Methoden zur Evaluation des neuronalen Zelltods in hippokampalen und kortikalen Primärkulturen der Ratte

The important medical and economic interest of apoplectic stroke resulted in intensive research over the past years. The delayed neurodegeneration, which proceeds in the penumbra, is a promising approach for the neuroprotective therapy. After the ischemic occurrence neurodegenerative processes proceed in this place, which can be affected pharmacological and may result in an advancement of prognosis (Lo, 2003). A focal point of this study was the investigation of comparability of aberrant laboratory techniques for the identification of the cell damage. A main interest was the simplification of the quantitative analysis of neuronal cell cultures with the photometric measurement. A further focal point was the investigation of the cell damage and the neuroprotective effect of diverse substances under the conditions of oxygen-glucose-deprivation. A trypanblue stained neuronal cell culture was analysed photometric, which showed, that there was no significant difference between 570 and 590 nm. There was also no significant difference between the cell damage of eight and thirteen day old cell cultures. Thus it appears that results, which base upon methodical differences, like the age of the cell culture or different wavelengths of photometric analysis, are very well comparable. Also by comparing the photometric measurement and the enumeration of a cell culture there were no significant differences between the methods. However the scatter of the results, which were detected with the photometric measurement, was considerably greater than the scatter of the results of enumeration. If methods are used, which give rise to expectations for a higher impreciseness, like the photometric measurement, it should be considered in the design of an experiment, for instance by increasing the number of trials. Concerning the damage caused by glutamate in this study the concentration was less deciding for the cell damage than the duration of exposure. This criterion should be approximate the same, when considering results. The substance Dizocilpin resulted in significant reduction of the cell damage under glutamate exposure. Also under NMDA exposure the substance affected neuroprotective. That shows, that Dizocilpin acts significant neuroprotective caused by blockade of the NMDA-receptors in line with excitatory cell damage. To appraise the efficacy of neuroprotective substances under ischemic conditions in cell culture, the model of oxygen-glucose-deprivation was more exacting analysed. Neuroprotective substances, which proved to be effective in the animal model (Culmsee et al., 2001, 2004), achieved also a reduction of the cell damage under the conditions of oxygen-glucose-deprivation. So in this study Pifithrin achieved a significant reduction of cell damage under these conditions. Memantine and Clenbuterol showed no intense protective effect in this study. On the one hand it could be caused by the short time of pre-treatment of only one hour, on the other hand it could be caused by the high basic-damage and the comparative low damage caused by oxygen-glucose-deprivation. The reason for the high basic-damage could be the different conditions of preparation and the usage of different cell cultures. The low damage could be caused by hypothermia of the cell cultures during the long period of treatment or by a deficient oxygen-deprivation. The model of oxygen-glucose-deprivation is an established model, which is able to give information about the ischemic cell damage under controlled conditions. However it is expedient to mind the same conditions of preparation and cell cultures and to detect the temperature and the oxygen content of the cultures under the conditions of oxygen-glucosedeprivation to eliminate such side effects.