Specific Features of SVZ Neurogenesis After Cortical Ischemia: a Longitudinal Study.

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.

Characterization of Gcf2/Lrrfip1 in experimental cerebral ischemia and its role as a modulator of Akt, mTOR and ß-catenin signaling pathways.

Leucine-rich repeat in Flightless-1 interaction protein 1 (Lrrfip1) is an up-regulated protein after cerebral ischemia whose precise role in the brain both in healthy and ischemic conditions is unclear. Different Lrrfip1 isoforms with distinct roles have been reported in human and mouse species. The present study aimed to analyze the Lrrfip1 transcriptional variants expressed in rat cortex, to characterize their expression patterns and subcellular location after ischemia, and to define their putative role in the brain. Five transcripts were identified and three of them (Lrrfip1, CRA_g and CRA_a' (Fli-I leucine-rich repeat associated protein 1 - Flap-1)) were analyzed by quantitative real-time polymerase chain reaction (qPCR). All the transcripts were up-regulated and showed differential expression patterns after in vivo and in vitro ischemia models. The main isoform, Lrrfip1, was found to be up-regulated from the acute to the late phases of ischemia in the cytoplasm of neurons and astrocytes of the peri-infarct area. This study demonstrates that Lrrfip1 activates ß-catenin, Akt, and mammalian target of rapamycin (mTOR) proteins in astrocytes and positively regulates the expression of the excitatory amino acid transporter subtype 2 (GLT-1). Our findings point to Lrrfip1 as a key brain protein that regulates pro-survival pathways and proteins and encourages further studies to elucidate its role in cerebral ischemia as a potential target to prevent brain damage and promote functional recovery after stroke.

Daidzein has neuroprotective effects through ligand-binding-independent PPARγ activation.

Phytoestrogens are a group of plant-derived compounds that include mainly isoflavones like daidzein. Phytoestrogens prevent neuronal damage and improve outcome in experimental stroke; however, the mechanisms of this neuroprotective action have not been fully elucidated. In this context, it has been postulated that phytoestrogens might activate the peroxisome proliferator-activated receptor-γ (PPARγ), which exerts neuroprotective effects in several settings. The aim of this study was to determine whether the phytoestrogen daidzein elicits beneficial actions in neuronal cells by mechanisms involving activation of PPARγ. Our results show that daidzein (0.05-5 µM) decreases cell death induced by exposure to oxygen-glucose deprivation (OGD) from rat cortical neurons and that improves synaptic function, in terms of increased synaptic vesicle recycling at nerve terminals, being both effects inhibited by the PPARγ antagonist T0070907 (1 µM). In addition, this phytoestrogen activated PPARγ in neuronal cultures, as shown by an increase in PPARγ transcriptional activity. Interestingly, these effects were not due to binding to the receptor ligand site, as shown by a TR-FRET PPARγ competitive binding assay. Conversely, daidzein increased PPARγ nuclear protein levels and decreased cytosolic ones, suggesting nuclear translocation. We have used the receptor antagonist (RE) fulvestrant to study the neuroprotective participation of daidzein via estrogen receptor and at least in our model, we have discarded this pathway. These results demonstrate that the phytoestrogen daidzein has cytoprotective properties in neurons, which are due to an increase in PPARγ activity not mediated by direct binding to the receptor ligand-binding domain but likely due to post-translational modifications affecting its subcellular location and not depending to the RE and it is not additive with the agonist rosiglitazone.

Tests neurológicos para la evaluación del pronóstico funcional en modelos de ictus isquémico en roedores / Neurological tests for functional outcome assessment in rodent models of ischaemic stroke

Un aspecto crítico en todos los modelos experimentales de patologías del sistema nervioso es la evaluación del pronóstico neurológico final. En el caso de una lesión cerebral isquémica focal, además de la determinación del tamaño de la lesión, una valiosa herramienta es la evaluación del déficit funcional final. Ello se debe al hecho de que el daño isquémico produce diferentes grados de deterioro sensoriomotor y cognitivo, que pueden proporcionar información sobre la ubicación y el tamaño de la lesión y sobre la eficacia de los tratamientos neuroprotectores después del daño agudo. Además, la magnitud de estas alteraciones también puede ser útil para predecir el resultado final y para evaluar terapias reparadoras a largo plazo. Con este fin se ha desarrollado una amplia gama de tests que permite la cuantificación de todos estos síntomas neurológicos. Esta revisión tiene como intención recopilar los tests de comportamiento más útiles diseñados para evaluar los síntomas neurológicos en los estudios de isquemia cerebral focal experimental en roedores inducida por oclusión de la arteria cerebral media, el modelo más utilizado para el estudio del ictus isquémico (AU)

A critical aspect in all models is the assessment of the final outcome of the modelling procedure. In the case of a focal ischaemic brain injury, apart from the determination of the size of the lesion, another valuable tool is the evaluation of the final functional deficit. Indeed, ischaemic damage leads to the appearance of different degrees of sensoriomotor and cognitive impairments, which may yield useful information on location and size of the lesion and on the efficacy of neuroprotective treatments after the acute injury. In addition, the magnitude of these impairments may also be useful to predict final outcome and to evaluate neuro-restorative therapies in a long-term scenario. To this aim, a wide range of tests has been developed which allow the quantification of all these neurological symptoms. This review intends to compile the most useful behavioural tests designed to assess neurological symptoms in studies of focal experimental cerebral ischemia in rodents induced by middle cerebral artery occlusion, the most commonly used model of ischaemic stroke (AU)

Lack of adrenomedullin, but not complement factor H, results in larger infarct size and more extensive brain damage in a focal ischemia model.

Adrenomedullin (AM) and its binding protein, complement factor H (FH), are expressed throughout the brain. In this study we used a brain-specific conditional knockout for AM and a complete knockout for FH to investigate the effect of these molecules on the pathophysiology of stroke. Following 48 h of middle cerebral artery permanent occlusion, there was a statistically significant infarct size increase in animals lacking AM when compared to their wild type littermates. In contrast, lack of FH did not affect infarct volume. To investigate some of the mechanisms by which lack of AM may augment brain damage, markers of nitrosative stress, apoptosis, and autophagy were studied at the mRNA and protein levels. There was a significant increase of inducible nitric oxide synthase (iNOS), matrix metalloproteinase-9 (MMP9), fractin, and Beclin-1 in the peri-infarct area of AM-deficient mice when compared to their wild type counterparts and to contralateral and sham-operated controls. These data suggest that AM exerts a neuroprotective action in the brain and that this protection may be mediated by regulation of iNOS, matrix metalloproteases, and inflammatory mediators. In the future, substances that increase AM actions in the central nervous system may be used as potential neuroprotective agents in stroke.

Delayed post-ischemic administration of CDP-choline increases EAAT2 association to lipid rafts and affords neuroprotection in experimental stroke.

Glutamate transport is the only mechanism for maintaining extracellular glutamate concentrations below excitotoxic levels. Among glutamate transporters, EAAT2 is responsible for up to 90% of all glutamate transport and has been reported to be associated to lipid rafts. In this context, we have recently shown that CDP-choline induces EAAT2 translocation to the membrane. Since CDP-choline preserves membrane stability by recovering levels of sphingomyelin, a glycosphingolipid present in lipid rafts, we have decided to investigate whether CDP-choline increases association of EAAT2 transporter to lipid rafts. Flotillin-1 was used as a marker of lipid rafts due to its known association to these microdomains. After gradient centrifugation, we have found that flotillin-1 appears mainly in fractions 2 and 3 and that EAAT2 protein is predominantly found colocalised with flotillin-1 in fraction 2. We have also demonstrated that CDP-choline increased EAAT2 levels in fraction 2 at both times examined (3 and 6 h after 1 g/kg CDP-choline administration). In agreement with this, [(3)H] glutamate uptake was also increased in flotillin-associated vesicles obtained from brain homogenates of animals treated with CDP-choline. Exposure to middle cerebral artery occlusion also increased EAAT2 levels in lipid rafts, an effect which was further enhanced in those animals receiving 2 g/kg CDP-choline 4 h after the occlusion. Infarct volume measured at 48 h after ischemia showed a reduction in the group treated with CDP-choline 4 h after occlusion. In summary, we have demonstrated that CDP-choline redistributes EAAT2 to lipid raft microdomains and improves glutamate uptake. This effect is also found after experimental stroke, when CDP-choline is administered 4 h after the ischemic occlusion. Since we have also shown that this delayed post-ischemic administration of CDP-choline induces a potent neuroprotection, our data provides a novel target for neuroprotection in stroke.

A chronic treatment with CDP-choline improves functional recovery and increases neuronal plasticity after experimental stroke.

Chronic impairment of forelimb and digit movement is a common problem after stroke that is resistant to therapy. Although in the last years some studies have been performed to increase the efficacy of rehabilitative experience and training, the pharmacological approaches in this context remain poorly developed. We decided to study the effect of a chronic treatment with CDP-choline, a safe and well-tolerated drug that is known to stabilize membranes, on functional outcome and neuromorphological changes after stroke. To assess the functional recovery we have performed the staircase reaching test and the elevated body swing test (EBST), for studying sensorimotor integration and asymmetrical motor function respectively. The treatment with CDP-choline, initiated 24 h after the middle cerebral artery occlusion (MCAO) and maintained during 28 days, improved the functional outcome in both the staircase test (MCAO+CDP=87.0+/-6.6% pellets eaten vs. MCAO+SAL=40.0+/-4.5%; p<0.05) and the EBST (MCAO+CDP=70.0+/-6.8% vs. MCAO+SAL=88.0+/-5.4%; contralateral swing p<0.05). In addition, to study potential neuronal substrates of the improved function, we examined the dendritic morphology of layer V pyramidal cells in the undamaged motor cortex using a Golgi-Cox procedure. The animals treated with CDP-choline showed enhanced dendritic complexity and spine density compared with saline group. Our results suggest that a chronic treatment with CDP-choline initiated 24 h after the insult is able to increase the neuronal plasticity within noninjured and functionally connected brain regions as well as to promote functional recovery.

Targets of cytoprotection in acute ischemic stroke: present and future.

Although the management of stroke has improved remarkably over the last decade due mainly to the advent of thrombolysis, most neuroprotective agents, although successful in animal studies, have failed in humans. Our increasing knowledge concerning the ischemic cascade is leading to a considerable development of pharmacological tools suggesting that each step of this cascade might be a target for cytoprotection. Glutamate has long been recognized to play key roles in the pathophysiology of ischemia. However, although some trials are still ongoing, the results from several completed trials with drugs interfering with the glutamatergic pathway have been disappointing. Regarding the inhibition of glutamate release as a possible target for cytoprotection, it might be afforded either by decreasing glutamate efflux or by increasing glutamate uptake. In this context, it has been shown that glutamate transport is the primary and only mechanism for maintaining extracellular glutamate concentrations below excitotoxic levels. This transport is executed by the five high-affinity, sodium-dependent plasma membrane glutamate transporters. Among them, the transporter EAAT2 is responsible for up to 90% of all glutamate transport. We will discuss the effect of different neuroprotective tools (membrane stabilizers or endogenous neuroprotection) affecting glutamate efflux and/or expression of EAAT2. We will also describe the finding of a novel polymorphism in the EAAT2 promoter region which could be responsible for differences in both gene function and regulation under pathological conditions such as cerebral ischemia, and which might well account for the failure of glutamate antagonists in the clinical practice. These results may possess important therapeutic implications in the management of patients at risk of ischemic events, since it has been demonstrated that those patients with progressing stroke have higher plasma concentrations of glutamate which remain elevated up to 24 h when compared to the levels in patients without neurological deterioration.

TNFR1 mediates increased neuronal membrane EAAT3 expression after in vivo cerebral ischemic preconditioning.

A short ischemic event (ischemic preconditioning) can result in subsequent resistance to severe ischemic injury (ischemic tolerance). Glutamate is released after ischemia and produces cell death. It has been described that after ischemic preconditioning, the release of glutamate is reduced. We have shown that an in vitro model of ischemic preconditioning produces upregulation of glutamate transporters which mediates brain tolerance. We have now decided to investigate whether ischemic preconditioning-induced glutamate transporter upregulation takes also place in vivo, its cellular localization and the mechanisms by which this upregulation is controlled. A period of 10 min of temporary middle cerebral artery occlusion was used as a model of ischemic preconditioning in rat. EAAT1, EAAT2 and EAAT3 glutamate transporters were found in brain from control animals. Ischemic preconditioning produced an up-regulation of EAAT2 and EAAT3 but not of EAAT1 expression. Ischemic preconditioning-induced increase in EAAT3 expression was reduced by the TNF-alpha converting enzyme inhibitor BB1101. Intracerebral administration of either anti-TNF-alpha antibody or of a TNFR1 antisense oligodeoxynucleotide also inhibited ischemic preconditioning-induced EAAT3 up-regulation. Immunohistochemical studies suggest that, whereas the expression of EAAT3 is located in both neuronal cytoplasm and plasma membrane, ischemic preconditioning-induced up-regulation of EAAT3 is mainly localized at the plasma membrane level. In summary, these results demonstrate that in vivo ischemic preconditioning increases the expression of EAAT2 and EAAT3 glutamate transporters the upregulation of the latter being at least partly mediated by TNF-alpha converting enzyme/TNF-alpha/TNFR1 pathway.

Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo.

We report for the first time that primary human neutrophils can undergo persistent, directionally biased movement away from a chemokine in vitro and in vivo, termed chemorepulsion or fugetaxis. Robust neutrophil chemorepulsion in microfluidic gradients of interleukin-8 (IL-8; CXC chemokine ligand 8) was dependent on the absolute concentration of chemokine, CXC chemokine receptor 2 (CXCR2), and was associated with polarization of cytoskeletal elements and signaling molecules involved in chemotaxis and leading edge formation. Like chemoattraction, chemorepulsion was pertussis toxin-sensitive and dependent on phosphoinositide-3 kinase, RhoGTPases, and associated proteins. Perturbation of neutrophil intracytoplasmic cyclic adenosine monophosphate concentrations and the activity of protein kinase C isoforms modulated directional bias and persistence of motility and could convert a chemorepellent to a chemoattractant response. Neutrophil chemorepulsion to an IL-8 ortholog was also demonstrated and quantified in a rat model of inflammation. The finding that neutrophils undergo chemorepulsion in response to continuous chemokine gradients expands the paradigm by which neutrophil migration is understood and may reveal a novel approach to our understanding of the homeostatic regulation of inflammation.

Isquemia intestinal por estenosis de la arteria mesentérica superior. Tratamiento con angioplastia e implante de stent / Intestinal ischemia due to stenosis of the superior mesenteric artery. Treatment with angioplasty and stent implantation

La isquemia intestinal crónica es un cuadro poco frecuente que se encuentra asociado a una alta morbilidad y mortalidad. La causa más frecuente es la arterioesclerosis. Los pacientes sufren de dolor abdominal periumbilical o en epigastrio, que aparece de 10 a 30 minutos luego de la ingestión de alimentos. Presentamos un caso de isquemia intestinal crónica diagnosticado por colonoscopia con biopsias. Se practicó arteriografía que mostró estenosis de la arteria mesentérica superior y se trató con angioplastia percutánea e implante de stent. El paciente presentó completa mejoría clínica y endoscópica. Se comentan los hallazgos clínicos, endoscópicos, radiológicos y el procedimiento terapéutico. Creemos que el tratamiento con arteriografía, angioplastia y colocación de stent es una alternativa que puede mejorar los resultados clínicos disminuyendo la morbimortalidad en esta patología.

Chronic mesenteric arterial ischemia is an uncommon condition associated with a high morbidity and mortality. It is most commonly caused by atherosclerotic occlusive disease. Patients may suffer epigastric or periumbilical post prandial pain, ten to thirty minutes after eating. A case of chronic mesenteric ischemia is presented due to superior mesenteric artery stenosis. The diagnosis was performed with colonoscopy and biopsy. We present a case report of a patient with chronic mesenteric ischemia. Mesenteric arteriography was performed and documented estenosis of the mesenteric superior artery. Then, percutaneus arteriography with angioplasty and implant of stent was performed. The patient became completely asymptomatic and normal colon mucosa is observed in a control colonoscopy. The purpose of this report is to present the case, endoscopic, clinic and radiological features and to describe the percutaneous angioplasty and implant of stent.We believe that angioplasty treatment offers an improvement in this pathology with a low complication rate, and increasing patient confort degree.

Role of nitric oxide after brain ischaemia.

Ischaemic stroke is the second or third leading cause of death in developed countries. In the last two decades substantial research and efforts have been made to understand the biochemical mechanisms involved in brain damage and to develop new treatments. The evidence suggests that nitric oxide (NO) can exert both protective and deleterious effects depending on factors such as the NOS isoform and the cell type by which NO is produced or the temporal stage after the onset of the ischaemic brain injury. Immediately after brain ischaemia, NO release from eNOS is protective mainly by promoting vasodilation; however, after ischaemia develops, NO produced by overactivation of nNOS and, later, NO release by de novo expression of iNOS contribute to the brain damage. This review article summarizes experimental and clinical data supporting the dual role of NO in brain ischaemia and the mechanisms by which NO is regulated after brain ischaemia. We also review NO-based therapeutic strategies for stroke treatment, not only those directly linked with the NO pathway such as NO donors and NOS inhibitors but also those partially related like statins, aspirin or lubeluzole.

Up-regulation of TNF-alpha convertase (TACE/ADAM17) after oxygen-glucose deprivation in rat forebrain slices.

Tumour necrosis factor-alpha (TNF-alpha) is a major immunomodulatory and proinflammatory cytokine which is shed in its soluble form by a membrane-anchored zinc protease, identified as a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). The role of this protease in the adult nervous system remains poorly understood. During cerebral ischemia and subsequent reperfusion, expression and release of TNF-alpha have been shown. We have investigated the expression and activity of TACE in an in vitro model of brain ischemia consisting of rat forebrain slices exposed to oxygen-glucose deprivation (OGD). OGD caused the release of TNF-alpha, an effect which was inhibited by a hydroxamate-based metalloprotease inhibitor, BB-3103, with an IC(50) of 0.1 microM, suggesting that TNF-alpha release results selectively from TACE activity. Assay of TACE enzymatic activity on a fluorescein-labelled peptide spanning the cleavage site in pro-TNF-alpha, as well as Western blot and RT-PCR analyses showed that TACE is present in control forebrain and, more interestingly, that TACE expression is increased in OGD-exposed tissue. TACE enzymatic activity from OGD-exposed slices was significantly inhibited by cycloheximide, suggesting that de novo synthesis of TACE contributes to TNF-alpha release after ischaemia. Moreover, it was also inhibited by bisindolylmaleimide I, indicating that TACE activity is regulated by PKC. These findings posed the question of what was its function therein. Among other actions, TNF-alpha has been described to be involved in the expression of inducible nitric oxide synthase (iNOS), a high-output NOS isoform associated to cellular damage, but the link between TNF-alpha release after brain ischaemia and iNOS expression in this condition has not been shown. We have now found that iNOS expression in OGD-subjected brain slices is inhibited by BB-3103 at concentrations below 1 microM, indicating that shedding of TNF-alpha by TACE plays a necessary part in the induction of this NOS isoenzyme after OGD. Taken together, these data demonstrate that (1) TACE/ADAM17 activity accounts for the majority of TNF-alpha shedding after OGD in rat forebrain slices, (2) an increase in TACE expression contributes, at least in part, to the rise in TNF-alpha after OGD and (3) iNOS expression in OGD-subjected brain slices results from TACE activity and subsequent increase in TNF-alpha levels.

Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis.

Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.

Fructose-1,6-bisphosphate inhibits the expression of inducible nitric oxide synthase caused by oxygen-glucose deprivation through the inhibition of glutamate release in rat forebrain slices.

Fructose-1,6-bisphosphate (FBP) is a glycolytic pathway intermediate with a neuroprotective action in animal models of brain ischaemia. We addressed the question of whether FBP acts through inhibiting inducible nitric oxide synthase (iNOS) expression via reduction of glutamate release, since we have recently demonstrated that glutamate is involved in the expression of iNOS. FBP (5 mM) added to the incubation solution of rat forebrain slices subjected to oxygen-glucose deprivation (OGD) inhibited glutamate release significantly (around 40%). FBP also inhibited the induction of the calcium-independent NOS activity and reduced the levels of iNOS protein in rat forebrain slices subjected to OGD. We conclude that the action of FBP by reducing glutamate release and iNOS expression, both of which have been implicated in cell damage, is a reason for further evaluation of FBP as a neuroprotectant.

Microfabricated elastomeric stencils for micropatterning cell cultures.

Here we present an inexpensive method to fabricate microscopic cellular cultures, which does not require any surface modification of the substrate prior to cell seeding. The method utilizes a reusable elastomeric stencil (i.e., a membrane containing thru holes) which seals spontaneously against the surface. The stencil is applied to the cell-culture substrate before seeding. During seeding, the stencil prevents the substrate from being exposed to the cell suspension except on the hole areas. After cells are allowed to attach and the stencil is peeled off, cellular islands with a shape similar to the holes remain on the cell-culture substrate. This solvent-free method can be combined with a wide range of substrates (including biocompatible polymers, homogeneous or nonplanar surfaces, microelectronic chips, and gels), biomolecules, and virtually any adherent cell type.

Implication of glutamate in the expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices.

Nitric oxide synthesis by inducible nitric oxide synthase (iNOS) has been postulated to contribute to ischemia-reperfusion neurotoxicity. The expression of this enzyme has been demonstrated in cells present in the postischemic brain. The mechanisms of iNOS expression after cerebral ischemia are a subject of current research. We therefore decided to investigate whether glutamate, which is released in ischemia and is implicated in neurotoxicity, might be involved in the mechanisms by which oxygen and glucose deprivation (OGD) leads to the expression of iNOS in rat forebrain slices. In this model, we have shown previously that 20 min of OGD causes the expression of iNOS. We have now found that the NMDA receptor antagonist MK-801 blocks the expression of iNOS, suggesting that the activation of the NMDA subtype of glutamate receptor is implicated in the mechanisms that lead to the expression of this isoform. Moreover, we have found that glutamate alone could trigger the induction process, as shown by the appearance of a Ca(2+)-independent NOS activity and by the detection of iNOS mRNA and protein in slices exposed to glutamate. Glutamate-dependent iNOS expression was concentration-dependent and was blocked by EGTA and by the inhibitors of nuclear factor kappaB (NF-kappaB) activation pyrrolidine dithiocarbamate and MG132. In addition, glutamate induced NF-kappaB translocation to the nucleus, an effect that was inhibited by MG132. Taken together, our data suggest that activation of NMDA receptors by glutamate released in ischemia is involved in the expression of iNOS in rat forebrain slices via a Ca(2+)-dependent activation of the transcription factor NF-kappaB. To our knowledge, this is the first report showing an implication of excitatory amino acids in the expression of iNOS caused by ischemia.

Molding of deep polydimethylsiloxane microstructures for microfluidics and biological applications.

Here we demonstrate the microfabrication of deep (> 25 microns) polymeric microstructures created by replica-molding polydimethylsiloxane (PDMS) from microfabricated Si substrates. The use of PDMS structures in microfluidics and biological applications is discussed. We investigated the feasibility of two methods for the microfabrication of the Si molds: deep plasma etch of silicon-on-insulator (SOI) wafers and photolithographic patterning of a spin-coated photoplastic layer. Although the SOI wafers can be patterned at higher resolution, we found that the inexpensive photoplastic yields similar replication fidelity. The latter is mostly limited by the mechanical stability of the replicated PDMS structures. As an example, we demonstrate the selective delivery of different cell suspensions to specific locations of a tissue culture substrate resulting in micropatterns of attached cells.

Cintigrafía de paratiroides con Tc99m Sestamibi / Parathyroid scintigraphy with Tc99m Sestamibi

Background: parathyroid scintigraphy with Tc99m Sestamibi, a tracer that is taken up by byperfuncttoning parathyroid tissue, has a high yield in the diagnosis of abnormal parathyroid grouths. Aim: to assess the usefulness of parathyroid scintigraphy with Tc99m Sestamibi in patients with suspected hyperparathyroidism. Patients and methods: we analyzed retrospectively 29 patients with suspected hyperparathyroidism, in whom a scintigraphy with Tc99m Sestamibi was performed. Results: twenty four of 25 patients in whom hyperfuntioning parathyroid tissue was evidenced with scintigraphy, were operated. Histological diagnosis of the escised parathyroid gland, showed 23 adenomas and 1 hyperplasia. Anatomical location disclosed by scintigraphy was concordant with surgical finding in all cases, including a mediastinic adenoma. Of the four patients with negative scintigraphic findings, two had transitory PTH elevations and in two the presence of adenoma or hyperplasia could not be demonstrated with other diagnostic procedures. Conclusions: parathyroid scintigraphy with Tc99m Sestamibi had an 86 percent sensitivity for the detection of hyperfunctioning parathyroid tissue in patients with elevated PTH