RESUMEN
OBJECTIVE: To define the anti-inflammatory effects of PPARbeta/delta activation by use of the selective PPARbeta/delta ligand (GW0742) in a model of lipopolysaccharide (LPS)-induced pulmonary inflammation. METHODS: Male BALB/c mice were pretreated for three days with the PPARbeta/delta agonist, GW0742, prior to induction of LPS-mediated pulmonary inflammation. Bronchial alveolar lavage fluid (BALF) was analyzed for inflammatory cell influx and for levels of pro-inflammatory mediators. BALF-derived inflammatory cells were also collected for mRNA analysis. RESULTS: Pretreatment with GW0742 resulted in a significant decrease in leukocyte recruitment into the pulmonary space. Protein and mRNA levels of the pro-inflammatory cytokines IL-6, IL-1beta and TNFalpha in BALF were found to be significantly decreased in GW0742-treated animals (30 mg/kg). A significant decrease in granulocyte macrophage-colony stimulating factor (GM-CSF), a major regulator of neutrophil chemotaxis (via its downstream actions on TNFalpha and other cytokines/chemokines), activation and survival, was also noted in the BALF levels of GW0742-treated animals. CONCLUSIONS: The present study demonstrates that activation of PPARbeta/delta attenuates the degree of inflammation in a model of LPS-induced pulmonary inflammation and may therefore represent a novel therapeutic approach for the treatment of inflammation-mediated pathologies.
Asunto(s)
Citocinas/metabolismo , Lipopolisacáridos/metabolismo , Neutrófilos/metabolismo , PPAR delta/metabolismo , PPAR-beta/metabolismo , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Inflamación , Leucocitos/efectos de los fármacos , Ligandos , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Tiazoles/farmacologíaRESUMEN
Evidence suggests that endothelin receptor antagonists may have therapeutic potential for the chronic treatment of heart failure. In the current study, the effects of an orally active mixed endothelin-A/endothelin-B (ETA /ETB ) receptor antagonist (enrasentan) were assessed in a model of cardiac hypertrophy and dysfunction (spontaneously hypertensive stroke prone rats) maintained on a high-salt/high-fat diet. Echocardiography was used to quantify cardiac performance and left ventricular dimensions. Enrasentan (1,200 and 2,400 parts per million in the high-salt/high-fat diet) had no significant effects on body weight and systolic blood pressure. However, increases in heart rate were not observed in the enrasentan-treated groups at 12 weeks (p < 0.05). Enrasentan-treated groups exhibited significantly improved survival (90-95% vs. 30% [control rats] at 18 weeks; p < 0.001). Enrasentan treatments also increased stroke volume (at 8, 12, and 16 weeks) and cardiac index (at 8 and 16 weeks) 33-50% and 45-63%, respectively. Enrasentan treatments reduced the relative wall thickness (14-27% at 8 and 12 weeks), ratio of left ventricular mass to body weight (20% at 12 weeks), and ratio of terminal heart weight to body weight (16-23%, p < 0.05). Finally, circulating aldosterone concentration (54-57%) and proANF fragment (33%) were reduced in enrasentan-treated groups (54-57% and 33%, respectively). Mixed ETA /ETB receptor antagonism improves cardiac performance and attenuates ventricular remodeling and premature mortality in an aggressive hypertension model.
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Ácidos Carboxílicos/uso terapéutico , Hipertensión/tratamiento farmacológico , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Indanos/uso terapéutico , Remodelación Ventricular/efectos de los fármacos , Aldosterona/sangre , Animales , Factor Natriurético Atrial/sangre , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Antagonistas de los Receptores de Endotelina , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Hipertensión/sangre , Hipertensión/mortalidad , Hipertrofia Ventricular Izquierda/sangre , Hipertrofia Ventricular Izquierda/mortalidad , Masculino , Miocardio/metabolismo , Precursores de Proteínas/sangre , Ratas , Ratas Endogámicas SHR , Tasa de Supervivencia , Remodelación Ventricular/fisiologíaRESUMEN
Failure of several putative neuroprotectants in large multicentred clinical trials has re-focussed attention on the predictability of pre-clinical animal models of stroke. Model characterisation and relationship to heterogeneous patient sub-groups remains of paramount importance. Information gained from magnetic resonance imaging (MRI) signatures indicates that the Zea Longa model of rat middle cerebral artery occlusion may be more representative of slowly evolving infarcts. Understanding the molecular changes over several hours following cerebral ischaemia will allow detailed characterisation of the adaptive response to brain injury. Using a fully characterised model of Zea Longa middle cerebral artery occlusion we have used the representational difference analysis (RDA) subtractive hybridisation method to identify transcripts that accumulate in the ischaemic cortex. Along with a number of established ischaemia-induced gene products (including MCP-1, TIMP-1, hsp 70) we were also able to identify nine genes which have not previously been shown to accumulate following focal ischaemia (including SOCS-3, GADD45gamma, Xin).
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Química Encefálica/genética , Infarto de la Arteria Cerebral Media/fisiopatología , Hibridación de Ácido Nucleico/métodos , Compuestos Orgánicos , Animales , Antígenos de Superficie/genética , Benzotiazoles , Citocinas/genética , Diaminas , Colorantes Fluorescentes , Expresión Génica/fisiología , Biblioteca de Genes , Proteínas de Choque Térmico/genética , Masculino , Reacción en Cadena de la Polimerasa , Quinolinas , Ratas , Ratas Sprague-DawleyRESUMEN
This study tested the hypothesis that increased nitric oxide (NO) inactivation and concurrent peroxynitrite formation is responsible for endothelial dysfunction in the spontaneously hypertensive stroke-prone rat (SHRSP). In SHRSP, the aortic vasorelaxation to acetylcholine (ACh) was decreased (p < 0.05), but NO production was unchanged. Nitrotyrosine staining, a footprint of peroxynitrite (ONOO(-)) formation, was detected. Exposure of SHRSP to a high-salt, high-fat diet (SFD) further exacerbated hypertension and accelerated end-organ disease. A severe endothelial dysfunction [maximal ACh relaxation: 49.8 +/- 2.1 versus 94.5 +/- 1.8% in Wistar-Kyoto rats (WKY), p < 0.01], increased basal NO production (482 +/- 17 versus 356 +/- 21 nM, p < 0.01), decreased ACh-stimulated NO production (57 +/- 6 versus 112 +/- 6 nM, p < 0.01), extensive inducible NO synthase and nitrotyrosine staining, elevated nitrotyrosine content (21-fold increase over WKY), and a high percentage of cells with DNA damage were observed in the aortic tissues from these animals. Treatment of SHRSP on SFD with carvedilol restored ACh-induced vasorelaxation and NO production, inhibited nitrotyrosine formation, reduced vascular cell DNA damage, and reduced end-organ injury. These data demonstrate that endothelial dysfunction was caused by increased NO inactivation alone (SHRSP) or in combination with decreased NO production from endothelial NO synthase (SHRSP on SFD). Antioxidant treatment with carvedilol exerted significant vascular protective effects, attenuated end-organ damage, and decreased mortality under these conditions.
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Endotelio Vascular/metabolismo , Óxido Nítrico/metabolismo , Accidente Cerebrovascular/metabolismo , Tirosina/análogos & derivados , Acetilcolina/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Apoptosis/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Carbazoles/farmacología , Carvedilol , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/sangre , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Oxidación-Reducción , Propanolaminas/farmacología , Ratas , Ratas Endogámicas SHR , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/mortalidad , Tirosina/metabolismoRESUMEN
Sequencing of the human genome is nearing completion and biologists, molecular biologists, and bioinformatics specialists have teamed up to develop global genomic technologies to help decipher the complex nature of pathophysiologic gene function. This review will focus on differential gene expression in ischemic stroke. It will discuss inheritance in the broader stroke population, how experimental models of spontaneous stroke might be applied to humans to identify chromosomal loci of increased risk and ischemic sensitivity, and also how the gene expression induced by stroke is related to the poststroke processes of brain injury, repair, and recovery. In addition, we discuss and summarise the literature of experimental stroke genomics and compare several approaches of differential gene expression analyzes. These include a comparison of representational difference analysis we have provided using an experimental stroke model that is representative of stroke evolution observed most often in man, and a summary of available data on stroke differential gene expression. Issues regarding validation of potential genes as stroke targets, the verification of message translation to protein products, the relevance of the expression of neuroprotective and neurodestructive genes and their specific timings, and the emerging problems of handling novel genes that may be discovered during differential gene expression analyses will also be addressed.
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Expresión Génica , Accidente Cerebrovascular/genética , Animales , Encefalopatías/etiología , Encefalopatías/genética , Isquemia Encefálica/complicaciones , Isquemia Encefálica/genética , Mapeo Cromosómico , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Mutación , Hibridación de Ácido Nucleico , Accidente Cerebrovascular/complicacionesAsunto(s)
Benzodiazepinas , Caspasas/metabolismo , Fibrinolíticos/farmacología , Corazón/efectos de los fármacos , Miocardio/enzimología , Oligopéptidos/farmacología , Piperidinas , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Animales , Caspasa 3 , Células Cultivadas/efectos de los fármacos , Cumarinas/metabolismo , Activación Enzimática , Fibrinolíticos/toxicidad , Miocardio/citología , Oligopéptidos/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/toxicidad , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Eprosartan is a selective angiotensin II type I receptor antagonist approved for the treatment of hypertension. In the present studies, eprosartan's ability to provide end-organ protection was evaluated in a model of cardiomyopathy and renal failure in stroke-prone rats (SP). METHODS: SP were fed a high fat (24.5% in food) and high salt (1% in water) diet (SFD). Eprosartan (60 mg/kg/day) or vehicle (saline control) (n = 25/group) was administered by intraperitoneally-implanted minipumps to these SP on the SFD for 12 weeks. Normal diet fed SP and WKY rats (n = 25/group) were also included for comparison (i.e. served as normal controls). Mortality, hemodynamics, and both renal and cardiac function and histopathology were monitored in all treatment groups. RESULTS: Eprosartan decreased the severely elevated arterial pressure (-12%; P < 0.05) produced by SFD but did not affect heart rate. Vehicle-treated SP-SFD control rats exhibited significant weight loss (-13%; P < 0.05) and marked mortality (50% by week 6 and 95% by week 9; P < 0.01). Eprosartan-treated SP-SFD rats maintained normal weight, and exhibited zero mortality at week 12 and beyond. Eprosartan prevented the increased urinary protein excretion (P < 0.05) that was observed in vehicle-treated SP-SFD rats. Echocardiographic (i.e. 2-D guided M-mode) evaluation indicated that SP-SFD vehicle control rats exhibited increased septal (+22.2%) and posterior left ventricular wall (+30.0%) thickness, and decreased left ventricular chamber diameter (-15.9%), chamber volume (-32.7%), stroke volume (-48.7%) and ejection fraction (-22.3%), and a remarkable decrease in cardiac output (-59.3%) compared to controls (all P < 0.05). These same parameters in eprosartan-treated SP-SFD rats were normal and differed markedly and consistently from vehicle-treated SP-SFD rats (i.e. treatment prevented pathology; all P < 0.05). Cardiac-gated MRI data confirmed the ability of eprosartan to prevent cardiac pathology/remodeling (P < 0.05). Histopathological analysis of hearts and kidneys indicated that eprosartan treatment significantly reduced end-organ damage (P < 0.01) and provided corroborative evidence that eprosartan reduced remodeling of these organs. Vehicle-treated SP-SFD rats exhibited a 40% increase in the plasma level of pro-atrial natiuretic factor that was reduced to normal by eprosartan (P < 0.05). CONCLUSION: These data demonstrate that eprosartan, at a clinically relevant dose, provides significant end-organ protection in the severely hypertensive stroke-prone rat. It preserves cardiac and renal structural integrity, reduces cardiac hypertrophy and indices of heart failure, maintains normal function of the heart and kidneys, and eliminates premature mortality due to hypertension-induced end-organ failure.
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Acrilatos/uso terapéutico , Antihipertensivos/uso terapéutico , Cardiomegalia/tratamiento farmacológico , Hipertensión/tratamiento farmacológico , Imidazoles/uso terapéutico , Tiofenos , Animales , Factor Natriurético Atrial/sangre , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Riñón/patología , Imagen por Resonancia Magnética , Masculino , Miocardio/patología , Natriuresis/fisiología , Tamaño de los Órganos/efectos de los fármacos , Fragmentos de Péptidos/sangre , Precursores de Proteínas/sangre , Proteinuria/prevención & control , Ratas , Ratas Endogámicas SHR , Accidente Cerebrovascular/prevención & control , Tasa de Supervivencia , Remodelación Ventricular/efectos de los fármacosRESUMEN
Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2'-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 microm DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDP-induced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death.
Asunto(s)
Caspasas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Oxidantes/farmacología , Canales de Potasio/metabolismo , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Benzamidas/farmacología , Inhibidores de Caspasas , Células Cultivadas , Disulfuros/antagonistas & inhibidores , Disulfuros/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neuronas/citología , Neuronas/efectos de los fármacos , Niacinamida/farmacología , Oxidantes/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Técnicas de Placa-Clamp , Fosforilación/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Compuestos de Sulfhidrilo/metabolismo , Reactivos de Sulfhidrilo/antagonistas & inhibidores , Reactivos de Sulfhidrilo/farmacología , Zinc/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The binding of Tyr-D-Arg(2)-Phe-sarcosine(Sar)(4) (TAPS), a proposed mu-opioid receptor-selective tetrapeptide analog of dermorphin to opioid receptors, was studied using selective binding assays for subtypes of mu-, delta- and kappa-opioid receptors. Subtype specific mu-opioid receptor binding was further characterized in the presence of sodium and guanosine nucleotides and the activity of TAPS in isolated guinea pig ileum was compared to other mu-opioid receptor-selective ligands. Further, the antinociceptive properties of TAPS following intrathecal (i.t.) administration in rats, as a model of spinal antinociception, were evaluated. The K(i)-values for TAPS at the mu(1)- and mu(2)-opioid receptor sites were 0.4 and 1.3 nM, respectively, suggesting high affinity binding to mu-opioid receptor binding sites with an increased selectivity to mu(1)-opioid receptor sites. The attenuated reduction of TAPS binding at the mu(2)-opioid receptor subtype in the presence of the stable guanosintriphosphate analog 5'-guanylylimidodiphosphate and sodium suggests a potential partial antagonist mode of action at this site.
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Analgésicos/farmacología , Naloxona/análogos & derivados , Oligopéptidos/farmacología , Receptores Opioides/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Catalepsia/inducido químicamente , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Cobayas , Íleon/efectos de los fármacos , Íleon/fisiología , Técnicas In Vitro , Inyecciones Espinales , Masculino , Contracción Muscular/efectos de los fármacos , Naloxona/farmacología , Nociceptores/efectos de los fármacos , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptidos Opioides , Ventilación Pulmonar/efectos de los fármacos , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Mitogen-activated protein kinases (MAPKs) are involved in many cellular processes. The stress-activated MAPK, p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Here, we demonstrate focal ischemic stroke-induced p38 enzyme activation (i.e., phosphorylation) in the brain. The second generation p38 MAPK inhibitor SB 239063 was identified to exhibit increased kinase selectivity and improved cellular and in vivo activity profiles, and thus was selected for evaluation in two rat models of permanent focal ischemic stroke. SB 239063 was administered orally pre- and post-stroke and intravenously post-stroke. Plasma concentration levels were achieved in excess of those that effectively inhibit p38 activity. In both moderate and severe stroke, SB 239063 reduced infarct size by 28-41%, and neurological deficits by 25-35%. In addition, neuroprotective plasma concentrations of SB 239063 that reduced p38 activity following stroke also reduced the stroke-induced expression of IL-1beta and TNFalpha (i.e., cytokines known to contribute to stroke-induced brain injury). SB 239063 also provided direct protection of cultured brain tissue to in vitro ischemia. This robust SB 239063-induced neuroprotection emphasizes a significant opportunity for targeting MAPK pathways in ischemic stroke injury, and also suggests that p38 inhibition be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Imidazoles/uso terapéutico , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Isquemia Encefálica/metabolismo , Células Cultivadas , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Humanos , Imidazoles/administración & dosificación , Imidazoles/farmacocinética , Interleucina-1/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacocinética , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The aim of the present study was to evaluate p38 MAPK activation following focal stroke and determine whether SB 239063, a novel second generation p38 inhibitor, would directly attenuate early neuronal injury. Following permanent middle cerebral artery occlusion (MCAO), brains were dissected into ischemic and non-ischemic cortices and Western blots were employed to measure p38 MAPK activation. Neurologic deficit and MR imaging were utilized at various time points following MCAO to monitor the development and resolution of brain injury. Following MCAO, there was an early (15 min) activation of p38 MAPK (2.3-fold) which remained elevated up to 1 h (1.8-fold) post injury compared to non-ischemic and sham operated tissue. Oral SB 239063 (5, 15, 30, 60 mg/kg) administered to each animal 1 h pre- and 6 h post MCAO provided significant (P<0.05) dose-related neuroprotection reducing infarct size by 42, 48, 29 and 14%, respectively. The most effective dose (15 mg/kg) was further evaluated in detail and SB 239063 significantly (P<0.05) reduced neurologic deficit and infarct size by at least 30% from 24 h through at least 1 week. Early (i.e. observed within 2 h) reductions in diffusion weighted imaging (DWI) intensity following treatment with SB 239063 correlated (r=0.74, P<0.01) to neuroprotection seen up to 7 days post stroke. Since increased protein levels for various pro-inflammatory cytokines cannot be detected prior to 2 h in this stroke model, the early improvements due to p38 inhibition, observed using DWI, demonstrate that p38 inhibition can be neuroprotective through direct effects on ischemic brain cells, in addition to effects on inflammation.
Asunto(s)
Infarto Cerebral/prevención & control , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Ataque Isquémico Transitorio/fisiopatología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/efectos de los fármacos , Pirimidinas/farmacología , Animales , Muerte Celular/efectos de los fármacos , Infarto Cerebral/patología , Ataque Isquémico Transitorio/patología , Masculino , Arteria Cerebral Media , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neuronas/patología , Ratas , Ratas Endogámicas SHR , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The stress-activated mitogen-activated protein kinase (MAPK) p38 has been linked to the production of inflammatory cytokines/mediators/inflammation and death/apoptosis following cell stress. In these studies, a second-generation p38 MAPK inhibitor, SB 239063 (IC(50) = 44 nM), was found to exhibit improved kinase selectivity and increased cellular (3-fold) and in vivo (3- to 10-fold) activity over first-generation inhibitors. Oral SB 239063 inhibited lipopolysaccharide-induced plasma tumor necrosis factor production (IC(50) = 2.6 mg/kg) and reduced adjuvant-induced arthritis (51% at 10 mg/kg) in rats. SB 239063 reduced infarct volume (48%) and neurological deficits (42%) when administered orally (15 mg/kg, b.i.d.) before moderate stroke. Intravenous SB 239063 exhibited a clearance of 34 ml/min/kg, a volume of distribution of 3 l/kg, and a plasma half-life of 75 min. An i.v. dosing regimen that provided effective plasma concentrations of 0.38, 0.75, or 1.5 microg/ml (i.e., begun 15 min poststroke and continuing over the initial 6-h p38 activation period) was used. Significant and dose-proportional brain penetration of SB 239063 was demonstrated during these infusion periods. In both moderate and severe stroke, intravenous SB 239063 produced a maximum reduction of infarct size by 41 and 27% and neurological deficits by 35 and 33%, respectively. No effects of the drug were observed on cerebral perfusion, hemodynamics, or body temperature. Direct neuroprotective effects from oxygen and glucose deprivation were also demonstrated in organotypic cultures of rat brain tissue. This robust in vitro and in vivo SB 239063-induced neuroprotection emphasizes the potential role of MAPK pathways in ischemic stroke and also suggests that p38 inhibition warrants further study, including protection in other models of nervous system injury and neurodegeneration.
Asunto(s)
Encéfalo/patología , Inhibidores Enzimáticos/uso terapéutico , Imidazoles/uso terapéutico , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/patología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Temperatura Corporal/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Hipocampo/patología , Inflamación/patología , Inflamación/prevención & control , Técnicas de Cultivo de Órganos , Piridinas/uso terapéutico , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas SHR , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Increased levels of endothelin (ET) have been demonstrated in the ischemic brain, and ET receptor antagonism has been shown to improve outcome in cerebral ischemia. However, no previous work has been carried out evaluating the role of ET and its antagonism in brain trauma as compared to experimental stroke. In this study, we evaluated changes in brain ET levels following closed head injury (CHI) and the effects of SB 234551, an endothelin-A- (ET(A)) selective antagonist, and SB 209670, a mixed endothelin-A- and -B- (ET(A)/ET(B)) antagonist, on outcome in CHI and focal stroke. Male Sabra rats were subjected to CHI (weight drop model). Male Sprague Dawley rats were subjected to focal stroke (intraluminal suture model). Motor function(s) were assessed and immunoreactive ET (irET) and the degree of cerebral edema were measured for 24 h after CHI. Brain swelling (edema), neurological deficits and forebrain infarct volumes were measured 24 h after focal stroke. Antagonists (total doses of 7.5, 15, 30 or 60 mg/kg) were administered intravenously for 6-24 h (beginning 15 min after injury). Control rats were infused with vehicle. CHI resulted in increased ET levels in the directly contused hemisphere at 12 and 24 h. In addition, SB 234551 significantly reduced neurological deficits (decreased 30%) and brain edema (decreased 40%) following CHI (p < 0.05 at 60 mg/kg dose). SB 209670 had no effects on CHI outcome. Focal stroke studies yielded similar results. SB 234551 reduced focal stroke-induced neurological deficits by 50%, brain swelling by 54% and the degree of infarction by 36% (p < 0.05 at 30 mg/kg). SB 209670 did not provide any neuroprotection in focal stroke. These data indicate that ET plays a significant role in the pathophysiology of CHI, and that selectively targeting ET(A)-receptors similarly in both CHI and stroke might be a therapeutic opportunity.
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Traumatismos Craneocerebrales/tratamiento farmacológico , Dioxoles/uso terapéutico , Antagonistas de los Receptores de Endotelina , Indanos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Pirazoles/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Endotelina-1/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina ARESUMEN
The importance of cytokines, especially TNF-alpha and IL-1beta, are emphasised in the propagation and maintenance of the brain inflammatory response to injury. Much data supports the case that ischaemia and trauma elicit an inflammatory response in the injured brain. This inflammatory response consists of mediators (cytokines, chemokines and adhesion molecules) followed by cells (neutrophils early after the onset of brain injury and then a later monocyte infiltration). De novo upregulation of pro-inflammatory cytokines, chemokines and endothelial-leukocyte adhesion molecules occurs soon after focal ischaemia and trauma, as well as at the time when the tissue injury is evolving. The significance of this brain inflammatory response and its contribution to brain injury is now becoming more understood. In this review, we discuss the role of TNF-alpha and IL-1beta in traumatic and ischaemic brain injury and associated inflammation and the co-operative actions of chemokines and adhesion molecules in this process. We also address novel approaches to target cytokines and reduce the brain inflammatory response and thus brain injury, in stroke and neurotrauma. The mitogen-activated protein kinase (MAPK), p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Stroke-induced p38 enzyme activation in the brain has been demonstrated and treatment with a second generation p38 MAPK inhibitor, SB-239063, provides a significant reduction in infarct size, neurological deficits and inflammatory cytokine expression produced by focal stroke. SB-239063 can also provide direct protection of cultured brain tissue to in vitro ischaemia. This robust SB-239063-induced neuroprotection emphasises a significant opportunity for targeting MAPK pathways in ischaemic stroke injury and also suggests that p38 inhibition should be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.
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Antiinflamatorios/uso terapéutico , Lesiones Encefálicas/tratamiento farmacológico , Moléculas de Adhesión Celular/metabolismo , Citocinas/metabolismo , Expresión Génica/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Lesiones Encefálicas/fisiopatología , Moléculas de Adhesión Celular/efectos de los fármacos , Citocinas/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Accidente Cerebrovascular/fisiopatologíaRESUMEN
The pharmacological and pharmacokinetic profile of SB-222200 [(S)-(-)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-car boxami de], a human NK-3 receptor (hNK-3R) antagonist, was determined. SB-222200 inhibited (125)I-[MePhe(7)]neurokinin B (NKB) binding to Chinese hamster ovary (CHO) cell membranes stably expressing the hNK-3 receptor (CHO-hNK-3R) with a K(i) = 4.4 nM and antagonized NKB-induced Ca(2+) mobilization in HEK 293 cells stably expressing the hNK-3 receptor (HEK 293-hNK-3R) with an IC(50) = 18.4 nM. SB-222200 was selective for hNK-3 receptors compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 250 nM). In HEK 293 cells transiently expressing murine NK-3 receptors (HEK 293-mNK-3R), SB-222200 inhibited binding of (125)I-[MePhe(7)]NKB (K(i) = 174 nM) and antagonized NKB (1 nM)-induced calcium mobilization (IC(50) = 265 nM). In mice oral administration of SB-222200 produced dose-dependent inhibition of behavioral responses induced by i.p. or intracerebral ventricular administration of the NK-3 receptor-selective agonist, senktide, with ED(50) values of approximately 5 mg/kg. SB-222200 effectively crossed the blood-brain barrier in the mouse and rat. The inhibitory effect of SB-222200 against senktide-induced behavioral responses in the mouse correlated significantly with brain, but not plasma, concentrations of the compound. Pharmacokinetic evaluation of SB-222200 in rat after oral administration (8 mg/kg) indicated sustained plasma concentrations (C(max) = about 400 ng/ml) and bioavailability of 46%. The preclinical profile of SB-222200, demonstrating high affinity, selectivity, reversibility, oral activity, and central nervous system penetration, suggests that it will be a useful tool compound to define the physiological and pathophysiological roles of NK-3 receptors, in particular in the central nervous system.
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Encéfalo/efectos de los fármacos , Quinolinas/farmacología , Receptores de Neuroquinina-3/antagonistas & inhibidores , Animales , Encéfalo/metabolismo , Células CHO , Calcio/metabolismo , Cricetinae , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Iris/efectos de los fármacos , Iris/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/farmacología , Quinolinas/farmacocinética , Conejos , Ratas , Ratas Sprague-Dawley , Sustancia P/análogos & derivados , Sustancia P/farmacologíaRESUMEN
Increasing evidence supports a role for oxidative stress, proinflammatory cytokines, and apoptosis in the pathophysiology of focal ischemic stroke. Previous studies have found that the multi-action drug, carvedilol, is a mixed adrenergic antagonist, and that it behaves as an antioxidant and inhibits apoptosis. In the current study, the authors investigated whether carvedilol provides protection in focal cerebral ischemia and whether this protection is associated with reduced apoptosis and the downregulation of the inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin- 1beta (IL-1beta). Male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) by an intraluminal filament technique. Carvedilol (1, 3, and 10 mg/kg) was injected daily subcutaneously 2 or 4 days before the induction of ischemia. Neurologic scores, infarct volumes, TUNEL staining, and mRNA levels of TNF-alpha and IL-1beta were assessed at 24 hours reperfusion. The effect of carvedilol on microvascular cortical perfusion was studied with continuous laser-Doppler flowmetry. Twenty-four hours after MCAO, carvedilol at all three doses reduced infarct volumes by at least 40% and reduced neurologic deficits on average by 40% compared with vehicle-treated controls when given 2 or 4 days before the induction of ischemia. This protection was not mediated by changes in temperature or blood flow. Treatment with all three dose regimens resulted in fewer TUNEL positive cells compared with controls. At 24 hours reperfusion, carvedilol decreased TNF-alpha and IL-1beta expression by 40% to 50% in the ipsilateral ischemic cortex compared with the contralateral controls. The results of the current study indicate that carvedilol is neuroprotective in focal cerebral ischemia and may protect the ischemic brain by inhibiting apoptosis and attenuating the expression of TNF-alpha and IL-1beta.
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Antagonistas Adrenérgicos beta/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Carbazoles/farmacología , Ataque Isquémico Transitorio/patología , Fármacos Neuroprotectores/farmacología , Propanolaminas/farmacología , Accidente Cerebrovascular/patología , Animales , Apoptosis , Carvedilol , Corteza Cerebral/irrigación sanguínea , Infarto Cerebral/patología , Circulación Cerebrovascular , Interleucina-1/metabolismo , Masculino , Sistema Nervioso/fisiopatología , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
For progression to clinical trials in stroke, putative neuroprotective compounds should show robust efficacy post-ischaemia in several experimental models of stroke. This paper describes the characterisation of (+)(1S, 2R)-cis-1-[4-(1-methyl-1-phenylethyl)phenoxy]-2-methylamino indane hydrochloride (SB-221420-A), a Ca(2+) and Na(+) channel antagonist. SB-221420-A inhibited (IC(50)=2.2 microM) N-type voltage-operated Ca(2+) channel currents in cultured superior cervical ganglion neurons, which were pretreated with 10 microM nimodipine to block L-type voltage-operated Ca(2+) channel currents. In dorsal root ganglion neurons pretreated with 1 microM omega-conotoxin GVIA to block N-type voltage-operated Ca(2+) channel currents, SB-221420-A inhibited the residual Ca(2+) current with an IC(50) of 7 microM. SB-221420-A also inhibited Na(+) currents in dorsal root ganglion neurons with an IC(50) of 8 microM. In rats, the pharmacokinetic profile of SB-221420-A shows that it has a half-life of 6.4 h, a high volume of distribution, is highly brain penetrating, and has no persistent metabolites. Following bilateral carotid artery occlusion in gerbils, SB-221420-A significantly reduced the level of ischaemia-induced hyperlocomotor activity and the extent of hippocampal CA1 cell loss compared to the ischaemic vehicle-treated group. SB-221420-A was also effective in focal models of ischaemia. In the mouse permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously, post-ischaemia significantly (P<0.05) reduced lesion volume compared to the ischaemic vehicle-treated group. In the normotensive rat permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously over 1 h, beginning 30 min postmiddle cerebral artery occlusion, significantly (P<0.05) reduced lesion volume from 291+/-16 to 153+/-30 mm(3), compared to ischaemic vehicle-treated controls when measured 24 h postmiddle cerebral artery occlusion. Efficacy was maintained when the same total dose of SB-221420-A was infused over a 6-h period, beginning 30 min postmiddle cerebral artery occlusion. SB-221420-A also significantly (P<0.05) reduced lesion volume following transient middle cerebral artery occlusion in normotensive rats and permanent middle cerebral artery occlusion in spontaneously hypertensive rats (SHR). Investigation of the side effect profile using the Irwin screen in mice revealed that, at neuroprotective doses, there were no overt behavioural or cardiovascular changes. These data demonstrate that robust neuroprotection can be seen post-ischaemia with SB-221420-A in both global and focal ischaemia with no adverse effects at neuroprotective doses, and indicate the potential utility of a mixed cation blocker to improve outcome in cerebral ischaemia.
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Bloqueadores de los Canales de Calcio/farmacología , Indanos/farmacología , Fármacos Neuroprotectores/farmacología , Bloqueadores de los Canales de Sodio , Accidente Cerebrovascular/prevención & control , Anestesia , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Estenosis Carotídea/fisiopatología , Estenosis Carotídea/prevención & control , Células Cultivadas , Estado de Conciencia , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Gerbillinae , Hemodinámica/efectos de los fármacos , Hipertensión/fisiopatología , Indanos/farmacocinética , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/prevención & control , Ataque Isquémico Transitorio/fisiopatología , Ataque Isquémico Transitorio/prevención & control , Masculino , Potenciales de la Membrana/efectos de los fármacos , Tasa de Depuración Metabólica , Ratones , Actividad Motora/efectos de los fármacos , Neuronas Aferentes/citología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Accidente Cerebrovascular/fisiopatología , Distribución TisularRESUMEN
Two relatively well characterised kinase signalling pathways are those involving MAPK/ERK and p38/SAPK2, that are known to be activated in vitro by various factors known to increase following stroke, such as glutamate, IL-1 and TNF. The present study was designed to investigate the activation and cellular distribution of phosphorylated-ERK1/2, -p38 and the transcription factor CREB following focal cerebral ischaemia using phosphospecific antibodies. Up to 24 h following transient MCAO (90 min) and 6 h following permanent MCAO, phospho-ERK1/2 staining was markedly increased within the cytoplasm of neuronal perikarya in 'penumbral-like' regions. In contrast, phospho-p38 immunostaining was markedly increased in cells with astrocyte-like morphology in both 'core' and 'penumbral-like' regions. Phospho-p38 staining was also detected in some neurones within 'penumbral-like' regions up to 24 h following transient MCAO. CREB activation was confined to neurones in 'penumbral-like' regions. Increased phospho-p38 immunoreactivity was detected in astrocyte-like cells present in the subcortical white matter ipsilateral to the occluded MCAO, while phospho-CREB and -ERK1/2 staining was localised to cells with the morphological appearance of oligodendrocytes. This study demonstrates phosphorylation, indicative of activation, of both the MAPK and p38 pathways following transient and permanent MCAO. However, each pathway shows a distinct cellular and spatial distribution within ischaemic tissue. Together these data indicate that neuroprotection offered by agents directed towards the ERK1/2 pathway may act directly through protection of neurones and oligodendrocytes, while those directed towards the p38 pathway kinase signalling pathways may be indirectly via inhibition of cytokines and other mediators involved in the brains response to injury.
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Isquemia Encefálica/enzimología , Encéfalo/enzimología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ataque Isquémico Transitorio/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroglía/enzimología , Neuronas/enzimología , Animales , Encéfalo/fisiopatología , Isquemia Encefálica/fisiopatología , Corteza Cerebral/enzimología , Corteza Cerebral/fisiopatología , Activación Enzimática , Ataque Isquémico Transitorio/fisiopatología , Masculino , Proteína Quinasa 3 Activada por Mitógenos , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
We describe here the cloning and characterization of a rat homolog of the chemokine receptor CXCR3. The predicted amino acid sequence of rat CXCR3 contains 367 amino acid residues, sharing 96 and 87% amino acid sequence identity to the murine and human CXCR3, respectively. Among a large panel of chemokines tested, only interferon-inducible protein-10 (IP-10), interferon-gamma-induced monokine, and interferon-inducible T cell alpha-chemoattractant demonstrated specific abilities to induce an intracellular calcium mobilization response in human embryonic kidney 293 cells transfected with rat CXCR3 expression vector. (125)I-IP-10 competition binding studies to the CXCR3-transfected human embryonic kidney 293 cells demonstrated that human IP-10 and interferon-inducible T cell alpha-chemoattractant are more potent ligands than human interferon-gamma-induced monokine. Following our previous observation for the induced expression of IP-10 in focal stroke, we demonstrate here the time-dependent up-regulation of CXCR3 mRNA in the rat ischemic cortex after permanent occlusion of the middle cerebral artery. A significant increase in (125)I-IP-10-specific binding to ischemic cerebral cortical samples was obtained and paralleled the increase in CXCR3 mRNA expression. The changes in receptor expression and ligand binding correlate highly with known changes in leukocyte accumulation, and gliosis occurred after focal stroke. These data suggest that CXCR3/IP-10 may be a potential novel therapeutic target in focal stroke. In addition, the cloning of rat CXCR3 provides an important tool for the investigation of the pathophysiological role of CXCR3 in other rodent disease models.
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Quimiocinas CXC/metabolismo , Receptores de Quimiocina/genética , Accidente Cerebrovascular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Isquemia Encefálica/metabolismo , Células Cultivadas , Enfermedades Arteriales Cerebrales/metabolismo , Corteza Cerebral/metabolismo , Quimiocina CXCL10 , Clonación Molecular , ADN Complementario/análisis , Humanos , Radioisótopos de Yodo , Masculino , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Ensayo de Unión Radioligante , Ratas , Ratas Endogámicas SHR , Receptores CXCR3 , Receptores de Quimiocina/biosíntesis , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
We have established a focal preconditioning (PC) paradigm that produces significant and prolonged ischemic tolerance (IT) of the brain to subsequent permanent middle cerebral artery occlusion (MCAO). PC using 10 min of MCAO induces brain tolerance at 1-7 days of reperfusion that requires active protein synthesis. The protective protein(s) involved are unknown. In these studies the increased transcription and translation of the inducible 70-kDa heat shock protein (Hsp70) and the 27-kDa heat shock protein (Hsp27), and astrogliosis/glial fibrillary acidic protein (GFAP) were determined by Northern analysis and immunohistochemistry following PC. Cellular localization of proteins was determined by double labeling. PC produced no brain injury but did increase Hsp70 mRNA transiently at 6 h and increased Hsp27 mRNA later at 24 h for at least 5 days. Protein expression induced by PC exhibited a similar profile. Hsp70 protein was primarily expressed in neurons from 1 to 5 days post-PC throughout the PC cortex. Hsp27 protein expression was initiated later for a much longer period of time. A remarkable astroglyosis was verified with increased astrocytic Hsp27 from 1 to 7 days after PC. Gliosis with increased Hsp27 in the PC cortex was still present but reduced 4 weeks after PC. Therefore, PC that results in brain tolerance/neuroprotection increases neuronal Hsp70 in the PC cortex and activated astrocytic Hsp27 in the PC cortex in a temporal fashion associated with developing IT. The short duration of benign ischemia (PC) that produces IT produces a robust, long-lived cellular and protein synthetic response that extends throughout the entire cortex (i.e. well beyond the MCA perfusion territory). The resulting IT is associated with changes in astrocyte-activation that might provide increased support and protection from injury. Although both Hsp70 and Hsp27 may participate in the neuroprotection/brain tolerance induced by PC, the temporal expression patterns of these proteins indicate that they are not solely responsible for the tolerance to brain injury.