, this inhibitory effect is weakened after the use of a SIRT3 inhibitor. In conclusion, the protective effect of MeBavaC against CIRI may be achieved by inhibiting parthanatos of ischemic penumbra neurons through the SIRT3-PARP-1 axis.

Inhibition of Notch1 signal promotes brain recovery by modulating glial activity after stroke.

BACKGROUND: Notch1 signaling inhibiton with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester] (DAPT) treatment could promote brain recovery and the intervention effect is different between striatum (STR) and cortex (CTX), which might be accounted for different changes of glial activities, but the in-depth mechanism is still unknown. The purpose of this study was to identify whether DAPT could modulate microglial subtype shifts and astroglial-endfeet aquaporin-4 (AQP4) mediated waste solute drainage. METHODS: Sprague-Dawley rats (n=10) were subjected to 90min of middle cerebral artery occlusion (MCAO) and were treated with DAPT (n=5) or act as control with no treatment (n=5). Two groups of rats underwent MRI scans at 24h and 4 week, and sacrificed at 4 week after stroke for immunofluorescence (IF). RESULTS: Compared with control rats, MRI data showed structural recovery in ipsilateral STR but not CTX. And IF showed decreased pro-inflammatory M1 microglia and increased anti-inflammatory M2 microglia in striatal lesion core and peri-lesions of STR, CTX. Meanwhile, IF showed decreased AQP4 polarity in ischemic brain tissue, however, AQP4 polarity in striatal peri-lesions of DAPT treated rats was higher than that in control rats but shows no difference in cortical peri-lesions between control and treated rats. CONCLUSIONS: The present study indicated that DAPT could promote protective microglia subtype shift and striatal astrocyte mediated waste solute drainage, that the later might be the major contributor of waste solute metabolism and one of the accounts for discrepant recovery of STR and CTX.

Exploring the intricacies of calcium dysregulation in ischemic stroke: Insights into neuronal cell death and therapeutic strategies.

Calcium ion (Ca2+) dysregulation is one of the main causes of neuronal cell death and brain damage after cerebral ischemia. During ischemic stroke, the ability of neurons to maintain Ca2+ homeostasis is compromised. Ca2+ regulates various functions of the nervous system, including neuronal activity and adenosine triphosphate (ATP) production. Disruptions in Ca2+ homeostasis can trigger a cascade of events, including activation of the unfolded protein response (UPR) pathway, which is associated with endoplasmic reticulum (ER) stress and mitochondrial dysfunction. This response occurs when the cell is unable to manage protein folding within the ER due to various stressors, such as a high influx of Ca2+. Consequently, the UPR is initiated to restore ER function and alleviate stress, but prolonged activation can lead to mitochondrial dysfunction and, ultimately, cell death. Hence, precise regulation of Ca2+ within the cell is mandatory. The ER and mitochondria are two such organelles that maintain intracellular Ca2+ homeostasis through various calcium-operating channels, including ryanodine receptors (RyRs), inositol trisphosphate receptors (IP3Rs), sarco/endoplasmic reticulum calcium ATPases (SERCAs), the mitochondrial Na+/Ca2+ exchanger (NCLX), the mitochondrial calcium uniporter (MCU) and voltage-dependent anion channels (VDACs). These channels utilize Ca2+ sequestering and release mechanisms to maintain intracellular Ca2+ homeostasis and ensure proper cellular function and survival. The present review critically evaluates the significance of Ca2+ and its physiological role in cerebral ischemia. We have compiled recent findings on calcium's role and emerging treatment strategies, particularly targeting mitochondria and the endoplasmic reticulum, to address Ca2+ overload in cerebral ischemia.

Molecular Pathways of Vulnerable Carotid Plaques at Risk of Ischemic Stroke: A Narrative Review.

The concept of vulnerable carotid plaques is pivotal in understanding the pathophysiology of ischemic stroke secondary to large-artery atherosclerosis. In macroscopic evaluation, vulnerable plaques are characterized by one or more of the following features: microcalcification; neovascularization; lipid-rich necrotic cores (LRNCs); intraplaque hemorrhage (IPH); thin fibrous caps; plaque surface ulceration; huge dimensions, suggesting stenosis; and plaque rupture. Recognizing these macroscopic characteristics is crucial for estimating the risk of cerebrovascular events, also in the case of non-significant (less than 50%) stenosis. Inflammatory biomarkers, such as cytokines and adhesion molecules, lipid-related markers like oxidized low-density lipoprotein (LDL), and proteolytic enzymes capable of degrading extracellular matrix components are among the key molecules that are scrutinized for their associative roles in plaque instability. Through their quantification and evaluation, these biomarkers reveal intricate molecular cross-talk governing plaque inflammation, rupture potential, and thrombogenicity. The current evidence demonstrates that plaque vulnerability phenotypes are multiple and heterogeneous and are associated with many highly complex molecular pathways that determine the activation of an immune-mediated cascade that culminates in thromboinflammation. This narrative review provides a comprehensive analysis of the current knowledge on molecular biomarkers expressed by symptomatic carotid plaques. It explores the association of these biomarkers with the structural and compositional attributes that characterize vulnerable plaques.

The role of dorsolateral striatum in the effects of deep cerebellar stimulation-mediated motor recovery following ischemic stroke in rodents.

Despite great advances in acute care and rehabilitation, stroke remains the leading cause of motor impairment in the industrialized world. We have developed a deep brain stimulation (DBS)-based approach for post-stroke rehabilitation that has shown reproducible effects in rodent models and has been recently translated to humans. Mechanisms underlying the rehabilitative effects of this novel therapy have been largely focused on the ipsilesional cortex, including cortical reorganization, synaptogenesis, neurogenesis and greater expression of markers of long-term potentiation. The role of subcortical structures on its therapeutic benefits, particularly the striatum, remain unclear. In this study, we compared the motor rehabilitative effects of deep cerebellar stimulation in two rodent models of cerebral ischemia: a) cortical ischemia; and b) combined striatal and cortical ischemia. All animals underwent the same procedures, including implantation of the electrodes and tethered connections for stimulation. Both experimental groups received four weeks of continuous lateral cerebellar nucleus (LCN) DBS and each was paired with a no stimulation, sham, group. Fine motor function was indexed using the pasta matrix task. Brain tissue was harvested for histology and immunohistochemical analyses. In the cortical-only ischemia, the average pasta matrix performance of both sham and stimulated groups reduced from 19 to 24 pieces to 7-8 pieces following the stroke induction. At the end of the four-week treatment, the performance of stimulated group was significantly greater than that of sham group (14 pieces vs 7 pieces, p < 0.0001). Similarly, in the combined cortical and striatal ischemia, the performance of both sham and stimulated groups reduced from 29 to 30 pieces to 7-11 pieces following the stroke induction. However, at the end of the four-week treatment, the performance of stimulated group was not significantly greater than that of sham group (15 pieces vs 11 pieces, p = 0.452). In the post-mortem analysis, the number of cells expressing CaMKIIα at the perilesional cortical and striatum of the LCN DBS treated animals receiving cortical-only stroke elevated but not those receiving cortical+striatal stroke. The current findings suggested that the observed, LCN DBS-enhanced motor recovery and perilesional plasticity may involve striatal mechanisms.

Autophagy-related 5 in acute ischemic stroke: Variation and linkage with neurofunction, and survival.

OBJECTIVE: Autophagy-related 5 (ATG5) facilitates the pathologic process of acute ischemic stroke (AIS) via multiple ways. This study aimed to identify the association of serum ATG5 with clinical outcomes in AIS patients. METHODS: Serum ATG5 from 280 AIS patients were detected at admission, Day (D)1, D3, D7, D30, and D90 after admission by enzyme-linked immunosorbent assay. The median (interquartile range) follow-up was 21.1 (5.9-43.9) months. Another 50 healthy controls (HCs) were also enrolled for serum ATG5 determination. RESULTS: ATG5 was elevated (p < 0.001) (vs. HCs), and positively correlated with hyperlipidemia (p = 0.016), and the national institutes of health stroke scale score (p = 0.001) in AIS patients. Interestingly, ATG5 was increased from admission to D1, but gradually decreased until D90 (p < 0.001). Besides, 85 (30.4%) and 195 (69.6%) AIS patients were assessed as modified Rankin Scale (mRS) >2 and mRS ≤2 at D90, respectively. ATG5 at admission, D1, D3, D30, and D90 was elevated in AIS patients with mRS >2 versus those with mRS ≤2 (all p < 0.050). ATG5 at admission, D1, D3, D7, D30, or D90 was elevated in relapsed (vs. non-relapsed) or died (vs. survived) AIS patients (all p < 0.050). Recurrence-free survival was shortened in AIS patients with high (≥52.0 ng/mL) ATG5 versus those with low (<52.0 ng/mL) ATG5 at admission, D3, D7, and D30 (all p < 0.050); overall survival was shorter in AIS patients with high (vs. low) ATG5 at D7 and D30 (both p < 0.050). INTERPRETATION: Serum ATG5 elevates at first, thereafter gradually declines, whose elevation associates with neurological dysfunction, recurrence, and death risk in AIS patients.

Matrilin-3 supports neuroprotection in ischemic stroke by suppressing astrocyte-mediated neuroinflammation.

In the brain, the role of matrilin-3, an extracellular matrix component in cartilage, is unknown. Here, we identify that matrilin-3 decreased in reactive astrocytes but was unchanged in neurons after ischemic stroke in animals. Importantly, it declined in serum of patients with acute ischemic stroke. Genetic or pharmacological inhibition or supplementation of matrilin-3 aggravates or reduces brain injury, astrocytic cell death, and glial scar, respectively, but has no direct effect on neuronal cell death. RNA sequencing demonstrates that Matn3-/- mice display an increased inflammatory response profile in the ischemic brain, including the nuclear factor κB (NF-κB) signaling pathway. Both endogenous and exogenous matrilin-3 reduce inflammatory mediators. Mechanistically, extracellular matrilin-3 enters astrocytes via caveolin-1-mediated endocytosis. Cytoplasmic matrilin-3 translocates into the nucleus by binding to NF-κB p65, suppressing inflammatory cytokine transcription. Extracellular matrilin-3 binds to BMP-2, blocking the BMP-2/Smads pathway. Thus, matrilin-3 is required for astrocytes to exert neuroprotection, at least partially, by suppressing astrocyte-mediated neuroinflammation.

Lifibrate attenuates blood-brain barrier damage following ischemic stroke via the MLCK/p-MLC/ZO-1 axis.

Dysfunction of tight junction proteins-associated damage to the blood-brain barrier (BBB) plays an important role in the pathogenesis of ischemic stroke. Lifibrate, an inhibitor of cholinephosphotransferase (CPT), has been used as an agent for serum lipid lowering. However, the protective effects of Lifibrate in ischemic stroke and the underlying mechanism have not been clearly elucidated. Here, we employed an in vivo mice model of MCAO and an OGD/R model in vitro. In the mice models, neurological deficit scores and infarct volume were assessed. Evans Blue solution was used to detect the BBB permeability. The TEER was examined to determine brain endothelial monolayer permeability. Here, we found that Lifibrate improved neurological dysfunction in stroke. Additionally, increased BBB permeability during stroke was significantly ameliorated by Lifibrate. Correspondingly, the reduced expression of the tight junction protein ZO-1 was restored by Lifibrate at both the mRNA and protein levels. Using an in vitro model, we found that Lifibrate ameliorated OGD/R-induced injury in human bEnd.3 brain microvascular endothelial cells by increasing cell viability but reducing the release of LDH. Importantly, Lifibrate suppressed the increase in endothelial monolayer permeability and the reduction in TEER induced by OGD/R via the rescue of ZO-1 expression. Mechanistically, Lifibrate blocked activation of the MLCK/ p-MLC signaling pathway in OGD/R-stimulated bEnd.3 cells. In contrast, overexpression of MLCK abolished the protective effects of Lifibrate in endothelial monolayer permeability, TEER, as well as the expression of ZO-1. Our results provide a basis for further investigation into the neuroprotective mechanism of Lifibrate during stroke.

Knockdown of RGMA improves ischemic stroke via Reprogramming of Neuronal Metabolism.

Neuronal energy metabolism dysregulation is involved in various pathologies of Ischemia-reperfusion (I/R), yet the role of RGMA in neuronal metabolic reprogramming has not been reported. In this study, we found that RGMA expression significantly increased after I/R, and compared to control mice, mice with MCAO/R showed an increase in glycolytic metabolic products and the expression of glycolytic pathway proteins. Furthermore, RGMA levels are closely related to neuronal energy metabolism. We discovered that knockdown of RGMA can shift neuronal energy metabolism towards oxidative phosphorylation and the pentose phosphate pathway, thereby protecting mice from ischemic reperfusion injury. Mechanistically, knockdown of RGMA can downregulate PGK1 expression, reducing the increase in glycolytic flux following ischemia reperfusion. Moreover, we found that knockdown of RGMA can reduce the interaction between USP10 and PGK1, thus affecting the ubiquitination degradation of PGK1. In summary, our data suggest that RGMA may regulate neuronal energy metabolism by inhibiting the USP10-mediated deubiquitination of PGK1, thus protecting it from I/R injury. This study provides new ideas for clarifying the intrinsic mechanism of neuronal damage after I/R.

Discovery of Novel Hybrids of Edaravone and 6-Phenyl-4,5-dihydropyridazin-3(2H)-one with Antiplatelet Aggregation and Neuroprotection for Ischemic Stroke Treatment.

Drugs with anti-platelet aggregation and neuroprotection are of great significance for the treatment of ischemic stroke. A series of edaravone and 6-phenyl-4,5-dihydropyridazin-3(2H)-one hybrids were designed and synthesized. Among them, 6g showed the most effective cytoprotective effect against oxygen-glucose deprivation/reoxygenation-induced damage in BV2 cells and an excellent inhibitory effect on platelet aggregation induced by adenosine diphosphate and arachidonic acid. Additionally, 6g could prevent thrombosis caused by ferric chloride in rats and pose a lower risk of causing bleeding compared with aspirin. It provides better protection against ischemia/reperfusion injury in rats compared with edaravone and alleviates the oxidative stress related to cerebral ischemia/reperfusion by increasing the GSH and SOD levels and decreasing the MDA concentration. Finally, molecular docking results showed that 6g probably acts on PDE3 A and plays an anti-platelet aggregation effect. Overall, 6g could be a potential candidate compound for the treatment of ischemic stroke.

Absence in CX3CR1 receptor signaling promotes post-ischemic stroke cognitive function recovery through suppressed microglial pyroptosis in mice.

BACKGROUND: Post-stroke cognitive impairment (PSCI) is a major source of morbidity and mortality after stroke, but the pathological mechanisms remain unclear. Previous studies have demonstrated that the CX3CR1 receptor plays a crucial role in maintaining an early protective microenvironment after stroke, but whether it persistently influences cognitive dysfunction in the chronic phase requires further investigation. METHODS: Mouse was used to establish a middle cerebral artery occlusion (MCAO)/reperfusion model to study PSCI. Cognitive function was assessed by the Morris water maze (MWM) and the novel object recognition test. Neurogenesis was assessed by immunofluorescence staining with Nestin+ /Ki67+ and DCX+ /BrdU+ double-positive cells. The cerebral damage was monitored by [18 F]-DPA-714 positron emission tomography, Nissel, and TTC staining. The pyroptosis was histologically, biochemically, and electron microscopically examined. RESULTS: Upon MCAO, at 28 to 35 days, CX3CR1 knockout (CX3CR1-/- ) mice had better cognitive behavioral performance both in MWM and novel object recognition test than their CX3CR1+/- counterparts. Upon MCAO, at 7 days, CX3CR1-/- mice increased the numbers of Nestin+ /Ki67+ and DCX+ /BrdU+ cells, and meanwhile it decreased the protein expression of GSDMD, NLRP3 inflammasome subunit, caspase-1, mature IL-1ß/IL-18, and p-P65 in the hippocampus as compared with CX3CR1+/- mice. In addition, CX3CR1-/- mice could reverse infarct volume in the hippocampus region post-stroke. CONCLUSION: Our study demonstrated that CX3CR1 gene deletion was beneficial to PSCI recovery. The mechanism might lie in inhibited pyroptosis and enhanced neurogenesis. CX3CR1 receptor may serve as a therapeutic target for improving the PSCI.

Pericyte Microvesicles as Plasma Biomarkers Reflecting Brain Microvascular Signaling in Patients With Acute Ischemic Stroke.

BACKGROUND: Blood-based biomarkers have the potential to reflect cerebrovascular signaling after microvascular injury; yet, the detection of cell-specific signaling has proven challenging. Microvesicles retain parental cell surface antigens allowing detection of cell-specific signaling encoded in their cargo. In ischemic stroke, the progression of pathology involves changes in microvascular signaling whereby brain pericytes, perivascular cells wrapping the microcapillaries, are one of the early responders to the ischemic insult. Intercepting the pericyte signaling response peripherally by isolating pericyte-derived microvesicles may provide not only diagnostic information on microvascular injury but also enable monitoring of important pathophysiological mechanisms. METHODS: Plasma samples were collected from patients with acute ischemic stroke (n=39) at 3 time points after stroke onset: 0 to 6 hours, 12 to 24 hours, and 2 to 6 days, and compared with controls (n=39). Pericyte-derived microvesicles were isolated based on cluster of differentiation 140b expression and quantified by flow cytometry. The protein content was evaluated using a proximity extension assay, and vascular signaling pathways were examined using molecular signature hallmarks and gene ontology. RESULTS: In this case-control study, patients with acute ischemic stroke showed significantly increased numbers of pericyte-derived microvesicles (median, stroke versus controls) at 12 to 24 hours (1554 versus 660 microvesicles/µL; P=0.0041) and 2 to 6 days after stroke (1346 versus 660 microvesicles/µL; P=0.0237). Their proteome revealed anti-inflammatory properties mediated via downregulation of Kirsten rat sarcoma virus and IL (interleukin)-6/JAK/STAT3 signaling at 0 to 6 hours, but proangiogenic as well as proinflammatory signals at 12 to 24 hours. Between 2 and 6 days, proteins were mainly associated with vascular remodeling as indicated by activation of Hedgehog signaling in addition to proangiogenic signals. CONCLUSIONS: We demonstrate that the plasma of patients with acute ischemic stroke reflects (1) an early and time-dependent increase of pericyte-derived microvesicles and (2) changes in the protein cargo of microvesicles over time indicating cell signaling specifically related to inflammation and vascular remodeling.