RESUMEN
An automated system, TRAKCELL, was developed for the quantitation of cells in culture. It enabled cell counting, classification according to morphological cell characteristics and measurement of cell proliferation and differentiation. The system was tested on the toxic effect of ascorbic acid on rat brain catecholaminergic neurons in primary culture. In parallel, the effects of nerve growth factor, dexamethasone and forskolin on cell differentiation were studied using rat pheochromocytoma PC12 cells. The results show that the system permits rapid and reproducible measurements of cell density and of the morphological changes observed following various drug treatments.
Asunto(s)
Recuento de Células/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Neuronas/efectos de los fármacos , Animales , Ácido Ascórbico/toxicidad , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , Medios de Cultivo/química , Dexametasona/farmacología , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Células PC12 , Ratas , Ratas Wistar , Programas Informáticos , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
This paper describes the coupling between a scanning electron microscope (SEM) and an image analysis workstation. The system was designed in order to drive the SEM and to analyse any sample. It allows automatic (edge detection) or semiautomatic (pointing, marking, drawing) object detection. Two types of data can be obtained: (1) topographical information, such as the location of the object within a region of interest drawn at any magnification of the microscope, or (2) quantitative data, such as morphometric characteristics of objects. In addition, high resolution maps of the section, regions of interest, and objects can be obtained with a laser printer. This software was first applied to quantitate the adhesion of the bacteria Pseudomonas aeruginosa to human respiratory epithelial cells in culture. P. aeruginosa was shown associated with ciliated cells. The second application concerned the study of the distribution of specific carbohydrate residues at the surface of the respiratory cells. The gal residues were revealed using the lectin Ricinus communis agglutinin II, adsorbed to colloidal gold particles. A relationship between the presence of adherent bacteria and labelling was shown.
Asunto(s)
Adhesión Bacteriana/fisiología , Microscopía Electrónica de Rastreo/métodos , Pseudomonas aeruginosa/ultraestructura , Sistema Respiratorio/ultraestructura , Carbohidratos/análisis , Células Cultivadas , Células Epiteliales , Epitelio/fisiología , Epitelio/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador , Pseudomonas aeruginosa/fisiología , Fenómenos Fisiológicos Respiratorios , Sistema Respiratorio/citologíaRESUMEN
Human nasal polyps in outgrowth culture were used to study the Pseudomonas aeruginosa adhesion to respiratory cells. By scanning electron microscopy, P. aeruginosa were seen associated with ciliated cells, but by transmission electron microscopy, bacteria were never seen at the interciliary spaces or attached along cilia, but were identified trapped at the extremities of cilia, usually as bacterial aggregates. A fibronectin-containing fibrillar material was seen associated with aggregated bacteria. By time-lapse video microscopy, bacteria were seen to aggregate in the culture medium following their addition to the culture wells. Progressively, these aggregates were trapped by cilia or attached to migrating cells of a lower cell layer that protruded beneath the upper layer cells, at the outgrowth periphery. P. aeruginosa adhesion to these lower cell layer migrating cells was significantly higher than to ciliated or nonciliated cells of the upper cell layer. Migrating cells were intensely labeled by the complexes Con A and arachis hypogea agglutinin (PNA)-FITC, in contrast to the other cells. The percentage of PNA-labeled cells with attached bacteria was significantly higher than that without bacteria. These results suggest that changes of cell surface glycoconjugates related with cell migration may favor P. aeruginosa adhesion to respiratory cells.