Improving stroke prognosis by TLR4 KO to enhance N2 neutrophil infiltration and reduce M1 macrophage polarization.

Cerebral ischemic stroke remains a leading cause of mortality and morbidity worldwide. Toll-like receptor 4 (TLR4) has been implicated in neuroinflammatory responses poststroke, particularly in the infiltration of immune cells and polarization of macrophages. This study aimed to elucidate the impact of TLR4 deficiency on neutrophil infiltration and subsequent macrophage polarization after middle cerebral artery occlusion (MCAO), exploring its role in stroke prognosis. The objective was to investigate how TLR4 deficiency influences neutrophil behavior poststroke, its role in macrophage polarization, and its impact on stroke prognosis using murine models. Wild-type and TLR4-deficient adult male mice underwent MCAO induction, followed by various analyses, including flow cytometry to assess immune cell populations, bone marrow transplantation experiments to evaluate TLR4-deficient neutrophil behaviors, and enzyme-linked immunosorbent assay and Western blot analysis for cytokine and protein expression profiling. Neurobehavioral tests and infarct volume analysis were performed to assess the functional and anatomical prognosis poststroke. TLR4-deficient mice exhibited reduced infarct volumes, increased neutrophil infiltration, and reduced M1-type macrophage polarization post-MCAO compared to wild-type mice. Moreover, the depletion of neutrophils reversed the neuroprotective effects observed in TLR4-deficient mice, suggesting the involvement of neutrophils in mediating TLR4's protective role. Additionally, N1-type neutrophils were found to promote M1 macrophage polarization via neutrophil gelatinase-associated lipocalin (NGAL) secretion, a process blocked by TLR4 deficiency. The study underscores the protective role of TLR4 deficiency in ischemic stroke, delineating its association with increased N2-type neutrophil infiltration, diminished M1 macrophage polarization, and reduced neuroinflammatory responses. Understanding the interplay between TLR4, neutrophils, and macrophages sheds light on potential therapeutic targets for stroke management, highlighting TLR4 as a promising avenue for intervention in stroke-associated neuroinflammation and tissue damage.

Insight into the pathophysiological advances and molecular mechanisms underlying cerebral stroke: current status.

Molecular pathways involved in cerebral stroke are diverse. The major pathophysiological events that are observed in stroke comprises of excitotoxicity, oxidative stress, mitochondrial damage, endoplasmic reticulum stress, cellular acidosis, blood-brain barrier disruption, neuronal swelling and neuronal network mutilation. Various biomolecules are involved in these pathways and several major proteins are upregulated and/or suppressed following stroke. Different types of receptors, ion channels and transporters are activated. Fluctuations in levels of various ions and neurotransmitters have been observed. Cells involved in immune responses and various mediators involved in neuro-inflammation get upregulated progressing the pathogenesis of the disease. Despite of enormity of the problem, there is not a single therapy that can limit infarction and neurological disability due to stroke. This is because of poor understanding of the complex interplay between these pathophysiological processes. This review focuses upon the past to present research on pathophysiological events that are involved in stroke and various factors that are leading to neuronal death following cerebral stroke. This will pave a way to researchers for developing new potent therapeutics that can aid in the treatment of cerebral stroke.

A Nonlinear Relation between Body Mass Index and Long-Term Poststroke Functional Outcome-The Importance of Insulin Resistance, Inflammation, and Insulin-like Growth Factor-Binding Protein-1.

Both high serum insulin-like growth factor-binding protein-1 (s-IGFBP-1) and insulin resistance (IR) are associated with poor functional outcome poststroke, whereas overweight body mass index (BMI; 25-30) is related to fewer deaths and favorable functional outcome in a phenomenon labeled "the obesity paradox". Furthermore, IGFBP-1 is inversely related to BMI, in contrast to the linear relation between IR and BMI. Here, we investigated s-IGFBP-1 and IR concerning BMI and 7-year poststroke functional outcome. We included 451 stroke patients from the Sahlgrenska Study on Ischemic Stroke (SAHLSIS) with baseline measurements of s-IGFBP1, homeostasis model assessment of IR (HOMA-IR), BMI (categories: normal-weight (8.5-25), overweight (25-30), and obesity (>30)), and high-sensitivity C-reactive protein (hs-CRP) as a measure of general inflammation. Associations with poor functional outcome (modified Rankin scale [mRS] score: 3-6) after 7 years were evaluated using multivariable binary logistic regression, with overweight as reference due to the nonlinear relationship. Both normal-weight (odds-ratio [OR] 2.32, 95% confidence interval [CI] 1.30-4.14) and obese (OR 2.25, 95% CI 1.08-4.71) patients had an increased risk of poor functional outcome, driven by deaths only in the normal-weight. In normal-weight, s-IGFBP-1 modestly attenuated (8.3%) this association. In the obese, the association was instead attenuated by HOMA-IR (22.4%) and hs-CRP (10.4%). Thus, a nonlinear relation between BMI and poor 7-year functional outcome was differently attenuated in the normal-weight and the obese.

Current evidence of synaptic dysfunction after stroke: Cellular and molecular mechanisms.

BACKGROUND: Stroke is an acute cerebrovascular disease in which brain tissue is damaged due to sudden obstruction of blood flow to the brain or the rupture of blood vessels in the brain, which can prompt ischemic or hemorrhagic stroke. After stroke onset, ischemia, hypoxia, infiltration of blood components into the brain parenchyma, and lysed cell fragments, among other factors, invariably increase blood-brain barrier (BBB) permeability, the inflammatory response, and brain edema. These changes lead to neuronal cell death and synaptic dysfunction, the latter of which poses a significant challenge to stroke treatment. RESULTS: Synaptic dysfunction occurs in various ways after stroke and includes the following: damage to neuronal structures, accumulation of pathologic proteins in the cell body, decreased fluidity and release of synaptic vesicles, disruption of mitochondrial transport in synapses, activation of synaptic phagocytosis by microglia/macrophages and astrocytes, and a reduction in synapse formation. CONCLUSIONS: This review summarizes the cellular and molecular mechanisms related to synapses and the protective effects of drugs or compounds and rehabilitation therapy on synapses in stroke according to recent research. Such an exploration will help to elucidate the relationship between stroke and synaptic damage and provide new insights into protecting synapses and restoring neurologic function.

Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo.

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

The cerebroprotection and prospects of FNDC5/irisin in stroke.

Stroke, the leading cause of disability and cognitive impairment, is also the second leading cause of death worldwide. The drugs with multi-targeted brain cytoprotective effects are increasingly being advocated for the treatment of stroke. Irisin, a newly discovered myokine produced by cleavage of fibronectin type III domain 5, has been shown to regulate glucose metabolism, mitochondrial energy, and fat browning. A large amount of evidence indicated that irisin could exert anti-inflammatory, anti-apoptotic, and antioxidant properties in a variety of diseases such as myocardial infarction, inflammatory bowel disease, lung injury, and kidney or liver disease. Studies have found that irisin is widely distributed in multiple brain regions and also plays an important regulatory role in the central nervous system. The most common cause of a stroke is a sudden blockage of an artery (ischemic stroke), and in some circumstances, a blood vessel rupture can also result in a stroke (hemorrhagic stroke). After a stroke, complicated pathophysiological processes lead to serious brain injury and neurological dysfunction. According to recent investigations, irisin may protect elements of the neurovascular unit by acting on multiple pathological processes in stroke. This review aims to outline the currently recognized effects of irisin on stroke and propose possible directions for future research.

Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Mastoparan M promotes functional recovery in stroke mice by activating autophagy and inhibiting ferroptosis.

Neuronal ferroptosis and autophagy are crucial in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). Mastoparan M (Mast-M), extracted from the crude venom of Vespa magnifica (Smith), comprises 14 amino acid residues. Previous studies suggested that Mast-M reduces neuronal damage following global CIRI, but its protective mechanisms remain unclear. The present study examined the effect of Mast-M on middle cerebral artery occlusion/reperfusion (MCAO/R) induced neurological deficits using Grip, Rotarod, Longa test, and TTC staining, followed by treating the mice for three days with Mast-M (20, 40, and 80 µg/kg, subcutaneously). The results demonstrate that Mast-M promotes functional recovery in mice post-ischemic stroke, evidenced by improved neurological impairment, reduced infarct volume and neuronal damage. Meanwhile, the level of iron (Fe2+) and malonyldialdehyde was decreased in the ischemic hemisphere of MCAO/R mice at 24 hours or 48 hours by Mast-M (80 µg/kg) treatment, while the expression of NRF2, x-CT, GPX4, and LC3B protein was increased. Furthermore, these findings were validated in three models-oxygen-glucose deprivation/ reoxygenation, H2O2-induced peroxidation, and erastin-induced ferroptosis-in hippocampal neuron HT22 cells or primary neurons. These data suggested that Mast-M activates autophagy as well as inhibits ferroptosis. Finally, autophagy inhibitors were introduced to determine the relationship between the autophagy and ferroptosis, indicating that Mast-M alleviates ferroptosis by activating autophagy. Taken together, this study described that Mast-M alleviates cerebral infarction, neurologic impairment, and neuronal damage by activating autophagy and inhibiting ferroptosis, presenting a potential therapeutic approach for CIRI.

Unveiling the role of HIST2H2AC in stroke through single-cell and transcriptome analysis.

Stroke is a leading cause of death and disability, and genetic risk factors play a significant role in its development. Unfortunately, effective therapies for stroke are currently limited. Early detection and diagnosis are critical for improving outcomes and developing new treatment strategies. In this study, we aimed to identify potential biomarkers and effective prevention and treatment strategies for stroke by conducting transcriptome and single-cell analyses. Our analysis included screening for biomarkers, functional enrichment analysis, immune infiltration, cell-cell communication, and single-cell metabolism. Through differential expression analysis, enrichment analysis, and protein-protein interaction (PPI) network construction, we identified HIST2H2AC as a potential biomarker for stroke. Our study also highlighted the diagnostic role of HIST2H2AC in stroke, its relationship with immune cells in the stroke environment, and our improved understanding of metabolic pathways after stroke. Overall, our research provided important insights into the pathogenesis of stroke, including potential biomarkers and treatment strategies that can be explored further to improve outcomes for stroke patients.

Analysis of ischemic stroke-mediated effects on blood-brain barrier properties along the arteriovenous axis assessed by intravital two-photon imaging.

Early breach of the blood-brain barrier (BBB) and consequently extravasation of blood-borne substances into the brain parenchyma is a common hallmark of ischemic stroke. Although BBB breakdown is associated with an increased risk of cerebral hemorrhage and poor clinical prognosis, the cause and mechanism of this process are largely unknown. The aim of this study was to establish an imaging and analysis protocol which enables investigation of the dynamics of BBB breach in relation to hemodynamic properties along the arteriovenous axis. Using longitudinal intravital two-photon imaging following photothrombotic induction of ischemic stroke through a cranial window, we were able to study the response of the cerebral vasculature to ischemia, from the early critical hours to the days/weeks after the infarct. We demonstrate that disruption of the BBB and hemodynamic parameters, including perturbed blood flow, can be studied at single-vessel resolution in the three-dimensional space as early as 30 min after vessel occlusion. Further, we show that this protocol permits longitudinal studies on the response of individual blood vessels to ischemia over time, thus enabling detection of (maladaptive) vascular remodeling such as intussusception, angiogenic sprouting and entanglement of vessel networks. Taken together, this in vivo two-photon imaging and analysis protocol will be useful in future studies investigating the molecular and cellular mechanisms, and the spatial contribution, of BBB breach to disease progression which might ultimately aid the development of new and more precise treatment strategies for ischemic stroke.

STING inhibition suppresses microglia-mediated synapses engulfment and alleviates motor functional deficits after stroke.

Ischemic stroke is the leading cause of adult disability. Ischemia leads to progressive neuronal death and synapse loss. The engulfment of stressed synapses by microglia further contributes to the disruption of the surviving neuronal network and related brain function. Unfortunately, there is currently no effective target for suppressing the microglia-mediated synapse engulfment. Stimulator of interferon genes (STING) is an important participant in innate immune response. In the brain, microglia are the primary cell type that mediate immune response after brain insult. The intimate relationship between STING and microglia-mediated neuroinflammation has been gradually established. However, whether STING affects other functions of microglia remains elusive. In this study, we found that STING regulated microglial phagocytosis of synapses after photothrombotic stroke. The treatment of STING inhibitor H151 significantly improved the behavioral performance of injured mice in grid-walking test, cylinder test, and adhesive removal test after stroke. Moreover, the puncta number of engulfed SYP or PSD95 in microglia was reduced after consecutive H151 administration. Further analysis showed that the mRNA levels of several complement components and phagocytotic receptors were decreased after STING inhibition. Transcriptional factor STAT1 is known for regulating most of the decreased molecules. After STING inhibition, the nucleus translocation of phosphorylated STAT1 was also suppressed in microglia. Our data uncovered the novel regulatory effects of STING in microglial phagocytosis after stroke, and further emphasized STING as a potential drug-able target for post-stroke functional recovery.

Inhibition of OGFOD1 by FG4592 confers neuroprotection by activating unfolded protein response and autophagy after ischemic stroke.

BACKGROUND: Acute ischemic stroke is a common neurological disease with a significant financial burden but lacks effective drugs. Hypoxia-inducible factor (HIF) and prolyl hydroxylases (PHDs) participate in the pathophysiological process of ischemia. However, whether FG4592, the first clinically approved PHDs inhibitor, can alleviate ischemic brain injury remains unclear. METHODS: The infarct volumes and behaviour tests were first analyzed in mice after ischemic stroke with systemic administration of FG4592. The knockdown of HIF-1α and pretreatments of HIF-1/2α inhibitors were then used to verify whether the neuroprotection of FG4592 is HIF-dependent. The targets predicting and molecular docking methods were applied to find other targets of FG4592. Molecular, cell biological and gene knockdown methods were finally conducted to explore the potential neuroprotective mechanisms of FG4592. RESULTS: We found that the systemic administration of FG4592 decreased infarct volume and improved neurological defects of mice after transient or permanent ischemia. Meanwhile, FG4592 also activated autophagy and inhibited apoptosis in peri-infarct tissue of mice brains. However, in vitro and in vivo results suggested that the neuroprotection of FG4592 was not classical HIF-dependent. 2-oxoglutarate and iron-dependent oxygenase domain-containing protein 1 (OGFOD1) was found to be a novel target of FG4592 and regulated the Pro-62 hydroxylation in the small ribosomal protein s23 (Rps23) with the help of target predicting and molecular docking methods. Subsequently, the knockdown of OGFOD1 protected the cell against ischemia/reperfusion injury and activated unfolded protein response (UPR) and autophagy. Moreover, FG4592 was also found to activate UPR and autophagic flux in HIF-1α independent manner. Blocking UPR attenuated the neuroprotection, pro-autophagy effect and anti-apoptosis ability of FG4592. CONCLUSION: This study demonstrated that FG4592 could be a candidate drug for treating ischemic stroke. The neuroprotection of FG4592 might be mediated by inhibiting alternative target OGFOD1, which activated the UPR and autophagy and inhibited apoptosis after ischemic injury. The inhibition of OGFOD1 is a novel therapy for ischemic stroke.

Acetylation of c-Myc at Lysine 148 Protects Neurons After Ischemia.

This study focuses on understanding the role of c-Myc, a cancer-associated transcription factor, in the penumbra following ischemic stroke. While its involvement in cell death and survival is recognized, its post-translational modifications, particularly acetylation, remain understudied in ischemia models. Investigating these modifications could have significant clinical implications for controlling c-Myc activity in the central nervous system. Although previous studies on c-Myc acetylation have been limited to non-neuronal cells, our research examines its expression in perifocal cells during stroke recovery to explore regulatory mechanisms via acetylation. We found that in peri-infarct neurons, c-Myc is upregulated with acetylation at K148 but not K323 during the acute phase of stroke, with SIRT2 deacetylase primarily affecting K148 acetylation. Molecular dynamics simulations suggest that lysine 148 plays a crucial role in stabilizing c-Myc spatial structure. Increased acetylation at K148 reduces c-Myc compaction, potentially limiting its nuclear penetration, promoting calpain-mediated cleavage, and decreasing nuclear localization. Additionally, cytoplasmic acetylation at K148 may alter c-Myc's interaction with unidentified proteins, potentially influencing its pro-apoptotic effects and promoting cytoplasmic accumulation. Targeting SIRT2 with selective inhibitors could be a promising avenue for future stroke therapy strategies.

Myricetin Oligomer Triggers Multi-Receptor Mediated Penetration and Autophagic Restoration of Blood-Brain Barrier for Ischemic Stroke Treatment.

Restoration of blood-brain barrier (BBB) dysfunction, which drives worse outcomes of ischemic stroke, is a potential target for therapeutic opportunities, whereas a sealed BBB blocks the therapeutics entrance into the brain, making the BBB protection strategy paradoxical. Post ischemic stroke, hypoxia/hypoglycemia provokes the up-regulation of transmembrane glucose transporters and iron transporters due to multiple metabolic disorders, especially in brain endothelial cells. Herein, we develop a myricetin oligomer-derived nanostructure doped with Ce to bypass the BBB which is cointermediated by glucose transporters and iron transporters such as glucose transporters 1 (GLUT1), sodium/glucose cotransporters 1 (SGLT1), and transferrin(Tf) reporter (TfR). Moreover, it exhibits BBB restoration capacity by regulating the expression of tight junctions (TJs) through the activation of protective autophagy. The myricetin oligomers scaffold not only acts as targeting moiety but is the prominent active entity that inherits all diverse pharmacological activities of myricetin. The suppression of oxidative damage, M1 microglia activation, and inflammatory factors makes it a multitasking nanoagent with a single component as the scaffold, targeting domain and curative components.

Chemogenetic activation of astrocytic Gi signaling promotes spinogenesis and motor functional recovery after stroke.

Ischemic stroke is the leading cause of adult disability. The rewiring of surviving neurons is the fundamental process for functional recovery. Accumulating evidence implicates astrocytes in synapses and neural circuits formation, but few studies have further studied how to enhance the effects of astrocytes on synapse and circuits after stroke and its impacts on post-stroke functional recovery. In this study, we made use of chemogenetics to specifically activate astrocytic Gi signaling in the peri-infarcted sensorimotor cortex at different time epochs in a mouse model of photothrombotic stroke. We found that early activation of astrocytic hM4Di after stroke by CNO modulates astrocyte activity and upregulates synaptogenic molecules including thrombospondin-1 (TSP1) as revealed by bulk RNA-sequencing, but no significant improvement was observed in dendritic spine density and behavioral performance in grid walking test. Interestingly, when the manipulation was initiated at the subacute phase of stroke, the recovery of spine density and motor function could be effectively promoted, accompanied by increased TSP1 expression. Our data highlight the important role of astrocytes in synapse remodeling during the repair phase of stroke and suggest astrocytic Gi signaling activation as a potential strategy for synapse regeneration, circuit rewiring, and functional recovery.

Covalent organic framework based cytoprotective therapy after ischemic stroke.

Cytoprotection has emerged as an effective therapeutic strategy for mitigating brain injury following acute ischemic stroke (AIS). The sulfonylurea receptor 1-transient receptor potential M4 (SUR1-TRPM4) channel plays a pivotal role in brain edema and neuroinflammation. However, the practical use of the inhibitor glyburide (GLB) is hindered by its low bioavailability. Additionally, the elevated reactive oxygen species (ROS) after AIS exacerbate SUR1-TRPM4 activation, contributing to irreversible brain damage. To overcome these challenges, GLB and superoxide dismutase (SOD) were embedded in a covalent organic framework (COF) with a porous structure and great stability. The resulting S/G@COF demonstrated significant improvements in survival and neurological functions. This was achieved by eliminating ROS, preventing neuronal loss and apoptosis, suppressing neuroinflammation, modulating microglia activation, and ameliorating blood-brain barrier (BBB) disruption. Mechanistic investigations revealed that S/G@COF concurrently activated the Wnt/ß-catenin signaling pathway while suppressing the upregulation of SUR1-TRPM4. This study underscores the potential of employing multi-target therapy and drug modification in cytoprotective strategies for ischemic stroke.

Therapeutic gene delivery by mesenchymal stem cell for brain ischemia damage: Focus on molecular mechanisms in ischemic stroke.

Cerebral ischemic damage is prevalent and the second highest cause of death globally across patient populations; it is as a substantial reason of morbidity and mortality. Mesenchymal stromal cells (MSCs) have garnered significant interest as a potential treatment for cerebral ischemic damage, as shown in ischemic stroke, because of their potent intrinsic features, which include self-regeneration, immunomodulation, and multi-potency. Additionally, MSCs are easily obtained, isolated, and cultured. Despite this, there are a number of obstacles that hinder the effectiveness of MSC-based treatment, such as adverse microenvironmental conditions both in vivo and in vitro. To overcome these obstacles, the naïve MSC has undergone a number of modification processes to enhance its innate therapeutic qualities. Genetic modification and preconditioning modification (with medications, growth factors, and other substances) are the two main categories into which these modification techniques can be separated. This field has advanced significantly and is still attracting attention and innovation. We examine these cutting-edge methods for preserving and even improving the natural biological functions and therapeutic potential of MSCs in relation to adhesion, migration, homing to the target site, survival, and delayed premature senescence. We address the use of genetically altered MSC in stroke-induced damage. Future strategies for improving the therapeutic result and addressing the difficulties associated with MSC modification are also discussed.

Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex.

BACKGROUND: REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke. METHODS AND RESULTS: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's t test was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05. CONCLUSIONS: If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.

Retinoic acid attenuates ischemic injury-induced activation of glial cells and inflammatory factors in a rat stroke model.

Stroke is a leading cause of death and long-term disability which can cause oxidative damage and inflammation of the neuronal cells. Retinoic acid is an active metabolite of vitamin A that has various beneficial effects including antioxidant and anti-inflammatory effects. In this study, we investigated whether retinoic acid modulates oxidative stress and inflammatory factors in a stroke animal model. A middle cerebral artery occlusion (MCAO) was performed on adult male rats to induce focal cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected into the peritoneal cavity for four days before MCAO surgery. The neurobehavioral tests were carried out 24 h after MCAO and cerebral cortex tissues were collected. The cortical damage was assessed by hematoxylin-eosin staining and reactive oxygen species assay. In addition, Western blot and immunohistochemical staining were performed to investigate the activation of glial cells and inflammatory cytokines in MCAO animals. Ionized calcium-binding adapter molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP) were used as markers of microglial and astrocyte activation, respectively. Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were used as representative pro-inflammatory cytokines. Results showed that MCAO damage caused neurobehavioral defects and histopathological changes in the ischemic region and increased oxidative stress. Retinoic acid treatment reduced these changes caused by MCAO damage. We detected increases in Iba-1 and GFAP in MCAO animals treated with vehicle. However, retinoic acid alleviated increases in Iba-1 and GFAP caused by MCAO damage. Moreover, MCAO increased levels of nuclear factor-κB and pro-inflammatory cytokines, including TNF-α and IL-1ß. Retinoic acid alleviated the expression of these inflammatory proteins. These findings elucidate that retinoic acid regulates microglia and astrocyte activation and modulates pro-inflammatory cytokines. Therefore, this study suggests that retinoic acid exhibits strong antioxidant and anti-inflammatory properties by reducing oxidative stress, inhibiting neuroglia cell activation, and preventing the increase of pro-inflammatory cytokines in a cerebral ischemia.