Middle Cerebral Artery Occlusion Model Research Articles

Tetrahydropiperine, a natural alkaloid with neuroprotective effects in ischemic stroke

BackgroundIschemic stroke (IS) is a life-threatening neurological disease with various pathological mechanisms. Tetrahydropiperine (THP) is a natural alkaloid with protective effects against multiple diseases, such as seizure, and pain. This study was to examine the impact of THP on IS and investigate its potential mechanism. Material and methodsWe employed network pharmacology and molecular docking techniques to identify the target proteins of THP for intervention in IS. Adult male Sprague-Dawley rats were used to create a permanent middle cerebral artery occlusion model. PC-12 cells were chosen to establish an oxygen–glucose deprivation (OGD) cell model. Disease modeling followed by nimodipine (NIMO); 3-methyladenine (3-MA) and rapamycin (RAP) interventions. Open field test, Longa score, balance beam test, and forelimb grip test were used to measure motor and neurological functions. The degree of neurological damage recovery was assessed through behavioral analysis, and cerebral infarction volume was determined using TTC staining. Morphological changes were examined through HE and Nissl staining, and ultrastructural changes in neurons were observed using transmission electron microscopy. The protein expression of autophagy and related pathways was analyzed through Western blot (WB). The appropriate hypoxia time and drug concentration were determined using CCK-8 assay, which also measured cell survival rate. ResultsThe network pharmacology findings indicated that the impact of THP on IS was enhanced in the PI3K/Akt signaling pathway. THP demonstrated robust docking capability with proteins associated with the autophagy and PI3K/Akt/mTOR, as indicated by the molecular docking outcomes. THP significantly improved behavioral damage, reduced the area of cerebral infarction, ameliorated histopathological damage from ischemia, increase neuronal survival, and alleviated ultrastructural damage in neurons (P < 0.05). THP enhanced the survival of PC-12 cells induced by OGD and ameliorated the morphological harm to the cells (P < 0.05). THP was found to elevate the quantities of P62, LC3-Ⅰ, PI3K, P-AKt/Akt, and P-mTOR/mTOR proteins while reducing the levels of Atg7 and Beclin1 proteins. The results of transmission electron microscopy showed no autophagosomes in the THP, 3-MA, and 3-MA + THP groups. ConclusionThe activation of the PI3K/Akt/mTOR signaling pathway by THP inhibits autophagy and provides relief from neurological damage in IS.

Long noncoding RNA KCNQ1OT1 aggravates cerebral infarction by regulating PTBT1/SIRT1 via miR-16-5p.

Cerebral infarction (CI) is one of the leading causes of disability and death. LncRNAs are key factors in CI progression. Herein, we studied the function of long noncoding RNA KCNQ1OT1 in CI patient plasma samples and in CI models. Quantitative real-time PCR and Western blotting tested gene and protein expressions. The interactions of KCNQ1OT1/PTBP1 and miR-16-5p were analyzed using dual-luciferase reporter and RNA immunoprecipitation assays; MTT assays measured cell viability. Cell migration and angiogenesis were tested by wound healing and tube formation assays. Pathological changes were analyzed by triphenyltetrazolium chloride and routine staining. We found that KCNQ1OT1 and PTBP1 were overexpressed and miR-16-5p was downregulated in CI patient plasma and in oxygen-glucose deprived (OGD) induced mouse brain microvascular endothelial (bEnd.3) cells. KCNQ1OT1 knockdown suppressed pro-inflammatory cytokine production and stimulated angiogenic responses in OGD-bEnd.3 cells. KCNQ1OT1 upregulated PTBP1 by sponging miR-16-5p. PTBP1 overexpression or miR-16-5p inhibition attenuated the effects of KCNQ1OT1 knockdown. PTBP1 silencing protected against OGD-bEnd.3 cell injury by enhancing SIRT1. KCNQ1OT1 silencing or miR-16-5p overexpression also alleviated ischemic injury in a mice middle cerebral artery occlusion model. Thus, KCNQ1OT1 silencing alleviates CI by regulating the miR-16-5p/PTBP1/SIRT1 pathway, providing a theoretical basis for novel therapeutic strategies targeting CI.

Annexin A6 mitigates neurological deficit in ischemia/reperfusion injury by promoting synaptic plasticity.

Alleviating neurological dysfunction caused by acute ischemic stroke (AIS) remains intractable. Given Annexin A6 (ANXA6)'s potential in promoting axon branching and repairing cell membranes, the study aimed to explore ANXA6's potential in alleviating AIS-induced neurological dysfunction. A mouse middle cerebral artery occlusion model was established. Brain and plasma ANXA6 levels were detected at different timepoints post ischemia/reperfusion (I/R). We overexpressed and down-regulated brain ANXA6 and evaluated infarction volume, neurological function, and synaptic plasticity-related proteins post I/R. Plasma ANXA6 levels were measured in patients with AIS and healthy controls, investigating ANXA6 expression's clinical significance. Brain ANXA6 levels initially decreased, gradually returning to normal post I/R; plasma ANXA6 levels showed an opposite trend. ANXA6 overexpression significantly decreased the modified neurological severity score (p = 0.0109) 1 day post I/R and the infarction area at 1 day (p = 0.0008) and 7 day (p = 0.0013) post I/R, and vice versa. ANXA6 positively influenced synaptic plasticity, upregulating synaptophysin (p = 0.006), myelin basic protein (p = 0.010), neuroligin (p = 0.078), and tropomyosin-related kinase B (p = 0.150). Plasma ANXA6 levels were higher in patients with AIS (1.969 [1.228-3.086]) compared to healthy controls (1.249 [0.757-2.226]) (p < 0.001), that served as an independent risk factor for poor AIS outcomes (2.120 [1.563-3.023], p < 0.001). This study is the first to suggest that ANXA6 enhances synaptic plasticity and protects against transient cerebral ischemia.

Abstract WP307: Combinatory Therapy With a PAF-Receptor Antagonist Plus a Lipid Mediator Provides Long-Lasting Protection in Experimental Stroke

Objective: Neuroprotective efficacy observed during short survival periods may not necessarily apply to more extended survival periods, and some stroke-impaired behaviors recover naturally in rodent models 1-2 weeks after stroke. The objective of the present study was to test the hypothesis that acute LAU-0901 plus NPD1-induced neuroprotection endures in animals allowed to survive for several weeks after focal ischemic insult. Two small bioactive molecules were investigated: LAU-0901, a PAF-R antagonist that blocks pro-inflammatory signaling, and neuroprotectin D1 (NPD1), which activates cell-survival pathways, and their combination exerts potent anti-inflammatory activity in the brain. Methods: Male SD rats (290-360g) were subjected to 2h of middle cerebral artery occlusion (MCAo) by the intraluminal filament and treated with vehicle, LAU-0901 (60 mg/kg, IP), NPD1 (222 μg/kg, IV), LAU-0901+NPD1 at 3 h after onset of MCAo. Rats received neurobehavioral examinations during MCAo (60 min) and then on days 1, 2, 3, 7, and weekly during eight weeks survival period, followed by ex vivo MRI on weeks 4 and 8. Results: Physiological variables showed no significant differences among groups. No adverse behavioral side effects were observed after administering LAU-0901, NPD1, or LAU-0901+NPD1. LAU-0901 and NPD1 treatments alone significantly improved the behavior compared to the vehicle group on day 1 (by 32 and 33%), day 7 (38 and 40%), week 4 (38 and 39%), and persisting throughout eight weeks of survival period (by 58 and 44%, respectively). Combinatory treatment with LAU-0901+NPD1 dramatically improved behavior on days 1 (by 37), 2 (by 37%), 3 (by 36%), 7 (40%), and on weeks 4 (by 40%), 5 (44%), 6 (44%), 7 (44%) and 8 (41%) compared to vehicle group. Treatments with LAU-0901, NPD1, and NPD1+RvD1 reduced T2WI lesion volumes on week 4 by 32, 41, and 50% and tissue loss on week 8 by 83, 51, and 50%, respectively, compared to vehicles. Conclusion: These data suggest that LAU-0901 and NPD1 alone provide high-grade neuroprotection in the MCAo model. Combinatory therapy with LAU-0901 plus NPD1 is more effective than the single therapy affording synergistic neuroprotection with improved neurological recovery when administered at 3h after focal cerebral ischemia in rats.

Abstract WP312: Microglial Tmem119 Inhibits IRAK4 Activation and Confers Neuroprotection After Ischemic Stroke

Introduction: Ischemic microglia trigger post-stroke inflammation leading to secondary neuronal death. Microglial responses are regulated through membrane protein signals that in turn activate inflammatory transcription factors. Transmembrane 119 (Tmem119) is a type 1A membrane protein that is exclusively expressed by microglia. It has been suggested that Tmem119 interferes with myeloid differentiation primary response 88 (MyD88)-interleukin-1 receptor-associated kinase 4 (IRAK4) axis that is involved in microglial activation. However, the exact role of Tmem119 in microglial activation has been elusive. Hypothesis: Tmem119 inhibits IRAK4 activation to suppress microglial pro-inflammatory responses and is protective against ischemic injury. Methods: Young (6-8-week-old) C57BL/6 WT mice were injected stereotaxically Lenti-Tmem119 to induce overexpression in microglia. Six weeks later, the mice were subjected to a 60-min middle cerebral artery occlusion (MCAO) model. Oxygen-glucose deprivation (OGD) was also performed in primary microglia cultures treated with Lenti-Tmem119 or Tmem119 siRNA. Western blotting was performed on brain homogenates or microglia cells respectively for expression of p-IRAK4. ELISA was conducted to measure cytokine levels in the plasma and cell culture supernatant. Results: p-IRAK4 protein levels were significantly lower in ischemic brains from Lenti-Tmem119 vs. Lenti-GFP injected mice (p=0.0035) at 3d after MCAO (n=4-6), accompanied by smaller infarct size in Lenti-Tmem119 injected mice. Cultured microglia treated with Lenti-Tmem119 or Tmem119 siRNA had decreased and increased expression of p-IRAK4 respectively than controls after OGD (p=0.0090 and p=0.0152 respectively, n=3-4). Pro-inflammatory cytokine (TNF-α, IL-1β, IL-6) levels were significantly lower in the plasma of Lenti-Tmem119 treated mice compared to controls. Levels of these cytokines were decreased or increased in microglia culture medium treated with Lenti-Tmem119 or Tmem119 siRNA respectively than in control treated medium. Conclusion: Tmem119 signaling regulates microglial inflammatory responses through interaction with IRAK4 and is beneficial to stroke. Keywords: Stroke; microglia; Tmem119; IRAK4; inflammation

Dexmedetomidine Alleviates Brain Ischemia/Reperfusion Injury by Regulating Metastasis-associated Lung Adenocarcinoma Transcript 1/MicroRNA-140-5p/ Nuclear Factor Erythroid-derived 2-like 2 Axis.

Dexmedetomidine (Dex) is widely used in perioperative anesthesia, and recent studies have reported that it protects organs from ischemia/reperfusion (I/R) injury. This study was performed to investigate the role of Dex in alleviating cerebral I/R injury and its regulatory effects on metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-140-5p (miR-140-5p)/nuclear factor erythroid-derived 2-like 2 (Nrf2) axis. In vivo rat middle cerebral artery occlusion (MCAO) model and in vitro oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced neuronal injury model were constructed. Dex was injected into the animals or used to culture HT22 cells to observe the pharmacological effects. The neurological defect, brain water content, infarct volume of the rats, and neuron viability were evaluated. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were detected. Besides, the regulatory effects of Dex on MALAT1, miR-140-5p, and Nrf2 expression levels and regulatory relationships among them were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and dual- luciferase reporter assay. Dex significantly alleviated the neurological injury of rats with MCAO and promoted the viability of neurons. Dex treatment suppressed miR-140-5p expression, but elevated MALAT1 and Nrf2 expressions. MALAT1 knockdown down-regulated Nrf2 expression and promoted oxidative stress in neurons. Additionally, miR-140-5p directly targeted Nrf2, and it also functioned as a downstream target miRNA of MALAT1. Dex, via regulating MALAT1/miR-140-5p/Nrf2 axis, plays a neuroprotective role against I/R-induced brain injury.

Abstract 21: Similarity in the Changes of Brain Myeloid Transcriptome Signature is Shared Between Ischemic Stroke and Chronic Inflammation Caused by Diabetes Mellitus

Introduction: Ischemic stroke (IS) is a pressing global health concern. Diabetes mellitus not only heightens the risk of IS but also aggravates its outcomes. There is established evidence that neutrophil infiltration is involved in inflammation after stroke through various mechanisms, such as activating neutrophil extracellular traps. We aimed to examine the factors that contribute to the deterioration of IS in diabetic mice focusing on mechanisms underlying immune cell infiltration by single cell RNA sequencing (scRNAseq) of brain before and after stroke. Methods: Experimental stroke was induced using the distal middle cerebral artery occlusion (dMCAO) model. db/db mice were used to model type 2 diabetes and obesity, while db/+ mice served as euglycemic controls. Mononuclear cells from ipsilateral mouse brains were isolated 3 days post-dMCAO or sham surgery with the Percoll gradient following enzymatic digestion. Transcriptomic analysis was performed using scRNAseq from db/+ sham, db/db sham, db/+ stroke, and db/db stroke. Fluorescence-activated cell sorting (FACS) and immunofluorescence (IF) staining were conducted to validate the changes detected in the transcriptome results. Results: Prior to the onset of stroke, db/db mice exhibited changes in gene expression in endothelial cells and myeloid cells including macrophages, monocytes, and microglia, mirroring patterns of changes after stroke. Among the macrophage subpopulation in particular, db/db sham and db/+ stroke mice shared an overlap of 638/901 (70.8%) differentially expressed genes compared to db/+ sham. Gene enrichment analysis indicated predominant upregulation of genes linked to leukocyte migration and neutrophil chemotaxis. FACS revealed a heightened neutrophil presence in the brain parenchyma of diabetic mice before stroke, and further elevated after stroke compared to control mice. Specifically, the increased expression of anaphylatoxin receptor C3aR and complement C3 shown by IF corroborated with the transcriptomic signature, supporting a role of complement activation and myeloid cell transmigration. Conclusions: Our findings provide valuable insights into the mechanisms whereby diabetes potentially escalates neutrophil infiltration post-stroke.

Abstract TP309: Cortical Extravasation of Neutrophils and Unexpected Persistence Following Ischemia/Reperfusion in a Murine Ischemic Stroke Model

Background and Purpose: Neutrophils are the first recruited leukocytes following ischemia/reperfusion injury in the brain; however, when and where neutrophils enter the brain parenchyma to exert their effects is controversial. Understanding the spatiotemporal underpinnings of leukocyte extravasation is required to advance therapeutic interventions. Here we describe the complex evolution of neutrophil recruitment and extravasation following ischemic stroke. Methods: Circulating neutrophils were pulse-labeled with EdU to track specific cells recruited post stroke. The transient middle cerebral artery occlusion (tMCAO) model was used to simulate large vessel occlusion and reperfusion. Coronal sections were examined at various timepoints and the position of EdU-labeled neutrophils was precisely determined by fluorescence microscopy. PECAM antibody treatment was used to disrupt neutrophil extravasation at the cortical surface and alter migration kinetics. Results: At early timepoints, 12 h and 24 h, neutrophil recruitment was unexpectedly superficial. Over 120 h, neutrophils were found increasingly deeper into the subcortex. Timing of tMCAO surgery with EdU labeling allowed for specific labeling of the initial wave of neutrophils. Surprisingly, many EdU positive neutrophils were still observed at 72 h, indicating they had survived for several days. The number and distribution of EdU positive neutrophils confirms the majority of neutrophil recruitment occurs before 24 h across the infarct. Disrupting leukocyte extravasation with PECAM function-blocking antibodies restricted neutrophils to the cortical surface. Conclusions: Our findings demonstrate that neutrophil infiltration post stroke evolves over several days. This data suggests neutrophils are recruited to the infarct within the first 24 h, enter the brain at the cortical surface, distribute throughout the infarct, and persist over 72 h. Furthermore, disrupting extravasation with PECAM antibodies modulates neutrophil progression into the infarct. A better understanding of leukocyte spatiotemporal infiltration and its regulators will help inform future therapeutic interventions targeting leukocytes.

Comparative prototypes and metabolites of Du-zhi pill in normal and cerebral ischemia rats by UHPLC-Q-TOF-MS/MS method

Du-Zhi pill (DZP) is widely used as a Chinese medicine in treating cerebral ischemia. UHPLC-Q-TOF-MS/MS techniques were used to detect and identify the metabolites in rat brain samples of normal and middle cerebral artery occlusion (MCAO) model rats administered with DZP. It was tentatively found that 43 prototypes and 93 metabolites could be identified in rat brain samples. Normal and MCAO model rat brain samples contained 19 prototype components. Eight prototype components were only detected in normal rat brain samples, while 16 were found only in MCAO model rat brain samples. It was determined that 47 metabolites had been identified in the normal rats, while 86 had been placed in MCAO model rats. There were 40 common metabolites in both normal and MCAO model rat brain samples. Seven metabolites were only detected in normal rat brain samples, while 46 were found only in MCAO rat brain samples. The comparison of metabolites in brain samples of normal and MCAO rats showed apparent differences. It was discovered that glucuronidation, methylation, acetylation, and sulfation are phase II metabolic routes of DZP, while hydrogenation, hydroxylation, and dehydroxylation are phase I metabolic routes. Moreover, hydrogenation, glucuronidation, hydroxylation, and methylation were the main metabolic pathways because of the number of metabolites identified in these metabolic pathways. The results provide a valuable reference for further research into effective substances of DZP for treating cerebral ischemia.

Validation of a C57/BL6J mouse model of focal cerebral ischemia established by electrocoagulation of the middle cerebral artery

To modify the method for establishing mouse models of middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia using electrocoagulation. Forty-six C57/BL6J male mice were divided into MCAO model group (n=34) and sham-operated group (n=12). In the model group, MCAO was induced by permanent coagulation of the right middle cerebral artery (MCA) using a coagulator, and cerebral blood flow perfusion was monitored before and at 20 min and 1 day after modeling. Neurological deficits of the mice at 1, 7, and 14 days after modeling were evaluated using Longa score, mNSS score, beam walking test, cylinder test and corner test. TTC staining was used to measure the cerebral infarct size, and Western blotting was performed to detect the expressions of BDNF, GFAP and DCX proteins in the ischemic cortex. The mice in the model group showed significantly reduced cerebral blood flow in the MCA on the ischemic side and obvious neurological deficits with increased forelimb use asymmetry on days 1, 7 and 14 after modeling (P < 0.05). In the cerebral cortex on the ischemic side of the model mice, the expressions of GFAP and DCX increased significantly at 1, 7, and 14 days (P < 0.05) and the expression of BDNF increased at 1 day after modeling ischemia (P < 0.05). We successfully prepared mouse models of MCAO using a modified method by changing the electrocoagulation location from the distal location of the junction between the MCA and the inferior cerebral vein to a 2 mm segment medial to the junction between the MCA and the olfactory bundle.

Matrix Metalloproteinase-9 Contributes to Epilepsy Development after Ischemic Stroke in Mice.

Epilepsy, a neurological disorder affecting over 50 million individuals globally, is characterized by an enduring predisposition and diverse consequences, both neurobiological and social. Acquired epilepsy, constituting 30% of cases, often results from brain-damaging injuries like ischemic stroke. With one third of epilepsy cases being resistant to existing drugs and without any preventive therapeutics for epileptogenesis, identifying anti-epileptogenic targets is crucial. Stroke being a leading cause of acquired epilepsy, particularly in the elderly, prompts the need for understanding post-stroke epileptogenesis. Despite the challenges in studying stroke-evoked epilepsy in rodents due to poor long-term survival rates, in this presented study the use of an animal care protocol allowed for comprehensive investigation. We highlight the role of matrix metalloproteinase-9 (MMP-9) in post-stroke epileptogenesis, emphasizing MMP-9 involvement in mouse models and its potential as a therapeutic target. Using a focal Middle Cerebral Artery occlusion model, this study demonstrates MMP-9 activation following ischemia, influencing susceptibility to seizures. MMP-9 knockout reduces epileptic features, while overexpression exacerbates them. The findings show that MMP-9 is a key player in post-stroke epileptogenesis, presenting opportunities for future therapies and expanding our understanding of acquired epilepsy.

The effect of extra virgin olive oil on HSP27 expression in the cerebral cortex of male Rattus norvegicus middle cerebral artery occlusion model

Background: Ischemic stroke produces oxidative stress in neurons and neuroglial, triggering a cellular response with HSP27 as one of the markers. Extra virgin olive oil (EVOO) is reported to act as a neuroprotection by inhibiting reactive oxygen species, which can modulate HSP27.&#x0D; Objective: To analyze the potential neuroprotection of EVOO containing 7-g MUFA on the expression of HSP27 in the cerebral cortex of Rattus norvegicus Wistar strain middle cerebral artery occlusion (MCAO) model.&#x0D; Design: MCAO-Rattus norvegicus Wistar male rodents (n = 32), randomly divided into negative control (sham), positive control (P0), MCAO added EVOO 0.5 mL/kg body weight (P1), and MCAO added EVOO 2 mL/kg body weight (P2); the experiment was conducted 14 days. The nuclei of neurons, glial, and expression of HSP27 on the cerebral cortex were analyzed using Image J software, data were compared between groups with a significance level of P &lt; 0.05.&#x0D; Results: There were significant differences in the neuron and glia nuclei numbers between groups (P = 0.01). There was also a significant difference in cerebral cortex HSP27 expression between groups (P = 0.006).&#x0D; Conclusion: There was a significant down-regulated of HSP27 in the cerebral cortex of the male Rattus norvegicus Wistar strain MCAO model given EVOO with 7 g of MUFA content at a dose of 0.5–2 mL/kgBW.

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

JOURNAL/nrgr/04.03/01300535-202503000-00029/figure1/v/2024-06-17T092413Z/r/image-tiff 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.

Monomeric CXCL12-Engineered Adipose-Derived Stem Cells Transplantation for the Treatment of Ischemic Stroke.

Adipose-derived stem cells (ASCs) possess therapeutic potential for ischemic brain injury, and the chemokine CXCL12 has been shown to enhance their functional properties. However, the cumulative effects of ASCs when combined with various structures of CXCL12 on ischemic stroke and its underlying molecular mechanisms remain unclear. In this study, we genetically engineered mouse adipose-derived ASCs with CXCL12 variants and transplanted them to the infarct region in a mice transient middle cerebral artery occlusion (tMCAO) model of stroke. We subsequently compared the post-ischemic stroke efficacy of ASC-mCXCL12 with ASC-dCXCL12, ASC-wtCXCL12, and unmodified ASCs. Neurobehavior recovery was assessed using modified neurological severity scores, the hanging wire test, and the elevated body swing test. Changes at the tissue level were evaluated through cresyl violet and immunofluorescent staining, while molecular level alterations were examined via Western blot and real-time PCR. The results of the modified neurological severity score and cresyl violet staining indicated that both ASC-mCXCL12 and ASC-dCXCL12 treatment enhanced neurobehavioral recovery and mitigated brain atrophy at the third and fifth weeks post-tMCAO. Additionally, we observed that ASC-mCXCL12 and ASC-dCXCL12 promoted angiogenesis and neurogenesis, accompanied by an increased expression of bFGF and VEGF in the peri-infarct area of the brain. Notably, in the third week after tMCAO, the ASC-mCXCL12 exhibited superior outcomes compared to ASC-dCXCL12. However, when treated with the CXCR4 antagonist AMD3100, the beneficial effects of ASC-mCXCL12 were reversed. The AMD3100-treated group demonstrated worsened neurological function, aggravated edema volume, and brain atrophy. This outcome is likely attributed to the interaction of monomeric CXCL12 with CXCR4, which regulates the recruitment of bFGF and VEGF. This study introduces an innovative approach to enhance the therapeutic potential of ASCs in treating ischemic stroke by genetically engineering them with the monomeric structure of CXCL12.

Bat-derived oligopeptide LE6 inhibits the contact-kinin pathway and harbors anti-thromboinflammation and stroke potential.

Thrombosis and inflammation are primary contributors to the onset and progression of ischemic stroke. The contact-kinin pathway, initiated by plasma kallikrein (PK) and activated factor XII (FXIIa), functions bidirectionally with the coagulation and inflammation cascades, providing a novel target for therapeutic drug development in ischemic stroke. In this study, we identified a bat-derived oligopeptide from Myotis myotis (Borkhausen, 1797), designated LE6 (Leu-Ser-Glu-Glu-Pro-Glu, 702 Da), with considerable potential in stroke therapy due to its effects on the contact kinin pathway. Notably, LE6 demonstrated significant inhibitory effects on PK and FXIIa, with inhibition constants of 43.97 μmol/L and 6.37 μmol/L, respectively. In vitro analyses revealed that LE6 prolonged plasma recalcification time and activated partial thromboplastin time. In murine models, LE6 effectively inhibited carrageenan-induced mouse tail thrombosis, FeCl 3-induced carotid artery thrombosis, and photochemically induced intracerebral thrombosis. Furthermore, LE6 significantly decreased inflammation and stroke injury in transient middle cerebral artery occlusion models. Notably, the low toxicity, hemolytic activity, and bleeding risk of LE6, along with its synthetic simplicity, underscore its clinical applicability. In conclusion, as an inhibitor of FXIIa and PK, LE6 offers potential therapeutic benefits in stroke treatment by mitigating inflammation and preventing thrombus formation.

Cerebral Angiography and Neurobehavioral Patterns in a Non-human Primate Middle Cerebral Artery Occlusion Model.

Ischemic stroke is a major health concern globally and developing reliable animal models is crucial for understanding its pathophysiology. This study evaluated the relationship between cerebral angiographic findings and neurologic dysfunction in an acute non-human primate thromboembolic stroke model and determined the minimum clot length for suitable middle cerebral artery (MCA) occlusion. A thromboembolic stroke model was developed by injecting autologous blood clots (length: 1, 2, 3, 4, 5, and 10 cm, n=1 to 3, 14 monkeys in total) into the internal carotid artery of male cynomolgus monkeys. Digital subtraction angiography (DSA) and neurologic deficit observation were performed pre-; immediately after (DSA only); and 1, 3, 6, and 24 h after embolization, and the relationship between clot length, neurologic deficits, and cerebral infarction was assessed. DSA confirmed MCA occlusion in all animals after the clot injection. Recanalization of the MCA was observed within 6 h post-embolization in animals with shorter clots (≤3 cm). Neurologic deficits were evident in animals with MCA occlusion and correlated with the clot length. Larger clots (≥5 cm) led to permanent MCA occlusion, significant neurologic deficits, and extensive cerebral infarction. Histopathological examination revealed ischemic damage in brain regions corresponding to the infarcted areas. Clot length is critical in determining the extent of neurologic dysfunction and cerebral infarction, with larger clots producing more severe outcomes. Furthermore, the minimum clot length required for model creation is 5 cm.

Function and mechanism of the human SOD2 gene in mice cerebral ischemia/ reperfusion injury.

To investigate the neuroprotective effects of the SOD2 gene in cerebral ischemia reperfusion injury function and the underlying mechanisms in a mice model of middle cerebral artery ischemia reperfusion. SOD2 transgenic mice were engineered using transcription activator-like effector nucleases, and the genotype was identified using PCR after every three generations. Transgenic and C57BL/6J wild type mice were simultaneously subjected to the middle cerebral artery occlusion model. SOD2 expression in the brain, heart, kidney, and skeletal muscle of transgenic mice was significantly higher than that in the wild type. Following ischemia reperfusion, the infarct volume of wild type mice decreased after treatment with fenofibrate compared to the CMC group. Infarction volume in SOD2 transgenic mice after CMC and fenofibrate treatment was significantly reduced. The recovery of cerebral blood flow in wild type mice treated with fenofibrate was significantly enhanced compared with that in the CMC group. The expression of SOD2 in transgenic mice was significantly higher than that in wild type mice, the neuroprotective role of fenofibrate depends on an increase in SOD2 expression.

Microglia Autophagy Mediated by TMEM166 Promotes Ischemic Stroke Secondary to Carotid Artery Stenosis.

Ischemic stroke can be a serious complication of selective carotid endarterectomy (CEA) in patients with carotid artery stenosis (CAS). The underlying risk factors and mechanisms of these postoperative strokes are not completely understood. Our previous study showed that TMEM166-induced neuronal autophagy is involved in the development of secondary brain injury following cerebral ischemia-reperfusion injury in rats. This current study aimed to investigate the role of TMEM166 in ischemic stroke following CEA. In the clinical part of this study, the quantitative analysis demonstrated circulating TMEM166, interleukin 6 (IL-6), and C-reactive protein (CRP) levels were significantly elevated in patients who suffered an ischemic stroke after CEA compared to those who did not. Furthermore, non-survivors exhibited higher levels of these proteins than survivors. In the preclinical part of this study, a middle cerebral artery occlusion (MCAO) model was implemented following CAS simulation in TMEM166-/- mice. We found TMEM166 expression was positively correlated with the degree of ischemic brain injury. Ad5-TMEM166 transfection aggravated ischemic brain injury by inducing microglial autophagy activation and release of inflammatory cytokines. Accordingly, TMEM166 deficiency reduced brain inflammation and inhibited excessive microglial autophagy through the mammalian target of rapamycin (mTOR) pathway. These findings suggest that TMEM166 may play a key role in the development of ischemic injury after CEA and may serve as a biomarker for risk assessment of postoperative ischemic stroke.

Netrin-1 protects blood-brain barrier (BBB) integrity after cerebral ischemia-reperfusion by activating the Kruppel-like factor 2 (KLF2)/occludin pathway.

Ischemia/reperfusion (I/R)-induced neural damage and neuroinflammation have been associated with pathological progression during stroke. Netrin-1 is an important member of the family of laminin-related secreted proteins, which plays an important role in governing axon elongation. However, it is unknown whether Netrin-1 possesses a beneficial role in stroke. Here, we employed the middle cerebral artery occlusion (MCAO)model to study the function of Netrin-1 in alleviating brain injuries. Our results demonstrate that Netrin-1 rescued poststroke neurological deficits and inhibited production of the inflammatory cytokines such as interleukin 6 (IL-6)and endothelial chemokine (C-X-C motif) ligand 1 (Cxcl1). Importantly, Netrin-1 protected against MCAO-induced dysfunction of the blood-brain barrier (BBB)in mice and a reduction in the expression of the tight junction (TJ)protein occludin. Additionally, we report that Netrin-1 could ameliorate oxygen-glucose deprivation/reoxygenation (OGD/R)-inducedinjury and prevent aggravation in endothelial monolayer permeability in bEnd.3 human brain microvascular endothelial cells (HBMVECs).Mechanistically, Netrin-1 ameliorated OGD/R-induced decrease in occludin and Kruppel-likefactor 2 (KLF2) in HBMVECs. Notably, silencing of KLF2abolished the beneficial effects of Netrin-1 in protecting endothelial permeability and occludin expression, suggesting that these effects are mediated by KLF2. In conclusion, our findings suggest that Netrin-1 could constitute a novel therapeutic strategy for ischemic stroke.

Molecular Mechanisms of Phyllanthus urinaria in Cerebral Ischemia-Reperfusion Injury

Phyllanthus urinaria (P. urinaria), a prominent herb in traditional Zhuang medicine, is characterized by its heat clearance, detoxification, diuresis, and detumescence. This study investigates the therapeutic effects and molecular mechanisms of P. urinaria on cerebral ischemia-reperfusion injury (CIRI). 60 adult SD rats were randomly divided into four groups: a sham operation group, a model group of right middle cerebral artery occlusion (MCAO), a low-dose group treated with P. urinaria at 5 g/kg, and a high-dose group treated with P. urinaria at 10 g/kg, each consisting of 15 rats. After seven days of continuous administration, a rat model of the right MCAO was established to simulate CIRI. Nerve function was assessed using the Longa scoring method, the cerebral infarction area was evaluated with TTC staining, and cytokine levels (IL-1β, TNF-α, IL-6) were measured via ELISA. PI3K and AKT protein expression in brain tissues was analyzed by Western blot. The results revealed a significant improvement in neurological function scores in both low and high-dose P. urinaria groups compared to the MCAO model group. Treatment with P. urinaria led to a notable reduction in cerebral infarction area after 72 hours of cerebral ischemia. The levels of inflammatory cytokines (IL-1β, TNF-α, IL-6) were decreased in both treatment groups compared to the MCAO model group. There was a significant increase in the expression of PI3K and AKT proteins in both treatment groups. In conclusion, P. urinaria exhibits a protective effect against CIRI by inhibiting inflammatory responses, improving neurological function, and reducing brain injury.