Semi-automated analysis of NMDA-mediated toxicity in digitised colour images from rat hippocampus

The evaluation of neuronal cell survival after, for example, mechanical, hypoxic or drug-mediated injury requires the analysis of a high number of histological specimens. Since this is a time-consuming occupation, we have developed a semi-automated analysis routine for the determination of the distribution of live and dead cells. After digitalization of the histological preparations, 8-bit colour bitmaps were assessed using a compiled image-analysis programme of the software package Khoros. In the current study a detailed example of the application of this image-processing approach is described for the investigation of the cell survival after intraventricular application of N-methyl-D-aspartate (NMDA). The samples were prepared as fuchsin acid/toluidine blue stained hippocampal thin slices. The calculated areas of the live and dead cells were highly correlated with manual counts of live and dead cells in the 100 samples examined in this study. Twenty-four hours following NMDA-treatment animals (n = 5) were found to have significantly fewer live and more dead hippocampal cells than the saline-treated animals (n = 5), using either automated or manual examination techniques. The automated technique also revealed that NMDA treatment resulted in a reduction in the density of live cell distribution.