Focal Cerebral Ischemia-reperfusion Research Articles

Effect of Diammonium Glycyrrhizinate in Improving Focal Cerebral Ischemia-Reperfusion Injury in Rats Through Multiple Mechanisms.

Effect of Diammonium Glycyrrhizinate in Improving Focal Cerebral Ischemia-Reperfusion Injury in Rats Through Multiple Mechanisms.

Isolation of a novel isoprenylated phenolic compound and neuroprotective evaluation of Dodonaea viscosa extract against cerebral ischaemia–reperfusion injury in rats

Dodonaea viscosa grows widely in Saudi Arabia, but studies evaluating its neuroprotective activity are lacking. Thus, this study aimed to isolate and identify the secondary metabolites and evaluate the neuroprotective effects of D. viscosa leaves. The isolation and identification of phytochemicals were performed using chromatographic and spectroscopic techniques. The neuroprotective potential of the extract was evaluated against focal cerebral ischaemia–reperfusion injury in rat model. Neurobehavioural deficits in the rats were evaluated, and their brains were harvested to measure infarct volume and oxidative biomarkers. Results revealed the presence of three compounds: a novel isoprenylated phenolic derivative that was elucidated as 4-hydroxy-3-(3ʹ-methyl-2ʹ-butenyl) phenyl 1-O-β-D-apiosyl-(1ʹʹʹ → 6ʹʹ)- β-D-glucopyranoside (named Viscomarfadol) and two known compounds (isorhamnetin-3-O-rutinoside and epicatechin (4–8) catechin). Pre-treatment of the rats with the extract improved neurological outcomes. It significantly reduced neurological deficits and infarct volume; significantly reduced lipid peroxidation, as evidenced by decreased malondialdehyde levels; and significantly elevated antioxidant (superoxide dismutase, catalase, and glutathione) activities. These results indicate that D. viscosa is a promising source of bioactive compounds that can improve neurological status, decrease infarct volume, and enhance antioxidant activities in rats with cerebral ischaemic injury. Thus, D. viscosa could be developed into an adjuvant therapy for ischaemic stroke and other oxidative stress-related neurodegenerative disorders. Further investigations are warranted to explore other bioactive compounds in D. viscosa and evaluate their potential neuroprotective activities.

Effects of electroacupuncture on imaging and behavior in rats with ischemic stroke through miR-212-5p

BackgroundIschemic stroke is a serious disease leading to significant disability in humans worldwide. Increasing evidence suggests that some microRNAs (miRNAs) participate in the pathophysiology of ischemic stroke. A key role for MiR-212 has been found in neuronal function and synaptic plasticity. Ischemic stroke can be effectively treated with electroacupuncture (EA); however, there is a lack of understanding of the relevant mechanisms. In this study, we employed behavioral test and resting-state functional magnetic resonance imaging (rs-fMRI) to detect behavioral and brain function alterations in rats suffering from ischemic stroke. The efficacy of EA therapy and miR-212-5p’s role in this process were also evaluated.Methods and resultsForty rats were randomly divided into the following groups: Sham, middle cerebral artery occlusion/reperfusion (MCAO/R), MCAO/R + EA, MCAO/R + EA + antagomir-negative control and MCAO/R + EA + antagomir-212-5p groups. Behavioral changes were assessed by Catwalk gait analysis prior to and after modeling. Rs-fMRI was performed at one week after EA treatment, amplitude of low-frequency fluctuations (ALFF) and regional homogeneity (ReHo) were calculated to reveal neural activity. Furthermore, neuronal apoptosis in the ischemic penumbra was analyzed using a TUNEL assay. Treatment with EA significantly improved the performance of rats in the behavioral test. The motor and cognition-related brain regions showed decreased ALFF and ReHo following focal cerebral ischemia-reperfusion, and EA treatment could reactivate these brain regions. Moreover, EA treatment significantly decreased MCAO/R-induced cell death. However, the transfection of antagomir-212-5p attenuated the therapeutic effect of EA.ConclusionsIn conclusion, the results suggested that EA improved the behavioral and imaging outcomes of ischemic stroke through miR-212-5p.

Retracted: Influence of Melatonin on Behavioral and Neurological Function of Rats with Focal Cerebral Ischemia-Reperfusion Injury via the JNK/FoxO3a/Bim Pathway

Retracted: Influence of Melatonin on Behavioral and Neurological Function of Rats with Focal Cerebral Ischemia-Reperfusion Injury via the JNK/FoxO3a/Bim Pathway

O-GlcNAcylation is essential for therapeutic mitochondrial transplantation

BackgroundTransplantation of mitochondria is increasingly explored as a novel therapy in central nervous system (CNS) injury and disease. However, there are limitations in safety and efficacy because mitochondria are vulnerable in extracellular environments and damaged mitochondria can induce unfavorable danger signals.MethodsMitochondrial O-GlcNAc-modification was amplified by recombinant O-GlcNAc transferase (OGT) and UDP-GlcNAc. O-GlcNAcylated mitochondrial proteins were identified by mass spectrometry and the antiglycation ability of O-GlcNAcylated DJ1 was determined by loss-of-function via mutagenesis. Therapeutic efficacy of O-GlcNAcylated mitochondria was assessed in a mouse model of transient focal cerebral ischemia-reperfusion. To explore translational potential, we evaluated O-GlcNAcylated DJ1 in CSF collected from patients with subarachnoid hemorrhagic stroke (SAH).ResultsWe show that isolated mitochondria are susceptible to advanced glycation end product (AGE) modification, and these glycated mitochondria induce the receptor for advanced glycation end product (RAGE)-mediated autophagy and oxidative stress when transferred into neurons. However, modifying mitochondria with O-GlcNAcylation counteracts glycation, diminishes RAGE-mediated effects, and improves viability of mitochondria recipient neurons. In a mouse model of stroke, treatment with extracellular mitochondria modified by O-GlcNAcylation reduces neuronal injury and improves neurologic deficits. In cerebrospinal fluid (CSF) samples from SAH patients, levels of O-GlcNAcylation in extracellular mitochondria correlate with better clinical outcomes.ConclusionsThese findings suggest that AGE-modification in extracellular mitochondria may induce danger signals, but O-GlcNAcylation can prevent glycation and improve the therapeutic efficacy of transplanted mitochondria in the CNS.

Study on the anti-mitochondrial apoptosis mechanism of Erigeron breviscapus injection based on UPLC-Q-TOF-MS metabolomics and molecular docking in rats with cerebral ischemia-reperfusion injury

Ethnopharmacological relevanceErigeron breviscapus is a common medicine of eight ethnic minorities, including Miao, Naxi, and Yi. As early as the Ming Dynasty (AD 1368–1644), Lanmao's Materia Medica of Southern Yunnan (AD 1436) recorded that the medicine is used for the treatment of “Zuo tan you huan.” In modern pharmacological research, Erigeron breviscapus injection is the most commonly used preparation in the treatment of ischemic stroke caused by acute cerebral infarction, but its mechanism of action in the treatment of ischemic stroke is not well understood. Aim of the studyIn this study, a metabonomics study based on ultraperformance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) was used in investigating the effect of a traditional Chinese medicine preparation Erigeron breviscapus injection on the rat model of focal cerebral ischemia-reperfusion and the affinity of its main components with the targets of mitochondrial apoptotic pathways. Materials and methodsThis study used molecular docking technology to verify the effective binding ability of main effective components of Erigeron breviscapus injection to target proteins related to mitochondrial apoptosis pathway. This study developed a metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of focal cerebral ischemia-reperfusion model group and healthy control group was achieved. ResultsErigeron breviscapus injection can significantly reduce the area of cerebral infarction, improve tissue morphological lesion in rats, and can increase the number of Nissl bodies. It may be a promoting factor by inhibiting hippocampal nerve cell apoptosis and Bax protein expression and by exerting effects against ischemia reperfusion after the induction of apoptosis. Thus, it plays a role in brain protection. Moreover, it can considerably promote the recovery of neurological deficiency signs in advance. Meanwhile, Erigeron breviscapus decreased malondialdehyde content and T-NOS activity. Its curative effect from strong to weak order: low dose > high dose > medium dose. The representative components of Erigeron breviscapus have good affinity with the active sites of mitochondrial apoptosis-related proteins. Metabolomics found that the potential biomarkers regulated by breviscapine are kynurequinolinic acid, succinylornithine, and leucine proline. It is speculated that it may participate in TRP–kynurequinolinic acid and succinylornithine–urea cycle–NO metabolic pathways. ConclusionsThis paper revealed the potential biomarkers and metabolic pathways regulated by Erigeron breviscapus. It was speculated that the mechanism is related to its inhibition of mitochondrion-mediated apoptosis. Erigeron breviscapus could restore the metabolic profiles of the model animals to normal animal levels. The mechanism may be related to the potential biomarkers of quinolinic acid, succinylornithine, and leucine proline and the metabolic pathways involved. However, the exact mechanism by which Erigeron breviscapus inhibits mitochondrion-mediated apoptosis remains to be further explored.

Gas Chromatography-Mass Spectrometry-Based Cerebrospinal Fluid Metabolomics to Reveal the Protection of Coptisine against Transient Focal Cerebral Ischemia-Reperfusion Injury via Anti-Inflammation and Antioxidant.

Coptisine (Cop) exerts a neuroprotective effect on central nervous system disease, particularly ischemic stroke. However, its protective mechanism is still unclear. This study aimed to investigate the protective effect of Cop on cerebral ischemia-reperfusion (IR) rats with a middle cerebral artery occlusion model by integrating a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach with biochemical assessment. Our results showed that Cop could improve neurobehavioral function and decrease the ischemia size in IR rats. In addition, Cop was found to decrease inflammatory mediators (e.g., prostaglandin D2 (PGD2) and tumor necrosis factor-α (TNF-α) and attenuate oxidative stress response (e.g., increase the superoxide dismutase (SOD) expression and decrease 8-iso-PGF2α level). Furthermore, the GC-MS-based cerebrospinal fluid (CSF) metabolomics analysis indicated that Cop influenced the level of glycine, 2,3,4-trihydroxybutyric acid, oleic acid, glycerol, and ribose during IR injury. Cop exhibited a good neuroprotective effect against cerebral IR injury and metabolic alterations, which might be mediated through its antioxidant and anti-inflammatory properties.

Screening Serum Biomarkers for Rats Preconditioned with Hyperbaric Oxygen: Potential of Predicting Prognosis for Stroke.

Stroke is a major health concern and a leading cause of mortality and morbidity. We and other groups have documented that hyperbaric oxygen preconditioning could significantly alleviate neuronal damage in ischemia‒reperfusion models through various mechanisms. However, we found that some of the subjects did not benefit from preconditioning with hyperbaric oxygen. The preconditioning phenomenon is similar to vaccination, in which the endogenous survival system is activated to fight against further injuries. However, with vaccine inoculations, we could test for specific antibodies against the pathogens to determine if the vaccination was successful. Likewise, this experiment was carried out to explore a biomarker that can reveal the effectiveness of the preconditioning before neuronal injury occurs. Middle cerebral artery occlusion (MCAO) was used to induce focal cerebral ischemia-reperfusion injury. 2D-DIGE-MALDI-TOF-MS/MS proteomic technique was employed to screen the differentially expressed proteins in the serum of rats among the control (Con) group (MCAO model without hyperbaric oxygen (HBO) preconditioning), hyperbaric oxygen protective (HBOP) group (in which the infarct volume decreased after HBO preconditioning vs. Con), and hyperbaric oxygen nonprotective (HBOU) group (in which the infarct volume remained the same or even larger after HBO preconditioning vs. Con). Candidate biomarkers were confirmed by western blot and enzyme linked immunosorbent assay (ELISA), and the relationship between the biomarkers and the prognosis of cerebral injury was further validated. Among the 15 differentially expressed protein spots detected in the HBOP group by Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), 3 spots corresponding to 3 different proteins (haptoglobin, serum albumin, and haemopexin) products were identified by MALDI-TOF-MS/MS. Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group (p < 0.05 vs. Con and HBOU groups). After the western blot study, only the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and enzyme linked immunosorbent assay (ELISA) analysis. The serum level of the hemopexin (HPX) at 2 h after HBO preconditioning was correlated with the infarct volume ratio after MCAO. Haemopexin may be developed as a predictive biomarker that indicated the effectiveness of a preconditioning strategy against cerebral ischaemic injury.

Isolation of new compound and neuroprotective studies from Dodonaea viscosa

ObjectiveNeurological disorders is an increasing problem in the society. Our study aims to explore the secondary metabolites and evaluate the neuroprotective effect of leave extract of Dodonaea viscosa. MethodsThe ethyl acetate extract was chromatographed and produced one new entity which was characterized by spectroscopy (IR, UV, NMR, MS) studies. This entity was named as (6S, 9R)-vomifoliol-9-β-D-glucopyranosyle (1 -3)- O-α-L-rhamnopyranoside (DVE-8) while the known entities were identified as viscosine (DVE-12), and catechin (DVE-18). The neuroprotective potential of D. viscosa leaves extract was evaluated using the transient middle cerebral artery occlusion (MCAO) method to induce focal cerebral ischemia–reperfusion injury in rats. ResultsThe levels of proinflammatory cytokines such as TNF-α, IL-6, and NF-κB gene expression along with the level of anti-apoptotic Bcl–2, BAX, and caspase–3 gene expression was evaluated. Neurological findings showed a significant reduction in the levels of pro-inflammatory cytokines (TNF-α and, IL-6; both P < 0.05). Also, the NF-κB gene expression was significantly downregulated (P < 0.05). Furthermore, the level of anti-apoptotic Bcl–2 gene expression was significantly upregulated (P < 0.05), whereas the level of BAX and, caspase–3 gene expression was significantly downregulated (P < 0.05). ConclusionThe neuroprotective effect of D. viscosa leaves extract on MCAO-induced ischemic stroke may be due to the anti-inflammation, and anti-apoptosis activities. The findings of the present study may suggest that D. viscosa leaves extract supplementation may serve as valuable adjuvant therapy for neuronal protection.

Retraction: Dexmedetomidine and Phosphocreatine Post-treatment Provides Protection against Focal Cerebral Ischemia-reperfusion Injury in Rats.

[This retracts the article DOI: 10.1267/ahc.21-00040.].

Electroacupuncture alleviates cerebral ischemia injury in rats by regulating melatonin-NLRP3 and inhibiting pyroptosis

To investigate the mechanism of electroacupuncture in alleviating cerebral ischemia injury in cerebral ischemia-reperfusion rats by regulating melatonin - NOD-like receptor protein 3 (NLRP3) mediated pyroptosis. A total of 48 SD rats were randomly divided into sham operation group, model group, electroacupuncture (EA) group and EA +Luz group, with 12 rats in each group. The focal cerebral ischemia-reperfusion injury model was established by middle cerebral artery embolization. Rats of the EA group was treated with EA stimulation (4 Hz/20 Hz, 0.5 mA,20 min) at "Baihui" (GV20) and "Shenting" (GV24) once a day for 7 consecutive days; rats of EA+Luz group were given the same EA treatment and intraperitoneally administered melatonin receptor antagonist (luzindole, 30 mg/kg), once a day for 7 consecutive days. The neurological impairment was evaluated by Zea Longa score. The level of serum melatonin content at 12:00 and 24:00 was detected by ELISA. The percentage of cerebral infarction volume was evaluated by MRI of small animals. The apoptosis rate of nerve cells in cerebral cortex of infarct side was detected by TUNEL staining. The activation of microglia cells was detected by immunofluorescence staining. The expression levels of pyroptosis-related proteins NLRP3, Caspase-1 and interleukin (IL) -1β were detected by Western blot. Compared with the sham operation group, the neural function score was significantly increased (P<0.01); the melatonin content was significantly decreased at 24:00 (P<0.01); the percentage of cerebral infarction volume, apoptosis rate of nerve cells in cerebral cortex area of infarction side, the expressions of NLRP3, Caspase-1 and IL-1β proteins were significantly increased (P<0.01); and microglia cells were significantly activated in the model group.Compared with the model and EA +Luz groups, the nerve function score was significantly decreased (P<0.05); the percentage of cerebral infarction volume, the nerve cell apoptosis rate, the activation level of microglia cells, the expression levels of NLRP3, Caspase-1 and IL-1β were significantly decreased (P<0.01, P<0.05) in the EA group. Compared with the model and EA+Luz groups, the melatonin content at 24:00 was significantly increased (P<0.01, P<0.05) in the EA group. EA at GV20 and GV24 can reduce the neurolo-gical injury in cerebral ischemia reperfusion model rats, which may be related to regulating the expression of endogenous melatonin, inhibiting cell scorchification and reducing cerebral ischemia injury.

Electroacupuncture reverses endothelial cell death and promotes angiogenesis through the VEGF/Notch signaling pathway after focal cerebral ischemia-reperfusion injury.

Angiogenesis is an important mechanism of recovery from ischemic stroke. Recent studies have found that there is a close relationship between the VEGF/Notch pathway and angiogenesis. It is unknown whether EA can exert a brain protection effect and promote angiogenesis by acting on the VEGF/Notch signaling pathway after focal cerebral ischemia-reperfusion injury (CIRI). The Middle Cerebral Artery occlusion/Reperfusion (MCAo/R) model was established, in which rats were subjected to occlusion with ischemic intervention for 30min, followed by reperfusion for 8h, 1 day, 3 days, and 7 days. The first EA treatment was performed 90 min after the animal model was successfully established, and then EA treatments were performed once a day for 7 days. The 2,3,5-triphenyltetrazolium chloride staining and neurological deficit examination were performed to assess the level of CIRI and neuroprotection by EA. Expression levels of VEGFA, Notch1, and Hes1 proteins were measured via western blotting, while the morphological changes of ECs and microvasculature in the cortex were determined using an ultrastructural observation method. EA treatment of PC6, GV26, and SP6 can significantly improve the neurological function of MCAO/R rats, reduce the volume of cerebral infarction, and modulate the ultrastructure of ECs and microvessels in pathological states. Western blotting revealed that EA increased VEGFA protein expression at 8h and 3 days after CIRI, as well as Notch1 protein expression at 1 and 7 days. Subsequently, EA activated the VEGF/Notch pathway, increasing the expression of the downstream target protein Hes1, reversing EC death, and promoting angiogenesis. Our findings showed that EA plays a role in promoting angiogenesis following focal CIRI, and we hypothesized that this was due to the regulation of ECs by the EA-activated VEGF/Notch signaling pathway.

Effect of lipoxin A4 methyl ester from arachidonic acid on JAK2/STAT3 pathway after cerebral ischemia-reperfusion injury

It was determined if lipoxin A4 methyl ester (LXA4 ME) affects JAK2/STAT3 pathway following transient focal cerebral ischemia-reperfusion injury (CIRI) in rats. Adult male SD rats were randomly assigned to sham-operated, CIRI group, DMSO + CIRI, AG490 + CIRI, and LXA4 ME + CIRI. Neurologic deficit was evaluated in rats after 24 h reperfusion. IL-6 levels of brain tissue were determined 6 and 24 h post-reperfusion using an ELISA assay. STAT3 and SOCS3 mRNA levels were analyzed via real-time quantitative PCR. P-JAK2, p-STAT3, and SOCS3 were measured via Western blotting, and localized by fluorescent immunohitochemistry. Transient cerebral ischemia in rats caused a significantly elevated concentration of IL-6, levels of STAT3 and SOCS3 mRNA, phosphorylation of JAK2 and STAT3, and immunoreactive glia of ipsilateral cortex 24 h post-reperfusion versus sham rats. The AG490 intervention downregulated cerebral ischemia-induced JAK2 and STAT3 phosphorylation, and their immunoreactive glia located in the ischemic cortex. LXA4 ME reduced the concentration of IL-6, increased SOCS3 expression, inhibited phosphorylation and immunoreactive glia of JAK2 and STAT3, as well as ameliorated neurologic dysfunction. In conclusions, LXA4 ME inhibited JAK2/STAT3 activation, perhaps via upregulating SOCS3, as well as suppressed IL-6 expression to ameliorate cerebral ischemic injury.

Acupuncture combined with exercise training at different time points on nerve repair of cerebral ischemia-reperfusion injury in rats and its effects on the expressions of Nestin, bFGF and EGF.

The aim of the study was to observe the neuroreparative effect of electroacupuncture in rats with cerebral ischemia-reperfusion injury, and to explore the difference in the therapeutic effect of acupuncture on different acupoint groups after cerebral ischemia-reperfusion. Experimental rats were randomly divided into: sham operation group, model group, electroacupuncture group, rehabilitation group, and Diankang group (electroacupuncture + rehabilitation training). There were 24 rats in each group, and the focal cerebral ischemia-reperfusion model was established by Zea-Longa suture method. After modeling, it took 4 hours to electroacupuncture at Baihui and Dazhui points, which was used to observe the changes of nerve function in rats with signs of keel nerve function defect. Protein expression was detected by immunohistochemistry. Compared with the model group, the EA 3d, 7d, 10d groups and the rehabilitation group had no significant difference in promoting the expression of Nestin (p>0.05). There was a significant difference (p<0.01). After cerebral ischemia-reperfusion injury, the expression of bFGF and EGF on the ischemic side was stronger. The peak of bFGF expression appeared earlier, and the peak of EGF expression appeared later. The expression of bFGF and EGF in cerebral ischemic cortex at different time points of ischemia in electroacupuncture group, rehabilitation group and Diankang group was increased, and the response was enhanced. The effect of Diankang group on the upregulation of bFGF and EGF was more significant (p<0.01, p<0.05). Under the influence of different effects, Diankang is superior to simple treatment in improving ischemic neurological dysfunction. This may be related to the fact that Diankang can promote the proliferation of neural stem cells and the expression of neurotrophic factors on the ischemic side of the rat brain.

Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.

Dexmedetomidine treatment prevents cerebral ischemic reperfusion injury through HIF-1α/Beclin1-mediated autophagy

ABSTRACT Objective Cerebral ischemic reperfusion injury (CIRI) is a common cerebrovascular disorder with high disability and morbidity that threatens human health. Former investigations found that dexmedetomidine (DEX) has a protective effect against CIRI, but regulatory mechanism is unclear. Methods The current study utilized C57BL/6 mice to establish a focal cerebral ischemic reperfusion model. Cerebral infarct volume was defined by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. BV2 cells and primary neurons were utilized for molecular mechanism studies after treatment with DEX or autophagy inhibitor 3-Methyladenine (3-Ma). Results Data revealed that DEX pretreatment protected nerves against CIRI. In vitro studies also found that DEX pretreatment enhanced microglial M2 polarization and protected against neuronal apoptosis by autophagy activation. Downregulation of hypoxia inducible factor (HIF)-1α or Beclin-1 inhibited the promotional effects of DEX on microglial M2 polarization and inhibited the protective effects of DEX against neuronal apoptosis. Conclusion The present study found that DEX treatment protects against CIRI by modulating microglial polarization via HIF-1α/Beclin1-mediated autophagy.

Transcutaneous Electrical Acupoint Stimulation Pretreatment Alleviates Cerebral Ischemia-Reperfusion Injury in Rats by Modulating Microglia Polarization and Neuroinflammation Through Nrf2/HO-1 Signaling Pathway.

Cerebral ischemia-reperfusion injury (CIRI) may lead to severe disability even death, but the strategies for prevention and treatment are still limited. Transcutaneous electrical acupoint stimulation (TEAS) has been reported to have a significant neuroprotection against CIRI, but the underlying mechanisms remain obscure. In this study, we established a focal cerebral ischemia-reperfusion model in male Sprague-Dawley rats. TEAS pretreatment was applied to Baihui (GV20), Sanyinjiao (SP6) and Zusanli (ST36) acupoints for 5 consecutive days before CIRI. After 24h reperfusion, the brain damage was assessed using Zea-Longa score, brain water content (BWC) and infarct volume. Meanwhile, the number of activated microglia and the TNF-α were detected by immunofluorescence and ELISA respectively. Moreover, Western Blot and RT-qPCR were conducted to detect the proteins and mRNA expressions of Nrf2, HO-1, iNOS and Arg-1. We found that TEAS pretreatment significantly reduced Longa score, BWC, infarct volume and the number of activated microglia. Besides, TEAS pretreatment increased Nrf2 and HO-1 levels, while lowered the expression of TNF-α. Subsequently, we also discovered that the microglia M1 phenotype maker iNOS decreased and the M2 maker Arg-1 increased after TEAS pretreatment. However, these effects of TEAS pretreatment were markedly eliminated by brusatol. These findings clearly suggested that TEAS pretreatment exerted neuroprotection against CIRI, which might be related to modulating microglia polarization and neuroinflammation via Nrf2/HO-1 pathway.

Effect of Diammonium Glycyrrhizinate in Improving Focal Cerebral Ischemia-Reperfusion Injury in Rats Through Multiple Mechanisms.

Acute ischemic stroke is a current major disabling and killer disease worldwide. We aimed to investigate the protective effect and mechanism of diammonium glycyrrhizinate in alleviating acute ischemic stroke. Ninety male Sprague Dawley (SD) rats (weighing 250-300g) were randomly allocated into three groups: sham operation group (sham group), diammonium glycyrrhizinate group (DG group) and model group (model group) each with 30 individuals. A rat model of focal CIR injury was established by reversible middle cerebral artery occlusion. Zea-Longa scores for the rats in the DG group and model group were 7-fold and 8-fold higher than those of the sham group 2 h post-surgery (2.90 ± 0.99 vs. 0.30 ± 0.53, P < .05; 2.80 ± 0.61 vs. 0.30 ± 0.53, P < .05, respectively). Three days after model establishment, the scores of DG group were 26.92% lower compared with those of the model group (1.90 ± 0.76 vs. 2.60 ± 0.62, P < .05). In addition, compared with the sham group, the number of Nissl bodies and Akt-positive cells in were 27.35% and 30.42% lower in the hippocampus of the DG group (Nissl bodies: 83.40 ± 7.01 vs. 115.60 ± 11.97, p < 0.05; Akt-positive cells: 94.70 ± 8.23 vs. 136.10 ± 10.37, P < .05) and 58.65% and 57.31% lower in the model group (Nissl bodies: 47.80 ± 4.91 vs. 115.60 ± 11.97, P < .05; Akt-positive cells: 58.10 ± 4.98 vs. 136.10 ± 10.37, P < 0.05), respectively. However, the number of Nissl bodies and Akt-positive cells in the hippocampus of DG group were 74.48% and 62.9% higher compared with the model group, respectively (Nissl bodies: 83.40 ± 7.01 vs. 47.80 ± 4, P < 0.05; Akt-positive cells: 94.70 ± 8.23 vs. 58.10 ± 4.98, P < .05). In addition, compared with the sham group, the number of caspase-3-positive cells, the expression level of p38 mitogen-activated protein kinase (MAPK) and the expression of matrix metallopeptidase 9 (MMP-9) were 2-fold, 34.38%, 64.78% higher in the DG group (caspase-3-positive cells: 78.70 ± 6.52 vs. 27.10 ±3.00, P < .05; p-38MAPK: 0.43 ± 0.15 vs. 0.32 ± 0.10, P < .05; MMP-9: 14.83 ± 1.18 vs. 9.00 ± 2.05, P < .05, respectively), and more than 3-fold, 1-fold and 1-fold higher in model group (caspase-3-positive cells: 121.10 ± 11.04 vs. 27.10 ± 3.00, P < .05; p-38MAPK: 0.70 ± 0.12 vs. 0.32 ± 0.10, P < .05; MMP-9: 19.00 ± 1.90 vs. 9.00 ± 2.05, P < .05), respectively. However, the number of caspase-3-positive cells and the expression levels of p-38MAPK and MMP-9 were 35.01%, 38.57% and 28.12% lower in DG group compared with the model group (caspase-3-positive cells: 78.70 ± 6.52 vs. 121.10 ± 11.04, P < .05; p-38MAPK: 0.43 ± 0.15 vs. 0.70 ± 0.12, P < .05; MMP-9: 14.83 ± 1.18 vs. 19.00 ± 1.90, P < .05). Our study showed that diammonium glycyrrhizinate at 20mg/kg/day had a protective effect on cerebral ischemia-reperfusion injury in rats by promoting formation of Nissl bodies and increasing protein expression of Akt while decreasing that of caspase-3, p38 MAPK and MMP-9, either directly or indirectly, by inhibiting apoptosis and reducing neuroinflammation. All these mechanisms resulted in improved overall neurological function.

Enhanced autophagy interacting proteins negatively correlated with the activation of apoptosis-related caspase family proteins after focal ischemic stroke of young rats

BackgroundNeuronal injury induced in young rats by cerebral ischemia reperfusion (CIR) is known to differ substantially from that in adult rats. In the present study, we investigated the specific differences in neuronal injury induced by focal CIR between young and adult rats.Results2, 3, 5-triphenyl tetrazolium chloride (TTC) staining revealed a gradual increase in the infarct volume of both young and adult rats in accordance with I/R times and was significantly lower in young rats than in adult rats under the same conditions. The number of cells in the cortex showing immunoreactivity for neuronal nuclei (NeuN) gradually decreased in both young and adult rats in accordance with I/R times; these numbers were significantly higher in young rats than in adult rats under the same conditions. Similarly, as the duration of I/R increased, the degree of glial activation in the cortex penumbra region became more severe in both young and adult groups; however, glial activation was significantly lower in the cortex penumbra region of young rats when compared with that in adult rats. In addition, the expression of Beclin-1 was significantly higher in the infarct penumbra of young rats than adult rats and was more frequently co-expressed with neurons. The levels of autophagy-related proteins increased significantly in the penumbra region after I/R in both young and adult groups, this increase was more pronounced in young rats than in adult rats. Following CIR, analysis revealed significantly lower levels of pro-apoptosis-related factors and significantly higher levels of anti-apoptosis-related proteins in the young rats than in adult rats.ConclusionsCollectively, the present results suggest that the the reduced levels of neuronal death after CIR in young rats were closely related to enhanced levels of autophagy and reduced levels of pro-apoptosis in neurons.

Mitochondrial surface coating with artificial lipid membrane improves the transfer efficacy

Extracellular mitochondria are present and act as non-cell-autonomous signals to support energetic homeostasis. While mitochondria allograft is a promising approach in rescuing neurons, glia, and vascular cells in CNS injury and disease, there are profound limitations in cellular uptake of mitochondria together with the efficacy. Here, we modified mitochondria by coating them with cationic DOTAP mixed with DOPE via a modified inverted emulsion method to improve mitochondrial transfer and efficacy. We initially optimized the method using control microbeads and liposomes followed by using mitochondria isolated from intact cerebral cortex of male adult C57BL/6J mice. After the coating process, FACS analysis indicated that approximately 86% of mitochondria were covered by DOTAP/DOPE membrane. Moreover, the artificial membrane-coated mitochondria (AM-mito) shifted the zeta-potential toward positive surface charge, confirming successful coating of isolated mitochondria. Mitochondrial proteins (TOM40, ATP5a, ACADM, HSP60, COX IV) and membrane potentials were well maintained in AM-mito. Importantly, the coating improved mitochondrial internalization and neuroprotection in cultured neurons. Furthermore, intravenous infusion of AM-mito immediately after focal cerebral ischemia-reperfusion amplified cerebroprotection in vivo. Collectively, these findings indicate that mitochondrial surface coating with artificial lipid membrane is feasible and may improve the therapeutic efficacy of mitochondria allograft.