Experimental Stroke Models Research Articles

The exo-microRNA (miRNA) signaling pathways in pathogenesis and treatment of stroke diseases: Emphasize on mesenchymal stem cells (MSCs).

A major factor in long-term impairment is stroke. Patients with persistent stroke and severe functional disabilities have few therapy choices. Longnoncoding RNAs (lncRNAs) may contribute to the regulation of the pathophysiologic processes of ischemic stroke as shown by altered expression of lncRNAsand microRNA (miRNAs) in blood samples of acute ischemic stroke patients. On the other hand, multipotent mesenchymal stem cells (MSCs) increase neurogenesis, and angiogenesis, dampen neuroinflammation, and boost brain plasticity to improve functional recovery in experimental stroke models. MSCs can be procured from various sources such as the bone marrow, adipose tissue, and peripheral blood. Under the proper circumstances, MSCs can differentiate into a variety of mature cells, including neurons, astrocytes, and oligodendrocytes. Accordingly, the capability of MSCs to exert neuroprotection and also neurogenesis has recently attracted more attention. Nowadays, lncRNAs and miRNAs derived from MSCs have opened new avenues to alleviate stroke symptoms. Accordingly, in this review article, we examined various studies concerning the lncRNAs and miRNAs'role in stroke pathogenesis and delivered an overview of the therapeutic role of MSC-derived miRNAs and lncRNAs in stroke conditions.

Abstract WP322: GluN2A-NMDAR Mediated Increased Neuronal Prostaglandin E2 Release and Microglial Activation Exacerbates Ischemic Brain Injury Under Hyperhomocysteinemic Condition

Hyperhomocysteinemia (Hhcy), a metabolic disorder is recognized as a risk factor for ischemic stroke. However, the impact of Hhcy on stroke outcome is not known. Using a rodent model of experimental stroke, we recently showed that predisposition to Hhcy exacerbates ischemic brain injury. The findings also showed that Hhcy triggers GluN2A subunit containing NMDA receptor (GluN2A-NMDAR) stimulation that act in conjunction with ischemia-induced pathways involving GluN2B subunit containing NMDAR (GluN2B-NMDAR) stimulation to enhance stroke pathology. The current study evaluated whether GluN2A-NMDAR-mediated increased expression of cyclooxygenase-2 (COX-2) and excessive release of the pro-inflammatory mediator prostaglandin E2 (PGE2) from neurons results in increased microglial activation and exacerbated ischemic brain injury. HHcy was induced by implantation of Alzet pumps with L-homocysteine in adult male Wistar rats. Cerebral ischemia was induced by middle cerebral artery occlusion (60 min) in control and Hhcy rats. In a subset of Hhcy rats, NVP-AAM077, a pharmacological inhibitor of GluN2A-NMDAR was injected intravenously at the onset of stroke. At specified times following reperfusion, brain tissues were processed for FluroJade staining, immunoblotting with COX2 antibody, immunohistochemistry with COX2/NeuN and Iba1 antibodies, prostaglandin E2 (PGE2) assay and RT-PCR. FluroJade staining shows an early onset and exacerbation of ischemic injury in Hhcy rats. This involves increased expression of COX-2 in neurons, release of PGE2, and a shift in microglial morphology from ramified state to active amoeboid state, 6h after reperfusion. Administration of NVP-AAM077 attenuates the increase in COX2 expression and PGE2 levels as well as microglial activation. The findings demonstrate that an early onset of neuroinflammatory response mediated through GluN2A-NMDAR stimulation could contribute to the exacerbation of ischemic brain injury under Hhcy conditions. As GluN2A-NMDAR stimulation is primarily involved in promoting synaptic plasticity, neuronal growth, and survival, establishing their role in promoting neuroinflammatory response could lead to a paradigm shift in our understanding of the consequences of NMDAR signaling.

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed invivo and exvivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction exvivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.

MiR-497-5p promoted neuronal injury in ischemic stroke by inhibiting the BDNF/TrkB/PI3K/Akt pathway.

The aim of this study was to investigate the molecular mechanism by which miR-497-5p regulates neuronal injury after ischemic stroke through the BDNF/TrkB/Akt signaling pathway. PC12 cells were used to construct a stroke injury model by oxygen-glucose deprivation/reoxygenation (OGD/R). The expression level of miR-497-5p was measured by RT-qPCR. CCK-8 kit was used to detect cell viability. Cell apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. MDA and SOD detection kits were used to detect MDA content and SOD activity. A double luciferase reporter system was used to verify the targeting relationship between miR-497-5p and BDNF. The expression of BDNF, TrkB, p-TrkB, Akt and p-Akt was detected by Western blot. We have found that miR-497-5p expression was inhibited after treatment with OGD/R. Simultaneously, cell apoptosis, MDA content and ROS were upregulated, while cell viability and SOD were significantly decreased in PC12 cells. The effects of OGD/R on PC12 cells were reversed with the downregulation of miR-497-5p. A double luciferase reporter assay demonstrated that miR-497-5p negatively targets BDNF. BDNF inhibited cell apoptosis and oxidative stress injury in PC12 cells. These findings suggest that miR-497-5p aggravates neuronal injury in experimental model of ischemic stroke by inhibiting the BDNF/TrkB/PI3K/Akt signaling pathway.

Role of NLRP3 Inflammasome in Stroke Pathobiology: Current Therapeutic Avenues and Future Perspective.

Neuroinflammation is a key pathophysiological feature of stroke-associated brain injury. A local innate immune response triggers neuroinflammation following a stroke via activating inflammasomes. The nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome has been heavily implicated in stroke pathobiology. Following a stroke, several stimuli have been suggested to trigger the assembly of the NLRP3 inflammasome. Recent studies have advanced the understanding and revealed several new players regulating NLRP3 inflammasome-mediated neuroinflammation. This article discussed recent advancements in NLRP3 assembly and highlighted stroke-induced mitochondrial dysfunction as a major checkpoint to regulating NLRP3 activation. The NLRP3 inflammasome activation leads to caspase-1-dependent maturation and release of IL-1β, IL-18, and gasdermin D. In addition, genetic or pharmacological inhibition of the NLRP3 inflammasome activation and downstream signaling has been shown to attenuate brain infarction and improve the neurological outcome in experimental models of stroke. Several drug-like small molecules targeting the NLRP3 inflammasome are in different phases of development as novel therapeutics for various inflammatory conditions, including stroke. Understanding how these molecules interfere with NLRP3 inflammasome assembly is paramount for their better optimization and/or development of newer NLRP3 inhibitors. In this review, we summarized the assembly of the NLRP3 inflammasome and discussed the recent advances in understanding the upstream regulators of NLRP3 inflammasome-mediated neuroinflammation following stroke. Additionally, we critically examined the role of the NLRP3 inflammasome-mediated signaling in stroke pathophysiology and the development of therapeutic modalities to target the NLRP3 inflammasome-related signaling for stroke treatment.

Involvement of CKS1B in the anti-inflammatory effects of cannabidiol in experimental stroke models

We have previously demonstrated that treatment with cannabidiol (CBD) ameliorates mitochondrial dysfunction and attenuates neuronal injury in rats following cerebral ischemia. However, the role of CBD in the progression of ischemic stroke-induced inflammation and the molecules involved remain unclear. Here, we found that CBD suppressed the production of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), reduced the activation of microglia, ameliorated mitochondrial deficits, and decreased the phosphorylation of nuclear factor κ-B (NF-κB) in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Cyclin-dependent kinase regulatory subunit 1B (CKS1B) expression was decreased in BV-2 cells following OGD/R and this reduction was blocked by treatment with CBD. Knockdown of CKS1B increased the activation of microglia and enhanced the production of IL-1β and TNF-α in BV-2 cells treated with CBD. Moreover, CKS1B knockdown exacerbated mitochondrial deficits and increased NF-κB phosphorylation. CBD treatment also ameliorated brain injury, reduced neuroinflammation, and enhanced the protein levels of mitochondrial transcription factor A and CKS1B in rats following middle cerebral artery occlusion/reperfusion. These data identify CKS1B as a novel regulator of neuroinflammation; and reveals its involvement in the anti-inflammatory effects of CBD. Interventions targeting CKS1B expression are potentially promising for treating in ischemic stroke.

Elovanoids Suppress Lipid Peroxidation after Experimental Stroke: An LC-MS-based Untargeted Lipidomics Analysis

At the onset and progression of neurodegenerative diseases, enhanced reactive oxygen species (ROS) and an overall uncompensated oxidative stress environment evolve. Oxidized phospholipids (OxPLs) cause detrimental perturbations to synaptic membranes and other parts of the nervous system. We tested if the lipid mediators Elovanoids (ELVs) that exert neuroprotective signaling also counteract lipid peroxidation using an experimental ischemic stroke model. Male Sprague-Dawley rats after 2 hours of middle cerebral artery occlusion (MCAo) were used. ELV-N34 was administered intranasally at 1 hour, 24 hours and 48 hours after, and rats were sacrificed on day 3; the brains were removed and sampled into the right and left hemispheres and the cortex, subcortex. Untargeted lipidomics were performed using a Waters Xevo G2-XS QTof system under MSE mode, and collected MS/MS spectra for the OxPLs was processed and searched against a reference spectral library by MS-DIAL software. ELV-N34 reduced oxidized phosphatidylcholine (OxPC) and phosphatidylethanolamine (OxPE) molecular species. We suggest that ELVs target lipid peroxidation as part of their neuroprotective bioactivity.

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Endogenous Protein C Activation with AB002 Improves Outcomes in a Rat Model of Acute Ischemic Stroke

Background: The hemorrhagic side-effects of current thrombolytic agents limit their usefulness to a small subset of acute ischemic stroke patients. An early, effective, and hemostatically safe antithrombotic/neuroprotectant in addition to standard of care could improve outcomes. AB002 (E-WE thrombin) is an engineered recombinant thrombin analog (W215A/E217A) that activates cytoprotective and antithrombotic endogenous protein C on intravascular surfaces without any measurable procoagulant activity. In renal failure patients undergoing hemodialysis, AB002 treatment reduced dialyzer clotting without increasing markers of hemostasis impairment or coagulation (NCT03963895). Preclinical studies also showed that AB002 exhibits potent antithrombotic/thrombolytic effects in a non-human primate model of acute vascular graft thrombosis, as well as reduces infarct size and improves outcomes in an experimental acute myocardial ischemia-reperfusion model in mice. Aims: Here, we evaluated whether neuroprotective effects of AB002 can be observed following middle cerebral artery occlusion-recanalization (MCAO-R) in rats. Methods: The local animal care and use committee approved all studies. 8 to 9-week-old male rats (275-377 g) were anesthetized under isoflurane and a small laser-Doppler probe was affixed to the skull to monitor cortical perfusion and verify vascular occlusion and reperfusion. A silicone-coated 4-0 nylon monofilament was inserted into the right internal carotid artery (ICA) via the external carotid artery (ECA) until the laser-Doppler signal dropped to < 30 % of baseline. To evaluate the effect of AB002 on outcomes, rats (n = 4/ group) were treated with 25 µg/kg (0.25 mL/kg; i.v.) or saline placebo, administered 30 min after induction of ischemia. Since prolongation of the activated partial plasminogen time (aPTT) is a pharmacologic effect of high dose drug exposure, a 0.45 mL blood sample was collected at 20 min post treatment from the right jugular vein, processed to plasma and frozen for subsequent aPTT evaluation. At 90 min, the filament was removed, the surgical incisions were sutured closed and postoperative animals were monitored during recovery. Neurological performance scores and weight were assessed daily for 4 days. Rats were sacrificed on day 7 post-surgery and infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. To account for the effect of edema, the infarcted area was estimated indirectly by subtracting the non-infarcted area in the ipsilateral hemisphere from the contralateral hemisphere and expressing infarct volume as a percentage of contralateral hemisphere. APTT was measured in duplicate using a KC-4 coagulometer. All analyses were performed by researchers blinded to treatment. Results: Compared to placebo, rats that received AB002 exhibited less ischemic damage, evidenced by a statistically significant reduction in infarct volume of the cortex (32.3% versus 8.9%, P < 0.05) and hemisphere (28.0% versus 9.8%, P < 0.01). In addition, rats had better neurological scores and greater weight gains during recovery following treatment with AB002, compared to placebo. Mean aPTT values were higher in AB002 treated rats compared to placebo (34 ± 2.2 s versus 21 ± 1.6 s), confirming drug exposure. Conclusions: Treatment with AB002 during ischemia reduced infarct volume and improved outcomes in the rat experimental stroke model. Considering its neuroprotective and antithrombotic properties, we propose that early treatment with AB002 may confer outcome benefit for patients experiencing acute thrombotic emergencies.

The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

Spatiotemporal expression patterns of ZBP1 in the brain of mouse experimental stroke model

Z-DNA binding protein 1 (ZBP1) is a cytosolic nucleic acid sensor, functioning as a critical mediator of inflammation and cell death pathways. Since neuroinflammation could occur in response to damage-associated molecular patterns (DAMPs), ZBP1 might be involved in neuroinflammation after stroke. However, the spatiotemporal expression profile of ZBP1 in the post-stroke brain remains to be elucidated. The aim of this study is to demonstrate the spatiotemporal expression patterns of ZBP1 in the post-stroke brain using a mouse photothrombotic stroke model. Real-time PCR assays showed that ZBP1 is induced on days 3–14 post stroke. ZBP1 immunoreactivity was observed in Iba1-positive microglia/macrophages in peri-infarct regions by immunohistochemistry. ZBP1-positive cells were spread in layers surrounding the infarct core by 7–14 days post stroke. Interestingly, ZBP1 immunoreactivity was also detected in CD206-positive border-associated macrophages (BAMs) in the meninges. Furthermore, ZBP1-expressing cells were positive for antibodies against inflammatory mediators such as Toll-like receptor 4 (TLR4), Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Morphological analysis with confocal microscopy showed that the co-localization signals of ZBP1 and its adaptor, TRIF, are increased by glucose oxidase (GOx) treatment, which has been reported to induce mitochondrial DNA (mtDNA) release. These results suggest that ZBP1 is induced in peri-infarct microglia/macrophages and may be involved in DAMPs-mediated neuroinflammation involving mtDNA in the post-infarct brain.

Abstract 17854: Imaging Intravascular Macrophages Using Micro-Sized Superparamagnetic Decoys

Introduction: During inflammatory processes, intravascular macrophages are capable of phagocytizing circulating debris in the circulation. Hypothesis: Intravenous injection of contrast-loaded decoys could be used to label intravascular macrophages and map their distribution by non-invasive imaging. Methods: We synthetized superparamagnetic decoys with hydrodynamic diameters from 20 nm to 4.5 μm. Experimental models of neuroinflammation (intrastriatal injection of LPS or TNF) and ischemic stroke (thrombin model and ferric chloride model) were performed in mice. High resolution 3D T2*-weighted MRI sequences were performed after intravenous injection of the superparamagnetic decoys. Results: In experimental models of neuroinflammation, intravenous injection of superparamagnetic decoys with a diameter of 1 μm (but neither smaller nor larger particles) reveals intravascular phagocytic cells in the cerebral circulation, in a dose dependent manner. In ischemic stroke, we found that intravenous injection of the decoys leads to numerous signal voids in the vessels in and around the infarct core at 48 hours (Figure 1a-b). Interestingly, the intravascular phagocytic activity appears much higher in permanent rather than transient models of ischemic stroke. Pre-treatment with anti-CD11a blocking antibodies significantly reduced the number of intravascular macrophages in stroke. Histological studies confirmed that injected decoys are phagocytized by intravascular cells which express typical markers of the monocyte/macrophage lineage including CX3CR1 dependent GFP expression and CD68 (Figure 1c). Conclusions: Intravenous injection of superparamagnetic decoys in the micrometer size range allows to image intravascular macrophages. This new method allows to study the pathophysiological roles of intravascular macrophages in experimental models and could be easily translated to humans.

Decreased long-chain acylcarnitine content increases mitochondrial coupling efficiency and prevents ischemia-induced brain damage in rats

Long-chain acylcarnitines (LCACs) are intermediates of fatty acid oxidation and are known to exert detrimental effects on mitochondria. This study aimed to test whether lowering LCAC levels with the anti-ischemia compound 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB) protects brain mitochondrial function and improves neurological outcomes after transient middle cerebral artery occlusion (MCAO). The effects of 14 days of pretreatment with methyl-GBB (5 mg/kg, p.o.) on brain acylcarnitine (short-, long- and medium-chain) concentrations and brain mitochondrial function were evaluated in Wistar rats. Additionally, the mitochondrial respiration and reactive oxygen species (ROS) production rates were determined using ex vivo high-resolution fluorespirometry under normal conditions, in models of ischemia-reperfusion injury (reverse electron transfer and anoxia-reoxygenation) and 24 h after MCAO. MCAO model rats underwent vibrissae-evoked forelimb-placing and limb-placing tests to assess neurological function. The infarct volume was measured on day 7 after MCAO using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Treatment with methyl-GBB significantly reduced the LCAC content in brain tissue, which decreased the ROS production rate without affecting the respiration rate, indicating an increase in mitochondrial coupling. Furthermore, methyl-GBB treatment protected brain mitochondria against anoxia–reoxygenation injury. In addition, treatment with methyl-GBB significantly reduced the infarct size and improved neurological outcomes after MCAO. Increased mitochondrial coupling efficiency may be the basis for the neuroprotective effects of methyl-GBB. This study provides evidence that maintaining brain energy metabolism by lowering the levels of LCACs protects against ischemia-induced brain damage in experimental stroke models.

Safety and efficacy of glibenclamide combined with rtPA in acute cerebral ischemia with occlusion/stenosis of anterior circulation (SE-GRACE): a randomized, double-blind, placebo-controlled trial

Glibenclamide alleviates brain edema and improves neurological outcomes in experimental models of stroke. We aimed to assess whether glibenclamide improves functional outcomes in patients with acute ischemic stroke treated with recombinant tissue plasminogen activator (rtPA). In this randomized, double-blind, placebo-controlled trial, patients with acute ischemic stroke were recruited to eight academic hospitals in China. Patients were eligible if they were aged 18-74 years, presented with a symptomatic anterior circulation occlusion with a deficit on the NIHSS of 4-25, and had been treated with rtPA within 4.5h of symptom onset. We used web-based randomization (1:1) to allocate eligible participants to the glibenclamide or placebo group, stratified according to endovascular treatment and baseline stroke severity. Glibenclamide or placebo was taken orally or via tube feeding at a loading dose of 1.25mg within 10h after symptom onset, followed by 0.625mg every 8h for 5 days. The primary outcome was the proportion of patients with good outcomes (modified Rankin Scale of 0-2) at 90 days, assessed in all randomly assigned patients who had been correctly diagnosed and had begun study medication. The study is registered with ClinicalTrials.gov, NCT03284463, and is closed to new participants. Between January 1, 2018, and May 28, 2022, 305 patients were randomly assigned, of whom 272 (142 received glibenclamide and 130 received placebo) were included in the primary efficacy analysis. 103 (73%) patients in the glibenclamide group and 94 (72%) in the placebo group had a good outcome (adjusted risk difference 0.002, 95% CI-0.098 to 0.103; p=0.96). 12 (8%) patients allocated to glibenclamide and seven (5%) patients allocated to placebo died from any cause at 90 days (p=0.35). The number and type of adverse events were similar between the two groups. There were no drug-related adverse events and no drug-related deaths. The addition of glibenclamide to thrombolytic therapy did not increase the proportion of patients who achieved good outcomes after stroke compared with placebo, but it did not lead to any safety concerns. Southern Medical University and Nanfang Hospital.

E. globulus leaf EO exhibits anti-inflammatory effects by regulating GSDMD-mediated pyroptosis, thereby alleviating neurological impairment and neuroinflammation in experimental stroke mice

Ethnopharmacological relevanceAromatic and medicinal plants continue to be a major component of alternative and traditional medicine in the developing countries. Eucalyptus globulus (Labill.) is being employed to cultivation and production in China. However, few studies have reported the chemical composition and anti-inflammatory activity of Eucalyptus globulus (Labill.) leaf essential oil (E. globulus leaf EO) extracted from Eucalyptus globulus. Aim of the studyThis study aimed to assess the composition of E. globulus leaf EO and identify its bacteriostatic action as well as anti-inflammatory activity. Importantly, we evaluated the effect of E. globulus leaf EO on neurological impairment and neuroinflammation in experimental stroke mice. Materials and methodsGas Chromatography-Mass Spectrometer (GC-MS) analyses was employed to evaluate the chemical components of E. globulus leaf EO, and the relative content of each component was determined by area normalization method. The antimicrobial activity of E. globulus leaf EO was determined by Oxford cup method and microbroth dilution assay. Cytotoxic activity of E. globulus leaf EO on THP-1 cells or BV2 cells in vitro was determined by CCK8 assay. In addition, the lipopolysaccharide (LPS)/ATP-induced inflammation model in THP-1 cells or BV2 cells were established, and the relative expression of TNF-α, IL-1β, MCP-1and IL-6 were confirmed by RT-PCR. Furthermore, the expression of protein GSDMD, IL-lβ, NLRP3 and NFκB signaling pathway were assessed by immunoblotting. In vivo,the experimental stroke model constructed by middle cerebral artery occlusion/reperfusion (MCAO/R) in mice was employed and subsequently treated with E. globulus leaf EO (50,100 mg/kg, subcutaneous injection) for 3 days to assess neurological impairment and neuroinflammation. Behavioral and neuronal damage were assessed using grip strength test, rod trarod test, and Nissl staining. Pro-inflammatory factors in serum or ischemic brain tissue was detected by ELISA kits. ResultsGC-MS analyses revealed that the major compound in E. globulus leaf EO was eudesmol (71.967%). E. globulus leaf EO has antimicrobial activity against Staphylococcus aureus (gram positive bacteria, MIC = 0.0625 mg/mL), Escherichia coli (gram negative bacteria, MIC = 1 mg/mL), and Candida albicans (MIC = 4 mg/mL). E. globulus leaf EO (0.5312, 1.0625, and 2.15 mg/mL) significantly decreased the expression of inflammation-related genes, including IL-1β, TNF-α, MCP-1, and IL-6. Furthermore, reduced levels of TLR4, Myd88, phosphorylated NF-κB P65, and IκBα were found in the E. globulus leaf EO group for BV2 cells (1.025, and 2.125 mg/mL). In addition, the expression levels of GSDMD, NLRP3, IL-1β and AIM2 were significantly decreased in the E. globulus leaf EO group when compared with the LPS -stimulated group, regulating GSDMD-mediated pyroptosis. In vivo, E. globulus leaf EO improved neurological functional deficits, inhibited excessive activation of microglia, and reduced the secretion of pro-inflammatory factors IL-1β, TNF-α in the ischemic tissue and serum after MCAO/R. ConclusionE. globulus leaf EO has strong antibacterial and anti-inflammatory activity, which has been implicated in blocking GSDMD-mediated pyroptosis. Moreover, E. globulus leaf EO could exert neuroprotective effect on cerebral ischemia-reperfusion injury.

Growth arrest specific protein 6 alleviated white matter injury after experimental ischemic stroke

Ischemic white matter injury leads to long-term neurological deficits and lacks effective medication. Growth arrest specific protein 6 (Gas6) clears myelin debris, which is hypothesized to promote white matter integrity in experimental stroke models. By the middle cerebral artery occlusion (MCAO) stroke model, we observed that Gas6 reduced infarcted volume and behavior deficits 4 weeks after MCAO. Compared with control mice, Gas6-treatment mice represented higher FA values in the ipsilateral external capsules by MRI DTI scan. The SMI32/MBP ratio of the ipsilateral cortex and striatum was profoundly alleviated by Gas6 administration. Gas6-treatment group manifested thicker myelin sheaths than the control group by electron microscopy. We observed that Gas6 mainly promoted OPC maturation, which was closely related to microglia. Mechanically, Gas6 accelerated microglia-mediated myelin debris clearance and cholesterol transport protein expression (abca1, abcg1, apoc1, apoe) in vivo and in vitro, accordingly less myelin debris and lipid deposited in Gas6 treated stroke mice. HX531 (RXR inhibitor) administration mitigated the functions of Gas6 in speeding up debris clearance and cholesterol transport protein expression. Generally, we concluded that Gas6 cleared myelin debris and promoted cholesterol transportation protein expression through activating RXR, which could be one critical mechanism contributing to white matter repair after stroke.

Neurovascular Inflammation and Complications of Thrombolysis Therapy in Stroke.

Intravenous thrombolysis via tPA (tissue-type plasminogen activator) is the only approved pharmacological treatment for acute ischemic stroke, but its benefits are limited by hemorrhagic transformation. Emerging evidence reveals that tPA swiftly mobilizes immune cells which extravasate into the brain parenchyma via the cerebral vasculature, augmenting neurovascular inflammation, and tissue injury. In this review, we summarize the pronounced alterations of immune cells induced by tPA in patients with stroke and experimental stroke models. We argue that neuroinflammation, triggered by ischemia-induced cell death and exacerbated by tPA, compromises neurovascular integrity and the microcirculation, leading to hemorrhagic transformation. Finally, we discuss current and future approaches to attenuate thrombolysis-associated hemorrhagic transformation via uncoupling immune cells from the neurovascular unit.

Transient Middle Cerebral Artery Occlusion Model of Stroke.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models. In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Nicotinamide Deteriorates Post-Stroke Immunodepression Following Cerebral Ischemia-Reperfusion Injury in Mice.

(1) Background: Inducing experimental stroke leads to biphasic immune responses, where the early activation of immune functions is followed by severe immunosuppression accompanied by spleen and thymus atrophy. Nicotinamide, a water-soluble B-group vitamin, is a known neuroprotectant against brain ischemia in animal models. We examined the effect of nicotinamide on the central and peripheral immune response in experimental stroke models. (2) Methods: Nicotinamide (500 mg/kg) or saline was intravenously administered to C57BL/6 mice during reperfusion after transiently occluding the middle cerebral artery or after LPS injection. On day 3, the animals were examined for behavioral performance and were then sacrificed to assess brain infarction, blood-brain barrier (BBB) integrity, and the composition of immune cells in the brain, thymus, spleen, and blood using flow cytometry. (3) Results: Nicotinamide reduced brain infarction and microglia/macrophage activation following MCAo (p < 0.05). Similarly, in LPS-injected mice, microglia/macrophage activation was decreased upon treatment with nicotinamide (p < 0.05), suggesting a direct inhibitory effect of nicotinamide on microglia/macrophage activation. Nicotinamide decreased the infiltration of neutrophils into the brain parenchyma and ameliorated Evans blue leakage (p < 0.05), suggesting that a decreased infiltration of neutrophils could, at least partially, be the result of a more integrated BBB structure following nicotinamide treatment. Our studies also revealed that administering nicotinamide led to retarded B-cell maturation in the spleen and subsequently decreased circulating B cells in the thymus and bloodstream (p < 0.05). (4) Conclusions: Cumulatively, nicotinamide decreased brain inflammation caused by ischemia-reperfusion injury, which was mediated by a direct anti-inflammatory effect of nicotinamide and an indirect protective effect on BBB integrity. Administering nicotinamide following brain ischemia resulted in a decrease in circulating B cells. This warrants attention with respect to future clinical applications.

Phosphodiesterase type 5 Inhibitors (PDE-5I) as a potential therapeutic drug in stroke: A systematic narrative review

Objectives. Stroke is an acute cerebrovascular disease with high morbidity and mortality rate. Many stroke management strategies can improve prognosis and quality of life, but only to a certain extent. Phosphodiesterase inhibitor-5 (PDE-5i) has shown benefits in numerous preclinical studies, but human studies have been inconclusive. Material and Methods. For this narrative review, we conducted a systematic search following the PRISMA statement guideline from inception until November 2022. The search was conducted in PubMed, ScienceDirect, EBSCOhost, and ProQuest. We included all human and animal studies on this topic, including preclinical studies, randomized clinical trials, and case studies. All the excluded studies are reviews or non-English studies. Review. Many PDE-5i have shown benefits in anatomical and functional outcomes in preclinical acute experimental stroke models. PF-03049423 was safe and well tolerated in humans but had no significant impact on neurological or functional outcomes. Sildenafil on acute/subacute was deemed safe to use and demonstrated improvement over baseline, but power remains unknown due to the lack of a control group. Tadalafil use resulted in a reduction in regional cerebral blood flow in subjects with a history of stroke. Conclusions. Despite promising results in preclinical studies, current evidence shows that PDE-5i does not affect clinical or functional recovery in people with acute stroke. The disparity in the intricacy of stroke pathophysiology between human and animal models was crucial. Further research in a larger population with more consistent stroke onset, medicines, and doses is required for a more conclusive result.