Sodium Pumps and Fixation

by Charles W Scouten, Ph. D.

Living animal cells, every type studied so far, have cell membrane proteins that serve to pump sodium out and potassium in. Sodium continually leaks through the semi-permeable cell membrane, and is continually pumped out, like a pump in a leaking boat. And potassium slowly leaks out of a cell down a concentration gradient, and is pumped back in. More sodium is pumped than potassium, so this process requires metabolic energy, and builds electrical and concentration gradients.

Due to this pumping process, sodium is at a much higher concentrations outside the cell relative to inside (roughly 10 to 1). The inside of a cell is sodium depleted. Anything that disrupts function of the sodium pump proteins will result in a rapid rise of intracellular sodium concentration, as sodium leaks in down the steep gradient, and a slower depletion of intracellular potassium ions, as these leak out down a shallower gradient.

Formaldehyde and other cross-linking fixatives disable the sodium pump proteins on the outside of the cell membrane as soon as it arrives at the cell membrane during a vascular perfusion. Sodium leaks in, and is not pumped out. Potassium leaks out at a slower rate. As a consequence, tonicity increases inside the cell relative to outside, and water must flow in to balance osmolarity.

The cell volume swells, stretching the membrane, and eliminating any extracellular space. Fixation and crosslinking of adjoining proteins continues as cell membranes are pressed against their neighbors. Proteins in one cell membrane are crosslinked to proteins in the membrane of neighboring cells. Later, membranes are made fully permeable by the actions of fixatives, tonicity reaches equilibrium, and cells shrink back to their normal size, but are now linked to neighboring cells and pull them in with them as they shrink. As a consequence, whole organs are shrunk in proportion to their previous extracellular space, in the brain about 20%, and distorted from original position in the living animal. Electron micrographs show no extracellular space in perfusion fixed material.

Suppose all sodium ions were removed from the extracellular fluids? Inside the cell would then be relatively sodium rich. Fixatives would disable the sodium pump, but there would be no influx, just gradual leaking out. It is possible to replace the extracellular fluid during a sacrifice perfusion by replacing it with isotonic sucrose (just under 10%). Perfusion at normal pressures accomplishes this goal in most tissues, but not brain.

The blood-brain barrier prevents vascular fluids from replacing extracellular fluids. Pumping up to 300 mm Hg breaches this barrier, and permits washout of brain extracellular fluids, rapid replacement with isotonic sucrose, and shrinkage free perfusions.

Posted with Permission: the original author is my father