RESUMEN
Hypertension (HTN), a disease afflicting over one billion individuals worldwide, is a leading cause of cognitive impairment, the mechanisms of which remain poorly understood. In the present study, in a mouse model of HTN, we find that the neurovascular and cognitive dysfunction depends on interleukin (IL)-17, a cytokine elevated in individuals with HTN. However, neither circulating IL-17 nor brain angiotensin signaling can account for the dysfunction. Rather, IL-17 produced by T cells in the dura mater is the mediator released in the cerebrospinal fluid and activating IL-17 receptors on border-associated macrophages (BAMs). Accordingly, depleting BAMs, deleting IL-17 receptor A in brain macrophages or suppressing meningeal T cells rescues cognitive function without attenuating blood pressure elevation, circulating IL-17 or brain angiotensin signaling. Our data unveil a critical role of meningeal T cells and macrophage IL-17 signaling in the neurovascular and cognitive dysfunction in a mouse model of HTN.
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Disfunción Cognitiva , Hipertensión , Ratones , Animales , Interleucina-17 , Angiotensina II , Linfocitos T , Cloruro de Sodio DietéticoRESUMEN
Stroke is a leading cause of mortality and disability. It occurs when cerebral blood flow is disrupted via vascular occlusion or rupture, causing tissue damage. Research has extensively examined the role of the immune response in stroke pathophysiology, focusing on infiltrated immune cells and inflammatory molecules. However, the stroke's impact on immune physiology remains underexplored. While initially stroke triggers the activation of peripheral inflammation, a subsequent profound immunosuppression occurs in a matter of hours/days. This response, potentially shielding the brain from excessive inflammation, significantly affects stroke patients. Beyond rendering patients more susceptible to infections, immunosuppression generates diverse consequences by disrupting immune system functions that are crucial for organ homeostasis. This review explores the effects of immunosuppression on stroke patients, shedding light on potential issues in immune organs such as the spleen and bone marrow, as well as non-immune organs like the small intestine, liver and heart. By synthesizing existing literature and offering additional insights, this manuscript highlights the multifaceted impact of post-stroke immunosuppression.
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Accidente Cerebrovascular , Humanos , Encéfalo , Terapia de Inmunosupresión , Sistema Inmunológico , InflamaciónRESUMEN
In order to understand the preferred modes of chelation in metal-binding peptides, quantum mechanical calculations can be used to compute energies, resulting in a hierarchy of binding affinities. These calculations often produce increasing stabilization energies the higher the coordination of the complex. However, as the coordination of a metal increases, the conformational freedom of the polypeptide chain is inevitably reduced, resulting in an entropic penalty. Estimating the magnitude of this penalty from the many different degrees of freedom of biomolecular systems is very challenging, and as a result this contribution to the free energy is often ignored. Here we explore this problem focusing on a family of phosphorylated neuropeptides that bind to aluminum. We find that there is a general negative correlation between both stabilization energy and entropy. Our results suggest that a subtle interplay between enthalpic and entropic forces will determine the population of the most favourable species. Additionally, we discuss the requirements for a possible "Metal Ion Hypothesis" based on our findings.
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Aging is associated to progressive changes impairing fundamental cellular and tissue functions, and the relationships amongst them through the vascular and immune systems. Aging factors are key to understanding the pathophysiology of stroke since they increase its risk and worsen its functional outcome. Most currently recognised hallmarks of aging are also involved in the cerebral responses to stroke. Notably, age-associated chronic low-grade inflammation is related to innate immune responses highlighted by induction of type-I interferon. The interferon program is prominent in microglia where it interrelates cell damage, danger signals, and phagocytosis with immunometabolic disturbances and inflammation. Microglia engulfment of damaged myelin and cell debris may overwhelm the cellular capacity for waste removal inducing intracellular lipid accumulation. Acute inflammation and interferon-stimulated gene expression are also typical features of acute stroke, where danger signal recognition by microglia trigger immunometabolic alterations underscored by lipid droplet biogenesis. Aging reduces the capacity to control these responses causing increased and persistent inflammation, metabolic dysregulation, and impaired cellular waste disposal. In turn, chronic peripheral inflammation during aging induces immunosenescence further worsening stroke-induced immunodepression, thus increasing the risk of post-stroke infection. Aging also alters gut microbiota composition inducing dysbiosis. These changes are enhanced by age-related diseases, such as atherosclerosis and type-II diabetes, that further promote vascular aging, predispose to stroke, and exacerbate brain inflammation after stroke. Current advances in aging research suggest that some age-associated alterations may be reversed. Future work will unravel whether such evolving anti-aging research may enable designing strategies to improve stroke outcome in the elderly.
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Accidente Cerebrovascular , Humanos , Anciano , Accidente Cerebrovascular/etiología , Inmunidad Innata , Envejecimiento/metabolismo , Inflamación/etiología , InterferonesRESUMEN
Recruitment of stem cells is crucial for tissue repair. Although stem cell niches can provide important signals, little is known about mechanisms that coordinate the engagement of disseminated stem cells across an injured tissue. In Drosophila, adult brain lesions trigger local recruitment of scattered dormant neural stem cells suggesting a mechanism for creating a transient stem cell activation zone. Here, we find that injury triggers a coordinated response in neuro-glial clusters that promotes the spread of a neuron-derived stem cell factor via glial secretion of the lipocalin-like transporter Swim. Strikingly, swim is induced in a Hif1-α-dependent manner in response to brain hypoxia. Mammalian Swim (Lcn7) is also upregulated in glia of the mouse hippocampus upon brain injury. Our results identify a central role of neuro-glial clusters in promoting neural stem cell activation at a distance, suggesting a conserved function of the HIF1-α/Swim/Wnt module in connecting injury-sensing and regenerative outcomes.
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Drosophila , Células-Madre Neurales , Animales , Mamíferos , Ratones , Neuroglía , Neuronas , Nicho de Células MadreRESUMEN
Stroke is an acute neurological disease with a strong inflammatory component that can be regulated by the intestinal microbiota and intestinal immune cells. Although stroke has been shown to alter immune cell populations in the gut, the dynamics of cell trafficking have not been elucidated. To study the trafficking of gut-derived immune cells after stroke, we used mice expressing the photoconvertible protein Kikume Green-Red, which turns form green to red when exposed to violet light. Mice underwent laparotomy and the small intestine was exposed to violet laser light. Immune cells were isolated from the small intestine immediately after photoconversion and 2 days later. Percentage of immune cells (CD45+KikR+) that expressed the red variant of the protein (KikR) was higher immediately after photoconversion than 2 days later, indicating cell egress from the small intestine. To investigate whether intestinal immune cells traffic to the periphery and/or the central nervous system (CNS) after stroke, we analyzed KikR+ immune cells (2 days after photoconversion) in peripheral lymphoid organs, meninges and brain, 3 and 14 days after transient occlusion of the middle cerebral artery (tMCAo) or sham-surgery. Although migration was observed in naïve and sham animals, stroke induced a higher mobilization of gut KikR+ immune cells, especially at 3 days after stroke, to all the organs analyzed. Notably, we detected a significant migration of CD45hi immune cells from the gut to the brain and meninges at 3 days after stroke. Comparison of cell trafficking between organs revealed a significant preference of intestinal CD11c+ cells to migrate from the small intestine to brain and meninges after stroke. We conclude that stroke increases immune cell trafficking from the small intestine to peripheral lymphoid organs and the CNS where they might contribute to post-stroke inflammation.
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Isquemia Encefálica , Accidente Cerebrovascular , Animales , Encéfalo , Inflamación , Intestino Delgado , Ratones , Ratones Endogámicos C57BLRESUMEN
Background and Purpose- Commensal gut bacteria have a profound impact on stroke pathophysiology. Here, we investigated whether modification of the microbiota influences acute and long-term outcome in mice subjected to stroke. Methods- C57BL/6 male mice received a cocktail of antibiotics or single antibiotic. After 4 weeks, fecal bacterial density of the 16S rRNA gene was quantitated by qPCR, and phylogenetic classification was obtained by 16S rRNA gene sequencing. Infarct volume and hemispheric volume loss were measured 3 days and 5 weeks after middle cerebral artery occlusion, respectively. Neurological deficits were tested by the Tape Test and the open field test. Results- Mice treated with a cocktail of antibiotics displayed a significant reduction of the infarct volume in the acute phase of stroke. The neuroprotective effect was abolished in mice recolonized with a wild-type microbiota. Single antibiotic treatment with either ampicillin or vancomycin, but not neomycin, was sufficient to reduce the infarct volume and improved motorsensory function 3 days after stroke. This neuroprotective effect was correlated with a specific microbial population rather than the total bacterial density. In particular, random forest analysis trained for the severity of the brain damage revealed that Bacteroidetes S24.7 and the enzymatic pathway for aromatic metabolism discriminate between large versus small infarct size. Additionally, the microbiota signature in the ampicillin-treated mice was associated with a reduced gut inflammation, long-term favorable outcome shown by an amelioration of the stereotypic behavior, and a reduction of brain tissue loss in comparison to control and was predictive of a regulation of short-chain fatty acids and tryptophan pathways. Conclusions- The findings highlight the importance of the intestinal microbiota in short- and long-term outcomes of ischemic stroke and raises the possibility that targeted modification of the microbiome associated with specific microbial enzymatic pathways may provide a preventive strategy in patients at high risk for stroke. Visual Overview- An online visual overview is available for this article.
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Bacterias/crecimiento & desarrollo , Isquemia Encefálica , Microbioma Gastrointestinal , Enfermedad Aguda , Animales , Bacterias/clasificación , Bacterias/genética , Isquemia Encefálica/microbiología , Isquemia Encefálica/prevención & control , Masculino , Ratones , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Accidente Cerebrovascular/microbiología , Accidente Cerebrovascular/prevención & controlRESUMEN
Stroke is a devastating disease with a strong inflammatory component. It has been shown that part of this response is mediated by IL17+ γδT cells. γδT cells constitute a lymphocyte population with innate features that mainly populates epithelial surfaces including skin, intestine, and airways. We have shown that in the context of stroke, T cells migrate from the small intestine to the meninges but whether they can migrate from other epithelial surfaces is still unknown. Because of its proximity, one possible source of stroke-associated IL17+ γδT cells could be the Nasal-Associated Lymphoid Tissue (NALT) from which T cells could migrate along olfactory nerve sheaths through the cribriform plate into the brain and/or meninges. In order to study the role of NALT as a source for immune cells and/or inflammatory mediators in the context of stroke, we analyzed the effect of NALT ablation on immune cell infiltration and infarct volume after stroke. Infarct volume analysis did not show any significant difference between sham and NALT-ablated animals. In addition, no significant differences were found in immune cell infiltration in the brain or meninges of stroke animals subjected to NALT or Sham-ablation surgery. In conclusion, NALT ablation does not affect ischemic brain damage or immune cell infiltration in the meninges or brain after stroke.
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Infarto de la Arteria Cerebral Media/patología , Tejido Linfoide/cirugía , Mucosa Nasal/cirugía , Animales , Encéfalo/inmunología , Encéfalo/patología , Infarto de la Arteria Cerebral Media/veterinaria , Mediadores de Inflamación/metabolismo , Interleucina-17/metabolismo , Linfocitos Intraepiteliales/citología , Linfocitos Intraepiteliales/metabolismo , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/metabolismo , Tejido Linfoide/inmunología , Masculino , Meninges/inmunología , Meninges/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infiltración NeutrófilaRESUMEN
Exposure to low-dose lipopolysaccharide (LPS) before cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning (PC). Although it is well established that LPS-PC induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by PC and tested whether monocytes may be reprogrammed by ex vivo LPS exposure, thus modulating inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose LPS induces a Ly6Chi monocyte response that protects the brain after transient middle cerebral artery occlusion (MCAO) in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 h after transient MCAO reduced brain injury. Gene expression and functional studies showed that IL-10, inducible nitric oxide synthase, and CCR2 in monocytes are essential for neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to LPS and then injected into male mice after stroke. Cell-tracking studies showed that protective monocytes are mobilized from the spleen and reach the brain and meninges, where they suppress postischemic inflammation and neutrophil influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable of protecting the brain with an extended therapeutic window and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes in patients after ischemic stroke.SIGNIFICANCE STATEMENT Inflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using lipopolysaccharide (LPS) preconditioning (PC) as an approach to induce ischemic tolerance in mice, we found generation of neuroprotective monocytes within the spleen, from which they traffic to the brain and meninges, suppressing postischemic inflammation. Importantly, systemic LPS-PC can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of BM mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.
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Precondicionamiento Isquémico/métodos , Lipopolisacáridos/farmacología , Monocitos , Neuroprotección/inmunología , Accidente Cerebrovascular , Traslado Adoptivo , Animales , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/trasplante , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/patologíaRESUMEN
A diet rich in salt is linked to an increased risk of cerebrovascular diseases and dementia, but it remains unclear how dietary salt harms the brain. We report that, in mice, excess dietary salt suppresses resting cerebral blood flow and endothelial function, leading to cognitive impairment. The effect depends on expansion of TH17 cells in the small intestine, resulting in a marked increase in plasma interleukin-17 (IL-17). Circulating IL-17, in turn, promotes endothelial dysfunction and cognitive impairment by the Rho kinase-dependent inhibitory phosphorylation of endothelial nitric oxide synthase and reduced nitric oxide production in cerebral endothelial cells. The findings reveal a new gut-brain axis linking dietary habits to cognitive impairment through a gut-initiated adaptive immune response compromising brain function via circulating IL-17. Thus, the TH17 cell-IL-17 pathway is a putative target to counter the deleterious brain effects induced by dietary salt and other diseases associated with TH17 polarization.
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Trastornos Cerebrovasculares/inducido químicamente , Trastornos del Conocimiento/inducido químicamente , Intestino Delgado/patología , Cloruro de Sodio Dietético/toxicidad , Células Th17/efectos de los fármacos , Acetilcolina/farmacología , Amidas/farmacología , Animales , Antihipertensivos/farmacología , Diferenciación Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Trastornos Cerebrovasculares/tratamiento farmacológico , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Interleucina-17/administración & dosificación , Interleucina-17/sangre , Interleucina-17/genética , Interleucina-17/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Acoplamiento Neurovascular/efectos de los fármacos , Fosforilación/efectos de los fármacos , Piridinas/farmacologíaRESUMEN
Experimental reproducibility between laboratories is a major translational obstacle worldwide, particularly in studies investigating immunomodulatory therapies in relation to brain disease. In recent years increasing attention has been drawn towards the gut microbiota as a key factor in immune cell polarization. Moreover, manipulation of the gut microbiota has been found effective in a diverse range of brain disorders. Within this study we aimed to test the impact of microbiota differences between mice from different sources on the post-stroke neuroinflammatory response. With this rationale, we have investigated the correlation between microbiota differences and the immune response in mice from three commercial breeders with the same genetic background (C57BL/6). While overall bacterial load was comparable, we detected substantial differences in species diversity and microbiota composition on lower taxonomic levels. Specifically, we investigated segmented filamentous bacteria (SFB)-which have been shown to promote T cell polarization-and found that they were absent in mice from one breeder but abundant in others. Our experiments revealed a breeder specific correlation between SFB presence and the ratio of Treg to Th17 cells. Moreover, recolonization of SFB-negative mice with SFB resulted in a T cell shift which mimicked the ratios found in SFB-positive mice. We then investigated the response to a known experimental immunotherapeutic approach, CD28 superagonist (CD28SA), which has been previously shown to expand the Treg population. CD28SA treatment had differing effects between mice from different breeders and was found to be ineffective at inducing Treg expansion in SFB-free mice. These changes directly corresponded to stroke outcome as mice lacking SFB had significantly larger brain infarcts. This study demonstrates the major impact of microbiota differences on T cell polarization in mice during ischemic stroke conditions, and following immunomodulatory therapies.
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Microbioma Gastrointestinal , Accidente Cerebrovascular/inmunología , Linfocitos T/fisiología , Animales , Antígenos CD28/administración & dosificación , Antígenos CD28/inmunología , Encefalitis/inmunología , Encefalitis/metabolismo , Femenino , Inmunoterapia , Activación de Linfocitos , Masculino , Ratones Endogámicos C57BL , Reproducibilidad de los Resultados , Accidente Cerebrovascular/terapiaRESUMEN
BACKGROUND: A key feature of the inflammatory response after cerebral ischemia is the brain infiltration of blood monocytes. There are two main monocyte subsets in the mouse blood: CCR2+Ly6Chi "inflammatory" monocytes involved in acute inflammation, and CX3CR1+Ly6Clo "patrolling" monocytes, which may play a role in repair processes. We hypothesized that CCR2+Ly6Chi inflammatory monocytes are recruited in the early phase after ischemia and transdifferentiate into CX3CR1+Ly6Clo "repair" macrophages in the brain. METHODS: CX3CR1GFP/+CCR2RFP/+ bone marrow (BM) chimeric mice underwent transient middle cerebral artery occlusion (MCAo). Mice were sacrificed from 1 to 28 days later to phenotype and map subsets of infiltrating monocytes/macrophages (Mo/MΦ) in the brain over time. Flow cytometry analysis 3 and 14 days after MCAo in CCR2-/- mice, which exhibit deficient monocyte recruitment after inflammation, and NR4A1-/- BM chimeric mice, which lack circulating CX3CR1+Ly6Clo monocytes, was also performed. RESULTS: Brain mapping of CX3CR1GFP/+ and CCR2RFP/+ cells 3 days after MCAo showed absence of CX3CR1GFP/+ Mo/MΦ but accumulation of CCR2RFP/+ Mo/MΦ throughout the ischemic territory. On the other hand, CX3CR1+ cells accumulated 14 days after MCAo at the border of the infarct core where CCR2RFP/+ accrued. Whereas the amoeboid morphology of CCR2RFP/+ Mo/MΦ remained unchanged over time, CX3CR1GFP/+ cells exhibited three distinct phenotypes: amoeboid cells with retracted processes, ramified cells, and perivascular elongated cells. CX3CR1GFP/+ cells were positive for the Mo/MΦ marker Iba1 and phenotypically distinct from endothelial cells, smooth muscle cells, pericytes, neurons, astrocytes, or oligodendrocytes. Because accumulation of CX3CR1+Ly6Clo Mo/MΦ was absent in the brains of CCR2 deficient mice, which exhibit deficiency in CCR2+Ly6Chi Mo/MΦ recruitment, but not in NR4A1-/- chimeric mice, which lack of circulating CX3CR1+Ly6Clo monocytes, our data suggest a local transition of CCR2+Ly6Chi Mo/MΦ into CX3CR1+Ly6Clo Mo/MΦ phenotype. CONCLUSIONS: CX3CR1+Ly6Clo arise in the brain parenchyma from CCR2+Ly6Chi Mo/MΦ rather than being de novo recruited from the blood. These findings provide new insights into the trafficking and phenotypic diversity of monocyte subtypes in the post-ischemic brain.
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Encéfalo/patología , Movimiento Celular/fisiología , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Monocitos/fisiología , Animales , Proteínas de Unión al Calcio/metabolismo , Movimiento Celular/genética , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Regulación de la Expresión Génica/fisiología , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Infarto de la Arteria Cerebral Media/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismoRESUMEN
Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17-positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal IL-17(+) γδ T cells on ischemic injury.
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Encéfalo/inmunología , Células Dendríticas/inmunología , Disbiosis/inmunología , Microbioma Gastrointestinal/inmunología , Infarto de la Arteria Cerebral Media/inmunología , Intestinos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Animales , Antibacterianos/farmacología , Conducta Animal , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatología , Isquemia Encefálica/inmunología , Isquemia Encefálica/microbiología , Isquemia Encefálica/fisiopatología , Disbiosis/microbiología , Trasplante de Microbiota Fecal , Citometría de Flujo , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Inmunidad Mucosa/inmunología , Inmunohistoquímica , Infarto de la Arteria Cerebral Media/microbiología , Infarto de la Arteria Cerebral Media/fisiopatología , Interleucina-10/inmunología , Interleucina-17/inmunología , Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Intestino Delgado/microbiología , Intestinos/microbiología , Leucocitos/inmunología , Linfocitos/inmunología , Ratones , ARN Mensajero/metabolismo , ARN Ribosómico 16S/genética , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/microbiología , Accidente Cerebrovascular/fisiopatología , Linfocitos T Reguladores/inmunologíaRESUMEN
In this work, we report our study of protein expression in rat peri-infarct tissue, 48 h after the induction of permanent focal cerebral ischemia. Two proteomic approaches, gel electrophoresis with mass spectrometry and combined fractional diagonal chromatography (COFRADIC), were performed using tissue samples from the periphery of the induced cerebral ischemic lesions, using tissue from the contra-lateral hemisphere as a control. Several protein spots (3408) were identified by gel electrophoresis, and 11 showed significant differences in expression between peri-infarct and contra-lateral tissues (at least 3-fold, p < 0.05). Using COFRADIC, 5412 proteins were identified, with 72 showing a difference in expression. Apart from blood-related proteins (such as serum albumin), both techniques showed that the 70 kDa family of heat shock proteins were highly expressed in the peri-infarct tissue. Further studies by 1D and 2D western blotting and immunohistochemistry revealed that only one member of this family (the inducible form, HSP72 or HSP70i) is specifically expressed by the peri-infarct tissue, while the majority of this family (the constitutive form, HSC70 or HSP70c) is expressed in the whole brain. Our data support that HSP72 is a suitable biomarker of peri-infarct tissue in the ischemic brain.
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Isquemia Encefálica/patología , Animales , Western Blotting , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Modelos Animales de Enfermedad , Electroforesis en Gel Bidimensional , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas del Choque Térmico HSP72/metabolismo , Inmunohistoquímica , Masculino , Espectrometría de Masas , Proteómica , Ratas , Ratas Sprague-DawleyRESUMEN
Brain ischaemia (stroke) triggers an intense inflammatory response predominately mediated by the accumulation of inflammatory cells and mediators in the ischaemic brain. In this context, regulatory T (Treg) cells, a subpopulation of CD4(+) T cells with immunosuppressive and anti-inflammatory properties, are activated in the late stages of the disease. To date, the potential therapeutic usefulness of Treg cells has not been tested. In this study, we aimed to investigate whether Treg cells exert protection/repair following stroke. Both the adoptive transfer of Treg cells into ischaemic rats and the stimulation of endogenous T-cell proliferation using a CD28 superagonist reduced the infarct size at 3-28 days following the ischaemic insult. Moreover, T cell-treated animals had higher levels of FoxP3 and lower levels of IL-1ß, CD11b+ and CD68+ cells in the infarcted hemisphere when compared with control animals. However, T-cell treatment did not alter the rate of proliferation of NeuN-, NCAM- or CD31-positive cells, thereby ruling out neurogenesis and angiogenesis in protection. These results suggest that adoptive transfer of T cells is a promising therapeutic strategy against the neurological consequences of stroke.
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Isquemia Encefálica/prevención & control , Infarto de la Arteria Cerebral Media/prevención & control , Inflamación/inmunología , Neovascularización Patológica/inmunología , Células-Madre Neurales/inmunología , Accidente Cerebrovascular/prevención & control , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Western Blotting , Isquemia Encefálica/etiología , Isquemia Encefálica/inmunología , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnicas para Inmunoenzimas , Inmunosupresores , Infarto de la Arteria Cerebral Media/etiología , Infarto de la Arteria Cerebral Media/inmunología , Inflamación/patología , Activación de Linfocitos , Imagen por Resonancia Magnética , Masculino , Células-Madre Neurales/patología , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
In order to provide sufficient sensibility for detection, selection of an adequate payload of imaging probe is critical, during the design of MRI theranostic nanoplatforms. This fact is particularly crucial for in vivo applications in the brain, where delivery of macromolecules is limited by the blood-brain barrier. Here we report a simple and quick process for the estimation of adequate payloads of gadolinium in liposomes with potential to act as theranostic agents, for in vivo MRI applications in the brain. Our studies show that an excessive payload of gadolinium in liposomes may actually have a negative influence on in vivo T1 contrast. By preparing and characterizing 4 different liposomal compositions of increasing Gadolinium loads, we show that a superior sensitivity for in vivo detection of MRI theranostic molecules can be quickly improved by adjusting the payload of imaging probe in the molecules. FROM THE CLINICAL EDITOR: This team of authors report the development of a simple and quick process for the estimation of adequate payloads of gadolinium in liposomes as theranostic agents for in vivo brain MRI studies, using a rodent model.
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Encéfalo/diagnóstico por imagen , Medios de Contraste/farmacología , Gadolinio/farmacología , Imagen por Resonancia Magnética/métodos , Nanopartículas , Animales , Medios de Contraste/química , Gadolinio/química , Liposomas , Masculino , Radiografía , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Clinical-Diffusion mismatch (CDM; NIHSS score ≥8 & DWI lesion volume ≤25 mL) and Perfusion-Diffusion mismatch (PDM; difference >20% between initial DWI and MTT lesion volumes) have been proposed as surrogates for ischemic brains that are at risk of infarction. However, their utility to improve the selection of patients for thrombolytic treatment remains controversial. Our aim was to identify molecular biomarkers that can be used with neuroimaging to facilitate the selection of ischemic stroke patients for systemic thrombolysis. METHODS: We prospectively studied 595 patients with ischemic stroke within 12 h of the stroke onset. A total of 184 patients received thrombolytic treatment according to the SITS-MOST criteria. DWI and MTT volumes were measured at admission. The main outcome variable was good functional outcome at 3 months (modified Rankin scale <3). Serum levels of glutamate (Glu), IL-10, TNF-α, IL-6, NSE, and active MMP-9 also were determined at admission. RESULTS: Patients treated with t-PA who presented with PDM had higher IL-10 levels at admission (p < 0.0001). In contrast, patients with CDM had higher levels of IL-10 (p < 0.0001) as well as Glu and TNF-α (all p < 0.05) and lower levels of NSE and active MMP-9 (all p < 0.0001). IL-10 ≥ 30 pg/mL predicts good functional outcome at 3 months with a specificity of 88% and a sensitibity of 86%. IL-10 levels ≥30 pg/mL independently in both patients with PDM (OR, 18.9) and CDM (OR, 7.5), after an adjustment for covariates. CONCLUSIONS: Serum levels of IL-10 facilitate the selection of ischemic stroke patients with CDM and PDM for systemic thrombolysis.
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Isquemia Encefálica , Imagen de Difusión por Resonancia Magnética/métodos , Fibrinolíticos/uso terapéutico , Interleucina-10/sangre , Angiografía por Resonancia Magnética/métodos , Accidente Cerebrovascular , Terapia Trombolítica/normas , Activador de Tejido Plasminógeno/uso terapéutico , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Isquemia Encefálica/sangre , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patología , Terapia Trombolítica/métodosRESUMEN
BACKGROUND: Sevoflurane is an anesthetic agent which also participates in protective mechanisms in sepsis, likely due to anti-inflammatory properties. A key tissue in sepsis is the endothelium, which expresses TLR2 and TLR4 receptors, known regulators of inflammatory mechanisms and potential therapeutic targets for this pathology. In this context, we explored the effect of sevoflurane postconditioning in an in vitro sepsis model. METHODS: Primary cultures of human umbilical vein endothelial cells were used for two different experiments. In the first set, cultures were placed in an airtight incubation chamber and exposed to different concentrations of sevoflurane (0,1,3 or 7% vol,) for 1 hour. In the second set, lipopolysaccharide from Escherichia coli 0111:B4 (1 µg/mL) was added to culture medium for 3 hours and cells were subsequently exposed to sevoflurane (0,1,3 or 7% vol,) for 1 hour as explained before. In both cases, cell viability was measured by MTT and Trypan blue assays, TLR2 and TLR4 expression were analyzed by flow cytometry, and TNFα and IL-6 levels were quantified in cell culture media by an immunoassay immediately after exposure, at 6 and 24 hours. RESULTS: Exposure to 3% sevoflurane decreased TLR2 at 24 hours and TLR4 at 6 and 24 hours (both p<0.05), whereas exposure to 7% decreased TLR4 expression at 6 hours (p<0.05). Both 3 and 7% sevoflurane decreased TNF-α and IL-6 levels at 24 hours (both p<0.05). In LPS-stimulated cultures, exposure to 3% sevoflurane was cytoprotective at 6 and 24 hours (p<0.05) compared with control, and decreased TLR2 and TLR4 expression at 24 hours (p<0.05); whereas 7% decreased TLR4 expression at 24 hours (p<0.05). Both 3% and 7% sevoflurane decreased TNF-α and IL-6 levels at 24 hours (both p<0.05). CONCLUSIONS: Postconditioning with the halogenated anesthetic agent sevoflurane after LPS stimulation shows a cytoprotective effect in an in vitro model, decreasing cell death and reducing TLR2 and TLR4 expression as well as levels of the inflammatory mediators TNF-α and IL-6 in human endothelial cells.
Asunto(s)
Muerte Celular , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Inflamación/metabolismo , Éteres Metílicos/farmacología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Supervivencia Celular , Medios de Cultivo/farmacología , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Sepsis/metabolismo , Sevoflurano , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The use of theranostics in neurosciences has been rare to date because of the limitations imposed on the free delivery of substances to the brain by the blood-brain barrier. Here we report the development of a theranostic system for the treatment of stroke, a leading cause of death and disability in developed countries. We first performed a series of proteomic, immunoblotting and immunohistological studies to characterize the expression of molecular biomarkers for the so-called peri-infarct tissue, a key region of the brain for stroke treatment. We confirmed that the HSP72 protein is a suitable biomarker for the peri-infarct region, as it is selectively expressed by at-risk tissue for up to 7 days following cerebral ischemia. We also describe the development of anti-HSP72 vectorized stealth immunoliposomes containing imaging probes to make them traceable by conventional imaging techniques (fluorescence and MRI) that were used to encapsulate a therapeutic agent (citicoline) for the treatment of cerebral ischemia. We tested the molecular recognition capabilities of these nano-platforms in vitro together with their diagnostic and therapeutic properties in vivo, in an animal model of cerebral ischemia. Using MRI, we found that 80% of vectorized liposomes were located on the periphery of the ischemic lesion, and animals treated with citicoline encapsulated on these liposomes presented lesion volumes up to 30% smaller than animals treated with free (non-encapsulated) drugs. Our results show the potential of nanotechnology for the development of effective tools for the treatment of neurological diseases.