Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E2 (PGE2) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE2 and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE2 production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE2, and suggest that the coordinated down-regulation of COX-1 facilitates PGE2 production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke

Estudio de la lesión por reperfusión en la isquemia cerebral experimental y su tratamiento

[spa] El ictus es un fenómeno agudo que causa una interrupción del aporte sanguíneo al cerebro, causando muerte de las células por la falta de oxígeno. El ictus es la segunda causa de muerte para personas mayores de 60 años y la causa más frecuente de discapacidad permanente. Debido al creciente envejecimiento de la población y al gran impacto que tiene el ictus, existe una gran necesidad de investigar sobre esta enfermedad, los factores de riesgo, sus consecuencias y por supuesto, nuevos tratamientos. El principal objetivo del tratamiento del ictus es el retorno del FSC al tejido. Aunque la reperfusión es indispensable para salvar el tejido cerebral hipoperfundido, esta puede tener efectos nocivos que se conocen como daño por reperfusión. Diversos estudios han demostrado las alteraciones provocadas por la reperfusión, como el aumento de la producción de ROS y RNS, aumento de expresión de moléculas de adhesión intercelular, inflamación, disminución de la función y ruptura de la BHE, transformación hemorrágica del infarto y desarrollo de un fenotipo procoagulante/protrombótico de la pared vascular. Esta tesis se ha enfocado en el estudio de dos principales consecuencias del daño por reperfusión como son el estrés oxidativo y la transformación hemorrágica (TH) del infarto en modelos animales. Sin embargo, los seres humanos que sufren un ictus suelen presentar enfermedades concomitantes que exacerban el daño por isquemia/reperfusión y es importante que en los modelos animales también se evalúen estos factores de riesgo o situaciones clínicas relacionadas con el ictus isquémico humano. Teniendo en cuenta estas circunstancias, este trabajo de tesis se ha centrado en el estudio del estrés oxidativo aumentado por hiperglucemia y en la trasformación hemorrágica del infarto en condiciones de linfopenia. Así mismo, estudiamos la posibilidad de modular el estrés oxidativo y la respuesta inflamatoria como métodos para potenciar el tratamiento del ictus y minimizar los daños por reperfusión. En el primer trabajo mostramos que los ratones a los que se les indujo hiperglucemia previamente a la I/R podían beneficiarse del tratamiento con el antioxidante natural ácido úrico (AU). Este estudio ha demostrado que las altas concentraciones sanguíneas de glucosa exacerban el daño por isquemia/reperfusión. Y que el AU puede contribuir a reducir el volumen de infarto en los ratones hiperglucémicos a través de su efecto antioxidante con una gran eficacia en la limpieza de radicales libres derivados del peroxinitrito y sus efectos vasculoprotectores limitando la toxicidad de la glucosa y la muerte celular secundaria. El segundo trabajo se centra en el estudio de las células T y su relación con la TH. Este estudio demuestra que los linfocitos T reducen el riesgo de TH. Confirma la relevancia del volumen del infarto como un factor crítico que incrementa el riesgo de TH. A su vez, los resultados de este estudio resaltan la comunicación bidireccional que existe entre las células T y las plaquetas creando un puente entre la inmunidad y la hemostasia para prevenir la TH tras un ictus isquémico agudo. Estos resultados pueden determinar nuevas dianas celulares y moleculares para la prevención de la TH secundaria al ictus isquémico. En el tercer trabajo, hemos intentado reducir la TH mediante un tratamiento farmacológico con el inmunomodulador Fingolimod. Los resultados de este estudio muestran una activación de la vía de señalización S1P en el cerebro después de la I/R, esta activación sugiere que los fármacos moduladores del receptor S1P pueden desempeñar un papel en el parénquima independiente de los efectos del fármaco sobre el sistema inmune. También demostró que el Fingolimod puede atenuar la TH del infarto cerebral por una vía independiente de los linfocitos, pero el tratamiento no es efectivo en condiciones de trombocitopenia.[eng] Stroke is an acute phenomenon caused by the interruption of the blood supply to the brain. Stroke is one of the leading causes of death and permanent disability in the world. Due to the huge impact of stroke in our society, there is a great need to investigate this disease, the risk factors, their consequences and, of course, new treatments. The main objective of stroke treatment is the return of cerebral blood flow to the brain tissue. Although reperfusion is essential to save hypoperfused brain tissue, it may have unwanted complications that are known as reperfusion damage. This thesis has focused on the study of two negative consequences of reperfusion, such as oxidative stress and hemorrhagic transformation (HT) of infarction in animal models. Likewise, we studied the possibility of modulating oxidative stress and the inflammatory response in order to minimize reperfusion damage. In the first work we showed that mice that had hyperglycemia induced before ischemia/reperfusion could benefit from treatment with uric acid (AU) through its antioxidant effect, limiting glucose toxicity and secondary cell death. The second work focuses on the study of T cells and their relationship with HT. The results of this study highlight the bidirectional communication that exists between T cells and platelets, creating a bridge between immunity and hemostasis to prevent HT after an acute ischemic stroke. These results can determine new cellular and molecular targets for the prevention of HT secondary to ischemic stroke. In the third work, we have tried to reduce HT through a pharmacological treatment with Fingolimod. It also showed that Fingolimod can attenuate the HT of cerebral infarction through a lymphocyte-independent pathway, but the treatment is not effective in thrombocytopenia conditions. Overall this thesis identifies cellular and molecular mechanisms underlying complications of reperfusion therapies following ischemic stroke, and suggests pharmacological therapies to increase the benefits of reperfusion treatments

Development of an HSP70-regulated far-red fluorescent reporter mouse for imaging the ischemic penumbra

Trabajo presentado al XXVth International Symposium on Cerebral Blood Flow, Metabolism and Function y a la Xth International Conference on Quantification of Brain Function with PET celebrados del 25 al 28 de mayo en Barcelona.[Objectives]: Neurons surrounding the ischemic area express Hsp-70 and this is regarded as a marker of the ischemic penumbra. Hsp-70 is also strongly induced in blood vessels within the ischemic territory. Our aim was to obtain transgenic mice expressing a fluorescent reporter to image the induction of Hsp70. For such applications, the use of a far-red fluorescent protein reporter is desirable so that fluorescence absorption and scattering by the cerebral parenchyma are reduced. [Methods]: An Hsp70 promoter-driven reporter vector for the far-red fluorescent protein mPlum was constructed with Invitrogen's Multisite Gateway Vector Construction kit. NIH3T3 cells were transfected with this vector and stimulated with sodium arsenite (50 uM) to induce Hsp-70 expression mPlum and Hsp70 protein expression was detected with specific antibodies by Western blotting and immunocytochemistry. mPlum fluorescence was detected with a fluorescence inverted-microscope. Transgenic mice were generated at the “Norsk Transgen Senter” (Norway). Transient middle cerebral artery occlusion (MCAO) was performed on adult transgenic or wt mice as previously described. A cranial window sealed with a cover slip was opened on ketamine/xylacine-anesthetised mice over the ipsilateral hemisphere for live observation under high speed confocal microscope (590nm ex, 649nm em) 24h after ischemia. Mice were killed thereafter and the brain was processed for immunohistochemistry. In another set of animals, brain samples were obtained 24h after MCAO and were processed for either quantitative real time RT-PCR or Western blotting. [Results]: Cells transfected with the Hsp-70 reporter vector showed induction of mPlum when Hsp-70 expression was stimulated, showing the usefulness of our construct to track Hsp-70 induction. In transgenic mice, MCAO induced expression of mPlum and hsp70 mRNAs and proteins in the ipsilateral hemisphere. Intravital confocal microscopy showed mPlum fluorescence in superficial blood vessels of the ipsilateral hemisphere. Immunohistochemistry on post-mortem brain sections revealed expression of mPlum in the infarct area, which colocalised with endogenous Hsp70 expression in blood vessels and neurons. Despite mPlum's limited intensity of fluorescence signal, our studies reveal that mPlum fluorescence can be detected in live animals after brain ischemia, mainly in cortical blood vessels that show the strongest induction of Hsp-70 and mPlum. [Conclusions]: Our results show that our Hsp70-mPlum reporter vector in the transgenic mice responds to ischemia in a parallel way to endogenous Hsp70, and that it is possible to track Hsp-70 expression in vivo through mPlum fluorescence.We thank Roger Tsien (UCSD) for providing the mPlum cDNA. X. R. has a fellowship from the Spanish Ministry of Science and Innovation (MICINN). T.S. participates in the Program for Stabilization of Investigators from the “Generalitat de Catalunya”, Spain. Financed in part by the EC (FP6 Diagnostic Molecular Imaging) and MICINN (SAF2008-04515).Peer Reviewe

ENGIU: Encuentro Nacional de Grupos de Investigación de UNIMINUTO.

El desarrollo del prototipo para el sistema de detección de Mina Antipersona (MAP), inicia desde el semillero ADSSOF perteneciente al programa de Administración en Seguridad y Salud en el trabajo de la UNIMINUTO, se realiza a partir de un detector de metales que emite una señal audible, que el usuario puede interpretar como aviso de presencia de un objeto metálico, en este caso una MAP. La señal audible se interpreta como un dato, como ese dato no es perceptible a 5 metros de distancia, se implementa el transmisor de Frecuencia Modulada FM por la facilidad de modulación y la escogencia de frecuencia de transmisión de acuerdo con las normas y resolución del Ministerio de Comunicaciones; de manera que esta sea la plataforma base para enviar los datos obtenidos a una frecuencia establecida. La idea es que el ser humano no explore zonas peligrosas y buscar la forma de crear un sistema que permita eliminar ese riesgo, por otro lado, buscar la facilidad de uso de elementos ya disponibles en el mercado