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

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

Mechanism of ameliorating cerebral ischemia/reperfusion injury by antioxidant inhibition of autophagy based on network pharmacology and experimental verification.

Cerebral ischemia-reperfusion injury (CIRI) is one of the most difficult challenges in cerebrovascular disease research. It is primarily caused by excessive autophagy induced by oxidative stress. Previously, a novel compound X5 was found, and the excellent antioxidant activity of it was verified in this study. Moreover, network pharmacological analysis suggested that compound X5 was closely associated with autophagy and the mTOR pathway. In vitro, X5 could significantly inhibit the expression of autophagy proteins Beclin-1 and LC3-ß, which are induced by H2O2, and promote the expression of SIRT1. In vivo, compound X5 significantly reduced the infarct size and improved the neurological function scores in the middle cerebral artery occlusion (MCAO) model of rats. In conclusion, ROS-induced autophagy is closely related to mTOR, SIRT1 and others, and X5 holds promise as a candidate for the treatment of CIRI.

Caspase-1 deletion reveals pyroptosis participates in neural damage induced by cerebral ischemia/reperfusion in tMCAO model mice.

Pyroptosis, a form of programmed cell death, drives inflammation in the context of cerebral ischemia/reperfusion. The molecular mechanism of pyroptosis underlying ischemia/reperfusion, however, is not fully understood. The transient middle cerebral artery occlusion was applied to wild-type and caspase-1 knockout mice. 2,3,5-Triphenyltetrazolium chloride-staining and immunohistochemistry were used to identify the ischemic region, and western blot and immunofluorescence for the examination of neuronal pyroptosis. The expression of inflammatory factors and the behavioral function assessments were further conducted to examine the effects of caspase-1 knockout on protection against ischemia/reperfusion injury. Ischemia/reperfusion injury increased pyroptosis-related signals represented by the overexpression of pyroptosis-related proteins including caspase-1 and gasdermin D (GSDMD). Meanwhile, the number of GSDMD positive neurons increased in penumbra by immunofluorescence staining. Compared with wild-type mice, those with caspase-1 knockout exhibited decreased levels of pyroptosis-related proteins following ischemia/reperfusion. Furthermore, ischemia/reperfusion attack-induced brain infarction, cerebral edema, inflammatory factors, and neurological outcomes were partially improved in caspase-1 knockout mice. The data indicate that pyroptosis participates in ischemia/reperfusion induced-damage, and the caspase-1 might be involved, it provides some new insights into the molecular mechanism of ischemia.

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

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

Long-Term Accumulation of T Cytotoxic 1, T Cytotoxic 17, and T Cytotoxic 17/1 Cells in the Brain Contributes to Microglia-Mediated Chronic Neuroinflammation After Ischemic Stroke.

Post-stroke neuroinflammation affects the damage and recovery of neurological functions. T cells including CD8+ T cells were present in the ipsilateral hemisphere in the subacute and late phases of ischemic stroke. However, the potential roles of CD8+ T cell subsets in the progression of neuroinflammation have not been characterized. In the current mouse transient middle cerebral artery occlusion model, we investigated the existence of CD8+ T cell subsets in the ipsilateral hemisphere in the subacute and late phases of stroke. We found that ipsilateral CD8+ T cells were present on post-stroke day 3 and increased on post-stroke day 30. The day-3 ipsilateral CD8+ T cells predominantly produced interferon-γ (IFN-γ), while the day-30 ipsilateral CD8+ T cells co-expressed IFN-γ and interleukin-17A (IL-17A). In addition, evaluation of cytokines and transcription factors of the day-30 ipsilateral CD8+ T cells revealed the presence of T cytotoxic 1 (Tc1), T cytotoxic 17 (Tc17), and T cytotoxic 17/1 (Tc17/1) cells. Furthermore, based on the expression of a series of chemokine/cytokine receptors, viable ipsilateral Tc1, Tc17, and Tc17.1 cells were identified and enriched from the day-30 ipsilateral CD8+ T cells, respectively. Co-culture of microglia with ipsilateral Tc1, Tc17, or Tc17.1 cells indicated that the three CD8+ T cell subsets up-regulated the expression of pro-inflammatory mediators by microglia, with Tc17.1 cells being the most potent cell in doing so. Collectively, this study sheds light on the contributions of Tc1, Tc17, and Tc17.1 cells to long-term neuroinflammation after ischemic stroke.

N6022 attenuates cerebral ischemia/reperfusion injury-induced microglia ferroptosis by promoting Nrf2 nuclear translocation and inhibiting the GSNOR/GSTP1 axis.

Stroke poses a significant risk of mortality, particularly among the elderly population. The pathophysiological process of ischemic stroke is complex, and it is crucial to elucidate its molecular mechanisms and explore potential protective drugs. Ferroptosis, a newly recognized form of programmed cell death distinct from necrosis, apoptosis, and autophagy, is closely associated with the pathophysiology of ischemic stroke. N6022, a selective inhibitor of S-nitrosoglutathione reductase (GSNOR), is a "first-in-class" drug for asthma with potential therapeutic applications. However, it remains unclear whether N6022 exerts protective effects in ischemic stroke, and the precise mechanisms of its action are unknown. This study aimed to investigate whether N6022 mitigates cerebral ischemia/reperfusion (I/R) injury by reducing ferroptosis and to elucidate the underlying mechanisms. Accordingly, we established an oxygen-glucose deprivation/reperfusion (OGD/R) cell model and a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to mimic cerebral I/R injury. Our data, both in vitro and in vivo, demonstrated that N6022 effectively protected against I/R-induced brain damage and neurological deficits in mice, as well as OGD/R-induced BV2 cell damage. Mechanistically, N6022 promoted Nrf2 nuclear translocation, enhancing intracellular antioxidant capacity of SLC7A11-GPX4 system. Furthermore, N6022 interfered with the interaction of GSNOR with GSTP1, thereby boosting the antioxidant capacity of GSTP1 and attenuating ferroptosis. These findings provide novel insights, showing that N6022 attenuates microglial ferroptosis induced by cerebral I/R injury through the promotion of Nrf2 nuclear translocation and inhibition of the GSNOR/GSTP1 axis.

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

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

Neuroprotective effects of VCE-004.8 in a rat model of neonatal stroke.

BACKGROUND: Currently there is no effective treatment for neonatal stroke, an acute neurologic syndrome with sequelae, due to focal ischemic, thrombotic, or hemorrhagic event occurring in the perinatal period. VCE-004.8, an aminoquinone exhibiting activity on CB2 and PPARγ receptors, is neuroprotective in adult mice models of acute and chronic brain damaging conditions. We hereby aimed to study VCE-004.8 neuroprotection in a rat model of neonatal stroke. METHODS: 7-day-old (P7) Wistar rats of both sexes were submitted to Middle Cerebral Artery Occlusion (MCAO), receiving i.p. 30 min after vehicle (MCAO + VEH) or VCE-004.8 5 mg/kg (MCAO + VCE). Non-occluded rats served as controls (SHAM). MCAO consequences were assessed at P14 by MRI, histological (TUNEL staining), biochemical (lactate/n-acetyl aspartate ratio by 1H-NMR spectroscopy) and motor studies (grasp test), and at P37 assessing myelination (MBP signal), hemiparesis and hyperlocomotion. Effects of VCE-004.8 on excitotoxicity (glutamate/n-acetyl aspartate, 1H-NMR), oxidative stress (protein nitrosylation, Oxyblot) and neuroinflammation (Toll-like receptor 4 and TNFa expression, Western blot) were assessed at P14. Therapeutic window was assessed by delaying drug administration for 12 or 18 h. RESULTS: Post-MCAO administration of VCE-004.8 reduced the volume of infarct and histological and biochemical brain damage, reducing hyperlocomotion, restoring motor performance and preserving myelination, in a manner linked to the modulation of excitotoxicity, oxidative stress and neuroinflammation. VCE-004.8 was still effective being administered 12-18 h post-insult. CONCLUSIONS: These data suggest that this drug could be effective for the treatment of stroke in newborns.

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

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

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.

Shikonin attenuates cerebral ischemia/reperfusion injury via inhibiting NOD2/RIP2/NF-κB-mediated microglia polarization and neuroinflammation.

OBJECTIVES: Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, which still lacks effective therapeutic agents. Shikonin possesses anti-inflammatory and neuroprotective properties. However, its underlying mechanism remains elusive. This study aimed to investigate whether Shikonin confers protection against cerebral ischemia/reperfusion (I/R) injury by modulating microglial polarization and elucidate the associated mechanisms. METHODS: This study employed an oxygen-glucose deprivation and reoxygenation (OGD/R) BV2 microglial cellular model and a middle cerebral artery occlusion/reperfusion (MCAO/R) animal model to investigate the protection and underlying mechanism of Shikonin against ischemic stroke. RESULTS: The results demonstrated that Shikonin treatment significantly reduced brain infarction volume and improved neurological function in MCAO/R rats. Simultaneously, Shikonin treatment significantly reduced microglial proinflammatory phenotype and levels of proinflammatory markers (inducible-NO synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6), increased microglial anti-inflammatory phenotype and levels of anti-inflammatory markers (Arginase-1 (Arg1), transforming growth factor-beta (TGF-ß), and IL-10), reversed the expression of Nucleotide-binding oligomerization domain 2 (NOD2) and phosphorylation receptor interacting protein 2 (p-RIP2), and suppressed nuclear factor kappa-B (NF-κB) signaling activation in the ischemic penumbra regions. These effects of Shikonin were further corroborated in OGD/R-treated BV2 cells. Furthermore, overexpression of NOD2 markedly attenuated the neuroprotective effects of Shikonin treatment in MCAO/R rats. NOD2 overexpression also attenuated the regulatory effects of Shikonin on neuroinflammation, microglial polarization, and NF-κB signaling activation. CONCLUSION: This study illustrates that Shikonin mitigates inflammation mediated by microglial proinflammatory polarization by inhibiting the NOD2/RIP2/NF-κB signaling pathway, thereby exerting a protective role. The findings uncover a potential molecular mechanism for Shikonin in treating ischemic stroke.

Neuroprotective effects of the salidroside derivative SHPL-49 via the BDNF/TrkB/Gap43 pathway in rats with cerebral ischemia.

Ischemic stroke is a common intravascular disease and one of the leading causes of death and disability. The salidroside derivative SHPL-49, which we previously synthesized, significantly attenuates cerebral ischemic injury in a rat model of permanent middle cerebral artery occlusion. To explore the neuroprotective mechanism of SHPL-49, the effects of SHPL-49 on the expression levels of neurotrophic factors in neurons and microglia and the polarization of microglia were investigated in the present study. SHPL-49 activated the brain-derived neurotrophic factor (BDNF) pathway, decreased the number of degenerated neurons, and accelerated neurogenesis in rats with cerebral ischemia. In addition, SHPL-49 promoted the polarization of microglia toward the M2 phenotype to alleviate neuroinflammation. In BV2 cells, SHPL-49 upregulated CD206 mRNA and protein levels and inhibited CD86 mRNA and protein levels. SHPL-49 also increased neurotrophic factor secretion in BV2 cells, which indirectly promoted the survival of primary neurons after oxygen-glucose deprivation (OGD). Proteomics analysis revealed that SHPL-49 promoted growth-associated protein 43 (Gap43) expression. SHPL-49 enhanced synaptic plasticity and increased Gap43 protein levels via activation of the BDNF pathway in the OGD primary neuron model. These results indicate that SHPL-49 prevents cerebral ischemic injury by activating neurotrophic factor pathways and altering microglial polarization. Thus, SHPL-49 is a potential neuroprotective agent.

Hypochlorous acid derived from microglial myeloperoxidase could mediate high-mobility group box 1 release from neurons to amplify brain damage in cerebral ischemia-reperfusion injury.

Myeloperoxidase (MPO) plays critical role in the pathology of cerebral ischemia-reperfusion (I/R) injury via producing hypochlorous acid (HOCl) and inducing oxidative modification of proteins. High-mobility group box 1 (HMGB1) oxidation, particularly disulfide HMGB1 formation, facilitates the secretion and release of HMGB1 and activates neuroinflammation, aggravating cerebral I/R injury. However, the cellular sources of MPO/HOCl in ischemic brain injury are unclear yet. Whether HOCl could promote HMGB1 secretion and release remains unknown. In the present study, we investigated the roles of microglia-derived MPO/HOCl in mediating HMGB1 translocation and secretion, and aggravating the brain damage and blood-brain barrier (BBB) disruption in cerebral I/R injury. In vitro, under the co-culture conditions with microglia BV cells but not the single culture conditions, oxygen-glucose deprivation/reoxygenation (OGD/R) significantly increased MPO/HOCl expression in PC12 cells. After the cells were exposed to OGD/R, MPO-containing exosomes derived from BV2 cells were released and transferred to PC12 cells, increasing MPO/HOCl in the PC12 cells. The HOCl promoted disulfide HMGB1 translocation and secretion and aggravated OGD/R-induced apoptosis. In vivo, SD rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus different periods of reperfusion. Increased MPO/HOCl production was observed at the reperfusion stage, accomplished with enlarged infarct volume, aggravated BBB disruption and neurological dysfunctions. Treatment of MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH) and HOCl scavenger taurine reversed those changes. HOCl was colocalized with cytoplasm transferred HMGB1, which was blocked by taurine in rat I/R-injured brain. We finally performed a clinical investigation and found that plasma HOCl concentration was positively correlated with infarct volume and neurological deficit scores in ischemic stroke patients. Taken together, we conclude that ischemia/hypoxia could activate microglia to release MPO-containing exosomes that transfer MPO to adjacent cells for HOCl production; Subsequently, the production of HOCl could mediate the translocation and secretion of disulfide HMGB1 that aggravates cerebral I/R injury. Furthermore, plasma HOCl level could be a novel biomarker for indexing brain damage in ischemic stroke patients.

Inhibition of Anaplastic Lymphoma Kinase Protects From Ischemic Stroke.

BACKGROUND: Ischemic stroke is often accompanied by oxidative stress and inflammatory response, both of which work synergistically to exacerbate the disruption of the blood-brain barrier and ischemic brain injury. ALK (anaplastic lymphoma kinase), a cancer-associated receptor tyrosine kinase, was found to play a role in oxidative stress and inflammation. In this study, we investigated the role of ALK inhibition in a murine model of ischemic stroke. METHODS: Focal cerebral ischemia was induced by temporary occlusion of the right middle cerebral artery in mice with a filament. The ALK inhibitor alectinib was administered following the stroke. ALOX15 (arachidonic acid 15-lipoxygenase) was overexpressed by adenovirus injection. The immunohistochemistry, Western blot, oxidative stress, inflammation, blood-brain barrier leakage, infarct volume, and functional outcomes were determined. RESULTS: We found that the expression of ALK was markedly increased in the neurovascular unit after cerebral ischemia. Treatment with the ALK inhibitor alectinib reduced the accumulation of reactive oxygen species, lipid peroxidation, and oxidative DNA, increased the vascular levels of antioxidant enzymes, inactivated the vascular NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome pathway, and reduced vascular inflammation (ICAM-1 [intercellular adhesion molecule-1] and MCP-1 [monocyte chemoattractant protein-1]) after ischemia. Moreover, alectinib reduced the loss of cerebrovascular integrity and blood-brain barrier damage, consequently decreasing brain infarction and neurological deficits. Furthermore, alectinib reduced stroke-evoked ALOX15 expression, whereas virus-mediated overexpression of ALOX15 abolished alectinib-dependent inhibition of oxidative stress and vascular inflammation, blood-brain barrier protection, and neuroprotection, suggesting the protective effects of alectinib for stroke may involve ALOX15. CONCLUSIONS: Our findings demonstrated that alectinib protects from stroke by regulating ischemic signaling cascades and suggest that ALK may be a novel therapeutic target for ischemic stroke.

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.

Chrysin inhibits ferroptosis of cerebral ischemia/reperfusion injury via regulating HIF-1α/CP loop.

Chrysin is a natural flavonoid with powerful neuroprotective capacity. Cerebral ischemia/reperfusion injury (CIRI) is associated with oxidative stress and ferroptosis. Hypoxia-inducible factor 1α (HIF-1α) and ceruloplasmin (CP) are the critical targets for oxidation reactions and iron transport. But the regulatory mechanism between them is still unclear. Transient middle cerebral artery occlusion (tMCAO) model in rats and oxygen and glucose deprivation/re-oxygenation (OGD/R) model in PC12 cells were applied. Pathological tissue staining and biochemical kit were used to evaluate the effect of chrysin. The relationship between HIF-1α and CP was verified by transcriptomics, qRT-PCR and Western blot. In CIRI, HIF-1α/CP loop was discovered to be the regulatory pathway of ferroptosis. CIRI led to activation and nuclear translocation of HIF-1α, which promoted CP transcription and translation, and downstream ferroptosis. Inhibition of HIF-1α had opposite effect on CP and ferroptosis regulation. Overexpression of CP increased the expression of HIF-1α, nevertheless, inhibited the nuclear translocation of HIF-1α and alleviated CIRI. Silencing CP promoted HIF-1α elevation in nucleus and aggravated CIRI. Mechanistically, chrysin restrained HIF-1α nuclear translocation, thereby inhibiting CP transcription and translation, which in turn reduced downstream HIF-1α expression and mitigated ferroptosis in CIRI. Our results highlight chrysin restrains ferroptosis in CIRI through HIF-1α/CP loop.

BRCC3 mediates inflammation and pyroptosis in cerebral ischemia/reperfusion injury by activating the NLRP6 inflammasome.

AIMS: Neuroinflammation and pyroptosis are key mediators of cerebral ischemia/reperfusion (I/R) injury-induced pathogenic cascades. BRCC3, the human homolog of BRCC36, is implicated in neurological disorders and plays a crucial role in neuroinflammation and pyroptosis. However, its effects and potential mechanisms in cerebral I/R injury in mice are unclear. METHODS: Cellular localization of BRCC3 and the interaction between BRCC3 and NLRP6 were assessed. Middle cerebral artery occlusion/reperfusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R) models were established in mice and HT22 cells, respectively, to simulate cerebral I/R injury in vivo and in vitro. RESULTS: BRCC3 protein expression peaked 24 h after MCAO and OGD/R. BRCC3 knockdown reduced the inflammation and pyroptosis caused by cerebral I/R injury and ameliorated neurological deficits in mice after MCAO. The effects of BRCC3 on inflammation and pyroptosis may be mediated by NLRP6 inflammasome activation. Moreover, both BRCC3 and its N- and C-terminals interacted with NLRP6, and both BRCC3 and its terminals reduced NLRP6 ubiquitination. Additionally, BRCC3 affected the interaction between NLRP6 and ASC, which may be related to inflammasome activation. CONCLUSION: BRCC3 shows promise as a novel target to enhance neurological recovery and attenuate the inflammatory responses and pyroptosis caused by NLRP6 activation in cerebral I/R injury.

Microvascular Network Remodeling in the Ischemic Mouse Brain Defined by Light Sheet Microscopy.

BACKGROUND: Until now, the analysis of microvascular networks in the reperfused ischemic brain has been limited due to tissue transparency challenges. METHODS: Using light sheet microscopy, we assessed microvascular network remodeling in the striatum from 3 hours to 56 days post-ischemia in 2 mouse models of transient middle cerebral artery occlusion lasting 20 or 40 minutes, resulting in mild ischemic brain injury or brain infarction, respectively. We also examined the effect of a clinically applicable S1P (sphingosine-1-phosphate) analog, FTY720 (fingolimod), on microvascular network remodeling. RESULTS: Over 56 days, we observed progressive microvascular degeneration in the reperfused striatum, that is, the lesion core, which was followed by robust angiogenesis after mild ischemic injury induced by 20-minute middle cerebral artery occlusion. However, more severe ischemic injury elicited by 40-minute middle cerebral artery occlusion resulted in incomplete microvascular remodeling. In both cases, microvascular networks did not return to their preischemic state but displayed a chronically altered pattern characterized by higher branching point density, shorter branches, higher unconnected branch density, and lower tortuosity, indicating enhanced network connectivity. FTY720 effectively increased microvascular length density, branching point density, and volume density in both models, indicating an angiogenic effect of this drug. CONCLUSIONS: Utilizing light sheet microscopy together with automated image analysis, we characterized microvascular remodeling in the ischemic lesion core in unprecedented detail. This technology will significantly advance our understanding of microvascular restorative processes and pave the way for novel treatment developments in the stroke field.

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

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

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.