A Study of Extracellular Space in Central Nervous Tissue by Freeze-substitution

It was attempted to preserve the water distribution in central nervous tissue by rapid freezing followed by substitution fixation at low temperature. The vermis of the cerebellum of white mice was frozen by bringing it into contact with a polished silver mirror maintained at a temperature of about -207 degrees C. The tissue was subjected to substitution fixation in acetone containing 2 per cent OsO(4) at -85 degrees C for 2 days, and then prepared for electron microscopy by embedding in Maraglas, sectioning, and staining with lead citrate or uranyl acetate and lead. Cerebellum frozen within 30 seconds of circulatory arrest was compared with cerebellum frozen after 8 minutes' asphyxiation. From impedance measurements under these conditions, it could be expected that in the former tissue the electrolyte and water distribution is similar to that in the normal, oxygenated cerebellum, whereas in the asphyxiated tissue a transport of water and electrolytes into the intracellular compartment has taken place. Electron micrographs of tissue frozen shortly after circulatory arrest revealed the presence of an appreciable extracellular space between the axons of granular layer cells. Between glia, dendrites, and presynaptic endings the usual narrow clefts and even tight junctions were found. Also the synaptic cleft was of the usual width (250 to 300 A). In asphyxiated tissue, the extracellular space between the axons is either completely obliterated (tight junctions) or reduced to narrow clefts between apposing cell surfaces.