ADAMTS13 deficiency exacerbates neuroinflammation by targeting matrix metalloproteinase-9 in ischemic brain injury.

Our design aimed to explore the potential involvement of matrix metalloproteinase-9 (MMP-9) in the inflammatory response associated with acute ischemic stroke (AIS). We also aimed to preliminarily examine the potential impact of a disintegrin-like and metalloprotease with thrombospondin type I repeats-13 (ADAMTS13) on MMP-9 in AIS. We conducted oxygen-glucose deprivation models of microglia cells and mice models of AIS with middle cerebral artery occlusion (MCAO). We assessed the expression pattern of MMP-9 with western blotting (WB) and real-time quantitative PCR both in vivo and in vitro. MMP-9 downregulation was achieved by using ACE inhibitors such as trandolapril. For the MCAO model, we used ADAMTS13-deficient mice. We then evaluated the related neurological function scores, cerebral edema and infarct volume. The levels of inflammation-related proteins, such as COX2 and iNOS, were assessed using WB, and the expression of inflammatory cytokines was measured via enzyme-linked immuno sorbent assay in vivo. Our findings indicated that MMP-9 was up-regulated while ADAMTS13 was down-regulated in the MCAO model. Knockdown of MMP-9 reduced both inflammation and ischemic brain injury. ADAMTS13 prevented brain damage, improved neurological function and decreased the inflammation response in mice AIS models. Additionally, ADAMTS13 alleviated MMP-9-induced neuroinflammation in vivo. It showed that ADAMTS13 deficiency exacerbated ischemic brain injury through an MMP-9-dependent inflammatory mechanism. Therefore, the ADAMTS13-MMP-9 axis could have therapeutic potential for the treatment of AIS.

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.

Dihydroergotamine protects against ischemic stroke by modulating microglial/macrophage polarization and inhibiting inflammation in mice.

OBJECTIVES: The search for drugs that can protect the brain tissue and reduce nerve damage in acute ischemic stroke has emerged as a research hotspot. We investigated the potential protective effects and mechanisms of action of dihydroergotamine against ischemic stroke. METHODS: C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO), and dihydroergotamine at a dose of 10 mg/kg/day was intraperitoneally injected for 14 days. Adhesive removal and beam walking tests were conducted 1, 3, 5, 7, 10, and 14 days after MCAO surgery. Thereafter, the mechanism by which dihydroergotamine regulates microglia/macrophage polarization and inflammation and imparts ischemic stroke protection was studied using enzyme-linked immunosorbent assay, immunofluorescence staining, and western blotting. RESULTS: From the perspective of a drug repurposing strategy, dihydroergotamine was found to inhibit oxygen-glucose deprivation damage to neurons, significantly improve cell survival rate, and likely exert a protective effect on ischemic brain injury. Dihydroergotamine significantly improved neural function scores and survival rates and reduced brain injury severity in mice. Furthermore, dihydroergotamine manifests its protective effect on ischemic brain injury by reducing the expression of TNF-α and IL-1ß in mouse ischemic brain tissue, inhibiting the polarization of microglia/macrophage toward the M1 phenotype and promoting polarization toward the M2 phenotype. CONCLUSION: This study is the first to demonstrate the protective effect of dihydroergotamine, a first-line treatment for migraine, against ischemic nerve injury in vitro and in vivo.

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.

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.

Epigallocatechin gallate improves neuronal damage in animal model of ischemic stroke and glutamate-exposed neurons via modulation of hippocalcin expression.

Epigallocatechin gallate (EGCG) is a polyphenolic component of green tea that has anti-oxidative and anti-inflammatory effects in neurons. Ischemic stroke is a major neurological disease that causes irreversible brain disorders. It increases the intracellular calcium concentration and induces apoptosis. The regulation of intracellular calcium concentration is important to maintain the function of the nervous system. Hippocalcin is a neuronal calcium sensor protein that controls intracellular calcium concentration. We investigated whether EGCG treatment regulates the expression of hippocalcin in stroke animal model and glutamate-induced neuronal damage. We performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. EGCG (50 mg/kg) or phosphate buffered saline was injected into the abdominal cavity just before MCAO surgery. The neurobehavioral tests were performed 24 h after MCAO surgery and cerebral cortex tissue was collected. MCAO damage induced severe neurobehavioral disorders, increased infarct volume, and decreased the expression of hippocalcin in the cerebral cortex. However, EGCG treatment improved these deficits and alleviated the decrease in hippocalcin expression in cerebral cortex. In addition, EGCG dose-dependently alleviated neuronal cell death and intracellular calcium overload in glutamate-exposed neurons. Glutamate exposure reduced hippocalcin expression, decreased Bcl-2 expression, and increased Bax expression. However, EGCG treatment mitigated these changes caused by glutamate toxicity. EGCG also attenuated the increase in caspase-3 and cleaved caspase-3 expressions caused by glutamate exposure. The effect of EGCG was more pronounced in non-transfected cells than in hippocalcin siRNA-transfected cells. These findings demonstrate that EGCG protects neurons against glutamate toxicity through the regulation of Bcl-2 family proteins and caspase-3. It is known that hippocalcin exerts anti-apoptotic effect through the modulation of apoptotic pathway. Thus, we can suggest evidence that EGCG has a neuroprotective effect by regulating hippocalcin expression in ischemic brain damage and glutamate-exposed cells.

Rat Model of Middle Cerebral Artery Occlusion.

Stroke is the third-leading cause of death and the leading cause of acquired adult disability worldwide. Several ischemic stroke models are currently available. However, mimicking focal cerebral ischemia (FCI) is the most common. The formation of an embolic or thrombotic occlusion at or near the middle cerebral artery causes most events in FCI. The current protocol closely mimics the etiology of human stroke and ensures that the results obtained are highly relevant. The method described in this protocol yields reproducible results. The success of this model in ischemic research can be examined through the utilization of Doppler blood flow imaging equipment.

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.

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

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

Suppression of cerebral ischemia injury induced blood brain barrier breakdown by dexmedetomidine via promoting CCN1.

BACKGROUND: Blood-brain barrier (BBB) could aggravate cerebral ischemia injury. Dexmedetomidine (Dex) has been believed to play a protective role in cerebral ischemia injury-induced BBB injury. METHODS: Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) models were established to simulate cerebral ischemia injury. Animal experiments included 4 groups, Sham, MCAO, MCAO+Dex, MCAO+Dex+sh-CCN1. Generally applicable gene set enrichment analysis was performed to analyze gene expression difference. Total collagen content and Evans blue staining were performed to measure infarct ratio and BBB breakdown, respectively. The cell apoptosis, mRNA and protein expression were measured through flow cytometry, PCR, and western blotting, respectively. The levels of IL-1ß, TNF-α, and IL-6 in serum were measured with commercial ELISA kits. RESULTS: Dex greatly promoted the expression level of CCN1. Dex suppressed cerebral ischemia injury, increased tight junction protein expression, improved the memory ability and neurological function of MCAO rats through targeting CCN1. The significant increase of inflammatory factors in the serum of MCAO rats were suppressed by Dex. Dex suppressed OGD induced increase of HRP permeability and promoting tight junction protein expression in vitro through regulating CCN1. The neurological function evaluation was performed with Neurological Severity Score (NSS) and Longa Score Scale. CONCLUSIONS: Dex could remarkably alleviate cerebral ischemia injury by inhibiting BBB breakdown, inflammatory response, and promoting neurological function and tight junction protein expression via up-regulating CCN1. This study might provide a novel therapeutic target for the prevention and treatment of cerebral ischemia injury-induced BBB.

Dl-3-n-Butylphthalide Alleviates Secondary Brain Damage and Improves Working Memory After Stroke in Cynomolgus Monkeys.

BACKGROUND: Remote secondary neurodegeneration is associated with poststroke cognitive impairment (PSCI). Dl-3-n-butylphthalide (NBP) improves PSCI clinically. However, whether it ameliorates PSCI by alleviating secondary neurodegeneration remains uncertain. Nonhuman primates provide more relevant models than rodents for human stroke and PSCI. This study investigated the effects of NBP on PSCI and secondary neurodegeneration in cynomolgus monkeys after permanent left middle cerebral artery occlusion (MCAO). METHODS: Thirteen adult male cynomolgus monkeys were randomly assigned to sham (n=4), MCAO+placebo (n=5), and MCAO+NBP groups (n=4). The MCAO+placebo and MCAO+NBP groups received saline and NBP injections intravenously, respectively, starting at 6-hour postsurgery for 2 weeks, followed by soybean oil and NBP orally, respectively, for 10 weeks after MCAO. Infarct size was assessed at week 4 by magnetic resonance imaging. Working memory and executive function were evaluated dynamically using the delayed response task and object retrieval detour task, respectively. Neuron loss, glia proliferation, and neuroinflammation in the ipsilateral dorsal lateral prefrontal cortex, thalamus, and hippocampus were analyzed by immunostaining 12 weeks after MCAO. RESULTS: Infarcts were located in the left middle cerebral artery region, apart from the ipsilateral dorsal lateral prefrontal cortex, thalamus, or hippocampus, with no significant difference between the MCAO+placebo and MCAO+NBP group. Higher success in delayed response task was achieved at weeks 4, 8, and 12 after NBP compared with placebo treatments (P<0.05), but not in the object retrieval detour task (all P>0.05). More neurons and less microglia, astrocytes, CD68-positive microglia, tumor necrosis factor-α, and inducible NO synthase were observed in the ipsilateral dorsal lateral prefrontal cortex and thalamus after 12 weeks of NBP treatment (P<0.05), but not in the hippocampus (P>0.05). CONCLUSIONS: Our findings indicate that NBP improves working memory by alleviating remote secondary neurodegeneration and neuroinflammation in the ipsilateral dorsal lateral prefrontal cortex and thalamus after MCAO in cynomolgus monkeys.

Impact of the pandemic and concomitant COVID-19 on the management and outcomes of middle cerebral artery strokes: a nationwide registry-based study.

OBJECTIVES: To investigate the impact of the COVID-19 pandemic as well as concomitant COVID-19 itself on stroke care, focusing on middle cerebral artery (MCA) territory infarctions. DESIGN: Registry-based study. SETTING: We used the National Inpatient Sample (NIS) database, which covers a wide range of hospitals within the USA. PARTICIPANTS: The NIS was queried for patients with MCA strokes between 2016 and 2020. In total, 35 231 patients were included. OUTCOME MEASURES: Outcome measures were postprocedural complications, length of stays (LOSs), in-hospital mortality and non-routine discharge. Propensity score matching using all available baseline variables was performed to reduce confounders when comparing patients with and without concomitant COVID-19. RESULTS: Mechanical thrombectomy (MT) was performed in 48.4%, intravenous thrombolysis (IVT) in 38.2%, and both MT and IVT (MT+IVT) in 13.4% of patients. A gradual increase in the use of MT and an opposite decrease in the use of IVT (p<0.001) was detected during the study period. Overall, 25.0% of all patients were admitted for MCA strokes during the pandemic period (2020), of these 209 (2.4%) were concomitantly diagnosed with COVID-19. Patients with MCA strokes and concomitant COVID-19 were significantly younger (64.9 vs 70.0; p<0.001), had significantly worse NIH Stroke Severity scores, and worse outcomes in terms of LOS (12.3 vs 8.2; p<0.001), in-hospital mortality (26.3% vs 9.8%; p<0.001) and non-routine discharge (84.2% vs 76.9%; p=0.013), as compared with those without COVID-19. After matching, only in-hospital mortality rates remained significantly higher in patients with COVID-19 (26.7% vs 8.5%; p<0.001). Additionally, patients with COVID-19 had higher rates of thromboembolic (12.3% vs 7.6%; p=0.035) and respiratory (11.3% vs 6.6%; p=0.029) complications. CONCLUSIONS: Among patients with MCA stroke, those with concomitant COVID-19 were significantly younger and had higher stroke severity scores. They were more likely to experience thromboembolic and respiratory complications and in-hospital mortality compared with matched controls.

GPR55 Inactivation Diminishes Splenic Responses and Improves Neurological Outcomes in the Mouse Ischemia/Reperfusion Stroke Model.

Although strokes are frequent and severe, treatment options are scarce. Plasminogen activators, the only FDA-approved agents for clot treatment (tissue plasminogen activators (tPAs)), are used in a limited patient group. Moreover, there are few approaches for handling the brain's inflammatory reactions to a stroke. The orphan G protein-coupled receptor 55 (GPR55)'s connection to inflammatory processes has been recently reported; however, its role in stroke remains to be discovered. Post-stroke neuroinflammation involves the central nervous system (CNS)'s resident microglia activation and the infiltration of leukocytes from circulation into the brain. Additionally, splenic responses have been shown to be detrimental to stroke recovery. While lymphocytes enter the brain in small numbers, they regularly emerge as a very influential leukocyte subset that causes secondary inflammatory cerebral damage. However, an understanding of how this limited lymphocyte presence profoundly impacts stroke outcomes remains largely unclear. In this study, a mouse model for transient middle cerebral artery occlusion (tMCAO) was used to mimic ischemia followed by a reperfusion (IS/R) stroke. GPR55 inactivation, with a potent GPR55-specific antagonist, ML-193, starting 6 h after tMCAO or the absence of the GPR55 in mice (GPR55 knock out (GPR55ko)) resulted in a reduced infarction volume, improved neurological outcomes, and decreased splenic responses. The inhibition of GPR55 with ML-193 diminished CD4+T-cell spleen egress and attenuated CD4+T-cell brain infiltration. Additionally, ML-193 treatment resulted in an augmented number of regulatory T cells (Tregs) in the brain post-tMCAO. Our report offers documentation and the functional evaluation of GPR55 in the brain-spleen axis and lays the foundation for refining therapeutics for patients after ischemic attacks.

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

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

Echinatin protects from ischemic brain injury by attenuating NLRP3-related neuroinflammation.

BACKGROUND: Microglia-mediated neuroinflammation is the major contributor to the secondary brain injury of ischemic stroke. NLRP3 is one of the major components of ischemia-induced microglial activation. Echinatin, a chalcone found in licorice, was reported to have the activity of anti-inflammation and antioxidant. However, the relative study of echinatin in microglia or ischemic stroke is still unclear. METHODS: We intravenously injected echinatin or vehicle into adult ischemic male C57/BL6J mice induced by 60-min transient middle cerebral artery occlusion (tMCAO). The intraperitoneal injection was performed 4.5 h after reperfusion and then daily for 2 more days. Infarct size, blood brain barrier (BBB) leakage, neurobehavioral tests, and microglial-mediated inflammatory reaction were examined to assess the outcomes of echinatin treatment. LPS and LPS/ATP stimulation on primary microglia were used to explore the underlying anti-inflammatory mechanism of echinatin. RESULTS: Echinatin treatment efficiently decreased the infarct size, alleviated blood brain barrier (BBB) damage, suppressed microglial activation, reduced the production of inflammatory factors (e.g., IL-1ß, IL-6, IL-18, TNF-α, iNOS, COX2), and relieved post-stroke neurological defects in tMCAO mice. Mechanistically, we found that echinatin could suppress the NLRP3 assembly and reduce the production of inflammatory mediators independently of NF-κB and monoamine oxidase (MAO). CONCLUSION: Based on our study, we have identified echinatin as a promising therapeutic strategy for the treatment of ischemic stroke.

Movement disorders following mechanical thrombectomy resulting in ischemic lesions of the basal ganglia: An emerging clinical entity.

BACKGROUND AND PURPOSE: Post-stroke movement disorders (PMDs) following ischemic lesions of the basal ganglia (BG) are a known entity, but data regarding their incidence are lacking. Ischemic strokes secondary to proximal middle cerebral artery (MCA) occlusion treated with thrombectomy represent a model of selective damage to the BG. The aim of this study was to assess the prevalence and features of movement disorders after selective BG ischemia in patients with successfully reperfused acute ischemic stroke (AIS). METHODS: We enrolled 64 consecutive subjects with AIS due to proximal MCA occlusion treated with thrombectomy. Patients were clinically evaluated by a movement disorders specialist for PMDs onset at baseline, and after 6 and 12 months. RESULTS: None of the patients showed an identifiable movement disorder in the subacute phase of the stroke. At 6 and 12 months, respectively, 7/25 (28%) and 7/13 (53.8%) evaluated patients developed PMDs. The clinical spectrum of PMDs encompassed parkinsonism, dystonia and chorea, either isolated or combined. In most patients, symptoms were contralateral to the lesion, although a subset of patients presented with bilateral involvement and prominent axial signs. CONCLUSION: Post-stroke movement disorders are not uncommon in long-term follow-up of successfully reperfused AIS. Follow-up conducted by a multidisciplinary team is strongly advisable in patients with selective lesions of the BG after AIS, even if asymptomatic at discharge.

PI3Kδ inhibition alleviates the brain injury during cerebral ischemia reperfusion via suppressing pericyte contraction in a TNF-α dependent manner.

The pericytes (PCs) surrounding capillaries are vital regulators of capillary constriction. Persistent PC contraction results in the increased capillary constriction, therefore leading to the impaired cerebral blood flow (CBF) recovery after reperfusion and worsening the clinical outcomes in stroke patients. However, the potential determinants of PC functions during ischemia/reperfusion are poorly understood. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit Delta (PIK3CD/PI3Kδ) is a crucial factor involved with neuronflammation during ischemic stroke. PI3Kδ has shown the expression in PCs, while its effect on PC functions has not been explored yet. In this study, a rodent ischemia/reperfusion model was established in C57BL/6 mice via transient middle cerebral artery occlusion and reperfusion (MCAO/R). The PI3Kδ expression in ischemic penumbra was remarkably upregulated following MCAO/R induction. PI3Kδ inhibitor CAL-101 improved the CBF recovery, ischemic brain injury, and suppressed capillary constriction in MCAO/R mice. Besides, the production of tumor necrosis factor alpha (TNF-α), an inducer for tissue injury, and the expression of transient receptor potential vanilloid type 2 (TRPV2), a channel protein permitting calcium (Ca2+) uptake, were significantly reduced in ischemic penumbra after CAL-101 treatment. In vitro, oxygen-glucose deprivation and reoxygenation (OGD/R) enhanced the expression of PI3Kδ and TRPV2 in primary mouse PCs. CAL-101 suppressed the TNF-α-induced TRPV2 expression in OGD/R-treated PCs, thus inhibiting the Ca2+ uptake and PC contraction. Collectively, this study suggests that PI3Kδ is a critical regulator of PC function during ischemic stroke.

Social isolation initiated post-weaning augments ischemic brain injury by promoting pro-inflammatory responses.

Social isolation is associated with poor stroke outcome, but the underlying molecular mechanisms were largely unknown. In male Balb/C mice exposed to transient middle cerebral artery occlusion (MCAo), we examined the effects of social isolation initiated post-weaning on ischemic injury, cytokine/chemokine responses and cell signaling using a broad panel of techniques that involved immunocytochemistry, cytokine/chemokine array and Western blots. Social isolation initiated post-weaning elevated infarct size, brain edema and neuronal injury in the ischemic brain tissue 3 days after MCAo, and increased microglia/ macrophage and leukocyte accumulation. In line with the increased immune cell recruitment, levels of several proinflammatory cytokines (e.g., IL-1α, IL-1ß, IL-13, IL-17, TNF-α, IFN-γ), chemokines (e.g., CCL3, CCL4, CCL12, CXCL2, CXCL9, CXCL12) and adhesion molecules (i.e., ICAM-1) were increased in the ischemic brain tissue of socially isolated compared with paired housing mice, whereas levels of selected cytokines (IL-5, IL-6, IL-16) and colony-stimulating factors (G-CSF, GM-CSF) were reduced. The activity of the transcription factor nuclear factor-ĸB (NF-ĸB), which promotes cell injury via pro-inflammatory responses, was increased by social isolation, whereas that of nuclear factor erythroid related factor-2 (Nrf-2), which mediates anti-oxidative responses under oxidative stress conditions, was reduced. Our study shows that social isolation profoundly alters post-ischemic cell signaling in a way promoting pro-inflammatory responses. Our results highlight the importance of social support in preventing deleterious health effects of social isolation.

YTHDF2-regulated matrilin-3 mitigates post-reperfusion hemorrhagic transformation in ischemic stroke via the PI3K/AKT pathway.

Hemorrhagic transformation can complicate ischemic strokes after recanalization treatment within a time window that requires early intervention. To determine potential therapeutic effects of matrilin-3, rat cerebral ischemia-reperfusion was produced using transient middle cerebral artery occlusion (tMCAO); intracranial hemorrhage and infarct volumes were assayed through hemoglobin determination and 2,3,5-triphenyltetrazoliumchloride (TTC) staining, respectively. Oxygen-glucose deprivation (OGD) modeling of ischemia was performed on C8-D1A cells. Interactions between matrilin-3 and YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) were determined using RNA immunoprecipitation assay and actinomycin D treatment. Reperfusion after tMCAO modeling increased hemorrhage, hemoglobin content, and infarct volumes; these were alleviated by matrilin treatment. Matrilin-3 was expressed at low levels and YTHDF2 was expressed at high levels in ischemic brains. In OGD-induced cells, matrilin-3 was negatively regulated by YTHDF2. Matrilin-3 overexpression downregulated p-PI3K/PI3K, p-AKT/AKT, ZO-1, VE-cadherin and occludin, and upregulated p-JNK/JNK in ischemic rat brains; these effects were reversed by LY294002 (a PI3K inhibitor). YTHDF2 knockdown inactivated the PI3K/AKT pathway, inhibited inflammation and decreased blood-brain barrier-related protein levels in cells; these effects were reversed by matrilin-3 deficiency. These results indicate that YTHDF2-regulated matrilin-3 protected ischemic rats against post-reperfusion hemorrhagic transformation via the PI3K/AKT pathway and that matrilin may have therapeutic potential in ischemic stroke.

Polygalasaponin F ameliorates middle cerebral artery occlusion-induced focal ischemia / reperfusion injury in rats through inhibiting TXNIP/NLRP3 signaling pathway.

BACKGROUND: Polygalasaponin F (PGSF), an oleanane triterpenoid saponin extracted from Polygala japonica, has been demonstrated with neuroprotective effect. However, the therapeutic effects and mechanisms of PGSF on focal ischemia remain unknown; METHODS: In this study, male Sprague Dawley (SD) rats aged 6-8 weeks were initially selected to establish a rat model of middle cerebral artery occlusion (MCAO) to evaluate the therapeutic effect of PGSF intervention and to investigate the impact of PGSF on the thioredoxin-interacting protein/NOD-, LRR-, and pyrin domain-containing protein 3 (TXNIP/NLRP3) inflammatory pathway. Secondly, brain neuron cells were isolated, and the cells received oxygen-glucose deprivation/reoxygenation (OGD/R) culture to establish the cell injury model in vitro. The mechanism of PGSF on the TXNIP/NLRP3 pathway was further validated; RESULTS: Our results showed that PGSF treatment reduced neurological scores, brain tissue water content and infarct volume and ameliorated the pathological changes in cerebral cortex in MCAO-induced focal ischemia rats. The TNF-α, IL-1ß and IL-6 levels decreased in MCAO-induced focal ischemia rats after PGSF treatment. Moreover, PGSF down-regulated the protein expressions of TXNIP, NLRP3, ASC, cleaved caspase-1, IL-1ß, and IL-18 in MCAO-induced focal ischemia rats. Meanwhile, PGSF treatment inhibited apoptosis, and reduced the levels of ROS, inflammatory cytokine and TXNIP/NLRP3 pathway-related proteins (TXNIP, NLRP3, ASC, cleaved caspase-1, IL-1ß, and IL-18) in OGD/R-induced neuronal injury cells. Finally, PGSF treatment also disrupted the interaction between NLRP3 and TXNIP in vitro; CONCLUSIONS: Our study demonstrated the therapeutic effects of PGSF on MCAO-induced focal ischemia rats. Moreover, the neuroprotective mechanism of PGSF on focal ischemia was associated with the inhibition of TXNIP/NLRP3 signaling pathway.