RESUMEN
The functions of sleep and its links with neuropsychiatric diseases have long been questioned. Among the numerous hypotheses on sleep function, early studies proposed that sleep helps to replenish glycogen stores consumed during waking. Later studies found increased brain glycogen after sleep deprivation, leading to "glycogenetic" hypothesis, which states that there is a parallel increase in synthesis and utilization of glycogen during wakefulness, whereas decrease in the excitatory transmission creates an imbalance causing accumulation of glycogen during sleep. Glycogen is a vital energy reservoir to match the synaptic demand particularly for re-uptake of potassium and glutamate during intense glutamatergic transmission. Therefore, sleep deprivation-induced transcriptional changes may trigger migraine by reducing glycogen availability, which slows clearance of extracellular potassium and glutamate, hence, creates susceptibility to cortical spreading depolarization, the electrophysiological correlate of migraine aura. Interestingly, chronic stress accompanied by increased glucocorticoid levels and locus coeruleus activity and leading to mood disorders in which sleep disturbances are prevalent, also affects brain glycogen turnover via glucocorticoids, noradrenaline, serotonin and adenosine. These observations altogether suggest that inadequate astrocytic glycogen turnover may be one of the mechanisms linking migraine, mood disorders and sleep.
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Depresión , Glucógeno , Encéfalo/metabolismo , Glucógeno/metabolismo , Cefalea , Humanos , SueñoRESUMEN
Autoimmune and dysimmune inflammatory mechanisms on a genetically susceptible background are implicated in the etiology of Behçet's Disease (BD). Heat-shock protein-65 (HSP-65) derived from Streptococcus sanguinis was proposed as a triggering factor based on its homology with human HSP-60. However, none of the autoantigens identified so far in sera from BD share common epitopes with bacterial HSP-65 or has a high prevalence. Here, we report that sera from BD patients are immunoreactive against filamentous neuronal processes in the mouse brain, retina and scrotal skin in great majority of patients. By using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting, Western blotting and peptide blocking experiments, we have identified neurofilament medium (NF-M) as the probable antigen for the serologic response observed. Clustal Omega analyses detected significant structural homology between the human NF-M and bacterial HSP-65 corresponding to amino acids 111-126, 213-232 and 304-363 of mycobacterial HSP-65, which were previously identified to induce proliferation of lymphocytes obtained from BD patients. We also found that sera immunoreactive against NF-M cross-reacted with bacterial HSP-65. These findings suggest that NF-M may be involved in autoimmunity in BD due to its molecular mimicry with bacterial HSP-65.
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Autoantígenos/inmunología , Proteínas Bacterianas/inmunología , Síndrome de Behçet/inmunología , Chaperonina 60/inmunología , Epítopos de Linfocito B/inmunología , Proteínas de Choque Térmico/inmunología , Proteínas de Neurofilamentos/inmunología , Neuronas/fisiología , Streptococcus sanguis/inmunología , Adulto , Animales , Anticuerpos/sangre , Autoantígenos/genética , Proteínas Bacterianas/genética , Encéfalo/patología , Células Cultivadas , Chaperonina 60/genética , Reacciones Cruzadas , Epítopos de Linfocito B/genética , Femenino , Predisposición Genética a la Enfermedad , Proteínas de Choque Térmico/genética , Humanos , Masculino , Ratones , Persona de Mediana Edad , Homología Estructural de Proteína , Adulto JovenRESUMEN
Aquaporins (AQPs), members of the water-channel protein family, are highly expressed in brain tissue especially in astrocytic end-feet. They are important players for water hemostasis during development of cytotoxic as well as vasogenic edema. Increased expression of AQPs is important in pathophysiology of neurological diseases such as neuroinflammation and ischemia. Unfortunately, there are a few pharmacological inhibitors of AQP4 with several side effects limiting their translation as a drug for use in clinical conditions. Another therapeutic approach is using antisense oligonucleotides (ASOs) to block AQP4 activity. These are short, synthetic, modified nucleic acids that bind RNA to modulate its function. However, they cannot pass the blood brain barrier (BBB). To overcome this obstacle we designed a nanoparticulate system made up of chitosan nanoparticles surface modified with PEG and conjugated with monoclonal anti transferrin receptor-1 antibody via streptavidin-biotin binding. The nanocarrier system could be targeted to the transferrin receptor-1 at the brain endothelial capillaries through monoclonal antibodies. It is hypothesized that the nanoparticles could pass the BBB via receptor mediated transcytosis and reach brain parenchyma. Particle size, zeta potential, loading capacity and release profiles of nanoparticles were investigated. It was observed that all types of chitosau (CS) nanoparticles had positive zeta potential values and nanoparticle particle size distribution varied between 100 and 800 nm. The association efficiency of ASOs into the nanoparticles was between 80-97% and the release profiles of the nanoparticles exhibited an initial burst effect followed by a controlled release. The results showed that the designed chitosan based nanocarriers could be a promising carrier system to transport nucleic acid based drugs to brain parenchyma.
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Acuaporina 4/antagonistas & inhibidores , Acuaporina 4/genética , Encéfalo/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , Edema Encefálico/tratamiento farmacológico , Química Farmacéutica , Quitosano , Composición de Medicamentos , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Electroquímica , Nanopartículas , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Similarities between laboratory animals and humans in anatomy and physiology of the cephalic nociceptive pathways have allowed scientists to create successful models that have significantly contributed to our understanding of headache. They have also been instrumental in the development of novel anti-migraine drugs different from classical pain killers. Nevertheless, modelling the mechanisms underlying primary headache disorders like migraine has been challenging due to limitations in testing the postulated hypotheses in humans. Recent developments in imaging techniques have begun to fill this translational gap. The unambiguous demonstration of cortical spreading depolarization (CSD) during migraine aura in patients has reawakened interest in studying CSD in animals as a noxious brain event that can activate the trigeminovascular system. CSD-based models, including transgenics and optogenetics, may more realistically simulate pain generation in migraine, which is thought to originate within the brain. The realization that behavioural correlates of headache and migrainous symptoms like photophobia can be assessed quantitatively in laboratory animals, has created an opportunity to directly study the headache in intact animals without the confounding effects of anaesthetics. Headache and migraine-like episodes induced by administration of glyceryltrinitrate and CGRP to humans and parallel behavioural and biological changes observed in rodents create interesting possibilities for translational research. Not unexpectedly, species differences and model-specific observations have also led to controversies as well as disappointments in clinical trials, which, in return, has helped us improve the models and advance our understanding of headache. Here, we review commonly used headache and migraine models with an emphasis on recent developments.
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Modelos Animales de Enfermedad , Cefalea , Trastornos Migrañosos , Animales , Degranulación de la Célula , Cefalea/tratamiento farmacológico , Cefalea/fisiopatología , Humanos , Mastocitos/fisiología , Arterias Meníngeas/fisiología , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/fisiopatología , Inflamación Neurogénica , Neuropéptidos/sangre , Proteínas Proto-Oncogénicas c-fos/metabolismo , Nervio Trigémino/fisiologíaRESUMEN
Mesial temporal lobe epilepsy (MTLE) is often characterized pathologically by severe neuronal loss in the hippocampus. In this study we investigated concomitant appearance of the pro-apoptotic and anti-apoptotic mechanisms in injured neurons in epileptic human hippocampi. Postsurgical hippocampal specimens of randomly selected 25 patients with MTLE were studied with standard immunohistochemical techniques to detect the below markers of cell death pathways: truncated Bid - tBid, mitochondrial translocation of Bax (markers of pro-apoptotic Bcl-2 protein activation) and nuclear translocation of AIF (caspase-independent pro-apoptotic pathway). For cell survival pathways, we investigated the expression of c-IAP1, c-IAP2 and Hsp70 (heat shock protein). Immunopositive cells were counted in different regions of the hippocampus. We also verified IAP (inhibitor of apoptosis) expression with Western blotting. The results were statistically compared with hippocampi from non-epileptic autopsy controls. In patient hippocampi, Bax and tBid immunoreactivity were significantly increased and Bax staining was consistent with mitochondrial translocation. AIF was not translocated to the nucleus. c-IAP1 and c-IAP2 were barely detectable in control hippocampi, whereas their expression was dramatically increased in the patients in all hippocampal subfields. Interestingly, these neurons were also positively co-labeled for tBid and translocated Bax. Hsp70 immunreactivity was significantly increased in all surviving neurons in patient hippocampi whereas degenerating neurons failed to express Hsp70. Our findings are consistent with both pro-apoptotic and anti-apoptotic mechanisms being active within the same hippocampal neurons of patients with MTLE, illustrating an ongoing struggle between cell death and survival mechanisms in neurons under stress.
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Apoptosis/fisiología , Epilepsia del Lóbulo Temporal/patología , Hipocampo/patología , Neuronas/patología , Adolescente , Adulto , Factor Inductor de la Apoptosis/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Supervivencia Celular/fisiología , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Neuronas/metabolismo , Grabación en Video , Adulto Joven , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Alpha-phenyl-n-tert-butyl nitrone (PBN) shows its major effect by scavenging free radicals formed in the ischemia and it has the ability to penetrate through the blood brain barrier easily. The in vivo stability of PBN is very low and when administered systemically, it has a mean plasma half life of about three hours. Therefore, formulations which are able to prolong the plasma residence time of PBN are of major interest, because oxygen radicals are usually continuously formed under pathological conditions. In this study, PBN, a nitrone compound having neuroprotective properties, was encapsulated in chitosan (CS) and chitosan-poly(ethylene glycol) (CS-PEG) nanoparticles for treatment of diseases such as stroke, in which sustained free radical production is reported. The nanoparticles were characterized through particle size determination, zeta potential, encapsulation efficiency, surface morphology determinations and in vitro release studies. The surface morphologies were evaluated by transmission electron microscopy (TEM) and nanoparticles having spherical shapes were characterized. The particle size distribution was between approximately 97 nm and approximately 322 nm; and the zeta potentials varied between approximately 9 mV and approximately 33 mV. Size of the nanoparticle formulations was important for the release of PBN from nanoparticles. The quantitative determination of PBN has been evaluated by a validated analytical HPLC method. The presented chitosan-based nanotechnology opens new perspectives for testing antioxidant activity in vivo.
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Óxidos N-Cíclicos/administración & dosificación , Óxidos N-Cíclicos/química , Depuradores de Radicales Libres/administración & dosificación , Depuradores de Radicales Libres/química , Química Farmacéutica , Quitosano , Composición de Medicamentos , Electroquímica , Excipientes , Nanopartículas , Tamaño de la Partícula , Polietilenglicoles/químicaRESUMEN
Migraine headaches have a complex pathophysiology; both vascular and neuronal mechanisms have been proposed. One possible scenario begins with brain-initiated events evolving to cortical spreading depression (CSD), which in turn activates the trigeminal nerve to cause headaches. Experimental evidence supports a relationship between CSD as a cause of migraine aura as well as CSD as a cause of trigeminal activation. Susceptibility to CSD and to migraine appears to be genetically determined. In some migraine subtypes, genes controlling translocation of calcium, sodium and potassium have been implicated, perhaps altering the susceptibility to CSD. This chapter briefly reviews current knowledge pertaining to migraine pathophysiology with emphasis on current notions linking disturbances in ion flux to the genesis of headache.
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Circulación Cerebrovascular/fisiología , Depresión de Propagación Cortical/fisiología , Migraña con Aura/fisiopatología , Nervio Trigémino/fisiopatología , Humanos , Inflamación NeurogénicaRESUMEN
Hippocampal sclerosis constitutes the most frequent neuropathological finding in patients with medically intractable mesial temporal lobe epilepsy. Serial analysis of gene expression was used to get a global view of the gene profile in human hippocampus in control condition and in epileptic condition associated with hippocampal sclerosis. Libraries were generated from control hippocampus, obtained by rapid autopsy, and from hippocampal surgical specimens of patients with mesial temporal lobe epilepsy and the classical pattern of hippocampal sclerosis. More than 50,000 tags were analyzed (28,282, control hippocampus; 25,953, hippocampal sclerosis) resulting in 9206 (control hippocampus) and 9599 (hippocampal sclerosis) unique tags (genes), each representing a specific mRNA transcript. Comparison of the two libraries resulted in the identification of 143 transcripts that were differentially expressed. These genes belong to a variety of functional classes, including basic metabolism, transcription regulation, protein synthesis and degradation, signal transduction, structural proteins, regeneration and synaptic plasticity and genes of unknown identity of function. The database generated by this study provides an extensive inventory of genes expressed in human control hippocampus, identifies new high-abundant genes associated with altered hippocampal morphology in patients with mesial temporal lobe epilepsy and serves as a reference for future studies aimed at detecting hippocampal transcriptional responses under various pathological conditions.
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Epilepsia del Lóbulo Temporal/genética , Regulación de la Expresión Génica , Hipocampo/fisiopatología , Secuencia de Bases , Cartilla de ADN , Enzimas/genética , Etiquetas de Secuencia Expresada , Hipocampo/patología , Humanos , Regeneración Nerviosa/genética , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/genética , ARN/genética , ARN/aislamiento & purificación , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Treatment of central nervous system (CNS) diseases with potentially useful pharmaceuticals is prevented by the blood-brain barrier (BBB). The BBB is a unique protective barrier in the body. It is formed by epithelial-like tight junctions, which are expressed by the brain capillary endothelial cells. Although most molecules are potentially active in the CNS, they cannot readily enter the brain because of their properties. Antisense oligonucleotides (ODNs) have a great potential as neuropharmaceuticals; however, the large size and polar nature of nucleic acid drugs prevent these molecules from bypassing the BBB and readily entering the CNS following systemic administration. One approach to improve both the pharmacokinetics and the pharmacodynamics of ODNs involves the use of sustained-release polymer formulations, such as poly(lactide-co-glycolide) (PLGA) nanoparticulate systems. In this study, nanospheres were prepared by the emulsification diffusion technique and characterized in terms of particle size, surface morphology, encapsulation efficiency, in vitro release profiles and ODN stability. The nanospheres produced were spherical with homogenous size distribution. Nanospheres were prepared with different encapsulation efficiency. Release profiles of formulations were also evaluated. The results show that formulations with different ODN content exhibited different release profiles. Moreover, the chemical integrity of ODN during the processes was conserved. These results demonstrate that a stable ODN formulation could be prepared utilizing PLGA nanospheres as a potential delivery system for the treatment of CNS diseases.
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Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Nanotubos/química , Oligonucleótidos Antisentido/administración & dosificación , Receptores de N-Metil-D-Aspartato/genética , Química Farmacéutica/métodos , Preparaciones de Acción Retardada , Portadores de Fármacos , Estabilidad de Medicamentos , Electroforesis en Gel de Poliacrilamida , Humanos , Ácido Láctico/química , Microscopía Electrónica de Rastreo , Oligonucleótidos Antisentido/farmacocinética , Tamaño de la Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/químicaRESUMEN
In the present study, the preparation and characterization of bovine serum albumin (BSA) microspheres and the evaluation of the in vitro cytotoxicity of these microspheres on acute promyelocytic leukaemia (HL-60) cells were described. Mitoxantrone (MTZ)-incorporated microspheres were evaluated for particle size, drug loading, release characteristics and surface morphology. The biological effect of MTZ released from BSA microspheres was determined on an in vitro cultured HL-60 cell line, showing that, after encapsulation, MTZ still retains cytotoxic activity. For this purpose, methyl-thiazol-tetrazolium (MTT) assay was used to evaluate the in vitro cytotoxicity of MTZ-loaded microspheres. Particle size of BSA microspheres was determined between 17.61-20.38 microm and they were smooth and spherical in shape. Encapsulation efficiency of the drug-loaded microspheres was between 22.26-60.50%. For MTZ-containing microspheres, the cell death ratios were greater than 80% for all formulations. This study demonstrate that BSA microspheres were well suited for the controlled release of MTZ and were promising for anti-cancer chemotherapy.
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Antibióticos Antineoplásicos/uso terapéutico , Células HL-60/efectos de los fármacos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Mitoxantrona/uso terapéutico , Animales , Antibióticos Antineoplásicos/farmacocinética , Portadores de Fármacos , Composición de Medicamentos/métodos , Humanos , Microscopía Electrónica de Rastreo , Microesferas , Mitoxantrona/farmacocinética , Tamaño de la Partícula , Albúmina Sérica Bovina , Propiedades de SuperficieAsunto(s)
Lesiones Encefálicas/patología , Propidio/administración & dosificación , Animales , Apoptosis , Biomarcadores , Bisbenzimidazol , Encéfalo/metabolismo , Encéfalo/patología , Muerte Celular , Hipoxia-Isquemia Encefálica/diagnóstico , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/patología , Etiquetado Corte-Fin in Situ , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Inyecciones Intravenosas , Inyecciones Intraventriculares , Ratones , Microscopía Fluorescente , Necrosis , Propidio/farmacocinética , Juego de Reactivos para DiagnósticoRESUMEN
A research proposal should start with defining its aims. A synopsis of the literature and observations that the hypothesis is grounded should be given together with relevant references. The significance of the project, expected contributions to the field should also be indicated. If available, preliminary data enhance the impact of the proposal. A detailed description of the methods and subjects to be used is an important part of the proposal. What data are to be collected, the method of collecting data, and selection criteria for subjects should be indicated. Variables and how the measurements will be taken, have to be defined precisely. Data processing and analysis tools should be described. Additionally, the expected time table, project cost, the ethical and legal issues should be included in the proposal. It should be kept in mind that a scientist's primary responsibility is to create conditions such that the hypothesis can be tested objectively rather than proving his hypothesis.
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Ensayos Clínicos como Asunto/métodos , Neurocirugia , Proyectos de Investigación , Ensayos Clínicos como Asunto/ética , Recolección de Datos/ética , Ética en Investigación , Humanos , Apoyo a la Investigación como Asunto/organización & administraciónRESUMEN
Brief cerebral ischemia is reported to cause selective neuronal necrosis, apoptotic cell death, silent infarcts and, when recurrent, cognitive decline. Acute administration of selegiline and EGb 761 have been shown to have anti-apoptotic and neuroprotective effects in experimental ischemia. Their daily use is currently advised to slow down cognitive decline in patients with vascular dementia. Hence, unlike previous studies, we studied the neuroprotective action of chronic daily administration of these drugs in Swiss mice subjected to 30-min middle cerebral artery occlusion and 72 h of reperfusion since this model was reported to induce a slowly evolving infarct with profuse apoptotic cell death. Infarct area was evaluated by H&E staining on coronal brain sections and, apoptotic cells were identified by histological criteria, terminal transferase-mediated d-UTP nick-end labeling (TUNEL) and by immunohistochemical detection of caspase-cleaved actin fragments (fractin). Fifty-one mice received daily intraperitoneal injections of 10 mg/kg selegiline (n=18) or 50 mg/kg EGb 761 (n=17) or equal volume of saline (n=16) for 10-14 days before but not on the day of insult. The infarct volume, number of TUNEL- and fractin-positive cells were significantly reduced in treatment groups by 30, 42 and 51% (selegiline) and, 27, 27 and 29% (EGb 761), respectively. These data suggest that prophylactic use of selegiline and EGb 761 could increase the brain's resistance to mild ischemic injury.
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Isquemia Encefálica/prevención & control , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/administración & dosificación , Selegilina/administración & dosificación , Selegilina/farmacología , Actinas/química , Animales , Apoptosis , Caspasas/fisiología , Infarto Cerebral/patología , Esquema de Medicación , Ginkgo biloba , Inmunidad Innata , Etiquetado Corte-Fin in Situ , Ratones , Neuronas/metabolismo , Neuronas/fisiología , Extractos Vegetales/farmacologíaRESUMEN
Nitric oxide can promote or inhibit apoptosis depending on the cell type and coexisting metabolic or experimental conditions. We examined the impact of nitric oxide on development of apoptosis 6, 24, and 72 h after permanent middle cerebral artery occlusion in mutant mice that lack the ability to generate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal sections passing through the anterior commissure were stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Immunoblotting was used to identify changes in the anti- and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of caspases was assessed by appearance of actin cleavage products using a novel antiserum directed against 32-kDa actin fragment (fractin). In the neuronal nitric oxide synthase mutant mouse, infarct size and TUNEL positive apoptotic neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 levels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-mediated actin cleavage appeared in the ischemic hemisphere at this time point, and was significantly less in mutant brains at 72 h compared to the wild-type. The reduction in the number of TUNEL and fractin positive apoptotic cells appears far greater than anticipated based on the smaller lesion size in mutant mice.Hence, from these data we suggest that a deficiency in neuronal nitric oxide production slows the development of apoptotic cell death after ischemic injury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases.
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Apoptosis/fisiología , Isquemia Encefálica/enzimología , Encéfalo/enzimología , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/fisiología , Actinas/metabolismo , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Caspasas/metabolismo , Colorantes/farmacología , Fragmentación del ADN/fisiología , Eosina Amarillenta-(YS)/farmacocinética , Femenino , Hematoxilina/farmacocinética , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Noqueados , Neuronas/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2RESUMEN
We tested the hypothesis that combined use of trophic factors and caspase inhibitors increases brain resistance to ischaemia in mice. Intracerebroventricular administration of bFGF (>10 ng) 30 min after MCA occlusion decreased infarct size and neurological deficit in a dose-dependent manner following 2 h ischemia and reperfusion (20 h). Combined administration of the subthreshold doses of bFGF (3 ng) and caspase inhibitors (z-VAD.FMK, 27 ng or z-DEVD.FMK, 80 mg) reduced infarct volume by 60%, and reduced neurological deficit. Treatment with a subthreshold dose of bFGF (3 ng) extended the therapeutic window for z-DEVD.FMK (480 ng) from 1 to 3 h after reperfusion. Caspase-3 activity in the ischaemic brain was increased 30 min and 2 h after reperfusion but, was significantly reduced in bFGF-treated animals by 29 and 16%, respectively. Caspase-3 activity was not reduced by a direct bFGF effect because addition of bFGF (10 nM - 2 microM) did not decrease recombinant caspase-3 activity, in vitro. Our data show that combining caspase inhibitors and bFGF lengthens the treatment window for the second treatment, plus lowers the dosage requirements for neuroprotection. These findings are important because low doses of caspase inhibitors or bFGF reduce the possibility of side effects plus extend the short treatment window for ischaemic stroke.
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Isquemia Encefálica/tratamiento farmacológico , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Cisteína Proteinasa/uso terapéutico , Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Clorometilcetonas de Aminoácidos/farmacología , Clorometilcetonas de Aminoácidos/uso terapéutico , Animales , Isquemia Encefálica/enzimología , Isquemia Encefálica/patología , Caspasa 3 , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/administración & dosificación , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Masculino , Ratones , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Oligopéptidos/uso terapéuticoRESUMEN
BACKGROUND: In recent studies increased amounts of nitric oxide (NO) and apoptosis have been implicated in various pathological conditions in the kidney. We have studied the role of NO and its association with apoptosis in an experimental model of nephrotic syndrome induced by a single injection of adriamycin (ADR). METHODS: The alteration in the NO pathway was assessed by measuring nitrite levels in serum/urine and by evaluating the changes in vascular reactivity of the isolated perfused rat kidney (IPRK) system. Rats were stratified into control groups and ADR-induced nephropathy groups. These two groups were then divided into: group 1, animals receiving saline; and group 2, animals receiving aminoguanidine (AG) which is a specific inhibitor of inducible-NO synthase. On day 21, rats were sacrificed after obtaining material for biochemical analysis. RESULTS: Histopathological examination of the kidneys of rats treated with ADR revealed focal areas of mesangial proliferation and mild tubulointerstitial inflammation. They also had significantly higher levels of proteinuria compared with control and treatment groups (P < 0.05). Urine nitrite levels were significantly increased in the ADR-nephropathy group (P < 0.05). In the IPRK phenylephrine and acetylcholine related responses were significantly impaired in the ADR-nephropathy group. Apoptosis was not detected in controls. However, in the ADR-nephropathy group, numerous apoptotic cells were identified in the tubulointerstitial areas. Double staining revealed numerous interstitial apoptotic cells to stain for ED1, a marker for monocytes/macrophages. Treatment with AG prevented the impairment of renal vascular bed responses and reduced both urine nitrite levels and apoptosis to control levels. CONCLUSION: We suggest that interactions between NO and apoptosis are important in the pathogenesis of the ADR-induced nephrosis.
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Apoptosis/fisiología , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Óxido Nítrico/biosíntesis , Animales , Doxorrubicina/toxicidad , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Técnicas In Vitro , Síndrome Nefrótico/inducido químicamente , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Perfusión , Fenilefrina/farmacología , Ratas , Ratas Wistar , Circulación Renal/efectos de los fármacos , Circulación Renal/fisiologíaRESUMEN
The association between Escherichia coli endotoxin-induced organ damage and nitric oxide-related mechanisms was investigated in the spleen of male Swiss albino mice (20-40 g) by using (1) Pt/Ir electrochemical sensor connected to an amperometric detection system (NO-501, InterMedical Co., Japan), (2) nitrotyrosine immunohistochemistry, (3) conventional light microscopy and (4) immunoblotting techniques in parallel. 1 h before endotoxin injection, animals were pretreated with either nitric oxide synthase inhibitor, L-N(G)-nitroarginine methyl ester (L-NAME, 20 mg kg(-1), i.p.) or inducible nitric oxide synthase expression inhibitor, dexamethasone (5 mg kg(-1), i.p.) or the inhibitor of murine inducible nitric oxide synthase in vivo, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 1 mg kg(-1), i.p.). 5 h after endotoxin treatment, electrochemically detected concentration of nitric oxide was significantly elevated (nM, endotoxin: 716.6 +/- 178.2, n = 10 vs saline: 209.4 +/- 127.8, n = 9, P = 0.0312, unpaired Student's t-test) and remained so throughout the 30 min monitorization period. Neither dexamethasone nor AMT blocked the endotoxin-induced overproduction of nitric oxide indicating that the enhanced inducible nitric oxide synthase activity cannot be the only explanation. When dexamethasone and L-NAME combination was used to block both the constitutive and the inducible isoforms, nitric oxide production was virtually abolished, indicating a significant contribution from the constitutive isoform of nitric oxide synthase. The results of nitrotyrosine immunohistochemistry and the conventional light microscopy were also in agreement with the amperometric method while immunoblotting revealed the expression of both the endothelial and the inducible isoforms of nitric oxide synthase were induced endotoxaemic animals. Thus, conclude that endotoxin-induced splenic damage in endotoxaemia can be explained by enhanced production of nitric oxide due to the induction of both endothelial and inducible nitric oxide synthases while causal relationship and the roles of other deleterious mediators such as oxygen-derived free radicals are yet to be established.
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Endotoxemia/patología , Óxido Nítrico/fisiología , Bazo/patología , Animales , Dexametasona/farmacología , Endotoxemia/metabolismo , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Tiazinas/farmacologíaRESUMEN
A buccoadhesive controlled-release system for delivery of carbamazepine (CBZ) was prepared by compression of hydroxypropyl methylcellulose (HPMC) and carbomer, incorporating a penetration enhancer, sodium glycodeoxycholate (GDC). The release behaviour of systems containing CBZ and various amounts of the two polymers with and without GDC was found to be non-Fickian. Formation of an interpolymer complex between HPMC and carbomer was confirmed in acidic medium by turbidity, viscosity and FT-IR measurements. Addition of the drug to the buccoadhesive formulation reduced the adhesion force significantly (p < 0.1). GDC did not have any effect on bioadhesion. Permeability of bovine buccal mucosa to CBZ was determined using Ussing diffusion chambers [1]. In vivo interaction between the tablet and tissue was examined histologically as well as by scoring mucosal irritation. Histological changes observed in the buccal epithelium after 4 h contact with the tablets containing GDC recovered completely within 24 h after removal. No measurable plasma level of CBZ was obtained either in the absence or presence of GDC.
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Anticonvulsivantes/administración & dosificación , Carbamazepina/administración & dosificación , Mucosa Bucal/metabolismo , Absorción , Adhesivos , Adulto , Animales , Anticonvulsivantes/efectos adversos , Anticonvulsivantes/farmacocinética , Carbamazepina/efectos adversos , Carbamazepina/farmacocinética , Bovinos , Femenino , Humanos , Técnicas In Vitro , Irritantes , Cinética , Lactosa/análogos & derivados , Masculino , Metilcelulosa/análogos & derivados , Oxazinas , Solubilidad , ComprimidosRESUMEN
BACKGROUND AND PURPOSE: Reperfusion injury is one of the factors that unfavorably affects stroke outcome and shortens the window of opportunity for thrombolysis. Surges of nitric oxide (NO) and superoxide generation on reperfusion have been demonstrated. Concomitant generation of these radicals can lead to formation of the strong oxidant peroxynitrite during reperfusion. METHODS: We have examined the role of NO generation and peroxynitrite formation on reperfusion injury in a mouse model of middle cerebral artery occlusion (2 hours) and reperfusion (22 hours). The infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride staining; blood-brain barrier permeability was evaluated by Evans blue extravasation. Nitrotyrosine formation and matrix metalloproteinase-9 expression were detected by immunohistochemistry. RESULTS: Infarct volume was significantly decreased (47%) in animals treated with the nonselective nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA) at reperfusion. The specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), given at reperfusion, showed no protection, although preischemic treatment with 7-NI decreased infarct volume by 40%. Interestingly, prereperfusion administration of both NOS inhibitors decreased tyrosine nitration (a marker of peroxynitrite toxicity) in the ischemic area. L-NA treatment also significantly reduced vascular damage, as indicated by decreased Evans blue extravasation and matrix metalloproteinase-9 expression. CONCLUSIONS: These data support the hypothesis that in addition to the detrimental action of NO formed by neuronal NOS during ischemia, NO generation at reperfusion plays a significant role in reperfusion injury, possibly through peroxynitrite formation. Contrary to L-NA, failure of 7-NI to protect against reperfusion injury suggests that the source of NO is the cerebrovascular compartment.
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Endotelio Vascular/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Nitratos/metabolismo , Óxido Nítrico/biosíntesis , Oxidantes/metabolismo , Daño por Reperfusión/metabolismo , Animales , Biomarcadores , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiología , Colorantes , Inhibidores Enzimáticos/farmacología , Azul de Evans , Indazoles/farmacología , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/prevención & control , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratones , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroarginina/farmacología , Permeabilidad/efectos de los fármacos , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Tirosina/análogos & derivados , Tirosina/biosíntesisRESUMEN
We recently demonstrated that a long-lasting transmission defect in cortical synapses caused motor dysfunction after brief middle cerebral artery (MCA) occlusion in the rat despite rapid recovery of axons. In this experimental study, we have examined the impact of differential recovery of synapses and axons on generation of motor-evoked potentials (MEP) recorded from contralateral paralyzed and ipsilateral unaffected muscles, to gain insight into mechanisms of MEPs recorded from stroke patients by transcranial magnetic stimulation (TMS). MEPs generated by focal electrical stimulation of the forelimb area of motor cortex were simultaneously recorded from the brain stem, contra- and ipsilateral forelimb and contralateral hindlimb muscles in rats subjected to transient MCA occlusion. The effect of ischemia on cortical activity and axonal conduction was differentially studied by proximal or distal occlusion of the MCA. Regional cerebral blood flow changes in the forelimb area were monitored by laser-Doppler flowmetry during ischemia and reperfusion. In addition, synaptic transmission within the forelimb area of motor cortex was examined by intracellular and extracellular recording of potentials generated by stimulation of the premotor area. No MEP response was recorded during ischemia. Upon reperfusion: (i) motor axons readily regained their excitability and cortical stimulation caused successive pyramidal volleys (recorded as D waves from the brain stem) and a MEP from contralateral paralytic muscles although synaptic activation of motor pathways was not feasible; (ii) the amplitude of pyramidal volley was increased; (iii) MEPs with a longer latency were recorded from the ipsilateral forelimb. In conclusion, differential recovery of synapses and axons after ischemia may account for some previously unexplained findings (such as preserved MEPs in paralysed muscles) observed in cortical stimulation studies of stroke patients.