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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., Depto. de Farmacología y Toxicología, Depto. de Biología Celular, Fac. de Medicina, Instituto Universitario de Investigación en Neuroquímica (IUIN), TRUE, pub, APC financiada por la UCM

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., Depto. de Biología Celular, Depto. de Farmacología y Toxicología, Fac. de Medicina, Instituto Universitario de Investigación en Neuroquímica (IUIN), TRUE, pub, APC financiada por la UCM

Background: Aging is not just a risk factor of stroke, but it has also been associated with poor recovery. It is known that stroke-induced neurogenesis is reduced but maintained in the aged brain. However, there is no consensus on how neurogenesis is affected after stroke in aged animals. Our objective is to determine the role of aging on the process of neurogenesis after stroke. Methods: We have studied neurogenesis by analyzing proliferation, migration, and formation of new neurons, as well as inflammatory parameters, in a model of cerebral ischemia induced by permanent occlusion of the middle cerebral artery in young- (2 to 3 months) and middle-aged mice (13 to 14 months). Results: Aging increased both microglial proliferation, as shown by a higher number of BrdU(+) cells and BrdU/Iba1(+) cells in the ischemic boundary and neutrophil infiltration. Interestingly, aging increased the number of M1 monocytes and N1 neutrophils, consistent with pro-inflammatory phenotypes when compared with the alternative M2 and N2 phenotypes. Aging also inhibited (subventricular zone) SVZ cell proliferation by decreasing both the number of astrocyte-like type-B (prominin-1(+)/epidermal growth factor receptor (EGFR)(+)/nestin(+)/glial fibrillary acidic protein (GFAP)(+) cells) and type-C cells (prominin-1(+)/EGFR(+)/nestin(-)/Mash1(+) cells), and not affecting apoptosis, 1 day after stroke. Aging also inhibited migration of neuroblasts (DCX(+) cells), as indicated by an accumulation of neuroblasts at migratory zones 14 days after injury; consistently, aged mice presented a smaller number of differentiated interneurons (NeuN(+)/BrdU(+) and GAD67(+) cells) in the peri-infarct cortical area 14 days after stroke. Conclusions: Our data confirm that stroke-induced neurogenesis is maintained but reduced in aged animals. Importantly, we now demonstrate that aging not only inhibits proliferation of specific SVZ cell subtypes but also blocks migration of neuroblasts to the damaged area and dec, Unión Europea, Ministerio de Ciencia e Innovación, Comunidad de Madrid, Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

Background and purpose: Hemorrhagic transformation is the main complication of revascularization therapies after stroke. Toll-like receptor 4 (TLR4) is implicated in cerebral damage and inflammation in stroke. This study was designed to determine the role of TLR4 in hemorrhagic transformation development after tissue plasminogen activator (tPA) administration. Methods: Mice expressing (TLR4+/+) or lacking functional TLR4 (TLR4-/-) were subjected to middle cerebral artery occlusion using an in situ thromboembolic model by thrombin injection into the middle cerebral artery, and tPA (10 mg/kg) was administered 20 minutes or 3 hours after ischemia. Infarct size, hemorrhages, IgG extravasation, matrix metalloproteinase 9 expression, and neutrophil infiltration were assessed 24 hours after ischemia. Results: In TLR4+/+, early reperfusion (tPA at 20 minutes) resulted infarct volume, whereas late recanalization (tPA at 3 hours) did not modify lesion size and increased the rate of the most severe hemorrhages. In TLR4-/- mice, both early and late reperfusion did not modify lesion size. Importantly, late tPA administration did not result in worse hemorrhages and in an increased bleeding area as occurred in TLR4+/+ group. In TLR4-/- animals, late reperfusion produced a lesser increase in matrix metalloproteinase 9 expression when compared with TLR4+/+ animals. Conclusions: Our results demonstrate TLR4 involvement in hemorrhagic transformation induced by delayed tPA administration, very likely by increasing matrix metalloproteinase 9 expression., Unión Europea, Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

This work was supported by the grants from Spanish Ministry of Science and Innovation, PID2019-106581RB-I00 (MM); Leducq Foundation for Cardiovascular Research, TNE-19CVD01 (MM); Fundación La Caixa, HR17_00527 (MM); 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); by contracts FJC-039343-I (AG-C) from the Spanish Ministry of Science and Innovation; and FPU01405265 (VM) and FPU19/02989 (EF) from the Spanish Ministry of Universities. 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)., The substantial clinical burden and disability after stroke injury urges the need to explore therapeutic solutions. Recent compelling evidence supports that neurogenesis persists in the adult mammalian brain and is amenable to regulation in both physiological and pathological situations. Its ability to generate new neurons implies a potential to contribute to recovery after brain injury. However, post-stroke neurogenic response may have different functional consequences. On the one hand, the capacity of newborn neurons to replenish the damaged tissue may be limited. In addition, aberrant forms of neurogenesis have been identified in several insult settings. All these data suggest that adult neurogenesis is at a crossroads between the physiological and the pathological regulation of the neurological function in the injured central nervous system (CNS). Given the complexity of the CNS together with its interaction with the periphery, we ultimately lack in-depth understanding of the key cell types, cell–cell interactions, and molecular pathways involved in the neurogenic response after brain damage and their positive or otherwise deleterious impact. Here we will review the evidence on the stroke-induced neurogenic response and on its potential repercussions on functional outcome. First, we will briefly describe subventricular zone (SVZ) neurogenesis after stroke beside the main evidence supporting its positive role on functional restoration after stroke. Then, we will focus on hippocampal subgranular zone (SGZ) neurogenesis due to the relevance of hippocampus in cognitive functions; we will outline compelling evidence that supports that, after stroke, SGZ neurogenesis may adopt a maladaptive plasticity response further contributing to the development of post-stroke cognitive impairment and dementia. Finally, we will discuss the therapeutic potential of specific steps in the neurogenic cascade that might ameliorate brain malfunctioning and the development of post-stroke, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2015-68632-R to MAM), the Instituto de Salud Carlos III (ISCIII) (FIS PI17/01601 to IL), and ISCIII cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa” RETICS (RD12/0014/0003 to IL), and the Canadian Institutes of Health Research (FDN143227to PWF)., Poststroke cognitive impairment is considered one of the main complications during the chronic phase of ischemic stroke. In the adult brain, the hippocampus regulates both encoding and retrieval of new information through adult neurogenesis. Nevertheless, the lack of predictive models and studies based on the forgetting processes hinders the understanding of memory alterations after stroke. Our aim was to explore whether poststroke neurogenesis participates in the development of long-term memory impairment. Here, we show a hippocampal neurogenesis burst that persisted 1 month after stroke and that correlated with an impaired contextual and spatial memory performance. Furthermore, we demonstrate that the enhancement of hippocampal neurogenesis after stroke by physical activity or memantine treatment weakened existing memories. More importantly, stroke-induced newborn neurons promoted an aberrant hippocampal circuitry remodeling with differential features at ipsi- and contralesional levels. Strikingly, inhibition of stroke-induced hippocampal neurogenesis by temozolomide treatment or using a genetic approach (Nestin-CreERT2/NSE-DTA mice) impeded the forgetting of old memories. These results suggest that hippocampal neurogenesis modulation could be considered as a potential approach for treatment of poststroke cognitive impairment., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

Stroke is a devastating disease with an increasing prevalence. Part of the current development in stroke therapy is focused in the chronic phase, where neurorepair mechanisms such as neurogenesis, are involved. In the adult brain, one of the regions where neurogenesis takes place is the subventricular zone (SVZ) of the lateral ventricles. Given the possibility to develop pharmacological therapies to stimulate this process, we have performed a longitudinal analysis of neurogenesis in a model of cortical ischemia in mice. Our results show an initial decrease of SVZ proliferation at 24 h, followed by a recovery leading to an increase at 14d and a second decrease 28d after stroke. Coinciding with the 24 h proliferation decrease, an increase in the eutopic neuroblast migration towards the olfactory bulb was observed. The analysis of the neuroblast ectopic migration from the SVZ toward the lesion showed an increase in this process from day 14 after the insult. Finally, our data revealed an increased number of new cortical neurons in the peri-infarct cortex 65d after the insult. In summary, we report here critical check-points about post-stroke neurogenesis after cortical infarcts, important for the pharmacological modulation of this process in stroke patients., Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

Background and Purpose- After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-like receptor 4) on neutrophil infiltration and polarization in this setting. Methods- Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice. Results- As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils (YM1+ cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage. Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma, and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets. Conclusions- TLR4 deficiency increased the levels of alternative neutrophils (N2)-an effect associated with neuroprotection after stroke-supporting that modulation of neutrophil polarization is a major target of, Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role., Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

Cognitive dysfunction is a frequent stroke sequela but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodelling has been proposed but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. Stroke was induced by permanent occlusion of the middle cerebral artery (MCAO) in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analysed longitudinally by magnetic resonance spectroscopy (MRS). Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts and interneurons were analysed by immunofluorescence. Approximately 50% of mice exhibited progressive post-MCAO cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the non-impaired group. Using MRS, significant bilateral changes in hippocampal metabolites such as or N-acetylaspartic acid (NAA) 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 neurogenesis and remodelling processes. Specifically, MCAO 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. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. Hippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for post-stroke cognitive impairment., European Commission, Ministerio de Ciencia e Innovación (España), Leducq Foundation for Cardiovascular Research, Instituto de Salud Carlos III, Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

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., Ministerio de Ciencia e Innovacion (España) MCIN, Instituto de Salud Carlos III, Leducq Foundation for Cardiovascular Research, Fondos FEDER (Unión Europea), Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

El ictus es una de las enfermedades más prevalentes en el mundo, afectando a 15millones de personas cada año. Actualmente, para el ictus agudo, sólo existen dos tratamientos aprobados por la EMA y la FDA: la trombolisis farmacológica con t-PA (tissue-Plasminogen Activator por sus siglas en inglés) y la trombectomía mecánica. Sin embargo, estos tratamientos cuentan con grandes inconvenientes, entre ellos, la baja incidencia de reperfusión y el fenómeno de la transformación hemorrágica (TH) (Larrue et al., 1997; Kaesmacher et al., 2017).Actualmente se desconocen las causas de la no-reperfusión, pero se cree que la composición del trombo juega un papel importante, siendo el porcentaje de éxito, en el caso de los trombos ricos en plaquetas, de un 6% (Tomkins et al., 2015). De hecho, la composición del trombo ha sido de interés para la comunidad científica durante la última década, siendo los NETs (Neutrophil Extracellular Traps por sus siglas en inglés) el último componente en ser identificado (Gardiner & Andrews, 2012; Laridan et al.,2017)..., Stroke is one of the most prevalent diseases worldwide, affecting 15 million people every year. Only two treatment regimens are currently approved by the EMA and the FDA: pharmacological thrombolysis using t-PA (tissue plasminogen activator) and mechanical thrombectomy. Unfortunately, these two treatments have some limitations: a low rate of arterial recanalization and the phenomenon of hemorrhagic transformation (HT), among others (Larrue et al., 1997; Kaesmacher et al., 2017). On the one hand, the reasons of this non-reperfusion phenomenon are still unknown. However, considering that only 6% occlusive platelet-rich thrombi are successfully reperfused (Tomkins et al., 2015), it is believed that thrombus composition might have an important role on this process. In fact, during the last few years, scientists have been focused on this issue who have had identified a new thrombus component called NETs (Neutrophil Extracellular Traps) (Gardiner & Andrews, 2012; Laridan et al., 2017)...

El ictus es una de las enfermedades más prevalentes en el mundo, afectando a 15millones de personas cada año. Actualmente, para el ictus agudo, sólo existen dos tratamientos aprobados por la EMA y la FDA: la trombolisis farmacológica con t-PA (tissue-Plasminogen Activator por sus siglas en inglés) y la trombectomía mecánica. Sin embargo, estos tratamientos cuentan con grandes inconvenientes, entre ellos, la baja incidencia de reperfusión y el fenómeno de la transformación hemorrágica (TH) (Larrue et al., 1997; Kaesmacher et al., 2017).Actualmente se desconocen las causas de la no-reperfusión, pero se cree que la composición del trombo juega un papel importante, siendo el porcentaje de éxito, en el caso de los trombos ricos en plaquetas, de un 6% (Tomkins et al., 2015). De hecho, la composición del trombo ha sido de interés para la comunidad científica durante la última década, siendo los NETs (Neutrophil Extracellular Traps por sus siglas en inglés) el último componente en ser identificado (Gardiner & Andrews, 2012; Laridan et al.,2017)...

Poststroke cognitive impairment is considered one of the main complications during the chronic phase of ischemic stroke. In the adult brain, the hippocampus regulates both encoding and retrieval of new information through adult neurogenesis. Nevertheless, the lack of predictive models and studies based on the forgetting processes hinders the understanding of memory alterations after stroke. Our aim was to explore whether poststroke neurogenesis participates in the development of long-term memory impairment. Here, we show a hippocampal neurogenesis burst that persisted 1 month after stroke and that correlated with an impaired contextual and spatial memory performance. Furthermore, we demonstrate that the enhancement of hippocampal neurogenesis after stroke by physical activity or memantine treatment weakened existing memories. More importantly, stroke-induced newborn neurons promoted an aberrant hippocampal circuitry remodeling with differential features at ipsi- and contralesional levels. Strikingly, inhibition of stroke-induced hippocampal neurogenesis by temozolomide treatment or using a genetic approach (Nestin-CreERT2/NSE-DTA mice) impeded the forgetting of old memories. These results suggest that hippocampal neurogenesis modulation could be considered as a potential approach for treatment of poststroke cognitive impairment.

Background and Purpose—After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-likereceptor 4) on neutrophil infiltration and polarization in this setting., Methods—Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice., Results—As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils(YM1+ cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage.Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma,and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets., Conclusions—TLR4 deficiency increased the levels of alternative neutrophils (N2)—an effect associated with neuroprotection after stroke—supporting that modulation of neutrophil polarization is a major target of TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke.

Newborn granule cells are continuously produced in the subgranular zone of dentate gyrus throughout life. Once these cells mature, they integrate into pre-existing circuits modulating hippocampus-dependent memory. Subsequently, mechanisms controlling generation and maturation of newborn cells are essential for proper hippocampal function. Therefore, we have studied the role of aryl hydrocarbon receptor (AhR), a ligand-activated bHLH-PAS transcription factor, in hippocampus-dependent memory and granule neuronal morphology and function using genetic loss-of-function approaches based on constitutive and inducible-nestin AhR–/– mice. The results presented here show that the impaired hippocampus-dependent memory in AhR absence is not due to its effects on neurogenesis but to aberrant dendritic arborization and an increased spine density, albeit with a lower number of mature mushrooms spines in newborn granule cells, a finding that is associated with an immature electrophysiological phenotype. Together, our data strongly suggest that AhR plays a pivotal role in the regulation of hippocampal function, by controlling hippocampal granule neuron morphology and synaptic maturation., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

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)..., In the Western countries, stroke is the second cause of death (the first cause among women in Spain), the second cause of dementia, and the leading cause of severe disability in adults. Despite these data, the only therapeutic approaches are recanalization of blocked arteries by fibrinolytic treatment through tissue plasminogen activator (t-PA) (only useful in 5-10% of patients), or endovascular thrombectomy (Goyal et al., 2016; Hacke et al., 1995). Therefore, it is necessary the development of new neuroprotective therapies for the treatment in the acute phase of this pathology. On the one hand, treatment with t-PA is used in very restrictive conditions to avoid the hemorrhagic transformation phenomenon (TH), which is the main complication of thrombolysis. Since HT is also a serious complication after mechanical thrombectomy (Goyal et al., 2016), it is necessary to investigate the mechanisms of HT and the potential therapies that could reduce the risk of it, improving the prognosis of patients with this disease. In addition, the occurrence of ischemic stroke involves a cascade of inflammatory events (ischemic cascade) that begins in the cerebral microvasculature, where oxidative stress and reactive oxygen species induce the activation of the complement system, platelets and endothelial cells (for review see (Iadecola and Anrather, 2011)). In turn, at the perivascular level and the parenchyma an innate immune response occurs that triggers a strong inflammatory response. One of the major cell types responsible for the brain response is microglia, which triggers the inflammatory response by producing inflammatory mediators, which is done - in part - thanks to intracellular signaling that starts up after activation of toll-like receptors (TLR)...

Bovine neosporosis control programs are currently based on herd management and serodiagnosis because effective treatments and vaccines are unavailable. Although a wide variety of serological tools have been developed, enzyme-linked immunosorbent assays (ELISAs) are the most commonly commercialized tests. Partial comparative studies have been performed in the past, and the panel of available ELISAs has notably changed in the last few years. Therefore, diagnostic laboratories are requesting updated information about the performance of these tests. Accordingly, the aim of this study was to compare all of the commercially available ELISAs (n = 10) by evaluating their performance and to re-standardize them based on TG-ROC analyses when necessary. For this purpose, a well-characterized serum panel from experimentally and naturally infected bovines and non-infected bovines (n = 458) was used. Two different definitions of gold standard were considered: (i) the result of the majority of tests and (ii) pre-test information based on epidemiological, clinical and serological data. Most of the tests displayed high sensitivity (Se) and specificity (Sp) values when both gold standard criteria were considered. Furthermore, all the tests showed near perfect agreement, with the exception of the pair-wise comparisons that included the VMRD and SVANOVIR. The best-adjusted ELISAs were the HIPRA-CIVTEST, IDVET, BIOVET and IDEXX Rum (Se and Sp > 95%). After the TG-ROC analyses, higher Se and Sp values were obtained for the BIO-X, LSI Bov, LSI Rum and IDEXX Bov, though the increases were more significant for the SVANOVIR and VMRD. The Kappa values also increased with the new adjusted cut-offs. This is the first study that offers updated performance evaluations of commercially available ELISAs. Such analyses are essential for diagnostic laboratories and are valuable to the companies that develop and distribute these tests., Depto. de Sanidad Animal, CAI Ciencias de la Tierra y Arqueometría, TRUE, pub