RESUMEN
Accurate assessment of post-stroke deficits is crucial in translational research. Recent advances in machine learning offer precise quantification of rodent motor behavior post-stroke, yet detecting lesion-specific upper extremity deficits remains unclear. Employing proximal middle cerebral artery occlusion (MCAO) and cortical photothrombosis (PT) in mice, we assessed post-stroke impairments via the Staircase test. Lesion locations were identified using 7 T-MRI. Machine learning was applied to reconstruct forepaw kinematic trajectories and feature analysis was achieved with MouseReach, a new data-processing toolbox. Lesion reconstructions pinpointed ischemic centers in the striatum (MCAO) and sensorimotor cortex (PT). Pellet retrieval alterations were observed, but were unrelated to overall stroke volume. Instead, forepaw slips and relative reaching success correlated with increasing cortical lesion size in both models. Striatal lesion size after MCAO was associated with prolonged reach durations that occurred with delayed symptom onset. Further analysis on the impact of selective serotonin reuptake inhibitors in the PT model revealed no clear treatment effects but replicated strong effect sizes of slips for post-stroke deficit detection. In summary, refined movement analysis unveiled specific deficits in two widely-used mouse stroke models, emphasizing the value of deep behavioral profiling in preclinical stroke research to enhance model validity for clinical translation.
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Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media , Accidente Cerebrovascular , Animales , Ratones , Masculino , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/fisiopatología , Infarto de la Arteria Cerebral Media/complicaciones , Accidente Cerebrovascular/fisiopatología , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/complicaciones , Ratones Endogámicos C57BL , Imagen por Resonancia Magnética/métodos , Aprendizaje Automático , Movimiento/fisiologíaRESUMEN
Acid sphingomyelinase (ASM) inhibitors are widely used for the treatment of post-stroke depression. They promote neurological recovery in animal stroke models via neurorestorative effects. In a previous study, we found that antidepressants including amitriptyline, fluoxetine, and desipramine increase cerebral angiogenesis post-ischemia/reperfusion (I/R) in an ASM-dependent way. To elucidate the underlying mechanisms, we investigated the effects of the functional ASM inhibitor amitriptyline in two models of I/R injury, that is, in human cerebral microvascular endothelial hCMEC/D3 cells exposed to oxygen-glucose deprivation and in mice exposed to middle cerebral artery occlusion (MCAO). In addition to our earlier studies, we now show that amitriptyline increased mitochondrial reactive oxygen species (ROS) formation in hCMEC/D3 cells and increased ROS formation in the vascular compartment of MCAO mice. ROS formation was instrumental for amitriptyline's angiogenic effects. ROS formation did not result in excessive endothelial injury. Instead, amitriptyline induced a profound metabolic reprogramming of endothelial cells that comprised reduced endothelial proliferation, reduced mitochondrial energy metabolism, reduced endoplasmic reticulum stress, increased autophagy/mitophagy, stimulation of antioxidant responses and inhibition of apoptotic cell death. Specifically, the antioxidant heme oxygenase-1, which was upregulated by amitriptyline, mediated amitriptyline's angiogenic effects. Thus, heme oxygenase-1 knockdown severely compromised angiogenesis and abolished amitriptyline's angiogenic responses. Our data demonstrate that ASM inhibition reregulates a complex network of metabolic and mitochondrial responses post-I/R that contribute to cerebral angiogenesis without compromising endothelial survival.
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Amitriptilina , Células Endoteliales , Mitocondrias , Estrés Oxidativo , Especies Reactivas de Oxígeno , Daño por Reperfusión , Esfingomielina Fosfodiesterasa , Animales , Esfingomielina Fosfodiesterasa/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Humanos , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Especies Reactivas de Oxígeno/metabolismo , Amitriptilina/farmacología , Ratones , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/metabolismo , Supervivencia Celular/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Línea Celular , AngiogénesisRESUMEN
BACKGROUND: Stroke is a type of acute brain damage that can lead to a series of serious public health challenges. Demonstrating the molecular mechanism of stroke-related neural cell degeneration could help identify a more efficient treatment for stroke patients. Further elucidation of factors that regulate microglia and nuclear factor (erythroid-derived 2)-like 1 (Nrf1) may lead to a promising strategy for treating neuroinflammation after ischaemic stroke. In this study, we investigated the possible role of pterostilbene (PTS) in Nrf1 regulation in cell and animal models of ischaemia stroke. METHODS: We administered PTS, ITSA1 (an HDAC activator) and RGFP966 (a selective HDAC3 inhibitor) in a mouse model of middle cerebral artery occlusion-reperfusion (MCAO/R) and a model of microglial oxygenâglucose deprivation/reperfusion (OGD/R). The brain infarct size, neuroinflammation and microglial availability were also determined. Dual-luciferase reporter, Nrf1 protein stability and co-immunoprecipitation assays were conducted to analyse histone deacetylase 3 (HDAC3)/Nrf1-regulated Nrf1 in an OGD/R-induced microglial injury model. RESULTS: We found that PTS decreased HDAC3 expression and activity, increased Nrf1 acetylation in the cell nucleus and inhibited the interaction of Nrf1 with p65 and p65 accumulation, which reduced infarct volume and neuroinflammation (iNOS/Arg1, TNF-α and IL-1ß levels) after ischaemic stroke. Furthermore, the CSF1R inhibitor PLX5622 induced elimination of microglia and attenuated the therapeutic effect of PTS following MCAO/R. In the OGD/R model, PTS relieved OGD/R-induced microglial injury and TNF-α and IL-1ß release, which were dependent on Nrf1 acetylation through the upregulation of HDAC3/Nrf1 signalling in microglia. However, the K105R or/and K139R mutants of Nrf1 counteracted the impact of PTS in the OGD/R-induced microglial injury model, which indicates that PTS treatment might be a promising strategy for ischaemia stroke therapy. CONCLUSION: The HDAC3/Nrf1 pathway regulates the stability and function of Nrf1 in microglial activation and neuroinflammation, which may depend on the acetylation of the lysine 105 and 139 residues in Nrf1. This mechanism was first identified as a potential regulatory mechanism of PTS-based neuroprotection in our research, which may provide new insight into further translational applications of natural products such as PTS.
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Histona Desacetilasas , Accidente Cerebrovascular Isquémico , Ratones Endogámicos C57BL , Microglía , Enfermedades Neuroinflamatorias , Estilbenos , Animales , Histona Desacetilasas/metabolismo , Microglía/metabolismo , Microglía/efectos de los fármacos , Ratones , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/metabolismo , Masculino , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Estilbenos/farmacología , Estilbenos/uso terapéutico , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/complicaciones , Transducción de Señal/efectos de los fármacos , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismoRESUMEN
BACKGROUND: Clinically, ischemic reperfusion injury is the main cause of stroke injury. This study aimed to assess the effectiveness of fingolimod in suppressing inflammation caused by ischemic brain injury and explore its pharmacological mechanisms. METHODS: In total, 75 male Sprague-Dawley rats were randomly and equally assigned to five distinct groups: sham, middle cerebral artery occlusion/reperfusion (MCAO/R) surgery, fingolimod low-dose (F-L), fingolimod medium-dose (F-M), and fingolimod high-dose (F-H). Neurobehavioral tests, 2,3,5-triphenyltetrazolium chloride staining, and the brain tissue drying-wet method were conducted to evaluate neurological impairment, cerebral infarction size, and brain water content. Enzyme-linked immunosorbent assay was employed to quantify pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) protein levels. Western blotting and immunohistochemical staining were performed to assess high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), and nuclear factor kappa-B p65 (NF-κBp65) levels. RESULTS: Rats in the F-L, F-M, and F-H groups exhibited lower Longa scores, reduced infarction volumes, and decreased brain edema than those in the MCAO/R group. Additionally, the F-L, F-M, and F-H groups exhibited lower serum levels of IL-1ß, IL-6, and TNF-α than those of the MCAO/R group. Additionally, F-L, F-M, and F-H treatments resulted in decreased HMGB1, TLR4, and NF-κBp65 protein expression levels in the hippocampus of MCAO/R rats. CONCLUSIONS: Fingolimod was found to reduce ischemic brain injury in a dose-dependent manner. Moreover, it was also found to alleviate inflammation following ischemic brain injury via the HMGB1/TLR4/NFκB signaling pathway.
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Isquemia Encefálica , Clorhidrato de Fingolimod , Transducción de Señal , Animales , Masculino , Ratas , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod/farmacología , Clorhidrato de Fingolimod/administración & dosificación , Proteína HMGB1/metabolismo , Proteína HMGB1/efectos de los fármacos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/etiología , FN-kappa B/metabolismo , FN-kappa B/efectos de los fármacos , Ratas Sprague-Dawley , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/efectos de los fármacosRESUMEN
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.
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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/terapiaAsunto(s)
Infarto de la Arteria Cerebral Media , Trastornos de la Percepción , Humanos , Trastornos de la Percepción/etiología , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/complicaciones , Masculino , Persona de Mediana Edad , Anciano , FemeninoRESUMEN
Endoplasmic reticulum stress (ERS) and ferroptosis are linked to cerebral ischemia reperfusion injury (CIRI). The neuroprotective properties of 1α, 25-dihydroxyvitamin D3 (VitD3 or 1,25-D3) have been well established; however, the mechanism by which VitD3 treats CIRI through ERS and ferroptosis has not been examined. Hence, we developed middle cerebral artery occlusion/reperfusion (MCAO/R) model in SD rats to ascertain if VitD3 preconditioning mediates ERS and ferroptosis involving of p53 signaling. In this study, we observed that VitD3 can reduce infarction volume and cerebral edema, which leads to the improvement of nerve function. HE, Nissl and Tunel staining showed that VitD3 treatment significantly improved the morphology of neuronal cells and reduced their death. The expression and activation of Vitamin D receptor (VDR), PKR-like ER kinase (PERK), C/EBP-homologous protein (CHOP), p53, nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4) and reactive oxygen species (ROS) in the ischemic penumbral area were detected by real-time qPCR, Western-blotting and Elisa. The results showed that after VitD3 treatment, VDR increased, ERS-related indices (PERK, CHOP) significantly decreased and ferroptosis-related indices (Nrf2, GPX4) increased. As a VDRs antagonist, pyridoxal-5-phosphate (P5P) can partially block the neuroprotective effects of VitD3. Therefore, CIRI can induce ERS and ferroptosis in the ischemic penumbra area and VitD3 may ameliorate nerve damage in CIRI rats by up-regulating VDR, alleviating p53-associated ERS and ferroptosis.
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Estrés del Retículo Endoplásmico , Ferroptosis , Receptores de Calcitriol , Transducción de Señal , Proteína p53 Supresora de Tumor , Animales , Masculino , Ratas , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Isquemia Encefálica/tratamiento farmacológico , Calcitriol/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ferroptosis/efectos de los fármacos , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Factor 2 Relacionado con NF-E2/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptores de Calcitriol/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal/efectos de los fármacos , Factor de Transcripción CHOP/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Huanglian Jiedu decoction (HLJD) has been used to treat ischemic stroke in clinic. However, the detailed protective mechanisms of HLJD on ischemic stroke have yet to be elucidated. The aim of this study is to elucidate the underlying pharmacological mechanisms of HLJD based on the inhibition of neuroinflammation and the amelioration of nerve cell damage. A middle cerebral artery occlusion reperfusion (MCAO/R) model was established in rats and received HLJD treatment. Effects of HLJD on neurological function was assessed based on Bederson's score, postural reflex test and asymmetry score. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining, Hematein and eosin (HE) and Nissl staining were used to observe the pathological changes in brain. Then, transcriptomics was used to screen the differential genes in brain tissue in MCAO/R model rats following HLJD intervention. Subsequently, the effects of HLJD on neutrophil extracellular trap (NET) formation-related neuroinflammation, gamma-aminobutyric acid (GABA)ergic synapse activation, nerve cell damage and proliferation were validated using immunofluorescence, western blot and enzyme-linked immunosorbent assay (ELISA). Our results showed that HLJD intervention reduced the Bederson's score, postural reflex test score and asymmetry score in MCAO/R model rats. Pathological staining indicated that HLJD treatment decreased the cerebral infarction area, mitigated neuronal damage and increased the numbers of Nissl bodies. Transcriptomics suggested that HLJD affected 435 genes in MCAO/R rats. Among them, several genes involving in NET formation and GABAergic synapses pathways were dysregulated. Subsequent experimental validation showed that HLJD reduced the MPO+CitH3+ positive expression area, reduced the protein expression of PAD4, p-P38/P38, p-ERK/ERK and decreased the levels of IL-1ß, IL-6 and TNF-α, reversed the increase of Iba1+TLR4+, Iba1+p65+ and Iba1+NLRP3+ positive expression area in brain. Moreover, HLJD increased GABA levels, elevated the protein expression of GABRG1 and GAT3, decreased the TUNEL positive expression area and increased the Ki67 positive expression area in brain. HLJD intervention exerts a multifaceted positive impact on ischemia-induced cerebral injury in MCAO/R rats. This intervention effectively inhibits neuroinflammation by mitigating NET formation, and concurrently improves nerve cell damage and fosters nerve cell proliferation through activating GABAergic synapses.
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Isquemia Encefálica , Medicamentos Herbarios Chinos , Ratas Sprague-Dawley , Sinapsis , Animales , Medicamentos Herbarios Chinos/farmacología , Ratas , Masculino , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Modelos Animales de Enfermedad , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Daño por Reperfusión/complicaciones , Fármacos Neuroprotectores/farmacología , Encéfalo/patología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacosRESUMEN
BACKGROUND: Stroke is a globally dangerous disease capable of causing irreversible neuronal damage with limited therapeutic options. Meldonium, an inhibitor of carnitine-dependent metabolism, is considered an anti-ischemic drug. However, the mechanisms through which meldonium improves ischemic injury and its potential to protect neurons remain largely unknown. METHODS: A rat model with middle cerebral artery occlusion (MCAO) was used to investigate meldonium's neuroprotective efficacy in vivo. Infarct volume, neurological deficit score, histopathology, neuronal apoptosis, motor function, morphological alteration and antioxidant capacity were explored via 2,3,5-Triphenyltetrazolium chloride staining, Longa scoring method, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, rotarod test, transmission electron microscopy and Oxidative stress index related kit. A primary rat hippocampal neuron model subjected to oxygen-glucose deprivation reperfusion was used to study meldonium's protective ability in vitro. Neuronal viability, mitochondrial membrane potential, mitochondrial morphology, respiratory function, ATP production, and its potential mechanism were assayed by MTT cell proliferation and cytotoxicity assay kit, cell-permeant MitoTracker® probes, mitochondrial stress, real-time ATP rate and western blotting. RESULTS: Meldonium markedly reduced the infarct size, improved neurological function and motor ability, and inhibited neuronal apoptosis in vivo. Meldonium enhanced the morphology, antioxidant capacity, and ATP production of mitochondria and inhibited the opening of the mitochondrial permeability transition pore in the cerebral cortex and hippocampus during cerebral ischemia-reperfusion injury (CIRI) in rats. Additionally, meldonium improved the damaged fusion process and respiratory function of neuronal mitochondria in vitro. Further investigation revealed that meldonium activated the Akt/GSK-3ß signaling pathway to inhibit mitochondria-dependent neuronal apoptosis. CONCLUSION: Our study demonstrated that meldonium shows a neuroprotective function during CIRI by preserving the mitochondrial function, thus prevented neurons from apoptosis.
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Apoptosis , Supervivencia Celular , Metilhidrazinas , Mitocondrias , Neuronas , Fármacos Neuroprotectores , Ratas Sprague-Dawley , Daño por Reperfusión , Animales , Fármacos Neuroprotectores/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Daño por Reperfusión/patología , Daño por Reperfusión/tratamiento farmacológico , Masculino , Supervivencia Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Metilhidrazinas/farmacología , Metilhidrazinas/uso terapéutico , Isquemia Encefálica/patología , Isquemia Encefálica/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , RatasAsunto(s)
Encéfalo , Accidente Cerebrovascular , Trombectomía , Humanos , Encéfalo/irrigación sanguínea , Encéfalo/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Accidente Cerebrovascular Isquémico/etiología , Accidente Cerebrovascular Isquémico/cirugía , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/cirugíaAsunto(s)
Accidente Cerebrovascular , Trombectomía , Humanos , Encéfalo/diagnóstico por imagen , Infarto Cerebral/etiología , Infarto Cerebral/cirugía , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/cirugía , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/diagnóstico por imagenRESUMEN
BACKGROUND: Angiogenesis is crucial in neuroprotection of secondary thalamic injury after cortical infarction. The p75 neurotrophin receptor (p75NTR) plays a key role in activating angiogenesis. However, the effects of p75NTR on angiogenesis in the thalamus after cortical infarction are largely unknown. Herein we investigate whether p75NTR facilitates angiogenesis to attenuate secondary thalamic damage via activating hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway mediated by Von Hippel-Lindau (VHL) after distal middle cerebral artery occlusion (dMCAO). METHODS: The male rat model of dMCAO was established. The effects of p75NTR on the angiogenesis was evaluated using RNA-sequencing, immunohistochemistry, western blot, quantitative real-time polymerase chain reaction, magnetic resonance imaging, behavior tests, viral and pharmacological interventions. RESULTS: We found that the p75NTR and vessel density were decreased in ipsilateral thalamus after dMCAO. The p75NTR-VHL interaction was reduced, which promoted the ubiquitination degradation of HIF-1α and reduced VEGF expression after dMCAO. Notably, p75NTR overexpression restrained the ubiquitination degradation of HIF-1α by inhibiting VHL-HIF-1α interaction, further promoted angiogenesis, increased cerebral blood flow of ipsilateral thalamus and improved neurological function after dMCAO. CONCLUSION: For the first time, we highlighted that the enhancement of p75NTR-VHL interaction promoted angiogenesis in attenuating secondary thalamic damage after dMCAO.
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Infarto de la Arteria Cerebral Media , Neovascularización Fisiológica , Ratas Sprague-Dawley , Tálamo , Animales , Masculino , Ratas , Tálamo/metabolismo , Tálamo/patología , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Neovascularización Fisiológica/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Infarto Cerebral/patología , Angiogénesis , Proteínas del Tejido Nervioso , Receptores de Factores de CrecimientoRESUMEN
Vasogenic brain edema, a potentially life-threatening consequence following an acute ischemic stroke, is a major clinical problem. This research aims to explore the therapeutic benefits of nimodipine, a calcium channel blocker, in mitigating vasogenic cerebral edema and preserving blood-brain barrier (BBB) function in an ischemic stroke rat model. In this research, animals underwent the induction of ischemic stroke via a 60-min blockage of the middle cerebral artery and treated with a nonhypotensive dose of nimodipine (1 mg/kg/day) for a duration of five days. The wet/dry method was employed to identify cerebral edema, and the Evans blue dye extravasation technique was used to assess the permeability of the BBB. Furthermore, immunofluorescence staining was utilized to assess the protein expression levels of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). The study also examined mitochondrial function by evaluating mitochondrial swelling, succinate dehydrogenase (SDH) activity, the collapse of mitochondrial membrane potential (MMP), and the generation of reactive oxygen species (ROS). Post-stroke administration of nimodipine led to a significant decrease in cerebral edema and maintained the integrity of the BBB. The protective effects observed were associated with a reduction in cell apoptosis as well as decreased expression of MMP-9 and ICAM-1. Furthermore, nimodipine was observed to reduce mitochondrial swelling and ROS levels while simultaneously restoring MMP and SDH activity. These results suggest that nimodipine may reduce cerebral edema and BBB breakdown caused by ischemia/reperfusion. This effect is potentially mediated through the reduction of MMP-9 and ICAM-1 levels and the enhancement of mitochondrial function.
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Barrera Hematoencefálica , Edema Encefálico , Bloqueadores de los Canales de Calcio , Accidente Cerebrovascular Isquémico , Metaloproteinasa 9 de la Matriz , Nimodipina , Animales , Nimodipina/farmacología , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/etiología , Edema Encefálico/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Masculino , Ratas , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Modelos Animales de Enfermedad , Especies Reactivas de Oxígeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas Sprague-Dawley , Molécula 1 de Adhesión Intercelular/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/complicaciones , Dilatación Mitocondrial/efectos de los fármacos , Succinato Deshidrogenasa/metabolismoRESUMEN
INTRODUCTION: Mangiferin is a Chinese herbal extract with multiple biological activities. Mangiferin can penetrate the bloodâbrain barrier and has potential in the treatment of nervous system diseases. These findings suggest that mangiferin protects the neurological function in ischemic stroke rats by targeting multiple signaling pathways. However, little is known about the effect and mechanism of mangiferin in alleviating poststroke cognitive impairment. METHODS: Cerebral ischemia/reperfusion (I/R) rats were generated via middle cerebral artery occlusion. Laser speckle imaging was used to monitor the cerebral blood flow. The I/R rats were intraperitoneally (i.p.) injected with 40 mg/kg mangiferin for 7 consecutive days. Neurological scoring, and TTC staining were performed to evaluate neurological function. Behavioral experiments, including the open field test, elevated plus maze, sucrose preference test, and novel object recognition test, were performed to evaluate cognitive function. Metabolomic data from brain tissue with multivariate statistics were analyzed by gas chromatographyâmass spectrometry and liquid chromatographyâmass spectrometry. RESULTS: Mangiferin markedly decreased neurological scores, and reduced infarct areas. Mangiferin significantly attenuated anxiety-like and depression-like behaviors and enhanced learning and memory in I/R rats. According to the metabolomics results, 13 metabolites were identified to be potentially regulated by mangiferin, and the differentially abundant metabolites were mainly involved in lipid metabolism. CONCLUSIONS: Mangiferin protected neurological function and relieved poststroke cognitive impairment by improving lipid metabolism abnormalities in I/R rats.
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Disfunción Cognitiva , Metabolismo de los Lípidos , Ratas Sprague-Dawley , Daño por Reperfusión , Xantonas , Animales , Xantonas/farmacología , Xantonas/uso terapéutico , Masculino , Metabolismo de los Lípidos/efectos de los fármacos , Ratas , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/etiología , Disfunción Cognitiva/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/complicaciones , Daño por Reperfusión/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Conducta Animal/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Modelos Animales de Enfermedad , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/complicaciones , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Cognición/efectos de los fármacosRESUMEN
OBJECTIVE: To explore the neuroprotective effects and mechanism of Tanreqing Injection (TRQ) on treating ischemic stroke based on network pharmacology and in vivo experimental validation. METHODS: The chemical compounds of TRQ were retrieved based on published data, with targets retrieved from PubChem, Therapeutic Target Database and DrugBank. Network visualization and analysis were performed using Cytoscape, with protein-protein interaction networks derived from the STRING database. Enrichment analysis was performed using Kyoto Encyclopedia of Genes Genomes pathway and Gene Ontology analysis. In in vivo experiments, the middle cerebral artery occlusion (MCAO) model was used. Infarct volume was determined by 2,3,5-triphenyltetrazolium hydrochloride staining and protein expressions were analyzed by Western blot. Molecular docking was performed to predict ligand-receptor interactions. RESULTS: We screened 81 chemical compounds in TRQ and retrieved their therapeutic targets. Of the targets, 116 were therapeutic targets for stroke. The enrichment analysis showed that the apelin signaling pathway was a key pathway for ischemic stroke. Furthermore, in in vivo experiment we found that administering with intraperitoneal injection of 2.5 mL/kg TRQ every 6 h could significantly reduce the infarct volume of MCAO rats (P<0.05). In addition, protein levels of the apelin receptor (APJ)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway were increased by TRQ (P<0.05). In addition, 41 chemical compounds in TRQ could bind to APJ. CONCLUSIONS: The neuroprotective effect of TRQ may be related to the APJ/PI3K/AKT signaling pathway. However, further studies are needed to confirm the findings.
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Medicamentos Herbarios Chinos , Accidente Cerebrovascular Isquémico , Simulación del Acoplamiento Molecular , Farmacología en Red , Fármacos Neuroprotectores , Ratas Sprague-Dawley , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Animales , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/patología , Masculino , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/complicaciones , Transducción de Señal/efectos de los fármacos , Mapas de Interacción de Proteínas/efectos de los fármacos , Ratas , Modelos Animales de Enfermedad , Inyecciones , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
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.
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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/complicacionesRESUMEN
BACKGROUND AND PURPOSE: The pathophysiological features of acute ischemic stroke (AIS) often involve dysfunction of the blood-brain barrier (BBB), characterized by the degradation of tight junction proteins (Tjs) leading to increased permeability. This dysfunction can exacerbate cerebral injury and contribute to severe complications. The permeability of the BBB fluctuates during different stages of AIS and is influenced by various factors. Developing effective therapies to restore BBB function remains a significant challenge in AIS treatment. High levels of vascular endothelial growth factor (VEGF) in the early stages of AIS have been shown to worsen BBB breakdown and stroke progression. Our study aimed to investigate the protective effects of the VEGF receptor inhibitor Axitinib on BBB dysfunction and cerebral ischemia/reperfusion-induced injury. METHODS: BEnd3 cell exposed to oxygen-glucose deprivation (OGD) model was constructed to estimate pharmacological activity of Axitinib (400 ng/ml) on anti-apoptosis and pathological barrier function recovery. In vivo, rats were subjected to a 1 h transient middle cerebral artery occlusion and 23 h reperfusion (tMCAO/R) to investigate the permeability of BBB and cerebral tissue damage. Axitinib was administered through the tail vein at the beginning of reperfusion. BBB integrity was assessed by Evans blue leakage and the expression levels of Tjs claudin-5 and occludin. RESULTS: Our research revealed that co-incubation with Axitinib enhanced the cell viability of OGD-insulted bEnd3 cells, decreased LDH leakage rate, and suppressed the expression of apoptosis-related proteins cytochrome C and Bax. Axitinib also mitigated the damage to Tjs and facilitated the restoration of transepithelial electrical resistance in OGD-insulted bEnd.3 cells. In vivo, Axitinib administration reduced intracerebral Evans blue leakage and up-regulated the expression of Tjs in the penumbra brain tissue in tMCAO/R rats. Notably, 10 mg/kg Axitinib exerted a significant anti-ischemic effect by decreasing cerebral infarct volume and brain edema volume, improving neurological function, and reducing pro-inflammatory cytokines IL-6 and TNF-α in the brain. CONCLUSIONS: Our study highlights Axitinib as a potent protectant of blood-brain barrier function, capable of promoting pathological blood-brain barrier recovery through VEGF inhibition and increased expression of tight junction proteins in AIS. This suggests that VEGF antagonism within the first 24 h post-stroke could be a novel therapeutic approach to enhance blood-brain barrier function and mitigate ischemia-reperfusion injury.
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Axitinib , Barrera Hematoencefálica , Accidente Cerebrovascular Isquémico , Ratas Sprague-Dawley , Daño por Reperfusión , Animales , Axitinib/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Ratas , Masculino , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Ratones , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/tratamiento farmacológicoRESUMEN
BACKGROUND: The inflammatory response is a key factor in the pathogenesis of cerebral ischemia/reperfusion injury (CIRI), and anti-inflammatory interventions may offer a promising therapeutic strategy. Forsythoside B (FB) is a phenylethanoid glycoside isolated from Forsythiae fructus, which has been reported to have anti-inflammatory effects. However, the mechanism of the neuroprotective effect of FB on CIRI remains unclear. METHODS: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). FB was administered intraperitoneally for 3 days prior to MCAO/R. Cerebral infarct volume and neurological deficit score were used as indices to evaluate MCAO/R injury. The serum levels of inflammatory factors and antioxidant enzymes were measured. The activation of silent information regulator 2 homolog 1 (Sirt1) and the inhibition of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) pathway were assessed through western blot and immunohistochemistry analysis. Furthermore, the rats were treated with Sirt1 shRNA 3 days before MCAO/R by stereotactical injection into the ipsilateral hemispheric region to assess the impact of Sirt1 knockdown on the protection of FB during MCAO/R. RESULTS: FB reduced cerebral infarct volume and neurological deficit score in MCAO/R rats. FB reduced pathological changes and cell apoptosis in the hippocampal CA1 region and cortex on the ischemic side of rats. FB inhibited the serum levels of inflammatory factors and increased the activities of antioxidant enzymes. Further study showed that FB inhibited the activation of the NLRP3 pathway and induced Sirt1 activation. CONCLUSION: FB demonstrated neuroprotective and anti-inflammatory effects by inhibiting the NLRP3 pathway through Sirt1 activation in CIRI.
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Infarto de la Arteria Cerebral Media , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Ratas Sprague-Dawley , Daño por Reperfusión , Sirtuina 1 , Animales , Sirtuina 1/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Masculino , Inflamasomas/metabolismo , Ratas , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/complicaciones , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Ácidos Cafeicos , GlucósidosRESUMEN
AIMS: Infection is a complication after stroke and outcomes vary by sex. Thus, we investigated if sepsis affects brain from ischemic stroke and sex involvement. MAIN METHODS: Male and female Wistar rats, were submitted to middle cerebral artery occlusion (MCAO) and after 7 days sepsis to cecal ligation and perforation (CLP). Infarct size, neuroinflammation, oxidative stress, and mitochondrial activity were quantified 24 h after CLP in the prefrontal cortex and hippocampus. Survival and neurological score were assessed up to 15 days after MCAO or 8 days after CLP (starting at 2 h after MCAO) and memory at the end. KEY FINDINGS: CLP decreased survival, increased neurological impairments in MCAO females. Early, in male sepsis following MCAO led to increased glial activation in the brain structures, and increased TNF-α and IL-1ß in the hippocampus. All groups had higher IL-6 in both tissues, but the hippocampus had lower IL-10. CLP potentiated myeloperoxidase (MPO) in the prefrontal cortex of MCAO male and female. In MCAO+CLP, only male increased MPO and nitrite/nitrate in hippocampus. Males in all groups had protein oxidation in the prefrontal cortex, but only MCAO+CLP in the hippocampus. Catalase decreased in the prefrontal cortex and hippocampus of all males and females, and MCAO+CLP only increased this activity in males. Female MCAO+CLP had higher prefrontal cortex complex activity than males. In MCAO+CLP-induced long-term memory impairment only in females. SIGNIFICANCE: The parameters evaluated for early sepsis after ischemic stroke show a worse outcome for males, while females are affected during long-term follow-up.
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Accidente Cerebrovascular Isquémico , Ratas Wistar , Sepsis , Caracteres Sexuales , Animales , Masculino , Femenino , Sepsis/complicaciones , Sepsis/metabolismo , Ratas , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/complicaciones , Accidente Cerebrovascular Isquémico/patología , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Estrés Oxidativo , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Recuperación de la Función , Factores Sexuales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/complicaciones , Peroxidasa/metabolismoRESUMEN
Microglia mediated neuroinflammation is one of the major contributors to brain damage in cerebral ischemia reperfusion injury (CI/RI). Recently, RNA modification was found to contribute to the regulation of microglia polarization and the subsequent development of cerebral I/R neuroinflammation. Herein, we investigated the effect and mechanism of m5C RNA modification in the microglia induced CI/RI neuroinflammation. We found that the m5C RNA modification levels decreased in the primary microglia isolated from a mouse model of intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) and the BV2 microglial cells subjected to oxygen-glucose deprivation and reoxygenation (OGD/R), and this change was accompanied by an increase in the M1/M2 polarization ratio. Furthermore, the expression of m5C demethylase TET1 in microglia increased, which promoted M1 polarization but impeded M2 polarization. Mechanistically, the higher TET1 expression decreased the m5C modification level of RelB and enhanced its mRNA stability, which subsequently increased the M1/M2 polarization ratio. In conclusion, this study provides insight into the role of m5C RNA modification in the pathogenesis of cerebral I/R neuroinflammation and may deepen our understanding on clinical therapy targeting the TET1-RelB axis.