Ischemic Cortex Research Articles

Protective Effect of Adenosine A2B Receptor Agonist, BAY60-6583, Against Transient Focal Brain Ischemia in Rat.

Cerebral ischemia is a multifactorial pathology characterized first by an acute injury, due to excitotoxicity, followed by a secondary brain injury that develops hours to days after ischemia. During ischemia, adenosine acts as an endogenous neuroprotectant. Few studies have investigated the role of A2B receptor in brain ischemia because of the low potency of adenosine for it and the few selective ligands developed so far. A2B receptors are scarcely but widely distributed in the brain on neurons, glial and endothelial cells and on hematopoietic cells, lymphocytes and neutrophils, where they exert mainly anti-inflammatory effects, inhibiting vascular adhesion and inflammatory cells migration. Aim of this work was to verify whether chronic administration of the A2B agonist, BAY60-6583 (0.1 mg/kg i.p., twice/day), starting 4 h after focal ischemia induced by transient (1 h) Middle Cerebral Artery occlusion (tMCAo) in the rat, was protective after the ischemic insult. BAY60-6583 improved the neurological deficit up to 7 days after tMCAo. Seven days after ischemia BAY60-6583 reduced significantly the ischemic brain damage in cortex and striatum, counteracted ischemia-induced neuronal death, reduced microglia activation and astrocytes alteration. Moreover, it decreased the expression of TNF-α and increased that of IL-10 in peripheral plasma. Two days after ischemia BAY60-6583 reduced blood cell infiltration in the ischemic cortex. The present study indicates that A2B receptors stimulation can attenuate the neuroinflammation that develops after ischemia, suggesting that A2B receptors may represent a new interesting pharmacological target to protect from degeneration after brain ischemia.

Protective Effects of 28-O-Caffeoyl Betulin (B-CA) on the Cerebral Cortex of Ischemic Rats Revealed by a NMR-Based Metabolomics Analysis.

28-O-caffeoyl betulin (B-CA) has been demonstrated to reduce the cerebral infarct volume caused by transient middle cerebral artery occlusion (MCAO) injury. B-CA is a novel derivative of naturally occurring caffeoyl triterpene with little information associated with its pharmacological target(s). To date no data is available regarding the effect of B-CA on brain metabolism. In the present study, a 1H-NMR-based metabolomics approach was applied to investigate the therapeutic effects of B-CA on brain metabolism following MCAO in rats. Global metabolic profiles of the cortex in acute period (9h after focal ischemia onset) after MCAO were compared between the groups (sham; MCAO + vehicle; MCAO + B-CA). MCAO induced several changes in the ipsilateral cortex of ischemic rats, which consequently led to the neuronal damage featured with the downregulation of NAA, including energy metabolism dysfunctions, oxidative stress, and neurotransmitter metabolism. Treatment with B-CA showed statistically significant rescue effects on the ischemic cortex of MCAO rats. Specifically, treatment with B-CA ameliorated the energy metabolism dysfunctions (back-regulating the levels of succinate, lactate, BCAAs, and carnitine), oxidative stress (upregulating the level of glutathione), and neurotransmitter metabolism disturbances (back-regulating the levels of γ-aminobutyric acid and acetylcholine) associated with the progression of ischemic stroke. With the administration of B-CA, the levels of three phospholipid related metabolites (O-phosphocholine, O-phosphoethanolamine, sn-glycero-3-phosphocholine) and NAA improved significantly. Overall, our findings suggest that treatment with B-CA may provide neuroprotection by augmenting the metabolic changes observed in the cortex following MCAO in rats.

Identification of regulated proteins by epigallocatechin gallate treatment in an ischemic cerebral cortex animal model: a proteomics approach.

Ischemic stroke is a fatal disease that has long-term disability. It induces excessive oxidative stress generation and cellular metabolic disorders, result in tissue damage. Epigallocatechin gallate (EGCG) is a naturally derived flavonoid with strong antioxidant property. We previously reported the neuroprotective effect of EGCG in ischemic stroke. The defensive mechanisms of stroke are very diverse and complex. This study investigated specific proteins that are regulated by EGCG treatment in the ischemic brain damage. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia. EGCG (50 mg/kg) or vehicle was intraperitoneally administered just prior to MCAO. MCAO induced severe neurological deficits and disorders. EGCG treatment alleviated these neurological disorder and damage. Cerebral cortex was used for this study. Two-dimensional gel electrophoresis and mass spectrometry were performed to detect the proteins altered by EGCG. We identified various proteins that were changed between vehicle- and EGCG-treated animals. Among these proteins, isocitrate dehydrogenase, dynamin-like protein 1, and γ-enolase were decreased in vehicle-treated animals, while EGCG treatment prevented these decreases. However, pyridoxal-5’-phosphate phosphatase and 60 kDa heat shock protein were increased in vehicle-treated animals with MCAO injury. EGCG treatment attenuated these increases. The changes in these proteins were confirmed by Western blot and reverse transcription-PCR analyses. These proteins were associated with cellular metabolism and neuronal regeneration. Thus, these findings can suggest that EGCG performs a defensive mechanism in ischemic damage by regulating specific proteins related to energy metabolism and neuronal protection.

Increased apoptosis, tumor necrosis factor-α, and DNA damage attenuated by 3',4'-dihydroxyflavonol in rats with brain İschemia-reperfusion.

OBJECTIVES:This research was aimed to find out the effects of 3',4'-dihydroxyflavonol (DiOHF) on apoptosis, DNA damage, and tumor necrosis factor-α (TNF-α) levels in the frontal cortex of rats with induced experimental brain ischemi reperfusion.MATERIALS AND METHODS:A total of 38 Wistar albino male rats were used. Groups were created as 1-Sham; 2-Ischemia-reperfusion (I/R); 3-I/R + DiOHF (10 mg/kg); 4-Ischemia + DiOHF + reperfusion; 5-DiOHF + I/R. I/R was performed by carotid artery ligation for 30 min in anesthesized animals. Following experimental applications, blood samples were taken from anesthetized rats to obtain erythrocyte and plasma. Later, the rats were killed by cervical dislocation, and frontal cortex samples were taken and stored at − 80° C for the analysis.RESULTS:In the ischemic frontal cortex tissue sections degenerate neuron numbers, Terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) positive cell ratio and caspase-3 positive cell ratio increased. Malondialdehyde, TNF-α, and 8-OHdG levels were increased in both plasma and tissue in ischemia group, whereas tissue and erythrocyte glutathione levels were significantly suppressed. However, these values were significantly reversed by DiOHF treatment.CONCLUSION:The results of the study showed that I/R significantly increased apoptosis, TNF-α, and DNA damage in rats with brain I/R. However, 10 mg/kg intraperitoneal DiOHF treatment improved deterioted parameters.

The novel Nrf2 activator CDDO-EA attenuates cerebral ischemic injury by promoting microglia/macrophage polarization toward M2 phenotype in mice.

The aim of present study was to explore whether 2‐cyano‐3, 12‐dioxooleana‐1, 9‐dien‐28‐oic acid (CDDO)‐ethylamide (CDDO‐EA) attenuates cerebral ischemic injury and its possible mechanisms using a middle cerebral artery occlusion (MCAO) model in C57BL/6 mice. Our results showed that intraperitoneal injection (i.p.) of CDDO‐EA (2 and 4 mg/kg) augmented NFE2‐related factor 2 (Nrf2) and heme oxygenase‐1 (HO‐1) expression in ischemic cortex after MCAO. Moreover, CDDO‐EA (2 mg/kg, i.p.) significantly enhanced Nrf2 nuclear accumulation, associated with increased cytosolic HO‐1 expression, reduced neurological deficit and infarct volume as well as neural apoptosis, and shifted polarization of microglia/macrophages toward an antiinflammatory M2 phenotype in ischemic cortex after MCAO. Using an in vitro model, we confirmed that CDDO‐EA (100 μg/mL) increased HO‐1 expression and primed microglial polarization toward M2 phenotype under inflammatory stimulation in BV2 microglial cells. These findings suggest that a novel Nrf2 activator CDDO‐EA confers neuroprotection against ischemic injury.

Perioperative Dexmedetomidine attenuates brain ischemia reperfusion injury possibly via up-regulation of astrocyte Connexin 43

BackgroundAstrocyte Connexin 43 (Cx43) is essential for the trophic and protective support of neurons during brain ischemia reperfusion (I/R) injury. It is believed that dexmedetomidine participates in Cx43-mediated effects. However, its mechanisms remained unclear. This study aims to address the relationship and regulation among them.MethodsAdult male Sprague-Dawley rats were allocated to the 90-min right middle cerebral arterial occlusion with or without dexmedetomidine pretreatment (5 μg/kg). Neurological functions were evaluated and brain lesions, as well as inflammatory factors (IL-1β, IL-6, TNF-α), were assessed. Ischemic penumbral cortex was harvested to determine the expression of astrocyte Cx43. Primary astrocytes were cultured to evaluate the effect of dexmedetomidine on Cx43 after oxygen-glucose deprivation.ResultsDexmedetomidine pretreatment attenuated neurological injury, brain lesions and expression of inflammatory factors (IL-1β, IL-6, TNF-α) after brain ischemia (P < 0.05). Astrocyte Cx43 was down-regulated by brain I/R injury, both in vivo and in vitro, which were reversed by dexmedetomidine (P < 0.05). This effect was mediated by the phosphorylation of Akt and GSK-3β. Further studies with LY294002 (PI3K inhibitor) or SB216763 (GSK-3β inhibitor) confirmed the effect of dexmedetomidine on astrocyte Cx43.ConclusionsPerioperative dexmedetomidine administration attenuates neurological injury after brain I/R injury, possibly through up-regulation of astrocyte Cx43. Activation of PI3K-Akt-GSK-3β pathway might contribute to this protective effect.

Effect of Electroacupuncture on Neurological Deficit and Activity of Clock and Bmal1 in Cerebral Ischemic Rats.

Acute focal cerebral ischemic stroke (IS) is a leading cause of morbidity and mortality worldwide. Acupuncture is an emerging alternative therapy that has been beneficial to acute brain ischemia. However, the underlying protective mechanism of its neuroprotective effect remains unclear. Human original circadian rhythm will be lost after IS, which seriously affects the quality of life and functional recovery of stroke patients. We hypothesize that acupuncture treats IS by regulating the balance of Clock and Bmal1. This study aims to explore the effect of acupuncture at acupoints GV20 and BL23 on neuroprotection and anti-apoptosis in middle cerebral artery occlusion (MCAO) rats and expression of apoptosis and circadian rhythm related proteins. Male Sprague-Dawley (SD) rats were randomly divided into five groups: normal group (Normal), sham model group (Sham MCAO), MCAO model group (MCAO), sham electroacupuncture group (Sham EA) and electroacupuncture group (EA). The MCAO model was prepared by electrocoagulation. The first acupuncture treatment was performed within 2 h after surgery, and then acupuncture therapy was performed on 1st day, 2nd day and 3rd day respectively. After their neurological examination at 72 h of ischemia, the rats from each group were sacrificed. Triphenyltetrazolium chloride (TTC) staining was used to evaluate the brain infarct size. Ultrastructural observation on cerebral ischemic cortex and serum inflammatory cytokines were evaluated. TUNEL staining was used to detect cell apoptosis of brain tissue. The expression levels of proteins Bax, bcl-2, caspase-3, Clock and Bmal1 in the cerebral ischemic region were detected by immunofluorescence staining. Here, we presented evidence that EA at GV20 and BL23 could significantly improve the neurological deficit score and infarct size, and alleviate the cell apoptosis of brain tissue. Moreover, acupuncture treatment upregulated the anti-apoptotic Bcl-2/Bax ratio and reversed the upregulation of caspase-3 following 72-h cerebral ischemia. In addition, the expression levels of circadian proteins Clock and Bmal1 were upregulated in EA group while compared with MCAO group. Our study demonstrated that acupuncture exerted neuroprotective effect against neuronal apoptosis after stroke and the mechanism might be related with regulation of circadian rhythm proteins Clock and Bmal1.

Electroacupuncture improves learning and memory functions in a rat cerebral ischemia/reperfusion injury model through PI3K/Akt signaling pathway activation.

Electroacupuncture has been widely used to treat cognitive impairment after cerebral ischemia, but the underlying mechanism has not yet been fully elucidated. Studies have shown that autophagy plays an important role in the formation and development of cognitive impairment, and the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays an important role in autophagy regulation. To investigate the role played by the PI3K/Akt signaling pathway in the electroacupuncture treatment of cerebral ischemia/reperfusion rat models, we first established a rat model of cerebral ischemia/reperfusion through the occlusion of the middle cerebral artery using the suture method. Starting at 2 hours after modeling, electroacupuncture was delivered at the Shenting (GV24) and Baihui (GV20) acupoints, with a dilatational wave (1-20 Hz frequency, 2 mA intensity, 6 V peak voltage), for 30 minutes/day over 8 consecutive days. Our results showed that electroacupuncture reduced the infarct volume in a rat model of cerebral ischemia/reperfusion injury, increased the mRNA expression levels of the PI3K/Akt signaling pathway-related factors Beclin-1, mammalian target of rapamycin (mTOR), and PI3K, increased the protein expression levels of phosphorylated Akt, Beclin-1, PI3K, and mTOR in the ischemic cerebral cortex, and simultaneously reduced p53 mRNA and protein expression levels. In the Morris water maze test, the latency to find the hidden platform was significantly shortened among rats subjected to electroacupuncture stimulation compared with rats without electroacupuncture stimulation. In the spatial probe test, the number of times that a rat crossed the target quadrant was increased in rats subjected to electroacupuncture stimulation compared with rats without electroacupuncture stimulation. Electroacupuncture stimulation applied to the Shenting (GV24) and Baihui (GV20) acupoints activated the PI3K/Akt signaling pathway and improved rat learning and memory impairment. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine, China (approval No. 8150150901) on March 10, 2016.

Characterization of the Blood Brain Barrier Disruption in the Photothrombotic Stroke Model.

Blood brain barrier (BBB) damage is an important pathophysiological feature of ischemic stroke which significantly contributes to development of severe brain injury and therefore is an interesting target for therapeutic intervention. A popular permanent occlusion model to study long term recovery following stroke is the photothrombotic model, which so far has not been anatomically characterized for BBB leakage beyond the acute phase. Here, we observed enhanced BBB permeability over a time course of 3 weeks in peri-infarct and core regions of the ischemic cortex. Slight increases in BBB permeability could also be seen in the contralesional cortex, hippocampus and the cerebellum at different time points, regions where lesion-induced degeneration of pathways is prominent. Severe damage of tight and adherens junctions and loss of pericytes was observed within the peri-infarct region. Overall, the photothrombotic stroke model reproduces a variety of features observed in human stroke and thus, represents a suitable model to study BBB damage and therapeutic approaches interfering with this process.

Acute ischemic stroke induces magnetic resonance susceptibility signs dominated by endothelial nitric oxide synthase activation.

Acute ischemic stroke induces deoxyhemoglobin accumulation around the ischemic region while activating endothelial nitric oxide synthase (eNOS) coupling and the subsequent release of nitric oxide (NO). Because deoxyhemoglobin is a natural NO spin trap, its interplay with NO could be prominent during acute stroke. Its interaction with NO has been shown to induce overt paramagnetic signals in vitro; our goal was to investigate whether this interplay can be detected using MRI. To verify the in vivo image effects using the deoxyhemoglobin-NO interaction during acute stroke, eNOS states were manipulated in an animal model of acute ischemia, and the susceptibility signals, cerebral perfusion, and infarction were assessed noninvasively via MR susceptibility weighted imaging (SWI). Occlusion of the right middle cerebral artery increased eNOS coupling and susceptibility signals in the ischemic cortex while abolishing regional cerebral blood flow. Pharmacological eNOS blockage led to weakened susceptibility signals in the ischemic cortex as well as worsened tissue survival. Consistently, abolishment of eNOS coupling through genetic editing reduced the regional susceptibility signals in the ischemic cortex, causing large infarcts. Upregulation of eNOS during acute ischemia sustains tissue viability through the interaction between NO and deoxyhemoglobin. This interplay can be traced in vivo using SWI and can be considered a sensitive marker revealing the delicate oxygenation status of the ischemic tissue, therefore, guiding the management of acute stroke in clinical settings.

Expression and Distribution Pattern of Pnn in Ischemic Cerebral Cortex and Cultured Neural Cells Exposed to Oxygen-Glucose Deprivation.

Pinin (Pnn), a multifunctional protein, participates in embryonic development as well as in cellular apoptosis, proliferation, and migration through regulating mRNA alternative splicing and gene transcription. Previous studies have shown that Pnn plays important roles in neural system development and the expression level of Pnn in astrocytes is altered by ischemic stress and associated with cellular apoptosis. In the present study, we further utilized primary cultured rat neurons and astrocytes with oxygen-glucose deprivation (OGD) and a mouse model with middle cerebral artery occlusion (MCAO)-induced ischemic stroke to examine the effect of ischemic stress on Pnn expression and distribution in different types of neural cells. Under normoxia, Pnn is mainly localized in the nuclear speckle of primary cultured neurons. The expression level of Pnn was increased after the OGD treatment and then decreased in the reoxygenation period. Moreover, the cytoplasmic expression of Pnn was observed in neurons with OGD and reoxygenation (OGD/R). Unlike that in neurons, the Pnn expression in astrocytes was decreased after OGD treatment and then gradually increased during the reoxygenation period. Of interest, the nuclear–cytoplasmic translocation of Pnn was not observed in astrocytes with OGD/R. In the MCAO mouse model, the neuronal expression of Pnn in the peri-ischemic region was reduced by three days post induction of ischemic stroke. However, the Pnn expression in astrocytes was not altered. Moreover, the nuclear speckle distribution of Pnn in neurons was also diminished following ischemic stroke. In conclusion, the Pnn expression and distribution after OGD and during reoxygenation showed distinct manners in neurons and astrocytes, implying that Pnn may play different roles in different types of neural cells in the stress response to ischemic injury.

Induced mRNA expression of matrix metalloproteinases Mmp-3, Mmp-12, and Mmp-13 in the infarct cerebral cortex of photothrombosis model mice

A strong therapeutic target of ischemic stroke is controlling brain inflammation. Recent studies have implicated the critical role of C-C chemokine receptor 5 (CCR5) in neuroinflammation during ischemic stroke. It has been reported that the expression of the matrix metalloproteinases, MMP-3, MMP-12, and MMP-13, is controlled by CCR5; however, their expressional regulation in the infarct brain has not been clearly understood. This study investigated the mRNA expression of Mmp-3, -12, and -13 in the ischemic cerebral cortex of photothrombosis mouse model. The three Mmps were highly upregulated in the early stages of ischemic stroke and were expressed in different types of cells. Mmp-3 and Mmp-13 were expressed in blood vessel endothelial cells after ischemia-induction, whereas Mmp-12 was expressed in activated microglia. The expression of Mmp-13 in resting microglia and in neurons of uninjured cerebral cortex was lost in the infarct region. Therefore, the MMPs responding to CCR5 are differentially regulated during ischemic stroke.

Inflammatory mechanism of cerebral ischemia-reperfusion injury with treatment of stepharine in rats

BackgroundIncreasing evidence has shown that microglia-induced neuroinflammation is involved in the pathogenesis of ischemic stroke. Stepharine, one of the alkaloids extracted from Stephania japonica (Thunb.) Miers, exhibited strong inhibitory effect on microglial overactivation. However, it is not known whether it has the potential to prevent ischemic stroke. MethodsThe neuroprotective and anti-neuroinflammatory effects of stepharine were investigated in vivo and in vitro, using a rat model of middle cerebral artery occlusion (MCAO) and lipopolysaccharide (LPS)-stimulated BV-2 cells, respectively. ResultsIn vivo, stepharine (500 μg/kg) suppressed neurological deficits scores, brain water content and cerebral infarct volume induced by MCAO. Moreover, stepharine (500 μg/kg) inhibited NeuN+ cells loss and Iba-1+ cells increase in the MCAO ischemic cortex. In vitro, stepharine (10, 30 μM) substantially inhibited nitric oxide release as well as the mRNA and protein expression of pro-inflammatory mediators [inducible nitric oxide synthase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β] in LPS-activated BV-2 cells. LPS-induced increase of TLR4 expression, IκBα phosphorylation, and NF-κB p65 nuclear translocation was inhibited by stepharine (10, 30 μM). Molecular docking analysis showed that stepharine directly interacted with TLR4. SPR assay further confirmed that stepharine could bind to the TLR4/MD2 complex. Meanwhile, stepharine exhibited neuroprotective effects on SH-SY5Y cells cultured with LPS-treated conditioned medium. ConclusionOur study demonstrated for the first time that stepharine improved the outcomes in MCAO rats, reduced neuronal loss, and suppressed microglial overactivation via the inhibition of TLR4/NF-κB pathway. These results suggest that stepharine might be a potential therapeutic agent for the treatment of ischemic stroke.

Exosomes from MSCs overexpressing microRNA-223-3p attenuate cerebral ischemia through inhibiting microglial M1 polarization mediated inflammation

AimsTo explore the therapeutic effect and possible mechanism of exosomes from MSCs overexpressing miR-223 on cerebral ischemia and microglia polarization mediated inflammation. Main methodsRats after middle cerebral artery occlusion and reperfusion (MCAO/R) surgery and microglia BV-2 exposed to oxygen and glucose deprivation (OGD) and cysteinyl leukotrienes (CysLTs) stimulation were subject to exosomes from miR-223-3p transfected MSCs treatment, respectively. Behavioral tests were applied to assess the rats' neurological function. FACS was used to analyze M1/M2 microglia BV-2. production of cytokines in the ischemic hemisphere and BV-2 was detected by ELISA or qRT-PCR. Western blotting and qRT-PCR were also used to examine the expression of cysteinyl leukotriene receptor 2 (CysLT2R) in vivo and in vitro. Key findingsExosomes from MSCs over expressing miR-223-3p decreased MCAO/R induced cerebral infarct volume, improved neurological deficits, promoted learning and memorizing abilities. They suppressed pro-inflammatory factors expression and promoted anti-inflammatory factors secretion in the ischemic cortex and hippocampus. In vitro, exosomal miR-223-3p exhibited a more evident impact on modulating mRNA expression and protein production of cytokines. It promoted M2 microglia transformation of M1 microglia induced by NMLTC4 with a concentration-dependent manner. Western blot and qRT-PCR also revealed exosomal miR-223-3p decreased mRNA and protein expression of CysLT2R in vitro and in vivo. SignificanceExosomal miR-223-3p from MSCs attenuated cerebral ischemia/reperfusion injury through inhibiting microglial M1 polarization mediated pro-inflammatory response, which may be related with inhibitory effect of exosomal miR-223-3p on CysLT2R.

Genetic Deletion or Pharmacological Inhibition of Cyclooxygenase-2 Reduces Blood-Brain Barrier Damage in Experimental Ischemic Stroke.

Cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 are two crucial mediators contributing to blood-brain barrier (BBB) damage during cerebral ischemia. However, it is not known whether MMP-9 activation is involved in COX-2-mediated BBB disruption in ischemic stroke. In this study, we hypothesized that genetic deletion or pharmacological inhibition of COX-2 reduces BBB damage by reducing MMP-9 activity in a mouse model of ischemic stroke. Male COX-2 knockout (COX-2−/−) and wild-type (WT) mice were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Genetic deletion of COX-2 or post-ischemic treatment with CAY10404, a highly selective COX-2 inhibitor, significantly reduced BBB damage and hemorrhagic transformation, as assessed by immunoglobulin G (IgG) extravasation and brain hemoglobin (Hb) levels, respectively. Immunoblotting analysis showed that tight junction proteins (TJPs) zonula occludens (ZO)-1 and occludin as well as junctional adhesion molecule-A (JAM-A) and the basal lamina protein collagen IV were dramatically reduced in the ischemic brain. Stroke-induced loss of these BBB structural proteins was significantly attenuated in COX-2−/− mice. Similarly, stroke-induced loss of ZO-1 and occludin was significantly attenuated by CAY10404 treatment. Ischemia-induced increase in MMP-9 protein levels in the ipsilateral cerebral cortex was significantly reduced in COX-2−/− mice. Stroke induced a dramatic increase in MMP-9 enzymatic activity in the ischemic cortex, which was markedly reduced by COX-2 gene deficiency or pharmacological inhibition with CAY10404. Levels of myeloperoxidase (MPO, an indicator of neutrophil infiltration into the brain parenchyma), neutrophil elastase (NE), and lipocalin-2 (LCN2, also known as neutrophil gelatinase-associated lipocalin), measured by western blot and specific ELISA kits, respectively, were markedly increased in the ischemic brain. Increased levels of markers for neutrophil infiltration were significantly reduced in COX-2−/− mice compared with WT controls following stroke. Altogether, neurovascular protective effects of COX-2 blockade are associated with reduced BBB damage, MMP-9 expression/activity and neutrophil infiltration. Our study shows for the first time that MMP-9 is an important downstream effector contributing to COX-2-mediated neurovascular damage in ischemic stroke. Targeting the COX-2/MMP-9 pathway could represent a promising strategy to reduce neuroinflammatory events in order to preserve the BBB integrity and ameliorate ischemic stroke injury.

Melatonin Enhances the Therapeutic Effect of Plasma Exosomes Against Cerebral Ischemia-Induced Pyroptosis Through the TLR4/NF-κB Pathway

IntroductionIschemic stroke-induced inflammation and inflammasome-dependent pyroptotic neural death cause serious neurological injury. Nano-sized plasma exosomes have exhibited therapeutic potential against ischemia and reperfusion injury by ameliorating inflammation. To enhance its therapeutic potential in patients with ischemic injury, we isolated exosomes from melatonin-treated rat plasma and assessed the neurological protective effect in a rat model of focal cerebral ischemia.MethodsBasal plasma exosomes and melatonin-treated plasma exosomes were isolated and intravenously injected into a rat model of focal cerebral ischemia. Neurological recovery was evaluated by determining the modified neurological severity score (mNSS), infarct volume, and brain water content. Pyroptosis in the ischemic cortex was detected through dUTP nick-end labeling (TUNEL) assay, lactate dehydrogenase (LDH) release, and gasdermin D (GSDMD) cleavage. NLRP3 inflammasome assembly and global inflammatory cytokine secretion were detected by enzyme-linked immunosorbent assay (ELISA) and Western blot assay. In immunized Sprague–Dawley rats, microglia pyroptosis was determined through a positive percentage of IBA1+ and caspase-1 (p20)+ cells. Finally, the microRNA (miRNA) profiles in melatonin-treated plasma exosomes were analyzed by exosome miRNA microarray analysis.ResultsMelatonin treatment enhanced plasma exosome therapeutic effects against ischemia-induced inflammatory responses and inflammasome-mediated pyroptosis. In addition, we confirmed that ischemic stroke-induced pyroptotic cell death occurred in the microglia and neuron, while the administration of melatonin-treated exosomes further effectively decreased the infarct volume and improved recovery of function via regulation of the TLR4/NF-κB signaling pathway. Finally, the altered miRNA profiles in the melatonin-treated plasma exosomes demonstrated the regulatory mechanisms involved in neurological recovery after ischemic injury.ConclusionThis study suggests that nano-sized plasma exosomes with melatonin pretreatment might be a more effective strategy for patients with ischemic brain injury. Further exploration of key molecules in the plasma exosome may provide increased therapeutic value for cerebral ischemic injury.

Antagonistic effects of IL-17 and Astragaloside IV on cortical neurogenesis and cognitive behavior after stroke in adult mice through Akt/GSK-3\u03b2 pathway

We aimed to investigate the exact effect of IL-17 on regulating neural stem cells (NSCs) stemness and adult neurogenesis in ischemic cortex after stroke, how Astragaloside IV(As-IV) regulated IL-17 expression and the underlying mechanism. Photochemical brain ischemia model was established and IL-17 protein expression was observed at different time after stroke in WT mice. At 3 days after stroke, when IL-17 expression peaked, IL-17 knock out (KO) mice were used to observe cell proliferation and neurogenesis in ischemic cortex. Then, As-IV was administered intravenously to assess cell apoptosis, proliferation, neurogenesis, and cognitive deficits by immunochemistry staining, western blots, and animal behavior tests in WT mice. Furthermore, IL-17 KO mice and As-IV were used simultaneously to evaluate the mechanism of cell apoptosis and proliferation after stroke in vivo. Besides, in vitro, As-IV and recombinant mouse IL-17A was administered, respectively, into NSCs culture, and then their diameters, viable cell proliferation and pathway relevant protein was assessed. The results showed knocking out IL-17 contributed to regulating PI3K/Akt pathway, promoting NSCs proliferation, and neurogenesis after ischemic stroke. Moreover, As-IV treatment helped inhibit neural apoptosis, promote the neurogenesis and eventually relieve mice anxiety after stroke. Unsurprisingly, IL-17 protein expression could be downregulated by As-IV in vivo and in vitro and they exerted antagonistic effect on neurogenesis by regulating Akt/GSK-3β pathway, with significant regulation for apoptosis. In conclusion, IL-17 exerts negative effect on promoting NSCs proliferation, neurogenesis and cognitive deficits after ischemic stroke, which could be reversed by As-IV.

PPAR-γ Mediates Ta-VNS-Induced Angiogenesis and Subsequent Functional Recovery after Experimental Stroke in Rats.

Background Neoangiogenesis after cerebral ischemia in mammals is insufficient to restore neurological function, illustrating the need to design better strategies for improving outcomes. Our previous study has suggested that transcutaneous auricular vagus nerve stimulation (ta-VNS) induced angiogenesis and improved neurological functions in a rat model of cerebral ischemia/reperfusion (I/R) injury. However, the mechanisms involved need further exploration. Peroxisome proliferator-activated receptor-γ (PPAR-γ), well known as a ligand-modulated nuclear transcription factor, plays a crucial role in the regulation of cerebrovascular structure and function. Hence, the present study was designed to explore the role of PPAR-γ in ta-VNS-mediated angiogenesis and uncover the possible molecular mechanisms against ischemic stroke. Methods Adult male Sprague–Dawley rats were transfected with either PPAR-γ small interfering RNA (siRNA) or lentiviral vector without siRNA prior to surgery and subsequently received ta-VNS treatment. The expression and localization of PPAR-γ in the ischemic boundary after ta-VNS treatment were examined. Subsequently, neurological deficit scores, neuronal damage, and infarct volume were all evaluated. Additionally, microvessel density, endothelial cell proliferation condition, and the expression of angiogenesis-related molecules in the peri-infarct cortex were measured. Results We found that the expression of PPAR-γ in the peri-infarct cortex increased at 14 d and reached normal levels at 28 d after reperfusion. Ta-VNS treatment further upregulated PPAR-γ expression in the ischemic cortex. PPAR-γ was mainly expressed in neurons and astrocytes. Furthermore, ta-VNS-treated I/R rats showed better neurobehavioral recovery, alleviated neuronal injury, reduced infarct volume, and increased angiogenesis, as indicated by the elevated levels of brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and phosphorylated endothelial nitric oxide synthase (P-eNOS). Surprisingly, the beneficial effects of ta-VNS were weakened after PPAR-γ silencing. Conclusions Our results suggest that PPAR-γ is a potential mediator of ta-VNS-induced angiogenesis and neuroprotection against cerebral I/R injury.

Modulation of Cerebral Store-operated Calcium Entry-regulatory Factor (SARAF) and Peripheral Orai1 Following Focal Cerebral Ischemia and Preconditioning in Mice

Store-operated Ca2+ entry (SOCE) contributes to Ca2+ refilling of endoplasmic reticulum (ER), but also provides Ca2+ influx involved in physiological and pathological signalling functions. Upon depletion of Ca2+ store, the sensor protein stromal interaction molecule (STIM) activates Orai1, forming an ion-conducting pore highly selective for Ca2+. SOCE-associated regulatory factor (SARAF) associates with STIM1 to facilitate a slow form of Ca2+-dependent inactivation of SOCE or interacts with Orai1 to stimulate SOCE in STIM1-independent manner. We have investigated whether cerebral ischemic damage and neuroprotection conferred by ischemic preconditioning (PC) in mouse are associated with changes in the expression of the molecular components of SOCE.Ischemic PC induced by 15-min occlusion of the middle cerebral artery (MCAo) resulted in significant amelioration of histological and functional outcomes produced, 72 h later, by a more severe ischemia (1 h MCAo). Neither ischemia, nor PC affected the expression of Orai1 in the frontoparietal cortex. However, the number of Orai1-immunopositive cells, mostly corresponding to Ly-6G+ neutrophils, was significantly elevated in the blood after the ischemic insult, regardless of previous PC. The expression of Stim1 and SARAF, mainly localised in NeuN-immunopositive neurons, was reduced in the ischemic cortex. Interestingly, neuroprotection by ischemic PC prevented the reduction of SARAF expression in the lesioned cortex and this could be interpreted as a compensatory mechanism to restore ER Ca2+ refilling in neurons in the absence of STIM1. Thus, preventing SARAF downregulation may represent a pivotal mechanism implicated in neuroprotection provided by ischemic PC and should be exploited as an original target for novel stroke therapies.

Therapeutic Effects of Simultaneous Delivery of Nerve Growth Factor mRNA and Protein via Exosomes on Cerebral Ischemia

Stroke is the leading neurological cause of death and disability all over the world, with few effective drugs. Nerve growth factor (NGF) is well known for its multifaceted neuroprotective functions post-ischemia. However, the lack of an efficient approach to systemically deliver bioactive NGF into ischemic region hinders its clinical application. In this study, we engineered the exosomes with RVG peptide on the surface for neuron targeting and loaded NGF into exosomes simultaneously, with the resultant exosomes denoted as NGF@ExoRVG. By systemic administration of NGF@ExoRVG, NGF was efficiently delivered into ischemic cortex, with a burst release of encapsulated NGF protein and de novo NGF protein translated from the delivered mRNA. Moreover, NGF@ExoRVG was found to be highly stable for preservation and function efficiently for a long time in vivo. Functional study revealed that the delivered NGF reduced inflammation by reshaping microglia polarization, promoted cell survival, and increased the population of doublecortin-positive cells, a marker of neuroblast. The results of our study suggest the potential therapeutic effects of NGF@ExoRVG for stroke. Moreover, the strategy proposed in our study may shed light on the clinical application of other neurotrophic factors for central nervous system diseases.