Preservation of extracellular space during fixation of the brain for electron microscopy

Adult mammalian brain contains about 20% extracellular space, but fixatives cause the cellular processes to ingest the extracellular fluid, and the space is not preserved in electron micrographs prepared by any of the conventional methods. This distortion can be prevented by replacing part of the sodium chloride in the extracellular fluid by an impermeant solute such as sucrose. To do this, the blood-brain barrier can be opened by vascular perfusion at 300 mmHg pressure, or the barrier can be bypassed by immersion of thin slices of fresh brain in the impermeant solution. In either case, addition of aldehyde fixatives and conventional processing then leads to the preservation of extracellular space in electron micrographs. Both procedures are as easy to use for routine fixation as conventional methods. In well fixed tissue the cellular processes are different in size, shape and electron density from the inflated profiles seen after the ingestion of extracellular fluid that accompanies conventional fixation. Moreover, extracellular space is found to separate widely some cellular elements, while leaving others contiguous.