Examinando por Autor "Lizasoain, Ignacio"

Mostrando 1 - 6 de 6

Iron Overload Exacerbates the Risk of Hemorrhagic Transformation After tPA (Tissue-Type Plasminogen Activator) Administration in Thromboembolic Stroke Mice
(2018-06-13) García-Yébenes, Isaac; García-Culebras, Alicia; Peña-Martínez, Carolina; Fernández-López, David; Díaz-Guzmán, Jaime; Negredo, Pilar; Avendaño, Carlos; Castellanos, Mar; Gasull, Teresa; Dávalos, Antoni; Moro, María; Lizasoain, Ignacio
Background and Purpose—Recanalization with tPA (tissue-type plasminogen activator) is the only pharmacological therapy available for patients with ischemic stroke. However, the percentage of patients who may receive this therapy is limited by the risk of hemorrhagic transformation (HT)—the main complication of ischemic stroke. Our aim is to establish whether iron overload affects HT risk, to identify mechanisms that could help to select patients and to prevent this devastating complication. Methods—Mice fed with control or high-iron diet were subjected to thromboembolic stroke, with or without tPA therapy at different times after occlusion. Blood samples were collected for determination of malondialdehyde, matrix metalloproteinases, and fibronectin. Brain samples were collected 24 hours after occlusion to determine brain infarct and edema size, hemorrhage extension, IgG extravasation, and inflammatory and oxidative markers (neutrophil infiltration, 4-hydroxynonenal, and matrix metalloproteinase-9 staining). Results—Despite an increased rate of recanalization, iron-overload mice showed less neuroprotection after tPA administration. Importantly, iron overload exacerbated the risk of HT after early tPA administration, accelerated ischemia-induced serum matrix metalloproteinase-9 increase, and enhanced basal serum lipid peroxidation. High iron increased brain lipid peroxidation at most times and neutrophil infiltration at the latest time studied. Conclusions—Our data showing that iron overload increases the death of the compromised tissues, accelerates the time of tPA-induced reperfusion, and exacerbates the risk of HT may have relevant clinical implications for a safer thrombolysis. Patients with stroke with iron overload might be at high risk of HT after fibrinolysis, and, therefore, clinical studies must be performed to confirm our results.
Neutrophil Extracellular Trap Targeting Protects Against Ischemic Damage After Fibrin-Rich Thrombotic Stroke Despite Non-Reperfusion
(Frontiers in Immunology, 2022-02-16) Peña-Martínez, Carolina; Durán-Laforet, Violeta; García-Culebras, Alicia; Cuartero, María Isabel; Moro, María; Lizasoain, Ignacio
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.
Pharmacological Modulation of Neutrophil Extracellular Traps Reverses Thrombotic Stroke tPA (Tissue-Type Plasminogen Activator) Resistance
(2019-07-16) Peña-Martínez, Carolina; Durán-Laforet, Violeta; García-Culebras, Alicia; Ostos, Fernando; Hernández-Jiménez, Macarena; Bravo-Ferrer, Isabel; Pérez-Ruiz, Alberto; Ballenilla, Federico; Díaz-Guzmán, Jaime; Pradillo, Jesús; Lizasoain, Ignacio; Moro, María
Background and Purpose—Recanalization of the occluded artery is a primary goal in stroke treatment. Unfortunately, endovascular treatment is not always available, and tPA (tissue-type plasminogen activator) therapy is limited by its narrow therapeutic window; importantly, the rate of early arterial recanalization after tPA administration is low, especially for platelet-rich thrombi. The mechanisms for this tPA resistance are not well known. Since neutrophil extracellular traps (NETs) have been implicated in this setting, our aim was to study whether NET pharmacological modulation can reverse tPA resistance and the role of TLR4 (Toll-like receptor 4), previously related to NET formation, in thrombosis. Methods—To this goal, we have used a mouse photothrombotic stroke model, which produces a fibrin-free thrombus composed primarily of aggregated platelets and thrombi obtained from human stroke patients. Results—Our results demonstrate that (1) administration of DNase-I, which promotes NETs lysis, but not of tPA, recanalizes the occluded vessel improving photothrombotic stroke outcome; (2) a preventive treatment with Cl-amidine, impeding NET formation, completely precludes thrombotic occlusion; (3) platelet TLR4 mediates NET formation after photothrombotic stroke; and (4) ex vivo fresh platelet-rich thrombi from ischemic stroke patients are effectively lysed by DNase-I. Conclusions—Hence, our data open new avenues for recanalization of platelet-rich thrombi after stroke, especially to overcome tPA resistance.
Role of TLR4 (Toll-Like Receptor 4) in N1/N2 Neutrophil Programming After Stroke
(2019-07-26) García-Culebras, Alicia; Durán-Laforet, Violeta; Peña-Martínez, Carolina; Moraga, Ana; Ballesteros, Iván; Cuartero, María Isabel; de la Parra, Juan; Palma-Tortosa, Sara; Hidalgo, Andrés; Corbí, Ángel; Moro, María; Lizasoain, Ignacio
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 TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke.
Test repositioning for functional assessment of neurological outcome after experimental stroke in mice
(Public Library of Science, 2017-05-04) Hernández Jiménez, Macarena; Peña Martínez, Carolina; Godino, Maria del Carmen; Diaz Guzman, Jaime; Moro, María Ángeles; Lizasoain, Ignacio
Stroke is a cerebrovascular pathology for which the only approved treatment is fibrinolysis. Several studies have focused on the development of new drugs but none has led to effective therapies to date, due, among others, to the difficulty to evaluate clinical deficits in experimental animal models. The present study aims to explore the applicability of known behavioral tests not commonly used in ischemia for the neurological assessment of mice after experimental stroke in different brain areas. A total of 225 CD1 male mice were randomly assigned to permanent middle cerebral artery occlusion by ligature (pMCAO) or permanent anterior cerebral artery occlusion by photothrombosis (pACAO) models. Modified neuroseverity score, footprint test, forced swim test and elevated plus maze were performed. Under these experimental conditions, modified neuroseverity score showed neurological impairment early after experimental stroke in both models. By contrast, the footprint test and the elevated plus maze detected short-term neurological deterioration in the pMCAO model but not in the pACAO model. Furthermore, the forced swim test identified depression-like behavior in mice after ischemia only when the left hemisphere was affected. In conclusion, we propose the repositioning of known neurobehavioral tests, but not commonly used in the stroke field, for the fast detection of neurological impairments early after ischemia, and even specific to discriminate the territory affected by arterial occlusion as well as the hemisphere where brain damage occurs. All these findings may prove useful to improve the experimental design of neuroprotective drugs in order to bridge the gap between experimental studies and clinical trials.
TLR4-Binding DNA aptamers show a protective effect against acute stroke in animal models
(2018-08-01) Fernández, Gerónimo; Moraga, Ana; Cuartero, María; García-Culebras, Alicia; Peña-Martínez, Carolina; Pradillo, Jesús; Hernández-Jiménez, Macarena; Sacristán, Silvia; Ayuso, Irene; Gonzalo-Gobernado, Rafael; Fernández López, David; Martín, Elena; Moro, María; González, Víctor; Lizasoain, Ignacio
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.