Perfusion Fixation With Glutaraldehyde and Post-Fixation With Osmium Tetroxide for Electron Microscopy

Abstract

The conductivity of cerebral cortex drops during perfusion with glutaraldehyde in 5 min to about 60% of the original value, to remain unchanged during the subsequent 10-15 min of perfusion. Circulatory arrest causes a similar drop in the tissue conductivity. Perfusion of asphyxiated tissue with glutaraldehyde does not produce additional major changes in the conductivity. Perfusion of the cortex with an osmium tetroxide solution causes an initial drop in conductivity. However, after about 3 min this trend is reversed and the conductivity increases again to close to the pre-perfusion value. Perfusion of asphyxiated cortex with OsO4 causes a marked increase of the conductivity. So does perfusion with an OsO4 solution of tissue previously treated with glutaraldehyde. One interpretation of these impedance changes is that glutaraldehyde perfusion causes, like asphyxiation, a transport of extracellular material into the intracellular compartment and that during OsO4 perfusion an extracellular space is again created. This possibility is supported by electron micrographs made of this material. Cerebral cortex perfused with glutaraldehyde and post-fixed with OsO4 shows electron-transparent dendritic elements and to a lesser extent pre-synaptic terminals, which seem to be swollen. When the cortex is flooded with a salt solution during glutaraldehyde perfusion the dendrites exhibit ballooning in the surface layer of the cortex, suggesting that the fluid on the cortex participates in the swelling. The tissue elements in the glutaraldehyde-perfused and OsO4 post-fixed cortex are separated by narrow extracellular spaces. The latter may have been produced by the OsO4 perfusion as is suggested by a comparison of micrographs prepared by freeze substitution (which tends to preserve the water distribution) of glutaraldehyde-perfused but not post-fixed cortex with micrographs of cortex treated with OsO4 after the glutaraldehyde perfusion. In accordance with the conductivity changes, the former micrographs showed very little extracellular space, and in many places tight junctions, whereas the latter showed clefts between the tissue elements.

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