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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

The influence of toll-like receptor 4 on neurogenesis and inflammation has been scarcely explored so far by using neuroimaging techniques. For this purpose, we performed magnetic resonance imaging and positron emission tomography with 3'-deoxy-3'-[(18)F]fluorothymidine and [(11)C]PK11195 at 2, 7, and 14 days following cerebral ischemia in TLR4(+/+)and TLR4(-/-)mice. MRI showed similar infarction volumes in both groups. Despite this, positron emission tomography with 3'-deoxy-3'-[(18)F]fluorothymidine and [(11)C]PK11195 evidenced an increase of neurogenesis and a decrease of inflammation in TLR4(-/-)mice after ischemia. These results evidence the versatility of neuroimaging techniques to monitor the role of toll-like receptor 4 after cerebral ischemia., Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage. Swiss mice were fed with a standard diet or with a diet supplemented with 2.5% carbonyl iron to produce iron overload. Mice were submitted to permanent (by ligature and by in situ thromboembolic models) or transient focal ischemia (by ligature for 1 or 3h). Treatment with iron diet produced an increase in the basal levels of ferritin in all the groups. However, serum ferritin did not change after ischemia. Animals submitted to permanent ischemia had the same infarct volume in the groups studied. However, in mice submitted to transient ischemia followed by early (1h) but not late reperfusion (3h), iron overload increased ischemic damage and haemorrhagic transformation. Iron worsens ischemic damage induced by transient ischemia and early reperfusion. In addition, ferritin is a good indicator of body iron levels but not an acute phase protein after ischemia., Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

CDP-choline has shown neuroprotective effects in cerebral ischemia. In humans, although a recent trial International Citicoline Trial on Acute Stroke (ICTUS) has shown that global recovery is similar in CDP-choline and placebo groups, CDP-choline was shown to be more beneficial in some patients, such as those with moderate stroke severity and not treated with t-PA. Several mechanisms have been proposed to explain the beneficial actions of CDP-choline. We have now studied the participation of Sirtuin1 (SIRT1) in the neuroprotective actions of CDP-choline. Fischer rats and Sirt1⁻/⁻ mice were subjected to permanent focal ischemia. CDP-choline (0.2 or 2 g/kg), sirtinol (a SIRT1 inhibitor; 10 mg/kg), and resveratrol (a SIRT1 activator; 2.5 mg/kg) were administered intraperitoneally. Brains were removed 24 and 48 h after ischemia for western blot analysis and infarct volume determination. Treatment with CDP-choline increased SIRT1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP-choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP-choline. CDP-choline failed to reduce infarct volume in Sirt1⁻/⁻ mice. Our present results demonstrate a robust effect of CDP-choline like SIRT1 activator by up-regulating its expression. Our findings suggest that therapeutic strategies to activate SIRT1 may be useful in the treatment of stroke. Sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions. Regarding stroke, there is no direct evidence. We have demonstrated that citicoline increases SIRT1 protein levels in brain concomitantly to neuroprotection. Citicoline fails to reduce infarct volume in Sirt1⁻/⁻ mice. Our findings suggest that therapeutic strategies acting on SIRT1 may be useful in the treatment of stroke., Depto. de Farmacología y Toxicología, Fac. de Medicina, TRUE, pub

This work was supported by grants from Instituto de Salud Carlos III and cofinanced by the European Development Regional Fund “A Way to Achieve Europe” (PI17/01601 and RETICS RD16/0019/0009; I.L.), from Regional Madrid Government B2017/BMD-3688 (I.L.); from Spanish Ministry of Economy and Competitiveness SAF2015-68632-R (M.A.M.); from PICATA Project (Campus of international excellence of Moncloa; J.M.P.) and from M+Vision-COFUND Project (J.M.P.) (RTI2018-098682-B-I00 (MCIU/AEI/FEDER, EU) funded by the Spanish Ministry of Science and Innovation (MJLC)., Stroke affects primarily aged and co-morbid people, aspects not properly considered to date. Since angiogenesis/vasculogenesis are key processes for stroke recovery, we purposed to determine how different co-morbidities affect the outcome and angiogenesis/vasculogenesis, using a rodent model of metabolic syndrome, and by dynamic enhanced-contrast imaging (DCE-MRI) to assess its non-invasive potential to determine these processes. Twenty/twenty-two month-old corpulent (JCR:LA-Cp/Cp), a model of metabolic syndrome and lean rats were used. After inducing the experimental ischemia by transient MCAO, angiogenesis was analyzed by histology, vasculogenesis by determination of endothelial progenitor cells in peripheral blood by flow cytometry and evaluating their pro-angiogenic properties in culture and the vascular function by DCE-MRI at 3, 7 and 28 days after tMCAO. Our results show an increased infarct volume, BBB damage and an impaired outcome in corpulent rats compared with their lean counterparts. Corpulent rats also displayed worse post-stroke angiogenesis/vasculogenesis, outcome that translated in an impaired vascular function determined by DCE-MRI. These data confirm that outcome and angiogenesis/vasculogenesis induced by stroke in old rats are negatively affected by the co-morbidities present in the corpulent genotype and also that DCE-MRI might be a technique useful for the non-invasive evaluation of vascular function and angiogenesis processes., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, 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

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

La isquemia cerebral o ictus es una de las principales causas de muerte en países desarrollados así como la primera causa de discapacidad en mayores de 65 años (Sousa et al., 2009). Asimismo, entre los pacientes que sobreviven a un infarto cerebral, una gran parte suelen desarrollar una serie de secuelas como son demencia, afasia, hemiparesia o algún grado de dependencia (Kalaria et al., 2016; Kelly-Hayes et al., 2003). Debido a lo anterior, el ictus supone un gran gasto socioeconómico, lo que hace necesario obtener avances en prevención y tratamiento de la enfermedad. El ictus se puede clasificar, según su naturaleza, en isquémico (mayoritario, un 85% de los casos) y hemorrágico (15% de los casos). Desde el punto de vista mecanístico, el ictus isquémico se produce por la oclusión de una arteria cerebral, dando lugar a un una falta de aporte sanguíneo focal que causa un daño irreversible en un área delimitada denominada core, que corresponde a la zona más afectada, y un daño parcialmente reversible, denominada área de penumbra, la cual, si se consigue restaurar el flujo sanguíneo, será susceptible de recuperación (Astrup et al., 1977; Astrup et al., 1981). Como consecuencia de la reducción, total o parcial, del flujo sanguíneo cerebral, la llegada de oxígeno y glucosa a las células del área irrigada por el vaso ocluido se ve comprometida, induciéndose una serie de procesos bioquímicos conocidos como cascada isquémica que lleva, entre muchos otros eventos, a una desviación del metabolismo de la glucosa a la vía anaerobia debido a la reducción de la disponibilidad de oxígeno, lo que lleva a un incremento de ácido láctico y a la consiguiente acidosis...

La isquemia cerebral o ictus es una de las principales causas de muerte en países desarrollados así como la primera causa de discapacidad en mayores de 65 años (Sousa et al., 2009). Asimismo, entre los pacientes que sobreviven a un infarto cerebral, una gran parte suelen desarrollar una serie de secuelas como son demencia, afasia, hemiparesia o algún grado de dependencia (Kalaria et al., 2016; Kelly-Hayes et al., 2003). Debido a lo anterior, el ictus supone un gran gasto socioeconómico, lo que hace necesario obtener avances en prevención y tratamiento de la enfermedad. El ictus se puede clasificar, según su naturaleza, en isquémico (mayoritario, un 85% de los casos) y hemorrágico (15% de los casos). Desde el punto de vista mecanístico, el ictus isquémico se produce por la oclusión de una arteria cerebral, dando lugar a un una falta de aporte sanguíneo focal que causa un daño irreversible en un área delimitada denominada core, que corresponde a la zona más afectada, y un daño parcialmente reversible, denominada área de penumbra, la cual, si se consigue restaurar el flujo sanguíneo, será susceptible de recuperación (Astrup et al., 1977; Astrup et al., 1981). Como consecuencia de la reducción, total o parcial, del flujo sanguíneo cerebral, la llegada de oxígeno y glucosa a las células del área irrigada por el vaso ocluido se ve comprometida, induciéndose una serie de procesos bioquímicos conocidos como cascada isquémica que lleva, entre muchos otros eventos, a una desviación del metabolismo de la glucosa a la vía anaerobia debido a la reducción de la disponibilidad de oxígeno, lo que lleva a un incremento de ácido láctico y a la consiguiente acidosis...

La isquemia cerebral o ictus es una de las principales causas de muerte en países desarrollados así como la primera causa de discapacidad en mayores de 65 años (Sousa et al., 2009). Asimismo, entre los pacientes que sobreviven a un infarto cerebral, una gran parte suelen desarrollar una serie de secuelas como son demencia, afasia, hemiparesia o algún grado de dependencia (Kalaria et al., 2016; Kelly-Hayes et al., 2003). Debido a lo anterior, el ictus supone un gran gasto socioeconómico, lo que hace necesario obtener avances en prevención y tratamiento de la enfermedad. El ictus se puede clasificar, según su naturaleza, en isquémico (mayoritario, un 85% de los casos) y hemorrágico (15% de los casos). Desde el punto de vista mecanístico, el ictus isquémico se produce por la oclusión de una arteria cerebral, dando lugar a un una falta de aporte sanguíneo focal que causa un daño irreversible en un área delimitada denominada core, que corresponde a la zona más afectada, y un daño parcialmente reversible, denominada área de penumbra, la cual, si se consigue restaurar el flujo sanguíneo, será susceptible de recuperación (Astrup et al., 1977; Astrup et al., 1981). Como consecuencia de la reducción, total o parcial, del flujo sanguíneo cerebral, la llegada de oxígeno y glucosa a las células del área irrigada por el vaso ocluido se ve comprometida, induciéndose una serie de procesos bioquímicos conocidos como cascada isquémica que lleva, entre muchos otros eventos, a una desviación del metabolismo de la glucosa a la vía anaerobia debido a la reducción de la disponibilidad de oxígeno, lo que lleva a un incremento de ácido láctico y a la consiguiente acidosis..., Cerebral ischemia, also known as stroke, is one of the leading causes of death in developed countries and the first cause of disability in elderly people. Moreover, the majority of surviving stroke patients may develop sequels as dementia, aphasia, hemiparesis or some dependence degree (Kalaria et al., 2016; Kelly-Hayes et al., 2003). Assuming those facts, stroke represents a huge socioeconomic burden revealing the necessity to improve prevention strategies and disease treatments. According to its nature, a stroke can be classified into ischemic stroke (85%) or hemorrhagic stroke (15%). From the mechanical point of view, ischemic stroke is caused by the occlusion of a cerebral artery. That insult results in a focal ischemic process that causes irreversible damage in a delimited area called core, which corresponds to the most affected area; and a partially reversible damage, called penumbra area, where, if blood flow is restored, there could be some recovery (Astrup et al., 1977; Astrup et al., 1981). As a consequence of the total or partial reduction of cerebral blood flow, the oxygen and glucose input is compromised. This situation induces a series of biochemical processes known as ischemic cascade, leading to a deviation of glucose metabolism to an anaerobic pathway due to the reduced oxygen availability. This scenario promotes an increase in lactic acid and the consequent acidosis. Nowadays, the only approved treatments for stroke are limited to the mechanical thrombus removal by thrombectomy and the thrombus dissolution by thrombolysis. Thrombus dissolution is achieved by the administration of the only FDA-approved pharmacological treatment (r-tPA, recombinant Plasminogen Tissue Activator). Nevertheless, this treatment has a very restrictive inclusion criteria and a high risk of hemorrhagic transformation. That implies that only an average of 5% of patients can benefit from it (Larrue et al., 2001; McArthur et al., 2011)...

La isquemia cerebral o ictus es una de las principales causas de muerte en países desarrollados así como la primera causa de discapacidad en mayores de 65 años (Sousa et al., 2009). Asimismo, entre los pacientes que sobreviven a un infarto cerebral, una gran parte suelen desarrollar una serie de secuelas como son demencia, afasia, hemiparesia o algún grado de dependencia (Kalaria et al., 2016; Kelly-Hayes et al., 2003). Debido a lo anterior, el ictus supone un gran gasto socioeconómico, lo que hace necesario obtener avances en prevención y tratamiento de la enfermedad. El ictus se puede clasificar, según su naturaleza, en isquémico (mayoritario, un 85% de los casos) y hemorrágico (15% de los casos). Desde el punto de vista mecanístico, el ictus isquémico se produce por la oclusión de una arteria cerebral, dando lugar a un una falta de aporte sanguíneo focal que causa un daño irreversible en un área delimitada denominada core, que corresponde a la zona más afectada, y un daño parcialmente reversible, denominada área de penumbra, la cual, si se consigue restaurar el flujo sanguíneo, será susceptible de recuperación (Astrup et al., 1977; Astrup et al., 1981). Como consecuencia de la reducción, total o parcial, del flujo sanguíneo cerebral, la llegada de oxígeno y glucosa a las células del área irrigada por el vaso ocluido se ve comprometida, induciéndose una serie de procesos bioquímicos conocidos como cascada isquémica que lleva, entre muchos otros eventos, a una desviación del metabolismo de la glucosa a la vía anaerobia debido a la reducción de la disponibilidad de oxígeno, lo que lleva a un incremento de ácido láctico y a la consiguiente acidosis..., Cerebral ischemia, also known as stroke, is one of the leading causes of death in developed countries and the first cause of disability in elderly people. Moreover, the majority of surviving stroke patients may develop sequels as dementia, aphasia, hemiparesis or some dependence degree (Kalaria et al., 2016; Kelly-Hayes et al., 2003). Assuming those facts, stroke represents a huge socioeconomic burden revealing the necessity to improve prevention strategies and disease treatments. According to its nature, a stroke can be classified into ischemic stroke (85%) or hemorrhagic stroke (15%). From the mechanical point of view, ischemic stroke is caused by the occlusion of a cerebral artery. That insult results in a focal ischemic process that causes irreversible damage in a delimited area called core, which corresponds to the most affected area; and a partially reversible damage, called penumbra area, where, if blood flow is restored, there could be some recovery (Astrup et al., 1977; Astrup et al., 1981). As a consequence of the total or partial reduction of cerebral blood flow, the oxygen and glucose input is compromised. This situation induces a series of biochemical processes known as ischemic cascade, leading to a deviation of glucose metabolism to an anaerobic pathway due to the reduced oxygen availability. This scenario promotes an increase in lactic acid and the consequent acidosis. Nowadays, the only approved treatments for stroke are limited to the mechanical thrombus removal by thrombectomy and the thrombus dissolution by thrombolysis. Thrombus dissolution is achieved by the administration of the only FDA-approved pharmacological treatment (r-tPA, recombinant Plasminogen Tissue Activator). Nevertheless, this treatment has a very restrictive inclusion criteria and a high risk of hemorrhagic transformation. That implies that only an average of 5% of patients can benefit from it (Larrue et al., 2001; McArthur et al., 2011)...

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 TFG