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

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

[Effect of Naotaifang on microglial polarization and glial scar following cerebral ischemia reperfusion injury].

This study aims to investigate the effect of Naotaifang(NTF) on the proteins associated with microglial polarization and glial scar in the rat model of cerebral ischemia reperfusion injury(CIRI). The CIRI model was established by middle cerebral artery occlusion/reperfusion. The 48 successfully modeled rats were randomized into model 7 d, model 14 d, NTF 7 d, and NTF 14 d groups(n=12). In addition, 12 SD rats were selected as the sham group. The NTF group was administrated with NTF suspension at 27 g·kg~(-1)·d~(-1) by gavage, and the sham, model 7 d, and model 14 d groups were administrated with the same volume of normal saline every day by gavage for 7 and 14 days, respectively. After the intervention, Longa score was evaluated. The infarct volume was measured by 2,3,5-triphenyl-2H-tetrazolium chloride(TTC) staining. Morris water maze and open field tests were carried out to evaluate the spatial learning, memory, cognitive function, and anxiety degree of rats. Hematoxylin-eosin(HE) staining was employed to observe the morphological structure and damage of the brain tissue. The immunofluorescence assay was employed to measure the expression of glial fibrillary acidic protein(GFAP) and glial scar. Western blot was employed to determine the protein levels of GFAP, neurocan, phosphacan, CD206, arginase-1(Arg-1), interleukin(IL)-1ß, IL-6, and IL-4. Compared with the sham, model 7 d and model 14 d groups showed cerebral infarction of different degrees, severe pathological injury of cerebral cortex and hippocampus, neurological impairment, reduced spatial learning and memory, cognitive dysfunction, severe anxiety, astrocyte hyperplasia, thickening penumbra glial scar, and up-regulated protein levels of IL-1ß, IL-6, GFAP, neurocan, phosphacan, CD206, and Arg-1(P<0.01). Compared with the model group, NTF 7 d and NTF 14 d groups improved spatial learning, memory, and cognitive function, reduced anxiety, improved nerve function, reduced cerebral infarction volume, reduced astrocyte hyperplasia, thinned penumbra glial scar, down-regulated the protein levels of GFAP, neurocan, phosphacan, IL-6, and IL-1ß, and up-regulated the protein levels of IL-4, CD206, and Arg-1(P<0.05 or P<0.01). NTF exerts a neuroprotective effect on CIRI by inducing the M2 polarization of microglia, inhibiting inflammatory response, and reducing the formation of glial scar.

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

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

Impact of inflammatory preconditioning on murine microglial proteome response induced by focal ischemic brain injury.

Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).

[PI3K/Akt/Erk signaling pathway mediates neuroprotection of CaMK⠡γ and CaMK⠡δ against ischemic reperfusion injury in mice].

OBJECTIVE: To observe neuroprotective effects of Ca2+/calmodulin-dependent kinase Ⅱ (CaMK Ⅱ)γ and CaMkII δ against acute neuronal ischemic reperfusion injury in mice and explore the underlying mechanism. METHODS: Primary cultures of brain neurons isolated from fetal mice (gestational age of 18 days) were transfected with two specific siRNAs (si-CAMK2G and si-CAMK2D) or a control sequence (si-NT). After the transfection, the cells were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) conditions for 1 h followed by routine culture. The expressions of phosphatidylinositol-3-kinase/extracellular signal-regulated kinase (PI3K/Akt/Erk) signaling pathway components in the neurons were detected using immunoblotting. The expressions of the PI3K/Akt/Erk signaling pathway proteins were also detected in the brain tissues of mice receiving middle cerebral artery occlusion (MCAO) or sham operation. RESULTS: The neuronal cells transfected with siCAMK2G showed significantly lower survival rates than those with si-NT transfection at 12, 24, 48, and 72 h after OGD/R (P < 0.01), and si-CAMK2G transfection inhibited OGD/R-induced upregulation of CaMKⅡγ expression. Compared to si-NT, transfection with si-CAMK2G and si-CAMK2D both significantly inhibited the expressions of PI3K/Akt/Erk signaling pathway components (P < 0.01). In the mouse models of MCAO, the expressions of CaMKⅡδ and CaMKⅡγ were significantly increased in the brain, where activation of the PI3K/Akt/Erk signaling pathway was detected. The expression levels of CaMKⅡδ, CaMKⅡγ, Erk, phosphorylated Erk, Akt, and phosphorylated Akt were all significantly higher in MCAO mice than in the sham-operated mice at 24, 48, 72, and 96 h after reperfusion (P < 0.05). CONCLUSION: The neuroprotective effects of CaMKⅡδ and CaMKⅡγ against acute neuronal ischemic reperfusion injury are mediated probably by the PI3K/Akt/Erk pathway.

Poly(ADP-ribose) mediates bioenergetic defects and redox imbalance in neurons following oxygen and glucose deprivation.

PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP-1 and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drive bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.

Progress of researches on mechanisms of acupuncture underlying improvement of cerebral ischemia-reperfusion injury by regulating calcium overload. / é’ˆåˆºè°ƒæŽ§è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤åŽé’™è¶ è½½æœºåˆ¶ç ”ç©¶è¿›å±•.

Ischemic stroke is currently the most common type of stroke, and the key pathological link is cerebral ischemia-reperfusion injury (CIRI), while the key factor leading to apoptosis and necrosis of ischemic nerve cells is calcium overload. Current studies have confirmed that acupuncture therapy has a good modulating effect on calcium homeostasis and can reduce cerebral ischemia-reperfusion induced damage of neuronal cells by inhibiting calcium overload. After reviewing the relevant literature published in the past 15 years, we find that acupuncture plays a role in regulating the pathological mechanism of calcium overload after CIRI by inhibiting the opening of connexin 43 hemichannels, regulating the intracellular free calcium ion concentration, suppressing the expression of calmodulin, and blocking the function of L-type voltage-gated calcium channels, thereby inhibiting calcium overload, regulating calcium homeostasis and antagonizing neuronal damage resulted from cerebral ischemia-reperfusion, which may provide ideas for future research.

Probucol protects against brain damage caused by intra-neural pyroptosis in rats with vascular dementia through inhibition of the Syk/Ros pathway.

BACKGROUND: Neuronal injury in chronic cerebral hypoperfusion (CCH) is the main pathogenic factor of vascular dementia (VD). Clinically, there isn't a drug specifically for VD; instead, the majority of medications used to treat Alzheimer's disease (AD) are also used to treat VD. Based on the proven anti-inflammatory and antioxidant effects of Probucol, we hypothesized that it may have therapeutic effects on VD, but more research is required to determine its exact mechanism of action. METHODS: In vivo experiment: We used SD rats and most commonly used bilateral carotid artery occlusion (2-VO) in VD for modeling. After successful modeling, SD rats were given Probucol 3.5 mg/kg/day for 8 weeks to evaluate the therapeutic effect. In vitro experiment: BV-2 microglia of rats were cultured and divided into Control group and Probucol group. Each group was treated with hypoxia-hypoglycemia, hypoxia-hypoglycemia hydrogen peroxide and hypoxia-hypoglycemia hydrogen peroxide Syk inhibitor respectively. RESULTS: The results of immunofluorescence and Western blot showed that Probucol could significantly improve the cognitive impairment induced by CCH, and the neuronal damage was also attenuated. On the one hand, the underlying mechanism of Probucol was to reduce oxidative stress and cell apoptosis of hippocampal neurons by inhibiting the expression of phosphorylated spleen tyrosine kinase (P-Syk); On the other hand, it exerted a protective effect by reducing NLRP3-dependent cell pyroptosis and inhibiting neuroinflammation induced by microglia activation. CONCLUSION: Probucol could reduce oxidative stress and cell apoptosis by inhibiting the Syk/ROS signaling pathway, thereby improving CCH-induced cognitive impairment in vitro and in vivo.

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.

Trans- and Cis-Phosphorylated Tau Protein: New Pieces of the Puzzle in the Development of Neurofibrillary Tangles in Post-Ischemic Brain Neurodegeneration of the Alzheimer's Disease-like Type.

Recent evidence indicates that experimental brain ischemia leads to dementia with an Alzheimer's disease-like type phenotype and genotype. Based on the above evidence, it was hypothesized that brain ischemia may contribute to the development of Alzheimer's disease. Brain ischemia and Alzheimer's disease are two diseases characterized by similar changes in the hippocampus that are closely related to memory impairment. Following brain ischemia in animals and humans, the presence of amyloid plaques in the extracellular space and intracellular neurofibrillary tangles was revealed. The phenomenon of tau protein hyperphosphorylation is a similar pathological feature of both post-ischemic brain injury and Alzheimer's disease. In Alzheimer's disease, the phosphorylated Thr231 motif in tau protein has two distinct trans and cis conformations and is the primary site of tau protein phosphorylation in the pre-entanglement cascade and acts as an early precursor of tau protein neuropathology in the form of neurofibrillary tangles. Based on the latest publication, we present a similar mechanism of the formation of neurofibrillary tangles after brain ischemia as in Alzheimer's disease, established on trans- and cis-phosphorylation of tau protein, which ultimately influences the development of tauopathy.

Neuroprotective Effects of Aucubin against Cerebral Ischemia and Ischemia Injury through the Inhibition of the TLR4/NF-κB Inflammatory Signaling Pathway in Gerbils.

Aucubin, an iridoid glycoside, possesses beneficial bioactivities in many diseases, but little is known about its neuroprotective effects and mechanisms in brain ischemia and reperfusion (IR) injury. This study evaluated whether aucubin exhibited neuroprotective effects against IR injury in the hippocampal CA1 region through anti-inflammatory activity in gerbils. Aucubin (10 mg/kg) was administered intraperitoneally once a day for one week prior to IR. Neuroprotective effects of aucubin were assessed by neuronal nuclei (NeuN) immunofluorescence and Floro-Jade C (FJC) histofluorescence. Microgliosis and astrogliosis were evaluated using immunohistochemistry with anti-ionized calcium binding adapter protein 1 (Iba1) and glial fibrillary acidic protein (GFAP). Protein levels of proinflammatory cytokines interleukin1 beta (IL1ß) and tumor necrosis factor alpha (TNFα) were assayed using enzyme-linked immunosorbent assay and Western blot. Changes in toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway were assessed by measuring levels of TLR4, inhibitor of NF-κB alpha (IκBα), and NF-κB p65 using Western blot. Aucubin treatment protected pyramidal neurons from IR injury. IR-induced microgliosis and astrogliosis were suppressed by aucubin treatment. IR-induced increases in IL1ß and TNFα levels were significantly alleviated by the treatment. IR-induced upregulation of TLR4 and downregulation of IκBα were significantly prevented by aucubin treatment, and IR-induced nuclear translocation of NF-κB was reversed by aucubin treatment. Briefly, aucubin exhibited neuroprotective effects against brain IR injury, which might be related to the attenuation of neuroinflammation through inhibiting the TLR-4/NF-κB signaling pathway. These results suggest that aucubin pretreatment may be a potential approach for the protection of brain IR injury.

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.

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 acupuncture in attenuating cerebral ischaemia-reperfusion injury based on nuclear receptor coactivator 4 mediated ferritinophagy.

OBJECTIVE: To explore the effect of acupuncture treatment on cerebral ischaemia-reperfusion injury (CIRI) and reveal the underlying mechanism of the effect based on nuclear receptor coactivator 4 (NCOA4) mediated ferritinophagy. METHODS: Sprague-Dawley male rats were divided into four groups: the sham group, model group, acupuncture group, and sham acupuncture group. After 2 h of middle cerebral artery occlusion (MCAO), reperfusion was performed for 24 h to induce CIRI. The rats were treated with acupuncture at the Neiguan (PC6) and Shuigou (GV26) acupoints. Their neurological function was evaluated by taking their Bederson scores at 2 h after ischaemia and 24 h after reperfusion. Triphenyltetrazolium chloride staining was applied to assess the cerebral infarct volume at 24 h after reperfusion. The malondialdehyde (MDA) and ferrous iron (Fe2+) levels were observed after 24 h of reperfusion using an assay kit. Western blotting was performed to detect the expression of NCOA4 and ferritin heavy chain 1 (FTH1) at 24 h after reperfusion. Moreover, the colocalization of ferritin with neurons, NCOA4 with microtubule-associated protein 1 light chain 3 (LC3), and NCOA4 with ferritin was visualized using immunofluorescence staining. RESULTS: Acupuncture significantly improved neurological function and decreased cerebral infarct volume in the acupuncture group. Following CIRI, the expression of NCOA4, LC3 and FTH1 was increased, which enhanced ferritinophagy and induced an inappropriate accumulation of Fe2+ and MDA in the ischaemic brain. However, acupuncture dramatically downregulated the expression of NCOA4, LC3 and FTH1, inhibited the overactivation of ferritinophagy, and decreased the levels of MDA and Fe2+. CONCLUSIONS: Acupuncture can inhibit NCOA4-mediated ferritinophagy and protect neurons against CIRI in a rat model.

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.

Fat mass and obesity associated protein inhibits neuronal ferroptosis via the FYN/Drp1 axis and alleviate cerebral ischemia/reperfusion injury.

OBJECTIVES: FTO is known to be involved in cerebral ischemia/reperfusion (I/R) injury. However, its related specific mechanisms during this condition warrant further investigations. This study aimed at exploring the impacts of FTO and the FYN/DRP1 axis on mitochondrial fission, oxidative stress (OS), and ferroptosis in cerebral I/R injury and the underlying mechanisms. METHODS: The cerebral I/R models were established in mice via the temporary middle cerebral artery occlusion/reperfusion (tMCAO/R) and hypoxia/reoxygenation models were induced in mouse hippocampal neurons via oxygen-glucose deprivation/reoxygenation (OGD/R). After the gain- and loss-of-function assays, related gene expression was detected, along with the examination of mitochondrial fission, OS- and ferroptosis-related marker levels, neuronal degeneration and cerebral infarction, and cell viability and apoptosis. The binding of FTO to FYN, m6A modification levels of FYN, and the interaction between FYN and Drp1 were evaluated. RESULTS: FTO was downregulated and FYN was upregulated in tMCAO/R mouse models and OGD/R cell models. FTO overexpression inhibited mitochondrial fission, OS, and ferroptosis to suppress cerebral I/R injury in mice, which was reversed by further overexpressing FYN. FTO overexpression also suppressed mitochondrial fission and ferroptosis to increase cell survival and inhibit cell apoptosis in OGD/R cell models, which was aggravated by additionally inhibiting DRP1. FTO overexpression inhibited FYN expression via the m6A modification to inactive Drp1 signaling, thus reducing mitochondrial fission and ferroptosis and enhancing cell viability in cells. CONCLUSIONS: FTO overexpression suppressed FYN expression through m6A modification, thereby subduing Drp1 activity and relieving cerebral I/R injury.

Pretreatment with a dual antiplatelet and anticoagulant (APAC) reduces ischemia-reperfusion injury in a mouse model of temporary middle cerebral artery occlusion-implications for neurovascular procedures.

BACKGROUND: Several neurovascular procedures require temporary occlusion of cerebral arteries, leading to ischemia of unpredictable length, occasionally causing brain infarction. Experimental models of cerebral ischemia-reperfusion injury have established that platelet adhesion and coagulation play detrimental roles in reperfusion injury following transient cerebral ischemia. Therefore, in a model of cerebral ischemia-reperfusion injury (IRI), we investigated the therapeutic potential of a dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic which is able to bind to vascular injury sites. METHODS: Brain ischemia was induced in mice by transient occlusion of the right middle cerebral artery for 60 min. APAC, unfractionated heparin (UFH) (both at heparin equivalent doses of 0.5 mg/kg), or vehicle was intravenously administered 10 min before or 60 min after the start of ischemia. At 24 h later, mice were scored for their neurological and motor behavior, and brain damage was quantified. RESULTS: Both APAC and UFH administered before the onset of ischemia reduced brain injury. APAC and UFH pretreated mice had better neurological and motor functions (p < 0.05 and p < 0.01, respectively) and had significantly reduced cerebral infarct sizes (p < 0.01 and p < 0.001, respectively) at 24 h after transient occlusion compared with vehicle-treated mice. Importantly, no macroscopic bleeding complications were observed in either APAC- or UFH-treated animals. However, when APAC or UFH was administered 60 min after the start of ischemia, the therapeutic effect was lost, but without hemorrhaging either. CONCLUSIONS: Pretreatment with APAC or UFH was safe and effective in reducing brain injury in a model of cerebral ischemia induced by transient middle cerebral artery occlusion. Further studies on the use of APAC to limit ischemic injury during temporary occlusion in neurovascular procedures are indicated.

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.

Intracerebroventricular BDNF infusion may reduce cerebral ischemia/reperfusion injury by promoting autophagy and suppressing apoptosis.

Here, it was aimed to investigate the effects of intracerebroventricular (ICV) Brain Derived Neurotrophic Factor (BDNF) infusion for 7 days following cerebral ischemia (CI) on autophagy in neurons in the penumbra. Focal CI was created by the occlusion of the right middle cerebral artery. A total of 60 rats were used and divided into 4 groups as Control, Sham CI, CI and CI + BDNF. During the 7-day reperfusion period, aCSF (vehicle) was infused to Sham CI and CI groups, and BDNF infusion was administered to the CI + BDNF group via an osmotic minipump. By the end of the 7th day of reperfusion, Beclin-1, LC3, p62 and cleaved caspase-3 protein levels in the penumbra area were evaluated using Western blot and immunofluorescence. BDNF treatment for 7 days reduced the infarct area after CI, induced the autophagic proteins Beclin-1, LC3 and p62 and suppressed the apoptotic protein cleaved caspase-3. Furthermore, rotarod and adhesive removal test times of BDNF treatment started to improve from the 4th day, and the neurological deficit score from the 5th day. ICV BDNF treatment following CI reduced the infarct area by inducing autophagic proteins Beclin-1, LC3 and p62 and inhibiting the apoptotic caspase-3 protein while its beneficial effects were apparent in neurological tests from the 4th day.