Estudio del papel de TLR4 en la transformación hemorrágica e infiltración aguda tras ictus experimental

En los países occidentales, el ictus es la segunda causa de muerte (primera causa entre mujeres en España), la segunda causa de demencia, y la principal causa de discapacidad grave entre los adultos. A pesar de estos datos, las únicas aproximaciones terapéuticas consisten en la recanalización de las arterias obstruidas mediante el tratamiento fibrinolítico a través del activador del plasminógeno tisular (t-PA) (sólo útil entre el 5-10% de los pacientes), o la trombectomía endovascular (Goyal et al., 2016; Hacke et al., 1995). Por ello, es necesario el desarrollo de nuevas terapias neuroprotectoras para el tratamiento en fase aguda de esta patología. Por un lado, el tratamiento con t-PA es utilizado en condiciones muy restrictivas para evitar el fenómeno de transformación hemorrágica (TH), que es la principal complicación de la trombólisis. Dado que la TH, también es una grave complicación después de la trombectomía mecánica (Goyal et al., 2016) se hace necesario la investigación en los mecanismos de TH y en las terapias potenciales que podrían reducir el riesgo de sufrirla, mejorando el pronóstico de los pacientes con esta enfermedad. Además, la aparición de un ictus isquémico, conlleva una cascada de eventos inflamatorios (cascada isquémica) que se inicia en la microvasculatura cerebral, donde el estrés oxidativo y las especies reactivas de oxígeno inducen la activación del sistema del complemento, las plaquetas y las células endoteliales (para revisión ver (Iadecola and Anrather, 2011)). A su vez, a nivel perivascular y del parénquima se produce una respuesta inmune innata que pone en marcha una fuerte respuesta inflamatoria. Uno de los principales tipos celulares responsables de la respuesta cerebral es la microglía, que participa en esta respuesta mediante la producción de mediadores inflamatorios, en gran medida gracias a la señalización intracelular que se pone en marcha tras la activación de los receptores toll-like (TLR)..

Neutrophil Extracellular Trap Targeting Protects Against Ischemic Damage After Fibrin-Rich Thrombotic Stroke Despite Non-Reperfusion.

Stroke is one of the most prevalent diseases worldwide caused primarily by a thrombotic vascular occlusion that leads to cell death. To date, t-PA (tissue-type plasminogen activator) is the only thrombolytic therapy approved which targets fibrin as the main component of ischemic stroke thrombi. However, due to its highly restrictive criteria, t-PA is only administrated to less than 10% of all stroke patients. Furthermore, the research in neuroprotective agents has been extensive with no translational results from medical research to clinical practice up to now. Since we first described the key role of NETs (Neutrophil Extracellular Traps) in platelet-rich thrombosis, we asked, first, whether NETs participate in fibrin-rich thrombosis and, second, if NETs modulation could prevent neurological damage after stroke. To this goal, we have used the thromboembolic in situ stroke model which produces fibrin-rich thrombotic occlusion, and the permanent occlusion of the middle cerebral artery by ligature. Our results demonstrate that NETs do not have a predominant role in fibrin-rich thrombosis and, therefore, DNase-I lacks lytic effects on fibrin-rich thrombosis. Importantly, we have also found that NETs exert a deleterious effect in the acute phase of stroke in a platelet-TLR4 dependent manner and, subsequently, that its pharmacological modulation has a neuroprotective effect. Therefore, our data strongly support that the pharmacological modulation of NETs in the acute phase of stroke, could be a promising strategy to repair the brain damage in ischemic disease, independently of the type of thrombosis involved.This work was supported by grants from Instituto de Salud Carlos III and co-financed by the European Development Regional Fund “A Way to Achieve Europe” PI20/00535 and RETICS RD16/0019/ 0009 (IL), from Regional Madrid Government B2017/BMD- 3688 (IL), from Spanish Ministry of Science and Innovation PID2019- 106581RB-I00 (MÁM), from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MÁM), from Fundación La Caixa HR17_00527 (MM). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG152/2 macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogensThis work was supported by grants from the Spanish Ministry of Health FIS2011-00127, Comunidad de Madrid UAM-CM-CCG10-4911 and UAM-Banco de Santander to SG. This work was also partly supported by NIAID grant U19AI083025 and by CRIP (Center for Research on Influenza Pathogenesis, HHSN266200700010C), a NIAID Center of Excellence for Influenza Research and Surveillance (CEIRS) to AGS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

The role of gut microbiota in cerebrovascular disease and related dementia.

In recent years, increasing evidence suggests that commensal microbiota may play an important role not only in health but also in disease including cerebrovascular disease. Gut microbes impact physiology, at least in part, by metabolizing dietary factors and host-derived substrates and then generating active compounds including toxins. The purpose of this current review is to highlight the complex interplay between microbiota, their metabolites. and essential functions for human health, ranging from regulation of the metabolism and the immune system to modulation of brain development and function. We discuss the role of gut dysbiosis in cerebrovascular disease, specifically in acute and chronic stroke phases, and the possible implication of intestinal microbiota in post-stroke cognitive impairment and dementia, and we identify potential therapeutic opportunities of targeting microbiota in this context.Instituto de Salud Carlos III (ISCIII), Grant/Award Number: PI20/00535; Leducq Foundation for Cardiovascular Research Grant/Award Numbers: TNE-21CVD04 (MAM, IL), TNE19CVD01 (MAM, MIC), TNE21CVD04, TNE19CVD01; Ministerio de Ciencia e Innovacion (MCIN), Grant/Award Numbers: PID2020-117765RB-I00, PID2019-106581RB-I00; RICORS-ICTUS, Grant/Award Number: RD21/0006/0001.N

Myeloid cells in vascular dementia and Alzheimer's disease: Possible therapeutic targets?

Growing evidence supports the suggestion that the peripheral immune system plays a role in different pathologies associated with cognitive impairment, such as vascular dementia (VD) or Alzheimer's disease (AD). The aim of this review is to summarize, within the peripheral immune system, the implications of different types of myeloid cells in AD and VD, with a special focus on post-stroke cognitive impairment and dementia (PSCID). We will review the contributions of the myeloid lineage, from peripheral cells (neutrophils, platelets, monocytes and monocyte-derived macrophages) to central nervous system (CNS)-associated cells (perivascular macrophages and microglia). Finally, we will evaluate different potential strategies for pharmacological modulation of pathological processes mediated by myeloid cell subsets, with an emphasis on neutrophils, their interaction with platelets and the process of immunothrombosis that triggers neutrophil-dependent capillary stall and hypoperfusion, as possible effector mechanisms that may pave the way to novel therapeutic avenues to stop dementia, the epidemic of our time.Spanish Ministry of Science and Innovation (MCIN), Grant/Award Number: PID2019-106581RB-I00; Leducq Foundation for Cardiovascular Research, Grant/Award Numbers: TNE19CVD01, TNE-21CVD04; Instituto de Salud Carlos III (ISCIII) and cofinanced by the European Development Regional Fund “A Way to Achieve Europe”, Grant/Award Numbers: PI20/00535, RICORSICTUSRD21/0006/0001; Severo Ochoa Center of Excellence, Grant/Award Number: CEX2020-001041-S.S

Caracterización de la neurogénesis maladaptativa y el deterioro cognitivo post-ictus en un modelo experimental de isquemia cerebral inducida mediante cloruro férrico en ratón

El ictus isquémico es considerado una de las principales causas de demencia y discapacidad en el mundo. La neurogénesis hipocampal que ocurre tras ictus en diversos modelos experimentales de la enfermedad se ha asociado previamente al deterioro cognitivo a largo plazo. Sin embargo, dada la alta heterogeneidad de la patología es necesario estudiar la respuesta neurogénica en nuevos modelos experimentales de ictus isquémico. El objetivo de este estudio fue caracterizar la respuesta neurogénica hipocampal adulta y su relación con el deterioro cognitivo post-ictus en un modelo experimental de isquemia cerebral permanente por cloruro férrico en ratón. Se llevó a cabo la evaluación de la lesión en fase aguda y crónica tras la isquemia a través de imagen por resonancia magnética. Adicionalmente, se evaluó la respuesta neurogénica hipocampal tras isquemia mediante inmunofluorescencia, así como el deterioro cognitivo mediante pruebas de comportamiento. Nuestros resultados muestran que el modelo de isquemia por cloruro férrico produce atrofia e hipoperfusión cortical y no afecta directamente al hipocampo. Además, el modelo de isquemia estudiado resulta en un aumento de la neurogénesis hipocampal tras la isquemia. Concretamente, demostramos un incremento de neuroblastos y neuronas inmaduras en la zona subgranular del giro dentado 7 días tras la isquemia. Asimismo, nuestros resultados muestran que este modelo resulta en un aumento del número de nuevas neuronas con morfología aberrante en ambos hemisferios del cerebro. Además, observamos que parte de los animales desarrollan déficit cognitivo a largo plazo. Los resultados preliminares presentados en este estudio sugieren que el ictus isquémico inducido por cloruro férrico es un modelo experimental adecuado para el estudio de la neurogénesis hipocampal adulta y el deterior cognitivo post-ictus

Defective hippocampal neurogenesis underlies cognitive impairment by carotid stenosis-induced cerebral hypoperfusion in mice.

Chronic cerebral hypoperfusion due to carotid artery stenosis is a major cause of vascular cognitive impairment and dementia (VCID). Bilateral carotid artery stenosis (BCAS) in rodents is a well-established model of VCID where most studies have focused on white matter pathology and subsequent cognitive deficit. Therefore, our aim was to study the implication of adult hippocampal neurogenesis in hypoperfusion-induced VCID in mice, and its relationship with cognitive hippocampal deficits. Mice were subjected to BCAS; 1 and 3 months later, hippocampal memory and neurogenesis/cell death were assessed, respectively, by the novel object location (NOL) and spontaneous alternation performance (SAP) tests and by immunohistology. Hypoperfusion was assessed by arterial spin labeling-magnetic resonance imaging (ASL-MRI). Hypoperfused mice displayed spatial memory deficits with decreased NOL recognition index. Along with the cognitive deficit, a reduced number of newborn neurons and their aberrant morphology indicated a remarkable impairment of the hippocampal neurogenesis. Both increased cell death in the subgranular zone (SGZ) and reduced neuroblast proliferation rate may account for newborn neurons number reduction. Our data demonstrate quantitative and qualitative impairment of adult hippocampal neurogenesis disturbances associated with cerebral hypoperfusion-cognitive deficits in mice. These findings pave the way for novel diagnostic and therapeutic targets for VCID.This work was supported by grants from Spanish Ministry of Science and Innovation (MCIN) PID2019-106581RB-I00 (MM), from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MM) and TNE-21CVD04 (MM and IL), and from Instituto de Salud Carlos III (ISCIII) and co-financed by the European Development Regional Fund “A Way to Achieve Europe” PI20/00535 and RICORS-ICTUS RD21/0006/0001 (IL). CNIC was supported by ISCIII, MCIN, and ProCNIC Foundation, and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). The microscopy experiments were performed in Unidad de Microscopía e Imagen Dinámica, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER “Una manera de hacer Europa” (#ICTS-2018- 04-CNIC-16). Part of the research work included in this publication has been carried out in the ReDIB ICTS infrastructure BioImaC, MCIN.S

Ipsilesional Hippocampal GABA Is Elevated and Correlates With Cognitive Impairment and Maladaptive Neurogenesis After Cortical Stroke in Mice.

BACKGROUND Cognitive dysfunction is a frequent stroke sequela, but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodeling has been proposed, but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. METHODS Stroke was induced by permanent occlusion of the middle cerebral artery occlusion in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analyzed longitudinally by in vivo magnetic resonance spectroscopy. Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts, interneurons, γ-aminobutyric acid (GABA), and c-fos were analyzed by immunofluorescence. RESULTS Approximately 50% of mice exhibited progressive post-middle cerebral artery occlusion cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the nonimpaired group. Using magnetic resonance spectroscopy, significant bilateral changes in hippocampal metabolites, such as myo-inositol or N-acetylaspartic acid, were found that correlated, respectively, with numbers of glia and immature neuroblasts in the ischemic group. Importantly, some metabolites were specifically altered in the ipsilateral hippocampus suggesting its involvement in aberrant hippocampal neurogenesis and remodeling processes. Specifically, middle cerebral artery occlusion animals with higher hippocampal GABA levels displayed worse cognitive outcome. Implication of GABA in this setting was supported by the amelioration of ischemia-induced memory deficits and aberrant hippocampal neurogenesis after blocking pharmacologically GABAergic neurotransmission, an intervention which was ineffective when neurogenesis was inhibited. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. CONCLUSIONS Hippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for poststroke cognitive impairment.S

Perspectiva de género en la asignatura de farmacología

En este proyecto de innovación docente hemos trabajo para comenzar a poner las bases de posibles diferencias que pueden existir en la farmacología de los medicamentos, atendiendo a si la persona que los toma es un hombre o una mujer. Con alumnos del grado de Nutrición Humana y Dietética y de Odontología hemos buscado bibliografía que nos demuestre si efectivamente se dan estas diferencias en la farmacocinética, en la farmacodinamia y en los efectos adversos. Los resultados encontrados nos han permitido presentar 2 comunicaciones en congresos y realizar un TFGDepto. de Farmacología y ToxicologíaFac. de MedicinaFALSEsubmitte

Role of TLR4 in Neutrophil Dynamics and Functions: Contribution to Stroke Pathophysiology.

BACKGROUND AND PURPOSE The immune response subsequent to an ischemic stroke is a crucial factor in its physiopathology and outcome. It is known that TLR4 is implicated in brain damage and inflammation after stroke and that TLR4 absence induces neutrophil reprogramming toward a protective phenotype in brain ischemia, but the mechanisms remain unknown. We therefore asked how the lack of TLR4 modifies neutrophil function and their contribution to the inflammatory process. METHODS In order to assess the role of the neutrophilic TLR4 after stroke, mice that do not express TLR4 in myeloid cells (TLR4loxP/Lyz-cre) and its respective controls (TLR4loxP/loxP) were used. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery and infarct size was measured by MRI. A combination of flow cytometry and confocal microscopy was used to assess different neutrophil characteristics (circadian fluctuation, cell surface markers, cell complexity) and functions (apoptosis, microglia engulfment, phagocytosis, NETosis, oxidative burst) in both genotypes. RESULTS As previously demonstrated, mice with TLR4 lacking-neutrophils had smaller infarct volumes than control mice. Our results show that the absence of TLR4 keeps neutrophils in a steady youth status that is dysregulated, at least in part, after an ischemic insult, preventing neutrophils from their normal circadian fluctuation. TLR4-lacking neutrophils showed a higher phagocytic activity in the basal state, they were preferentially engulfed by the microglia after stroke, and they produced less radical oxygen species (ROS) in the first stage of the inflammatory process. CONCLUSIONS TLR4 is specifically involved in neutrophil dynamics under physiological conditions as well as in stroke-induced tissue damage. This research contributes to the idea that TLR4, especially when targeted in specific cell types, is a potential target for neuroprotective strategies.This work was supported by grants from Instituto de Salud Carlos III and co-financed by the European Development Regional Fund “A Way to Achieve Europe” PI20/00535 and RETICS RD16/0019/ 0009 (IL), from Regional Madrid Government B2017/BMD- 3688 (IL), from Spanish Ministry of Science and Innovation PID2019- 106581RB-I00 (MM), from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MM), from Fundació n La Caixa HR17_00527 (MM). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation.S