RESUMEN
Homoeostatic regulation of the acid-base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid-base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.e., GPR30, which leads to Gq-coupled calcium responses. Gpr30-Venus knock-in mice reveal predominant expression of GPR30 in brain mural cells. Primary culture and fresh isolation of brain mural cells demonstrate bicarbonate-induced, GPR30-dependent calcium responses. GPR30-deficient male mice are protected against ischemia-reperfusion injury by a rapid blood flow recovery. Collectively, we identify a bicarbonate-sensing GPCR in brain mural cells that regulates blood flow and ischemia-reperfusion injury. Our results provide a perspective on the modulation of GPR30 signalling in the development of innovative therapies for ischaemic stroke. Moreover, our findings provide perspectives on acid/base sensing GPCRs, concomitantly modulating cellular responses depending on fluctuating ion concentrations under the acid-base homoeostasis.
Asunto(s)
Isquemia Encefálica , Daño por Reperfusión , Accidente Cerebrovascular , Masculino , Ratones , Animales , Bicarbonatos , Calcio/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
There are no effective treatments for post-stroke glial scar formation, which inhibits axonal outgrowth and functional recovery after stroke. We investigated whether astrocytic extracellular vesicles (AEVs) regulated by microglia modulate glial scars and improve stroke recovery. We found that peri-infarct glial scars comprised reactive astrocytes with proliferating C3d and decreased S100A10 expression in chronic stroke. In cultured astrocytes, microglia-conditioned media and treatment with P2Y1 receptor antagonists increased and reduced the area of S100A10- and C3d-expressing reactive astrocytes, respectively, by suppressing mitogen-activated protein kinase/nuclear factor-κß (NF-κB)/tumor necrosis factor-α (TNF-α)/interleukin-1ß signaling after oxygen-glucose deprivation. Intracerebral administrations of AEVs enriched miR-146a-5p, downregulated NF-κB, and suppressed TNF-α expressions, by transforming reactive astrocytes to those with S100A10 preponderance, causing functional recovery in rats subjected to middle cerebral artery occlusion. Modulating neuroinflammation in post-stroke glial scars could permit axonal outgrowth, thus providing a basis for stroke recovery with neuroprotective AEVs.
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Vesículas Extracelulares , Accidente Cerebrovascular , Ratas , Animales , Microglía/metabolismo , FN-kappa B/metabolismo , Astrocitos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Gliosis/patología , Accidente Cerebrovascular/patología , Vesículas Extracelulares/metabolismoRESUMEN
AIMS: White matter lesions (WMLs) are involved in the pathological processes leading to cognitive decline and dementia. We examined the mechanisms underlying the exacerbation of ischemia-induced cognitive impairment and WMLs by diet-induced obesity, including lipopolysaccharide (LPS)-triggered neuroinflammation via toll-like receptor (TLR) 4. METHODS: Wild-type (WT) and TLR4-knockout (KO) C57BL/6 mice were fed a high-fat diet (HFD) or low-fat diet (LFD), and subjected to bilateral carotid artery stenosis (BCAS). Diet groups were compared for changes in gut microbiota, intestinal permeability, systemic inflammation, neuroinflammation, WML severity, and cognitive dysfunction. RESULTS: In WT mice, HFD induced obesity and increased cognitive impairment and WML severity compared with LFD-fed mice following BCAS. HFD caused gut dysbiosis and increased intestinal permeability, and plasma LPS and pro-inflammatory cytokine concentrations. Furthermore, HFD-fed mice had higher LPS levels and higher neuroinflammatory status, including increased TLR4 expression, in WMLs. In TLR4-KO mice, HFD also caused obesity and gut dysbiosis but did not increase cognitive impairment or WML severity after BCAS. No difference was found between HFD- and LFD-fed KO mice for LPS levels or inflammatory status in either plasma or WMLs. CONCLUSION: Inflammation triggered by LPS-TLR4 signaling may mediate obesity-associated exacerbation of cognitive impairment and WMLs from brain ischemia.
Asunto(s)
Isquemia Encefálica , Estenosis Carotídea , Disfunción Cognitiva , Sustancia Blanca , Ratones , Animales , Lipopolisacáridos/toxicidad , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Ratones Obesos , Enfermedades Neuroinflamatorias , Sustancia Blanca/patología , Disbiosis , Ratones Endogámicos C57BL , Inflamación/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Disfunción Cognitiva/patología , Isquemia Encefálica/complicaciones , Isquemia Encefálica/patología , Dieta Alta en Grasa/efectos adversos , Estenosis Carotídea/patologíaRESUMEN
Background Mucosal-associated invariant T (MAIT) cells have been associated with inflammation in several autoimmune diseases. However, their relation to ischemic stroke remains unclear. This study attempted to elucidate the role of MAIT cells in acute ischemic stroke in mice. Methods and Results We used MR1 knockout C57BL/6 (MR1-/-) mice and wild-type littermates (MR1+/+). After performing a transient middle cerebral artery occlusion (tMCAO), we evaluated the association with inflammation and prognosis in the acute cerebral ischemia. Furthermore, we analyzed the tMCAO C57BL/6 mice administered with the suppressive MR1 ligand and the vehicle control. We also evaluated the infiltration of MAIT cells into the ischemic brain by flow cytometry. Results showed a reduction of infarct volume and an improvement of neurological impairment in MR1-/- mice (n=8). There was a reduction in the number of infiltrating microglia/macrophages (n=3-5) and in their activation (n=5) in the peri-infarct area of MR1-/- mice. The cytokine levels of interleukin-6 and interleukin-17 at 24 hours after tMCAO (n=3-5), and for interleukin-17 at 72 hours after tMCAO (n=5), were lower in the MR1-/- mice. The administration of the suppressive MR1 ligand reduced the infarct volume and improved functional impairment (n=5). Flow cytometric analysis demonstrated there was a reduction of MAIT cells infiltrating into the ischemic brain at 24 hours after tMCAO (n=17). Conclusions Our results showed that MAIT cells play an important role in neuroinflammation after focal cerebral ischemia and the use of MAIT cell regulation has a potential role as a novel neuroprotectant for the treatment of acute ischemic stroke.
Asunto(s)
Inmunidad Celular , Inflamación/inmunología , Accidente Cerebrovascular Isquémico/inmunología , Células T Invariantes Asociadas a Mucosa/inmunología , Enfermedad Aguda , Animales , Modelos Animales de Enfermedad , Accidente Cerebrovascular Isquémico/metabolismo , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Stroke is the leading cause of disability, and stroke survivors suffer from long-term sequelae even after receiving recombinant tissue plasminogen activator therapy and endovascular intracranial thrombectomy. Increasing evidence suggests that exosomes, nano-sized extracellular membrane vesicles, enhance neurogenesis, angiogenesis, and axonal outgrowth, all the while suppressing inflammatory reactions, thereby enhancing functional recovery after stroke. A systematic literature review to study the association of stroke recovery with exosome therapy was carried out, analyzing species, stroke model, source of exosomes, behavioral analyses, and outcome data, as well as molecular mechanisms. Thirteen studies were included in the present systematic review. In the majority of studies, exosomes derived from mesenchymal stromal cells or stem cells were administered intravenously within 24 h after transient middle cerebral artery occlusion, showing a significant improvement of neurological severity and motor functions. Specific microRNAs and molecules were identified by mechanistic investigations, and their amplification was shown to further enhance therapeutic effects, including neurogenesis, angiogenesis, axonal outgrowth, and synaptogenesis. Overall, this review addresses the current advances in exosome therapy for stroke recovery in preclinical studies, which can hopefully be preparatory steps for the future development of clinical trials involving stroke survivors to improve functional outcomes.
Asunto(s)
Exosomas , Células Madre Mesenquimatosas/metabolismo , Accidente Cerebrovascular , Animales , Axones/metabolismo , Modelos Animales de Enfermedad , Exosomas/metabolismo , Exosomas/trasplante , Humanos , MicroARNs/metabolismo , Neovascularización Fisiológica , Neurogénesis , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/terapia , Sinapsis/metabolismoRESUMEN
As oligodendrocyte precursor cells (OPCs) are vulnerable to ischemia, their differentiation to oligodendrocytes (OLG) is impaired in chronic cerebral hypoperfusion. Astrocyte-OLG interaction is important for white matter homeostasis. Recently, reactive astrocytes were separated into two types, A1 (cytotoxic) and A2 (neurotrophic). However, their role in prolonged cerebral hypoperfusion remains unclear. We analyzed the effects of interaction between A1-A2 astrocytes and OPC-OLG under hypoperfusion, focusing on mitochondrial migration. As an in vivo model, chronic hypoperfusion model mice were created by bilateral common carotid artery stenosis (BCAS) using microcoils. As a matching in vitro study, rat primary cells were cocultured with a nonlethal concentration of CoCl2 . At 28 days after hypoperfusion, the number of OPC and astrocytes increased, whereas that of OLG decreased. Increased astrocytes were mainly A1-like astrocytes; however, the number of A2-like type decreased. In cell culture, OPC differentiation was interrupted under mimic chronic ischemia, but improved after astrocyte-conditioned medium (ACM) was added. However, injured-ACM was unable to improve OPC maturation. Incubation with CoCl2 changed astrocytes from A2-like to A1-like, and mitochondrial migration was also reduced. A Trkß agonist was able to maintain astrocytes from A1-like to A2-like even under hyperperfused conditions, and aided in OPC maturation and memory impairment via mitochondrial migration and drug effects in cell culture study and BCAS model. The reduction of A1-like astrocytes protects against white matter injury. Trkß agonists may play an important role in the impairment under chronic ischemic conditions. Mitochondrial migration may be a broad therapeutic strategy for cerebrovascular diseases. MAIN POINTS: Prolonged cerebral hypoperfusion leads to impaired oligodendrocyte (OLG) maturation and increased numbers of A1 astrocytes. Mitochondria migration maintained A2 astrocyte morphology, mature OLG, and myelinated white matter in vivo/vitro.
Asunto(s)
Isquemia Encefálica , Estenosis Carotídea , Sustancia Blanca , Animales , Astrocitos , Medios de Cultivo Condicionados/farmacología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Oligodendroglía , RatasRESUMEN
Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and a potent inflammatory stimulus for the innate immune response via toll-like receptor (TLR) 4 activation. Type 2 diabetes is associated with changes in gut microbiota and impaired intestinal barrier functions, leading to translocation of microbiota-derived LPS into the circulatory system, a condition referred to as metabolic endotoxemia. We investigated the effects of metabolic endotoxemia after experimental stroke with transient middle cerebral artery occlusion (MCAO) in a murine model of type 2 diabetes (db/db) and phenotypically normal littermates (db/+). Compared to db/+ mice, db/db mice exhibited an altered gut microbial composition, increased intestinal permeability, and higher plasma LPS levels. In addition, db/db mice presented increased infarct volumes and higher expression levels of LPS, TLR4, and inflammatory cytokines in the ischemic brain, as well as more severe neurological impairments and reduced survival rates after MCAO. Oral administration of a non-absorbable antibiotic modulated the gut microbiota and improved metabolic endotoxemia and stroke outcomes in db/db mice; these effects were associated with reduction of LPS levels and neuroinflammation in the ischemic brain. These data suggest that targeting metabolic endotoxemia may be a novel potential therapeutic strategy to improve stroke outcomes.
Asunto(s)
Isquemia Encefálica/metabolismo , Endotoxemia/metabolismo , Lipopolisacáridos/metabolismo , Inflamación Neurogénica/metabolismo , Administración Oral , Animales , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Isquemia Encefálica/patología , Estudios de Casos y Controles , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Endotoxemia/tratamiento farmacológico , Endotoxemia/fisiopatología , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/fisiología , Bacterias Gramnegativas/metabolismo , Inmunidad Innata/efectos de los fármacos , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/veterinaria , Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Masculino , Ratones , Modelos Animales , Inflamación Neurogénica/tratamiento farmacológico , Inflamación Neurogénica/fisiopatología , Polimixina B/administración & dosificación , Polimixina B/uso terapéutico , Accidente Cerebrovascular/metabolismo , Tasa de Supervivencia , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
White matter lesions due to cerebral hypoperfusion may be an important pathophysiology in vascular dementia and stroke, although the inherent mechanisms remain to be fully elucidated. The present study, using a mouse model of chronic cerebral hypoperfusion, examined the white matter protective effects of levetiracetam, an anticonvulsant, via the signaling cascade from the activation of cAMP-responsive element binding protein (CREB) phosphorylation. Mice underwent bilateral common carotid artery stenosis (BCAS), and were separated into the levetiracetam group (injected once only after BCAS [LEV1] or injected on three consecutive days [LEV3]), the vehicle group, or the anti-epileptic drugs with different action mechanisms phenytoin group (PHT3; injected on three consecutive days with the same condition as in LEV3). Cerebral blood flow analysis, Y-maze spontaneous alternation test, novel object recognition test, immunohistochemical and Western blot analyses, and protein kinase A assay were performed after BCAS. In the LEV3 group, SV2A expression was markedly increased, which preserved learning and memory after BCAS. Moreover, as the protein kinase A level was significantly increased, pCREB expression was also increased. The activation of microglia and astrocytes was markedly suppressed, although the number of oligodendrocyte precursor cells (OPCs) and GST-pi-positive-oligodendrocytes was markedly higher in the cerebral white matter. Moreover, oxidative stress was significantly reduced. We found that 3-day treatment with levetiracetam maintained SV2A protein expression via interaction with astrocytes, which influenced the OPC lineage through activation of CREB to protect white matter from ischemia.
Asunto(s)
Isquemia Encefálica/complicaciones , Encéfalo/efectos de los fármacos , Estenosis Carotídea/complicaciones , Disfunción Cognitiva/prevención & control , Levetiracetam/farmacología , Fármacos Neuroprotectores/farmacología , Sustancia Blanca/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Estenosis Carotídea/metabolismo , Estenosis Carotídea/patología , Cognición/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Levetiracetam/uso terapéutico , Glicoproteínas de Membrana/metabolismo , Memoria/efectos de los fármacos , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Fosforilación/efectos de los fármacos , Sustancia Blanca/metabolismo , Sustancia Blanca/patologíaRESUMEN
Background and Purpose- Exosomes play a pivotal role in neurogenesis. In the peri-infarct area after stroke, axons begin to regenerate but are inhibited by astrocyte scar formation. The direct effect and underlying molecular mechanisms of astrocyte-derived exosomes on axonal outgrowth after ischemia are not known. Methods- Using a semaphorin 3A (Sema3A) inhibitor, we explored neuronal signaling during axonal outgrowth after ischemia in rats subjected to middle cerebral artery occlusion and in cultured cortical neurons challenged with oxygen-glucose deprivation. Furthermore, we assessed whether this inhibitor suppressed astrocyte activation and regulated astrocyte-derived exosomes to enhance axonal outgrowth after ischemia. Results- In rats subjected to middle cerebral artery occlusion, we administered a Sema3A inhibitor into the peri-infarct area from 7 to 21 days after occlusion. We found that phosphorylated high-molecular weight neurofilament-immunoreactive axons were increased, glial fibrillary acidic protein-immunoreactive astrocytes were decreased, and functional recovery was promoted at 28 days after middle cerebral artery occlusion. In cultured neurons, the Sema3A inhibitor decreased Rho family GTPase 1, increased R-Ras, which phosphorylates Akt and glycogen synthase kinase 3ß (GSK-3ß), selectively increased phosphorylated GSK-3ß in axons, and thereby enhanced phosphorylated high-molecular weight neurofilament-immunoreactive axons after oxygen-glucose deprivation. In cultured astrocytes, the Sema3A inhibitor suppressed activation of astrocytes induced by oxygen-glucose deprivation. Exosomes secreted from ischemic astrocytes treated with the Sema3A inhibitor further promoted axonal elongation and increased prostaglandin D2 synthase expression on microarray analysis. GSK-3ß+ and prostaglandin D2 synthase+ neurons were robustly increased after treatment with the Sema3A inhibitor in the peri-infarct area. Conclusions- Neuronal Rho family GTPase 1/R-Ras/Akt/GSK-3ß signaling, axonal GSK-3ß expression, and astrocyte-derived exosomes with prostaglandin D2 synthase expression contribute to axonal outgrowth and functional recovery after stroke.
Asunto(s)
Astrocitos/metabolismo , Exosomas/efectos de los fármacos , Prostaglandinas/farmacología , Semaforina-3A/antagonistas & inhibidores , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Astrocitos/efectos de los fármacos , Axones/efectos de los fármacos , Axones/metabolismo , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas Wistar , Accidente Cerebrovascular/metabolismoRESUMEN
Survivin, a member of the inhibitors of apoptosis protein gene family, inhibits the activity of caspase, leading to a halt of the apoptotic process. Our study focused on the neuroprotective effect of survivin after transient middle cerebral artery occlusion (MCAO) with intraparenchymal administration of an adeno-associated virus (AAV) vector. His-tagged survivin was cloned and packaged into the AAV-rh10 vector. Four-week-old Sprague-Dawley rats were injected with 4 × 109 vg of AAV-GFP or AAV-His-survivin into the right striatum and were treated 3 weeks later with transient MCAO for 90 min. Twenty-four hours after MCAO, functional and histological analyses of the rats were performed. The result showed that rats that had been treated with AAV-green fluorescent protein (GFP) and those that had been treated with AAV-His-survivin did not show a significant difference in neurological scores 24 hr after MCAO, however, infarction volume was significantly reduced in the AAV-His-survivin group compared to that in the AAV-GFP group. Although the neutrophil marker myeloperoxidase did not show a significant difference in the ischemic boundary zone, cells positive for active caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling were significantly decreased in the AAV-His-survivin group. In conclusion, survivin overexpression in the ischemic boundary zone induced by using an AAV vector has the potential for amelioration of ischemic damage via an antiapoptotic mechanism.
Asunto(s)
Isquemia Encefálica/virología , Infarto de la Arteria Cerebral Media/virología , Fármacos Neuroprotectores/farmacología , Survivin/metabolismo , Animales , Apoptosis/efectos de los fármacos , Isquemia Encefálica/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Etiquetado Corte-Fin in Situ/métodos , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Ratas Sprague-Dawley , Survivin/genéticaRESUMEN
AIMS: Rats subjected to transient focal ischemia and cultured neuronal cells subjected to oxygen-glucose deprivation (OGD) were treated with clarithromycin (CAM) to evaluate the effects of CAM in protecting against neuronal damage. MAIN METHODS: Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 90min and then reperfused. Each animal was given an oral dose clarithromycin (CAM, 100mg/kg) or vehicle alone just after the ischemia was commenced. The infarct volume, edema index and neurological performance were assessed after 24 and 72h of reperfusion. The cerebral blood flow (CBF) was measured with an MRI system at 90min after MCAO. After 24 and 72h, oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) were assessed by immunohistochemical analyses and degenerative cells were assessed in the cortex by Fluoro-Jade C (FJC) labeling. The cultured neuronal cells were also used to examine the effects of CAM exposure on the viability of the cells after OGD. KEY FINDINGS: CBF was unchanged between the two groups. Significant reductions of the infarct volume and edema index, an improved neurological deficit score, a significant suppression of 4-HNE and 8-OHdG expression, marked reductions of Iba-1 and TNF-α expression, and a significant reduction of FJC-positive cells were also observed in the CAM-treated animals at both time points. Treatment with 10µM and 100µM CAM in vitro significantly reduced cell death after OGD. SIGNIFICANCE: CAM appears to provide antioxidant and anti-inflammatory effects and protect against neuronal damage after cerebral ischemia and OGD.
Asunto(s)
Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Claritromicina/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Animales , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Encéfalo/irrigación sanguínea , Encéfalo/patología , Isquemia Encefálica/patología , Células Cultivadas , Circulación Cerebrovascular/efectos de los fármacos , Claritromicina/farmacología , Glucosa/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Masculino , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Oxígeno/metabolismo , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: This study aims to determine if erythromycin provides neuroprotective effects against ischemic injury following permanent focal cerebral ischemia. METHODS: Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO). Each animal received a single subcutaneous injection of erythromycin lactobionate (EM, 50 mg/kg) or vehicle immediately after ischemia. The infarct volume, edema index and neurological performance were evaluated at 24 and 72 h after MCAO. The cerebral blood flow (CBF) was measured with an MRI system at 30 min after MCAO. TUNEL staining and immunohistochemical analyses for oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) in the cortex were conducted at 24 and 72 h after MCAO. RESULTS: The CBF did not differ between the EM-treated and vehicle-treated groups. The EM treatment significantly reduced the infarct volume (p < 0.01) at 24 and 72 h after MCAO and significantly reduced the edema index (p < 0.01) at 24 h. The EM treatment significantly improved the neurological deficit scores (p < 0.05) at 24 and 72 h. EM also significantly suppressed the accumulation of 4-HNE (p < 0.01) and 8-OHdG (p < 0.01) and markedly reduced Iba-1 (p < 0.01) and TNF-α expression (p < 0.05) at both time points. The EM treatment significantly reduced TUNEL-positive cells (p < 0.01) at both time points. CONCLUSION: These findings suggest that EM can protect against the neuronal damage caused by cerebral ischemia by alleviating inflammation and reducing oxidant stress.
Asunto(s)
Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/etiología , Eritromicina/uso terapéutico , Infarto de la Arteria Cerebral Media/complicaciones , Fármacos Neuroprotectores/uso terapéutico , 8-Hidroxi-2'-Desoxicoguanosina , Aldehídos/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/etiología , Infarto Encefálico/tratamiento farmacológico , Infarto Encefálico/etiología , Lesiones Encefálicas/diagnóstico por imagen , Proteínas de Unión al Calcio/metabolismo , Circulación Cerebrovascular/efectos de los fármacos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Modelos Animales de Enfermedad , Etiquetado Corte-Fin in Situ , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Imagen por Resonancia Magnética , Proteínas de Microfilamentos/metabolismo , Ratas , Estadísticas no Paramétricas , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: This study aims to determine if macrolide antibiotics have neuroprotective effects against transient cerebral ischemia. METHODS: Sprague-Dawley rats were subjected to cerebral ischemia for 90 minutes followed by 24 or 72 hours of reperfusion. An oral suspension of roxithromycin (RXM), clarithromycin (CAM), erythromycin (EM), azithromycin (AZM), or kitasamycin (INN) was given at 10 or 100 mg/kg for 7 days before ischemia. The infarct volume, edema volume, and neurological performance were evaluated after 24 and 72 hours of reperfusion. The cerebral blood flow (CBF) was measured with a magnetic resonance imaging (MRI) system after 90 minutes of ischemia. Another experiment was conducted to investigate how the ischemic injury was affected by the interval from the antibiotic pretreatment to the ischemia in rats pretreated with CAM. RESULTS: Roxithromycin, CAM, AZM, and INN significantly reduced the infarct volume in the high-dose group after 24 and 72 hours of reperfusion. All of the agents significantly decreased the edema in the high-dose groups at 24 and 72 hours, while only CAM and AZM significantly reduced the edema volume in the low-dose groups at 24 hours. All of the macrolide antibiotics at the high dose significantly improved neurological deficit scores at 24 and 72 hours. There were no differences in the CBF between the vehicle and respective antibiotic groups. In the experiment examining the interval, the 24-hour interval group exhibited the strongest neuroprotective effect. DISCUSSION: These results demonstrate that the macrolide antibiotics RXM, CAM, EM, AZM, and INN may confer neuroprotective effects against ischemic damage following cerebral ischemia without affecting the CBF.
Asunto(s)
Ataque Isquémico Transitorio/tratamiento farmacológico , Macrólidos/farmacología , Fármacos Neuroprotectores/farmacología , Daño por Reperfusión/tratamiento farmacológico , Animales , Antibacterianos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Encéfalo/fisiopatología , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/patología , Edema Encefálico/fisiopatología , Circulación Cerebrovascular/efectos de los fármacos , Circulación Cerebrovascular/fisiología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Ataque Isquémico Transitorio/patología , Ataque Isquémico Transitorio/fisiopatología , Masculino , Distribución Aleatoria , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Factores de TiempoRESUMEN
This study aims to determine if erythromycin has neuroprotective effects against transient ischemia and oxygen-glucose deprivation (OGD) in cultured neuronal cells. Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 90 min, followed by reperfusion. The animals received a subcutaneous single injection of erythromycin lactobionate (EM, 50mg/kg) or vehicle immediately after ischemia. Infarct volume, edema index, and neurological performance were evaluated at 24 and 72 h after reperfusion. Immunohistochemical analyses for oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) were conducted in the cortex at 24h. Primary cortical neuronal cell cultures were prepared from the cerebral cortices of the animals and then subjected to OGD for 3h. Ten or 100 µM EM was added before OGD to determine the effect of EM on cell viability after OGD. EM significantly reduced infarct volume (p<0.01) and edema volume (p<0.05) and improved neurological deficit scores (p<0.05) at 24 and 72 h. EM significantly suppressed the accumulation of 4-HNE (p<0.01) and 8-OHdG (p<0.01) and markedly reduced Iba-1 (p<0.01) and TNF-α expression (p<0.01). Treatment with 100 µM EM in vitro significantly reduced cell death after OGD. EM reduces neuronal damage following cerebral ischemia and OGD and may have antioxidant and anti-inflammatory effects.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Corteza Cerebral/efectos de los fármacos , Eritromicina/administración & dosificación , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Daño por Reperfusión/tratamiento farmacológico , Animales , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/patología , Edema Encefálico/fisiopatología , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Modelos Animales de Enfermedad , Glucosa/deficiencia , Hemiplejía/etiología , Hemiplejía/prevención & control , Infarto de la Arteria Cerebral Media , Masculino , Neuroinmunomodulación/efectos de los fármacos , Neuroinmunomodulación/fisiología , Neuronas/patología , Neuronas/fisiología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Distribución Aleatoria , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatologíaRESUMEN
OBJECTIVES: Among several anti-platelet drugs to prevent recurrent stroke, cilostazol has shown various effects besides its anti-platelet activity. We examined whether 7 days of oral administration of cilostazol protects against subsequent cerebral ischemia, and whether or not the effect of combination therapy with aspirin is more protective. METHODS: We used Sprague-Dawley (SD) rats and assigned them to four groups: vehicle, aspirin, cilostazol, and aspirin plus cilostazol combination therapy. After oral administration of anti-platelets for 7 days, we performed transient middle cerebral artery occlusion (MCAO) for 90 minutes, and examined infarct volume, neurological symptoms, and regional cerebral blood flow (rCBF). Immunostaining of Bax, Bcl-2, TUNEL, 4-HNE, 8-OHdG, and COX-2 was performed 24 hours after ischemia. RESULTS: The cilostazol group and the combination therapy group showed significant decreases of infarct volume and significant improvements of rCBF during ischemia, compared with the vehicle or aspirin group. Significant decreases of Bax, TUNEL, 8-OHdG, and 4-HNE expression in the combination therapy group, compared with those in the vehicle or aspirin group, were shown in the boundary zone. COX-2 expression was unexpectedly increased in the combination therapy group. DISCUSSION: Aspirin co-administration did not inhibit this effect. The addition of the oral administration of cilostazol either alone or with aspirin administration may be beneficial for subsequent cerebral ischemic damage in terms of reducing infarct volume, improving rCBF during ischemia, inhibiting the apoptotic pathway, and reducing oxidative stress.
Asunto(s)
Aspirina/uso terapéutico , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/prevención & control , Fármacos Neuroprotectores/uso terapéutico , Tetrazoles/uso terapéutico , Aldehídos/metabolismo , Animales , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Cilostazol , Ciclooxigenasa 2/metabolismo , Quimioterapia Combinada , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Proteína X Asociada a bcl-2/metabolismoRESUMEN
AIMS: Pre-treatment with statins is known to ameliorate ischemic brain damage after experimental stroke, and is independent of cholesterol levels. We undertook pre- vs post-ischemic treatment with atorvastatin after focal cerebral ischemia in rats. MAIN METHODS: Male Sprague-Dawley rats underwent transient 90-min middle cerebral artery occlusion (MCAO). Atorvastatin (20mg/kg/day) or vehicle was administered orally. Rats were divided into vehicle-treated, atorvastatin pre-treatment, atorvastatin post-treatment, and atorvastatin continuous-treatment groups. In the pre-treatment, rats were given atorvastatin or vehicle for 7 days before MCAO. In the post-treatment, rats received atorvastatin or vehicle for 7 days after MCAO. Measurement of infarct volume, as well as neurological and immunohistochemical assessments, were done 24h and 7 days after reperfusion. KEY FINDINGS: Each atorvastatin-treated group demonstrated significant reductions in infarct and edema volumes compared with the vehicle-treated group 24h after reperfusion. Seven days after reperfusion, infarct volumes in the post-treatment group and continuous-treatment group (but not the pre-treatment group) were significantly smaller than in the vehicle-treated group. Only the continuous-treatment group had significantly improved neurological scores 7 days after reperfusion compared with the vehicle group. Post-treatment and continuous-treatment groups had significantly decreased lipid peroxidation, oxidative DNA damage, microglial activation, expression of tumor necrosis factor-alpha, and neuronal damage in the cortical ischemic boundary area after 7 days of reperfusion. SIGNIFICANCE: These results suggest that continuous oral administration (avoiding withdrawal) with statins after stroke may reduce the extent of post-ischemic brain damage and improve neurological outcome by inhibiting oxidative stress and inflammatory responses.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Ácidos Heptanoicos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Pirroles/uso terapéutico , Administración Oral , Animales , Atorvastatina , Encéfalo/patología , Encéfalo/fisiopatología , Edema Encefálico/etiología , Edema Encefálico/patología , Isquemia Encefálica/complicaciones , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Daño del ADN/efectos de los fármacos , Ácidos Heptanoicos/administración & dosificación , Peroxidación de Lípido/efectos de los fármacos , Imagen por Resonancia Magnética , Masculino , Microglía/efectos de los fármacos , Microglía/patología , Neuroimagen , Fármacos Neuroprotectores/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Pirroles/administración & dosificación , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
AIMS: Transplantation of bone marrow mononuclear cells (BMMCs) exerts neuroprotection against cerebral ischemia. We examined the therapeutic timepoint of allogeneic BMMC transplantation in a rat model of focal cerebral ischemia, and determined the effects of repeated transplantation outside the therapeutic window. MAIN METHODS: Male Sprague-Dawley rats were subjected to 90 minute focal cerebral ischemia, followed by intravenous administration of 1 × 10(7) allogeneic BMMCs or vehicle at 0, 3 or 6 h after reperfusion or 2 × 10(7) BMMCs 6 h after reperfusion. Other rats administered 1 × 10(7) BMMCs at 6 h after reperfusion received additional BMMC transplantation or vehicle 9 h after reperfusion. Infarct volumes, neurological deficit scores and immunohistochemistry were evaluated 24 or 72 h after reperfusion. KEY FINDINGS: Infarct volumes at 24 h were significantly decreased in transplantation rats at 0 and 3 h, but not at 6 h, after reperfusion, compared to vehicle-treatment. Even high dose BMMC transplantation at 6h after reperfusion was ineffective. Repeated BMMC transplantation at 6 and 9h after reperfusion reduced infarct volumes and significantly improved neurological deficit scores at 24 and 72 h. Immunohistochemistry showed repeated BMMC transplantation reduced ionized calcium-binding adapter molecule 1, 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine expression at 24 and 72 h after reperfusion. SIGNIFICANCE: Intravenous allogeneic BMMCs were neuroprotective following transient focal cerebral ischemia, and the therapeutic time window of BMMC transplantation was >3 h and <6 h after reperfusion in this model. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in ischemic brains, resulting in improved neuroprotection.
Asunto(s)
Trasplante de Médula Ósea/métodos , Lesiones Encefálicas/terapia , Ataque Isquémico Transitorio/complicaciones , Leucocitos Mononucleares/trasplante , 8-Hidroxi-2'-Desoxicoguanosina , Aldehídos/metabolismo , Animales , Lesiones Encefálicas/etiología , Proteínas de Unión al Calcio/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Inflamación/etiología , Inflamación/terapia , Masculino , Proteínas de Microfilamentos/metabolismo , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Trasplante HomólogoRESUMEN
Long-chain n-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have been shown to reduce ischemic neuronal injury. We investigated the effects of ethyl-EPA (EPA-E) on ischemic brain damage using a rat transient focal cerebral ischemia model. Male Sprague-Dawley rats (n=105) were subjected to 90 min of focal cerebral ischemia. EPA-E (100mg/kg/day) or vehicle was administered once a day for 3, 5 or 7 days prior to ischemia. Different withdrawal intervals of 3, 5, and 7 days prior to ischemia following 7-day pretreatment with EPA-E or vehicle were also examined. In addition, post-ischemic administration of EPA-E was investigated. Pretreatment with EPA-E for 7 and 5 days, but not 3 days, showed significant infarct volume reduction and neurological improvements when compared with vehicle pretreatment. In addition, withdrawal of EPA-E administration for 3 days, but not 5 and 7 days, also demonstrated significant infarct volume reduction and neurological improvements when compared with vehicle treatment. Post-ischemic treatment of EPA-E did not show any neuroprotection. Immunohistochemistry revealed that 7-day pretreatment with EPA-E significantly reduced cortical expression of 8-hydroxydeoxyguanosine (maker for oxidative DNA damage), 4-hydroxy-2-nonenal (maker for lipid peroxidation), phosphorylated adducin (marker for Rho-kinase activation) and von Willebrand factor (endothelial marker) when compared with vehicle pretreatment. In addition, phosphorylated adducin expression co-localized with von Willebrand factor immunoreactivity. The present study established the neuroprotective effect of EPA-E on ischemic brain damage following transient focal cerebral ischemia in rats, which may be involved in the suppression of oxidative stress and endothelial Rho-kinase activation.
Asunto(s)
Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/etiología , Ácido Eicosapentaenoico/uso terapéutico , Ataque Isquémico Transitorio/complicaciones , Fármacos Neuroprotectores/uso terapéutico , 8-Hidroxi-2'-Desoxicoguanosina , Aldehídos/metabolismo , Análisis de Varianza , Animales , Infarto Encefálico/etiología , Infarto Encefálico/prevención & control , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Imagen por Resonancia Magnética , Masculino , Examen Neurológico , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Factor de von Willebrand/metabolismoRESUMEN
OBJECTIVES: Intra-arterial transplantation of bone marrow mononuclear cells (BMMCs) effectively improves neuronal function and limits the infarct size. We monitored the fate of BMMCs labeled with super paramagnetic iron oxide (SPIO) until 7 days after the transplantation using high-field magnetic resonance imaging (MRI). METHODS: Male Sprague-Dawley rats were subjected to 90-minute focal ischemia using the intraluminal suture technique followed by transplantation of 1 × 10(7) BMMCs or vehicle only via the ipsilateral carotid artery immediately after reperfusion. Autologous BMMCs were labeled with SPIO by electroporation prior to ischemia. MRI studies were performed at 1 hour, 24 hours, 3 days, and 7 days after reperfusion on each rat. The total infarct volume and the volume of negative dots were measured on T2-weighted images and T2*-weighted images, respectively. RESULTS: One hour after BMMC transplantation, we confirmed wide spread distribution of BMMCs in the ischemic hemisphere as a negative dot. The volume of negative dot normalized by hemispheric volume decreased rapidly and was seldom seen at the seventh day after transplantation. The infarct volume was significantly smaller in the transplanted group than the vehicle group at 24 hours and 7 days after reperfusion. DISCUSSION: The present study established In vivo monitoring of intra-arterial transplanted SPIO-labeled BMMCs immediately after reperfusion using MRI of a rat transient focal ischemia model. The accumulation of BMMCs in ischemic lesion at the acute stage of ischemia can be part of the conditions to limit the infarct size.