RESUMEN
Dementia is one of the big medical challenges of our time with Alzheimer's, Huntington's and Parkinson's disease among its most common forms. In year 2000, 4.5 million people were diagnosed with Alzheimer's disease in the United States. In the case of Alzheimer's disease one of many contributing factors is a metabolic imbalance that leads to elevated oxidative stress levels. Consequences of this imbalance can be symptoms like apraxia, agnosia or sundowning. The use of field-effect transistors is a novel approach to study the effects of external stimuli on cells in vitro to provide researchers with a new tool for high resolution and high throughput studies to better understand cellular interaction and the effects of pharmacological compounds. In our study we use ion-sensitive field-effect transistors (FETs) to analyze the apoptosis inducing effects of hydrogen peroxide treatment on primary cells obtained from the subventricular zone of postnatal BALB/c mice. Upon apoptosis, the cell-substrate adhesion of the neurons is gradually weakened until complete detachment. In former studies we used our FET devices to conduct Electrical Cell-substrate Impedance Sensing (ECIS) experiments on the single cell level using morphologically different cell lines. Here we demonstrate that our novel approach of ECIS using FET devices can be expanded to primary neuronal tissue with high prospects for further studies in the field of pharmacological research.
Asunto(s)
Apoptosis/efectos de los fármacos , Técnicas Biosensibles , Ventrículos Laterales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Espectroscopía Dieléctrica , Peróxido de Hidrógeno/administración & dosificación , Ventrículos Laterales/citología , Ratones , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/patología , Cultivo Primario de CélulasRESUMEN
We introduce a novel technique of impedimetric sensing of cellular adhesion, which might have the potential to supplement the well-known technique of Electrical Cell-substrate Impedance Sensing (ECIS) in cell culture assays. In contrast to the already commercialized ECIS method, we are using ion-sensitive field-effect transistor (ISFET) devices. The standard gold microelectrode size in ECIS is in the range of 100-250 µm in diameter. Reason for this limitation is that when downscaling the sensing electrodes, their effective impedance governed by the metal-liquid interface impedance is becoming very large and hence the currents to be measured are becoming very small reaching the limit of standard instrumentation. This is the main reason why typical assays with ECIS are focusing on applications like cell-cell junctions in confluent cultures. Single cell resolution is barely reachable with these systems. Here we use impedance spectroscopy with ISFET devices having gate dimensions of only 16 × 2 µm(2), which is enabling a real single cell resolution. We introduce an electrically equivalent circuit model, explain the measured effects upon single cell detachment, and present different cellular detachment scenarios. Our approach might supplement the field of ECIS with an alternative tool opening up a route for novel cell-substrate impedance sensing assays with so far unreachable lateral resolution.
Asunto(s)
Espectroscopía Dieléctrica , Análisis de la Célula Individual , Transistores Electrónicos , Adhesión Celular , Técnicas de Cultivo de Célula/instrumentación , Células Cultivadas , Espectroscopía Dieléctrica/instrumentación , Impedancia Eléctrica , Oro/química , Células HEK293 , Humanos , Microelectrodos , Análisis de la Célula Individual/instrumentaciónRESUMEN
BACKGROUND: The microelectrode array (MEA) was used to investigate the pharmacological relevance of chloride (Cl-) ions in antigen-dependent mast cell activation and the inhibitory effect of disodium cromoglycate (DSCG) on mast cell activation. METHODS: The movements of ions across the cellular membrane and the potential relationship between Cl- channels and DSCG during immunological activation were investigated using the MEA. The results were then subsequently compared with the amount of histamine released from anti-IgE activated peritoneal mast cells. RESULTS: The inclusion of charybdotoxin (ChTX) in Cl--free buffer showed that the measured field potentials during antigen-stimulated peritoneal mast cell were a combination of Cl- influx and K+ efflux. The delayed onset time of Cl- influx indicated the presence of a delayed outwardly-rectifying Cl- current in the antigen-stimulated peritoneal mast cells. The use of 5-nitro-2-(3-phenylpropylamino) benzoic acid demonstrated that the activated mast cell membrane potential can be stabilised, thereby reducing the amount of histamine released from the anti-IgE activated mast cells. The correlation between the results of the histamine release assay and the electrophysiological measurements demonstrated the importance of Cl- to anti-IgE dependent mast cell activation. The inhibitory effect of DSCG on anti-IgE activated cells, however, did not correlate with the presumed influx of Cl-. CONCLUSIONS: The MEA data suggest that Cl- influx is crucial to IgE-dependent mast cell degranulation. GENERAL SIGNIFICANCE: While the MEA cannot yield information about single channel properties, it is convenient to use and can provide information on the global changes in electrophysiological responses of non-excitable cells.
Asunto(s)
Cloruros/farmacología , Cromolin Sódico/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Receptores de IgE/fisiología , Animales , Anticuerpos Antiidiotipos/farmacología , Caribdotoxina/farmacología , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/metabolismo , Liberación de Histamina/genética , Masculino , Microelectrodos , Nitrobenzoatos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de IgE/efectos de los fármacosRESUMEN
The microelectrode array (MEA) was used to evaluate the cardioprotective effects of adenosine triphosphate sensitive potassium (K(ATP)) channel activation using potassium channel openers (KCOs) on HL-1 cardiomyocytes subjected to acute chemically induced metabolic inhibition. Beat frequency and extracellular action potential (exAP) amplitude were measured in the presence of metabolic inhibitors (sodium azide (NaN(3)) or 2-deoxyglucose (2-DG)) or KCOs (pinacidil (PIN, a cyanoguanidine derivative, activates sarcolemmal K(ATP) channels) or SDZ PCO400 (SDZ, a benzopyran derivative, activates mitochondrial K(ATP) channels)). The protective effects of these KCOs on metabolically inhibited HL-1 cells were subsequently investigated. Signal shapes indicated that NaN(3) and 2-DG reduced the rate of the sodium (Na(+)) influx signal as reflected by a reduction in beat frequency. PIN and SDZ appeared to reduce both rate of depolarization and extent of the Na(+) influx signals. Pre-treating cardiomyocytes with PIN (0.1 mM), but not SDZ, prevented the reduction of beat frequency associated with NaN(3)- or 2-DG-induced metabolic inhibition. The exAP amplitude was not affected by either KCO. The cardioprotective effect of PIN relative to SDZ may be due to the opening of different K(ATP) channels. This metabolic inhibition model on the MEA may provide a stable platform for the study of cardiac pathophysiology in the future.