Molecular Pathogenesis of Ischemic and Hemorrhagic Strokes: Background and Therapeutic Approaches.

Stroke represents one of the neurological diseases most responsible for death and permanent disability in the world. Different factors, such as thrombus, emboli and atherosclerosis, take part in the intricate pathophysiology of stroke. Comprehending the molecular processes involved in this mechanism is crucial to developing new, specific and efficient treatments. Some common mechanisms are excitotoxicity and calcium overload, oxidative stress and neuroinflammation. Furthermore, non-coding RNAs (ncRNAs) are critical in pathophysiology and recovery after cerebral ischemia. ncRNAs, particularly microRNAs, and long non-coding RNAs (lncRNAs) are essential for angiogenesis and neuroprotection, and they have been suggested to be therapeutic, diagnostic and prognostic tools in cerebrovascular diseases, including stroke. This review summarizes the intricate molecular mechanisms underlying ischemic and hemorrhagic stroke and delves into the function of miRNAs in the development of brain damage. Furthermore, we will analyze new perspectives on treatment based on molecular mechanisms in addition to traditional stroke therapies.

ARMC10 regulates mitochondrial dynamics and affects mitochondrial function via the Wnt/ß-catenin signalling pathway involved in ischaemic stroke.

Mitochondrial dynamics has emerged as an important target for neuronal protection after cerebral ischaemia/reperfusion. Therefore, the aim of this study was to investigate the mechanism by which ARMC10 regulation of mitochondrial dynamics affects mitochondrial function involved in ischaemic stroke (IS). Mitochondrial morphology was detected by laser scanning confocal microscopy (LSCM), and mitochondrial ultrastructural alterations were detected by electron microscopy. The expression of mitochondrial dynamics-related genes Drp1, Mfn1, Mfn2, Fis1, OPA1 and ARMC10 and downstream target genes c-Myc, CyclinD1 and AXIN2 was detected by RT-qPCR. Western blot was used to detect the protein expression of ß-catenin, GSK-3ß, p-GSK-3ß, Bcl-2 and Bax. DCFH-DA fluorescent probe was to detect the effect of ARMC10 on mitochondrial ROS level, Annexin V-FITC fluorescent probe was to detect the effect of ARMC10 on apoptosis, and ATP assay kit was to detect the effect of ARMC10 on ATP production. Mitochondrial dynamics was dysregulated in clinical IS samples and in the OGD/R cell model, and the relative expression of ARMC10 gene was significantly decreased in IS group (p < 0.05). Knockdown and overexpression of ARMC10 could affect mitochondrial dynamics, mitochondrial function and neuronal apoptosis. Agonist and inhibitor affected mitochondrial function and neuronal apoptosis by targeting Wnt/ß-Catenin signal pathway. In the OGD/R model, ARMC10 affected mitochondrial function and neuronal apoptosis through the mechanism that regulates Wnt/ß-catenin signalling pathway. ARMC10 regulates mitochondrial dynamics and protects mitochondrial function by activating Wnt/ß-catenin signalling pathway, to exert neuroprotective effects.

Overexpression of olfactory receptor 78 ameliorates brain injury in cerebral ischaemia-reperfusion rats by activating Prkaca-mediated cAMP/PKA-MAPK pathway.

Ischemic stroke is one of the main causes of disability and death. However, recanalization of occluded cerebral arteries is effective only within a very narrow time window. Therefore, it is particularly important to find neuroprotective biological targets for cerebral artery recanalization. Here, gene expression profiles of datasets GSE160500 and GSE97537 were downloaded from the GEO database, which were related to ischemic stroke in rats. Olfactory receptor 78 (Olfr78) was screened, and which highly associated with Calcium signalling pathway and MAPK pathway. Interacting protein of Olfr78, Prkaca, was predicted by STRING, and their interaction was validated by Co-IP analysis. Then, a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) and a neuronal cell model stimulated by oxygen-glucose deprivation/reoxygenation (OGD/R) were constructed, and the results showed that expression of Olfr78 and Prkaca was downregulated in MCAO rats and OGD/R-stimulated neurons. Overexpression of Olfr78 or Prkaca inhibited the secretion of inflammatory factors, Ca2+ overload, and OGD/R-induced neuronal apoptosis. Moreover, Overexpression of Prkaca increased protein levels of cAMP, PKA and phosphorylated p38 in OGD/R-stimulated neurons, while SB203580, a p38 inhibitor, treatment inhibited activation of the cAMP/PKA-MAPK pathway and counteracted the effect of Olfr78 overexpression on improvement of neuronal functions. Meanwhile, overexpression of Olfr78 or Prkaca markedly inhibited neuronal apoptosis and improved brain injury in MCAO/R rats. In conclusion, overexpression of Olfr78 inhibited Ca2+ overload and reduced neuronal apoptosis in MCAO/R rats by promoting Prkaca-mediated activation of the cAMP/PKA-MAPK pathway, thereby improving brain injury in cerebral ischaemia-reperfusion.

p39 Affects Myelin Formation in Cerebral Ischemic Injury.

Stroke is a significant public health issue, and research has consistently focused on studying the mechanisms of injury and identifying new targets. As a CDK5 activator, p39 plays a crucial role in various diseases. In this article, we will explore the role and mechanism of p39 in cerebral ischemic injury. We measured the level of p39 using western blot and QPCR at various time points following cerebral ischemia-reperfusion (I/R) injury. The results indicated a significant reduction in the level of p39. TTC staining and behavioral results indicate that the knockout of p39 (p39KO) provides neuroprotection in the short-term. Interestingly, the behavioral dysfunction in p39KO mice was exacerbated after the repair phase of I/R. Further study revealed that this deterioration may be due to demyelination induced by elevated p35 levels. In summary, our study offers profound insights into the significance of p39 in both the acute and repair stages of ischemic injury recovery and a theoretical foundation for future therapeutic drug exploration.

SLC22A17 as a Cell Death-Linked Regulator of Tight Junctions in Cerebral Ischemia.

BACKGROUND: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. METHODS: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. RESULTS: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. CONCLUSIONS: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

Apoptosis, Autophagy, and Mitophagy Genes in the CA3 Area in an Ischemic Model of Alzheimer's Disease with 2-Year Survival.

Background: Currently, no evidence exists on the expression of apoptosis (CASP3), autophagy (BECN1), and mitophagy (BNIP3) genes in the CA3 area after ischemia with long-term survival. Objective: The goal of the paper was to study changes in above genes expression in CA3 area after ischemia in the period of 6-24 months. Methods: In this study, using quantitative RT-PCR, we present the expression of genes associated with neuronal death in a rat ischemic model of Alzheimer's disease. Results: First time, we demonstrated overexpression of the CASP3 gene in CA3 area after ischemia with survival ranging from 0.5 to 2 years. Overexpression of the CASP3 gene was accompanied by a decrease in the activity level of the BECN1 and BNIP3 genes over a period of 0.5 year. Then, during 1-2 years, BNIP3 gene expression increased significantly and coincided with an increase in CASP3 gene expression. However, BECN1 gene expression was variable, increased significantly at 1 and 2 years and was below control values 1.5 years post-ischemia. Conclusions: Our observations suggest that ischemia with long-term survival induces neuronal death in CA3 through activation of caspase 3 in cooperation with the pro-apoptotic gene BNIP3. This study also suggests that the BNIP3 gene regulates caspase-independent pyramidal neuronal death post-ischemia. Thus, caspase-dependent and -independent death of neuronal cells occur post-ischemia in the CA3 area. Our data suggest new role of the BNIP3 gene in the regulation of post-ischemic neuronal death in CA3. This suggests the involvement of the BNIP3 together with the CASP3 in the CA3 in neuronal death post-ischemia.

[Effect of Xuming Decoction in Records of Proved Prescriptions, Ancient a nd Modern on cerebral ischemic injury and angiogenesis in rat model of acute cerebral infarction].

This paper aims to explore the effect of Xuming Decoction in the Records of Proved Prescriptions, Ancient and Modern on cerebral ischemic injury and angiogenesis in the rat model of acute cerebral infarction. SD rats were randomized into 6 groups: sham group, model group, low-, medium-, and high-dose(5.13, 10.26, and 20.52 g·kg~(-1), respectively) Xuming Decoction groups, and butylphthalide(0.06 g·kg~(-1)) group. After the successful establishment of the rat model by middle cerebral artery occlusion(MCAO), rats in the sham and model groups were administrated with distilled water and those in other groups with corresponding drugs for 7 consecutive days. After the neurological function was scored, all the rats were sacrificed, and the brain tissue samples were collected. The degree of cerebral ischemic injury was assessed by the neurological deficit score and staining with 2,3,5-triphenyltetrazolium chloride. Hematoxylin-eosin staining was performed to observe the pathological changes in the brain. Transmission electron microscopy was employed to observe the ultrastructures of neurons and microvascular endothelial cells(ECs) on the ischemic side of the brain tissue. Immunofluorescence assay was employed to detect the expression of von Willebrand factor(vWF) and hematopoietic progenitor cell antigen CD34(CD34) in the ischemic brain tissue. Real-time PCR and Western blot were employed to determine the mRNA and protein levels, respectively, of Runt-related transcription factor 1(RUNX1), vascular endothelial growth factor(VEGF), angiopoietin-1(Ang-1), angiopoietin-2(Ang-2), and VEGF receptor 2(VEGFR2) in the ischemic brain tissue. The results showed that compared with the sham group, the model group showed increased neurological deficit score and cerebral infarction area(P<0.01), pathological changes, and damaged ultrastructure of neurons and microvascular ECs in the ischemic brain tissue. Furthermore, the modeling up-regulated the mRNA levels of RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P<0.01) and the protein levels of vWF, CD34, RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P<0.05 or P<0.01). Compared with the model group, high-dose Xuming Decoction and butylphthalide decreased the neurological deficit score and cerebral infarction area(P<0.01) and alleviated the pathological changes and damage of the ultrastructure of neurons and microvascular ECs in the ischemic brain tissue. Moreover, they up-regulated the mRNA levels of RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P<0.01) and the protein levels of vWF, CD34, RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P<0.01). The results suggest that Xuming Decoction in the Records of Proved Prescriptions, Ancient and Modern can promote the angiogenesis and collateral circulation establishment to alleviate neurological dysfunction of the ischemic brain tissue in MCAO rats by regulating the RUNX1/VEGF pathway.

Microglial CMPK2 promotes neuroinflammation and brain injury after ischemic stroke.

Neuroinflammation plays a significant role in ischemic injury, which can be promoted by oxidized mitochondrial DNA (Ox-mtDNA). Cytidine/uridine monophosphate kinase 2 (CMPK2) regulates mtDNA replication, but its role in neuroinflammation and ischemic injury remains unknown. Here, we report that CMPK2 expression is upregulated in monocytes/macrophages and microglia post-stroke in humans and mice, respectively. Microglia/macrophage CMPK2 knockdown using the Cre recombination-dependent adeno-associated virus suppresses the inflammatory responses in the brain, reduces infarcts, and improves neurological outcomes in ischemic CX3CR1Cre/ERT2 mice. Mechanistically, CMPK2 knockdown limits newly synthesized mtDNA and Ox-mtDNA formation and subsequently blocks NLRP3 inflammasome activation in microglia/macrophages. Nordihydroguaiaretic acid (NDGA), as a CMPK2 inhibitor, is discovered to reduce neuroinflammation and ischemic injury in mice and prevent the inflammatory responses in primary human monocytes from ischemic patients. Thus, these findings identify CMPK2 as a promising therapeutic target for ischemic stroke and other brain disorders associated with neuroinflammation.

Electro-scalp acupuncture regulates the expression of CYP27a1/b1, CYP24a and related inflammatory cytokines in ischemic cortex of rats with middle cerebral artery occlusion. / ç”µå¤´é’ˆè°ƒæŽ§å¤§è„‘ä¸­åŠ¨è„‰æ “å¡žå¤§é¼ ç¼ºè¡€å¤§è„‘çš®å±‚åŒºCYP27a1/b1、CYP24aåŠç›¸å ³ç‚Žæ€§ç»†èƒžå› å­è¡¨è¾¾çš„ç ”ç©¶.

OBJECTIVES: To observe the effect of electro-scalp acupuncture (ESA) on the expression of cytochrome P450a1/b1 (CYP27a1/b1), cytochrome P45024a (CYP24a), signal transducer and activator of transcription (STAT)4, STAT6, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-4 in ischemic cerebral cortex of rats with acute ischemic stroke, so as to explore its mechanism in alleviating inflammatory reaction of ischemic stroke. METHODS: Sixty SD rats were randomly divided into sham-operation, model, vitamin D3 and ESA groups, with 15 rats in each group. The middle cerebral artery occlusion rat model was established with thread ligation according to Zea-Longa's method. Rats in the vitamin D3 group were given 1, 25-VitD3 solution (3 ng·100 g-1·d-1) by gavage, once daily for 7 days. Rats in the ESA group were treated at bilateral anterior parietotemporal slash (MS6) with ESA (2 Hz/100 Hz, 1 mA), 30 min a day for 7 days. Before and after interventions, the neurological deficit score and neurobehavioral score were evaluated. TTC staining was used to detect the volume of cerebral infarction in rats. The positive expressions of CYP24a, CYP27a1 and CYP27b1 in the cerebral cortex of ischemic area were detected by immunofluorescence. The mRNA expressions of STAT4 and STAT6 in the cerebral cortex of ischemic area were detected by quantitative real-time PCR. The protein expression levels of TNF-α, IL-1ß and IL-4 in the cerebral cortex of ischemic area were detected by Western blot. RESULTS: Compared with the sham-operation group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were increased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA, protein expression level of IL-4 were decreased (P<0.01) in the model group. After the treatment and compared with the model group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were decreased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA expression level, protein expression level of IL-4 were increased (P<0.01) in the ESA and vitamin D3 groups. CONCLUSIONS: ESA can alleviate the inflammatory response in ischemic stroke, which maybe related to its function in regulating the balance between CYP27a1/b1 and CYP24a, converting vitamin D into active vitamin D3, inhibiting vitamin D3 degradation, and regulating Th1/Th2 balance.

FOXP3 gene is associated with susceptibility to ischemic stroke in the Chinese population.

AIM: Immunoinflammatory response plays an important role in the pathophysiological process of ischemic stroke (IS). Forkhead box P3 (FOXP3) is a master regulator for immune cells. Polymorphisms of FOXP3 gene might contribute to the susceptibility of IS. This study aimed to explore the association between FOXP3 gene polymorphisms (rs3761548 and rs2232365) and IS susceptibility in the Chinese Han population. METHODS: Polymerase chain reaction and Sanger sequencing were used to detect the genotype of FOXP3 gene rs3761548 and rs2232365 polymorphisms. RESULTS: Smoking, diabetes mellitus (DM), and HBP histories, higher TG and HDL-C levels were more frequently observed in IS patients than in controls. In comparison with rs3761548 GG genotype, GT genotype (OR = 1.573, 95 %CI = 1.030-2.402; adjusted: OR = 1.736, 95 %CI = 1.070-2.817) and GT + TT vs. GG model (OR = 1.581, 95 %CI = 1.0449-2.382; adjusted: OR = 1.720, 95 %CI = 1.074-2.755) of rs3761548 polymorphism was significantly correlated with elevated ischemic stroke susceptibility both at prior and after adjusted by smoking, HBP, DM, TG and HDL-C. Recessive model of rs2232365 polymorphism could elevate the susceptibility of ischemic stroke (OR = 11.962, 95 %CI = 1.144-3.3363; adjusted: OR = 1.876, 95 %CI = 1.016-3.463). Besides, rs3761548 dominant model (OR = 2.757, 95 %CI = 1.379-5.552; adjusted: OR = 2.601, 95 %CI = 1.268-5.336) and rs2232365 recessive model (OR = 3.103, 95 %CI = 1.463-6.583; adjusted: OR = 3.545, 95 %CI = 1.600-7.855) were related to the severity of ischemic stroke. CONCLUSION: FOXP3 gene rs3761548 and rs2232365 polymorphisms were risk factors for susceptibility and severity of IS.

Pharmacological inhibition of mTORC1 reduces neural death and damage volume after MCAO by modulating microglial reactivity.

Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation.

Effect of electroacupuncture on proBDNF/mBDNF and synaptic plasticity in rats with learning and memory impairment after cerebral ischemia-reperfusion. / ç”µé’ˆå¯¹è„‘ç¼ºè¡€å†çŒæ³¨å­¦ä¹ è®°å¿†éšœç¢å¤§é¼ æµ·é©¬è„‘æºæ€§ç¥žç»è¥å »å› å­è½¬åŒ–å’Œçªè§¦å¯å¡‘æ€§çš„å½±å“.

OBJECTIVES: To observe the effect of electroacupuncture (EA) at "Baihui" (GV20) and "Shenting" (GV24) on the rats' behavior and the transforming precursor of brain-derived neurotrophic factor (proBDNF) into mature brain-derived neurotrophic factor (mBDNF) in the hippocampus of rats with learning and memory impairment induced by cerebral ischemia-reperfusion (IR), so as to explore its mechanisms underlying improvement of learning and memory ability. METHODS: SD rats were randomly divided into blank, sham operation, model, and EA groups, with 6 rats in each group. The model of IR was established by occlusion of the middle cerebral artery. EA (1 Hz/20 Hz) was applied to GV24 and GV20 for 30 min, once daily for 14 days. The neurological function was evaluated according to the Zea Longa's score criteria 24 h after modeling and after intervention. Morris water maze test was used to detect the learning and memory function of the rats. TTC staining was used to evaluate the cerebral infarction volume on the affected side. The protein expression levels of proBDNF, mBDNF, tissue plasminogen activator (tPA), tyrosine kinase receptor B (TrkB) and p75 neurotrophin receptor (p75NTR) in hippocampal tissue were detected by Western blot. RESULTS: Compared with the sham operation group, the neurological function score, the percentage of cerebral infarction volume and the expression levels of proBDNF and p75NTR protein in hippocampus were increased (P<0.01), while the times of crossing the original platform and the total distance in the target quadrant, the expression levels of mBDNF, TrkB and tPA protein and the ratio of mBDNF/proBDNF were decreased (P<0.01, P<0.05) in the model group. Compared with the model group, the neurological function score, the percentage of cerebral infarction volume, and the expression levels of proBDNF and p75NTR protein in hippocampus were decreased (P<0.01, P<0.05), while the times of crossing the original platform, the total distance in the target quadrant, and the expression levels of mBDNF, TrkB and tPA protein and the ratio of mBDNF/proBDNF were increased (P<0.05, P<0.01) in the EA group. CONCLUSIONS: EA can alleviate learning and memory impairment in IR rats, which may be related to its function in up-regulating the expression of tPA protein and promoting the transformation of proBDNF to mBDNF, thus improving the synaptic plasticity.

Genetically Determined Plasma Hepatocyte Growth Factor Levels Are Associated With the Risk and Prognosis of Ischemic Stroke.

BACKGROUND: Observational studies suggest that hepatocyte growth factor (HGF) is associated with the risk and prognosis of ischemic stroke, but the causality of these associations remains unclear. Therefore, we conducted Mendelian randomization (MR) analyses to explore the associations of genetically determined plasma HGF levels with the risk and prognosis of ischemic stroke. METHODS: A total of 13 single-nucleotide polymorphisms associated with plasma HGF were selected as genetic instruments based on the data from a genome-wide association study with 21 758 European participants. Summary data about the risk of ischemic stroke were obtained from the MEGASTROKE (Multiancestry Genome-Wide Association Study of Stroke) Consortium with 34 217 ischemic stroke cases and 406 111 controls of European ancestry, and summary data about the prognosis of ischemic stroke were obtained from the GISCOME study (Genetics of Ischaemic Stroke Functional Outcome) with 6165 European patients with ischemic stroke. We conducted an inverse-variance weighted Mendelian randomization analysis followed by a series of sensitivity analyses to evaluate the associations of genetically determined plasma HGF with the risk and prognosis of ischemic stroke. RESULTS: The primary analyses showed that genetically determined high HGF was associated with an increased risk of ischemic stroke (odds ratio per SD increase, 1.11 [95% CI, 1.04-1.19]; P=1.10×10-3) and poor prognosis of ischemic stroke (odds ratio per SD increase, 2.43 [95% CI, 1.76-3.52]; P=6.35×10-8). In the secondary analysis, genetically determined plasma HGF was associated with a high risk of large atherosclerotic stroke (odds ratio per SD increase, 1.39 [95% CI, 1.18-1.63]; P=5.08×10-5) but not small vessel stroke and cardioembolic stroke. Mendelian randomization-Egger regression showed no directional pleiotropy for all associations, and the sensitivity analyses with different Mendelian randomization methods further confirmed these findings. CONCLUSIONS: We found positive associations of genetically determined plasma HGF with the risk and prognosis of ischemic stroke, suggesting that HGF might be implicated in the occurrence and development of ischemic stroke.

Differential DNA methylation associated with delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage: a systematic review.

Recent studies suggest that differential DNA methylation could play a role in the mechanism of cerebral vasospasm (CVS) and delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH). Considering the significance of this matter and a lack of effective prophylaxis against DCI, we aim to summarize the current state of knowledge regarding their associations with DNA methylation and identify the gaps for a future trial. PubMed MEDLINE, Scopus, and Web of Science were searched by two authors in three waves for relevant DNA methylation association studies in DCI after aSAH. PRISMA checklist was followed for a systematic structure. STROBE statement was used to assess the quality and risk of bias within studies. This research was funded by the National Science Centre, Poland (grant number 2021/41/N/NZ2/00844). Of 70 records, 7 peer-reviewed articles met the eligibility criteria. Five studies used a candidate gene approach, three were epigenome-wide association studies (EWAS), one utilized bioinformatics of the previous EWAS, with two studies using more than one approach. Methylation status of four cytosine-guanine dinucleotides (CpGs) related to four distinct genes (ITPR3, HAMP, INSR, CDHR5) have been found significantly or suggestively associated with DCI after aSAH. Analysis of epigenetic clocks yielded significant association of lower age acceleration with radiological CVS but not with DCI. Hub genes for hypermethylation (VHL, KIF3A, KIFAP3, RACGAP1, OPRM1) and hypomethylation (ALB, IL5) in DCI have been indicated through bioinformatics analysis. As none of the CpGs overlapped across the studies, meta-analysis was not applicable. The identified methylation sites might potentially serve as a biomarker for early diagnosis of DCI after aSAH in future. However, a lack of overlapping results prompts the need for large-scale multicenter studies. Challenges and prospects are discussed.

Electroacupuncture Promotes Angiogenesis in Mice with Cerebral Ischemia by Inhibiting miR-7.

OBJECTIVE: To observe the angiogenesis effect of electroacupuncture (EA) at Shuigou acupoint (GV 26) in the treatment of cerebral ischemia, and explore the value of miRNA-7 (miR-7) in it. METHODS: First, 48 mice were randomly divided into sham operation, middle cerebral artery occlusion (MCAO) model, and EA treatment groups. Then 9 mice were divided into carrier control group, miR-7 knockout group and miR-7 overexpression group (n=3 each group). Finally, 20 mice were divided into model and carrier control group, model and miR-7 knockout group, EA treatment and carrier control group and EA treatment and miR-7 overexpression group, with 3-6 mice in each group. The MCAO model was established in the MCAO and EA groups. Neurological deficit score and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to evaluate the severity of cerebral ischemia. Hematoxylin-eosin staining was used to describe basic pathological changes. Immunohistochemistry was used to quantify cerebral microvessel density. Real-time PCR and Western blot were used to detect the expression of miR-7 and its downstream target genes Krüppel-like factor 4/vascular endothelial growth factor (KLF4/VEGF) and angiopoietin-2 (ANG-2) in the ischemic cerebral cortex. RESULTS: After EA, neurological deficit scores and infarction volumes decreased, and the density of cerebral microvessels increased. In the MCAO group, miR-7 expression was higher than that in the sham group (P<0.01). After EA at GV 26, miR-7 expression decreased (P<0.01) and the expression of downstream target genes KLF4/VEGF and ANG-2 increased as compared with the MCAO group (P<0.01). After EA combined with overexpression of miR-7, the expression of downstream target genes KLF4/VEGF and ANG-2 decreased compared to the control EA group (P<0.01). After miR-7 knockdown, the expression of KLF4/VEGF and ANG-2 increased (P<0.05 or P<0.01). CONCLUSIONS: EA could promote angiogenesis in MCAO mice likely by inhibiting the expression of miR-7 and relieving inhibition of downstream target genes KLF4/VEGF and ANG-2.

TIGAR reduces neuronal ferroptosis by inhibiting succinate dehydrogenase activity in cerebral ischemia.

Ischemia Stroke (IS) is an acute neurological condition with high morbidity, disability, and mortality due to a severe reduction in local cerebral blood flow to the brain and blockage of oxygen and glucose supply. Oxidative stress induced by IS predisposes neurons to ferroptosis. TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits the intracellular glycolytic pathway to increase pentose phosphate pathway (PPP) flux, promotes NADPH production and thus generates reduced glutathione (GSH) to scavenge reactive oxygen species (ROS), and thus shows strong antioxidant effects to ameliorate cerebral ischemia/reperfusion injury. However, in the current study, prolonged ischemia impaired the PPP, and TIGAR was unable to produce NADPH but was still able to reduce neuronal ferroptosis and attenuate ischemic brain injury. Ferroptosis is a form of cell death caused by free radical-driven lipid peroxidation, and the vast majority of ROS leading to oxidative stress are generated by mitochondrial succinate dehydrogenase (SDH) driving reverse electron transfer (RET) via the mitochondrial electron transport chain. Overexpression of TIGAR significantly inhibited hypoxia-induced enhancement of SDH activity, and TIGAR deficiency further enhanced SDH activity. We also found that the inhibitory effect of TIGAR on SDH activity was related to its mitochondrial translocation under hypoxic conditions. TIGAR may inhibit SDH activity by mediating post-translational modifications (acetylation and succinylation) of SDH A through interaction with SDH A. SDH activity inhibition reduces neuronal ferroptosis by decreasing ROS production, eliminating MitoROS levels and attenuating lipid peroxide accumulation. Notably, TIGAR-mediated inhibition of SDH activity and ferroptosis was not dependent on the PPP-NADPH-GPX4 pathways. In conclusion, mitochondrial translocation of TIGAR in prolonged ischemia is an important pathway to reduce neuronal ferroptosis and provide sustainable antioxidant defense for the brain under prolonged ischemia, further complementing the mechanism of TIGAR resistance to oxidative stress induced by IS.

Mechanism of astragaloside IV regulating NLRP3 through LOC102555978 to attenuate cerebral ischemia reperfusion induced microglia pyroptosis.

Astragaloside IV(ASⅣ), the main component of Radix Astragali, has been used to treat cerebral ischemia reperfusion injury (CIRI). However, the molecular mechanism of ASIV in CIRI needs to be further elucidated. Long non-coding RNA (lncRNA) is considered to be an important kind of regulatory molecule in CIRI. In this work, the biological effect and molecular mechanism of ASIV in CIRI through lncRNA were analyzed by using rat middle cerebral artery occlusion and reperfusion (MCAO/R) model and primary rat microglia (RM) cells oxygen and glucose deprivation/reoxygenation (OGD/R) model. The neurological deficit score was evaluated, the volume of cerebral infarction was calculated, and pyroptosis related molecules were detected by qPCR and western blot. Then, high-throughput sequencing was performed in sham and MCAO/R groups. The competitive endogenous RNA (ceRNA) networks associated with pyroptosis were constructed by functional enrichment analysis. CCK-8 detection of cell survival rate, qPCR and western blot were used to determine the specific molecular mechanism of ASⅣ through ceRNA in vitro. Results showed thatASⅣ could decrease the neurological deficit score, reduce the volume of cerebral infarction, inhibit inflammatory reaction and pyroptosis in MCAO/R model rats. Next, the ceRNA network was established, including the LOC102555978/miR-3584-5p/NLRP3 regulatory network. In vitro experiments showed that LOC102555978 promotes NLRP3 mediated pyroptosis of RM cells through sponge adsorption of miR-3584-5p, which may provide a potential therapeutic target for post-CIRI inflammation regulation. ASⅣ could inhibit pyroptosis of RM cells by down-regulating LOC102555978. LOC102555978/miR-3584-5p/NLRP3 may be the molecular mechanism of ASⅣ's CIRI protective effect.

Utilizing RNA sequencing to identify gene expression markers of stroke-causing thrombi origin: A pilot study.

INTRODUCTION: Stroke embolic source have an unknown origin in 30-40% of cases. Mechanical thrombectomy for acute large vessel occlusion stroke has provided us with a method to directly retrieve the thrombi from patients for analysis. By collecting stroke-causing thrombi from known sources, we can then use high-throughput RNA sequencing (RNAseq) technology to directly measure the gene expression signatures of these clots. This may allow us to identify genetic markers to predict the cause of cryptogenic embolism. METHODS: This is a prospective study in which RNAseq was used to analyze cerebral thrombi retrieved by mechanical thrombectomy devices in acute ischemic stroke patients. Samples were separated into two groups based on known stroke thrombus etiology, including Carotid group (patients with ipsilateral >70% carotid stenosis) and Atrial fibrillation (AF) group (patients with atrial fibrillation). Gene expression was compared by RNAseq analysis between the groups. RESULTS: From October 2016 to September 2017, 8 thrombi (4 in Carotid group, 4 in Afib group) were included in this study. There were 131 genes that were significantly up- or down-regulated between the two groups defined as a false discovery rate ≤ 0.05 and a fold change ≥ 2. Twenty-six genes were selected as candidate gene biomarkers based on the criteria in the methods section. Candidate genes HSPA1B, which encodes a heatshock protein, and GPRC5B, which encodes a G-protein, showed the greatest fold differences in expression between the two groups. CONCLUSION: This study has shown that RNA sequencing of acute ischemic stroke thrombi is feasible and indentified potential novel biomarkers for identifying stroke-causing thrombi origin, especially in cryptogenic stroke.

Differential Vulnerability and Response to Injury among Brain Cell Types Comprising the Neurovascular Unit.

The neurovascular unit (NVU) includes multiple different cell types, including neurons, astrocytes, endothelial cells, and pericytes, which respond to insults on very different time or dose scales. We defined differential vulnerability among these cell types, using response to two different insults: oxygen-glucose deprivation (OGD) and thrombin-mediated cytotoxicity. We found that neurons are most vulnerable, followed by endothelial cells and astrocytes. After temporary focal cerebral ischemia in male rats, we found significantly more injured neurons, compared with astrocytes in the ischemic area, consistent with differential vulnerability in vivo. We sought to illustrate different and shared mechanisms across all cell types during response to insult. We found that gene expression profiles in response to OGD differed among the cell types, with a paucity of gene responses shared by all types. All cell types activated genes relating to autophagy, apoptosis, and necroptosis, but the specific genes differed. Astrocytes and endothelial cells also activated pathways connected to DNA repair and antiapoptosis. Taken together, the data support the concept of differential vulnerability in the NVU and suggest that different elements of the unit will evolve from salvageable to irretrievable on different time scales while residing in the same brain region and receiving the same (ischemic) blood flow. Future work will focus on the mechanisms of these differences. These data suggest future stroke therapy development should target different elements of the NVU differently.