Treatment of PC12 cells by nerve growth factor, dexamethasone, and forskolin. Effects on cell morphology and expression of neurotensin and tyrosine hydroxylase.

Several lines of anatomical, neurochemical, electrophysiological, and behavioral evidence suggest the existence of physiological interactions between neurotensin (NT) and the brain dopaminergic systems. Thus, NT has been shown to exert a neuroleptic-like action and could be implicated in the pathogenesis and treatment of schizophrenia. It is thus of particular importance to develop in vitro cell culture systems as models to study such interactions. Rat adrenal pheochromocytoma PC12 cells, which expressed high levels of tyrosine hydroxylase, were used in the present study. In contrast to rat brain cells in primary cultures, PC12 cells did not express functional NT receptors. However, they were able to express both NTmRNA and NT in response to NGF, forskolin, and dexamethasone. Those neurochemical modifications furthermore may be related to changes in the morphology of the PC12 cells in response to NGF, forskolin, and dexamethasone alone or in combination. These data suggest that PC12 cells may provide a useful model to study in vitro the regulation of both catecholamine and neurotensin phenotypes.

Automated image analyzing system for the quantitative study of living cells in culture.

A fully automated image analyzing system was developed for the quantitative study of cells in culture. It was able to count cells, to classify cells according to their morphological characteristics and to follow cell culture development. A specific procedure was designed to process Hoffman modulation contrast images. It detects local gray level differences while using conditional dilation techniques. We were able to successfully detect aggregated unstained cells, presently a technical limit in image segmentation. Living cells can be studied in a noninvasive and nondestructive way with this system. An improved automatic focusing algorithm was developed which ensured an accurate prediction of the optimal focus position. A strictly defined sampling procedure was applied to estimate unbiasedly cell density and obtain precisely cell contours. The evaluation of the system was carried out on Chinese hamster ovary (CHO-NTR) cell cultures treated with a newly developed neurotensin agonist JMV449. Chinese hamster ovary cell division was found to be retarded 20 hours after the JMV449 treatment, while the morphology of CHO-NTR cells has already undergone significant changes 12 hours after the treatment. This image analyzing system provides the possibility to follow cell culture development (e.g., cell density evolution, cell morphological changes) under various experimental conditions.

Antiproliferative activity of a liposomal delivery system of mitoxantrone on rabbit subconjunctival fibroblasts in an ex-vivo model.

Wound healing is the main cause of the failure of filtering surgery in glaucoma. We developed a liposomal delivery system of mitoxantrone (MITX), an anthracyclin derivative, to allow a single adjuvant administration and to lessen ocular side-effects of the drug. In order to evaluate the antiproliferative activity of liposomal MITX, an ex vivo model consisting in the culture of subconjunctival tissue explants from rabbits pretreated with subconjunctival injections of free or liposomal MITX was used. We found that both forms of MITX decreased the growth rate as well as the explant proliferation surfaces 15 days or 1 month after a single administration of the drug in vivo. A morphometric analysis of the cells showed that the surface of the fibroblasts exposed to both forms of MITX was from 10 to 12 times as important as that of the control cells exposed to the empty liposomes and to the control buffer. A radioautographic study showed that more than 95% of the fibroblasts exposed to both forms of MITX were in the G1 phase of the cell cycle, while the control cell population was equally distributed among the different phases of the cell cycle.

Automatic cell culture quantitation with TRAKCELL: application to cell toxicology and differentiation.

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.