RESUMEN
The inflammatory response plays an important role in neuroprotection and regeneration after ischemic insult. The use of non-steroidal anti-inflammatory drugs has been a matter of debate as to whether they have beneficial or detrimental effects. In this context, the effects of the anti-inflammatory agent meloxicam have been scarcely documented after stroke, but its ability to inhibit both cyclooxygenase isoforms (1 and 2) could be a promising strategy to modulate post-ischemic inflammation. This study analyzed the effect of meloxicam in a transient focal cerebral ischemia model in rats, measuring its neuroprotective effect after 48 hours and 7 days of reperfusion and the effects of the treatment on the glial scar and regenerative events such as the generation of new progenitors in the subventricular zone and axonal sprouting at the edge of the damaged area. We show that meloxicam's neuroprotective effects remained after 7 days of reperfusion even if its administration was restricted to the two first days after ischemia. Moreover, meloxicam treatment modulated glial scar reactivity, which matched with an increase in axonal sprouting. However, this treatment decreased the formation of neuronal progenitor cells. This study discusses the dual role of anti-inflammatory treatments after stroke and encourages the careful analysis of both the neuroprotective and the regenerative effects in preclinical studies.
RESUMEN
The development of RNA-based anti-infectives has gained interest with the successful application of mRNA-based vaccines. Small RNAs are molecules of RNA of <200 nucleotides in length that may control the expression of specific genes. Small RNAs include small interference RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), or microRNAs (miRNAs). Notably, the role of miRNAs on the post-transcriptional regulation of gene expression has been studied in detail in the context of cancer and many other genetic diseases. However, it is also becoming apparent that some human miRNAs possess important antimicrobial roles by silencing host genes essential for the progress of bacterial or viral infections. Therefore, their potential use as novel antimicrobial therapies has gained interest during the last decade. The challenges of the transport and delivery of miRNAs to target cells are important, but recent research with exosomes is overcoming the limitations in RNA-cellular uptake, avoiding their degradation. Therefore, in this review, we have summarised the latest developments in the exosomal delivery of miRNA-based therapies, which may soon be another complementary treatment to pathogen-targeted antibiotics that could help solve the problem caused by multidrug-resistant bacteria.
RESUMEN
Stroke is one of the main causes of death and disability worldwide. Ischemic stroke results in unfolded/misfolded protein accumulation in endoplasmic reticulum (ER), a condition known as ER stress. We hypothesized that previously reported neuroprotection of celecoxib, a selective inhibitor of cyclooxygenase-2, in transient middle cerebral artery occlusion (tMCAO) model, relies on the ER stress decrease. To probe this hypothesis, Sprague-Dawley rats were subjected to 1 h of tMCAO and treated with celecoxib or vehicle 1 and 24 h after ischemia. Protein and mRNA levels of the main hallmarks of ER stress, unfolded protein response (UPR) activation, UPR-induced cell death, and ubiquitin proteasome system (UPS) and autophagy, the main protein degradation pathways, were measured at 12 and 48 h of reperfusion. Celecoxib treatment decreased polyubiquitinated protein load and ER stress marker expression such as glucose-related protein 78 (GRP78), C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP), and caspase 12 after 48 h of reperfusion. Regarding the UPR activation, celecoxib promoted inositol-requiring enzyme 1 (IRE1) pathway instead of double-stranded RNA-activated protein kinase-like ER kinase (PERK) pathway. Furthermore, celecoxib treatment increased proteasome catalytic subunits transcript levels and decreased p62 protein levels, while the microtubule-associated protein 1 light chain 3 (LC3B) II/I ratio remained unchanged. Thus, the ability of celecoxib treatment on reducing the ER stress correlates with the enhancement of IRE1-UPR pathway and UPS degradation. These data support the ability of anti-inflammatory therapy in modulating ER stress and reveal the IRE1 pathway as a promising therapeutic target in stroke therapy.Graphical abstract.
Asunto(s)
Celecoxib/farmacología , Infarto de la Arteria Cerebral Media/patología , Neuroprotección , Complejo de la Endopetidasa Proteasomal/metabolismo , Respuesta de Proteína Desplegada , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Biomarcadores/metabolismo , Isquemia Encefálica/complicaciones , Isquemia Encefálica/patología , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/metabolismo , Proteínas de Choque Térmico/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Masculino , Complejos Multienzimáticos/metabolismo , Neuroprotección/efectos de los fármacos , Poliubiquitina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Proteolisis/efectos de los fármacos , Ratas Sprague-Dawley , Respuesta de Proteína Desplegada/efectos de los fármacos , eIF-2 Quinasa/metabolismoRESUMEN
Ischemic stroke is one of the most important causes of death and disability worldwide. Subroutines underlying cell death after stroke are largely unknown despite their importance in the design of novel therapies for this pathology. Necroptosis, a recently described form of regulated cell death, has been related with inflammation and, in some models, with endoplasmic reticulum (ER) stress. We hypothesize that alleviation of ER stress following a salubrinal treatment will reduce the ischemic-dependent necroptosis. To probe the hypothesis, we measured, at 48 and 72 h after transient global cerebral ischemia in rat, in cerebral cortex and cornu ammonis 1, the main hallmarks of necroptosis: mRNA levels and phosphorylation of mixed lineage kinase domain like pseudokinase as well as receptor interacting serine/threonine protein kinase 3, along the years 2017-2018. Selective neuronal loss after 7 days of the ischemic insult, and other markers related with the inflammatory response were also measured. This study shows that necroptosis in cerebral cortex can be detected after 72 h of the insult and seems to be elicited before 48 h of reperfusion. The type of necroptosis here observed seems to be tumor necrosis factor receptor 1 independent. Necroptotic response is less evident in the cornu ammonis 1 hippocampal area than in cerebral cortex. The treatment with salubrinal administered 1 and 24 h after the ischemia, decreased the necroptotic marker levels and reduced the areas of selective neuronal loss, supporting the presence of ischemic-dependent necroptosis, and the notion that ER stress is involved in the necroptotic response. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.
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Isquemia Encefálica/patología , Isquemia Encefálica/prevención & control , Cinamatos/uso terapéutico , Modelos Animales de Enfermedad , Necroptosis/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Tiourea/análogos & derivados , Animales , Isquemia Encefálica/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Cinamatos/farmacología , Masculino , Necroptosis/fisiología , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Tiourea/farmacología , Tiourea/uso terapéuticoRESUMEN
Brain-derived neurotrophic factor (BDNF) is considered as a putative therapeutic agent against stroke. Since BDNF role on oxidative stress is uncertain, we have studied this role in a rat brain slice ischemia model, which allows BDNF reaching the neural parenchyma. Hippocampal and cerebral cortex slices were subjected to oxygen and glucose deprivation (OGD) and then returned to normoxic conditions (reperfusion-like, RL). OGD/RL increased a number of parameters mirroring oxidative stress in the hippocampus that were reduced by the BDNF presence. BDNF also reduced the OGD/RL-increased activity in a number of antioxidant enzymes in the hippocampus but no effects were observed in the cerebral cortex. In general, we conclude that alleviation of oxidative stress by BDNF in OGD/RL-exposed slices relies on decreasing cPLA2 activity, rather than modifying antioxidant enzyme activities. Moreover, a role for the oxidative stress in the differential ischemic vulnerability of cerebral cortex and hippocampus is also supported.
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Factor Neurotrófico Derivado del Encéfalo/farmacología , Encéfalo/patología , Glucosa/deficiencia , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Oxígeno/toxicidad , Animales , Antioxidantes/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citosol/enzimología , Peroxidación de Lípido/efectos de los fármacos , Masculino , NADPH Oxidasas/metabolismo , Fosfolipasas A2/genética , Fosfolipasas A2/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Reperfusión , Transcripción Genética/efectos de los fármacosRESUMEN
Areas of selective neuronal loss (SNL) represent the first morphologic signs of damage in the penumbra region and are considered putative targets for ischemic stroke therapy. We performed a novel assessment of measuring the effects of the anti-inflammatory agent celecoxib by analyzing simultaneously the different neural populations (neurons, astrocytes, and microglia cells) in SNL and non-SNL areas. Rats were subjected to 1 hour of middle cerebral artery occlusion (MCAO) and treated with celecoxib 1 and 24 hours after ischemia. Infarct volume measurements and triple immunostaining of neurons (neuronal nuclear antigen), microglia (ionized calcium-binding adaptor molecule 1), and astroglia were performed after 12 and 48 hours of reperfusion. Motor response was tested by standard behavioral assays at 3, 12, 24, and 48 hours. Confocal analysis revealed that the percentage of SNL areas, microglia densities, and glial activation increased at 48 hours of reperfusion. Celecoxib treatment improved the neurologic deficit, reduced the infarct volume by 50% after 48 hours of reperfusion, and resulted in a reduced percentage of SNL areas and microglia and astroglia reactivity after 48 hours of reperfusion. This study proves, for the first time, that celecoxib presents postischemic neuroprotective effects in a transient MCAO model, prevents or delays the presence of SNL areas, and reduces glial activation.
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Celecoxib/farmacología , Infarto de la Arteria Cerebral Media/complicaciones , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/etiología , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Modelos Animales de Enfermedad , Masculino , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/tratamiento farmacológico , Accidente Cerebrovascular/tratamiento farmacológicoAsunto(s)
Enfermedades del Sistema Nervioso/prevención & control , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Metabolismo Energético , Humanos , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/patología , Fármacos Neuroprotectores/uso terapéutico , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vitamina D/uso terapéuticoRESUMEN
We previously demonstrated that the non-steroidal anti-inflammatory agent meloxicam has neuroprotective effects in an oxygen and glucose deprivation model (OGD) of rat organotypic hippocampal slice cultures. We wondered if GABAergic transmission changed the neuroprotective effects of meloxicam and if meloxicam was able to modulate endoplasmic reticulum stress (ER stress) in this model. Mortality was measured using propidium iodide. Western blot assays were performed to measure levels of cleaved and non-cleaved caspase-3 to quantify apoptosis, while levels of GRP78, GRP94 and phosphorylated eIF2α were used to detect unfolded protein response (UPR). Transcript levels of GRP78, GRP94 and GABAergic receptor α, ß, and γ subunits were measured by real-time quantitative polymerase chain reaction (qPCR). In the present study, we show that the presence of meloxicam in a 30â¯min OGD assay, followed by 24â¯h of normoxic conditions, presented an antiapoptotic effect. The simultaneous presence of the GABAA receptor antagonist, bicuculline, in combination with meloxicam blocked the neuroprotective effect provided by the latter. However, in light of its effects on caspase 3 and PARP, bicuculline did not seem to promote the apoptotic pathway. Our results also showed that meloxicam modified the unfolded protein response (UPR), as well as the transcriptional response of different genes, including the GABAA receptor, alpha1, beta3 and gamma2 subunits. We concluded that meloxicam has a neuroprotective anti-apoptotic action, is able to enhance the UPR independently of the systemic anti-inflammatory response and its neuroprotective effect can be inhibited by blocking GABAA receptors.
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Bicuculina/farmacología , Glucosa/deficiencia , Hipocampo/metabolismo , Meloxicam/farmacología , Fármacos Neuroprotectores/farmacología , Oxígeno , Animales , Antiinflamatorios no Esteroideos/farmacología , Muerte Celular/fisiología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Relación Dosis-Respuesta a Droga , Antagonistas de Receptores de GABA-A/farmacología , Glucosa/metabolismo , Hipocampo/efectos de los fármacos , Técnicas de Cultivo de Órganos , Oxígeno/metabolismo , Ratas , Ratas Wistar , Resultado del TratamientoRESUMEN
BACKGROUND: Blood reperfusion of the ischemic tissue after stroke promotes increases in the inflammatory response as well as accumulation of unfolded/misfolded proteins in the cell, leading to endoplasmic reticulum (ER) stress. Both Inflammation and ER stress are critical processes in the delayed death of the cells damaged after ischemia. The aim of this study is to check the putative synergic neuroprotective effect by combining anti-inflammatory and anti-ER stress agents after ischemia. METHODS: The study was performed on a two-vessel occlusion global cerebral ischemia model. Animals were treated with salubrinal one hour after ischemia and with robenacoxib at 8â¯h and 32â¯h after ischemia. Parameters related to the integrity of the blood-brain barrier (BBB), such as matrix metalloproteinase 9 and different cell adhesion molecules (CAMs), were analyzed by qPCR at 24â¯h and 48â¯h after ischemia. Microglia and cell components of the neurovascular unit, including neurons, endothelial cells and astrocytes, were analyzed by immunofluorescence after 48â¯h and seven days of reperfusion. RESULTS: Pharmacologic control of ER stress by salubrinal treatment after ischemia, revealed a neuroprotective effect over neurons that reduces the transcription of molecules involved in the impairment of the BBB. Robenacoxib treatment stepped neuronal demise forward, revealing a detrimental effect of this anti-inflammatory agent. Combined treatment with robenacoxib and salubrinal after ischemia prevented neuronal loss and changes in components of the neurovascular unit and microglia observed when animals were treated only with robenacoxib. CONCLUSION: Combined treatment with anti-ER stress and anti-inflammatory agents is able to provide enhanced neuroprotective effects reducing glial activation, which opens new avenues in therapies against stroke.
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Isquemia Encefálica/tratamiento farmacológico , Cinamatos/uso terapéutico , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Difenilamina/análogos & derivados , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Fenilacetatos/uso terapéutico , Tiourea/análogos & derivados , Animales , Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/diagnóstico por imagen , Isquemia Encefálica/inmunología , Cinamatos/administración & dosificación , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Difenilamina/administración & dosificación , Difenilamina/uso terapéutico , Esquema de Medicación , Quimioterapia Combinada , Inflamación , Masculino , Fármacos Neuroprotectores/administración & dosificación , Fenilacetatos/administración & dosificación , Ratas Sprague-Dawley , Tiourea/administración & dosificación , Tiourea/uso terapéuticoRESUMEN
This study describes the neuroprotective effect of treatment with salubrinal 1 and 24 h following 15 min of ischemia in a two-vessel occlusion model of global cerebral ischemia. The purpose of this study was to determine if salubrinal, an enhancer of the unfolded protein response, reduces the neural damage modulating the inflammatory response. The study was performed in CA1 and CA3 hippocampal areas as well as in the cerebral cortex whose different vulnerability to ischemic damage is widely described. Characterization of proteins was made by western blot, immunofluorescence, and ELISA, whereas mRNA levels were measured by Quantitative PCR. The salubrinal treatment decreased the cell demise in CA1 at 7 days as well as the levels of matrix metalloprotease 9 (MMP-9) in CA1 and cerebral cortex at 48 h and ICAM-1 and VCAM-1 cell adhesion molecules. However, increases in tumor necrosis factor α and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inflammatory markers were observed at 24 h. Glial fibrillary acidic protein levels were not modified by salubrinal treatment in CA1 and cerebral cortex. We describe a neuroprotective effect of the post-ischemic treatment with salubrinal, measured as a decrease both in CA1 cell demise and in the blood-brain barrier impairment. We hypothesize that the ability of salubrinal to counteract the CA1 cell demise is because of a reduced ability of this structure to elicit unfolded protein response which would account for its greater ischemic vulnerability. Data of both treated and non-treated animals suggest that the neurovascular unit present a structure-dependent response to ischemia and a different course time for CA1/cerebral cortex compared with CA3. Finally, our study reveals a high responsiveness of endothelial cells to salubrinal in contrast to the limited responsiveness of astrocytes. The alleviation of ER stress by enhancing UPR with salubrinal treatment reduces the ischemic damage. This effect varies across the different neurovascular unit cell types. The salubrinal neuroprotective effect on CA1 supports differences in neurovascular unit for different brain regions and involves the inflammatory response and its time course. Thus, UPR modulation could be a therapeutic target in cerebral ischemia.
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Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Fármacos Neuroprotectores/farmacología , Animales , Astrocitos/metabolismo , Isquemia Encefálica/patología , Infarto Cerebral/metabolismo , Infarto Cerebral/patología , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/metabolismo , Masculino , Ratas Sprague-DawleyRESUMEN
AIMS: To evaluate the neuroprotective role of autophagy in the cerebral cortex and hippocampus using an ex vivo animal model of stroke in brain slices. METHODS: Brain slices were maintained for 30 min in oxygen and glucose deprivation (OGD) followed by 3 h in normoxic conditions to simulate the reperfusion that follows ischaemia in vivo (RL, reperfusion-like). Phagophore formation (Beclin 1 and LC3B) as well as autophagy flux (p62/SQSTM1, Atg5, Atg7 and polyubiquitin) markers were quantified by Western blot and/or qPCR. The release of lactate dehydrogenase (LDH) and glutamate in the medium was used as a measure of the mortality in the absence and in the presence of the autophagy inhibitor 3-methyladenine. RESULTS: Striking differences in the autophagy markers were observed between the hippocampus and cerebral cortex in normoxic conditions. OGD/RL induced increases both in the phagophore formation and in the autophagy flux in the first three hours in the cerebral cortex that were not observed in the hippocampus. The blocking of autophagy increased the OGD/RL-induced mortality, increased the glutamate release in both the cerebral cortex and hippocampus and abolished the OGD-induced decrease in the polyubiquitinated proteins in the cerebral cortex. CONCLUSIONS: We conclude that OGD induces a rapid autophagic response in the cerebral cortex that plays a neuroprotective role. Polyubiquitination levels and control of the glutamate release appear to be involved in the neuroprotective role of autophagy.
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Autofagia , Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Hipoxia de la Célula , Glucosa/deficiencia , Ácido Glutámico/metabolismo , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismoRESUMEN
Stroke is one of the leading causes of death and permanent disability in the elderly. However, most of the experimental studies on stroke are based on young animals, and we hypothesised that age can substantially affect the stroke response. The two-vessel occlusion model of global ischemia by occluding the common carotid arteries for 15 min at 40 mmHg of blood pressure was carried out in 3- and 18-month-old male Sprague-Dawley rats. The adhesion molecules E- and P-selectin, cell adhesion molecules (CAMs), both intercellular (ICAM-1) and vascular (VCAM-1), as well as glial fibrillary acidic protein (GFAP), and cleaved caspase-3 were measured at 48 h after ischemia in the cerebral cortex and hippocampus using Western blot, qPCR and immunofluorescence techniques. Diametric expression of GFAP and a different morphological pattern of caspase-3 labelling, although no changes in the cell number, were observed in the neurons of young and old animals. Expression of E-selectin and CAMs was also modified in an age- and ischemia/reperfusion-dependent manner. The hippocampus and cerebral cortex had similar response patterns for most of the markers studied. Our data suggest that old and young animals present different time-courses of neuroinflammation and apoptosis after ischemic damage. On the other hand, these results suggest that neuroinflammation is dependent on age rather than on the different vulnerability described for the hippocampus and cerebral cortex. These differences should be taken into account in searching for therapeutic targets.
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Envejecimiento/metabolismo , Isquemia Encefálica/metabolismo , Selectina E/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Reperfusión/métodos , Molécula 1 de Adhesión Celular Vascular/genética , Envejecimiento/genética , Envejecimiento/patología , Animales , Apoptosis , Western Blotting , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Modelos Animales de Enfermedad , Selectina E/biosíntesis , Proteína Ácida Fibrilar de la Glía , Masculino , Proteínas del Tejido Nervioso/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
Assays on "ex vivo" sections of rat hippocampus and rat cerebral cortex, subjected to oxygen and glucose deprivation (OGD) and a three-hour reperfusion-like (RL) recovery, were performed in the presence of either GABA or the GABA(A) receptor binding site antagonist, bicuculline. Lactate dehydrogenase (LDH) and propidium iodide were used to quantify cell mortality. We also measured, using real-time quantitative polymerase chain reaction (qPCR), the early transcriptional response of a number of genes of the glutamatergic and GABAergic systems. Specifically, glial pre- and post-synaptic glutamatergic transporters (namely GLAST1a, EAAC-1, GLT-1 and VGLUT1), three GABAA receptor subunits (α1, ß2 and γ2), and the GABAergic presynaptic marker, glutamic acid decarboxylase (GAD65), were studied. Mortality assays revealed that GABAA receptor chloride channels play an important role in the neuroprotective effect of GABA in the cerebral cortex, but have a much smaller effect in the hippocampus. We also found that GABA reverses the OGD-dependent decrease in GABA(A) receptor transcript levels, as well as mRNA levels of the membrane and vesicular glutamate transporter genes. Based on the markers used, we conclude that OGD results in differential responses in the GABAergic presynaptic and postsynaptic systems.