RESUMEN

Gene therapy using a protein-based CRISPR system in the brain has practical limitations due to current delivery systems, especially in the presence of arterial occlusion. To overcome these obstacles and improve stability, we designed a system for intranasal administration of gene therapy for the treatment of ischemic stroke. Methods: Nanoparticles containing the protein-based CRISPR/dCas9 system targeting Sirt1 were delivered intranasally to the brain in a mouse model of ischemic stroke. The CRISPR/dCas9 system was encapsulated with calcium phosphate (CaP) nanoparticles to prevent them from being degraded. They were then conjugated with ß-hydroxybutyrates (bHb) to target monocarboxylic acid transporter 1 (MCT1) in nasal epithelial cells to facilitate their transfer into the brain. Results: Human nasal epithelial cells were shown to uptake and transfer nanoparticles to human brain endothelial cells with high efficiency in vitro. The intranasal administration of the dCas9/CaP/PEI-PEG-bHb nanoparticles in mice effectively upregulated the target gene, Sirt1, in the brain, decreased cerebral edema and increased survival after permanent middle cerebral artery occlusion. Additionally, we observed no significant in vivo toxicity associated with intranasal administration of the nanoparticles, highlighting the safety of this approach. Conclusion: This study demonstrates that the proposed protein-based CRISPR-dCas9 system targeting neuroprotective genes in general, and SIRT1 in particular, can be a potential novel therapy for acute ischemic stroke.

Asunto(s)

Administración Intranasal , Encéfalo , Modelos Animales de Enfermedad , Terapia Genética , Accidente Cerebrovascular Isquémico , Nanopartículas , Sirtuina 1 , Animales , Ratones , Humanos , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/genética , Nanopartículas/administración & dosificación , Terapia Genética/métodos , Sirtuina 1/genética , Sirtuina 1/metabolismo , Encéfalo/metabolismo , Masculino , Fosfatos de Calcio , Sistemas CRISPR-Cas , Ratones Endogámicos C57BL , Células Endoteliales/metabolismo , Isquemia Encefálica/terapia , Isquemia Encefálica/genética , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/genética , Células Epiteliales/metabolismo

RESUMEN

Acute ischemic stroke (AIS) is a major global health concern due to its high mortality and disability rates. Hemorrhagic transformation, a common complication of AIS, leads to poor prognosis yet lacks effective treatments. Preclinical studies indicate that hyperbaric oxygen (HBO) treatment within 12 h of AIS onset alleviates ischemia/reperfusion injuries, including hemorrhagic transformation. However, clinical trials have yielded conflicting results, suggesting some underlying mechanisms remain unclear. In this study, we confirmed that HBO treatments beginning within 1 h post reperfusion significantly alleviated the haemorrhage and neurological deficits in hyperglycemic transient middle cerebral arterial occlusion (tMCAO) mice, partly due to the inhibition of the NLRP3 inflammasome-mediated pro-inflammatory response in microglia. Notably, reactive oxygen species (ROS) mediate the anti-inflammatory and protective effect of early HBO treatment, as edaravone and N-Acetyl-L-Cysteine (NAC), two commonly used antioxidants, reversed the suppressive effect of HBO treatment on NLRP3 inflammasome-mediated inflammation in microglia. Furthermore, NAC countered the protective effect of early HBO treatment in tMCAO mice with hyperglycemia. These findings support that early HBO treatment is a promising intervention for AIS, however, caution is warranted when combining antioxidants with HBO treatment. Further assessments are needed to clarify the role of antioxidants in HBO therapy for AIS.

Asunto(s)

Oxigenoterapia Hiperbárica , Hiperglucemia , Microglía , Especies Reactivas de Oxígeno , Animales , Microglía/metabolismo , Microglía/efectos de los fármacos , Oxigenoterapia Hiperbárica/métodos , Ratones , Especies Reactivas de Oxígeno/metabolismo , Hiperglucemia/metabolismo , Hiperglucemia/complicaciones , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Modelos Animales de Enfermedad , Accidente Cerebrovascular/terapia , Accidente Cerebrovascular/metabolismo , Antioxidantes/farmacología , Ratones Endogámicos C57BL , Infarto de la Arteria Cerebral Media/terapia , Edaravona/farmacología , Daño por Reperfusión/metabolismo

RESUMEN

In recent years, the mechanism of acupuncture in the treatment of post-stroke cognitive impairment (PSCI) has not been fully elucidated. The balance between mitochondrial fission and fusion is important for PSCI. Our previous research demonstrated that electroacupuncture can improve learning and memory in middle cerebral artery ischemia reperfusion (MCAO/R) rats. However, the specific mechanism by which electroacupuncture improves learning and memory in MCAO/R rats by regulating mitochondrial fission and fusion needs to be further investigated. The MCAO/R rats was developed using the line-bolt method. The rats were randomly divided into sham-operated (Sham), model (MCAO/R), electroacupuncture (MCAO/R + EA) and sham-electroacupuncture (MCAO/R + sham EA) groups. Investigating the effects of EA on the expression of Sirtuin1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), Optic atrophy 1R + (OPA1) and Dynamin-related protein 1 (DRP1) in hippocampal neurons and on the morphology and function of hippocampal neurons and mitochondria. EA was able to reduce neurologic deficit scores and cerebral infarct volume and improve new object discrimination in MCAO/R rats, but there were no significant changes in these indices in the sham-electroacupuncture group. Moreover, EA increased the expression of SIRT1, PGC-1α, and OPA1 in hippocampal tissues, inhibited the expression of DRP1, attenuated neuronal and mitochondrial damage, and reduced mitochondrial fragmentation. The mechanism by which EA improves learning memory deficits in MCAO/R rats may be related to the inhibition of SIRT1/PGC-1α expression, the enhancement of mitochondrial fusion and the obstruction of its fission, and the reduction of hippocampal neuronal damage.

Asunto(s)

Disfunción Cognitiva , Electroacupuntura , Hipocampo , Infarto de la Arteria Cerebral Media , Accidente Cerebrovascular Isquémico , Dinámicas Mitocondriales , Animales , Masculino , Ratas , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Isquemia Encefálica/complicaciones , Disfunción Cognitiva/terapia , Disfunción Cognitiva/etiología , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Dinaminas/metabolismo , Electroacupuntura/métodos , GTP Fosfohidrolasas/metabolismo , Hipocampo/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/complicaciones , Mitocondrias/metabolismo , Dinámicas Mitocondriales/fisiología , Neuronas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Sirtuina 1/metabolismo , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/terapia

RESUMEN

BACKGROUND: Ischemic stroke is one of the leading causes of chronic disability worldwide, and stroke-induced heart damage can lead to death. According to research, patients with a variety of brain disease have good clinical results after vagus nerve stimulation (VNS). After ischemic stroke, mast cells (MCs) degranulate and release a large number of mediators, which may cause systemic inflammation. Chymase secreted by MCs can increase the levels of pathological angiotensin II (AngⅡ), which plays a crucial role in the deterioration of heart disease. Our goal was to develop a minimally invasive, targeted, and convenient VNS approach to assess the impact of VNS and to clarify the relationship between VNS and MCs in the prognosis of patients with myocardial atrophy after acute ischemic stroke. METHODS: In this study, we verified the role of VNS in the treatment of myocardial atrophy after stroke and its molecular mechanism using a rat model of middle cerebral artery occlusion (MCAO/r). Behavioral studies were assessed using neurobehavioral deficit scores. Enzyme-linked immunosorbent assays, immunofluorescence staining, Western blotting and qRT-PCR were used to analyze the expression levels of myocardial atrophy, MC and inflammatory markers in rat hearts. RESULTS: VNS improved myocardial atrophy in MCAO/r rats, inhibited MC activation, reduced the expression of chymase and AngⅡ, and inhibited the expression of proinflammatory factors. The chymase activator C48/80 reversed these effects of VNS. Chymase activation inhibited the effect of VNS on myocardial atrophy in MCAO/r rats, increased AngⅡ expression and aggravated inflammation and autophagy. The myocardial atrophy of MCAO/r rats was improved after chymase inhibition, and AngⅡ expression, inflammation and autophagy were reduced. Our results suggest that VNS may reduce the expression of chymase and AngⅡ by inhibiting MC activation, thereby improving myocardial atrophy and reducing inflammation and autophagy in MCAO/r rats. Inhibition of MC activation may be an effective strategy for treating myocardial atrophy after stroke. CONCLUSIONS: VNS inhibits MC activation and reduces the expression of chymase and AngII, thereby alleviating myocardial atrophy, inflammation and autophagy after stroke.

Asunto(s)

Quimasas , Infarto de la Arteria Cerebral Media , Accidente Cerebrovascular Isquémico , Mastocitos , Ratas Sprague-Dawley , Estimulación del Nervio Vago , Animales , Mastocitos/inmunología , Masculino , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/inmunología , Accidente Cerebrovascular Isquémico/patología , Ratas , Quimasas/metabolismo , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/inmunología , Miocardio/patología , Miocardio/inmunología , Atrofia , Modelos Animales de Enfermedad , Angiotensina II/metabolismo

RESUMEN

Basi-parallel anatomic scanning has been widely used for assessing the vascular morphology of vertebral basilar arteries. Previous studies have demonstrated its efficacy in evaluating the morphology of the MCA, which we refer to as MCA parallel anatomic scanning MR imaging (MCPAS). In this study, we present our experience with the application of MCPAS in patients with MCA occlusion. Endovascular treatment was performed on the patients with intact MCA morphology visible in on MCPAS, with no intracranial hemorrhage, occlusion, or other complications observed. No severe stenosis or re-occlusion was observed at the 12-month postoperative follow-up. In conclusion, MCPAS is an effective method for assessing the outer contour of an occlusive MCA. Endovascular treatment can be considered a safe and efficient option for patients who show a favorable MCA through MCPAS assessment.

Asunto(s)

Procedimientos Endovasculares , Infarto de la Arteria Cerebral Media , Humanos , Masculino , Femenino , Procedimientos Endovasculares/métodos , Persona de Mediana Edad , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/cirugía , Infarto de la Arteria Cerebral Media/terapia , Resultado del Tratamiento , Anciano , Enfermedad Crónica , Angiografía por Resonancia Magnética/métodos , Imagen por Resonancia Magnética/métodos

RESUMEN

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells (BECs) results in long-term neurological dysfunction post-stroke. We previously reported data from a pilot study where intravenous administration of human BEC (hBEC)-derived mitochondria-containing extracellular vesicles (EVs) showed a potential efficacy signal in a mouse middle cerebral artery occlusion (MCAo) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species (e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC (mBEC)-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated potential differences in the mitochondria transfer of EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) vs. cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa). Our results showed that while both hBEC- and mBEC-EVs transferred EV mitochondria, mBEC-EVs outperformed hBEC-EVs in increasing ATP levels and improved recipient mBEC mitochondrial function via increasing oxygen consumption rates. mBEC-EVs significantly reduced brain infarct volume and neurological deficit scores compared to vehicle-injected MCAo mice. The superior therapeutic efficacy of mBEC-EVs in MCAo mice support the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical mouse models.

Asunto(s)

Encéfalo , Células Endoteliales , Vesículas Extracelulares , Infarto de la Arteria Cerebral Media , Accidente Cerebrovascular Isquémico , Ratones Endogámicos C57BL , Mitocondrias , Animales , Vesículas Extracelulares/trasplante , Vesículas Extracelulares/metabolismo , Mitocondrias/metabolismo , Humanos , Células Endoteliales/metabolismo , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/metabolismo , Infarto de la Arteria Cerebral Media/terapia , Encéfalo/metabolismo , Masculino , Ratones , Células Cultivadas , Barrera Hematoencefálica/metabolismo

RESUMEN

INTRODUCTION: Cerebral ischemia reperfusion injury (CIRI) often leads to deleterious complications after stroke patients receive reperfusion therapy. Exercise preconditioning (EP) has been reported to facilitate brain function recovery. We aim to explore the specific mechanism of EP in CIRI. METHODS: Sprague-Dawley rats were randomized into Sham, middle cerebral artery occlusion (MCAO), and EP groups (n = 11). The rats in the EP group received adaptive training for 3 days (10 m/min, 20 min/day, with a 0° incline) and formal training for 3 weeks (6 days/week, 25 m/min, 30 min/day, with a 0° incline). Then, rats underwent MCAO surgery to establish CIRI models. After 48 h, neurological deficits and cerebral infarction of the rats were measured. Neuronal death and apoptosis in the cerebral cortices were detected. Furthermore, RNA sequencing was conducted to investigate the specific mechanism of EP on CIRI, and qPCR and Western blotting were further applied to confirm RNA sequencing results. RESULTS: EP improved neurological deficit scores and reduced cerebral infarction in MCAO rats. Additionally, pre-ischemic exercise also alleviated neuronal death and apoptosis of the cerebral cortices in MCAO rats. Importantly, 17 differentially expressed genes (DEGs) were identified through RNA sequencing, and these DEGs were mainly enriched in the HIF-1 pathway, cellular senescence, proteoglycans in cancer, and so on. qPCR and Western blotting further confirmed that EP could suppress TIMP1, SOCS3, ANGPTL4, CDO1, and SERPINE1 expressions in MCAO rats. CONCLUSION: EP can improve CIRI in vivo, the mechanism may relate to TIMP1 expression and HIF-1 pathway, which provided novel targets for CIRI treatment.

Asunto(s)

Infarto de la Arteria Cerebral Media , Condicionamiento Físico Animal , Ratas Sprague-Dawley , Daño por Reperfusión , Animales , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & control , Daño por Reperfusión/terapia , Ratas , Masculino , Condicionamiento Físico Animal/fisiología , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Análisis de Secuencia de ARN , Modelos Animales de Enfermedad , Apoptosis , Precondicionamiento Isquémico/métodos

RESUMEN

Brain injury caused by stroke has a high rate of mortality and remains a major medical challenge worldwide. In recent years, there has been significant attention given to the use of human Umbilical cord-derived Mesenchymal Stem Cells (hUC-MSCs) for the treatment of stroke in different adult and neonate animal models of stroke. However, using hUC-MSCs by systemic administration to treat ischemic stroke has not been investigated sufficiently. In this study, we conducted various experiments to explore the neuroprotection of hUC-MSCs in rats. Our findings demonstrate that an intravenous injection of a high dose of hUC-MSCs at 2 × 10^7 cells/kg markedly ameliorated brain injury resulting from ischemic stroke. This improvement was observed one day after inducing transient middle cerebral artery occlusion (MCAO) and subsequent reperfusion in rats. Notably, the efficacy of this single administration of hUC-MSCs surpassed that of edaravone, even when the latter was used continuously over three days. Mechanistically, secretory factors derived from hUC-MSCs, such as HGF, BDNF, and TNFR1, ameliorated the levels of MDA and T-SOD to regulate oxidative stress. In particular, TNFR1 also improved the expression of NQO-1 and HO-1, important proteins associated with oxidative stress. More importantly, TNFR1 played a significant role in reducing inflammation by modulating IL-6 levels in the blood. Furthermore, TNFR1 was observed to influence the permeability of the blood-brain barrier (BBB) as demonstrated in the evan's blue experiment and protein expression of ZO-1. This study represented a breakthrough in traditional methods and provided a novel strategy for clinical medication and trials.

Asunto(s)

Accidente Cerebrovascular Isquémico , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Estrés Oxidativo , Ratas Sprague-Dawley , Cordón Umbilical , Animales , Estrés Oxidativo/fisiología , Humanos , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/citología , Masculino , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/terapia , Ratas , Inflamación/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/terapia , Neuroprotección/fisiología , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/metabolismo

RESUMEN

We have previously reported that the expression of miR-34c-5p was up-regulated during acupuncture treatment in the setting of a cerebral ischemia/reperfusion injury (CIRI), indicating that miR-34c-5p plays an important role in healing from a CIRI-induced brain injury. This study sought to evaluate the effects of acupuncture on miR-34c-5p expression and autophagy in the forward and reverse directions using a rat focal cerebral ischemia/reperfusion model. After 120 minutes of middle cerebral artery occlusion and reperfusion, rats were treated with acupuncture at the "Dazhui" (DU20), "Baihui" (DU26) and "Renzhong" (DU14) points. Neurologic function deficit score, cerebral infarct area ratio, neuronal apoptosis and miR-34c-5p expression were evaluated 72 hr after treatment. The autophagy agonist RAPA and the antagonist 3MA were used to evaluate the neuro protective effects of autophagy-mediated acupuncture. We found that acupuncture treatment improved autophagy in the brain tissue of CIRI rats. Acupuncture reversed the negative effects of 3MA on CIRI, and acupuncture combined with RAPA further enhanced autophagy. We also found that acupuncture could increase miR-34c-5p expression in hippocampal neurons after ischemia/reperfusion. Acupuncture and a miR-34c agomir were able to enhance autophagy, improve neurologic deficits, and reduce the cerebral infarct area ratio and apoptosis rate by promoting the expression of miR-34c-5p. Silencing miR-34c resulted in a significantly reduced activating effect of acupuncture on autophagy and increased apoptosis, neurologic deficit symptoms, and cerebral infarct area ratio. This confirms that acupuncture can upregulate miR-34c-5p expression, which is beneficial in the treatment of CIRI.

Asunto(s)

Terapia por Acupuntura , Autofagia , Isquemia Encefálica , MicroARNs , Ratas Sprague-Dawley , Daño por Reperfusión , Animales , MicroARNs/metabolismo , MicroARNs/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/terapia , Autofagia/fisiología , Masculino , Terapia por Acupuntura/métodos , Ratas , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/metabolismo , Apoptosis/fisiología , Neuronas/metabolismo , Modelos Animales de Enfermedad , Encéfalo/metabolismo

RESUMEN

Acupuncture is a traditional Chinese therapy with treating potential against cognitive dysfunction. MicroRNA-21-3p (miR-21-3p) is well characterized for its benefits on neural tissues. The current study hypothesizes that the acupuncture aiming "Du" channel could attenuate IS-induced neural disorders by modulating the function of REST/miR-21-3p axis. Complications associated with IS are induced by a middle cerebral artery occlusion (MCAO) model in vivo. The disorders are then handled with the acupuncture with nimodipine as the positive control. It is found that the acupuncture improved cognitive function, reduced brain apoptosis, and increased the viable neuron number of model rats. Additionally, the production of cytokines is also suppressed by the acupuncture. At the molecular level, the level of miR-21-3p was up-regulated, while the level of REST was down-regulated by the acupuncture. The changes in miR-REST/21-3p contributed to the inhibition of PDCD4. Collectively, the findings in the current study highlight that miR-21-3p is associated with the anti-IS function of the acupuncture, which is mediated by the inhibition of REST.

Asunto(s)

Terapia por Acupuntura , Proteínas Reguladoras de la Apoptosis , Infarto de la Arteria Cerebral Media , MicroARNs , Transducción de Señal , Animales , Masculino , Ratas , Terapia por Acupuntura/métodos , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Encéfalo/metabolismo , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/genética , MicroARNs/genética , MicroARNs/metabolismo , Ratas Sprague-Dawley , Proteínas Represoras , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética

RESUMEN

Neuroinflammation caused by excessive microglial activation plays a key role in the pathogenesis of ischemic stroke. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive neuromodulatory technique that has recently been reported to regulate microglial functions and exert anti-inflammatory effects. The intermittent burst stimulation (iTBS) regimen in rTMS improves neuronal excitability. However, whether iTBS exerts its anti-inflammatory effects by stimulating neurons and thereby modulating microglial polarization remains unclear. Motor function was assessed after 1 week of rTMS (iTBS regimen) treatment in adult male mice with occlusion/reperfusion of the middle cerebral artery (MCAO/r) injury. We also investigated the molecular biological alterations associated with microglial polarization using a cell proliferation assay, multiplex cytokine bioassays, and immunofluorescence staining. iTBS regimen can improve balance and motor coordination function, increase spontaneous movement, and improve walking function in mice with early cerebral ischemia injury. Expression levels of IL-1ß, TNF-α, and IL-10 increased significantly in mice with MCAO injury. Especially, rTMS significantly increased the number of proliferating cells in the infarcted cortex. The fluorescence intensity of MAP2 in the peri-infarct area of MCAO injured mice was low, but the signal was broader. Compared with MCAO group, the fluorescence intensity of MAP2 in rTMS group was significantly increased. rTMS inhibited pro-inflammatory M1 activation (Iba1+/CD86+) and improved anti-inflammatory M2 activation (Iba1+/CD206+) in the peri-infarct zone, thus significantly changing the phenotypic ratio M1/M2. rTMS improves motor dysfunction and neuroinflammation after cerebral I/R injury in mice by regulating microglial polarization.

Asunto(s)

Accidente Cerebrovascular Isquémico , Microglía , Estimulación Magnética Transcraneal , Animales , Masculino , Estimulación Magnética Transcraneal/métodos , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/fisiopatología , Ratones , Microglía/metabolismo , Ratones Endogámicos C57BL , Infarto de la Arteria Cerebral Media/terapia , Actividad Motora/fisiología , Plasticidad Neuronal/fisiología

RESUMEN

An ischemic stroke, one of the leading causes of morbidity and mortality, is caused by ischemia and hemorrhage resulting in impeded blood supply to the brain. According to many studies, blueberries have been shown to have a therapeutic effect in a variety of diseases. Therefore, in this study, we investigated whether blueberry-treated mesenchymal stem cell (MSC)-derived extracellular vesicles (B-EVs) have therapeutic effects in in vitro and in vivo stroke models. We isolated the extracellular vesicles using cryo-TEM and characterized the particles and concentrations using NTA. MSC-derived extracellular vesicles (A-EVs) and B-EVs were round with a lipid bilayer structure and a diameter of ~150 nm. In addition, A-EVs and B-EVs were shown to affect angiogenesis, cell cycle, differentiation, DNA repair, inflammation, and neurogenesis following KEGG pathway and GO analyses. We investigated the protective effects of A-EVs and B-EVs against neuronal cell death in oxygen-glucose deprivation (OGD) cells and a middle cerebral artery occlusion (MCAo) animal model. The results showed that the cell viability was increased with EV treatment in HT22 cells. In the animal, the size of the cerebral infarction was decreased, and the behavioral assessment was improved with EV injections. The levels of NeuN and neurofilament heavy chain (NFH)-positive cells were also increased with EV treatment yet decreased in the MCAo group. In addition, the number of apoptotic cells was decreased with EV treatment compared with ischemic animals following TUNEL and Bax/Bcl-2 staining. These data suggested that EVs, especially B-EVs, had a therapeutic effect and could reduce apoptotic cell death after ischemic injury.

Asunto(s)

Arándanos Azules (Planta) , Vesículas Extracelulares , Accidente Cerebrovascular Isquémico , Células Madre Mesenquimatosas , Vesículas Extracelulares/metabolismo , Animales , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/patología , Arándanos Azules (Planta)/química , Masculino , Modelos Animales de Enfermedad , Supervivencia Celular/efectos de los fármacos , Línea Celular , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/metabolismo

RESUMEN

BACKGROUND: This study aimed to investigate whether moxibustion could affect PI3K/Akt pathway to regulate Transforming acidic coiled-coil containing protein 3 (TACC3) and promote axonal regeneration to improve learning and memory function in middle cerebral artery occlusion (MCAO) rats. METHODS: Sixty SD rats were randomly divided into 4 groups: sham-operated control group (SC), model control group (MC), model + moxibustion group (MM), and model + inhibitor + moxibustion group (MIM). The rats in MC, MM, and MIM groups were made into MCAO models, and PI3K inhibitor LY294002 was injected into the rats in MIM group before modeling; while the rats in SC group were only treated with artery separation without monofilament inserting. After that, the rats in MM and MIM groups were intervented with moxibustion. We used the Zea-Longa scale, micro-Magnetic Resonance Imaging (micro-MRI), Morris water maze (MWM), TUNEL, western blot (WB), immunofluorescence and immunohistochemistry to evaluate the neurological deficits, cerebral infarct volume, learning and memory, apoptotic cell percentage in the hippocampal, the expression level of axonal regeneration and PI3K/AKt related proteins, the expression level of TACC3. The detection of 2 h after surgery showed the result before moxibustion and 7 days after the intervention showed the results after moxibustion. RESULTS: After 7 d of intervention, the scores of Zea-Longa and the cerebral infarct volume, the escape latency, the percentage of apoptosis cells of MM group were lower than that of MC and MIM groups; the frequency of rats crossed the previous platform location, PI3K, p-Akt/t-Akt and TACC3, the level of GAP-43 in MM group was more than MC and MIM groups (P < 0.05). While no statistical difference existed between MIM group and MC group (P > 0.05). CONCLUSION: Moxibustion can promote axonal regeneration and improve learning and memory of Post-stroke cognitive impairment via activating the PI3K/AKT signaling pathway and TACC3.

Asunto(s)

Axones , Disfunción Cognitiva , Memoria , Proteínas Asociadas a Microtúbulos , Regeneración Nerviosa , Transducción de Señal , Animales , Masculino , Ratas , Axones/fisiología , Disfunción Cognitiva/terapia , Disfunción Cognitiva/etiología , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/complicaciones , Péptidos y Proteínas de Señalización Intercelular , Memoria/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Moxibustión/métodos , Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Accidente Cerebrovascular/terapia , Accidente Cerebrovascular/complicaciones

RESUMEN

Exosomes (Exo) generated from mesenchymal stem cells (MSCs) have great therapeutic potential in ischemia-reperfusion treatment. For best therapeutic effect, high quality Exo product and effective delivery system are indispensable. In this study, we developed a new strategy for ischemia-reperfusion recovery by combining MSCs 3D (3D-MSC) culturing technology to generate Exo (3D-MSC-Exo) and microneedle for topical delivery. Firstly, primary MSCs from neonatal mice were isolated and 3D cultured with gelatin methacryloyl (GelMA) hydrogel to prepare 3D-MSC-Exo. The 3D-MSC showed better viability and 3D-MSC-Exo exhibited more effective effects of reducing neuroinflammation, inhibiting glial scarring, and promoting angiogenesis. Subsequently, the biocompatible GelMA was used to construct microneedles for 3D-Exo delivery (GelMA-MN@3D-Exo). The results demonstrated GelMA microneedles had excellent 3D-Exo loading capacity and enabled continuous 3D-Exo release to maintain effective therapeutic concentrations. Furthermore, the rat middle cerebral artery occlusion (MCAO) model was established to evaluate the therapeutic effect of GelMA-MN@3D-Exo in ischemia-reperfusion in vivo. Animal experiments showed that the GelMA-MN@3D-Exo system could effectively reduce the local neuroinflammatory reaction, promote angiogenesis and minimize glial scar proliferation in ischemia-reperfusion. The underlying reasons for the stronger neuroprotective effect of 3D-Exo was further studied using mass spectrometry and transcriptome assays, verifying their effects on immune regulation and cell proliferation. Taken together, our findings demonstrated that GelMA-MN@3D-Exo microneedle can effectively attenuate ischemia-reperfusion cell damage in the MCAO model, which provides a promising therapeutic strategy for ischemia-reperfusion recovery.

Asunto(s)

Exosomas , Gelatina , Células Madre Mesenquimatosas , Agujas , Daño por Reperfusión , Animales , Gelatina/química , Ratones , Ratas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Daño por Reperfusión/prevención & control , Daño por Reperfusión/terapia , Metacrilatos/química , Masculino , Modelos Animales de Enfermedad , Hidrogeles/química , Ratas Sprague-Dawley , Infarto de la Arteria Cerebral Media/terapia

RESUMEN

This study aims to investigate how electroacupuncture regulates the learning and memory abilities of poststroke cognitive impairment (PSCI) rats through the TLR4/NF-κB/NLRP3 signaling pathway on the hippocampal microglia. Thirty male rats were randomly divided into three groups: sham surgery group, PSCI model group, and electroacupuncture group, with 10 rats in each group. Middle cerebral artery occlusion was used to establish the PSCI model. The Zea Longa method was used to score the rats' neurological function. Electroacupuncture was utilized for 21 days to improve PSCI. The learning and memory abilities of rats were tested using the Morris water maze. Hematoxylin-eosin staining and immunofluorescence were used to find the hippocampus' pathological changes. The concentration of interleukin-1ß, interleukin-6, tumor necrosis factor-α, and interleukin-18 were detected by ELISA. The mRNA expression levels of associated inflammatory corpuscles were measured by quantitative real-time PCR. The protein expression levels of TLR4, MyD88, NF-κB, and NLRP3 were measured using western blotting. Electroacupuncture improved not only the learning and memory abilities of PSCI rats but also hippocampal morphology. Electroacupuncture inhibited the activation of microglia and the TLR4/NF-κB/NLRP3 signaling pathway. Electroacupuncture also reduced proinflammatory factors and restrained the mRNA levels of NLRP3-associated inflammatory cytokines. Its mechanism was related to inhibiting the expression of the TLR4/NF-κB/NLRP3 signaling pathway, attenuating the release of inflammatory factors, and regulating the activation of hippocampal microglia in the brain.

Asunto(s)

Electroacupuntura , Hipocampo , Microglía , FN-kappa B , Proteína con Dominio Pirina 3 de la Familia NLR , Ratas Sprague-Dawley , Transducción de Señal , Receptor Toll-Like 4 , Animales , Electroacupuntura/métodos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Receptor Toll-Like 4/metabolismo , Masculino , Hipocampo/metabolismo , Ratas , FN-kappa B/metabolismo , Microglía/metabolismo , Transducción de Señal/fisiología , Memoria/fisiología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/terapia , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/metabolismo

RESUMEN

BACKGROUND: Electroacupuncture (EA) has been shown to facilitate brain plasticity-related functional recovery following ischemic stroke. The functional magnetic resonance imaging technique can be used to determine the range and mode of brain activation. After stroke, EA has been shown to alter brain connectivity, whereas EA's effect on brain network topology properties remains unclear. An evaluation of EA's effects on global and nodal topological properties in rats with ischemia reperfusion was conducted in this study. METHODS AND RESULTS: There were three groups of adult male Sprague-Dawley rats: sham-operated group (sham group), middle cerebral artery occlusion/reperfusion (MCAO/R) group, and MCAO/R plus EA (MCAO/R + EA) group. The differences in global and nodal topological properties, including shortest path length, global efficiency, local efficiency, small-worldness index, betweenness centrality (BC), and degree centrality (DC) were estimated. Graphical network analyses revealed that, as compared with the sham group, the MCAO/R group demonstrated a decrease in BC value in the right ventral hippocampus and increased BC in the right substantia nigra, accompanied by increased DC in the left nucleus accumbens shell (AcbSh). The BC was increased in the right hippocampus ventral and decreased in the right substantia nigra after EA intervention, and MCAO/R + EA resulted in a decreased DC in left AcbSh compared to MCAO/R. CONCLUSION: The results of this study provide a potential basis for EA to promote cognitive and motor function recovery after ischemic stroke.

Asunto(s)

Electroacupuntura , Infarto de la Arteria Cerebral Media , Imagen por Resonancia Magnética , Ratas Sprague-Dawley , Daño por Reperfusión , Animales , Electroacupuntura/métodos , Masculino , Ratas , Daño por Reperfusión/fisiopatología , Daño por Reperfusión/terapia , Daño por Reperfusión/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/fisiopatología , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Encéfalo/fisiopatología , Encéfalo/diagnóstico por imagen , Isquemia Encefálica/terapia , Isquemia Encefálica/fisiopatología , Isquemia Encefálica/diagnóstico por imagen , Modelos Animales de Enfermedad , Red Nerviosa/fisiopatología , Red Nerviosa/diagnóstico por imagen , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/fisiopatología , Accidente Cerebrovascular Isquémico/diagnóstico por imagen , Hipocampo/diagnóstico por imagen , Hipocampo/fisiopatología

RESUMEN

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

Asunto(s)

Infarto de la Arteria Cerebral Media , Vitamina D3 24-Hidroxilasa , Animales , Humanos , Masculino , Ratas , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Puntos de Acupuntura , Isquemia Encefálica/terapia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/genética , Corteza Cerebral/metabolismo , Colestanotriol 26-Monooxigenasa/genética , Colestanotriol 26-Monooxigenasa/metabolismo , Citocinas/metabolismo , Citocinas/genética , Electroacupuntura , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Vitamina D3 24-Hidroxilasa/genética , Vitamina D3 24-Hidroxilasa/metabolismo

RESUMEN

This study aimed to investigate the intervention effect of tetramethylpyrazine(TMP) combined with transplantation of neural stem cells(NSCs) on middle cerebral artery occlusion(MCAO) rat model and to explore the mechanism of TMP combined with NSCs transplantation on ischemic stroke based on the regulation of stem cell biological behavior. MCAO rats were randomly divided into a model group, a TMP group, an NSCs transplantation group, and a TMP combined with NSCs transplantation group according to neurological function scores. A sham group was set up at the same time. The neurological function score was used to evaluate the improvement of neurological function in MCAO rats after TMP combined with NSCs transplantation. The proliferation, migration, and differentiation of NSCs were evaluated by BrdU, BrdU/DCX, BrdU/NeuN, and BrdU/GFAP immunofluorescence labeling. The protein expression of stromal cell-derived factor 1(SDF-1), C-X-C motif chemokine receptor 4(CXCR4), as well as oxidative stress pathway proteins nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(KEAP1), heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1) was detected by Western blot to study the migration mechanism of TMP combined with NSCs. The results showed that TMP combined with NSCs transplantation significantly improved the neurological function score in MCAO rats. Immunofluorescence staining showed a significant increase in the number of BrdU~+, BrdU~+/DCX~+, BrdU~+/NeuN~+, and BrdU~+/GFAP~+ cells in the TMP, NSCs transplantation, and combined treatment groups, with the combined treatment group showing the most significant increase. Further Western blot analysis revealed significantly elevated expression of CXCR4 protein in the TMP, NSCs transplantation, and combined treatment groups, along with up-regulated protein expression of Nrf2, HO-1, and NQO1, and decreased KEAP1 protein expression. This study showed that both TMP and NSCs transplantation can promote the recovery of neurological function by promoting the proliferation, migration, and differentiation of NSCs, and the effect of TMP combined with NSCs transplantation is superior. The mechanism of action may be related to the activation of the Nrf2/HO-1/CXCR4 pathway.

Asunto(s)

Isquemia Encefálica , Proteína Doblecortina , Factor 2 Relacionado con NF-E2 , Células-Madre Neurales , Pirazinas , Ratas Sprague-Dawley , Receptores CXCR4 , Animales , Pirazinas/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/trasplante , Células-Madre Neurales/metabolismo , Ratas , Masculino , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Isquemia Encefálica/terapia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Trasplante de Células Madre/métodos , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Humanos , Daño por Reperfusión/terapia , Daño por Reperfusión/metabolismo , Infarto de la Arteria Cerebral Media/terapia , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética

RESUMEN

Mitochondria play a crucial role in maintaining cellular energy supply and serve as a source of energy for repairing nerve damage following a stroke. Given that exercise has the potential to enhance energy metabolism, investigating the impact of exercise on mitochondrial function provides a plausible mechanism for stroke treatment. In our study, we established the middle cerebral artery occlusion (MCAO) model in Sprague-Dawley rats and implemented early exercise intervention. Neurological severity scores, beam-walking test score, and weight were used to evaluate neurological function. The volume of cerebral infarction was measured by MRI. Nerve cell apoptosis was detected by TUNEL staining. Mitochondrial morphology and structure were detected by mitochondrial electron microscopy. Mitochondrial function was assessed using membrane potential and ATP measurements. Western blotting was used to detect the protein expression of AMPK/PGC-1α/GLUT4. Through the above experiments, we found that early exercise improved neurological function in rats after MCAO, reduced cerebral infarction volume and neuronal apoptosis, promoted the recovery of mitochondrial morphology and function. We further examined the protein expression of AMPK/PGC-1α/GLUT4 signaling pathway and confirmed that early exercise was able to increase its expression. Therefore, we suggest that early exercise initiated the AMPK/PGC-1α/GLUT4 signaling pathway, restoring mitochondrial function and augmenting energy supply. This, in turn, effectively improved both nerve and body function in rats following ischemic stroke.

Asunto(s)

Proteínas Quinasas Activadas por AMP , Mitocondrias , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Condicionamiento Físico Animal , Ratas Sprague-Dawley , Transducción de Señal , Animales , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Transducción de Señal/fisiología , Masculino , Proteínas Quinasas Activadas por AMP/metabolismo , Mitocondrias/metabolismo , Condicionamiento Físico Animal/fisiología , Condicionamiento Físico Animal/métodos , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/terapia , Isquemia Encefálica/metabolismo , Ratas , Modelos Animales de Enfermedad , Apoptosis/fisiología

RESUMEN

Ischemic stroke involves various pathological processes, among which ferroptosis is crucial. Previous studies by our group have indicated that electroacupuncture (EA) mitigates ferroptosis after ischemic stroke; however, the precise mechanism underlying this effect remains unclear. In the present study, we developed a rat model of middle cerebral artery occlusion/reperfusion. We chose the main acupoint of the treatment methods of the "Awakening and Opening of the Brain". Rats' neurological function and motor coordination were evaluated by neurological function score and the rotarod test, respectively, and the volume of cerebral infarction was analyzed by 2,3,5-triphenyltetrazolium chloride Staining. The cerebrovascular conditions were visualized by time-of-flight magentic resonance angiography. In addition, we detected changes in lipid peroxidation and endogenous antioxidant activity by measuring the malondialdehyde, glutathione, superoxide dismutase activities, glutathione/oxidized glutathione and reduced nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide phosphate ratios. Inductively coupled plasma-mass spectrometry, western blot, reverse transcription-polymerase chain reaction, fluoro-jade B staining, immunofluorescence analysis, and transmission electron microscopy were utilized to examine the influence of EA. The results indicate that EA treatment was effective in reversing neurological impairment, neuronal damage, and protecting mitochondrial morphology and decreasing the cerebral infarct volume in the middle cerebral artery occlusion/reperfusion rat model. EA reduced iron levels, inhibited lipid peroxidation, increased endogenous antioxidant activity, modulated the expression of several ferroptosis-related proteins, and promoted nuclear factor-E2-related factor 2 (Nrf2) nuclear translocation. However, the protective effect of EA was hindered by the Nrf2 inhibitor ML385. These findings suggest that EA can suppress ferroptosis and decrease damage caused by cerebral ischemia/reperfusion by activating Nrf2 and increasing the protein expression of solute carrier family 7 member 11 and glutathione peroxidase 4.

Asunto(s)

Electroacupuntura , Ferroptosis , Infarto de la Arteria Cerebral Media , Factor 2 Relacionado con NF-E2 , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Ratas Sprague-Dawley , Animales , Ferroptosis/fisiología , Electroacupuntura/métodos , Factor 2 Relacionado con NF-E2/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Masculino , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/terapia , Daño por Reperfusión/metabolismo , Ratas , Neuronas/metabolismo , Regulación hacia Abajo/fisiología