Rat Model Of Cerebral Artery Occlusion Research Articles

Electro-scalp acupuncture regulates the expression of CYP27a1/b1, CYP24a and related inflammatory cytokines in ischemic cortex of rats with middle cerebral artery occlusion.

To observe the effect of electro-scalp acupuncture (ESA) on the expression of cytochrome P450a1/b1 (CYP27a1/b1), cytochrome P45024a (CYP24a), signal transducer and activator of transcription (STAT)4, STAT6, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-4 in ischemic cerebral cortex of rats with acute ischemic stroke, so as to explore its mechanism in alleviating inflammatory reaction of ischemic stroke. Sixty SD rats were randomly divided into sham-operation, model, vitamin D3 and ESA groups, with 15 rats in each group. The middle cerebral artery occlusion rat model was established with thread ligation according to Zea-Longa's method. Rats in the vitamin D3 group were given 1, 25-VitD3 solution (3 ng·100 g-1·d-1) by gavage, once daily for 7 days. Rats in the ESA group were treated at bilateral anterior parietotemporal slash (MS6) with ESA (2 Hz/100 Hz, 1 mA), 30 min a day for 7 days. Before and after interventions, the neurological deficit score and neurobehavioral score were evaluated. TTC staining was used to detect the volume of cerebral infarction in rats. The positive expressions of CYP24a, CYP27a1 and CYP27b1 in the cerebral cortex of ischemic area were detected by immunofluorescence. The mRNA expressions of STAT4 and STAT6 in the cerebral cortex of ischemic area were detected by quantitative real-time PCR. The protein expression levels of TNF-α, IL-1β and IL-4 in the cerebral cortex of ischemic area were detected by Western blot. Compared with the sham-operation group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1β in cerebral cortex were increased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA, protein expression level of IL-4 were decreased (P<0.01) in the model group. After the treatment and compared with the model group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1β in cerebral cortex were decreased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA expression level, protein expression level of IL-4 were increased (P<0.01) in the ESA and vitamin D3 groups. ESA can alleviate the inflammatory response in ischemic stroke, which maybe related to its function in regulating the balance between CYP27a1/b1 and CYP24a, converting vitamin D into active vitamin D3, inhibiting vitamin D3 degradation, and regulating Th1/Th2 balance.

MSC Promotes the Secretion of Exosomal lncRNA KLF3-AS1 to Regulate Sphk1 Through YY1-Musashi-1 Axis and Improve Cerebral Ischemia-Reperfusion Injury.

Stroke remains the 3rd leading cause of long-term disability globally. Over the past decade, mesenchymal stem cell (MSC) transplantation has been proven as an effective therapy for ischemic stroke. However, the mechanism of MSC-derived exosomal lncRNAs during cerebral ischemia/reperfusion (I/R) remains ambiguous. The oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion (MCAO) rat model were generated. MSCs were isolated and characterized by flow cytometry and histochemical staining, and MSC exosomes were purified and characterized by transmission electron microscopy, flow cytometry and Western blot. Western blot, RT-qPCR and ELISA assay were employed to examine the expression or secretion of key molecules. CCK-8 and TUNEL assays were used to assess cell viability and apoptosis. RNA immunoprecipitation and RNA pull-down were used to investigate the direct association between krüppel-like factor 3 antisense RNA 1 (KLF3-AS1) and musashi-1(MSI1). Yin Yang 1 (YY1)-mediated transcriptional regulation was assessed by chromatin immunoprecipitation and luciferase assays. The histological changes and immunoreactivity of key molecules in brain tissues were examined by H&E and immunohistochemistry. MSCs were successfully isolated and exhibited directionally differential potentials. MSC exosomal KLF3-AS1 alleviated OGD/R-induced inflammation in SK-N-SH and SH-SY5Y cells via modulating Sphk1. Mechanistical studies showed that MSI1 positively regulated KLF3-AS1 expression through its direct binding to KLF3-AS1. YY1 was identified as a transcription activator of MSI1 in MSCs. Functionally, YY1/MSI1 axis regulated the release of MSC exosomal KLF3-AS1 to modulate sphingosine kinase 1 (Sphk1)/NF-κB pathway, thereby ameliorating OGD/R- or cerebral I/R-induced injury. MSCs promote the release of exosomal KLF3-AS1 to regulate Sphk1 through YY1/MSI axis and improve cerebral I/R injury.

High-frequency rTMS alleviates cognitive impairment and regulates synaptic plasticity in the hippocampus of rats with cerebral ischemia

Poststroke cognitive impairment (PSCI) is a common complication of stroke, but effective treatments are currently lacking. Repetitive transcranial magnetic stimulation (rTMS) is gradually being applied to treat PSCI, but there is limited evidence of its efficacy. To determine rTMS effects on PSCI, we constructed a transient middle cerebral artery occlusion (tMCAO) rat model. Rats were then grouped by random digital table method: the sham group (n = 10), tMCAO group (n = 10) and rTMS group (n = 10). The shuttle box and Morris water maze (MWM) tests were conducted to detect the cognitive functions of the rats. In addition, synaptic density and synaptic ultrastructural parameters, including the active zone length, synaptic cleft width, and postsynaptic density (PSD) thickness, were quantified and analyzed using an electron microscope. What’s more, synaptic associated proteins, including PSD95, SYN, and BDNF were detected by western blot. According to the shuttle box and MWM tests, rTMS improved tMCAO rats’ cognitive functions, including spatial learning and memory and decision-making abilities. Electron microscopy revealed that rTMS significantly increased the synaptic density, synaptic active zone length and PSD thickness and decreased the synaptic cleft width. The western blot results showed that the expression of PSD95, SYN, and BDNF was markedly increased after rTMS stimulation. Based on these results, we propose that 20 Hz rTMS can significantly alleviate cognitive impairment after stroke. The underlying mechanism might be modulating the synaptic plasticity and up-regulating the expression PSD95, SYN, and BDNF in the hippocampus.

Ecdysterone improves oxidative damage induced by acute ischemic stroke via inhibiting ferroptosis in neurons through ACSL4

Ethnopharmacological relevanceAcute ischemic stroke (AIS) is a prominent cause of disability and mortality around the world. Achyranthes bidentata Blume, a regularly prescribed traditional Chinese herb, plays a significant role in traditional Chinese stroke therapy due to its ability to promote blood circulation and remove stasis. Ecdysterone (EDS) is one of the key active components in Achyranthes bidentata Blume, which exhibits antioxidant and anti-cerebral hypoxia properties. However, whether EDS improves AIS and the mechanism of action of AIS is still unclear. Aim of the studyThe objective of this study was to observe whether EDS ameliorates oxidative damage caused by AIS by inhibiting ferroptosis in neurons via ACSL4. Materials and methodsIn vivo, the Middle cerebral artery occlusion (MCAO) rat model was established for research. After treatment with EDS, Neurologic score, TTC, HE and FJC staining were performed, followed by measurements of oxidative stress-related indicators, the content of Fe2+, iron deposition levels and expression of ACSL4, NCOA4 and FTH1 in brain tissue. In vitro, oxygen-glucose deprivation and reperfusion (OGD/R) cell model was established. After treatment with EDS, cell viability, oxidative stress-related indicators, the content of Fe2+ and expression of ACSL4, NCOA4 and FTH1 were detected. In addition, the overexpression of ACSL4 and CETSA technology further elucidated that EDS improves AIS through ACSL4. ResultsThe results showed that the treatment of EDS could improve the oxidative damage of MCAO rats by inhibiting ferroptosis, and then improve AIS. Importantly, EDS inhibited ferroptosis via ACSL4, thereby inhibiting oxidative stress in MCAO rats or OGD/R-induced PC12 cells. ConclusionsThese results provide evidence that EDS ameliorates oxidative damage caused by AIS by inhibiting ferroptosis via ACSL4, and provide new insights into the potential use of EDS as an effective drug development candidate for AIS.

Luteolin-7-O-β-d-glucuronide Ameliorates Cerebral Ischemic Injury: Involvement of RIP3/MLKL Signaling Pathway.

Luteolin-7-O-β-d-glucuronide (LGU) is a major active flavonoid glycoside compound that is extracted from Ixeris sonchifolia (Bge.) Hance, and it is a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the neuroprotective effect of LGU was investigated in an oxygen glucose deprivation (OGD) model and a middle cerebral artery occlusion (MCAO) rat model. In vitro, LGU was found to effectively improve the OGD-induced decrease in neuronal viability and increase in neuronal death by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) leakage rate assay, respectively. LGU was also found to inhibit OGD-induced intracellular Ca2+ overload, adenosine triphosphate (ATP) depletion, and mitochondrial membrane potential (MMP) decrease. By Western blotting analysis, LGU significantly inhibited the OGD-induced increase in expressions of receptor-interacting serine/threonine-protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). Moreover, molecular docking analysis showed that LGU might bind to RIP3 more stably and firmly than the RIP3 inhibitor GSK872. Immunofluorescence combined with confocal laser analyses disclosed that LGU inhibited the aggregation of MLKL to the nucleus. Our results suggest that LGU ameliorates OGD-induced rat primary cortical neuronal injury via the regulation of the RIP3/MLKL signaling pathway in vitro. In vivo, LGU was proven, for the first time, to protect the cerebral ischemia in a rat middle cerebral artery occlusion (MCAO) model, as shown by improved neurological deficit scores, infarction volume rate, and brain water content rate. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia.

Salidroside derivative SHPL-49 attenuates glutamate excitotoxicity in acute ischemic stroke via promoting NR2A-CAMKⅡα-Akt /CREB pathway

BackgroundIschemic stroke is a significant cause of death and disability with a limited treatment time window. The reduction of early glutamate excitotoxicity using neuroprotective agents targeting N-methyl-D-aspartic acid (NMDA) receptors have attracted recent research attention. SHPL-49, a structurally modified derivative of salidroside, was synthesized by our team. Previous studies have confirmed the neuroprotective efficacy of SHPL-49 in rats with ischemic stroke. However, the underlying mechanisms need to be clarified. MethodsWe conducted in vivo experiments using the permanent middle cerebral artery occlusion rat model to investigate the role of SHPL-49 in glutamate release at different time points and treatment durations. Glutamate transporters and receptor proteins and neural survival proteins in the brain were also examined at the same time points. In vitro, primary neurons and the coculture system of primary neurons–astrocytes were subjected to oxygen–glucose deprivation and glutamate injury. Proteomics and parallel reaction monitoring analyses were performed to identify potential therapeutic targets of SHPL-49, which were further confirmed through in vitro experiments on the inhibition and mutation of the target. ResultsSHPL-49 significantly reduced glutamate release caused by hypoxia–ischemia. One therapeutic pathway of SHPL-49 was promoting the expression of glutamate transporter-1 to increase glutamate reuptake and further reduce the occurrence of subsequent neurotoxicity. In addition, we explored the therapeutic targets of SHPL-49 and its regulatory effects on glutamate receptors for the first time. SHPL-49 enhanced neuroprotection by activating the NMDA subunit NR2A, which upregulated the cyclic-AMP response binding protein (CREB) neural survival pathway and Akt phosphorylation. Since calcium/calmodulin-dependent kinase IIα (CaMKIIα) is necessary for synaptic transmission of NMDA receptors, we explored the interaction between CaMKIIα and SHPL-49, which protected CaMKIIα from hypoxia–ischemia-induced autophosphorylation damage. ConclusionOverall, SHPL-49 enhanced neuronal survival and attenuated acute ischemic stroke by promoting the NR2A–CAMKⅡα–Akt/CREB pathway. Our study provides the first evidence demonstrating that the neuroprotective effect of SHPL-49 is achieved by promoting the NR2A subunit to extend the treatment time window, making it a promising drug for ischemic stroke.

METTL3 Mediates Microglial Activation and Blood-Brain Barrier Permeability in Cerebral Ischemic Stroke by Regulating NLRP3 Inflammasomes Through m6A Methylation Modification.

Cerebral ischemic stroke (CIS) is the main cause of disability. METTL3 is implicated in CIS, and we explored its specific mechanism. Middle cerebral artery occlusion (MCAO) rat model and oxygen-glucose deprivation/reperfusion (OGD/R) HAPI cell model were established and treated with LV-METTL3 or DAA, oe-METTL3, miR-335-3p mimics, or DAA, to assess their effects on MCAO rat neurological and motor function, cerebral infarction area, brain water content, microglial activation, blood-brain barrier (BBB) permeability, and NLRP3 inflammasome activation. METTL3, pri-miR-335-3p, mature miR-335-3p, and miR-335-3p mRNA levels were assessed by RT-qPCR; M1/M2 microglial phenotype proportion and M1/M2 microglia ratio, inflammatory factor levels, and m6A modification were assessed. MCAO rats manifested cerebral ischemia injury. METTL3 was under-expressed in CIS. METTL3 overexpression inhibited microglial activation and M1 polarization and BBB permeability in MCAO rats and inhibited OGD/R-induced microglial activation and reduced M1 polarization. METTL3 regulated miR-335-3p expression and inhibited NLRP3 inflammasome activation. m6A methylation inhibition averted METTL3's effects on NLRP3 activation, thus promoting microglial activation in OGD/R-induced cells and METTL3's effects on BBB permeability in MCAO rats. Briefly, METTL3 regulated miR-335-3p expression through RNA m6A methylation and inhibited NLRP3 inflammasome activation, thus repressing microglial activation, BBB permeability, and protecting against CIS.

Maintaining moderate levels of hypochlorous acid promotes neural stem cell proliferation and differentiation in the recovery phase of stroke

Abstract It has been shown clinically that continuous removal of ischemia/reperfusion-induced reactive oxygen species is not conducive to the recovery of late stroke. Indeed, previous studies have shown that excessive increases in hypochlorous acid after stroke can cause severe damage to brain tissue. Our previous studies have found that a small amount of hypochlorous acid still exists in the later stage of stroke, but its specific role and mechanism are currently unclear. To simulate stroke in vivo, a middle cerebral artery occlusion rat model was established, with an oxygen-glucose deprivation/reoxygenation model established in vitro to mimic stroke. We found that in the early stage (within 24 hours) of ischemic stroke, neutrophils produced a large amount of hypochlorous acid, while in the recovery phase (10 days after stroke), microglia were activated and produced a small amount of hypochlorous acid. Further, in acute stroke in rats, hypochlorous acid production was prevented using a hypochlorous acid scavenger, taurine, or myeloperoxidase inhibitor, 4-aminobenzoic acid hydrazide. Our results showed that high levels of hypochlorous acid (200 μM) induced neuronal apoptosis after oxygen/glucose deprivation/reoxygenation. However, in the recovery phase of the middle cerebral artery occlusion model, a moderate level of hypochlorous acid promoted the proliferation and differentiation of neural stem cells into neurons and astrocytes. This suggests that hypochlorous acid plays different roles at different phases of cerebral ischemia/reperfusion injury. Lower levels of hypochlorous acid (5 and 100 μM) promoted nuclear translocation of β-catenin. By transfection of single-site mutation plasmids, we found that hypochlorous acid induced chlorination of the β-catenin tyrosine 30 residue, which promoted nuclear translocation. Altogether, our study indicates that maintaining low levels of hypochlorous acid plays a key role in the recovery of neurological function.

Uncovering Mechanism and Efficacy of Salvia Miltiorrhiza-Safflower in Cerebral Ischemia-Reperfusion Injury

Cerebral ischemia (CI) is the main cause of stroke morbidity and disability. This study aims to identify the early molecular regulation responsible for the therapeutic effectiveness of the Herb pair Danshen-Honghua (DH) for CI. The major targets of DH were identified by searching the public database of traditional Chinese medicine (TCM). In addition, GeneCards, Disgenet, and GeneMap databases in OMIM were used to determine the disease targets of CI. A total of 88 common targets of DH and CI were selected, a protein–protein interaction (PPI) network was established by Cytoscape, and 19 core targets were screened. These genes were primarily enriched in biological processes including wound healing, reaction to oxidative stress, and response to peptides, lipid and atherosclerosis, Age-rage signaling pathway, and TNF signaling pathway by KEGG and GO enrichments. The effective components of DH had stable binding to these key targets by molecular docking. Finally, it was verified that the mechanism of DH on CI treatment may be related to the activation of the TNF-α/JNK signaling pathway by establishing the middle cerebral artery occlusion (MCAO) rat model.

Modulation of muscarinic receptors by anisodine hydrobromide in cerebral ischemia.

Ischemic cerebrovascular diseases pose significant challenges due to their high mortality, disability rates, and recurrence risk, imposing substantial societal and healthcare burdens. Current treatment modalities, including medication and surgical interventions, have limitations. This study explores the therapeutic potential of anisodine hydrobromide, a neuroprotective compound, with a focus on its interaction with muscarinic receptors (M1-M5) in cerebral ischemic diseases, employing a middle cerebral artery occlusion (MCAO) rat model, and microglial HM cells and astrocytes SVG12 as models. Immunohistochemistry comprehensively assessed M1-M5 receptor expression in cerebral arteries, hippocampus, and parenchymal tissues in MCAO rats before and after anisodine hydrobromide administration. Additionally, a hypoxia/reoxygenation (H/R) model validated our findings using SVG12 and HM cells. M receptor mechanisms under hypoxia, including calcium ion influx, reactive oxygen species (ROS) levels, and aspartate expression were explored. Anisodine hydrobromide effectively reduced exacerbated M1, M2, M4, and M5 receptor expression in hypoxia/reoxygenation (H/R)-treated brain tissues and M2 receptors in H/R-treated cells. Concentration-dependent inhibition of calcium ion influx and ROS levels was observed, elucidating its neuroprotective mechanisms. Under H/R conditions, HM cells exhibited decreased aspartate levels by anisodine hydrobromide, Atropine, and M2 inhibitor treatments. These findings shed light on the modulation of muscarinic receptors, particularly the M2 subtype, by anisodine hydrobromide in cerebral ischemia. The neuroprotective effects observed in this study highlight the promising clinical prospects of anisodine hydrobromide as a potential therapeutic agent for ischemic brain diseases, warranting further investigation into its mechanisms of action.

The brain protection of MLKL inhibitor necrosulfonamide against focal ischemia/reperfusion injury associating with blocking the nucleus and nuclear envelope translocation of MLKL and RIP3K.

Mixed lineage kinase like protein (MLKL) is a key mediator of necroptosis. While previous studies highlighted the important role of MLKL as one of the central regulators of brain damage against acute ischemic neuronal injury, how the activation of MLKL mediates brain injuries and cell death remains unclear, especially in astrocytes. In a transient middle cerebral artery occlusion (tMCAO) rat model in vivo, and an oxygen-glucose deprivation and reoxygenation (OGD/Re) injury model in both primary cultured astrocytes and human astrocytes, we show that necrosulfonamide (NSA), a MLKL specific inhibitor, reduces infarction volume and improves neurological deficits in tMCAO-treated rats. In addition, NSA treatment, as well as RIP1K inhibitor Nec-1 or RIP3K inhibitor GSK-872 treatment, decreases the OGD/Re-induced leakage of LDH in both primary cultured astrocytes and human astrocytes. NSA treatment also reduces the number of propidium iodide (PI)-positive cells, and prevents the upregulation of necroptotic biomarkers such as MLKL/p-MLKL, RIP3K/p-RIP3K, and RIP1K/p-RIP1K in ischemic penumbra of cerebral cortex in tMCAO-treated rats or in OGD/Re-treated human astrocytes. Importantly, NSA treatment blocks both the nucleus and nuclear envelope localization of MLKL/p-MLKL and RIP3K/p-RIP3K in ischemic cerebral cortex induced by tMCAO. Similarly, Co-immunoprecipitation assay shows that NSA treatment decreases tMCAO- or OGD/Re- induced increased combination of MLKL and RIP3K in nuclear envelope of ischemic penumbra of cerebral cortex or of primary cultured astrocytes, respectively. RIP3K inhibitor GSK-872 also reduces tMCAO-induced increased combination of MLKL and RIP3K in nuclear envelope of ischemic penumbra of cerebral cortex. These data suggest NSA exerts protective effects against focal ischemia/reperfusion injury via inhibiting astrocytic necroptosis through preventing the upregulation of necroptotic kinases as well as blocking both the nucleus and nuclear envelope co-localization of p-MLKL and p-RIP3K. The translocation of p-MLKL, along with p-RIP3K, to the nuclear envelope and the nucleus may play a crucial role in MLKL-mediated necroptosis under ischemic conditions.

Dynamic assessment of brain perfusion in a middle cerebral artery occlusion rat model by contrast-enhanced ultrasound imaging: a pilot study.

The middle cerebral artery occlusion model (MCAo) is a commonly used animal model for cerebral ischemia studies but lacks accessible imaging techniques for the assessment of hemodynamic changes of the model. The study aims to explore the value of contrast-enhanced ultrasound (CEUS) in evaluating brain perfusion in the early stages after MCAo surgery. In total, 18 adult male Sprague-Dawley rats were subjected to right MCAo using an intraluminal filament model, and CEUS was performed at the three following timepoints: before (T0), immediately after (T1), and 6 h after permanent MCAo (T2). Twelve rats successfully completed the study, and their brains were removed and stained using 2, 3, 5-triphenyltetrazolium chloride (TTC). CEUS video images were visualized offline, and the time-intensity curves (TICs) were analyzed. Different cerebrovascular patterns and manifestations of the contrast enhancement in rat ischemic hemispheres were observed. Semi-quantitative parameters of TICs in ischemic areas (ROIi) and the surrounding normal- or hypo-perfused areas (ROIn) were calculated and compared between T0, T1, and T2, and also between ROIi and ROIn. A significant correlation was found between the lesion volume (%) determined by TTC and CEUS parameters (r = -0.691, P = 0.013 for peak intensity; r = -0.742, P = 0.006 for area under the curve) at T2. After the same occlusion, there were differences in contrast perfusion in each group. This study suggests that CEUS could be an effective imaging tool for studying cerebral ischemia and perfusion in small animals as long as the transcranial acoustic window allows it.

BRAP silencing protects against neuronal inflammation, oxidative stress and apoptosis in cerebral ischemia-reperfusion injury by promoting PON1 expression.

BRCA1 associated protein (BRAP) participates in the regulation of myocardial infarction and atherosclerosis. But the function of BRAP in cerebral ischemia-reperfusion (CIR) injury has not been elucidated yet. BRAP expression in PC12 cells in response to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment was examined with Western blot assay. PC12 cells underwent OGD/R-treatment and were subsequently transfected with pcDNA-BRAP or sh-BRAP, followed by determination of viability, lactate dehydrogenase (LDH) production, apoptosis, inflammatory cytokine secretion, and oxidative stress marker protein levels. Paraoxonase 1 (PON1) promoter methylation was evaluated with methylation-specific PCR assay. the effect of BRAP/PON1 axis on CIR injury was investigated by rescue experiments. Additionally, sh-BRAP was injected into a middle cerebral artery occlusion (MCAO) rat model, and the changes of neurological damage were evaluated. BRAP overexpression exacerbated OGD/R-induced viability reduction, LDH production, apoptosis, inflammatory cytokine secretion and oxidative stress in PC12 neuronal cells. In contrast, BRAP silencing showed the opposite results. Mechanistically, BRAP reduced PON1 expression by promoting DNA methyl transferase1 (DNMT1)-mediated PON1 promoter methylation. PON1 silencing reversed BRAP-mediated neuroprotection. Additionally, BRAP silencing alleviated CIR-induced neurological damage in MCAO rats. BRAP silencing suppressed OGD/R-induced neuronal apoptosis, inflammation, and oxidative stress, and alleviated CIR-induced neurological damage in MCAO rats through facilitating PON1 expression.

Lithium chloride promotes neural functional recovery after local cerebral ischaemia injury in rats through Wnt signalling pathway activation.

Lithium chloride (LiCl) has a significant neuroprotective effect in cerebral ischaemia. However, to date, there is a paucity of evidence on the role of LiCl in neural restoration after brain ischaemia and the signalling pathways involved remain unclear. Therefore, to address this gap, the middle cerebral artery occlusion (MCAO) rat model was used to simulate human ischaemia stroke. Male Sprague-Dawley rats were given MCAO for 90 min followed by reperfusion, and Dickkopf-1 (DKK1, 5.0 μg/kg) was administered half an hour before MCAO. Rats were then treated with hypodermic injection of LiCl (2.0 mmol/kg) twice a day for 1 week. After treatment, cognitive impairment was assessed by the Morris water maze test. Neurological deficit score, 2,3,5-triphenyl tetrazolium chloride staining, brain water content, and histopathology were used to evaluate brain damage. Enzyme-linked immunosorbent assay was used to measure oxidative stress damage and inflammatory cytokines. Apoptosis of the hippocampal neurons was tested by western blot. The key factors of Wnt signalling pathway in the ischaemic penumbra were detected by immunofluorescence staining and quantitative real-time polymerase chain reaction. Current experimental results showed that LiCl treatment significantly improved the impaired spatial learning and memory ability, suppressed oxidative stress, inflammatory reaction, and neuron apoptosis accompanied by attenuating neuronal damage, which subsequently decreased the brain oedema, infarct volume and neurological deficit. Furthermore, the treatment of LiCl activated Wnt signalling pathway. Interestingly, the aforementioned effects of LiCl treatment were markedly reversed by administration of DKK1, an inhibitor of Wnt signalling pathway. These results indicate that LiCl exhibits neuroprotective effects in focal cerebral ischaemia by Wnt signalling pathway activation, and it might have latent clinical application for the prevention and treatment of ischaemic stroke.

Enhanced Cerebroprotection of Xenon-Loaded Liposomes in Combination with rtPA Thrombolysis for Embolic Ischemic Stroke.

Xenon (Xe) has shown great potential as a stroke treatment due to its exceptional ability to protect brain tissue without inducing side effects. We have previously developed Xe-loaded liposomes for the ultrasound-activated delivery of Xe into the cerebral region and demonstrated their therapeutic efficacy. At present, the sole FDA-approved thrombolytic agent for stroke treatment is recombinant tissue plasminogen activator (rtPA). In this study, we aimed to investigate the potential of combining Xe-liposomes with an intravenous rtPA treatment in a clinically relevant embolic rat stroke model. We evaluated the combinational effect using an in vitro clot lysis model and an in vivo embolic middle cerebral artery occlusion (eMCAO) rat model. The treatment groups received intravenous administration of Xe-liposomes (20 mg/kg) at 2 h post-stroke onset, followed by the administration of rtPA (10 mg/kg) at either 2 or 4 h after the onset. Three days after the stroke, behavioral tests were conducted, and brain sections were collected for triphenyltetrazolium chloride (TTC) and TUNEL staining. Infarct size was determined as normalized infarct volume (%). Both in vitro and in vivo clot lysis experiments demonstrated that Xe-liposomes in combination with rtPA resulted in effective clot lysis comparable to the treatment with free rtPA alone. Animals treated with Xe-liposomes in combination with rtPA showed reduced TUNEL-positive cells and demonstrated improved neurological recovery. Importantly, Xe-liposomes in combination with late rtPA treatment reduced rtPA-induced hemorrhage, attributing to the reduction of MMP9 immunoreactivity. This study demonstrates that the combined therapy of Xe-liposomes and rtPA provides enhanced therapeutic efficacy, leading to decreased neuronal cell death and a potential to mitigate hemorrhagic side effects associated with late rtPA treatment.

Comparative transcriptome findings reveal the neuroinflammatory network and potential biomarkers to early detection of ischemic stroke

IntroductionIschemic stroke accounts for 70–80% of all stroke cases, leading to over two million people dying every year. Poor diagnosis and late detection are the major causes of the high death and disability rate.MethodsIn the present study, we used the middle cerebral artery occlusion (MCAO) rat model and applied comparative transcriptomic analysis, followed by a systematic advanced bioinformatic analysis, including gene ontology enrichment analysis and Ingenuity Pathway Analysis (IPA). We aimed to identify novel biomarkers for the early detection of ischemic stroke. In addition, we aimed to delineate the molecular mechanisms underlying the development of ischemic stroke, in which we hoped to identify novel therapeutic targets for treating ischemic stroke.ResultsIn the comparative transcriptomic analysis, we identified 2657 differentially expressed genes (DEGs) in the brain tissue of the MCAO model. The gene enrichment analysis highlighted the importance of these DEGs in oxygen regulation, neural functions, and inflammatory and immune responses. We identified the elevation of angiopoietin-2 and leptin receptor as potential novel biomarkers for early detection of ischemic stroke. Furthermore, the result of IPA suggested targeting the inflammasome pathway, integrin-linked kinase signaling pathway, and Th1 signaling pathway for treating ischemic stroke.ConclusionThe results of the present study provide novel insight into the biomarkers and therapeutic targets as potential treatments of ischemic stroke.

Human Serum Albumin-enriched Clopidogrel Bisulfate Nanoparticle Alleviates Cerebral Ischemia-Reperfusion Injury in Rats.

Cerebral ischemia-reperfusion (I/R) injury remains a leading cause of mobility and mortality among patients with ischemic stroke. This study aims to develop a human serum albumin (HSA)-enriched nanoparticle platform for solubilizing clopidogrel bisulfate (CLP) for intravenous administration, and to explore the protective effect of HSA-enriched nanoparticles loaded with CLP (CLP-ANPs) against cerebral I/R injury in transient middle cerebral artery occlusion (MCAO) rat model. CLP-ANPs were synthesized via a modified nanoparticle albumin-bound technology, lyophilized, and then characterized by morphology, particle size, zeta potential, drug loading capacity, encapsulation efficiency, stability and in vitro release kinetics. In vivo pharmacokinetic studies were conducted using Sprague-Dawley (SD) rats. Also, an MCAO rat model was established to explore the therapeutic effect of CLP-ANPs on cerebral I/R injury. CLP-ANPs remained spherical particles with a layer of proteins forming protein corona. Lyophilized CLP-ANPs after dispersion displayed an average size of about 235.6 ± 6.6nm (PDI = 0.16 ± 0.08) with a zeta potential of about - 13.5 ± 1.8mV. CLP-ANPs achieved sustained release for up to 168h in vitro. Next, a single injection of CLP-ANPs dose-dependently reversed the histopathological changes induced by cerebral I/R injury possibly via attenuating apoptosis and reducing oxidative damages in the brain tissues. CLP-ANPs represent a promising and translatable platform system for the management of cerebral I/R injury during ischemic stroke.

Protective effect and underlying mechanism of muscone on acute cerebral ischemia-reperfusion injury in rats.

Musk is a widely used traditional Chinese medicine, which has resuscitation, activating blood, and disperse swelling effects. Musk is commonly used in the prevention of myocardial infarction and ischemic stroke, and muscone is its main active component. The effect and mechanism of muscone to improve the condition of ischemic stroke is not clear, accordingly, we verified its efficacy in ischemia-reperfused rats, and investigated its mechanism by PC12 and THP-1cells. A transient middle cerebral artery occlusion (tMCAO) rat model was established for in vivo experiments. 2,3,5-Triphenyl Tetrazolium Chloride (TTC) staining was used to calculate infarct rate. Neuroprotection and angiogenesis were assessed by Hematoxylin-eosin (HE) staining, nissl staining, immunofluorescence staining, and quantitative real-time PCR (qRT-PCR). Oxygen glucose deprivation-reperfusion (OGD/R) model of PC12cells was established for neuroprotection analysis, where CCK-8 assay was used to measure cell viability, flow cytometry and Hoechst 33258 staining were used to demonstrate apoptosis, and protein levels were detected by Western blot. For angiogenesis analysis, enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were used to detect angiogenic factors expressed by THP-1. Cell viability assay, scratch wound assay, and tube formation assay were used to evaluate angiogenic effect of HUVECs treated with medium of THP-1. And the angiogenic pathway in HUVECs was detected by Western blot. According to the results, in cerebral ischemia-reperfusion rats, the infarct rate and tissue damage were significantly reduced by muscone, and the expression of neurotrophic factors and angiogenesis-related factors were all elevated. In OGD/R-PC12cell models, muscone could increase cell viability and inhibit apoptosis via Bax/Bcl-2/Caspase-3 pathway. In THP-1-mediated angiogenesis of HUVECs, muscone promoted the secretion of angiogenesis-related factors in THP-1 and thus indirectly promoted the proliferation, migration and tube formation of HUVECs, and then regulated phosphorylation of VEGFR2 and Akt in HUVECs. Our study indicated that muscone may be a potential neuroprotective and proangiogenic agent in cerebral ischemia.

NLRP3 Inflammasome-Targeting Nanomicelles for Preventing Ischemia–Reperfusion-Induced Inflammatory Injury

Ischemia-reperfusion (I/R) injury is a disease process that affects several vital organs. There is widespread agreement that the NLRP3 inflammasome pathway plays a crucial role in the development of I/R injury. We have developed transferrin-conjugated, pH-responsive nanomicelles for the entrapment of MCC950 drug. These nanomicelles specifically bind to the transferrin receptor 1 (TFR1) expressed on the cells of the blood-brain barrier (BBB) and thus help the cargo to cross the BBB. Furthermore, the therapeutic potential of nanomicelles was assessed using in vitro, in ovo, and in vivo models of I/R injury. Nanomicelles were injected into the common carotid artery (CCA) of a middle cerebral artery occlusion (MCAO) rat model to achieve maximum accretion of nanomicelles into the brain as blood flows toward the brain in the CCA. The current study reveals that the treatment with nanomicelles significantly alleviates the levels of NLRP3 inflammasome biomarkers which were found to be increased in oxygen-glucose deprivation (OGD)-treated SH-SY5Y cells, the I/R-damaged right vitelline artery (RVA) of chick embryos, and the MCAO rat model. The supplementation with nanomicelles significantly enhanced the overall survival of MCAO rats. Overall, nanomicelles exerted therapeutic effects against I/R injury, which might be due to the suppression of the activation of the NLRP3 inflammasome.

Transmission of NLRP3-IL-1β Signals in Cerebral Ischemia and Reperfusion Injury: from Microglia to Adjacent Neuron and Endothelial Cells via IL-1β/IL-1R1/TRAF6.

The pyrin domain-containing protein 3 (NLRP3) inflammasome drives the profound cerebral ischemia and reperfusion injury (I/R) and mediates the secretion of IL-1β (interleukin-1β), which exerts a subsequent cascade of inflammatory injury. The NLRP3-activated-microglial manipulation in adjacent neuronal and endothelial NLRP3 activation has been confirmed in our previous studies. In the present study, we extended the cognition of how microglia mediated neuronal and endothelial NLRP3-IL-1β signaling during cerebral ischemia and reperfusion injury. In vitro, Neuro-2a and bEND3 cells were cultured alone or co-cultured with BV2 cells and oxygen-glucose deprivation/reoxygenation (OGD/R) was performed. In vivo, transient middle cerebral artery occlusion (tMCAO) rat models and lentiviral silencing targeting IL-1R1 were performed. The NLRP3 inflammasome activation was evaluated by enzyme-linked immunosorbent assay, western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence. In the co-culture system after OGD/R treatment, NLRP3 inflammasomes in neurons and endothelial cells were activated by microglial IL-1β via IL-1β/IL-1R1/TRAF6 signaling pathway, with the basal protein level of NLRP3. In addition, ruptured lysosomes engulfing ASC specks which were possibly secreted from microglia triggered the enhanced NLRP3 expression. In cortices of tMCAO rats at 24h of reperfusion, silencing IL-1R1, mainly presented in neurons and endothelial cells, was efficient to block the subsequent inflammatory damage and leukocyte brain infiltration, leading to better neurological outcome. Neuronal and endothelial NLRP3 inflammasomes were activated by microglia in cerebral ischemia and reperfusion injury mainly via IL-1β/IL-1R1/TRAF6 signaling, which might be therapeutically targetable.