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Reactive oxygen species generated via reperfusion cause lipid damage and induce lipid peroxidation, leading to cerebral ischemia/reperfusion injury and exacerbation of cerebral infarction. Lipid radicals are key molecules generated during lipid peroxidation. Therefore, understanding the spatiotemporal behavior of lipid radicals is important to improve the therapeutic outcomes of cerebral infarction. However, the behaviors of lipid radicals in the brain remain unclear. In this study, we aimed to evaluate the distribution of radioactivity in a transient middle cerebral artery occlusion (tMCAO) model using lipid radical detection probe [125I] 1 to assess the behaviors of lipid radicals after cerebral ischemia/reperfusion. The tMCAO model administered [125I] 1 exhibited significant differences in the timing and location of radioactivity accumulation between the ischemic and non-ischemic regions. Liquid chromatography/mass spectrometry analysis identified the lipid radical adducts formed by the reaction of 1 with the lipid radicals generated after reperfusion. More adducts were detected in the ischemic region samples than in the non-ischemic region samples. Therefore, 1 successfully detected the lipid radicals generated after cerebral ischemia/reperfusion. Overall, this study demonstrates the potential of nuclear medical imaging using radiolabeled 1 to detect the lipid radicals generated after cerebral ischemia/reperfusion. Our approach can aid in the development of new therapeutic agents scavenging lipid radicals after cerebral reperfusion by facilitating the determination of therapeutic efficacy and optimal administration period. [Display omitted] • Radioiodinated probe detects lipid radicals after cerebral ischemia-reperfusion. • Ex vivo ARG maps lipid radical distribution post-ischemia. • Lipid radical generation varies in timing and location across brain regions. • LC/MS identifies lipid radical adducts at different timepoints after reperfusion. • Edaravone's effectiveness in suppressing lipid radicals evaluated spatiotemporally. [ABSTRACT FROM AUTHOR]

Ischemic stroke is a leading cause of disability and mortality worldwide. Recently, increasing evidence implicates microRNAs (miRs) in the pathophysiology of ischemic stroke. Studies have shown that miR-15a/16-1 is abnormally expressed in brains after ischemic stroke, and its upregulation may increase ischemic damage. Given that sex and age are significant modifiers of stroke outcomes, here we investigated whether inhibiting miR-15a/16-1 with antagomirs mitigates cerebral ischemia/reperfusion (I/R) injury in a sex- and age-dependent manner. Young (3 months) and aged (18 months) male and female C57/BL mice underwent 1-h middle cerebral artery occlusion and 3–7 days reperfusion (tMCAO). We administered miR-15a/16-1 antagomir (30 pmol/g) or control antagomir (NC, 30 pmol/g) via tail vein 2 h post-MCAO. Neurobehavioral testing and infarct volume assessment were performed on days 3 and 7. Compared to controls, antagomir treatment significantly improved neurobehavioral outcomes and reduced infarct volume in tMCAO mice at day 7, with the effects being more pronounced in young mice. Notably, young female mice exhibited superior survival and sensorimotor function compared to young male mice. These results were also replicated in a permanent MCAO (pMCAO) mice model. This suggests miR-15a/16-1 antagomir and estradiol may synergistically regulate genes involved in neurovascular cell death, inflammation, and oxidative stress, with sex and age-dependent expression of miR-15a/16-1 and its targets likely underlying the observed variations. Overall, our findings identify miR-15a/16-1 antagomir as a promising therapeutic for ischemic stroke and suggest that sex and age should be considered when developing miR-based therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Aquaporin‐4 (AQP4), predominantly expressed in astrocytes, has been implicated in the development of brain edema following ischemic events. However, its role in post‐stroke neuroinflammation is not fully understood. Methods: Using a middle cerebral artery occlusion (MCAO) mouse model, we assessed AQP4's role in post‐stroke inflammation. Brain tissue slices from male C57BL/6 mice were subjected to immunohistochemistry and western blot post‐MCAO. Additionally, primary astrocytes were isolated for quantitative real‐time PCR and immunofluorescence assays to evaluate the expression of inflammatory markers glial fibrillary acidic protein (GFAP) and AQP4. AQP4 modulation was achieved using viral knockdown and overexpression methods. Neuronal damage was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests in co‐culture studies. Results: MCAO mice exhibited a significant upregulation in GFAP. This reactive astrogliosis corresponded with an elevation in inflammatory markers. AQP4 expression responded to this inflammatory trend, peaking at 6 h after OGD and returning to baseline levels at 24 and 48 h. Co‐culture experiments revealed that AQP4(+) astrocytes exacerbated injury in OGD‐treated neurons, as evidenced by increased TUNEL positivity and apoptotic events. Conversely, AQP4(−) astrocytes appeared to have a protective effect. Knockdown of AQP4 resulted in reduced post‐OGD inflammatory response, whereas AQP4 overexpression intensified the injury to neurons post‐OGD. In vivo experiments also confirmed that AQP4 inhibitor TGN‐020 reduced and overexpression of AQP4 increased behavioral abnormalities and brain infarcts. Conclusion: Our findings underscore AQP4's pivotal role in modulating post‐stroke neuroinflammation. Targeting AQP4 may present a novel therapeutic avenue for mitigating ischemia‐induced neuronal damage. [ABSTRACT FROM AUTHOR]

Ling Zhao,1– 3,* Shichao Li,1,2,4,* Xiaoyu Wang,1,2 Lingyan Zhang,1,2 Jingge Zhang,1,2 Xiyun Liu,1,2 Yuyan Hu,1,2 Xiaohui Xian,1,2 Feng Zhang,5 Wenbin Li,1,2 Min Zhang1,2,6 1Department of Pathophysiology, Hebei Medical University, Shijiazhuang, Hebei Province, 050017, People’s Republic of China; 2Hebei Key Laboratory of Critical Disease Mechanism and Intervention, Shijiazhuang, Hebei Province, 050017, People’s Republic of China; 3Department of Obstetrics, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, 050000, People’s Republic of China; 4Experimental Diagnostic Center for Infectious Diseases, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, 050000, People’s Republic of China; 5Department of Rehabilitation Medicine, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, 050051, People’s Republic of China; 6The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Shijiazhuang, Hebei Province, 050017, People’s Republic of China*These authors contributed equally to this workCorrespondence: Min Zhang, Department of Pathophysiology, Key Laboratory of Critical Disease Mechanism and intervention of Hebei Province, The Key Laboratory of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, No. 361 Zhongshan East Road, Shijiazhuang, Hebei, 050017, People’s Republic of China, Email hebmuzhangmin@hebmu.edu.cnBackground: Obesity is recognized as a primary risk factor for cerebral ischemia, which has shown a significant increase in its incidence among obese patients. The exact mechanism by which obesity exacerbates cerebral ischemic injury is not fully understood though. The present study validated the hypothesis that obesity mediates pyroptosis by the AGEs/RAGE signaling pathway to exacerbate cerebral ischemic injury.Methods: Leptin receptor knockout (Lepr−/−) rats were used in this study to construct an obesity model, and the middle cerebral artery occlusion (MCAO) models of ischemic stroke were established in Lepr−/− obese rats and their wild-type (WT) littermates respectively. Zea-Longa score, TTC and H&E staining were utilized to evaluate the neurological impairment. Western Blot, immunohistochemistry, and immunofluorescence were used to detect protein expressions. Transmission electron microscopy was used to observe the pores in the neuronal cell membrane in the ischemic penumbra cortex.Results: Compared with WT littermates, Lepr−/− obese rats exhibited exacerbated neuronal injury after MCAO, with higher expressions of NLRP3 inflammasome and pyroptosis-related proteins in the cortical tissue of the penumbra. Moreover, more GSDMD pores were observed on the neuronal cell membranes of Lepr−/− obese rats according to the electron microscopy. Inhibition of NLRP3 inflammasome expression with MCC950 inhibited neuronal pyroptosis after cerebral ischemia in Lepr−/− obese rats, thus reducing neuronal injury. We also found that compared with WT littermates, the levels of AGEs and RAGE in the cortex of Lepr−/− obese rats are significantly higher, with further increase after cerebral ischemia. Inhibition of AGEs/RAGE signaling pathway with FPS-ZM1 reduced the NLRP3 inflammasome-mediated neuronal pyroptosis in Lepr−/− obese rats, thereby mitigating the neuronal damage after cerebral ischemia.Conclusion: The AGEs/RAGE signaling pathway is involved in the exacerbation of cerebral ischemic injury in Lepr−/− obese rats via regulating NLRP3-mediated neuronal pyroptosis. Keywords: MCAO, neuronal pyroptosis, NLRP3, Lepr−/− obese rats, AGEs/RAGE signaling pathway

Stroke is a cause of death and disability in the world. Animal research needs animal models that are as similar as possible to the pathophysiological processes that occur in humans. The established ischemic stroke model is the middle cerebral artery occlusion (MCAO) model. The creation of this model requires sophisticated equipment and precision. Our study aims to describe the MCAO modeling procedure using simple tools and to identify causes of death. Creation of ischemic stroke animal models with MCAO procedure with simple equipment and using monofilament nylon suture 4.0 to clog the middle cerebral artery and reperfusion for 90 minutes. Neurological deficits are clinically tested with Bederson parameters after one hour of MCAO. 2.3.5 triphenyl tetrazolium chloride staining was performed to determine the area of infarction after 24 hours of MCAO. The histological picture is examined with hematoxylin-eosin staining. Two rats succeeded in modeling ischemic stroke, two rats failed in modeling ischemic stroke, two rats died during preparation, four rats died during surgery, and 10 rats died several hours after occlusion. The most common cause of death was bleeding from 4.0 nylon sutures, which are too long to pass through the middle cerebral artery, thereby puncturing the blood vessels. When creating a model of ischemic stroke, it is essential to consider the elements that contribute to rat failure and death.

BackgroundSignificant advances have been made in our understanding of the endolysosomal cycle. Disruption of this cycle leads to cell death. The objective of this study aims to investigate the role of disrupted endolysosomal cycle in brain ischemia-reperfusion injury.MethodsA total of 57 mice were randomly assigned into four experimental groups: (i) wildtype (wt) sham control; (ii) wt middle cerebral artery occlusion (MCAO); (iii) cathepsin B (CTSB) knockout (KO) sham control; and (iv) CTSB KO MCAO. Mice were subjected either to 0 min (sham) or 40 min of MCAO, followed by reperfusion for 1 or 7 days. Physical and behavioral examinations were conducted in the 7-day reperfusion group for 7 consecutive days after MCAO. Confocal microscopy was used to assess the levels, redistributions, and co-localizations of key endolysosomal cycle-related proteins. Histopathology was examined by light microscopy.ResultsConfocal microscopy revealed a significant accumulation of CTSB in post-ischemic penumbral neurons relative to those in the sham group. In addition, N-ethylmaleimide sensitive factor ATPase (NSF) was irreversibly depleted in these neurons. Furthermore, CTSB-immunostained structures were enlarged and diffusely distributed in both the cytoplasm and extracellular space, indicating the release of CTSB from post-ischemic neurons. Compared to wt mice, CTSB KO mice showed a significant decrease in hippocampal injury area, a significant increase in the number of survival neurons in the striatal core area, and a significant improvement in physical and functional performance in post-MCAO mice.ConclusionBrain ischemia leads to a cascade of events leading to inactivation of NSF, disruption of the endolysosomal cycle, endolysosomal structural buildup and damage, and the release of CTSB, eventually resulting in brain ischemia reperfusion injury. CTSB KO in mice protected the brain from ischemia-reperfusion injury.

Mitophagy influences the progression and prognosis of ischemic stroke (IS). However, whether DNA methylation in the brain is associated with altered mitophagy in hypoxia-injured neurons remains unclear. Here, miR-138–5p was found to be highly expressed in exosomes secreted by astrocytes stimulated with oxygen and glucose deprivation/re-oxygenation (OGD/R), which could influence the recovery of OGD/R-injured neurons through autophagy. Mechanistically, miR-138–5p promotes the stable expression of Ras homolog enriched in brain like 1(Rhebl1) through DNA-methyltransferase-3a (DNMT3A), thereby enhancing ubiquitin-dependent mitophagy to maintain mitochondrial homeostasis. Furthermore, we employed glycosylation engineering and bioorthogonal click reactions to load mirna onto the surface of microglia and deliver them to injured region utilising the inflammatory chemotactic properties of microglia to achieve drug-targeted delivery to the central nervous system (CNS). Our findings demonstrate miR-138–5p improves mitochondrial function in neurons through the miR-138–5p/DNMT3A/Rhebl1 axis. Additionally, our engineered cell vector-targeted delivery system could be promising for treating IS. In this study, we demonstrated that miR-138–5p in exosomes secreted by astrocytes under hypoxia plays a critical role in the treatment of hypoxia-injured neurons. And we find a new target of miR-138–5p, DNMT3A, which affects neuronal mitophagy and thus exerts a protective effect by regulating the methylation of Rbebl1. Furthermore, we have developed a carrier delivery system by combining miR-138–5p with the cell membrane of microglia and utilized the inflammatory chemotactic properties of microglia to deliver this system to the brain via intravenous injection. This groundbreaking study not only provides a novel therapeutic approach for ischemia-reperfusion treatment but also establishes a solid theoretical foundation for further research on targeted drug delivery for central nervous system diseases with promising clinical applications. [Display omitted] [ABSTRACT FROM AUTHOR]

Repulsive guidance molecule A (RGMa) is an inhibitor of neuronal growth and survival which is upregulated in the damaged central nervous system following acute spinal cord injury (SCI), traumatic brain injury, acute ischemic stroke (AIS), and other neuropathological conditions. Neutralization of RGMa is neuroprotective and promotes neuroplasticity in several preclinical models of neurodegeneration and injury including multiple sclerosis, AIS, and SCI. Given the limitations of current treatments for AIS due to narrow time windows to intervention (TTI), and restrictive patient selection criteria, there is significant unmet need for therapeutic agents that enable tissue survival and repair following acute ischemic damage for a broader population of stroke patients. In this preclinical study, we evaluated whether elezanumab, a human anti-RGMa monoclonal antibody, could improve neuromotor function and modulate neuroinflammatory cell activation following AIS with delayed intervention times up to 24 h using a rabbit embolic permanent middle cerebral artery occlusion model (pMCAO). In two replicate 28-day pMCAO studies, weekly intravenous infusions of elezanumab, over a range of doses and TTIs of 6 and 24 h after stroke, significantly improved neuromotor function in both pMCAO studies when first administered 6 h after stroke. All elezanumab treatment groups, including the 24 h TTI group, had significantly less neuroinflammation as assessed by microglial and astrocyte activation. The novel mechanism of action and potential for expanding TTI in human AIS make elezanumab distinct from current acute reperfusion therapies, and support evaluation in clinical trials of acute CNS damage to determine optimal dose and TTI in humans. A: Ramified/resting astrocytes and microglia in a normal, uninjured rabbit brain. B: Rabbit pMCAO brain illustrating lesion on right side of brain (red), surrounded by penumbra (pink) during acute phase post stroke, with minimal injury to left brain hemisphere. Penumbra characterized by activated astrocytes and microglia (region in crosshair within circle), with upregulation of free and bound RGMa. C: Elezanumab binds to both free and bound RGMa, preventing full activation of astrocytes and microglia. D: Elezanumab is efficacious in rabbit pMCAO with a 4 × larger TTI window vs. tPA (6 vs. 1.5 h, respectively). In human AIS, tPA is approved for a TTI of 3-4.5 h. Elezanumab is currently being evaluated in a clinical Ph2 study of AIS to determine the optimal dose and TTI (NCT04309474). [ABSTRACT FROM AUTHOR]

ABSTRACT Background Aquaporin‐4 (AQP4), predominantly expressed in astrocytes, has been implicated in the development of brain edema following ischemic events. However, its role in post‐stroke neuroinflammation is not fully understood. Methods Using a middle cerebral artery occlusion (MCAO) mouse model, we assessed AQP4's role in post‐stroke inflammation. Brain tissue slices from male C57BL/6 mice were subjected to immunohistochemistry and western blot post‐MCAO. Additionally, primary astrocytes were isolated for quantitative real‐time PCR and immunofluorescence assays to evaluate the expression of inflammatory markers glial fibrillary acidic protein (GFAP) and AQP4. AQP4 modulation was achieved using viral knockdown and overexpression methods. Neuronal damage was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests in co‐culture studies. Results MCAO mice exhibited a significant upregulation in GFAP. This reactive astrogliosis corresponded with an elevation in inflammatory markers. AQP4 expression responded to this inflammatory trend, peaking at 6 h after OGD and returning to baseline levels at 24 and 48 h. Co‐culture experiments revealed that AQP4(+) astrocytes exacerbated injury in OGD‐treated neurons, as evidenced by increased TUNEL positivity and apoptotic events. Conversely, AQP4(−) astrocytes appeared to have a protective effect. Knockdown of AQP4 resulted in reduced post‐OGD inflammatory response, whereas AQP4 overexpression intensified the injury to neurons post‐OGD. In vivo experiments also confirmed that AQP4 inhibitor TGN‐020 reduced and overexpression of AQP4 increased behavioral abnormalities and brain infarcts. Conclusion Our findings underscore AQP4's pivotal role in modulating post‐stroke neuroinflammation. Targeting AQP4 may present a novel therapeutic avenue for mitigating ischemia‐induced neuronal damage.

Abstract Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation.

Motor rehabilitation strategies after unilateral stroke suggest that the immobilization of the healthy, unimpaired limb can promote the functional recovery of a paretic limb. In rodents, this has been modeled using casts, harnesses, and other means of restricting the use of the non-paretic forelimb in models of experimental stroke. Here, we evaluated an alternative approach, using botulinum toxin injections to limit the function of the non-paretic forelimb. Adult male rats were subjected to permanent ligation of the left distal middle cerebral artery, resulting in right forelimb paresis. The rats were then subjected to: (1) no treatment; (2) botulinum toxin injections 1 day post stroke; or (3) cast placement 5 days post stroke. Casts were removed after 5 weeks, while the botulinum toxin injection effectively immobilized subjects for approximately the same duration. Rats with bilateral forelimb impairment due to the stroke plus casting or botulinum injections were still able to feed and groom normally. Both immobilization groups showed modest recovery following the stroke compared to those that did not receive immobilization, but the casting approach led to unacceptable levels of animal stress. The botulinum toxin approach to limb immobilization had both advantages and disadvantages over traditional physical limb immobilization. The major advantage was that it was far less stress-inducing to the subject animals and appeared to be well tolerated. A disadvantage was that the paresis took roughly 10 weeks to fully resolve, and any degree of residual paresis could confound the interpretation of the behavioral assessments.

Metabolic disorders are risk factors for stroke exacerbating subsequent complications. Rapidly after brain injury, a glial scar forms, preventing excessive inflammation and limiting axonal regeneration. Despite the growing interest in wound healing following brain injury, the formation of a glial scar in the context of metabolic disorders is poorly documented. In this study, we used db/db mice to investigate the impact of metabolic perturbations on brain repair mechanisms, with a focus on glial scarring. First, we confirmed the development of obesity, poor glucose regulation, hyperglycaemia and liver steatosis in these mice. Then, we observed that 3 days after a 30‐min middle cerebral artery occlusion (MCAO), db/db mice had larger infarct area compared with their control counterparts. We next investigated reactive gliosis and glial scar formation in db/+ and db/db mice. We demonstrated that astrogliosis and microgliosis were exacerbated 3 days after stroke in db/db mice. Furthermore, we also showed that the synthesis of extracellular matrix (ECM) proteins (i.e., chondroitin sulphate proteoglycan, collagen IV and tenascin C) was increased in db/db mice. Consequently, we demonstrated for the first time that metabolic disorders impair reactive gliosis post‐stroke and increase ECM deposition. Given that the damage size is known to influence glial scar, this study now raises the question of the direct impact of hyperglycaemia/obesity on reactive gliosis and glia scar. It paves the way to promote the development of new therapies targeting glial scar formation to improve functional recovery after stroke in the context of metabolic disorders. [ABSTRACT FROM AUTHOR]

This study utilized middle cerebral artery occlusion (MCAO) mouse models and HT‐22 cell oxygen and glucose deprivation/reoxygenation (OGD/R) models to investigate the therapeutic effects of melatonin on ischemic brain injury. In the experiments, MCAO mice were treated with 5 and 10 mg/kg doses of melatonin, and H‐T22 cells underwent OGD/R treatment and were administered different concentrations of melatonin. The results showed that melatonin significantly reduced ischemic brain area, neural damage, cerebral edema, and neuronal apoptosis in MCAO mice. In the HT‐22 cell model, melatonin also improved cell proliferation ability, reduced apoptosis, and ROS production. Further mechanistic studies found that melatonin exerts protective effects by inhibiting ferroptosis, an iron‐dependent form of regulated cell death, through regulation of the ACSL4/CYP1B1 pathway. In MCAO mice, melatonin decreased lipid peroxidation, ROS production, and ACSL4 protein expression. Overexpression of CYP1B1 increased ACSL4 ubiquitination and degradation, thereby increasing cell tolerance to ferroptosis, reducing ACSL4 protein levels, and decreasing ROS production. CYP1B1 knockdown obtained opposite results. The CYP1B1 metabolite 20‐HETE induces expression of the E3 ubiquitin ligase FBXO10 by activating PKC signaling, which promotes ACSL4 degradation. In the OGD/R cell model, inhibition of CYP1B1 expression reversed the therapeutic effects of melatonin. In summary, this study demonstrates that melatonin protects the brain from ischemic injury by inhibiting ferroptosis through regulation of the ACSL4/CYP1B1 pathway, providing evidence for new therapeutic targets for ischemic brain injury. [ABSTRACT FROM AUTHOR]

Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation. [ABSTRACT FROM AUTHOR]

Cerebral ischemia/reperfusion causes high disability, recurrence, and mortality. Ischemic stroke is a powerful stimulus that triggers significant microglia activation. Ginsenoside Rb1 (GS-Rb1) has been demonstrated to have neuroprotective effects in the central nervous system. In this study, the effects of GS-Rb1 against cerebral ischemia/reperfusion were explored. A mouse model of middle cerebral artery occlusion (MCAO) was used to mimic the cerebral ischemia/reperfusion. Mice in MCAO + GS-Rb1 groups received 5, 10, or 20 mg/kg GS-Rb1 through intraperitoneal injection. Modified neurological severity scoring (mNSS) showed neurological function, while the open field test tested the anxiety-like behaviors. Cognitive impairment was evaluated by Morris water maze. Protein levels were evaluated by ELISA and Western blot and mRNA levels were analyzed by qRT-PCR. When compared to the MCAO mice, mice in the MCAO + GS-Rb1 group had significantly lower mNSS scores and less brain water content. GS-Rb1 alleviated both cognitive impairment and anxiety and inhibited microglial activation in the cerebral ischemia/reperfusion model. GS-Rb1 enhanced M2-type microglia polarization while inhibiting M1-type microglia polarization. In summary, we observed that GS-Rb1 had neuro-protective effects in a cerebral ischemia/reperfusion mouse model through regulating the microglia polarization. [ABSTRACT FROM AUTHOR]

Background: Hibiscus sabdariffa var. altissima, which is known for its high-quality fiber, is commonly used in Traditional Chinese Medicine (TCM) for its diuretic, choleretic, analgesic, antitussive, and hypotensive effects. Furthermore, the flowers have been used to treat diabetes, hypertension, atherosclerosis, and obesity. However, there are no studies assessing its neuroprotective effects on cerebral ischemia, unlike the sabdariffa variety known for its neuroprotective effects. Therefore, this study was aimed to investigate the potential efficacy of Hibiscus sabdariffa var. altissima extract (HAS) and its underlying mechanism on oxidative stress and neuroinflammation during cerebral ischemia-reperfusion (I/R) injury. Methods: A model of cerebral ischemia was established in male Wistar rats through middle cerebral artery occlusion (MCAO) and reperfusion. Rats were randomly divided into the sham, IR, IR + HSA100, IR + HSA200, IR + HSA400 and IR + Eda groups and were treated for 14 consecutive days. The neurological deficit score and the cylinder test were performed to assess neurological impairment. Oxidative stress was determined by measuring the levels of malondialdehyde (MDA) and antioxidant markers such as reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) were measured to evaluate antioxidant activities. In addition, the levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), were also determined. Results: Our results demonstrated that HSA significantly ameliorated neurological impairment and reduced the volume of brain infarct. HSA also decreased the levels of MDA and enhanced the antioxidant activities of GSH, SOD and CAT in brain tissues. Furthermore, HSA decreased the expression of the proinflammatory cytokines TNF-α, IL-1β and IL-6 in the serum. Conclusion: These findings demonstrated that HSA exhibited a potential neuroprotective effect against cerebral I/R injury, possibly by improving oxidative stress and attenuating inflammatory responses. Therefore, HSA could be used as a potential therapeutic option for cerebral ischemic injuries.

Background: Buyang Huanwu Decoction (BHD) is widely used in Chinese clinical practice for the treatment and prevention of ischemic cerebral vascular diseases. This study was designed to investigate the effects of BHD on ischemic stroke (IS) and its underlying mechanism. Methods: The middle cerebral artery occlusion (MCAO) rat model and oxygen-glucose deprivation and reoxygenation (OGD/R) rat brain microvascular endothelial cell (RBMVEC) models were established. Brain infarction size and neurological score were calculated following MCAO surgery. Evans blue was used to measure blood brain barrier (BBB) permeability. Cell counting kit-8 (CCK-8) and TUNEL assays were performed to evaluate the cell viability and apoptosis of RBMVECs. Dual-luciferase reporter assay was used to analyze the transcriptional activities of apoptosis-related genes. Results: Results showed that higher infarction volume, neurological scores, and BBB permeability in the MCAO group rats were reduced after BHD treatment. Drug serum (DS) treatment had no impact on the normal RBMVECs’ cell viability and cell apoptosis. Besides, DS treatment decreased the lactate production, glucose uptake, and extracellular acidification rate in normal and OGD/R-induced RBMVECs. DS treatment downregulated the protein levels of pan-lysine lactylation (kla), histone H3 lysine 18 lactylation (H3K18la), and the transcriptional of apoptotic protease activating factor-1 (Apaf-1) in OGD/R-treated RBMVECs. In addition, Apaf-1 overexpression decreased cell viability and increased apoptosis and glycolysis activity of OGD/R-treated RBMVECs. Conclusion: In summary, BHD inhibited glycolysis and apoptosis via suppressing the pan-kla and H3K18la protein levels and the Apaf-1 transcriptional activity, thus restraining the progression of IS.

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. 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. 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 < 0.05. 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). 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.

This study aimed to clarify the roles and underlying mechanisms of luteolin in the progression of cerebral ischemia/reperfusion injury (CIRI).

ObjectiveWe tested the hypothesis that blocking pro-inflammatory platelet-activating factor receptor (PAFR) with LAU-0901 (LAU) plus administering a selected docosanoid, aspirin-triggered neuroprotectin D1 (AT-NPD1), which activates cell-survival pathways after middle cerebral artery occlusion (MCAo), would lead to neurological recovery. Dose-response and therapeutic window were investigated.Materials and methodsMale SD rats were subjected to 2 hours of MCAo. Behavior testing (days 1-7) and ex vivo MRI on day 7 were conducted. In dose-response, rats were treated with LAU (45 and 60 mg/kg; IP), AT-NPD1 (111, 222, 333 µg/kg; IV), LAU+AT-NPD1 (LAU at 3 hours and AT-NPD1 at 3.15 hours) or vehicle. In the therapeutic window, vehicle, LAU (60 mg/kg), AT-NPD1 (222 µg/kg), and LAU+AT-NPD1 were administered at 3, 4, 5, and 6 hours after onset of MCAo.ResultsLAU and AT-NPD1 treatments alone improved behavior by 40-42% and 20-30%, respectively, and LAU+AT-NPD1 by 40% compared to the vehicle group. T2-weighted imaging (T2WI) volumes were reduced with all doses of LAU and AT-NPD1 by 73-90% and 67-83% and LAU+AT-NPD1 by 94% compared to vehicle. In the therapeutic window, LAU+AT-NPD1, when administered at 3, 4, 5, and 6 hours, improved behavior by 50, 56, 33, and 26% and reduced T2WI volumes by 93, 90, 82, and 84% compared to vehicle.ConclusionsWe have shown here for the first time that LAU plus AT-NPD1 treatment affords high-grade neuroprotection in MCAo, equaling or exceeding that afforded by LAU or AT-NPD1 alone at considerably moderate doses. It has a broad therapeutic window extending to 6 hours after stroke onset.

The mechanisms of cerebral ischemia–reperfusion injury (IRI) limitation by remote ischemic postconditioning (RPC) are of interest because the latter may positively influence functional recovery after cerebral ischemia. The study was aimed to assess the role of plasma proteins syndecan-1 (SDC-1) and annexin-5 (ANXA-5) in limiting cerebral IRI by RPC in a middle cerebral artery occlusion (MCAO) model in male Wistar rats. The animals were randomized into three groups: (1) control sham-operated rats (SO group), (2) rats with 30-min MCAO-induced ischemia (MCAO group), (3) rats with 30-min MCAO-induced ischemia followed by RPC (MCAO + RPC group). SDC-1 plasma levels, as assessed by ELISA, in MCAO (41.4 ± 1.3 ng/mL) and MCAO + RPC (67.8 ± 5.8 ng/mL) rats were, respectively, by 30% (p < 0.05) and 112% (p < 0.01) higher than in SO control (31.9 ± 1.1 ng/mL). No intergroup differences in ANXA-5 plasma levels were detected. Cerebral infarct volume in MCAO and MCAO + RPC rats accounted for 32.0 ± 2.5 and 13.6 ± 1.3% of the total brain volume, respectively (p < 0.05). Cerebral edema volume in MCAO and MCAO + RPC rats were 47.0 ± 3.3 and 16.0 ± 2.1%, respectively (p < 0.05). In MCAO + RPC animals, correlation analysis revealed a high direct correlation between infarct size and right forelimb muscular strength (grip strength test, 3.9 ± 0.8 H, p > 0.5; KK = 0.72, p < 0.05) and a very high inverse correlation between infarct size and right hindlimb capillary blood flow (Doppler flowmetry, r = –0.98, p < 0.01). Animals' neurologic deficits were assessed by the Garcia scoring scale, while their locomotor coordination was assessed in the Rotarod test. It is assumed that SDC-1 plasma protein may serve as a potential regulator of the infarct size-limiting effect of remote ischemic postconditioning, which is determinative for functional recovery. [ABSTRACT FROM AUTHOR]

Aims: To distinguish between the genuine cellular impact of the ischemic cascade by leukocytes and unspecific effects of edema and humoral components, two knock‐in mouse lines were utilized. Mouse lines Y731F and Y685F possess point mutations in VE‐cadherin, which lead to a selective inhibition of transendothelial leukocyte migration or impaired vascular permeability. Methods: Ischemic stroke was induced by a model of middle cerebral artery occlusion. Analysis contained structural outcomes (infarct volume and extent of brain edema), functional outcomes (survival analysis, rotarod test, and neuroscore), and the extent and spatial distribution of leukocyte migration (heatmaps and fluorescence‐activated cell sorting (FACS) analysis). Results: Inhibition of transendothelial leukocyte migration as in Y731F mice leads to smaller infarct volumes (52.33 ± 4719 vs. 70.43 ± 6483 mm3, p =.0252) and improved motor skills (rotarod test: 85.52 ± 13.24 s vs. 43.06 ± 15.32 s, p =.0285). An impaired vascular permeability as in Y685F mice showed no effect on structural or functional outcomes. Both VE‐cadherin mutations did not influence the total immune cell count or spatial distribution in ischemic brain parenchyma. Conclusion: Selective inhibition of transendothelial leukocyte migration by VE‐cadherin mutation after ischemic stroke in a mouse model leads to smaller infarct volumes and improved motor skills. [ABSTRACT FROM AUTHOR]

Ischemic stroke refers to the sudden loss of blood flow in a specific area of the brain. It is the fifth leading cause of mortality and the leading cause of permanent disability. The transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2) controls the production of several antioxidants and protective proteins and it has been investigated as a possible pharmaceutical target for reducing harmful oxidative events in brain ischemia. Each cell type exhibits different roles and behaviors in different phases post‐stroke, which is comprehensive yet important to understand to optimize management strategies and goals for care for stroke patients. In this review, we comprehensively summarize the protective effects of Nrf2 in experimental ischemic stroke, emphasizing the role of Nrf2 in different cell types including neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells during acute and chronic phases of stroke and providing insights on the neuroprotective role of Nrf2 on each cell type throughout the long term of stroke care. We also highlight the importance of targeting Nrf2 in clinical settings while considering a variety of important factors such as age, drug dosage, delivery route, and time of administration. [ABSTRACT FROM AUTHOR]

Numerous studies have shown that an enriched environment can promote ischemic stroke and improve cognitive function. In addition, white matter is closely related to cognitive function. The effects and mechanisms of the enriched environment on white matter recovery after stroke have not been elucidated. This study will analyse the effects of the enriched environment on white matter and cognitive function in the post-stroke brain from the perspective of astrocytes and their secretions. Stroke models were used for middle cerebral artery occlusion model. post-operative rats were divided into sham-operated, standard and enriched environment groups. The degree of cerebral infarction was assessed by TTC staining and the degree of white matter damage was assessed by Luxol-Fast Blue staining. The prognosis after stroke was assessed using the longa score and Morris water maze test. Western Blot and immunofluorescence were used to quantify and localize astrocytes and their associated secretory factors and myelin protein markers. We found that ischemic stroke can cause severe demyelination. After EE treatment, there was a significant increase in cerebral remyelination and a significant improvement in neurological and cognitive functions. Astrocyte, BDNF, and VEGF expression were significantly higher than in rats in the standard circumstances of stroke model. These data suggest that the enriched environment contributes to brain white matter recovery and improvement of cognitive function after stroke. The mechanism is related to astrocytes and their secretions. EE can activate astrocytes to secrete BDNF and VEGF, which may be crucial to promote white matter recovery. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Cerebral ischemia (CI) is a condition in which metabolic stress increases when blood flow is interrupted in a part of the brain, resulting in oxygen and glucose deprivation. It is known that asprosin (Asp), secreted from adipose tissue during fasting, has an effect on some metabolic processes such as apoptosis, autophagy, and glucose metabolism. This study aimed to explain which of the cell death/survival Asp induces in the CI/reperfusion model. MATERIALS AND METHODS: In the study, 48 male Wistar Albino rats were divided into 6 groups: Sham, CI, Asp+CI, CI+Asp, CI+Asp+3- MA, and Asp+CI+3-MA (n=48). CI was created using the intraluminal filament technique for 60 minutes, autophagy inhibitor 3-MA (15 mg/kg/ day) and Asp (1 µ g/kg/day) injections were administered 3 days before or 3 days during reperfusion. Beclin-1, ATG5, ATG7, p62, Bcl-2, Bax, active-caspase-3, and active-caspase-9 protein levels from brain tissues were determined by the Western-Blot method. The infarct area was determined by triphenyl tetrazolium chloride (TTC) staining. The Kruskal-Wallis' test was used to compare differences between groups. p<0.05 was considered statistically significant. RESULTS: Compared to the Sham group, the increase in ischemic area and the decrease in Beclin-1, ATG-5, ATG-7, Bcl-2, Bax, active-caspase-3 and active-caspase-9 levels in the CI groups are statistically significant (p<0.05). The increase of Beclin-1, ATG-7, Bcl-2, and Bax levels in the Asp groups is statistically significant compared to the CI group (p<0.05). When Asp+CI groups and CI+Asp groups are compared, an increase in Beclin-1 levels in the Asp+CI group and the increase in Bcl-2, Bax, active- caspase-3/9 and ATG-5 levels in the CI+Asp groups are statistically significant (p<0.05). CONCLUSIONS: Asp has protective and therapeutic effects against CI/R damage. While applying Asp before ischemia activates the autophagy pathway more, applying it after ischemia protects the neuronal death/survival balance by activating the apoptosis pathway more. [ABSTRACT FROM AUTHOR]

[Display omitted] • Urolithin B effectively mitigates cerebral ischemia–reperfusion injury in rats. • Urolithin B attenuates oxidative stress via activation of the Nrf2 pathway. • We consider Urolithin B may also be beneficial in a clinical setting. Ischemia-reperfusion (IR) induces a wide range of irreversible injuries. Cerebral IR injury (IRI) refers to additional brain tissue damage that occurs after blood flow is restored following cerebral ischemia. Currently, no established methods exist for treating IRI. Oxidative stress is recognized as a primary mechanism initiating IRI and a crucial focal target for its treatment. Urolithin B, a metabolite derived from ellagitannins, antioxidant polyphenols, has demonstrated protective effects against oxidative stress in various disease conditions. However, the precise mechanism underlying UB's effect on IRI remains unclear. In our current investigation, we assessed UB's ability to mitigate neurological functional impairment induced by IR using a neurological deficit score. Additionally, we examined cerebral infarction following UB administration through TTC staining and neuron Nissl staining. UB's inhibition of neuronal apoptosis was demonstrated through the TUNEL assay and Caspase-3 measurement. Additionally, we examined UB's effect on oxidative stress levels by analyzing malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, and immunohistochemistry analysis of inducible nitric oxide synthase (iNOS) and 8-hydroxyl-2′-deoxyguanosine (8-OHdG). Notably, UB demonstrated a reduction in oxidative stress levels. Mechanistically, UB was found to stimulate the Nrf2/HO-1 signaling pathway, as evidenced by the significant reduction in UB's neuroprotective effects upon administration of ATRA, an Nrf2 inhibitor. In summary, UB effectively inhibits oxidative stress induced by IR through the activation of the Nrf2/HO-1 signaling pathway. These findings suggest that UB holds promise as a therapeutic agent for the treatment of IRI. [ABSTRACT FROM AUTHOR]

• Activation of the innate immune system drives outcomes after stroke. • PD-1+ monocytes in the blood of stroke patients correlate with cerebral edema. • PD-L1 administration decreases edema, improves survival, and promotes recovery. • Myeloid-specific PD-1 knockout abrogates the PD-L1 treatment effect. • PD-1 signaling skews blood monocytes away from an inflammatory phenotype prior to tissue infiltration. Acute cerebral ischemia triggers a profound inflammatory response. While macrophages polarized to an M2-like phenotype clear debris and facilitate tissue repair, aberrant or prolonged macrophage activation is counterproductive to recovery. The inhibitory immune checkpoint Programmed Cell Death Protein 1 (PD-1) is upregulated on macrophage precursors (monocytes) in the blood after acute cerebrovascular injury. To investigate the therapeutic potential of PD-1 activation, we immunophenotyped circulating monocytes from patients and found that PD-1 expression was upregulated in the acute period after stroke. Murine studies using a temporary middle cerebral artery (MCA) occlusion (MCAO) model showed that intraperitoneal administration of soluble Programmed Death Ligand-1 (sPD-L1) significantly decreased brain edema and improved overall survival. Mice receiving sPD-L1 also had higher performance scores short-term, and more closely resembled sham animals on assessments of long-term functional recovery. These clinical and radiographic benefits were abrogated in global and myeloid-specific PD-1 knockout animals, confirming PD-1+ monocytes as the therapeutic target of sPD-L1. Single-cell RNA sequencing revealed that treatment skewed monocyte maturation to a non-classical Ly6Clo, CD43hi, PD-L1+ phenotype. These data support peripheral activation of PD-1 on inflammatory monocytes as a therapeutic strategy to treat neuroinflammation after acute ischemic stroke. [ABSTRACT FROM AUTHOR]

Aims: Sleep disorders are prevalent among stroke survivors and impede stroke recovery, yet they are still insufficiently considered in the management of stroke patients, and the mechanisms by which they occur remain unclear. There is evidence that boosting phasic GABA signaling with zolpidem during the repair phase improves stroke recovery by enhancing neural plasticity; however, as a non‐benzodiazepine hypnotic, the effects of zolpidem on post‐stroke sleep disorders remain unclear. Method: Transient ischemic stroke in male rats was induced with a 30‐minute middle cerebral artery occlusion. Zolpidem or vehicle was intraperitoneally delivered once daily from 2 to 7 days after the stroke, and the electroencephalogram and electromyogram were recorded simultaneously. At 24 h after ischemia, c‐Fos immunostaining was used to assess the effect of transient ischemic stroke and acute zolpidem treatment on neuronal activity. Results: In addition to the effects on reducing brain damage and mitigating behavioral deficits, repeated zolpidem treatment during the subacute phase of stroke quickly ameliorated circadian rhythm disruption, alleviated sleep fragmentation, and increased sleep depth in ischemic rats. Immunohistochemical staining showed that in contrast to robust activation in para‐infarct and some remote areas by 24 h after the onset of focal ischemia, the activity of the ipsilateral suprachiasmatic nucleus, the biological rhythm center, was strongly suppressed. A single dose of zolpidem significantly upregulated c‐Fos expression in the ipsilateral suprachiasmatic nucleus to levels comparable to the contralateral side. Conclusion: Stroke leads to suprachiasmatic nucleus dysfunction. Zolpidem restores suprachiasmatic nucleus activity and effectively alleviates post‐stroke sleep disturbances, indicating its potential to promote stroke recovery. [ABSTRACT FROM AUTHOR]

Background: Ischemic stroke (IS) is a cerebrovascular disease with high incidence and mortality. White matter repair plays an important role in the long‐term recovery of neurological function after cerebral ischemia. Neuroprotective microglial responses can promote white matter repair and protect ischemic brain tissue. Aims: The aim of this study was to investigate whether hypoxic postconditioning (HPC) can promote white matter repair after IS, and the role and mechanism of microglial polarization in white matter repair after HPC treatment. Materials & Methods: Adult male C57/BL6 mice were randomly divided into three groups: Sham group (Sham), MCAO group (MCAO), and hypoxic postconditioning group (HPC). HPC group were subjected to 45 min of transient middle cerebral artery occlusion (MCAO) immediately followed by 40 min of HPC. Results: The results showed that HPC reduced the proinflammatory level of immune cells. Furthermore, HPC promoted the transformation of microglia to anti‐inflammatory phenotype on the third day after the procedure. HPC promoted the proliferation of oligodendrocyte progenitors and increased the expression of myelination‐related proteins on the 14th day. On the 28th day, HPC increased the expression of mature oligodendrocytes, which enhanced myelination. At the same time, the motor neurological function of mice was restored. Discussion: During the acute phase of cerebral ischemia, the function of proinflammatory immune cells was enhanced, long‐term white matter damage was aggravated, and motor sensory function was decreased. Conclusion: HPC promotes protective microglial responses and white matter repair after MCAO, which may be related to the proliferation and differentiation of oligodendrocytes. [ABSTRACT FROM AUTHOR]

Neuroinflammation is closely related to prognosis in ischemic stroke. Microglia are the main immune cells in the nervous system. Under physiological conditions, microglia participate in clearance of dead cells, synapse pruning and regulation of neuronal circuits to maintain the overall health of the nervous system. Once ischemic stroke occurs, microglia function in the occurrence and progression of neuroinflammation. Therefore, the regulation of microglia-mediated neuroinflammation is a potential therapeutic strategy for ischemic stroke. The anti-inflammatory activity of gypenosides (GPs) has been confirmed to be related to the activity of microglia in other neurological diseases. However, the role of GPs in neuroinflammation after ischemic stroke has not been studied. In this study, we investigated whether GPs could reduce neuroinflammation by regulating microglia and the underlying mechanism through qRT-PCR and western blot. Results showed that GPs pretreatment mitigated blood–brain barrier (BBB) damage in the mice subjected to middle cerebral artery occlusion (MCAO) and improved motor function. According to the results of immunofluorescence staining, GPs pretreatment alleviated neuroinflammation in MCAO mice by reducing the number of microglia and promoting their phenotypic transformation from M1 to M2. Furthermore, GPs pretreatment reduced the number of astrocytes in the penumbra and inhibited their polarization into the A1 type. We applied oxygen and glucose deprivation (OGD) on BV2 cells to mimic ischemic conditions in vitro and found similar effect as that in vivo. At the molecular level, the STAT-3/HIF1-α and TLR-4/NF-κB/HIF1-α pathways were involved in the anti-inflammatory effects of GPs in vitro and in vivo. Overall, this research indicates that GPs are potential therapeutic agents for ischemic stroke and has important reference significance to further explore the possibility of GPs application in ischemic stroke. [ABSTRACT FROM AUTHOR]

Ischemic stroke remains a major cause of disability and death. Kaempferol (Kae) is a neuroprotective flavonoid compound. Thus, this study aimed to explore the impact of Kae on cerebral infarction. We generated the middle cerebral artery occlusion (MCAO) mouse model to study the effects of Kae on infarction volume and neurological function. The oxygen and glucose deprivation (OGD)/ reoxygenation (R) model of neural stem cells (NSCs) was established to study the effects of Kae on cell viability, migration, and apoptosis. Cell processes were assessed by cell counting kit-8, Transwell assay, flow cytometry, and TUNEL analysis. The molecular mechanism was assessed using the Western blot. The results indicated that Kae attenuated MCAO-induced cerebral infarction and neurological injury. Besides, Kae promoted cell viability and migration and inhibited apoptosis of OGD/R-treated NSCs. Moreover, OGD/R suppressed total O‐GlcNAcylation level and O‐GlcNAcylation of β-catenin, thereby suppressing the Wnt/β-catenin pathway, whereas Kae reversed the suppression. Inactivation of the Wnt/β-catenin pathway abrogated the biological functions of NSCs mediated by Kae. In conclusion, Kae suppressed cerebral infarction by facilitating NSC viability, migration, and inhibiting apoptosis. Mechanically, Kae promoted O‐GlcNAcylation of β-catenin to activate the Wnt/β-catenin pathway. Kae may have a lessening effect on ischemic stroke. [ABSTRACT FROM AUTHOR]

The fetus is strongly dependent on nutrients from the mother, including polyunsaturated fatty acids (PUFA). In adult animals, n-3 PUFA ameliorates stroke-mediated brain injury, but the modulatory effects of different PUFA content in maternal diet on focal arterial stroke in neonates are unknown. This study explored effects of maternal n-3 or n-6 enriched PUFA diets on neonatal stroke outcomes. Pregnant mice were assigned three isocaloric diets until offspring reached postnatal day (P) 10-13: standard, long-chain n-3 PUFA (n-3) or n-6 PUFA (n-6) enriched. Fatty acid profiles in plasma and brain of mothers and pups were determined by gas chromatography-mass spectrometry and cytokines/chemokines by multiplex protein analysis. Transient middle cerebral artery occlusion (tMCAO) was induced in P9-10 pups and cytokine and chemokine accumulation, caspase-3 and calpain-dependent spectrin cleavage and brain infarct volume were analyzed. The n-3 diet uniquely altered brain lipid profile in naïve pups. In contrast, cytokine and chemokine levels did not differ between n-3 and n-6 diet in naïve pups. tMCAO triggered accumulation of inflammatory cytokines and caspase-3-dependent and -independent cell death in ischemic-reperfused regions in pups regardless of diet, but magnitude of neuroinflammation and caspase-3 activation were attenuated in pups on n-3 diet, leading to protection against neonatal stroke. In conclusion, maternal/postnatal n-3 enriched diet markedly rearranges neonatal brain lipid composition and modulates the response to ischemia. While standard diet is sufficient to maintain low levels of inflammatory cytokines and chemokines under physiological conditions, n-3 PUFA enriched diet, but not standard diet, attenuates increases of inflammatory cytokines and chemokines in ischemic-reperfused regions and protects from neonatal stroke.

Abstract In experimental stroke, ovariectomized (OVX) adult rats have larger infarct volumes and greater sensory-motor impairment as compared to ovary-intact females and is usually interpreted to indicate that ovarian hormones are neuroprotective for stroke. Previous work from our lab shows that middle-aged, acyclic reproductively senescent (RS) females have worse stroke outcomes as compared to adult (normally cycling) females. We hypothesized that if loss of ovarian estrogen is the critical determinant of stroke outcomes, then ovary-intact middle-aged acyclic females, who have reduced levels of estradiol, should have similar stroke outcomes as age-matched OVX. Instead, the data demonstrated that OVX RS animals showed better sensory-motor function after stroke and reduced infarct volume as compared to ovary-intact females. Inflammatory cytokines were decreased in the aging ovary after stroke as compared to non-stroke shams, which led to the hypothesis that immune cells may be extravasated from the ovaries post-stroke. Flow cytometry indicated reduced overall T cell populations in the aging ovary after middle cerebral artery occlusion (MCAo), with a paradoxical increase in regulatory T cells (Tregs) and M2-like macrophages. Moreover, in the brain, OVX RS animals showed increased Tregs, increased M2-like macrophages, and increased MHC II + cells as compared to intact RS animals, which have all been shown to be correlated with better prognosis after stroke. Depletion of ovary-resident immune cells after stroke suggests that there may be an exaggerated response to ischemia and possible increased burden of the inflammatory response via extravasation of these cells into circulation. Increased anti-inflammatory cells in the brain of OVX RS animals further supports this hypothesis. These data suggest that stroke severity in aging females may be exacerbated by the aging ovary and underscore the need to assess immunological changes in this organ after stroke.

Abstract Aims To distinguish between the genuine cellular impact of the ischemic cascade by leukocytes and unspecific effects of edema and humoral components, two knock‐in mouse lines were utilized. Mouse lines Y731F and Y685F possess point mutations in VE‐cadherin, which lead to a selective inhibition of transendothelial leukocyte migration or impaired vascular permeability. Methods Ischemic stroke was induced by a model of middle cerebral artery occlusion. Analysis contained structural outcomes (infarct volume and extent of brain edema), functional outcomes (survival analysis, rotarod test, and neuroscore), and the extent and spatial distribution of leukocyte migration (heatmaps and fluorescence‐activated cell sorting (FACS) analysis). Results Inhibition of transendothelial leukocyte migration as in Y731F mice leads to smaller infarct volumes (52.33 ± 4719 vs. 70.43 ± 6483 mm3, p = .0252) and improved motor skills (rotarod test: 85.52 ± 13.24 s vs. 43.06 ± 15.32 s, p = .0285). An impaired vascular permeability as in Y685F mice showed no effect on structural or functional outcomes. Both VE‐cadherin mutations did not influence the total immune cell count or spatial distribution in ischemic brain parenchyma. Conclusion Selective inhibition of transendothelial leukocyte migration by VE‐cadherin mutation after ischemic stroke in a mouse model leads to smaller infarct volumes and improved motor skills.

Background: Numerous studies showed that electroacupuncture at Shenting (GV24) and Baihui (GV20) could improve cognitive function in MCAO rats. However, the underlying molecular protein mechanisms need to be further investigated, including the effects of electroacupuncture at Shenting and Baihui points on MCAO-related gene and protein expression. Methods: Sprague-Dawley (SD) adult male rats (n = 40) were randomly divided into the following four groups: sham group (SHAM/S), Middle cerebral artery occlusion (MCAO/M) group, electroacupuncture at Shenting and Baihui group (EA/E) and acupuncture at non-meridian and non-acupuncture points (NON/N) (n = 10 per group). After one week of electroacupuncture treatment, the protein expression of hippocampus-related genes in the brain regions of rats in all was measured. Three samples from each groups were used to identify potential biomarkers in the hippocampus of the rat brain region using tandem mass labeling (TMT), liquid chromatography and functional enrichment clustering analysis of differentially expressed proteins (DEPs). The protein interactions network was analysed by enrichment clustering to explore the major and potential protein targets of electroacupuncture at Shenting and Baihui for the treatment of MCAO rats. Results: (1) Protein functional annotation and repeated quantitative analysis showed that protein expression in the E groups was closer to that in the S group, while the N group similarly to the M group. These demonstrated that electroacupuncture could regulate protein expression in the hippocampal region of MCAO rats to normalize the expression. (2) The difference in protein expression between groups quantified by the specific peptide could be hypothesized that the improvement of learning memory in MCAO rats after electroacupuncture of Shenting and Baihui was achieved by the downregulation of a class or a specific protein in hippocampus. (3) It was observed that the inter-synaptic regulatory proteins were the structural basis for the improvement of protein expression in MCAO rats by electroacupuncture, which could provide a new idea for the localization and characterization of specific proteins. Conclusions: Our findings provided valuable protein biomarkers and new ideas for future mechanistic studies on improving cognitive function in post-stroke cognitive impairment.

Background: Estimation of brain damage following an ischemic stroke is most often performed within the first few days after the insult, where large amounts of oedematous fluid have accumulated. This can potentially hamper correct measurement of infarcted area, since oedema formation poorly reflects infarct size. This study presents a non-invasive, easily applicable and reliable method to accurately predict long-term evolution and late-stage infarction. Objective: We performed a longitudinal analysis of brain infarct evolution after MCAO in mice, in order to determine whether water-compensated N-Acetylaspartate (NAA) levels in the infarct area, measured 24 h after the insult, is a suitable marker for late-stage infarction and thereby prognosis. Methods: Twenty mice were divided into 4 groups and scanned longitudinally at different time-points after MCAO, followed by euthanisation for histology: Group 1) MRI/MRS at day 1 after MCAO (n = 4), Group 2) MRI/MRS at days 1 and 7 after MCAO (n = 5), Group 3) MRI/MRS at days 1, 7, and 14 after MCAO (n = 3), and Group 4) MRI/MRS at days 1, 7, 14, and 28 after MCAO (n = 4). At days 1, 7, 14, and 28, NAA levels were correlated with histological determination of neuronal death based on Nissl and H&E stainings. Results: Twenty-four hours after the insult, NAA levels in the infarcted area decreased by 35 %, but steadily returned to normal after 28 days. In the acute phases, NAA levels strongly correlated with loss of Nissl substance (r2 = −0.874, p = 0.002), whereas NAA levels in later stages reflect glial metabolism and tissue reorganisation. Most importantly, NAA levels 24 h after MCAO was highly correlated with late stage infarction at days 14 and 28 (r2 = 0.73, p = 0.01), in contrast to T2 (r2 = 0.06, p = 0.59). Conclusions: By using a fixed voxel, which is easily positioned in the affected area, it is possible to obtain reliable measures of the extent of neuronal loss at early time points independent of oedema and brain deformation. Importantly, NAA levels 24 h after MCAO accurately reflects late-stage infarction, suggesting that NAA is a useful prognostic biomarker early after an ischemic stroke.

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

Neuroinflammation and oxidative stress damage are involved in the pathogenesis of cerebral ischemia–reperfusion injury (CIRI). Ferroptosis emerged as a new player in the regulation of lipid peroxidation processes. This study aimed at exploring the potential involvement of ciprofol on ferroptosis-associated CIRI and subsequent neurological deficits in the mouse model of transient cerebral ischemia and reperfusion. Cerebral ischemia was built in male C57BL/6 J wild-type (WT) and Nrf2-knockout (Nrf2 KO) mice in the manner of middle cerebral artery occlusion (MCAO) followed by reperfusion. Ciprofol improved autonomic behavior, alleviated reactive oxygen species output and ferroptosis-induced neuronal death by nucleus transportation of NFE2 like BZIP transcription factor 2 (Nrf2) and the promotion of heme oxygenase 1 (Ho-1), solute carrier family 7 member 11 (SLC7A11/xCT), and glutathione peroxidase 4 (GPX4). Additionally, ciprofol improved neurological scores and reduced infarct volume, brain water content, and necrotic neurons. Cerebral blood flow in MCAO-treated mice was also improved. Furthermore, absence of Nrf2 abrogated the neuroprotective actions of ciprofol on antioxidant capacity and sensitized neurons to oxidative stress damage. In vitro, the primary-cultured cortical neurons from mice were pre-treated with oxygen–glucose deprivation/reperfusion (OGD/R), followed by ciprofol administration. Ciprofol effectively reversed OGD/R-induced ferroptosis and accelerated transcription of GPX4 and xCT. In conclusion, we investigated the ciprofol-induced inhibition effect of ferroptosis-sheltered neurons from lipid preoxidation in the pathogenesis of CIRI via Nrf2-xCT-GPX4 signaling pathway. [ABSTRACT FROM AUTHOR]

Background. Cell therapy using neural progenitor cells (NPCs) is a promising approach for ischemic stroke treatment according to the results of multiple preclinical studies in animal stroke models. In the vast majority of conducted animal studies, the therapeutic efficacy of NPCs was estimated after intracerebral transplantation, while the information of the effectiveness of systemic administration is limited. Nowadays, several clinical trials aimed to estimate the safety and efficacy of NPCs transplantation in stroke patients were also conducted. In these studies, NPCs were transplanted intracerebrally in the subacute/chronic phase of stroke. The results of clinical trials confirmed the safety of the approach, however, the degree of functional improvement (the primary efficacy endpoint) was not sufficient in the majority of the studies. Therefore, more studies are needed in order to investigate the optimal transplantation parameters, especially the timing of cell transplantation after the stroke onset. This study aimed to evaluate the therapeutic effects of intra-arterial (IA) and intravenous (IV) administration of NPCs derived from induced pluripotent stem cells (iNPCs) in the acute phase of experimental stroke in rats. Induced pluripotent stem cells were chosen as the source of NPCs as this technology is perspective, has no ethical concerns and provides the access to personalized medicine. Methods. Human iNPCs were transplanted IA or IV into male Wistar rats 24 h after the middle cerebral artery occlusion stroke modeling. Therapeutic efficacy was monitored for 14 days and evaluated in comparison with the cell transplantation-free control group. Additionally, cell distribution in the brain was assessed. Results. The obtained results show that both routes of systemic transplantation (IV and IA) significantly reduced the mortality and improved the neurological deficit of experimental animals compared to the control group. At the same time, according to the MRI data, only IA administration led to faster and prominent reduction of the stroke volume. After IA administration, iNPCs transiently trapped in the brain and were not detected on day 7 after the transplantation. In case of IV injection, transplanted cells were not visualized in the brain. The obtained data demonstrated that the systemic transplantation of human iNPCs in the acute phase of ischemic stroke can be a promising therapeutic strategy. [ABSTRACT FROM AUTHOR]

Acute ischemic stroke is a catastrophic medical condition that causes severe disability and mortality if the sufferer escapes treatment within a stipulated timeframe. While timely intervention with clot-bursting agents like tissue-plasminogen activators abrogates some post-stroke neurologic deficits, no neuroprotective therapy is yet promisingly addresses the post-recanalization neuroinflammation in post-stroke survivors. Herein, we investigated the effect of partial blood replacement therapy (BRT), obtained from healthy and treadmill-trained donor rats, on neurological deficits, and peripheral and central inflammatory cascades using the ischemia-reperfusion animal paradigm. The cerebral ischemia-reperfusion was induced in rats by occlusion of the middle cerebral artery (MCAO) for 90 min, followed by reperfusion. Rats underwent MCAO surgery displayed remarkable sensorimotor and motor deficits in rotarod, foot fault, adhesive removal, and paw whisker tests till 5 days post-surgery. These behavior abnormalities were ameliorated in the BRT-recipient MCAO rats. BRT also reduced the infarct volume and neuronal death in the ipsilateral hemisphere revealed by TTC and cresyl violet staining compared to the MCAO group. Rats received BRT infusion exhibited the reduced expression of glial fibrillary acidic protein, ionized calcium-binding adaptor molecule-1 (Iba-1), and MyD88 on day 5 post-MCAO in immunohistochemistry and immunofluorescent assays. Moreover, elevated levels of toll-like receptor 4 (TLR4) and mRNA expression of IL-1β, TNF-α, matrix metalloproteinase-9 and NLRP3, and decreased levels of zonula occludens-1 in MCAO rats, were reversed following BRT. These findings suggest that the partial BRT may rescind MCAO-induced neurological dysfunctions and cerebral injury by intervening in the TLR4 and NLRP3 pathways in rats. [ABSTRACT FROM AUTHOR]

Microglia polarization and microglia-mediated inflammation play a crucial role in the development of ischaemic brain injury. Electroacupuncture (EA) has the function of anti-inflammatory, which has been thoroughly validated and utilized to treat ischemic brain damage. The fundamental mechanism by which EA alleviates ischemic brain damage by decreasing microglia polarization and microglia-mediated inflammation, however, remains unknown. In the current study, the activation of microglia and inflammatory cytokines was analyzed to confirm the anti-inflammatory function of EA in middle cerebral artery occlusion (MCAO) rats. Whole-transcriptome sequencing was used to examine the differentially expressed lncRNAs in the control, MCAO, and MCAO +EA groups. Our findings demonstrated that EA treatment reduced microglia activation and inflammatory cytokine production. In addition, there are 44 lncRNAs were found significantly different in three groups, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of the predicted targets of these lncRNAs suggested that the Hippo pathway may contribute to the development of ischaemic brain injury and to the anti-inflammatory function of EA. Moreover, our data showed that lncRNA TCONS_00022826 (Lnc826) was upregulated in MCAO group, whereas blocked by EA treatment. Furthermore, in vitro OGD cell model data showed that Lnc826 promoted M1 polarization of microglia by regulating the Hippo pathway. Our data suggested that regulating microglia polarization via Lnc826-mediated hippo pathway is a possible mechanism of the EA treatment on ischemic brain injury. [ABSTRACT FROM AUTHOR]

Ischemic stroke is a worldwide disease that seriously threatens human health, and there are few effective drugs to treat it. Dihydromyricetin (DHM) has anti-inflammatory, antioxidant, and antiapoptotic functions. We identified pyroptosis following ischemic stroke. Here, we investigated the effect of DHM on ischemic stroke and pyroptosis. In the first part of the experiment, Sprague-Dawley rats were randomly divided into the sham group and MCAO group. The MCAO model was established by occlusion of the middle cerebral artery for 90 min using a silica gel suture. The ischemic penumbra was used for mRNA sequencing 1 day after reperfusion. In the second part, rats were divided into the sham group, MCAO group, and DHM group. DHM was injected intraperitoneally at the same time as reperfusion starting 90 min after embolization for 7 consecutive days. The changes in pyroptosis were observed by morphological and molecular methods. The transcriptomics results suggested the presence of NLRP3-mediated pyroptotic death pathway activation after modeling. The Longa score was increased after MCAO and decreased after DHM treatment. 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that DHM could reduce the infarct volume induced by MCAO. Nissl staining showed disordered neuronal arrangement and few Nissl bodies in the MCAO group, but this effect was reversed by DHM treatment. Analysis of pyroptosis-related molecules showed that the MCAO group had serious pyroptosis, and DHM effectively reduced pyroptosis. Our results demonstrate that DHM has a neuroprotective effect on ischemic stroke that is at least partly achieved by reducing pyroptosis. [ABSTRACT FROM AUTHOR]

Background: Ischemic stroke remains one of the leading causes of mortality and disability in the population. Middle cerebral artery occlusion (MCAO) followed by reperfusion is widely accepted to mimic stroke in basic medical research. Trehalose is a non-reducing disaccharide compound found naturally in most organisms such as bacteria, yeasts, invertebrates and even in plants except for vertebrates. Its protective role has been shown in various neurological disorders. In the current study, the MCAO model was used to investigate the therapeutic efficacy of trehalose. Methods: In this experimental study, 32 Male Wistar rats were subjected to MCAO following 24 h of reperfusion. Wistar rats were randomly divided into four groups: Sham-operated control group (Sham), middle cerebral artery occlusion (MCAO) group, trehalose (50 and 100 mg/kg) treatment + MCAO groups. Trehalose was intraperitoneally injected 3- and 6 h post-ischemia. After 24 h of reperfusion and evaluation of the intensity of neurological dysfunction via a modified Rogers scale, the brains were removed and the volume of ischemia was stained using 2,3,5-triphenyl tetrazolium chloride. Results: Compared with the control group, trehalose administration with dosages of 50 and 100 mg/kg significantly decreased the modified Rogers score (p

Background Cell therapy using neural progenitor cells (NPCs) is a promising approach for ischemic stroke treatment according to the results of multiple preclinical studies in animal stroke models. In the vast majority of conducted animal studies, the therapeutic efficacy of NPCs was estimated after intracerebral transplantation, while the information of the effectiveness of systemic administration is limited. Nowadays, several clinical trials aimed to estimate the safety and efficacy of NPCs transplantation in stroke patients were also conducted. In these studies, NPCs were transplanted intracerebrally in the subacute/chronic phase of stroke. The results of clinical trials confirmed the safety of the approach, however, the degree of functional improvement (the primary efficacy endpoint) was not sufficient in the majority of the studies. Therefore, more studies are needed in order to investigate the optimal transplantation parameters, especially the timing of cell transplantation after the stroke onset. This study aimed to evaluate the therapeutic effects of intra-arterial (IA) and intravenous (IV) administration of NPCs derived from induced pluripotent stem cells (iNPCs) in the acute phase of experimental stroke in rats. Induced pluripotent stem cells were chosen as the source of NPCs as this technology is perspective, has no ethical concerns and provides the access to personalized medicine. Methods Human iNPCs were transplanted IA or IV into male Wistar rats 24 h after the middle cerebral artery occlusion stroke modeling. Therapeutic efficacy was monitored for 14 days and evaluated in comparison with the cell transplantation-free control group. Additionally, cell distribution in the brain was assessed. Results The obtained results show that both routes of systemic transplantation (IV and IA) significantly reduced the mortality and improved the neurological deficit of experimental animals compared to the control group. At the same time, according to the MRI data, only IA administration led to faster and prominent reduction of the stroke volume. After IA administration, iNPCs transiently trapped in the brain and were not detected on day 7 after the transplantation. In case of IV injection, transplanted cells were not visualized in the brain. The obtained data demonstrated that the systemic transplantation of human iNPCs in the acute phase of ischemic stroke can be a promising therapeutic strategy.

The protective effects of remote ischemic conditioning (RIC) on acute ischemic stroke have been reported. However, the protective mechanisms of RIC have not been fully elucidated. This study aimed to investigate whether RIC could reduce oxidative stress and inflammatory responses in middle cerebral artery occlusion (MCAO)-reperfusion mice via the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. C57BL/6 mice were subjected to MCAO and underwent RIC twice daily at 1, 3, and 7 days after MCAO. ML385 was used to specifically inhibit Nrf2 in MCAO mice. Neurological deficit scores, infarct volume, and hematoxylin-eosin (HE) staining were assessed. Oxidative stress levels were assessed based on total antioxidant capacity (TAC), malonaldehyde (MDA), superoxide dismutase (SOD), and glutathione/glutathione disulfide (GSH/GSSG). mRNA levels were detected using real-time polymerase chain reaction (PCR), and protein levels were detected using western blotting and enzyme-linked immunosorbent assay (ELISA). Protein localization was investigated using immunofluorescence staining. RIC significantly reduced infarct volume and improved neurological function and histological changes after MCAO. RIC significantly increased TAC, SOD, and GSH/GSSG levels and decreased MDA levels. RIC significantly increased Nrf2 and HO-1 mRNA levels and decreased Keap1, NLRP3, and Cleaved Caspase-1 mRNA levels. RIC significantly increased Nrf2, HO-1, and NQO1 protein expression and decreased Keap1, NLRP3, Cleaved Caspase-1, Cleaved IL-1β, IL-6, and TNF-α protein expression. RIC promoted the activation and translocation of Nrf2 into the nucleus. The protective effects of RIC were abolished by ML385 treatment. In conclusion, our findings suggest that RIC alleviates oxidative stress and inflammatory responses via the Nrf2/HO-1 pathway, which in turn improves neurobehavioral function. RIC may provide novel therapeutic options for acute ischemic stroke.

Background: Ischemic stroke, the most common stroke type, has threatened human life and health. Currently, intravenous thrombolysis and endovascular thrombectomy are the mainstream treatment methods, but they may cause cerebral ischemia-reperfusion injury (CIRI), which aggravates brain injury. Consequently, it is worthwhile to start with a study of CIRI mechanism to identify better prevention and treatment methods. Applying single-cell RNA sequencing (scRNA-seq) technology to further understand the biological functions of various cell types in CIRI will facilitate the intervention of CIRI. Methods: This study aimed to establish a rat middle cerebral artery occlusion (MCAO) model to simulate cerebral ischemia-reperfusion, perform enzymatic hydrolysis, and suspend cerebral cortex tissue edema. Single-cell transcriptome sequencing was used, combined with cluster analysis, t-distributed stochastic neighbor embedding (t-SNE) visualization, and other bioinformatics methods to distinguish cell subgroups while using gene ontology (GO) function enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment to reveal the biological function of each cell subgroup. Results: We identified 21 brain clusters with cell type-specific gene expression patterns and cell subpopulations, as well as 42 marker genes representing different cell subpopulations. The number of cells in clusters 0-3 increased significantly in MCAO group compared to that in the sham group, and nine-cell subpopulations exhibited remarkable differences in the number of genes. Subsequently, GO and KEGG analyses were performed on the top 40 differentially expressed genes (DEGs) in the six cell subpopulations with significant differences. These results indicate that biological processes and signaling pathways are involved in different cell subpopulations. Conclusions: ScRNA-seq revealed the diversity of cell differentiation and the unique information of cell subpopulations in the cortex of rats with acute ischemic stroke, providing novel insight into the pathological process and drug discovery in stroke. [ABSTRACT FROM AUTHOR]

Background: Epigenetic histone methylation plays a crucial role in cerebral ischemic injury, particularly in the context of ischemic stroke. However, the complete understanding of regulators involved in histone methylation, such as Enhancer of Zeste Homolog 2 (EZH2), along with their functional effects and underlying mechanisms, remains incomplete. Methods: Here, we employed a rat model of MCAO (Middle cerebral artery occlusion) and an OGD (Oxygen‐Glucose Deprivation) model of primary cortical neurons to study the role of EZH2 and H3K27me3 in cerebral ischemia‐reperfusion injury. The infarct volume was measured through TTC staining, while cell apoptosis was detected using TUNEL staining. The mRNA expression levels were quantified through quantitative real‐time polymerase chain reaction (qPCR), whereas protein expressions were evaluated via western blotting and immunofluorescence experiments. Results: The expression levels of EZH2 and H3K27me3 were upregulated in OGD; these expression levels were further enhanced by GSK‐J4 but reduced by EPZ‐6438 and AKT inhibitor (LY294002) under OGD conditions. Similar trends were observed for mTOR, AKT, and PI3K while contrasting results were noted for UTX and JMJD3. The phosphorylation levels of mTOR, AKT, and PI3K were activated by OGD, further stimulated by GSK‐J4, but inhibited by EPZ‐6438 and AKT inhibitor. Inhibition of EZH2 or AKT effectively counteracted OGD‐/MCAO‐induced cell apoptosis. Additionally, inhibition of EZH2 or AKT mitigated MCAO‐induced infarct size and neurological deficit in vivo. Conclusions: Collectively, our results demonstrate that EZH2 inhibition exerts a protective effect against ischemic brain injury by modulating the H3K27me3/PI3K/AKT/mTOR signaling pathway. The results provide novel insights into potential therapeutic mechanisms for stroke treatment. [ABSTRACT FROM AUTHOR]

• Down-regulation of SPRED1 expression was induced by cerebral ischemia–reperfusion. • Oxidative stress exhibited a fluctuating regulatory effect on SPRED1 level in BV2 microglia. • Oxidative stress enhances the induction to p65 upon SPRED1 over-expression in BV2 microglia. • Oxidative stress-mediated SPRED down-regulation contributes to resisting oxidative injury in microglia. Microglia play an ambiguous role in injury or repair after ischemia–reperfusion, and the induced oxidative stress serves as an important signal, mediates direct toxicity to nerve cells, and eventually simulates complex physiological processes such as activation of microglia to repair the damaged area. Herein, we show that sprouty-related protein with an EVH1 domain 1 (SPRED1) may act as a regulatory node in this phenomenon. The ischemic brain of an ischemia–reperfusion rat model constructed by middle cerebral artery occlusion (MCAO) showed an increase in oxidative stress and downregulation of SPRED1 expression. Hydrogen peroxide (H 2 O 2)-simulated oxidative damage exerted a fluctuating regulatory effect on SPRED1 level in BV2 microglia, which is highly consistent with its regulatory effect on nuclear factor kappa B (NF-κB) transcription factor p65. Interestingly, SPRED1 overexpressed in BV2 cells did not exert any regulatory effect on p38 mitogen-activated protein kinase (MAPK), NF-κB p65, and pro-inflammatory cytokines. However, treatment of BV2 cells overexpressing SPRED1 with H 2 O 2 led to significant changes in the above phenomena as well as their viability and apoptosis. In the absence of H 2 O 2 induction, SPRED overexpression alone did not mediate such an effect. These findings indicate that SPRED1 tends to maintain intracellular homeostasis of signals, but the oxidative stress derived from ischemia-reperfusion can easily degrade SPRED1 and consequently re-activate these restricted signals and alter the behavior of microglia. Thus, our study reveals a novel role of SPRED1 in microglia in response to cerebral ischemia-induced oxidative stress. [ABSTRACT FROM AUTHOR]

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA's effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA's selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy. [ABSTRACT FROM AUTHOR]