In an attempt to elucidate the problem of extracellular space within the cerebral cortex of the rat, several physiological parameters were analyzed during perfusion fixation with glutaraldehyde and acrolein for electron microscopy. These parameters were electroencephalography, electromyography, electrocardiography, cerebrospinal fluid pressure, impedance of cerebral cortex, perfusion pressure, and flow rate of the perfusate.
The first portion of this investigation dealt with the problem of asphyxiation of tissues during fixation. A substituted blood containing fluorocarbon which was oxygenated served as the means to study this problem. The last portion of this investigation dealt with buffers and molecular size in relationship to the extracellular space. Buffer curves were drawn and conductance studies performed on the solutions prior to perfusion. Since simulated animal studies revealed an insufficient perfusion pressure via gravity within the limitations of laboratory space, and the signs of organ distress developed with a constant head of pressure, a. pulsatile perfusion apparatus was developed which utilized an oxygen tank as the driving force. Impedance measurements were recorded continuously as sensed by a monopolar stainless steel electrode placed in the cerebral cortex. The cerebrospinal fluid pressure in the subarachnoid space was recorded by utilizing a specially prepared hollow screw traversing the parietal bone.
The pertinent findings can be summarized as follows:
1) The electroencephalographic activity flattened at a variable time during perfusion, depending upon the type of buffer used and presence of glucose and/or oxygen.
2) The electromyographic activity was intensified in both frequency and amplitude at the time of fixation except when choline chloride was the buffer.
3) The cerebrospinal fluid pressure was markedly increased when the strong fixative reached the animal, and produced massive muscular depolarization; however, this effect was diminished when oxygenated fluorocarbons were used in the fixative perfusate.
4) The cerebral impedance gradually increased in all animals dying without exsanguination prior to perfusion, and gradually increased in most animals following perfusion. The oxygenated fluorocarbons slowed down the rate of increase which occurred during perfusion.
5) Gravity flow with perfusion bottle at ceiling height failed to maintain adequate perfusion pressures within the rat's physiological blood pressure range.
6) Pulsatile perfusion was necessary to maintain adequate flow rate throughout the time fluids were being administered.
7) The electron micrographs revealed minimal extracellular space except when a phosphate buffer prewash of pH 2 preceded the usual fixation.
The present investigation is in agreement with the work of other investigators who observed a sizeable extracellular space from physiological evidence. The anatomical evidence revealed that the quantity of the extracellular space is dependent upon the conditions of the perfusion fixation procedure. The multiple monitoring approach-- recording relevant variables before, during, and after perfusion fixation-demonstrated considerable promise in improving fixation methods, as well as in helping to clarify the functional morphology of the brain.
Robert L. Schultz
Bernell E. Baldwin
Guy M. Hunt
Walter H. B. Roberts
E. Harold Shryock
William H. Taylor
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Central Nervous System -- physiology; Rats -- anatomy & histology
Loma Linda University Libraries
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Witzel, Everet W., "Physiological Parameters During Perfusion Fixation of the Rat Central Nervous System for Electron Microscopy" (1972). Loma Linda University Electronic Theses, Dissertations & Projects. 1134.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives