Induced Mesenchymal Stem Cells-Small Extracellular Vesicles Alleviate Post-stroke Cognitive Impairment by Rejuvenating Senescence of Neural Stem Cells.

Ischemic stroke is typified by hypoxia and a cascade of pathophysiological events, including metabolic dysfunction, ionic dysregulation, excitotoxicity, inflammatory infiltration, and oxidative stress. These ultimately result in neuronal apoptosis or necrosis with constrained neuroregenerative capabilities. In this study, neural stem cells (NSCs) under conditions of oxygen-glucose deprivation (OGD) in vitro and following middle cerebral artery occlusion (MCAO) in vivo were explored. Transcriptome sequencing revealed a decline in NSC differentiation and neurogenesis after OGD exposure, which was related to cellular senescence. This observation was corroborated by increased senescence markers in the MCAO mouse model, reduction in NSC numbers, and decline in neurogenesis. Importantly, iMSC-sEVs (induced mesenchymal stem cells-small extracellular vesicles) have the therapeutic potential to alleviate NSC senescence and rejuvenate their regenerative capacities both in vitro and in vivo. Moreover, iMSC-sEVs contribute to the recovery of cognitive function and synapse loss caused by MCAO.

Identification of Hub Genes and Pathways of Middle Cerebral Artery Occlusion in Aged Rats Using the Gene Expression Omnibus Database.

Stroke remained the leading cause of disability in the world, and the most important non-modifiable risk factor was age. The treatment of stroke for elder patients faced multiple difficulties due to its complicated pathogenesis and mechanism. Therefore, we aimed to identify the potential differentially expressed genes (DEGs) and singnalling pathways for aged people of stroke. To compare the DEGs in the aged rats with or without middle cerebral artery occlusion (MCAO) and to analyse the important genes and the key signaling pathways involved in the development of cerebral ischaemia in aged rats. The Gene Expression Omnibus (GEO) analysis tool was used to analyse the DEGs in the GSE166162 dataset of aged MCAO rats compared with aged sham rats. Differential expression analysis was performed in aged MCAO rats and sham rats using limma. In addition, the 74 DEGs (such as Fam111a, Lcn2, Spp1, Lgals3 and Gpnmb were up-regulated; Egr2, Nr4a3, Arc, Klf4 and Nr4a1 were down-regulated) and potential compounds corresponding to the top 20 core genes in the Protein-Protein Interaction (PPI) network was constructed using the STRING database (version 12.0). Among these 30 compounds, resveratrol, cannabidiol, honokiol, fucoxanthin, oleandrin and tyrosol were significantly enriched. These DEGs were subjected to Gene Ontology (GO) function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to determine the most significantly enriched pathway in aged MCAO rats. Moreover, innate immune response, the complement and coagulation cascades signaling pathway, the IL-17 and other signaling pathways were significantly correlated with the aged MCAO rats. Our study indicates that multiple genes and pathological processes involved in the aged people of stroke. The immune response might be the key pathway in the intervention of cerebral infarction in aged people.

Tanshinone IIA Alleviates Traumatic Brain Injury by Reducing Ischemia‒Reperfusion via the miR-124-5p/FoxO1 Axis.

Background: Cerebral ischemia-reperfusion injury is a common complication of ischemic stroke that affects the prognosis of patients with ischemic stroke. The lipid-soluble diterpene Tanshinone IIA, which was isolated from Salvia miltiorrhiza, has been indicated to reduce cerebral ischemic injury. In this study, we investigated the molecular mechanism of Tanshinone IIA in alleviating reperfusion-induced brain injury. Methods: Middle cerebral artery occlusion animal models were established, and neurological scores, tetrazolium chloride staining, brain volume quantification, wet and dry brain water content measurement, Nissl staining, enzyme-linked immunosorbent assay, flow cytometry, western blotting, and reverse transcription-quantitative polymerase chain reaction were performed. The viability of cells was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assays, while cell damage was measured by lactate dehydrogenase release in the in vitro oxygen glucose deprivation model. In addition, enzyme-linked immunosorbent assay, flow cytometry, western blotting, and reverse transcription-quantitative polymerase chain reaction were used to evaluate the therapeutic effect of Tanshinone IIA on ischemia/reperfusion (I/R) induced brain injury, as well as its effects on the inflammatory response and neuronal apoptosis, in vivo and in vitro. Furthermore, this study validated the targeting relationship between miR-124-5p and FoxO1 using a dual luciferase assay. Finally, we examined the role of Tanshinone IIA in brain injury from a molecular perspective by inhibiting miR-124-5p or increasing FoxO1 levels. Results: After treatment with Tanshinone IIA in middle cerebral artery occlusion-reperfusion (MCAO/R) rats, the volume of cerebral infarction was reduced, the water content of the brain was decreased, the nerve function of the rats was significantly improved, and the cell damage was significantly reduced. In addition, Tanshinone IIA effectively inhibited the I/R-induced inflammatory response and neuronal apoptosis, that is, it inhibited the expression of inflammatory cytokines IL-1ß, IL-6, TNF-α, decreased the expression of apoptotic protein Bax and Cleaved-caspase-3, and promoted the expression of antiapoptotic protein Bcl-2. In vitro oxygen-glucose deprivation/reoxygenation (OGD/R) cell model, Tanshinone IIA also inhibited the expression of inflammatory factors in neuronal cells and inhibited the occurrence of neuronal apoptosis. In addition, Tanshinone IIA promoted the expression of miR-124-5p. Transfection of miR-124-5p mimic has the same therapeutic effect as Tanshinone IIA and positive therapeutic effect on OGD cells, while transfection of miR-124-5p inhibitor has the opposite effect. The targeting of miR-124-5p negatively regulates FoxO1 expression. Inhibition of miR-124-5p or overexpression of FoxO1 can weaken the inhibitory effect of Tanshinone IIA on brain injury induced by I/R, while inhibition of miR-124-5p and overexpression of FoxO1 can further weaken the effect of Tanshinone IIA. Conclusion: Tanshinone IIA alleviates ischemic-reperfusion brain injury by inhibiting neuroinflammation through the miR-124-5p/FoxO1 axis. This finding provides a theoretical basis for mechanistic research on cerebral ischemia-reperfusion injury.

Ginsenoside Rd enhances blood-brain barrier integrity after cerebral ischemia/reperfusion by alleviating endothelial cells ferroptosis via activation of NRG1/ErbB4-mediated PI3K/Akt/mTOR signaling pathway.

The incidence of ischemic stroke is increasing year by year and showing a younger trend. Impaired blood-brain barrier (BBB) is one of the pathological manifestations caused by cerebral ischemia, leading to poor prognosis of patients. Accumulating evidence indicates that ferroptosis is involved in cerebral ischemia/reperfusion injury (CIRI). We have previously demonstrated that Ginsenoside Rd (G-Rd) protects against CIRI-induced neuronal injury. However, whether G-Rd can attenuate CIRI-induced disruption of the BBB remains unclear. In this study, we found that G-Rd could upregulate the levels of ZO-1, occludin, and claudin-5 in ipsilateral cerebral microvessels and bEnd.3 cells, reduce endothelial cells (ECs) loss and Evans blue (EB) leakage, and ultimately improve BBB integrity after CIRI. Interestingly, the expressions of ACSL4 and COX2 were upregulated, the expressions of GPX4 and xCT were downregulated, the levels of GSH was decreased, and the levels of MDA and Fe2+ were increased in ischemic tissues and bEnd.3 cells after CIRI, suggesting that ECs ferroptosis occurred after CIRI. However, G-Rd can alleviate CIRI-induced BBB disruption by inhibiting ECs ferroptosis. Mechanistically, G-Rd prevented tight junction loss and BBB leakage by upregulating NRG1, activating its tyrosine kinase ErbB4 receptor, and then activating downstream PI3K/Akt/mTOR signaling, thereby inhibiting CIRI-induced ferroptosis in ECs. Taken together, these data provides data support for G-Rd as a promising therapeutic drug for cerebral ischemia.

FGF17 protects cerebral ischemia reperfusion-induced blood-brain barrier disruption via FGF receptor 3-mediated PI3K/AKT signaling pathway.

Maintaining blood-brain barrier (BBB) integrity is critical components of therapeutic approach for ischemic stroke. Fibroblast growth factor 17 (FGF17), a member of FGF8 superfamily, exhibits the strongest expression throughout the wall of all major arteries during development. However, its molecular action and potential protective role on brain endothelial cells after stroke remains unclear. Here, we observed reduced levels of FGF17 in the serum of patients with ischemic stroke, as well as in the brains of mice subjected to middle cerebral artery occlusion (MCAO) injury and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cells (bEnd.3) cells. Moreover, treatment with exogenous recombinant human FGF17 (rhFGF17) decreased infarct volume, improved neurological deficits, reduced Evans Blue leakage and upregulated the expression of tight junctions in MCAO-injured mice. Meanwhile, rhFGF17 increased cell viability, enhanced trans-endothelial electrical resistance, reduced sodium fluorescein leakage, and alleviated reactive oxygen species (ROS) generation in OGD/R-induced bEnd.3 cells. Mechanistically, the treatment with rhFGF17 resulted in nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear accumulation and upregulation of heme oxygenase-1 (HO-1) expression. Additionally, based on in-vivo and in-vitro research, rhFGF17 exerted protective effects against ischemia/reperfusion (I/R) -induced BBB disruption and endothelial cell apoptosis through the activation of the FGF receptor 3/PI3K/AKT signaling pathway. Overall, our findings indicated that FGF17 may hold promise as a novel therapeutic strategy for ischemic stroke patients.

Formononetin alleviates cerebral ischemia-reperfusion injury in rats by targeting the PARP-1/PARG/Iduna signaling pathway.

Formononetin has been demonstrated to protect against cerebral ischemia-reperfusion injury, however its mechanism has to be further researched. This study examined the effect of formononetin on cerebral ischemia-reperfusion injury in rats using the PARP-1/PARG/Iduna signaling pathway. In male SD rats, a model of cerebral ischemia-reperfusion injury was developed. Animals were randomly assigned to one of eight groups: Sham operation, Sham operation + formononetin, MCAO, MCAO + formononetin, PARP inhibitor (PJ34) + MCAO, formononetin + PJ34 + MCAO, PARG inhibitor (Ethacridine lactate) + MCAO, and ethacridine lactate + formononetin. The neurological deficit test, TTC staining, HE staining, Nissl staining, TUNEL staining, and western blotting were utilized to assess formononetin's protective effects in MCAO rats. The data show that formononetin can effectively alleviate neurological dysfunction and pathological changes in brain tissue in rats with cerebral ischemia-reperfusion injury, reduce the area of cerebral infarction and neuronal apoptosis, decrease the protein levels of PARP-1, PARG, Caspase-3, P53, and AIF in brain tissue, and increase the protein levels of Iduna and p-AKT. As a result, we concluded that formononetin improves brain ischemia-reperfusion injury in rats by modulating the PARP-1/PARG/Iduna signaling pathway.

A Brain Ischemia-Reperfusion Model for the Study of Tau Phosphorylation and O-GlcNAcylation.

Cerebral ischemia produces a decrease, loss, or instability of the assembly processes in the neuronal cytoskeleton, related to the alteration in the normal processes of phosphorylation of the Tau protein, triggering its hyperphosphorylation and altering the normal processes of formation of neuronal microtubules. Here we describe the methods used to study the impact of middle cerebral artery occlusion (MCAo) on neurological functions and Tau phosphorylation in Wistar rat brain.

FDA compound library screening Baicalin upregulates TREM2 for the treatment of cerebral ischemia-reperfusion injury.

Acute ischemic stroke (AIS) is a leading cause of global incidence and mortality rates. Oxidative stress and inflammation are key factors in the pathogenesis of AIS neuroinjury. Therefore, it is necessary to develop drugs that target neuroinflammation and oxidative stress in AIS. The Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), primarily expressed on microglial cell membranes, plays a critical role in reducing inflammation and oxidative stress in AIS. In this study, we employed a high-throughput screening (HTS) strategy to evaluate 2625 compounds from the (Food and Drug Administration) FDA library in vitro to identify compounds that upregulate the TREM2 receptor on microglia. Through this screening, we identified Baicalin as a potential drug for AIS treatment. Baicalin, a flavonoid compound extracted and isolated from the root of Scutellaria baicalensis, demonstrated promising results. Next, we established an in vivo mouse model of cerebral ischemia-reperfusion injury (MCAO/R) and an in vitro microglia cell of oxygen-glucose deprivation reperfusion (OGD/R) to investigate the role of Baicalin in inflammation injury, oxidative stress, and neuronal apoptosis. Our results showed that baicalin effectively inhibited microglia activation, reactive oxygen species (ROS) production, and inflammatory responses in vitro. Additionally, baicalin suppressed neuronal cell apoptosis. In the in vivo experiments, baicalin not only improved neurological functional deficits and reduced infarct volume but also inhibited microglia activation and inflammatory responses. Overall, our findings demonstrate the efficacy of Baicalin in treating MCAO/R by upregulating TREM2 to reduce inflammatory responses and inhibit neuronal apoptosis.

Cepharanthine maintains integrity of the blood-brain barrier (BBB) in stroke via the VEGF/VEGFR2/ZO-1 signaling pathway.

Dysfunction of tight junctions such as zonula occludens protein-1 (ZO-1)-associated aggravation of blood-brain barrier (BBB) permeability plays an important role in the progression of stroke. Cepharanthine (CEP) is an extract from the plant Stephania cepharantha. However, the effects of CEP on stroke and BBB dysfunction have not been previously reported. In this study, we report that CEP improved dysfunction in neurological behavior in a middle cerebral artery occlusion (MCAO) mouse model. Importantly, CEP suppressed blood-brain barrier (BBB) hyperpermeability by increasing the expression of ZO-1. Notably, we found that CEP inhibited the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) in the cortex of MCAO mice. Additionally, the results of in vitro experiments demonstrate that treatment with CEP ameliorated cytotoxicity of human bEnd.3 brain microvascular endothelial cells against hypoxia/reperfusion (H/R). Also, CEP attenuated H/R-induced aggravation of endothelial permeability in bEND.3 cells by restoring the expression of ZO-1. Further study proved that the protective effects of CEP are mediated by inhibition of VEGF-A and VEGFR2. Based on the results, we conclude that CEP might possess a therapeutic prospect in stroke through protecting the integrity of the BBB mediated by the VEGF/VEGFR2/ZO-1 axis.

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.

Long noncoding RNA KCNQ1OT1 aggravates cerebral infarction by regulating PTBT1/SIRT1 via miR-16-5p.

Cerebral infarction (CI) is one of the leading causes of disability and death. LncRNAs are key factors in CI progression. Herein, we studied the function of long noncoding RNA KCNQ1OT1 in CI patient plasma samples and in CI models. Quantitative real-time PCR and Western blotting tested gene and protein expressions. The interactions of KCNQ1OT1/PTBP1 and miR-16-5p were analyzed using dual-luciferase reporter and RNA immunoprecipitation assays; MTT assays measured cell viability. Cell migration and angiogenesis were tested by wound healing and tube formation assays. Pathological changes were analyzed by triphenyltetrazolium chloride and routine staining. We found that KCNQ1OT1 and PTBP1 were overexpressed and miR-16-5p was downregulated in CI patient plasma and in oxygen-glucose deprived (OGD) induced mouse brain microvascular endothelial (bEnd.3) cells. KCNQ1OT1 knockdown suppressed pro-inflammatory cytokine production and stimulated angiogenic responses in OGD-bEnd.3 cells. KCNQ1OT1 upregulated PTBP1 by sponging miR-16-5p. PTBP1 overexpression or miR-16-5p inhibition attenuated the effects of KCNQ1OT1 knockdown. PTBP1 silencing protected against OGD-bEnd.3 cell injury by enhancing SIRT1. KCNQ1OT1 silencing or miR-16-5p overexpression also alleviated ischemic injury in a mice middle cerebral artery occlusion model. Thus, KCNQ1OT1 silencing alleviates CI by regulating the miR-16-5p/PTBP1/SIRT1 pathway, providing a theoretical basis for novel therapeutic strategies targeting CI.

Electroacupuncture ameliorates neuroinflammation by inhibiting TRPV4 channel in ischemic stroke.

AIMS: We investigated the potential mechanisms underlying the therapeutic efficacy of electroacupuncture (EA) at the Shuigou (GV26) and Baihui (GV20) acupoints in the treatment of ischemic stroke. METHODS: We assessed the therapeutic effects of EA on MCAO mice through behavioral studies and TTC staining. Various techniques, such as RT-PCR, immunofluorescence, and Western blots, were employed to evaluate the activation and polarization of microglia/macrophages, and changes in the TRPV4 ion channel. We used the TRPV4 antagonist GSK2193874 (GSK219) to verify the involvement of TRPV4 in the therapeutic effects of EA. RESULTS: EA effectively improved neurological impairments and reduced cerebral infarction volume in MCAO mice. It suppressed activated microglia/macrophages and inhibited their polarization toward the M1 phenotype post-MCAO. EA also downregulated the expression of pro-inflammatory cytokines, including Tnf-α, Il-6, Il-1ß, and Ccl-2 mRNA. Furthermore, EA reduced the elevated expression of TRPV4 following MCAO. Treatment with the TRPV4 antagonist GSK219 mirrored the effects of EA in MCAO mice. Notably, the combination of EA and GSK219 did not demonstrate an additive or synergistic effect. CONCLUSION: EA may inhibit neuroinflammation and exhibit a protective effect against ischemic brain injury by suppressing TRPV4 and the subsequent M1 polarization of microglia/macrophages.

Sophoricoside ameliorates cerebral ischemia-reperfusion injury dependent on activating AMPK.

AIMS: Ischemic stroke accounts for 87% of all strokes, and its death and disability bring a huge burden to society. Brain injury caused by ischemia-reperfusion (I/R) is also a major difficulty in clinical treatment and prognosis. Sophoricoside (SOP) is an isoflavone glycoside isolated from the seed of medical herb Sophora japonica L. Previously, SOP was found to be effective in anti-inflammation and glucose-lipid metabolism-related diseases. In order to investigate whether SOP has a regulatory effect on cerebral I/R injury, we conducted this study. METHODS: Here, by application of SOP into MCAO (transient middle cerebral artery occlusion)-induced mice and OGD/R (oxygen glucose deprivation/reperfusion)-induced primary neurons, the regulation effects of SOP was analyzed by detecting neurological score of post-stroke mice, phenotypes of brains and brain sections, cell viabilities, and apoptosis- and inflammation-regulation. RNA sequencing and molecular biology experiments were performed to explore the mechanism of SOP regulating cerebral I/R injury. RESULTS: SOP administration decreased the infarct size, neurological deficit score, neuronal cell injury, inflammation and apoptosis. Mechanistically, SOP exerted its protective effect by activating the AMP-activated protein kinase (AMPK) signaling pathway. CONCLUSION: SOP inhibits cerebral I/R injury by promoting the phosphorylation of AMPK.

Effect of Cyclosporin H on ischemic injury and neutrophil infiltration in cerebral infarct model of rats via PET imaging.

BACKGROUND: Brain ischemia-reperfusion injury is a complex process, and neuroinflammation is an important secondary contributing pathological event. Neutrophils play major roles in ischemic neuroinflammation. Once activated, neutrophils express formyl peptide receptors (FPRs), which are special receptors of a class of chemoattractants and may be potential targets to regulate the activity of neutrophils and control cerebral ischemic injury. This study was aimed to explore the ameliorating effect of Cyclosporin H (CsH), a potent FPR antagonist, on brain ischemic injury by inhibiting the activation and migration of neutrophils, and improving cerebral blood flow. METHODS: We employed a middle cerebral artery occlusion (MCAO) Model on rats and performed behavioral, morphological, and microPET imaging assays to investigate the potential restoring efficacy of CsH on cerebral ischemic damages. Peptide N-cinnamoyl-F-(D)L-F-(D)L-F (cFLFLF), an antagonist to the neutrophil FPR with a high binding affinity, was used for imaging neutrophil distribution. RESULTS: We found that CsH had similar effect with edaravone on improving the neurobehavioral deficient symptoms after cerebral ischemia-reperfusion, and treatment with CsH also alleviated ischemic cerebral infarction. Compared with the MCAO Model group, [18F]FDG uptake ratios of the CsH and edaravone treatment groups were significantly higher. The CsH-treated groups also showed significant increases in [18F]FDG uptake at 144 h when compared with that of 24 h. This result indicates that like edaravone, treatment with both doses of CsH promoted the recovery of blood supply after cerebral ischemic event. Moreover, MCAO-induced cerebral ischemia significantly increased the radiouptake of [68Ga]Ga-cFLFLF at 72 h after ischemia-reperfusion operation. Compared with MCAO Model group, radiouptake values of [68Ga]-cFLFLF in both doses of CsH and edaravone groups were all decreased significantly. These results showed that both doses of CsH resulted in a similar therapeutic effect with edaravone on inhibiting neutrophil infiltration in cerebral infarction. CONCLUSION: Potent FPR antagonist CsH is promisingly beneficial in attenuating neuroinflammation and improving neurobehavioral function against cerebral infarction. Therefore, FPR may become a novel target for regulating neuroinflammation and improving prognosis for ischemic cerebrovascular disorders.

Sinigrin improves cerebral ischaemia-reperfusion injury by inhibiting the TLR4 pathway-mediated oxidative stress.

Cerebral ischaemia-reperfusion (CIR) injury occurs in stroke patients after the restoration of cerebral perfusion. Sinigrin, a phytochemical found in cruciferous vegetables, exhibits strong antioxidant activity. This study investigated the role of sinigrin in oxidative stress using a CIR injury model. The effects of sinigrin were studied in middle cerebral artery occlusion (MCAO) rats and oxygen-glucose deprivation/reoxygenation (OGD/R)-injured SH-SY5Y cells. Sinigrin treatment improved brain injury and neurological deficits induced by MCAO surgery in rats. Sinigrin inhibited apoptosis in brain tissues and SH-SY5Y cells following OGD/R induction. Additionally, sinigrin elevated the levels of superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) while reducing malondialdehyde (MDA) levels. Furthermore, sinigrin inhibited the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signalling pathway. The anti-apoptotic and antioxidant activities of sinigrin in OGD/R-injured SH-SY5Y cells were reversed by TLR4 overexpression. In conclusion, sinigrin inhibits oxidative stress in CIR injury by suppressing the TLR4/MyD88 signalling pathway.

Blocking cerebral lymphatic system reduces central and peripheral inflammatory response in ischemic stroke.

Reduced blood supply to the brain activates the intracranial inflammatory response, a key contributor to secondary brain damage in ischemic stroke. Post-stroke, activation of peripheral immune cells leads to systemic inflammatory responses. Usingin vivo approaches, we investigated meningeal lymphatics' role in central immune cell infiltration and peripheral immune cell activation. The bilateral deep cervical lymph nodes (dCLNs) were removed 7 days before right middle cerebral artery occlusion in Sprague Dawley (SD) rats. At 3, 24, and 72 h post-intervention, brain immune cell infiltration and microglial and astrocyte activation were measured, while immune cells were classified in the spleen and blood. Inflammatory factor levels in peripheral blood were analyzed. Simultaneously, reverse verification was conducted by injecting AAV-vascular endothelial growth factor C (AAV-VEGFC) adenovirus into the lateral ventricle 14 days before middle cerebral artery occlusion (MCAO) induction to enhance meningeal lymph function. Blocking meningeal LVs in MCAO rats significantly reduced infarct area and infiltration, and inhibited microglia and pro-inflammatory astrocytes activation. After removing dCLNs, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, macrophages, and neutrophils in the spleen and blood of MCAO rats decreased significantly at different time points. The levels of inflammatory factors IL-6, IL-10, IL-1ß, and TNF-α in plasma decreased significantly. Tests confirmed the results, and AAV-VEGFC-induced MCAO rats provided reverse validation.

Thymoquinone regulates microglial M1/M2 polarization after cerebral ischemia-reperfusion injury via the TLR4 signaling pathway.

Acute ischemic stroke followed by microglia activation, and the regulation of neuroinflammatory responses after ischemic injury involves microglia polarization. microglia polarization is involved in the regulation of neuroinflammatory responses and ischemic stroke-related brain damage. Thymoquinone (TQ) is an anti-inflammatory agent following ischemic stroke onset. However, the significance of TQ in microglia polarization following acute ischemic stroke is still unclear. We predicted that TQ might have neuroprotective properties by modulating microglia polarization. In this work, we mimicked the clinical signs of acute ischemic stroke using a mouse middle cerebral artery ischemia-reperfusion (I/R) model. It was discovered that TQ treatment decreased I/R-induced infarct volume, cerebral oedema, and promoted neuronal survival, as well as improved the histopathological changes of brain tissue. The sensorimotor function was assessed by the Garica score, foot fault test, and corner test, and it was found that TQ could improve the motor deficits caused by I/R. Secondly, real-time fluorescence quantitative PCR, immuno-fluorescence, ELISA, and western blot were used to detect the expression of M1/M2-specific markers in microglia to explore the role of TQ in the modulation of microglial cell polarization after cerebral ischemia-reperfusion. We found that TQ was able to promote the polarization of microglia with extremely secreted inflammatory factors from M1 type to M2 type. Furthermore, TQ could block the TLR4/NF-κB signaling pathway via Hif-1α activation which subsequently may attenuate microglia differentiation following the cerebral ischemia, establishing a mechanism for the TQ's beneficial effects in the cerebral ischemia-reperfusion model.

Edaravone dexborneol alleviates ischemic injury and neuroinflammation by modulating microglial and astrocyte polarization while inhibiting leukocyte infiltration.

Poststroke inflammation is essential in the mechanism of secondary injury, and it is orchestrated by resident microglia, astrocytes, and circulating immune cells. Edaravone dexborneol (EDB) is a combination of edaravone and borneol that has been identified as a clinical protectant for stroke management. In this study, we verified the anti-inflammatory effect of EDB in the mouse model of ischemia and investigated its modulatory action on inflammation-related cells. C57BL/6 male mice, which had the transient middle cerebral artery occlusion (tMCAO), were treated (i.p.) with EDB (15 mg/kg). EDB administration significantly reduced the brain infarction and improved the sensorimotor function after stroke. And EDB alleviated the neuroinflammation by restraining the polarization of microglia/macrophages and astrocyte toward proinflammatory phenotype and inhibiting the production of proinflammatory cytokines (such as IL-1ß, TNF-α, and IL-6) and chemokines (including MCP-1 and CXCL1). Furthermore, EDB ameliorated the MCAO-induced impairment of Blood-brain barrier (BBB) by suppressing the degradation of tight junction protein and attenuated the accumulation of peripheral leukocytes in the ischemic brain. Additionally, systemic EDB administration inhibited the macrophage phenotypic shift toward the M1 phenotype and the macrophage-dependent inflammatory response in the spleen and blood. Collectively, EDB protects against ischemic stroke injury by inhibiting the proinflammatory activation of microglia/macrophages and astrocytes and through reduction by invasion of circulating immune cells, which reduces central and peripheral inflammation following stroke.

Enriched environment treatment promotes neural functional recovery together with microglia polarization and remyelination after cerebral ischemia in rats.

BACKGROUND: Microglia activation and oligodendrocyte maturation are critical for remyelination after cerebral ischemia. Studies have shown that enriched environment (EE) can effectively alleviate stroke-induced neurological deficits. However, little is known about the mechanism associated with glial cells underlying the neuroprotection of EE. Therefore, this study focuses on investigating the effect of EE on activated microglia polarization as well as oligodendrogenesis in the progress of remyelination following cerebral ischemia. METHODS: The ischemia/reperfusion (I/R) injury model was established by middle cerebral artery occlusion (MCAO) in rats. Animals executed 4 weeks of environmental intervention after performing MCAO or sham surgery and were divided into sham, MCAO, and MCAO+EE groups. Cognitive function, myelin damage, microglia activation and polarization, inflammation, oligodendrogenesis, remyelination, and protein expression of the PI3K/AKT/GSK3ß signaling pathway were determined. RESULTS: The staining of NeuN indicated that the infarct size of MCAO rats was decreased under EE. EE intervention improved animal performance in the Morris water maze test and novel object recognition test, promoting the recovery of cognitive function after I/R injury. EE treatment alleviated myelin damage in MCAO rats, as evidenced by the lower fluorescence intensity ratio of SMI-32/MBP in MCAO+EE group. EE increased the fluorescence intensity ratio of NG2+/Ki67+/Olig2+, MBP, and MOG, enhancing the proliferation and differentiation of OPCs and oligodendrogenesis after MCAO. In terms of remyelination, more myelinated axons and lower G/ratio were detected in MCAO+EE rats compared with MCAO group. Moreover, EE treatment decreased the number of Iba1+/CD86+ M1 microglia, increased the number of Iba1+/CD206+ M2 microglia, and suppressed the inflammation response after I/R injury, which could be attributed to the augmented expression of PI3K/AKT/GSK3ß axis. CONCLUSION: EE improved long­term recovery of cognitive function after cerebral I/R injury, at least in part by promoting M2 microglia transformation through activation of the PI3K/AKT/GSK3ß signaling pathway, inhibiting inflammation to provide a favorable microenvironment for oligodendrocyte maturation and remyelination. The effect of the EE on myelin and inflammation could account for the neuroprotection provided by EE.

Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke.

Neutrophil infiltration has been linked to worse clinical outcomes after ischemic stroke. Microglia, a key type of immune-competent cell, engage in cross-talk with the infiltrating immune cells in the inflamed brain area, yet the molecular mechanisms involved remain largely unexplored. In this study, we investigated the mechanisms of how canonical transient receptor potential 1 (TRPC1) modulated neutrophil infiltration in male mouse cerebral ischemia and reperfusion injury (CIRI) models. Our findings revealed a notable upregulation of TRPC1 in microglia within both middle cerebral artery occlusion reperfusion (MCAO/R) and in vitro oxygen-glucose deprivation/regeneration (OGD/R) model. Conditional Trpc1 knockdown in microglia markedly reduced infarct volumes and alleviated neurological deficits. Microglia conditional Trpc1 knockdown mice displayed less neutrophil infiltration in peri-infarct area. Trpc1 knockdown microglia exhibited a reduced primed proinflammatory phenotype with less secretion of CC-Chemokines ligand (CCL) 5 and CCL2 after MCAO/R. Blocking CCL5/2 significantly mitigated neutrophil infiltration in microglia/neutrophil transwell co-culture system upon OGD/R condition. Trpc1 knockdown markedly reduced store-operated calcium entry and nuclear factor of activated T-cells c1 (NFATc1) level in OGD/R treated microglia. Overexpression of Nfatc1 reversed the CCL5/2 reducing effect of Trpc1 knockdown, which is mediated by small interfering RNA in BV2 cells upon OGD/R. Our data indicate that upregulation of TRPC1 in microglia stimulates the production of CCL5/2 through the Ca2+/NFATc1 pathway. Upregulated CCL5/2 leads to an increase in neutrophil infiltration into the brain, thereby aggravating reperfusion injury. Our results demonstrate the importance of TRPC1 in microglia-mediated neuroinflammation and suggest a potential means for reducing CIRI induced neurological injury.