RESUMO
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.
Assuntos
Administração Intranasal , Encéfalo , Modelos Animais de Doenças , Terapia Genética , AVC Isquêmico , Nanopartículas , Sirtuína 1 , Animais , Camundongos , Humanos , AVC Isquêmico/terapia , AVC Isquêmico/genética , Nanopartículas/administração & dosagem , Terapia Genética/métodos , Sirtuína 1/genética , Sirtuína 1/metabolismo , Encéfalo/metabolismo , Masculino , Fosfatos de Cálcio , Sistemas CRISPR-Cas , Camundongos Endogâmicos C57BL , Células Endoteliais/metabolismo , Isquemia Encefálica/terapia , Isquemia Encefálica/genética , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/genética , Células Epiteliais/metabolismoRESUMO
This study investigated the neuroprotective effects of peripheral nerve microcurrent stimulation therapy in a rat model of middle cerebral artery occlusion (MCAO). Twenty 8-week-old male Sprague Dawley rats weighing 300-330 g were categorised into group A, serving as the healthy control; group B, including rats subjected to MCAO; group C, including rats receiving microcurrent therapy immediately after MCAO, which was continued for one week; and group D, including rats receiving microcurrent therapy one week before and one week after MCAO. A gross morphological analysis, behavioural motion analysis, histological examination, immunohistochemistry, and Western blotting were conducted. Microcurrent therapy significantly reduced ischaemic damage and pyramidal cells of the hippocampus CA1 region. Haematoxylin and eosin staining revealed infarction areas/viable pyramidal cell numbers of 0%/94.33, 28.53%/40.05, 17.32%/80.13, and 5.38%/91.34 in groups A, B, C, and D, respectively (p < 0.001). A behavioural analysis revealed that the total distances moved were 1945.24 cm, 767.85 cm, 1781.77 cm, and 2122.22 cm in groups A, B, C, and D, respectively (p < 0.05), and the mean speeds were 6.48 cm/s, 2.50 cm/s, 5.43 cm/s, and 6.82 cm/s, respectively (p < 0.05). Inflammatory markers (cluster of differentiation 68, interleukin-6, and tumour necrosis factor-α) significantly decreased in the treated groups (p < 0.001). Western blotting revealed reduced proinflammatory, oxidative stress, and apoptosis-related protein levels, along with increased angiogenic factors and mitogen-activated protein kinase (MAPK) pathway modulation in the treated groups. Peripheral nerve microcurrent stimulation therapy effectively mitigates ischaemic damage, promotes recovery, reduces inflammation, and modulates protein expression, emphasising its potential as a therapeutic strategy for ischaemic stroke.
Assuntos
Modelos Animais de Doenças , Terapia por Estimulação Elétrica , Infarto da Artéria Cerebral Média , Ratos Sprague-Dawley , Animais , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Ratos , Terapia por Estimulação Elétrica/métodos , NeuroproteçãoRESUMO
The blood-brain barrier (BBB) strictly limits the entry of most exogenous therapeutic drugs into the brain, which brings great challenges to the drug treatment of refractory central diseases, including the treatment of ischemic stroke. Our previous studies have shown that specific mode electroacupuncture stimulation (SMES) can temporarily open the BBB, but with the mechanisms largely unknown. This study explored whether SMES opens the BBB in the infarcted border zone of rats during middle cerebral artery occlusion/reperfusion recovery, and whether this is related to p65 or vascular endothelial growth factor A (VEGFA) modulation of tight junction protein expression through in vivo and in vitro studies. Evans blue, FITC-dextran, mouse-derived nerve growth factor (NGF), and transendothelial electrical resistance values were used to evaluate the permeability of the BBB. Additionally, microvascular endothelial cells and astrocytes were utilized for in vitro study. Immunofluorescence, immunohistochemistry, western blot, and ELISA were employed to assess related protein expression. SMES significantly increased vascular permeability for Evans blue and NGF in the infarcted border zone, and increased the expression of VEGFA by activating p-p65, thereby reducing the expression of tight junction proteins Occludin and ZO-1. Correspondingly, oxygen glucose deprivation/reoxygenation activated p-p65 in and induced VEGFA secretion from astrocytes in vitro. Their conditioned medium reduced the expression of Occludin in bEnd.3 cells and increased the permeability of FITC-dextran. The mechanism of SMES opening infarcted border zone BBB is partly related to its actions on p65, VEGFA, and tight junction proteins.
Assuntos
Barreira Hematoencefálica , Eletroacupuntura , Infarto da Artéria Cerebral Média , Ratos Sprague-Dawley , Proteínas de Junções Íntimas , Fator A de Crescimento do Endotélio Vascular , Animais , Eletroacupuntura/métodos , Barreira Hematoencefálica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Masculino , Proteínas de Junções Íntimas/metabolismo , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Ratos , NF-kappa B/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/terapia , Células Endoteliais/metabolismo , Astrócitos/metabolismoRESUMO
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.
Assuntos
Oxigenoterapia Hiperbárica , Hiperglicemia , Microglia , Espécies Reativas de Oxigênio , Animais , Microglia/metabolismo , Microglia/efeitos dos fármacos , Oxigenoterapia Hiperbárica/métodos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Hiperglicemia/metabolismo , Hiperglicemia/complicações , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Modelos Animais de Doenças , Acidente Vascular Cerebral/terapia , Acidente Vascular Cerebral/metabolismo , Antioxidantes/farmacologia , Camundongos Endogâmicos C57BL , Infarto da Artéria Cerebral Média/terapia , Edaravone/farmacologia , Traumatismo por Reperfusão/metabolismoRESUMO
Cerebral ischemia reperfusion injury is a severe neurological impairment that occurs after blood flow reconstruction in stroke, and microglia cell pyroptosis is one of its important mechanisms. Electroacupuncture has been shown to be effective in mitigating and alleviating cerebral ischemia reperfusion injury by inhibiting neuroinflammation, reducing cellular pyroptosis, and improving neurological function. In this experiment, we divided the rats into three groups, including the sham operation (Sham) group, the middle cerebral artery occlusion/reperfusion (MCAO/R) group, and the pre-electroacupuncture (EAC) group. Pre-electroacupuncture group was stimulated with electroacupuncture of a certain intensity on the Baihui (GV 20) and Dazhui (GV 14) of the rat once a day from the 7th day to the 1st day before the MCAO/R operation. The extent of cerebral infarction was detected by TTC staining. A modified Zea-Longa five-point scale scoring system was used to determine neurologic function in MCAO rats. The number of neurons and morphological changes were accessed by Nissl staining and HE staining. The cellular damage was detected by TUNEL staining. In addition, the expression levels of RhoA, pyrin, GSDMD, Caspase1, cleaved-Caspase1, Iba-1, CD206, and ROCK2 were examined by western blotting and immunofluorescence. The results found that pre-electroacupuncture significantly attenuated neurological impairment and cerebral infarction compared to the post-MCAO/R rats. In addition, pre-electroacupuncture therapy promoted polarization of microglia to the neuroprotective (M2) phenotype. In addition, pre-electroacupuncture inhibited microglia pyroptosis by inhibiting RhoA/pyrin/GSDMD signaling pathway, thereby reducing neuronal injury and increasing neuronal survival in the MCAO/R rats. Taken together, these results demonstrated that pre-acupuncture could attenuate cerebral ischemia-reperfusion injury by inhibiting microglial pyroptosis. Therefore, pre-electroacupuncture might be a potential preventive strategy for ischemic stroke patients.
Assuntos
Eletroacupuntura , Microglia , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Transdução de Sinais , Animais , Eletroacupuntura/métodos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/terapia , Traumatismo por Reperfusão/prevenção & controle , Microglia/metabolismo , Masculino , Transdução de Sinais/fisiologia , Ratos , Proteínas de Ligação a Fosfato/metabolismo , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Piroptose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Gasderminas , Proteínas rho de Ligação ao GTPRESUMO
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.
Assuntos
Disfunção Cognitiva , Eletroacupuntura , Hipocampo , Infarto da Artéria Cerebral Média , AVC Isquêmico , Dinâmica Mitocondrial , Animais , Masculino , Ratos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Isquemia Encefálica/complicações , Disfunção Cognitiva/terapia , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Modelos Animais de Doenças , Dinaminas/metabolismo , Eletroacupuntura/métodos , GTP Fosfo-Hidrolases/metabolismo , Hipocampo/metabolismo , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , AVC Isquêmico/metabolismo , AVC Isquêmico/complicações , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Neurônios/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Sirtuína 1/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/terapiaRESUMO
Previous studies have shown that cerebral ischemia can cause white matter injury in the brain. This study aimed to investigate the potential mechanism of electroacupuncture (EA) at the Baihui (GV20) and Zusanli (ST36) acupoints in protecting white matter. Sprague-Dawley rats were used to establish permanent middle cerebral artery occlusion (pMCAO) rat models. Comprehensive motor functions were assessed using the mesh experiment. Morphological changes in the myelin sheath were assessed with Luxol fast blue staining. Morphological changes in oligodendrocytes and myelinated axons were evaluated using Nissl staining. The expressions of high-mobility group box 1 protein (HMGB1) and the receptor for advanced glycation end products (RAGE) in the corpus callosum were detected by immunohistochemical staining and Western blot analysis. pMCAO caused severe injury to the corpus callosum, evidenced by significant loss of white matter fibers and myelinated axons, and induced overexpression of HMGB1 and RAGE in the corpus callosum. EA treatment significantly improved comprehensive motor function alleviated white matter damage, and downregulated the expression of HMGB1 and RAGE. Its effects were comparable to those of FPS-ZM1, a RAGE receptor inhibitor. In conclusion, EA effectively improves comprehensive motor function in rats with cerebral infarction and alleviates corpus callosum injury. This effect may be related to the inhibition of HMGB1 and RAGE overexpression.
Assuntos
Corpo Caloso , Eletroacupuntura , Proteína HMGB1 , Ratos Sprague-Dawley , Receptor para Produtos Finais de Glicação Avançada , Animais , Eletroacupuntura/métodos , Proteína HMGB1/metabolismo , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Masculino , Ratos , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/terapia , BenzamidasRESUMO
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 (Angâ ¡), 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 Angâ ¡, 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 Angâ ¡ expression and aggravated inflammation and autophagy. The myocardial atrophy of MCAO/r rats was improved after chymase inhibition, and Angâ ¡ expression, inflammation and autophagy were reduced. Our results suggest that VNS may reduce the expression of chymase and Angâ ¡ 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.
Assuntos
Quimases , Infarto da Artéria Cerebral Média , AVC Isquêmico , Mastócitos , Ratos Sprague-Dawley , Estimulação do Nervo Vago , Animais , Mastócitos/imunologia , Masculino , AVC Isquêmico/terapia , AVC Isquêmico/imunologia , AVC Isquêmico/patologia , Ratos , Quimases/metabolismo , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/imunologia , Miocárdio/patologia , Miocárdio/imunologia , Atrofia , Modelos Animais de Doenças , Angiotensina II/metabolismoRESUMO
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.
Assuntos
Procedimentos Endovasculares , Infarto da Artéria Cerebral Média , Humanos , Masculino , Feminino , Procedimentos Endovasculares/métodos , Pessoa de Meia-Idade , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/cirurgia , Infarto da Artéria Cerebral Média/terapia , Resultado do Tratamento , Idoso , Doença Crônica , Angiografia por Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/métodosRESUMO
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.
Assuntos
Encéfalo , Células Endoteliais , Vesículas Extracelulares , Infarto da Artéria Cerebral Média , AVC Isquêmico , Camundongos Endogâmicos C57BL , Mitocôndrias , Animais , Vesículas Extracelulares/transplante , Vesículas Extracelulares/metabolismo , Mitocôndrias/metabolismo , Humanos , Células Endoteliais/metabolismo , AVC Isquêmico/terapia , AVC Isquêmico/metabolismo , Infarto da Artéria Cerebral Média/terapia , Encéfalo/metabolismo , Masculino , Camundongos , Células Cultivadas , Barreira Hematoencefálica/metabolismoRESUMO
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.
Assuntos
AVC Isquêmico , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Estresse Oxidativo , Ratos Sprague-Dawley , Cordão Umbilical , Animais , Estresse Oxidativo/fisiologia , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical/citologia , Masculino , AVC Isquêmico/metabolismo , AVC Isquêmico/terapia , Ratos , Inflamação/metabolismo , Lesões Encefálicas/metabolismo , Lesões Encefálicas/terapia , Neuroproteção/fisiologia , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismoRESUMO
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.
Assuntos
Infarto da Artéria Cerebral Média , Condicionamento Físico Animal , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/terapia , Ratos , Masculino , Condicionamento Físico Animal/fisiologia , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Análise de Sequência de RNA , Modelos Animais de Doenças , Apoptose , Precondicionamento Isquêmico/métodosRESUMO
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.
Assuntos
Terapia por Acupuntura , Proteínas Reguladoras de Apoptose , Infarto da Artéria Cerebral Média , MicroRNAs , Transdução de Sinais , Animais , Masculino , Ratos , Terapia por Acupuntura/métodos , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Encéfalo/metabolismo , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , AVC Isquêmico/metabolismo , AVC Isquêmico/terapia , AVC Isquêmico/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Ratos Sprague-Dawley , Proteínas Repressoras , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
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 120â¯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 72â¯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.
Assuntos
Terapia por Acupuntura , Autofagia , Isquemia Encefálica , MicroRNAs , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/terapia , Autofagia/fisiologia , Masculino , Terapia por Acupuntura/métodos , Ratos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Apoptose/fisiologia , Neurônios/metabolismo , Modelos Animais de Doenças , Encéfalo/metabolismoRESUMO
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.
Assuntos
Exossomos , Gelatina , Células-Tronco Mesenquimais , Agulhas , Traumatismo por Reperfusão , Animais , Gelatina/química , Camundongos , Ratos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/terapia , Metacrilatos/química , Masculino , Modelos Animais de Doenças , Hidrogéis/química , Ratos Sprague-Dawley , Infarto da Artéria Cerebral Média/terapiaRESUMO
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 21â 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.
Assuntos
Eletroacupuntura , Hipocampo , Microglia , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ratos Sprague-Dawley , Transdução de Sinais , Receptor 4 Toll-Like , Animais , Eletroacupuntura/métodos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptor 4 Toll-Like/metabolismo , Masculino , Hipocampo/metabolismo , Ratos , NF-kappa B/metabolismo , Microglia/metabolismo , Transdução de Sinais/fisiologia , Memória/fisiologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/terapia , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismoRESUMO
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.
Assuntos
AVC Isquêmico , Microglia , Estimulação Magnética Transcraniana , Animais , Masculino , Estimulação Magnética Transcraniana/métodos , AVC Isquêmico/terapia , AVC Isquêmico/fisiopatologia , Camundongos , Microglia/metabolismo , Camundongos Endogâmicos C57BL , Infarto da Artéria Cerebral Média/terapia , Atividade Motora/fisiologia , Plasticidade Neuronal/fisiologiaRESUMO
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.
Assuntos
Axônios , Disfunção Cognitiva , Memória , Proteínas Associadas aos Microtúbulos , Regeneração Nervosa , Transdução de Sinais , Animais , Masculino , Ratos , Axônios/fisiologia , Disfunção Cognitiva/terapia , Disfunção Cognitiva/etiologia , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/complicações , Peptídeos e Proteínas de Sinalização Intercelular , Memória/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Moxibustão/métodos , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/terapia , Acidente Vascular Cerebral/complicaçõesRESUMO
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.
Assuntos
Mirtilos Azuis (Planta) , Vesículas Extracelulares , AVC Isquêmico , Células-Tronco Mesenquimais , Vesículas Extracelulares/metabolismo , Animais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , AVC Isquêmico/metabolismo , AVC Isquêmico/terapia , AVC Isquêmico/patologia , Mirtilos Azuis (Planta)/química , Masculino , Modelos Animais de Doenças , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismoRESUMO
Ischemic stroke is a devastating medical condition with poor prognosis due to the lack of effective treatment modalities. Transplantation of human neural stem cells or primary neural cells is a promising treatment approach, but this is hindered by limited suitable cell sources and low in vitro expansion capacity. This study aimed (1) use small molecules (SM) to reprogram gingival mesenchymal stem cells (GMSCs) commitment to the neural lineage cells in vitro, and (2) use hyaluronic acid (HA) hydrogel scaffolds seeded with GMSCs-derived neural lineage cells to treat ischemic stroke in vivo. Neural induction was carried out with a SM cocktail-based one-step culture protocol over a period of 24 h. The induced cells were analyzed for expression of neural markers with immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The Sprague-Dawley (SD) rats (n = 100) were subjected to the middle cerebral artery occlusion (MCAO) reperfusion ischemic stroke model. Then, after 8 days post-MCAO, the modeled rats were randomly assigned to six study groups (n = 12 per group): (1) GMSCs, (2) GMSCs-derived neural lineage cells, (3) HA and GMSCs-derived neural lineage cells, (4) HA, (5) PBS, and (6) sham transplantation control, and received their respective transplantation. Evaluation of post-stroke recovery were performed by behavioral tests and histological assessments. The morphologically altered nature of neural lineages has been observed of the GMSCs treated with SMs compared to the untreated controls. As shown by the qRT-PCR and immunocytochemistry, SMs further significantly enhanced the expression level of neural markers of GMSCs as compared with the untreated controls (all p < 0.05). Intracerebral injection of self-assembling HA hydrogel carrying GMSCs-derived neural lineage cells promoted the recovery of neural function and reduced ischemic damage in rats with ischemic stroke, as demonstrated by histological examination and behavioral assessments (all p < 0.05). In conclusion, the SM cocktail significantly enhanced the differentiation of GMSCs into neural lineage cells. The HA hydrogel was found to facilitate the proliferation and differentiation of GMSCs-derived neural lineage cells. Furthermore, HA hydrogel seeded with GMSCs-derived neural lineage cells could promote tissue repair and functional recovery in rats with ischemic stroke and may be a promising alternative treatment modality for stroke.