RESUMEN
Four commonly used methods for the assessment of neuronal (or glial) cell viability are described in this unit. The MTT assay is sensitive to the function of labile mitochondrial enzymes, which typically lose activity early in the progression towards death. The lactate dehydrogenase (LDH) assay measures the appearance of this cytosolic enzyme in the bathing medium, providing a measure of plasma membrane integrity. Loss of plasma membrane integrity is also the basis of the trypan blue dye assay and the propidium iodide assay. Trypan blue staining is assessed by cell counts; propidium iodide labeling can be assessed either by cell counts, typically in conjunction with fluorescein diacetate counterstaining to identify intact cells containing adequate levels of functional esterases, or with a fluorescence plate reader.
Asunto(s)
Supervivencia Celular , Neuronas/citología , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Permeabilidad de la Membrana Celular , Células Cultivadas/química , Células Cultivadas/citología , Células Cultivadas/ultraestructura , Colorimetría/métodos , Medios de Cultivo Condicionados/química , Colorantes Fluorescentes/análisis , L-Lactato Deshidrogenasa/análisis , Microscopía Fluorescente/métodos , Mitocondrias/metabolismo , Neuronas/química , Neuronas/ultraestructura , Preservación Biológica/métodos , Propidio/análisis , Coloración y Etiquetado , Sales de Tetrazolio/análisis , Tiazoles/análisis , Azul de Tripano/análisisRESUMEN
In light of recent evidence implicating the upregulation of outward K+ current in mediating several forms of neuronal apoptosis, we tested the hypothesis that such an upregulation might specifically contribute to the pathogenesis of beta-amyloid peptide (A beta)-induced neuronal death. Exposure to A beta fragment 25-35 (20 microM) or 1-42 (20 microM) enhanced the delayed rectifier K+ current IK, shifting its activation voltage relationship toward hyperpolarized levels and increasing maximal conductance, but did not affect the transient K+ current IA or charybdotoxin-sensitive BK current. Reducing IK by adding the channel blocker tetraethylammonium (TEA, 5 mM) or raising extracellular K+ to 25 mM attenuated A beta-induced neuronal death, even in the presence of nifedipine or gadolinium to block associated increases in Ca2+ influx. The IA blocker 4-aminopyridine (4-AP, 5 mM) and the CI- channel blocker anthracene-9-carboxylic acid (ACA, 500 microM) were not neuroprotective. These data raise the intriguing possibility that manipulations aimed at reducing outward K+ current may provide an approach to reducing neuronal degeneration in patients with Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Corteza Cerebral/fisiología , Neuronas/fisiología , Fragmentos de Péptidos/farmacología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Técnicas de Cocultivo , Canales de Potasio de Tipo Rectificador Tardío , Feto , Ratones , Neuroglía , Neuronas/efectos de los fármacos , Potasio/fisiología , Canales de Potasio/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacologíaRESUMEN
The extracellular acidity that accompanies brain hypoxia-ischemia is known to reduce both NMDA and AMPA-kainate receptor-mediated currents and NMDA receptor-mediated neurotoxicity. Although a protective effect of acidic pH on AMPA-kainate receptor-mediated excitotoxicity has been assumed, such has not been demonstrated. Paradoxically, we found that lowering extracellular pH selectively increased AMPA-kainate receptor-mediated neurotoxicity in neocortical cell cultures, despite reducing peak elevations in intracellular free Ca2+. This injury potentiation may, at least in part, be related to a slowed recovery of intracellular Ca2+ homeostasis, observed after AMPA-kainate receptor activation, but not after NMDA receptor activation or exposure to high K+. The ability of acidic pH to selectively augment AMPA-kainate receptor-mediated excitotoxicity may contribute to the prominent role that these receptors play in selective neuronal death after transient global ischemia.
Asunto(s)
Ácidos/metabolismo , Corteza Cerebral/fisiología , Espacio Extracelular/metabolismo , Neuronas/fisiología , Receptores AMPA/fisiología , Anaerobiosis/fisiología , Animales , Calcio/metabolismo , Muerte Celular/fisiología , Corteza Cerebral/citología , Agonistas de Aminoácidos Excitadores/farmacología , Glucosa/deficiencia , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Ácido Kaínico/farmacología , Ratones/embriología , Neuronas/efectos de los fármacos , Neuronas/patología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
The biophysical mechanism(s) underlying diffusion-weighted MRI contrast following brain injury remains to be elucidated. Although it is generally accepted that water apparent diffusion coefficient (ADC) decreases after brain injury, it is unknown whether this is associated with a decrease in intracellular or extracellular water displacement, or both. To address this question, 2-[19F]luoro-2-deoxyglucose-6-phosphate (2FDG-6P) was employed as a compartment-specific marker in normal and globally ischemic rat brain. Through judicious choice of routes of administration, 2FDG-6P was confined to the intra- or extracellular space. There was no statistical difference between intra- and extracellular 2FDG-6P ADCs in normal or in globally ischemic brain (P > 0.16), suggesting that water ADCs in both compartments are similar. However, ischemia did result in a 40% ADC decrease in both compartments (P < 0.001). Assuming that 2FDG-6P reflects water motion, this study shows that water ADC decreases in both spaces after ischemia, with the reduction of intracellular water motion being the primary source of diffusion-weighted contrast.
Asunto(s)
Isquemia Encefálica/diagnóstico , Encéfalo/metabolismo , Espacio Extracelular/metabolismo , Glucosa-6-Fosfato/análogos & derivados , Líquido Intracelular/metabolismo , Espectroscopía de Resonancia Magnética , Animales , Biomarcadores/análisis , Isquemia Encefálica/metabolismo , Células Cultivadas/metabolismo , Difusión , Modelos Animales de Enfermedad , Flúor , Glucosa-6-Fosfato/análisis , Masculino , Ratones , Neuroglía/metabolismo , Neuronas/metabolismo , Fantasmas de Imagen , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
Apoptosis of mouse neocortical neurons induced by serum deprivation or by staurosporine was associated with an early enhancement of delayed rectifier (IK) current and loss of total intracellular K+. This IK augmentation was not seen in neurons undergoing excitotoxic necrosis or in older neurons resistant to staurosporine-induced apoptosis. Attenuating outward K+ current with tetraethylammonium or elevated extracellular K+, but not blockers of Ca2+, Cl-, or other K+ channels, reduced apoptosis, even if associated increases in intracellular Ca2+ concentration were prevented. Furthermore, exposure to the K+ ionophore valinomycin or the K+-channel opener cromakalim induced apoptosis. Enhanced K+ efflux may mediate certain forms of neuronal apoptosis.
Asunto(s)
Apoptosis , Neuronas/citología , Canales de Potasio/metabolismo , Potasio/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Benzopiranos/farmacología , Calcio/metabolismo , Corteza Cerebral/citología , Cromakalim , Cicloheximida/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Gadolinio/farmacología , Ratones , N-Metilaspartato/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Nifedipino/farmacología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Pirroles/farmacología , Estaurosporina/farmacología , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Veratridina/farmacologíaRESUMEN
This study examined the possibility that the excitotoxin-induced death of cultured cortical neurons might occur by apoptosis, specifically focusing on the slowly triggered death induced by low concentrations of excitotoxin. Cultured murine cortical neurons (days in vitro 10-12) were exposed continuously to N-methyl-D-aspartate (10-15 microM), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (3-100 microM) or kainate (30-60 microM) over 24 h. Within 2 h of exposure onset, neuronal cell body swelling was visible under phase-contrast optics. At this point, transmission electron microscopy revealed disruption of cell membranes and organelles, mitochondrial swelling and scattered chromatin condensation at the periphery of nuclei. By 8 h after exposure onset, many neurons were devoid of cytoplasmic structures, but nuclear membranes remained relatively intact. This excitotoxic degeneration was not blocked by the protein synthesis inhibitor, cycloheximide, or the growth factors, brain-derived neurotrophic factor or insulin-like growth factor-1, agents that did block serum deprivation-induced apoptosis death in other cultures. DNA agarose gel electrophoresis, however, revealed the transient occurrence of internucleosomal DNA fragmentation, appearing 4-8 h after exposure onset, but absent 24 h after exposure onset. The present results suggest that even slowly triggered excitotoxicity occurs by necrosis, and raise a cautionary note in interpreting internucleosomal DNA fragmentation in isolation as evidence for apoptosis.
Asunto(s)
Corteza Cerebral/fisiopatología , Aminoácidos Excitadores/toxicidad , Neuronas/fisiología , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Cicloheximida/farmacología , Fragmentación del ADN , Electroforesis en Gel de Poliacrilamida , Ratones , Microscopía Electrónica , Necrosis , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Neuronas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacologíaRESUMEN
Cultured mouse cortical neurons undergo apoptosis when exposed to staurosporine. The cell-permeable caspase inhibitor Z-Val-Ala-Asp fluoromethylketone (Z-VAD.FMK) attenuated this death, without altering overall protein synthesis. Z-VAD.FMK also attenuated cortical neuronal apoptosis induced by removal of serum. However, Z-VAD.FMK did not attenuate the excitotoxic necrosis induced by 5-min exposure to 100 microM NMDA, 24-h exposure to 100 microM kainate, or 90-min exposure to oxygen-glucose deprivation. We have previously shown that blockade of the excitotoxic component of oxygen-glucose deprivation-induced neuronal death with glutamate antagonists unmasks an apoptotic death. Treatment with Z-VAD.FMK, but not the cathepsin-B protease inhibitor Z-Phe-Ala fluoromethylketone (Z-FA.FMK), also attenuated this oxygen-glucose deprivation-induced neuronal apoptosis. These data support the idea that brain caspases mediate the apoptotic component of oxygen-glucose deprivation-induced neuronal death and raise the possibility that combining caspase inhibitors with glutamate antagonists might attenuate brain damage induced by hypoxic-ischemic insults in vivo.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Muerte Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Neuronas/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glucosa/fisiología , Técnicas In Vitro , Ratones , Oxígeno/fisiología , Estaurosporina/farmacologíaRESUMEN
Recent studies have suggested that rats subjected to transient global brain ischemia develop depressed expression of GluR-B in CA1 hippocampal neurons. The present study was performed to determine whether a similar change in AMPA receptor expression could be triggered in vitro by sublethal oxygen-glucose deprivation in rat hippocampal neuronal cultures. mRNA was extracted from individual hippocampal neurons via patch electrodes and amplified by RT-PCR 24-48 hr after sublethal oxygen-glucose deprivation. Compared with controls, insulted neurons expressed increased levels of GluR-D flop. As an indication that this change in receptor expression was functionally significant, insulted cultures exhibited increased AMPA- or kainate-induced 45Ca2+ accumulation sensitive to Joro spider toxin and increased vulnerability to kainate-induced death. These data support the hypothesis that exposure to ischemia may enhance subsequent hippocampal neuronal vulnerability to AMPA receptor-mediated excitotoxicity by modifying the relative expression of AMPA receptor subunits in a manner that promotes Ca2+ permeability.
Asunto(s)
Glucosa/farmacología , Neuronas/química , Neuronas/citología , Receptores AMPA/genética , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula/fisiología , Células Cultivadas , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Expresión Génica/fisiología , Hipocampo/citología , Precondicionamiento Isquémico , Ácido Kaínico/farmacología , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/efectos de los fármacos , Neurotoxinas/farmacología , Quinoxalinas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores AMPA/agonistas , Venenos de Araña/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
We used the ratioable fluorescent dye mag-fura-5 to measure intracellular free Zn2+ ([Zn2+]i) in cultured neocortical neurons exposed to neurotoxic concentrations of Zn2+ in concert with depolarization or glutamate receptor activation and identified four routes of Zn2+ entry. Neurons exposed to extracellular Zn2+ plus high K+ responded with a peak cell body signal corresponding to a [Zn2+]i of 35-45 nM. This increase in [Zn2+]i was attenuated by concurrent addition of Gd3+, verapamil, omega-conotoxin GVIA, or nimodipine, consistent with Zn2+ entry through voltage-gated Ca2+channels. Furthermore, under conditions favoring reverse operation of the Na+-Ca2+ exchanger, Zn2+ application induced a slow increase in [Zn2+]i and outward whole-cell current sensitive to benzamil-amiloride. Thus, a second route of Zn2+ entry into neurons may be via transporter-mediated exchange with intracellular Na+. Both NMDA and kainate also induced rapid increases in neuronal [Zn2+]i. The NMDA-induced increase was only partly sensitive to Gd3+ or to removal of extracellular Na+, consistent with a third route of entry directly through NMDA receptor-gated channels. The kainate-induced increase was highly sensitive to Gd3+ or Na+ removal in most neurons but insensitive in a minority subpopulation ("cobalt-positive cells"), suggesting that a fourth route of neuronal Zn2+ entry is through the Ca2+-permeable channels gated by certain subtypes of AMPA or kainate receptors.
Asunto(s)
Neuronas/química , Neuronas/metabolismo , Zinc/análisis , Zinc/metabolismo , Animales , Unión Competitiva/fisiología , Transporte Biológico/fisiología , Calcimicina/análogos & derivados , Calcimicina/farmacología , Calcio/metabolismo , Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Células Cultivadas , Estimulación Eléctrica , Electrofisiología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Colorantes Fluorescentes , Fura-2/análogos & derivados , Ionóforos/farmacología , Ácido Kaínico/farmacología , Magnesio/metabolismo , Magnesio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , N-Metilaspartato/farmacología , Neocórtex/citología , Neuronas/citología , Nimodipina/farmacología , Quinoxalinas/farmacología , Intercambiador de Sodio-Calcio/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
The effect of DL-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, on colon tumor development was studied in rats. Weanling male Sprague-Dawley rats were randomly assigned to one of three groups following a week of adaptation. Group 1 rats received BSO (4.5 mM) daily in the drinking water one week before 1,2-dimethylhydrazine (DMH) injections and continued to receive BSO daily until sacrificed; group 2 rats received BSO (4.5 mM) after the last DMH injection, and continued to receive BSO daily until sacrificed; group 3 rats did not receive BSO. All experimental rats received 20 weekly subcutaneous injections of DMH (20 mg/kg body wt.) for colon tumor induction. The tumor incidence was lower in group 1 (54%) than in group 2 (97%) or group 3 (96%). The median tumor size is significantly smaller in group 1 (11 mm2) than group 3 (46 mm2). The group 2 had the largest median tumor size (65 mm2).