Vagus nerve stimulation as a promising neuroprotection for ischemic stroke via α7nAchR-dependent inactivation of microglial NLRP3 inflammasome.

Ischemic stroke is a major cause of disability and death worldwide, and its management requires urgent attention. Previous studies have shown that vagus nerve stimulation (VNS) exerts neuroprotection in ischemic stroke by inhibiting neuroinflammation and apoptosis. In this study, we evaluated the timing for VNS intervention in ischemic stroke, and the underlying mechanisms  of VNS-induced neuroprotection. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min. The left vagus nerve at cervical level was exposed and attached to an electrode connected to a low-frequency electrical stimulator. Vagus nerve stimulation (VNS) was given for 60 min before, during and after tMCAO (Pre-VNS, Dur-VNS, Post-VNS). Neurological function was assessed 24 h after reperfusion. We found that all the three VNS significantly protected against the tMCAO-induced injury evidenced by improved neurological function and reduced infarct volume. Moreover, the Pre-VNS was the most effective against the ischemic injury. We found that tMCAO activated microglia in the ischemic core and penumbra regions of the brain, followed by the NLRP3 inflammasome activation-induced neuroinflammation, which finally triggered neuronal death. VNS treatment preserved α7nAChR expression in the penumbra regions, inhibited NLRP3 inflammasome activation and ensuing neuroinflammation, rescuing cerebral neurons. The role of α7nAChR in microglial NLRP3 inflammasome activation in ischemic stroke was further validated using genetic manipulations, including Chrna7 knockout mice and microglial Chrna7 overexpression mice, as well as pharmacological interventions using the α7nAChR inhibitor methyllycaconitine and agonist PNU-282987. Collectively, this study demonstrates the potential of VNS as a safe and effective strategy to treat ischemic stroke, and presents a new approach targeting microglial NLRP3 inflammasome, which might be therapeutic for other inflammation-related diseases.

Predictors of post-stroke depression: the perspective from the social convoy model.

BACKGROUND: Post-stroke depression (PSD) as one of the most common neuropsychiatric disorders after a stroke and is caused by many factors. However, the relationships among different factors and their potential contributions to PSD remain unclear. METHODS: Two hundred and seventy-six patients were recruited into this study. The general information questionnaire, the Patient Health Questionnaire-9, the Perceived Social Support Scale, the Family Assessment Device, the General Well-Being Scale, the Barthel Index, and the modified Rankin Scale were used to assess the condition of patients. Subsequently, we identify the main causes associated with the PSD and then performed a path analysis to clarify the direct, indirect and total effects among the variables. RESULTS: We found that age, stroke with coronary heart disease, neurological function, family function, social support, and general well-being had a significant impact on PSD (P < 0.05). Of these, neurological function had the largest total effect on PSD (ß = 0.451), social support contributed the most as a direct effect (ß = -0.306), and family function showed the largest indirect effect (ß = -0.264). CONCLUSION: Individual, disease, and social-psychological factors all contributed to the development of PSD. We should pay more attention to comprehensive assessment, especially for those with poor neurological function, and lacking family or social support. In addition, it would be preferable to provide them with necessary support and care strategies to reduce the incidence of PSD.

Iptakalim improves cerebral microcirculation in mice after ischemic stroke by inhibiting pericyte contraction.

Pericytes are present tight around the intervals of capillaries, play an essential role in stabilizing the blood-brain barrier, regulating blood flow and immunomodulation, and persistent contraction of pericytes eventually leads to impaired blood flow and poor clinical outcomes in ischemic stroke. We previously show that iptakalim, an ATP-sensitive potassium (K-ATP) channel opener, exerts protective effects in neurons, and glia against ischemia-induced injury. In this study we investigated the impacts of iptakalim on pericytes contraction in stroke. Mice were subjected to cerebral artery occlusion (MCAO), then administered iptakalim (10 mg/kg, ip). We showed that iptakalim administration significantly promoted recovery of cerebral blood flow after cerebral ischemia and reperfusion. Furthermore, we found that iptakalim significantly inhibited pericytes contraction, decreased the number of obstructed capillaries, and improved cerebral microcirculation. Using a collagen gel contraction assay, we demonstrated that cultured pericytes subjected to oxygen-glucose deprivation (OGD) consistently contracted from 3 h till 24 h during reoxygenation, whereas iptakalim treatment (10 µM) notably restrained pericyte contraction from 6 h during reoxygenation. We further showed that iptakalim treatment promoted K-ATP channel opening via suppressing SUR2/EPAC1 complex formation. Consequently, it reduced calcium influx and ET-1 release. Taken together, our results demonstrate that iptakalim, targeted K-ATP channels, can improve microvascular disturbance by inhibiting pericyte contraction after ischemic stroke. Our work reveals that iptakalim might be developed as a promising pericyte regulator for treatment of stroke.

Oridonin prevents oxidative stress-induced endothelial injury via promoting Nrf-2 pathway in ischaemic stroke.

Oridonin, a natural diterpenoid compound extracted from a Chinese herb, has been proved to exert anti-oxidative stress effects in various disease models. The aim of the present study was to investigate the protective effects of oridonin on oxidative stress-induced endothelial injury in ischaemic stroke. We found oridonin repaired blood-brain barrier (BBB) integrity presented with upregulation of tight junction proteins (TJ proteins) expression, inhibited the infiltration of periphery inflammatory cells and neuroinflammation and thereby reduced infarct volume in ischaemic stroke mice. Furthermore, our results showed that oridonin could protect against oxidative stress-induced endothelial injury via promoting nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf-2). The specific mechanism could be the activation of AKT(Ser473)/GSK3ß(Ser9)/Fyn signalling pathway. Our findings revealed the therapeutic effect and mechanism of oridonin in ischaemic stroke, which provided fundamental evidence for developing the extracted compound of Chinese herbal medicine into an innovative drug for ischaemic stroke treatment.

S1PR3 is essential for phosphorylated fingolimod to protect astrocytes against oxygen-glucose deprivation-induced neuroinflammation via inhibiting TLR2/4-NFκB signalling.

Fingolimod (FTY720) is used as an immunosuppressant for multiple sclerosis. Numerous studies indicated its neuroprotective effects in stroke. However, the mechanism remains to be elucidated. This study was intended to investigate the mechanisms of phosphorylated FTY720 (pFTY720), which was the principle active molecule in regulating astrocyte-mediated inflammatory responses induced by oxygen-glucose deprivation (OGD). Results demonstrated that pFTY720 could protect astrocytes against OGD-induced injury and inflammatory responses. It significantly decreased pro-inflammatory cytokines, including high mobility group box 1 (HMGB1) and tumour necrosis factor-α (TNF-α). Further, studies displayed that pFTY720 could prevent up-regulation of Toll-like receptor 2 (TLR2), phosphorylation of phosphoinositide 3-kinase (PI3K) and nuclear translocation of nuclear factor kappa B (NFκB) p65 subunit caused by OGD. Sphingosine-1-phosphate receptor 3 (S1PR3) knockdown could reverse the above change. Moreover, administration of TLR2/4 blocker abolished the protective effects of pFTY720. Taken together, this study reveals that pFTY720 depends on S1PR3 to protect astrocytes against OGD-induced neuroinflammation, due to inhibiting TLR2/4-PI3K-NFκB signalling pathway.

Potential role of microRNA-7 in the anti-neuroinflammation effects of nicorandil in astrocytes induced by oxygen-glucose deprivation.

BACKGROUND: It is generally recognized that the inflammatory reaction in glia is one of the important pathological factors in brain ischemic injury. Our previous study has revealed that opening ATP-sensitive potassium (K-ATP) channels could attenuate glial inflammation induced by ischemic stroke. However, the detailed mechanisms are not well known. METHODS: Primary cultured astrocytes separated from C57BL/6 mice were subjected to oxygen-glucose deprivation (OGD); cellular injuries were determined via observing the changes of cellular morphology and cell viability. MicroRNA (miR) and messenger RNA (mRNA) level was validated by real-time PCR. The interaction between microRNA and the target was confirmed via dual luciferase reporter gene assay. Expressions of proteins and inflammatory cytokines were respectively assessed by western blotting and enzyme-linked immunosorbent assay. RESULTS: OGD resulted in astrocytic damage, which was prevented by K-ATP channel opener nicorandil. Notably, we found that OGD significantly downregulated miR-7 and upregulated Herpud2. Our further study proved that miR-7 targeted Herpud2 3'UTR, which encoded endoplasmic reticulum (ER) stress protein-HERP2. Correspondingly, our results showed that OGD increased the levels of ER stress proteins along with significant elevations of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). Pretreatment with nicorandil could remarkably upregulate miR-7, depress the ER-related protein expressions including glucose-regulated protein 78 (GRP78), C/EBP-homologous protein (CHOP), and Caspase-12, and thereby attenuate inflammatory responses and astrocytic damages. CONCLUSIONS: These findings demonstrate that opening K-ATP channels protects astrocytes against OGD-mediated neuroinflammation. Potentially, miR-7-targeted ER stress acts as a key molecular brake on neuroinflammation.

Gut microbiota is necessary for pair-housing to protect against post-stroke depression in mice.

The goal of this study is to investigate the role of microbiota-gut-brain axis involved in the protective effect of pair-housing on post-stroke depression (PSD). PSD model was induced by occluding the middle cerebral artery (MCAO) plus restraint stress for four weeks. At three days after MCAO, the mice were restrained 2 h per day. For pair-housing (PH), each mouse was pair housed with a healthy isosexual cohabitor for four weeks. While in the other PH group, their drinking water was replaced with antibiotic water. On day 35 to day 40, anxiety- and depression-like behaviors (sucrose consumption, open field test, forced swim test, and tail-suspension test) were conducted. Results showed pair-housed mice had better performance on anxiety- and depression-like behaviors than the PSD mice, and the richness and diversity of intestinal flora were also improved. However, drinking antibiotic water reversed the effects of pair-housing. Furthermore, pair-housing had an obvious improvement in gut barrier disorder and inflammation caused by PSD. Particularly, they showed significant decreases in CD8 lymphocytes and mRNA levels of pro-inflammatory cytokines (TNF-a, IL-1ß and IL-6), while IL-10 mRNA was upregulated. In addition, pair-housing significantly reduced activated microglia and increased Nissl's body in the hippocampus of PSD mice. However, all these improvements were worse in the pair-housed mice administrated with antibiotic water. We conclude that pair-housing significantly improves PSD in association with enhanced functions of microbiota-gut-brain axis, and homeostasis of gut microbiota is indispensable for the protective effect of pair-housing on PSD.

PD-1/PD-L1 axis is involved in the interaction between microglial polarization and glioma.

The tumor microenvironment plays a vital role in glioblastoma growth and invasion. PD-1 and PD-L1 modulate the immunity in the brain tumor microenvironment. However, the underlying mechanisms remain unclear. In the present study, in vivo and in vitro experiments were conducted to reveal the effects of PD-1/PD-L1 on the crosstalk between microglia and glioma. Results showed that glioma cells secreted PD-L1 to the peritumoral areas, particularly microglia containing highly expressed PD-1. In the early stages of glioma, microglia mainly polarized into the pro-inflammatory subtype (M1). Subsequently, the secreted PD-L1 accumulated and bound to PD-1 on microglia, facilitating their polarization toward the microglial anti-inflammatory (M2) subtype primarily via the STAT3 signaling pathway. The role of PD-1/PD-L1 in M2 polarization of microglia was partially due to PD-1/PD-L1 depletion or application of BMS-1166, a novel inhibitor of PD-1/PD-L1. Consistently, co-culturing with microglia promoted glioma cell growth and invasion, and blocking PD-1/PD-L1 significantly suppressed these processes. Our findings reveal that the PD-1/PD-L1 axis engages in the microglial M2 polarization in the glioma microenvironment and promotes tumor growth and invasion.

The oxytocin receptor is essential for the protective effect of pair housing on post-stroke depression in mice.

The beneficial effect of social interaction in mitigating the incidence of post-stroke depression (PSD) and ameliorating depressive symptoms has been consistently demonstrated through preclinical and clinical studies. However, the underlying relationship with oxytocin requires further investigation. In light of this, the present study aimed to explore the protective effect of pair housing on the development of PSD and the potential relationship with oxytocin receptors. The PSD model was induced by middle cerebral artery occlusion (MCAO) for 50 min, followed by 4-week isolated housing and restrained stress. Subsequently, each mouse in the pair-housing group (PH) was pair-housed with an isosexual healthy partner. Another group was continuously administrated fluoxetine (10 mg/Kg, i.p, once a day) for 3 weeks. To elucidate the potential role of oxytocin, we subjected pair-housed PSD mice to treatment with an oxytocin receptor (OXTR) antagonist (L368,889) (5 mg/Kg, i.p, once a day) for 3 weeks. At 31 to 32 days after MCAO, anxiety- and depressive-like behaviors were assessed using sucrose consumption, forced swim test, and tail-suspension test. The results showed that pair housing significantly improved post-stroke depression to an extent comparable to that of fluoxetine treatment. Furthermore, pair housing significantly decreased corticosterone in serum, increasing OXT mRNA expression in the hypothalamus. Treatment with L368,889 essentially reversed the effect of pair housing, with no discernible sex differences apart from changes in body weight. Pair housing increased hippocampal serotonin (5-HT), but treatment with L368,889 had no significant impact. Additionally, pair housing effectively reduced the number of reactive astrocytes and increased Nissl's body in the cortex and hippocampal CA3 regions. Correspondingly, treatment with L368,889 significantly reversed the changes in the Nissl's body and reactive astrocytes. Moreover, pair housing downregulated mRNA levels of TNF-α, IL-1ß, and IL-6 in the cortex caused by PSD, which was also reversed by treatment with L368,889. In conclusion, pair housing protects against the development of PSD depending on OXT and OXTR in the brain, with no significant divergence based on sex. These findings provide valuable insights into the potential of social interaction and oxytocin as therapeutic targets for PSD. Further research into the underlying mechanisms of these effects may contribute to the development of novel treatments for PSD.

Disorders in the gut and liver are involved in depression contagion between isosexual post-stroke depression mice and the healthy cohabitors.

Despite the accumulated evidence that pair housing could attenuate post-stroke depression (PSD), but less attention has been paid to the healthy cohabitors, and the underlying mechanisms remain unclear. This study aimed to determine whether there is depressive contagion between PSD mice and their healthy cohabitors. PSD was induced by middle cerebral artery occlusion (MCAO) plus restraint stress for four weeks. Three days after MCAO, the mice were restrained two hours per day and isosexually pair-housed for four weeks. The results showed that, compared with the partners pair housed with normal control mice (Ctrl group), the partners pair housed with PSD mice (CH group) displayed depressive-like behaviors, including decreased sucrose preference rate, significantly shorter duration in the center arena and reduced total distance in the open-field test, and extended immobile time in forced swimming test and tail-suspension test without sex differences. Regarding the change in the body weight, only the males showed a significant reduction on days 17 and 24 after treatment. Furthermore, the CH group showed significantly increased corticosterone and decreased oxytocin (OXT) levels in serum, while the mRNA levels of OXT, vasopressin and oxytocin receptor were remarkably upregulated in the hypothalamus of the CH group. However, there was no significant change in the vasopressin receptor V1a. Interestingly, compared with the Ctrl group, there was a significant decrease in butyrate in serum of the CH group. Consistently, they had mild liver dysfunction with increased alanine transaminase, extended hepatic sinus surrounded by enhanced SLC22A9, and significantly increased Iba1-positive macrophages. Moreover, the expression of tight junction protein (Occludin and ZO-1) obviously decreased in the colon with increasing Iba1-positive cells. These results suggest that isosexual pair-housing with PSD mice causes the healthy partners to develop depressive-like behaviors with disturbances in the gut and liver.

Lipidomic analysis identifies long-chain acylcarnitine as a target for ischemic stroke.

INTRODUCTION: Lipid metabolism dysfunction is widely involved in the pathological process of acute ischemic stroke (AIS). The coordination of lipid metabolism between neurons and astrocytes is of great significance. However, the full scope of lipid dynamic changes and the function of key lipids during AIS remain unknown. Hence, identifying lipid alterations and characterizing their key roles in AIS is of great importance. METHODS: Untargeted and targeted lipidomic analyses were applied to profile lipid changes in the ischemic penumbra and peripheral blood of transient middle cerebral artery occlusion (tMCAO) mice as well as the peripheral blood of AIS patients. Infarct volume and neurological deficits were assessed after tMCAO. The cell viability and dendritic complexity of primary neurons were evaluated by CCK8 assay and Sholl analysis. Seahorse, MitoTracker Green, tetramethyl rhodamine methyl ester (TMRM), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSOX were used as markers of mitochondrial health. Fluorescent and isotopic free fatty acid (FFA) pulse-chase assays were used to track FFA flux in astrocytes. RESULTS: Long-chain acylcarnitines (LCACs) were the lipids with the most dramatic changes in the ischemic penumbra and peripheral blood of tMCAO mice. LCACs were significantly elevated on admission in AIS patients and associated with poor outcomes in AIS patients. Increasing LCACs through a bolus administration of palmitoylcarnitine amplified stroke injury, while decreasing LCACs by overexpressing carnitine palmitoyltransferase 2 (CPT2) ameliorated stroke injury. Palmitoylcarnitine aggravated astrocytic mitochondrial damage after OGD/R, while CPT2 overexpression in astrocytes ameliorated cocultured neuron viability. Further study revealed that astrocytes stimulated by OGD/R liberated FFAs from lipid droplets into mitochondria to form LCACs, resulting in mitochondrial damage and lowered astrocytic metabolic support and thereby aggravated neuronal damage. CONCLUSION: LCACs could accumulate and damage neurons by inducing astrocytic mitochondrial dysfunction in AIS. LCACs play a crucial role in the pathology of AIS and are novel promising diagnostic and prognostic biomarkers for AIS.

Resolvin D1 reprograms energy metabolism to promote microglia to phagocytize neutrophils after ischemic stroke.

Neutrophil aggregation and clearance are important factors affecting neuroinflammatory injury during acute ischemic stroke. Emerging evidence suggests that energy metabolism is essential for microglial functions, especially microglial phagocytosis, which determines the degree of brain injury. Here, we demonstrate that Resolvin D1 (RvD1), a lipid mediator derived from docosahexaenic acid (DHA), promotes the phagocytosis of neutrophils by microglia, thereby reducing neutrophil accumulation in the brain and alleviating neuroinflammation in the ischemic brain. Further studies reveal that RvD1 reprograms energy metabolism from glycolysis to oxidative phosphorylation (OXPHOS), providing sufficient energy for microglial phagocytosis. Moreover, RvD1 enhances microglial glutamine uptake and stimulates glutaminolysis to support OXPHOS to boost ATP production depending on adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation. Overall, our results reveal that RvD1 reprograms energy metabolism to promote the microglial phagocytosis of neutrophils after ischemic stroke. These findings may guide perspectives for stroke therapy from modulating microglial immunometabolism.

Nicorandil inhibits inflammasome activation and Toll-like receptor-4 signal transduction to protect against oxygen-glucose deprivation-induced inflammation in BV-2 cells.

BACKGROUND AND PURPOSE: Our previous studies have demonstrated adenosine triphosphate-sensitive potassium channel (KATP channel) openers could protect against inflammatory response in brain disease, but little is known about the mechanisms involved in KATP channel openers inhibiting neuroinflammation. METHODS AND RESULTS: In the present study, we found that oxygen-glucose deprivation (OGD) resulted in BV-2 cells activation, significantly increased tumor necrosis factor-alpha and interleukin-1beta (IL-1ß) levels, accompanied by downregulating Kir6.1 subunit. Pretreatment with nicorandil, a KATP channel opener, could attenuate OGD-induced BV-2 cells activation and inhibit pro-inflammatory factors release. Further study demonstrated that OGD activated Toll-like receptor-4 (TLR4) signaling pathway and NOD-like receptor pyrin domain containing three inflammasome, thereby increased IL-1ß production. Pretreatment with nicorandil could reverse the two pathways involved in IL-1ß production. CONCLUSIONS: Our findings reveal that KATP channel openers could protect against OGD-induced neuroinflammation via inhibiting inflammasome activation and TLR4 signal transduction.

Kir6.1 knockdown aggravates cerebral ischemia/reperfusion-induced neural injury in mice.

BACKGROUND AND PURPOSE: ATP-sensitive potassium (K-ATP) channels couple energy metabolism with electric activity, which play important roles in brain diseases including stroke. However, the impacts of Kir6.1-containing K-ATP channels that mainly expressed on glia in stroke remain unclear. METHODS AND RESULTS: In this study, we found that expression of Kir6.1 was significantly decreased in the ischemic brain area of C57BL/6J mice after 1-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. Then, we subjected Kir6.1 heterozygote knockout (Kir6.1(+/-) ) mice to cerebral ischemia/reperfusion (I/R) injury and found that Kir6.1(+/-) mice exhibited exacerbated neurological disorder and enlarged infarct size, companied by glial over-activation and blood-brain barrier (BBB) damages. Furthermore, we showed that Kir6.1 knockdown aggravated endoplasmic reticulum (ER) stress and thereby increased the levels of proinflammatory factors tumor necrosis factor-α and interleukin-1ß (TNF-α and IL-1ß) in mouse brain. CONCLUSIONS: Our findings reveal that Kir6.1 knockdown exacerbates cerebral I/R-induced brain damages via increasing ER stress and inflammatory response, indicating that Kir6.1-containing K-ATP channels may be a potential therapeutic target for stroke.