Middle Cerebral Artery Occlusion Surgery Research Articles

Dihydroergotamine protects against ischemic stroke by modulating microglial/macrophage polarization and inhibiting inflammation in mice

ABSTRACT Objectives The search for drugs that can protect the brain tissue and reduce nerve damage in acute ischemic stroke has emerged as a research hotspot. We investigated the potential protective effects and mechanisms of action of dihydroergotamine against ischemic stroke. Methods C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO), and dihydroergotamine at a dose of 10 mg/kg/day was intraperitoneally injected for 14 days. Adhesive removal and beam walking tests were conducted 1, 3, 5, 7, 10, and 14 days after MCAO surgery. Thereafter, the mechanism by which dihydroergotamine regulates microglia/macrophage polarization and inflammation and imparts ischemic stroke protection was studied using enzyme-linked immunosorbent assay, immunofluorescence staining, and western blotting. Results From the perspective of a drug repurposing strategy, dihydroergotamine was found to inhibit oxygen-glucose deprivation damage to neurons, significantly improve cell survival rate, and likely exert a protective effect on ischemic brain injury. Dihydroergotamine significantly improved neural function scores and survival rates and reduced brain injury severity in mice. Furthermore, dihydroergotamine manifests its protective effect on ischemic brain injury by reducing the expression of TNF-α and IL-1β in mouse ischemic brain tissue, inhibiting the polarization of microglia/macrophage toward the M1 phenotype and promoting polarization toward the M2 phenotype. Conclusion This study is the first to demonstrate the protective effect of dihydroergotamine, a first-line treatment for migraine, against ischemic nerve injury in vitro and in vivo.

Epigallocatechin gallate improves neuronal damage in animal model of ischemic stroke and glutamate-exposed neurons via modulation of hippocalcin expression.

Epigallocatechin gallate (EGCG) is a polyphenolic component of green tea that has anti-oxidative and anti-inflammatory effects in neurons. Ischemic stroke is a major neurological disease that causes irreversible brain disorders. It increases the intracellular calcium concentration and induces apoptosis. The regulation of intracellular calcium concentration is important to maintain the function of the nervous system. Hippocalcin is a neuronal calcium sensor protein that controls intracellular calcium concentration. We investigated whether EGCG treatment regulates the expression of hippocalcin in stroke animal model and glutamate-induced neuronal damage. We performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. EGCG (50 mg/kg) or phosphate buffered saline was injected into the abdominal cavity just before MCAO surgery. The neurobehavioral tests were performed 24 h after MCAO surgery and cerebral cortex tissue was collected. MCAO damage induced severe neurobehavioral disorders, increased infarct volume, and decreased the expression of hippocalcin in the cerebral cortex. However, EGCG treatment improved these deficits and alleviated the decrease in hippocalcin expression in cerebral cortex. In addition, EGCG dose-dependently alleviated neuronal cell death and intracellular calcium overload in glutamate-exposed neurons. Glutamate exposure reduced hippocalcin expression, decreased Bcl-2 expression, and increased Bax expression. However, EGCG treatment mitigated these changes caused by glutamate toxicity. EGCG also attenuated the increase in caspase-3 and cleaved caspase-3 expressions caused by glutamate exposure. The effect of EGCG was more pronounced in non-transfected cells than in hippocalcin siRNA-transfected cells. These findings demonstrate that EGCG protects neurons against glutamate toxicity through the regulation of Bcl-2 family proteins and caspase-3. It is known that hippocalcin exerts anti-apoptotic effect through the modulation of apoptotic pathway. Thus, we can suggest evidence that EGCG has a neuroprotective effect by regulating hippocalcin expression in ischemic brain damage and glutamate-exposed cells.

Enriched environment treatment promotes neural functional recovery together with microglia polarization and remyelination after cerebral ischemia in rats

BackgroundMicroglia activation and oligodendrocyte maturation are critical for remyelination after cerebral ischemia. Studies have shown that enriched environment (EE) can effectively alleviate stroke-induced neurological deficits. However, little is known about the mechanism associated with glial cells underlying the neuroprotection of EE. Therefore, this study focuses on investigating the effect of EE on activated microglia polarization as well as oligodendrogenesis in the progress of remyelination following cerebral ischemia. MethodsThe ischemia/reperfusion (I/R) injury model was established by middle cerebral artery occlusion (MCAO) in rats. Animals executed 4 weeks of environmental intervention after performing MCAO or sham surgery and were divided into sham, MCAO, and MCAO+EE groups. Cognitive function, myelin damage, microglia activation and polarization, inflammation, oligodendrogenesis, remyelination, and protein expression of the PI3K/AKT/GSK3β signaling pathway were determined. ResultsThe staining of NeuN indicated that the infarct size of MCAO rats was decreased under EE. EE intervention improved animal performance in the Morris water maze test and novel object recognition test, promoting the recovery of cognitive function after I/R injury. EE treatment alleviated myelin damage in MCAO rats, as evidenced by the lower fluorescence intensity ratio of SMI-32/MBP in MCAO+EE group. EE increased the fluorescence intensity ratio of NG2+/Ki67+/Olig2+, MBP, and MOG, enhancing the proliferation and differentiation of OPCs and oligodendrogenesis after MCAO. In terms of remyelination, more myelinated axons and lower G/ratio were detected in MCAO+EE rats compared with MCAO group. Moreover, EE treatment decreased the number of Iba1+/CD86+ M1 microglia, increased the number of Iba1+/CD206+ M2 microglia, and suppressed the inflammation response after I/R injury, which could be attributed to the augmented expression of PI3K/AKT/GSK3β axis. ConclusionEE improved long‑term recovery of cognitive function after cerebral I/R injury, at least in part by promoting M2 microglia transformation through activation of the PI3K/AKT/GSK3β signaling pathway, inhibiting inflammation to provide a favorable microenvironment for oligodendrocyte maturation and remyelination. The effect of the EE on myelin and inflammation could account for the neuroprotection provided by EE.

Electronic Cigarette Vape Exposure Exacerbates Post-Ischemic Outcomes in Female but Not in Male Rats.

Nicotine-containing electronic cigarette (EC) vaping has become popular worldwide, and our understanding of the effects of vaping on stroke outcomes is elusive. Using a rat model of transient middle cerebral artery occlusion, the current exploratory study aims to evaluate the sex-dependent effects of EC exposure on brain energy metabolism and stroke outcomes. Adult Sprague-Dawley rats of both sexes were randomly assigned to air/EC vapor (5% nicotine Juul pods) exposure for 16 nights, followed by randomization into 3 cohorts. The first cohort underwent exposure to air/EC preceding randomization to transient middle cerebral artery occlusion (90 minutes) or sham surgery, followed by survival for 21 days. During the survival period, rats underwent sensorimotor and Morris water maze testing. Subsequently, brains were collected for histopathology. A second cohort was exposed to air/EC after which brains were collected for unbiased metabolomics analysis. The third cohort of animals was exposed to air/EC and received transient middle cerebral artery occlusion/sham surgery, and brain tissue was collected 24 hours later for biochemical analysis. In females, EC significantly increased (P<0.05) infarct volumes by 94% as compared with air-exposed rats, 165±50 mm3 in EC-exposed rats, and 85±29 mm3 in air-exposed rats, respectively, while in males such a difference was not apparent. Morris water maze data showed significant deficits in spatial learning and working memory in the EC sham or transient middle cerebral artery occlusion groups compared with the respective air groups in rats of both sexes (P<0.05). Thirty-two metabolites of carbohydrate, glycolysis, tricarboxylic acid cycle, and lipid metabolism were significantly altered (P≤0.05) due to EC, 23 of which were specific for females. Steady-state protein levels of hexokinase significantly decreased (P<0.05) in EC-exposed females; however, these changes were not seen in males. Even brief EC exposure over 2 weeks impacts brain energy metabolism, exacerbates infarction, and worsens poststroke cognitive deficits in working memory more in female than male rats.

Sinigrin improves cerebral ischaemia-reperfusion injury by inhibiting the TLR4 pathway-mediated oxidative stress.

Cerebral ischaemia-reperfusion (CIR) injury occurs in stroke patients after the restoration of cerebral perfusion. Sinigrin, a phytochemical found in cruciferous vegetables, exhibits strong antioxidant activity. This study investigated the role of sinigrin in oxidative stress using a CIR injury model. The effects of sinigrin were studied in middle cerebral artery occlusion (MCAO) rats and oxygen-glucose deprivation/reoxygenation (OGD/R)-injured SH-SY5Y cells. Sinigrin treatment improved brain injury and neurological deficits induced by MCAO surgery in rats. Sinigrin inhibited apoptosis in brain tissues and SH-SY5Y cells following OGD/R induction. Additionally, sinigrin elevated the levels of superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) while reducing malondialdehyde (MDA) levels. Furthermore, sinigrin inhibited the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signalling pathway. The anti-apoptotic and antioxidant activities of sinigrin in OGD/R-injured SH-SY5Y cells were reversed by TLR4 overexpression. In conclusion, sinigrin inhibits oxidative stress in CIR injury by suppressing the TLR4/MyD88 signalling pathway.

Abstract TP298: Cellular Senescence and Microglial Dysfunction are Linked to Progressive Cognitive Deficits in the Chronic Phase of Ischemic Stroke

Improvements in acute stroke treatment, including endovascular thrombectomy and critical care management, have increased survival rates after stroke. However, many survivors have significant neurological deficits, as stroke remains one of the leading causes of long-term disability. Despite this, the chronic progression and long-term sequelae of ischemic stroke pathology remains woefully understudied. This study examined long-term progressive deficits in young adult male C57Bl/6 mice at 3 and 6 months after a 60-minute transient middle cerebral artery occlusion (MCAO) or sham surgery to identify secondary injury processes, and neuropathology of chronic stroke human brains. Depressive-like behavior persisted for 6 months post-stroke (PS) (p= &lt;0.0001). Cognitive function progressively worsened from 3 to 6 months as seen by a continual reduction in the discrimination index in the novel object recognition task (p=.0063). Deficits in memory retention in mice 6-months PS were observed using the fear-conditioning test (p=.0084). Brain atrophy was evident by MicroCT imaging. Histopathological assessment revealed increased microglial activation in the hippocampus and frontal cortex in both mouse and human brains chronically after stroke. Interestingly, we also observed microglia activation proximal to apoptotic neurons in the brains of stroke mice, as shown by TUNEL assay. TUNEL co-labeling with a neuronal marker showed the same neuronal apoptosis in human samples from patients with chronic stroke, in both the hippocampus and thalamus. Disease-associated microglial (DAM) phenotypes were marked by both increased proliferative status and senescent-like phenotypes, including elevated production of cytokines and other proinflammatory molecules. Our findings demonstrate that experimental stroke accelerates inflammaging in the brain, which induces premature senescence within the chronic infarct microenvironment. Cellular senescence and chronic neurodegenerative signatures likely contribute to progressive cognitive impairment following stroke. The chronic and evolving neurological consequences of stroke may offer viable targets for delayed treatment strategies to reduce risk of vascular dementia and neurodegenerative disease.

Abstract 105: Sex Difference in Brain Energy Metabolism After Electronic Cigarette Vape Exposure May Contribute to Worsened Stroke Injury in Rats

Introduction: Electronic cigarettes (EC) are battery-powered nicotine delivery devices that have gained popularity as alternatives to tobacco cigarettes for young smokers and smokers trying to cease. Because of its relative novelty, our understanding about the effects of EC vaping on stroke outcome is limited. Here we aim to investigate the effects of EC on energy metabolism in the brain and on stroke outcome in animals of both sexes. Methods: Female and male Sprague-Dawley rats (2-3 months old; n=8) were randomly assigned to either air or EC vapor (5% nicotine Juul pods) exposure for 16 nights using the EcigAero-TM Aerosol Exposure Apparatus. After the exposure period, the rats were divided into two cohorts. The brains were collected from the first cohort of rats for unbiased metabolomic (Metabolon Inc.) or Western blot analysis. The second cohort underwent transient middle cerebral artery occlusion (tMCAO; 90 min) or sham surgery and was survived for 21 days. During the post-tMCAO/sham survival, cognition was assessed using the Morris water maze followed by brain collection for histopathological analysis. Results: Metabolomic analysis showed substantial changes in glycolytic metabolites in EC-exposed female rats in relation to air controls, implying altered brain energetics. In EC-exposed female animals, a significant increase (p&lt;0.05) in glucose, glucose 6-phosphate, fructose-6-phosphate, and fructose 1-6 diphosphate was noted compared to air controls. However, such changes were not seen in EC-exposed male rats. Quantification of infarct volume demonstrated that, compared to air rats, EC-exposed rats displayed a significantly increased infarct volume in female rats while showing a trend of increase in male rats. In female rats, infarct volumes were 86 ± 19 mm 3 and 151 ± 25 mm 3 for air and EC groups, respectively, a significant difference (p&lt;0.05). In the male rat air group, tMCAO resulted in an infarct volume of 80 ± 37 mm 3 , while EC yielded 130 ± 38 mm 3 . Behavioral testing showed worsened post-stroke cognitive outcome in EC groups compared to air groups in rats of both sexes. Conclusion: EC vape exposure, even for as short as two weeks, impacts glucose metabolism and worsens post-stroke cognitive deficits in female more than male rats.

Abstract WP316: Single-Cell RNA Sequencing Reveals a Massive Alteration in the Molecular Landscape of Microglia After Ischemic Stroke

Introduction: Ischemic stroke, a prevalent and often devastating disease, remains poorly treated. Neuroinflammation is a key element of stroke pathophysiology and represents a promising therapeutic target. Among the central components driving neuroinflammation, microglia - the primary resident immune cells within the brain parenchyma - are key players in mediating the immune response in the stroke brain. However, how microglia respond to stroke is incompletely understood. In this study, we took advantage of a mouse genetic tool (ie, Tmem119-CreERT2), sorted microglia, and performed single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of permanent stroke. Moreover, as stroke exhibits sexual dimorphism, we included both male and female mice. Methods: Young male and female Ai6/TMEM119-CreERT2;Ai6 (Tmem119-Ai6) mice with microglia labeled with ZsGreen underwent permanent middle cerebral artery occlusion (pMCAO) or sham surgery. On day 3 after stroke, ZsGreen+ cells (microglia) in the brain were isolated by FACS, and used for scRNA-seq analysis. Results: First, we confirmed that in Tmem119-Ai6 mice, expression of fluorescent ZsGreen was almost exclusively in microglia in the brain, with minimal presence in blood cells. Our unbiased scRNA-seq analysis of ZsGreen+ cells revealed 13 distinct microglial clusters after stroke. Importantly, 4 major stroke-related microglial clusters were identified. One cluster shared a gene signature with the reported disease-associated microglia (DAM) subset. The other 3 clusters were characterized by an interferon (IFN) response signature, a highly proliferative state, and a macrophage-like profile, respectively. RNA in situ hybridization analysis showed that cells from these clusters mainly resided within the stroke boundaries. Notably, our scRNA-seq data suggest no major sexual dimorphism in the microglial response to permanent stroke. Conclusions: This is the first scRNA-seq study with a focus on microglia in the context of sex and stroke. We propose that 4 microglial subsets may be deemed as common stroke-associated microglia. Future work will be designed to elucidate the functional significance of these subsets and their potential implications for stroke therapy.

Abstract WP301: STAT3 Inhibition Reduces Infarct and Neuroinflammation in Experimental Stroke

Signal Transducer and Activator of Transcription 3 (STAT3) activation is increased in the brain following cerebral ischemia. STAT3, a transcription factor, is a positive regulator of cell survival and is implicated in cytoprotection following ischemia in multiple cell types, including neurons and endothelial cells. We hypothesized that pharmacological inhibition of STAT3 activation in mice during the reperfusion phase after middle cerebral artery occlusion (MCAO) will lead to increased infarct size. We subjected mice to 1-hour MCAO, following which they received STAT3 inhibitor Stattic (20 mg/kg), or vehicle, for 24 hours delivered subcutaneously via osmotic pump. Twenty-four hours later, infarct was measured by TTC and inflammatory cells were measured by flow cytometry. For experiments involving splenectomy, spleens were surgically removed 14 days prior to MCAO surgery. Surprisingly, we found that STAT3 inhibition reduces infarct after MCAO, with infarct in the hemisphere reducing from 49.90 ± 2.44 % in vehicle to 32.86 ± 1.55 % in Stattic-treated mice. Since STAT3 is involved in inflammatory processes, as well as cytoprotection, this led us to hypothesize that the reduction in infarct we observe may be due to decreased inflammation. We carried out FACS analysis and observed that Stattic inhibits MCAO-induced infiltration of B-, T- and dendritic cells into the brain. Neutrophil and monocyte infiltration into the brain were unaffected by Stattic. Since MCAO is known to induce splenic atrophy, with spleen size being inversely proportional to infarct volume, we measured spleen weights. Interestingly Stattic-treated mice had larger spleens (66.31 ± 5.40 g) compared to vehicle-treated mice (46.43 ± 4.60 g), indicating a dampened peripheral immune response upon inhibition of STAT3. Furthermore, Stattic was no longer effective in reducing infarct size in splenectomized mice. We conclude that pharmacological inhibition of STAT3 reduces infarct following MCAO, with the mechanism of protection involving differential regulation of the peripheral immune response.

TRIM59 suppresses the brain ischaemia/reperfusion injury and pyroptosis of microglial through mediating the ubiquitination of NLRP3

Cerebral ischaemia/reperfusion (I/R) injury induces irreversible brain injury and causes functional impairment. Ubiquitination plays a crucial role in protein degradation, but its role in cerebral I/R injury remains unclear. Differentially expressed genes in stroke were identified by analysing the microarray dataset GSE119121. Cerebral I/R was simulated in vitro by treating human microglial HMC3 cells with oxygen–glucose deprivation/reperfusion (OGD/R). Cell viability was tested by Cell Counting Kit 8 (CCK-8) assays, and pyroptosis was examined by flow cytometry. Lactate dehydrogenase (LDH) and inflammatory cytokine secretion were measured by LDH cytotoxicity assays and enzyme-linked immunosorbent assay (ELISA), respectively. The cerebral I/R animal model was established by middle cerebral artery occlusion (MCAO) surgery in rats. Bioinformatic analysis indicated that tripartite motif-containing protein 59 (TRIM59) is downregulated in stroke, which was verified in cerebral I/R models. The upregulation of TRIM59 promoted viability and inhibited pyroptosis in OGD/R-treated microglia and alleviated cerebral I/R injury in vivo. TRIM59 attenuated NOD-like receptor family pyrin domain containing 3 (NLRP3) protein expression through ubiquitination, thus degrading NLRP3 and alleviating OGD/R-induced injury. TRIM59 relieves cerebral I/R injury in vivo and in vivo. Mechanistically, TRIM59 directly interacts with NLRP3 and inhibits NLRP3 through ubiquitination. Targeting the TRIM59/NLRP3 signalling axis may be an effective therapeutic strategy for cerebral I/R.

Effect of Edible Bird’s Nest on Protecting against Cognitive Deficit and Ameliorating Beta‐Amyloid in Hippocampal Rats’ Model of Cerebral Ischemia

Cerebral ischemia has been identified as the primary cause of global mortality, but there is currently no effective therapy for treating this condition. Consequently, the search for novel neuroprotective agents that can guard against stroke remains essential. Recent research has demonstrated the importance of alternative treatments in the pathology of cerebral ischemia leading to vascular dementia. The purpose of this study was to determine the effect of neurocognitive behavior and beta‐amyloid markers in terms of the neuroprotective effects of Edible Bird’s Nest (EBN). Male adult Wistar rats were orally administered EBN at concentrations of 10, 50, and 100 mg/kg for 2 weeks prior to and 3 weeks after middle cerebral artery occlusion surgery to mimic vascular dementia. The rodents were then classified based on their neurological score, beta‐amyloid accumulation, volume of cerebral infarction, and acetylcholinesterase activity. We assessed the animals’ spatial memory and administered the Morris water maze and radial arm maze tests. We found that EBN substantially decreased beta‐amyloid in the hippocampus, decreased acetylcholinesterase activity, and enhanced the animals’ neurological scores and spatial memory. We conclude that EBN contains potential substances that promote learning and memory pathways. However, additional research is still necessary to confirm these findings.

Retinoic acid alleviates the reduction of Akt and Bad phosphorylation and regulates Bcl-2 family protein interactions in animal models of ischemic stroke.

Ischemic stroke causes a lack of oxygen and glucose supply to brain, eventually leads to severe neurological disorders. Retinoic acid is a major metabolic product of vitamin A and has various biological effects. The PI3K-Akt signaling pathway is an important survival pathway in brain. Phosphorylated Akt is important in regulating survival and apoptosis. We examined whether retinoic acid has neuroprotective effects in stroke model by regulating Akt and its downstream protein, Bad. Moreover, we investigated the relationship between retinoic acid and Bcl-2 family protein interactions. Animals were intraperitoneally administered vehicle or retinoic acid (5 mg/kg) for four days before surgery and ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Neurobehavioral tests were performed 24 h after MCAO and cerebral cortical tissues were collected. Cresyl violet staining and TUNEL histochemistry were performed, Western blot and immunoprecipitation analysis were performed to elucidate the expression of various proteins. Retinoic acid reduced neurological deficits and histopathological changes, decreased the number of TUNEL-positive cells, and alleviated reduction of phospho-PDK1, phospho-Akt, and phospho-Bad expression caused by MCAO damage. Immunoprecipitation analysis showed that MCAO damage reduced the interaction between phospho-Bad and 14-3-3, which was attenuated by retinoic acid. Furthermore, retinoic acid mitigated the increase in Bcl-2/Bad and Bcl-xL/Bad binding levels and the reduction in Bcl-2/Bax and Bcl-xL/Bax binding levels caused by MCAO damage. Retinoic acid alleviated MCAO-induced increase of caspase-3 and cleaved caspase-3 expression. We demonstrate that retinoic acid prevented apoptosis against cerebral ischemia through phosphorylation of Akt and Bad, maintenance of phospho-Bad and 14-3-3 binding, and regulation of Bcl-2 family protein interactions. .

Huatanmaitong tablet alleviate cerebral ischemic reperfusion injury with hyperlipidaemia in rats by regulating OATPs/VEGF axis.

Stroke is the top priority pathogenesis of disability and death globally, affecting people worldwide. The presence of high levels of lipids in the blood has been confirmed as a vital factor of ischemic stroke. We aim to examine the effectiveness of Huatanmaitong tablet in hyperlipidemia rats that have experienced an ischemic stroke. We created a rat model of middle cerebral artery occlusion (MCAO) with hyperlipidemia as a basis. Following 8 weeks of high-fat diet, the model rats underwent MCAO surgery. Subsequently, the rats were administered huatanmaitong tablets and lipitor tablets as treatments. Therefore there are five groups, CONTROL, MCAO, hyperlipidemia (HLP), Huatanmaitong tablet (HTMTT) and Lipitor (LIPITOR) groups respective ly. To assess the efficacy of the medication, the serum lipid levels of rats were measured both prior to and following administration. Hematoxylin eosin staining was used to observe the alterations in the brain and liver structures within each group. VEGF and OATPs related factors were detected in brain, liver by using immunohistochemistry, Western blotting, and Quantitative PCR. After the model was established successfully, the infarct volume and behavioral scores of the model group, hyperlipidemia group, Huatan Maitong tablet group and Lipitor group had statistical differences (P<0.05). Blood lipid levels of rats were measured before and after treatment, and it was found that Huatanmaitong tablets effectively reduced these levels. Hematoxylin and eosin staining of the brain and liver showed that huatanmaitong tablets maintained the microstructure stability. Western blotting and real-time PCR revealed that Huatanmaitong tablets improved the expression level of organic anion transport (OATP1B1, OATP2B1) in rat tissues with ischemic stroke, enhancing the transmembrane transport of exogenous substances and maintaining homeostatic balance. Additionally, it down-regulated the expression of VEGF in various organs such as the brain, and liver, demonstrating the ability of Huatanmaitong tablets to remove phlegm, blood stasis, and promote circulation by regulating serum lipid levels, organic anion transport peptide, and VEGF in rats. The behavioral score of ischemic stroke rats can be improved and the neurological impairment symptoms of rats can be alleviated by Huatanmaitong tablet through the regulation of OATPS/VEGF axis.

Recombinant human brain natriuretic peptide attenuates ischemic brain injury in mice by inhibiting oxidative stress and cell apoptosis via activation of PI3K/AKT/Nrf2/HO-1 pathway.

Ischemic stroke followed by cerebral artery occlusion is a main cause of chronic disability worldwide. Recombinant human brain natriuretic peptide (rhBNP) has been reported to alleviate sepsis-induced cognitive dysfunction and brain I/R injury. However, the function and molecular mechanisms of rhBNP in ischemic brain injury have not been clarified. For establishment of an animal model of ischemic brain injury, C57BL/6 mice were treated with middle cerebral artery occlusion (MCAO) surgery for 1h and reperfusion for 24h. After subcutaneous injection of rhBNP into model mice, neurologic deficits were assessed by evaluating behavior of mice according to Longa scoring system, and TTC staining was utilized to determine the brain infarct size of mice. The levels of oxidative stress markers, superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA), were detected in hippocampal tissues of mice by corresponding kits. Cell apoptosis in hippocampus tissues was examined by TUNEL staining. Protein levels of antioxidant enzymes (HO-1 and NQO1) in cerebral cortex, apoptotic markers (Bax, Bcl-2, and cleaved caspase), and PI3K/AKT pathway-associated factors in hippocampus were tested by western blot analysis. The results revealed that injection of rhBNP decreased neurologic deficit scores, the percent of brain water content, and infarct volume. Additionally, rhBNP downregulated MDA level, upregulated the levels of SOD, CAT, and GSH in hippocampus of mice, and increased protein levels of HO-1 and NQO1 in the cortex. Cell apoptosis in hippocampus tissues of model mice was inhibited by rhBNP which was shown as the reduced TUNEL-positive cells, the decreased Bax, cleaved caspase-3, and cleaved caspase-9 protein levels, and the enhanced Bcl-2 protein level. In addition, rhBNP treatment activated the PI3K/AKT signaling pathway and upregulated the protein levels of HO-1 and NRF2. Overall, rhBNP activates the PI3K/AKT/HO-1/NRF2 pathway to attenuate ischemic brain injury in mice after MCAO by suppression of cell apoptosis and oxidative stress.

Mesenchymal Stem Cells Overexpressing FGF21 Preserve Blood-Brain Barrier Integrity in Experimental Ischemic Stroke.

Blood-brain barrier (BBB) disruption is a prominent pathophysiological mechanism in stroke. Transplantation of mesenchymal stem cells (MSCs) preserves BBB integrity following ischemic stroke. Fibroblast growth factor 21 (FGF21) has been shown to be a potent neuroprotective agent that reduces neuroinflammation and protects against BBB leakage. In this study, we assessed the effects of transplantation of MSCs overexpressing FGF21 (MSCs-FGF21) on ischemia-induced neurological deficits and BBB breakdown. MSCs-FGF21 was injected into the rat brain via the intracerebroventricular route 24 h after middle cerebral artery occlusion (MCAO) surgery. The behavioral performance was assessed using modified neurological severity scores and Y-maze tests. BBB disruption was measured using Evans blue staining, IgG extravasation, and brain water content. The levels of tight junction proteins, aquaporin 4, and neuroinflammatory markers were analyzed by western blotting and immunohistochemistry. The activity of matrix metalloproteinase-9 (MMP-9) was determined using gelatin zymography. At day-5 after MCAO surgery, intraventricular injection of MSCs-FGF21 was found to significantly mitigate the neurological deficits and BBB disruption. The MCAO-induced loss of tight junction proteins, including ZO-1, occludin, and claudin-5, and upregulation of the edema inducer, aquaporin 4, were also remarkably inhibited. In addition, brain infarct volume, pro-inflammatory protein expression, and MMP-9 activation were effectively suppressed. These MCAO-induced changes were only marginally improved by treatment with MSCs-mCherry, which did not overexpress FGF21. Overexpression of FGF21 dramatically improved the therapeutic efficacy of MSCs in treating ischemic stroke. Given its multiple benefits and long therapeutic window, MSC-FGF21 therapy may be a promising treatment strategy for ischemic stroke.

Combination of fMRI and PET reveals the beneficial effect of three-phase enriched environment on post-stroke memory deficits by enhancing plasticity of brain connectivity between hippocampus and peri-hippocampal cortex.

The three-phase enriched environment (EE) intervention paradigm has been shown to improve learning and memory function after cerebral ischemia, but the neuronal mechanisms are still unclear. This study aimed to investigate the hippocampal-cortical connectivity and the metabolic interactions between neurons and astrocytes to elucidate the underlying mechanisms of EE-induced memory improvement after stroke. Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) or sham surgery and housed in standard environment or EE for 30 days. Memory function was examined by Morris water maze (MWM) test. Magnetic resonance imaging (MRI) was conducted to detect the structural and functional changes. [18 F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) was conducted to detect brain energy metabolism. PET-based brain connectivity and network analysis was performed to study the changes of hippocampal-cortical connectivity. Astrocyte-neuron metabolic coupling, including gap junction protein connexin 43 (Cx43), glucose transporters (GLUTs), and monocarboxylate transporters (MCTs), was detected by histological studies. Our results showed EE promoted memory function improvement, protected structure integrity, and benefited energy metabolism after stroke. More importantly, EE intervention significantly increased functional connectivity between the hippocampus and peri-hippocampal cortical regions, and specifically regulated the level of Cx43, GLUTs and MCTs in the hippocampus and cortex. Our results revealed the three-phase enriched environment paradigm enhanced hippocampal-cortical connectivity plasticity and ameliorated post-stroke memory deficits. These findings might provide some new clues for the development of EE and thus facilitate the clinical transformation of EE.

Exercise improves cardiac function and attenuates myocardial inflammation and apoptosis by regulating APJ/STAT3 in mice with stroke

Stroke can induce cardiac dysfunction without a primary cardiac disease. Exercise can promote the overall rehabilitation of stroke patients and be beneficial for all kinds of heart diseases. However, the mechanisms underlying the protective effects of exercise in stroke-induced cardiac dysfunction are poorly understood. Hence, we aimed to distinguish the different effects of acute and long-term exercise and further study the mechanism of protection against cardiomyopathy caused by stroke. Mice underwent a single acute session or long-term exercise for 30 days, followed by middle cerebral artery occlusion surgery. The expression of apoptosis-related proteins and proinflammatory factors in the heart was evaluated. Then, overexpression of apelin peptide jejunum (APJ) transfected adeno-associated virus type 9 (AAV9) and inhibition of signal transducer and activator of transcription 3 (STAT3) by Stattic were used in stroke mice or hypoxic cardiomyocytes. ML221 were used to inhibit APJ activity in exercise mouse. Thereafter, changes in apoptotic and proinflammatory factors were evaluated. The results demonstrated that chronic exercise prevented myocardial inflammation, apoptosis and cardiac dysfunction after stroke. However, acute exercise did not have similar effects. Exercise maintained the levels of APJ expression and decreased phosphorylated-STAT3 (p-STAT3) activation to protect cardiomyocytes. Moreover, APJ overexpression promoted cardiomyocyte survival and reduced p-STAT3 levels. STAT3 inhibition also reduced apoptosis and proinflammatory factors in mice hearts. Conversely, the protective effect of exercise was eliminated by APJ inhibition. This study showed that exercise can maintain APJ expression and inhibit p-STAT3, thus, conferring protection against myocardial inflammation and apoptosis induced by stroke.

Ruscogenin timing administration mitigates cerebral ischemia-reperfusion injury through regulating circadian genes and activating Nrf2 pathway

BackgroundRuscogenin (Rus), a steroidal sapogenin extracted from Ophiopogon japonicus (L. f.) Ker-Gawl., has the effect of alleviating cerebral ischemia-reperfusion injury (IRI), acute lung injury. At present, the chronopharmacological effects of Rus are still unknown. PurposeThis study explored the alleviating effect and mechanism of Rus timing administration on mice cerebral IRI. MethodsThe animals in different groups were administrated Rus (10 mg/kg) by gavage at four time points (23:00–01:00, 05:00–07:00, 11:00–13:00, 17:00–19:00) respectively for 3 days. On the 4th day, middle cerebral artery occlusion (MCAO) surgery was operated during 5:00–7:00. Behavioral tests were executed and the brain was collected for infarct volume, qPCR and immunoblot detection. The levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin-1beta (IL-1β) and inducible nitric oxide synthase (iNOS) were detected by qPCR. Glutathione (GSH), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in serum and cerebral cortex were detected. The clock genes were tested by western blot. Based on these results, 17:00–19:00 was selected to administrate Rus for further mechanism study and Nrf2 blocker group was administrated all-trans-retinoic acid (ATRA) at 14:00 for 3 days. ResultsAdministration of Rus reduced cerebral infarcted volume, ameliorated the behavior score and upregulated the mRNA and protein expression of Per1, Bmal1, Clock, Rev-erbα, transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), quinone oxidoreductase 1 (NQO1). Administration of Rus during 17:00–19:00 had better preventive effect than other three time points. Combined administration of ATRA blunted the preventive effect of Rus. ConclusionThe preventive effect of Rus is affected by the time of administration, which was regulated by Nrf2 pathway. Taken together, we provide solid evidence to suggest that different administration time point affect the effectiveness of Rus in alleviating IRI.

Remote limb ischemic postconditioning inhibits microglia pyroptosis by modulating HGF after acute ischemia stroke.

The repetitive inflation-deflation of a blood pressure cuff on a limb is known as remote limb ischemic postconditioning (RIPostC). It prevents brain damage induced by acute ischemia stroke (AIS). Pyroptosis, executed by the pore-forming protein gasdermin D (GSDMD), is a type of regulated cell death triggered by proinflammatory signals. It contributes to the pathogenesis of ischemic brain injury. However, the effects of RIPostC on pyroptosis following AIS remain largely unknown. In our study, linear correlation analysis confirmed that serum GSDMD levels in AIS patients upon admission were positively correlated with NIHSS scores. RIPostC treatment significantly reduced GSDMD level compared with patients without RIPostC at 3 days post-treatment. Besides, middle cerebral artery occlusion (MCAO) surgery was performed on C57BL/6 male mice and RIPostC was induced immediately after MCAO. We found that RIPostC suppressed the activation of NLRP3 inflammasome to reduce the maturation of GSDMD, leading to decreased pyroptosis in microglia after AIS. Hepatocyte growth factor (HGF) was identified using the high throughput screening. Importantly, HGF siRNA, exogenous HGF, and ISG15 siRNA were used to reveal that HGF/ISG15 is a possible mechanism of RIPostC regulation in vivo and in vitro.

M2 Microglia Extracellular Vesicle miR-124 Regulates Neural Stem Cell Differentiation in Ischemic Stroke via AAK1/NOTCH.

Small extracellular vesicles (sEVs) derived from M2 microglia (M2-microglia-derived small extracellular vesicles [M2-sEVs]) contribute to central nervous system repair, although the underlying mechanism remains unknown. In this study, we aimed to identify the mechanism through which microRNA-124 (miR-124) carried in sEVs promotes neural stem cell (NSC) proliferation and neuronal differentiation in the ischemic mouse brain. M2-sEVs with or without miR-124 knockdown were injected intravenously for 7 consecutive days after transient middle cerebral artery occlusion surgery. The atrophy volume, neurological score, and degree of neurogenesis were examined at different time points after ischemic attack. NSCs treated with different sEVs were subjected to proteomic analysis. Target protein concentrations were quantified, and subsequent bioinformatic analysis was conducted to explore the key signaling pathways. M2-sEV transplantation promoted functional neurological recovery following transient middle cerebral artery occlusion injury. M2-sEV treatment decreased the brain atrophy volume, neurological score, and mortality rate. The effect was reserved by knockdown of miR-124 in M2-sEVs. M2-sEVs promoted proliferation and differentiation of mature neuronal NSCs in vivo. Proteomic analysis of NSC samples treated with M2-sEVs with and without miR-124 knockdown revealed that AAK1 (adaptor-associated protein kinase 1) was the key responding protein in NSCs. The binding of AAK1 to Notch promoted the differentiation of NSCs into neurons rather than astrocytes. Our data suggest that AAK1/Notch is the key pathway in NSCs that responds to the miR-124 carried within M2-sEVs in the ischemic brain. M2-sEVs carrying ample quantities of miR-124 promote functional recovery after ischemic stroke by enhancing NSC proliferation and differentiation. Targeting of M2-sEVs could represent a potential therapeutic strategy for brain recovery.