Morphology of non-vascular intracerebral fluid spaces.

In the electron microscope the value for the extracellular space (ECS) in the mammalian CNS was suggested of several percent to about one third dependent on the method how it was evaluated. Since von Harreveld introduced 1965 cryofixation to estimate the extension of the ECS, the method has been never applied in brain edema research. We carried out improved low temperature methods to measure the extracellular space of the mammalian CNS in physiological conditions. Small samples of brain tissue were cryofixed by slam freezing on a precooled metal mirror and substituted with ethanol at -95 degrees C over 17 hours. The embedding procedure was carried out at -22 degrees C with LR-White under UV-irradiation. ECS was measured computer assisted with Bioquant Software. The values for the ECS of the cryofixed normal rat brain were more than twice compared to the usual transmission electron microscopy (16.3% to 7.4%, p < 0.01) and close to those estimated by von Harreveld (18.1-25.5%, 1965). It was interesting that the data obtained in cryofixed normal rat brain correspond to the extension measured in rat brain with irradiation edema, which was conventionally treated for EM. Greater variance of ECS in cryofixed brain (16.3% +/- 3.4) demonstrate that it is far more variable than expected. This data correspond closely to the in vivo ECS. The morphological evaluation of brain edema should be revised under this premise.